Isotopic labeling kinetic isotope effects

Many data have been provided in support of addition-elimination characteristic of an 5 n2(P) process, and include information from studies of reaction kinetics, isotopic labelling, kinetic isotope effects and stereochemical changes. Green and Hudson demon-... 

In most cases, more 1,4- than 1,2-addition product is obtained. This may be a consequence of thermodynamic control of products, as against kinetic. In most cases, under the reaction conditions, 15 is converted to a mixture of 15 and 16, which is richer in 16. That is, either isomer gives the same mixture of both, which contains more 16. It was found that at low temperatures, butadiene and HCl gave only 20-25% 1,4 adduct, while at high temperatures, where attainment of equilibrium is more likely, the mixture contained 75% 1,4 product. 1,2 Addition predominated over 1,4 in the reaction between DCl and 1,3-pentadiene, where the intermediate was the symmetrical (except for the D label) HjCHC—CH—CHCH2D. Ion pairs were invoked to explain this result, since a free ion would be expected to be attacked by Cl equally well at both positions, except for the very small isotope effect. 

A disadvantage of this technique is that isotopic labeling can cause unwanted perturbations to the competition between pathways through kinetic isotope effects. Whereas the Born-Oppenheimer potential energy surfaces are not affected by isotopic substitution, rotational and vibrational levels become more closely spaced with substitution of heavier isotopes. Consequently, the rate of reaction in competing pathways will be modified somewhat compared to the unlabeled reaction. This effect scales approximately as the square root of the ratio of the isotopic masses, and will be most pronounced for deuterium or... 

The kinetic parameters are E = 6.3 kcal.mole" and AS = —38.4 eu, and at 25 °C the reaction exhibits a primary kinetic isotope effect of 6.6. When 0-labelled MnO was employed, no labelled oxygen appeared in the benzophenone. The mechanism involves abstraction of hydrogen, either as a hydride ion or a hydrogen atom, from the anion of the alcohol... 

A kinetic isotope effect 160/180 of 2% in the spontaneous hydrolysis of the 2,4-dinitrophenyl phosphate dianion, whose ester oxygen is labeled, suggests a P/O bond cleavage in the transition state of the reaction, and thus also constitutes compelling evidence for formation of the metaphosphate 66,67). The hydrolysis behavior of some phosphoro-thioates (110) is entirely analogous 68). 

The authors have also synthesized134 fatty acids labelled with deuterium and carbon-11 in order to investigate if kinetic isotope effects related to fatty acid metabolism can be observed in vivo by pet133,135-137. In vitro, the large kinetic deuterium isotope effects are observed in the oxidation of deuteriated aliphatic carboxylic acids with alkaline permanganate and manganate135-139. 

Labelling experiments provided the evidence that the Fe1- and Co1-mediated losses of H2 and 2H2 from tetralin are extremely specific. Both reactions follow a clear syn- 1,2-elimination involving C(i)/C(2) and C(3)/C(4), respectively. In the course of the multistep reaction the metal ions do not move from one side of the rr-surface to the other. The kinetic isotope effect associated with the loss of the first H2 molecule, k( 2)/k(Y)2) = 3.4 0.2, is larger than the KIE, WFLj/ATHD) = 1.5 0.2, for the elimination of the second H2 molecule. A mechanism of interaction of the metal ion with the hydrocarbon n-surface, ending with arene-M+ complex 246 formation in the final step of the reaction, outlined in equation 100, has been proposed241 to rationalize the tandem MS studies of the unimolecular single and double dehydrogenation by Fe+ and Co+ complexes of tetraline and its isotopomers 247-251. 

The crystal structure of an isopropylamine complex of Ru of this type has been reported [78]. Surprisingly, a negligible kinetic isotope effect (kRuHOH/kRUDOD= 1.05 0.14) was found when D labels on both the OH and RuH sites were used,... 

Reduction of aldonolactones and their derivatives with isotopically modified reducing agents leads to sugars labeled at the anomeric center. Glycosides substituted with deuterium or labeled with tritium are widely employed for kinetic isotope-effect measurements, mechanistic studies, isotope-tracing experiments, and so on. 

It may be concluded that for reactions where the proton is less or more than one-half transferred in the transition state, i.e. the A—H and H—B force constants are unequal, the primary hydrogen-deuterium kinetic isotope effect will be less than the maximum of seven. The maximum isotope effect will be observed only when the proton is exactly half-way between A and B in the transition state. This relationship is also found for carbon kinetic isotope effects where the isotopically labelled carbon is transferred between two atoms in the reaction10,11. This makes interpreting carbon isotope effects difficult. 

Nitrogen, carbon-13 and carbon-14 kinetic isotope effects have been determined38 for the analogous acid-catalyzed ortho,ortho -rearrangement of the Af-2-naphthyl-Ar/-phenylhydrazine (equation 24). The labelled compounds required for this study were prepared by the sequence of reactions shown in Schemes 20-22. 

The nitrogen kinetic isotope effect of 1.0197 found using the substrate with the natural abundance of nitrogen isotopes corresponds to an isotope effect of 1.04 for the reaction of the doubly labelled compound. Thus, the nitrogen isotope effects found using two different analytical techniques to measure the isotope effect are in excellent agreement. 

The first report of this new type of kinetic isotope effect in a Menshutkin reaction was published by Matsson and coworkers in 198744. In this study, the alpha carbon kll/ku kinetic isotope effect was measured for the Menshutkin reaction between N,N-dimethyl-para-toluidine and labelled methyl iodide in methanol at 30 °C (equation 35). The carbon-11 labelled methyl iodide required for this study was prepared from the nC atoms produced in the cyclotron in three steps45 (equation 37). 

TABLE 11. The carbon-ll/carbon-14 kinetic isotope effects for the SN2 reactions between several amine nucleophiles and the labelled methyl iodide in dimethoxyethane... 

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 haloalkane dehalogenase DhlA mechanism takes place in two consecutive Sn2 steps. In the first, the carboxylate moiety of the aspartate Aspl24, acting as a nucleophile on the carbon atom of DCE, displaces chloride anion which leads to formation of the enzyme-substrate intermediate (Equation 11.86). That intermediate is hydrolyzed by water in the subsequent step. The experimentally determined chlorine kinetic isotope effect for 1-chlorobutane, the slow substrate, is k(35Cl)/k(37Cl) = 1.0066 0.0004 and should correspond to the intrinsic isotope effect for the dehalogenation step. While the reported experimental value for DCE hydrolysis is smaller, it becomes practically the same when corrected for the intramolecular chlorine kinetic isotope effect (a consequence of the two identical chlorine labels in DCE). 

---