Promising general solution to the problem of ligating peptides and glycopeptides

Article abstract of DOI:10.1021/ja1084628

Our global goal is that of synthesizing complex polypeptides and glycopeptides in homogeneous form. Chemistry-derived access to homogeneous biologics could well have useful consequences in the discovery of drugs and vaccines. The key finding in this study

Full text of DOI:10.1021/ja1084628

Published on Web 11/04/2010
Promising General Solution to the Problem of Ligating
Peptides and Glycopeptides
Ping Wang† and Samuel J. Danishefsky*,†,‡
Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York
AVenue, New York, New York 10065, and Department of Chemistry, Columbia UniVersity,
HaVemeyer Hall, 3000 Broadway, New York, New York 10027, United States
Received September 19, 2010; E-mail: s-danishefsky@ski.mskcc.org
Abstract: Our global goal is that of synthesizing complex polypeptides and glycopeptides in homogeneous
form. Chemistry-derived access to homogeneous biologics could well have useful consequences in the
discovery of drugs and vaccines. The key finding in this study is that thio acids can become highly competent
acyl donors following even trace levels of oxidative activation, thereby undergoing amide bond formation
upon reaction with N-terminal peptides. Though our data set does not establish the specific mechanism of
this reaction, a framework to account for the fact that minute levels of oxidation actuate amide bond formation
with high turnover is offered. An apparently general coupling of thio acids (including complex peptide thio
acids with N-termini of complex peptides) has thus been realized. These ligations are conducted with minimal
R-epimerization in the C-terminal group and allow for the coupling of N-terminal and C-terminal glycopeptides
en route to homogeneous glycoproteins.
Introduction
the accessibility of a class of highly complex biologics to
chemical synthesis.5
There is a sharp division in current modalities of development
between small-molecule-based drugs and large-molecule agents,
which are often referred to as “biologics”. Small-molecule
prospects are seen to arise from “chemistry”. By contrast,
biologics (cf. vaccines, antibodies, enzymes, and factors) are
perceived to be derivable from strictly biological means. It is
our view that recent advances in the scope and depth of organic
chemistry raise the possibility that chemical synthesis could well
play a valuable role in fashioning biologic-level candidate
structures.1 For such a goal to be feasible in the molecular space
of biologics, complex issues associated with the assembly of
key biolevel repeating building blocks must be mastered.
Biologically active glycopeptides and glycoproteins are of
particular interest to our laboratory.2 A formidable challenge
in reaching such compounds via synthesis is that of joining and
managing two differing biolevel domains (polysaccharides3
and polypeptides), each with their own chemical personalities
and vulnerabilities. Since target glycopeptides or glycoproteins
tend to arise in nature as horrific mixtures of glycoforms,
chemical synthesis might well provide the best prospect for
reaching and evaluating homogeneous glycopeptides for
structure-activity relationship (SAR) studies. We have de-
scribed strategies and enabling methodologies for assembling
complex oligosaccharides with high levels of convergence and
stereocontrol.4 These advances have, for instance, been used in
the building of fully synthetic vaccines, thereby establishing
A massive advance in the capacity to synthesize homogeneous
polypeptides, and even modestly sized proteins, arose from the
seminal discovery of native chemical ligation (NCL) by Kent
and colleagues.6 In NCL, a C-terminal acyl donor is initially
joined to the SH group of an N-terminal cysteine site. Following
S f N acyl transfer, a peptide bond is fashioned (Figure 1a).
Our laboratory has extended the inherent logic of NCL by
exploiting metal-free chemospecific dethiolation of SH groups,
thereby allowing Ala ligation to become a practical option via
an N-terminal Cys.7 By installing thiol groups into otherwise
proteogenic amino acids through chemical synthesis, the elegant
concept of NCL has been extended to enable ligations at
N-terminal Phe, Val, Thr, and Leu sites.8 Helpful as such
advances have been, there is still a huge unmet need for a broadly
based method to enable the ligation of peptides, including glyco-
(4) For reviews, see: (a) Seeberger, P. H.; Bilodeau, M. T.; Danishefsky,
S. J. Aldrichimica Acta 1997, 30, 75–92. (b) Zhu, J.; Warren, J. D.;
Danishefsky, S. J. Expert ReV. Vaccines 2009, 8, 1399–1413.
(5) For two particularly interesting approaches to amide bond formation from
nonobvious coupling partners, see: (a) Carrillo, N.; Davalos, E. A.; Russak,
J. A.; Bode, J. W. J. Am. Chem. Soc. 2006, 128, 1452–1453. (b) Shen,
B.; Makley, D. M.; Johnston, J. N. Nature 2010, 465, 1027–1033.
(6) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776–779. (b) Dawson, P. E.; Churchill, M. J.; Ghadiri,
M. R.; Kent, S. B. H. J. Am. Chem. Soc. 1997, 119, 4325–4329.
(7) Wan, Q.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2007, 46, 9248–
9252.
† Sloan-Kettering Institute for Cancer Research.
‡ Columbia University.
(8) (a) Yang, L.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526–533.
(b) Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007, 129, 10064–
10065. (c) Haase, C.; Rohde, H.; Seitz, O. Angew. Chem., Int. Ed.
2008, 47, 6807–6810. (d) Chen, J.; Wan, Q.; Yuan, Y.; Zhu, J.;
Danishefsky, S. J. Angew. Chem., Int. Ed. 2008, 47, 8521–8524. (e)
Chen, J.; Wang, P.; Zhu, J.; Wan, Q.; Danishefsky, S. J. Tetrahedron
2010, 66, 2277–2283. (f) Harpaz, Z.; Siman, P.; Kumar, K. S. A.;
Brik, A. ChemBioChem 2010, 11, 1232–1235.
(1) Nagorny, P.; Fasching, B.; Li, X.; Chen, G.; Aussedat, B.; Danishefsky,
S. J. J. Am. Chem. Soc. 2009, 131, 5792–5799.
(2) Tan, Z.; Shang, S.; Halkina, T.; Yuan, Y.; Danishefsky, S. J. J. Am.
Chem. Soc. 2009, 131, 5424–5431.
(3) Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem., Int. Ed. Engl. 1996,
35, 1830–1419.
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Wang and Danishefsky
peptides, independent of the logic of NCL. It is this goal to which
the research described in this paper is addressed.
Results and Discussion
Before describing our findings, we relate the etiology of the
discovery progression. It will thus be appreciated that hap-
penstance played no small role in mediating our advances. The
organizing concept started with the reaction of a thio acid, 1,
with an isonitrile, 2, in the presence of an amine-based acyl
acceptor, of type 4 (Figure 1b),9 giving rise to a presumed thio
formimidate carboxylate mixed anhydride (termed a thio-FCMA,
3). The latter can be interdicted by amines to generate even
complex amides. However, attempts to extend the scope of this
coupling to a C-terminal thio acid of even a dipeptide failed to
provide useful yields of the desired product. It was surmised
that the dipeptide thio-FCMA intermediate suffers rapid conver-
sion to its corresponding oxazolone, which is not a competent
acyl donor under these conditions (Figure 1c). We asked whether
the presumed thio-FCMA (3) could be diverted to produce a
more functional acyl donor than the presumed oxazolone. For
instance, peptide bond formation via 1-hydroxy-1H-benzotria-
zole (HOBT) esters tends to result in markedly reduced levels
of C-terminal, oxazolone-promoted epimerization.10 Accord-
ingly, we investigated the possible formation of peptidic bonds
Figure 1. (a) Native chemical ligation. (b) Thio-FCMA ligation. (c)
Challenge: thio-FCMA ligation in peptide couplings.
a
Table 1
a Reagents and conditions: DMSO, 5-8 equiv of cyclohexylisonitrile, 2.0 equiv of HOBT, 4 Å molecular sieves, room temperature. HOBT )
1-hydroxy-1H-benzotriazole.
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Table 2. HOBT-Mediated Peptide Couplinga
a Reagents and conditions: DMSO, 2 equiv of HOBT, 4 Å molecular sieves, air, room temperature; (a) DMSO, 0.3 equiv of HOBT, 4 Å molecular
sieves, air, room temperature.
via the reaction of a C-terminal thio acid with cyclohexylisoni-
trile and N-terminal peptide in the presence of HOBT. The hope
was that this FCMA would give way to an HOBT ester. The
results, shown in Table 1, seemed quite encouraging.
Phe, Leu, and Val, was accomplished smoothly. Even Pro-Val
ligations could be achieved in excellent yield (Table 2, entry 3,
87%). Ligations of large peptide partners can be readily realized.
The method is also applicable to macrolactamization (Table 2,
entries 6 and 7). Although significantly longer reaction times
were required (48 h), product yields were not compromised.
Clearly, the finding that the isonitrile component is not
required for ligation does not rule out the validity of the pathway
and mechanism advanced above when isonitrile is present.
However, it does establish the existence of a pathway to amide
bond formation from the reaction of thio acids and amines
independent of isonitrile chemistry.
We next addressed the question of C-terminal epimerization
during the course of ligation in more discriminating settings. A
series of thio acid peptides, presenting C-terminal residues such
as Phe, Ala, Leu, and Val, were evaluated as acyl donors (Table
3). In the simplest case, thio acid 21 readily undergoes amino
acid extension with 22 to provide 23 in 89% yield with no
detectable loss of stereointegrity (<3%). This result is quite
encouraging, since Phe is particularly prone to C-terminal
epimerization.12 An even more discriminating case, involving
coupling of the C-terminal Phe-bearing thio acid 24 with peptide
13, furnished adduct 25 in 82% yield with only a modest degree
In a control experiment, we asked whether C-terminal thio
acids11 could couple with N-terminal peptides in the presence
of HOBT, but in the absence of “throwaway” isonitrile. It was
indeed fortunate that this control experiment was conducted.
As seen in Table 2, coupling had occurred even more effectively
than was the case with the isonitrile present (compare Tables 1
and 2, entries 1-5). Though chromatographic criteria suggest
coupling efficiencies of ca. 90%, Table 2 reports only yields of
purified isolated products. As seen, both C-terminal Gly and
Pro residues were readily accommodated. Moreover, coupling
of a range of hindered N-terminal amino acid residues, including
(9) (a) Yuan, Y.; Zhu, J.; Li, X.; Wu, X.; Danishefsky, S. J. Tetrahedron
Lett. 2009, 50, 2329–2333. (b) Rao, Y.; Li, X.; Danishefsky, S. J.
J. Am. Chem. Soc. 2009, 131, 12924–12926.
(10) Ko¨nig, W.; Geiger, R. Chem. Ber. 1970, 103, 788–798.
(11) The C-terminal thioacids were prepared by well-known simple
methods, as described in the Supporting Information. Cf. inter alia
the following: (a) Crich, D.; Sharma, I. Angew. Chem., Int. Ed. 2009,
48, 2355–2358. (b) Crich, D.; Sharma, I. Angew. Chem., Int. Ed. 2009,
48, 1–5. (c) Vetter, S. Synth. Commun. 1998, 28, 3219–3223.
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Wang and Danishefsky
Table 3. HOBT-Mediated Peptide Couplinga
a Reagents and conditions: general procedure, DMSO, 2 equiv of HOBT, 4 Å molecular sieves, room temperature, 12 h; (a) CH2Cl2, 2 equiv of
HOBT, 4 Å molecular sieves, room temperature, 12 h; (b) DMSO, 2 equiv of HOOBT, 4 Å molecular sieves, room temperature, 12 h; (c) DMSO, 0.3
equiv of HOBT, 4 Å molecular sieves, room temperature, 20 h. HOOBT ) hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine.
of epimerization at the Phe site (Table 3, entry 2). By contrast,
C-terminal Ala-presenting thio acids seemed to be markedly
more resistant to epimerization (Table 3, entries 3-5). Even
when peptide thio acids bearing C-terminal Leu and Val residues
were employed, epimerization levels were only 4% and 5%,
respectively (Table 3, entries 6 and 7). We noted some
improvement in the suppression of C-terminal epimerization via
the use of HOOBT (seen in Table 3, entries 2 and 7).
The thio acid/HOBT method was shown to be effective in
ligating a glycopeptide with a C-terminal thio acid, as demon-
strated in the synthesis of 18-mer 36, containing a hexasaccha-
ride of the high-mannose type. As shown in Figure 2, in the
presence of 2.0 equiv of HOBT, peptide thio acid 12 and
glycopeptide 35 undergo condensation to provide adduct 36 in
80% yield. Moreover, in the presence of 2.0 equiv of HOBT,
glycopeptide thio acid 37 and glycopeptide 38 readily undergo
fragment condensation to provide adduct 39 in 81% yield
(Figure 2). The capability of ligating peptides to glycopeptides
and glycopeptides to glycopeptidessso critical to synthesizing
homogeneous glycoproteins in the laboratoryshas thus been
realized.
harbingers of such a reaction in very simple systems could be
found early.13 A later paper by Rosen et al.14 also described
such a reaction in a simple setting. Of course, the most obvious
interpretation of such findings would be to suppose that, unlike
carboxylic acids, their thio counterparts have strong inherent
acyl donor character. While we could not rule this out, we began
to suspect that there could be an oxidative component in these
ligations. We first probed this possibility by conducting the
couplings in an argon atmosphere rather than in air, but with
no other precautions to ensure exclusion of air. Entries 1 and
2 in Table 4 are instructive. Under these “less oxidizing”
conditions, the yield of coupling was clearly reduced. Not
surprisingly, suppression of ligation was even more successful
when HOBT was also omitted (see Table 4, entry 2). The same
trend is also supported by entries 3 and 4 (Table 4). Attempted
suppression of oxygen in the absence of HOBT still provided
a 28% yield of tetrapeptide (see Table 4, entry 4). We note
that, in larger peptide-peptide settings, omission of HOBT or
conduction of the reaction under argon results in, at best, trace
amounts of peptide bond formation with simple unhindered
substrates (Table 4, entry 5). It is not clear whether the
suppressed couplings under attempted anaerobic conditions
reflect residual acyl donor competence of the thio acid itself.
We now turn to experiments which were aimed toward
gaining some mechanistic insight into this amide bond forming
reaction between thio acids and amines. Indeed, in retrospect,
(13) A more complete review of the relevant literature is provided in the
Supporting Information. Selected references: (a) Pawlewski, B. Chem.
Ber. 1898, 31, 661–663. (b) Gunter, H. Chem. Ber. 1950, 83, 258–
261.
(12) Kovacs, J. Racemization and Coupling Rates of N-R-Protected Amino
Acid and Peptide Active Esters: Predictive Potential. In The Peptides;
Gross, E., Meienhofer, J., Eds.; Academic: New York, 1980; Vol. 2,
pp 485-539.
(14) Rosen, T.; Lico, I. M.; Chu, D. T. W. J. Org. Chem. 1988, 53, 1580–
1582.
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Figure 2
In principle, in a nonoxidative coupling, the sulfur should
formally emerge at the H2S oxidation level, rather than as
elemental sulfur, which would be reflective of oxidation (vide
infra).
stoichiometric level oxidation were to occur, elemental sulfur
would be extruded. Hitherto, the oxidative version of the
reaction15,16 had been predicated, quite reasonably, on the use
of an established oxidizing agent at stoichiometric levels or in
excess.
Having shown that minimization of opportunities for
adventitious oxidation does indeed attenuate coupling, we
asked whether inclusion of an established oxidizing agent
would promote amidation. The apparent validation of this
expectation is shown in Table 5. While the yields of isolated
peptide coupling are not substantially increased, the rates are
enhanced (see the conditions governing Table 5) and C-
terminal epimerization is still suppressed (Table 5, entries 4
and 5). Remarkably (see Table 5, entries 1-3), several
otherwise highly oxidation vulnerable amino acids (Cys, Met,
Trp, Tyr, and His) appear to survive nicely in the context of
the peptides employed.
In retrospect, we note once again that the concept of
constructing amides from the coupling of thio acids and amines,
albeit in very simple cases, goes back more than a century. In
earlier work, when such amidation reactions were periodically
described, the operating assumption had been that a thio acid
is an inherent C-acyl donor.13,14 A provocative step forward
was provided in 1952 by J. C. Sheehan, who showed that the
rate of amide formation from an apparent thio acid acyl donor
could be significantly increased by the use of an oxidizing agent
(I2) and could be extended to a simple N-terminal amine.15 The
notion of oxidation-mediated coupling of an amino acid to a
thio acid was demonstrated more recently in conjectures about
amide bond formation under prebiotic conditions by Orgel (using
potassium ferricyanide as the oxidizing agent).16 Formally, if
However, there occurred to us an alternative explanation
which could better account for our findings. We came to
envision a chain reaction, involving low levels of oxidative
initiation with turnover to reconstitute competent C-terminal
acyl donor capacity (vide infra). The findings provided herein
clearly point to a substoichiometric oxidative component in
the amide construction, since couplings occur much more
readily and efficiently when conducted in the presence of
air as opposed to under attempted oxygen exclusion. More-
over, they occur somewhat more rapidly in the presence of
an established oxidizing agent, such as iodine. To account
for the excellent yields reported above, it is necessary to chart
a scenario by which low levels of oxidative initiation are
magnified through an efficient pathway for reconstituting acyl
donor capacity.
The exact nature of the proposed oxidative initiation still
awaits definition. Presently, we can only provide a type of
framework by which small levels of oxidation can be amplified
to lead to what would otherwise, perhaps simplistically, be
considered a simple acylation. Certainly, many variations of that
projected in Figure 3 can be entertained. Among the possibilities
which have been invoked for oxidative activation (as opposed
to inherent acyl donor competence of a thio acid) is a diacyl
disulfide of type 50.17 While there is nothing in our data set
which establishes 50 as the oxidatively induced acyl donor
species, we suggest in Figure 3 a model for a type of high-
turnover pathway to recycle the competent acyl donor (i.e., 50).
Thus, reaction of 50 with HOBT followed by amine 51 leads
(15) Sheehan, J. C.; Johnson, D. A. J. Am. Chem. Soc. 1952, 74, 4726–
4727.
(16) Liu, R.; Orgel, L. E. Nature 1997, 389, 52–54.
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Table 4. Attempted Suppression of Possibilities for Oxidationa
a Reagents and conditions: (a) solvent, 2 equiv of HOBT, 4 Å molecular sieves, argon, room temperature; (b) solvent, no HOBT, 4 Å molecular
sieves, argon, room temperature.
to amide and perthio acid 52. Reaction of 52 with thio acid 49
reconstitutes acyl donor 50 (and H2S). Of course, reaction of
extruded thio acid 49 with 52 at the SH group (see the dotted
line pathway) would constitute a nonproductive (invisible)
reaction.
It would be expected that this type of trace oxidative pathway
would be accelerated by enhanced opportunities for oxidation.
As described above, this too was observed. Extensive experi-
mentation will be necessary to pin down the actual operative
details among a continuum of possibilities. Not to be excluded
from such a continuum is the notion that thio acids themselves
have some marginal intrinsic acyl donor capacity. While direct
classical acylation has the virtue of simplicity (Occam’s razor!),
our data suggest that it is unlikely to be a major participant in
the chemistry we have described above, particularly for the
complex cases.
of HOBT holds considerable promise for accomplishing new
fragment coupling that is independent of the Cys-associated
logic of native chemical ligation. The method has been
successfully demonstrated in a range of challenging peptide
bond constructions. Ligation occurs with manageably low
levels of C-terminal epimerization. The method has been
demonstrated to work well in the contexts of macrolactam-
ization and glycopeptide-peptide and glycopeptide-glyco-
peptide constructions. The C-terminal thio acid is cleanly
differentiated from O-carboxy groups on side chains. At this
stage, side chain amines do require protection.
Of course, much work will be required to establish how this
method of peptide bond formation best augments the current
menu of NCL-based strategies,6-8 SPPS,18 and other non-
cysteine-related ligations.19 The optimal fitting of the protocols
described above to particular sequences intended for ligation
will also require considerable fact gathering and field-tested
data sets. However, we are already confident that ligation of
C-terminal thio acids with N-terminal peptides (in the presence
Conclusion
Mechanistic uncertainties notwithstanding, the coupling of
C-terminal thio acids with N-terminal peptides in the presence
(18) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149–2154.
(19) (a) Blake, J.; Li, C. H. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4055–
4058. (b) Chen, G.; Wan, Q.; Tan, Z.; Kan, C.; Hua, Z.; Ranganathan,
K.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2007, 46, 7383–7387.
(c) Payne, R. J.; Ficht, S.; Greenberg, W. A.; Wong, C. H. Angew.
Chem., Int. Ed. 2008, 47, 4411–4415.
(17) (a) Yamashiro, D.; Li., C. H. Int. J. Pept. Protein Res. 1988, 31, 322–
334. (b) Høeg-Jensen, T.; Olsen, C. E.; Holm, A. J. Org. Chem. 1988,
59, 1257–1263. For a different but related variation of the framework
suggested in Figure 3, see: (c) Liu, C.-F.; Rao, C.; Tam, J. P.
Tetrahedron Lett. 1996, 37, 933–936.
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Table 5. I2,HOBT-Mediated Peptide Couplinga
a Reagents and conditions: (a) DMSO, 2 equiv of HOBT, 4 Å molecular sieves, I2 (0.6 equiv), DIPEA (1.5 equiv), room temperature, 30 min; (b)
DMF, 2 equiv of HOBT, 4 Å molecular sieves, I2 (0.6 equiv), DIPEA (1.5 equiv), room temperature, 30 min; (c) DMF, 2 equiv of HOOBT, 4 Å
molecular sieves, I2 (0.6 equiv), DIPEA (1.5 equiv), room temperature, 30 min.
chemistry and biology will be of value in pharmaceutical level
discovery research.
Acknowledgment. Support for this research was provided by
the National Institutes of Health (Grant CA103823 to S.J.D.). We
thank Drs. Jin Chen, Xuechen Li, Yu Yuan, and Jianglong Zhu for
help and advice.
Supporting Information Available: General experimental
procedures, including spectroscopic and analytical data for new
compounds. This material is available free of charge via the
Figure 3
of HOBT or HOOBT) will be a valuable method for building
homogeneous biologics, including highly complex glycopep-
tides, by chemical synthesis. We expect that synergy between
Internet at http://pubs.acs.org.
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