Novel halogenation and pallado-biology strategies for biological probes

Thesis

Post-translational modifications (PTMs), which biochemically modify proteins to generate diversity and control functionality, are important in the field of chemical biology research. However, typical bioconjugation methods involving nucleophilic addition of cysteine and lysine residues are limited by their lack of site-specificity. In this thesis, we have demonstrated the site-selective chemical modification of a variety of proteins through protein halogenation and subsequent pallado-biology strategies.

The "Tag-and-Modify" approach, developed previously in our group and involving the creation of a protein tag for further manipulation, is a useful tool for site-selective chemical modification. Here, we envisioned extending its utility to a novel concept of [¹⁸F]-radiolabelling of proteins involving direct electrophilic fluorination, thus forming a C-F bond at a specific modification site and leading to the generation of protein species applicable for in vivo [¹⁸F]-PET imaging and diagnosis. We also explored several alternative methods for the installation of C-Br/C-I bonds onto a protein of interest. Our first attempts involved the biosynthetic incorporation of a synthetic bromotryptophan into the protein, however these processes were unsuccessful. As a workaround, we found IPy₂BF₄, an iodinating agent, to be very effective as a direct method for the site-specific installation of C-I bonds on tyrosine/histidine residues of proteins. We subsequently demonstrated novel and efficient palladium-catalysed cross-couplings of the resultant iodinated tyrosine/histidine moieties, leading to the creation of new C-C bonds. This approach was compatible with a wide range of functionally diverse boronic acids, using a water-soluble and phosphine-ligand-free Pd-complex catalyst under fully aqueous conditions.

Our novel, successful method for C-C bond formation onto proteins is potentially applicable to the initial investigation of challenging in vivo Pd cross-coupling of thyroglobulin, based on our achievement of protein labelling under ex vivo conditions. In summary, we are able to create new chemical tools for the site-selective/site-specific chemical modification of proteins, enabling their use as biological probes.

Authors: Phanumartwiwath, A

• DOI: 10.5287/ora-nomjrye17
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Protein modification
• Subjects: Chemical Biology