Quantitative analysis of the slow exchange process by 19F NMR in the presence of scalar and dipolar couplings: applications to the ribose 2′-19F probe in nucleic acids

Solution NMR spectroscopy is a particularly powerful technique for characterizing the functional dynamics of biomolecules, which is typically achieved through the quantitative characterization of chemical exchange processes via the measurement of spin relaxation rates. In addition to the conventional nuclei such as ¹⁵N and ¹³C, which are abundant in biomolecules, fluorine-19 (¹⁹F) has recently garnered attention and is being widely used as a site-specific spin probe. While ¹⁹F offers the advantages of high sensitivity and low background, it can be susceptible to artifacts in quantitative relaxation analyses due to a multitude of dipolar and scalar coupling interactions with nearby ¹H spins. In this study, we focused on the ribose 2′-¹⁹F spin probe in nucleic acids and investigated the effects of ¹H-¹⁹F spin interactions on the quantitative characterization of slow exchange processes on the millisecond time scale. We demonstrated that the ¹H-¹⁹F dipolar coupling can significantly affect the interpretation of ¹⁹F chemical exchange saturation transfer (CEST) experiments when ¹H decoupling is applied, while the ¹H-¹⁹F interactions have a lesser impact on Carr-Purcell-Meiboom-Gill relaxation dispersion applications. We also proposed a modified CEST scheme to alleviate these artifacts along with experimental verifications on self-complementary RNA systems. The theoretical framework presented in this study can be widely applied to various ¹⁹F spin systems where ¹H-¹⁹F interactions are operative, further expanding the utility of ¹⁹F relaxation-based NMR experiments.