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

In our previous studies, we found a relationship between the fluorescence c haracteristics (the fluorescence intensity, the maximum excitation and emis sion wavelengths) and Hammett substituent constants (sigma p) of the substi tuent groups at the 4- and 7-positions of benzofurazan compounds, which ena bled us to predict the fluorescence characteristics of the benzofurazan com pounds. This prompted us to design a new fluorogenic derivatization reagent having a benzofurazan skeleton for carboxylic acids along this line of tho ught. Accordingly, the fluorogenic reagent, 7-acetylamino-4-mercapto-2,1,3- benzoxadiazole (AABD-SH) was synthesized. The reagent itself fluoresced ver y weakly but fluoresced strongly after the reaction with carboxylic acids. AABD-SH reacted with carboxylic acids within 5 min at room temperature in t he presence of triphenylphosphine and 2,2'-dipyridyl disulfide. The derivat ives of five carboxylic acids were separated on a reversed phase column and detected fluorimetrically at 524 nm with excitation at 368 nm. The detecti on limits were 10-20 fmol (signal-to-noise ratio of 3).