The reaction of oxalyl thiolesters with nucleophiles, especially thiols

Since there is evidence that oxalyl thiolesters (RSCOCOO⁻) are present in animal cells, and possibly may participate in the control of metabolism, the present study was undertaken to characterize their reactivity with nucleophiles so that one could gain a better understanding of how they might be affecting the activities of enzymes. At 25°C and neutral pH, N-acetyl-S-oxalyl-2-aminoethanethiol (NAC-S-Ox) reacts rapidly with cysteamine (2-aminoethanethiol) to give N-acetylcysteamine and N-oxalylcysteamine. Under similar conditions, other aminothiols, such as cysteine, homocysteine, penicillamine, and cysteine ethyl ester, also react rapidly with NAC-S-Ox, but non-thiol-containing amines, such as alanine, alanine ethyl ester, glycine, and S-methylcysteine, react more than four orders of magnitude less rapidly. The aminothiol reactions apparently proceed by rate-determining oxalyl transfer to the thiol followed by a rapid intramolecular S- to N-oxalyl migration. The reactions follow second-order kinetics with the thiolate anion being the reactive nucleophile. At 25°C and ionic strength 1.0 , k_N, defined in the equation, rate = k_NRS⁻]NAC-S-Ox], has the following values ( ⁻¹ s⁻¹) for the anion of the reacting thiol: cysteamine, 170; cysteine, 260; cysteine ethyl ester, 76; homocysteine, 380. Rate data for the reaction of NAC-S-Ox with hydroxylamine, imidazole, hydroperoxide, and hydroxide were also obtained. The reaction of S-oxalyl-p-thiocresol with thiol anions under the same conditions gives the following values for k_N ( ⁻¹ s⁻¹ × 10⁻³): glutathione, 5.6; N-acetylcysteamine, 3.7; pantetheine, 4.8; 8-mercaptooctanoic acid, 4.5; 6-mercaptooctanoic acid, 1.0; dihydrolipoic acid, 8.2. These results indicate that oxalyl transfers from oxalyl thiolesters to thiol anions occur more than two orders of magnitude more rapidly than corresponding acetyl transfers, and that under physiological conditions any in vivo oxalyl thiolester would equilibrate within minutes with virtually every thiol in the cell, including those attached to enzymes. Consequently, it is proposed that one mechanism by which oxalyl thiolesters may function in vivo to alter the catalytic activities of enzymes is to covalently modify enzymic thiols by acylation with an oxalyl group.