2'-O-Trifluoromethylated RNA - a powerful modification for RNA chemistry and NMR spectroscopy

New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics, and of RNA-ligand interactions has attracted considerable interest. To this end, fluorine labeling in conjunction with ¹⁹F-NMR spectroscopy has emerged as a powerful strategy. Appropriate probes for RNA previously focused on single fluorine atoms attached to the 5-position of pyrimidine nucleobases or at the ribose 2'-position. To increase NMR sensitivity, trifluoromethyl labeling approaches have been developed, with the ribose 2'-SCF₃ modification being the most prominent one. A major drawback of the 2'-SCF₃ group, however, is its strong impact on RNA base pairing stability. Interestingly, RNA containing the structurally related 2'-OCF₃ modification has not yet been reported. Therefore, we set out to overcome the synthetic challenges toward 2'-OCF₃ labeled RNA and to investigate the impact of this modification. We present the syntheses of 2'-OCF₃ adenosine and cytidine phosphoramidites and their incorporation into oligoribonucleotides by solid-phase synthesis. Importantly, it turns out that the 2'-OCF₃ group has only a slight destabilizing effect when located in double helical regions which is consistent with the preferential C3'-endo conformation of the 2'-OCF₃ ribose as reflected in the ³ J (H1'-H2') coupling constants. Furthermore, we demonstrate the exceptionally high sensitivity of the new label in ¹⁹F-NMR analysis of RNA structure equilibria and of RNA-small molecule interactions. The study is complemented by a crystal structure at 0.9 Å resolution of a 27 nt hairpin RNA containing a single 2'-OCF₃ group that well integrates into the minor groove. The new label carries high potential to outcompete currently applied fluorine labels for nucleic acid NMR spectroscopy because of its significantly advanced performance.