A selective and atom-economic rearrangement of uridine by cascade biocatalysis for production of pseudouridine

As a crucial factor of their therapeutic efficacy, the currently marketed mRNA vaccines feature uniform substitution of uridine ( U ) by the corresponding C -nucleoside, pseudouridine ( Ψ ), in 1-N-methylated form. Synthetic supply of the mRNA building block (1-N-Me- Ψ −5’-triphosphate) involves expedient access to Ψ as the principal challenge. Here, we show selective and atom-economic 1 N -5 C rearrangement of β- d -ribosyl on uracil to obtain Ψ from unprotected U in quantitative yield. One-pot cascade transformation of U in four enzyme-catalyzed steps, via d -ribose (Rib)-1-phosphate, Rib-5-phosphate (Rib5P) and Ψ -5’-phosphate ( Ψ MP), gives Ψ . Coordinated function of the coupled enzymes in the overall rearrangement necessitates specific release of phosphate from the Ψ MP, but not from the intermediary ribose phosphates. Discovery of Yjjg as Ψ MP-specific phosphatase enables internally controlled regeneration of phosphate as catalytic reagent. With driving force provided from the net N - C rearrangement, the optimized U reaction yields a supersaturated product solution (∼250 g/L) from which the pure Ψ crystallizes (90% recovery). Scale up to 25 g isolated product at enzyme turnovers of ∼10 5 mol/mol demonstrates a robust process technology, promising for Ψ production. Our study identifies a multistep rearrangement reaction, realized by cascade biocatalysis, for C -nucleoside synthesis in high efficiency.