Cationic N-confused porphyrin derivative as a better molecule scaffold for G-quadruplex recognition

Article abstract of DOI:10.1016/j.bmc.2009.12.049

One N-confused porphyrin derivative was prepared and its first observation that it could stabilize G-quadruplex and possessed high selectivity over duplex DNA was made, furthermore, it was also proved to possess the capacity to induce a structural transition from the antiparallel to the mixed-type hybrid G-quadruplex structure.

Full text of DOI:10.1016/j.bmc.2009.12.049

Bioorganic & Medicinal Chemistry 18 (2010) 1111–1116
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cationic N-confused porphyrin derivative as a better molecule scaffold
for G-quadruplex recognition
Yuhao Du a, , Dan Zhang a, , Wei Chen a, Ming Zhang a, Yangyang Zhou a, Xiang Zhou a,b,c,d,
*
a College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, Minist Educ, Key Lab Biomed, Wuhan University, Hubei, Wuhan 430072, PR China
b State Key Laboratory of Bio-Organic & Natural Products Chemistry, Shanghai Institute of Organic Chemistry Chinese Academy of Sciences, Shanghai 200032, PR China
c State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, PR China
d State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
One N-confused porphyrin derivative was prepared and its first observation that it could stabilize G-
quadruplex and possessed high selectivity over duplex DNA was made, furthermore, it was also proved
to possess the capacity to induce a structural transition from the antiparallel to the mixed-type hybrid G-
quadruplex structure.
Received 13 November 2009
Revised 15 December 2009
Accepted 16 December 2009
Available online 24 December 2009
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cationic N-confused
Porphyrin
G-quadruplex
1. Introduction
mode. For instance, the c-myc promoter region has a parallel
mode7 while the bcl-2 promoter region has a mixed parallel/anti-
It has been reported by numerous groups that guanine-rich nu-
cleic acid sequences are able to form G-quadruplex structures via
Hoogsteen base pair.1–3 A large number of putative sequences in
many biologically pivotal genomic regions that might form quadru-
plex structures have been identified, such as the chromosomal telo-
meres,4 the promoters of oncogenes,5–9 and the immunoglobulin
switch region.10 Among these reports, small molecules targeting
the G-quadruplex have aroused a significant amount of interest
due to their potential biological and clinical applications in drug de-
sign and disease diagnosis.11–29 Therefore, we have more opportuni-
ties to design specific molecules that specifically recognize and
stabilize G-rich sequences.
The human telomeric quadruplex has been extensively investi-
gated, and it has been reported that the formation of G-quadruplex
structures will lead to the inhibition of telomerase activity and
interfere with telomere extension.30,31 Moreover, such structures,
if stabilized, may lead to tumor cell death. Several convincing
reports have indicated that quadruplex motifs are particularly en-
riched in the promoter regions of genes.5–9 This suggests that
G-quadruplex formation may be involved in the regulation of gene
expression.32–35 The promoter regions of genes such as c-myc and
bcl-2 have an inherent conformational G-quadruplex structure
parallel mode.8 Thus, the development of new G-quadruplex li-
gands, especially the sequence and mode selective ligands, is a
promising approach for the development of cancer chemothera-
peutics and for cancer diagnosis.
N-Confused porphyrin (NCP) has a nitrogen atom pointing out-
ward and a carbon atom located in its internal core. In addition,
NCP contains a confused pyrrole ring connected to the meso-car-
bons at the
a
and b positions.36–38 The inner and outer nitrogen
atoms can undergo NH-tautomerism generating two types of tau-
tomers (Scheme 1).38 Because NCP has an additional external func-
tional site for protonation or coordination, this molecule garners
particular interest to a variety of researchers. However, few reports
have been published in regard to the medicinal or biological appli-
cations of NCP.39
Ph
Ph
NH
N
N
NH
Ph
Ph
Ph
Ph
NH
N
N
HN
Ph
* Corresponding author. Tel.: +86 27 61056559; fax: +86 27 87336380.
E-mail address: xzhou@whu.edu.cn (X. Zhou).
Ph
These two authors contributed equally to this work.
Scheme 1. NH-tautomerism of NCP.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.049

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Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 1111–1116
Herein, we report the synthesis of a water soluble cationic NCP
The binding ability was further elucidated using the Taq DNA
polymerase stop assay to determine the NCP-induced stability of
the quadruplex based on the concentration of the template strand
at low K+ concentration.43 The primer extension assay using Taq
polymerase stopped at the location of the DNA substrate that cor-
responded to the G-quadruplex structure. These results indicated
that the pPyNCP led to the significant inhibition observed for Taq
polymerase at the G-quadruplex site (Fig. S4, see Supplementary
data). Also, with increasing concentrations of the ligand, there
was enhanced pausing at the G-quadruplex site on the DNA tem-
plate. This suggests that pPyNCP could bind to and stabilize the
G-quadruplex.
derivative and investigate its interaction with a G-quadruplex
structure. The basis of our design preserves the planar macrocyclic
ring skeleton to enable interactions with G-quartets. The cationic
side chains of this molecule are introduced to enhance the water
solubility and binding ability of NCP via grooves of DNA in addition
to the negatively charged sugar–phosphate backbone. For the first
time, we have identified an NCP derivative that has sufficient se-
quence and mode selective binding to G-quadruplex structures
as evaluated by CD spectroscopy, a Taq polymerase stop assay,
SPR experiments, CD melting curves, and FRET melting curves
coupled with competitor and qualitative molecular modeling
(Scheme 2).
2.2. Detection of the conformation of telomere sequence
induced by ligands
2. Results and discussion
Circular dichroism (CD) spectroscopy was used to confirm the
above results and to investigate the conformation of the quadru-
plex induced by the ligand. Without the addition of any metal cat-
ions, the human telomeric sequence (T2AG3)4 generated a positive
peak near 256 nm. Interestingly, the human telomere sequence is
highly polymorphic and can exist as parallel,44 antiparallel,45 and
mixed-type parallel/antiparallel structures.46 With the addition of
increasing concentrations of pPyNCP, there was a sharp decrease
in the 257 nm band accompanied by the detection of a small band
with positive ellipticity near 270 nm and a dramatic increase in the
band ellipticity observed around 295 nm (Fig. 1a). Thus the resul-
tant CD spectroscopic profile indicated that pPyNCP could induce
the formation of hybrid parallel/antiparallel G-quadruplex struc-
tures.23,47 In a control experiment, pPyP was only prone to induce
the formation of the G-quadruplex, but the conformational change
2.1. Stabilization of ligands to G-quadruplex
The ligand’s ability to stabilize a G-quadruplex structure was
investigated by the analysis of CD melting curves using the G4C
oligomer (sequence 50-CATGGTGGTTTGGGTTAGGGTTAGGGTTAGG
GTTACCAC-30). The results indicated that pPyNCP was able to in-
duce the stabilization of the G-quadruplex in a physiologically rel-
evant K+ buffer. The
DTm values increased sharply with increasing
concentrations of pPyNCP, approximately reaching its maxima at
r = 5 (r = [pPyNCP]/[G4C], Table 1, Figs. S1–S3 of Supplementary
data). The binding affinity of pPyNCP is similar to the well-investi-
gated G-quadruplex stabilizer TMPyP440–42 and has stronger
affinity than the pPyP (5,10,15,20-tetrakis(a-pyridinio-p-tolyl)por-
phyrin tetrabromide salt), which possesses the same side chains as
pPyNCP.
N
Br
N
NH
NH
b
N
Br
Br
N
N
N
HN
NH
N
a
4Br
pPyNCP
Br
N
H
N
2
+
Br
N
Br
c
CHO
1
NH
N
d
N
HN
N
Br
Br
N
N
N
HN
NH
4Br
Br
N
3
pPyP
Scheme 2. Synthesis of pPyNCP. Reagents and conditions: (a) methanesulfonic acid (MSA), 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), CH2Cl2, 40 °C, 30 min; (b)
pyridine, 40 °C, 7 days; (c) BF3ꢀEt2O, chloranil, CHCl3, 2 h; (d) pyridine, 110 °C, 1.5 h.

Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 1111–1116
1113
Table 1
CD melting data of 10
l
M oligonucleotide (G4C) at different concentrations of pPyNCP and TMPyP4 in 10 mM Tris–HCl buffer (1 mM EDTA, pH 7.4), 100 mM KCl
Samples
Control (°C)
r = [pPyNCP]/[G4C]
1
2
3
4
5
pPyNCP
TMPyP4
pPyP
Tm
56.6 0.8
60.4 0.3
3.8 0.4
60.8 1.9
4.2 0.8
58.8 0.7
2.2 0.1
63.2 0.6
6.6 0.2
64.1 1.3
7.5 0.3
59.9 1.1
3.3 0.2
64.9 0.4
8.3 0.3
66.2 0.9
9.6 0.0
61.6 1.2
5.0 0.2
65.8 1.0
9.2 0.1
67.8 0.6
11.2 0.2
63.1 1.3
6.5 0.3
65.9 1.7
9.3 0.6
66.6 1.1
10.0 0.2
64.2 0.9
7.6 0.0
D
Tm
Tm
D
Tm
Tm
D
Tm
+
control
Na control
4
3
2
1
0
pPyNCP r=1
pPyNCP r=2
pPyNCP r=3
pPyNCP r=5
pPyNCP r=1
pPyNCP r=2
pPyNCP r=3
pPyNCP r=5
2
1
0
-1
220
-1
220
240
260
280
300
320
340
240
260
280
300
320
340
wavelength (nm)
wavelength (nm)
Figure 1. (a) CD titration of (T2AG3)4 in 10 mM Tris–HCl 1 mM EDTA, pH 7.4; (b) CD titration of (T2AG3)4 in 10 mM Tris–HCl, 1 mM EDTA, pH 7.4, 100 mM NaCl. Arrows from
bottom to up indicated increments in r values from 0 to 5 (r = [pPyNCP]/[(T2AG3)4]).
was not characteristic of an antiparallel or a hybrid of a parallel/
antiparallel G-quadruplex structure (Fig. S5).
mixed antiparallel/parallel quadruplex mode as determined from
the CD analysis, since the bcl2 promoter sequence had the same
structural conformation. When compared with TMPyP4 and pPyP,
pPyNCP showed a greater selectivity and preference for the hybrid
of a parallel/antiparallel G-quadruplex structure (Figs. S9 and S10
of Supplementary data).
To further confirm the binding selectivity observed using the
SPR analysis, the interaction of pPyNCP with G-quadruplex DNA
in the presence of various concentrations of competitor dsDNA
was studied using a competitive FRET melting assay.52,53 The re-
sults clearly showed that a high level of stabilization of the G-
quadruplex by pPyNCP was maintained even with a large excess
The results shown in Figure 1b indicated that pPyNCP was
prone to inducing the structural transition from the antiparallel
structure formed by Na+ to the mixed-type hybrid G-quadruplex,
as indicated by the increase in the CD signal at 265 nm from a neg-
ative peak to a small positive band. To our knowledge, only a few
ligands are known to induce the structural transition of the human
telomere sequence.21,22,48,49 As for pPyP, the DNA sequence cannot
change to the characteristic band known for a hybrid of a parallel/
antiparallel G-quadruplex structure (Fig. S6). In the presence of a
K+ buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.4, and 100 mM
KCl), the telomeric sequence (T2AG3)4 formed a hybrid of paral-
lel/antiparallel G-quadruplex structures in the absence of any com-
pound.47 It was easy to identify, when the addition of pPyNCP, the
hybrid G-quadruplex structure still retained (Fig. S7, see Supple-
mentary data). Therefore, it was reasonable to conclude that
pPyNCP possessed the ability to induce and maintain the hybrid
G-quadruplex structure formed from a telomeric sequence.
of competitor ds22 (10 lM). This indicated that pPyNCP possessed
high selectivity for G-quadruplex DNA versus dsDNA (Fig. 2).
To understand the strong interaction between NCP and G-quad-
ruplex, a close examination of the interaction between pPyNCP and
G-quadruplex DNA was studied by qualitative molecular modeling
using molecular dynamics and energy minimization methods. The
results clearly illuminated sufficient overlap between the aromatic
macrocycle scaffold and the guanine quartets. This binding mode is
2.3. The binding selectivity between ligands and different kinds
of G-quadruplex
characterized by favorable p–p stacking interactions between
pPyNCP and the G-quadruplex structure (Fig. 3). The four cationic
side chains were properly oriented and directed into the DNA
groove toward the negatively charged sugar–phosphate backbone.
We further investigated the binding abilities between G-quad-
ruplex DNA and pPyNCP using surface plasmon resonance
(SPR).8,17,25,50,51 The human telomeric G-quadruplex sequence,
the well-characterized c-myc and bcl2 promoter G-quadruplex se-
quences, and a duplex DNA hybridized with its complementary
strand, which served as a control, had been chosen to evaluate
whether pPyNCP could distinguish among various modes of quad-
ruplex formation and between dsDNA. The SPR results showed that
pPyNCP possessed high binding ability, significant selectivity for G-
quadruplex DNA over dsDNA, and 108-fold selectivity for bcl2 over
dsDNA (see Table 2 and Fig. S8 of Supplementary data). These re-
sults were also consistent with the preference of pPyNCP for the
3. Conclusion
To our knowledge, these data demonstrate for the first time that
NCP can serve as G-quadruplex structure stabilizer. A variety of
experimental results demonstrated that pPyNCP could induce
and stabilize the formation of G-quadruplex DNA. For example,
SPR sensorgrams showed the high affinity and significant selectiv-
ity of pPyNCP for G-quadruplex DNA over dsDNA, and CD titration
experiment indicated that it possess the capacity to induce a

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Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 1111–1116
Table 2
Kinetic rate constants and the chi2 (v2) for DNA–drug interactions using kinetics simultaneous algorithm by the model of binding with mass transfer
Compd
DNA
ka (Mꢁ1 sꢁ1
a
)
kd (sꢁ1
a
)
KA (Mꢁ1
)
b
KA(G4)/KA(ds)
v2c
pPyNCP
bcl2
1.43 ꢂ 105
8.79 ꢂ 104
2.45 ꢂ 104
4.21 ꢂ 102
1.01 ꢂ 10ꢁ2
1.17 ꢂ 10ꢁ2
4.6 ꢂ 10ꢁ3
3.2 ꢂ 10ꢁ3
1.42 ꢂ 107
7.54 ꢂ 106
5.32 ꢂ 106
1.32 ꢂ 105
108
57
40
1
1.6
2.1
1.1
1.3
c-myc
htelo
dsDNA
TMPyP4
pPyP
bcl2
1.42 ꢂ 106
7.86 ꢂ 105
1.33 ꢂ 105
3.64 ꢂ 103
1.86 ꢂ 104
2.64 ꢂ 105
3.53 ꢂ 104
1.7 ꢂ 103
4.6 ꢂ 10ꢁ2
7.78 ꢂ 10ꢁ3
2.87 ꢂ 10ꢁ3
1.02 ꢂ 10ꢁ3
1.07 ꢂ 10ꢁ3
4.54 ꢂ 10ꢁ3
1.49 ꢂ 10ꢁ3
2.48 ꢂ 10ꢁ3
3.09 ꢂ 107
1.01 ꢂ 108
4.65 ꢂ 107
3.62 ꢂ 106
1.74 ꢂ 107
5.82 ꢂ 107
2.37 ꢂ 107
6.85 ꢂ 105
9
1.4
1.5
0.8
1.8
c-myc
htelo
dsDNA
28
13
1
bcl2
25
85
34
1
0.9
1.4
1.7
1.2
c-myc
htelo
dsDNA
a
Kinetic constants (ka and kd are the corresponding association and dissociation rate constants) determined from BIAcore analysis by using 1:1 binding with mass-transfer
fitting of 240 s association and 240 s disassociation.
b
KA determined from ka/kd, KD determined from kd/ka.
c
The chi2 (v2) value is a standard statistical measure of the closeness of fit. v2 is of the same order of magnitude as the noise in RU. And the fitting process is terminated
automatically when a minimum value is found for v2
.
K++ F21T 0.5 µM
4. Experimental section
100
75
50
25
0
+ pPyNCP 2.5 µM
+ ds 3 µM
+ ds 10 µM
4.1. Synthesis of compound 1, 2, pPyNCP and pPyp
4.1.1. Synthesisofcompound1(4-(bromomethyl)benzaldehyde)54
A solution of p-xylylene dibromide (4 g, 15 mmol) and activated
manganese dioxide (6 g, 69 mmol) in chloroform (150 mL) was re-
fluxed for 3 days. The reaction mixture was filtered and washed
with chloroform. The combined filtrates were evaporated and iso-
lated by silica gel with petroleum ether–dichloromethane (3:1) as
eluent. Compound was obtained as white solid in 21% yield
(627 mg). 1H NMR (300 MHz, CDCl3): d 4.46 (s, 2H), 7.50 (d, 2H,
J = 7.8 Hz), 7.81 (d, 2H, J = 7.8 Hz), 9.95 (s, 1H).
º
T1/2 (no dsDNA)=16.4 C
º
T1/2 (3 µ M dsDNA)=17.3 C
º
T1/2 (10 µ M dsDNA)=18.5 C
30
40
50
60
70
80
90
100
4.1.2. Synthesis of compound 2 (5,10,15,20-tetrakis(40-bromo-
methylphenyl)-2-aza-21-carbaporphyrin)39
Temperature (ºC)
Pyrrole (138
solved in 200 mL of CH2Cl2 and the reaction was initiated by add-
ing methanesulfonic acid (MSA) (90 L, 1.4 mmol). The reaction
lL, 2 mmol) and 1 (398 mg, 2 mmol) were dis-
Figure 2. FRET melting curves of 0.5 lM labeled oligonucleotide F21T with 2.5 lM
pPyNCP in the absence or presence of competitor ds26 in 10 mM lithium cacodylate
buffer, 100 mM KCl.
l
mixture was stirred at room temperature for 30 min. 2,3-Di-
chloro-5,6-dicyano-p-benzoquinone (DDQ) (400 mg, 1.76 mmol)
was added and the mixture was allowed to stir for 1 min and then
structural transition from the antiparallel to the mixed-type hybrid
G-quadruplex structure, which could implicate its recognition to
different G-quadruplex. In addition, when compared with pPyP,
which possessed the same substitution patterns, the N-confused
porphyrin derivative could enhance the binding potential and
selectivity relative to the normal porphyrin derivative. These stud-
ies suggested that the NCP derivative was a better class of quadru-
plex binding ligands. Further investigation of its mechanism,
biological activities and structural relationship is under going.
the reaction was quenched by adding 360 lL triethylamine. The
solvent was evaporated and purified by silica gel column chroma-
tography with CH2Cl2–CH3OH (v/v = 100: 3) as eluents and precip-
itated from CH2Cl2–CH3OH. Compound 2 was obtained as dark
green solid, which was subjected for subsequent reaction as soon
as possible after precipitation without further drying because 2
was unstable under condensed conditions. 1H NMR (300 MHz,
Figure 3. Minimized model of a complex between pPyNCP and the human parallel d(AG3[T2AG3]3) quadruplex. pPyNCP is colored in red, the bases G are colored in green, and
the other bases are colored in gray. (a) The side view of complex; (b) the top view of complex.

Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 1111–1116
1115
CDCl3): d ꢁ5.04 (s, 1H), ꢁ2.56 (br, 2H), 4.74 (s, 2H), 4.77 (s, 6H),
7.72 (d, 4H, J = 6.9 Hz), 7.82 (m, 4H), 8.07 (m, 4H), 8.23–8.31 (m,
4H), 8.48–8.54 (m, 4H), 8.65 (s, 1H), 8.88 (d, 1H, J = 3.6 Hz), 8.93
(d, 1H, J = 5.7 Hz). ESI-MS: m/z: calculated for: M+H = 987.4, found:
986.9.
quently added to increase the concentration ratio of pPyNCP/
DNA strand. All the CD spectra were baseline corrected for signal
contributions from the buffer. In CD melting experiment, the sam-
ples contain 10 mM Tris–HCl buffer (1 mM EDTA, pH 7.4), 100 mM
KCl, 20 lM G4C and different concentrations of pPyNCP, pPyP or
TMPyP4. Before the melting experiment, the samples were boiled
at 95 °C for 5 min, and then cooled down to room temperature.
The CD melting curves were obtained by monitoring absorbance
at 290 nm, while the temperature was ramped from 25 °C to
95 °C at about 1.5 °C/min.
4.1.3. Synthesis of compound pPyNCP (5,10,15,20-tetrakis(a-
pyridinio-p-tolyl)-2-aza-21-carbaporphyrin tetrabromide salt)39
Compound 2 was prepared from 2 mmol pyrrole and 2 mmol
aldehyde and was used directly after precipitation. Compound 2
was dissolved in 150 mL of pyridine. The mixture was vigorously
stirred for 7 days at 40 °C. The solution was filtrated. A dark green
solid of pPyNCP was obtained as a hygroscopic solid in 5.1% yield
from 1 (36 mg, 25 mmol as 7H2O adduct). 1H NMR(300 MHz,
DMSO-d6): d ꢁ3.01 (s, 2H), 2.06 (s, 1H), 6.18 (s, 8H), 7.50 (s, 2H),
7.81 (m, 6H), 7.91 (m, 9H), 7.99 (m, 5H), 8.30 (s, 9H), 8.71 (s,
4.4. Taq DNA polymerase stop assay
General procedures were described by our and other group.43,56
Then various concentrations of compounds pPyNCP, dNTPs (final
concentration 200 lM) and Taq DNA polymerase (1.5 U/reaction,
4H), 9.41 (s, 8H), 12.94 (s, 1H); UV–vis (CH3OH) kmax/nm (log
e
)
Bioready) were added, the mixture was incubated at 55 °C for
30 min. Finally, the products were separated on a 20% denaturing
polyacrylamide gel for electrophoresis and autoradiographed on
a Typhoon phosphor imager (Amersham Biosciences, AB, Uppsala,
Sweden).
411 (4.53), 542 (3.39), 591 (3.42), 725 (3.35); HRMS, m/z: calcu-
lated for: (Mꢁ4Br–)/4 = 245.6112, found: 245.6106. Elemental
analysis calcd for C68H54N8Br4ꢀ7H2O: C, 57.16; H, 4.80; N, 7.84.
Found: C, 57.10; H, 4.73; N, 8.06%.
Compound of 5,10,15,20-tetrakis(40-bromomethylphenyl)por-
55
phyrin was synthesized according to the Ref.
4.5. Surface plasmon resonance
4.1.4. Synthesis of compound pPyP (5,10,15,20-tetrakis(
dinio-p-tolyl)porphyrin tetrabromide salt)
a
-pyri-
The measurements were finished by using BIAcore 3000 optical
biosensor system (BIAcore AB, Uppsala, Sweden) using a CM5 sen-
sor chip covered streptavidin by an Amine Coupling Kit. Experi-
mental procedures were described in the reported papers.25,57
Compound 5,10,15,20-tetrakis(40-bromomethylphenyl)porphy-
rin (98 mg, 0.1 mmol) was dissolved in 20 mL of pyridine. The mix-
ture was refluxed with vigorous stirring for 1.5 h. After cooling to
room temperature, the solution was filtrated and precipitated from
CH3OH–Et2O. Compound pPyP was obtained as purple solid in 91%
yield (118 mg). 1H NMR(600 MHz, DMSO-d6): d 0.42 (s, 1H), 6.27 (s,
8H), 7.97 (d, 8H, J = 7.2 Hz), 8.28 (d, 8H, J = 7.8 Hz), 8.36 (d, 8H,
J = 6.6 Hz), 8.78 (m, 12H), 9.53 (d, 8H, J = 6 Hz); ESI-MS, m/z: calcu-
lated for: (Mꢁ4Br–)/4 = 245.8, found: 245.9.
4.6. FRET melting assay53
Fluorescence melting curves were determined with a real-time
PCR machine (Rotor-Gene 2000, Corbett, Australia) using F21T,
which was performed as a high-throughput screen in a 36-well for-
mat. The total reaction volume of samples was 20
lL, and it con-
tained 0.5 M of labeled oligonucleotide F21T in a buffer of
l
4.2. Biology
10 mM Cacodylate Lithium, pH 7.4 and 100 mM KCl. The fluores-
cence DNA solution without compounds were boiled at 95 °C for
5 min first and then cooling down to room temperature to form
G-quadruplex structures completely. Then adding compounds
and variable concentrations of double-stranded competitor ds22
into different samples and lay them at 4 °C for more than 6 h.
The temperature procedure in real-time PCR was as following:
35 °C for 5 min, and then a stepwise increase of 1 °C every minute
from 35 °C to reach 95 °C was performed and measurements of
FAM were made after each stepwise.
4.2.1. Materials
All oligonucleotides/primers except fluorescent oligonucleo-
tides P18 and F21T were synthesized and purified by Invitrogen
Technology (Shanghai, China). P18 and F21T were purchased from
TaKaRa, Dalian, China. Taq DNA polymerase was purchased from
Toyobo (China). The oligonucleotides Telo24 (50-TTAGGGT-
TAGGGTTAGGGTTAGGG-30) was used in CD spectroscopy, the se-
quence HT4 (50-TCCAA CTATG TATAC TTAGGG TTAGGG TTAGGG
TTAGGG ACATA TCGAT GAAAT TGCTA TAGTG AGTCG TATTA-30)
and 50Tamra labeled P18 (50-TAATA CGACT CACTATAG-30) were
used for DNA polymerase stop assay, the sequence G4C (50-CATGG
TGGTTTGGGTTAGGGTTAGGGTTAGGGTTACCAC-30) was used in the
CD melting experiments, F21T (FAM-G3[TTAG3]3-Tamra, FAM: 6-
carboxyfluorescein, Tamra: 6-carboxytetramehtylrhodamine) and
ds22 (the self-complementary 50-GGCATAGTGCGTGGGCGTTAGC-30)
was used in FRET melting assay. The human telomeric DNA
(50-biotin-AGGGTTAGGG TTAGGGTTAGGG-30), the two promoter
DNA c-myc (50-biotin-GAGGGTGGGGAGGGTGGGG-30) and bcl2
d(50-biotin-GGGCGCGGGAGGAATTGGGCGGG-30), and the duplex
DNA d(50-biotin-GGCATAGTGCGTGGGCGTTAGC-30) hybridized
with its complementary were used in SPR.
4.7. Molecular modeling
The structure of porphyrin was built manually and energy min-
imized by GAUSSION0358 using DFT method at 6-31G level. The coor-
dinates of the parallel G-quadruplex were obtained from the
Brookhaven Protein Data Bank (PDB ID: 1KF1). Discovery STUDIO
2.1 package was used for molecular dynamics calculation. Porphy-
rin was dock manually to the G-quadruplex and the complex was
allowed to minimized using CHARMm force field. First 1000 steps
steepest descent followed by 1000 steps conjugate gradient, then
the system was healing from 50 k to 300 k in 2000 steps, equili-
brate 2000 steps, and product 5000 steps. Then the final conforma-
tion was minimized by 500 steps steepest descent and 500 steps
conjugate gradient.
4.3. CD experiments
Experimental conditions were set up according to our previous
reported procedures.23,51 In the absence or presence of 100 mM KCl
or NaCl, we observed changes of the oligomer Telo24 d(T2AG3)4.
Various amounts of a stock solution of the pPyNCP were subse-
Acknowledgments
This work was partially supported by the National Science of
Foundation of China
(Nos. 20672084, 90813031, 20621502,

1116
Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 1111–1116
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