Houszka, N. (2019). Intracellular retargeting via bioorthogonal substitution : synthesis of precursors for a proof-of-concept study [Diploma Thesis, Technische Universität Wien]. reposiTUm. http://hdl.handle.net/20.500.12708/79399
Bioorthogonal bond cleavage reactions have recently emerged as a thriving area of chemical research. The fastest known bioorthogonal reaction, the inverse electron demand Diels-Alder reaction between a tetrazine and a trans cyclooctene (TCO) has recently been modified to allow a click-to-release approach (Figure Ia) using a carbamate-functionalized “release”-TCO. In this controlled reaction, upon click, tautomerization triggers the release of a caged molecule. The click-to-release toolkit can further be utilized to enable bioorthogonal substitution and thus exchange of a ligand (Figure Ib). The concept is highly modular, further expanding the scope of bioorthogonal chemistry. Efficient click-to-release reactions are hampered, as fast click kinetics seemingly go hand in hand with slow and incomplete release. A novel release-TCO was developed in our group to enhance formation of the releasing tautomer. This system does not only exhibit a fast click reaction but also achieves efficient release, which has not been achieved so far. In this proof-of-concept study for the investigation of bioorthogonal substitution in living cells, the required chemical tools and several building blocks were synthesized, including water-soluble fluorophores for monitoring of in vitro experiments using fluorescence microscopy, but also targeting ligands. We aim to pave the way for so-called target-hopping approaches using bioorthogonal retargeting to increase the efficiency of drug or radionuclide delivery in cancer therapy (Figure II). There is only a limited number of abundant and sufficiently internalizing cancer cell receptors that can be targeted. Aiming for a non internalizing receptor in the first step leads to a higher concentration of the drug or radionuclide on the outside of the cell. Reaction with a second ligand modified with a tetrazine can then be used to achieve retargeting of the drug or radionuclide to an internalizing receptor, and thus cell uptake. Therefore, tumor-specific non-internalizing receptors can be used to target the tumor, while retargeting to non-specific internalizing receptors then leads to a stepwise drug delivery into cancer cells.