Note: Descriptions are shown in the official language in which they were submitted.
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BRANCHED POLYMERIC SUGARS AND
NUCLEOTIDES THEREOF
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention resides in the field of modified sugars and
nucleotides thereof.
Background
[0003] Post-expression in vitro modification of peptides is an attractive
strategy to remedy
the deficiencies of methods that rely on controlling glycosylation by
engineering expression
systems; including both modification of glycan structures or introduction of
glycans at novel
sites. A comprehensive toolbox of recombinant eukaryotic glycosyltransferases
is becoming
available, making in vitro enzymatic synthesis of mammalian glycoconjugates
with custom
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designed glycosylation patterns and glycosyl structures possible. See, for
example,
U.S. Patent No. 5,876,980; 6,030,815; 5,728,554; 5,922,577; and WO/9831826;
US2003180835; and WO 03/031464.
[0004] Enzyme-based syntheses have the advantages of regioselectivity and
stereoselectivity.
Moreover, enzymatic syntheses are performed using unprotected substrates.
Three principal
classes of enzymes are used in the synthesis of carbohydrates,
glycosyltransferases (e.g.,
sialyltransferases, oligosaccharyltransferases, N-
acetylglucosaminyltransferases), and
glycosidases. The glycosidases are further classified as exoglycosidases
(e.g.,I3-
mannosidase,I3-glucosidase), and endoglycosidases (e.g., Endo-A, Endo-M). Each
of these
classes of enzymes has been successfully used synthetically to prepare
carbohydrates. For a
general review, see, Crout et al., Curr. Opin. Chem. Biol. 2: 98-111(1998).
[0005] Glycosyltransferases modify the oligosaccharide structures on
glycopeptides.
Glycosyltransferases are effective for producing specific products with good
stereochemical
and regiochemical control. Glycosyltransferases have been used to prepare
oligosaccharides
and to modify terminal N- and 0-linked carbohydrate structures, particularly
on
glycopeptides produced in mammalian cells. For example, the terminal
oligosaccharides of
glycopeptides have been completely sialylated and/or fucosylated to provide
more consistent
sugar structures, which improves glycopeptide pharmacodynamics and a variety
of other
biological properties. For example, 0-1,4-galactosyltransferase was used to
synthesize
lactosamine, an illustration of the utility of glycosyltransferases in the
synthesis of
carbohydrates (see, e.g., Wong etal., J. Org. Chem. 47: 5416-5418 (1982)).
Moreover,
numerous synthetic procedures have made use of a-sialyltransferases to
transfer sialic acid
from cytidine-5'-monophospho-N-acetylneuraminic acid to the 3-0H or 6-0H of
galactose
(see, e.g., Kevin etal., Chem. Eur. J. 2: 1359-1362 (1996)).
Fucosyltransferases are used in
synthetic pathways to transfer a fucose unit from guanosine-5'-diphosphofucose
to a specific
hydroxyl of a saccharide acceptor. For example, Ichikawa prepared sialyl Lewis-
X by a
method that involves the fucosylation of sialylated lactosamine with a cloned
fucosyltransferase (Ichikawa etal., J. Am. Chem. Soc. 114: 9283-9298 (1992)).
For a
discussion of recent advances in glycoconjugate synthesis for therapeutic use
see, Koeller et
al., Nature Biotechnology 18: 835-841 (2000). See also, U.S. Patent No.
5,876,980;
6,030,815; 5,728,554; 5,922,577; and WO/9831826.
[0006] In addition to manipulating the structure of glycosyl groups on
polypeptides, interest
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has developed in preparing glycopeptides that are modified with one or more
non-saccharide
modifying group, such as water soluble polymers. Poly(ethyleneglycol) ("PEG")
is an
exemplary polymer that has been conjugated to polypeptides. The use of PEG to
derivatize
peptide therapeutics has been demonstrated to reduce the immunogenicity of the
peptides.
For example, U.S. Pat. No. 4,179,337 (Davis et al.) discloses non-immunogenic
polypeptides,
such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or
polypropylene glycol. Between 10 and 100 moles of polymer are used per mole
polypeptide.
Although the in vivo clearance time of the conjugate is prolonged relative to
that of the
polyp eptide, only about 15% of the physiological activity is maintained.
Thus, the prolonged
circulation half-life is counterbalanced by the dramatic reduction in peptide
potency.
[0007] The loss of peptide activity is directly attributable to the non-
selective nature of the
chemistries utilized to conjugate the water-soluble polymer. The principal
mode of
attachment of PEG, and its derivatives, to peptides is a non-specific bonding
through a
peptide amino acid residue. For example, U.S. Patent No. 4,088,538 discloses
an
enzymatically active polymer-enzyme conjugate of an enzyme covalently bound to
PEG.
Similarly, U.S. Patent No. 4,496,689 discloses a covalently attached complex
of a-1
proteinase inhibitor with a polymer such as PEG. Abuchowski et al. (I Biol.
Chem. 252:
3578 (1977) discloses the covalent attachment of MPEG to an amine group of
bovine serum
albumin. U.S. Patent No. 4,414,147 discloses a method of rendering interferon
less
hydrophobic by conjugating it to an anhydride of a dicarboxylic acid, such as
poly(ethylene
succinic anhydride). PCT WO 87/00056 discloses conjugation of PEG and
poly(oxyethylated) polyols to such proteins as interferon-0, interleukin-2 and
immunotoxins.
EP 154,316 discloses and claims chemically modified lymphokines, such as IL-2
containing
PEG bonded directly to at least one primary amino group of the lymphokine.
U.S. Patent No.
4,055,635 discloses pharmaceutical compositions of a water-soluble complex of
a proteolytic
enzyme linked covalently to a polymeric substance such as a polysaccharide.
[0008] Another mode of attaching PEG to peptides is through the non-specific
oxidation of
glycosyl residues on a glycopeptide. The oxidized sugar is utilized as a locus
for attaching a
PEG moiety to the peptide. For example M'Timkulu (WO 94/05332) discloses the
use of an
amino-PEG to add PEG to a glycoprotein. The glycosyl moieties are randomly
oxidized to
the corresponding aldehydes, which are subsequently coupled to the amino-PEG.
[0009] In each of the methods described above, poly(ethyleneglycol) is added
in a random,
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= non-specific manner to reactive residues on a peptide backbone. For the
production of
therapeutic peptides, it is clearly desirable to utilize a derivitization
strategy that results in the
formation of a specifically labeled, readily characterizable, essentially
homogeneous product.
A promising route to preparing specifically labeled peptides is through the
use of enzymes,
such as glycosyltransferases to append a modified sugar moiety onto a peptide.
The modified
sugar moiety must function as a substrate for the glycosyltransferase and be
appropriately
activated. Hence, synthetic routes that provide facile access to activated
modified sugars are
desirable. The present invention provides such a route.
SUMMARY OF THE INVENTION
[0010] The present invention provides polymeric species, sugars and activated
sugars
conjugated to these polymeric species and nucleotide sugars that include these
polymers. The
polymeric species include both water-soluble and water-insoluble species.
Moreover, the
polymers are either branched- or straight-chain polymers. Exemplary sugar
moieties include
straight-chain and cyclic structures and aldoses and ketoses.
[0011] The polymeric modifying group can be attached at any position of the
sugar moiety.
In the discussion below, the invention is exemplified by reference to an
embodiment in which
the polymeric modifying group is attached to C-5 of a furanose or C-6 of a
pyranose. Those
of skill will appreciate that the focus of the discussion is for clarity of
illustration, the
polymeric moiety can be attached to other positions of both pyranoses and
furanoses using
the methods set forth herein and art-recognized methods.
[0012] In an exemplary embodiment, the invention provides a sugar or a sugar
nucleotide
that is conjugated to a polymer:
(R6)d
(Rnd
0 R2
R6
; and
R5 R3
R4 R3
R4
I II
[0013] In Formulae I and II, R1 is H, CH2OR7, COOR7 or OR7, in which R7
represents H,
substituted or unsubstituted alkyl or substituted or unsubstituted
heteroalkyl. R2 is H, OH or
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a moiety that includes a nucleotide. An exemplary R2 species according to this
embodiment
has the formula:
0 \
0 _________________________________ PI I 0 __ R8
/ 1 _3
in which R8 is a nucleoside.
[0014] The symbols R3, R4, R5, R6 and R6' independently represent H,
substituted or
unsubstituted alkyl, OR9, NHC(0)R10. The index d is 0 or 1. R9 and R1 are
independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl or
sialic acid. At least one of R3, R4, R5, R6 or R6' includes the polymeric
modifying moiety e.g.,
PEG. In an exemplary embodiment, R6 and R6', together with the carbon to which
they are
attached are components of the side chain of sialic acid. In a further
exemplary embodiment,
this side chain is functionalized with the polymeric modifying moiety.
[0015] In an exemplary embodiment, the polymeric moiety is bound to the sugar
core,
generally through a heteroatom on the core, through a linker, L, as shown
below:
(R11 )w
R" is the polymeric moiety and L is selected from a bond and a linking group.
The index w
represents and integer selected from 1-6, preferably 1-3 and more preferably 1-
2. Exemplary
linking groups include substituted or unsubstituted alkyl, substituted or
unsubstituted
heteroalkyl moieties and sialic acid. An exemplary component of the linker is
an acyl
moiety.
[0016] When L is a bond it is formed between a reactive functional group on a
precursor of
R" and a reactive functional group of complementary reactivity on a precursor
of L. L can
be in place on the saccharide core prior to reaction with R11. Alternatively,
R11 and L can be
incorporated into a preformed cassette that is subsequently attached to the
saccharide core.
As set forth herein, the selection and preparation of precursors with
appropriate reactive
functional groups is within the ability of those skilled in the art. Moreover,
coupling the
precursors proceeds by chemistries that are well understood in the art.
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[0017] In an exemplary embodiment L is a linking group that is formed from an
amino acid,
or small peptide (e.g., 1-4 amino acid residues) providing a modified sugar in
which the
polymeric modifying moiety is attached through a substituted alkyl linker. An
exemplary
linker is glycine.
[0018] In an exemplary embodiment, R6 includes the polymeric modifying moiety.
In
another exemplary embodiment, R6 includes both the polymeric modifying moiety
and a
linker, L, joining the modifying moiety to the remainder of the molecule.
[0019] In an exemplary embodiment, the polymeric modifying moiety is a
branched structure
that includes two or more polymeric chains attached to central moiety. An
exemplary
structure of a useful polymeric modifying moiety precursor according to this
embodiment of
the invention has the formula:
R12¨X2
X5-C-X3'
R13_x4
The sugars and nucleotide sugars according to this formula are essentially
pure water-soluble
polymers. X3' is a moiety that includes an ionizable (e.g., COOH, etc.) or
other reactive
functional group, see, e.g., infra. C is carbon. X5 is preferably a non-
reactive group (e.g., H,
unsubstitited alkyl, unsubstituted heteroalkyl). R12 and R13 are independently
selected
polymeric arms, e.g., nonpeptidic, nonreactive polymeric arms. X2 and X4 are
linkage
fragments that are preferably essentially non-reactive under physiological
conditions, which
may be the same or different. Alternatively, these linkages can include one or
more moiety
that is designed to degrade under physiologically relevant conditions, e.g.,
esters, disulfides,
etc. X2 and X4 join polymeric arms R12 and R13 to C. When X3' is reacted with
a reactive
functional group of complementary reactivity on a linker, sugar or linker-
sugar cassette, X3'
is converted to a component of linkage fragment X3.
[0020] By reaction of the precursor with a suitable sugar or sugar linker
species the invention
provides sugars and nucleotide sugars that have the formulae:
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(R9d
R12_x2
0 RIR12¨X2 (R9d
X5¨C¨LaR2
R- and X5_¨La
R13¨X3 ;
R5R3 R13¨X3
R4 R3
R4
Iv V
=
in which the identity of the radicals represented by the various symbols is
the same as that
discussed hereinabove. La is a substituted or unsubstituted alkyl or
substituted or
unsubstituted heteroalkyl moiety. In an exemplary embodiment, La is a moiety
of the side
chain of sialic acid that is functionalized with the polymeric modifying
moiety as shown.
[0021] The polymeric modifying moiety comprises two or more repeating units
that can be
water-soluble or essentially insoluble in water. Exemplary water-soluble
polymers of use in
the compounds of the invention include PEG, e.g., m-PEG, PPG, e.g., m-PPG,
polysialic
acid, polyglutamate, polyaspartate, polylysine, polyethyeleneimine,
biodegradable polymers
(e.g., polylactide, polyglyceride), and functionalized PEG, e.g., terminal-
functionized PEG.
[0022] The sugar moiety of the polymeric conjugates of the invention is
selected from both
natural and unnatural furanoses and hexanoses. The unnatural saccharides
optionally include
an alkylated or acylated hydroxyl and/or amine moiety, e.g., ethers, esters
and amide
substituents on the ring. Other unnatural saccharides include an H, hydroxyl,
ether, ester or
amide substituent at a position on the ring at which such a substituent is not
present in the
natural saccharide. Alternatively, the carbohydrate is missing a substituent
that would be
found in the carbohydrate from which its name is derived, e.g., deoxy sugars.
Still further
exemplary unnatural sugars include both oxidized (e.g., -onic and ¨uronic
acids) and reduced
(sugar alcohols) carbohydrates. The sugar moiety can be a mono-, oligo- or
poly-saccharide.
[0023] Exemplary natural sugars of use in the present invention include
glucose,
glucosamine, galactose, galactosamine, fucose, mannose, mannosamine, xylanose,
ribose, N-
acetyl glucose, N-acetyl glucosamine, N-acetyl galactose, N-acetyl
galactosamine, and sialic
acid.
[0024] An exemplary sialic acid-based conjugate has the formula:
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HO
OH
OH 0 ON P
R12-x2 0
COON
N
R13-X4
0 OH
in which AA is that portion of an amino acid residue that does not include the
carboxyl
moiety and NP is a nucleotide phosphate. ONP can also be replaced by an
activating moiety
to form an activated sugar. As will be appreciated by those of skill in the
art, the polymeric
modifying moiety-linker can also be attached to the sialic acid side chain at
C-6, C-7 and/or
C-9.
[0025] Also provided is a synthetic method for producing an activated sialic
acid-PEG
conjugate that is an appropriate substrate for an enzyme, e.g., a
glycosyltransferase. The
method includes the steps: (a) contacting mannosamine with an activated, N-
protected amino
acid (or an amino acid functionalized with a polymeric modifying moiety, a
linker precursor
or a linker-polymeric modifying moiety cassette) under conditions appropriate
to form an
amide conjugate between the maimosamine and the N-protected amino acid; (b)
contacting
the amide conjugate with pyruvate and sialic acid aldolase under conditions
appropriate to
convert the amide conjugate to a sialic acid amide conjugate; (c) contacting
the sialic acid
amide conjugate with cytidine triphosphates, and a synthetase under conditions
appropriate to
form a cytidine monophosphate sialic acid amide conjugate; (d) removing the N-
protecting
group from the cytidine monophosphate sialic acid amide conjugate, thereby
producing a free
amine; and (e) contacting the free amine with an activated PEG (straight-chain
or branched),
thereby forming the cytidine monophosphate sialic acid-poly(ethylene glycol).
[0026] The nucleoside can be selected from both natural and unnatural
nucleosides.
Exemplary natural nucleosides of use in the present invention include
cytosine, thymine,
guanine, adenine and uracil. The art is replete with structures of unnatural
nucleosides and
methods of making them.
[0027] Exemplary modified sugar nucleotides of the invention include
polymerically-
modified GDP-Man, GDP-Fuc, LTDP-Gal, LTDP-GalNAc, UDP-Glc, UDP-G1cNAc, UDP-
Glc, UDP-GlcUA and CMP-SA and the like. Examples include UDP-Gal-2'-NH-PEG,
UDP-Glc-2'-NH-PEG, CMP-5'-PEG-SA and the like. Compounds encompassed by the
invention include those in which the L-R11 moiety is conjugated to a furanose
or a pyranose,
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e.g., at C-5 of a furanose or at C-6 of a pyranose, generally through a
heteroatom attached to
this carbon atom.
[0028] When the compound of the invention is a nucleotide sugar, or activated
sugar, the
polymeric conjugates of the nucleotide sugars are generally substrates for an
enzyme that
transfers the sugar moiety and its polymeric substituent onto an appropriate
acceptor moiety
of a substrate. Accordingly, the invention also provides substrates modified
by
glycoconjugation using a polymeric conjugate of a nucleotide sugar, or
activated sugar, and
an appropriate enzyme. Substrates that can be glycoconjugated using a compound
of the
invention include peptides, e.g., glycopeptides, peptides, lipids, e.g.,
glycolipids and
aglycones (sphingosines, ceramides).
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a table of sialyltransfcrases for which selected modified
sialic acid
nucleotides and activated sugars are substrates.
[0030] FIG. 2 is a general synthetic scheme of the invention for preparing a
sialic acid-
poly(ethylene glycol) conjugate.
[0031] FIG. 3 is a synthetic scheme of the invention for preparing a sialic
acid-glycyl-
poly(ethylene glycol) conjugate.
Figures 4 and 5 depict cytidine monophosphate sialic acid poly(ethylene
glycol)
compounds of the invention.
DETAILED DESCRIPTION OF THE INVENTION
AND THE EMBODIMENTS
Abbreviations
[0032] Branched and unbranched PEG, poly(ethyleneglycol), e.g., m-PEG, methoxy-
poly(ethylene glycol); Branched and unbranched PPG, poly(propyleneglycol),
e.g., m-PPG,
methoxy-poly(propylene glycol); Fuc, fucosyl; Gal, galactosyl; GalNAe, N-
acetylgalactosaminyl; Glc, glucosyl; GIcNAc, N-acetylglucosaminyl; Man,
mannosyl;
ManAc, mannosaminyl acetate; Sia, sialic acid; and NeuAc, N-acetylneuraminyl.
Definitions
[0033] The term "sialic acid" refers to any member of a family of nine-carbon
carboxylated
sugars. The most common member of the sialic acid family is N-acetyl-
neuraminic acid (2-
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keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid
(often
abbreviated as Neu5Ac, NeuAc, or NANA). A second member of the family is N-
glycolyl-
neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is
hydroxylated.
A third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN)
(Nadano et al.
(1986)J Biol. Chem. 261: 11550-11557; Kanamori et al., I Biol. Chem. 265:
21811-21819
(1990)). Also included are 9-substituted sialic acids such as a 9-0-C1-C6 acyl-
Neu5Ac like
9-0-lactyl-Neu5Ac or 9-0-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-
deoxy-
Neu5Ac. For review of the sialic acid family, see, e.g., Varki, Glycobiology
2: 25-40 (1992);
Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-
Verlag, New
York (1992)). The synthesis and use of sialic acid compounds in a sialylation
procedure is
disclosed in international application WO 92/16640, published October 1, 1992.
[0034] As used herein, the term "modified sugar," refers to a naturally- or
non-naturally-
occurring carbohydrate that is enzymatically added onto an amino acid or a
glycosyl residue
of a peptide in a process of the invention. The modified sugar is selected
from a number of
enzyme substrates including, but not limited to sugar nucleotides (mono-, di-,
and tri-
phosphates), activated sugars (e.g., glycosyl halides, glycosyl mesylates) and
sugars that are
neither activated nor nucleotides. The "modified sugar" is covalently
functionalized with a
"modifying group." Useful modifying groups include, but are not limited to,
water-soluble
polymers, therapeutic moieties, diagnostic moieties, biomolecules and the
like. The
modifying group is preferably not a naturally occurring, or an unmodified
carbohydrate. The
locus of functionalization with the modifying group is selected such that it
does not prevent
the "modified sugar" from being added enzymatically to a peptide.
[0035] The term "water-soluble" refers to moieties that have some detectable
degree of
solubility in water. Methods to detect and/or quantify water solubility are
well known in the
art. Exemplary water-soluble polymers include peptides, saccharides,
poly(ethers),
poly(amines), poly(carboxylic acids) and the like. Peptides can have mixed
sequences or be
composed of a single amino acid, e.g., poly(lysine). An exemplary
polysaccharide is
poly(sialic acid). An exemplary poly(ether) is poly(ethylene glycol), e.g., m-
PEG.
Poly(ethylene imine) is an exemplary polyamine, and poly(acrylic) acid is a
representative
poly(carboxylic acid). Exemplary polymers are typically comprised of 2-8
polymeric units.
[0036] The polymer backbone of the water-soluble polymer can be poly(ethylene
glycol) (i.e.
PEG). However, it should be understood that other related polymers are also
suitable for use
CA 02554466 2012-08-13
in the practice of this invention and that the use of the term PEG or
poly(ethylene glycol) is
intended to be inclusive and not exclusive in this respect. The term PEG
includes
poly(ethylene glycol) in any of its forms, including alkoxy PEG, difunctional
PEG,
multiarmed PEG, forked PEG, branched PEG, 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.
[00371 The polymer backbone can be linear or branched. Branched polymer
backbones are
generally known in the art. 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 commonly
used in branched forms that can be prepared by addition of ethylene oxide to
various polyols,
such as glycerol, pentaerythritol and sorbitoI. 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)õ, in which R represents the core
moiety, such as
glycerol or pentaerythritol, and m represents the number alarms. Multi-armed
PEG
molecules, such as those described in U.S. Pat. No. 5,932,462,
can also be used as the polymer backbone.
[0038) Many other polymers are also suitable for the invention. Polymer
backbones that are
non-peptidic and water-soluble, with from 2 to about 300 termini, are
particularly useful in
the invention. Examples of suitable polymers include, but are not limited to,
other
poly(alkylene glycols), such as poly(propylene glycol) ("PPG"), copolymers of
ethylene
glycol and propylene glycol and the like, poly(oxyethylated polyol),
poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxypropyhnethacrylamide), poly(oc-hydroxy
acid),
poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-
acryloylmoiPholine), such as
described in U.S. Pat. No. 5,629,384,
and copolymers, telpolymers, and mixtures thereof. Although the molecular
weight of each
chain of the polymer backbone can vary, it is typically in the range of from
about 100 Da to
about 100,000 Da, often from about 6,000 Da to about 80,000 Da.
[0039] The term "glycoconjugation," as used herein, refers to the
enzymatically mediated
conjugation of a modified sugar species to an amino acid or glycosyl residue
of a
polypeptide, e.g., a mutant human growth hormone of the present invention. A
subgenus of
"glycoconjugation" is "glycol-PEGylation," in which the modifying group of the
modified
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sugar is poly(ethylene glycol), and alkyl derivative (e.g., m-PEG) or reactive
derivative (e.g.,
H2N-PEG, HOOC-PEG) thereof.
[0040] The term, "glycosyl linking group," as used herein refers to a glycosyl
residue to
which a modifying group (e.g., PEG moiety, therapeutic moiety, biomolecule) is
covalently
attached; the glycosyl linking group joins the modifying group to the
remainder of the
conjugate. In the methods of the invention, the "glycosyl linking group"
becomes covalently
attached to a glycosylated or unglycosylated peptide, thereby linking the
agent to an amino
acid and/or glycosyl residue on the peptide. A "glycosyl linking group" is
generally derived
from a "modified sugar" by the enzymatic attachment of the "modified sugar" to
an amino
acid and/or glycosyl residue of the peptide. The glycosyl linking group can be
a saccharide-
derived structure that is degraded during formation of modifying group-
modified sugar
cassette (e.g., oxidation¨>Schiff base formation¨>reduction), or the glycosyl
linking group
may be intact. An "intact glycosyl linking group" refers to a linking group
that is derived
from a glycosyl moiety in which the saccharide monomer that links the
modifying group and
to the remainder of the conjugate is not degraded, e.g., oxidized, e.g., by
sodium
metaperiodate. "Intact glycosyl linking groups" of the invention may be
derived from a
naturally occurring oligosaccharide by addition of glycosyl unit(s) or removal
of one or more
glycosyl unit from a parent saccharide structure.
[0041] Where substituent groups are specified by their conventional chemical
formulae,
written from left to right, they equally encompass the chemically identical
substituents, which
would result from writing the structure from right to left, e.g., -CH20- is
intended to also
recite ¨OCH2-.
[0042] The term "alkyl," by itself or as part of another substituent means,
unless otherwise
stated, a straight or branched chain, or cyclic hydrocarbon radical, or
combination thereof,
which may be fully saturated, mono- or polyunsaturated and can include di- and
multivalent
radicals, having the number of carbon atoms designated (i.e. C1-C10 means one
to ten
carbons). Examples 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, homologs and isomers of, for example, n-
pentyl, n-
hexyl, 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-
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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 below, such as "heteroalkyl." Alkyl groups that are
limited to
hydrocarbon groups are termed "homoalkyl".
[0043] The term "alkylene" by itself or as part of another substituent means a
divalent radical
derived from an alkane, as exemplified, but not limited, by ¨CH2CH2CH2CH2-,
and further
includes those groups described below as "heteroalkylene." Typically, an alkyl
(or alkylene)
group will have from 1 to 24 carbon atoms, with those groups having 10 or
fewer carbon
atoms being preferred in the present invention. A "lower alkyl" or "lower
alkylene" is a
shorter chain alkyl or alkylene group, generally having eight or fewer carbon
atoms.
[0044] The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are
used in their
conventional sense, and refer to those alkyl groups attached to the remainder
of the molecule
via an oxygen atom, an amino group, or a sulfur atom, respectively.
[0045] The term "heteroalkyl," by itself or in combination with another term,
means, unless
otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon
radical, or
combinations thereof, consisting of the stated number of carbon atoms and at
least one
heteroatom selected from the group consisting of 0, N, Si and 5, and wherein
the nitrogen
and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may
optionally be
quaternized. The heteroatom(s) 0, N and S and Si may be 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, -CH2-C112-0-CH3, -
CH2-CH2-NH-
CH3, -CH2-CH2-N(CH3)-C113, -CH2-S-CH2-CH3, -CH2-CH2,-S(0)-CH3, -CH2-CH2-S(0)2-
CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and ¨CH=CH-N(CH3)-CH3. Up to
two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and
¨CH2-0-
Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another
substituent
means a divalent radical derived from heteroalkyl, as exemplified, but not
limited by, -CH2-
CH2-S-CH2-CH2- and ¨CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups,
heteroatoms
can also occupy either or both of the chain termini (e.g., alkyleneoxy,
alkylenedioxy,
alkyleneamino, alkylenediamino, 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. For example, the formula ¨C(0)2R'-
represents both
¨C(0)2R'- and ¨R'C(0)2-.
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[0046] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in
combination with
other terms, represent, unless otherwise stated, cyclic versions of "alkyl"
and "heteroalkyl",
respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at
which the heterocycle is attached to the remainder of the molecule. Examples
of cycloalkyl
include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-
cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not
limited to, 1 -
(1,2,5,6-tetrahydropyridy1), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-
morpholinyl, 3-
morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,
tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.
[0047] The terms "halo" or "halogen," by themselves or as part of another
substituent, mean,
unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
Additionally, terms
such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For
example, the
term "halo(Ci-C4)alkyl" is mean to include, but not be limited to,
trifluoromethyl, 2,2,2-
trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
[0048] The term "aryl" means, unless otherwise stated, a polyunsaturated,
aromatic,
substituent that can be a single ring or multiple rings (preferably from 1 to
3 rings), which are
fused together or linked covalently. The term "heteroaryl" refers to aryl
groups (or rings) that
contain from one to four heteroatoms selected from N, 0, and S, wherein the
nitrogen and
sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally
quaternized. A
heteroaryl group can be attached to the remainder of the molecule through a
heteroatom.
Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-
naphthyl, 2-naphthyl,
4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-
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-pyrimidyl, 5-
benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-
quinoxalinyl, 3-
quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl, 2,3-
dihydrobenzo[1,4]dioxin-6-yl,
benzo[1,3]dioxo1-5-y1 and 6-quinolyl. Substituents for each of the above noted
aryl and
heteroaryl ring systems are selected from the group of acceptable substituents
described
below.
[0049] For brevity, the term "aryl" when used in combination with other terms
(e.g., aryloxy,
arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined
above. Thus, the
14
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WO 2005/072371 PCT/US2005/002522
term "arylalkyl" is meant to include those radicals in which an aryl group is
attached to an
alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including
those alkyl groups
in which a carbon atom (e.g., a methylene group) has been replaced by, for
example, an
oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,
and the
like).
[0050] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl" and
"heteroaryl") is
meant to include both substituted and unsubstituted forms of the indicated
radical. Preferred
substituents for each type of radical are provided below.
[0051] Substituents for the alkyl and heteroalkyl radicals (including those
groups often
referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl,
cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are generically
referred to as "alkyl
group substituents," and they can be one or more of a variety of groups
selected from, but not
limited to: -OR', =0, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R", -
0C(0)R', -
C(0)R', -CO2R', -CONR'R", -0C(0)NR'R", -NR"C(0)R', -NW-C(0)NR"R", -
NR"C(0)2R', -NR-C(NR'R"R'")=NR'", -NR-C(NR'R")=NR'", -S(0)R', -S(0)2R', -
S(0)2NR'R", -NRSO2R', -CN and ¨NO2 in a number ranging from zero to (2m'+1),
where
in' is the total number of carbon atoms in such radical. R', R", R" and R'"
each preferably
independently refer to hydrogen, substituted or unsubstituted heteroalkyl,
substituted or
unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or
unsubstituted alkyl,
alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the
invention
includes more than one R group, for example, each of the R groups is
independently selected
as are each R', R", R" and R'" groups when more than one of these groups is
present. When
R' and R" 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, -NR'R" is meant to
include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of
substituents,
one of skill in the art will understand that the term "alkyl" is meant to
include groups
including carbon atoms bound to groups other than hydrogen groups, such as
haloalkyl (e.g.,
-CF3 and ¨CH2CF3) and acyl (e.g., -C(0)CH3, -C(0)CF 3, -C(0)CH2OCH3, and the
like).
[0052] Similar to the substituents described for the alkyl radical,
substituents for the aryl and
heteroaryl groups are generically referred to as "aryl group substituents."
The substituents
are selected from, for example: halogen, -OR', =0, =NR', =N-OR', -NR'R", -SR',
-halogen,
-SiR'R"R'", -0C(0)R', -C(0)R', -CO2R', -CONR'R", -0C(0)NR'R", -NR"C(0)R',
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
-NR'-C(0)NR"R", -NR"C(0)2R', -NR-C(NR'R"R'")=NR'", -NR-C(NR'R")=NR'", -
S(0)R', -S(0)2R', -S(0)2NR'R", -NRSO2R', -CN and ¨NO2, -N3,
-CH(Ph)2, fluoro(Ci-
C4)alkoxy, and fluoro(Ci-C4)alkyl, in a number ranging from zero to the total
number of open
valences on the aromatic ring system; and where R', R", R" and R'" are
preferably
independently selected from hydrogen, substituted or unsubstituted alkyl,
substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted
or unsubstituted
heteroaryl. When a compound of the invention includes more than one R group,
for example,
each of the R groups is independently selected as are each R', R", R" and R"
groups when
more than one of these groups is present. In the schemes that follow, the
symbol X
represents "R" as described above.
[0053] Two of the substituents on adjacent atoms of the aryl or heteroaryl
ring may
optionally be replaced with a substituent of the formula ¨T-C(0)-(CRR')q-U-,
wherein T and
U are independently ¨NR-, -0-, -CRR'- or a single bond, and q is an integer of
from 0 to 3.
Alternatively, two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may
optionally be replaced with a substituent of the formula ¨A-(CH2),-B-, wherein
A and B are
independently ¨CRR'-, -0-, -NR-, -S-, -S(0)-, -S(0)2-, -S(0)2NR'- or a single
bond, and r is
an integer of from 1 to 4. One of the single bonds of the new ring so formed
may optionally
be replaced with a double bond. Alternatively, two of the substituents on
adjacent atoms of
the aryl or heteroaryl ring may optionally be replaced with a substituent of
the formula ¨
(CRR'),-X-(CR"R'")d-, where s and d are independently integers of from 0 to 3,
and X is ¨0-
, -NR'-, -S-, -S(0)-, -S(0)2-, or ¨S(0)2NR'-. The substituents R, R', R" and
R" are
preferably independently selected from hydrogen or substituted or
unsubstituted (Ci-C6)alkyl.
[0054] As used herein, the term "heteroatom" is meant to include oxygen (0),
nitrogen (N),
sulfur (S) and silicon (Si).
[0055] The use of reactive derivatives of PEG (or other linkers) to attach one
or more peptide
moieties to the linker is within the scope of the present invention. The
invention is not
limited by the identity of the reactive PEG analogue. Many activated
derivatives of
poly(ethyleneglycol) are available commercially and in the literature. It is
well within the
abilities of one of skill to choose, and synthesize if necessary, an
appropriate activated PEG
derivative with which to prepare a substrate useful in the present invention.
See, Abuchowski
etal. Cancer Biochem. Biophys., 7: 175-186 (1984); Abuchowski et al., .I.
Biol. Chem., 252:
3582-3586 (1977); Jackson et al., Anal. Biochem., 165: 114-127 (1987); Koide
et al.,
16
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
Biochem Biophys. Res. Commun., 111: 659-667 (1983)), tresylate (Nilsson et
al., Methods
Enzyniol., 104: 56-69 (1984); Delgado et al., Biotechnol. App!. Biochem., 12:
119-128
(1990)); N-hydroxysuccinimide derived active esters (Buckmann et al.,
Makromol. Chem.,
182: 1379-1384 (1981); Joppich et al., Makromol. Chem., 180: 1381-1384 (1979);
Abuchowski et al., Cancer Biochem. Biophys., 7: 175-186 (1984); Katreet al.
Proc. Natl.
Acad. Sci. U.S.A., 84: 1487-1491 (1987); Kitamura et al., Cancer Res., 51:
4310-4315
(1991); Boccu et al., Z. Naturforsch., 38C: 94-99 (1983), carbonates (Zalipsky
et al.,
POLY(ETHYLENE GLYCOL) CHEMISTRY: BIOTECHNICAL AND BIOMEDICAL APPLICATIONS,
Harris, Ed., Plenum Press, New York, 1992, pp. 347-370; Zalipsky etal.,
Biotechnol. App!.
Biochem., 15: 100-114 (1992); Veronese etal., Appl. Biochem. Biotech., 11: 141-
152
(1985)), imidazolyl formates (Beauchamp et al., Anal. Biochem., 131: 25-33
(1983); Berger
et al., Blood, 71: 1641-1647 (1988)), 4-dithiopyridines (Woghiren etal.,
Bioconjugate
Chem., 4: 314-318 (1993)), isocyanates (Byun etal., ASAIO Journal, M649-M-653
(1992))
and epoxides (U.S. Pat. No. 4,806,595, issued to Noishiki etal., (1989). Other
linking groups
include the urethane linkage between amino groups and activated PEG. See,
Veronese, et al.,
App!. Biochem. Biotechnol., 11: 141-152 (1985).
[0056] The term "amino acid" refers to naturally occurring and synthetic amino
acids, as well
as amino acid analogs and amino acid mimetics. Naturally occurring amino acids
are those
encoded by the genetic code, as well as those amino acids that are later
modified, e.g.,
hydroxyproline, 7-carboxyglutamate, and 0-phosphoserine. Amino acid analogs
refers to
compounds that have the same basic chemical structure as a naturally occurring
amino acid,
i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino
group, and an R
group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl
sulfonium.
Such analogs have modified R groups (e.g., norleucine) or modified peptide
backbones, but
retain the same basic chemical structure as a naturally occurring amino acid.
"Amino acid
mimetics" refers to chemical compounds having a structure that is different
from the general
chemical structure of an amino acid, but that functions in a manner similar to
a naturally
occurring amino acid.
[0057] "Peptide" refers to a polymer in which the monomers are amino acids,
amino acid
analogues and/or amino acid mimetics and are joined together through amide
bonds,
alternatively referred to as a polypeptide. Additionally, unnatural amino
acids, for example,
13-alanine, phenylglycine and homoarginine are also included. Amino acids that
are not gene-
17
CA 02554466 2006-07-26
WO 2005/072371
PCT/US2005/002522
encoded may also be used in the present invention. Furthermore, amino acids
that have been
modified to include reactive groups, glycosylation sites, polymers,
therapeutic moieties,
biomolecules and the like may also be used in the invention. All of the amino
acids used in
the present invention may be either the D - or L -isomer. The L -isomer is
generally preferred.
In addition, other peptidomimetics are also useful in the present invention.
As used herein,
"peptide" refers to both glycosylated and unglycosylated peptides. Also
included are petides
that are incompletely glycosylated by a system that expresses the peptide. For
a general
review, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS,
PEPTIDES
AND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).
[0058] The term "nucleoside" refers to a glycosylamine that is a component of
a nucleic acid
and that comprises a nitrogenous base linked either to p-D-ribofuranose to
form a
ribonucleoside, or to 2-deoxy-p-D-ribofuranose to form a deoyribonucleoside.
The base may
be a purine e.g., adenine or guanosine, or a pyrimidine e.g., thymidine,
cytidine, uridine or
pseudouridine. Nucleoside also includes the unusual nucleoside used by
microorganisms.
[0059] The term "targeting moiety," as used herein, refers to species that
will selectively
localize in a particular tissue or region of the body. The localization is
mediated by specific
recognition of molecular determinants, Molecular size of the targeting agent
or conjugate,
ionic interactions, hydrophobic interactions and the like. Other mechanisms of
targeting an
agent to a particular tissue or region are known to those of skill in the art.
Exemplary
targeting moieties include antibodies, antibody fragments, transferrin, HS-
glycoprotein,
coagulation factors, serum proteins, 0-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO
and the
like.
[0060] As used herein, "therapeutic moiety" means any agent useful for therapy
including,
but not limited to, antibiotics, anti-inflammatory agents, anti-tumor drugs,
cytotoxins, and
radioactive agents. "Therapeutic moiety" includes prodrugs of bioactive
agents, constructs in
which more than one therapeutic moiety is bound to a carrier, e.g, multivalent
agents.
Therapeutic moiety also includes proteins and constructs that include
proteins. Exemplary
proteins include, but are not limited to, Erythropoietin (EPO), Granulocyte
Colony
Stimulating Factor (GCSF), Granulocyte Macrophage Colony Stimulating Factor
(GMCSF),
Interferon (e.g., Interferon-a, -p,
Interleukin (e.g., Interleukin II), serum proteins (e.g.,
Factors VII, VIIa, VIII, IX, and X), Human Chorionic Gonadotropin (HCG),
Follicle
18
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WO 2005/072371 PCT/US2005/002522
Stimulating Hormone (FSH) and Lutenizing Hormone (LH) and antibody fusion
proteins
(e.g. Tumor Necrosis Factor Receptor ((TNFR)/Fc domain fusion protein)).
[0061] As used herein, "anti-tumor drug" means any agent useful to combat
cancer including,
but not limited to, cytotoxins and agents such as antimetabolites, alkylating
agents,
anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea,
asparaginase,
corticosteroids, interferons and radioactive agents. Also encompassed within
the scope of the
ten-n "anti-tumor drug," are conjugates of peptides with anti-tumor activity,
e.g. TNF-a.
Conjugates include, but are not limited to those formed between a therapeutic
protein and a
glycoprotein of the invention. A representative conjugate is that formed
between PSGL-1
and TNF-a.
[0062] As used herein, "a cytotoxin or cytotoxic agent" means any agent that
is detrimental to
cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine,
mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin,
daunorubicin, dihydroxy anthracinedione, mitoxantrone, mithramycin,
actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and
puromycin and analogs or homologs thereof. Other toxins include, for example,
ricin, CC-
1065 and analogues, the duocarmycins. Still other toxins include diptheria
toxin, and snake
venom (e.g., cobra venom).
[0063] As used herein, "a radioactive agent" includes any radioisotope that is
effective in
diagnosing or destroying a tumor. Examples include, but are not limited to,
indium-111,
cobalt-60. Additionally, naturally occurring radioactive elements such as
uranium, radium,
and thorium, which typically represent mixtures of radioisotopes, are suitable
examples of a
radioactive agent. The metal ions are typically chelated with an organic
chelating moiety.
[0064] Many useful chelating groups, crown ethers, cryptands and the like are
known in the
art and can be incorporated into the compounds of the invention (e.g., EDTA,
DTPA, DOTA,
NTA, HDTA, etc. and their phosphonate analogs such as DTPP, EDTP, HDTP, NTP,
etc).
See, for example, Pitt et al., "The Design of Chelating Agents for the
Treatment of Iron
Overload," In, INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE; Martell, Ed.;
American
Chemical Society, Washington, D.C., 1980, pp. 279-312; Lindoy, THE CHEMISTRY
OF
MACROCYCLIC LIGAND COMPLEXES; Cambridge University Press, Cambridge,1989;
Dugas,
19
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WO 2005/072371 PCT/US2005/002522
BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, and references
contained
therein.
[0065] Additionally, a manifold of routes allowing the attachment of chelating
agents, crown
ethers and cyclodextrins to other molecules is available to those of skill in
the art. See, for
example, Meares et al., "Properties of In Vivo Chelate-Tagged Proteins and
Polypeptides."
In, MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICAL ASPECTS;"
Feeney, et al., Eds., American Chemical Society, Washington, D.C., 1982, pp.
370-387;
Kasina et al., Bioconjugate Chem., 9: 108-117 (1998); Song et al.,
Bioconjugate Chem., 8:
249-255 (1997).
INTRODUCTION
[0066] The present invention provides polymeric species, and sugars, activated
sugars, and
nucleotide sugars that are conjugated to these polymers. The polymeric
conjugates of the
nucleotide sugars are generally substrates for an enzyme that transfers the
sugar moiety and
its polymeric substituent onto an appropriate acceptor moiety of a substrate.
Accordingly, the
invention also provides substrates modified by glycoconjugation using a
polymeric conjugate
of a nucleotide sugar and an appropriate enzyme. Substrates that can be
glycoconjugated
using a compound of the invention include peptides, e.g., glycopeptides,
lipids, e.g.,
glycolipids and aglycones (sphingosines, ceramides).
[0067] As discussed in the preceding sections, art-recognized chemical methods
of covalent
PEGylation rely on chemical conjugation through reactive groups on amino acids
or
carbohydrates. Through careful design of the conjugate and the reaction
conditions, useful
conjugates have been prepared using chemically-mediated conjugation
strategies. A major
shortcoming of chemical conjugation of polymers to proteins or glycoproteins
is the lack of
selectivity of the activated polymers, which often results in attachment of
polymers at sites
implicated in protein or glycoprotein bioactivity. Several strategies have
been developed to
address site selective conjugation chemistries, however, only one universal
method suitable
for a variety of recombinant proteins has been developed.
[0068] In contrast to art-recognized methods, the present invention provides
compounds that
are of use in a novel strategy for highly selective, site-directed
glycoconjugation of branched
water-soluble polymers, e.g., glyco-PEGylation. In an exemplary embodiment of
the
invention, site directed attachment of branched water-soluble polymers is
accomplished by in
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
vitro enzymatic glycosylation of specific peptide sequences using a nucleotide
sugar or
activated sugar of the invention. Glyco-conjugation can be performed
enzymatically utilizing
a glycosyltransferase, e.g., a sialyltransferase, capable of transferring the
species branched
water-soluble polymer-sugar, e.g., PEG-sialic acid, to a glycosylation site
("glyco-
PEGylation").
[0069] As discussed above, the present invention provides a conjugate between
a sugar
having any desired carbohydrate structure, modified with a polymeric moiety.
Sugar
nucleotides and activated sugars based on these sugar structures are also a
component of the
invention. The polymeric modifying moiety is attached to the sugar moiety by
enzymatic
means, chemical means or a combination thereof, thereby producing a modified
nucleotide
sugar. The sugars are substituted with the polymeric modifying moiety at any
desired
position. In an exemplary embodiment, the sugar is a furanose that is
substituted at one or
more of C-1, C-2, C-3, C-4 or C-5. In another embodiment, the invention
provides a
pyranose that is substituted with the polymeric modifying moiety at one or
more of C-1, C-2,
C-3, C-4, C-5 or C-6. Preferably, the polymeric modifying moiety is attached
directly to an
oxygen, nitrogen or sulfur pendent from the carbon. Alternatively, the
polymeric modifying
moiety is attached to a linker that is interposed between the sugar and the
modifying moiety.
The linker is attached to an oxygen, nitrogen or sulfur pendent from the
selected carbon.
[0070] In a presently preferred embodiment, the polymeric modifying moiety is
appended to
a position, that is selected such that the resulting conjugate functions as a
substrate for an
enzyme used to ligate the modified sugar moiety to another species, e.g.,
peptide,
glycopeptide, lipid, glycolipid, etc. Exemplary enzymes are discussed in
greater detail herein
and include glycosyl transferases (sialyl transferases, glucosyl transferases,
galactosyl
transferases, N-acetylglucosyl transferases, N-acetylgalactosyl transferases,
mannosyl
transferases, fucosyl transferases, etc.). Exemplary sugar nucleotide and
activated sugar
conjugates of the invention also include substrates for mutant glycosidases
and mutant
glycoceramidases that are modified to have synthetic, rather than hydrolytic
activity.
[0071] In an exemplary embodiment, the conjugate of the invention includes a
sugar,
activated sugar or nucleotide sugar that is conjugated to one or more polymer,
e.g. a branched
polymer. Exemplary polymers include both water-soluble and water-insoluble
species.
[0072] In an exemplary embodiment, the polymeric modifying group is directly
or indirectly
attached to a pyranose or a furanose . For example:
21
CA 02554466 2012-08-13
=
(R6.)d
R1
R2 Re."
; and
ReR3
R4 R3
R4
I It
In Formulae I and It, RI is H, CH2OR7, COOR7 or OR7, in which R7 represents H,
substituted
or =substituted alkyl or substituted or =substituted heteroalkyl. R2 is H, OH,
NH or a
moiety that includes a nucleotide. An exemplary R2 species according to this
embodiment
has the formula:
0
0- 1.3
in which Xi represents 0 or NH and R8 is a nucleoside.
100731 100741 The symbols R3, R4, R5, R6 and R6'
independently represent H, substituted or
=substituted alkyl, OR9, NHC(0)R1 . The index d is 0 or 1. R9 and RI are
independently
selected from H, substituted or =substituted alkyl, substituted or
unsubstituted heteroalkyl or
sialic acid. At least one of R3, R4, R5, R6, and R6' includes the polymeric
modifying moiety,
e.g., PEG. In an exemplary embodiment, R6 and R6', together with the carbon to
which they
are attached are components of the side chain of sialic acid. In still a
further exemplary
embodiment, this side chain is modified with the polymeric modifying moiety
(or a linker-
polymeric modifying moiety) at one or more of C-6, C-7 or C-9.
[00751 The symbols R3, R4, R5 and R6 independently represent H, OR9, NHC(0)RI0
. R9 and
Rt are independently selected from H, substituted or unsubstituted alkyl or
substituted or
=substituted heteroalkyl. At least one of R3, R4, R5, R6, or R6' include the
polymeric
modifying moiety.
[00761 In another exemplary embodiment, the sugar moiety is a sialic acid
moiety that has
been oxidized and conjugated to a polymeric modifying moiety.
22
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WO 2005/072371 PCT/US2005/002522
[0077] In an exemplary embodiment, the polymeric modifying moiety is joined to
the sugar
core through a linker:
(R11)õõ
in which R11 is the polymeric moiety and L is selected from a bond and a
linking group, and
w is an integer from 1-6, preferably 1-3 and more preferably, 1-2.
[0078] When L is a bond it is formed between a reactive functional group on a
precursor of
R11 and a reactive functional group of complementary reactivity on a precursor
of L. As set
forth herein, the selection and preparation of precursors with appropriate
reactive functional
groups is within the ability of those skilled in the art. Moreover, combining
the precursors
proceed by chemistries that are well-understood in the art.
[0079] In an exemplary embodiment L is a linking group that is formed from an
amino acid,
an amino acid mimetic, or small peptide (e.g., 1-4 amino acid residues)
providing a modified
sugar in which the polymeric modifying moiety is attached through a
substituted alkyl linker.
The linker is formed through reaction of the amine moiety and carboxylic acid
(or a reactive
derivative, e.g., active ester, acid halide, etc.) of the amino acid with
groups of
complementary reactivity on the precursors to L and R11. The elements of the
conjugate can
be conjugated in essentially any convenient order. For example the precursor
to L can be in
place on the saccharide core prior to conjugating the precursors of R11 and L.
Alternatively,
an R11-L cassette, bearing a reactive functionality on L can be prepared and
subsequently
linked to the saccharide through a reactive functional group of complementary
reactivity on
this species.
[0080] In an exemplary embodiments, the polymeric modifying moiety is R3
and/or R6. In
another exemplary embodiment, R3 and/or R6 includes both the polymeric
modifying moiety
and a linker, L, joining the polymeric moiety to the remainder of the
molecule. In another
exemplary embodiment, the polymeric modifying moiety is R3. And, in a further
exemplary
embodiment, R3 includes both the polymeric modifying moiety and a linker, L,
joining the
polymeric moiety to the remainder of the molecule. In yet another exemplary
embodiment in
which the sugar is a sialic acid, the polymeric modifying moiety is at R5 or
attached at a
position of the sialic acid side chain, e.g., C-9.
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Linear Polymer Conjugates
[0081] In an exemplary embodiment, the present invention provides a sugar or
activated
sugar conjugate or nucleotide sugar conjugate that is formed between a linear
polymer, such
as a water-soluble or water-insoluble polymer. In the conjugates of the
invention, the
polymer is attached to a sugar, activated sugar or sugar nucleotide. As
discussed herein, the
polymer is linked to the sugar moiety, either directly or through a linker.
[0082] An exemplary compound according to this embodiment has a structure
according to
Formulae I or II, in which at least one of R1, R3, R4, R5 or R6 has the
formula:
N R
[0083] Another example according to this embodiment has the formula:
HC(0)(CH2)5¨N HC(0)_Ri
in which s is an integer from 0 to 20 and R11 is a linear polymeric modifying
moiety.
[0084] PEG moieties of any molecular weight, e.g., 2 Kda, 5 Kda, 10 Kda, 20
Kda, 30 Kda
and 40 Kda are of use in the present invention.
Branched Polymer Conjugates
[0085] In an exemplary embodiment, the polymeric modifying moiety is a
branched structure
that includes two or more polymeric chains attached to central moiety, having
the formula:
in which R11 and L are as discussed above and w' is an integer from 2 to 6,
preferably from 2
to 4 and more preferably from 2 to 3.
[0086] An exemplary precursor of use to form the conjugates according to this
embodiment
of the invention has the formula:
R12¨x2
X5¨C¨X3'
R13¨X4
(III).
[0087] The branched polymer species according to this formula are essentially
pure
24
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WO 2005/072371 PCT/US2005/002522
water-soluble polymers. X3' is a moiety that includes an ionizable, e.g.,
COOH, H2PO4,
HS03, HP03, etc.) or other reactive functional group, e.g., infra. C is
carbon. X5 is
preferably a non-reactive group (e.g., H, unsubstituted alkyl, unsubstituted
heteroalkyl), and
can be a polymeric arm. R12 and 1213 are independently selected polymeric
arms, e.g.,
nonpeptidic, nonreactive polymeric arms. X2 and X4 are linkage fragments that
are
preferably essentially non-reactive under physiological conditions, which may
be the same or
different. Alternatively, these linkages can include one or more moiety that
is designed to
degrade under physiologically relevant conditions, e.g., esters, disulfides,
etc. X2 and X4 join
polymeric arms R12 and R13 to C. When X3' is reacted with a reactive
functional group of
complementary reactivity on a linker, sugar or linker-sugar cassette, X3' is
converted to a
component of linkage fragment X3.
[0088] Exemplary linkage fragments for X2 and X4 include S, SC(0)NH, HNC(0)S,
SC(0)0, 0, NH, NHC(0), (0)CNH and NHC(0)0, and OC(0)NH, CH2S, CH20 ,
CH2CH20, CH2CH2S, (CH2)a0, (CH2)aS or (CH2)aY'-PEG or (CH2)aY'-PEG wherein Y'
is S
or 0 and a is an integer from 1 to 50.
[0089] In an exemplary embodiment, the precursor (III), or activated
derivative thereof, is
bound to the sugar, activated sugar or sugar nucleotide through a reaction
between X3' and a
group of complementary reactivity on the sugar moiety. Alternatively, X3'
reacts with a
reactive functional group on a precursor to linker, L. One or more of R1, R3,
R4, R5 or R6 of
Formulae I and II can include the branched polymeric modifying moiety.
[0090] In an exemplary embodiment, the moiety:
X5¨C¨X3-4
¨X14
is the linker arm, L. In this embodiment, an exemplary linker is derived from
a natural or
unnatural amino acid, amino acid analogue or amino acid mimetic, or a small
peptide formed
from one or more such species. For example, certain branched polymers found in
the
compounds of the invention have the formula:
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
o
R12.__x2, a
X
X4
R13
[0091] Xa is a linking moiety that is formed by the reaction of a reactive
functional group on
a precursor of the branched polymeric modifying moiety and the sugar moiety,
or a precursor
to a linker. For example, when X3' is a carboxylic acid, it can be activated
and bound directly
to an amine group pendent from an amino-saccharide (e.g., Ga1NH2, G1eNH2,
ManNH2, etc.),
forming an Xa that is an amide. Additional exemplary reactive functional
groups and
activated precursors are described hereinbelow. The index c represents an
integer from 1 to
10. The other symbols have the same identity as those discussed above.
[0092] In another exemplary embodiment, Xa is a linking moiety formed with
another linker:
in which Xb is a linking moiety and is independently selected from those
groups set forth for
Xa, and L1 is a bond, substituted or unsubstituted alkyl or substituted or
unsubstituted
heteroalkyl.
[0093] Exemplary species for Xa and Xb include S, SC(0)NH, HNC(0)S, SC(0)0, 0,
NH,
NHC(0), (0)CNH and NHC(0)0, and OC(0)NH.
[0094] For example,
¨NHC(0)(CH2)s¨NHC(0)¨R11
in which s is an integer from 0 to 20 and R11 is a linear polymeric modifying
moiety.
[0095] In another exemplary embodiment, X4 is a peptide bond to R13, which is
an amino
acid, di-peptide or tri-peptide in which the alpha-amine moiety and/or side
chain heteroatom
is modified with a polymer.
[0096] In a further exemplary embodiment, R6 includes the branched polymeric
modifying
group and the modified sugar or nucleotide sugar has a formula that is
selected from:
26
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WO 2005/072371 PCT/US2005/002522
( dR6')
R12¨x2
(R9d
X5--C¨LaC)RIR
0 2
R13-4(3 ; and
R5R3 R13¨X13
R4 R3
R4
Iv V
in which the identity of the radicals represented by the various symbols is
the same as that
discussed hereinabove. La is a substituted or unsubstituted alkyl or
substituted or
unsubstituted heteroalkyl moiety. In an exemplary embodiment, La is a moiety
of the side
chain of sialic acid that is fimctionalized with the polymeric modifying
moiety as shown.
[0097] In yet another exemplary embodiment, the invention provides sugars and
nucleotide
sugars that have the formula:
0
R12.-)(2. L- 6 0 Ra R12__)(2" La.7yR2
X4 R2 X4
R13 R5 R3 R13 R4
;and R3
R4
VII
VI
The identity of the radicals represented by the various symbols is the same as
that discussed
hereinabove. As those of skill will appreciate, the linker arm in Formulae VI
and VIII is
equally applicable to other modified sugars set forth herein.
[0098] The embodiments of the invention set forth above are further
exemplified by
reference to species in which the polymer is a water-soluble polymer,
particularly
poly(ethylene glycol) ("PEG"), e.g., methoxy-poly(ethylene glycol) ("m-PEG").
Those of
skill will appreciate that the focus in the sections that follow is for
clarity of illustration and
the various motifs set forth using PEG as an exemplary polymer are equally
applicable to
species in which a polymer other than PEG is utilized.
Water-Soluble Polymers
[0099] Many water-soluble polymers are known to those of skill in the art and
are useful in
practicing the present invention. The term water-soluble polymer encompasses
species such
as saccharides (e.g., dextran, amylose, hyalouronic acid, poly(sialic acid),
heparans, heparins,
27
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WO 2005/072371 PCT/US2005/002522
etc.); poly (amino acids), e.g., poly(aspartic acid) and poly(glutamic acid);
nucleic acids;
synthetic polymers (e.g., poly(acrylic acid), poly(ethers), e.g.,
poly(ethylene glycol);
peptides, proteins, and the like. A polymer typically comprises at least two
polymeric units.
In an exemplary embodiment the polymer is from 2-25 units. In another
exemplary
embodiment the polymer comprises 2-8 polymeric units. The present invention
may be
practiced with any water-soluble polymer with the sole limitation that the
polymer must
include a point at which the remainder of the conjugate can be attached.
[0100] Methods for activation of polymers can also be found in WO 94/17039,
U.S. Pat. No.
5,324,844, 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. 5,28 1,698, and more WO 93/15189, and for
conjugation
between activated polymers and peptides, e.g. Coagulation Factor VIII (WO
94/15625),
hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No. 4,412,989),
ribonuclease and superoxide dismutase (Veronese at al., App. Biochem. Biotech.
11: 141-45
(1985)).
[0101] Preferred water-soluble polymers are those in which a substantial
proportion of the
polymer molecules in a sample of the polymer are of approximately the same
molecular
weight; such polymers are "homodisperse."
[0102] The present invention is further illustrated by reference to a
poly(ethylene glycol)
conjugate. Several reviews and monographs on the functionalization and
conjugation of PEG
are available. See, for example, Harris, Macronol. Chem. Phys. C25: 325-373
(1985);
Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme 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); and Bhadra, et
al.,
Pharmazie, 57:5-29 (2002). Routes for preparing reactive PEG molecules and
forming
conjugates using the reactive molecules are known in the art. For example,
U.S. Patent No.
5,672,662 discloses a water soluble and isolatable conjugate of an active
ester of a polymer
acid selected from linear or branched poly(alkylene oxides), poly(oxyethylated
polyols),
poly(olefinic alcohols), and poly(acrylomorpholine).
[0103] U.S. Patent No. 6,376,604 sets forth a method for preparing a water-
soluble
1-benzotriazolylcarbonate ester of a water-soluble and non-peptidic polymer by
reacting a
terminal hydroxyl of the polymer with di(1-benzotriazoyl)carbonate in an
organic solvent.
The active ester is used to form conjugates with a biologically active agent
such as a protein
or peptide.
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WO 2005/072371 PCT/US2005/002522
[0104] WO 99/45964 describes a conjugate comprising a biologically active
agent and an
activated water soluble polymer comprising a polymer backbone having at least
one terminus
linked to the polymer backbone through a stable linkage, wherein at least one
terminus
comprises a branching moiety having proximal reactive groups linked to the
branching
moiety, in which the biologically active agent is linked to at least one of
the proximal reactive
groups. Other branched poly(ethylene glycol's) are described in WO 96/21469,
U.S. Patent
No. 5,932,462 describes a conjugate formed with a branched PEG molecule that
includes a
branched terminus that includes reactive functional groups. The free reactive
groups are
available to react with a biologically active species, such as a protein or
peptide, forming
conjugates between the poly(ethylene glycol) and the biologically active
species. U.S. Patent
No. 5,446,090 describes a bifunctional PEG linker and its use in forming
conjugates having a
peptide at each of the PEG linker termini.
[0105] Conjugates that include degradable PEG linkages are described in WO
99/34833; and
WO 99/14259, as well as in U.S. Patent No. 6,348,558. Such degradable linkages
are
applicable in the present invention.
[0106] The art-recognized methods of polymer activation set forth above are of
use in the
context of the present invention in the formation of the branched polymers set
forth herein
and also for the conjugation of these branched polymers to other species,
e.g., sugars, sugar
nucleotides and the like.
[0107] Exemplary modifying groups are discussed below. The modifying groups
can be
selected for their ability to impart to a peptide one or more desirable
property. Exemplary
properties include, but are not limited to, enhanced pharmacokinetics,
enhanced
pharmacodynamics, improved biodistribution, providing a polyvalent species,
improved
water solubility, enhanced or diminished lipophilicity, and tissue targeting.
[0108] Exemplary poly(ethylene glycol) molecules of use in the invention
include, but are
not limited to, those having the formula:
Ya
Xa¨ (CH2CH20)e(CH2)d¨ A1¨ A2
in which A2 is H, OH, NH2, substituted or unsubstituted alkyl, substituted or
unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl,
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WO 2005/072371 PCT/US2005/002522
substituted or unsubstituted heteroalkyl, e.g., acetal, OHC-, H2N-(CH2)q-, HS-
(CH2)q, or
-(CH2)qC(Yb)Zb. The index "e" represents an integer from 1 to 2500. The
indices b, d, and q
independently represent integers from 0 to 20. The symbols Za and Zb
independently
represent OH, NH2, leaving groups, e.g., imidazole, p-nitrophenyl, HOBT,
tetrazole, halide,
S-Ra, the alcohol portion of activated esters; -(CH2)pC(Yb)V, or -
(CH2)pU(CH2),C(Yb)v. The
symbol Ya represents H(2), =0, =S, =N-Rb. The symbols Xa, ya, yb, Al, and
independently represent the moieties 0, S, N-Rc. The symbol V represents OH,
NH2,
halogen, S-i, the alcohol component of activated esters, the amine component
of activated
amides, sugar-nucleotides, and proteins. The indices p, q, s and v are members
independently
selected from the integers from 0 to 20. The symbols Ra, Rb, and Re
independently represent
H, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heterocycloalkyl and
substituted or
unsubstituted heteroaryl.
[0109] Specific embodiments of linear and branched polymers, e.g., PEGs, of
use in the
invention include:
e
OH
H2N)Y
0 ;
e
OH
H2N
0 ;and
me' 0 )y
OH
HN
and carbonates and active esters of these species, such as:
HN io F
0 F
F ;and
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WO 2005/072371 PCT/US2005/002522
me0
f:r/rAy
0
HN)
hfle0/( )`0A0 F =
can be used to form the linear and branched polymeric species, linker arm
conjugates of these
species and conjugates between these compounds and sugars and nucleotide
sugars. The
indices e and f are independently selected from 1 to 2500.
[0110] Other exemplary activating, or leaving groups, appropriate for
activating linear PEGs
of use in preparing the compounds set forth herein include, but are not
limited to the species:
0 0
N=Nx
N--0 0-1 ;
0
0
N.=N
0o ;
0)r-
),zL
0 1,
0
0
F F 0
F =
0
;and 0
F F
It is well within the abilities of those of skill in the art to select an
appropriate activating
group for a selected moiety on the precursor to the polymeric modifying
moiety.
[0111] PEG molecules that are activated with these and other species and
methods of making
the activated PEGs are set forth in WO 04/083259.
[0112] In exemplary embodiments, the branched polymer is a PEG based upon a
cysteine,
serine, lysine, di- or tri-lysine core. Thus, further exemplary branched PEGs
include:
31
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WO 2005/072371
PCT/US2005/002522
NHC(0)0CH2CH2(OCH2CH2LOCH3
HO
NH2
HN
NHC(0)0CH2CH2(0CH2CH2)rOCH3
0
0
NHC(0)CH2CH2(OCH2CH2LOCH3
HN
HO
NH2
NHC(0)CH2CH2(OCH2CH2LOCH3
0
0 0
HOS¨(CH2CH20)eCH3
NHC(0)CH2CH2(OCH2CH2)rOCH3 NHC(0)0CH2CH2(OCH2CH2)/0CH3
0 0
1.100¨(CH2CH20)CH3 ; HO0¨(CH2CH20)0CH3
NHC(0)CH2CH2(OCH2CH2)100H3 NHC(0)0CH2CH2(OCH2CH2),OCH3
0 0
H0S¨(CH2CH20)CH3
NHC(0)CH2CH2OCH3 NHC(0)0CH3
, and
0
HOS¨(CH2CH20)õCH3
NHC(0)CH3
The indices e and fare independently selected from 1 to 2500.
[0113] In yet another embodiment, the branched PEG moiety is based upon a tri-
lysine
peptide. The tri-lysine can be mono-, di-, tri-, or tetra-PEG-ylated.
Exemplary species
according to this embodiment have the formulae:
0
HO ))22(223
0
NH )1\ ro.....,.............____NHC(0)0CH2CH2(OCH2CH2),OCH3
HN NH2 q"
0 HC(0 )0CH2C H2(OCH2CH2)rOCH3
; and
q.
NHc(o)cH2cH2(ocH2cH2LocH3
HO
0
NH
HN NH2 q"
HC(0)CH2CH2(OCH2CH2)r0CH3
0 q'
32
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WO 2005/072371 PCT/US2005/002522
in which e, f and? are independently selected integers from 1 to 2500; and q,
q' and q" are
independently selected integers from 0 to 20.
[0114] In exemplary embodiments of the invention, the PEG is m-PEG (5 kD, 10
kD, 20kD,
30 kD or 40IcD). An exemplary branched PEG species is a lysine, serine- or
cysteine-(m-
PEG)2 in which the na-PEG is a 20 Id) m-PEG.
[0115] As will be apparent to those of skill, the branched polymers of use in
the invention
include variations on the themes set forth above. For example the di-lysine-
PEG conjugate
shown above can include three polymeric subunits, the third bonded to the a-
amine shown as
unmodified in the structure above. Similarly, the use of a tri-lysine
fimctionalized with three
or four polymeric subunits is within the scope of the invention.
[0116] Those of skill in the art will appreciate that one or more of the m-PEG
arms of the
branched polymer can be replaced by a PEG moiety with a different terminus,
e.g., OH,
COOH, NH2, C2-Cio-alkyl, etc. Moreover, the structures above are readily
modified by
inserting alkyl linkers (or removing carbon atoms) between the oLcarbon atom
and the
functional group of the side chain. Thus, "homo" derivatives and higher
homologues, as well
as lower homologues are within the scope of cores for branched PEGs of use in
the present
invention,
[0117] The branched PEG species set forth herein are readily prepared by
methods such as
that set forth in the scheme helcw:.
NH2
HXbssrty0 NH2
H (Cok )VOTs "U'Mr µNo-(\/ cbsiOH
0 1 r
0
0
0
0-hs.-AYK-s.'0A0 II NO2
ir -0 NH
CH2C121TEA yOH
0
2
in which Xb is 0, NH or S and r is an integer from 1 to 10. The indices e and
fare
independently selected integers from 1 to 2500. Exemplary branched PEG species
are
10,000, 15,000, 20,000, 30,000 and 40,000 daltons.
33
SUBSTITUTE SHEET (RULE 26)
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[0118] Thus, according to this scheme, a natural or unnatural amino acid is
contacted with an
activated m-PEG derivative, in this case the tosylate, forming 1 by alkylating
the side-chain
heteroatom Xb. The mono-functionalized m-PEG amino acid is submitted to N-
acylation
conditions with a reactive m-PEG derivative, thereby assembling branched m-PEG
2. As one
of skill will appreciate, the tosylate leaving group can be instead any
suitable leaving group,
e.g., halogen, mesylate, triflate, etc. Similarly, the reactive carbonate
utilized to acylate the
amine can be instead an active ester, e.g., N-hydroxysuccinimide, etc., or the
acid can be
activated in situ using a dehydrating agent such as dicyclohexylcarbodiimide,
carbonyldiimidazole, etc.
[0119] In the exemplary scheme set forth above, the modifying group is a
linear PEG moiety,
however, any modifying group, e.g., water-soluble polymer, water-insoluble
polymer,
branched polymer, therapeutic moiety, etc., can be incorporated in a glycosyl
moiety through
[0120] Further branched polymeric species of use in the compounds of the
invention are
exemplified by branched cores functionalized with PEG, such as the examples
set forth
below:
o o
H3C' (:)0
; and
R14 RI 4
0 0
H30(00
C H3
'YcH HN
f
R14
in which R14 is OH or another reactive functional group. An exemplary reactive
functional
group is C(0)Q', in which Q' is selected such that C(0)Q' is a reactive
functional group.
Exemplary species for Q' include halogen, NHS, pentafluorophenyl, HOBT, HOAt,
and p-
nitrophenyl. The index "e" and the index "f' are integers independently
selected from 1 to
2500.
[0121] The branched compounds set forth above, and additional branched
compounds of use
in the compounds of the invention are readily prepared from such starting
materials as:
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WO 2005/072371 PCT/US2005/002522
02 y
HO OH HOOC C
CI H21\I iNH2
00OON Br Br
HO ; y; \_cc,
= ; and
OH OH OH 0-S02 OH
OH
Polymer Modified Sugar Species
[0122] The sugar moiety of the nucleotide sugars of the invention can be
selected from both
natural and unnatural furanoses and hexanoses. The unnatural saccharides
optionally include
an alkylated or acylated hydroxyl and/or amine moiety, e.g., ethers, esters
and amide
substituents on the ring. Other unnatural saccharides include an H, hydroxyl,
ether, ester or
amide substituent at a position on the ring at which such a substituent is not
present in the
natural saccharide. The sugar moiety can be a mono-, oligo- or poly-
saccharide.
[0123] Exemplary natural sugars of use in the present invention include
glucose, galactose,
fucose, mannose, xylanose, ribose, N-acetyl glucose, sialic acid and N-acetyl
galactose.
[0124] Similarly, the nucleoside can be selected from both natural and
unnatural or unusual
nucleosides. Exemplary natural nucleosides of use in the present invention
include cytosine,
thymine, guanine, adenine and uracil. Unusual nucleosides may include but are
not limited to
such molecules as spongouridin and spongothymidin. The art is replete with
structures of
unnatural and unusual nucleosides and methods of making them.
[0125] Exemplary modified sugar nucleotides of the invention include GDP-Man,
GDP-Fuc,
UDP-Gal, UDP-Gal-NH2,UDP-GalNAc, UDP-Glc, UDP-Glc-NH2, UDP-G1cNAc, LTDP-Glc,
LTDP-GlcUA and CMP-Sia. As with the sugars of the invention discussed above,
the sugar
nucleotides of the invention can be substituted with a polymeric modifying
moiety (or linker-
modifying moiety) at any position of the saccharide. For example, compounds
encompassed
by the invention include those in which the L-R11 moiety is conjugated to C-5
of a furanose-
based nucleotide sugar or C-6 of a pyranose-based nucleotide sugar.
[0126] Exemplary moieties attached to the conjugates disclosed herein include,
but are not
limited to, PEG derivatives (e.g., alkyl-PEG, acyl-PEG, acyl-alkyl-PEG, alkyl-
acyl-PEG
carbamoyl-PEG, aryl-PEG), PPG derivatives (e.g., alkyl-PPG, acyl-PPG, acyl-
alkyl-PPG,
alkyl-acyl-PPG carbamoyl-PPG, aryl-PPG), therapeutic moieties, diagnostic
moieties,
mannose-6-phosphate, heparin, heparan, SLex, mannose, mannose-6-phosphate,
Sialyl Lewis
X, FGF, VFGF, proteins, chondroitin, keratan, dermatan, albumin, integrins,
antennary
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oligosaccharides, peptides and the like. Methods of conjugating the various
modifying
groups to a saccharide moiety are readily accessible to those of skill in the
art (POLY
(ETHYLENE GLYCOL CHEMISTRY: BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, J.
Milton
Harris, Ed., Plenum Pub. Corp., 1992; POLY (ETHYLENE GLYCOL) CHEMICAL AND
BIOLOGICAL APPLICATIONS, J. Milton Harris, Ed., ACS Symposium Series No. 680,
American Chemical Society, 1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic
Press, San Diego, 1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG
DELIVERY
SYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society, Washington,
D.C.
1991).
[0127] Exemplary sugar nucleotides that of the present invention, in their
modified form,
include nucleotide mono-, di- or triphosphates or analogs thereof of a LTDP-
glycoside, CMP-
glycoside, or a GDP-glycoside. Even more preferably, the modified sugar
nucleotide is
selected from an UDP-galactose, LTDP-galactosamine, UDP-glucose, LTDP-
glucosamine,
GDP-mannose, GDP-facose, CMP-sialic acid, or CMP-NeuAc. N-acetylamine
derivatives of
the sugar nucleotides are also of use in the method of the invention.
[0128] In other embodiments, the modified sugar is an activated sugar.
Activated modified
sugars, which are useful in the present invention are typically glycosides
which have been
synthetically altered to include an activated leaving group. As used herein,
the term
"activated leaving group" refers to those moieties, which are easily displaced
in enzyme-
regulated nucleophilic substitution reactions. Many activated sugars are known
in the art.
See, for example, Vocadlo et al., In CARBOHYDRATE CHEMISTRY AND BIOLOGY, Vol.
2, Ernst
et al. Ed., Wiley-VCH Verlag: Weinheim, Germany, 2000; Kodama et al.,
Tetrahedron Lett.
34: 6419 (1993); Lougheed, et al., J. Biol. Chem. 274: 37717 (1999)).
[0129] Examples of activating groups (leaving groups) include fluoro, chloro,
bromo,
tosylate ester, mesylate ester, triflate ester and the like. Preferred
activated leaving groups,
for use in the present invention, are those that do not significantly
sterically encumber the
enzymatic transfer of the glycoside to the acceptor. Accordingly, preferred
embodiments of
activated glycoside derivatives include glycosyl fluorides and glycosyl
mesylates, with
glycosyl fluorides being particularly preferred. Among the glycosyl fluorides,
a-galactosyl
fluoride, a-mannosyl fluoride, a-glucosyl fluoride, a-fucosyl fluoride, a-
xylosyl fluoride, a-
sialyl fluoride, a-N-acetylglucosaminyl fluoride, a-N-acetylgalactosaminyl
fluo.ride, 13-
galactosyl fluoride, p-mannosyl fluoride, P-glucosyl fluoride, P-fucosyl
fluoride, P-xylosyl
36
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fluoride, 13-sialy1 fluoride, 13-N-acetylglucosaminyl fluoride and P-N-
acetylgalactosaminyl
fluoride are most preferred.
[0130] By way of illustration, glycosyl fluorides can be prepared from the
free sugar by first
acetylating the sugar and then treating it with HF/pyridine. This generates
the
thermodynamically most stable anomer of the protected (acetylated) glycosyl
fluoride (i.e.,
the a-glycosyl fluoride). If the less stable anom.er (i.e., the )3-glycosyl
fluoride) is desired, it
can be prepared by converting the peracetylated sugar with HBr/HOAc or with
HCI to
generate the anomeric bromide or chloride. This intermediate is reacted with a
fluoride salt
such as silver fluoride to generate the glycosyl fluoride. Acetylated glycosyl
fluorides may
be deprotected by reaction with mild (catalytic) base in methanol (e.g.
Na0Me/Me0H). In
addition, many glycosyl fluorides are commercially available.
[0131] Other activated glycosyl derivatives can be prepared using conventional
methods
known to those of skill in the art. For example, glycosyl mesylates can be
prepared by
treatment of the fully benzylated hemiacetal form of the sugar with mesyl
chloride, followed
by catalytic hydrogenation to remove the benzyl groups.
[0132] In a further exemplary embodiment, the modified sugar is an
oligosaccharide having
an antennary structure. In another embodiment, one or more of the termini of
the antennae
bear the modifying moiety. When more than one modifying moiety is attached to
an
oligosaccharide having an antennary structure, the oligosaccharide is useful
to "amplify" the
modifying moiety; each oligosaccharide unit conjugated to the peptide attaches
multiple
copies of the modifying group to the peptide. The general structure of a
typical conjugate of
the invention as set forth in the drawing above, encompasses multivalent
species resulting
from preparing a conjugate of the invention utilizing an antennary structure.
Many antennary
saccharide structures are known in the art, and the present method can be
practiced with them
without limitation.
[0133] In an exemplary embodiment, the activated, modified sugar is a
substrate for a mutant
enzyme that transfers the sugar onto an appropriate acceptor moiety of a
substrate.
Exemplary mutant enzymes include, e.g., those set forth in commonly assigned
PCT
publications W003/046150 and W003/045980
[0134] Water-soluble polymer modified sugar, activated sugar and nucleotide
sugar species
in which the sugar moiety is modified with a water-soluble polymer, e.g., a
water-soluble
37
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WO 2005/072371 PCT/US2005/002522
polymer, are of use in the present invention. An exemplary modified sugar
nucleotide bears a
sugar group that is modified through an amine moiety on the sugar. Modified
sugar
nucleotides, e.g., saccharyl-amine derivatives of a sugar nucleotide, are also
of use in the
methods of the invention. For example, a saccharyl amine (without the
modifying group) can
be enzymatically conjugated to a peptide (or other species) and the free
saccharyl amine
moiety subsequently conjugated to a desired modifying group. Alternatively,
the modified
sugar nucleotide can function as a substrate for an enzyme that transfers the
modified sugar to
a saccharyl acceptor on a substrate, e.g., a peptide, glycopeptide, lipid,
aglycone, glycolipid,
etc.
[0135] In one embodiment, the sugar is conjugated to a branched polymeric
species, such as
those set forth herein.
[0136] In another embodiment, the sugar moiety is a modified sialic acid. When
sialic acid is
the sugar, the sialic acid is substituted with the modifying group at either
the 9-position on
the pyruvyl side chain or at the 5-position on the amine moiety that is
normally acetylated in
sialic acid.
[0137] In another embodiment, in which the saccharide core is galactose or
glucose, R5 is
NHC(0)Y.
[0138] In an exemplary embodiment, the modified sugar is based upon a 6-amino-
N-acetyl-
glycosyl moiety. As shown below for N-acetylgalactosamine, the 6-amino-sugar
moiety is
readily prepared by standard methods:
38
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0
OH
0 NH
HO
AcNH 0,Y1ii
N 0
P..
0 0
0' CT
________________ ROH HO) OH
0
=
a
________________ R=42 R= HN)L0)-A'eCH3
________________ R= In
0
)Veo
HN
n
0
a. galactose oxidase ; N1440Ac, NaBH3CN ; b. 3
0 n
C. )L,
A 0"1
in
[0162] In the scheme above, the index n represents an integer from 1 to 2500,
preferably
from 10 to 1500, and more preferably from 10 to 1200. The symbol "A"
represents an
activating group, e.g., a halo, a component of an activated ester (e.g., a N-
hydroxysuccinimide ester), a component of a carbonate (e.g., p-nitrophenyl
carbonate) and
the like. Those of skill in the art will appreciate that other PEG-amide
nucleotide sugars are
readily prepared by this and analogous methods. Moreover, a branched polymer,
as set forth
herein, can be substituted for the linear PEG.
[0139] Another exemplary polymerically modified nucleotide sugar of the
invention in which
the C-6 position is modified has the formula:
0
NHC(0)(CH2),N1H-J¨(CH2CH20),CH3
/Y
NHC(0)X6CH2CH2(00H2CH2)fOCH3
0 0
NNH
0
0 HN-----NNF12
0
0- 0-
HO OH
in which X6 is a bond or 0, J is S or 0, and y is 0 or 1. The indices e and
fare independently
selected from 1 to 2500.
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WO 2005/072371 PCT/US2005/002522
[0140] In other exemplary embodiments, the amide moiety is replaced by a group
such as a
urethane or a urea.
[0141] In the discussion that follows, a number of specific examples of
modified sugars that
are useful in practicing the present invention are set forth. In the exemplary
embodiments, a
sialic acid derivative is utilized as the sugar nucleus to which the modifying
group is
attached. The focus of the discussion on sialic acid derivatives is for
clarity of illustration
only and should not be construed to limit the scope of the invention. Those of
skill in the art
will appreciate that a variety of other sugar moieties can be activated and
derivatized in a
manner analogous to that set forth using sialic acid as an example. For
example, numerous
methods are available for modifying galactose, glucose, N-acetylgalactosamine
and fucose to
" name a few sugar substrates, which are readily modified by art recognized
methods. See, for
example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and Schafer et al.,
J. Org. Chem.
65: 24 (2000)).
[0142] In FIG. 2, a general scheme according to the present invention is set
forth. Thus,
according to FIG. 2, an amide conjugate between mannosamine and a protected
amino acid is
formed by contacting mannosamine with an N-protected amino acid under
conditions
appropriate to form the conjugate. The carboxyl terminus of the protected
amino acid is
activated in situ or it is optionally converted to a reactive group that is
stable to storage, e.g.,
N-hydroxy-succinimide. The amino acid can be selected from any natural or non-
natural
amino acid. Those of skill in the art understand how to protect side-chain
amino acids from
undesirably reacting in the method of the invention. The amide conjugate is
reacted with
pyruvate and sialic acid aldolase under conditions appropriate to convert the
amide conjugate
to a sialic acid amide conjugate, which is subsequently converted to a
nucleotide phosphate
sialic acid amide conjugate by reaction of the sialic acid amide conjugate
with a precursor of
the nucleotide phosphate and an appropriate enzyme. In an exemplary
embodiment, the
precursor is cytidine triphosphate and the enzyme is a synthetase. Following
the formation of
the nucleotide sugar, the amino acid amine is deprotected, providing a free,
reactive amine
amine. The amine serves as a locus for conjugating the modifying moiety to the
nucleotide
sugar. In FIG. 2, the modifying moiety is exemplified by a water-soluble
polymer, i.e.,
poly(ethylene glycol), e.g., PEG, m-PEG, etc.
[0143] The present invention is further exemplified in FIG. 3, which sets
forth a scheme for
preparing sialic acid-glycyl-PEG-cytidine monophosphate. Similar to the scheme
set forth in
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PCT/US2005/002522
FIG. 2, that of FIG. 3 originates with mannosamine. The sugar is conjugated
with FM0C-
glycine, using the N-hydroxysuccinimide activated derivative of the protected
amino acid.
The resulting amide conjugate is converted to the corresponding sialic acid by
the action of
sialic acid aldolase on the conjugate and pyruvate. The resulting sialic acid
conjugate is
converted to the cytidine monophosphate analogue using cytidine triphosphate
and a
synthetase. The CMP-analogue is deprotected by removing the protecting group
from the
amino acid amine moiety, converting this moiety to a reactive locus for
conjugation. The
amine moiety is reacted with an activated PEG species (m-PEG-0-nitrophenyl
carbonate),
thereby forming the sialic acid-glycyl-PEG-cytidine monophosphate.
[0144] Exemplary sugar cores based upon sialic acid have the formula:
00H
HO XC
R2
G¨ N7/
OH
in which D is -OH or
L-. The symbol G represents H, (1211)¨L- or -C(0)(C1-
C6)alkyl. R" is as is as described above. At least one of D and G is R11-L-.
[0145] In another embodiment, the invention provides a sugar, activated sugar
or sugar
nucleotide that comprises the structure:
OH
HOH2C¨ 000H
H 0
HO' N 0 __
\ HO
0
e
in which L2 is as described above in the context of L, e.g., a bond,
substituted or
unsubstituted alkyl or substituted or unsubstituted heteroalkyl group. The
index e represents
an integer from 1 to about 2500.
[0146] In another embodiment, the sugar or sugar nucleotide comprises the
structure:
41
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WO 2005/072371 PCT/US2005/002522
OH
HOH2C _______ COOH
HC":71-1\110-i
0 __
H/
S
NH
,CH3
0
in which s is selected from the integers from 0 to 20, and e is 1 to 2500.
[0147] Selected sialic acid-based nucleotide sugars functionalized with a
branched polymer
have the formula:
HO
OH
OH
ONIP
R12-x2
HAA(2, N 0 c00H
x5iN
R13-X4
0 OH
in which AA is an amino acid residue, PEG is poly(ethylene glycol) or methoxy-
poly(ethylene glycol) and NP is a nucleotide, which is linked to the glycosyl
moiety via a
phosphodiester bond ("nucleotide phosphate"). Those of skill will appreciate
that ONP can
be replaced by an activating moiety as discussed herein.
[0148] In still further embodiments, the sialic acid derivative has a
structure that is a member
selected from:
42
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WO 2005/072371
PCT/US2005/002522
HOOCX0 CH(OH)CH(OH)CH2OH
\ / 0
HO
y---- NI HC(0)(CH2)aN HC(0)X6(CH2)b( OCH2CH2)c0(CH2)dN ht'''''''-
''.'.*.***'=I'"----J ¨ (CH,CH,O).CH3 =
,
OH
NHC(0)XOCH2CH2(OCH2CH2)1OCH,
>
HOOC7 0 CH(OH)CH(OH)CH2OH
0
HO
NHC(0)(CH2).NH ------.7---- \J¨(CH2CH20).CH3 .
,
OH
NHC(0)X6CH2CH2(OCH2CH2)OCH3
0
HOOC 0 CH (OH)CH(OH)CH2NHJ ¨(CH,CH20).CH,
.
X \7
HO NI HC(0)X6CH,CHAOCH2CH2),OCH3
NHC(0)CH3
OH
0
HOOC 0 CH
(OH)CH(OH)CH2NHC(0)0(C FIA(OCH2CH2)GO(CH2)dN J¨(CH2CH20).CH,
X\ 7 ; and
HO NHC(0)X6CH2CH2(OCH2CH2),OCH3
N HC(0)CH3
OH
0
HOOC 0 CH(OH)CH(OH)CH2NHC(0)X6(CH2)b(OCH2CH2)cO(CH2)dNHJ¨(CH,CH20).CH,
X \ 7
HO NHC(0)X6CH2C1-1,(OCH2CH2)OCH3
.=NHC(0)CH3
OH
in which X6 is a bond or 0, and J is S or 0. The indices a, b and c are
independently selected
from 0 to 20, and e and fare independently selected from 1 to 2500.
[0149] Moreover, as discussed above, the present invention provides nucleotide
sugars that
are modified with a water-soluble polymer, which is either straight-chain or
branched. For
example, compounds having the formula shown below are within the scope of the
present
invention:
,
43
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PCT/US2005/002522
HOOC 0 / CH(OH)CH(OH)CH2OH
\..
0 0
0
H2N---C\N 0
OH
OH NHC(0)X6CH2CH2(OCH2CH2)(0CH3 ;
and
HO
0
HOOC 0 CH(OH)CH(OH)CH2NHJ¨(CH,CH20).CH3
X
0
0
NHC(0)XaCH2CH2(OCH2CH2),OCH3
µ1=3
H N
2 -----C \µ0-
N-- OH
HO
in which X6 is 0 or a bond, and J is S or 0. The indices e and fare
independently selected
from 1 to 2500.
[0150] Also provided are conjugates of peptides and glycopeptides, lipids and
glycolipids
that include the compositions of the invention. The conjugates are formed by
combining a
nucleotide sugar or activated sugar of the invention and a substrate with an
appropriate
acceptor moiety for the sugar moiety and an enzyme for which the modified
nucleotide sugar
is a substrate under conditions appropriate to transfer the modified sugar
from the nucleotide
sugar onto the acceptor moiety. For example, the invention provides conjugates
having the
following formulae:
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WO 2005/072371 PCT/US2005/002522
HOOC..oCH(OH)CH(OH)CH2OH
0
0
/
\ ''...'''...***NHC(0)(CH2)aNHC(0)X4(CH2)b(OCH2CH2),O(CH2)dN
J¨(CHzCH20),,CH3 .=
OH
NHC(0)CH2CH2(OCH2CH2)10CH3
HOOC 0 HC (OH)CH(OH)CH2OH
0
0
/ V
NHC(0)(OH2)aNH J¨(CH2CH20).CH, =
OH
NHC(0)CH2CH2(OCH2CH2)10CH3
0
HOOC 0 CH(OH)CH(OH)CH2NH J¨(CH2CH20).CH,
; and
(:) NHC(0)CH2CH2(OCH2CH2),OCH3
'211,/, NHC(0)CH3
OH
0
HOO .)]..,
CH(OH)CH(OH)CH2NHC(0)X6(CH2)b(OCH2CH2)c0(CH2)dNH J¨(CH,CH20).CH3
Co'\./
(D NHC(0)CH2CH2(OCH2CH2),OCH,
L.) /
-,..,O
y-----NHC(0)CH3
OH .
wherein J and X6 are as discussed above. The indices a, b, c, e and fare as
discussed above.
[0151] Selected compounds of the invention are based on species having the
stereochemistry
of mannose, galactose and glucose. The general formulae of these compounds
are:
R6
______ 0
R6iii.- OH
R54-.)--C1-1 R511.. OH
R4 R3 = R4 R3 =
, and R4 R3
in which one of R3-R6 is the modifying moiety, e.g., polymeric modifying
moiety or the
polymeric modifying moiety-linker construct.
[0152] As discussed above, certain compounds of the present invention are
polymeric
modified sugar nucleotides. Exemplary sugar nucleotides that are used in the
present
invention in their modified form include nucleotide mono-, di- or
triphosphates or analogs
thereof. In a preferred embodiment, the modified sugar nucleotide is selected
from a UDP-
glycoside, CMP-glycoside, or a GDP-glycoside. Even more preferably, the
modified sugar
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
nucleotide is selected from an UDP-galactose, UDP-galactosamine, UDP-glucose,
UDP-
glucosamine, GDP-mannose, GDP-fu.cose, CMP-sialic acid, or CMP-Sia. In an
exemplary
embodiment, the nucleotide mono- di- or tri-phosphate is attached to C-1.
[0153] The saccharyl-amine derivatives of the sugar nucleotides are also of
use in the method
of the invention. For example, the saccharyl amine (without the modifying
group) can be
enzymatically conjugated to a peptide (or other species) and the free
saccharyl amine moiety
subsequently conjugated to a desired modifying group.
[0154] The sugar nucleotide conjugates of the invention are described
generically by the
formula:
R6
RJ0
R4 0-(11)Y 0 0
11 Base
R3 1:1)0 171-X
0" 0"
HO OH
in which the symbols represent groups as discussed above. When the sugar core
is mannose,
the polymeric modifying moiety is preferably at R3, R4 or R6. For glucose, the
polymeric
modifying moiety is optionally at R5 or R6. The index "u" is 0, 1 or 2.
[0155] A further exemplary nucleotide sugar of the invention, based on GDP
mannose has
the structure:
o / \
/ 0 3
f HN s e
0
R5/õ,, /N NH
0
I
N----NNH
*Th)
I='= ..-P 2
0- 0-
-'
HO 'pH'
[0156] In a still further exemplary embodiment, the invention provides a
conjugate, based on
UDP galactose having the structure:
46
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WO 2005/072371 PCT/US2005/002522
HN
0--CH3
s 0
R5j0 HN)ti
(1211 0 Nr--
R-3
0' 0'
HO -0H
[0157] In another exemplary embodiment, the nucleotide sugar is based on
glucose and has
the formula:
0
0,CH3
FINAC\
0
R54õ,,f
HN)\
0
R40fil
ON
.AD
R-3 1 0 1 0
0' 0-
()H H.
In each of the three preceding formulae, the identity of the radicals and
indices is as discussed
above.
[0158] As is apparent to those of skill in the art, the linear PEG moiety can
be replaced by a
branched polymeric or other linear polymeric species as described herein.
[0159] In one embodiment in which the saccharide core is galactose or glucose,
R5 is
NHC(0)Y.
Water-insoluble polymers
[0160] In another embodiment, analogous to those discussed above, the modified
sugars
include a water-insoluble polymer, rather than a water-soluble polymer. A
water-insoluble
polymer, like a water soluble polymer is typically comprised of at least two
polymeric units.
In one exemplary embodiment the polymer is comprised of from 2 to 25 polymeric
units. In
another exemplary embodiment the polymer is comprised of 2 to 8 polymeric
units. The
conjugates of the invention may also include one or more water-insoluble
polymers. This
embodiment of the invention is illustrated by the use of the conjugate as a
vehicle with which
to deliver a therapeutic peptide in a controlled manner. Polymeric drug
delivery systems are
known in the art. See, for example, Dunn et al., Eds. POLYMERIC DRUGS AND DRUG
47
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
DELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society,
Washington, D.C. 1991. Those of skill in the art will appreciate that
substantially any known
drug delivery system is applicable to the conjugates of the present invention.
[0161] Representative water-insoluble polymers include, but are not limited
to,
polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates,
polyalkylenes,
polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyalkylene
terephthalates,
polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,
polyglycolides,
polysiloxanes, polyurethanes, poly(methyl methacrylate), poly(ethyl
methacrylate),
poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl
methacrylate),
poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate),
poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),
poly(octadecyl
acrylate) polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene
oxide), poly
(ethylene terephthalate), poly(vinyl acetate), polyvinyl chloride,
polystyrene, polyvinyl
pyrrolidone, pluronics and polyvinylphenol and copolymers thereof.
[0162] Synthetically modified natural polymers of use in conjugates of the
invention include,
but are not limited to, alkyl celluloses, hydroxyalkyl celluloses, cellulose
ethers, cellulose
esters, and nitrocelluloses. Particularly preferred members of the broad
classes of
synthetically modified natural polymers include, but are not limited to,
methyl cellulose,
ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
hydroxybutyl
methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate
butyrate, cellulose
acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose
sulfate sodium salt,
and polymers of acrylic and methacrylic esters and alginic acid.
[0163] These and the other polymers discussed herein can be readily obtained
from
commercial sources such as Sigma Chemical Co. (St. Louis, MO.), Polysciences
(Warrenton,
PA.), Aldrich (Milwaukee, WI.), Fluka (Ronkonkoma, NY), and BioRad (Richmond,
CA), or
else synthesized from monomers obtained from these suppliers using standard
techniques.
[0164] Representative biodegradable polymers of use in the conjugates of the
invention
include, but are not limited to, polylactides, polyglycolides and copolymers
thereof,
poly(ethylene terephthalate), poly(butyric acid), poly(valeric acid),
poly(lactide-co-
caprolactone), poly(lactide-co-glycolide), polyanhydrides, polyorthoesters,
blends and
copolymers thereof. Of particular use are compositions that form gels, such as
those
including collagen, pluronics and the like.
,
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[0165] The polymers of use in the invention include "hybrid' polymers that
include water-
insoluble materials having within at least a portion of their structure, a
bioresorbable
molecule. An example of such a polymer is one that includes a water-insoluble
copolymer,
which has a bioresorbable region, a hydrophilic region and a plurality of
crosslinkable
functional groups per polymer chain.
[0166] For purposes of the present invention, "water-insoluble materials"
includes materials
that are substantially insoluble in water or water-containing environments.
Thus, although
certain regions or segments of the copolymer may be hydrophilic or even water-
soluble, the
polymer molecule, as a whole, does not to any substantial measure dissolve in
water.
[0167] For purposes of the present invention, the term "bioresorbable
molecule" includes a
region that is capable of being metabolized or broken down and resorbed and/or
eliminated
through normal excretory routes by the body. Such metabolites or break down
products are
preferably substantially non-toxic to the body.
[0168] The bioresorbable region may be either hydrophobic or hydrophilic, so
long as the
copolymer composition as a whole is not rendered water-soluble. Thus, the
bioresorbable
region is selected based on the preference that the polymer, as a whole,
remains water-
insoluble. Accordingly, the relative properties, i.e., the kinds of functional
groups contained
by, and the relative proportions of the bioresorbable region, and the
hydrophilic region are
selected to ensure that useful bioresorbable compositions remain water-
insoluble.
[0169] Exemplary resorbable polymers include, for example, synthetically
produced
resorbable block copolymers of poly(a-hydroxy-carboxylic
acid)/poly(oxyalkylene, (see,
Cohn et al., U.S. Patent No. 4,826,945). These copolymers are not crosslinked
and are water-
soluble so that the body can excrete the degraded block copolymer
compositions. See,
Younes et al., J Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J
Biomed.
Mater. Res. 22: 993-1009 (1988).
[0170] Presently preferred bioresorbable polymers include one or more
components selected
from poly(esters), poly(hydroxy acids), poly(lactones), poly(amides),
poly(ester-amides),
poly (amino acids), poly(anhydrides), poly(orthoesters), poly(carbonates),
poly(phosphazines), poly(phosphoesters), poly(thioesters), polysaccharides and
mixtures
thereof. More preferably still, the bioresorbable polymer includes a
poly(hydroxy) acid
49
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
component. Of the poly(hydroxy) acids, polylactic acid, polyglycolic acid,
polycaproic acid,
polybutyric acid, polyvaleric acid and copolymers and mixtures thereof are
preferred.
[0171] In addition to forming fragments that are absorbed in vivo
("bioresorbed"), preferred
polymeric coatings for use in the methods of the invention can also form an
excretable and/or
metabolizable fragment.
[0172] Higher order copolymers can also be used in the present invention. For
example,
Casey et al., U.S. Patent No. 4,438,253, which issued on March 20, 1984,
discloses tri-block
copolymers produced from the transesterification of poly(glycolic acid) and an
hydroxyl-
ended poly(alkylene glycol). Such compositions are disclosed for use as
resorbable
monofilament sutures. The flexibility of such compositions is controlled by
the incorporation
of an aromatic orthocarbonate, such as tetra-p-tolyl orthocarbonate into the
copolymer
structure.
[0173] Other polymers based on lactic and/or glycolic acids can also be
utilized. For
example, Spinu, U.S. Patent No. 5,202,413, which issued on April 13, 1993,
discloses
biodegradable multi-block copolymers having sequentially ordered blocks of
polylactide
and/or polyglycolide produced by ring-opening polymerization of lactide and/or
glycolide
onto either an oligomeric diol or a diamine residue followed by chain
extension with a di-
functional compound, such as, a diisocyanate, diacylchloride or
dichlorosilane.
[0174] Bioresorbable regions of coatings useful in the present invention can
be designed to
be hydrolytically and/or enzymatically cleavable. For purposes of the present
invention,
"hydrolytically cleavable" refers to the susceptibility of the copolymer,
especially the
bioresorbable region, to hydrolysis in water or a water-containing
environment. Similarly,
"enzymatically cleavable" as used herein refers to the susceptibility of the
copolymer,
especially the bioresorbable region, to cleavage by endogenous or exogenous
enzymes.
[0175] When placed within the body, the hydrophilic region can be processed
into excretable
and/or metabolizable fragments. Thus, the hydrophilic region can include, for
example,
polyethers, polyalkylene oxides, polyols, poly(vinyl pyrrolidine), poly(vinyl
alcohol),
poly(alkyl oxazolines), polysaccharides, carbohydrates, peptides, proteins and
copolymers
and mixtures thereof. Furthermore, the hydrophilic region can also be, for
example, a
poly(alkylene) oxide. Such poly(alkylene) oxides can include, for example,
poly(ethylene)
oxide, poly(propylene) oxide and mixtures and copolymers thereof.
CA 02554466 2006-07-26
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[0176] Polymers that are components of hydrogels are also useful in the
present invention.
Hydrogels are polymeric materials that are capable of absorbing relatively
large quantities of
water. Examples of hydrogel forming compounds include, but are not limited to,
polyacrylic
acids, sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl
pyrrolidine, gelatin,
carrageenan and other polysaccharides, hydroxyethylenemethacrylic acid (HEMA),
as well as
derivatives thereof, and the like. Hydrogels can be produced that are stable,
biodegradable
and bioresorbable. Moreover, hydrogel compositions can include subunits that
exhibit one or
more of these properties.
[0177] Bio-compatible hydrogel compositions whose integrity can be controlled
through
crosslinking are known and are presently preferred for use in the methods of
the invention.
For example, Hubbell et al., U.S. Patent Nos. 5,410,016, which issued on April
25, 1995 and
5,529,914, which issued on June 25, 1996, disclose water-soluble systems,
which are
crosslinked block copolymers having a water-soluble central block segment
sandwiched
between two hydrolytically labile extensions. Such copolymers are further end-
capped with
photopolymerizable acrylate functionalities. When crosslinked, these systems
become
hydrogels. The water soluble central block of such copolymers can include
poly(ethylene
glycol); whereas, the hydrolytically labile extensions can be a poly(a-hydroxy
acid), such as
polyglycolic acid or polylactic acid. See, Sawhney et al., Macromolecules 26:
581-587
(1993).
[0178] In another embodiment, the gel is a thermoreversible gel.
Thermoreversible gels
including components, such as pluronics, collagen, gelatin, hyalouronic acid,
polysaccharides, polyurethane hydrogel, polyurethane-urea hydrogel and
combinations
thereof are presently preferred.
[0179] In yet another exemplary embodiment, the conjugate of the invention
includes a
component of a liposome. Liposomes can be prepared according to methods known
to those
skilled in the art, for example, as described in Eppstein et al., U.S. Patent
No. 4,522,811,
which issued on June 11, 1985. For example, liposome formulations may be
prepared by
dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine,
stearoyl
phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an
inorganic
solvent that is then evaporated, leaving behind a thin film of dried lipid on
the surface of the
container. An aqueous solution of the active compound or its pharmaceutically
acceptable
salt is then introduced into the container. The container is then swirled by
hand to free lipid
51
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WO 2005/072371 PCT/US2005/002522
material from the sides of the container and to disperse lipid aggregates,
thereby forming the
liposomal suspension.
[0180] The above-recited microparticles and methods of preparing the
microparticles are
offered by way of example and they are not intended to define the scope of
microparticles of
use in the present invention. It will be apparent to those of skill in the art
that an array of
microparticles, fabricated by different methods, is of use in the present
invention.
[0181] The structural formats discussed above in the context of the water-
soluble polymers,
both straight-chain and branched are generally applicable with respect to the
water-insoluble
polymers as well. Thus, for example, the cysteine, senile, dilysine, and
trilysine branching
cores can be functionalized with two water-insoluble polymer moieties. The
methods used to
produce these species are generally closely analogous to those used to produce
the water-
soluble polymers.
[0182] The in vivo half-life of therapeutic glycopeptides can also be enhanced
with PEG
moieties such as polyethylene glycol (PEG). For example, chemical modification
of proteins
with PEG (PEGylation) increases their molecular size and decreases their
surface- and
functional group-accessibility, each of which are dependent on the size of the
PEG attached
to the protein. This results in an improvement of plasma half-lives and in
proteolytic-
stability, and a decrease in immunogenicity and hepatic uptake (Chaffee et al.
I Clin. Invest.
89: 1643-1651 (1992); Pyatak et al. Res. Commun. Chem. Pathol Pharmacol. 29:
113-127
(1980)). PEGylation of interleukin-2 has been reported to increase its
antitumor potency in
vivo (Katre et al. Proc. Natl. Acad. Sci. USA. 84: 1487-1491 (1987)) and
PEGylation of a
F(ab')2 derived from the monoclonal antibody A7 has improved its tumor
localization
(Kitamura et al. Biochem. Blophys. Res. Commun. 28: 1387-1394 (1990)). Thus,
in another
embodiment, the in vivo half-life of a peptide derivatized with a PEG moiety
by a method of
the invention is increased relevant to the in vivo half-life of the non-
derivatized peptide.
[0183] The increase in peptide in vivo half-life is best expressed as a range
of percent
increase in this quantity. The lower end of the range of percent increase is
about 40%, about
60%, about 80%, about 100%, about 150% or about 200%. The upper end of the
range is
about 60%, about 80%, about 100%, about 150%, or more than about 250%.
52
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
Preparation of Modified Sugars
[0184] In general, the sugar moiety and the modifying group are linked
together through
the use of reactive groups, which are typically transformed by the linking
process into a new
organic functional group or unreactive species. The sugar reactive functional
group(s), is
located at any position on the sugar moiety. Reactive groups and classes of
reactions useful
in practicing the present invention are generally those that are well known in
the art of
bioconjugate chemistry. Currently favored classes of reactions available with
reactive sugar
moieties are those, which proceed under relatively mild conditions. These
include, but are
not limited to nucleophilic substitutions (e.g., reactions of amines and
alcohols with acyl
halides, active esters), electrophilic substitutions (e.g., enamine reactions)
and additions to
carbon-carbon and carbon-hetero atom multiple bonds (e.g., Michael reaction,
Diels-Alder
addition). These and other useful reactions are discussed in, for example,
March, ADVANCED
ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,
BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal.,
MODIFICATION OF PROII,INS; Advances in Chemistry Series, Vol. 198, American
Chemical
Society, Washington, D.C., 1982.
[0185] Useful reactive functional groups pendent from a sugar nucleus, linker
precursor or
polymeric modifying moiety precursor include, but are not limited to:
(a) carboxyl groups and various derivatives thereof including, but not limited
to,
N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl
imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and
aromatic esters;
(b) hydroxyl groups, which can be converted to, e.g., esters, ethers,
aldehydes, etc.
(c) haloalkyl groups, wherein the halide can be later displaced with a
nucleophilic
group such as, for example, an amine, a carboxylate anion, thiol anion,
carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of
a
new group at the functional group of the halogen atom;
(d) dienophile groups, which are capable of participating in Diels-Alder
reactions
such as, for example, maleimido groups;
(e) aldehyde or ketone groups, such that subsequent derivatization is possible
via
formation of carbonyl derivatives such as, for example, imines, hydrazones,
53
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
semicarbazones or oximes, or via such mechanisms as Grignard addition or
alkyllithium addition;
(f) sulfonyl halide groups for subsequent reaction with amines, for example,
to form
sulfonamides;
(g) thiol groups, which can be, for example, converted to disulfides or
reacted with
acyl halides;
(h) amine or sulfhydryl groups, which can be, for example, acylated, alkylated
or
oxidized;
(i) alkenes, which can undergo, for example, cycloadditions, acylation,
Michael
addition, etc; and
(j) epoxides, which can react with, for example, amines and hydroxyl
compounds.
[0186] The reactive functional groups can be chosen such that they do not
participate in, or
interfere with, the reactions necessary to assemble the reactive sugar nucleus
or modifying
group. Alternatively, a reactive functional group can be protected from
participating in the
reaction by the presence of a protecting group. Those of skill in the art
understand how to
protect a particular functional group such that it does not interfere with a
chosen set of
reaction conditions. For examples of useful protecting groups, see, for
example, Greene et
al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York,
1991.
[0187] In the discussion that follows, a number of specific examples of
modified sugars
that are useful in practicing the present invention are set forth. In the
exemplary
embodiments, a sialic acid derivative is utilized as the sugar nucleus to
which the modifying
group is attached. The focus of the discussion on sialic acid derivatives is
for clarity of
illustration only and should not be construed to limit the scope of the
invention. Those of
skill in the art will appreciate that a variety of other sugar moieties can be
activated and
derivatized in a manner analogous to that set forth using sialic acid as an
example. For
example, numerous methods are available for modifying galactose, glucose, N-
acetylgalactosamine and fucose to name a few sugar substrates, which are
readily modified
by art recognized methods. See, for example, Elhalabi et al., Curr. Med. Chem.
6: 93 (1999);
and Schafer et al., I Org. Chem. 65: 24 (2000)).
54
CA 02554466 2006-07-26
WO 2005/072371
PCT/US2005/002522
[0188] In Scheme 1 below, the amino glycoside 1, is treated with the active
ester of a
protected amino acid (e.g., glycine) derivative, converting the sugar amine
residue into the
corresponding protected amino acid amide adduct. The adduct is treated with an
aldolase to
form a-hydroxy carboxylate 2. Compound 2 is converted to the corresponding CMP
derivative by the action of CMP-SA synthetase, followed by catalytic
hydrogenation of the
CMP derivative to produce compound 3. The amine introduced via formation of
the glycine
adduct is utilized as a locus of PEG or PPG attachment by reacting compound 3
with an
activated (m-) PEG or (m-) PPG derivative (e.g-., PEG-C(0)NHS, PPG-C(0)NHS),
producing 4 or 5, respectively.
OH 1. CMP-SA synthetase, CTP
HO NH 2 1. FMOC-Glycine-NHS HO _.9H 2. H2/Pd/C
HO 2. NeuAc Aldolase, pyruvate HO
HO ) FM00., N..tr.NH OH 0
1
OH NH, 8 H 2 1
NH,
CIN
0 1
0 II N 0
II N
H 'o 0
0
0----13-0--NO
0¨P-NO II I
1 (m-) PEG-C-NHS O'Na
HOE1 ehr-0-+Na HO OH
0 HO H O'Na
ii -+Na0---" Ho OH
0 0 -..( __
0
NN
PEG-c o ._ _, NH OH
HN 11 Thr NH OH 2 3
H0 4 0
CMP-SA-5-NHCOCH2NH¨PEG (m-PEG)1
(m-) PPG-C-NHS CMP-SA-5-NHCOCH2NH2
CMP-SA-5-NHCOCH2NH¨PPG (in-PPG)
Scheme 1
As those of skill will appreciate, the polymeric modifying moiety can also be
a branched
moiety, such as those described herein.
[0189] An exemplary scheme for preparing the branched polymerically-modified
sugars of
the invention is provided below:
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
H 0 0
NH2 HCI Fmoc-Lys(Fmoc)-CO'N'-)cNH0H
OH '
0
H3Cyji"0_ *Na.1,1a HO.õ....).õ.....r.
HO- ._. HO. ,.... 0 Fmoc-Lys(Fmoc)-C11.õ HNi 0
COOH
N-Ir HO
Me0H, Et3N HO ---4=======4õ H 0
OH H 0 OH Aldolase )
Fmoc-Lys(Fmoc)-CO- 0.1M HEPES
OPfp CTP,
C CMP-sialic acid Synthetase,
pH 7.5 37
Buffer, MgC12
NH2 NH2
0 0
' II (LNN 0 n
HO 0H 0' 1 '0
CLN
I Me2NH P I
01*() N 0
0"+Na-yi ., water, THF
0 HO H 2H 0"+Na
II
, _ Fmoc-Lys(Fmoc)-C ...-..õ_HN 0
c00-+Na = _
HO HO OH HO H8 OH
H 0 .1 g
NO2
NH2
0
II CL,N
0 HO 0H
0-+Nasb
HOõ......l......i.,
C).1 1' jLNH LN_____TrHN 0 COU+Na .1 ..
n
HO HO OH
H 0
H..5. .
0-1--10--N
n 0 .
[0190] Another exemplary scheme for preparing the polymerically-modified
sugars of the
invention is set forth below:
o
Fmoc-NH
activating group
NH2 1.
0
II
N
P I
O'' I 0 N 0 NH-Fmoc NH2
HO OH
Cr+Na
HOõõ.3.....r, ________________________________ >
HN 0 ) 0
II
C-IN
2. Me2NH P I
H2N------r H0 CO0aH8 8H e-I0 NO
o HO 0H
0-+Naj
0 HOõ,=õ.1õ......r.
H2N 0 0o0-+NaA
..1 _
N -ThrHN7HO HO OH
H 0
0 H2N
OF--- V0-1Cactivating group
n
NH2
w 0
II N
0 HO 0H
0-+NaAOJ
HOõ,..3........
CII V' .-1t-NH ' N "¨ 0 o00-+Na _
" IvHN7HOr HO OH
H 0
H
N
(211(310¨
11 0 .
[0191] Table 1 sets forth representative examples of sugar monophosphates that
are
derivatized with a polymeric modifying moiety, e.g., a branched- or straight-
chain PEG or
56
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WO 2005/072371 PCT/US2005/002522
PPG moiety. Certain of the compounds of Table 1 are prepared by the method of
Scheme 1.
Other derivatives are prepared by art-recognized methods. See, for example,
Keppler et al.,
Glycobiology 11: 11R (2001); and Charter et al., Glycobiology 10: 1049
(2000)). Other
amine reactive polymeric modifying moiety precursors and components, e.g., PEG
and PPG
analogues are commercially available, or they can be prepared by methods
readily accessible
to those of skill in the art.
Table 1
NH2 NH2
01
N
0 0 I
II NO II N o
0¨P--o--01 0- o¨P-o¨c))
+Na 1 ,
Ho ..QH HO sOH 0- Na ,
HO OH R-0 T..
,-0 0-+Na HO OH
R-NH ___ OH AcNH OH o
CMP-SA-5-NH-R CMP-NeuAc-9-0-R
NH2 NH2
(1\4
-=N
O o I
II NO II (N o
0¨P-0---NO 0¨P-0---\0
01-+Na 0I-+Na
HO OH HO opH
HO,-0-+Na HO OH R-NH,-/7,O7")1-0-+Na HO OH
R-0 ____ OH AcNH¨fri.i 0
CMP-NeuAc-9-NH-R NH2
CMP-KDN-5-0-R
C)N
NH2 0 I
II N o
eN 01 0¨Pi-0---.C.1)
0 R-NH
II N-0 0-+Na
0¨P-0-
,s,,1
R-0
01-+Na HO ::- OVhr-0-+Na
HO OH
j ,-1 0
HO C
::- )r0-+Na Ho OH ___ AcNH oH
AcNH oH CMP-NeuAc-8-NH-R
NH2
CMP-NeuAc-8-0-R
NH2
CN
0 I
(ly II N 0
0 O¨P1-0--\c0)
II 1\10
0¨Ir.-Cr-NO HO NH-R Na
HO 0-R 0-+Na Ho :="- o 0-+Na No OH
HO OH AcNH __ OH 0
AcNH OH 0
CMP-NeuAc-7-NH-R NH2
NH2
CMP-NeuAc-7-0-R C"N
el
0 0 I
II N 0
II N 0
O¨Pi-0---\c.0)
HO ..91-1 v))701-+Na
HO OH 4)r0-+Na
HO.,--1-0 0-+Na HO OH
HO.......--0 0-+Na HO OH 0
o AcNH
AcNH NH-R
O-R
CMP-NeuAc-4-NH-R
CMP-NeuAc-4-0-R
in which R is the polymeric (branched or straight-chain) modifying moiety.
57
CA 02554466 2013-07-25
10192] The modified sugar phosphates of use in practicing the present
invention can be
substituted in other positions as well as those set forth above. Presently
preferred
substitutions of sialic acid are set forth in the formula below:
NH2
(61;1
0
Rd1);"Fec 0 0. N a r--0-2;" \SI/
F HO OH
Rt_ye 0
Z-119
in which one or more of r, Y9, Yb, Y` and Z is a linking group, which is
preferably selected
from -0-, -N(H)-, -S, CH2-, and N(R)2. When r, Y, yb, y. and Z is a linking
group, it is
attached to the polymeric modifying moiety as represented by RC, Rd, Re, R1
and R.
Alternatively, these symbols represent a linker that is bound to a branched-
or straight-chain
water-soluble or water-insoluble polymer, therapeutic moiety, biomolecule or
other moiety.
When le, Rd, R9, R' or R8 is not a polymeric modifying moiety, the combination
of rle,
yak% yK b- e,
YeRf or ZR8 is H, OH or NC(0)C113.
101931 Also provided is a synthetic method for producing an activated sialic
acid-polymeric
modifying group conjugate that is an appropriate substrate for an enzyme that
transfers the
modified sugar moiety onto an acceptor, e.g., a glycosyltransferase. The
method includes the
steps: (a) contacting mannosamine with an activated, N-protected amino acid
(or an amino
acid functionalized with a polymeric modifying moiety, a linker precursor or a
linker-
polymeric modifying moiety cassette) under conditions appropriate to form an
amide
conjugate between the mannosamine and the N-protected amino acid; (b)
contacting the
amide conjugate with pyruvate and sialic acid aldolase under conditions
appropriate to
convert the amide conjugate to a sialic acid amide conjugate; (c) contacting
the sialic acid
amide conjugate with cytidine triphosphates, and a synthetase under conditions
appropriate to
form a cytidine monophosphate sialic acid amide conjugate; (d) removing the N-
protecting
group from the cytidine monophosphate sialic acid amide conjugate, thereby
producing a free
amine; and (e) contacting the free amine with an activated PEG (straight-chain
or branched),
thereby forming the cytidine monophosphate sialic acid-poly(ethylene glycol).
Cross-linking Groups
101941 Preparation of the modified sugar for use in the methods of the present
invention
includes attachment of a modifying group to a sugar residue and forming a
stable adduct,
58
CA 02554466 2012-08-13
which is a substrate for a glycosyltransferase. The sugar and modifying group
can be coupled
by a zero- or higher-order cross-linking agent. Exemplary bifunctional
compounds which
can be used for attaching modifying groups to carbohydrate moieties include,
but are not
limited to, bifunctional poly(ethyleneglycols), polyamides, polyethers,
polyesters and the
like. General approaches for linking carbohydrates to other molecules are
known in the
literature. See, for example, Lee et al., Biochemistyy 28: 1856 (1989); Bhatia
et al., Anal.
Biochem. 178: 408 (1989); Janda et al., J. Am. Chem. Soc. 112: 8886 (1990) and
Bednarski et
al., WO 92/18135. In the discussion that follows, the reactive groups are
treated as benign on
the sugar moiety of the nascent modified sugar. The focus of the discussion is
for clarity of
illustration. Those of skill in the art will appreciate that the discussion is
relevant to reactive
groups on the modifying group as well.
101951 An exemplary strategy involves incorporation of a protected sulfhydryl
onto the
sugar using the heterobifunctional crosslinker SPDP (n-succinimidy1-3-(2-
pyridyldithio)propionate and then deprotecting the sulfhydryl for formation of
a disulfide
bond with another sulithydryl on the modifying group.
[0196] A variety of reagents are used to modify the components of the modified
sugar with
intramolecular chemical crosslinks (for reviews of crosslinking reagents and
crosslinking
procedures see: Wold, F., Meth. EnzymoL 25: 623-651, 1972; Weetall, H. H., and
Cooney, D.
A., In: ENZYMES AS DRUGS. (Holcenberg, and Roberts, eds.) pp. 395-442, Wiley,
New York,
1981; ji, T. H., Meth. Darla 91: 580-609, 1983; Mattson etal., MoL Biol. Rep.
17: 167-
183, 1993). Preferred crosslinking reagents
are derived from various zero-length, homo-bifunctional, and hetero-
bifunctional crosslinking
reagents. Zero-length crosslinking reagents include direct conjugation of two
intrinsic
chemical groups with no introduction of extrinsic material.
Conjugation of Modified Sugars to Peptides
[01971 The modified sugars are conjugated to a glycosylated or non-
glycosylated peptide
using an appropriate enzyme to mediate the conjugation. Thus, the compounds of
the
invention, particularly the nucleotide sugars are preferably substrates for
enzymes that
transfer sugar moieties from a nucleotide sugar onto an amino acid, glycosyl,
or aglycone
acceptor moiety. Nucleotide sugars that act as sugar donors for acceptors,
e.g., galactosyl
acceptors, e.g., GaINAc, Galf31,4G1cNAc, Ga1131,4GalNAc, Ga1131,3GalNAc, lacto-
N-
tetraose, Gali31,3G1cNAc, Gall31,3Ara, Ga1131,6G1cNAc, Ga1131,4G1c (lactose),
and other
59
CA 02554466 2012-08-13
acceptors well known to those of skill in the art (see, e.g., Paulson et al.,
J. Biol. Chem. 253:
5617-5624 (1978)).
[0198] Exemplary enzymes for which the modified nucleotide sugars of the
invention are
substrates include glycosyltransferases. The glycosyltransferase can be
cloned, or isolated
from any source. Many cloned glycosyltransferases are known, as are their
polynucleotide
sequences. Glycosyltransferase amino acid sequences and
nucleotide sequences encoding glycosyltransferases from which the amino acid
sequences
can be deduced are also found in various publicly available databases,
including GenBank,'
Swiss-Prof:EMI:if, and others.
[01991 Glycosyltransferases for which the compounds of the invention are
substrates
include, but are not limited to, galactosyltransferases, fucosyltransferases,
glucosyltransferases, N-acetylgalactosaminyltransferases, N-
acetylglucosaminyltransferases,
glucuronyltransferases, sialyltransferases, mannosyltransferases, glucuronic
acid transferases,
galacturonic acid transferases, and oligosaccharyltransferases. Suitable
glycosyltransferases
include those obtained from eukaryotes, as well as from prokaryotes.
[02001 In some embodiments, the compound of the invention is a substrate for a
fucosyltransferase. Fucosyltransferases are generally known to those of skill
in the art, and
are exemplified by enzymes that transfer L-fitcose from GDP-fucose to a
hydroxy position of
an acceptor sugar.
[02011 In another group of embodiments, the compound is a substrate for a
galactosyltransferase. Exemplary galactosyltransferases include ct(1 ,3)
galactosyltransferases
(B.C. No. 2.4.1.151, see, e.g., Dabkowsld et al., Transplant Proc. 25:2921
(1993) and
Yamamoto et al, Nature 345: 229-233 (1990), bovine (GenBank j04989, Joziasse
et al., J.
Biol. Chem. 264: 14290-14297 (1989)), murine(GenBank m26925; Larsen et al.,
Proc. Nat'l.
Acad. Sci. USA 86: 8227-8231 (1989)), porcine (GenBank L36152; Strahan et al.,
linnuinogenetics 41: 101-105 (1995)). Another suitable a1,3
galactosyltransferase is that
which is involved in synthesis of the blood group B antigen (EC 2.4. 1.37,
Yamamoto et al., J.
Biol. Chem. 265: 1146-1151 (1990) (human)). Yet a further exemplary
galactosyltransferase
is core Gal-T1. Still further examples include 1(1,4) galactosyltransferases,
which include,
for example, EC 2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose
synthetase) (bovine
(D'Agostaro et al., Eur. J. Biochem. 183: 211-217 (1989)), human (Masri et aL,
Biochem.
CA 02554466 2006-07-26
WO 2005/072371 PCT/US2005/002522
Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa et al., J.
Biochem. 104:
165-168 (1988)), as well as E.C. 2.4.1.38 and the ceramide
galactosyltransferase (EC
2.4.1.45, Stahl et al., J. Neurosci. Res. 38: 234-242 (1994)). Other suitable
galactosyltransferases include, for example, a1,2 galactosyltransferases (from
e.g.,
Schizosaccharomyces pombe, Chapell et al., MoL Biol. Cell 5: 519-528 (1994)).
Also
suitable in the practice of the invention are soluble forms of al, 3-
galactosyltransferase such
as that reported by Cho et al., J. Biol. Chem., 272: 13622-13628 (1997).
a) Sialyltransferases
[0202] Sialyltransferases are another type of glycosyltransferase for which
the compounds
of the invention are substrates. Examples include ST3Ga1 III (e.g., a rat or
human 5T3 Gal
III), ST3Ga1 IV, ST3Ga1 I, ST6Ga1 I, ST3Ga1 V, ST6Ga1 II, ST6Ga1NAc I,
ST6Ga1NAc II,
and ST6Ga1NAc III (the sialyltransferase nomenclature used herein is as
described in Tsuji et
al., Glycobiology 6: v-xiv (1996)). An exemplary a(2,3)sialyltransferase
referred to as
a(2,3)sialyltransferase (EC 2.4.99.6) transfers sialic acid to the non-
reducing terminal Gal of
a Ga1131-->3G1c disaccharide or glycoside. See, Van den Eijnden et al., J.
Biol. Chem. 256:
3159 (1981), Weinstein etal., J. Biol. Chem. 257: 13845 (1982) and Wen et al.,
.1: Biol.
Chem. 267: 21011 (1992). Another exemplary a2,3-sialyltransferase (EC
2.4.99.4) transfers
sialic acid to the non-reducing terminal Gal of the disaccharide or glycoside.
see, Rearick et
al., J. Biol. Chem. 254: 4444 (1979) and Gillespie etal., J. Biol. Chem. 267:
21004 (1992).
Further exemplary enzymes include Gal-13-1,4-GleNAc a-2,6 sialyltransferase
(See,
Kurosawa et al. Eur. I Biochem. 219: 375-381 (1994)). Other sialyltransferases
for which
the compounds of the invention are substrates include those that form
polysialic acids.
Examples include the a-2,8-polysialyltransferases, e.g., ST8SiaI, ST8Siall,
ST8SiaIII,
ST8SialV and ST8SiaV. See for example, Angata et al. J. Biol. Chem. 275: 18594-
18601
(2000); Kono et al., J. Biol. Chem. 271: 29366-29371 (1996); Greiner et al.,
Infect. Immun.
72: 4249-4260 (2004); and Jones et al., J. Biol. Chem. 277: 14598-14611(2002).
[0203] An example of a sialyltransferase that is useful in the claimed methods
is ST3Gal
III, which is also referred to as a(2,3)sialyltransferase (EC 2.4.99.6). This
enzyme catalyzes
the transfer of sialic acid to the Gal of a Ga1131,3G1cNAc or Galf31,4G1cNAc
glycoside (see,
e.g., Wen etal., I. Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J.
Biol. Chem.
61
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WO 2005/072371 PCT/US2005/002522
256: 3159 (1991)). Still further sialyltransferases include those isolated
from Campylobacter
jejuni, including the a(2,3). See, e.g, W099/49051.
[0204] Preferably, the compounds of the invention are substrates for an enzyme
that
transfers the modifies sialic acid to the sequence Galf31,4G1cNAc-, the most
common
penultimate sequence underlying the terminal sialic acid on fully sialylated
carbohydrate
structures.
b) GalNAc transferases
[0205] Selected compounds of the invention are substrates for N-
acetylgalactosaminyltransferases. Exemplary N-acetylgalactosaminyltransferases
include,
but are not limited to, a(1,3) N-acetylgalactosaminyltransferase, 13(1,4) N-
acetylgalactosaminyltransferases (Nagata et al., J. Biol. Chem. 267: 12082-
12089 (1992) and
Smith et al., I Biol Chem. 269: 15162 (1994)) and polypeptide N-
acetylgalactosaminyltransferase (Homa et al., I Biol. Chem_ 268: 12609
(1993)).
Glycosidases
[0206] This invention also encompasses substrates for wild-type and mutant
glycosidases.
Mutant13-galactosidase enzymes have been demonstrated to catalyze the
formation of
disaccharides through the coupling of a-glycosyl fluoride to a galactosyl
acceptor molecule.
(Withers, U.S. Pat. No. 6,284,494; issued Sept. 4, 2001). Other glycosidases
of use in this
invention include, for example, 0-glucosidases, (3-galactosidases, (3-
mannosidases, (3-acetyl
glucosaminidases, (3-N-acetyl galactosaminidases, (3-xylosidases,13-
fucosidases, cellulases,
xylanases, galactanases, mannanases, hemicellulases, amylases, glucoamylases,
a-
glucosidases, a-galactosidases, a-mannosidases, a-N-acetyl glucosaminidases, a-
N-acetyl
galactose-aminidases, a-xylosidases, a-fucosidases, and
neuraminidases/sialidases,
endoglycoceramidases.
[0207] The following examples are provided to illustrate selected
embodiments of the
invention and are not to be construed as limiting its scope.
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CA 02554466 2012-08-13
EXAMPLES
Example 1 .
Preparation of UDP-GalNAe-6'-CHO
[0344] UDP-GaINAc (200 mg, 0.30 mmoles) was dissolved in a 1 triM CuSO4
solution (20
mL) and a 25 mM NaH2PO4 solution (pH 6.0; 20 mL). Galactose oxidase (240 U;
240 L)
and catalase (13000 U; 130 L) were then added, the reaction system equipped
with a balloon
filled with oxygen and stirred at room temperature for seven days. The
reaction mixture was
then filtered (spin cartridge; MWCO 5Krand the filtrate (-40 mL) was stored at
4 C until
required. TLC (silica; Et0H/water (7/2); Rf = 0.77; visualized with
anisaldehyde stain).
Example 2
Preparation of UDP-GaiNAe-6'-N112):
[0345) Ammonium acetate (15 mg, 0.194 mmoles) and NaBH3CN (1M THE solution;
0.17
mL, 0.17 mmoles) were added to the UDP-GaINAc-6'-CHO solution from above (2 mL
or ¨
20 mg) at 0 C and allowed to warm to room temperature overnight. The reaction
was filtered
through a 0-10 column with water and the product collected. The appropriate
fractions were
freeze-dried and stored frozen. TLC (silica; ethanol/water (7/2); Rf --- 0.72;
visualized with
ninhydrin reagent).
Example 3
Preparation of UDP-GalNAe-6-NHCO(CH2)-r0-PEG-0Me (1 I<Da).
[0346] The galactosaminy1-1-phosphate-2-NHCO(CH2)2-0-PEG-0Me (1 KDa) (58 mg,
0.045 mmoles) was dissolved in DMF (6 mL) and pyridine (1.2 mL). UMP-
morpholidate
(60 mg, 0.15 mmoles) was then added and the resulting mixture stirred at 70 C
for 48 h. The
solvent was removed by bubbling nitrogen through the reaction mixture and the
residue
purified by reversed phase chromatography (C-18 silica, step gradient between
10 to 80%,
methanol/water). The desired fractions were collected and dried at reduced
pressure to yield
a white solid. TLC (silica, propanol/H20/NH4OH, (30/20/2), Rf = 0.54). MS
(MALDI):
Observed, 1485, 1529, 1618, 1706.
63
CA 02554466 2012-08-13
Example 4
Preparation of Cysteine-PEG2 ( 2 )
NH2
NO'N'7COTs ___________________________ KOH, Me0H H2
in OH
0
1
I NO2
CH2C12rreA
-
2
4.1 Synthesis of Compound 1
[0347) Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was added to a
solution of
L-cysteine (93.7mC0.75 mmol) in anhydrous methanol (20L) under argon. The
mixture was
stirred at room temperature for 30 min, and then inPEG-0-tosylate of molecular
mass 20
kilodalton (Ts; 1.0 g, 0.05 mmol) was added in several portions over 2 hours.
The mixture
was stirred at room temperature for 5 days, and concentrated by rotary
evaporation. The
residue was diluted with water (30 mL), and stirred at room temperature for 2
hours to
destroy any excess 20 kilodalton mPEG- 0-tosylate. The solution was then
neutralized with
acetic acid, the pH adjusted to pH 5.0 and loaded onto a reversed phase
chromatography (C-
18 silica) column. The column was eluted with a gradient of methanol/water
(the product
elutes at about 70% methanol), product elution monitored by-evaporative light
scattering, and
- the appropriate fractions collected and diluted with water (500 mL).
This solution was
chromatographed (ion exchange, XK 50 (jm, BIG Bead; 300 nil, hydroxide form;
gradient of
water to water/acetic acid-0.75N) and the pH of the appropriate fractions
lowered to 6.0 with
acetic acid. This solution was then captured on a reversed phase column (C-18
silica) and
eluted with a gradient of methanol/water as described above. The product
fractions were
pooled, concentrated, redissolved in water and freeze-dried to afford a white
solid (1).
Structural data for the compound were as follows: 1H-NMR (500 MHz; D20) 5 2.83
(t, 2H, -
0-C-Cf_12-S), 3.05 (q, 1H, S-CHH-CHN), 3.18 (q, 111, (q, 1H, S-CHH-CHN), 3.38
(s, 3H,
CO), 3.7 (t, OCLI2Cf_120), 3.95 (q, 1H, CHN). The purity of the product was
confirmed by
SDS PAGE.
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PCT/US2005/002522
4.2 Synthesis of Compound 2 (Cysteine-PEG2)
[0348] Triethylamine (-0.5 mL) was added dropwise to a solution of compound 1
(440 mg,
22 mop dissolved in anhydrous CH2C12 (30 mL) until the solution was basic. A
solution of
20 kilodalton mPEG-0-p-nitrophenyl carbonate (660 mg, 33 mop and N-
hydroxysuccinimide (3.6 mg, 30.8 [tmol) in CH2C12 (20 mL) was added in several
portions
over 1 hour at room temperature. The reaction mixture was stirred at room
temperature for
24 hours. The solvent was then removed by rotary evaporation, the residue was
dissolved in
water (100 mL), and the pH adjusted to 9.5 with 1.0 N NaOH. The basic solution
was stirred
at room temperature for 2 hours and was then neutralized with acetic acid to a
p1-1 7Ø The
solution was then loaded onto a reversed phase chromatography (C-18 silica)
column. The
column was eluted with a gradient of methanol/water (the product elutes at
about 70%
methanol), product elution monitored by evaporative light scattering, and the
appropriate
fractions collected and diluted with water (500 mL). This solution was
chromatographed (ion
exchange, XI( 50 Q, BIG Beads, 300 mL, hydroxide form; gradient of water to -
water/acetic
acid-0.75N) and the pH of the appropriate fractions lowered to 6.0 with acetic
acid. This
solution was then captured on a reversed phase column (C-18 silica) and.eluted
with a
gradient of methanol/water as described above. The product fractions were
pooled,
concentrated, redissolved in water and freeze-dried to afford a white solid
(2). Structural data
for the compound were as follows: 11-1-NMR (500 MHz; D20) 5 2.83 (t, 2H, O-C-
CH2-S),
2.95 (t, 2H, 0-C-CH2-S), 3.12 (q, 1H, S-CHH-CHN), 3.39 (s, 3H CH30), 3.71 (t,
OCH2CH20). The purity of the product was confirmed by SDS PAGE.
Example 5
Preparation of UDP-GaINAc-6-NHCO(CH2)2-0-PEG-0Me (1 KDa).
[0349] Galactosaminy1-1-phosphate-2-NHCO(CH2)2-0-PEG-0Me (1 kilodalton) (58
mg,
0.045 mmoles) was dissolved in DMF (6 mL) and pyridine (1.2 mL). LTMP-
morpholidate
(60 mg, 0.15 mmoles) was then added and the resulting mixture stirred at 70 C
for 48 h. The
solvent was removed by bubbling nitrogen through the reaction mixture and the
residue
purified by reversed phase chromatography (C-18 silica, step gradient between
10 to 80%,
methanol/water). The desired fractions were collected and dried at reduced
pressure to yield
a white solid. TLC (silica, propanol/H20/NH4OH, (30/20/2), Rf = 0.54). MS
(1\4ALDI):
Observed, 1485, 1529, 1618, 1706.
CA 02554466 2012-08-13
SDS PAGE Procedure
[0208] The purity of the products, 1 and 2, were confirmed by SDS PAGE. A 4-
20% Tris-
Glycine SDS PAGE gel (Invitrogen) was used. The sample was mixed 1:1 with SDS
Sample Buffer, and was run in Tris-Glycine Running Buffer (LC2675-5) at a
constant voltage
(125 V) for 1 hr 50 min. After electrophoresis, the gel was washed with water
(100 mL) for
min followed by a wash with a 5% barium chloride aqueous solution (100 mL) for
10 min.
Products 1 or 2 were visualized by staining the gels with 0.1 N iodine
solution (4.0 mL) at
room temperature and the staining process stopped by washing the gels with
water. The .
visualized product bands were scanned with an HP Scanjet 7400C: and the image
of the gel
was optimized with the HP Precision Scan Prograe
[0209] While this invention has been disclosed with reference to specific
embodiments, it is
apparent that other embodiments and variations of this invention may be
devised by others
skilled in the art. The scope of the claims should not be limited by the
preferred
embodiments and the examples, but should be given the broadest interpretation
consistent with the description as a whole.
66
