Rapid high‐yield N ‐acylation of aminothiols: N ‐acetylglutathione and N ‐acetylhomocysteine and their thiol p K a 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 di N ‐acetylated in ~100% yield in 0.1 M aqueous NaHCO 3 (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, di N ‐acetylGSSG was further purified and desalted on a strong anion‐exchange (SAX) cartridge. Di N ‐acetylhomocystine was simultaneously purified and desalted on a C 18 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 p K a (SH) values of importance for biological studies. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.