Solvent Deuterium Kinetic Isotope Effects Research Articles

Studies on the activation energy and deuterium isotope effect of human skin collagenase on homologous collagen substrates.

The activation energy (EA) and solvent-deuterium kinetic isotope effect (kH/kD) of human skin fibroblast collagenase were studied on the homologous human type I, II, and III collagens in both native and denatured states. Values for EA on human type I and II collagens in solution were 47,000 and 61,000 cal, respectively. The Arrhenius plot for type III collagen, unlike that for the other types, was characterized by a break in EA at approximately 26 degrees C. At temperatures below this point, EA was 42,500 cal; at higher temperatures, EA fell to 29,500 cal. This latter value, intermediate between type I collagen monomers and denatured random gelatin alpha chains, appears to result from a further opening in the already loosened helix of the type III collagen molecule in the region of the 3/4:1/4 collagenase cleavage site. The EA of trypsin on native human type III collagen was also measured and found to be 70,000 cal. This high value calls into question the role of serine proteases in the physiologic degradation of this substrate; a much higher energy expenditure was required for trypsin to cleave type III collagen than for the fibroblast collagenase. Reaction velocity on human collagen types I-III in solution was slowed 15-35% (kH/kD = 1.2-1.5) by the substitution of deuterium for hydrogen in the solvent buffer. This value was far lower than that observed following the aggregation of solution monomers into insoluble fibrils (kH/kD = 9). Denaturation of triple helical monomers into random gelatin alpha chains eliminated any slowing by deuterium, and kH/kD was 1.0 in all cases. Since the same peptide bond hydrolysis accompanies the cleavage of all these forms of the collagen substrate, it would appear that the role of water at the rate-limiting step of collagen degradation may not reside in the hydrolysis of a peptide bond per se, but rather may reflect the difficulty in transporting water molecules to the site of such catalysis, especially following fibril aggregation.

Effect of an aromatic ester conjugate base on E1cB ester hydrolysis. Alkaline hydrolysis of fluorene-9-carboxylate esters

A series of alkyl and substituted-aryl esters of fluorene-9-carboxylic acid have been synthesised and their alkaline hydrolyses studied. The pH profiles for hydrolysis indicated substrate ionisation at higher pH values (confirmed by spectral studies) and kinetic pK values for a number of these esters were obtained at 25 °C. At high pH (pKa) the rate of hydrolysis became independent of pH with the observed rate constant in this high pH plateau region being called k′. A plot of the logarithm (to base 10) of k′versus the pKa of the conjugate acid of the appropriate leaving group consisted of two limbs, A and B, flanking a minimum. The slopes of the plots described above for regions A and B were –1.01 ± 0.05 and +0.11 ± 0.01, respectively. Region A, of high negative slope, was shown to correspond to an E1cB reaction of the title esters on the basis of linear free energy relationship arguments, the solvent deuterium kinetic isotope effect, activation parameters, and comparison with the 9-methyl-blocked analogues, as well as by the observation of saturation kinetics in aniline buffers at low pH. Region B was shown to correspond to a rate-determining step of attack of hydroxide ion on the limiting amount of ester in its neutral form. The low eliminative reactivity of fluorene ester anions was discussed along with factors considered in the literature for explaining elimination rates of ester anions.

An E1cB mechanism for the alkaline hydrolysis of thiolesters of fluorene-9-carboxylic acid: effect of ester conjugate base structure on differential eliminative reactivity of oxygen and sulphur nucleofuges

The alkaline hydrolysis of a series of thiolesters of fluorene-9-carboxylic acid has been studied and pseudo-first-order rate constants, kobs(hydroxide ion in excess), showed saturation with respect to hydroxide ion concentration for any given ester. The kinetic expression describing such behaviour was found to be kobs=k′/(1 +Kw/Ka[HO–]) where k′ is the limiting rate constant at high base concentrations and Ka is the acid dissociation constant of the acidic ester. Values of log10k′ followed a Bronsted correlation (with the pKa of the conjugate acid of the appropriate leaving group) with equation log10k′= 6.10 – 1.14 pK1.g. (r 0.9700). There was no significant steric effect on k′ as the 2′-methoxythiophenyl ester obeyed the above relationship. Esters with poorer leaving groups (S-benzyl, S-n-propyl) deviated positively from the correlation. For active thiolesters of fluorene-9-carboxylic acid the highly negative value of β1.g. for the k′ term (slope of the Bronsted plot), along with the saturation kinetics and a positive/zero value (+4.6 ± 6.1 J K–1 mol–1) for the entropy of activation for the S-phenyl ester was evidence of an E1cB alkaline hydrolytic mechanism. In support of this were the low solvent deuterium kinetic solvent isotope effect on k′(kH/kD= 1.37) and the rate inhibition on 9-methylation. Comparison of fluorene-9-esters with acetoacetic esters showed that there was less difference in reactivity between oxygen and sulphur esters for the former, an effect explained as lying in the highly delocalised nature of the fluorene ester conjugate bases.

The kinetics and mechanism of the aminolysis of phenethyl nitrite

Kinetic studies on the ammonolysis and aminolysis of phenethyl nitrite were carried out in 61% dioxan–water. Good second-order rate constants were obtained for the reactions except that with trimethylamine which is autocatalysed by the trimethylammonium ion formed. The rate of the trimethylammonium ion catalysed reaction was obtained from the slope of the plots of apparent second-order rate constants against concentration of trimethylammonium ion. There was no general base catalysis in any of the reactions. Solvent kinetic deuterium isotope effects were found to be 1.96 and 2.21 for the reactions with methylamine and morpholine, respectively, and 2.44 for the trimethylammonium ion catalysed reaction with trimethylamine. Bronsted plots are scattered, since substitution of hydrogen by a methyl group on NH3 markedly increases the nucleophilicity, e.g. the relative rates NH3 : MeNH2 : Me2NH : Me3N were 1 : 5.5 × 102 : 5.1 × 105 : 5.3 × 106. However, logarithms of the rate constants are correlated linearly with the vertical ionization potentials of these amines. All these results suggest that the reaction is ‘orbital controlled’ and proceeds via concerted displacement of alkoxide by amine rather than addition–elimination. The results are rationalized in terms of the high electronegativity of nitrogen and the presence of a lone pair of electrons on nitrogen.