➨
➨ 3 ❶➨ 1 ⠇㛵ࡍࡿᐇ㦂
Scheme 3-1㛵ࡍࡿᐇ㦂
3′,5′-Di-O-(tert-butyldiphenylsilyl)thymidine (3) ࡢྜᡂ
Thymidine (2) (1.21 g, 5.00 mmol) imidazole (1.05 g, 15.4 mmol) ࢆDMF (20 mL)
⁐ゎࡋࠊị෭ୗࠊtert-buthyldiphenylsilyl chloride (3.9 mL, 15.0 mmol) ࢆຍ࠼ࠊᐊ ࡛12 㛫᧠ᢾࡋࡓࠋ⁐፹ࢆῶᅽ␃ཤࡋࠊ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝ␃Ỉ࡛ᢳฟࡋࡓࠋ᭷
ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩ
ࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 5:1~2:1) ࡚⢭〇ࡋࠊ 3′,5′-O-di(tert-butyldiphenylsilyl)thymidine (3) (3.66 g, quant) ࢆ↓Ⰽ⤖ᬗࡋ࡚ᚓࡓࠋ
3,5-Di-O-(tert-butyldiphenylsilyl-1,2-didehydro-1,2-dideoxy-D-ribofuranose (4) ࡢ ྜ ᡂ
᳨ウ
3′,5′-Di-O-(tert-butyldiphenylsilyl)thymidine (3) (7.73 g, 10.8 mmol) (NH4)2SO4 (2.27 g, 17.2 mmol) HMDS (78 mL) ࢆຍ࠼ࠊ150 °C࡛4㛫᧠ᢾࡋࡓࠋ⁐፹ࢆῶᅽ␃ཤࡋࠊ࣊
࢟ࢧ࡛ࣥᕼ㔘ᚋࠊ࣊࢟ࢧࣥỈ࡛ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺࣜ
࣒࡛࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane) ࡚⢭〇ࢆ᳨ウࡋࡓࡇࢁࠊ3,5-di-O-(tert-butyldiphenylsilyl-didehydro- 1,2-dideoxy-D-ribofuranose (4) ࡛ࡣ࡞ࡃࠊ5-O-(tert-butyldiphenylsilyl)methylfuran (5) (2.42 g, 38%) ࡀᚓࡽࢀࡓࠋ
Scheme 3-3㛵ࡍࡿᐇ㦂
5′-O-(tert-Butyldiphenylsilyl)thymidine (6) ࡢྜᡂ
Thymidine (2) (486 mg, 2.01 mmol) DMAP (49.2 mg, 403 μmol) ࢆpyridine (10 mL)
⁐ゎࡋࠊị෭ୗࠊtert-buthyldiphenylsilyl chloride (600 μL, 2.34 mmol) ࢆຍ࠼ࠊᐊ ࡛15 㛫᧠ᢾࡋࡓࠋ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬ሷ
ࣥࣔࢽ࣒࢘Ỉ⁐ᾮ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃
ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 3:1~0:1) ࡚⢭〇 ࡋࠊ5′-O-(tert-butyldiphenylsilyl)thymidine (6) (882 mg, 91%) ࢆ↓Ⰽ⤖ᬗࡋ࡚ᚓࡓࠋ1H NMR (CDCl3) δ 9.25 (s, 1H, NH), 7.67–7.64 (m, 4H, H-Ph), 7.50 (d, J = 0.9 Hz, 1H, H-6), 7.47–7.37 (m, 6H, H-Ph), 6.43 (dd, J = 8.4 Hz, 5.8 Hz, 1H, H-1′), 4.58–4.56 (m, 1H, H-3′), 4.05–4.03 (m, 1H, H-4′), 3.98 (dd, J = 11.8 Hz, 2.7 Hz, 1H, H-5′α), 3.85 (dd, J = 11.8 Hz, 2.7 Hz, 1H, H-5β), 2.43 (ddd, J = 13.8 Hz, 5.8 Hz, 2.3 Hz, 1H, H-2′α), 2.20 (ddd, J = 13.8 Hz, 8.4 Hz, 5.8 Hz,1H, H-2′β), 1.62 (d, J = 0.9 Hz, 3H, CH3 of thymine), 1.08 (s, 9H, C(CH3)3); 13CNMR (CDCl3) δ 163.8, 150.5, 135.5, 135.3, 132.8, 132.3, 130.1, 130.0, 128.0, 127.9, 111.2, 87.1,
46
84.7, 72.3, 64.1, 41.0, 27.0, 19.3, 12.1; MS (DART) m/z 504 [M+Na]+, HRMS(DART) Calcd.
for C26H32N2NaO5Si [M+Na]+: 503.6180. Found: 503.6163.
5-O-(tert-butyldiphenyl)-1,2-didehydro-1,2-dideoxy-3-O-trimethylsilyl-D- ribofuranose (7) ࡢྜᡂ᳨ウ
5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (969 mg, 2.02 mmol) (NH4)2SO4 (536 mg, 4.06 mmol) HMDS (29 mL) ࢆຍ࠼ࠊ150 °C࡛2㛫᧠ᢾࡋࡓࠋ⁐፹ࢆῶᅽ␃ཤࡋࠊ㓑 㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝ␃Ỉ࡛ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲
㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮
(n-hexane) ὶࡋ࡚⢭〇ࢆ᳨ウࡋࡓࡇࢁࠊ5-O-(tert-butyldiphenylsilyl)methylfuran (5) ࡢኚࡀ㉳ࡇࡾࠊ5-O-tert-butyldiphenylsilyl-1,2-didehydro-1,2-dideoxy-3-O- trimethylsilyl-D -ribofuranose (7) ࡢ༢㞳ࡣᅔ㞴࡛࠶ࡗࡓࠋ
Scheme 3-4㛵ࡍࡿᐇ㦂
5-O-(tert-butyldiphenyl)-1,2-didehydro-1,2-dideoxy-D-ribofuranose (8) ࡢྜᡂ᳨ウ 5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (2.41 g, 5.01 mmol) (NH4)2SO4 (1.33 g, 10.0 mmol) HMDS (37 mL) ࢆຍ࠼ࠊ150 °C࡛1.5㛫᧠ᢾࡋࡓࠋ⁐፹ࢆῶᅽ␃ཤᚋࠊ㓑㓟࢚
ࢳࣝ␃Ỉ࡛ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐
፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´K2CO3 (141 mg, 1.02 mmol) ࡢMeOH (40 mL) ᠱ⃮ᾮࢆຍ࠼ࠊ ᐊ ࡛12㛫᧠ᢾࡋࡓࠋ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷
ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩ
ࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 5:1~2:1) ὶࡋ࡚⢭〇ࢆ᳨ウࡋࡓ
ࡇ ࢁ ࠊ5-O-(tert-butyldiphenylsilyl)methylfuran (5) ࡢ ኚ ࡀ ㉳ ࡇ ࡾ ࠊ 5-O-tert-butyldiphenylsilyl-1,2-didehydro-1,2-dideoxy-D-ribofuranose (8) ࡢ༢㞳ࡣᅔ㞴࡛࠶ࡗࡓࠋ
Scheme 3-5㛵ࡍࡿᐇ㦂
5-O-(tert-butyldiphenyl)-1,2-dideoxy-D-ribofuranose (9) ࡢྜᡂ᳨ウ
5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (969 mg, 2.02 mmol) (NH4)2SO4 (535 mg, 4.05 mmol) HMDS (14 mL) ࢆຍ࠼ࠊ150 °C࡛1.5㛫᧠ᢾࡋࡓࠋ⁐፹ࢆῶᅽ␃ཤᚋࠊ 㓑㓟࢚ࢳࣝ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒ࣜ࢘
࡛⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´K2CO3 (8.0 mg, 57.9 μmol) ࡢMeOH (4 mL) ᠱ⃮
ᾮࢆຍ࠼ࠊᐊ ࡛12 㛫᧠ᢾࡋࡓࠋ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝ␃Ỉࢆ⏝࠸࡚ᢳ ฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋ ṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane:EtOAc=50:1~20:1) ࡚⢭〇ࡋࠊ 5-O-tert-butyldiphenyl-1,2-didehydro-1,2-dideoxy-D-ribofuranose (8) ࢆྵࡴΰྜ≀ (621 mg, crude) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋṧ´10% Pd/C (62.7 mg, 10 wt%)ࠊMeOH (4 mL) ࢆ
47
ຍ࠼ࡓᚋࠊỈ⣲⨨ࡋࠊᐊ ࡛12㛫᧠ᢾࡋࡓࠋゐ፹ࢆࢭࣛࢺℐ㐣࡚㝖ཤᚋࠊ⁐፹ࢆ
ῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 50:1~10:1)
࡚⢭〇ࡋࡓࡇࢁࠊ5-O-tert-butyldiphenyl-1,2-dideoxy-D-ribofuranose (9) (224 mg, 36%)
ࢆ 㯤 Ⰽ ࢜ ࣝ ࡋ ࡚ ᚓ ࡓ ࠋ ࡲ ࡓ ࠊ5-O-tert-butyldiphenylsilyl-2,3-dideoxy-1-O-methyl-D -ribofuranose (10) (148 mg, 31%) ࡀ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡽࢀࡓࠋ
Scheme 3-6㛵ࡍࡿᐇ㦂
5-O-(tert-butyldiphenyl)-1,2-dideoxy-D-ribofuranose (9) ࡢྜᡂ
5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (962 mg, 2.00 mmol) (NH4)2SO4 (530 mg, 4.01 mmol) HMDS (15 mL) ࢆຍ࠼ࠊ150 °C࡛1.5㛫᧠ᢾࡋࡓࠋ⁐፹ࢆῶᅽ␃ཤᚋࠊ 㓑㓟࢚ࢳࣝ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒ࣜ࢘
࡛⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´K2CO3 (55.3 mg, 400 μmol) ࡢMeOH (17 mL) ᠱ
⃮ᾮࢆຍ࠼ࠊᐊ ࡛12 㛫᧠ᢾࡋࡓࠋ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝ␃Ỉࢆ⏝࠸࡚
ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋ ࡓࠋṧ´10% Pd/C (32.0 mg, 5 wt%)ࠊi-PrOH (4 mL) ࢆຍ࠼ࡓᚋࠊỈ⣲⨨ࡋࠊᐊ ࡛ 12㛫᧠ᢾࡋࡓࠋゐ፹ࢆࢭࣛࢺℐ㐣࡚㝖ཤᚋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ
࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 10:1~3:1) ࡚⢭〇ࡋࡓࡇࢁࠊ5-O-tert-butyldiphenyl-1,2-dideoxy-D-ribofuranose (9) (499 mg, 70%) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (CDCl3) δ 7.70–7.66 (m, 4H, H-Ph), 7.46–7.37 (m, 6H, H-Ph), 4.43–4.40 (m, 1H, H-3), 3.96 (dd, J = 8.4 Hz, 5.7 Hz, 2H, H-5), 3.85–3.82 (m, 1H, H-4), 3.78 (dd, J = 10.7 Hz, 4.3 Hz, 1H, H-1α), 3.60 (dd, J = 10.7 Hz, 6.4 Hz, 1H, H-1β), 2.20–2.10 (m, 1H, H-2α), 1.93–1.86 (m, 1H, H-2β), 1.07 (s, 9H, C(CH3)3); 13C NMR (CDCl3) δ 135.6, 135.5, 133.2, 133.1, 129.8, 129.8, 127.7, 86.1, 77.2, 74.3, 67.1, 64.8, 34.8, 26.8, 19.2; MS (DART) m/z 379 [M+Na]+, HRMS (DART) Calcd. for C21H28NaO3Si [M+Na]+: 379.1705. Found: 379.1750.
Scheme 3-7㛵ࡍࡿᐇ㦂
5-O-(tert-butyldiphenyl)-1,2-dideoxy-D-ribofuranose (9) ࡢྜᡂ
5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (482 mg, 1.00 mmol) ammonium sulfate (265 mg, 2.01 mmol) HMDS (8 mL) ࢆຍ࠼ࠊ150 °C࡛1.5㛫᧠ᢾࡋࡓࠋ⁐፹ࢆῶᅽ␃ཤᚋࠊ 㓑㓟࢚ࢳࣝ␃Ỉ࡛ᢳฟࡋࡓࠋᚓࡽࢀࡓ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺࣜ࢘
࣒࡛⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋᚓࡽࢀࡓṧ´K2CO3 (28.5 mg, 206 μmol) ࡢMeOH
(15 mL) ᠱ⃮ᾮࢆຍ࠼ࠊᐊ ࡛2㛫᧠ᢾࡋࡓࠋ㓑㓟࢚ࢳࣝ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࠊᚓࡽ
ࢀࡓ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ᚋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋᚓࡽ
ࢀࡓṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/Et2O, 3:1~2:1) ࡛⢭〇ࡋࠊ5-O-tert-butyldiphenyl-1,2-didehydro-1,2-dideoxy-D-ribofuranose (8) ࢆྵࡴΰྜ≀ (340 mg, mixture) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ10% Pd/C (53.6 mg, 15 wt%) ࢆຍ࠼ࠊi-PrOH (10 mL)
48
ࢆୗᚋࠊỈ⣲㞺ᅖẼୗࠊᐊ ࡛24㛫᧠ᢾࡋࡓࠋPd/Cࢆࢭࣛࢺℐ㐣࡚㝖ཤᚋࠊ⁐፹
ࢆῶᅽ␃ཤࡋࡓࠋᚓࡽࢀࡓṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 7:1~2:1) ࡚⢭〇ࡋࠊ5-O-(tert-butyldiphenyl)-1,2-dideoxy-D-ribofuranose (9) (338 mg, 94%)
ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ
Scheme 3-8㛵ࡍࡿᐇ㦂
1,2-Dideoxy-D-ribofuranose (1) ࡢࡢྜᡂ
5-O-(tert-butyldiphenyl)-1,2-dideoxy-D-ribofuranose (9) (215 mg, 603 μmol) ࢆTHF (2.7 mL) ⁐ゎᚋࠊ1 M TBAF in THF (660 μL, 660 μmol) ࢆຍ࠼ࠊᐊ ࡛12㛫᧠ᢾࡋࡓࠋ⁐
፹ࢆῶᅽ␃ཤᚋࠊᚓࡽࢀࡓṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (CH2Cl2/acetone, 5:1~2:1) ࡚⢭〇ࡋࠊ1,2-dideoxy-D-ribofuranose(1)(68.8 mg, 97%) ࢆ㯤Ⰽ࢜ࣝࡋ࡚
ᚓࡓࠋ1H NMR (CD3OD) δ 4.21–4.18 (m, 1H, H-3), 3.97–3.87 (m, 2H, H-1), 3.75–3.72 (m, 1H, H-4), 3.57–3.49 (m, 2H, H-5), 2.18–2.03 (m, 1H, H-2α), 1.88–1.81 (m, 1H, H-2β); 13C NMR (CD3OD) δ 88.1, 73.8, 68.0, 63.6, 35.9; MS (DART) m/z 141 [M+Na]+, HRMS (DART) Calcd. for C5H10NaO3 [M+Na]+: 141.1209. Found: 141.1250.
➨ 3 ❶➨ 2 ⠇㛵ࡍࡿᐇ㦂
Scheme 3-10㛵ࡍࡿᐇ㦂1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (11) ࡢྜᡂ
1,2-Dideoxy-D-ribofuranose (1) (101 mg, 853 μmol) DMTrCl (376 mg, 1.11 mmol) ࡢ
pyridine (3.3 mL) ⁐ゎᾮࢆୗࡋࠊᐊ ࡛12㛫᧠ᢾࡋࡓࠋࢪࢡ࣓ࣟࣟࢱ࡛ࣥᕼ㔘ᚋࠊࢪ
ࢡ࣓ࣟࣟࢱࣥ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺࣜ࢘
࣒ ࡛ ⇱ ࡋ ࠊ ⁐ ፹ ࢆ ῶ ᅽ ␃ ཤ ࡋ ࡓ ࠋ ṧ ´ ࢆ ࢩ ࣜ ࢝ ࢤ ࣝ ࢝ ࣛ ࣒ ࢡ ࣟ ࣐ ࢺ ࢢ ࣛ ࣇ ࣮ (CH2Cl2/acetone, 20:1~10:1) ࡚ ⢭ 〇 ࡋ ࠊ 1,2-dideoxy-5-O-(4,4′-dimethoxytrityl)-D -ribofranose (11) (265 mg, quant) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.8 Hz, 2H, H-Ph), 7.36–7.16 (m, 7H, H-Ph), 6.82 (d, J = 9.2 Hz, 4H, H-Ph), 4.30–4.27 (m, 1H, H-3), 3.99–3.96 (m, 2H, H-1), 3.90–3.86 (m, 1H, H-4), 3.79 (s, 6H, OCH3), 3.23 (dd, J = 9.8 Hz, 4.8 Hz, 1H, H-5α), 3.07 (dd, J = 9.8 Hz, 6.2 Hz, 1H, H-5β), 2.19–2.10 (m, 1H, H-2α), 1.92–1.84 (m, 1H, H-2β); 13CNMR (CDCl3) δ 158.4, 144.8, 136.0, 136.0, 130.0, 128.1, 127.8, 126.8, 113.1, 86.2, 85.0, 74.6, 67.0, 64.5, 55.2, 34.8; MS (EI) m/z 420 (M+)
3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1,2-dideoxy-5-O-(4,4′- dimethoxytrityl)-D-ribofuranose (12) ࡢྜᡂ
1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (11) (292 mg, 694 μmol) ࢆTHF
⁐ ゎ ࡋ ࠊDIPEA (730 μL, 4.18 mmol) ࢆ ຍ ࠼ ࡓ ᚋ ࠊ2-cyanoethyldiisopropylchloro
49
phosphoamidite (250 μL, 1.12 mmol) ࢆୗࡋࠊᐊ ࡛2㛫᧠ᢾࡋࡓࠋࢡ࣒࡛ࣟࣟ࣍ࣝᕼ 㔘ᚋࠊࢡ࣒ࣟࣟ࣍ࣝ㣬Ⅳ㓟Ỉ⣲ࢼࢺ࣒ࣜ࢘Ỉ⁐ᾮࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷ Ỉ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛ
ࢡ ࣟ ࣐ ࢺ ࢢ ࣛ ࣇ ࣮ (n-hexane/EtOAc, 4:1) ࡚ ⢭ 〇 ࡋ ࠊ 3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1,2-dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (12) (451 mg, quant) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ31P NMR (160 MHz, CDCl3) δ 148.3, 148.6.
dRH㐃㐃⤖ᅛ┦ᢸయ (14) ࡢྜᡂ
1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (11) (102 mg, 243 μmol) ࢆpyridine (2.4 mL) ⁐ゎࡋࠊDMAP (30.4 mg, 249 μmol) succinic anhydride (112 mg, 1.12 mmol)
ࢆຍ࠼ࠊᐊ ࡛23㛫᧠ᢾࡋࡓࠋ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝ㣬Ⅳ㓟Ỉ⣲ࢼࢺࣜ
࣒࢘Ỉ⁐ᾮࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ
⁐፹ࢆῶᅽ␃ཤࡋࡓࠋ┿✵⇱ᚋࠊDMF (5.3 mL) ⁐ゎࡋࠊCPGᶞ⬡ (546 mg, 53.5 μmol)
EDC·HCl (41.6 mg, 217 μmol) ࢆຍ࠼ࠊᐊ ࡛72㛫࠺ࡋࡓࠋ࢞ࣛࢫࣇࣝࢱ࣮࡛
ᶞ⬡ྜ≀ࢆ྾ᘬࢁྲྀࡋࠊࣆࣜࢪࣥ (20 mL) ࡛Ὑίࡋࡓࠋ⇱ᚋࠊ100 mM DMAP ⁐ᾮ [DMAP (370 mg, 3.03 mmol), acetic anhydride (3 mL), pyridine (27 mL)] ࢆຍ࠼ࠊᐊ ࡛72
㛫࠺ࡋࡓࠋ࢞ࣛࢫࣇࣝࢱ࣮࡛ᶞ⬡ྜ≀ࢆ྾ᘬࢁྲྀࡋࠊࣆࣜࢪࣥ (20 mL)ࠊ࢚ࢱࣀ
࣮ࣝ (20 mL)ࠊࢭࢺࢽࢺࣜࣝ (20 mL) ࡢ㡰࡛Ὑίࡋࡓࠋ┿✵⇱ᚋࠊdRH㐃⤖ᅛ┦ᢸయ (14) (479 mg) ࢆᚓࡓࠋ14 (6.4 mg) ࢆ࢞ࣛࢫࣇࣝࢱ࣮ධࢀࠊ㐣ሷ⣲㓟/࢚ࢱࣀ࣮ࣝ (3:1)
ࢆຍ࠼ࠊࢁᾮࢆ25 mL࣓ࢫࣇࣛࢫࢥ࡛ཷࡅࡓࠋᾮ㔞ࡀ25 mL࡞ࡿࡲ࡛ΰྜᾮࢆຍ࠼ࡓᚋࠊ
ࢁᾮࡢ྾ගᗘࢆ ᐃࡋࠊ௨ୗࡢᘧࢆ⏝࠸࡚άᛶ್ࢆ⟬ฟࡋࡓ (άᛶ್:54.5 μmol/g)ࠋ άᛶ್ (μmol/g) = Abs498 ∙ Vol (HClO4) ∙ ε498 ∙ Weight (mg)-1
dRHࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ dRHࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ
᰾㓟⮬ືྜᡂᶵ (Applied Biosystems Model 3400) ࢆ⏝࠸࡚1 μmol ࢫࢣ࣮࡛ࣝ⾜ࡗࡓࠋ ኳ↛ࡢ࣍ࢫ࣑࣍ࣟࢲࢺࡣ100 mMࠊdRH࣍ࢫ࣑࣍ࣟࢲࢺయ12ࡣ150 mM࡞ࡿࡼ
࠺MeCN⁐ᾮࢆㄪᩚࡋࠊࡲࡓdRH㐃⤖ᅛ┦ᢸయ (14) ࢆ1 μmolศ (18.3 mg) ࢆ࣒࢝ࣛ
㔞ࡾࡗ࡚⏝ࡋࡓࠋ
ྜᡂᚋࠊᅛ┦ᢸయࢆࢧࣥࣉࣝࢫࢺࢵࢡࢳ࣮ࣗࣈ⛣ࡋࠊࣥࣔࢽỈ/࢚ࢱࣀ࣮ࣝ (3:1) ΰ
ྜ⁐ᾮ (1.2 mL) ࢆຍ࠼ࠊᐊ ࡛12 㛫࠺ࡋ࡚ᅛ┦ᢸయࡽࡢษࡾฟࡋሷᇶ㒊ಖㆤ ᇶࡢ㝖ཤࢆ⾜ࡗࡓࠋୖΎࢆ1.5 mL࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈ⛣ࡋࠊᅛ┦ᢸయࢆ࢚ࢱࣀ࣮ࣝ
/⁛⳦Ỉ (3:1) ⁐ᾮ࡛Ὑίࡋࡓࠋ⁐፹ࢆῶᅽ␃ཤࡋ࡚ᅛᚋࠊTBAF (1 M solution in THF, 1 mL) ࢆຍ࠼ࠊᐊ ࡛12 㛫࠺ࡋ࡚⢾㒊ಖㆤᇶࢆ㝖ཤࡋࡓࠋᛂᾮࢆ100 mM TEAA *1
buffer࡚30 mLᕼ㔘ࡋࠊᖹ⾮ࡋࡓ㏫┦࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮㏻ࡋࠊ࣒࢝ࣛ
྾╔ࡉࡏࡓࠋࡇࡇ࡛ࠊ࣒࢝ࣛࢆ⁛⳦Ỉ (15 mL) ࡛Ὑίࡋ࡚ሷࢆ㝖ཤࡋࠊ50% CH3CN in ⁛
50
⳦Ỉ (4 mL) ࡛⁐ฟࡋࠊ⁐፹ࢆ㐲ᚰῶᅽ␃ཤࡋࡓࠋṧ´ࢆloading solution *2 (180 μL) ⁐
ゎࡋࠊ20% PAGE*3 (500 V, 20 mA) ࡼࡾ┠ⓗࡢ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆศ㞳ࡋࡓࠋ┠ⓗࡢ࢜
ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢࣂࣥࢻࢆษࡾฟࡋࠊ⁐ฟᾮ*4 (20 mL) ᾐࡋ୍࡚ᬌ࠺ࡋࡓࠋࢁᾮࢆ
ᖹ⾮ࡋࡓSep-Pak㏻ࡋࠊ࣒࢝ࣛ྾╔ࡉࡏࡓᚋࠊ⁛⳦Ỉ (15 mL) ࡛Ὑίࡋ࡚ሷࢆ㝖ཤ ᚋࠊ50% CH3CN in ⁛⳦Ỉ (3 mL) ࡛⁐ฟࡋࠊ⁐፹ࢆ㐲ᚰῶᅽ␃ཤࡋࡓࠋ
*1 2 M TEAA buffer [㓑㓟 (114.38 mL) ࢺ࢚ࣜࢳ࣑ࣝࣥ (277.6 mL) ࢆΰྜࡋࠊ㉸⣧Ỉ (Milli-QỈ) ࡛1 Lmess upࡋࠊpH 7.0ㄪᩚࡋࡓ] ࢆ⁛⳦Ỉ࡛20ಸᕼ㔘ࡋࡓ⁐ᾮࠋ
*2 Formamide (9 mL) 1 × TBE buffer*5 (1 mL) ࡢΰྜ⁐ᾮࠋ
*3 40%ࢡ࣑ࣜࣝࢻ (19 : 1) ⁐ᾮ*6 (40 mL)ࠊᒀ⣲ (33.6 g)ࠊ10 × TBE buffer*5 (8 mL) ࢆ ΰྜࡋࠊMilli-QỈ࡛80 mLmess up ࡋࡓᚋࠊammonium peroxodisulfate (55 mg) ࢆຍ
࠼࡚⁐ゎࡋࠊ᭱ᚋTEMED (40 μL) ࢆຍ࠼࡚ࡾΰࡐࡓᚋࠊ1.5 mm ࢫ࣮࣌ࢧ࣮ࢆᣳࢇ࡛
ᅛᐃࡋࡓ 2 ᯛ࢞ࣛࢫ∧ࡢ㛫ὶࡋ㎸ࡳࠊ1 㛫௨ୖ㟼⨨ࡋ࡚ᅛࡉࡏࡓࠋࡲࡓࠊ1 × TBE buffer*5ࢆὋື⦆⾪ᾮࡋ࡚⏝࠸ࡓࠋ
*4 2 M TEAA buffer (1 mL) 0.1 M EDTAỈ⁐ᾮ [EDTA-4Na (1.81 g) ࢆ⁛⳦Ỉ࡛40 mL mess upࡋࡓ⁐ᾮ] (0.2 mL) ࢆΰྜࡋࠊ⁛⳦Ỉ࡛20 mLmess upࡋࡓ⁐ᾮࠋ
*5 10 × TBE buffer [Tris(hydroxymethyl)aminomethane (108 g)ࠊ࣍࢘㓟 (55 g)ࠊEDTA-4Na (7.43 g) ࢆỈ⁐ゎࡋ࡚1 Lࡋࡓ⁐ᾮ] ࢆ␃Ỉ࡛10ಸᕼ㔘ࡋࡓ⁐ᾮࠋ
*6 ࢡ࣑ࣜࣝࢻ (190 g) N,N′-ࣅࢫࢡ࣑ࣜࣝࢻ (10 g) ࢆMilli-QỈ⁐ゎࡋࠊ500 mLࡋࡓ⁐ᾮࠋ
࢜࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢ⢭〇
HPLC ࢆ⏝࠸࡚ୗグ♧ࡍࢢࣛࢪ࢚ࣥࢺࢧࢡ࡛ࣝྜᡂࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆ⢭〇
ࡋࡓࠋ
Aᾮ : 5% ࢭࢺࢽࢺࣜࣝ/0.1 M TEAA ⦆⾪ᾮ (pH 7.0) Bᾮ : 50% ࢭࢺࢽࢺࣜࣝ/0.1 M TEAA ⦆⾪ᾮ (pH 7.0)
⁐ฟ㏿ᗘ 1 mL/min ᐃἼ㛗 260 nm
Time ( min. ) B solution
0 0
5 10
20 30
30 100
35 10
40 0
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UV྾྾ࢫ࣌ࢡࢺࣝࡼࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢOD260 ್ࡢ⟬ฟ
࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆ㉸⣧Ỉ⁐ゎࡋࠊἼ㛗260 nm࡛ࡢ྾ගᗘ (Abs260) ࡀ྾ගᗘィࡢ
᭷ຠ⠊ᅖ (0.2-1.0) ࡞ࡿࡼ࠺100ಸᕼ㔘ࡋࡓࠋග㊰㛗 ( l ) 1 cmࡢ྾ගᗘ ᐃ⏝▼ⱥࢭ
ࣝࢆ⏝࠸࡚ࠊᐊ ࡚Abs260ࢆ ᐃࡋࡓࠋOD260್ࡢィ⟬ࡣ௨ୗࡢᘧࢆ⏝࠸ࡓࠋࡲࡓࠊV ࡣ⁐ᾮ㔞ࢆ♧ࡍࠋ
OD260 (Mε-1mL-1cm-1) = Abs260 (Mε-1) · V -1 (mL) · l -1 (cm-1)
Sequence OD260
5′-r(gua gga gua gug aaa ggc c dRHdRH)-3′ 0.147 5’-r(ggc cuu uca cua cuc cua c dRHdRH)-3′ 0.145 5′-F-r(gua gga gua gug aaa ggc c dRHdRH)-3′ 0.251
࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢε್ཬࡧ⃰ᗘ⟬ฟἲ
N1pN2pN3p···Nn-1pNn࡛⾲ࡉࢀࡿ୍ᮏ㙐࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢࣔࣝ྾ගᗘಀᩘ ε260 ࡢ⟬ฟࡣ௨ୗࡢᘧࢆ⏝࠸ࡓࠋ
ε = 2 { ε (N1pN2) +ε (N2pN3) + ···+ε (Nn-1pNn) } - { ε (N2) +ε (N3) + ···+ε (Nn-1) } ࡇࡇ࡛ࠊε (Nn) ࡣ࠶ࡿ᰾㓟Nnࡢε260ࢆ♧ࡋࠊε (Nn-1pNn) ࡣ࠶ࡿ᰾㓟㔞యNn-1pNnࡢε260
ࢆ♧ࡍࠋ
ࡲࡓࠊ⃰ᗘC ( mol/L )ࡢ⟬ฟࡣࠊḟᘧࢆ⏝࠸ࡓࠋ C = Abs260 ·ε260 -1 · 1-1
࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢᵓ㐀☜ㄆ
࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆ⁛⳦Ỉ (1 mL) ⁐ゎࡉࡏࠊ260 nm࠾ࡅࡿ྾ගᗘࢆ ᐃࡋࠊ
㔞ࢆồࡵࠊ30 pmolศࡢ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢ⁐፹ࢆ㐲ᚰῶᅽ␃ཤࡉࡏࠊ⁛⳦Ỉ (3 mL) ཬ ࡧ࣐ࢺࣜࢵࢡࢫ⁐ᾮ (3 μL) ΰࡋࠊࣉ࣮ࣞࢺୖ࡛ᅛࡋࡓᚋࠊMALDI-TOF/MS ࡛ᵓ㐀
☜ㄆࢆ⾜ࡗࡓࠋ
Sequence Calculated Observed
5′-r(gua gga gua gug aaa ggc c dRHdRH)-3′ 6559.9 6559.8 5′-r(ggc cuu uca cua cuc cua c dRHdRH)-3′ 6250.8 6251.0 5′-F-r(gua gga gua gug aaa ggc c dRHdRH)-3′ 7097.1 7096.6
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➨
➨ 3 ❶➨ 3 ⠇㛵ࡍࡿᐇ㦂
ศゎ㓝⣲⪏ᛶホ౯5′ᮎ➃ࢆࣇࣝ࢜ࣞࢭࣥಟ㣭ࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ (Renilla luciferase ࢆᶆⓗࡋࡓ
siRNAࡢࣥࢳࢭࣥࢫ㙐㓄ิ) ࢆ300 pmolศ1.5 mL࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈྲྀࡾࠊ
ᅛࡉࡏࡓࠋᅛࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ PBS (-) ࡛ᕼ㔘ࡋࡓ࣊ࣅẘ࣍ࢫ࣍ࢪ࢚ࢫࢸ࣮ࣛ
ࢮ (1 unit/mL, 100 μL)ࢆຍ࠼ࠊ37 °C࡛࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋ5 min, 10 min, 15 min, 30 min,
60 minᚋ࠶ࡽࡌࡵ1.5 mL࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈศὀࡋࡓᛂṆᾮ (Ⰽ⣲࣐࣮࢝
࣮, 15 μL) ᛂᾮࢆ5 μLຍ࠼ࠊ100 °C࡛10ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡍࡿࡇ࡛ᛂࢆṆ ࡉࡏࡓࠋ0 min (control) ࡣ㓝⣲ࢆຍ࠼࡞࠸ࡶࡢࡋࡓࠋ
ᚓࡽࢀࡓᛂ⁐ᾮࢆ20%PAGE (Urea)ࠊ500 Vࠊ20 mA࡛ศ㞳ࡋࠊศᯒࡋࡓࠋ
➨ 3 ❶➨ 4 ⠇㛵ࡍࡿᐇ㦂
⇕ⓗᏳᐃᛶホ౯
࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢ┦⿵㙐ྠኈࢆ 600 pmolศࡣࡾࡾࠊ㐲ᚰῶᅽ␃ཤࡋ࡚ᅛᚋࠊ ᐃ⏝⦆⾪ᾮ (10 mM NaH2PO4/Na2HPO4, 100 mM NaCl (pH 7.0)) (200 μL) ⁐ゎࡋࠊ3 μMࡢ⁐ᾮࢆㄪ〇ࡋࡓࠋἛ㦐ࡋࡓ࠾࡛5ศ㛫ຍ⇕ᚋࠊ1㛫௨ୖᨺ⨨ࡋ࡚ࢽ࣮ࣜࣥࢢࡋ ࡓࠋ3ศ㛫⬺Ẽᚋࠊ160 μLࢆᑓ⏝ࢭࣝධࢀࠊ50%⼥ゎ ᗘ (Tm) ᐃ⨨࡛260 nmࡢ྾
ගᗘࡢ ᗘኚࢆ ᐃࡋࡓࠋᚓࡽࢀࡓࢢࣛࣇࡽࠊ୰⥺ἲࡼࡾTmࢆ⟬ฟࡋࡓࠋ
➨ 3 ❶➨ 5 ⠇㛵ࡍࡿᐇ㦂
㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯96 well plateHeLa⣽⬊ࡀ40000 cell/mL࡞ࡿࡼ࠺ᕼ㔘ࡋࡓ⣽⬊ᠱ⃮ᾮࢆ100 μLࡎ ࡘຍ࠼ࠊ24 hᇵ㣴ࡋࡓࠋホ౯⏝࠸ࡿsiRNAࡣࠊTE buffer (pH7.0, 100 mM Nacl) ࡛⁐ゎ ࡋࡓྛࣥࢳࢭࣥࢫ㙐ࢭࣥࢫ㙐ࢆࢽ࣮ࣜࣥࢢࡍࡿࡇࡼࡾㄪ〇ࡋࡓࠋ
⣽⬊ࢆᇵ㣴ࡋ࡚24 㛫ᚋࠊsiRNA (1.75 nM, 17.5 nM, 175 nM, ྛ10 μL) ཬࡧpsi-CHECK (0.1 μg/μL, 1 μL)ࠊtransfast (1.0 μg/μL, 1.5 μL)ࠊOpti-MEM (115 μL) ࢆࡑࢀࡒࢀ1.5 mL࢚
ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈຍ࠼࡚ΰྜࡋࠊ96 well plateࡢྛwellࡢᇵᆅࢆ㝖ཤᚋࠊㄪᩚࡋࡓ ΰྜ⁐ᾮ (35 μL) ࢆຍ࠼ࡓࠋ1 㛫ᇵ㣴ᚋࠊᇵᆅࢆ100 μLຍ࠼ࠊࡉࡽ24 㛫ᇵ㣴ࡋࡓࠋ ᇵᆅࢆ㝖ཤࡋࠊ-80 °C (෭ᗜ) ࡛12 㛫௨ୖಖᏑࡋࡓࠋᐊ ᡠࡋࠊ96 well plateࡢྛ
wellDual glo substrate (25 μL) ࢆຍ࠼ࠊ10ศ㛫ᨺ⨨ࡋࠊྛࢧࣥࣉࣝࢆⓎගィ ⏝96 well plate⛣ࡋࠊFirefly luciferaseࢆ ᐃࡋࡓࠋ ᐃᚋࠊStop and glo substrate (25 μL) ࢆຍ
࠼ࠊ10ศ㛫ᨺ⨨ࡋࠊRenilla luciferaseࢆ ᐃࡋࡓࠋ ᐃ⤖ᯝࡣࠊRenilla luciferaseࡢⓎග㔞
ࢆFirefly luciferaseࡢⓎග㔞࡛ࡾࠊ% of controlࢆ⟬ฟࡋࡓࠋ
53
➨
➨ 3 ❶➨ 6 ⠇㛵ࡍࡿᐇ㦂
⾑ΎෆᏳᐃᛶホ౯
㉸⣧Ỉ࡛⁐ゎࡋࡓྛࣥࢳࢭࣥࢫ㙐ࢭࣥࢫ㙐ࢆࢽ࣮ࣜࣥࢢࡋࡓᚋࠊ㐲ᚰῶᅽ␃ཤࡋ
ᅛᚋࠊD-MEM (50 μL, Phenol red࡞ࡋ) ࢆຍ࠼࡚⁐ゎࡋࠊ300 pmolศ (50 μL) ࢆ1.5 mL
࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈศὀࡋࠊBS (50 μL) ࢆຍ࠼ࠊ37 °C࡛࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋྛ
(1, 3, 6, 12, 24) 㛫ᚋࠊ࠶ࡽࡌࡵ600 μL࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈศὀࡋࡓᛂṆ ᾮ (Ⰽ⣲࣐࣮࣮࢝, 15 μL) ᛂᾮࢆ5 μLຍ࠼ࠊ-80 °C (ᜏ ᛂ⨨) ධࢀ࡚ᛂࢆ
Ṇࡋࡓࠋᚓࡽࢀࡓᛂ⁐ᾮࢆ20% Native PAGEࠊ500 Vࠊ20 mA࡛ศ㞳ࠊGelRed࡛ᰁⰍࡋࠊ
LAS-4000ࢆ⏝࠸࡚⺯ග⏬ീࢆᙳࡋࡓࠋ࡞࠾ࠊBSࢆຍ࠼࡞࠸ࡶࡢࢆࢥࣥࢺ࣮ࣟࣝࡋࡓࠋ
➨ 4 ❶➨ 1 ⠇➨ 1 㡯㛵ࡍࡿᐇ㦂
Scheme 4-2㛵ࡍࡿᐇ㦂3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17) ࡢྜᡂ 1-Deoxy-D-ribofuranose (16) (1.34 g, 11.0 mmol) imidazole (2.74 g, 40.2 mmol) ࢆDMF (15 mL) ⁐ゎࡋࠊị෭ୗࠊ1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (3.47 g, 11.0 mmol)
ࡢDMF (15 mL) ⁐ᾮࢆୗࡋࠊᐊ ࡛18 㛫᧠ᢾࡋࡓࠋ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳ
ࣝ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ
⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 10:1)
࡚⢭ 〇ࡋ ࠊ3,5-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17) (3.07 g, 82%) ࢆ↓Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (CDCl3) δ 4.25–4.18 (m, 2H, H-1α, H-2), 4.08 (dd, J = 10.0 Hz, 4.4 Hz, 1H, H-1β), 4.01 (dd, J = 12.4 Hz, 3.6 Hz, 1H, H-4), 3.89 (dd, J
= 12.4 Hz, 5.6 Hz, 1H, H-3), 3.82–3.77 (m, 2H, H-5), 2.85 (s, 1H, OH), 1.09–0.95 (m, 28H, CH(CH3)2 of TIPDS); 13C NMR (CDCl3) δ 80.7, 73.5, 73.0, 71.0,62.5, 17.4, 17.3, 17.2, 17.0, 17.0, 13.4, 13.1, 12.8, 12.6; MS (ESI) m/z 399 [M+Na]+,HRMS (ESI) Calcd. for C17H36NaO5Si2 [M+Na]+: 399.1999. Found: 399.1961.
Scheme 4-3㛵ࡍࡿᐇ㦂
2-O-Benzyl-1-deoxy-D-ribofuranose (15) ࡢྜᡂ
3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17) (1.49 g, 3.96 mmol) ࢆDMF (20 mL) ⁐ゎࡋࠊị෭ୗࠊ60% NaH (338 mg, 8.45 mmol) ࢆຍ࠼ࡓᚋࠊ benzyl bromide (0.8 mL, 6.74 mmol) ࢆୗࡋࠊᐊ ࡛4.5㛫ᨩᢾࡋࡓࠋị෭ୗࠊ࣓ࢱࣀ
࣮ࣝࢆຍ࠼ࠊ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗 ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆTHF (10 mL) ࡛
⁐ゎࡋࡓᚋࠊTBAF (1 M solution in THF, 15 mL) ࢆຍ࠼ࠊᐊ ࡛19㛫᧠ᢾࡋࡓࠋ⁐፹
ࢆῶᅽ␃ཤࡋࠊṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (CH2Cl2/acetone, 30:1~2:1)
54
࡚⢭〇ᚋࠊ࣊࢟ࢧ࡛ࣥ⤖ᬗࡋࠊ2-O-benzyl-1-deoxy-D-ribofuranose (15) (237 mg, 27%)
ࢆ↓Ⰽ⤖ᬗࡋ࡚ᚓࡓࠋ1H NMR (DMSO-d6) δ 7.37–7.25 (m, 5H, H-Ph), 4.84–4.76 (m, 1H, 3-OH), 4.68–4.61 (m, 2H, OCH2αPh, 5-OH), 4.54–4.46 (m, 1H, OCH2βPh), 3.97–3.82 (m, 3H, H-1, H-3), 3.66–3.64 (m, 1H, H-2), 3.57–3.34 (m, 3H, H-4, H-5); 13C NMR (CDCl3) δ 137.2, 128.7, 128.2, 127.9, 83.5, 78.2, 72.4, 71.3, 70.5, 62.5; MS (ESI) m/z 247 [M+Na]+, HRMS (ESI) Calcd. for C12H16NaO4 [M+Na]+: 247.0946. Found: 247.0927.
Scheme 4-4㛵ࡍࡿᐇ㦂
2-O-Benzyl-1-deoxy-D-ribofuranose (15) ࡢࡢྜᡂ
3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17) (62.1 mg, 165 μmol) ࢆCH3NO2 (1 mL) ⁐ゎࡋࠊbenzaldehyde (14 μL, 137 μmol)ࠊEt3SiH (26 μL, 163 μmol) ࢆຍ࠼ࡓࠋ-20 ºC࡛FeCl3 (1.1 mg, 7 μmol) ࡢCH3NO2 (70 μL) ⁐ᾮࢆୗࡋࠊ ᐊ ࡛41 㛫᧠ᢾᚋࠊEt3N·3HF (100 μL, 613 μmol) ࢆຍ࠼ࠊᐊ ࡛4 㛫᧠ᢾࡋࡓࠋ⁐
፹ࢆῶᅽ␃ཤࡋࠊṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (CH2Cl2/acetone, 50:1~3:1)
࡚⢭〇ࡋࠊ2-O-benzyl-1-deoxy-D-ribofuranose (15) (14.5 mg, 47%) ࢆ↓Ⰽ⤖ᬗࡋ࡚ᚓ ࡓࠋ
➨ 4 ❶➨ 1 ⠇➨ 2 㡯㛵ࡍࡿᐇ㦂
Scheme 4-5㛵ࡍࡿᐇ㦂2-O-Benzyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (19) ࡢྜᡂ
2-O-Benzyl-1-deoxy-D-ribofuranose (15) (218 mg, 972 μmol) ࢆpyridine (5 mL) ࡛⁐ゎ ࡋࠊị෭ୗࠊ4,4′-dimethoxytrityl cloride (401 mg, 1.18 mmol) ࡢpyridine (5 mL) ⁐ᾮࢆୗ
ࡋࡓᚋࠊᐊ ࡛14㛫᧠ᢾࡋࡓࠋࢪࢡ࣓ࣟࣟࢱ࡛ࣥᕼ㔘ᚋࠊࢪࢡ࣓ࣟࣟࢱࣥ␃Ỉࢆ⏝
࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬Ⅳ㓟Ỉ⣲ࢼࢺ࣒ࣜ࢘Ỉ⁐ᾮ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼ ࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮
(CH2Cl2/acetone, 40:1~20:1) ࡚⢭〇ࡋࠊ2-O-benzyl-1-deoxy-5-O- (4,4′-dimethoxytrityl)-D -ribofuranose (19) (499 mg, 65%) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (CDCl3) δ 7.43 (d, J = 7.4 Hz, 2H, CH of DMTr), 7.39–7.17 (m, 7H, CH of DMTr), 6.81 (d, J = 7.4 Hz, 4H, CH of DMTr), 4.63 (dd, J = 24.8 Hz, 11.6 Hz, 2H, OCH2Ph), 4.18–4.06 (m, 3H, H-1α, H-2, H-3), 3.98–3.95 (m, 1H, H-4), 3.90 (dd, J = 9.6 Hz, 4.6 Hz, 1H, H-1β), 3.78 (s, 6H, OCH3), 3.32 (dd, J = 10.1 Hz, 3.4 Hz, 1H, H-5α), 3.11 (dd, J = 10.1 Hz, 4.4 Hz, 1H, H-5β), 2.65 (d, J = 6.4 Hz, 1H, OH); 13C NMR (CD3OD) δ 158.4, 144.9, 137.3, 136.1, 136.0, 130.1, 128.6, 128.2, 128.1, 127.9, 127.8, 126.7, 113.0, 86.0, 83.3, 78.2, 77.2, 72.3, 72.3, 70.1, 64.3, 55.2; MS (ESI) m/z 549 [M+Na]+, HRMS (ESI) Calcd. for C33H34NaO6 [M+Na]+: 549.2253. Found: 549.2270.
55
2-O-Benzyl-3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1-dideoxy- 5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (20) ࡢࡢྜᡂ
2-O-Benzyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (19) (196 mg, 372 μmol) ࢆ THF (2 mL) ⁐ ゎ ࡋ ࠊDIPEA (450 μL, 2.65 mmol) ࢆ ຍ ࠼ ࡓ ᚋ ࠊ2-cyanoethyl diisopropylchlorophosphoamidite (200 μL, 897 μmol) ࢆୗࡋࠊᐊ ࡛3 㛫᧠ᢾࡋࡓࠋ ࢡ࣒࡛ࣟࣟ࣍ࣝᕼ㔘ᚋࠊࢡ࣒ࣟࣟ࣍ࣝ㣬Ⅳ㓟Ỉ⣲ࢼࢺ࣒ࣜ࢘Ỉ⁐ᾮࢆ⏝࠸࡚ᢳฟࡋࡓࠋ
᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆ
ࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 3:1) ࡚⢭〇ࡋࠊ2-O-benzyl-3-
O-[(2-cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (20) ࢆ㯤Ⰽ࢜ࣝ (262 mg, 97%) ࡋ࡚ᚓࡓࠋ31P NMR (160 MHz, CDCl3) δ 149.6, 149.1.
RHOBnࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ RHOBnࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ
᰾㓟⮬ືྜᡂᶵ (NTS H-6) ࢆ⏝࠸࡚1 μmol ࢫࢣ࣮࡛ࣝ⾜ࡗࡓࠋኳ↛ࡢ࣍ࢫ࣑࣍ࣟࢲ
ࢺࡣ50 mMࠊRHOBnࡢ࣍ࢫ࣑࣍ࣟࢲࢺయ 20 ࡣ75 mM࡞ࡿࡼ࠺MeCN⁐ᾮࢆ
ㄪᩚࡋ࡚⏝ࡋࡓࠋ
ྜᡂᚋࠊᅛ┦ᢸయࢆࢧࣥࣉࣝࢫࢺࢵࢡࢳ࣮ࣗࣈ⛣ࡋࠊࣥࣔࢽỈ/40% ࣓ࢳ࣑ࣝࣥ
⁐ᾮ (1:1) (1.2 mL) ࢆຍ࠼ࠊ60 ºC࡛ 30ศ㛫࠺ࡋ࡚ᅛ┦ᢸయࡽࡢษࡾฟࡋሷᇶ㒊 ಖㆤᇶࡢ㝖ཤࢆ⾜ࡗࡓࠋୖΎࢆ1.5 mL࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈ⛣ࡋࠊᅛ┦ᢸయࢆ࢚ࢱࣀ
࣮ࣝ/⁛⳦Ỉ (3:1) ࡛Ὑίࡋࡓࠋ⁐፹ࢆῶᅽ␃ཤࡋ࡚ᅛᚋࠊDMSO (100 μL) ⁐ゎࡋࠊ Et3N·3HF⁐ᾮ (125 μL) ࢆຍ࠼࡚ࠊ65 ºC࡛2.5㛫࣮ࣥ࢟ࣗ࣋ࢺࡋ࡚⢾㒊ಖㆤᇶࢆ㝖ཤ ࡋࡓࠋ3 M 㓑㓟ࢼࢺ࣒ࣜ࢘Ỉ⁐ᾮ (25 μL) ࢆຍ࠼࡚15 ⛊㛫࠺ᚋࠊࣈࢱࣀ࣮ࣝ (1 mL)
ࢆຍ࠼30 ⛊㛫࠺ࡋࠊ-80 ºC෭༷ࡋࡓࠋ15000 rpmࠊ10 minࠊ4 ºC࡛㐲ᚰࡋࠊỿẊࢆ
ᚓࡓࠋỿẊࢆ࢚ࢱࣀ࣮ࣝ (750 μL) ࡛Ὑίࡋࡓᚋࠊloading solution(200 μL) ⁐ゎࡋࠊ20%
PAGE*3 (500 V, 20 mA) ࡼࡾ┠ⓗࡢ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆศ㞳ࡋࡓࠋ┠ⓗࡢ࢜ࣜࢦࢾࢡࣞ
࢜ࢳࢻࡢࣂࣥࢻࢆษࡾฟࡋࠊ⁐ฟᾮ(20 mL) ᾐࡋ୍࡚ᬌ࠺ࡋࡓࠋࢁᾮࢆᖹ⾮ࡋࡓ
Sep-Pak ㏻ࡋࠊ࣒࢝ࣛ྾╔ࡉࡏࡓࠋ࣒࢝ࣛࢆ⁛⳦Ỉ (15 mL) ࡛Ὑίࡋ࡚ሷࢆ㝖ཤᚋࠊ
50% CH3CN in ⁛⳦Ỉ (3 mL) ࡛⁐ฟࡋࠊ⁐፹ࢆ㐲ᚰῶᅽ␃ཤࡋࡓࠋ
56
UV྾྾ࢫ࣌ࢡࢺࣝࡼࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢOD260 ್ࡢ⟬ฟ
➨3❶➨2⠇ྠᵝࡢ᪉ἲ࡛ࠊ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢOD260್ࢆ⟬ฟࡋࡓࠋ
OD260 (Mε-1mL-1cm-1) = Abs260 (Mε-1) · V -1 (mL) · l -1 (cm-1)
Sequence OD260
5′-r(gua gga gua gug aaa ggc c RHOBnRHOBn)-3′ 0.373 5’-r(ggc cuu uca cua cuc cua c RHOBnRHOBn)-3′ 0.238 5′-F-r(gua gga gua gug aaa ggc c RHOBnRHOBn)-3′ 0.322
࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢε್ཬࡧ⃰ᗘ⟬ฟἲ
➨3❶➨2⠇ྠᵝࡢ᪉ἲ࡛ࠊ⃰ᗘࢆ⟬ฟࡋࡓࠋ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢᵓ㐀☜ㄆ
➨3❶➨2⠇ྠᵝࡢ᪉ἲ࡛ࠊMALDI-TOF/MS ࢆ⏝࠸࡚ᵓ㐀☜ㄆࢆ⾜ࡗࡓࠋ
Sequence Calculated Observed
5′-r(gua gga gua gug aaa ggc c RHOBnRHOBn)-3′ 6772.0 6772.8 5’-r(ggc cuu uca cua cuc cua c RHOBnRHOBn)-3′ 6462.9 6463.0 5′-F-r(gua gga gua gug aaa ggc c RHOBnRHOBn)-3′ 7309.1 7309.1
➨ 5 ❶➨ 1 ⠇➨ 3 㡯㛵ࡍࡿᐇ㦂
᰾㓟ศゎ㓝⣲⪏ᛶホ౯
➨3❶➨3⠇ྠᵝࡢ᪉ἲ࡛⾜ࡗࡓࠋ
➨ 5 ❶➨ 1 ⠇➨ 4 㡯㛵ࡍࡿᐇ㦂
⇕ⓗᏳᐃᛶホ౯
➨3❶➨4⠇ྠᵝࡢ᪉ἲ࡛⾜ࡗࡓࠋ
➨ 5 ❶➨ 1 ⠇➨ 5 㡯㛵ࡍࡿᐇ㦂
㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯➨3❶➨5⠇ྠᵝࡢ᪉ἲ࡛⾜ࡗࡓࠋ
57
➨
➨ 4 ❶➨ 1 ⠇➨ 6 㡯㛵ࡍࡿᐇ㦂
࢘ࢩ⾑Ύ୰Ᏻᐃᛶホ౯
➨3❶➨6⠇ྠᵝࡢ᪉ἲ࡛⾜ࡗࡓࠋ
➨ 4 ❶➨ 1 ⠇➨ 7 㡯㛵ࡍࡿᐇ㦂
ࣄࢺArgonaute2-PAZࢻ࣓ࣥࢱࣥࣃࢡ㉁ࡢぶᛶホ౯
hAgo2-PAZࢱࣥࣃࢡ㉁ࡢⓎ⌧ࠊ⢭〇
LB/Amp ᇵᆅ (20 mL) Hs Argonaute 2 PAZ Ⓨ⌧ࣉࣛࢫ࣑ࢻ࡛ᙧ㉁㌿ࡋࡓ⭠⳦
BL21(DE3) ᰴࢆ᳜⳦ࡋࠊ37 ˚C୍࡛ᬌᇵ㣴ࡋࠊ๓ᇵ㣴ᾮࢆㄪ〇ࡋࡓࠋ⩣᪥ࠊ๓ᇵ㣴ᾮࢆ2 ᮏ
ࡢLB/Ampᇵᆅ (1 L) 10 mLࡎࡘຍ࠼ࠊ37˚Cࠊ180 rpm࡛⃮ᗘࡀOD600 = 0.8࡞ࡿࡲ
࡛࠺ᇵ㣴ࡋࡓࠋᇵ㣴ᾮ1M IPTG (1 mL) ࢆῧຍࡋ࡚20˚Cࠊ180 rpm࡛ࡉࡽ6 㛫 ᇵ㣴ࡋࡓࠋᇵ㣴ᾮࢆ4˚Cࠊ6000 rpm࡛10ศ㛫㐲ᚰࡋ࡚⳦యࢆᅇࡋࠊBuffer A [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM imidazole, 1 mM phenylmethylsulfonyl fluoride] ᠱ⃮ࡋ ࡓࠋࢯࢽࢣ࣮ࢱ࣮ࢆ⏝࠸࡚ịୖ࡛5 sec×20ᅇࡢ㉸㡢Ἴ◚○ࢆ⾜ࡗࡓᚋࠊ4˚Cࠊ10000 rpm࡛ 15 ศ㛫㐲ᚰࡋࠊୖΎࢆ⣽⬊ᢳฟᾮࡋ࡚ᅇࡋࡓࠋࡇࡢ⣽⬊ᢳฟᾮࢆBuffer Aᖹ⾮ࡋࡓ TALON® Metal Affinity Resinࣉࣛࡋࠊ┠ⓗࢱࣥࣃࢡ㉁ࢆ࣒࢝ࣛ྾╔ࡉࡏࡓࠋBuffer A (5 bet௨ୖ) ࢆ⏝࠸࡚࣒࢝ࣛࢆὙίࡋࡓᚋࠊWash Buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride] (5 bet௨ୖ) ࢆ⏝࠸࡚࢝ࣛ
࣒ࢆ୰㛫ὙίࡋࠊElution Buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 200 mM imidazole, 1 mM phenylmethylsulfonyl fluoride] (5 bet௨ୖ) ࢆ⏝࠸࡚┠ⓗࢱࣥࣃࢡ㉁ࢆ⁐ฟࡉࡏࡓࠋ⁐
ฟᾮࡣ㝈እℐ㐣⭷ (30 kDa, 10 kDa) ࢆ⏝࠸࡚⢭〇ࠊ⃰⦰ࡋࡓࠋྛᐇ㦂᧯స࡛ࠊ100 μLࢆྲྀ
ࡾศࡅࡓࠋᚓࡓ⣽⬊ᢳฟᾮࡢࢱࣥࣃࢡࡢⓎ⌧ࢆ☜ㄆࡍࡿࡓࡵࠊSDS-PAGEࢆ⾜ࡗࡓࠋ 30%ࢡ࣑ࣜࣝࢻ⁐ᾮ (3.6 mL)ࠊ1.5 M Tris-HCl pH 8.8/0.4% SDS (1.8 mL)ࠊMilli-QỈ (1.8 mL)ࠊ10% APS (40 μL)ࠊTEMED (6 μL) ࢆΰࡋࠊ15%ࢡ࣑ࣜࣝࢻ⃰ᗘࡢศ㞳ࢤ
ࣝࢆసᡂࡋࡓࠋศ㞳ࢤࣝࡀᅛࡋࡓᚋࠊ30%ࢡ࣑ࣜࣝࢻ⁐ᾮ (252 μL)ࠊ0.5 M Tris-HCl (pH 6.8)/0.4% SDS (420 μL)ࠊMilli-QỈ (1 mL)ࠊ10% APS (8 μL)ࠊTEMED (4 μL)ࢆΰࡋࠊ ศ㞳ࢤࣝࡢୖ⃰⦰ࢤࣝࢆసᡂࡋࡓࠋࢧࣥࣉࣝ➼㔞ࡢ Loading dye ࢆῧຍࡋࠊ100˚C ࡛ 10ศ㛫ຍ⇕ࡋࡓࠋࡇࢀࢆ㐺㔞ࡗ࡚࢙࢘ࣝࣉࣛࡋࠊᐃ㟁ὶ (500 V, 20 mA) ࡛1 㛫ὋືࡋࡓࠋὋື⏝⦆⾪ᾮࡣRunning Bufferࢆ⏝ࡋࡓࠋὋື⤊ᚋࠊࢤࣝࢆᅛᐃᾮ
10ศ㛫ᾐࡋࠊCBBᰁⰍᾮࢆ⏝࠸࡚30ศ㛫ᰁⰍࡋࡓࠋ᭱ᚋMilliQ࡛⬺ⰍࡋࠊὋືᚋࡢࢤ
ࣝࡢ┿ࢆLAS-4000࡛ᙳࡋࡓࠋ
hAgo2–PAZࢱࣥࣃࢡ㉁ࢆ⏝࠸ࡓぶᛶホ౯
1.5 mL࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈdsRNA (10 μM, 1μL) ࢺࣜࢫሷ㓟ࣂࢵࣇ࣮ (20 mM Tris-HCl, 100 mM NaCl, 14 μL) ࢆຍ࠼ࠊΰྜࡋࡓᚋࠊhAgo2-PAZࢱࣥࣃࢡ㉁⁐ᾮ (10 μM,
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5 μL) ࢆຍ࠼ࠊ37 ºC࡛10ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋ࣮ࣥ࢟ࣗ࣋ࢺᚋࠊ50%ࢢࣜࢭ࣮ࣟ
ࣝ⁐ᾮ (20 μL) ࢆຍ࠼࡚ΰྜࡋࡓᚋị෭ࡋ࡚ᛂṆࡋࡓࠋ10ศ㛫ị෭ࡋࠊ10% native PAGE (500 V, 21 mA) ࡛30ศ㛫ὋືࡋࠊGelRed࡛15ศ㛫ᰁⰍࡋࡓᚋࠊLAS-4000ࢆ⏝࠸
࡚⺯ග⏬ീࢆᙳࡋࡓࠋ
➨
➨ 4 ❶➨ 2 ⠇➨ 1 㡯㛵ࡍࡿᐇ㦂
Scheme 4-6㛵ࡍࡿᐇ㦂2-O-Propargyl-1-deoxy-D-ribofuranose (21) ࡢྜᡂ
3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17) (1.91 g, 5.07 mmol) ࢆDMF (25 mL) ⁐ゎࡋࠊị෭ୗࠊ60% NaH (223 mg, 5.58 mmol) ࢆຍ࠼ࡓᚋࠊ propargyl bromide (500 μL, 6.60 mmol) ࢆຍ࠼ࠊᐊ ࡛3 㛫᧠ᢾࡋࡓࠋị෭ୗࠊ㣬ሷ
ࣥࣔࢽ࣒࢘Ỉ⁐ᾮࢆຍ࠼ࡓᚋࠊ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝỈ࡛ᢳฟࡋࡓࠋ᭷ᶵ ᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆTHF (10 mL) ⁐ゎࡋࡓᚋࠊTBAF (1 M solution in THF, 15 mL) ࢆຍ࠼ࠊᐊ ࡛1㛫᧠ᢾࡋ ࡓࠋ⁐፹ࢆῶᅽ␃ཤࡋࠊṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (CHCl3/MeOH, 50:1~10:1) ࡚⢭〇ࡋࠊ2-O-propargyl-1-deoxy-D-ribofuranose (21) (330 mg, 38%) ࢆ〓Ⰽ
࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (CDCl3) δ 4.38–3.65 (m, 5H), 3.92–3.65 (m, 3H), 3.74–3.65 (m, 1H), 2.72–2.66 (m, 1H), 2.58 (d, 1H), 2.50 (t, 1H); 13C NMR (CDCl3) δ 83.4, 78.2, 75.4, 71.3, 62.3, 58.8, 57.7.
➨ 4 ❶➨ 2 ⠇➨ 2 㡯㛵ࡍࡿᐇ㦂
Scheme 4-7㛵ࡍࡿᐇ㦂2-O-Propargyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (22) ࡢྜᡂ
2-O-Propargyl-1-deoxy-D-ribofuranose (21) (259 mg, 1.50 mmol) ࢆpyridine (5 mL) ⁐
ゎࡋࠊị෭ୗࠊ4,4′-dimethoxytrityl chloride (611 mg, 1.80 mmol) ࡢpyridine (5 mL) ⁐ᾮࢆ
ୗࡋࡓᚋࠊᐊ ࡛14 㛫᧠ᢾࡋࡓࠋ㓑㓟࢚ࢳ࡛ࣝᕼ㔘ᚋࠊ㓑㓟࢚ࢳࣝ␃Ỉࢆ⏝࠸࡚
ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬Ⅳ㓟Ỉ⣲ࢼࢺ࣒ࣜ࢘Ỉ⁐ᾮ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺࣜ
࣒࡛࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 5:1~2:1) ࡚⢭〇ࡋࠊ2-O-propargyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D -ribofuranose (22) (316 mg, 44%) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (CDCl3) δ 7.46–7.18 (m, 9H), 6.89–6.81 (m, 4H), 4.35–4.23 (m, 2H), 4.19–4.07 (m, 2H), 3.97–3.88 (m, 2H), 3.80–
3.79 (m, 7H), 3.35–3.12 (m, 2H), 2.52–2.48 (m, 2H); 13C NMR (CDCl3) δ 158.4, 144.9, 136.1, 136.0, 130.1, 128.2, 127.8, 126.7, 113.5, 113.1, 86.1, 83.1, 79.1, 78.3, 75.2, 72.3, 69.9, 64.3, 57.7, 55.2. MS (ESI) m/z 497 [M+Na]+, HRMS (ESI) Calcd. for C29H30NaO6 [M+Na]+ : 497.1940. Found 497.1918.
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2-O-Propargyl-3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1-dideoxy- 5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (23) ࡢࡢྜᡂ
2-O-Propargyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (22) (196 mg, 372 μmol)
ࢆ THF (2 mL) ⁐ ゎ ࡋ ࠊDIPEA (0.4 mL, 2.35 mmol) ࢆ ຍ ࠼ ࡓ ᚋ ࠊ2-cyanoethyl diisopropylchlorophosphoamidite (200 μL, 897 μmol) ࢆୗࡋࠊᐊ ࡛1 㛫᧠ᢾࡋࡓࠋ ࢡ࣒࡛ࣟࣟ࣍ࣝᕼ㔘ᚋࠊࢡ࣒ࣟࣟ࣍ࣝ㣬Ⅳ㓟Ỉ⣲ࢼࢺ࣒ࣜ࢘Ỉ⁐ᾮࢆ⏝࠸࡚ᢳฟࡋࡓࠋ
᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓ỈὶỈࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆ
ࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/EtOAc, 5:1~2:1) ࡚⢭〇ࡋࠊ2-O-
propargyl-3-O-[(2-cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1-dideoxy-5-O-(4,4′dimethoxytrityl)-D-ribofuranose (23) (200 mg, 77%) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ31P NMR (160 MHz, CDCl3) δ 150.00, 149.86.
RHOPgࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ RHOPgࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ
᰾㓟⮬ືྜᡂᶵ (NTS H-6) ࢆ⏝࠸࡚1 μmol ࢫࢣ࣮࡛ࣝ⾜ࡗࡓࠋኳ↛ࡢ࣍ࢫ࣑࣍ࣟࢲ
ࢺࡣ50 mMࠊRHOPgࡢ࣍ࢫ࣑࣍ࣟࢲࢺయ 23 ࡣ75 mM࡞ࡿࡼ࠺MeCN⁐ᾮࢆ
ㄪᩚࡋ࡚⏝ࡋࡓࠋྜᡂᚋࠊ➨5❶➨2⠇ྠᵝࡢ᪉ἲ࡛⢭〇ࡋࡓࠋ
UV྾ࢫ࣌ࢡࢺࣝࡼࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢOD260 ್ࡢ⟬ฟ
࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆ㉸⣧Ỉ⁐ゎࡋࠊἼ㛗260 nm࡛ࡢ྾ගᗘ (Abs260) ࡀ྾ගᗘィࡢ
᭷ຠ⠊ᅖ (0.2-1.0) ࡞ࡿࡼ࠺100ಸᕼ㔘ࡋࡓࠋග㊰㛗 ( l ) 1 cmࡢ྾ගᗘ ᐃ⏝▼ⱥࢭ
ࣝࢆ⏝࠸࡚ࠊᐊ ࡚Abs260ࢆ ᐃࡋࡓࠋOD260್ࡢィ⟬ࡣ௨ୗࡢᘧࢆ⏝࠸ࡓࠋࡲࡓࠊV ࡣ⁐ᾮ㔞ࢆ♧ࡍࠋ
OD260 (Mε-1mL-1cm-1) = Abs260 (Mε-1) · V -1 (mL) · l -1 (cm-1)
Sequence OD260
5′-r(gua gga gua gug aaa ggc c RHOPg)-3′ 0.371
࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢε್ཬࡧ⃰ᗘ⟬ฟἲ
➨3❶➨2⠇ྠᵝࡢ᪉ἲ࡛ࠊ⃰ᗘࢆ⟬ฟࡋࡓࠋ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢᵓ㐀☜ㄆ
➨3❶➨2⠇ྠᵝࡢ᪉ἲ࡛ࠊMALDI-TOF/MS࡛ᵓ㐀☜ㄆࢆ⾜ࡗࡓࠋ
Sequence Calculated Observed
5′-r(gua gga gua gug aaa ggc c RHOPg)-3′ 6433.9 6433.6
60
➨
➨ 4 ❶➨ 2 ⠇➨ 3 㡯㛵ࡍࡿᐇ㦂
Scheme 4-8㛵ࡍࡿᐇ㦂4-Fluorobenzyl azide (25) ࡢྜᡂ
4-Fluorobenzyl chloride (520 mg, 3.60 mmol) ࢆDMF (7 mL) ⁐ゎࡋࠊNaI (55.1 mg, 368 μmol) NaN3 (465 mg, 7.15 mmol) ࢆຍ࠼ࠊ60 ºC࡛3 㛫᧠ᢾࡋࡓࠋị෭ୗࠊ␃
Ỉࢆຍ࠼ࡓᚋࠊࢪ࢚ࢳ࢚࣮ࣝࢸ࡛ࣝᕼ㔘ᚋࠊࢪ࢚ࢳ࢚࣮ࣝࢸࣝ␃Ỉ࡛ࢆ⏝࠸࡚ᢳฟࡋࡓࠋ
᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆ
ࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ (n-hexane/CH2Cl2, 50:1~20:1) ࡚⢭〇ࡋࠊ 4-fluorobenzyl azide (25) (431 mg, 79%) ࢆ↓Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (CDCl3) δ 7.32–
7.27 (m, 2H), 7.10–7.05 (m, 2H), 7.50–7.44 (m, 2H), 4.32 (s, 2H); 13C NMR (CDCl3) δ 162.6 (d, J = 245 Hz), 131.1, 130.0 (d, J = 7.6 Hz), 115.8 (d, J = 21.9 Hz), 54.0.
1-Azido-1-deoxy-2,3,4,6-tetra-O-acetyl-β-D-glucopyranose (26) ࡢྜᡂ
Penta-O-acetyl-β-D-glucopyranose (30) (783 mg, 3.01 mmol) ࢆCH2Cl2 (10 mL) ⁐ゎ ࡋࠊị෭ୗࠊFeCl3 (36.8 mg, 227 μmol) ࢆຍ࠼ࠊ5ศ㛫᧠ᢾࡋࡓᚋࠊị෭ୗ࡛TMSN3 (400
μL, 3.04 mmol) ࢆຍ࠼ࠊᐊ ࡛2 㛫᧠ᢾࡋࡓࠋị෭ୗࠊ␃Ỉࢆຍ࠼ࡓᚋࠊࢪࢡ࣓ࣟࣟ
ࢱ࡛ࣥᕼ㔘ᚋࠊࢪࢡ࣓ࣟࣟࢱࣥ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑίᚋࠊ
↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛ
ࣇ࣮ (n-hexane/EtOAc, 5:1~2:1) ࡚⢭〇ࡋࠊ1-azido-1-deoxy-2,3,4,6-tetra-O-acetyl-β-D- glucopyranose (26) (700 mg, 94%) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (CDCl3) δ 5.22 (t, J
= 9.6 Hz, 1H), 5.11 (t, J = 9.6 Hz, 1H), 4.65 (d, J = 8.4 Hz, 1H), 4.28 (dd, J = 12.4, 4.8 Hz, 1H), 4.17 (dd, J = 12.8, 2.4 Hz, 1H), 3.79 (ddd, J = 7.4, 5.0, 2.2 Hz, 1H), 3.82–3.77 (m, 1H), 2.10 (s, 3H), 2.08 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H); 13C NMR (CDCl3) δ 170.6, 170.1, 169.3, 169.2, 87.9, 74.0, 72.6, 70.6, 67.8, 61.6, 20.7, 20.5.
1-Azido-1-deoxy-β-D-glucopyranose (27) ࡢྜᡂ
1-Azido-1-deoxy-2,3,4,6-tetra-O-acetyl-β-D-glucopyranose (26) (61.7 mg, 165 μmol) ࢆ
9.4 mM ࣥࣔࢽᛶ࣓ࢱࣀ࣮ࣝ (18 mL) ⁐ゎࡋࠊᐊ ࡛2 㛫᧠ᢾࡋࡓࠋࣝࢦࣥ࢞
ࢫࢆࣂࣈࣜࣥࢢࡋࠊࣥࣔࢽࢆ㝖࠸ࡓᚋ⁐፹ࢆῶᅽ␃ཤࡋࠊ1-azido-1-deoxy-β-D -glucopyranose (27) (29.3 mg, 86%) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (DMSO-d6) δ 4.90 (d, J = 4.9 Hz, 1H), 4.87 (d, J = 4.9 Hz, 1H), 4.85 (d, J = 4.9 Hz, 1H), 4.08 (d, J = 4.1 Hz, 1H), 3.77–3.63 (m, 2H), 3.43–3.39 (m, 2H), 3.37–3.33 (m, 2H), 2.94–2.91 (m, 1H).
1-Naphtalenemethylazide (22) ࡢྜᡂ
1–Naphthaldehyde (31) (510 mg, 3.27 mmol) ࢆMeOH (10 mL) ⁐ゎࡋࠊị෭ୗࠊNaBH4
(269 mg, 7.11 mmol) ࢆຍ࠼ࠊᐊ ࡛2 㛫᧠ᢾࡋࡓࠋị෭ୗࠊ␃Ỉࢆຍ࠼ࡓᚋࠊࢪࢡࣟ
61
࣓ࣟࢱ࡛ࣥᕼ㔘ᚋࠊࢪࢡ࣓ࣟࣟࢱࣥ␃Ỉࢆ⏝࠸࡚ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗ሷỈ࡛Ὑί ᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆDMF (10 mL) ⁐ゎࡋࠊ ị෭ୗࠊCBr4 (2.18 g, 6.57 mmol) PPh3 (1.72 g, 6.56 mmol) ࢆຍ࠼ࠊ5ศ㛫᧠ᢾࡋࡓᚋࠊ ị෭ୗ࡛NaN3 (429 mg, 6.60 mmol) ࢆຍ࠼ࠊᐊ ࡛14 㛫᧠ᢾࡋࡓࠋị෭ୗࠊ␃Ỉࢆ
ຍ࠼ࡓᚋࠊࢪࢡ࣓ࣟࣟࢱ࡛ࣥᕼ㔘ᚋࠊࢪࢡ࣓ࣟࣟࢱࣥ␃Ỉ࡛ᢳฟࡋࡓࠋ᭷ᶵᒙࢆ㣬㣗 ሷỈ࡛Ὑίᚋࠊ↓Ỉ◲㓟ࢼࢺ࣒࡛ࣜ࢘⇱ࡋࠊ⁐፹ࢆῶᅽ␃ཤࡋࡓࠋṧ´ࢆࢩࣜ࢝ࢤࣝ࢝ࣛ
࣒ ࢡ ࣟ ࣐ ࢺ ࢢ ࣛ ࣇ ࣮ (n-hexane/CH2Cl2, 50:1~20:1) ࡚ ⢭ 〇 ࡋ ࠊ 1-naphtalenemethylazide (28) (465 mg, 78%) ࢆ㯤Ⰽ࢜ࣝࡋ࡚ᚓࡓࠋ1H NMR (CDCl3) δ 8.04 (d, J = 8.8 Hz, 2H), 7.92–7.85 (m, 2H), 7.61–7.52 (m, 2H), 7.50–7.44 (m, 2H), 4.78 (s, 2H); 13C NMR (CDCl3) δ 133.9, 131.3, 130.9, 129.4, 128.8, 127.3, 126.7, 126.2, 125.2, 123.5, 53.0.
RHOPgྵ᭷࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢪࢻྜ≀ࡢCuAACᛂ
ࢪࢻྜ≀ࢆ⏝࠸ࡓCuAACᛂࡢ᳨ウ
1.5 mL࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈ3′ᮎ➃RHOPgࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ (100 μM solution in Milli-QỈ, 20 μL) ࢆศྲྀࡋࠊࢪࢻྜ≀ (10 mM solution in DMSO, 1 μL)ࠊ
ࣜࣥ㓟ࣂࢵࣇ࣮ (1M solution in Milli-QỈ, pH 7.0)ࠊCH3CN (1 μL) ࡢ㡰ຍ࠼࣎ࣝࢸࢵࢡ ࢫࠊࢫࣆࣥࢲ࢘ࣥࡋࡓᚋࠊCuSO4•5H2O (100 mM solution in Milli-QỈ, 1 μL)ࠊNa ascorbate (100 mM solution in Milli-QỈ, 1 μL) ࡢ㡰ຍ࠼ࠊ࣎ࣝࢸࢵࢡࢫࠊࢫࣆࣥࢲ࢘ࣥࡋᐊ ࡛15 ศ㛫㟼⨨ࡋࡓࠋMilli-QỈ (300 μL) ࢆຍ࠼ࠊࣇࣝࢱ࣮ (TOSMIC, 0.2 μmぶỈᛶPVDF) ℐ 㐣ࡋࡓᚋHPLC࡚ᛂࡢ㐍⾜ࢆ☜ㄆࡋࡓࠋ
Aᾮ : 5% ࢭࢺࢽࢺࣜࣝ/0.1 M TEAA ⦆⾪ᾮ (pH 7.0) Bᾮ : 50% ࢭࢺࢽࢺࣜࣝ/0.1 M TEAA ⦆⾪ᾮ (pH 7.0)
⁐ฟ㏿ᗘ 1 mL/min ᐃἼ㛗 260 nm
Time ( min. ) B solution
0 5
5 5
30 10
35 30
40 100
62
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ᘬ⏝ᩥ⊩
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ᮏᏛㄽᩥࡣࠊୗグࡢཎⴭㄽᩥࢆᇶసᡂࡉࢀࠊᒱ㜧ᏛᏛ㝔㐃ྜ⸆་⒪
ሗ◊✲⛉ᥦฟࡉࢀࡓࡶࡢ࡛࠶ࡿࠋ
1. Practical and Reliable Synthesis of 1,2-Dideoxy-D-ribofuranose and its Application in RNAi Studies
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2. Introduction of 2-O-benzyl abasic nucleosides to the 3′-overhang regions of siRNAs greatly improves nuclease resistance
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Bioorg. Med. Chem. Lett. 2017, 27, 5454−5456.
NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
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