Deuterium Isotope Effects on Acid-Base Equilibrium of Organic Compounds

Deuterium isotope effects on acid–base equilibrium have been investigated using a combined path integral and free-energy perturbation simulation method. To understand the origin of the linear free-energy relationship of ΔpKa=pKaD2O−pKaH2O versus pKaH2O, we examined two theoretical models for computing the deuterium isotope effects. In Model 1, only the intrinsic isotope exchange effect of the acid itself in water was included by replacing the titratable protons with deuterons. Here, the dominant contribution is due to the difference in zero-point energy between the two isotopologues. In Model 2, the medium isotope effects are considered, in which the free energy change as a result of replacing H2O by D2O in solute–solvent hydrogen-bonding complexes is determined. Although the average ΔpKa change from Model 1 was found to be in reasonable agreement with the experimental average result, the pKaH2O dependence of the solvent isotope effects is absent. A linear free-energy relationship is obtained by including the medium effect in Model 2, and the main factor is due to solvent isotope effects in the anion–water complexes. The present study highlights the significant roles of both the intrinsic isotope exchange effect and the medium solvent isotope effect.

Epoxidation and late-stage C-H functionalization by P450 TamI is mediated by variant heme-iron oxidizing species

P450-catalyzed hydroxylation reactions are well understood mechanistically including the identity of the active oxidizing species. However, the catalytically active heme-iron species in P450 iterative oxidation cascades that involve mechanistically divergent pathways and distinct carbon atoms within a common substrate remains unexplored. Recently, we reported the enzymatic synthesis of tri-functionalized tirandamycin O (9) and O’ (10) using a bacterial P450 TamI variant and developed mechanistic hypotheses to explore their formation. Here, we report the ability of bacterial P450 TamI L295A to shift between different oxidizing species as it catalyzes the sequential epoxidation, hydroxylation and radical-catalyzed epoxide-opening cascade to create new tirandamycin antibiotics. We also provide evidence that the TamI peroxo-iron species could be a viable catalyst to enable nucleophilic epoxide opening in the absence of iron-oxo Compound I. Using site-directed mutagenesis, kinetic solvent isotope effects, artificial oxygen surrogates, end-point assays, and density functional theory (DFT) calculations, we provide new insights into the active oxidant species that P450 TamI employs to introduce its unique pattern of oxidative decorations.

Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases

Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis. Graphic abstract

Rate and Product Studies with 1-Adamantyl Chlorothioformate under Solvolytic Conditions

A study was carried out on the solvolysis of 1-adamantyl chlorothioformate (1-AdSCOCl, 1) in hydroxylic solvents. The rate constants of the solvolysis of 1 were well correlated using the Grunwald–Winstein equation in all of the 20 solvents (R = 0.985). The solvolyses of 1 were analyzed as the following two competing reactions: the solvolysis ionization pathway through the intermediate (1-AdSCO)+ (carboxylium ion) stabilized by the loss of chloride ions due to nucleophilic solvation and the solvolysis–decomposition pathway through the intermediate 1-Ad+Cl− ion pairs (carbocation) with the loss of carbonyl sulfide. In addition, the rate constants (kexp) for the solvolysis of 1 were separated into k1-Ad+Cl− and k1-AdSCO+Cl− through a product study and applied to the Grunwald–Winstein equation to obtain the sensitivity (m-value) to change in solvent ionizing power. For binary hydroxylic solvents, the selectivities (S) for the formation of solvolysis products were very similar to those of the 1-adamantyl derivatives discussed previously. The kinetic solvent isotope effects (KSIEs), salt effects and activation parameters for the solvolyses of 1 were also determined. These observations are compared with those previously reported for the solvolyses of 1-adamantyl chloroformate (1-AdOCOCl, 2). The reasons for change in reaction channels are discussed in terms of the gas-phase stabilities of acylium ions calculated using Gaussian 03.

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>

Substrate-Dependent Sensitivity of SIRT1 to Nicotinamide Inhibition

SIRT1 is the most extensively studied human sirtuin with a broad spectrum of endogenous targets. It has been implicated in the regulation of a myriad of cellular events, such as gene transcription, mitochondria biogenesis, insulin secretion as well as glucose and lipid metabolism. From a mechanistic perspective, nicotinamide (NAM), a byproduct of a sirtuin-catalyzed reaction, reverses a reaction intermediate to regenerate NAD+ through “base exchange”, leading to the inhibition of the forward deacetylation. NAM has been suggested as a universal sirtuin negative regulator. Sirtuins have evolved different strategies in response to NAM regulation. Here, we report the detailed kinetic analysis of SIRT1-catalyzed reactions using endogenous substrate-based synthetic peptides. A novel substrate-dependent sensitivity of SIRT1 to NAM inhibition was observed. Additionally, SIRT1 demonstrated pH-dependent deacetylation with normal solvent isotope effects (SIEs), consistent with proton transfer in the rate-limiting step. Base exchange, in contrast, was insensitive to pH changes with no apparent SIEs, indicative of lack of proton transfer in the rate-limiting step. Consequently, NAM inhibition was attenuated at a high pH in proteated buffers. Our study provides new evidence for “activation by de-repression” as an effective sirtuin activation strategy.