4.1.12  (2S)-2-amino-4-({[(2R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl}(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)amino)butanamide (4)
Compound 22 (116 mg, 0.10 mmol, 1.00 eq) was treated at room temperature with TFA (1.44 mL) in water (360 μL) and stirred for 3 h at room temperature. The solvents were removed and the residue was treated with 7 M NH3 in MeOH (10 mL). After stirring for 24 h at 30 °C, solvents were removed and the residue was purified by flash column chromatography on a reversed-phase silica gel column C18 (4 g, 40 μm) with a 0–15% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7. The fractions containing the pure compound were pooled, concentrated and lyophilized with water to give 4 (23 mg, 38%) as a white powder with 98% purity determined by HPLC analysis at 260 nm. 1H NMR (600 MHz, D2O) δ 8.12 (s, 1H), 8.08 (s, 1H), 7.99 (s, 1H), 7.90 (s, 1H), 5.90 (d, J = 2.7 Hz, 1H), 5.79 (d, J = 3.5 Hz, 1H), 4.53 (dd, J = 5.2 Hz, J = 2.7 Hz, 1H), 4.52–4.48 (m, 1H), 4.38 (dd, J = 5.0 Hz, J = 3.6 Hz, 1H), 4.35–4.28 (m, 1H), 4.18–4.07 (m, 3H), 3.98 (td, J = 10.7 Hz, J = 2.6 Hz, 1H), 3.88–3.80 (m, 2H), 3.72 (dd, J = 12.9 Hz, J = 3.5 Hz, 1H), 3.16–2.98 (m, 3H), 2.88–2.76 (m, 2H), 2.62 (dt, J = 14.0 Hz, J = 2.7 Hz, 1H), 2.17–1.98 (m, 2H). 13C NMR (150 MHz, D2O) δ 173.0, 155.0 (2C) 152.5, 152.3, 147.8, 147.6, 139.4, 138.7, 118.6, 118.3, 88.9, 86.8, 84.9, 81.8, 79.8, 73.8, 71.1, 69.0, 67.6, 60.4, 55.3, 53.9, 52.6, 52.4, 27.0. HRMS (ESI+): m/z calcd for C26H38N13O8 [M+H]+: 660.2961, found: 660.2966.