Highly efficient endoglycosidase-catalyzed synthesis of glycopeptides using oligosaccharide oxazolines as donor substrates

Article abstract of DOI:10.1021/ja051715a

A highly efficient chemoenzymatic synthesis of N-glycopeptides was achieved. It was found that using synthetic oligosaccharide oxazolines, the mimics of the presumed oxazolinium ion intermediate formed in a retaining mechanism of substrate-assisted cataly

Full text of DOI:10.1021/ja051715a

Published on Web 06/15/2005
Highly Efficient Endoglycosidase-Catalyzed Synthesis of Glycopeptides Using
Oligosaccharide Oxazolines as Donor Substrates
Bing Li, Ying Zeng, Steven Hauser, Haijing Song, and Lai-Xi Wang*
Institute of Human Virology, UniVersity of Maryland Biotechnology Institute, UniVersity of Maryland,
725 West Lombard Street, Baltimore, Maryland 21201
Received March 17, 2005; E-mail: wangx@umbi.umd.edu
Scheme 1. Chemical Synthesis of the Di- and Tetrasaccharide
Oxazolines
Glycosylation is a common post-translational modification of
proteins in eukaryotes.1 The oligosaccharide components of gly-
coproteins affect a wide range of protein functions and are involved
in many important cellular recognition processes.2 However, natural
glycoproteins typically exist as a mixture of glycoforms differing
in oligosaccharide structures, and pure individual glycoforms are
difficult to isolate for detailed structural and functional studies.3
Thus, a variety of synthetic methods have been explored to generate
homogeneous glycopeptides and glycoproteins.4 Among others, the
chemoenzymatic approach using the endo-â-N-acetylglucosamini-
dase (ENGase)-catalyzed transglycosylation seems particularly
promising.5 ENGases are a class of endoglycosidases that hydrolyze
the â-1,4-glycosidic bond in the core N,N′-diacetylchitobiose moiety
of N-glycoproteins to release the N-glycans. However, some
ENGases, such as Endo-A from Arthrobacter protophormiae6 and
Endo-M from Mucor hiemalis,7 possess transglycosylation activity
and are able to transfer the releasing N-glycan to a GlcNAc-peptide
acceptor to form a new glycopeptide.5 In contrast to common
glycosyltransferases and exoglycosidases that transfer only monosac-
charides, Endo-A and Endo-M can transfer a large intact oligosac-
charide to a GlcNAc-peptide acceptor in a single step to form a
new glycopeptide, thus allowing a highly convergent glycopeptide
synthesis without the need of protecting groups. A number of large
N-glycopeptides were synthesized by the chemoenzymatic method
for structural and functional studies.8-11 Nevertheless, the chemoen-
zymatic method suffers with a low transglycosylation yield (gener-
ally 5-20%), the product hydrolysis, and the limitations of using
only natural N-glycans as the donor substrates. To solve these
problems, we report in this paper the use of synthetic oligosaccha-
ride oxazolines, the mimics of the presumed oxazolinium ion
intermediate formed in a retaining mechanism, as donor substrates
for glycopeptide synthesis, which not only broadened the substrate
availability but also led to a high-yield synthesis of large N-
glycopeptides.
The method was based on the assumption that the ENGase-
catalyzed reaction proceeds via a mechanism of the substrate-
assisted catalysis involving an oxazolinium ion intermediate, as
demonstrated for some chitinases12 and N-acetyl-â-hexosamini-
dases.13 Although a detailed mechanism of ENGase-catalyzed
transglycosylation is yet to be characterized, Fujita and co-workers
recently reported that a disaccharide oxazoline of Manâ1,4GlcNAc
could serve as a substrate for ENGase-catalyzed transglycosyla-
tion.14 This observation suggested that the Endo-A- and Endo-M-
catalyzed transglycosylation might indeed proceed via an oxazo-
linium ion intermediate. To test whether oligosaccharide oxazolines
would be kinetically more favorable substrates for an efficient
N-glycopeptide synthesis than natural N-glycans, we synthesized
the di- and tetrasaccharide oxazolines corresponding to the core of
N-glycans (Scheme 1). The synthesis of the Manâ1,4GlcN disac-
charide core was achieved through stereocontrolled â-glycosylation
of intermediates 1 and 2, followed by selective inversion of the
Glc C-2 configuration to give 4. Compound 4 was changed to 6
via protecting group manipulations and was glycosidated with the
mannosyl imidate to give the tetrasaccharide 7, which was then
converted to the fully acetylated derivative 8. Finally, treatment of
8 with TMS-Br/BF3 Et2O for oxazoline formation,15 followed by
de-O-acetylation gave the desired tetrasaccharide oxazoline 9. The
disaccharide oxazoline 11 was synthesized from 4 by similar
protecting group manipulations and oxazoline formation (Scheme
1).
To examine the synthetic oligosaccharide oxazolines as donor
substrates for constructing N-glycopeptides, we synthesized two
typical GlcNAc-peptides, a GlcNAc-heptapeptide 12 derived from
HIV-1 gp120 and a 34-mer peptide GlcNAc-C34 derived from
HIV-1 gp41 to serve as the glycosyl acceptors.9,11 It was found
that the Endo-A-catalyzed reaction between the oxazoline 11 and
the GlcNAc-heptapeptide 12 (3:1) in phosphate buffer (pH 6.5)
proceeded smoothly to form the glycopeptide 13, which was isolated
in 82% yield (Scheme 2). Endo-M was also effective in catalyzing
the reaction to give the glycopeptide 13 in 78% yield. The newly
formed glycosidic bond was unambiguously determined to be the
expected GlcNAcâ1,4GlcNAc-Asn linkage by detailed NMR
(TOCSY and NOESY) analysis of 13 (see Supporting Information).
A doublet at δ 4.65 with a large coupling constant (J ) 8.5 Hz)
for H-1′ indicated a â-glycosidic bond, and an apparent NOE
correlation between H-1′ and the H-4 of the Asn-linked GlcNAc
indicated that the newly formed glycosidic linkage was a â-1,4-
type. The results confirm that the ENGase-catalyzed transglyco-
sylation using oligosaccharide oxazoline as the donor substrate
proceeds in both a stereo- and regiospecific manner to afford the
desired glycopeptide, as previously observed when natural N-
glycans were used as the donor substrates.9-11,16
9
9692
J. AM. CHEM. SOC. 2005, 127, 9692-9693
10.1021/ja051715a CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. ENGase-Catalyzed Synthesis of a HIV-1 gp120
Fragment Carrying the Core Trisaccharide
enables efficient overproduction of the acceptor GlcNAc-protein
in E. coli,17 the high-yield transglycosylation may be also very
useful for total glycoprotein synthesis and remodeling.
Acknowledgment. We thank Prof. K. Takegawa for kindly
providing the pGEX-2T/Endo-A plasmid, and Prof. K. Yamamoto
for kindly providing Endo-M. The work was supported by the
National Institutes of Health (AI054354 and AI051235).
Supporting Information Available: Experimental procedures for
the synthesis of oligosaccharide oxazolines; ESI-MS spectra of gly-
copeptides 13, 14, and 15; and detailed NMR analysis (TOCSY and
NOESY) of glycopeptide 13. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Apweiler, R.; Hermjakob, H.; Sharon, N. Biochim. Biophys. Acta 1999,
1473, 4-8.
Scheme 3. ENGase-Catalyzed Synthesis of HIV-1 gp41
Fragments Carrying the Core Tri- and Pentasaccharides
(2) (a) Varki, A. Glycobiology 1993, 3, 97-130. (b) Dwek, R. A. Chem.
ReV. 1996, 96, 683-720. (c) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson,
I. A.; Dwek, R. A. Science 2001, 291, 2370-2376.
(3) Fukuda, M.; Hindsgaul, O. Molecular and Cellular Glycobiology; Oxford
University Press: New York, 2000.
(4) (a) Arsequell, G.; Valencia, G. Tetrahedron: Asymmetry 1999, 10, 3045-
3094. (b) Sears, P.; Wong, C. H. Science 2001, 291, 2344-2350. (c)
Grogan, M. J.; Pratt, M. R.; Marcaurelle, L. A.; Bertozzi, C. R. Annu.
ReV. Biochem. 2002, 71, 593-634. (d) Davis, B. G. Chem. ReV. 2002,
102, 579-601. (e) Geng, X.; Dudkin, V. Y.; Mandal, M.; Danishefsky,
S. J. Angew. Chem., Int. Ed. 2004, 43, 2562-2565. (f) Warren, J. D.;
Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am. Chem. Soc. 2004,
126, 6576-6578.
(5) (a) Yamamoto, K. J. Biosci. Bioeng. 2001, 92, 493-501. (b) Wang, L.
X.; Singh, S.; Ni, J. In Synthesis of Carbohydrates through Biotechnology;
Wang, P. G., Ichikawa, Y., Eds.; ACS Symposium Series 873; American
Chemical Society: Washington, DC, 2004; pp 73-92.
(6) Takegawa, K.; Yamaguchi, S.; Kondo, A.; Iwamoto, H.; Nakoshi, M.;
Kato, I.; Iwahara, S. Biochem. Int. 1991, 24, 849-855.
(7) Yamamoto, K.; Kadowaki, S.; Watanabe, J.; Kumagai, H. Biochem.
Biophys. Res. Commun. 1994, 203, 244-252.
(8) (a) Takegawa, K.; Tabuchi, M.; Yamaguchi, S.; Kondo, A.; Kato, I.;
Iwahara, S. J. Biol. Chem. 1995, 270, 3094-3099. (b) Haneda, K.; Inazu,
T.; Yamamoto, K.; Kumagai, H.; Nakahara, Y.; Kobata, A. Carbohydr.
Res. 1996, 292, 61-70. (c) Wang, L. X.; Tang, M.; Suzuki, T.; Kitajima,
K.; Inoue, Y.; Inoue, S.; Fan, J. Q.; Lee, Y. C. J. Am. Chem. Soc. 1997,
119, 11137-11146. (d) Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto,
I.; Kawakami, T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am. Chem.
Soc. 1999, 121, 284-290. (e) O’Connor, S. E.; Pohlmann, J.; Imperiali,
B.; Saskiawan, I.; Yamamoto, K. J. Am. Chem. Soc. 2001, 123, 6187-
6188.
We next tested the Endo-A-catalyzed transglycosylation of the
di- and tetrasaccharide oxazolines with the large acceptor, GlcNAc-
C34.11 It was found that the oligosaccharides could also be
effectively transferred to the large GlcNAc-C34 by Endo-A to form
the glycopeptides 14 (73%) and 15 (75%), respectively (Scheme
3). The glycopeptides were again characterized by ESI-MS and
NMR analysis. Further structural characterization of glycopeptide
15 was performed by Pronase digestion that yielded a single Asn-
linked oligosaccharide, which was identical to the authentic Asn-
(9) Singh, S.; Ni, J.; Wang, L. X. Bioorg. Med. Chem. Lett. 2003, 13, 327-
330.
(10) Li, H.; Singh, S.; Zeng, Y.; Song, H.; Wang, L. X. Bioorg. Med. Chem.
Lett. 2005, 15, 895-898.
(11) Wang, L. X.; Song, H.; Liu, S.; Lu, H.; Jiang, S.; Ni, J.; Li, H.
ChemBioChem 2005, 6, 1068-1074.
(12) (a) Brameld, K. A.; Shrader, W. D.; Imperiali, B.; Goddard, W. A., III.
J. Mol. Biol. 1998, 280, 913-923. (b) Terwisscha van Scheltinga, A. C.;
Armand, S.; Kalk, K. H.; Isogai, A.; Henrissat, B.; Dijkstra, B. W.
Biochemistry 1995, 34, 15619-15623. (c) Tews, I.; Terwisscha van
Scheltinga, A. C.; Perrakis, A.; Wilson, K. S.; Dijkstra, B. W. J. Am.
Chem. Soc. 1997, 119, 7954-7959.
(13) (a) Mark, B. L.; Vocadlo, D. J.; Knapp, S.; Triggs-Raine, B. L.; Withers,
S. G.; James, M. N. J. Biol. Chem. 2001, 276, 10330-10337. (b) Williams,
S. J.; Mark, B. L.; Vocadlo, D. J.; James, M. N.; Withers, S. G. J. Biol.
Chem. 2002, 277, 40055-40065.
(14) Fujita, M.; Shoda, S.; Haneda, K.; Inazu, T.; Takegawa, K.; Yamamoto,
K. Biochim. Biophys. Acta 2001, 1528, 9-14.
(15) Colon, M.; Staveski, M. M.; Davis, J. T. Tetrahedron Lett. 1991, 32,
4447-4450.
1
linked core pentasaccharide Man3GlcNAc2Asn by H NMR, ESI-
MS, and Dionex HPAEC analysis. It was also observed that while
the Manâ1,4GlcNAc-oxazoline and Man3GlcNAc-oxazoline acted
as an efficient substrate for transglycosylation, the resulting
glycopeptide ManGlcNAc2-C34 (14) was resistant to Endo-A
hydrolysis, and the glycopeptide Man3GlcNAc2-C34 (15) was
hydrolyzed only slowly by Endo-A (data not shown). This suggests
that the oligosaccharide oxazolines are much more active substrates
than the ground state N-glycopeptides, thus being kinetically
favorable for product accumulation.
In conclusion, a highly efficient chemoenzymatic synthesis of
N-glycopeptides was achieved. The use of synthetic oligosaccharide
oxazolines as the donor substrates for the ENGase-catalyzed
transglycosylation not only expanded the substrate availability but
also resulted a substantial enhancement of the synthetic efficiency,
allowing a high-yield synthesis of large N-glycopeptides. When
combined with the novel in vivo suppressor tRNA technology that
(16) Fan, J. Q.; Takegawa, K.; Iwahara, S.; Kondo, A.; Kato, I.; Abeygunawar-
dana, C.; Lee, Y. C. J. Biol. Chem. 1995, 270, 17723-17729.
(17) (a) 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. (b) Xu, R.;
Hanson, S. R.; Zhang, Z.; Yang, Y. Y.; Schultz, P. G.; Wong, C. H. J.
Am. Chem. Soc. 2004, 126, 15654-15655.
JA051715A
9
J. AM. CHEM. SOC. VOL. 127, NO. 27, 2005 9693