Abstract
This chapter describes a rapid and efficient approach for the solid-phase synthesis of N-linked glycopeptides that utilizes on-resin glycosylamine coupling to produce N-linked glycosylation sites. In this method, the full-length nonglycosylated peptide is first synthesized on a solid-phase support using standard Fmoc chemistry. The glycosylation site is then introduced through an orthogonally protected 2-phenylisopropyl (PhiPr) aspartic acid (Asp) residue. After selective deprotection of the Asp residue, a high mannose type oligosaccharide glycosylamine is coupled on-resin to the free Asp side chain to form a N-glycosidic bond. Subsequent protecting group removal and peptide cleavage from the resin ultimately yields the desired glycopeptide. This strategy provides an effective route for conducting glycosylation reactions on a solid-phase support, simplifies the process of glycopeptide purification relative to solution-phase glycopeptide synthesis strategies, and enables the recovery of potentially valuable, un-reacted oligosaccharides. This approach has been applied to the solid-phase synthesis of the N-linked high mannose glycosylated form of peptide T (ASTTTNYT), a fragment of the HIV-1 envelope glycoprotein gp120.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dove, A. (2001) The bittersweet promise of glycobiology. Nature Biotech. 19, 913–917.
Scanlan, C. N., Offer, J., Zitzmann, N., and Dwek, R. A. (2007) Exploiting the defensive sugars of HIV-1 for drug and vaccine design. Nature 446, 1038–1045.
Lee, J. C., Greenberg, W. A., and Wong, C. H. (2006) Programmable reactivity-based one-pot oligosaccharide synthesis. Nat. Protoc. 1, 3143–3152.
Lis, H., and Sharon, N. (1978) Soybean agglutinin--a plant glycoprotein. Structure of the carboxydrate unit. J. Biol. Chem. 253, 3468–3476.
Dudkin, V. Y., Miller, J. S., and Danishefsky, S. J. (2004) Chemical synthesis of normal and transformed PSA glycopeptides. J. Am. Chem. Soc. 126, 736–738.
Demchenko, A. V. (2005) Strategic approach to the chemical synthesis of oligosaccharides. Lett. Org. Chem. 2, 580–589.
Chen, R., Pawlicki, M. A., Hamilton, B. S., and Tolbert, T. J. (2008) Enzyme-catalyzed synthesis of a hybrid N-linked oligosaccharide using N-acetylglucosaminyltransferase I. Adv. Synth. Catal. 350, 1689–1695.
Seeberger, P. H. (2008) Automated oligosaccharide synthesis. Chem. Soc. Rev. 37, 19–28.
Kan, C., Trzupek, J. D., Wu, B., Wan, Q., Chen, G., Tan, Z., Yuan, Y., and Danishefsky, S. J. (2009) Toward homogeneous erythropoietin: Chemical synthesis of the Ala(1)-Gly(28) glycopeptide domain by “alanine” ligation. J. Am. Chem. Soc. 131, 5438–5443.
Tan, Z., Shang, S., Halkina, T., Yuan, Y., and Danishefsky, S. J. (2009) Toward Homogeneous Erythropoietin: Non-NCL-Based Chemical Synthesis of the Gln(78)-Arg(166) Glycopeptide Domain. J. Am. Chem. Soc. 131, 5424–5431.
Chen, R., and Tolbert, T. J. (2010) Study of On-resin Convergent Synthesis of N-linked Glycopeptides Containing a Large High Mannose Oligosaccharide. J. Am. Chem. Soc. 132, 3211–3216.
Hojo, H., and Nakahara, Y. (2007) Recent progress in the field of glycopeptide synthesis. Biopolymers 88, 308–324.
Cohen-Anisfeld, S. T., and Lansbury, P. T. (1993) A practical, convergent method for glycopeptide synthesis. J. Am. Chem. Soc. 115, 10531–10537.
Mandal, M., Dudkin, V. Y., Geng, X. D., and Danishefsky, S. (2004) In pursuit of carbohydrate-based HIV vaccines, Part 1: The total synthesis of hybrid-type gp120 fragments. Angew. Chem. Int. Ed. Engl. 43, 2557–2561.
Yue, C. W., Thierry, J., and Potier, P. (1993) 2-Phenyl isopropyl esters as carboxyl terminus protecting groups in the fast synthesis of peptide-fragments. Tetrahedron Lett. 34, 323–326.
Kates, S. A., Delatorre, B. G., Eritja, R., and Albericio, F. (1994) Solid-phase N-glycopeptide synthesis using allyl side-chain protected Fmoc-amino acids. Tetrahedron Lett. 35, 1033–1034.
Offer, J., Quibell, M., and Johnson, T. (1996) On-resin solid-phase synthesis of asparagine N-linked glycopeptides: Use of N-(2-acetoxy-4-methoxybenzyl) (AcHmb) aspartyl amide-bond protection to prevent unwanted aspartimide formation. J. Chem. Soc., Perkin Trans. 1, 175–182.
Packman, L. C. (1995) N-2 Hydroxy-4-Methoxybenzyl (Hmb) Backbone protection strategy prevents double aspartimide formation in a difficult peptide sequence. Tetrahedron Lett. 36, 7523–7526.
Pert, C. B., Hill, J. M., Ruff, M. R., Berman, R. M., Robey, W. G., Arthur, L. O., Ruscetti, F. W., and Farrar, W. L. (1986) Octapeptides deduced from the neuropeptide receptor-like pattern of antigen T4 in brain potently inhibit human immunodeficiency virus receptor binding and T-cell infectivity. Proc. Natl. Acad. Sci. USA 83, 9254–9258.
D’Ursi, A., Caliendo, G., Perissutti, E., Santagada, V., Severino, B., Albrizio, S., Bifulco, G., Spisani, S., and Temussi, P. A. (2007) Conformation-activity relationship of peptide T and new pseudocyclic hexapeptide analogs. J. Pept. Sci. 13, 413–421.
Kaiser, E., Colescot, RL, Bossinge,CD, and Cook, P. I. (1970) Color test for detection of free terminal amino groups in solid-phase synthesis of peptides. Anal. Biochem. 34, 595–598.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356.
Likhosherstov, L. M., Novikova, O. S., Derevitskaja, V. A., and Kochetkov, N. K. (1986) A new simple synthesis of amino sugar beta-d-glycosylamines, Carbohydr. Res. 146, C1–C5.
Kay, C., Lorthioir, O. E., Parr, N. J., Congreve, M., McKeown, S. C., Scicinski, J. J., and Ley, S. V. (2000) Solid-phase reaction monitoring – Chemical derivatization and off-bead analysis, Biotechnol. Bioeng. 71, 110–118.
Acknowledgment
This work was supported by Award Number R01GM090080 from the National Institute of General Medical Sciences.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Chen, R., Tolbert, T.J. (2011). On-Resin Convergent Synthesis of a Glycopeptide from HIV gp120 Containing a High Mannose Type N-Linked Oligosaccharide. In: Mark, S. (eds) Bioconjugation Protocols. Methods in Molecular Biology, vol 751. Humana Press. https://doi.org/10.1007/978-1-61779-151-2_22
Download citation
DOI: https://doi.org/10.1007/978-1-61779-151-2_22
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-150-5
Online ISBN: 978-1-61779-151-2
eBook Packages: Springer Protocols