Remote isotope effects

Lewis et al. (entry 11 of Table 2) examined the temperature-dependence of isotope effects in the action of both the human enzyme and the soybean enzyme, by measuring the relative amounts of per-protio and per-deuterio-13-hydroperoxy-products by HLPC. The observed effects are, therefore, composed of primary, secondary, and perhaps remote isotope-effect contributions. Isotope effects on fecat/ M for both enzymes (determined by competition between labeled substrates) are increased by high total substrate concentration, an effect previously observed but stiU ill-understood. At 100 /rM substrate, the effects are roughly independent of temperature below about 15 °C, and are about 60 (H/D) for the human enzyme and 100 (H/D) for the soybean enzyme. Above 15 °C, the effects decline to about 50 for the human enzyme and about 60 for the soybean enzyme, perhaps because non-isotope-sensitive steps become more nearly rate-limiting (see Chart 4). 

Subsequent work confirmed this apparently abnormal behaviour. Deuteriation at remote sites (the S- or e-position) induces small inverse secondary isotope effects in a-cleavages occurring in the ion source, but normal isotope effects in the decomposition of metastable ions in the field-free regions94,95. The time dependence of the isotope effect was also studied by field ionization kinetics, which permit the analysis of fragmentations occurring after lifetimes as short as 10 12 s-1. It was found that the inverse isotope effect favouring loss of the deuteriated radical operates at times shorter than 10 9 s95. 

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 ... 

Comparison of the results allows calculation of kj6/ki8. Obviously there are drawbacks to this procedure. The major one is the necessity of a costly and tedious isotopic synthesis of labeled materials. Optimally those compounds should be as close as possible to 100% enriched. This can seldom be achieved and using partially enriched samples requires substantial corrections to the raw data and increases experimental uncertainty. A rule of thumb used in remote labeling experiments is that the remote (reporting) position should be reasonably far from the reaction center (the phenolic oxygen in the present example). For the case where there is no isotope effect at the reporting site (e.g. no 15N-KIE), the double-label experiment leads directly to the isotope effect of interest. This is more probable when the reporting site is remote, (i.e. well isolated from the reaction coordinate). 

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). 

Secondary kinetic isotope effects are observed if an isotopic label is located adjacent to or remote from the bond that is being broken or formed during the reaction. Again, these depend on the internal energy of the decomposing ions. Secondary kinetic isotope effects, 4ec, are generally much smaller than their primary analogues. 

Example Secondary kinetic isotope effects on the a-cleavage of tertiary amine molecular ions occurred after deuterium labeling both adjacent to and remote from the bond cleaved (Chap. 6.2.5). They reduced the fragmentation rate relative to the nonlabeled chain by factors of 1.08-1.30 per D in case of metastable ion decompositions (Fig. 2.18), but the isotope effect vanished for ion source processes. [78] With the aid of field ionization kinetic measurements the reversal of these kinetic isotope effects for short-lived ions (lO -lO" s) could be demonstrated, i.e., then the deuterated species decomposed slightly faster than their nonlabeled isoto-pomers (Fig. 2.17). [66,76]... 

Ingemann, S. Hammerum, S. Derrick, P.J. Fokkens, R.H. Nibbering, N.M.M. Energy-Dependent Reversal of Secondary Isotope Effects on Simple Cleavage Reactions Tertiary Amine Radical Cations With Deuterium at Remote Positions. Org. Mass Spectrom. 1989, 24, 885-889. 

A very remote secondary H/D isotope effect has been measured for the 2 + 2-cycloaddition of TCNE to 2,7-dimethylocta-2,fran -4,6-triene. The reaction of nitric oxide with iV-benzylidene-4-methoxyaniline to produce 4-methoxybenzenediazonium nitrate and benzaldehyde is thought to proceed via a 2 + 2-cycloaddition between nitric oxide and the imine double bond. A novel mechanism for the stepwise dimerization of the parent silaethylene to 1,3-disilacyclobutane involves a low-barrier [1,2]-sigmatropic shift. Density functional, correlated ab initio calculations, and frontier MO analysis support a concerted 2 + 2-pathway for the addition of SO3 to alkenes. " The enone cycloaddition reactions of dienones and quinones have been reviewed. The 2 + 2-photocycloadditions of homochiral 2(5H)-furanones to vinylene carbonate are highly diastereoisomeric. ... 

It is a basic assumption in tracer work that labelled and unlabelled molecules have identical reactivities. If the labelling involves one of the atoms attached directly to the valency bond concerned in the reaction, this assumption is not valid. The differences between the reactivities is referred to as an isotope effect. When the isotopes involved are those of hydrogen, the isotope effect is quite large for heavier elements, the effects are much smaller but in the case of carbon they can be detected in careful work. To some extent, isotope effects impose a limitation upon the accuracy of results obtained from tracer studies, but they can also lead to a fuller understanding of the mechanisms of reactions of certain types. In routine tracer work, it is advisable whenever possible to label molecules at sites remote from the points of reactivity. 

In polyatomic molecules several electronic factors contribute to the isotope effect on /. The primary isotope effect involves largely the primary derivative of the coupling constant, the secondary isotope effect on the coupling involves largely the secondary derivative of the coupling constant with respect to the stretching of a remote bond. 

Birck MR, Schramm VL (2004) Binding causes the remote [5 -3H]thymidine kinetic isotope effect in human thymidine phosphorylase. J. Am. Chem. Soc. 126 6882-6883... 

Secondary kinetic isotope effects provide one of the most subtle probes of reaction mechanism currently available as the perturbation of the system under study is small. Unfortunately, the interpretation of isotope effects is far from straightforward. There is no general agreement about the mechanistic significance of a-deuterium isotope effects in solvolytic reactions. The interpretation of more remote deuterium isotope effects appears to be even more complex. 

HEAVY ATOM AND REMOTE HYDROGEN KINETIC ISOTOPE EFFECTS IN GLYCOSYL TRANSFER... 

Table 3.3 Kinetic isotope effects for hydrolysis of various glycosyl derivatives. The isotopic quasi-racemate method was used for all measurements except those for AMP, where the remote label method was used.

Breakdown in the rule of the geometric mean (RGM) can contribute to inflation of the RS exponent. The RGM states that isotope effects are insensitive to remote labels [58], For example, the magnitude of the secondary kn/kx ratio will be independent of the primary label (H or D) so long as the isotope effects arise solely from vibrational modes, which can be expressed as the following exponential relationship ... 

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