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

1,2-<i>trans</i>-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<i> </i>4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect of <i>k</i>(H₃O⁺)/<i>k</i>(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 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 <i>k</i>_H/<i>k</i>_D = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect <i>k</i>(H₂O)/<i>k</i>(D₂O) = 1.1, 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, 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 <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.5 and a strongly negative entropy of activation (D<i>S</i>^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.6 indicates that the formation of 1,2-anhydrosugar is the rate determining step.<br>

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

1,2-<i>trans</i>-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<i> </i>4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect of <i>k</i>(H₃O⁺)/<i>k</i>(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 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 <i>k</i>_H/<i>k</i>_D = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect <i>k</i>(H₂O)/<i>k</i>(D₂O) = 1.1, 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, 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 <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.5 and a strongly negative entropy of activation (D<i>S</i>^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.6 indicates that the formation of 1,2-anhydrosugar is the rate determining step.<br>

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

1,2-<i>trans</i>-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<i> </i>4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect of <i>k</i>(H₃O⁺)/<i>k</i>(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 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 <i>k</i>_H/<i>k</i>_D = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect <i>k</i>(H₂O)/<i>k</i>(D₂O) = 1.1, 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, 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 <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.5 and a strongly negative entropy of activation (D<i>S</i>^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.6 indicates that the formation of 1,2-anhydrosugar is the rate determining step.<br>

pH-Rate Profile for Hydrolysis of 4-Nitrophenyl β-D-Glucopyranoside: Unimolecular, Bimolecular and Intramolecular Cleavage Mechanisms

1,2-<i>trans</i>-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<i> </i>4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect of <i>k</i>(H₃O⁺)/<i>k</i>(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 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 <i>k</i>_H/<i>k</i>_D = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect <i>k</i>(H₂O)/<i>k</i>(D₂O) = 1.1, 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, 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 <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.5 and a strongly negative entropy of activation (D<i>S</i>^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.6 indicates that the formation of 1,2-anhydrosugar is the rate determining step.<br>

pH-Rate Profile for Hydrolysis of 4-Nitrophenyl β-D-Glucopyranoside: Unimolecular, Bimolecular and Intramolecular Cleavage Mechanisms

1,2-<i>trans</i>-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<i> </i>4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect of <i>k</i>(H₃O⁺)/<i>k</i>(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 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 <i>k</i>_H/<i>k</i>_D = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect <i>k</i>(H₂O)/<i>k</i>(D₂O) = 1.1, 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, 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 <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.5 and a strongly negative entropy of activation (D<i>S</i>^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.6 indicates that the formation of 1,2-anhydrosugar is the rate determining step.<br>

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

1,2-<i>trans</i>-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<i> </i>4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect (<i>k</i>(H₃O⁺)/<i>k</i>(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 exhibit general catalysis with a single proton in flight, a normal solvent isotope effect of <i>k</i>_H/<i>k</i>_D = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect <i>k</i>(H₂O)/<i>k</i>(D₂O) = 1.1, 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, 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 <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.5 and a strongly negative entropy of activation (D<i>S</i>^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.6 indicates that the formation of 1,2-anhydrosugar is the rate determining step.<br>

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

1,2-<i>trans</i>-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<i> </i>4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect (<i>k</i>(H₃O⁺)/<i>k</i>(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 exhibit general catalysis with a single proton in flight, a normal solvent isotope effect of <i>k</i>_H/<i>k</i>_D = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect <i>k</i>(H₂O)/<i>k</i>(D₂O) = 1.1, 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, 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 <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.5 and a strongly negative entropy of activation (D<i>S</i>^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.6 indicates that the formation of 1,2-anhydrosugar is the rate determining step.<br>

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

1,2-<i>trans</i>-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<i> </i>4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect (<i>k</i>(H₃O⁺)/<i>k</i>(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 exhibited general catalysis with a single proton in flight with a normal solvent isotope effect of <i>k</i>_H/<i>k</i>_D = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect <i>k</i>(H₂O)/<i>k</i>(D₂O) = 1.1, 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 an inverse solvent isotope effect of <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.5 and a strongly negative entropy of activation (delta<i>S</i>^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of <i>k</i>(HO^–)/<i>k</i>(DO^–) = 0.6 indicates that the formation of 1,2-anhydrosugar is the rate determining step.<br>

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

<p>1,2-<i>trans</i>-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<i> </i>4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect (<i>k</i>(H₃O⁺)/<i>k</i>(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 <i>k</i>(H₂O)/<i>k</i>(D₂O) = 1.1 and a positive entropy of activation (D<i>S</i>^‡ = 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 <i>k</i>(HO^-)/<i>k</i>(DO^-) = 0.5 and a strongly negative entropy of activation (D<i>S</i>^‡ = –13.6 cal mol^–1 K^–1). Finally, at high pH, an inverse solvent isotope effect of <i>k</i>(HO^-)/<i>k</i>(DO^-) = 0.6 and a weakly negative entropy of activation (D<i>S</i>^‡ = –5.5 cal mol^–1 K^–1) indicates that the formation of 1,2-anhydrosugar is the rate determining step. <b></b></p>

Inverse Solvent Isotope Effects in Enzyme-Catalyzed Reactions

Solvent isotope effects have long been used as a mechanistic tool for determining enzyme mechanisms. Most commonly, macroscopic rate constants such as kcat and kcat/Km are found to decrease when the reaction is performed in D2O for a variety of reasons including the transfer of protons. Under certain circumstances, these constants are found to increase, in what is termed an inverse solvent kinetic isotope effect (SKIE), which can be a diagnostic mechanistic feature. Generally, these phenomena can be attributed to an inverse solvent equilibrium isotope effect on a rapid equilibrium preceding the rate-limiting step(s). This review surveys inverse SKIEs in enzyme-catalyzed reactions by assessing their underlying origins in common mechanistic themes. Case studies for each category are presented, and the mechanistic implications are put into context. It is hoped that readers may find the illustrative examples valuable in planning and interpreting solvent isotope effect experiments.