Chemo-enzymatic Syntheses of Bioactive Glycoconjugates Using Microbial Endoglycosidase.

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Endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase) is a unique endoglycosidase that hydrolytically cleaves the N, N′-diacetylchitobiose moiety of asparagine-linked oligosaccharides of various glycoproteins. A novel endo-β-GlcNAc-ase in the culture fluid of Mucor hiemalis isolated from soil was found to have transglycosylation activity. This endo-β-GlcNAc-ase, Endo-M, couldtransfer the intact complex-type oligosaccharide from glycopeptide to suitable acceptors with an Nacetylglucosamine (GlcNAc) residue during hydrolysis of the glycopeptide. Using Endo-M, the chemo-enzymatic synthesis of a bioactive glycopeptide by chemical synthesis of N-acetylglucosaminyl peptide and enzymatic transfer of oligosaccharide was attempted. Peptide T can block HIV infection of human T cells. We added the sialo complex-type oligosaccharide to chemically synthesized N-acetylglucosaminyl Peptide T using the transglycosylation activity of Endo-M. The glycopeptide T thus produced showed a higher degree of resistance to protease digestion. We also prepared calcitonin glycopeptide by the chemo-enzymatic method described above. Calcitonin is a calcium-regulating hormone which is widely used in therapy for hyper calcemia. This glycopeptide demonstrated sufficient physiological activity. N-Acetylglucosaminyl glutamine was also a good glycoside acceptor of Endo-M. We were able to add oligosaccharides to the glutamine residues of Substance P neuropeptide and a-mating factor of yeast Saccharomyces cerevisiae using Endo-M. The glycosylated Substance P was biologically active and stable against peptidase digestion. On the other hand, the bioactivity of the glycosylated a-mating factor was lower than that of native one, although that of N-acetylglucosaminyl a-mating factor was higher. Using the transglycosylation activity of Endo-M, we prepared alkyl compound having oligosaccharide of glycoprotein. We used it as an antigen for preparing monoclonal antibody against oligosaccharide of glycoprotein.
日本応用糖質科学会の論文

Chemo-enzymatic Syntheses of Bioactive Glycoconjugates Using Microbial Endoglycosidase.

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