Establishing a chemoenzymatic flow-based strategy for nucleoside synthesis

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 STUDENTSHIP

The 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.