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(4H)-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(4H)-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(4H)-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-beta-(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.(ABSTRACT TRUNCATED AT 250 WORDS)