Studies on the nitrosation of thiols in relation to vasodilatory action

Abstract

A kinetic study of the nitrosation of L-cysteine, L-cysteine methyl and ethyl esters, N-acetyl-L-cysteine and glutathione by isopropyl nitrite in acid solution at 25 C was undertaken. The thiols exhibited identical rate laws and in all cases the observed rate constant was reduced by added isopropyl alcohol. The results were found to be consistent with the mechanism in which a rapid reversible acid-catalysed hydrolysis of isopropyl nitrite occurs to give nitrous acid, which then in its protonated form effects nitrosation. The third-order rate constant for the nitrosation of the thiols by isopropyl nitrite and the equilibrium constant for the formation of isopropyl nitrite were found to be in good agreement with the literature values obtained by direct measurement. A similar kinetic study of the nitrosation of L-cysteine, L-cysteine methyl and ethyl esters, N-acetyl-L-cysteine, thioglycolic acid and glutathione by various alkyl nitrites, in water at 25 C , in the pH range 6-13 was undertaken. The pH dependence of the rate constant is consistent with a mechanism involving a direct nitrosation by alkyl nitrites with the thiolate anion (RS(^-)) of the thiol. A quantitative kinetic analysis yielded macroscopic and microscopic pKa values for RSH ionisation in good agreement with the literature values. One exception is L-cysteine where the microscopic, pKp, value (for NH(_2)RSH → NH(_2)RS(^-)) differs significantly from the literature value. In the case of simple alkyl nitrites (ethyl, isopropyl, isoamyl and t-butyl nitrites) steric effects appear to be the major influence in reactivity whereas electron-withdrawing substituents in the β-position greatly enhanced the rate constant. The results were found to satisfy Taft's equation and thus a correlation between structure and reactivity of the alkyl nitrites with the thiols was established. This work shows that at least in vitro a direct and rapid reaction occurs between alkyl nitrites and thiols at pH values likely to be encountered in vivo. This confirms that such reactions could occur in vivo and could be an important feature of the chain of events occurring during the vasodilatory action of alkyl nitrites. Finally a preliminary investigation of the reaction of glyceryl trinitrate with cysteine, in oxygen and oxygen-free nitrogen atmosphere, in the pH range 6-13 was undertaken. In this case no evidence was found for the formation of S-nitrosocysteine or nitric oxide from glyceryl trinitrate in the presence or absence of cysteine. Thus the results could not confirm the hypothesis that glyceryl trinitrate owes its vasodilatory action to the formation of the intermediate, S-nitrosocysteine, from the reaction of nitric oxide (formed from glyceryl trinitrate) and cysteine.