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 <i>galacto</i>-like configured cyclohexenyl carbasugars form reversible covalent complexes with both α-galactosidase and β-galactosidase. In addition, we show that the β-galactosidase from <i>Aspergillus oryzae</i> reacts with three different carbasugar inhibitors, with three different second-order rate constants (<i>k</i>_inact/<i>K</i>_i), to give the same enzyme-carbasugar covalent intermediate. The surprising observation that the α-galacto-configured inhibitor covalently labels the <i>A. oryzae</i> β-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.