4.1.20  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)butanoic acid (8)
Compound 25 (140 mg, 0.15 mmol, 1.00 eq) was treated with TFA (1.37 mL) and water (0.34 mL). After stirring for 5.5 h, solvents were removed under vacuum. The residue was dissolved in water (0.5 mL) and 2 M LiOH aqueous solution (3.5 mL) was added. After stirring at 0 °C for 30 min, pH was adjusted to ≈3 or 4 by addition of 1 M HCl aqueous solution. Solvents were removed under vacuum and the residue was purified by flash column chromatography on a reversed-phase silica gel column C18 (4 g, 40 μm) with a 0–20% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7. The fractions containing the pure compound were pooled, concentrated and lyophilized with water and 1,4-dioxane to give 8 (35 mg, 36%) as a white powder with 99% purity determined by HPLC analysis at 260 nm. 1H NMR (600 MHz, D2O) δ 8.24 (s, 1H), 8.13 (s, 1H), 8.05 (s, 1H), 8.01 (s, 1H), 5.98 (d, J = 4.3 Hz, 1H), 5.94 (d, J = 4.6 Hz, 1H), 4.68–4.59 (m, 1H), 4.51–4.44 (m, 2H), 4.44–4.38 (m, 1H), 4.38–4.30 (m, 1H), 4.23–4.15 (m, 1H), 4.09–3.95 (m, 2H), 3.91 (dd, J = 12.9, J = 2.7 Hz, 1H), 3.80 (dd, J = 12.9, J = 3.8 Hz, 1H), 3.69–3.33 (m, 4H), 3.20 (m, 2H), 2.29 (td, J = 7.0 Hz, J = 2.7 Hz, 2H), 2.02–1.85 (m, 2H). 13C NMR (150 MHz, D2O) δ 174.5, 154.9 (2C), 152.5, 152.3, 147.7, 147.5, 139.3, 138.5, 118.6, 118.3, 89.0, 86.9, 84.8, 81.8, 80.1, 73.9, 71.1, 69.0, 67.8, 60.3, 56.2, 55.3, 53.8, 52.5, 26.1. HRMS (ESI+): m/z calcd for C26H37N12O9 [M+H]+: 661.2806, found: 661.2811.