Thymidine Phosphorothioates with (R) or (S) Configurations at Phosphorus
FULL PAPER
residue was dissolved in dichloromethane (1.0 mL) and poured into
petroleum ether (100 mL), and the precipitates were collected by
filtration and dried to give the target compound 5 (66.5 mg, 88%
saturated aqueous solution (50 mL). The aqueous layer was ex-
tracted with ethyl acetate (100 mL, 2×), and the organic layers were
collected and washed with brine (50 mL). After drying, concentra-
tion of the organic solution afforded a residual material, which was
subjected to silica gel column chromatography with elution with a
1:9 mixture of methanol and dichloromethane followed by a
1:30:10 mixture of methanol, ethyl acetate, and hexane to give 10
(389 mg, 62% yield): TLC Rf = 0.60 (methanol/dichloromethane,
1
yield): TLC Rf = 0.21 (methanol/dichloromethane, 1:9). H NMR
([D6]DMSO): δ = 1.17 (t, J = 7.2 Hz, 9 H), 1.37 (s, 3 H), 2.25–2.38
(m, 2 H), 3.07 (q, J = 7.3 Hz, 6 H), 3.15 (dd, J = 2.7 and 10.2 Hz,
1 H), 3.27–3.29 (m, 1 H), 3.74 (s, 6 H), 4.11 (d, J = 2.4 Hz, 1 H),
5.00–5.03 (m, 1 H), 6.18 (dd, J = 5.9 and 8.3 Hz, 1 H), 6.88–6.90
(m, 4 H), 7.22–7.41 (m, 9 H), 7.48 (s, 1 H), 9.49 (br. s, 1 H), 11.32
1
1:9), Rf = 0.38 (methanol/ethyl acetate/hexane, 1:30:10). H NMR:
(s, 1 H) ppm. 13C NMR: δ = 8.62, 11.6, 39.8 (d, J = 4.1 Hz), 45.7, δ = 1.46 (s, 3 H), 1.95 (s, 3 H), 2.11 (s, 3 H), 2.16–2.25 (m, 1 H),
52.9 (d, J = 6.5 Hz), 55.2, 63.9, 76.5 (d, J = 4.9 Hz), 84.7, 85.1 (d,
J = 5.8 Hz), 87.0, 111.1, 113.3, 127.0, 128.2, 130.1, 135.4, 135.6,
135.8, 144.4, 150.4, 158.7, 163.8 ppm. 31P NMR ([D6]DMSO): δ =
54.3 ppm. HRMS (ESI–): m/z calcd. for C32H34N2O9PS [M–H]–:
653.1728; found 653.2584.
2.37–2.50 (m, 2 H), 2.58 (dd, J = 5.6 and 14.0 Hz, 1 H), 3.45 (br.,
2 H), 3.69 (d, J = 14.0 Hz, 3 H), 3.80 (s, 6 H), 4.18–4.23 (m, 2 H),
4.28–4.38 (m, 2 H), 5.28–5.30 (m, 1 H), 5.35–5.39 (m, 1 H), 6.34–
6.41 (m, 2 H), 6.85 (d, J = 8.8 Hz, 4 H), 7.23–7.70 (m, 11 H), 8.65
(s, 1 H), 8.73 (s, 1 H) ppm. 13C NMR: δ = 11.8, 12.5, 20.9, 37.2,
39.0 (d, J = 5.8 Hz), 54.8 (d, J = 4.9 Hz), 55.3, 63.3, 67.7 (d, J =
5.8 Hz), 74.4, 79.7 (d, J = 3.3 Hz), 82.7 (d, J = 9.1 Hz), 84.4, 84.6
(d, J = 6.6 Hz), 84.8, 87.3, 111.7 (2 C), 113.4, 127.2, 128.1, 130.1,
135.0 (2 C), 135.1, 135.2, 144.1, 150.5, 150.6, 158.8, 163.8 (2 C),
170.6 ppm. 31P NMR: δ = 68.0 ppm. HRMS (ESI+): m/z calcd. for
C44H49N4O14PSNa [M+Na]+: 943.2596; found 943.3270.
Synthesis of 3Ј-O-(Acetyl)thymidin-5Ј-yl Allyl (Sp)-5Ј-O-(p,pЈ-Di-
methoxytrityl)thymidin-3Ј-yl Phosphorothioate (7): A solution of
DEAD in toluene (40%, 0.35 mL, 0.77 mmol) was added at 0 °C
to a solution of triphenylphosphane (202 mg, 0.77 mmol) in THF
(20 mL). After the mixture had been stirred at 0 °C for 30 min, 4
(460 mg, 0.59 mmol) and the nucleoside 6 (335 mg, 1.18 mmol)
were added at the same temperature and the reaction mixture was
stirred at 60 °C for 3 h. According to 31P NMR analysis, this reac-
tion mixture included not only the desired compound 13 but also
Preparation of Allyl (Sp)-5Ј-O-(p,pЈ-Dimethoxytrityl)thymidin-3Ј-yl
Thymidin-5Ј-yl Phosphorothioate (8) by Deacetylation of (Sp)-3Ј-O-
(Acetyl)thymidin-5Ј-yl Allyl 5Ј-O-(p,pЈ-Dimethoxytrityl)thymidin-3Ј-
yl Phosphorothioate (7): Sodium methoxide in methanol solution
(1.0 , 55.0 µL, 55.0 µmol) was slowly added at 0 °C to a solution
of 7 (103 mg, 109 µmol) in methanol (4.00 mL). The reaction mix-
ture was warmed up to 25 °C and stirred at this temperature for
4 h, diluted with ethyl acetate (20 mL), and washed with an ammo-
nium chloride-saturated aqueous solution (5.0 mL). The aqueous
layer was extracted with ethyl acetate (20 mL, 2×), and the com-
bined organic layers were washed with brine (10 mL), dried with
Na2SO4 (ca. 30 g), and concentrated to give an oily product. This
crude product was purified by silica gel column chromatography
with a 1:9 mixture of methanol and dichloromethane as eluent to
afford 8 (86.8 mg, 88% yield): TLC Rf = 0.53 (methanol/dichloro-
a considerable amount (ca. 42%) of the by-product 13 (R1
=
CH2=CHCH2), showing a 31P NMR signal at δ = 26.5 ppm. The
reaction mixture was diluted with ethyl acetate (50 mL) and washed
with an ammonium chloride-saturated aqueous solution (20 mL).
The aqueous layer was extracted with ethyl acetate (50 mL, 2×),
and the collected organic solutions were washed with brine
(20 mL), dried with Na2SO4 (ca. 50 g), and concentrated. The re-
sulting residue was subjected to column chromatography on silica
gel with a 1:9 mixture of methanol and dichloromethane, followed
by a 1:30:10 mixture of methanol, ethyl acetate, and hexane as el-
uents to afford 7 (301 mg, 54% yield): TLC Rf = 0.57 (methanol/
dichloromethane, 1:9), Rf = 0.43 (methanol/ethyl acetate/hexane,
1
1:30:10). H NMR: δ = 1.47 (s, 3 H), 1.94 (s, 3 H), 2.10–2.19 (m, methane, 1:9). 1H NMR: δ = 1.48 (s, 3 H), 1.92 (s, 3 H), 2.10–2.17
4 H), 2.35–2.44 (m, 2 H), 2.60 (dd, J = 5.6 and 12.8 Hz, 1 H), 3.46
(m, 1 H), 2.35–2.45 (m, 2 H), 2.60 (dd, J = 5.6 and 13.2 Hz, 1 H),
(d, J = 2.4 Hz, 2 H), 3.80 (s, 6 H), 4.13–4.35 (m, 4 H), 4.60 (dd, J 3.46 (d, J = 2.4 Hz, 2 H), 3.80 (s, 6 H), 4.08–4.32 (m, 4 H), 4.46
= 5.6 and 10.8 Hz, 2 H), 5.21–5.41 (m, 4 H), 5.88–5.97 (m, 1 H),
6.36 (dd, J = 5.6 and 9.2 Hz, 1 H), 6.41 (dd, J = 5.2 and 8.4 Hz, 1
H), 6.85 (d, J = 8.8 Hz, 4 H), 7.22–7.40 (m, 10 H), 7.58 (s, 1 H),
(t, J = 2.8 Hz, 1 H), 4.59 (dd, J = 5.6 and 10.8 Hz, 2 H), 5.26–5.40
(m, 3 H), 5.87–5.97 (m, 1 H), 6.32 (t, J = 6.4 Hz, 1 H), 6.41 (dd, J
= 5.2 and 8.8 Hz, 1 H), 6.85 (d, J = 9.2 Hz, 4 H), 7.22–7.40 (m, 10
8.81 (s, 1 H), 8.85 (s, 1 H) ppm. 13C NMR: δ = 11.8, 12.6, 21.0, H), 7.56 (d, J = 1.2 Hz, 1 H), 9.36 (s, 1 H), 9.53 (s, 1 H) ppm. 13C
37.1, 39.1 (d, J = 5.8 Hz), 55.3, 63.3, 67.5 (d, J = 5.8 Hz), 69.4 (d,
J = 4.9 Hz), 74.4, 79.6 (d, J = 4.1 Hz), 82.6 (d, J = 9.1 Hz), 84.4,
84.6, 84.7 (d, J = 5.8 Hz), 87.3, 111.6, 111.9, 113.4, 119.5, 127.2,
NMR: δ = 11.8, 12.6, 39.0 (d, J = 4.9 Hz), 40.1, 55.3, 63.4, 67.6
(d, J = 6.6 Hz), 69.3 (d, J = 4.1 Hz), 71.3, 79.6 (d, J = 4.9 Hz),
84.5, 84.6 (d, J = 8.2 Hz), 84.7 (d, J = 4.1 Hz), 85.1, 87.3, 111.5,
128.1, 130.1, 131.7 (d, J = 7.4 Hz), 134.8, 135.1 (2 C), 135.2, 144.1, 111.9, 113.4, 119.3, 127.2, 128.1, 130.1, 131.8 (d, J = 6.9 Hz), 135.1,
150.4, 150.5, 158.8, 163.6, 163.7, 170.4 ppm. 31P NMR: δ =
67.0 ppm. HRMS (ESI+): m/z calcd. for C46H51N4O14PSNa
[M+Na]+: 969.2752; found 969.3589.
135.2, 135.6, 144.2, 150.7, 150.9, 158.8, 164.0, 164.2 ppm. 31P
NMR:
65.7 ppm. HRMS (ESI+): m/z calcd. for
C44H49N4O13PSNa [M+Na]+: 927.2647; found 927.3500.
δ
=
Synthesis of 3Ј-O-(Acetyl)thymidin-5Ј-yl (Rp)-5Ј-O-(p,pЈ-Dimeth- Preparation
of (Rp)-5Ј-O-(p,pЈ-Dimethoxytrityl)thymidin-3Ј-yl
Methyl Thymidin-5Ј-yl Phosphorothioate (11) by Deacetylation of
toluene solution (40%, 0.40 mL, 0.88 mmol) was added at 0 °C (Rp)-3Ј-O-(Acetyl)thymidin-5Ј-yl 5Ј-O-(p,pЈ-Dimethoxytrityl)thymi-
oxytrityl)thymidin-3Ј-yl Methyl Phosphorothioate (10): A DEAD/
with stirring to
a
solution of triphenylphosphane (231 mg,
din-3Ј-yl Methyl Phosphorothioate (10): Deacetylation of 10, afford-
ing 11, was carried out in a manner similar to that described above
for preparation of 8 from 7. From 19.0 mg (21.0 µmol) of 10,
15.5 mg of 11 (84% yield) was produced: TLC Rf = 0.54 (methanol/
0.88 mmol) in THF (23 mL). Stirring at this temperature was con-
tinued for 30 min. To this mixture, at the same temperature, were
added the phosphorothioate 5 (510 mg, 0.68 mmol) and the nucleo-
side 6 (387 mg, 1.36 mmol). The reaction mixture was stirred at
60 °C for 3 h. 31P NMR analysis showed that this reaction mixture
included the desired compound 10 and a considerable amount (ca.
33%) of the by-product 13 (R1 = CH3), showing a 31P NMR signal
at δ = 27.1 ppm. Ethyl acetate (100 mL) was added to this reaction
mixture, and the mixture was washed with an ammonium chloride-
1
dichloromethane, 1:9). H NMR: δ = 1.50 (s, 3 H), 1.92 (s, 3 H),
2.16–2.23 (m, 1 H), 2.31–2.38 (m, 1 H), 2.44 (ddd, J = 3.4, 5.8 and
9.8 Hz, 1 H), 2.68 (dd, J = 4.9 and 13.6 Hz, 1 H), 3.40 (dd, J = 3.4
and 10.7 Hz, 1 H), 3.69 (d, J = 6.8 Hz, 3 H), 3.79 (s, 6 H), 4.13–
4.15 (m, 1 H), 4.24–4.35 (m, 3 H), 4.52–4.55 (m, 1 H), 5.26–5.29
(m, 1 H), 6.29–6.33 (m, 2 H), 6.85 (d, J = 8.8 Hz, 4 H), 7.22–7.41
Eur. J. Org. Chem. 2006, 3834–3844
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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