CARACTERIZACIÓN PROCESOS DE EXPLOTACIÓN DE ASFALTITA

As showed in figure 6.1, to replace the 4-oxygen with selenium, we activated the partially protected thymidine derivative (1) at the 4-position via the formation of triazolide 2 in two reactions (87% yield). We also developed di(2-cyanoethyl) diselenide reagent for the Se introduction.(112) After reducing the diselenide reagent to the corresponding

selenol with NaBH4, the protected Se functionality was introduced to the 4-position of 2 by displacing the 4-triazolyl activating group, achieving the simultaneous Se incorporation and protection for the Se-T intermediate (3). Due to the undesired removal of the 2-cyanoethyl group on Se while removing the 3’-TMS group by the fluoride or base treatment, we developed a mild condition to remove the 3’-TMS group by using 10% triethylamine in methano (81% yield). Alternatively, the 3’-TMS group could also firstly be removed by the fluoride treatment, followed by the Se incorporation (85% yield in two reactions). Finally, Se-thymidine derivative 3 was converted to the corresponding phosphoramidite 4 in 92% yield. The detailed experiment and characterization for each compound were listed in the following.

O OH DMTr-O NH N O O O DMTr-O N N O N N N TMS-O O OH DMTr-O N N Se O NC 1 2 3 5 a b d c O P DMTr-O N N Se O NC 4 N _O-CH 2CH2CN e P O -_O O O O P O -_O Oligonucleotide-3' f Oligonucleotide 5'- NH N Se O

Scheme 6.1: Synthesis of 4-Se-thymidine phosphoramidite (4) and oligonucleotides containing 4-Se-T (5). (a) TMS-Im and CH3CN; (b) triazole-POCl3-Et3N; (c) (NCCH2CH2Se)2/NaBH4, EtOH; (d) 10% Et3N in MeOH; (e) 2-cyanoethyl N,N- diisopropylchlorophosphor-amidite and N,N-diisopropylethylamine in CH2Cl2; (f) the solid phase synthesis.

4-(1,2,4-triazol-1-yl)-5’-O-(4,4’-dimethoxytriphenylmethyl)-3’-O-trimethylsilylthymidine(2).

Phosphorus oxychloride (0.92 mL, 10 mmol) was added to a solution of 1,2,4-triazole (2.77 g, 40 mmol) in dry acetonitrile (32 mL) under argon, and the reaction was stirred for 45 min at room temperature. Dry triethylamine (11.2 mL, 79 mmol) was then injected into the reaction and the reaction was stirred for another 45 min. Without exposing to air, the suspension was filtered directly into a round bottle flask (100 mL) containing compound 1 (1.82 g, 3.35 mmol) reacted with 1-(trimethylsilyl)imidazole (0.98 ml, 6.67

mmol) in dry acetonitrile (15 mL) under argon. The reaction mixture was allowed to run for 1 hr at room temperature. The formation of N4-triazolide 2 was revealed by a

fluorescent spot on TLC (5% MeOH in CH2Cl2, product Rf = 0.25). After the reaction was

complete, the reaction mixture was poured into water (150 mL), and extracted with ethylacetate (3x 80 mL). The combined organic layer was washed with saturated NaCl (100 mL) and dried over MgSO4 before evaporation. After loading the crude product on a silica gel column equilibrated with hexanes/CH2Cl2 (1:1), the column was eluted with hexanes/CH2Cl2 (1:1), pure CH2Cl2, and then with a methanol/CH2Cl2 gradient (0, 0.5% and 1% methanol in CH2Cl2). After solvent evaporation and dry on high vacuum, the pure compound (2) was obtained as a white foam product (1.94 g, 87% yield). 1H-NMR

(300 MHz, CDCl3) δ: 0.61 [s, 9H, (CH3)3Si], 1.98 (s, 3H, CH3), 2.26-2.35 and 2.63-2.71 (2x m, 2H, H-2’), 3.30-3.60 (2x dd, J = 2.7, 11.1 Hz, 2H, H-5’), 3.79 (s, 6H, 2xCH3O), 4.07-4.10 (m, 1H, H-3’), 4.46-4.52 (q, J = 4.8 Hz, 1H, H-4’), 6.30 (t, J = 6.3 Hz, 1H, H-1’), 6.84 (dd, J = 1.5, 9.0 Hz, 4H, aromatic), 7.22-7.42 (m, 9H, aromatic), 8.09 (s, 1H, H-6), 8.42 (s, 1H, H-Im), 9.29 (s, 1H, H-Im). 13C-NMR (100 MHz, CDCl3) δ: -0.06 (CH3-Si), 16.54 (CH3), 42.20 (C-2’), 55.26 (OCH3), 62.12 (C-5’), 70.58 (C-3’), 86.88, 135.31,

135.33, 144.23, 158.16 (Ar-C), 87.16 (C-4’), 87.65 (C-1’), 105.71 (C-5), 113.29, 113.31, 127.14, 128.02, 128.13, 130.06, 130.07 (Ar-CH), 146.70 (C-6), 153.36 (CH-Im), 153.95 (C-2), 158.79 (C-4). HRMS (ESI-TOF): molecular formula, C36H41N5O6Si; [M+Na]+: 690.2724 (calc. 690.2718).

4-(2-cyanoethyl)seleno-5’-O-(4,4’-dimethoxytriphenylmethyl) thymidine (3).

The NaBH4 suspension (250 mg NaBH4 in 5 mL of EtOH) was injected into a flask containing di(2-cyanoethyl) diselenide [(NCCH2CH2Se)2, 0.45 mL, d= 1.8 g/mL, 3.0 mmol] and ethanol (8 mL) on an ice bath and under argon. After injection of the NaBH4 suspension for 15 minutes, the ice bath was removed. The yellow color of the diselenide disappeared in approximately 15 min, giving an almost colorless solution of sodium selenide (NCCH2CH2SeNa). The solution of 2 (1.34 g, 2 mmol) in THF (4 mL) was injected to the solution of sodium selenide. After the selenium incorporation was completed in 15 min. (monitored on TLC, 5% MeOH in CH2Cl2, product Rf = 0.58), water

(100 mL) was added to the reaction flask. The solution was adjusted to pH 7-8 using CH3COOH (10%), and was then extracted with ethyl acetate (3 x 100 mL). The organic phases were combined, washed with NaCl (sat., 100 mL), dried over MgSO4 (s) for 30 min., and evaporated to minimum volume under reduced pressure. Deprotecting solution (5 mL, 10% triethylamine in methanol) was added to the crude product, and the reaction was stirred for 4 hours to completely remove the 3’-TMS group (monitored on TLC, 5% MeOH in CH2Cl2, product Rf = 0.46). The deprotecting solution was

evaporated under reduced pressure. The crude product was then dissolved in methylene chloride (5 mL) and purified on a silica gel column equilibrated with hexanes/methylene chloride (1:1). The column was eluded with a gradient of methylene

chloride (CH2Cl2, 0.5%, 1%, and 2% MeOH in CH2Cl2, 500 mL each). After solvent evaporation and dry on high vacuum, the pure compound (3) was obtained as a white

foamy product (1.07 g, 81% yield). 1H-NMR (400 MHz, CDCl3) δ: 1.62 (s, 3H, CH3), 2.27-2.35 and 2.71-2.77 (2x m, 2H, H-2’), 3.00 (dd, J= 6.5 and 6.7 Hz, 2H, CH2-CN), 3.32 (br, 1H, OH), 3.37-3.41 (m+t, J= 6.7 Hz, 3H, 1H-5’, CH2-Se), 3.52 (dd, J5’-5’ = 10.6 Hz, J5’-4’ = 3.0 Hz, 1H, 1H-5’), 3.81 (s, 6H, 2x OCH3), 4.17-4.22 (m, 1H, H-3’), 4.57-4.62 (m, 1H, H-4’), 6.34 (t, J1’-2’ = 6.3 Hz, 1H, H-1’), 6.85 (dd, J = 8.8 Hz, 4H, aromatic), 7.23- 7.42 (m, 9H, aromatic), 7.91 (s, 1H, H-6). 13C-NMR (100 MHz, CDCl3) δ: 14.30 (CH3), 18.73 (CH2-CN), 20.53 (CH2-CH2-CN), 42.17 (C-2’), 55.29 (OCH3), 63.18 (C-5’), 71.71 (C-3’), 86.90, 135.37, 135.41, 144.36, 158.73 (Ar-C), 86.73 (C-4’), 87.01 (C-1’), 113.31, 127.17, 128.03, 128.10, 130.07 (Ar-CH), 114.38 (C-5), 119.00 (CN), 137.56 (C-6), 153.35 (C-2), 177.20 (C-4). HRMS (MALDI-TOF): molecular formula, C34H35N3O6Se; [M+Na]+: 684.1583 (calc. 684.1583).

Alternative approach: Et3N•3HF (7.5 mL, 1 M in THF, 7.5 mmol) was added to the

solution of 2 (4.98 g, 7.47 mmol) in THF (10 mL). The reaction was completed after

stirring under argon for 15 minutes (monitored by TLC). The solvent of the reaction was removed by rotary evaporation and the crude intermediate was then dried on high vacuum. The NaBH4 suspension (933 mg NaBH4 in 10 mL of EtOH) was injected into a flask containing di(2-cyanoethyl) diselenide [(NCCH2CH2Se)2, 1.66 mL, d= 1.8 g/mL, 11.2 mmol, 3 eq.] and ethanol (10 mL) on an ice bath and under argon. After adding the NaBH4 suspension and stirring it for 15 minutes, the ice bath was removed. The brown- yellow color of the diselenide disappeared in approximately 15 min, giving an almost colorless solution of sodium selenide (NCCH2CH2SeNa). The sodium selenide solution

was then injected into the flask containing the crude intermediate under argon. After the selenium incorporation reaction was completed in 45~60 minutes (monitored on TLC, 5% MeOH in CH2Cl2, product Rf = 0.58), CH2Cl2 (100 mL) was added to the reaction flask, followed by addition of NaCl solution (100 mL, saturated) and addition of CH3COOH (5%) to adjust the pH to 7-8. After removing the organic phase, the aqueous phase was extracted again with CH2Cl2 (2 x 100 mL). The organic phases were combined, washed with NaCl (sat., 50 mL), dried over MgSO4 (s) for 25 min., and evaporated to minimum volume under reduced pressure. The crude product was dissolved in CH2Cl2 (5 mL) and purified on a silica gel column equilibrated with CH2Cl2 and eluded with a gradient (CH2Cl2, 0.5%, 1.0%, 2.0% MeOH in CH2Cl2, 400 mL each). After solvent evaporation and dry on high vacuum, the pure compound (3) was obtained

as a white foamy product (4.20 g, 85% yield).

4-(2-cyanoethyl)seleno-5’-O-(4,4’-dimethoxytriphenylmethyl)-thymidine3’-O-(2-cyanoet- hyl) diisopropylamino phosphoramidite (4).

To the flask (25 mL) containing 3 (453 mg, 0.68 mmol) under argon, dry methylene

chloride (2.5 mL), N,N-diisopropylethylamine (0.17 mL, 1.03 mmol, 1.5 eq.), and 2- cyanoethyl N,N-diisopropyl-chlorophosphoramidite (195 mg, 0.83 mmol, 1.2 eq.) were added sequentially. The reaction mixture was stirred at -10 °C in an ice-salt bath under argon for 10 minutes, followed by removal of the bath. The reaction was completed in 45 minutes at room temperature (indicated by TLC, 5% MeOH in CH2Cl2, product Rf =

0.63 and 0.68), generating a mixture of two diastereisomers. The reaction was then quenched with NaHCO3 (2 mL, sat.), stirred for 5 min, and the product was then extracted with CH2Cl2 (3 x 5 mL). The combined organic layer was washed with NaCl

(10 mL, sat.) and dried over MgSO4 (s) for 20 min, followed by filtration. The solvent was then evaporated under reduced pressure and the crude product was re-dissolved in CH2Cl2 (2 mL). This solution was added drop-wise to petroleum ether (100 mL) under vigorous stirring, generating a white precipitate. The petroleum ether solution was decanted. The crude product was re-dissolved again in CH2Cl2 (2 mL) and then loaded into an Al2O3 column (neutral) that was equilibrated with CH2Cl2/Hexanes (1:1). The column was eluded with a gradient of methylene chloride and ethyl acetate [CH2Cl2 to CH2Cl2/EtOAc (7:3)]. After solvent evaporation and dry over high vacuum, the pure 4 (538 mg) was obtained as a white foamy product (92% yield). 1H-NMR (400 MHz, CDCl3, two sets of signals from a mixture of two diastereomers) δ: 0.85-1.20 (m, 24H, 8x CH3-ipr), 1.55 and 1.57 (2x s, 6H, 2x CH3), 2.30-2.38 and 2.70-2.82 (2x m, 4H, 2x H- 2’), 2.34 and 2.64 (2x t, J= 6.4 Hz, 4H, 2x O-CH2-CH2-CN), 3.00-3.04 (m, 4H, 2x Se- CH2-CH2-CN), 3.32-3.44 (m, 6H, 2x H-5’, 2x Se-CH2), 3.52-3.64 (m, 8H, 4x CH-ipr, 2x O-CH2-CH2-CN), 3.73-3.84 (m, 2H, 2x H-5’), 3.82 and 3.83 (2x s, 12H, 4x OCH3), 4.18- 4.24 (m, 2H, 2x H-3’), 4.63-4.72 (m, 2H, 2x H-4’), 6.28 and 6.32 (2x t, J1’-2’ = 6.3 Hz, 2H, 2x H-1’), 6.83-3.88 (m, 8H, aromatic), 7.27-7.43 (m, 18H, aromatic), 7.82 and 7.94 (2x s, 2H, 2x H-6). 13_{C-NMR (100 MHz, CDCl}

3, two sets of signals from a mixture of two diastereomers) δ: 14.18 (CH3), 18.78 (Se-CH2-CH2-CN), 20.15 and 20.23 (O-CH2-CH2- CN), 20.43 (Se-CH2-CH2-CN), 24.48, 24.52, 24.55, 24.59, 24.67 (CH3-ipr), 40.85, 40.89 and 41.17, 41.19 (C-2’), 43.17, 43.25, 43.29, 43.38 (CH-ipr), 55.27 and 55.31 (OCH3), 58.15 and 58.34 (O-CH2-CH2-CN), 62.30 and 62.65 (C-5’), 72.31 and 72.87 (C-3’), 85.68 and 85.86 (C-4’), 86.74 and 86.76 (C-1’), 86.87, 135.28, 135.31, 144.25, 158.77 (Ar-C), 113.27, 127.18, 127.23, 128.00, 128.18, 128.26, 130.13, 130.19, 130.21 (Ar-CH),

114.04 and 114.08 (C-5), 117.38 and 117.53 (O-CH2-CH2-CN), 119.03 (Se-CH2-CH2- CN), 137.41 and 137.44 (C-6), 153.10 (C-2), 176.98 (C-4). HRMS (MALDI-TOF): molecular formula, C43H52N5O7PSe; [M+H]+: 862.2836 (calc. 862.2842).