Unimolecular, bimolecular and intramolecular hydrolysis mechanisms of 4-nitrophenyl β-D-glucopyranoside

1,2- trans -Glycosides hydrolyze through different mechanisms at different pH values, 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 of k (H 3 O + )/ k (D 3 O) + = 0.65 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 exhibit general catalysis with a single proton in flight, a normal solvent isotope effect of k H / k D = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect k (H 2 O)/ k (D 2 O) = 1.1, consistent with water attack through a dissociative mechanism. In the basic region, solvolysis in 18 O-labelled water and H 2 O/MeOH mixtures allowed detection of bimolecular hydrolysis and neighboring group participation, with a minor contribution of nucleophilic aromatic substitution. Under mildly basic conditions, a bimolecular concerted mechanism is implicated through an inverse solvent isotope effect of k (HO – )/ k (DO – ) = 0.5 and a strongly negative entropy of activation (D S ‡ = –13.6 cal mol –1 K –1 ). Finally, at high pH, an inverse solvent isotope effect of k (HO – )/ k (DO – ) = 0.6 indicates that the formation of 1,2-anhydrosugar is the rate determining step.