Semi-automatic amino acid sequencing and D/L-configuration determination of peptides with detection of liberated N-terminal phenylthiocarbamoylamino acids

Abstract

A semi-automatic method for the simultaneous determination of the sequence and the D/L-configuration of amino acids in peptides is reported. The automated sequencing program was set up based both on the yield in the cyclization/cleavage reaction with 40 mmol dm^–3 boron trifluoride (BF₃) etherate at 48 °C for 5 min and on the recovery of anilinothiazolinone- (ATZ-) amino acids extracted with ethyl acetate and 1-chlorobutane. The D/L-configuration of phenylthiocarbamoylamino acids was determined manually after hydrolysis of ATZ-amino acids by HPLC using a combination of a reversed phase and a chiral stationary phase of β-cyclodextrin. The hydrolysis conditions for ATZ-amino acids were examined with HCl to suppress the amino acid racemization, and 0.1 mol dm^–3 HCl at room temperature for 5 min was selected for hydrolysis. The sequence and D/L-configuration determination of a synthetic β-amyloid 1–40 peptide was carried out and up to 10 residues could be successfully analyzed by the semi-automated sequencing procedure without racemization.