Efficient Synthetic Access to Stable Isotope Labelled Pseudouridine Phosphoramidites for RNA NMR Spectroscopy.

Here we report the efficient synthetic access to C/N-labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75 nt E. coli tRNA. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis.

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 1N-5C rearrangement of β-D-ribosyl on uracil to obtain Ψ from unprotected U in quantitative yield.