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Preparation of a cyanine-based fluorescent probe for highly selective detection of glutathione and its use in living cells and tissues of mice

Abstract

Glutathione (GSH) is a major endogenous antioxidant that has a central role in cellular defense against toxins and free radicals. This protocol describes the preparation of CPDSA, a cyanine-based near-infrared (NIR) fluorescent probe for the detection of GSH in cells and in vivo. CPDSA is prepared with high yield through a simple two-step process. The first step is to react commercially available IR-780 iodide with excess anhydrous piperazine in anhydrous N,N-dimethyl formamide at 85 °C to form cyanine-piperazine (CP). The second step is the sulfonylation of CP with dansyl chloride in anhydrous dichloromethane. CPDSA selectively detects GSH in cells, and it has been shown to not react with other biothiols such as cysteine (Cys) and homocysteine (Hcy). This probe can also be used to monitor the GSH level of mouse bone marrow–derived neutrophils (BMDNs). The preparation of probe CPDSA takes 2 d, and experiments in cells and mice take 12–13 d.

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Figure 1: The GSH-based fluorescence probe CPDSA.
Figure 2
Figure 3
Figure 4: Confocal microscopy images of CPDSA in HeLa cells.
Figure 5: Confocal microscopy images of the CPDSA probe in HeLa cells.
Figure 6: Confocal microscopy images of probe CPDSA in RAW 264.7 cells.
Figure 7: Confocal microscopy images of probe CPDSA in mouse bone marrow–derived neutrophils.
Figure 8: Flow cytometry analysis images of the CPDSA probe in peritoneal neutrophils derived from the CLP mouse model.

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References

  1. 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  Google Scholar 

  2. Yang, X.-F. et al. A dual emission fluorescent probe enables simultaneous detection of glutathione and cysteine/homocysteine. Chem. Sci. 5, 2177–2183 (2014).

    CAS  PubMed  Google Scholar 

  3. Yin, C. et al. Thiol-addition reactions and their applications in thiol recognition. Chem. Soc. Rev. 42, 6032–6059 (2013).

    CAS  PubMed  Google Scholar 

  4. Yin, L.-L., Chen, Z.-Z., Tong, L.-L., Xu, K.-H. & Tang, B. Progress on fluorescent probes for thiols. Chin. J. Anal. Chem. 37, 1073–1081 (2009).

    CAS  Google Scholar 

  5. Jung, H.S., Chen, X., Kim, J.S. & Yoon, J. Recent progress in luminescent and colorimetric chemosensors for detection of thiols. Chem. Soc. Rev. 42, 6019–6031 (2013).

    CAS  PubMed  Google Scholar 

  6. 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  Google Scholar 

  7. Iş1k, M. et al. Designing an intracellular fluorescent probe for glutathione: two modulation sites for selective signal transduction. Org. Lett. 16, 3260–3263 (2014).

    PubMed  Google Scholar 

  8. Zhai, D., Lee, S.-C., Yun, S.-W. & Chang, Y.-T. A ratiometric fluorescent dye for the detection of glutathione in live cells and liver cancer tissue. Chem. Commun. 49, 7207–7209 (2013).

    CAS  Google Scholar 

  9. Shao, J. et al. A highly selective red-emitting FRET fluorescent molecular probe derived from BODIPY for the detection of cysteine and homocysteine: an experimental and theoretical study. Chem. Sci. 3, 1049–1061 (2013).

    Google Scholar 

  10. Niu, L.-Y. et al. A turn-on fluorescent sensor for the discrimination of cysteine from homocysteine and glutathione. Chem. Commun. 49, 1294–1296 (2013).

    CAS  Google Scholar 

  11. Wang, H., Zhou, G., Gai, H. & Chen, X. A fluorescein-based probe with high selectivity to cysteine over homocysteine and glutathione. Chem. Commun. 48, 8341–8343 (2012).

    CAS  Google Scholar 

  12. Jung, H.S. et al. Molecular modulated cysteine-selective fluorescent probe. Biomaterials 33, 945–953 (2012).

    CAS  PubMed  Google Scholar 

  13. Hammers, M.D. & Pluth, M.D. Ratiometric measurement of hydrogen sulfide and cysteine/homocysteine ratios using a dual-fluorophore fragmentation strategy. Anal. Chem. 86, 7135–7140 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Jung, H.S. et al. A cysteine-selective fluorescent probe for the cellular detection of cysteine. Biomaterials 33, 8495–8502 (2012).

    CAS  PubMed  Google Scholar 

  15. Liu, J. et al. Simultaneous fluorescence sensing of Cys and GSH from different emission channels. J. Am. Chem. Soc. 136, 574–577 (2014).

    CAS  PubMed  Google Scholar 

  16. Zhou, X., Jin, X., Sun, G., Li, D. & Wu, X. A cysteine probe with high selectivity and sensitivity promoted by response-assisted electrostatic attraction. Chem. Commun. 48, 8793–8795 (2012).

    CAS  Google Scholar 

  17. Lee, H.Y. et al. Selective homocysteine turn-on fluorescent probes and their bioimaging applications. Chem. Commun. 50, 6967–6969 (2014).

    CAS  Google Scholar 

  18. Hu, Y. et al. One-photon and two-photon sensing of biothiols using a bis-pyrene-Cu (II) ensemble and its application to image GSH in the cells and tissues. Anal. Chem. 87, 3308–3313 (2015).

    CAS  PubMed  Google Scholar 

  19. Lee, D., Kim, G., Yin, J. & Yoon, J. Aryl-thioether substituted nitrobenzothiadiazole probe for selective detection of cysteine and homocysteine. Chem. Commun. 51, 6518–6520 (2015).

    CAS  Google Scholar 

  20. 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  Google Scholar 

  21. Camera, E. & Picardo, M. Analytical methods to investigate glutathione and related compounds in biological and pathological processes. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 781, 181–206 (2002).

    CAS  PubMed  Google Scholar 

  22. Toyo'oka, T. Recent advances in separation and detection methods for thiol compounds in biological samples. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877, 3318–3330 (2009).

    CAS  PubMed  Google Scholar 

  23. Monostori, P., Wittmann, G., Karg, E. & Túri, S. Determination of glutathione and glutathione disulfide in biological samples: an in-depth review. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877, 3331–3346 (2009).

    CAS  PubMed  Google Scholar 

  24. Szaciłowski, K., Macyk, W., Drzewiecka-Matuszek, A., Brindell, M. & Stochel, G. Bioinorganic photochemistry: frontiers and mechanisms. Chem. Rev. 105, 2647–2694 (2005).

    PubMed  Google Scholar 

  25. Guo, Z., Park, S., Yoon, J. & Shin, I. Recent progress in the development of near-infrared fluorescent probes for bioimaging applications. Chem. Soc. Rev. 43, 16–29 (2014).

    PubMed  Google Scholar 

  26. Yuan, L., Lin, W., Zheng, K., He, L. & Huang, W. Far-red to near infrared analyte-responsive fluorescent probes based on organic fluorophore platforms for fluorescence imaging. Chem. Soc. Rev. 42, 622–661 (2013).

    CAS  PubMed  Google Scholar 

  27. 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  Google Scholar 

  28. Wu, X. et al. In vivo and in situ tracking cancer chemotherapy by highly photostable NIR fluorescent theranostic prodrug. J. Am. Chem. Soc. 136, 3579–3588 (2014).

    CAS  PubMed  Google Scholar 

  29. Peng, X. et al. Fluorescence ratiometry and fluorescence lifetime imaging: using a single molecular sensor for dual mode imaging of cellular viscosity. J. Am. Chem. Soc. 133, 6626–6635 (2011).

    CAS  PubMed  Google Scholar 

  30. Karton-Lifshin, N. et al. A unique paradigm for a Turn-ON near-infrared cyanine-based probe: noninvasive intravital optical imaging of hydrogen peroxide. J. Am. Chem. Soc. 133, 10960–10965 (2011).

    CAS  PubMed  Google Scholar 

  31. Karton-Lifshin, N., Albertazzi, L., Bendikov, M., Baran, P.S. & Shabat, D. 'Donor–two-acceptor' dye design: a distinct gateway to NIR fluorescence. J. Am. Chem. Soc. 134, 20412–20420 (2012).

    CAS  PubMed  Google Scholar 

  32. Redy-Keisar, O., Kisin-Finfer, E., Ferber, S., Satchi-Fainaro, R. & Shabat, D. Synthesis and use of QCy7-derived modular probes for the detection and imaging of biologically relevant analytes. Nat. Protocol. 9, 27–36 (2014).

    CAS  Google Scholar 

  33. Myochin, T. et al. Rational design of ratiometric near-infrared fluorescent pH probes with various pKa values, based on aminocyanine. J. Am. Chem. Soc. 133, 3401–3409 (2011).

    CAS  PubMed  Google Scholar 

  34. Takahashi, S. et al. Reversible off–on fluorescence probe for hypoxia and imaging of hypoxia–normoxia cycles in live cells. J. Am. Chem. Soc. 134, 19588–19591 (2012).

    CAS  PubMed  Google Scholar 

  35. Yang, Z. et al. Folate-based near-infrared fluorescent theranostic gemcitabine delivery. J. Am. Chem. Soc. 135, 11657–11662 (2013).

    CAS  PubMed  Google Scholar 

  36. Yu, F., Li, P., Wang, B. & Han, K. Reversible near-infrared fluorescent probe introducing tellurium to mimetic glutathione peroxidase for monitoring the redox cycles between peroxynitrite and glutathione in vivo. J. Am. Chem. Soc. 135, 7674–7680 (2013).

    CAS  PubMed  Google Scholar 

  37. Kundu, K. et al. Hydrocyanines: a class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo. Angew. Chem. Int. Ed. 48, 299–303 (2009).

    CAS  Google Scholar 

  38. Liu, Y. et al. A cyanine-modified nanosystem for in vivo upconversion luminescence bioimaging of methylmercury. J. Am. Chem. Soc. 135, 9869–9876 (2013).

    CAS  PubMed  Google Scholar 

  39. Edgington, L.E. et al. Functional imaging of legumain in cancer using a new quenched activity-based probe. J. Am. Chem. Soc. 135, 174–182 (2013).

    CAS  PubMed  Google Scholar 

  40. Tang, b. et al. Near-infrared neutral pH fluorescent probe for monitoring minor pH changes: imaging in living HepG2 and HL-7702 cells. J. Am. Chem. Soc. 131, 3016–3023 (2009).

    CAS  PubMed  Google Scholar 

  41. Fu, N., Xiong, Y. & Squier, T.C. Synthesis of a targeted biarsenical Cy3-Cy5 affinity probe for superresolution fluorescence imaging. J. Am. Chem. Soc. 134, 18530–18533 (2012).

    CAS  PubMed  Google Scholar 

  42. Guo, Z., Nam, S., Park, S. & Yoon, J. A highly selective ratiometric near-infrared fluorescent cyanine sensor for cysteine with remarkable shift and its application in bioimaging. Chem. Sci. 3, 2760–2765 (2012).

    CAS  Google Scholar 

  43. Zhang, J. et al. A near-infrared fluorescence probe for thiols based on analyte-specific cleavage of carbamate and its application in bioimaging. Eur. J. Org. Chem. 2015, 1711–1718 (2015).

    CAS  Google Scholar 

  44. Sanlioglu, S. et al. Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-α secretion through IKK regulation of NF-κB. J. Biol. Chem. 276, 30188–30198 (2001).

    CAS  PubMed  Google Scholar 

  45. Park, H.S. et al. Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-κB. J. Immunol. 173, 3589–3593 (2004).

    CAS  PubMed  Google Scholar 

  46. Witko-Sarsat, V., Rieu, P., Descamps-Latscha, B., Lesavre, P. & Halbwachs-Mecarelli, L. Neutrophils: molecules, functions and pathophysiological aspects. Lab. Invest. 80, 617–653 (2000).

    CAS  PubMed  Google Scholar 

  47. Rosen, H. & Klebanoff, S.J. Bactericidal activity of a superoxide anion-generating system. J. Exp. Med. 149, 27–39 (1979).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This research was supported by a grant from the National Creative Research Initiative programs of the National Research Foundation of Korea (NRF) funded by the Korean government (grant no. 2012R1A3A2048814), by the Basic Science Research Program through the NRF funded by the Ministry of Education (grant no. 2013R1A1A2008511), and by the Yonsei University College of Medicine (grant no. 6-2014-0135 to J.-H.R.) and the National Natural Science Foundation of China (grant no. 21402057).

Author information

Authors and Affiliations

Authors

Contributions

J. Yin, Y.K. and G.K. conducted experiments; D.K., D.L. and Y.H. measured partial data; and J. Yin, Y.K., G.K., J.-H.R. and J. Yoon designed experiments and wrote the paper.

Corresponding authors

Correspondence to Ji-Hwan Ryu or Juyoung Yoon.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Fluorescent titration of probe CPDSA.

Fluorescent titration of probe CPDSA (10 uM) upon addition of GSH in HEPES (10 mM, pH = 7.4) containing 10% DMSO. Each spectrum was recorded at 20 min after the addition of GSH. (λex = 730 nm, λem = 736 nm, slit: 10/10 nm)

Supplementary Figure 2 Confocal microscope images following addition of probe CPDSA to HeLa cells.

Confocal microscope images following addition of probe CPDSA to HeLa cells (The data is collected from FV1200, Olympus, Japan). Cell images were obtained using an excitation wavelength of 635 nm and a band‐path (655−755 nm) emission filter. (A) Fluorescence image of HeLa cells; (B) Fluorescence image of HeLa cells incubated with probe CPDSA (20 μM) for 20 min; (C) Fluorescence image of HeLa cells pretreated with N-methylmaleimide (NMM, 1 mM) for 20 min and incubated with probe CPDSA (20 μM) for 20 min; (D) Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min, and then added with cysteine (100 μM) and incubated with probe CPDSA (20 μM) for 20 min, (E) Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min, and then added with homocysteine (100 μM) and incubated with probe CPDSA (20 μM) for 20 min; (F) Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min, and then added with GSH-MEE (100 μM) and incubated with probe CPDSA (20 μM) for 20 min. Scale bar: 15 μm.

Supplementary Figure 3 The time-dependent confocal microscope images following addition of probe CPDSA to HeLa cells.

Confocal microscope images following addition of probe CPDSA to HeLa cells (The data is collected from FV1200, Olympus, Japan). HeLa cells were incubated with 20 μM probe CPDSA for 20 min and washed with DPBS and exchanged with new media. The time-dependent fluorescence images of probe CPDSA were acquired by confocal microscopy. Cell images were obtained using an excitation wavelength of 635 nm and a band-path (655−755 nm) emission filter.

Supplementary Figure 4 The time-dependent fluorescence intensity of probe CPDSA in HeLa cells.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Methods (PDF 836 kb)

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Yin, J., Kwon, Y., Kim, D. et al. Preparation of a cyanine-based fluorescent probe for highly selective detection of glutathione and its use in living cells and tissues of mice. Nat Protoc 10, 1742–1754 (2015). https://doi.org/10.1038/nprot.2015.109

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