A new insight into using chlorine leaving group and nucleophile carbon kinetic isotope effects to determine substituent effects on the structure of SN2 transition states

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.     

Transition-state structure of human 5'-methylthioadenosine phosphorylase

Kinetic isotope effects (KIEs) and computer modeling using density functional theory were used to approximate the transition state of human 5'-methylthioadenosine phosphorylase (MTAP). KIEs were measured on the arsenolysis of 5'-methylthioadenosine (MTA) catalyzed by MTAP and were corrected for the forward commitment to catalysis. Intrinsic KIEs were obtained for [1'-(3)H], [1'-(14)C], [2'-(3)H], [4'-(3)H], [5'-(3)H(2)], [9-(15)N], and [Me-(3)H(3)] MTAs. The primary intrinsic KIEs (1'-(14)C and 9-(15)N) suggest that MTAP has a dissociative S(N)1 transition state with its cationic center at the anomeric carbon and insignificant bond order to the leaving group. The 9-(15)N intrinsic KIE of 1.039 also establishes an anionic character for the adenine leaving group, whereas the alpha-primary 1'-(14)C KIE of 1.031 indicates significant nucleophilic participation at the transition state. Computational matching of the calculated EIEs to the intrinsic isotope effects places the oxygen nucleophile 2.0 Angstrom from the anomeric carbon. The 4'-(3)H KIE is sensitive to the polarization of the 3'-OH group. Calculations suggest that a 4'-(3)H KIE of 1.047 is consistent with ionization of the 3'-OH group, indicating formation of a zwitterion at the transition state. The transition state has cationic character at the anomeric carbon and is anionic at the 3'-OH oxygen, with an anionic leaving group. The isotope effects predicted a 3'-endo conformation for the ribosyl zwitterion, corresponding to a H1'-C1'-C2'-H2' torsional angle of 33 degrees. The [Me-(3)H(3)] and [5'-(3)H(2)] KIEs arise predominantly from the negative hyperconjugation of the lone pairs of sulfur with the sigma (C-H) antibonding orbitals. Human MTAP is characterized by a late S(N)1 transition state with significant participation of the phosphate nucleophile.     

Determining the transition-state structure for different SN2 reactions using experimental nucleophile carbon and secondary alpha-deuterium kinetic isotope effects and theory

Nucleophile (11)C/ (14)C [ k (11)/ k (14)] and secondary alpha-deuterium [( k H/ k D) alpha] kinetic isotope effects (KIEs) were measured for the S N2 reactions between tetrabutylammonium cyanide and ethyl iodide, bromide, chloride, and tosylate in anhydrous DMSO at 20 degrees C to determine whether these isotope effects can be used to determine the structure of S N2 transition states. Interpreting the experimental KIEs in the usual fashion (i.e., that a smaller nucleophile KIE indicates the Nu-C alpha transition state bond is shorter and a smaller ( k H/ k D) alpha is found when the Nu-LG distance in the transition state is shorter) suggests that the transition state is tighter with a slightly shorter NC-C alpha bond and a much shorter C alpha-LG bond when the substrate has a poorer halogen leaving group. Theoretical calculations at the B3LYP/aug-cc-pVDZ level of theory support this conclusion. The results show that the experimental nucleophile (11)C/ (14)C KIEs can be used to determine transition-state structure in different reactions and that the usual method of interpreting these KIEs is correct. The magnitude of the experimental secondary alpha-deuterium KIE is related to the nucleophile-leaving group distance in the S N2 transition state ( R TS) for reactions with a halogen leaving group. Unfortunately, the calculated and experimental ( k H/ k D) alpha's change oppositely with leaving group ability. However, the calculated ( k H/ k D) alpha's duplicate both the trend in the KIE with leaving group ability and the magnitude of the ( k H/ k D) alpha's for the ethyl halide reactions when different scale factors are used for the high and the low energy vibrations. This suggests it is critical that different scaling factors for the low and high energy vibrations be used if one wishes to duplicate experimental ( k H/ k D) alpha's. Finally, neither the experimental nor the theoretical secondary alpha-deuterium KIEs for the ethyl tosylate reaction fit the trend found for the reactions with a halogen leaving group. This presumably is found because of the bulky (sterically hindered) leaving group in the tosylate reaction. From every prospective, the tosylate reaction is too different from the halogen reactions to be compared.     

Transition-state analysis of S. pneumoniae 5'-methylthioadenosine nucleosidase

Kinetic isotope effects (KIEs) and computer modeling are used to approximate the transition state of S. pneumoniae 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN). Experimental KIEs were measured and corrected for a small forward commitment factor. Intrinsic KIEs were obtained for [1'-3H], [1'-14C], [2'-3H], [4'-3H], [5'-3H(2)], [9-15N] and [Me-3H(3)] MTAs. The intrinsic KIEs suggest an SN1 transition state with no covalent participation of the adenine or the water nucleophile. The transition state was modeled as a stable ribooxacarbenium ion intermediate and was constrained to fit the intrinsic KIEs. The isotope effects predicted a 3-endo conformation for the ribosyl oxacarbenium-ion corresponding to H1'-C1'-C2'-H2' dihedral angle of 70 degrees. Ab initio Hartree-Fock and DFT calculations were performed to study the effect of polarization of ribosyl hydroxyls, torsional angles, and the effect of base orientation on isotope effects. Calculations suggest that the 4'-3H KIE arises from hyperconjugation between the lonepair (n(p)) of O4' and the sigma* (C4'-H4') antibonding orbital owing to polarization of the 3'-hydroxyl by Glu174. A [methyl-3H(3)] KIE is due to hyperconjugation between np of sulfur and sigma* of methyl C-H bonds. The van der Waal contacts increase the 1'-3H KIE because of induced dipole-dipole interactions. The 1'-3H KIE is also influenced by the torsion angles of adjacent atoms and by polarization of the 2'-hydroxyl. Changing the virtual solvent (dielectric constant) does not influence the isotope effects. Unlike most N-ribosyltransferases, N7 of the leaving group adenine is not protonated at the transition state of S. pneumoniae MTAN. This feature differentiates the S. pneumoniae and E. coli transition states and explains the 10(3)-fold decrease in the catalytic efficiency of S. pneumoniae MTAN relative to that from E. coli.     

Kinetic isotope effects in the active site of B. subtilis chorismate mutase

Kinetic isotope effects are determined for the enzyme‐catalyzed Claisen rearrangement of chorismate to prephenate using computational methods. The calculated kinetic isotope effects (KIEs) compare reasonably with the few available experimental values with both the theory and experiment obtaining a large KIE for the ether oxygen, indicating large polarization of the transition‐state geometry. Because there is a question of the extent that the experimental rate constants are for chemistry as the rate‐limiting step, the KIEs for all the atoms of the substrate are reported with the exception of the carboxylate groups. A substantial number of large regular and inverse isotope effects are predicted for the hydrogens on the cyclohexadienyl ring related to activation of the reactant and charge reorganization in the transition state. A large KIE is predicted for the hydrogen atom bound to the ether carbon atom because the largest valency change and charge transfer occurs at the ether bond in both the reactant and tansition state. Observation of the overall pattern of predicted KIEs would ensure that conditions are favorable for the rate‐limiting chemistry. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 94: 287–292, 2003     

Probing the transition states of four glucoside hydrolyses with 13C kinetic isotope effects measured at natural abundance by NMR spectroscopy

Kinetic isotope effects (KIEs) were measured for methyl glucoside (4) hydrolysis on unlabeled material by NMR. Twenty-eight (13)C KIEs were measured on the acid-catalyzed hydrolysis of alpha-4 and beta-4, as well as enzymatic hydrolyses with yeast alpha-glucosidase and almond beta-glucosidase. The 1-(13)C KIEs on the acid-catalyzed reactions of alpha-4 and beta-4, 1.007(2) and 1.010(6), respectively, were in excellent agreement with the previously reported values (1.007(1), 1.011(2): Bennet and Sinnott, J. Am. Chem. Soc. 1986, 108, 7287). Transition state analysis of the acid-catalyzed reactions using the (13)C KIEs, along with the previously reported (2)H KIEs, confirmed that both reactions proceed with a stepwise D(N)A(N) mechanism and showed that the glucosyl oxocarbenium ion intermediate exists in an E(3) sofa or (4)H(3) half-chair conformation. (13)C KIEs showed that the alpha-glucosidase reaction also proceeded through a D(N)*A(N) mechanism, with a 1-(13)C KIE of 1.010(4). The secondary (13)C KIEs showed evidence of distortions in the glucosyl ring at the transition state. For the beta-glucosidase-catalyzed reaction, the 1-(13)C KIE of 1.032(1) demonstrated a concerted A(N)D(N) mechanism. The pattern of secondary (13)C KIEs was similar to the acid-catalyzed reaction, showing no signs of distortion. KIE measurement at natural abundance makes it possible to determine KIEs much more quickly than previously, both by increasing the speed of KIE measurement and by obviating the need for synthesis of isotopically labeled compounds.     

Kinetic isotope effects of L-Dopa decarboxylase

A mixed centroid path integral and free energy perturbation method (PI-FEP/UM) has been used to investigate the primary carbon and secondary hydrogen kinetic isotope effects (KIEs) in the amino acid decarboxylation of L-Dopa catalyzed by the enzyme L-Dopa decarboxylase (DDC) along with the corresponding uncatalyzed reaction in water. DDC is a pyridoxal 5'-phosphate (PLP) dependent enzyme. The cofactor undergoes an internal proton transfer between the zwitterionic protonated Schiff base configuration and the neutral hydroxyimine tautomer. It was found that the cofactor PLP makes significant contributions to lowering the decarboxylation barrier, while the enzyme active site provides further stabilization of the transition state. Interestingly, the O-protonated configuration is preferred both in the Michaelis complex and at the decarboxylation transition state. The computed kinetic isotope effects (KIE) on the carboxylate C-13 are consistent with that observed on decarboxylation reactions of other PLP-dependent enzymes, whereas the KIEs on the α carbon and secondary proton, which can easily be validated experimentally, may be used as a possible identification for the active form of the PLP tautomer in the active site of DDC.     

Primary semiclassical kinetic hydrogen isotope effects in identity carbon-to-carbon proton- and hydride-transfer reactions, an ab initio and DFT computational study

Enthalpies of activation, transition state (ts) geometries, and primary semiclassical (without tunneling) kinetic isotope effects (KIEs) have been calculated for eleven bimolecular identity proton-transfer reactions, four intramolecular proton transfers, four nonidentity proton-transfer reactions, eleven identity hydride transfers, and two 1,2-intramolecular hydride shifts at the HF/6-311+G, MP2/6-311+G, and B3LYP/6-311++G levels. We find the KIEs to be systematically smaller for hydride transfers than for proton transfers. This outcome is not the result of "bent" transition states, although extreme bending can lower the KIE. Rather, it is a consequence of generally greater total bonding in a hydride-transfer ts than in a proton-transfer ts, most prominently manifested as a reduced contribution from the zero-point vibrational energy difference between reactant and transition states (the DeltaZPVE factor) for hydride transfers relative to proton transfers. This and other differences between proton and hydride transfers are rationalized by modeling the central .C...H...C unit of a proton-transfer ts as a 4-electron, 3-center (4-e 3-c) system and the same unit of a hydride-transfer ts as a 2-e 3-c system. Inclusion of tunneling is most likely to magnify the observed differences between proton-transfer and hydride-transfer KIEs, leaving our qualitative conclusions unchanged.     

Multidimensional tunneling in the [1,5] shift in (Z)-1,3-pentadiene: how useful are Swain-Schaad exponents at detecting tunneling?

Rates, kinetic isotope effects (KIE), and Swain-Schaad exponents (SSE) have been calculated for a variety of isotopologues for the [1,5] shift in (Z)-1,3-pentadiene using mPW1K/6-31+G(d,p). Quantum mechanical effects along the reaction coordinate were incorporated with the zero-curvature tunneling (ZCT) model and with the multidimensional small curvature tunneling (SCT) model, which allows for coupling of modes perpendicular to the reaction coordinate. The latter model gives the best agreement with experimental rates and primary KIEs. The small quasiclassical primary KIE (2.6) is rationalized in terms of a nonlinear transition state. For sp3 to sp2 rehybridization, the quasiclassical alpha-secondary KIE shows an unusual inverse effect due to compression of the nonbonding hydrogens in the suprafacial transition state. SCT transmission coefficients (kappa) increase the rates by as much as one order of magnitude. Tunneling allows the reactant to evade 1-2.5 kcal/mol of the barrier depending on the isotope. Inclusion of tunneling in the secondary KIE increases it beyond the equilibrium isotope effect and converts the inverse effect (0.95) into a normal KIE (1.12). Tunneling was found to deflate the primary y SSE but by an amount too small to distinguish it from the quasiclassical SSE. On the other hand, when a specific labeling pattern is used, the difference between the quasiclassical secondary SSE (4.1) and the tunneling secondary SSE (2.3) may be sufficiently large to detect tunneling. The mixed secondary SSE shows even larger differences.     

Deep tunneling dominates the biologically important hydride transfer reaction from NADH to FMN in morphinone reductase

The temperature dependence of the primary kinetic isotope effect (KIE), combined temperature-pressure studies of the primary KIE, and studies of the alpha-secondary KIE previously led us to infer that hydride transfer from nicotinamide adenine dinucleotide to flavin mononucleotide in morphinone reductase proceeds via environmentally coupled hydride tunneling. We present here a computational analysis of this hydride transfer reaction using QM/MM molecular dynamics simulations and variational transition-state theory calculations. Our calculated primary and secondary KIEs are in good agreement with the corresponding experimental values. Although the experimentally observed KIE lies below the semiclassical limit, our calculations suggest that approximately 99% of the reaction proceeds via tunneling: this is the first "deep tunneling" reaction observed for hydride transfer. We also show that the dominant tunneling mechanism is controlled by the isotope at the primary rather than the secondary position: with protium in the primary position, large-curvature tunneling dominates, whereas with deuterium in this position, small-curvature tunneling dominates. Also, our study is consistent with tunneling being preceded by reorganization: in the reactant, the rings of the nicotinamide and isoalloxazine moieties are stacked roughly parallel to each other, and as the system moves toward a "tunneling-ready" configuration, the nicotinamide ring rotates to become almost perpendicular to the isoalloxazine ring.     

Ab initio path‐integral calculations of kinetic and equilibrium isotope effects on base‐catalyzed RNA transphosphorylation models

Detailed understandings of the reaction mechanisms of RNA catalysis in various environments can have profound importance for many applications, ranging from the design of new biotechnologies to the unraveling of the evolutionary origin of life. An integral step in the nucleolytic RNA catalysis is self‐cleavage of RNA strands by 2′‐O‐transphosphorylation. Key to elucidating a reaction mechanism is determining the molecular structure and bonding characteristics of transition state. A direct and powerful probe of transition state is measuring isotope effects on biochemical reactions, particularly if we can reproduce isotope effect values from quantum calculations. This article significantly extends the scope of our previous joint experimental and theoretical work in examining isotope effects on enzymatic and nonenzymatic 2′‐O‐transphosphorylation reaction models that mimic reactions catalyzed by RNA enzymes (ribozymes), and protein enzymes such as ribonuclease A (RNase A). Native reactions are studied, as well as reactions with thio substitutions representing chemical modifications often used in experiments to probe mechanism. Here, we report and compare results from eight levels of electronic‐structure calculations for constructing the potential energy surfaces in kinetic and equilibrium isotope effects (KIE and EIE) computations, including a “gold‐standard” coupled‐cluster level of theory [CCSD(T)]. In addition to the widely used Bigeleisen equation for estimating KIE and EIE values, internuclear anharmonicity and quantum tunneling effects were also computed using our recently developed ab initio path‐integral method, that is, automated integration‐free path‐integral method. The results of this work establish an important set of benchmarks that serve to guide calculations of KIE and EIE for RNA catalysis. © 2014 Wiley Periodicals, Inc.     

Small temperature dependence of the kinetic isotope effect for the hydride transfer reaction catalyzed by Escherichia coli dihydrofolate reductase

The H/D primary kinetic isotope effect (KIE) for the hydride transfer reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) is calculated as a function of temperature employing ensemble-averaged variational transition-state theory with multidimensional tunneling. The calculated KIEs display only a small temperature dependence over the temperature range of 5 to 45 degrees C. We identify two key features that contribute to canceling most of the temperature dependence of the KIE that would be expected on the basis of simpler models. Related issues such as the isotope effects on Arrhenius preexponential factors, large differences between free energies of activation and Arrhenius activation energy, and fluctuations of effective barriers are also discussed.     

Experimental and theoretical multiple kinetic isotope effects for an SN2 reaction. An attempt to determine transition-state structure and the ability of theoretical methods to predict experimental kinetic isotope effects

The secondary alpha-deuterium, the secondary beta-deuterium, the chlorine leaving-group, the nucleophile secondary nitrogen, the nucleophile (12)C/(13)C carbon, and the (11)C/(14)C alpha-carbon kinetic isotope effects (KIEs) and activation parameters have been measured for the S(N)2 reaction between tetrabutylammonium cyanide and ethyl chloride in DMSO at 30 degrees C. Then, thirty-nine readily available different theoretical methods, both including and excluding solvent, were used to calculate the structure of the transition state, the activation energy, and the kinetic isotope effects for the reaction. A comparison of the experimental and theoretical results by using semiempirical, ab initio, and density functional theory methods has shown that the density functional methods are most successful in calculating the experimental isotope effects. With two exceptions, including solvent in the calculation does not improve the fit with the experimental KIEs. Finally, none of the transition states and force constants obtained from the theoretical methods was able to predict all six of the KIEs found by experiment. Moreover, none of the calculated transition structures, which are all early and loose, agree with the late (product-like) transition-state structure suggested by interpreting the experimental KIEs.     

Intramolecular kinetic isotope effect in hydride transfer from dihydroacridine to a quinolinium ion. Rejection of a proposed two-step mechanism with a kinetically significant intermediate

The intramolecular kinetic isotope effect (KIE) for hydride transfer from 10-methyl-9,10-dihydroacridine to 1-benzyl-3-cyanoquinolinium ion has been found to be 5-6 by both (1)H NMR and mass spectrometry. This KIE is consistent with other hydride transfers. It is inconsistent with the high intermolecular KIEs derived by fitting to a two-step mechanism with a kinetically significant intermediate complex, and it is inconsistent with the strong temperature dependence of those KIEs. We therefore reject the two-step mechanism for this reaction, and we suggest that other cases proposed to follow this mechanism are in error.     

13C kinetic isotope effects in the copper(I)-mediated living radical polymerization of methyl methacrylate

Carbon-13 kinetic isotope effects (KIEs) have been determined for free-radical and copper-mediated living radical polymerizations of methyl methacrylate at 60 degrees C. While free-radical polymerization shows only one primary 13C KIE, on the least-substituted double bond carbon (k12/k13 = 1.045), two significant KIEs are observed, one on each double bond carbon, for copper-mediated polymerization (k12/k13(H2C=) = 1.050, k12/k13(=C <) = 1.010), showing that copper-mediated living radical polymerization does not propagate via a simple free radical process.     

The effect of solvent on the structure of the transition state for the S(N)2 reaction between cyanide ion and ethyl chloride in DMSO and THF probed with six different kinetic isotope effects

The secondary alpha- and beta-deuterium, the alpha-carbon, the nucleophile carbon, the nucleophile nitrogen, and the chlorine leaving group kinetic isotope effects for the S(N)2 reaction between cyanide ion and ethyl chloride were determined in the very slightly polar solvent THF at 30 degrees C. A comparison of these KIEs with those reported earlier for the same reaction in the polar solvent DMSO shows that the transition state in THF is only slightly tighter with very slightly shorter NC-C(alpha) and C(alpha)-Cl bonds. This minor change in transition state structure does not account for the different transition structures that were earlier suggested by interpreting the experimental KIEs and the gas-phase calculations, respectively. It therefore seems unlikely that the different transition states suggested by the two methods are due to the lack of appropriate solvent modeling in the theoretical calculations. Previously it was predicted that the transition state of S(N)2 reactions where the nucleophile and the leaving group have the same charge would be unaffected by a change in solvent. The experimental KIEs support this view.     

Hydride reduction of NAD+ analogues by isopropyl alcohol: kinetics, deuterium isotope effects and mechanism

Observed pseudo-first-order rate constants (k(obs)) of the hydride-transfer reactions from isopropyl alcohol (i-PrOH) to two NAD(+) analogues, 9-phenylxanthylium ion (PhXn(+)) and 10-methylacridinium ion (MA(+)), were determined at temperatures ranging from 49 to 82 degrees C in i-PrOH containing various amounts of AN or water. Formations of the alcohol-cation ether adducts (ROPr-i) were observed as side equilibria. The equilibrium constants for the conversion of PhXn(+) to PhXnOPr-i in i-PrOH/AN (v/v = 1) were determined, and the equilibrium isotope effect (EIE = K(i-PrOH)/K(i-PrOD)) at 62 degrees C was calculated to be 2.67. The k(H) of the hydride-transfer step for both reactions were calculated on the basis of the k(obs) and K. The corresponding deuterium kinetic isotope effects (e.g., KIE(OD)(H) = k(H)(i-PrOH)/k(H)(i-PrOD) and KIE(beta-D6)(H) = k(obs)(i-PrOH)/k(obs)((CD3)2CHOH)), as well as the activation parameters, were derived. For the reaction of PhXn(+) (62 degrees C) and MA(+) (67 degrees C), primary KIE(alpha-D)(H) (4.4 and 2.1, respectively) as well as secondary KIE(OD)(H) (1.07 and 1.18) and KIE(beta-D6)(H) (1.1 and 1.5) were observed. The observed EIE and KIE(OD)(H) were explained in terms of the fractionation factors for deuterium between OH and OH(+)(OH(delta+)) sites. The observed inverse kinetic solvent isotope effect for the reaction of PhXn(+) (k(obs)(i-PrOH)/k(obs)(i-PrOD) = 0.39) is consistent with the intermolecular hydride-transfer mechanism. The dramatic reduction of the reaction rate for MA(+), when the water or i-PrOH cosolvent was replaced by AN, suggests that the hydride-transfer T.S. is stabilized by H-bonding between O of the solvent OH and the substrate alcohol OH(delta+). This result suggests an H-bonding stabilization effect on the T.S. of the alcohol dehydrogenase reactions.     

Origin of the temperature dependence of isotope effects in enzymatic reactions: the case of dihydrofolate reductase

The origin of the temperature dependence of kinetic isotope effects (KIEs) in enzyme reactions is a problem of general interest and a major challenge for computational chemistry. The present work simulates the nuclear quantum mechanical (NQM) effects and the corresponding KIE in dihydrofolate reductase (DHFR) and two of its mutants by using the empirical valence bond (EVB) and the quantum classical path (QCP) centroid path integral approach. Our simulations reproduce the overall observed trend while using a fully microscopic rather than a phenomenological picture and provide an interesting insight. It appears that the KIE increases when the distance between the donor and acceptor increases, in a somewhat counter intuitive way. The temperature dependence of the KIE appears to reflect mainly the temperature dependence of the distance between the donor and acceptor. This trend is also obtained from a simplified vibronic treatment, but as demonstrated here, the vibronic treatment is not valid at short and medium distances, where it is essential to use the path integral or other approaches capable of moving seamlessly from the adiabatic to the diabatic limits. It is pointed out that although the NQM effects do not contribute to catalysis in DHFR, the observed temperature dependence can be used to refine the potential of mean force for the donor and acceptor distance and its change due to distanced mutations.     

Mechanistic Insights into RNA Transphosphorylation from Kinetic Isotope Effects and Linear Free Energy Relationships of Model Reactions

Phosphoryl transfer reactions are ubiquitous in biology and the understanding of the mechanisms whereby these reactions are catalyzed by protein and RNA enzymes is central to reveal design principles for new therapeutics. Two of the most powerful experimental probes of chemical mechanism involve the analysis of linear free energy relations (LFERs) and the measurement of kinetic isotope effects (KIEs). These experimental data report directly on differences in bonding between the ground state and the rate‐controlling transition state, which is the most critical point along the reaction free energy pathway. However, interpretation of LFER and KIE data in terms of transition‐state structure and bonding optimally requires the use of theoretical models. In this work, we apply density‐functional calculations to determine KIEs for a series of phosphoryl transfer reactions of direct relevance to the 2′‐O‐transphosphorylation that leads to cleavage of the phosphodiester backbone of RNA. We first examine a well‐studied series of phosphate and phosphorothioate mono‐, di‐ and triesters that are useful as mechanistic probes and for which KIEs have been measured. Close agreement is demonstrated between the calculated and measured KIEs, establishing the reliability of our quantum model calculations. Next, we examine a series of RNA transesterification model reactions with a wide range of leaving groups in order to provide a direct connection between observed Brønsted coefficients and KIEs with the structure and bonding in the transition state. These relations can be used for prediction or to aid in the interpretation of experimental data for similar non‐enzymatic and enzymatic reactions. Finally, we apply these relations to RNA phosphoryl transfer catalyzed by ribonuclease A, and demonstrate the reaction coordinate–KIE correlation is reasonably preserved. A prediction of the secondary deuterium KIE in this reaction is also provided. These results demonstrate the utility of building up knowledge of mechanism through the systematic study of model systems to provide insight into more complex biological systems such as phosphoryl transfer enzymes and ribozymes.     

The effect of solvent on a Lewis acid catalyzed Diels-Alder reaction,using computed and experimental kinetic isotope effects

A new transition structure for the Diels-Alder reaction between isoprene and acrolein catalyzed by Et(2)AlCl is found to reconcile reported discrepancies between computed and observed secondary kinetic isotope effects (KIEs). Including the effect of solvent realigns the computed results with experiment demonstrating the importance of nonbond interactions at transition structures. Comparison of experimental and newly predicted KIE data reaffirms the ability of theory and experiment to probe the mechanism and transition structure geometry of organic reactions.