Triplex-Forming Molecules
A R T I C L E S
1 H, C5′OH), 5.16 (bs, 2 H, NH2), 6.24 (d, J ) 6.5, 1 H, C1′H), 6.52
(d, J ) 6.5, 1 H, C2′H), 7.38-7.53 (m, 10 H), 7.80-7.86 (m, 3 H),
9.38 (s, 1 H).
1 H, C3′OH), 5.89 (dd, J ) 4.0, 7.5, 1 H, C1′H), 6.86 (d, J ) 9.0, 4
H), 7.20-7.28 (m, 7 H), 7.38 (d, J ) 8.5, 2 H), 7.65 (d, J ) 7.5, 1 H),
7.72 (d, J ) 8.5, 1 H), 7.87 (dd, J ) 7.5, 8.5, 1 H), 9.69 (s, 1 H),
10.70 (s, 1 H); HRMS (FAB) m/z 656.2735 ([M + Na]+), 656.2737
(calcd).
5-[â-D-Glyceropentofuran-3′-ulos-1′-yl]-2-aminoquinazoline (6).
Tetrabutylammonium fluoride (3.5 mL, 1 M in THF) was added to a
cooled (ice bath) solution of nucleoside 5 in THF (10 mL). Then a
catalytic amount of concentrated AcOH (250 µL) was added and the
reaction stirred at 0 °C under N2 for 1 h and then concentrated under
reduced pressure. The product was purified by flash chromatography
(EtOAc: MeOH, 9:1). The final product was a pale ivory colored
powder (0.43 g, 98% yield): 1H NMR (DMSO-d6) δ 2.98 (dd, J )
12.0, 5.5, 2 H, C2′H, C2′′H), 3.69-3.72 (m, 2 H, C5′H, C5′′H), 4.08
(t, J ) 3.1, 1 H, C4′H), 5.81 (dd, J ) 5.0, 5.5, 1 H, C1′H), 6.80 (s, 2
H, NH2), 7.38 (d, J ) 7.5, 1 H), 7.45 (d, J ) 6.5, 1 H), 7.65 (dd, J )
7.5, 6.5, 1 H), 9.46 (s, 1 H); HRMS (FAB) m/z 259.0960 (M+),
259.0957 (calcd).
N2-Isobutyryl-5-[2′-deoxy-â-D-threo-pentofuranosyl-3′-O-(2-cya-
noethoxy)(diisopropylamino)phosphino-5′-O-(4,4′-dimethoxytrityl)]-
2-aminoquinazoline (10). Dry trityl compound 9 (95 mg) was dissolved
in dry CH2Cl2 (4 mL) and cooled in an ice bath under N2. Then Hunig
base (120 µL) was added followed by 2-cyanoethyl-N,N-diisopropyl-
phosphoramidite (60 µL). The reaction was stirred at 0 °C for 10 min
and then at room temperature for 1 h. The final reaction was
concentrated under reduced pressure and the residue chromatographed
(Et2O containing 1% Et3N). The final product was a white powder (0.1
g, 79% yield): 1H NMR (DMSO-d6) δ 1.12 (d, J ) 7.0, 6 H), 1.17-
1.28 (m, 12 H), 2.07-2.10 (m, 1 H, C2′H), 2.46-2.48 (m, 1 H), 2.50-
2.54 (m, 1 H, C2′′H), 2.65-2.68 (m, 1 H), 3.30-3.36 (m, 2 H, C5′H,
C5′′H), 3.50 (dd, J ) 7.5, 6.5, 1 H), 3.50-3.68 (m, 4 H), 3.78 (s, 6
H), 4.32 (bs, 1 H, C4′H), 4.59 (bs,1 H, C3′H), 5.84 (m, 1 H, C1′H),
6.78 (m, 4 H), 7.20-7.35 (m, 7 H), 7.43-7.46 (m, 2 H), 7.69 (m, 1
H), 7.70-7.80 (m, 2 H), 8.04 (s, 1 H), 9.65 (s, 1 H); HRMS (FAB)
m/z 856.3837 ([M + Na]+), 856.3815 (calcd).
Oligonucleotide Synthesis and Purification. The synthesis of the
oligomers was performed on a 200 nmol scale using standard solid-
phase phosphoramidite methodology on an ABI 394 instrument. For
200 nmol scale synthesis, a 0.1 M MeCN solution of phosphoramidite
(10) was used. On the basis of preliminary runs, the coupling time
was set to 7 min with greater than 99% coupling efficiency as
determined by trityl assay. After the synthesis, the resin inside the
cartridge was mixed with 1 mL of concentrated NH4OH, placed in a
sealed bottle, and deprotected at 55 °C for 8 h. The crude oligomers
were purified by HPLC: flow rate, 4 mL/min; solvent, 0.1 M TEAA
(pH 7.0)/MeCN (9:1) for 10 min, to 0.1 M TEAA (pH 7.0)/MeCN
(3:2) over 15 min, and then to 100% MeCN over 5 min. The HPLC
fractions containing the oligomer were combined and lyophilized and
the residue detritylated with 80% acetic acid solution (30 µL per OD)
at room temperature for 20 min. The final product was purified on a
Sephadex G-25M column. The products were analyzed by MALDI-
MS.
5-[2′-Deoxy-â-D-threo-pentofuranosyl]-2-aminoquinazoline (7).
Ketone 6 (0.65 g) was dissolved in 80 mL of AcOH:CH3CN (1:1) and
stirred under N2 at -23 °C (dry ice/CCl4). NaHB(OAc)3 (0.6 g) was
added to the cooled solution and the mixture stirred under N2 at -23
°C for 1 h at which time 3% H2O2 (0.5 mL) was added. The reaction
was concentrated and stirred with Na2CO3 (3 g) in 10 mL of CH2Cl2/
MeOH (1:1) at room temperature for 1 h. The product was purified by
flash chromatography with EtOAc:MeOH (4:1). The final product was
obtained as a pale yellow powder (0.38 g, 59% yield): 1H NMR
(DMSO-d6) δ 1.90-1.95 (m, 1 H, C2′H), 2.25-2.28 (m, 1 H, C2′′H),
3.46-3.55 (m, 2 H, C5′H, C5′′H), 3.86 (dd, J ) 3.0, 5.0, 1 H, C4′H),
4.23 (br s, 1 H, C3′H), 5.66 (dd, J ) 4.5, 6.0, 1 H, C1′H), 6.78 (s, 2
H, NH2), 7.31 (d, J ) 7.5, 1 H), 7.33 (d, J ) 8.5, 1 H), 7.61 (dd, J )
8.5, 7.5, 1 H), 9.34 (s, 1 H); HRMS (FAB) m/z 262.1189 (MH+),
262.1192 (calcd).
N2-Isobutyryl-5-[2′-deoxy-â-D-threo-pentofuranosyl]-2-amino-
quinazoline (8). Alcohol 7 (0.38 g) was dissolved in dry pyridine (20
mL) and cooled in an ice bath. Then TMS-Cl (1 mL) was added to the
cooled solution under N2. The reaction was stirred at 0 °C for 30 min
at which time isobutyric anhydride (1.3 mL) was added and the reaction
stirred for an additional 2 h at room temperature. After 2 h, the reaction
was again cooled in an ice bath and 2.5 mL of cold water added. The
reaction was stirred for another 15 min; then concentrated NH4OH (2.5
mL) was added to give a final concentration of approximately 2 M in
ammonia. This mixture was stirred for another 30 min in an ice bath
and then concentrated under reduced pressure to afford an oil. The oil
was dissolved in a very small amount of water, which was washed
with Et2O. The aqueous layer was collected and concentrated and the
residue purified by flash chromatography (EtOAc:MeOH, 9:1) to give
the desired product as a yellow powder (0.3 g, 63% yield): 1H NMR
(DMSO-d6) δ 1.12 (d, J ) 7.0, 6 H), 1.95-2.01 (m, 1 H, C2′H), 2.31-
2.35 (m, 1 H, C2′′H), 2.88-2.94 (m, 1 H), 3.50-3.60 (m, 2 H, C5′H,
C5′′H), 3.91 (dd, J ) 3.0, 5.0, 1 H, C4′H), 4.26 (bs, 1 H, C3′H), 4.86
(t, J ) 5.5, 1 H, C5′OH), 5.19 (d, J ) 4.0, 1 H, C3′OH), 5.77 (dd, J
) 4.5, 6.0, 1 H, C1′H), 7.67 (d, J ) 7.5, 1 H), 7.70 (d, J ) 8.5, 1 H),
7.89 (dd, J ) 8.5, 7.5, 1 H), 9.71 (s, 1 H), 10.68 (s, 1 H); HRMS
(FAB) m/z 331.1532 (M+), 331.1535 (calcd).
Thermal Denaturation Studies. Melting curve transition measure-
ments were performed on an AVIV 14 DS UV/vis spectrophotometer
equipped with an IBM computer as thermoprogrammer. Oligomers
(3-6 µM) were dissolved in 10 mM sodium phosphate (pH 7.0)
containing 50 or 200 mM NaCl, and when specified with netropsin.
The oligomers were heated to 80 °C and slowly cooled to 0 °C and
held at that temperature for 30 min. The temperature was scanned from
0 to 80 °C at a heating rate of 0.2 °C/min with data-point collection in
intervals of 0.2 °C. Below 20 °C, the cell compartment was flushed
with N2 to avoid condensation of water on the UV cells. The unfolding
of the oligomers were measured by the change in absorbance at 260
and 350 nm versus temperature. The transition temperature TM was
determined by shape analysis of the melting curves.8
Fluorescent Studies. Fluorescence emission spectra were recorded
from 330 to 600 nm with data-point collection in intervals of 2 nm on
an AVIV Automated Titrating Differential/Ratio Spectroflurometer
(Model ATF105) with a fixed excitation wavelength at 350 nm. The
emission and excitation slits were set at 5 nm. DNA melting curve
transitions measured by fluorescence change as a function of temper-
ature were conducted with the same spectroflurometer with a fixed
excitation wavelength at 350 nm and a fixed emission wavelength at
425 nm. OL-1, -2, and -5 (3-6 µM) and ketone 6 (4 µM) were
dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 200
mM NaCl. The oligomers were heated to 75 °C, then slowly cooled to
0 °C, and held at 0 °C for 1 h. The temperature was scanned from 0 to
N2-Isobutyryl-5-[2′-deoxy-â-D-threo-pentofuranosyl-5′-O-(4,4′-
dimethoxytrityl)]-2-aminoquinazoline (9). Dry 8 (0.215 g) was
dissolved in anhydrous pyridine (5 mL) under N2. Then 4,4′-dimethox-
ytrityl chloride (0.28 g), DMAP (6 mg), and Et3N (0.2 mL) were added
to the solution at room temperature. After 5 h, additional aliquots of
4,4′-dimethoxytrityl chloride, DMAP, and Et3N were added. The
reaction was stirred under N2 at room temperature for a total of 24 h
and the reaction concentrated under reduced pressure and quickly flash
chromatographed (EtOAc containing 1% Et3N). The final product was
a white powder (0.25 g, 61% yield): 1H NMR (DMSO-d6) δ 1.12 (d,
J ) 7.0, 6 H), 2.07-2.10 (m, 1 H, C2′H), 2.41-2.44 (m, 1 H, C2′′H),
2.91-2.95 (m, 1 H), 3.12-3.19 (m, 2 H, C5′H, C5′′H), 3.72 (s, 6 H),
4.01-4.05 (m, C4′H), 4.22 (bs,1 H, C3′H), 5.25 (d, J ) 4.5,
(8) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601-1620.
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J. AM. CHEM. SOC. VOL. 125, NO. 8, 2003 2087