A green synthesis of vidarabine 5’-monophosphate via a one-pot multienzymatic reaction catalyzed by immobilized biocatalysts

In nature, enzyme cascades can be found in many metabolic pathways. The idea of using multienzymatic systems to mimic these processes is gaining interest for production of chemical compounds. A type of multi-enzymatic application is the use of multiple enzymes for shifting reaction equilibria. This strategy relies on removing intermediates, inhibitory products or byproducts, via a second enzymatic reaction. In the context of a multi-enzymatic system, a one-pot process uses more than one enzyme in a single reactor.1 We here describe a three-step sequential enzymatic reaction for the one-pot synthesis of vidarabine 5’-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade) and adenosine triphosphate (ATP) as precursors. To this aim, three immobilized biocatalysts involved in the biosynthesis of nucleosides and nucleotides were used: uridine phosphorylase from Clostridium perfringens (CpUP),2 a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP),2 and deoxyadenosine kinase from Dictyostelium discoideum (DddAK).3 Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-D-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK which produces the 5’-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor (Scheme 1). Reaction conditions (i.e. medium, temperature, immobilization carriers) and biocatalyst stability have been balanced and optimized to achieve the highest productivity. Vidarabine 5’-monophosphate was obtained in 95.5% conversion. Optimization of the purification step is in progress.