Normal Isotope Effect Research Articles

Evidence for the Mechanism of Hydroxylation by 4-Hydroxyphenylpyruvate Dioxygenase and Hydroxymandelate Synthase from Intermediate Partitioning in Active Site Variants

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) each catalyze similar complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. The reactions differ in that HPPD hydroxylates at the ring C1 and HMS at the benzylic position. The HPPD reaction is more complex in that hydroxylation at C1 instigates a 1,2-shift of an aceto substituent. Despite that multiple intermediates have been observed to accumulate in single turnover reactions of both enzymes, neither enzyme exhibits significant accumulation of the hydroxylating intermediate. In this study we employ a product analysis method based on the extents of intermediate partitioning with HPP deuterium substitutions to measure the kinetic isotope effects for hydroxylation. These data suggest that, when forming the native product homogentisate, the wild-type form of HPPD produces a ring epoxide as the immediate product of hydroxylation but that the variant HPPDs tended to also show the intermediacy of a benzylic cation for this step. Similarly, the kinetic isotope effects for the other major product observed, quinolacetic acid, showed that either pathway is possible. HMS variants show small normal kinetic isotope effects that indicate displacement of the deuteron in the hydroxylation step. The relatively small magnitude of this value argues best for a hydrogen atom abstraction/rebound mechanism. These data are the first definitive evidence for the nature of the hydroxylation reactions of HPPD and HMS.

Kinetics and Mechanism of Hydrogen‐Atom Abstraction from Rhodium Hydrides by Alkyl Radicals in Aqueous Solutions

The kinetics of the reaction of benzyl radicals with [L(1)(H(2)O)RhH{D}](2+) (L(1)=1,4,8,11-tetraazacyclotetradecane) were studied directly by laser-flash photolysis. The rate constants for the two isotopologues, k=(9.3±0.6) × 10(7) M(-1) s(-1) (H) and (6.2±0.3) × 10(7) M(-1) s(-1) (D), lead to a kinetic isotope effect k(H)/k(D)=1.5±0.1. The same value was obtained from the relative yields of PhCH(3) and PhCH(2)D in a reaction of benzyl radicals with a mixture of rhodium hydride and deuteride. Similarly, the reaction of methyl radicals with {[L(1)(H(2)O)RhH](2+) + [L(1)(H(2)O)RhD](2+)} produced a mixture of CH(4) and CH(3)D that yielded k(H)/k(D)=1.42±0.07. The observed small normal isotope effects in both reactions are consistent with reduced sensitivity to isotopic substitution in very fast hydrogen-atom abstraction reactions. These data disprove a literature report claiming much slower kinetics and an inverse kinetic isotope effect for the reaction of methyl radicals with hydrides of L(1)Rh.

Kinetics and mechanism of the anilinolyses of aryl dimethyl, methyl phenyl and diphenyl phosphinates

The reactions of Z-aryl dimethyl (1), methyl phenyl (2), and diphenyl (3) phosphinates with X-anilines in dimethyl sulfoxide at 60.0 °C are studied kinetically. Kinetic results yield the primary normal deuterium kinetic isotope effects (DKIEs) involving deuterated aniline (XC(6)H(4)ND(2)) nucleophiles, k(H)/k(D) = 1.03-1.17, 1.15-1.29, and 1.24-1.51, and the cross-interaction constants (CICs), ρ(XZ) = 0.37, 0.34, and 0.65 for 1, 2, and 3, respectively. The steric effects of the ligands (R(1) and R(2)) on reaction rates play a role, but are relatively much smaller compared to other phosphinate systems. A stepwise mechanism with a rate-limiting leaving group expulsion from the intermediate is proposed on the basis of the CICs positive signs. The dominant frontside nucleophilic attack through a hydrogen-bonded, four-center-type transition state is proposed on the basis of primary normal DKIEs and large magnitudes of the CICs for 2 and 3, while both frontside and backside attack are proposed on the basis of relatively small primary normal DKIEs for 1.

Isotope Effects on the Nonequilibrium Dynamics of Ultrafast Photoinduced Proton-Coupled Electron Transfer Reactions in Solution.

The hydrogen/deuterium isotope effects on the ultrafast dynamics of photoinduced proton-coupled electron transfer (PCET) are investigated with a recently developed nonadiabatic dynamics approach. An ensemble of surface hopping trajectories is propagated according to a Langevin equation on electron-proton vibronic free energy surfaces that depend on a collective solvent coordinate. The calculations illustrate that ultrafast PCET reactions could exhibit a significant normal isotope effect, where PCET is faster for hydrogen than deuterium, but could also exhibit a negligible isotope effect or even a slight inverse isotope effect. The isotope effect is very small or absent when highly excited electron-proton vibronic states dictate the nonadiabatic dynamics and increases with greater participation of lower vibronic states. Thus, although the presence of a significant isotope effect strongly suggests that proton motion is coupled to electron transfer, the absence of an isotope effect does not exclude the possibility that proton transfer accompanies electron transfer in ultrafast photoinduced charge transfer processes.

Accumulation of Tetrahedral Intermediates in Cholinesterase Catalysis: A Secondary Isotope Effect Study

In a previous communication, kinetic β-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic β-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 ± 0.03, (D3)k(cat) = 1.12 ± 0.02 and (D3)βk(cat) = 0.97 ± 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.

Dehydrogenation of propane over Pt/KL catalyst: Investigating the role of L-zeolite structure on catalyst performance using isotope labeling

Dehydrogenation of propane using an equimolar mixture of propane- d 0 and propane- d 8 was investigated over 1%Pt/KL and 1%Pt/SiO 2 at atmospheric pressure and different reaction temperatures. A normal kinetic isotope effect exists ( k H/ k D = 1.4–1.5) when the reaction is conducted on Pt/KL at different temperatures (400, 500, and 600 °C), suggesting that C–H bond activation is involved in the kinetically relevant steps. Furtheremore, there is hardly any H–D exchange in the recovered propane during dehydrogenation at temperatures above 400 °C, suggesting that adsorption and subsequent dehydrogenation of propane are essentially irreversible and that C–H bond activation is the rate determining step. Unlike the case of hexane aromatization, the unique structure of L-zeolite does not help in controlling the entry of propane molecules into the lobes of the L-zeolite containing the active sites; hence, bimolecular reactions do occur, leading to coke formation and catalyst deactivation.

Isotope effects in the chlorination of naphthalene

The chlorination of naphthalene shows an inverse deuterium isotope effect, the reaction of naphthalene being slower than that of octadeuterionaphthalene by a factor of 0.85. The product-forming stages are also subject to a deuterium isotope effect which influences the partitioning between addition and substitution; the most direct interpretation of the results is that the values of kH/kD for the stages of proton-loss are subject to a normal deuterium isotope effect, with kH/kD ⦔ 1.4.

A secondary isotope effect study of equine serum butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine

β-Secondary deuterium isotope effects have been measured for equine serum butyrylcholinesterase-catalyzed hydrolysis of acetyl-L 3-thiocholine (L = H or 2H). The dependencies of initial rates on isotopic substrate concentrations show close adherence to Michaelis–Menten kinetics, and yield the following isotope effects: D3 k cat/ K m = 0.98 ± 0.02 and D3 k cat = 1.10 ± 0.02. The modestly inverse isotope effect on k cat/ K m is consistent with partial rate limitation by a step that converts the sp 2-hybridized ester carbonyl of the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis. On the other hand, the markedly normal isotope effect on k cat indicates that the Michaelis complex that accumulates at substrate saturation of the active site during catalytic turnover is a tetrahedral intermediate, whose decomposition is the rate-limiting step. These results compliment a previous report [J.R. Tormos et al., J. Am. Chem. Soc. 127 (2005) 14538–14539] that showed that substrate-activated hydrolysis of acetylthiocholine (ATCh), catalyzed by recombinant human butyrylcholinesterase, is also rate limited by decomposition of an accumulating tetrahedral intermediate.

Kinetics and Mechanism of the Aminolysis of Aryl N-Allyl Thiocarbamates in Acetonitrile

The aminolysis reactions of phenyl N-benzyl thiocarbamate with benzylamines in acetonitrile at are investigated. The reactions are first order in both the amine and the substrate. Under amine excess, pseudo-first coefficient () are obtained, plot of vs free amine concentration are linear. The signs of ( and with respect to the sustituent in the substrate and large value indicate that the reactions proceed concerted mechanism. The normal kinetic isotope effects ( = 1.3 ~ 1.5) involving deuterated benzylamine nucleophiles suggest a hydrogen-bonded, four-centered-type transition state. The activation parameters, and , are consistent with this transition state structure.

Mechanism of general acid–base catalysis in transesterification of an RNA model phosphodiester studied with strongly basic catalysts

Using 80% vol aqueous DMSO as the reaction medium for transesterification of an RNA model substrate 2-hydroxypropyl 4-nitrophenyl phosphate allows one to observe catalysis in buffer mixtures composed of highly basic components such as guanidines, amidines or alkylamines, which provide up to 10(3)-fold accelerations over the background reaction in the 0.01-0.1 M concentration range. The rate law k(obs) = k(1)[B] + k(2)[B][BH(+)] was established indicating contributions from both simple general base catalysis and the reaction involving concerted action of neutral (B) and protonated (BH(+)) forms of the buffer. The catalytic efficiency of guanidinium and amidinium cations is 10 times larger than that of more acidic ammonium cations. Rate constants k(1) and k(2) obey the Brønsted equations with the slopes 0.77 and 0.69 respectively. Proton inventory for k(2) (B = guanidine) in D(2)O/H(2)O mixtures gives two fractionation factors phi(1) = 0.48 and phi(2) = 1.26 for normal and inverse isotope effects respectively. The former results from the proton transfer to B and the latter from the binding of guanidinium cation to the phosphate group as follows from observation of an inverse solvent isotope effect for the binding of guanidinium and amidinium cations to a phosphodiester anion. The results of kinetic studies together with analysis of transition state stabilization free energies for guanidinium and amidinium cations show that the protonated buffer component acts via electrostatic transition state stabilization rather than proton transfer, which may be possible for a guanidinium assisted hydroxide catalyzed reaction.

Revisiting the mechanism of dioxygen activation in soluble methane monooxygenase from M. capsulatus (Bath): evidence for a multi-step, proton-dependent reaction pathway.

Stopped-flow kinetic investigations of soluble methane monooxygenase (sMMO) from M. capsulatus (Bath) have clarified discrepancies that exist in the literature regarding several aspects of catalysis by this enzyme. The development of thorough kinetic analytical techniques has led to the discovery of two novel oxygenated iron species that accumulate in addition to the well-established intermediates H(peroxo) and Q. The first intermediate, P*, is a precursor to H(peroxo) and was identified when the reaction of reduced MMOH and MMOB with O(2) was carried out in the presence of >or=540 microM methane to suppress the dominating absorbance signal due to Q. The optical properties of P* are similar to those of H(peroxo), with epsilon(420) = 3500 M(-1) cm(-1) and epsilon(720) = 1250 M(-1) cm(-1). These values are suggestive of a peroxo-to-iron(III) charge-transfer transition and resemble those of peroxodiiron(III) intermediates characterized in other carboxylate-bridged diiron proteins and synthetic model complexes. The second identified intermediate, Q*, forms on the pathway of Q decay when reactions are performed in the absence of hydrocarbon substrate. Q* does not react with methane, forms independently of buffer composition, and displays a unique shoulder at 455 nm in its optical spectrum. Studies conducted at different pH values reveal that rate constants corresponding to P* decay/H(peroxo) formation and H(peroxo) decay/Q formation are both significantly retarded at high pH and indicate that both events require proton transfer. The processes exhibit normal kinetic solvent isotope effects (KSIEs) of 2.0 and 1.8, respectively, when the reactions are performed in D(2)O. Mechanisms are proposed to account for the observations of these novel intermediates and the proton dependencies of P* to H(peroxo) and H(peroxo) to Q conversion.

Kinetic Analysis of Cysteine Desulfurase CD0387 from Synechocystis sp. PCC 6803: Formation of the Persulfide Intermediate

Stopped-flow absorption and isotope effect experiments have been used to dissect the mechanism of formation of the enzyme cysteinyl persulfide intermediate in the reaction of a cysteine desulfurase (CD), CD0387 from Synechocystis sp. strain PCC 6803. Seven accumulating intermediates have been identified and tentatively mapped onto the CD chemical mechanism originally proposed by Dean, White, and co-workers [Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720]. The first intermediate with lambda(max) approximately 350 nm is assigned as either a gem-diamine complex or a thiol adduct formed by nucleophilic attack of either the amine group or the sulfhydryl group of the substrate on the internal aldimine form of the pyridoxal 5'-phosphate (PLP) cofactor. The second intermediate, with absorption features at approximately 417 and approximately 340 nm, is assigned as Cys aldimine and Cys ketimine forms in rapid equilibrium. In agreement with this assignment, a significant substrate alpha-deuterium equilibrium isotope effect ((2)H-EIE) favoring the aldimine form (417 nm) is observed in the second state produced in either wild-type CD0387 or the inactive C326A variant protein, which lacks the nucleophilic cysteine residue and is thus unable to proceed beyond this state unless "rescued" by a high concentration of an exogenous thiol. The third intermediate has an additional approximately 506 nm feature, characteristic of a quinonoid form, along with the features of the previous state. Its assignment as Ala aldimine, quinonoid, and ketimine forms in rapid equilibrium, which associates its formation with C-S bond cleavage and persulfide formation, is supported by its failure to develop in the C326A variant and the normal kinetic isotope effect ((2)H-KIE) on its formation, which is similar in magnitude to the (2)H-EIE disfavoring Cys-ketimine (from which the third state forms) in the second state. Decay of the Ala quinonoid absorption is tentatively attributed to a conformational change by the enzyme that disfavors this form in its equilibrium with Ala aldimine and Ala ketimine. Subsequent decay of the ketimine absorption ( approximately 340 nm) is attributed to release of Ala from the cofactor with an observed rate constant of 10 s(-1), the slowest step in the persulfide-forming half-reaction. The enzyme-persulfide.Ala complex dissociates rapidly with a K(d) of 98 mM. The final state with lambda(max) approximately 350 nm is assigned as a dead-end complex between the enzyme-persulfide and a second l-cysteine, which adds to the cofactor via its sulfhydryl group, possibly forming a cyclic thiazolidine species.

ChemInform Abstract: Temperature‐Dependent Transitions Between Normal and Inverse Isotope Effects Pertaining to the Interaction of H—H and C—H Bonds with Transition Metal Centers

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Kinetic Mechanism of Pyranose 2-Oxidase from Trametes multicolor

Pyranose 2-oxidase (P2O) from Trametes multicolor is a flavoprotein oxidase that catalyzes the oxidation of aldopyranoses by molecular oxygen to yield the corresponding 2-keto-aldoses and hydrogen peroxide. P2O is the first enzyme in the class of flavoprotein oxidases, for which a C4a-hydroperoxy-flavin adenine dinucleotide (FAD) intermediate has been detected during the oxidative half-reaction. In this study, the reduction kinetics of P2O by d-glucose and 2-d-d-glucose at pH 7.0 was investigated using stopped-flow techniques. The results indicate that d-glucose binds to the enzyme with a two-step binding process; the first step is the initial complex formation, while the second step is the isomerization to form an active Michaelis complex (E-Fl(ox):G). Interestingly, the complex (E-Fl(ox):G) showed greater absorbance at 395 nm than the oxidized enzyme, and the isomerization process showed a significant inverse isotope effect, implying that the C2-H bond of d-glucose is more rigid in the E-Fl(ox):G complex than in the free form. A large normal primary isotope effect (k(H)/k(D) = 8.84) was detected in the flavin reduction step. Steady-state kinetics at pH 7.0 shows a series of parallel lines. Kinetics of formation and decay of C-4a-hydroperoxy-FAD is the same in absence and presence of 2-keto-d-glucose, implying that the sugar does not bind to P2O during the oxidative half-reaction. This suggests that the kinetic mechanism of P2O is likely to be the ping-pong-type where the sugar product leaves prior to the oxygen reaction. The movement of the active site loop when oxygen is present is proposed to facilitate the release of the sugar product. Correlation between data from pre-steady-state and steady-state kinetics has shown that the overall turnover of the reaction is limited by the steps of flavin reduction and decay of C4a-hydroperoxy-FAD.

Temperature-Dependent Transitions Between Normal and Inverse Isotope Effects Pertaining to the Interaction of H−H and C−H Bonds with Transition Metal Centers

Deuterium kinetic isotope effects (KIEs) serve as versatile tools to infer details about reaction mechanisms and the nature of transition states, while equilibrium isotope effects (EIEs) associated with the site preferences of hydrogen and deuterium enable researchers to study aspects of molecular structure. Researchers typically interpret primary deuterium isotope effects based on two simple guidelines: (i) the KIE for an elementary reaction is normal (k(H)/k(D) > 1) and (ii) the EIE is dictated by deuterium preferring to be located in the site corresponding to the highest frequency oscillator. In this Account, we evaluate the applicability of these rules to the interactions of H-H and C-H bonds with a transition metal center. Significantly, experimental and computational studies question the predictability of primary EIEs in these systems based on the notion that deuterium prefers to occupy the highest frequency oscillator. In particular, the EIEs for (i) formation of sigma-complexes by coordination of H-H and C-H bonds and (ii) oxidative addition of dihydrogen exhibit unusual temperature dependencies, such that the same system may demonstrate both normal (i.e., K(H)/K(D) > 1) and inverse (i.e., K(H)/K(D) < 1) values. The transition between a normal and inverse EIE indicates that these systems do not demonstrate the typical monotonic variation predicted by the van't Hoff relationship. Instead, the calculated EIEs in these systems are 0 at 0 K, increase to a value greater than 1, and then decrease to unity at infinite temperature. This unusual behavior may be rationalized by considering the individual factors that contribute to the EIE. Specifically, the EIE may be expressed in the form EIE = SYM x MMI x EXC x ZPE (where SYM is the symmetry factor, MMI is the mass-moment of inertia term, EXC is the excitation term, and ZPE is the zero-point energy term), and the distinctive temperature profile results from the inverse ZPE (enthalpy) and normal [SYM x MMI x EXC] (entropy) components opposing each other and having different temperature dependencies. At low temperatures, the ZPE component dominates and the EIE is inverse, while at high temperatures, the [SYM x MMI x EXC] component dominates and the EIE is normal. The inverse nature of the ZPE term is a consequence of the rotational and translational degrees of freedom of RH (R = H, CH(3)) becoming low-energy isotopically sensitive vibrations in the product, while the normal nature of the [SYM x MMI x EXC] component results from deuterium substitution having a larger impact on the moment of inertia of the smaller molecule.

Dissection of the Stepwise Mechanism to β-Lactam Formation and Elucidation of a Rate-determining Conformational Change in β-Lactam Synthetase

Clavulanic acid is a widely used beta-lactamase inhibitor whose key beta-lactam core is formed by beta-lactam synthetase. beta-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by beta-lactam formation. 32PPi-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 +/- 0.04), it was concluded that beta-lactam synthesis contributes at least partially to kcat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pKa approximately 8.1 apparent in the pH-kcat profile that stabilizes the beta-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on kcat attenuating the observed solvent isotope effect on beta-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 +/- 0.08 from a plot of log(o kcat/kcat) versus log(eta/eta(o)) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of > 1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 +/- 0.15) on kcat/Km. The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (DeltaH approximately 20 kcal/mol) comparable to the chemical barrier of beta-lactam formation.

Reversible Intramolecular Hydrogen Transfer between Cysteine Thiyl Radicals and Glycine and Alanine in Model Peptides: Absolute Rate Constants Derived from Pulse Radiolysis and Laser Flash Photolysis

The intramolecular reaction of cysteine thiyl radicals with peptide and protein alphaC-H bonds represents a potential mechanism for irreversible protein oxidation. Here, we have measured absolute rate constants for these reversible hydrogen transfer reactions by means of pulse radiolysis and laser flash photolysis of model peptides. For N-Ac-CysGly6 and N-Ac-CysGly2AspGly3, Cys thiyl radicals abstract hydrogen atoms from Gly with k(f) = (1.0-1.1 x 10(5) s(-1), generating carbon-centered radicals, while the reverse reaction proceeds with k(r) = (8.0-8.9) x 10(5) s(-1). The forward reaction shows a normal kinetic isotope effect of k(H)/k(D) = 6.9, while the reverse reaction shows a significantly higher normal kinetic isotope effect of 17.6, suggesting a contribution of tunneling. For N-Ac-CysAla2AspAla3, cysteine thiyl radicals abstract hydrogen atoms from Ala with k(f) = (0.9-1.0) x 10(4) s(-1), while the reverse reaction proceeds with k(r) = 1.0 x 10(5) s(-1). The order of reactivity, Gly > Ala, is in accord with previous studies on intermolecular reactions of thiyl radicals with these amino acids. The fact that k(f) < k(r) suggests some secondary structure of the model peptides, which prevents the adoption of extended conformations, for which calculations of homolytic bond dissociation energies would have predicted k(f) > k(r). Despite k(f) < k(r), model calculations show that intramolecular hydrogen abstraction by Cys thiyl radicals can lead to significant oxidation of other amino acids in the presence of physiologic oxygen concentrations.

Thermodynamic and Kinetic Studies of H Atom Transfer from HMo(CO)3(η5-C5H5) to Mo(N[t-Bu]Ar)3 and (PhCN)Mo(N[t-Bu]Ar)3: Direct Insertion of Benzonitrile into the Mo−H Bond of HMo(N[t-Bu]Ar)3 forming (Ph(H)C═N)Mo(N[t-Bu]Ar)3

Synthetic studies are reported that show that the reaction of either H2SnR2 (R = Ph, n-Bu) or HMo(CO)3(Cp) (1-H, Cp = eta(5)-C5H5) with Mo(N[t-Bu]Ar)3 (2, Ar = 3,5-C6H3Me2) produce HMo(N[t-Bu]Ar)3 (2-H). The benzonitrile adduct (PhCN)Mo(N[t-Bu]Ar)3 (2-NCPh) reacts rapidly with H2SnR2 or 1-H to produce the ketimide complex (Ph(H)C=N)Mo(N[t-Bu]Ar)3 (2-NC(H)Ph). The X-ray crystal structures of both 2-H and 2-NC(H)Ph are reported. The enthalpy of reaction of 1-H and 2 in toluene solution has been measured by solution calorimetry (DeltaH = -13.1 +/- 0.7 kcal mol(-1)) and used to estimate the Mo-H bond dissociation enthalpy (BDE) in 2-H as 62 kcal mol(-1). The enthalpy of reaction of 1-H and 2-NCPh in toluene solution was determined calorimetrically as DeltaH = -35.1 +/- 2.1 kcal mol(-1). This value combined with the enthalpy of hydrogenation of [Mo(CO)3(Cp)]2 (1(2)) gives an estimated value of 90 kcal mol(-1) for the BDE of the ketimide C-H of 2-NC(H)Ph. These data led to the prediction that formation of 2-NC(H)Ph via nitrile insertion into 2-H would be exothermic by approximately 36 kcal mol(-1), and this reaction was observed experimentally. Stopped flow kinetic studies of the rapid reaction of 1-H with 2-NCPh yielded DeltaH(double dagger) = 11.9 +/- 0.4 kcal mol(-1), DeltaS(double dagger) = -2.7 +/- 1.2 cal K(-1) mol(-1). Corresponding studies with DMo(CO)3(Cp) (1-D) showed a normal kinetic isotope effect with kH/kD approximately 1.6, DeltaH(double dagger) = 13.1 +/- 0.4 kcal mol(-1) and DeltaS(double dagger) = 1.1 +/- 1.6 cal K(-1) mol(-1). Spectroscopic studies of the much slower reaction of 1-H and 2 yielding 2-H and 1/2 1(2) showed generation of variable amounts of a complex proposed to be (Ar[t-Bu]N)3Mo-Mo(CO)3(Cp) (1-2). Complex 1-2 can also be formed in small equilibrium amounts by direct reaction of excess 2 and 1(2). The presence of 1-2 complicates the kinetic picture; however, in the presence of excess 2, the second-order rate constant for H atom transfer from 1-H has been measured: 0.09 +/- 0.01 M(-1) s(-1) at 1.3 degrees C and 0.26 +/- 0.04 M(-1) s(-1) at 17 degrees C. Study of the rate of reaction of 1-D yielded kH/kD = 1.00 +/- 0.05 consistent with an early transition state in which formation of the adduct (Ar[t-Bu]N)3Mo...HMo(CO)3(Cp) is rate limiting.

Evaluation of Functional Groups Responsible for Chloroform Formation during Water Chlorination Using Compound Specific Isotope Analysis

Compound-specific isotope analysis was used to monitor the delta(13)C signature of chloroform produced upon the chlorination of model compounds representing natural organic matter functional groups (resorcinol, acetylacetone, acetophenone, phenol, and 2,4,6-trichlorophenol) and a natural water sample. For each model compound, a different apparent kinetic isotope effect was found for chloroform formation. Normal isotope effects were found for resorcinol, acetylacetone, and acetophenone, and ranged from 1.009 +/- 0.002 to 1.024 +/- 0.004. For the two phenols, an inverse effect was found (0.980 +/- 0.004). Lake Zürich water also had a inverse effect (0.997 +/- < 0.001) indicating that phenols are likely chloroform precursors in NOM, but that other functional groups may also participate. The apparent 13C kinetic isotope effect for the addition/ elimination reaction of 1,1,1-trichloropropanone mediated by OH- to yield chloroform is 1.014 +/- 0.002. A comparison of this value to those found for the chlorination of the model precursors and an evaluation of the differences in chloroform production kinetics for the different model precursors argue against a mechanism in which all NOM precursors react via a common intermediate. Compound specific isotope analysis may give additional insights into chloroform formation mechanisms beyond those allowed by current techniques.

Anilinolysis of Diphenyl Thiophosphinic Chloride and Theoretical Studies on Various R1R2P(O or S)Cl

The aminolysis of diphenyl thiophosphinic chloride (2) with substituted anilines in acetonitrile at 55.0 oC is investigated kinetically. Kinetic results yield large Hammett ρX (ρnuc = ?3.97) and Bronsted βX (βnuc = 1.40) values. A concerted mechanism involving a partial frontside nucleophilic attack through a hydrogen-bonded, four-center type transition state is proposed on the basis of the primary normal kinetic isotope effects (kH/kD = 1.0-1.1) with deuterated aniline (XC6H4ND2) nucleophiles. The natural bond order charges on P and the degrees of distortion of 42 compounds: chlorophosphates [(R1O)(R2O)P(=O)Cl], chlorothiophosphates [(R1O)(R2O)P(=S)Cl], phosphonochloridates [(R1O)R2P(=O)Cl], phosphonochlorothioates [(R1O)R2P(=S)Cl], chlorophosphinates [R1R2P(=O)Cl], and chlorothiophosphinates [R1R2P(=S)Cl] are calculated at the B3LYP/ 6-311+G(d,p) level in the gas phase.