Thioacetylation method of protein sequencing: Derivatization of 2-methyl-5(4 H)-thiazolones for high-performance liquid chromatographic detection

Abstract

A reinvestigation of the thioacetylation method of protein sequencing ( G. A. Mross and R. F. Doolittle (1971) Fed. Proc. 30, 1241. G. A. Mross and R. F. Doolittle (1977) in Advanced Methods in Protein Sequence Determination (Needleman, S. B., Ed.), pp. 1–20, Springer, Berlin) has revealed that 2-methyl-5(4 H)-thiazolones, prepared by trifluoroacetic acid-catalyzed cleavage of the N-terminal amino acid from a N-thioacetylated polypeptide, were found to react instantaneously with one equivalent of carboxylic acid chloride, sulfonic acid chloride, or chloroformate to yield stable derivatives suitable for identification by high-performance liquid chromatography. NMR studies confirmed the products of the derivatization to be the corresponding 5- O-substituted-2-methylthiazoles. 2-Methyl-5(4 H)-thiazolones were derivatized by reaction with 3,5-dinitrobenzoyl chloride, 4-nitrophenylchloroformate, 4-nitrobenzenesulfonyl chloride, or 4- N-dimethylaminoazobenzene-4′-sulfonyl chloride (dabsyl chloride) in dichloromethane in the presence of triethylamine. Analytical standards were prepared by 1,3-dicyclohexylcarbodiimide-catalyzed cyclization of N-thioacetyl amino acids to 2-methyl-5(4 H)-thiazolones followed by derivatization with 4-nitrobenzenesulfonyl chloride. Stable crystalline 2-methyl-5- O-(4′-nitrobenzenesulfonyl)thiazole standards were obtained for 15 amino acids. Cysteine, serine, and threonine proved recalcitrant toward derivatization with 4-nitrobenzenesulfonyl chloride due to the dehydration of their respective thiazolones. Alkylated cysteine derivatives including ( S-β-(4-pyridylethyl)cysteine and S-ethylcysteine were derivatized without difficulty. Cyclization of N-thioacetylproline afforded a mesoionic compound which resisted derivatization, but could be detected directly. A preliminary high-performance liquid chromatographic separation was developed and the feasibility of this approach to protein sequencing demonstrated by solid-phase degradation of the oxidized insulin B chain. An average repetitive efficiency of 95% was obtained for 20 cycles of manual degradation. As compared to the products of the Edman degradation, 2-methyl-5- O-(4′-nitrobenzenesulfonyl)thiazole derivatives were obtained free of interferences arising during the coupling reaction.