Development of an amino acid sequence and D/L configuration determination method of peptide with a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate

On the basis of the relationship between the fluorescence characteristics o f the benzofurazan compounds and the Hammett constants (sigma(p)), a new fl uorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocya nate (MTBD-NCS) was designed and synthesized. MTBD-thiohydantoin (TH)-amino acid derivatives produced by the Edman sequencing method gave fluorescence , whereas other degradation byproducts such as MTBD-thiocarbamoyl (TC)- or carbamoyl (CA)-amino acids did not fluoresce. MTBD-NCS was applicable as an Edman sequencing reagent to the simultaneous determination of both the seq uence and D/L-configuration of amino acids in peptides. Boron trifluoride ( BF3) and HCl/methanol were adopted as the cyclization/cleavage and conversi on reagents to suppress the amino acid residue racemization. The MTBD-TH-am ino acids were separated on a reversed-phase column for amino acid sequenci ng, and their enantiomers were resolved on two types of polysaccharide-base d chiral stationary phases for D/L-configuration determination. The method was successfully applied to the sequence and D/L-configuration determinatio n of D-amino acid-containing peptide [D-Ala(2)]-deltorphin II.