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

This article has been updated

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Si-rhodamine as a candidate for rapid and reversible detection of GSH.
Figure 2: Properties of the QuicGSH probes (buffer: 0.2 M sodium phosphate buffer, pH 7.4, containing 5% DMSO).
Figure 3: Quantification of GSH in several cell lines.
Figure 4: Real-time GSH imaging with QG3.0 (1 μM) in A549 cells upon H2O2 treatment with a perfusion system.
Figure 5: Real-time GSH imaging with QG3.0 (1 μM) in A549 cells under glucose-deprivation with a perfusion system.

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.

References

  1. 1

    Meister, A. & Anderson, M. E. Glutathione. Annu. Rev. Biochem. 52, 711–760 (1983).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2

    Wu, G., Fang, Y.-Z., Yang, S., Lupton, J. R. & Turner, N. D. Glutathione metabolism and its implications for health. J. Nutr. 134, 489–492 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3

    Townsend, D. M., Tew, K. D. & Tapiero, H. The importance of glutathione in human disease. Biomed. Pharmacother. 57, 145–155 (2003).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4

    Balendiran, G. K., Dabur, R. & Fraser, D. The role of glutathione in cancer. Cell Biochem. Fucnt. 22, 343–352 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Estrela, J. M., Ortega, A. & Obrador, E. Glutathione in cancer biology and therapy. Crit. Rev. Clin. Lab. Sci. 43, 143–181 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6

    Ishimoto, T. et al. CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc and thereby promotes tumor growth. Cancer Cell 19, 387–400 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. 7

    Fierro, S. et al. In vivo assessment of cancerous tumors using boron doped diamond microelectrode. Sci. Rep. 2, 901 (2012).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. 8

    Chen, X., Zhou, Y., Peng, X. & Yoon, J. Fluorescent and colorimetric probes for detection of thiols. Chem. Soc. Rev. 39, 2120–2135 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. 9

    Kim, G.-J., Lee, K., Kwon, H. & Kim, H.-J. Ratiometric fluorescence imaging of cellular glutathione. Org. Lett. 13, 2799–2801 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10

    Niu, L.-Y. et al. BODIPY-based ratiometric fluorescent sensor for highly selective detection of glutathione over cysteine and homocysteine. J. Am. Chem. Soc. 134, 18928–18931 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11

    Yin, J. et al. Cyanine-based fluorescent probe for highly selective detection of glutathione in cell cultures and live mouse tissues. J. Am. Chem. Soc. 136, 5351–5358 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12

    Lim, S. Y., Hong, K.-H., Kim, D. I., Kwon, H. & Kim, H.-J. Tunable heptamethine–azo dye conjugate as an NIR fluorescent probe for the selective detection of mitochondrial glutathione over cysteine and homocysteine. J. Am. Chem. Soc. 136, 7018–7025 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13

    Yoshida, M., Kamiya, M., Yamasoba, T. & Urano, Y. A highly sensitive, cell-membrane-permeable fluorescent probe for glutathione. Bioorg. Med. Chem. Lett. 24, 4363–4366 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14

    Ahn, Y. H., Lee, J. S. & Chang, Y. T. Combinatorial rosamine library and application to in vivo glutathione probe. J. Am. Chem. Soc. 129, 4510–4511 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15

    Xu, K. et al. A near-infrared reversible fluorescent probe for real-time imaging of redox status changes in vivo. Chem. Sci. 4, 1079–1086 (2013).

    CAS  Article  Google Scholar 

  16. 16

    Chen, J., Jiang, X., Carroll, S. L., Huang, J. & Wang, J. Theoretical and experimental investigation of thermodynamics and kinetics of thiol-Michael addition reactions: a case study of reversible fluorescent probes for glutathione imaging in single cells. Org. Lett. 17, 5978–5981 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17

    Jiang, X. et al. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe. ACS Chem. Biol. 10, 864–874 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18

    Kenmoku, S., Urano, Y., Kojima, H. & Nagano, T. Development of a highly specific rhodamine-based fluorescence probe for hypochlorous acid and its application to real-time imaging of phagocytosis. J. Am. Chem. Soc. 129, 7313–7318 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. 19

    Sakabe, M. et al. Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization. J. Am. Chem. Soc. 135, 409–414 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. 20

    Uno, S. et al. A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging. Nat. Chem. 6, 681–689 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21

    Mayr, H. Reactivity scales for quantifying polar organic reactivity: The benzhydrylium methodology. Tetrahedron 71, 5095–5111 (2015).

    CAS  Article  Google Scholar 

  22. 22

    Appel, R. & Mayr, H. Quantification of the electrophilic reactivities of aldehydes, imines, and enones. J. Am. Chem. Soc. 133, 8240–8251 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23

    Mayr, H. et al. Reference scales for the characterization of cationic electrophiles and neutral nucleophiles. J. Am. Chem. Soc. 123, 9500–9512 (2001).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24

    Lavis, L. D. & Raines, R. T. Bright building blocks for chemical biology. ACS Chem. Biol. 9, 855–866 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25

    Umezawa, K., Citterio, D. & Suzuki, K. New trends in near-infrared fluorophores for bioimaging. Anal. Sci. 30, 327–349 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26

    Kushida, Y., Nagano, T. & Hanaoka, K. Silicon-substituted xanthene dyes and their applications to bioimaging. Analyst 140, 685–695 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27

    Koide, Y., Urano, Y., Hanaoka, K., Terai, T. & Nagano, T. Evolution of group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer. ACS Chem. Biol. 6, 600–608 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28

    Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  29. 29

    Fu, M. Y., Xiao, Y., Qian, X. H., Zhao, D. F. & Xu, Y. F. A design concept of long-wavelength fluorescent analogs of rhodamine dyes: replacement of oxygen with silicon atom. Chem. Commun. 1780–1782 (2008).

  30. 30

    Streidl, N., Denegri, B., Kronja, O. & Mayr, H. A practical guide for estimating rates of heterolysis reactions. Acc. Chem. Res. 43, 1537–1549 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  31. 31

    Mayr, H. et al. Scales of Lewis basicities toward C-centered Lewis acids (carbocations). J. Am. Chem. Soc. 137, 2580–2599 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32

    Marí, M., Morales, A., Colell, A., García-Ruiz, C. & Fernández-Checa, J. C. Mitochondrial glutathione, a key survival antioxidant. Antioxid. Redox Signal. 11, 2685–2700 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  33. 33

    Pompella, A., De Tata, V., Paolicchi, A. & Zunino, F. Expression of γ-glutamyltransferase in cancer cells and its significance in drug resistance. Biochem. Pharmacol. 71, 231–238 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34

    Urano, Y. et al. Rapid cancer detection by topically spraying a γ-glutamyltranspeptidase–activated fluorescent probe. Sci. Transl. Med. 3, 110ra119 (2011).

    Article  CAS  Google Scholar 

  35. 35

    Hino, H. et al. Rapid cancer fluorescence imaging using a γ-glutamyltranspeptidase-specific probe for primary lung cancer. Transl. Oncol. 9, 203–210 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  36. 36

    Anderson, M. E., Powrie, F., Puri, R. N. & Meister, A. Glutathione monoethyl ester: Preparation, uptake by tissues, and conversion to glutathione. Arch. Biochem. Biophys. 239, 538–548 (1985).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. 37

    Schirmer, R. H., Müller, J. G. & Krauth-Siegel, R. L. Disulfide-reductase inhibitors as chemotherapeutic agents: the design of drugs for trypanosomiasis and malaria. Angew. Chem. Int. Ed. 34, 141–154 (1995).

    CAS  Article  Google Scholar 

  38. 38

    Zhao, Y. et al. Increase in thiol oxidative stress via glutathione reductase inhibition as a novel approach to enhance cancer sensitivity to X-ray irradiation. Free Radical Biol. Med. 47, 176–183 (2009).

    CAS  Article  Google Scholar 

  39. 39

    Ahmad, I. M. et al. Mitochondrial and H2O2 mediate glucose deprivation-induced stress in human cancer cells. J. Biol. Chem. 280, 4254–4263 (2005).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40

    Aykin-Burns, N., Ahmad, I. M., Zhu, Y., Oberley, L. W. & Spitz, D. R. Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer cells versus normal cells to glucose deprivation. Biochem. J. 418, 29–37 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41

    Seebacher, N. A., Richardson, D. R. & Jansson, P. J. Glucose modulation induces reactive oxygen species and increases P-glycoprotein-mediated multidrug resistance to chemotherapeutics. Br. J. Pharmacol. 172, 2557–2572 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42

    Gutscher, M. et al. Real-time imaging of the intracellular glutathione redox potential. Nat. Methods 5, 553–559 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43

    Nordberg, J. & Arnér, E. S. J. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radical Biol. Med. 31, 1287–1312 (2001).

    CAS  Article  Google Scholar 

  44. 44

    Darby Weydert, C. J. et al. Inhibition of oral cancer cell growth by adenovirusMnSOD plus BCNU treatment. Free Radical Biol. Med. 34, 316–329 (2003).

    CAS  Article  Google Scholar 

Download references

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

Affiliations

Authors

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.

Corresponding authors

Correspondence to Mako Kamiya or Yasuteru Urano.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 3408 kb)

Supplementary movie

Supplementary movie 1 (AVI 2249 kb)

Supplementary movie

Supplementary movie 2 (AVI 2122 kb)

Supplementary movie

Supplementary movie 3 (AVI 2025 kb)

Supplementary movie

Supplementary movie 4 (AVI 1337 kb)

Supplementary movie

Supplementary movie 5 (AVI 1862 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing