Effects of the substituent groups at the 4- and 7-positions on the fluorescence characteristics of benzofurazan compounds

To develop new fluorogenic reagents having the benzofurazan structure, we investigated the effects of the substituent groups at the 4- and 7-positions of the benzofurazan skeleton on the fluorescence characteristics (fluorescence intensity, maximum excitation wavelength and maximum emission wavelength). Seventy benzofurazan compounds substituted at the 4- and 7-positions were obtained for this purpose. The Hammett substituent constant (sigma(p)) was adopted as a parameter for electronic effects by substituent groups. The study using the sum and the difference of the Hammett substituent constants (sigma(p)) at the 4- and 7-positions revealed that the highly fluorescent benzofurazan compounds were classified into two groups and that singlet excitation energies, calculated by the maximum excitation and emission wavelengths, of the benzofurazan compounds were different between these two groups, The fluorescence characteristics of benzofurazan compounds substituted at the 4- and 7-positions were empirically predictable by these relationships and sigma(p) values. A new fluorogenic reagent, 4-phenylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole, for amines was developed based on this method and applied to the amino acids analysis.