Comparison of four fluorescence Edman reagents with benzofurazan structure for the detection of thiazolinone amino acid derivatives

Abstract

Two newly synthesized fluorescence Edman reagents with the benzofurazan structure, 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (PSBD-NCS) and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MSBD-NCS), were compared with 7-aminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (ABD-NCS) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) for peptide and protein sequence analysis by the generation of fluorescent 2,1,3-benzoxadiazolylthiazolinone (TZ)-amino acids. The effects of the substituent group at the para position to the isothiocyanate moiety of these reagents on the rate of the cyclization/cleavage reaction, the repetitive yield and the fluorescence quantum yield and stability of TZ amino acids were investigated. MSBD-TZ-amino acids were most sensitively detected and the detection limit for MSBD-TZ-Pro was 7 fmol (S/N = 3). ABD-NCS afforded the highest repetitive yield in the sequencing analysis. Fewer interfering peaks were observed in the chromatogram with DBD-NCS.