A new fluorogenic reagent for carboxylic acids, 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH), derived from an empirical method for predicting fluorescence characteristics

Abstract

In our previous studies, we found a relationship between the fluorescence characteristics (the fluorescence intensity, the maximum excitation and emission wavelengths) and Hammett substituent constants (ςp) of the substituent groups at the 4- and 7-positions of benzofurazan compounds, which enabled us to predict the fluorescence characteristics of the benzofurazan compounds. This prompted us to design a new fluorogenic derivatization reagent having a benzofurazan skeleton for carboxylic acids along this line of thought. Accordingly, the fluorogenic reagent, 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH) was synthesized. The reagent itself fluoresced very weakly but fluoresced strongly after the reaction with carboxylic acids. AABD-SH reacted with carboxylic acids within 5 min at room temperature in the presence of triphenylphosphine and 2,2′-dipyridyl disulfide. The derivatives of five carboxylic acids were separated on a reversed phase column and detected fluorimetrically at 524 nm with excitation at 368 nm. The detection limits were 10–20 fmol (signal-to-noise ratio of 3).