3 Conclusive summary 69
5.2 Chemical synthesis
5.2.1 5-‐Nitro-‐1-‐indolyl-‐2′-‐deoxyriboside-‐5′-‐triphosphate (dNITP)
The 5-‐nitro-‐1-‐indolyl-‐2’-‐deoxyriboside (dNI) was purchased from Berry & Associates Inc., USA. 20 mg (71.9 µmol) nucleoside and 23 mg (107.8 µmol) proton sponge was dissolved in previously distilled trimethylphosphate. The addition of POCl3 was carried out under ice bad cooling. The reaction was monitored by reverse phase TLC in isopropanol / water/ ammonia = 3 / 1 / 1. After the addition of 1.2 equivalents of POCl3 719 µl (359 µmol) pyrophosphate and 190 µl (720 µmol) tributylamine was added simultaneously and fast. The solution was stirred for 30 min and afterwards quenched by adding 5 ml 0.1 M TEAB buffer. The reaction mixture was three times extracted against ethyl acetate. The aqueous solution was further purified by ion exchange chromatography (see chapter 5.1.2). The remaining sample was desalted by RP-‐MPLC using 0.05 M TEAA buffer and acetonitrile (see chapter 5.1.2) to afford dNITP (13.7 µmol, 19% yield).
1H-‐NMR (400 MHz, D2O): δ = 8.72 (d, 3J = 2.2 Hz, 1H, H-‐4), 8.25 (dd, 3J = 2.3, 9.2 Hz, 1H, H-‐6), 7.89 (d, 3J = 3.5 Hz, 1H, H-‐2), 7.80 (d, 3J = 9.2 Hz, 1H, H-‐7), 6.97 (d, 3J = 5.9 Hz, 1H, H-‐3), 6.70 (dd, 3J = 6.1, 8.2 Hz, 1H, H-‐
1’), 4.86 (dt, 3J = 2.8, 5.9 Hz, 1H, H-‐3’), 4.35 – 4.31 (m, 1H, H-‐4’), 4.30 – 4.17 (m, 2H, H-‐5’), 3.26 (q, 3J = 7.3 Hz, 24H, NEt3); 2.87 (ddd, 3J = 6.2, 8.2 Hz, 2J = 14.3 Hz 1H, H-‐2’), 2.57 (ddd, 3J = 2.9, 6.0 Hz, 2J = 14.0 Hz, 1H, H-‐2’), 1.99 (s, 3H, acetic acid), 1.34 ppm (t, 3J = 7.3 Hz, 36H, NEt3).
31P NMR (162 MHz, D2O): δ = -‐10.76 (d, 2J = 19.8 Hz, 1P, Pγ), -‐11.30 (d, 2J = 20.1 Hz, 1P, Pα), -‐23.21 ppm (t, 2J
= 20.1 Hz, 1P, Pβ)
HRMS: m/z: calcd for [C13H16N2O14P3-‐]: 516.9809; found: 516.9804.
5.2.2 5’-‐Tert-‐butyldiphenylsilyl-‐2’-‐deoxyuridine
To protect 2’-‐deoxyuridine at the 5’ position 1.5637 g (6.85 mmol) nucleoside was dissolved in 15 ml pyridine. Next 1.96 ml (7.54 mmol) TBDPS-‐Cl (tert-‐butyldiphenylsilylchloride) was added. The reaction
mixture was stirred over night. Pyridine was removed and the reaction residue was purified by flash chromatography (solvent: hexane / ethyl acetate = 1 / 3) to afford 5’-‐TBDPS proteced 2’-‐deoxyuridine (5.49 mmol, 80% yield).
1H-‐NMR (400 MHz, CDCl3): δ = 8.41 (br s, 1H, NH), 7.78 (d, 3J = 8.2 Hz, 1H, H-‐6), 7.69 – 7.59 (m, 4H, ArH), 7.51 – 7.37 (m, 6H, ArH), 6.33 (t, 3J = 6.4 Hz, 1H, H-‐1’), 5.46 (dd, 4J = 2.1 Hz, 3J = 8.1 Hz, 1H, H-‐5), 4.60 – 4.49 (m, 1H, H-‐3’), 4.04 – 3.95 (m, 2H, H-‐4’, H-‐5’), 3.90 – 3.80 (m, 1H, H-‐5’), 2.43 (ddd, 3J = 4.0, 6.1 Hz, 2J = 13.6 Hz, 1H, H-‐2’), 2.29 – 2.15 (m, 1H, H-‐2’), 1.09 ppm (s, 9H, C(CH3)3).
13C NMR (101 MHz, CDCl3): δ = 163.0, 150.2, 140.6, 135.8, 135.5, 132.8, 132.4, 130.4, 130.3, 128.2, 128.2, 102.5, 87.0, 85.1, 71.5, 63.7, 41.4, 27.1, 19.5 ppm.
5.2.3 2’,3’-‐dideoxyuridine
200 mg (0.43 mmol) 5’-‐TBDPS protected 2’-‐deoxyuridine and 199mg (1.63 mmol) DMAP (4-‐
(dimethylamino)-‐pyridine) were dissolved in 5 ml acetonitrile. 89 µl (0.64 mmol) PhOCSCl (o-‐
phenylchlorothionoformate) was added drop wise. The reaction mixture was stirred at room temperature and monitored by TLC. After 2.5 h acetonitrile was removed and the reaction residue was resolved in CH2Cl2 following an extraction against 5% citric acid. The organic layer was dried over MgSO4 and the solvent was removed. After two times of co-‐evaporation with toluene the sample was solved in 3 ml toluene. Next 14 mg (0.086 mmol) AIBN (2,2’-‐azobis(2-‐methylpropionitrile)) and 346 µl (1.29 mmol) Bu3SnH (tributylstannane) were dissolved in toluene and heated to 85°C. The solution was added drop wise to this solution. The reaction mixture was refluxed for 3 h. Afterwards toluene was removed and the reaction sample was filtered over a short column (3 cm; displaced with 1% KF) using ethyl acetate. The filtrate was concentrated and resolved in THF for further chemical reaction. 136 µl (0.47 mmol) TBAF solution was added. After complete turnover monitored by TLC the reaction mixture was purified by flash chromatography (solvent: CH2Cl2 and MeOH; stepwise gradient 0-‐5% MeOH) to afford 2’,3’-‐
dideoxyuridine (0.15 mmol, 35% yield).
1H-‐NMR (400 MHz, CDCl3): δ = 7.86 (d, 3J = 8.1 Hz, 1H, H-‐6), 5.86 (dd, 3J = 3.5, 6.6 Hz, 1H, H-‐1’), 5.50 (d, 3J = 8.1 Hz, 1H, H-‐5), 4.01 – 3.95 (m, 1H, H-‐4’), 3.73 (dd, 3J = 2.9, 2J = 12.2, 1H, H-‐5’), 3.50 (dd, 3J = 3.7, 2J = 12.2, 1H, H-‐5’), 2.23 (dddd, 3J = 6.8, 8.5, 9.8 Hz, 2J = 13.5 Hz, 1H, H-‐2’), 1.90 (ddd, 3J = 3.9,7.8 Hz, 2J = 13.2 Hz, 1H, H-‐2’), 1.83 – 1.74 ppm (m, 2H, H-‐3’).
13C NMR (101 MHz, CDCl3): δ = 164.7, 150.6, 140.9, 101.1, 86.3, 82.0, 62.4, 32.7, 24.5 ppm.
5.2.4 5-‐Iodo-‐2’,3’-‐dideoxyuridine
100 mg (0.47 mmol) 2’,3’-‐dideoxyuridine was dissolved in MeOH. 33 µl (0.66 mmol) ICl (iodine monochloride) was added. The reaction was monitored by TLC. The reaction mixture was stirred at room temperature over night. After an addition of in total 1.9 equivalents of ICl the reaction mixture was neutralized with 10% NH3 solution. Afterwards the excess of iodine was destroyed by adding 0.5 M NaHSO3 solution until the solutions was discolored. The solvent was removed by the rotation evaporator.
The residue was purified by MPLC using ethyl acetate and methanol as solvent to afford 5-‐Iodo-‐2’,3’-‐
dideoxyuridine (0.28 mmol, 59% yield).
1H-‐NMR (400 MHz, CDCl3): δ = 8.51 (s, 1H, H-‐6), 5.97 (dd, 3J = 2.8, 6.6 Hz, 1H, H-‐1’), 4.14 (qd, J = 2.6, 5.9, 1H, H-‐4’), 3.95 (dd, 3J = 2.4, 2J = 12.2, 1H, H-‐5’), 3.66 (dd, 3J = 3.0, 2J = 12.2, 1H, H-‐5’), 2.23 (dt, 3J = 8.3, 2J = 15.7, 1H, H-‐2’), 2.11 – 2.00 (m, 1H, H-‐2’), 1.99 – 1.89 ppm (m, 2H, H-‐3’).
13C NMR (101 MHz, CDCl3): δ = 145.7, 87.0, 82.4, 62.3, 33.3, 24.3ppm.
5.2.5 5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’,3’-‐dideoxyuridine (ddT
alkyne)
80 mg (0.24 mmol) 5-‐iodo-‐2’,3’-‐dideoxyuridine, 90 mg (0.70 mmol) 1,4-‐diethynylbenzene and 9 mg (0.047 mmol) CuI (copper(I) iodine) were dissolved in DMF. The solution was degassed before 27 mg (0.024 mmol) Pd(PPh3)4 (tetrakis(triphenylphosphine)palladium (0)) catalyst was added. After a second time of degassing 66 µl (0.47 mmol) previously distilled Et3N was added. The reaction mixture was stirred at room temperature and monitored by TLC (CH2Cl2 and 10% MeOH). After 2 h the solvent was removed and the product was purified by flash chromatography (solvent: CH2Cl2 and MeOH; stepwise gradient 0-‐
2% MeOH) to yield 5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’,3’-‐dideoxyuridine (0.17 mmol, 71% yield).
1H-‐NMR (400 MHz, MeOD): δ = 8.60 (s, 1H, H-‐6), 7.50 – 7.38 (m, 4H, ArH), 6.04 (dd, 3J = 2.9, 6.6 Hz, 1H, H-‐
1’), 4.18 (ddt, 3J = 3.0, 6.2, 9.1 Hz, 1H, H-‐4’), 3.96 (dd, 3J = 2.8, 12.3 Hz, 1H, H-‐5’), 3.71 (dd, 3J = 3.2, 12.3 Hz, 1H, H-‐5’), 3.47 (s, 1H, C≡CH), 2.43 (m, 1H, H-‐2’), 2.19 – 2.09 (m, 1H, H-‐2’), 2.07 – 1.92 ppm (m, 2H, H-‐3’).
13C-‐NMR (101 MHz, MeOD): δ = 164.0, 150.8, 145.2, 132.4, 132.1, 124.4, 123.2, 99.4, 93.0, 87.9, 83.7, 83.7, 83.6, 80.1, 62.8, 59.3, 33.9, 25.0 ppm.
HRMS: m/z: calcd for [C19H15N2O4-‐]: 335.1026; found: 335.1034.
5.2.6 5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’,3’-‐dideoxycytidine (ddC
alkyne)
For the conversion of the modified dideoxyuridine to the corresponding modified dideoxycytidine 14 mg (42 µmol) of the modified dideoxyuridine and 8.7 mg (71 µmol) DMAP (4-‐(dimethylamino)-‐pyridine) were dissolved in acetonitrile. Under ice bath cooling 9,8 µl (71 µmol) Et3N and subsequently 20.4 mg (67 µmol) TPSCl (triisopropyl benzenesulfonyl chloride) were added. The reaction mixture was stirred for 2 h at 0°C. Next 1 ml of a mixture of 33% NH4OH / CH3CN = 1 / 1 was added to the reaction mixture. After 1.5 h stirring at 0°C the reaction mixture was allowed to warm up to room temperature and stirring was continued for another 1.5 h. The reaction mixture was diluted with CH2Cl2 and extracted against 1M aqueous KHSO4 solution. After three times extraction of the aqueous phase with CH2Cl2 the organic layer were combined, dried over MgSO4, and concentrated. The desired cytidine derivative (23 µmol, 57%
yield) was obtained following purification by flash chromatography (solvent: ethyl acetate and MeOH;
stepwise gradient 0-‐5% MeOH).
1H-‐NMR (600 MHz, DMSO-‐d6): δ = 8.57 (s, 1H, H-‐6), 7.70 (br s, 1H, NH2), 7.62 – 7.58 (m, 2H, ArH), 7.53 – 7.47 (m, 2H, ArH), 7.03 (br s, 1H, NH2), 5.90 (dd, 3J = 2.7, 6.7 Hz, 1H, H-‐1’), 5.23 (t, 3J = 5.0 Hz, 1H, OH), 4.32 (s, 1H, C≡CH), 4.11 – 4.05 (m, 1H, H-‐4’), 3.79 (ddd, 3J = 3.0, 5.4 Hz, 2J = 12.0 Hz, 1H, H-‐5’), 3.61 – 3.55 (m, 1H, H-‐5’), 2.36 – 2.27 (m, 1H, H-‐2’), 1.98 – 1.91 (m, 1H, H-‐2’), 1.87 – 1.79 ppm (m, 2H, H-‐3’).
13C NMR (151 MHz, DMSO-‐d6): δ = 163.9, 163.8, 153.4, 145.6, 132.5, 131.8, 131.4, 131.3, 123.2, 121.3, 86.5, 84.3, 84.2, 83.1, 82.6, 82.2, 61.3, 33.0, 23.8 ppm.
HRMS: m/z: calcd for [C19H16N3O3-‐]: 334.1186; found: 334.1193.
5.2.7 5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’,3’-‐dideoxyuridine-‐5’-‐triphophate (ddT
alkyneTP)
6 mg (17.7 µmol) modified dideoxyuridine and 5.7 mg (26.8 µmol) proton sponge were dissolved in previously distilled trimethylphosphate. The addition of 2 µl (21.4 µmol) POCl3 was carried out under ice bad cooling. The reaction was monitored by reverse phase TLC in isopropanol / water/ ammonia = 3 / 1 / 1. After 2 h stirring 173 µl (83.2 µmol) pyrophosphate and 47 µl (173 µmol) tributylamine were added fast and simultaneously. The solution was stirred for 2 min and afterwards quenched by adding 5 ml 0.1 M TEAB buffer. The reaction mixture was extracted three times against ethyl acetate. The aqueous solution was further purified by ion exchange chromatography (see chapter 5.1.2). The remaining sample was desalted by RP-‐MPLC using 0.05 M TEAA buffer and acetonitrile (see chapter 5.1.2) to afford the modified dideoxyuridine-‐5’-‐triphosphate (5.2 µmol, 29% yield).1H-‐NMR (400 MHz, D2O): δ = 8.10 (s, 1H, H-‐6), 7.59 (d, 3J = 8.3 Hz, 2H, ArH), 7.53 (d, 3J = 8.3 Hz, 2H, ArH), 6.10 (dd, 3J = 3.6, 6.9 Hz, 1H, H-‐1’), 4.43 – 4.35 (m, 1H, H-‐4’), 4.30 – 4.23 (m, 1H, H-‐5’), 4.18 – 4.10 (m, 1H, H-‐5’), 3.58 (s, 1H, C≡CH), 3.14 (m, 24H, NEt3), 2.53 – 2.42 (m, 1H, H-‐2’), 2.21 – 2.09 (m, 2H, H-‐2’, H-‐3’), 2.02 – 1.92 (m, 1H, H-‐3’), 1.90 (s, 3H, acetic acid), 1.25 ppm (t, 3J = 5.9 Hz, 36H, NEt3).
31P NMR (162 MHz, D2O): δ = -‐10.84 (d, 2J = 19.7 Hz,1P, Pγ), -‐11.25 (d, 2J = 19.9 Hz, 1P, Pα), -‐23.31 ppm (t, 2J
= 19.5 Hz 1P, Pβ).
HRMS: m/z: calcd for [C19H18N2O13P3-‐]: 575.0016; found: 575.0022.
5.2.8 5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’,3’-‐dideoxycytidine-‐5’-‐triphosphate (ddC
alkyneTP)
10 mg (23.8 µmol) modified dideoxycytidine and 9.6 mg (44.7 µmol) proton sponge were dissolved in previously distilled trimethylphosphate. The addition of 3.3 µl (35.7 µmol) POCl3 was carried out under ice bad cooling. The reaction was monitored by reverse phase TLC in isopropanol / water/ ammonia = 3 / 1 / 1. After 2 h stirring 293 µl (143 µmol) pyrophosphate and 79 µl (297 µmol) tributylamine were added fast and simultaneously. The solution was stirred for 2 min and afterwards quenched by adding 5 ml 0.1 M TEAB buffer. The reaction mixture was extracted three times against ethyl acetate. The aqueous solution was further purified by ion exchange chromatography (see chapter 5.1.2). The remaining sample was desalted by RP-‐MPLC using 0.05 M TEAA buffer and acetonitrile (see chapter 5.1.2) to afford the modified dideoxycytidine-‐5’-‐triphosphate (0.4 µmol, 1.7% yield).
1H-‐NMR (400 MHz, D2O): δ = 8.22 (s, 1H, H-‐6), 7.60 (d, 3J = 8.3 Hz, 2H, ArH), 7.54 (d, 3J = 8.3 Hz, 2H, ArH), 6.08 (dd, 3J = 2.6, 6.8 Hz, 1H, H-‐1’), 4.45 – 4.35 (m, 1H, H-‐4’), 4.34 – 4.25 (m, 1H, H-‐5’), 4.23 – 4.13 (m, 1H, H-‐5’), 3.59 (s, 1H, C≡CH), 3.17 (q, 3J = 7.3 Hz, 24H, NEt3), 2.54 – 2.42 (m, 1H, H-‐2’), 2.17 – 2.05 (m, 2H, H-‐2’, H-‐3’), 1.89 – 1.87 (m, 1H, H-‐3’), 1.25 ppm (t, 3J = 7.2 Hz, 36H, NEt3).
31P NMR (162 MHz, D2O): δ = -‐8.66 – -‐9.33 (m,1P, Pγ), -‐10.65 – -‐11.08 (m, 1P, Pα), -‐21.82 – -‐22.50 ppm (m, 1P, Pβ)
HRMS: m/z: calcd for [C19H19N3O12P3-‐]: 574.0176; found: 574.0176.
5.2.9 3’-‐O-‐acetyl-‐5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’-‐deoxyuridine (dT
alkyne)
200 mg (0.51 mmol) 5-‐iodo-‐2’,3’-‐dideoxyuridine, 127 mg (1.0 mmol) 1,4-‐diethynylbenzene and 19 mg (0.1 mmol) CuI (copper(I) iodine) were dissolved in DMF. The solution was degassed before 5.8 mg (5.1 µmol) Pd(PPh3)4 (tetrakis(triphenylphosphine)palladium (0)) catalyst was added. After a second time of
degassing 140 µl (1.0 mmol) previously distilled Et3N was added. The reaction was stirred at room temperature and monitored by TLC (ethyl acetate / petrol ether = 3 / 1). After 5 h the reaction mixture was extracted against saturated NaHCO3 solution. The aqueous solution was washed several times with diethyl ether. The combined organic layers were dried over MgSO4. Next the solvent was removed and the product was purified by flash chromatography (solvent: ethyl acetate and petrol ether; stepwise gradient 2/1 – 1/1 – 1/2 – 1/3) to yield 5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’-‐deoxyuridine (0.13 mmol, 27% yield).
1H-‐NMR (400 MHz, MeOD): δ = 8.45 (s, 1H, H-‐6), 7.54 – 7.40 (m, 4H, ArH), 6.28 (dd, 3J = 5.9, 8.2 Hz, 1H, H-‐
1’), 5.33 (dt, 3J = 2.0, 6.1 Hz, 1H, H-‐3’), 4.14 (q, 3J = 2.7 Hz, 1H, H-‐4’), 3.91 – 3.78 (m, 2H, H-‐5’), 3.62 (s, 1H, C≡CH), 2.52 – 2.31 (m, 2H, H-‐2’), 2.10 ppm (s, 3H, OAc).
13C NMR (101 MHz, MeOD): δ = 172.2, 151.2, 145.1, 133.0, 132.5, 124.7, 123.8, 101,4, 100.6, 93.4, 87.1, 87.0, 83.9, 83.8, 80.6, 76.4, 62.8, 39.1, 20.9 ppm.
HRMS: m/z: calcd for [C21H17N2O6-‐]: 393.1081; found: 393.1088.
5.2.10 3’-‐O-‐acetyl-‐5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’-‐deoxycytidine (dC
alkyne)
For the conversion of the modified deoxyuridine to the corresponding modified deoxycytidine 64 mg (0.16 mmol) of the modified deoxyuridine and 39.7 mg (0.33 mmol) DMAP (4-‐(dimethylamino)-‐pyridine) were dissolved in acetonitrile. Under ice bath cooling 45 µl (0.33 mmol) Et3N and subsequently 93.4 mg (0.31 mmol) TPSCl (triisopropyl benzenesulfonyl chloride) were added. The reaction mixture was stirred for 1.5 h at 0°C. Next 1 ml of a mixture of 33% NH4OH / CH3CN = 1 / 1 was added to the reaction mixture.
After 1.5 h stirring at 0°C the reaction mixture was allowed to warm up to room temperature and stirring was continued for another 1.5 h. The reaction mixture was diluted with CH2Cl2 and extracted against 1M aqueous KHSO4 solution. After three times extraction of the aqueous phase with CH2Cl2 the organic layers were combined, dried over MgSO4, and concentrated. The desired cytidine derivative (95 µmol, 59%
yield) was obtained following purification by flash chromatography (solvent: ethyl acetate and MeOH;
stepwise gradient 0-‐4% MeOH).
1H-‐NMR (600 MHz, DMSO-‐d6): δ = 8.32 (s, 1H, H-‐6), 7.85 (br s, 1H, NH2), 7.68 – 7.59 (m, 2H, ArH), 7.55 – 7.48 (m, 2H, ArH), 7.16 (br s, 1H, NH2), 6.17 (dd, 3J = 5.8, 8.3 Hz, 1H, H-‐1’), 5.35 – 5.24 (m, 1H, OH), 5.21 (dt,
3J = 2.0, 6.2 Hz, 1H, H-‐3’), 4.34 (s, 1H, C≡CH), 4.08 – 4.01 (m, 1H, H-‐4’), 3.73 – 3.62 (m, 2H, H-‐5’), 2.33 (ddd,
3J = 1.9, 5.7 Hz, 2J = 14.0 Hz, 1H, H-‐2’), 2.22 (ddd, 3J = 6.3, 8.3 Hz, 2J = 14.3 Hz, 1H, H-‐2’), 2.07 ppm (s, 3H, OAc).
13C NMR (151 MHz, DMSO-‐d6): δ = 177.7, 170.1, 163.7, 153.3, 145.1, 135.8, 131.8, 131.4, 123.0, 121.5, 93.4, 89.7, 85.5, 85.0, 83.7, 83.1, 82.7, 74.6, 61.2, 37.9, 20.9 ppm.
HRMS: m/z: calcd for [C21H18N3O5-‐]: 392.1241; found: 392.1247.
5.2.11 5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’-‐deoxyuridine-‐5’-‐triphosphate (dT
alkyneTP)
14 mg (35.5 µmol) modified deoxyuridine and 11.4 mg (53.2 µmol) proton sponge were dissolved in previously distilled trimethylphosphate. The addition of 3.9 µl (42.6 µmol) POCl3 was carried out under ice bad cooling. The reaction was monitored by reverse phase TLC in isopropanol / water/ ammonia = 3 / 1 / 1. After 1 h stirring 355 µl (1.78 mmol) pyrophosphate and 94 µl (355 µmol) tributylamine were added fast and simultaneously. The solution was stirred for 15 min and afterwards quenched by adding 5 ml 0.1 M TEAB buffer. The reaction mixture was extracted three times against ethyl acetate. The samplewas concentrated and resolved in 10 ml water. To remove the 3’ protecting group 20 ml 33% ammonia solution was added. The reaction mixture was stirred for 1 h and was afterwards concentrated. The aqueous solution was further purified by ion exchange chromatography (see chapter 5.1.2). The remaining sample was desalted by RP-‐MPLC using 0.05 M TEAA buffer and acetonitrile (see chapter 5.1.2) to afford the modified deoxyuridine-‐5’-‐triphosphate (3.6 µmol, 10% yield).
1H-‐NMR (400 MHz, D2O): δ = 8.20 (s, 1H, H-‐6), 7.56 (d, 3J = 8.0 Hz, 2H, ArH), 7.50 (d, 3J = 8.0 Hz, 2H, ArH), 6.28 (t, 3J = 6.7 Hz, 1H, H-‐1’), 4.67 – 4.62 (m, 1H, H-‐3’), 4.27 – 4.16 (m, 3H, H-‐4’, H-‐5’), 3.58 (s, 1H, C≡CH), 3.16 (q, 3J = 7.3 Hz, 24H, NEt3), 2.53 – 2.42 (m, 1H, H-‐2’), 2.43 – 2.37 (m, 2H, H-‐2’), 1.24 ppm (t, 3J = 7.3 Hz, 36H, NEt3).
31P NMR (162 MHz, D2O): δ = -‐7.78 – -‐8.25 (m, 1P, Pγ), -‐11.23 (d, 2J = 20.0 Hz, 1P, Pα), -‐22.04 – -‐22.50 ppm (m, 1P, Pβ).
HRMS: m/z: calcd for [C19H18N2O14P3-‐]: 590.9965; found: 590.9972.
5.2.12 5-‐(2-‐(4-‐ethynylphenyl)ethynyl)-‐2’-‐deoxycytidine-‐5’-‐triphosphate (dC
alkyneTP)
15 mg (38 µmol) modified deoxycytidine and 12.3 mg (57.2 µmol) proton sponge were dissolved in previously distilled trimethylphosphate. The addition of 4.2 µl (45.8 µmol) POCl3 was carried out under ice bad cooling. The reaction was monitored by reverse phase TLC in isopropanol / water/ ammonia = 3 / 1 / 1. After 1 h stirring 381 µl (191 µmol) pyrophosphate and 101 µl (380 µmol) tributylamine were added fast and simultaneously. The solution was stirred for 15 min and afterwards quenched by adding 5 ml 0.1 M TEAB buffer. The reaction mixture was extracted three times against ethyl acetate. The sample was concentrated and resolved in 10 ml water. To remove the 3’ protecting group 20 ml 33% ammonia solution was added. The reaction mixture was stirred for 1 h and was afterwards concentrated. The aqueous solution was further purified by ion exchange chromatography (see chapter 5.1.2). The remaining sample was desalted by RP-‐MPLC using 0.05 M TEAA buffer and acetonitrile (see chapter 5.1.2) to afford the modified deoxycytidine-‐5’-‐triphosphate (4.5 µmol, 12% yield).1H-‐NMR (400 MHz, D2O): δ = 8.22 (s, 1H, H-‐6), 7.64 (d, 3J = 7.0 Hz, 2H, ArH), 7.57 (d, 3J = 7.0 Hz, 2H, ArH), 6.31 (t, 3J = 6.4 Hz, 1H, H-‐1’), 4.69 – 4.60 (m, 1H, H-‐3’), 4.27 (br s, 3H, H-‐4’, H-‐5’), 3.65 (s, 1H, C≡CH), 3.23 (q, 3J = 7.1 Hz, 6H, NEt3), 2.57 – 2.46 (m, 1H, H-‐2’), 2.42 – 2.30 (m, 2H, H-‐2’), 1.31 ppm (t, 3J = 7.2 Hz, 9H, NEt3).
31P NMR (162 MHz, D2O): δ = -‐8.40 – -‐10.63 (m, 1P, Pγ), -‐10.62 – 12.00 (m, 1P, Pα), -‐20.43 – -‐23.90 ppm (m, 1P, Pβ).
HRMS: m/z: calcd for [C19H19N3O13P3-‐]: 590.0125; found: 590.0143.