Low Isotope Effect Research Articles

Variable carbon and chlorine isotope fractionation in TCE co-metabolic oxidation.

Identifying co-metabolic TCE oxidation in polluted groundwater is challenging due to lack of indicative by-products. This challenge may theoretically be resolved if the oxidation process can be characterized by a distinct dual isotope enrichment. In this work, we aimed to explore the carbon and chlorine isotope effects associated with TCE oxidation by a variety of oxygenases. These included pure strains and enrichment cultures of methane, toluene and ammonia oxidizers, as well as experiments with crude extracts. Isotope effects determined for TCE oxidation by toluene and ammonia oxidizers were mostly in line with expected values for epoxidation mechanism (ϵ13C-11.0 ± 0.7 to-24.8 ± 0.2‰ and ϵ37Cl+0.9±0.5 to+1.0±0.4‰), whereas, the methanotrophs resulted in distinctively different isotope effects (ϵ13C-2.4 ± 0.4 to-3.4 ± 0.8‰ and ϵ37Cl-1.8 ± 0.2 to-2.9 ± 0.9‰). It is suggested that in TCE oxidation by methanotrophs, substrate binding rather than bond cleavage is rate limiting, leading to this unexpected isotope effect. On the environmental level, our results imply that the oxidative process can be differentiated if catalyzed by toluene and ammonia oxidizers or by methanotrophs. Additionally, the oxidative process can be distinguished from the reductive one. However, using dual isotope analysis in the field may result in an under-estimation of the overall co-metabolic process if methanotrophs are to be excluded due to low isotope effects.

Microbial oxidation of tri-halogenated phenols - Multi-element isotope fractionation

The tri-halogenated phenols, 2,4,6-trichlorophenol (TCP) and 2,4,6-tribromophenol (TBP), are known as groundwater contaminants. Indigenous bacteria may degrade these compounds aerobically or anaerobically, with the aerobic process initiated by ring hydroxylation at the para position. Stable isotope analysis can be proposed as a tool for determining biodegradation process of these compounds in the environment. However, to date isotope fractionation in microbial oxidation of trihalogenated phenols has not been reported. This work aimed to investigate carbon and halogen (chlorine or bromine) isotope effects associated with the process by two microbial strains: Achromobacter piechaudii strain TBPZ which is capable of degrading both TCP and TBP, and Ralstonia eutropha strain JMP 134 which was extensively studied in the past to characterize the TCP oxidation mechanism. Low carbon isotope effects were observed in all degradation experiments. Opposite fractionation trends were detected for TCP and TBP by strain TBPZ (εC = −0.51 ± 0.28‰ and εC = +0.68 ± 0.26‰, respectively). Strain JMP presented a low inverse carbon isotope effect for TCP oxidation (εC = +0.26 ± 0.13‰), contrary to that of strain TBPZ. For the halogen atoms, low yet significant normal halogen isotope effects were detected in all cases (−0.49 to −1.43‰ for chlorine, and −0.42‰ for bromine). The results suggest that the observed isotope effects do not reflect the ring hydroxylation. Instead, they likely represent rate-limiting steps preceding the catalysis of hydroxylation. Dual-element isotope effects observed in this study did not enable distinguishing between oxidation and reduction of TCP. The oxidation and reduction dual-element trends were distinct for TBP. However, it is uncertain if this difference can be extrapolated from the laboratory to the field, since the isotope effects in TBP degradation are strongly influenced by slow steps prior to hydroxylation.

Close coupling of N‐cycling processes expressed in stable isotope data at the redoxcline of the Baltic Sea

AbstractOver the past decades, the hypoxic state of the central Baltic Sea has deteriorated because of eutrophication, but little is known about the extent to which related factors such as nitrogen removal have been altered. The Baltic Sea is a stratified semi‐enclosed basin with a large, anoxic bottom water mass in its central Gotland Basin and highly active microbial nitrogen transformation processes at the redoxcline, the interface between oxic and anoxic waters. In this study, we identified and quantified the dominant transformation processes of reactive nitrogen by exploiting fine resolution profiles of δ15NNO3, δ18ONO3, and δ15NNH4 through the pelagic redoxcline between 60 and 140 m. Our results showed increasing δ15NNO3 and δ18ONO3 values with decreasing nitrate concentrations, but the associated low apparent isotope effect (ε = ~5‰), as inferred from a closed system Rayleigh model, was not consistent with the high ε (~25‰) characteristic of denitrification in the water column. These findings could be explained by substrate limitation. The observed δ18ONO3:δ15NNO3 ratio of 1.38:1 rather than the usual 1:1 ratio typical for denitrification‐dominated systems could be explained by the occurrence of both nitrification and denitrification. We then developed a numeric reaction‐diffusion model, according to which a realistic denitrification rate of 14 nmol N L−1 d−1 was estimated, and a nitrification rate of 6.6 nmol N L−1 d−1 confirmed. Our study not only demonstrates the value of stable isotope data for investigating nitrogen transformation processes but also highlights that care is needed in interpreting systems with closely coupled processes such as those at ocean redoxclines.

The contributions of nitrate uptake and efflux to isotope fractionation during algal nitrate assimilation

In order to strengthen environmental application of nitrate N and O isotopes, we measured the N and O isotopic fractionation associated with cellular nitrate uptake and efflux in the nitrate-assimilating marine diatom Thalassiosira weissflogii. We isolated nitrate uptake and efflux from nitrate reduction by growing the cells in the presence of tungsten, which substitutes for molybdenum in assimilatory nitrate reductase, yielding an inactive enzyme. After growth on ammonium and then N starvation, cells were exposed to nitrate. Numerical models fit to the evolution of intracellular nitrate concentration and N and O isotopic composition yielded distinct N isotope effects (15ε) for nitrate uptake and nitrate efflux (2.0±0.3‰ and 1.2±0.4‰, respectively). The O isotope effects (18ε) for nitrate uptake and nitrate efflux were indistinguishable (2.8±0.6‰), yielding a ratio of O to N isotopic fractionation for uptake of 1.4±0.4 and for efflux of 2.3±0.9. The 15ε for nitrate uptake can account for at most 40% of the organism-level N isotope effect (15εorg) measured in laboratory studies of T. weissflogii and in the open ocean (typically 5‰ or greater). This observation supports previous evidence that most isotope fractionation during nitrate assimilation is due to intracellular nitrate reduction, with nitrate efflux allowing the signal to be communicated to the environment. An O to N fractionation ratio (18εorg:15εorg) of ∼1 has been measured for nitrate assimilation in algal cultures and linked to the N and O isotope effects of nitrate reductase. Our results suggest that the ratios of O to N fractionation for both nitrate uptake and efflux may be distinct from a ratio of 1, to a degree that could cause the net 18εorg:15εorg to rise appreciably above 1 when 15εorg is low (e.g., yielding a ratio of 1.1 when 15εorg is 5‰). However, field and culture studies have consistently measured nearly equivalent fractionation of N and O isotopes in association with low isotope effects, calling for isotopic studies of nitrate transport by other phytoplankton strains.

Anomalous isotopic effect in multibubble sonoluminescence of aqueous solutions of terbium chloride

An anomalously low isotope effect has been discovered in 20-kHz sonoluminescence of terbium chloride solutions in H2O-D2O mixtures. The intensity of luminescence in the characteristic 488-and 545-nm lines of the Tb3+ ion, which are observed against the solvent continuum (230–700 nm), increases with the content of D2O to a maximum value of 4.0 ± 0.4, whereas the isotope effect in photoluminescence of the same solutions reaches 10 ± 1.0. The result is explained using the model of nonexponential decay of sonoluminescence of Tb3+ ions. These ions, which are formed in an excited state in cavitation bubbles, first undergo radiative and radiationless deactivation in the gas phase. However, some excited ions enter the solution bulk, because the excitation lifetime is longer than the average bubble lifetime. At the first stage, the isotope effect is small, because the density of the gas phase is low and quenching by solvent molecules is weak. At the second stage, the isotope effect coincides with the effect in usual photoluminescence in the solution. The total decrease in the effect in sonoluminescence depends on the weight of the “gas” stage of deactivation of excited Tb3+ ions.

Effect of pressure on deuterium isotope effects of yeast alcohol dehydrogenase: evidence for mechanical models of catalysis.

Moderate pressure accelerates hydride transfer catalyzed by yeast alcohol dehydrogenase, indicative of a large negative volume of activation [Cho and Northrop (1999) Biochemistry 38, 7470-7475]. A comparison of the effects of pressure on the oxidation of normal versus dideuteriobenzyl alcohol generates a monophasic decrease in the intrinsic isotope effect; therefore, the volume of activation for the transition-state of deuteride transfer must be even more negative, by 10.4 mL/mol. This finding appears consistent with hydrogen tunneling previously proposed for this dehydrogenase [Cha, Y., Murray, C. J., and Klinman, J. P. (1989) Science 243, 1325-1330]. However, a global fit of the primary data shows that the entire isotope effect arises from a transition-state phenomenon, unlike normal isotope effects, which arise from different vibrational frequencies in reactant states, and tunneling isotope effects, which arise from a mixture of both states. Assuming the phenomenon is tunneling, the isotopic data are consistent with a Bell tunneling correction factor of Q(H) = 12 and an imaginary frequency of nu(H) = 1220 cm(-1), the first so calculated from experimental enzymatic data. This excessively large correction factor and the large difference in the isotopic activation volumes, plus the low isotope effects at extrapolated pressures, challenge traditional applications of physical organic chemistry and transition-state theory to enzymatic catalysis. They suggest instead that something other than transition-state stabilization or tunneling is responsible for the rate acceleration, something unique to the enzymatic transition state that does not occur in nonenzymatic reactions. Arguments for the vibrational model of coupled atomic motions and the fluctuating enzyme model of protein domain motion are put forward as possible interpretations.

On the Mechanism of C−H Bond Activation in the Photochemical Arylation of Rhenium(V) Oxo Iodide Complexes

Photolysis of the Re(V) oxo−iodide compound (HBpz3)ReO(I)Cl (1) in arene solvents gives the aryl complexes (HBpz3)ReO(Ar)Cl. Substituted arenes react with electrophilic selectivity exclusively at aromatic C−H bonds, and a variety of functional groups are tolerated. Yields are improved when photolysis is carried out in the presence of pyridine. Photolysis of the diiodide complex, (HBpz3)ReOI2 (2), gives a mixture of mono- and disubstituted Re−aryl complexes, and the diphenyl derivative (HBpz3)ReOPh2 has been structurally characterized. The isotope effect found when 1 is photolyzed with a 1:1 mixture of C6H6 and C6D6 was found to depend on the concentration of 1. Lower concentrations (∼0.5 mM) show a 1.2(2):1 ratio of d0 to d5 products, while higher concentrations (∼20 mM) show higher d0:d5 ratios of 1.8(2):1. The apparent isotope effects increase with increasing conversion and are also affected by the presence of additives such as pyridine or iodine. Photolysis of 1 with 1,3,5-trideuteriobenzene shows a 4.0(4):1 ratio of C−H vs C−D activation, which is independent of reaction conditions. A mechanism for arene activation is proposed that involves initial arene binding, which discriminates intermolecularly between arenes with a low isotope effect, followed by C−H bond cleavage, which discriminates intramolecularly within an arene with a higher isotope effect. The reactive species appears to be a rhenium(IV) oxo complex which can add aromatic C−H bonds across the ReO linkage.

Mechanism-based inactivation of human cytochrome P450 1A2 by furafylline: detection of a 1:1 adduct to protein and evidence for the formation of a novel imidazomethide intermediate.

The rapid loss of human CYP1A2 (cytochrome P450 1A2) activity caused by the 8-methylxanthine furafylline is investigated with the aim of determining whether a stable covalent adduct of the xanthine to the enzyme could be identified. Metabolic studies employing expressed CYP1A2 with radiolabeled furafylline and a close analogue, cyclohexylline, where the furan ring is replaced with cyclohexane, indicate that these xanthines are bound in a 1:1 ratio to CYP1A2 protein. This result, combined with earlier kinetic studies, verifies that these compounds are mechanism-based inhibitors of the enzyme. The 8'-methyl carbinols are the only metabolites formed by CYP1A2, and substantial (70-80%) incorporation of oxygen from the medium into the carbinols is observed. Carbinol formation is further characterized by high intramolecular isotope effects (kH/kD > 9) and low intermolecular isotope effects (DV/K < 2). Overall partition ratios are low (5.0 and 7.6, respectively), confirming our previous conclusion that furafylline is an efficient inactivator. By contrast, the N7-methyl-8-methylxanthines are good substrates for CYP1A2 but are not themselves inactivating agents. In addition to other metabolic products, the 8'-methyl carbinols of these N7-methyl-8-methylxanthines are formed in substantial amounts with equally high intramolecular isotope effects; however, the carbinol oxygen is derived exclusively from molecular oxygen. We conclude that oxidation of the 8-methyl group of furafylline and cyclohexylline, but not their N7-methyl analogues, by CYP1A2 promotes a major fraction of the inactivating xanthines to a two electron oxidized intermediate which either terminates enzyme activity by reaction with an active site amino acid or is decomposed by reaction with the medium to give carbinol.

Tritium Isotope Effects in Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase

Methylmalonyl-CoA mutase from Propionibacterium shermanii is an adenosylcobalamin-dependent enzyme which catalyzes the reversible isomerization of methylmalonyl-CoA and succinyl-CoA. The rate of tritium loss from 5'-[3H]adenosylcobalamin during the enzymic reaction and the relative rates of tritium appearance in substrate and product were examined. Upon the addition of methylmalonyl-CoA to a solution of holoenzyme, tritium was completely released from the cofactor within about 500 ms. No tritium was found either bound to the enzyme or released into the water. The radioactivity was found in methylmalonyl-CoA and succinyl-CoA in a constant ratio of 1 to 3, which did not change during the first 300 ms of the reaction. Upon the addition of succinyl-CoA to a solution of holoenzyme, tritium was released at essentially the same rate, and the radioactivity was found in methylmalonyl-CoA and succinyl-CoA in the identical constant ratio of 1 to 3. The tritium isotope effect on the enzyme-catalyzed hydrogen transfer, measured using 14C-labeled methylmalonyl-CoA as substrate, was kH/kT = 4.9. This low value shows that hydrogen transfer is only partly rate limiting and that at least one subsequent slow step, such as product release, contributes substantially to the overall reaction velocity. The identical partitioning of tritium, regardless of the substrate used, shows that the rearrangement of the substrate radical into the product radical is not rate limiting. The very low tritium isotope effect and the fact that all the tritium is found bound either to the CoA esters or to the cofactor make it very unlikely that a protein radical is an intermediate in the methylmalonyl-CoA mutase-catalyzed rearrangement.

Radical cation intermediates in N-dealkylation reactions.

1. A number of mechanistic possibilities exist for P450-catalysed N-dealkylation and have been considered over the years, including C- and N-hydroxylation and sequential electron transfer (SET). With peroxidases the evidence strongly favours SET and free radicals can be detected. Any mechanism must account for lack of incorporation of label from H218O into product by P450s and the high kinetic deuterium isotope effects that are seen in N-dealkylation reactions catalysed by peroxidases but not P450s. 2. Several lines of evidence support a role for SET in P450 amine oxidations, including Hammett analysis, products of dihydropyridine oxidations, and products of mechanism-based inhibition by strained cycloalkylamines. 3. The hypothesis was considered that the P450s act via base catalysis to deprotonate the aminium radical generated by SET, since the pKa has been estimated to be approximately 9. Dihydropyridine aminium radicals have low pKa (< 4) and are generally considered to have considerable kinetic acidity. None of the haemoproteins under consideration (including the peroxidases and haemoglobin) showed high kinetic hydrogen isotope effects for the oxidation of [4-2H]- or [4-3H]-labelled 1,4-dihydropyridines. These results are consonant with the view that P450s catalyse the deprotonation of N,N-dialkylaniline aminium radicals. 4. Since low isotope effects were seen with biomimetic metalloporphyrin models as well as P450s, the deprotonation is attributed to the (FeO)2+ entity, expected to be a strong base, and not the apoprotein. Thus, the FeO moiety of peroxidases is shielded, consistent with evidence by others that SET occurs through the porphyrin edge. Both P450s and peroxidases catalysed the oxidative N-demethylation of aminopyrine and N,N-dimethylaminothioanisole; however, only the peroxidases generated the stable coloured aminium radicals. 5. The rates of N-demethylation of variously para-substituted N,N-dimethylanilines can be used to undertake Hammett or Marcus analysis. The former yields rho = -0.6 and the latter an apparent E1/2 of approximately 1.8 for the formal (FeO)3+ entity of P4502B1. 6. Even in the oxidation of N,N-dialkylanilines, a finite rate of N-oxidation is seen (approximately 0.1% of N-dealkylation). The simplest paradigm has N-oxygenation and N-dealkylation both proceeding from a common aminium radical intermediate.

Interpretations of cytochrome P450 mechanisms from kinetic studies

The catalytic mechanism of cytochrome P450 (P450) enzymes has generally been understood in terms of a classic cycle in which electron donation is often limiting and catalysis is understood in terms of hydrogen abstraction and rapid oxygen rebound. In the course of detailed investigations with kinetic hydrogen isotope effects we have studied two systems in which somewhat unusual isotope effects have been interpreted in terms of modifications of the general paradigm. The low isotope effects observed for N-demethylation reactions are in contrast to high values seen with P450-catalyzed C-hydroxylation and peroxidase-catalyzed N-demethylation and are consonant with a role for the P450 FeO 2+ entity in base-catalyzed deprotonation of an aminium radical. With P450 2E1, kinetic deuterium isotope effects are seen on the apparent K m for the substrate (increased) but not on V max. The results are interpreted in terms of a mechanism where CH bond cleavage is sensitive to deuterium substitution but a step following this is rate-limiting. This step may be product release.

PH-dependent one- and two-electron oxidation of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine catalyzed by horseradish peroxidase.

The porphyrinogenic agent 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) is known to inactivate hepatic cytochrome P450 (P450) enzymes 2C11, 2C6, and 3A1 [Correia et al. (1987) Arch. Biochem. Biophys. 258, 436-451] by different mechanisms. The inactivation of P450 2C11 and 2C6 appears to be due to the ethylation of the heme in the active sites of the enzymes [Augusto et al. (1982) J. Biol. Chem. 257, 11288-11295], whereas the inactivation of P450 3A1 appears to involve the covalent binding of the heme to the apoprotein [Correia et al. (1987)]. Moreover, we have found that DDEP inactivates horseradish peroxidase (HRP) pretreated with hydrogen peroxide. In this system, DDEP was oxidized predominately to 3,5-dicarbethoxy-2,6-dimethyl-4-ethylpyridine (EDP) under weakly acidic conditions and predominately to 3,5-dicarbethoxy-2,6-dimethylpyridine (DP) under basic conditions. The loss of heme and the formation of altered heme products were also pH-dependent and were correlated with the formation of DP and the inactivation of HRP. Thus the inactivation of HRP appears to depend on the formation of an ethyl radical, which presumably reacts with the heme in the active site of the enzyme. Similar product ratios were obtained for the oxidation of DDEP by K3Fe(CN)6, indicating that product ratios of DP over EDP are mainly determined by the pH of buffer. These results, in addition to semiemperical calculations (AM1) for the oxidation of DDEP in the gas phase, are consistent with the idea that the inhibitor undergoes a single-electron oxidation to form the DDEP radical cation, the fate of which depends on the environment of the active site of the enzyme. The proposed formation of a radical cation by the abstraction of an electron from nitrogen is consistent with the finding of low intramolecular isotope effects of the metabolism of 3,5-dicarbethoxy-2,6-dimethyl-[4-2H,4-1H]-1,4-dihydropyridine by P450 2C11 and 3A4. Under basic or aprotic conditions, the radical dissociates to form DP and the ethyl radical, which reacts with the heme, thereby inactivating the enzyme. Under acidic or polar conditions, the radical undergoes an additional one-electron oxidation to form EDP.(ABSTRACT TRUNCATED AT 400 WORDS)

Low Temperature Isotope Effect in Ammonium Hexachloroselenate(IV) Studied by 35Cl NQR

Abstract The temperature dependence o f the 35Cl NQR frequency and spin-lattice relaxation time T1Q of (NH4)2SeCl6 and (ND4)2SeCl6 were measured from 400 K to 24.8 and 53.8 K, respectively. The disappearance of NQR signals in the low temperature region of both salts is attributed to phase transitions. We concluded from the temperature behavior of just above the transition point that the operative mechanism o f the transition is different in these salts: The transition of (ND4)2SeCl6 seems to be associated with rotary soft modes, while in the natural salt non-rotary soft modes seem to play an important role at the transition.

Collisional Deactivation of O2(1Δg) by Solvent Molecules. Comparative Experiments with 16O2 and 18O2

AbstractPhosphorescence spectra of natural singlet oxygen 16O2(1Δg) and its heavier isotope 18O2(1Δg) have been recorded in several O2(1Δg) weakly deactivating solvents. Lifetimes of singlet oxygen were determined for both isotopes in 21 solvents. From these data rate constants kXY of deactivation of singlet oxygen by terminal oscillators X—Y of the solvent molecules are calculated revealing a graduated isotope effect of 1.0 ≤ 16kXY/18kXY ≤ 1.9. The unexpectedly low isotope effect points to the important role of overtone excitations of X—Y during O2(1Δg) deactivation. A modified collisional deactivation model including overtone excitation of the energy accepting oscillator reproduces the experimental findings: the tremendeous graduation of kXY of almost 5 orders of magnitude, the heavy atom effect, the O2 isotope effect and the very weak temperature effect on kXY.

Low kinetic hydrogen isotope effects in the dehydrogenation of 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (nifedipine) by cytochrome P-450 enzymes are consistent with an electron/proton/electron transfer mechanism

Cytochrome P-450 enzymes have been postulated to oxidize amines through a variety of mechanisms. One of the means of distinguishing among potential pathways involves the use of kinetic hydrogen isotope effects: low isotope effects are characteristic of aminium radical mechanisms while high values are consistent with hydrogen atom abstraction, a process documented in alkane hydroxylation. Nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic+ ++ acid dimethyl ester] was prepared with isotopic substitution at the 4-position and utilized in determinations of the deuterium and tritium kinetic isotope effects. The noncompetitive intermolecular deuterium [DV, D(V/K)] and competitive intermolecular tritium [T(V/K)] isotope effects observed in the oxidation of nifedipine with liver microsomes prepared from untreated male rats were less than 2. Similar low deuterium and tritium isotope effects (less than 2) were also found with six other types of rat liver microsomal preparations and with three different human liver microsomal preparations, even though the rates of oxidation (expressed per nanomole of total cytochrome P-450) varied by an order of magnitude and other investigations indicate that several different cytochrome P-450 enzymes can catalyze the reaction. Similar low deuterium and tritium isotope effects were measured with a purified rat liver cytochrome P-450 preparation and horseradish peroxidase. These results are interpreted in terms of an electron/proton/electron transfer mechanism previously postulated for the oxidation of other dihydropyridines by cytochrome P-450 enzymes and model one-electron acceptors and argue against a mechanism involving hydrogen atom abstraction from nifedipine.

Kinetic deuterium isotope effects on deamination and N-hydroxylation of cyclohexylamine by rabbit liver microsomes

Deuterium isotope effects on the kinetic parameters for deamination and N-hydroxylation of cyclohexylamine (CHA) catalyzed by rabbit liver microsomes with NADPH are investigated. Both reactions are inhibited by carbon monoxide and have the characteristics of typical cytochrome P450-dependent monooxygenase reactions. A small and significant deuterium isotope effect operates in the oxidative deamination of CHA. The apparent isotope effects i.e., V H V D and ( V K) H ( V K ratios for deamination, are 1.75 and 1.8–2.3, respectively. On the basis of N-hydroxylation, the V H V D and V K ratios are 0.8–0.9. The N-hydroxylation rate of α-deuterated CHA (D-CHA) is somewhat higher than that of CHA. The increased increment of hydroxylamine formation seems to coincide with the decreased amount of deamination. Substitution of deuterium in the α-position of CHA results in metabolic switching of cytochrome P450 from deamination to N-hydroxylation with low deuterium isotope effects. The data are interpreted in terms of an initial one-electron abstraction from the nitrogen to form an aminium cation radical followed by recombination with iron-bound hydroxyl radical leading to N-hydroxylamine, or followed by α-carbon deprotonation to form a neutral carbon radical. The latter can lead to a carbinolamine intermediate for deamination by way of imine or recombination with nascent iron-bound hydroxyl radical. The relative rates of the reactions depend on the α-carbon deprotonation rates of amines.

Primary2H kinetic isotope effects in the base-catalysed 1,3-prototropic rearrangement of 1-methylindene and 1,3-dimethylindene

Rate constants and primary deuterium kinetic isotope effects (p.d.k.i.e.s.) have been determined for the base-catalysed rearrangement of 1-methylindene (1) and 1,3-dimethylindene (3) in the solvent o-dichlorobenzene at 30°C. The structurally similar tertiary amines 1-aza-adamantane, quinuclidine, 1,3-diaza-adamantane, DABCO, and 1,3,5-triaza-adamantane were used as catalysts. Substrate (3) gave a lower isotope effect than (1). The response in p.d.k.i.e. due to a change in basicity of the amine catalyst is very similar for (1) and (3). Despite its intermediate basicity, DABCO showed a significantly lower p.d.k.i.e. for both substrates. The p.d.k.i.e.s. obtained for the other bases, with a pK at or around 2.6, were almost constant. The consequences of a possible influence of internal ion-pair return on the rearrangement of the substrates are discussed and related to the measured p.d.k.i.e.s. The similarity between the substrates and the symmetry in the rearrangement of (3) permits some rationalizations to be made.

Studies on internal nitroso group transfer. Nitrosation of thiomorpholine

The kinetics of nitrosation of thiomorpholine have been studied under different experimental conditions. At low nitrite concentration and high acidity the reaction rate is first order with respect to nitrite and thiomorpholine and independent of acidity. The results are interpreted through a mechanism in which the S-nitroso intermediate with a deprotonated amino group must acquire a boat conformation to facilitate the migration of the NO group from the sulphur to the nitrogen atom. The mechanism is supported by the low isotope effect observed when the reaction is studied in D2O. In the pH range 2.3–3.6 and high nitrite concentration, the reaction rate is second order with respect to nitrite, the accepted mechanism being the nitrosation of deprotonated thiomorpholine by dinitrogen trioxide.

Isotope Effects in Liquid Metals

Abstract The areas treated in this article are: (i) The Haeffner effect; where the light isotope moves towards the anode in all cases so far investigated. (ii) Self diffusion and mutual diffusion. (iii) Shear viscosity of pure isotopes of Li6 and Li7. In the course of the discussion, the Haeffner effect is shown to be directly related to electrical resistively at the level of the lowest order Born approximation. Models used for treating isotopic mass effects are then considered; limitations and usefulness being assessed by comparison with experimental data on liquid metals. The marked contrast with low temperature isotope effects is finally commented on.

Alkaline hydrolysis of arylesters of diphenylphosphorodiamidates—evidence consistent with an elimination—addition mechanism

The alkaline hydrolysis of a series of aryl phosphorodiamidates has been studied in aqueous ethanol and aqueous DMSO mixtures. Although the reactions do not show any saturation kinetics with respect to the hydroxide ion, the low solvent isotope effect, the highly negative Bronsted β value, the large Hammett ρ for the leaving aryloxy group and the decrease in rate with increase in the proportion of DMSO in the solvent mixtures indicate that the hydrolysis proceeds by an elimination-addition pathway. Comparison of rate data with other hydrolytic E1CB reactions show that the title reaction belongs to the (E1CB)R category.