Organelle-selective di-(2-picolyl)amine-appended water-soluble fluorescent sensors for Cu(II): synthesis, photophysical and in vitro studies

Original Article


A novel water-soluble fluorescent naphthalimide derivative as a sensor for Cu(II) has been synthesized. The sensor, conjugated with DPA (as the Cu(II)-binding moiety) and TPP (as the mitochondrial-targeting moiety) was further decorated with a galactose unit at the fluorophore’s terminal using the click reaction. Although confocal fluorescence imaging revealed that probe 1 was not localized in mitochondria, and was neither quenched under Cu-overloading conditions, probe 1 did exhibit a high selectivity for Cu(II) ions over various metal ions in HEPES-buffered solutions. Furthermore the fluorescence was dramatically quenched upon the addition 1 equivalent of Cu(II), and the response was stable in the range of pH 6–10.

Graphical abstract

A fluorophore conjugated with DPA (as the Cu(II)-binding moiety) and TPP (as the mitochondrial-targeting moiety) was further decorated with a galactose unit at the fluorophore’s terminal. The sensor exhibited selectivity over other biologically relevant ions.


Fluorescence Copper ions Cell-imaging Subcellular environment 



This work was supported by CRI project (No. 2009-0081566) from the National Research Foundation of The Ministry of Science, ICT & Future Planning in Korea (JSK) and the Korea University Researcher Support Program (PV).

Supplementary material

10847_2015_482_MOESM1_ESM.docx (326 kb)
Supplementary material 1 (DOCX 325 kb)


  1. 1.
    Giepmans, B.N.G., Adams, S.R., Ellisman, M.H., Tsien, R.Y.: The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006)CrossRefGoogle Scholar
  2. 2.
    Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P.: Molecular Biology of the Cell. Garland Science, New York (2002)Google Scholar
  3. 3.
    Lesnefskya, E.J., Moghaddas, S., Tandler, B., Kerner, J., Hoppel, C.L.: Mitochondrial dysfunction in cardiac disease: ischemia–reperfusion, aging, and heart failure. J. Mol. Cell. Cardiol. 33, 1065–1089 (2001)CrossRefGoogle Scholar
  4. 4.
    McBride, H.M., Neuspiel, M., Wasiak, S.: Mitochondria: more than just a powerhouse. Curr. Biol. 16, R551–R560 (2006)CrossRefGoogle Scholar
  5. 5.
    Pasquali, C., Fialka, I., Huber, L.A.: Subcellular fractionation, electromigration analysis and mapping of organelles. J. Chromatogr. B 722, 89–102 (1999)CrossRefGoogle Scholar
  6. 6.
    Broz, P., Benito, S.M., Saw, C., Burger, P., Heider, H., Pfisterer, M., Marsch, S., Meier, W., Hunziker, P.: Cell targeting by a generic receptor-targeted polymer nanocontainer platform. J. Control. Release 102, 475–488 (2005)CrossRefGoogle Scholar
  7. 7.
    Lin, S., Zhang, L., Lei, K., Zhang, A., Liu, P., Liu, J.: Development of a multifunctional luciferase reporters system for assessing endoplasmic reticulum-targeting photosensitive compounds. Cell Stress Chaperones 19, 927–937 (2014)CrossRefGoogle Scholar
  8. 8.
    Fahrni, C.J.: Biological applications of X-ray fluorescence microscopy: exploring the subcellular topography and speciation of transition metals. Curr. Opin. Chem. Biol. 11, 121–127 (2007)CrossRefGoogle Scholar
  9. 9.
    Kurishita, Y., Kohira, T., Ojida, A., Hamachi, I.: Organelle-localizable fluorescent chemosensors for site-specific multicolor imaging of nucleoside polyphosphate dynamics in living cells. J. Am. Chem. Soc. 134, 18779–18789 (2012)CrossRefGoogle Scholar
  10. 10.
    Lee, M.H., Han, J.H., Lee, J.-H., Choi, H.G., Kang, C., Kim, J.S.: Mitochondrial thioredoxin-responding off-on fluorescent probe. J. Am. Chem. Soc. 134, 17317–17319 (2012)Google Scholar
  11. 11.
    Walkup, G.K., Burdette, S.C., Lippard, S.J., Tsien, R.Y.: A new cell-permeable fluorescent probe for Zn2+. J. Am. Chem. Soc. 122, 5644–5645 (2000)CrossRefGoogle Scholar
  12. 12.
    Fernandez-Carneado, J., Van Gool, M., Martos, V., Castel, S., Prados, P., de Mendoza, J., Giralt, E.: Highly efficient, nonpeptidic oligoguanidinium vectors that selectively internalize into mitochondria. J. Am. Chem. Soc. 127, 869–874 (2005)CrossRefGoogle Scholar
  13. 13.
    Horton, K.L., Stewart, K.M., Fonseca, S.B., Guo, Q., Kelley, S.O.: Mitochondria-penetrating peptides. Chem. Biol. 15, 375–382 (2008)CrossRefGoogle Scholar
  14. 14.
    Yousif, L.F., Stewart, K.M., Kelley, S.O.: Targeting mitochondria with organelle-specific compounds: strategies and applications. ChemBioChem 10, 1939–1950 (2009)CrossRefGoogle Scholar
  15. 15.
    Colston, J., Horobin, R.W., Rashid-Doubell, F., Pediani, J., Johal, K.K.: Why fluorescent probes for endoplasmic reticulum are selective: an experimental and QSAR-modelling study. Biotech. Histochem. 78, 323–332 (2003)CrossRefGoogle Scholar
  16. 16.
    Yang, Z., Cao, J., He, Y., Yang, J.H., Kim, T., Peng, X., Kim, J.S.: Macro-/micro-environment-sensitive chemosensing and biological imaging. Chem. Soc. Rev. 43, 4563–4601 (2014)CrossRefGoogle Scholar
  17. 17.
    Li, X., Gao, X., Shi, W., Ma, H.: Design strategies for water-soluble small molecular chromogenic and fluorogenic probes. Chem. Rev. 114, 590–659 (2014)CrossRefGoogle Scholar
  18. 18.
    Kim, H.J., Lee, S.J., Park, S.Y., Jung, J.H., Kim, J.S.: Detection of Cu(II) by a chemodosimeter-functionalized monolayer on mesoporous silica. Adv. Mater. 20, 3229–3234 (2008)CrossRefGoogle Scholar
  19. 19.
    Kim, H.J., Hong, J., Hong, A., Ham, S., Lee, J.H., Kim, J.S.: Cu2+-induced intermolecular static excimer formation of pyrenealkylamine. Org. Lett. 10, 1963–1966 (2008)CrossRefGoogle Scholar
  20. 20.
    Kim, Y.-R., Kim, H.J., Kim, J.S., Kim, H.: Rhodamine-based “turn-on” fluorescent chemodosimeter for Cu(II) on ultrathin platinum films as molecular switches. Adv. Mater. 20, 4428–4432 (2008)CrossRefGoogle Scholar
  21. 21.
    Jung, H.S., Kwon, P.S., Lee, J.W., Kim, J.I., Hong, C.S., Kim, J.W., Yan, S., Lee, J.Y., Lee, J.H., Joo, T., Kim, J.S.: Coumarin-derived Cu2+ selective fluorescence sensor: synthesis, mechanisms, and applications in living cells. J. Am. Chem. Soc. 131, 2008–2012 (2009)CrossRefGoogle Scholar
  22. 22.
    Kim, H.J., Kim, S.H., Kim, J.H., Anh, L.N., Lee, J.H., Lee, C.-H., Kim, J.S.: ICT-based Cu(II) sensing 9,10-anthraquinonecalix[4]crown. Tetrahedron Lett. 50, 2782–2786 (2009)CrossRefGoogle Scholar
  23. 23.
    Jung, H.S., Park, M., Han, D.Y., Kim, E., Lee, C., Ham, S., Kim, J.S.: Cu2+ ion-induced self-assembly of pyrenylquinoline with a pyrenyl excimer formation. Org. Lett. 11, 3378–3381 (2009)CrossRefGoogle Scholar
  24. 24.
    Zhang, J.F., Zhou, Y., Yoon, J., Kim, Y., Kim, S.J., Kim, J.S.: Naphthalimide modified rhodamine derivative: ratiometric and selective fluorescent sensor for Cu2+ based on two different approaches. Org. Lett. 12, 3852–3855 (2010)CrossRefGoogle Scholar
  25. 25.
    Ko, K.C., Wu, J.-S., Kim, H.J., Kwon, P.S., Kim, J.W., Bartsch, R.A., Lee, J.Y., Kim, J.S.: Rationally designed fluorescence ‘turn-on’ sensor for Cu2+. Chem. Commun. 47, 3165–3167 (2011)CrossRefGoogle Scholar
  26. 26.
    Jung, H.S., Park, M., Han, J.H., Lee, J.H., Kang, C., Jung, J.H., Kim, J.S.: Selective removal and quantification of Cu(II) using fluorescent iminocoumarin-functionalized magnetic nanosilica. Chem. Commun. 48, 5082–5084 (2012)CrossRefGoogle Scholar
  27. 27.
    Jang, J.H., Bhuniya, S., Kang, J., Yeom, A., Hong, K.S., Kim, J.S.: Copper ion responsive bimodal (optical/MRI) contrast agent for cellular imaging. Org. Lett. 15, 4702–4708 (2013)CrossRefGoogle Scholar
  28. 28.
    Lee, Y.H., Park, N., Park, Y.B., Hwang, Y.J., Kang, C., Kim, J.S.: Organelle-selective fluorescent Cu2+ ion probes: revealing endoplasmic reticulum as reservoir for Cu-overloading. Chem. Commun. 50, 3197–3200 (2014)CrossRefGoogle Scholar
  29. 29.
    Kang, D.E., Lim, C.S., Kim, J.Y., Kim, E.S., Chun, H.J., Cho, B.R.: Two-photon probe for Cu2+ with an internal reference: quantitative estimation of Cu2+ in human tissues by two-photon microscopy. Anal. Chem. 86, 5353–5359 (2014)CrossRefGoogle Scholar
  30. 30.
    McCranor, B.J., Szmacinski, H., Zeng, H.H., Stoddard, A.K., Hurst, T., Fierke, C.A., Lakowicz, J.R., Thompson, R.B.: Fluorescence lifetime imaging of physiological free Cu(II) levels in live cells with a Cu(II)-selective carbonic anhydrase-based biosensor. Metallomics 6, 1034–1042 (2014)CrossRefGoogle Scholar
  31. 31.
    Kroemer, G., Galluzzi, L., Brenner, C.: Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 87, 99–163 (2007)CrossRefGoogle Scholar
  32. 32.
    Dodani, S.C., Leary, S.C., Cobine, P.A., Winge, D.R., Chang, C.J.: A targetable fluorescent sensor reveals that copper-deficient SCO1 and SCO2 patient cells prioritize mitochondrial copper homeostasis. J. Am. Chem. Soc. 133, 8606–8616 (2011)CrossRefGoogle Scholar
  33. 33.
    Lee, M.H., Han, J.H., Kwon, P.-S., Bhuniya, S., Kim, J.Y., Sessler, J.L., Kang, C., Kim, J.S.: Hepatocyte-targeting single galactose-appended naphthalimide: a tool for intracellular thiol imaging in vivo. J. Am. Chem. Soc. 134, 1316–1322 (2012)CrossRefGoogle Scholar
  34. 34.
    Maryanoff, B.E., Reitz, A.B., Duhl-Emswiler, B.A.: Stereochemistry of the Wittig reaction. Effect of nucleophilic groups in the phosphonium ylide. J. Am. Chem. Soc. 107, 217–226 (1985)CrossRefGoogle Scholar
  35. 35.
    Kopple, J.D., Swendseid, M.E.: Evidence that histidine is an essential amino acid in normal and chronically uremic man. J. Clin. Invest. 55, 881–891 (1975)CrossRefGoogle Scholar
  36. 36.
    Pasternak, K., Kocot, J., Horecka, A.: Biochemistry of magnesium. J. Elementol. 15, 601–616 (2010)Google Scholar
  37. 37.
    Mersch-Sundermann, V., Knasmüller, S., Wu, X.J., Darroudi, F., Kassie, F.: Use of a human-derived liver cell line for the detection of cytoprotective, antigenotoxic and cogenotoxic agents. Toxicology 198, 329–340 (2004)CrossRefGoogle Scholar
  38. 38.
    Spiess, M.: The asialoglycoprotein receptor: a model for endocytic transport receptors. Biochemistry 29, 10009–10018 (1990)CrossRefGoogle Scholar
  39. 39.
    Ross, M.F., Kelso, G.F., Blaikie, F.H., James, A.M., Cochemé, H.M.: Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology. Biochemistry (Mosc.) 70, 222–230 (2005)CrossRefGoogle Scholar
  40. 40.
    Chen, Y., Zhu, C., Cen, J., Li, J., He, W., Jiao, Y., Guo, Z.: A reversible ratiometric sensor for intracellular Cu2+ imaging: metal coordination-altered FRET in a dual fluorophore hybrid. Chem. Commun. 49, 7632–7634 (2013)CrossRefGoogle Scholar
  41. 41.
    Lim, C.S., Han, J.H., Kim, C.W., Kang, M.Y., Kang, D.W., Cho, B.R.: A copper(I)-ion selective two-photon fluorescent probe for in vivo imaging. Chem. Commun. 47, 7146–7148 (2011)CrossRefGoogle Scholar
  42. 42.
    Moriya, M., Ho, Y.H., Grana, A., Nguyen, L., Alvarez, A., Jamil, R., Ackland, M.L., Michalczyk, A., Hamer, P., Ramos, D., Kim, S., Mercer, J.F., Linder, M.C.: Copper is taken up efficiently from albumin and alpha2-macroglobulin by cultured human cells by more than one mechanism. Am. J. Physiol. Cell Physiol. 295, C708–C721 (2008)CrossRefGoogle Scholar
  43. 43.
    Cui, C.T., Uriu-Adams, J.Y., Tchaparian, E.H., Keen, C.L., Rucker, R.B.: Metavanadate causes cellular accumulation of copper and decreased lysyl oxidase activity. Toxicol. Appl. Pharmacol. 199, 35–43 (2004)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Department of ChemistryKorea UniversitySeoulKorea
  2. 2.Korea Research Institute of Standards and ScienceDaejeonKorea

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