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CA 02672205 2009-06-09
WO 2008/077079 PCT/US2007/088011
COMPOSITIONS CONTAINING, METHODS INVOLVING, AND USES OF NON-NATURAL
AMINO ACIDS AND POLYPEPTIDES
RELATED APPLICATIONS
[00011 This application claims the benefit of U.S. Non-Provisional Patent
Application No. 60/870,594 filed
December 18, 2006.
FIELD OF THE INVENTION
100021 Non-natural amino acids, polypeptides containing at least one non-
natural amino acid, methods for
producing such. non-natural amino,acids and polypeptides, and uses of such non-
natural amino acids and
polypeptides for diagnostic, environmental, industrial, and therapeutic uses.
BACKGROUND OF THE INVENTION
(00031 The ability to incorporate non-genetically,encoded amino acids (i.e.,
"non-natural amino acids") into
proteins permits the introduction of chemical functional groups that could
provide valuable alternatives to the
naturally-occurring functional groups, such as the epsilon -NH2 of lysine, the
sulthydryl -SH of cysteine, the
imino group of histidine, etc. Certain cheniical functional groups are
documented as inert to the functional
groups found in the 20 common, genetically-encoded amino acids but react
cleanly and efficiently to form
stable linkages with fttnctional groups that can be incorporated onto non-
natural amino acids.
100041 Methods are now available to selectively introduce chemical functional
groups that are not found in
proteins, that are chemically inert to all of the functional groups found in
the 20 comnion, genetically-encoded
amino acids and that may be used to react efficiently and selectively with
reagents comprising certain functional
groups to form stable covalent linkages.
SUMMARY OF THE INVENTION
100051 Described herein are methods, conipositions, techniques and strategies
for making, purifying,
characterizing, and using non-natural amino acids, non-natural amino acid
polypeptides and modified non-
natural amino acid polypeptides. In one aspect are methods, compositions,
techniques and strategies for
derivatizing.a non-natural amino acid and/or a non-natural amino acid
polypeptide. In one, embodiment, such
methods, compositions, techniques and strategies involve chemical
derivatization, in other embodiments,
biological derivatization, in other embodiments, physical derivatizationõin
other embodiments a cornbination of
derivatizations. In further or additional embodiments, such derivatizations
are regioselective. In further or
additional embodiments, such derivatizations are regiospecific. In further or
additional embodiments, such
dcrivations are stoichiometric or near stoichiometric in both the non-natural
amino acid containing reagent and
the derivitizing reagent. In further or additional embodiments, such
derivatizations are rapid at ambient
temperature. In further or additional embodiments, such derivatizations occur
in aqueous solutions. In further or
additional embodiments, such derivatizations occur at a pH between about 4 and
about 10. In further or
additional embodiments, such derivatizations are stoichiometric, near
stoichiometric or stoichiometric -like in
both the non-natural amino acid containing reagent and the derivatizing
reagent. In further or additional
embodiments are provided methods which allow the stoichiometric, near
stoichiometric or stoichiometric-like
incorporation of a desired group onto a non-natural amino acid polypeptide. In
further or additional
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embodinients are provided strategies, "reaction mixtures, synthetic conditions-
which allow the stoichiometric,
near stoichiometric or stoichiometric -like incorporation of a desired group
onto a non-natural 'amino acid
polypeptide.
[00061 In one aspect are non-natural aniino acids for the chemical
derivatization of peptides and proteins
based'upon the reactivity of a carbonyl group or masked carbonyl group,
including a group containing.at least
one.ketone;group, and/or at least one aldehyde groups. In further or
additional aspects are non-natural amino
acids for the chemical derivatization of peptides and proteins based upon the
reactivity ofa hydrazine group or
masked hydrazine group. In further or additional embodiments, at least one of
the aforementioned non-natural
amino acids is incorporated into a polypeptide, that is, such embodiments are
non-natural amino acid
polypeptides. In further or additional embodiments, the non-natural amino
acids are functionalized on their
sidechains such that their reaction with a derivatizing molecule generates an
indole containing linkage. In
further or additional embodiments are non-natural amino acid polypeptides that
can react with a derivatizing
niolecule to generate a non-natural amino acid polypeptide containing an
indole linkage. In further or additional
embodinients, the non-natural amino acids are selected from amino acids having
carbonyl and/or hydrazine
sidechains. In further or additional embodimeints, the non-nathiral amino
acids comprise a masked sidechain,
including a masked hydrazine group and/or a masked carbonyl group.
100071 In further or additional embodiments, the non-natural aniino. acids
comprise carbonyl sidechains where
the carbonyl is selected from a ketone or an aldehyde. In another
embodiment:are non-natural amino acids
containing a functional group that is capable of forming an indole upon
treatment with an appropriately
functionalized reagent. In some embodiments are non-natural amino acids
containing an indole moiety. In a
further embodiment are-non=natural amino acids containing an indole-moiety for
the treatment of disorders,
conditions or diseases. In a further or additional embodinient, the non-
natural amino acids resemble a natural
amino aci`d in structure but contains one. of the aforementioned functional
groups. In another or further
embodinient the non-natural amino acids resemble phenylalanine or. tyrosine
(aromatic amino acids); while in a
separate embodiment, the non-natural amino acid's resemble alanine and leucine
(hydrophobic amino acids). In
onc embodiment, the non-natural amino acids have properties that are distinct
from those of the natural amino
acids. In one embodiment, such distinct properties are. the chemical
reactivity of the side chain, in a further
embodiment this distinct chemical reactivity permits the side chain of the non-
natural amino acid to undergo a
reaction while being a unit of a polypeptide even though the side chains of
the naturally-occurring amino acid
units in the same polypeptide do not undergo the aforementioned reaction. In a
further embodiment, the side
chain of the non-natural amino acid has chemistry orthogonal to those of the
naturally-occurring amino acids. In
a further embodiment, the side chain-of the non-natural anuno acid,comprises
an electrophile-containing inoiety;
in a further embodiment, the electrophile-containing moiety on.the sidechain
of the non-natural amino acid can
undergo, nucleophilic attack to generate, a heterocycle-derivatized protein,
including a nitrogen-containing
heterocycle-derivatized protein (e.g., containing an indole moiety). In any
ofthe aforementioned ernbodiments
in this paragraph, the.non-natural amino=acid are either separate molecules or
incorporated into a polypeptide of
any length; if thelatter, then the polypeptide, in.some:embodiments, further
incorporates.additional naturally-
occurring or non-natural amino acids.
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100081 In another aspect are hydrazine-substituted molecules for the
production of derivatized non-natural
anlino acid polypeptides based upon an indole-containing heterocycle linkage.
In a further embodiment are
hydrazine-substituted molecules used to derivatize carbonyl-containing non-
natural amino acid polypeptides via
the formation ofan indole-containing:heterocycle linkage. In
furtherembodiments the aforementioned carbonyl-
S containing non-natural amino acid polypeptides are ketone-containing
non=natural.amino acid polypeptides. In
further or additional embodiments, the carbonyl-containing non-natural aniino
acids, comprise sidechains where
the carbonyl.is selected from a ketone, or an aldehyde. In further or
additional embodiments, the hydrazine-
substituted molecules comprise a. desired functionality: In further or
additional embodiments,,the hydrazine-
substitutedmolecules are hydrazine-substituted polyethylene glycol
(PEG).molecules. In a further embodiment,
the sidechain of the non-natural anuno acid has:a chetrustry orthogonal to
those of the naturally-occurring amino
acids that allows the non-natural anuno acid to. reacC selective,ly with the
hydrazine-substituted molecules. In a
further embodiment, the sidechain of the non-natural amino acid corriprises an
electrophile-containing moiety
that reacts selectively with the hydrazine-containing. molecule; in a further
embodiment, the electrophile-
containing moiety on the sidechain of the non-natural amino acid can undergo
nucleophilic attack to generate a
heterocycle-derivatized protein, including a nitrogen-containing heterocycle-
derivatized protein. In a further
aspect related to the embodiments described in this paragraph are the modified
non-natural amino acid
polypeptides that result from the reaction of the derivatizing molecule with
the non-natural amino acid
polypeptides. Further embodiments include any further modifications of the
already modified inon-natural arnino
acid polypeptides.
100091 In another aspect are carbonyl-substituted molecules for the production
of derivatized non-natural
amino. acid polypepti,des based upon a heterocycle, including. a nitrogen-
containing heterocycle (e.g., an indole
or a multi-cyclic structure containing an indole portion), linkage. In a
further embodiment are carbonyl-
substituted molecules used to derivatize hydrazine-containingnon-natural amino
acid polypeptides via. the
formation of an indole-containfng heterocycle linkage. In a-further embodiment
are carbonyl-substituted
molecules that can form such heterocycle tivith a hydrazine-containing non-
natural amino acid potypeptide in a
pH range between about 1 and about 6. In a further embodiment are carbonyl-
substituted. molecules used to
derivatize hydrazine-containing non-natural amino acid polypeptid.es via the
formation of an indole-containing
heterocycle linkage between the" derivatizing molecule and the hydrazine-
containing non-natural amino acid
polypeptides. In a ftirther embodiment the carbonyl-substituted molecules are
ketone-substitued molecules, in
otlter aspects aldehyde-substituted molecules. In further embodiments, the
carbonyl-substituted molecules
comprise a desired functionality. ln further or additional embodiments, the
aldehyde-substituted molecules are
aldeltyde-substituted polyethylene glycol (PEG) molecules. In a furtlier
enibodiment, the sidechain of the non-
natural amino acid has a chetnistry orthogonal to those of the naturally-
occurring amino acids that allows the
non-natural amino acid to react selectively with the carbonyl-substituted
molccules. In a further embodiment,
the sidechain of tlie non-natural amino acid comprises a moiety (e.g.,
hydrazine group) that reacts selectively
witli the carbonyl-containing molecule; in a further cmbodiment, the
nucleophilic moiety on the sidechain of the
non-natural amino acid can undergo electrophilic attack to generate a
heterocyclic-derivatized proteui, including
a nitrogen-containing heterocycle-derivatized protein. In a further aspect
related to the embodiments described
in this paragraph are the modified non-natural amino acid polypeptides that
result from the reaction of the
derivatizing molecule with the non-natural aniino acid.polypeptides. Further
embodiments include any further
modificatioris of the already modified non-natural amino acid polyp,eptides.
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100101 In another aspect are inono-, bi- and multi-funct9onal linkers for the
generatioit of derivatized non-
natural anvno acid "polypeptides based upon an indole-containing heterocycle
linkage. In one embodiment are
molecular linkers (bi- and niulti-funetional) that can be used to. connect
carbonyl-containing non-natural amino
acid polypeptides to other molecules. In another embodiment are molecular
linkers (bi- and niulti-functional)
that can be used to connect hydrazine-containing non-natural anrino acid
polypeptides to other molecules. In
another embodiment.. the carbonyl-containing non-natural amino acid
polypeptides comprise a ketone, or an
aldehyde. In an embodinient utilizing a hydrazine-containing non-natural amino
acid polypeptide, the molecular
linker contains a carbonyl group at one of its termini; in further
embodiments, the carbonyl group is selected
from an aldehyde group, or a ketone group. In further or additional
embodiments, the hydrazine-substituted
linker molecules are hydrazine-substituted polyethylene glycol (PEG) linker
molecules. In fiutlter or additional
embodiments; the: carbonyl-substituted linker molecules are
carbonyl=substituted polyethylene glycol (PEG)
linker molecules. In further cmbodiments, the phrase "other molecules"
includes, by way of example only;
proteins, other polymers and small molecules. In further or. additional
embodiments, the hydrazine=containing.
inolecular linkers comprise the same orequivalent groups on all termini'so
that. upon reaction with a carbonyl-
containing non-natural amino acid polypep6de, the xesulting product is the
homo-niultimerization of the
carbonyl-containing non-natural amino acidpolypeptide. In further embodiments,
the homo-multimerization is a
homo-dimerization. In further or additional embodiments, the carbonyl-
containing molecular linkers comprise
the same or equivalent groups on all termini so that upon reaction with a
hydrazine-containing non-natural
amino acid polypeptide, the resulting product is the homo-multimerization of
the hydrazine-containing non-
natural amino acid polypeptide. In further embodiments, the homo-
multimerization is a homo-dimerization. In a
further embodiment, the sidechain of the non-natural amino acid has a
chemistry orthogonal to those of the
naturally-occutring amino acids that allows the non-natural amino acid to
react selectively with the hydrazine-
substituted linker molecules. In a further embodiment, the sidechain of the
non-natural amino acid has a
chemistry orthogonal to those of the naturally-occuning amino acids that
allows the non-natural amino acid to
react selectively with the carbonyl-substituted linker molecules. In a further
embodiment, the sidechain of the
non-natural amino acid comprises an electrophile-containing moiety that reacts
selectively with the hydrazine-
containing linker molecule; -in a further embodiment; the electrophile-
containing moiety on the sidechain of the
non-iiatural aniino acid can undergo,:nucleophilic attack by the hydrazine-
containing linker molecule to generate
a heterocycle-derivatized protein, including a nitrogen-containing heterocycle-
derivatized protein. In a further
aspect related to, the embodiments described im this paragraph are the linked
(modified) non-natural amino acid
polypeptides that resul.t from the reaction of the, linker-.molecule with the;
non-natural. amino acid polypeptides.
Further embodiments include=any-further modifications of the alreadylinked
(iiiodified) non-natural amino acid
polypeptides.
100111 In one aspect~are methods to derivatize proteins via the reaction of
carbonyl and hydrazine reactants to
generate a heterocycle-derivatized protein, including a nitrogen-containing.
heterocycle-derivatized protein.
included within this aspect are methods for the derivatization of proteins
based upon the condensation of
carbonyl- and hydrazine-containing reactants to generate a heterocycle-
derivatized protein adduct, including a
nitrogen-containing heterocycle-derivatized protein adduct. In additional or
further embodiments are methods to
derivatize ketone-containing proteins or aldehyde-containing proteins with
hydrazine-functionalized
polyethylene glycol (PEG) molecules. In yet additional or further aspects, the
hydrazine-substituted molecule
can include proteins, other polymers, and small molecules.
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100121 In another aspect are methods for the chemical synthesis of hydrazine-
substituted molecules for the
derivatization of carbonyl-substituted proteins. In, one embodiment, the
hydrazine-substituted molecule can
comprise peptides, other polymers (non-branched and branched) and small
niolecules. In one embodiment are
methods for the preparation of hydrazine-substituted molecules suitable for
the derivatization of carbonyl-
containing non-natural amino acid polypeptides, including by way of example
only, ketone-, or aldehyde-
containing non-natural amino acid polypeptides. In a further or additional
embodiment, the non-natural amino
acids arc incorporated site-specifically during the in vivo translation of
proteins. In a further or additional
embodiment, the hydrazine-substituted molecules allow for the site-specific
derivatization of carbonyl-
containing non-natural amino acids via nucleophilic attack of each carbonyl
group to form a heterocycle-
derivatized polypeptide, including a nitrogen-containing heterocycle-
derivatized polypeptide in a site-specific
fashion. In a further or additional entbodiment, the method for the
preparation of hydrazine-substituted
molecules provides access to a wide variety of site-specifically derivatized
polypeptides. In a further or
additional embodiment are methods for synthesizing hydrazine-functionalized
polyethyleneglycol (PEG)
molecules.
100131 In another aspect are methods for the chemical synthesis of carbonyl-
substituted molecules for the
derivatization.of hydrazine-substituted non-natural amino acid polypeptides.
In one embodiment, the carbonvl-
substituted niolecule is a ketone-, and/or an aldehyde-substituted molecule.
In another embodiment, the
carbonyl-substituted molecules include proteins, polymers (non-branched and
branched) and small molecules.
In a further or additional embodiment, such methods complement technology that
enables the site-specific
incorporation of non-iiatural amino acids during the in vivo translation of
proteins. In a further or additional
embodiment are general methods for the preparation of carbonyl-substituted
molecules suitable for reaction with
hydrazine-containing non-natural amino acid polypeptides to provide site-
specifically derivatized non-natural
amino acid polypeptides. In a further or additional embodiment are methods for
synthesizing carbonyl-
substituted polyethylene glycol (PEG) molecules.
100141 In another aspect are methods for the chemical derivatization of
carbonyl-substituted non-natural
amino acid polypeptides using a liydrazine-containing bi-functional linker. In
one embodiment are methods for
attaching a hydrazine-substituted linker to a carbonyl-substituted protein via
a condensation reaction to generate
a heterocycle, including a nitrogen-containing heterocycle, linkage. In
further or additional embodiments, the
carbonyl-substituted non-natural amino acid is a ketone-, and/or an aldehyde-
substituted non-natural amino
acid. In further or additional embodiments, the non-natural amino acid
polypeptides are derivatized site-
specifically and/or with precise control of three-dimensional structure, using
a hydrazine-containing bi-
functional linker. In one embodiment, such methods are used to attach
motecular linkers (mono- bi- and multi-
functional) to carbonyl-containing (including by way of example ketone-,
and/or an aldehyde-containing) non-
natural amino acid polypeptides, wherein at least one of the linker termini
contains a hydrazine group which can
link to the carbonyl-containing rion-natural amino acid polypeptides via a
heterocycle, including a nitrogen-
containing heterocycle, linkage. In a further or additional embodiment, these
linkers are used to connect the
carbonyl-containing non-natural amino acid polypeptides- to other molecules,,
including by way of example,
proteins, other polymers (branched and non-branched) and small molecules.
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[00151 In some embodiments, the non-natural amino acid polypeptide is linked
to a water soluble polymer. In
some embodiments, the water soluble polymer comprises a polyethylene glycol
moiety. In some embodiments,
the polyethylene glycol molecule is a bifunctional polymer. In some
embodiments, the bifunctional polymer is
linked to a second polypeptide. In some embodiments, the second polypeptide is
identical to the first
polypeptide, in other embodiments, the second polypeptide is a different
polypeptide. In some embodiments, the
non-natural amino acid polypeptide comprises at least two amino acids. linked
to a water soluble polymer
comprising a poly(ethylene glycol) moiety.
100161 In some embodiments, the non-natural anuno acid polypeptide comprises a
substitution, addition or
deletion that increases af6nity of the non-natural amino acid polypeptide for
a receptor. In some embodiments,
the non-natural amino acid polypeptide comprises a substitution, addition, or
deletion that increases the stability
of the non-natural amino acid polypeptide. In some embodiments, the non-
natural amino acid polypeptide
comprises a substitution, addition, or deletion that increases the aqueous
solubility of the non-natural amino acid
polypeptide. In some embodiments, the non-natural amino acid polypeptide
comprises a substitution, addition,
or deletion that increases the solubility of the non-natural amino acid
polypeptide produced in a host cell. In
some embodiments, the non-natura] amino acid polypeptide comprises a
substitution, addition, or deletion that
modulates protease resistance, serum half-life, immunogenicity, and/or
expression relative to the amino-acid
polypeptide without the substitution, addition or deletion.
100I7J In some embodiments, the non-natural amino acid polypeptide is an
agonist, partial agonist, antagonist,
partial antagonist, or inverse agonist. In some embodiments, the agonist,
partial agonist, antagonist, partial
antagonist, or inverse agonist comprises a non-natural amino acid linked to a
water soluble polymer. In some
enibodinients, the water polymer comprises a polyethylene glycol moiery. In
some embodiments, the
polypeptide comprising a non-natural amino acid linked to a water soluble
polymer prevents dimerization of the
corresponding receptor. In some embodiments, the polypeptide contprising a non-
natural amino acid linked to a
water soluble polymer modulates binding of the polypeptide to a binding
partner, ligand or receptor. In some
embodiments, the polypeptide comprising a non-natural amino acid linked to a
water soluble polymer modulates
one or more properties or activities of the polypeptide.
100181 ln some embodiments, the selector codon is selected from the group
consisting of an amber codon,
ochre codon, opal codon, a unique codon, a rare codon, an unnatural codon, a
five-base codon, and a four-base
codon.
100191 Also described herein are methods of making a non-natural amino acid
polypeptide litilced to a water
solublc polymer. In some embodiments, the niethod comprises contacting an
isolated polypeptide comprising a
non-natural amino acid with a water soluble polymer comprising a moiety that
reacts with the non-natural
amino acid. In some embodiments, the non-natural amino acid incorporated into
is reactive toward a water
soluble polymer that is otherwise unreactive toward any of the 20 conunon
amino acids. In some embodiments,
the water polymer comprises a polyethylene glycol moiety. The molecular weight
of the polymer optionally is
within a desired polymer molecular weight range.
100201 Also described herein are compositions comprising a polypeptide
comprising at least one of the non-
natural amino acids described herein and a pharmaceutically acceptable
canrier. In some embodiments, the non-
natural amino acid is linked to a water soluble polymer. Also described herein
are pharmaceutical compositions
comprising a pharmaceutically acceptable carrier and a polypeptide, wherein at
least one amino acid is
substituted by a non-natural aniino acid. In some embodiments, the non-natural
amino acid comprises a
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saccharide moiety. In sonie embodiments, the water soluble polymer is linked
to the polypeptide via a
saccharide moiety. Also described herein are prodrugs of the non-naturalamino
acids, non-natural amino acid
polypeptides, and modified non-natural amino acid polypeptides; further
described herein are compositions
coniprising such prodrugs and a pharmaceutically acceptable carrier. Also
described herein are metabolites of
the non-natural amino acids, non-natural amino acid polypeptides, and modified
non-natural amino acid
polypeptides; in some embodiments, such metabolites have a desired activity
that complements or synergizes
with the activity of the non-natural amino acids, non-natural aniino acid
polypeptides, and modified non-natural
amino acid polypeptides. Also described herein are the use of the non-natural
amino acids, non-natural amino
acid polypeptides, and modified non-natural amino acid polypeptides described
herein to provide a desired
metabolite to an organism, including a patient in need of sucli metabolite.
100211 Also described herein are cells comprising a polynucleotide encod'uig
the polypeptide comprising a
selector codon. In some embodinients, the cells comprise_an orthogonal RNA
synthetase, and/or an orthogonal
tRNA for substitutinga,non-natural amino acid into the polypeptide. In
some,embodiments the cells are in a cell
culture, whereas in other embodiments the cells of part of a multicellular
organism, including amphibians,
reptiles, birds, and mamnials. In any of the cell embodiments, further
enibodiments include expression of the
polynucleotide to produce the.non-natural amino acid polypeptide. In other
embodiments are organisms that can
utilize the non-natural amino acids described herein to produce a non-natural
amino acid polypeptide, including
a modified non-natural amino acid polypeptide. In other embodiments are
organisms containing the non-natural
anuno acids, the non-natural amino acid polypeptides, and/or the modified non-
natural amino acid polypeptides
described herein. Such organisms include unicellular and multicellular
organisms, including amphibians,
reptiles, birds, and niammals. In some embodiments, the non-natural amino acid
polypeptide is produced in
vitro. In some embodiments, the non-natural amino acid polypeptide is produced
in cell lysate. In some
embodiments, the non-natural anuno acid polypeptide is produced byribosomal
translation.
100221 Also described herein are methods of making a polypeptide comprising a
non-natural aniino acid. In
soine embodiments, the methods comprise culturing cells comprising a
polynucleotide or polynucleotides
encoding a polypeptide, an orthogonal.RNA synthetase and/or an orthogonal tRNA
under conditions to permit
expression of the polypeptide; and purifying the polypeptide from the cells
and/or culture medium.
100231 Also described herein are libraries of the non-natural amino acids
described herein or libraries of the
non-natural amino acid polypeptides described herein, or libraries of the
modified non-natural amino acid
polypeptides described herein, or combination libraries thereof. Also
described herein are arrays containing at
least one non-natural amino acid, at least one non-natural aniino acid
polypeptide, and/or at least one modified
non-natural aniino acid. Also described herein are arrays containing at least
one polynucleotide encoding a
polypeptide comprising a selector codon. The arrays described herein are used,
for exaniple, to screen for the
production of the non-natural amino acid polypeptides in an organism (either
by detecting transcription of the
polynucleotide encoding the polypeptide or by detecting the translation of the
polypeptide).
100241 Also described herein are methods for screening libraries described
herein for a desired activity, or for
using the arrays described herein to screen the libraries described herein, or
for other libraries of compounds
and/or polypeptides and/or polynucleotides for a desired activity. Also
desccibed herein is the use of such
activity data from library screening to develop and discover new therapeutic
agents, as well as the therapeutic
agents themselves.
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100251 Also described herein are niethods of increasing therapeutic half life,
serum half-life or circulation
time of a polypeptide. In some embodiments, the methods comprise substituting
at least one non-natural amino
acid for any one or niore aniino acids in a naturally occurring polypeptide
and/or coupling the polypeptide to a
water soluble polymer.
[00261 Also described herein are methods of treating a patient in need of such
treatment with an effective
amount of a pharmaceutical composition which comprises a polypeptide
comprising a non-natural amino acid
and a pharmaceutically acceptable carrier. In some embodiments, :the non-
natural amino acid is coupled to a
water soluble polymer.
100271 In further or alternative embodiments are methods for treating a,
disorder, condition or disease, the
method comprising administering a therapeutically effective.amount of`a non-
natural amino acid polypeptide
comprising at least one non-natural amino acid selected from the group
consisting of an indole-containing.non-
natural amino acid, a carbonyl-containing non-natural amino acid, and a
hydrazine-containing non-natural
ainino acid. In other embodiments such non-natural amino acids have been
synthetically incorporated into the
polypeptide as described herein. In further or alternative embodiments
such.non-natural amino acid polypeptide
comprises at least one non-natural amino acid selected from amino acids of
Formula I-XV, In another
embodiment, such non-natural amino acid polypeptide comprises at least one
natural aniino acid selected from
amino acids of compounds 1-4.
100281 In further or altetnative embodiments are methods for treating, a
disorder, condition or disease, the
method comprising administering a therapeutically effective amount of a non-
natural amino acid polypeptide
comprising at least one non-natural amino acid having the structure of
compounds:
R H
R R3 A, ~~ N R3 R3 A' af\ N R.
3 B Rs B n
R'\ R` R, RZ Rs
H~O Rs ~0 2~ m~ `
Rs
Ra N Rs Rs R
s
~ N Rs
R3 R A~~/-~ Rs R3 R A,13wt
B m Rs Rs
Rj. T-r RZ Ra Rj~ N Rz
H Rd 0 3 H R4.0 q Rs Rs
wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene,
lower cycloalkylene,
substituted lower cycloalkylene, lower alkenylene, substituted lower
alkenylene, alkynylene,
substituted alkynylene, lower heteroalkylene, substituted heteroalkylene,
lower
heterocycloalkylene, substituted lower heterocycloalkylene, arylene,
substituted arylene,
heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene, or
substituted aralkylene;
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B is optional, and when present is a linker, linked at one end to an indole-
containing moiety, the linker
selected from the group consisting of lower alkylene, substituted lower
alkylene, lower
alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted
lower
heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-,
-O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted
alkylene)-, -S(O)k-
where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -
NS(0)2-, -OS(O)Z-,
-C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or
substituted alkylene)-, -
N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-
(alkylene or
substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-
, -N(R')CO-
(alkylenc or substituted alkylene)-, -1`'(R')C(O)O-, -S(O)kN(R')-, -
N(R')C(O)N(R')-,
-N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNO-,)N(R')-, -
N(R')C(NCOOR')N(R')-,
-N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z-N=N-, and -C(R')2-N(R')-
N(R')-
and each R' is independently I-I, alkyl, or substituted alkyl;
R, is H, an amino protecting.group, resin, at least one aniino acid,
polypeptide, or polynucleotide; and
R2 is OH, an esterprotecting group, resin, atleast one amino acid,
polypeptide, or polynucleotide;
n is 0, .1, 2, or 3, and m is 0, 1, 2, or 3, provided that at least one of n
or m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R,
group can contain 0, 1, or 2 R.
groups and each R. is independently selected from the group consisting of H,
halogen, alkyl,
substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)L.R", where k
is 1, 2, or 3, where
each R" is independently H; alkyl, or substituted alkyl; or when more than one
R. group is present, two
R, optionally form an aryl, cycloalkyl or heterocycloalkyl;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted
lower alkyl, or R3 and R4 or
two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
cach R5 is independently H, halogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, alkylalkbxy, substituted
alkylalkoxy,
polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl,
heteroaryl,
substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted
aralkyl, -(alkylene or
substituted alkylene)-ON(R")2, OH, NH2, CN, NO2, -(alkylene or substituted
alkylene)-
C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -
C(O)R",
-C(O)ZR", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl,
substituted
alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl,
substituted aryl,
heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or
when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or R3 is L-X; where, X is a selected from the group consisting of: a label; a
dye; a polymer; a water-
soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a
cytotoxic
compound; a drug; an affinity label; a photoaffinity label; a. reactive
compound; a resin; a
second protein or polypeptide or polypeptide analog; an antibody or antibody
fragment; a
metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a
DNA; a RNA; an
antisense polynucleotide; a saccharide, a water-soluble dendrimer, a
cyclodextrin, a
biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing
moiety; a
radioactive moiety; a novel functional group; a group that covalently or
noncovalently
9
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interacts with other molecules; a photocaged moiety; an actinic radiation.
excitable moiety; a
ligand; a photoisomerizable moiety; biotin; a biotin.analogue; a moiety
incoiporating a heavy
atom; a chemically cleavable group; a photocleavable group; an elongated side
chain; a
carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety;
an isotopically
labeled moiety; a biophysical probe; a phosphorescent group; a
chemiluminescent group; an
electron densegroup; a.magnetic group; an intercalating group; a chromophore;
an energy
transfer agent; a biologically active agent; a detectable label; a small
molecule; an inhibitory
ribonucleic acid;.a radionucleotide; a neutron-capture agent; a derivative of
biotin; quantuni
dot(s); a nanotransmitter; a radiotransmitter; an abzyme, an activated complex
activator, a
virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone,
an antioxidant, an
aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecttle, a
mimotope, a receptor,
a reverse micelle, and any combination thereof; and L is optional, and when
present.is.a linker
selected from the group consisting of'alkylene, substituted alkylene,
alkenylene, substituted
alkenylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or
substituted
alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted
alkylene)-, -C(O)-,
-C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or
substituted alkylene)-, -
N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-
(alkylene or
substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substittited
alkylene)-, -N(R')CO-
(alkylene or substituted alkylene)-, -N(R')C(O)0-,.-(alkylene or substituted
alkylene)-O-
N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(allcylene or substituted
alkylene)-, -
(alkylene. or substituted alkylene)=S(O)k-( alkylene or substituted alkylene)-
S-, -(alkylene or
substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-,
-N(R')S(O)kN(R!)-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-
N=N-,
and -C(R')z-N(R')-N(R')-, where each R' is independently H, alkyl, or
substituted alkyl;
when more than one RS group is present, two ortho R5 groups can optionally
form a heterocycloalkyl or
an aromatic heterocycloalkyl;
or the -B-itidole containing moiety together forni a bicyclic or tricyclic
cycloalkyl or heterocycloalkyl
coniprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or
solvate thereof.
100291 In one embodiment, X is selected from a water-soluble polymer; a
polyalkylene oxide; a polyethylene
glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one
amino acid; at least one sugar group;
at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin
analogue; a detectable label; and any
combination thereof;
(00301 In one embodiment, both A and B are bonds, each R3 is H and R4 is H. In
a further embodiment, each
of Ri and R2 are at least one_ amino acid. In a further embodiment, each of R,
and R2 are at least two amino
acids. In a further embodiment, each of R, and R, are at least three.amino
acids. In a further embodiment, each
of R, and R2 are at least four amino acids. In a further embodiment, each of
R, and R2 are at least five. amino
acids: In a fttrther embodiment, each of R, and R2 are at least eix amino
acids.
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100311 In another embodiment are methods for treating a disorder, condition
or, disease, the method
comprising administering a therapeutically effective amount of a non-natuial
amino acid polypeptide with a
pharniaceutically acceptable carrier. In a further embodiment is a method for
treating a disorder, condition or
disease, wherein X is a water-soluble polymer. In another embodiment is a
method for treating a disorder,
condition or disease, wherein X is a derivative of polyethylene glycol. ln a
further embodiment, is a method for
treating a disorder, condition or disease, wherein X is a cytotoxic compound.
In a fitrther embodiment is a
metliod for treating a disorder, condition or disease, wherein X is a drug.
100321 In some embodiments are methods for treating a disorder, condition or
disease, wherein X is a second
polypeptide. In a further embodiment are methods for treating a disorder,
condition ordisease, wherein the
second polypeptide is a peptide containing a non-natural amino acid
polypeptide. In a further embodiment, are
methods for treating a disorder, condition or disease, wherein the second
polypeptide has the same amino acid
structure as the non-natural amino acid polypeptide of compounds having the,
structures 1-4. In another
embodiment is a method for treating.a disorder; condition or disease, wherein
X is a detectable label. In yet
another embodiment are methods for treating a disorder, condition or disease,
wherein the at least one non-
natural amino acid of compounds.1-4 is incorporated at a specific site withiin
the polypeptide. In.yet a further
embodimejit are methods for treating a disorder, condition or disease, wherein
the non-natural amino acid of
compounds 1-0 is incorporated using a translation system.
(00331 In another embodiment is a method for treating a disorder, condition or
disease comprising
administering a therapeutically effective amount of a polypeptide comprising
at least one non-natural amino
acid selected from the group consisting of:
Ra
k ~ NH
~ H N
R5 Ri, N R2 n
R5 H R4 O Ra m Rs
Ri.N Rz RS
5 R5
Rs 6 Rs
H Rs
P-a~
ti n 1/ R5 R5
m RS R5
Rt, N R 2 ~ RS R
RS
5 Ri.N R
H RaO H RaO
8
wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene,
lower cycloalkylene,
substituted lower cycloalkylene, lower alkenylene; substituted lower
alkenylene, alkynylene,
substituted alkynylene, lower heteroalkylene, substituted heteroalkylene,
lower
heterocycloalkylene, substituted lower heterocycloalkylene, arylene,
substituted arylene,
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heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene, or
substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole-
containing moiety, the liril:er
selected from the group consisting of lower alkylene, substituted lower
alkylene, lower
alkenylene; substituted lower alkenylene,.lower heteroalkylene, substituted
lower
heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-,.
-O-(alkylene or substituted alkylene)-,.-S-, -S-(alkylene or substituted
alkylene)-, -S(O)k-
where k is 1, 2; or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -
NS(O)2-, -OS(O)Z-,
-C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or
substituted alkylene)-. -
N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-
(alkylene or
substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-
, -N(R')CO-
(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -
N(R')C(O)N(R')-,
-N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNO2)N(R')-, -
N(R')C(NCOOR')N(R')-,
-NT(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')a-N-N-, and -C(R')rN(R')-
N(R')-
and each R' is independently H, alkyl, or substituted alkyl;
R, is H, an amino protecting group, resin, at least one aniino acid,
polypeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one aniino acid,
polypeptide, or polynucleotide;
n is 0, 1, 2; or 3, and m is 0, 1, 2, or 3, providedthat at least one of n or
m is not 0;
wherein, each ring in structures 1,;2, 3, and 4 that has an associated R9
group can contain 0, 1, or 2 R.
groups and each R, is independently selected from the group consisting of H,
halogen, alkyl,
substituted alkyl, -N(R")Z, -C(O)R", -C(O)N(R")2, -OR", and -S(O)rR", where k
is 1, 2, or 3, where
each R" is independently H, alkyl, or substituted alkyl; or when more than one
R. group is present, two
R, optionally form an aryl, cycloalkyl or heterocycloalkyl;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted
lower alkyl, or R3 and R4 or
two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
each RS is independently H, halogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted
alkylalkoxy,
polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl,
heteroaryl,
substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted
aralkyl, -(alkylene or
substituted alkylene)-ON(R")2, OH, NH2, CN, NO2, -(alkylene or substituted
alkylene)-
C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -
C(O)R",
-C(O)ZR", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl,
substituted
alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl,
substittited aryl,
heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,. or
when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or R5 is L-X, where, X is a selected from the group consisting of: a label; a
dye; a polymer=, a water-
soluble polynter; a derivative of polyethylene.glycol; a photocrosslinker; a
cytotoxic
compound; a drug; an affinity label; a photoaffinity label; a reactive
compound; a resin; a
second protein or polypeptide or polypeptide analog; an antibody or antibody
fragment; a
metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a
DNA; a RNA; an
antisense polynucleotide; a saccharide, a water-soluble dendrimer, a
cyclodextrin, a
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biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing
moiety; a
radioactive moiety.; a novel functional group; a group that covalently or
noncovalently
interacts with other molecules; a photocaged moiety; an actinic radiation
excitable moiety; a
ligand; a photoisomerizable moiety; biotin; a biotin analogue; a nioiety
incorporating a heavy
atom; a chemically cleavable group; a photocleavable group; an elongated side
chain; a
carbon-linked sugar;:a redox-active agent; an amino thioacid; a toxic moiety;
an isotopically
labeled moiety; a biophysical probe; a phosphorescent group; a
chemiluminescent group; an
electron dense group; a magnetic group; an intercalating group; a chromophore;
an energy
transfer agent; a biologically active agent; a detectable label; a small
molecule; an inhibitory
ribonucleic acid; axadionucleotide; a netitron-capture agent; a derivative of
biotin; quantum
dot(s); a nanotransmitter; a radiotransmitter; an abzyme, an activated complex
activator, a
virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone,
an antioxidant, an
aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a
mimotope, a receptor,
a reverse micelle, and any combination thereof; and L is optional, and when
present is a linker
selected from the group consisting of alkylene, substituted alkylene,
alkenylene, substituted
alkenylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or
substituted
alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted
alkylene)-, -C(O)-,
-C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or
substituted alkylene)-, -
N(R')-, -NR'-(alkylene or substituted alkylene)-,-C(O)N(R')-, -CON(R')-
(alkylene or
substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or. substituted
alkylene)-, -N(R')CO-
(alkylene or substituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted
alkylene)-O-
N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted
alkylene)-, -
(alkylene or substituted alkylene)-S(O)k-( alkylene:or substituted alkylene)-S-
, -(alkylene or
substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-; -N(R')C(S)N(R')-,,
-N(R')S(O)k-N(R')-, -N(R')-N=, -C(R')=NT-, -C(R')=N-N(R')-, -C(R')=N-N=, -
C(R')2-N=N-,
and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or
substituted alkyl;
when more than one R5 group is present, two ortho R5 groups can optionally
form a heterocycloalkyl or
an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic
cycloalkyl or ccterocycloalkyl
comprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or
solvate thereof.
100341 In one embodiment, X is selected from a water-soluble polymer; a
polyalkylene oxide; a polyethylene
glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one
amino acid; at least one sugar group;
at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin
analogue; a detectable label; and any
combination thereof;
100351 In one embodiment, both A and B are bonds, each R3 is H and R4 is H. In
a further embodiment, each
of Ri and R2 are at least one amino acid. In a further. embodiment, each of R,
and R, are at least two amino
acids. In a further embodiment, each of Ri and RZ. are at least three amino
acids. In a further embodiment, each
of R, and R., are at least four amino acids. In a fnrther embodiment, each of
R, and R2 are at least five amino
acids. In a further embodiment, each of R, and R2 are at least eix amino
acids.
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100361 In yet another embodiment, is a niethod for treating a disorder,
condition or disease further comprising
administering a pharmaceutically acceptable carrier with the therapeutically
effective amount of the polypeptide
having the compounds of structures 5-8. In a further embodiment, is a method
for treating a disorder, condition
or disease, wherein Ri and R2 are both polypeptides. In yet another
embodiment, is a method for treating a
disorder, condition or disease, wherein X is a water-soluble polymer. In
another einbodiment, is a method for
treating a disorder, condition or disease, wherein X is a derivative of
polyethylene glycol. In yet another
embodiment, is a method.for tieating a disorder, condition or disease, wherein
X is a cytotoxic compound. In
yet a further embodiment,: is a method for treating a disorder, condition or
disease, wherein X is a drug. In yet
another embodiment, is a method for treating a disorder, condition or disease,
wherein X is a second
polypeptide. In yet another embodiment, is a method for treating a disorder,
condition or disease, wherein the
second polypeptide is a peptide containing a non-natural amino acid
polypeptide.
100371 In further or alternative embodiments are methods for treating a
disorder, condition or disease, the
method comprising administering a therapeutically effective amount of a non-
natural amino acid polypeptide
comprising at least one non-natural amino acid having the structure of
compounds 1-4, wherein the polypeptide
is a protein homologous to a therapeutic protein selected from the group
consisting of: alpha- I antitrypsin,
angiostatin, antihemolytic factor, antibody, antibody fragment,
apolipoprotein, apoprotein, atrial natriuretic
factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine,
T39765, NAP-2, EIv'A-78, gro-a, gro-b,
gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand,
cytokine, CC chemokine, monocyte
chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte
chemoattractant protein-3, monocyte
int7ammatory protein-1 alpha, monocyte inllammatory protein-i beta, RANTES,
1309, R83915, R91733, HCCI,
T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony
stimulating factor (CSF),
complement factor 5a, coniplement inhibitor, complement receptor 1, cytokine,
epithelial neutrophil activating
peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelial
neutrophil activating peptide,
erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII,
Factor X, fibroblast growth factor
(FGF), fibrinogen, fibronectin, four-helical bundle protein, G-CSF,, glp-1, GM-
CSF, glucocerebrosidase;
gonadotropin, growth factor,,growth factor receptor, grf, hedgehog protein,
hemoglobin, hepatocyte growth
factor (hGF), hirudin, human growth horinone (hGH), human serum albumin, ICAM-
1, ICAM- I receptor, LFA-
1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II,
interferon (IFN), IFN-alpha, IFN-
beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL41, IL-12,
keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor,
luciferase, neurturin, neutrophil
inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene product,
paracitonin, parathyroid hormone,
PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth,
pyrogenic exotoxin A, pyrogenic
exotoxiit B, pyrogenic exotozin C, pyy, relaxin, renin, SCF, small
biosynthetic protein, soluble complement
receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF
receptor, somatomedin, somatostatin,
somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA,
SEB, SECI, SEC2, SEC3, SED,
SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome
toxin, thymosin alpha I, tissue
plasniinogen activator, tumor growth factor (TGF), tumor necrosis factor,
tumor necrosis. factor alpha, tumor
necrosis factor beta; tumor necrosis factor receptor (TNFR), urotensin, VLA-4
protein, VCAM-1 protein,
vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,
tat, fos, myc, jun, myb, rel,
estrogen receptor, progesterone receptor, testosterone receptor, aldosterone
receptor, LDL receptor, and
corticosterone receptor.
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100381 In a further embodiment are methods for treating a disorder, condition,
or disease, the method
comprising administering compounds having the structures 5-8, wherein the at
least one non-natural acid is
incorporated at a specific site within the polypeptide. In a further
embodiment are methods for treating a
disorder, condition, or disease, the method comprising administering compounds
having the structures 5-8,
wherein the non-natural amino acid is incorporated using a translation system.
In a further enibodiment are
methods for treating a disorder, condition, or disease, the method comprising
administering compounds having
the structures 5-8, wherein the non-natural amino acid is incorporated into
the polypeptide using a translation
system and a post translation modification system. In a further embodiment are
methods for treating a disorder,
condition, or disease, the method comprising administering compounds having
the structures 5-8, wherein the
polypeptide comprising at least one non-natural amino acid is stable for at
least 1 month. In a further
embodiment are methods for treating a disorder, condition, or disease, the
method comprising administering
compounds having the structures 5-8, wherein the polypeptide comprising at
least one non-natural amino acid is
stable for at least 2 weeks.
100391 In further or alternative embodiments are methods for treating a
disorder, condition or disease, the
15, method comprising administering: a therapeutically effective amount of a
non-natural ,amino acid polypeptide
comprising at least one indole-containing non-natural amino acid and the
resulting indole-containing non-
natural amino acid polypeptide increases the bioavailability-of the
polypeptide relative to the homologous
naturally-occurring amino acid polypeptide.
100401 In further or alternative embodiments are methods for treating a
disorder, condition or disease, the
method comprising administering a therapeutically effective amount of a non-
natural anuno acid polypeptide
comprising at least one indole-containing non-natural amino acid and the
resulting indole-containing non-
natural amino acid polypeptide increases the safety profile of the polypeptide
relative to the homologous
naturally-occurring ainino acid polypeptide.
100411 In fiu-ther or alternative embodiments are methods for treating a
disorder, condition or disease, the
method comprising administering a therapeutically effective amount of a non-
natural amino acid polypeptide
comprising at least one indole-containing non-natural amino acid and the
resulting indole-containing non-
natural amino acid polypeptide increases the water solubility of the
polypeptide relative to the homologous
naturally-occurring.amino acid polypeptide.
100421 In further or alternative. embodiments are methods for treating a
disorder; condition or disease, the
metliod comprising administering a therapeutically effective amount of a non-
natural amino acid polypeptide
comprising at least one indole-containing non-natural amino acid and the
resulting indole-containing non-
natural amino acid polypeptide increases the therapeutic half-life of the
polypeptide relative to the homologous
naturally-occurring amino acid polypeptide.
100431 In further or alternative embodiments are metliods for treatntg a
disorder, condition or disease,. the
method comprising administering a tlierapeutically effective amount bf a
non=natural amino acid polypeptide
comprising at least one indole-containing non-natural amino acid and the
resulting indole-containing non-
natural amino acid polypeptide increases the serum half-life of the
polypeptide relative to the homologous
naturally-occurring amino acid polypeptide.
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100441 In further or alternative embodiments are methods for treating a
disorder, condition or disease, the
method comprising administering;a therapeutically effective:amount of a:non-
natural amino acid polypeptide
coniprising at least one indole-containing non-natural amino acid and the
resulting indole-containing non-
natural, amino acid polypeptide extends the circulation, time of the
polypeptide relative to the homologous
naturally-occurring amino.acid polypeptide.
(,0045) In further or alternative embodiments are methods for treating a
disordei, condition or disease, the
method comprising administering a, therapeutically effective amount of a non-
natural amino acid polypeptide
comprising at least one indole-containing non-natural amino acid and the
resulting, indole-containing non-
iiatural ainino acid polypepride nlodulates the biological, activity of
the.polypeptide relative to the homologous
naturally-occurring amino acid polypeptide.
100461 In further or alternative embodiments are methods for treating a
disorder, condition or disease, the
method comprising administering a therapeutically effective ~amount of a-non-
natural amino acid polypeptide
comprising at least one indole-containing non-natural amino acid and the
resulting indole-containing non-
natural amino acid polypeptide modulates the immunogenicity of the polypeptide
relative to the homologous
naturally-occurring amino acid polypeptide.
100471 It is to be understood that the methods and compositions described
herein are not limited to the
particular metliodology, protocols, cell lines, constructs, and reagents
described herein and as such optionally
vary. It is also to be understood that the terminology used herein is for the
purpose of describing particular
enibodiments only, and is not intended to limit the scope of the methods and
compositions described herein,
which, will be limited only by the appended claims.
100481 As used herein and in the appended claims, the singular forms "a,"
"anand "the" include plural
reference unless the context clearly indicates otherwise.
100491 Unless defined otherwise, all technical and scientific xerms used
herein have the same meaning as
commonly understood to one of ordinary skill in the. art to which the
inventions described herein belong.
Although any methods, devices, and materials similar'or equivalent to those
described heiein can be used in the
practice or testing of the. inventions described herein, the preferred
methods, devices and materials are now
described.
100501 The publications discussed herein are provided solely for theii=
disclosure prior to the filing date of the
present application. Notbing herein is to be construed as an admission that
the inventors described herein are not
entitled to antedate such disclosure by virtue of prior invention or for any
other reason.
100511 The term "affinity label," as used heiein, refers to a label which
reversibly or irreversibly binds another
molecule, either to modify it, destroy it, or form a compound with it. By way
of example, affinity labels include
enzymes and their substrates, or antibodies and their antigens.
[0052) The terni.s "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy)
refer to those alkyl groups linked to
molecules via an oxygen atom, an amino group, or a sulfuuatom, respectively.
100531 The term "alkyl," by itself or as part of another molecule means,
unless otherwise stated, a.straight or
branched chain, or cyclic hydiocarbon radical, or: combination thereof, which
optionally is fully saturated,
mono- or polyunsaturated and can include di- and multivalent radicals, having
the number of carbon atoms
designated (i.e. Cl-C1o rneans one to ten'carbons). Exainples of saturated
hydrocarbon radicals include, but are
not limited to, groups such as methyl, ethyl; n-propyl, isopropyl, n-butyl; t-
butyl, isobutyl, sec-butyl,
cyclohexyl; (cyclohexyl)methyl, cyclopropylmethyl, horriologs.and isomefs of,
for example, n-pentyl, n-hexyl,
16
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WO 2008/077079 PCT/US2007/088011
n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one
or more double bonds or triple
bonds. Examples of unsaturated alkyl groups include, but are not limited to,
vinyl, 2-propenyl, crotyl, 2-
isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the
higher homologs and isomers. The term "alkyl," unless otherwise noted, is also
meant to include those
derivatives of alkyl defined in more detail herein, such as "heteroalkyl",
"haloalkyl" and "homoalkyl".
100541 The term. "alkylene" by itself or as part of another molecule means a
divalent radical derived from an
alkane, as exemplified, by (-CHZ-),,, wherein n is 1 to about 24. By way of
example only, such groups include,
but are not limited to, groups having 10 or fewer carbon atoms such as the
structures -CHZCHZ- and -
CH2CH2CH2CI-Ii-. A "lower alkyl" or "lower alkylene" is a shorter chain.alkyl
or alkylene group, generally
having eight or fewer carbon atoms. The term "alkylene," unless otherwise
noted, is also meant to, include those
groups described hercin as "heteroalkylene."
[0055[ The terni "amino acid" refers to naturallyoccurring and non-natural
amino acids, as well as amino acid
analogs and aniino acid mimetics that function in a manner similar to the
naturally occurring amino acids.
Naturally encoded amino acids are the 20 common amino acids (alariine,
arginine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,
lysine, methionine,, phenylalaninc,
proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine
and selenocysteine: Amino acid
analogs refers to compounds that have the same basic chemical structure as a
naturally occurring amino acid, by
way of example only, an cr-carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R
group. Such analogs optionally have modified R groups (by way of example,
norleucine) or optionally inodi6ed
peptide backbones while still retaining the same basic chemical structure as a
naturally occurring amino acid.
Non-limiting examples of amino acid analogs include homoserine, norleucine,
methionine sulfoxide,
methionine methyl sulfonium.
100561 Amino acids may be. referred to herein by either their name, their
three letter symbols or by the one-
letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature
Commission. Additionally,
nucleotides, may be referred to by their commonly accepted single-letter
codes.
100571 By "antibody fragment" is meant any form of an antibody other than the
full-length fotni. Antibody
fragments herein include antibodies that are smaller .comporients that exist
within full-length antibodies, and
antibodies that have been engineered. Antibody fragments include but are not
limited to Fv, Fc, Fab, and
(Fab')2, single chain Fv (scFv), diabodies, triabodies, tetrabodies,
bifunctional hybrid antibodies, CDRI, CDR2,
CDR3, combinations of CDR's, variable regions, framework regions, constant
regions, heavy chains, light
chains, and variable regions, and alternat.ive scaffold non-antibody
molecules, bispecific antibodies, and the like
(Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998,
Curr. Opin. Biotechnol.
9:395-402). Another functional substructure is a single chain Fv (scFv),
comprised of the variable regions of the
irnmunoglobulin heavy and light chain, covalently connected by a peptide
linker (S-z Hu et al., 1996, Cancer
Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain
specificity and affinity for antigen
in a single polypeptide and can provide a convenient building block for
larger, antigen-specific molecules.
Unless specifically noted otherwise, statements and claims that use the term
"antibody" or "antibodies"
specifically includes "antibody fragment" and "antibody fragnients."
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100581 The term "aromatic" or "aryl", as used herein, refers to a closed ring
structure which has at least one
ring having a conjugated pi electron system and includes both carbocyclic aryl
and heterocyclic aryl (or
"heteroaryP" or "heteroaromatic") groups. The carbocyclic or heterocyclic
aromatic group optionally contains
from 5 to 20 ring atonu. The term includes monocyclic rings linked covalently
or fused-ring polycyclic (i.e.,
rings which share adjacent pairs of carbon atoms) groups. An aromatic group
can be unsubstituted or
substituted. Non-limiting examples of "aroniatic" or "aryl", groups include
phenyl, 1-naphthyl, 2-naphthyl, 4-
biphenyl, anthracenyl, and phenantliracenyl. Substituents for each of the
above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents described
herein.
100591 For brevity, the term "aromatic" or "aryl" when used in combination
with other terms (including but
not limited to, aryloxy, arylthioxy, aralkyl) includes both aryl and
heteroaryl rings as defined above. Thus, the
term "aralkyl" or "alkaryl" is meant to include those radicals in which an
aryl group is attached to an alkyl
group (including but not liniited to, benzyl, phenethyl, pyridylmethyl and the
like) including those alkyl groups
in which a carbon atom (including but not limited to, a methylene group) has
been replaced by a heteroatom, by
way of example only, by an oxygen atom. Examples of such aryl groups include,
but are not limited to,
phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like.
[00601 The term "arylene", as used herein, refers to a divalent aryl radical.
Non-limiting examples of
"arylene" include phenylene, pyridinylene, pyrimidinylene and thiophenylene.
Substituents for arylene groups
are selected from the group of acceptable substituents described herein.
100611 The term "at least one amino acid" refers to a single amino acid, a
multiplicity of aniino acids, an
oligopeptide, an amino acid dimer, an aniino acid trimer, an amino acid
tetramer, a polypeptide, a protein, an
antibody, or any other connected chain of amino acids.
100621 A"bifunct.ional polymer", also referred to as a "bifunctional linker",
refers to a polymer comprising
two functional groups that are capable of reacting specifically with other
moieties to form covalent or non-
covalent linkages. Such moieties include, but are not limited to, the side
groups on natural or non-natural amino
acids or peptides which contain such natural or non-natural amino acids. By
way of example only, a bifunctional
linker has a functional group reactive with a group on a first peptide, and
another functional group which is
reactive with a group on a second peptide, whereby forming a conjugate that
includes the first peptide, the
bifunctional linker and the second peptide. Procedures and linker molecules
for attachment of various
compounds to peptides include, e.g., European Patent Application No. 188,256;
U.S. Patent Nos. 4,671,958,
4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789. A "multi-functional
polymer" also referred to as a
"multi-functional linker", refers to a polymer comprising two or more
functional groups that are capable of
reacting with other moieties. Such moieties include, but are not limited to,
the side groups on natural or non-
natural aniino acids or peptides which contain such natural or non-natural
amino acids. (including but not
linuted to, amino acid.side groups) to form covalent or non-covalent linkages.
A bi-functional polymer or multi-
functional polymer is optionally any desired length or molecular weight, and
is optionally selected to provide a
particular desired spacing or conformation between one or more molecules
linked to a compound and molecules
it binds to or the compound.
100631 'fhe term "bioavailability," as used herein, refers to the rate and
extent to which a substance or its
active moiety is delivered from a pharmaceutical dosage form and becomes
available at the site of action or in
the general circulation. Increases in bioavailability refers to increasing the
rate and extent a substance or its
active moiety is delivered from a pharmaceutical dosage form and becomes
available at the site of action or in
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WO 2008/077079 PCT/US2007/088011
the gencral circulation. By way of exaniple, an increase in bioavailability is
indicated as an increase in
concentration of the substance or its active moiety in the blood when compared
to other substances or active
moieties. This method is optionally used for evaluating the bioavailability of
any polypeptide.
(0064) The term "biologically active molecule", "biologically active nioiety"
or "biologically active agent"
when used herein means any substance which can affect any physical or
biochemical properties of a biological
system, pathway, molecule, or interaction relating to an organism, including
but not limited to, viruses, bacteria,
bacteriophage, transposon, prion, insects, fungi, plants, animals, and humans.
In particular, as used herein,
biologically active molecules include but are not limited to any substance
intended for diagnosis, cure,
mitigation, treatment, or prevention of disease in hunians or other animals,
or to otherwise enhance physical or
mental well-being of humans or animals. Examples of biologically active
molecules include, but are not limited
to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs,
carbohydrates, inorganic atoms or
molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins,
cells, viruses, liposomes,
microparticles and micelles. Classes of biologically active agents that are
suitable for use with the methods and
compositions described herein include, but are not limited to, drugs,
prodrugs, radionuclides, imaging agents,
polymers,:antibiotics, fungicides, anti-viral agents, ant'r-inflammatory
agents, anti-tumor agents, cardiovascular
agents, anti-anxiety agents, hormones, growth factors, steroidal agents,
microbially derived toxins, and the like.
100651 By "modulating biological activity" is meant increasing or decreasing
the reactivity of a polypeptide,
altering the selectivity of the polypeptide, enhancing or decreasing the
substrate selectivity of the polypeptide.
Analysis of modified biological activity can be performed by comparing the
biological activity of the non-
natural polypeptide to that of the natural polypeptide.
100661 The tenn "biontaterial," as used herein, refers to a biologically-
derived material, including but not
liniited to material obtained from bioreactors and/or from recombinant methods
and techniques:
100671 The term `-`biophysical probe,=" as used herein; refers to probes which
can detect or monitor structural
changes in molecules. Such molecules include, but are not limited to,
proteins; and the "biophysical probe" is
optionally used to detect or monitor interaction of proteins with other
macromolecules. Exantples of biophysical
probes include, but are not limited to, spin-labels, a fluorophores, and
photoactivatible groups.
100681 The term "biosynthetically," as used herein, refers to any method
utilizing a translation system (cellular
or non-cellular), including use of at least one of the following components: a
polynucleotide, a codon, a tRNA,
and a ribosome. By way of example., non-natural amino acids are
"biosynthetically incorporated" into non-
natural amino acid polypeptides using the methods and techniques described in
section VIII "ln vivo generation
of polypeptides comprising non-natural amino acids".
100691 The term "biotin analogue," or also referred to as "biotin mimic", as
used herein, is any molecule, other
than biotin, which bind witli high affinity to avidin and/or streptavidin.
100701 The term "carbonyl" as used herein refers to a group containing at a
moiety selecting from the group
consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including, but not limited
to, groups containing a least one
ketone group, and/or at least one aldehyde groups, and/or at least one ester
group, and/or at least one carboxylic
acid group, and/or at least one thioestergroup. Such carbonyl groups include
ketones, aldehydes, carboxylic
acids, esters, and thioesters. In addition, such groups are optionally part of
linear, branched, or cyclic molecules.
100711 The term "chemically cleavable group," also referred to as "chemically
labile", as used herein, refers to
a group which breaks or cleaves. upon exposure to acid, base, oxidizing
agents, reducing agents, chemical
inititiators, or radical initiators.
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100721 The term "chemiluminescent group," as used herein, refers to a group
which emits light as a result of a
chemical reaction without the addition of heat. By way of example only,
luminol (5-amino-2,3-dihydro-l,4-
phthalazinedione) reacts with oxidants like hydrogen peroxide (H202) in the
presence of a base and a metal
catalyst to produce an excited state product (3-aminophthalate, 3-APA).
100731 The term "cliromophore," as used herein, refers to a molecule which
absorbs light of visible
wavelengths, UV wavelengths or IR wavelengths.
100741 The term "cofactor," as used herein, refers to an atom or molecule
essential for the action of a large
molecule. Cofactors include, but arenot limited to, inorganic ions, coenzymes,
proteins, or some other factor
necessary for the activity of enzymes. Examples include, heme in hemoglobin,
magnesium in chlorophyll, and
metal ions for proteins.
100751 A"con-parison wrindow," as used herein, refers a segment of any one of
contiguous positions used to
compare a sequence to a reference sequence of the same number of contiguous
positions after the two sequences
are optimally aligned. Such contiguous positions include, but are pot limited
to a group consisting of from about
to about 600 sequential units, including about 50 to about 200 sequential
tinits, and about 100 to about 150
15 sequential units. By way of example only, such sequences include
polypeptides and polypeptides containing
non-natural amino acids, with the sequential units include, but are not
limited to natural and non-natural amino
acids. In addition, by way of example only, such sequences include
polynucleotides with nucleotides being the
corresponding sequential units. Methods of alignment of sequences for
comparison include, but are not liniited
to, the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.
2:482c, by the homology
20 alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method
of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by
computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics
Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and
visual inspection (see, e.g.,
Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).
100761 By way of example; an algorithm which is used to determine percent
sequence identity and sequence
similarity are the BLAST and BLAST 2.0 algorithms, which are described in
Altschul et al. (1997) Nuc. Acids
Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410;
respectively. Software for performing
BLAST analyses is publicly available through the National Center for
Biotechnology Information. The BLAST
algorithm parameters W; T, and X determine the sensitivity and speed of the
alignment. The BLASTN program
(for nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=4 and a.
comparison of both strands. For aniino acid sequences, the BLASTP program uses
as defaults a wordlength of
3, and expectation (E)' of 10, and the BLOSUM62 scoring matrix (see
Henikoffand Henikoff (1992) Proc. Natl.
Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-
4, and a comparison of both
strands. The BLAST algorithm is typically performed with the "low complexity"
filter turned off.
100771 The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see,
e.g., Karlin and Altschul (1993) Proc. Nati. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided
by the BLAST algorithm is the smallest sum probability (P(N)), which provides
an indication of the probability
by which a match between two nucleotide or amino acid sequences would occur by
chance. For example, a
nucleic acid is considered similar to a reference sequence if the smallest sum
probability in a comparison of the
test nucleic acid to the reference nucleic acid is less than about 0.2, or
less than about 0.01, or less than about
0.001.
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100781 The term "conservatively modified variants" applies to both natural and
non-natural amino acid and
natural and non-natural nucleic acid sequences, and combinations thereof. With
respect to particular nucleic
acid sequences, "conservatively modified variants" refers to those natural and
non-natural nucleic acids which
encode identical or essentially identical natural and non-natural amino acid
sequences, or where the natural and
non-natural nucleic acid does not encode a natural and non-natural amino acid
sequence, to essentially identical
sequences. By way of example, becauseof the degeneracy of the genetic code, a
large number of functionally
identical nucleic acids encode any given protein. For instance, the codons
GCA, GCC, GCG and GCU all
encode the aniino acid alanine. Thus, at every position where an alanine is
specified by a codon, the codon can
be altered to any of the corresponding codons described without altering the
encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Thus by way
of example every natural or non-natural nucleic acid sequence herein which
encodes a natural or non-natural
polypeptide also describes every possible silent.variation of the natural or
non-natural nucleic acid. Each codon
in a natural or non-natural. nucleic acid (except AUG, which is ordinarily the
only codon for methionine, and
TGG, which is ordinarily the only codon for tryptophan) can be modified to
yield a functionally identical
tnolecule. Accordingly, each silent variation of a.natural and non-natural
nucleic acid.which encodes a natural
and non-natural polypeptide is implicit in each described sequence.
100791 As to amino acid sequences,, individual substitutions, deletions or
additions to a nucleic acid, peptide,
polypeptide, or protein sequence which alters, adds or deletes a single
natttral and non-natural amino acid or a
small percentage of natural and non-natural amino acids in the encoded
sequence is a "conservatively modified
variant" where the alteration results in the deletion of an anvno acid,
addition of an amino acid, or substitution
of a natural and non-natural amino acid with a chemically similar.aniino acid.
Conservative substitution tables,
available in the scientific literature, provide funetionally sinular natural
amino acids. Such conservatively
modified variants are in addition to and do not exclude polymorphicvariants,
interspecies homologs, and alleles
of the methods and compositions described herein.
100801 The following eight groups each contain amino acids that are
conservative substitutions for one
another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) .Isoleucine (I)õLeucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine:(T); and
8) Cysteine (C), Methioriine (M)
(see, e.g., Creighton, Proteins:Structures and Molecular Properties (W H
Freeman & Co.; 2nd edition
(December 1993).
100811 The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in
combination with other terms,
represent, unless otherwise stated, cyclic versions of "alkyl" and
"heteroalkyl", respectively. Thus, a cycloalkyl
or heterocycloalkyl include saturated, partially unsaturated and fully
unsaturated ring linkages. Additionally, for
heterocycloalkyl, a heteroatom can occupy the position at which the
heterocycle is attached to the remainder of
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the molecule. The heteroatom includes, but is not limited to, oxygen, nitrogen
or sulfur. Examples of cycloalkyl
include,:but are not limited to, cyclopcntyl, cyclohexyl, 1-cyclohexenyl, 3-
cyclohexenyl, cycioheptyl, and the
like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5;6-
tetrahydropyridyl),.1-piperidinyl,
2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-
y1, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,. 2-piperazinyl, and
the like: Additionally, the term
encompasses. multicyclic structures, including but not limited to;
liicy.clicand tricyolic ring structures. Similarly,
the terni "heterocycloalkylene" by itself or, as, part of-another molecule
means a divalent radical derived from
heterocycloalkyl,and the term "cycloalkylene" by itself or as part.of another
niolecule means a divalent radical
derived from cycloalkyl.
100821 The term "cyclodextrin," as used herein, refers to-cyclic caibohydrates
consisting of at least six to eight
glucose molecules in a ring formation. The outer part of the ring contains
water soluble groups; at the center of
the ring:is a relatively nonpolar cavity able to accommodate small molecules.
100831 The term "cytotoxic," as used herein, refers to a compound which harms
cells.
100841 The term "desired functionality," as used herein refers to any one of
the following goups: a label; a
dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol;
a photocrosslinker; a cytotoxic
compound; a drug; an affinity label; a photoaffinity label; a reactive
compound; a resin; a second protein or
polypeptide or polypeptide analog; an antibod.y or antibody fragment; a metal
chelator; a cofactor; a fatty acid; a
carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a
saccharide, a water-soluble
dendrimer, a cyclodextrin, a bioniaterial; a nanoparticle; a spin ,1abe1; a
fluorophore; a metal-containing moiety;
a radioactive moiety; a novel functional group; a group that covaleiitly or
noncovalently interacts with other
molecules; a pliotocaged moiety; an actinic, radiation excitable moiety; a
ligand; ;a photoisomerizable nwiety;
biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically
cleavable group; a photocleavable
group; an elongated side chain; a carbon-linked sugar; a redox-active agent;,
an amino thioacid; a toxic moiety;
an isotopically labeled moiety; a biophysical probe; a. phosphorescent group;
a chemiluminescent group; an
electron dense gro.up; a magnetic group; an intercalating group; a
chromophore; an energy transfer agent; a
biologically active agent (in which case, the biologically active agent
can.include an agent with therapeutic
activity and the non-natural amino acid polypeptide or modified non-natural
anlino acid can serve either as a co-
therapeutic agent with the attached therapeutic agent or as a means for
delivery the therapeutic agent to a
desired site within an organism); a detectable label; a small molecule; an
inhibitory ribonucleic acid; a
radionucleotide; a neutron-capture agent; a derivative of biotin; quantum
dot(s); a nanotransntitter; a
radiotransnritter; an abzyme, an activated coniplex activator, a virus, an
adjuvant, an aglycan, an allergan, an
angiostatin, an antihormone, an antioxidant, an aptamer, a iguide R.NA, a
saponin, a shuttle vector, a
niacromolecule, a mimotope, a receptor, a reverse micelle, and any combination
thereof.
100851 The term "hydrazine,"as used herein, refers to groups/molecules
comprising at least one hydrazine
functional group.
100861 The term "detectable label," as used herein, refers to a label. which
is optionally observable using
analytical, techniques including, but not limited. to, fluorescence,
chemiluminescence,._electron-spin resonance;
ultraviolet/visible absorbance spectroscopy, mass spectrome`try, nuclear
magnetic resonance, magnetic
resonance,:and electxochemical methods.
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100871 The term "carbonyl" as used herein refers to a groups/molecules
containing at least one aldehyde or
one ketone.
100881 7'he term "drug," as used herein, refers to any substance used in the
prevention, diagnosis, alleviation,
treatment, or cure of a disease or condition.
[00891 The term "dye," as used herein, refers to a soluble, coloring substance
which contains a chromophore.
100901 The term "effective amount," as used herein, refers to a sufficient
amount of an agent or a compound
being adininistered which will relieve: to some extent one or more of the
symptonis of the disease or condition
being treated. The result can be reduction and/or alleviation of the signs,
`symptoms, or causes of a disease, or
any other desired alteration of a biological system. By way of example,. an
agent. or a cornpound being
administered includes, but' is not limited to, a natural amino acid
polypeptide, non-natural amino acid
polypeptide, niodified natural amino acid polypeptide, or modified non-amino
acid polypeptide. Compositions
containing such natural amino acid polypeptides, non-natural amino acid
polypeptides, modified natural amino
acid polypeptides, or modified non-natural amino acid polypeptides can be
administered for prophylactic,
enhancing, and/or therapeutic treatments. An appropriate "effective" amount in
any individual case is optionally
detemtined using techniques, such as a dose escalation study.
100911 The term "electron dense group," as used herein, refers to a group
which scatters electrons when
irradiated with an electron beam. Such groups include, but are not limited to,
ainmoniuni molybdate, bismuth
subnitrate cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate,
hexamethylene tetramine, 98.5%,
indiuni trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate,
lead citrate trihydrate, lead nitrate,
periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium
ferricyanide, potassium ferrocyanide,
ruthenium red, silver nitrate, silver proteinate (Ag Assay: 8.0-8.5%)
"Strong", silver tetraphenylporphin (S-
TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate,
thiosemicarbazide (TSC), uranyl acetate, uranyl
nitrate, and vanadyl sulfate.
[00921 The term "energyiransfer agent," as used herein, refers to ainolecule
which can either donate or accept
energy from another molecule. By way of example only, fluorescence resonance
energy transfer (FRET) is a
dipole-dipole coupling process by which the excited-state energy of a:
fluoreseence donor molecule is non-
radiatively tratisferred to an unexcited.acceptor molecule which then
tluorescently enrits the donated energy at a
longer wavelength.
100931 The terms "enhance" or "enhancing" means to increase or prolong either
in potency or duration a
desired effect. By way of example, "enhancing" the effect of therapeutic
agents refers to the ability to increase
or prolong, either in potency or duration, the effect of therapeutic agents on
during treatment of a disease,
disorder or condition. An "enhancing-effective amount," as used herein, refers
to an amount adequate to
enhance the effect of a therapeutic agent in the treatment of a disease,
disorder or condition. When used in a
patient, amounts effective for this use will depend on the severity and course
of the disease, disorder or
condition, previous therapy, the patient's health status and response to the
drugs, and the judgment of the
treating physician.
100941 As used herein, the term "eukaryote" refers to organisms belonging to
the :phylogenetic domain
Eucarya, including but not limited to animals (including but not limited to,
mammals, insects, reptiles, birds,
etc.), ciliates, plants (including but not limited to, monocots, dicots, and
algae), fungi, yeasts, flagellates,
microsporidia, and protists.
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100951 The term "fatty acid," as used herein, refers to carboxylic acids with
about C6 or longer hydrocarbon
side chain.
100961 The term "fluoropliore," as used herein, refers to a molecule which
upon excitation emits photons and
is thereby fluorescent.
100971 The terms "functional group", "active moiety", "activating group",
"leaving group", "reactive site",
"chenucally reactive group" and "chemically reactive moiety," as used herein,
refer to portions or units of a
molecule at which chemical reactions occur. The terms are somewhat synonymous
in the chenucal arts and are
used herein to indicate the portions of molecules that perform some function
or activity and are reactive with
other molecules.
[0098] The term "halogen" includes fluorine, chlorine, iodine, and broniine.
100991 The term "haloacyl," as used herein, refers to acyl groups which
contain halogen moieties, including,
but not limited to, -C(O)CH3, -G(O)CF3i -C(O)CHZOCH3, and the like.
.1001001 The term."haloalkyl," as used herein,.refers to alkyl groups which
contain halogen moieties, including,
but not linuted to, -CF3 and -CH2CF3 and the like.
[001011 The term "heteroalkyl," as used herein, refers to, straight or
branched chain, or cyclic hydrocarbon
radicals, or combinations thereof, consisting of an alkyl group and at least
one. heteroatom. selected from the
group consisting of 0, N, Si and S, and wherein the nitrogen and sulfur atoms
are optionally oxidized and the
nitrogen heteroatom is optionally quaternized. The heteroitom(s) 0, N and S
and Si are optionally placed at any
interior position of the heteroalkyl group or at the position. at which the
alkyl group is attached to the remainder
of the molecule. Examples include, but are not limited to, -CHZ-CHZ-O-CH3, -
CH2-CH2-NH-CH3, -CHZ-CH2-
N(CH,3)-CH3, -CH2-S-CHZ-CH3, -CH2-CHZ,-S(O)-CH3, -CH2_-CH2-S(0)2-CH3i -CH=CH-O-
CH3, -Si(CH3)3, -
CHZ-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. In addition, up to two heteroatoms are
optionally consecutive,
such as, by way of example, -CH2-NH-OCH3 and -CHZ-O-Si(CH3)3.
1001021 The terms "heterocyclic-based linkage" or "heterocycle linkage" refers
to a moiety formed from the
reaction of a carbonyl group with a hydrazine group. The resulting reaction
product is a heterocycle, including a
heteroaryl group or a heterocycloalkyl group. The resulting heterocycle group
serves as a chemical link between
a non-natural amino acid or non-natural amino acid polypeptide and another
fiuictional group. In one
einbodiment, the heterocycle linkage includes a nitrogen-containing
heterocycle linkage, including by way of
example only a a pyrrole linkage, 'an indole linkage, a benzodiazepine
linkage, and a pyrazalone linkage.
1001031 Similarly, the term ' heteroalkylene" Tefers to. a divalent radical
derived from heteroalkyl, as
exemplified, but not linvted by, -CH2-CH2-S-CHZ-CHZ- and -CH2-S-CH2-CH2-NH-CH2-
. For heteroalkylene
groups, the same or different heteroatoms can also occupy either or both of
the chain termini (including but not
limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,
aminooxyalkylene, and the like). Still
further, for alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the
direction in which the formula of the linking -group is written. By way of
example, the formula -C(0)2R'-
represents both -C(0)2R'- and -R'C(O)z-.
1001041 The term "heteroaryl" or "heteroaromatic," as used herein, refers to
aryl groups which contain at least
one heteroatom selected from N, 0, and S; wherein the nitrogen and sulfur
atoms are optionally oxidized, and
the nitrogen atom(s) areoptionally quatemized. Heteroaryl groups are
optionally substituted or unsubstituted. A
heteroaryl group is optionally attached to the remainder of the molecule
through a heteroatom. Non-limiting
exaniples of heteroaryl groups include I-pyrrolyl, 2-pyrrolyl, -3-pyrrolyl, 3-
pyrazolyl, 2-imidazolyl, 4-
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imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-
oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-
isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-
thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-
pyridyl, 2-pyrimidyl, 4-pyriniidyl, 5-benzothiazolyl, purinyl, 2-
benzinvdazolyl, 5-indolyl, 1-isoquinolyl, 5-
isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
1001051 The term "homoalkyl," as used herein refers to alkyl groups which are
hydrocarbon groups.
(00106( The term "identical," as used herein, refers to two or more sequences
or subsequences which are the
same. In addition, the term "substantially identical," as used herein, refers
to two or more sequences which have
a percentage of sequential units which are the same when compared and aligned
for maximum correspondence
over a comparison window, or designated region as measured using comparison
algorithms or by manual
alignnient and visual inspection. By way of example only, two or more
sequences are "substantially identical" if
the sequential units are about 60% identical, about 65% identical, about 70%
identical, about 75% identical,
about 80% identical, about 85% identical, about 90% identical, or about 95%
identical over a specified region.
Such percentages to describe the "percent identity" of two or more sequences.
The identity of a sequence can
exist over a region that is at least about 75-100 sequential units in length,
over a region that is about 50
sequential units in length, or, where not specified, across the entire
sequence. This definition also refers to the
complement of a test sequence. By way of example only, two or more polypeptide
"sequences are identical when
the amino acid residues are the same, while two or more polypeptide sequences
are "substantially identical" if
the amino acid residues are about 60% identical, about 65 /n identical, about
70% identical, about 75% identical,
about 80% identical, about 85% identical, about 90% identical, or about 95%
identical over a specified region.
The identity can exist over a region. that is at least about 75-100 amino
acids in length, over a region that is
about 50 anvno acids in length, or, where not specified, across the entire
sequence of a polypeptide sequence. In
addition, by way of example only, two or more polynucleotide sequences are
identical when the nucleic acid
residues are the same, while two or more polynucleotide sequences are
"substantially identical" if the nucleic
acid residues are about 60% identical, about 65% identical, about 70%
identical, about 75% identical, about
80% identical, about 85% identical, about 90% identical, or about 95%
identical over a specified region. The
identity can exist over a region that is at least about 75-100 nucleic acids
in length, over a region that is about 50
nucleic acids in length, or, where not specified, across the entire sequence
of a polynucleotide sequence.
1001071 For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences
are compared. When using a sequence comparison algorithm, test and reference
sequences are entered into a
computer, subsequence coordinates are designated, if necessary, and sequence
algorithm program parameters
are designated. Default program parameters can be used, or altemative
parameters can be designated. The
sequence comparison algorithm then calculates the percent sequence identities
for the test sequences relative to
the reference sequence, based on the program parameters.
1001081 The terni "immunogenicity," as used herein, refers to an antibody
response to administration of a
therapeutic drug. The immunogenicity toward therapeutic non-natural amino acid
polypeptides can be obtained
using quantitative and-qualitative assays for detection of anti-non-natural
amino acid polypeptides antibodies in
biological fluids. Such assays include, but are not litnited to,
Radioinimunoassay (RIA), Enzyme-linked
immunosorbent assay ( ELISA), luminescent immunoassay (LIA), and fluorescent
inununoassay (FIA).
Analysis of immunogenicity toward therapeutic non-natural amino acid
polypeptides involves comparing the
antibody response upon administration of therapeutic non-natural amino acid
polypeptides to the antibody
response upon administration of therapeutic natural amino acid polypeptides.
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1001091 The term "intercalating agent," also referred to as "intercalating
group," as used herein, refers to a
chemical that can insert into the intramolecular space of a molecule or the
intermolecular space between
molecules. By way of example only an intercalating agent or:group.is a
molecule which inserts into the stacked
bases of the DNA double helix.
1001101 The term "isolated," as used herein, refers to separating, and
removing a component of interest from
components not of interest. Isolated substances can be in either a dry or semi-
dry state, or in solution, including
but not liniited to an aqueous solution. The isolated component can be in a
homogeneous state or the isolated
component can be a part of a pharmaceutical composition that comprises
additional pharmaceutically acceptable
carriers and/or excipients: Purity and homogeneity are detennined using
analytical chemistry techniques
including, but not limited to, polyacrylamide gel electrophoresis or high
performance liquid chromatography. In
addition, when a component of interest is isolated and is the predominant
species present in a preparation, the
component is described herein as substantially purified. The term "purified,"
as used herein, refers to a
component of interest which is at least 85% pure, at least 90% pure, at least
95% pure, at least 99% or greater
pure. By way of example only, nucleic acids or proteins are "isolated" when
such nucleic acids or proteins are
free of at Ieast some of the cellular components with which it is associated
in the natural state, or that the nucleic
acid or protein has been concentrated to a level greater than the
concentration of its in vivo or in vitro
production. Also, by way of exaniple, a gene is isolated when separated from
open reading frames which flank
the gene and encode a protein other than the gene of interest.
1001111 The term "label," as used.herein, refers to asubstance which is
incorporated into a conipound and is
readily detected, whereby its physical distribution is optionally detected
and/or monitored.
1001121 The term "linkage;" as used herein to refer to.bonds or chemical
moiety formed from a chemical
reaction between the functional group of a linker and anothermolecule. Such
bonds include, but are_not limited
to, covalent linkages aiid non-covalent bonds, while such chemical moieties
include, but are not limited to,
esters, carbonates, imines phospliate esters, hydrazones, acetals,
orthoesters, peptide linkages, and
oligonucleotide.linkages. Hydrolytically stable linkages means that the
linkages are-substantially stable in water
and do not react with water at useful pH values, including but not limited to;
under physiological conditions for
an extended period of time, perhaps even indefinitely. Hydrolytically unstable
or degradable linkages nleans
that the linkages are degradable in water or in aqueous solutions, including
for example, blood. Enzyrnatically
unstable or degradable linkages means that the linkage can be degraded by one
or more enzymes. By way of
example only, PEG and related polymers include degradable linkages in the
polymer backbone or in the linker
group between the polymer backbone and one or more of the temiinal functional
groups of the polymer
molecule. Such degradable linkages include, but are not limited to, ester
linkages formed by the reaction of PEG
carboxylic acids or activated PEG carboxylic acids with alcohol groups on a
biologically active agent, wherein
such ester groups generally hydrolyze under physiological conditions to
release the biologically active agent.
Other hydrolytically degradable linkages include but are not limited to
carbonate linkages; imine linkages
resulted from reaction of an amine and an aldehyde; phosphate ester linkages
formed by reacting an alcohol
with a phosphate group; hydrazone linkages which are reaction product of a
hydrazide and an aldehyde; acetal
linkages that are the reaction product of an aldehyde and an alcohol;
orthoester linkages that are the reaction
productof a formate and an alcohol; peptide linkages formed by an amine group,
including but not.limited to, at
an end of a polymer such as PEG, and a carboxyl group of a peptide; and
oligonucleotide linkages formed by a
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phosphoramidite group, including but not limited to, at the end of a polytner,
and a 5' hydroxyl group of an
6ligonucleo6de.
1001131 The terms "medium" or "media," as used herein, refer to any culture
medium used to grow and harvest
cells and/or products expressed and/or secreted by such cells. Such "medium"
or "media" include, but are not
limited to, solution, solid, semi-solid, or rigid supports that support or
contain any host cell, including, by way
of example, bacterial host cells,~yeast host cells, insect host cells, plant
host cells, eukaryotic host cells,
mammalian host cells, CHO cells, prokaryotic host.cells, E. coli, or
Pseudomonas host cells, and cell.contents.
Such "medium' ' or. "inedia" includes,, but is,not limited to,. medium or.
media in which the host cell has been
grown into which a polypeptide has been secreted, including medium either
before or'after a.proliferation step.
Such "medium" or "media" also includes; but is not limited to, buffers or
reagents'that contain host cell.lysates,
by way of "example a polypeptide produced intraeel[ularly-and the host cells
are lysed or disrupted to release the
polypeptide.
[001141 The terni "metabolite," as used herein, refers to a derivative of
acompound, by way of example natural
aniino acid polypeptide, 'a non-natural amino acid polypeptide, a
modified'natural amino acid polypeptide, or a
modified non-natural amino acid polypeptide, that is formed when the compound,
by way of example natural
amino acid polypeptide, non-natural amino acid polypeptide, modified natural
aniino acid polypeptide, or
modified non-natural amino acid polypeptide, is metabolized. The term
"pharmaceutically active metabolite" or
"active metabolite" refers to a biologically active derivative of a compound,
by -way of example natural amino
acid polypeptide, a non-natural amino acid polypeptide, a modified natural
amino acid polypeptide, or a
modified non-natural amino acid polypeptide, that is formed when such a
compound, by way of example a
natural amino acid polypeptide, non-natural amino acid polypeptide, niodified
natural amino acid polypeptide,
or modified non-natural anlino acid polypeptide, is nietabolized.
1001.151 The terni "metabolized," as used herein, refers to the sum of the
processcs by which a particular
substance is changed by an organism. Such processes include, but are not
limited to, hydrolysis reactions and
reactions catalyzed by enzymes. Further information on metabolism is obtained
from The Pharmacological
Basis of:Therapeutics, 9th Edition, McGraw-Hill (1996). By way of example
only, metabolites of natural amino
acid polypeptides, non-natural amino acid polypeptides, modified natural amino
acid polypeptides, or modified
non-natural amino acid polypeptides, are identified either by administration
of the natural anuno acid
polypeptides, non-natural amino acid polypeptides, modified natural amino acid
polypeptides, or modified non-
natural amino acid.polypeptides to a host and analysis of tissue- samples from
the host, or by incubation of
natural artmino acid polypeptides, non-natural amino acid polypeptides,
modified natural anuno acid
polypeptides, or modified non-natural amino acid polypeptides with hepatic
cells in vitro and analysis of the
resulting compounds.
1001161 The tenn "metal chelator," as used herein, refers to a molecule which
fornrnis a metal complex with
metal ions. By way of example, such molecules form two or more coordination
bonds with a central inetal ion
and form ring structures.
100117] The term "metal-containing moiety," as used herein, refers to a group
which contains a metal ion, atom
or particle. Such moieties include, but.are not limited to, cisplatin,
chelated metals ions (such as nickel, iron, and
platinuni), and metal nanoparticles (such as nickel, iron, and platinum).
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1001181 The term "moiety incorporating a heavy atom,!' as used herein, refers
to a group which incorporates an
ion. of atom which is usually heavier than carbon. Such ions or atoms include,
but are not, timited to, silicon,
tungsten, gold, lead, and uranium.
(001191 The term "modified," as used herein refers to the presence of a change
to a natural amino acid, a non-
natural amino acid, a natural amino acid polypeptide or a non-natural amino
acid polypeptide. Such changes, or
modifications, are optionally obtained by posYsynthesis modifications of
natural amino acids, non-natural amino
acids, natural amino acid polypeptides or non-natural amino acid polypeptides,
or by co-translational, or by
post-translational modification of natural anuno acids, non-natural amino
acids, natural amino acid polypeptides
or non-natural amino acid polypeptides. The -form "modified or unmodified"
means that the natural amino acid,
non-natural amino acid, natural amino acid polypeptide or non-natural amino
acid polypeptide being discussed
are optionally modified, that is, he natural amino acid, non-natural amino
acid, natural amino acid polypeptide
or non-natural amino acid polypeptide under discussion can be modified or
unmodified.
1001201 As used herein, the tcrm.`modulated serum half-life" refers to
positive or negative changes in the
circulating half-life of a modified biologically active molecule relative: to
its non-modified form. By way of
example, the iriodified biologically active, molecules include, ,but are not
limited to,.natural amino acid, non-
natural amino acid, natural amino acid polypeptide or non-natural amino acid
polypeptide. By way ofexample,
serum half-life, is measured by, taking blood samples at, various time points
after administration of the
biologically active-molecule or modified biologically active molecule, and
deternvning<the concentration of that
molecule in each.sampie. Correlation,of the. serum concentration with time
allows calculation of the serum half-
life. By way of example, modulated serum half=.life is an increased in serum
half-life, which enables an
imp,roved dosing regimens or avoid toxic effects. Such increases in serum are
at:least about two fold, at least
about three-fold, at least about five-fold, or at least about ten-fold. This
method is optionally used for evaluating
the serum half-life of any polypeptide.
1001211 The term "modulated therapeutic half-life," as used herein, refers to
positive or negative change in the
half-life of-the therapeutically effective amount of a modified biologically
active molecule, relative to its non-
modified form. By way of exaniple, the modified biologically active molecules
include, but are not linuted to,
natural amino acid, non-natural amino acid, natural amino acid polypeptide or
non-naturat amino acid
polypeptide. By way of example, therapeutic half-life is, measured by
measuring pharmacokinetic and/or
pharmacodynamic properties of the, molecule at various time points after
administration. Increased therapeutic
half-life optionally enable a particular beneficial dosing regimen, a
particular beneficial total dose, or avoids an
undesired effect. By way of example, the increased therapeutic half-life
results from increased potency,
increased or decreased binding of,,the modified niolecule to its target, an
increase or decrease in another
paranieter,or mechanism of action of the, non-modified molecule, or an
increased or decreased breakdown of the
molecules by enzymes such as, by way of example .only, proteases. This method
is used for evaluating the
therapeotic ha1F life.of any polypeptide.
1001221 The term "nanoparticle;" as used herein, refers to a particle which
has a particle size between about 500
nm'to about I nm.
1001231 The term "near-stoichiometric," as used herein, refers to the ratio of
the moles of compounds
participating in a chemical reaction being about 0.75 to about 1.5.
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1001241 As used herein, the term "non-eukaryote" refers to non-eukaryotic
organisms. By way of example, a
non-eukaryotic organism belongs to the Eubacteria, (which includes but is not
limited to, Escherichia coli,
Thertnus thermophilus, or Bacillus stearothermophilus, Pseudomonas
fluorescens, Pseudomonas aeruginosa,
Pseudomonas putida), phylogenetic domain, or the Archaea, which includes, but
is not lirnited to,
Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus
fulgidus, Pyrococcus
furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, or Halobacterium such as
Haloferax volcanii and
Halobacterium species NRC-l, or phylogenetic doniain.
1001251 A, "non-natural aniino acid" refers to an amino acid that is not one
of the 20 common amino acids or
pyrolysitte or selenocysteine. Other synonymous terms are "non-naturally
encoded amino acid," "unnatural
amino acid," "non-naturally-occurring amino acid," and variously hyphenated
and non-hyphenated versions
thereof. The term "non-natural amino acid" includes, but is not limited to,
amino acids which occur naturally by
modifcation of a naturally encoded amino acid (including but not lirnited to,
the 20 common amino acids or
pyrrolysine and selenocysteine) but are not themselves incorporated into a
growing polypeptide chain by the
translation complex. Exaniples of naturally-occurring amino acids that are not
naturally-encoded include, but
are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-
threonine, and 0-phosphotyrosine.
Additionally, the term "non-natural amino acid" includes, but is not limited
to, amino acids which do not occur
naturally and are obtained synthetically or are obtained by modification of
non-natural amino acids.
1001261 'llte term "nucleic acid," as used herein, refers to
deoxyribonucleotides, deoxyribonucleosides,
ribonucleosides or ribonucleotides and polymers thereof in either single- or
double-stranded form. By way of
example only, such nucleic acids and nucleic acid polymers include, but are
not limited to, (i) analogues of
natural nucleotides which have similar binding properties as a reference
nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides; (ii) oligonucleotide
analogs including; but are not limited to,
PNA (peptidonucleic acid), analogs of DNA used in antisense technology
(phosphorothioates,
phosphoroamidates, and the like); (iii) conservatively modified variants
thereof (including but not limited to,
degenerate codon substitutions) and complementary sequences and sequence
explicitly indicated. By way of
example, degenerate codon substitutions are achieved by generating sequences
in which the third position of
one or more selected (or all) codons is substituted with mixed-base and/or
deoxyinosine residues (Batzer et al.,
Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Rossolini et al.,
Mol. Cell. Probes 8:91-98 (1994)).
1001271 The term "oxidizing agent," as used herein, refers to a compound or
material which removes an
electron from a compound being oxidized. By way of example oxidizing agents
include, but are not limited to,
oxidized glutathione, cystine, cystanvne, oxidized dithiothreitol, oxidized
erythreitol, and oxygen. Axvide
variety of oxidizing agents are suitable for use in the methods and
compositions described herein.
1001281 The term "pharmaceutically acceptable", as used hcrein, refers to a
material, including but not limited,
to a salt, carrier or diluent, which does not abrogate the biological activity
or properties of the compound, and is
relatively nontoxic, i.e., the material is administered to an individual
without causing undesirable biological
effects or interacting in a deleterious nianner with any of the components of
thecomposition: in which it is
contained.
1001291 The term "photoaffinity label," as used herein, refers to a label with
a- group, which, upon exposure to
light, forms a linkage with a molecule for which the label has an affinity: By
way of example only, such a
linkage is covalent or non-covalent.
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1001301 The term "photocaged moiety," as used herein, refers to a group which,
upon illumination at certain
wavelengths, covalently or non-covalently binds other ions or molecules.
1001311 The term "photocleavable group," as used herein, refers to a group
which breaks upon exposure to
light.
1001321 The term "photocrosslinker," as used herein, refers to a conipound
comprising two or more functional
groups which, upon exposure to light, are reactive and form a covalent or non-
covalent linkage with two or
more monomeric or polymeric molecules.
1001331 The term "photoisomerizable moiety," as used herein, refers to a group
wherein upon illumination with
light changes from one isomeric form to another.
1001341 The term "polyalkylene glycol," as used herein, refers to linear or
branched polymeric polyether
polyols. Such, polyall.ylene glycols, including, but are not limited to,
polyethylene glycol, polypropylene glycol,
polybutylene glycol, and derivatives thereof. Other exemplary embodiments are
listed, for exatnple, in
commercial supplier catalogs, such as Shearwater Corporation's catalog
"Polyethylene Glycol and Derivatives
for Biomedical Applications" (2001). By way of example only, such polymeric
polyether polyols have average
molecular weights within a desired polymer molecular weight range.
1001351 The term "within a desired polymer molecular weight range," as used
herein means between about 0.1
kDa to about 100 kDa. By way of example, between about 100 Da and about
100,000 Da or more. The
niolecular weight of the polymer is between, for example, about 100 Da and
about 100,000 Da, including but
not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000
Da, 70,000 Da, 65,000 Da,
60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,
25,000 Da, 20,000 Da, 15,000
Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,
3,000 Da, 2,000 Da, 1,000 Da,
900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In
some embodiments, the
molecular weight of the polymer is between about 100 Da and about 50,000 Da.
In some enibodiments, the
molecular weigllt of the polymer is between about 100 Da and about 40,000 Da.
In some embodiments, the
molecular weight of the polymer is between about 1,000 Da and about 40,000 Da.
In some embodiments, the
molecular weight of the polymer is between about 5,000 Da and about 40,000 Da.
In sonie embodiments, the
molecular weight of the polymer is between about 10,000 Da and about 40,000
Da. In some embodiments, the
polymer niolecule is a branched polymer. The molecular weight of the branched
chain polymer is between, for
example, about 1,000 Da and about 100,000 Da, including but not limited to,
100,000 Da, 95,000 Da, 90,000
Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000
Da, 50,000 Da, 45,000 Da,
40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da,
9,000 Da, 8,000 Da, 7,000 Da,
6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some
embodiments, the molecular weight
of the branched chain polymer is between about 1,000 Da and about 50,000 Da.
In some embodiments, the
molecular weight of the branched chain polymer is between about 1,000 Da and
about 40,000 Da. In some
enibodiments, the molecular weight of the branched chain polymer is between
about 5,000 Da and about 40,000
Da. In sonie embodiments, the molecular weight of the branched chain polymer
is between about 5,000 Da and
about 20,000 Da.
1001361 The term "polymer," as used herein, refers to a molecule composed of
repeated subunits. Such
niolecules include, but are not limited to, polypeptides, polynucleotides, or
polysaccharides or polyalkylene
glycols.
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100137] The terms''polypeptide;"'`peptide" and "protein" are used
interchangeably herein to refer to a polymer
of amino acid residues. That is, a description directed to a polypeptide
applies equally to a description of a
peptide and a description of a protein, and vice versa. The terms apply to
naturally occurring amino acid
polymers as well as amino acid polymers in which one or more amino acid
residues is a non-natural amino acid.
Additionally, such "polypeptides; " "peptides" and "proteins" include aniino
acid chains of any length, including
full length proteins, wherein the amino acid residues are linked by covalent
peptide bonds.
1001381 The term "post-translationally modified" refers to any modification of
a natural or non-natural amino
acid which occurs after such an amino acid has been translationally
incorporated into a polypeptide chain. Such
inodifications include, but are not limited to, co-translational in vivo
modifications, co-translational in vitro
modifications (such as in a cell-free translation system), post-translational
in vivo modifications, and post-
translational in vitro niodifications.
1001391 The terms "prodrug" or "pharmaceutically acceptable prodrug," as used
herein, refers to an agent that
is converted into the parent drug in vivo or in vitro, wherein which does not
abrogate the biological activity or
properties of the drug, and is relatively nontoxic, i.e., the material is
administered to an individual without
causing undesirable biological effects or interacting in a deleterious manner
with any of the components of the
composition in which it is contained. Prodrugs are generally drug precursors
that, following administration to a
subject and subsequent absorption, are converted to an active, or a more
active species via some process, such as
conversion by a metabolic pathway. Some prodrugs have a chemical group present
on the prodrug that renders it
less active and/or confers solubility or some other property to the drug. Once
the chemical group has been
cleaved and/or modified from the prodrug the active. drug is generated.
Prodrugs are converted into active drug
within the body through enzymatic or non-enzymatic reactions. Prodrugs, for
example, provide iniproved
physiochemical properties such as better solubility, enhanced delivery
characteristics, such as specifically
targeting a particular cell, tissue, organ or ligand, and iniproved
therapeutic value of the drug. The benefits of
such prodrugs include, but are not limited to, (i) ease of administration
compared with the parent drug; (ii) the
prodrug is bioavailable by oral administration whereas the parent is not; and
(iii) the prodrug has improved
solubility in pharmaceutical compositions compared with the parent drug. A pro-
drug includes a
pharmacologically inactive, or reduced-activity, derivative of an active diug.
Prodrugs are designed, for
example, to modulate the amount of a drug or biologically active molecule that
reaches a desired site of action
through the manipulation of the properties of a drug, such as physiochemical,
biopharmaceutical, or
pharmacokinetic properties. An example, without limitation, of a prodrug would
be a non-natural amino acid
polypeptide which is administered as an ester (the "prodrug") to facilitate
transmittal across a cell membrane
where water solubility is detrimental to mobility but which then is
metabolically hydrolyzed to the carboxylic
acid, the active entity, once inside the cell where water-solubility is
beneficial. Prodrugs are also designed, for
example, as reversible drug derivatives, for use as modifiers to enhance drug
transport to site-specific tissues.
1001401 The term "prophylactically effective amount," as used herein, refers
that amount of a composition
containing at least one non-natural amino acid polypeptide or at least one
modified non-natural amino acid
polypeptide prophylactically applied to a patient which will relieve to some
extent one or more of the symptoms
of a disease, condition or disorder being treated. In such prophylactic
applications, such amounts depend, for
example, on the patient's state of health, weight, and the like.
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1001411 The term "protected," as used lierein, refers to the presence of a
"protecting group" or moiety that
prevents reaction of the chemically reactive functional group under certain
reaction conditions. The protecting
group will vary depending on the type of chemically reactive group being
protected. By way of example only,
(i) if the chemically reactive group is an amine or a hydrazide, the
protecting group is selected from tert-
butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the
chemically reactive group is a
thiol,,the protecting group is orthopyridyldisulfide; and (iii) if the
chemically reactive group is a carboxylic acid,
such as butanoic or propionic acid, or a hydroxyl group, the-protecting group
is benzyl or an.alkyl group such as
methyl, ethyl, or tert-butyl.
1001421 By way of example only,=blocldng/protecting groups are selected from:
Hz H2 0
H C~CC~H~ C~O H2C C~HO` H3C
2 H2 2 ~O(
allyl Bn Cbz alloc Me
H2 H3C\ CH3 0
H3C' C-_ (H3C)3C-~ (H3C)3C- SI-I (H3C)3&\~ O
Et t-butyl TBDMS Teoc
0
H2 ~
C~ O HzC-O
(CH3)IC'0~ / I (C8H513C'_ H3C~
O. H3C0 ~ ~
BOC pMBn trityl acetyl
Fmoc
1001431 Additionally, protecting groups include, but are not liniited to,
including photolabile groups such as
Nvoc and MeNvoc and other protecting groups, such as those described in Greene
and Wuts, Protective Groups
in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY,.1999.
1001441 The term "radioactive moiety," as used herein, refers to a group whose
nuclei spontaneously give off
nuclear radiation, such as alpha, beta, or gamma particles; wherein, alpha
particles are helium nuclei, beta
particles are electrons, and gamma particles are high energy photons.
1001451 The term "reactive compound," as used herein, refers to a compound
which under appropriate
conditions is reactive toward another atom, molecule or compound.
1001461 The term "recombinant host cell," also referred to as "host cell,"
refers to a cell which includes an
exogenous polynucleotide, wherein the methods used to insert the exogenous
polynucleotide into a cell include,
but are not limited to, direct uptake, transduction, orf-mating, to create
reconibinant host cells. By way of
exaniple only, such exogenous polynucleotide is a nonintegrated vector,
including but not limited to a plasmid,
or is integrated into the host genome.
1001471 'rhe term "redox-active agent," as used herein, refers to a molecule
which oxidizes or reduces another
molecule, whereby the redox active agent becomes reduced or oxidized. Examples
of redox active agent
include, but are not limited to, ferrocene, quinones, Ru2'13+ complexes,
Co2'/3+ complexes, and Os2"3'
complexes.
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1001481 The tenn "reducing agent," as used herein, refers to a compound or
material which is capable of adding
an electron to a compound being reduced. By way of example reducing agents
include, but are not limited to,
dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine,
cysteamine (2-aminoethanethiol), and
reduced glutathione. Such reducing agents are used, by way of example only, to
niaintain sulfhydryl groups in
the reduced state and to reduce intra- or intermolecular disulfide bonds.
1001491 The term "resin," as used herein, refers to high molecular weight,
insoluble polymer beads. By way of
example only, such beads are used as supports for solid phase peptide
synthesis,, or sites for attachment of
molecules prior to purification.
[001501 The term "saccharide," as used herein,, refers to a series of
carbohydrates including but not limited to
sugars, monosaccharides, oligosaccharides, and polysaccharides.
1001511 The term "safety" or "safety profile;" as used herein, refers to side
effects that are related to
administration of a drug relative to the number of times the drug has been
adrriinistered. By way of example, a
drug which has been administered many times and produced only mild or no side
effects is said to have an
excellent safety profile. This method is used, for example, for evaluating the
safety profile of any polypeptide.
1001521 The term "spin label," as used herein, refers to molecules which
contain an atom or a group of atoms
exhibiting an unpaired electron spin (i.e. a stable paramagnetic group) that
can be detected by electron spin
resonance spectroscopy and can be attached to another molecule. Such spin-
label molecules include, but are not
liniited to, nitryl radicals and nitroxides, and are single spin-labels or
double spin-labels.
1001531 The term "stoichiometric," as used herein, refers to the ratio of the
moles of compounds participating
in a chemical reaction being about 0.9 to about I.I.
1001541 The tenn "stoichionietric-like," as used herein, refers to a cheniical
reaction which becomes
stoichiometric or near-stoichiometric upon changes in reaction conditions or
in the presence of additives. Such
changes in reaction conditions include, but are not limited to, an increase in
temperature or change in pH. Such
additives include, but are not limited to, accelerants.
1001551 The term "subject" as used herein, refers"to an animal which is the
object of treatment, observation or
experiment. By way of example only, a subject is, but is not limited to, a
mammal including, but not limited to,
a human.
1001561 The term "substantially purified," as used hereinõrefers to a
component of interest that is substantially
or essentially free of other components which normally accompany or interact
with the component of interest
prior to purification. By way of example only, a component of interest is
"substantially purified" when the
preparation of the coniponent of interest contains less than about.30%; less
than about 25%, less than about
20%, less than about 15%, less than about 10%, less than about 5%, less than
about 4%, less than about 3%, less
than about 2%, or less than about 1% (by dry weight) of contaminating
components. Thus, a "substantially
purified" component of interest has a purity level of about 70%, about 75%,
about 80%, about 85%, about 90%,
about 95%, about 96%, about 97%, about 98%, about 99% or greater. By way of
example only, a natural amino
acid polypeptide or a non-natural amino acid polypeptide is purified from a
native cell, or host cell in the case of
recombinantly produced natural amino acid polypeptides or non-natural amino
acid polypeptides. By way of
example a preparation of a natural anuno acid polypeptide or a non-natural
amino acid polypeptide is
"substantially purified" when the preparation contains less than about 30%,
less than about 25%, less than about
20%, less than about 15%, less than about 10%, less than about 5%, less than
about 4%, less than about 3%, less
than about 2%, or less than about 1% (by dry weight) of contanunating
material. By way of example when a
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natural amino acid polypeptide or a non-natural amino acid polypeptide is
recombinantly produced by host
cells, the natural amino acid polypeptide or non-natural amino acid
polypeptide is present at about 30%, about
25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%,
or about 1% or less of the
dry weight of the cells. By way of example when a natural amino acid
polypeptide or a non-natural amino acid
polypeptide is recombinantly produced by host cells, the natural amino acid
polypeptide or non-natural amino
acid polypeptide is present in the culture medium at about 5g/L, about 4g/L,
about 3g/L, about 2g/L, about lg/L,
about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L,
about I Omg/L, or about I mg/L
or less of the dry weight of the cells. By way of example, "substantially
purified" natural aniino acid
polypeptides or non-natural amino acid polypeptides has a purity level of
about 30%, about 35%, about 40%,
about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%,
about 90%, about 95%, about'99% or greater as deterniined by appropriate
methods, including, but not limited
to, SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
1001571 The tetm "substituents" also referred to as "non-interfering
substituents" "refers to groups which are
used to replace another group on a molecule. Such groups include, but are not
limited to, halo, Cl-Clo alkyl, C2-
CQ alkenyl, C2-Clo alkynyl, C-Cio alkoxy, C5-C12 aralkyl, C3-C12 cycloalkyl,
C4-Cõ cycloalkenyl, phenyl,
substituted phenyl, toluolyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, CS-Cõ
alkoxyaryl, CS-.Ci2 aryloxyalkyl, C7-
C12 oxyaryl, CI-C6 alkylsulfinyl, Cl-Clo alkylsulfonyl, -(CHZ),n O-(Cj-Co
alkyl) wherein m is.from 1 to 8, aryl,
substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical,
substituted heterocyclic radical, nitroalkyl,
-NO2, -CN, -NRC(O)-(Ci-C,o alkyl), -C(O)-(CI-COalkyl), C2-C10 alkthioalkyl, -
C(O)0-(Ci-Clo alkyl), -OH, -
SO2, =S, -COOH, -NR2, carbonyl, -C(O)-(CI-Cio alkyl)-CF3, -C(O)-CF3, -C(O)NR2,
-(CI-Clo aryl)-S-(C6-CIO
aryl), -C(O)-(C6-Cio aryl), -(CH,),,; O-(CH2),,; O-(Cj-Cio alkyl) wherein each
m is from 1 to 8, -C(O)NR2, -
C(S)NR2, -SOZNR2, -NRC(O)NR2, -NRC(S)NR2, salts thereof, and the like. Each R
group in the preceding list
includes, but is not limited to, H, alkyl or substituted alkyl, aryl or
substituted aryl, or alkaryl. Where substituent
groups are specified by their conventional chemical formulas, written from
left to right, they equally encompass
the chemically identical substituents that would result from writing the
structure from right to left; for example,
-CH2O- is equivalent to -OCH2-.
(001581 By way of example only, substituents for alkyl and heteroalkyl
radicals (including those groups
referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl,
cycloalkyl, heterocycloalkyl,
cycloalkcnyl, and heterocycloalkenyl) includes, but is not limited to: -OR,
=0, =NR, =N-OR, -NR2, -SR, -
halogen, -SiR3, -OC(O)R, -C(O)R, -COZR, -CONR2, -OC(O)NR2, -NRC(O)R, -
NRC(O)NR,, -NR(O)zR, -NR-
C(NRz)=NR, -S(O)R, -S(O)2R, -S(O)_NR,, -NRSO2R, -CN and -NO2. Each R group in
the preceding list
includes, but is not limited to, hydrogen, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted
aryl, including but not limited to, aryl substituted with 1 or 2 halogens,
substituted or unsubstituted.alkyl, alkoxy
or thioalkoxy groups, or aralkyl groups. When two R groups are attached to the
same nitrogen atom, they can be
combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For
example, -NR2 is meant to include,
but not be liniited to, i-pyrrolidinyl and 4-morpholinyl.
1001591 By way of example, substituents for aryl and heteroaryl groups
include, but are not linzited to, -OR,
=0, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -C02R, -CONR2, -
OC(O)NR2, -NRC(O)R, -
NRC(O)NRZ, -NR(O),R, -NR-C(NR2)=NR, -S(O)R, -S(0)2R, =S(O)ZNRZ, -NRSOZR, -CN, -
NO2, -R, -N3, -
CH(Ph)2, iluoro(Ci-C4)alkoxy, and fluoro(Ci-C4)alkyl, in a number ranging from
zero to the total number of
34
CA 02672205 2009-06-09
WO 2008/077079 PCT/US2007/088011
open valences on the aroniatic ring system; and where each R group in the
preceding list includes, but is not
limited to, hydrogen, alkyl, heteroalkyl, aryl and heteroaryl.
1001601 The term "therapeutic protein," as used herein, refers to any one or
all of the following
polypeptides/proteins: alpha-I antitrypsin, angiostatin, antihemolytic.
factor, antibody, antibody fragment,
apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic
polypeptide, atrial pep6de, C-X-C
chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b,.gTo-c, IP-10, GCP-2, NAP-4,
SDF-1, PF4, MIG, calcitonin,
c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1,
monocyte chemoattractant protein-
2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha,
monocyte inflammatory
protein-i beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262,
CD40, CD40 ligand, c-kit
ligand, collagen, colony stimulating factor (CSF), complement facto'r 5a,
complement inhibitor, complement
receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-
l, epidermal growth factor
(EGF), epithelial neutrophil activating peptide, erythropoietin (EPO),
exfoliating toxin, Factor IX, Factor VII,
Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen,
fibronectin, four-helical bundle protein, G-
CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth
factor receptor, grf, hedgehog
protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth
hormone (hGH), human serum
albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like
growth factor (IGF), IGF-I,
IGF-ll, interferon (IFN), IFN-alpha, IFN-beta, IFN-ganuna, any interferon-like
molecule or meniber of the IFN
fanuly, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-10, 1L-11, IL-12, keratinocyte
growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase,
neurturin, neutrophil inhibitory factor
(NIF), oncostatin M, osteogenic protein, oncogene pcoduct, paracitonin,
parathyroid hormone, PD-ECSF,
PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic
exotoxin A, pyrogenic exotoxin B,
pyrogenic exotozin C, pyy, relaxin; renin, SCF, small biosynthetic. protein,
soluble complement receptor 1,
soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor,
somatomedin, somatostatin, somatotropin,
streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SECI,
SEC2, SEC3, SED, SEE, steroid
hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin
alpha 1, tissue plasminogen
activator, tumor growth factor (TGF), tunior necrosis factor, tumor necrosis
factor alpha, tumor necrosis factor
beta, tumor necrosis factor receptor ('ITIFR), VLA-4 protein, VCAM-1 protein,
vascular endothelial growth
factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb,
rel, estrogen receptor, progesterone
receptor, testosterone receptor, aldosterone receptor, LDL receptor, and
corticosterone.
1001611 The term "therapeutically effective amount," as used herein, refers to
the amount of a composition
containing at least one non-natural anuno acid polypeptide and/or at least one
modified non-natural amino acid
polypeptide administered to a patient already suffering from a disease,
condition or disorder, sufficient to cure
or at least partially arrest, or relieve to some extent one or nlore of the
symptonis of the disease, disorder or
condition being treated. The effectiveness of such compositions depend
conditions including, but not limited to,
the severity and course of the disease, disorder or condition, previous
therapy, the patient's health status and
response to the drugs, and the judgment of the treating physician. By way of
example only, therapeutically
effective amounts are determined by methods, including but not limited to a
dose escalation clinical trial.
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[001621 The term "thioalkoxy," as used herein, refers to sulfur containing
alkyl groups linked to molecules via
an oxygen atom.
1001631 7'he term "thermal melting point" or Tm is the temperature (under
defined ionic strength, pH, and
nucleic concentration) at which 50% of probes complementary to a target
hybridize to the target sequence at
equilibrium.
1001641 The term "toxic moiety," as used herein, refers to a compound which
can cause harm or death.
1001651 The terms "treat," "treating" or "treatment", as used herein, include
alleviating, abating or ameliorating
a disease or condition symptonis, preventing additional symptoms,,
ameliorating or preventing the underlying
metabolic causes of symptoms, inhibiting the disease or condition, e.g.,
arresting the development of the disease
1.0 or condition, relieving the disease or condition, causing regression of
the disease or condition, relieving a
condition caused by'the disease or condition, or stopping the symptoms of the
disease or condition. The terms
"treat," "treating" or "treatment", include, but are not limited to,
prophylactic and/or therapeutic treatments.
[001661 As used herein, the term "water soluble polymer" refers to any polymer
that is soluble.in aqueous
solvents. Such water soluble polymers include, but are not limited to,
polyethylene glycol, polyethylene. glycol
propionaldehyde, mono Cl-Cln alkoxy or aryloxy derivatives thereof (described
in U.S. Patent No. 5,252,714
which is incorporated by reference herein for the disclosure of such water
soluble polymers), monomethoxy-
polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino
acids, divinylether maleic anhydride,
N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives including
dextran sulfate, polypropylene
glycol, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol,
heparin, heparin fragments,
polysaccharides, oligosaccharides, glycans, cellulose and cellulose
derivatives, including but not limited to
methylcellulose and carboxymethyl cellulose, serum albumin, starch and starch
derivatives, polypeptides,
polyalkylene glycol and derivatives thereof, copolymers of polyalkylene
glycols and derivatives thereof,
polyvinyl ethyl ethers, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide,
and the like, or mixtures thereof.
By way of example only, coupling of such water soluble polymers to natural
amino acid polypeptides or non-
natural polypeptides results in changes including, but not limited to,
increased water solubility, increased or
modulated serum half-life; increased or modulated therapeutic half-life
relative to the unmodified form,
increased bioavailability, modulated biological activity, extended circulation
time, modulated immunogenicity,
modutated physical association characteristics including, but not limited to,
aggregation and multitner
formation, altered receptor binding, altered binding to one or more binding
partners, and altered receptor
dimerization or multimerization. In addition, such water soluble polymers
optionally have their own biological
activity.
1001671 Unless otherwise indicated, conventional methods of mass spectroscopy,
NMR, HPLC, protein
chemistry, biochemistry, recombinant DNA techniques and pharmacology, are
employed.
1001681 Compounds, (including, but not limited to non-natural amino acids, non-
natural amino acid
polypeptides, modified non-natural amino acid polypeptides, and reagents for
producing the aforementioned
compounds) presented herein include isotopically-labeled compounds, which are
identical to those recited in the
various formulas and structures presented herein, but for the fact that one or
more atoms are replaced by an
atom having an atomic mass or mass number different from the atomic mass or
mass number usually found in
nature. Examples of isotopes that can be incorporated into the present
compounds include isotopes of hydrogen,
carbon, nitrogen, oxygcn, fluorine and chlorine, such as ZH, 'H, 13C, 14C,
15N, 18O, 170, 35S iaF 36CI
respectively. Certain isotopically-labeled compounds described herein, for
example those into which radioactive
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CA 02672205 2009-06-09
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isotopes such as 3H and 14C are incorpbrated, are useful in drug and/or
substrate tissue distribution assays.
Further, substitution with isotopes such as deuterium, i.e., 2H, can afford
certain therapeutic advantages
resulting from greater metabolic stability, for example increased in vivo half-
life or reduced dosage
requirements.
1001691 Some of the compounds herein (including, but not limited to non-
natural amino acids, non-natural
amino acid polypeptides and modified non-natural amino acid polypeptides, and
reagents for producing the
aforementioned compounds) have asymmetric carbon atoms and can therefore exist
as enantiomers or
diastereomers. Diasteromeric mixtures can be separated into their individual
diastereomers on the basis of their
physical chemical differences by documented methodologies, for example, by
chromatography and/or fractional
crystallization. Enantiomers can be separated by converting the enantiomeric
mixture into a diastereomeric
mixture by reaction with an appropriate optically active compound (e.g.,
alcohol), separating the diastereomers
and converting (e.g., hydrolyzing) the individual diastereomers to the
corresponding pure enantiomers. All such
isomers, including diastereomers, enantioniers, and mixtures thereof are
considered as part of the compositions
described herein.
1001701 In additional or further embodiments, the compounds described herein
(including, but not lirruted to
non-natural amino acids, non-natural amino acid polypeptides and modified non-
natural amino acid
polypeptides, and reagents for producing the aforementioned compounds) are
used in the form of pro-drugs. In
additional or further embodiments, the compounds described herein (including,
but not limited to non-natural
amino acids, non-natural amino acid polypeptides and modified non-natural
amino acid polypeptides, and
reagents for producing the aforementioned conipounds) are metabolized upon
administration to an organism in
need to produce a metabolite that is then used to produce a desired effect,
including a desired therapeutic effect.
In further or additional embodiments are active metabolites of non-natural
amino acids and "modified or
unmodified" non-natural amino acid polypeptides.
1001711 The methods and formulations described herein include the use of N-
oxides, crystalline forms (also
known as polymorphs), or pharmaceutically acceptable salts of non-natural
amino acids, non-natural anutto acid
polypeptides and modified non-natural amino acid polypeptides. In addition,
the non-natural amino acids, non-
natural amino acid polypeptides and modified non-natural amino acid
polypeptides described herein can exist in
unsolvated as well as solvated forms with pharmaceutically acceptable solvents
such as water, ethanol, and the
like. 7'he solvated forms of the non-natural amino acids, non-natural amino
acid polypeptides and modified non-
natural aniino acid polypeptides presented herein are also considered to be
disclosed herein.
1001721 Some of the compounds herein (including, but not limited to non-
natural amino acids, non-natural
amino acid polypeptides and modified non-natural amino acid polypeptides and
reagents for producing the
aforementioned compounds).may exist in several tautomeric forms. All such
tautomeric. forms are considered as
part of the contpositions described.herein. Also, for example all enol-keto
forms of any compounds (including,
but not linuted to non-natural amino acids, non-natural amino acid
polypeptides and modified non-natural
amino acid polypeptides and reagents for producing the aforementioned
compounds) herein are considered as
part of the compositions described herein.
1001731 Some of the compounds herein (including, but not limited to non-
natural amino acids, non-natural
amino acid polypeptides and modified non-natural amino acid polypeptides and
reagents for producing either of
the aforementioned compounds) are acidic and form a salt with a
pharmaceutically acceptable cation. Some of
the compounds herein (including, but not limited to non-natural amino acids,
non-natural aniino acid
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polypeptides and modified non-natural amino acid polypeptides and reagents for
producing the aforementioned
compounds) are basic and accordingly, form a salt with a pharmaceutically
acceptable anion. All such salts,
including di-salts are within the scope of the compositions described herein
and are prepared by documented
methodologies. For example, salts are optionally prepared by contacting the
acidic and basic entities, in either
an aqueous, non-aqueous or partially aqueous medium. The salts are recovered
by using at least one of the
following techniques_ "filtration, precipitation with a non-solvent followed
by filtration, evaporation of the
solvent, or, in the case of aqueous solutions, lyophilization.
1001741 Pharmaceutically :acceptable salts of the non-natural anlino acid
polypeptides disclosed herein are
optionally formed when an acidic proton present in the parent non-natural
amino acid polypeptides either is
replaced by a metal ion, by way of example an alkali metal ion, an alkaline
earth ion, or an aluminum ion; or
coordinates with an organic base. In addition, the, salt forms of the
disclosed non-natural amino acid
polypeptides are optionally prepared using salts of the starting materials or
intermediates. The non-natural
amino acid polypeptides described herein are optionally prepared as a
pharmaceutically acceptable acid addition
salt (which is a type of a pharmaceutically acceptable salt) by reacting the
free base form of non-natural amino
acid polypeptides described herein with a pharmaceutically acceptable
inorganic or organic acid. Altematively,
the non-natural amino acid polypeptides described herein are prepared as
pharmaceutically acceptable base
addition salts (which are a type of a pharmaceutically acceptable salt) by
reacting the free acid form of non-
natural amino acid polypeptides described herein witli a pharniaceutically
acceptable inorganic or organic base.
1001751 The type of pharmaceutical acceptable salts, include, but are not
limited to: (]) acid addition salts,
fornied with inorganic acids=such as hydrochloric acid, hydrobroni.ic
acid,sulfuric acid, nitric acid, phosphoric
acid, and the like; or formed with organic acids such as acetic acid,
propionic acid, hexanoic acid,
cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic
acid; succinic acid, malic acid,
maleic, acid, fumaric. acid, tartaric acid, citric acid, benzoic acid, 3-(4-
hydroxybenzoyl)benzoic acid, cinnamic
acid,. mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-
ethanedisulfonic acid, 2-
hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid,
4-methylbicyclo-[2.2.2]oct-2-
ene-l-carboxylic acid, glucoheptonic acid, 4;4'-methylenebis-(3-hydroxy-2-ene-
1 -carboxylic acid), 3-
phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl
sulfuric acid, gluconic acid, glutamic
acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and
the like; (2) salts formed when an
acidic proton present in the parent compound either is replaced by a metal
ion, e.g., an alkali metal ion, an
alkaline earth ion, or an aluminum ion; or coordinates with an organic base.
Acceptable organic bases include
ethanolamine, diethanolamine, triethanolamine, troniethamine, N-
niethylglucamine, and the like. Acceptable
inorganic bases include aluminum hydroxide, calcium hydroxide, potassium
hydcoxide, sodium carbonate,
sodium hydroxide, and the like.
1001761 'rhe corresponding counterions of the non-natural amino acid
polypeptide pharmaceutical acceptable
salts are analyzed and identified using various methods including, but not
limited to, ion exchange
chromatography, ion chromatography, capillary electrophoresis, inductively
coupled plasma, atomic absorption
spectroscopy, mass spectrometry, or any combination thereof. In addition, the
therapeutic activity of such non-
natural amino acid polypeptide pharmaceutical acceptable salts are tested
using the techniques and methods
described in examples 9-17.
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1001771 It should be understood that a reference; to a salt includes the
solvent addition forms or crystal forms
thereof, particularly solvates or. polymorphs..Solvates contain either
stoichiometric or non-stoichiometric
ainounts of a solvent, and are often formed during the process of
crystallization with pharmaceutically
acceptable solvents such as water, ethanol, and the like. Hydrates are formed
when the solvent is water, or
alcoholates are formed when the solvent is alcohol. Polymorphs include the
different crystal packing
arrangements of the same elem_ental composition of.a compound. Polymorphs
usually have different X-ray
diffraction patterns, infrared spectra, melting points, density, hardness,
crystal shape, optical and electrical
properties, stability, and solubility. Various factors.such as the
recrystallization solvent, rate of crystallization,
and.storage temperature are expected to cause a single crystal form,to
dominate.
[00178) The screening and characterization of non-natural:amino acid
,polypeptide pltarmaceutical acceptable
salts polymorphs and/or solvates is accomplished using a variety of
techniquesincluding, but not limited to;
thermal analysis, x-ray diffraction, spectroseopy, vapor soiption; and
microscopy. Thermal analysis methods
address thermo chemical degradation or thermo physical processes including,
but not limited to, polymorphic
transitions, and such methods are used to analyze the relationships between
polymorphie forms; determine
weight loss, to find the glass transition temperature, or for excipient
compatibility studies. Such methods
include, but are not Iimited.`to, Differential scanning calorimetry (DSC),
Modulated Differential Scanning
Calorimetry (MDCS), Thermogravimetric analysis (TGA), and Thermogravi-metric
and Infrared analysis
(TG/IR). X-ray diffraction methods include, but are not limited to, single
crystal and powder diffractometers
and synchrotron sources. The various spectroscopic techniques used include,
but are not limited to, Raman,
FTIR, UVIS, and NMR (liquid and solid state). The various microscopy
techniques include, but are not limited
to, polarized light microscopy, Scanning Electron Microscopy (SEM) with Energy
Dispersive X-Ray Analysis
(EDX), Environmental Scanning Electron Microscopy with EDX (in gas or water
vapor atmosphere), IR
nucroscopy, and Raman microscopy.
BRIEF DESCRIPTION. OF THE FIGURES
1001791 A better understanding of the features and advantages of the present
methods;and compositions. maybe
obtained by reference to the following detailed description that sets forth
illustrative embodiinents, in which the
principles of our methods, coiripositions, devices and apparatuses. are
utilized, and the accompanying drawings
of :which:
1001801 FIG. I presents a non-linuting schematic representation of the
relationship.of certain aspects of the
methods, compositions, strategies and techniques described herein.
1001811 FIG. 2 presents a non limiting schematic representation of the
mechanism of Fischer indole synthesis.
[001821 FIG. 3 presents illustrative, non-limiting examples of the synthetic
methodology used to niake the non-
natural aniino, acids described herein.
1001831 FIG. 4 presents illustrative, non-limiting examples of the synthetic
methodology used to make the non-
natural amino.acids described herein.
1001841 FIG. 5 presents an illustrative, non-limiting example of the synthetic
methodology used to make the
non-natura( amino acids described herein.
1001851 FIG. 6 presents illustrative, non-limiting examples of the synthetic
methodology used to make the non-
natural amino acids described herein.
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1001861 FIG. 7 presents illustrative, non-limiring,examples of the synthetic
methodology used to make the non-
natural anuno acids described. herein.
1001871 FIG. 8 presents illustrative, non-limiting.examples of the synthetic
niethodology used.to make the non-
natural aniino acids described herein.
1001881 FIG. 9 presents illustrative, non-limiting examples of the synthetic
methodology used to make the non-
natural amino acids described herein.
1001891 FIG. 10 presents illustrative, non-limiting examples of the synthetic
methodology used to make the
non-natural amino acids described herein.
1001901 FIG. I1 presents illustrative, non-limiting examples of the synthetic
methodology used to make the
non-natural amino acids described herein.
[00191] FIG. 12 presents illustrative, non-limiting examples of the effect of
metal ion on the Fisher indole
synthesis.
(00192] FIG. 13 presents illustrative, non-limiting examples of the
accelerating effect of nickel metal ion on the
synthetic methodology used to make the non-natural anuno acids described
herein.
1001931 FIG. 14 presents illustrative, non-limiting examples of the effect of
the solvent on the synthetic
methodology used to make the non-naniral aminoacids described herein.
1001941 FIG. 15 presents illustrative, non-limiting examples of hydrazine-
containing non-natural amino acid
reagents used in the synthesis of indole-containing;non-amino acids
described.herein.
1001951 FIG. 16; presents illustrative, non-limiting examples of carbonyl-
containing and masked carbonyl-
containing non-natural amino acid reagents used in the synthesis of indole-
eontaining non-natural amino acids
described herein.
1001961 FIG. 17 presents illustrative, non-limiting examples of carbonyl-
containing and masked carbonyl-
containing non-natural amino acid reagents used in the synthesis of indole-
containing non-natural amino acids
described herein.
1001971 FIG. 18 presents illustrative, non-limiting examples of carbonyl-
containing non-natural amino acid
reagents used in the synthesis of indole-containing non-natural aniino acids
described herein.
1001981 FIG. 19 presents illustrative, non-limiting examples of the post-
translational modification of carbonyl-
containing non-natural amino acid polypeptides with hydrazine-containing
reagents to form modifed indole-
containing non-natural amino acid polypeptides.
1001991 FIG. 20 presents illustrative, non-limiting examples of the post-
translational modification of
hydrazine-containing non-natural amino acid polypeptides with carbonyl-
containingreagents to form modifed
indole-containing non-natural amino acid polypeptides.
1002001 FIG. 21 presents illustrative, non-limiting examples of carbonyl-
containing and masked carbonyl-
containing proteins labeling or modification.with hydrazine-containing
reagents to form indole-containing non-
natural amino:acid proteins.
1002011 FIG. 22 presents illustrative, non-lin-iting exantples of hydrazine-
containing proteins labeling or
niodification with carbonyl-containing reagents to form indole-containing non-
natural amino acid proteins.
1002021 Fig. 23 presents illustrative, non-limiting examples of (A) the
modification of non-natural amino
acid polypeptides by chemical conversion into carbonyl-containing non-natural
amino acid polypeptides and (B)
the niodification of non-natural amino acid polypeptides by chemical
conversion into hydrazine-containing non-
natural amino acid polypeptides. Such non-natural amino acid polypeptides are
used in or incorporated into any
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of the methods, compositions, techniques and strategies for making, purifying,
characterizing, and using non-
natural amino acids, non-natural amino acid polypeptides and modified non-
natural amino acid polypeptides
described herein.
1002031 FIG. 24A represents illustrative, non-limiting examples of the
modification of hydrazine and
carbonyl non-natural amino acid.containing polypeptides or proteins to
introduce the desired functionality. Such
non-natural amino acid polypeptides are used in or incorporated into any of
the methods, compositions,
techniques and strategies for making, purifying, characterizing, and using non-
natural amino acids, non-natural
amino acid polypeptides and modified non-natural amino acid polypeptides
described herein.
1002041 FIG. 24B represents illustrative, non-limiting examples of the
reaction of functional group
containing polypeptides or proteins with PEG derivatives. Such non-natural
amino acid polypeptides are used in
or incorporated into any of the methods, compositions, techniques and
strategies for making, purifying,
characterizing, and using non-natural amino acids, non-natural amino acid
polypeptides and modified non-
natural amino acid polypeptides described herein.
1002051 FIG. 25 presents an illustrative, non-liniiting representation of the
use of a bifunctional linker
group to link protein or polypeptide containing non-natural amino acid with
PEG derivatives through the
formation of indole.
1002061 FIG. 26 presents an illustrative, non-linuting examples of the the
synthesis a bifunctional linker
group containing hydrazine at both ends.
1002071 FIG. 27 presents an illustrative, non-limiting example of the use of a
bifunctional linker to form a
homodimer of two non-natural amino acids polypeptides.
1002081 FIG. 28 represents illustrative, non-limiting examples of the reaction
between branched PEG
containing reagents and carbonyl non-natural amino acid containing
polypeptides to form indole modified
polypeptides. Such non-natural amino acid polypeptides are used in or
incorporated into any of the methods,
compositions, techniques and strategies for making, purifying, characterizing,
and using non-natural amino
acids, non-natural amino acid polypeptides and modified non-natural amino acid
polypeptides described herein.
1002091 FIG. 29 represents illustrative, non-limiting examples of the reaction
between branched PEG
containing reagents and hydrazine non-natural amino acid containing
polypeptides to form indole modified
polypeptides. Such non-natural anuno acid polypeptides are used in or
incorporated into any of the methods,
compositions, tecluiiques and strategies for making, purifying,
characterizing, and using non-natural amino
acids, non-natural amino acid polypeptides and niodified non-natural amino
acid polypeptides described herein.
(00210) FIG. 30 represents illustrative, non-limiting examples of PEG
derivatives containing hydrazine
and carbonyl groups.
DETAILED DESCRIPTION OF THE INVEN7'ION
I. Introduction
1002111 Recently, an entirely new technology in the protein sciences has been
reported, which overcome many
of the limitations associated with site-specific modifications of proteins.
Speci6cally, new components have
been added to the protein biosynthetic niachinery of the prokaryote
Escherichia coli (E. coli) (e.g., L. Wang, et
al., (2001), Science 292:498-500) and the eukaryote Sacclrornyces cerevisiae
(S. cerevisiae) (e.g., J. Chin et al.,
Science 301:964-7 (2003)), which has enabled the incorporation of non-natural
amino acids to proteins in vivo.
A number of new aniino acids with novel chemical, physical or biological
properties, including photoaffinity
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labels and photoisoinerizable amino acids, keto amino acids, and glycosylated
amino acids have been
incorporated efficiently and with high fidelity into proteins in E. coli and
in yeast in response to the amber
codon, TAG, using this methodology. See, e.g., J. W. Chin et al., (2002),
Journal of the American Chemical
Socie 124:9026-9027 (incorporated by reference in its entirety); J. W. Chin, &
P. G. Schultz, (2002),
CheniBioChem 3(11):1135-1137 (incorporated by reference in its entirety); J.
W. Chin, et al., (2002), PNAS
United States of America 99(71):11020-1 1024 (incorporated by reference in its
entirety); and, L. Wang, & P. G.
Scliultz, (2002), Chem. Comm., 1-11 (incorporated by reference in its
entirety). These studies have
demonstrated that it is possible to selectively introduce chemical functional
groups that are not found in
proteins, that are chemically inert to all of the functional groups found in
the 20 common, genetically-encoded
amino acids and that may be used to react efficiently and selectively to form
stable covalent linkages.
IL Overview
1002121 FIG. 1 is a non-limiting example of the compositions, methods,
techniques and strategies that are
described herein. At one level, described herein are the tools (methods,
compositions, techniques) for creating
and using a polypeptide comprising at least one non-natural amino acid or
modified non-natural amino acid with
a carbonyl, hydrazine, or heterocycle, including a nitrogen-containing
heterocycle group. The carbonyl group
includes, but is not limted to, ketones or aldehydes,. Such non-natural amino
acids optionally contain further
functionality, including but not limited to a desired functionality. Note that
the various aforenientioned
functionalities are not meant to imply that the members of one functionality
can not be classified as menibers of
another functionality. Indeed, there will be overlap depending upon the
particular circumstances. By way of
example only, a water-soluble polymer overlaps in scope with a derivative of
polyethylene glycol, however the
overlap is not complete and thus both functionalities are cited above.
1002131 As shown in FIG. 1, in one aspect are niethods for selecting and
designing a polypeptide to be
modified using the methods, compositions and techniques described herein. The
new polypeptide is optionally
designed de novo, including by way of exantple only, as part of liigh-
throughput screening process (in which
case numerous polypeptides are designed, synthesized, cliaracterized and/or
tested) or based on the interests of
the researcher. Alternativly, the new polypeptide is optionally designed based
on the structure of a known or
partially characterized polypeptide. By way of example only, the Growth
Hormone Gene Superfamily (see
infra) has been the subject of intense study by the scientific conununity; in
one embodiment, a new polypeptide
is designed based on the structure of a member or members of this gene
superfamily. The principles for
selecting which amino acid(s) to substitute and/or modify are described
separately herein. The choice of which
modification to employ is also described herein, and is used to nieet the need
of the experimenter or end user.
Such needs include, but are not limited to, manipulating the therapeutic
effectiveness of the polypeptide,
improving the safety profile of the polypeptide, adjusting the
pharmacokinetics, pharmacologics and/or
pharmacodynamics of the polypeptide, such as, by way of example only,
increasing water solubility,
bioavailability, increasing serum half-life, increasing therapeutic. half-
life, niodulating immunogenicity,
modulating biological activity, or extending the circulation time. In
addition, such modifications include, by
way of example only, providing additional. functionality to the polypeptide,
incorporating a tag, label or
detectable signal into the polypeptide, easing the isolation properties of the
polypeptide, and any combination of
the aforementioned modifications.
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[002141 Also described herein are non-natural amino acids that have been or
are optionally modified to contain
a hydrazine, carbonyl, or heterocycle, including a nitrogen-containirig
heterocycle group. The carbonyl group
iitcludes, but is not limted to, ketones, and aldehydes. Included with this
aspect are methods for producing,
purifying, characterizing and using such non-natural amino acids. In another
aspect described herein are
methods, strategies and techniques for incorporating at least one such non-
natural amino acid into a polypeptide.
Also included with this aspect are methods for producing, purifying,
characterizing and using such polypeptides
containing at least one such non-natural amino acid. Also included with this
aspect are compositions of and
methods for producing, purifying, characterizing and using oligonucleotides
(including DNA and RNA) that can
be used to produce, at least in part, a polypeptide containing at least one
non-natural amino acid. Also included
with this aspect are compositions of and methods for producing, purifying,
characterizing and using cells that
can express such oligonucleotides that can be used to produce, at least in
part, a polypeptide containing at least
one non-natural amino acid.
1002151 Thus, polypeptides comprising at least one non-natural amino acid or
modified non-natural amino acid
with a hydrazine, carbonyl, or heterocycle, including a nitrogen-containing
heterocycle group are provided and
described herein. Carbonyl modified non-natural amino acids include, but are
not limted to, ketones and
aldehydes. ln certain embodiments, polypeptides with at least one non-natural
amino acid or modified tton-
natural amino acid with a hydrazine, carbonyl, or heterocycle, including, a
nitrogen-containing heterocycle
group include at least one co-translational or post-translational modification
at some position on the
polypeptide. In such emdodiments, the carbonyl modified non-natural amino
acids further include, but are not
linited to, ketones, and aldehydes. In some embodiments the co-translational
or post-translational modification
occurs via the cellular machinery (e.g., glycosylation, acetylation,.
acylation, lipid-modification, palmitoylation,
palmitate addition, phosphorylation, glycolipid-linkage modification, and the
like), in many instances, such
cellular-machinery-based co-translational or post-translational modifications
occur at the naturally occurring
amino acid sites on the polypeptide, however, in certain embodiments, the
cellular-niachinery-based co-
translational or post-translational modifications occur on the non-natural
amino acid site(s) on the polypeptide.
(00216[ In other embodiments. the post-translational modification does not
utilize the cellular machinery, but
the futictionality is instead provided by attachment of a molecule (including
but not limited to, a desired
functionality) comprising a second reactive group to the at least one non-
natural amino acid comprising a first
reactive group (including but not limited to, non-natural amino acid
containing a ketone, an aldehyde, a
hydrazine, or a lieterocycle, including a nitrogen-containing heterocycle,
functional group) utilizing chemistry
metliodology described herein, or others suitable for the particular reactive
groups. In certain embodiments, the
co-translational or post-translational modification is made in vivo in a
eukaryotic cell or in a non-eukaryotic cell.
In certain embodiments, the co-translational or post-translational
modification is made in vitro not utilizing the
cellular machinery. Also included with this aspect are methods for producing,
purifying, characterizing and
using such polypeptides containing at least one such post-translationally
modified non-natural amino acids.
1002171 Also included within the scope of the methods, compositions,
strategies and techniques described
herein are reagents capable of reacting with a non-natural amino acid
(containing either a carbonyl group, a
ketone, a ketoaldehyde, a hydrazine, or: protected forms thereof) that is part
of a polypeptid'e so as to produce
any of the aforementioned post-translational modifications. In general, the
resulting post-translationally
modified non-iiatural amino acid will produce at least one indole derivative.
The resulting modified indole-
based non-natural amino acid, in some embodiments, undergoes subsequent
modification reactions. Also
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included with this aspect are methods for producing, purifying, characterizing
and using such reagents that are
capable of any such post-translational modifications of such non-natural
aniino acid(s).
1002181 In certain embodiments, the polypeptide includes at least one co-
translational or post-translational
modification that is made in vivo by one host cell, where the post-
translational modification is not normally
made by another host cell type. In certain embodiments, the polypeptide
includes at least one co-translational or
post-translational modification that is made in vivo by a eukaryotic cell,
where the co-translational or post-
translational modification is not normally made by a non-eukaryotic cell.
Examples of such co-translational or
post-translational modifications include, but are not limited to,
glycosylation, acetylation, acylation, lipid-
modification, palmitoylation, palmitate addition,,phosphorylation, glycolipid-
linkage modification, and the like.
In one embodiment, the co-translational or post-translational modification
comprises attachment of an
oligosaccharide to an asparagine by a GIcNAc-asparagine linkage (including but
not limited to, where the
oligosaccharide comprises (GIcNAc-Man)2-Man-GIcNAc-G1cNAc, and the like). In
another embodiment, the
co-translational or post-translational modification comprises attachment of an
oligosaccharide (including but not
limited to, Gal-GaINAc, Gal-GIcNAc, etc.) to a serine or threonine by a GaINAc-
serine, a GaINAc-threonine, a
G1cNAc-serine, or a G1cNAc-threonine linkage. Examples of secretion signal
sequences include, but are not
limited to, a prokaryotic secretion signal sequence, a eukaryotic secretion
signal sequence, a eukaryotic
secretion signal sequence 5'-optimized forbacterial expression, a novel
secretion signal sequence, pectate lyase
secretion signal sequence, Omp A secretion signal sequence, and a phage
secretion signal sequence. Examples
of secretion signal sequences, include, but are not limited to, S"TII
(prokaryotic), Fd GIII and M 13 (phage), Bg12
(yeast), and the signal sequence bla derived from a transposon. In certain
embodiments, a protein or polypeptide
can comprise a secretion or localization sequence, an epitope tag, a FLAG tag,
a polyhistidine tag, a GST
fusion, and/or the like. Also included with this aspect are methods for
producing, purifying, characterizing and
using such polypeptides containing atleast one such co-translational or post-
translational modification. In other
embodiments, the glycosylated non-natural amino acid polypeptide is produced
in a non-glycosylated form.
Such a non-glycosylated form of a glycosylated non-natural amino acid are
optionally produced by methods that
include chemical or enzymatic removal of oligosaccharidegroups from an
isolated or substantially purified or
unpurified glycosylated non-natural amino acid polypeptide; production of ihe
non-natural amino acid in a host
that does not glycosylate such a non-natural amino acid polypeptide (such a
host including, prokaryotes or
eukaryotes engineered or mutated to not glycosylate such a polypeptide), the
introduction of a glycosylation
inhibitor into the cell culture medium in which such a non-natural amino acid
polypeptide is being produced by
a eukaryote that normally would glycosylate such a polypeptide, or a
combination of any such methods. Also
described herein are such non-glycosylated forms of normally-glycosylated non-
natural amino acid polypeptides
(by normally-glycosylated is meant a polypeptide that would be glycosylated
when produced ttnder conditions
in which naturally-occurring polypeptides are glycosylated). Of course, such
non-glycosylated forms of
norinally-glycosylated non-natural amino acid polypeptides are optionally in
an unpurified form, a substantially
purified form, or in an isolated form.
1002191 The non-natural anvno acid polypeptide contains, in alternative
embodiments, at least: one, at least two,
at least three, at least four, at ]east.five, at least six, at least seven, at
least eight, at least nine, or ten or niore non-
natural amino acids containing a carbonyl group, a ketone, an aldehyde, a
hydrazine, heterocycle, including a
nitrogen-containing heterocycle group, or protected forms thereof. The non-
natural amino acids can be the same
or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, or more
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different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or
more different non-natural amino acids. ln certain embodiments, at least one,
but fewer than all, of a particular
amino acid present in a naturally occurring version of the protein is
substituted with a non-natural amino acid.
[00220[ The methods and compositions provided and described herein include
polypeptides comprising at least
one non-natural amino acid containing a carbonyl group, a ketone, an aldehyde;
a hydrazine, heterocycle,
including a nitrogen-containing heterocycle group, or protected or masked
forms thereof. Introduction of at least
one non-natural amino acid into a polypeptide can allow for the application of
conjugation chemistries that
involve specific chemical reactions, including, but not limited to, with one
or niore non-natural amino acids
while not reacting with the commonly occurring 20 aniino acids. Once
incorporated, the non-naturally occurring
amino acid side chains can also be modified by utilizing chemistry
methodologies described herein or suitable
for the particular functional groups or substituents present in the naturally
encoded amino acid.
1002211 The non-natural amino acid methods and compositions described herein
provide conjugates of
substances having a wide variety of functional groups, substituents or
moieties, with other substances including
but not limited to a desired functionality.
1002221 In certain embodiments the non-natural amino acids, non-natural amino
acid polypeptides, linkers and
reagents described herein, including compounds of Formulas I-XV, and compounds
1-0 are stable in aqueous
solution under mildly acidic conditions (including but not lintited to pH of
about I to about 6). In other
embodiments, such compounds are stable for at least one month under mildly
acidic conditions. In other
embodiments, such compounds are stable for at least 2 weeks under mildly
acidic conditions. In other
embodiments, such compounds are stable for at least 5 days under mildly acidic
conditions
1002231 In another aspect of the compositions, methods, techniques and
strategies described herein are methods
for studying or using any of the aforementioned modified or ttttmodified non-
natural amino acid polypeptides.
Included within this aspect, by way of example only, are therapeutic,
diagnostic, assay-based, industrial,
cosmetic, plant biology, environmental, energy-production, consumer products
and/or military uses which
would benefit from a polypeptide comprising a modified or unmodified non-
natural amino acid polypeptide or
protein.
M. Location of non-natural amino acids iu po/ypeptides
[002241 The methods and compositions described herein include incorporation of
one or more non-natural
aniino acids into a polypeptide. One or rnore non-natural amino acids are, in
certain embodiments, incorporated
at one or more particular positions which does not disrupt activity of the
polypeptide. This is optionally
achieved by making "conservative" substitutions, including but not limited to,
substituting hydrophobic amino
acids with non-natural or natural hydrophobic amino acids, bulky amino acids
with non-natural or natural bulky
amino acids, hydrophilic amino acids with non-natural or natural hydrophilic
amino acids) and/or inserting the
non-natural amino acid in a location that is not required for activity.
Although such substitions are not known
for the non-natural amino acids described herein, the practice of making
conservative substitutions within the
group of naturally-occurring aniino acids has been documented. Similar
approaches are optionally used for the
non-natural amino acids described herein.
1002251 A variety of biochemical and structural approaches can be employed to
select the desired sites for
substitution with a non-natural amino acid within the polypeptide. Any
position of the polypeptide chain is
suitable for selection to incorporate a non-natural amino acid, and selection
is optionally based on rational
design or by random selection for any or no particular desired purpose.
Selection of desired sites is optionally
CA 02672205 2009-06-09
WO 2008/077079 PCT/US2007/088011
based on producing a non-natural amuio acid polypeptide (which is optionally
further nwdified or remain
unmodified) having any desired property or activity, including but not
liinited to agonists, super-agonists, partial
agonists, inverse agonists, antagonists, receptor binding modulators,
receptor, activity modulators, modulators of
binding to binder partners, binding partner activity modulators, biitding
partner coiiformation modulators, dimer
or multimer formation, no change to activity or property compared to the
native molecule, or manipulating any
physical or chemical property of the polypeptide such as solubility,
aggregation, or stability. For example,
locations in the polypeptide required for biological activity of a polypeptide
can be identified using methods
includ'uig, but not liniited to, point mutation analysis, alanine scanning or
homolog scanning methods. Residues
other than those identified as critical to biological activity by methods
including, but not limited to, alanine or
homolog scanning mutagenesis are good candidates for substitution with a non-
natural amino acid depending on
the desired activity sought for the polypeptide. Altematively, the sites
identified as critical to biological activity
are also good candidates for substitution with a non-natural amino acid, again
depending on the desired activity
sought for the polypeptide. Another altemative would be to simpty make serial
substitutions in each position on
the polypeptide chain with a non-natural amino acid and observe the effect on
the activities of the polypeptide.
Any means, technique, or method for selecting a position for substitution with
a non-natural amino acid into any
polypeptide is suitable for use in the methods, techniques and compositions
described.herein.
1002261 The structure and activity of naturally-occurring mutants of a
polypeptide that contain deletions can
also be examined to deterniine regions of the protein that are likely to be
tolerant of substitution with a non-
natural amino acid. Once residues that are likely to be intolerant to
substitution with non-natural amino acids
have been eliminated, the impact of proposed substitutions at each of-the
remaining positions can be examined
using methods including, but not limited to, the three=dimensional structure
of the relevant polypeptide, and any
associated ligands or binding proteins. X-ray crystallographic and NMR
siructures of many polypeptides are
available in the Protein Data Bank (PDB, www.rcsb.org), a centralized database
containing three-dimensional
structural data of large molecules of proteins and nucleic acids, one can be
used to identify amino acid positions
that can be substituted with non-natural amino acids. . In addition, models
are optionally made investigating the
secondary and tertiary structure of polypeptides, if three-dimensional
structural data is not available. Thus, the
identity of amino acid positions that are available for substitution with non-
natural amino acids is readily
obtained.
1002271 Exemplary sites of incorporation of a non-natural amino acid include,
but are not limited to, those that
are excluded from potential receptor binding regions, or regions for binding
to binding proteins or ligands, are
fully or partially solvent exposed, have minimal or no hydrogen-bonding
inteiactions with nearby residues, are
minimally exposed to nearby reactive residues, and/or are in regions that are
highly flexible as predicted by the
three-diniensional crystal structure of a particular polypeptide with its
associated receptor, ligand or binding
proteins.
1002281 A wide variety of non-natural amino acids are optionally substituted
for, or incorporated into, a given
position in a polypeptide. By way of example, a particular non-natural amino
acid is selected for incorporation
based on an examination of the three dimensional crystal structure of a
polypeptide, with its associated ligand,
receptor and/or binding proteins, a preference for conservative substitutions
46
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1002291 In one embodiment, the methods described herein include incorporating
into the polypeptide the non-
natural amino acid, where the non-natural amino acid comprises a first
reactive group; and contacting the
polypeptide with a molecule (including but not limited to a desired
functionality) that coniprises a second
reactive group. In certain embodiments, the first reactive group is a carbonyl
moiety and the second reactive
group is a hydrazine moiety, whereby an indole linkage is formed. In certain
embodiments, the first reactive
group is a hydrazine moiety and the second reactive group is carbonyl moiety,
whereby an indole linkage is
formed.
1002301 In some cases, the non-natural amino acid substitution(s) or
incorporation(s) will be combined with
other additions, substitutions, or deletions within the polypeptide to affect
other chemical, physical,
pharmacologic and/or biological traits. In some cases, the other additions,
substitutions or deletions increase the
stability (including but not limited to, resistance to proteolytic
degradation) of the polypeptide or increase
affinity of the polypeptide for its appropriate receptor, ligand and/or
binding proteins. In some cases, the other
additions, substitutions or deletions increase the solubility (including but
not limited to, when expressed in E.
coli or other host cells) of the polypeptide. In some embodiments sites are
selected for substitution with a
naturally encoded or non-natural anvno acid in addition to another site for
incorporation of a non-natural amino
acid for the purpose of increasing the polypeptide solubility following
expression in E. coli, or other
recotnbinant host cells. In some embodiments, the polypeptides comprise
another addition, substitution, or
deletion that modulates affinity for the associated ligand, binding proteins,
and/or receptor, modulates
(including but not limited to, increases or decreases) receptor dimerization,
stabilizes receptor dimers,
niodulates circulating half-life, modulates release or bio-availability,
facilitates purification, or improves or
alters a particular route of administration. Siniilarly, the non-natural amino
acid polypeptide can comprise
chemical or enzyme cleavage sequences, protease cleavage sequences, reactive
groups, antibody-binding
doniains (including butnot linuted to, FLAG or poly-His) or other affinity
based sequences (,including but not
limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not
limited to, biotin) that improve
detection (including but r(ot limited to, GFP), traasport thru tissues or cell
membranes, prodrug release or
activation, size reduction,purification or other traits of the polypeptide.
IV. Growth Horn:one Supergene Family as Exemplar
1002311 The methods, compositions, strategies and techniques described herein
are not limited to a particular
type, class or family of polypeptides or proteins. Indeed, virtually any
polypeptide is optionally designed or
modified to include at least one modified or unmodified non-natural amino
acids described herein. By way of
example only, the polypeptide is homologous to a therapeutic protein.
1002321 Thus, the following description of the growth hormone (GH) supergene
family is provided for
illustrative purposes and by way of example only and not as a limit on the
scope of the methods, compositions,
strategies and techniques described herein. Further, reference to GH
polypeptides in this application is intended
to use the generic term as an example of any member of the GH supergene
family. Thus, it is understood that
the modifications and chentistries described herein with reference to GH
polypeptides or protein can be equally
applied to any member of the GH supergene family, including those specifically
listed herein.
1002331 The following proteins include those encoded by genes of the growth
hormone (GH) supergene family
(Bazan, F., Immunology Today 11: 350-354 (1990); Bazan, J. F. Science 257: 410-
411 (1992); Mott, H. R. and
Campbell, I. D., Current Opinion in Structural Biology 5: 114-121 (1995);
Silvennoinen, 0. and Ihle, J. N.,
SIGNALLING BY THE: HGMATOROIGTIC CYTOKINE RECCPTOR5 (1996)): growth hormone,
prolactin, placental
47
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lactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2 (IL-2), IL-
3, IL-4, IL-5, IL-6, IL-7, IL-9,
I.L-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary
neurotrophic factor, leukeniia inhibitory
factor, alpha interferon, beta interferon, epsilon interferon, gamma
interferon, omega interferon, tau interferon,
granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony
stimulating factor (GM-CSF),
macropliage colony stimulating factor (M-CSF) and cardiotrophin-l (CT-1) ("the
GH supergene family"). It is
anticipated that additional members of this gene family will be identified in
the future through gene cloning and
sequencing. Members of the GH supergene family have similar secondary and
tertiary structures, despite the
fact that they generally have liniited amino acid or DNA sequence identity.
The shared structural featuresallow
new members of the gene family to be readily identified and the non-natural
amino acid methods and
conipositions described herein sinularly applied.
1002341 Structtues of a number of cytokines, including G-CSF (Zink et al.,
FEBS Lett. 314:435 (1992); Zink et
al., Biochemistry 33:8453 (1994); Hill et al., Proc.Natl.Acad.Sci.USA 90:5167
(1993)), GM-CSF (Diederichs,
K., et al. Science 154: 1779-1782 (1991); Walter et al., J. Mol. Biol.
224:1075-1085 (1992)), IL-2 (Bazan, J. F.
and McKay, D. B., Science 257: 410-413 (1992); IL-4 (Redfield et al.,
Bioc{remistty 30: 11029-11035 (1991);
Poweis et al., Science 256:1673-1677 (1992)), and IL-5 (Milbum et al., Nature
363: 172-176 (1993)) have been
determined by X-ray diffraction and NMR studies and show striking conservation
with the GH stnicture, despite
a lack of significant primary sequence homology. IFN is considered to be. a
member of this family based upon
modeling and other studies (Lee et al., J. Interferon Cytokine Res. 15:341
(1995); Murgolo et al., Proteins 17:62
(1993); Radhakrishnan et al., Structure 4:1453 (1996); Klaus et al., J. Mol.
Biol. 274:661 (1997)). A large
number of additional cytokines and growth factors including ciliary
neurotrophic factor (CNTF), leukemia
inhibitory factor (LIF), thrombopoietin (TPO), oncostatin M, macrophage colony
stimulating factor (M-CSF),
IL-3, IL-6, IL-7, IL-9, IL-l2, IL-13, IL-15, and granulocyte-colony
stimulating factor (G-CSF), as well as the
IFN's such as alpha, beta, omega, tau, epsilon, and gamma interferon belong to
this family (reviewed in Mott
and Campbell, Current Opinion in Structural Biology 5: 114-121 (1995);
Silvennoinen and Ihle (1996)
SIGNALLING E3Y THE HEMATOPOIETIC CYTOKiNE RECEPTORS). All of the above
cytokines and growth factors are
now considered to comprise one large gene family.
(002351 In addition to sharing similar secondary and tertiary structures,
members of this family share the
property that they must oligomerize cell surface receptors to activate
intracellular signaling pathways. Some GH
family members, including but not linuted to; GH and EPO, bind a single type
of receptor and cause it to form
homodimers. Other family members, including but not limited to, IL-2, IL4. and
IL-6, bind more than one type
of receptor and cause the receptors to form heterodimers or higher order
aggregates (Davis et al., (1993) Science
260: 1805-1808; Paonessa et al., 1995) EMBO J. 14: 1942-1951; Mott and
Campbell, Current Opinia: in
Sti-uctura! Biology 5: 114-121 (1995)). Mutagenesis studies have shoNvn that,
like GH, these other cytokines and
growth factors contain multiple receptor binding sites, typically two, and
bind their cognate receptors
sequentially (Mott and Campbell, Current Opinion in Structural Biology 5: 114-
121 (1995); Matthews et al.,
(1996) Proc. Natl. Acad. Sci. USA 93: 9471-9476). Like GH, the primary
receptor binding sites for these other
family members occur priinarily in the four alpha helices and the A-B loop.
The specific amino acids in the
helical bundles that participate in receptor binding differ amongst the family
niembers. Most of the cell surface
receptors that interact with niembers of the GH supergenc family are
struchually related and comprise a second
large multi-gene family. See, e.g. U.S. Patent No. 6,608,183, which is herein
incorporated by reference for the
description of the GH supergene family.
48
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WO 2008/077079 PCT/US2007/088011
1002361 A general conclusion reached from mutational studies of various
members of the GH supergene family
is that the loops joining the alpha helices generally tend to not be involved
in receptor binding. In particular the
short B-C loop appears to be non-essential for receptor binding in most, if
not all, family members. For this
reason, the B-C loop are optionally substituted with non-natural amino acids
as described herein in members of
the GH supergene family. The A-B loop, the C-D loop (and D-E loop of
interferon/ IL-10-like members of the
GH superfamily) are also opttionally substituted with a non-natural amino
acid. Amino acids proximal to
helix A and distal to the final helix also tend not to be involved in receptor
binding and also are optional sites
for introducing non-natural amino acids. In sonie embodiments, a non-natural
amino acid is substituted at any
position within a loop structure including but not limited to the first 1, 2,
3, 4, 5, 6, 7, or more amino acids of the
A-B, B-C, C-D or D-E loop. In some embodiments, a non-natural aniino acid is
substituted within the last 1, 2,
3. 4, 5, 6, 7, or more amino acids of the A-B, B-C, C-D or D-E loop.
1002371 Certain members of the GH family, including but not limited to, EPO,
IL-2, IL-3, IL-4, IL-6, IFN, GM-
CSF, TPO, IL-10, II,-12 p35, IL-13, IL-15 and beta interferon contain N-linked
and/or 0-linked sugars. The
glycosylation sites in the proteins occur almost exclusively in the loop
regions and not in the alpha helical
bundles. Because the loop regions generally are not involved in receptor
binding and because they are sites for
the covalent attachment of sugar groups, they are useful sites for introducing
non-natural amino acid
substitutions into the proteins. Amino acids that comprise the N- and 0-linked
glycosylation sites in the proteins
are sites for non-natural amino acid substitutions because these amino acids
are surface-exposed. Therefore, the
natural protein tolerates:bulky sugar groups attached to the proteins at these
sites and the glycosylation sites tend
to be located away from the receptor binding sites.
1002381 Additional members of the GH gene family are likely to be discovered
in the future. New members of
the GH supergene family can be identified through computer-aided secondary and
tertiary structure analyses of
the predicted protein sequences, and by selection techniques designed to
identify molecules that bind to a
particular target. Members of the GH supergene family typically possess four
or five amphipathic helices joined
by non-helical amino acids (the loop regions). In some embodiments, the
proteins contain a hydrophobic signal
sequence at their N-terminus to promote secretion from the cell. Such later
discovered niembers of the GH
supergene family also are included within the methods and compositions
described herein.
V. Non-nalura! Amino Acids
1002391 The non-natural amino acids used in the methods and compositions
described herein have at least one
of the following four properties: (1) at least one functional group on the
sidechain of the non-natural amino acid
has at least one characteristic and/or activity and/or reactivity orthogonal
to the chemical reactivity of the 20
common, genetically-encoded amino acids (i.e., alanine, arginine, asparagine,
aspartic acid, cysteine, glutamine,
glutanuc acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine,
threonine, 1ryptophan, tyiosine, and valine), or at least orthogonal to. the
chemical reactivity of the naturally
occurring amino acids present in the polypeptide that includes the non-natural
amino acid; (2) the introduced
non-natural amino acids.are substantially chemically inert toward the 20
conunon, genetically-encoded amino
acids; (3) the non-natural amino acid can be stably incorporated into a
polypeptide, preferably with the stability
conunensurate with the naturally-occurring amino acids or under typical
physiological conditions, and further
prcferably such incorporation can occur via an in vivo system; and (4) the non-
natural amino acid includes a
carbonyl group, a ketone group, an aldehyde group, a hydrazine group,
heterocycle, including a nitrogen-
49
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WO 2008/077079 PCT/US2007/088011
containing heterocycle group (including, an indolyl group), or a functional
group that can be transformed into an
carbonyl group, a ketone group, an aldehyde group, a hydrazine group,
heterocycle, including a nitrogen-
containing heterocycle group (including an indolyl group), by reacting with a
reagent, preferably under
conditions that do not destroy the biological properties of the polypeptide
that includes the non-natural amino
acid (unless of course such a destruction of biological properties is the
purpose of the
modification/transformation), or preferably where the transformation can occur
under aqueous conditions at a
pH between about I and about 6, or preferably. where the reactive site on the
non-natural amino acid is an
electrophilic site. Illustrative, non-limiting examples of amino acids that
satisfy these four properties for non-
natural amino acids that can be used with the compositions and methods,
described herein are presented in FIGS.
15-18. Any number of non-natural amino acids can be introduced into the
polypeptide:
1002401 Note that, even though FIGS. 15, 16, and 18 present amino acids having
a substituted carbocyclic aryl
sidechain, it is to be understood that these FIGS. also disclose substituted
heteroaryl sidechains. By way of
exainple, each of the compounds of FIG. 15 has a substittited phenyl
sidechain; however, in some embodiments,
the phenyl group is replaced by a pyridinyl group, a pyrmidinyl group, a
pyrazinyl group, a thiofuranyl group,
or a fttranyl group. Thus, the carbocyclic aryl sidechains are merely
illustrative exaniples of the variety of
aromatic groups included within the present disclosure.
1002411 Non-natural amino acids optionally also include a carbonyl group or a
protected or masked groups that
can be transformed into a carbonyl group, a carbonyl group after deprotection
of the protected group or
unmasldng of the masked group, a hydrazine group or a protected or masked
group that can be transformed into
a hydrazine group.
1002421 Non-natural amino acids -that are optionally used in the methods and
compositions described herein
include, but are not limited to, amino acids comprising a photoactivatable
cross-linker, spin-labeled amino
acids, fluorescent amino acids, metal binding amino acids, metal-containing
amino acids, radioactive amino
acids, amino acids with novel functional groups, amino acids that covalently
or noncovalently interact with
other molecules, photocaged and/or photoisomerizable amino acids, amino acids
comprising biotin or a biotin
analogue, glycosylated amino acids such as a sugar substituted serine, other
carbohydrate modified amino acids,
keto-containing amino acids, amino acids comprising polyethylene glycol or
other polyethers, heavy atom
substituted amino acids, chemically cleavable and/or photocleavable amino
acids, amino acids with an
elongated side chains as compared to natural amino acids, including but not
limited to, polyethers or long chain
hydrocarbons, including but not limited to, greater than about 5 or greater
than about 10 carbons, carbon-linked
sugar-containing amino acids, redox-active anuno acids, amino thioacid
containing amino acids, and amino
acids comprising one or niore toxic moiety.
1002431 In some embodiments, non-natural amino acids comprise a saccharide
moiety. Examples of stich amino
acids include N-acetyl-L-glucosanunyl-L-serine, N-acetyl-L-galaetosaminyl-L-
serine, N-acetyl-L-glucosaminyl-
L-threonine, N-acetyl-L-glucosaminyl-L-asparagine and 0-mannosantinyl-L-
serine. Examples of such amino
acids also include examples where the naturally-occurring N- or 0- linkage
between the amino acid and the
saccharide is replaced by a covalent linkage not commonly found in nature. -
including but not limited to, an
alkene, an oxime, a thioether, an amide, a heterocycle, including a nitrogen-
containing heterocycle, a carbonyl
and the like. Examples of such amino acids also include saccharides that are
not commonly found in naturally-
occurring proteins such as 2-deoxy-glucose, 2-deoxygalactose and the like.
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1002441 The chenucal inoieties incorporated into polypeptides via
incorporation of non-natural amino acids into
such polypeptides offer a variety of advantages and manipulations of the
polypeptides. For example, the unique
non-natural amino acids (including but not limited to, amino acids with
benzophenone and arylazides (including
but not limited to, phenylazide side chains), for example, allow for efficient
in vivo and in vitro
photocrosslinking of protein. Examples of photoreactive non-natural anuno
acids include, but are not limited to,
p-azido-phenylalanineand p-benzoyl-phenylalanine. The polypeptide with the
photoreactive non-natural amino
acids is then optionally crosslinked at will by excitation of the
photoreactive group-providing temporal control.
In a non-limiting example, the methyl group of a non-natural amino is
substituted with an isotopically labeled,
including but not limited to, with a methyl group, as a probe of local
structure and dynamics, including but not
limited to, with the use of nuclear magnetic resonance and
vibrational'spectroscopy.
A. Structure and Synthesis of Non-Natrtral Amino Acids: Hydrazine, Hydraziue-
like, Masked
Hydrazine, asd Protected Hydrazine Groups
1002451 Non-natural amino acids containing a hydrazine group allow for
reaction with a variety of carbonyl and
carbonyl equivalent groups to form conjugates via indole linkage. Thus, in
certain embodiments described
herein are non-natural amino acids with sidechains comprising a hydrazine
group, a hydrazine like group (which
has reactivity similar to a hydrazine group and is structurally similar to a
hydrazine group), a niasked hydrazine
group (which can be readily converted into a hydrazine group), or a protected
hydrazine group (which has
reactivity similar to a hydrazine group upon deprotection). Such amino acids
include amino acids having the
structureofFormula (1):
R3
R} ~ JR
H R;A,B
R2
0 (I),
wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene;
lower cycloalkylene, substituted
lower cycloalkyl"ene, lower alkenylene, substituted lower alkenylene,
alkynylene, substituted alkynylene,
lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,
substituted lower
heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene,
substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting
of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-, -0-(alkylene or
substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-
where k is 1, 2, or 3, -
S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)Z-, -OS(O)z-, -C(O)-
(alkylene or substituted
alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-
(alkylene or substituted
alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -
CSN(R')-, -CSN(R')-(alkylene or
substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -
N(R')C(O)O-, -S(O)kN(R')-,
-N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNO2)N(R')-,
-N(R')C(NCOOR')N(R')-, -N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-
N=N-, and
-C(R')2-N(R')-N(R')-;. and each R' is independently.H, alkyl, or substituted
alkyl;
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WO 2008/077079 PCT/US2007/088011
N Ny N ~N N
\NHt N%~ I ~NH= i ~ . .
~~ I NH= NH=
NHp r I (
(y (\ N N
Jis~', or
R is H, lower alkenylene, substituted lower alkenylene,,alkynylene;
substituted alkynylene; carbonyl, or
substituted. carbonyl;
R' is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R2.is OH, an ester protecting group, resin, at least.one amino acid,
polypeptide,, or polynucleotide;
each of R3 and R 4 is independently H, halogen, lower alkyl, or substituted
lower alkyl, or R3 and R4 taken
together or two R3 groups taken together optionally form a cycloalkyl or a
heterocycloalkyl;
or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or
heterocycloalkyl comprising a
hydrazine group, a protected hydrazine group or a masked hydrazine group;
It should be noted that, where I appears in structure J, this means that J can
be attached to B and R at any
position. As a non-limiting example, where J is a hydrazinephenyl derivative,
B and J are optionally positioned
around the ring, as illustrated below:
N..NH2 - \ ~ N`NHz N.NHZ R q N_NH2 R NNH2 R NNH2 R N,NHZ
R g~ ~g
I
H N
H R
/ I N' NH2 /~ N.NH2 g/' NNH2 g/ N,NH2 g/ N.NH2 ,g &,N~NH,
g \ R g~ ~ I R R~ ~ R R
It should also be further noted that the ring is optionally further optionally
substituted. In addition it should be
noted that at least one of the adjacent substituent to hydrazine group is
hydrogen. Such non-natural amino acids
are optionally in the form of a salt, or incorporated into a non-natural amino
acid polypeptide, polynier,
polysaccharide,. or a polynucleotide and optionally post translationally
modified.
1002461 In onc embodiment, both A and B are bonds, each R3 is H and R, is H.
In a further embodiment, each
of R, and R2 are at least one amino acid. In a further embodiment, each of R,
and R_, are at least two amino
acids. In a further embodiment, each of Ri and R,-are at least three amino
acids. In a further embodiment, each
of Ri and R2 are at least four amino acids. In a further embodiment, each of
R, and R2 are at least five amino
acids. In a further embodiment, each of R, and R, are at least eix amino
acids.
1002471 In certain embodiments, compounds of Formula (I) are stable in aqueous
solution for at least 1
month under mildly acidic conditions. In certain embodiments, compounds of
Fomiula (I) are stable for at least
2 weeks under mildly acidic conditions. In certain embodiments, compound of
Formula (I) are stable for at least
5 days under nuldly acidic conditions. In certain embodiments, such acidic
conditions are pH about 2 to about 8.
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WO 2008/077079 PCT/US2007/088011
1002481 In addition, the following amino acids having the structure of Formula
(11) are included:
Ra
H AB , \ Ra
R' N~ NN.Ra
R N
0 R, . (II)
wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene,
lower cycloalkylene, substituted
lower cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, substitutedalkynylene,
lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,
substituted lower
heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene,
substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting
of lower alkylene, substituted
lower alkylene, lower alkenyiene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-(alkylene or
substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where
k is 1, 2, or 3, -S(O)k(alkylene
or sutistituted alkylene)-, -C(O)-,. -NS(O)2-, -OS(Q)2-, -C(O)-(alkylene or
substituted alkylene)-; -C(S)-,
-C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted
alkylene)-, -C(O)N(R')-,
-CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or
substituted alkylene)-,
=N-O-(alkylene or substituted alkylene), -N(R')CO-(alkylene or substituted
alkylene)-, -N(R')C(O)O-,
-S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, and -C(R')2-N(R')-
N(R')-;,and each R' is
independently H, alkyl, or substituted alkyl;
R, is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R3 and R4 is independently selected from the group consisting of hydrogen or
from an amine protecting group,
including, but not limited to,
H
~YO1_~CCI3 .,IryO,,,~/'
O ~ O O O OzN CZHS
/~
CN
r-rl yO~CH ' ~yC%CN =~~ = -C~ W3 or ~~0./~SiMe3
~,O ~ 1
O ~ O H3C ,. ~ O ~ CH; O ; and
each Re is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl, CN, NOZ, -
N(R.')Z, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2 or 3 and
each R' is independently H,
alkyl, or substituted alkyl.
Such non-natural amino acids are optionally 'in the form of a salt, or are
incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally
post translationally modified.
53
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1002491 In one.embodiment; both A and B are bonds. In a further embodiment,
each of R1 and R,_ are at least
one amino acid. In a further embodiment, each of Ri and R2 are at least two
amino acids. In a further
embodinient, each of R, and R2 are at least three amino acids. In a further
embodiment, each of Ri and R2 are at
least four amino acids. In a further embodiment, each of R, and R2 are at
least five amino acids. In a further
embodiment, each of R, and R, are at least eix amino acids.
1002501 In addition, the following, amino acids having the structure of
Formula (Ill) are included:
R4
R' N (CRaa)n.--B N-N,R
3
0 R2 (III),
wherein:
B is optional, and wlien present is a linker selected from the group
consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-, -N(R')-, -S-,
-O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-
, -S(O)k- where k is l, 2, or 3, -
S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)Z-, -OS(O)Z-, -C(O)-
(alkylene or substituted
alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-. -NR'-(alkylene
or substituted alkylene)-,
-C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -
CSI`T(R')-(alkylene or substituted
alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)0-, -
S(O)kN(R')-, -C(R')=N-, -
C(R')=N-N(R')-, -C(R')rN=I`'-, and -C(R')2-N(R')-N(R')-; -S(O)kN(R')-, -
C(R')=N-, -C(R')=N-N(R')-,
-C(R')Z-N=N-, and -C(R')2-N(R')-N(R')-; and each R' is independently H, alkyl,
or substituted alkyl;
R' is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R3 and R4 is independently selected from the group consisting of hydrogen or
from an amine protecting group,
including, but not limited to,
i ~ H
; \
~\ yO.V CCl3 ~~0~ ~~O \ ~ fY0 ~ l C
j(
O ~ O O O OZN CZHS
~\
O-~.~,( CH3 ~
-~~ (I CN ~~/ iCH3 Ur ~
. ~ O./~ SiMe3
O ~
~ p ~ 1II 1~ O H3C CHs CN O ,~=c CH3 O ; and
each R, is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl, -N(R')2, -
C(G)R', -C(O)N(R')2, -OR', and -S(O)kR',:where k is 1, 2 or 3 and each R' is
independently H, alkyl, or
substituted alkyl; and
nis0to8.
Such non-natural amino acids niay be in the form of a salt, or niay be
incorporated into a non-natural amino acid
polypeptide, polynier, polysaccharide, or a polynucleotide and optionally post
translationally modified.
54
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1002511 In yet another embodiment is a conipound of Formula (II) having the
structure:
R.
(CH2)nXI(CO)yX2(CI-12)m'$ R4
Rl-, N R2 13 R~ I/ N.R3
H R4O R. H
wherein, n is 6, 1, 2, 3, or 4; m is 0, 1, 2, 3, or 4; provided that n + m is
1, 2, 3, 4, or 5; Xi and X2 are
independently a bond, 0, or NH; and y is.0 or 1.
1002521 In addition, the following.amino acids are included:
H
HN N, NH2
H 0 H ZOH
HN O N N
~`H .NHZ H N`NH
H N OH HZN OH H2N H s
z 0 0 0 , and
, .
H
II NNHZ
N a
HNJ O
HN--'Z~'N' CN
H2N OH
0 , wherein such compounds are optionally amino protected and carboxyl
protected,
or a salt thereof, or niay be incorporated into a non-natural amino acid
polypeptide, polyiner, polysaccharide, or
a polynucleotide and optionally post translationally modified.
1002531 In.addition, the following amino acids having the.structure of Formula
(IV) aie included:
Ra
H
Ra N, N" R4
I
H R3
Rl,,N R 2
H
0 (IV)
wherein:
R' is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R3 and R4 are independently selected from the group consisting of hydrogen or
from an amine protecting group,
including, but not limited to,
i
/Y OvCCij J~O I ~=C H
0 0 ; 0 O OZN CzHs
O CN
~ S ~/
~ O y ~CH3 S=C~ II ~_ ;GH3 or ~0,tiSiMey
~ 0 H3C CH3 ; SSS CN 0 CH3 0 0 ; and
CA 02672205 2009-06-09
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each Ra is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl, CN, NO2, -
N(R'),,, -C(O)R', -C(O)N(R'),, -OR', and -S(O)kR', where k:is 1,2 or 3 and
each R' is independently H,
alkyl, or substituted alkyl.
Such non-naturai anuno acids are optionally in the form of a salt, or
incorporated into a non-natural 'amino acid
polypeptide, polynier, polysaccharide, or a polynucleotide and optionally post
translationally modified.
1002541 In addition, the following amino acids are included:
0
N H ~ H H
NH2 I N, N/ \ N_N~ N- N
H
\ \ ~ \
OH OH OH OH
H2N H2N HZN H2N
O O O , O
CN
I NNCN HZN N,
I
OH OH
HyN
o ; and o
wherein such compounds are optionally amino protected, optionally carboxyl
protected, optionally amino
protected and carboxyl protected, or a salt thereof, or optionally
incorporated into a non-natural aniino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and optionally
post.translationally modified.
B Structure and Synthesis of Non-Natttral Amino Acids: carbonyl, carbonyl-
like, Masked
carbonyl,, and Protected carbonyl Grottps
[002551 Amino acids with an electrophilic reactive group allow for a variety
of reactions to link molecules
via various chemical reactions, including, but not limited to, nucleophilic
addition reactions. Such electrophilic
reactive groups include a carbonyl group (including a keto- or aldehyde
group), a carbonyl-like group (which
has reactivity similar to a carbonyl group and is structurally similar to a
carbonyl group), a masked carbonyl
group (which can be readily converted into a carbonyl group), or a protected
carbonyl group (which has
reactivity similar to a carbonyl group upon deprotection). Such amino acids
include amino acids having the
structure of Formula (V):
R3
R3 A,_B/J\R
R~\ R2
N R4
H
0 (v),
wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene,
lower cycloalkylene, substituted
lower cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene,
substituted heteroalkylene, lower heterocycloalkylene, substituted lower
heterocycloalkylene, arylene,
substituted arylene, heteroarylene,, substituted heteroarylene, alkarylene,
substituted alkarylene, aralkylene,
or substituted aralkylene;
56
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B is optional, and when present is a linker selected from the group consisting
of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -O-(alkylene or
substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -
S(O)k(alkylene or substituted alkylene)-,
where k is 1, 2, or 3, -C(O)-(alkylene or substituted alkylene)-, -C(S)-
(alkylene or substituted alkylene)-,
-NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted
alkylene)-, -CSN(R')-(alkylene
or substituted alkylene)-, and -N(R')CO-(alkylene or substituted alkylene)-,
where each R' is independently
H, alkyl, or substituted alkyl;
0
SR' R" O+N Rõ
O R" Rõ
OR"
~
J - ~s ~ O
o > , ~~
R" R" R" R"
R O S s s
N ; or R is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl;
each R" is independently H, alkyl, substittited alkyl, or a protecting group,
or when more than one R" group is
present, two R" optionally form a heterocycloalkyl;
Ri is 14, an anvno protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted
lower alkyl, or R3 and R4 or two R3
groups optionally form a cycloalkyl or a heterocycloalkyl;
or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or
heterocycloalkyl comprising at least
one carbonyl group, including a dicarbonyl group, protected carbonyl
group,.including a protected
dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl
group;
or the -J-R group together forms a monocyclic or bicyclic cycloalkyl or
heterocycloalkyl comprising at least
one carbonyl group, including a dicarbonyl group, protected carbonyl group,
including a protected
dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl
group;
with a proviso that when A is phenylene and each R3 is H, B is present; and
that when A is -(CHZ)4- and each
R, is H, B is not -NHC(O)(CH2CH2)-; and that when A and B are absent and each
R3 is H, R is not methyl.
Such non-natural amino acids are optionally in the form of a salt, or
incorporated into a non-natural amino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post
translationally modified.
1002561 In certain embodiments, compounds of Formula (V) are stable in aqueous
solution for at least I
month under mildly acidic conditions. In certain embodiments, compounds of
Formula (V) are stable for at least
2 weeks under mildly acidic conditions. In certain embodiinents, compound of
Formula (V) are stable for at
least 5 days under mildly acidic conditions. In certain embodiments, such
acidic conditions are pH of about 2 to
about 8.
57
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(00257( In certain embodiments of compounds of Formula (V), B is lower
alkylene, substituted lower
alkylene, -O-(alkylene or substituted alkylene)-, arylene,. substituted
arylene, heteroarylene, substituted
heteroarylene, -C(O)-(alkylene or substituted alkylene)-, -CON(R')-(alkylene
or substituted alkylene)-, -
S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-
, or -S(O)2(alkylene or substituted
alkylene)-. ]n certain embodiments of compounds of Fomtula (1), B is -O(CH2)-,
-CH=N-, -CH=N-NH-, -
NHCH2-, -NHCO-, -C(O)-,_ -C(O)-(CHz)-,, -CONH-(CHZ)-, -SCH2-, -S(=O)CH2-, or -
S(O)2CHZ-. In certain
embodiments of compounds of Formula (V), R is Ci_6 alkyl or cycloalkyl. In
certain embodiments of
compounds of Formula (V) R is -CH3, -CH(CH3)2, or cyclopropyl. In certaui
embodiments of compounds of
Formula (V), R, is H, tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl
(Fmoc), N-acetyl,
tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of
compounds of Formula (V), Ri
is a resin, at least one amino acid, polypeptide, or polynucleotide. In
certain embodiments of compounds of
Forniula (V), R2 is OH, 0-methyl, O-ethyl, or 0-t-butyl. In certain
embodiments of compounds of Formula (V),
R2 is a resin, at least one amino acid, polypeptide, or polynucleotide. In
certain embodiments of compounds of
Formula (V), R2 is a polynucleotide. In certain embodiments of conYpounds of
Formula (V), R2 is ribonucleic
acid (RNA). In certain embodiments of compounds of Formula (V), R2 is tRNA. In
certain embodiments of
compounds of Formula (V), ibe tRNA specifically recognizes a selector codon.
In certain embodiments of
compounds of Formula (V) the selector codon is selected from the group
consisting of an amber codon, ochre
codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-
base codon, and a four-base codon.
In certain embodiments of compounds of Formula (V), R2 is a suppressor tR.N'A.
1002581 In certain embodiments of compounds of Formula (V), B is selected from
the
group consisting of:
(i) A is substituted lower alkylene, C4-arylene, substituted arylene,
heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted
aralkylene;
B is optional, and when present is a divalent linker selected from the group
consisting of
lower alkylene, substituted lower alkylene, lower alkenylene, substituted
lower alkenylene,
arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -
0-(alkylene or
substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -N'S(O)z-, -OS(O)z-, -C(O)-, -
C(O)-(alkylene or
substituted alkylene)-, -C(S)-, -N(R')-, -C(O)N(R')-, -CON(R')-(alkylene or
substituted
alkylene)-, -CSN(R')-, -N(R')CO-(alkylene or substituted alkylene)-, -
N(R')C(O)O-, -
N(R')C(S)-, -S(O)N(R'.), -S(O)zN(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-,
-N(R')S(O)N(R')-, -N(R')S(O)ZN(R')-, -C(R')=N-N(R')-,~-C(R')Z-N=N-,
-N(R')C(NCN)N(R')-, -N(R')C(NNOZ)N(R')-, -N(R')C(NCOOR')N(R')-, and
-C(R')2-N(R')-N(R' )-;
(ii) A is optional, and when present is substituted lower alkylene, C4-
arylene, substituted arylene,
heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene, or
substituted aralkylene;
B is a divalent linker selected from the group consisting of lower alkylene,
substituted lower
alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted
arylene,
heteroarylene, substituted heteroarylene, -0-, -O-(alkylene or substituted
alkylene)-, -S-, -
S(O)-, -S(O)Z-, -NS(O)Z-, -OS(O)2-, -C(O)-, -C(O)-(alkylene or substituted
alkylene)-, -C(S)-1
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-N(R')-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-,
-N(R')CO-
(alkylene or substituted alkylene)-, -N(R')C(O)O-, -N(R')C(S)-, -S(O)N(R'), -
S(O)2N(R'),
-N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O)2N(R')-, -
C(R')=N-
N(R')-, -C(R')2-N=N-, -N(R')C(NCN)N(R')-, -N(R')C(NNOZ)N(R')-,
-N(R')C(NCOOR')N(R')-, and -C(R')2-N(R')-N(R')-;
(iii) A is lower alkylene;
B is optional, and when present is a divalent littker selected from the group
consisting of
lower alkylene, substituted lower alkylene, lower alkenylene, substituted
lower alkenylene,
arylene, substituted arylene, heteroarylene, substituted heteroarylene, -0-, -
O-(alkylene or
substituted alkylene)-, -C(O)-(alkylene or substituted alkylene)-, and -
CON(R')-(alkylene or
substituted alkylene)-, and
(iv) A is pbenylene;
B is a divalent linker selected from the group consisting of lower alkylene,
substituted lower
alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted
arylene,
heteroarylene, substituted heteroarylene, -O-(alkylene or substituted
alkylene)-, -C(O)-
(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted
alkylene)-, and
-N(R')CO-(alkylene or substituted alkylene)-;
o c O R" > ~,~ ~
J is o/ ~
~ f
Rõ R" Rõ Rõ
R O s s s
;or
each R' is independently H, alkyl, or substituted alkyl;
each R" is independently H, alkyl, substituted alkyl, or'a protecting group,
or when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
R, isoptional, and when present, is H, an amino protecting group, resin, at
least one amino acid,
polypeptide, or polynucleotide; and
R2 is optional, and when present, is OH, an ester protecting group,.resin, at
least one amino acid,
polypeptide, or polynucleotide; and
each R3 and Ra is independently H, halogen, lower alkyl, or substituted lower
alkyl;
R' is H, alkyl, or substituted alkyl;
R is H. alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or
heterocycloalkyl comprising
at least one carbonyl.
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1002591 In addition, amino acids having the structure of Formula (VI) are
included:
O
B'k R
Rl-, N RZ
H
O (VI),
wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene,
lower cycloalkylene, substituted
lower cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene,
substituted heteroalkylene, lower heterocycloalkylene, substituted lower
heterocycloalkylene, arylene,
substituted arylene, heteroarylene, substituted heteroarylene, alkarylene,
substituted alkarylene, aralkylene,
or substituted aralkylene;
B is optioual, and when present is a linker selected from the group consisting
of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -O-(alkylene or
substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where
k is 1, 2, or 3, -S(O)k(alkylene
or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)z-, -C(O)-(alkylene or
substituted alkylene)-, -C(S)-,
-C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted
alkylene)-, -C(O)N(R')-,
-CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or
substituted alkylene)-,
-N(R')CO-(alkylene or substituted alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-
(alkylene or substituted
alkylene), -N(R.')C(S)NR'-(alkylene or. substituted alkylene), -N(R')S(O)kNR'-
(alkylene or substituted
alkylene), -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, -N(R')C(NCN)NR'-(alkylene
or substituted
alkylene), -N(R')C(NNOZ)NR'-(alkylene or substituted alkylene), -
N(R')C(NCOOR')NR'-(alkylene or
substituted alkylene), and -C(R')2-N(R')-I`T(R')-, where each R' is
independently H, alkyl, or substituted
alkyl;
R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
R, is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
with a proviso that when A is phenylene, B is present; and that when A is -
(CH2)4-, B is not -
NHC(O)(CH2CH2)-; and that when A and B are absent, R is not methyl. Such non-
natural amino acids are
optionally in the form of a salt, or incorporated into a non-natural amino
acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post translationally
modified.
1002601 In addition, amino acids having the structure of Formula (VII) are
included:
Ra
Ra By R
O
Ra
Ra
R, ~, N R2
H
0 (VII),
CA 02672205 2009-06-09
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wherein:
B is a linker selected from the group consisting of lower alkylene,
substituted lower alkylene,.lower alkenylene,
substituted lower alkenylene, lower heteroalkylene, substituted lower
heteroalkylene, arylene, substituted
arylene, heteroarylene, substituted heteroarylene, -O-(alkylene or substituted
alkylene)-, -S-(alkylene or
substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or
substituted alkylene)-, -C(O)-, -
NS(O)z-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-
(alkylene or substituted
alkylene)-, -NR'-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or
substituted alkylene)-,
-CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or
substituted alkylene)-, -S(O)kN(R')-
(alkylene or substituted alkylene)-, -N(R')C(O)NR'-(alkylene or substituted
alkylene), -N(R')C(S)NR'-
1.0 (alkylene or substituted alkylene), -N(R')S(O)kNR'-(alkylene or
substituted alkylene), -C(R')=N-, -
C(R')=N-N(R')-, -C(R')2-N=N-, -N(R')C(NCN)NR'-(alkylene or substituted
alkylene),
-N(R')C(NNO,)NR'-(alkylene or substituted alkylene), -N(R')C(NCOOR')NR'-
(alkylene or substituted
alkylene), and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl,
or substituted alkyl;
R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
Ri is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
each R. is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl, NO2, CN, -
N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where
each R' is independently
H, alkyl, or substituted alkyl; or at least two R, taken together form a
heterocycle, heteroaryl or aryl.
Such non-natural amino acids are optionally in the form of a salt, or
incorporated into a non-natural aniino acid
polypeptide, polymer, polysaccharide,'or a polynucleotide and optionally post
translationally modified.
1002611 In addition, the following amino acids are included:
G 0
H2 H H2N OH H HZN OH 12 O O O O
O Nj O
O ~) Q
O O~(\ O eOH N~% -NH 0
~ O I~ I~ O II ~\/
H2N OH H~ OH O HzN HyN OH
O O O O
O SJ, NJ,
O
HZ OH H2N H H2N H
HzN COOH O O
F ~
i0
F
H2N OH HzN OH
0 and 0
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Such non-natural anuno acids are optionally amino protected group, carboxyl
protected and/or in the form of a
salt, or incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide
and optionally post translationally modified.
1002621 In addition, the following amino acids having the structure of Formula
(VIII) are included:
O
(CRa)n\Blj~ R
Ri-, N RZ
H
0 (VIII)
wherein
- B is optional, and when present is a linker selected from the. group
consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenytene, lower
heteroalkylene, substituted lower
heteroalkylene, arylene; substituted arylene, heteroarylene; substituted
he,teroarylene, -O-(alkylene or
substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where
k is 1, 2, or 3, -S(O)k(alkylene
or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or
substituted alkylene)-, -C(S)-,
-C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted
alkylene)-, -CON(R')-(alkylene or
substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-
(alkylene or substituted
alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -
N(R')C(S)NR'-(alkylene or
substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -
C(R')=N-, -C(R')=N-N(R')-,
-C(R')2-N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -
N(R')C(NNO2)NR'-(alkylene or
substituted alkylene), -N(R')C(NCOOR')NR'-(alkylene or substituted alkylene),
and -C(R')Z-N(R')-N(R')-
, where each R' is independently H, alkyl, or substituted alkyl;
R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
R, is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
each R,,is independently selected from the group consisting of H,
halogen,.alkyl, substituted alkyl, CN, NO2, -
N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where
each R' is independently
H, alkyl, or substituted alkyl; and n is 0 to 8;
with a proviso that when A is -(CH2)4-, B is not NHC(O),(CH2CH2)-.-Such non-
natural amino:acids are
optionally in.the form of a salt, or are incorporated into a non-natural amino
acid polypeptide, polymer,
polysaccharide, ar apolynucleotide and optionally post translationally
modified.
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1002631 In addition, the following amino acids are included:
~ O O O
p ~ O 000
s
HZN OH H2Nf~H Hz H H2N OH H2N OH HZN OH H2N H
O , . ~ O ~ = O
HN p N N"
O
HzN ~.{~N
H2N H HZ OH H2N OH H2N OH
p O O 0 OH OH
O~
p N" `
~O FO
N (N
NH N
HZN N
HzN H2N F OH
H2N
0 OH , 0 OH , p OH , and 0 , wherein such compounds are
optionally amino protected, optionally carboxyl protected, optionally amino
protected and carboxyl
protected, or a salt thereof, or are incorporated into a non-natural amino
acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post tTanslationally
modified.
(002691 In addition, the following amino acids having thestructure,ofFormula
(IX) are included:
Z
A" B'-'~ Y
Ri R2-
H
0 (IX),
wherein,
A is optional, and when present is lower alkylene, substituted lower alkylene,
lower cycloalkylene, substituted
lower cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene,
substituted heteroalkylene, lower heterocycloalkylene, substituted lower
heterocycloalkylene, arylene,
substituted arylene, heteroarylene, substituted heteroarylene, alkarylene,
substituted alkarylene; aralkylene,
or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting
of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylenc, -O-(alkylene or
substituted alkylene)-, -S-(alkylene or substituted alkylene)-,-S(O)k- where k
is 1, 2, or 3, -S(O)k(alkylene
or substituted alkylene)-, -C(O)-, -NS(O)Z-, -OS(O)Z-, -C(O)-(alkylene or
substituted alkylene)-, -C(S)-,
-C(S)-(alkylene, or substituted alkylene)-, -NR'-(alkylene or substituted
alkylene)-, -CON(R')-(alkylene or
substituted alkylene)-, -CSN(R')-(alkylene.or substituted alkylene)-, -N(R')CO-
(alkylene or substituted
alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -
N(R')C(S)NR'-(alkylene or
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substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -
C(R')=N-, -C(R')=N-N(R')-,
-C(R')2-N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -
N(R')C(NNOZ)NR'-(alkylene or
substituted alkylene),-N(R')C(NCOOR')NR'-(alkylene or substituted alkylene),
and -C(R')Z-N(R')-N(R')-
, where each R' is independently H, alkyl, or substituted alkyl;
R, is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R2 is OH,. an ester protecting,group, resin,. at least one amino acid,
polypeptide, or polynucleotide; and
Y and Z are independently selected from the group consisting of -OH, alkyl
substituted oxygen, -SH or alkyl
substituted sulfur and when Y and Z taken together can form a cycloalkyl ring.
Such non-natural amino acids are optionally in the form of a salt, or are
incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally
post translationally modified.
1002651 In addition, the following amino acids having the structure of
Forniula (X) are included:
Ra
Ra B'T-z
Y
Ra
Ra
Ri, N Rz
H
O (X),
wherein,
B is optional, and when present is a linker selected fromthe_group
consistingof.lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene,substituted lower
heteroalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-(alkylene or
substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where
k is 1, 2, or 3, -S(O)k(alkylene
or substituted alkylene)-, -C(O)-, -NS(O)z-, -OS(O)2-, -C(O)-(alkylene or
substituted alkylene)-, -C(S)-,
-C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted
alkylene)-, -CON(R')-(alkylene or
substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-
(alkylene or substituted
alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -
N(R')C(S)NR'-(alkylene or
substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -
C(R')=N-, -C(R')=N-N(R')-,
-C(R')2-N=N-, -N(R')C(NCN)I`TR'-(alkylene or substituted alkylene), -
N(R')C(NNOZ)NR'-(alkylene or
substituted alkylene), -N(R')C(NCOOR')NR'-(alkylene or substituted alkylene),
and -C(R')2-N(R')-N(R')-
, where each R' is independently H, alkyl, or substituted alkyl;
R, is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
Each Re is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl, CN, NO2i -
N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3,where
each R' is independently H,
alkyl, or substituted alkyl; or at least two Ra taken together form a
heterocycle, hetoroaryl or aryl; and
Y and Z are independently selected from the group consisting of -OH, alkyl
substituted oxygen, -SH or alkyl
substituted sulfur and when Y and Z taken together can form.a cycloalkyl ring.
Such non-natural amino acids are optionally in the form of a salt, or are
incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally
post translationally modified.
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(002661 In addition, the following amino acids are included:
\ ~ O \ ___~ I/ sJ ~/ I
H2 H HZN OH HzN H H2N OH
0 0
QH
\ I ~ O~ O
H2N H HZN OH H2N H HZN OH
O O 0 O
O O~
O~.
\ N eOH N/!~O
/ I "
H2N H HZ H ~N H2N OH
O O O
O e
ro O~ H~ OH H2N o and
wherein such compounds are optionally amino protected, optiotially carboxyl
protected, optionally aniino
protected and carboxyl protected, or a salt thereof, or are incorporated into
a non-natural amino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post
translationally modified.
1002671 In addition, the following amino acids having the structure of
Forniula (XI) are included:
Ra
Re B.~N,~,
~ R~
Ra
Re
RI, N R2
H
0 (XI)
B is optional, and when present is a linker selected from the group
consisting=of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -O-(alkylene or
substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where
k is 1, 2, or 3, -S(O)k(alkylene
or substituted alkylene)-, -C(O)-, -NS(0)2-, -OS(0)2-, -C(O)-(alkylene or
substituted alkylene)-, -C(S)-,
-C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted
alkylene)-, -CON(R')-(alkylene or
substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-
(alkylene or substituted
alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -
N(R')C(S)NR'-(alkylene or
substitutcd alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -
C(R')=N-, -C(R')=N-N(R')-,
-C(R')Z=N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -
N(R')C(NNO2)NR'-(alkylene or
substituted alkylene), -I`'(R')C(NCOOR')NR'-(alkylene or substituted
alkylene), and -C(R')Z-N(R')-N(R')-
, where each R' is independently H, alkyl, or substituted alkyl;
R is fl, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
CA 02672205 2009-06-09
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R, is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R, is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
Each Ra is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl, CN, NOZ, -
N(R')2, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where
each R' is independently H,
alkyl, or substituted alkyl; or at least two R, taken together form a
heterocycle, hetoroaryl.or aryl; and
Y independently selected from the group consisting of OR", NR"R' ; NC(O)R"
where each R" is is
independently H, alkyl, substituted alkyl.
Such non-natural amino_acids are optionally in the form of a salt, or are
incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, `or a polynucleotide and optionally
post translationally modified.
1002681 In addition the following amino acids are included:
O O
N
\ ~ N.N ~ i N.On N=N N,II
H N
I~ ~ o
H2N OH H2N OH H2N OH H2N OH
0 0 o and o
wherein such compounds are optionally amino protected, optionally carboxyl
protected, optionally amino
protected and carboxyl protected, or a salt thereof, or are incorporated into
a non-natural amino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post
translationally modified.
1002691 In addition, the following amino acids having the structure of Formula
(X[I) are included:
R3
R ~
a O
\ ~ x )n
Ra ~
R, 'N RZ R4
H O (XIl)
R, is H, an amino protecting group, resin, at least one aniino acid,
polypeptide, or polynucleotide;
R-is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
Each R. is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl,CN, NOz, -
N(R'),, -C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where
each R' is
independently H, alkyl, or substituted alkyl; or at least two R0 taken
together form a heterocycle,
hetoroaryl or aryl;
R3 and R4 are independently H, halogen, CN, NOz, alkyl, substituted alkyl,
N(R')2, C(O)R', -C(O)N(R')2, -OR',
and -S(O)kR', where k is 1, 2, or 3, where each R' is independently H, alkyl,
or substituted alkyl;
X is C, N, 0, S; with the proviso that when X is 0, or S, R4 cannot be H,
halogen, CN, NO2, alkyl, substituted
alkyl, N(R')2, C(O)R', -C(O)N(R')2, -OR', and -S(O)kR'; where k is 1, 2, or 3,
and n is 0, 1 or 2.
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1002701 In addition the following ainino acids are included:
OMe
I O / ~ O /.I O Me0 ./ I O
o o I ~ ~
H2N OH H2N OH H2N OH F H~ OH HZN OH H2N OH
O O O O 0 O
O Mo0
O
F
H2N OH HzN OH H2N OH
0 0 and o
wherein such compounds are optionally amino protected, optionally carboxyl
protected, optionally amino
protected and carboxyl protected, or a salt thereof, or are incorporated into
a non-natural amino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post
translationally modified.
1002711 In addition, the following amino acids having the structure of Formula
(XIII) are included:
Z
(CRa)n-0 B /\y
Ri, N R2
H
0 (XIII),
wherein,
B is optional, and when present is a linker selected from the group consisting
of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -O-(alkylene or
substituted alkylene)-,, -S-(alkylene or substituted alkylene)-, -S(O)k- where
k is 1, 2, or 3, -S(O)k(alkylene
or substituted alkylene)-, -C(O)-, -NS(O)Z-, -OS(O)z-, -C(O)-(alkylene or
substituted alkylene)-, -C(S)-,
-C(S)-(alkylene or substituted alkylene)-, -NR'-(alkylene or substituted
alkylene)-, -CON(R')-(alkylene or
substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-
(alkylene or substituted
alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -
N(R')C(S)NR'-(alkylene or
substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -
C(R')=IN'-, -C(R')=N-N(R')-,
-C(R')Z-N=N-, -N(R')C(NCN)NR'-(alkylene or substituted alkylene), -
N(R')C(NNOZ)NR'-(alkylene or
substituted alkylene), -N(R')C(NCOOR')NR'-(alkylene or substituted alkylene),
and -C(R')Z-N(R')-N(R')-
, where each R' is independently N, alkyl, or substituted alkyl;
R, is 11, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
each R, is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl, -N(R')2, -
C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where each R'
is independeutly H, alkyl,
or substituted alkyl; and n is 0 to 8, and
Y and Z are independently selected from the group consisting of -OH, alkyl.
substituted oxygen, -SH or alkyl
substituted sulfur and when Y and Z taken together can form a cycloalkyl ring.
Such non-natural amino acids are optionally in the form of a salt, or are
incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally
post translationally modified.
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(00272) In addition, the following amino acids are included:
o o_~
O O o
PO ~
pp
Oy NH NH NH NH
H2N H H2N H H2N H H2N~O HZN~OH HZN OH HN OH
O O O O
0. 7 \
O O O
H2N H H2N H H2 OH H2N OH H2NI~OH H2N~OH ~N OH
, o 0 0 (0~ o and o
wherein such conipounds are optionally amino protected, optionally carboxyl
protected, optionally amino
protected and carboxyl protected, or a salt thereof, or are incorporated into
a non-natural ainino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post
translationally modified.
1002731 In addition, the following atnino acids having the>structure of
Formula (XIV) are included:
R
CRa)k, g ~N /Y
R, .N R2
H 0 (XIV),
wherein,
B is optional, and when present is a linker selected from the group consisting
of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -O-(alkylene or
substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -S(O)k- where
k is 1, 2, or 3, -S(O)k(alkylene
or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)Z-, -C(O)-(alkylene or
substituted alkylene)-, -C(S)-,
-C(S)-(alkylene.or substituted alkylene)-, -NR'-(alkylene or substituted
alkylene)-, -CON(R')-(alkylene or
substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-
(alkylene or substituted
alkylene)-, -S(O)kN(R')-, -N(R')C(O)NR'-(alkylene or substituted alkylene), -
N(R')C(S)NR'4alkylene or
substituted alkylene), -N(R')S(O)kNR'-(alkylene or substituted alkylene), -
N(R')-N=, -C(R')=N-, -
C(R')=N-N(R')-, -C(R')=N-N=, -C(R')Z-N=N-, -N(R')C(NCN)NR'-(alkylene. or
substituted alkylene),
-N(R')C(NNO2)NR'-(alk.ylene or substituted alkylene), -N(R')C(NCOOR')NR'-
(alkylene or substituted
alkylene), and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl,
or substituted alkyl;
R is I=1, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
Ri is H, an aniino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
R2 is OH, an ester protecting group, resin,.at least one amino acid,
polypeptide, or polynucleotide;
each R. is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl, -N(R')2, -
C(O)R', -C(O)N(R')2, -OR', and -S(O)kR', where k is 1, 2, or 3, where each R'
is independently H, alkyl,
or substituted alkyl; and n is 0 to 8; and
Y independently selected from the group consisting of OR", NR"R", NC(O)R"
where each R" is is
independently H, alkyl, substituted alkyl.
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Such non-natural amino acids are optionally in the form of a salt, or are
incorporated into a non-natural amino
acid polypepdde, polymer, polysaccharide, or a polynucleotide and optionally
post.translationally modified.
1002741 In addition, the following amino acids are included:
0
N,O-,_:..~_N,N1k1
NNi N,O^ N=N^ H
H~NH HzNH ~N _ OH H H2N OH
on no o and 0
wherein such compounds are optionally 'amino protected, optionally earboxyl
protected, optionally amino
protected and carboxyl protected, or a salt thereof,. or are incorporated into
a non=natural amino acid
polypeptide, polymer, polysaccharide, or a polynucleotide~and optionally
posttranslationallymodified.
C. Non-Natural Amino Acids Containing an Indolyl Functional Group
1002751 Non-natural amino acids containing an indole group are produced by
reaction of either a non-
natural amino acid containing a hydrazine with a reagent containing a carbonyl
group, or a non-natural ainino
acid containing a carbonyl with a reagent containing a hydrazine group. This
reaction is called the Fisher indole
synthesis. This reaction is traditionally carried out under very harsh
condition in the presence of strong acids and
/or metal ion accelerators, or uder reflux in organic solvents. Fig. 2
describes the mechanisim of this reaction
which involves arylhdrazone intermediate formation between aryllrydrazine and
carbonyl compound, followed
by [3,3] sigmatropic rearrangement to form the indole product after
elimination of ammonia.
100276) In one enibodiment the reactions between carbonyl and arylhydrazines
is perfomed in aqueous
buffer at room temperature. In another embodiment the reactions between
carbonyl and arylhydrazines is
perfonied at pH of about 1 to about 6 and about 2 to about 4. In addition,.
the reaction is accelerated, for
example, by performing the hydrazone intermediate formation and the
rearrangement step to form the indole at
different pH. In one embodiment the formation of hydrazone intermediate is
realized at pH 5. In another
embodiment the rearrangement step is perfomed at pH 1. Fig. 3-6, and 8-11
describe the effect of the pH on
Fisher irtdole synthesis.
1002771 In certain:embodiments, metal ions are,used to accelerate_the rate of
formation of indole product.
Fig. 12 illustrates non limiting examples of metal ions that have.
accelerating effect on the rate of formation of
the indole product. In another embodiment,, the amount of organic solvent in
the reaction milieu has an effect on
tlie rate of formation of indole product. Fig. 14 describes the effect of the
solvent on the rate of formation of the
indole product.
1002781 The reagents used in this reaction are optionally further linked to,a
desired functionality. In some
enibod'nnents, the non-natural amino acid is incorporated into a polypeptide,
wheretipon reaction with the
appropriate reagent. aconjugate is formed between the polypeptide and molecule
of interest.
1002791 Such aniino acids include amino acids having the structure of Formula
(XV):
R3
R3 A_ B_1 ]
R i _ R2
N R4
H
0 (XV)
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wherein:
A is optional, and when present is lower alkylene, substituted lower
alkylene;_ lower cycloalkylene, substituted
lower cycloalkylene, lower alkenylene, substituted lower
alkenylene,.alkynylene, substituted alkynylene,
lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,
substituted lower
heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene,
substituted alka .rylene, aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting
of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower
heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-, -0-(alkylene or
substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k-
where k is 1, 2, or 3, -
S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-
(alkylene or substituted
alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-
(alkylene or substituted
alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -
CSN(R')-, -CSN(R')-(alkylene or
substituted alkylene)-, -N(R')CO{alkylene or substituted alkylene)-, -
N(R')C(O)O-, -S(O)kN(R')-,
-N(R')C(O)N(R')-, -N(R')C(S)i`'(R')-, -N(R')C(NCN)N(R')-, -NI(R')C(NNOz)N(R')-
,
-N(R')C(NCOOR')N(R')-, -N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z-
N=N-, and
-C(R')2-N(R')-N(R')- and each R' is independently H,. alkyl, or substituted
alkyl;
Ra N Re
( ~ )a RS Ra N
R H H
N H Rs
N R5 ~ RS RS ~-, I R5 Ra \' ~~ R5
R5 ~ R Ra R5 R6
J is R5 Rs R5 5 or
Ri is 1-1, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted
lower alkyl, or R3 and R4 or two R3
groups optionally form a cycloalkyl or a heterocycloalkyl;
each R5 is independently H, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl,
alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,
polyalkylene.oxide, substituted
polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, alkaryl, substituted alkaryl,
aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-0N(R")2, CN,
NOz, -(alkylene or substituted
alkylene)-C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or
substituted:aryl), -C(O)R", -C(O)2R",
or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted
alkyl, alkenyl, substituted
alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl,.heteroaryl,
alkaryl, substituted alkaryl, aralkyl,
substituted aralkyl, L-Y, or when more than. one R" group is present, two R"
optionally form a
heterocycloalkyl;
when more than one R5 group is present, two R5 optionally form a
heterocycloalkyl or an aromatic
heterocycloalkyl;
n is 0, 1, 2, or 3 and m is 0, 1, 2, or 3, provided that at least one of n or
m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R.
group can contain 0, 1, or 2 R.
groups and each R. independently selected from the group cojisisting of H,
halogen, alkyl, subsituted alkyl,
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-N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)kR", where each R" is
independently H, alkyl, or
substituted alkyl; or, when more than one R, group is present; two R.
opfionally form an aryl, cycloalkyl or
heterocycloalkyl;
Y is selected from: a label; a dye; a polymer; a water-soluble polymer; a
derivative of polyethylene glycol; a
photocrosslinker; a cytotoxic compound; a drug; an affinity label; a
photoaffinity label; a reactive
compound; a resin; a second protein or polypeptide or polypeptide analog; an
antibody or antibody
fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a
polynucleotide; a DNA; a RNA; an
antisense polynucleotide; a saccharide, a water-soluble dendrimer, a
cyclodextrin, a biomaterial; a
nanoparticle; a spin label; a fluorophore; a metal-containing moiety; a
radioactive moiety; a novel
fiuictional group; a group that covalently or noncovalently interacts with
other molecules; a photocaged
moiety; an actinic radiation excitable moiety; a ligand; a photoisomerizable
moiety; biotin; a biotin
analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a
photocleavable group; an
elongated side chain; a carbon-Iinked sugar; a redox-active agent; an amino
thioacid; a toxic moiety; an
isotopically labeled moiety; a biophysical probe; a phosphorescent group; a
chemiluminescent group; an
electron dense group; a magnetic group; an interealating group; a chromophore;
an energy transfer agent; a
biologically active agent (in which case, the biologically active agent can
include an agent with therapeutic
activity and the non-natural amino acid polypeptide or modified non-natural
amino acid can serve either as
a co-therapeutic agent with the attached therapeutic agent or as a means for
delivery the therapeutic agent to
a desired site within an organism); a detectable label; a small molecule; an
inhibitory ribonucleic acid; a
radionucleotide; a neutron-capture agent; a derivative ofbiotin; quantum
dot(s); a nanotransmitter; a
radiotransmitter; an abzyme, an activated complex activator, a virus, an
adjuvant, an aglycan, an allergan,
an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a
saponin, a shuttle vector, a
macromolecule, a mimiotope, a receptor, a reverse micelle, and any combination
thereof;
L is optional, and when present is a linker selected from the group consisting
of alkylene, substituted alkylene,
alkenylene, substituted alkenylene, -0-, -O-(alkylene or substituted alkylene)-
, -S-, -S-(alkylene or
substituted alkylene)-, -S(O)k-, -S(O)k(alkylene or substituted alkylene)-, -
C(O)-, -C(O)-(alkylene or
substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -
N(R')-, -NR'-(alkylene or
substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted
alkylene)-, -CSN(R')-, -CSN(R')-
(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or subsfituted
alkylene)-, -N(R')C(O)O-, -(alkylene
or substituted alkylene)-O-N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-
(alkylene or substituted
alkylene)-, -(alkylene or substituted alkylene)-S(O)k-( alkylene or
substituted alkylene)-S-, -(alkylene or
substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -
N(R')S(O)kN(R')-,
-N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')Z-N=N-, and.-C(R')2-
I~'(R')-N(R')-, where
k is 1, 2 or 3 and each R' is independently H, alkyl, or substituted allcyl
or the -A-B-J groups together form a bicyclic or tricyclic cycloalkyl or
heterocycloalkyl comprising an indole
portion.
1002801 In one enibodiment, Y is selected from a water-soluble polymer; a
polyalkylene oxide; a polyethylene
glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one
amino acid; at least one sugar group;
at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin
analogue; a detectable label; and any
combination thereof;
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1002811 In a further embodiment is a compound having the structures of
compounds 1-4:
Ra H R3 R. R.
N
R RA. N R3 A
3 B R B~/
R. R21 ~ R, ~N RZ n Rs
j H R4 0 R5 H~ O 2~ m~ R
s
R5 Rs
Re N Rj
i $
R3 3 A`B/ R5 R3 3 A,H,, R5
RR. ! Rs R N R
m Rs
R Rj~ R2
Rt~H R4 O Z 3 e H R40 4 R5 R5
wherein:
A is optional, and wlten present is lower alkylene, substituted lower
alkylene, lower cycloalkylene,
substituted lower cycloalkylene, lower alkenylene, substituted lower
alkenylene, alkynylene,
substituted alkynylene, lower heteroalkylene, substituted heteroalkylene,
lower
heterocycloalkylene, substituted lower heterocycloalkylene, arylene,
substituted arylene,
heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene, or
substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole-
containing moiety, the linker
selected from the group consisting of lower alkylene, substituted lower
alkylene; lower
alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted
lower
heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-,
-O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted
alkylene)-, -S(O)k-
where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -
NS(O)Z-, -OS(O)2-,
-C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene'or
substituted alkylene)-, -
N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(.R')-
(alkylene or
substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-
, -N(R')CO-
(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -
N(R')C(O)N(R')-,
-N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNOZ)N(R')-, -
N(R')C(NCOOR')N(R')-,
-N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')2-N=N-, and -C(R')2-N(R')-
N(R')-
and each R' is independently H, alkyl, or substituted alkyl;
R, is H, an aniino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
n is 0, 1, 2, or 3, and m is 0, 1, 2, or 3, provided that at least one of n or
m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R.
group can contain 0, 1, or 2 R.
groups and each R. is independently selected from the group consisting of H,
halogen, alkyl,
substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")2, -OR", and -S(O)LR", where k
is 1, 2, or 3, where
each R" is independently H, alkyl, or substituted all.yl; or when niore than
one R, group is present, two
R, optionally form an aryl, cycloalkyl or heterocycloalkyl;
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each of R3 and R4 is independently H, halogen, lower alkyl, or substituted
lower alkyl, or R3 and R, or
two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;
each R$ is independently H, halogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted
alkylalkoxy,
polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl,
heteroaryl,
substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted
aralkyl, -(alkylene or
substituted alkylene)-ON(R")2, OH, NH2, CN, NO., -(alkylene or substituted
alkylene)-
C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -
C(O)R",
-C(O)2R", or -C(O)N(R )Z, wherein each R" is independently hydrogen, alkyl,
substituted
alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl,
substituted aryl,
heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or
when more than one RW'
group is present, two R" optionally form a heterocycloalkyl;
or R5 is.L-X, where, X is a selected from the group consisting of: a label; a
dye; a polymer; a water-
soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a
cytotoxic
compound; a drug; an affinity label; a photoaffinity label; a reactive
compound; a resin; a
second protein or polypeptide or polypeptide analog; an antibody or antibody
fragment; a
metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a
DNA; a RNA; an
antisense polynucleotide; a saccharide, a water-soluble dendrimer, a
cyclodextrin, a
biomaterial; a nanoparticle; a spin label; a fluoropltore, a metal-containing
moiety; a
radioactive moiety; a novel functional group; a group that covalently or
noncovalently
interacts with other molecules; a photocaged moiety; an actinic radiation
excitable moiety; a
ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety
incorporating a heavy
atom; a chemically cleavable group; a photocleavable group; an elongated side
chain; a
carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety;
an isotopically
labeled moiety; a biophysical probe; a phosphorescent group; a
chetniluniinescent group; an
electron dense group; a magnetic group; an intercalating group; a
chroniophore; an energy
transfer agent; a biologically active agent; a detectable label; a small
molecule; an inhibitory
ribonucleic acid; a radionucleotide; a neutron-capture agent; a derivative of
biotin; quantum
dot(s); a nanotransmitter; a radiotransmitter; an abzyme,. an activated
complex activator, a
virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone;
an antioxidant, an
aptamer', a guide RNA, a saponin, a shuttle vector, a macromolecule, a
mimotope, a receptor,
a reverse micelle, and any combination thereof; and L is optional, and when
present is a linker
selected from the group consisting of alkylene, substituted alkylene,
alkenylene, substituted
alkenylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or
substituted
alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)t(alkylene or substituted
alkylene)-, -C(O)-,
-C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or
substituted alkylene)-, -
N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-
(alkylene or
substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-
, -N(R')CO-
(alkylene or substituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted
alkylene)-O-
N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted
alkylene)-, -
(alkylene or substituted alkylene)-S(O)k-( alkylene or substituted alkylene)-S-
, -(alkylene or
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substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-õ-N(R')C(S)N(R')-,
-N(R')S(0)kN(R')-, -N(R')-N=, -C(R')=N-. -C(R')=N-N(R')'.-, -C(R').=N-N=, -
C(R')z-N=N-,
and -C(R')2=N(R') N(R')-, where each R' is independently.H, alkyl, or
substituted-alkyl;
when more than one R5 group is present,. two ortho R$ groups can optionally
form aheterocycloalkyl or
an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic
cycloalkyl or heterocycloalkyl
coniprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable-prodrug or
solvate thereof.
1002821 In one emboditnent, X is selected from a water-soluble polymer; a
polyalkylene oxide; a polyethylene
glycol; a derivative of polyethylene glycol; a photocrosslinker;at least one
amino acid; at least one sugar group;
at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin
analogue; a detectable label; and any
combination thereof;
1002831 In a further enibodinient is a compound having the structures of
compounds 1-4, wherein A is a
bond, substituted. or unsubstituted lower alkylene, or an unsubsimted or
substituted arylene selected from the
group consisting of a phenylene, pyridinylene, pyrimidinylene or
thiophenylene. In a. further embodiment is a
compound having the structures of compounds 1-4, wherein A is bond. In a
further embodiment is a compound
having the. structures of compounds 1-4, wherein A is substituted or
uns"ubstituted lo.werbeteroalkylene. In a
further embodiment is a compound having the structures of comp.ounds 1-4,
wherein A is a phenylene. In a
further embodiment` is a compound having tlie structures of compounds 1-4,
wherein A is a substituted lower
heteroalkylene,, whereinthe.subsituent is a single =O group. In a further
embodiment is a compound having the
structures of compounds 1-4, wherein A is a substituted lower alkylene,.
wherein the substituent is the single =0
group. In yet a fiu-ther embodiment is a compound having the structures of
compounds 1-4, wherein B is a bond,
lower alkylene, substituted lower alkylene, -O-(alkylene or substituted
alkylene)-, -CON(R")-, -NR'-(;alkylene
or substituted alkylene)- -N(R")CO-, -C(O)-, -C(O)-(alkylene or substituted
alkylene)-, -CON(R")-(atkylene or
substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene
or substituted" alkylene)-, or -
S(O)2(alkylene or substituted alkylene)-. In a further embodiment is a
compound having the structures of
compounds 1-4, wherein B is a bond. In a further embodiment is a cornpound
having the structures of
compounds 1-4, wherein B is -O(CHZ)-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CHZ)-, -
CONH-(CH+, -SC112-, -
S(=O)CH2-, or -S(O)ZCH,-. In sonte embodiments are compounds having the
structures of compounds 1-4,
wherein R5 is -OH, -NH2, or NOz. In another embodiment is a compound having
the structures of compounds 1-
4, wherein Ri is H, tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl
(Fmoc), N-acetyl,
tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In a further embodiment
is a compound having the
structures of compounds 1-4, wherein Ri is a resin,_at least one amino acid,
polypeptide, or polynucleotide. In
another enibodiinent is a compound having the structures of compounds 1-41
wherein R2 is OH, 0-methyl, 0-
ethyl, or O-t-butyl. In a further embodiment is a.compound having the
structures of compounds 1-4, wherein R2
is a resin, at least. one amino acid,.polypeptide, or polynucleotide. In yet=a
,further embodiment is a compound
having the structures `of compounds 1-4, wherein R2 is a polynucleotide. In
;another embodiment is a compound
having the structures of compounds 1-4, wherein R,. is ribonucleic acid (RNA).
In a further embodintent is a
compound having the 'stivctures of compounds 1-4, wherein R2 is tRNA. In a
further embodiment is a
compound having the structures of compounds 1.-0, wherein said tRNA
specifically recognizes a.selector codon.
In a further emboditnent is a compound having the structures of compounds 1-4,
wherein said selector codon is
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selected from the group consisting of an amber codon, ochre codon, opal codon,
a unique codon, a rare codon,
an unnatural codon, a five-base codon, and a four-base codon. In a further
embodiment is a compound having
the structures of compounds 1-4, wherein R2 is a suppressor tRNA. In a further
embodiment is a compound
having the structures ;of compounds 1-4, wherein X is a biologically active
agent selected from the group
consisting of a peptide, protein, enzyme, antibody, drug, dye, lipid,
nucleosids, oligonucleotide, cell, virus,
liposome, microparticle, and .micelle,_ In a further embodiment is a compourid
having the structures of
compounds 1-4, wherein X is a drug., selected. from the group consisting of an
antibiotic, fungicide, anti-viral
agent, anti-inflammatory agent; :anti-aumor agent, cardiovascular agent, anti-
anxiety agent, hormone, growth
factor, and steroidal agent. Iin'a further embodiment is a compound having the
.structures of compounds 1-4,
wherein X is an enzyme selected from ihe group consisting of horseradish
peroxidase; alkaline phosphatase, (3-
galactosidase, and glucose.oxidase. In another embodiment is a compound.having
the structures of compounds
14, wherein X is a detectable label selected from the group consisting of a
fluorescent, phosphorescent,
chemiluminescent, chelating, electron dense, magnetic, intercalating,
radioactive, chroniophoric, and energy
transfermoiety.ln a furtlier embodiment..is a compound having the structures
of compounds 1-4, wherein X is a
polymer comprising alkyl, substituted alkyl, alkenyt, substituted alkenyl,
alkynyl, substituted alkynyl, alkoxy,
substituted alkoxy, alkylaikoxy, substituted alkylalkoxy, polyalkylene oxide,
substituted polyalkylene oxide,
aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,
substituted alkaryl, aralkyl, or substituted
aralkyl. In a further embodiment is a compound having the structures of
compounds 1-4, wherein said polymer
comprises polyalkylene oxide or substituted polyalkylene oxide. In another
enibodiment is a compound having
the structures of compounds 1-4, wherein said polymer comprises -[(alkylene or
substituted alkylene)-O-(
hydrogen, alkyl, or substituted alkyl)],;, wherein x is from 20-10,000. In a
further embodiment is a compound
having the structures of compounds 1-4, wherein said polymer is m-PEG lsaving
a molecular weight ranging
from about 2 to about 40 KDa. In certain embodiments, compounds of
Formula.(XV) are stable in aqueous
solution for at least 1 month under mildly acidic conditions. In certain
embodiments, compounds of Formula
(XV) are stable for, at least 2 weeks under mildly acidic conditions. In
certain embodiments, compound of
Formula (XV) are stable for at least; 5 days under mildly acidic conditions.
In certain embodiments, =such acidic
conditions are pH about 2 to about 8.
1002841 Non=limiting examples oTsuch amino acids include amino acids having
the following structures:
O\N NM 1 NH
NH NH F
OMe
H=N OH HZN OH H2N OH H-N oM H2N Oli
0 0 0 0 O
/ ,
~ \
~ \
Mel7 ~ N
\ NH
I / I ~ ~ I /
HxN Of{ MzN OH
0 , or 0 ; or a salt thereof; or a polypeptide incorporating at any position
of the compounds. Such non-natural amino acids are optionally in the form of a
salt, or are incorporated into a.
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WO 2008/077079 PCT/US2007/088011
non-natural amino acid polypeptide, polymer, polysaccharide, or a
polynucleotide and optionally post
translationally modified.
1002851 ln some embodiments are compounds further having the structures 5-8:
Ra
R N H /H Ra
a~
t' R5 Rt.~T RZ n
H Ro m RS
r Rs
Rj. R2 R5 O Rs
H R10 5 6 RS
Ra
R H RS
a
n ~~ RS RS
m Rg R5
Ri.~T R2~ R5 R Ri~N R2 RS
H R4C1 5 H R4 0
7
8
n is 0, 1, 2, or 3, and m is 0, 1, 2, or 3,, provided that at least one of n
or m is not 0;
wherein, each ring in structures 5, 6, 7 and 8 that has an'associated R0 group
can contain 0, 1, or 2 R,
groups and each R. is, independently selected from the group consisting of H,
halogen, alkyl,
substituted alkyl, -N(R")z, -C(O)R", -C(O)N(R")Z, -OR", and -S(O)kR", where k
is 1, 2, or 3, where
each.R" is independently H, alkyl, or substituted alkyl; or when more than one
R. group is present, two
R, optionally form an aryl, cycloalkyl or heterocycloalkyl.
1002861 In another embodiment are compounds having the structures 9-12:
(CHz)nX,(CO)yX2(CHz)m~.\ N (CHz)nXt(CO)y~+z(CHz)m~ H Ra
Ri`N 4.1R2 9 ~~ / RS Ri, N Rz 10 R/~ ~ ~n +
H O Ra R$ H R4 O m R5
RS
R. Rs
RS H
(CH,)õXI(CO),xz(CHz),,, n I NH RS (CH2)oX,(CO)yXz(CHz),õ- N Rs
R5
Ri.NR, 11 R. Ra Rs Ri,N, Rz 12
H R~40 RS RS H R4 0 RS Rs
wherein n is 0, 1, 2, 3, or 4; m is 0, 1, 2, 3, or 4; provided that n + m is
1, 2, 3, 4, or 5; X, and X2 are
independently a bond, 0, or NH; and y is 0 or 1.
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1002871 In yet another embodiment are conipounds, selected from the group
consisting of:
O O
N N O
HZN OFI H ( / / I{zN ~OH NI
O O 112N OH
O
OH NOZ
r '
H H
O N~~~N
O O O N
Oli OH ~O ' OH
HzN f' HZN HxN OH H21`
O O O O
1Rv
H2N OH H-N Ofl
0 , or 0 or a salt thereof; or a polypeptide incorporating at any position any
of the compounds.
C. Cellular Uptake of Non-Natural Amino Acids
1002881 Non-natural amino acid uptake by a eukaryotic cell is one issue that
is typically considered when
designing and selecting non-natural amino acids, including but not limited to,
for incorporation into a protein.
For example, the high charge density of a-amino acids suggests that these
conipounds are unlikely to be cell
permeable. Natural amino acids are taken up into the eukaryotic cell via a
collection of protein-based transport
systems. A rapid screen can be done which assesses which non-natural amino
acids, if any, are taken up by
cells. See, e.g., the toxicity assays in, e.g., the U.S. Patent Publication
No. 2004/198637 entitled "Protein
Arrays," which is herein incorporated by reference in its entirety, and Liu,
D.R. & Schultz, P.G. (1999)
Progress.toward the evolution of an organism with an expanded genetic code.
PNAS United States 96:4780-
4785. Although uptake is easily analyzed with various assays, an altemative to
designing non-natural amino
acids that are amenable to cellular uptake pathways is to provide biosynthetic
pathways to create amino acids in
viva.
1002891 Typically, the non-natural amino acid produced via cellular uptake as
described hcrcin is produced in a
concentration sufficient for efficient protein biosynthesis, including but not
limited to, a natural cellular amount,
but not to such a degree as to affect the concentration of the other amino
acids or exhaust cellular resources.
Typical concentrations produced in this manner are about 10 mM to about 0.05
mM.
D. Biosyntliesis of Non-Natural Amino Acids
1002901 Many biosynthetic pathways already exist in cells for the production
of amino acids and other
compounds. While a biosynthetic method for a particular non-natural amino acid
may not exist in nature,
including but not limited to, in a cell, the methods and compositions
described herein provide such methods. For
example, biosynthetic pathways for non-natural amino acids are optionally
generated in host cell by adding new
enzymes or niodifying existing host cell pathways. Additional new enzymes
include naturally occurring
enzymes or artificially evolved enzymes. For exaniple, the biosyntliesis of p-
aminophenylalanine (as presented
in an example in WO 2002/085923 entitled "In vivo incorporation of t-nnatural
amino acids") relies on the
addition of a combination of known enzymes from other organisms. The genes for
these enzymes can be
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introduced into a eukaryotic cell by transforming the cell with a plasmid
comprising the genes. The genes, when
expressed in the cell, provide an enzymatic pathway to synthesize the desired
compound. Examples of the types
of enzymes that are optionally added are provided herein. Additional enzymes
sequences are found, for
exaniple, in Genbank. Artificially evolved enzymes can be added into a cell in
the same manner. In this manner,
the cellular machinery and resources of a cell are manipulated to produce non-
natural amino acids.
1002911 A variety of methods are available for producing novel enzymes for use
in biosynthetic pathways or for
evolution of existing pathways. For example, recursive recombination,
including but not limited to, as
developed by Maxygen, Inc. (available on the world wide web at
www.rrtaxygen.com), can be used to develop
novel enzymes and pathways. See, e.g., Stemmer (1994), Rapid evolution of a
protein in vitro by DNA shuffling,
Nature 370(4):389-391; and, Sterruner, (1994), DNA shuffling by random frag-
nentation and reassembly: hi
vitro reconibination for ntolecular evolution, Proc. Natl. Acad. Sci. USA.,
91:1 0747-1 075 1. Similarly
DesignPathTM, developed by Genencor (available on the world wide web at
genencor.com) is optionally used
for nietabolic pathway engineering, including but not limited to, to engineer
a pathway to create a non-natural
amino acid in a cell. This technology reconstructs existing pathways in host
organisms using a combination of
new genes, including but not limited to, those identified through functional
genomics, and molecular evolution
and design. Diversa Corporation (available on the world wide web at
diversa.com) also provides technology for
rapidly screening libraries of genes and gene pathways, including but not
limited to, to create new pathways for
biosynthetically producing non-natural amino acids.
1002921 Typically, the non-nattual amino acid produced with an engineered
biosynthetic pathway as described
herein is produced in a concentration sufficient for efficient protein
biosynthesis, including but not limited to, a
natural cellular amount, but not to such a degree as to affect the
concentration of the other aniino acids or
exhaust cellular resources. Typical concentrations produced in vivo in this
manner are about 10 niM to about
0.05 mM. Once a cell is transformed with a plasmid comprising the genes used
to produce enzynies desired for
a specific pathway and a non-natural amino acid is generated, in vivo
selections are optionally used to further
optimize the production of the non-natural amino acid for both ribosomal
protein synthesis and cell growth.
E. Additional Syntlietic Methodology
1002931 "Che non-natural amino acids described herein are optionally
synthesized using documented
methodologies or using the techniques described herein or by a combination
thereof. As an aid, the following
table provides various starting electrophiles and nucleophiles which are
optionally combined to create a desired
functional group. The information provided is meant to be illustrative and not
limiting to the synthetic
tec}miques described herein.
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Table 1: Examples of Covalent Linkages and Precursors Thereof
Covalent Linka e Pfoduct . J Electio `hile : : . :. . .: Nucleo hile
Carboxamides Activated esters amines/anilines
Carboxaniides acyl azides amines/anilines
Carboxacnides acyl halides amines/anilines
Esters acyl halides alcohols/ henots
Esters acyl nitriles alcohols/phenols
Carboxarnides acyl nitriles amines/anilines
Imines Aldehydes amines/anilines
Hydrazones aldehydes or ketones Hydrazines
Oximes aldeh des or ketones H drox lamines
Alk 1 amines alkyl halides amines/anilines
Esters alkyl halides carboxylic acids
Thioethers alkyl halides Thiols
Ethers alkyl halides alcohols! henols
'Thioethers alkyl sulfonates Thiols
Esters alkyl sulfonates carboxylic acids
Ethers alkyl sulfonates alcohols! henols
Esters Anhydrides alcohols/phenols
Carboxamides Anhydrides amines/anilines
Thiophenols aryl halides Thiols
Aryl an-tines aryl halides Amines
Thioethers Azindines Thiols
Boronate esters Boronates Glycols
Carboxamides carboxylic acids amines/anilines
Esters carboxylic acids Alcohols
h drazines Hydrazides carboxylic acids
N-acylureas, or Anhydrides carbodiimides carboxylic acids
Esters diazoalkanes carboxylic acids
Thioethers Epoxides Thiols
Thioethers haloacetamides 'I'hiols
Ammotriazines halotriazines amines/anilines
Triazinyl ethers halotriazines alcohols/phenols
Amidines inudo esters amines/anilines
Ureas Isoc anates amines/anilines
Urethanes Isocyanates alcohols/phenols
Thioureas isothiocyanates aniineslanilittes
Thioethers Maleimides Thiols
Phosphite esters phosphoramidites Alcohols
Silyl ethers silyl halides Alcohols
Alkyl amines sulfonate esters amines/anilines
Thioethers sulfonate esters Thiols
Esters sulfonate esters carboxylic acids
Ethers sulfonate esters Alcohols
Sulfonamides sulfonyl halides amines/anilines
Sulfonate esters sulfon 1'halides phenols/alcohols
1002941 In general, carbon electrophiles are susceptible to attack by
complementary nucleophiles, including
carbon nucleophiles, wherein an attacking nucleophile brings an electron pair
to the carbon electrophile in order
to form a new bond between the nucleophile and the carbon electrophile.
1002951 Non-limiting examples of carbon nucleophiles include, but are not
limited to alkyl, alkenyl, aryl and
alkynyl Grignard, organolithium, organozinc, alkyl-, alkenyl , aryl- and
alkynyl-tin reagents (organostannanes),
alkyl-, alkenyl-, aryl- and alkynyl-borane reagents (organoboranes and
organoboronates); these carbon
nucleophiles have the advantage of being kinetically stable in water or polar
organic solvents. Other non-
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limiting examples of carbon nucleophiles include phosphorus ylids, enol and
enolate reagents; these carbon
nucleophiles have. the advantage of being relatively easy to generate from
precursors. Carbon nucleophiles,
when used in conjunction with carbon electrophiles, engender new carbon-carbon
bonds between the carbon
nucleophile and carbon electrophile.
1002961 Non-limiting examples of non-carbon nucleophiles suitable for coupling
to carbon electrophiles
include but are not limited to primary and secondary amines, thiols,
thiolates, and thioethers, alcohols,
alkoxides, azides, semicarbazides, and the like. These non-carbon
nucleophiles, when used in conjunction with
carbon electrophiles, typically generate heteroatom linkages (C-X-C), wherein
X is a hetereoatom, including,
but'not limited to, oxygen, sulfu.r, or nitrogen.
1002971 In one embodiment is a method of making a compound of structures *1 or
2 comprising reacting a
compound of Formula (11) with a carbonyl-containing compound, wherein the
compound of Formula (li) is,
Ra
H A B I R4
Ri N Ra H' N.R3
Ra
0 R, (11)
wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene,
lower cycloalkylene,
substituted lower cycloalkylene, lower alkenylene, substituted lower
alkenylene, alkynylene,
substituted alkynylene, lower heteroalkylene, substituted heteroalkylene,
lower
heterocycloalkylene, substituted lower heterocycloalkylene, arylene,
substituted arylene,
heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene; or
substituted aralkylene;
B is optional, and when present is a Iinker, linked at one end to an indole
containing moiety, the linker
selected from the group consisting of lower alkylene, substituted lower
alkylene, lower
alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted
lower
heteroalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-
(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -
S(O)k- where k is
1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)z-, -
OS(O)Z-, -C(O)-
(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylenc or substituted
alkylene)-, -NR'-
(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or
substituted
alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, =N-O-
(alkylene or
substituted alkylene), -N(R')CO-(alkylene or substituted alkylene)-, -
N(R')C(O)O-,
-S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z-N=N-, and -C(R')2-N(R')-
N(R')-;,and
each R' is independently H, alkyl, or substituted alkyl;
R' is Fl, an aniino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and
RZ is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
each R. is independently selected from the group consisting. of H,:halogen,
alkyl; substituted alkyl, CN,
NO2, -N(R')2, -C(O)R', -C(O)N(R')Z, -OR', and -S(O)kR', where k is 1, 2 or 3
and each R' is
independently H, alkyl, or substituted alkyl;
R3 and R4 are independently hydrogen or amine protecting group, including, but
not limited to,
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i
-3-1 y0 OCC13 ,Sf\ /Q~ ~ ~ =C
1Q( Q I0l NQ2 CZHS
S3J CN
yQ_~<CCH =G CN Y' ~=C~ H3 or ~rQ~/~SiMc3
0 O H3C 3, + Q CH7 O
1002981 In a further embodiment is a method of making a compound of structures
1 or 2, wherein A is
substituted or unsubstittited lower alkylene, or an unsubsituted or
substituted arylene selected from the group
consisting of a phenylene, pyridinylene, pyrimidinylene or thiophenylene. In
another embodiment is a method
of making a compound of structures 1 or 2, wherein A is substituted or
unsubstituted lower heteroalkylene.
1002991 In a further embodiment is a method of making a compound of structures
I or 2, wherein A is a
substituted lower heteroalkylene, wherein the subsituent is a single =0 group.
In yet a further embodiment is a
method of making a compound of structures 1 or 2, wherein A is a substituted
lower alkylene, wherein the
substituent is the single =0 group.
1003001 In a further embodiment is a method of making a compound of structures
1 or 2, wherein B is lower
alkylene, substituted lower alkylene, -0-(alkylene or substituted alkylene)-, -
CON(R")-, -NR"-(alkylene or
substituted'alkylene)-, -N(R")CO-, -C(O)-, -C(O)-(alkylene or substituted
alkylene)-, -CON(R")-(alkylene or
substituted alkylene)-, -S(atkylene or substituted alkylene)-, -S(O)(alkylene
or substituted alkylene)-, or -
S(0)2(alkylene or substituted alkylene)-; wherein each R" is independently H,
alkyl, or substituted alkyl.
1003011 In a further embodiment is a method of making a compound of structures
l or 2, wherein B is -
O(CH2)-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CH2)-, -CONH-(CHZ)-, =SCHz-, -S(-
0)CH2-, or -S(0)2CH2-.
1003021 In a further embodiment is a method of making a compound of structures
I or 2, wherein Ri is H, tert-
butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl,
tetrafluoroacetyl (TFA), or
benzyloxycarbonyl (Cbz).
1003031 In a further embodiment is a method of niaking a compound of
structures I or 2, wherein R3 and R. are
~
iCH, /CN _ C H
hydrogen, CCH3, =C,CN, ~ 0 , or C2H5.
1003041 In a further embodiment is a method of making a compound of structures
1 or 2, wherein Ri is a resin,
at least one amino acid, polypeptide, or polynucleotide. In another embodiment
is a method of making a
compound of structures I or 2, wherein R, is OH, 0-methyl, O-ethyl, or O-t-
butyl. In-yet a further embodiment
is a method of making a compound ofstructures. l or 2, wherein RZ is a resin,
at least one amino acid,
polypeptide, or polynucleotide.
1003051 In some embodiments are methods of making a compound of structures 1
or'2, wherein the compound
of Formula (II) has the structure:
R.
(CH2)nXI(C0)yX2(CH2)m-B ~
I ~
RI~N ~ R2 13 R N'
0 ~
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wherein, n is 0, 1, 2, 3, or 4; m is 0, 1, 2, 3, or 4; provided that n+ m is
1, 2, 3, 4, or 5; XI and X, are
independently a bond, 0, or NH; and y is 0 or 1.
1003061 In a further embodiment is a method of making a.compound of structures
I or 2, wherein R2 is a
polynucleotide. In another embodiment is a method of making a compound of
structures 1 or 2, wherein R, is
ribonucleic acid (RNA). In a further embodiment is a method of making a
compound of structures 1 or 2,
whereinR2 is tRNA. In a further embodiment is a method of making.a compound of
structures I or 2, wherein
said tRNA specifically recognizes a selector codon. In yet a further
enibodiment is a method of making a
compound of structures I or 2, wherein said selector codon is selected from
the group consisting of an amber
codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural
codon, a five-base codon, and a
four-base codon. In a further embodiment is a method of making a compound of
structures 1 or 2; wherein R~ is
a suppressor tRNA.
1003071 In afurther embodiment is a method of making a compound of structures
1 or 2, selected from the
group consisting of
H
HN N, NH:
NHz \-~\
H ~ NH
H N OH HiN 1't Hz,~ 2
~ O 0
O~~~y~ O~/~y \
H N H H N H A OH H I/ N'~Y
Z H , or Z H I
1003081 In sonie embodiments are niethods of making a compound of structures I
or 2, corresponding to
Formula (IV):
Ra
H
Ra N~ ,Rd
I I
Rj
H
Rt,, Ra
N H R2
0 (IV)
wherein, each R, is independently selected from the gtoup consisting-of H,
halogen, alkyl, substituted alkyl,
CN, NO2, -N(R")2, -C(O)R", -C(O)N(R")Z, -OR", and -S(O)kR", where k is 1, 2,
or 3, where each
R" is independently H, alkyl, or substituted alkyl.
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1003091 In a further embodiment is a method of making a conipound of
structures 1 or 2, selected from the
group consisting of:
N
~ H
NHZ N"N~
( }~ (
~ \ \
OH OII OH
HaN -H3N H2N
0
GN
N~y~
\ ~ \ I N/ CN
OH OH
HZN HzN
o ;or
1003101 In a further enibodiment is a method of making a compound of
structures 1 or 2, further contprising
reacting a compound of Formula (V) with a hydrazine containing agent; wherein
the conipound of Formula (V)
is:
R3
R A \B1-1 jR
R, R:
H EQ (v),
wherein:
A is optional, and when present is lower alkylene,.substituted lower alkylene,
lower cycloalkylene, substituted
lower cycloalkylene, lower alkenytene, substituted lower alkenylene,
alkynylene, lower heteroalkylene,
substituted heteroalkylene, lower heteroeycloalkylene, substituted lower
heterocycloalkylene, arylene,
substituted arylene, heteroarylene, substituted heteroarylene, alkarylene,
substituted alkarylene,
aralkylene, or substituted aralkylene;
B is optional, and when present is a linker selected from the group consisting
of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted
lower heteroalkylene, arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-, -0-
(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-,
where..k is 1, 2, or 3, -
S(O)k(alkylene or substituted alkylene)-, -C(O)-(alkylene or substituted
alkylene)-, -C(S)-(alkylene or
substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -
CON(R')-(alkylene or
substituted alkylene)-, -CSN(R')-(alkylene or substituted alkylene)-, and -
N(R')CO-(alkylene or
substituted alkylene)-, where each R' is independently H, alkyl, or
substituted alkyl;
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0 R'
0 R,' Rõ SR" R~~ +N
0 S O o ORn
N
y,,/~r'" ,s~~ \ 0 /
`~,
Jis O
Rõ R" Rõ Rõ
s . s b
or
R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
each R" is independently H, alkyl, substituted alkyl, or a protecting group,
or when more than one R" group is
present, two R" optionally form a heterocycloalkyl;
R, is H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
each of R3 and R4 is independently H, halogen, lower alkyl, or substituted
lower alkyl, or R3 and R4 or two R3
groups optionally form a cycloalkyl or a heterocycloalkyl;
or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or
heterocycloalkyl comprising at least
one carbonyl goup, including a carbonyl group, protected carbonyl group,
including a protected
carbonyl group, or masked carbonyl group, including a masked carbonyl group;
or the -J-R group together forms a monocyclic or bicyclic cycloalkyl or
heterocycloalkyl comprising at least
one carbonyl group, including a carbonyl group, protected carbonyl group,
including a protected
carbonyl group, or masked carbonyl group, including a masked carbonyl group;
with a proviso that when A is phenylene and each R3 is H, B is present; and
that when A is -(CH2)4- and each
Rj.is H, B is not -NHC(O)(CHZCH2)-; and that when A and B are absent and each
R3 is H, R is not
methyl.
1003111 In a further.embodiment is a method of making a compound of structures
1 or 2, corresponding to
Formula (VI):
0
~R
Rj~N H
;RB2
0 (VI)
1003121 In a further embodiment is a method of making a compound of structures
1 or 2, wherein A is
substituted or unsubstituted lower alkylene, or an unsubsituted or substituted
arylene selected from the group
consisting of a plienylene, pyridinylene, pyrimidinylene or method.
1003131 In a further embodiment is a method of making a compound of structtues
I or 2, wherein B is lower
alkylene, substituted lower alkylene, -O-(alkylene or substituted alkylene)-, -
CON(R")-, -NR".-(alkylene or
substituted alkylene)-, -N(R")CO-, -C(O)-, -C(O)-(alkylene or substituted
alkylene)-, -CON(R")-(alkylene or
substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene
or substituted alkylene)-, or -
S(O)2(alkylene or substituted alkylene)-. In a further embodiment is a method
of making a compound of
structures ] or 2, wherein B is -O(CH2)-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CH2)-
, -CONH-(CHZ)-, -SCH2-, -
S(=O)CH2-, or -S(O)2CH2-.
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1003141 In a further embodiment is a inethod of making a compound of
structures 1 or 2, selected from the
group consisting of:
0 fi ~ ~
~
H
H,N Oil H1 j N OI{ HN H H NH H OH
a HZN H2N H7N
O
O O
O H^t
~ FtN'~O
F1zN OH HN Oil H,N OH H2N OFi
o , , , or
1003151 In a further embodiment is a method of making a compound.of structures
I or 2, corresponding to
Formula (VII):
Ra
R, By R
0
R.
R.
RI~N RZ
H
0 (VII),
wherein, each R. is independently selected from the group consisting of H,
halogen, alkyl, substituted
alkyl, -N(R"),, -C(O)R", -C(O)N(R")2, -OR", and -S(O)kR", where k is 1, 2, or
3, where each R" is
independently H, alkyl, or substituted alkyl.
(00316) In a further embodiment is a niethod of making a compound of
structures 1 or 2, selected from the
group consisting of:
o
oa cl o,
~ ~;~ ~~
`~~ ~/ ~'
On
Hc~ H>N ~OH .~ OH 14,N OH
O O O
e > , ,
0'I O
10 ~ O~/'`. \
I/ I, o^ y H0 ~/
H-N Oil oti 0 I,hN H
O Q HZN COOH
> , > >
~ ~ i
OA
N-y ' / I /
H; H,N OH H2N H H=N OH
, or
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(00317) In a further embodiment is a method of making a cornpound of
structures l or 2, corresponding to
Formula (VIII):
Ra
BYz
Y
R,
R.
R,, N R.Z
H
O
wherein, each R. is independently selected from.the group consistingof H,
halogen, alkyl, substituted
alkyl, -N(R")2, -C(O)R",--C(O)N(R")2, -OR";.and -S(O)kR", where.k is 1, 2, or
3, where each R" is
independently H, alkylõor substituted alkyl;
Y and Z are independently selected from the group consisting of=OI-I, alkyl
substituted oxygen, -SH or
alkyl substituted sulfur and when Y and Z taken together can form a cycloalkyl
ring.
1003181 In a further embodiment is a method of making a compound of structures
1 or 2, selected from the
group consisting of:
HzA tiN }t ttN H oH
H
O
H
\ \ \ ~/ ~ \
H y H H,N H H2N tl tizN H
H ~ O Qj
\ `S~ \ '~ i ~~/~
0
/ \ HHz h H [1z Oli H2N H HxN Oti
r O
O>
O\
iO
H
Hz1 ~OH H,1\T
O or
1003191 In a further embodiment is a method of making a conipound of
structures I or 2, corresponding to
Formula (IX):
R,
l3vN, ~.
RI
a
RF
Ri~ N H
0 (IX)
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wherein, each Ra is independently selected from the group consisting of H,
halogen, alkyl, substituted
alkyl, -N(R")2, -C(O)R", -C(O)N(R")Z, -OR", and -S(O)kR", where k is 1, 2, or
3, where each R" is
independently H, alkyl, or substituted alkyl;
Y is independently selected from the group consisting of OR", NR"R", NC(O)R"
where each R" is
independently H, alkyl, or substituted alkyl.
1003201 In=a further embodiment is a method of making a compound of structures
1 or 2, selected from the
grotip consisting, o f:
.?J,N~
~ .1 I ~ .Nbn I ~ H
i i
HzN pH H2N H2N H
0 Oi
N'
O
H3N OH
O
1003211 In a fui-ther enibodiment is a method of making a compound of
structures 1 or 2, corresponding to
Formula (X):
R3
R' O
R,
1 X
R, I
Ri, N RZ R~
H 0 (X)
wherein, each Ra is independently selected from the group consisting of H,
halogen, alkyl, substituted
alkyl, -N(R")2, -C(O)R", -C(O)N(R")Z, -OR", and -S(O)kR", where k is 1, 2, or
3, where each R" is
independently H, alkyl, or substituted alkyl; or when more than one R. group
is present, two R.
optionally form a cycloalkyl or heterocycloalkyl;
R3. and R4 are independently H, halogen, CN, NO2, alkyl, substitutcd alkyl,
N(R')z, C(O)R', -
C(O)N(R');; -OR', and -S(O)kR', where k is 1, 2, or 3, where each R' is
independently H, alkyl, or
substituted alkyl;
X is.C, N, or S, with the proviso that when X is 0 or S, Ra cannot be H,
halogen, CI`?, NOZ, alkyl,
.substituted alkyl, N(R')2, C(O)R', -C(O)N(R')Z, -OR', and -S(O)kR'; where k
is 1, 2, or 3, and n is 0,
lor2.
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1003221 In a fitrther embodiment is a method of making a compound of
structures I or 2, selected from the
group consisting of:
OMe
$jo 0
H2N H HZN OH HzN OH F H2\ OH HaN OFI
0 Q O , 0 , 0 MeO O Me0 O
/ F
\ I \ ( \ I O
H=N -ly OH H2N OH H'N OH 112N OH
O 0 O Or 0
1003231 In yet a further embodiment is a method of niaking a compound of
structures 1 or 2, corresponding to
Formula (XI):
z
~CRa)n\ / ~~,
B
RI\N R2
H
0 (Xl)
wherein, each Rais independently selected from the group consisting of H,
halogen, alkyl, substituted
all.yl;
Y and Z are independently selected from the group consisting of -(OH, alkyl
substituted oxygen, =.SI-I 'or
alkyl substituted sulfur and when Y and Z taken together canform a.cycloalkyl
ring.
1003241 In a further embodiment is a method of making a compound of structures
1 or 2, selected from the
group consisting of:
^
po
O
~ ~ O O~ N}i Jl
O
NH Nll NH
HZV 11 Hzn~fi HZN~OH H=N~O HzN ~OH 112N OH
a 0 , 0 0 0 iO
s
H2N UH H,N OH tl,N OH HIN fl l1=N H H2N Oli
I
o 0
0\
H
fIZNOH H2N
or
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1003251 In a ftirther embodiment is a method of making a comp.ound of
structures I or 2, corresponding.to
Formula (XII):
B -r N_ Y
(CR ) n R
R~~ Rz
O
(XII)
wherein, each R. is independently selected from the group consisting of H,
halogen, alkyl, substituted
alkyl, -N(R")2. -C(O)R", -C(O)N(R"),, -OR", and -S(O)kR", where k is 1, 2, or
3, where each R" is
independently H, alkyl, or substituted alkyl;
wherein B further comprises -CH=N-O-(alkylene or substituted alkylene)-;
n is 1, 2, or 3; and
Y is independently selected from the group consisting of OR", NR"R", NC(O)R"
where each R" is
independently H, alkyl,,substituted alkyl.
1003261 In a furthcr embodiment is a.method of making. a conipoundof
structures 1 or 2, selected from the
group consisting of:
1
~1'Ni, i;J~ iN.N
I
"2N OH N,NH H,N~ H2N Oll
0 0 or O
1003271 In a further embodiment is a method of making a compound of structures
1 or 2, wherein the
compound is reactive with a carbonyl cotitaining agent in aqueous solution
under mild conditions.
1003281 In a further embodiment is a method of making a compound of structures
1 or 2, wherein the reaction
of the compotmd with the carbonyl containing or protected carbonyl containing
agent has at least one of the
following characteristics: (i) occurs in a pH range of about 1 to about 6,
(ii) generates an indole linkage which is
stable under biological conditions; (iii) is site-specific; (iv) does not
irreversibly destroy the tertiary structure of
a polypeptide; (v) occurs at room termperature; (vi) occurs readily in aqueous
conditions; or (vii) is
regioselective and/or regiospecific.
I003291 In yet a further embodiment is a method of making a compound of
structures I or 2, wherein the mild
conditions are pH about I to about 6. In another embodiment is a ntethod of
making a compound of structures I
or 2, wherein the mild conditions are pH about 3 to about 6. In a further
embodiment is a method of making a
compound of structures 1 or 2, the reaction is in an aqueous solution iunder
mild conditions.
1003301 In a further embodiment is a method of making a compound of structures
1 or 2, wherein reacting a
compound of Formula (V) with a hydrazine containing agent has at least one of
the following characteristics: (i)
occurs in a pH range of about I to about 6, (ii) generates an indole linkage
which is stable under biological
conditions; (iii) is site-specific; (iv) does not irreversibly destroy the
tertiary structure of a polypeptide; (v)
occurs rapidly at room termperature; (vi) occurs readily is aqueous
conditions; or (vii) is regioselective and/or
regiospecific.
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VI. Polypeptides with Non-natural Amino Acids
1003311 For convenience, the form, properties and other characteristics of the
compounds described in this
section have been described generically and/or with specific examples.
However, die forni, properties and other
characteristics described in this section should not be limited to just the
generic descriptions or specific example
provided in this section, but rather the form, properties and other
characteristics described in this section apply
equally well to all compounds that fall within the scope of Formulas I-XV, and
compounds having the structures
1-4, including. any sub-forntulas or specific compounds that fall within the
scope of Formulas I-XV and
conipounds having the structures 1-4 that are described in the specification,
claims and figures herein.
[003321 The compositions and methods described herein provide for the
incorporation of at least one non-
natural aniino acid into a polypeptide. The non-natural aniino acid are
optionally present at any location on the
polypeptide, including any terminal position or any internal position of the
polypeptide. Preferably, the non-
natural amino acid does not destroy the activity and/or the tertiary structure
of the potypeptide relative to the
honiologous naturally-occurring amino acid polypeptide, unless such
destruction of the activity and/or tertiary
stntcture was one of the purposes of incorporating the non-natural amino acid
into the polypeptide. Further, the
incorporation of the non-natural amino acid into thepolypcptide optionally
modifies to some extent the activity
(e.g., manipulating the therapeutic effectiveness of the polypeptide,
improving the safety profile of the
polypeptide, adjusting the pharmacokinetics,. pharmacologics and/or
pharmacodynamics of the polypeptide
(e.g., increasing water solubility, bioavailability, increasing serum half-
life, increasing therapeutic half-life,
modulating immunogenicity, modulating biological activity, or extending the
circulation time), providing
additional functionality to the polypeptide, incorporating a tag, label or
detectable signal into the polypeptide,
easing the isolation properties of the polypepfide, and any contbination of
the aforementioned modifications)
and/or tertiary structure of the polypeptide relative to the homologous
naturally-occurring amino acid
polypeptide without fully causing destruction of the activity and/or tertiary
structure. Such modifications of the
activity and/or tertiary structure are often one of the goals of effecting
such incorporations, although the
incorporation of the non-natural aniino acid into the polypeptide optionally
has little effect on the activity and/or
tertiary structure of the polypeptide relative to the homologous naturally-
occurring amino acid polypeptide.
Correspondingly, non-natural amino acid polypeptides, conipositions comprising
non-natural amino acid
polypeptides, methods for making such polypeptides and polypeptide
compositions, methods for purifying,
isolating, and characterizing such polypeptides and polypeptide compositions,
and methods for using such
polypeptides and polypeptide compositions are considered within the scope of
the present disclosure. Further,
the non-natural amino acid polypeptides described herein are optionally
ligated to another polypeptide
(including, by way of exaniple, a non-natural amino acid polypeptide or a
naturally-occurring amino acid
polypeptide).
1003331 The polypeptide is selected, for example, froni any known therapeutic
protein, that is a protein which is
known to have a therapeutic effect on a person having a disease, disorder or
condition. By way of example only,
the polypeptide is selected froin alpha-I antitrypsin, angiostatin,
antihemolytic factor, antibody, antibody
fragment, apolipoprotein, apoprotein, atrial natriuretic factor, atrial
natriuretic polypeptide, atria] peptide, C-X-C
chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b; gro-c, IP-10, GCP-2, NAP-4,
SDF-1, PF4; MIG, calcitonin,
c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1,
monocyte chemoattractant protein-
2, monocyte chemoattractant protein-3; monocyte inflammatory protein-1 alpha,
nionocyte inflammatory
protein-i beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262,
CD40, CD40 ligand, c-kit
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WO 2008/077079 PCT/US2007/088011
ligand, collagen, colony stimulating factor (CSF), contplement factor 5a,
complement inhibitor, complement
receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-
1, epidernial growth factor
(EGF), epithelial neutrophil activating peptide, crythropoietin (EPO),
exfoliating toxin, Factor IX, Factor VII,
Factor VIII, Factor X, fibroblast growth factor (FGF), 6brinogen, fibronectin,
four-helical bundle protein, G-
CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth
factor receptor, grf, hedgehog
protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth
hormone (hGH), human serum
albumin, ICAM-1, ICAM-1 receptor, LFA-I, LFA-1 receptor, insulin, insulin-like
growth factor (IGF), IGF-I,
IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-garnma, interleukin (IL),
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF),
lactoferrin, leukemia inhibitory factor,
luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M,
osteogenic protein, oncogene product,
paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,
protein A, protein G, pth,
pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy,
relaxin, renin, SCF, small biosynthetic
proteiii, soluble complement receptor I, soluble I-CAM 1, soluble interleukin
receptor, soluble TNF receptor,
somatomedin, soniatostatin, somatotropin, streptokinase, superaritigens,
staphylococcal enterotoxin, SEA, SEB,
SECI, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase,
toxic shock syndrome toxin,
thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF),
tumor necrosis factor, tumor
necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor
receptor (TNFR), urotensin, VLA-4
protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase,
mos, ras, raf, met, p53, tat,
fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,
testosterone receptor, aldosterone receptor,
LDL receptor, and corticosterone receptor.
1003341 The non-natural amino acid polypeptides described herein are
optionally produced biosynthetically or
non-biosynthetically. By biosynthetically is meant any method utilizing a
translation system (cellular or non-
cellular), including use of at least one of the following components: a
polynucleotide, a codon, a tRNA, and a
ribosome. By non-biosynthetically is meant any method not utilizing a
translation system: this approach can be
further divided into methods utilizing solid state peptide synthetic methods,
solid phase peptide synthetic
methods, methods that utilize at least one enzyme, and methods that do not
utilize at least one enzyme; in
addition any of these sub-divisions may overlap with another sub-division and
many methods optionally utilize
a combination of these sub-divisions.
1003351 The methods, compositions, strategies and techniques described herein
are not limited to a particular
type, class or family of polypeptides or proteins. Indeed, the scope of the
compositions described herein allows
for virtually any polypeptide to include at least one non-natural amino acids
described herein. By way of
example only, the polypeptide is homologous to a therapeutic protein. In a
related or further embodiment, the
non-natural ainino acid polypeptide is homologous to any polypeptide member of
the growth honnone
supergene family.
1003361 The non-natural amino acid polypeptides are optionally further
modified as described elsewhere in this
disclosure or the non-natural amino acid polypeptide is optionally used
without further modification.
Incorporation of a non-natural amino acid into a polypeptide is done for a
variety of purposes, including but not
liniited to, tailoring changes in protein structure and/or function, changing
size, acidity, nucleophilicity,
hydrogen bonding, hydrophobicity, accessibility of protease target sites,
targeting to a moiety (including but not
limited to, for a polypeptide array), etc. Polypeptides that include a non-
natural amino acid can have enhanced
or even entirely new catalytic or biophysical properties. By way of example
only, the following properties can
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WO 2008/077079 PCT/US2007/088011
be modified by inclusion of a non-natural amino acid into a polypeptide:
toxicity,. biodistribution, structwal
properties, spectroscopic properties, chen-iical and/or photochemical
properties, catalytic ability, half-life
(including but not limited to, serum half-life), ability to react with other
molecules, including but not limited to,
covalently or noncovalently, and the like. Compositions with polypeptides that
include at least one non-natural
aniino acid are useful for, including but not linuted to, novel tlierapeutics,
diagnostics, catalytic enzymes,
industrial enzymes, binding proteins (including but not limited to,
antibodies), and research including, but not
limited to, the study of protein structure and function. See, e.g., Dougherty,
(2000) Unnatural Amino Acids as
Probes of Protein Structure and Function, Current Opinion in Chemical Biology,
4:645-652.
1003371 Further, the sidechain of the non-natural amino acid component(s) of a
polypeptide provides a wide
range of additional functionality to the polypeptide; by way of example only,
and not as a limitation, the
sidechain of the non-natural amino acid portion of a polypeptide includes any
desired functionality.
1003381 In one aspect, a composition includes at least one polypeptide with at
least one, including but not
liniited to, at least two, at least three, at least four, at least five, at
least six, at least seven, at least eight, at least
nine, or at least ten or more non-natural amino acids. Such non-natural amino
acids are optionally the same or
different. In addition, there is optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, or more
different sites in the polypeptide which comprise 1, 2, 3, 4, 5, 6, 7, 8; 9,
10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20,
or more different, or the same, non-natural amino acids. In another aspect, a
composition includes a polypeptide
with at least one, but fewer than all, of a particular amino acid present in
the polypeptide is substituted with a
non-natural amino acid(s). For a given polypeptide with more than one non-
natural amino acids, the non-natural
amino acids can be identical or different (such as; by way of exaniple only,
the polypeptide can include two or
more different types of non-natural amino acids, or can include two of the
same non-natural amino acid). For a
given polypeptide with more than two non-natural amino acids, the non-natural
amino acids can be the same,
different or a combination of a multiple number of non-natural amino acids of
the same kind with at least one
different non-natural amino acid.
1003391 Although embodiments of the non-natural amino acid polypeptides
described herein are optionally
chemically synthesized via solid phase peptide synthesis methods (such as, by
way of example only, on a solid
resin), by solution phase peptide synthesis niethods, and/or without the aid
of enzymes, other embodiments of
the non-natural ainino acid polypeptides described herein allow synthesis via
a cell membrane, cellular extract,
or lysate system or via an in vivo system, such as, by way of example only,
using the cellular machinery of a
prokaryotic or eukaryotic cell. In fiuther or additional embodinlents, one of
the key features of the non-natural
amino acid polypeptides described herein is that they are synthesized
utilizing ribosomes. In further or
additional embodiments of the non-natural amino acid polypeptides described
herein are, the non-natural amino
acid polypeptides are synthesized by a combination.of the methods including,
but.not limited to, a combination
of solid resins, without the aid of eiizynies, via the aid of ribosomes,
and/or via an in vivo system.
1003401 Synthesis of non-natural amino acid polypeptides via ribosomes, and/or
an in vivo system has distinct
advantages and characteristic from a non-natural amino acid polypeptide
synthesized on a solid resin or without
the aid of enzymes. These advantages or characteristics include different
impurity profiles: a system utilizing
ribosomes and/or an in vivo system will have impurities stemming from the
biological system utilized,
including host cell proteins, membrane portions, and lipids, whereas the
impurity profile from a system utilizing
a solid resin and/or without the aid of enzymes may include organic solvents,
protecting groups, resin materials,
coupling reagents and other chemicals used in the synthetic procedures. In
addition, the isotopic pattern of the
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CA 02672205 2009-06-09
WO 2008/077079 PCT/US2007/088011
non-natural amino acid polypeptide synthesized via the use of ribosomes and/or
an in vivo system may mirror
the isotopic pattern of the feedstock utilized for the cells; on, the other
hand, the isotopic. pattern of the non-
natural amino acid polypeptide synthesized on a solid resin. and/or without
the aid of enzymes may mirror the
isotopic pattern of the amino acids utilized in the synthesis. Further, the
non-natural amino acid synthesized via
the use of ribosomes and/or an in vivo system may be substantially free of the
D-isomers of the amino acids
and/or may be able to readily incorporate intetnal cysteine amirio acids into
the'stxucture of the polypeptide,
and/or may rarely provide,intemal anuno acid deletion polypeptides. On the
other hand, a non-natural amino
acid polypeptide synthesized via a solid resin and/or without.the use of
enzymes may have a higher content of
D-isomers of the amino acids and/or a lower content of internal cysteine amino
acids and/or a higher percentage
of internal amino acid deletion polypeptides. Furthermore, one will be able to
differentiate a non-natural amino
acid polypeptide synthesized by use of a ribosome and/or an in vivo system
from a non-natural amino acid
polypeptide synthesized via a solid resin and/or without the use of enzymes.
VIL Con:positions and Methods Conrprising Nucleic Acids and Oligonucleotides
A. General Recontbinant Nucleic Acid Methods For Use Herein
1003411 In numerous embodiments of the methods and compositions described
herein, nucleic acids encoding a
polypeptide of interest (including by way of example a GH polypeptide) will be
isolated, cloned and often
altered using recombinant methods. Such embodiments are used, including but
not limited to, for protein
expression or during the generation of variants, derivatives, expression
cassettes, or other sequences derived
from a polypeptide. In some embodiments, the sequences encoding the
polypeptides are operably linked to a
heterologous promoter.
[00342) Also described herein are cells that can producenon-natural amino acid
polypeptides wherein at least
one non-natural amino acid on-the polypeptide comprises a side-chain having a
carbonyl, a hydrazine; an indole
linkage.. Such cells produce such non-natural amino acid polypeptides using
the methods described herein or
variants thereof, but biosynthetically produce at least one non-natural amino.
Cells that biosynthesize at least
one non-natural amino acid may be produced using the techniques, methods,
compositions and strategies
described herein or variants thereof
1003431 A nucleotide sequence encoding a polypeptide comprising a non-natural
amino acid may be
synthesized on the basis of the amino acid sequence of the parent polypeptide,
and then changing the nucleotide
sequence so as to effect introduction (i.e., incorporation or substitution) or
removal (i.e., deletion or substitution)
of the relevant amino acid residue(s). The nucleotide sequence may be
conveniently modified by site-directed
mutagenesis in accordance witb documented niethodologies. Altematively, the
nucleotide sequence may be
prepared by chemical synthesis, including but not limited to, by using an
oligonucleotide synthesizer, wherein
oligonucleotides are designed based on the amino acid sequence of the desired
polypeptide, and preferably
selecting those codons that are favored in the host cell in which the
recombinant polypeptide will be produced.
For example, several small oligonucleotides coding for portions of the desired
polypeptide may be synthesized
and assembled by PCR, ligation or ligation chain reaction. See, e.g., Barany,
ec al., Proc. Natl. Acad. Sci. 88.
189-193 (1991); U.S. 6,521,427 which are incorporated by reference herein for
disclosure of the
aforementioned.
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CA 02672205 2009-06-09
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(00344) The non-natural amino acid methods and compositions described herein
utilize techniques in the field
of reconibinant genetics. Basic texts disclosing the general methods of use
for the non-natural amino acid
methods and compositions described herein include Sanibrook et al., Molecular
Cloning, A Laboratory Manual
(3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual
(1990); and Current Protocols
in Molecular Biology (Ausubel et al., eds., 1994)).
1003451 General texts which describe molecular biological techniques include
Berger and Kinimel, Guide to
,v volume 152 Academic Press, Inc., San Diego, CA
Molecular Clonint; Techniques Methods in Enzymolop
(Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed.).
Vol. 1 or 2, Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current
Protocols in Molecular
Bioloev, F.M. Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (supplemented through 1999) ( Ausubel")).
These texts describe
mutagenesis, the use of vectors, promoters and many other relevant topics
related to, including but not limited
to, the generation of or polynucleotides which include selector codoii.s for
production of proteins that include
non-natural amino acids, orthogonal tRNAs, orthogonal synthetases, and pairs
thereof.
1003461 Various types of mutagenesis are used in the non-natural amino acid
methods and compositions
described herein for a variety of purposes, including but not limited to, to
produce novel synthetases or tRNAs,
to mutate tRNA molecules, to mutate polynucleotides encoding synthetases,
libraries of tRNAs, to produce
libraries of synthetases, to produce selector codons, to insert selector
codons that encode non-natural amino
acids in a protein or polypeptide of interest. They include but are not
limited to site-directed mutagenesis,
random point mutagenesis, homologous reconibination, DNA shuffling or other
recursive mutagenesis methods,
chimeric construction, mutagenesis using uracil containing, templates,
oligonucleotide-directed niutagenesis,
phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA
or the like, or any
combination thereof. Additional suitable methods include point mismatch
repair, mutagenesis using repair-
deficient liost strains, restriction-selection and restriction-purification,
deletion mutagenesis, mutagenesis by
total gene synthesis., double-strand break repair, and the like. Mutagenesis,
including but not limited to,
involving chilneric constructs, are also included in the non-natural amino
acid methods and compositions
described herein. In one embodiment, niutagenesis can be guided by documented
information of the naturally
occurring molecule or altered,or mutated naturally occurring molecule,
including but not liniited to, sequence
comparisons, physical properties, crystal structure or the like.
1003471 The texts and examples found herein describe these and other relevant
procedures. Additional
information is found in the following publications and references
cited.within: Ling et al., Approaches to DNA
ntutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,
Oligonucleotide-directed
random n:utagenesis using the phosphorothioate method, Methods Mol. Biol.
57:369-374 (1996); Smith, In
vitro mutagenesis, Ann. Rev. Genet. 19:423-462(1985); Botstein & Shortle,
Strategies and applications of in
vitro mutagenesis, Science 229:1193-1201(1985); Carter, Site-directed
mtrtagenesis, Biochem. J. 237:1-7
(1986); Kunkel, The efficiency of oligomtcleotide directed mutagenesis, in
Nucleic Acids & Molecular Bioloav
(Eckstein, F. and Lilley, D.M.J. eds., Springer Verlag, Berlin)) (1987);
Kunkel, Rapid and efficient site-specific
tnutagenesis without phenotypic selection, Proc. Natl. Acad. Sci, USA 82:488-
492 (1985); Kunkel et al., Rapid
and efficient site-specific inutagenesis without phenotypic selection, Methods
in EnzMiol. 154, 367-382 (1987);
Bass et al., Mutant Trp repressors with new DNA-binding spe.cifrci.ties,
Science 242:240-245 (1988); Methods in
Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987); Zoller
& Smith, Oligonucleotirle-
94
CA 02672205 2009-06-09
WO 2008/077079 PCT/US2007/088011
directed niulagenesis using M73-der.ived vectors: an efficient and general
procedure for the production of point
mutations in any DNA fragntent, Nucleic Acids Res. 10:6487-6500 (1982); Zoller
& Snuth, Oligonucleotide-
directed mutagenesis of DNA fragments cloned into M13 vectors, Methods in
Enzyniol. 100:468-500 (1983);
Zoller & Smith, Oligonucleotide-directed mutagenesis: a simple metliod using
two oligonucleotide primers and
a single-stranded DNA template, Methods in Enz ny iol. 154:329-350 (1987);
Taylor et al., The use of
phosphorothioate-modified DNA in t-estriction enzyme reactiatis to prepare
nicked DNA, Nucl. Acids Res. 13:
8749-8764 (1985); Taylor et al., The rapid generation of oligonucleotide-
directed nnitations at high freguency
using phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8785 (1985);
Nakamaye & Eckstein,
Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate
groups and its application to
oligonucleotide-directed mutagenesis, Nucl. Acids Res. 14: 9679-9698 (1986);
Sayers et al., 5'-3' Exonucleases
in phosphorothioate-based oligonucleotide-directed mutrtgenesis, Nucl. Acids
Res. 16:791-802 (1988); Sayers
et al., Strand specific cleavage of phosphorothioate-containing DNA by
reaction with restriction endonucleases
in the presence of ethidium bromide, (1988) Nucl. Acids Res. 16: 803-814;
Kramer et al., The gapped duplex
DNA approach to oligottucleotide-directed mutatiott construction, Nucl. Acids
Res. 12: 9441-9456 (1984);
Kramer & Fritz Oligonucleotide-directed construction of mutations via gapped
duplex DN.4, Methods in
Enzymol. 154:350-367 (1987); Kramer et al., Intproved enzymatic in vitro
reactions in the gapped duplex DNA
approach to oligonucleotide-directed construction of ntutations, Nucl. Acids
Res. 16: 7207 (1988); Fritz et al.,
Oligottucleotide-directed construction of trttttations: a gapped duplex DNA
procedure witliout enzymatic
reactions in vitro, Nucl. Acids Res. 16: 6987-6999 (1988); Kramer et al.,
Point Mistnatch Repair, Cell 38:879-
887 (1984); Carter et al., Improved oligonucleotide site-directed mutagenesis
using M13 vectors, Nucl. Acids
Res. 13: 4431-4443 (1985); Carter, Intproved oligonucleotide-directed
mutagenesis using M13 vectors, Methods
in Enzymot. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use of
oligonucleotides to generate large
deletions, Nucl. Acids Res. 14: 5115 (1986); Wells et al., Importance of
liydrogen-bonrl formation in stabilizing
the transition state of subtilisin, Phil. Trans. R. Soc. Lond. A 317: 415-423
(1986); Nambiar et al., Total
synthesis and cloning of a gene codingfor the ribonuclease S proteiii, Science
223: 1299-1301 (1984); Sakmar
and Khorana, Total s_vnthesi.s and expression of a gene for the alpha-subunit
of bovine rod outer segment
guanine nucleotide-binding protein (transducin), Nucl. Acids Res. 14: 6361-
6372 (1988); Wells et al., Cassette
mutagenesis: an efficient method for generation of tnultiple mtdations at
defined sites, Gene 34:315-323 (1985);
Grundstrom et al., Oligonucleotide-directed mutagenesis by inicroscale 'shot-
gun' gene synthesis, Nucl. Acids
Res. 13: 3305-3316 (1985); Mandecki, Oligonucleotide-directed double-strand
break repair in plasntids of
Escherichia coli: a method for site-specific rnutagenesis, Proc. Natl. Acad.
Sci. USA, 83:7177-7181 (1986);
Arnold, Protein e gineering for unusual environments, Current Oainion in
Biotechnology 4:450-455 (1993);
Sieber, et al., Nattire.Biotechnology, 19:456-460 (2001). W. P. C. Stemmer,
Nature 370, 389-91 (1994); and, I.
A. Lorimer, 1. Pastan, Nucleic Acids Res. 23, 3067-8 (1995). Additional
details on niany of such methods can
be found in Methods in Enzymology Volume 154, which also describes useful
controls for trouble-shooting
problems with various mutagenesis methods.
1003481 The niethods and compositions described herein also include use of
eukaryotic host cells, non-
eukaryotic host cells, and organisms for the in vivo incorporation of a non-
natural amino acid via orthogonal
tRNA/RS pairs. Host cells are genetically engineered (including but not
limited to, transformed, transduced or
transfected) with the polynucleotides corresponding to the polypeptides
described herein or constructs which
include a polynucleotide conesponding to the polypeptides described herein,
including but not limited to, a
CA 02672205 2009-06-09
WO 2008/077079 PCT/US2007/088011
vector corresponding to the polypeptides described herein, which can be, for
example, a cloning vector or an
expression vector. For example, the coding regions for the orthogonal tRNA,
the orthogonal tRNA synthetase,
and the protein to be derivati2ed are operably linked to gene expression
control elements that are functional in
the desired host cell. The vector can be, for example, in the form of a
plasmid, cosmid, a phage, a bacterium, a
virus, a naked polynucleotide, or a conjugated polynucleotide. The vectors are
introduced into cells and/or
microorganisms by methods including electroporation (Fromm et al., Proc: Natl.
Acad. Sci. USA 82, 5824
(1985)), infection by viral vectors, high velocity ballistic penetration by
small particles with the nucleic acid
either within the matrix of small beads or particles, or on the surface (Klein
et al., Nature 327, 70-73 (1987)),
and/or the like.
1003491 The engineered host cells can be cultured in conventional nutrient
media modified as appropriate for
such activities as, for example, screening steps, activating promoters or
selecting transformants. These cells can
optionally be cultured into transgenic organisms. Other useful references,
including but not limited to for cell
isolation and culture (e.g., for subsequent nucleic acid isolation) include
Freshney (1994) Culture of Animal
Cells, a Manual of Basic Techniaue, third edition, Wiley- Liss, New York and
the references cited therein;
Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley
& Sons, Inc. New York, NY;
Ganiborg and Phillips (eds) (1995) Plant Cell Tissue and Organ Culture;
Fundamental Methods Springer Lab
Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds)
The Handbook of
Microbiological Media (1993) CRC Press, Boca Raton, FL.
1003501 Several methods of introducing target nucleic acids into cells are
available, any of which can be used in
methods and compositions described herein. These include: fusion of the
recipient cells with bacterial
protoplasts containing the DNA, electroporation, projectile bonibardment,. and
infection with viral vectors
(discussed further, herein), etc. Bacterial cells can be used to amplify the
number of plasn-tids containing DNA
constructs corresponding to the polypeptides described herein. The bacteria
are grown to log phase and the
plasmids within the bacteria can be isolated by a variety of inethods (see,
for instance, Sambrook). In addition, a
plethora of kits are commercially available for the purification of plasmids
from bacteria, (see, e.g., EasyPrepT"t,
FlexiPrepTM, both from Pharmacia Biotech; StrataCleanT"', from Stratagene;
and, QlAprepTM from Qiagen). The
isolated and purified plasmids are then further manipulated to produce other
plasniids, used to transfect cells or
incorporated into related vectors to infect organisms. Typical vectors contain
transcription and translation
terminators, transcription and translation initiation sequences, and promoters
useful for regulation of the
expression of the particular target nucleic acid. The vectors optionally
comprise generic expression cassettes
containing at least one independent terminator sequence, sequences permitting
replication of the cassette in
eukaryotes, or prokaryotes, or both, (including but not limited to, shuttle
vcctors) and selection markers for both
prokaryotic and eukaryotic systems. Vectors are suitable for replication and
integration in prokaryotes,
eukaryotes, or preferably both. See, Gillam & Smith, Gene 8:81 (1979);
Roberts, et al., Nature, 328:731 (1987);
Schneider, E., et al., Protein Expr. Purif. 6(l):10-14 (1995); Ausubel,
Sambrook, Berger (all supra). A catalogue
of bacteria and bacteriophages useful for cloning is provided, e.g., by the
ATCC, e.g., The ATCC Catalogue of
bacteria and bacteriophage (1992) Ghema et al. (eds) published by the ATCC.
Additional basic procedures for
sequencing, cloning and other aspects of molecular biology and underlying
theoretical considerations are also
found in Watson et al. (1992) Recombinant DNA Second Edition Scientific
American Books, NY. In addition,
essentially any nucleic acid (and virtually any labeled nucleic acid, whether
standard or non-standard) can be
custom or standard ordered from any of a variety of commercial sources, such
as the Midland Certified Reagent
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Company (Midland, TX mcrc.com), The Great American Gene Company (Ramona, CA
available on the World
Wide Web at genco.com), ExpressGen Inc. (Chicago, IL available on the World
Wide. Web at expressgen.com),
Operon Technologies Inc. (Alameda, CA) and many others.
B. Selector Codons
1003511 Selector codons encompassed within the niethods and compositions
described herein expand the
genetic codon framework of protein biosynthetic machinery. For example, a
selector codon includes, but is not
limited to, a unique three base codon, a nonsense codon, such as a stop codon,
including but not limited to, an
amber codon (UAG), or an opal codon (UGA), a unnatural codon, a four or more
base codon, a rare codon, or
the like. There is a wide range in the number of selector codons that can be
introduced into a desired gene or
polynucleotide, including but not limited to, one or more, two or more, tluee
or more, 4, 5, 6, 7, 8, 9, 10 or more
in a single polynucleotide encoding at least a portion of a polypeptide of
interest.
1003521 In one enibodinient, the methods involve the use of a selector codon
that is a stop codon for the
incorporation of one or more non-natural amino acids in vivo. For exaniple, an
O-tRNA is produced that
recognizes the stop codon, including but not limited to, UAG, and is
aminoacylated by an O-RS with a desired
non-natural amino acid. This O-tRNA is not recognized by the naturally
occurring host's aminoacyl-tRNA
synthetases. Site-directed mutagenesis can be used to introduce the stop
codon, including but not limited to,
UAG, at the site of interest in a polypeptide of interest. See. e.g.. Sayers,
J.R., et al. (1988), 5.3' Exonuclease in
phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids
Res. 16(3):791-802. When the 0-
RS, 0-tRNA and the nucleic acid that encodes the polypeptide of interest are.
combined in vivo, the non-natural
amino acid is incorporated in response to the UAG codon to give a polypeptide
containing the non-natural
amino acid at the specified position.
1003531 Non-natural amino acids can also be encoded with rare codons. For
example, when the arginine
concentration in an in vitro protein synthesis reaction is reduced, the rare
arginine codon, AGG, has proven to
be efficient for insertion of Ala by a synthetic tRNA acylated with alanine.
See, e.g., Ma et al., Biochenustry,
32:7939 (1993). In this case, the synthetic tRNA competes with the naturally
occurring tRNAArg, which exists
as a minor species in Escherichia coli. Some organisms do not use all triplet
codons. An unassigned codon AGA
in Micrococcus luteus has been utilized for insertion of amino acids in an in
vitro transcription/translation
extract. See, e.g., Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). In one
embodiment, components of the
compositions and methods described herein can be generated to use these rare
codons in vivo.
1003541 The incorporation of non-natural amino acids in vivo can be done
without significant perturbation of
the eukaryotic host cell. For example, because the suppression efficiency for
the UAG codon depends upon the
competition between the O-tRNA, including but not limited to, the amber
suppressor tRNA, and a eukaryotic
release factor (including but not limited to, eRF) (which binds to a stop
codon and initiates release of the
growing peptide from the ribosome), the suppression efficiency can be
modulated by, including but not limited
to, increasing the expression level of O-tRNA, and/or the suppressor tRNA.
1003551 Selector codoiis also comprise extended codons, including but not
limited to, four or more base codons,
such as, four, five, six or more base codons. Examples of four base codons
include, but are not limited to,
AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codons include, but
are not limited to,
AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. A feature of the
methods and
compositions described herein includes using extended codons based on
frameshift suppression. Four or more
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base codons can insert, including but not limited to, one or multiple non-
natural amino acids into the same
protein. For example, in the presence of mutated O-tRNAs, including but.not
limited to, a special frameshift
suppressor tRNAs, with anticodon loops, for example, with at least 8-10 nt
anticodon loops, the four or more
base codon is read as single amino acid. In other embodiments, the anticodon
loops can decode, including but
not liniited to, at least a four-base codon, at least a five-base codon, or at
least a six-base codon or more. Since
there are 256 possible four-base codons, multiple non-natural amino acids can
be encoded in the same cell using
a four or niore base codon. See, Anderson et al., (2002) Exploring the Limits
of Codon and Anticodon Size,
Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:
Selection of Effrcient
Suppressors of Four-base Codoais and Identifcation of "Shifty" Four-base
Codons with a Library Approach in
Escherichia coli, J. Mol. Biol. 307: 755-769.
(00356) For example, four-base codons have been used to incorporate non-
natural amino acids into proteins
using in vitro biosynthetic methods. See, e.g., Ma et al., (1993)
Biochemistry, 32:7939-7945; and Hohsaka et
al., (]999) J. Am. Chem. Soc., .121:34-40. CGGG and AGGU were used to
simultaneously incorporate 2-
naphthylalanine and an NBD derivative of lysine into, streptavidin in vitro
with two chemically acylated
frameshift suppressor tRNAs. See, e:g., Hohsaka et al., (I 999). J. Am. Chem.
Soc., 121:12194-12195. In an in
vivo study, Moore et al. examined the ability of tRNALeu derivatives with NCUA
anticodons to suppress
UAGN codons (N can be U, A, G, or C), and found that the quadruplet UAGA can
be decoded by a tRNALeu
with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in
the 0 or -1 frame. See, Moore et
al., (2000) J. Mol. Biol., 298:195-205. In one embodiment, extended codons
based on rare codons or nonsense
codons can be used in the methods and compositions described herein, which can
reduce missense readthrough
and frameshift suppression at other unwanted sites.
1003571 For a given system, a selector codon can also include one of the
natural three base codons, where the
endogenous system does not use (or rarely uses) the natural base codon. For
example, this includes a system that
is lacking a tRNA that recognizes the natural three base codon, and/or a
system where the three base codon is a
rare codon.
1003581 Selector codons optionally include unnatural base pairs. These
unnatural base pairs further expand the
existing genetic alphabet. One extra base pair increases the number of triplet
codons from 64 to 125. Properties
of third base pairs include stable and selective base pairing, efficient
enzymatic incorporation into DNA with
high fidelity by a polymerase, and the efficient continued primer extension
after synthesis of the nascent
unnatural base pair. Descriptions of unnatural base pairs which can be adapted
for methods and compositions
include, e.g., Hirao, et al., (2002) An unnatural base pair for incorporating
amino acid analogues into protein,
Nature Biotechnology, 20:177-182, and see also, Wu, Y., et. al. (2002) J: Am.
Chem. Soc. 124:14626-14630.
Other relevant publications are listed herein.
100359] For in vivo usage, the unnatural nucleoside is membrane permeable and
is phosphorylated to form the
corresponding triphosphate. In addition, the increased genetic information is
stable and not destroyed by cellular
enzymes. Previous efforts by Benner and others took advantage. of hydrogen
bonding patterns that are different
from those in canonical Watson-Crick pairs, the most noteworthy example of
which is the iso-C:iso-G pair. See,
e.g., Switzer et al., (1989) J. Am. Chem. Soc., 111:8322-8322; and Piccirilli
et al., (1990) Nature, 343:33-37;
Kool, (2000) Curr. Opin. Chem. Biol., 4:602-608. These bases in general
mispair to some degree with natural
bases and cannot be enzytnatically replicated. Kool and co-workers
demonstrated that hydrophobic packing.
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interactions between bases can replace hydrogen bonding to drive the formation
of base pair. See, Kool, (2000)
Curr. Opin. Chem. Biol., 4:602-608; and Guckian and Kool, (1998) Angew. Chem.
Int. Ed. Engl.,.36(24): 2825-
2828. In an effort to develop an unnatural base pair satisfying all the above
requirements, Schultz, Romesberg
and co-workers have systematically synthesized and studied a series of
unnatural hydrophobic bases. A
PICS:PICS self-pair is found to be more stable than natural base pairs, and
can be efficiently incorporated into
DNA by Klenow fragment of Escherichia coli DNA polymerase I(KF). See, e.g.,
McMinn et al., (1999) J. Am.
Chem. Soc., 121:11585-11586; and Ogawa et al., (2000) J. Am. Chem. Soc.,
122:3274-3278. A 3MN:3MN self-
pair can be synthesized by KF with efficiency and selectivity sufficient for
biological function. See, e.g., Ogawa
et al., (2000) J. Am. Chem. Soc., 122:8803-8804. However, both bases act as a
chain terminator for further
replication. A mutant DNA polymerase has been recently evolved that can be
used to replicate the PICS self
pair. In addition, a 7AI self pair can be replicated. See, e.g., Tae et al.,
(2001) J. Am. Chem. Soc., 123:7439-
7440. A novel metallobase pair, Dipic:Py, has also been developed, which fonns
a stable pair upon binding
Cu(II). See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714-10715.
Because extended codons and
unnatural codons are intrinsically orthogonal to natural codons, the non-
natural amino acid methods described
herein can take advantage of this property to generate orthogonal tRNAs for
them.
1003601 A translational bypassing system can also be used to incorporate a non-
natural amino acid in a desired
polypeptide. In a translational bypassing system, a large sequence is
incorporated into a gene but is not
translated into protein. The sequence contains a structure that serves as
a.cue io induce the ribosome to hop over
the sequence and resume translation downstream of the insertion.
1003611 In certain embodiments, the protein or polypeptide of interest (or
portion thereof) in the methods
and/or compositions described herein is encoded by a nucleic acid. Typically,
the nucleic acid comprises at least
one selector codon, at least two selector codons, at least three selector
codons, at least four selector codons, at
least five selector codons, at least six selector codons, at least seven
selector codons, at least eight selector
codons, at least nine selector codons, ten or more selector codons.
1003621 Genes coding for proteins or polypeptides of interest can be
mutagenized using, for exaniple, methods
described herein under "Mutagenesis and Other Molecular Biology Techniques" to
include, for example, one or
more selector codons for the incorporation of a non-natural amino acid. For
example, a nucleic acid for a protein
of interest is mutagenized to include one or more selector codons, providing
for the incorporation of the one or
more non-natural amino acids. The methods and conipositions described herein
include any such variant,
including but not limited to, niutant, versions of any protein, for example,
including at least one non-natural
amino acid. Similarly, the methods and compositions described herein include
corresponding nucleic acids, i.e.,
any nucleic acid with one or more selector codons that encodes or allows for
the in vivo incorporation of one or
more non-natural amino acid.
1003631 Nucleic acid molecules encoding a polypeptide of interest, including
by way of example only, GH
polypeptide may be readily mutated to introduce a cysteine at any desired
position -of the polypeptide. Cysteine
is widely used to introduce reactive molecules, water soluble polymers,
proteins, or a wide variety of other
molecules, onto a protein of interest. Methods suitable for the incorporation
of cysteine into a desired position of
a polypeptide include those described in U.S. Patent No. 6,608,183, which is
herein incorporated by reference
for the aforementioned disclosure, and mutagenesis techniques. The use of such
cysteine-introducing and
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utilizing techniques can be used in conjunction with the non-natural amino
acid introducing and utilizing
techniques described herein.
VIIL Ii: vivo generation of polypeptides con:prising non-natural amino acids
1003641 For convenience, the in vivo generation of polypeptides comprising non-
natural amino acids described
in this section have been described generically and/or with specific examples.
However, the in vivo generation
of polypeptides comprising non-natural amino acids described in this section
should not be limited to just the
generic descriptions or specific example provided in this section, but rather
the in vivo generation of
polypeptides comprising.non-natural amino acids described in'this section
apply equally well to.all compounds
that fall within the scope of Fonnulas I-XV, including any sub-formulas or
specific compounds that fall within
the scope of Formulas I-XV that are described in the specification, claims and
figures herein.
1003651 The polypeptides described herein can be generated in vivo using
modified tRNA and tRNA
synthetases to add to or substitute amino acids that are notencoded in
naturally-occurring systems.
1003661 Methods for generating tRNAs and tRNA synthetases which use amino
acids that are not encoded in
naturally-occurring systems are described in, e.g., U.S. Patent No. 7,045,337,
entitled "In vivo incorporation of
unnatural amino acids" and U.S. Patent No. 7,083,970, entitled "Methods and
compositions for the production
of orthogonal tRNA-aminoacyl tRNA synthetase pairs" which are incorporated by
reference herein. These
niethods involve generating a translational machinery that functions
independently of the synthetases and
tRNAs endogenous to the translation system (and are therefore sometimes
referred to as "orthogonal"). In'one
embodinient the translation system comprises a polynucleotide encoding the
polypeptide; the. polynucleotide
can be mR.NA that was transcribed from the corresponding DNA, or the mRNA may
arise from an RNA viral
vector; further the polynucleotide comprises a selector codon corresponding to
the predesignated site of
incorporation for the non-natural amino acid. The translation further
comprises a tRNA comprising the non-
natural amino acid, where the tRNA is specific to the aforementioned selector
codon;. in further embodiments,
the non-natural amino acid is aminoacylated. In further or additional
embodiments, the translation system
comprises an aminoacyl synthetase specific for the tRNA; and in other or
further embodiments, the translation
system comprises an orthogonal tRNA and an orthogonal anzinoacyl tRNA
synthetase. In further or additiorial
embodiments, the -translation system comprises at least one of the following:
a plasmid comprising the
aforementioned polynucleotide (typically in the form of DNA), genomic DI`TA
comprising the aforementioned
polynucleotide (typically in the form of DNA), or genomic DNA into which the-
aforementioned polynucleotide
has been integrated (in further embodiments, the integration is stable
integration). In further or additional
embodiments of the translation system, the selector codon is selected from the
group consisting of an amber
codon, ochre codon, opal codon, a unique codon, a rare codon, an unnatural
codon, a five-base codon, and a
four-base codon. In further or additional embodiments of the translation
system, the tRNA is a suppressor
tRNA. In further or additional embodiments, the non-natural amino acid
polypeptide is synthesized by a
ribosome.
1003671 In further or additional embodiments, the translation system comprises
an orthogonal tRNA (O-tRNA)
and an orthogonal aminoacyl tRNA synthetase (O-RS). Typically, the O-RS
preferentially anunoacylates the 0-
tRNA with at least one non-natural amino acid in the translation system and
the O-tRNA recognizes at least one
selector codon that is not recognized by other tRNAs in the system. The
translation system thus inserts the non-
natural amino acid into a polypeptide produced in the system, in response to
an encoded selector codon, thereby
"substituting" a, non-natural amino acid into a position in the encoded
polypeptide.
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1003681 A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases for
inscrting particular synthetic
amino acids into polypeptides are generally suitable for use in the methods
described herein to produce the non-
natural amino acid polypeptides described herein. For example, keto-specific O-
tRNA/aminoacyl-tRNA
synthetases are described in Wang, L., et al., Proc. Natl. Acad. Sci. USA
l00(i):56-61 (2003) and Zhang, Z. et
al., Biochem. 42(22):6735-6746 (2003). Exemplary O-RS, or portions thereof,
are encoded by polynucleotide
sequences and include amino acid sequences disclosed in U.S. Patent Nos.
7,045,337 and 7,083,970, each
incorporated herein by reference in their entirety. Corresponding O-tRNA
molecules for use with the O-RSs are
also described in U.S. Patent Nos. 7,045,337 and 7,083,970 which are
incorporated by reference in their entirety
herein. In addition, Mehl et al. in J. Am. Chem Soc. 2003; 125:935-939 and
Santoro et al. Nature
Biotechnology 2002 Oct; 20:1044-1048, which are incorporated by reference in
their entirety herein, discuss
screening methods and aminoacyl tRNA synthetase and tRNA molecules for the
incorporation of p-
aniinophenylalanine into polypeptides
1003691 Exemplary O-tRNA sequences suitable for use in the methods described
herein include, but are not
limited to, nucleotide sequences SEQ ID NOs: I or 2 as disclosed in U.S.
Patent No. 7,045,337 which is
incorporated by reference herein. Other examples of O-tRNA/aminoacyl-tRNA
synthetase pairs specific to
particular non-natural amino acids are described in U.S. Patent No. 7,083,970
which is incorporated by
reference in its entirety herein. O-RS and O-tRNA that incorporate both keto-
and azide-containing amino acids
in S. cerevisiae are described in Chin, J. W., et al., Science 301:964-967
(2003).
1003701 Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of a
specific codon which encodes the
non-natural amino acid. While any codon can be used, it is generally desirable
to select a codon that is rarely or
never used in the cell in which the O-tRNA/aminoacyl-tRNA synthetase is
expressed. By way of example only,
exeniplary codons include nonsense codon such as stop codons (amber, ochre,
and opal), four or more base
codons and other natural three-base codons that are rarely or unused.
1003711 Specific selector codon(s) can be introduced into appropriate
positions in the polynucleotide coding
sequence using mutagenesis methods (including but not limited to, site-
specific mutagenesis, cassette
mutagenesis, restriction selection mutagenesis, etc.).
1003721 Methods for generating components of the protein biosynthetic
machinery, such as O-RSs, O-tRNAs,
and ortliogonal O-tRNA/O-RS pairs that can be used to incorporate a non-
natural amino acid are described in
Wang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J. Am.
Chem Soc. 124:9026-9027 (2002);
Zhang, Z. et al., Biochemistry 42: 6735-6746 (2003). Methods and compositions
for the in vivo incorporation of
non-natural amino acids are dcscribed in U.S. Patent No. 7,045,337 which is
incorporated by reference in its
entirety herein. Methods for selecting an orthogonal tRNA-tRNA synthetase pair
for use in vivo translation
system of an organism are also described in U.S. Patent Nos. 7,045,337 and
7,083,970 which are incorporated
by reference in its entirety herein. In addition PCT Publication No. WO
04/035743 entitled "Site Specific
Incorporation of Keto Amino Acids into proteins, which is incorporated by
reference in its entirety, describes
orthogonal RS and tRNA pairs for the incorporation of keto amino acids. PCT
Publication No. WO 04/094593
entitled "Expanding the Eukaryotic Genetic Code," which is incorporated by
reference herein in its entirety,
describes orthogonal RS and tRNA pairs for the incorporation of non-naturally
encoded amino acids in
eukaryotic host cells.
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1003731 Methods for producing at -least. one recombinant orthogonal aminoacyl-
tRNA syitthetase (O-RS)
contprise; (a) generating a library .of (optionally mutant) RSs derived from
at least one aminoacyl-tRNA
synthetase (RS) from a first organism, including but not liniited to, a
prokaryotic organism, such as, by way of
example only, Methanococcus jannaschii, Methanobacterium thermoautotrophicum,
Halobacterium,
Escherichia coli, A. fulgidus, P. firriosus, P. horikoshii, A. pernix. T.
thermaphilus, or the like, or a eukaryotic
organism; (b) selecting (and/or screening), the library of RSs (optionally
mutant RSs) for meinbers that
anunoacylate an orthogonal tRNA (O-tRNA) in the presence of a non-natural
atnino acid and a natural amino
acid, thereby providing a pool of active (optionally mutant) RSs; and/or,_ (c)
selecting (optionally through
negative selection) the pool for active RSs (including but not limited to,
mutant RSs) that preferentially
aminoacylate the O-tRNA in the absence of the non-natural anuno acid, thereby
providing the at least one
recombinant O-RS; wherein the at least one recombinant O-RS preferentially
aminoacylates the O-tRNA witli
the non-natural amino acid.
1003741 In one embodiment, the RS is an inactive RS. The inactive RS can be
generated by mutating an active
RS. By way of example only, the inactive RS can be generated by mutating at
least about 1, at least about 2, at
least about 3, at least about 4, at least about 5, at least about 6, or at
least about 10 or more amino acids to
different anuno acids, including but not limited,to, alanine.
[003751 Libraries of mutant RSs can be generated using various techniques,
including but not.limited to rational
design based on protein three dimensional RS structure, or mutagenesis of RS
nucleotides in a random or
rational design technique. By way of example only, the mutant RSs can be
generated by site-specific mutations,
random mutations, diversity generating recombination mutations, chimeric
constructs, rational design and by
other methods described herein.
(003761 In one embodiment, selecting (and/or screening) the library of RSs
(optionally mutant RSs) for
members that are active, including but not limited to, those which
aniinoacylate an orthogonal tRNA (O-tRNA)
in the presence of a non-natural amino acid.and a natural amino acid,
includes, but is not limited to: introducing
a positive selection or screening marker, including but not limited to, an
antibiotic resistance gene, or the like,
and the library of (optionally mutant) RSs into a plurality of cells, wherein
the positive selection and/or
screening marker comprises at least one selector codori, including but not
limited to, an amber codon, ochre
codon, opal codon, a unique codon, a rare codon, an unnatural codon, a five-
base codon, and a four-base codon;
growing the plurality of cells in the presence of a selection agent;
identifying cells that survive (or show a
specific response) in the presence of the selection andlor screening agent:by
suppressing the at least one selector
codon in the positive selection or screening marker, thereby providing a
subset of positively selected cells that
contains the pool of active (optionally mutant) RSs. Optionally, the selection
and/or screening agent
concentration can be varied.
1003771 In one aspect, the positive selection marker is_a chloramphenicol
acetyltransferase (CAT) gene and the
selector codon is an amber stop codon in the CAT gene. Additional selection
markers include, but are not
limited to, a neomycin resistance gene, a blasticidin resistance gene, a
hygromycin resistance gene, or any other
available resistance genes. Optionally, the positive selection marker is a(3-
lactamase gene and the selector
codon is an atnber stop codon in the (3-lactamase gene. In another aspect the
positive screening marker
comprises a fluorescent or luminescent screening marker or an affinity based
screening marker (including but
not limited to, a cell surface marker).
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1003781 In one embodiment, negatively selecting or screening the pool for
active RSs (optionally mutants)
including, but not limited to, those which preferentially aminoacylate the O-
tRNA in the absence of the non-
natural amino acid includes, but is not limited to: introducing a negative
selectiotror screening marker with the
pool of active (optionally mutant) RSs from the positive selection or
screening into a plurality of cells of a
second organism, wherein the negative selection or screening marker comprises
at least one selector codon
(including but not limited to, an -antibiotic resistance gene, including but
not liniited to, a chloramphenicol
acetyltransferase (CAT) gene); and, identifying cells that survive or show a
specific screening response in a first
medium supplemented with the non-natural amino acid and a screening or
selection agent, but fail to survive or
to show the specific response in a second medium not supplemented with the non-
natural amino acid and the
selection or screening agent, thereby providing surviving cells or screened
cells with the at least one
recombinant O-RS. By way of example only, a CAT identification protocol
optionally acts as a positive
selection and/or a negative screening in determination of appropriate O-RS
recombinants. For instance, a pool
of clones is optionally replicated on growth plates containing CAT (which
comprises at least one selector
codon) either with or without one or more non-natural amino acid. Colonies
growing exclusively on the plates
containing non-natural amino acids are thus regarded as containing recombinant
O-RS. In one aspect, the
concentration of the selection (and/or screening) agent is varied. In some
aspects the first and second organisms
are different. Thus, the first and/or second organism optionally comprises: a
prokaryote, a eukaryote, a mammal,
an Escherichia coli, a fungi, a yeast, an archaebacterium, a eubacterium, a
plant, an insect, a protist, etc. In other
embodiments, the screening marker comprises a fluorescent or lunvnescent
screening marker or an affinity
based screening niarker.
1003791 In another embodiment, screening or selecting (including but not
limited to, negati,vely selecting) the
pool for active (optionally mutant) RSs includes, but is not limited to:
isolating the pool of active mutant RSs
from the positive selection step (b); introducing a negative selection or
screening marker, wherein the negative
selection or screening marker comprises at least one selector codon (including
but not limited to, a toxic marker
gene, including but not limited to, a ribonuclease barnase gene, comprising at
least one selector codon), and the
pool of active (optionally mutant) RSs into a plurality of cells of a second
organism; and identifying cells that
survive or show a specific screening response in a first medium not
supplemented witlt the non-natural amino
acid, but fail to survive or show a specific screening response in a second
medium supplemented with the non-
natural amino acid, thereby providing surviving or screened cells with the at
least one recombinant O-RS,
wlierein the at least one recombinant O-RS is specific for the non-natural
amino acid. In one aspect, the at least
one selector codon comprises about two or more selector codons. Such
embodiments optionally can include
wherein the at least one selector codon comprises two or more selector codons,
and wherein the first and second
organism are different (including but not limited to, each organism is
optionally, including but not limited to, a
prokaryote, a eukaryote, a mammal, an Escher=ichiu. coli, a fungi, a yeast, an
archaebacteria, a eubacteria, a
plant, an irtsect, a protist, etc.). Also, some aspects include wherein the
negative selection marker comprises a
ribonuclease barnase gene (which comprises at least one selector codon). Other
aspects include wherein the
screening niarker optionally comprises a fluorescent or luminescent screening
marker or an affinity based
screening marker. In the embodiments herein, the screenings and/or selections
optionally include variation of
the screening and/or selection stringency.
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1003801 In another embodiment, the methods for producing at least one
recombinant orthogonal aminoacyl-
tRNA synthetase (O-RS) may further comprise: (d) isolating the at least one
reeombinant O-RS; (e) generating a
second set of O-RS (optionally mutated) derived from the at least one
recombinant O-RS; and, (f) repeating
steps (b) and (c) until a inutated O-RS is obtained that comprises an ability
to preferentially aminoacylate the 0-
tRNA. Optionally, steps (d)-(f) are repeated, including but not limited to, at
least about two times. In one aspect,
the second-set of mutated O-RS derived from at,least one.recombinant O-RS.oan
be generated by mutagenesis,
including but not limited to, random mutagenesis, site-specific mutagenesis,.
recombination or a combination
thereof
1003811 The stringency of the selection/screening steps, including but not
limited to, the positive
selection/screening step (b), the negative selection/screening step (c) or
both the positive and negative
selection/screening steps (b) and (c), in the above-described methods,
optionally includes varying the
selectionlscreening stringency. In another embodiment, the positive
selection/screening step (b), the negative
selection/screening step (c) or both the positive and negative
selection/screening steps (b) and (c) comprise
using a reporter, wherein the reporter is detected by fluorescence-activated
cell sorting (FACS) or wherein the
reporter is detected by luminescence. Optionally, the reporter is displayed on
a cell surface, on a phage display
or the like and selected based upon affinity or catalytic activity involving
the non-natural amino acid or an
analogue. In one embodinient, the mutated synthetase is displayed on a cell
surface, on a phage display or the
like.
1003821 Methods for producing a recombinant orthogonal tRNA (O-tRNA) include,
but are not limited to: (a)
generating a library ofmutant tRNAs derived from:at least one tRNA, including
but not limited to, a suppressor
tRNA, from a first organism; (b) selecting (including but not limited to,
negatively selecting) or screening the
library for (optionally mutant) tRNAs that are aminoacylated by an aminoacyl-
tRNA synthetase (RS) froni a
second organisin in the absence'of a RS from the first organisni, thereby
providing a pool of tRNAs (optionally
mutant); and, (c) selecting or screening the pool of tRNAs (optionally mutant)
for inenibers that are
aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at
least one recombinant O-tRNA;
wherein the at least one recombinant .0-tRNA recognizes-a selector codon and
is not efficiency recognized by
the RS from the.second organism and is preferentially aminoacylated by the O-
RS. In some embodiments the at
least one tRNA is a suppressor tRNA and/or comprises a. wiique three base
codon of natural and/or unnatural
bases, or is a nonsense codon, a rare codon, an unnatural codon, :a codon
comprising at least 4 bases, an aniber
codon, an ochre codon, or an opal stop codon. In one embodiment, the
recoinbinant O-tRNA possesses an
improvement of orthogonality. It will be appreciated that in some embodiments,
O-tRNA is optionally imported
into a first organism from a second organism without the need for
modification. In various embodiments, the
first and second organisms are either the same or different and are optionally
chosen from, including but not
limited to, prokaryotes (including but not limited to, Methanococcus
jannaschii, Metlranobacteriuni
thern7oautotrophicuni, Escherichia coli, Halobacteriuni, etc.), eukaryotes,
mammals, fungi, yeasts,
archaebacteria, eubacteria, plants, insects, protists, etc. Additionally, the
recombinant tRNA is optionally
aniinoacylated by a non-natural amino acid, wherein the non-natural amino acid
is biosynthesized in vivo either
naturally or through genetic manipulation. The non-natural amino acid is
optionally added to a growth medium
for.at least the first or second organism, wherein the non-natural amino acid
is capable of achieving appropriate
intracellular concentrations to allow incorporation into the non-natural anuno
acid polypeptide
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1003831 In one aspect, selecting (including but not liniited to, negatively
selecting) or screening the library for
(optionally mutant) tRNAs that are aminoacylated by an aminoacyl-tRNA
synthetase (step (b)) includes:
introducing a toxic marker gene, wherein the toxic marker gene comprises at
least one of the selector codons (or
a gene that leads to the production of a toxic or static agent or a gene
essential to the organism wherein such
marker gene comprises at least one selector codon) and the library of
(optionally mutant) tRNAs into a plurality
of cells from the second organism; and, selecting surviving cells, wherein the
surviving cells contain the pool of
(optionally mutant) tRNAs comprising at least one orthogonal tRNA or
nonfunctional tRNA. For example,
surviving cells can be selected by using a comparison ratio cell density
assay.
1003841 In another aspect, the toxic marker gene can include two or more
selector codons. In another
embodiment of the. methods described herein, the toxic marker gene is a
ribonuclease baniase gene, where the
ribonuclease bamase gene comprises at least one amber codon. Optionally,
theribonuclease barnase gene can
include two or more amber codons.
1003851 In another embodiment, selecting or screening the pool of (optionally
mutant) tRNAs for members that
are aminoacylated by an introduced orthogonal RS (O-RS) can include:
introducing a positive selection or
screening marker gene, wherein the positive marker.gene. comprises a drug
resistance gene (including but not
liniited to, 0-lactamase gene, comprising at least one of ihe selector codons,
such as at least one amber stop
codon) or a gene essential to the organism, or a gene that leads to
detoxification of a toxic agent, along with the
O-RS, and the pool of (optionally mutant) tRNAs into a plurality of cells from
the second organism; and,
identifying surviving or screened cells grown in the presence of a selection
or screening agent, including but not
lirr-ited to, an antibiotic, thereby providing a pool of cells possessing the
at least one recombinant tRNA, where
the at least one recombinant tRNA is aminoacylated by the O-RS and inserts an
amino acid into a translation
product encoded by the positive marker gene, in response to the at least one
selector codons. In another
enibodiment, the concentration of the selection and/or screening agent is
varied.
1003861 Methods for generating specific O-tRNA/O-RS pairs are provided.
Methods include, but are not
limited to: (a) generating a library of mutant tRNAs derived from at least one
tRNA from a first organism; (b)
negatively selecting or screening the library for (optionally mutant) tRNAs
that are aminoacylated by an
aminoacyl-tRNA synthetase (RS) from a second organism in the absence of =a RS
from the first organism,
thereby providing a pool of (optionally mutant) tRNAs; (c) selecting or
screening the pool of (optionally
mutant) tRNAs for members that are aminoacylated by an introduced orthogonal
RS (O-RS), thereby providing
at least one recombinant O-tRNA. The at least one recombinant O-tRNA
recognizes a selector codon and is not
efficiently recognized by the RS from the second organism and is
prefereintially aminoacylated by the O-RS.
The method also includes (d) generating a library of (optionally mutant) RSs
derived from at least one
aminoacyl-tRNA synthetase (RS) from a third organism; (e) selecting or
screening the library of mutant RSs for
members that preferentially aminoacylate the at least one recombinant O-tRNA
in the presence of a non-natural
anvno acid and a natural aniino acid, thereby providing a pool of active
(optionally mutant) RSs; and, (f)
negatively selecting or screening the pool for active (optionally mutant) RSs
that preferentially aminoacylate the
at least one reconibinant O-tRNA in the absence of the non-natural amino acid,
thereby providing the at least
one specific O-tRNA/O-RS pair, wherein the at least one specific O-tRNA/O-RS
pair comprises at least one
reconibinant O-RS that is specific for the non-natural anuno acid and the at
least one recombinant O-tRNA.
Specific O-tRNA/O-RS pairs produced by the methods described herein are
included within the scope and
methods described herein. For example, the specific O-tRNA/O-RS pair can
include, including but not limited
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to, a mutRNATyr-mutTyrRS pair, such as a mutRNATyr-SS12TyrRS pair, a mutRNALeu-
mutLeuRS pair, a
mutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.
Additionally,, such methods include
wherein the first and third organism are the same (including but not limited
to, Methanococcus jannaschit).
1003871 Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use
in an in vivo translation
system of a second organism are also included in the methods described herein.
The methods include, but are
not limited to: introducing a. marker gene, a tRNA and an aminoacyl-tRNA
synthetase (RS) isolated or derived
from a first organism into a first set of cells from the second organism;
introducing the marker gene and the
tRNA into a duplicate cell set from.a second organism; and, selecting for
surviving cells in the first set that fail
to survive in the duplicate cell set or screening for cells shoxving a
specific screening response that fail to give
such response in the duplicate cell set, wherein the first set and the
duplicate cell set are grown in the presence
of a selection or screening agent, wherein the surviving or screened cells
comprise the orthogonal tRNA-tRNA
synthetase pair for use in the in.the in vivo translation system of -he second
organism. In one embodiment,
comparing and selecting or screening includes an in vivo coniplementation
assay. The concentration of the
selection or screening agent can be varied.
(00388] The organisms described herein comprise a variety of organism and a,
variety of combinations. In one
embodiment, the organisms are optionally a prokaryotic organism, including but
not limited to, Methanococcus
jannaschii, /l9ethanobacteriunt thermoautotrophicunt; Halobacterium,
Escherichia coli, A. fulgidus, P. furiosus,
P. horikoshii, A. pernix, T. thermophiltis, or -the.like. Alternatively, the
organisms are a eukaryotic organism,
including but not limited to, plants (including but not linmited to, complex
plants such as monocots, or dicots),
algae, protists, fungi (including but not limited to, yeast, etc), animals
(including but not limited to, manunals,
insects, arthropods, etc.), or the like.
A. Expression in Non-eukaryotes and Eukaryotes
1003891 The techniques disclosed in this section can be applied to the
expression in non-eukaryotes and
eukaryotes of the non-natural amino acid polypeptides described herein. Such
expression systems are further
described in U.S. Patent Application No.
1003901 A eukaryotic host cell or non-eukaryotic host cell as described herein
provides the ability to synthesize
polypeptides which comprise non-natural amino acids in large useful
quantities. In one aspect, the composition
optionally includes, but is not litnited to, at least 10 tnicrograms, at least
50 micrograms, at least 75 micrograms,
at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at
least 500 micrograms, at least 1
milligram, at least 10 milligrams, at least 100 milligrams, at least one gram,
or more of the polypeptide that
comprises a non-natural amino acid, or an amount that can be achieved with in
vivo polypeptide production
methods (details on recombinant protein production and purification are
provided herein).. In another aspect, the
polypeptide is optionally present- in the composition at a concentration of,
including but not limited to, at least
10 niicrograms of polypeptide per liter, at least 50 micrograms of polypeptide
per liter, at least 75 nucrograms
of polypeptide per liter, at least 100 micrograms of polypeptide per liter, at
least 200 microgrants of polypeptide
per liter, at 7east 250 nucrograms of polypeptide per liter, at least 500
mictograms of polypeptide per liter, at
least 1 milligram of polypeptide per liter, or at least 10 milligrams of
polypeptide per liter or more, in, including
but not limited to, a cell lysate, a buffer, a pharmaceutical buffer, or other
liquid suspension (including but not
liniited to, in a volume of anywhere from about 1 nl to about 100 L or more).
The production of large quantities
(including but not limited to, greater that than typical. with other methods,
including but not limited to, in vitro
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translation) of a protein in a eukaryotic cell including at least one non-
natural aniino acid is a feature of the
methods, techniques and compositions described herein.
1003911 A eukaryotic host cell or non-eukaryotic host cell as described herein
provides the ability to
biosynthesize polypeptides that comprise non-natural amino acids in large
useful quantities. For example,
polypeptides comprising a non-natural aniino acid can be produced at a
concentration of, including but not
limited to, at least 10 g/liter, at least 50 pg/liter, at least 75 pg/liter,
at.least 100 g/liter, at least 200 pg/liter, at
least 250 pg/liter, or at least 500 g/liter, at least lmg/liter, at least
2mg/liter, at least 3 mg/liter, at least 4
mg/liter, at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at
least 8 mg/liter, at least 9 mg/liter, at least 10
mg/liter, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400. 500,
600, 700, 800, 900 mg/liter, I g/liter, 5
g/liter, 10 g/liter or more of protein in a cell extract, cell lysate, culture
medium, a buffer, and/or the like.
[003921 The techniques disclosed in this section can be applied to the
expression systems, culture and isolation
of the non-natural amino acid polypeptides described herein. Non-natural amino
acid polypeptides may be
expressed in any number of suitable expression systenis including, but not
limited to, yeast, insect cells,
mammalian cells, and bacteria.
1003931 Once a recombinant host cell strain has been established (i.e., the
expression construct has been
introduced into the host cell and host cells with the proper expression
constntct are isolated), the recombinant
host cell strain is cultured under conditions appropriate for production of
polypeptides. The method of culture of
the recombinant host cell strain will be dependent on the nature of the
expression construct utilized and the
identity of the host cell. Recombinant host strains are normally cultured
using documented methodologies.
Recombinant ltost cells are typically cultured in liquid medium containing
assimilatable sources of carbon,
nitrogen, and inorganic salts and, optionally, containing vitaniins, amino
acids, growth factors, and other
proteinaceous culture supplements. Liquid media for culture of host cells may
optionally contain antibiotics or
anti-fungals to prevent the growth of undesirable microorganisms and/or
compounds including, but not limited
to, antibiotics to select for host cells containing the expression vector.
1003941 Recombinant host cells may be cultured in batch or continuous formats,
with either cell harvesting (in
the case where the desired polypeptide accumulates intracellularly) or
harvesting of culture supematant in either
batch or continuous formats. For production in prokaryotic host cells, batch
culture and cell harvest are
preferred. Where protein expression is accomplished via a cell or cell line
expression system, cells can be
propagated in vitro in a variety of modes including, but not limited to, non-
anchorage dependent cells growing
in suspension throughout the bulk of the culture or as anchorage-dependent
cells requiring attachment to a solid
substrate for their propagation (i.e., a monolayer type of cell growth). Non-
anchorage dependent or suspension
cultures from continuous established cell lines are the most widely used means
of large scale production of cells
and cell products. Again, cell type and propagation mode may be selected based
on a variety of production
considerations as described above.
1003951 In one embodinient, the non-natural amino acid polypeptides described
herein are purified after
expression in recombinant systenis. The polypcptides may be ptirified from
host cells or culture medium by a
variety of documented methodologies. Normally, many polypeptides produced in
bacterial host cells are poorly
soluble or insoluble (in the form of inclusion bodies). In one embodiment,
anuno acid substitutions are readily
made in the polypeptides that are selected for the purpose of increasing the
solubility of the recombinantly
produced polypeptide utilizing the methods disclosed herein. In the case of
insoluble polypeptides, the
polypeptides may be collected from host cell lysates by centrifugation or
filtering and may further be followed
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by homogenization of the cells. In the case of poorly soluble polypeptides,
compounds including, but not
limited to, polyethylene imine (PEI) may be added to induce . the
precipitation of partially soluble polypeptides.
The precipitated polypeptides may then be conveniently collected by
centrifugation or filtering. Recombinant
host cells may be disrupted or homogenized to release the inclusion bodies
from within the cells using
documented methods. Host cell disruption or homogenization may be performed
using well documented
methodologies including, but not liniited to, enzymatic cell disruption,
sonication, dounce homogenization, or
high pressure release disruption. In one embodiment of the methods described
and encompassed herein, the high
pressure release technique is used to disrupt the E. coli host cells to
release the inclusion bodies of the
polypeptides. When hatidling inclusion bodies of polypeptides, it is
advantageous to minitnize the
Itomogenization time on repetitions in order to maximize the yield of
inclusion bodies without loss due to
factors such as solubilization, mechanical shearing or proteolysis.
(00396) Insoluble or precipitated polypeptides may then be solubilized using
any of a number of documented
suitable solubilization agents. By way of example, the polypeptides are
solubilized with urea or guanidine
hydrochloride. The volume of the solubilized polypeptides should be.
minimized. so that large batches may be
produced using'conveniently manageablebatch sizes. This factor may be
significant in a large-scale cotnmercial
setting where the recombinant host may be grown in batches that are thousands
of liters in volume. In addition,
when manufacturing polypeptides in a large-scale commercial setting, in
particular for human pharmaceutical
uses, the avoidance of harsh chemicals that can damage the machinery and
container, or the polypeptide product
itself, should be avoided, if possible. It has been shown in the methods
described and encompassed herein that
the milder denaturing agent urea can be used to solubilize the polypeptide
inclusion bodies in place of the
harsher denaturing agent guanidine hydrochloride. The use of urea
significantly reduces the risk of damage to
stainless steel equipment utilized in the manufacturing and purification
process of a polypeptide while
efficiently solubilizing the polypeptide inclusion bodies.
1003971 In the case of soluble polypeptides, the peptides may be secreted into
the periplasmic space or into the
culture mediurn. In addition, soluble, peptides may be present. in the
cytoplasm of the host cells. The soluble
peptide may be concentrated prior to perfonning, purification steps.
documented niethodologies, including but
not limited to those described herein, may be used to concentrate soluble
peptide from, by way of example, cell
lysates or culture medium. In addition, documented methodologies,. including,
but not limited to those described
lterein, may be used to disrupt host cells and release soluble peptide from
the cytoplasm or periplasmic space of
the host cells.
1003981 When the polypeptide is produced as a fusion protein, the, fusion
sequerice is preferably removed.
Removal of a fusion sequence may be accomplished by tnethods including, but
not limited to, enzymatic or
chemical cleavage, wherein etizymatic. cleavage is preferred. Enzymatic
removal of fusion sequences may be
accomplished using-documented.methodologies. The choice of enzyme for removal
of the fusion sequence will
be determined by the identity of the fusion, and the reaction conditions will
be specified by the choice of
enzyme. Chemical cleavage may be accomplished using reagents, including but
not limited to, cyanogen
broniide, TEV protease, and other reagents. The cleaved polypeptide is
optionally purified froni the cleaved
fusion sequence by documented methodologies. Such methods will be determined
by the identity and properties
of the fusion sequence and the polypeptide. Methods for purification may
include, but are not limited to, size-
exclusion chromatography, hydrophobic interaction chrotnatography, ion-
exchange chroniatography or dialysis
or any combination thereof.
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1003991 The polypeptide is also optionally purified to remove DNA from the
protein solution. DNA may be
removed by documented methodologies, including, but not liniited to,
precipitation or ion exchange
chromatography. In one embodiment, DNA is removed by precipitation with a
nucleic acid precipitating agent,
such as, but not limited to, protamine sulfate. The polypeptide may be
separated from die precipitated DNA
using documented niethodologies including, but not limited to, centrifugation
or filtration. Removal of host
nucleic acid molecules is an-important factor in a setting where the
polypeptide is to be used to treat humans and
the methods described herein reduce host cell DNA to pharmaceutically
acceptable levels.
1004001 Methods for small-scale or large-scale fermentation may also be used
in protein expression, including
but not limited to, fermentors, shake flasks, fluidized bed bioreactors,
hollow fiber bioreactors, roller bottle
culture systems, and stirred tank bioreactor systems. Each of these methods
can be performed in a batch, fed-
batch, or continuous niode process.
1004011 Human forms of the non-natural amino acid polypeptides described
herein can generally be recovered
using documented methodologies. For example, culture medium or cell lysate can
be centrifuged or filtered to
reinove cellular debris. The supernatant may be concentrated or diluted to a
desired volume or diafiltered into a
suitable buffer to condition the preparation for further purification. Further
purification of the non-natural anuno
acid polypeptides described herein include, but are not limited to, separating
deamidated and clipped forms of a
potypeptide variant from the corresponding intact form.
1004021 Polypeptides encompassed within the methods and compositions described
herein, including but not
limited to, polypeptides comprising non-natural amino acids, antibodies to
polypeptides comprising non-natural
amino acids, binding partners for polypeptides comprising non-natural aniino
acids, may be purified, either
partially or substantially to homogeneity, according to documented
inethodologies. Accordingly, polypeptides
described herein may be recovered and purified by documented methodologies,
including but not limited to,
amrnonium sulfate or ethanol precipitation, acid or base extraction, column
chromatography, affinity column
chromatography, anion or cation exchange chromatography, phosphocellulose
chromatography, hydrophobic
interaction chromatography, hydroxylapatite chromatography, lectin
chromatography, gel electrophoresis and
any combination thereof. Protein refolding steps can be used, as desired, in
making correctly folded mature
proteins. High performance liqiiid chromatography (HPLC), affinity
chromatography or other suitable methods
can be eniployed in final purification steps where high purity is desired. In
one embodiment, antibodies made
against non-natural amino acids (or polypeptides comprising non-natural amino
acids) are used as purification
reagents, including but not limited to, for affinity-based purification of
polypeptides comprising one or more
non-natural amino acid(s). Once purified, partially or to homogeneity, as
desired, the polypeptides are
optionally used for a wide variety of utilities, including but not liniited
to, as assay components, therapeutics,
prophylaxis, diagnostics, research reagents, and/or as immunogens for antibody
production.
1004031 One advantage of producing polypeptides comprising at least one non-
natural amino acid in a
eukaryotic host cell or non-eukaryotic host cell is that typically the
polypeptides will be folded in their native
conformations. However, in certain embodiments of the methods and compositions
described herein, after
synthesis, expression and/or purification, the polypeptides may possess a
conformation different from the
desired conformations of the relevant polypeptides. In one aspect of the
methods and compositions described
herein, the expressed protein is optionally denattired and then renatured.
This optional denaturation and
renaturation is accomplished utilizing documented methodologies, including but
not liniited to, by adding a
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chaperonin to the polypeptide of interest, and by solubilizing the
polypeptides in a chaotropic agent including,
but not limited to, guanidine HCI, and utilizing protein disulfide isomerase.
1004041 In general, it is occasionally desirable to denature and reduce
expressed polypeptides and then to cause
the polypeptides to re-fold into the preferred conformation. By way of
example, such re-folding. may be
accomplished with the addition guanidine, urea, DTT, DTE, and/or a chaperonin
to a translation product of
interest: Methods of reducing, denaturing and renaturing proteins include, the
references above, and Debinski, et
al. (1993) J. Biol. Chem., 268: 14065-14070;.Kreitman and Pastan (1993)
Bioconjug. Chem.,4: 581-585;:and
Buchner, et al., (1992) Anal. Biochem., 205: 263=270. Debinski, et al., for
example, describe the denaturation
and ieduction of inclusion body proteins in guanidine-DTE. The proteins can be
refolded in a redox buffer
containing, including but not limited to, oxidized glutathione and L-arginine.
Refolding reagents can be flowed
'or otherwise moved into contact with the one or more polypeptide or other
expression product, or vice-versa.
1004051 In the case of prokaryotic production of a non-natural amino acid
polypeptide, the polypeptide thus
produced tnay be misfolded and thus lacks or has reduced biological activity.
The bioactivity of the protein may
be restored by "refolding". In one embodiment, a misfolded polypeptide is
refolded by solubilizing (where the
polypeptide is also insoluble), unfolding and reducing the polypeptide chain
using, by way of example, one or
more chaotropic agents (including , but not limited to, urea and/or guanidine)
and a reducing agent capable of
reducing disulfide bonds (including , but not linuted to, dithiothreitol, DTT
or 2-mercaptoethanol, 2-ME). At a
moderate concentration of chaotrope, an oxidizing agent is then added
(including, but not limited to, oxygen,
cystine or cystamine), which allows the reformation of disulfide bonds. An
unfolded or misfolded polypeptide
may be refolded using doctunented methodologies, such as those described in
U.S. Pat. Nos. 4,511,502,
4,511,503, and 4,512,922, each of which is herein incorporated by reference
for the aforementioned disclosure.
The polypeptide may also be cofolded with other proteins to form beterodimers
or heteromultimers. After
refolding or cofolding, the polypeptide is optionally further purified.
[004061 After purification, the non-natural amino acid polypeptides may be
exchanged into different buffers
and/or concentrated by=documented methodologies, including, but not limited
to, diafiltration and dialysis. hGH
that is provided as a single purified protein may be subject to aggregation
and precipitation. In certain
embodiments the purified non-natural amino acid polypeptides may be at least
90% pure (as measured by
reverse phase high performance liquid chromatography, RP-HPLC, or sodium
dodecyl sulfate-polyacrylamide
gel electrophoresis, SDS-PAGE). In certain other embodiments the purified non-
natural amino acid
polypeptides may be at least 95% pure, or at least 98% pure, or at least 99%
or greater purity. Regardless of the
exact numerical value, of the purity of the non-natural amino acid
polypeptides, the non-natural aniino acid
polypeptides is sufficiently pure for use as a pharmaceutical product or for
further processing, including but not
linvted to, conjugation with a water soluble polymer such as PEG.
1004071 In certain embodiments the non-natural amino acid polypeptides
molecules may be used as therapeutic
agents in the absence of other active ingredients or proteins (other than
excipients, carriers, and stabilizers,
serum albuinin and the like), and in certain embodiments the non-natural amino
acid polypeptides molecules
they may be complexed with another polypeptide or a polymer.
1004081 A wide variety of methods and procedures can be used to assess the
yield and purity of a polypeptide
containing one or more non-natural amino acids, including but not limited to,
SDS-PAGE coupled with protein
staining methods, inununoblotting, mass spectrometry, matrix assisted laser
desorption/ionization-mass
spectrometry (MALDI-MS), liquid chromatography/mass spectrometry, isoelectric
focusing, analytical anion
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exchange, chromatofocusing, and circular dichroism. By way of exantple such
methods and procedures for
characterizing proteins include, but are not limited to, the Bradford assay,
SDS-PAGE, and silver stained SDS-
PAGE, coomassie stained SDS-PAGE. Additional methods include, but are not
limited to, steps to remove
endotoxins. Endotoxins are lipopoly-saccharides (LPSs) which are located on
the outer membrane of Gram-
negative host cells, such as, for example, Escherichia coli. Methods for
reducing endotoxin levels include, but
are. not limited to, purification techniques using silic.a supports, glass
powder or hydroxyapatite, reverse-phase,
affinity, size-exclusiou, anion-exchange chromatography, hydrophobic
interaction chromatography, a
conibination of these methods, and the like. Modifications or additional
methods may be required to remove
contaminants such as co-migrating proteins from the polypeptide of interest.
Methods for measuring endotoxin
levels include, but are.not limited to, Limulus Amebocyte Lysate (LAL) assays.
1004091 In certain embodiments amino acids of Formulas I-XV, including any sub-
formulas or specific
compounds that fall within the scope of Formulas I-XV, may be biosynthetically
incorporated into polypeptides,
thereby making non-natural amino acid polypeptides. In other embodiments, such
amino acids are incorporated
at a specific site within the polypeptide. In other embodiments, such amino
acids incorporated into the
polypeptide using atranslation system. In other embodiments, such translation
systems comprise: (i) a
polynucleotide encoding the polypeptide, wherein the polynucleotide comprises
a selector codon corresponding
to the pre-designated site of incorporation of the above amino acids, and (ii)
a tRNA comprising the aniino acid,
wherein the tRNA is specific to the selector codon. In other embodiments of
such translation systems, the
polynucleotide is mRNA produced in the translation system. In other
embodinients of such translation systems,
the translation system comprises a plasmid or a phage comprising the
polynucleotide. In other embodiments of
such translation systems, the translation system comprises genomic DNA
comprising the polynucleotide. In
other embodiments of such-translation systems, the polynucleotide is stably
integrated into the genomic DNA.
In other embodiments of such translation systems, the translation system
comprises tRNA specific for a selector
codon selected from the group consisting of an amber codon, ochre codon, opal
codon, a unique codon, a rare
codon, an unnatural codon, a five-base codon, and a four-base codon. In other
embodinients of such translation
systems, the tRNA is a suppressor tRI`'A. In other embodiments of such
translation systems, the translation
system comprises a tRNA that is aminoacylated to the amino acids above. In
other embodiments of such
translation systems, the translation system comprises an aminoacyl synthetase
specific for the tRNA. In other
embodiments of such translation systems, the translation system comprises an
orthogonal tRNA and an
ortltogonal aminoacyl tRNA synthetase. In other embodiments of such
translation systems, the polypeptide is
synthesized by a ribosome, and in further embodiments the translation system
is an in vivo translation system
comprising a cell selected from the group consisting of a bacterial cell,
archeaebacterial cell, and eukaryotic
cell. In other embodiments the cell is an Escherichia coli cell, yeast cell, a
cell from a species of Pseudomonas,
mammalian cell, plant cell, or an insect cell. In other embodiments of such
translation systems, the translation
system is an in vitro translation systeni comprising cellular extract from a
bacterial cell, archeaebacterial cell, or
eukaryotic cell. In other embodiments, the cellular extract is from an
Escherichia coli cell, a cell from a species
of Pseudomonas, yeast-cell; mammalian cell, plant cell, or an insect cell. In
other enibodinients at least:a portion
of the polypeptide is synthesized by solid phase or solution phase peptide
synthesis, or a combination thereof,
while in other embodiments further comprise ligating the polypeptide to
another polypeptide. In other
embodiments amino acids of Formulas I-XV, including any sub-formulas or
specific compounds that fall within
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the scope of Formulas I-XV, may be biosynthetically incorporated into
polypeptides, wherein the polypeptide is
a protein homologous to a therapeutic-protein.
B. In vivo Post-Translationa/ Modrfications
1004101 By producing polypeptides of interest with at least one non-natural
amino acid in eukaryotic cells, such
polypeptides may include eukary.otic post-translational modifications. In
certain embodiments,, a polypeptide
includes at least one non-natural amino acid and'at least one post-
translational modification that is made in vivo
by a eukaryotic cell, where the post-translational niodification is not made
by a prokaryotic cell. By way of
example, the post-traiislation modification includes, including but not
limited to, acetylation, acylation, lipid-
modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-
linkage modification,
glycosylation, and the like. In one aspect, the post-translational
nlodification includes attachment of an
oligosaccharide (including but not linuted to, (GIcNAc-Man)2-Man-GIcNAc-
G1cNAc)) to an asparagine by a
GlcNAc-asparagine linkage. See Table 1 which lists some examples of N-linked
oligosaccharides of eukaryotic
proteins (additional residues can also be present, which are not shown). In
another aspect, the post-translational
modification includes attachment of an oligosaccharide (including but not
limited to, Gal-GaINAc, Gal-
G1cNAc, etc.) to a serine or threonine by a GaINAc-serine or GaINAc-threonine
linkage, or a G1cNAc-serine or
a GIcNAc-threonine linkage.
SECTION 1.01 TABLE 1: EXAMPLES OF OLIGOSACCHARIDES THROUGH GIcNAc-LINKAGE
Type Base Structure
Mana1-6
ARTICLE lI. / Mana1-6
High-mannose Mana1-3 > ManR1-4GIcNAc~31-4GIcNAcR1-Asn
Mana1-3
Mana1-6
Article III. ~ ManR1-4GIcNAc~31-4GIcNAcR1-Asn
Hybrid GIcNAcR1-2 Mana1-3
Article lv. GIcNAc(31-2 Mana1-6
Complex > Man(31-4GIcNAc(31-4GIcNAc~31-Asn
GIcNAc~31-2 Mana1-3
Article V. Mana1-6
Xylose > Manp1-4GIcNAcp1-4GIcNAcR1-Asn
xyl
G31-2
(00411 1 In yet another aspect, the post-translation modification includes
proteolytic processing of precursors
(including but not limited to, calcitonin precursor, calcitonin gene-related
peptide precursor,. preproparathyroid
hornnone, preproinsulin, proinsulin, prepro-opionielanocortin, pro-
opiomelanocortin and the like), assembly into
a multisubunit protein or macromolecular assembly, translation to another site
in the cell (including but not
limited to, to organelles, such as the endopiasmic reticulum, the golgi
apparatus, the nucleus, lysosomes,
peroxisomes, mitochondria, chloroplasts, vacuoles, etc., or through the
secretory pathway). In certain
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embodiments, the protein comprises a secretion or localization sequence, an
epitope tag, a FLAG tag, a
polyhistidine tag, a GST fusion, or the like.
1004121 One advantage of a non-natural amino acid is that it presents
additional chemical moieties that can be
used to add additional molecules. These modifications can be made in vivo in a
eukaryotic or non-eukaryotic
cell, or in vitro. Thus, in certain embodiments, the post-translational
niodification is through the non-natural
amino acid. For example, the post-translational modification can be through a
nucleophilic-electrophilic
reaction. Most reactions currently used for the selective modification of
proteins involve covalent bond
formation betweeu nucleophilic and electrophilic reaction partners, including
but not limited to the reaction of
a-haloketones with histidine or cysteine side chains. Selectivity in these
cases is determined by the number and
accessibility of the nucleophilic residues in the protein. In polypeptides
described herein or produced using the
methods described herein, other more selective reactions can be used,
including but not limited to, the reaction
of a non-natural carbonyl amino acid with hydrazine, in vitro and in vivo.
Illustrative exaniples may be found in
the following references. Comish, et al., (1996) J. Am. Chem. Soc., 118:8150-
8151; Mahal, et al., (1997)
Science, 276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et
al., (2002) J. Am. Chem. Soc.
124:9026-9027; Chin, et al., (2002) Proc. Natl. Acad. Sci., 99:11020-11024;
Wang, et al., (2003) Proc. Natl.
Acad. Sci., 100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and,
Chin, et al., (2003) Science,
300:964-967. This allows the selective labeling of virtually any protein with
a host of reagents including
ftuorophores, crosslinking agents, saccharide derivatives and cytotoxic
molecules. See also, U.S.Patent
Application Serial No. 10/686,944 entitled "Glycoprotein synthesis" filed
January 16, 2003, which is
incorporated by reference for the aforementioned disclosure. Post-
translational modifications, including but not
limited to, through an azido amino acid, can also made through the Staudinger
ligation (including but not
limited to, with triarylphosphine reagents). See, e.g.. Kiick et al., (2002)
Incorporation of azides into
recombinant proteins for chemoselective modificatioir by the Stauditrger
ligtation, PNAS 99(1):19-24.
IX. Alternate Systenrs For Producing Non-Natural Amino Acid Polypeptides
1004131 Several strategies have been employed to introduce non-natural ainino
acids into proteins in non-
recombinant host cells, mutagenized host cells,.or in cell-free systems.
Further information in this regard can be
found in U.S. Patent Application No. 11/316,534.
X. Post-Translational Modifications of Non-Natural Amino Acid Components of a
Polypeptide
1004141 For convenience, the post-translational modifications of non-natural
amino acid components of a
polypeptide described in this section (XA to XJ) have been described
generically and/or with specific examples.
However, the post-translational modifications of non-natural amino acid
components of a polypeptide described
in this section should not be limited to just the generic descriptions or
specific example provided in this section,
but rather the post-translational modifications of non-natural amino acid
components of a polypeptide described
in this section apply equally well to all compounds that fall within the scope
of Formulas I-XV and compounds
having the structures 1-4, including any sub-formulas or specific compounds
that fall within the scope of
Forinulas I-XV and compounds having the structures 1-4, that are described in
the specification, claims and
figures herein.
1004151 Methods, compositions, techniques and strategies have been developed
to site-specifically incorporate
non-natural aniino acids during the in vivo translation of proteins. By
incorporating a non-natural amino acid
with a sidechain cheniistry that is orthogonal to those of the naturally-
occurring amino acids, this technology
allows the site-specific derivatization of recombinant proteins. As a result,
a major advantage of the rimethods,
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compositions, techniques and strategies described herein is that derivatized
proteins can now be prepared as
defined homogeneous products. However, the methods, compositions, reaction
mixtures, techniques and
strategies described herein are not limited to non-natural aniino acid
polypeptides formed by in vivo protein
translation techniques, but includes non-natural amino acid polypeptides
formed by any technique, including by
way of example only expressed protein ligation, chemical synthesis, ribozyme-
based techniques (see, e.g.,
section herein entitled "Expression in Alternate Systems").
1004161 The ability to incorporate non-natural amino acids into reconibinant
proteins broadly expands the
chemistries which may be implemented for post-translational derivatization,
wherein such derivatization occurs
either in vivo or in vitro. More specifically, polypeptide derivatization
utilizing the reaction of a carbonyl and a
hydrazine to an indole linkage on a non-natural aniino acid portion of a
polypeptide offers several advantages.
First, the naturally occurring amino acids do not (a) contain carbonyl groups
that can react with hydrazine
groups to form indole linkage and (b) hydrazine groups that can react with
carbonyl groups to form indole
linkages, and thus reagents designed to form such linkages will react site-
specifically with the non-natural
amino acid component of the polypeptide (assuming of course that the non-
natural, amino acid and the
corresponding.reagent have been designed to form such a linkage), thus the
ability to site-selectively derivatize
proteins provides a single homogeneous product as opposed to the mixtures of
derivatized proteins produced
using documented methodologies. Second, such indole linkages are stable under
biological conditions,
suggesting that proteins derivatized by such indole linkages are valid
candidates for therapeutic applications.
Third, the stability of the resulting indole linkage can be manipulated based
on the identity (i.e., the functional
groups and/or structure) of the non-natural amino acid to which the indole
linkage has been formed. In some
embodiments, the indole linkage to the non-natural amino acid polypeptide has
a decomposition half life less
than one hour, in other embodiments less than 1 day, in other embodiments less
than 2 days, in other
embodiments less than I week and in other embodiments more than I week. In yet
other enibodiments, the
resulting indole linkage is stable for at least two weeks under mildly acidic
conditions, in other embodiments the
resulting indole linkage is stable for at least 5 days under mildly acidic
conditions. In other embodiments, the
non-natural aniino acid polypeptide is stable for at least I day in a pH
between about 2 and about 8; in other
embodiments, from a pH of about 2 to about 6; in other embodiment, in a pH of
about 2 to about 4. In other
embodiments,. using the strategies, methods, compositions and techniques
described herein, an indole linkage to
a non-natural amino acid polypeptide is synthesized with a decomposition half-
life tuned to the situation at hand
(e.g., for a therapeutic use such as sustained release, or a diagnostic use,
or an industrial use or a military use).
1004171 The non-natural amino acid polypeptides described above are useful
for, including but not linvted to,
novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes,
binding proteins (including but not
limited to, antibodies and antibody fragments), and including but not limited
to, the study of protein structure
and function. See; e.g., Dougherty, (2000) Unnatural Amino Acids as Probes of
Protein Structetre and Function,
Current Opinion in Cheniical Bioloev,, 4:645-652. Other uses for the non-
natural amino acid polypeptides
described above include, by way of example only, assay-based, cosmetic, plant
biology, environmental, energy-
production, and/or military uses. However, the non-natural amino acid
polypeptides described above can
undergo further modifications so as to incoiporate new or modified
functionalities, including manipulating the
therapeutic effectiveness of the polypeptide, improving the safety profile of
the polypeptide, adjusting the
pharmacokinetics, pharmacologics and/or pharmacodynamics of the polypeptide
(e.g., increasing water
solubility, bioavailability, increasing senim half-life, increasing
therapeutic half-life, modulating
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immunogenicity, modulating biological activity, or extending the circulation
time), providing additional
functionality to the polypeptide; incorporating a tag, label or detectable
signal into the polypeptide, easing the
isolation properties ofxhe polypeptide; and any combiiaation of the
aforementioned modifications.
(00418.1 In certain 'embodiments are metliods for easing the isolation
properties of a polypeptide comprising
utilizing a homologous non-natural an-ino acid polypeptide comprising at least
one, non-natural amino acid
selected from the. group consisting _of a carbonyl-containing_ non-natural
amino acid, a hydrazine-containing
non-natural amino acid. In other embodiments such non-natural amino acids have
been biosynthetically
incorporated into the polypeptide. as described herein. In further or
alternative embodiments such non-natural
amino acid polypeptides comprise at least one non-natural amino acid selected
froni amino acids of Formula I-
XV. In further or alternative enibodiments such non-natural aniino acid
polypeptides comprise at least one non-
natural amino acid selected from amino acids of compounds having structures 1-
4.
1004191 The methods, compositions, strategies and techniques described herein
are not limited to a particular
type, class or farnily of polypeptides. Virtually any polypeptide may include
at least one non-natural amino
acids described herein. By way of example only, the polypeptide can be
homologous to a therapeutic protein.
'rhe non-natural amino acid polypeptide may also be homologous to any
polypeptide member of the growth
hormone supergene family.
[004201 Such modifications include the incorporation of fuztlter'
functionality onto the non-natural amino acid
component of the polypeptide, including but not.limited to, a desired
functionality.
1004211 In addition, non-natural amino acid polypeptides may contain moieties
which may be converted
into other functional groups, such as, by way of exantiple only, carbonyls or
hydrazines. FIGS. 23 illustrates the
chemical conversion of non-natural amino acid polypeptides into carbonyl-
containiiig non-natural amino acid
polypeptides and hydrazine containing non-natural an-dno acid polypeptides.
The resulting hydrazine- and
carbonyl-containing non-natural amino acid polypeptides may be used in or
incorporated into any of the
methods, compositions, techniques and strategies for making, purifying,
characterizing, and using non-natural
aniino acids, non-natural amino acid polypeptides and modified non-natural
amino acid polypeptides described
herein. The chcmical conversion of chemical moieties into other functional
groups, such as, by way of example
only, carbonyls, or hydrazinc can be achieved using documented methodologies,
such as described, for example,
in March, ADVANCED ORGANIC CHEMISTRY 5`s Ed., (Wiley 2001); and Carey and
Sundberg, ADVANCED
ORGANIC CHEMISTRY 4th Ed., Vols. A and B (Plenum 2000, 2001).
1004221 Furthermore, the chemical modification of carbonyl-containing non-
natural amino acid
polypeptides with hydrazine containing reagents can be used to generate highly
fluorescent indole derivative
containing non-natural amino acid polypeptides under the appropriate
excitation. Fig. 19 and 21 illustrate the
chemical modification of carbonyl containing non-natural amino acid
polypeptides with hydrazine containing
reagents. In addition, hydrazine containing non-natural anzino acid
polypeptides can chemically react with
carbonyl containing reagents to form highly fluorescent. indole derivative
containing non-natural amino acid
poypeptides under the appropriate excitation. Fig. 20 and 22 illustrate the
chemical modification of hydrazine
containing non-natural amino aeidpolypeptide with carbonyl containing
reagents.
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A -Methods for Post-Translationally Modifying Non-Natural Amino Acid
Polypeptides:
Syntl:esis of indole-containing non-natural amino acid polypeptides
1004231 The incorporation of substituted carbonyl and substituted hydrazine-
containing non-natural aniino
acids to polypeptides provides the site-specific derivatization via the
forrnation of an indole linkage. The
methods for derivatizing and/or further rriodifying may be conducted with a
polypeptide that has been purified
prior.to -the derivatization step or after the derivatization step. In
addition, the methods for derivatizing and/or
further modifying 'may be conducted with synthetic polymers, polysaccharides,
or polynucleotides which have
been purified before or after such modifications. In addition, derivatization
step can occur efficiently under
mildly acidic,. including by way of example, between a pH of about Ito about
6.
[004241 Furthermore, certain indole linkages allow the production of
fluorescent non-natural. amino acid
polypeptides that are used in a variety of detection methods.
1004251 Fig. 21 illustrates the site specific labeling of carbonyl containing
non-natural amino acid
polypeptides with hydrazine containing reagents. Fig. 22 illustrates the site
specific labeling of hydrazine
containing non-natural amino acid polypeptides with carbonyl containing
reagents:
1004261 In addition, the derivatization may be performed using reagents
containing carbonyl or hydrazine
groups on one end and functional groups on the other. The resulting indole-
containing non-natural amino acid.
polypeptides can be further modified to introduce molecules, including by a
way of example only polymers,
polysaccharides, or polynucleotides. Fig. 24B represents illustrative, non-
limiting examples of the reaction of
functional group containing polypeptides with PEG derivatives.
1004271 By way of example only, the reagents of formula (XVI) are the type of
carbonyl- or hydrazine-
containing reagents that can be used to form indole-containing non-natural
aniino acid polypeptides and can
further be modified to introduce other molecules. In one embodiment, hydrazine-
containing compounds of
Formula I to IV can react with reagent of Formula XVI containing carbonyl
group to form indole-containing.
non-natural amino acid polypeptides. In another embodiment, carbonyl-
containing compound of Formula V to
XIV can react with reagent of Formula XVI containing hydrazine group to form
indole-containing non-natural
amino acid polypeptides.
~X L L, W
(XVI)
wherein:
each X is independently H, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl,
alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene
oxide, substituted
polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, alkaryl, substituted alkaryl,
aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")Z, -
(alkylene or substituted alkylene)-
C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -
C(O)R", -C(O)ZR", or
-C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted
alkyl, alkenyl, substituted
alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl,
alkaryl, substituted alkaryl, aralkyl, or
substituted aralkyl;
or each X is independently a desired functionality;
eacli L is independently selected from the group consisting of alkylene,
substituted alkylene, alkenylene,
substituted alkenylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-
(alkylene or substituted
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alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted
alkylene)-, -C(O)-, -C(O)-(alkylene
or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -
N(R')-, -NR'-(alkylene or
substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted
alkylene)-, -(alkylene or substituted
alkylene)NTR'C(O)O-(alkylene or substituted alkylene)-, -O-CON(R'){alkylene or
substituted alkylene)-, -
CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or
substituted alkylene)-,
-N(R')C(O)O-, -N(R')C(O)O-(alkylene or substituted alkylene)-, -S(O)kN(R')7, -
N(R')C(O)N(R')-,
-N(R')C(O)N(R')-(alkylene or substituted alkylene)-, -N(R')C(S)N(R')-, -
N(R')S(O)kN(R')-, -N(R')-N=, -
C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')Z-N=N-, and -C(R')z-N(R=')-N(R')-
;
Li is optional, and when present, is -C(R')P NR'-C(O)O-(alkylene or
substituted alkylene)- where p is 0, 1, or
2;
each R' is independently H, alkyl, or substituted alkyl;
R"rO H
~! \ N`NHZ
W is ~- , and R is H. alkyl, or substituted alkyl;
1004281 In certain einbodiments of compounds of Formula (XVI), X is a polymer
comprising alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
alkoxy, substituted alkoxy,
alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted
polyalkylene oxide, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or
substituted aralkyl. In certain
embodiinents of conipounds of Fomiula (XVI), X is a polymer comprising
polyalkylene oxide or substituted
polyalkylene oxide. In certain embodiments of conipounds of Formula (XVI), X
is a polymer coniprising -
[(alkylene or substituted alkylene)-O-( hydrogen, alkyl, or substituted
alkyl)],x, wherein x is from about 20 to
about 10,000. In certain embodiments of compounds of Formula (XVI), X is m-PEG
having a molecular weight
ranging from about 2 to about 40 KDa. In certain embodiments of compounds of
Formula (XVI), X is a
biologically active agent selected from the group consisting of a peptide,
protein, enzyme, antibody, drug, dye,
lipid, nucleoside, oligonucleotide, cell, virus, liposonie, microparticle, and
micelle. In certain embodiments of
compounds of Formula (XVI), X is a drug selected from the group consisting of
an andbiotic, fungicide, anti-
viral agent, anti-inflamrnatory agent, anti-tumor agent, cardiovascular agent,
anti-anxiety agent, hormone,
growth factor, and steroidal agent. In certain embodiments of compounds of
Formula (XVI), X is an enzyme
selected from the group consisting of horseradish peroxidase, alkaline
phosphatase, 0-galactosidase, and glucose
oxidase. In certain embodiments of compotuids of Formula (XVI),, X is a
detectable label selected from the
group consisting of a fluorescent, phosphorescent, chemiluminescent,
chelating, electron dense, magnetic,
intercalating, radioactive, chromophoric, and energy: transfer moiety. In
certain embodiments of compounds of
Formula (XVI), X is a reactive group consisting of carbonyl containing moiety
and hydrazine containing
moiety. In certain embodiments of compounds of Formula (XVI), X is a indole
derivatives. In certain
embodiments of compounds of Formula (XVI), L is selected froni the.group
consisting of -N(R')CO-(alkylene
or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -
N(R')C(O)N(R')-(alkylene or
substi(uted alkylene)-, -O-CON(R')-(alkylene or substituted alkylene)-, -O-
(alkylene or substituted alkylene)-,
-C(O)N(R')-, and -N(R')C(O)O-(alkylene or substituted alkylene)-.
1004291 In certain enibodiments of compounds of Formula (XVI), are compounds
having the structure of
Formula (XVTI):
X-L-W (XVII)
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wherein:
H
R` O NH2
W is - , and ;_R is H, alkyl, or substituted alkyl.
1004301 In certain embodiments of compounds of formula (XVII), are compounds
having the structure of
Formula (XVIII):
H
" / N`NHZ O
~-o ~,'N~`' 0
m-PEG N N ~ I m-PEG
H
/ N`NH2
m-PEG N ~ ~ m-PEG N~/~/
O O OI and
H O
I m-PEG OuN~~O~
0 ; where in other embodiments such m-PEG or PEG groups have a
molecular weight ranging from about 5 to about 30 kDa.
1004311 In certain embodiments of compounds of Formula (XVII), are compounds
having the stnicture of
Formula (XIX):
x
~ O
NJ" O'~Y\W
X H
(XIX).
wlierein:
H
N
R O NH2
I
W is , and ~ ; R is H, alkyl, or substituted alkyl.
Y when present is alkyl, or substituted alkyl.
L is -(alkylene or substituted alkylene)-N(R')C(O)O-(alkylene or substituted
alkylene)-. In certain embodiments
of conipounds of Formula (XVII), are compounds having the structure of Formula
(XX):
Im-P EG
L O
~N"J~O"'N~\W
H m-PEG L
(XX).
wherein other embodiments of compounds of Formula (XX) such m-PEG groups have
a molecular weight
ranging from about 5 to about 30 kDa.
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(00432) In certain embodiments of compounds of Formula (XVII), are compounds
having the structure of
Formula (XXI):
x
~ O
X-L
L'~"J' N O/ W
X H
(XXI).
wherein:
H
R O 0(NH2
W is , and ; R is H, alkyl, or substituted alkyl.
Y when is present is alkyl, or substituted alkyl.
L is -(alkylene or substituted alkylene)-N(R')C(O)O-(alkylene or substituted
alkylene)-. In certain embodiments
of compounds of Formula (XXI), are compounds having the stnicture of Formula
(XXII):
m-PEG
1
L 0
Lm-PEG L H O/Y\ W
L
I
m-PEG
(XXII).
wherein other embodiments of compounds of Fomiula (XXII) such m-PEG groups
have a molecular weight
ranging from about 5 to about 30 kDa.
1004331 In certain embodiments, linkers of Formula (XVII) are reactive with
carbonyl- or hydrazine-
containing polypeptide in aqueous solution under acidic conditions. In certain
embodiments, such acidic
conditions are pH about 1 to about 6.
1004341 In ccrtain embodiments of compounds of Formula (XVII), are conipounds
having the structure of
Formula (XXIII):
O O
W Z O"J Z W
(XXIII)
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wherein:
ZisOorNHandnis 1,2,3and4
H
~ , NHZ
R'T '0 N
W is and R is H, alkyl, or substituted alkyl. ln certain embodiments of
compounds.of Formula (XXIII), are compounds having the structure of Fomiula
(XXIV):
O O
eN-__~0_1~04n ~/
ON I HyN~ ~ /NHZ
H H
(XXIV)
1004351 In other embodiments of compounds of Formula (XXIII), are compounds
having the structure of
Formula (XXV)
0 0
p O
H/ v O
(XXV)
1004361 In certain embodiments are methods for derivatizing a polypeptide
comprising amino acids of
Formulas I-XV and compounds having the structures 1-4, including any sub-
formulas or specific compounds
that fall within the scope of Formulas I-XV, wherein the method comprises
contacting the polypeptide
comprising at least one amino acid of Formula I-XV with a reagent of Formula
(XVI). In certain embodiments
the polypeptide is purified prior to or after contact with the reagent of
Formula (XVI). In other embodiments are
resulting polypeptide coniprises at least one carbonyl- or one hydrazine-
containing amino acid of formula I-XV.
In other embodiments are resulting polypeptide comprises at least one indole-
containing polypeptide generated
from the coupling of compounds of formula I-XV with the reagent of formula
(XVI).
(004371 Figure 26 provides an illustrative example of the synthesis of
bifunctional linker of formula
(XXIV). Wherrin the method comprises coupling a spacer reagent containing on
both ends an amine or
hydroxyl group to acid containing Boc-protected hydrazine. The cleavage of
Boc:group leds to linkers of
formula (XXIV).
1004381 Fig 27 provides a schematic representation of post-translational
modification of polypeptide
containing carbonyl non-natural amino acid with reagent of formula (XXIV) to
form indole containing
polypeptide.
1004391 Fig 28 provides a scheniatic representation of post-translational
modification of polypeptide
containing carbonyl non-natural amino acid with reagent of formula XX and XXII
to form indole containing
polypeptide.
1004401 Fig 29 provides a schematic representation of post-translational
modification of polypeptide
containing hydrazine non-natural amino acid with reagent of formula XX and
XXII to form indole containing
polypeptide.
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1004411 Fig 30 illtistrates examples of reagents of formula (XVIII).
1004421 In certain embodiments are methods for producing a polypeptide dimer
via indole linkage, wherein
the method consists of the reaction of a linker of formula (XXIII) with
carbonyl- or hydrazine-containing non-
natural amino acid polypeptide. Fig. 27 provides a representative ezample of
the formation of such dimmer
using condensation of linker of formula (XXIV) with carbonyl-containing non-
natural amino, acid polypeptide.
(00443] In certain embodiments are methods for preparing a polypeptides
containing an indole moiety via
the use of bifunctional linker, wherein the metliod comprises:
(i) derivatizing a first polypeptide comprising an atnino acid of Formula.(1)
with a bifunctional linker, and
(ii) contacting the resulting_derivatized protein of step (i) with a second
second reagent, such as;PEG. In
certain embodiments the polypeptides are purified prior to or after contact
with the bifunctional linker.
1004441 Fig. 24 shows a illustrative euample of such bifunctional linker. and
its use to produce indole
containing polypeptide attached to PEG group.
1004451 By way of example only, the following are representative examples of
bifnnctional linkcrs of
formula (XXVI).
0 0
SR 0~S~ S-3
W-L-O-NHZ W-L~ W-L-/ W--L--f Ar
(XXVI)
Wherein;
H
N
R~O i ~ ~NH2
W is ^^^- , and R is H, alkyl, or substituted alkyl; and
L is -(alkylene orsubstituted alkylene)=N(R')C(O)O-(alkylene,or substituted
alkylene)-.
(00446) In one embodiment, multiple linker ehemistries can react site-
specifically with a carbonyl- or a
hydrazine-contaitiing non-natural amino acid polypeptide. In one embodiment,,
the linker methods described
herein utilize linkers containing the hydrazine functionality on at least one
liiilcer terinini (mono, bi- or multi-
functional). The, reaction of an hydrazine-derivatized linker with a carbonyl-
substituted protein generates an
indole substituted non-natural protein. ln other embodiments,, the linker
methods described herein utilize linkers
containing the carbonyl functionalityon at least one linker termini (mono, bi-
or multi-functional). The reaction
of carbonyl-derivatized linker with a hydrazine-substituted protein generates
an indole substituted non-natural
protein.
100447E In certain embodiments are methods for derivatizing a chemically
synthesized polypeptide
comprising carbonyl- or hydrazine-containing non-natural polypeptide with
carbonyl or hydrazine containing
reagents to form indole derivatives.
1004481 Figure 19 provides illustrative exaniples of the derivatization of
carbonyl-containing Urotensin
with hydrazine containing reagents. In this illustrative embodiment, hydrazine-
containing reagents are added to
a buffered solution (pH 1-5) of carbonyl-containing Urotensin analogs. The
reaction proceeds at ambient
teinperature for hours to days.
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1004491 Figure 20 provides illustrative exainples of the derivatization of
hydrazine-containing Urotensin
with carbonyl containing reagents. In this illustrative embodiment, carbonyl-
containing reagents are added to a
buffered solution (pH 1-5) of hydrazine-containing Urotensin analogs. The
reaction proceeds at ambient
temperature for hours to days.
1004501 In other embodiments such derivatized polypeptides are stable in
aqueous solution for at least 1
month under mildly acidic conditions. In other embodiments such
derivatized.polypeptides are stable for at least
2 weeks under mildly acidic conditions. In other embodiments such derivatized
polypeptides are stable for at
least 5 days under mildly acidic conditions. In other embodiments.such
conditions are pH about 1 to about 6. In
certain embodiments the tertiary structure of the.derivatized potypeptide is
preserved. In other embodiments
such derivatization of polypeptides further comprises ligating the derivatized
polypeptide to another
polypeptide. Iri-other embodiments stich polypeptides being derivatized are
homologous to a therapeutic protein.
B. E.ranrple of Adding Functionalily: Macron:olecular Polynters Coupled to Non-
Natural
Amino Acid Pulypeptides
1004511 Various modifications to the non-natural amino acid polypeptides
described herein can be effected
using the compositions, methods, techniques andstrategies described herein.
These modifications include the
incorporation of further functionality onto the non-natural amino acid
component of the polypeptide, including
but not limited to, a desired functionality. As an illustrative, non-limiting
example of the compositions,
methods, techniques and strategies described herein, the following description
will focus on adding
macromolecular polymers to the non-natural amino acid polypeptide with the
understanding that the
compositions, methods, techniques and strategies described thereto are also
applicable to adding other
functionalities, including but not limited to those listed above.
1004521 A wide variety of macromolecular polymers and other moleculcs can be
coupled to the non-natural
amino acid polypeptides described hercin to modulate biological properties of
the non-natural amino acid
polypeptide (or the corresponding natural amino acid polypeptide), and/or
provide new biological properties to
the non-natural arriino acid polypeptide (or the corresponding natural amino,
acid polypeptide). These
macromolecular polymers can be coupled to the non-natural amino acid
polypeptide via_ the non-natural amino
acid, or any functional substituent of the non-natural amino acid, or any
substituent or fimctional group added to
the non-natural amino acid.
1004531 Water soluble polymers can be coupled to the non-natu.ral amino acids
incorporated into polypeptides
(natural or synthetic), polynucleotides, poly saccharides or synthetic
polymers described herein. The water
soluble polymers may be coupled via a-non-natural amino acid incorporated in
the polypeptide or any functional
group or substituent of a non-natural amino acid, or any functional group or
substituent added to a non-natural
amino acid. ln some cases, the-non-natural amino acid polypeptides described
herein comprise one or more non-
natural amino acid(s) coupled to water soluble polymers and one or more
naturally-occurring amino acids linked
to water soluble polyniers. Covalent attachment of hydrophilic polymers to a
biologically active molecule
represents one approach to increasing water solubility (such as in a
physiological environment), bioavailability,
increasing serum half-life, increasing therapeutic half-life, modulating
immunogenicity, modulating biological
activity, or extending the circulation time of the biologically active
molecule, including proteins, peptides, and
particularly hydrophobic molecules. Additional important features of such
hydrophilic polymers include
biocompatibility, lack of toxicity, and lack of immunogenicity. Preferably,
for therapeutic use of the end-
product preparation, the polymer will be pharmaceutically acceptable.
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[004541 Examples of hydrophilic polyniers include, but are not limited to:
polyalkyl ethers and'alkoxy-capped
analogs thereof (e.g:, polyoxyethylene glycol, =polyoxyethylene/propylene
glycol, and methoxy or ethoxy-
capped analogs thereof, especially polyoxyethylene glycol, the latter is also
known as polyethylene glycol or
PEG); polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyl
oxazolines and
polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl.acrylamides, and
polyhydroxyalkyl acrylamides (e.g.,
polyhydroxypropylmethacrylamide and derivatives thereof); polyliydroxyalkyl
acrylates; polysialic acids and
analogs thereof; hydrophilic peptide sequences; polysaccharides and their
derivatives, including dextran and
dextran derivatives, e.g., carboxymethyldextran, deztran sulfates,
aminodextran; cellulose and its derivatives,
e.g., carboxymethyl cellulose, hydroxyalkyl celluloses; chitin and its
derivatives, e.g., chitosan, succinyl
chitosan, carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and its
derivatives; starches; alginates;
chondroitin sulfate; albumin; pullulan and carboxvmethyl pullulan;
polyaminoacids and derivatives thereof, e.g.,
polyglutamic acids, polylysines, polyaspartic acids, polyaspartamides; maleic
anhydride copolymers such as:
styrene nialeic anhydride copolymer, divinylethyl ether maleic anhydride
copolymer; polyvinyl alcohols;
copolymers thereof,=, terpolymers thereof; mixtures thereof; and derivatives
of the foregoing. The water soluble
polymer may be any structural form including but not.limited to linear, forked
or branched. In. sonie
embodiments, polymer backbones'that are water-soluble, with from 2 to about.
300 termini, are particularly
useful. Multifunctional polymer derivatives include, buk,are not limited to,
linear polyn-ers having two termini,
each terminus being bonded to a functional group which may be the same or
different: In soineembodiments,
the water polymer comprises a poly(ethylene :glycol) moiety. The molecular
weight of the polymer may be
within a desired polymer:molecular weight range. The foregoing list for
substantially water soluble backbones is
by no means exhaustive and is merely illustrative, and that all polymeric
materials having the qualities described
above are contemplated as being suitable for use in methods and compositions
described herein.
1004551 As described above, one exaniple of a hydrophilic polymer is
polyethylene glycol, abbreviated PEG,
which has been used extensively in pharmaceuticals, on artificial implants,
and in other applications where
biocompatibility, lack of toxicity, and lack of inununogenicity are of
iniportance. The polymer:polyeptide
embodiments described herein will use PEG as an example hydrophilic polymer
with the understanding that
other hydrophilic polymers may be siniilarly utilized in such embodiments.
1004561 PEG is water soluble polymer that is conunercially available or can be
prepared by ring-opening
polymerization of ethylene glycol according to documented methodologies
(Sandier and Karo, Polymer
Synthesis, Academic Press, New York, Vol. 3, pages 138-161). PEG is typically
clear, colorless, odorless,
soluble in water, stable to heat, inert to many chemical agents, does not
hydrolyze or deteriorate, and is
generally non-toxic. Poly(ethylene..=glycol) is considered to be
biocompatible; which is to.say that PEG is
capable of :coexistence, with. living tissues or organisms without causing
harm. More specifically, PEG is
substantially non-immunogenic, which i's tosay that PEG does not tend to
produce an immune response.in the
body. When attached to a molecule having some desirable function in the body,
such as a biologically active
agent; the PEG tends to mask the agent and can reduce or eliminate any
immune.responseso that an organism
can tolerate the presence of the agent. PEG conjugates tend not to produce a
substantial. immune response or
cause clotting or other undesirable effects.
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[004571 The term "PEG" is used broadly to encompass any polyethylene glycol
molecule, without regard to
size or to modification at an end of the PEG, and can be represented as linked
to a non-natural amino acid
polypeptide by the formula:
XO-(CHZCHzO)õ-CHZCH2-Y
where, n is 2 to 10,000 arid X is H or a terminal modification, including but
not Gmited to, a C1.4 alkyl, a
protecting group, or a terminal functional group. The. term PEG includes, but
is not limited to, polyethylene
glycol in any of its forms, including bifunctional PEG, multiarmed PEG,
derivatized PEG, forked PEG,
branched PEG (with each chain having a molecular weight of from about 1 kDa to
about 100 kDa, from about I
kDa to about 50 kDa, or from about 1 kDa to about 20 kDa), pendent PEG (i.e.
PEG or related polymers having
one or more functional groups pendent to the polymer backbone), or PEG with
degradable linkages therein. In
one embodiment, PEG in which n is from about 20 to about 2000 is suitable for
use in the methods and
compositions described herein. In some embodiments, the water polymer
comprises a polyethylene glycol
moiety. The molecular weight of the PEG polynier may be of a wide range
including but not limited to, between
about 100 Da and about 100,000 Da or more. The molecular weiglit of the PEG
polymer may be within a
desired polymer molecular weight range. A wide range of PEG molecules are
described in, including but not
limited to, the Shearwater Polymers, Inc. catalog, Nektar Therapeutics
catalog, incorporated herein by reference.
1004581 Specific examples of terminal functional groups in the literature
include, but are not limited to, N-
succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), anune
(see, e.g., Buckmann et al.
Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177
(1983)), hydrazide (Sec, e.g.,
Andresz et al. Makromol. Chem. 179:301 (1978)), succininiidyl propionate and
succinimidyl butanoate (see,
e:g., Olson et al. inPoly(ethylene glycol) Cliemistry & Biological
Applications, pp 170-18 1, Harris & Zalipsky
Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.. No. 5,672,662),
succinimidyl succinate (See, e:g.,
Abuchowski et al. Cancer Biochem. Biophys. 7:_175 (1984) and Joppich et al.
Makromol. Chem. 180:1381
(1979), succinimidyl ester (see, e.g., U.S. Pat. No. 4,670,417), benzotriazole
carbonate (see, e.g., U.S. Pat. No.
5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur..J Biochem. 94:11
(1979), Elling et al., Biotech. Appl.
Biochem. 13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al.,
Anal. Biochem. 131:25 (1983),
Tondelli et al. J. Controlled Release 1:251 (1985)), p-nitrophenyl carbonate
(see, e.g., Veronese, et al., Appl.
Biochem: Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem Biotech.,
27:45 (1991)), aldehyde (see,
e.g., Harris et al. J. Polym. Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No.
5,824,784, U.S. Pat. No. 5,252,714),
nialeimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romani et
al. in Chemistry of Peptides and
Proteins 2:29 (1984)), and Kogan, Synthetic Conun. 22:2417 (1992)),
orthopyridyl-disulfide (see, e.g.,
Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et
al., Macromolecules, 26:581
(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of the above
references and patents. are
incorporated herein by reference in their entirety.
1004591 In some cases, a PEG terminates on one end with hydroxy or metlioxy,
i.e., X is H or CH3 ("methoxy
PEG"). Alternatively, the PEG can terminate with a reactive group, thereby
forming a bifunctional polynier.
Typical reactive groups can include those reactive groups that are commonly
used to react with the functional
groups found in the 20 common amino acids (including but not limited to,
maleimide groups, activated
carbonates (including but not limited to, p-nitrophenyl ester), activated
esters (including but not limited to, N-
hydroxysuccinimide, p-nitrophenyl ester and aldehydes) as well as functional
groups that are inert to the 20
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WO 2008/077079 PCT/US2007/088011
conunon amino acids but that react specifically with complementary functional
groups present in non-natural
amino acids (including but not liniited to, phenyl hydrazine and carbonyl
groups).
1004601 It is noted that the other end of the PEG, which is shown in the above
formula by Y, will attach either
directly or indirectly to a polypeptide (synthetic or natural),
polynucleotide, polysaccharide or synthetic polymer
via a non-natural amino acid. When Y is a phenyl liydrazine group, then the
phenyl hydrazine-containing PEG
reagent can react with a carbonyl-containing non-natural amino acid in a
polypeptide to form a PEG group
linked to the polypepride via an indole linkage. When Y is a carbonyl group,
then the carbonyl-containing PEG
reagent can react with a phenyl hydrazine-containing non-natural amino acid in
a polypeptide to form a PEG
group linked to the polypeptide via an indole linkage: FIG. 30 presents non-
limiting examples carbonyl- and
hydrazine-containing PEG reagents.
1004611 In some embodiments, a hydrazine can be reacted with a carbonyl group
present in a non-natural amino
acid to form an indole. Alternatively, the hydrazine can be incorporated into
the polypeptide via a non-natural
amino acid and used to react preferentially with a carbonyl group present in
the water soluble polymer.
Generally, at least one terminus of the PEG molecule is available for reaction
with the non-natural amino acid.
1004621 Thus, in some embodiments, the polypeptide comprising the non-natural
amino acid is linked to a
water soluble polymer, such as polyethylene glycol (PEG), via the side chain
of the non-nattiral aniino acid. The
non-natural amino acid methods and compositions described herein provide a
highly efficient method for the
selective modification of proteins with PEG derivatives, which involves the
selective incorporation of non-
natural amino acids, including but not limited to, those amino acids
containing functional groups or substituents
not found in the 20 naturally incorporated ainino acids, into proteins in
response to a selector codon and the
subsequent modification of those amino acids with a suitably reactive PEG
derivative. Documented
methodologies of a wide variety are suitable for use with the non-natural
amino acid methods and compositions
descnbed herein to incorporate a water soluble polymer into the protein.
1004631 The polymer backbone can be linear or branched. Branched polymer
backbones have been generally
documented. Typically, a branched polymer has a central branch core moiety and
a plurality of linear polymer
chains linked to the central branch core. PEG is used in branched forms that
can be prepared by addition of
ethylene oxide to various polyols, such as glycerol, glycerol oligomers,
pentaerythritol and sorbitol. The central
branch moiety can also be derived from several amino acids, such as lysine.
The branched poly(ethylene glycol)
can be represented in general form as R(-PEG-OH)m in which R is derived from a
core moiety, such as glycerol,
glycerol oligomers, or pentaerythritol, and m represents the nuniber of arms.
Multi-armed PEG molecules, such
as ttiose described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;
4,289,872; U.S. Pat. Appl. 2003/0143596;
WO 96/21469; and WO 93/21259, each of which is incorporated by reference
herein for the aforementioned
disclosure, can also be used as the polymer backbone.
1004641 Branched PEG can also be in the form of a forked PEG represented by
PEG(-YCHZ,),,, where Y is a
linking group and Z is an activated terminal group linked to CH by a chain of
atoms of defined length. Yet
another branched form, the pendant PEG, has reactive groups, such as carboxyl,
along the PEG backbone rather
than at the end of PEG chains.
1004651 In addition to these forms of PEG, the polymer can also be prepared
with weak or degradable linkages
in the backbone. For example, PEG can be prepared with ester linkages in the
polymer backbone that are subject
to liydrolysis. As shown herein, this hydrolysis results in cleavage of the
polynier into fragments of lower
molecular weight:
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-PEG-COZ-PEG-+HZO 4 PEG-CO2H+HO-PEG-
The term polyethylene glycol or PEG represents or includes all documented
fornis including but not limited to
those disclosed herein. The niolecular weight of the polymer may be of a wide
range, including but not limited
to, between about 100 Da and about 100,000 Da or more. The molecular weight of
the polymer may be within a
desired polymer molecular weight range.
1004661 In order to nuzxintize the desired properties of PEG, the total
molecular weight and hydration state of
the PEG polyiner or polymers attached to the biologically active molecule must
be sufficiently high to impart
the advantageous characteristics typically associated with PEG polymer
attachment, such as increased water
solubility and circulating half life, while not adversely impacting the
bioactivity of the parent molecule.
(004671 The methods and compositions described herein may be used to produce
substantially homogenous
preparations of polymer:protein conjugates. "Substantially honiogenous" as
used herein means that
polymer:protein conjugate molecules arc observed to be greater than half of
the total protein. The
polymer:protein conjugate has biological activity and the present
"substantially homogenous" PEGylated
polypeptide preparations provided herein are those which are homogenous enough
to display the advantages of
a homogenous preparation, e.g., ease in clinical application in predictability
of lot to lot pharmacokinetics.
1004681 One may also choose to prepare a mixture of polymei=:protein conjugate
molecules, and the advantage
provided herein is that one may select the proportion of mono-polymer:protein
conjugate to include in the
mixture. Thus, if desired, one may prepare a mixture of various proteins with
various numbers of polymer
moieties attached (i.e., di-, tri-, tetra-, etc.) and combine said conjugates
with the mono-polymer:protein
conjugate prepared using the methods described herein, and have a mixture with
a predeternvned proportion of
mono-polymer:protein conjugates.
1004691 The proportion of polyethylene glycol molecules to protein molecules
will vary, as will their
concentrations in the reaction mixture. In general, the optimum ratio (in
terms of efficiency of reaction in that
there is minimal excess unreacted protein or polymer) may be determined by the
molecular weight of the
polyethylene glycol selected and on the number of available reactive groups
available. As relates to molecular
weight, typically the higher the molecular weight of the polymer, the fewer
number of polymer molecules which
niay be attached to the protein. Sirnilarly, branching of the polymer should
be taken into account when
optimizing these parameters. Generally, the higher the molecular weight (or
the more branches) the higher the
polymer:protcin ratio.
1004701 As used herein, and when contemplating hydrophilic
polymer:polypeptide/protein conjugates, the term
"therapeutically effective amount" further refers to an amount which gives an
increase in desired benefit to a
patient. The amount will vary from one individual to another and will depend
upon a number of factors,
including the' overall physical condition of the patient and the underlying
cause of the disease, disorder or
condition to be treated.
1004711 The number of water soluble polymers linked to a modified or
unmodified non-natural amino acid
polypeptide (i.e., the extent of PEGylation or glycosylation) described herein
can be adjusted to provide an
altered (including but not limited to, increased or decreased) pharmacologic,
pharmacokineuc or
pharmacodynamic characteristic such as in vivo half-life. In some embodiments,
the half-life of the polypeptide
is increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, two
fold, five-fold, 10-fold, 50-fold, or at
least about 100-fold over an unmodified polypeptide.
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1004721 In one embodiment, a polypeptide comprising a carbonyl-contaiiiing non-
natural amino acid is
modified with a PEG derivative that contains a terminal arylhydrazine moiety
that is linked directly to the PEG
backbone.
1004731 In one embodiment, a polypeptide comprising a carbonyl-containing non-
natural amino acid
is modified with a PEG derivative that contains-a terminal hydrazine moiety
that is linked directly to the PEG
backbone.
1004741 In another embodinient, a polypeptide comprising a hydrazine-
containing amino acid is
modified with a PEG derivative that contains a terminal carbonyl moiety that
is linked directly to the PEG
backbone.
1004751 In some.embodiments, the carbonyl-terminal PEG derivatives have the
structure:
RO-(CHZCH2O),,-0-(CHZ)Z-NH-C(O)(CH2),,; C(O)-R
where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is
100-1,000 (i.e., average molecular
weight is between 5-40 kDa). The molecular weight of the polymer may be of a
wide range, including but not
limited to, between about 100 Da and about 100,000 Da or more. The molecular
weight of the polymer may be
within a desired polymer molecular weight range.
(004761 Several reviews and monographs on the functionalization and
conjugation of PEG are
available. See, for example, Harris, Macromol. Chem. Pliys. C25: 325-373
(1985); Scouten, Methods in
Enzyrnology 135: 30-65 (1987); Wong et, al., Ettzyme Microb. Technol. 14: 866-
874 (1992); Delgado et al.,
Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);
Zalipsky, Bioconjugate Chem. 6:
150-165 (1995).
1004771 Methods for activation of polymers can also be found. in WO 94/17039,
U.S. Pat. No.
5,324,844õ WO 94/18247, WO 94/04193, U.S. Pat. No. 5;219,564; U.S. Pat. No.
5,122,614, WO 90/13540, U.S.
Pat. No. 5,281,698, and more WO 93/15189, and for conjugation between
activated polymers and enzymes
including but, not limited to Coagulation Factor VIII (WO 94/15625),
haemoglobin (WO 94/09027), oxygen
carrying molecule (U.S. Pat. No. 4,412,989), ribonuclease and superoxide
dismutase (Veronese at al., App.
Biochein. Biotech. 11: 141-152 (1985)), all of which are herein incorporated
by reference for the
aforementioned disclosure.
1004781 If necessary, the PEGylated non-natural amino acid polypeptides
described herein obtained
from the hydrophobic chroina.tography can be purified.further by documented
methodologies including, but are
not limited to, affinity chromatography; anion- or cation-exchange
chromatography (using, including but not
limited to, DEAE SEPHAROSE); chromatography on silica; reverse phase HPLC; gel
filtration (using,
including but not limited to, SEPHADEX G-75); hydrophobic interaction
chromatography; size-exclusion
chromatography, metal-chelate chromatography; ultrafiltration/diafiltration;
ethanol precipitation; ammonium
sulfate precipitation; cliromatofocusing; displacement chromatography;
electrophoretic procedures (including
but not limited to preparative isoelectric focusing), differential solubility
(including but not limited to
ammonium sulfate precipitation), or extraction. Apparent niolecular weight may
be estimated by GPC by
coinparison to globular protein standards (Preneta AZ, PROTEIN PURIFICATION
METHODS, A PRACTICAL
APPROACfi (Harris & Angal, Eds.) IRL Press 1989,, 293-306). The purity of the
(non-natural amino acid
polypeptide):PEG conjugate can be assessed by proteolytic degradation
(including but not limited to, trypsin
cleavage) followed by niass spectrometry analysis. Pepinsky R.B., et.al., J.
Pharmacal. & Exp. Ther.
297(3):1059-66(2001).
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[004791 A water soluble polymer linked to a non-natural amino acid of a
polypeptide described herein
can be further derivatized or substituted without limitation.
C. Enbanci-ig affinityfor seruni albumin
(00480) Various molecules can also be fused to the non-natural amino acid
polypeptides described
herein to modulate the half-life in serurn In some embodiments, molecules are
linked or fused to the modified
or unmodified non-natural amino acid polypeptid8s describ"ed herein to enhance
affinity for endogenous serum
albumin in an aninial.
1004811 For example, in some cases, a recombinant fusion of a polypeptide and
an albumin binding
sequence is made. Exemplary albumin binding.sequences include, but are not
limited to, the albumin binding
domain from streptococcal protein G(see. e.g., Makrides et al., J. Pharmacol.
Exp. Ther. .277(1):534-542
(1996) and Sjolander el al., J, /mmunol. Methods 201:115-123 (1997)), or
albumin-binding peptides such as
those described in, e.g., Dennis, et al., J. Biol. Chem. 277(38):35035-35043
(2002).
(00482) In other embodiments, the modified or unmodified non-natural amino
acid polypeptides
described herein are acylated with fatty acids. In some cases, the fatty acids
promote binding to serum albumin.
See, e.g., Kurtzhals, et al., Biochem. J. 312:725-731 (1995).
1004831 In other embodiments, the modified or unmodified non-natural amino
acid polypeptides
described herein are fused directly with serum albumin (including but not
limited to, human serum albumin). A
wide variety of other niolecules can also be linked to non-natural arimino
acid polypeptides, modified or
unmodified, as described herein, to modulate binding to serum albumin or other
serum components.
A. Glycosylation of non-natural aniino acid polypeptides described Irerein
100484j The methods and compositions described herein include polypeptides
incorporating one or
niore non-natural amino acids bearing saccharide residues. The saccharide
residues may be either natural
(including but not limited to, N-acetylglucosamine) or non-natural (including
but not limited to, 3-
fluorogalactose). The saccharides may be linked to the non-natural,amino acids
either by an N- or 0-linked
glycosidic linkage (including but not limited to, N-acetylgalactose-L-serine)
or a non-natural linkage (including
but not lirnited to, a heterocycle, including a nitrogen-containing
heterocycle, linkage or the corresponding C- or
S-Iinked glycoside).
(00485) The saccharide (including but not limited to, glycosyl) moieties can
be added to the non-
natural amino acid polypeptides either in vivo or in vitro. In some
embodiments, "a polypeptide comprising a
carbonyl-containing non-natural amino acid is. modified with a saccharide
derivatized with an arylhydrazine
group to generate the corresponding glycosylated polypeptide linked via an
indole linkage. In other
enibodiments, a polypeptide comprising an arylhydrazine-containing non-natural
amino acid is modified with a
saccharide derivatized with a carbonyl group to generate. the corresponding
glycosylated polypeptide linked via
a linkage. Once attached to the non-natural amino acid, the saccharide may be
further elaborated by treatment
with glycosyltransferases and other enzymes to generate an oligosaccharide
bound to the non-natural amino acid
polypeptide. See, e.g., H. Liu, et al. J. Ani. Client. Soc. 125: 1702-1703
(2003).
1004861 FIG. 26 presents an illustrative example of the synthesis of a
bifunctional homolinker in
which the linker has two identical ends, i.e., hydrazine groups. Such a linker
may be used to form a homodimer
o1' a carbonyl-containing non-natural amino acid polypeptide to form two
indole linkages. Alternatively, if one
end of such a linker is protected, then such a partially protected linker can
be used to bind the unprotected
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hydrazine end to a carbonyl-containing non-natural aniino acid polypeptide via
an indole linkage, leaving the
other protected end available for further linking reactions following
deprotection. Alternatively, careful
manipulation of the stoichiometry of the reagents inay provide a similar
result (a heterodirner), albeit a result in
which the desired heterodimer will likely be contaminated with some homodimer.
1004871 FIG. 27 presents an illustrative example of protein dimerization by
coupling two proteins via a
b'-functional homolinker.
[004881 FIG. 25 presents an illustrative example of the use of a
heterobifunctional linker in which the linker has
two different cnds, by way of example only a carbonyl group and a reactive
group. In addition, FIG. 25. presents
illustrative example of the use of a heterobifunctional linker to attach a PEG
group to a non-natural amino acid
polypeptide in a multi-step synthesis. In the first step, as depicted in this
illustrative figure, a hydrazine-
containing non-natural amino acid polypeptide reacts with a carbonyl-
containing bifunctional linker to form an
indole-containing non-natural an-tino acid polypeptide. However, the
bifunctional linker still retains a reactive
functional group which reacts in a second step with a PEG reagent to form
a~PEGylated non-natural amino acid
polypeptide via a heterocycle linkage.
1004891 The methods and compositions described herein also provide for
polypeptide combinations,
such as homodimers, heterodimers, homomultimers, or. heteromultimers (i.e.,
trimers, tetramers,, etc:). By way
of example only, the follovving description focuses on the- GH supergene
family members, however, the
methods, techniques and compositions described in this section can be applied
to virtually any other polypeptide
which can provide benefit iri the form of dimers and multimers, including by
way of example only a therapeutic
protein.
1004901 Thus, encompassed within the methods, techniques and coanpositions
described herein are a
GH supergene family member polypeptide containing one or more non-natural
amino acids bound to another
GH supergene family member or variant thereof or any other polypeptide that is
a non-GH supergene family
mcmber or variant thereof, either directly to the polypeptide backbone or via
a linker. Due to its increased
molecular weight conipared to monomers, the GH supergene family-member dimer
or multimer conjugates may
exhibit new or desirable properties, including but not limited to different
pharmacological, pharmacokinetic,
pharmacodynamic, modulated therapeutic half-life, or modulated plasma half-
life relative to the monomeric GH
supergene family nlember: In some embodiments, the GH supergene family meniber
dimers described herein
will modulate the dimerization of the GH supergene family member receptor. In
other embodiments, the GH
supergene family member dimers or multimers described herein will -act as a GH
supergene family member
receptor aritagonist; agonist; or modulator.
1004911 In some embodiments, the- GH supergene family member polypeptides are
linked directly,
including but not:limited to, via an Asn-Lys.amide linkage or Cys-Cys
disulfide linkage. In some embodiments,
the linked GH supergene family member polypeptides, and/or the linked non-GH
supergene family member,
will 'comprise different non-natural `amino acids to facilitate dimerization,
including but not limited to, a first
GH supergene family member,,and/or the linked.non-GH supergene family member,
polypeptide comprising a
carbonyl-containing- non-natural amino acid conjugated to a second GH
supergene family member polypeptide
comprising a hydrazine-containing non-natural amino acid and the polypeptides
are reacted via formation of the
corresponding indole.
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1004921 Alternatively, the two GH supergene fanuly member polypeptides, and/or
the linked non-GH
supergenc family member, are linked via a linker. Any hetero- or homo-
bifunctional linker can be used to link
the two GH supeigene family member, and/or the linked non-GH supergene family
meniber, polypeptides,
which can.have the same or different primary sequence. In some. cases, the
linker used to tether the GH
supergene family member, and/or the linked non-GH supergene family member,
polypeptides together can be a
bifunctional PEG reagent.
1004931 In some embodiments, the methods and compositions described herein
provide for water-
soluble bifunctional Gnkers that have a dumbbell structure that includes: a)
an azide, an alkyne, a hydrazine, a
diamine, a hydrazide, a hydroxylamine, or a carbonyl (including a dicarbonyl)-
containing moiety on at least a
first end of a polymer backbone; and b) at least a second functional group on
a second end of the polymer
backbone. 'fhe second functional group can be the same or different as the
first functional group. The second
functional group, in some embodiments, is not reactive with the first
functional group. The methods and
compositions described herein provide, in some embodiments, water-soluble
compounds that comprise at least
one arm of a branched molecular structure. For example, the branched molecular
structure can be dendritic.
1004941 In some embodiments, the methods and compositions described herein
provide multimers
comprising one or niore GI-I supergene family member formed by reactions with
water soluble activated
polymers that have the structure:
R-(CHaCH2O)n-O-(CHZ)n; X
1004951 wherein n is from about 5 to about 3,000, m is 2-10, X can be an
azide, an alkyne, a hydrazine, a
diamine, a hydrazide, a hydroxylamine, a acetyl, or carbonyl (including a
dicarbonyl)-containing moiety, and R
is a capping group, a functional group, or a leaving group that can be the
same or different as X. R can be, for
example, a functional group selected from the group consisting of hydroxyl,
protected hydroxyl, alkoxyl, N-
hydroxysuccinimidyl ester, 1-benzotriazolyl ester, N-hydroxysuccinimidyl
carbonate, 1-benzotriazolyl
carbonate, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,
methacrylate, acrylamid'e, active sulfone,
amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected
thiol, carboxylic acid, protected
carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,
dithiopyridine, vinylpyridine,
iodoacetamide; epoxide, glyoxals, diones, mesylates, tosylates, and tresylate,
alkene, and ketone. In a further
embodiment, linker groups can be used to link transcription factors. Genes
require multiple transcription factors
to efficiently initiate expression of the encoded protein. Transcription
factors synthesized with non-natural
aniino acids can be linked, via linkers as described above, and used to
enhance artificial activation of targeted
genes. The linked transcription factors can bind target DNA and promote
recruitment of RNA polymerase in the
absence of the nornial activation signal cascade, thus expressing genes
without the required signal. In yet a
further embodiment, ligands for cell receptors can be linked for efficient
activation of the receptors. Platelet-
derived Growth Factor (PDGF) forms dimers in order to bind its receptor. PDGF
which contains non-natural
amino acids can be linked in dimer formation via linkers as described above
and administered to provide
efficient binding of the PDGF receptor. Still further embodiments of linked
proteins include linked antibodies.
Two different antibodies, each specific for unique epitopes on the same or
adjacent =targets. can be linked for
enhanced stimulation, binding, or neutralization. For example, antibodies
specific for two different epitopes
found on gp120 and associated gp4O of HIV can be linked to provide more:
effectiveneutralization of the target.
Similarly, linked antibodies can be used to stimulate cell surface receptors.
For example, antibodies to CD3 as
well as CD4 of the T-cell receptor can be linked to provide the necessary
stimuli for activation of the receptor.
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A further embodiment includes peptides linked to nucleic acid. For example,
ligands for cell receptors or
proteins which bind cell surfaces can be linked to a therapeutic nucleic acid
that is administered to a desired
target. The linked ligand facilitates the uptake of the nucleic acid that is
then expressed within the cell to exert
its therapeutic effect. Similarly, peptides may be linked to nucleic acids to
facilitate packaging or condensation
of the nucleic acid.
1004961 The functional groups on the linker do not have to be identical, nor
do they have to be phenyl
hydrazine groups. The chemistry detailed throughout this specification allows
the design of a linker in which at
least one functional group can form an indole group with a non-natural amino
acid polypeptide; the other
functional groups on the linker, in some. embodiments, utilize other
documented methodologies, including
nucleophile/electrophile based cheniistry.
C. Exanrple ofAdding Functionality: Easing the Isolation Properties of a
Polypept[de
1004971 A naturally-occutring or non-natural amino acid polypeptide may be
difficult to isolate from a sample
for a number of reasons, including but not limited to the solubility or
binding characteristics of the polypeptide.
For example, in the preparation of a polypeptide for therapeutic use; such a
polypeptide may be isolated from a
recombinant system that lias been engineered to overproduce the polypeptide.
However, because of the
solubility or binding characteristics of the polypeptide, achieving a desired
level of purity often proves difficult.
The methods, compositions, techniques and strategies described herein provide
a solution to this situation.
1004981 The methods, compositions, techniques and strategies described herein,
allow production of a
heterocycle-, including a nitrogen-containing heterocycle, containing non-
natural amino acid polypeptide that is
homologous to the desired polypeptide, wherein the heterocycle-, including a
nitrogen-containing heterocycle,
containing non-natural amino acid polypeptide has improved isolation
characteristics. In one embodiment, a
homologous non-natural amino acid polypeptide is produced biosynthetically. In
a further or additional
embodinient, the non-natural amino acid has incorporated into its structure
one of the non-natural amino acids
described herein. In a further or additional embodiment, the non-natural
aniino acid is incorporated at a terminal
or internal position and is further incorporated site specifically.
1004991 In one embodiment, the resulting non-natural amino acid, as produced
biosynthetically, already has the
desired improved isolation characteristics. In further or additional
embodiments, the non-natural amino acid
comprises a heterocycle, including a nitrogen-containing heterocycle, linkage
to a group that provides the
improved isolation characteristics. In further or additional embodiments, the
non-natural amino acid is further
modified to form a modified heterocycle-, including a nitrogen-containing
heterocycle, containing non-natural
amino acid polypeptide, wherein the modification provides a heterocycle,
including a nitrogen-containing
heterocycle, linkage to a group that provides the improved isolation
characteristics. ln some embodiments, such
a group is directly linked to the non-natural amino acid, and in other
embodiments, such a group is linked via a
linker group to the non-natural amino acid. In certain embodiments, such a
group is connected to the non-natural
amino acid by a single chemical reaction, in other embodiments a series of
chemical reactions is required to
connect such a group to the non-natural amino acid. Preferably, the group
imparting improved isolation
characteristics is linked site specifically to the non-natural amino acid in
the non-natural amino acid polypeptide
and is not linked to a naturally occuring amino acid under the reaction
conditions utilized.
1005001 In further or additional embodiments the resulting non-natural amino
acid polypeptide is homologous
to the GH supergene family members, however, the methods, techniques and
composirions described in this
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section can be applied to virtually any other polypeptide which can benefit
from improved isolation
characteristics, including by way of example only, a therapeutic protein.
1005011 In further or additional embodiments, the group imparting improved
isolation characteristics improves
the water solubility of the polypeptide; in other embodiments, the group
improves the binding properties of the
polypeptide; in other embodiments, the group provides new binding properties
to the polypeptide (including, by
way of example only, a biotin group or a biotin-binding group). In embodiments
wherein the group improves
the water solubility of the polypeptide, the group is selected from the water
soluble polymers described herein,
including by way of exarnple only, any of the PEG polymer groups described
herein.
B. Example of Adding Fiuictiojtality: Detecting the Presence of a Polypeptide
1005021 A naturally-occurring or non-natural amino acid polypeptide may be
difficult to detect in a sample
(including an in vivo sample and an in vitro sample) for a number of reasons,
including but not limited to the
lack of a reagent or label that can readily bind to the polypeptide. The
methods, compositions, techniques and
strategies described herein provide a solution to this situation.
[005031 The methods, contpositions, techniques and strategies described herein
allow production of a
heterocycle-, including a nitrogen-containing heterocycle, containing non-
natural amino acid polypeptide that is
homologous to the desired polypeptide, wherein the heterocycle-, including a
nitrogen-containing heterocycle,
containing non-natural anvno acid polypeptide allows the detection of the
polypeptide in an in vivo sample and
an in vitro sample. In one embodiment, a homologous non-natural amino acid
polypeptide is produced
biosynthetically. In a further or additional embodiment, the non-natural amino
acid has incorporated into its
structure one of the non-natural amino acids described herein. In a further or
additioal embodiment, the non-
natural amino acid is incorporated at a terminal or internal position and is
further incorporated site specifically.
1005041 In one embodiment, the resulting non-natural amino acid polypeptide,
as produced biosynthetically,
already has the desired detection characteristics. In further or additional
embodiments, the non-natural amino
acid polypeptide comprises at least one non-natural amino acid selected from
the group consisting of a
carbonyl-containing non-natural amino acid, a hydrazine-containing non-natural
amino acid, including an
indole-containing amino acid to provide improved detection characteristics. In
other embodiments such non-
natural aniino acids have been biosynthetically incorporated into the
polypeptide as described herein. In further
or alternative enibodiments non-natural amino acid polypeptide comprises at
]east one non-natural ainino acid
selected from amino acids of Formula I-XV. In further or alternative
embodiments non-natural amino acid
polypeptide comprises at least on e non-natural amino acid selected from amino
acids of compounds of
structures 1-4. In further or additional embodiments, the non-natural amino
acid comprises an indole linkage
that provides the improved detection characteristics. In further or additional
embodiments, the non-natural
amino acid is further nlodified to form a modified indole-containing non-
natural amino acid polypeptide,
wherein the modification provides an indole-containing linkage to a group that
provides the improved detection
characteristics. In some embodiments, such a group is directly linked to the
non-natural amino acid, and in other
embodiments, such a group is linked via a linker group to the non-natural
aniino acid. In certain einbodiments,
such a group is connected to the non-natural aniino acid by a single chemical
reaction, in other embodinients a
series of chemical reactions is required to connect such a group to the non-
natural amino acid. Preferably, the
group imparting improved detection characteristics is linked site specifically
to the non-natural amino acid in
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the non-natural amino acid polypeptide and is not linked to a naturally
occurring amino acid under the reaction
conditions utilized.
1005051 In further or additional embodiments the resulting non-natural amino
acid polypeptide is homologous
to the GH supergene family members, however, the methods, techniques and
compositions described in this
section can be applied to virtually any other polypeptide which needs to be
detected.in an in vivo sample and an
in vitro sample, including.by way of example only, a therapeutic protein.
1005061 In further or additional enibodiments, the group imparting improved
detection characteristics is
selected froni the group consisting of a label; a dye; an affinity label; a
photoaffinity label; a spin label; a
fluorophore; a radioactive moiety; a moiety incorporating a heavy atom; an
isotopically labeled moiety; a
biophysical probe; a phosphorescent group; a chemiluminescent group; an
electron dense group; a magnetic
group; a chromophore; an energy transfer agent; a detectable label; and any
combination thereof.
1005071 In one embodiment, an antibody is engineered to contain an indole non-
natural amino acid, and the
antibody recognizes a unique antigen on a cancerous cell. After labeling/or
modifying the antibody with the
indole-via methods described herein and purifying the labelled antibody, it is
administered to a subject
suspected of having a cancer that can be recognizcd by the labelled antibody.
Following administration of the
labelled antibody, presence and location of the labelled antibody within the
patient can identify the presence of
cancerous tissues. Administration of the labelled antibody allows for the
detection of the cancer within the
patient, metasteses within the subject, and/or efficacy of treatments for the
cancer within the subject.
1005081 In another embodiment, a peptide that binds to antigens on the surface
of cells is engineered to contain
a dye, inlcuding but not linuted to fluorescent dyes which can be used to
track the peptide following
administration of the peptide to a subject. The dye is attached to the peptide
via the non-natural amino acid
located within the peptide, and the peptide is administered to the subject.
Localization or binding of the peptide
to its ligand(s) is accomplished with documented imaging or detection
techniques.
1005091 In another embodiment is a method for detecting the presence of a
polypeptide in a patient, the method
comprising administering an effective amount of a homologous non-natttral
atnino acid polypeptide comprising
at least one non-natural amino acid having the structures of conipounds 1-4:
H
R K3 AR.~\ N R3 R3 A B af\ \ N Ra
R 3 R- ~/ ~ R,.N RZ n Rs
rH~ 1 RS HRaG R. m~ R
O 2 s
Ra N Rs Rs R
R s
Ra n\ s N Rs
R3
R3 R3 A, R5 R3 A'B'~
B m R Rs
s
Rl~ R, Ra R, ~N RZ
H~ O 3 H R4 0 4 R5 Rs
wherein:
A is optional, and when present is lower alkylene, substituted lower alkylene,
lower cycloalkylene,
substituted lower cycloalkylene, lower alkenylene, substituted lower
alkenylene, alkynylene,
substituted alkynylene, lower heteroalkylene, substituted heteroalkylene,
lower
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heterocycloalkylene, substituted lower heterocycloalkylene, arylene,
substituted arylene,
heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene, or
substituted aralkylene;
B is optional, and when present is a linker, linked at one end to an indole-
containing nioiety, the linker
selected from the group consisting of lower alkylene, substituted lower
alkylene, lower
alkenylene, substituted lower alkenylene, lower.heteroalkylene, substituted
lower
heteroalkylene, - arylene, substituted arylene, heteroarylene, substituted
heteroarylene, -0-,
-O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted
alkylene)-, -S(O)k-
where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -
NS(O)z-, -OS(O)2-,
-C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or
substituted alkylene)-, -
N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-
(alkylene or
substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or-subs6tuted alkylene)-
, -N(R')CO-
(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)kN(R')-, -
N(R')C(O)N(R')-,
-N(R')C(S)N(R')-, -N(R')C(NCN)N(R')-, -N(R')C(NNOZ)N(R')-, -
N(R')C(NCOOR')N(R')-,
-N(R')S(O)kN(R')-, -C(R')=N-, -C(R')=N-N(R')-, -C(R')Z-N=N-, and -C(R'),-N(R')-
N(R')-
and each R' is independently H, alkyl, or substituted alkyl;
R~ is H, an amino protecting group, resin, at least one amino acid,
polvpeptide, or polynucleotide; and
R2 is OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide;
n is 0, I, 2, or 3, and m is 0, 1, 2, or 3, provided that at least one of n or
m is not 0;
wherein, each ring in structures 1, 2, 3, and 4 that has an associated R.
group can contain 0, 1, or 2 Re
groups and each R. is independently selected from the group consisting of H,
halogen, alkyl,
substituted alkyl, -N(R")2, -C(O)R", -C(O)N(R")z, -OR", and -S(O)kR", where.k
is 1, 2, or 3, where
each R" is independently H, alkyl, or substituted alkyl; or when more than one
R. group is present, two
R. may optionally form an aryl, cycloalkyl or heterocycloalkyl;
each of R3.and R4 is independently H, halogen, lower alkyl, or substituted
lower alkyl, or R3 and .R4 or
two R, groups optionally form a cycloalkyl or a heterocycloalkyl;
each R5 is independently H, halogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl,
substituted.alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted
alkylalkoxy,
polyalkylene oxide, substituted polyalkylene oxide,.aryl, substituted
aryl,heteroaryl,
substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted
aralkyl, -(alkylene or
substituted alkylene)-ON(R")2, OH, NH2, CN; NOZ, -(alkylene or substituted
alkylene)-
C(O)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -
C(O)R",
-C(0)2R", or -C(O)N(R")Z, wherein each R" is independently liydrogen, alkyl,
substituted
alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl,
substituted aryl,
heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or
when more than one R"
group is present, two R" optionally form a heterocycloalkyl;
or RS is L-X, where, X is a selected from the group consisting of: a label; a
dye; a polymer; a water-
soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a
cytotoxic
contpound; a drug; an affinity label; a photoaffinity label; a reactive
compound; a resin; a
second protein or polypcptide or polypeptide analog; an antibody or antibody
fragment; a
nietal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a
DNA; a RNA; an
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antisense polynucleotide; a saccharide, a water-soluble dendrimer, a
cyclodextrin, a
biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing
moie.ty; a
radioactive moiety; a novel functional group; agroup that covalently or
noncovalently
interacts with other molecules; a photocaged moiety; an actinic radiation
excitable moiety; a
ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety
incorporating a heavy
atom; a chemically cleavable group; a photocleavable group; an elongated side
chain; a
carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety;
an isotopically
labeled moiety; a biophysical probe; a phosphorescent group; a
chemiluminescent group; an
electron dense group; a magnetic group; an intercalating,group; a chromophore;
an energy
transfer agent; a biologically active agent; a detectable label; a small
molecule; an inhibitory
ribomicleic acid; a radionucleotide; a neutron-capture agent; a derivative of
biotin; quantum
dot(s); a nanotransmitter; a radiotransmitter; an abzyme, an activated complex
activator,-a
vinis, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone,
an antioxidant, an
aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a
mimotope, a receptor,
a reverse micelle, and any combination thereof; and L is optional, and when
present is a linker
selected from the group consisting of alkylene, substituted alkylene,
alkenylene, substituted
alkenylene, -0-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or
substituted
alkylene)-, -S(O)k- where k is 1,2, or 3, -S(O)k(alkylene orsubstituted
alkylene)-, -C(O)-,
-C(O)-(alkylene or substituted alkylene)-, -C(S)-,. -C(S)-(alkylene or
substituted alkylene)-, -
N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-
(alkylene or
substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-
, -N(R')CO-
(alkylene or substituted alkylene)-, -N(R')C(O)O-, -(alkylene or substituted
alkylene)-O-
N=CR'-, -(alkylene or substituted alkylene)-C(O)NR'-(alkylene or substituted
alkylene)-, -
(alkylene or substituted alkylene)-S(O)k-( alkylene or substituted alkylene)-S-
, -(alkylene or
substituted alkylene)-S-S-, -S(O)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-,
-N(R')S(O)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R')2-
N=N-,
and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or
substituted alkyl;
when more than one R$ group is present, two ortho R5 groups can optionally
form a heterocycloalkyl or
an aromatic heterocycloalkyl;
or the -B-indole containing moiety together form a bicyclic or tricyclic
cycloalkyl or heterocycloalkyl
comprising at least one indole portion;
or an active metabolite, salt, or a pharmaceutically acceptable prodrug or
solvate thereof.
1005101 In another enibodiment is a method for detecting the presence of a
polypeptide in a patient, the method
comprising adntinistering an effective amount of a homologous non-natural
amino acid polypeptide comprising
at least one non-natural amino acid having the structures of compounds 1-4,
wherein the at least one non-natural
acid is incorporated at a specific site within the polypeptide. In yet another
embodiment is a method for
detecting the presence of a polypeptide in a patient, the method comprising
administering an effective amount of
-a homologous non-natural amino acid polypeptide comprising at least one non-
natural amino acid having the
stnictures of compounds 1-4, whercin the non-natural amino acid is
incorporated using a translation system. In
another embodiment is a method for detecting the presence of a polypeptide in
a patient, the method comprising
administering an effective amount of a homologous non-natural amino acid
polypeptide comprising at least one
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non-natural amino acid having the structures of compounds 1-4, wherein the non-
natural amino acid is
incorporated into the polypeptide using a translation system and a post
translation modification system. In
another embodiment is a method for detecting the presence of a polypeptide in
a patient, the method comprising
administering an effective=amount.of a homologous non-natural amino acid
polypeptide cotriprising at least one
non-natural amino acid having the structures of compounds 1-4, wherein the at
least one non-natural amino acid
is_stable: in aqueous.solution for at least I month. ln another eniboditnent
is a method for detecting the presence
of a polypeptide in a patient, the method coniprising administeringan
effective amount of a homologous non-
natural amino acid polypeptide comprising at least one non-natural amino acid
having the.stiuctttres of
compounds 1-4, wherein the at least one non-natural amino acid is stable for
at least 2 weeks. In another
embodiment is a method for detecting the presence ofapolypeptide in a patient,
the method-comprising
administering an effective amount of a homologous non-natural aniino acid
polypeptide comprising at least one
non-natural amino acid having the structures of compounds 1-4, wherein the at
least one non-natural amino acid
is stable for at least 5 days.
1005111 In another embodiment is a method for detecting the presence of a
polypeptide in a patient, the method
comprising administering an effective amount of a homologous non-natural amino
acid polypeptide comprising
at least one non-natural amino acid having the structures of compounds 1-4,
wherein the polypeptide is a protein
homologous to a therapeutic protein selected from the group consisting of:
alpha-I antitrypsin., angiostatin,
antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic
factor, atrial natriuretic polypeptide,
atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c,
IP-10, GCP-2, NAP-4, SDF-1,
PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte
chemoattractant protein-1, monocyte
chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte
inflammatory protein-1 alpha,
monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733, HCC1,
T58847, D31065, T64262,
CD40, CD401igand, c-kit ligand, collagen, colony stimulating factor (CSF),
complement factor 5a, complement
inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating
peptide-78, MIP-16, MCP-1,
epidernial growth factor (EGF), epithelial neutrophil activating peptide,
erythropoietin (EPO), exfoliating toxin,
Factor IXjactor VA, Factor VIII, Factor X, fibroblasrgrowth factor (FGF),
fibrinogen, fibronectin, four-helical
bundle protein, G-CSF, glp-l, GM-CSF,;glucocerebrosidase,,gonadotropin, growth
factor, growth factor
receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF),
hirudin, human growth hormone
(hGH), hurnan serutn albuniin, ICAM-1, ICAM-I receptor, LFA-1, LFA-1 receptor,
insulin, insulin-like growth
factor (IGF), IGF-1, IGF-I1, interferon (WN), IFN-alpha, IFN-beta, IFN-gamma,
interieukin (IL), IL-1, IL-2, IL-
3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, (L-11, IL-12, keratinocyte
growth factor (KGF), lactoferrin,
leukenva inhibitory factor, luciferase, neurturin, neutrophil inhibitory
factor (NIF), oncostatin M, osteogenic
protein, oncogene product, paracitonin, parathyroid hormone, PD-ECSF, PDGF,
peptide homione, pleiotropin,
protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,
pyrogenic exotoxin C, pyy, relaxin,
renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble
I-CAM 1, soluble interleukin
receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin,
streptokinase, superantigens,
staphylococcal enterotoxin, SEA, SEB, SECI, SEC2, SEC3, SED, SEE, steroid
hormone receptor, superoxide
dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen
activator, tumor growth factor
(TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis
factor beta, tumor necrosis factor
receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth
factor (VEGF), urokinase,
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mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor,
progesterone receptor, testosterone
receptor, aldosterone receptor, LDL receptor, and corticosterone.
1005121 In yet another enibodiment, an indole-containing moiety is attached to
a peptide, polypeptide, or
protein via non-natural amino acids located within the peptide, polypeptide,
or protein. Appropriately labelled
peptides, polypeptides, or proteins are adniinistered to a desired subject for
detection and imaging via
documented methodologies. Through these labelled peptides,. polypeptides, or
proteins, a variety of diseases,
metabolic pathways, physiological structures or cellular components can be
imaged. By way of example,
fluorescent imaging microscopy can be used to detect the presence of labelled
peptides, polypeptides, or
proteins within a subject.
C. Example of Adding Functionality: I-nproving the Tlrerapeutic Properties of
a Polypeptide
1005131 A naturally-occurring or non-natural amino acid polypeptide will be
able to provide a certain
therapeutic benefit to a patient with a particular disorder, disease or
condition. Such a therapeutic benefit will
depend upon a number of factors, including by way of example only: the safety
profile.of the polypeptide, and
the pharmacokinetics, pharmacologics and/or pharmacodynamics of the
polypeptide (e.g., water solubility,
bioavailability, serum half-life, therapeutic half-life, immunogenicity,
biological activity, or circulation time). In
addition, it may be advantageous to provide additional functionality to the
polypeptide, such as an attached
cytotoxic compound or drug, or it may be desirable to attach additional
polypeptides to form the homo- and
heteromultimers described herein. Such niodifications preferably do not
destroy the activity and/or tertiary
structure of the original polypeptide. The methods, compositions, techniques
and strategies described herein
provide solutions to these issues.
1005141 The methods, compositions, techniques and strategies described allow
production of a heterocycle-,
including a nitrogen-containing heterocycle, containing non-natural amino acid
polypeptide that is homologous
to the desired polypeptide, wherein the heterocycle-, including a nitrogen-
containing heterocycle, containing
non-natural amino acid polypeptide has improved therapeutic characteristics.
In one embodiment, a honiologous
non-natural amino acid polypeptide is produced biosynthetically. In a further
or additional embodiment, the
non-natural amino acid has incorporated into its structure one of the non-
natural amino acids described herein.
In a further or additional embodiment, the non-natural anrino acid is
incorporated at a terminal or internal
position and is further incorporated site specifically.
1005151 In one embodiment, the resulting non-natural amino acid, as produced
biosynthetically, already has the
desired improved therapeutic characteristics. In further or additional
embodiments, the non-natural amino acid
comprises a heterocycle, including a nitrogen-containing heterocycle, linkage
to a group that provides the
improved therapeutic characteristics. In further or additional embodiments,
the non-natural amino acid is further
niodified to form a modified heterocycle-, including a nitrogen-containing
heterocycle, containing non-natural
atnino acid polypeptide, wherein the modification provides a heterocycle,
including a nitrogen-containing
heterocycle, linkage to a group that provides the iniproved therapeutic
characteristics. In some embodiments,
such a group is directly linked to the non-natural amino acid, and in other
embodiments, such a group is liiilced
via a linker group to the non-natural amino acid. In certain embodiments,
sucli a group is connected to the non-
natural amino acid by a single chemical reaction, in other embodintents a
series of chcniical reactions is
required to connect such a group to the non-natural amino acid. Preferably,
the group imparting improved
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therapeutic characteristics is linked site specifically to the non-natural
amino acid in the non-natural amino acid
polypeptide and is not linked to a naturally occurring amino acid under the
reaction conditions utilized.
1005161 In further or additional embodiments the resulting non-natural amino
acid polypeptide is homologous
to the GH supergene family members, however, the methods, techniques and
compositions described in this
section can be applied to virtually any other polypeptide which can benefit
from improved therapeutic
characteristics, including by way of example only, a therapeutic protein.
1005171 In further or additional embodiments, the group imparting improved
therapeutic characteristics
improves the water solubility of the polypeptide; in other embodiments, the
group improves the binding
properties of the polypeptide; in other embodiments, the group provides new
binding properties to the
polypeptide (including, by way of example only, a biotin group or a biotin-
binding group). In embodiments
wherein the group improves the water solubility of the polypeptide, the group
is selected from the water soluble
polyniers described herein, including by way of example only the PEG polymer
groups. In further or additional
embodiments the group is a cytotoxic compound, whereas in other embodiments
the group is a drug. In further
emboditnents the linked drug or cytotoxic compound can be cleaved from the non-
natural amino acid
polypeptide so as to deliver the drug or cytotoxic compound to a desired
therapeutic location. In othe
embodiments, the group is a second polypeptide, including by way of example, a
heterocycle-, including a
nitrogen-containing heterocycle, containing non-natural amino acid
polypeptide, further including by way of
example, a polypeptide that has the same amino acid structure as the first non-
natural amino acid polypeptide.
[005181 In further or additional embodiments, the heterocycle-, including a
nitrogen-containing heterocycle,
containing non-natural amino acid polypeptide is a modified heterocycle-,
including a nitrogen-containing
heterocycle, containing non-natural amino acid polypeptide. In further or
additional embodiments, the
heterocycle-, including a nitrogen-containing heterocycle, containing non-
natural amino acid polypeptide
increases the bioavailability of the polypeptide relative to the liomologous
naturally-occumng amino acid
polypeptide. In further or additional embodiments, the heterocycle-, including
a nitrogen-containing
heterocycle, containing non-natural amino acid polypeptide increases the
safety profile of the polypeptide
relative to the homologous naturally-occurring atnino acid polypeptide. In
further or additional embodinients,
the heterocycle-, including a nitrogen-containing heterocycle, containing non-
natural amino acid polypeptide
increases the water solubility of the polypeptide relative to the homologous
naturally-occurring amino acid
polypeptide. In further or additional embodiments, the heterocycle-, including
a nitrogen-containing
heterocycle, containing non-natural amino acid polypeptide increases the
therapeutic half-life of the polypeptide
relative to the homologous naturally-occurring amino acid polypeptide. In
further or additional embodiments,
the heterocycle-, including a nitrogen-containing heterocycle, containing non-
natural amino acid polypeptide
increases the serum half-life of the polypeptide relative to the homologous
naturally-occurring amino acid
polypeptide. In further or additional embodiments, the heterocycle-, including
a nitrogen-containing
heterocycle, containing non-nahiral amino acid polypeptide extends the
circulation time of the polypeptide
relative to the. homologous naturally-occurring amino acid polypeptide. In
further or additional embodiments,
the heterocycle-, including a nitrogen-containing heterocycle, containing non-
natural amino acid polypeptide
modulates the biological activity=of the polypeptide relative to the
homologous naturally-occurring amino acid
polypeptide. In further or additional embodiments, the heterocycle-, including
a nitrogen-containing
heterocycle, containing non-natural amino acid polypeptide modulates the
immunogenicity of the polypeptide
relative to the homologous naturally-occurring amino acid polypeptide.
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XI. Therapeutic Uses of Modified Polypeptides
1005191 For convenience, the modified or unmodified non-natural. amino acid
polypeptides described in this
section have been described generically and/or with specific examples.
However, the modified or unmodified
non-natural amino acid polypeptides described in this section should not. be
limited to just the generic
descriptions or specific example provided in this section, but rather the
modified or unmodified non-natural
amino acid polypeptides described in this section apply equally well to all
modified or unmodified non-natural
amino acid polypeptides comprising at least one non-natural amino acid which
falls within the scope of
Formulas I-XV and compounds of structures 1-4, including any sub-formulas or
specific compounds that fall
within the scope of Formulas I-LXV, that are described:in the specification,
claims and figures herein.
1005201 The modified or unmodified non-natural amino acid polypeptides
described herein, including homo-
and hetero-multimers thereof find tnu7tiple uses, including but not limited.
to: therapeutic, diagnostic, assay-
based, industrial, cosmetic, plant biology, environmental, energy-production,
consumer products, and/or
military uses. As a non-liiniting illustration, the following therapeutic uses
of modified or unmodified non-
natural amino acid polypeptides are provided.
1005211 The modified or unmodified non-natural amino acid polypeptides
described herein are useful for
treating a wide range of disorders, conditions or diseases. Administration of
the modified or unmodified non-
natural amino acid polypeptide products described herein results in any of the
activities demonstrated by
comnlercially available polypeptide preparations in humans. Average
qttantities of the modified or unmodified
non-natural amino acid polypeptide product nzay vary and in particular should
be based upon the
recommendations and prescription of a qualified physician. The exact amount of
the modified or unmodified
non-natural amino acid polypeptide is a matter of preference subject to such
factors as the exact type of
condition being treated, the condition of the patient being treated, as well
as the other ingredients in the
composition. The amount to be given may be determined based upon therapy with
the modified or unmodified
non-natural amino acid polypeptide.
A. rldarinistration and Pharneaceutical Compositions
1005221 The non-natural amino acid polypeptides, modified or unmodified, as
described lierein (including but
not liniited to, synthetases, proteins comprising one or more non-natural
anuno acid, etc.) are optionally
employed for therapeutic uses, including but not lirnited to, in combination
with a suitable pharmaceutical
carrier. Such compositions, for example, comprise a therapeutically effective
amount of the non-natural amino
acid polypeptides, modified or unmodified, as described herein, and a
pharmaceutically acceptable carrier or
excipient. Such a carrier or excipient includes, but is not limited to;
saline, buffered saline, dextrose, water,
glycerol, ethanol, and/or combinations thereof. The fornlulation is tnade to
suit the mode of administration. In
general, methods of adnunistering proteins can be applied to administration of
the non-natural amino acid
polypeptides, modified or unmodified, as described herein.
1005231 Therapeutic compositions comprising one or more of the non-natural
amino acid polypeptides,
tnodified or unniodified, as described herein are optionally tested in one or
more appropriate in vitro and/or in
vivo animal models of disease, to confirm efficacy, tissue metabolism, and to
estimate dosages, according to
documented methodologies. In particular, dosages can be initially determined
by activity, stability or other
suitable measures of non-natural to natural amino acid homologues (including
but not limited to, cotnparison of
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a polypeptide modified to include one or more non-natural amino acids to a
natural amino acid polypeptide),
i.e., in a relevant assay.
1005241 Administration is by any of the routes normally used for introducing a
molecule into ultimate contact
with blood or tissue cells. The non-natural amino acid polypeptides, modified
or unmodified, as described
herein, are administered in any suitable manner, optionally with one or more
pharniaceutically acceptable
carriers. Suitable methods of administering the non-natural amino acid
polypeptides, modified or unmodified, as
described herein, to a patient are available, and, although more than one
route can be used to administer a
particular composition, a particular route can often provide a more immediate
and more effective action or
reaction than another route.
1005251 Pharmaceutically acceptable carriers are detemiined in part by the
particular composition being
administered, as well as by the particular niethod used to administer the
composition. Accordingly, there is a
wide variety of suitable formulations of pharmaceutical compositions described
herein.
1005261 The non-natural anuno acid polypeptides described herein and
compositions comprising such
polypeptides may be administered by any route suitable for proteins or
peptides, including, but not limited to
parenterally, e:g: injections including, but not limited to, subcutaneously or
intraveriously or any other form of
injections or.infusions. Polypeptide pharmaceutical compositions (including
the various non-riatural amino acid
polypeptides described herein) can be administered by a number of routes
including, but not limited.,to oral,
intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous,
topical, sublingual, or rectal means.
Compositions comprising non-natural amino.acid polypeptides, modified or
unmodified, as described herein,
can also be administered via liposomes. The non-natural amino acid
polypeptides described herein may be used
alone or in combination with other suitable components, including but not
limited to, a pharmaceutical carrier.
1005271 The non-natural amino acid polypeptides, modified or unmodified, as
described herein, alone or in
combination with other suitable components, can also be made into aerosol
formulations (i.e., they can be
"nebulized") to be administered via inhalation. Aerosol formulations can be
placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
1005281 Formulations suitable for parenteral administration, such as, for
example, by intraarticular (in the
joints), intravenous, intramuscular, intradermal, intraperitoneal, and
subcutaneous routes, include aqueous and
non-aqueous, isotonic sterile injection solutions, which can contain
antioxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the. intended
recipient, and aqueous and non-
aqueous sterile suspensions thatcan include-suspending agents,,solubilizers,
thickening agents, stabilizers, and
preservatives. The formulations of packaged nucleic acid can be presented in
unit-dose or multi-dose sealed
containers, such as ampules and vials.
1005291 Parenteral administration and'intravenous administration are.
preferred methods of adnunistration. In
particular, the routes of administration already in use for natural anuno acid
homologue therapeutics (including
but not limited to, those typically used for EPO, IFN, GH, G-CSF, GM-CSF,
IFNs, interleukins, antibodies,
and/or any other pharmaceutically delivered protein), along with formulations
in current use, provide preferred
routes of administration and formulation for the non-natural amino acid
polypeptides, modified or unmodified,
as described herein.
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1005301 The. dose adtninistered to a patient, in the context compositions and.
methods described herein, is
sufficient to have a beneficial therapeutic response in the, patient over
time. The dose is deternuned by the
efficacy of the particular formulation, and the activity, stability or serum
half-life of the non-natural amino acid
polypeptides, modified or unmodified, employed and the condition of the
patient, as well as the body weight or
surface area of the patient to be treated. The size of the dose is also
determined by the existence, nature, and
extent of any adverse side-effects-that accompany the administration of a
particular formulation, or the like in a
particular patient.
1005311 Tn determining the effective amount of the formulation to be
administered in the treatment or
prophylaxis.of disease (including but not limited to, cancers, inherited
diseases, diabetes, AIDS, or the like), the
physician evaluates circulating plasnia levels, formulation toxicities,
progression of the disease, and/or where
relevant, the production of anti-non-natural amino acid polypeptide
antibodies.
1005321 The dose adniinistered, for example, to a 70 kilogram patient, is
typically in the range equivalent to
dosages of currently-used therapeutic proteins, adjusted for the altered
activity or serum half-life of the relevant
composition. The pharmaceutical formulations described herein can supplement a
variety of therapies, including
antibody administration, vaccine administration, administration of cytotoxic
agents, natural amino acid
polypeptides, nucleic acids, nucleotide analogues, biologic response
modifiers, and the like.
1005331 For adnunistration, the pharmaceutical fotYrtulations described herein
are administered at a rate
determined by the LD-50 or ED-50 of the relevant formulation, and/or
observation of any side-effects of the
non-natural amino acid polypeptides, modified or unmodified, at various
concentrations, including but not
limited to, as applied to the mass and overall health of the patient.
Administration can be. accomplished via
single or divided doses.
1005341 If a patient undergoing infusion of a formulation develops fevers,
chills, or muscle aches, he/she
receives the appropriate dose of aspirin, ibuprofen, acetaminophen or other
pain/fever controlling drug. Patients
who experience reactions to the infusion such as fever, muscle aches, and
chills are. premedicated 30 minutes
prior to the future infusions with either aspirin, acetatninophen, or,
including but not limited to,
diphenhydramine. Meperidine is used for more severe chills and muscle aches
that do not quickly respond to
antipyretics and antihistamines. Cell infusion is slowed or discontinued
depending upon the severity of the
reaction.
1005351 Non-natural amino acid polypeptides, modified or unmodified, as
described herein, can be
administered directly to a mammalian subject. Administration is by any of the
routes normally used for
introducing a polypeptide to a subject. The non-natural amino acid
polypeptides, modified or unmodified, as
described herein, include those suitable for oral, rectal, topical, inhalation
(including but not limited to, via an
aerosol), buccal (including. but not limited to, sub-lingual), vaginal,
parenteral (including but not limited to,
subcutaneous, intramuscular, intradermal, intraarticular, intrapleural,
intraperitoneal, inracerebral, intraarterial,
or intravenous), topical (i.e_ both skin and mucosal surfaces, including
airway surfaces) and transdermal
administration, although the.most suitable route in any given case will depend
on the nature and severity of the
condition being treated. Administration can be either local or' systemic. T7le
formulations can be presented in
unit-dose or multi-dose sealed containers, such as ampoules and vials. The non-
natural amino acid polypeptides,
modified or unmodified, as described herein, can be prepared in a mixture in a
unit dosage injectable form
(including but not limited to, solution, suspension, or emulsion) with a
pharmaceutically acceptable carrier. The
non-natural amino acid polypeptides, modified or unmodified, as described
herein, can also be administered by
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continuous infusion (using, including but not limited to, minipumps such as
osmotic pumps), single bolus or
slow-release depot.formulations.
1005361 Formulatioils suitable for administration include aqueous and:non-
aqueous solutions, isotonicsterile
solutions, which can;contain antioxidants, buffers, bacteriostats, and solutes
that render the formulation isotonic,
and aqueous and non-aqueous sterile suspensions that can include suspending
agents, solubilizers, thickening
agents, stabilizers, and preservatives. Solutions and.suspensions can
be.prepared from sterile:powders, granules,
and tablets of the kind previously described.
1005371 Freeze-drying is a commonly employed technique for presenting proteins
which serves to remove
water from the, protein preparation of interest. Freeze-drying, or
lyophilization, is a process by which the
material to be dried is first frozen and then the ice or frozen solvent is
removed by sublimation in a vacuum
environmerit. An excipient may be included in pre-lyophilized fornwlations to
enhance stability during the
freeze-drying process and/or to improve stability of the lyophilizcd product
upon storage. Pikal, M. Biopharm.
3(9) 26-30 (1990) and Arakawa et al. Pharm. Res. 8(3):285-291 (1991).
1005381 The spray drying of phannaceuticals is documented. For example, see
Broadhead, J. et al., "The Spray
Drying of Pharmaceuticals," in Drug Dev. Ind. Pharm, 18 (11 & 12), 1169-1206
(1992). In addition to small
molecule pharmaceuticals, a variety of biological materials have been spray
dried and these include: enzymes,
sera, plasma, micro-organisms and yeasts. Spray drying is a useful technique
because it can convert a liquid
pharmaceutical preparation into a fine, dustless or agglomerated powder 'in a
one-step process. The basic
technique comprises the following four.steps: a) atomization of the feed
solution into a spray; b) spray-air
20. contact; c) drying of the spray; and d) separation of the dried product
from the drying air. U:S.. Patent Nos.
6,235,710 and 6,001,800, which. are herein incorporated by reference for the
aforementioned disclosure,
describe the: preparation of recombinant erythropoietin by spray drying.
1005391 The pkiarmaceutical compositions described herein may 'comprise
apharmaceutically acceptable
carrier, excipient or stabilizer. Pharmaceutically acceptable carriers are
deternuned in part by the particular
composition being administered, as well as by the particular method used to
administer the composition.
Accordingly, there is a wide variety of suitable formulations of
pharmaceutical compositions (including optional
pharmaceutically acceptable carriers, excipients, or stabilizers) for the non-
natural amino acid polypeptides,
niodiGed or unmodified, described herein, (see, for example, in Remington: The
Science and Practice of
Phannacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover,
John E., Remington's
Pharmaceutical.Sciences, Mack Publishing Co., Easton, Pennsylvania 19.75;
Liberman, H.A. and Lachinan, L.,
Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and
Pliarmaceutical Dosage Forms
and Drug Delivery Systenis, Seventh Ed. (Lippincott Willianis & Wilkins,
1999)). Suitable carriers include
buffers containing succinate, phosphate, borate, HEPES, citrate, imidazole,
acetate; bicarbonate, and other
organic acids; antioxidants including but not limited to, ascorbic acid; low
molecular weight polypeptides
including but not limited to those less than about 10 residues; proteins,
including but not limited to, serum.
albumin, getatin, or immunoglobulins; hydrophilic polymers including but not
limited to, polyvinylpyrrolidone;
amino acids including but not linvted to, glycine, glutamine,. asparagine,
arginine, histidine or histidine
derivatives, methionine, glutamate, or lysine; monosaccharides, disaccharides,
and other carbohydrates,
including but not limited to, trehalose, sucrose, glucose, mannose, or
dextrins; chelating agents including but not
limited to, EDTA and edentate disodium; divalent metal ions including but not
limited to,, zinc, cobalt; or
copper; sugar alcohols including but not limited to, mannitol or sorbitol;
salt-forming'counter ions including but
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not limited to, sodium; and/or nonionic surfactants, including but not limited
to TweenTM (including but not
limited to, Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20),
PluronicsT"' and other pluronic acids,
including but not limited to, and other pluronic acids, including but not
limited to, pluronic acid F68 (poloxamer
188), or PEG. Suitable surfactants include for example but are not limited to
polyethers based upon
poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., (PEO-
PPO-PEO), or poly(propylene
oxide)-poly(ethylene oxide)-poly(propylene oxide), i.e., (PPO-PEO-PPO), or a
combination thereof. PEO-PPO-
PEO and PPO-PEO-PPO are commercially available under the trade names
PluronicsTM, R-PluronicsTM,
TetronicsTM and R-TetronicsTM (BASF Wyandotte Corp., Wyandotte, Mich.) and are
further described in U.S.
Pat. No. 4,820,352 incorporated herein in its entirety by reference. Other
ethylene/polypropylene block
polymers may be suitable surfactants. A surfactant or a combination of
suifactants may be used to stabilize
PEGylated non-natural amino acid polypeptides against.one or more stresses
including but not limited to stress
that results frotn agitation. Some of the above may be referred to as "bulking
agents." Some may also be
referred to as "tonicity modifiers." Antimicrobial preservatives may also .be
applied for product stability and
antimicrobial effectiveness; suitable preservatives include but are not
liniited to, benzyl alcohol, benzalkonium
chloride, metacresol, methyl/propyl parabene, cresol, and phenol, or a
combination thereof.
1005401 The non-natural amino acid polypeptides, modified or unmodified, as
described herein, including those
linked to water soluble polymers such as PEG can also be administered by or as
part of sustained-release
systems. Sustained-release compositions include, including but not limited to,
semi-permeable polymer inatrices
in the form of shaped articles, including but not limited to, films, or
microcapsules. Sustained-release matrices
include from biocompatible materials such as poly(2-hydroxyethyl methacrylate)
(Langer et al., J. Bianed.
Mater. Res., 15: 267-277 (1981); Langer, Clieui. Tech., 12: 98-105 (1982),
ethylene vinyl acetate (Langer et al.,
supra) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988), polylactides
(polylactic acid) (U.S. Patent No.
3,7731919; EP 58,481), polyglycolide (polymer of glycolic acid), polylactide
co-glycolide (copolymers of lactic
acid and glycolic acid) polyanhydrides, copolymers of L-glutamic acid and
gamma-ethyl=L-glutamate (U.
Sidman et al.., Biopolymers, 22, 547-556 (1983), poly(ortho)esters,
polypeptides, hyaluronic acid, collagen,
chondroitin sulfate, carboxylic acids, fatty acids, phospholipids,
polysaccharides, nucleic acids, polyamino
acids, amino acids such as phenylalaiiine, tyrosine,. isoleucine,
polynucleotides, polyvinyl propylene,
polyvinylpyrrolidone and silicone. Sustained-release compositions also include
a liposomally entrapped
compound. Liposomes containing the compound are prepared by methods such as:
DE 3,218,121; Eppstein et
a1., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc.
Natl. Acad. Sci. U.S.A., 77: 4030-
4034 (1980); EP 52,322; EP 36,676; EP 143,949; EP 142,641; Japanese Pat.
Appln. 83-1 1 8008; U.S. Pat. Nos.
4,485,045, 4,619,794, and 4,544,545; and EP 102,324.
1005411 Liposonially entrapped polypeptides can be prepared by methods
described in, e.g., DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et
al., Proc. Natl. Acad. Sci. U.S.A.,
77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; Japanese
Pat. Appln. 83-118008; U.S.
Patent Nos. 4,485,045, 5,021,234, and 4,544,545; and EP 102,324. Composition
and size of liposomes are
documented methodologies. Some examples of liposomes as described in, e.g.,
Park JW, et al., Proc. Natl.
Acad. Sci. USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D (eds):
MEDICAI. APPLICATIONS OF
LiPosoMEs (1998); Drunimond DC, et al., Liposomal drug delivery systems for
cancer therapy, in Teicher B
(ed): CANC6R DRUG DISCOVERY AND DrVELOPMENT (2002); Park JW, et al., Clin.
Cancer Res. 8:1172-1181
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(2002); Nielsen UB, et al., Biochim. Biophys: Acta 1591(l or 2):109-118
(2002); Mamot C, et al., Cancer Res.
63:3154-3161 (2003).
The dose administered to a patient in the context of the compositions,
formulations and methods described
herein, should be sufficient to cause a beneficial response in the subject
over time. Generally, the total
pharmaceutically effective amount of the non-natural amino acid polypeptides,
modified or unmodified, as
described herein, administered parenterally per dose is in the range of about
0.01 g/kg/day to about 100 g/kg,
or abotit 0.05 mg/kg to about 1 mg/kg, of patient body weight, although this
is subject to therapeutic discretion.
The frequency of dosing is also subject to therapeutic discretion, and may be
more frequent or less frequent than
the commercially available products approved for use in humans. Generally, a
polymer:polypeptide conjugate,
including by way of example only, a PEGylated polypeptide, as described
herein, can be administered by any of
the routes of administration described above.
XII. Structure-Function Relationship of Modified Polypeptides
1005421 The non-natural amino acid polypeptides, modified or unmodified, as
described herein (including but
not limited to, synthetases, proteins comprising one or more non-natural amino
acids, etc.) will confer different
physical and chemical characteristics on the polypeptide in which it resides.
The usefulness of such
characteristics will depend upon the structure of the inon-natural anuno acid,
the structure of the modification on
the non-natural amino acid, or both, and can be evaluated via experimental
models that assess structure-function
relationships of test polypeptides.
1005431 In any given experimental model, a non-natural amino acid is
substituted for a natural amino acid in a
desired polypeptide or protein. After expression of the non-natural amino acid
containing peptide or protein, the
protein is derivatized with a library of alternate R groups. These R groups
are reacted with the non-natural
amino acid contained within the polypeptide or protein. The library of R
groups is chosen by their structural or
chemical similarity to the R-group of the replaced amino acid. Following the
addition of novel R groups to the
non-natural amino acid within the protein, the protein is then screened for
function or activity within the
appropriate test system. By way of example, phenylalanine is replaced with a
non-natural aniino acid within a
protein. A library of alternative R. groups with similar characteristics to
the R group of phenylalanine are then
added to the non-natural amino acid. A single altentative R group is added to
a non-natural R groups added
include rings, hetero-rings, conjugated rings, or other chemical moieties that
confer, but not limited to, similar
chemical and structural characteristics. The derivitized protein is then
screened for function or functions related
to the addition of the newly substituted non-natural amino acid by testing in
an appropriate experimental model.
Examples of experimental models include, but are not limited to, based assays,
cell free assays, cell-based
assays, tissue culture models, and animal models.
1005441 In a further embodiinent, indoles are substituted on the non-natural
amino acid for pharmacophore
activity in drug discovery or as fluorescent cores useful in detection. To
facilitate such addition, indole-based R
groups or R groups suitable for indole synthesis are added to the non-natural
amino acid by conducting indole
formations in aqueous buffers at room temperature with an optomized two-step
reaction. Following this
reaction, the derivitized'protein is screened for its desired activity.
1005451 By way of example, the effect of non-natural amino acid substitutions
in the acid alpha-glucosidase
enzyme (GAA) on alleviation of Pompe disease can be evaluated in a mouse model
for Pompe disease. A
library of GAA molecules which contain various amino acid substitutions at
chosen sites within the enzyme can
be created and expressed via the methods and compositions disclosed herein.
The non-natural amino acid
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containing enzymes can thcn be evaluated for their activity in a mouse model
for Pompe disease (mice which
are bred to be genetically difficient for GAA (GAA-/-)), either in unmodified
or post-translationally modified
forms as disclosed herein. The non-natural amino acid containing enzymes can
be administered intravenously,
orally, or any other route of administration that allows for efficient protein
transport and absorption. Efficacy of
administration, enzyme half-life, and alleviation of Pompe disease can be
evaluated, for example, by
measurements of glycogen degredation and/or clearance in the mice, assessment
of serum levels of GAA,
changes or reduction in cardiomegaly, cardio myopathy, skeletal niyopathy.
1005461 The modified or unmodified non-naturalamino acid polypeptides
described herein are useful in a wide
range of industiral applications. Use,of the modified or unniodified non-
natural amino acid polypeptide:products
described herein results in any of the activities demonstrated by
conunercially available polypeptide
preparations in industrial applications.
1005471 By way of example, enzymes for the production of ethanol can be:
modi6ed with non-natural amino
acids and assayed for changes iin function:.A library of alcohol dehydrogenase
II and pyruvate decarboxylase
enzymes which contain various non-natural amino acid substitutions can be
created and expressed via the
niethods and compositions disclosed herein. The non-natural aniino acid
modified enzymes can then be
screened for changes in their efficiency of ethanol production conferred.by or
as a result of the non-natural
amino acid substitions. Increases in, but not limited to, affinity for
substrate and rate of conversion can be
screened by documented methodologies, and applied to the industrial production
of ethanol.
1005481 Further examples of industrial applieatibn of the. methods and
compositions disclosed herein include
environmental clean-up of herbicides and pesticides. The removal of a commonly
used herbicide, atrazine, from
contaminated soil is facilitated by enzymes that metabolize the atrazine, thus
rendering it non-toxic. A library of
modified antrazine chlorohydrolase enzymes which contain non-natural amino
acid substitutions can be created
and expressed via the methods and compositions disclosed herein. The library
of non-natural amino acid
modified atrazine cholorhydrolase enzymes can then be screened for changes in
ability to dechlorinate atrazine
found in the environment as well as any new modes of atrazine metabolism
conferred by or as a result of the
non-natural amino-acid substitutions. As described previously, changes in
enzyme efficiency can be assessed
via documented methodologies; including but not liniited to, increases in
metabolism of atrazine or
intermediates.
EXAMPLES
H
N-NH2
Example 1 Synthesis of H2N COOH
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1005491 The synthesis used is described in the following reaction scheme:
ac ~
~
~ N~ AciO.~. ~ N.N.AC NB3.AIBN I~ N.N.Ac
~/ B k ~ ~ Ac CG< & Ac
A,~,~ (COZEI EIONa; EtOH
" -' ~COiEt 3596 for 2 steps
~.{ AC
N-NHZ N;N:Ac
FCI. diamne
~ i Ac
81%
Cp:Et
HpN COpH AcHN ~Et
AC
N.NAc
~
a) Synthesis of
1005501 To a solution of 1-p-tolylhydrazine (5.0 g, 31 mmol) irrpyridine (50
mL) at 0 C was added Ac20 (30
mL, 318 mmol)). The nuxture was stirred at room temperature overnight and
quenched with MeOH (100 mL).
After the solvent was renioved in vacuo, the residue was purified by flash
chromatography (silica, 20-50%
EtOAc/hexanes) to afford a colorless oil (6.72. g, 87%): 'H NMR (500 MHz,
CDCI,) 8 7.28 (d, J= 8.4 Hz, 2H),
7.24 (d, J= 8.4 Hz, 2H), 2.47 (s, 6H), 2.40 (s, 3H), 2.14 (s, 3H); 13C NMR
(125 MHz, CDCI3) S 171.8, 169.5,
139.1, 138.8, 130.4, 126.4, 25.4, 22.3, 21.3.
Ac
N,N.Ac
b) Synthesis of Br ~Z
[005511 To a solution of N',N'-diacetyl-N-p-tolylacetohydrazide (6.4 g, 25.8
mmol) in CCh (300 mL) was
added N-bromo succinimide (5.1 g, 28.7 mmol). The mixture was heated at
reflux. 2,2'-Azobisisobutyronitrile
(AIBN, 0.2 g, 1.2 mmol) was added. The resultant mixture was stirred at reflux
for 36 h and cooled to room
temperature. The mixtuie was washed with H20 and brine, dried over anhydrous
Na2SO4i filtered and
concentrated to afford bromide (8.62 g) as a brown oil. The crude product was
directly used for the next step
without purification.
Ac
F N.N.Ac Ac
c) Synthesis of ACHN co2Et
1005521 To a solution of'EtONa (2.3 g, 32.1 nimol) in EtOH (80 mL) was added
diethyl 2-acetamidomalonate
(6.3 g, 29.0 mmol). The resultant mixture was stirred at. 0 C for 20 min.
N',N'-diacetyl-N-(4-
(bromomethyl)phenyl)acetohydrazide (8.62 g, 26.4 nunol) was added in one
potion. The mixture was heated at
80 C overnight and cooled to room temperature. Citric acid (10 g, 50 mmol)
was added to the reaction mixture.
After most solvent was removed, the residue was diluted with EtOAc (500 mL).
The mixture was washed with
H20 and brine, dried over anhydrous NaZSO4, filtered and concentrated. The
residue was purified by flash
chromatography (silica, 15-80% EtOAc/hexanes) to afford diethyl 2-(4-
(ace(amido)benzyl)-2-
acetamidotnalonate (4.17 g, 35% for two steps) as a yellow oil: 11=1 NMR (500
MHz, CDC13) S 7.23 (d, J = 8.0
Hz, 2H), 7.03 (d, J = 8.0 Hz, 2H), 6.57 (s, I H.), 4.29-4.20 (m, 4H), 3.65 (m,
2H), 2.41 (s, 6H), 2.08 (s, 3H), 2.01
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WO 2008/077079 PCT/US2007/088011
(s, 3H), 1.27 (t, J= 3.6 Hz, 6H); "C NMR (125 MHz, CDC13) 5 171.7, 169.3,
169:2, 167.4,,140.3, 136.4, 131.3,
126.2, 67.2, 63.0, 37.4, 25.3,,23.2, 22.3, 14.2.
H
N-NH2
d) Synthesis of H2N COOH
1005531 To a solution of diethyl 2-(4-(acetaniido)benzyl)-2-acetamidomalonate
(572 mg, 1.24 mmol) in
dioxane (15 mL) was added HCI (12 N, 15 mL). The resultant mixture was heated
at reflux overnight and
concentrated in vacuo. To the residue was added MeOH (1 ml:.). Ether (200 mL)
was added to precipitate the
product (231 mg, 81%) as a solid: 'H NMR (500 MHz, D20) 8 7.28 (d, J = 8.5 Hz,
2H), 7.00 (d, J = 8.5 Hz,
2H), 4.21 (dd, J = 7.4, 5.7 Hz, IH), 3.26 (dd, J = 9.2, 5.7 Hz, 1 H), 3.15
(dd,-J = 14.7, 7.4 Hz, 1H); 13C NMR
(125 MHz, D20) 5 171.5, 142.9, 130.3, 129.0, 115.7, 54.1, 34.7.
Example 2
1005541 The synthesis of this carbonyl-containing amino acid follows standard
methodology.
"lO
H2N COOH
Example 3
1005551 The synthesis of this carbonyl-containing amino acid follows standard
methodology.
O
H2N COOH
Example 4: Reaction between an aryl hydrazine and an aldehyde
1005561 Presented in Figures 3, 4, 5, 6, and 7 is a niodel reaction between
two molecules to form an indole.
Either of the two starting materials can be present as a sidechain on a non-
natural amino acid polypeptide
provided that the other reagent represents a functional group on a compound
for derivatizing the aforementioned
sidechain. That is, the sidechain on a non-natural amino acid polypeptide can
be an aryl hydrazine group, and
the reactive moiety is an aldehyde connected to a water-soluble polymer, at
least one amino acid or a detectable
label. Conversely, the sidecliain on a non-natural amino acid polypeptide can
be an aldehyde group, and the
reactive moiety is an aryl hydrazine connected to a water-soluble polymer, at
least one amino acid or a
detectable label.
pH 3.6
_ + NNH2 30 H H
4`~
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WO 2008/077079 PCT/US2007/088011
Exaniple 5: Reaction between an aryl hydrazine and an ketone
1005571 Presented in Figures 8, 9, 10, 11, 13 and 14 is a model reaction
between two molecules to form an
indole. Eitiier of the two starting materials can be present as a sidechain on
a non-natural amino acid
polypeptide provided that the other reagent represents a functional group on a
compound for derivatizing the
aforementioned. sidechain. That is, the sidechain on a non-natural antino acid
polypeptide can be an aryl
hydrazine group, and the reactive moiety is a ketone connected to a water-
soluble polymer, at least one amino
acid or a. detectable label. Conversely, tlie, sidechain on a non=natural:
amino acid polypeptide can be a ketone
group, and the reactive moiety is an aryl hydrazine connected to a water-
soluble polymer, at least one amino
acid or a detectable label.
Example 6: Urotensin Analogs Containing an Aryl Hydrazine Non-Natural Amino
Acid
1005581 Presented in Figure 20 is a Urotensin analog that includes, at the "X"
position, a non-natural amino
acid with an aryl hydrazine sidechain. The compound was chemically synthesized
using standard techniques. It
is to be understood that the sidechain alternatively can include a ketone, an
aldehyde, or a protected carbonyl
sidecliain (show-n schematically in Figure 21).
Example 7: Indole Formation for the Derivatization of Non-Natural Amino Acids
1005591 Presentcd in Figures 20, 21, 22, 24, 28 and 29 are examples of how the
formation of indole moieitics
from the reaction of aryl hydrazines with carbonyl groups can be used to
derivatize non-natural amino acid
polypeptides. The protein `cartoon' shown in these figures can be urotensin,
human growth hormone, insulin, an
antibody, a kinase, erythropoietin, or any other polypeptide or protein
described herein or available in the
literature.
Example 8
1005601 This example details cloning and expression of a modified polypeptide
in E. coli. An introduced
translation system that comprises an brtliogonal tRNA (O-tRNA) and an
orthogonal aminoacyl tRNA synthetase
(O-RS) is used to express the polypeptide containing anon-natural amino acid.
The O-RS. preferentially
aminoacylates the O-tR1NA with a non-natural amino acid. In turtrn the
translation system inserts the non-natural
amino acid into the polypeptide, in response to an encoded selector codon.
Amino acid and polynucleotide
sequences of.O-tRNA and O-RS useful for'the incorporation of non-natural amino
acids are described in U.S.
Patent application serial no. 10/126,927 entitled "In Vivo Incorporation of
Unnatural Amino Acids" and U.S.
Patent application serial no. 10/126,931 entitled "Methods and Compositions
for the Production of Orthogonal
tRNA-Aniinoacyl tRNA Synthetase Pairs," which are incorporated by reference
herein.
SEQ ID NO:1 pq, janna.schii n1tRNA~uA tRNA
SEQ ID NO:2 HLAD03; an o timized antber suppressor tRNA tRNA
SEQ ID NO:3 HL325A; ait o timized AGGA ranreshi t su ressor tRNA tRNA
SEQ ID NO:4 Aminoacyl tRNA syn[hetase for tlie incorporation of p-azido-L-
phenylalanine RS
Az-PheRS(6)
SEQ ID NO:5 Aminoaevl tRNA synthetase for the incorporatiaz of p-benzoyl-L
phenylalanine RS
B aRS l
SEQ ID NO:6 Aminoacyl IRNA synt/ietase_/'or the incorporation of propargyl-
phenylalanine RS
Prv ar l-PheRS
SEQ ID NO:7 Anzinoacyl tRNA synthetase for the incorporation of propargyl-
pheny/alanine RS
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Pro ar 1-Phe.RS
SEQ ID NO:8 Aminoacyl tRNA synthetase jor the incotporcitinn ojpropargyl,-
phenylalanine RS
Pro ar 1-PheRS
SEQ ID NO:9 Aminoacyl tRNA synthetase for the incorporation of p-
azidophenylalanine RS
Az-PheRS I
SEQ ID NO: 10 Aminoacyl tRNA synthetase for the incorporation of p-azido
phenylalanine RS
p-Az-PheRS(3)
SEQ ID NO:11 Aminoacyl tRNA synthetase for the incorporation of p-azido
phenylalcmine RS
Az-PheRS(4
SEQ ID NO: 12 Atninoacy! IRNA synthetase for the incorporation of p-azido
phenylalanine RS
-Az-PheRS(2)
SEQ ID NO: 13 Aminoacyl tRNA synthetase for the incorporation of p-azido
phenylalanine RS
LWI
SEQ ID NO: 14 Aminoacvl tRNA synthetase for the incorporation of p-azido
phenylalanine RS
LWS
SEQ ID NO: 15 Arninoacyl tRNA synthetase for the incorporation of p-aziclo-
phenylalanine RS
(LW6
SEQ ID NO:16 Aminoacyl tRNA synthetase for tlte incorporation of p-azido
phenylalanine RS
(AzPheRS-S
SEQ ID NO'17 Aniinoacyl tRNA synthe.tase for the incorporation of p-azido
phenylalanitte RS
(AzPheRS-6)
1005611 The transfomiation of E. coli with plasmids containing the modified
gene and the orthogonal
aminoacyl tRNA synthetase/tRNA pair (specific for the desired non-natural
amino acid) allows the site-specific
incorporation of non-natural amino acid into the polypeptide. The transformed
E. coli, grown at 37 C in media
containing between 0.01 - 100 mM of the particular non-natural amino acid,
expresses modified polypeptide
with high fidelity and efficiency. The His-tagged polypeptide containing a non-
natural amino acid is produced
by the E. coli host cells as inclusion bodies or aggregates. The aggregates
are solubilized and affinity purified
under denaturing conditions in 6M guanidine HCI. Refolding is performed by
dialysis at 4`C ovemight in 50mM
TRIS-HCI, pH8.0, 40 M CuSO4, and 2% (w/v) Sarkosyl. The material is then
dialyzed against 20mM TRIS-
HCI, pH 8.0, 100mM NaC1, 2mM CaC12, followed by removal of the His-tag. See
Boissel et al., (1993)
268:15983-93. Methods for purification of polypeptides are are confirmed by
SDS-PAGE, Western Blot
analyses, or electrospray-ionization ion trap mass spectrometry and the like.
1005621 The following examples describe methods to measure and compare the in
vitro and in vivo activity of a
modified therapeutically active non-natural amino acid polypeptide to the in
vitro and in vivo activity of a
therapeutically active natural amino acid polypept9de.
Example 9: Cell Binding Assays
1005631 Cells (3x106) are incubated in duplicate in PBS/1% BSA (100 l) in the
absence or presence of various
concentrations (volume: 10 l) of unlabeled GH, hGH or GM-CSF and in the
presence of 125 I-GH (approx.
100,000 cpm or I ng) at 0 C for 90 minutes (total volume: 120 l). Cells are
then resuspended and layered over
200 l ice cold FCS in a 350 lcl plastic centrifuge tube and centrifuged (1000
g; I minute). The pellet is
collected by cutting off the end of the tube and pellet and supernatant
counted separately in a gamma counter
(Packard).
[005641 Specific binding (cpm) is deterrrtined as total binding in the absence
of a competitor (inean of
duplicates) minus binding(cpm) in the presence of 100-fold excess of unlabeled
GH. (non-specific binding). The
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non-specific binding is measured for each of the cell types used. Experiments
are run on separate days using the
same preparation of 1ZSI-GH and should display internal consistency. 1251-GH
demonstrates binding to the GH
receptor-producing cells. The binding is inhibited in a dose dependent manner
by unlabeled natural GH or hGH,
but not by GM-CSF or other negative control. The ability of hGH to compete for
the binding of natural 125 1-
GH, similar to natural GH, suggests that the receptors recognize both forms
equally well.
Example 10: In Vivo Studies of hGH PEGylated via a indole linkage
1005651 PEG-hGH, unmodified hGH and buffer solution are administered to niice
or rats. The results show
superior activity and prolonged half life of the PEGylated hGH described
herein compared to urunodified hGH
which is indicated by significantly increased bodyweight.
Example 11: Measurement of the in vivo Half-life of Conjueated and Non-
conjuaated hGH and Variants
Thereof.
1005661 All animal experimentation is conducted in an AAALAC accredited
facility and under protocols
approved by the Institutional Aninial Care and Use Committee of St. Louis
University. Rats are housed
individually in cages in rooms with a 12-hour light/dark cycle. Aninials are
provided access to certified Purina
rodent chow 5001 and water ad libitum. For hypophysectomized rats, the
drinking water additionally contains
5% glucose.
Example 12: Pharmacokinetic studies
1005671 The quality of each PEGylated mutant hGH is evaluated by three assays
before entering animal
experiments. The purity of the PEG-hGH (PEGylated via an indole linkage) is
examined by running a 4-12%
acrylamide NuPAGE Bis-Tris gel with MES SDS ntnning buffer under non-reducing
conditions (Invitrogen,
Carlsbad, CA). The gels are stained with Coomassie blue. The PEG-hGH band is
greater than 95% pure based
on densitometry scan. The endotoxin level in each PEG-hGH is tested by a
kinetic LAL assay using the KTA''
kit from Charles River Laboratories (Wihnington, MA), and is less than 5 EU
per dose. The biological activity
of the PEG-hGH is assessed with a IM-9 pSTAT5 bioassay, and the EC50 value
confirmed to be less than 15
nM.
1005681 Pharmacokinetic properties of PEG-modified growth hormone compounds
are compared to each otlier
and to nonPEGylated growth hormone in niale Sprague-Dawley rats (261-425g)
obtained from Charles River
I_aboratories. Catheters are surgically installed into the carotid artery for
blood collection. Following successful
catheter installation, animals are assigned to treatment groups (three to six
per group) prior to dosing. Animals
are dosed subcutaneously with 1 mg/kg of compound in a dose volume of 0.41-
0.55 nil/kg. Blood samples are
collected at various time points via the indwelling catheter and into EDTA-
coated microfuge tubes. Plasma is
collected after centrifugation, and stored at -80 C until analysis. Compound
concentrations are measured using
antibody sandwich growth hormone ELISA kits froni either BioSource
International (Caniarillo, CA) or
Diagnostic Systenis Laboratories (Webster, TX). Concentrations are calculated
using standards corresponding to
the analog that is dosed. Pharmacokinetic parameters are estimated using the
modeling program WinNonlin
(Pharsight, version 4.1). Noncompart mental analysis with linear-up/log-down
trapezoidal integration is used,
and concentration data is unifornily weighted.
1005691 Plasma concentrations are obtained at regular intervals following a
single subcutaneous dose in rats.
Rats (n=3-6 per group) are given a single bolus dose of 1 mg/kg protein. hGH
wild-type protein (WHO hGH),
His-tagged hGH polypeptide (his-hGH), or His-tagged hGH polypeptide comprising
non-natural amino acid
indole covalently linked to 30 k.Da PEG at each of six different positions are
compared to WHO hGH and (his)-
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CA 02672205 2009-06-09
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hGH. Plasma samples are taken over the regular time intervals and assayed for
injected compound as described.
Concentration vs time curves are evaluated by noncompartmental analysis
(Pharsight,. version 4.1). Values
shown are averages (+/- standard deviation). Cmax: maximum concentration;
terminal ~l/_,: terniinal half-life;
AUCo.>;,,f: area under the concentration-time curve extrapolated to infnity;
MRT: mean residence time; Cl/f:
apparent total, plasma clearance; Vz/f apparent volume of distribution during
terminal phase.
Example 13: Pharmacod,+namic studies
1005701 Hypophysectomized niale Sprague-Dawley rats are obtained from Charles
River Laboratories.
Pituitaries are surgically removed at 3-4 weeks of age. Animals re, allowed to
acclimate for a period of three
weeks, during which time bodyweight was monitored. Atumals with a bodyweight
gain of 0-8g over a period of
seven days before the start of the study are included and randonrized to
treatment groups. Rats are administered
either a bolus dose or daily dose subcutaneously. Throughout the study rats re
daily and sequentially weighed,
anesthetized, bled, and dosed (when applicable). Blood is collected from the
orbital sinus using a heparinized
capillary tube and placed into an EDTA coated microfuge tube. Plasma is
isolated by centrifugation and stored
at -80 C until analysis. The mean (+/- S.D.) plasma concentrations are plotted
versus time intervals.
1005711 The peptide IGF-1 is a meniber of the family of son-atomedins or
insulin-like growth factors. IGF-1
mediates many of the growth-promoting effects of growth hormone. IGF-1
concentrations are measured using a
conipetitive binding enzyme immunoassay k-it against the provided rat/mouse
IGF-1. standards (Diagnosic
Systems Laboratories). Hypophysectomized rats. Rats (n= 5-7 per group) are
given either a single dose or daily
dose subcutaneously. Animals are sequentially weighed, anesthetized, bled, and
dosed (when applicable) daily.
Bodyweight results are taken for placebo, treatments, wild'type hGH (hGH), His-
tagged hGH ((his)hGH), and
hGH polypeptides comprising an indole covalently-linked to 30 kDa PEG at
positions 35 and 92.
Example 14: Human Clinical Trial of the Safety and/or Efficacy of PEGvlated
hGH (PEGylated via an
indole linkage) Comprising a Non-Naturally Encoded Amino Acid.
[005721 The following example of a clinical trial is used to treat childhood
and adult growth hormone
deficiency, Tumer syndrome, chronic renal failure, Prader-Willi syndrome,
children with intrauterine growth
retardation, idiopathic short stature, growth failure associated with clironic
high dose glucocorticoid use, post-
transplant growth failure, X-linked hypophosphatemic rickets, inflammatory
bowel disease, Noonan syndrome,
bone dysplasia, growth failure associated with Celiac's disease, muscle
wasting associated, e.g., with advance
acquired imrnunodeficiency syndrome, promote healing of burns, side effects of
severe dieting for obese
individuals, fybromyalgia, chronic fatigue syndrome, debilities associated
with aging, and other uses of human
growth hornione.
1005731 Obiective To compare the safety and pharmacokinetics of subcutaneously
administered PEGylated
recombinant human hGH comprising a non-naturally encoded aniino acid with one
or more of the commercially
available. hGH products (including, but not limited to HumatropeT"f (Eli Lilly
& Co.), NutropinTb (Genentech),
NorditropinThl (Novo-Nordisk), GenotropinTM (Pfizer) and Saizen/SerostimTM
(Serono)).
1005741 Patients Eighteen healthy volunteers ranging between 8-40 years of age
and weighing betweett 20-90
kg are enrolled in the study (e.g., children for pediatric indications and
adults for adult indications). The subjects
will have no clinically significant abnormal laboratory values for hematology
or serum chemistry, and a
negative urine toxicology screen, HIV screen, and hepatitis B surface antigen.
They should not have any
evidence of the following: hypertension; a. history. of any primary
heniatologic disease; history of significant
hepatic, renal, cardiovascular, gastrointestinal, genitourinary, metabolic,
neurologic disease; a history of anemia
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or seizure disorder; a known sensitivity to bacterial or manunalian-derived
products, PEG, or huntan serum
albumin; habitual and heavy consumer to beverages containing caffeine;
participation in any other clinical trial
or had blood transfused or donated within 30 days of study entry; had exposure
to hGH within three months of
study entry; had an illness within seven days of study entry; and have
significant abnormalities on the pre-study
physical examination or the clinical laboratory evaluations within 14 days of
study entry. All subjects are
evaluated for safety. and all blood collections. for pharmacokinetic analysis
are collected as scheduled. All
studies are performed with institutional ethics conunittee approval and
patient consent.
[005751 Study Design This will be a Phase I, single-center, open-label,
randomized,.two-period crossover study
in healthy nzale volunteers. Eighteen subjects are randomly assigned to one of
two treatment sequence groups
(nine subjects/group). GH is adtninistered over two separate dosing periods as
a bolus scc. injection in.the upper
thigh using equivalent doses of the PEGylated hGH comprising a non-naturally
encoded aniino acid and the
commercially available,product chosen: The dose and frequency
of;administration of the commercially available
product is as instructed in the package Iabe1. Additiot>al..do'sing, dosing
frequency, or other parameter as desired,
using the, commercially. available products may be added to the'study by
including additional groups of subjects.
Each dosing period is separated by a 14-day washoutperiod. Subjects are
confined to the study center at least 12
houis prior to and. 72 hours following dosing for each of the two dosing
periods, but not between dosing
periods. Additional.groups of subjects may be added if there are to be
additional dosing, frequency, or other
parameter, to be tested for the. PEGylated hGH as well. Multiple formulations
of GH that are approved for
human use niay be used in this study. HumatropeT" (Eli Lilly & Co.),
NutropinTM (Genentech), NorditropinTM
(Novo-Nordisk), GenotropinTM (Pfizer) and Saizen/SerostimT"' (Serono)) are
commercially available Gl-1
products approved for human use. The experimental formulation of hGH is the
PEGylated hGH comprising a
non-naturally encoded amino acid.
1005761 Blood Samnline Serial blood is drawn by direct vein puncture before
and after administration of hGH.
Venous blood samples (5 mL) for determi.nation of serum GH concentrations are
obtained at about 30, 20, and
10 minutes prior to dosing (3 baseline samples) and at approximately the
following times. after dosing: 30
minutes and at 1, 2, 5, 8,. 12, 15, 18, 24, 30, 36, 48, 60 and 72 hours. Each
serum sample is divided into two
aliquots. All serum samples are stored' at -20 C. Serum samples are. shipped
on dry ice: Fasting clinical
laboratory, tests, (heniatology, serum chemistry, and urinalysis) are
performed inunediately prior to the initial
dose on day 1, the morning of day 4,immediately prior to dosing on,day 16, and
the niorning'of day 19.
1005771 Bioanalytical Methods An ELISA kit procedure (Diagnostic Systems
Laboratory [DSL], Webster TX),
is used for the deterniination of serum GH concentrations.
[005781 Safety Determinations Vital signs, are recorded immediately prior to
each dosing (Days I and 16), and
at 6, 24õ 48, and 72 hours after each dosing. Safety determinations are based
on the incidence and type of
adverse events and the changes in clinical laboratory tests from baseline. In
addition, changes froni pre-study in
vital sign measurements, including blood pressure, and physical examination
results are evaluated.
1005791 Data Analysis Post-dose serum concentration values are corrected. for
pre-dose baseline Gl=I
concentrations by subtracting from each of the post-dose values the mean
baseline GH concentration determined
from averaging the GH levels from the three saniples collected at 30, 20, and
10 minutes. before dosing. Pre-
dose seruni GI3 conccntrations are not included in the calculation of the mean
value if they are below the
quantification levcl of the assay. Pharmacokinetic parameters are determined
from serum concentration data
corrected for baseline GI-I concentrations. Pharmacokinetic parameters are
calculated by model independent
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methods on a Digital Equipment Corporation VAX 8600 computer system using the
latest version of the
BIOAVL software. The following pharmacokinetics parameters are determined:
peak serum concentration
(C,,,,X); time to peak. serart concentration (t,,,,); area under
the,concentration-rirtte curve (AUC) from time zero
to the last blood sampling time (AUCa.>>) calculated .with the use of the
linear trapezoidal rule; and terminal
elimination half-life (tin), computed from the elimination rate constant. The
elimination rate constant is
estimated by linear regression of consecutive data points in the terminal
linear region of the log-linear
concentration-time plot. The mean, standard deviation (SD), and coefficient of
variation (CV) of the
pharmacokinetic parameters are calculated for each treatment. The ratio of the
parameter means (preserved
formulation/non-preserved formulation) is calculated.
[005801 Safety Results The incidence of adverse events is equally distributed
across the treatment groups.
There are no clinically significant changes from baseline or pre-study
clinical laboratory tests or blood
pressures, and no notable changes from pre-study in physical examination
results and vital sign measurements.
The safety profiles for the two treatment groups should appear similar.
1005811 Pharmacokinetic Results Mean senim GH concentration-time profiles
(uncorrected for baseline GH
lcvels), in all 18 subjects after receiving a single dose of one or more of
comniercially available hGH products
(including, but not limited to HumatropeTM (Eli Lilly & Co:), NutropinTm
(Genentech), NorditropinT"' (Novo-
Nordisk), GenotropinTM (Pfizer) and Saizen/SerostimTM (Serono)) are compared
to the PEGylated hGH
comprising a non-naturally encoded amino acid at each time point measured. All
subjects should have pre-dose
baseline GH concentrations within the normal physiologic range.
Pharrnacokinetic parameters are determined
from serum data corrected for pre-dose mean baseline GH concentrations and the
C. and t,õ,x are determined.
The mean t,,,ax for the clinical comparator(s) chosen (HumatropeTM (Eli Lilly
&-Co.), NutropinTM (Genentech),
NorditropinTM (Novo-Nordisk), GenotropinTM (Pfizer), Saizen/SerostimTM
(Serono)) is significantly shorter than
the tõm,t for the PEGylated hGH comprising the non-naturally encoded amino
acid. Terminal half-life values are
significantly shorter for the conunercially available hGH products tested
compared with the terminal half-life
for the PEGylated hGH comprising a non-naturally encoded amino acid.
1005821 Although the present study is conducted in healthy male subjects,
similar absorption characteristics and
safety profiles would be anticipated in other patient populations; such as
male or female patients with cancer or
chronic renal failure, pediatric renal failure patients, patients in
autologous predeposit programs, or patients
scheduled for elective surgery.
1005831 In conclusion, subcutaneously adniinistered single doses of PEGylated
hGH comprising non-naturally
encoded amino acid will be safe and well tolerated by healthy male subjects.
Based on a comparative incidence
of adverse events, clinical laboratory values, vital signs, and physical
examination results, the safety profiles of
the commercially available forms of hGH and PEGylated hGH "comprising non-
naturally encoded amino acid
will be equivalent. The PEGylated hGH comprising non-naturally encoded amino
acid potentially provides
large clinical utility to patients and health care providers.
Example 15: Comparison of water solubility of PEGylated hGH and non-PEGylated
hGH
1005841 The=water solubility of hGH wild-type protein (WHO hGH), His-tagged
hGH polypeptide (his-hGH),
or His-tagged hGH polypeptide comprising non-natural amino acid indole
covalently linked to 30 kDa PEG at
position 92 are obtained by determining the quantity of the respective
polypeptides which can dissolve on 100
L of water. The quantity of PEGylated hGH is larger than the quantities:for
WHO hGH and hGH which shows
a that PEGylation ofnon-natural amino acid polypeptides increases the water
solubility.
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Examnle 16: In Vivo Studies of modified therapeutically active non-natural
amino acid polypentide
1005851 Prostate cancer tumor xenografts are implanted uito mice which are
"then separated into two groups.
One group is treated daily with a modified therapeutically active non=natural
amino acid polypeptide and the
other group is treated daily with therapeutically active natural amino acid
polypeptide. The tumor size is
measured daily and the modified therapeutically active non-natural anuno acid
polypeptide has improved
therapeutic effectiveness compared to the therapeutically active natural amino
acid polypeptide as indicated by a
decrease in tumor size for the group treated with the modi6ed therapeutically
active non-natural amino acid
polypeptide.
Example 17: Measurement of non-natural amino acid polVneptide activity and
affinity
(005861 This example details the measurement of non-natural amino acid
polypeptide activity and affinity of
non-natural amino acid polypeptides for their receptor, binding partner, or
ligand.
1005871 Protein for the non-natural amino acid polypeptide receptor, binding
partner, or ligand is expressed and
isolated according to documented methodologies. The BiocoreTM system is used
to analyze the binding of non-
natural amino acid polypeptide to its receptor. Similarly, a binding partner
or ligand may be used in this assay.
1005881 Approximately 600-800 RUs of soluble receptor is immobilized on a
BiacoreTM CM5 chip, using an
amine-coupling procedure, as recommended by the manufacturer. Various
concentrations of wild type or
modified or unmodified non-natural amino acid polypeptide in HBS-EP buffer
(BiacoreT"t, Pharmacia) are
injected over the surface at a flow rate of 40 l/min for 4-5 minutes, and
dissociation was monitored for 15
minutes post-injection. The surface is regenerated by a 15 second pulse of
4.5M MgCIZ. Only a minimal loss of
binding affinity (1-5%) is observed after at least 100 regeneration cycles: A
reference cell with no receptor
inunobilized is used to subtract any buffer bulk effects and non-specific
binding.
1005891 Kinetic binding data obtained from modified or unmodified non-natural
amino acid polypeptide
titration experiments is processed with BiaEvaluation 4.1 software
(BIACORETM). Equilibrium dissociation
constants (Kd) are calculated as ratios of individual rate constants (k
n/kaõ).
1005901 Stable Cell Lines are established expressing receptor, binding
partner, or ligand for the non-natural
amino acid polypeptide. Cells are electroporated with a coristruct that
containing the receptor, binding partner,
or ligand cDNA. Transfected cells are allowed to recover for 48 hours before
cloning. Receptor, binding
partner, or ligand expressing transfectants are identified by surface staining
with antibody against the receptor
and are analyzed on a FACS Array (BD Biosciences, San Diego, CA). Stably
transfected cell clones are
established upon further rounds of repeated subcloning of desired
transfectants. Such cells are used in cell
binding assays.
1005911 Cells (3x106) are incubated in duplicate in PBS/1% BSA (100 l) in the
absence or presence of various
concentrations (volunie: 10 l) of unlabeled natural amino acid polypeptide or
a negative control polypeptide
and in the presence of 1251-(modified) non-natural amino acid polypeptide
(approx. 100,000 cpm or I ng) at 0 C
for 90 minutes (total volume: 120 l). Cells are then resuspended and layered
over 200 l ice cold FCS in a 350
l plastic centrifuge tube and centrifuged (1000 g; 1 minute). The pellet is
collected by cutting off the end of the
tube and pellet and supernatant counted separately in a ganuna counter
(Packard).
1005921 Specific binding (cpm) is determined as total binding in the absence
of a competitor (mean of
duplicates) minus non-specific binding. The non-specific binding is measured
for each of the cell types used.
Experiments are run on separate days using the same preparation of 125I-
(modified) non-natural amino acid
polypeptide and should display internal consistency. '251-(modified) non-
natural aniino acid polypeptide
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demonstrates binding to the receptor, binding protein, or ligand-producing
cells. The binding is inhibited in a
dose dependent manner by unlabeled natural amino acid polypeptide, but not by
a negative control polypeptide.
1005931 It is understood that the examples and embodiments described herein
are for illustrative purposes only
and that various modifications or changes in light thereof are to be included
within the spirit and purview of this
application and scope of the appended claims.
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