A Novel Site-Selective Bioconjugation Strategy for the Synthesis of Energy Harvesting Protein-Conjugates

Site-selective bioconjugation is a powerful technique that is important for production of well-defined conjugates, which is needed in many research fields, including biology, pharmacology and electro chemistry. It is, however, challenging to achieve good site-selectivity due to the high number of each amino acid in a protein of interest. There are around 300 amino acids in a protein, and with only 21 available amino acids to choose from, there will unavoidably be unselectivity. Another strategy involves the recombinant installation of unnatural amino acids. However, this is time consuming and often give low yields. In addition, the installation of an unnatural amino acid can alter the function of a protein. In this thesis is presented two novel strategies to achieve site-selective bioconjugation on a protein containing only natural amino acids. Both strategies utilize the site-specificity achieved by the phosphorylating enzyme PKA. In one strategy, an azido handle is site-selectively installed by utilizing an azide-functionalized ATP-analogues as PKA substrate. The other strategy makes use of the natural enzyme substrate ATP followed by a selective pyrophosphorylation reaction to install the azido functionality.
Connecting proteins to electrodes is important for varies applications, with energy harvesting being one important field. However, the effect of the resulting conjugate is highly dependent on the protein having the right orientation on the electrode, as well as a good bridging molecule to ensure efficient electron transfer. In this thesis, an anthraquinone-based bridge molecule is investigated, which contains a thiol handle for electrode binding and a handle allowing future conjugation to the protein.
These are the themes of this PhD thesis.