A route to cyclic peptides and glycopeptides by native chemical ligation using in situ derived thioesters

Article abstract of DOI:10.1016/j.tetlet.2006.01.077

The synthesis of cyclic peptides and glycopeptides by native chemical ligation using in situ derived thioesters is described.

Full text of DOI:10.1016/j.tetlet.2006.01.077

Tetrahedron Letters 47 (2006) 1969–1972
A route to cyclic peptides and glycopeptides by native
chemical ligation using in situ derived thioesters
Jiehao Chen,a J. David Warren,a Bin Wu,a Gong Chen,a
Qian Wana and Samuel J. Danishefskya,b,
*
aLaboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY 10021, USA
bDepartment of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
Received 22 December 2005; accepted 13 January 2006
Available online 3 February 2006
Abstract—The synthesis of cyclic peptides and glycopeptides by native chemical ligation using in situ derived thioesters is described.
Ó 2006 Elsevier Ltd. All rights reserved.
We have initiated a program seeking to enable the full
chemical synthesis of glycopolypeptides and ultimately
glycoproteins. We were initially drawn to this problem
by the intellectual and experimental challenges of develo-
ping a workable chemical estate that would accommo-
date the multifaceted nuances of two of the three major
classes of biomolecular building blocks. Moreover, there
were specific target structures (i.e., isoforms of prostate
specific antigen1 as well as candidate antigens around
which to generate gp120-directed HIV vaccines2), which
served to focus our efforts. A central premise of our
enterprise is that Nature’s elaborate program to achieve
post-translational glycosidation of proteins is presum-
ably driven by important advantages accruing from
the elaboration of saccharide domains in proteins.3
Therefore, access to homogeneous complex glycopoly-
peptides could be of considerable value in understanding
the biology-level consequences of protein glycosidation.
total synthesis of prostate specific antigen glycoforms,1
as well as to the carbohydrate domain of gp120 linked
to a small polypeptide domain.2
More recently we have described a new method to
generate, in situ, an active thioester containing a fully
synthetic oligosaccharide domain (Scheme 1).7 The
incipient acyl donor can then be coupled, through native
chemical ligation, to a glycopolypeptide through its
N-terminal cysteine moiety. There was thus provided a
totally synthetic polypeptide with two N-linked oligo-
saccharide domains.
In their linear form, biologically relevant peptides can
assume a baffling number of conformations. However,
it is likely that only select conformations bind to their
receptor with high affinity. Conformational constraints
are often applied in order to evaluate the structural
and dynamic properties that are important to the selec-
tivity and potency of peptides. Surely one of the most
informational modifications that can impart significant
amounts of conformational rigidity is brought about
by cyclization. Through ring formation, one has the
ability to systematically study the biologically active
conformations and perhaps infer their significance in
the linear peptides. Moreover, cyclic peptides have many
advantages over their open-chain counterparts, includ-
ing increased bioavailability, metabolic stability and
receptor selectivity.8 Accordingly, cyclic peptides have
become a common starting point in drug development.9
We had initiated our program by learning how to build
N-linked glycopeptides.4 In these early studies, we
focused on constructing fully synthetic complex oligo-
saccharides and attaching them to small peptide
domains through adaptation of the Kochetkov–Lans-
bury anomeric amination–aspartylation methodology5
to total synthesis contexts. In the next phase, we learned
how to ligate fully synthetic oligosaccharide-small pep-
tide domains using (cysteine-based) native chemical liga-
tion.6 These methodologies were then applied to the
*
Tam et al. has demonstrated the applicability of native
chemical ligation to the synthesis of cyclic peptides.10,11
Corresponding author. Tel.: +1 212 639 5501; fax: +1 212 772
8691; e-mail: danishes@mskcc.org
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.01.077

1970
J. Chen et al. / Tetrahedron Letters 47 (2006) 1969–1972
SEt
S
O
O
H2N
S
R'
disulfide
reduction
HS
H2N
HS
R'
O
N
O
N
H
H
R
R
O
R
O
StBu
intramolecular
acyl transfer
O
O
O
H
N
R'
H N
O2
R'
HO
H2N
HS
R'
N
N
O
N
H
H
H
O
HS
R
S
R
S
Scheme 1. Presumed mechanism of in situ thioester generation and subsequent ligation.
In this case, the linear peptides of interest were gener-
ated using standard Boc (tert-butyloxycarbonyl)-based
solid phase synthesis, and were obtained as discrete
peptide alkyl thioesters. Cyclization then proceeded at
pH P 6.4 in phosphate buffer containing a 3–5-fold
excess of 3-mercaptopropionic acid. However, given
the expected incompatibility of many glycosidic linkages
to Boc-based SPPS,12 we initially set out to determine if
our proposed method could be extended to provide a
simple entry into this class of compound. Thus, peptide
acid 1 was prepared using standard Fmoc-based SPPS
and then converted into the cyclization precursor 3, by
direct acylation with 2 followed by acidic removal of
the Boc and Pbf protecting groups (Scheme 2).
been of concern since both ends of the linear peptide
precursor are highly reactive and head to tail cyclization
of peptides containing less than seven residues is often
problematic.13 The cyclic nature of the peptide was
confirmed by formation of disulfide 5 upon exposure
of 3 to hydroxylamine. No trace of products arising
from hydroxylamine addition was observed by LCMS
analysis of the disulfide14 (Scheme 3).
The pH dependence on the rate and yield of the reaction
was examined at three discrete values (pH 4.5, 6.0 and
7.4). Tam has reported that, at low pH, the concentra-
tion of anionic thiolate is not high enough to compete
with hydrolysis of the thioester.15 We found this to be
the case in our system as well. The reaction was slowest
and accompanied by the highest amount of hydrolysis at
pH 4.5, although starting material was still present even
after stirring for 15 h at room temperature. At pH 6.0,
the reaction occurred much faster (completion after
7 h), however a small amount of the hydrolysis product
was still observed. At pH 7.4, the reaction proceeded so
rapidly that virtually no hydrolysis occurred.
With 3 in hand, we investigated the applicability of
various cyclization conditions. In the event, treatment
of 3 with 2-mercaptoethanesulfonic acid sodium salt
(MesNa) in phosphate buffered saline (pH 7.4) afforded
cyclic peptide 4 in an encouraging 78% yield. The
reaction itself occurred quite rapidly, with complete
consumption of the starting material within 3 h. LCMS
analysis during and after the reaction gave no indica-
tion of thioester hydrolysis. Moreover, we were quite
excited to find that there were no oligomerized products
detected in the reaction mixture. This possibility had
Covalently bound oligosaccharides can significantly
affect the biophysical properties displayed by a given
protein.16 Indeed, the clinical relevancy of the pleiotro-
H
PbfN
H
N
NH
O
O
O
H
N
H
N
BocN
H
O
+
N
H
N
H
OH
H2N
O
O
O
tBuSS
S
1
2
SEt
a,b
H
N
H2N
O
NH
O
O
H
N
H
H2N
N
O
N
H
N
N
H
H
O
O
O
tBuSS
S
3
SEt
Scheme 2. Reagents and conditions: (a) EDCI, HOBt, DMF, CH2Cl2; (b) TFA, H2O, PhOH (67% over two steps). EDCI = N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide, HOBt = 1-hydroxybenzotriazole, DMF = dimethylformamide, TFA = trifluoroacetic acid.

J. Chen et al. / Tetrahedron Letters 47 (2006) 1969–1972
1971
H
H2N
O
N
NH
O
O
H
N
H
N
H2N
O
N
H
N
H
N
H
O
O
O
tBuSS
S
3
SEt
a
H
H
N
H
N
N
HN
SH
NH
HN
S
S
NH
O
O
O
O
O
O
b
O
O
O
O
O
NH
HN
O
O
O
O
HN
O
HN
O
O
NH
NH
NH
HN
N
H
N
H
N
H
HN
NH
NH2
HN
NH
NH2
HN
NH
NH2
5
4
Scheme 3. Reagents and conditions: (a) MesNa, PBS (pH = 7.4), then TCEP (78%); (b) NH2OH, H2O. MesNa = 2-mercaptoethane sulfonic acid,
TCEP = tris(carboxyethyl)phosphine, PBS = phosphate buffered saline.
phic cytokine, erythropoietin is amplified upon glycosyla-
tion.17 Furthermore, the mannopeptimycin family of
antibiotics is an important emerging class of cyclic gly-
copeptides active against vancomycin-resistant bacte-
ria.18 Importantly, it is believed that the carbohydrate
domain of these compounds is responsible for their
biological activity.19 Glycosylation of in place cyclic
peptides is not considered a viable option for the
generation of such compounds, as the peptidic sub-
strates are often insoluble under the conditions typically
used for successful glycosidation.20 Having demon-
strated the applicability of our methodology to the syn-
thesis of cyclic peptides, we felt it appropriate to extend
the scope of the reaction to include cyclic glycopeptides.
In doing so, we minimize solubility concerns since the
carbohydrate is already attached to the peptide prior
to cyclization. Moreover, issues such as microhetero-
geneity in the glycodomain are avoided because well-
tested methodologies can be employed in the synthesis
of the linear glycopeptide.21
H2N
O
O
O
H
N
H
N
H2N
O
N
H
N
H
N
H
O
O
O
tBuSS
O
S
AcHN
O
6
SEt
HO
HO
OH
a
H
N
HN
SH
O
O
O
O
O
NH
HN
O
NH
N
H
7
NH2
O
Thus, glycopeptide 6, containing a single O-linked galac-
tosamine moiety was synthesized using a procedure simi-
lar to that of 1. The carbohydrate was incorporated
using the cassette method for glycopeptide synthesis.21
As before, smooth cyclization was observed following
treatment of the glycopeptide with MesNa in PBS
(pH 7.4), providing cyclic glycopeptide 7. Again, the
reaction proceeded rapidly with complete consumption
of the starting material in 3 h (Scheme 4).
AcHN
O
HO
HO
OH
Scheme 4. Reagents and conditions: (a) MesNa, PBS (pH = 7.4) then
TCEP (73%).
Acknowledgements
This work was supported by the NIH (CA103823 to
S.J.D.). We thank Dr. George Sukenick, Ms. Sylvi Rusli
and Ms. Hui Fang of the Sloan-Kettering Institute’s
NMR core facility for mass spectral and NMR
spectroscopic analysis (SKI core Grant No.: CA02848).
With these milestones accomplished, the program to
reach homogeneous glycopolypeptides and even com-
plex glycoproteins moves to encompass more challeng-
ing systems.

1972
J. Chen et al. / Tetrahedron Letters 47 (2006) 1969–1972
10. (a) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997, 119,
2363; (b) Tam, J. P.; Lu, Y.-A. Tetrahedron Lett. 1997, 38,
5599.
Postdoctoral fellowship support is gratefully acknowl-
edged by J.D.W. (NIH, CA62948).
11. For other examples of the use of NCL in the synthesis of
cyclic peptides, please see: (a) Shao, Y.; Kent, S. B. H.
Tetrahedron Lett. 1998, 39, 3911; (b) Meutermans, W. D.
F.; Golding, S. W.; Bourne, G. T.; Miranda, L. P.;
Dooley, M. J.; Alweood, P. F.; Smythe, M. L. J. Am.
Chem. Soc. 1999, 121, 9790.
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