A Modular Nucleoside Kinase Cascade for the Synthesis of Ribonucleoside Triphosphates

The increasing demand for affordable nucleoside 5'-triphosphates (NTPs) necessitates efficient and scalable methods of synthesis. Herein, we report standardized one-pot enzymatic cascades for the production of all canonical NTPs (ATP, CTP, GTP, UTP). Central to these cascades is the Saccharomyces cerevisiae (Saccharomyces cerevisiae UMP kinase (ScURA6), which efficiently phosphorylates AMP, CMP, and UMP. ScURA6 is inactive toward GMP, so Saccharomyces cerevisiae GMP kinase (ScGMPK) was used to synthesize GTP. NDPs were phosphorylated by Escherichia coli acetate kinase (EcAcK), using acetyl phosphate as the phosphate donor in the presence of catalytic amounts of ATP or GTP. Substrate-specific kinases were used to phosphorylate the nucleoside starting material. The resulting four cascades were optimized for flux through each step to afford the NTP product in the absence of NMP and NDP intermediates. The reactions were intensified for full conversion (≥ 93%) of 50-100 mM substrate within 60-120 min. Alternatively, a modified cascade employing the product NTP as a phosphate shuttle was established, achieving an NTP conversion of ≥ 84%. Preparative-scale syntheses (10 mL) reached up to 89% isolated yield (≥ 150 mg product), demonstrating a robust and scalable platform for NTP production. The kinases used are active with noncanonical nucleosides and nucleoside analogs, offering flexibility for broad synthetic applicability of the cascade transformations used.