Fluorogenic and fluorescent labeling reagents having a benzofurazan (2,1,3-benzoxadiazole) skeleton such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), 4-N,N-dimethylaminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole (DBD-H), 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ), 4-N,N-dimethylaminosulfonyl-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ), 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) are reviewed in terms of synthetic method, reactivity, fluorescence characteristics, sensitivity and application to analytes.

We have elucidated the relationship between the fluorescence quantum yield (Φ) of 4-monosubstituted benzofurazan (2,1,3-benzoxadiazole) compounds and the substituent group at the 4-position using a group of eleven such compounds. The absorption and fluorescence spectra of these compounds were measured in fifteen different solvents. Semi-empirical PM3 calculations were carried out to obtain the optimized geometry, energies, charges and dipole moments of this group in the ground and excited states. The S 1 -T 2 energy gaps obtained from the PM3-CAS/CI calculations, which reflected the probability of S 1 →T 2 intersystem crossing, related well to the fluorescence quantum yield (Φ) of the 4-monosubstituted benzofurazan compounds in nonpolar solvents. It also appeared that the decrease in the S 1 -T 1 energy gaps was related to the change in the fluorescence quantum yield (Φ) due to solvent effects. In highly polar solvents, non-radiative S 1 →T 1 intersystem crossing occurs, in addition to non-radiative S 1 →T 2 intersystem crossing, in 4-monosubstituted benzofurazan compounds, as they have a larger dipole moment in the S 1 state than in the T 2 state. These studies have enabled us to predict the fluorescence quantum yield (Φ) for a series of 4-monosubstituted benzofurazan compounds.

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An automated fluorescence protein sequencer using 7-methylthio-5-(2,1,3-benzoxadiazolyl) isithiocyanate (MTBD-NCS), a fluorescent Edman reagent, is developed by the modification of a commercial protein sequencer. The generated MTBD-thiohydantoin amino acids fluoresced strongly, whereas the by-products such as MTBD-thiocarbamoyl amino acids and MTBD-carbamoly amino acids did not fluoresce. A few interfering peaks were observed in the chromatogram and amino acid sequence was easily determined. The coupling and cyclization/cleavage reaction conditions and extraction conditions of generated MTBD-thiazolinone amino acids were optimized using an autonalyzer. Finally, the sequence of a synthetic peptide (25 pmol), leucine-enkephalin-Thr-amide, was determined and up to six residues were successively analyzed.

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