CHEMICAL CARBOXY-TERMINAL SEQUENCE-ANALYSIS OF PEPTIDES USING ACETYL ISOTHIOCYANATE

A new derivatizing reagent, acetyl isothiocyanate (AITC), is applied f or C-terminal peptide sequencing. It has been successfully used to seq uence six C-terminal residues of a synthetic peptide at low nanomole l evels. According to the mechanism study of the derivatization of C-ter minal amino acid with various reagents, the derivatizing reagents (R-N =C=S or SCN-) were classified into three types: type I, the ionic comp ound (e.g., HSCN, NH4SCN, KSCN); type II, R is a good leaving group (e .g., TMS-ITC, TBSn-ITC); type III, R is reactive (e.g., AITC, BITC, DP P-ITC). Type III reagents are superior to other reagents because their double-function of activation and derivatization. Unlike type I and t ype II reagents, type III reagent chemistry does not require oxazolino ne formation which can cause racemization, Compared with benzoyl isoth iocyanate, diphenyl phosphoroisothiocyanatidate, trimethylsilyl isothi ocyanate, and ammonium thiocyanate, AITC is the most effective derivat izing reagent, As a type III reagent, AITC possesses some features suc h as no need for oxazolinone formation, no requirement for separate ac tivation step, high reactivity, easy preparation, and low absorption a t 260-270 nm. Different reaction conditions were investigated for opti mization and the chemical mechanism of AITC chemistry is illustrated. A convenient and efficient approach for synthesis of amino acid thiohy dantoins as reference standards has also been developed. (C) 1997 Acad emic Press.