Development of Benzofurazan−bearing Fluorescence Labeling Reagents for Separation and Detection in High−performance Liquid Chromatography

Abstract

This review summarizes the synthesis, features and application of benzofurazan (i.e., 2,1,3−benzoxadiazole)−bearing fluorescence labeling reagents for the determination of biologically important molecules, such as biogenic amines, amino acids, carboxylic acids, thiols and drugs. Ammonium 4−fluoro−2,1,3−benzoxadiazole−7−sulfonate (SBD−F), 4−fluoro−7−aminosulfonyl−2,1,3−benzoxadiazole (ABD−F) and 4−fluoro−7−(N,N−dimethylaminosulfonyl)−2,1,3−benzoxadiazole (DBD−F) were synthesized from the investigation of various substituted groups at the 7−position of the 4−fluoro−2,1,3−benzoxadiazole. The fluorescence property of these derivatives was almost the same, but the reactivity and solubility were different for each reagent. The synthesized reagents were applied to the sensitive determination of biological thiols and amines, such as cysteine, homocysteine and cysteinylglycine in human plasma, glutathione in human blood cells, α−lipoic acid in animal tissues, and histamine and polyamines in hair. Furthermore, the chiral derivatization reagents, e.g., 4−(3−aminopyrrolidin−1−yl)−7−(N,N−dimethylaminosulfonyl)−2,1,3−benzoxadiazole (DBD−APy), 4−(3−aminopyrrolidin−1−yl)−7−aminosulfonyl−2,1,3−benzoxadiazole (ABD−APy), 4−(3−aminopyrrolidin−1−yl)−7−nitro−2,1,3−benzoxadiazole (NBD−APy), 4−(3−isothiocyanatopyrrolidin−1−yl)−7−(N,N−dimethylaminosulfonyl)−2,1,3−benzoxadiazole (DBD−PyNCS), 4−(3−isothiocyanatopyrrolidin−1−yl)−7−nitro−2,1,3−benzoxadiazole (NBD−PyNCS), 4−(2−chloroformylpyrrolidin−1−yl)−7−(N,N−dimethylaminosulfonyl)−2,1,3−benzoxadiazole (DBD−Pro−COCl), and 4−(2−chloroformylpyrrolidin−1−yl)−7−nitro−2,1,3−benzoxadiazole (NBD−Pro−COCl), were developed for the resolution of various chiral molecules, in terms of reactivity, separatability, handling easiness, sensitivity and selectivity. The chiral separation of various racemates was efficiently performed by reversed−phase chromatography after labeling with the chiral reagents. Some applications utilizing these reagents for the analyses of bioactive chiral compounds and drugs are also described in this paper.