Theoretical Study on the Gas‐Phase SN2 Reaction of Microhydrated Fluoride with Methyl Fluoride

The rate constants for the gas‐phase S_N2 reaction of F^?(H₂O) with CH₃F have been calculated using the dual‐level variational transition state theory including multidimensional tunneling from 50 to 500 K. Tunneling was found to dominate the reaction below 200 K. The deuterium, ¹³C, and ¹⁴C kinetic isotope effects (KIEs) and solvent (D₂O) isotope effects (SKIEs) were also calculated in the same temperature range. The results indicated that the deuterium and heavy water substitutions resulted in inverse KIEs (0.6～0.8 ) while the ¹³C and ¹⁴C substitutions resulted in normal KIEs (1.0～1.2) at room temperature. The calculated carbon KIEs increased significantly below 80 K due to the differences in the magnitude of the tunneling effects for different isotopic substitutions.     

Synthesis of linoleic acids combinatorially labeled at the vinylic positions as substrates for lipoxygenases

Mammalian lipoxygenases have been implicated in a number of inflammation-related human diseases. Soybean lipoxygenase-1 is the archetypical example of known lipoxygenases. Here we report the synthesis of linoleic acid and (11,11)-d2-linoleic acid which are combinatorially labeled at the vinylic positions (9, 10, 12, and 13). Combinatorial labeling schemes allow for the simultaneous determination of KIEs in enzymatic reactions using NMR. Substrates are, thus, available as probes of detailed mechanism in kinetic isotope effect (KIE) studies of lipoxygenases.     

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.     

Tunneling in C-H oxidation reactions by an oxoiron(IV) porphyrin radical cation: direct measurements of very large H/D kinetic isotope effects

Rate constants for oxidations of benzyl alcohol-d0 and -d7 by oxoiron(IV) tetramesitylporphyrin radical cation perchlorate in acetonitrile were measured in single turnover kinetic studies. The kinetic isotope effect (kH/kD) increased from 28 at 23 degrees C to 360 at -30 degrees C due to extensive hydrogen atom tunneling that was analyzed in terms of a parabolic energy barrier to tunneling. Similarly, large KIE values were found for oxidations of ethylbenzene-d0 and -d10 at room temperature. The large KIE values are a function of the porphyrin identity, and porphyrins containing electron-withdrawing groups display normal KIEs. KIEs found under catalytic turnover conditions are somewhat smaller than those obtained in single turnover reactions. The results should serve as benchmarks for computational studies of C-H oxidations by porphyrin and heme-iron-oxo systems.     

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.     

Isotope effects and heavy-atom tunneling in the Roush allylboration of aldehydes

Intermolecular (13)C kinetic isotope effects (KIEs) for the Roush allylboration of p-anisaldehyde were determined using a novel approach. The experimental (13)C KIEs fit qualitatively with the expected rate-limiting cyclic transition state, but they are far higher than theoretical predictions based on conventional transition state theory. This discrepancy is attributed to a substantial contribution of heavy-atom tunneling to the reaction, and this is supported by multidimensional tunneling calculations that reproduce the observed KIEs.     

Hydrogen tunneling steps in cyclooxygenase-2 catalysis

Cyclooxygenases-1 and -2 are tyrosyl radical (Y·)-utilizing hemoproteins responsible for the biosynthesis of lipid-derived autocoids. COX-2, in particular, is a primary mediator of inflammation and believed to be up-regulated in many forms of cancer. Described here are first-of-a-kind studies of COX-2-catalyzed oxidation of the substrate analogue linoleic acid. Very large (≥20) temperature-independent deuterium kinetic isotope effects (KIEs) on the rate constant for enzyme turnover were observed, due to hydrogen atom abstraction from the bisallylic C-H(D) of the fatty acid. The magnitude of the KIE depends on the O(2) concentration, consistent with reversible H/D tunneling mediated by the catalytic Y·. At physiological levels of O(2), retention of the hydrogen initially abstracted by the catalytic tyrosine results in strongly temperature-dependent KIEs on O-H(D) homolysis, also characteristic of nuclear tunneling.     

Turnover is rate-limited by deglycosylation for Micromonospora viridifaciens sialidase-catalyzed hydrolyses: conformational implications for the Michaelis complex

A panel of seven isotopically substituted sialoside natural substrate analogues based on the core structure 7-(5-acetamido-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonic acid)-(2→6)-β-D-galactopyranosyloxy)-8-fluoro-4-methylcoumarin (1, Neu5Acα2,6GalβFMU) have been synthesized and used to probe the rate-limiting step for turnover by the M. viridifaciens sialidase. The derived kinetic isotope effects (KIEs) on k(cat) for the ring oxygen ((18)V), leaving group oxygen ((18)V), anomeric carbon ((13)V), C3-carbon ((13)V), C3-R deuterium ((D)V(R)), C3-S deuterium ((D)V(S)), and C3-dideuterium ((D)(2)V) are 0.986 ± 0.003, 1.003 ± 0.005, 1.021 ± 0.006, 1.001 ± 0.008, 1.029 ± 0.007, 0.891 ± 0.008, and 0.890 ± 0.006, respectively. The solvent deuterium KIE ((D(2)O)V) for the sialidase-catalyzed hydrolysis of 1 is 1.585 ± 0.004. In addition, a linear proton inventory was measured for the rate of hydrolysis, under saturating condition, as a function of n, the fraction of deuterium in the solvent. These KIEs are compatible with rate-determining cleavage of the enzymatic tyrosinyl β-sialoside intermediate. Moreover, the secondary deuterium KIEs are consistent with the accumulating Michaelis complex in which the sialosyl ring of the carbohydrate substrate is in a (6)S(2) skew boat conformation. These KIE measurements are also consistent with the rate-determining deglycosylation reaction occurring via an exploded transition state in which synchronous charge delocalization is occurring onto the ring oxygen atom. Finally, the proton inventory and the magnitude of the solvent KIE are consistent with deglycosylation involving general acid-catalyzed protonation of the departing tyrosine residue rather than general base-assisted attack of the nucleophilic water.     

The mechanism of base-promoted HF elimination from 4-fluoro-4-(4'-nitrophenyl)butan-2-one: a multiple isotope effect study including the leaving group (18)F/(19)F KIE.

Leaving-group fluorine as well as the primary and secondary deuterium kinetic isotope effects (KIEs) have been determined for the base-promoted elimination of hydrogen fluoride from 4-fluoro-4-(4'-nitrophenyl)butan-2-one in aqueous solution. The elimination was studied for formate, acetate, and imidazole as the catalyzing base. The fluorine KIEs were determined using the accelerator-produced short-lived radionuclide (18)F in combination with natural (19)F. The (19)F substrate was labeled with (14)C in a remote position to enable radioactivity measurement of both isotopic substrates. The elimination reaction exhibits large primary deuterium KIEs: 3.2, 3.7, and 7.5 for formate, acetate, and imidazole, respectively, thus excluding the E1 mechanism. The corresponding C(4)-secondary deuterium KIEs are 1.038, 1.050 and 1.014 and the leaving group fluorine KIEs are 1.0037, 1.0047 and 1.0013, respectively. The size of the fluorine KIEs corresponds to 5-15% of the estimated maximum of 1.03 for complete C-F bond breakage. No H/D exchange is observed during the reaction. The size and trends of the KIEs for the different bases are consistent with an E1cB-like E2 or an E1cB(ip) mechanism.     

Large Isotope Effects in Organometallic Chemistry

The kinetic isotope effect (KIE) is key to understanding reaction mechanisms in many areas of chemistry and chemical biology, including organometallic chemistry. This ratio of rate constants, k_H/k_D, typically falls between 1–7. However, KIEs up to 105 have been reported, and can even be so large that reactivity with deuterium is unobserved. We collect here examples of large KIEs across organometallic chemistry, in catalytic and stoichiometric reactions, along with their mechanistic interpretations. Large KIEs occur in proton transfer reactions such as protonation of organometallic complexes and clusters, protonolysis of metal–carbon bonds, and dihydrogen reactivity. C−H activation reactions with large KIEs occur with late and early transition metals, photogenerated intermediates, and abstraction by metal-oxo complexes. We categorize the mechanistic interpretations of large KIEs into the following three types: (a) proton tunneling, (b) compound effects from multiple steps, and (c) semi-classical effects on a single step. This comprehensive collection of large KIEs in organometallics provides context for future mechanistic interpretation.     

Temperature dependence of carbon-13 kinetic isotope effects of importance to global climate change

We report here a theoretical study of the 13C kinetic isotope effect (KIE) and its temperature dependence for the reaction OH + CH4 --> H2O + CH3, the major sink of atmospheric methane in the troposphere. The KIE values at various atmospherically significant temperatures were determined by direct dynamics using variational transition state theory with multidimensional tunneling contributions (VTST/MT). The potential energy surfaces (PESs) were generated by hybrid density functional theory as well as by recently developed doubly hybrid density functional theory methods. Comparisons of our calculated KIEs with experimental data and theoretical values in the literature reveal the critical contributions due to multidimensional tunneling and torsion anharmonicity as well as the critical issue of the choice of internal rotational axis.     

Mechanism of the

Stereochemical studies on [2 + 2] photoaddition of cis-/trans-4-propenylanisole (cis-1 and trans-1) and cis-1-(p-methoxyphenyl)ethylene-2-d(1) (cis-3-d(1)) to C(60) exhibit stereospecificity in favor of the trans-2 cycloadduct in the former case and nonstereoselectivity in the latter. The observed stereoselectivity in favor of the cis-6-d(3) [2 + 2] diastereomer by 12% in the case of the photochemical addition of (E)-1-(p-methoxyphenyl)-2-methyl-prop-1-ene-3,3,3-d(3) (trans-5-d(3)) to C(60) is attributed to a steric kinetic isotope effect (k(H)/k(D) = 0.78). The loss of stereochemistry in the cyclobutane ring excludes a concerted addition and is consistent with a stepwise mechanism. Intermolecular secondary kinetic isotope effects of the [2 + 2] photocycloaddition of 3-d(0) vs 3-d(1), and 3-d(6) as well as 5-d(0) vs 5-d(1), and 5-d(6) to C(60) were also measured. The intermolecular competition due to deuterium substitution of both vinylic hydrogens at the beta-carbon of 3 exhibits a substantial inverse alpha-secondary isotope effect k(H)/k(D) = 0.83 (per deuterium). Substitution with deuterium at both vinylic methyl groups of 5 yields a small inverse k(H)/k(D) = 0. 94. These results are consistent with the formation of an open intermediate in the rate-determining step.     

A stepwise solvent-promoted SNi reaction of α-D-glucopyranosyl fluoride: mechanistic implications for retaining glycosyltransferases

The solvolysis of α-d-glucopyranosyl fluoride in hexafluoro-2-propanol gives two products, 1,1,1,3,3,3-hexafluoropropan-2-yl α-d-glucopyranoside and 1,6-anhydro-β-D-glucopyranose. The ratio of these two products is essentially unchanged for reactions that are performed between 56 and 100 °C. The activation parameters for the solvolysis reaction are as follows: ΔH(++) = 81.4 ± 1.7 kJ mol(-1), and ΔS(++) = -90.3 ± 4.6 J mol(-1) K(-1). To characterize, by use of multiple kinetic isotope effect (KIE) measurements, the TS for the solvolysis reaction in hexafluoro-2-propanol, we synthesized a series of isotopically labeled α-d-glucopyranosyl fluorides. The measured KIEs for the C1 deuterium, C2 deuterium, C5 deuterium, anomeric carbon, ring oxygen, O6, and solvent deuterium are 1.185 ± 0.006, 1.080 ± 0.010, 0.987 ± 0.007, 1.008 ± 0.007, 0.997 ± 0.006, 1.003 ± 0.007, and 1.68 ± 0.07, respectively. The transition state for the solvolysis reaction was modeled computationally using the experimental KIE values as constraints. Taken together, the reported data are consistent with the retained solvolysis product being formed in an S(N)i (D(N)(++)*A(Nss)) reaction with a late transition state in which cleavage of the glycosidic bond is coupled to the transfer of a proton from a solvating hexafluoro-2-propanol molecule. In comparison, the inverted product, 1,6-anhydro-β-D-glucopyranose, is formed by intramolecular capture of a solvent-equilibrated glucopyranosylium ion, which results from dissociation of the solvent-separated ion pair formed in the rate-limiting ionization reaction (D(N)(++) + A(N)). The implications that this model reaction have for the mode of action of retaining glycosyltransferases are discussed.     

Hydroxylation by the hydroperoxy-iron species in cytochrome P450 enzymes

Intramolecular and intermolecular kinetic isotope effects (KIEs) were determined for hydroxylation of the enantiomers of trans-2-(p-trifluoromethylphenyl)cyclopropylmethane (1) by hepatic cytochrome P450 enzymes, P450s 2B1, Delta2B4, Delta2B4 T302A, Delta2E1, and Delta2E1 T303A. Two products from oxidation of the methyl group were obtained, unrearranged trans-2-(p-trifluoromethylphenyl)cyclopropylmethanol (2) and rearranged 1-(p-trifluoromethylphenyl)but-3-en-1-ol (3). In intramolecular KIE studies with dideuteriomethyl substrates (1-d(2)) and in intermolecular KIE studies with mixtures of undeuterated (1-d(0)) and trideuteriomethyl (1-d(3)) substrates, the apparent KIE for product 2 was consistently larger than the apparent KIE for product 3 by a factor of ca. 1.2. Large intramolecular KIEs found with 1-d(2) (k(H)/k(D) = 9-11 at 10 degrees C) were shown not to be complicated by tunneling effects by variable temperature studies with two P450 enzymes. The results require two independent isotope-sensitive processes in the overall hydroxylation reactions that are either competitive or sequential. Intermolecular KIEs were partially masked in all cases and largely masked for some P450s. The intra- and intermolecular KIE results were combined to determine the relative rate constants for the unmasking and hydroxylation reactions, and a qualitative correlation was found for the unmasking reaction and release of hydrogen peroxide from four of the P450 enzymes in the absence of substrate. The results are consistent with the two-oxidants model for P450 (Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555), which postulates that a hydroperoxy-iron species (or a protonated analogue of this species) is a viable electrophilic oxidant in addition to the consensus oxidant, iron-oxo.     

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

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

Secondary alpha isotope effects on deuterium tunneling in triplet o-methylanthrones: extraordinary sensitivity to barrier width

The rates of deuterium transfer in the photoenolization of triplet 1,4-dimethyl-10H-anthracen-9-one (1) with varying degrees of deuterium label in their methyl groups (1-d3, 1-d2, and 1-d) have been investigated as a function of temperature between 5 and 77 K. Measurable rate constants in the case of 1-d3 and 1-d2 were used to construct Arrhenius plots which illustrate the expected curvature and leveling off of rate constant versus temperature. The difference in tunneling rate constants of 1-d3 and 1-d2 yields a tunneling isotope effect, TIE = 2.4, which is attributed to the secondary alpha isotopic substitution. Density functional theory (DFT, B3LYP/6-31G*) calculations were carried out to obtain structural and energetic information for the H(D) transfer along the triplet state zero-point energy levels. The temperature dependence of the rate constants for each isotopologue was simulated with a model that considers the frequency of the C-D stretching mode and the quantum mechanical permeability determined from calculated energy parameters. The model suggests that a difference in barrier width of only 0.015 A between 1-d3 and 1-d2 leads to the observed 2-fold difference between tunneling rates.     

Heavy-atom kinetic isotope effects,cocatalysts, and the propagation transition state for polymerization of 1-hexene using the rac-(C(2)H(4)(1-indenyl)(2))ZrMe(2) catalyst precursor

Using 13C NMR techniques, the 12C/13C kinetic isotope effects (KIEs) for the polymerization of 1-hexene catalyzed by rac-(C2H4(1-indenyl)2)ZrMe2 in the presence of four different cocatalysts (tris(pentfluorophenyl)borane, tris(pentafluorophenyl)alane, anilinium tetrakis(pentafluorophenyl)borate, and methylalumoxane) have been determined. All cocatalysts yield similar KIEs within experimental uncertainty, with values of 1.009(1) and 1.017(2) at C1 and C2, respectively. Ab initio DFT computational modeling of the polymerization KIE indicates that alkene binding to the catalyst must be reversible, with the majority of the KIE developing in the subsequent migratory insertion reaction.     

Ensemble-averaged QM/MM kinetic isotope effects for the S(N)2 reaction of cyanide anions with chloroethane in DMSO solution

The existence of solvent fluctuations leads to populations of reactant-state (RS) and transition-state (TS) configurations and implies that property calculations must include appropriate averaging over distributions of values for individual configurations. Average kinetic isotope effects 〈KIE〉 for NC(-) + EtCl → NCEt + Cl(-) in DMSO solution at 30?°C are best obtained as the ratio 〈f(RS)〉/〈f(TS)〉 of isotopic partition function ratios separately averaged over all RS and TS configurations. In this way the hybrid AM1/OPLS-AA potential yields 〈KIE〉 values for all six isotopic substitutions (2° α-(2)H(2), 2° β-(2)H(3), α-(11)C/(14)C, leaving group (37)Cl, and nucleophile (13)C and (15)N) for this reaction in the correct direction as measured experimentally. These thermally-averaged calculated KIEs may be compared meaningfully with experiment, and only one of them differs in magnitude from the experimental value by more than one standard deviation from the mean. This success contrasts with previous KIE calculations based upon traditional methods without averaging. The isotopic partition function ratios are best evaluated using all (internal) vibrational and (external) librational frequencies obtained from Hessians determined for subsets of atoms, relaxed to local minima or saddle points, within frozen solvent environments of structures sampled along molecular dynamics trajectories for RS and TS. The current method may perfectly well be implemented with other QM or QM/MM methods, and thus provides a useful tool for investigating KIEs in relation to studies of chemical reaction mechanisms in solution or catalyzed by enzymes.     

Deuterium kinetic isotope effects on the gas-phase reactions of C2H with H2(D2) and CH4(CD4)

Kinetics of the ethynyl (C(2)H) radical reactions with H(2), D(2), CH(4) and CD(4) was studied over the temperature range of 295-396 K by a pulsed laser photolysis/chemiluminescence technique. The C(2)H radicals were generated by ArF excimer-laser photolysis of C(2)H(2) or CF(3)C(2)H and were monitored by the chemiluminescence of CH(A(2)Δ) produced by their reaction with O(2) or O((3)P). The measured absolute rate constants for H(2) and CH(4) agreed well with the available literature data. The primary kinetic isotope effects (KIEs) were determined to be k(H(2))/k(D(2)) = 2.48 ± 0.14 and k(CH(4))/k(CD(4)) = 2.45 ± 0.16 at room temperature. Both of the KIEs increased as the temperature was lowered. The KIEs were analyzed by using the variational transition state theory with semiclassical small-curvature tunneling corrections. With anharmonic corrections on the loose transitional vibrational modes of the transition states, the theoretical predictions satisfactorily reproduced the experimental KIEs for both C(2)H + H(2)(D(2)) and C(2)H + CH(4)(CD(4)) reactions.