Synthesis of an Atom-Specifically ² H/¹³ C-Labeled Uridine Ribonucleoside Phosphoramidite

A combined enzymatic and chemical synthesis of a 2'-O-cyanoethoxymethyl (CEM) protected [1',6-¹³ C₂ , 5-² H]-uridine phosphoramidite is described herein. This is the first report of an atom-specific nucleobase and ribose labeled 2'-O-CEM protected ribonucleoside phosphoramidite. Importantly, the CEM 2'-OH protecting group permits the efficient solid-phase synthesis of large (>60 nucleotides) RNAs with good yield and purity. The new isotope-labeled phosphoramidite can therefore be applied to nuclear magnetic resonance (NMR) spectroscopy studies. Specifically, the [1',6-¹³ C₂ , 5-² H]-uridine phosphoramidite can be used to make position-specifically labeled RNAs for NMR analysis without complications from resonance overlap and scalar and dipolar couplings. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the ribonucleoside 6 Basic Protocol 2: Synthesis of the ribonucleoside phosphoramidite 11.