Integrated Chemoenzymatic Synthesis of the mRNA Vaccine Building Block <i>N</i>¹‐Methylpseudouridine Triphosphate

Pseudouridine-5'-triphosphate (ΨTP) and its N¹-methylated derivative (m¹ΨTP) are critical monomer building blocks of mRNA therapeutics, yet efficient, scalable methods of their synthesis from readily accessible substrates remain underdeveloped. m¹ΨTP is a major cost factor of production of the current COVID-19 vaccines. We herein report a notably atom-economic and high-yielding biocatalytic route toward ΨTP and present two chemoenzymatic routes for producing m¹ΨTP at ∼200 mg scale of isolated compound. Biocatalytic cascade rearrangement of uridine delivered ΨMP or Ψ in high yields. Acetonide-protected ΨMP was selectively N¹-methylated using dimethyl sulfate and subsequently converted to the triphosphate through efficient kinase cascade reaction. Saccharomyces cerevisiae uridine 5'-monophosphate kinase was shown for ATP-dependent phosphorylation of m¹ΨMP to m¹ΨDP and m¹ΨTP synthesis was catalyzed by Escherichia coli acetate kinase which also served to regenerate ATP by acetyl phosphate. Benchmarked against chemical route converting the enzymatically produced Ψ into m¹ΨTP, the novel chemoenzymatic route from ΨMP offered improved metrics of reaction efficiency and sustainability. The synthetic ΨTP and m¹ΨTP replaced UTP for mRNA synthesis by in vitro transcription. Overall, this study shows the productive integration of chemical methylation with enzymatic cascade reactions for C─C coupling and phosphorylation toward an efficient preparation of m¹ΨTP.