Aqueous solvent isotope effects

Abstract This chapter introduces the basic principles used in applying isotope effects to studies of the kinetics and mechanisms of enzyme catalyzed reactions. Following the introduction of algebraic equations typically used for kinetic analysis of enzyme reactions and a brief discussion of aqueous solvent isotope effects (because enzyme reactions universally occur in aqueous solutions), practical examples illustrating methods and techniques for studying enzyme isotope effects are presented. Finally, computer modeling of enzyme catalysis is briefly discussed. 

It is reasonable to expect that isotopic substitution on solvent molecules will affect both equilibrium and rate constants. This is especially true for reactions in aqueous media, many of which are acid or base catalyzed and therefore sensitive to pH or pD. Furthermore H/D aqueous solvent isotope effects often display significant nonlinearity when plotted against isotope fraction of the solvent. The analysis of this effect can yield mechanistic information. The study of aqueous solvent isotope effects is particularly important in enzyme chemistry because enzyme reactions universally occur in aqueous media and are generally pH sensitive. 

Further substrate and solvent isotope effects were measured by Batts and Gold472 for the dedeuteration and detritiation of labelled 1,3,5-trimethoxy-benzene in aqueous protium- and deuterium-containing perchloric acid. Contrary to the observations above, they found the rate coefficients for dedeuteration to detritiation to be independent of the concentration of the catalysing acid (Table 125). Detritiation in the deuterium-containing aqueous perchloric acid media occurred 1.68 times faster than in the protium-containing media. 

RATE COEFFICIENTS AND SOLVENT ISOTOPE EFFECT FOR REACTION OF 4-ClC6H4SiMe3 WITH AQUEOUS TRIFLUOROACETIC ACID AT 25 "C471... 

Kinetic solvent isotope effect as a measure of electrophilic assistance to bromide ion departure limiting values rate data in ethanol, methanol and their aqueous mixtures using Bentley s TBr scale its decrease corresponds to the involvement of nucleophilic assistance. R = (/caqhtOII//cAcoH)r as a measure of nucleophilic solvent assistance. Model for a limiting bromination mechanism. Ruasse et al. (1991). /Ruasse and Zhang (1984). 9Argile and Ruasse (to be published). Modro et al. (1979). 

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. 

Christensen (1966, 1967) found that aromatic sulfonic anhydrides undergo rapid, uncatalyzed hydrolysis in either acetone or aqueous dioxan (t1/2= 17 s at 25°C for Ar =p-tolyl in 65% dioxan). Added strong acids (or added salts like NaCl or LiCl) have no effect on the rate at concentrations up to 0.01 M. The solvent isotope effect associated with this spontaneous hydro-... 

The mechanism of the spontaneous hydrolysis of aryl cr-disulfones (188) in aqueous dioxan has been studied in some detail (Kice and Kasperek, 1969). The reaction is approximately 104 times slower under a given set of conditions than the very rapid spontaneous hydrolysis of aryl sulfinyl sulfones (135) discussed earlier in Section 5. The large difference in rate arises because AH for the spontaneous hydrolysis of a given cr-disulfone is about 6 kcal mol-1 larger than AH for the spontaneous hydrolysis of the corresponding sulfinyl sulfone. However, despite the large difference in rate and AH, the two spontaneous hydrolyses show a remarkable similarity in (a) Hammett p, (b) increase in rate with increasing water content of the solvent, (c) solvent isotope effect, and (d) AS. ... 

Interpretation of KIEs on enzymatic processes (see Chapter 11) has been frequently based on the assumption that the intrinsic value of the kinetic isotope effect is known. Chemical reactions have long been used as models for catalytic events occurring in enzyme active sites and in some cases this analogy has worked quite well. One example is the decarboxylation of 4-pyridylacetic acid presented in Fig. 10.9. Depending on the solvent, either the zwitterionic or the neutral form dominates in the solution. Since the reaction rates in D20/H20 solvent mixtures are the same (see Section 11.4 for a discussion of aqueous D/H solvent isotope effects), as are the carbon KIEs for the carboxylic carbon, it is safe to assume that this is a single step reaction. The isotope effects on pKa are expected to be close to the value of 1.0014 determined for benzoic acid. This in mind, changes in the isotope effects have been attributed to changes in solvation. 

The solvent isotope effect on the dissociation constant of an acidic proton in aqueous solution has been used to deduce a value for the fractionation... 

A concentration scale for solutes in aqueous solutions, equal to moles of solute/55.51 mol water. It is frequently used in studies of solvent isotope effects. As pointed out by Schowen and Schowen the choice of standard states can change the sign for the free energy of transfer of a species from one solvent to another, even from HOH and DOD. The commonly used concentration scales are molarity, mole fraction, aquamolality, and molality. Free energies tend to be nearly the same on all but the molality scale, on which they are about 63 cal mol more positive at 298 K than on the first three scales. The interested reader should consult Table I of Schowen and Schowen ... 

Williams and co-workers provided solvent isotope effect data that supported the mechanism of Scheme 4P The rearrangement of N-phenyl-hydroxylamine (pAl = 1.9) in aqueous H2SO4 exhibits an inverse solvent isotope effect at pH > 2 and a normal solvent isotope effect of 1.5 in the plateau region at pH < 1.0. This is expected for the mechanism of Scheme 4... 

Increasing numbers of nitrogen atoms increase not only the kinetic susceptibility toward attack but also the thermodynamic stability of the adducts. Reversible covalent hydration of C = N bonds has been observed in a number of heterocyclic compounds (76AHC(20)117). Pyrimidines with electron-withdrawing groups and most quinazolines show this phenomenon of covalent hydration . Thus, in aqueous solution the cation of 5-nitropyrimidine exists as (164) and quinazoline cation largely as (165). These cations possess amidinium cation resonance. The neutral pteridine molecule is covalently hydrated in aqueous solution. Solvent isotope effects on the equilibria of mono- (166) and dihydration (167) of neutral pteridine as followed by NMR are near unity (83JOC2280). The cation of 1,4,5,8-tetraazanaphthalene exists as a bis-covalent hydrate (168). 

Stoichiometry (28) is followed under neutral or in alkaline aqueous conditions and (29) in concentrated mineral acids. In acid solution reaction (28) is powerfully inhibited and in the absence of general acids or bases the rate of hydrolysis is a function of pH. At pH >5.0 the reaction is first-order in OH but below this value there is a region where the rate of hydrolysis is largely independent of pH followed by a region where the rate falls as [H30+] increases. The kinetic data at various temperatures both with pure water and buffer solutions, the solvent isotope effect and the rate increase of the 4-chloro derivative ( 2-fold) are compatible with the interpretation of the hydrolysis in terms of two mechanisms. These are a dominant bimolecular reaction between hydroxide ion and acyl cyanide at pH >5.0 and a dominant water reaction at lower pH, the latter susceptible to general base catalysis and inhibition by acids. The data at pH <5.0 can be rationalised by a carbonyl addition intermediate and are compatible with a two-step, but not one-step, cyclic mechanism for hydration. Benzoyl cyanide is more reactive towards water than benzoyl fluoride, but less reactive than benzoyl chloride and anhydride, an unexpected result since HCN has a smaller dissociation constant than HF or RC02H. There are no grounds, however, to suspect that an ionisation mechanism is involved. 

Kinetic evidence for the involvement of a-hydroxydialkylnitrosamines (142) in the pH-independent solvolysis of the a-(acyloxy)dialkylnitrosamines (141) has been obtained.120 The aminolysis in benzene of 0-(2,4-dinitrophenyl)-/7,/ -disubstituted benzophenone oximes (143) with pyrrolidine and piperidine are third order in amine.121 Hir st s mechanism involving electrophilic catalysis operates and can explain the various effects observed. The bis(pentamethylphenyl)-A-isopropylketenimine (144) undergoes pre-equilibrium /V-protonation in aqueous acetonitrile followed by water attack. An inverse solvent isotope effect and the observation of the diol (145) confirm this.122... 

A study of the decomposition of /f-hydroxy-/V-chloroamines in aqueous medium has established that pre-equilibrium formation of the conjugate alcoholate is a prerequisite feature of the competing fragmentation and intramolecular elimination paths (Scheme 13).98 A very high effective molarity (EM = 2 x 105 M) has been estimated for the intramolecular process, which cannot occur in the case of (/V-chloro)butylethanolaminc. For reaction of (A-c h I o ro )ct hy I cth an o I am i nc kmln/k g = 6.1 and the solvent isotope effect ( oh- /koo- )obs = 0.68 is consistent with pre-equilibrium deprotonation followed by a unimolecular reaction in which there is no participation by solvent. 

There are two areas in which it seems that substantial advances could be made even on the basis of first-order theory. These are the field of spontaneous reactions where isotope effects are sometimes large and where the existence of many closely related systems makes it likely that a useful framework of generalizations could be found. The second field is that of strictly non-aqueous solvent systems where a comparison of solvent isotope efFects with those in aqueous solution is likely to throw light on essential differences in chemistry. 

As a mechanistic tool in the investigation of acid- or base-catalysed reactions in aqueous solution, the measurements in isotopically mixed solvents are most useful for reactions where a certain amount is already known about the mechanism. In particular, the study of mixed solvents is also a good deal more informative whenever it is possible to measure product isotope effects in addition to rate isotope effects. In such cases (and A-Sb2 reactions spring to mind as a good example) solvent isotope effect studies can add considerably to the detailed picture of a transition state. The phenomena are as yet less suited to the ah initio assignment of reaction mechanism, such as the decision between weak nucleophilic participation of water in an acid-catalysed reaction and an A-l mechanism, when no information beyond the kn-n relation is available. For these reasons it is likely that mechanistic investigation by this method will increasingly be directed towards systems where both rate and product isotope effects are obtainable. 

Two 67Zn (natural abundance = 4.12% / = f) n.m.r. studies have been reported.9,10 The chemical shift of 67Zn (4.81 MHz at 1.807 Tesla) in aqueous zinc chloride, bromide, and iodide solutions was found to be strongly concentration dependent, while no such dependence was noted in solutions of the perchlorate, nitrate, or sulphate. This behaviour resembles that found for analogous cadmium systems, and is attributed to the formation of mono- and poly-halogeno- complexes even at low salt concentrations. In addition, the zinc halide solutions show an anomalous shift to higher frequencies for their solutions in D20, compared with those in H20. The perchlorate, nitrate and sulphate show no solvent isotope effect. 

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