Rapid high-yield N-acylation of aminothiols: N-acetylglutathione and N-acetylhomocysteine and their thiol pKa values

Methodology for the rapid N‐acylation of aminothiols in aqueous solution using procedures commonly employed in biochemical studies is described here. Glutathione disulfide (GSSG) and homocystine were diN‐acetylated in ~100% yield in 0.1 M aqueous NaHCO3 (pH 8.5) at room temperature by 2.5 equiv of the activated ester, N‐hydroxysulfosuccinimidyl acetate, an efficient water‐soluble acetylating reagent. Following acetone precipitation, diN‐acetylGSSG was further purified and desalted on a strong anion‐exchange (SAX) cartridge. DiN‐acetylhomocystine was simultaneously purified and desalted on a C18 cartridge. The N‐acetylated aminothiols were generated using gel‐immobilized tris(2‐carboxyethyl)phosphine as a reductant, which obviated the need for further purification. Alternatively, disulfide exchange with dissolved dithiothreitol yielded N‐acetylglutathione, which was purified on the SAX cartridge. pH titrations of N‐acetylglutathione (8.99) and N‐acetylhomocysteine (9.66) as well as those of commercially available N‐acetylcysteine (9.53) and N‐acetylpenicillamine (10.21) yielded pKa(SH) values of importance for biological studies. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Rapid, high‐yield N‐acetylation of aminodisulfides in aqueous medium under mild conditions is accomplished using the activated ester, N‐hydroxysulfosuccinimidyl acetate. Heterogeneous reduction of the diN‐acetylated aminodisulfides by agarose‐bound tris(2‐carboxyethyl)phosphine yields the corresponding N‐acetylated aminothiols in ~1 h without further purification. The acid dissociation constants of the thiol groups of N‐acetylglutathione and N‐acetylhomocysteine prepared under these conditions were determined, which expands the set of known thiol pKa values of biological interest.