Detection of DBD-Carbamoyl Amino Acids in Amino Acid Sequence andd/lConfiguration Determination of Peptides with Fluorogenic Edman Reagent 7-[(N,N-Dimethylamino)sulfonyl]-2,1,3- benzoxadiazol-4-yl Isothiocyanate

Abstract A method for amino acid sequence and d/l configuration identification of peptides by using fluorogenic Edman reagent 7-[( N,N -dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF 3 , a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO 2 /H + to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their d/l configurations. Combination of the two HPLC systems, the amino acid sequence and d/l configuration of peptides could be determined. This method will be useful for searching d -amino-acid-containing peptides in animals.