4.1  Chemistry

4.1.1  General procedures
All dry solvents and reagents were purchased from commercial suppliers and were used without further purification. DIEA was distilled over calcium hydride. Thin-layer chromatography (TLC) analyses were carried out on silica plate 60 F254. Purifications by column chromatography were performed using Biotage Isolera 1 system with FlashPure cartridges (Buchi). NMR experiments were recorded on Bruker 400, 500 or 600 MHz spectrometers at 20 °C. HRMS analyses were obtained with electrospray ionization (ESI) in positive mode on a Q-TOF Micromass spectrometer. Analytical HPLC was performed on a UHPLC Thermoscientific Ultimate 3000 system equipped with a LPG-3400RS pump, a DAD 3000 detector and an WPS-3000TBRS Autosampler, Column Oven TCC-3000SD. Dinucleosides 1–16 were analyzed by RP-HPLC on a Column Nucleodur C18 ec 100-3, 4.6 × 75 mm (Macherey Nagel) at 30 °C. The following HPLC solvent systems were used: 1% CH3CN in 12.5 mM TEAAc (buffer A), 80% CH3CN in 12.5 mM TEEAc (buffer B). Flow rate was 1 mL/min. UV detection was performed at 260 nm. Lyophilized compounds 1–16 were stored at −20 °C for several months without any degradation.

4.1.2  General method A for the synthesis of compounds 18, 26, 27 and 29
To a solution of 5′-amino-2′,3′-isopropylidene adenosine in anhydrous DMF (conc. 0.2 M) was added Et3N (2.00 eq) and the corresponding nitrobenzenesufonyl chloride (1.50 eq) at 0 °C. The reaction mixture was stirred for 4 h at room temperature under argon then was diluted with AcOEt and brine. The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated under vacuum. The precipitate was washed with Et2O and DCM to give the desired compound as a colored solid.

4.1.3  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-4-nitrobenzene-1-sulfonamide (18)
Following method A with 5′-amino-2′,3′-isopropylidene adenosine (2.21 g, 7.22 mmol, 1.00 eq) and 4-nitrobenzenesufonyl chloride, 18 (2.64 g, 74%) was obtained as a beige solid. Rf 0.50 (8:92 MeOH/DCM). 1H NMR (600 MHz, DMSO‑d 6) δ 8.55 (t, J = 5.8 Hz, 1H), 8.33–8.27 (m, 2H), 8.26 (s, 1H), 8.13 (s, 1H), 7.96–7.88 (m, 2H), 7.37 (br. s, 2H), 6.09 (d, J = 2.8 Hz, 1H), 5.32 (dd, J = 6.3 Hz, 2.9 Hz, 1H), 4.89 (dd, J = 6.3 Hz, 3.0 Hz, 1H), 4.16 (td, J = 5.9 Hz, 3.0 Hz, 1H), 3.24–3.10 (m, 2H), 1.50 (s, 3H), 1.27 (s, 3H). 13C NMR (600 MHz, DMSO‑d 6) δ 156.3, 152.7, 149.5, 148.4, 146.0, 140.2, 128.0, 124.5, 119.3, 113.6, 89.4, 84.4, 83.0, 81.6, 44.6, 27.0, 25.2. HRMS (ESI+): m/z calcd for C19H22N7O7S [M+H]+: 492.1301, Found 492.1311.

4.1.4  General method B for the synthesis of compounds 19, 30-33
To a solution of 18 or 26–29 in anhydrous DMF (conc. 0.13 M) was successively added 17 (1.10 eq), KI (0.30 eq) and K2CO3 (3.00 eq). After stirring for 24 h at 50 °C under argon, the reaction was diluted in AcOEt and water. The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0–8% MeOH in DCM) to give the desired compound as colored solid.

4.1.5  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-3-yl]oxy}ethyl)-4-nitrobenzene-1-sulfonamide (19)
Following method B using 18 (290 mg, 0.59 mmol, 1.00 eq), 19 (450 mg, 74%) was obtained as a pale yellow solid. Rf 0.60 (10:90 MeOH/DCM). 1H NMR (600 MHz, CDCl3) δ 8.30 (2s, 2H), 8.14 (s, 1H), 8.03–7.98 (m, 2H), 7.74 (s, 1H), 7.70–7.66 (m, 2H), 6.11–5.93 (m, 5H), 5.89 (d, J = 1.9 Hz, 1H), 5.35 (dd, J = 6.4 Hz, J = 1.9 Hz, 1H), 5.04 (dd, J = 6.4 Hz, J = 3.8 Hz, 1H), 4.51 (dd, J = 6.1 Hz, J = 4.6 Hz, 1H), 4.43 (dt, J = 8.5 Hz, J = 4.2 Hz, 1H), 4.22 (dd, J = 4.7 Hz, J = 3.2 Hz, 1H), 4.05 (dt, J = 5.9 Hz, J = 3.0 Hz, 1H), 3.99 (dd, J = 11.5 Hz, J = 3.5 Hz, 1H), 3.85–3.41 (m, 7H), 1.57 (s, 3H), 1.35 (s, 3H), 0.92 (s, 9H), 0.91 (s, 9H), 0.14–0.04 (m, 12H). 13C NMR (150 MHz, CDCl3) δ 155.8, 155.5, 153.2, 153.0, 149.7, 149.5, 148.9, 145.4, 140.2, 139.5, 128.4, 123.8, 120.5, 120.2, 114.7, 90.7, 87.3, 85.0, 84.5, 84.2, 82.8, 82.6, 69.7, 69.6, 61.5, 50.7, 47.7, 27.3, 26.2, 25.9, 25.4, 18.6, 18.2, −4.4, −4.7, −5.2, −5.3. HRMS (ESI+): m/z calcd for C43H65N12O11SSi2 [M+H]+: 1013.4155, found: 1013.4155.

4.1.6  9-[(2R,5R)-3-[2-({[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}amino)ethoxy]-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-2-yl]-9H-purin-6-amine (20)
To a solution of 19 (210 mg, 0.20 mmol, 1.00 eq) in anhydrous DMF (10 mL) was added K2CO3 (85 mg, 0.61 mmol, 3.00 eq) and thiophenol (11 μL, 1.14 mmol, 5.70 eq). After stirring for 2 h at room temperature under argon, the reaction mixture was diluted with DCM (20 mL) and washed with 1 M aqueous NaOH solution (10 mL). The aqueous layer was extracted with DCM (3 × 20 mL) and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0–15% MeOH in DCM) as the eluent to give 20 (129 mg, 76%) as a white solid. Rf 0.31 (8:92 MeOH/DCM). 1H NMR (500 MHz, CDCl3) δ 8.32 (s, 1H), 8.31 (s, 1H), 8.17 (s, 1H), 7.93 (s, 1H), 6.17–6.06 (m, 5H), 6.03 (d, J = 2.9 Hz, 1H), 5.39 (dd, J = 6.5 Hz, J = 3.0 Hz, 1H), 4.99–4.92 (m, 1H), 4.51–4.45 (m, 1H), 4.33–4.25 (m, 2H), 4.08 (dt, J = 5.6 Hz, J = 3.2 Hz, 1H), 3.98 (dd, J = 11.5 Hz, J = 3.6 Hz, 1H), 3.75 (dd, J = 11.6 Hz, J = 2.8 Hz, 1H), 3.71–3.63 (m, 2H), 2.95–2.69 (m, 4H), 1.61 (s, 3H), 1.37 (s, 3H), 0.92 (s, 9H), 0.88 (s, 9H), 0.13–0.04 (m, 12H). 13C NMR (125 MHz, CDCl3) δ 155.8, 155.7, 153.3, 153.1, 149.7, 149.6, 139.8, 139.4, 120.5, 120.3, 114.8, 90.7, 87.2, 85.8, 85.1, 83.9, 82.6, 82.4, 70.3, 70.13, 62.0, 51.5, 49.5, 27.4, 26.2, 25.8, 18.6, 18.2, −4.5, −4.8, −5.2, −5.3. HRMS (ESI+): m/z calcd for C37H62N11O7Si2 [M+H]+: 828.4372, found: 828.4371.

4.1.7  General method C for the synthesis of compounds 1, 3, 5–7, 9, 10-16
The synthesis intermediate was treated at room temperature with TFA in water (8/2, conc. 0.14 M). After stirring for 3–6 h until completion of reaction (TLC DCM/MeOH), solvents were removed and the residue was coevaporated three times with 7 M NH3 in MeOH. The residue was purified by flash column chromatography on a reversed-phase silica gel column C18 (4 g, 40 μm) with a linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7. The fractions containing the pure compound were pooled, concentrated and lyophilized with water/dioxane to give desired compound as a powder.

4.1.8  (2R,5R)-2-(6-amino-9H-purin-9-yl)-5-{[(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)amino]methyl}oxolane-3,4-diol (1)
Following method C with 20 (117 mg, 0.14 mmol, 1.00 eq) and a 0–25% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 1 (60 mg, 76%) was obtained as a white powder with 99% purity determined by HPLC analysis at 260 nm. 1H NMR (600 MHz, DMSO‑d 6) δ 8.38 (s, 1H), 8.34 (s, 1H), 8.15 (s, 1H), 8.14 (s, 1H), 7.36 (br.s, 2H), 7.29 (br. s, 2H), 5.97 (d, J = 6.4 Hz, 1H), 5.85 (d, J = 5.9 Hz, 1H), 5.58–4.98 (m, 4H), 4.70–4.63 (m, 1H), 4.52 (dd, J = 6.4 Hz, J = 4.7 Hz, 1H), 4.33 (dd, J = 4.7 Hz, J = 2.6 Hz, 1H), 4.12 (dd, J = 5.2 Hz, J = 3.8 Hz, 1H), 4.02–3.93 (m, 2H), 3.70–3.48 (m, 4H), 2.86–2.58 (m, 4H). 13C NMR (150 MHz, DMSO‑d 6) δ 156.2, 156.1, 152.6, 152.5, 149.4, 149.0, 140.1, 139.9, 119.4, 119.3, 87.6, 86.3 (2C), 83.7, 81.5, 72.8, 71.5, 69.5, 69.1, 61.7, 51.2, 49.0. HRMS (ESI+): m/z calcd for C22H30N11O7 [M+H]+: 560.23242, found: 560.23188.

4.1.9  Methyl (2S)-4-({[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-3-yl]oxy}ethyl)amino)-2-{bis[(tert-butoxy)carbonyl]amino}butanoate (22)
To a solution of 20 (250 mg, 0.30 mmol, 1.00 eq) in anhydrous DCM (9 mL) was added aldehyde 21 (120 mg, 0.36 mmol, 1.20 eq) in anhydrous DCM (9 mL) and acetic acid (20 μL, 0.36 mmol, 1.20 eq). After stirring for 20 min at room temperature under argon, sodium triacetoxyborohydride (96 mg, 0.45 mmol, 1.5 eq) was added. After stirring for 2.5 h, the reaction mixture was diluted with DCM (30 mL) and washed with saturated aqueous NaHCO3 (30 mL). The aqueous layer was extracted with DCM (3 × 30 mL) and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0–8% MeOH in DCM) to give 22 (321 mg, 93%) as a white solid. Rf 0.71 (MeOH/DCM 5:95). 1H NMR (600 MHz, CDCl3) δ 8.33 (s, 1H), 8.30 (s, 1H), 8.17 (s, 1H), 7.93 (s, 1H), 6.09 (d, J = 3.3 Hz, 1H), 6.01 (d, J = 2.4 Hz, 1H), 5.97 (m, 4H), 5.42 (dd, J = 6.5 Hz, J = 2.4 Hz, 1H), 4.93–4.86 (m, 2H), 4.54–4.49 (m, 1H), 4.33 (td, J = 6.8 Hz, J = 3.3 Hz, 1H), 4.29–4.24 (m, 1H), 4.08 (dt, J = 6.2 Hz, J = 3.2 Hz, 1H), 3.99 (dd, J = 11.5 Hz, J = 3.7 Hz, 1H), 3.78–3.63 (m, 6H), 2.84–2.74 (m, 3H), 2.71–2.51 (m, 3H), 2.33–2.23 (m, 1H), 1.91–1.82 (m, 1H), 1.58 (s, 3H), 1.46 (s, 18H), 1.36 (s, 3H), 0.91 (s, 9H), 0.90 (s, 9H), 0.10–0.05 (m, 12H). 13C NMR (125 MHz, CDCl3) δ 171.6, 155.8, 155.7, 153.3, 153.2, 152.3, 149.7, 149.5, 140.1, 139.8, 120.5, 120.4, 114.5, 91.1, 87.4, 85.45, 84.8, 84.1, 83.3, 83.3, 82.5, 69.9, 69.1, 61.9, 56.3, 56.3, 53.7, 52.4, 52.1, 28.1, 28.1, 27.3, 26.2, 26.0, 25.6, 18.7, 18.3, −4.3, −4.6, −5.2, −5.2. HRMS (ESI+): m/z calcd for C52H87N12O13Si2 [M+H]+: 1143.6049, found: 1143.6043.

4.1.10  (2S)-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)-2-azaniumylbutanoate (2)
Compound 22 (86 mg, 75.3 μmol, 1.00 eq) was treated at room temperature with TFA (830 μL) in water (210 μL). After stirring for 3 h, solvents were removed under vacuum. The residue was dissolved in water (0.5 mL) and 2 M aqueous LiOH solution (4 mL) was added. After stirring at 0 °C for 30 min, pH ≈ 3 or 4 was adjusted by addition of 1 M aqueous HCl solution. The solvent was 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 0–15% 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 2 (16 mg, 32%) as a white powder with 99% purity determined by HPLC analysis at 260 nm. 1H NMR (600 MHz, D2O) δ 8.11 (s, 1H), 8.05 (s, 1H), 7.98 (s, 1H), 7.86 (s, 1H), 5.89 (d, J = 2.5 Hz, 1H), 5.77 (d, J = 3.3 Hz, 1H), 4.51–4.47 (m, 2H), 4.36 (dd, J = 5.1 Hz, J = 3.4 Hz, 1H), 4.34–4.29 (m, 1H), 4.14–4.08 (m, 2H), 3.97 (td, J = 10.6 Hz, J = 2.4 Hz, 1H), 3.89–3.80 (m, 3H), 3.71 (dd, J = 13.0, J = 3.4 Hz, 1H), 3.15–3.04 (m, 2H), 3.03–2.81 (m, 2H), 2.76 (d, J = 14.2 Hz, 1H), 2.58 (d, J = 13.8 Hz, 1H), 2.14–1.99 (m, 2H). 13C NMR (150 MHz, D2O) δ 174.5, 154.9 (2C) 152.5, 152.2, 147.7, 147.5, 139.3, 138.5, 118.5, 118.2, 89.0, 86.8, 84.8, 81.7, 80.0, 73.9, 71.1, 68.9, 67.7, 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.

4.1.11  Methyl (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)butanoate (3)
Following method C with 22 (140 mg, 0.12 mmol, 1.00 eq) and a 0–30% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 3 (28 mg, 35%) was obtained as a white powder with 98% purity determined by HPLC analysis at 260 nm. 1H NMR (600 MHz, D2O) δ 8.08 (s, 1H), 8.05 (s, 1H), 7.98 (s, 1H), 7.84 (s, 1H), 5.89 (d, J = 2.5 Hz, 1H), 5.75 (d, J = 3.3 Hz, 1H), 4.55–4.50 (m, 1H), 4.47 (dd, J = 5.1 Hz, J = 2.3 Hz, 1H), 4.38 (dd, J = 5.0 Hz, J = 3.2 Hz, 1H), 4.36–4.30 (m, 1H), 4.26 (dd, J = 10.5 Hz, J = 2.8 Hz, 1H), 4.13 (dd, J = 7.9 Hz, J = 5.1 Hz, 1H), 4.07 (dt, J = 6.2 Hz, J = 3.1 Hz, 1H), 4.05–3.86 (m, 2H), 3.84 (s, 3H), 3.82–3.64 (m, 2H), 3.22–3.01 (m, 3H), 2.88 (dt, J = 13.7, J = 3.9, 1H), 2.78 (d, J = 13.9 Hz, 1H), 2.66–2.58 (m, 1H), 2.19–2.02 (m, 2H). 13C NMR (150 MHz, D2O) δ 170.6, 154.9 (2C), 152.5, 152.2, 147.7, 147.4, 139.1, 138.3, 118.5, 118.1, 89.1, 86.8, 84.6, 81.8, 79.6, 73.9, 70.9, 69.1, 60.2, 54.8, 54.1, 53.5, 53.3, 52.3, 25.0. HRMS (ESI+): m/z calcd for C27H39N12O9 [M+H]+: 675.2957, found: 675.2944.

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.

4.1.13  General method D for the synthesis of compounds 23, 24 and 25
To a solution of 20 in anhydrous NMP (conc. 0.03 M) was added the corresponding bromide reagent (3.00 eq) and DIEA (3.00 eq). After stirring for 4 h at 110 °C under microwaves, the reaction mixture was diluted with AcOEt and brine. The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0–4% MeOH in DCM) to give the desired compound as a colorless solid.

4.1.14  9-[(2R,5R)-3-[2-({[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}(butyl)amino)ethoxy]-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-2-yl]-9H-purin-6-amine (23)
Following method D with 20 (80 mg, 97 μmol, 1.00 eq) and 1-bromobutane, 23 (40 mg, 47%) was obtained as a white solid. Rf 0.48 (MeOH/DCM 5:95). 1H NMR (600 MHz, CDCl3) δ 8.33 (s, 2H), 8.16 (s, 1H), 7.92 (s, 1H), 6.11 (d, J = 3.8 Hz, 1H), 6.03 (d, J = 2.3 Hz, 1H), 5.77 (br. s, 4H), 5.47 (dd, J = 6.4 Hz, J = 2.2 Hz, 1H), 4.91 (dd, J = 6.4 Hz, J = 3.2 Hz, 1H), 4.51 (t, J = 5.1 Hz, 1H), 4.37–4.24 (m, 2H), 4.09 (dt, J = 5.9 Hz, J = 3.0 Hz, 1H), 3.99 (dd, J = 11.5 Hz, J = 3.7 Hz, 1H), 3.76 (dd, J = 11.5 Hz, J = 2.8 Hz, 1H), 3.70–3.61 (m, 2H), 2.77–2.68 (m, 3H), 2.63 (dd, J = 13.5 Hz, J = 7.0 Hz, 1H), 2.46–2.37 (m, 2H), 1.59 (s, 3H), 1.38 (s, 3H), 1.31–1.22 (m, 2H), 1.22–1.12 (m, 2H), 0.92 (s, 9H), 0.91 (s, 9H), 0.82 (t, J = 7.3 Hz, 3H), 0.10–0.08 (m, 12H). 13C NMR (150 MHz, CDCl3) δ 155.6, 155.5, 153.2, 153.1, 149.8, 149.4, 140.2, 139.7, 120.5, 120.3, 114.4, 91.2, 87.2, 85.8, 84.9, 84.0, 83.4, 82.4, 69.9, 69.3, 62.0, 56.7, 55.1, 53.9, 29.2, 27.2, 26.1, 25.9, 25.5, 20.5, 18.6, 18.2, 14.1, −4.4, −4.6, −5.2, −5.2. HRMS (ESI+): m/z calcd for C46H72N11O7Si2 [M+H]+: 884.4998, found: 884.5002.

4.1.15  9-[(2R,5R)-3-[2-({[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}(3-phenylpropyl)amino)ethoxy]-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-2-yl]-9H-purin-6-amine (24)
Following method D with 20 (120 mg, 0.15 mmol, 1.00 eq) and 1-bromo-3-phenylpropane, 24 (75 mg, 53%) was obtained as a white solid. Rf 0.53 (MeOH/DCM 5:95).1H NMR (600 MHz, CDCl3) δ 8.31 (s, 1H), 8.28 (s, 1H), 8.15 (s, 1H), 7.90 (s, 1H), 7.25–7.08 (m, 5H), 6.11 (d, J = 3.7 Hz, 1H), 6.02 (d, J = 2.4 Hz, 1H), 5.81 (br. s, 4H), 5.46 (dd, J = 6.5 Hz, J = 2.1 Hz, 1H), 4.92 (dd, J = 6.5 Hz, J = 3.2 Hz, 1H), 4.50 (t, J = 5.1 Hz, 1H), 4.33–4.24 (m, 2H), 4.08 (dt, J = 5.9 Hz, J = 3.2 Hz, 1H), 3.98 (dd, J = 11.4 Hz, J = 3.6 Hz, 1H), 3.76 (dd, J = 11.5 Hz, J = 2.8 Hz, 1H), 3.72–3.60 (m, 2H), 2.80–2.70 (m, 3H), 2.64 (dd, J = 13.7 Hz, J = 6.7 Hz, 1H), 2.56–2.43 (m, 4H), 1.69–1.62 (m, 2H), 1.58 (s, 3H), 1.37 (s, 3H), 0.92 (s, 9H), 0.90 (s, 9H), 0.13–0.04 (m, 12H).13C NMR (150 MHz, CDCl3) δ 155.6, 155.5, 153.2, 153.1, 149.7, 149.4, 142.2, 140.2, 139.6, 128.4, 128.4, 125.8, 120.5, 120.3, 114.4, 91.1, 87.2, 85.7, 84.8, 83.9, 83.3, 82.5, 69.8, 69.3, 61.8, 56.7, 54.9, 53.9, 33.5, 28.8, 27.2, 26.1, 25.8, 25.5, 18.6, 18.2, −4.4, −4.6, −5.2, −5.2. HRMS (ESI+): m/z calcd for C46H72N11O7Si2 [M+H]+: 946.5155, found: 946.5149.

4.1.16  Methyl 4-({[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-3-yl]oxy}ethyl)amino)butanoate (25)
Following method D with 20 (600 mg, 0.73 mmol, 1.00 eq) and methyl-4-bromobutyrate, 25 (390 mg, 58%) was obtained as a white solid. Rf 0.34 (MeOH/DCM 5:95). 1H NMR (600 MHz, CDCl3) δ 8.32 (s, 1H), 8.31 (s, 1H), 8.17 (s, 1H), 7.90 (s, 1H), 6.11 (d, J = 3.6 Hz, 1H), 6.02 (m, 5H), 5.45 (dd, J = 6.5 Hz, J = 2.2 Hz, 1H), 4.91 (dd, J = 6.6 Hz, J = 3.3 Hz, 1H), 4.51 (t, J = 5.3 Hz, 1H), 4.32–4.23 (m, 2H), 4.08 (dt, J = 6.1 Hz, J = 3.2 Hz, 1H), 3.99 (dd, J = 11.5 Hz, J = 3.6 Hz, 1H), 3.76 (dd, J = 11.5 Hz, J = 2.8 Hz, 1H), 3.71–3.60 (m, 5H), 2.77 (dd, J = 13.5 Hz, J = 6.9 Hz, 1H), 2.74–2.69 (m, 2H), 2.64 (dd, J = 13.5 Hz, J = 6.8 Hz, 1H), 2.51–2.44 (m, 2H), 2.32–2.18 (m, 2H), 1.69–1.61 (m, 2H), 1.58 (s, 3H), 1.37 (s, 3H), 0.92 (s, 9H), 0.90 (s, 9H), 0.13–0.04 (m, 12H). 13C NMR (150 MHz, CDCl3) δ 174.0, 155.7, 155.6, 153.2, 153.1, 149.7, 149.4, 140.1, 139.5, 120.5, 120.3, 114.4, 91.1, 87.2, 85.7, 84.8, 83.9, 83.3, 82.5, 69.8, 69.3, 61.9, 56.7, 54.3, 53.7, 51.6, 31.5, 27.2, 26.1, 25.8, 25.5, 22.6, 18.6, 18.2, −4.4, −4.6, −5.2, −5.2. HRMS (ESI+): m/z calcd for C42H70N11O9Si2 [M+H]+: 928.4897, found: 928.4900.

4.1.17  (2R,5R)-2-(6-amino-9H-purin-9-yl)-5-{[(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)(butyl)amino]methyl}oxolane-3,4-diol (5)
Following method C with 23 (40 mg, 0.45 mol, 1.00 eq) and a 0–50% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 5 (20 mg, 72%) was obtained as a white powder with 98% purity determined by HPLC analysis at 260 nm.1H NMR (500 MHz, DMSO‑d 6) δ 8.36 (s, 1H), 8.32 (s, 1H), 8.14 (s, 1H), 8.13 (s, 1H), 7.33 (br. s, 2H), 7.25 (br. s, 2H), 5.97 (d, J = 6.3 Hz, 1H), 5.85 (d, J = 5.2 Hz, 1H), 5.49–5.39 (m, 3H), 5.14 (d, J = 5.3 Hz, 1H), 4.62 (q, J = 5.4 Hz, 1H), 4.52 (dd, J = 6.5, 4.7 Hz, 1H), 4.29 (dd, J = 4.8, 2.8 Hz, 1H), 4.06 (q, J = 4.9 Hz, 1H), 4.00–3.92 (m, 2H), 3.68–3.46 (m, 4H), 2.79 (dd, J = 14.0, 4.6 Hz, 1H), 2.70–2.51 (m, 3H), 2.41–2.30 (m, 2H), 1.22 (q, J = 7.4, 2H), 1.07 (tdd, J = 13.4 Hz, J = 10.5 Hz, J = 6.3 Hz, 2H), 0.73 (t, J = 7.3 Hz, 2H). 13C NMR (125 MHz, DMSO‑d 6) δ 156.2, 156.0, 152.6, 152.4, 149.3, 148.9, 139.8, 139.8, 119.3, 119.1, 87.5, 86.3, 86.2, 82.4, 81.2, 72.5, 71.9, 69.2, 67.9, 61.5, 56.1, 54.0, 53.7, 28.3, 19.8, 13.7. HRMS (ESI+): m/z calcd for C26H38N11O7 [M+H]+: 616.2956, Found 616.2955.

4.1.18  (2R,5R)-2-(6-amino-9H-purin-9-yl)-5-{[(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)(3-phenylpropyl)amino]methyl}oxolane-3,4-diol (6)
Following method C with 24 (40 mg, 0.42 μmol, 1.00 eq) and a 0–50% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 6 (8 mg, 28%) was obtained as a white powder with 99% purity determined by HPLC analysis at 260 nm. 1H NMR (500 MHz, DMSO‑d 6) δ 8.36 (s, 1H), 8.32 (s, 1H), 8.14 (s, 1H), 8.13 (s, 1H), 7.33 (br. s, 2H), 7.25 (br. s, 2H), 5.97 (d, J = 6.3 Hz, 1H), 5.85 (d, J = 5.2 Hz, 1H), 5.49–5.39 (m, 3H), 5.14 (d, J = 5.3 Hz, 1H), 4.62 (q, J = 5.4 Hz, 1H), 4.52 (dd, J = 6.5, 4.7 Hz, 1H), 4.29 (dd, J = 4.8, 2.8 Hz, 1H), 4.06 (q, J = 4.9 Hz, 1H), 4.00–3.92 (m, 2H), 3.68–3.46 (m, 4H), 2.79 (dd, J = 14.0, 4.6 Hz, 1H), 2.70–2.51 (m, 3H), 2.41–2.30 (m, 2H), 1.22 (q, J = 7.4, 2H), 1.07 (tdd, J = 13.4 Hz, J = 10.5 Hz, J = 6.3 Hz, 2H), 0.73 (t, J = 7.3 Hz, 2H). 13C NMR (125 MHz, DMSO‑d 6) δ 156.1, 156.0, 152.5, 152.4, 149.3, 149.0, 139.8, 139.8, 119.3, 119.2, 87.6, 86.3, 86.2, 82.4, 81.2, 72.5, 71.9, 69.2, 68.0, 62.0, 56.2, 54.0, 53.7, 28.3, 19.8, 13.8. HRMS (ESI+): m/z calcd for C26H38N11O7 [M+H]+: 616.2956, Found 616.2955.

4.1.19  Methyl 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)butanoate (7)
Following method C with 25 (116 mg, 0.13 mmol, 1.00 eq) and a 0–50% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 7 (51 mg, 60%) was obtained as a white powder with 97% purity determined by HPLC analysis at 260 nm. 1H NMR (500 MHz, DMSO‑d 6) δ 8.37 (s, 1H), 8.33 (s, 1H), 8.14 (s, 1H), 8.13 (s, 1H), 7.38 (br. s, 2H), 7.29 (br. s, 2H), 5.97 (d, J = 6.3 Hz, 1H), 5.84 (d, J = 5.3 Hz, 1H), 5.52–5.32 (m, 3H), 5.19 (d, J = 5.3 Hz, 1H), 4.62 (q, J = 5.4 Hz, 1H), 4.49 (dd, J = 6.3, 4.7 Hz, 1H), 4.30 (dd, J = 4.8, 2.8 Hz, 1H), 4.04 (q, J = 4.9 Hz, 1H), 3.99–3.96 (m, 1H), 3.95–3.87 (m, 1H), 3.66 (dt, J = 12.1, 4.1 Hz, 1H), 3.62–3.45 (m, 6H), 2.79 (dd, J = 14.1, 4.6 Hz, 1H), 2.71–2.52 (m, 3H), 2.45–2.28 (m, 2H), 2.14 (td, J = 7.4, 5.9 Hz, 2H), 1.58–1.46 (m, 2H). 13C NMR (125 MHz, DMSO‑d 6) δ 173.3, 156.2, 156.1, 152.6, 152.5, 149.4, 148.9, 139.9, 139.8, 119.4, 119.2, 87.6, 86.4, 86.3, 82.4, 81.3, 72.6, 71.9, 69.2, 68.0, 61.6, 56.3, 53.5, 53.4, 51.2, 30.9, 21.8. HRMS (ESI+): m/z calcd for C27H38N11O9 [M+H]+: 660.2854, Found 660.2855.

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.

4.1.21  N-{[(2R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)-4-nitrobenzene-1-sulfonamide (9)
Using method C with 19 (100 mg, 97.4 μmol, 1.00 eq) and a 0–40% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 9 (25 mg, 34%) was obtained as a white powder with 99% purity determined by HPLC analysis at 260 nm. 1H NMR (600 MHz, DMSO‑d 6) δ 8.32 (s, 1H), 8.29 (s, 1H), 8.22–8.18 (m, 2H), 8.14 (s, 1H), 8.13 (s, 1H), 7.96–7.91 (m, 2H), 7.32 (br. s, 2H), 7.26 (br. s, 2H), 5.89 (d, J = 5.4 Hz, 1H), 5.79 (d, J = 5.6 Hz, 1H), 5.73–5.20 (m, 4H), 4.70 (t, J = 5.4 Hz, 1H), 4.34 (t, J = 5.1 Hz, 1H), 4.30–4.26 (m, 1H), 4.15 (t, J = 4.6 Hz, 1H), 4.09 (dt, J = 8.5 Hz, J = 4.3 Hz, 1H), 3.89 (q, J = 3.6 Hz, 1H), 3.75–3.70 (m, 2H), 3.66–3.63 (m, 1H), 3.60–3.29 (m, 5H). 13C NMR (150 MHz, DMSO‑d 6) δ 156.2, 156.2, 152.8, 152.7, 149.6, 149.3, 149.0, 145.0, 140.2, 139.6, 128.5, 124.3, 119.3, 119.3, 87.9, 86.3, 86.0, 82.9, 81.6, 72.5, 71.4, 70.0, 68.2, 61.2, 50.4, 47.5. HRMS (ESI+): m/z calcd for C28H33N12O11S [M+H]+: 745.2112, Found 745.2104.

4.1.22  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-4-methoxy-2-nitrobenzene-1-sulfonamide (26)
Following method A with 5′-amino-2′,3′-isopropylidene adenosine (280 mg, 0.92 mmol, 1.00 eq) and 4-methoxy-2-nitrobenzenesufonyl chloride, 26 (191 mg, 40%) was obtained as a beige solid. Rf 0.59 (8:92 MeOH/DCM). 1H NMR (600 MHz, DMSO‑d 6) δ 8.43 (t, J = 5.8 Hz, 1H), 8.28 (s, 1H), 8.12 (s, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.51 (d, J = 2.6 Hz, 1H), 7.40 (br. s, 2H), 7.20 (dd, J = 8.9 Hz, J = 2.6 Hz, 1H), 6.11 (d, J = 3.0 Hz, 1H), 5.36 (dd, J = 6.3 Hz, J = 3.0 Hz, 1H), 4.93 (dd, J = 6.4 Hz, J = 2.9 Hz, 1H), 4.23 (td, J = 5.8 Hz, J = 2.9 Hz, 1H), 3.87 (s, 3H), 3.27–3.14 (m, 2H), 1.52 (s, 3H), 1.28 (s, 3H). 13C NMR (150 MHz, DMSO‑d 6) δ 162.5, 156.2, 153.0, 149.0, 148.3, 140.2, 131.3, 123.8, 119.3, 117.2, 113.5, 109.8, 89.5, 84.1, 82.8, 81.5, 56.6, 44.5, 27.0, 25.1. HRMS (ESI+): m/z calcd for C20H24N7O8S [M+H]+: 522.1407, Found 522.1407.

4.1.23  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-2-methoxy-4-nitrobenzene-1-sulfonamide (27)
Following method A with 5′-amino-2′,3′-isopropylidene adenosine (280 mg, 0.92 mmol, 1.00 eq) and 2-methoxy-4-nitrobenzenesufonyl chloride, 27 (200 mg, 42%) was obtained as a beige solid. Rf 0.59 (8:92 MeOH/DCM). 1H NMR (600 MHz, DMSO‑d 6) δ 8.30 (t, J = 5.4 Hz, 1H), 8.23 (s, 1H), 8.12 (s, 1H), 7.88–7.82 (m, 2H), 7.77 (dd, J = 8.5 Hz, J = 2.2 Hz, 1H), 7.37 (br. s, 2H), 6.06 (d, J = 2.9 Hz, 1H), 5.31 (dd, J = 6.3 Hz, J = 3.0 Hz, 1H), 4.90 (dd, J = 6.4 Hz, J = 2.9 Hz, 1H), 4.16 (td, J = 5.7 Hz, J = 2.9 Hz, 1H), 3.88 (s, 3H), 3.24–3.12 (m, 2H), 1.49 (s, 3H), 1.27 (s, 3H). 13C NMR (150 MHz, DMSO‑d 6) δ 156.7, 156.2, 152.6, 150.8, 148.3, 140.1, 133.5, 130.3, 119.3, 114.8, 113.4, 107.7, 89.5, 84.2, 82.9, 81.5, 56.8, 44.7, 26.9, 25.1. HRMS (ESI+): m/z calcd for C20H24N7O8S [M+H]+: 522.1407, Found 522.1407.

4.1.24  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-2-nitro-4-(trifluoromethyl)benzene-1-sulfonamide (28)
To a solution of 5′-amino-2′,3′-isopropylidene adenosine (280 mg, 0.92 mmol, 1.00 eq) in anhydrous DMF (4.6 mL) was added Et3N (250 μL, 1.83 mmol, 2.00 eq). 2-nitro-4-trifluoromethylbenzenesufonyl chloride (396 mg, 1.37 mmol, 1.50 eq) was added at 0 °C and the reaction mixture was stirred for 4 h at room temperature under argon. The reaction mixture was diluted with AcOEt (30 mL) and water (30 mL). The aqueous layer was extracted with AcOEt (3 × 30 mL) and the combined organic extracts were washed with brine (30 mL), dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0–5% MeOH in DCM) to give 28 (230 mg, 45%) as a dark red solid. Rf 0.34 (8:92 MeOH/CH2Cl2). 1H NMR (500 MHz, DMSO‑d 6) δ 8.93 (t, J = 5.1 Hz, 1H), 8.50 (d, J = 1.8 Hz, 1H), 8.28 (s, 1H), 8.14 (s, 1H), 8.10 (dd, J = 1.6 Hz, J = 8.6 Hz, 1H), 8.04 (d, J = 8.3 Hz), 7.45 (br. s, 2H), 6.11 (d, J = 2.9 Hz, 1H), 5.34 (dd, J = 6.3 Hz, J = 2.9 Hz, 1H), 4.92 (dd, J = 6.3 Hz, J = 3.1 Hz, 1H), 4.20 (td, J = 5.9 Hz, J = 3.1 Hz, 1H), 3.37–3.25 (m, 2H), 1.50 (s, 3H), 1.27 (s, 3H). 13C NMR (125 MHz, DMSO‑d 6) δ 156.1, 152.4, 148.4, 147.6, 140.2, 136.4, 133.8, 133.5, 133.2, 133.0, 130.8, 129.4, 125.5, 123.4, 121.2, 119.0, 122.0, 122.0, 119.3, 113.5, 107.7, 89.4, 84.3, 83.0, 81.5, 44.7, 26.9, 25.0. HRMS (ESI+): m/z calcd for C20H21N7O7SF3 [M+H]+: 560.1175, Found 560.1180.

4.1.25  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-4-chloro-3-nitrobenzene-1-sulfonamide (29)
Following method A with 5′-amino-2′,3′-isopropylidene adenosine (280 mg, 0.92 mmol, 1.00 eq) and 4-chloro-3-nitrobenzenesufonyl chloride, 29 (346 mg, 72%) was obtained as a brown solid. Rf 0.42 (8:92 MeOH/CH2Cl2). 1H NMR (400 MHz, DMSO‑d 6) δ 8.56 (s, 1H), 8.39 (d, J = 2.1 Hz, 1H), 8.27 (s, 1H), 8.14 (s, 1H), 7.96 (dd, J = 8.5 Hz, J = 2.1 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.37 (br. s, 2H), 6.12 (d, J = 2.8 Hz, 1H), 5.34 (dd, J = 6.3 Hz, J = 2.9 Hz, 1H), 4.89 (dd, J = 6.3 Hz, J = 3.1 Hz, 1H) 4.17 (td, J = 5.9 Hz, J = 3.0 Hz, 1H), 3.33–3.18 (m, 2H), 1.51 (s, 3H), 1.29 (s, 3H). 13C NMR (100 MHz, DMSO‑d 6) δ 156.7, 153.0, 148.9, 147.8, 141.1, 140.6, 133.4, 131.6, 129.8, 124.4, 119.7, 114.0, 107.7, 89.8, 84.7, 83.4, 82.0, 44.9, 27.4, 25.6. HRMS (ESI+): m/z calcd for C19H21N7O7SCl [M+H]+: 526.0912, Found 526.0914.

4.1.26  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-3-yl]oxy}ethyl)-4-methoxy-2-nitrobenzene-1-sulfonamide (30)
Following method B with 26 (161 mg, 0.31 mmol, 1.00 eq), 30 (166 mg, 52%) was obtained as a pale white solid. Rf 0.67 (10:90 MeOH/DCM). 1H NMR (500 MHz, CDCl3)) δ 8.286 (s, 1H), 8.292 (s, 1H), 8.14 (s, 1H), 7.82 (s, 1H), 7.66 (d, J = 8.9 Hz, 1H), 6.92 (d, J = 2.6 Hz, 1H), 6.74 (dd, J = 9.0 Hz, J = 2.6 Hz, 1H), 6.01 (d, J = 3.2 Hz, 1H), 5.97–5.88 (m, 5H), 5.31 (dd, J = 6.5 Hz, J = 2.1 Hz, 1H), 4.99 (dd, J = 6.4 Hz, J = 3.7 Hz, 1H), 4.52 (dd, J = 6.1 Hz, J = 4.6 Hz, 1H), 4.43 (dt, J = 8.4 Hz, J = 4.2 Hz, 1H), 4.22 (dd, J = 4.7 Hz, J = 3.2 Hz, 1H), 4.06 (dt, J = 6.1 Hz, J = 3.1 Hz, 1H), 3.97 (dd, J = 11.5 Hz, J = 3.5 Hz, 1H), 3.86–3.49 (m, 10H), 1.55 (s, 3H), 1.33 (s, 3H), 0.90 (br. s, 18H), 0.11–0.05 (m, 12H). 13C NMR (125 MHz, CDCl3) δ 162.8, 155.7, 155.5, 153.2, 152.9, 149.6, 149.1, 149.0, 140.2, 139.7, 133.0, 125.0, 120.4, 120.3, 116.2, 114.7, 109.9, 90.6, 87.4, 84.5, 84.5 (2C), 84.3, 82.6, 82.5, 69.9, 69.6, 61.6, 50.2, 47.2, 27.3, 26.2, 25.9, 25.4, 18.6, 18.2, −4.4, −4.7, −5.3. HRMS (ESI+): m/z calcd for C47H67N12O12SSi2 [M+H]+: 1043.4261, found: 1043.4265.

4.1.27  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-3-yl]oxy}ethyl)-2-methoxy-4-nitrobenzene-1-sulfonamide (31)
Following method B with 27 (185 mg, 0.36 mmol, 1.00 eq), 31 (160 mg, 43%) was obtained as a pale white solid. Rf 0.67 (10:90 MeOH/DCM). 1H NMR (500 MHz, CDCl3) δ 8.30 (s, 1H), 8.29 (s, 1H), 8.16 (br. s, 1H), 7.82 (s, 1H), 7.67 (m, 2H), 7.57–7.43 (m, 2H), 6.16–5.90 (m, 5H), 5.88 (s, 1H), 5.27 (br. s, 1H), 4.94 (dd, J = 6.7 Hz, J = 3.7 Hz, 1H), 4.52 (t, J = 5.4 Hz, 1H), 4.42 (dt, J = 8.7 Hz, J = 4.2 Hz, 1H), 4.20 (dd, J = 4.7 Hz, J = 3.1 Hz, 1H), 4.07 (dt, J = 6.1 Hz, J = 3.0 Hz, 1H), 3.99 (dd, J = 11.5 Hz, J = 3.4 Hz, 1H), 3.92–3.44 (m, 10H), 1.54 (s, 3H), 1.32 (s, 3H), 0.921 (br. s, 9H), 0.918 (br. s, 9H), 0.15–0.05 (m, 12H). 13C NMR (125 MHz, CDCl3) δ 157.1, 155.8, 155.5, 153.2, 152.9, 150.9, 149.5, 148.9, 139.7, 139.6, 134.2, 131.5, 120.4, 120.2, 114.7 (2C), 106.7, 90.5, 87.5, 84.5, 84.4, 84.4, 82.8, 82.6, 70.7, 69.6, 61.5, 56.8, 50.2, 47.1, 27.2, 26.2, 25.9, 25.4, 18.6, 18.2, −4.3, −4.8, −5.2. HRMS (ESI+): m/z calcd for C47H67N12O12SSi2 [M+H]+: 1043.4261, found: 1043.4255.

4.1.28  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-3-yl]oxy}ethyl)-2-nitro-4-(trifluoromethyl)benzene-1-sulfonamide (32)
Following method B with 28 (77 mg, 0.14 mmol, 1.00 eq), 32 (68 mg, 46%) was obtained as a pale white solid. Rf 0.47 (10:90 MeOH/DCM). 1H NMR (500 MHz, CDCl3) δ 8.33 (s, 1H), 8.30 (s, 1H), 8.15 (s, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 1.7 Hz, 1H), 7.76 (s, 1H), 7.54 (dd, J = 8.4 Hz, J = 1.8 Hz, 1H), 5.97 (d, J = 2.8 Hz, 1H), 5.93 (d, J = 1.9 Hz, 1H), 5.74 (br. s, 4H), 5.33 (dd, J = 6.4 Hz, J = 2.0 Hz, 1H), 5.01 (dd, J = 6.4 Hz, J = 3.8 Hz, 1H), 4.50 (dd, J = 6.4 Hz, J = 4.6 Hz, 1H), 4.43 (dt, J = 8.3 Hz, J = 4.0 Hz, 1H), 4.18 (dd, J = 4.7 Hz, J = 2.8 Hz, 1H), 4.01–3.96 (m, 2H), 3.94–3.78 (m, 4H), 3.77–3.66 (m, 2H), 3.56 (dt, J = 15.5 Hz, J = 5.1 Hz, 1H), 1.55 (s, 3H), 1.34 (s, 3H), 0.91 (s, 9H), 0.89 (s, 9H), 0.13–0.02 (m, 12H). 13C NMR (125 MHz, CDCl3) δ 155.6, 155.4, 153.3, 153.0, 149.5, 149.1, 147.9, 140.2, 139.5, 137.4, 135.6, 135.3, 135.0, 134.7, 132.0, 128.3, 128.3, 125.3, 123.2, 121.0, 118.8, 121.7, 121.6, 133.0, 120.4, 120.3, 116.2, 114.8, 109.9, 90.6, 87.5, 84.7, 84.3, 84.1, 82.9, 82.6, 69.6, 69.4, 61.3, 50.2, 47.4, 27.2, 26.2, 25.9, 25.4, 18.6, 18.2, −4.4, −4.8, −5.3. HRMS (ESI+): m/z calcd for C44H64N12O11SF3Si2 [M+H]+: 1081.4029, found: 1081.4034.

4.1.29  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-3-yl]oxy}ethyl)-4-chloro-3-nitrobenzene-1-sulfonamide (33)
Following method B with 29 (230 mg, 0.44 mmol, 1.00 eq), 33 (160 mg, 43%) was obtained as a pale white solid. Rf 0.52 (10:90 MeOH/DCM). 1H NMR (600 MHz, CDCl3) δ 8.29 (s, 1H), 8.28 (s, 1H), 8.116 (2s, 2H), 7.79 (s, 1H), 7.65 (dd, J = 8.4 Hz, J = 2.1 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 6.02–5.93 (m, 6H), 5.36 (dd, J = 6.4 Hz, J = 1.8 Hz, 1H), 5.05 (dd, J = 6.4 Hz, J = 3.8 Hz, 1H), 4.51 (dd, J = 6.1 Hz, J = 4.7 Hz, 1H), 4.44 (dt, J = 8.5 Hz, J = 4.3 Hz, 1H), 4.19 (dd, J = 4.7 Hz, J = 3.0 Hz, 1H), 4.05–3.97 (m, 2H), 3.82 (t, J = 5.5 Hz, 2H), 3.79–3.65 (m, 3H), 3.46 (q, J = 5.3 Hz, 2H), 1.56 (s, 3H), 1.35 (s, 3H), 0.92 (br. s, 9H), 0.90 (br. s, 9H), 0.12–0.05 (m, 12H). 13C NMR (150 MHz, CDCl3) δ 155.8, 155.6, 153.2, 153.1, 149.5, 149.0, 147.6, 140.3, 140.2, 139.4, 132.4, 131.3, 131.2, 124.6, 120.5, 120.2, 114.7, 90.7, 87.4, 85.0, 84.3 (2C), 82.9, 82.6, 69.7, 69.5, 61.4, 50.5, 47.7, 27.2, 26.2, 25.9, 25.4, 18.6, 18.2, −4.4, −4.7, −5.2. HRMS (ESI+): m/z calcd for C43H64N12O11SSi2Cl [M+H]+: 1047.3765, found: 1047.3766.

4.1.30  N-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N-(2-(((2R,3R,4R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)ethyl)-4-methoxy-2-nitrobenzenesulfonamide (10)
Using method C with 30 (167 mg, 0.16 mmol, 1.00 eq) and a 0–40% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 10 (25 mg, 20%) was obtained as a white powder with 97% purity determined by HPLC analysis at 260 nm. 1H NMR (500 MHz, DMSO‑d 6) δ 8.32 (s, 1H), 8.28 (s, 1H), 8.13 (s, 2H), 7.77 (d, J = 9.0 Hz, 1H), 7.44 (dd, J = 8.5 Hz, J = 2.6 Hz, 1H), 7.34 (br. s, 2H), 7.29 (br. s, 2H), 6.99 (dd, J = 9.0 Hz, J = 2.6 Hz, 1H), 5.95 (d, J = 5.2 Hz, 1H), 5.83 (d, J = 5.5 Hz, 1H), 5.65 (br. s, 1H), 5.49 (br. s, 1H), 5.38 (t, J = 5.7 Hz, 1H), 5.27 (br. s, 1H), 4.66 (t, J = 5.2 Hz, 1H), 4.37 (t, J = 5.1 Hz, 1H), 4.33–4.26 (m, 1H), 4.17–4.08 (m, 2H), 3.93 (q, J = 3.6 Hz, 1H), 3.84 (s, 3H), 3.80–3.62 (m, 3H), 3.60–3.37 (m, 5H). 13C NMR (125 MHz, DMSO‑d 6) δ 162.6, 156.1, 156.2, 152.6, 152.6, 149.3, 149.0, 148.9, 139.9, 139.8, 131.5, 123.3, 119.2 (2C), 116.7, 109.7, 87.8, 86.3, 85.8, 81.8, 81.5, 72.5, 71.5, 68.1, 66.4, 61.1, 56.6, 50.3, 47.1. HRMS (ESI+): m/z calcd for C29H35N12O12S [M+H]+: 775.2218, Found 775.2225.

4.1.31  N-{[(2R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)-2-methoxy-4-nitrobenzene-1-sulfonamide (11)
Using method C with 31 (160 mg, 0.15 mmol, 1.00 eq) and a 0–40% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 11 (15 mg, 13%) was obtained as a white powder with 98% purity determined by HPLC analysis at 260 nm. 1H NMR (500 MHz, DMSO‑d 6) δ 8.32 (s, 1H), 8.28 (s, 1H), 8.13 (s, 2H), 7.77 (d, J = 9.0 Hz, 1H), 7.44 (dd, J = 8.5 Hz, J = 2.6 Hz, 1H), 7.34 (br. s, 2H), 7.29 (br. s, 2H), 6.99 (dd, J = 9.0 Hz, J = 2.6 Hz, 1H), 5.95 (d, J = 5.2 Hz, 1H), 5.83 (d, J = 5.5 Hz, 1H), 5.65 (br. s, 1H), 5.49 (br. s, 1H), 5.38 (t, J = 5.7 Hz, 1H), 5.27 (br. s, 1H), 4.66 (t, J = 5.2 Hz, 1H), 4.37 (t, J = 5.1 Hz, 1H), 4.33–4.27 (m, 1H), 4.17–4.08 (m, 2H), 3.93 (q, J = 3.6 Hz, 1H), 3.84 (s, 3H), 3.80–3.62 (m, 3H), 3.60–3.37 (m, 5H). 13C NMR (125 MHz, DMSO‑d 6) δ 156.9, 156.1, 155.9, 152.5, 152.4, 150.6, 149.0, 148.8, 134.0, 139.5, 133.7, 131.0, 119.2, 119.2, 114.4, 107.4, 87.8, 86.2, 85.9, 81.5, 81.4, 72.5, 71.3, 68.9, 66.7, 61.1, 56.8, 50.2, 47.1. HRMS (ESI+): m/z calcd for C29H35N12O12S [M+H]+: 775.2218, Found 775.2220.

4.1.32  N-{[(2R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)-2-nitro-4-(trifluoromethyl)benzene-1-sulfonamide (12)
Using method C with (120 mg, 0.11 mmol, 1.00 eq) and a 0–40% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 12 (18 mg, 20%) was obtained as a white powder with 99% purity determined by HPLC analysis at 260 nm. 1H NMR (500 MHz, DMSO‑d 6) δ 8.47 (d, J = 1.7 Hz, 1H), 8.32 (s, 1H), 8.28 (s, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.12 (s, 1H), 8.11 (s, 1H), 7.88 (dd, J = 8.6 Hz, J = 1.8 Hz, 1H), 7.36 (br. s, 2H), 7.31 (br. s, 2H), 5.90 (d, J = 5.2 Hz, 1H), 5.82 (d, J = 5.4 Hz, 1H), 5.61 (d, J = 6.0 Hz, 1H), 5.43 (d, J = 5.1 Hz, 1H), 5.38 (dd, J = 6.6 Hz, J = 4.8 Hz, 1H), 5.30 (d, J = 5.3 Hz, 1H), 4.65 (q, J = 5.4 Hz, 1H), 4.37 (t, J = 5.1 Hz, 1H), 4.31 (q, J = 4.7 Hz, 1H), 4.19–4.10 (m, 2H), 3.91 (q, J = 3.6 Hz, 1H), 3.87–3.63 (m, 4H), 3.61–3.45 (m, 4H). 13C NMR (125 MHz, DMSO‑d 6) δ 156.2, 156.1, 152.7, 152.6, 149.2, 148.9, 147.7, 139.9, 139.4, 135.9, 133.9, 133.6, 133.3, 133.1, 131.1, 128.9, 128.9, 152.5, 123.3, 121.1, 119.0, 122.0, 121.9, 119.2 (2C), 87.8, 86.3, 85.9, 81.6, 81.5, 72.5, 71.4, 68.8, 67.7, 61.0, 50.0, 47.3. 19F NMR (378 MHz, DMSO‑d 6): δ −62.8. HRMS (ESI+): m/z calcd for C29H32N12O11SF3 [M+H]+: 813.1986, Found 813.1988.

4.1.33  N-{[(2R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)-4-chloro-3-nitrobenzene-1-sulfonamide (13)
Using method C with 33 (121 mg, 0.12 mmol, 1.00 eq) and a 0–40% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 13 (14 mg, 16%) was obtained as a white powder with 98% purity determined by HPLC analysis at 260 nm. 1H NMR (500 MHz, DMSO‑d 6) δ 8.42 (d, J = 2.2 Hz, 1H), 8.34 (s, 1H), 8.32 (s, 1H), 8.144 (s, 1H), 8.140 (s, 1H), 7.96 (dd, J = 8.5 Hz, J = 2.2 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.41 (br. s, 2H) 7.35 (br. s, 2H), 5.90 (d, J = 5.2 Hz, 1H), 5.83 (d, J = 5.8 Hz, 1H), 5.59 (d, J = 6.0 Hz, 1H), 5.48–5.31 (m, 2H), 5.25 (d, J = 5.3 Hz, 1H), 4.72 (q, J = 5.5 Hz, 1H), 4.36 (t, J = 5.0 Hz, 1H), 4.29 (q, J = 4.6 Hz, 1H), 4.22–4.08 (m, 2H), 3.87 (q, J = 3.6 Hz, 1H), 3.78–3.70 (m, 2H), 3.67 (dd, J = 12.2 Hz, J = 3.4 Hz, 1H), 3.56–3.29 (m, 5H). 13C NMR (125 MHz, DMSO‑d 6) δ 156.1, 156.0, 152.6, 152.5, 149.3, 148.9, 147.4, 140.1, 139.5, 139.5, 132.7, 131.6, 129.6, 124.3, 119.3, 119.3, 87.6, 86.2, 85.8, 81.8, 81.5, 72.3, 71.3, 68.8, 68.1, 61.4, 50.8, 48.1. HRMS (ESI+): m/z calcd for C28H32N12O11SCl [M+H]+: 779.1723, Found 779.1730.

4.1.34  N-{[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-3-yl]oxy}ethyl)-4-chlorobenzene-1-sulfonamide (34)
To a solution of 20 (171 mg, 0.21 mmol, 1.00 eq) in anhydrous DCM (2 mL) was added Et3N (60 μL, 0.41 mmol, 2.00 eq). 4-chlorobenzenesulfonyl chloride (65 mg, 0.31 mmol, 1.50 eq) was added at 0 °C and the reaction mixture was stirred for 3 h at room temperature under argon. Solvents were removed under vacuum and the residue was diluted with DCM (40 mL) and washed with saturated aqueous NaHCO3 (40 mL). The aqueous layer was extracted with DCM (3 × 30 mL) and the combined organic extracts were washed with brine (30 mL), dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0–4% MeOH in DCM) to give 34 (188 mg, 90%) as a white solid. Rf 0.67 (10:90 MeOH/CH2Cl2). 1H NMR (500 MHz, CDCl3) δ 8.32 (s, 1H), 8.29 (s, 1H), 8.12 (s, 1H), 7.81 (s, 1H), 7.59–7.46 (m, 2H), 7.34–7.22 (m, 2H), 5.97 (d, J = 3.3 Hz, 1H), 5.96–5.88 (m, 5H), 5.41 (dd, J = 6.4 Hz, J = 2.0 Hz, 1H), 5.07 (dd, J = 6.4 Hz, J = 3.5 Hz, 1H), 4.50 (dd, J = 5.9 Hz, J = 4.6 Hz, 1H), 4.46–4.34 (m, 1H), 4.21 (dd, J = 4.7 Hz, J = 3.3 Hz, 1H), 4.03 (dt, J = 6.0 Hz, J = 3.0 Hz, 1H), 3.98 (dd, J = 11.4 Hz, J = 3.5 Hz, 1H), 3.80–3.72 (m, 3H), 3.68 (dd, J = 14.6 Hz, J = 5.6 Hz, 1H), 3.46–3.31 (m, 3H), 1.57 (s, 3H), 1.36 (s, 3H), 0.91 (s, 9H), 0.89 (s, 9H), 0.11–0.03 (m, 12H). 13C NMR (125 MHz, CDCl3) δ 155.8, 155.6, 153.2, 153.1, 149.5, 149.1, 140.2, 139.5, 139.1, 138.0, 129.2, 128.6, 120.5, 120.2, 114.6, 90.9, 87.3, 85.3, 84.5, 84.1, 82.6 (2C), 69.6 (2C), 61.6, 50.7, 48.1, 27.2, 26.1, 25.9, 25.4, 18.6, 18.2, −4.5, −4.7, −5.3. HRMS (ESI+): m/z calcd for C43H65N11O9SSi2Cl [M+H]+: 1002.3909, found: 1002.3928.

4.1.35  9-[(2R,5R)-3-[2-({[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}[(4-chloro-3-nitrophenyl)methyl]amino)ethoxy]-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-2-yl]-9H-purin-6-amine (35)
To a solution of 20 (100 mg, 0.12 mmol, 1.00 eq) in anhydrous DCE (3.5 mL) was added a solution of 4-chloro-3-nitrobenazaldehyde (27 mg, 0.18 mmol, 1.50 eq) in anhydrous DCE (3.5 mL) and acetic acid (10 μL, 0.18 mmol, 1.50 eq). After stirring for 20 min at 40 °C under argon, sodium triacetoxyborohydride (38 mg, 0.18 mmol, 1.50 eq) was added. After stirring at 40 °C for 16 h, the solution was diluted with DCM (30 mL) and washed with saturated aqueous NaHCO3. The aqueous layer was extracted with DCM (3 × 30 mL) and the combined organic extracts were washed with brine (60 mL), dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0–5% MeOH in DCM) to give 35 as a white solid (105 mg, 87%). Rf 0.71 (MeOH/DCM 5:95). 1H NMR (500 MHz, CDCl3) δ 8.27 (s, 1H), 8.18 (s, 1H), 8.15 (s, 1H), 7.82 (br. s, 2H), 7.29–7.27 (m, 2H), 6.22 (br. s, 2H), 6.20 (br. s, 2H), 6.10 (d, J = 3.6 Hz, 1H), 5.99 (d, J = 2.1 Hz, 1H), 5.37 (dd, J = 6.5 Hz, J = 2.1 Hz, 1H), 4.91 (dd, J = 6.5 Hz, J = 3.7 Hz, 1H), 4.48 (t, J = 5.1 Hz, 1H), 4.30 (td, J = 6.7 Hz, J = 3.6 Hz, 1H), 4.22 (t, J = 4.2 Hz, 1H), 4.07 (dt, J = 5.8 Hz, J = 3.0 Hz, 1H), 3.99 (dd, J = 11.5 Hz, J = 3.4 Hz, 1H), 3.81–3.72 (m, 2H), 3.71–3.66 (m, 1H), 3.65–3.61 (m, 2H), 2.90–2.70 (m, 4H), 1.57 (s, 3H), 1.36 (s, 3H), 0.92 (s, 9H), 0.86 (s, 9H), 0.14 – - 0.02 (m, 12H). 13C NMR (125 MHz, CDCl3) δ 155.8, 155.7, 153.1, 153.0, 149.6, 149.0, 147.8, 140.6, 140.1, 139.3, 133.0, 131.4, 125.6, 125.2, 120.3, 120.1, 114.6, 90.8, 87.0, 85.7, 84.9, 84.0, 83.4, 82.8, 69.6, 69.3, 61.8, 58.2, 56.4, 53.5, 27.2, 26.2, 25.8, 25.4, 18.6, 18.2, −4.5, −4.7, −5.2, −5.3. HRMS (ESI+): m/z calcd for C44H66N12O9Si2Cl [M+H]+: 997.4297, found: 997.4327.

4.1.36  9-[(2R,5R)-3-[2-({[(4R,6R)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyl-tetrahydro-2H-furo[3,4-d] [1,3]dioxol-4-yl]methyl}[(4-chlorophenyl)methyl]amino)ethoxy]-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}oxolan-2-yl]-9H-purin-6-amine (36)
To a solution of 20 (210 mg, 0.25 mmol, 1.00 eq) in anhydrous DCE (7 mL) was added 4-chlorobenzaldehyde (43 mg, 0.38 mmol, 1.50 eq) in anhydrous DCE (7 mL) and acetic acid (18 μL, 0.38 mmol, 1.50 eq). After stirring for 20 min at 40 °C under argon, sodium triacetoxyborohydride (81 mg, 0.38 mmol, 1.50 eq) was added. After stirring at 40 °C for 16 h, the solution was diluted with DCM (50 mL) and washed with saturated aqueous NaHCO3. The aqueous layer was extracted with DCM (3 × 50 mL) and the combined organic extracts were washed with brine (70 mL), dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, linear gradient 0–5% MeOH in DCM) to give 36 as a white solid (172 mg, 0.18 mmol, 72%). Rf 0.71 (MeOH/CH2Cl2 5:95). 1H NMR (600 MHz, CDCl3) δ 8.30 (s, 1H), 8.20 (s, 1H), 8.15 (s, 1H), 7.83 (s, 1H), 7.19–7.07 (m, 4H), 6.11 (d, J = 3.7 Hz, 1H), 5.99 (d, J = 2.2 Hz, 1H), 5.97–5.83 (m, 4H), 5.37 (dd, J = 6.5 Hz, J = 2.2 Hz, 1H), 4.85 (dd, J = 6.4 Hz, J = 3.4 Hz, 1H), 4.49 (t, J = 5.1 Hz, 1H), 4.32 (td, J = 6.7 Hz, J = 3.3 Hz, 1H), 4.26 (t, J = 4.2 Hz, 1H), 4.08 (dt, J = 5.9 Hz, J = 3.1 Hz, 1H), 3.98 (dd, J = 11.5 Hz, J = 3.6 Hz, 1H), 3.79–3.47 (m, 5H), 2.84–2.66 (m, 4H), 1.58 (s, 3H), 1.36 (s, 3H), 0.92 (s, 9H), 0.88 (s, 9H), 0.13–0.02 (m, 12H). 13C NMR (150 MHz, CDCl3) δ 155.7, 155.6, 153.1, 153.1, 149.7, 149.3, 140.0, 139.5, 137.8, 132.8, 130.1, 128.4, 120.5, 120.3, 114.4, 91.0, 87.2, 85.7, 84.9, 84.0, 83.5, 82.7, 69.8, 69.5, 61.3, 58.6, 56.4, 53.6, 27.3, 26.2, 25.9, 25.5, 18.6, 18.2, −4.5, −4.7, −5.2, −5.3. HRMS (ESI+): m/z calcd for C44H67N11O7Si2Cl [M+H]+: 952.4447, found: 952.4426.

4.1.37  N-{[(2R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl}-N-(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)-4-chlorobenzene-1-sulfonamide (14)
Using method C with 34 (150 mg, 0.15 mmol, 1.00 eq) and a 0–40% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 14 (55 mg, 48%) was obtained as a white powder with 96% purity determined by HPLC analysis at 260 nm. 1H NMR (600 MHz, DMSO‑d 6) δ 8.33 (s, 1H), 8.32 (s, 1H), 8.150 (s, 1H), 8.147 (s, 1H), 7.75–7.68 (m, 2H), 7.53–7.49 (m, 2H), 7.35 (br. s, 2H), 7.28 (br. s, 2H), 5.95 (d, J = 5.5 Hz, 1H), 5.84 (d, J = 5.9 Hz, 1H), 5.52 (d, J = 6.1 Hz, 1H), 5.36 (m, 2H), 5.20 (d, J = 5.2 Hz, 1H), 4.74 (q, J = 5.6 Hz, 1H), 4.39 (t, J = 5.2 Hz, 1H), 4.29 (q, J = 4.7 Hz, 1H), 4.16 (q, J = 4.6 Hz, 1H), 4.11–4.05 (m, 1H), 3.93 (q, J = 3.6 Hz, 1H), 3.73–3.64 (m, 3H), 3.56–3.46 (m, 2H), 3.37–3.29 (m, 3H), 3.22 (dt, J = 14.7 Hz, 6.4 Hz, 1H). 13C NMR (150 MHz, DMSO‑d 6) δ 156.1, 156.0, 152.5, 152.5, 149.3, 148.9, 140.1, 139.5, 138.0, 137.7, 129.2, 128.7, 119.3, 119.3, 87.7, 86.2, 85.9, 82.1, 81.4, 72.2, 71.3, 69.9, 68.3, 61.1, 50.8, 47.7. HRMS (ESI+): m/z calcd for C28H33N11O9SCl [M+H]+: 734.1868, Found 734.1866.

4.1.38  (2R,5R)-2-(6-amino-9H-purin-9-yl)-5-{[(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)[(4-chloro-3-nitrophenyl)methyl]amino]methyl}oxolane-3,4-diol (15)
Using method C with 35 (140 mg, 0.14 mmol, 1.00 eq) and a 0–40% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 15 (25 mg, 25%) was obtained as a white powder with 99% purity determined by HPLC analysis at 260 nm. 1H NMR (600 MHz, DMSO‑d 6) δ 8.35 (s, 1H), 8.23 (s, 1H), 8.11 (s, 1H), 8.07 (s, 1H), 7.89 (d, J = 1.9 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.45 (dd, J = 8.3 Hz, J = 2.0 Hz, 1H), 7.32 (br. s, 2H), 7.24 (br. s, 2H), 5.99 (d, J = 5.9 Hz, 1H), 5.83 (d, J = 5.1 Hz, 1H), 5.51–5.32 (m, 2H), 5.24 (br. s, 1H), 5.13 (br. s, 1H), 4.57 (t, J = 5.2 Hz, 1H), 4.45 (dd, J = 6.1 Hz, J = 4.8 Hz, 1H), 4.30 (t, J = 4.1 Hz, 1H), 4.05 (t, J = 5.0 Hz, 1H), 4.02–3.95 (m, 2H), 3.73–3.63 (m, 4H), 3.58–3.51 (m, 2H), 2.83 (dd, J = 14.0 Hz, J = 4.4 Hz, 1H), 2.73 (dd, J = 14.0 Hz, J = 7.2 Hz, 1H), 2.69–2.63 (m, 2H). 13C NMR (150 MHz, DMSO‑d 6) δ 156.1, 156.0, 152.5 (2C), 149.3, 148.9, 147.2, 140.1, 139.8, 139.6, 133.7, 131.0, 125.0, 122.9, 119.3, 119.2, 87.8, 86.2, 86.1, 82.2, 81.3, 72.4, 71.8, 68.9, 68.1, 61.3, 56.8, 56.0, 53.0. HRMS (ESI+): m/z calcd for C29H34N12O9Cl [M+H]+: 729.2255, Found 729.2256.

4.1.39  (2R,5R)-2-(6-amino-9H-purin-9-yl)-5-{[(2-{[(2R,5R)-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy}ethyl)[(4-chlorophenyl)methyl]amino]methyl}oxolane-3,4-diol (16)
Using method C with 36 (172 mg, 0.18 mmol, 1.00 eq) and a 0–40% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7 for purification, 16 (45 mg, 37%) was obtained as a white powder with 98% purity determined by HPLC analysis at 260 nm. 1H NMR (600 MHz, DMSO‑d 6) δ 8.36 (s, 1H), 8.25 (s, 1H), 8.13 (s, 1H), 8.11 (s, 1H), 7.34 (br. s, 2H), 7.25 (br. s, 2H), 7.23–7.17 (m, 2H), 7.17–7.11 (m, 2H), 6.00 (d, J = 6.2 Hz, 1H), 5.84 (d, J = 5.0 Hz, 1H), 5.45–5.39 (m, 2H), 5.27 (d, J = 4.8 Hz, 1H), 5.12 (d, J = 5.4 Hz, 1H), 4.58 (q, J = 5.5 Hz, 1H), 4.48 (dd, J = 6.2 Hz, J = 4.8 Hz, 1H), 4.30 (td, J = 4.6 Hz, J = 2.9 Hz, 1H), 4.04 (q, J = 5.1 Hz, 1H), 4.02–3.96 (m, 2H), 3.70–3.63 (m, 2H), 3.60–3.48 (m, 4H), 2.80 (dd, J = 14.0 Hz, J = 4.5 Hz, 1H), 2.67 (dd, J = 13.9 Hz, J = 7.0 Hz, 1H), 2.61 (q, J = 6.1 Hz, 2H). 13C NMR (150 MHz, DMSO‑d 6) δ 156.2, 156.0, 152.6, 152.5, 149.3, 149.0, 139.8, 139.7, 138.2, 131.1, 130.4, 127.8, 119.3, 119.16, 87.67, 86.3, 86.1, 82.4, 81.2, 72.4, 71.8, 69.0, 67.5, 61.5, 57.5, 55.8, 52.9. HRMS (ESI+): m/z calcd for C29H35N11O7Cl [M+H]+: 684.2404, Found 684.2398.