Combined chemical and enzymatic synthesis of a C-glycopeptide and its inhibitory activity toward glycoamidases

A novel chemoenzymatic approach to synthesizing high-mannose-type N-glycopeptide and its C-linked glycopeptide analog is described. The synthesis consists of two steps: a chemical synthesis of GlcNAc-containing peptides and an enzymatic glycosyl transfer of Man(9)GlcNAc to the terminal GlcNAc in the peptides in an aqueous medium containing organic solvents. The essential enzyme used is an endo-beta-N-acetyl-glucosaminidase from Arthrobacter protophormiae (Endo-A). This approach should be generally applicable to the synthesis of both natural and designed high-mannose-type glycopeptides. It has been found that, while the natural high-mannose-type N-glycopeptide 2 can be rapidly hydrolyzed by glycoamidases [commonly called N-glycanase or, systematically, peptide-N-4-(N-acetyl-beta-D-glucosaminylasparagine amidase], the synthetic C-glycopeptide 1 with an insertion of a methylene group at the crucial asparagine-GlcNAc linkage is resistant to the enzyme-catalyzed hydrolysis and shows apparent inhibitory activity toward glycoamidases of plant, bacterial, and animal origin, with the K-i values ranging from 1 to 160 mu M for different enzymes, The C-glycopeptide 1 is the first, broad spectrum inhibitor for glycoamidases, which is expected to be a useful tool in the study of the mechanism and biological functions of the enzymes.