Apparent Isotope Effect Research Articles

Apparent isotope effects of reversible enzymatic reactions

Accurately predicting apparent isotope effects of biogeochemical processes can aid the interpretation of laboratory batch experiments and field observations. It has been observed that the apparent instantaneous isotope fractionation factor changes with residual substrate concentration during a reaction process. The change can be attributed to the reversibility of a biogeochemical reaction, which is often overlooked for convenience. Here, we explore the influence of reverse reactions on high-dimensional isotope effects in the framework of Reverse Michaelis-Menten (RMM) kinetics. The apparent instantaneous isotope fractionation factors, as well as the isotope fractionation relationships between two elements of the same nonlabile compound in the RMM model, are functions of specific affinities of the product and substrate to an enzyme and the product-substrate ratios for both isotopes. In our cases, the influence of reaction reversibility is negligible when the substrate consumption rate is less than 63%. The analysis of the simultaneous stable isotope fractionation of two elements in a compound and of the relationship between the associated isotope fractionation factors can serve as a more effective indicator for characterizing the reaction mechanism.

Marine N2O cycling from high spatial resolution concentration, stable isotopic and isotopomer measurements along a meridional transect in the eastern Pacific Ocean

Nitrous oxide (N2O) is a potent greenhouse gas and ozone depleting substance, with the ocean accounting for about one third of global emissions. In marine environments, a significant amount of N2O is produced by biological processes in Oxygen Deficient Zones (ODZs). While recent technological advances are making surface N2O concentration more available, high temporal and spatial resolution water-column N2O concentration data are relatively scarce, limiting global N2O ocean models’ predictive capability. We present a N2O concentration, stable isotopic composition and isotopomer dataset of unprecedently large spatial coverage and depth resolution in the broader Pacific, crossing both the eastern tropical South and North Pacific Ocean ODZs collected as part of the GO-SHIP P18 repeat hydrography program in 2016/2017. We complement these data with dissolved gases (nitrogen, oxygen, argon) and nitrate isotope data to investigate the pathways controlling N2O production in relation to apparent oxygen utilization and fixed nitrogen loss. N2O yield significantly increased under low oxygen conditions near the ODZs. Keeling plot analysis revealed different N2O sources above the ODZs under different oxygen regimes. Our stable isotopic data and relationships between the N2O added by microbial processes (ΔN2O) and dissolved inorganic nitrogen (DIN) deficit confirm increased N2O production by denitrification under low oxygen conditions near the oxycline where the largest N2O accumulations were observed. The slope for δ18O-N2O versus site preference (SP, the difference between the central (α) and outer (β) N atoms in the linear N2O molecule) in the eastern tropical North Pacific ODZ was lower than expected for pure N2O reduction, likely because of the observed decrease in δ15Nβ. This trend is consistent with prior ODZ studies and attributed to concurrent production of N2O from nitrite with a low δ15N or denitrification with a SP >0‰. We estimated apparent isotope effects for N2O consumption in the ETNP ODZ of 3.6‰ for 15Nbulk, 9.4‰ for 15Nα, -2.3‰ for 15Nβ, 12.0‰ for 18O, and 11.7‰ for SP. These values were generally within ranges previously reported for previous laboratory and field experiments.

Perceptions on the treatment of apparent isotope effects during the analyses of reaction rate and mechanism.

Isotope substitution, a fascinating tool of physical chemistry, has been broadly applied in the research field of heterogeneous catalysis. In general, due to the differences in the mass-related atomic vibrational frequencies and zero-point energy of isotopic molecules, the apparent isotope effect (AIE) or observed kinetic isotope effect (observed KIE) from isotope substitution examination could provide unique knowledge regarding the reaction rate and mechanism of a catalytic reaction, such as the rate-determining step, key reaction intermediate, or catalyst design and synthesis. However, the treatment of the AIE is not as straightforward as the isotopic switch experiment, and needs sufficient care and comprehensive identification to deal with the influences from the equilibrium isotope effects (EIEs) of quasi-equilibrium elementary steps, kinetic isotope effect (KIE) of the pseudo rate-determining step, transition states, intrinsic reaction barriers, etc. Fundamentally, the key factors affecting the AIE could be the partition function part and the zero-point energy part of each single elementary step. Theoretically, the classification of the KIE could be based on the quantity of KIE (including normal KIE and inverse KIE) or the molecular transformation (including primary KIE, secondary KIE, tunneling KIE, and solvent KIE) involved. This article presents a recap of the fundamental concepts and relations of KIE, EIE and AIE, and a concise review on the selected applications of isotope effects throughout heterogeneous catalysis. Lastly, the meaningful perspectives regarding the critical factors that impact the AIE and the appropriate treatment of the AIE are discussed meticulously.

Observation of varied characteristics of chlorine isotope effects of organochlorines in dechlorination reactions on different types of electron ionization mass spectrometers

Dechlorination reactions of organochlorines can commonly take place in fragmentation on electron ionization mass spectrometry (EI-MS) and collision-induced dissociation (CID) on electron ionization tandem mass spectrometry (EI-MS/MS), possibly accompanied with chlorine isotope effects. However, it remains unclear whether characteristics of chlorine isotope effects of individual organochlorines occurring on different types of mass spectrometers are similar or different. Revelation of characteristics of chlorine isotope effects on different mass spectrometers may help to elucidate mechanisms of dechlorination reactions and fragmental pathways of organochlorines. This study investigated the characteristics of chlorine isotope effects of two model organochlorines, i.e., tetrachloroethylene and trichloroethylene, during fragmentation/CID on EI-double focusing magnetic-sector high resolution MS (EI-HRMS), EI-quadrupole MS (EI-qMS) and EI-triple-quadrupole MS/MS (EI-MS/MS) that were all coupled with gas chromatography (GC). The patterns of chlorine isotope ratios measured by different instruments were different, showing different characteristics of chlorine isotope effects. On EI-HRMS, both normal and inverse chlorine isotope effects were observed; while on EI-qMS, only normal isotope effects were found, and on EI-MS/MS merely inverse isotope effects were discovered. On EI-HRMS and EI-qMS, inter-ion and intra-ion chlorine kinetic isotope effects (Cl-KIEs) might participate in isotope fractionation processes and contribute to inverse inter-ion and normal intra-ion isotope effects, respectively, and the dominant isotope effects determined the direction of observed (apparent) isotope effects. On EI-MS/MS, both inter-ion and intra-ion Cl-KIEs were inverse at high collision energies (including 30 and 45 eV) and contributed to normal inter-ion and inverse intra-ion isotope effects, respectively, and the inverse intra-ion isotope effects were the dominant, leading to the inverse apparent isotope effects in all dechlorination reactions. Particularly, the chlorine isotope ratios measured by EI-qMS exponentially decreased with the increasing Cl atoms of ions, implying that the intra-ion isotope effects rose as broken C–Cl bonds increased. Moreover, fundamentals of chlorine isotope effects in the stepwise multibond dechlorination reactions were revealed with the aid of density functional theory calculations. The different measurement timescales and geometries of the mass spectrometers and the difference in EI-induced fragmentation and CID might result in the different characteristics of chlorine isotope effects. This study may be conducive to elucidating isotope fractionation behaviors and fragmental pathways of organochlorines on EI-MS(/MS), and provides new insights into the mechanisms of dechlorination reactions losing multiple chlorine atoms.

Assimilation and nitrification in pelagic waters: insights using dual nitrate stable isotopes (δ15N, δ18O) in a shallow lake

Nitrate dual stable isotopes (δ15N and δ18O of NO3 −) have proven to be a powerful technique to elucidate nitrogen (N) cycling pathways in aquatic systems. We applied this technique for the first time in the pelagic zone of a small temperate meso-eutrophic lake to identify the dominant N cycling pathways, and their spatial and temporal variability. We measured the lake NO3 − δ15N and δ18O signatures over an annual cycle and compared them to that of the watershed. Both δ15N and δ18O of NO3 − in the lake increased during summer relative to the inputs. Relationships between lake NO3 − isotopic composition and concentrations were different across thermal strata with an apparent isotope effect in the epilimnion of 15eepi = 4.6‰ and 18eepi = 10.9‰. We found a strong deviation of the lake NO3 − δ18O and δ15N from the expected 1:1 line for assimilation (slope = 1.73) suggesting that nitrification was co-occurring. We estimated that nitrification could support between 5 and 30% of nitrate-based production during the growing season, but was negligible in early spring and fall, and probably more dominant under ice. We showed that the technique is promising to study N processes at the ecosystem scale in shallow lakes, particularly during winter. Our results suggest that recycled NO3 − could support primary productivity and influence phytoplankton composition in the surface waters of small lakes.

Nitrite consumption and associated isotope changes during a river flood event

Abstract. In oceans, estuaries, and rivers, nitrification is an important nitrate source, and stable isotopes of nitrate are often used to investigate recycling processes (e.g. remineralisation, nitrification) in the water column. Nitrification is a two-step process, where ammonia is oxidised via nitrite to nitrate. Nitrite usually does not accumulate in natural environments, which makes it difficult to study the single isotope effect of ammonia oxidation or nitrite oxidation in natural systems. However, during an exceptional flood in the Elbe River in June 2013, we found a unique co-occurrence of ammonium, nitrite, and nitrate in the water column, returning towards normal summer conditions within 1 week. Over the course of the flood, we analysed the evolution of δ15N–NH4+ and δ15N–NO2− in the Elbe River. In concert with changes in suspended particulate matter (SPM) and δ15N SPM, as well as nitrate concentration, δ15N–NO3− and δ18O–NO3−, we calculated apparent isotope effects during net nitrite and nitrate consumption. During the flood event, > 97 % of total reactive nitrogen was nitrate, which was leached from the catchment area and appeared to be subject to assimilation. Ammonium and nitrite concentrations increased to 3.4 and 4.4 µmol L−1, respectively, likely due to remineralisation, nitrification, and denitrification in the water column. δ15N–NH4+ values increased up to 12 ‰, and δ15N–NO2− ranged from −8.0 to −14.2 ‰. Based on this, we calculated an apparent isotope effect 15ε of −10.0 ± 0.1 ‰ during net nitrite consumption, as well as an isotope effect 15ε of −4.0 ± 0.1 ‰ and 18ε of −5.3 ± 0.1 ‰ during net nitrate consumption. On the basis of the observed nitrite isotope changes, we evaluated different nitrite uptake processes in a simple box model. We found that a regime of combined riparian denitrification and 22 to 36 % nitrification fits best with measured data for the nitrite concentration decrease and isotope increase.

Dielectric Anomaly in Ice near 20 K: Evidence of Macroscopic Quantum Phenomena.

H2O is one of the most important substances needed in sustaining life; but not much is known about its ground state. Here a previously unidentified anomaly is identified in the form of a minimum in the imaginary part of the dielectric constant with respect to temperature near 20 K, while the real part remains monotonic. Isothermal dispersion and absorption measurements show coinciding results. For the case of heavy ice (D2O), no anomaly was identified, confirming an apparent isotope effect. Concerted quantum tunneling of protons is believed to be the main cause behind the reported anomaly. Our findings identify another system that exhibits macroscopic quantum phenomena that rarely occur in nature.

Oxidative uncoupling in cysteine dioxygenase is gated by a proton-sensitive intermediate.

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine (Cys) to produce cysteine sulfinic acid (CSA). This enzyme catalyzes the first committed step in Cys catabolism; thus, it is central to mammalian sulfur metabolism and redox homeostasis. Ironically, despite nearly 45 years of continued research on CDO, essentially no information has been reported with respect to its kinetic mechanism. In this work, the timing of chemical steps in the CDO kinetic mechanism is investigated by pH/pD-dependent steady-state kinetics and solvent isotope effects on kcat, kcat/KM, and (O2/CSA) coupling. Normal solvent kinetic isotope effects of 1.45 ± 0.05 and 2.0 ± 0.1 are observed in kcat-pL and kcat/KM-pL profiles, respectively. Proton inventory experiments within the pL-independent region (pL 8.5) suggest multiple solvent-exchangeable protons in flight for both kcat and kcat/KM data. The influence of solvent viscosity was also investigated to probe non-chemical steps and to verify that the apparent isotope effects were not attributable to increased solvent viscosity of D2O reactions relative to H2O. Although solvent viscosity did have a modest influence on kcat and kcat/KM, the response is not sufficient to account for the observed solvent isotope effects. This suggests that product release is only partially rate-limiting for CDO catalysis. Most crucially, proton inventory of (O2/CSA) coupling indicates that a proton-sensitive transition state directly follows O2 activation. Thus, protonation of a transient species preceding Cys oxidation is gated by protons in flight. This behavior provides valuable insight into the kinetically masked transients generated during catalysis.

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.

Implications of nitrate and nitrite isotopic measurements for the mechanisms of nitrogen cycling in the Peru oxygen deficient zone

Oceanic oxygen deficient zones (ODZs) are important regions for oceanic primary productivity, nitrous oxide (N2O) production, and the marine nitrogen (N) budget. These areas are recognized as hotspots for fixed N loss, although the rates and mechanisms of N loss have been difficult to quantify. Stable isotopes of nitrate and nitrite integrate the effects of a complex suite of processes occurring in these regions. Here we examine the distributions of nitrate δ15N and δ18O and nitrite δ15N in the Peruvian ODZ. Our data reveal elevated nitrate δ15N and δ18O values, particularly after correcting for the isotopic contribution of nitrite. Moreover, the isotopic composition of nitrite, a central intermediate in the marine N cycle, provides an additional constraint on the processes occurring in the Peru ODZ. A simple finite difference model is used to interpret the mechanisms and relative rates of N transformation in the waters sampled off the coast of Peru. Nitrite oxidation is found to be an important sink for nitrite, in many cases exceeding the rate of nitrite reduction. In model results, the apparent isotope effect for nitrate reduction, as inferred from a closed system Rayleigh model of nitrate concentration and δ15N, is greater than the prescribed value due to the effects of nitrite oxidation. Accordingly, the isotope effect for nitrate reduction that best fits the data is 12‰, much lower than the commonly inferred 25‰. Furthermore, nitrite oxidation may mediate the δ15N of N2 produced in this water column suboxic zone through its effect on the δ15N values of NO2− and NO3−.

Hydrogen/deuterium storage properties of Pd nanoparticles

The Pd nanoparticles of 10 nm have been prepared by dealloying method. They can absorb/desorb hydrogen or deuterium completely in a few seconds at room temperature. There is no apparent isotope effect on the sorption kinetics. The activation energies of hydrogen and deuterium absorption are 23.2 and 23.7 kJmol−1, respectively. The diffusion coefficients of hydrogen and deuterium in the Pd nanoparticles are similar at 423 K. However, the deuterium isotherm shows higher plateau pressure and narrower gap than the hydrogen isotherm at the same temperature. When the temperature increases to 473 K, no phase transformation can be detected for both hydrogen and deuterium. The particle size and structure effects on the hydrogen/deuterium sorption process are discussed.

Deuterium isotope effects for the oxidation of 1-methyl-3-phenyl-3-pyrrolinyl analogues by monoamine oxidase B

The parkinsonian inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a cyclic tertiary allylamine exhibiting good monoamine oxidase B (MAO-B) substrate properties. MAO-B catalyzes the ring α-carbon 2-electron bioactivation of MPTP to yield the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP +). The corresponding 5-membered ring MPTP analogue, 1-methyl-3-phenyl-3-pyrroline, also undergoes MAO-B-catalyzed oxidation to give the 2-electron oxidation product, 1-methyl-3-phenylpyrrole. Here we report the kinetic deuterium isotope effects on V max and V max/ K m for the steady-state oxidation of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-fluorophenyl)-3-pyrroline by baboon liver MAO-B, using the corresponding pyrroline-2,2,4,5,5- d 5 analogues as the deuterated substrates. The apparent isotope effects for the two substrates were 4.29 and 3.98 on V max, while the isotope effects on V max/ K m were found to be 5.71 and 3.37, respectively. The values reported for the oxidation of MPTP by bovine liver MAO-B with MPTP-6,6- d 2, as deuterated substrate, are D ( V max) = 3.55; D ( V max/ K m) = 8.01. We conclude that the mechanism of the MAO-B-catalyzed oxidation of pyrrolinyl substrates is similar to that of the tetrahydropyridinyl substrates and that a carbon–hydrogen bond cleavage step is, at least partially, rate determining.

Carbon isotope fractionation during aerobic biodegradation of n-alkanes and aromatic compounds in unsaturated sand

Microcosm experiments were conducted to quantify carbon isotope fractionation during aerobic biodegradation of n-alkanes (from C 3 to C 10) and monoaromatic hydrocarbons in unsaturated alluvial sand. In single compound experiments with n-alkanes, the largest enrichment factor was obtained for propane (−10.8 ± 0.7‰). The magnitude of the enrichment factor decreased with increasing number of carbon atoms from propane to n-decane (−0.2 ± 0.1‰). This trend can partly be explained by the decreasing probability that a 13C is located at the reacting site in the molecule with increasing chain length. After correcting for the presence of non-reacting positions, a chain length dependence of the calculated apparent isotope effect persisted. This observation suggests that transport and binding steps before the actual reaction step become increasingly rate limiting with increasing chain length. For aromatic compounds tested individually, the enrichment factor was the largest (−1.4 ± 0.1‰) for benzene (B), followed by toluene (T) (−0.8 ± 0.1‰) and m-xylene (X) (−0.6 ± 0.1‰). Enrichment factors for BTX were systematically smaller than for n-alkanes with equivalent number of carbons, which is likely related to different biodegradation mechanisms. The study demonstrates that significant carbon isotope fractionation occurs during aerobic biodegradation of n-alkanes and aromatic compounds under unsaturated conditions and that the magnitude of isotope enrichment is linked to molecule size and molecule structure.

Probing stratospheric transport and chemistry with new balloon and aircraft observations of the meridional and vertical N<sub>2</sub>O isotope distribution

Abstract. A comprehensive set of stratospheric balloon and aircraft samples was analyzed for the position-dependent isotopic composition of nitrous oxide (N2O). Results for a total of 220 samples from between 1987 and 2003 are presented, nearly tripling the number of mass-spectrometric N2O isotope measurements in the stratosphere published to date. Cryogenic balloon samples were obtained at polar (Kiruna/Sweden, 68° N), mid-latitude (southern France, 44° N) and tropical sites (Hyderabad/India, 18° N). Aircraft samples were collected with a newly-developed whole air sampler on board of the high-altitude aircraft M55 Geophysica during the EUPLEX 2003 campaign. For mixing ratios above 200 nmol mol−1, relative isotope enrichments (δ values) and mixing ratios display a compact relationship, which is nearly independent of latitude and season and which can be explained equally well by Rayleigh fractionation or mixing. However, for mixing ratios below 200 nmol mol−1 this compact relationship gives way to meridional, seasonal and interannual variations. A comparison to a previously published mid-latitude balloon profile even shows large zonal variations, justifying the use of three-dimensional (3-D) models for further data interpretation. In general, the magnitude of the apparent fractionation constants (i.e., apparent isotope effects) increases continuously with altitude and decreases from the equator to the North Pole. Only the latter observation can be understood qualitatively by the interplay between the time-scales of N2O photochemistry and transport in a Rayleigh fractionation framework. Deviations from Rayleigh fractionation behavior also occur where polar vortex air mixes with nearly N2O-free upper stratospheric/mesospheric air (e.g., during the boreal winters of 2003 and possibly 1992). Aircraft observations in the polar vortex at mixing ratios below 200 nmol mol−1 deviate from isotope variations expected for both Rayleigh fractionation and two-end-member mixing, but could be explained by continuous weak mixing between intravortex and extravortex air (Plumb et al., 2000). However, it appears that none of the simple approaches described here can capture all features of the stratospheric N2O isotope distribution, again justifying the use of 3-D models. Finally, correlations between 18O/16O and average 15N/14N isotope ratios or between the position-dependent 15N/14N isotope ratios show that photo-oxidation makes a large contribution to the total N2O sink in the lower stratosphere (possibly up to 100% for N2O mixing ratios above 300 nmol mol−1). Towards higher altitudes, the temperature dependence of these isotope correlations becomes visible in the stratospheric observations.

Proton-Transfer Reactions between Nitroalkanes and Hydroxide Ion under Non-Steady-State Conditions. Apparent and Real Kinetic Isotope Effects [J. Am. Chem. Soc. 2000, 123, 1579−1586

Proton-Transfer Reactions between Nitroalkanes and Hydroxide Ion under Non-Steady-State Conditions. Apparent and Real Kinetic Isotope Effects [<i>J. Am. Chem. Soc</i>. <b>2000</b>, <i>123</i>, 1579−1586

Complex-Induced Proximity Effects in Directed Lithiations: Analysis of Intra- and Intermolecular Kinetic Isotope Effects in Directed Aryl and Benzylic Lithiations

The mechanism of proton transfers in directed lithiations has been investigated by measuring the intra- and intermolecular kinetic isotope effects for the benzylic lithiation of N-benzyl-N,N‘-dimethyl urea (6) and the ortho lithiations of the tertiary amide N,N-diisopropylbenzamide (7) and the secondary amide N-isopropylbenzamide (8) by sec-BuLi/TMEDA in THF. For the lithiation of 6, a large primary kinetic isotope effect is observed for intramolecular competition of monodeuterated substrate, and a much smaller apparent isotope effect is seen for the intermolecular competition between diprotiated and dideuterated substrates. These results support a two-step mechanism of largely irreversible initial complexation between the substrate and the organolithium reagent, which is followed by hydrogen transfer to the organolithium reagent for the directed lithiation of urea 6. Indistinguishable limits for large intermolecular and intramolecular isotope effects are measured for the lithiations of 7 and 8. The directed lithiations of amides 7 and 8 are suggested to proceed by a two-step mechanism in which initial complexation is largely reversible.

Ultrafast photodissociation dynamics of the S1 and S2 states of acetone

The photodissociation dynamics for the two lowest excited electronic states (S1 valence state and the S2, 3s Rydberg state) of acetone (h6 and d6) have been studied using femtosecond mass-resolved photoionization spectroscopy. The S1 state dynamics was investigated by near ultraviolet (UV) pump (∼265 nm) and deep UV (205 nm) or visible (410 nm) probe. The primary dissociation time is instrument-limited, providing a 200 fs upper limit to the lifetime. The acetyl ion signal exhibits a subpicosecond decay and a persistent signal. The fast decay is consistent with results from Kim et al. [J. Chem. Phys. 103, 477 (1995)] for two-photon excitation to near the 4s state. The persistent signal is due to probe-induced ionization of acetyl radicals that are stable with respect to secondary dissociation. The S2 excited state lifetime measured for acetone-d6 using 194 pump and 259 nm probe is 13.5±1.0 ps. This is almost three times longer than we previously determined for this state in acetone-h6, 4.7±0.2 ps. The secondary dissociation time for acetyl-d3 measured with two-photon ionization probe at 388 nm is 3.0±1.0 ps. This is the same (within the experimental uncertainty) as our result for acetyl-h3 (3.1±0.5 ps), so that there is no apparent isotope effect. The calculated RRKM (Rice–Ramsperger–Kassel–Marcus) rate, however, is significantly faster for acetyl-h3 at the same internal energy, so that the isotopic dependence of the rate deviates from the RRKM predictions. Consequently, there is either an isotope dependence in the energy partitioning for primary dissociation or a reverse isotope effect in the secondary dissociation, or both. In the latter, more likely case, this indicates that the secondary dissociation does not conform to a statistical, RRKM-type unimolecular dissociation.

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.

The N-Acetyl Neuraminyl Oxecarbenium Ion Is an Intermediate in the Presence of Anionic Nucleophiles

Solvolysis of CMP N-acetyl neuraminate (CMP-NeuAc) in 1.8 M acetate buffer at pH 5 containing 0.9 M azide results in the formation of both anomers of 2-deoxy-2-azido N-acetyl neuraminic acid in addition to N-acetyl neuraminic acid as determined by 1H-NMR product analysis. A rate dependence on [azide] was observed with an apparent bimolecular rate constant of (2.1 ± 0.3) × 10-3 M-l min-l which could only account for half of the azido-NeuAc formed. Comparison of rate, product ratio, and stereochemical data indicate that concurrent pathways for formation of N3-NeuAc are operative, with 17% of product forming from reaction of azide and the tight ion pair, 12% via the solvent separated ion pair, and 6% from the free NeuAc oxocarbenium ion. From the corrected product ratio data, the lifetime of the oxocarbenium ion was estimated to be ≥3 × 10-11 s. Solvolysis of CMP-NeuAc at pL = 5.0 afforded an observed solvent deuterium isotope effect (SDIE) kH2O/kD2O = 0.45, consistent with specific acid catalysis of glycosidic bond cleavage. A SDIE of 0.66 for the apparent bimolecular azide trapping pathway was also observed. An apparent isotope effect of ∼1.1 for trapping of the N-acetyl neuraminyl oxocarbenium ion by water was determined by product analysis of azide trapping in H2O and D2O. An ab initio transition state for attack of water on an N-acetyl neuraminyl oxocarbenium ion model was located which featured a hydrogen bond between the oxocarbenium ion carboxylate and water; proton transfer was not part of the reaction coordinate. It is proposed that the N-acetyl neuraminate carboxylate group stabilizes an intermediate oxocarbenium ion, but the barrier for capture by water is lowered by a transition state hydrogen bond.

Isotope Effects during Metabolism of (+)- and (−)- Trans Tramadol Isotopomers by Human Liver Microsomes

Abstract As for the unlabelled (±)-1(e)-(m-methoxyphenyl)-2(e)-(dimethylaminomethyl)cyclohexane 1(a)-ol, tramadol, (T), pronounced stereochemical effects were observed for the isotopomers with regard to the extent of formation of O- and N-demethylated metabolites. The pharmacological active O-demethylated metabolite M1 was formed from the unlabelled as well as from the labelled (+)-tramadols about 6 times less than from the corresponding (−)-T. In contrast, the N-demethylated metabolite M2 was formed from the (+)-tramadols about 1.5 times more than from the corresponding (−)-T. These steric effects were modulated by the smaller apparent isotope effects for the isotopomers. Isotope effects were expressed as kH/kD (calculated from concentrations) and as DV, DV/K and HKm/DKm (from Eadie-Hofstee plots).