Benzyl 2-hydroxyoctadecanoate (4)
To a solution of α-hydroxy stearic acid 3 (10 g, 33.28 mmol), triethylamine (6.73 g, 9.25 ml, 66.56 mmol), TBAI (1.23 g, 3.33 mmol) in toluene (150 mL), benzyl bromide (5.7 g, 5.03 mL, 33.3 mmol) is added, and held at 90°C overnight. After the completion of the reaction, the reaction mixture is triturated with diethyl ether, the precipitate formed was washed with ether and dried to obtain the desired compound 4 as white solid (7.25 g, 55.7%). 1H NMR (300 MHz, CDCl3, ppm): δ 7.37 (m, 5H, Ar), 5.25 (q, 2H, CH2), 4.23 (m, 1H), 2.79 (br, 1H, -OH), 1.82-1.73 (m, 1H), 1.69 (m, 1H), 1.25 (s, 28H, -CH2), 0.90 (t, -CH2). 13C NMR (300 MHz, CDCl3, ppm): δ 175.41(C = O), 135.37(-C-), 128.75 (-C-Ar), 128.65 (-C-Ar), 128.44 (-C-Ar), 70.64 (-C-H), 67.36 (Ar-CH2), 34.52 (-CH2), 32.05 (-CH2), 29.82 (-CH2), 29.79 (-CH2), 29.66 (-CH2), 29.58 (-CH2), 29.49 (-CH2), 24.78 (-CH2), 22.81(-CH2), 14.24 (-CH3). HRMS (ESI+) m/z Calculated for C25H43O3 (MH+): 391.3212 found 391.2869. C25H42O3Na (MNa+): 413.3031 found 413.2918.
Benzyl 3-hydroxy stearate (9)
To a solution of β-hydroxy stearic acid 8 (2 g, 6.65 mmol), triethylamine (1.2 ml, 8.65 mmol), and TBAI (0.25 g, 0.66 mmol) in toluene, benzyl bromide (1.14 g, 0.8 mL, 6.65 mmol) was added, and stirred at 90°C overnight. After the completion of the reaction, the reaction mixture is triturated with diethyl ether, and the precipitate formed was washed with diethyl ether (2 × 50 mL). The residue obtained was purified on silica gel column chromatography using dichloromethane to obtain the desired compound 9[46] as white solid (1.47 g, 56%). 1H NMR (300 MHz, CDCl3): 7.34(m, 5H-Ph), 5.14 (s, 2H, CH2-Ph), 4.01 (m, 1H), 2.9 (br s, 1H, OH), 2.58-2.41 (m, 2H, CH2), 1.43(m, 2H, CH2), 1.41(m, 2H, CH2), 1.25 (s, 24H, CH2), 0.9 (t, 3H, CH3). 13C (300 MHz, CDCl3):172.50 (C = O), 135.32(C-Ph), 128.28(C-Ph), 128.02(C-Ph),127.92(C-Ph), 67.73(C, C-OH), 66.12(C, CH2-Ph), 41.08 (C, CH2),36.24 (C, CH2), 31.60 (C, CH2), 29.37 (C, CH2), 29.34 (C, CH2), 29.26 (C, CH2), 29.24 (C, CH2), 29.20 (C, CH2), 29.03 (C, CH2), 25.14 (C, CH2), 22.36 (C, CH2), 13.78 (C, CH3). HRMS(ES+) m/z Calculated for C25H42O3 (MH+): 391.3206, found 391.3199.
Benzyl, (benzyl stearate)-2-yl, N, N-diisopropyl phosphoramidite [(±)-12]
To a stirring mixture of benzyloxy bis(N, N’-diisopropylamino)phosphine (10 g, 29.55 mmol) and alpha-hydroxy benzyl stearate (5.77 g, 14.77 mmol) in dry DCM (100 mL), was added 0.5 M solution of 1H-Tetrazole (29.6 mL, 14.77 mmol) in ACN dropwise at 0°C. After the addition, cooling bath was removed and the reaction mixture was allowed to stir at rt for 15 min. The reaction mixture was diluted with DCM (200 mL), and the organic layer was washed with 1 M TEAB solution. The extracts were dried over Na2SO4 and concentrated in vacuum and purified by flash column chromatography using eluent 400:50:10-Hexane:EtOAc:TEA (Rf = 0.5) to obtain the phosphoramidite 12 (7.10 g, 71%). P31 NMR (CDCl3): δ 149.10, 148.69.
Benzyl, (benzyl stearate)-3-yl, N, N-diisopropyl phosphoramidite [(±)-13]
To a stirring mixture of benzyloxy Bis(N, N’-diisopropylamino) phosphine (11, 1.74 g, 5.12 mmol) and β-hydroxy stearic benzyl ester (9, 1 g, 2.56 mmol) in dry DCM (10 mL) was added 0.5 M solution of 1H-tetrazole (5.12 mL, 2.56 mmol) in ACN dropwise at 0°C. After the addition, cooling bath was removed and the reaction mixture was allowed to stir at room temperature for 15 min. The reaction mixture was diluted with DCM (150 mL), and was washed with 1 M TEAB (100 mL) solution. Dichloromethane layer was dried over Na2SO4 and concentrated in vacuo, and the obtained oil was purified by silica gel flash column chromatography using the eluent 400:50:10-Hexane:EtOAc:TEA (Rf = 0.5) to get 13 as an pale yellow oil (0.985 g, 61%). 31P NMR (300 MHz, CDCl3, ppm): δ 147.63, 147.07.
3-O-benzyl-1,2:5,6-Di-O-isopropylidene-α-D-glucofuranose (17)
To a stirring solution of diacetone glucose (10 g, 38.42 mmol) in anhydrous DMF at 0°C, was added sodium hydride (60% in mineral oil (w/w), total 2.3 g, 57.63 mmol) portion wise. Stirring was continued for 1 h at 0°C, and then benzyl bromide (5.51 g, 46.10 mmol) was added drop wise. After addition, ice bath was removed, and stirring continued overnight at room temperature. After completion of the reaction, excess of sodium hydride in the reaction mixture was quenched by the addition of ice cold water (25 mL). The reaction mixture was extracted with EtOAc (4 × 150 mL) and the combined organic phase was dried (Na2SO4) and concentrated under vacuum, and the residue was purified by silica gel column chromatography using eluents hexane:ethylacetate-8:2 to give the title compound 17 (13 g, 96%) as oil. 1H NMR (300 MHz, CDCl3, ppm): 7.33-7.28 (m, 5H, Ar), 5.88 (d, 1H, 1′-H), 4.68-4.62 (m, 2H, CH2-Ar), 4.56 (d, 1H, 2′-H), 4.37-4.33 (m, 1H, 5′-H), 4.16-4.06 (m, 2H, 6′-H, 6″-H), 4.01-3.96 (m, 2H, 3′-H, 4′-H), 1.47 (s, 3H, CH3), 1.41(s, 3H, CH3), 1.35(s, 3H, CH3), 1.28(s, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 137.66, 128.33, 127.76, 127.58, 111.65, 108.86, 108.86, 105.27, 82.64, 81.68, 81.31, 72.50, 72.29, 67.35, 26.81, 26.75, 26.20, 25.40.
3-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranose (18)
In a 250 mL round bottom flask 3-O-benzyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (7.00 g, 19.97 mmol) was dissolved in 1:1 methanol/1% aqueous sulfuric acid (110 mL), and the resulting solution stirred at rt. After completion of the reaction (approximately 5 h, monitored by TLC), the reaction mixture was quenched with triethylamine (pH7). The residue was concentrated in vacuo to afford the crude residue as syrup, which was purified by silica gel flash column chromatography using 5% MeOH in dichloromethane, affording the title compound 18[33] (5.05 g, 81%) as a colorless solid. 1H NMR (300 MHz, CDCl3, ppm): 7.31-7.28 (m, 5H, Ar), 5.91 (m, 1H, 1′-H), 4.71 (dd, 2H, CH2-Ar), 4.51 (s, 1H), 4.13 (m, 2H), 4.09 (m, 1H), 3.81 (dd, 2H), 3.03 (br, 1H), 2.02, (s, 1H), 1.47 (s, 3H), 1.29 (s, 3H). 13C NMR (300 MHz, CDCl3, ppm): δ171.29, 137.41, 128.70, 128.64, 128.11, 127.81, 111.80, 105.16, 82.21, 81.97, 80.01, 69.11, 64.33, 60.45, 26.73, 26.22, 21.03, 14.19. HRMS (ESI+) m/z Calculated for C16H22O6 (MH+): 311.1489, found 311.1486.
3-O-benzyl-1,2-O-isopropylidene-β-D-xylofuranose (20)
To a stirring solution of 3-O-benzyl-1,2- isopropylidene-α-D-glucofuranose (5 g, 16.11 mmol) in water (50 mL) was added sodium meta-periodate (4.13 g, 19.33) at room temperature. Stirring continued until consumption of starting material. The reaction mixture was diluted with ethanol (80 mL) and stirring continued for another 30 min. The reaction mixture is filtered on celite and the celite pad washed with ethanol. The filtrate was transferred to a 500 mL round bottom flask, and cooled to 0°C. Sodium borohydride (0.67 g, 17.72 mmol) was added in small portions to the filtrate. After completion of the addition, ice bath was removed and stirring continued for 2 h at room temperature. The reaction mixture was neutralized by drop wise addition of acetic acid, concentrated in vacuo and the residue is purified by silica gel column chromatography to get the title compound 20[33],[47] (4 g, 88.5%). 1H NMR (300 MHz, CDCl3, ppm): δ 7.37-7.28 (m, 5H, Ar-H), 5.99 (d, 1H, 1′-H), 4.73 (dd, CH2-Ar), 4.64 (s, 1H, 4-′H), 4.30 (dd, 1H, 2′-H), 3.92 (d, 1H, 3′-H), 3.91-3.1 (m, 2H, 5′-H, 5″-H), 2.24 (dd, 1H), 1.48 (s, 3H, CH3), 1.33 (s, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 137.19 (-C-, Ar), 128.78(CH, Ar), 128.31 (CH, Ar), 127.85 (CH, Ar), 111.90 (C), 105.20(C-1′), 82.87 (C-4′), 82.60 (C-2′), 80.22 (C-3′), 72.04 (CH2-Ar), 61.09 (C-5′), 26.93 (CH3), 26.43(CH3). HRMS (ESI+) m/z Calculated for C15H20O5 [M + Na]+ : 303.1203, found 303.1207.
3-O-benzyl-1,2-O-isopropylidene-β-D-xylofuranose (21)
To a suspension of 3-O-Benzyl-1,2-O-isopropylidene-β-D-xylofuranose (4 g, 14.26 mmol) in benzoyl chloride (1.64 g, 2 mL,17.12 mmol), was added sulfamic acid (0.55 g, 5.70 mmol). The reaction mixture was stirred at 60°C for 3 h, allowed to come to room temperature and the reaction mixture was poured into ice cold saturated NaHCO3 solution, and extracted with minimum amount of diethyl ether. The organic solvents were dried on Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using eluents hexane:ethyl acetate (8:2) to obtain the compound 21[36] (4.8 g, 91%). 1H NMR (300 MHz, CDCl3, ppm): δ 8.01 (d, 2H), 7.54-7.51 (m, 1H), 7.40-7.37 (m, 2H), 7.30-7.26 (m, 4H), 7.24 (d, 1H), 5.99 (d, 1H), 4.70-4.60 (m, 3H), 4.57-4.48 (m, 3H), 4.04 (br s, 1H), 1.49 (s, 3H, CH3), 1.32 (s, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ166.21, 137.16, 133.03, 129.88, 129.75, 128.53, 128.31, 128.07, 127.77, 111.82, 105.32, 82.13, 81.52, 78.14, 71.88, 26.83, 26.25.
(9-(2′-O-acetyl-3′-O-benzyl-5′-O-benzoyl) β-D-xylofuranosyl) 6-N-benzoyladenine (23)
To a cooled solution of 5-O-benzoyl-3-O-benzyl-1,2-O-isopropylidene β-D-xylofuranose (4 g, 10.40 mmol) in AcOH (40 mL) and Ac2O (15 mL) was added drop wise sulfuric acid (1.5 mL). Stirring was continued at room temperature until TLC analysis shows disappearance of the starting material, the mixture was poured into ice-water, extracted with CHCl3 (3 × 100mL), washed with saturated NaHCO3 (200 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the residue is used directly in the next step. TLC-(7:3 hexanes–EtOAc-Rf = 0.4).
To a suspension of 22 (5 g, 11.67 mmol) and N6-benzoyladenine (4.2 g, 17.50 mmol) in anhydrous acetonitrile (60 mL) was added drop wise 1 M SnCl4 in dichloromethane (23.5 mL, 23.34 mmol) under argon. The resulting mixture was allowed to stir for 4 h at room temperature. After completion of the reaction, saturated aq NaHCO3 was added slowly until the evolution of carbon dioxide ceased. Then the mixture was filtered through a pad of Celite 545, that was subsequently washed with CHCl3 (3x100 mL). The combined filtrate was washed successively with saturated aq NaHCO3 (3 × 100 mL) and brine (2x100 mL), dried (Na2SO4). The filtrates were concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography using 3%MeOH in CHCl3 as eluent (Rf-0.5), affording nucleoside 23[36] (6.12 g, 86%) as a colorless solid. 1H NMR (500 MHz, DMSO- DMSO-d6, ppm): δ 9.36 (br s, 1H, -NH), 8.77 (s, 1H, H-2), 8.42 (s, 1H, H-8), 8.03 (dd, 5H, Ar), 7.59 (q, 2H, Ar), 7.50 (t, 2H, Ar), 7.43 (t, 2H, Ar), 7.27 (m, 5H, Ar), 6.44 (s, 1H, H-1′), 5.54 (s, 1H, H-2′), 4.76 (dd, 2H), 4.74 (dd, 1H), 4.65 (m, 1H), 4.16 (d, 1H),2.18 (s, 3H, CH3). 13C NMR (500 MHz, DMSO- d6, ppm): δ 169.69 (C = O, CH3), 166.38 (C = O, Ar), 164.63 (C = O, Ar-NH), 153.03 (C-2), 151.57 (C-6), 149.58 (C-4), 141.81 (C-8), 136.24 (CH-Ar), 133.84 (CH-Ar), 133.45 (CH-Ar), 132.92 (CH-Ar), 129.88 (CH-Ar), 129.68 (CH-Ar), 129.02 (CH-Ar), 128.84 (CH-Ar), 128.59 (CH-Ar), 128.39 (CH-Ar), 127.99 (CH-Ar), 122.92 (CH-Ar), 87.82(1′-C), 81.18 (4′-C), 79.98 (3′-C), 79.75 (C-2′), 72.36 (CH2-Ar), 62.43 (C-5′), 20.99 (CH3). HRMS (ESI+) m/z Calculated for C33H29N5O7 (MH+): 608.2139, found 608.2135.
9-(3′-O-benzyl) β-D-xylofuranosyl)-adenine (24)
To a solution of nucleoside 23 (2 g, 3.29 mmol) in MeOH (20 mL) was added saturated methanolic ammonia (100 mL) in a sealed tube and the mixture was stirred at 85°C for 3 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure and the residue was coevaporated with toluene (5x10 mL). The residue obtained was purified by column chromatography using 5% MeOH in CHCl3 to afford nucleoside 24 (0.8 g, 68%). 1H NMR (500 MHz, DMSO-d6, ppm): δ 8.17 (s, 1H, H-2), 8.14 (s, 1H, H-8), 7.35-7.27 (m, 7H, Ar, -NH2), 6.04 (d, 1H, 2′-OH), 5.95 (s, 1H, 1′-H), 4.94 (br, 2H, 5′-OH), 4.68 (s, 1H, 2′-H), 4.67 (dd, 2H, CH2), 4.34 (m, 1H, 4′-H), 4.05 (br s, 1H, 3′-H), 3.81-3.73 (m, 5′-H, 5″-H). 13C NMR (500 MHz, DMSO- DMSO-d6, ppm): δ 156.04 (C-6), 152.67(C-2), 149.21 (C-4), 139.01 (C-8), 138.04(-C-, Ar), 128.29(-CH, Ar), 127.56 (-CH, Ar), 127.40 (-CH, Ar), 118.67(C-5), 88.74 (1′-C), 82.68 (3′-C), 77.26 (2′-C), 71.29 (CH2-Ar), 59.44 (5′-C). HRMS (ESI+) m/z Calculated for C17H19N5O4 (MH+): 358.1509, found 358.1510.
9-(5′-O-tert-butyldimethylsilyl-3′-O-benzyl) β-D-xylofuranosyl) adenine (25)
To a cooled suspension of 9-(5′-O-tert-butyldimethylsilyl-3-O-Benzyl-β-D-xylofuranosyl)-adenine (6 g, 16.78 mmol) and imidazole (2.85 g, 41.97 mmol) in anhydrous N, N- dimethylformamide was added tert-butyldimethylchlorosilane (3.03 g, 16.78 mmol) in anhydrous DMF under argon. The reaction mixture was allowed to stir for 20 h at room temperature. After completion of the reaction, the organic solvent was removed under high vacuum. The residue was dissolved in 150 mL of ethyl acetate, the solution was washed with two times 80 mL of water, and one time with 30 mL of brine and the extract was dried on Na2SO4 and the organic solvent was concentrated under reduced pressure. The residue obtained was purified on silica gel column chromatography using 2% MeOH in DCM to obtain the compound 25 as colorless solid (7.56 g, 95%). 1H NMR (500 MHz, CDCl3, ppm): δ 8.16 (s, 1H, H-2), 8.11 (s, 1H, H-8), 7.33 (m, 7H, Ar, -NH2), 6.05 (d, 1H, 2′-OH), 5.96 (d, 1H, 1′-H), 4.69 (s, 2H, 2′-H), 4.66-4.53 (m, Ar-CH2), 4.32 (m, 1H, 4′-H), 4.06 (m, 1H, 3′-H), 4.00-3.86 (m, 2H, 5′-H, 5″-H), 0.85 (s, 9H, -(CH3)3), 0.02 (s, 6H, -(CH3)2). 13C NMR (500 MHz, DMSO-d6, ppm): δ 155.85(C-6), 153.79 (-C = O, Cbz) 153.16(C-2), 149.52 (C-4), 139.04(C-8), 136.70 (-C-), 134.41 (-C-), 128.83 (-CH-, Ar), 128.68 (-CH-, Ar), 128.53 (-CH-, Ar), 128.50, (-CH-, Ar) 128.17 (-CH-, Ar), 127.91 (-CH-, Ar), 119.30 (C-5), 86.89 (C-1′), 83.31 (C-4′), 82.91 (C-3′), 79.92 (C-2′), 72.45 (-CH2, Bn), 70.57 (-CH2, Cbz), 60.37 (C-5′), 25.88 (-(CH3)3), 18.29 (-C-), -5.31 (CH3), -5.42 (CH3). HRMS (ESI+) m/z Calculated for C23H33N5O4Si1 (MH+): 472.2374, found: 472.2379.
(9-(5′-O-tert-butyldimethylsilyl-3′-O-benzyl-2′-O-benzyloxycarbonyl) β-D-xylofuranosyl) adenine (26)
To a solution of 9-(5′-O-tert-butyldimethylsilyl-3′-O-Benzyl-2′-O-benzyloxycarbonyl-β-D-xylofuranosyl)-adenine (2.5 g, 53 mmol) and DMAP (1.3 g, 10.60 mmol) in 50 mL of anhydrous dichloromethane was added benzyl chloroformate (CbzCl, 1.8 g, 1.78 mL, 10.60 mmol) at 0°C under argon atmosphere. After stirring for 72 hours at room temperature, the reaction mixture was diluted with DCM (200 mL) and washed with cold 1.0 M HCl aqueous solution (50 mL) and then with water (100 mL). The organic layer was dried over anhydrous NaSO4, filtered, and concentrated under reduced pressure. The residue obtained, was purified by silica gel column chromatography (hexane/EtOAc-10:1 to 1:1 v/v) to obtain 26 (2.85 g, 89%). 1H NMR (500 MHz, CDCl3, ppm): δ 8.25 (s, 1H, H-2), 8.05 (s, 1H, H-8), 7.28-7.17 (m, 10H, Ar), 6.38 (br, m, 2H, -NH2), 6.29 (s, 1H, 1′-H), 5.11 (s, 2H, CH2-Cbz) 4.64 (s, 2H, 2′-H), 4.61-4.52 (m, Ar-CH2), 4.27 (m, 1H, 4′-H), 4.10 (d, 1H, 3′-H), 3.97-3.87 (m, 2H, 5′-H, 5″-H), 0.82 (s, 9H, -(CH3)3), 0.01 (s, 6H, -(CH3)2). 13C NMR (500 MHz, CDCl3, ppm): 155.85, 153.79, 153.16, 149.52, 139.04, 136.70, 134.41, 128.83, 128.68, 128.53, 128.50, 128.17, 127.91, 119.30, 86.89, 83.31, 82.91, 79.92, 72.45, 70.57, 60.37, 25.88, 18.29, -5.31, -5.42. HRMS (ESI+) m/z Calculated for C31H39N5O6Si1 (MH+): 606.2742, found: 606.2729.
(9-(3′-O-benzyl-2′-O-benzyloxycarbonyl) β-D-xylofuranosyl) adenine (27)
To a solution of 26 (2 g, 33 mmol) in 30 mL of anhydrous THF was added 1 M TBAF in THF (8.63 g, 330 mmol) at 0°C under N2 atmosphere. The solution was stirred for 12 h at room temperature, and all volatiles were removed using a rotary evaporator. The residue was dissolved in EtOAc (100 mL) and washed with cold saturated NaHCO3 solution (30 mLx2), and brine (30 mL). The organic solvent was dried over Na2SO4 and filtered. The filtrate was concentrated and the obtained residue was purified by silica gel column chromatography (CH2Cl2/MeOH-9:1) to give 27 (1.41 g) as white solid in 86% yield. 1H NMR (500 MHz, CDCl3, ppm): δ 8.15 (s, 1H, H-2), 8.14 (s, 1H, H-8), 7.37-7.28 (m, 10H, 2xAr), 6.20 (s, 1H, 1′-H), 5.71 (t, 1H, 2′-H), 5.17 (s, 2H, -NH2), 5.03 (t, 1H, 5′-OH), 4.75 (dd, 2H, CH2-Ar), 4.38 (m, 1H, 4′-H), 4.30 (m, 1H, 3′-H), 3.78 (m, 2H, 5′-H, 5″-H). 13C NMR (500 MHz, CDCl3, ppm): 156.05, 153.24, 152.81, 149.14, 138.68, 137.57, 134.96, 128.53, 128.51, 128.32, 128.31, 127.70, 127.53, 118.52, 85.67, 82.58, 81.98, 79.90, 71.46, 69.75, 59.07. HRMS (ESI+) m/z Calculated for C25H25N5O6 (MH+):492.1877, found: 492.1881.
3′-O-benzyloxycarbonyl-2′-deoxyadenosine-5′-(O-(benzyl stearate)-2-yl, O-benzyl) phosphate (29)
To a stirred solution of 14 (1 g, 25.94 mmol) and 12 (2.45 g, 38.92 mmol) in 5 mL dry DCM was added 0.5 M solution of 1H-Tetrazole (25.94 mL, 129.74 mmol) drop wise at 0°C. The reaction mixture was allowed to stir at room temperature for 4 h. Then the reaction mixture was cooled down to -78°C and hydrogen peroxide 35% (W/V, 10 mL) was added. After stirring for 5 min at -78°C, cooling bath was removed and the reaction mixture was allowed to stir at room temperature for further 30 min. The reaction mixture is diluted with DCM (150 mL) and washed with 1 M phosphoric acid (70 mL), 5% aq. sodium bicarbonate (70 mL) and with brine (60 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The obtained residue was purified by column chromatography using EtOAc to obtain the title compound 29 as oil. 1H NMR (300 MHz, DMSO-d6, ppm): δ 8.33 (m, 1H, H-2), 8.18 (m, 1H, H-8), 7.38 -7.26 (m, 15H, Ar-H), 5.64 (br s, 1H), 5.34-5.04 (m, 6H, 3xCH2), 4.90-4.82 (m, 1H), 4.32 (m, 2H), 2.17 (s, 1H), 1.78 (m, 2H), 1.25 (m, 28H, CH2), 0.89 (t, 3H, CH3). 13C NMR (500 MHz, DMSO-d6, ppm): δ 170.16, 155.59, 154.37, 153.28, 139.08, 138.93, 134.83, 128.85, 128.72, 128.55, 128.18, 128.13, 128.01, 127.97, 120.04, 84.20, 83.03, 82.92, 70.31, 67.40, 37.55, 33.09, 32.06, 29.84, 29.79, 29.65, 29.49, 19.16, 24.66, 22.82, 14.24. HRMS (ESI+) m/z Calculated for C50H67N5O10P1 (MH+): 928.4619, found 928.4582.
2′-deoxyadenosine-5′-(O-stearic acid-3-yl) phosphate (30)
To the solution of 29 (0.2 g, 0.21 mmol) in THF, K2CO3 (60 mg, 4.31 mmol) and water (2 mL) is added, followed by Palladium (10%) on charcoal, and stirring continued at room temperature under Hydrogen for 72 h. After completion of the reaction, the mixture is filtered on celite 545, and the celite pad was washed with THF:Water-1:1, and the filtrate was evaporated and purified by column chromatography using eluent DCM:MeOH:H2O-17:7:1 to obtain the potassium salt of 30 as white solid. 1H NMR (500 MHz, DMSO-d6, ppm): δ 8.37 (d, 1H, H-2), 8.12 (d, 1H, H-8), 7.25 (t, 2H), 6.37 (m, 1H), 5.43 (br s, 1H), 4.44 (br, 1H) 4.29 (m, 1H), 3.96 (t, 1H), 3.90-3.85 (m, 1H), 3.78 (m, 1H), 2.74 (m, 1H), 2.28 (m, 1H), 1.66 (m, 1H), 1.511 (m, 1H), 1.23 (m, 28H), 0.86 (t, 3H). 13C NMR (500 MHz, DMSO-d6, ppm): 172.98, 172.89, 156.12, 152.02, 152.53, 152.39, 149.22, 149.17, 148.91, 139.57, 139.24, 139.11, 119.28, 119.00, 118.94, 88.02, 85.97, 83.96, 83.17, 73.49, 71.17, 71.14, 71.00, 65.44, 65.18, 61.92, 50.01, 31.31, 29.06, 28.91, 28.85, 28.83, 28.72, 24.65, 22.12, 13.99. 31P NMR (500 MHz, DMSO-d6, ppm): 1.31, 1.21. HRMS (ESI-) m/z Calculated for C28H47N5O8P1 (MH-): 612.3167, found 612.3171.
3′-O-benzyloxycarbonyl-thymidine-5′-(O-(benzyl stearate)-2-yl, O-benzyl) phosphate (32)
To the stirring solution of 15 (2 g, 5.31 mmol) and 12 (5 g, 8 mmol) in dry DCM (5 mL), was added 0.5 M solution of 1H-Tetrazole (53 mL, 26.57 mmol) drop wise at 0°C and the reaction mixture was stirred at rt for 4 h. Then the reaction mixture was cooled down to -78°C and hydrogen peroxide 35% (W/V, 15 mL) was added. After stirring for 5 min at -78°C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM (200 mL) and washed with 1 M phosphoric acid (100 mL), dilute sodium bicarbonate (100 mL) and with brine (100 mL), dried over sodium sulfate, filtered, concentrated in vacuo. The obtained pale yellow oil was purified by column chromatography using EtOAc as eluent to give the desired compound 32 as oil (4.35 g, 89%). H1 NMR (CDCl3, 300 MHz, ppm): δ 8.85 (br s, 1H, -NH), 7.38 (m, 15H, 3xAr), 6.38 (m, 1H), 5.20 (m, 7H), 4.89 (m, 1H), 4.30 (m, 3H), 1.87 (m, 5H), 1.25 (m, 29H), 0.89 (t, 3H). 13C NMR (CDCl3, 300 MHz, ppm): δ 171.15, 169.83, 169.73, 163.87, 163.78, 154.36, 154.33, 150.55, 150.51, 150.49, 150.47, 135.40, 135.36, 135.14, 135.08, 135.01, 134.93, 134.79, 134.72, 128.97, 128.93, 128.85, 128.76, 128.68, 128.51, 128.45, 128.14, 128.11, 127.99, 127.95, 111.86, 111.84, 111.63, 84.44, 82.60, 70.22, 70.17, 69.83, 67.41, 60.42, 37.26, 31.97, 29.75, 29.71, 29.67, 29.56, 29.40, 29.09, 24.67, 24.58, 22.74, 21.07, 21.05, 19.12, 14.24, 14.16, 12.40. 31P NMR (500 MHz, CDCl3, ppm): δ -1.00, -1.24, -1.80, -1.83. HRMS (ESI+) m/z Calculated for C50H67N2O12P1 (MH+): 919.4504, found 919.4512.
Thymidine-5′-(O-stearic acid-2-yl) phosphate (33)
To a stirring solutions of 32 (2 g, 2.17 mmol) in THF: MeOH-1:1 (30 mL) was added Palladium on Charcoal (10%), and kept for stirring under hydrogen for 6 h at room temperature. After completion of the reaction, the reaction mixture is passed through celite pad and the celite pad is washed with THF-MeOH mixture (200 mL). The organic solvents were removed under vacuum, and the obtained white residue was purified by silica gel chromatography using DCM:MeOH:H2O-17:7:1. The organic solvents were removed with a rotavapor and the aqueous solvent was removed with a lyophilizer to get the desired product 33 as white solid (0.81 g, 61%). 1H NMR (300 MHz, D2O, ppm): δ 7.82 (d, 1H, NH), 6.28 (d, 1H, H-5), 4.55 (br m, 2H), 4.14 (br m, 3H), 2.37 (m, 2H, 5′-H), 1.94 (m, 3H, CH3), 1.77 (br s, 2H, CH2), 1.42 (br, 2H), 1.18 (m, 26H, 13xCH2), 0.81 (t, 3H, CH3). 13C NMR (300 MHz, D2O, ppm): δ 173.02, 165.43, 150.90, 136.63, 110.88, 110.77, 85.53, 84.89, 74.38, 70.84, 70.60, 64.69, 39.23, 38.98, 33.09, 31.58, 29.53, 29.40, 29.27, 29.09, 24.74, 22.27, 13.53, 11.69, 11.61. 31P NMR (300 MHz, D2O, ppm): δ -0.80, -0.95. HRMS (ESI+) m/z Calculated for C28H49N2O10P1 (MH-): 603.3051, found 603.3054.
2′,3′-O-bisbenzyloxycarbonyl, adenosine-5′-((O-benzyl stearate)-2-yl, O-benzyl) phosphate (35)
To the stirring solution of 16 (2.5 g, 4.668 mmol) and 0.5 M solution of 1H-Tetrazole (46.7 mL, 23.34 mmol) in anhydrous dichloromethane, was added 12 (0.88 g, 7 mmol) in dry dichloromethane, drop wise at 0°C and the reaction mixture was stirred at rt for 4 h. Then the reaction mixture was cooled down to -78°C and hydrogen peroxide 35% (W/V) (15 mL) was added. After stirring for 5 min at -78°C, cooling bath was removed and the reaction mixture was allowed to stir at room temperature for 30 min. The reaction mixture is diluted with DCM (200 mL) and washed with 1 M phosphoric acid (100 mL), 5% aqueous sodium bicarbonate (100 mL) and with brine (100 mL). Organic layer was separated and dried over sodium sulfate, filtered and concentrated in vacuo. The obtained pale yellow oil was purified by silica gel column chromatography with eluent EtOAc to give 35 (4.55 g, 90%) as oil. 1H NMR (500 MHz, CDCl3, ppm): δ 8.28 (d, 1H, H-2), 8.07 (d, 1H, H-8), 7.34-7.29 (m, 20H, Ar), 6.21 (dd, 1H), 5.92 (m, 1H), 5.85 (br s, 2H), 5.65 (m, 1H), 5.18 (m, 1H), 5.12 (m, 6H), 5.87(m, 1H), 4.38 (m, 2H), 1.78 (m, 2H), 1.25 (m, 28H), 0.89 (t, 3H, CH3). 13C NMR (500 MHz, CDCl3, ppm): δ 170.13, 155.65, 154.09, 153.73, 153.47, 150.02, 139.38, 139.28, 135.27, 134.75, 134.59, 128.88, 128.85, 128.77, 128.71, 128.67, 128.600, 128.52, 128.11, 127.98, 127.92, 120.13, 85.63, 80.64, 76.17, 76.06, 73.98, 73.79, 70.63, 70.57, 69.83, 67.43, 66.2633.02, 32.97, 32.05, 29.83, 29.79, 29.66, 29.48, 29.45, 29.15, 24.64, 24.59, 22.81, 14.24. 31P NMR (500 MHz, CDCl3):-1.57,-1.61, -2.09. HRMS (ESI+) m/z Calculated for C58H72N5O13P1 (MH+): 1078.4936, found 1078.4946.
Adenosine-5′-(O-stearic acid-2-yl) phosphate (36)
To a solution of 35 (2 g, 1.85 mmol) in THF, K2CO3 (0.52 g, 37 mmol) and water (2 mL) was added. To this, Pd (10%) on Charcoal is added and kept for stirring at room temperature under hydrogen for 72 h. After the completion of the reaction, the mixture is filtered on celite 545, filtrate was washed with THF:Water-1:1. The filtrate was concentrated in vacuo and purified by silica gel column chromatography (DCM:MeOH:H2O-17:7:1) to obtain the potassium salt of 33 as white solid (0.62 g, 53%). 1H NMR (500 MHz, CDCl3, ppm): δ 8.4 (br s, 1H, H-2), 8.13 (s, 1H, H-8), 7.21 (br s, 2H, -NH2), 5.92 (d, 1H, H-1′), 5.32 (m, 1H), 4.61 (br, 1H), 4.21 (br s, 1H), 4.04 (s, 1H), 3.90 (br s, 1H), 2.08 (s, 1H), 1.68 (br, 1H), 1.52 (br, 1H), 1.22 (m, 28H), 0.86 (t, 3H, CH3).
3′-O-benzyloxycarbonyl, deoxyadenosine-5′-(O-benzyl, O-(benzyl stearate)-2-yl) phosphate (38)
To the stirring solution of 14 (0.3 g, 0.77 mmol) and 13 (0.734 g, 1.16 mmol) in 5 mL dry DCM was added 0.5 M solution of 1H-tetrazole (12.5 mL, 6.22 mmol) drop wise at 0°C, and the reaction mixture was stirred at room temperature for 12 h. Then the reaction mixture was cooled down to -78°C and hydrogen peroxide 35% (W/V) was added. After stirring for 5 min at -78°C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM and washed with 1 M phosphoric acid, 5% aq. sodium bicarbonate and with brine, dried over sodium sulfate, filtered and concentrated on vacuo, and purified by column chromatography (EtOAc) which gives 38 as an oil. 1H NMR (300 MHz, CDCl3, ppm): δ7.37-7.28 (m, 15H), 6.45 (m, 2H), 5.31 (d, 1H), 5.13-4.98 (m, 6H, 3 × CH2), 4.29-4.20 (m, 1H), 2.78-2.69 (m, 1H), 2.68-2.54 (m, 1H), 1.65 (m, 2H, CH2), 1.25 (m, 26H, 13xCH2), 0.89 (t, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 169.89, 155.84, 154.25, 153.16, 149.69, 138.63, 135.73, 134.76, 128.77, 128.69, 128.57, 128.52, 128.40, 128.35, 128.26, 128.24, 128.13, 127.99, 127.96, 127.88, 127.81, 119.89, 84.08, 83.97, 82.93, 82.82, 78.32, 78.25, 76.52, 76.45, 76.38, 70.12, 69.62, 69.54, 69.46, 66.63, 40.15, 37.46, 35.25, 31.91, 29.69, 29.65, 29.54, 29.43, 29.41, 29.35, 29.28, 24.79, 22.68, 14.12. 31 P NMR (500 MHz, CDCl3, ppm): δ -1.92, -1.98, -2.17, -2.31. HRMS (ESI+) m/z Calculated for C50H66N5O10P1 (MH+): 928.4619, found 928.4615.
2′-deoxyadenosine-5′-(O-stearic acid-3-yl) phosphate (39)
To a stirring solution of 38 (0.2 g, 0.21 mmol) in THF, K2CO3 (60 mg, 4.31 mmol) and water (2 mL) is added. To this Pd (10%) on charcoal is added and kept for stirring at rt under hydrogen for 12 h. After completion, the reaction mixture is filtered on celite-545 and the solvent is evaporated and purified by column chromatography (DCM:MeOH:H2O-17:7:1) to obtain the potassium salt of 39 as white solid. 1H NMR (500 MHz, D2O, ppm): δ 8.48 (d, 1H), 8.09 (d, 1H), 6.42 (d,1H), 4.61 (br s, 1H), 4.26 (br s, 1H), 4.16 (br m, 2H, -NH2), 2.64 (m, 3H), 1.68 (m, 2H), 1.25 (m, 2H), 1.31-1.22 (m,2H), 1.01 (m, 18H, CH2), 0.66 (t, 3H, CH3). 13C NMR(500 MHz, D2O, ppm): 176.53, 154.04, 150.78, 147.64, 139.41, 117.66, 85.66, 83.89, 74.11, 70.77, 70.49, 64.73, 61.14, 58.56, 41.62, 40.05, 39.94, 35.24, 35.05, 33.61, 31.40, 29.34, 29.23, 28.91, 28.02, 24.66, 22.09, 13.37. 31P NMR(500 MHz, D2O, ppm): δ -1.04, -1.12. HRMS (ESI-) m/z Calculated for C28H48N5O8P1 (MH-): 612.3167, found: 612.3173.
3′-O-benzyloxycarbonyl thymidine 5′-(O-(benzyl stearate)-3-yl, O-benzyl)-phosphate (41)
To a stirring solution of 15 (0.3 g, 0.77 mmol) and 13 (0.734 g, 1.16 mmol) in 5 mL dry DCM was added 0.5 M solution of 1H-tetrazole (12.5 mL, 6.22 mmol) drop wise at 0°C, and the reaction mixture was stirred at room temperature for 12 h. Then the reaction mixture was cooled down to -78°C and hydrogen peroxide 35% (W/V) was added. After stirring for 5 min at -78°C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM and washed with 1 M phosphoric acid, 5% aq. sodium bicarbonate and with brine, dried over sodium sulfate, filtered and concentrated on vacuo, purified by column chromatography (EtOAc) which gives 41 as an oil. 1H NMR (500 MHz, CDCl3, ppm): δ 9.27 (br s, 1H, NH), 7.43-7.31 (m, 15H, 3 × Ar-H), 6.37 (m, 1H), 5.16 (s, 2H, Ar-CH2), 5.12-5.03 (m, 4H, Ar-CH2), 4.86 (m, 1H), 4.18 (m, 2H), 2.74-2.64 (m, 2H), 2.36 (m, 1H),2.03 (m, 1H), 1.89 (d, 2H), 1.69 (m, 2H), 1.25 (m, 26H, -CH2), 0.89 (t, 3H, CH3). 1H NMR (300 MHz, CDCl3, ppm): δ 169.87, 163.75, 154.36, 150.49, 135.63, 135.59, 135.11, 135.02, 134.76, 134.73, 128.85, 128.76, 128.68, 128.63, 128.50, 128.46, 128.36, 128.28, 128.09, 128.01, 127.95, 111.81, 111.73, 84.52, 84.44, 82.59, 82.48, 78.07, 78.02, 70.22, 70.20, 69.87, 69.80, 69.70, 69.63, 67.13, 67.06, 67.00, 66.72, 66.68, 40.25, 40.18, 40.12, 37.28, 37.20, 35.39, 31.97, 29.75, 29.71, 29.69, 29.60, 29.51, 29.41, 29.38, 24.91, 24.85, 22.71, 14.18, 12.44. 31P NMR(300 MHz, CDCl3): δ -1.68, -1.86, -2.06, -2.25. HRMS (ES+) m/z Calculated for C50H67N2O12P1 (MH+): 919.4504, found 919.4498.
Thymidine-5′-(O-stearic acid-3-yl)-phosphate (42)
To the stirring solutions of 41 (2 g, 2.17 mmol) in THF:MeOH-1:1 (30 mL) was added Pd (10%) on charcoal, and kept for stirring under hydrogen for 6 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture is passed through celite pad and the celite pad is washed with THF-MeOH mixture (200 mL). The organic solvents were removed under vacuum, and the obtained white residue was purified by silica gel chromatography using DCM:MeOH:H2O-17:7:1. The organic solvents were removed in the rotavapor and the aqueous solvent was removed by lyophilization to obtain the desired product 42 as white solid (0.94 g, 71%). 1H NMR (500 MHz, D2O, ppm): δ 7.84 (br m, 1H), 6.32 (br m, 1H), 4.60 (br m, 2H), 4.16 (m, 3H), 2.61 (m, 2H), 2.39 (m, 2H), 1.94 (s, 3H), 1.72 (m, 2H), 1.21 (m, 26H, CH2), 0.83 (s, 3H, CH3). 13C NMR (500 MHz, D2O, ppm): δ 174.93, 165.58, 151.16, 136.86, 111.04, 85.69, 85.20, 84.89, 73.60, 71.08, 70.80, 65.06, 64.67, 40.54, 39.33, 35.21, 31.78, 29.70, 29.60, 29.28, 24.80, 22.46, 13.74, 11.86. 31P NMR(500 MHz, CDCl3): δ -1.13, -1.15. HRMS (ES+) m/z Calculated for C28H49N2O10P1 (MH-): 603.3051, found 603.3048.
2′,3′-O-dibenzyloxycarbonyl, adenosine-5′-(O-(benzyl stearate)-3-yl, O-benzyl) phosphate (44)
To a stirring solution of 16 (2 g, 3.73 mmol) and 13 (2.81 g, 4.48 mmol) in dry DCM was added 0.5 M solution of 1H-tetrazole (0.5 M in dry acetonitrile (75 ml, 37.34 mmol) dropwise at 0°C and the reaction mixture was stirred at rt for 12 h. Then the reaction mixture was cooled down to -78°C and hydrogen peroxide 35% was added. After stirring for 5 min at -78°C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM and washed with 1 M phosphoric acid, dilute sodium bicarbonate and with brine, dried over sodium sulfate, filtered and concentrated in vacuo, purified by column chromatography with eluent EtOAC to give 44 as oil. 1H NMR (500 MHz, CDCl3, ppm) : δ 8.27 (s, 1H, H-2), 8.01-7.97 (s, 1H, H-8), 7.33-7.25 (m, 20H, 4xAr-H), 6.19 (d, 1H), 5.93-5.91 (m, 3H), 5.62 (m, 1H), 5.12-5.01 (m, 8H, CH2-Ar), 4.81 (m, 1H), 4.38 (m, 1H), 4.30-4.28 (m, 1H), 4.24-4.22 (m, 1H), 2.7-2.70 (m, 1H), 2.61-2.57 (m, 1H), 2.21 (br s, 1H), 1.67-1.60 (m, 2H), 1.25 (m, 26H), 0.89 (t, 3H, CH3).13C NMR (500 MHz, CDCl3, ppm): 170.05, 155.70, 154.00, 153.72, 153.48, 149.90, 139.26, 135.78, 134.69, 134.54, 128.85, 128.75, 128.70, 128.64, 128.54, 128.52, 128.40, 128.06, 127.98, 120.16, 85.83, 85.79, 85.63, 80.65, 80.59, 76.66, 73.79, 70.65, 70.53, 69.67, 66.64, 65.94, 40.22, 35.31, 32.03, 29.81, 29.77, 29.68, 29.61, 29.54, 29.47, 29.40, 24.86, 22.79, 14.23. 31P NMR (500 MHz, CDCl3, ppm): δ (q -1.96, -2.07, -2.25, -2.39). HRMS(ES+) m/z Calculated for C58H72N5O13P1 (MH+): 1078.4937, found 1078.4946.
Adenosine-5′-(O-stearic acid-3-yl)-phosphate (45)
To the solution of 44 (0.3 g, 0.27 mmol) in THF, K2CO3 (77 mg, 0.55 mmol) and water 2 mL was added. To this Pd (10%) on charcoal is added and held for stirring at rt under hydrogen for 12 h. After completion of the reaction, the reaction mixture is filtered on celite and the solvent is evaporated and purified by column chromatography (DCM: MeOH: H2O-17:7:1) to obtain the potassium salt of 45 as white solid. 1H NMR (500 MHz, D2O, ppm): δ 8.42 (d, 1H, H-2), 8.12 (s, 1H, H-8), 7.24 (br s, 2H, -NH2), 5.91 (d, 1H), 4.53 (m, 1H), 4.19 (m, 2H), 4.01 (br s, 1H), 3.93 (m, 2H), 2.68 (m, 1H), 2.40 (ddd, 1H), 1.91 (m, 1H), 1.60 (m, 1H), 1.45 (m, 1H), 1.23 (m, 26H, CH2), 0.86 (t, 3H, CH3). 13C NMR (500 MHz, D2O, ppm): δ 172.50, 165.45, 155.98, 152.55, 149.58, 139.32, 139.19, 127.96, 126.94, 118.83, 86.93, 83.74, 74.01, 73.88, 71.17, 70.82, 70.64, 67.48, 64.73, 64.41, 60.48, 53.30, 47.85, 43.37, 42.56, 35.52, 31.28, 29.04, 28.69, 24.89, 22.08, 13.95. 31P NMR (500 MHz, D2O): δ -0.69. HRMS(ES-) m/z Calculated for C28H48N5O9P1 (MH-): 628.3116, found 594.3061.
9-((O-benzyl, O-(O-benzyl stearate)-2-yl-5′ phospho), 3′-O-benzyl, 2′-O-benzyloxycarbonyl xylofuranosyl) adenine (46)
To a stirred solution of 27 (1.2 g, 24.41 mmol) and 12 (2.3 g, 36.62 mmol) in 12 mL dry DCM was added 0.5 M solution of 1H-Tetrazole (24.41 mL, 122.07 mmol) drop wise at 0°C, and the reaction mixture was stirred at room temperature for 12 h. Then the reaction mixture was cooled down to -78°C and hydrogen peroxide 35% (W/V) was added. After stirring for 5 min at -78°C, cooling bath was removed and the reaction mixture was allowed to stir at room temperature for further 30 min. The reaction mixture is diluted with DCM (150 mL) and washed with 1 M phosphoric acid (100 mL), 5% aq. sodium bicarbonate (100 mL) and with brine (80 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The obtained residue was purified by column chromatography (EtOAc) giving 46 (100 mg, 4%) as an oil. 1H NMR (300 MHz, CDCl3, ppm): δ 8.34 (s, 1H, H-2), 8.09 (m, 1H, H-8), 7.38-7.21 (m, 20H, Ar-H), 6.33 (m, 1H), 5.93 (br s, 1H), 5.41 (br s, 1H), 5.20-5.02 (m, 6H, CH2-Ar), 4.83 (m, 1H), 4.67 (m, 1H), 4.54 (m, 1H), 4.44 (m, 3H), 4.09 (br m, 1H), 1.80 (m, 2H), 1.25 (m, 26H, CH2), 0.87 (m, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 170.03, 155.58, 153.84, 153.37, 149.69, 139.24, 139.15, 136.38, 136.27, 135.73, 135.65, 135.27, 134.46, 129.04, 128.85, 128.67, 128.57, 128.47, 128.42, 128.19, 128.12, 128.06, 127.92, 119.54, 87.38, 87.33, 82.60, 82.52, 81.34, 81.23, 81.16, 80.02, 79.94, 76.04, 75.97, 72.40, 70.80, 69.87, 69.82, 69.72, 69.65, 67.25, 65.37, 65.30, 65.21, 65.10, 65.03, 33.06, 32.97, 32.02, 29.80, 29.75, 29.71, 29.60, 29.45, 29.41, 29.11, 24.60, 22.78, 14.22. 31P NMR (300 MHz, CDCl3, ppm): δ -1.58, -1.68, -1.82, -1.91. HRMS (ESI+) m/z Calculated for C57H72N5O11P1 (MH+):1034.5038, found: 1034.5048.
9-xylofuranosyl adenine-5′-(O-stearic acid-2-yl) phosphate (47)
To a solution of 46 (100 mg, 0.09 mmol) in THF, K2CO3 (27 mg, 0.18 mmol) and water (2 mL) is added. To this Palladium (10%) on charcoal (10 mg) is added and held for stirring at rt under hydrogen for 12 h. After the completion of the reaction, the mixture is filtered on celite and the solvent is removed in vacuo. The obtained residue was purified by column chromatography with the eluent (DCM:MeOH:H2O-17:7:1) to obtain the potassium salt of 47 as white solid. 1H NMR (300 MHz, CDCl3, ppm): δ 8.35 (m, 1H, H-2), 8.13 (s, 1H, H-8), 7.31 (d, 2H), 5.91 (br s, 1H), 4.31 (m, 2H), 4.12 (m, 2H), 4.05 (m, 1H), 3.87 (m, 1H), 3.80-3.61 (m, 1H), 1.63 (m, 2H), 1.19 (m, 24H, CH2), 0.85 (t, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 156.06, 155.96, 152.43, 149.02, 148.78, 139.79, 118.44, 89.42, 83.62, 82.01, 80.93, 75.05, 74.87, 60.64, 59.27, 54.96, 31.34, 29.20, 28.78, 28.28, 22.12, 13.92. 31P NMR (300 MHz, CDCl3, ppm): δ 1.67, 1.36. HRMS (ESI-) m/z Calculated for C28H48N5O9P1 (MH-): 628.3116, found: 628.3123.
9-((O-benzyl, O-(O-benzyl stearate)-3-yl-5′ phospho), 3′-O-benzyl, 2′-O-benzyloxycarbonyl xylofuranosyl) adenine (48)
To a stirred solution of 27 (1.2 g, 2.44 mmol) and 13 (2.3 g, 3.66 mmol) in 20 mL dry DCM was added 0.5 M solution of 1H-Tetrazole (54 mL, 24.4 mmol) drop wise at 0°C, and the reaction mixture was stirred at room temperature for 12 h. Then the reaction mixture was cooled down to -78°C and hydrogen peroxide 35% (W/V) was added. After stirring for 5 min at -78°C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM and washed with 1 M phosphoric acid, 5% aq. sodium bicarbonate and with brine, dried over sodium sulfate, filtered and concentrated in vacuo, purified by column chromatography (EtOAc) which gives 48 as an oil. 1H NMR (500 MHz, CDCl3, ppm): δ 8.33 (s, 1H, H-2), 8.10 (m, 1H, H-8), 7.38-7.19 (m, 20H, 4 × Ar), 6.33 (d, 1H, H-1′), 6.14 (br s, 2H, -NH2), 5.42 (br s, 1H), 5.19 (m, 2H), 5.08-5.00 (m, 4H), 4.83 (m, 1H), 4.66 (dd, 1H), 4.54 (dd, 1H), 4.38 (m, 1H), 4.35 (m, 2H), 4.10 (m, 1H), 3.55-3.46 (m, 1H), 2.77-2.72 (m, 1H, H-5′), 2.63-58 (m, 1H, H-5″), 1.66 (m, 2H, CH2), 1.25 (m, 28H, CH2), 0.89 (t, 3H, CH3). 13C NMR (500 MHz, CDCl3, ppm): δ 169.91, 155.68, 153.75, 153.22, 149.53, 139.02, 136.24, 135.82, 135.62, 134.36, 128.95, 128.75, 128.63, 128.58, 128.57, 128.54, 128.36, 128.27, 128.12, 128.10, 128.00, 127.92, 128.86, 119.41, 87.25, 82.44, 81.24, 79.91, 76.28, 72.27, 70.69, 69.47, 66.54, 64.92, 40.33, 35.26, 31.94, 29.71, 29.71, 29.68, 29.64, 29.56, 29.45, 29.37, 29.31, 24.76, 22.98, 22.71, 14.16. HRMS (ES-) m/z Calculated for C57H72N5O11P1 (MH+): 1034.5038, found: 1034.5026.
β-D-xylofuranosyladenine-5′-(O-stearic acid-3-yl)-phosphate (49)
To the solution of 48 (0.2 g, 0.21 mmol) in THF, K2CO3 (60 mg, 4.31 mmol) and water (2 mL) is added. To this Pd (10%) on charcoal is added and held for stirring at rt under hydrogen for 12 h. After the completion of the reaction, the mixture is filtered on celite-545 and the solvent is evaporated and the residue is purified on column chromatography (DCM:MeOH:H2O-17:7:1) to obtain the potassium salt of 49 as white solid. H1 NMR (500 MHz, DMSO-d6, ppm): δ 8.26 (m, 1H), 8.11 (d, 1H, H-8), 5.98 (br s, 1H), 4.63 (m, 2H), 4.46 (m, 2H), 4.25 (m, 2H), 3.60 (t, 1H), 2.44 (m, 2H), 1.78 (t, 1H), 1.55 (m, 2H), 1.06 (m, 26H, CH2), 0.74 (t, 3H, CH3). 13C NMR (600 MHz, DMSO-d6, ppm): δ 179.31, 162.09, 154.98, 152.10, 152.03, 148.07, 147.75, 139.95, 139.86, 118.24, 118.09, 89.68, 88.46, 81.43, 81.39, 80.54, 80.11, 79.59, 75.66, 75.53, 74.57, 62.98, 62.75, 61.24, 48.57, 44.01, 35.17, 33.73, 31.45, 29.33, 29.24, 28.93, 28.12, 24.69, 24.63, 22.97, 22.17, 13.51. 31P NMR (500 MHz, D2O, ppm): -0.94, -1.09. HRMS (ES-) m/z Calculated for C28H48N5O9P (MH+): 628.3117, found: 628.3119.
2′-deoxyadenosine-5′-(N-(methyl stearate)-2-yl)-phosphoramidate (51)
In a 100 mL two neck flask, 2′-deoxyadenosine 5′-O-monophosphate (0.3 g, 0.9 mmol) and methyl α-aminostearate 50 (1.42 g, 4.52 mmol) were dissolved in a mixture of t- butanol and water (5:1). Triethylamine (0.5 mL), and a freshly prepared solution of N, N’-dicyclohexylcarbodiimide (DCC) in t-butanol (0.5 g/mL) was added to the reaction mixture under argon atmosphere, and the reaction mixture was allowed to reflux for 2 h. The progress of the reaction was monitored by TLC. Another 5 eq of DCC was added to the reaction mixture, and was continued refluxed under argon for 1 h. Upon completion, the reaction mixture was cooled down to room temperature and the solvent was concentrated under vacuo. The residue obtained was purified by silica gel column chromatography (DCM:MeOH:H2O-17:7:1) to obtain 51 (0.44 g, 77%) as white solid. 1H NMR (500 MHz, DMSO, ppm): δ 8.38 (d, 1H, H-2), 8.12 (s, 1H, H-8), 7.22 (br s, 2H), 6.35 (t, 1H), 4.41 (s, 1H), 3.92 (br s, 1H), 3.78-3.74 (m, 2H), 3.67 (m, 1H), 3.55 (s, 2H), 3.52 (s, 1H), 3.02 (dd, 3H), 2.69-2.63 (m, 1H), 1.47 (m, 2H), 1.22-1.14 (m, 28H, 14xCH2), 0.85 (t, 3H, CH3). 13C NMR (500 MHz, DMSO, ppm): δ 175.11, 155.95, 152.50, 149.18, 139.20, 118.86, 117.45, 82.97, 71.34, 64.14, 54.47, 51.28, 45.20, 31.26, 29.01, 28.87, 28.73, 28.67, 24.94, 22.07, 13.93, 8.46. 31P NMR (500 MHz, DMSO): δ 4.51. HRMS (ESI+) m/z Calculated for C29H50N6O7P1(MH-): 625.3483 found 625.3453.
2′-deoxyadenosine-5′-(N-stearic acid-2-yl)-phosphoramidate (52)
A solution of 51 (0.4 g, 0.63 mmol) in MeOH/H2O (4:1 v/v, 3 mL, containing 0.4 M of NaOH (0.051 g, 1.27 mmol) was stirred at room temperature under nitrogen for 2 h. After completion of the reaction, the solvents were removed under reduced pressure. The resulted crude reaction mixture was purified by silica gel chromatography (DCM:MeOH:H2O-17:7:1) to obtain 52 (0.28 g, 71%) as white solid. 1H NMR (500 MHz, DMSO, ppm): δ 8.42 (d, 1H, H-2), 8.11 (d, 1H, H-8), 7.23 (br s, 2H, -NH2), 6.37 (m, 1H), 4.44 (d, 1H), 3.97 (m, 1H), 3.82-3.71 (m, 3H), 2.69 (m, 1H), 2.27 (m, 1H), 1.65 (br s, 1H), 1.21 (m, 28H), 0.85 (t, 3H). 13C NMR (500 MHz, DMSO, ppm): δ 176.18, 156.08, 152.59, 149.31, 139.31, 119.01, 86.45, 83.21, 71.62, 64.31, 64.09, 55.13, 54.78, 31.87, 31.39, 29.14, 25.90, 25.87, 22.20, 14.06. 31P NMR (500 MHz, DMSO): δ 7.08, 6.96. HRMS (ES-) m/z Calculated for C28H49N6O7P1 (MH-): 611.3327, found: 611.3323.
Thymidine-5′-(N-(methyl stearate)-2-yl)-phosphoramidate (53)
In a 100 mL two neck flask, 2′-deoxythymidine 5′-O-monophosphate (0.5 g, 1.55 mmol) and methyl α-aminostearate 50 (2 g, 6.20 mmol) were dissolved in a mixture of t- butanol and water (5:1). Triethylamine (0.5 mL), and a freshly prepared solution of N, N’-dicyclohexylcarbodiimide (DCC, 1.6 g, 7.75 mmol) in t- butanol (0.5 g/mL) was added to the reaction mixture under argon atmosphere, and the reaction was allowed to reflux for 2 h. Another 5 eq of DCC was added to the reaction mixture which was refluxed for 1 h. Upon completion, the reaction mixture was cooled down to room temperature and the reaction mixture was concentrated in vacuo. The residue obtained was purified by silica gel column chromatography (DCM:MeOH:H2O-17:7:1) to obtain 53 (0.69 g, 72%) as white solid. 1H NMR (500 MHz, DMSO): δ 10.82 (br s, 1H, -NH), 7.28 (d, 1H), 5.70 (m, 1H), 5.24 (s, 1H), 3.76 (br s, 1H), 2.67 (s, 2H), 1.56 (m, 2H), 1.32 (br s, 2H), 0.71 (m, 28H, 14xCH2), 0.34 (m, 3H, CH3).
13C NMR (300 MHz, DMSO, ppm): δ 175.26, 163.79, 150.50, 136.26, 109.62, 86.03, 85.92, 83.74, 70.97, 63.82, 54.54, 54.44, 51.19, 49.19, 45.10, 34.30, 31.28, 30.21, 29.03, 28.75, 28.70, 25.01, 24.62, 23.83, 22.07, 13.87, 12.06, 8.36. 31P NMR (300 MHz, DMSO, ppm): δ 3.70. HRMS (ES-) m/z Calculated for C29H52N3O9P1 (MH-): 616.3368, found: 616.3371.
Thymidine-5′-(N-stearic acid-2-yl)-phosphoramidate (54)
A solution of 53 (0.5 g, 0.8 mmol) in MeOH/H2O (4:1-v/v), was added 0.4 M NaOH (0.065 g, 1.61 mmol), and the mixture was stirred at room temperature under nitrogen for 2 h. The solvent was removed under reduced pressure. The resulting crude material was purified by chromatography (DCM:MeOH:H2O-17:7:1) to obtain 54 (0.34 g, 69%) as white solid. 1H NMR (500 MHz, DMSO): δ11.24 (s, 1H), 7.84 (s, 1H), 6.22 (q, 1H), 5.36 (br s, 1H), 4.31 (d, 1H), 3.86-3.72 (m, 1H), 2.12-2.01 (m, 2H), 1.80 (m, 1H), 1.76 (), 1.23 (m, 28H), 0.86 (t, 3H, CH3). 13C NMR (500 MHz, DMSO): 176.13, 163.89, 150.58, 136.39, 109.79, 86.12, 83.84, 71.35, 71.00, 31.38, 29.12, 26.03, 25.85, 22.18, 14.05, 12.16, 12.12. 31P NMR (500 MHz, DMSO): δ 7.29, 7.00. HRMS (ES-) m/z Calculated for C28H49N3O9P1 (MH-): 602.3211, found 602.3210.
Bodipy (55)
8-S-Methyl Bodipy (120 mg, 0.5 mmol, prepared according to ref. [41]) is dissolved in DMSO/DCM (1/1; v/v, 5 ml) and mixed with taurine (60 mg, 0.5 mmol) and NaHCO3 (42 mg, 0.5 mmol). The resulting mixture is stirred at room temperature until TLC indicates complete consumption of the starting material, and the formation of a highly polar compound displaying blue fluorescence. The reaction mixture is diluted with water (10 ml) and dichloromethane (10 ml) and extracted. The aqueous layer is collected and lyophilized to yield the desired product 55 as a pale yellow solid. 1H NMR(300 MHz, DMSO, ppm): δ 7.47 (br s, 1H, -CH), 7.36 (d, 1H, -CH), 7.24 (br s, 1H, -CH), 7.11 (d, 1H, -CH), 6.49-6.47 (m, 1H, -CH), 6.31-6.29 (m, 1H, -CH), 3.99 (t, 2H, -CH2), 2.92 (t, 2H, -CH2). 13C NMR (500 MHz, DMSO, ppm): δ 147.79, 132.15, 129.05, 122.45, 122.18, 114.57, 113.77, 112.42, 40.42.
Vesicles preparation and visualization
Nucleolipid aggregate/vesicle formation was performed by dissolving the nucleolipids in water or DMSO and dilute with water or THF/dioxane (1:1) in a glass vial. To this solution Bodipy fluorescent dye either in water, THF or chloroform was added, vortexed for 10 seconds to stimulate vesicle formation and set aside for 5 min. The pH of the nucleolipids emulsion is found to be 6.82. If the pH was lowered further, the emulsion appeared opalescent. An aliquot of the mixture (100 μL) was pipetted out from this reaction mixture and spin coated at 3000 rpm on a microscope glass plate for 2 min, resulting in a thin layer adequate for optical microscopy.
Vesicles formed in presence of Bodipy fluorescent dye were monitored under fluorescent microscopy and the images were recorded with Olympus Fluoview FV1000 by carrying out excitation wavelength readings at 532 nm with 100 × zoom. Two fluorescent dyes were used for the vesicle encapsulation: aqueous soluble dye 55 (soluble in both water) and organic soluble dye 56 (chloroform/THF soluble), which were used at a concentration of 0.5 μM.
Stability study by NMR
Samples were prepared in D2O or DMSO and the pD of the sample was adjusted by the addition of a small volume (a few μL) of HCl or NaOH solutions in D2O (0.1 M). 31P NMR was used to study the stability of the nucleolipids in acidic (pH4) and in base (pH12) environment. One-dimensional 31P spectra were used as a fast screening experiment to monitor degradation of the conjugates. Two-dimensional 1H-31P correlation spectra were used to characterize the 31P containing products formed by degradation of the nucleolipids. Correlations were established using a Proton-detected hetero-TOCSY experiment [24] with a DIPSI spinlock of 50 ms, allowing correlations of 31P resonances with several 1H resonances of adjacent spin systems.