Primary and secondary kinetic isotope effects

The kinetic solvent-isotope effects on these reactions are made up of primary and secondary kinetic isotope effects as well as a medium effect, and for either scheme it is difficult to estimate the size of these individual contributions. This means that the value of the isotope effect does not provide evidence for a choice between the two schemes (Kresge, 1973). The effect of gradual changes in solvent from an aqueous medium to 80% (v/v) Me2SO—H20 on the rate coefficient for hydroxide ion catalysed proton removal from the monoanions of several dicarboxylic acids was interpreted in terms of Scheme 6 (Jensen et al., 1966) but an equally reasonable explanation is provided by Scheme 5. 

Experimental data on primary and secondary kinetic isotope effects in the hydride-transfer step in liver alcohol dehydrogenase, LADH, were analyzed using canonical variational transition theory (CVT) for overbarrier dynamics and the optimized multidimentional path (OMT) for the nuclear tunneling (Alhambra et al., 2000 and references therein). This work demonstrates somewhat better agreement of theoretical values of primary and secondary Schaad- Swein exponents calculated by combining CVT/OMT methods with the experimental values instead of CVT and classical transition states (TST). 

Bach, R. D., Braden, M. L. Primary and secondary kinetic isotope effects in the Cope and Hofmann elimination reactions. J. Org. Chem. 1991,56, 7194-7195. 

Primary and secondary kinetic isotope effects are of general importance in the study of neighboring group participation. Isotopic substitution a to the incipient carbo-cation produces a secondary isotope effect whereas 0 and y substituents may give rise to both primary and secondary effects. For example, if the rate determining step of a solvolytic reaction involves a hydrogen shift or elimination, primary deuterium isotope effects are clearly implicated. 

The primary and secondary kinetic isotope effects of a given reaction step ij can be written in the form... 

Mixed labeling experiments with specifically isotopically substituted 4R- and 4S-NADPH cofactors established the primary and secondary kinetic isotope effects and their temperature dependence for the hydride transfer reaction. Indeed, resulting data could be rationalized only by a reaction model featuring an extensive tunneling contribution that is environmentally coupled. The difference in the observed and calculated intrinsic kinetic isotope effects requires a commitment factor arising from dissecting the pre-steady state hydride step into kinetic steps, one the actual hydride transfer step itself and the other a motion of the protein and/or nicotinamide associated with the hydride transfer step [17]. 

The latter method, called the PI-FEP/UM approach, allows accurate primary and secondary kinetic isotope effects to be computed for enzymatic reactions. These methods are illustrated by applications to three enzyme systems, namely, the proton abstraction and reprotonation process catalyzed by alanine race-mase, the enhanced nuclear quantum effects in nitroalkane oxidase catalysis, and the temperature (in)dependence of the wild-type and the M42W/G121V double mutant of dihydrofolate dehydrogenase. These examples show that incorporation of quantum mechanical effects is essential for enzyme kinetics simulations and that the methods discussed in this chapter offer a great opportunity to more accurately model the mechanism and free energies of enzymatic reactions. 

INFLUENCE OF TUNNELING ON THE PRIMARY AND SECONDARY KINETIC ISOTOPE EFFECTS... 

Important additional evidence for aryl cations as intermediates comes from primary nitrogen and secondary deuterium isotope effects, investigated by Loudon et al. (1973) and by Swain et al. (1975 b, 1975 c). The kinetic isotope effect kH/ki5 measured in the dediazoniation of C6H515N = N in 1% aqueous H2S04 at 25 °C is 1.038, close to the calculated value (1.040-1.045) expected for complete C-N bond cleavage in the transition state. It should be mentioned, however, that a partial or almost complete cleavage of the C — N bond, and therefore a nitrogen isotope effect, is also to be expected for an ANDN-like mechanism, but not for an AN + DN mechanism. 

The primary hydrogen-deuterium kinetic isotope effect is obtained from the percent cw-2-butene obtained from the deuterated and undeuterated stannanes. This is possible because a hydride and a deuteride are transferred to the carbocation when the undeuterated and deuterated stannane, respectively, forms c -2-butene. The secondary deuterium kinetic isotope effect for the hydride transfer reaction is obtained from the relative amounts of fraws-2-butene in each reaction. This is because a hydride is transferred from a deuterated and undeuterated stannane when trans-2-butene is formed. 

The primary hydrogen-deuterium kinetic isotope effect for the reaction was 3.7 and the secondary alpha-deuterium kinetic isotope effect was found to be 1.1. It is worth noting that the primary hydrogen-deuterium kinetic isotope effect of 3.7 is in excellent agreement... 

Similar conclusions attend the insertions of CCI2 (from the thermolysis of ClsCCOONa at 120 °C) into a-deuteriocumene and cumene in which the primary fen/feo = 2.6, similar to Seyferth s finding with 32, and the p-secondary kinetic isotope effect is 1.20-1.25 for six deuteriums. Here, hyperconjugation at the p-CH (CD) bonds is thought to stabilize the partial cationic charge at the reaction center in transition state 33. 

Denu, J. M., and Fitzpatrick, P. F., 1994, Intrinsic primary, secondary, and solvent kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase evidence against a concerted mechanism. Biochemistry 33 400194007. 

Sialosides have a distinct mechanism of hydrolysis for its unusual sugar structure of sialic acid. For example, the large 8-dideuterium and small primary kinetic isotope effects observed at the anomeric carhon and the large secondary kinetic isotope effect observed at the carboxylate carbon in the acid-catalyzed solvolysis of CMP-Af-acetyl neuraminate 24 support an oxocarbenium ion-like transition state 25 having the 5S conformation without nucleophilic participation of carboxylate and with the carboxylate anion in a looser environment than in the ground state [15] (O Fig. 3). Such a zwitterion structure is consistent with the results from calculations using the COSMO-AMI method for aqueous solutions [16]. 

Unlike primary kinetic isotope effects, secondary kinetic isotope effects arise due to presence of stable isotope at sites close to point of reaction and consequently influence the geometry of the reaction intermediates. Although the absolute values are much lower than those observed for primary effects, secondary kinetic isotope effects yield important information pertaining to the progress of the reaction and transition states involved therein [100,124,125],... 

Recognition should be made of the fact that includes both a primary and a secondary kinetic isotope effect, but the latter is usually much smaller than the former and should not contribute greatly to the large difference in activation energies (for normal primary hydrogen kinetic isotope effects, the difference in activation energies is usually around 1 kcal/mol). 

The primary isotope effects discussed so far take place when the isotopic substitution occurs at the bond broken in the reaction in question, and we have pointed out that, for ku > ko, the C-H or C-D bond breakage is significantly advanced in the rate-limiting transition state. Numerous studies have also been made on the effect of isotopic substitution of deuterium or tritium at a bond adjacent to the reacting bond, such that the C-D or C-T bond is not itself broken during the reaction. Isotopic substitution at such positions can lead to either slower or faster rates, leading to secondary kinetic isotope effects (Dahlquist et al., 1968 Jencks, 1969 Smith etai, 1973 Melander Saunders, 1980 Cook, 1991). 

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