Allylic Carbocyclic Inhibitors Covalently Bind Glycoside Hydrolases

[Image: see text] Allylic cyclitols were investigated as covalent inhibitors of glycoside hydrolases by chemical, enzymatic, proteomic, and computational methods. This approach was inspired by the C(7) cyclitol natural product streptol glucoside, which features a potential carbohydrate leaving group in the 4-position (carbohydrate numbering). To test this hypothesis, carbocyclic inhibitors with leaving groups in the 4- and 6- positions were prepared. The results of enzyme kinetics analyses demonstrated that dinitrophenyl ethers covalently inhibit α-glucosidases of the GH13 family without reactivation. The labeled enzyme was studied by proteomics, and the active site residue Asp214 was identified as modified. Additionally, computational studies, including enzyme homology modeling and density functional theory (DFT) calculations, further delineate the electronic and structural requirements for activity. This study demonstrates that previously unexplored 4- and 6-positions can be exploited for successful inhibitor design.