DEVELOPMENT OF NITRIC OXIDE FLUORESCENT PROBES FOR BIOIMAGING AND THEIR APPLICATION

我们开发了二氨基荧光素（daf），如DAF-2，作为一氧化氮（NO）的荧光指示剂。在好氧条件下，daf可以通过亚硝化和脱水捕获NO生成高荧光三唑荧光素（DAF-Ts）。然而，在将DAF-2用于血管内皮细胞NO成像的过程中，我们遇到了DAF-Ts荧光强度的pH依赖性问题，这使得在刺激改变细胞内pH水平后，很难监测到细胞内NO水平的微小变化。因为我们预料到了这个问题，所以我们合成了DAF的二氯衍生物。氯原子的电负性降低了邻酚羟基的pKa值。然而，由于它们在中性ph附近的荧光强度不稳定，因此出乎意料地不如DAF-2有用。这种不稳定性可能是由三唑质子引起的，因此我们引入了甲基。各项检测结果表明，DAF-4 M2对NO最敏感。三唑型daf - 4m2的荧光强度在pH 5.8以上稳定。据报道，氟化荧光素衍生物比荧光素具有更强的抗光漂白能力，可在共聚焦激光扫描显微镜用氩离子激光器的488 nm光谱线上有效激发。因此，我们制备了DAF-FM，其中DAF-4 M2的氯原子被氟原子取代。同时考察了pH值对DAF-FM T荧光强度的影响。从7 micromol DAF-FM标定的NO标准曲线来看，NO的检出限为3 nM。DAF-FM的灵敏度是DAF-2的1.4倍。据认为，这种灵敏度的提高是由于甲基的给电子作用使与NO+等价物的反应速率更高。对于NO的生物成像，我们合成了DAF-FM DA，它能很好地渗透到细胞中，并在胞浆中被酯酶迅速转化为水溶性的DAF-FM，并且染料能在胞浆中停留很长时间。荧光显微镜下应用DAF-FM DA指标测定体外培养牛主动脉内皮细胞NO含量。总之，我们认为DAF-FM是可视化细胞内NO时空分布的有用工具。少

We have developed diaminofluoresceins (DAFs), such as DAF-2, as fluorescent indicators for nitric oxide (NO). Under aerobic conditions, DAFs can trap NO to yield highly fluorescent triazolofluoresceins (DAF-Ts) by nitrosation and dehydaration. However, during the application of DAF-2 for NO imaging in vascular endothelial cells, we encountered the problem of pH dependency of the fluorescence intensity of DAF-Ts, which makes it difficult to monitor small changes in intracellular NO levels after a stimulus that shifts the intracellular pH level. Since we had anticipated this problem, we synthesized dichloro derivatives of DAF. The chlorine atoms lower the pKa value of the adjacent phenolic OH group due to their electronegativity. However, they were unexpectedly less useful than DAF-2 owing to their unstable fluorescence intensity around neutral pH. The instability may arise from a triazole proton, so we introduced a methyl group. As the resiults of various examinations, DAF-4 M2 was the … More most sensitive to NO. The fluorescence intensity of the triazole form of DAF-4 M2 was stable above pH 5.8.It was reported that fluorinated fluorescein derivatives are more resistant to photobleaching than fluorescein and can be efficiently excited with 488 nm spectral line of the argon ion laser used in confocal laser scanning microscopes. Therefore, we prepared DAF-FM, in which the chlorine atoms of DAF-4 M2 are replaced by fluorine atoms. Also, the effect of pH on the fluorescence intensity of DAF-FM T was examined. Judging from NO standard curves calibrated with 7 micromol DAF-FM, the detection limit of NO was 3 nM. The sensitivity of DAF-FM was 1.4 times higher than that of DAF-2. It is considered that this increase in sensitivity results from the higher rate of the reaction with NO+ equivalents due to the electron-donating effect of the methyl group.For the bioimaging of NO, we synthesized DAF-FM DA, which permeates well into cells and is quickly transformed into water-soluble DAF-FM by esterases in the cytosol, where the dye can remain for a long time. The indicator DAF-FM DA was applied to the imagine of NO in cultured bovine aortic endothelial cells by using a fluorescence microscope. In conclusion, we suggest that DAF-FM is a useful tool for visualizing the temporal and spatial distribution of intracellular NO. Less