10.1002/cbic.201800399
ChemBioChem
FULL PAPER
TLC Rf = 0.4 (10% MeOH in CH2Cl2). 1H-NMR (400 MHz, CDCl3): δ 7.98
(s, 1H), 7.39-7.37 (m, 2H), 7.34-7.25 (m, 7H), 6.87-6.84 (m, 4H), 5.96 (d,
J = 3.2 Hz, 1H). 4.48-4.32 (m, 2H), 4.20-4.18 (m, 1H), 3.78 (s, 6H), 3.56-
3.53 (m, 1H), 3.47-3.44 (m, 1H), 2.59 (d, J = 17.2 Hz, 1H), 2.46 (d, J =
17.2 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ162.4, 158.8, 158.8, 150.8,
144.2, 139.3, 135.1, 135.0, 130.1, 130.1, 128.2, 128.1, 127.4, 127.4,
5.72-5.69 (m, 1H), 4.80 (m, 3H). HRMS (ESI-TOF) [M+NH4]+ = 599.1830
(calc. 599.1778). Chemical formula: C31H23N3O9.
5-cyanouridine 12.21 Compound 11 (5.81 g, 10 mmol) was dissolved in
50 mL of 7N NH3/MeOH at room temperature, and stirred for 12 h.
Solvent was removed by repeat evaporation with readdition of MeOH to
remove all ammonia. The residue was purified by silica gel
chromatography to give compound 12 (1.80 g, 6.69 mmol, 67% yield) as
a white solid. TLC Rf = 0.4 (25% MeOH in CH2Cl2). 1H NMR (400 MHz,
MeOD) δ 9.02 (s, 1H), 5.83 (d, J = 2.8 Hz, 1H), 4.21-4.16 (m, 2H), 4.07-
4.03 (m, 1H), 3.95 (dd, J = 2.4 Hz, 12.4 Hz, 1H), 3.77 (dd, J = 2.4 Hz,
12.4 Hz, 1H). HRMS (ESI-TOF) [M+Na]+ = 292.0564 (calc. 292.0546).
Chemical formula: C10H11N3O6.
113.4, 105.2, 87.2, 83.9, 55.2, 15.0. HRMS (ESI-TOF) [M+Na]+
608.2024 (calc. 608.2009). Chemical formula: C32H31N3O8.
=
1-(2’-O-tert-butyldimethylsilyl-5’-O-4,4’-dimethoxytrityl-beta-D-
ribofuranosyl)-5-cyanomethyluridine 7. To a solution of compound 6 (200
mg, 0.342 mmol) in THF (3.5 mL) was added pyridine (81 µL) and AgNO3
(92.75 mg, 0.546 mmol). The reaction was stirred at room temperature in
the dark for 30 min. Then, TBDMSCl (90.28 mg, 0.6 mmol) was added
and the resulting solution was stirred at room temperature in the dark for
another 12 h. After removing the solvent and the residue was purified by
silica gel column chromatography to give compound 7 (100 mg, 42%) as
a white solid. TLC Rf = 0.7 (5% MeOH in CH2Cl2). 1H-NMR (400 MHz,
CDCl3): δ 7.98 (s, 1H), 7.39-7.25 (m, 9H), 6.89-6.86 (m, 4H), 6.0 (d, J =
3.2 Hz, 1H), 4.47-4.44 (m, 1H), 4.37-4.35 (m, 1H), 3.81 (s, 6H), 3.57-3.36
(m, 2H), 2.53 (d, J = 17.2 Hz, 1H), 2.28 (d, J = 17.2 Hz, 1H), 0.94 (s, 9H),
0.18 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 159.0, 158.9, 149.8, 144.1,
138.7, 134.9, 134.7, 130.2, 130.1, 129.1, 128.1, 127.5, 116.1, 113.5,
113.3, 113.2, 105.3, 88.7, 87.3, 83.8, 62.8, 55.2, 25.6, 18.0, -4.7, -5.2.
HRMS (ESI-TOF) [M+H]+ = 700.2960 (calc. 700.3054). Chemical formula:
C38H45N3O8Si.
1-(2’-O-tert-butyldimethylsilyl-3’,5’-O-di-tert-butylsilylene-beta-D-
ribofuranosyl)-5-cyanouridine 13. Compound 12 (1.40 g, 5.2 mmol) was
suspended in DMF (20 mL) and cooled to 0 oC. Then di-tert-butylsilyl
bis(trifluoromethanesulfonate) (2.4 mL, 6.24 mmol) was added dropwise
and the resulting solution was stirred at 0 oC for 1 h. Subsequently,
imidazole (2.04 g, 26 mmol) was added and the mixture was warmed to
room temperature at which TBDMS-Cl (1.1 g, 6.24 mmol) was added.
The reaction was allowed to proceed at 60 oC for 2 h. Then, the reaction
was quenched with water (50 mL) and extracted with DCM (3 x 50 mL).
The organic layer was dried by anhydrous sodium sulfate, filtered and
evaporated under reduced pressure. The residue was purified by silica
gel chromatography to give compound 13 (2.40 g, 4.59 mmol, 88% yield)
as a white solid. TLC Rf = 0.7 (30% ethyl acetate in hexane). 1H NMR
(400MHz, CDCl3) δ 7.91 (s, 1H), 5.64 (s, 1H), 4.58-4.53 (m, 1H), 4.30-
4.23 (m, 2H), 4.03-3.98 (m, 1H), 3.78-3.73 (m, 1H), 1.06 (s, 9H), 1.03 (s,
9H), 0.94 (s, 9H), 0.19 (s, 3H), 0.15 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 159.52, 159.47, 148.2, 147.5, 112.8, 94.1, 90.1, 75.3, 75.1, 22.6, 20.3,
18.1, -4.1, -4.3, -5.0, -5.2. HRMS (ESI-TOF) [M+H]+ = 524.2641 (calc.
524.2612). Chemical formula: C24H41N3O6Si2.
1-[2’-O-tert-butyldimethylsilyl-3’-O-(2-cyanoethyl-N,N-
diisopropylamino)phosphoramidite-5’-O-(4,4’-dimethoxytrityl-beta-D-
ribofuranosyl)]-5-cyanomethyluridine 8. To a solution of compound 7 (70
mg, 0.1 mmol) in THF (1 mL) was added DIPEA (0.14 mL, 0.8 mmol) and
stirred at room temperature for 30 min. (i-Pr)2NPClOCH2CH2CN (0.05 mL,
0.2 mmol) was added and the mixture was stirred at room temperature
for 12 h. The reaction was quenched with water and extracted with DCM.
The organic layer was dried by anhydrous sodium sulfate, filtered and
evaporated under reduced pressure. The residue was purified by flash
silica gel chromatography to give compound 8 (73 mg, 0.08 mmol, 80%
yield) as a white solid. TLC Rf = 0.7 (5% MeOH in CH2Cl2). 1H NMR
(400MHz, CDCl3) δ 8.06-7.94 (m, 1H), 7.44-7.26 (m, 9H), 6.89-6.85 (m,
4H), 6.18-5.94 (m, 1H), 4.52-4.44 (m, 1H), 4.39-4.29 (m, 2H), 4.29-4.14
(m, 1H), 3.98-3.90 (m, 1H), 3.57-3.50 (m, 1H), 3.44-3.32 (m, 1H), 2.75-
2.54 (m, 3H), 2.46-2.40 (m, 1H), 1.17 (s, 12H), 0.91 (s, 9H), 0.16 (s, 6H).
1-(2’-O-tert-butyldimethylsilyl-beta-D-ribofuranosyl)-5-cyanouridine 14.
To a solution of compound 13 (2.10 g, 4.00 mmol) in tetrahydrofuran (20
mL) at 0 oC was added a solution of hydrogen fluoride-pyridine complex
(hydrogen fluoride ~70%, pyridine ~30%) (0.4 mL) in pyridine (2 mL).
After 1 h at 0 oC the reaction was complete and pyridine (15 mL) was
added. The reaction mixture was washed with saturated NaHCO3, dried
over Na2SO4 and evaporated. The residue was purified by silica gel
chromatography to give compound 14 (1.10 g, 2.87 mmol, 75% yield) as
a white solid. TLC Rf = 0.4 (10% MeOH in CH2Cl2). 1H NMR (400MHz,
CD3OD) δ 9.12 (s, 1H), 5.76 (s, 1H), 4.27 (m, 1H), 4.15-4.04 (m, 2H),
4.01-3.97 (m, 1H), 3.81-3.77 (m, 1H), 0.94 (s, 9H), 0.15 (d, 6H). 13C NMR
(100 MHz, CDCl3) δ 160.61, 160.60, 149.7, 149, 4, 149, 1, 113.2, 90.8,
88.3, 76.7, 76.4, 17.6, -6.0. HRMS (ESI-TOF) [M+Na]+ = 406.1411
(calc.406.1410). Chemical formula: C16H25N3O6Si.
31P NMR (CDCl3) δ 149.89, 149.57. HRMS (ESI-TOF) [M+NH4]+
=
900.4235 (calc. 900.4054). Chemical formula: C47H62N5O9PSi.
1-(2’,3’,5’-tri-O-benzoate-beta-D-ribofuranosyl)-5-cyanouridine 11. To a
solution of compound 9 (8.22 g, 60 mmol) in hexamethyldisilazane
(HMDS, 500 mL) was added trimethylsiylchloride (TMSCl, 15.2 mL, 120
mmol). The mixture was stirred at 130 oC for 20 h until the mixture turned
clear. Then, the solution evaporated to remove excess HMDS and
compound 10 was obtained and immediately used without further
purification. At room temperature, to a solution of compound 10 and
2,3,5-tri-O-benzoyl-β-D-ribofuranose (33.26 g, 66 mmol) in 1,2-
dichloroethane (DCE, 500 mL) was added SnCl4 (7.8 mL, 66 mmol)
1-(2’-O-tert-butyldimethylsilyl-5’-O-4,4’-dimethoxytrityl-beta-D-
ribofuranosyl)-5-cyanouridine 15. To a solution of compound 14 (766 mg,
2 mmol) in dry pyridine (10 mL) was added 4,4’-Dimethoxytrityl chloride
(812 mg, 2.4 mmol) under Ar. The resulting solution was stirred at room
temperature for 12 h. The reaction was quenched with methanol (1 mL)
slowly at
0
oC. After 30 min, the reaction was brought to room
and stirred for another
5 min. The reaction mixture was then
temperature and continued for another 2 h. Then, the reaction was
quenched with saturated NaHCO3 aqueous solution (500 mL) at 0 oC and
extracted with DCM (3 x 500 mL). The organic layer was dried by
anhydrous sodium sulfate, filtered and evaporated under reduced
pressure. The residue was purified by silica gel chromatography to give
compound 11 (30 g, 51.64 mmol, 86% yield) as a white solid. TLC Rf =
0.6 (10% MeOH in CH2Cl2). 1H NMR (400MHz, CDCl3) δ 8.14 (s, 1H),
8.10-7.88 (m, 6H), 7.64-7.33 (m, 9H), 6.23 (d, J = 5.2 Hz), 5.86 (m, 1H),
concentrated to dryness under vacuum. The residue was purified by
silica gel chromatography to give compound 15 (1.20 g, 1.75 mmol, 73%
yield) as a white solid. TLC Rf = 0.5 (50% ethyl acetate in hexane). 1H
NMR (400MHz, CDCl3) δ 8.42 (s, 1H), 7.42-7.39 (m, 2H), 7.35-7.24 (m,
7H), 6.89-6.86 (m, 4H), 5.90 (d, J = 3.2 Hz, 1H), 4.50 (m, 1H), 4.43 (m,
1H), 4.22 (m, 1H), 3.80 (s, 6H), 3.58-3.55 (m, 1H), 3.42-3.35 (m, 1H),
0.95 (s, 9H), 0.19 (d, 6H). 13C NMR (100 MHz, CDCl3) δ 159.1, 158.8,
158.7, 148.6, 147,8, 147.68, 147,67, 144.0, 135.1, 134.7, 113.6, 113.4,
111.6, 90.8, 89.8, 89.76, 87.4, 80.3, 70.7, 18.0, -4.6, -5.1. HRMS (ESI-
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