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

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Abstract
On the basis of the relationship between the fluorescence characteristics of the benzofurazan compounds and the Hammett constants (sigma p), a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (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 sequence and D/L-configuration of amino acids in peptides. Boron trifluoride (BF3) and HC1/methanol were adopted as the cyclization/cleavage and conversion reagents to suppress the amino acid residue racemization. The MTBD-TH-amino acids were separated on a reversed-phase column for amino acid sequencing, and their enantiomers were resolved on two types of polysaccharide-based chiral stationary phases for D/L-configuration determination. The method was successfully applied to the sequence and D/L-configuration determination of D-amino acid-containing peptide [D-Ala2]-deltorphin II.

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