Team:TU Eindhoven/Background/SPAAC Reaction

SPAAC Reaction: Bio-Orthogonal Click Chemistry

In order to functionalize bacterial membranes with polymers, two strategies can be followed: one can either engineer the bacteria in such a way that they produce the entire polymer, this strategy is further discussed under Zwitterionic Antifouling Protein, or only produce an ‘anchor’ on which polymers can be reacted. 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:

1. Staudinger Ligation
2. Copper catalysed [3+2] azide-alkyne cycloaddition
3. Strain promoted [3+2] azide-alkyne cycloaddition

The main advantage of these reactions is that they can be 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)

Figure 1. A schematic overview of the Staudinger ligation.

Copper Catalysed [3+2] Azide-Alkyne Cycloaddition (CuAAC)

The reaction often referred to as ‘the click reaction’, is the extremely selective copper catalysed [3+2] cycloaddition of an azide and an alkyne (CuAAC). (Figure 2) CuAAC is a variant of the classical [3+2] cycloaddition discovered by Huisgen, in which copper serves as catalyst, creating a reaction that proceeds rapidly at room temperature. The copper is also the biggest disadvantage of this reaction, since this heavy metal is toxic to cells and organisms. (Meldal & Tornoe, 2008) Nowadays, several suitable Cu(I) ligands are available that minimize the cytotoxicity while maintaining or even further increasing the rate of CuAAC. Therefore, CuAAC has become a very feasible option for in vivo studies. (Yang, Li, & Chen, 2014)

Figure 2. A schematic overview of CuAAC.

Strain Promoted [3+2] Azide-Alkyne Cycloaddition (SPAAC)

In order to avoid the disadvantages of copper, a copper free version of CuAAC called SPAAC has been designed. In SPAAC, instead of copper, ring strain is used to activate the alkyne. (Figure 3) Agard et al. showed that an appropriately derivatized cyclooctyne can efficiently and selectively react with azides in biological mixtures. (Agard, Prescher, & Bertozzi, 2004)

Figure 3. A schematic overview of SPAAC.

Because of the efficiency and selectivity of SPAAC and the fact that no further reagents have to be added to the reaction mixture, SPAAC was chosen to further explore as a platform to functionalize bacterial membranes. Currently, the fastest cyclooctyne known is BARAC. However, this cyclooctyne has shown to be susceptible to Michael addition by thiols , which is not favourable when applying the system into a protein, since proteins contain lots of thiols (cysteines). Dibenzocycloocytne (DBCO) has a lower rate constant, but does not show susceptibility to Michael addition. (Debets, et al., 2014) Furthermore, there are already several DBCO-functionalized polymers and fluorophores commercially available. Therefore, the SPAAC reaction between DBCO-functionalized molecules and azide-functionalized proteins that have been incorporated into the membrane of E. coli has been chosen. (Figure 4)

Figure 4. A schematic overview of the bio-orthogonal click chemistry that will be conducted in our project: SPAAC using a DBCO-functionalized polymer A and an azide-functionalized amino acid B.

Bibliography

[1] 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.

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

[3] Chin, Jason W., Stephen W. Santoro, Andrew B. Martin, David S. King, Lei Wang, and Peter G. Schultz. Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichia coli. J. Am. Chem. Soc. 124.31 (2002): 9026-9027.

[4] 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.

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

[6] 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.

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