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 k_H/k_D = 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.