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


Correspondence to: Ann English, Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke West, Montreal, Quebec, Canada H4B 1R6. E-mail: ann.english@concordia.ca


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.