J.H. Ryu et al. / Tetrahedron 68 (2012) 72e78
77
purified chromatographically (SiO2; hexane/EtOAc, 1:2) to yield 3
(104 mg, 61%): mp >133 ꢀC dec; IR (film):
3421, 3180, 3060, 2929,
of-flight (MALDI-TOF) mass spectra of the ODNs were recorded
using a Kratos Analytical AXIMA LNR MALDI-TOF mass spectrom-
eter operated in the linear mode with an 8:1 mixture of 3-
hydroxypicolinic acid (0.35 M) and ammonium citrate (0.1 M) as
the matrix; the accelerating voltage was 20 kV.
n
2837, 2216, 1711, 1661, 1607, 1508, 1454, 1388, 1356, 1302, 1276,
1249, 1176, 1095, 1059, 1032, 965, 913, 878, 830, 792, 741, 701, 663,
645, 585 cmꢁ1 1H NMR (400 MHz, CDCl3):
; d 8.27 (s, 1H; H-6),
7.65e7.63 (m, 1H; fluoreneeH), 7.50e7.44 (m, 4H; fluoreneeH),
7.37e7.23 (m, 9H; fluoreneeH and DMTreH), 7.19e7.15 (m, 1H;
fluoreneeH), 7.12 (dd, J¼7.8, 1.4 Hz, 1H; DMTreH), 6.80 (dd, J¼9.2,
1.6 Hz, 4H; DMTreH), 6.37 (dd, J¼7.2, 6.0 Hz, 1H; H-10), 4.62e4.60
(m, 1H; H-30), 4.14e4.10 (m, 1H; H-40), 3.71 and 3.70 (2s, 6H; OCH3),
3.50 (dd, J¼11.2, 2.4 Hz, 1H; H-50), 3.33 (dd, J¼10.8, 3.2 Hz, 1H; H-
50), 2.54 (ddd, J¼14.9, 6.9, 2.7 Hz, 1H; H-20), 2.38 (ddd, J¼13.8, 7.4,
4.4. Melting temperature (Tm) measurements
All values of Tm of the ODNs (1.5 mM) were recorded in 10 mM
TriseHCl buffer (pH 7.2) containing 100 mM NaCl and 20 mM
MgCl2. Absorbance-versus-temperature profiles were measured at
260 nm using a Cary 100 Conc UVevis spectrophotometer equip-
ped with a temperature controller (cell path length: 1 cm). The
absorbance of the samples was monitored at 260 nm from 5 to
90 ꢀC at a heating rate of 1 ꢀC/min. Melting temperatures were
determined using a derivative method and Cary Win UV thermal
application software. Each measurement was run in triplicate.
6.2 Hz, 1H; H-20); 13C NMR (75 MHz, CD2Cl2):
d 193.2, 161.6, 159.3,
149.6, 145.2, 144.4, 143.3, 138.3, 136.2, 136.0, 135.5, 134.9, 134.5,
130.5, 130.4, 130.0, 128.6, 128.4, 127.5, 124.7, 123.8, 121.3, 120.7,
113.8, 100.3, 93.0, 87.6, 87.3, 86.4, 72.7, 64.0, 55.7, 42.1; HRMSeFAB
(m/z): [MþH]þ calcd for C45H37N2O8, 733.2551; found, 733.2543.
4.2.3. 50-O-[Bis(4-methoxyphenyl)phenylmethyl]-20-deoxy-5-(2-
ethynyl-9H-fluoren-9-onyl)-30-[2-cyanoethylbis(1-methylethyl)
4.5. UV and fluorescence measurements
phosphoramidyl]uridine
chlorophosphoramidite (72.2 mg, 0.306 mmol) was added drop-
wise to a solution of 3 (170 mg, 0.232 mmol) and 4-methyl-
morpholine (70 mL, 0.637 mmol) in CH2Cl2 at room temperature.
(4). 2-Cyanoethyl
N,N-diisopropyl-
ODN solutions were prepared as described above for the Tm
measurements. Absorption spectra were recorded using a Cary 100
Conc UVevis spectrophotometer (cell path length: 1 cm). Fluores-
cence spectra were recorded using a Hitachi F4500 spectrofluo-
rometer (cell path length: 1 cm; excitation at 340 nm). The
excitation and emission bandwidth was 1 nm.
After the reaction had reached completion (30 min), the mixture
was concentrated in vacuo and the residue purified chromato-
graphically through a short column (SiO2; hexane/EtOAc, 2:1) to
yield 4 (194 mg, 90%): mp 64e66 ꢀC; IR (film):
n 3179, 3065, 2966,
4.6. CD spectroscopy
2929, 2871, 2252, 2216, 1712, 1608, 1509, 1453, 1409, 1364, 1303,
1276, 1249, 1180, 1157, 1114, 1080, 1029, 975, 940, 879, 832, 790,
Mixtures of ODNs were equilibrated by cooling to 10 ꢀC; after
30 min, the CD spectra were recorded using a JASCO J-715 CD
spectropolarimeter. The temperature was controlled using a JASCO
PTC-348WI temperature controller.
767, 739, 702, 645, 581, 563 cmꢁ1 1H NMR (300 MHz, CDCl3):
;
d
8.34 and 8.29 (2s, 1H; NH), 7.64 and 7.62 (2s, 1H; H-6), 7.58e7.43
(m, 4H; fluoreneeH), 7.54e7.02 (m, 12H; fluoreneeH and
DMTreH), 6.87e6.70 (m, 4H; DMTreH), 6.40e6.31 (m, 1H; H-10),
4.72e4.56 (m, 1H; H-30), 4.29e4.18 (m, 1H; H-40), 3.82e3.79 (m,
1H; OCH2), 3.70 and 3.96 (2s, 6H; OCH3), 3.63e3.46 (m, 4H; NCH,
OCH2, H-50), 3.36e3.26 (m, 1H; H-50), 2.80e2.56 (m, 2H; CH2CN,
H-20), 2.46e2.36 (m, 2H; CH2CN, H-20), 1.32e1.02 (m, 12H;
Acknowledgements
We thank the National Research Foundation of Korea (NRF) for
financial support through the WCU program (R31-10105) and the
EPB center (2011-0001019). This study was also supported by the
Basic Science Research Program through the NRF, funded by the
Ministry of Education, Science, and Technology (2010-0007605).
NCHCH3); 31P NMR (121 MHz, CDCl3):
d 151.7, 151.3; HRMSeFAB
(m/z): [MþNa]þ calcd for C54H53N4O9PNa, 955.3451; found,
955.3448.
4.3. Synthesis of oligonucleotides
Supplementary data
ODNs were prepared using the
method on controlled pore glass supports (1
b
-cyanoethylphosphoramidite
mol) with a POLYGEN
Supplementary data associated with this article can be found, in
m
Professional 12-Column DNA synthesizer and standard methods.19
After automated synthesis, the oligonucleotides were cleaved from
the solid support and deprotected through treatment with 30%
aqueous NH4OH (1.0 mL) for 10 h at 55 ꢀC. The crude products from
the automated ODN synthesis were lyophilized and diluted with
distilled water (1 mL); they were then purified using high-
performance liquid chromatography (HPLC; Grace VyDACÔ C18
References and notes
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