Two new mixed copper(ii)-dipeptide complexes of N, N -donor heterocycle ligands: Studies on their non-covalent DNA binding, chemical nuclease, antioxidant and anticancer activities

Article abstract of DOI:10.1039/c6ra01868h

Two novel mononuclear mixed ligand copper(ii) complexes, [Cu(Gly-l-val)(HPB)(H₂O)]·ClO₄·1.5H₂O (1) and [Cu(Gly-l-val)(PBT)(H₂O)]·ClO₄ (2) (Gly-l-val = glycyl-l-valine, HPB = 2-(2′-pyridyl)benzimidazole, PBT = 2-(2′-pyridyl)benzothiazole), have been synthesized and characterized using various analytical and spectroscopic methods. The interactions of the complexes with DNA have been explored by viscometry, thermal denaturation, cyclic voltammetry (CV), agarose gel electrophoresis and spectroscopic means (UV absorption, circular dichroism (CD) and fluorimetry), as well as molecular docking techniques. These studies confirmed the mode of the complexes bound to calf thymus DNA (CT-DNA) through insertion with certain affinity (K_b = 3.211 × 10⁵ M^-1 for 1 and 4.734 × 10⁴ M^-1 for 2). In the fluorimetric experiments of thermodynamics (K_SV = 1.145 × 10⁴ M^-1 for 1 and 2.634 × 10³ M^-1 for 2), the changes in enthalpy (ΔH > 0), entropy (ΔS > 0) and Gibbs free energy (ΔG < 0) in the interactions between the complexes with DNA suggested that the process occurred spontaneously through hydrophobic interactions. The complexes displayed oxidative cleavage of pBR322 plasmid DNA in the presence of ascorbic acid, probably induced by ^?OH as a reactive oxygen species. Furthermore, the molecular docking technique was applied to ascertain the mode of action for the complexes towards DNA. Moreover, superoxide dismutase (SOD) activity studies were performed using the photoreduction of nitroblue tetrazolium (NBT) under a non-enzymatic system and the antioxidant activities of 1 and 2 determined with IC₅₀ values of 0.337 and 0.146 μM, respectively. The cytotoxicity of the Cu(ii) complexes against A549, HeLa, PC-3 tumor cell lines and NIH3T3 (non-tumor cell line) was studied by an MTT assay and it was found that 1 exhibited better cytotoxicity against A549 and PC-3 than 2 and the widely used drug cisplatin.

Full text of DOI:10.1039/c6ra01868h

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: Q. Gan, C. Zhang,
B. Wang, Y. Xiong, Y. Fu, Z. MAO and X. Le, RSC Adv., 2016, DOI: 10.1039/C6RA01868H.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
1
2
3
4
5
6
7
8
9
Two new mixed copper(II)ꢀdipeptide complexes of
N,Nꢀdonor heterocycle ligands: studies on nonꢀcovalent DNA
binding, chemical nuclease, antioxidant and anticancer
activities
a
a
a
*
a
b
Qian Gan , ChunꢀLian Zhang , BingꢀFeng Wang , YaꢀHong Xiong , YinꢀLian Fu ,
ac
a*
ZongꢀWan Mao , XueꢀYi Le
a
Department of Applied Chemistry, South China Agricultural University, Guangzhou 510642, PR China
Department of Applied Mathematics, South China Agricultural University, Guangzhou 510642, PR China
School of Chemistry and Chemical Engineering, Sun yatꢀsen University, Guangzhou 510275, PR China
b
c
1
1
1
1
1
1
1
1
1
1
2
2
0
1
2
3
4
5
6
7
8
9
0
1
Abstract:
Two
novel
mononuclear
mixed
ligand
copper(II)
complexes
[Cu(GlyꢀLꢀval)(HPB)(H O)]·ClO ·1.5H O (1) and [Cu(GlyꢀLꢀval)(PBT)(H O)]·ClO4 (2)
2
4
2
2
(GlyꢀLꢀval
=
glycylꢀLꢀvaline,
HPB
=
2ꢀ(2′ꢀpyridyl)benzimidazole,
PBT
=
2ꢀ(2′ꢀpyridyl)benzothiazole), have been synthesized and characterized by various analytical and
spectral methods. The interactions of the complexes with DNA have been explored by viscometry,
thermal denaturation, cyclic voltammetry (CV), agarose gel electrophoresis and spectroscopic
means (UV absorption, circular dichroism (CD) and fluorimetry), as well as molecular docking
technique. These studies confirmed the mode of the complexes bound to calf thymus DNA
5
−1
4
−1
(CTꢀDNA) through insertion with certain affinity (K
b
= 3.211 × 10 M for 1, 4.734 × 10 M for
4
−1
3
2). In the fluorimetric experiments of thermodynamics (KSV = 1.145 × 10 M for 1, 2.634 × 10
−1
M
for 2), the changes of enthalpy (ꢁH > 0), entropy (ꢁS > 0) and Gibbs free energy (ꢁG < 0) in
the interactions between the complexes with DNA suggested that the process occurred
*
Corresponding authors: Department of Applied Chemistry, South China Agricultural University,
Guangzhou 510642, PR China. Tel.: +8602085280319. Eꢀmail addresses: lexyfu@163.com
(XueꢀYi Le), wbfeng@scau.edu.cn (BingꢀFeng Wang).
1

RSC Advances
Page 2 of 41
View Article Online
DOI: 10.1039/C6RA01868H
2
2
2
2
2
2
2
2
3
3
3
2
3
4
5
6
7
8
9
0
1
2
spontaneously through hydrophobic interaction. The complexes displayed oxidative cleavage of
pBR322 plasmid DNA in the presence of ascorbic acid, probably induced by •OH as reactive
oxygen species. Furthermore, the molecular docking technique was applied to ascertain the action
mode of the complexes towards DNA. Besides, superoxide dismutase (SOD) like activity studies
were performed using the mean of nitroblue tetrazolium (NBT) photoreduction under a
nonenzymatic system, the antioxidant activities of 1 and 2 with the values of IC50 were 0.337 and
0.146 ꢂM, respectively. The cytotoxicity of the Cu(II) complexes against A549, HeLa, PCꢀ3 tumor
cell lines and NIH3T3 (nonꢀtumor cell line) were studied by MTT assay. And it was found that 1
exhibited better cytotoxicity against A549 and PCꢀ3 than 2 and the widely used drug cisplatin.
Keywords: Nꢀaromatic heterocycles; Dipeptide Cu(II) complexes; DNA interaction; Molecular
docking; Antioxidation; Antitumor activity
3
3
3
3
3
3
3
4
4
4
4
3
4
5
6
7
8
9
0
1
2
3
Introduction
Nowadays, cancer has become one of the most serious and dreadful diseases as the leading cause
1
of death in the world. The clinical applied success of cisplatin for treating malignancies attracted
a lot of scientists towards the development of new metal complexes in order to overcome the
2
, 3
drawbacks of cisplatin as anticancer drug. Thus a large number of transition metal complexes
4
ꢀ7
were synthesized and screened for their antitumor activities.
Copper(II) complexes are
considered the most promising alternatives to cisplatin because copper is a bioꢀessential trace
8
, 9
element
and plays an important role in vital life processes including iron transport, energy
1
0
metabolism and respiration. Encouragingly, Lena Ruiz and coꢀworkers developed a series of
medicine named Casiopeinas® based on copper(II) complexes, which have approved for clinical
1
1, 12
trials as antitumor drugs.
In addition, the unique spectroscopic and redox properties of
2

Page 3 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
copper(II) complexes compared to small organic compounds are beneficial to cleavage DNA
under physiological conditions, because these features are crucial for generating the reactive
13
oxygen species (ROS) necessary for DNA cleavage. The ligands (HPB and PBT) belong to
heterocyclic aromatic compounds, which are important fragments in medicinal molecules because
14, 15
of their wide range of biological and pharmaceutical activities such as antifungal,
1
4, 15
7, 14ꢀ16
17
18
antibacterial,
antitumor,
antiꢀinflammatory, antiviral, etc. Additionally, peptides could
work as an ancillary ligand contributing to improve the biological compatibility and recognition of
the complexes in the physiological system due to their structure similar to protein. Importantly,
some dipeptides such as glycylꢀLꢀvaline have been known for their clinical and nutritional
19, 20
importance,
the valine is the essential amino acid for human body and has wide applications in
21
the field of pharmaceutical and food industries, and hence, glycylꢀLꢀvaline can be a suitable
research model as ancillary ligand. Therefore, it is of great interest and significance to explore the
biochemical properties of the benzoheterocycle derivatives based copper(II) complexes with
peptides, in consideration of the possible synergistic effect.
As we all know, DNA plays a vital role in the life process. Extensive studies have proven that
DNA is the primary intracellular target of most anticancer drugs. Since it contains all genetic
information required for cellular function, and various mutagens can damage its replication
22, 23
machinery which may block the division of cell and lead to programmed cell death.
Thus, the
study of interactions between small molecules and DNA is essential in new antitumor drugs
discovery research. Metal complexes are known to bind to DNA via covalent or nonꢀcovalent
2
4
interactions which include intercalative, electrostatic and groove binding modes. And the trend
25
of current research is preferably nonꢀcovalent binding. It is worth noting that there have been
3

RSC Advances
Page 4 of 41
View Article Online
DOI: 10.1039/C6RA01868H
6
6
6
6
7
7
7
7
7
7
7
7
7
7
8
8
8
8
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
considerable research on the binding of metal complexes with DNA, but only limited
thermodynamic parameters about the interaction of complexes to DNA have been measured. It is
also important to study the thermodynamic relationship of DNAꢀcomplex interaction so as to
thoroughly understand the driving forces behind the binding of complexes with DNA and develop
26
novel, effective, DNAꢀtargeted drugs.
The present work stems from our continued interest in researching the DNAꢀbinding
27, 28
interactions of aromatic heterocyclic derivatives based Cu(II)ꢀdipeptide complexes.
We herein
complexes
synthesized and characterized two new copper(II)
2 4 2 2 4
[Cu(GlyꢀLꢀval)(HPB)(H O)]·ClO ·1.5H O (1) and [Cu(GlyꢀLꢀval)(PBT)(H O)]·ClO (2). The
interactions of the complexes with DNA were studied by multispectroscopic methods (UV, CD
and fluorimetry), cyclic voltammetry, viscosity, thermal denaturation and agarose gel
electrophoresis as well as the molecular docking technique. The thermodynamic relationship of
DNAꢀcomplex was revealed. Besides, the antioxidant activities of the complexes were evaluated
using the photoreduction of nitroblue tetrazolium (NBT) assays, and the in vitro cytotoxicity were
assessed by MTT assay, against three human carcinoma cell lines (A549, HeLa, and PCꢀ3) and
mouse embryonic fibroblast (NIH3T3). We hope that the research results will be valuable in
understanding the DNAꢀbinding properties of the complexes as well as laying a foundation for the
rational design of newer and more effective anticarcinogen of targeting DNA.
8
8
8
8
4
5
6
7
Experimental
Materials and instruments
The reagents and chemicals were obtained from commercial sources and used without further
29,
purification. The ligands HPB and PBT were synthesized using previously reported procedures.
4

Page 5 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
3
0
8
8
9
9
9
9
9
9
9
9
9
9
8
9
0
1
2
3
4
5
6
7
8
9
GlycylꢀLꢀvaline was purchased from Aladdin. Calf thymus DNA (CTꢀDNA) (stored at 4 °C)
and ethidium bromide (EthBr) were purchased from Sigma. pBR322 DNA was obtained from
MBI Fermentas (Lithuania). 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT),
nitroblue
tetrazolium
(NBT),
sodium
azide
(NaN
3
),
riboflavin
(VB
2
),
and
tetramethylethylꢀenediamine (TEMED) were purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Shanghai, China). Human cancer cell lines of HeLa (cervical), A549 (pulmonary), PCꢀ3
(prostatic) and normal cell line of NIH3T3 (mouse embryonic fibroblast) were obtained from the
Laboratory Animal Center of Sun YatꢀSen University (Guangzhou, China). Deionized water was
used in all experiments.
All experiments involving CTꢀDNA were performed in TrisꢀHCl buffer solution (pH 7.2)
containing 5 mM TrisꢀHCl and 50 mM NaCl. Solutions of CTꢀDNA in TrisꢀHCl buffer gave a
ratio of UV absorbance at 260 and 280 nm (A260/A280) of ca. 1.8−1.9, indicating that the DNA
3
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
was sufficiently free of protein. The concentration of the DNA was determined by UV
−1
−1
absorbance at 260 nm using the molar absorption coefficient of 6600 M cm . DNA solutions
were used after no more than 4 days.
Elemental analyses (carbon, hydrogen and nitrogen) were performed on a Vario EL elemental
analyzer (Elementar, Germany). ESIꢀMS were carried out on a API4000 triple quadrupole mass
spectrometer (AB Sciex, USA). Molar conductance was measured with a DDSꢀ11A digital
conductometer (LeiCi, Shanghai). Electronꢀspinꢀresonance (ESR) spectra for the complexes were
obtained on a Bruker EMX A300 spectrometer (Bruker, Germany). IR (KBr discs, 4000 − 400
−
1
cm ), UVꢀvis, CD and emission spectra were recorded using a VERTEX 70 FTꢀIR spectrometer
(Bruker, Germany), Pharmacia 2550 spectrophotometer (Shimadzu, Japan), Chirascan CD
5

RSC Advances
Page 6 of 41
View Article Online
DOI: 10.1039/C6RA01868H
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
spectropolarimeter (Applied Photophysics, UK) and Hitachi Fꢀ4500 fluorescence
spectrophotometer (Hitachi, Japan), respectively. Cyclic voltammetry measurements were
performed on a CHI 660A Electrochemical Workstation using a standard threeꢀelectrode system
with a Ptꢀwire as the auxiliary electrode, a glass carbon electrode as the working electrode, and a
saturated calomel electrode (SCE) as the reference electrode.
Synthesis of [Cu(GlyꢀLꢀval)(HPB)(H O)]·ClO ·1.5H O (1)
2
4
2
GlyꢀLꢀval (0.087 g, 0.5 mmol) dissolved in water (5 ml), which was deprotonated by using a
solution of NaOH (0.020 g, 0.5 mmol), was added to a solution of Cu(ClO ·6H O (0.185 g, 0.5
4
)
2
2
mmol) with stirring. A solution of HPB (0.098 g, 0.5 mmol) in MeOH (20 ml), was added
dropwise to the above solution and then stirring was continued at 50 °C for 2 h. The resulting
solution was filtered and kept aside for slow evaporation at room temperature until blue
microcrystalline product formed. It was then recrystallized with 80% MeOH.
27 5
Yield: 0.204 g (71%). Anal. calc. for C19H N O9.5ClCu (1): C, 39.58; H, 4.72; N, 12.15; found:
−
1
C, 39.29; H, 4.70; N, 11.81. IR bands (KBr pellet, cm ): 3420 (b), 3322 (m), 3092 (m), 2960 (m),
−1
−1
1611 (s), 1459 (s), 1382 (m), 1101 (s), 624 (s), 440 (s). UVꢀvis (MeOH) [λmax/nm (ε/M cm )]:
−
1
2
−1
−3
336 (25142), 664 (71.03). Molar conductance, Λ /ꢃ cm mol (1.0 × 10 M, MeOH, 28 °C) =
M
+
113.8. ESIꢀMS (MeOH): m/z = 431.0 [Cu(GlyꢀLꢀval)(HPB)] . ESR (MeOH): g|| = 2.2801, g
⊥
=
2.0589.
2 4
Synthesis of [Cu(GlyꢀLꢀval)(PBT)(H O)]·ClO (2)
Complex 2 was prepared by the same procedure used for 1, but replacing HPB with PBT (0.106 g,
0.5 mmol).
6

Page 7 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Yield: 0.221 g (78%). Anal. calc. for C H N O ClSCu (2): C, 40.28; H, 4.09; N, 9.89; found:
19 23 4 8
−
1
C, 40.51; H, 3.97; N, 10.06. IR bands (KBr pellet, cm ): 3424 (b), 3336 (m), 3093 (m), 2964 (m),
−1
−1
1625 (s), 1496 (s), 1389 (s), 1096 (s), 624 (s), 419 (s). UVꢀvis (MeOH) [λmax/nm (ε/M cm )]:
−
1
2
−1
−3
308 (18694), 642 (66.75). Molar conductance, Λ
M
/ꢃ cm mol (1.0 × 10 M, MeOH, 28 °C) =
+
98.8. ESIꢀMS (MeOH): m/z = 450.2 [Cu(GlyꢀLꢀval)(PBT)] . ESR (MeOH): g|| = 2.2798, g
⊥
=
2.0651.
DNA binding experiments
Electronic absorption titration
Absorption spectra titration was carried out with fixed concentrations of the Cu(II) complexes (50
ꢂM), and gradually increasing the concentration of DNA. To eliminate the impact of absorbance
changes due to DNA, an equal volume of DNA was added to both the sample and reference cells.
After addition of DNA to the metal complexes, the resulting solutions were kept for 5 min to
equilibrate at room temperature, and then the absorption spectra were recorded in the range of
200–500 nm. In order to quantitatively compare the DNA binding affinities, the intrinsic binding
32
b
constant K was determined using the Wolfe–Shimmer equation:
[DNA]/(ε – ε ) = [DNA]/(ε – ε ) + 1/K (ε – ε )
a
f
b
f
b
b
f
a f b
where [DNA] is the concentration of DNA in base pairs, ε , ε and ε correspond to the apparent
extinction coefficient (Aobsd/[Cu]), the extinction coefficient of the free (unbound) and fully bound
compound, respectively. From the plots of [DNA]/(ε
given by the ratio of the slope to the intercept.
Fluorimetric studies
a f b
– ε ) vs. [DNA], the binding constants K are
7

RSC Advances
Page 8 of 41
View Article Online
DOI: 10.1039/C6RA01868H
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
EthBr emits intense fluorescence in the presence of DNA due to its strong intercalation between
adjacent DNA base pairs. The fluorescence can be quenched when the second molecule was added
to the solution and replaced EthBr. In the fluorescence quenching studies, the concentrations of
DNA and EthBr were kept constant (10 ꢂM and 8 ꢂM, respectively), while varying the complexes
−5
concentration from 0 to 11.7 × 10 M at three different temperatures (300, 305, 310 K). The
fluorescence spectra were measured using an excitation wavelength at 525 nm and emission
wavelength in the range of 540 to 680 nm. For each addition, the solutions were kept for 10 min to
equilibrate.
Viscosity experiments
Viscosity experiments were performed using an Ubbelohde viscometer maintained at a constant
temperature (29.0 ± 0.1 °C) in a thermostatic waterꢀbath. Flow time was measured by a digital
stopwatch, and each sample was measured three times, and an average flow time was calculated.
1
/3
The data of (η/η
and absence of the complexes, respectively, are plotted against r ([Complex]/[DNA] = 0.0 − 0.35).
Viscosity values were calculated according to the relation η = (t − t )/t , where t and t are the
0 0
) , where η and η represent the viscosity of CTꢀDNA (200 ꢂM) in the presence
0
0
0
observed flow time in the presence and absence of the samples, respectively.
DNA melting experiments
DNA melting experiments were carried out with a PerkinꢀElmer Lambda 35 spectrophotometer
equipped with a Peltier temperatureꢀcontrolling programmer (± 0.1 °C). The absorbance at 260 nm
was continuously monitored for solutions of CTꢀDNA (50 ꢂM) in the absence and presence of the
–
1
Cu(II) complexes (2.5 ꢂM). The temperature was increased at a rate of 1 °C min from 30 to
m
90 °C. The melting temperature (T ) was taken as the midꢀpoint of the hyperchromic transition.
8

Page 9 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
The data were presented as (A – A )/(A – A ) versus temperature, where A, A , and A are the
1 f 1 1 f
observed, the initial, and the final absorbance at 260 nm, respectively.
Circular dichroism (CD) studies
CD spectra of CTꢀDNA (100 ꢂM) were determined in 5 mM TrisꢀHCl/50 mM NaCl buffer
solution (pH 7.2) at room temperature by increasing [Complex]/[CTꢀDNA] ratios (r = 0.0, 0.2, 0.4,
0.6, 0.8, 1.0). The test system was protected by nitrogen atmosphere during the experiment.
Spectra were measured as the average of three scans from 220 to 320 nm and subtracting the
buffer background.
Cyclic voltammetry
All cyclic voltammetric experiments were carried out in a single compartment cell at 25 °C. The
supporting electrolyte consisted of 10 mM TrisꢀHCl/50 mM NaCl buffer solution (pH 7.2). The
experiments were performed in 0.3 mM complexes solution in the absence and presence of
2
CTꢀDNA. Before the experiments, all solutions were deoxygenated with N for 10 min and were
2
kept under a N atmosphere throughout the experiments.
DNA cleavage experiments
The cleavage of supercoiled pBR322 plasmid DNA (250 ng) was monitored by agarose gel
electrophoresis. The samples were incubated for 1 h at 37 °C in the dark. A loading buffer was
added and electrophoresis performed at 100 V for 40 min in TrisꢀBoric acidꢀEDTA (TBE) buffer
(pH 8.3) using 0.8% agarose gel containing 5 ꢂL GoldView. Control experiments were carried out
4 2 2
using 50 ꢂM ascorbic acid, 20 ꢂM Cu(ClO ) ·6H O and 20 ꢂM ligands. After electrophoresis, the
bands were visualized by UV light and photographed with a BioꢀRad LaboratoriesꢀSegrate Gel
Imaging System.
9

RSC Advances
Page 10 of 41
View Article Online
DOI: 10.1039/C6RA01868H
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
The DNA cleavage mechanisms of the complexes were investigated in the presence of typical
reactive oxygen species scavengers like DMSO, tertꢀbutyl alcohol, and ethanol (hydroxyl radical),
3
sodium azide (NaN ) (singlet oxygen) and superoxide dismutase (SOD) (superoxide anion radical).
Each sample was treated and analyzed according to the procedure described above.
Molecular docking studies
33
The molecular docking studies were performed using the AutoDock Vina1.1.2
program to
investigate the interactions between the complexes and DNA containing iodinated 8ꢀmer
3
4, 35
oligonucleotides d(5’ꢀGꢀdIUꢀTGCAACꢀ3’) (PDB ID: 454D).
This DNA fragment included a
cavity between GC/GC consecutive base pairs. Meanwhile, the water molecules and the substrate
were deleted. Polar hydrogens were added and Gasteiger charges were calculated. The DNA was
enclosed in a grid box with 60 × 60 × 60 points in which almost involved the entire DNA
molecule and the binding site was located in the center of DNA molecule. Other parameters were
default settings. The structures of the complexes were sketched by Gaussian viewer. And the full
geometry optimizations were performed with the Gaussian09 program package using the hybrid
density functional theory (B3LYP) method and 6ꢀ31G(D) basis set. All calculatiosn were carried
out on Dell T7500 server with dual XEON 5660 cores, RedHat linux operating system.
Visualization of the docked pose has been done by using PyMol molecular graphics program.
Superoxide radical scavenging activity
－
2
The superoxide radical (O •) scavenging activity of the title complexes were determined by the
28
modified nitroblue tetrazolium (NBT) photoreduction method. A nonenzymatic system of
TEMEDꢀVB ꢀNBT containing the tested complexes (0.05–0.80 ꢂM), TEMED (100 ꢂM), VB
2
2
(6.80 ꢂM), and NBT (93.20 ꢂM) in 0.05 M phosphate buffer (pH 7.8) was applied. The reactions
1
0

Page 11 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
were monitored by detecting the concentration of the reduced NBT (blue formazan) at 560 nm.
The inhibition ratio (I) of NBT reduction was calculated according to the following equation:
0 i 0
I (%) = (A – A )/A × 100
0 i
where A and A represent the slopes of the straight line of absorbance values as a function of time
in the absence and presence of the title complexes, respectively. The IC50 values were the
concentration of the complexes which caused 50% inhibition of NBT reduction. Each sample was
measured in three parallel times.
In vitro cytotoxicity assays (MTT)
In vitro cytotoxicity tests were carried out using MTT colorimetric assay. Three different human
tumor cell lines of HeLa (cervical), A549 (pulmonary), PCꢀ3 (prostatic) and mouse embryonic
4
fibroblast NIH3T3 (normal cell line) were seeded into 96ꢀwell microtiter plates (1 × 10 cells per
2
well) and incubated overnight in a humidified atmosphere containing 5% CO at 37 °C. The tested
compounds were dissolved in DMSO and diluted with RPMI 1640 medium supplemented with 10%
−
1
−1
fetal bovine serum (FBS), 100 units mL penicillin and 50 units mL streptomycin to the
required concentrations (ranging from 1.56 to 100 ꢂM) prior to use. Control wells were prepared
by the addition of culture medium (200 ꢂL). The plates were incubated in a 5% CO incubator at
2
37 °C for 48 h. Then stock MTT dye solution (20 ꢂL, 5 mg/mL) was added to each well after 4 h
incubation. The formed MTT formazan were then dissolved in 150 ꢂL DMSO, and the optical
density (OD) of each well was then measured at 595 nm on a microplate spectrophotometer. The
antiꢀgrowth effects of the tested compounds are expressed as GI50 values, which indicate the
concentration of the compounds that inhibits 50% of the cell growth. GI50 value is calculated from
36,37
the equation: 100 × (T
i
0
– T )/(C – T
0
) = 50,
0 i
where T and T indicate the OD values at time zero
11

RSC Advances
Page 12 of 41
View Article Online
DOI: 10.1039/C6RA01868H
2
2
40
41
and after treatment period, respectively, and C indicates the OD value measured in untreated cells
(control) after an incubation period equal to the treatment period.
2
2
2
2
2
2
2
2
2
2
42 Results and discussion
43
44
45
46
47
48
49
50
51
Synthesis and characterization
The titled complexes were isolated by the reaction of copper(II) perchlorate hexahydrate with
glycylꢀLꢀvaline and HPB/PBT in equimolar quantities using 80% MeOH as solvent (Scheme 1).
The complexes were characterized by elemental analyses, IR, UV–vis, ESIꢀMS, ESR and molar
conductance measurements. The obtained results for the elemental analyses were found to be in
good agreement with the calculated values, which confirmed the formation of the complexes. The
molar conductance values of the two complexes in methanol indicate that the complexes are 1:1
38
type electrolytes. The ESIꢀMS of the complex 1 displayed a peak at m/z = 431.0 in methanol
+
+
matching exactly with [Cu(GlyꢀLꢀval)(HPB)] (m/z = 450.2 for 2, [Cu(GlyꢀLꢀval)(PBT)] ).
2
2
52
53
Scheme 1: Synthetic route for the ligands and complexes.
2
2
2
54
55
56
The electronic absorption spectra of the complexes were recorded in methanol solution at room
temperature. The intense bands at 316 nm for 1 and 309 nm for 2 were assigned to the intraligand
(π → π*) transitions of ligands HPB and PBT, respectively. The broad and weak bands were
1
2

Page 13 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
observed at the lower frequency of 664 and 642 nm in the spectrum for 1 and 2, respectively,
corresponding to the d → d transitions of copper(II) and indicating approximate square pyramidal
3
9
geometry around copper(II).
−
1
In the IR region, the peak near at 3420 cm can be attributed to the stretching vibration ν(−OH)
−
1
−1
of the water molecule. The band near at 3322 cm for 1 (3336 cm for 2) was the stretching
vibration ν(−NH) of the secondary amide in dipeptide. And the two bands at approximately 3092
−
1
−1
and 2920 cm for 1 (3093 and 2964 cm for 2) were attributed to asymmetric νas(−NH
) and
2
symmetric ν
s 2
(−NH ) stretching frequencies, respectively. The absence of any band in the region of
−
1
1700–1750 cm for the complexes suggests coordination of the carboxylate group of the
−
−1
dipeptide to the central copper ion. Besides, the difference values ꢁν = [νas(−COO ) (1, 1611 cm ;
−
1
−
−1
−1
−1
2, 1625 cm ) − ν (−COO ) (1, 1382 cm ; 2, 1389 cm )] > 200 cm can be explained by the fact
s
−
2+ 40
that the COO group as a unidentate coordinated to the central Cu . Moreover, the band at 1459
−
1
−1
cm for 1 (1496 cm for 2) can be assigned to the ring stretching frequencies ν(C=N) of ligand
2
+
HPB/PBT, indicating that HPB/PBT was coordinated to the central Cu . The complexes showed
−
1
−1
very strong bands at 1101 cm for 1 and 1096 cm for 2, which can be attributed to ν(Cl−O) of
−
−1
−1
ClO . The bands at 440 and 624 cm for 1 (419 and 624 cm for 2) most likely belong to the
4
ν(Cu–N) and ν(Cu–O) stretching vibrations, respectively.
The Xꢀband ESR spectra of the complexes were recorded in MeOH (100 K) at frequency of
9.46 GHz under the magnetic field strength 3000 ± 1000 gauss using tetracyanoethylene (TCNE)
as field marker. The ESR spectra revealed an anisotropic signal with g|| = 2.2801, g
⊥
= 2.0589,
and A = 167 G for 1 and g = 2.2798, g
⊥
= 2.0651, and A = 163 G for 2. The parameters g > g
⊥
>
||
||
||
||
e
g (2.0023) revealed that the unpaired electron was located in the dx2–y2 orbital of the Cu(II) ions
1
3

RSC Advances
Page 14 of 41
View Article Online
DOI: 10.1039/C6RA01868H
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
characteristic of local symmetry, indicating nearly square pyramidal geometry for the
4
1
complexes, which is in agreement with the result obtained by the electronic absorption spectra.
Therefore, from the above results, a tentative structure was proposed for the two Cu(II)
complexes with an approximate squareꢀpyramidal geometry where four equatorial positions were
2
occupied by HPB/PBT (N, N) and dipeptide (N, O), and the axial position was occupied by a H O
molecule.
Electronic absorption spectra studies
Electronic absorption spectroscopy is an efficient and widely employed method to study the
binding mode of metal complexes to DNA. In general, the nonꢀcovalent binding of small
molecules to DNA involves three interaction modes: groove binding, intercalation and
electrostatic interactions. Intercalative binding is the most effective mode of the drugs targeted to
DNA, which is associated with the DNA damage or antineoplastic activity of the complexes. The
effect of hypochromism and red shift can be observed with the intercalative mode of the
complexes to DNA involving a stacking interaction between aromatic chromophore and the base
pairs of DNA. The absorption spectra of the two complexes incubated with increasing
concentrations of CTꢀDNA are given in Fig. 1. The absorption bands of complexes 1 and 2
decreased in molar absorptivity (hypochromism, 1: 28.9%, 2: 17.8%) as well as 1~2 nm
bathochromism, which might be attributed to the intercalation between the heteroaromatic rings of
the complexes and DNA base pairs. The extent of the hypochromism commonly parallels the
42
affinity of a compound binding to DNA and the strength of the intercalative interaction. The
5
−1
4
−1
binding constants (K ) are calculated as 3.211 × 10 M (1) and 4.734 × 10 M (2), respectively,
b
43
b
which are smaller than that of the classical intercalator EthBr. The higher K value for 1
1
4

Page 15 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
compared to 2 shows that the binding ability of 1 to DNA is stronger than 2. This may be related
to the difference of the benzoheterocycle moiety of the complexes. In addition, compared to 2, 1
owns higher number of hydrogen bonds to DNA, just as seen from molecular docking studies (cf.
below).
Fig. 1
Fluorescence studies
Fluorescence spectroscopy was employed to investigate the interaction characteristics between
chromophore and other compounds. The fluorescence quenching efficiency is evaluated by the
Stern–Volmer constant (KSV) according to the classical Stern–Volmer equation:
F /F = 1 + K [Q]
0
SV
where F
0
and F correspond to the fluorescence intensities of EthBrꢀDNA in the absence and
presence of the complexes, respectively, and [Q] is the concentration of the complexes. KSV is a
linear Stern–Volmer quenching constant obtained from the linear regression of F /F with [Q]. As
0
shown in Fig. 2, the emission band of the EthBr–DNA system decreased in intensity with an
increase in the concentration of the two complexes, which suggested that the complexes could
displace EthBr at their DNA binding sites, characteristic for the intercalative binding of the
complexes to DNA. The obtained KSV values in 300K for complexes 1 and 2 were found to be
4
−1
3
−1
1.145 × 10 M and 2.634 × 10 M , respectively. The complex 1 owns a stronger DNA binding
affinity compared with 2. The result is consistent with that of absorption spectroscopic studies.
Fig. 2
1
5

RSC Advances
Page 16 of 41
View Article Online
DOI: 10.1039/C6RA01868H
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Fluorescence quenching studies
Fluorescence quenching refers to the process in which the fluorescence intensity of fluorophore
decreases after interaction with quencher, including collisional quenching, excitedꢀstate reactions,
energy transfer groundꢀstate complex formation, and molecular rearrangements. The mechanisms
of quenching are divided into two types: dynamic and static quenching. The two mechanisms can
be distinguished according to the relationship of temperature dependence of the K . In general,
SV
dynamic quenching depends on diffusion, higher temperatures lead to larger diffusion coefficients,
which cause more collision probability, the KSV can be increased by rising the temperature. In
contrast, increased temperatures decreases compounds stability, and thus lower values of the KSV
4
4
were obtained in the static quenching. As seen in Fig. 3, the Stern–Volmer plots were linear
which showed that only one type of quenching process occurs, either dynamic or static quenching.
4
−1
4
−1
Obviously, the KSV increased with the temperature (1.145 × 10 M (300 K), 1.173 × 10 M (305
4
−1
3
−1
3
−1
3
K), 1.203 × 10 M (310 K) for 1; 2.634 × 10 M (300 K), 3.102 × 10 M (305 K), 3.474 × 10
−1
M
(310 K) for 2, Table 1), indicating that the fluorescence quenching process is dynamic
quenching.
Fig. 3
Table 1
Thermodynamic studies
In order to have a better understanding of the driving forces behind the binding of metal
complexes to DNA, the thermodynamic parameters (such as enthalpy (ꢁH), entropy (ꢁS) and
Gibbs free energy (ꢁG)) with three different temperatures were obtained and analyzed. The main
1
6

Page 17 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
interaction forces about the nonꢀcovalent binding contain hydrogen bonding interaction, van der
45
Waals forces, electrostatic interaction and hydrophobic interaction. If ꢁH > 0 and ꢁS > 0, the
main force is hydrophobic interaction; if ꢁH < 0 and ꢁS < 0, the major force is the Van der Waals
force or hydrogen bond; if ꢁH < 0 or ꢁH ≈ 0 and ꢁS > 0, electrostatic force plays major role in the
46
reaction. The values of ꢁH, ꢁS and ꢁG for a binding reaction were calculated from the vant’t
4
7
Hoff equation and Gibbs–Helmholtz equation:
ln K = －ꢁH/RT + ꢁS/R
ꢁG = ꢁH － TꢁS = －RT ln K
where K is the Stern–Volmer quenching constant at the corresponding temperature and R is the gas
constant. The ꢁH can be regarded as a constant if the temperature does not vary significantly. The
thermodynamic parameters were listed in Table 1. The positive ꢁH and ꢁS values suggested that
the binding of the complexes to DNA is an endothermic and entropy increasing process. And the
hydrophobic interaction plays a major role in this process. Moreover, the negative value of ꢁG
reveals that the interaction process is spontaneous. The binding process of the complexes with
CTꢀDNA is entropyꢀdriven due to |ꢁH| < |TꢁS|.
Viscosity measurements
The viscosity of CTꢀDNA upon treatment with varying concentrations of complexes was measured
to clarify the interaction nature between the complexes and DNA. As it is sensitive to the change
in the length of DNA molecule, hydrodynamic measurement (i.e. viscosity) is regarded as the least
ambiguous and the most critical tests of a DNA binding mode in solution in the absence of
4
8
crystallographic or NMR structural data. The intercalation mode will cause an increase in
relative viscosity of DNA, but the nonꢀintercalation binding mode has little effect on the relative
1
7

RSC Advances
Page 18 of 41
View Article Online
DOI: 10.1039/C6RA01868H
4
9, 50
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
viscosity of DNA in the binding process.
As shown in Fig. 4, the relative viscosity of DNA
steadily increases upon increasing the concentration of complexes. This may be explained by the
fact that the complexes can insert the DNA adjacent base pairs, leading to an increase in the
separation of base pairs at insertion sites, as a consequence, an increase in overall DNA contour
length. The increased degree of the viscosity of DNA related to the interaction is in the order 1 > 2,
indicating that the DNA binding affinity of 1 is greater than 2. It is consistent with the results of
the absorption and emission spectral studies.
Fig. 4
DNA thermal denaturation studies
The hydrogen bonding and base stacking interactions between the strands of the double helix in
DNA can be disrupted by heating, resulting in the separation of the double helix into two single
strands. DNA melting experiments can further give insight into DNA conformational changes
when the temperature is raised and offer information about the interaction strength of complexes
51
m
with DNA. The melting temperature (T ) of DNA is defined as the temperature at which half of
5
2
the double helical DNA strands are unfolded to single strands. Generally, intercalation of natural
or synthesized organicꢀ and metalloꢀintercalators into the DNA double helix increases the thermal
m
stability of the helix and thereby causes an appreciable enhancement in the T of DNA. However,
m
groove binding leads to an imperceptible variation in the value of T . The melting profiles of
m
CTꢀDNA in the absence and presence of the Cu(II) complexes is provided in Fig. 5. The T of free
CTꢀDNA was determined to be 60.1 °C. The melting point increased by 10.8 °C for complex 1 and
5.1 °C for complex 2 at a concentration ratio of [Cu]/[DNA] = 0.05, respectively. The obvious
1
8

Page 19 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
increases (~5 or 11 °C) in the T indicate the intercalative binding of the two complexes to DNA.
m
And the interaction strength of 1 is greater than 2, in conformity with the above results by
absorption spectra and viscosity.
Fig. 5
Circular dichroism spectra
Circular dichroism (CD) is a powerful technique to monitor the possible conformational changes
of biomolecules (DNA or proteins) upon interaction with metal complexes, as the CD signals are
quite sensitive to the interaction mode. The observed CD spectrum of CTꢀDNA exhibited a
positive band at 274 nm and a negative band at 246 nm, owing to the π–π stacking of base pairs
53
and rightꢀhand helicity, respectively, which is the characteristic of Bꢀform DNA. As shown in
Fig. 6, the intensities of both the positive and negative bands decreased significantly (shifting to
zero levels) with increasing [Complex]/[DNA] ratio, with the former being affected slightly more
than the latter. Meanwhile, the positive and negative bands both showed red shifts (6–7 nm and 2–
3 nm for the positive and negative bands, respectively). This suggested that the interactions of the
complexes with DNA disturbed the right handed helicity and base stacking of DNA, and thus
induced certain conformational changes of BꢀDNA, such as the conversion from B → C like
5
4
structure. The larger decrease of positive bands indicated that the complexes intercalated into the
55, 56
DNA base pairs and thereby unwinding the double helix.
Moreover, the changing intensity of
1 exceeded 2, in agreement with the results obtained by the above methods.
Fig. 6
1
9

RSC Advances
Page 20 of 41
View Article Online
DOI: 10.1039/C6RA01868H
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Redox behavior
In order to further understand the binding nature of the Cu(II) complexes to DNA, the
electrochemical methods were employed to study the redox behavior between the metal
complexes and DNA. The typical cyclic voltammograms of the copper(II) complexes in the
absence and presence of CTꢀDNA are shown in Fig. 7, and the related data are summarized in
Tables 2.
The ratio of the oxidation peak current to reduction peak current (Ipa/Ipc) ≈ 1, indicated that the
redox process was quasiꢀreversible. No new peaks appeared after the addition of CTꢀDNA to the
complexes with R ([DNA]/[complex]) = 3. Complexes 1 and 2 all experienced a significant
reduction in Ipa and Ipc, indicating that the complexes are bound strongly to DNA. According to
Kelly and coworkers, complex bounded to DNA and the product was nonꢀelectroactive,
decreasing the concentration of electroactive species in solution and resulting in the decline of the
5
7
peak currents. The observed decline of the voltammetric current in the presence of DNA can be
attributed to slow diffusion of the complexes bound to DNA. Meanwhile, the anodic peak
potential (Epa) and formal potential (E1/2) showed positive shifts. Generally, when the metal
complex binds DNA via electrostatic interaction, the electrochemical potential of the complex will
5
8
shift in a negative direction. If it intercalates into DNA, the potential will shift positively.
Therefore, the positive shifts in the CV peak potentials of the complexes indicated an intercalative
binding mode of the complexes to DNA. This binding was considered principally due to the
5
9
hydrophobic interaction.
0
’
0’
The separation between Eb and Ef can be used to estimate the ratio of binding constants for
54
the reduced and oxidized forms to DNA using the following equation:
2
0

Page 21 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
0
’
0’
4
4
4
4
4
4
4
4
4
4
4
33
34
35
36
37
38
39
40
41
42
43
E
b
－ E
f
+
= 0.059log[K /K2+]
0
f
’
0’
b
where E and E are the formal potentials of the Cu(I)/Cu(II) couple in the free and bound
+
species, respectively, and K and K2+ are the corresponding binding constants for the binding of
+
the Cu(I) and Cu(II) species to DNA, respectively (Scheme 2). The values of K /K2+ were 13.6 for
1 and 1.4 for 2, suggesting that the interaction of Cu(I) form with DNA was stronger than Cu(II)
form. The more prominent decrease of the peak currents and more shift of the peak potentials
observed for 1 over 2 upon addition of DNA indicate that the binding affinity of the complexes to
DNA follow the order of 1 > 2. The result is consistent with the above experiment data.
Fig. 7
Table 2
4
4
44
45
Scheme 2
4
4
4
4
4
4
46
47
48
49
50
51
Chemical nuclease activities
In order to assess the chemical nuclease activities of the complexes for DNA strand scission,
pBR322 DNA was incubated with the complexes, and the cleavage reactions were monitored by
agarose gel electrophoresis. When plasmid DNA is subjected to electrophoresis, the fastest
migration will be observed for the intact supercoiled DNA (Form I). If scission occurs on one
2
1

RSC Advances
Page 22 of 41
View Article Online
DOI: 10.1039/C6RA01868H
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
strand, the supercoiled DNA will relax to generate a slowerꢀmoving open circular form (Form II).
Once both strands are cleaved, a linear form (Form III) will be generated that migrates between
60
Form I and Form II. As shown in Fig. 8, control experiments performed using only ascorbic acid,
4 2 2
Cu(ClO ) ·6H O or ligands alone in separate lanes showed no prominent cleavage of DNA.
Meanwhile, no apparent DNA cleavage was observed for the complexes (20 ꢂM) without ascorbic
acid (lane 6). The complexes converted the Form I to Form II and III with the increase in the
concentration of the complexes in the presence of 50 ꢂM ascorbic acid (lanes 7–10), providing a
clear evidence for direct doubleꢀstrand DNA cleavage. Doubleꢀstrand breaks are thought to be
more significant than singleꢀstrand breaks as sources of cell lethality, because they are apparently
6
1
less readily repaired by DNA repair mechanisms. Under identical experimental conditions,
complex 1 shows higher cleavage ability (Fig. 9), which is traced to its higher DNA binding
affinity and Cu(II)/Cu(I) redox potential (cf. above).
Fig. 8
Fig. 9
Copper complexes can cleave DNA through oxidative mechanism or hydrolytic mechanism. In
the process of oxidative DNA cleavage, it may lead to the formation of reactive hydroxyl radical
－
1
2 2
(•OH), superoxide anion radical (O •), and/or singlet oxygen ( O ) species, which cause damage
6
2
to the base or/and sugar of DNA. To reveal among the reactive oxygen species (ROS) which was
responsible for the DNA cleavage, the ROS scavengers were used and the results are illustrated in
Fig. 10. When the hydroxyl radical (•OH) scavengers (DMSO, tertꢀbutyl alcohol, and ethanol)
were added to the reaction mixture respectively, inhibition of DNA cleavage was observed
2
2

Page 23 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
1
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
revealing that the cleavage reaction involve •OH. The presence of NaN ( O quencher) didn’t
3 2
significantly reduce the efficiency of DNA cleavage, ruling out the possibility of involvement of
1
diffusible O
2
or singlet oxygenꢀlike entities in the cleavage. Furthermore, DNA cleavage was
－
promoted by adding superoxide dismutase (O
2
• scavenger) to the reaction mixture (lane 8), and
－
2
free SOD did not exhibit any DNA cleavage (lane 2), which validates that O • could be involved
in the DNA strand scission in a roundꢀabout way, in which the complexes can catalyze the
－
dismutation O
2 2 2 2
• to generate H O and O . Moreover, it has been known that the Cu(I) species
binds to DNA with affinity higher than Cu(II) species from the above electrochemical test. And
63
thus DNA is made more accessible for the ROS (•OH) produced by Fenton type reaction,
resulting in higher DNA cleavage.
Fig. 10
Molecular docking analysis
Molecular docking technique is an attractive scaffold to better understand the interaction of a
macromolecule (receptor) and a small molecule (ligand). It can contribute to the rational drug
design and the mechanistic study by placing a small molecule into the binding site of the DNA
6
4
target specific region mainly in a nonꢀcovalent mode. As shown in Fig. 11, the resulting model
showed that 1 and 2 could intercalate into the cavity between GC/GC consecutive base pairs
through heteroaromatic rings and stabilized by hydrophobic interactions and hydrogen bonds.
There are certain hydrogenꢀbonding interactions between complex 1 and DNA, such as complex
1:H16…454D:DGꢀ4:N7 (1.8 Å), complex 1:H18…454D:DCꢀ13:O4’ (2.6 Å), complex
1:H19…454D:DCꢀ5:N4 (2.6 Å), complex 1:H19…454D:DGꢀ12:O6 (1.8 Å). And the hydrophobic
2
3

RSC Advances
Page 24 of 41
View Article Online
DOI: 10.1039/C6RA01868H
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
interaction was the predominant forces between complex 2 and DNA in the absence of hydrogen
bonds. The resulting relative binding energy of docked 1 and 2 with DNA were found to be −34.31
−
1
and −30.12 kJ mol , respectively. The more negative the relative binding energy, the more potent
the binding as between complex and DNA. Thus, the result indicated that 1 binds to the DNA
stronger than 2, which is correlated well with the experimental DNA binding results.
Fig. 11
Antioxidant activity
－
2
The superoxide anion radical (O •) was one of the most reactive species of ROS, which could
result in cell membrane and DNA damage, aging, chromosomal aberrations, carcinogenesiands
6
5, 66
and further initiate or propagate the development of many diseases.
Therefore, it's important
－
2
for further investigation of the O • scavenging activities of the metal complexes. As shown in Fig.
12, the inhibition ratio was concentrationꢀdependent and increased with the sample concentration.
1 and 2 exhibited excellent antioxidant activities with IC50 values of 0.337 and 0.146 ꢂM,
2
+
respectively. This could be related to the flexible geometry around the center Cu contributing to
－
67
O • approach, with fast exchange of axial weakly coordinated H O. Moreover, the electrons of
2
2
nitrogen heteroatomic ligands, such as pyridine and thiazole, can facilitate stability of complex–
－
O
2
• interaction in favour of high SOD activity. The complexes are potent SOD mimics due to
their much lower molecular weight compared with that of the native Cu, ZnꢀSOD (MW 32000 Da),
6
8
although the activities of the complexes are slightly lower than the SOD (IC50 ∼ 0.04 ꢂM). As
－
shown in Scheme 3, the mechanism for the complexes scavenging O • was considered that a six–
2
coordinate octahedral adduct initially formed, which is quite unstable due to the influence of the
2
4

Page 25 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
6
9
5
5
5
5
5
5
5
5
5
5
5
5
5
18
19
20
21
22
23
24
25
26
27
28
29
30
Jahn–Teller effect, then the rapid internal transformation of Cu(II) and Cu(I) was made possible
via electron transfer between copper and reactive oxygen radical anion, and eventually the
－
reaction of catalyzing the dismutation O
2
• was realized. The redox property of copper plays a
0
crucial role in this process. In addition, an adequate Cu(II)/Cu(I) redox potential between E =
－
0
－
2 2 2 2 2
−0.405 V vs. SCE for O /O • and E = 0.645 V vs. SCE for O •/H O was considered necessary
70
for effective catalysis of superoxide radical. From the above results of electrochemical
experiments, the redox potentials of the complexes were all in the allowed ranges of an SOD
mimic, hence both of them have relatively high SODꢀlike activity. Meanwhile, the difference in
Cu(II)/Cu(I) redox potential provides a possible explanation about SODꢀlike activity order of the
7
0
complexes: 1 < 2. Hence, the present work would be helpful to develop new potential
6
6
antioxidants and new therapeutic reagents for some diseases.
Fig. 12
Formazane
VB2
hv
ꢀ
O ·
2
TEMED
NBT
scavenge
H O
2
H O
H O
2
2
N
O
N
N
N
O
N
N
N
O
N
ꢀ
Cu(II)
+
O ·
+ O2
2
Cu(II)
Cu(I)
N
O2
ꢀ
O ·
2
H O
2
H O
2
N
N
O
N
N
O
N
2
H+
Cu(II)
+
H O
2 2
Cu(I)
N
O2
5
5
31
32
Scheme 3: Possible mechanism for SODꢀlike activity of the Cu(II) complexes
2
5

RSC Advances
Page 26 of 41
View Article Online
DOI: 10.1039/C6RA01868H
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Cytotoxic assays
The in vitro cytotoxic activities of the complexes were evaluated against cell lines of HeLa
(cervical), A549 (pulmonary), PCꢀ3 (prostatic) and the control cell line of NIH3T3 (mouse
embryonic fibroblast) by MTT assay. The GI50 values for the cell lines incubated with the
compounds were summarized in Table 3. Complex 1 exhibited higher in vitro antiꢀproliferative
effects against A549 and PCꢀ3 than cisꢀplatinum, one of the most used anticancer drugs against
malignancies. And 2 displayed lower antiꢀproliferative activity than cisꢀplatinum, but its
antiꢀproliferative activity against HeLa cells is the highest among the selected tumor cell lines.
Additionally, 1 displayed higher antiꢀproliferative effects against tumor cell lines (A549 and PCꢀ3)
than normal cell line (NIH3T3). Hence, the above results make a clear indication that 2 is active
on specific cancer cells and 1 has the better potential to act as an effective antitumor agent against
A549 and PCꢀ3. The relative better antiꢀproliferative effects of 1 may be attributed to its stronger
ability to bind and cleave DNA as compared with 2.
Table 3
5
5
5
5
5
5
49 Conclusions
50
51
52
53
54
Two new mononuclear mixed ligand copper(II) complexes with the compositions
[Cu(GlyꢀLꢀval)(HPB)(H O)]·ClO ·1.5H O (1) and [Cu(GlyꢀLꢀval)(PBT)(H O)]·ClO (2) have
2
4
2
2
4
been prepared and characterized by various analytical and spectral methods. Their DNAꢀbinding
behaviors were examined by UV, fluorimetry, CD, viscosity measurements, thermal denaturation
and cyclic voltammetric experiments as well as molecular docking technique. The corroborative
2
6

Page 27 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
5
5
5
5
5
5
5
5
5
5
5
5
5
55
56
57
58
59
60
61
62
63
64
65
66
67
results of these experiments validated that the copper(II) complexes bind to DNA via nonꢀcovalent
interaction with an intercalative mode. On the basis of the fluorimetric experiments, we found that
the interaction processes of the complexes with DNA were dynamic quenching and occurred
spontaneously through hydrophobic interaction. Besides, the complexes exhibited efficient activity
of oxidative cleavage DNA at ~5 ꢂM and were capable of double strand cleavage with a certain
concentration range in the presence of ascorbic acid, probably induced by •OH. Furthermore, the
complexes also displayed anticancer properties against three selected human tumor cell lines.
Especially, 1 displayed higher antiꢀproliferative effects against A549 and PCꢀ3 than that of the
widely used antiꢀcancer drug cisplatin. Meanwhile, the cytotoxicity correlated with the DNA
binding and cleavage activities of the complexes, which showed the sequence 1 > 2. As a whole,
the biochemical properties of the Cu(II) complexes were linked, not isolated. This work will be
valuable in the development probes of DNA structure and potential anticancer drugs.
5
5
5
5
5
68 Acknowledgments
69
70
71
72
We acknowledge the Program of Natural Science Foundation of Guangdong Province
(2015A030313423) and Guangdong Province College Students' Innovation and Entrepreneurship
Training Program (No. 1056413042) for the financial support.
5
73 References:
5
5
5
5
5
5
74
75
76
77
78
79
1. R. Siegel, J. Ma, Z. Zou and A. Jemal, CAꢀCancer J. Clin., 2014, 64, 9ꢀ29.
2. K. Zheng, L. Jiang, Y. T. Li, Z. Y. Wu and C. W. Yan, RSC Adv., 2015, 5, 51730ꢀ51744.
3. G. Bologna, P. Lanuti, P. D Ambrosio, L. Tonucci, L. Pierdomenico, C. D Emilio, N. Celli, M.
Marchisio, N. D Alessandro, E. Santavenere, M. Bressan and S. Miscia, Biometals, 2014, 27,
575ꢀ589.
4. A. Basu, D. Thiyagarajan, C. Kar, A. Ramesh and G. Das, RSC Adv., 2013, 3, 14088ꢀ14098.
2
7

RSC Advances
Page 28 of 41
View Article Online
DOI: 10.1039/C6RA01868H
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
5. I. Correia, S. Roy, C. P. Matos, S. Borovic, N. Butenko, I. Cavaco, F. Marques, J. Lorenzo, A.
Rodríguez, V. Moreno and J. C. Pessoa, J. Inorg. Biochem., 2015, 147, 134ꢀ146.
6. M. J. Li, T. Y. Lan, X. H. Cao, H. H. Yang, Y. P. Shi, C. Q. Yi and G. N. Chen, Dalton Trans.,
2014, 43, 2789ꢀ2798.
7. S. K. Mal, M. Mitra, G. Kaur, V. M. Manikandamathavan, M. S. Kiran, A. R. Choudhury, B. U.
Nair and R. Ghosh, RSC Adv., 2014, 4, 61337ꢀ61342.
8. W. Kaim and J. Rall, Angew. Chem. Int. Ed., 1996, 35, 43ꢀ60.
9. R. Borthakur, A. Kumar, A. Lemtur and R. A. Lal, RSC Adv., 2013, 3, 15139ꢀ15147.
10. C. Marzano, M. Pellei, F. Tisato and C. Santini, AntiꢀCancer Agent. Me., 2009, 9, 185ꢀ211.
11. M. E. BravoꢀGómez, J. C. GarcíaꢀRamos, I. GraciaꢀMora and L. RuizꢀAzuara, J. Inorg. Biochem.,
2009, 103, 299ꢀ309.
12. A. I. ValenciaꢀCruz, L. I. UribeꢀFigueroa, R. GalindoꢀMurillo, K. BacaꢀLópez, A. G. Gutiérrez, A.
VázquezꢀAguirre, L. RuizꢀAzuara, E. HernándezꢀLemus and C. Mejía, PLoS ONE, 2013, 8,
e54664.
13. S. Anbu and M. Kandaswamy, Inorg. Chim. Acta, 2012, 385, 45ꢀ52.
14. R. M. Kumbhare, T. L. Dadmal, R. Pamanji, U. B. Kosurkar, L. R. Velatooru, K. Appalanaidu, Y.
K. Rao and J. V. Rao, Med. Chem. Res., 2014, 23, 4404ꢀ4413.
15. D. Seenaiah, P. R. Reddy, G. M. Reddy, A. Padmaja, V. Padmavathi and N. S. Krishna, Eur. J.
Med. Chem., 2014, 77, 1ꢀ7.
16. P. Xiang, T. Zhou, L. Wang, C. Y. Sun, J. Hu, Y. L. Zhao and L. Yang, Molecules, 2012, 17,
873ꢀ883.
17. K. C. S. Achar, K. M. Hosamani and H. R. Seetharamareddy, Eur. J. Med. Chem., 2010, 45,
2048ꢀ2054.
18. Y. F. Li, G. F. Wang, P. L. He, W. G. Huang, F. H. Zhu, H. Y. Gao, W. Tang, Y. Luo, C. L. Feng,
L. P. Shi, Y. D. Ren, W. Lu and J. P. Zuo, J. Med. Chem., 2006, 49, 4790ꢀ4794.
19. S. Mandal, G. Das and H. Askari, New J. Chem., 2015, 39, 5208ꢀ5217.
20. R. Steininger, J. Karner, E. Roth and K. Langer, Metabolism, 1989, 38, 78ꢀ81.
21. T. Bartek, B. Blombach, E. Zoennchen, P. Makus, S. Lang, B. J. Eikmanns and M. Oldiges,
Biotechnol. Progr., 2010, 26, 361ꢀ371.
22. R. Singh, M. Afzal, M. Zaki, M. Ahmad, S. Tabassum and P. K. Bharadwaj, RSC Adv., 2014, 4,
43504ꢀ43515.
23. R. S. Kumar and S. Arunachalam, Eur. J. Med. Chem., 2009, 44, 1878ꢀ1883.
24. S. K. Jana, S. K. Seth, H. Puschmann, M. Hossain and S. Dalai, RSC Adv., 2014, 4, 57855ꢀ57868.
25. G. J. Chen, X. Qiao, P. Q. Qiao, G. J. Xu, J. Y. Xu, J. L. Tian, W. Gu, X. Liu and S. P. Yan, J.
Inorg. Biochem., 2011, 105, 119ꢀ126.
26. J. B. Chaires, Curr. Opin. Struc. Biol., 1998, 8, 314ꢀ320.
27. X. B. Fu, G. T. Weng, D. D. Liu and X. Y. Le, J. Photoch. Photobio. A., 2014, 276, 83ꢀ95.
28. X. B. Fu, D. D. Liu, Y. Lin, W. Hu, Z. W. Mao and X. Y. Le, Dalton Trans., 2014, 43,
8721ꢀ8737.
29. S. M. Yue, H. B. Xu, J. F. Ma, Z. M. Su, Y. H. Kan and H. J. Zhang, Polyhedron, 2006, 25,
635ꢀ644.
30. S. Tzanopoulou, I. C. Pirmettis, G. Patsis, C. Raptopoulou, A. Terzis, M. Papadopoulos and M.
Pelecanou, Inorg. Chem., 2006, 45, 902ꢀ909.
31. M. E. Reichmann, S. A. Rice, C. A. Thomas, P. Doty and M. Afzal, J. Am. Chem. Soc., 1954, 76,
2
8

Page 29 of 41
RSC Advances
View Article Online
DOI: 10.1039/C6RA01868H
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
3047ꢀ3053.
32. A. Wolfe, G. H. J. Shimer and T. Meehan, Biochemistry, 1987, 26, 6392ꢀ6396.
33. O. Trott and A. J. Olson, J. Comput. Chem., 2010, 31, 455ꢀ461.
34. C. L. Kielkopf, K. E. Erkkila, B. P. Hudson, J. K. Barton and D. C. Rees, Nat. Struct. Mol. Biol.,
2000, 7, 117ꢀ121.
35. W. Hu, S. W. Deng, J. Y. Huang, Y. M. Lu, X. Y. Le and W. X. Zheng, J. Inorg. Biochem., 2013,
127, 90ꢀ98.
36. M. D. Coskun, F. Ari, A. Y. Oral, M. Sarimahmut, H. M. Kutlu, V. T. Yilmaz and E. Ulukaya,
Bioorgan. Med. Chem., 2013, 21, 4698ꢀ4705.
37. A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J. Langley, P.
Cronise and A. VaigroꢀWolff, J. Natl. Cancer Inst., 1991, 83, 757ꢀ766.
38. W. J. Geary, Coordin. Chem. Rev., 1971, 7, 81ꢀ122.
39. P. R. Reddy, N. Raju and B. Satyanarayana, Chem. Biodivers., 2011, 8, 131ꢀ144.
40. K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds (Part B),
John Wiley and Sons, New York, 6th edn, 2009, vol. 58, p.65.
41. B. J. Hathaway, J. Chem. Soc. Dalton Trans., 1972, 1196ꢀ1199.
42. V. K. Verma, A. K. Asatkar, T. A. Jain, S. K. Tripathi, R. Singh, P. B. Hitchcock, S. Nigam and S.
K. Gupta, Polyhedron, 2009, 28, 2591ꢀ2598.
43. J. B. LePecq and C. Paoletti, J. Mol. Biol., 1967, 27, 87ꢀ106.
44. B. L. Fei, W. S. Xu, H. W. Tao, W. Li, Y. Zhang, J. Y. Long, Q. B. Liu, B. Xia and W. Y. Sun, J.
Photoch. Photobio. B., 2014, 132, 36ꢀ44.
45. I. M. Klotz, Ann. Ny Acad. Sci., 1973, 226, 18ꢀ35.
46. H. A. Azab, E. M. Mogahed, F. K. Awad, R. M. A. El Aal and R. M. Kamel, J. Fluoresc., 2012,
22, 971ꢀ992.
47. N. Shahabadi, Z. M. Kalar and N. H. Moghadam, Spectrochim. Acta A., 2012, 96, 723ꢀ728.
48. S. Satyanarayana, J. C. Dabrowiak and J. B. Chaires, Biochemistry, 1993, 32, 2573ꢀ2584.
49. S. Satyanarayana, J. C. Dabrowiak and J. B. Chaires, Biochemistry, 1992, 31, 9319ꢀ9324.
50. I. Haq, P. Lincoln, D. Suh, B. Norden, B. Z. Chowdhry and J. B. Chaires, J. Am. Chem. Soc.,
1995, 117, 4788ꢀ4796.
51. L. Xu, N. J. Zhong, Y. Y. Xie, H. L. Huang, G. B. Jiang and Y. J. Liu, PLoS ONE, 2014, 9,
e96082.
52. S. Das and G. S. Kumar, J. Mol. Struct., 2008, 872, 56ꢀ63.
53. V. I. Ivanov, L. E. Minchenkova, A. K. Schyolkina and A. I. Poletayev, Biopolymers, 1973, 12,
89ꢀ110.
54. S. Mahadevan and M. Palaniandavar, Inorg. Chem., 1998, 37, 693ꢀ700.
55. X. Y. Xu, D. D. Wang, X. J. Sun, S. Y. Zeng, L. W. Li and D. Z. Sun, Thermochim. Acta, 2009,
493, 30ꢀ36.
56. G. Y. Li, K. J. Du, J. Q. Wang, J. W. Liang, J. F. Kou, X. J. Hou, L. N. Ji and H. Chao, J. Inorg.
Biochem., 2013, 119, 43ꢀ53.
57. J.M. Kelly, E.G. Lyons, J.M.V. Putten, and R.E. SmythM, Analytical Chemistry, Symposium
Series, Electrochemistry, Sensors and Analysis, vol. 25, Elsevier, Amsterdam, 1986, p. 205.
58. M. T. Carter, M. Rodriguez and A. J. Bard, J. Am. Chem. Soc., 1989, 111, 8901ꢀ8911.
59. K. Nagaraj, K. S. Murugan, P. Thangamuniyandi and S. Sakthinathan, RSC Adv., 2014, 4,
56084ꢀ56094.
2
9