Heavy Atom Kinetic Isotope Effects Research Articles

Heavy-Atom Kinetic Isotope Effects: Primary Interest or Zero Point?

Chemists have many options for elucidating reaction mechanisms. Global kinetic analysis and classic transition-state probes (e.g., LFERs, Eyring) inevitably form the cornerstone of any strategy, yet their application to increasingly sophisticated synthetic methodologies often leads to a wide range of indistinguishable mechanistic proposals. Computational chemistry provides powerful tools for narrowing the field in such cases, yet wholly simulated mechanisms must be interpreted with great caution. Heavy-atom kinetic isotope effects (KIEs) offer an exquisite but underutilized method for reconciling the two approaches, anchoring the theoretician in the world of calculable observables and providing the experimentalist with atomistic insights. This Perspective provides a personal outlook on this synergy. It surveys the computation of heavy-atom KIEs and their measurement by NMR spectroscopy, discusses recent case studies, highlights the intellectual reward that lies in alignment of experiment and theory, and reflects on the changes required in chemical education in the area.

Hopping Conductance in Molecular Wires Exhibits a Large Heavy-Atom Kinetic Isotope Effect.

We report a large kinetic isotope effect (KIE) for intramolecular charge transport in π-conjugated oligophenyleneimine (OPI) molecules connected to Au electrodes. 13C and 15N substitution on the imine bonds produces a conductance KIE of ∼2.7 per labeled atom in long OPI wires >4 nm in length, far larger than typical heavy-atom KIEs for chemical reactions. In contrast, isotopic labeling in shorter OPI wires <4 nm does not produce a conductance KIE, consistent with a direct tunneling mechanism. Temperature-dependent measurements reveal that conductance for a long 15N-substituted OPI wire is activated, and we propose that the exceptionally large conductance KIEs imply a thermally assisted, through-barrier polaron tunneling mechanism. In general, observation of large conductance KIEs opens up considerable opportunities for understanding microscopic conduction mechanisms in π-conjugated molecules.

Theoretical Simulations of Heavy-Atom Kinetic Isotope Effects in Aliphatic Claisen Rearrangement.

The aliphatic Claisen rearrangement of allyl vinyl ether has attracted great interest for its broad applications in chemical synthesis and biosynthesis. Although it is well agreed that this reaction proceeds via a concerted, "chair-like" transition state, certain inconsistencies of kinetic isotope effect (KIE) data between experimental measurements and theoretical simulations or between independent experiments indicate that the nature and mechanism of this important reaction need to be investigated in more detail. In order to verify two independent sets of experimental data, we present theoretical calculations on heavy-atom KIE values of alipahtic Claisen rearrangement, using our recently developed path-integral method with the second-order Kleinert's variational perturbation theory, which goes beyond the traditional method for computing KIE values by employing the Bigeleisen equation. Amazingly, the results demonstrate that both sets of experimental measurements are correct, while the inconsistency originates from the fact that the aliphatic Claisen rearrangement undergoes similar but different mechanisms in gas and solution phases. Different experimental conditions will alter the actual reactant state by tuning the population distribution of reactant conformers. According to the comparison between experimental and theoretical results, a more clear reaction mechanism of aliphatic Claisen rearrangement is revealed.

Transition State Analysis of the Reaction Catalyzed by the Phosphotriesterase from Sphingobium sp. TCM1.

Organophosphorus flame retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 ( Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the p Ka of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Brønsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates.

Second-Shell Hydrogen Bond Impacts Transition-State Structure in Bacillus subtilis Oxalate Decarboxylase.

There is considerable interest in how "second-shell" interactions between protein side chains and metal ligands might modulate Mn(II) ion redox properties and reactivity in metalloenzymes. One such Mn-dependent enzyme is oxalate decarboxylase (OxDC), which catalyzes the disproportionation of oxalate monoanion into formate and CO2. Electron paramagnetic resonance (EPR) studies have shown that a mononuclear Mn(III) ion is formed in OxDC during catalytic turnover and that the removal of a hydrogen bond between one of the metal ligands (Glu101) and a conserved, second-shell tryptophan residue (Trp132) gives rise to altered zero-field splitting parameters for the catalytically important Mn(II) ion. We now report heavy-atom kinetic isotope effect measurements on the W132F OxDC variant, which test the hypothesis that the Glu101/Trp132 hydrogen bond modulates the stability of the Mn(III) ion during catalytic turnover. Our results suggest that removing the Glu101/Trp132 hydrogen bond increases the energy of the oxalate radical intermediate from which decarboxylation takes place. This finding is consistent with a model in which the Glu101/Trp132 hydrogen bond in WT OxDC modulates the redox properties of the Mn(II) ion.

Heavy Atom Secondary Kinetic Isotope Effect on H-Tunneling.

Although frequently employed, heavy atom kinetic isotope effects (KIE) have not been reported for quantum mechanical tunneling reactions. Here we examine the secondary KIE through 13C-substitution of the carbene atom in methylhydroxycarbene (H3C-C̈-OH) in its [1,2]H-tunneling shift reaction to acetaldehyde (H3C-CHO). Our study employs matrix-isolation IR spectroscopy in various inert gases and quantum chemical computations. Depending on the choice of the matrix host gas, the KIE varies within a range of 1.0 in xenon to 1.4 in neon. A KIE of 1.1 was computed using the Wentzel-Kramers-Brillouin (WKB) CVT/SCT, and instanton approaches for the gas phase at the B3LYP/cc-pVTZ level of theory. Computations with explicit consideration of the noble gas environment indicate that the surrounding atoms influence the tunneling reaction barrier height and width. The tunneling half-lives computed with the WKB approach are in good agreement with the experimental results in the different noble gases.

Measurement of Kinetic Isotope Effects by Continuously Monitoring Isotopologue Ratios Using NMR Spectroscopy.

Nuclear magnetic spectroscopic (NMR) methods are discussed for the measurement of heavy atom (13C, 18O, 15N) and secondary deuterium kinetic isotope effects. The discussion focuses primarily on the NMR methods that enable the measurement of quantitative spectra and not on methods to make labeled substrates. Two main techniques are considered: single-point determinations on natural abundance material and the continuous monitoring of isotopically enriched materials. The second method is described in more detail, and we include a discussion of the current state of instrumentation and computer programs for data acquisition and analysis.

An investigation into the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine using heavy atom kinetic isotope effects

Heavy atom kinetic isotope effects (KIEs) were determined for the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine (FTC). The leaving-S, carbonyl-C, and carbonyl-O KIEs are 34k=0.994±0.004, 13k=1.0148±0.0007, and 18k=0.999±0.002, respectively. The observed KIEs support a mechanism for both acylation and deacylation where the steps up to and including the formation of the tetrahedral intermediate are at least partially rate determining. These results, in contrast to previous studies with acetylthiocholine, suggest that the decomposition of a tetrahedral intermediate is not rate-determining for FTC hydrolysis. Structural differences between the two substrates are likely responsible for the observed mechanism change with FTC.

A mechanistic study of thioester hydrolysis with heavy atom kinetic isotope effects.

The carbonyl-C, carbonyl-O, and leaving-S kinetic isotope effects (KIEs) were determined for the hydrolysis of formylthiocholine. Under acidic conditions, (13)k(obs) = 1.0312, (18)k(obs) = 0.997, and (34)k(obs) = 0.995; for neutral conditions, (13)k(obs) = 1.022, (18)k(obs) = 1.010, and (34)k(obs) = 0.996; and for alkaline conditions, (13)k(obs) = 1.0263, (18)k(obs) = 0.992, and (34)k(obs) = 1.000. The observed KIEs provided helpful insights into a qualitative description of the bond orders in the transition state structure.

Cytochrome P450 Monooxygenase-Catalyzed Ring Opening of the Bicyclic Amine, Nortropine: An Experimental and DFT Computational Study

An approach combining experimental kinetic isotope effects (KIEs) and quantum chemistry calculations at the DFT level was used to explore the mechanism by which the CN bond of the bicyclic amine nortropine (2) was cleaved during the catabolism of tropine (1) by Pseudomonas bacteria. The working model is that the ring opening involves the oxidation of nortropine at one bridgehead carbon position by cytochrome P450 monooxygenase, followed by the protonation of the nitrogen, which leads to heterolytic bond cleavage. 15N and 2H heavy-atom KIEs for the reaction were determined both experimentally and theoretically (DFT). Transition states were described for both oxidation and ring cleavage. Calculation of the heavy-atom KIE values indicated a high 2H, a moderate 13C, and a negligible 15N effect for the oxidation and a strong inverse 15N and normal 13C KIEs at N and 1C for the 1CN bond cleavage. Experimental KIE values for the overall reaction were a strongly inverse 15N and negligible 2H effect, which indicated that the catalytic rate depended on the bond-breaking part of the reaction. Thus, from the experimental and calculated results, it could be proposed that the enzyme-dependent activation of the 1C position to destabilize the structure is a prerequisite for bond cleavage, but the driving force behind the carbon–nitrogen bond fission is the simultaneous protonation of the nitrogen of the intermediate species [3] and sp3 to sp2 transition at the 1C position. Thus, a mechanism that satisfactorily fits both the experimental and theoretical results could be proposed. It is unusual in that it indicates that the cytochrome P450 monooxygenase-catalyzed hydrogen abstraction—though essential for the overall reaction to occur—is not the kinetically limiting partial reaction.

Path‐integral calculations of heavy atom kinetic isotope effects in condensed phase reactions using higher‐order trotter factorizations

A convenient approach to compute kinetic isotope effects (KIEs) in condensed phase chemical reactions is via path integrals (PIs). Usually, the primitive approximation is used in PI simulations, although such quantum simulations are computationally demanding. The efficiency of PI simulations may be greatly improved, if higher-order Trotter factorizations of the density matrix operator are used. In this study, we use a higher-order PI method, in conjunction with mass-perturbation, to compute heavy-atom KIE in the decarboxylation of orotic acid in explicit sulfolane solvent. The results are in good agreement with experiment and show that the mass-perturbation higher-order Trotter factorization provides a practical approach for computing condensed phase heavy-atom KIE.

Rational Design of Transition-State Analogues as Potent Enzyme Inhibitors with Therapeutic Applications

The structures of the transition states for a variety of enzyme-catalyzed ribosyl group transfer reactions, determined by computational evaluation of multiple tritium and heavy atom kinetic isotope effects on these enzymatic reactions, have been found to show a considerable variation in the extent of bond cleavage at the ribosyl anomeric carbon. The calculated transition-state structures have been used to guide the design of high-affinity transition-state analogue inhibitors for 5'-methylthioadenosine nucleosidases with potential as therapeutic agents.

Supermolecule density functional calculations suggest a key role for solvent in alkaline hydrolysis of p-nitrophenyl phosphate

Supermolecule density functional theory calculations show that solvent is responsible for the concerted transition state in alkaline hydrolysis of p-nitrophenyl phosphate suggested by heavy atom kinetic isotope effects.

Transition state analysis of adenosine nucleosidase from yellow lupin ( Lupinus luteus)

The transition state of adenosine nucleosidase (EC 3.2.2.7) isolated from yellow lupin ( Lupinus luteus) was determined based upon a series of heavy atom kinetic isotope effects. Adenosine labeled with 13C, 2H, and 15N was analyzed by liquid chromatography/electrospray mass spectrometry to determine kinetic isotope effects. Values of 1.024 ± 0.004, 1.121 ± 0.005, 1.093 ± 0.004, 0.993 ± 0.006, and 1.028 ± 0.005 were found for [1′- 13C], [1′- 2H], [2′- 2H], [5′- 2H], and [9- 15N] adenosine, respectively. Using a bond order bond energy vibrational analysis, a transition state consisting of a significantly broken C–N bond, formation of an oxocarbenium ion in the ribose ring, a conformation of C3- exo for the ribose ring, and protonation of the heterocyclic base was proposed. This transition state was found to be very similar to the transition state for nucleoside hydrolase, another purine metabolizing enzyme, isolated from Crithidia fasciculata.

Physical Organic Perspectives on Phospho Group Transfer from Phosphates and Phosphinates

AbstractFor Abstract see ChemInform Abstract in Full Text.

Physical Organic Perspectives on Phospho Group Transfer From Phosphates and Phosphinates

As phosphoryl transfer reactions are ubiquitous in biological chemistry, organic chemists have been very interested in the mechanisms of phosphate and phosphinate esters. Physical organic chemistry methods, including stereochemical studies, linear free energy relationships, and, most recently, heavy-atom kinetic isotope effects, have been used in the quest for mechanistic information about the chemistry of these compounds. This review summarizes what has been learned about the uncatalyzed phosphoryl transfer reactions of phosphate and phosphinate esters. Keywords: dianions, hydrolysis, phosphorothioate analogs, isotope, solvolysis

Inverse Heavy-Atom Kinetic Isotope Effects in Chloroalkanes

Intramolecular heavy-atom kinetic isotope effects (KIEs) are reported for reactions of some gas-phase ions relevant to atmospheric chemistry. The anionic complexes carbon tetrachloride/Cl-, chloroform/Cl-, and methylene chloride/Cl- (CHnCl5-n-; n = 0−2) were generated by various ionization methods and subjected to single-stage and tandem mass spectrometric analysis in a variety of instruments. Strongly pressure dependent, anomalous chloride abundances were observed in single-stage mass spectra under self-chemical ionization conditions for CHCl4- and CCl5- in a triple quadrupole and for CHCl4- in a quadrupole ion trap. Further examination of these complexes in triple quadrupole MS/MS experiments confirmed the presence of an unusually large inverse kinetic isotope effect. We suggest that the selective loss of 37Cl- is simply due to the difference in centrifugal barriers associated with the competitive reaction channels leading to the two chloride isotopes. This effect is greatly magnified near the threshold for dissociation, and an extreme value of an inverse kinetic isotope effect (0.026 ± 0.01) is displayed by the isotopomeric chloride adduct of chloroform, CH35Cl337Cl-, under single-collision conditions with xenon at 27 eV lab collision energy. The methylene chloride adduct, CH235Cl237Cl-, had a KIE close to unity (0.90 ± 0.05), and the carbon tetrachloride adduct displayed no apparent KIE, under these same conditions. Remarkably, in addition to the heavy-atom KIEs for chlorinated chloroform, (i) substantial H/D isotope effects are associated with chloride loss, (ii) the chlorine KIE values are strongly dependent on the particular isotopomer selected for examination, and (iii) the chlorine KIE for CHCl4- is not monotonic with collision energy. In sum, the results are indicative of a complex potential energy surface and of a mixture of dissociating structures or independent dissociation channels of the precursor.

Heavy atom isotope effects on the reaction catalyzed by the oxalate decarboxylase from Bacillus subtilis.

Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate. Like the native OxDC isolated from Aspergillus niger, the recombinant, bacterial OxDC from Bacillus subtilis contains Mn(II) in its resting state and requires catalytic dioxygen for activity. The most likely mechanism for OxDC-catalyzed C-C bond cleavage involves the participation of free radical intermediates, although this hypothesis remains to be unequivocally demonstrated. Efforts to delineate the catalytic mechanism have been placed on a firm foundation by the high-resolution crystal structure of recombinant, wild type B. subtilis OxDC (Anand et al., Biochemistry 2002, 41, 7659-7669). We now report the results of heavy-atom kinetic isotope effect measurements for the OxDC-catalyzed decarboxylation of oxalate, in what appear to be the first detailed studies of the mechanism employed by OxDC. At pH 4.2, the OxDC-catalyzed formation of formate and CO(2) have normal (13)C isotope effects of 1.5% +/- 0.1% and 0.5% +/- 0.1%, respectively, while the (18)O isotope effect on the formation of formate is 1.1% +/- 0.2% normal. Similarly at pH 5.7, the production of formate and CO(2) exhibits normal (13)C isotope effects of 1.9% +/- 0.1% and 0.8% +/- 0.1%, respectively, and the (18)O isotope effect on the formation of formate is 1.0% +/- 0.2% normal. The (18)O isotope effect on the formation of CO(2), however, 0.7% +/- 0.2%, is inverse at pH 5.7. These results are consistent with a multistep model in which a reversible, proton-coupled, electron transfer from bound oxalate to the Mn-enzyme gives an oxalate radical, which decarboxylates to yield a formate radical anion. Subsequent reduction and protonation of this intermediate then gives formate.

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.

Carbon Isotope Fractionation in the Thermal Decomposition of Uranyl Formate

Thermal decompositions of uranium(VI) chelates have been used in the past for quantitative determinations of uranium. The mechanisms of these pyrolyses have been investigated several times by the method of differential thermal analyses to clear up the inconsistencies in the literature [l-7]. The discrepant results have been reported also in the case of the much simpler thermal decompositions of uranyl formates and discussed also [8-13]. The irresolvable mechanistic problems of reactions in solutions are usually studied by supplementing the classical kinetic observations with kinetic isotope effects (KIEs) determinations. The much more specific solid state reactions are less frequently probed by the heavy atom KIEs methods. We investigated recently the carbon kinetic isotope effects (13C KIEs) in the decarbonylation (and oxidation) of liquid formic acid as such [14,14a] and in the presence of uranium oxide, uranyl formate (UF) and uranium peroxide [15,16]. The experimental 13C KIEs were found to be constituent with 13C KIEs calculated assuming that in the case of decarbonylations taking place in liquid phase they are determined by the rupture of the single carbonoxygen bond. In the case of oxidation of liquid formic acid with uranium peroxide the 13C/12C fractionation was found to be governed by the carbon-hydrogen bond rupture [16]. The differences between solid phase and liquid phase decompositions of formic acid and formates should be more precisely revealed by 13C KIEs determinations. We therefore initiated 13C KIEs studies in the thermal decomposition of uranyl formate in vacuum glass reaction vessel in a 260-390°C temperature interval. We quantitatively isolated carbon dioxide, carbon monoxide, volatile formic acid and water as decomposition products (Eq. 1). The [CO2]/[CO] ratio was increasing with the increase of the decomposition temperature from 0.90±0.02 (at 275-316°C) to 2.06 (at 382°C). At “low” temperatures the [C02]/[CO] ratio was increasing also with the increase of the degree “f” of decomposition of solid formate. The first portion of gaseous products collected at 275 "C contained carbon dioxide and carbon monoxide in a ratio (0.163 1 /0.5723) = 0.285 only.