title: Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics.
abstract: 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 Kdvalues (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.
authors.affiliation: Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan.|Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Umezawa
authors.affiliation: Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan.|Department of Otolaryngology - Head and Neck Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yoshida
authors.affiliation: Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan.|PRESTO, Japan Science and Technology (JST) Agency, Honcho, Kawaguchi-shi, Saitama 332 0012, Japan.
authors.forename: Mako
authors.initials: M
authors.lastname: Kamiya
authors.affiliation: Department of Otolaryngology - Head and Neck Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Yamasoba
authors.affiliation: Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan.|Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan.|CREST, Japan Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100 0004, Japan.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
mesh_terms: D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D005978:Glutathione / Q000032:analysis* / Q000737:chemistry; D006207:Half-Life; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D012235:Rhodamines / Q000737:chemistry; D012825:Silicon / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; D006861:Hydrogen Peroxide; D005978:Glutathione; D012825:Silicon