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Here we report the efficient synthetic access to 13C/15N‐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 tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo‐enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5′‐monophosphate (ΨMP) by using YeiN, a Ψ‐5′‐monophosphate C‐glycosidase. Removal of the 5′‐phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ ‐building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63 nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75 nt E. coli tRNAGly CCmnm5U, identifying the TΨC‐loop 5‐methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI‐labelled Ψ building blocks will allow the solution and solid‐state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ‐modified nucleic acids. [ABSTRACT FROM AUTHOR]

Here we report the efficient synthetic access to 13 C/ 15 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 Gly . The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis. 13 C/ 15 N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5'-monophosphate (ΨMP) by using YeiN, a Ψ-5'-monophosphate C-glycosidase. Removal of the 5'-phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ -building block carrying both a 13 C and a 15 N label was incorporated into a product RNA and the complex formation with a 63 nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75 nt E. coli tRNA Gly CCmnm 5 U, identifying the TΨC-loop 5-methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI-labelled Ψ building blocks will allow the solution and solid-state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ-modified nucleic acids., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)