One-pot enzyme cascade production of therapeutic deoxyribonucleoside analogs from glycerol and acetaldehyde

In this study, we demonstrated a novel one-pot enzyme cascade for the production of deoxyribonucleoside analogs using glycerol and acetaldehyde (AcH) as feedstocks. In the initial stage of the cascade, 2-deoxy-D-ribose 5-phosphate (D-dRib 5P) was generated in situ from glycerol via a non-hydrolytic aldol addition between D-glyceraldehyde 3-phosphate (D-GAP) and AcH, catalyzed by aldolase. D-dRib 5P was subsequently converted into target deoxyribonucleosides through a retrosynthetic enzyme cascade that effectively reverses the natural deoxyribonucleoside salvage pathway, with the choice of nucleobases and nucleoside phosphorylases determining the final products. A key limitation-yield reduction due to the accumulation of free hydrogen phosphate (HPO₄^2-)-was strategically addressed by modulating HPO₄^2- levels through a free HPO₄^2--scavenging system employing sucrose phosphorylase and excess sucrose. Using this approach, we successfully synthesized four commercially relevant therapeutic deoxyribonucleosides-floxuridine, idoxuridine, decitabine, and cladribine-by varying the nucleobases and nucleoside phosphorylases. These findings highlight the potential of this biocatalytic platform for the sustainable and cost-effective production of deoxyribonucleoside analogs from simple, renewable feedstocks.