Nitric oxide is a gaseous, free radical which plays a role as an intracellular second messenger and a diffusable intercellular messenger. To obtain direct evidence for NO functions in vivo, we have designed and synthesized diaminofluoresceins (DAFs) as novel fluorescent indicators for NO. The fluorescent chemical transformation of DAFs is based on the reactivity of the aromatic vicinal diamines with NO in the presence of dioxygen. The N-nitrosation of DAFs, yielding the highly green-fluorescent triazole form, offers the advantages of specificity, sensitivity, and a simple protocol for the direct detection of NO (detection limit 5 nM). The fluorescence quantum efficiencies are increased more than 100 times after the transformation of DAFs by NO. Fluorescence detection with visible light excitation and high sensitivity enabled the practical assay of NO production in living cells. Membrane-permeable DAF-2 diacetate (DAF-2 DA) can be used for real-time bioimaging of NO with fine temporal and spatial resolution. The dye was loaded into activated rat aortic smooth muscle cells, where the ester bonds are hydrolyzed by intracellular esterase, generating DAF-2. The fluorescence in the cells increased in a NO concentration-dependent manner.

The membrane-permeating indicator DAF-FM DA is transformed by intracellular esterases into the highly water-soluble dye DAF-FM, which traps NO produced by NO synthase (NOS) to yield a highly fluorescent triazole compound in cells (see schematic diagram). Monitoring with a fluorescence microscope should allow direct identification of intracellular production and location of NO.

Boron dipyrromethene (BODIPY) is known to have a high quantum yield (phi) of fluorescence in aqueous solution but has not been utilized much for biological applications, compared to fluorescein. We developed 8-(3,4-diaminophenyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (DAMBO-P(H)), based on the BODIPY chromophore, as a highly sensitive fluorescence probe for nitric oxide (NO). DAMBO-P(H) had a low phi value of 0.002, whereas its triazole derivative (DAMBO-P(H)-T), the product of the reaction of DAMBO-P(H) with NO, fluoresced strongly (phi = 0.74). The change of the fluorescence intensity was found to be controlled by an intramolecular photoinduced electron transfer (PeT) mechanism. The strategy for development of DAMBO-P(H) was as follows: (1) in order to design a highly sensitive probe of NO, the reactivity of o-phenylenediamine derivatives as NO-reactive moieties was examined using 4,5-diaminofluorescein (DAF-2, a widely used NO fluorescence probe), (2) in order to avoid pH-dependency of the fluorescence intensity, the PeT process was controlled by modulating the spectroscopic and electrochemical properties of BODIPY chromophores according to the Rehm-Weller equation based on measurement of excitation energies of chromophores, ground-state reduction potentials of PeT acceptors (BODIPYs), and calculation of the HOMO energy level of the PeT donor (o-phenylenediamine moiety) at the B3LYP/6-31G level, (3) in order to avoid quenching of fluorescence by stacking of the probes and to obtain probes suitable for biological applications, hydrophilic functional groups were introduced. This strategy should be applicable for the rational design of other novel and potentially useful bioimaging fluorescence probes.

We systematically examined the mechanism of the solvent polarity dependence of the fluorescence ON/OFF threshold of the BODIPY (boron dipyrromethene) fluorophore and the role of photoinduced electron transfer (PeT). In a series of BODIPY derivatives with variously substituted benzene moieties at the 8-position, the oxidation potential of the benzene moiety became more positive and the reduction potential of the BODIPY fluorophore became more negative as the solvent polarity was decreased; consequently, the free energy change of PeT from the benzene moiety becomes larger in a more nonpolar environment. Utilizing this finding, we designed and synthesized a library of probes in which the threshold of fluorescence ON/OFF switching corresponds to different levels of solvent polarity. These environment-sensitive probes were used to examine bovine serum albumin (BSA) and living cells. The polarity at the surface of albumin was concluded to be similar to that of acetone, while the polarity of the internal membranes of HeLa cells was similar to that of dichloromethane.

The measurement of nitric oxide (NO) is important for direct examination of the regulatory roles of NO in various biological systems. Diaminofluoresceins (DAFs), new fluorescence indicators for NO, were applied to detect the release of NO from bovine aortic endothelial cells (ECs). DAFs react with NO to yield the corresponding green-fluorescent triazolofluoresceins, which provide the advantages of specificity, sensitivity and a simple protocol for the direct detection of NO. Using these DAFs, we could detect the generation of NO not only from inducible NO synthase expressed in macrophages, but also from constitutive NO synthase expressed in ECs.z 1998 Federation of European Biochemical Societies.