Integrated Chemoenzymatic Synthesis of the mRNA Vaccine Building Block N-Methylpseudouridine Triphosphate.

Pseudouridine-5'-triphosphate (ΨTP) and its N-methylated derivative (mΨTP) are critical monomer building blocks of mRNA therapeutics, yet efficient, scalable methods of their synthesis from readily accessible substrates remain underdeveloped. mΨTP 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 mΨTP at ∼200 mg scale of isolated compound. Biocatalytic cascade rearrangement of uridine delivered ΨMP or Ψ in high yields. Acetonide-protected ΨMP was selectively N-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 mΨMP to mΨDP and mΨTP 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 mΨTP, the novel chemoenzymatic route from ΨMP offered improved metrics of reaction efficiency and sustainability. The synthetic ΨTP and mΨTP 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 mΨTP.