4′-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine as a Sensitive ¹⁹F NMR Sensor for the Detection of Oligonucleotide Secondary Structures

Abstract

4'-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine was synthesized and incorporated as a phosphoramidite into oligonucleotide sequences. Its applicability as a sensor for the (19)F NMR spectroscopic detection of DNA and RNA secondary structures was demonstrated. On DNA, the (19)F NMR measurements were focused on monitoring of duplex-triplex conversion, for which this fluorine-labeled 2'-deoxynucleoside proved to be a powerful sensor. This sensor seemingly favors DNA, but its behavior in the RNA environment also turned out to be informative. As a demonstration, invasion of a 2'-O-methyl oligoribonucleotide into an RNA hairpin model (HIV-1 TAR) was monitored by (19)F NMR spectroscopy. According to the thermal denaturation studies by UV spectroscopy, the effect of the 4'-C-(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl moiety on the stability of these DNA and RNA models was marginal.

Synthesis of C-5, C-2′ and C-4′-neomycin-conjugated triplex forming oligonucleotides and their affinity to DNA-duplexes

“…We have recently shown that 4'-C-(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl thymidine is a good 19 F NMR sensor (F) for this purpose. 43 It does not affect the DNA-triplex stabilities, but its 19 F resonance shift is highly sensitive to environmental changes that take place when the initial duplex minor groove is converted to the triplex Watson-Crick groove. 34 The three magnetically equivalent fluorine atoms in a quasiisolated spin system allows 19 F NMR measurements to be carried out in relatively low oligonucleotide concentrations without the need for fluorine-proton coupling.…”

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

“…However, uniform labeling of the RNA with 2 0 F-substituted nucleotides can disrupt tertiary interactions that involve the 2 0 OH group and shift the conformational preferences of the ribofuranosyl sugar unit to preferentially adopt the 3 0 -endo conformation and therefore influences the structural behavior (Reif et al 1997). The use of other labeling positions than 2 0 can overcome the 3 0 -endo preference in uniformly labeled RNAs without losing the advantage of 19 F-NMR spectroscopy (Granqvist and Virta 2014;Puffer et al 2009). 19 F-labeled nucleotides are often introduced by chemical synthesis which provides a single introduction of the label at a defined position within the RNA of interest.…”

2′-O-[(4-CF3-triazol-1-yl)methyl] Uridine – A Sensitive 19F NMR Sensor for the Detection of RNA Secondary Structures

“…0.1 mol L -1 aqueous Et 3 N + AcO -(6.0 mL, pH = 7.0) was eluted though the cartridges, the crude RNAs were released by elution with 60% aqueous acetonitrile and the RNA -fractions were evaporated to dryness. 20 Similarly, the 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 UV-and CD-measurements. The melting curves (absorbance versus temperature) were measured at 260 nm on a UV-Vis spectrometer equipped with a multiple cell holder and a Peltier temperaturecontroller.…”

“…Sensor 2 was, in turn, primarily designed for the DNA environment and its applicability has previously been described. 20 The superiority of 1 (the large shift dispersion, the simple synthesis of 10 and its efficient coupling) among these three sensors (1, 2 and 3) seemed obvious and its functionality was then further evaluated.…”

“…3'-O-[(2-cyanoethoxy)-(N,N-diisopropylamino)phosphinyl]-5'-O-(4,4'-dimetoxytrityl)-4'-C-(4-trifluoromethylphenyl)-2'-O-(triisopropylsilyloxymethyl)uridine(20) 19. (50 mg, 57 µmol) was dried over P 2 O 5 in a vacuum desiccator and dissolved in dry dichloromethane (0.3 mL).Triethylamine (55 µL, 0.4 mmol) and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (32 µL, 0.14 mmol) were added and the mixture was stirred overnight at ambient temperature under nitrogen.…”