Note: Descriptions are shown in the official language in which they were submitted.
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EXTENDED NATIVE CHEMICAL LIGATION
Cross Reference to Related Applications
This application claims benefit to provisional application U.S. Serial No.
60/231,339, filed September 8, 2000.
Acknowledgments
This invention was supported in part by National Institute of Health
Postdoctoral Fellowship grant GN190402. The United States government may
have certain rights.
Technical Field
The present invention relates to methods and compositions for extending
the technique of native chemical ligation to permit the ligation of a wide
range of
peptides, polypeptides, other polymers and other molecules via an amide bond.
Background
Chemical ligation involves the formation of a selective covalent linkage
between a first chemical component and a second chemical component. Unique,
mutually reactive, functional groups present on the first and second
components
can be used to render the ligation reaction chemoselective. For example, the
chemical ligation of peptides and polypeptides involves the chemoselective
reaction of peptide or polypeptide segments bearing'compatible unique,
mutually
reactive, C-terminal and N-terminal amino acid , residues. Several different
a
chemistries have been utilized for this purpose, examples of which include
native
chemical ligation (Dawson, et al., Science (1994) 266:776-779; Kent, et al.,
WO
96/34878; Kent, et al., WO 98/28434), oxime forming chemical ligation (Rose,
et
al., J. Amer. Chem. Soc. (1994) 116:30-34), thioester forming ligation
(Schnolzer,
et al., Science (1992) 256:221-225), thioether forming ligation (Englebretsen,
et
al., Tet. Letts. (1995) 36(48):8871-8874), hydrazone forming ligation
(Gaertner, et
al., Bioconj. Chem. (1994) 5(4):333-338), and thiazolidine forming ligation
and
oxazolidine forming ligation (Zhang, et al., Proc. Natl. Acad. Sci. (1998)
95(16):9184-9189; Tam, et al., WO 95/00846; US Patent No. 5,589,356).
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Of these methods, only the native chemical ligation approach yields a
ligation product having a native amide (i.e. peptide) bond at the ligation
site. The
original native chemical ligation methodology (Dawson et al., supra; and WO
96/34878) has proven a robust methodology for generating a native amide bond
at the ligation site. Native chemical ligation involves a chemoselective
reaction
between a first peptide or polypeptide segment having a C-terminal a-
carboxythioester moiety and a second peptide or polypeptide having an N-
terminal cysteine residue. A thiol exchange reaction yields an initial
thioester-
linked intermediate, which spontaneously rearranges to give a native amide
bond
at the ligation site while regenerating the cysteine side chain thiol. The
primary
drawback of the original native chemical ligation approach is that it requires
an N-
terminal cysteine, i.e., it only permits the joining of peptides and
polypeptide
segments possessing a cysteine at the ligation site.
Notwithstanding this drawback, native chemical ligation of peptides with N
terminal amino acids other than cysteine has been reported (W098/28434). In
this approach, the ligation is performed using a first peptide or polypeptide
segment having a C-terminal a-carboxythioester and a second peptide or
polypeptide segment having an N-terminal N-{thiol-substituted auxiliary} group
represented by the formula HS-CHZ-CH2-O-NH-[peptide] . Following ligation, the
N-~thiol substituted auxiliary} group is removed by cleaving the HS-CHI-CHZ-O-
auxiliary group to generate a native amide bond at the ligation site. One
limitation
of this method is that the use of a mercaptoethoxy auxiliary group can
successfully lead to amide bond formation only at a glycine residue. This
produces a ligation product that upon cleavage generates a glycine residue at
the
position of the N-substituted amino acid of the second peptide or polypeptide
segment. As such, this embodiment of the method is only suitable if one
desires
the ligation product of the reaction to contain a glycine residue at this
position, and
in any event can be problematic with respect to ligation yields, stability of
precursors, and the ability to remove the O-linked auxiliary group. Although
other
auxiliary groups may be used, for example the HSCHZCH2NH-[peptide], without
limiting the reaction to ligation at a glycine residue, such auxiliary groups
cannot
be removed from the ligated product.
Accordingly, what is needed is a broadly applicable and robust chemical
ligation system that extends native chemical ligation to a wide variety of
different
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amino acid residues, peptides, polypeptides, polymers and other molecules by
means of an effective, readily removable thiol-containing auxiliary group, and
that
joins such molecules together with a native amide bond at the ligation site.
The
present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The invention is directed to methods and compositions related to extended
native chemical ligation. The extended native chemical ligation method of the
invention comprises: generating an N-substituted amide-linked initial ligation
product of the formula:
J1-C(O)-N(C1(R1)-C2-SH)-J2 I
or
J1-C(O)-N(C1 (R1 )-C2(R2)-C3(R3)-SH)-J2 I I
where J1 is a peptide or polypeptide having one or more optionally protected
amino acid side chains, or a moiety of such peptide or polypeptide, a polymer,
a
dye, a suitably functionalized surface, a linker or detectable marker, or any
other
chemical moiety compatible with chemical peptide synthesis or extended native
chemical ligation; R1, R2 and R3 are independently H or an electron donating
group conjugated to C1; with the proviso that at least one of R1, R2 and R3
comprises an electron donating group conjugated to C1; and J2 is a peptide or
polypeptide having one or more optionally protected amino acid side chains, or
a
moiety of such peptide or polypeptide, a polymer, a dye, a suitably
functionalized
surface, a linker or detectable marker; or any other chemical moiety
compatible
with chemical peptide synthesis or extended native chemical ligation.
The ligation product is produced by the process of ligating a first
component comprising a carboxyl thioester of the formula J1-C(O)SR to a second
component comprising an acid stable N-substituted 2 or 3 carbon chain amino
alkyl or aryl thiol of the formula:
HS-C2-C1 (R1 )-HN-J2 III
or
HS-C3(R3)-C2(R2)-C1(R1)-HN-J2 IV
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where J2, R1, R2, and R3, are as defined above, and then optionally removing
the
2 or 3 carbon chain alkyl or aryl thiol from the N-substituted amide-linked
ligation
product. In a preferred embodiment, such cleavage is facilitated by forming a
resonance stabilized cation at C1 under peptide compatible cleavage
conditions.
The removal of the alkyl or aryl thiol chain from the N generates a final
ligation
product of the formula:
J1-C(O)-HN-J2 V
where J1, J2, R1, R2, and R3 are as defined above.
The invention also is directed to compositions for effecting such extended
native chemical ligation, and to cartridges and kits that comprise them. The
compositions comprise a fully protected, partially protected or fully
unprotected
acid stable N-substituted, and preferably Na-substituted, 2 or 3 carbon chain
amino alkyl or aryl thiol of the formula:
SX2-C2-C1 (R1 )-X1 N-CH(Z2)-C(O)-J2 VI
or
SX2-C3(R3)-C2(R2)-C1 (R1 )-X1 N-CH(Z2)-C(O)-J2 VII
where X1 is H or an amino protecting group; X2 is H or a thiol protecting
group;
J2, R1, R2 and R3 are as defined above; and Z2 is any chemical moiety
(including, without limitation, an amino acid side chain) compatible with
chemical
peptide synthesis or extended native chemical ligation. The invention also is
directed to chiral forms of such compounds of the invention that are
substantially
free of racemates or diasterioisomers.
The invention is further directed to solution phase and solid phase
methods of producing such fully protected, partially protected or fully
unprotected
N-substituted 2 or 3 carbon chain amino alkyl or aryl thiols. The methods for
producing these compounds include halogen-mediated amino alkylation, reductive
amination, and preparation of Na-protected, N-alkylated, S-protected, amino
alkyl-
or aryl- thiol amino acid precursors compatible with solid phase peptide
synthesis
methods.
The J1 moiety of the carboxythioester component can comprise any
chemical moiety compatible with the carboxythioester and reaction conditions
for
extended native chemical ligation, and the N-substituted component of the
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invention can be provided alone or joined to a wide range of chemical
moieties,
including amino acids, peptides, polypeptides, nucleic acids or other chemical
moieties such as dyes, haptens, carbohydrates, lipids, solid support,
biocompatible polymers or other polymers and the like. The extended native
chemical ligation method of the invention is robust and can be perFormed in an
aqueous system near neutral pH and at a range of temperature conditions. The
methods of producing the N-substituted components of the invention also are
robust, providing a wide range of synthetic routes to these novel compounds in
surprisingly high and pure yields. Na-protected, N-alkylated, S-protected,
amino
alkyl- or aryl- thiol amino acid precursors of the invention are particularly
useful for
rapid automated synthesis using conventional peptide synthesis and other
organic
synthesis strategies. Moreover, the protected N-substituted components of the
invention expand the utility of chemical ligation to multi-component ligation
schemes, such as when producing a polypeptide involving multiple ligation
strategies, such as a three or more segment ligation scheme or convergent
ligation synthesis schemes. For example, the methods and compositions of the
present invention permit one to use a first pair of carboxythioester and N-
substituted components to synthesize a first portion of a desired molecule,
and to
use additional pairs of carboxythioester and N-substituted components to
synthesize additional portions of the molecule. The ligation products of each
such
synthesis can then be ligated together (after suitable deprotection and/or
modification) to form the desired molecule.
Accordingly, the methods and compositions of the invention greatly
expand the scope of native chemical ligation, and the starting, intermediate
and
final products of the invention find a wide range of uses.
Brief Description Of The Drawings:
Figure 1 illustrates the present invention by showing its ability to mediate
the extended native chemical ligation of peptides; the same schemes could be
employed to effect the ligation of any suitable molecule. As shown in the
Figure, a
first component containing an a-carboxyl thioester of the formula J1-HN-CH(Z1)-
aC0-SR, and a second component containing an N-terminal acid stable Na-
substituted 2 carbon chain alkyl or aryl thiol of the formula HS-C2-C1(R1)-NHa-
CH(Z2)-C(O)-J2. The components J1 and J2 can be any chemical moiety
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compatible with the chemoselective ligation reaction, such as a protected or
unprotected amino acid, peptide, polypeptide, other polymer, dye, linker and
the
like. Z1 is any side chain group compatible with the aC0-SR thioester, such as
a
protected or unprotected side chain of an amino acid. Z2 is any side chain
group
compatible with an Na-substituted amino acid, such as a protected or
unprotected
side chain of an amino acid. R1 is a benzyl moiety (benzyl when referred to in
the
context of C1, otherwise referred to as phenyl) substituted with an electron-
donating group preferably in the ortho or para position relative to C1; or a
picolyl
(unsubstituted or substituted with hydroxyl or thiol in the ortho or para
position
relative to C1 ).
Thiol exchange occurs between the aCOSR thioester component and the
amino N-{alkyl thiol} component. The exchange generates a thioester-linked
intermediate ligation product that after spontaneous rearrangement through a 5-
membered ring intermediate generates a first ligation product of the formula
J1-
HN-CH(Z1 )-C(O)-Na(C1 (R1 )-C2-SH)-CH(Z2)-C(O)-J2 having a removable Na
substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-] at the ligation
site.
The Na-substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-] at the
ligation site is amenable to being removed, under peptide-compatible
conditions,
to generate a final ligation product of the formula J1-HN-CH(Z1)-CO-NH-CH(Z2)
CO-J2 having a native amide bond at the ligation site.
Figure 2 illustrates the present invention by showing its ability to mediate
the extended native chemical ligation of peptides; the same schemes could be
employed to effect the ligation of any suitable molecule. As shown in the
Figure, a
first component containing a-carboxyl thioester of the formula J1-HN-CH(Z1)-
aC0-SR, and a second component containing an acid stable Na-substituted 3
carbon chain alkyl or aryl thiol of the formula HS-C3(R3)-C2(R2)-C1 (R1 )-NHa-
CH(Z2)-C(O)-J2. The components J1 and J2 can be any chemical moiety
compatible with the chemoselective ligation reaction, such as a protected or
unprotected amino acid, peptide, polypeptide, other polymer, dye, linker and
the
like. Z1 is any side chain group compatible with the aC0-SR thioester, such as
a
protected or unprotected side chain of an amino acid. Z2 is any side chain
group
compatible with an Na-substituted amino acid, such as a protected or
unprotected
side chain of an amino acid. When R1 is other than hydrogen, R2 and R3 are
hydrogen, and R1 is a phenyl moiety, unsubstituted or substituted with an
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electron-donating group in the ortho or para position relative to C1; a
picolyl
(unsubstituted or substituted with hydroxyl or thiol in the ortho or para
position
relative to C1); a methanethiol; or a sulfoxymethyl. When R2 and R3 are other
than hydrogen, R1 is hydrogen, and R3 and R2 form a benzyl group that is
substituted with an electron-donating group in the ortho or para position
relative to
C1; or a picolyl (unsubstituted or substituted with hydroxyl or thiol in the
ortho or
para position relative to C1).
Thiol exchange occurs between the COSR thioester component and the
amino alkyl thiol component. The exchange generates a thioester-linked
intermediate ligation product that after spontaneous rearrangement through a 6-
membered ring intermediate generates a first ligation product of the formula
J1-
HN-CH(Z1)-C(O)-Na(C1-C2(R2)-C3(R3)-SH)-CH(Z2)-J2 having a removable Na-
substituted 3 carbon chain alkyl or aryl thiol [HS-C3(R3)-C2(R2)-C1 (R1 )-] at
the
ligation site. The Na-substituted 3 carbon chain aryl thiol [HS-C3(R3)-C2(R2)-
C1(R1)-] at the ligation site is amenable to being removed, under peptide-
compatible conditions, to generate a final ligation product of the formula J1-
HN-
CH(Z1)-CO-NH-CH(Z2)-CO-J2 having a native amide bond at the ligation site.
Figure 3 illustrates a multi-component extended native chemical ligation
scheme. A polypeptide a-carboxyl thioester with an Na-protected N-terminal
polypeptide Na-substituted 2 carbon chain alkyl or aryl thiol of the formula
HS-C2-
C1 (R1 )-Na(PG1 )-CH(Z2)-C(O)-J2 as embodied in Figure 1 is reacted with a
peptide that contains an N-terminal Cys residue. R1 is a phenyl,
unsubstituted, or
substituted with an electron-donating group, preferably in the ortho or para
position relative to C1; or a picolyl (unsubstituted or substituted with
hydroxyl or
thiol in the ortho or para position relative to C1). The protecting group
(PG1) may
be any suitable protecting group, such as an alkylcarbonyl protecting group
(e.g.,
benzyloxycarbonyl (Z), Boc, Bpoc, Fmoc, etc.), a triphenylmethyl protecting
group
(Trt), a 2-nitrophenylsulfenyl protecting group (Nps), etc. The protecting
group is
removed after the first ligation reaction.
A first native chemical ligation reaction is carried out between the
polypeptide a-carboxyl thioester with an Na-protected N-terminal polypeptide
Na-
substituted 2 carbon chain alkyl or aryl thiol of the formula HS-C2-C1(R1)-
Na(PG1)-CH(Z2)-C(O)-J2 as embodied in Figure 1 and the N-terminal Cys-
peptide to give a first ligation product of formula: HS-C2-C1(R1)-Na(PG1)-
CH(Z2)-
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C(O)-Peptide2-Peptide3 . The protecting group PG1 is then removed to give the
ligation product of formula HS-C2-C1(R1)-Na(H)-CH(Z2)-C(O)-Peptide2-Peptide3.
This species is then reacted with a third, thioester-containing component.
Thiol
exchange occurs between the COSR thioester component and the amino N-{alkyl
thiol~ component. The exchange generates a thioester-linked intermediate
ligation
product that after spontaneous rearrangement through a 5-membered ring
intermediate generates a second ligation product of the formula Peptide1-C(O)-
Na(C1 (R1 )-C2-SH)-CH(Z2)-C(O)Peptide2-Cys-Peptide3, having a removable Na-
substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-] at the second
ligation site. The Na-substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1
(R1 )-]
at the second ligation site is amenable to being removed, under peptide-
compatible conditions, to generate a final ligation product of the formula
peptide1-
C(O)-NaH-CH(Z2)-C(O)Peptide2-Cys-Peptide3, having a native amide bond at
the first and second ligation sites.
Figure 4 illustrates a general ligation strategy employing two different 1-
phenyl-2-mercaptoethyl auxiliaries of the invention.
Figures 5A and 5B shows analytical High Performance Liquid
Chromatography (HPLC) results of a ligation reaction for cytochrome b562 as
described in Example 21 using an Na-1-(4-methoxyphenyl)-2-mercaptoethyl
auxiliary. Figure 5A shows the status of the ligation reaction at time = 0.
Figure
5B shows the status of the ligation after the reaction is allowed to proceed
overnight. As also shown in Figure 5B, two ligation products are observed that
result from the achiral center at C1 of the Na-1-(4-methoxyphenol)-2-
mercaptoethyl auxiliary.
Figure 6A and 6B shows reconstructed electrospray mass spectra (MS) of
the ligation product Cytochrome b562 residues 1-106 formed by using extended
native chemical ligation with an Na-{1-(4-methoxyphenyl) 2-mercaptoethano}-
modified N-terminal segment. Cytochrome b562 residues 1-63 bearing a C-
terminal athioester was ligated with Cytochrome b562 residues 64-1.06 bearing
an
N-terminal Na-{1-(4-methoxyphenyl) 2-mercaptoethano~ glycine. Figure 6A
shows MS reconstruct of the initial ligation product that includes a removable
Na-
{1-(4-methoxyphenyl) 2-mercaptoethano} group at the ligation site. Figure 6B
shows a MS reconstruct of ligation product following hydrogen fluoride (HF)
treatment to remove the Na-{1-(4-ri~ethoxyphenyl) 2-mercaptoethano} group to
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generate a native amide bond at the ligation site. The observed masses were
11948~1 Da (before HF treatment) and 11781~1 Da (after HF treatment), i.e. a
loss
of 167~2Da, in good agreement with the 166Da loss expected for removal of the
1-(4-methoxyphenyl) 2-mercaptoethano auxiliary group.
Figure 7A and 7B illustrate a representative analytical HPLC of linear
cytochrome b562 material (Figure 7A) depicted in Figure 6B, and an ion
exchange chromatogram (Figure 7B) of the material following folding.
DESCRIPTION OF SPECIFIC EMBODIMENTS
The invention is directed to methods and compositions related to extended
native chemical ligation. In general, the method involves ligating a first
component comprising a carboxyl thioester, and more preferably, an a-carboxyl
thioester with a second component comprising an acid stable N-substituted, and
preferably, Na-substituted, 2 or 3 carbon chain amino alkyl or aryl thiol.
Chemoselective reaction between the carboxythioester of the first component
and
the thiol of the N-substituted 2 or 3 carbon chain alkyl or aryl thiol of the
second
component proceeds through a thioester-linked intermediate, and resolves into
an
initial ligation product. More specifically, the thiol exchange occurring
between the
COSR thioester component and the amino alkyl thiol component generates a
thioester-linked intermediate ligation product that after spontaneous
rearrangement through a 5-membered or 6-membered ring intermediate
generates an amide-linked first ligation product of the formula:
J1-C(O)-N(C1 (R1 )-C2-SH)-J2 I
or
J1-C(O)-N(C1 (R1 )-C2(R2)-C3(R3)-SH)-J2 II
where J1, J2, R1, R2 and R3 are as defined above.
The N-substituted 2 or 3 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-] or
[HS-(C3(R3)-C2(R2)-C1(R1)-] at the ligation site is amenable to being removed,
under peptide-compatible conditions, without damage to the product, to
generate
a final ligation product of the formula:
J 1-C(O)-H N-J2 V
where J1, J2, R1, R2, and R3 are as defined above.
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The final ligation product has a native amide bond at the ligation site
More particularly, the extended native chemical ligation method of the
invention comprises chemical ligation of: (i) a first component comprising an
a-
carboxyl thioester of the formula J1-C(O)SR and (ii) a second component
comprising an acid stable N-substituted 2 or 3 carbon chain amino alkyl or
aryl
thiol of the formula:
J1-C(O)-N(C1 (R1 )-C2-SH)-J2 I
or
J1-C(O)-N(C1 (R1 )-C2(R2)-C3(R3)-SH)-J2 II
where J1, J2, R1, R2, and R3 are as defined above.
The R1, R2 and R3 groups are selected to facilitate cleavage of the N-C1
bond under peptide compatible cleavage conditions. For example, electron
donating groups, particularly if conjugated to C1, can be used to form a
resonance
stabilized cation at C1 that facilitates cleavage. The chemical ligation
reaction
preferably includes as an excipient a thiol catalyst, and is carried out
around
neutral pH conditions in aqueous or mixed organic-aqueous conditions. Chemical
ligation of the first and second components may proceed through a five or six
member ring that undergoes spontaneous rearrangement to yield an N-substituted
amide linked ligation product. Where the first and second components are
peptides or polypeptides, the N-substituted amide linked ligation product has
the
formula:
J1-C(O)-Na(C1 (R1 )-C2-SH)-CH(Z2)-C(O)-J2 VII I
or
J1-C(O)-Na(C1 (R1 )-C2(R2)-C3(R3)-SH)-CH(Z2)-C(O)-J2 IX
where J1, J2 and R1, R2, R3 and Z2 are as defined above
The conjugated electron donating groups R1, R2 or R3 of the N-
substituted amide bonded ligation product facilitate cleavage of the N-C1 bond
and removal of the 2 or 3 carbon chain alkyl or aryl thiol from the N-
substituted
amide-linked ligation product. Removal of the alkyl or aryl thiol chain of the
N
under peptide-compatible cleavage conditions generates a ligation product
having
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a native amide bond at the ligation site. If the first and second components
were
peptides or polypeptides, the ligation product will have the formula:
J1-CONocH-CH(Z2)-C(O)-J2 X
The present invention provides multiple advantages over previous
chemical ligation approaches. Several such advantages relate to the finely
tuned
nature of the N-substituted 2 or 3 carbon chain alkyl or aryl thiol component
of the
present invention. First, the unligated N-substituted component is stable to
acidic
conditions, which permits its robust synthesis and storage. Second, it
selectively
reacts with the carboxythioester component to generate an initial ligation
product
having an N-substituted amide bond at the ligation site. Third, the
regenerated
alkyl or aryl thiol moiety at the Na position of the ligation site of the
initial ligation
product can be selectively removed under conditions fully compatible with
unprotected, partially protected or fully protected peptides, polypeptides or
other
moieties, i.e., the alkyl or aryl thiol moiety can be removed without damaging
the
desired ligation product. The selective cleavage reaction can be readily
performed under standard peptide-compatible cleavage conditions such as
acidic,
photolytic, or reductive conditions, depending on the particular N-substituted
alkyl
or aryl thiol moiety chosen for ligation. Thus, another advantage of the
invention
is that one or more groups on remaining portions of the ligation components,
if
present, can be unprotected, partially protected or fully protected depending
on
the intended end use. Moreover, given the chemoselective nature and solubility
properties of the carboxyl thioester and N-substituted 2 or 3 carbon chain
alkyl or
aryl thiol, the ligation reaction can be carried out rapidly and cleanly to
give high
product yields at around pH 7 under aqueous conditions at around room
temperature. This makes the invention particularly flexible for ligating
partially or
fully unprotected peptides, polypeptides or other polymers under mild
conditions.
For a peptide component that comprises the N-substituted 2 carbon chain
alkyl or aryl thiol component of the invention, this compound has the formula:
HS-C2-C1 (R1 )-NHa-CH(Z2)-C(O)-J2 XI
as depicted below in Table I. J2 and R2 are as described above; Z2 is any side
chain group compatible with an N-substituted amino acid, such as a side chain
of
an amino acid. R1 is preferably a phenyl group substituted with an electron-
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donating group in the ortho or para position relative to C1; or a picolyl
group
(unsubstituted or substituted with hydroxyl or thiol in the ortho or para
position
relative to C1).
Table I
Formula I
z
HNI CH-~~ J2
C~~ O
HS C2 R~
R1 Substituent Groups for Formula I (C1 included for reference)
' ~~ R1i
N ~ /N
R5~ R3 R3~ R5.
Positioning of the phenyl and picolyl electron-donating substituents R1', R3'
and
R5' in the ortho or para positions is necessary to maintain electronic
conjugation
to the C1 carbon to enhance cleavage of the N-C1 bond following ligation.
Preferred electron-donating groups for R1', R3' and R5' include strong
electron-
donating groups such as methoxy (-OCH3), thiol (-SH), hydroxyl (-OH),
methylthio
(-SCH3), and moderate electron-donators such as methyl (-CH3), ethyl (-CH2-
CH3), propyl (-CH2-CH2-CH3), isopropyl (-CH2(CH3)3). Provided that any or all
of R1', R3' and R5' maybe H. A general observation is that the strong electron-
donating groups enhance the sensitivity of the 2-carbon chain alkyl or aryl
thiol to
cleavage following ligation. When a single electron-donating group is present
as a
R1', R3' or R5' substituent, the ligation reaction may proceed at a faster
rate,
whereas cleavage is slower or requires more stringent cleavage conditions.
When two or more electron-donating groups are present as a R1', R3' or R5'
substituent, the (igation reaction may be slower, whereas cleavage is faster
or
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requires less stringent cleavage conditions. Thus a particular electron-
donating
group can be selected accordingly.
Another embodiment of the invention relates to the N-substitufied 2 carbon
chain compounds, which include a thiol as a substituent of R1 in the R1' and
R5'
positions. In addition to being an electron donating group conjugated to C1,
introduction of a thiol at one or both of these locations enables the
compounds to
ligate through a 6-member ring mediated through the R1 group (as well as
through a 5-member ring by the Na-2 carbon chain alkyl thiol). It also
increases
the local concentration of available thiols for reacting with the a-carboxy
thioester,
and provides for additional conformations in terms of structural constraints
that
can improve ligation.
Referring to the Na-substituted 3 carbon chain alkyl or aryl thiol
component of the invention, this compound has the formula HS-C3(R3)-C2(R2)-
C1 (R1 )-NHa-CH(Z2)-C(O)-J2, which is depicted below in Table II.
Table II
Formula II
Zz
HN ~ CH-~I J2
HS\ /C~\ O
C3-C2 R~
R~ Rz
R1, R2 and R3 Substituents (C1 included for reference)
1 1
C~~R1
N ~ ~ R~
N
R3 R '
3
R~ R~~ R~
N /i N
Rs Rs ~ Rs, Rs /i
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As described above, J2 can be any chemical moiety compatible with the chemical
peptide synthesis or extended native chemical ligation, Z2 is any side chain
group
compatible with an N-substituted amino acid, such as a side chain of an amino
acid. When R1 is other than hydrogen, R2 and R3 are hydrogen, and R1 is a
phenyl moiety, unsubstituted, or more preferably, substituted with an electron-
donating group in the ortho or para position relative to C1; or a picolyl
(unsubstituted or substituted with hydroxyl or thiol in the ortho or para
position
relative to C1). When R2 and R3 are other than hydrogen, R1 is hydrogen, and
R3 and R2 form a benzyl group that is substituted with an electron-donating
group
in the ortho or para position relative to C1; or a picolyl (unsubstituted or
substituted with hydroxyl or thiol in the ortho or para position relative to
C1 ).
As with the N-substituted 2 carbon chain compounds, positioning of the
phenyl and picolyl electron-donating substituents R1', R3' and R5' in the
ortho or
para positions is necessary to maintain electronic conjugation to the C1
carbon for
robust cleavage of the Na-C1 bond following ligation. However, when R2 and R3
form a benzyl group with C2 and C3, at least one of R1' and R3' comprises a
strong electron donating group, where R1' or R3' is selected from methoxy (-
OCH3), thiol (-SH), hydroxyl (-OH), and thiomethyl (-SCH3). For the N-
substituted
3 carbon chain thiols in which R2 and R3 are hydrogens, R1 comprises a phenyl
or picolyl group in which R1', R3' and R5' include either strong or moderate
electron-donating groups, or a combination thereof. As with the N-substituted
2
carbon chain alkyl or aryl thiols, the strong electron-donating groups enhance
the
sensitivity of the 3 carbon chain alkyl or aryl thiol to cleavage following
ligation.
Thus a particular electron-donating group or combination thereof can be
selected
accordingly.
Similar to the N-substituted 2 carbon chain compounds, the N-substituted
3 carbon chain compounds of the present invention may include a thiol as a
substituent of R1 in the R1' and R5' positions when available for substitution
in a
construct of interest. Here again the electron-donating thiol group is
conjugated to
C1 and its introduction at these locations enables the compounds to have two
routes for the 6-member ring forming ligation event. It also increases the
local
concentration of available thiols for reacting with the a-carboxy thioester,
and
provides for additional conformations in terms of structural constraints that
can
improve ligation.
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Synthesis of the N-terminal N-substituted 2 or 3 carbon chain alkyl or aryl
thiol amino acids of the invention can carried out as described herein, for
example, in Scheme I and Scheme (l, the Examples, and in accordance with
standard organic chemistry techniques known in the art. See, e.g., "Advanced
Organic Chemistry, Reactions, Mechanisms, and Structure," 4t" Edition, J.
March
(Ed.), John Wiley & Sons, New York, NY, 1992; "Comprehensive Organic
Transformations, A Guide to Functional Group Preparations," R. Larock (Ed.),
VCH Publishers, New York, NY, 1989. They may be synthesized in solution, by
polymer-supported synthesis, or a combination thereof. The preferred approach
employs N alpha protected N alkylated S-protected amino alkyl- or aryl- thiol
amino acid precursors. The reagents utilized for synthesis can be obtained
from
any number of commercial sources. Also, it will be well understood that the
starting components and various intermediates, such as the individual amino
acid
derivatives can be stored for later use, provided in kits and the like.
In preparing the N-terminal Na-substituted 2 or 3 carbon chain alkyl or aryl
thiol amino acids of the invention, protecting group strategies are employed.
The
preferred protecting groups (PG) utilized in the various synthesis strategies
in
general are compatible with Solid Phase Peptide Synthesis ("SPPS"). In some
instances, it also is necessary to utilize orthogonal protecting groups that
are
removable under different conditions. Many such protecting groups are known
and suitable for this purpose (See, e.g., "Protecting Groups in Organic
Synthesis",
3rd Edition, T.W. Greene and P.G.M. Wuts, Eds., John Wiley & Sons, Inc., 1999;
NovaBiochem Catalog 2000; "'Synthetic Peptides, A User's Guide," G.A. Grant,
Ed., W.H. Freeman & Company, New York, NY,1992; "Advanced Chemtech
Handbook of Combinatorial & Solid Phase Organic Chemistry," W.D.. Bennet,
J.W. Christensen, L.K. Hamaker, M.L. Peterson, M.R.Rhodes, and H.H. Saneii,
Eds., Advanced Chemtech, 1998; "Principles of Peptide Synthesis, 2nd ed.," M.
Bodanszky, Ed., Springer-Verlag, 1993; "The Practice of Peptide Synthesis, 2nd
ed.," M. Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; and
"Protecting Groups," P.J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart,
Germany, 1994). Examples include benzyloxycarbonyl (Z), Boc, Bpoc, Trt, Nps,
FmocCl-Z, Br-Z; NSC; MSC, Dde, etc. For sulfur moieties, examples of suitable
protecting groups include, but are not limited to, benzyl, 4-methylbenzyl, 4-
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methoxybenzyl, trityl, Acm, TACAM, xanthyl, disulfide derivatives, picolyl,
and
phenacyl.
More particularly, the Na-substituted 2 or 3 carbon chain alkyl or aryl thiols
can be prepared in accordance with Scheme I (Solid-Phase preparation of the
Na-substituted precursor), Scheme II (Solution-Phase preparation of the Na
substituted precursor). In Scheme I, Na-substituted 2 or 3 carbon chain alkyl
or
aryl thiols are assembled directly on the solid phase using standard methods
of
polymer-supported organic synthesis, while the Na-protected, N-alkylated, S-
protected, aminoalkyl or arylthiol amino acid precursor of Scheme II are
coupled
to the resin using standard coupling protocols. In Scheme I, X is a halogen,
R1
and R2 are as described above and can be preformed as protected or
unprotected moieties or elaborated on-resin, and J2 is preferably attached to
halogen as X-CH(R)-J2-Resin, where R is hydrogen or other side chain. It will
be
appreciated that J2 can be a variety of groups, for example where halogen X
and
J2-Resin are separated by more than one carbon, such as in synthesis of beta
or
gamma amino acids or similar molecules. Where glyoxalic moiety (HC(O)-C(O)-
J2-Resin) is employed, resulting side chain R is hydrogen. In Scheme II, X is
a
halogen, R1 and R2 are as described above and can be preformed as protected
or unprotected moieties or elaborated in solution or on-resin, and where R is
hydrogen or other side chain. Where glyoxalic acid moiety (HC(O)-C(O)-OH) is
employed, the resulting side chain R is hydrogen. As noted above, it will be
appreciated that Schemes I and II can be applied in the synthesis of the 3
carbon
chain alkyl or aryl thiols. Where racemic or. diastereomeric products are
produced, it may be necessary to separate these by standard methods before use
in extended native chemical ligation.
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Scheme I
NHz X
S S
PG~/ \R~ PG~~ \R~
Rz p Rz
HOC~Jz-Resin
Resin J -X ~r HN-Jz-Resin
- z
HN-Jz-Resin
S
PG~/ \R~
Rz
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Scheme II
R X
OR' /g
X PGf Rt
R'
NHz O Rz R
OR OR NH OR'
PG~ R~
O
O 0
Rz= H
~ PGt~s R~
OHC' -OH r
Rz
,Jz-resin
Referring to the carboxy thioester moiety of the first component utilized for
the
extended native chemical ligation method of the invention, this component has
the
formula J1-CO-SR. The more preferred carboxy thioester component comprises
an a-carboxyl thioester amino acid of the formula J1-NH-C(Z1)-CO-SR. The group
J1 can be any chemical moiety compatible with the chemoselective ligation
reaction, such as a protected or unprotected amino acid, peptide, polypeptide,
other polymer, dye, linker and the like. Z1 is any side chain group compatible
with
the aC0-SR thioester, such as a side chain of an amino acid. R is any group
compatible with the thioester group, including, but not limited to, aryl,
benzyl, and
alkyl groups. Examples of R include 3-carboxy-4-nitrophenyl thioesters, benzyl
thioesters, and mercaptopropionic acid leucine thioesters (See, e.g., Dawson
et
al., Science (1994) 266:776-779; Canne et al. Tetrahedron Lett. (1995) 36:1217-
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1220; Kent, et al., WO 96/34878; Kent, et al., WO 98/28434; Ingenito et al.,
JACS
(1999) 121 (49):11369-11374; and Hackeng et al., Proc. Natl. Acad. Sci. U.S.A.
(1999) 96:10068-10073). Other examples include dithiothreitol, or alkyl or
aryl
thioesters, which can be produced by intein-mediated biological techniques,
which .
also are well known (See, e.g., Chong et al., Gene (1997) 192:277-281; Chong
et
al., Nucl. Acids Res. (1998) 26:5109-5115; Evans et al., Protein Science
(1998)
7:2256-2264; and Cotton et al., Chemistry & Biology (1999) 6(9):247-256) .
The a-carboxythioesters can be generated by chemical or biological
methods following standard techniques known in the art, such as those
described
herein, including the Examples. For chemical synthesis, a-carboxythioester
peptides can be synthesized in solution or from thioester-generating resins,
which
techniques are well known (See, e.g., Dawson et al., supra; Canne et al.,
supra;
Hackeng et al., supra, Hojo H, Aimoto, S. (1991 ) Bull Chem Soc Jpn 64:111-
117).
For instance, chemically synthesized thioester peptides can be made from the
corresponding peptide a-thioacids, which in turn, can be synthesized on a
thioester-resin or in solution, although the resin approach is preferred. The
peptide-a-thioacids can be converted to the corresponding 3-carboxy-4-
nitrophenyl thioesters, to the corresponding benzyl ester, or to any of a
variety of
alkyl thioesters. All of these thioesters provide satisfactory leaving groups
for the
ligation reactions, with the 3-carboxy-4-nitrophenyl thioesters demonstrating
a
somewhat faster reaction rate than the corresponding benzyl thioesters, which
in
turn may be more reactive than the alkyl thioesters. As another example, a
trityl-
associated mercaptoproprionic acid leucine thioester-generating resin can be
utilized for constructing C-terminal thioesters (Hackeng et al., supra). C-
terminal
thioester synthesis also can be accomplished using a 3-
carboxypropanesulfonamide safety-catch linker by activation with diazomethane
or iodoacetonitrile followed by displacement with a suitable thiol (Ingenito
et al.,
supra; Shin et al., (1999) J. Am. Chem. Soc., 121, 11684-11689).
Peptide or polypeptide C-terminal a-carboxythioesters also can be made
using biological processes. For instance, intein expression systems, with or
without labels such as affinity tags can be utilized to exploit the inducible
self
cleavage activity of an "intein" protein-splicing element in the presence of a
suitable thiol to generate a C-terminal thioester peptide or polypeptide
segment.
In particular, the intein undergoes specific self cleavage in the presence of
thiols
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such as DTT, a-mercaptoethanol, [i-mercaptoethanesulfonic acid, or cysteine,
which generates a peptide segment bearing a C-terminal thioester. See, e.g.,
Chong et al., (1997) supra; Chong et al., (1998) supra; Evans et al., supra;
and
Cotton et al., supra.
Ligation of the N-substituted 2 or 3 carbon chain alkyl or aryl thiol
components of the invention with the first carboxythioester component
generates
a ligation product having an N-substituted amide bond at the ligation site, as
depicted in Figures 1, 2 and 3. The ligation conditions of the reaction are
chosen
to maintain the selective reactivity of the thioester with the N-substituted 2
or 3
carbon chain alkyl or aryl thiol moiety. In a preferred embodiment, the
ligation
reaction is carried out in a buffer solution having pH 6-8, with the preferred
pH
range being 6.5-7.5. The buffer solution may be aqueous, organic or a mixture
thereof. The ligation reaction also may include one or more catalysts and/or
one
or more reducing agents, lipids, detergents, other denaturants or solubilizing
reagents and the like. Examples of preferred catalysts are thiol and phosphine
containing moieties, such as thiophenol, benzylmercaptan, TCEP and alkyl
phosphines. Examples of denaturing and/or solubilizing agents include
guanidinium, urea in water or organic solvents such as TFE, HFIP, DMF, NMP,
acetonitrile admixed with water, or with guanidinium and urea in water. The
temperature also may be utilized to regulate the rate of the ligation
reaction, which
is usually between 5°C and 55°C, with the preferred temperature
being between
15°C and 40°C. As an example, the ligation reactions proceed
well in a reaction
system having 2% thiophenol in 6M guanidinium at a pH between 6.8 and 7.8.
For the N-substituted 2 carbon chain alkyl or aryl thiols, the ligation event
results from a thiol exchange that occurs between the COSR thioester component
and the amino alkyl thiol component. The exchange generates a thioester-linked
intermediate ligation product that after spontaneous rearrangement through a 5
membered ring intermediate generates a first ligation product of the formula
J1
HN-CH(Z1)-C(O)-Na(C1(R1)-C2-SH)-CH(Z2)-J2 having a removable N
substituted 2 carbon chain alkyl or aryl thiol [HS-C2-C1(R1)-] at the ligation
site,
where the substituents are as defined above. The N-substituted 2 carbon chain
alkyl or aryl thiol [HS-C2-C1(R1)-] at the ligation site is amenable to being
removed, under peptide-compatible conditions, to generate a final ligation
product
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of the formula J1-HN-CH(Z1)-CO-NH-CH(Z2)-CO-J2 having a native amide bond
at the ligation site.
For the N-substituted 3 carbon chain aryl or alkyl thiols, the thiol exchange
between the COSR thioester component and the amino alkyl thiol component
generates a thioester-linked intermediate ligation product that after
spontaneous
rearrangement through a 6-membered ring intermediate generates a first
ligation
product of the formula J1-HN-CH(Z1)-C(O)-Na(C1-C2(R2)-C3(R3)-SH)-CH(Z2)-J2
having a removable N-substituted 3 carbon chain alkyl or aryl thiol [HS-C3(R3)-
C2(R2)-C1(R1)-] at the ligation site. The N-substituted 3 carbon chain aryl
thiol
[HS-C3(R3)-C2(R2)-C1(R1)-] at the ligation site is amenable to being removed,
under peptide-compatible conditions, to generate a final ligation product of
the
formula J1-HN-CH(Z1)-CO-NH-CH(Z2)-CO-J2 having a native amide bond at the
ligation site.
Removal of the N-substituted alkyl or aryl thiol group is preferably
performed in acidic conditions to facilitate cleavage of the N-C1 bond,
yielding a
stabilized, unsubstituted amide bond at the ligation site. By "peptide-
compatible
cleavage conditions" is intended physical-chemical conditions compatible with
peptides and suitable for cleavage of the N-linked alkyl or aryl thiol moiety
from
the ligation product. Peptide-compatible cleavage conditions in general are
selected depending on the Na-alkyl or aryl thiol moiety employed, which can be
readily deduced through routine and well known approaches (See, e.g.,
"Protecting Groups in Organic Synthesis", 3rd Edition, T.W. Greene and P.G.M.
Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem Catalog 2000;
"Synthetic Peptides, A User's Guide," G.A. Grant, Ed., W.H. Freeman & Company,
New York, NY,1992; "Advanced Chemtech Handbook of Combinatorial & Solid
Phase Organic Chemistry," W.D.. Bennet, J.W. Christensen, L.K. Hamaker, M.L.
Peterson, M.R.Rhodes, and H.H. Saneii, Eds., Advanced Chemtech, 1998;
"Principles of Peptide Synthesis, 2nd ed.," M. Bodanszky, Ed., Springer-
Verlag,
1993; "The Practice of Peptide Synthesis, 2nd ed.," M. Bodanszky and A.
Bodanszky, Eds., Springer-Verlag, 1994; and "Protecting Groups," P.J.
Kocienski,
Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994).
For example, where the R1', R2' or R3' substituents comprises a methoxy,
hydroxyl, thiol or thiomethyl, methyl and the like, the more universal method
for
removal involves acidic cleavage conditions typical for peptide synthesis
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chemistries. This includes cleavage of the N-C1 bond under strong acidic
conditions or water-acidic conditions, with or without reducing reagents
and/or
scavenger systems (e.g., acid such as anhydrous hydrogen fluoride (NF),
triflouroacetic acid (TFA), or trifluoromethanesulfonic acid (TFMSA) and the
like).
More specific acidic cleavage systems can be chosen to optimize cleavage of
the
Na-C1 bond to remove the aryl or alkyl thiol moiety for a given construct.
Such
conditions are well known and compatible with maintaining the integrity of
peptides. Another method for cleavage involves the inclusion of a thiol
scavenger
where tryptophans are present in a peptide or polypeptide sequence to avoid
reaction of the tryptophan side chain with the liberated aryl or alkyl thiol
moiety.
Examples of thiol scavengers include ethanediol, cysteine, beta-
mercaptoethanol
and thiocresol. Accordingly, another embodiment of the invention is the
addition
of a thiol scavenger when cleaving the N-C1 bond to remove the aryl or alkyl
thiol
moiety.
Other specialized cleavage conditions include light or reductive-cleavage
conditions when the picolyl group is the substituent. As an example, when the
R1,
or R2 and R3 substituents comprise a picolyl moiety, photolysis (e.g.,
ultraviolet
light), zinc/acetic acid or electrolytic reduction may be used for cleavage
following
standard protocols. Where R1 of the N-substituted 2 carbon chain thiol
comprises
a thiomethane at R1, then mercury(II) or HF cleavages can be used. The
cleavage system also can be used for simultaneous cleavage from a solid
support
and/or as a deprotection reagent when the first or second ligation components
comprise other protecting groups. For instance, N-picolyl groups can be
removed
by dissolving the polypeptide in a 10% acetic acid/water solution, with
activated
zinc (~0.5g/ml). Thiomethane groups, such as 2-mercapto, 1-
methylsulfinylethane
groups (HS-C2-C1 (S(O)-CH3)-Na), can be removed after ligation by reduction
and mercuric, mercaptan-mediated cleavage. As an example, the
methylsulfinylethane group can be removed by dissolving the polypeptide in an
aqueous 3% acetic acid solution containing N-methylmercaptoacetamide (MMA)
(e.g., 1 mg polypeptide in 0.5m1 of acetic acid/water and 0.05 ml of MMA), for
reduction to the thiomethane form, followed by freezing and lyophilization of
the
mixture after overnight reaction. The reduced auxiliary can then be removed in
an
aqueous solution of 3% acetic acid containing mercury acetate (Hg(OAC)2)
(e.g.,
0.5m1 of acetic acid in water and 10 mg of Hg(OAC)z for about 1 hour),
followed by
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addition of beta-mercaptoethanol (e.g., 0.2m1 beta-mercaptoethanol). Products
can then be purified by standard methods, such as reverse phase HPLC
(RPHPLC).
As can be appreciated, one or more catalysts and/or excipients may also
be utilized in the cleavage system, such as one or more scavengers,
detergents,
solvents, metals and the like. In general, selection of specific scavengers
depends upon the amino acids present. For instance, the presence of scavengers
can be used to suppress the damaging effect that the carbonium ions, produced
during cleavage, can have on certain amino acids (e.g., Met, Cys, Trp, and
Tyr).
Other additives like detergents, polymers, salts, organic solvents and the
like also
may be employed to improve cleavage by modulating solubility. Catalysts or
other
chemicals that modulate the redox system also can be advantageous. It also
will
be readily apparent that a variety of other physical-chemical conditions such
as
buffer systems, pH and temperature can be routinely adjusted to optimize a
given
cleavage system.
The present invention also provides protected forms of the Na-substituted
2 or 3 carbon chain alkyl or aryl thiols of the invention. These compounds are
especially useful for automated peptide synthesis and orthogonal and
convergent
ligation strategies. These compositions comprise a fully protected, partially
protected or fully unprotected acid stable Na-substituted 2 or 3 carbon chain
amino alkyl or aryl thiol of the formula (PG2)S-C2-C1(R1)-Na(PG1)-CH(Z2)-C(O)-
J2 or (PG2)S-C3(R3)-C2(R2)-C1(R1)-Na(PG1)-CH(Z2)-C(O)-J2, which are
depicted below in Table III and Table IV. In particular, one or more of R1, R2
and
R3 comprises an electron donating group conjugated to C1 that, following
conversion of the Na-substituted amino alkyl or aryl thiol to an Na-
substituted
amide alkyl or aryl thiol, is capable of forming a resonance stabilized cation
at C1
that facilitates cleavage of the Na-C1 bond under peptide compatible cleavage
conditions. PG1 and PG2 are protecting groups that are present individually or
in
combination or are absent and can be the same or different, where Z2 is any
chemical moiety compatible with chemical peptide synthesis or extended native
chemical ligation, and where J2 is any chemical moiety compatible with
chemical
peptide synthesis or extended native chemical ligation.
PG1 (or X1 ) is a group for protecting the amine. PG2 (or X2) is a group for
protecting the thiol. Many such protecting groups are known and suitable for
this
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purpose (See, e.g., "Protecting Groups in Organic Synthesis", 3rd Edition,
T.W.
Greene and P.G.M. Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem
Catalog 2000; "Synthetic Peptides, A User's Guide," G.A. Grant, Ed., W.H.
Freeman & Company, New York, NY,1992; "Advanced Chemtech Handbook of
Combinatorial & Solid Phase Organic Chemistry," W.D.. Bennet, J.W.
Christensen, L.K. Hamaker, M.L. Peterson, M.R.Rhodes, and H.H. Saneii, Eds.,
Advanced Chemtech, 1998; "Principles of Peptide Synthesis, 2nd ed.," M.
Bodanszky, Ed., Springer-Verlag, 1993; °The Practice of Peptide
Synthesis, 2nd
ed.," M. Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; and
"Protecting Groups," P.J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart,
Germany, 1994).
Table III
HNa CH-C J2 PG1-Na CH-C J~
C1 O C1 O
PGZ S C2 ~R1 HS-C~ ~R1
Z2
PG1-Na CH-C Jz
C1 O
PG2-S-C~ ~ R1
Examples of preferred protecting groups for PG1 and X1 include, but are
not limited to [Boc(t-Butylcarbamate), Troc(2,2,2,-Trichloroethylcarbamate),
Fmoc(9-Fluorenylmethylcarbamate), Br-Z or CI-Z(Br- or CI-Benzylcarbamate),
Dde(4,4,-dimethyl-2,6-dioxocycloex1-ylidene), MsZ(4-
Methylsulfinylbenzylcarbamate), Msc(2-Methylsulfoethylcarbamate) Nsc(4-
nitrophenylethylsulfonyl-ethyloxycarbonyl]. Preferred PG1 and X1 protecting
groups are selected from "Protective Groups in Organic Synthesis," Green and
Wuts, Third Edition,Wiley-Interscience, (1999) with the most preferred being
Fmoc
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and Nsc. Examples of preferred protecting groups for PG2 include, but are not
limited to [Acm(acetamidomethyl), MeOBzI or Mob(p-Methoxybenzyl), MeBzl(p-
Methylbenzyl), Trt(Trityl),Xan(Xanthenyl),tButhio(s t-butyl),Mmt(p-
Methoxytrityl),2
or 4 Picolyl(2 or 4 pyridyl)),Fm(9-Fluorenylmethyl), tBu(t-
Butyl),Tacam(Trimethylacetamidomethyl)] Preferred protecting groups PG2 and
X2 are selected from "Protective Groups in Organic Synthesis," Green and Wuts,
Third Edition,Wiley-Interscience, (1999), with the most preferred being Acm,
Mob,
MeBzl, Picolyl .
Orthogonal protection schemes involves two or more classes or groups
that are removed by differing chemical mechanisms, and therefore can be
removed in any order and in the presence of the other classes. Orthogonal
schemes offer the possibility of substantially milder overall conditions,
because
selectivity can be attained on the basis of differences in chemistry rather
than
reaction rates.
Table IV
z
PG~ ~ a CH ~ ~ J2 ~ GZ HN ~ CH-~ ~ J2
HS\ ~C~ \ O S\ C~ \ 0
Cs-C2 R1 Cs-C ~ R1
R3 R~ R3 \R2
Z2
PG~ ~ a CH ~ ~ J2
PG2 S\ C~ \ O
C3-C ~ R~
R3 R~
The protected forms of the Na-substituted 2 or 3 chain alkyl or aryl thiols of
the invention can be prepared as in Schemes I and II above.
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The compounds of the present invention may be produced by any of a
variety of means, including halogen-mediated amino alkvlation, reductive
amination, and by the preparation of Na-protected, N-alkylated, S-protected,
amino alkyl- or aryl- thiol amino acid precursors compatible with solid phase
or
solution amino acid or peptide synthesis methods. When desirable, resolution
of
the racemates or diastereisomers produced to give compounds of acceptable
chiral purity can be carried out by standard methods.
As noted above, Na-substituted 2 or 3 carbon chain alkyl or aryl thiols of
acceptable chiral purity are preferred in some instances. As shown in Example
21, and in Figure 5B, use of the Na-1-(4-methoxyphenyl)-2-mercaptoethyl
auxiliary in the preparation of cytochrome b562 yielded two ligation products
(diastereoisomers) with overlapping purification profiles. Although removal of
the
Na-auxiliary yields a single major product, a small percentage of deletion and
side-reactant products will be present in the final product, which may be
undesirable. For instance, the reductive amination synthetic route as
described in
Examples 4 through 6 employed for synthesis the Na-1-(4-methoxyphenyl)-2-
mercaptoethyl auxiliary employed in the cyt b562 synthesis inherently results
in
the production of both epimers at the chiral center C1. As noted above, when
desirable, resolution of the racemates or diastereisomers produced to give
compounds of acceptable chiral purity can be carried out by standard methods.
Standard approaches for obtaining Na-auxiliaries of the invention of
acceptable chiral purity are: (1) chiral chromatography; (2) chiral synthesis;
(3)
use of a covalent diasteriomeric conjugate; and (4) crystallization ' or other
traditional separation methods to give enantiomerically pure chiral auxiliary.
(See,
e.g., Ahuja, Satinder. 'Chiral separations. An overview.' ACS Symp. Ser. (1991
),
471 (Chiral Sep. Liq. Chromatogr.), 1-26; Collet, Andre. "Separation and
Purification of Enantiomers by Crystallization Methods", In: Enantiomer (1999)
4:157-172; Lopata et al., J. Chem. Res. Minipprint (1984) 10:2930-2962; Lopata
et
al., J. Chem. Res. (1984) 10:2953-2973; Ahuja, Satinder. 'Chiral separations
and
technology: an overview.' Chiral Sep. (1997), 1-7; Chiral Separations:
Applications and Technology. Ahuja, Satinder; Editor. USA. (1997), 349 pp.
Publisher: (ACS, Washington, D. C.) ). All of these standard methods
approaches can be used for resolution of racemates or diastereisomers to give
compounds of acceptable chiral purity. For instance, crystallization can be
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employed for optical resolution of enantiomers. For chiral chromatography, it
is
well known that racemic mixtures can be separated into chirally pure
enantiomers
by means of preparative chromatography using chiral media. Thus, a racemic
mixture produced by the reductive amination route for the total synthesis of
chiral
Na-auxiliaries can be used to prepare each enantiomer in chirally pure form,
for
example, as illustrated below for an amino acid auxiliary (e.g., where R is
amino
acid side chain):
N~CH(R)COOH NHoCH(R)COOH NHoCH(R)COOH
~H]
\ ~ \ + ~~''~rr \
PG2 S Rs v Ra' pG2 S Rs'~Rs' PGZ IS R5
~1:1 molar ratio
Either enantiomer may be obtained in chirally pure form, or both may be
obtained in chirally pure form. Either enantiomer may be used to form chirally-
pure
auxiliary modified components, such as peptide segments (i.e. two chirally
pure
epimers), which can be rigorously purified without interference from the
presence
of the other epimer and its impurities. Note that unless some provision is
made
for using both enantiomers, 50% of the total mass of the auxiliary will be
wasted.
For example, the two chirally pure auxiliary-modified peptide segments can
then
used in separate ENCL reactions, to give chirally pure auxiliary-modified
ligation
product mixtures. After separate purifications, the auxiliary group is removed
from
the epimer ligation products (either separately or after being combined) to
give the
same native structure, ligated product, which is then subjected to
purification.
For chiral synthesis, a preferred method employs an enantiomerically pure,
chiral starting material, as illustrated below for a para-methoxyphenyl
substituted
Na-2 carbon auxiliary:
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OCH3 OCH3 OCH
3
/ ~ ~ \
/
PG~NH COOH PG~NH CH20H pG~NH ~CHZSPGZ
OCH3
--a
NHa ~CHZSPGz
[PG~ = Boc or Fmoc; PG2 = (4Me)Benzyl or (4Me0)Benzyl]
The resulting chirally pure precursor compound can then be used to make either
a
protected (N-substituted) amino acid, viz.: ,
OCH3 OCH3 OCH3
\ ~ \
BrCH~COOH Boc~O
/ ' / '~ /
NHZ ~CH~SPG~ H i CH~SPGz Boc_ i ~CH~SPGZ
HZC HZC~
~COOH COON
or used directly in the 'sub-monomer' peptoid route, viz:
BrCH2COOH
H2N-(PEPTIDE2)-RESIN +activatingagent grCH2C0-NH-(PEP/TIDE2)-RESIN
'(PG)n
~(PG)n
--~ HN-CH2C0-NH-(PEP/TIDE2)-RESIN
-(PG)n
PG2SCH2
~CHg
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to form the auxiliary-modified peptide on a polymer support. Subsequent
deprotection/cleavage gives the auxiliary-modified peptide segment in chirally-
pure form, viz.:
Deprotection
HN-CH~CO-NH-(PEPTIDE)-RESIN ~ HN-CHzCO-NH-PEPTIDE2
Cleavage
PG2SCHz ~ - . HSCHZ I / ocH
oCfi3 3
Thus chirally pure Na-auxiliaries of the invention can be made from the
readily
available para-substituted phenylgylcine(s) of known chirality, thus
predetermining
the chirality and chiral purity of the resulting auxiliary.
Alternatively, another preferred embodiment employs enantioselective
synthesis employing asymmetric reduction to yield the auxiliary, for example
as
illustrated below:
O NOR
Rt' I R~~ NH2 R~
\ \
> -~ \
PGz S Rs' ~ Ra' PG2 S Rs' s R3' PG ~S Rs' I Rs
R = -H, or -CH3, or -CHZCOOH
(or, opposite enantiomer)
Asymmetric reduction can also be used for enantioselective synthesis to yield
an
Na-auxiliary-modified amino acid, such as for glycine illustrated below, viz.:
O N.CH2COOH sCH~COOH
Rt' I R~' NH Rt
----~ \
PGZ S Rs' ~ R3 PG2 S Rs' ~ R3 PGZ S R5'\%'R3
(or, other enantiomer)
While a suitably executed asymmetric synthesis will give a considerable excess
of
one enantiomer over the other, nonetheless it is expected that there will be
present amounts of the other enantiomer. This can be addressed using a chiral
purification step in order to obtain the majority enantiomer in pure form. The
main
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benefits of an enantioselective synthetic route are that the chiral
chromatographic
separation is easier, and that large amounts of material are not discarded
(wasted).
Another preferred standard technique is resolution by use of a covalent
diasteriomeric conjugate. In general, this approach employs a chiral amino
acid
(e.g. Ala) to modify a racemic auxiliary mixture, and separation of the
resulting
diastereomers by standard (non-chiral) chromatography methods, such as
illustrated below. For instance, the racemic auxiliary 1 can be converted to a
mixture of diastereomers by covalent incorporation of a second chiral center:
COOH
NH2 _1 HN/
°CH3
PGaSCH2 ~~~ COOH PG2SCH~ ~~
(S) ~ OCH3 Br~ (S'S) ~ OCH3
~CH3
--~ COOH
1
H2 (R) 2-Br-propionic acid HN
~~CH3
PGzSCH~ "'~n~~ \ PGZSCH ~~.~~~°'~ \
R OCH3
()
(R S) OCH3
ENANTIOMERS DIASTEREOMERS
(racemic mixture)
In this case, the racemic mixture is reacted, by means of an SN2 nucleophilic
reaction (with inversion), with (R)2-Br-propionic acid, to yield the pair of
diastereomers shown. In effect, we have generated L-Ala with an N-linked
chiral
thiol-containing auxiliary.
As diastereomers, these two compounds will typically exhibit different
chromatographic behavior under achiral chromatography conditions, and thus be
separable under practical preparative conditions to give the pure, distinct
epimers.
After suitable protection of the Na moiety, each compound can be used to
generate an unprotected or partially protected chirally pure auxiliary-
modified
peptide segment with an N-terminal Ala residue.
The protected Na-substituted components of the invention are particularly
useful for rapid automated synthesis using conventional peptide synthesis and
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other organic synthesis strategies. They also expand the utility of chemical
ligation to multi-component ligation schemes, such as when producing a
polypeptide involving orthogonal ligation strategies, such as a three or more
segment ligation scheme or convergent ligation synthesis schemes.
For instance, the extended native chemical ligation method and
compositions of the invention can be employed in conjunction with nucleophile
stable thioester generating methods and thioester safety-catch approaches,
such
as the orthothioloester and carboxyester thiols described in co-pending
application
PCT application Serial No. [Not yet assigned] filed August 31, 2001, and U.S.
provisional patent application Serial No. 60/229,295 filed September 1, 2000,
which are incorporated herein by reference. Briefly, the nucleophile-stable
thioester generating compounds comprise an orthothioloester or a carboxyester
thiol; these compounds have wide applicability in organic synthesis, including
the
generation of peptide-, polypeptide- and other polymer-thioesters. The
nucleophile-stable thioester generating compounds are particularly useful for
generating activated thioesters from precursors that are made under conditions
in
which strong nucleophiles are employed, such as peptides or polypeptides made
using Fmoc SPPS, as well as multi-step ligation or conjugation schemes that
require (or benefit from the use of) compatible selective-protection
approaches for
directing a specific ligation or conjugation reaction of interest. The
nucleophile-
stable orthothioloesters have the formula X-C(OR')2-S-R, where X is a target
molecule of interest optionally comprising one or more nucleophile cleavable
protecting groups, R' is a nucleophile-stable protecting group that is
removable
under non-nucleophilic cleavage conditions, and R is any group compatible with
the orthothioloester -C(OR')-S-. Nucleophile-stable orthothioloester thioester-
generating resins also are provided, and have the formula X-C(OR')~-S-R-linker-
resin or X-C((OR~'-linker-resin )(OR2'))-SR, where X, R' and R are as above,
and
where the linker and resin are any nucleophile-stable linker and resin
suitable for
use in solid phase organic synthesis, including safety-catch linkers that can
be
subsequently converted to nucleophile-labile linkers for cleavage. The
nucleophile-stable orthothioloesters can be converted to the active thioester
by a
variety of non-nucleophilic. conditions, such as acid hydrolysis conditions.
The
nucleophile-stable carboxyester thiols have the formula X-C(O)-O-CH(R")-(CHZ)~
S-R"', where X is a target molecule of interest comprising one or more
nucleophile-labile protecting groups, R" is a non-nucleophile stable group, n
is 1
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or 2, with n=1 preferred, and R"' is hydrogen, a protecting group or an acid-
or
reduction-labile or safety catch linker attached to a resin or protecting
group that is
removable under non-nucleophilic conditions. Nucleophile-stable carboxyester
thiol-based thioester-generating resins also are provided, and have the
formula X-
C(O)-O-CH(R")-CH2n-S-linker-resin or X-C(O)-O-CH(R"-linker-resin)-CH2n-S-R"',
where X, R", n and R"' are as above, and where the linker and resin are any
nucleophile-stable linker and resin suitable for use in solid phase organic
synthesis. The nucleophile-stable carboxyester thiols can be converted to the
active thioester by addition of a thiol catalyst, such as thiophenol. Thus the
extended native chemical ligation methods and compositions of the present
invention can be employed in multi-segment convergent ligation techniques,
where a one end of a target compound can bear a protected or unprotected Na,-2
or 3 carbon chain alkyl or aryl thiol of the present invention, and the other
end a
orthothioloester or carboxyester thiol moiety for subsequent conversion to the
active thioester and ligation.
It will also be appreciated that the Na-2 or 3 carbon chain alkyl or aryl
thiol
of the present invention can be employed in combination with other ligation
methods, for example, such as native chemical ligation (Dawson, et al.,
Science
(1994) 266:776-779; Kent, et al., WO 96/34878), extended general chemical
ligation (Kent, et al., WO 98/28434), oxime-forming chemical ligation (Rose,
et al.,
J. Amer. Chem. Soc. (1994) 116:30-33), thioester forming ligation (Schnolzer,
et
al., Science (1992) 256:221-225), thioether forming ligation (Englebretsen, ef
al.,
Tet. Letts. (1995) 36(48):8871-8874), hydrazone forming ligation (Gaertner, et
al.,
Bioconj. Chem. (1994) 5(4):333-338), and thiazolidine forming ligation and
oxazolidine forming ligation (Zhang, et al., Proc. Nat!. Acad. Sci. (1998)
95(16):9184-9189; Tam, et al., W0.95/00846) or by other methods (Yan, L.Z. and
Dawson, P.E., "Synthesis of Peptides and Proteins without Cysteine Residues by
Native Chemical Ligation Combined with Desulfurization," J. Am. Chem. Soc.
2001, 123, 526-533, herein incorporated by reference; Gieselnan et al., Org.
Lett.
2001 3(9):1331-1334; Saxon, E. et al., "'Traceless" Staudinger Ligation for
the
Chemoselective Synthesis of Amide Bonds. Org. Lett. 2000, 2, 2141-2143). Also
contemplated by the present invention is the substitution of selenium in place
of
the thiol sulfur in the Na-2 or 3 carbon chain alkyl or aryl thiol of the
invention.
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The methods and compositions of the invention have many uses. The
methods and compositions of the invention are particularly useful for ligating
peptides, polypeptides and other polymers. The ability to carry out native
chemical ligation at practically any amino acid, including the naturally
occurring as
well as unnatural amino acids and derivatives expands the scope of native
chemical ligation to targets that are missing suitable cysteine ligation
sites. The
invention also can be used to ligate polymers in addition to peptide or
polypeptide
segments when it is desirable to join such moieties through a linker having an
Noc-
substituted or totally native amide bond at the ligation site. The invention
also
finds use in the production of a wide range of peptide labels for expressed-
protein
ligation (EPL) applications. For instance, EPL-generated thioester
polypeptides
can be ligated to a wide range of peptides via an Na-substituted alkyl or aryl
thiol
amide bond or a completely native amide bond, depending on the intended end
use. The invention also can be exploited to produce a variety of cyclic
peptides
and polypeptides, having a native amide bond at the point of cyclization even
for
peptides and polypeptides that do not contain cysteine. For instance, this is
significant as most cyclic peptides, such as antibiotics and other drugs
generated
by industry standards do not contain a cysteine residue that can be used to
form a
native amide bond at the cyclizing (i.e., head-to-tail) ligation site.
All publications and patent applications mentioned in this specification are
herein incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually indicated
to be
incorporated by reference.
EXAMPLES
The following preparations and examples are given to enable those skilled
in the art to more clearly understand and to practice the present invention.
They
should not be considered as limiting the scope of the invention, but merely as
being illustrative and representative thereof.
Abbreviations
Acm acetamidomethyl
Aloc allyoxycarbonyl
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BOP benzotriazol-1-yloxytris (dimethylamino)
phosphonium
hexafluorophosphate
Br,CI Z Br,CI Benzylcarbamate
DCM dichloromethane
DDE 4,4-dimethyl-2,6-dioxocycloex 1-ylidene
DIPCDI N, N-diisopropylcarbodiimide
DIPEA N, N-diisopropylethylamine
DMAP 4-dimethylaminopyridine
DMF N,N-dimethylformamide
10DMSO dimethylsulfoxide
EtOH ethanol
Fmoc 9-fluorenylmethoxycarbonyl
FM 9-Fluorenylmethyl
HATU (N-[(dimethylamino)-1 H-1, 2, 3-triazol [4,
5-b] pyridiylmethylene]-N-
methylmethanaminium hexafluorophosphate N-oxide).
HBTU N-[(1-H-benzotriazol-1-yl)(dimethylamine)
methylene]-N-
methylmethanaminium hexafluorophosphate N-oxide
previously
named 0-(benzotriazol-1-yl)-1, 1, 3, 3-tetramethyluronium
hexafluorophosphate
20HF hydrofluoric acid
HMP resin 4-hydroxymethylphenoxy resin; palleoxybenzyl
alcohol resin; or
Wang resin
HOAt 1-hydroxy-7-azabenzotriazole
HOBt 1-hydroxybenzotriazole
25Mbh dimethoxybenzhydryl
MBHA resin4-methylbenzhydrylamine resin
Meb p-MethyIBenzyl
MMA N-methylmercaptoacetamide
Mmt p-Methoxytriityl
30Mob p-MethoxyBenzyl
Msc 2-Methylsulfoethylcarbamate
Msz 4-Methylsulfinylbenzylcarbamate
Mtr 4-methoxy-2, 3, 6-trimethylbenzene sulfonyl
NMM Nmethylmorpholine
35NMP N-methylprrolidone, N methyl-2-pyrrolidone
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Nsc 4-nitrophenylethylsulfonyl-ethyloxycarbonyl
OPfp pentafluorophenyl ester
OtBu tart-butyl ester
PAC peptide acid linker
PAL peptide amide linker
Pbf 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl
PEG-PS polyethylene glycol-polystyrene
Picolyl methyl-pyridyl
Pmc 2, 2, 4, 6, 8-pentamethylchroman-6-sulfonyl
PyAOP 7-azabenzotroazol-1-1yloxtris (pyrrolidino)
phosphonium
hexafluorophosphate
S-tBu tart butyl-thio
Tacam Trimethylacetamidomethyl
tBoc tent butyloxycarbonyl
TBTU 0-(benzotriazol-1-yl)-1, 1, 3, 3-tetramethyl
uronium tetrafluoroborate
tBu tart butyl
TFA trifluoroacetic acid
Tis Trisisopropylsilane
Tmob 2, 4, 6-trimehoxybenzyl
TMOF trimethylorthoformate
Troc 2,2,2Trichloroethylcarbamate
Trt triphenylmethyl
Example 1: General Materials and Methods
Peptides were synthesized in stepwise fashion on a modified ABI 430A
peptide synthesizer by SPPS using in situ neutralization/HBTU activation
protocols for Boc-chemistry on PAM resin or thioester-generating resin
following
standard protocols (Hackeng et al., supra; Schnolzer et al., (1992)
Int.J.Pept.Prot.Res., 40:180-193; and Kent, S.B.H. (1988) Ann. Rev. Biochem.
57, 957-984). After chain assembly the peptides were deprotected and
simultaneously cleaved by treatment with anhydrous hydrogen fluoride (NF) with
5% p-cresol and lyophilized and purified by preparative HPLC. Boc protected
amino acids were obtained from Peptides International and Midwest Biotech.
Trifluoroacetic acid (TFA) was obtained by Halocarbon. Other chemicals were
from Fluka or Aldrich. Analytical and preparative HPLC were performed on a
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Rainin HPLC system with 214 nm UV detection using Vydac C4 analytical or
preparative. Peptide and protein mass spectrometry was performed on a Sciex
API-I electrospray mass spectrometer.
Example 2: Preparation of 2(4'methoxybenzylthio)benzylbromide
2-Hydroxymethylthiopheno1,10 mmol 1.4g, was reacted in 10m1 of DMF
with 10 mmol of 4-methoxybenzylchforide and 1.75 ml of DIEA at room
temperature. The reaction is completed in 10 minutes.50 ml of water at pH 3
were
added. The product was extracted with ethyl acetate and dried over sodium
sulfate. The obtained crude oil was then reacted with 11 mmol of carbon
tetrabromide(3.64g) and 11mmol of triphenylphosphine(2.88g) in 20 ml of THF.
After overnight reaction the THF was evaporated. The product was purified with
silica gel chromatography using hexanes/ethyl acetate 6/ 1 as mobile phase.
Recovered 1.8g.
Example 3: Preparation of Na (2-mercaptobenzyl) glycine-peptide
To a resin bound peptide with N-terminal Boc-protected Ala(78mg), neat
TFA was added to remove the Boc group. Using standard chemistry protocols
BocGIycineOSuccinimide was coupled to the resin. After the coupling was
completed Boc group was removed and the resin was neutalized with 2 washes
with 10% Diisopropylethylamine in DMF.The resin was then washed with DMF
and DMSO. Then 9mg of 2(4'methoxybenzylthio)benzylbromide in 0.2 ml of
DMSO and 0.01 ml of Diisopropylethylamine were added. The mixture reacted for
12 hrs at room temperature. The peptide was cleaved and deprotected in HF
conditions using standard protocols. The peak with correct mass of 2,079 Da
was
about 12% (measured by HPLC) of all peptidic material. The correct peptide was
purified using standard semi-preparative HPLC.
Example 4: Preparation of 4'-Methoxy 2(4'methylbenzylthio) acetophenone
4-methylbenzylmercaptan, 4 mmol, 0.542m1 and 4'methoxy
2bromoacetophenone 4 mmol, 916.3 mg were dissolved in 4 ml DMF. Then
diisopropylethylamine, 4mmol 0.7m1 was added. The mixture was stirred at room
temperature for one hour. The mixture was poured in diluted HCI and extracted
with ethylacetate and dried over sodium sulfate. The oil was dissolved in
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ethylacetate and precipitated by addition of petroleum ether, with recovery of
450mg of a white solid.
Example 5: Preparation of 1 amino,1(4-methoxyphenyl),2(4-
methylbenzylthio) ethane
4'-Methoxy 2(4'methylbenzylthio) acetophenone 1.44 mmol, 411 mg and
aminoxyacetic acid 4.3 mmol, 941 mg were dissolved in 20m1 of TMOF and
0.047m1 of methanesulfonic acid was added as catalyst at room temperature.
After 48 hours, the solvent was evaporated and the residue taken up in
ethylacetate, washed with 1 M monohydrogenpotassium sulfate and dried over
sodium sulfate. The crude product was purified with silica gel chromatography,
and 200mg of oxime complex obtained. T200mg of this oxime complex,
0.556mmol was dissolved in 2m1 of THF, followed by the addition of 1.67m1 of 1
M
BH3/THF complex. After 27 hours no starting material was left. 3m1 of water
were
added and 1.5 ml of 10N sodium hydroxide was added. The mixture was refluxed
for 1 hour. The mixture was then extracted with ethylacetate (4x) and dried
over
sodium sulfate. The final product (40 mg) was then purified using silica gel
chromatography.
Example 6: Preparation of Na 1-(4-Methoxyphenyl) 2-mercapto ethane
glycine-peptide
To a resin bound model peptide with an N-terminal Boc protected Ala
(78mg), neat TFA was added to remove Boc group. Using standard chemistry
protocols bromoacetic acid was coupled to the resin. Then 1 amino,1(4-
methoxyphenyl),2(4-methylbenzylthio) ethane 17mg in 0.3m1 of DMSO with
0.010m1 of diisopropylethylamine were added to the resin. After overnight
reaction the resin was washed and the peptide was cleaved and deprotected
using standard HF protocol. The desired product was then purified using semi-
preparative HPLC .
Preparation of another Na 1-(4-Methoxyphenyl) 2-mercapto ethane
glycine-peptide
To a model peptide resin of sequence S-Y-R-F-L-Polymer 0.1 mmol, bromo acetic
acid was coupled using standard coupling protocol. After coupling the resin
was
washed with DMSO. Then 1 amino,1(4-methoxyphenyl),2(4-methylbenzylthio)
ethane 32.5 mg, 0.12 mmol in 0.3 ml DMSO and 0.025m1 of diisopropylethylamine
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were added. The mixture was kept reacting overnight. The peptide was cleaved
and deprotected using standard HF procedure. The HPLC of the crude cleavage
showed the desired product (MW 2,122) in 60% of the total product. The n-
alkylethanethio group was found to be 97% stable in HF.
Example 7: Preparation of 2',4'-Dimethoxy 2(4'methylbenzylthio)
acetophenone
4-methylbenzylmercaptan, 3.94 mmol, 0.534 ml and 2',4' dimethoxy 2-
bromoacetophenone 3.86 mmol, 1g were dissolved in 4 ml DMF. Then
Diisopropylethylamine, 3.94mmol 0.688m1 was added. The mixture is stirred at
room temperature for 24 hrs. The mixture is poured in 1 M solution of
potassium
monohydrogensulfate and extracted with ethylacetate and dried over sodium
sulfate. After evaporation, the residual oil was dissolved in ethylacetate and
precipitated by addition of petroleum ether, which yielded 616mg of a white
solid.
Example 8: Preparation of 1 amino,1(2,4-dimethoxyphenyl),2(4-
methylbenzylthio) ethane
2',4'-Dimethoxy 2(4'methylbenzylthio) acetophenone 0.526mmol, 166mg
and aminoxyacetic acid 1.59 mmol, 345 mg were dissolved in 6 ml of TMOF and
0.034 ml of methanesulfonic acid was added as catalyst at room temperature.
After 31 hrs. the solvent was evaporated and taken up in ethylacetate, washed
with 1 M monohydrogenpotassium sulfate and dried over sodium sulfate. The
crude product was then purified with silica gel chromathography, yielding 126
mg
(61 % yield) of oxime complex. The 126 mg of oxime complex, 0.324 mmol was
dissolved in 1.5 ml of THF. Then 0.973 ml of 1 M BH3/THF complex was added.
After 54 hrs starting material was still found and 0.5 ml of 1M BH3/THF
complex was added. After 3 more days, total of 6days reaction 3m1 of water
were
added and 1 m1 of 10N sodium Hydroxide was added. The mixture was refluxed
for 1 h. The mixture was then extracted with ethylacetate (4x) and dried over
sodium sulfate. The final product ( 43 mg) was then purified using silica gel
chromatography.
Example 9: Preparation of Na 1-(2,4-Dimethoxyphenyl) 2-mercapto ethane
glycine-peptide
To a resin bound peptide of the sequence S-Y-R-F-L-Polymer, 0.1 mmol,
bromo acetic acid was coupled using a standard coupling protocol. After
coupling
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the resin was washed with DMSO. Then 1 amino,1 (2',4'-dimethoxyphenyl),2(4-
methylbenzylthio) ethane 36mg, 0.12 mmol in 0.3 ml DMSO and 0.025m1 of
diisopropylethylamine were added. The mixture was kept reacting overnight. The
peptide was cleaved and deprotected using standard HF procedure. The HPLC of
the crude cleavage showed the desired product (MW 938) in 42% of the total
product. The n-alkylethanethiol group was found to be 92% stable in HF.
Example 10: Ala-Gly chemical ligation of C-terminal SDF1-alanine-thioester
and N-terminal Na (2-mercaptobenzyl) glycine-peptide
For 6-member rearrangement ligation, 1 mg of C-terminal Ala thioester
fragment (MW 4429) of SDF1-a, and 0.6 mg of N-terminal Na (2-mercaptobenzyl)
glycine fragment (MW 2079) of SDF1-a, were dissolved in 100 p1 of 6 M
guanidinium buffer pH 7.0 and 1 p1 of thiophenol was added. After 2 days at
room
temperature (~25°C), formation of the desired ligation product (MW
5472) was
confirmed by ES-MS. The reaction mixture was then incubated at 40°C for
an
additional 24 hours, and the yield of desired ligation product determined
based on
the ratio between product and the non-reacted C-terminal fragment as measured
by HPLC integration. The observed yield was about 40%.
Example 11: Ala-Gly chemical ligation of C-terminal SDF1-alanine-thioester
and N-terminal Na 1-(4-methoxyphenyl) 2-mercaptoethane
glycine-peptide
For 5-member rearrangement ligation, 1 mg of C-terminal alanine-thioester
fragment (MW 4429) of SDF1, and 1 mg of an N-terminal peptide Model (MW
2122) having an N-terminal glycine comprising an Na 1-(4-methoxyphenyl) 2-
mercaptoethane group, were dissolved in 100 p,1 of 6 M guanidinium buffer pH
7.0
[and 1 p.1 of thiophenol]. The reaction mixture was incubated at room
temperature
(~25°C), and the ligation reaction monitored. After 8 hours, formation
of the
desired ligation product (MW 5515) was confirmed by ES-MS. After 3 days, yield
of the desired ligation product was about 45% based on the ratio between
product
and the non-reacted N-terminal fragment. After 3 days at room temperature,
followed by further incubation at 40°C for an additional 24 hours, the
yield was
65%. After 3 days at room temperature, followed by further incubation at
40°C for
an additional 48 hours, the yield increased to about 70%.
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For 5-member rearrangement ligation, 0.5 mg of C-terminal Ala thioester
fragment (MW 4429) of SDF1-a, and 0.5 mg of an N-terminal fragment (MW
2122) having an N-terminal glycine comprising an Na 1-(4-methoxyphenyl) 2-
mercaptoethane group, were dissolved in 100 p,1 of 6 M guanidinium buffer pH
8.2
and 1 p,1 of thiophenol. The reaction mixture was then incubated at room
temperature (rv25°C), followed by the addition of another 1 p,1 of
thiophenol after 6
hours. After 24 hours, the yield of desired product was about 60%.
Example 12: Gly-Gly chemical ligation of C-terminal glycine-thioester and
N-terminal Na-(2-mercaptobenzyl) glycine-peptide
For 6-member rearrangement ligation, 3.5 mg of C-terminal Gly thioester
fragment (MW 1357) of a decamermodel peptide, and 2 mg of N-terminal Na (2-
mercaptobenzyl) glycine fragment (MW 2079) of a model peptide with 3 HisDnp ,
were dissolved in 200 p1 of 6 M guanidinium buffer pH 7.9 and 2 p,1 of
thiophenol
was added. The mixture was incubated at 33°C for 60 hours. Formation of
the
desired ligation product (MW 2631 ) was confirmed by ES-MS, with an observed
yield of about 40% based on the ratio between product and the non-reacted N-
terminal fragment.
Chemical ligation of C-terminal glycine-thioester peptide and Na 1-(4-
methoxyphenyl) 2-mercaptoethane glycine-peptide
For 5-member rearrangement ligation, 2 mg of C-terminal Gly thioester
fragment (MW 1357) and 2.5 mg of an N-terminal fragment (MW 2122) of a model
peptide with 3 HisDnp comprising an Na 1-(4-methoxyphenyl) 2-mercaptoethane
group, were dissolved in 100 w1 of 6 M guanidinium buffer pH 7.0 with 1 p.1 of
thiophenol. The reaction mixture was incubated at room temperature
(~25°C),
and the ligation reaction monitored. Formation of the desired ligation product
(MW 2675.9) was confirmed by ES-MS after 3 and 8 hours of incubation. After 24
hours, yield of the desired ligation product was about 40% based on the ratio
between product and the non-reacted N-terminal fragment. The pH was then
raised to 8.2 by addition of solid sodium bicarbonate, and the reaction
mixture
incubated for an additional 24 hours, resulting in a yield of 88%.
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Example 13: Ala-Gly chemical ligation of Larc-alanine-thioester with Na 1-
,(4-methoxyphenyl) 2-mercapto ethane glycine-peptide
A mouse Larc 1-31 Ala C terminal peptide thioester 3mg (MW 3609) and
model peptide Na 1-,(4-methoxyphenyl) 2-mercapto ethane glycine-S-Y-R-F-L 1
mg (MW 908) were dissolved in 0.15 ml 6molar guanidinium buffer pH8.2 and
0.03 ml thiophenol. After overnight stirring the ligation was 81 % complete
and
after 40 hrs 92% complete based on consumption of peptide thioester. Expected
ligated product 4312Da, found 4312Da.
Example 14: Ala-Gly chemical ligation of Larc 1-31-alanine-thioester with
Na 1-(2,4-Dimethoxyphenyl) 2-mercapto ethane glycine
peptide
Mouse Larc 1-31 Ala C terminal peptide thioester 3mg (MW 3609) and
model peptide Na 1-(2,4-dimethoxyphenyl) 2-mercapto ethane glycine-S-Y-R-F-L
1 mg (MW 938) were dissolved in 0.15 ml 6molar guanidinium buffer pH8.2 and
0.03 ml thiophenol. After overnight stirring the ligation was 73% complete,
and
after 40 hrs 85% complete based on consumption of peptide thioester. The
calculated and experimental masses of the ligation product were both 4342Da.
Example 15: Gly-Gly chemical Ligation of C-terminal tripeptide glycine
thioester and N-terminal Na-1-(2,4-dimethoxyphenyl) 2
mercapto ethane glycine-peptide
Peptide fragment FGG-thioester 0.8mg and model peptide Na 1-(2,4-
dimethoxyphenyl) 2-mercapto ethane glycine-S-Y-R-F-L 1 mg (MW 938) were
dissolved in 0.1 ml of 6M guanidinium buffer pH 8.2 and 0.02 ml of thiophenol.
After overnight stirring the reaction was completed quantitatively. The
calculated
and experimental masses of the ligation product were 1199.4Da and 1195.5Da,
respectively.
Example 16: Removal of 1-(2,4-Dimethoxyphenyl) 2-mercapto ethane from
ligation product
1 mg of purified ligation product from Example 15 was dissolved in 0.95 ml
of TFA and 0.025 ml of water and 0.025 ml of TIS. After 1 h. the solvent was
evaporated and a 50% solution waterlacetonitrile was added and the mixture
lyophilized. The cleavage is greater than 95% complete by HPLC. The calculated
and experimental mass was 1003Da.
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Example 17: Gly-Gly chemical Ligation of C-terminal tripeptide glycine-
thioester and N-terminal Na 1-,(4-methoxyphenyl) 2-mercapto
ethane glycine-peptide
A tripeptide peptide fragment thioester, FGG-thioester 1.6mg and model
peptide Na 1-(4-methoxyphenyl) 2-mercapto ethane glycine-S-Y-R-F-L 2 mg (MW
908) were dissolved in 0.2 ml of 6M guanidinium buffer pH 8.2 and 0.04 ml of
thiophenol was added. After overnight stirring the reaction was completed
quantitatively. Expected MW for ligated product 1169.4Da, found 1169.5Da
Example 18: Removal of the 1-(,4-methoxyphenyl) 2-mercapto ethane group
after ligation
Purified ligation product from Example 17 was treated with HF 5% p cresol
at -2°C for 1 hour. After HF evaporation, the ligation product was
precipitated with
ether. The crude peptide was taken up in 50% water/acetonitrile 0.1 % TFA and
injected on HPLC. The major peak > 80% showed the expected molecular weight
for the cleaved peptide (expected mass 1003Da, found 1003Da).
Example 19: His-Gly chemical ligation of C-terminal histidine-thioester and
N-terminal Na 1-(2,4-Dimethoxyphenyl) 2-mercapto ethane
glycine-peptide
Peptide fragment TBP-A 1-67 CaHis thioester 4 mg and model peptide Na
1-(2,4-Dimethoxyphenyl) 2-mercapto ethane Glycine-S-Y-R-F-L 1 mg (MW 938)
were dissolved in 0.1 ml of 6M guanidinium buffer pH 8.2 and 0.02 ml of
thiophenol. After overnight stirring the reaction was 87% complete based on
the
consumption of the peptide thioester. Expected molecular weight for the
ligated
product 9220Da, and found 9220Da.
Example 20: Removal of the 1-(2,4-Dimethoxyphenyl) 2-mercapto ethane
group after ligation
Purified peptide fragment after H-G ligation 2 mg was dissolved in 0.95 ml
of TFA and 0.025 ml of water and 0.025 ml of TIS. After 1 h. the solvent was
evaporated and to the residue was added a 50% solution water/acetonitrile and
the mixture was lyophilized. The cleavage is >90% complete by HPLC. Expected
MW 9023Da, found 9024Da.
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Example 21: Synthesis of Cytochrome b562 by Extended Native Chemical
Ligation
3 mg of Cytochrome 1-63 C-terminal thioester MW 7349 0.4 pmol and 1.5
mg of N-terminal Na 1-,(4-methoxyphenyl) 2-mercapto ethane glycine
Cytochrome b562 residues 64-106 MW 4970 0.3 pmol were dissolved in 0.1 ml of
6M guanidinium ligation buffer pH 7with 0.002 ml of thiophenol as catalyst.
See
Figure 5A. After 24 hrs 0.025 ml of 2-mercapto ethanol were added to the
mixture
and kept reacting for 45 minutes, then 15 mg of TCEP were added and after
additional 30 minutes stirring the ligation mixture was loaded onto a semi-
preparative HPLC. After the break through was eluted and the mixture was then
desalted all the components of the ligation mixture were eluted by ramping the
gradient to 65% B and collected in a sole vial. The analytical HPLC of the
desalted
material showed the ligation was greater than 90% complete based on the
consumption of the C terminal peptide. The HPLC showed two major peaks
(diastereoisomers) with calculated and expected mass of 11,946Da. See Figures
5B and 6A.
The amino acid sequence for Cytochrome b562 (1-106) is shown below:
ADLEDNMETL NDNLKVIEKA DNAAQVKDAL TKMRAAALDA
QKATPPKLED KSPDSPEMKD FRHGFDILVG QIDDALKLAN
EGKVKEAQAA AEQLKTTRNA YHQKYR (SEQ ID N0:1)
Calculated Mass (average isotope composition) 11780.3Da
N-terminal Group: Hydrogen C-terminal Group: Free Acid
MH+ Monoisotopic Mass = 11774.0088 amu ~ HPLC Index =
249.80
MH+ Average Mass = 11781.2781 amu
Bull & Breese value = 1.5360
Elemental Composition: C508 H830 N147 0168 S3
User-Defined Amino Acid Residues: B-HisDNP
Example 22: Removal of the 1-,(4-methoxyphenyl) 2-mercapto ethane group
from ligated Cytochrome b562 residues 1-106 and generation
of the native protein
The desalted solution was then lyophilized prior to removal of the 1-,(4-
methoxyphenyl) 2-mercapto ethane group. Lyophilized material was treated with
a
HF 95% 5% anisol and 1 mmol of cysteine (121 mg) for 1 h at -2 ° C.
The HF
was evaporated using standard protocols and then 100 ml of 50% Buffer B were
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added and the mixture was lyophilized. The mixture was then purified using
semi-
preparative HPLC giving 2 mg of purified single peak native Cytochrome 1-106
(56% of yield after purification), with calculated and experimental mass of
11,780Da. See Figures 6B and 7A.
An analog of wild type cytochrome b562 was also synthesized in the same
manner, and designated SIm7 cyt b562. The SIm7 cyt b562 mutant differed from
the wild type by replacing methionine at position 7 with a selenomethionine
(sulfur
of methionine replaced with its lower cogener selenium). Circular dichroism
was
performed that indicated high a-helical content in both apo wild type b562 and
apo
SIm7 b562 (data not shown). ESMS also showed that both apo wild type b562
and apo SIm7 b562 had the expected molecular masses (data not shown). The
apo proteins were reconstituted with heme (heme pH7 NaPi overnight, room
temperature), and the resulting proteins purified with ion exchange FPLC (FPLC
purification Resource Q, Tris HCL pH 8, NaCI gradient). For example, see
Figure
7B. UV-visible (optical ) spectra of the heme-reconstituted proteins were
found to
be consistent with sulfur or selenium coordination to Fe (data not shown). A
cyt
b562 mutant with the non-coordinating isotere norleucine also is prepared in
the
same manner. Thus synthetic cytochromes were made using extended native
chemical ligation, reconstituted with their heme active sites and fully
characterized
by biophysical methods. Accordingly, this example further demonstrates that
peptides and proteins devoid of suitable cysteines for the original native
chemical
ligation approach can be made by extended native chemical ligation, and non-
standard amino acids incorporated therein. For instance, the folding and
reactivity
of many cyt b562 mutants have been studied, but thus far unnatural axial
ligands
have remained unexplored. Together with extended native chemical ligation, the
vast array of unnatural amino acids available should allow systematic tuning
of the
properties of these and other proteins.
Example 23: Lys-Gly chemical ligation of MCP 1-35-Lysine-thioester with
Na 1-,(4-methoxyphenyl) 2-mercapto ethane glycine-peptide
A MCP 1-35 Lys C terminal peptide thioester 3mg and model peptide Na
1-,(4-methoxyphenyl) 2-mercapto ethane glycine-S-Y-R-F-L 1 mg (MW 908) were
dissolved in 0.15 ml 6molar guanidinium buffer pH7 and adjusted to pH 7.2 by
addition of Triethylamine and 0.03 ml thiophenol. After overnight stirring the
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ligation was 69% complete and after 40 hrs 76% complete based on consumption
of peptide thioester. Expected ligated product 4893Da, found 4893Da
Example 24: Removal of the 1-(,4-methoxyphenyl) 2-mercapto ethane group
after ligation)
Lyophilized de-salted crude from Example 23 (Lys-Gly ligation) was
dissolved in 1 mg of TFA, 25p1 of Ethane di thiol , 50p1 of TIS. Then 150p1 of
bromotrimethylsilane was added. The reaction was allowed to proceed for 2 hrs
at room temperature ("'rt"). The volatile components of the mixture were
evaporated in vacuo, and the remaining oil was taken up in 6M Guanidinium
buffer pH 7.5. The organic material was extracted with CHC13. HPLC showed no
more starting material, therefore the auxiliary group was successfully
removed.
The expected mass for the native sequence was 4726Da, and a mass of 4725Da
was found.
Example 25: Comparison of ligation studies with GSYRFL peptides
Comparison of 5-member rearrangement ligations studies with GSYRFL
peptides from Examples 13-20 and 24 are summarized below in Table V.
Table V: Results of ligation studies with GSYRFL peptides
Auxiliary
Model C-terminal N-terminalReactionLigationRemoval
Peptide
Reactionthioester a Auxilia Time Yield Conditions
tide h %
1 Phe-GI -GI I 16 >98% HF
2 Phe-GI -GI II 16 >98 TFA
3 TBP-A 1-67 II 16 87 TFA
His
4 Mouse Larc I 16 81
1-31 (Ala)
40 92 HF
5 Mouse Larc II 16 73
1-31 (Ala)
40 85 TFA
6 MCP1 1-35 (Lys)I 16 69
40 76 TFA/TMSBr
Note: N-terminal auxiliary I - Na-1-(4-methoxyphenyl)-2-mercaptoethane glycine-
SYRFL, and N-terminal auxiliary II = Na-1-(2, 4-methoxyphenyl)-2-
mercaptoethane
glycine-SYRFL
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Example 26: Preparation of BocGlycine N-1 (4'-methoxyphenyl),2(4'-
methylbenzylthio) ethane
4'-Methoxy 2(4'methylbenzylthio) acetophenone 2mmol, 572 mg, and
glycine ethyl ester HCI salt 2mmol, 139.5 mg are suspended in 15m1 of DCM.
DIEA 6mmol, 1 g is added slowly and under nitrogen 1 ml of titanium
tetrachloride
(1 M solution) is added.
The reaction is kept for 2 days at room temperature. Then
sodiumcyanoborohydride 6mmol, 0.4g in 2.5 ml of anhydrous methanol are
added. The TLC shows approximately 40% of a spot of new product that after
purification is identified by NMR as N-1 (4-methoxyphenyl),2(4-
methylbenzylthio)
ethane Glycine ethyl ester.
1 mmol 374 mg of N-1 (4-methoxyphenyl),2(4-methylbenzylthio) ethane
Glycine ethyl ester is then dissolved in 2m1 of THF and 2mmol of LiOH hydrate
83mg are added to the solution. After overnight stirring the ester has been
completely hydrolyzed. THF is removed in vacuo and the product is taken up in
2m1 of DMF, 5mmol of ditbutyl dicarbonate 1.1g are added and finally 3mmol of
DIEA, 0.45m1. After overnight reaction diluted HCI water solution is added and
the
final product is extracted (3X) with ethyl acetate.
The invention now being fully described, it will be apparent to one of
ordinary skill in the art that many changes and modifications can be made
thereto
without departing from the spirit or scope of the appended claims.
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SEQUENCE LISTING
<110> Gryphon Sciences
Botti, Paolo
Bradburne, James A.
Kent, Stephen B.H.
Low, Donald W.
<120> Extended Native Chemical Ligation
<130> GRFN035/01 WO 03504.291
<140>
<141>
<150> 60/231,339
<151> 2000-09-08
<160> 1
<170> PatentIn Ver. 2.1
<210> 1
<211> 106
<z12> PRT
<213> Homo sapiens
<400> 1
Ala Asp Leu Glu Asp Asn Met Glu Thr Leu Asn Asp Asn Leu Lys Val
1 5 10 15
Ile Glu Lys Ala Asp Asn Ala Ala Gln Val Lys Asp Ala Leu Thr Lys
20 25 30
Met Arg Ala Ala Ala Leu Asp Ala Gln Lys Ala Thr Pro Pro Lys Leu
35 40 45
Glu Asp Lys Ser Pro Asp Ser Pro Glu Met Lys Asp Phe Arg His Gly
50 55 60
Phe Asp Ile Leu Val Gly Gln Ile Asp Asp Ala Leu Lys Leu Ala Asn
65 70 75 80
Glu Gly Lys Val Lys Glu Ala Gln Ala Ala Ala Glu Gln Leu Lys Thr
85 90 95
Thr Arg Asn Ala Tyr His Gln Lys Tyr Arg
100 105
