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 (HPO₄²⁻)—was strategically addressed by modulating HPO₄²⁻ levels through a free HPO₄²⁻-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.