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

Abstract

Abstract Pseudouridine‐5′‐triphosphate (ΨTP) and its N 1 ‐methylated derivative (m 1 ΨTP) are critical monomer building blocks of mRNA therapeutics, yet efficient, scalable methods of their synthesis from readily accessible substrates remain underdeveloped. m 1 ΨTP 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 m 1 ΨTP 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 1 ‐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 m 1 ΨMP to m 1 ΨDP and m 1 ΨTP 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 m 1 ΨTP, the novel chemoenzymatic route from ΨMP offered improved metrics of reaction efficiency and sustainability. The synthetic ΨTP and m 1 ΨTP 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 m 1 ΨTP.