Quantum dynamics of hydride transfer catalyzed by bimetallic electrophilic catalysis: synchronous motion of Mg(2+) and H(-) in xylose isomerase

Xylose isomerase exhibits a bridged-bimetallic active-site motif in which the substrate is bound to two metals connected by a glutamate bridge, and X-ray crystallographic studies suggest that metal movement is involved in the hydride transfer rate-controlling catalytic step. Here we report classical/quantal dynamical simulations of this step that provide new insight into the metal motion. The potential energy surface is calculated by treating xylose with semiempirical molecular orbital theory augmented by a simple valence bond potential and the rest of the system by molecular mechanics. The rate constant for the hydride-transfer step was calculated by ensemble-averaged dynamical simulations including both variational transition-state theory for determination of the statistically averaged dynamical bottleneck and optimized multidimensional tunneling calculations. The dynamics calculations include 25 317 atoms, with quantized vibrational free energy in 89 active-site degrees of freedom, and with 32 atoms moving through static secondary zone transition-state configurations in the quantum tunneling simulation. Our simulations show that the average Mg-Mg distance R increases monotonically as a function of the hydride-transfer progress variable z. The range of the average R along the reaction path is consistent with the X-ray structure, thus providing a dynamical demonstration of the postulated role of Mg in catalysis. We also predicted the primary deuterium kinetic isotope effect (KIE) for the chemical step. We calculated a KIE of 3.8 for xylose at 298 K, which is consistent with somewhat smaller experimentally observed KIEs for glucose substrate at higher temperatures. More than half of our KIE is due to tunneling; neglecting quantum effects on the reaction coordinate reduces the calculated KIE to 1.8.     

Mutagenesis of morphinone reductase induces multiple reactive configurations and identifies potential ambiguity in kinetic analysis of enzyme tunneling mechanisms

We have identified multiple reactive configurations (MRCs) of an enzyme-coenzyme complex that have measurably different kinetic properties. In the complex formed between morphinone reductase (MR) and the NADH analogue 1,4,5,6-tetrahydro-NADH (NADH4) the nicotinamide moiety is restrained close to the FMN isoalloxazine ring by hydrogen bonds from Asn-189 and His-186 as determined from the X-ray crystal structure. Molecular dynamic simulations indicate that removal of one of these hydrogen bonds in the N189A MR mutant allows the nicotinamide moiety to occupy a region of configurational space not accessible in wild-type enzyme. Using stopped-flow spectroscopy, we show that reduction of the FMN cofactor by NADH in N189A MR is multiphasic, identifying at least four different reactive configurations of the MR-NADH complex. This contrasts with wild-type MR in which hydride transfer occurs by environmentally coupled tunneling in a single kinetic phase [Pudney et al. J. Am. Chem. Soc. 2006, 128, 14053-14058]. Values for primary and alpha-secondary kinetic isotope effects, and their temperature dependence, for three of the kinetic phases in the N189A MR are consistent with hydride transfer by tunneling. Our analysis enables derivation of mechanistic information concerning different reactive configurations of the same enzyme-coenzyme complex using ensemble stopped-flow methods. Implications for the interpretation from kinetic data of tunneling mechanisms in enzymes are discussed.     

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.     

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.     

Nonperfect synchronization of reaction center rehybridization in the transition state of the hydride transfer catalyzed by dihydrofolate reductase

It has been suggested that the magnitudes of secondary kinetic isotope effects (2 degrees KIEs) of enzyme-catalyzed reactions are an indicator of the extent of reaction-center rehybridization at the transition state. A 2 degrees KIE value close to the corresponding secondary equilibrium isotope effects (2 degrees EIE) is conventionally interpreted as indicating a late transition state that resembles the final product. The reliability of using this criterion to infer the structure of the transition state is examined by carrying out a theoretical investigation of the hybridization states of the hydride donor and acceptor in the Escherichia coli dihydrofolate reductase (ecDHFR)-catalyzed reaction for which a 2 degrees KIE close to the 2 degrees EIE was reported. Our results show that the donor carbon at the hydride transfer transition state resembles the reactant state more than the product state, whereas the acceptor carbon is more productlike, which is a symptom of transition state imbalance. The conclusion that the isotopically substituted carbon is reactant-like disagrees with the conclusion that would have been derived from the criterion of 2 degrees KIEs and 2 degrees EIEs, but the breakdown of the correlation with the equilibrium isotope effect can be explained by considering the effect of tunneling.     

Hydride transfer catalyzed by xylose isomerase: mechanism and quantum effects

We have applied molecular dynamics umbrella-sampling simulation and ensemble-averaged variational transition state theory with multidimensional tunneling (EA-VTST/MT) to calculate the reaction rate of xylose-to- xylulose isomerization catalyzed by xylose isomerase in the presence of two Mg2+ ions. The calculations include determination of the free energy of activation profile and ensemble averaging in the transmission coefficient. The potential energy function is approximated by a combined QM/MM/SVB method involving PM3 for the quantum mechanical (QM) subsystem, CHARMM22 and TIP3P for the molecular mechanical (MM) environment, and a simple valence bond (SVB) local function of two bond distances for the hydride transfer reaction. The simulation confirms the essential features of a mechanism postulated on the basis of kinetics and X-ray data by Whitlow et al. (Whitlow, M.; Howard, A. J.; Finzel, B. C.; Poulos, T. L.; Winborne, E.; Gilliland, G. L. Proteins 1991, 9, 153) and Ringe, Petsko, and coworkers (Labie, A.; Allen, K.-N.; Petsko, G. A.; Ringe, D. Biochemistry 1994, 33, 5469). This mechanism involves a rate-determining 1,2-hydride shift with prior and post proton transfers. Inclusion of quantum mechanical vibrational energy is important for computing the free energy of activation, and quantum mechanical tunneling effects are essential for computing kinetic isotope effects (KIEs). It is found that 85% of the reaction proceeds by tunneling and 15% by overbarrier events. The computed KIE for the ratio of hydride to deuteride transfer is in good agreement with the experimental results. The molecular dynamics simulations reveal that proton and hydride transfer reactions are assisted by breathing motions of the mobile Mg2+ ion in the active site, providing evidence for concerted motion of Mg2+ during the hydride transfer step.     

Combined experimental and theoretical study on aromatic hydroxylation by mononuclear nonheme iron(IV)-oxo complexes

The hydroxylation of aromatic compounds by mononuclear nonheme iron(IV)-oxo complexes, [FeIV(Bn-tpen)(O)]2+ (Bn-tpen=N-benzyl-N,N',N'-tris(2-pyridylmethyl)ethane-1,2-diamine) and [FeIV(N4Py)(O)]2+ (N4Py=N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine), has been investigated by a combined experimental and theoretical approach. In the experimental work, we have performed kinetic studies of the oxidation of anthracene with nonheme iron(IV)-oxo complexes generated in situ, thereby determining kinetic and thermodynamic parameters, a Hammett rho value, and a kinetic isotope effect (KIE) value. A large negative Hammett rho value of -3.9 and an inverse KIE value of 0.9 indicate that the iron-oxo group attacks the aromatic ring via an electrophilic pathway. By carrying out isotope labeling experiments, the oxygen in oxygenated products was found to derive from the nonheme iron(IV)-oxo species. In the theoretical work, we have conducted density functional theory (DFT) calculations on the hydroxylation of benzene by [FeIV(N4Py)(O)]2+. The calculations show that the reaction proceeds via two-state reactivity patterns on competing triplet and quintet spin states via an initial rate determining electrophilic substitution step. In analogy to heme iron(IV)-oxo catalysts, the ligand is noninnocent and actively participates in the reaction mechanism by reshuttling a proton from the ipso position to the oxo group. Calculated kinetic isotope effects of C6H6 versus C6D6 confirm an inverse isotope effect for the electrophilic substitution pathway. Based on the experimental and theoretical results, we have concluded that the aromatic ring oxidation by mononuclear nonheme iron(IV)-oxo complexes does not occur via a hydrogen atom abstraction mechanism but involves an initial electrophilic attack on the pi-system of the aromatic ring to produce a tetrahedral radical or cationic sigma-complex.     

Tunneling in the 1,5-hydrogen shift reactions of 1,3-cyclopentadiene and 5-methyl-1,3-cyclopentadiene

MPW1K/6-31+G(d,p) calculations which include the effects of small curvature tunneling find that, around room temperature, thermally activated tunneling dominates the 1,5-hydrogen shift reactions of 1,3-cyclopentadiene (2a) and 5-methyl-1,3-cyclopentadiene (2c). The calculated temperature dependence of the H/D kinetic isotope effect (KIE) for the latter rearrangement agrees well with experimental measurements that were published nearly 40 years ago. It is argued that the experimental KIEs provide prima facie evidence for tunneling in this reaction. The calculations also predict that it should be possible, at least in principle, to confirm this conclusion by observing curvature in the Arrhenius plot for the rearrangement of 2c.     

Temperature dependence of the kinetic isotope effects in thymidylate synthase. A theoretical study

In recent years, the temperature dependence of primary kinetic isotope effects (KIE) has been used as indicator for the physical nature of enzyme-catalyzed H-transfer reactions. An interactive study where experimental data and calculations examine the same chemical transformation is a critical means to interpret more properly temperature dependence of KIEs. Here, the rate-limiting step of the thymidylate synthase-catalyzed reaction has been studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) simulations in the theoretical framework of the ensemble-averaged variational transition-state theory with multidimensional tunneling (EA-VTST/MT) combined with Grote-Hynes theory. The KIEs were calculated across the same temperature range examined experimentally, revealing a temperature independent behavior, in agreement with experimental findings. The calculations show that the H-transfer proceeds with ～91% by tunneling in the case of protium and ～80% when the transferred protium is replaced by tritium. Dynamic recrossing coefficients are almost invariant with temperature and in all cases far from unity, showing significant coupling between protein motions and the reaction coordinate. In particular, the relative movement of a conserved arginine (Arg166 in Escherichia coli ) promotes the departure of a conserved cysteine (Cys146 in E. coli ) from the dUMP by polarizing the thioether bond thus facilitating this bond breaking that takes place concomitantly with the hydride transfer. These promoting vibrations of the enzyme, which represent some of the dimensions of the real reaction coordinate, would limit the search through configurational space to efficiently find those decreasing both barrier height and width, thereby enhancing the probability of H-transfer by either tunneling (through barrier) or classical (over-the-barrier) mechanisms. In other words, the thermal fluctuations that are coupled to the reaction coordinate, together with transition-state geometries and tunneling, are the same in different bath temperatures (within the limited experimental range examined). All these terms contribute to the observed temperature independent KIEs in thymidylate synthase.     

Secondary kinetic isotope effect on the photoenolization of triplet o-methylanthrones. A microcanonical transition state theory calculation

The energy profile for the tautomerization reaction of 1,4-dimethylanthrone in the first triplet electronic state obtained through electronic calculations (B3LYP/ 6-31G(d)) is used to calculate the rate constants for the process at a wide range of energies using a modified RRKM microcanonical statistical formalism that takes into account tunneling. Through partial or total substitution of the hydrogen atoms of the methyl groups by deuterium atoms, it is possible to evaluate different primary and secondary kinetic isotope effects (KIE). These results can be compared with experimental data for these processes taking place in solid matrix at extremely low temperatures (4-50 K). Such a comparison allows us to conclude that the reaction is taking place at energies just slightly below (around 0.5 kcal/mol) the adiabatic potential energy barrier, a result that was previously found for other related molecules so that this mechanism may be extended to the photoenolization of other o-aryl methyl ketones. Analysis of the different factors contributing to the primary and secondary KIEs discloses that at energies not far below the adiabatic barrier, the tunneling effect is not the only factor that accounts for the large KIE but the differences in the energy level distribution upon isotopic substitution may be the predominant factor at a certain range of negative energies (this is especially so for the case of primary KIE). At positive energies (above the barrier) the levels factor is always the dominant factor in the total KIE.     

Conversion and origin of normal and abnormal temperature dependences of kinetic isotope effect in hydride transfer reactions

The effects of substituents on the temperature dependences of kinetic isotope effect (KIE) for the reactions of the hydride transfer from the substituted 5-methyl-6-phenyl-5,6-dihydrophenanthridine (G-PDH) to thioxanthylium (TX(+)) in acetonitrile were examined, and the results show that the temperature dependences of KIE for the hydride transfer reactions can be converted by adjusting the nature of the substituents in the molecule of the hydride donor. In general, electron-withdrawing groups can make the KIE to have normal temperature dependence, but electron-donating groups can make the KIE to have abnormal temperature dependence. Thermodynamic analysis on the possible pathways of the hydride transfer from G-PDH to TX(+) in acetonitrile suggests that the transfers of the hydride anion in the reactions are all carried out by the concerted one-step mechanism whether the substituent is an electron-withdrawing group or an electron-donating group. But the examination of Hammett-type free energy analysis on the hydride transfer reactions supports that the concerted one-step hydride transfer is not due to an elementary chemical reaction. The experimental values of KIE at different temperatures for the hydride transfer reactions were modeled by using a kinetic equation formed according to a multistage mechanism of the hydride transfer including a returnable charge-transfer complex as the reaction intermediate; the real mechanism of the hydride transfer and the root that why the temperature dependences of KIE can be converted as the nature of the substituents are changed were discovered.     

Ab initio molecular orbital study of the flavin-catalyzed dehydrogenation reaction of glycine - protein transport channel driving hydride-transfer mechanism

The reaction mechanism of flavin-catalyzed dehydrogenation of glycine has been studied by ab initio molecular orbital calculations using the 6-31G* basis set. 10-Methyl isoalloxazine (10-MIA) has been used as the flavin model compound. The results showed that when we assume a proton transport channel in amino acid oxidase, which is switched on by the substrate anion, the O12-protonated 10-MIA [10-MIAH⁺(O12)] is generated. The main structure of 10-MIAH⁺(O12) is one in which the central ring is expressed by an NAD⁺-like structure, which is favorable for driving the hydride-transfer reaction, i.e., the abstraction of the α-hydrogen of glycine by the hydride-transfer mechanism. We have found that this protonation results in a dramatic lowering of the activation energy of the reaction. The proposed mechanism is summarized as follows: the hydride transfer proceeds via two-electron transfer and synchronous intramolecular proton transfer → intermolecular proton transfer. Received: 10 August 1998 / Accepted: 17 September 1998 / Published online: 8 February 1999     

Tunneling and classical paths for proton transfer in an enzyme reaction dominated by tunneling: oxidation of tryptamine by aromatic amine dehydrogenase

Proton tunneling dominates the oxidative deamination of tryptamine catalyzed by the enzyme aromatic amine dehydrogenase. For reaction with the fast substrate tryptamine, a H/D kinetic isotope effect (KIE) of 55 +/- 6 has been reported-one of the largest observed in an enzyme reaction. We present here a computational analysis of this proton-transfer reaction, applying combined quantum mechanics/molecular mechanics (QM/MM) methods (PM3-SRP//PM3/CHARMM22). In particular, we extend our previous computational study (Masgrau et al. Science 2006, 312, 237) by using improved energy corrections, high-level QM/MM methods, and an ensemble of paths to estimate the tunneling contributions. We have carried out QM/MM molecular dynamics simulations and variational transition state theory calculations with small-curvature tunneling corrections. The results provide detailed insight into the processes involved in the reaction. Transfer to the O2 oxygen of the catalytic base, Asp128beta, is found to be the favored reaction both thermodynamically and kinetically, even though O1 is closer in the reactant complex. Comparison of quantum and classical models of proton transfer allows estimation of the contribution of hydrogen tunneling in lowering the barrier to reaction in the enzyme. A reduction of the activation free energy due to tunneling of 3.1 kcal mol-1 is found, which represents a rate enhancement due to tunneling by 2 orders of magnitude. The calculated KIE of 30 is significantly elevated over the semiclassical limit, in agreement with the experimental observations; a semiclassical value of 6 is obtained when tunneling is omitted. A polarization of the C-H bond to be broken is observed due to the close proximity of the catalytic aspartate and the (formally) positively charged imine nitrogen. A comparison is also made with the related quinoprotein methylamine dehydrogenase (MADH)-the much lower KIE of 11 that we obtain for the MADH/methylamine system is found to arise from a more endothermic potential energy surface for the MADH reaction.     

Density functional theory study of the mechanism of the BF(3)-catalyzed rearrangement of 2,3,3-trimethyl-1,2-epoxybutane to 2,3,3-trimethylbutanal

The potential energy surface for the rearrangement of BF(3)-coordinated 2,3,3-trimethyl-1,2-epoxybutane to 2,3, 3-trimethylbutanal has been investigated at the B3LYP/6-31G level of theory. SCRF(SCI-PCM) solvent calculations and theoretical primary and secondary kinetic isotope effects at the same level of theory provide support for a two-step process with ring opening of the BF(3)-coordinated epoxide to a tertiary carbocation intermediate followed by hydride/deuteride migration to give aldehyde. The experimentally measured primary isotope effect (k(H)(D)/k(D)(H)) requires a correction for an appropriate secondary isotope effect to give a true isotope effect k(H)(H)/k(D)(H). For the lowest energy pathway for hydride migration, the calculated secondary kinetic isotope effect is 0.92, which when applied to the experimentally measured isotope effect of k(H)(D)/k(D)(H) = 1.73 gives a revised "true" primary kinetic isotope effect of k(H)(H)/k(D)(H) = 1.59. This compares with a calculated value of 2.01. From intermediate 15, migration of the C1-H(a) proton via 19 is energetically favored over C1-H(b) migration via 18 and this result is consistent with the experimental results in which hydride migration of the proton cis to the methyl is favored.     

Calculations find that tunneling plays a major role in the reductive elimination of methane from hydridomethylbis(trimethylphosphine)platinum: how to confirm this computational prediction experimentally

DFT calculations, including the effects of small curvature tunneling, have been performed on the reductive elimination of methane from hydridomethylbis(trimethylphosphine)platinum (1d). The calculations find that at 250 K tunneling results in an increase in the rate constant for reductive elimination by a factor of 4, a lowering of Ea by 1.7 kcal/mol, and a decrease in A by a factor of nearly 10. Tunneling is also calculated to increase the primary H/D kinetic isotope effect (KIE) from k(1d)/k(1f) = 2.26 to k(1d)/k(1f) = 4.12 and to result in a large secondary KIE of k(1d)/k(1e) = 1.35. In addition, tunneling is predicted to result in a violation of the rule of the geometric mean, so that the secondary KIE for reductive elimination of methane-d1 from 1f is calculated to be k(1f)/k(1g) = 1.06, which is much smaller than the secondary KIE of k(1d)/k(1e) = 1.35 for reductive elimination of methane from 1d. Comparison of the measured values of k(1d)/k(1e) and k(1f)/k(1g) is therefore proposed as an experimental test of the prediction that tunneling plays an important role in the reductive elimination of methane from 1d.     

Pressure effects on enzyme-catalyzed quantum tunneling events arise from protein-specific structural and dynamic changes

The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions.     

The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

Background

The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent K _m of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude.

Results

We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold.

Conclusions

Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of K _m associated with kinase enzymes.     

Switchover of the Mechanism between Electron Transfer and Hydrogen‐Atom Transfer for a Protonated Manganese(IV)–Oxo Complex by Changing Only the Reaction Temperature

Hydroxylation of mesitylene by a nonheme manganese(IV)–oxo complex, [(N4Py)Mn^IV(O)]²⁺ ( 1 ), proceeds via one‐step hydrogen‐atom transfer (HAT) with a large deuterium kinetic isotope effect (KIE) of 3.2(3) at 293 K. In contrast, the same reaction with a triflic acid‐bound manganese(IV)‐oxo complex, [(N4Py)Mn^IV(O)]²⁺‐(HOTf)₂ ( 2 ), proceeds via electron transfer (ET) with no KIE at 293 K. Interestingly, when the reaction temperature is lowered to less than 263 K in the reaction of 2 , however, the mechanism changes again from ET to HAT with a large KIE of 2.9(3). Such a switchover of the reaction mechanism from ET to HAT is shown to occur by changing only temperature in the boundary region between ET and HAT pathways when the driving force of ET from toluene derivatives to 2 is around ?0.5 eV. The present results provide a valuable and general guide to predict a switchover of the reaction mechanism from ET to the others, including HAT.     

Kinetic isotope effects calculated with the instanton method

The ring-opening reaction of the cyclopropylcarbinyl radical proceeds via heavy-atom tunneling at low temperature. We used instanton theory to calculate tunneling rates and kinetic isotope effects with on-the-fly calculation of energies by density functional theory (B3LYP). The accuracy was verified by explicitly correlated coupled-cluster calculations (UCCSD(T)-F12). At cryogenic temperatures, we found protium/deuterium KIEs up to 13 and inverse KIEs down to 0.2. We also studied an intramolecular tautomerization reaction. A simple and computationally efficient method is proposed to calculate KIEs with the instanton method: the instanton path is assumed to be independent of the atomic masses. This results in surprisingly good estimates of the KIEs for the cyclopropylcarbinyl radical and for the secondary KIEs of the tautomerization. Challenges and capabilities of the instanton method for calculating KIEs are discussed.     

The gas-phase Meerwein reaction

A systematic investigation of a novel epoxide and thioepoxide ring expansion reaction promoted by gaseous acylium and thioacylium ions is reported. As ab initio calculations predict, and 18O-labeling and MS3 pentaquadrupole experiments demonstrate, the reaction proceeds by initial O(S)-acylation of the (thio)epoxides followed by rapid intramolecular nucleophilic attack that results in three-to-five-membered ring expansion, and forms cyclic 1,3-dioxolanylium, 1,3-oxathiolanylium, or 1,3-dithiolanylium ions. This gas-phase reaction is analogous to a condensed-phase reaction long since described by H. Meerwein (Chem. Ber. 1955, 67, 374), and is termed as "the gas-phase Meerwein reaction"; it occurs often to great extents or even exclusively, but in some cases, particularly for the most basic (thio)epoxides and the most acidic (thio)acylium ions, proton transfer (eventually hydride abstraction) competes efficiently, or even dominates. When (thio)epoxides react with (thio)-acylium ions, the reaction promotes O(S)-scrambling; when epoxides react with thioacylium ions and the adducts are dissociated, it promotes S/O replacement. An analogous four-to-six-membered ring expansion also occurs predominantly in reactions of trimethylene oxide with acylium and thioacylium ions.