Targeting tyrosine: a catch-and-release approach to protein modification
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Protein modification is an essential tool in Chemical Biology, allowing a functional biomolecule to be equipped with a small molecule tag or label. However, as proteins are constructed from a limited palette of around 20 canonical amino acids, achieving selective modification can be problematic. Previously reported methods for protein modification will be discussed in Chapter 1; these often rely on alteration of the protein sequence to introduce a uniquely reactive (often non-canonical) amino acid which may then be covalently modified in a bioorthogonal manner. An alternative approach is to identify a uniquely reactive site within the native protein sequence, such as the protein N-terminus or the reactive side chain of an amino acid with low frequency, and modify this using selective chemistry. In this project, modification of a native sequence protein was achieved by targeting a low abundance residue, tyrosine (Tyr), in a selective manner. Tyr was identified as the ideal candidate as it displays only ~3% frequency in the proteome and, due to its electron-rich aryl ring, it can be selectively modified by electrophilic aromatic substitution. Using a diazonium salt as the tuned electrophile, modification results in formation of an azobenzene motif which may be orthogonally cleaved under mild reducing conditions. The resulting cleavage product bears an o-aminophenol modification on the Tyr side chain, which can then be conjugated to a fluorescent label using established chemistry. This system has been developed on a solid-phase platform to give further control over the extent of modification achieved. In Chapter 2, the component parts of this method are developed through reactions performed in-solution on small molecule substrates. In Chapter 3, this work is then moved onto a solid-phase resin in order to ‘catch-and-release’ small molecule and peptide substrates. Finally in Chapter 4, the resin-based catch-and-release system is optimised for use in protein modification, and analysis of the modification site is explored.