Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

Abstract We present the access to [5‐19F, 5‐13C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐19F, 5‐13C]‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre‐microRNA (miRNA) 21 and the 59 nt full length pre‐miRNA 21. The main stimulus to introduce the aromatic 19F–13C‐spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic 13C–19F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5‐19F, 5‐13C]/[5‐19F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial 19F–13C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [19F, 13C]‐labeling of the SAM VI aptamer domain and the pre‐miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.

The product was dried in high vacuum. Yield: 2.52 g (4.80 mmol, 70%); TLC: ethyl acetate/n-hexane = 3/7) Rf = 0.5; 1 H-NMR (400 MHz, DMSO-d6, 25°C): δ 11.93 (bs, 1H, NH); 7.92 (dd, 1H, 3   (1.29 g, 2.50 mmol) was dissolved in 20 mL of dry methylene chloride. To this solution was added dropwise at 0°C under stirring a premixed solution of 225 µL HF-Pyridine (70 % hydrogen fluoride basis, 30 % pyridine basis) and 1.30 mL pyridine. After stirring 2h at 0°C thin layer chromatography showed complete conversion (ethyl acetate/n-hexane = 7/3; Rf = 0.2). The mixture was allowed to warm to room temperature, and was diluted with chloroform and washed once with saturated sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The crude product was dried in high vacuum and used without further purification for the next step. The crude product (1.0 eq., 2.50 mmol) together with one spatula tip of 4-(dimethylamino)pyridine was co-evaporated twice with anhydrous pyridine and then dissolved in 25 mL of dry pyridine. Then 4,4'-dimethoxytrityl chloride (1.1 eq., 932 mg, 2.75 mmol) was added in three portions within 1h and the mixture was stirred for 3h at room temperature. The mixture was quenched with 1 mL of methanol, evaporated to an oily residue and two times co-evaporated with toluene. The residue was dissolved in chloroform and washed two times with 5% citric acid and once with saturated sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The crude product was applied to a silica gel column with methylene chloride and eluted using a gradient from 0 to 3% methanol in methylene chloride to give 6 as a yellowish foam.

4-N-acetyl-3',5'-O-bis(tert.-butylsilyl)-2'-O-(tert.-butyldimethylsilyl)-[5-19 F,5-13 C]-cytidine (9).
Compound 8 (1.0 eq., 1.68 g, 3.25 mmol) was dissolved in 15 mL of dry N,N-dimethylformamide (DMF) and acetic anhydride (1.5 eq., 499 mg, 461 µL) was added under argon. The reaction was stirred for 24h at room temperature and then quenched by the addition of 2 mL methanol. The reaction mixture was evaporated under high vacuum and at 60°C. The residue was taken up in methylene chloride and the organic phase was washed with saturated bicarbonate solution, followed by washing with brine (three times). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The crude product was applied to a silica gel column with n-hexanes/ethyl acetate from 9/1 4/6 to give 9 as a colorless foam. The product was dried in high vacuum. Yield

4-N-acetyl-2'-O-(tert.-butyldimethylsilyl)-5'-O-(4,4'-dimethoxytrityl)-[5-19 F,5-13 C]-cytidine (10).
Compound 9 (1.0 eq., 1.52 g, 2.71 mmol) was dissolved in 30 mL of dry methylene chloride and cooled to 0°C. Then, 10% HF-pyridine (3.85 eq., 10.75mmol, 2 mL,) was added under argon and stirring was continued for 2.5h at 0°C. The reaction mixture was diluted with methylene chloride and the organic phase was washed with saturated bicarbonate solution, water and 5% citric acid. The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The crude product was dried in high vacuum for 1h. The crude product was co-evaporated three times with anhydrous pyridine and then 4,4'-dimethoxytrityl chloride (1.5 eq., 1.4 g, 4.08 mmol) was added in three portions within 1h and the mixture was stirred for 16h at room temperature. The mixture was quenched with 1 mL of methanol, evaporated to an oily residue and two times co-evaporated with toluene. The residue was dissolved in chloroform and washed two times with 5% citric acid and once with saturated sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The crude product was applied to a silica gel column and eluted using a gradient from 30 to 60% ethyl acetate in n-hexanes to give 10 as a yellowish foam. The product was dried in   nm on a NanoPhotometer (Implen).

LC-ESI mass spectrometry
All RNAs were analyzed on Finnigan LCQ Advantage MAX ion trap instrumentation connected to a

NMR spectroscopy
RNA samples were lyophilized as sodium salts and dissolved in 280 µL NMR buffer (15 mM sodium phosphate, 25 mM NaCl, 0.1% NaN3, pH 6.9) and transferred into restricted volume Shigemi tubes giving 0.2 to 1 mM sample concentrations. Experiments were run at 298 K unless otherwise stated. All NMR experiments were conducted on a Bruker 600 MHz Avance II+ NMR with a Prodigy TCI probe.
The 13 C-detected, 19 F-13 C out-and-stay TROSY experiment is available from Arthanari Laboratory at the Dana Farber Cancer Institute (https://artlab.dana-farber.org/19f_13c_aromatictrosy.html). For the HIV TAR 2 RNA 12 the following parameters were used: spectral width in the indirect 19 F dimension was set to 10 ppm, and the spectral width in the direct 13 C dimension was set to 10 ppm. A total of 64 complex points was collected in the indirect 19  Results and discussion on the chemical solid phase synthesis of [5-19 F, 5-13 C]-pyrimidine modified RNA. With the building blocks 7 and 11 available their performance was checked in solid phase RNA synthesis. Phosphoramidites 7 and 11 were used in combination with RNA TBS phosphoramidites and were incorporated with a high coupling efficiency (> 98%). The standard deprotection procedure using aqueous methylamine and aqueous ammonia solutions (AMA) at an elevated temperature followed by treatment with triethylamine hydrogen fluoride in dimethylsulfoxide and quenching with a triethylammonium acetate buffer could be applied. We obtained high quality crude products for both the 30nt HIV TAR 2 RNA 12, the 61nt hHBV e RNA 13, the SAM VI aptamer 14 and the 59nt and 29nt pre-miR 21 RNAs 15 and 15a and after purification by anion exchange chromatography the correct sequence assembly was confirmed by mass spectrometry (Supporting Table 1 and Supporting Figure S1). To summarize, the [5- 19  sensing riboswitch [2] . The global stability of the RNA very likely remains unchanged by a compensatory effect -the reduced hydrogen bonding stability by an increased acidity of the uridine imino proton [3] is counteracted by an increased base stacking interaction due to the fluorine substitution. [4] Supporting Table 1. Sequence information and mass spectrometric data for RNAs used in this study.