Click Chemistry Design and Protocols

Click Chemistry Design / Protocol

This section contains an example of a Copper(I)-catalyzed click reaction. This protocol may be used as a starting point for optimization of your particular click chemistry procedures.

I. Preparation of the 'Click Solution'

1. NOTE: The 'click solution' (0.1 M CuBr / 0.1 M TBTA 1:2 (v/v) in DMSO/t-BuOH 3:1 (v/v)) must always be freshly prepared prior to use!
2.Dissolve 1 mg CuBr in 70 μl DMSO/t-BuOH 3:1 (v/v) to obtain a 0.1 M solution. This solution must be freshly prepared and cannot be stored.
3.Dissolve 54 mg TBTA in 1 ml DMSO/t-BuOH 3:1 (v/v) for a 0.1 M solution. This solution can be stored at -20C.
4.Add 1 volume of the 0.1 M CuBr solution quickly to 2 volumes of the 0.1 M TBTA solution to obtain the click solution, which is ready to use.

II. Click Procedure for Short DNA Oligos

Procedure using CuBr: To 5 μl of a 2 mM DNA solution (10 nmol) in water, 2 μl of an azide solution (50 mM, 50 nmol, 5 eq. in DMSO or in 3:1 (v/v) DMSO/t-BuOH), 3 μl of a freshly prepared solution containing 0.1 M CuBr and 0.1 M TBTA ligand in a 1:2 (v/v) ratio in 3:1 (v/v) DMSO/t-BuOH is added. The mixture is thoroughly mixed and shaken at 25C for 3 h. The reaction is subsequently diluted with 0.3 M NaOAc (100 μl) and the DNA is precipitated using 1 ml cold EtOH. The supernatant is then removed and the residue is washed twice with 1 ml cold EtOH. The washed residue is re-dissolved in pure water (20 μl) and can be used without further purification.

References

1. 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. (2002), 41: 2596-2599.
2. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
3. Akhtar, S., Hughes, M.D., Khan, A., Bibby, M., Hussain, M., Nawaz, Q., Double, J., Sayyed, P. The delivery of antisense therapeutics. Adv. Drug. Deliv. Rev. (2000), 44: 3-21.
4. Lundin, P., Johansson, H., Guterstam, P., Holm, T., Hansen, M., Langel, A., El Andaloussi, S. Distinct uptake routes of cell-penetrating peptide conjugates. Bioconjug. Chem. (2008), 19: 2535-2542.
5. Frederic, H., May Catherine, M., Gilles, D., Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br. J. Pharmacol. (2009), 157: 195-206.
6. Lu, K., Duan, Q-P., Ma, L., Zhao, D-X. Chemical strategies for the synthesis of peptide-oligonucleotide conjugates. Bioconjug. Chem. (2010), 21: 187-202.
7. Gogoi, K., Mane, M.V., Kunte, S.S., Vaijayanti, A.K. A versatile method for the preparation of conjugates of peptides with DNA/PNA/analog by employing chemo-selective click reaction in water. Nucleic Acids Res., (2007), 35: e139.
8. Brown, S.D., Graham, D. Conjugation of an oligonucleotide to Tat, a cell-penetrating peptide, via click chemistry. Tet. Lett., (2010), 51: 5032-5034.
9. Wenska, M., Alvira, M., Steunenberg, P., Stenberg, A., Murtola, M., Stromberg, R. An activated triple bond linker enables ‘click’ attachment of peptides to oligonucleotides on solid support. Nucleic Acids Res., (2011), 39: 9047-9059.
10. Seo, T.S., Li, Z., Ruparel, H., Ju, J. Click chemistry to construct fluorescent oligonucleotides for DNA sequencing. J. Org. Chem., (2003), 68: 609-612.
11. Hall, L.M., Gerowska, M., Brown, T. A highly fluorescent DNA toolkit: synthesis and properties of oligonucleotides containing new Cy3, Cy5 and Cy3B monomers. Nucleic Acids Res., (2012), 40: e108.
12. Wengel, J., Astakhova, I.K. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes. Chemistry, (2013), 19: 1112-1122.