Deuterium Kinetic Isotope Effects Research Articles

Investigating Inner-Sphere Reorganization via Secondary Kinetic Isotope Effects in the C−H Cleavage Reaction Catalyzed by Soybean Lipoxygenase: Tunneling in the Substrate Backbone as Well as the Transferred Hydrogen

This work describes the application of NMR to the measurement of secondary deuterium (2° (2)H) and carbon-13 ((13)C) kinetic isotope effects (KIEs) at positions 9-13 within the substrate linoleic acid (LA) of soybean lipoxygenase-1. The KIEs have been measured using LA labeled with either protium (11,11-h2-LA) or deuterium (11,11-d2-LA) at the reactive C11 position, which has been previously shown to yield a primary deuterium isotope effect of ca. 80. The conditions of measurement yield the intrinsic 2° (2)H and (13)C KIEs on k(cat)/K(m) directly for 11,11-d2-LA, whereas the values for the 2° (2)H KIEs for 11,11-h2-LA are obtained after correction for a kinetic commitment. The pattern of the resulting 2° (2)H and (13)C isotope effects reveals values that lie far above those predicted from changes in local force constants. Additionally, many of the experimental values cannot be modeled by electronic effects, torsional strain, or the simple inclusion of a tunneling correction to the rate. Although previous studies have shown the importance of extensive tunneling for cleavage of the primary hydrogen at C11 of LA, the present findings can only be interpreted by extending the conclusion of nonclassical behavior to the secondary hydrogens and carbons that flank the position undergoing C-H bond cleavage. A quantum mechanical method introduced by Buhks et al. [J. Phys. Chem. 1981, 85, 3763] to model the inner-sphere reorganization that accompanies electron transfer has been shown to be able to reproduce the scale of the 2° (2)H KIEs.

Kinetics of Reactions of the Actinomadura R39 dd-Peptidase with Specific Substrates

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

Catalytic Mechanism of a Heme and Tyrosyl Radical-Containing Fatty Acid α-(Di)oxygenase

The steady-state catalytic mechanism of a fatty acid α-(di)oxygenase is examined, revealing that a persistent tyrosyl radical (Tyr379(•)) effects O(2) insertion into C(α)-H bonds of fatty acids. The initiating C(α)-H homolysis step is characterized by apparent rate constants and deuterium kinetic isotope effects (KIEs) that increase hyperbolically upon raising the concentration of O(2). These results are consistent with H(•) tunneling, transitioning from a reversible to an irreversible regime. The limiting deuterium KIEs increase from ∼30 to 120 as the fatty acid chain is shortened from that of the native substrate. In addition, activation barriers increase in a manner that reflects decreased fatty acid binding affinities. Anaerobic isotope exchange experiments provide compelling evidence that Tyr379(•) initiates catalysis by H(•) abstraction. C(α)-H homolysis is kinetically driven by O(2) trapping of the α-carbon radical and reduction of a putative peroxyl radical intermediate to a 2(R)-hydroperoxide product. These findings add to a body of work which establishes large-scale hydrogen tunneling in proteins. This particular example is novel because it involves a protein-derived amino acid radical.

Cyclooxygenase reaction mechanism of prostaglandin H synthase from deuterium kinetic isotope effects

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS) is thought to involve a multistep mechanism with several radical intermediates. The proposed mechanism begins with the transfer of the C13 pro-(S) hydrogen atom from the substrate arachidonic acid (AA) to the Tyr385 radical in PGHS, followed by oxygen insertion and several bond rearrangements. The importance of the hydrogen-transfer step to controlling the overall kinetics of cyclooxygenase catalysis has not been directly examined. We quantified the non-competitive primary kinetic isotope effect (KIE) for both PGHS-1 and -2 using several deuterated AAs, including 13- pro-( S) d-AA, 13,13-d 2-AA and 10, 10, 13,13-d 4-AA. The primary KIE for steady-state cyclooxygenase catalysis, D k cat , ranged between 1.8 and 2.3 in oxygen electrode measurements. The intrinsic KIE of AA radical formation by C13 pro-(S) hydrogen abstraction in PGHS-1 was estimated to be 1.9–2.3 using rapid freeze-quench EPR kinetic analysis of anaerobic reactions and computer modeling to a mechanism that includes a slow formation of a pentadienyl AA radical and a rapid equilibration of the AA radical with a tyrosyl radical, NS1c. The observation of similar values for steady-state and pre-steady state KIEs suggests that hydrogen abstraction is a rate-limiting step in cyclooxygenase catalysis. The large difference of the observed KIE from that of plant lipoxygenases indicates that PGHS and lipoxygenases have very different mechanisms of hydrogen transfer.

Oxidative Addition of Ethyl Iodide to a Dimethylplatinum(II) Complex: Unusually Large Kinetic Isotope Effects and Their Transition-State Implications

The mechanism of oxidative addition of ethyl iodide to [PtMe2(2,2′-bipyridine)], 1, has been investigated by product analysis and by study of secondary deuterium kinetic isotope effects (KIEs), usi...

Evidence for Substrate Preorganization in the Peptidylglycine α-Amidating Monooxygenase Reaction Describing the Contribution of Ground State Structure to Hydrogen Tunneling

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme which catalyzes the post-translational modification of inactive C-terminal glycine-extended peptide precursors to the corresponding bioactive α-amidated peptide hormone. This conversion involves two sequential reactions both of which are catalyzed by the separate catalytic domains of PAM. The first step, the copper-, ascorbate-, and O(2)-dependent stereospecific hydroxylation at the α-carbon of the C-terminal glycine, is catalyzed by peptidylglycine α-hydroxylating monooxygenase (PHM). The second step, the zinc-dependent dealkylation of the carbinolamide intermediate, is catalyzed by peptidylglycine amidoglycolate lyase. Quantum mechanical tunneling dominates PHM-dependent C(α)-H bond activation. This study probes the substrate structure dependence of this chemistry using a set of N-acylglycine substrates of varying hydrophobicity. Primary deuterium kinetic isotope effects (KIEs), molecular mechanical docking, alchemical free energy perturbation, and equilibrium molecular dynamics were used to study the role played by ground-state substrate structure on PHM catalysis. Our data show that all Ν-acylglycines bind sequentially to PHM in an equilibrium-ordered fashion. The primary deuterium KIE displays a linear decrease with respect to acyl chain length for straight-chain N-acylglycine substrates. Docking orientation of these substrates displayed increased dissociation energy proportional to hydrophobic pocket interaction. The decrease in KIE with hydrophobicity was attributed to a preorganization event which decreased reorganization energy by decreasing the conformational sampling associated with ground state substrate binding. This is the first example of preorganization in the family of noncoupled copper monooxygenases.

Mechanism of the Cobalt Oxazoline Palladacycle (COP)-Catalyzed Asymmetric Synthesis of Allylic Esters

The catalytic enantioselective S(N)2' displacement of (Z)-allylic trichloroacetimidates catalyzed by the palladium(II) complex [COP-OAc](2) is a broadly useful method for the asymmetric synthesis of chiral branched allylic esters. A variety of experiments aimed at elucidating the nature of the catalytic mechanism and its rate- and enantiodetermining steps are reported. Key findings include the following: (a) the demonstration that a variety of bridged-dipalladium complexes are present and constitute resting states of the COP catalyst (however, monomeric palladium(II) complexes are likely involved in the catalytic cycle); (b) labeling experiments establishing that the reaction proceeds in an overall antarafacial fashion; (c) secondary deuterium kinetic isotope effects that suggest substantial rehybridization at both C1 and C3 in the rate-limiting step; and (d) DFT computational studies (B3-LYP/def2-TZVP) that provide evidence for bidentate substrate-bound intermediates and an anti-oxypalladation/syn-deoxypalladation pathway. These results are consistent with a novel mechanism in which chelation of the imidate nitrogen to form a cationic palladium(II) intermediate activates the alkene for attack by external carboxylate in the enantiodetermining step. Computational modeling of the transition-state structure for the acyloxy palladation step provides a model for enantioinduction.

Kinetics and Inhibition of Nicotinamidase from Mycobacterium tuberculosis

Nicotinamidase/pyrazinamidase (PncA) is involved in the NAD+ salvage pathway of Mycobacterium tuberculosis and other bacteria. In addition to hydrolyzing nicotinamide into nicotinic acid, PncA also hydrolyzes the prodrug pyrazinamide to generate the active form of the drug, pyrazinoic acid, which is an essential component of the multidrug treatment of TB. A coupled enzymatic activity assay has been developed for PncA that allows for the spectroscopic observation of enzyme activity. The enzyme activity was essentially pH-independent under the conditions tested; however, the measurement of the pH dependence of iodoacetamide alkylation revealed a pK value of 6.6 for the active site cysteine. Solvent deuterium kinetic isotope effects revealed an inverse value for kcat of 0.64, reconfirming the involvement of a thiol group in the mechanism. A mechanism is proposed for PncA catalysis that is similar to the mechanisms proposed for members of the nitrilase superfamily, in which nucleophilic attack by the active site cysteine generates a tetrahedral intermediate that collapses with the loss of ammonia and subsequent hydrolysis of the thioester bond by water completes the cycle. An inhibitor screen identified the competitive inhibitor 3-pyridine carboxaldehyde with a Ki of 290 nM. Additionally, pyrazinecarbonitrile was found to be an irreversible inactivator of PncA, with a kinact/KI of 975 M(−1) s(−1).

QM/MM Studies of the Matrix Metalloproteinase 2 (MMP2) Inhibition Mechanism of (S)-SB-3CT and its Oxirane Analogue

SB-3CT, (4-phenoxyphenylsulfonyl)methylthiirane, is a potent, mechanism-based inhibitor of the gelatinase sub-class of the matrix metalloproteinase (MMP) family of zinc proteases. The gelatinase MMPs are unusual in that there are several examples where both enantiomers of a racemic inhibitor have comparable inhibitory abilities. SB-3CT is one such example. Here, the inhibition mechanism of the MMP2 gelatinase by the (S)-SB-3CT enantiomer and its oxirane analogue is examined computationally, and compared to the mechanism of (R)-SB-3CT. Inhibition of MMP2 by (R)-SB-3CT was shown previously to involve enzyme-catalyzed C-H deprotonation adjacent to the sulfone, with concomitant opening by β-elimination of the sulfur of the three-membered thiirane ring. Similarly to the R enantiomer, (S)-SB-3CT was docked into the active site of MMP2, followed by molecular dynamics simulation to prepare the complex for combined quantum mechanics and molecular mechanics (QM/MM) calculations. QM/MM calculations with B3LYP/6-311+G(d,p) for the QM part (46 atoms) and the AMBER force field for the MM part were used to compare the reaction of (S)-SB-3CT and its oxirane analogue in the active site of MMP2 (9208 atoms). These calculations show that the barrier for the proton abstraction coupled ring opening reaction of (S)-SB-3CT in the MMP2 active site is 4.4 kcal/mol lower than its oxirane analogue, and the ring opening reaction energy of (S)-SB-3CT is only 1.6 kcal/mol less exothermic than its oxirane analogue. Calculations also show that the protonation of the ring-opened products by water is thermodynamically much more favorable for the alkoxide obtained from the oxirane, than for the thiolate obtained from the thiirane. In contrast to (R)-SB-3CT and the R-oxirane analogue, the double bonds of the ring-opened products of (S)-SB-3CT and its S-oxirane analogue have the cis-configuration. Vibrational frequency and intrinsic reaction path calculations on a reduced size QM/MM model (2747 atoms) provide additional insight into the mechanism. These calculations yield 5.9 and 6.7 for the deuterium kinetic isotope effect for C-H bond cleavage in the transition state for the R and S enantiomers of SB-3CT, in good agreement with the experimental results.

Reaction of C2(a Π3u) with methanol: Temperature dependence and deuterium isotope effect

Bimolecular rate constants for the gas-phase reactions of C2(a Π3u) with a variety of methanol isotopomers including CH3OH (k1), CH3OD (k2), CD3OH (k3), and CD3OD (k4) have been measured over the temperature range of 293–673 K by means of pulsed laser photolysis/laser-induced fluorescence technique. The rate constants, in units of cm3 molecule−1 s−1, can be fitted by the normal Arrhenius expressions: k1(T)=(1.32±0.02)×10−11 exp[−(366.80±4.44)/T], k2(T)=(1.34±0.02)×10−11 exp[−(376.86±5.09)/T], k3(T)=(1.09±0.02)×10−11 exp[−(640.00±7.23)/T], and k4(T)=(1.12±0.01)×10−11 exp[−(666.37±4.63)/T], where all error estimates are ±2σ and represent the precision of the fit. The observed deuterium kinetic isotope effects, k1/k2 and k1/k3, along with the positive temperature dependences of k(T), allow us to reach a conclusion that the reaction of C2(a Π3u) with methanol in 293–673 K proceeds via a site-specific hydrogen abstraction mechanism, that is, H-atom abstraction from the methyl site rather than from the hydroxyl site dominating reactivity.

Oxidation reactions performed by soluble methane monooxygenase hydroxylase intermediates H(peroxo) and Q proceed by distinct mechanisms.

Soluble methane monooxygenase is a bacterial enzyme that converts methane to methanol at a carboxylate-bridged diiron center with exquisite control. Because the oxidizing power required for this transformation is demanding, it is not surprising that the enzyme is also capable of hydroxylating and epoxidizing a broad range of hydrocarbon substrates in addition to methane. In this work we took advantage of this promiscuity of the enzyme to gain insight into the mechanisms of action of H(peroxo) and Q, two oxidants that are generated sequentially during the reaction of reduced protein with O(2). Using double-mixing stopped-flow spectroscopy, we investigated the reactions of the two intermediate species with a panel of substrates of varying C-H bond strength. Three classes of substrates were identified according to the rate-determining step in the reaction. We show for the first time that an inverse trend exists between the rate constant of reaction with H(peroxo) and the C-H bond strength of the hydrocarbon examined for those substrates in which C-H bond activation is rate-determining. Deuterium kinetic isotope effects revealed that reactions performed by Q, but probably not H(peroxo), involve extensive quantum mechanical tunneling. This difference sheds light on the observation that H(peroxo) is not a sufficiently potent oxidant to hydroxylate methane, whereas Q can perform this reaction in a facile manner. In addition, the reaction of H(peroxo) with acetonitrile appears to proceed by a distinct mechanism in which a cyanomethide anionic intermediate is generated, bolstering the argument that H(peroxo) is an electrophilic oxidant that operates via two-electron transfer chemistry.

Concerted Proton−Electron Transfer to Dioxygen in Water

Concerted proton-electron transfer (CPET) is documented for the homogeneous reduction of O(2) to HO(2)(*) in water by the one-electron-reduced heteropolytungstate anion, alpha-PW(12)O(40)(4-) (1(1e)). At 0.01-0.3 M H(+), O(2) reduction occurs via outer-sphere electron transfer followed by proton transfer (ETPT, with rate constant k(ET)). Between 0.30 and 1.9 M H(+), rates increase linearly with [H(+)] due to a parallel CPET pathway in which H(+) is now a reactant: (1/2)k(obs) = k(ET) + k(CPET)[H(+)] (k(ET) = 1.2 M(-1) s(-1); k(CPET) = 0.8 M(-2) s(-1)). Control experiments rule out preassociation between H(+) and 1(1e). Analysis of plausible rate expressions shows that the first-order dependence on [H(+)] is uniquely consistent with multisite CPET, and a deuterium kinetic isotope effect of 1.7 is observed. Reductions of O(2) by alpha-SiW(12)O(40)(5-) confirm theoretical predictions that CPET decreases in significance as ET becomes less endergonic. Marcus analysis, including the temperature dependence of DeltaG(o), gives reorganization energies, lambda(ET) = 41.5 kcal mol(-1) and lambda(CPET) = 52.4 kcal mol(-1). At 1.5 M H(+), approximately 75% of the (1(1e),O(2)) encounter pairs form within 6 A of H(+) ions. This value (6 +/- 1 A) is the "reaction distance" for proton diffusion and probably close to that for CPET. Even so, the 70-200 ps lifetimes of the (1(1e),O(2)) pairs provide additional time for H(+) to diffuse closer to O(2). CPET is first-order in [H(+)] because k(e) for "cage escape" from (1(1e),O(2)) pairs is much larger than k(CPET), such that the rate expression for CPET becomes -(1/2)d[1(1e)]/dt = (k(d)/k(e))k(CPET)[1(1e)][O(2)][H(+)], where k(d) is the rate constant for (1(1e),O(2)) pair formation. Overall, the findings suggest that the emergence of CPET, with hydronium ion as the proton donor, may prove a general feature of sufficiently endergonic reductions of dioxygen by otherwise "outer-sphere" complexes (or electrode reactions) at sufficiently low pH values in water.

Brønsted Analysis of an Enzyme-Catalyzed Pseudo-Deglycosylation Reaction: Mechanism of Desialylation in Sialidases

The Micromonospora viridifaciens Y370G inverting mutant sialidase has been found to possess beta-sialidase activity with various fluoro-substituted phenyl beta-sialosides. A reagent panel of seven mono- and difluorophenyl beta-d-sialosides was synthesized, and these compounds were used, in conjunction with the parent phenyl beta-d-sialoside, to probe the mechanism of M. viridifaciens Y370G mutant sialidase-catalyzed hydrolyses. These hydrolysis reactions mimic the deglycosylation reaction step of the crucial tyrosinyl enzyme-bound intermediate that is formed during the corresponding wild-type sialidase reactions. The derived Brønsted parameter (beta(lg)) on k(cat)/K(m) is -0.46 +/- 0.02 for the four substrates that display significant activity, and these span a range of leaving group abilities (as judged by the pK(a) of their conjugate acids being between 7.09 and 9.87). The 4-fluoro, 2,3- and 2,5-difluorosubstrates display a diminished activity, whereas the 3,5-difluoro compound undergoes catalyzed hydrolysis exceedingly slowly. These observations, taken with solvent deuterium kinetic isotope effects (k(H(2))(O)/k(D(2))(O)) on the catalyzed hydrolysis of the 2-fluorophenyl substrate of 0.88 +/- 0.24 (k(cat)/K(m)) and 1.16 +/- 0.12 (k(cat)) and the poor inhibition shown by phenol (IC(50) > 1 mM), are consistent with glycosidic C-O cleavage being rate determining for both k(cat)/K(m) and k(cat) with little or no protonation of the departing aryloxide leaving group. The kinetic data reported herein are consistent with rate-limiting glycoside hydrolysis occurring via two distinct transition states that incorporates a nonproductive binding component for the tighter binding substrates.

Proton and proton‐coupled electron transfer with paradigms towards single‐molecule systems

AbstractProton (PT), H‐atom and proton coupled electron transfer (PCET) are ubiquitously encountered in chemical and biological processes. PT and H‐atom transfer can belong to the partially or totally adiabatic limits representing ‘weak’ or ‘strong’ interactions between the donor and acceptor, reflected most conspicuously in large, i.e. ≫1 and small, i.e. 1–2, kinetic deuterium isotope effects (KIE). In view of the short proton/H‐atom transfer distances, the electronically adiabatic limit prevails in either case. The PCET notion applies from sequential PT and ET events to fully synchronous ET/PT as in H‐atom transfer. We first overview these notions. We then address several classes of PT reactions not commonly addressed in analytical condensed matter PT/H‐atom transfer theory. These include viscosity (relaxation) controlled PT and KIE < 1 in protein systems. Other classes are PT in strongly hydrogen bonded systems such as excess proton conduction in aqueous solution or in biological or synthetic membranes, and PT in the ‘inverted’ free energy region where excited proton vibrational states and a maximum in the Brønsted relation are important. We finally invoke an approach to single‐molecule PT and H‐atom transfer where the PT/H‐atom transferring molecules are enclosed between the substrate and tip in electrochemical (in situ) scanning tunnelling microscopy (STM) or between a pair of nanoscale electrodes. No data are presently available but could be within reach considering the recent success of in situ STM in single‐molecule electron transfer. Elusive notions such as PT/H‐atom transfer distances and distance dependent KIEs would become accessible based on this approach. Copyright © 2010 John Wiley & Sons, Ltd.

Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification

Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate-subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by yeast, particularly when the carbon source is acid-treated lignocellulose, which contains furfural at a relatively high concentration. NADPH is Ari1p's best known hydride donor. Here we report the stereochemistry of the hydride transfer step, determined by using (4R)-[4-(2)H]NADPD and (4S)-[4-(2)H]NADPD and unlabeled furfural in Ari1p-catalyzed reactions and following the deuterium atom into products 2-furanmethanol or NADP(+). Analysis of the products demonstrates unambiguously that Ari1p directs hydride transfer from the si face of NADPH to the re face of furfural. The singular orientation of substrates enables construction of a model of the Michaelis complex in the Ari1p active site. The model reveals hydrophobic residues near the furfural binding site that, upon mutation, may increase specificity for furfural and enhance enzyme performance. Using (4S)-[4-(2)H]NADPD and NADPH as substrates, primary deuterium kinetic isotope effects of 2.2 and 2.5 were determined for the steady-state parameters k(cat)(NADPH) and k(cat)/K(m)(NADPH), respectively, indicating that hydride transfer is partially rate limiting to catalysis.

The kinetic and solvent deuterium isotope effects in the 4- and 5-positions of the indole ring on the enzymatic decomposition of L-tryptophan

The kinetic and solvent deuterium isotope effects in the 4- and 5-positions of the indole ring on the enzymatic decomposition of l-tryptophan catalysed by the enzyme TPase (EC. 4.1.99.1) were determined. The isotope effects were investigated by the non-competitive method using [4′-2H]-l-tryptophan, which was enriched in deuterium in 70% in the 4-position. The numerical values of isotope effects for 100% enrichment in deuterated label in that position were calculated by approximation. Those same isotope effects were determined for [5′-2H]-l-tryptophan fully deuteriated in the 5′ -position.

Pyrolysis of α- and β-Heteroatoms Substituted Ethyl Phenyl Sulfoxides

A study on the mechanism of the thermal decomposition of α- and β-heteroatoms substituted ethyl phenyl sulfoxides was carried out using 1-chloroethyl phenyl sulfoxide (1); two diastereomeric 1-acetoxyethyl (substituted phenyl) sulfoxides (2a) and (2b); and 2-chloroethyl phenyl, 2-bromoethyl phenyl, and 2-methoxyethyl phenyl sulfoxides (3, 4, 5). The rate of pyrolysis of 1 was 4.8 times faster at 160°C than that of ethyl phenyl sulfoxide used as a reference, while those of 2a and 2b were 107 and 155 times faster, respectively. The results indicate that the lone pair of electrons on the α-heteroatoms has a larger rate acceleration effect than the electronegativity of them. The substituent effects of the phenyl group of 2a and 2b gave positive Hammett ρ-values (ρ a = 0.76 and ρ b = 0.80 vs. σ). Activation parameters for 2a and 2b are as follows: 2a, ΔH ‡ = 112 kJmol−1, ΔS ‡ = −20 JK−1mol−1; 2b, ΔH ‡ = 107 kJmol−1, ΔS ‡ = −29 JK-1mol−1. Large deuterium kinetic isotope effects for 1-acetoxyethyl-2,2,2-d3 phenyl sulfoxides (2ad and 2bd) were observed (kH/kD = 3.5 ∼ 4.1). These results suggest that the pyrolysis of α-heteroatom substituted ethyl phenyl sulfoxides proceeds via a five-membered transition state deviated to E1-like in character. On the other hand, from the results of kinetics for the pyrolysis of 3, 4, and 5, no effect by the β-halogen atoms or some deceleration effect by the β-methoxy group was observed. Thus the reaction seems to proceed via an E1-like mechanism.

Anilinolysis of Diethyl Phosphinic Chloride in Acetonitrile

Table 1. The second-order rate constants (kH(D) × 10 /M‒1 s‒1), selectivity parameters (ρX and βX), and deuterium kinetic isotope effects (kH/kD) for the reactions of diethyl phosphinic chloride (2) with XC6H4NH2(D2) in MeCN at 50.0 C X kH × 10 /M s kD × 10 /M s kH/kD 4-MeO 44.4 ± 0.2 45.6 ± 0.6 0.974 ± 0.001 4-Me 29.8 ± 0.9 31.2 ± 0.9 0.955 ± 0.001 3-Me 23.0 ± 0.6 25.1 ± 0.7 0.931 ± 0.001 H 16.2 ± 0.6 17.7 ± 0.3 0.915 ± 0.001 3-MeO 11.8 ± 0.3 13.2 ± 0.4 0.894 ± 0.003 4-Cl 6.89 ± 0.04 8.08 ± 0.20 0.853 ± 0.003 3-Cl 4.24 ± 0.06 5.12 ± 0.06 0.828 ± 0.003 –ρX 1.59 ± 0.05 1.48 ± 0.05 βX 0.56 ± 0.03 0.52 ± 0.03 The σ values were taken from Hansch, C.; Leo, A.; Taft, R. W. Chem. Rev. 1991, 91, 165. The pKa values of X-anilines in water were taken from Streitwieser, A. Jr.; Heathcock, C. H.; Kosower, E. M. Introduction to Organic Chemistry, 4th ed.; Macmillan: New York, 1992; p 735. Standard deviation. Calculated from kH. Correlation coefficient, r = 0.998. r = 0.998. Calculated from kD. r = 0.993. r = 0.993. Standard error {= 1/kD [(∆kH) + (kH/kD) × (∆kD)]} from Crumpler, T. B.; Yoh, J. H. Chemical Computations and Errors; John Wiley: New York, 1940; p 178. Nucleophilic substitution at a phosphoryl (P=O) or thiophosphoryl (P=S) center generally proceeds either through stepwise mechanism with a trigonal bipyramidal pentacoordinate (TBP-5C) intermediate or a concerted SN2 mechanism with a TBP-5C transition state (TS). Continuing the studies on phosphoryl transfer reactions, the reactions of diethyl phosphinic chloride [Et2P(=O)Cl; 2] with substituted anilines (XC6H4NH2) are investigated kinetically in acetonitrile at 50.0 C (eq 1). The deuterium kinetic isotope effects (DKIEs; kH/kD) involving deuterated aniline (XC6H4ND2) nucleophiles are also investigated, since the DKIEs are one of the strong tools to clarify the reaction mechanism. The DKIEs have provided a useful means to determine the TS structures in nucleophilic substitution reactions, and how the reactants, especially through changes in substituents, alter the TS structures. Incorporation of deuterium in the nucleophile has an advantage in that the α-DKIEs reflect only the degree of bond formation. When partial deprotonation of the aniline occurs in a rate-limiting step by hydrogen bonding, the kH/kD values are greater than unity, primary normal (kH/kD > 1.0). In contrast, the DKIEs can only be secondary inverse (kH/kD < 1.0) in a normal SN2 reaction, since the N-H(D) vibrational frequencies invariably increase upon going to the TS because of an increase in steric congestion in the bond-making process. This work aims to gain further information into the phosphoryl transfer reactions, as well as to compare the mechanism and DKIEs of the anilinolyses of dimethyl [Me2P(=O)Cl; 1], methyl phenyl [MePhP(=O)Cl; 3], and diphenyl [Ph2P (=O)Cl; 4] phosphinic chlorides in MeCN.

Tunneling Effect in Regeneration Reaction of Vitamin E by Ubiquinol

A kinetic study of the regeneration reaction of vitamin E by ubiquinol was carried out by means of double-mixing stopped-flow spectroscopy. A substantial deuterium kinetic-isotope effect was observed on the second-order rate constant and the activation energy. In the regeneration reaction of alpha-tocopherol, deuteration of ubiquinol increased and decreased the activation energy and the second-order rate constant by 6.1 kJ/mol and a factor of 18.3, respectively. From this result, it is considered that proton tunneling plays an important role in the regeneration reaction of vitamin E by ubiquinol. The conditions under which the tunneling effect becomes an important factor were discussed in conjunction of our experimental results.

Measurement of the Intramolecular Isotope Effect on Aliphatic Hydroxylation by Chromobacterium violaceum Phenylalanine Hydroxylase

The non-heme iron enzyme phenylalanine hydroxylase from Chromobacterium violaceum has previously been shown to catalyze the hydroxylation of benzylic and aliphatic carbons in addition to the normal aromatic hydroxylation reaction. The intrinsic isotope effect for hydroxylation of 3-cyclochexylalanine by the enzyme was determined in order to gain insight into the reactivity of the iron center. With 3-[(2)H(11)-cyclohexyl]alanine as the substrate, the isotope effect on the k(cat) value was 1, consistent with an additional step in the overall reaction being significantly slower than hydroxylation. Consequently, the isotope effect was determined as an intramolecular effect by measuring the amount of deuterium lost in the hydroxylation of 3-[1,2,3,4,5,6-(2)H(6)-cyclohexyl]alanine. The ratio of 4-HO-cyclohexylalanine that retained deuterium to that which lost one deuterium atom was 2.8. This gave a calculated value of 12.6 for the ratio of the primary deuterium kinetic isotope effect to the secondary isotope effect. This value is consistent with hydrogen atom abstraction by an electrophilic Fe(O) center and a contribution of quantum-mechanical tunneling to the reaction.