Substrate kinetic isotope effects

Because the zero-point energy of carbon-hydrogen bonds decreases in the order 

It follows that when the second step is fast, fci/fclJB] is large ( 1) and equation (16) reduces to 

Kinetic isotope effects also show a dependence upon the reactivity of the electrophile. Thus some reactions, e.g. positive chlorination, show no isotope effect whereas others, e.g. sulphonation, do show an isotope effect. There are two ways of visualising the reasons for this and they are closely related. Very 

This effect is observable within a series of very similar electrophiles. Zollinger27 found that in reactions of diazonium ions substituted with 4-C1, 3-C1, and 3-N02 substituents (i.e. the reactivity and electron-withdrawing power of the ion increased along the series) the respective kinetic isotope effects were 6.55, 5.48 and 4.78. 

It is apparent from equation (16) that if k x becomes much larger than k 2, the rate will depend upon k 2 and so a kinetic isotope effect will be observed. Now kL j will become large if there is steric hindrance to formation of the intermediate, and a number of examples are now known where an electrophile which normally gives no isotope effect, does so if formation of the intermediate is hindered. 

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NADH or aldehyde) in excess of the other reactant. Under these conditions, the chemical conversion of aldehyde to alcohol occurs with a (saturated) apparent first-order rate constant of 200 to 400 sec i. This process, as measured either by the disappearance of NADH or by the disappearance of chromophoric aldehyde, has been shown by McFarland and Bernhard (80) to be subject to a primary, kinetic isotope effect ksjkj) =2 to 3 when stereospecifically labeled (4-R)-deuterio NADH is compared to isotopically normal NADH. Shore and Gutfreund (84) earlier had investigated substrate kinetic isotope effects on the pre-steady-state phase of ethanol oxidation. Their studies demonstrated that the rate of the presteady-state burst production of NADH is subject to a primary kinetic isotope effect, kulkj) 4—6 when 1,1-dideuterio ethanol is compared to isotopically normal ethanol, and that there is no primary kinetic isotope effect on the steady-state rate. It can be concluded from these studies (a) that the rate of interconversion of ternary complexes e.g., Eq. (19) above], as already mentioned, is rapid relative to turnover, and (b) that the transition-state for the rate-limiting step in the interconversion of ternary complexes involves carbon-hydrogen bond scission and/or carbon-hydrogen bond formation. 

A special type of substituent effect which has proved veiy valuable in the study of reaction mechanisms is the replacement of an atom by one of its isotopes. Isotopic substitution most often involves replacing protium by deuterium (or tritium) but is applicable to nuclei other than hydrogen. The quantitative differences are largest, however, for hydrogen, because its isotopes have the largest relative mass differences. Isotopic substitution usually has no effect on the qualitative chemical reactivity of the substrate, but often has an easily measured effect on the rate at which reaction occurs. Let us consider how this modification of the rate arises. Initially, the discussion will concern primary kinetic isotope effects, those in which a bond to the isotopically substituted atom is broken in the rate-determining step. We will use C—H bonds as the specific topic of discussion, but the same concepts apply for other elements. 

Bromination has been shown not to exhibit a primary kinetic isotope effect in the case of benzene, bromobenzene, toluene, or methoxybenzene. There are several examples of substrates which do show significant isotope effects, including substituted anisoles, JV,iV-dimethylanilines, and 1,3,5-trialkylbenzenes. The observation of isotope effects in highly substituted systems seems to be the result of steric factors that can operate in two ways. There may be resistance to the bromine taking up a position coplanar with adjacent substituents in the aromatization step. This would favor return of the ff-complex to reactants. In addition, the steric bulk of several substituents may hinder solvent or other base from assisting in the proton removal. Either factor would allow deprotonation to become rate-controlling. 

The reaction between permangante ion and neutral formic acid follows similar bimolecular kinetics with k2 = 1.1 x 10 exp(—16.4x 10 /lt7 )l.mole . sec . No primary kinetic isotope effect was found for this path either in light or heavy water. However, Mocek and Stewart have reported that in very strong sulphuric acid the oxidations of neutral substrate by both HMnO and MnOj display substantial isotope effects. 

Kinetic data exist for all these oxidants and some are given in Table 12. The important features are (i) Ce(IV) perchlorate forms 1 1 complexes with ketones with spectroscopically determined formation constants in good agreement with kinetic values (ii) only Co(III) fails to give an appreciable primary kinetic isotope effect (Ir(IV) has yet to be examined in this respect) (/ ) the acidity dependence for Co(III) oxidation is characteristic of the oxidant and iv) in some cases [Co(III) Ce(IV) perchlorate , Mn(III) sulphate ] the rate of disappearance of ketone considerably exceeds the corresponding rate of enolisation however, with Mn(ril) pyrophosphate and Ir(IV) the rates of the two processes are identical and with Ce(IV) sulphate and V(V) the rate of enolisation of ketone exceeds its rate of oxidation. (The opposite has been stated for Ce(IV) sulphate , but this was based on an erroneous value for k(enolisation) for cyclohexanone The oxidation of acetophenone by Mn(III) acetate in acetic acid is a crucial step in the Mn(II)-catalysed autoxidation of this substrate. The rate of autoxidation equals that of enolisation, determined by isotopic exchange , under these conditions, and evidently Mn(III) attacks the enolic form. 

The oxidations of formic acid by Co(III) and V(V) are straightforward, being first-order with respect to both oxidant and substrate and acid-inverse and slightly acid-catalysed respectively. The primary kinetic isotope effects are l.Sj (25°C)forCo(IU)and4.1 (61.5 C°)for V(V). The low value for Co(lII) is analogous to those for Co(IIl) oxidations of secondary alcohols, formaldehyde and m-nitrobenzaldehyde vide supra). A djo/ h20 for the Co(III) oxidation is about 1.0, which is curiously high for an acid-inverse reaction . The mechanisms clearly parallel those for oxidation of alcohols (p. 376) where Rj and R2 become doubly bonded oxygen. 

Kinetic isotope effects have also been given in terms of e for reactions at a single carbon atom kPk = 1/(1 + e/1000) where k and are rates for and substrates. 

Today a good understanding of transition state structure can be obtained through a combination of experimental measurements of kinetic isotope effects (KIE) and computational chemistry methods (Schramm, 1998). The basis for the KIE approach is that incorporation of a heavy isotope, at a specific atom in a substrate molecule, will affect the enzymatic reaction rate to an extent that is correlated with the change in bond vibrational environment for that atom, in going from the ground state to the... 

The results from these experiments also allowed Hannon and Traylor to determine the primary and secondary hydrogen deuterium kinetic isotope effects for the hydride abstraction reaction. If one assumes that there is no kinetic isotope effect associated with the formation of 3-deutero-l-butene, i.e. that CH2=CHCHDCH3 is formed at the same rate (k ) from both the deuterated and undeuterated substrate (Scheme 25), then one can obtain both the primary (where a deuteride ion is abstracted) and the secondary deuterium... 

A secondary deuterium kinetic isotope effect is observed when substitution of a deuterium atom(s) for a hydrogen atom(s) in the substrate changes the rate constant but the bond to the deuterium atom is neither broken nor formed in the transition state of the rate-determining step of the reaction. Several types of secondary hydrogen-deuterium (deuterium) KIEs are found. They are characterized by the position of the deuterium relative to the reaction centre. Thus, a secondary a-deuterium KIE is observed when an a-hydrogen(s) is replaced by deuterium [equations (1) and (2)], where L is either hydrogen or deuterium. 

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. 

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