Kinetic Hydrogen Isotope Effects Research Articles

Laboratory Studies of the Hydrogen Kinetic Isotope Effects (KIES) of the Reaction of Non-Methane Hydrocarbons with the OH Radical in the Gas Phase

The hydrogen kinetic isotope effects (KIEs) of the reactions of 15 non-methane hydrocarbons (NMHCs) with the OH radical were measured at 298 ± 2 K. The measurements were made using NMHCs without artificial isotopic labeling or enrichment. The following average hydrogen KIE values, in per mil (‰), were obtained: 29.8 ± 2.1 (toluene),51.6 ± 2.1 (n-butane), 97.3± 12.5 (i-butane), 63.2 ± 5.9 (cyclopentane), 10.5 (p-xylene), 26.8 ± 3.5 (ethylbenzene), 65.9± 7.0 (n-pentane), 79.5 ± 9.6 (cyclohexane), 52.8 ± 5.0(n-hexane), 38.9 ± 7.8 (n-heptane), 33.4 ± 3.1 (n-octane), 29.6 ± 1.6(n-nonane), and 29.0 ± 5.3 (n-decane). The KIEs for reactions of two alkenes (cyclohexene and 1-heptene) could not be determined accurately due to interference from reaction with ozone, but nevertheless the results clearly show that the KIEs for reaction of alkenes with OH are significantly lower than those for saturated hydrocarbons. The KIEs for reaction of alkanes are smaller than isotope effects reported in literature for the reactions of NMHCs artificially labeled with deuterium. The main reason for this difference is the reduced probability for reaction at a labeled site for compounds with close to natural deuterium abundance, although some impact of secondary isotope effects cannot be ruled out. Still, the KIEs for NMHCs with natural or close to natural abundance of deuterium are of sufficient magnitude to allow determination of the extent of chemical processing of hydrocarbons in the atmosphere using methods analogous to stable carbon KIE studies. Furthermore, it is shown that combining stable hydrogen and stable carbon isotope ratio data has the potential to also provide valuable information regarding the stable isotope ratios of emissions, and specifically to test one of the key assumptions of the stable isotope hydrocarbon clock, the absence of significant variations of the stable isotope ratio for the emitted NMHCs.

Spin-forbidden deprotonation of aqueous nitroxyl (HNO).

The first mechanistic study of a spin-forbidden proton-transfer reaction in aqueous solution is reported. Laser flash photolysis of alkaline trioxodinitrate (N(2)O(3)(2)(-), Angeli's anion) is used to generate a nitroxyl anion in its excited singlet state ((1)NO(-)). Through rapid partitioning between protonation by water and electronic relaxation, (1)NO(-) produces (1)HNO (ground state, yield 96%) and (3)NO(-) (ground state, yield 4%), which comprise a unique conjugate acid-base couple with different ground-state multiplicities. Using the large difference between reactivities of (1)HNO and (3)NO(-) in the peroxynitrite-forming reaction with (3)O(2), the kinetics of spin-forbidden deprotonation reaction (1)HNO + OH(-) --> (3)NO(-) + H(2)O is investigated in H(2)O and D(2)O. Consistent with proton transfer, this reaction exhibits primary kinetic hydrogen isotope effect k(H)/k(D) = 3.1 at 298 K, which is found to be temperature-dependent. Arrhenius pre-exponential factors and activation energies of the second-order rate constant are found to be: log(A, M(-)(1) s(-)(1)) = 10.0 +/- 0.2 and E(a) = 30.0 +/- 1.1 kJ/mol for proton transfer and log(A, M(-)(1) s(-)(1)) = 10.4 +/- 0.1 and E(a) = 35.1 +/- 0.7 kJ/mol for deuteron transfer. Collectively, these data are interpreted to show that the nuclear reorganization requirements arising from the spin prohibition necessitate significant activation before spin change can take place, but the spin change itself must occur extremely rapidly. It is concluded that a synergy between the spin prohibition and the reaction energetics creates an intersystem barrier and is responsible for slowness of the spin-forbidden deprotonation of (1)HNO by OH(-); the spin prohibition alone plays a minor role.

Multiple hydrogen kinetic isotope effects for enzymes catalyzing exchange with solvent: application to alanine racemase.

Alanine racemase catalyzes the pyridoxal phosphate-dependent interconversion of the D- and L-isomers of alanine. Previous studies have shown that the enzyme employs a two-base mechanism in which Lys39 and Tyr265 are the acid/base catalysts. It is thus possible that stereoisomerization of the external aldimine intermediates occurs through a concerted double proton transfer without the existence of a distinct carbanionic intermediate. This possibility was tested by the application of multiple kinetic isotope effect (KIE) methodology to alanine racemase. The mutual dependence of primary substrate and solvent deuterium KIEs has been measured using equilibrium perturbation-type experiments. The conceptually straightforward measurement of the substrate KIE in H(2)O is complemented with a less intuitive protium washout perturbation-type measurement in D(2)O. The primary substrate KIE in the D --> L direction at 25 degrees C is reduced from 1.297 in H(2)O to 1.176 in D(2)O, while in the L --> D direction it is reduced from 1.877 in H(2)O to 1.824 in D(2)O. Similar reductions are also observed at 65 degrees C, the temperature to which the Bacillus stearothermophilus enzyme is adapted. These data strongly support a stepwise racemization of stereoisomeric aldimine intermediates in which a substrate-based carbanion is an obligatory intermediate. The ionizations observed in k(cat)/K(M) pH profiles have been definitively assigned based on the DeltaH(ion) values of the observed pK(a)'s with alanine and on the pH dependence of k(cat)/K(M) for the alternative substrate serine. The acidic pK(a) in the bell-shaped curve is due to the phenolic hydroxyl of Tyr265, which must be unprotonated for reaction with either isomer of alanine. The basic pK(a) is due to the substrate amino group, which must be protonated to react with Tyr265-unprotonated enzyme. A detailed reaction mechanism incorporating these results is proposed.

A Birch-like mechanism in enzymatic benzoyl-CoA reduction: a kinetic study of substrate analogues combined with an ab initio model.

Benzoyl-CoA reductase from the anaerobic bacterium Thauera aromatica catalyzes the ATP-driven two-electron reduction of the aromatic moiety of benzoyl-CoA. A Birch mechanism involving alternate one-electron and one-proton transfer steps to the aromatic ring was previously proposed for benzoyl-CoA reductase. Due to the high redox barrier, the first electron transfer step yielding a radical anion is considered the rate-limiting step in this reaction. Focusing on the mechanism of substrate reduction, this work combines the kinetic analysis of a number of substrate analogues with a model based on the ab initio calculated electron density of the radical anion of benzoyl-CoA, a transition state model of the proposed Birch mechanism. Both K(m) and k(cat) of ortho-substituted benzoyl-CoA increased in parallel with the substituent's acceptor strength (F > Cl = H > OH > NH(2)). Among the isomers of monofluorobenzoyl-CoA, reduction rates decreased in the following order: ortho > meta > para; the K(m) values increased in the following order: meta > ortho > para. Five-ring heteroaromatic acid thiol esters were reduced in the following order: thiophene > furan > pyrrole; the 2-isomers are reduced much faster than the 3-isomers. Most of these results could be rationalized by the model. A Hammett plot indicated that the reaction mechanism is only slightly polar, suggesting the involvement of a partial protonation of the carbonyl oxygen of benzoyl-CoA and/or a simultaneous transfer of the first electron and proton. Surprisingly, benzoyl-CoA reductase exhibited a hydrogen kinetic isotope effect on k(cat) for pyridine-2-carbonyl-CoA (2.1) but only a negligible one for benzoyl-CoA (1.2), indicating that pyridine-2-carbonyl-CoA reduction proceeds according to a varied mechanism.

Theoretical evaluation of the hydrogen kinetic isotope effect on the first step of the methylmalonyl-CoA mutase reaction

We have calculated hydrogen kinetic isotope effects (KIEs) for the first step of the methylmalonyl-CoA mutase reaction, including multidimensional tunneling correction at the zero curvature (ZCT) level, and compared them with the experimental values. Both alternative mechanisms of this step, concerted and stepwise, can be accommodated. It turned out to be essential to include Arg207 hydrogen-bonded to the reactant in the mechanism predicting simultaneous breaking of the Co–C bond of AdoCbl and hydrogen atom transfer. The consequence of the stepwise mechanism is a much larger facilitation of the homolytic dissociation of the carbon–cobalt bond by the enzyme than currently appreciated; our results suggest lowering of the activation energy by about 23 kcal mol−1. We have also shown that large hydrogen KIEs of tunneling origin do not necessarily break the Swain–Schaad equation. Furthermore, when this equation does not hold, the exponent may be smaller in the presence of tunneling than it is at the semi-classical limit, indicating that nonclassical behavior may be a more common phenomenon than expected.

Origin of the Primary Kinetic Hydrogen Isotope Effects on N-dealkylation from N-alkylamine by Hemoproteins

Abstract The atomic level reaction mechanism for N-dealkylation of N,N-dimethylaniline (PhNMe2) by hemoproteins, in which a kinetic hydrogen isotope effect (KHIE) was observed, was investigated by quantum mechanical theoretical calculations. Using quantum mechanical theory, one can determine the primary KHIE by the difference in zero-point vibrational energies on normal vibrational modes which are related to the reaction coordinate of H (D or T) atom abstraction from a C–H (C–D or C–T) bond. On the basis of this idea, we theoretically determined the intramolecular primary KHIE for two cases of hydrogen atom abstraction in N-dealkylation of PhNMe2; i.e., hydrogen atom abstraction from a neutral state and proton abstraction from a cation radical state. When the intramolecular primary KHIE obtained by theoretical calculation was compared with experimental results of the intramolecular KHIE on N-dealkylation of PhNMe2, which was catalyzed by some hemoproteins, it was found that four experimental values can be interpreted only from the primary KHIE. However, many other experimental values can not be interpreted only from the primary KHIE.

Chiral diamines 4: a computational study of the enantioselective deprotonation of Boc-pyrrolidine with an alkyllithium in the presence of a chiral diamine.

The enantioselective deprotonation of N-Boc-pyrrolidine (1) with i-PrLi-(-)-sparteine has been studied at theoretical levels up through B3P86/6-31G. Four low-energy intermediate complexes involving i-PrLi-(-)-sparteine and 1 were located via geometry optimizations; two of these complexes would lead to abstraction of the pro-S hydrogen from 1, and the other two complexes would lead to loss of the pro-R hydrogen. The lowest-energy intermediate complex was found to lead to loss of the pro-S hydrogen as observed experimentally. Transition states for the deprotonations were located using the synchronous transit-guided quasi-Newton method. The calculated activation enthalpy for transfer of the pro-S hydrogen within the lowest-energy intermediate complex, 10.8 kcal/mol, is reasonable for a reaction that occurs at a relatively low temperature, and the calculated kinetic hydrogen isotope effect is in agreement with experimental data. The lower enantioselectivity observed experimentally for deprotonation of 1 using t-BuLi-(-)-sparteine is attributed to a transition-state effect due to increased steric interaction engendered by the bulky t-BuLi. Replacement of the tert-butoxycarbonyl group in 1 by a methoxycarbonyl is predicted to result in a slower deprotonation with somewhat decreased enantioselectivity. Asymmetric deprotonation of 1 using i-PrLi in combination with the C(2)-symmetric diamine, (S,S)-1,2-bis(N,N-dimethylamino)cyclohexane, was calculated to be much less selective than is the deprotonation mediated by (-)-sparteine as observed experimentally. The relative energies of the intermediate complexes were fairly well-reproduced by ONIUM calculations in which the sparteine ligand less its nitrogen atoms was treated by molecular mechanics and the remainder of the complex was treated by quantum mechanics.

Carbon and hydrogen kinetic isotope effects for the reaction of Cl with CH4: Consolidating chemical kinetics and molecular dynamics measurements

In order to estimate carbon and hydrogen kinetic isotope effects for the reaction Cl+ CH4 at atmospheric temperatures, measured values of the rates of Cl reacting with 13CH4, 12CH3D, 12CH2D2, 12CHD3, and 12CD4 relative to those of Cl+12CH4 were fit using a model that accounts for quantum mechanical tunneling. The model also includes vibrational enhancement of the reaction rate by excitation of the bending and stretching modes of CH4. These fits allow extrapolation of measured values to temperatures outside the range in which they were measured. In addition, the kinetic isotope effect for Cl+14CH4 was predicted using this model based on the results for Cl+ 13CH4. The model results for Cl+14CH4, Cl+13CH4, and Cl+12CH3D were parameterized for use in atmospheric models.

Theoretical Modeling of the Hydrogen Abstraction Reaction of Fluoromethane by the Hydroxyl Radical

We have performed theoretical modeling of the reaction rate constants of the hydrogen abstraction reaction of fluoromethane (CH3F) by the hydroxyl radical (OH) using dual-level variational transition state theory calculation including multidimensional tunneling corrections from 200 to 1000 K. Correlated electronic structure theory with extended basis set calculation was applied for both the low-level reaction-path and the high-level stationary-point calculation. An improved interpolated correction scheme was used for better estimating the width of energy barrier by performing an intermediate-level electronic structure calculation. The calculated rate constants are in good agreement with available experimental values at most temperatures. The hydrogen kinetic isotope effects (KIEs) were also evaluated and analyzed. The current study suggested that the reaction has a relatively wide barrier, and the tunneling effects are thus not very important even at low temperatures. The current study also showed that th...

Hydride shift in substituted phenyl glyoxals: Interpretation of experimental rate data using electronic structure and variational transition state theory calculations

Experimental rate data for hydride transfer in some p-substituted phenyl glyoxals in 80: 20 dioxan:water are presented including kinetic hydrogen isotope effects. The roles of different substituents are discussed using electronic structure calculations of the potential energy surfaces at the SM5.4/PM3 level together with calculations employing variational transition state theory and multidimensional tunneling. p-NO2 substitution has the most pronounced effect on the rate parameters, which can be understood in terms of the relative destabilisation of the reactant and reduced quantum mechanical tunneling through the barrier. The use of different models for tunneling suggests that when coupled with the SM5.4/PM3 method, the higher level of theories tend to overestimate the degree of tunneling.

Rate-determining steps in phenacetin oxidations by human cytochrome P450 1A2 and selected mutants.

Mutants with altered activities were obtained from random libraries of human cytochrome P450 (P450) 1A2 with the putative substrate recognition sequences (SRS) mutated [Parikh, A., Josephy, P. D., and Guengerich, F. P. (1999) Biochemistry 38, 5283-5289]. Six mutants from SRS 2 (E225I, E225N, F226I, and F226Y) and 4 (D320A and V322A) regions were expressed as oligohistidine-tagged proteins, purified to homogeneity, and used to analyze kinetics of individual steps in the catalytic cycle, to determine which reaction steps have been altered. When the wild-type, E225I, E225N, F226I, F226Y, D320A, and V322A proteins were reconstituted with NADPH-P450 reductase, rates of 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation were in accord with those expected from membrane preparations. Within each assay, the values of k(cat)/K(m) varied by 2-3 orders of magnitude, and in the case of E225I and E225N, these parameters were 7-8-fold higher than for the wild-type enzyme. The coupling efficiency obtained from the rates of product formation and NADPH oxidation was low (<20%) in all enzymes. No correlation was found between activities and several individual steps in the catalytic cycle examined, including substrate binding, reduction kinetics, NADPH oxidation, and H(2)O(2) formation. Quench reactions did not show a burst for either phenacetin O-deethylation or formation of the acetol, a minor product, indicating that rate-determining steps occur prior to product formation. Inter- and intramolecular kinetic deuterium isotope effects for phenacetin O-deethylation were 2-3. In the case of phenacetin acetyl hydroxylation (acetol formation), large isotope effects [(D)k(cat) or (D)(k(cat)/K(m)) > 10] were observed, providing evidence for rate-limiting C-H bond cleavage. We suggest that the very high isotope effect for acetol formation reflects rate-limiting hydrogen atom abstraction; the lower isotope effect for O-deethylation may be a consequence of a 1-electron transfer pathway resulting from the low oxidation potential of the substrate phenacetin. These pre-steady-state, steady-state, and kinetic hydrogen isotope effect studies indicate that the rate-limiting steps are relatively unchanged over an 800-fold range of catalytic activity. We hypothesize that these SRS mutations alter steps leading to the formation of the activated Michaelis complex following the introduction of the first electron.

Experimentally determined kinetic isotope effects in the reaction of CH4 with Cl: Implications for atmospheric CH4

We report experimental values for the carbon and hydrogen kinetic isotope effects (KIEs) in the reaction of CH4 with Cl at temperatures between 273 and 350 K. Isotope ratio mass spectrometry was utilized to measure 13CH4/12CH4 and CH3D/CH4 ratios on samples taken from a 5870‐ L reaction chamber. At 299 K, kc12/kc13 = 1.0621±0.0001 (2σ) and kh/kd = 1.474±0.020 (2σ). For both KIEs, the ratio decreased with increasing temperature over the range studied. These results agree well with experimental studies using tunable diode laser absorption spectroscopy and FTIR absorption spectroscopy and with a recent theoretically‐calculated set of values.

Hydrogen and carbon kinetic isotope effects during soil uptake of atmospheric methane

The hydrogen and carbon kinetic isotope effects (KJEs) occurring during uptake of atmospheric methane (CH4) by soils were measured using in situ static flux chambers in a native grassland and a temperate forest in Washington State. The hydrogen KIE was αDsoil =k(CH4)/k(CH3D) = 1.099 ± 0.030 and 1.066 ± 0.007 for the grassland and forest, respectively. The carbon KIE of αCsoil =k(12CH4)/k(13CH4) = 1.0173 ± 0.0010 and 1.0181 ± 0.0004 for the grassland and forest, respectively, compares well to previous determinations in other ecosystems. Local spatial variability in αsoil was as large as the between‐ecosystem variability. The dependence of αsoil on αox and the KIE during diffusion is described. The apparent KIE associated with microbial oxidation, αox, was determined from αsoil and the relative rates of CH4 oxidation and diffusion in the soil column, derived from observed steady state profiles of soil air CH4 concentration. The apparent αox ranged from 1.094 to 1.209 for αDox and from 1.0121 to 1.0183 for αCox. These are the first determinations of the hydrogen KIEs during soil uptake of atmospheric CH4 and during aerobic microbial oxidation of CH4 at or below atmospheric concentrations. The KIE during uptake of atmospheric CH4 by soils is significantly different than the KIEs associated with the other sinks of atmospheric CH4. The interhemispheric asymmetry in the strength of the soil sink of atmospheric CH4 suggests a difference of ∼6‰ between the overall hydrogen KIEs in the two hemispheres. Modeling studies of the global atmospheric CH4 budget using deuterium as a tracer must therefore include αDsoil.

Hydrogen Isotope Effects in Electrochemical Reductions of Organic Chloro Compounds

The kinetic hydrogen isotope effects in electrochemical reductions of CCl3COOD, CDCl2COOD, CCl4, CDCl3, benzyl chloride, 1-chloronaphthalene and 4-chlorobenzonitrile in deuterated reaction media were determined. The aliphatic chloro compounds are reduced with rather small isotope effects kH/kD = 1.2…1.7, as expected if anionic intermediates are formed and protonated. Benzyl chloride is reduced with an apparent kH/kD = 2.1, which is probably too high due to radical reactions. Aromatic chloro compounds are reduced with significant participation of a second mechanism via radical intermediates which abstract carbon-bound hydrogen atoms from the components of the reaction system. The resulting isotope effects are kH/kD = 1.2 for the part of the reduction which proceeds via anions and kH/kD ∼ 2.5 for the competing radical reaction.

Kinetics of Cytochrome P450 2E1-Catalyzed Oxidation of Ethanol to Acetic Acid via Acetaldehyde

The P450 2E1-catalyzed oxidation of ethanol to acetaldehyde is characterized by a kinetic deuterium isotope effect that increases K(m) with no effect on k(cat), and rate-limiting product release has been proposed to account for the lack of an isotope effect on k(cat) (Bell, L. C., and Guengerich, F. P. (1997) J. Biol. Chem. 272, 29643-29651). Acetaldehyde is also a substrate for P450 2E1 oxidation to acetic acid, and k(cat)/K(m) for this reaction is at least 1 order of magnitude greater than that for ethanol oxidation to acetaldehyde. Acetic acid accounts for 90% of the products generated from ethanol in a 10-min reaction, and the contribution of this second oxidation has been overlooked in many previous studies. The noncompetitive intermolecular kinetic hydrogen isotope effects on acetaldehyde oxidation to acetic acid ((H)(k(cat)/K(m))/(D)(k(cat)/K(m)) = 4.5, and (D)k(cat) = 1.5) are comparable with the isotope effects typically observed for ethanol oxidation to acetaldehyde, and k(cat) is similar for both reactions, suggesting a possible common catalytic mechanism. Rapid quench kinetic experiments indicate that acetic acid is formed rapidly from added acetaldehyde (approximately 450 min(-1)) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, approximately 90% of the acetaldehyde intermediate is directly converted to acetic acid without dissociation from the enzyme active site. Competition experiments suggest that P450 2E1 binds acetic acid and acetaldehyde with relatively high K(d) values, which preclude simple tight binding as an explanation for rate-limiting product release. The existence of a rate-determining step between product formation and release is postulated. Also proposed is a conformational change in P450 2E1 occurring during the course of oxidation and the discrimination of P450 2E1 between acetaldehyde and its hydrated form, the gem-diol. This multistep P450 reaction is characterized by kinetic control of individual reaction steps and by loose binding of all ligands.

Acid-catalyzed decomposition of diazodiphenylmethane in dimethylsulfoxide

Acid-catalyzed decomposition of diazodiphenylmethane (DDM) has been studied in DMSO containing varying amounts of water. The reaction was found to be first-order in DDM and first-order in acid. The Brønsted plot for a series of carboxylic acids is curved with the H+ point falling below the curve defined by the carboxylic acids. In near-anhydrous DMSO, kinetic hydrogen isotope effects (KIEs) are 1.7 and 1.6 for acetic acid and chloroacetic acid, respectively. The chloroacetic acid KIE increases with increasing water concentration, rising to 2.9 at 3.6 M water. As a function of [H2O], kHA for chloroacetic acid shows a sharply defined minimum occurring at 1 M water. This behavior and the water effect on KIE suggest that the carboxylic acid-catalyzed reactions in near-anhydrous DMSO do not use the ASE-2 mechanism attributed to the reaction of DDM in hydroxylic solvents. A mechanism, leading to an azoalkane, is suggested. Either directly or indirectly, this may lead to diphenylcarbene, which would account for the observed products: benzophenone, benzhydrol, and benzhydryl esters. For the H+ catalyzed decomposition of DDM in near-anhydrous DMSO, benzophenone is not found among the products, and we suggest that this reaction does not undergo a change in mechanism and continues to use the ASE-2 mechanism.Key words: decomposition mechanism, kinetics, solvent effect, reaction with carboxylic acids.

Acid-catalyzed decomposition of diazodiphenylmethane in dimethylsulfoxide

Acid-catalyzed decomposition of diazodiphenylmethane (DDM) has been studied in DMSO containing varying amounts of water. The reaction was found to be first-order in DDM and first-order in acid. The Bronsted plot for a series of carboxylic acids is curved with the H + point falling below the curve defined by the carboxylic acids. In near-anhydrous DMSO, kinetic hydrogen isotope effects (KIEs) are 1.7 and 1.6 for acetic acid and chloroacetic acid, respectively. The chloroacetic acid KIE increases with increasing water concentration, rising to 2.9 at 3.6 M water. As a function of (H2O), kHA for chloroacetic acid shows a sharply defined minimum occurring at 1 M water. This behavior and the water effect on KIE suggest that the carboxylic acid-catalyzed reactions in near-anhydrous DMSO do not use the ASE-2 mechanism attributed to the reaction of DDM in hydroxylic solvents. A mechanism, leading to an azoalkane, is suggested. Either directly or indirectly, this may lead to diphenylcarbene, which would account for the observed products: benzophenone, benzhydrol, and benzhydryl esters. For the H + catalyzed decomposition of DDM in near- anhydrous DMSO, benzophenone is not found among the products, and we suggest that this reaction does not undergo a change in mechanism and continues to use the ASE-2 mechanism.

Kinetic Isotope Effects for Dialkylglycine Decarboxylase via a High-Precision Continuous-Flow Method

Kinetic Isotope Effects for Dialkylglycine Decarboxylase via a High-Precision Continuous-Flow Method

Evidence for a 1-Electron Oxidation Mechanism in N-Dealkylation of N,N-Dialkylanilines by Cytochrome P450 2B1: KINETIC HYDROGEN ISOTOPE EFFECTS, LINEAR FREE ENERGY RELATIONSHIPS, COMPARISONS WITH HORSERADISH PEROXIDASE, AND STUDIES WITH OXYGEN SURROGATES

Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects ( approximately 7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.

Ab initio quantum mechanical studies of the kinetics and mechanisms of quartz dissolution: OH − catalysis

The generally accepted mechanisms of quartz dissolution in basic pH solutions can be summarized as (a) direct attack by H 2O on a negatively charged surface site and (b) catalysis by hydroxide ion (OH −) on a neutral surface site. In order to test these proposals and to understand the full dynamics of the dissolution processes from first principles, we have carried out high level ab initio molecular orbital calculations to study the following reactions: (HO) 3SiOSi(OH) 2O − + H 2O → (HO) 3SiOH + −OSi(OH) 3 (HO) 3SiOSi(OH) 3 + OH − → (HO) 3SiOH + −OSi(OH) 3 Disilicic acid, (HO) 3SiOSi(OH) 3, and its −1 deprodonated form, (HO) 3SiOSi(OH) 2O −, have been chosen to simulate the neutral and negatively charged quartz surface site, respectively. Their reactions with OH − and H 2O, which lead to the hydrolysis of the SiO bond, have then been thoroughly investigated. Based on our ab initio calculations, we have discovered some interesting consequences: 1. 1) OH − attack on the hydroxyl surface will lead to the deprotonation of the surface by transferring one H to the OH −, resulting in a H-bonded H 2O adsorption onto a negatively charged SiO − site. This process is equivalent to H 2O attack on the negatively charged SiO − surface. 2. 2) The next step involves the formation of a negatively charged fivefold coordinated Si species; this step has to overcome a large energy barrier (18.91 kcal/mol). The fivefold coordinated Si species has almost the same potential energy as the reaction precursor—the hydrogen-bonded H 2O adsorption minimum—but it significantly weakens the SiO bond. 3. 3) The final step is the rupture of the SiOSi bond to form SiOH⋯ −OSi, with a much smaller energy barrier (4.49 kcal/mol). Therefore, our ab initio results uncover two adsorption minima in series before the cleavage of the SiO bond and suggest that it is the formation of the adsorption fivefold coordinated Si species, not the actual hydrolysis step, that controls the activation energy and the rate of quartz dissolution in basic pH solutions. The calculated activation energy, 18.91 kcal/mol, is similar to the one (24 kcal/mol) in H 3O + catalyzed hydrolysis of SiOSi bond, while much smaller than that (29 kcal/mol) in pure H 2O hydrolysis (see Xiao and Lasaga, 1994a), indicating the role that OH − as well as H 3O + play in catalyzing quartz dissolution. The normal mode analysis of the relevant transition states indicate that, even though H transfer has been involved in the overall reaction, the dominant mode in the rate determining step is the forming and breaking of the SiO bond. This will lead to a small hydrogen kinetic isotope effect, which agrees well with recent experimental results at basic pH.