Development of water-soluble fluorogenic reagents having a 2,1,3-benzoxadiazole structure and their application to the determination of peptides

Abstract

Water-soluble fluorogenic reagents having a 2,1,3-benzoxadiazole (benzofurazan) structure, 2-(7-fluoro-2,1,3-benzoxadiazole-4-sulfonamido)ethanesulfonic acid (ES-ABD-F), 4-(7-fluoro-2,1,3-benzoxadiazole-4-sulfonamido)benzenesulfonic acid (p-BS-ABD-F) and 3-(7-fluoro-2,1,3-benzoxadiazole-4-sulfonamido)benzenesulfonic acid (m-BS-ABD-F), were synthesized and evaluated for their fluorescence characteristics and reactivities with peptides. Although these reagents showed no fluorescence, their derivatives with valine exhibited fluorescence at maximum wavelengths from 562 to 566 nm with excitation from 424 to 426 nm. The derivatives with ES-ABD-F and m-BS-ABD-F showed a similar sensitivity to that of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), a previously developed fluorogenic reagent for amino compounds. The fluorescence intensity obtained with the p-BS-ABD-F derivative was about one-third of that of the others. Angiotensin II (ANG II), an octapeptide, was successfully derivatized with ES-ABD-F and m-BS-ABD-F and determined by using high performance liquid chromatography (HPLC) with fluorescence detection. Good linearity was obtained between the amount of peptide (1–200 pmol) and the peak area of their derivatives (correlation coefficients >0.999). The detection limits of the ANG II derivatives with ES-ABD-F and m-BS-ABD-F were 210 and 270 fmol, respectively. m-BS-ABD-F, however, showed some disadvantages in the simultaneous determination of peptides: a possible interference with the separation of derivatized peptides due to some blank peaks, and its complicated and changeable pH-reactivity profile. From these results, ES-ABD-F was recommended for the sensitive and simultaneous determination of peptides. The derivatization reaction with ES-ABD-F commonly reached a plateau at 60 °C and pH > 8.5 for several hours. The peptide derivatives of ES-ABD-F (ANG I, ANG II, ANG III, bradykinin, and substance P) were well separated by reversed phase HPLC. In addition, the amyloid β-peptide (1–40) [Aβ(1–40)] derivative with ES-ABD-F was successfully eluted from a YMC-pack C4 (150 × 4.6 mm id, 5 μm particle size) under conditions such that underivatized Aβ(1–40) was not eluted, suggesting that the derivatization with ES-ABD-F decreased the adsorption of hydrophobic peptides to the HPLC stationary phase.