6
H. Ozaki et al. / Tetrahedron xxx (2017) 1e8
was added to the solution, and the resulting mixture was stirred at
room temperature for 1 h under Ar gas. Satl. sodium hydrogen
carbonate aq. solution was added to the reaction mixture for
neutralization at 0 ꢀC, and the product was extracted with ethyl
acetate. The organic layer was dried with sodium sulfate and
concentrated in vacuo. The crude product was purified by silica gel
product 10 as a white solid (0.225 g, 81.1%): mp 177e178 ꢀC; 1H
NMR (300 MHz, CDCl3)
d
8.14 (d, J ¼ 2.4 Hz, 1H), 7.82 (m, 2H), 7.31
(m, 2H), 7.01 (m, 3H), 5.73 (dd, J ¼ 5.7 and 9.6 Hz, 1H), 4.67 (s, 2H),
4.25 (m, 1H), 3.91 (m, 1H), 3.89 (s, 3H), 3.86 (d, J ¼ 7.5 Hz, 1H), 2.46
(m, 1H), 1.78 (m, 1H); 13C NMR (125.65 MHz, CD3OD)
d165.01,
154.54, 136.88, 133.28, 126.02, 125.24,123.43, 120.84, 118.23, 114.84,
111.23, 103.86, 84.00, 73.80, 69.50, 63.86, 59.21, 48.02, 40.20; ESI-
MS (POS) m/z (MþNa)þ: Calcd for C21H23NO7Na: 424.1, found:
424.2.
column chromatography (ethyl acetate:methanol
¼
100:0 to
99.7:0.3 v/v) to obtain the desired product 1 as a brown gum
22
(0.155 g, 26.5%): [
(300 MHz, CD3OD)
a
d
]
¼ þ75 (c ¼ 0.20, CH3OH); 1H NMR
D
7.30 (d, J ¼ 8.4 Hz, 1H), 6.81 (dd, J ¼ 2.4 and
8.7 Hz, 1H), 6.80 (s, 1H), 5.19 (dd, J ¼ 5.4 and 10.5 Hz, 1H), 4.22 (m,
1H), 3.82 (m, 1H), 3.57 (d, J ¼ 9.6 Hz, 1H), 2.12 (m, 1H), 1.85 (m, 1H);
2.1.10. 6-(2-Deoxy-b-D-ribofranosyl)-3-aminobenzoic acid (2)
Compound 10 (11 mg, 0.027 mmol) was incubated in 1 M so-
dium hydroxide (3 mL) at 55 ꢀC for two days. The reaction mixture
was neutralized by acetic acid and triethylamine. The crude product
was purified by reversed-phase HPLC to obtain the desired product
13C NMR (125.65 MHz, CD3OD)
d 148.21, 132.47, 127.69, 119.08,
117.55,116.81,110.28, 87.65, 77.81, 72.88, 62.38, 42.72; ESI-MS (POS)
m/z (MþNa)þ: Calcd for C12H14N2O3Na: 257.1, found: 257.1.
as a yellow gum of a triethylammonium salt (4.5 mg, 47%):
22
2.1.8. 6-(2-Deoxy-
b-
D-ribofranosyl)-N-phenoxyacetyl-3-
[a
]
¼ þ12 (c ¼ 0.14, CH3OH); 1H NMR (300 MHz, CD3OD)
d 7.31
D
aminobenzonitrile (9)
(d, J ¼ 8.4 Hz,1H), 6.82 (d, J ¼ 2,4 Hz,1H), 6.71 (dd, J ¼ 8.4 Hz, 2.4 Hz,
1H), 5.53 (dd, J ¼ 5.9 and 10.0 Hz, 1H), 4.21 (m, 1H), 3.83 (m, 1H),
3.67 (d, J ¼ 5.1 Hz, 2H), 3.13 (q, J ¼ 7.3 Hz, 6H, -CH2CH3-), 2.36e2.30
(m, 1H), 1.89e1.81 (m, 1H), 1.27 (t, J ¼ 7.4 Hz, 9H, CH2CH3); 13C NMR
Phenoxyacetyl dehydrate (0.112 g, 0.426 mmol) was added to a
solution of compound 1 (0.121 g, 0.478 mmol) in DMF (3.5 mL)
under Ar gas at 0 ꢀC. After stirring under Ar gas at room tempera-
ture for 2 h, the reaction mixture was concentrated in vacuo to
dryness. The crude product was purified by silica gel column
chromatography (ethyl acetate:hexane ¼ 95:5 to 100:0 v/v) to
obtain the desired product 9 as a brown solid (0.105 g, 59.6%): mp
(150.91 MHz, CD3OD) d 177.74, 147.57, 140.88, 129.86, 127.92, 117.19,
115.00, 88.48, 79.24, 74.51, 64.22, 47.63, 45.09, 9.21; ESI-MS (POS)
m/z (MþH)þ: Calcd for C18H31N2O5: 355.2, found: 355.3.
143e146 ꢀC; 1H NMR (300 MHz, CDCl
)
d
8.43 (s, 1H), 8.04 (d,
2.1.11. 50-Dimethoxytrityl-6-(2-deoxy-
phenoxyacetyl-)3-aminobenzonitrile (11)
b-D-ribofranosyl)-(N-
3
J ¼ 7.2 Hz,1H), 7.77 (d, J ¼ 8.7 Hz, 1H), 7.54 (d, J ¼ 9.0 Hz, 1H), 7.36 (t,
J ¼ 7.7 Hz, 2H), 7.08 (t, J ¼ 7.5 Hz, 1H), 6.99 (d, J ¼ 8.7 Hz, 2H), 5.36
(dd, J ¼ 5.4 and 10.5 Hz, 1H), 4.64 (s, 2H), 4.54 (m, 1H), 4.06 (m, 1H),
3.83 (m, 2H), 2.39 (m, 1H), 2.03 (m, 1H); 13C NMR (150.91 MHz,
A solution of dimethoxytrityl chloride (0.111 g, 0.325 mmol) and
triethylamine (0.028 mL) in pyridine (1 mL) was added dropwise to
a solution of compound 9 (0.075 g, 0.204 mmol) and dimethyla-
minopyridine (0.005 g) in pyridine (2 mL) under Ar gas. After
stirring at room temperature for 2 h, 0.1 mL of methanol was added
to the reaction mixture. The solution was diluted with dichloro-
methane, washed with satl. sodium hydrogen carbonate aq. solu-
tion, dried with magnesium sulfate, and concentrated in vacuo. The
crude product was purified by silica gel column chromatography
(dichloromethane:hexane ¼ 7:3 to 10:0 v/v) to obtain the desired
product 11 as a white foam (0.100 g, 73.1%): 1H NMR (300 MHz,
CD3OD)
d 169.86, 159.21, 142.86, 139.07, 130.72, 128.57, 126.32,
125.11, 122.98, 122.47, 118.15, 115.94, 111.43, 89.46, 79.05, 74.34,
68.58, 63.82, 44.38; ESI-MS (POS) m/z (MþNa)þ: Calcd for
C
20H20N2O5Na: 391.1, found: 391.2.
2.1.9. Methyl 6-(2-deoxy-b-d-ribofranosyl)-(N-phenoxyacetyl-)3-
aminobenzoate (10)
A solution of palladium (II) acetate (0.063 g, 0.28 mmol) and
triphenylarsine (0.19 g, 0.062 mmol) in degassed DMF was stirred
under Ar gas at room temperature for 20 min. A solution of com-
pound 8 (0.980 g, 2.38 mmol) and 1,4-anhydro-3,5-bis-O-(tert-
CDCl3)
d 8.35 (s, 1H), 8.03 (s, 1H), 7.71e7.63 (m, 2H), 7.46e6.81 (m,
18H), 5.48 (dd, J ¼ 6.0 and 9.3 Hz, 1H), 4.63 (s, 1H), 4.45 (sbr, 1H),
4.10 (sbr, 1H), 3.79 (s, 6H), 3.36 (sbr, 1H), 2.52e2.45 (m, 1H),
butyldimethylsilyl)-2-deoxy-D-erythro-pent-1-enitol
(1.15
g,
2.02e1.93 (m, 1H); 13C NMR (150.91 MHz, CDCl3)
d 166.57,158.52,
3.34 mmol) in degassed DMF and then tributylamine (0.60 mL,
4.27 mmol) were sequentially added to the solution under Ar gas.
The mixture was stirred at 90 ꢀC for 210 min and then at 0 ꢀC for
15 min. Acetic acid (2.0 mL) and 1 M tetrabuthylammoniumfluoride
in THF (3.0 mL) were added to the reaction mixture at 0 ꢀC, and the
resulting mixture was stirred overnight at room temperature under
Ar gas. The reaction mixture was diluted with ethyl acetate, washed
with satl. sodium hydrogen carbonate aq. solution, dried with so-
dium sulfate, and concentrated in vacuo. The crude product was
purified by silica gel column chromatography (ethyl
acetate:hexane ¼ 3:7 to 10:0 v/v) to obtain methyl 6-(2R-5R-5-
hydroxymethyl-4-oxo-tetrahydrofuran-2-yl)-(N-phenozyacetyl-)
3-aminobnzoate (0.299 g, 31.5%).
A part of the obtained compound (0.276 g, 0.691 mmol) was
dissolved in acetonitrile (10 mL) and acetic acid (4 mL), and then
stirred at 0 ꢀC for 15 min. Sodium triacetoxyborohydrate (0.920 g,
4.34 mmol) was added to the solution, and the resulting mixture
was stirred at room temperature for 3 h under Ar gas. The reaction
mixture was diluted in ethyl acetate. The solution was washed with
satl. sodium hydrogen carbonate aq. solution, dried with sodium
sulfate, and concentrated in vacuo. The crude product was purified
156.74, 144.73, 142.41, 136.40, 135.90, 130.07, 130.01, 128.16, 127.88,
127.19, 126.88, 124.42, 123.52, 122.73, 116.82, 114.82, 113.16, 110.71,
86.34, 86.29, 74.30, 67.46, 64.03, 55.25, 43.44; ESI-MS (POS) m/z
(MþNa)þ: Calcd for C41H38N2O7Na: 693.3, found: 693.3.
2.1.12. 50-Dimethoxytrityl-6-(2-deoxy-
b-D-ribofranosyl)-(N-
phenoxyacetyl-)-methyl-3-aminobenzoate (12)
A solution of dimethoxytrityl chloride (0.175 g, 0.517 mmol) and
triethylamine (0.1 mL) in pyridine (1 mL) was added dropwise to a
solution of compound 10 (0.123 g, 0.306 mmol) and dimethylami-
nopyridine (0.01 g) in pyridine (2 mL) under Ar gas. After stirring at
room temperature for 2 h, 0.1 mL of methanol was added to the
reaction mixture. The solution was diluted with dichloromethane,
washed with satl. sodium hydrogen carbonate aq. solution, dried
with magnesium sulfate, and concentrated in vacuo. The crude
product was purified by silica gel column chromatography (hex-
ane:ethyl acetate ¼ 7:3 to 5:5 v/v) to obtain the desired product 12
as a white solid (0.196 g, 91.0%): mp 86e87 ꢀC; 1H NMR (300 MHz,
CDCl3)
d
8.34 (s, 1H), 8.08 (d, J ¼ 2.4 Hz, 1H), 7.84 (d, J ¼ 8.4 Hz, 1H),
7.75 (dd, J ¼ 2.4 and 8.7 Hz, 1H), 7.49e7.21 (m, 11H), 7.07 (t,
J ¼ 7.4 Hz, 1H), 7.00 (d, J ¼ 7.8 Hz, 2H), 6.84e6.82 (m, 4H), 5.81 (dd,
J ¼ 6.3 Hz, 8.5 Hz, 1H), 4.62 (s, 2H), 4.36 (m, 1H), 4.05 (m, 1H), 3.89
(s, 3H), 3.83 (s, 6H), 3.39 (m, 2H), 2.53 (m, 1H), 1.91 (m, 1H); 13C
by
silica
gel
column
chromatography
(ethyl
acetate:methanol ¼ 100:0 to 95:5 v/v) to obtain the desired
Please cite this article in press as: Ozaki H, et al., Synthesis and properties of microenvironment-sensitive oligonucleotides containing a small