Exploiting Halogenase Enzymes: New Reaction Pathways via Enzymatic CH Activation

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.

Halogens can dramatically improve the properties of organic molecules for selective modulation of biological targets in vivo and as a consequence >30% of pharmaceuticals and a high proportion of agrochemicals are halogenated. Halogen substituents also provide an orthogonal handle for derivatisation using a diverse range of cross-coupling reactions that are widely used in industry. Despite the importance of halogenated compounds, current halogenation methods use deleterious reagents/catalysts and solvents, lack regiocontrol and result in toxic by-products. This project aims to address these shortcomings by developing halogenase enzymes (Fl-Hal), using benign inorganic halides in water, for regioselective halogenation of structurally diverse bioactive scaffolds, leading to more efficient, economic and environmentally sustainable biomanufacture of pharmaceuticals, agrochemicals and other valuable materials.

We will mine genomes to discover new Fl-Hal. We will develop novel high-throughput mass spectrometry imaging and fluorescence screening approaches to rapidly evolve Fl-Hal, with improved properties, increasing the range of scaffolds that can be halogenated. The optimised Fl-Hal will then be used for the regioselective halogenation of industrially relevant compounds. We will also integrate Fl-Hal with chemocatalysts, affording a net C-H activation process, providing access to a wide range of cross-coupled derivatives with additional aryl-, alkenyl-, amino-, cyano-, fluoro-, or trifluoromethyl-substituents (also common in drugs, agrochemicals and other materials) in single 'one-pot' transformations. The combination of multiple catalytic transformations in a single reaction vessel represents a potential step-change improvement in manufacturing efficiency; avoiding work-up and purification between individual reactions, improves space-time yields, reduces energy-consuming and waste-generating solvent removal steps and eliminates the need for auxiliary chemicals.

WHO WILL BENEFIT: All pharmaceutical and agrochemical companies, or the chemical manufacturers that supply these companies, produce halogenated compounds at scale using traditional chemical methods that are inefficient and polluting, using deleterious reagents/catalysts and volatile organic solvents. Many of the traditional halogenation methods also lack regiocontrol and result in mixtures of toxic by products that are persistent in the environment and therefore need careful disposal which is costly. In light of this, many pharmaceutical and agrochemical companies, and their suppliers, could potentially benefit if we are successful in developing processes that deliver industrially important halogenated compounds, using halogenase enzymes in water, with benign inorganic halides (e.g. NaCl). In addition, halogenated compounds are widely used in the synthesis of polymers and other materials. For example halogenated aromatic compounds are used to synthesise polymers for the organic light-emitting diodes (OLEDs) in mobile phones, TVs and computer screens etc. Companies that manufacture such polymers and materials, could also potentially benefit from the methods we develop. The mass spectrometry imaging technology we develop, will involve development of new and novel DESI-IM-MS instrumentation in collaboration with Waters. Success in evolving this technology for colony imaging on plates could encourage other labs to adopt this technology. Consequently, it is possible that Waters and other mass spectrometry manufacturers could benefit through increased sales of DESI-IM-MS instruments and accessories (consumables and materials).

HOW WILL THEY BENEFIT: We will work closely with pharmaceutical and agrochemical companies to explore targets (halogenation challenges) that would be strategically important to them. We are members of the UK Centre of Excellence in Biocatalysis (CoEBio3) which includes industrial affiliates from a number of leading pharma, agro and biotech companies who have specific interest in the industrial applications of enzymes. Through collaborations, and meetings of CoEBio3, several companies have already provided us with targets for halogenation. Success in delivering scalable halogenation reactions can lead to more formal collaborations with these companies, and possible follow-on-funding for development work. Similarly our Michael Barber Centre for Mass Spectrometry (lead by PB), has strong links with Waters and other MS companies, which will provide the necessary interface to ensure that any MS technology developments we make can be exploited by these companies. To further disseminate our research to the broader academic and industrial community we plan to host two workshops/small meetings, a biohalogenation workshop and an integrated catalysis workshop on the more general topic of developing processes where enzymes are integrated with chemocatalysts. A key objective of these workshops is to bring together academic, industrial and other end-user groups, to establish new partnerships and collaborations, as well as to raise the profile of UK IB. Finally, we will work with the University of Manchester Intellectual Property Ltd. (UMIP) to file patents to protect IP generated in this project, and to explore longer term partnerships and licensing agreements with industry, to further develop any enzymes, products or technologies that are of commercial or strategic interest to industry.