Determining Isotope Effects

Choosing a method to determine isotope effects on rate constants, and selecting a particular set of techniques and instrumentation, will very much depend on the rate and kind of reaction to be studied, (i.e. does the reaction occur in the gas, liquid, or solid phase , is it 1st or 2nd order , fast or slow , very fast or very slow , etc.), as well as on the kind and position of the isotopic label, the level of enrichment (which may vary from trace amounts, through natural abundance, to full isotopic substitution). Also, does the isotopic substitution employ stable isotopes or radioactive ones, etc. With such a variety of possibilities it is useless to attempt to generate methods that apply to all reactions. Instead we will resort to discussing a few examples of commonly encountered strategies used to study kinetic isotope effects. 

The practical usefulness of Equations 11.46 through 11.53 has been demonstrated for the malic enzyme catalyzed conversion of L-malate to pyruvate (Equation 11.72). Table 11.1 lists experimentally determined isotope effects for this reaction. Comparison of carbon kinetic isotope effects for protio and deutero-malate substituted at position 2 (the carbon that undergoes sp3 to sp2 transition) rules out the possibility that the hydride transfer and the decarboxylation events are concerted. This conclusion follows from Equation 11.48 which, for a concerted reaction, predicts that 13(V/K) should be smaller than 13(V/K)D, which is opposite to the order observed experimentally. 

Mass spectrometry measures the abundance of ions versus their m/z ratio, and it is common practice to use the ratio /mH//mD = kn/ku as a direct measure of the isotope effect. The typical procedure for determining isotope effects from intensity ratios... 

A review of methods of synthesis of aromatic iodo compounds has appeared offering considerable information of potential value to research chemists wishing to prepare iodoheterocycles (84RCR343). Iodination differs from chlorination and bromination in that a much less reactive electrophile (and a much larger one) is involved. The second step of the reaction is usually at least partially rate-determining. Isotope effects are noted in the iodination of indole [68AC(R) 1435], and the transition state resembles the Wheland intermediate more than in chlorination and bromination. 

Computational QM/MM studies presented thus far provide better understanding of enzymatic catalysis and description of interactions within the active sites. Comparison of experimentally determined isotope effects with corresponding values predicted theoretically serves to indicate that theoretical methods yield meaningful results. In the remaining part of this contribution we will show how information about properties of the transitions state gathered collectively from molecular modeling and measurements of kinetic isotope effects can be effectively used in devising new compounds with therapeutic applications. 

Three methods for determining isotope effects exist. The first is direct comparison of reciprocal plots for unlabeled and deuterated substrate. This method is the least precise and is practical only for deuterium isotope effects of 1.1 or higher. However, it determines the isotope effect on the substrate whose concentration is varied (even if it is not the labeled one) and is the only method that gives the isotope effect on V. 

The second method is internal competition. This method is used for tritium and isotope effects where the label is a trace one and only determines isotope effects on V/K for the labeled substrate. It is also used for and isotope effects... 

Several papers in this volume deal with transport in various kinds of liquids others examine critically the fundamental statistical-mechanical theory determining isotope effects for both equilibrium and kinetic processes in condensed as well as gaseous systems. These studies are of interest not only because they serve as a framework for comparing the merits of different isotope separation processes, but they provide powerful tools for using isotope effect data to obtain an understanding of inter-molecular forces in condensed and adsorbed phases and changes in intramolecular forces in isolated molecules. The title of this volume has accordingly been broadened from that of the symposium to reflect the wider scope of its contents. 

The predictive capabilities of results of theoretical calculations of isotope effects have again been questioned, following an experimental and theoretical study of the decarboxylation of 3-carboxybenzisoxazole at room temperature (Kemp s reaction). The experimentally determined isotope effect in acetone is 1.0312 0.0006 and the C isotope effect (1.0448, 1.0445, 1.0472, and 1.0418 in 1,4-dioxane, acetonitrile, DMF, and water, respectively) is independent of solvent polarity even though the reaction rate is markedly solvent dependent. Theoretical models at the semiempirical (AMI, PM3, SAMI) and ab initio (up to B3LYP/6-3H- + G ) levels were all unable to predict the experimental results quantitatively. 

One may determine the Tafel constants, orders of reaction with respect to each reactant and product, the response to potential pulse jabs, and the results of attempts to introduce higher values of a s in favor of a specific mechanism. These criteria can then be compared with expected trends for the types of reactions concerned. From such data, an r.d.s. can sometimes be worked out or the number of possible r.d.s. s reduced to two or three possible sequences can be determined. If one can reduce the possible r.d.s. s to a low number, it is often possible to devise specific tests to distinguish between them. Such distinction methods may include the analysis of predicted and determined isotopic effects. Further, sometimes an r.d.s. can be eliminated on the basis of model calculations of the heat of reaction impractical high values showing a pathway that is too difficult. 

The main uses of deuterium are in tracer studies to follow reaction paths and in kinetic studies to determine isotope effects. " A good discussion with appropriate references is in Comprehensive Inorganic Chemistry, Vol. 1, pp. 99-116. The use of deuterated solvents is widespread in proton nmr studies to avoid interference from solvent hydrogen atoms, and deuteriated compounds are also valuable in structural studies involving neutron diffraction techniques. 

Among the tools used to study reaction mechanisms, the replacement of an atom by one of its isotopes has proven unique in its efficiency. In organic chemistry, which is mostly concerned with carbon and hydrogen containing compounds, replacing protium with deuterium has frequently been used, especially in tracer studies, to follow reaction paths and in kinetic studies and to determine isotope effects on reaction rates. In nature, hydrogen is essentially composed of atoms in which the nucleus is a single proton. However, it contains 0.0156% of deuterium, in which the nucleus also contains a neutron. A major source of deuterium is heavy water, D2O, which is prepared on industrial scale by the electrolytic enrichment of normal water. 

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