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

通过“亚胺”和“亚胺离子”催化合成生物碱开发新型生物催化剂

• 批准号: 1908707
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1908707
• 关键词: Development; novel; biocatalysts; synthesis; alkaloids

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.

生物碱是一个多样化的含氮天然产物家族，已被证明具有精致的生物活性，因此为开发新型药物和农用化学品提供了灵感。其中最著名的成员是化合物，如吗啡（止痛），尼古丁（兴奋剂）和毒芹碱（毒芹中发现的毒药）。在自然界中，这些生物碱主要在植物中通过生物合成途径产生，所述生物合成途径通常从简单的氨基酸开始，并且重要地涉及作为反应中间体的亚胺或亚胺离子的产生。虽然一些来自植物的关键酶已经被鉴定出来，但它们很难使用，并且通常具有低活性，这阻止了它们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，然后对其进行工程改造和进化，使其能够催化类似的反应，但具有更高的催化活性水平。这个跨学科项目的目标是（i）产生工程生物催化剂的“工具箱”，这些催化剂能够从相应的胺产生亚胺/亚胺离子，或者将亚胺/亚胺离子转化为亚胺/亚胺离子。亚胺离子转化为胺和其它更多官能化的分子;（ii）开发合成特定亚胺/亚胺鎓离子的合成方法，这些亚胺/亚胺鎓离子可作为这些生物催化剂的新基质，并将导致具有生物活性的天然/非天然产品的新合成路线。这是精心构建，以实现一系列的战略目标，将受益于尼克·特纳教授（生物催化）和罗杰·怀特黑德博士（有机合成）的共生研究专业知识。该计划的第一阶段将涉及使用“最先进的”合成和纯化技术合成一系列“定制”胺和亚胺底物。将选择底物以允许获得对在工业竞技场工作的科学家具有高价值的特权结构以及具有生物重要性的天然产物样化合物。与Turner教授小组密切合作，该计划的第二阶段将涉及筛选所选酶的新变体，以创建一套能够催化亚胺和亚胺离子合成和反应的生物催化剂，具有广泛的底物耐受性和高立体选择性。该项目的这一阶段将涉及酶变体库的生成以及高通量筛选。将鉴定能够催化所需反应的变体，以快速选择有效的生物催化剂。活性变体将使用定向进化进行优化，最终导致产生高活性生物催化剂的“工具箱”。酶耐受的高底物浓度将允许放大和分离所需化合物。为了进行HTS，我们将使用新开发的质谱方法和Perdita Barran教授的专业知识。