Primary and secondary isotope effects

In this review, primary isotope effects are defined as concerning reactions in which, at some stage, a bond to the isotopically substituted atom is either broken or formed. A secondary isotope effect will refer to reactions in which no bond to the isotopically substituted atom is broken and none is formed. The strengths of the bonds to the substituted atoms may, however, be altered and, in general, will be to some degree. 

Intermolecular kinetic isotope effects are likely to follow the same qualitative trends as the intramolecular, but as has been emphasised, reliable intermolecular kinetic isotope effects are rarely available in mass spectrometry. 

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For example, primary and secondary isotope effects in reduction by NADH Hr h... 

These data led to the model already described several times above. The enzyme executes a search for a tunneling sub-state, apparently 13 kcaFmol in energy above the principal state from this state the hydrogen atom tunnels with no further vibrational excitation. Probably motion of the secondary center is coupled into the tunneling coordinate. The result is large, temperature-independent primary and secondary isotope effects in the context of an isotope-independent activation energy. 

Rickert, K.W. and Klinman, J.P. (1999). Nature of hydrogen transfer in soybean lipoxygenase 1 separation of primary and secondary isotope effects. Biochemistry 38, 12218-12228... 

In studying both primary and secondary isotope effects, if the secondary effect is significantly reduced on the replacement of the primary H for D (i.e., by the primary effect), then tunneling occurs. Note that this method assumes that the observed primary and secondary isotope effects occur in the same step of the reaction. A number of methods have been developed to characterize tunneling phenomenal... 

A value of kA is available from measurements of hydrogen isotope exchange (kx) corrected for primary and secondary isotope effects. This is illustrated in Scheme 8 for the detritiation of tritiated benzene. In the lower part of the scheme, the reacting isotope of the benzenonium ion intermediate (9-t) is indicated as a superscript on the rate constant kv and a secondary isotope effect is neglected. 

The numerical factors in equations (70) to (73) are again statistical. The factors involving l raised to a power imply kinetic secondary isotope effects. The replacement of one protium atom by one deuterium atom increases the rate constant by i . This inverse isotope effect is qualitatively intelligible since the non-transferred nuclei are more tightly bound in the transition state than they were in the reactants. The existence of the secondary isotope effect also means that the ratio of rate constants in H20 and D20 ( H/fcD) Is not a measure of the primary isotope effect, whereas h o/3 h,do is- To obtain the primary isotope effect it is necessary to divide H/feD by Z2 - (For experimental evaluations of primary and secondary isotope effects see Kreevoy et al.t 1964 ... 

A requirement for an a/m-orientation of the hydridic p-C—H and C—metal bonds as in [10] is indicated by the reaction of threo-3-deuterio-2-(trimethylstannyl)butane with triphenylcarbenium tetrafluoroborate in methylene chloride at 24° which yields a mixture of 3-deuterio-l -butene, /ra v-2-deuterio-2-butene, and undeuteriated c/.v-2-butene as the major product (Hannon and Traylor, 1981). Comparison of the product distributions for the protio- and deuterio-stannanes yields primary and secondary isotope effects of 3.7 and 1.1 respectively. These reactions appear to avoid the complications of adduct formation between the triarylcarbenium salt and the hydride donor, but the preferential formation of the cw-2-butenes is not fully explained. The requirement for the anti-orientation is also shown by the relatively low hydride-donating properties of tris[(triphenylstannyl)methyl-methane (Ducharme et ai, 1984a) which adopts a C3-conformation with the P-C—H gauche to all three C—Sn bonds. In contrast, 1,3,5-triphenyl-2,4,6-trithia-1,3,5-tristannyladamantane, in which anti-orientations with respect to the bridgehead C—H bond are locked, shows high reactivity (Ducharme et al., 1984b). 

KIE calculated from this equation corresponds to the overall effect including primary and secondary isotope effects. For reaction with a C-H bond breakage in a methyl or methylene group the primary isotope effect (KIEr) is observed for... 

Combination of Equations (24) and (25) gives formulas for primary and secondary isotope effects ... 

The recovered 44 was analysed by means of 2H and 13C NMR and changes of composition were related to C4 methyl group as internal standard. From these changes the isotope effects were calculated. The overall isotope effect at C5 represents a combination of primary and secondary isotope effects of proton... 

The simplicity of eq. (11) is useful for discussion, but in no way limits the generality of the following discussion. Cases with internal rotation will exhibit the same trends.201 The primary and secondary isotope effects are discussed separately since their nature and origin are different. 

There are several rough experimental values for the decomposition of chemically activated CH4. Some older data on the reaction D + CH3 — CH3D, studied at 25°C. by Taylor and co-workers,38 correspond to (tf) 3 keal. (the zero-point energy difference for C—H and C—D is 2 keal.). These experiments correspond to the low-pressure limit, for which the calculated value in Table XI is kao 1.7 X 1010 sec.-1 at this energy. Marcus14 analyzed these data to obtain ka = 8 X 108 sec.-1 which, if we correct for the presence of a primary and secondary isotope effect due to the D atom, would be ka 1.5 X 109 sec.-1. He estimated a possible error of a factor of 5-10 in these values. We believe that the collision number used by Marcus in the calculation of ka is too low by a factor of 3-5 which would raise the experimental value to >5 X 10 sec.-1. The agreement is adequate, but the desirability of redoing these experiments with improved techniques is evident. 

Ethyl C2H6 -> C2H4 + H. The general kinetic behavior is discussed first, and then some primary and secondary isotope effects are very briefly summarized, since detailed discussions of this system have appeared recently. 17 20b M... 

In this study, we have shown that both alcohol and D20 have an Important effect on the nucleation and crystal growth of zeolites with Si/Al ratios between 1-2. In the case of alcohol, the formation of large pore zeolites such as zeolites X or Y is markedly accelerated at low alcohol levels. We attribute this to a stabilization of the cation-water complex and structured H20 which act as templates. However, at high alcohol levels, the structure of water disintegrates and leads to the formation of more condensed zeolites such as sodalite or cancrinite. Synthesis of zeolite A in D20 is slower than that in water, which primarily arises from the primary and secondary isotope effect during the condensation polymerization reactions necessary for zeolite growth. 

Thus the primary and secondary isotope effects are all within the semiclassical limits and their relationship is in full accord with the semiclassical Swain-Schaad relationship. There is no indication from the magnitudes of the secondary isotope effects in particular of any coupling between motion at the secondary center and the reaction-coordinate for hydride transfer. Thus the sole evidence taken to indicate tunneling is the rigorous temperature-independence of the primary isotope effects. 

A key line of evidence for a multistep mechanism, as opposed to the one-step hydride-transfer mechanism, had been derived from isotope effects measured in reduction of various substrates with monodeuterated analogs of NADH. One can compare the observed rate constants kHH and kno, which in the case of negligible secondary isotope effects should obey the relationship koH/kHM = (1 + kro/kyi])/2, allowing the calculation of the primary isotope effect kn/ku (if undeuterated, monodeuterated and dideuterated hydride donors are all used, both primary and secondary isotope effects can be obtained). In addition, for an oxidizing agent Acceptor one can determine the isotope ratio in the product Acceptor-H/Acceptor-D, called in these studies the product isotope effect Th/Td-... 

It is noteworthy that the Swain-Schaad exponents are temperature independent for both primary and secondary isotope effects at pH 6.1. Two scenarios can be considered. The first is that there is a significant commitment to catalysis which is obscuring the full value of the isotope effect on k st/Km- It is anticipated that, because the primary and secondary exponents are temperature independent, this commitment would be temperature independent. Jonsson et al. have used North-rop s expression [60] for correcting observed isotope effects based on the assumption of a temperature-independent commitment (Eq. (10.22)). A commitment of 0.6 for the oxidation of benzylamine brings the secondary exponent to about 3.3,... 

The exponents described by Saunders, sometimes called mixed isotopic exponents , are shown in Eq. (11.17). The exponent describes the relationship between the H/T isotope effect from substitution at site one (determined when protium is at site two), and the site-one D/T isotope effect (determined when deuterium is at site two). If the two sites are distinguished as giving primary and secondary isotope effects, the first exponent in Eq. (11.17) resembles the single-site Swain-Schaad exponent Eq. (11.9) for a primary isotope effect, and the second exponent in Eq. (11.17) resembles a single-site secondary Swain-Schaad exponent. However, the mixed isotopic exponents necessarily involve isotopic substitution at two sites and should not be confused with single-site Swain-Schaad exponents. 

Figure 11.11. Example of competitive experiments used by Saunders et al. [89, 97] to measure isotope effects for mixed-isotope exponent determinations. The primary and secondary isotope effects were determined using the starting tritium activities of the bromide substrate, the product styrene, and the solvent ethanol, all at a kno A/n fractional...

Appleman, J. R. (1997) Kinetic scheme for thymidylate synthase from Escherichia coli determination from measurements of ligand binding, primary and secondary isotope effects, and pre-steady-state catalysis. Biochemistry 36, 4212-4222. 

C,H) and V(H,H) in CH4 and its isotopomers, /(C,H) was found to increase by 0.088 Hz on raising the temperature from 180 K to 380 K, which agrees with the observed increase of 0.083 Hz on raising the temperature from 200 K to 370 The D primary and secondary isotope effects on /(C,H) were predicted to decrease with increasing temperature. Table 18 shows the total values, separated into stretching, bending and the second-order stretch-bend contributions to /(C,H) and 7 (C,H) in each of the five isotopomers at 300 K, as well as the zero-point corrections. 7 (X,H) is defined as in Eq. (85). 

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