Intra- and Intermolecular Triplexes of Mixed DNA Sequences
A R T I C L E S
100: 17:1) to give the desired desilylated (ketone) nucleoside, which
was recrystallized from CH2Cl2/iPrOH as an off-white solid (1.24 g,
96% yield), mp 164-166 °C. 1H NMR (CDCl3): δ 2.72 (dd, 1 H, J )
14.0, 11.5 Hz, C2′H), 2.95 (dd, 1 H, J ) 14.0, 11.5 Hz, C2′′H), 3.98
(bs, 2 H, C5′H, C5′′H), 4.13 (t, 1 H, J ) 3.5 Hz, C4′H), 4.89 (bs, 2 H,
NH2), 5.74 (dd, 1 H, J ) 11.5, 4.5 Hz, C1′H), 6.78 (d, 1 H, J ) 9.5
Hz), 7.45 (d, 1 H, J ) 9.0 Hz), 7.56 (t, 1 H, J ) 9.0 Hz), 7.66 (d, 1
H, J ) 9.0 Hz), 8.23 (c, 1 H, J ) 9.5 Hz). HRMS (FAB): m/z 259.1074
(MH+), 258.10044 (calcd).
amidite (0.76 mL). The reaction was stirred at 0 °C for 10 min and
then at room temperature for 75 min. The final reaction was
concentrated, and the residue was dissolved in CHCl3 (30 mL) and
washed three times with saturated NaHCO3 solution (30 mL) and three
times with water (30 mL). The volatiles were removed, and the residue
was chromatographed on silica gel with CH2Cl2/hexane/Et2O/Et3N (100:
200:300:6). The final product is a pale yellow powder (0.37 g, 88%
1
yield). H NMR (CDCl3): δ 1.09-1.32 (m, 14 H), 1.54 (d, 6 H, J )
7.0 Hz), 2.1-2.18 (m, 1 H, C2′H), 2.46-2.68 (m, 3 H, C2′′H), 3.38-
3.46 (m, 2 H, C5′H, C5′′H), 3.58-3.62 (m, 1 H), 3.78 (bs, 6 H), 4.26
(bs, 1 H, C4′H), 4.60 (bs, 1 H, C3′H), 5.76 (m, 1 H, C1′H), 6.78-6.86
(m, 3 H), 7.24-7.80 (m, 15 H), 8.08 (s, 1 H). HRMS (FAB): m/z
833.40221 (MH+), 832.39650 (calcd).
5-(2′-Deoxy-â-D-threo-pentofuranosyl)-2-aminoquinoline (8). 7
(1.29 g) was dissolved in 120 mL of AcOH:CH3CN (1:1) and stirred
under N2 at -23 °C. NaHB(OAc)3 (1.5 g) was then added to the cooled
solution and the mixture stirred for 75 min. The reaction was
concentrated and the product obtained by silica gel chromatography
with CH2Cl2/MeOH/Et3N (100:0:1 to 100: 35:1). The product was
recrystallized from CH2Cl2/iPrOH as a white powder (1.11 g, 85%
Determination of the pKa Value of antiCG Nucleoside (8). An
aqueous 1.22 mM solution of antiCG nucleoside was prepared as a
standard solution. NaOAc buffer at different pH values (pH ) 4.27,
5.26, 5.66, 6.02, 6.63, 7.72, 7.25, 7.44, 7.59, 8.17, 9.38, 9.54) was
prepared by adjusting the pH value with HOAc or NaOH. A 1 mL
aliquot of the antiCG nucleoside standard solution was diluted with
buffer solution to 25 mL, and the pH value of the final solution was
measured again to verify that the pH value did not change. The UV-
visible spectra of the diluted solutions were then recorded from 200 to
400 nm. The UV-visible spectra from all 10 buffer solutions were
plotted and analyzed. The pKa value of the antiCG nucleoside was
determined to be 7.2. At pH 7 and room temperature, the extinction
coefficient was 4626 and 2956 at 330 and 350 nm, respectively.
1
yield), mp 74-78 °C. H NMR (DMSO-d6/CDCl3): δ 2.03 (m, 1 H,
C2′H), 2.38 (m, 1 H, C2′′H), 3.76 (m, 2 H, C5′H, C5′′H), 4.07 (m, 1
H, C4′H), 4.38 (m, 1 H, C3′H), 564 (bs, 2 H, NH2), 5.68 (dd, 1 H, J
) 10.0, 4.5 Hz, C1′H), 6.82 (d, 1 H, J ) 9.5 Hz), 7.45-7.50 (m, 3 H),
8.17 (d, 1 H, J ) 9.5 Hz). HRMS (FAB): m/z 261.12300 (MH+),
260.11609 (calcd).
N2-Isobutyryl-5-[2′-deoxy-â-D-threo-pentofuranosyl]-2-amino-
quinoline (9). 8 (1.30 g, 5 mmol) was dissolved in dry pyridine (80
mL) and cooled in an ice bath under N2. TMS-Cl (6 mL) was then
added and the reaction stirred at 0 °C for 30 min, at which time
isobutyric anhydride (3.75 mL) was added. The reaction was stirred
for 2 h at room temperature under N2, and then it was cooled in an ice
bath and cold water (16 mL) added. The reaction mixture was stirred
for another 15 min, and then concentrated NH4OH (16 mL) was added
to give a solution approximately 2 M in ammonia. The final mixture
was stirred for another 30 min in an ice bath and then concentrated in
vacuo to afford an oil, which was chromatographed on silica gel with
CH2Cl2/MeOH/Et3N (100:0:1 to 100:9:1). The product obtained was
recrystallized from CH2Cl2/Et2O as a white powder (1.40 g, 85% yield),
NMR Conformational Studies. The conformational analysis of the
antiCG (8) TRIPside was determined by 1H NMR on a Varian INOVA
500-MHz spectrometer using NOESY with a presaturation field strength
γB1 of 50 Hz and a mixing time of 0.4 s at 25 °C in 10 mM sodium
phosphate buffer (pH 7.0) in D2O containing 50 mM NaCl. The sample
concentration was 18.4 mM, and the relaxation delay for the NOESY
experiment was 2.2 s.
Oligonucleotide Synthesis and Purification. The syntheses of the
oligomers were performed on a 200 nmol scale using standard solid-
phase phosphoramidite chemistry on an ABI 394 instrument. The
oligoTRIP was cleaved from the column and deprotected with 1 mL
of concentrated NH4OH, placed in a sealed bottle, and maintained at
55 °C for 8 h. The 5-DMTr-oligoTRIPs were purified by HPLC: flow
rate, 4 mL/min; solvent, 0.1 M TEAA (pH 7.0)/CH3CN (9:1) for 10
min, to 0.1 M TEAA (pH 7.0)/CH3CN (3:2) over 15 min, and then to
100% CH3CN over 5 min. The HPLC fractions containing the oligomer
were combined and lyophilized, and the residue was detritylated with
80% HOAc solution (30 µL per OD) at room temperature for 20 min.
The final product was purified on a Sephadex G-25M column, and the
products were analyzed by MALDI-MS.
1
mp 102-104 °C. H NMR (DMSO-d6): δ 1.12 (d, 6 H, J ) 7.0 Hz),
1.84-1.90 (m, 1 H, C2′H), 2.26-2.32 (m, 1 H, C2′′H), 2.78-2.86
(m, 1 H), 3.46-3.60 (m, 2 H, C5′H, C5′′H), 3.89 (m, 1 H, C4′H), 4.23
(bs, 1 H, C3′H), 4.82 (t, 1 H, J ) 2.5 Hz, C5′OH), 5.14 (d, 1 H, J )
4.0 Hz, C3′OH), 5.66 (dd, 1 H, J ) 10.0, 4.5 Hz, C1′H), 7.60-7.70
(m, 3 H), 8.30 (d, 1 H, J ) 9.5 Hz), 8.50 (d, 1 H, J ) 9.5 Hz) 10.73
(s, 1 H). HRMS (FAB): m/z 331.16541 (MH+), 330.15796 (calcd).
N2-Isobutyryl-5-[2′-deoxy-â-D-threo-pentofuranosyl-5′-O-(4,4′-
dimethoxytrityl)]-2-aminoquinoline (10). 9 (0.66 g, 2 mmol) was
dissolved in dried pyridine (30 mL) under N2. 4,4′-Dimethoxytrityl
chloride (DMTr-Cl) (0.70 g) and Et3N (1 mL) were then added to the
solution at room temperature. An additional aliquot each of DMTr-Cl
and Et3N were added after 3 h. The reaction was stirred under N2 at
room temperature for a total of 4 h. The reaction was then concentrated,
and the residue was dissolved in CHCl3 (30 mL) and washed three
times with saturated NaHCO3 solution (30 mL) and then three times
with water (30 mL). The solution was concentrated and the residue
chromatographed on silica gel with CH2Cl2/MeOH/Et3N (100:0:1 to
100:5:1). The product was recrystallized from Et2O/hexanes as a white
powder (0.92 g, 73% yield), mp 61-63 °C. 1H NMR (CDCl3): δ 1.12
(d, 6 H, J ) 7.0 Hz), 2.12-2.18 (m, 1 H, C2′H), 2.42-2.49 (m, 1 H,
C2′′H), 2.58-2.67 (m, 1 H), 3.36-3.46 (m, 2 H, C5′H, C5′′H, C5′′H),
3.78 (s, 6 H), 4.14 (m, 1 H, C4′H), 4.48 (bs, 1 H, C3′H), 5.80 (dd, 1
H, J ) 10.0, 4.5 Hz, C1′H), 6.82-6.90 (m, 3 H), 7.20-7.64 (m, 15
H), 8.44 (s, 1 H). HRMS (FAB): m/z 633.29435 (MH+), 632.28864
(calcd).
UV-Visible Melting Curves. Absorbance vs temperature profiles
were measured simultaneously at 250 and 330 nm with a thermoelec-
trically controlled AVIV 14 UV/vis spectrophotometer (Lakewood, NJ).
The absorbance was monitored in the temperature range of 0-110 °C,
increasing the temperature at a rate of ∼0.4 °C/min. In these
experiments, the 330 nm wavelength is used to monitor the absorbance
changes of the C-glycoside bases of the oligoTRIPs, while the 250 nm
wavelength follows the changes of the nucleobases of the Watson-
Crick duplex. Shape analysis of the resulting melting curves, using
procedures described earlier, yielded transition temperatures, TM, and
24
model-dependent van’t Hoff enthalpies, ∆HvH
.
Methylation Protection. An individual strand was 5′-end-labeled25
and purified by PAGE. The purified 5′-[32P]oligomers were heated to
95 °C for 3 min in 10 mM sodium phosphate buffer (pH 7.0) containing
1 M NaCl and then slowly cooled to room temperature. The oligoTRIP
(0-3 equiv) was added and the incubation maintained at 10 °C
overnight. The solution was cooled to 8 °C, and the G, G+A, and C
N2-Isobutyryl-5-[2′-deoxy-â-D-threo-pentofuranosyl-3′-O-(2-cya-
noethoxy)(diisopropylamino)phosphino-5′-O-(4,4′-dimethoxytrityl)]-
2-aminoquinoline (11). 10 (0.316 g, 0.5 mmol) was dissolved in dry
CH2Cl2 (10 mL) under N2 and cooled in an ice bath. Hunig base (0.8
mL) was added, followed by 2-cyanoethyl-N,N-diisopropylphosphor-
(24) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601-1620.
(25) Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980, 65, 499-560.
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J. AM. CHEM. SOC. VOL. 127, NO. 36, 2005 12659