Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Here we show that the carboxylic group of traditional fluorescein dyes, formerly considered indispensable, has been replaced with other substituents, affording various kinds of new fluoresceins. Further, by breaking out of the traditional structure of fluorescein, we developed the first and totally rational design strategy for novel fluorescence probes based on a strict photochemical basis. The value of this approach is exemplified by its application to develop a novel, highly sensitive, and membrane-permeable fluorescence probe for beta-galactosidase, which is the most widely used reporter enzyme.
Hydrogen sulfide (H(2)S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H(2)S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry to control the fluorescence. HSip-1 showed high selectivity and high sensitivity for H(2)S, and its potential for biological applications was confirmed by employing it for fluorescence imaging of H(2)S in live cells.
Rational design strategies based on practical fluorescence modulation mechanisms would enable us to rapidly develop novel fluorescence probes for target molecules. Here, we present a practical and general principle for modulating the fluorescence properties of fluorescein. We hypothesized that (a) the fluorescein molecule can be divided into two moieties, i.e., the xanthene moiety as a fluorophore and the benzene moiety as a fluorescence-controlling moiety, even though there is no obvious linker structure between them, and (b) the fluorescence properties can be modulated via a photoinduced electron transfer (PeT) process from the excited fluorophore to a reducible benzene moiety (donor-excited PeT; d-PeT). To evaluate the relationship between the reduction potential of the benzene moiety and the fluorescence properties, we designed and synthesized various derivatives in which the reduction potential of the benzene moiety was fine tuned by introducing electron-withdrawing groups onto the benzene moiety. Our results clearly show that the fluorescence properties of fluorescein derivatives were indeed finely modulated depending upon the reduction potential of the benzene moiety. This information provides a basis for a practical strategy for rational design of novel functional fluorescence probes.
Nitrative stress is implicated in various pathogenic processes, including neurodegenerative disorders, but there is no practical fluorescence probe which can monitor the generation of nitrative stress with high selectivity. To design a suitable fluorescence probe, we have first focused on the fluorescence quenching mechanism of the nitro group, which has been believed to be a unique quencher of fluorescent dyes. We found that nitro group-based fluorescence quenching could be explained in terms of an electron transfer process, from the excited fluorophore to the electron-deficient aromatic nitro moiety. By utilizing this result, we succeeded in developing novel fluorogenic probes, NiSPYs, which can selectively monitor the generation of nitrative stress based on aromatic nitration. NiSPYs showed strong fluorescence enhancement upon the reaction with nitrating agents, including peroxynitrite, but showed little or no fluorescence augmentation in the presence of other reactive oxygen species. NiSPYs should be potentially useful as tools to study the role of nitrative stress in various biological applications.
Using a newly synthesized gibberellin analog (GA3-AM) and its binding proteins, we developed a novel and efficient chemically induced dimerization (CID) system, that is completely orthogonal to the existing rapamycin-mediated protein dimerization. Combining the two systems should allow applications that were difficult or impossible with only one CID system. By using both chemical inputs (rapamycin and GA3-AM), we designed and synthesized Boolean logic gates in living mammalian cells. These gates produced output signals such as fluorescence and membrane ruffling on a timescale of seconds, a significant improvement over previous intracellular logic gates. The use of two orthogonal dimerization systems in the same cell also allows for finer modulation of protein perturbations than is possible with a single dimerizer.
Let it shine: New hypoxia-sensitive fluorescent probes were developed; they consist of a rhodamine moiety with an azo group directly conjugated to the fluorophore. Because of an ultrafast conformational change around the NN bond, the compounds are nonfluorescent under normoxia. However, under hypoxia, the azo group is reduced, and a strongly fluorescent rhodamine derivative is released.
Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (O), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate O only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to O generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of O to occur. In PDT in vitro, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azo-based strategy should be widely applicable to design a range of activatable photosensitizers.
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