Rapid high‐yield N‐acylation of aminothiols: N‐acetylglutathione and N‐acetylhomocysteine and their thiol pK_a values

Abstract

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 pK(a) (SH) values of importance for biological studies.

Context‐Dependence of the Reactivity of Cysteine and Lysine Residues

“…We found that thiol 2 has p K a =8.88(1) (Figure 2). Thiol 3 is less acidic with p K a =9.45(3), which is indistinguishable from the value obtained with potentiometric titrations [21,22] . The sulfhydryl group of thiol 1 has microscopic p K a values that depend on the protonation state of its amino group, [23] giving four relevant species, 1 a – 1 d (Scheme 1).…”

“…Thiol 3 is less acidic with pK a = 9.45(3), which is indistinguishable from the value obtained with potentiometric titrations. [21,22] The sulfhydryl group of thiol 1 has microscopic pK a values that depend on the protonation state of its amino group, [23] giving four relevant species, 1 a-1 d (Scheme 1). Applying a model that accounts for the interdependence of the sulfhydryl and ammonium pK a values (Equa-tion S1), we found that pK a SH,1 = 7.57 (20) and pK a SH,2 = 9.15(3) (Figure 2).…”

Practical and Selective Syntheses of S‐Acyl and N‐Acyl Glutathiones with N‐Acyl Imidazoles in Water

“…Conversely, reported methods to synthesize derivatives of N ‐acyl glutathiones remain underdeveloped and require smart manipulation of protecting group chemistry due to the greater reactivity of thiols over amines under standard reaction conditions [5] . That said, the following methods are worth mentioning: (a) N ‐acylation of glutathione disulfide (GSSG) with an activated ester followed by S−S reduction; [5a] (b) use of a trityl group to protect the thiol group of GSH, then N ‐acylation followed by trityl group deprotection; [5b] (c) use of 4‐nitrobenzoyl to protect the thiol group of GSH, then N ‐acylation with 1‐acyl‐1 H ‐benzotriazole followed by deprotection using pyrrolidine in dry tetrahydrofuran (THF)‐methanol for 4 h; [4c] and (d) a two‐step synthesis of N ‐butanoyl‐GSH using butyric anhydride, followed by selective thioester methanolysis of the S,N ‐dibutanoylated product [5c] . However, these methods have strong limitations for large‐scale applications because the reagents are highly corrosive and toxic, environmentally damaging solvents are necessary, and difficulties in the purification of the final S ‐ and N ‐acyl glutathiones are usually encountered.…”

An Improved Process for Preparation of S-Acetyl-l-glutathione

“…Previous studies have demonstrated that supplementation with a glutathione pro-drug, such as N -acetylcysteine (NAC), S -acetylcysteine (SAC), N -acetylglutathione ( N -GSH), and S -acetylglutathione ( S -GSH), is more efficient than GSH for intracellular GSH supplementation . However, N -acetyl derivatives may have neurotoxic effects on the nerves for patients with glutathione synthetase deficiency …”