19] Glycosidase inhibitors in study of glycoconjugates

Abstract

This chapter discusses the preparation of various oligosaccharide substrates with processing inhibitors in cell culture. The chapter also discusses preparation of radiolabeled Glc3Man7–9GlcNAc and Glc3Man9GlcNAc intermediates. Because castanospermine is a more effective inhibitor than other glucosidase inhibitors, it is a good choice for preparing a radiolabeled Glc3-Man7–9GlcNAc intermediate to use as an enzyme substrate. Deoxymannojirimycin, a mannosidase, is also added to the cell culture along with castanospermine to prevent removal of any mannose residues. The preparation of [2-3H]mannose-labeled Man9GlcNAc, Man5GlcNAc, and GlcNAc-Man5GlcNAc intermediates are also discussed. The oligosaccharide, [3H]Man9GIcNAc is prepared from influenza virus-infected Madin–Darby canine kidney (MDCK) cells. Among the various processing inhibitors, deoxynojirimycin, an inhibitor of glucosidase I, and deoxymannojirimycin, and inhibitor of mannosidase I, have been successfully used as affinity ligands to purify glucosidase I and mannosidase I respectively.