Self-activated DNA cleavage of a water-soluble mononuclear Cu(II) complex with polyquinolinyl ligand

Article abstract of DOI:10.1080/00958972.2014.973867

The synthesis and characterization of a water-soluble mononuclear Cu(II) complex, [CuLCl₂]·2CH₃CH₂OH, where L = bis(2-quinolinyl methyl)benzyl-amine has been reported. L is a tridentate polyquinolinyl ligand, coordinated to Cu(II) via NNN donors. The central copper ion of 1 has N₃Cl₂ donor set in a distorted trigonal-bipyramidal geometry. The dimer existing in the solid state resulted from hydrogen bonds and π-π accumulation between two mononuclear units. The interaction of 1 with CT-DNA has been explored by absorption and emission titration methods, revealing partial intercalation between 1 and CT-DNA. Moreover, 1 could make pBR322 plasmid DNA cleaved by a self-activated oxidative process; hydroxyl radical and singlet oxygen may be the main reactive oxygen species species in the process. Complex 1 may quench the intrinsic fluorescence of bovine serum albumin in a static quenching process, which has been investigated by UV-visible and fluorescence spectroscopic methods. 1 also demonstrates potent cytotoxity against Hela cells with IC₅₀ value of 2.84 M, which shows it to be a potential candidate as an anticancer metal-based drug.

Full text of DOI:10.1080/00958972.2014.973867

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Self-activated DNA cleavage of a water-
soluble mononuclear Cu(II) complex
with polyquinolinyl ligand
abd
abc
abc
abcd
Jun-Ling Li , Lin Jiang , Si-Tong Li , Jin-Lei Tian , Wen
ab
abc
ab
Gu , Xin Liu & Shi-Ping Yan
a
Department of Chemistry, Nankai University, Tianjin, PR China
Tianjin Key Laboratory of Metal and Molecule Based Material
b
Chemistry, Tianjin, PR China
c
Key Laboratory of Advanced Energy Materials Chemistry (MOE),
Tianjin, PR China
d
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Collaborative Innovation Center of Chemical Science and
Engineering, Tianjin, PR China
Accepted author version posted online: 13 Oct 2014.Published
online: 29 Oct 2014.
To cite this article: Jun-Ling Li, Lin Jiang, Si-Tong Li, Jin-Lei Tian, Wen Gu, Xin Liu & Shi-
Ping Yan (2014) Self-activated DNA cleavage of a water-soluble mononuclear Cu(II) complex
with polyquinolinyl ligand, Journal of Coordination Chemistry, 67:22, 3598-3612, DOI:
10.1080/00958972.2014.973867
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Journal of Coordination Chemistry, 2014
Vol. 67, No. 22, 3598–3612, http://dx.doi.org/10.1080/00958972.2014.973867
Self-activated DNA cleavage of a water-soluble mononuclear
Cu(II) complex with polyquinolinyl ligand
JUN-LING LI†‡¶, LIN JIANG†‡§, SI-TONG LI†‡§, JIN-LEI TIAN*†‡§¶, WEN GU†‡,
XIN LIU*†‡§ and SHI-PING YAN†‡
†
Department of Chemistry, Nankai University, Tianjin, PR China
Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry, Tianjin, PR China
Key Laboratory of Advanced Energy Materials Chemistry (MOE), Tianjin, PR China
Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, PR China
‡
§
¶
(Received 30 June 2014; accepted 17 September 2014)
The synthesis and characterization of a water-soluble mononuclear Cu(II) complex, [CuLCl
CH OH, where L = bis(2-quinolinyl methyl)benzyl-amine has been reported. L is a tridentate poly-
quinolinyl ligand, coordinated to Cu(II) via NNN donors. The central copper ion of 1 has N Cl
2 3
]·2CH -
2
3
2
donor set in a distorted trigonal-bipyramidal geometry. The dimer existing in the solid state resulted
from hydrogen bonds and π–π accumulation between two mononuclear units. The interaction of 1
with CT-DNA has been explored by absorption and emission titration methods, revealing partial
intercalation between 1 and CT-DNA. Moreover, 1 could make pBR322 plasmid DNA cleaved by a
self-activated oxidative process; hydroxyl radical and singlet oxygen may be the main reactive oxy-
gen species species in the process. Complex 1 may quench the intrinsic fluorescence of bovine
serum albumin in a static quenching process, which has been investigated by UV–visible and fluo-
rescence spectroscopic methods. 1 also demonstrates potent cytotoxity against Hela cells with IC50
value of 2.84 μM, which shows it to be a potential candidate as an anticancer metal-based drug.
Keywords: Cu(II) complex; Water soluble; Self-activation DNA cleavage; Protein binding
*
Corresponding authors. Email: tian@nankai.edu.cn (J.-L. Tian); liuxin64@nankai.edu.cn (X. Liu)
2014 Taylor & Francis
©

Cu(II) with polyquinolinyl
3599
1. Introduction
Metal complexes constitute an important class of compounds with biological interest [1]. In
964, the observation of cis-[Pt(NH ) Cl ] prompted a growing interest in the development
1
3
2
2
of metal-based drugs to suppress cell division and clinical use for treatment of cancers [2].
The platinum family, carboplatinum, and oxaliplatin have been used in clinical studies.
Recently, two ruthenium complexes, NAMI-A, [ImH][trans-RuCl (DMSO)(Im)] (where
4
Im = imidazole) and KP1019, [InH][trans-RuCl (In) ] (where In = indazole) have entered
4
2
clinical trials [3]. Transitional metal complexes have also attracted attention for its two
advantages as DNA-binding agents [4]: (i) well-defined coordination geometries to provide
attractive moieties for reversible recognition of nucleic acids; and (ii) distinctive electro-
chemical or photophysical properties thus enhancing the functionality of a binding agent.
Copper is an essential element for most aerobic organisms, so Cu-based complexes have
been investigated on the assumption that endogenous metals may be less toxic for normal
cells than cancer cells [5–8]. Recently, Carlo Santini and his co-workers have elaborated on
the advances of copper-based complexes as anticancer drugs [9]. Moreover, Kaushik Ghosh
et al. investigated a mononuclear Cu(II) complex, which shows self-activated DNA cleav-
age and affords excellent anticancer activity against MCF-7 cells. A ternary copper–terpyri-
dine complex, [Cu(ttpy)(Gly)(NO )](NO )·H O (ttpy = 4′-p-tolyl-2,2′:6,2″-terpyridine,
3
3
2
Gly = glycine), also displays remarkable DNA cleavage activity in the presence of Vc [10].
As neutral, non-deprotonated chelating ligands, di(picolyl)amine (dpa) and its derivatives
form many complexes, some of which show efficient DNA binding, cleavage activity, as
well as anticancer activities [11]. Especially, with N-benzyl di(pyridylmethyl)amine (phdpa),
Cu(II) complexes could effectively inhibit the proliferation of cancer cells, A549, Hela and
HepG-, with IC50 values lower than 5-Fluorouracil [12]. Compared with pyridine, quinoline
ring shows better planarity and stronger hydrophobic DNA interaction, which may enhance
DNA binding ability [6]. In addition, quinoline and its derivatives could be a privileged
scaffold in cancer drug discovery [13]. So, it will be interesting to investigate biological
activities of Cu(II) complexes with quinoline substituted di(picolyl)amines ligands.
A mononuclear Cu(II) complex, [CuLCl ]·2CH CH OH, where L = bis(2-quinolinyl
2
3
2
methyl)benzyl-amine, has been synthesized and structurally characterized. Complex 1
shows good water solubility, which provides a good start for further investigations. After
investigations of interaction between 1 and DNA by different techniques, 1 not only effi-
ciently binds with CT-DNA, but also reveals self-activated oxidative DNA cleavage. More-
over, 1 also could quench the intrinsic fluorescence of bovine serum albumin (BSA) in a
static quenching process. Complex 1 also demonstrates potent cytotoxicity against Hela
cells with IC50 of 2.84 μM.
2
2
. Experimental
.1. Materials and measurements
All reagents and chemicals were purchased from commercial sources and used as received.
Plasmid pBR322 DNA, agarose, ethidium bromide (EB), BSA, glutathione monoester
(
(
GSH-OEt), and calf thymus CT-DNA were obtained from Sigma. Stock solutions of 1
−
3
1.0 × 10 M in H O) were stored at 4 °C and prepared to required concentrations for all
2

3
600
J.-L. Li et al.
experiments. Ultra-pure MilliQ water (18.24 MΩ cm) was used in all experiments. Tris–
HCl and phosphate buffer solution were prepared using ultrapure water. Cell counting kit-8
(CCK-8) was purchased from Dojindo Molecular Technologies, Inc. Elemental analyses for
C, H, and N were obtained on a Perkin-Elmer analyzer model 240. Infrared spectra on KBr
pellets were performed on a Bruker Vector 22 FT-IR spectrophotometer from 4000 to
−
1
4
00 cm . Electrospray ionization mass spectrometry was obtained on an Agilent 6520
Q-TOF LC/MS. Electronic spectra were measured on a JASCO V-570 spectrophotometer.
Fluorescence spectral data were obtained on a MPF-4 fluorescence spectrophotometer at
room temperature. The gel imaging and documentation DigiDoc-It System was assessed
using Labworks Imaging and Analysis Software (UVI, UK).
2
2
.2. Synthesis of L and 1
.2.1. Preparation of ligand. L (scheme 1) was prepared according to a procedure
described in the literature [14] with some improvements. A mixture of 2-chloromethylquin-
oline hydrochloride (1.82 g, 8.4 mM), benzylamine (0.45 g, 4.2 mM), and potassium car-
bonate (3.5 g, 26 mM) in acetonitrile (30 mL) was refluxed for 48 h. After the solvent was
removed under reduced pressure, the residue was separated by chloroform/water and the
organic phase was dried, evaporated, and pale yellow powder was obtained, yield 1.05 g
−
1
(
(
(
64%). Selected IR data (KBr, ν, cm ): 3415(s), 3328(w), 1638(s), 1617(s), 1504(w), 1426
1
w), 1117(m), 824(m), 766(w), 620(s), 482(m), 411(w). H NMR (CDCl ): δ(ppm) 8.12
2H, d), 8.05 (2H, d), 7.79 (2H, t), 7.74 (2H, d), 7.67 (2H, m), 7.50 (2H, m), 7.44 (2H, d),
3
7
.32 (2H, m), 7.23 (1H, m), 4.02 (4H, s), 3.73 (2H, s).
Scheme 1. Schematic structure of L.
2
.2.2. Synthesis of [CuLCl ]·2CH CH OH. To an ethanol solution (10 mL) of L
2 3 2
0
.0392 g (0.1 mM), an aqueous solution (5 mL) of CuCl ·2H O 0.0172 g (0.1 mM) was
2 2
added. The resulting mixture was stirred for 3 h at room temperature. Cyan prism crystals
suitable for X-ray diffraction were obtained by slow evaporation of the filtrate after 15 days,
which were collected by filtration, washed with diethyl ether, and dried in air, yield

Cu(II) with polyquinolinyl
3601
0
5
3
.0137 g (22%). Elemental analysis (%): Anal Calcd for C H Cl CuN O : C, 60.44; H,
.73; N, 6.82. Found: C, 60.58; H, 5.62; N, 6.89. Selected IR data (KBr, ν, cm ): 3415(s),
328(w), 2026(m), 1638(s), 1617(s), 1456(w), 1129(m), 623(s), 484(m), 410(w).
3
1
35
2
3 2
−
1
2
.3. X-ray crystallographic studies
Single-crystal X-ray diffraction data were collected on a Bruker Smart 1000 CCD diffrac-
tometer using Mo K radiation (λ = 0.71073 Å) with the ω scan technique. The structure of
α
1
was solved by direct methods (SHELXS-97 and refined with full matrix least squares on
2
F using SHELXL-97 [15]). The hydrogens were added theoretically, riding on the con-
cerned atoms and refined with fixed thermal factors. The details of crystallographic data
and structure refinement parameters are summarized in table 1, and selected bond angles
and distances are listed in table 2. Crystallographic data for 1 have been deposited with the
Cambridge Crystallographic Data Center with the corresponding CCDC reference number
of 997352.
Table 1. Crystal data and structure refinement for 1.
Compound reference
Complex
Chemical formula
C
31
H
2 3 2
35Cl CuN O
Formula mass
T (K)
616.06
113.15
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
Monoclinic
P2(1)/n
9.2716(19)
15.010(3)
20.722(4)
90.00
β (°)
97.45(3)
90.00
2859.4(10)
4
γ (°) 3
V (Å )
No. of formula units per unit cell (Z)
Radiation type
Absorption coefficient (μ/mm
No. of reflections measured
Mo/Kα
0.985
26,792
5037
−
1
)
No. of independent reflections
R
int
0.0469
0.0438
0.1180
0.0494
0.1219
Final R
Final wR(F ) values [I > 2σ(I)]
Final R values (all data)
Final wR(F ) values (all data)
1
values [I > 2σ(I)]
2
1
2
Table 2. Selected bond lengths (Å) and angles (°) for 1.
Cu(1)–N(3)
Cu(1)–N(1)
Cu(1)–N(2)
N(3)–Cu(1)–N(1)
N(3)–Cu(1)–N(2)
N(1)–Cu(1)–N(2)
N(3)–Cu(1)–Cl(2)
N(1)–Cu(1)–Cl(2)
1.988(2)
1.994(2)
2.137(2)
163.38(10)
82.10(10)
81.62(10)
93.73(7)
92.41(7)
Cu(1)–Cl(2)
Cu(1)–Cl(1)
2.3277(9)
2.4266(9)
N(2)–Cu(1)–Cl(2)
N(3)–Cu(1)–Cl(1)
N(1)–Cu(1)–Cl(1)
N(2)–Cu(1)–Cl(1)
Cl(2)–Cu(1)–Cl(1)
123.21(7)
93.58(7)
93.99(7)
105.68(7)
131.11(3)

3
602
J.-L. Li et al.
2.4. DNA binding and cleavage activity studies
The UV absorbance at 260 and 280 nm of the CT-DNA solution in 5 mM Tris–HCl/50 mM
NaCl buffer (pH 7.2) gives a ratio of 1.8–1.9, indicating that CT-DNA was sufficiently free
of protein [16]. The concentration of CT-DNA was determined from its absorption intensity
−
1
−1
at 260 nm with a molar extinction coefficient of 6600 M cm . The absorption spectra of
binding to DNA were performed by increasing amounts of CT-DNA to 1 in Tris–HCl
1
buffer (pH 7.2).
The relative binding abilities of 1 to CT-DNA were studied with an EB-bound CT-DNA
solution in 5 mM Tris–HCl/50 mM NaCl buffer (pH 7.2). The fluorescence spectra were
recorded at room temperature with excitation at 510 nm and emission at 602 nm. The
experiments were carried out by titrating complex into EB-DNA solution containing
−6
−6
2
.4 × 10 M EB and 48 × 10 M CT-DNA.
The DNA cleavage experiments were carried out by agarose gel electrophoresis, which
was performed by incubating at 37 °C; pBR322 DNA (0.1 μg/μL) in 50 mM Tris–HCl
1
3
8 mM NaCl buffer (pH 7.2) was treated with complex. The samples were incubated for
h and loading buffer was added. Then the samples were electrophoresed for 2 h at 0.9%
agorose gel using Tris–boric acid–EDTA buffer. After electrophoresis, bands were visual-
ized by UV light and photographed.
Cleavage mechanism of pBR322 DNA was investigated in the presence of some radical
scavengers and reaction inhibitors. The reactions were conducted by adding standard radical
scavengers, KI, L-His, scavenger superoxide dismutase (SOD), EDTA, catalase, and groove
binding agents – Methyl Green (for major groove) and SYBR Green (for minor groove) to
pBR322 DNA after the addition of complex. Cleavage experiment was initiated by addition
of complex and quenched with 2 μL of loading buffer. Further analysis was carried out by
the above standard method.
2
.5. Protein binding studies
Fluorescence quenching experiments have been carried out to investigate the interaction
between BSA and 1 by using bovine serum albumin stock solution (BSA, 1.5 mM) in
1
0 mM phosphate buffer (pH 7.0). The fluorescence spectra were recorded at room temper-
ature with excitation of BSA at 280 nm and emission at 342 nm by keeping concentration
of BSA constant (29.4 μM) while varying the complex concentration from 0 to 40 μM.
2
.6. In vitro cell assay
The cytotoxicity assay of 1 against Hela cervical carcinoma cells was evaluated by a CCK-
kit assay. The Hela cells were cultured in a DMEM/F12 medium supplemented with 10%
8
fetal bovine serum (5% CO at 37 °C). The cells were seeded into 96 well plates at a den-
2
4
sity of 10 cell/well and incubated for 48 h to allow cell attachment. Then, the cells were
washed with PBS, and the medium was replaced with a fresh medium containing the indi-
cated concentrations of 1. Cells without pretreatment were used as the control. After a per-
iod of incubation, the cells were washed with PBS and incubated in DMEM/F12 with 10%
WST-8 solution for another 2 h. The absorbance of each well was measured at a wavelength
of 450 nm with a plate reader. The results were expressed as the mean values of three mea-
surements. The cell viability was calculated as follows,

Cu(II) with polyquinolinyl
3603
Isample ꢀ I
blank
cell viability ð%Þ ¼
ꢁ 100
Icontrol ꢀ I
blank
where Isample, Icontrol, and Iblank represent the absorbance intensity at 450 nm determined
for cells treated with different samples, for control cells (nontreated), and for blank wells
without cells (the same amounts of the CCK-8 solution and the sample solution as the
sample wells were added to the blank wells), respectively.
3
3
. Results and discussion
.1. Description of the crystal structure
The complex has been structurally characterized by X-ray crystallography (figure 1); details
of the data collection conditions and the parameters of refinement are given in table 1, and
selected bond lengths and angles are given in table 2.
Complex 1 crystallized in the monoclinic P2(1)/n space group with five-coordinate Cu
with the N Cl donor set is derived from one tertiary amine, two quinoline nitrogens, and
3
2
two chlorides. The τ value of Cu is 0.54, which indicates that the geometry of 1 is distorted
trigonal-bipyramidal. The basal plane is occupied by Cl(1), Cl(2), and N(2), while two
quinoline nitrogens occupy the axial positions, with Cu–N distance (Cu–N(1) 1.994(2) Å,
Cu–N(3) 1.988(2) Å) shorter than the equatorial distance (Cu–N(2) 2.137(2) Å). The bond
distances of Cu–N and Cu–Cl (Cu–Cl(1) 2.4266(9) Å, Cu–Cl(2) 2.3277(9) Å) fall in the
range of several reported Cu(II) complexes, [Cu(L6)Cl ](L6 = 2-(1H-benzimidazol-2-yl)
2
ethyl-(4,4adihydroquinolin-2-ylmethylene)amine) [6] (Cu–Nimine 2.050(2) Å, Cu–Nbzim
Figure 1. Molecular structure of 1. Hydrogens and solvent molecules have been omitted for clarity.

3
604
J.-L. Li et al.
Figure 2. Depiction of interactions between hydrogen bonds in 1.
1
[
.972(2) Å, Cu–Nquin 2.041(2) Å, Cu–Cl(1) 2.360(8) and Cu–Cl(2) 2.354(7) Å) and
CuCl (C H N )(H O)] (C H N {(1-methyl imidazol-2-yl)methylene}-2-amino
ethyl-pyridine) [17] (Cu–N1 2.053(2) Å, Cu–N2 2.032(2) Å, Cu–N3 2.035(2) Å, Cu–Cl1
=
2
12 14
4
2
12 14 4
2
.538(1) Å, and Cu–Cl2 2.302(1) Å).
A dimer exists between two mononuclear units in the solid state, which results from
−
hydrogen bonds and π–π accumulation. Hydrogen bonds between the equatorial Cl ligand
and O atom of ethanol, C atom of quinoline ring are presented in structure (figure 2). The
respective bond distance and angles are listed in table 3. In O–H…Cl, the corresponding
bond distances are similar with [CuCl (C H N )(H O)] [17]. The bond distances in C–
2
12 14
4
2
H…Cl also fall in the range of [Cu (L8)(Cl) ] (L8 = 1,4-bis[di(2-picolyl)aminomethyl]ben-
2
4
−
zene) [18]. The hydrogen bonding between quinoline ring and coordinated Cl is further
stabilized by aromatic π–π stacking interactions between two monomer units of quinoline
rings with a center-to-center distance of 3.257 Å.
3
3
.2. DNA-binding and cleavage activities
.2.1. Absorption spectra studies. Electronic absorption spectroscopy is a common
method to investigate the interaction between complex and DNA. If the compound binds to
DNA through intercalation, hypochromism with or without a small red shift occurs, due to
Table 3. Hydrogen bonds (Å) and (°) of 1.
D–H…A
d(D–H)
d(H…A)
d(D…A)
θ(\DHA)
O1–H1…Cl1
C14#–H14#…Cl2
0.82
0.93
2.873
2.853
3.266
3.669
111.68
147.07
Note: #, 1−x, 2−y, −z.

Cu(II) with polyquinolinyl
3605
Figure 3. Absorption spectra of 1 in the absence (dashed line) and presence (solid line) of increasing amounts of
CT-DNA (9.4–75.2 μM) in 5 mM Tris-HCl/50 mM NaCl buffer (pH 7.2). The insert shows the least-squares fit of
(ε −ε )/(ε −ε ) vs. [DNA] for 1.
a f b f
stacking between the planar aromatic chromophore and the base pair of DNA [19, 20].
From figure 3, upon incremental addition of DNA to complex, the obvious decrease in
molar absorptivity was observed, without a red shift, which is similar to several reported
complexes [6]. In the presence of DNA, the absorption band of 1 exhibited a hypochro-
mism about 11.7%. The result suggests that 1 may bind to DNA via intercalation. In order
to determine the binding strength of 1 with CT-DNA, intrinsic binding constant K was cal-
b
culated from a nonlinear fitting according to equations (1a) and (1b) [21].
ꢀ
ꢁ
À
Á
1=2
2
2
ðe ꢀ e Þ=ðe ꢀ e Þ ¼ b ꢀ b ꢀ 2K C ½DNAꢂ=s
=2K C
t
(1a)
(1b)
a
f
b
f
b
t
b
b ¼ 1 þ KbCt þ Kb½DNAꢂ=2s
Table 4. Absorption spectral properties and fluorescence spectral properties of 1 bound to CT-DNA.
−1
−1
app (M
Complex
K
b
(M
)
K
)
Ref.
5
6
4
4
4
4
5
Complex 1
9.18 × 10
1.06 × 10
3.69 × 10
8.20 × 10
3.40 × 10
7.60 × 10
This work
[
[
[
[
[
(phdpa)Cu(bpy)(ClO
4
)]
12
12
6
6
19
(phdpa)Cu(phen)(H O)]
2
3
Cu(L )Cl
2
]
]
6
3
5
Cu(L )Cl
2
6.60 × 10
2.98(±0.13) × 10
CuCl
2
(OQsc-H)]·H
2
O·CH
3
OH
2.91(±0.23) × 10
Notes: Phdpa: N-benzyl di(pyridylmethyl)amine.
bpy: 4,4-dimethyl-2,2-bipyridine.
phen: 1,10-phenanthroline.
3
L : (2-pyridin-2-yl-ethyl)pyridin-2-ylmethyleneamine.
6
L : 2-(1H -benzimidazol-2-yl)ethyl-(4,4a-dihydroquinolin-2-ylmethylene)amine.
OQsc-H: 2-oxo-1,2-dihydroquinoline-3-carbaldehyde semicarbazone.

3
606
J.-L. Li et al.
The DNA binding constant (K ) of 1, along with the binding site size (s), is given in
b
5
−1
table 4. The K value of 1 is 9.18 × 10 M , which is larger than several reported mononu-
b
1
clear Cu(II) complexes [6, 19]. However, the K value is smaller than [Cu(L )Br ]
b
2
6
−1
1
(
3.45 × 10 M ) (L = (2-((quinolin-8-ylimino)methyl)pyridine)) [22], which may be due to
the larger steric hindrance as a result of phenyl in L. The s value of 1 is 1.06, which may
be due to an aggregation of hydrophobic molecules on the DNA surface by π stacking [23].
The DNA binding propensity is also determined from other assays.
3
.2.2. Fluorescence spectral studies. Compound 1 shows no luminescence at room tem-
perature in aqueous solutions. As a result, the EB–DNA system was used to further investi-
gate the interaction between 1 and DNA. Emission spectra of EB bound to CT-DNA in the
absence and presence of 1 are predicted in figure 4. As the concentration of complex
increases, the emission band at 603 nm exhibited hypochromism up to 42.3% of the initial
fluorescence intensity. The remarkable reduction in the emission intensity clearly indicates
that EB molecules are replaced by 1 and released into solution [24]. Quenching data were
analyzed according to the Stern–Volmer equation: I /I = K [Q] + 1, where I and I represent
0
q
0
fluorescence intensities in the absence and presence of quencher and [Q] is the quencher
concentration. In the linear fit plot of I /I versus [Q], K is the Stern–Volmer dynamic
0
q
quenching constant. The apparent DNA binding constant (Kapp), which could express the
degree of affinity toward DNA, was calculated on the basis of the equation,
KEB ½EBꢂ ¼ Kapp ½complexꢂ
7
−1
Here, KEB = 1.0 × 10 M ([EB] = 2.4 μM). KEB is the binding constant of EB to DNA
and the complex concentration is the value at 50% reduction of the fluorescence intensity
6
−1
7
−1
of EB. The Kapp value of 1 is 1.06 × 10 M , which is smaller than K (1.0 × 10 M )
EB
Figure 4. Emission spectra of EB bound to CT-DNA in the absence (dashed line) and presence (solid lines) of 1
(
0
0–160 μM) in 50 mM Tris–HCl/18 mM NaCl buffer (pH 7.2). Inset: the plot of I /I vs. the complex concentration.

Cu(II) with polyquinolinyl
3607
−
1
Figure 5. Cleavage of plasmid pBR322 DNA (0.1 μg/μL ) at different concentration of 1 after 3 h incubation at
7 °C; Line 0: DNA control; Lines 1–6: DNA + complex (5, 20, 25, 30, 35, and 40 μM).
3
and suggests moderate interactions between 1 and DNA. However, the value is higher than
3
4
−1
two Cu(II) complexes with similar structures [6] (table 4), [Cu(L )Cl ] (3.40 × 10 M )
2
6
4
−1
and [Cu(L )Cl ] (7.60 × 10 M ), which suggests stronger DNA affinity than the two com-
2
plexes. The result may indicate that ligand plays the main role in interaction with DNA.
Moreover, the DNA binding ability of 1 is also stronger than [(phdpa)Cu(bpy)(ClO )]
4
4
−1
4
−1
(
3.69 × 10 M ) and [(phdpa)Cu(phen)(H O)] (8.20 × 10 M ) [12], which shares similar
2
ligand structure with incorporation of pyridyl instead of quinolyl. The result suggests that,
with stronger hydrophobic DNA interaction and larger aromaticity, the incorporation of qui-
nolyl moiety in L shows better DNA affinity.
3
.2.3. Cleavage of pBR322 DNA by complex without added reagents. To assess the
DNA cleavage ability of 1, supercoiled (SC) pBR322 DNA was incubated with varying
concentrations of 1 in a medium of 50 mM Tris–HCl/18 mM NaCl buffer (pH 7.2) for 3 h
(
figure 5). The cleavage reactions on plasmid DNA by 1 can be monitored by agarose gel
electrophoresis. Except a few examples of hydrolytic cleavage, most DNA cleavage agents
were mediated by Cu(II) complexes through an oxidative mechanism [25]. Activators [6,
1
0] or light [26] usually used in the reactions to generate reactive oxygen species (ROS) or
reactive metal-oxo species upon reaction with copper limit the development of such metal
agents in our body. However, different from reported complexes, 1 shows efficient DNA
cleavage without activators. It is clear from figure 5 that at 20 μM 1 converts 50% SC
DNA (Form I) into nicked circular NC DNA (Form II) with prominent DNA cleavage. As
the concentration of 1 increases, more SC DNA was converted into NC DNA, showing con-
centration dependence. When the concentration is 35 μM, 1 could completely degrade SC
DNA into NC DNA, though LC DNA (Form III) is still not found even at 40 μM. After
concentration-dependent DNA cleavage of 1, it is clear that 1 exhibits self-activated DNA
cleavage without activators, which may be conducive for in vivo applications.

3
608
J.-L. Li et al.
−
1
Figure 6. Cleavage of plasmid pBR322 DNA (0.1 μg/μL ) in the presence of 1 (30 μM) and different inhibitors
after 3 h incubation at 37 °C; Line 0: DNA control; Line 1: DNA + 1 (30 μM); Lines 2–10: DNA + 1 + (10 mM
KI, 10 mM L-His, 20 μ/mL SOD, 20 μ/mL Catalase, Methyl Green, SYBR Green, 10 mM EDTA).
3.2.4. DNA cleavage mechanism. The activity for DNA cleavage without an oxidant or
reducing agent is often related to a hydrolytic rather than oxidative mechanism. However,
without a co-reagent, there are still many complexes that act by an oxidative mechanism
[
8, 27]. In order to diagnose the reactive species responsible for DNA cleavage, the quench-
ing assay was monitored in the presence of different scavengers, and the results are depicted
in figure 6. The addition of hydroxyl radical scavenger KI and singlet-oxygen scavengers
L-His caused strong inhibition of the nuclease activity, and cleavage ability was also influ-
−
1
enced in the presence of O ˙ radical SOD, which indicated that ˙OH and O may be the
2
2
−
main ROS species in the reaction while O ˙ is also involved in the DNA cleavage [10].
2
Methyl green and EDTA efficiently inhibit the cleavage, which implies that 1 may bind to
DNA in the major groove and metal ions may participate in the cleavage process. In sum-
mary, hydroxyl radicals are the crucial ROS responsible for the cleavage activity of 1; sin-
−
glet oxygen and O ˙ are less influential in the cleavage.
2
3
.3. Protein binding activities
Investigation on the binding of a drug with protein facilitates interpretation of the metabo-
lism and transporting process of a drug and to explain the relationship between structures
and functions of protein. BSA was utilized extensively as a result of its structural homology
with human serum albumin. Qualitative analysis of binding of compounds to BSA is usu-
ally detected by inspecting the fluorescence spectra. BSA consists of three aromatic amino
acids (phenylalanine, tyrosine, and tryptophan), and its fluorescence can appear because of
tryptophan and tyrosine residues [28]. Figure 7 shows the effect of 1 on the fluorescence
intensity of BSA protein.
Upon addition of 1 to solution of BSA, a significant decrease of fluorescence intensity
has been observed. The fluorescence intensity of BSA at 350 nm decreased about 58.7%
from the initial fluorescence intensity of BSA, which suggested an interaction of the com-
pound with the BSA protein [29]. Quenching mechanisms are usually classified as either

Cu(II) with polyquinolinyl
3609
Figure 7. Fluorescence emission spectra of the BSA (29.4 μM) system in the absence (dashed line) and presence
solid lines) of 1 (0–40 μM, respectively). The insets (a) the plot of I /I vs. the concentration of complex; (b) plot
of log (I − I) /I vs. log[Q] for BSA in the presence of 1.
(
0
0
dynamic quenching or static quenching. Static quenching refers to fluorophore−quencher
complex formation, and if fluorophore and quencher come into contact during transient
existence of the exited state, dynamic quenching happens. To study the quenching process,
fluorescence quenching data have been analyzed by the Stern–Volmer equation:
I /I = 1 + K [Q] = 1 + K τ [Q], where I and I are fluorescence intensities of fluorophore in
0
sv
q 0
0
the absence and presence of quencher, respectively, τ is the average life time of biomole-
0
−
8
cule without quencher (about 10 s) and Ksv is the Stern–Volmer quenching constant. The
plot of I /I versus [Q] and the Stern–Volmer plots of fluorescence titration data for 1 are
0
shown in figure 7 and table 5. From table 5, the calculated bimolecular quenching rate con-
stants (K ) were larger than the maximum K value of various quenchers to biopolymers
q
q
1
0
−1 −1
(
2.0 × 10 M s ) [29], which suggests the formation of BSA-compound complex.
A simple method to explore the quenching mechanism is UV–visible absorption spectros-
copy. The electronic spectrum of BSA with 1 is shown in figure 8. In the presence of 1, the
absorption intensity was enhanced which further revealed the static interaction between
BSA and 1 [30].
Table 5. The quenching constant, binding constant, and number of binding sites for
interaction of 1 with BSA.
−
1
−1 −1
−1
Complex 1
K
sv (M
)
K
q
(M
s
)
K (M
)
n
4
12
4
3
.5 × 10
3.5 × 10
1.87 × 10
0.94

3
610
J.-L. Li et al.
Figure 8. Absorption spectra of BSA (15 μM) and BSA with 1 (1 μM).
For static quenching, if it is assumed that the binding of 1 with BSA could occur at equi-
librium, the binding constant (K) could be analyzed by the Scatchard equation:
log½ðI0 ꢀ IÞ=Iꢂ ¼ log K þ n log½Qꢂ
where K is the binding constant of the compound with BSA and n is the number of bind-
ing sites. The number of binding sites (n) and binding constant (K) have been obtained from
the plot of log(I – I)/I versus log[Q] and are listed in table 5. The K value of 1 is smaller
0
than several reported mononuclear Cu(II) complexes [19, 31], which may be due to large
steric effects as a result of the incorporation of phenyl. The value of n is approximately
equal to one, which indicates that there is just one independent binding site in BSA for 1.
3
.4. In vitro cell assay
The CCK-8 assay was carried out to evaluate the in vitro cytotoxicity of 1 against HeLa
cells. The CCK-8 assay is a well-established method for determination of cell viability in
cell proliferation and cytotoxicity assays [32, 33], which utilizes Dojindo’s highly water-
soluble tetrazolium salt, WST-8, to produce water-soluble formazan dye on reduction in the
living cells to determine the number of living cells. Complex 1 shows prominent cytotoxic-
ity, and the IC50 value (2.13 μM) obtained by plotting the cell viability against concentra-
tion of 1 reveals that 1 exhibits seven fold cytotoxicity higher than cisplatin (15 μM) [34]
for 48 h incubation. Notably, 1 shows prominent cytotoxicity against Hela cells even with
incubation for 24 h with IC50 of 2.84 μM, which indicates that our complex may be a new
candidate for metal-based anticancer agents. However, further investigations should be car-
ried out in order to apply the drug in clinic.

Cu(II) with polyquinolinyl
3611
4. Conclusion
With the tridentate polyquinolinyl ligand, bis(2-quinolinyl methyl)benzyl-amine, a new
water-soluble Cu(II) complex, has been prepared and characterized by structural and spec-
tral methods. The incorporation of quinoline not only increases the planarity but also the
hydrophilic interactions when compared to the reported pyridine analogs, which may con-
tribute to the good DNA binding ability. Further investigations, by absorption and fluores-
cence measurements, demonstrated that 1 may bind with DNA through partial intercalation.
Also, 1 shows good self-activated DNA cleavage ability, offering potential application
in vivo; complex 1 promotes DNA cleavage in an oxidative way; hydroxyl radical and sin-
glet oxygen may be the main ROS radicals in the process. According to the results obtained
from fluorescence and UV–visible spectrometry, we speculate that 1 could bind with BSA
in a static quenching process. Complex 1 shows a considerable cytotoxic activity
(IC50 = 2.84 μM) against Hela cells, better than cisplain, which suggests that 1 may be a
potential anticancer drug.
Funding
This work was supported by the National Natural Science Foundation of China [grant number 21171101], [grant
number 21371103], [grant number 21471085]; Tianjin Science Foundation [grant number 12JCYBJC13600];
NFFTBS [grant number J1103306]; MOE Innovation Team [grant number IRT13022] of China.
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