A fluorogenic reagent, 4-mercapto-7-methylthio-2,1,3-benzoxadiazole for carboxylic acids, designed by prediction of the fluorescence intensity

During the course of our studies of the development of fluorogenic reagents having a 4,7-disubstituted benzofurazan structure, we previously proposed 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH) as a fluorogenic re agent for carboxylic acids. Since then, progress has made it possible to es timate the fluorescence quantum yields of the 4,7-disubstituted benzofuraza n compounds on the basis of the PM3 calculation of their S-1-T-2 energies. Subsequently, a new fluorogenic :reagent, 4-mercapto-7-methylthio-2,1,3-ben zoxadiazole (MTBD-SH) was designed and synthesized. In the presence of cond ensation reagents, triphenylphosphine (TPP) and 2,2'-dipyridyl disulfide (D PDS), MTBD-SH readily reacted with n-caprylic acid within 1 min at room tem perature. The derivatives of five carboxylic acids (n-caprylic acid, n-capr ic acid, lauric acid, myristic acid, and palmitic acid) were well-separated on a reversed-phase column and were fluorimetrically detected at 519 nm wi th excitation at 391 mm. The detection limits (S/N = 3) were 2.4-5.0 fmol. Thus, MTBD-SH had properties that were considered to be superior. For carbo xylic acids, it was superior not only to AABD-SH, but also to many other co nventional reagents. The superiority was examined in terms of its reactivit y and sensitivity and the avoidance of interfering peaks that were derived from the reagent itself or degradation products in the chromatogram.