Glucosyl epi-cyclophellitol allows mechanism-based inactivation and structural analysis of human pancreatic alpha-amylase

As part of a search for selective, mechanism-based covalent inhibitors of human pancreatic alpha-amylase we describe the chemoenzymatic synthesis of the disaccharide analog alpha-glucosyl epi-cyclophellitol, demonstrate its stoichiometric reaction with human pancreatic alpha-amylase and evaluate the time dependence of its inhibition. X-ray crystallographic analysis of the covalent derivative so formed confirms its reaction at the active site with formation of a covalent bond to the catalytic nucleophile D197. The structure illuminates the interactions with the active site and confirms OH4' on the nonreducing end sugar as a good site for attachment of fluorescent tags in generating probes for localization and quantitation of amylase in vivo.