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Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound

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Abstract

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY’s reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7′-thiobis(benzo[c][1,2,5]oxadiazole-4,4′-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY’s reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY’s reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY’s reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy.

Live cell imaging and quantification of non-protein thiols by TBOP

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References

  1. Haugaard N. Reflections on the role of the thiol group in biology. Ann N Y Acad Sci. 2000;899:148–58.

    Article  CAS  Google Scholar 

  2. Moriarty-Craige SE, Jones DP. Extracellular thiols and thiol/disulfide redox in metabolism. Annu Rev Nutr. 2004;24:481–509. doi:10.1146/annurev.nutr.24.012003.132208.

    Article  CAS  Google Scholar 

  3. Coulter CV, Kelso GF, Lin TK, Smith RA, Murphy MP. Mitochondrially targeted antioxidants and thiol reagents. Free Radic Biol Med. 2000;28(10):1547–54.

    Article  CAS  Google Scholar 

  4. Rebrin I, Sohal RS. Comparison of thiol redox state of mitochondria and homogenates of various tissues between two strains of mice with different longevities. Exp Gerontol. 2004;39(10):1513–9. doi:10.1016/j.exger.2004.08.014.

    Article  CAS  Google Scholar 

  5. Rigobello MP, Folda A, Scutari G, Bindoli A. The modulation of thiol redox state affects the production and metabolism of hydrogen peroxide by heart mitochondria. Arch Biochem Biophys. 2005;441(2):112–22. doi:10.1016/j.abb.2005.07.007.

    Article  CAS  Google Scholar 

  6. Shipounova IN, Svinareva DA, Petrova TV, et al. Reactive oxygen species produced in mitochondria are involved in age-dependent changes of hematopoietic and mesenchymal progenitor cells in mice. A study with the novel mitochondria-targeted antioxidant SkQ1. Mech Ageing Dev. 2010;131(6):415–21. doi:10.1016/j.mad.2010.06.003.

    Article  CAS  Google Scholar 

  7. Nakamura T, Lipton SA. Redox regulation of mitochondrial fission, protein misfolding, synaptic damage, and neuronal cell death: potential implications for Alzheimer’s and Parkinson’s diseases. Apoptosis. 2010;15(11):1354–63. doi:10.1007/s10495-010-0476-x.

    Article  CAS  Google Scholar 

  8. Mari M, Morales A, Colell A, Garcia-Ruiz C, Fernandez-Checa JC. Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Signal. 2009;11(11):2685–700. doi:10.1089/ARS.2009.2695.

    Article  CAS  Google Scholar 

  9. Cacciatore I, Cornacchia C, Pinnen F, Mollica A, Di Stefano A. Prodrug approach for increasing cellular glutathione levels. Molecules. 2010;15(3):1242–64. doi:10.3390/molecules15031242.

    Article  CAS  Google Scholar 

  10. Ross EK, Gray JJ, Winter AN, Linseman DA. Immunocal(R) and preservation of glutathione as a novel neuroprotective strategy for degenerative disorders of the nervous system. Recent Pat CNS Drug Discov. 2012;7(3):230–5.

    Article  CAS  Google Scholar 

  11. Martin HL, Teismann P. Glutathione—a review on its role and significance in Parkinson’s disease. FASEB J. 2009;23(10):3263–72. doi:10.1096/fj.08-125443.

    Article  CAS  Google Scholar 

  12. Chen W, Zhao Y, Seefeldt T, Guan X. Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid. J Pharm Biomed Anal. 2008;48(5):1375–80. doi:10.1016/j.jpba.2008.08.033.

    Article  Google Scholar 

  13. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969;27(3):502–22.

    Article  CAS  Google Scholar 

  14. Shimada K, Mitamura K. Derivatization of thiol-containing compounds. J Chromatogr B Biomed Appl. 1994;659(1–2):227–41.

    Article  CAS  Google Scholar 

  15. Fu NN, Hong W, Li ML, Zheng GJ, Zhang HS, Liang SC. Spectrofluorimetric determination of thiols in biological samples with a new fluorescent probe 3-maleimidylbenzanthrone. Anal Lett. 2005;38:791–802.

    Article  CAS  Google Scholar 

  16. Chen SJ, Chang HT. Nile red-adsorbed gold nanoparticles for selective determination of thiols based on energy transfer and aggregation. Anal Chem. 2004;76(13):3727–34. doi:10.1021/ac049787s.

    Article  CAS  Google Scholar 

  17. Wang W, Rusin O, Xu X, et al. Detection of homocysteine and cysteine. J Am Chem Soc. 2005;127(45):15949–58. doi:10.1021/ja054962n.

    Article  CAS  Google Scholar 

  18. Durocher S, Rezaee A, Hamm C, Rangan C, Mittler S, Mutus B. Disulfide-linked, gold nanoparticle based reagent for detecting small molecular weight thiols. J Am Chem Soc. 2009;131(7):2475–7. doi:10.1021/ja808548x.

    Article  CAS  Google Scholar 

  19. Rao J, Dragulescu-Andrasi A, Yao H. Fluorescence imaging in vivo: recent advances. Curr Opin Biotechnol. 2007;18(1):17–25. doi:10.1016/j.copbio.2007.01.003.

    Article  CAS  Google Scholar 

  20. Rice GC, Bump EA, Shrieve DC, Lee W, Kovacs M. Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo. Cancer Res. 1986;46(12 Pt 1):6105–10.

    CAS  Google Scholar 

  21. Hedley DW, Chow S. Evaluation of methods for measuring cellular glutathione content using flow cytometry. Cytometry. 1994;15(4):349–58. doi:10.1002/cyto.990150411.

    Article  CAS  Google Scholar 

  22. Ahn YH, Lee JS, Chang YT. Combinatorial rosamine library and application to in vivo glutathione probe. J Am Chem Soc. 2007;129(15):4510–1. doi:10.1021/ja068230m.

    Article  CAS  Google Scholar 

  23. Lim CS, Masanta G, Kim HJ, Han JH, Kim HM, Cho BR. Ratiometric detection of mitochondrial thiols with a two-photon fluorescent probe. J Am Chem Soc. 2011;133(29):11132–5. doi:10.1021/ja205081s.

    Article  CAS  Google Scholar 

  24. Pullela PK, Chiku T, Carvan 3rd MJ, Sem DS. Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes. Anal Biochem. 2006;352(2):265–73. doi:10.1016/j.ab.2006.01.047.

    Article  CAS  Google Scholar 

  25. Lee MH, Han JH, Kwon PS, et al. Hepatocyte-targeting single galactose-appended naphthalimide: a tool for intracellular thiol imaging in vivo. J Am Chem Soc. 2012;134(2):1316–22. doi:10.1021/ja210065g.

    Article  CAS  Google Scholar 

  26. Tang B, Xing Y, Li P, Zhang N, Yu F, Yang G. A rhodamine-based fluorescent probe containing a Se-N bond for detecting thiols and its application in living cells. J Am Chem Soc. 2007;129(38):11666–7. doi:10.1021/ja072572q.

    Article  CAS  Google Scholar 

  27. Shibata A, Furukawa K, Abe H, Tsuneda S, Ito Y. Rhodamine-based fluorogenic probe for imaging biological thiol. Bioorg Med Chem Lett. 2008;18(7):2246–9. doi:10.1016/j.bmcl.2008.03.014.

    Article  CAS  Google Scholar 

  28. Hansen JM, Go YM, Jones DP. Nuclear and mitochondrial compartmentation of oxidative stress and redox signaling. Annu Rev Pharmacol Toxicol. 2006;46:215–34. doi:10.1146/annurev.pharmtox.46.120604.141122.

    Article  CAS  Google Scholar 

  29. Li Y, Yang Y, Guan X. Benzofurazan sulfides for thiol imaging and quantification in live cells through fluorescence microscopy. Anal Chem. 2012;84(15):6877–83. doi:10.1021/ac301306s.

    Article  CAS  Google Scholar 

  30. Yang Y, Guan X. Rapid and thiol-specific high-throughput assay for simultaneous relative quantification of total thiols, protein thiols, and nonprotein thiols in cells. Anal Chem. 2015;87(1):649–55. doi:10.1021/ac503411p.

    Article  CAS  Google Scholar 

  31. Allen S, Shea JM, Felmet T, Gadra J, Dehn PF. A kinetic microassay for glutathione in cells plated on 96-well microtiter plates. Methods Cell Sci. 2000;22(4):305–12.

    Article  CAS  Google Scholar 

  32. Lee JH, Lim CS, Tian YS, Han JH, Cho BR. A two-photon fluorescent probe for thiols in live cells and tissues. J Am Chem Soc. 2010;132(4):1216–7. doi:10.1021/ja9090676.

    Article  CAS  Google Scholar 

  33. Collins TJ. ImageJ for microscopy. Biotechniques. 2007;43(1 Suppl):25–30.

    Article  Google Scholar 

  34. Longin A, Souchier C, Ffrench M, Bryon PA. Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study. J Histochem Cytochem. 1993;41(12):1833–40.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank Professor Adam Hoppe of the Chemistry and Biochemistry Department, Professor Michael Hildreth of Biology and Microbiology, and Professor Hemachand Tummala of Pharmaceutical Sciences for technical assistance in fluorescence microscopy experiments and valuable discussion. The authors also would like to thank Professor Teresa Seefeldt for proofreading the manuscript. This work was supported by grants from the National Institutes of Health (1R15GM093678-01; 1R15GM107197-01A1).

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  1. Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Profession, South Dakota State University, 1055 Campanile Ave, Box 2202C, Brookings, SD, 57007, USA

    Yang Yang & Xiangming Guan

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  1. Yang Yang
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  2. Xiangming Guan
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Correspondence to Xiangming Guan.

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Yang, Y., Guan, X. Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound. Anal Bioanal Chem 409, 3417–3427 (2017). https://doi.org/10.1007/s00216-017-0285-y

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