Investigations on Fluorine‐Labeled Ribonucleic Acids by¹⁹F NMR Spectroscopy

5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19 F NMR spectroscopy

“…Again, at 300 K, the 1 H NMR imino proton resonance pattern of S4 and S5 was lacking one or two signals, respectively, that were attributed to 5-fluoro substituted Watson–Crick base pairs while all other imino protons provided chemical shift values comparable to that of the unmodified RNA counterpart. Our finding fits to a report by James and coworkers who showed in a temperature dependent series of 1 H NMR spectra for the self-complementary duplex of [d(GGAAT(dU 5F )CC)] 2 that the dU 5F N( 3 )–H resonance is extremely broad at low temperatures and becomes non-observable at room temperature ( 5 ). Also Hennig et al , observed for a uniformly 5-fluoro uridine labeled HIV TAR hairpin significant line broadening of 5-fluoro uridine N( 3 )–H imino protons together with a downfield chemical shift change between 0.6 and 0.7 ppm ( 24 ).…”

“…Our finding fits to a report by James and coworkers who showed in a temperature dependent series of 1 H NMR spectra for the self-complementary duplex of [d(GGAAT(dU 5F )CC)] 2 that the dU 5F N( 3 )–H resonance is extremely broad at low temperatures and becomes non-observable at room temperature ( 5 ). Also Hennig et al , observed for a uniformly 5-fluoro uridine labeled HIV TAR hairpin significant line broadening of 5-fluoro uridine N( 3 )–H imino protons together with a downfield chemical shift change between 0.6 and 0.7 ppm ( 24 ). The line broadening most likely reflects an increased exchange rate of dA:dU 5F and A:U 5F imino protons and relates to the considerable electronegativity of the 5-fluorine atom that lowers the apparent pK a of uridine N( 3 )–H from 9.18 ± 0.02 (unmodified uridine) to a value of 7.55 ± 0.02 (5-fluoro uridine) ( 43 ).…”

“…Interestingly, the 19 F NMR resonance of 5-fluoro uracil showed an increased line width when it was involved in base pairing compared to the situation when it was located in an unpaired strand region. Moreover, we stress that the 1 H NMR spectra of the bistable DNA S2 as well as the hairpin reference S2b were lacking the N( 3 )–H imino proton resonances of the respective dA:dU 5F base pairs at 300 K (compare Figure 2 A, top to Figure 2 B, top; and Figure 2 A, bottom to Figure 2 B, bottom). All other imino protons showed similar chemical shift values as the ones observed for the unmodified DNA counterparts, S1 and S1b .…”

“…acetic anhydride (for 2 ) or benzoic anhydride (for 6 ) in anhydrous ethanol, room temperature, 14 h, 99% ( 2 ) and 99% ( 6 ); ( b ) 1.1 eq. 4,4′-dimethoxytrityl chloride in pyridine, room temperature, 14 h, 60% ( 3 ) and 56% ( 7 ); ( c ) 1.8 eq. silver nitrate, 1.8 eq.…”

“…The use of fluorine labels in context with NMR spectroscopy represents an emerging tool to study structure and function of nucleic acids ( 1–3 ). In contrast to 1 H NMR spectroscopy, the problem of resonance degeneracy is practically absent for 19 F NMR spectroscopy.…”