Chem. Pharm. Bull. 53(7) 750—758 (2005)

for detecting organic and/or inorganic compounds and therefore it has been widely used in many scientific fields with improving analytical instruments such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), fluorescence confocal microscopy, etc. For these purposes, a great deal of effort has gone into the development of fluorescent reagents using various fluorophores, which can be utilized for derivatizing biologically active small molecules, labeling macromolecules such as proteins and DNAs, and probing ions such as biologically relevant metal cations and inorganic or organic anions. In the development of such fluorescent reagents, it should first be ensured that the fluorophore used has a high fluorescence quantum yield because of the reliably high sensitivity in the detection. Fluorescein is one of the most widely used fluorophore for biological experiments, since it has a high fluorescence quantum yield of 0.90 or grater in aqueous solution and its excitation and emission wavelengths are in the visible region. Recently, Nagano and coworkers have succeeded in developing fluorescent reagents for the specific detection of nitric oxide (NO), singlet oxygen (O2), 9,10) and others using fluorescein as the fluorophore. 4,4-Difluoro-4-bora-3a,4a-diazas-indacenes (BODIPYs) are also well-known fluorophores that have very sharp and narrow fluorescence bandwidths in addition to their high fluorescence quantum yield in aqueous solution. Therefore they have been used as mother fluorophores for many analytical purposes. However, the difference between the excitation and emission wavelengths of these derivatives are very small, less than approximately 30 nm for fluoresceins and 15 nm for BODIPYs, and it is necessary to correct their spectra from interferences such as Rayleigh or Raman scattering light. On the other hand, coumarin (2H-benzopyran-2-one), is also another interesting fluorophore, since its fluorescence changes drastically with substituents and their introduced positions. In previous papers, we reported the fluorescence characteristics of methoxycoumarins and discussed the structural features of strongly fluorescing methoxycoumarins from the viewpoint of intramolecular charge transfer (ICT) between pushand pull-substituents in the ground and the excited states. Based on the above findings, we succeeded in developing novel fluorescent reagents with both high sensitivity and functions such as self-catalytic reactivity. In the course of those studies, we suspected that most coumarin derivatives showed large Stokes shifts in their fluorescence spectra as compared with other fluorophores. These large Stokes shifts appear to be advantageous for escaping autofluorescence from biological molecules and reducing the self-absorption of chromophores. Further, if the emission bands of coumarins could be shifted to longer wavelengths with large Stokes shifts, analytical detection limits would markedly increase because of the clearing of interferences off and higher signal-to–noise (S/N) ratio. With this in mind, we began to design novel coumarin fluorophores that can fluoresce in the longer wavelength region while considering the relationship between the chemical structure of coumarins and their fluorescence properties. Here, we report the synthesis and the fluorescence properties of 6-methoxycoumarin and benzocoumarin (naphtopyranone) derivatives as candidate novel fluorophores emitting in the longer wavelength region, and the preliminary investigation of their application to fluorescence derivatization reagents using synthesized fluorophores as an example.