Derivatization and purification of bisecting tyrosinamide-oligosaccharides from ovotransferrin.

M L Corradi da Silva, T Tamura, K G Rice

Index: Arch. Biochem. Biophys. 315(2) , 460-6, (1994)

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Abstract

The major N-linked oligosaccharides from ovotransferrin were purified on a large scale. The oligosaccharides were released from 5 g of the glycoprotein using N-glycosidase F and isolated from a mixed bed ion exchange column. Reducing-oligosaccharides were reacted with ammonium bicarbonate to form a reducing-end glycosylamine which coupled with the N-hydroxysuccinimidyl ester of Boc-tyrosine, resulting in tyrosinamide-oligosaccharides. Resolution of tyrosinamide-oligosaccharides on reverse-phase (RP)-HPLC necessitated the removal of Boc by treatment with trifloroacetic acid. Two major tyrosinamide-oligosaccharides were isolated from preparative RP-HPLC and were characterized by 500 MHz proton NMR and FAB-MS. These were a biantennary and triantennary oligosaccharide, each possessing a bisecting GlcNAc residue extending from the central core Man residue. Thus, ovotransferrin represents an abundant glycoprotein source from which to prepare multi-micromole quantities of bisecting complex oligosaccharides which may find utility in exploring the ligand specificity of mammalian lectins or may be used as synthons to prepare novel glycoconjugates.