A Universal Nucleoside‐to‐Nucleoside‐5′‐Triphosphate Enzyme Cascade Driven by Polyphosphate

Nucleoside‐5′‐triphosphates (NTPs) are important building blocks for nucleic acids, but the efficiency of their chemical synthesis is limited by the low yield of the final 5′‐triphosphorylation reaction. Here, we present an enzymatic cascade reaction involving just two enzymes that enable nucleosides‐to‐NTPs conversion for all canonical rNTPs and dNTPs. The cascade is used in a single one‐pot operation driven by cost‐efficient sodium polyphosphate (polyP). The broad‐spectrum deoxynucleoside kinase (Dm‐dNK) from Drosophila melanogaster converts nucleosides into nucleoside‐5′‐monophosphates (NMPs) using ATP. The polyphosphate kinase family 2, class III (EbPPK) from an Erysipelotrichaceae bacterium simultaneously recycles ATP required by Dm‐dNK and converts the NMPs to NTPs in a polyP‐dependent reaction. Importantly, only trace amounts of catalytic ATP (0.0001 equivalents or less) are required. In addition to canonical NTPs, the cascade can generate ribose‐ and base‐modified NTP analogues. This is demonstrated through the synthesis of a set of eleven modified nucleotides, including the four 2′‐fluoro‐2′‐dNTPs and nucleotides with 2,6‐diaminopurine as the nucleobase. Production of an essential component for mRNA therapeutics, N 1‐methyl‐ΨTP (m 1 ΨTP), is demonstrated on a milligram scale.