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Fluorescence imaging is one of the most powerful techniques currently available for continuous observation of dynamic responses in living cells and animals. For highly efficient development of novel fluorescence probes, we have established several rational strategies for controlling the properties of visible light to NIR-excitable fluorophores based on the intramolecular photoinduced electron transfer. These strategies are quite powerful and versatile and, indeed, based on these strategies, we have succeeded to develop a series of fluorescence probes for reactive oxygen species, ions, and various enzymatic activities.

Recently, we have established another strategy to control the fluorescent properties of various fluorophores based on intramolecular spirocyclization. For example, if one of the amino groups of hydroxymethyl rhodaminegreen (HMRG), a new rhodaminegreen derivative bearing a hydroxymethyl group instead of the original carboxy group, is amidated with an amino acid, the resultant product is colorless in neutral pH buffer due to the preferred spirocyclization. This compound can be a highly sensitive fluorescence probe for the target aminopeptidase, because it is selectively reactive towards the target enzyme to yield highly fluorescent HMRG.

Based on these HMRG-based fluorescence probes, we have succeeded to establish a novel strategy for in vivo tumor imaging by utilizing upregulated enzymatic activities in cancer cells. Gamma-glutamyltranspeptidase (GGT) is well known to be significantly overexpressed in several human tumors, and we have developed a novel fluorescence probe (gGlu-HMRG) for GGT based on HMRG scaffold, which is quickly activated by GGT and yields an over 350-fold increase in fluorescence signal compared with the quenched state. gGlu-HMRG showed a large fluorescence increase in several cancer cell lines, but not in a normal cell line. Indeed, tumor cells in a mouse model of peritoneal metastases were successfully visualized with high signal contrast between the tumor and background. The activation occurred within 1 min of spraying the probe, and the signal was strong enough to be detected even with our naked eyes. This probe is believed to be practical for clinical application during surgical or endoscopic procedures.

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