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

生物碱是一类多种多样的含氮天然产物，已被证明具有良好的生物活性，因此为开发新型药物和农用化学品提供了灵感。这一类中最广为人知的成员是吗啡(止痛药)、尼古丁(兴奋剂)和可乐碱(在铁杉中发现的毒药)等化合物。在自然界中，这些生物碱主要是通过生物合成途径产生的，这些途径通常始于简单的氨基酸，重要的是涉及作为活性中间体的亚胺或亚胺离子的产生。尽管已经确定了植物中的一些关键酶，但它们很难处理，而且活性往往很低，使它们无法在实验室中用作制备生物催化剂。另一种方法是鉴定细菌酶，然后设计和进化它们，使它们能够催化类似的反应，但具有更高水平的催化活性。这个跨学科项目的目标是(I)产生一个工程生物催化剂的“工具箱”，这些生物催化剂要么能够从相应的胺产生亚胺/亚胺离子，要么将亚胺/亚胺离子转化为胺和其他功能更强的分子；(Ii)开发合成特定亚胺/亚胺/亚胺离子的方法，这些方法可以作为这些生物催化剂的新底物，并将导致具有生物活性的天然/非天然产品的新合成途径。该项目经过精心设计，以实现一系列战略目标，将受益于Nick Turner教授(生物催化)和Roger Whitehead博士(有机合成)的共生研究专长。该计划的第一阶段将涉及使用最先进的合成和提纯技术合成一系列“定制”的胺和亚胺底物。选择底物是为了让在工业领域工作的科学家能够获得具有高价值的特权结构，以及具有生物重要性的天然产品类化合物。该计划的第二阶段将与特纳教授的小组密切合作，包括筛选所选酶的新变种的产生，以创建一套能够催化亚胺和亚胺离子的合成和反应的生物催化剂，具有广泛的底物耐受性和高的立体选择性。该项目的这一阶段将涉及酶变异体文库的生成以及高通量筛选。将确定能够催化所需反应的变体，以便快速选择有效的生物催化剂。活性变异体将使用定向进化进行优化，最终导致高活性生物催化剂的“工具箱”的产生。这种酶能耐受的高底物浓度将允许放大和分离所需的化合物。为了进行HTS，我们将使用新开发的质谱学方法，以及Perdita Barran教授的专业知识。

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.