Observed Isotope Effect Research Articles

Kinetic solvent deuterium isotope effect in transesterification of RNA models

Abstract2‐Methylbenzimidazole ribonucleoside arylphosphates (1a,b) and alkylphosphates (2a,b) were synthesized as RNA model compounds containing a minimised number of exchangeable protons. Intramolecular transesterification of these substrates was studied in H2O and D2O solutions over a wide pL range and apparent kinetic solvent deuterium isotope effects of the alkaline cleavage of both substrates and of the cleavage and isomerisation of 2a under neutral and acidic conditions were determined. The observed k/k of 4.9 obtained for the alkaline cleavage of the arylphosphate 1b can be primarily attributed to the ΔpK of the attacking nucleophile. The alkyl leaving group in 2a brings about an additional 1.5‐fold isotope effect (k/k of 7.1 observed), which, considering the pL–dependence of the reaction, can not be explained by a process involving a proton transfer. Differences in solvation of the transition state are tentatively suggested as a source of the difference. In contrast to alkaline cleavage, under neutral and acidic conditions the cleavage and isomerisation of 2a showed no apparent solvent isotope effect. Several examples found in the literature show that intramolecular proton transfer from phosphate to the leaving group in pre‐equilibria may not necessarily result in an observable solvent isotope effect. This may also explain the results obtained in the present work, since intramolecular proton transfer processes take place in transesterification reactions of 2a under neutral and acidic conditions. Relevance of the results obtained in the base catalysed cleavage to hammerhead ribozyme reaction is briefly discussed. Copyright © 2004 John Wiley & Sons, Ltd.

Evidence for the crucial role of nuclear spins for the magnetic field effect in polarization echoes in glasses

A few years ago, pronounced magnetic field effects have been observed in the low-temperature dielectric properties of glasses and disordered crystals. In particular, dielectric susceptibility and coherent polarization echoes were reported to be strongly influenced by external magnetic fields. It is well-known that the lowtemperature dielectric properties of disordered solids are dominated by atomic tunneling. Recently, it has been suggested that the surprising magnetic field dependence is caused by the nuclear quadrupole moment associated with the tunneling particles. To investigate the role of the nuclear quadrupole we have studied the magnetic field dependence of the amplitude of spontaneous polarization echoes generated in ordinary and deuterated glycerol. The observed isotope effect gives striking evidence for the explanation involving nuclear quadrupole moments. In addition, the quantum beating of the nuclear spin states has been observed in our echo experiments. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The use of isotope effects to determine enzyme mechanisms

Isotope effects are one of the most powerful kinetic tools for determining enzyme mechanisms. There are three methods of measurement. First, one can compare reciprocal plots with labeled and unlabeled substrates. The ratio of the slopes is the isotope effect on V/ K, and the ratio of the vertical intercepts is the isotope effect on V max. This is the only way to determine V max isotope effects, but is limited to isotope effects of 5% or greater. The second method is internal competition, where the labeled and unlabeled substrates are present at the same time and the change in their ratio in residual substrate or in product is used to calculate an isotope effect, which is that on V/ K of the labeled reactant. This is the method used for tritium or 14C, or with the natural abundances of 13C, 15N, or 18O. The third method involves perturbations from equilibrium when a labeled substrate and corresponding unlabeled product are present at chemical equilibrium. This also gives just an isotope effect on V/ K for the labeled reactant. The chemistry is typically not fully rate limiting, so that the isotope effect on V/ K is given by: x( V/ K) = ( x k + c f + c r x K eq)/(1 + c f + c r) where x defines the isotope (D, T, 13, 15, 18 for deuterium, tritium, 13C, 15N, or 18O), and x( V/ K), x k, and x K eq are the observed isotope effect, the intrinsic one on the chemical step, and the isotope effect on the equilibrium constant, respectively. The constants c f and c r are commitments in forward and reverse directions, and are the ratio of the rate constant for the chemical reaction and the net rate constant for release from the enzyme of the varied substrate (direct comparison) or labeled substrate (internal competition and equilibrium perturbation) for c f, or the first product released or the one involved in the perturbation for c r. The intrinsic isotope effect, x k, can be estimated by comparing deuterium and tritium isotope effects on V/ K, or by comparing the deuterium isotope effect with 13C ones with deuterated and undeuterated substrates. Adding a secondary deuterium isotope effect and its effect on the 13C one can give an exact solution for all intrinsic isotope effects and commitments. The effect of deuteration on a 13C isotope effect allows one to tell if the two isotope effects are on the same or different steps. Applications of these methods to several enzyme systems will be presented.

Isotope Effects on H and D Coadsorptions on Si Surfaces

We studied isotope effects on D and H coadsorptions on the Si(100) and Si(111) surfaces, employing an atomic beam method combined with a desorption measurement technique. We evaluated D:H ratios to be approximately 6:4 for both the Si(100) and Si(111) surfaces exposed to the atomic H/D beam mixed with equal amounts of H and D atoms. This observed isotope effect was interpreted in terms of mass-dependent abstraction of surface adatoms by incident atoms. The observed D:H ratios could be well reproduced by the simulation based on the previously reported isotope effect on D(H) abstraction by H atoms.

Pressure, Isotope, and Water Co-solvent Effects in Liquid−Liquid Equilibria of (Ionic Liquid + Alcohol) Systems

Liquid-liquid phase splitting in ternary mixtures that contain a room-temperature ionic liquid and an alcohol aqueous solution-namely, [bmim] [PF6] + ethanol + water and [bmim] [NTf2] + 2-methylpropanol + water-is studied. Experimental cloud-point temperatures were obtained up to pressures of 400 bar, using a He-Ne laser light-scattering technique. Although pressurization favors mutual miscibility in the presence of high concentrations of alcohols, the contrary occurs in water-rich solutions. Both ternary mixtures exhibit a very pronounced water-alcohol co-solvent effect. Solvent isotope effects are also investigated. Phase diagrams are discussed using a phenomenological approach based on a polymer-like G(E) model coupled with the statistical-mechanical theory of isotope effects. The combined effect of a red shift of -15 cm(-1) for the O-H deformation mode of ethanol with a blue shift of +35 cm(-1) for the O-H stretching mode, both of which occurring after liquid infinite dilution in the ionic liquid, rationalizes the observed isotope effect in the phase diagram. Predicted excess enthalpy (H-E) values are inferred from the model parameters. Furthermore, using the Prigogine-Defay equation, an estimation of the excess volumes (V-E) is obtained.

Isotope effect on the phonon-drag component of the thermoelectric power of germanium

We have measured the thermoelectric power of several germanium single crystals with different isotopic compositions: natural, 96.3%, and 99.99% enriched ${}^{70}\mathrm{Ge}.$ N- and p-type Ge samples with the net concentration of electrically active impurities $<2\ifmmode\times\else\texttimes\fi{}{10}^{13}{\mathrm{cm}}^{3}$ were studied. Our experimental data show a decrease of thermopower with an increase in isotopic mass disorder. This isotope effect is evident at temperatures below approximately 70 K where a phonon-drag part of the thermopower dominates. At high temperatures, in the diffusion regime, the thermopower does not correlate with the isotopic composition within experimental error. The observed isotope effect can be explained with a special phonon-drag contribution induced by a momentum transfer from thermal phonons to low-energy phonons in the enriched samples.

Thermal conductivity of isotopically enriched 71GaAs crystal

A gallium arsenide bulk crystal highly enriched with gallium isotope 71Ga to 99.40 at% has been grown using the seedless horizontal Bridgman method. The crystal has a total impurity concentration of about 2 × 1017 atoms cm−3 and a high electrical resistivity ρ(300 K) ∼ 103–108 Ω cm. Thermal conductivity measurements have been made from 90 K to 300 K of the enriched 71GaAs sample and three different natGaAs samples with a natural isotope composition of gallium. The 71GaAs has a higher value of thermal conductivity due to reduced isotope scattering of phonons: the observed isotope effect amounts to 5% at room temperature, increasing to 13% with a temperature decrease down to 100 K.

Isotopic quantum effects on the structure of low density amorphous ice

Careful neutron diffraction measurements on deuterated low density amorphous(LDA) ice confirm that at 120 K it can be considered a fully ‘annealed’ structure,as no significant changes are observed in the amorphous spectra untilcrystallization occurred over time at 130 K. On this basis, the measurement ofstructural differences between the hydrogenated and deuterated forms of LDA iceat 120 K, have been carried out using 98 keV electromagnetic radiation diffractiontechniques. The maximum observed isotope effect in LDA ice is ∼ 3.4%at 40 K when compared to the magnitude of the first peak inthe electronic structure factor at Q = 1.70 Å−1.This compares to a maximum effect of ∼ 1.6%previously measured in liquid water at room temperature (Tomberli et al 2000 J.Phys.: Condens. Matter. 12 2597). The isotope effect is shown to be similarto a temperature shift in the structure of light LDA ice. However, theexistence of a first sharp diffraction peak at Q = 1.0 Å−1 inthe isotopic difference function is not reproduced in the temperature differenceand suggests that additional longer-range correlations are present in the moreordered deuterated form.

Estimation of the isotope effect on the lattice thermal conductivity of group IV and group III-V semiconductors

The isotope effect on the lattice thermal conductivity for group IV and group III-V semiconductors is calculated using the Debye-Callaway model modified to include both transverse and longitudinal phonon modes explicitly. The frequency and temperature dependences of the normal and umklapp phonon-scattering rates are kept the same for all compounds. The model requires as adjustable parameters only the longitudinal and transverse phonon Gr\"uneisen constants and the effective sample diameter. The model can quantitatively account for the observed isotope effect in diamond and germanium but not in silicon. The magnitude of the isotope effect is predicted for silicon carbide, boron nitride, and gallium nitride. In the case of boron nitride the predicted increase in the room-temperature thermal conductivity with isotopic enrichment is in excess of 100%. Finally, a more general method of estimating normal phonon-scattering rate coefficients for other types of solids is presented.

Isotope effects and the mechanism of an electron-transfer-catalyzed Diels-Alder reaction.

The electron-transfer-catalyzed Diels-Alder reaction of indole and 1,3-cyclohexadiene was studied by a combination of experimental and theoretical methods. The (13)C kinetic isotope effects were determined at natural abundance by NMR methodology. B3LYP/6-31G* calculations allow for the first time a quantitatively accurate description of the different possible pathways and provide the basis for an analysis of the experimentally observed isotope effects. The computational results, in conjunction with experimental observations, show that the reaction has a stepwise mechanism that is initiated by attack of the diene into the 3-position of the indole. Numerical simulation of the experimentally observed isotope effects shows that the first step is rate-determining and that the electron exchange in the reactant contributes partially to the overall isotope effect. The combination of electronic structure theory, experimental isotope effects, and numerical simulation thus allows a detailed analysis of a complex reaction mechanism.

Isotope Effects in Electron Exchange Reactions

It has been known that uranium chemical exchange gives relatively large isotope separation factors. The strange isotope effects of uranium gave a clue to solve the hidden isotope effects of electronic states. The present paper discusses recently observed isotope effects in two typical electron-exchange systems of amalgam formation and redox reactions in ion exchange chromatography. Attention has been placed on the temperature dependence of the isotopic fractionations. The results clearly show that the separation coefficient ε is expressed by the temperature dependence of ε=a/T 2-b/T. The unit mass separation coefficients εΔM were calculated and compared among the examined elements of Li, Rb, Mg, Ba in the amalgam system and Fe, Cu, Eu in the redox ion exchange systems.

Isotope Effects in Electron Exchange Reactions.

It has been known that uranium chemical exchange gives relatively large isotope separation factors. The strange isotope effects of uranium gave a clue to solve the hidden isotope effects of electronic states. The present paper discusses recently observed isotope effects in two typical electron-exchange systems of amalgam formation and redox reactions in ion exchange chromatography. Attention has been placed on the temperature dependence of the isotopic fractionations. The results clearly show that the separation coefficient ε is expressed by the temperature dependence of ε=a/T 2-b/T. The unit mass separation coefficients εΔM were calculated and compared among the examined elements of Li, Rb, Mg, Ba in the amalgam system and Fe, Cu, Eu in the redox ion exchange systems.

Benzonitrile assisted enolization of the acetone and acetamide radical cations: proton-transport catalysis versus an intermolecular H+/· transfer mechanism

The acetamide radical cation, CH3C(=O)NH2·+, can be induced to rearrange into its more stable enol isomer, CH2=C(OH)NH2·+, by an ion–molecule interaction with benzonitrile, C6H5C≡N, under conditions of chemical ionization. (This enolization does not occur unassisted because of a prohibitively high energy barrier: 26 kcal/mol, from a CBS-QB3 calculation.) The initially formed [C6H5C≡N ⋯ acetamide]·+ adduct ion isomerizes to a stable hydrogen bridged radical cation [C6H5C≡N ⋯ H-O-C(NH2)=CH2]·+ en route to its dissociation into the enol ion. Multiple collision and deuterium labeling experiments on the acetamide/benzonitrile and the previously reported acetone/benzonitrile systems, indicate that the acetone ion enolizes by way of a base-catalyzed 1,3-proton shift (“proton-transport catalysis”) but that a different mechanism must be operative in the acetamide system. Ab initio and density functional theory calculations at the PMP3//RHF/D95∗∗ and PMP3//B3LYP/D95∗∗ level of theory support a mechanism which can be described as a consecutive H+/H·transfer between the partners of the [C6H5C≡N·+ ⋯ acetamide] encounter complex. The calculations provide a rationale for the observed isotope effects and lead to a tentative explanation for the differences in interaction of the title ions with benzonitrile.

Kinetic Isotope Effects and Variable Reaction Coordinates in Barrierless Recombination Reactions

The factors affecting kinetic isotope effects in barrierless recombination reactions are considered from the perspective of variational transition state theory (VTST). Despite the broad application of VTST methods, a general consideration of kinetic isotope effect predictions of the theory has not previously been undertaken, especially for cases where changes in the internal structure and vibrational frequencies of the fragments (i.e., the conserved modes) can be assumed to be negligible. Use of the center-of-mass separation as the reaction coordinate in such a case entails some restriction on the range of kinetic isotope effects which can be accommodated. Larger effects are possible within a variable reaction coordinate implementation of transition state theory, and the predicted kinetic isotope effects are shown to be strongly dependent on the location of the pivot point. Illustrative model calculations demonstrate the feasibility of reproducing the experimentally observed kinetic isotope effects for the CH + O2, HCC + O2, CH + C2H2, and CH + C2H4 reactions with realistic deviations of the pivot points from the center-of-mass. In contrast, calculations restricted to center-of-mass pivot points predict isotope effects that are even inverted. For the CH + CH4 reaction, the isotope effects appear too large to be explained by the reaction coordinate variations, and changes in the conserved modes play a key role in the observed isotope effects, as demonstrated with ab initio based TST simulations. Overall, the experimentally observed kinetic isotope effects in CH addition reactions are strongly suggestive of an optimum reaction coordinate corresponding to a pivot point located near the center of the radical orbital.

Isotope effects in an electron cyclotron resonance ion source inmixtures of 15N/14N

Isotope effects in an electron cyclotron resonance ion source areinvestigated in mixtures of 15N and 14N. The ratio of the extractedcurrents I(15Nq + )/I(14Nq + ) increases with increasingcharge state q. This so-called `isotope anomaly' proves to be dependent onboth the mixture ratio of the gases as well as the microwave power fed intothe discharge. It is shown that the often-utilized `ion cooling' model cannotsolely explain the observed isotope effects as long as the temperature isassumed to be equal for all ion species. Since we observed the occurrence oflow-frequency noise in correlation with the isotope anomaly, we suggest thatthe ions are additionally heated by ion Landau damping. Due to the massdependence of this process, the lighter ion species will be heatedpredominantly. This results in an enhanced transport of lighter ions out ofthe plasma and a better confinement for the heavier component, which,consequently, may be more effectively ionized to higher charges states.

Isotope effects and possible pairing mechanism in optimally doped cuprate superconductors

We have studied the oxygen-isotope effects on ${T}_{c}$ and in-plane penetration depth ${\ensuremath{\lambda}}_{\mathrm{ab}}(0)$ in an optimally doped three-layer cuprate ${\mathrm{Bi}}_{1.6}{\mathrm{Pb}}_{0.4}{\mathrm{Sr}}_{2}{\mathrm{Ca}}_{2}{\mathrm{Cu}}_{3}{\mathrm{O}}_{10+y}$ ${(T}_{c}\ensuremath{\sim}107 \mathrm{K}).$ We find a small oxygen-isotope effect on ${T}_{c}$ $({\ensuremath{\alpha}}_{O}=0.019),$ and a substantial effect on ${\ensuremath{\lambda}}_{\mathrm{ab}}(0)$ $[\ensuremath{\Delta}{\ensuremath{\lambda}}_{\mathrm{ab}}(0)/{\ensuremath{\lambda}}_{\mathrm{ab}}(0)=2.5\ifmmode\pm\else\textpm\fi{}0.5%].$ The present results along with the previously observed isotope effects in single-layer and double-layer cuprates indicate that the isotope exponent ${\ensuremath{\alpha}}_{O}$ in optimally doped cuprates is small while the isotope effect on the in-plane effective supercarrier mass is substantial and nearly independent of the number of the ${\mathrm{CuO}}_{2}$ layers. A plausible pairing mechanism is proposed to explain the isotope effects, high-${T}_{c}$ superconductivity, and tunneling spectra in a consistent way.

Deuterium isotope effect on the intramolecular electron transfer in Pseudomonas aeruginosa azurin.

Intramolecular electron transfer in azurin in water and deuterium oxide has been studied over a broad temperature range. The kinetic deuterium isotope effect, k(H)/k(D), is smaller than unity (0.7 at 298 K), primarily caused by the different activation entropies in water (-56.5 J K(-1) mol(-1)) and in deuterium oxide (-35.7 J K(-1) mol(-1)). This difference suggests a role for distinct protein solvation in the two media, which is supported by the results of voltammetric measurements: the reduction potential (E(0')) of Cu(2+/+) at 298 K is 10 mV more positive in D(2)O than in H(2)O. The temperature dependence of E(0') is also different, yielding entropy changes of -57 J K(-1) mol(-1) in water and -84 J K(-1) mol(-1) in deuterium oxide. The driving force difference of 10 mV is in keeping with the kinetic isotope effect, but the contribution to DeltaS from the temperature dependence of E(0') is positive rather than negative. Isotope effects are, however, also inherent in the nuclear reorganization Gibbs free energy and in the tunneling factor for the electron transfer process. A slightly larger thermal protein expansion in H(2)O than in D(2)O (0.001 nm K(-1)) is sufficient both to account for the activation entropy difference and to compensate for the different temperature dependencies of E(0'). Thus, differences in driving force and thermal expansion appear as the most straightforward rationale for the observed isotope effect.

Dissociative Recombination of H3O+, HD2O+, and D3O+

We present an experimental study of the dissociative recombination (DR) of H3O+ and its isotopomers D3O+ and HD2O+ performed at the ASTRID storage ring. DR cross sections have been measured as a function of energy, while complete branching ratios have been measured at E = 0. The H3O+ data yield an accurate determination of the branching ratio for water formation (0.25 ± 0.01). The three molecular ions—H3O+, D3O+, and HD2O+—show a marked resemblance concerning cross sections and branching ratios. The only observed isotope effect is in the fragmentation pattern of HD2O+, where the release of a light fragment is favored over release of a heavier fragment. As a consequence, an enhanced production of deuterated molecules takes place as a result of the DR process.

Isotope effects and the mechanism of allylic hydroxylation of alkenes with selenium dioxide.

The mechanism of the allylic oxidation of 2-methyl-2-butene with selenium dioxide was explored by a combination of experimental and theoretical studies. A comparison of the experimental (13)C and (2)H kinetic isotope effects with predicted values shows that the observed isotope effects are consistent with an initial concerted ene step mediated by SeO(2). However, this comparison also does not rule out the involvement of a selenous ester in the ene reaction or a stepwise reaction involving reversible electrophilic addition of HSeO(2)(+) followed by rate-limiting proton abstraction. Becke3LYP calculations strongly favor SeO(2) over a selenous ester as the active oxidant, with the predicted barrier for reaction of 2-methyl-2-butene with SeO(2) being 21-24 kcal/mol lower than that for reaction with H(2)SeO(3). The possibility of a selenous ester being the active oxidant is also disfavored by the observation of oxidations in non-hydroxylic solvents. The involvement of HSeO(2)(+) does not appear consistent with a lack of dependence of the reaction on the basicity of the reaction mixture. A concerted ene reaction with SeO(2) as the active oxidant appears to be the major mechanistic pathway operative in these reactions.

Electron-induced dissociation of SiH complexes in hydrogenated Si-doped GaAs. Application to the fabrication of microstructures

We study the role of hot electron injection in the dissociation of the SiH complexes which appear in n-type Si-doped GaAs epilayer exposed to a hydrogen or deuterium plasma. Firstly, the results recently obtained in room-temperature aging experiments on hydrogenated or deuterated Schottky diodes submitted to high bias voltage are summarized and the role of hot carriers in the dissociation of donors and the observed isotope effect are described. Then, it is shown that SiH dissociation can also be achieved using hot carriers injected into the semiconductor by electron beam. Such electron-beam effects are finally used for the fabrication and characterization by cathodoluminescence of micronic conductive GaAs structures.