Chemo-enzymatic synthesis of 13C- and 19F-labeled uridine-5′-triphosphate for RNA NMR probing

Solution NMR of the magnetically active fluorine-19 ( 19 F) isotope offers tremendous advantages in the study of RNA structure and conformational changes. However, exploiting these advantages requires the synthesis of fluorinated RNA-building blocks, and this comes with certain challenges in overall yield of nucleotides, purity, and cost. Here, we detail a scalable and reliable method to produce [1′,5- 13 C 2 , 5- 19 F, 6- 2 H]- and [1′,5- 13 C 2 , 5- 19 F, 6- 2 H, 1,3- 15 N 2 ]-5-fluorouracil-5′-triphosphate. We further present the steps employed to characterize the labeled nucleotide before its incorporation into RNAs with relevant biological activities. Graphic abstract