Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Bis-triazolyl diguanosine derivatives as synthetic transmembrane ion channels

Nature Protocols volume 11, pages 1039–1056 (2016)Cite this article

Subjects

Abstract

In nature, ion channels facilitate the transport of ions across biological membranes. The development of artificial ion channels that can mimic the fundamental functions of the natural ones would be of great importance to biological research. Artificial ion channels based on nucleoside derivatives are expected to be biocompatible with functions that can be controlled by the presence or absence of biologically relevant molecules. This protocol describes the detailed procedures for the synthesis and ion-channel activity of four diguanosine derivatives, each made up of two guanosine moieties separated by a covalent linker (e.g., PEG). The procedure describes the preparation of guanosine azide and guanosine alkine building blocks, as well as the preparation of four distinct synthetic linkers each containing either two alkynes or two azides. The diguanosine derivatives are synthesized using a 'one-pot' modular synthetic approach based on Cu(I)-catalyzed azide and alkyne cycloaddition. The ion-channel activity of these diguanosine derivatives for the transportation of ions across a phospholipid bilayer is investigated using voltage-clamp experiment. By using the PEG-containing diguanosine as an example, we describe how to determine the ion-channel activity in the presence of different metal ions (e.g., Na+, K+ and Cs+) and the inhibition of the ion-channel activity using the nucleobase cytosine. The approximate time frame for the synthesis of the PEG dinucleoside is 3 d, and that for the experiments to evaluate its ability to transport K+ ion across a phospholipid bilayer is ∼8–10 h.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Guanine derivatives and their self-interactions and assembly as basis for the development of artificial, diguanosine-based ion channels.
Figure 2
Figure 3
Figure 4: Synthesis of diguanosine derivatives using Cu(I)-catalyzed azide-alkyne cycloaddition.
Figure 5: Traces of single-channel electrical conductance recordings obtained after the addition of a 20 μM solution of a diguanosine derivative to the cis side of the chamber after the planar lipid bilayer formation.
Figure 6: Results of voltage-clamp experiments showing representative states recorded after the addition of (G-G)-2 (20 μM) to the cis side of the chamber after planar lipid bilayer formation.
Figure 7: Inhibition of (G-G)-2-based ion channels using cytosine.
Figure 8: Formation of GUVs.
Figure 9: GUV preparation scheme.
Figure 10: Photographs.
Figure 11: Schematic illustration of a voltage-clamp experiment.

Similar content being viewed by others

References

  1. Alberts, B. et al. Molecular Biology of the Cell 5th edn. (Garland Science, 2007).

  2. Hille, B. Ion Channels of Excitable Membranes 3rd edn. (Sinauer Associates, 2001).

  3. Sisson, A.L., Shah, M.R., Bhosale, S. & Matile, S. Synthetic ion channels and pores (2004–2005). Chem. Soc. Rev. 35, 1269–1286 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Fyles, T.M. Synthetic ion channels in bilayer membranes. Chem. Soc. Rev. 36, 335–347 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Chui, J.K.W. & Fyles, T.M. Ionic conductance of synthetic channels: analysis, lessons, and recommendations. Chem. Soc. Rev. 41, 148–175 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. Gokel, G.W. & Carasel, I.A. Biologically active, synthetic ion transporters. Chem. Soc. Rev. 36, 378–389 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Szyman´ski, W., Beierle, J.M., Kistemaker, H.A.V., Velema, W.A. & Feringa, B.L. Reversible photocontrol of biological systems by the incorporation of molecular photoswitches. Chem. Rev. 113, 6114–6178 (2013).

    Article  CAS  Google Scholar 

  8. Itoh, H. & Inoue, M. Chemical construction and structural permutation of potent cytotoxin polytheonamide B: discovery of artificial peptides with distinct functions. Acc. Chem. Res. 46, 1567–1578 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. Otis, F., Auger, M. & Voyer, N. Exploiting peptide nanostructures to construct functional artificial ion channels. Acc. Chem. Res. 46, 2934–2943 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Fyles, T.M. How do amphiphiles form ion-conducting channels in membranes? Lessons from linear oligoesters. Acc. Chem. Res. 46, 2847–2855 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Gokel, G.W. & Negin, S. Synthetic ion channels: from pores to biological applications. Acc. Chem. Res. 46, 2824–2833 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Gong, B. & Shao, Z. Self-assembling organic nanotubes with precisely defined, sub-nanometer pores: formation and mass transport characteristics. Acc. Chem. Res. 46, 2856–2866 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Sakai, N., Mareda, J. & Matile, S. Ion channels and pores, made from scratch. Mol. Biosyst. 3, 658–666 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Sakai, N. et al. Dendritic folate rosettes as ion channels in lipid bilayers. J. Am. Chem. Soc. 128, 2218–2219 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Kaucher, M.S., Harrell, W.A. Jr. & Davis, J.T. A unimolecular G-quadruplex that functions as a synthetic transmembrane Na+ transporter. J. Am. Chem. Soc. 128, 38–39 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Ma, L., Melegari, M., Colombini, M. & Davis, J.T. Large and stable transmembrane pores from guanosine-bile acid conjugates. J. Am. Chem. Soc. 130, 2938–2939 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Ma, L., Harrell, W.A. Jr. & Davis, J.T. Stabilizing guanosine-sterol ion channels with a carbamate to urea modification in the linker. Org. Lett. 11, 1599–1602 (2009).

    Article  CAS  PubMed  Google Scholar 

  18. Kumar, Y.P. et al. Triazole-tailored guanosine dinucleosides as biomimetic ion channels to modulate transmembrane potential. Chemistry 20, 3023–3028 (2014).

    Article  CAS  PubMed  Google Scholar 

  19. Das, R.N., Kumar, Y.P., Schütte, O.M., Steinem, C. & Dash, J.A. DNA-inspired synthetic ion channel based on G-C base pairing. J. Am. Chem. Soc. 137, 34–37 (2015).

    Article  CAS  PubMed  Google Scholar 

  20. Langecker, M. et al. Synthetic lipid membrane channels formed by designed DNA nanostructures. Science 338, 932–936 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bayley, H. & Jayasinghe, L. Functional engineered channels and pores. Mol. Membr. Biol. 21, 209–220 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Davis, J.T. & Spada, G.P. Supramolecular architectures generated by self-assembly of guanosine derivatives. Chem. Soc. Rev. 36, 296–313 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Pieraccini, S. et al. Solvent-induced switching between two supramolecular assemblies of a guanosine-terthiophene conjugate. Org. Biomol. Chem. 8, 774–781 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Simeone, L. et al. Design, synthesis and characterization of guanosine-based amphiphiles. Chemistry 17, 13854–13865 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Lena, S., Neviani, P., Masiero, S., Pieraccini, S. & Spada, G.P. Triggering of guanosine self-assembly by light. Angew. Chem. Int. Ed. 49, 3657–3660 (2010).

    Article  CAS  Google Scholar 

  26. Lena, S. et al. Self-assembly of an alkylated guanosine derivative into ordered supramolecular nanoribbons in solution and on solid surfaces. Chemistry 13, 3757–3764 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Li, Y. et al. Influence of tunable external stimuli on the self-assembly of guanosine supramolecular nanostructures studied by atomic force microscope. Langmuir 25, 13432–13437 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. Arnal-Hrault, C. et al. Functional G-quartet macroscopic membrane films. Angew. Chem. Int. Ed. 46, 8409–8413 (2007).

    Article  CAS  Google Scholar 

  29. Sreenivasachary, N. & Lehn, J.M. Structural selection in G-quartet-based hydrogels and controlled release of bioactive molecules. Chem. Asian J. 3, 134–139 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Das, R.N., Kumar, Y.P., Pagoti, S., Patil, A.J. & Dash, J. Diffusion and birefringence of bioactive dyes in a guanosine supramolecular hydrogel. Chemistry 18, 6008–6014 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Buerkle, L.E., von Recum, H.A. & Rowan, S.J. Toward potential supramolecular tissue engineering scaffolds based on guanosine derivatives. Chem. Sci. 3, 564–572 (2012).

    Article  CAS  Google Scholar 

  32. Peters, G.M. et al. A G4·K+ hydrogel stabilized by an anion. J. Am. Chem. Soc. 136, 12596–12599 (2014).

    Article  CAS  PubMed  Google Scholar 

  33. Peters, G.M. et al. G4-quartet·M(+) borate hydrogels. J. Am. Chem. Soc. 137, 5819–5827 (2015).

    Article  CAS  PubMed  Google Scholar 

  34. Balasubramanian, S., Hurley, L.H. & Neidle, S. Targeting G-quadruplexes in gene promoters: a novel anticancer strategy? Nat. Rev. Drug Discov. 10, 261–275 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Collie, G.W. & Parkinson, G.N. The application of DNA and RNA G-quadruplexes to therapeutic medicines. Chem. Soc. Rev. 40, 5867–5892 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Zhang, B., Cui, Z. & Sun, L. Synthesis of 5′-deoxy-5′-thioguanosine- 5′-monophosphorothioate and its incorporation into RNA 5′-termini. Org. Lett. 3, 275–278 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Likhitsup, A., Yu, S., Ng, Y.H., Chai, C.L.L. & Tam, E.K.W. Controlled polymerization and self-assembly of a supramolecular star polymer with a guanosine quadruplex core. Chem. Commun. 4070–4072 (2009).

  38. Daz, D.D., Rajagopal, K., Strable, E., Schneider, J. & Finn, M.G. 'Click' chemistry in a supramolecular environment: stabilization of organogels by copper (I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 128, 6056–6057 (2006).

    Article  CAS  Google Scholar 

  39. Eissa, A.M. & Khosravi, E. Synthesis of a new smart temperature responsive glycopolymer via click-polymerisation. Eur. Polym. J. 47, 61–69 (2011).

    Article  CAS  Google Scholar 

  40. Thomas, J.R., Liu, X. & Hergenrother, P.J. Size-specific ligands for RNA hairpin loops. J. Am. Chem. Soc. 127, 12434–12435 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Rostovtsev, V.V., Green, L.G., Fokin, V.V. & Sharpless, K.B. A stepwise Huisgen cycloaddition process: copper (I)-catalyzed regioselective 'ligation' of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002).

    Article  CAS  Google Scholar 

  42. Kurz, A. et al. Lipid-anchored oligonucleotides for stable double-helix formation in distinct membrane domains. Angew. Chem. Int. Ed. 45, 4440–4444 (2006).

    Article  CAS  Google Scholar 

  43. Mathivet, L., Cribier, S. & Devaux, P.F. Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. Biophys. J. 70, 1112–1121 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Angelova, M.I. & Dimitrov, D.S. Liposome electroformation. Faraday Discuss. Chem. Soc. 81, 303–311 (1986).

    Article  CAS  Google Scholar 

  45. Itoh, H., Matsuoka, S., Kreir, M. & Inoue, M. Design, synthesis and functional analysis of dansylated polytheonamide mimic: an artificial peptide ion channel. J. Am. Chem. Soc. 134, 14011–14018 (2012).

    Article  CAS  PubMed  Google Scholar 

  46. Coppola, C. et al. Design, synthesis and characterisation of a fluorescently labelled CyPLOS ionophore. Chemistry 16, 13757–13772 (2010).

    Article  CAS  PubMed  Google Scholar 

  47. You, L. & Gokel, G.W. Fluorescent, synthetic amphiphilic heptapeptide anion transporters: evidence for self-assembly and membrane localization in liposomes. Chem. Eur. J. 14, 5861–5870 (2008).

    Article  CAS  PubMed  Google Scholar 

  48. Andersson, M. et al. Detection of single ion channel activity on a chip using tethered bilayer membranes. Langmuir 23, 2924–2927 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Krause, M.R. & Regen, S.L. The structural role of cholesterol in cell membranes: from condensed bilayers to lipid rafts. Acc. Chem. Res. 47, 3512–3521 (2014).

    Article  CAS  PubMed  Google Scholar 

  50. Hahn, F.E., Langenhahn, V. & Pape, T. Template synthesis of tungsten complexes with saturated N-heterocyclic carbene ligands. Chem. Commun. 5390–5392 (2005).

Download references

Acknowledgements

This work was supported by the Board of Research in Nuclear Sciences (BNRS), Department of Atomic Energy (DAE) and Department of Biotechnology (DBT) India. The DFG (SFB 803, project A01) is gratefully acknowledged. Y.P.K. and R.N.D. thank Council of Scientific and Industrial Research (CSIR), India, and R.N.D. thanks the Deutscher Akademischer Austauschdienst (DAAD) Exchange Programme for a research fellowship.

Author information

Authors and Affiliations

  1. Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata, India

    Y Pavan Kumar, Rabindra Nath Das & Jyotirmayee Dash

  2. Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India

    Rabindra Nath Das & Jyotirmayee Dash

  3. Institute of Organic and Biomolecular Chemistry, Georg-August-University Göttingen, Göttingen, Germany

    Ole Mathis Schütte & Claudia Steinem

Authors
  1. Y Pavan Kumar
    View author publications

    Search author on:PubMed Google Scholar

  2. Rabindra Nath Das
    View author publications

    Search author on:PubMed Google Scholar

  3. Ole Mathis Schütte
    View author publications

    Search author on:PubMed Google Scholar

  4. Claudia Steinem
    View author publications

    Search author on:PubMed Google Scholar

  5. Jyotirmayee Dash
    View author publications

    Search author on:PubMed Google Scholar

Contributions

J.D. and C.S. designed the experiments; Y.P.K. synthesized the compounds; R.N.D. and O.M.S. carried out the voltage-clamp experiments; and J.D. wrote the paper with the input from all the authors.

Corresponding authors

Correspondence to Claudia Steinem or Jyotirmayee Dash.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kumar, Y., Das, R., Schütte, O. et al. Bis-triazolyl diguanosine derivatives as synthetic transmembrane ion channels. Nat Protoc 11, 1039–1056 (2016). https://doi.org/10.1038/nprot.2016.045

Download citation

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/nprot.2016.045

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing