Halogenated WNA DeriVatiVes
3.6 Hz), 4.41-4.12 (m, 2H), 3.80 (s, 3.6H), 3.79 (s, 2.4H), 3.78-
3.64 (m, 1H), 3.62-3.39 (m, 4H), 3.24-3.19 (m, 1H), 3.10-3.05
(m, 1H), 2.66-2.29 (m, 3H), 1.14 (d, 2H, J ) 6.9 Hz), 1.08 (d,
4H, J ) 6.9 Hz), 0.91 (d, 2H, J ) 6.9 Hz), 0.88 (d, 4H, J ) 6.9
Hz). 31P NMR (161 MHz, CDCl3) δ 150.2, 149.5. FTIR (film) 3184,
3067, 2966, 2835, 1704, 1693, 1607, 1510, 1447 cm-1. ESIMS
(m/z) 949, 951 [M + Na]+.
1693, 1622, 1448, 1427 cm-1. HRMS (ESIMS) m/z calcd for
C33H37O7SiBrNa (M + Na)+ 675.1384, 677.1370, found 675.1352,
677.1335.
(1′S,3′R,4′R,5′R,7′S)-{4′-Acetoxy-1′-p-Bromophenyl-3′-(tert-
butyldiphenylsilyloxymethyl)-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl}-
thymine (pBr-WNA-âT, (29a(p)). N,O-Bis(trimethylsilyl)aceta-
mide (BSA; 0.4 mL, 1.66 mmol) and TMSOTf (0.15 mL, 0.83
mmol) were added to a suspension of thymine (105 mg, 0.83 mmol)
in CH3CN (5 mL). A solution of 26(p) (360 mg, 0.55 mmol) in
CH3CN (5 mL) was added to the above mixture. The reaction
mixture was stirred at room temperature for 1 h, quenched with
saturated NaHCO3, and extracted with AcOEt. The organic layer
was washed with brine, dried over Na2SO4, and evaporated. The
isomers were separated by flash chromatography (silica gel, CH3-
Cl/hexane/acetone ) 2:4:1) to give each isomer in total 98% yield.
pBr-WNA-âT (29a(p)) as a colorless foam (215 mg, 0.30 mmol,
Synthesis of pBr-WNA-âT (Scheme 2) (1RS,2R,3R,4R)-1-p-
Bromophenyl-5-O-(tert-butyldiphenylsilyl)-2,3-O-isopropyliden-
eribose (23(p)). A solution of n-BuLi (1.6 M in hexane, 9.35 mL,
15.0 mmol) was added slowly to a solution of p-dibromobenzene
(3.5 g, 15.0 mmol) in THF (30 mL) at -78 °C in portions. After
stirring for 1 h at -78 °C, a solution of 22 (4.3 g, 10.0 mmol) in
THF (30 mL) was added to the mixture. The reaction mixture was
stirred for 2 h, allowed to warm to 0 °C, quenched with saturated
NH4Cl solution, and extracted with AcOEt. The organic layer was
successively washed with water and brine, dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography
(silica gel, hexane/AcOEt ) 9:1) to give 23(p) as a colorless oil
(5.1 g, 8.7 mmol, 87%). 1H NMR (400 MHz, CDCl3/TMS) δ 7.71-
7.65 (m, 4H), 7.48-7.40 (m, 10H), 4.90 (d, 0.7H, J ) 5.6 Hz),
4.84 (d, 0.3H, J ) 6.7 Hz), 4.46 (d, 1H, J ) 6.7 Hz), 4.49-4.31
(m, 1H), 3.94 (dd, 0.7H, J ) 11.2, 3.4 Hz), 3.84 (dd, 0.3H, J )
10.8, 4.9 Hz), 3.76 (dd, 0.7H, J ) 11.2, 3.4 Hz), 3.70 (dd, 0.3H, J
) 10.8, 4.9 Hz), 1.37 (s, 3H), 1.24 (s, 3H), 1.12 (s, 9H). FTIR
(film) 3350 cm-1. HRMS (ESIMS) m/z calcd for C30H34O4BrSi
(M - OH)+ 565.1404, 567.1389, found 565.1404, 567.1353.
1
55%). H NMR (400 MHz, CDCl3/TMS) δ 8.46 (bs, 1H), 7.69-
7.56 (m, 4H), 7.48-7.28 (m, 10H), 7.18 (s, 1H), 6.23 (dd, 1H, J )
8.4, 5.8 Hz), 5.08 (d, 1H, J ) 4.1 Hz), 4.27-4.22 (m, 1H), 4.00
(dd, 1H, J ) 11.6, 3.0 Hz), 3.71 (dd, 1H, J ) 11.6, 3.4 Hz), 2.86
(dd, 1H, J ) 14.0, 5.8 Hz), 2.55 (dd, 1H, J ) 14.0, 8.4 Hz), 2.04
(s, 3H), 1.97 (s, 3H), 1.01 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
170.1, 163.3, 138.7, 135.8, 135.6, 133.0, 132.8, 131.7, 129.8, 127.8,
127.8, 127.1, 122.0, 111.5, 92.1, 88.8, 86.4, 80.8, 72.8, 62.3, 48.0,
26.8, 20.7, 19.1, 12.6. FTIR (film) 2931, 1682, 1674, 1651 cm-1
.
HRMS (ESIMS) m/z calcd for C36H40N2O7SiBr (M + H)+
719.1783, 721.1770, found 719.1734, 721.1807.
(1′S,3′R,4′R,5′R,7′S)-(1′-p-Bromophenyl-4′-hydroxy-3′-hy-
droxymethyl-2′,6′-dioxabicyclo[3.3.0]oct-7′-yl)-thymine (pBr-
(1R,3R,4R,5R,7RS)-1-p-Bromophenyl-3-(tert-butyldiphenylsi-
lyloxymethyl)-4,7-diacetoxy-2,6-dioxabicyclo[3.3.0]octane (26-
(p)). A solution of 25(p) (5.0 g, 8.6 mmol) and allyltrimethylsilane
(3.33 mL, 20.5 mmol) in CH3NO2 (24 mL) was added to a
suspension of zinc bromide (6.6 g, 29 mmol) in CH3NO2 (24 mL)
at 0 °C. After stirring for 2 h at room temperature, the reaction
mixture was quenched with saturated NaHCO3 solution and
extracted with EtOAc. The organic layer was successively washed
with water and brine, dried over Na2SO4, and then evaporated. The
residue was purified by flash chromatography (silica gel, hexane/
EtOAc ) 9:1) to give the corresponding allyated product as a
colorless oil (R- and â-isomer mixtures, 4.59 g, 7.57 mmol, 88%).
Aqueous solutions of OsO4 (0.131 M, 9.2 mL, 1.21 mmol) and
NaIO4 (0.6 M, 50.5 mL, 30.3 mmol) were added to a solution of
the colorless oil (4.59 g, 7.57 mmol) in pyridine (50 mL), and the
reaction mixture was stirred for 30 h at room temperature. The
reaction mixture was diluted with AcOEt and successively washed
with water and brine, dried over Na2SO4, and evaporated. A solution
of the residue in THF (100 mL)/5% H2SO4 (30 mL) was stirred
for 12 h at 60 °C, quenched by the addition of saturated NaHCO3
solution, and extracted with AcOEt. The organic layer was
successively washed with water and brine, dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography
(silica gel, CHCl3/AcOEt ) 5:1) to give a colorless foam (908 mg,
1.59 mmol, 21%, for two steps). Acetic anhydride (0.25 mL, 2.8
mmol) was added to a solution of the colorless foam (400 mg, 0.7
mmol) in pyridine (3.4 mL) at 0 °C and stirred for 12 h at room
temperature. The reaction mixture was diluted with EtOAc and
successively washed with water and brine, dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography
(silica gel, hexane/AcOEt ) 6:1) to give 26(p) as a colorless foam
1
WNA-âT, 29b(p)). A colorless oil (72%). H NMR (400 MHz,
CD3OD) δ 7.70 (bs, 1H), 7.69 (s, 1H), 7.64 (d, 2H, J ) 8.6 Hz),
7.49 (d, 2H, J ) 8.6 Hz), 6.25 (dd, 1H, J ) 8.0, 6.5 Hz), 4.01-
3.98 (m, 1H), 3.91-3.87 (m, 2H), 3.70-3.61 (m, 2H), 2.73-2.72
(m, 2H), 1.92 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 166.4, 152.2,
141.1, 139.0, 132.4, 128.8, 122.5, 111.7, 92.8, 90.6, 90.4, 84.7,
73.4, 63.1, 49.2, 12.3. FTIR (film) 3430, 1660, 1633, 1550 cm-1
.
ESIMS (m/z) 439, 441 (M + H)+, HRMS (ESIMS) m/z calcd for
C18H20N2O6Br (M + H)+ 439.0449, 441.0481, found 439.0458,
441.0507.
(1′S,3′R,4′R,5′R,7′S)-{1′-p-Bromophenyl-3′-dimethoxytrithyl-
oxymethyl-4′-O-(N,N-diisopropyl-â-cyanoethylphosphoramidyl)-2′,6′-
dioxabicyclo[3.3.0]oct-7′-yl}-thymine (pBr-WNA-âT, 29c(p)). A
1
white powder (46%). H NMR (400 MHz, CDCl3/TMS) δ 8.21
(bs, 1H), 7.65-7.41 (m, 6H), 7.35-7.15 (m, 8H), 6.81 (d, 4H, J
) 6.0 Hz), 6.33 (t, 0.5H, J ) 8.2 Hz), 6.11 (t, 0.5H, J ) 7.3 Hz),
5.03 (d, 0.5H, J ) 3.2 Hz), 4.92 (d, 0.5H, J ) 3.2 Hz), 4.36-4.34
(m, 0.5H), 4.32-4.25 (m, 1H), 4.13-4.11 (m, 4H), 3.81 (s, 3H),
3.80 (s, 3H), 3.79-3.71 (m, 2H), 3.57-3.44 (m, 4H), 3.20 (dd,
1H, J ) 10.7, 4.3 Hz), 2.97-2.90 (m, 1H), 2.59-2.50 (m, 1H),
2.39-2.31 (m, 1H), 2.00 (s, 1.5H), 1.96 (s, 1.5H), 1.13 (d, 4H, J
) 6.7 Hz), 1.10-1.05 (m, 6H), 0.92 (d, 2H, J ) 6.7 Hz). 31P NMR
(161.9 MHz, CDCl3) δ 150.0, 149.2. FTIR (film) 2359, 1693, 1681,
1674, 1564, 1486 cm-1. ESIMS (m/z) 941, 943 (M + H)+.
Synthesis of the TFO Containing the WNA Analog. The
triplex-forming oligodeoxynucleotides incorporating the WNA
analogue were synthesized by using an automated DNA synthesizer
(Applied Biosystems 394 DNA/RNA synthesizer) according to the
standard protocol except for the use of DCI as the activator.
Cleavage and deprotection of the synthesized oligomer were done
in 28% NH4OH at 55 °C for 5 h. HPLC conditions: column,
Nacalai Tesque COSMOSIL5C18-AR-II; buffer A, 0.1 M TEAA,
B, CH3CN. B: 10% to 40%/20 min, 40% to 100%/30min, linear
gradient; flow rate, 4 mL/min. A peak appeared at around tR ) 16
min and was collected and freeze-dried. The DMTr protecting group
was cleaved in 10% aqueous acetic acid at room temperature for
30 min, the resulting DMTr-OH was removed by washing with
ether, and the solvents were lyophilized. Structure and purity of
the synthesized TFO were confirmed by MALDI-TOF MS mea-
surements (Table 2).
1
(375 mg, 0.57 mmol, 82%). H NMR (400 MHz, CDCl3/TMS) δ
7.72-7.63 (m, 5H), 7.54 (d, 1H, J ) 8.6 Hz), 7.48-7.31 (m, 8H),
6.63 (dd, 0.7H, J ) 5.7, 1.6 Hz), 6.50 (d, 0.3H, J ) 5.8 Hz), 5.02
(dd, 1H, J ) 9.6, 4.3 Hz), 4.89 (d, 0.3H, J ) 4.9 Hz), 4.78 (d,
0.7H, J ) 4.3 Hz), 4.52-4.50 (m, 0.3H), 4.23-4.19 (m, 0.7H),
4.07 (dd, 1H, J ) 11.8, 2.4 Hz), 3.74 (dd, 1H, J ) 11.8, 3.0 Hz),
2.84 (dd, 0.7H, J ) 15.3, 5.7 Hz), 2.70 (dd, 0.3H, J ) 15.0, 5.8
Hz), 2.59 (dd, 1H, J ) 15.3, 1.6 Hz), 2.14 (s, 2H), 2.04 (s, 4H),
1.05 (s, 7H), 1.02 (s, 2H). FTIR (film) 2931, 2858, 1752, 1747,
J. Org. Chem, Vol. 71, No. 5, 2006 2121