Latent pH-responsive ratiometric fluorescent cluster based on self-assembled photoactivated SNARF derivatives

We have developed a self-assembled fluorescent cluster comprising a seminaphthorhodafluor (SNARF) derivative protected by a photoremovable o-nitrobenzyl group. Prior to UV irradiation, a colorless and nonfluorescent cluster was spontaneously assembled in aqueous solution. After UV irradiation, the self-assembled cluster remained intact and showed a large enhancement in pH-responsive fluorescence. The unique pH responsive fluorescent cluster could be used as a dual-emissive ratiometric fluorescent pH probe not only in the test tube but also in HeLa cell cultures

低酸素細胞特異的な蛍光イメージング剤の開発

We have designed and evaluated UTX-12 as a novel fluorescent pH probe for tumor hypoxia imaging. UTX-12 consists of a p-nitro benzyl moiety, which is a latent hypoxia-selective leaving group activated by nitro reduction, directly linked to SNARF. Although UTX-12 itself is colorless and non-fluorescent in aqueous solution, nitro reduction triggers the release of SNARF which has well-characterized long wavelength absorption and fluorescence that is sensitive to pH. The resultant SNARF, released intracellularly by enzymatic reduction of UTX-12, allows measurement of pH by pH-dependent dual emission shifts. UTX-12 showed clear differences in fluorescence behavior between hypoxic and aerobic conditions in liver microsomes and inside V79 cells. These data are confirmation that UTX-12 is biologically reduced inside tumor cells and the released SNARF should monitor intracellular pH of tumor cells selectively with reduced background signal

改良型pＨプロ－ブによる細胞内pHの定量的な計測方法の開発

SNARF is one of the most commonly used pH indicators for biological applications, owing to its unique fluorescent characteristics. For intracellular applications, esterase-substrate derivatives of SNARF, such as acetate or acetoxymethyl esters, have been employed previously as a generally accepted strategy to increase cell permeability. Unfortunately such cell-permeable SNARF derivatives retain significant fluorescence in aqueous solution, a property which results in a low signal-to-noise ratio. This in turn can lead to incorrect intracellullar pH measurements. Here we describe UTX-40, a newly designed SNARF derivative that successfully addresses these problems. In aqueous solution, UTX-40 is devoid of fluorescence before ester hydrolysis because it exists in aqueous environment as nano-scaled aggregates. As UTX-40 is converted into SNARF by enzymatic hydrolysis inside the cell, the aggregates become diffused and monomeric SNARF displays its characteristic fluorescent properties. The results of our studies reported in this communication demonstrate clearly the benefits of UTX-40 as an intracellular pH indicator. Since intracellular localized fluorescence was observed without cell-washing, the efficient uptake of intracellular fluorescence was confirmed. In addition, the actual intracellular pH and changes in intracellular pH caused by drug addition were monitored. The low background noise produced by UTX-40 is a property of this new pH probe that should be particularly advantageous for in vivo usage because, for this application, it is difficult to wash out the redundant probe

Latent pH-responsive ratiometric fluorescent cluster based on self-assembled photoactivated SNARF derivatives

We have developed a self-assembled fluorescent cluster comprising a seminaphthorhodafluor (SNARF) derivative protected by a photoremovable o-nitrobenzyl group. Prior to UV irradiation, a colorless and nonfluorescent cluster was spontaneously assembled in aqueous solution. After UV irradiation, the self-assembled cluster remained intact and showed a large enhancement in pH-responsive fluorescence. The unique pH responsive fluorescent cluster could be used as a dual-emissive ratiometric fluorescent pH probe not only in the test tube but also in HeLa cell cultures