Synthesis, characterization, and DNA binding of a novel ligand and its Cu(II) complex

Article abstract of DOI:10.1007/s00775-013-1048-7

A novel naphthalene-2,3-diamine-2-salicylaldehyde (NS) ligand and its mononuclear copper(II) complex (CuNS) have been synthesized and structurally characterized. The UV-vis absorption and emission spectra of NS showed obvious changes on addition of Cu

Full text of DOI:10.1007/s00775-013-1048-7

J Biol Inorg Chem (2013) 18:993–1003
DOI 10.1007/s00775-013-1048-7
ORIGINAL PAPER
Synthesis, characterization, and DNA binding of a novel ligand
and its Cu(II) complex
•
Mei-Jin Li Tao-Yu Lan Zhi-Shan Lin
•
•
•
Changqing Yi Guo-Nan Chen
Received: 12 June 2013 / Accepted: 4 September 2013 / Published online: 1 October 2013
Ó SBIC 2013
Abstract
A
novel naphthalene-2,3-diamine-2-salicy-
also studied, and they showed significant potential for
antineoplastic effects.
laldehyde (NS) ligand and its mononuclear copper(II)
complex (CuNS) have been synthesized and structurally
characterized. The UV–vis absorption and emission spectra
of NS showed obvious changes on addition of Cu2? solu-
tion. The interaction of the compounds with calf thymus
DNA and G-quadruplex DNA were investigated by spec-
troscopic methods and thermal melting assay. The nucle-
olytic cleavage activity of the compounds was investigated
on double-stranded circular pBR322 plasmid DNA and
G-quadruplex DNA by electrophoretic mobility shift assay.
The results show that CuNS has a greater ability to stabilize
G-quadruplex DNA over calf-thymus DNA. The cytotox-
icity of the compounds toward HpeG2 cancer cells was
Keywords Copper(II) complex ꢀ DNA binding ꢀ
Cytotoxicity ꢀ Nucleolytic cleavage
Introduction
One of the most widely applied ligands in coordination
chemistry is a Schiff base, in which the special structure of
the C=N bond and the oxygen atoms as the electron donor
in the hydroxyl group can complex well with metal ions.
Studies of a new kind of chemotherapeutic Schiff base are
now attracting the attention of biochemists [1, 2]. Earlier
work [3–5] reported that some drugs showed increased
activity when they were administered as metal complexes
rather than as organic compounds. Transition metal com-
plexes have shown great application prospects as antican-
cer drugs and DNA structural probes [6–12] in recent
years. DNA is the primary target molecule for most anti-
cancer and antiviral therapies. G-quadruplex DNA is a
functionally useful secondary DNA structure containing
G-quartets stabilized through Hoogsteen hydrogen bonding
[13–15]. G-quadruplexes have recently received great
attention because they can inhibit telomere maintenance
provided by telomerase activity, thus affecting the life span
of the cells of a number of cancer types [16, 17]. Investi-
gation of interactions of DNA with small molecules is
important in the development of DNA molecule probes and
chemotherapeutics. A number of promising small mole-
cules have also been devised to selectively promote the
formation and/or stabilization of G-quadruplex structures
[18–24]. Therefore, studying the mode and the mechanism
of interaction of transition metal complexes with DNA and
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-013-1048-7) contains supplementary
material, which is available to authorized users.
M.-J. Li (&) ꢀ T.-Y. Lan ꢀ Z.-S. Lin ꢀ G.-N. Chen
Key Laboratory of Analysis and Detection Technology for Food
Safety (Ministry of Education and Fujian Province),
Department of Chemistry,
Fuzhou University,
Fuzhou 350108, People’s Republic of China
e-mail: mjli@fzu.edu.cn
M.-J. Li
Beijing Synchrotron Radiation Laboratory,
Institute of High Energy Physics,
The Chinese Academy of Sciences,
Beijing 100039, China
C. Yi
School of Engineering,
Sun Yat-Sen University,
Guangzhou 510275,
People’s Republic of China
123

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J Biol Inorg Chem (2013) 18:993–1003
as Cu(CH3COO)2ꢀH2O and salicylaldehyde were of ana-
lytical grade and were used without further purification. TBS
buffer solution (5 mM Tris, 50 mM NaCl, pH 7.2) was used
for CT-DNA binding experiments and G-quadruplex bind-
ing experiments. Tris buffer (10 mM Tris–HCl, pH 7.6, and
1 mM EDTA) was used for gel electrophoresis experiments.
N
N
N
N
Cu
O
O
HO
OH
OH2
CuNS
NS
Synthesis of NS
Fig. 1 Chemical structures of the Schiff base ligand (NS) and its
copper(II) complex (CuNS)
The Schiff base NS was prepared by adding salicylalde-
hyde (80 lL, 0.639 mmol) to a solution of 2,3-diamino-
naphthalene (50 mg, 0.316 mM) in anhydrous ethanol
under a nitrogen atmosphere. The mixture was heated to
reflux for 4 h, and then a large amount of precipitate was
formed. After the mixture had been cooled to room tem-
perature, it was filtered, and an orange solid was obtained.
The resultant orange solid was dissolved in a few drops of
dichloromethane, and a small amount of n-hexane was
added. Orange crystals of NS were obtained in about
2–3 days. Positive-ion electrospray ionization mass spec-
trometry (ESI-MS) (CH2Cl2): m/z = 367.3 ([M ? H]?).
1H NMR (300 MHz, CD3Cl, Me4Si, d, ppm): 12.95 (b, 2H,
aromatic C–OH), 8.75 (s, 2H, –N=CH–), 7.86 (q, 2H,
J = 6.2 Hz, J = 2.9 Hz, naphthalene H), 7.60 (s, 2H,
naphthalene H), 7.48 (q, 2H, J = 7.7 Hz, J = 1.6 Hz
naphthalene H), 7.42 (d, 2H, J = 8.4 Hz, benzene H), 7.40
(t, 2H, J = 7.1 Hz, benzene H), 7.07 (d, 2H, J = 8.2 Hz
benzene H), 6.96 (t, 2H, J = 7.5 Hz, benzene H). IR (KBr
disk, m, cm-1): 1,607 (C=N). 1,567, 1,499, 1,468 (ArC).
exploring the application of metal complexes in antineo-
plastic medication, molecular biology, and bioengineering
have become hot topics in recent years.
Although metal-salen and metal–salphen complexes
have previously been shown to interact with duplex DNA
[25–27], there are relatively few reports on the stabilization
of quadruplex structures [28]. Here we report the synthesis,
structure, and characterization of the novel naphthalene-
2,3-diamine-2-salicylaldehyde (NS) ligand and its cop-
per(II) complex (CuNS) (Fig. 1). The molecular probe of
NS was investigated by UV–vis absorption and emission
spectroscopy on addition of Cu2?. The binding activities of
the complex with calf thymus DNA (CT-DNA) and
G-quadruplex DNA were studied by spectroscopic methods
and viscosity measurements. The nucleolytic cleavage
activity of the compounds was investigated by electro-
phoretic mobility shift assay (EMSA) experiments. The
results demonstrated that combining NS with the copper(II)
ion would result in stabilization of quadruplex DNA. The
cytotoxicity of the compounds toward HpeG2 cancer cells
was also studied, and it was found that they have significant
potential for antineoplastic effects.
Synthesis of CuNS
Cu(CH3COO)2ꢀH2O (15.3 mg, 0.0764 mmol) was added to
a solution of NS (28 mg, 0.0764 mmol) in anhydrous
ethanol under a nitrogen atmosphere. The mixture was
heated to reflux for 4 h, and then a brown precipitate was
formed. After the mixture had been cooled to room tem-
perature, it was filtered and washed with 95 % ethanol to
give an orange solid. Recrystallization from dimethyl
sulfoxide (DMSO)–acetonitrile afforded the desired CuNS
as needlelike brown crystals. Positive-ion ESI-MS
(CH2Cl2): m/z = 428.0 ([M ? H]?). Anal. Calcd for
C24H18N2O3Cuꢀ0.5H2O (%): C, 63.30; H, 4.18; N, 6.15.
Found (%): C, 63.01; H, 4.26; N, 6.39. IR (KBr disk, m,
cm-1): 1,605 (C=N), 1,583, 1,524, 1,445 (ArC).
Materials and methods
All reagents were used as received and solvents were
purified by standard methods. Naphthalene-2,3-diamine
was purchased from Alfa Aesar. CT-DNA was purchased
from Sigma-Aldrich (India). Ethidium bromide (EB) was
purchased from Aladin Chemistry. The supercoiled
pBR322 plasmid DNA and G-quadruplex DNA oligonu-
cleotide (50-CATGGTGGTTTGGGTTAGGGTTAGGGT
TAGGGTTACCAC-30) were obtained from Sangon
(Shanghai) Biotechnology, and G-quadruplex was purified
by denaturing polyacrylamide gel electrophoresis.
Intramolecular G-quadruplexes was formed as follows: the
G-quadruplex samples were annealed in tris(hydroxy-
methyl)aminomethane (Tris)–HCl–NaCl (TBS) buffer
solution (5 mM Tris, 50 mM NaCl, pH 7.2) at 95 °C for
5 min, which was slowly cooled to room temperature, and
was then incubated at 4 °C overnight. Other chemicals such
Physical measurements
1H NMR spectra were recorded with a Bruker AVANCE
III 300 MHz spectrometer with chemical shifts (parts per
million) relative to tetramethylsilane. Positive-ion ESI-MS
was performed with a Thermo Finnigan LCQ Deca XP
mass spectrometer (Finnigan, USA). Elemental analyses
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J Biol Inorg Chem (2013) 18:993–1003
995
Absorption and emission titrations
(C, H, N) were performed with a Vario MICRO instrument.
UV–vis absorption spectra were recorded with a Lambda
750 spectrophotometer. Emission spectra were recorded
with a Hitachi fluorescence F-4600 spectrophotometer
(photomultiplier tube voltage 700V). Circular dichroism
(CD) spectra were recorded with an AVIV model 420
spectropolarimeter. Fourier transform IR spectra were
recorded as KBr pellets with a Thermo Fisher Nicolet 6700
Fourier transform IR spectrophotometer.
The NS and CuNS stock solutions were prepared in DMSO,
and then they were diluted with buffer. The UV–vis
absorption experiments for DNA binding were performed in
6.6 % DMSO–TBS buffer solution (pH 7.2). The absor-
bance of NS at 335 nm and that of CuNS at 425 nm was
measured after titration with CT-DNA. The data were then
fit to Eq. 1 [30] to obtain the intrinsic binding constant Kb:
½DNAꢁ=½ea ꢂ efꢁ ¼ ½DNAꢁ=½eb ꢂ efꢁ þ 1=Kb½eb ꢂ efꢁ
ð1Þ
Single-crystal X-ray diffraction studies
where [DNA] is the concentration of DNA in base pairs, ea
is the extinction coefficient observed for the absorption
band at the given DNA concentration, ef is the extinction
coefficient of the complex free in solution, and eb is the
extinction coefficient of the complex when fully bound to
DNA.
The crystal structures of NS and CuNS were obtained by
the single-crystal X-ray diffraction technique. The crys-
tallographic data collection for NS and CuNS was per-
formed on beam line 3W1A at the Beijing Synchrotron
Radiation Facility with a mounted MarCCD-165 detector
˚
The emission experiment was performed in 6.6 %
DMSO-TBS solution with various concentrations of CT-
DNA (0–320 lM) or G-quadruplex DNA (0–16 lM) and a
constant concentration of NS solution (4 lM) at room
temperature (excitation at 400 nm).
using synchrotron radiation (k = 0.7500 A) at 20 °C. Data
reduction and numerical absorption correction were
applied using the HKL-2000 software package [29]. The
structures were solved by direct methods or the Patterson
procedure, and the heavy atoms were located from E-map.
The remaining non-hydrogen atoms were determined from
the successive difference Fourier syntheses. All non-
hydrogen atoms were refined anisotropically. The hydro-
gen atoms were generated geometrically with isotropic
thermal parameters. Crystal parameters and details of the
data collection and refinement are given in Table 1.
Circular dichroism
CD was measured with an AVIV model 420 spectropo-
larimeter at a scanning rate of 2.5 nm s-1 at 25 °C, using
1 mm path quartz cuvettes. The concentration of DNA was
1 9 10-4 M. The compounds were titrated into the DNA
solution stepwise, with the DNA-to-compound ratio rang-
ing from 10:0.5 to 10:5. The working solution was incu-
bated for 5 min after each addition. A solution of
G-quadruplex (10 lM) was prepared in TBS buffer, and
then solutions of NS and CuNS (0–40 lM) in DMSO were
added. The CD signals of TBS were subtracted as the
background.
Table 1 Crystallographic data for naphthalene-2,3-diamine-2-sali-
cylaldehyde (NS) and CuNS
NS
CuNS
Empirical formula
Formula weight
Space group
C24H18N2O2
366.40
C24H18N2O3Cu
445.95
P21
P2(1)/c
17.552 (3)
19.896 (4)
5.073 (1)
90
˚
a (A)
6.1450 (12)
9.2040 (18)
16.209 (3)
90
˚
b (A)
Thermal denaturation studies
˚
c (A)
a (°)
b (°)
c (°)
DNA thermal denaturation studies were performed by
monitoring the absorption intensity of CT-DNA (50 lM) in
1.6 % DMSO–TBS buffer solution (pH 7.2) at 266 nm by
varying the temperature from 30 to 95 °C in the absence
and in the presence of the different compounds with a
compound to CT-DNA molar ratio of 1:10. G-quadruplex
thermal denaturation studies were performed by monitor-
ing the absorption intensity of G-quadruplex (1 lM) in
5.5 % DMSO–TBS buffer solution (pH 7.2) at 295 nm by
varying the temperature from 40 to 90 °C in the absence
and in the presence of NS or CuNS at a molar ratio of
complex to G-quadruplex of 1:1. The Tm values were
determined graphically from the plots of absorbance versus
temperature.
93.57 (3)
90
90
90
3
˚
V (A )
915.0 (3)
2
1,771.6 (6)
4
Z
qcalcd (g cm-3
)
1.330
1.672
l (mm-1
)
0.086
1.462
F(000)
R1(Fo)a
384.0
916.0
0.0244 (2,615)
0.0673 (2,636)
1.065
0.0968 (2,725)
0.2201 (2,782)
1.309
2 b
)
wR2(Fo
Goodness of fit
P
P
a
R1 ¼ jFo ꢂ Fcj= jFoj
n
h
i
h
io
1=2
ꢀ
ꢁ
ꢂ
P
ꢀ
ꢁ
P
2
2
b
wR2 ¼
w Fo2 ꢂ Fc2
w Fo2
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J Biol Inorg Chem (2013) 18:993–1003
Chemical nuclease activity
dichloromethane–n-hexane. CuNS was synthesized directly
from the reaction of NS with Cu(CH3COO)2ꢀH2O in
anhydrous ethanol solution at 78 °C for 4 h, and was re-
crystallized from DMSO–acetonitrile. NS and CuNS were
characterized by ESI-MS, which showed molecular ion
fragments [M ? H]? as the principal peak with high
abundance. NS and CuNS were further characterized by IR
spectroscopy. NS showed vibration bands for C=N
(1,607 cm-1) and ring-breathing vibrations of the aromatic
rings (CC) (1,567, 1,499, and 1,368 cm-1). CuNS showed
vibration bands for C=N (1,605 cm-1) and ring-breathing
vibrations of the aromatic rings (CC) (1,584, 1,526,
The DNA cleavage studies were performed by agarose gel
electrophoresis on a 15-lL sample volume containing
pBR322 DNA (100 ng/lL) in 5 % dimethylformamide and
95 % Tris buffer (10 mM Tris–HCl, pH 7.6, and 1 mM
EDTA). For the gel electrophoresis experiments, super-
coiled pBR322 DNA was treated with different concen-
trations of the compounds or was treated with different
concentrations of the compounds and H2O2 as a reducing
agent. The mixtures were incubated in the dark for 30 min
at 37 °C, followed by addition of a loading buffer con-
taining 0.25 % bromophenol blue, 0.25 % xylene xyanol
FF, and 60 % glycerol (3 lL) to the mixture. The samples
were analyzed by 1 % agarose gel electrophoresis (Tris–
borate–EDTA buffer, pH 7.6) for 3 h at 60 V. The gels
were stained with EB at a concentration of 0.5 lg mL-1
and were visualized by UV light and photographed for
analysis.
1
1,445 cm-1). NS gave a well-defined H NMR spectrum,
which permitted unambiguous identification and assess-
ment of purity. A broad band corresponding to phenyl–OH
was observed at 12.95 ppm, and a singlet peak was
observed for the proton of –N=CH– at 8.75 ppm. CuNS
give a satisfactory elemental analysis corresponding to the
desired metal complex. NS and CuNS were also charac-
terized by the single-crystal X-ray structure.
Electrophoretic mobility shift assay
Crystal structures
EMSA was performed by maintaining the concentration of
G-quadruplex (0.7 lL, 10 lM), which was heated at 95 °C
for 5 min in TBS buffer (pH 7.2). After the DNA had
cooled to room temperature, a 10-mL stock solution of the
metal complex was added to each sample to produce the
specified concentrations in a total volume of 15 mL. The
reaction mixture was incubated for 12 h at room temper-
ature. 3 mL of loading buffer (0.25 % bromophenol blue,
0.25 % xylene xyanol FF, 60 % glycerol) was added to the
mixture, and it was analyzed by 20 % native polyacryl-
amide gel electrophoresis (the gel was prerun for 30 min).
Electrophoresis proceeded for 3 h in Tris–borate EDTA
running buffer. The gels were silver-stained according to a
previously reported protocol [31, 32].
Single crystals of NS suitable for X-ray diffraction were
grown by vapor diffusion of n-hexane into a concentrated
dichloromethane solution of NS. Single crystals of CuNS
suitable for X-ray diffraction were grown by vapor diffu-
sion of acetonitrile into a concentrated DMSO solution of
CuNS The solid-state structures of NS and CuNS were
determined by X-ray crystallography, and ORTEP drawings
of NS and CuNS are depicted in Fig. 2. The crystal system
of NS belongs to the P21 space group, and the N1–C7 and
˚
N2–C18 bond distances are 1.2902 and 1.2838 A, respec-
tively. The crystal system of CuNS belongs to the
P2(1)/c space group, with the metal center in a 4 ? 1
square-pyramidal CuN2O3 coordination geometry, the
apical site being occupied by a coordinated water molecule.
The donor atoms in each basal plane are two nitrogen atoms
(N1, N2) and two ortho oxygen atoms (O1, O2). The apical
site has a coordinated water molecule with a Cu1–O1W
Cytotoxicity studies
Cytotoxicity studies were conducted on the HepG2 cell
line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay method [33]. The pro-
cedure was the same as in a previous report [34], except
that NS and CuNS were used to treat HepG2 cells.
˚
distance of 2.220 A for CuNS compared with the equatorial
˚
Cu–O distance of 1.93–1.99 A. Selected bond lengths and
angles for NS and CuNS are listed in Table 2.
Photophysical properties
Results and discussion
The absorption spectra of NS and CuNS in DMSO solution
are shown in Fig. 3. The photophysical data for NS and
CuNS are listed in Table 3. The absorption bands of NS at
272 and 338 nm correspond to intraligand transitions of the
ligand. The absorption bands of CuNS at 269 and 321 nm
correspond to intraligand transitions of p to p* orbitals of
the ligand, and the band at 424 nm was assigned as the
Synthesis and characterization
NS was prepared by reaction of 2,3-diaminonaphtha-
lene with salicylaldehyde in anhydrous ethanol solution
at 78 °C for 4 h, and was recrystallized from
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997
Fig. 2 ORTEP drawings of
a NS and b CuNS with the atom
labeling scheme showing 30 %
thermal ellipsoids
˚
Table 2 Selected bond distances (A) and angles (°) for the NS and
CuNS
ligand-to metal-transition of CuNS. The low-energy band
around 600 nm for CuNS was assigned as the metal
d–d transition typical of copper(II) complexes [35, 36].
On addition of Cu2? (0.001–15 equiv) to the solution of
NS in DMSO, the absorption band at 272 nm increased in
intensity with no obvious shift, whereas the absorption
band at 338 nm increased in intensity and underwent a
blueshift of 17 nm. A new peak at 424 nm appeared on the
addition of Cu2? (Fig. 4a). The new band increased in
intensity and reached saturation at a molar ratio of ligand to
Cu2? of 1:1. All of these changes are possibly ascribed to
the coordination between the metal ion (Cu2?) and the
ligand (NS). The results also suggested that Cu2? could
easily insert itself into the hole in the ligand because of the
small steric hindrance of the ligand, which was able to
maximize the coordination effect. The fluorescence spec-
trum of NS (20 lM) in DMSO exhibited an emission band
at 490 nm (Fig. 4b), which can be attributed to a very fast
enol-imine to keto-amine tautomerism involving the phe-
nomenon of excited-state intramolecular proton transfer
(Fig. 5) [37]. The emission intensity decreased as Cu2?
was added to the solution of NS and reached saturation
after the addition of 5 equiv of Cu2?. This might be due to
the prohibition of the excited-state intramolecular proton
transfer phenomenon, and hence the fluorescence was
quenched. However, the fluorescence intensity of NS was
NS
CuNS
N(2)–C(18)
1.2838 (18) Cu(1)–O(1)
1.885 (8)
1.900 (8)
1.918 (9)
1.955 (8)
2.220 (9)
88.7 (4)
172.1 (4)
93.5 (3)
92.1 (3)
171.4 (4)
84.6 (4)
94.4 (4)
97.5 (3)
92.9 (4)
91.0 (4)
129.9 (7)
N(2)–C(17)
1.4143 (18) Cu(1)–O(2)
C(8)–N(1)
1.4256 (16) Cu(1)–N(2)
N(1)–C(7)
1.2902 (17) Cu(1)–N(1)
C(18)–N(2)–C(17)
C(16)–C(17)–N(2)
N(2)–C(17)–C(8)
C(9)–C(8)–N(1)
N(1)–C(8)–C(17)
C(7)–N(1)–C(8)
N(1)–C(7)–C(6)
N(1)–C(7)–H(7A)
N(2)–C(18)–C(19)
123.53 (11) Cu(1)–O(1W)
124.81 (12) O(1)–Cu(1)–O(2)
115.90 (11) O(1)–Cu(1)–N(2)
120.94 (11) O(2)–Cu(1)–N(2)
118.96 (11) O(1)–Cu(1)–N(1)
117.98 (11) O(2)–Cu(1)–N(1)
121.93 (12) N(2)–Cu(1)–N(1)
119.0
O(1)–Cu(1)–O(1W)
120.66 (12) O(2)–Cu(1)–O(1W)
N(2)–C(18)–H(18A) 119.7
N(2)–Cu(1)–O(1W)
N(1)–Cu(1)–O(1W)
C(1)–O(1)–Cu(1)
Cu(1)–O(1W)–H(1WA) 109.3
C(24)–O(2)–Cu(1)
C(7)–N(1)–Cu(1)
C(8)–N(1)–Cu(1)
C(18)–N(2)–Cu(1)
C(17)–N(2)–Cu(1)
125.9 (7)
123.5 (7)
111.6 (6)
126.0 (7)
113.0 (7)
Fig. 3 a UV–vis spectra of NS
(20 lM) and CuNS (20 lM).
b UV–vis spectra of CuNS
(1 mM)
0.6
0.8
(a)
(b)
NS
CuNS
0.5
0.4
0.3
0.2
0.1
0.0
300
CuNS
0.6
0.4
0.2
0.0
400
500
600
500
600
700
800
Wavelength/nm
Wavelength/nm
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J Biol Inorg Chem (2013) 18:993–1003
conformational and structural stabilization of CT-DNA
Table 3 UV–vis data for NS and CuNS
[39]. Kb of NS toward CT-DNA obtained from the regres-
sion analysis was approximately (1.8 0.3) 9 105 M-1
Compound
Absorbance, kmax (nm) [e (M-1 cm-1)]
,
NS
272 (60,284), 338 (35,579)
and Kb of CuNS toward CT-DNA obtained from the regres-
sion analysis was approximately (7.1 0.1) 9 104 M-1
CuNS
269 (49,639), 321 (47,483), 424 (49,331), 600 (307)
.
The values were lower than those obtained for typical in-
tercalators (e.g., EB–DNA, approximately 106 M-1) [40],
indicating that the affinity of NS and CuNS for CT-DNA
was weak and the partial intercalative binding mode
appeared likely.
not completely quenched after addition of Cu2? compared
with CuNS, because NS could not completely complex
with Cu2? at room temperature.
The binding abilities of NS and CuNS with a G-quadru-
plex were also examined by UV–vis absorption titration
experiments. An intramolecular G-quadruplex was prepared
by incubating an oligonucleotide [50-CATGGTGGTTT
(GGGTTA)4CCAC-30] containing four human telomeric
sequences in TBS buffer, which was heated to 95 °C for
5 min and then cooled slowly to room temperature, then kept
at 4 °C overnight. On addition of the G-quadruplex to the
solution of NS in TBS buffer, the absorption band at 340 nm
decreased in intensity (Fig. 7c). Kb of NS toward G-quad-
ruplex DNA obtained from the regression analysis was
approximately (3.3 0.1) 9 104 M-1. On addition of the
G-quadruplex to the solution of CuNS in TBS buffer, the
absorption band at 420 nm decreased in intensity and
underwent a slight redshift, and hypochromism (22 %) of the
band was observed (Fig. 7d). Kb of CuNS toward G-quad-
ruplex DNA obtained from the regression analysis was
approximately (6.5 0.2) 9 105 M-1, which was larger
than that of CT-DNA. The results demonstrated that CuNS
showed higher affinity for G-quadruplex DNA than for
nonquadruplex double-stranded DNA and the copper(II)
center plays an important role in stabilizing G-quadruplex
DNA.
Electrochemistry
Cyclic voltammetry of CuNS was performed with a glassy
carbon electrode at a scan rate of 0.1 V s-1 in dimethyl-
formamide containing n-Bu4NPF6 (0.1 M) as the support-
ing electrolyte; the cyclic voltammogram is shown in
Fig. 6. CuNS exhibited a quasi-reversible peak attributable
to Cu(II)/Cu(I) [38] at -1.46 V (DEP = 100 mV) versus
Ag/AgNO3. The redox potential was relatively negative
compared with that of a polypyridine copper(II) complex,
probably owing to the strong electron-donating ability of
oxygen in the hydroxyl groups after deprotonation.
Binding studies
Absorption spectroscopy studies
The DNA-binding ability of the compound was studied by
an electronic absorption spectrophotometric method. On
addition of CT-DNA to NS and CuNS solutions, the
intensity of peak at 270 nm increased owing to the absor-
bance of the CT-DNA itself. The absorption bands at
339 nm (NS) and at 320 and 425 nm (CuNS) also increased
in intensity, and hyperchromicity was observed, which
suggested that there was an interaction between NS and CT-
DNA (Fig. 7a) and between CuNS and CT-DNA (Fig. 7b).
The spectral changes were ascribed to the binding of NS and
CuNS to the base pairs of DNA, which made the base
stacking force, hydrophobic interaction, and the van
der Waals force change, and ultimately affected the
Emission spectroscopy studies
Emission spectroscopy studies were performed through the
successive additions of various concentration of DNA to
the NS solution. NS was emissive at 480 nm in the absence
of CT-DNA in aqueous 6.6 % DMSO–TBS solution. When
the concentration of CT-DNA solution was increased, the
Fig. 4 a Absorption spectra of
NS (20 lM) on the titration of
7000
1.6
(b)
(a)
6000
5000
4000
3000
2000
1000
1.4
Cu2? (0–15 equiv) in dimethyl
sulfoxide (DMSO) (solid lines)
and absorption spectrum of
CuNS
1.2
1.0
0.8
CuNS (20 lM, dashed line) in
DMSO. b Emission spectra of
0.6
0.4
NS (20 lM) on the titration of
Cu2? (0–15 equiv) in DMSO
0.2
0
CuNS
(solid lines) and emission
spectrum of CuNS (20 lM) in
DMSO (dashed line)
0.0
250
-1000
300
350
400
450
500
550
450
500
550
600
650
700
Wavelength/nm
Wavelength/nm
123

J Biol Inorg Chem (2013) 18:993–1003
999
CD spectral analysis
CD spectroscopy was used to assess whether nucleic
acids undergo conformational changes as a result of
complex formation or changes in the environment [42,
43], because the CD spectrum of DNA is very sensitive
to conformational changes. In the CD spectra, CT-DNA
exhibits a positive band at approximately 280 nm due to
base stacking and a negative band at approximately
245 nm due to the helicity, which is characteristic of
B-DNA [44]. The addition of NS to a solution of CT-
DNA induced a decrease in intensity for both the neg-
ative ellipticity band at 245 nm and the positive ellip-
ticity band at 280 nm, as shown in Fig. 9a. Intercalation
was the most probable mode of binding between NS and
CT-DNA because of the planar aromatic structure of NS.
After addition of CuNS, the ellipticity of the negative
band at 245 nm and that of the positive band at 280 nm
also decreased, as shown in Fig. 9b. Generally, covalent
binding and intercalative binding could influence the
tertiary structure of DNA and induce changes in the CD
spectra of DNA, whereas other noncovalent binding
modes such as electrostatic interaction or groove binding
could not significantly perturb the CD spectra [45]. But
the ability of CuNS for intercalation in DNA appeared
weak.
N
N
O
O
HO
OH
(a)
(b)
Fig. 5 Enol-imine (A) and keto-amine (B). ESIPT excited-state
intramolecular proton transfer
1.0
CuNS
0.5
0.0
-0.5
-1.0
-1.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
Potential/V
Fig. 6 Cyclic voltammogram of CuNS in dimethylformamide (0.1 M
n-Bu4NPF6) with a glassy carbon working electrode. The scan rate
was 0.1 Vs-1
The G-rich telomeric sequence forms intramolecular and
intermolecular G-quadruplexes in monomeric, dimeric, and
tetrameric structures through multiple methods [46]. CD
spectroscopy provides additional support for determining
the presence and to some degree the folding of G-quad-
ruplex structures. All guanine units in the parallel-stranded
G-quadruplex composed of anti glycosidic bond confor-
mations have a large positive band at 295 nm and a neg-
ative band at 240 nm, whereas guanine units in the
antiparallel-type G-quadruplex containing alternating syn
and anti glycosyl bond conformations along each DNA
strand have a characteristic positive band at 295 nm, a
negative minimum at 265 nm, and a smaller positive band
at 245 nm in the CD spectra [47]. Complex binding studies
were conducted in the presence of a stabilizing salt by CD
spectroscopy to investigate whether there was induction
and conversion among various kinds of human telomeric
quadruplexes. NS-induced and CuNS-induced formation of
G-quadruplex structure in the absence of Na? was moni-
tored using CD spectroscopy (Fig. 9c, d). The CD spectra
of the human telomeric quadruplexes at room temperature
exhibited a negative band centered at 239 nm and a rather
broad positive signal around 273–287 nm, which showed
that the quadruplex molecules exited as typical parallel
G-quadruplex conformations in the presence of Na?. On
titration of NS into the G-quadruplex solution, the intensity
of the CD signals corresponding to DNA was altered
emission intensity of NS was enhanced, as shown in Fig. 8a.
This is because the fluorescence of NS was weak when it
was in contact with the water molecules in 6.6 % DMSO–
TBS solution. After the CT-DNA had been added to the NS
solution, the prevention of the interacting moieties from
forming intermolecular hydrogen bonds by CT-DNA
increased the hydrophobility, which contributed to the
DNA-induced emission enhancement [41]. On addition of
the G-quadruplex to NS, the emission intensity at 480 nm of
NS decreased (Fig. 8b), and saturated at a G-quadruplex to
NS concentration ratio of 2 or greater. This was because
G-quadruplex was nonfluorescent, and the combination of
NS with G-quadruplex DNA would cover the fluorescent
part of NS. Competitive DNA binding experiments was also
performed by adding different concentrations of CuNS to a
solution of the EB–DNA system. However, there were no
obvious emission changes (Fig. S1), because EB molecules
could not be replaced completely by CuNS, which dem-
onstrated that CuNS showed weak binding ability toward
CT-DNA, in agreement with the findings from electronic
absorption titrations. It seemed that combining NS with the
copper(II) ion would not reinforce its binding to CT-DNA,
and so the partial intercalative binding interaction between
NS and CT-DNA played an important role in the high
cytotoxicity of the NS ligand.
123

1000
J Biol Inorg Chem (2013) 18:993–1003
1.0
0.8
0.6
0.4
0.2
0.0
(a)
0.5
0.4
0.3
0.2
0.1
0.0
(b)
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0
10
20
30
40
[DNA]/μM
0
10
20
30
40
[DNA]/μM
300
400
500
600
700
800
300
400
500
600
700
800
Wavelength/nm
Wavelength/nm
0.10
0.08
0.06
0.04
0.02
0.00
(c)
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
(d)
0.06
0.04
0.02
0.00
0
2
4
6
8
10 12 14 16 18
0
5
10
15
20
[DNA]/μM
[DNA]/μM
300
400
500
600
700
800
350
400
450
500
Wavelength/nm
Wavelength/nm
Fig. 7 a Absorption spectra of NS (20 lM) on the titration of calf
thymus DNA (CT-DNA; 0–40 lM) in 6.6 % DMSO– tris(hydroxy-
methyl)aminomethane (Tris)–HCl–NaCl (TBS) solution. The insert
shows the least-squares fits of (ea - ef)/(eb - ef) versus DNA
concentration for NS. b Absorption spectra of CuNS (20 lM) on
the titration of CT-DNA (0–40 lM) in 6.6 % DMSO–TBS solution.
The insert shows the least-squares fits of (ea - ef)/(eb - ef) versus
DNA concentration for CuNS. c Absorption spectra of NS (4 lM) on
the titration of G-quadruplex (0–16 lM) in 6.6 % DMSO–TBS
solution. The insert shows the least-squares fits of (ea - ef)/(eb - ef)
versus G-quadruplex concentration for NS. d Absorption spectra of
CuNS (4 lM) on the titration of G-quadruplex (0–20 lM) in 6.6 %
DMSO–TBS solution. The insert shows the least-squares fits of (ea -
ef)/(eb - ef) versus G-quadruplex concentration for CuNS
300
Fig. 8 a Emission spectra of
NS (20 lM) with increasing
concentration of DNA
(0–320 lM) in 6.6 % DMSO–
TBS solution. b Emission
spectra of NS (4 lM) with
increasing concentration of
G-quadruplex (0–16 lM) in
6.6 % DMSO–TBS solution
(b)
0.30
3000
(a)
5
4
3
2
1
0
0.25
250
0
0.20
I
2500
2000
1500
1000
500
/
)
I
0.15
-
0
I
200
(
0.10
0.05
0.00
150
0
2
4
6
8
10
12
14
16
18
-100
0
100 200 300 400 500 600 700 800
[DNA]/μM
[DNA]/μΜ
100
50
0
0
450
500
550
600
650
450
500
550
600
650
700
Wavelength/nm
Wavelength/nm
(Fig. 9c), although no significant spectral changes were
observed and the conformation of the G-quadruplex DNA
did not show a clear change [48]. The negative band at
238 nm decreased in intensity, and the rather broad posi-
tive signal in the CD spectra increased in intensity and
shifted to 289 nm on titration of CuNS into the
G-quadruplex solution. The results suggested that CuNS
could induce significant changes in the intensity of the CD
signals corresponding to G-quadruplex DNA in the pre-
sence of Na?, probably due to the structural conversion
between the intramolecular and the intermolecular
G-quadruplex [49–51].
123

J Biol Inorg Chem (2013) 18:993–1003
1001
ctDNA
[CuNS]/[ct]=0.5/10
[CuNS]/[ctDNA]=2/10
[CuNS]/[ctDNA]=5/10
Fig. 9 Circular dichroism (CD)
spectra of CT-DNA (200 lM)
in the presence of a NS and
b CuNS (0–100 lM) in TBS
buffer at room temperature, and
CD spectra of G-quadruplex
DNA (10 lM) in the presence
of c NS and d CuNS (0–40 lM)
in TBS buffer at room
ctDNA
1.0
0.5
(a)
(b)
1.0
0.5
[NS]/[ct]=0.5/10
[NS]/[ctDNA]=2/10
[NS]/[ctDNA]=5/10
0.0
0.0
-0.5
-1.0
-1.5
-2.0
-0.5
-1.0
-1.5
-2.0
temperature
240 260 280 300 320 340 360 380 400
Wavelength/nm
240 260 280 300 320 340 360 380 400
Wavelength/nm
5
(c)
(d)
5
4
G4
G4
4
3
G4+0.1CuNS
G4+0.5CuNS
G4+4.0CuNS
G4+0.1CuNS
G4+0.5CuNS
G4+2CuNS
G4+3CuNS
G4+4CuNS
3
2
2
1
1
0
0
-1
-2
-1
-2
240 260 280 300 320 340 360 380 400
Wavelength/nm
250
300
350
400
Wavelength/nm
DNA melting studies
DNA cleavage
DNA melting experiments was performed in the absence
and presence of NS and CuNS at different temperatures
(Fig. 10a). Such experiments could provide insight into
the conformational changes and information on the
strength of the interaction with DNA in the presence of
the compound. The melting temperature (Tm) of CT-DNA
increased by approximately 3 and 4 °C after addition of
NS and CuNS, respectively. Such minor changes sug-
gested the partial intercalative binding nature of the
compound [52].
The DNA cleavage ability of CuNS was studied by agarose
gel electrophoresis using supercoiled pBR322 plasmid
DNA as a substrate. Briefly, pBR322 DNA was mixed with
different concentrations of CuNS in aqueous buffer solu-
tion (10 mM Tris–HCl, pH 7.6, and 1 mM EDTA), and the
mixture was incubated at 37 °C for 30 min. With the
increase of concentration of CuNS, DNA was not con-
verted, and it did not show any cleavage activity (Fig. S1).
The DNA cleavage activities of CuNS (160 lM), NS
(160 lM), and Cu(CH3COO)2ꢀH2O (160 lM) using H2O2
or ascorbate as an activator were also observed under
similar conditions (Fig. S2). However, all of them did not
exhibit DNA scission activity, probably because of the low
redox potentials of these complexes.
Additional evidence for G-quadruplex stabilization was
provided by a thermal denaturation study. This was per-
formed in the absence and presence of NS and CuNS at
different temperatures (Fig. 10b). Tm of G-quadruplex in
TBS buffer was about 68 °C, but after the G-quadruplex
(10 lM) had been treated with an equimolar amount of NS,
Tm increased by 6 °C. When the G-quadruplex (10 lM)
was treated with an equimolar amount of CuNS, a marked
increase in Tm (approximately 12 °C) was observed, sug-
gesting CuNS showed higher affinity toward G-quadruplex
DNA than toward double-stranded DNA. This result agreed
with that from the UV–vis absorption and CD spectroscopy
studies. It seems that combining NS with copper(II) ions
reinforces its binding to G-quadruplex DNA probably
because of the square-pyramidal coordination geometry
[53].
Specificity for G-quadruplex DNA by EMSA
EMSA was performed to further identify whether the
compounds can facilitate the formation of G-quadruplexes
or facilitate conversion between the intramolecular and
intermolecular structures. In the absence of the compounds,
only the band corresponding to the monomer was observed
in the EMSAs, in accordance with the previous gel-shift
data [54]. When increasing amounts of NS were added to
G-quadruplex, no new bands formed, as shown in Fig. 11.
Increasing amounts of CuNS added to G-quadruplex
123

1002
J Biol Inorg Chem (2013) 18:993–1003
Fig. 10 a Thermal denaturation
graphs of CT-DNA (50 lM) in
the absence and presence of NS
(5 lM) and CuNS (5 lM).
b Thermal denaturation graphs
of G-quadruplex DNA (10 lM)
in the absence and presence of
NS and CuNS (10 lM). G4
G-quadruplex
0.55
0.54
0.53
0.52
0.51
0.50
0.49
0.48
0.47
0.46
0.45
0.44
0.43
0.42
0.41
0.40
0.39
(b)
(a)
0.55
0.50
0.45
0.40
0.35
0.30
G4
G4+NS
G4+CuNS
DNA
NS+DNA
CuNS+DNA
40
50
60
70
80
90
20
30
40
50
60
70
80
90
Temp/°C
Temp/°C
Fig. 11 Effects of NS (a) and
CuNS (b) on the assembly of
G-quadruplexes illustrated by
polyacrylamide gel
D
electrophoresis in TBS (pH 7.2).
Major bands were identified as
the monomer (M) and the dimer
(D)
M
M
(a)
(b)
resulted in the progressive appearance of new bands with
reduced mobility corresponding to the dimeric forms. The
result showed that CuNS could efficiently promote differ-
ent intermolecular quadruplex formation at high Na?
concentration, and had a stronger quadruplex affinity than
NS, showing that the copper(II) center played an important
role. This observation was in good agreement with the
results from CD spectroscopy, absorption spectroscopy,
and melting studies in a solution of Na? ions.
NS
CuNS
100
80
60
40
20
0
Control
5
20
0.1
10
0.5
Study of cytotoxicity by MTT assay
Concentration/μΜ
Fig. 12 The viability of HeLa cells on treatment with NS and CuNS
for 24 h
MTT assay was performed to check the antineoplastic effect
of NS and CuNS. Their effects on cellular viability are
shown in Fig. 12. Treatment of HepG2 cells with a series of
dilutions (0.1, 0.5, 5, 10, and 20 lM) of NS and CuNS
resulted in a decrease in cell viability. NS decreased cell
viability by 47 % in 24 h at the highest dose (20 lM),
whereas CuNS decreased cell viability by 83 % in 24 h at
the highest dose (20 lM). The cytotoxicity as assessed by
the 50% inhibitory concentration (IC50) was 22.1 and
13.9 lM for NS and CuNS, respectively. Cisplatin exhibited
an IC50 value of 28.8 lM in the control experiments. NS and
CuNS exhibited IC50 values that are more or less equal to
that of cisplatin for the same cell line, which indicates that
they are promising drugs for treatment of cancer. The
cytotoxicity of NS is probably due to the high affinity toward
CT-DNA or complexation with the metal ion in the cell. The
results revealed that the compounds, especially CuNS,
exhibited severe cytotoxicity toward HepG2 cells, indicat-
ing that synergy between the metal and the ligand results in a
significant enhancement of cell death. Although the exact
mechanism for the cytotoxicity is still unclear except for
cisplatin, the prominent cytotoxicity of the complex is
probably related to the strong DNA binding involving
hydrophobic interaction forces [55] or the dissociation of the
complexes in the cell, resulting in intracellular accumulation
of high amounts of copper and the chelation with biological
components such as proteins from the nucleus [56].
Conclusions
A novel NS ligand and its copper(II) complex (CuNS)
have been synthesized and characterized. The DNA bind-
ing properties of NS and CuNS were examined by spec-
troscopic methods and viscosity measurements. The larger
123

J Biol Inorg Chem (2013) 18:993–1003
1003
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binding constant demonstrated that CuNS showed much
higher affinity toward G-quadruplex DNA than toward CT-
DNA, probably owing to its square-pyramidal coordination
geometry. CD studies revealed that CuNS is a good
G-quadruplex stabilizer and could keep G-quadruplexes
parallel in the presence of Na? ions. EMSA further dem-
onstrated that CuNS can promote conversion of G-quad-
ruplex to the dimeric form. MTT assay showed that NS and
especially CuNS are cytotoxic to the HepG2 cell line. All
these findings suggest that the copper(II) center played a
very important role in stabilizing G-quadruplex DNA and
enhancing the cytotoxicity.
Acknowledgments This work was supported by the National Sci-
entific Foundation of China (no. 21171038) and the Program for
Changjiang Scholars and Innovative Research Team in University
(no. IRT1116).
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