Sugar-assisted glycopeptide ligation

Article abstract of DOI:10.1021/ja061165w

The chemical synthesis of glycopeptides and glycoproteins from readily available materials presents an attractive route to homogeneous products for structural and functional studies. Chemical synthesis of glycopeptides and glycoproteins based on native chemical ligation represents one of the useful methods for the synthesis of natural glycopeptide structures. Here we describe a method that allows for the synthesis of glycopeptides from cysteine-free peptides. This method utilizes a peptide thioester and a glycopeptide in which the sugar moiety is modified with a thiol handle at the C-2 position. Upon completion of the ligation reaction, the thiol handle can be reduced with H₂/metal to the acetamide moiety, furnishing the unmodified glycopeptides. Together, this sequence of reactions displays an attractive potential in glycopeptides and glycoproteins synthesis. Copyright

Full text of DOI:10.1021/ja061165w

Published on Web 04/08/2006
Sugar-Assisted Glycopeptide Ligation
Ashraf Brik, Yu-Ying Yang, Simon Ficht, and Chi-Huey Wong*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550
North Torrey Pines Road, La Jolla, California 92037
Received February 17, 2006; E-mail: wong@scripps.edu
The thiol capture strategy developed by Kemp and co-workers
has inspired the development of several ligation-based methods for
the preparation of large peptides.1 Among them, native chemical
ligation (NCL) represents one of the most useful tools for the
chemical synthesis of proteins.2 In this approach, an N-terminal
cysteine residue is required for capturing the thioester peptide,
followed by a spontaneous acyl transfer to form the amide bond at
the ligation junction. To overcome the restriction imposed by the
requirement for a cysteine residue at the ligation site, different
strategies are adopted to extend the repertoire of peptide conjugation
for non-cysteinyl ligation.3 The use of removable thiol-based
auxiliaries is an attractive approach to fulfill the same function as
the side chain of the cysteine. Peptides with NR -linked auxiliaries
based on cysteine, such as 1-phenyl-2-mercaptoethyl and 2-mer-
captobenzyl, have been investigated and successfully applied to the
synthesis of large peptides.4
The synthesis of glycopeptides and glycoproteins from readily
available materials represents an attractive route to homogeneous
products for structural and functional studies.5 Several approaches
are currently being used for the synthesis; one of particular interest
is the ligation method.6 Our interest in obtaining homogeneous
glycopeptides and glycoproteins via enzymatic and chemical
methods6d,7 prompted us to explore new ligation methods for
cysteine-free glycopeptides. Here we report that the sugar moiety
of a glycopeptide can mimic the cysteine function at the ligation
site once it is modified with a thiol handle at the C-2 position
(Figure 1). Moreover, the thiol handle can be reduced to the
acetamide moiety, which is often found in this position, furnishing
the unmodified glycopeptide (Figure 1).
Model studies were performed with small peptides to investigate
the new approach. Initially, we tested glycopeptide 1, in which the
serine residue was modified with unprotected â-thioacetamido
glucosamine (Figure 2). Reacting this peptide under NCL conditions
(6 M guanidine‚HCl, pH 8.0, 2% thiophenol, 2% benzyl mercaptan)
Figure 1. General strategy of sugar-assisted chemical synthesis of
glycopeptide.
with a model peptide thioester {AC-LYRAG-COS-(CH2)2-CONH2}
gave the ligated product after 6 h in ∼15% conversion. A second
model study was prepared in which glycopeptide 1 was extended
with a glycine residue to give glycopeptide 2 (Figure 2). Using
similar ligation conditions, we were pleased to see that the desired
product was formed rapidly and appeared as the major product in
the HPLC analysis (Figure 3). Preparative HPLC of the reaction
mixture yielded the desired glycopeptide in 76% yield (Table 1,
entry 1). Glycopeptide 3, which lacks the thiol handle, was also
prepared and tested under similar ligation conditions. However, only
a trace of ligation product was observed, indicating the crucial role
of the thiol handle in the reaction pathway and supporting the
proposed mechanism shown in Figure 1.
To investigate the effect of the C-terminal amino acid of thioester
peptide on the ligation rate, we performed initial studies by
preparing four peptides thioester bearing glycine, histidine, alanine,
and valine. The rate of the ligation reactions with glycopeptide 2
Figure 2. Glycopeptides that are used in the ligation reactions (for synthesis
of 1-3, see the Supporting Information).
is shown in Table 1.8 In the case of valine thioester (entry 4), which
reacted at a slower rate (t1/2 > 48 h) compared to glycine, histidine,
and alanine thioesters (entries 1-3), we were able to identify the
proposed thioester intermediate by the mass spectrometry frag-
mentation pattern (see Supporting Information). Initial study on the
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10.1021/ja061165w CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
The results reported here are surprising, considering the size of
the ring of the S-N acyl-transfer intermediate. It is plausible that
the sugar moiety affects the proximity of the N-terminal amine to
the thioester, which allows for the acyl transfer to occur despite
the large ring size. Efforts to determine the effects of the
configuration at the anomeric center and the N-linked sugar on the
ligation rate are currently being pursued in our laboratory.
In summary, we have demonstrated that the sugar moiety of a
glycopeptide modified with a thiol handle at the C-2 position can
assist in the ligation of a cysteine-free glycopeptide with a thioester
peptide. Upon completion, the thiol handle can be reduced to the
acetamide moiety. Together, this sequence of reactions displays
an attractive potential in glycopeptides and glycoproteins synthesis.
The scope, mechanism, and applications of the sugar-assisted
ligation in the synthesis of glycopeptides and glycoproteins are
currently under investigation.
Acknowledgment. We thank the NIH and Skaggs Institute for
Chemical Biology for financial support. S.F. is grateful for a
postdoctoral fellowship provided by the Deutsche Akademische
Austauschdienst (DAAD).
Figure 3. Analytical HPLC of the ligation reaction (Table 1, entry 1). (A)
Reaction at 0 h: peak a, glycopeptide 2; peak b, impurity from thioester
peptide substrate; peak c, thioester peptide substrate. (B) Ligation reaction
after 8 h: peak a, impurity from tris(2-carboxethyl)phosphine hydrochloride;
peak b, unreacted glycopeptide 2; peak c, thioester substrate hydrolysis
product; peak d, the desired product with the expected mass of 1147.2 Da
(see inset, MALDI-TOF/MS).
Supporting Information Available: Experimental procedures and
characterization data for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
Table 1. Effect of the C-Terminal Amino Acid of Peptide Thioester
on the Ligation Rate
(1) (a) Kemp, D. S. Biopolymers 1981, 20, 1793-1804. (b) Kemp D. S.;
Carey, R. I. J. Org. Chem. 1993, 58, 2216-2222.
(2) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994,
266, 776-779.
(3) (a) Beligere, G. S.; Dawson, P. E. J. Am. Chem. Soc. 1999, 121, 6332-
6333. (b) Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526-
533. (c) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2,
2141-2143. (d) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett.
2000, 2, 1939-1941.
(4) For selected examples, see: (a) Canne, L. E.; Bark, S. J.; Kent, S. B. H.
J. Am. Chem. Soc. 1996, 118, 5891-5896. (b) Low, D. W.; Hill, M. G.;
Carrasco, M. R.; Kent, S. B. H.; Botti, P. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 6554-6559. (c) Offer, J.; Boddy, C.; Dawson, P. E. J. Am.
Chem. Soc. 2002, 124, 4642-4646. (d) Macmillan, D.; Anderson, D. W.
Org. Lett. 2004, 6, 4659-4662. (e) Lu, Y.-A.; Tam, J. P. Org. Lett. 2005,
7, 5003-5006.
(5) For related reviews, see: (a) Seitz, O. ChemBioChem 2000, 1, 214-246.
(b) Grogan, M. J.; Pratt, M. R.; Marcaurelle, L. A.; Bertozzi, C. R. Annu.
ReV. Biochem. 2002, 71, 593-634. (c) Davis, B. G. Chem. ReV. 2002,
102, 579-602. (d) Liu, L.; Bennett, C. S.; Wong, C.-H. Chem. Commun.
2006, 1, 21-33.
ligation
junction
mass obsd,a
calcd (Da)
entry
-AA-
t1 2 (h)
/
1
2
4
4
Gly
His
Ala
Val
Gly-Gly
His-Gly
Ala-Gly
Val-Gly
∼5
∼5
1146.4 ( 0.2, 1146.52
1126.5 ( 0.2, 1226.55
1160.4 ( 0.2, 1160.53
1188.5 ( 0.2, 1188.56
∼10
>48
a
Characterized by ESI-MS.
(6) (a) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J. A.;
Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684-11689. (b) Warren,
J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am. Chem. Soc.
2004, 126, 6576-6578. (c) Macmillan, D.; Bertozzi, C. R. Angew. Chem.,
Int. Ed. 2004, 43, 1355-1359. (d) Tolbert, T. J.; Franke, D.; Wong, C.-
H. Bioorg. Med. Chem. 2005, 13, 909-915. (e) Hackenberger, C. P. R.;
Friel, C. T.; Radford, S. E.; Imperiali, B. J. Am. Chem. Soc. 2005, 127,
12882-12889.
generality of our approach showed that the N-terminal amino acid
is not limited only to a glycine residue. We have found that
extending peptide 1 with alanine and serine gave results similar to
those obtained in the glycine case.
By applying the desulfurization reaction developed by Dawson
and co-workers,3b we reduced the acetamido thiol handle in the
ligation product (Table 1, entry1) to the corresponding acetamide
moiety. The reaction was completed within 1 h using Pd/Al2O3
under hydrogen to give the unmodified glycopeptide in 80% isolated
yield. A mass decrease of 32 Da from the starting material was
observed, as expected for the loss of one sulfur atom (see Supporting
Information).
(7) For examples, see: (a) Liu, H.; Wang, L.; Brock, A.; Wong C.-H.; Schultz,
P. G. J. Am. Chem. Soc. 2003, 125, 1702-1703. (b) Zhang, Z.;
Gildersleeve, J.; Yang, Y.-Y.; Xu, R.; Loo, J. A.; Uryu, S.; Wong, C.-H.;
Schultz, P. G. Science 2004, 303, 371-373. (c) Xu, R.; Hanson, S. R.;
Zhang, Z.; Yang, Y.-Y.; Schultz, P. G.; Wong, C.-H. J. Am. Chem. Soc.
2004, 126, 15654-15655.
(8) We prepared the peptide R-phenyl thioester and used it directly in the
ligation mixture, which was superior in terms of rate acceleration (t1/2
2 h). However, we preferred preparing this peptide thioester in situ.
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