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

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

A new interpretation of chlorine leaving group kinetic isotope effects; a theoretical approach

The chlorine leaving group kinetic isotope effects (KIEs) for the S(N)2 reactions between methyl chloride and a wide range of anionic, neutral, and radical anion nucleophiles were calculated in the gas phase and, in several cases, using a continuum solvent model. In contrast to the expected linear dependence of the chlorine KIEs on the C(alpha)-Cl bond order in the transition state, the KIEs fell in a very small range (1.0056-1.0091), even though the C(alpha)-Cl transition state bond orders varied widely from approximately 0.32 to 0.78, a range from reactant-like to very product-like. This renders chlorine KIEs, and possibly other leaving-group KIEs, less useful for studies of reaction mechanisms than commonly assumed. A partial explanation for this unexpected relationship between the C(alpha)-Cl transition state bond order and the magnitude of the chlorine KIE is presented.     

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

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

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

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

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

Chlorine leaving group k(35)/k(37), nucleophile carbon k(11)/k(14), and secondary alpha-deuterium [(kH/kD)alpha] kinetic isotope effects (KIEs) have been measured for the SN2 reactions between para-substituted benzyl chlorides and tetrabutylammonium cyanide in tetrahydrofuran at 20 degrees C to determine whether these isotope effects can be used to determine the substituent effect on the structure of the transition state. The secondary alpha-deuterium KIEs indicate that the transition states for these reactions are unsymmetric. The theoretical calculations at the B3LYP/aug-cc-pVDZ level of theory support this conclusion; i.e., they suggest that the transition states for these reactions are unsymmetric with a long NC-C(alpha) and reasonably short C(alpha)-Cl bonds. The chlorine isotope effects suggest that these KIEs can be used to determine the substituent effects on transition state structure with the KIE decreasing when a more electron-withdrawing para-substituent is present. This conclusion is supported by theoretical calculations. The nucleophile carbon k(11)/k(14) KIEs for these reactions, however, do not change significantly with substituent and, therefore, do not appear to be useful for determining how the NC-C(alpha) transition-state bond changes with substituent. The theoretical calculations indicate that the NC-C(alpha) bond also shortens as a more electron-withdrawing substituent is placed on the benzene ring of the substrate but that the changes in the NC-C(alpha) transition-state bond with substituent are very small and may not be measurable. The results also show that using leaving group and nucleophile carbon KIEs to determine the substituent effect on transition-state structure is more complicated than previously thought. The implication of using both chlorine leaving group and nucleophile carbon KIEs to determine the substituent effect on transition-state structure is discussed.     

Deuterium kinetic isotope effects in microsolvated gas-phase E2 reactions: Methanol and ethanol as solvents

The gas-phase reactions of F(-)(CH(3)OH) and F(-)(C(2)H(5)OH) with t-butyl bromide have been investigated to explore the effect of the solvent on the E2 transition state. Kinetic isotope effects (KIEs) were measured using a flowing afterglow-selected ion flow tube (FA-SIFT) mass spectrometer upon deuteration of both the alkyl halide and the alcohol. Kinetic isotope effects are significantly more pronounced than those previously observed for similar reactions of F(-)(H(2)O) with t-butyl halides. KIEs for the reaction of F(-)(CH(3)OH) with t-butyl bromide are 2.10 upon deuteration of the neutral reagent and 0.74 upon deuteration of the solvent. KIEs for the reaction of F(-)(C(2)H(5)OH) with t-butyl bromide are 3.84 upon deuteration of the neutral reagent and 0.66 upon deuteration of the solvent. The magnitude of these effects is discussed in terms of transition-state looseness. Additionally, deuteration of the neutral regent and deuteration of the solvent do not produce completely separable isotope effects, which is likely due to a crowded transition state. These results are compared to our previous work on S(N)2 and E2 solvated systems.     

Transition-state variation in human, bovine, and Plasmodium falciparum adenosine deaminases

Adenosine deaminases (ADAs) from human, bovine, and Plasmodium falciparum sources were analyzed by kinetic isotope effects (KIEs) and shown to have distinct but related transition states. Human adenosine deaminase (HsADA) is present in most mammalian cells and is involved in B- and T-cell development. The ADA from Plasmodium falciparum (PfADA) is essential in this purine auxotroph, and its inhibition is expected to have therapeutic effects for malaria. Therefore, ADA is of continuing interest for inhibitor design. Stable structural mimics of ADA transition states are powerful inhibitors. Here we report the transition-state structures of PfADA, HsADA, and bovine ADA (BtADA) solved using competitive kinetic isotope effects (KIE) and density functional calculations. Adenines labeled at [6-13C], [6-15N], [6-13C, 6-15N], and [1-15N] were synthesized and enzymatically coupled with [1'-14C] ribose to give isotopically labeled adenosines as ADA substrates for KIE analysis. [6-13C], [6-15N], and [1-15N]adenosines reported intrinsic KIE values of (1.010, 1.011, 1.009), (1.005, 1.005, 1.002), and (1.004, 1.001, 0.995) for PfADA, HsADA, and BtADA, respectively. The differences in intrinsic KIEs reflect structural alterations in the transition states. The [1-15N] KIEs and computational modeling results indicate that PfADA, HsADA, and BtADA adopt early SNAr transition states, where N1 protonation is partial and the bond order to the attacking hydroxyl nucleophile is nearly complete. The key structural variation among PfADA, HsADA, and BtADA transition states lies in the degree of N1 protonation with the decreased bond lengths of 1.92, 1.55, and 1.28 A, respectively. Thus, PfADA has the earliest and BtADA has the most developed transition state. This conclusion is consistent with the 20-36-fold increase of kcat in comparing PfADA with HsADA and BtADA.     

Transition-state analysis of Trypanosoma cruzi uridine phosphorylase-catalyzed arsenolysis of uridine

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2'-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli , purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2'-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1'-(14)C = 1.103, 1,3-(15)N(2) = 1.034, 3-(15)N = 1.004, 1-(15)N = 1.030, 1'-(3)H = 1.132, 2'-(2)H = 1.086, and 5'-(3)H(2) = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S(N)2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.     

Transition state structure of E. coli tRNA-specific adenosine deaminase

Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single tRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNA(Arg2) was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1'-(3)H], [5'-(3)H2], [1'-(14)C], [6-(13)C], [6-(15)N], [6-(13)C, 6-(15)N] and [1-(15)N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-(13)C]-, [6-(15)N]-, [1-(15)N]-, [1'-(3)H]-, [5'-(3)H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late SNAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-(15)N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 A in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 A, respectively. The large inverse [5'-(3)H] KIE of -3.7% and modest normal [1'-(3)H] KIE of 1.4% indicate that significant ribosyl 5'-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes.     

Measurements of the 12C/13C kinetic isotope effects in the gas-phase reactions of light alkanes with chlorine atoms

The carbon kinetic isotope effects (KIEs) of the reactions of several light non-methane hydrocarbons (NMHC) with Cl atoms were determined at room temperature and ambient pressure. All measured KIEs, defined as the ratio of the Cl reaction rate constants of the light isotopologue over that of the heavy isotopologue (Clk12/Clk13) are greater than unity or normal KIEs. For simplicity, measured KIEs are reported in per mil according to Clepsilon=(Clk12/Clk13 -1)x1000 per thousand unless noted otherwise. The following average KIEs were obtained (all in per thousand): 10.73+/-0.20 (ethane), 6.44+/-0.14 (propane), 6.18+/-0.18 (methylpropane), 3.94+/-0.01 (n-butane), 1.79+/-0.42 (methylbutane), 3.22+/-0.17 (n-pentane), 2.02+/-0.40 (n-hexane), 2.06+/-0.19 (n-heptane), 1.54+/-0.15 (n-octane), 3.04+/-0.09 (cyclopentane), 2.30+/-0.09 (cyclohexane), and 2.56+/-0.25 (methylcyclopentane). Measurements of the 12C/13C KIEs for the Cl atom reactions of the C2-C8 n-alkanes were also made at 348 K, and no significant temperature dependence was observed. To our knowledge, these 12C/13C KIE measurements for alkanes+Cl reactions are the first of their kind. Simultaneous to the KIE measurement, the rate constant for the reaction of each alkane with Cl atoms was measured using a relative rate method. Our measurements agree with published values within+/-20%. The measured rate constant for methylcyclopentane, for which no literature value is available, is (2.83+/-0.11)x10-10 cm3 molecule-1 s-1, 1sigma standard error. The Clepsilon values presented here for the C2-C8 alkanes are an order of magnitude smaller than reported methane Clepsilon values (Geophys. Res. Lett., 2000, 27, 1715), in contrast to reported OHepsilon values for methane (J. Geophys. Res. (Atmos.), 2001, 106, 23, 127) and C2-C8 alkanes (J. Phys. Chem. A, 2004, 108, 11537), which are all smaller than 10 per thousand. This has important implications for atmospheric modeling of saturated NMHC stable carbon isotope ratios. 13C-structure reactivity relationship values (13C-SRR) for alkane-Cl reactions have been determined and are similar to previously reported values for alkane-OH reactions.     

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.     

The effect of inert salts on the structure of the transition state in the SN2 reaction between thiophenoxide ion and butyl chloride

The effect of inert salts on the structure of the transition state has been determined by measuring the secondary alpha deuterium and the chlorine leaving group kinetic isotope effects for the S(N)2 reaction between n-butyl chloride and thiophenoxide ion in both methanol and DMSO. The smaller secondary alpha deuterium isotope effects and very slightly larger chlorine isotope effects found in both solvents when the inert salt is present suggests that the S(N)2 transition state is tighter and more product-like, with a shorter S-C(alpha) and very a slightly longer C(alpha)-Cl bond when the added salt is present. The salt effect on the reaction in methanol where the reacting nucleophile is the solvent-separated ion-pair complex is much greater than the salt effect on the reaction in DMSO where the reacting nucleophile is the free ion. This greater change in transition-state structure found when the inert salt is present in methanol is consistent with the solvation rule for S(N)2 reactions. The greater change in the S-C(alpha) bond is predicted by the bond strength hypothesis. A rationale for the changes found in transition-state structure when the inert salt is present is suggested for both the free-ion and the ion-pair reactions.     

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

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

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.     

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.     

Deuterium kinetic isotope effects on the dissociation of a protein-fatty acid complex in the gas phase

Deuterium kinetic isotope effects (KIEs) are reported for the first time for the dissociation of a protein-ligand complex in the gas phase. Temperature-dependent rate constants were measured for the loss of neutral ligand from the deprotonated ions of the 1:1 complex of bovine β-lactoglobulin (Lg) and palmitic acid (PA), (Lg + PA)(n-) → Lg(n-) + PA, at the 6- and 7- charge states. At 25 °C, partial or complete deuteration of the acyl chain of PA results in a measurable inverse KIE for both charge states. The magnitude of the KIEs is temperature dependent, and Arrhenius analysis of the rate constants reveals that deuteration of PA results in a decrease in activation energy. In contrast, there is no measurable deuterium KIE for the dissociation of the (Lg + PA) complex in aqueous solution at pH 8. Deuterium KIEs were calculated using conventional transition-state theory with an assumption of a late dissociative transition state (TS), in which the ligand is free of the binding pocket. The vibrational frequencies of deuterated and non-deuterated PA in the gas phase and in various solvents (n-hexane, 1-chlorohexane, acetone, and water) were established computationally. The KIEs calculated from the corresponding differences in zero-point energies account qualitatively for the observation of an inverse KIE but do not account for the magnitude of the KIEs nor their temperature dependence. It is proposed that the dissociation of the (Lg + PA) complex in aqueous solution also proceeds through a late TS in which the acyl chain is extensively hydrated such that there is no significant differential change in the vibrational frequencies along the reaction coordinate and, consequently, no significant KIE.     

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

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

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

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

Isotope effects, dynamics, and the mechanism of solvolysis of aryldiazonium cations in water

The mechanism of the heterolytic solvolysis of p-tolyldiazonium cation in water was studied by a combination of kinetic isotope effects, theoretical calculations, and dynamics trajectories. Significant (13)C kinetic isotope effects were observed at the ipso (k(12)C/k(13)C = 1.024), ortho (1.017), and meta (1.013) carbons, indicative of substantial weakening of the C(2)-C(3) and C(5)-C(6) bonds at the transition state. This is qualitatively consistent with a transition state forming an aryl cation, but on a quantitative basis, simple S(N)1 heterolysis does not account best for the isotope effects. Theoretical S(N)2Ar transition structures for concerted displacement of N(2) by a single water molecule lead to poor predictions of the experimental isotope effects. The best predictions of the (13)C isotope effects arose from transition structures for the heterolytic process solvated by clusters of water molecules. These structures, formally saddle points for concerted displacements on the potential energy surface, may be described as transition structures for solvent reorganization around the aryl cation. Quasiclassical dynamics trajectories starting from these transition structures afforded products very slowly, compared to a similar S(N)2 displacement, and the trajectories often afforded long-lived aryl cation intermediates. Critical prior evidence for aryl cation intermediates is reconsidered with the aid of DFT calculations. Overall, the nucleophilic displacement process for aryldiazonium ions in water is at the boundary between S(N)2Ar and S(N)1 mechanisms, and an accurate view of the reaction mechanism requires consideration of dynamic effects.     

Transition state analysis of Vibrio cholerae sialidase-catalyzed hydrolyses of natural substrate analogues

A series of isotopically labeled natural substrate analogues (phenyl 5-N-acetyl-α-d-neuraminyl-(2→3)-β-d-galactopyranosyl-(1→4)-1-thio-β-d-glucopyranoside; Neu5Acα2,3LacβSPh, and the corresponding 2→6 isomer) were prepared chemoenzymatically in order to characterize, by use of multiple kinetic isotope effect (KIE) measurements, the glycosylation transition states for Vibrio cholerae sialidase-catalyzed hydrolysis reactions. The derived KIEs for Neu5Acα2,3LacβSPh for the ring oxygen ((18)V/K), leaving group oxygen ((18)V/K), C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.029 ± 0.002, 0.983 ± 0.001, 1.034 ± 0.002, and 1.043 ± 0.002, respectively. In addition, the KIEs for Neu5Acα2,6βSPh for C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.021 ± 0.001 and 1.049 ± 0.001, respectively. The glycosylation transition state structures for both Neu5Acα2,3LacβSPh and Neu5Acα2,6LacβSPh were modeled computationally using the experimental KIE values as goodness of fit criteria. Both transition states are late with largely cleaved glycosidic bonds coupled to pyranosyl ring flattening ((4)H(5) half-chair conformation) with little or no nucleophilic involvement of the enzymatic tyrosine residue. Notably, the transition state for the catalyzed hydrolysis of Neu5Acα2,6βSPh appears to incorporate a lesser degree of general-acid catalysis, relative to the 2,3-isomer.