Secondary Deuterium Isotope Effects Research Articles

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.

Large primary kinetic isotope effects in the abstraction of hydrogen from organic compounds by a fluorinated radical in water.

Isotope effects have been measured for the abstraction of hydrogen from a series of organic substrates by the perfluoro radical, Na+ -O3SCF2CF2OCF2CF2*, in water. Both primary and secondary deuterium isotope effects were measured, with the primary isotope effects ranging in value from 4.5 for isopropanol to 19.6 for acetic acid. The values for the alpha- and beta-secondary deuterium isotope effects were 1.06 and 1.035, respectively. It was concluded that tunneling contributes significantly to the production of the observed, large primary kinetic isotope effects in these C-H abstraction reactions.

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 (14)C, or with the natural abundances of (13)C, (15)N, or (18)O. 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, (13)C, (15)N, or (18)O), 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 (13)C ones with deuterated and undeuterated substrates. Adding a secondary deuterium isotope effect and its effect on the (13)C one can give an exact solution for all intrinsic isotope effects and commitments. The effect of deuteration on a (13)C 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.

The kinetic method reveals secondary deuterium isotope effects on the proton affinity and gas-phase basicity of glycine and alanine methyl esters

The kinetic method for measuring proton affinities (PA) and gas-phase basicities (GB) was applied to the methyl esters of simple amino acids. The experiments show that the GB and PA values for deuterium labeled glycine methyl ester are indeed greater than that of the corresponding unlabelled glycine methyl ester. The PA of l-Ala-OCD 3 is also slightly greater than that of the unlabeled alanine methyl ester. The secondary isotope effects originate, as shown by density functional theory, in differences in zero-point energies and thermal-energy corrections between H and D-bearing molecules.

Rearrangement Pathways of Five-Membered Ring Enlargement in Carbocations: Quantum Chemical Calculations and Deuterium Kinetic Isotope Effects

Three plausible routes for the five-membered ring expansion in the equilibrating 2-cyclopentyl-2-propyl and 1-(2-propyl)cyclopentyl cations 1A/1B were located on the PES, all calculated at the MP4/6-31G(d)//MP2/6-31G(d) level of theory. In pathway I, the six-membered transition structure (TS-I) connects the less stable cyclopentyl cation 1A and the 1,2-dimethylcyclohexyl carbocation (2) via a barrier of 16.4 kcal/mol. In pathway II, which has a barrier of 16.3 kcal/mol, the methyl migration occurs first in the more stable 1B via transition structure TS-II. Pathway III involves the uphill hydride shift and formation of the secondary cation 3, which undergoes Wagner-Meerwein 1,2-isopropyl shift via a transition structure TS-III and the protonated carbocation intermediate 4. The barrier pathway III is for 17.0 kcal/mol. Experimental secondary deuterium isotope effects of the rearrangement were measured for the hexadeuterated 1A-d6/1B-d6 (kH/kD = 2.40) and tetradeuterated 1A-d4/1B-d4 (kH/kD = 0.18) cations by means of 1H NMR. Comparison of the experimental data with the theoretical values (kH/kD = 2.40 for 1B-d6 and kH/kD = 0.24 for 1B-d4, respectively) obtained with QUIVER revealed that pathway II is a major reaction route.

Intrinsic deuterium isotope effects on benzylic hydroxylation by tyrosine hydroxylase.

Tyrosine hydroxylase (TyrH) is a mononuclear, non-heme iron monooxygenase that catalyzes the pterin-dependent hydroxylation of tyrosine to dihydroxyphenylalanine. When 4-methylphenylalanine is used as a substrate for TyrH, 4-hydroxymethylphenylalanine is one of the amino acid products. To examine the mechanism of benzylic hydroxylation, the products and their isotopic compositions were determined with 4-methylphenylalanines containing a mono-, di-, or trideuterated methyl group as substrates. Intrinsic primary and secondary deuterium isotope effects for benzylic hydroxylation of 9.6 +/- 0.9 and 1.21 +/- 0.08, respectively, were derived from the data. The magnitudes of these isotope effects are consistent with quantum mechanical tunneling of the hydrogen. The similarity of the effects to those seen for benzylic hydroxylation by other enzymes supports a mechanism where a high valence iron-oxo species, Fe(IV)=O, is the hydroxylating intermediate.

13C and deuterium isotope effects suggest an aldol cleavage mechanism for L-ribulose-5-phosphate 4-epimerase.

On the basis of (13)C and deuterium isotope effects, L-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of L-ribulose 5-phosphate to D-xylulose 5-phosphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group and condensation to the epimer at C-4. With the wild-type enzyme, (13)C isotope effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively. H97N and Y229F mutants at pH 7 gave values of 3.25 and 2.53% at C-3 and 2. 69 and 1.99% at C-4, respectively. Secondary deuterium isotope effects at C-3 were 2.5% at pH 7 and 3.1% at pH 5.5 with the wild-type enzyme, and 4.1% at pH 7 with H97N. At C-4, the corresponding values were 9.6, 14, and 19%. These data suggest that H97N shows no commitments, while the wild-type enzyme has an external commitment of approximately 1.4 at pH 7 and an internal commitment independent of pH of approximately 0.6. The Y229 mutant shows only the internal commitment of 0.6. The sequence of the epimerase is similar to those of L-fuculose-1-phosphate and L-rhamnulose-1-phosphate aldolases for residues in the active site of L-fuculose-1-phosphate aldolase, suggesting that Asp76, His95, His97, and His171 of the epimerase may be metal ion ligands, and Ser44, Gly45, Ser74, and Ser75 may form a phosphate binding pocket. The pH profile of V/K for L-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24. The CD spectra of L-ribulose 5-phosphate and D-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm.

NMR studies on hydrogen bonding and proton transfer in Mannich bases

A review of studies on the ortho Mannich bases containing various substituents in the phenyl ring on the basis of1H,13C and15N nuclear magnetic resonance (NMR) spectra in various solvents over the temperature range 110–298 K is presented. Some new results are also included. The data gathered so far show that there is some critical (inversion) range of ΔpK a (= pK a(NH+) − pK a(OH)) in which the proton transfer equilibrium appears. This inversion range is well reflected in the behaviour of secondary deuterium isotope effect in13C NMR spectra. A strong temperature effect on the strength of hydrogen bonding should be emphasized. The1H chemical shift for trichloroderivative increases from 13.5 at room temperature up to 17 ppm at 130 K when the proton is equally shared between the bridging atoms (1 J(1H,15N) = 30–40 Hz). The potential for the proton motion in such bridges is discussed taking into account the behaviour in the ultraviolet and infrared spectra. The role of dimerization in proton transfer equilibria is shown. In addition the rotation of OH groups involved in hydrogen bond formation and nitrogen pyramidal inversion was studied by the1H dynamical NMR spectra.

Aziridination of Alkenes and Amidation of Alkanes by Bis(tosylimido)ruthenium(VI) Porphyrins. A Mechanistic Study

Bis(tosylimido)ruthenium(VI) porphyrins, [RuVI(Por)(NTs)2] (Por = TPP, TTP, 4-Cl-TPP, 4-MeO-TPP, OEP), were prepared in 60−74% yields by treatment of [RuII(Por)(CO)(MeOH)] with (N-(p-tolylsulfonyl)imino)phenyliodinane (PhINTs) in dichloromethane. In dichloromethane containing pyrazole, they reacted with alkenes or alkanes to give tosylamidoruthenium(IV) porphyrins, [RuIV(Por)(NHTs)(pz)], in about 75% yields. The reactions of [RuVI(TPP)(NTs)2] and [RuVI(OEP)(NTs)2] with styrene, para-substituted styrenes, norbornene, cyclooctene, and β-methylstyrene afforded the corresponding N-tosylaziridines in 66−85% yields. The aziridination of cis-stilbene and cis-β-methylstyrene by [RuVI(Por)(NTs)2] is nonstereospecific with a partial loss of the alkene stereochemistry. Kinetic studies on the reactions between [RuVI(TPP)(NTs)2] and 16 alkenes (cyclooctene, norbornene, 2,3-dimethyl-2-butene, styrene, para-substituted styrenes, α- and β-methylstyrene, and α- and β-deuteriostyrene) gave the second-order rate constants (k2) ranging from (1.60 ± 0.06) × 10-3 to (90 ± 4) × 10-3 dm3 mol-1 s-1 at 298 K. The slope of the linear plot of log k2 vs E1/2 for eight representative alkenes was found to be −1.7 V-1. In the case of para-substituted styrenes, linear correlation between log kR (kR = relative rate) and σ+ gives a ρ+ value as small as −1.1. However, the effect of para substituents on kR can be best accounted for by considering both the polar and spin delocalization effect. Measurements on the secondary deuterium isotope effect revealed that only the β-carbon atom of styrene experienced a significant change in its hybridization in reaching the transition state. All these are consistent with rate-determining formation of a carboradical intermediate. The reactions of [RuVI(TPP)(NTs)2] and [RuVI(OEP)(NTs)2] with adamantane, cyclohexene, ethylbenzene, and cumene resulted in tosylamidation of these hydrocarbons and afforded the corresponding amides in 52−88% yields. For cyclohexane and toluene, the tosylamidation products were formed in poor yields (ca. 10%). Kinetic studies on the reactions between [RuVI(TPP)(NTs)2] and nine hydrocarbons (cumene, ethylbenzene, cyclohexene, and para-substituted ethylbenzenes) gave the second-order rate constants (k2) in the range of (0.330 ± 0.008) × 10-3 to (16.5 ± 0.3) × 10-3 dm3 mol-1 s-1. These reactions exhibit a large primary deuterium isotope effect, with a kH/kD ratio of 11 for the tosylamidation of ethylbenzene. In the case of para-substituted ethylbenzenes, both electron-donating and -withdrawing substituents moderately promote the reaction. There is an excellent linear correlation between log kR and a related carboradical parameter. On the basis of these observations, a mechanism involving the rate-limiting formation of a carboradical intermediate is postulated.

Electron Transfer Catalyzed [2 + 2] Cycloreversion of Benzene Dimers.

The catalysis of the [2 + 2] cycloreversion of the anti-o,o'-benzene dimer 1 and the syn-o,o'-naphthalene-benzene dimer 2 through thermal and photoinduced electron transfer is studied using experimental and computational methods. The reaction of the radical cations formed by electron transfer is at least 10(5) times faster than the thermal background reaction. It is demonstrated that the photoinduced electron transfer catalyzed reaction proceeds via an electron transfer sensitized pathway and that the observed inverse secondary deuterium isotope effect of 0.91 +/- 0.02 on the reaction is due to the equilibrium isotope effect on the electron transfer step. The relevance of these findings on the mechanism of the electron transfer catalyzed [2 + 2] cycloreversion of the biologically important cis,syn-cyclobutane-thymine dimer is also discussed.

Secondary Deuterium Kinetic Isotope Effects in Irreversible Additions of Hydride and Carbon Nucleophiles to Aldehydes: A Spectrum of Transition States from Complete Bond Formation to Single Electron Transfer

The competitive kinetics of hydride and organometallic additions to benzaldehyde-H and -D were determined at −78 °C using LiAlH4, LiBEt3H, NaBH4, LiBH4, LiAl(O-tert-butoxy)3H, NaB(OMe)3H, NaB(OAc)3H (at 20° C). methyl, phenyl, and allyl Grignard, and methyl-, phenyl-, n-butyl-, tert-butyl-, and allyllithium. The additions of hydride were found to have an inverse secondary deuterium kinetic isotope effects in all cases, but the magnitude of the effect varied inversely with the apparent reactivity of the hydride. In the additions of methyl Grignard reagent and of methyllithium and phenyllithium, inverse secondary deuterium isotope effects were observed; little if any isotope effect was observed with phenyl Grignard or n-butyl- and tert-butyllithium. With allyl Grignard and allyllithium, a normal secondary deuterium kinetic isotope effect was observed. The results indicate that rate-determining single-electron transfer occurs with allyl reagents, but direct nucleophilic reaction occurs with all of the other reagents, with the extent of bond formation dependent on the reactivity of the reagent. In the addition of methyllithium to cyclohexanecarboxaldehyde, a less inverse secondary deuterium kinetic isotope effect was observed than that observed in the addition of methyllithium to benzaldehyde, and allyllithium addition to cyclohexanecarboxaldehyde had a kinetic isotope effect near unity. The data with organometallic additions, which are not incompatible with observations of carbonyl carbon isotope effects, suggest that electrochemically determined redox potentials which indicate endoergonic electron transfer with energies less than ca. 13 kcal/mol allow electron-transfer mechanisms to compete well with direct polar additions to aldehydes, provided that the reagent is highly stabilized, like allyl species. Methyl- and phenyllithium and methyl and phenyl Grignard reagents are estimated to undergo electron transfer with endoergonicities greater than 30 kcal/mol with benzaldehyde, so these react by direct polar additions. A working hypothesis is that butyllithium reagents undergo polar additions, despite redox potentials which indicate less than 13 kcal/mol endoergonic electron transfer, because of the great exoergonicity associated with the two-electron addition, which is responsible for a low barrier for polar reactions.

The Behavior of Substrate Analogues and Secondary Deuterium Isotope Effects in the Phenylalanine Ammonia-Lyase Reaction

Metacresol and glycine can be thought as a dissection of metatyrosine, which is an excellent substrate of phenylalanine ammonia-lyase (PAL) (B. Schuster and J. Rétey,PNAS92, 8433, 1995). Whereas metacresol was a very weak inhibitor and glycine was inert, simultaneous addition of both compounds led to synergistic inhibition of PAL. [2H5]Phenylalanine as a substrate showed a kinetic deuterium isotope effect of 9% (kH/k2H= 1.09 ± 0.01) while itsKmvalue was identical to that of the unlabeled substrate. The following substrate analogues were synthesized and assayed with PAL: cyclooctatetraenyl (COT)-d,l)-alanine as well as 2-pyridyl-, 3-pyridyl-, and 4-pyridyl-(l)-alanines. While COT-(d,l)-alanine turned out to be a rather reluctant substrate, all three isomers of pyridyl-(l)-alanines were converted with a comparable or even higherVmaxthanl-phenylalanine into the corresponding pyridyl acrylic acids. TheirKmvalues were, however, an order of magnitude higher than that of the natural substrate. These results are discussed in terms of the novel mechanism which implies an electrophilic attack of the prosthetic dehydroalanine at the aromatic ring. The heats of formation of the putative sigma complexes of the electrophilic substitution at the pyridine ring have been calculated using semiempirical force-field methods. The results show the feasibility of the proposed mechanism also with the substrate analogues.

Theoretical Study on Carbocation Formation Reactions. Resonance Demands and Transition Structures

Abstract Ab initio molecular-orbital calculations were carried out for two carbocation-generating reactions, the SN1 reaction of protonated 1-phenylethyl alcohol (H2O leaving group) and the acid-catalyzed hydration of styrene. Separated reactants, reactant complexes, transition states (TSs), product complex, and separated products were optimized at MP2/6-31G* and the vibrational frequencies were analyzed. The SN1 TS lies halfway between the reactant and the product with respect to the bond lengths, charge distribution, and secondary deuterium isotope effects. The degree of the π-orbital overlap between the benzylic carbon and the phenyl ring is almost the same in the SN1 TS and the 1-phenylethyl cation. The proton-transfer TS of the hydration, on the other hand, reaches earlier, and the π-orbital overlap in the TS is less effective. These differences are responsible for the difference in the experimental substituent effect reported in the literature for the two reactions.

Ab Initio Density Functional Calculations of Deuterium Kinetic Isotope Effects for Decomposition of Dimethylnitramine

Ab initio harmonic force fields were computed using the Becke3−Lee−Yang−Parr hybrid density functional with the standard 6-31G* basis set for dimethylnitramine, for dimethylamino radical, and for the transition structure for five-center elimination of HONO from dimethylnitramine. These force fields were used to calculate the primary deuterium kinetic isotope effect for the HONO elimination, and the secondary isotope effect for the N−N bond homolysis of dimethylnitramine. The computed primary effect is kH/kD6 = 4.21, and the computed secondary effect is kH/kD6 = 1.40, both at 240 °C. Comparison with the experimentally observed isotope effect of 1.57 for decomposition of dimethylnitramine-d6 in solution at 240 °C suggests a significant part of the observed effect is due to the secondary effect on N−N bond homolysis. Similar β secondary deuterium isotope effects are expected for the N−NO2 cleavages that initiate the decompositions of the nitramine explosives HMX and RDX.

Deuterium isotope effect on 1H and 13C chemical shifts of intramolecularly hydrogen bonded perylenequinones

The primary and secondary deuterium isotope effects on proton and carbon chemical shifts were measured for a number of natural pigments and their derivatives, which contain the perylenequinone system and present a phenol–quinone tautomerism, cercosporin 1, isocercosporin 2, phleichrome 3, isophleichrome 4, cladochrome E 5, elsinochromes 6–8, hypocrellin 9, noranhydrocercosporin 10 and noranhydrophleichrome 11. Deuterium isotope effects on protons were also measured for 1,4-dihydroxyanthraquinone 12, methyl 4-(1,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)butanoate 13, N-acetyldaunomycin 15, daunomycinone 16, naphthazarin 17 and a number of intramolecularly hydrogen-bonded enols from β-diketones, β-ketoesters and o-hydroxyacyl aromatic compounds 19–24. The primary isotope effects Δδ(1H,2H) on OH proton shift and 1H chemical shifts of OH groups are correlated and can be used to estimate the strength of the hydrogen bonds in solution. The secondary isotope effects on proton and carbon nuclei are transmitted along the whole extended conjugated perylenequinone system. Long-range effects over eleven bonds and over seven bonds were observed in compounds 1–11 and in 12–18. The perturbation of the equilibrium due to the presence of deuterium was considered and calculations were performed in order to evaluate the amount of this contribution to the isotope effect. The variation with temperature of primary and secondary effects, from 25 °C down to –70 °C, was studied for compounds 1, 3, 6, 12 and 17, and for acetylacetone 22 and benzoylacetone 23. Parameters from a number of X-ray analyses, for example the O · · · O distances (as suggested by Gilli et al., J. Am. Chem. Soc., 1991, 113, 4917), gave evidence of a substantial parallelism between the liquid and the solid phase. The strength of the hydrogen bond in perylenequinones depends on the planarity of the two naphthalene units rather than on the distortion of the polycyclic ring.

Proton transfer and secondary deuterium isotope effect in the 13C NMR spectra of ortho-aminomethyl phenols

The temperature dependence of the deuterium isotope effect on the 13C NMR signals for various positions in the phenyl ring of 2-(N,N-diethylaminomethyl) tetrachlorophenol was studied in the moderately polar solvent CH 2Cl 2. The critical temperature, T c = 245 K, corresponds to the compensation of opposite effects for non-proton-transfer and proton-transfer (PT) forms existing in equilibrium. The UV spectra show that the contribution of the PT form at 245 K amounts to 20 ± 5%. Semi-empirical calculations taking into account the Onsager reaction field allowed us to discuss the role of permittivity and dimerization of polar PT forms in the charge separation.

Conformational dependence of deuterium-induced isotope effects on the olefinic one-bond13C–1H and three-bond1H–2H coupling constants in cis- and trans-stilbene

Secondary deuterium isotope effects on the olefinic one-bond 13C, 1H and three-bond 1H, 2H spin–spin coupling constant were determined in a series of specifically deuteriated cis- and trans-stilbene isotopomers. Inverse (1H detected) heteronuclear multiple quantum correlation (HMQC) NMR pulse sequence was used to measure the coupling constants in these π-conjugated systems of a moderate size. Complete spectral analyses were performed in cases where higher order perturbations took place. All secondary isotope effects on 1J(13Cα, 1H)/Hz are negative, i.e. the reduced values from –0.06 to –0.02 were observed. In contrast, the effects on 3J(1Hα, 2Hα′)/HZ are all positive from 0.04 to 0.13, thus being on average relatively higher than those on 1J(13Cα, 1H). Only two primary effects on 1J(13Cα, 1H) were determined to see whether the same trend holds as for secondary effects. The real values of olefinic coupling constants are given with a high accuracy. In order to relate isotope effects with structural parameters, AM1 semiempirical calculations were performed. Subtle conformational changes due to the deuteriation are discussed in terms of rovibrational averaging and overall geometry.

Kinetic isotope effects on metaphosphate generation from fragmentation of 2,3-oxaphosphabicyclo[2.2.2]octene derivatives

Kinetic isotope effects of deuterium and oxygen-18 were measured on fragmentation of syn-3-ethoxy (1a) and syn-3-(N,N-diethylamino) (1b) 2,3-oxaphosphabicyclo[2.2.2]octene derivatives in 1,2-dichloroethane at 100°C. The secondary deuterium isotope effect on hydrogen adjacent to the P(SINGLE BOND)C bond was found to be 1.060 ± 0.008 for 1a and 1.081 ± 0.009 for 1b. The kinetic oxygen isotope effect on the bridge P(SINGLE BOND)O(SINGLE BOND)C is 0.9901 ± 0.0016 for 1a. The data indicate an unsymmetrical transition state for retrocycloaddition extrusion of the metaphosphate moiety, with the breakage of the C(SINGLE BOND)P bond and formation of the P (DOUBLE BOND) O bond more advanced than the C(SINGLE BOND)O breakage. A synthesis of 1a and 1b labeled with deuterium is described. © 1996 John Wiley & Sons, Inc.

Nitrogen and deuterium kinetic isotope effects on the Menshutkin reaction

Nitrogen and deuterium kinetic isotope effects were measured in the Menshutkin reaction between methyl iodide and a series of para-substituted N,N-dimethylanilines in ethanol. The nitrogen kinetic isotope effect increases for the more electron-donating substituents [0·9989, 1·0032, and 1·0036 for 4-C(O)Me, H and 4-Me, respectively], in agreement with the Hammond postulate. The secondary deuterium isotope effect, however, exhibits the reverse trend (1·045, 0·989, 0·975 per deuterium, for the respective substituents). This discrepancy is rationalized in terms of solvent molecule participation in the transition state.

Secondary Deuterium Isotope Effects for Enolization Reactions

Secondary α- and β-deuterium isotope effects for enolization reactions and equilibria have been determined by ab initio calculations, 1H NMR spectroscopy, and triton exchange kinetics. Kinetic and equilibrium α-deuterium isotope effects for hydroxide ion-catalyzed enolization of acetaldehyde calculated by ab initio methods are normal and depend on the orientation of the secondary hydrogen with respect to the carbonyl group. The computed transition state structure indicates a small degree of bond rehybridization at the transition state. Experimentally measured secondary isotope effects on the deuteroxide ion-catalyzed proton exchange of acetophenone are kH/kD = 1.08 ± 0.07 for α-CH3 exchange and kH/kD = 0.96 ± 0.08 for α-CH2D exchange. For α-CH2T exchange in water, the corresponding secondary isotope effect is kH/kD = 1.06 ± 0.02, assuming the rule of the geometric mean is valid. These effects are smaller than the calculated equilibrium isotope effect for formation of the enolate ion−water complex: KH/KD ...