Mechanism-Based Allylic Carbasugar Chlorides That Form Covalent Intermediates with α- and β-Galactosidases

Glycoside hydrolases have been implicated in a wide range of human conditions including lysosomal storage diseases. Consequently, many researchers have directed their efforts towards identifying new classes of glycoside hydrolase inhibitors, both synthetic and from natural sources. A large percentage of such inhibitors are reversible competitive inhibitors that bind in the active site often due to them possessing structural features, often a protonatable basic nitrogen atom, that mimic the enzymatic transition state. We report that mechanism-based small molecule galacto-like configured cyclohexenyl carbasugars form reversible covalent complexes with both alpha-galactosidase and beta-galactosidase. In addition, we show that the beta-galactosidase from Aspergillus oryzae reacts with three different carbasugar inhibitors, with three different second-order rate constants (kinact/Ki), to give the same enzyme-carbasugar covalent intermediate. The surprising observation that the alpha-galacto-configured inhibitor covalently labels the A. oryzae beta-galactosidase highlights the catalytic versatility of glycoside hydrolases. We expect that cyclohexenyl covalent inhibitors will become an important class of compounds in the chemical biologist's tool box.