Evaluation of benzofurazan derivatives as fluorogenic reagents for thiols and amines using high-performance liquid chromatography

Abstract

The reactivities of three reagents, 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and 4-(fluorosulphonyl)-7-phenoxy-2,1,3-benzoxadiazole (PBD-SO₂F), towards thiols and amines were compared. All three reacted with thiol compounds to give derivatives which fluoresced at ca. 515 nm (excitation at ca. 388 nm). However, the PBD-SO₂ derivatives were not very stable. The rate of reaction of DBD-F with proline was faster than that of PBD-SO₂F or ABD-F. The three reagents and their hydrolysates exhibited extremely low fluorescence. The fluorescence intensities of the proline derivatives synthesised were higher in neutral and acidic solutions than in alkaline solution. The strength of the fluorescence intensities was in the order PBD-SO₂-proline > DBD-proline > ABD-proline at all pH values tested. The mean values of the maximum wavelengths of DBD-proline, ABD-proline and PBD-SO₂-proline were 596 nm (λ_ex. = 466 nm), 602 nm (λ_ex. = 466 nm) and 566 nm (λ_ex. 453 nm), respectively. Of the three reagents, DBD-F seemed to be applicable to the determination of amino acids. Nine amino acid derivatives of DBD-F were separated by reversed-phase high-performance liquid chromatography and detected with fluorescence at 590 nm (λ_ex. = 450 nm). The detection limits were in the sub-picomole range.