Exploiting Halogenase Enzymes: New Reaction Pathways via Enzymatic CH Activation

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The halogens fluorine, chlorine, and bromine are amongst the most reactive elements of the periodic table, combining vigorously with other molecules when used in their native, elemental form. As part of other molecules, however, they play a quite different role, imparting valuable stability and introducing favourable functional properties. Fluorine, chlorine, and to a lesser extent bromine, are found in numerous pharmaceuticals (including drugs critical to human health such as the antibiotics vancomycin, chloramphenicol, and ciproflaxin), agrochemicals that boost crop yields, polymers and other valuable materials. Molecules possessing halogen substituents (organohalogens) are also widely used intermediates for making drugs and other products, as the halogens can be readily substituted for a range of other functional groups.Currently the manufacture of organohalogen compounds involves multistep synthetic chemical methods which use deleterious solvents, harsh chemical halogenating reagents and expensive catalysts, as well as non-renewable petrochemical precursors, which have serious detrimental environmental impact. In this project we aim to develop alternative biotechnology based processes for more economic and environmentally sustainable production of halogenated molecules. To do this we will exploit enzymes that nature has evolved (halogenases) to selectively install halogens at specific positions within target molecules of industrial importance. Nature's halogenating enzymes have evolved to halogenate natural products at very low concentrations in living systems. As a result, they do not generally have the required activity and selectivity for halogenation of pharmaceutical or agrochemical target molecules on a useful scale. In light of this, we will determine the structures of promising halogenase enzymes using X-ray crystallography to obtain a 3D image of the enzyme's active site where the substrate binds. We will use this picture to change the 3D structure of the active site using a technique called mutagenesis, which will enable the enzyme to accommodate non-natural target molecules with enhanced efficiencies. In addition to targeted approaches we will also use more random mutagenesis techniques to create libraries (many millions) of mutant halogenase enzymes, followed by new mass spectrometry imaging technology and fluorescence assays to select mutants with the required activity and selectivity.These new, optimised, halogenase enzymes will then be used to produce the key halogenated building blocks that are required for the manufacture of pharmaceuticals including antiviral agents, anticancer agents and other drugs essential for human healthcare, as well as agrochemicals that urgently are required to boost crops yields and provide more food for the growing global population. Our enzymes can also be used to introduce halogens at new positions in biologically active molecules, creating new analogs that would be difficult or impossible to access by conventional chemical halogenation methods. This ability of halogenase enzymes to selectively place chlorine or bromine atoms into molecules opens up exciting opportunities to manipulate the halogenated products in further chemical transformations. Halogens are extremely versatile functional groups, enabling us to combine halogenases with other chemical catalysis steps to create new entities featuring (for example) carbon-nitrogen, carbon-carbon, or carbon-fluorine bonds. In each case, the exquisite selectivity of the halogenase enzyme provides a chemical shortcut to making valuable products that would otherwise be made via long-winded and expensive synthetic routes.