Chemical Carboxy-Terminal Sequence Analysis of Peptides Using Acetyl Isothiocyanate

Abstract

A new derivatizing reagent, acetyl isothiocyanate (AITC), is applied for C-terminal peptide sequencing. It has been successfully used to sequence six C-terminal residues of a synthetic peptide at low nanomole levels. According to the mechanism study of the derivatization of C-terminal amino acid with various reagents, the derivatizing reagents (R-N=C=S or SCN−) were classified into three types: type I, the ionic compound (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, DPP-ITC). Type III reagents are superior to other reagents because their double-function of activation and derivatization. Unlike type I and type II reagents, type III reagent chemistry does not require oxazolinone formation which can cause racemization. Compared with benzoyl isothiocyanate, diphenyl phosphoroisothiocyanatidate, trimethylsilyl isothiocyanate, and ammonium thiocyanate, AITC is the most effective derivatizing reagent. As a type III reagent, AITC possesses some features such as no need for oxazolinone formation, no requirement for separate activation step, high reactivity, easy preparation, and low absorption at 260–270 nm. Different reaction conditions were investigated for optimization and the chemical mechanism of AITC chemistry is illustrated. A convenient and efficient approach for synthesis of amino acid thiohydantoins as reference standards has also been developed.