Biosynthesis of five-membered heterocyclic rings

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We propose to investigate the mechanism of the formation of heterocycles by enzyme. These five membered rings are critical components of many important biologically active molecules. We have made significant progress in determining the chemical mechanism, which is the first step to harnessing the enzyme. We intend to pursue the mechanism by novel labeling strategies including stable isotope labeling of substrates. We will also probe the basis of recognition using NMR approaches to identify the crucial residues involved in binding substrate to the enzyme. This is important because in the long term we would wish to replace amino acids by non peptide like groups. We have shown that NMR appears to detect intermediates that form during the reaction. The oxidation state of the rings is important, since even this subtle modification controls activity and stability of the compounds. We have shown that air can be sued to replace in part the enzyme mechanism. We intend to develop further the chemical route but also to study the enzymatic route. There are important and puzzling differences between enzymes that catalyse this reaction. The ability to control the stereocentres of amino acids is also central the activity of these compounds. The mechanism by which residues adjacent to thiazolines are epimerised is unknown. We will determine whether it is spontaneous or enzyme catalysed. We have developed an approach using a combination of peptide synthesis and protein ligation that will allow us incorporate non natural amino acids. This will not only give us exquisite control of the biochemical experiments but lead to more interesting chemical scaffolds in the future.

Who might benefit from this research? In terms of economic impact the main beneficiaries will be the UK Industrial
Biotechnology sector. We see the technology as enabling new biologically active molecules. The adoption of new 'greener'
biotransformation processes will enable the difficult transformations required for the production of fine chemicals and
pharmaceuticals. The pharmaceutical industry in the UK generates a positive trade balance, and innovation in these
sectors is critical for the success of the UK as a whole. Society will benefit by the production of new pharmaceutical lead
molecules.
How might they benefit from this research? The Industrial Biotechnology sector will benefit from adopting new but de-risked
technology. Understanding this very useful flexible enzyme will give rise to whole new materials we test for bioactivity in a
number of disease targeted screens. These compounds can then be developed for commercial application and can be
licenced or co-developed with industry. There is an urgent need for a diverse arrays of complex molecules to refill the
pharmaceutical drug discovery pipelines. Our approach will produce materials that can be modified easily and thus tuned to
a particular application. New materials with unusual properties will also be produced as part of this work and these may
provide new products or ideas for new products. The production of heterocycles is difficult to achieve synthetically and
often gives low yields despite the use of large quantities of reagents. The use of efficient biotransformation enzymes will
reduce the use of chemical reagents, solvents, energy and waste products.