Team:TU Eindhoven/SPAAC

In order to functionalize bacterial membranes with polymers, two strategies can be followed; one can either engineer the bacteria in such a way that it produces the entire polymer, this strategy is further discussed under ??? , or only produces a ‘handle’ on which polymers can be reached. The latter strategy requires so-called bio-orthogonal chemistry; chemistry in which the two components are non-interacting (orthogonal) to the functionality presented in biological systems. Furthermore, the reaction conditions have to be viable for cells; in water, at (near-) neutral pH, at temperatures ranging from 25 to 37°C and without any cytotoxic reagents or by-products. (Baskin & Bertozzi, 2007)

A common functional group used in bio-orthogonal reactions is the azide, which does not exist among or reacts with functional groups in biology and is both kinetically stable and thermodynamically high in energy to specific reactivity. Using the azide functional group, two components can be linked together inherently efficient, therefore those types of reactions are called click reactions. (Baskin & Bertozzi, 2007) Until now, three bio-orthogonal click reactions are known: Staudinger ligation, Copper catalysed [3+2] azide-alkyne cycloaddition and Strain promoted [3+2] azide-alkyne cycloaddition.

The main advantage of these reactions is that they can applied to in vivo incorporation of unnatural amino acids containing azides or alkynes during translation and expression of proteins. In this way, in theory all bio-orthogonal molecules, such as fluorescent or chemical reporters, can be ‘clicked’ on proteins. (Meldal & Tornoe, 2008)

Staudinger ligation The Staudinger ligation is a modification of the classical Staudinger reaction, which implies the use of phosphines and azides. (Figure 1) The Staudinger ligation is arguably the first bio-orthogonal reaction and involves two fully abiotic functional groups and takes place at ambient temperature, in water and at neutral pH. (Baskin & Bertozzi, 2007) However, Vugts et al. showed that the Staudinger ligation is not bio-orthogonal and efficient enough in mice and that the slow reaction kinetics also severely restrict the applicability of the Staudinger ligation in vivo. (Vugts, et al., 2011)



Bibliography

Agard, N. J., Prescher, J. A., & Bertozzi, C. R. (2004). A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. Journal of the American Chemical Society, 126, 15046-15047.

Baskin, J. M., & Bertozzi, C. R. (2007). Bioorthogonal click chemistry: covalent labeling in living systems. QSAR & Combinatorial Science, 26(11-12), 1211 - 1219.

Debets, M. F., Prins, J. S., Merkx, D., van Berkel, S. S., van Delft, F. L., van Hest, J. C., & Rutjes, F. P. (2014). Synthesis of DIBAC analogues with excellent SPAAC rate constants. Organic & Biomolecular Chemistry, 12, 5031-5037.

Meldal, M., & Tornoe, C. W. (2008). Cu-catalyzed azide-alkyne cycloaddition. Chemical Reviews, 108, 2952-3015.

Vugts, D. J., Vervoort, A., Stigter-van Walsum, M., Visser, G. W., Robillard, M. S., Versteegen, R. M., . . . van Dongen, G. A. (2011). Synthesis of phosphine and antibody-azide probes for in vivo Staudinger ligation in a pretargeted imaging and therapy approach. Bioconjugate Chemistry, 22, 2072-2081.

Yang, M., Li, J., & Chen, P. R. (2014). Transition metal-mediated bioorthogonal protein chemistry in living cells. Chemical Society Reviews, 43(18), 6475-6660.