Click Chemistry Applications

Click Chemistry Applications

Although the copper(I)-catalyzed alkyne-azide [3+2] cycloaddition reaction has many potential uses as a method for synthesis of unique oligo-based research tools, two specific applications currently dominate, (A) conjugation of anti-sense/siRNA oligonucleotides to cell-penetrating peptides (CPP), and (B) labeling of oligonucleotides with biotin and/or fluorescent dyes.

The efficacy of anti-sense and siRNA oligos in vivo is severely limited due to their inability to cross the cell membrane (3). One method for significantly increasing cell uptake of these oligos is to covalently conjugate cell penetrating peptides (CPP) to the oligo, most commonly through amide or disulfide linkages (4,5). While relatively straightforward to perform, such conjugations show wide variability in final yield, and often require complex and/or multiple purification. In addition, if the peptide is highly cationic, achieving a successful conjugation reaction can itself be problematic, due to non-specific binding between the cationic peptide and the anionic phosphate backbone of the oligo (6). Using click chemistry to form the CPP-oligo conjugate by linking an azidopeptide to an alkyne-modified oligo can provide an effective solution to these problems, as yield is essentially quantitative, and because the conjugation can be performed on a solid support, the need for expensive, multi-step purification is often eliminated (7-9). Please see the references provided for more details.

Similar advantages (quantitative yield, simple purification) favor the use of click chemistry for labeling oligos with such moieties as biotin, fluorescent dyes, and haptens (10-12). In all these cases, the oligo is alkyne-modified and the labels all contain an active azide group. A number of azide-modified labels are available, including biotin, desthiobiotin, 6-FAM, HEX and TET, among others. Additional labels will become available over time.

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.