Development of novel biocatalysts for the synthesis of alkaloids via 'imine' and 'iminium ion' catalysis

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The alkaloids are a diverse family of nitrogen-containing natural products that have been shown to possess exquisite biological activity and hence have provided inspiration for the development of novel pharmaceuticals and agrochemicals. Amongst the most well-known members of this class are compounds such as morphine (pain relief), nicotine (stimulant) and coniine (poison found in hemlock). In nature these alkaloids are produced, mainly in plants, via biosynthetic pathways which usually start with simple amino acids and importantly involve the generation of imines or iminium ions as reactive intermediates. Although some of the key enzymes from plants have been identified they are difficult to work with and often have low activity preventing their use as preparative biocatalysts in the laboratory. An alternative approach is to identify bacterial enzymes and then engineer and evolve them so that they can catalyse similar reactions but with much higher levels of catalytic activity.The goal of this cross-disciplinary project is to (i) generate a "toolbox" of engineered biocatalysts that are either able to generate imines/imines ions from the corresponding amines or alternatively convert imines/iminium ions to amines and other more functionalised molecules; (ii) develop synthetic approaches for the synthesis of specific imine/iminium ions which can act as novel substrates for these biocatalysts and which will lead to novel synthetic routes to natural/unnatural products possessing biological activity.This project, which is carefully structured in order to accomplish a series of strategic goals, will benefit from the symbiotic research expertise of Professor Nick Turner (biocatalysis) and Dr Roger Whitehead (organic synthesis). The first stage of the programme will concern the synthesis of a "bespoke" array of amine and imine substrates using 'state of the art' synthetic and purification techniques. The substrates will be selected in order to allow access to privileged structures of high value to scientists working in the industrial arena as well as natural product-like compounds of biological importance. Working closely with the group of Professor Turner, the second stage of the programme will involve screening the generation of novel variants of the selected enzymes in order to create a suite of biocatalysts which are able to catalyse the synthesis and reaction of imines and iminium ions with broad substrate tolerance and high stereoselectivity. This phase of the project will involve the generation of libraries of enzyme variants coupled with high-throughput screening. Variants capable of catalysing the desired reaction will be identified to allow the rapid selection of effective biocatalysts. Active variants will be optimised using directed evolution leading ultimately to the generation of a "toolbox" of highly active biocatalysts. The high substrate concentrations tolerated by the enzyme will allow for scale-up and isolation of desired compounds. In order to perform the HTS we will use newly developed mass spectrometry approaches, and the expertise of Professor Perdita Barran.