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(H3O+)/k(D3O+) = 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 the 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 kH/kD = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect of k(H2O)/k(D2O) = 1.1, consistent with water attack through a dissociative mechanism. In the basic region, solvolysis in 18O-labeled water and H2O/MeOH mixtures allowed the 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 (ΔS‡ = -13.6 cal mol-1 K-1). Finally, at high pH, an inverse solvent isotope effect of k(HO-)/k(DO-) = 0.5 indicates that the formation of 1,2-anhydrosugar is the rate-determining step.