Integrated Chemoenzymatic Synthesis of the mRNA Vaccine Building Block N1‐Methylpseudouridine Triphosphate

Pseudouridine‐5’‐triphosphate (ΨTP) and the N1‐methylated derivative (m1ΨTP) are critical monomer building blocks of the currently used mRNA vaccines, yet efficient methods of their synthesis from readily accessible substrates remain underdeveloped. We herein report notably atom‐economic and high‐yielding biocatalytic route toward ΨTP (83% yield) and present two chemo‐enzymatic routes for producing m1ΨTP at ∼200 mg scale of isolated compound. Biocatalytic cascade rearrangement of uridine delivers ΨMP (∼320 mg/mL) or Ψ (∼240 mg/mL) in 95% yield each. The acetonide‐protected ΨMP is N1‐methylated selectively using dimethyl sulfate (4 eq, 85% yield) and is subsequently converted to the triphosphate (7.3 mg/mL; 83% yield) by kinase cascade reaction. Saccharomyces cerevisiae uridine 5’‐monophosphate kinase is shown for ATP‐dependent phosphorylation of (m1)ΨMP to (m1)ΨDP. The conversion to (m1)ΨTP is catalyzed by Escherichia coli acetate kinase. Benchmarked against chemical route converting the enzymatically produced Ψ into m1ΨTP (37% yield), the novel chemo‐enzymatic route from ΨMP offers greatly improved metrics of reaction efficiency (68% isolated yield) and sustainability (∼3‐fold less waste). The synthetic ΨTP and m1ΨTP replace 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 efficient preparation of m1ΨTP.