Assessing Enzyme-Catalysed Phosphorylation of Nucleosides to Aid Synthetic Approaches to Novel Antiviral Nucleotides

Nucleoside analogues acting as chain terminators of viral RNA and DNA polymerases represent an effective therapeutic strategy for the inhibition of viral replication and treatment of viral infections. These compounds must be anabolised into their triphosphate forms before inclusion into the nascent nucleic acid chain by the relevant polymerase thus their action is somewhat reliant on intracellular phosphorylation machinery. The synthetic addition of α-, β-, and γ-phosphate groups to the 5’-carbons of these compounds may provide a means to circumvent this reliance and produce more effective antiviral nucleoside analogues. This can be achieved through the utilisation of enzymes as a synthetic tool, enzymes capable of phosphorylating nucleoside analogues can be expressed in and purified from bacterial cell lines for use in the synthetic process.

The present study has optimised the catalytic reaction of human UCK1 for synthetic purpose and assessed the activity of the enzyme against a range of nucleoside analogues. A range of nucleoside kinase enzymes were recombinantly expressed and purified with the goal of developing an enzymatic reaction process with synthetic utility. A linear regression model capable of calculating the substrate turnover of UCK from 31P NMR spectra of reaction samples was developed, and optimal conditions for a PK-coupled UCK reaction were identified (12.5 mM substrate, 1.5 mg/mL UCK, 3 mg/mL PK) using this model. Under the previously stated reaction conditions UCK1 was found to phosphorylate 71.68% of cytidine and 14.12% of uridine in the sample in a thirty-minute duration, translating to a production of 2.90 mg of CMP and 0.57 mg of UMP from 1 mL reaction volume. Assessment of UCK1 activity against unnatural substrates of the enzyme revealed 13.67% conversion of 5-methyl-U to the corresponding monophosphate. UCK1 was also shown to have phosphorylative activity against aza-C and riboaminopyrrole via LCMS analysis, at a level below the sensitivity of the 31P NMR-based model. The results of this study reinforce the importance of the sugar moiety in nucleoside binding to UCK and indicate a low tolerability of the enzyme for modifications to the substrate at this moiety. These results also suggest the potential of UCK1 to be used as a tool in the synthesis of nucleobase-modified nucleoside analogues.

The reaction process developed in the present study represents a significant step towards the development of an efficient means of synthesising phosphorylated derivatives of nucleoside analogues through enzyme-catalysed reaction. Future efforts on this research should focus on the development of an effective means of isolating product compound from the reaction mixture and increasing the scale of reaction to be synthetically useful.