Solution-Phase Synthesis of Phosphorothioate Oligonucleotides
FULL PAPER
Synthesis of 5Ј-OH-dABz-dGiBu-3Ј-O-Lev Thiolatecyanoethyl Phos-
The organic layer was washed with water (2×160 mL), dried with
photriester: The monomer 5Ј-O-DMTr-dABz-3Ј-HP (987 mg, Na2SO4 and evaporated under reduced pressure to obtain 600 mg
1.2 mmol, 1.2 equiv.) was coevaporated twice with anhydrous pyri-
(0.34 mmol, 63%) of an oil of the desired phosphorylated trimer
dine (10 mL). 5Ј-OH-dGiBu-3Ј-O-Lev (435 mg; 1 mmol) was added (90% HPLC purity at 260 nm). MALDI-TOF MS (positive mode,
and two more coevaporations with anhydrous pyridine (10 mL)
were performed. The resulting foam was dissolved in CH2Cl2/pyri-
dine (7 mL, 1:1, v/v) and added to a round-bottomed flask contain-
ing polystyrene-supported acyl chloride (2.1 g, 4.75 mmol) swollen
in CH2Cl2/pyridine (5 mL, 1:1, v/v). The reaction was monitored
by reversed-phase HPLC. After shaking for 2 h 30 at room tem-
perature, N-(2-cyanoethylthio)succinimide (442 mg, 2.4 mmol) was
added. After stirring for 30 min, the resin was filtered off and
washed with CH2Cl2 (50 mL). The organic layer was washed with
deionized water (2×50 mL), dried with Na2SO4 and concentrated
under reduced pressure. The crude dimer was dissolved in cold
(0 °C) CH2Cl2/MeOH (14 mL, 7:3, v/v) and a solution of 10% ben-
zenesulfonic acid in CH2Cl2/MeOH (3.7 mL, 7:3, v/v) was added.
The reaction was stirred for 15 min at 0 °C (TLC monitoring:
CH2Cl2/MeOH 95:5, v/v). Then CH2Cl2 (50 mL), distilled water
(1 mL) and a saturated solution of NaHCO3 (50 mL) were success-
ively added to the solution. After phase separation, the organic
layer was washed with distilled water (3×50 mL), dried with
Na2SO4, and concentrated under vacuum. The residue was precipi-
tated in diethyl ether (100 mL) to give 760 mg (0.824 mmol, 83%)
of the desired product (92% HPLC purity at 260 nm). MALDI-
TOF MS (positive mode, THAP): calcd. 922.89; found 921.62.
Synthesis of 5Ј-O-DMTr-dCBz-dABz-dGiBu-3Ј-OH Thiolatecyano-
ethyl Phosphotriester: The monomer 5Ј-O-DMTr-dCBz-3Ј-HP
(799 mg, 0.99 mmol) was coevaporated twice with anhydrous pyri-
dine (10 mL). 5Ј-OH-dABz-dGiBu-3Ј-O-Lev (760 mg, 0.82 mmol)
was added and two more coevaporations with anhydrous pyridine
(10 mL) were performed. The resulting foam was dissolved in
CH2Cl2/pyridine (5 mL, 1:1, v/v) and added to a round-bottomed
flask containing polystyrene-supported acyl chloride (950 mg,
3.96 mmol) swollen in CH2Cl2/pyridine (3 mL, 1:1, v/v). The reac-
tion was monitored by reversed-phase HPLC. After shaking for
3 h at room temperature, N-(2-cyanoethylthio)succinimide (304 mg,
1.65 mmol) was added. After stirring for 30 min, the resin was fil-
tered off and washed with CH2Cl2 (50 mL). The organic layer was
washed with deionized water (2×50 mL), dried with Na2SO4 and
concentrated under reduced pressure. The residue was dissolved in
pyridine (16 mL) and acetic acid (4 mL). Then Amberlyst A15
NH3+-NH2 (3.1 g, 12.36 mmol) was added. The mixture was
shaken at room temperature for 2 h [TLC monitoring: CH2Cl2/
MeOH (9:1, v/v)] and the resin was filtered off and washed with
CH2Cl2 (100 mL). A cold (4 °C) saturated solution of NaHCO3
(55 mL) was added. After phase separation, the organic layer was
washed with distilled water (2×55 mL), dried with Na2SO4 and
concentrated under reduced pressure. The residue was precipitated
in cooled diethyl ether (400 mL) to obtain 860 mg (0.54 mmol,
66%) of the desired product (79% HPLC purity at 260 nm).
MALDI-TOF MS (positive mode, THAP): calcd. 1588.59; found
1587.27.
THAP): calcd. 1652.56; found 1652.69.
Synthesis of 5Ј-OH-dGiBu-dT-3Ј-O-Lev Thiolatecyanoethyl Phos-
photriester: The monomer 5Ј-O-DMTr-dGiBu-3Ј-HP (640 mg,
0.8 mmol, 1.2 equiv.) was coevaporated twice with anhydrous pyri-
dine (10 mL). 5Ј-OH-dT-3Ј-O-Lev (226 mg, 0.66 mmol) was added
and two more coevaporations with anhydrous pyridine (10 mL)
were performed. The resulting foam was dissolved in CH2Cl2/pyri-
dine (5 mL, 1:1, v/v) and added to a round-bottomed flask contain-
ing the polystyrene-supported acyl chloride (1.4 g, 3.12 mmol)
swollen in CH2Cl2/pyridine (3 mL, 1:1, v/v). After shaking for 3 h
at room temperature, N-(2-cyanoethylthio)succinimide (260 mg,
1.4 mmol) was added. After stirring for 30 min, the resin was fil-
tered off and washed with CH2Cl2 (50 mL). The organic layer was
washed with deionized water (2×50 mL), dried with Na2SO4 and
concentrated under reduced pressure. The residue was precipitated
in diethyl ether (70 mL) to give 580 mg (0.52 mmol, 79%) of the
desired product (92% HPLC purity at 260 nm). MALDI-TOF MS
(positive mode, THAP): calcd. 1112.14; found 1111.49.
The dimer (80 mg, 0.52 mmol) was dissolved in cold CH2Cl2/
MeOH (9 mL, 7:3, v/v) and 10% benzenesulfonic acid in CH2Cl2/
MeOH (2.4 mL, 7:3, v/v) was added at 0 °C. The reaction was
stirred for 15 min at 0 °C [TLC monitoring: CH2Cl2/MeOH (9:1,
v/v)] and then CH2Cl2 (50 mL), distilled water (1 mL) and a satu-
rated solution of NaHCO3 (50 mL) were successively added. After
phase separation, the organic layer was washed with distilled water
(3×50 mL), dried with Na2SO4 and concentrated under vacuum.
The residue was precipitated in diethyl ether (200 mL) to give
386 mg (0.48 mmol, 92%) of the desired product (95% HPLC pu-
rity at 260 nm). MALDI-TOF MS (positive mode, THAP): calcd.
809.77; found 808.93.
Synthesis of 5Ј-OH-dCBz-dGiBu-dT-3Ј-O-Lev Thiolatecyanoethyl
Phosphotriester: The monomer 5Ј-O-DMTr-dCBz-3Ј-HP (455 mg,
0.57 mmol, 1.2 equiv.) was coevaporated twice with anhydrous pyr-
idine (10 mL). 5Ј-OH-dGiBu-dT-3Ј-O-Lev (386 mg, 0.47 mmol) was
added and two more coevaporations with anhydrous pyridine
(10 mL) were performed. The resulting foam was dissolved in
CH2Cl2/pyridine (5 mL, 1:1, v/v) and added to a round-bottomed
flask containing polystyrene-supported acyl chloride (950 mg,
3.96 mmol) swollen in CH2Cl2/pyridine (3 mL, 1:1, v/v). After
shaking for 3 h at room temperature, N-(2-cyanoethylthio)suc-
cinimide (173 mg, 0.94 mmol) was added. After stirring for 30 min,
the resin was filtered off and washed with CH2Cl2 (50 mL). The
organic layer was washed with deionized water (2×50 mL), dried
with Na2SO4 and concentrated under reduced pressure. The residue
was precipitated in diethyl ether (50 mL) to give 650 mg
(0.41 mmol, 88%) of the desired product (85% HPLC purity at
260 nm). MALDI-TOF MS (positive mode, THAP): calcd.
1574.57; found 1571.00.
Synthesis of 5Ј-O-DMTr-dCBz-dABz-dGiBu-3Ј-HP Thiolatecyano-
ethyl Phosphotriester: The trimer 5Ј-O-DMTr-dCBz-dABz-dGiBu-3Ј-
OH (860 mg, 0.54 mmol) was coevaporated with pyridine
(2×6 mL) and dissolved in CH2Cl2/pyridine (10 mL, 1:1, v/v). Sal-
icylchlorophosphite (274 mg, 1.35 mmol) in CH2Cl2/pyridine
(10 mL, 1:1, v/v) was added at 0 °C and the reaction mixture was
stirred for 15 min at 0 °C and then at room temperature for 3 h.
Then water (150 µL) and sulfonyl resin as the triethylammonium
form (4.2 g, 13.5 mmol) were added and the mixture was shaken
The trimer (650 mg, 0.41 mmol) was dissolved in CH2Cl2/MeOH
(7 mL, 7:3, v/v) and 10% benzenesulfonic acid in CH2Cl2/MeOH
(1.9 mL, 7:3, v/v) was added at 0 °C. The reaction was stirred for
30 min at 0 °C [TLC monitoring: CH2Cl2/MeOH (9:1, v/v)] and
then CH2Cl2 (50 mL), distilled water (1 mL) and a saturated solu-
tion of NaHCO3 (50 mL) were successively added. After phase sep-
aration, the organic layer was washed with distilled water
(3×50 mL), dried with Na2SO4 and concentrated under vacuum.
The residue was precipitated in diethyl ether (200 mL) to give
for 1 h. The resin was filtered and washed with CH2Cl2 (50 mL). 440 mg (0.35 mmol, 84%) of the desired product (92% HPLC pu-
Eur. J. Org. Chem. 2006, 436–448
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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