Harnessing the Power of Diels-Alderases in Sustainable Chemoenzymatic Synthesis

Every chemical element is special, but some elements are more special than others! Carbon, the sixth element of the periodic table, is unique with respect to its versatility and impact on our lives. Carbon is the foundational element of all organic molecules including for example, materials, pharmaceuticals and fuels. No other element has shaped the world around us more than carbon. For this reason, the development of innovative methods to link carbon atoms together in desirable structures is of tremendous importance and is an overarching ambition in the field of organic chemistry. An important example of a reaction that can be used to link carbon atoms together is the Diels-Alder reaction, which since its discovery has been used to construct the complex carbon skeletons of numerous important molecules including pharmaceuticals, vitamins, hormones, agrochemicals and a raft of fragrance and flavour compounds. Historically, the Diels-Alder reaction has been performed using either chemical catalysts or high temperatures and pressures. Unfortunately, these approaches can give a mixture of products and have detrimental sustainability issues (use of energy and metal catalysts). However, biological catalysts for this reaction, so called 'Diels-Alderases', offer an attractive alternative, circumventing many of the complexities associated with chemical catalysis, and thus enabling Diels-Alder reactions to be performed under ambient conditions, with exquisite regio- and stereochemical control, and in an inherently 'greener' way. In this academic-industrial project, which builds on a strong foundation of interdisciplinary collaborative research by the applicants in the study of natural Diels-Alderases, the researchers will: i) Develop flow systems using immobilised enzymes to catalyse Diels-Alder reactions on gram scales; ii) investigate the ability of natural Diels-Alderases and rationally-engineered variants to catalyse both intramolecular (with both reactive groups within the same molecule) and intermolecular (with reactive groups in different molecules) cycloaddition reactions; iii) study hitherto uncharacterised natural Diels-Alderases and their associated natural products from cryptic biosynthetic gene clusters; iv) Deploy our portfolio of natural and engineered Diels-Alderases, in combination with auxiliary enzymes, to undertake chemoenzymatic total syntheses of high-value target compounds.

The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes and is considered the prototypical pericyclic reaction. Despite its unquestionable usefulness, detrimental features include general requirements for harsh reaction conditions (heat, pressure, metal catalysts) and may lead to mixtures of products. These complexities have fuelled recent efforts focused on the discovery and development of protein catalysts for this reaction, which hold promise in offering stereoselective 'green' routes to cyclohexenes at room temperature and pressure. Despite encouraging progress, the use of Diels-Alderases in the preparation of high-value molecules remains a nascent endeavour, with significant questions remaining regarding the structures and functions of these biocatalysts, along with their suitability for use in academic and industrial applications. In this project, which builds on a strong foundation of interdisciplinary research by the applicants in the identification, characterisation and exploitation of natural Diels-Alderases and associated biocatalysts, the researchers will: i) Develop and optimise a flow platform for biocatalytic Diels-Alder reactions using immobilised enzymes; ii) establish the molecular basis of Diels-Alderase catalysed intermolecular cycloaddition reactions, employing a range of chemical, molecular, structural and computational methods, undertaking structure and simulation-guided reengineering and directed evolution of these enzymes, to enable the diversification of their substrate specificities and catalytic activities; iii) build on our established knowledge and methods to identify and study hitherto uncharacterised natural Diels-Alderases and their associated natural products from cryptic biosynthetic gene clusters; iv) deploy our portfolio of natural and engineered Diels-Alderases, in combination with auxiliary enzymes, to undertake chemoenzymatic total syntheses of target compounds.