Article | Published:

Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics

Nature Chemistry volume 9, pages 279286 (2017) | Download Citation

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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 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.

  • Compound

    N-(6-(dimethylamino)-9-phenyl-3H-xanthen-3-ylidene)-N-methylmethanaminium

  • Compound

    N-(7-(dimethylamino)-5,5-dimethyl-10-phenyldibenzo[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium

  • Compound

    N-(7-(dimethylamino)-10-(2-fluorophenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium

  • Compound

    N-(10-(2-chlorophenyl)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium

  • Compound

    N-(7-(dimethylamino)-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium

  • Compound

    N-(7-(dimethylamino)-5,5-dimethyl-10-(2-(trifluoromethyl)phenyl)dibenzo[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium

  • Compound

    7-amino-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-iminium

  • Compound

    (E)-N-(7-amino-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-ylidene)methanaminium

  • Compound

    (E)-N-(5,5-dimethyl-7-(methylamino)-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-ylidene)methanaminium

  • Compound

    1,9,11,11-tetramethyl-5-(o-tolyl)-2,3,7,8,9,11-hexahydrosilino[3,2-f:5,6-f']diindol-1-ium

  • Compound

    N-(9-(4-(((1r,4r)-4-(4-(7-amino-3-iminio-5,5-dimethyl-3,5-dihydrodibenzo[o,e]silin-10-yl)-3-methylbenzamido)cyclohexyl)carbamoyl)-2-(methoxycarbonyl)phenyl)-6-(dimethylamino)-3H-xanthen-3-ylidene)-N-methylmethanaminium

  • Compound

    N-(9-(4-(((1r,4r)-4-(4-((E)-7-amino-5,5-dimethyl-3-(methyliminio)-3,5-dihydrodibenzo[o,e]silin-10-yl)-3-methylbenzamido)cyclohexyl)carbamoyl)-2-(methoxycarbonyl)phenyl)-6-(dimethylamino)-3H-xanthen-3-ylidene)-N-methylmethanaminium

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Change history

  • 13 December 2016

    In the version of this Article originally published, the y-axis of the middle panel in Fig. 1d was mislabelled. Molecular structures were also not present above Table 1. Both errors have been fixed in all versions of the Article.

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Acknowledgements

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.

Author information

Author notes

    • Keitaro Umezawa
    •  & Masafumi Yoshida

    These authors contributed equally to this work

Affiliations

  1. Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan

    • Keitaro Umezawa
    • , Masafumi Yoshida
    • , Mako Kamiya
    •  & Yasuteru Urano
  2. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan

    • Keitaro Umezawa
    •  & Yasuteru Urano
  3. 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

    • Masafumi Yoshida
    •  & Tatsuya Yamasoba
  4. PRESTO, Japan Science and Technology (JST) Agency, Honcho, Kawaguchi-shi, Saitama 332 0012, Japan

    • Mako Kamiya
  5. CREST, Japan Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100 0004, Japan

    • Yasuteru Urano

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Contributions

K.U., M.Y., M.K. and Y.U. designed the research. K.U. and M.Y. performed experiments and analysed the data. M.K., T.Y. and Y.U. supervised the project. All authors discussed the results and co-wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Mako Kamiya or Yasuteru Urano.

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DOI

https://doi.org/10.1038/nchem.2648

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