Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol

Article abstract of DOI:10.1002/anie.200600538

(Figure Presented) The missing link: Reiterative ligations based on non-cysteine and cysteine-based acyl acceptors have been used to prepare synthetic polypeptides and proteins with multiple sites of glycosylation. Highly complex positionally defined glyc

Full text of DOI:10.1002/anie.200600538

Communications
Glycoproteins
DOI: 10.1002/anie.200600538
Building Complex Glycopeptides: Development
of a Cysteine-Free Native Chemical Ligation
Protocol**
Bin Wu, Jiehao Chen, J. David Warren, Gong Chen,
Zihao Hua, and Samuel J. Danishefsky*
The development of increasingly efficient and general methods
for the merging of complex peptidic fragments remains a
central objective in the field of polypeptide and glycopolypep-
tide synthesis. A number of traditional native chemical ligation
(NCL) techniques have been applied to the problem of
polypeptide assembly through convergent ligation.[1] Nearly
all of these require the presence of an N-terminal cysteine
residue to function as the acyl acceptor. Given the relative
scarcity of cysteine residues in nature, a clear impetus arises for
the realization of new NCL capabilities.
Our laboratory has a major interest in the development of
methods for the preparation of homogeneous, fully synthetic
polypeptides, and even proteins, that display multiple sites of
glycosylation. A particularly relevant glycoprotein, which
serves to coordinate and focus our efforts in this field, is the
naturally occurring erythropoietin alpha (EPO; Figure 1).[2]
Figure 1. Structure of erythropoietin.
[*] Prof. S. J. Danishefsky
The Department of Chemistry
Columbia University
Havemeyer Hall, New York, NY 10027 (USA)
Fax: (+1) 212-772-8691
E-mail: s-danishefsky@ski.mskcc.org
Dr. B. Wu, Dr. J. Chen, Dr. J. D. Warren, Dr. G. Chen, Dr. Z. Hua,
Prof. S. J. Danishefsky
The Laboratory for Bioorganic Chemistry
Sloan-Kettering Institute for Cancer Research
1275 York Avenue, New York, NY 10021 (USA)
[**] This work was supported by the National Institutes of Health (Grant
CA28824 to S.J.D., and CA62948 to J.D.W.) and by New York State
Department of Health, New York State Breast Cancer Research and
Education Fund (to B.W.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Scheme 1. Cysteine-free ligation strategy. See text for details.
Scheme 2. Synthesis of compound 17. Reaction conditions, a) NaCNBH3, MeOH/DMF, 60%; b) 14, HATU, iPr2NEt, DMSO, 62%; c) 16, TFE/
CH2Cl2, 70%. Ac=acetyl, DMF=N,N-dimethylformamide, DMSO=dimethyl sulfoxide, HATU=O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyl-
uronium hexafluorophosphate, PMB=para-methoxybenzyl, TFE=2,2,2-trifluoroethanol.
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Scheme 3. Synthesis of 21. Reaction conditions, a) TCEP, PBS (pH 8.0), 37%. Aux=auxiliary, Dmab=4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexyl-
idene)-3-methylbutyl]-amino}benzyl, PBS=phosphate-buffered saline buffer solution, TCEP=tricarboxyethylphosphine.
Scheme 4. Ligation of 22 and 23. Reaction conditions, a) TCEP, DMF, Na2HPO4, 328C, 54%.
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This multiply glycosylated protein has found widespread
therapeutic application in the treatment of anemia. Despite
the clear-cut clinical importance of this compound, attempts
to rigorously evaluate the role of glycosylation on the activity
and stability of erythropoietin have thus far been complicated
by the daunting difficulties associated with isolating signifi-
cant quantities of homogeneous EPO.[3] On the basis of our
long-term involvement in the arena of carbohydrate and
glycopeptide total synthesis,[4] we reasoned that if the consid-
erable powers of chemical synthesis were brought to bear on
this problem in a principled and focused fashion, it might be
possible to gain access to fully synthetic, homogeneous
erythropoietins. Needless to say, such a capability would
likely enable access to a range of EPO analogues for
structure–activity-relationship investigations. Broadly speak-
ing, an undertaking of this magnitude could well lead to the
Scheme 5. Ligation of O-linked glycodomains. Reaction conditions, a) TFA, PhOH, H2O, TESH; b) 0.1n NaOH, MeOH; c) H2NNH2, MeOH, 61%
over 3 steps; d) 16, TFE, 67%; e) TCEP, DMF, Na2HPO4, 65%. TFA=trifluoroacetic acid, TESH=triethylsilane.
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development of generally useful strategies and protocols of
tide 12 provided 13 in 60% yield. This ability to connect a
substantial N-terminal domain to the auxiliary by reductive
amination is an important feature of this methodology. Next,
the disaccharide 14 was appended to the peptide through
reducing-end amination and aspartylation.[9] At this stage, we
utility to the entire field of glycoprotein synthesis.
Erythropoietin is a 166-residue protein possessing four
sites of glycosylation. Three of these are N linked to
asparagine residues and one is O linked to a serine residue.
In considering a strategy for the de novo synthesis of
erythropoietin, we took note of the paucity of cysteine
residues on the molecule. A maximally convergent route to
erythropoietin would involve the preparation of the four
different glycopeptide fragments, which would subsequently
be joined through some form of ligation to furnish the fully
glycosylated protein backbone. Thus, in light of the fact that
the four cysteine residues of erythropoietin do not segregate
into nearly equal-sized carbohydrate-bearing domains, we
were led to consider glycopeptide ligation methods that do
not require cysteine-based acyl acceptors. The development
of a repertoire of ligation methods could well be critical to the
success of our EPO-directed venture.
Scheme 1a shows, in the most general terms, a scenario
wherein two differentially glycosylated peptide fragments (1
and 2) would be temporarily engaged through an auxiliary
linker[5] such that the C-terminal coupling fragment would be
activated as a thioester (3). Having been coaxed into
proximity, the N-terminal coupling partner would attack the
thioester, thus forming the key amide bond (4). Removal of
the auxiliary would provide the doubly glycosylated peptide 5.
In this way, the limitation of a cysteine acyl acceptor situated
at the N-terminus would have been removed.
ꢀ
sought to convert the S PMB group into an aromatic
disulfide under mild conditions that would leave the sensitive
glycopeptide functionality intact. We were pleased to find
that, following treatment of 15 with sufenyl chloride 16 in
TFE/DCM, compound 17 was obtained.[10] Notably, this
transformation represents an appealing alternative to pre-
viously described conditions that typically require exposure to
harsh reagents such as anhydrous HF or Hg(OAc)2 and
TFA.[6a,7a]
With the auxiliary-bearing glycopeptide fragment 17 in
hand, we were now prepared to investigate the viability of our
cysteine-free coupling strategy. In this context, we first
examined a Gly–Ala ligation. As expected, upon exposure
to TCEP in PBS buffer solution (pH 8.0), glycopeptides 17
and 18 each underwent reductive disulfide cleavage (see
Scheme 3). Presumably, the C-terminal glycopeptide then
suffered an O!S acyl transfer of the type previously
described to generate the activated thioester (19).[11] As
anticipated, 19 and 20 were temporarily joined through a
thioester exchange reaction. Following intramolecular acyl
transfer, the fully functionalized glycopeptide 21 was isolated
in 37% yield.[12]
Encouraged by this early success, we next sought to
explore the generality of the method by attempting ligation at
a more challenging Gly–Gln center. Thus, glycopeptides 22
and 23 (Scheme 4) were prepared and subjected to ligation
conditions (TCEP in PBS buffer solution; pH 8.0). Unfortu-
nately, the yield of 24 was found to be quite low, and a
significant quantity of carboxylic acid arising from the
hydrolysis of 22 was observed. We postulated that, although
in the previous instance (17 + 18!21) the rate-determining
step of the sequence had been the joining of the two
fragments through transthioesterification, in the case at
hand, the increased steric hindrance around the reacting
center had caused the intramolecular acyl transfer to become
rate limiting. Consequently, hydrolysis of the tethered inter-
mediate had the opportunity to intervene as a competitive
side reaction. Although we were unable to improve upon the
product distribution by adjusting the pH of the system, we did
find that, by introducing DMF as a cosolvent with a small
amount of Na2HPO4, we were able to isolate the ligation
product 24 in a more-acceptable 54% yield.
One conceivable manifestation of this general strategy
would commence with the covalent appendage of a sulfur-
displaying auxiliary of the type 6 to the N terminus of an
appropriate peptide fragment (7) through reductive amina-
tion.[6] In this early feasibility demonstration, we benefited
from a recently disclosed clever idea of Dawson et al. for a
non-cysteine-based ligation, albeit by a different organizing
step.[7] The resultant intermediate would be advanced to the
glycopeptide 9. At this point, we envisioned joining the two
fully functionalized glycopeptide fragments under conditions
analogous to those that we had previously developed in the
context of a cysteine-based NCL method.[5a] Thus, the
coupling partner, 8, would be equipped with a C-terminal
phenolic ester, as shown in Scheme 1b. Under disulfide
reducing conditions, the C-terminal coupling partner, 8,
would be activated to form a thioester with the auxiliary
sulfur functionality of the N-terminal fragment, thus provid-
ing an intermediate of the type 10. Amide-bond formation
followed by auxiliary removal would provide the bifunctional
glycopeptide 5.
In the end, we elected to equip
the sulfur atom of the auxiliary with a
PMB protecting group.[8] We antici-
pated that deprotection of this group
could be accomplished under mild
conditions that are compatible with
survival of glycopeptide functionality
(see Scheme 2). Thus, aldehyde 11
was prepared through slight modifi-
cation of
Reductive amination with hexapep-
a
known procedure.[7a]
Scheme 6. Bifunctional glycopeptide.
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Having demonstrated the capacity of our methods to
successfully ligate two N-linked glycopeptide domains, we
next sought to investigate its compatibility with O-linked
glycodomains. Thus, intermediate 25 was advanced to 26
through a three-step sequence, as shown in Scheme 5.[13] The
latter was converted into the N-terminal coupling partner, 27,
according to previously developed reaction conditions. We
were pleased to find that, upon exposure to TCEP and DMF
with Na2HPO4, 27 and 28 readily underwent cysteine-free
native chemical ligation to provide glycopeptide 29, which
possesses both N- and O-linked carbohydrate domains.
Notably, no carbohydrate decomposition products were
observed.
We next validated our methodology in the context of
more-complex glycan fragments, including those containing
characteristic non-reducing and sialic acid moieties. The
Scheme 7. Synthesis of 32. Reaction conditions, a) methyl p-nitrobenzene sulfonate; b) 95% TFA.
Scheme 8. Trifunctional glycopeptide.
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extent of sialidation is apparently a determinant of EPO
auxiliary. First, intermediate 24 was treated with methyl p-
nitrobenzene sulfonate.[16] This step accomplished selective
methylation of the sulfur on the aromatic ring to provide
intermediate 31. The latter was not purified, rather, it was
exposed to the action of 95% TFA, thereby providing the
native glycopeptide 32, free of any observable thioester by-
product.
Having successfully field tested our novel cysteine-free
ligation protocol in the context of a convergent bis-domainal
glycopeptide synthesis, we next turned to the challenge of
synthesizing longer peptide chains that contain more than two
sites of glycosylation. Indeed, to consider the total synthesis of
a complex glycoprotein, such as EPO, it would be critical to be
able to couple, in a reiterative fashion, multiple glycopeptide
fragments. Along these lines, we recently disclosed a method
to generate differentially glycosylated trifunctional glycopep-
tides based on a cysteine-dependent native chemical ligation
protocol.[17] As a demonstration of the applicability of our
new non-cysteine NCL technology to complex targets, we
stability.[14] Thus, the coupling of two glycopeptide fragments,
each displaying an N-linked core pentasaccharide, was found
to proceed smoothly to provide the bifunctional glycopeptide
30 (Scheme 6).
The final phase of this investigation would be the
development of appropriately mild conditions for the cleav-
age of the thiol auxiliary. In this context, TFA with a
scavenger has been used in similar types of systems. However,
in our hands, the treatment of the ligation product with 95%
TFA with a triisopropyl silane (TIPS) scavenger resulted in a
mixture of the desired native glycopeptide along with another
compound of the same molecular weight as the starting
glycopeptide. The latter was tentatively assigned to be the
thioester intermediate, arising from acid-mediated intramo-
lecular N!S acyl transfer.[15] Presumably, the otherwise
endothermic step is driven by irreversible protonation of
the benzylic amine. In light of this finding, a two-step
sequence (Scheme 7) was devised for the removal of the
Scheme 9. Synthesis of 40. Reaction conditions, a) 14, HATU, iPr2NEt, DMSO, 72%; b) 14, HATU, iPr2NEt, DMSO, 83%; c) TFE, DCM, 16, 69%;
d) TCEP, DMF, Na2HPO4, 58% (38) + 11% (40); e) methyl p-nitrobenzene sulfonate; f) 95% TFA; g) PhSH or MesNa, PBS. DCM=dichloro-
methane, Fmoc=9-Fluorenylmethoxycarbonyl, MesNa=2-mercaptoethane sulfonic acid, sodium salt.
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next sought to apply a combination of our cysteine-dependent
and cysteine-free NCL protocols to the preparation of the
multiply glycosylated peptide, 33. Under our synthetic plan,
we would first prepare each of the three glycopeptide
fragments (34, 35, and 23) according to glycal assembly and
glycopeptide synthesis protocols that have been validated and
optimized over the course of many years in our laboratory
(Scheme 8).[4] Fragments 23 and 35 would then be joined
according to our newly developed cysteine-free ligation
method to form the Gly–Gln junction. Next, following
deprotection of the N-terminal cysteine residue, the bifunc-
tional peptide would be merged with glycopeptide 34 through
cysteine-based ligation to afford the fully functionalized
target compound.
Thus, polypeptides 36 and 37 were prepared for the
cysteine-free ligation event. It is noted that the termini have
been suitably equipped in anticipation of the reiterative
sequence. Thus, peptide 37 bears the requisite N-terminal
auxiliary for the cysteine-free coupling, whereas fragment 36,
which will serve as the middle glycopeptide component,
incorporates the C-terminal phenolic ester for the first
cysteine-free ligation as well as a 1,3-thiazolidine-4-carboxo
(Thz)-protected N-terminal cysteine residue, which is
unmasked prior to the second, cysteine-based ligation
event.[18]
Each peptide fragment was subjected to glycosylation
with disaccharide 14, and, following conversion of the N-
ꢀ
terminal auxiliary S PMB group to the requisite disulfide,
glycopeptides 35 and 23 were in hand. As hoped, the coupling
of the two fragments proceeded readily in the presence of
TCEP to afford the ligated product 38 in 58% yield, along
with 11% of the thioester 40. The latter could be converted
into 38 upon treatment with thiophenol or MesNa.[19] At this
stage, the thiol auxiliary could be removed according to the
two-step sequence shown in Scheme 9 (38!39); alternatively,
the auxiliary could also be maintained in the subsequent
ligation event without causing detriment.
As we had previously demonstrated, the cysteine residue
was readily unmasked through exposure of 38 to 10%
morpholine in DMF (to remove the Fmoc group) followed
by treatment with an aqueous solution of MeONH2·HCl
(Scheme 10). Native chemical ligation between 41 and 34 was
Scheme 10. Synthesis of 42. Reaction conditions, a) (i) 10% morpholine in DMF; (ii) 0.4m MeONH2·HCl, 60%; b) MesNa, TCEP, PBS (pH 8.0),
57%.
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carried out in the presence of MesNa and
TCEP and afforded the multifunctional
glycopeptide 42 in 57% yield.
Finally, the viability of our newly
developed cysteine-free ligation protocol
was demonstrated in another area of
peptide chemistry of great interest to
those at the forefront of chemistry and
glycobiology, that is, the synthesis of cyclic
peptides.[20] Cyclic peptides often possess
enhanced biological specificity, activity,
and metabolic stability in comparison to
their linear counterparts. This is as a
consequence of their constrained confor-
mations and their enhanced levels of
resistance to protease digestion. Although
traditional strategies for cyclic-peptide
formation are restricted to macrolactam
or disulfide formation, Tam and co-work-
ers[21] have disclosed that cyclic peptides
can be accessed through native chemical
ligation.[22] Recently, our research group
reported on a newly modified protocol for
native chemical ligation that allows for
formation of cyclic peptides possessing a
cysteine residue.[23] However, given the
scarcity of cysteine residues in nature, the
applicability of standard cysteine-based
ligation methods may be somewhat lim-
ited. Clearly, the development of
a
broadly useful, cysteine-independent liga-
tion protocol could well have profound
ramifications for the field of cyclic-pep-
tide synthesis.
The linear polypeptide 43 was pre-
pared through solid-phase peptide syn-
thesis. Reductive amination with alde-
hyde 11 served to introduce the N-termi-
nal auxiliary (44). The C terminus was
functionalized through HATU-mediated
esterification with phenol 45, providing
46. Following protecting-group removal
and exposure to 3-nitro-2-pyridinesul-
fenyl chloride, the requisite disulfide
cyclization precursor was in hand. Thus,
the linear peptide bis-disulfide (47) was
treated with TCEP and Na2HPO4 in DMF
to provide the desired cyclized peptide
(48) in good yield. Importantly, no dimers
or oligomers were observed in liquid
chromatograpy–MS
Scheme 11).
analysis
(see
Obviously, in undertaking a target of
the complexity of erythropoietin, oppor-
tunities for complications and even failure
are always inherent. However, we feel
that in principle the basis of a realistic
total synthesis of homogeneous erythro-
poietin has been set forth above. Always
Scheme 11. a) 11, NaCNBH3, MeOH, DMF, 66%; b) 45, HATU, DIPEA, DMF; c) TFA, PhOH,
TESH, H2O, 57% for 2 steps; d) 16, TFE, 60%; e) TCEP, Na2HPO4, DMF, 78%. DIPEA=N,N-
diisopropylethylamine, Pbf=2,2,4,6,7-pentymethyldihydrobenzofuran-5-sulfonyl.
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[7] a) J. Offer, C. N. C. Boddy, P. E. Dawson, J. Am. Chem. Soc.
mindful of the risks, we remain busily engaged in pursuing this
goal.
2002, 124, 4642 – 4646; b) J. Offer, P. E. Dawson, Org. Lett. 2000,
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[8] Other S-protecting groups such as 2-cyanoethyl group (remov-
able under basic conditions), and 2,4-dinitrophenyl (DNP;
removable with Mes-Na) were also successfully applied to this
approach to allow the incorporation of cysteine and tryptophan
residues in the glycopeptides.
[9] S. T. Cohen-Anisfeld, P. T. Lansbury, J. Am. Chem. Soc. 1993,
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[10] R. Matsueda, T. Kimura, E. T. Kaiser, G. R. Matsueda, Chem.
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Received: February 9, 2006
Revised: April 5, 2006
Published online: May 19, 2006
Keywords: cysteine-free ligation · erythropoietin · glycoproteins ·
.
native chemical ligation · peptides
[11] Manuscripts detailing the mechanism study of the postulated
O!S acyl shift are in preparation.
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establishing the ligation has a distinguished intellectual history.
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