Solid-Phase Oligosaccharide and Glycopeptide Synthesis Using Glycosynthases Jakob F. Tolborg, †,‡ Lars Petersen, †,‡ Knud J. Jensen,* ,†,§ Christoph Mayer, ⊥ David L. Jakeman, ⊥ R. Antony J. Warren, | and Stephen G. Withers* ,⊥ Department of Chemistry, Building 201, Kemitorvet, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark, and Protein Engineering Network of Centres of Excellence, Department of Chemistry and Department of Microbiology, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada kjj@kvl.dk Received December 5, 2001 Enzymatic approaches for the preparation of oligosaccharides are interesting alternatives to traditional chemical synthesis, the main advantage being the regio- and stereoselectivity offered without the need for protecting groups. The use of solid-phase techniques offers easy workup procedures and the prospect of automatability. Here, we report the first application of glycosynthases to solid-phase oligosaccharide synthesis by use of the 51 kDa serine and glycine mutants of Agrobacterium sp. -glucosidase, Abg E358S and E358G. Acceptors were linked to PEGA resin through a backbone amide linker (BAL), and using these mutated enzymes, a galactose moiety was transferred from a donor sugar, R-D-galactosyl fluoride, with high efficiency (>90%) together with excellent recovery of material. Furthermore, it was demonstrated that a resin-bound model glycopeptide was also an acceptor for the glycosynthase. Oligosaccharides play numerous roles in biological recognition processes through, for example, their location on cell surfaces. A better understanding of these recogni- tion events will assist in the design of new drug candi- dates against a wide range of illnesses including cancer and HIV. 1 A problem in studying carbohydrate interac- tions is the limited access to well-defined oligosaccharides and purification of oligosaccharides from natural sources is tedious. While the synthesis of complex oligosaccha- rides is quite feasible, no general glycosylation protocol has yet been developed, making each structure a chal- lenging synthetic target. 2 There is a need for parallel and combinatorial synthesis of oligosaccharides for the study of carbohydrate interactions, and the field of solid-phase oligosaccharide synthesis has gained much attention over the past decade. 3 Enzymatic approaches for the preparation of oligo- saccharides are interesting alternatives to traditional chemical synthesis, the main advantage being the regio- and stereoselectivity offered without the need for pro- tecting groups. Glycosidases have been employed for this task but typically give low yields due to competing product hydrolysis. Glycosyl transferases have seen only limited use until recently due to problems with their solubility and availability, though recombinant DNA technology is quickly solving this problem. Solution-phase enzymatic glycosylations 4 using glycosyl transferases have received increasing attention, and following early work by Zehavi, 5 the groups of Wong, 6 Meldal and Palcic, 7 Thiem, 8 Ko ¨pper, 9 Lee, 10 and Norberg 11 have successfully applied these enzymes to solid-phase oligosaccharide synthesis. 12 A glycosynthase is a mutated retaining glycosidase with the active-site carboxylate nucleophile replaced by a non-nucleophilic amino acid side chain. 13 The glycosi- dase thus loses its ability to hydrolyze glycosidic bonds, but it can instead catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors (Scheme 1). The first glycosynthase was reported by Withers, Warren, and co-workers in 1998, 13 and others have since been re- ported. 14,15 Here, we present the first application of † Technical University of Denmark. ‡ The first two authors contributed equally to this work. § Present address: Department of Chemistry, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark. ⊥ Department of Chemistry, University of British Columbia. | Department of Microbiology, University of British Columbia. (1) (a) Sharon, N.; Lis, H. Sci. Am. 1993, 82-89. (b) Varki, A. Glycobiology 1993, 3, 97-130. (c) Molecular Glycobiology; Fukuda, M., Hindsgaul, O., Eds.; IRL Press: Oxford, U.K., 1994. (2) (a) Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 155- 224. (b) Schmidt, R. R. Angew. Chem. 1986, 98, 213-236. (c) Davis, B. G. J. Chem. Soc., Perkin Trans. 1 2000, 2137-2160. (3) For reviews, see: (a) Seeberger, P. H.; Haase, W.-C. Chem. Rev. 2000, 100, 4349-4393. (b) St. Hilaire, P. M.; Meldal, M. Angew. Chem., Int. Ed. Engl. 2000, 39, 1162-1179. (c) Krepinski, J. J.; Douglas, S. P. In Carbohydrates in Chemistry and Biology; Ernst, B., Hart, G. W., Sinay ¨ , P., Eds.; Wiley-VCH: Weinheim, 2000; Chapter 10. (4) Fitz, W.; Wong, C.-H. In Preparative Carbohydrate Chemistry; Hanessian, S., Ed.; Marcel Dekker: New York, 1997; Chapter 22. (5) Zehavi, U.; Sadeh, S.; Herchmann, M. Carbohydr. Res. 1984, 124, 23-34. (6) (a) Schuster, M.; Wang, P.; Paulson, J. C.; Wong, C.-H. J. Am. Chem. Soc. 1994, 116, 1135-1136. (b) Halcomb, R. L.; Huang, H.; Wong, C.-H. J. Am. Chem. Soc. 1994, 116, 11315-11322. (7) Meldal, M.; Auzanneau, F.-I.; Hindsgaul, O.; Palcic, M. J. Chem. Soc., Chem. Commun. 1994, 1849-1850. (8) Wiemann, T.; Taubken, N.; Zehavi, U.; Thiem J. Carbohydr. Res. 1994, 257, C1-C6. (9) Ko ¨pper, S. Carbohydr. Res. 1994, 265, 161-166. (10) Nishimura, S.; Matsuoka, K.; Lee, Y. C. Tetrahedron Lett. 1994, 35, 5657-5660. (11) (a) Blixt, O.; Norberg, T. J. Carbohydr. Chem. 1997, 16, 143- 154. (b) Blixt, O.; Norberg, T. J. Org. Chem. 1998, 63, 2705-2710. (c) Blixt, O.; Norberg, T. Carbohydr. Res. 1999, 319, 80-91. (12) Auge ´, C.; Narvor, C. L.; Lubineau, A. In Carbohydrates in Chemistry and Biology; Ernst, B., Hart, G. W., Sinay ¨ , P., Eds.; Wiley- VCH: Weinheim, 2000; Chapter 28. (13) Mackenzie, L. F.; Wang, Q.; Warren, R. A. J.; Withers, S. G. J. Am. Chem. Soc. 1998, 120, 5583-5584. 4143 J. Org. Chem. 2002, 67, 4143-4149 10.1021/jo0163445 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/16/2002