Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t1/2 = 620 ms at [GSH] = 1 mM), as well as appropriate Kd values (1–10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.
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This research was supported in part by AMED-CREST, by JST, PRESTO, by MEXT/JSPS KAKENHI grant numbers JP16H02606 and JP26111012 (to Y.U.), JP15H05951 ‘Resonance Bio’ (to M.K.), by JSPS Core-to-Core Program, by a grant from Hoansha Foundation (to Y.U.). The authors thank H. Takahashi for advices on intracellular imaging and statistical analysis, A. Morozumi for providing 2′Me SiR620, and Y. Kagami for advice on synthesis of Si-rhodamines.
The authors declare no competing financial interests.
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Umezawa, K., Yoshida, M., Kamiya, M. et al. Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics. Nature Chem 9, 279–286 (2017). https://doi.org/10.1038/nchem.2648
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