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The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells

Nature Methods volume 3, pages 35–40 (2006)Cite this article

Abstract

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16–160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M−1 cm−1). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca2+ rapidly released Ca2+ (rate of 20,000 s−1) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of ∼0.6 GM and two-photon photolysis induced localized Ca2+-induced Ca2+ release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

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Figure 1: Synthesis of NDBF-EGTA.
Figure 2: Photophysical properties of NDBF-EGTA.
Figure 3: Two-photon photolysis of NDBF-EGTA in droplets and intact cardiac myocytes.
Figure 4: Tension recordings of successive activations of a single trabecula by UV laser photolysis of caged Ca2+.

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References

  1. Havinga, E., De Jongh, R.O. & Dorst, W. Photochemical acceleration of the hydrolysis of nitrophenyl phosphates and nitrophenyl sulfates. Recl. Trav. Chim. 75, 378–383 (1956).

    Article  CAS  Google Scholar 

  2. Baltrop, J.A., Plant, P.J. & Schofield, P. Photosensitive protecting groups. J. Chem. Soc. Chem. Commun. 822–823 (1966).

  3. Patchornik, A., Amit, B. & Woodward, R.B. Photochemical protecting groups. J. Am. Chem. Soc. 92, 6333–6335 (1970).

    Article  CAS  Google Scholar 

  4. Engels, J. & Schlaeger, E.-J. Synthesis, structure and reactivity of adenosine cyclic 3′,5′-phosphate benzyl triesters. J. Med. Chem. 20, 907–911 (1977).

    Article  CAS  Google Scholar 

  5. Kaplan, J.H., Forbush, B. & Hoffman, J.F. Rapid photolytic release of adenosine 5′-triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell ghosts. Biochemistry 17, 1929–1935 (1978).

    Article  CAS  Google Scholar 

  6. Ellis-Davies, G.C.R. Basics of photoactivation. In Imaging in Neuroscience and Development: A Laboratory Manual 2nd edn. (eds. Yuste, R. & Konnerth, A.) 367–373 (Cold Spring Harbor Laboratory Press, Cold spring Harbor, 2005).

    Google Scholar 

  7. Walker, J.W., Feeney, J. & Trentham, D.R. Photolabile precursors of inositol phosphates. Preparation and properties of 1-(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-trisphosphate. Biochemistry 28, 3272–3280 (1989).

    Article  CAS  Google Scholar 

  8. Wieboldt, R. et al. Photolabile precursors of glutamate: Synthesis, photochemical properties, activation of glutamate receptors in the microsecond time scale. Proc. Natl. Acad. Sci. USA 91, 8752–8756 (1994).

    Article  CAS  Google Scholar 

  9. Ellis-Davies, G.C.R. & Kaplan, J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc. Natl. Acad. Sci. USA 91, 187–191 (1994).

    Article  CAS  Google Scholar 

  10. Wootton, J.F. & Trentham, D.R. Caged compounds to probe the dynamics of cellular processes: synthesis and properties of some novel photosensitive P-2-nitrobenzyl esters of nucleotides. In Photochemical probes in biochemistry NATO ASI Ser C 272 (ed. Nielsen, P.E.) 277–296 (Kluwer Academic Publishers, Dordrecht, 1989).

    Chapter  Google Scholar 

  11. Nerbonne, J.M., Richard, S.Nargeot.J. & Lester, H.A. New photoactivatable cyclic nucleotides produce intracellular jumps in cyclic AMP and cyclic GMP concentrations. Nature 310, 74–76 (1984).

    Article  CAS  Google Scholar 

  12. Adams, S.R., Kao, J.P.Y., Grynkiewicz, G., Minta, A. & Tsien, R.Y. Biologically useful chelators that release Ca2+ upon illumination. J. Am. Chem. Soc. 110, 3212–3220 (1998).

    Article  Google Scholar 

  13. Kaplan, J.H. & Ellis-Davies, G.C.R. Photolabile chelators for the rapid photorelease of divalent cations. Proc. Natl. Acad. Sci. USA 85, 6571–6575 (1988).

    Article  CAS  Google Scholar 

  14. Ellis-Davies, G.C.R. Synthesis of photolabile EGTA derivatives. Tetrahedr. Lett. 39, 953–957 (1998).

    Article  CAS  Google Scholar 

  15. Papageorgiou, G. & Corrie, J.E.T. Effects of Aromatic substituents on the photocleavage of 1-acyl-7-nitroindolines. Tetrahedron 56, 8197–8205 (2001).

    Article  Google Scholar 

  16. Matsuzaki, M. et al. Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat. Neurosci. 4, 1086–1092 (2001).

    Article  CAS  Google Scholar 

  17. Denk, W., Stricker, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  CAS  Google Scholar 

  18. Lindegger, N. & Niggli, E. Paradoxical SR Ca2+ release in cardiac myocytes after β-adrenergic stimulation revealed by two-photon photolysis of caged Ca2+. J. Physiol. (Lond.) 565, 801–813 (2005).

    Article  CAS  Google Scholar 

  19. Martin, H., Bell, M.G., Ellis-Davies, G.C.R. & Barsotti, R.J. Activation of skinned cardiac muscle by laser photolysis of nitrophenyl-EGTA. Biophys. J. 86, 978–990 (2004).

    Article  CAS  Google Scholar 

  20. Pelliccioli, A.P. & Wirz, J. Photoremovable protecting groups: reaction mechanisms and applications. Photochem. Photobiol. Sci. 1, 441–458 (2002).

    Article  Google Scholar 

  21. Ellis-Davies, G.C.R. Development and application of calcium cages. Methods Enzymol. 360A, 226–238 (2003).

    Article  Google Scholar 

  22. Zucker, R.S. Effects of photolabile calcium chelators on fluorescent calcium indicators. Cell Calcium 13, 29–40 (1992).

    Article  CAS  Google Scholar 

  23. Adams, S.R., Lev-Ram, V. & Tsien, R.Y. A new caged Ca2+, azid-1, is far more photosensitive than nitrobenzyl-based chelators. Chem. Biol. 4, 867–878 (1997).

    Article  CAS  Google Scholar 

  24. Brown, E.B. et al. Photolysis of caged calcium in femtoliter volumes using two-photon excitation. Biophys. J. 76, 489–499 (1999).

    Article  CAS  Google Scholar 

  25. Niggli, E. Localized intracellular calcium signaling in muscle: calcium sparks and calcium quarks. Annu. Rev. Physiol. 61, 311–335 (1999).

    Article  CAS  Google Scholar 

  26. Denk, W. & Svoboda, K. Photo upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351–357 (1997).

    Article  CAS  Google Scholar 

  27. Trachtenberg, J.T. et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002).

    Article  CAS  Google Scholar 

  28. Brown, E.B. & Webb, W.W. Two-photon activation of caged calcium with submicron, submillisecond resolution. Methods Enzymol. 201, 356–380 (1998).

    Article  Google Scholar 

  29. Ellis-Davies, G.C.R., Kaplan, J.H. & Barsotti, R.J. Laser photolysis of caged calcium: rates of calcium release by nitrophenyl-EGTA and DM-nitrophen. Biophys. J. 70, 1006–1016 (1996).

    Article  CAS  Google Scholar 

  30. Lipp, P. & Niggli, E. Fundamental calcium release events revealed by two-photon photolysis of caged calcium in guinea-pig cardiac myocytes. J. Physiol. (Lond.) 508, 801–809 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by grants from the National Institutes of Health (GM53395), American Heart Association, McKnight Endowment Fund for Neuroscience, Human Frontiers Science Program, the Swiss National Science Foundation (3100-061344) and the PA Tobacco Fund.

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Author notes

  1. Atsuya Momotake

    Present address: Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba-shi Ibaraki-ken, 305-8577, Japan

Authors and Affiliations

  1. Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 North 15th St., Philadelphia, 19102, Pennsylvania, USA

    Atsuya Momotake & Graham C R Ellis-Davies

  2. Department of Physiology, University of Bern, Buhlplatz 5, Bern, CH-3012, Switzerland

    Nicolas Lindegger & Ernst Niggli

  3. Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust St., Philadelphia, 19107, Pennsylvania, USA

    Robert J Barsotti

Authors
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  5. Graham C R Ellis-Davies
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Corresponding author

Correspondence to Graham C R Ellis-Davies.

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Competing interests

G.C.R.E.-D. and A.M. have filed an application for a US patent for NBDF.

Supplementary information

Supplementary Fig. 1

NDBF-EGTA Ca2+ titration. (PDF 53 kb)

Supplementary Fig. 2

NDBF-EGTA Mg2+ titration. (PDF 287 kb)

Supplementary Fig. 3

NDBF-EGTA aci-nitro intermediate decay. (PDF 55 kb)

Supplementary Fig. 4

Generic nature of NDBF photochemistry. (PDF 24 kb)

Supplementary Table 1

Summary of the photochemical properties of coumarin caged compounds. (PDF 27 kb)

Supplementary Methods (PDF 64 kb)

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Momotake, A., Lindegger, N., Niggli, E. et al. The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells. Nat Methods 3, 35–40 (2006). https://doi.org/10.1038/nmeth821

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