Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

The use of enzymes and synthetic biology strategies hold significant potential for the synthesis of pharmaceutical intermediates, and sustainable syntheses of chiral amines are highly sought after since 70% of all pharmaceutical are derivatives of chiral amines. To date chiral amines have been generated using biocatalytic strategies predominantly via the kinetic resolution of racemic mixtures with hydrolytic enzymes, where only a 50% maximum yield can be achieved. A deracemization biocatalytic strategy has also been described [1,2], however, recently interest has focused of the use of transaminases (TAm) to generate chiral amines in a genuinely asymmetric transformation [3]. This has the potential for a process to be developed using transaminases that would yield 100% of the asymmetric product, resulting in less waste and a lower cost strategy to these important synthons. TAms catalyse the transfer of an amino group from a donor such as an amino acid, to an acceptor ketone or aldehyde moiety. While the alpha-TAms have a strong preference for an alpha-keto acid as the acceptor and the preferred donor is usually one of the 20 alpha-amino acids, the omega-transaminases can transfer an amino group to an aldehyde or a ketone and do not frequently have a requirement for the alpha-keto acid moiety [4]. They also have a broader range of amino donors that they can use. In previous work by Shin and co-workers [5] an omega-TAm used for the amination of a wide range of ketones and aldehydes, including aromatic substrates, was isolated from Vibrio fluvialis and we used the protein sequence of V. fluvialis JS17 omega-TAm to screen the genome databases for related enzymes [6]. This bioinformatics approach yielded several omega-TAms including one from Chromobacterium violaceum DSM30191 that can convert of a range of ketones and aldehydes, including aliphatic and aromatic 1,3-dihydroxy ketones, resulting in very high stereoselectivities in the product ((S)-amine) [6]. In a current, 12 month EPSRC Follow-on-Fund award (EP/G005834/1), this bioinformatics strategy to identify new TAms is being pursued to find further TAms that readily convert a range of aliphatic, cyclic and aromatic ketones and aldehydes. Our aim with a BBSRC industrial CASE project is to extend the bioinformatics screen to new groups of TAms and then use several of the TAms from the EPSRC project, and new TAms (including for example sugar-specific TAms and the omega, beta and gamma-TAms) which will be cloned and characterised as part of the project, for the synthesis of chiral amines of interest to the collaborating company Chirotech Technology Limited. This will enable application of the TAm biotransformation strategy in an industrial environment and establish key advantages and problems of translating this approach at a larger scale. References: [1] Turner, N.J. Curr. Opin. Biotechnol., 2003, 14, 401. [2] Pàmies, O.; Bäckvall, J.E. Trends Biotechnol., 2004, 22, 130. [3] Koszelewski, D.; Clay, D.; Rozzell, D.; Kroutil, W. Eur. J. Org. Chem., 2009, 2289. [4] Hwang, B.Y.; Byung-Kwan, C. B. K.; Yun, H.; Kinera, K. K.; Kim, J. Mol. Catal. B: Enz., 2005, 37, 47. [5] Shin, J.S.; Kim, B.G. Biosci. Biotechnol. Biochem., 2001, 65, 1782 [6] Kaulmann, U.; Smithies, K.; Smith, M.E.B.; Hailes, H.C.; Ward, J.M.; Enzyme Microb. Technol., 2007, 41, 628.

The pharmaceutical industry needs cleaner and greener ways to produce complex compounds that will form the basis of future pharmaceuticals. In this project we will use enzymatic methods to generate cyclic compounds which may be applied as potential treatments for a range of diseases (e.g. fungal infection, epilepsy). The enzymes are derived from a marine blue-green alga and have been modified to make them more efficient. Combining these enzymes enables us to generate the compounds of interest in a few days compared to chemical synthesis which may take months. One aim of this project is to optimise the production of these enzymes with industry input, as well as streamline the whole process, including practical purification procedures. A key aim is to generate some compounds in reasonable quantity for testing against a number of disease models. Using this process we anticipate starting a company based around the ability to generate these unique cyclic compounds in a sustainable way.

There are no examples of catalytic aymmetric 1,4-additions of Grignard reagents (RMgX) to unsaturated amides (Case 1) despite the high synthetic utility that this transformation would offer. Similarly, no catalytic addition of a diorganozinc species (ZnR2) to an unsaturated esters (Case 2) has ever been reported. In collaboration with DowPharma we have recently uncovered the first examples of these two transformations. We wish to carry out a highly focused proof of application study for these remarkable reactions.

Modern medicine has used drugs to cure disease, alleviate chronic pain and increase life spans. Drug companies must make drugs under very strict regulations - these have to be 100% pure. One complication is that Nature has evolved to make some chemicals look the same, weigh the same, are made of the exact same atoms but they are different in the fact that they are mirror images of each other - like a pair of hands - these are called "enantiomers". It turns out that Nature tends to work with only one enantiomer and it is often observed that the opposite one is toxic, this is the case with drugs, we desire only one enantiomer. Important types of natural molecules where handed-ness make a big impact are amino acids. These are the building blocks of the proteins inside every cell. Proteins are made up hundreds and thousands of amino acids, polymerised together head-to-tail like beads on a string. These chains fold up into specific 3 dimensional shapes that can carry out many essential functions in the cell. Enzymes are also proteins and they are the workhorses of the cell - enzymes are tiny catalysts that speed up the conversion of molecule A to molecule B. Without an enzyme these conversions would take years but an enzyme catalyst can accelerate the speed of a reaction over 10 billion times. Enzymes allow us to breakdown our food, provide us with energy and help us repair damaged tissue. It turns out that these enzymes can also be put to work to make the very molecules that drug companies want. Enzymes are very specific and only work with one particular hand/mirror image of a molecule. Pharmaceutical companies endeavour to make large amounts of drugs the cheapest, purest and least wasteful way they can. Drugs are complicated molecules, many are made from amino acid building blocks (only one mirror image) in a multi-step process. Because the process uses only one of the mirror images of the starting material, the other mirror image is not used and in thus 50% is wasted. Our project aims to tackle this fundamental problem. We aim to make key amino acid building blocks of only one hand or another and use up 100% of the starting material. We will use enzymes to carry out the conversion of amino acid precursors to the target amino acid. The enzymes themselves were not designed for this specific job so we have to engineer the enzymes at a molecular level. We can do this by rational design - with knowledge of the molecular structure we can make specific changes and hope that the new enzyme will have the desired characteristics - speed, efficiency and stability. We can also carry out a random approach then fish out the desired new enzyme from the mixture. The enzyme we study catalyses the interconversion of the mirror image of one amino acid precursor into the other mirror image - this is called a racemase. Once we have the ideal racemase we will pair it up with another enzyme - an acylase - this one converts the amino acid precursor into the final amino acid but is specific for only one of the mirror images. So, we will start with both starting amino acid precursors - 50% of each mirror image. The acylase will convert one half into the product until it is used up; at the same time the racemase will be doing its job converting the unused precursor into its mirror image and when this happens the acylase can convert it. In a perfect world all of the precursor will be used up (100% conversion) and there will be no precursors left. Moreover, the enzymes can be produced cheaply, re-cycled, are bio-degradable and they work in water. We have already made good progress and now require funds to optimise the whole process. We will do this at University in partnership with a company that are experts in making amino acid precursors and products for the pharmaceutical industry. As well as making valuable tools for drug production we will also gain fundamental knowledge about enzyme design that others can apply to numerous useful processes.