Visualizing Bioactive Small Molecules by Alkyne Tagging and Slit-Scanning Raman Microscopy

  • Jun Ando
  • Kosuke Dodo
  • Katsumasa Fujita
  • Mikiko SodeokaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1888)


An understanding of the intracellular distribution of bioactive small molecules provides insight into their target organelles and biomolecules, and throws light on their molecular mechanisms of action and specificity. Many studies in this area have employed fluorescence imaging, using molecules of interest labeled with fluorescent dyes. However, modification with a bulky fluorophore may significantly alter the properties, including bioactivity, of small molecules. To address this issue, we have recently developed Raman scattering microscopy of alkyne tags to visualize small molecules in biological systems. The tiny alkyne tag should have a minimal effect on the properties of the tagged molecule, and the unique alkyne vibrational properties allow observation of the Raman signals of the tagged molecules in a wavelength region where there is little interference from Raman signals of endogenous biomolecules. Here we summarize the methodology for Raman imaging of alkyne-tagged bioactive small molecules in living biological systems including the development of slit-scanning Raman microscope, which is useful for fast imaging of alkyne-tagged molecules.

Key words

Alkyne tag Raman scattering Small molecules Slit-scanning Raman microscopy 



This work was partially supported by JST-ERATO (Sodeoka Live Cell Chemistry Project), AMED-CREST (No. JP17gm0710004), RIKEN and JSPS KAKENHI Grant Number 26600117 (J.A.).


  1. 1.
    Parker FS (ed) (1983) Applications of Infrared, Raman, and Resonance Raman spectroscopy in biochemistry. Springer, HeidelbergGoogle Scholar
  2. 2.
    Carey P (1982) Biochemical applications of Raman and Resonance Raman spectroscopes. Academic Press, New YorkGoogle Scholar
  3. 3.
    Karthigeyan D, Siddhanta S, Kishore AH, Perumal SSRR, Ågren H, Sudevan S, Bhat AV, Balasubramanyam K, Subbegowda RK, Kundu TK, Narayana C (2014) SERS and MD simulation studies of a kinase inhibitor demonstrate the emergence of a potential drug discovery tool. Proc Natl Acad Sci U S A 111:10416–10421CrossRefPubMedCentralGoogle Scholar
  4. 4.
    Ando J, Asanuma M, Dodo K, Yamakoshi H, Kawata S, Fujita K, Sodeoka M (2016) Alkyne-tag SERS screening and identification of small-molecule-binding sites in protein. J Am Chem Soc 138:13901–13910CrossRefGoogle Scholar
  5. 5.
    Huang YS, Karashima T, Yamamoto M, Hamaguchi H (2003) Molecular-level pursuit of yeast mitosis by time- and space-resolved Raman spectroscopy. J Raman Spectrosc 34:1–3CrossRefGoogle Scholar
  6. 6.
    Palonpon AF, Ando J, Yamakoshi H, Dodo K, Sodeoka M, Kawata S, Fujita K (2013) Raman and SERS microscopy for molecular imaging of live cells. Nat Protoc 8:677–692CrossRefPubMedCentralGoogle Scholar
  7. 7.
    Ando J, Palonpon AF, Sodeoka M, Fujita K (2016) High-speed Raman imaging of cellular processes. Curr Opin Chem Biol 33:16–24CrossRefPubMedCentralGoogle Scholar
  8. 8.
    Hamada K, Fujita K, Smith NI, Kobayashi M, Inouye Y, Kawata S (2008) Raman microscopy for dynamic molecular imaging of living cells. J Biomed Opt 13:044027CrossRefPubMedCentralGoogle Scholar
  9. 9.
    Okada M, Smith NI, Palonpon AF, Endo H, Kawata S, Sodeoka M, Fujita K (2012) Label-free Raman observation of cytochrome c dynamic during apoptosis. Proc Natl Acad Sci U S A 109:28–32CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Ling J, Weitman SD, Miller MA, Moore RV, Bovik AC (2002) Direct Raman imaging techniques for study of the subcellular distribution of a drug. Appl Opt 41:6006–6017CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Yamakoshi H, Dodo K, Okada M, Ando J, Palonpon A, Fujita K, Kawata S, Sodeoka M (2011) Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy. J Am Chem Soc 133:6102–6105CrossRefPubMedCentralGoogle Scholar
  12. 12.
    Yamakoshi H, Dodo K, Palonpon A, Ando J, Fujita K, Kawata S, Sodeoka M (2012) Alkyne-tag Raman imaging for visualization of mobile small molecules in live cells. J Am Chem Soc 134:20681–20689CrossRefPubMedCentralGoogle Scholar
  13. 13.
    Ando J, Kinoshita M, Cui J, Yamakoshi H, Dodo K, Fujita K, Murata M, Sodeoka M (2015) Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy. Proc Natl Acad Sci U S A 112:4558–4563CrossRefPubMedCentralGoogle Scholar
  14. 14.
    Lee J, Zhang W, Zhang D, Yang Y, Liu B, Barker EL, Buhman KK, Slipchenko LV, Dai M, Cheng JX (2015) Assessing cholesterol storage in live cells and C. elegans by stimulated Raman scattering imaging of phenyl-diyne cholesterol. Sci Rep 5:7930CrossRefPubMedCentralGoogle Scholar
  15. 15.
    Wei L, Hu F, Shen Y, Chen Z, Yu Y, Lin CC, Wang MC, Min W (2014) Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering. Nat Methods 11:410–412CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Hong S, Chen T, Zhu Y, Li A, Huang Y, Chen X (2014) Live-cell stimulated Raman scattering imaging of alkyne-tagged biomolecules. Angew Chem Int Ed 53:5827–5831CrossRefGoogle Scholar
  17. 17.
    El-Mashtoly SF, Petersen D, Yosef HK, Mosig A, Reinacher-Schick A, Kötting C, Gerwert K (2014) Label-free imaging of drug distribution and metabolism in colon cancer cells by Raman microscopy. Analyst 139:1155–1161CrossRefPubMedCentralGoogle Scholar
  18. 18.
    Yamakoshi H, Palonpon A, Dodo K, Ando J, Kawata S, Fujita K, Sodeoka M (2015) A sensitive and specific Raman probe based on bisarylbutadiyne for live cell imaging of mitochondria. Bioorg Med Chem Lett 25:664–667CrossRefPubMedCentralGoogle Scholar
  19. 19.
    Ueda M, Egoshi S, Dodo K, Ishimaru Y, Yamakoshi H, Nakano T, Takaoka Y, Tsukiji S, Sodeoka M (2017) Noncanonical function of a small-molecular virulence factor coronatine against plant immunity: an in vivo Raman imaging approach. ACS Cent Sci 3:462–472CrossRefPubMedCentralGoogle Scholar
  20. 20.
    Chen Z, Paley DW, Wei L, Weisman AL, Friesner RA, Nuckolls C, Min W (2014) Multicolor live-cell chemical imaging by isotopically edited alkyne vibrational palette. J Am Chem Soc 136:8027–8033CrossRefPubMedCentralGoogle Scholar
  21. 21.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedCentralGoogle Scholar
  22. 22.
    Manen HJ, Kraan YM, Roos D, Otto C (2004) Intracellular chemical imaging of heme containing enzymes involved in innate immunity using resonance Raman microscopy. J Phys Chem B 108:18762–18771CrossRefGoogle Scholar
  23. 23.
    Lieber CA, Mahadevan-Jansen A (2003) Automated method for subtraction of fluorescence from biological Raman spectra. Appl Spectrosc 57:1363–1367CrossRefPubMedCentralGoogle Scholar
  24. 24.
    Mu ZM, Le XF, Vallian S, Glassman AB, Chang KS (1997) Stable overexpression of PML alters regulation of cell cycle progression in HeLa cells. Carcinogenesis 18:2063–2069CrossRefPubMedCentralGoogle Scholar
  25. 25.
    Doucet H, Hierso J-C (2007) Palladium-Based Catalytic Systems for the Synthesis of Conjugated Enynes by Sonogashira Reactions and Related Alkynylations. Angew Chem Int Ed 46:834–871CrossRefGoogle Scholar
  26. 26.
    Shealy YF, O’Dell CA, Arnett G, Shannon WM (1986) Synthesis and antiviral activity of the carbocyclic analogues of 5-Ethyl-2′-deoxyuridine and of 5-Ethynyl-2′-deoxyuridine. J Med Chem 29:79–84CrossRefPubMedCentralGoogle Scholar
  27. 27.
    Siemsen P, Livingston RC, Diederich F (2000) Acetylenic coupling: a powerful tool in molecular construction. Angew Chem Int Ed 39:2632–2657CrossRefGoogle Scholar
  28. 28.
    Pietruszka J, Witt A (2006) Synthesis of the Bestmann-Ohira reagent. Synthesis 24:4266–4268CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jun Ando
    • 1
  • Kosuke Dodo
    • 2
  • Katsumasa Fujita
    • 1
  • Mikiko Sodeoka
    • 2
    Email author
  1. 1.Department of Applied PhysicsOsaka UniversitySuitaJapan
  2. 2.Synthetic Organic Chemistry LaboratoryRIKEN Cluster for Pioneering ResearchWakoJapan

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