A modular in vitro multienzyme cascade for the sustainable synthesis of high-value vidarabine 5′-monophosphate from adenosine

Nucleoside analogues such as vidarabine (Ara-A) are vital therapeutics but are often limited by poor pharmacokinetics. Its phosphorylated form, vidarabine 5′-monophosphate (Ara-AMP), offers improved solubility and is used as a clinical alternative, although it can be dephosphorylated to Ara-A in vivo. Here, we report a modular multienzyme cascade for the sustainable synthesis of Ara-AMP directly from adenosine. The cascade integrates three functional modules: a core module (PNP, PPM, and RpiA) for the conversion of adenosine to Ara-A, a driver module (dNKδ20) for the selective phosphorylation of Ara-A to Ara-AMP, and a support module (PPK) for ATP regeneration from low-cost ADP and polyphosphate. The use of dNKδ20, a mutant deoxyribonucleoside kinase with high selectivity for Ara-A over adenosine, is critical for minimizing the formation of AMP byproduct, which would otherwise complicate downstream processing. Under optimized conditions, the cascade achieved an 80.2% conversion of 1 mM adenosine to Ara-AMP in 4 h using 0.05 mM ADP. The modular design was also applicable to guanosine and inosine, affording the corresponding arabinosyl nucleotides, albeit with lower yields. This work establishes a sustainable biocatalytic platform for nucleoside analogues, exemplifying how enzyme promiscuity, thermodynamic driving, and cofactor recycling enable green pharmaceutical manufacturing.