Phosphorylation-condensation cascade for biocatalytic synthesis of C-nucleosides

Abstract

C-nucleosides are important targets for synthesis as anti-infective agents and building blocks for therapeutic nucleic acids. Here, we show phosphorylation-condensation cascade reaction to produce pseudouridine 5′-phosphate (Ψ5P) from d-ribose (Rib) and uracil. Rib (∼1.0 M) was phosphorylated at O5 in aqueous acetyl phosphate (1.15 M) via ATP (5 mM) by coupled kinases. Using Ψ5P C-glycosidase, Rib5P intermediate (≥90% yield) was reacted with the mole equivalent of uracil, supplied as a solid, to give Ψ5P in quantitative yield. One-pot reaction optimized for reagent composition, automated pH control, and solid-liquid mass transfer yielded ∼2.2 g Ψ5P (productivity: 38 g/L/h) from 10-mL volume. Synthetic flexibility of the cascade reaction was shown with other pentoses (d-arabinose, 2-deoxy-Rib, d-xylose) and analogs of uracil (6-amino, 2-thio, 4-thio). Collectively, we show massive intensification (≥50-fold) of the pentose phosphorylation as well as the C–C bond-forming condensation step of the overall multienzyme cascade transformation for efficient C-nucleoside (monophosphate) synthesis.