Primary tritium isotope effects

The observation of a primary tritium isotope effect (H/T) that is substantially larger than the value predicted on the basis of the semiclassical Swain-Schaad relation (Chart 3) from a heavy-hydrogen (DAT) isotope effect. The same information can be expressed in terms of a Swain-Schaad exponent required to relate the two isotope effects that is substantially larger than the semiclassical value of 3.26. 

Primary tritium isotope effects have been determined by Schilf et alP at various temperatures for proton-nitrogen couplings in two Schifif bases obtained by the condensation of 4-methoxy- and 4,6-dimethoxysalicylaldehyde with methylamine. However, the results are not conclusive and it is not clear if they can be used as an additional probe of the hydrogen bond character. 

Primary tritium isotope effect 849 Propellanes, synthesis of 569, 570 Protecting groups for thiols 432 acetamidomethyl 675, 676 acetyl and benzoyl 677, 678 benzyl 671, 672 benzyloxycarbonyl 678 benzylthiomethyl and phenylthio-methyl 681... 

Primary tritium isotope effects can also be measured. As tritium is radioactive, this requires that the NMR tube is protected from breaking in the NMR instrument Referencing in this case can be either ghost referencing [15] or the use of CgHgT versus C5Hg [16]. 

Primary isotope effects, AT, can of course be also measured for tritium although it is experimentally more difficult to produce tritiated compounds because of the health hazards involved. However, a large survey has shown that primary tritium isotope effects are proportional to AD [31]. Primary isotope effects were early on pointed out as a means of distinguishing between double... 

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. 

The primary tritium KIE (k /l ) and the secondary tritium KIE ( h/ h) were determined in two different experiments carried out in the following way. If Ra is the radioactivity of the original reactant and / ROh is the radioactivity of ROH (ROT) determined at low (<5%) extents of reaction, the primary tritium KIE is given by equation (52). If the radioactivity of the styrene isolated at low extents of reaction, Rs, is also measured, the secondary tritium isotope effect can be calculated from (53). 

These reactions proceed through symmetrical transition states [H H H] and with rate constants kn,HH and kH,DH, respectively. The ratio of rate constants, kH,HH/kH,DH> defines a primary hydrogen kinetic isotope effect. More precisely it should be regarded as a primary deuterium kinetic isotope effect because for hydrogen there is also the possibility of a tritium isotope effect. The term primary indicates that bonds at the site of isotopic substitution the isotopic atom are being made or broken in the course of reaction. Within the limits of TST such isotope effects are typically in the range of 4 to 8 (i.e. 4 < kH,HH/kH,DH < 8). 

Quantitative relationships " between magnitudes of deuterium and tritium primary kinetic isotope effects on chemical reactivity. 

The primary deuterium, A( H, H), and tritium isotope effects, A( H, H), are proportional in general . The primary isotope effects are proportional to the hydrogen bond... 

It should be noted that in enols of (3-diketones (acetyl- and benzoylacetone, dibenzoylmethane) the largest primary negative isotope effect is observed, for deuterium between 0.6 and 0.7 ppm and for tritium between —0.8 and —1.0 ppm [70, 71]. It corroborates the double-well character of proton potential in compounds of this group. Even in the shortest intramolecular OHO bridge in 4-cyano-2,2,6, 6-tetramethyl-3,5-heptanedione [55], of 2.393 A length (see Sect. 10) it was evidenced, based on neutron diffraction and inelastic scattering, a double minimum potential with very low barrier. The authors of the cited paper believe the existence of single minimum potential to be unlikely in compounds of this type. 

In the competitive technique, the enzyme reacts with a mixture of labeled and unlabeled substrate, yielding isotope effects on k t/Ku [29]. Competitive measurements, while limited to kcat/KM isotope effects, are substantially more precise than noncompetitive measurements. In addition, they allow the use of tracer-level radioactive labels, permitting tritium isotope effects at the primary and secondary positions (kH/kj or ko/kj) to be determined. General methods for determining competitive isotope effects have been published [17b]. One drawback is that multiple isotopic labels must often be used, leading to extensive synthetic efforts. 

Large primary kinetic isotope effects have been measured for the H-atom transfer steps from substrate to dAdo and from dAdo to the product radical in a number of AdoCbl-dependent enzymes as indicated in Table 19.1. In methylmalonyl-CoA mutase, the steady-state deuterium isotope effect is 5-6 in the forward direction, and the intrinsic isotope effect of step (i) in Scheme 19.3 is masked by the kineti-cally coupled but slower later steps [37-39]. The steady-state tritium kinetic isotope effect kii/kj) in the forward direction has been reported to be 3.2 [38]. Note that the experiments with deuterium were performed with a fully deuterated methyl group, while those with tritium were carried out at the trace level and correspond to a single isotopic atom therefore these two isotope effects should not be directly compared. For the reverse reaction, the deuterium kinetic isotope effect is also par-... 

Isotope effects are called primary when a bond is made or broken to the isotopic atom during the reaction and secondary when it is not. Secondary deuterium or tritium isotope effects are called a when the isotopic hydrogen is attached to a primary carbon undergoing bond cleavage and 13 or y when the hydrogen is on a carbon once or twice removed from the primary carbon. Primary isotope effects are almost always normal, or greater than unity, whereas secondary isotope effects can be either normal or inverse (i.e., less than unity). 

The lack of a large intramolecular deuterium isotope effect in the reaction of hot tritium with partially deuteriated methanes has been explained by Rowland and coworkers by postulating the existence of a very large secondary deuterium isotope effect which counterbalances the normal primary replacement isotope effect. 

This is made possible through a combination of primary and secondary tritium isotope effects on the two methylene and one methyl groups. What is now required is a combination of developments that allows one to measure the concentration of the isotopomers as the reaction proceeds. 

Damgaard SE (1981) Primary deuterium and tritium isotope effects upon V/K in the liver alcohol dehydrogenase reaction with ethanol. Biochemistry 20 5662-5669... 

The method of analysis developed is for acetic acid and involves the use of two enzymes. Chiral acetic acid [as 1.26)] is irreversibly condensed as its CoA-derivative with glyoxylic acid (/.27), using the enzyme malate synthase, to give malic acid 1.28). The condensation occurs with loss of hydrogen isotope by a primary kinetic isotope effect (A h kj). This means that loss of H is favoured over loss of D which is in turn favoured over loss of T. The result is a high retention of tritium. 

Normal chemical removal of a hydrogen atom from C-1 will result in loss of H rather than H, by a primary kinetic isotope effect, i.e. high tritium retention ca. 90%. Stereospecific hydrogen removal from the... 

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