COMBINED CHEMICAL AND ENZYMATIC-SYNTHESIS OF A C-GLYCOPEPTIDE AND ITSINHIBITORY ACTIVITY TOWARD GLYCOAMIDASES

A novel chemoenzymatic approach to synthesizing high-mannose-type N-gl ycopeptide and its C-linked glycopeptide analog is described. The synt hesis consists of two steps: a chemical synthesis of GlcNAc-containing peptides and an enzymatic glycosyl transfer of Man(9)GlcNAc to the te rminal GlcNAc in the peptides in an aqueous medium containing organic solvents. The essential enzyme used is an endo-beta-N-acetyl-glucosami nidase 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 nat ural high-mannose-type N-glycopeptide 2 can be rapidly hydrolyzed by g lycoamidases [commonly called N-glycanase or, systematically, eptide-N -4-(N-acetyl-beta-D-glucosaminylasparagine amidase], the synthetic C-g lycopeptide 1 with an insertion of a methylene group at the crucial as paragine-GlcNAc linkage is resistant to the enzyme-catalyzed hydrolysi s 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, bro ad spectrum inhibitor for glycoamidases, which is expected to be a use ful tool in the study of the mechanism and biological functions of the enzymes.