pH Dependent Mechanisms of Hydrolysis of 4-Nitrophenyl β-D-Glucoside

1,2-trans-Glycosides hydrolyze through a range of mechanisms under conditions of different pH, but systematic studies are lacking. Here we report the pH-rate constant profile for the hydrolysis of 4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect (k(H₃O⁺)/k(D₃O⁺) = 0.63) in the acidic region indicates that the mechanism requires the formation of the conjugate acid of the substrate for the reaction to proceed, with heterolytic cleavage of the glycosidic C-O bond. Reactions in the pH-independent region extrapolated to zero buffer concentration show a small inverse solvent isotope effect k(H₂O)/k(D₂O) = 1.1 and a positive entropy of activation (DS^‡ = 3.07 cal mol^–1 K^–1), which is consistent with water attack through a dissociative mechanism. In the basic region, solvolysis in ¹⁸O-labelled water and H₂O/MeOH mixtures allowed detection of bimolecular hydrolysis and neighboring group participation, and to a minor degree, nucleophilic aromatic substitution. Under mildly basic conditions, a bimolecular dissociative mechanism is implicated through a solvent isotope effect of k(HO^-)/k(DO^-) = 0.5 and a strongly negative entropy of activation (DS^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of k(HO^-)/k(DO^-) = 0.6 and a weakly negative entropy of activation (DS^‡ = –5.5 cal mol^–1 K^–1) indicates that the formation of 1,2-anhydrosugar is the rate determining step.