2.46 (m, 8H), 1.52 (m, 8H), 1.40 (m, 4H); 13C-NMR (75 MHz,
DMSO-d6): d (ppm) 160.3 153.4, 148.2, 141.9, 136.8, 130.0, 128.7,
126.4, 119.7, 112.9, 64.1, 55.8, 52.7, 23.93, 22.2. ESI(+)-MS (m/z):
249.1 (100%), [a + 2H + Na + H2O + CH3OH]3+; 437.1 (90%), [a
function of the signal intensity to optimize data collection. Prior
to use the oligomer HTG22 d[AGGG(TTAGGG)3] dissolved in
corresponding buffer was heated to 95 ◦C for 5 min, then gradually
cooled to room temperature and incubated at 4 ◦C overnight.
HTG22 at a final concentration of 5 mM was resuspended in
10 mM Tris-HCl buffer (pH 7.4) with or without the specific
cations to be tested. CD titration was performed at a fixed
HTG22 concentration (5 mM) with various concentrations of the
compounds in the corresponding buffer. After each addition of
compound, the reaction was stirred and allowed to equilibrate
for at least 15 min (until no elliptic changes were observed) and
a CD spectrum was collected. A background CD spectrum of
corresponding buffer solution with or without compounds was
subtracted from the average scan for each sample. Final analysis
of the data was carried out using Origin 7.5 (OriginLab Corp.)
1
+ H + Na + H2O + 5CH3OH]2+. H and 13C NMR spectra were
shown in Fig. S6 and S7 (ESI†).
N,N¢-Bis(phenyl-4-ethoxypyrrole)-1,10-phenanthroline-2,9-
carboxamide (b). Compound b was prepared from ligand L
and N-(2-chloroethyl)pyrrolidine hydrochloride according to the
procedure described ◦for compound a above. Yellow solid (0.53 g,
64%); m.p. 158–160 C. IR (KBr, cm-1): 3446, 3321, 2956, 2925,
2775, 1678 (C O), 1596, 1542, 1512, 1475, 1245, 1132, 1107, 1033,
867, 827, 707, 667. 1H-NMR (300 MHz, DMSO-d6): d (ppm) 11.29
(s, 2H), 8.81 (d, J = 8.4 Hz, 2H), 8.56 (d, J = 8.1 Hz, 2H), 8.25 (s,
2H), 8.06 (d, J = 8.7 Hz, 4H), 7.09 (d, J = 9.0 Hz, 4H), 4.14 (t,
J = 5.7 Hz, 4H), 2.85 (t, J = 5.7 Hz, 4H), 2.58 (m, 8H), 1.72 (m,
8H); 13C-NMR (75 MHz, DMSO-d6): d (ppm) 162.6, 155.6, 150.4,
144.1, 139.1, 132.3, 130.9, 128.6, 122.0, 115.1, 67.4, 54.9, 54.6, 23.7;
ESI(+)-MS(m/z): 235.1 (100%), [b + 2H + Na + 2H2O]3+; 485.2
FRET assay
FRET assay was carried out on a Varian Cary Eclipse Fluores-
cence spectrometer equipped with a Peltier temperature control
accessory with excitation at 483 nm and detection at 500–700 nm.
Both excitation and emission slit are 5 nm. The fluorescent labeled
oligonucleotide F21T [5¢-FAM-d(GGG[TTAGGG]3)-TAMRA-
3¢], (donor fluorophore FAM: 6-carboxyfluorescein, and acceptor
fluorophore TAMRA: 6-carboxytetramethylrhodamine) used as
the FRET probes was diluted from stock to the correct concentra-
tion (400 nM) in either 10 mM sodium cacodylate buffer (pH 7.4)
containing 100 mM NaCl or 10 mM K2HPO4/KH2PO4 buffer
(pH 7.4) containing 100 mM KCl and then annealed by heating
to 90 ◦C for 5 min, followed by cooling to room temperature.
Samples were prepared by aliquoting 1 mL of the annealed F21T
(at 2¥ concentration, 400 nM) into cell, followed by 1 mL of buffer
or the compound solutions (at 2¥ concentration) and further
incubated for 1 h. The melting of G-quadruplex was monitored
alone or in the presence of various concentrations of compounds
and/or of either 2.0 or 5.0 mM double stranded competitor ds26
(5-CAATCGGATCGAATTCGATCCGATTG-3¢). Fluorescence
readings◦at 533 nm were taken at intervals of 2–3 ◦C over the range
of 5–95 C, with a constant temperature being maintained for 5
min prior to each reading to ensure a stable value. Final analysis
of the data was carried out using Origin 7.5 (OriginLab Corp.).
1
(90%), [b - (CH2)4N(CH2)2OC6H4NH - H + 2Na]+. H and 13C
NMR spectra were shown in Fig. S8 and S9 (ESI†).
N,N¢-Bis(phenyl-4-ethoxymorpholine)-1,10-phenanthroline-2,9-
carboxamide (c). Compound c was prepared from ligand L
and 4-(2-chloroethyl)morpholine hydrochloride according to the
procedure described ◦for compound a above. Yellow solid (0.61 g,
68%); m.p. 192–194 C. IR (KBr, cm-1): 3433, 3334, 3296, 2945,
1681 (C O), 1596, 1539, 1514, 1471, 1253, 1112, 1132, 1006,
873, 829, 709. 1H-NMR (300 MHz, DMSO-d6): d (ppm) 11.27 (s,
2H), 8.81 (s, J = 8.4 Hz, 2H), 8.56 (d, J = 8.4 Hz, 2H), 8.23 (s,
J = 10.1 Hz, 2H), 8.05 (d, J = 8.7 Hz, 4H), 7.08 (d, J = 8.7 Hz,
4H), 4.16 (t, J = 5.7 Hz, 4H), 3.60 (m, 8H), 2.73 (m, 4H), 2.49
(m, 8H); 13C-NMR (75 MHz, DMSO-d6): d (ppm) 162.5, 155.4,
150.3, 144.0, 138.9, 132.2, 130.8, 128.5, 121.8, 115.1, 66.5, 65.9,
57.5, 54.0. ESI(+)-MS(m/z): 672.2 (100%), [c + H]+; 472.2 (90%),
1
[c - (C6H4O(CH2)2C4H6ON) + H]+. H and 13C NMR spectra
were shown in Fig. S10 and S11 (ESI†).
N,N¢-Bis(phenyl-4-ethoxydimethylamino)-1,10-phenanthroline-
2,9-carboxamide (d). Compound d was prepared from ligand L
and 2-dimethylaminoethyl chloride hydrochloride according to
the procedure describe◦d for compound a above. Yellow solid (0.46
g, 59%); m.p. 176–178 C. IR (KBr, cm-1): 3454, 3323, 2956, 2927,
1666 (C O), 1595, 1542, 1514, 1467, 1384, 1250, 1172, 1134,
993, 867, 825, 810, 707, 667. 1H-NMR (300 MHz, methanol-d4):
d (ppm) 8.69 (d, J = 8.4 Hz, 2H), 8.56 (d, J = 8.4 Hz, 2H),
8.10 (s, 2H), 9.96 (d, J = 9.0 Hz, 4H), 7.18 (d, J = 9.0 Hz, 4H),
4.45 (m, 4H), 3.64 (m, 4H), 3.03 (m, 12H); 13C-NMR (75 MHz,
DMSO-d6): d (ppm) 164.0, 156.1, 151.7, 145.5, 140.5, 134.3,
132.4, 130.1, 123.3, 116.8, 65.1, 57.7, 45.2; ESI(+)-MS(m/z):
323.1 (100%), [d + Na + CH3OH]2+; 681.2 (90%), [d - 3H + 4Na]+.
1H and 13C NMR spectra were shown in Fig. S12 and S13 (ESI†).
TRAP assay
The ability of ligands to inhibit telomerase in a cell-free system
was assessed with the TRAP-LIG assay following previously pub-
lished procedures.12 Protein extracts from exponentially growing
HeLa cells were used. Briefly, 0.1 mg of TS forward primer (5¢-
AATCCGTCGAGCAGAGTT-3¢) was elongated by telomerase
(500 ng protein extract) in TRAP buffer (20 mM Tris-HCl [pH
8.3], 68 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, and 0.05%
Tween 20) containing 125 mM dNTPs and 0.05 mg BSA. The mix
was added to tubes containing freshly prepared ligand at various
concentrations and to a negative control containing no ligand.
The initial elongation step was carried out for 20 min at 30 ◦C,
followed by 94 ◦C for 5 min and a final maintenance of the mixture
at 20 ◦C. To purify the elongated product and to remove the bound
ligands, the QIA quick nucleotide purification kit (Qiagen) was
used according to the manufacturer’s instructions. The purified
CD spectroscopy
CD spectra were recorded on a dualbeam DSM 1000 CD
spectrophotometer (Olis, Bogart, GA). Each measurement was
the average of three repeated scans recorded from 220 to 320 nm
with a 0.1 mm quartz cell at 25 ◦C. The scanning rate (22
nm min-1) was automatically selected by the Olis software as a
2652 | Org. Biomol. Chem., 2011, 9, 2648–2653
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The Royal Society of Chemistry 2011
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