Establishing a chemoenzymatic flow-based strategy for nucleoside synthesis

核苷类似物是整个生物技术和制药工业中使用的基本构件。核苷类似物的应用是多种多样的，从将其掺入用于诊断的寡核苷酸（例如，PCR）和反义治疗，直到它们作为小分子抗病毒剂的效用。随着新型冠状病毒疫情的到来，全球新型核苷市场迅速增长，预计到2022年将达到8. 093亿美元。支撑这一增长（从2014年的1.133亿美元）的是创新战略的需要，以实现其有效和可持续的合成以及大规模生产。尽管核苷在整个工业和学术界的应用很普遍，但合成方法并没有跟上其下游应用的步伐。这主要是由于传统的合成方法会形成副产物以及使用有毒试剂，导致下游规模扩大在环境上不可持续。与使用纯合成途径的挑战性性质相反，使用例如嘌呤/嘧啶核苷磷酸化酶（PNP）的核苷合成的酶促方法更可持续、可扩展并且主要产生所需的立体异构体。这提供了相当多的机会，整合酶和化学合成，以产生核苷类似物不容易获得通过使用这些策略在隔离。为了集成各个过程中的每一个（即，通过化学合成和酶促糖基化来合成前体以形成核苷核心），我们提出使用流动化学来开发一种简易的合成“生产线”。流动化学提供了以相对于传统的基于批处理的过程更有效和更具成本效益的方式进行望远镜反应的潜力。此外，通过利用Burley和Hoskisson团队在开发用于生物催化的酶方面的经验，将使我们能够合作定制这些酶用于基于流动的合成。此外，利用白肋烟集团现有的经验以及与基于流程的工业合作伙伴（Vaportec）的联系，将为简化该项目的酶和合成化学部分的开发提供额外的机会。 这个合作研究项目的总体目标是建立一种新的基于流动的化学酶策略来合成核苷类似物。这项工作的核心是提供一种可持续的合成方法，其中构建模块将直接输入我们更大的BBSRC资助的网络（战略性更长-更大的资助，sLoLa，2020-2025），其中修饰的RNA寡核苷酸是询问基因表达如何调节的重要工具。该提案的具体目标是：（i）鉴定新型糖基化酶以制备核苷类似物。(ii)确定这些酶用于基于流动的反应器系统的相容性。(iii)建立基于流动的方法用于化学酶促合成新核苷以将其掺入寡核苷酸的效用。

Nucleoside analogues are essential building blocks used throughout the biotechnology and pharmaceutical industries. Applications of nucleoside analogues are diverse, ranging from their incorporation into oligonucleotides for diagnostics (e.g., PCR) and antisense therapeutics, through to their utility as small molecule anti-virals. With the advent of the Covid-19 pandemic, the global market for novel nucleosides is growing rapidly, expecting to reach US$809.3M by 2022. Underpinning this growth (from US$113.3M in 2014) is the need for innovative strategies to enable their efficient and sustainable synthesis, and large scale manufacture. Despite the pervasiveness of nucleosides used throughout industry and in academia, methods of synthesis have not kept abreast of their downstream applications. This is predominantly due to conventional synthetic methodology suffering from the formation of side-products as well as the use of toxic reagents which results in downstream scale-up being environmentally unsustainable. In contrast to the challenging nature of using pure synthetic routes, enzymatic methods of nucleoside synthesis using for example, purine/pyrimidine nucleoside phosphorylases (PNPs), are far more sustainable, scalable and predominantly produce the desired stereoisomer. This provides considerable opportunities to integrate enzymatic and chemical syntheses to produce nucleoside analogues not readily accessible by the use of these strategies in isolation. In order to integrate each of the respective processes (i.e., synthesis of precursors by chemical synthesis and enzymatic glycosylation to form the nucleoside core), we propose to develop a facile synthetic 'production line' using flow chemistry. Flow chemistry offers the potential to telescope reactions in a far more efficient and cost-effective manner relative to conventional batch-based processes. Furthermore, by exploiting experience of the Burley and Hoskisson groups in developing enzymes for applications in biocatalysis will enable our collaboration to tailor these enzymes for flow-based synthesis. In addition, using the Burley group's existing experience and links with a flow-based industrial partner (Vaportec) will provide additional opportunities to streamline the development of both the enzymatic and synthetic chemistry parts of the project. OVERALL OBJECTIVES OF THE STUDENTSHIPThe overall objective of this collaborative studentship proposal is to establish a new flow-based chemoenzymatic strategy to synthesise nucleoside analogues. Central to this work is delivering a sustainable synthetic method of synthesis in which the building blocks will feed directly into our larger BBSRC-funded network (strategic longer-larger grant, sLoLa, 2020-2025) in which modified RNA oligonucleotides are essential tools to interrogate how gene expression is regulated.The specific aims of the proposal are to: (i) identify novel glycosylation enzymes to prepare nucleoside analogues. (ii) determine the compatibility of these enzymes for use in a flow-based reactor system. (iii) establish the utility of the flow-based approach for the chemoenzymatic synthesis of novel nucleosides for their incorporation into oligonucleotides.