Accurate Single-Molecule Kinetic Isotope Effects.

Acetolysis of ferrocenylmethyl benzoate. High secondary α-deuterium kinetic isotope effect for primary carbon-oxygen cleavage

We have studied the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) and the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH); the substrate is 5-protonated 7,8-dihydrofolate, and the product is tetrahydrofolate. The potential energy surface is modeled by a combined quantum mechanical-molecular mechanical (QM/MM) method employing Austin model 1 (AM1) and a simple valence bond potential for 69 QM atoms and employing the CHARMM22 and TIP3P molecular mechanics force fields for the other 21 399 atoms; the QM and MM regions are joined by two boundary atoms treated by the generalized hybrid orbital (GHO) method. All simulations are carried out using periodic boundary conditions at neutral pH and 298 K. In stage 1, a reaction coordinate is defined as the difference between the breaking and forming bond distances to the hydride ion, and a quasithermodynamic free energy profile is calculated along this reaction coordinate. This calculation includes quantization effects on bound vibrations but not on the reaction coordinate, and it is used to locate the variational transition state that defines a transition state ensemble. Then, the key interactions at the reactant, variational transition state, and product are analyzed in terms of both bond distances and electrostatic energies. The results of both analyses support the conclusion derived from previous mutational studies that the M20 loop of DHFR makes an important contribution to the electrostatic stabilization of the hydride transfer transition state. Third, transmission coefficients (including recrossing factors and multidimensional tunneling) are calculated and averaged over the transition state ensemble. These averaged transmission coefficients, combined with the quasithermodynamic free energy profile determined in stage 1, allow us to calculate rate constants, phenomenological free energies of activation, and primary and secondary kinetic isotope effects. A primary kinetic isotope effect (KIE) of 2.8 has been obtained, in good agreement with the experimentally determined value of 3.0 and with the value 3.2 calculated previously. The primary KIE is mainly a consequence of the quantization of bound vibrations. In contrast, the secondary KIE, with a value of 1.13, is almost entirely due to dynamical effects on the reaction coordinate, especially tunneling.

Primary and secondary kinetic isotope effects for methoxy dehydrogenation and Cu(110): Absence of tunnelling corrections

Tunnelling and the kinetic isotope effect in [formula omitted]: An application of semiclassical transition state theory

Secondary kinetic deuterium isotope effects have recently come into widespread use as probes of reaction mechanism and transition state structure. Isotopic substitution at the reacting carbon gives rise to the so-called ..cap alpha.. effect due to differences in the energetics of out-of-plane bending of C-H and C-D bonds; these effects are usually interpreted in terms of changes in hybridization at the reacting carbon as the reaction processes. Alternately isotopic substitution at carbons adjacent to the reacting carbon leads to a ..beta.. effect presumably arising from differences in the ability of C-H and C-D bands to interact with orbitals on the reacting carbon by means of hyperconjugation. In connection with mechanistic studies of the epoxide-diol pathway secondary deuterium isotope effects were used to compare epoxidation of olefins by cytochrome P-450 with olefin epoxidation by m-chloroperbenzoic acid. Observations of secondary deuterium isotope effects on the acid-and base-catalyzed methanolysis of p-nitrostyrene oxide, a chemical model system for the epoxide hydrase system were reported. This is the first observation of an ..cap alpha.. effect at the ..beta.. carbon or a secondary effect at a neighboring carbon not attributable to hyperconjugation phenomena. Such effects might be expected but are not observed in the solvolysis ofmore » ..beta..-arglethyl tosylates and in the thermal cic-trans isomerization of cyclopropane derivatives. Another interesting result is the parallelism that the major reactions in acid and base both involve late or product-like transition states, while both minor reactions involve early or reactant-like transition states. These studies have shown that secondary deuterium isotope effects provide a sensitive and predictable probe for mechanistic studies of epoxide reactions. 3 tables. (DP)« less

The primary and secondary deuterium kinetic isotope effects as well as leaving-group fluorine kinetic isotope effects have been calculated for the base-promoted elimination of hydrogen fluoride from 4-fluoro-4-(4'-nitrophenyl)butane-2-one in 75% aqueous methanol solution. The elimination was studied for both formate and imidazole as the catalytic base; and reactant and transition state structures and vibrational frequencies have been calculated by including the base explicitly and by including the solvent by an implicit solvation model that includes both electrostatics by class IV charges and first-solvation-shell effects by atomic surface tensions. We used the M06-L density functional for all calculations. The optimized stationary points, the geometry changes along the solution-phase minimum free energy path, and the solution-phase free energy profile indicate that the elimination reaction occurs concertedly but asynchronously via an E1cb-like transition state. Reaction rates were calculated by the equilibrium solvation path method, using variational transition state theory with multidimensional tunneling. The primary deuterium kinetic isotope effects are calculated to be large: 1.67 and 5.13 for formate and imidazole, respectively. The corresponding C4-secondary deuterium kinetic isotope effects are 1.044 and 1.044, and the leaving group fluorine kinetic isotope effects are respectively 1.020 and 1.015.

Many of the enzymes that catalyze reactions at nucleotide glycosidic linkages proceed through either a reactive oxocarbenium-ion intermediate or a transition state with considerable oxocarbenium character. To investigate how an RNA active site deals with the catalytic challenge of nucleotide synthesis, we probed the transition state of a ribozyme able to promote the formation of a pyrimidine nucleotide. Primary and secondary kinetic isotope effects indicate that this ribozyme stabilizes a highly dissociative reaction with considerable sp2 hybridization and negligible bond order between the departing pyrophosphate leaving group and the anomeric carbon. The small primary 13C isotope effect of 1.002 +/- 0.003 indicates that the reaction is likely to be less concerted than that observed for protein nucleotide synthesis enzymes, which typically have primary 13C isotope effects of 1.02-1.03. The dissociative nature of the ribozyme reaction most resembles the reaction of some hydrolytic enzymes, such as uracil DNA glycosylase, which uses the negative charges found in the phosphodiester backbone of its DNA substrate to transiently stabilize an oxocarbenium ion during hydrolysis. The detectable hydrolysis observed in the ribozyme reaction indicates that shielding of this reactive intermediate from water is a significant challenge for RNA, which protein enzymes that synthesize nucleotides have managed to overcome during evolution, apparently by the utilization of more concerted chemistry.

Chlorine leaving group k(35)/k(37), nucleophile carbon k(11)/k(14), and secondary alpha-deuterium [(kH/kD)alpha] kinetic isotope effects (KIEs) have been measured for the SN2 reactions between para-substituted benzyl chlorides and tetrabutylammonium cyanide in tetrahydrofuran at 20 degrees C to determine whether these isotope effects can be used to determine the substituent effect on the structure of the transition state. The secondary alpha-deuterium KIEs indicate that the transition states for these reactions are unsymmetric. The theoretical calculations at the B3LYP/aug-cc-pVDZ level of theory support this conclusion; i.e., they suggest that the transition states for these reactions are unsymmetric with a long NC-C(alpha) and reasonably short C(alpha)-Cl bonds. The chlorine isotope effects suggest that these KIEs can be used to determine the substituent effects on transition state structure with the KIE decreasing when a more electron-withdrawing para-substituent is present. This conclusion is supported by theoretical calculations. The nucleophile carbon k(11)/k(14) KIEs for these reactions, however, do not change significantly with substituent and, therefore, do not appear to be useful for determining how the NC-C(alpha) transition-state bond changes with substituent. The theoretical calculations indicate that the NC-C(alpha) bond also shortens as a more electron-withdrawing substituent is placed on the benzene ring of the substrate but that the changes in the NC-C(alpha) transition-state bond with substituent are very small and may not be measurable. The results also show that using leaving group and nucleophile carbon KIEs to determine the substituent effect on transition-state structure is more complicated than previously thought. The implication of using both chlorine leaving group and nucleophile carbon KIEs to determine the substituent effect on transition-state structure is discussed.

In the pyrimidine biosynthetic pathway, N-carbamyl-L-aspartate (CA-asp) is converted to L-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 degrees C for hamster DHOase; 37, 50, and 60 degrees C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the beta-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO --> CA-asp have also been measured (secondary 13K(eq) = 1.0028 +/- 0.0002, primary 13K(eq) = 1.0053 +/- 0.0003, primary 15K(eq) = 1.0027 +/- 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the physiological reaction (CA-asp --> DHO) have been calculated. These values indicate that the carbon of the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the physiological reaction. It appears that the ring opening and closing is the slow step of the reaction.

The secondary α-deuterium kinetic isotope effect has been measured for the thermal reaction of 2-phenylethyldimethylamine oxide in 90 mol% DMSO–H2O at 60.0 °C. A large secondary α-deuterium isotope effect of 1.158 per α-D was found, which indicates significant [Formula: see text] bond rupture and very little double bond formation at the transition state for this concerted syn-elimination process. The observed large normal value for (kH/kD)α is discussed in terms of the use of secondary α-D isotope effects for the determination of stereochemistry for a concerted elimination process. Various isotope effects found for the syn elimination of the above amine oxide and those for the anti elimination of 2-arylethyltrimethylammonium salts with ethoxide are considered with respect to the relative transition state structures for syn- and anti-elimination reactions.

The effect of solvent composition in aqueous ethanol, trifluoroethanol and hexafluoropropan‐2‐ol on the rate constant and activation parameters for the uncatalysed hydrolysis of 2‐(p‐nitrophenoxy)tetrahydropyran (1) was investigated, and the m(YOTs) value is 0.60. This appreciable but less than maximal value is in accordance with an SN1 mechanism with rate‐limiting ionization. The α‐deuterium secondary kinetic isotope effect (α‐kie) for the uncatalysed hydrolysis of 1 is 1.17 in water (46°C), 1.15 in aqueous trifluoroethanol (50% mole fraction, 70.6°C) and 1.13 in aqueous ethanol (50% mole fraction, 70.6°C). These values correspond to about 1.19 at 25°C, which is characteristic of rate‐limiting ionization in an SN1 reaction and appreciably higher than values for enzyme‐catalysed glycolysis. The α‐kie is smaller under aqueous acidic conditions (1.07, 0.1 mol dm−3 hydrochloric acid, 20.2°C) when 1 hydrolyses with acid catalysis. The previously reported α‐kie for the hydrolysis of 1 in buffered aqueous dioxan (1.063, 25°C) is now seen to correspond to acid‐catalysed hydrolysis. These new results for 1 indicate that transition structures in enzyme‐catalysed glycolyses with α‐kie values of less than about 1.15 at 25°C involve a lower degree of carbenium ion character than has hitherto been assumed. Copyright © 2004 John Wiley & Sons, Ltd.

The E2 reactions of methyl- and fluoro-substituted ethyl chlorides with CH3-, NH2-, OH-, F-, SH-, and Cl- have been investigated at the MP2/6-31+G(d) level of theory for probing whether kinetic isotope effects are related to the geometry of the transition stucture. The primary kinetic isotope effects only display the expected maximum for the most symmetric transition structure when corrections for equilibrium isotope effects are taken into account. Such corrections are in general not possible from experimental data alone. Secondary kinetic isotope effects at the CH2Cl end of ethyl chloride correlate qualitatively with the carbon hybridization, although a quantitative relationship is lacking, even for closely related systems. The corresponding isotope effects at the carbon where a hydrogen is abstracted show little correlation with the geometry of the transition state.

Kinetic isotope effects were measured for oxidations of (S,S)-2-(p-trifluoromethylphenyl)cyclopropylmethane containing zero, two, and three deuterium atoms on the methyl group by Compounds I from the cytochrome P450 enzymes CYP119 and CYP2B4 at 22 degrees C. The oxidations displayed saturation kinetics, which permitted solution of both binding constants (K(bind)) and first-order oxidation rate constants (k(ox)) for both enzymes with the three substrates. The binding constant for CYP2B4 Compound I was about 1 order of magnitude greater than that for CYP119 Compound I, but the oxidation rate constants were similar for the two. In oxidations of 1-d(0), k(ox) = 10.4 s(-1) for CYP119 Compound I, and k(ox) = 12.4 s(-1) for CYP2B4 Compound I. Primary kinetic isotope effects (P) and secondary kinetic isotope effects (S) were obtained from the results with the three isotopomers. The primary KIEs were large, P = 9.8 and P = 8.9 for CYP119 and CYP2B4 Compounds I, respectively, and the secondary KIEs were small and normal, S = 1.07 and S = 1.05, respectively. Large intermolecular KIEs for 1-d(0) and 1-d(3) of k(H)/k(D) = 11.2 and 9.8 found for the two Compounds I contrast with small intermolecular KIEs obtained previously for the same substrate in P450-catalyzed oxidations; these differences suggest that a second electrophilic oxidant, presumably iron-complexed hydrogen peroxide, is important in cytochrome P450 oxidations under turnover conditions.

Secondary α-deuterium kinetic isotope effects for addition of phenyl radical to benzyl isocyanides: An evidence of concerted mechanism.

Kinetic ..cap alpha.. deuterium isotope effects have been measured for acid-catalyzed hydrolysis of inosine and adenosine. For inosine hydrolysis, values of k/sub H/k/sub D/ follow: in 1.0 M HCl, 1.21 and 1.20 at 25 and 50/sup 0/C, respectively; in 0.1 M HCl, 1.19 and 1.18 at 25 and 50/sup 0/C, respectively. For adenosine hydrolysis, k/sub H/k/sub D/ is 1.23 in 0.1 M HCl at 25/sup 0/C. The values require that the transition states for hydrolysis of both the monocation and dication of inosine and the dication of adenosine have marked oxocarbonium ion character. Detailed mechanisms which accord with this and other experimental observations include (1) a classical Al mechanism in which the C--N bond is largely cleaved in the transition state; (2) a mechanism involving some form of nucleophilic participation by solvent in which bond cleavage is advanced relative to bond formation in the transition state; or (3) complete C--N bond cleavage with rate-determining diffusion apart of oxocarbonium ion and purine base. 53 references, 1 figure, 2 tables.