All the reagents and solvents for the chemical syntheses were obtained from commercial sources and used without further purification. The 1H-, 19F- and 13C-NMR spectra were acquired on 400/700 MHz instruments (Bruker, Billerica, MA, USA) using CDCl3 as the solvent. The chemical shifts were reported in parts per million (δ) relative to the residual solvent signal (1H: CHCl3 7.27; 13C: CDCl3 77.0) and assigned by 2D-NMR experiments. All the NMR spectra were processed using the iNMR software package (Nucleomatica, Molfetta, Italy). The HRESI-MS spectra were recorded in positive mode on a Thermo Orbitrap XL mass spectrometer (ThermoFisher, Waltham, MA, USA). The column chromatography was performed by using silica gel 60, 70–230 mesh (Merck, Darmstadt, Germany). The TLC analyses were performed using 0.2 mm thick F254 silica gel plates (Merck, Darmstadt, Germany). The TLC spots were detected under UV light (254 nm). The high-performance liquid chromatography (HPLC) was performed on a UP-2075 Plus pump equipped with a UV-2075 Plus UV detector (Jasco, Cremella, Italy) using a 5 µm, 250-10 Si column (Purosphere® STAR, Merck, Darmstadt, Germany) eluted with n-hexane/AcOEt, 6:4 with a flow rate of 2.0 mL/min.
5′-Chloro-5′-deoxy-2′,3′-O-isopropylidene-6-fluoro nebularine (8). To a stirred solution of 4 (0.28 g, 0.86 mmol) in dry THF (7.5 mL), TsF (0.30 g, 1.7 mmol) and TBAF (2.6 mL of a 1.0 M solution in dry THF, 2.6 mmol) were added and the mixture refluxed for 16 h (TLC monitoring:
n-hexane/AcOEt; 1:1). The solvent was removed under reduced pressure and the crude residue purified by silica gel flash chromatography (
n-hexane/AcOEt, 6:4) to afford, in addition to the two expected compounds 5a and 5b [
19], the inseparable mixture of the two nucleosides 8 and 9 with Rf = 0.52 (10% yield). The mixture was purified by HPLC (see above), thus obtaining the pure compound 8. Colorless foam (3.5 mg, 1.2% yield).
1H-NMR (400 MHz; CDCl
3): δ 8.69 (s, 1H, 2-H), 8.29 (s, 1H, 8-H), 6.24 (d,
J = 2.6 Hz, 1H, 1′-H), 5.40 (dd,
J = 6.4, 2.7 Hz, 1H, 2′-H), 5.13 (dd,
J = 6.4, 3.1 Hz, 1H, 3′-H), 4.58-4.55 (m, 1H, 4′-H), 3.82 (dd,
J = 11.6, 6.1 Hz, 1H, 5′-H
a), 3.70 (dd,
J = 11.6, 4.9 Hz, 1H, 5′-H
b), 1.66 (s, 3H, CH
3), 1.42 (s, 3H, CH
3);
19F-NMR (377 MHz; CDCl
3): δ −68.4 (s);
13C-NMR (176 MHz; CDCl
3): δ 159.9 (d,
J = 261.8, 6-C), 154.5 (d,
J = 11.1, 4-C), 152.2 (d,
J = 14.3, 2-C), 144.0 (8-C), 121.15 (d,
J = 29.2, 5-C), 115.1 (C
q acetonide), 91.3 (1′-C), 86.2 (4′-C), 84.2 (2′-C), 82.3 (3′-C), 43.9 (CH
2Cl), 27.1 (CH
3), 25.3 (CH
3); HRESI-MS
m/z 329.0809, ([M + H]
+ calcd. for C
13H
15ClFN
4O
3 329.0817).