Creating Artificial Metallo-Enzymes for C-H Activation Chemistry

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Nature uses a standard alphabet of 20 amino acids, specified by the genetic code, to create enzymes capable of catalyzing a diverse array of complex transformations. As a consequence, our existing enzyme production and engineering strategies rely exclusively on these natural amino acid building blocks, which contain limited functionality and are not suitable for the creation of artificial enzymes with truly novel activities. Within this project, we will exploit state-of-the-art techniques in protein engineering to install 'chemically programmed' ligands and/or noble metal co-factors into existing metallo-enzyme scaffolds, in order to create artificial enzymes for selective 'catalyst controlled' functionalizations of unactivated C-H bonds. This strategy combines the benefits of small molecule- and enzyme- catalysis by allowing optimization of the local co-ordination environment surrounding the catalytic centre whilst maintaining the tunable protein environment required for selective substrate orientation and stabilization reactive intermediates. The recently discovered lytic polysaccharide monooxygenases (LPMOs) will be exploited as host templates to incorporate new chemically programmed active site environments. LPMOs utilize an unusual copper co-ordination environment to achieve the oxidation of normally inert C-H bonds, in which an N-terminal methylated residue serves as a bidentate ligand to the catalytic metal center. To identify suitable 'active site' environments for C-H activation chemistry, we will initially create small molecule copper and noble metal (e.g. Ir, Rh, Ru) complexes containing short synthetic peptides as functional ligands to mimic the bidentate co-ordination environment found in LPMOs. The catalytic properties of these 'enzyme mimics' will be evaluated towards a range of synthetically valuable C-H functionalizations to generate C-C, C-N, C-O and C-X (X = F, Cl, Br, I,) bonds. The experimental data will be compared with DFT calculations to explore the effects of non-standard amino acid ligands on the catalytic properties of these transition metal complexes. Finally we will exploit genetic code expansion technology to install optimized active site environments into LPMO protein scaffolds to create artificial enzymes for 'catalyst controlled' C-H functionalization. Significantly, these first generation biocatalysts are readily amenable to further optimization using established protein engineering technologies. This is a highly interdisciplinary project at the cutting edge of enzyme design and engineering research, and will provide the student with expertise in organic synthesis, molecular biology, protein expression / purification, biochemical assays, bioinorganic chemistry and computational enzymology.