Multiple Isotope Effects

In the approach above two different isotope effects of the same element substituted at the same position in the substrate have been compared. In similar fashion measurements of isotope effects of different elements on the same reaction can be used  

The third equation in Equation 11.47 represents a kinetic isotope effect of the first isotopomer pair measured in the presence of the second (which IE has perturbed the commitment). In order to make the changes in apparent commitment (cf/H2k3) sufficiently pronounced, deuterium is usually selected as the second isotope (H2). The first, (HI), on the other hand, is usually a heavy-atom (e.g. 13C, lsO, etc.). Most frequently this approach has been used for carbon kinetic isotope effects in which case Equation 11.47 becomes  

If the isotope sensitive step is reversible the equations get more complicated and cannot be solved explicitly for the intrinsic isotope effects (unless Cf = 0, or the equilibrium isotope effect is unity). The last two equations in Equation 11.48 demonstrate that a normal deuterium kinetic isotope effect diminishes the apparent commitment if both isotopes are present. Thus 13(V/K) is smaller than 13(V/K)d when both isotope effects are related to the same step. 

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An alternative way to obtain oxygen IE s is to take advantage of multiple isotope effects (see Sections 7.1.5 and 7.4). The method relates the isotopic composition of the atom of interest located at a specific position to an IE of another atom in the same molecule. The approach is called remote labeling. Remote labeling experiments are best explained using an example. Consider the / -nitrophenol anion ... 

It is not always possible to determine intrinsic isotope effects. However, other useful information about the reaction can still be obtained. Above we assumed a single rate determining step sensitive to each isotope substitution. More frequently, however, the isotope sensitivity is found in different steps. Studies with multiple isotope effects can be used to determine the sequence of steps. To illustrate, a more complicated reaction scheme is needed ... 

The results of a multiple isotope effect study of the acid-catalysed hydrolysis of HC02Me (Scheme 5) have provided a detailed picture of the transition-state structure for the reaction, which involves one hydronium ion and two water molecules (15).8 DFT calculations on the water-assisted neutral hydrolysis of MeC02Et indicated that a stepwise process involving four molecules of H20 is energetically favoured.9... 

Hermes JD, Roeske CA, O Leary MH, Cleland WW. Use of multiple isotope effects to determine enzyme mechanisms and intrinsic isotope effects. Malic enzyme and glucose-6-phosphate dehydrogenase. Biochemistry 1982 21 5106-5114. 

Hermes JD, Cleland WW. Evidence from multiple isotope effect determinations for coupled hydrogen motion and tunneling in the reaction catalyzed by glucose-6-phosphate dehydrogenase. J. Am. Chem. Soc. 1984 106 7263-7264. 

Marlier JF. Multiple isotope effects on the acyl group transfer... 

Cleland, W. W. (1991) Multiple isotope effects in enzyme-catalyzed reactions, in Enzyme Mechanism from Isotope Effects, Cook, P. F. (Ed.), pp. 247-268, CRC Press, Boca Raton, FI. 

The physical organic chemistry of very high-spin polyradicals, 40, 153 Thermodynamic stabilities of carbocations, 37, 57 Topochemical phenomena in solid-slate chemistry, 15, 63 Transition state analysis using multiple kinetic isotope effects, 37, 239 Transition state structure, crystallographic approaches to, 29, 87 Transition state structure, in solution, effective charge and 27, 1... 

Korzekwa KR, Trager WF, Gillette JR. Theory for the observed isotope effects from enzymatic systems that form multiple products via branched reaction pathways cytochrome P-450. Biochemistry 1989 28(23) 9012-9018. 

The statement applies not only to chemical equilibrium but also to phase equilibrium. It is obviously true that it also applies to multiple substitutions. Classically isotopes cannot be separated (enriched or depleted) in one molecular species (or phase) from another species (or phase) by chemical equilibrium processes. Statements of this truth appeared clearly in the early chemical literature. The previously derived Equation 4.80 leads to exactly the same conclusion but that equation is limited to the case of an ideal gas in the rigid rotor harmonic oscillator approximation. The present conclusion about isotope effects in classical mechanics is stronger. It only requires the Born-Oppenheimer approximation. 

Although the multiple isotope method is most frequently used with stable isotopes (for example studies of oxygen KIE s in biophosphates used 1SN at a remote nitro group, or 13C on a remote carboxy group, as reporting isotopes), the technique is not restricted to stable isotopes radioisotopes have been used as reporting sites for stable isotopes. In a practical sense this is the only method that allows the measurement of isotope effects for elements that have only one stable isotope (e.g. fluorine and phosphorus). In these cases doubly radiolabeled material is used (see Section 7.4). 

In complex systems that involve multiple Fe-bearing species and phases, such as those that are typical of biologic systems (Tables 1 and 2), it is often difficult or impossible to identify and separate all components for isotopic analysis. Commonly only the initial starting materials and one or more products may be analyzed for practical reasons, and this approach may not provide isotope fractionation factors between intermediate components but only assess a net overall isotopic effect. In the discussions that follow on biologic reduction and oxidation, we will conclude that significant isotopic fractionations are likely to occur among intermediate components. 

Isotope effects at different positions in a molecule are independent and multiplicative (the isotope effects on the free energy of reaction or activation are additive). 

It has been pointed out that routine accurate mass measurements are conducted at resolutions which are too low to separate isobaric isotopic compositions in most cases. Unfortunately, coverage of multiple isotopic compositions under the same signal distort the peak shape. This effect causes a loss of mass accuracy when elemental compositions have to be determined from such multicomponent peaks, e.g., if the monoisotopic peak is too weak as the case with many transition metals. The observed decrease in mass accuracy is not dramatic and the loss of mass accuracy is counterbalanced by the information derived from the isotopic pattern. However, it can be observed that mass accuracy decreases, e.g., from 2-3 mmu on monoisotopic peaks to about 4—7 mmu on multicomponent signals. 

While the concerted pathway has been preferred in early publications on the subject, evidence for a stepwise mechanism involving distonic ion intermediates is presented in more recent work taking kinetic isotope effects into account. [90] This is also in agreement with the postulation that reactions involving multiple bonds are generally stepwise processes. [37,91] Nevertheless, this question is still a matter of debate. [90]... 

It should incorporate 13C, 15N, and 180 instead of deuterium (2H) as the first choice to minimize the isotope effect and possibility of exchange. Multiple deuterium atoms can result in a significant isotope effect, to the point that the fully labeled form can be chromatographically resolved completely from the native. This means that the fully labeled form is not in the LC-MS or LC-MS/MS ion source at the same time as the analyte, and therefore this standard cannot control ion source events, limiting its ability to mimic the analyte behavior. 

Some other interesting observations regarding free radicals in these systems are noteworthy. In many instances, multiple conformations of radicals are found at lower but not higher temperatures. This indicates that the radicals exist in shallow energy wells at low temperature this phenomenon was observed very early, in the 4 K ENDOR investigation of radical formation in amino acids.23 Unlike the process in DNA. In which it is well understood that the thymine anion radical protonates at C6 to form T(C6)H-, in the crystalline state there is a not clear link between pyrimidine electron adducts and H-addition radicals. We finally note that a deuterium isotope effect of protonation/deprotonation processes was found in cytosine.HCl and 2 -deoxycytidine.HCl, as evidenced by a lower propensity for these processes to occur in partially deuterated systems than in predated ones. 

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