Effects of copper ions on DNA binding and cytotoxic activity of a chiral salicylidene Schiff base

Article abstract of DOI:10.1016/j.jphotobiol.2014.01.018

A chiral Schiff base HL N-(5-bromo-salicylaldehyde)dehydroabietylamine (1) and its chiral dinuclear copper complex [Cu₂L₄] ·4DMF (2) have been synthesized and fully characterized. The interactions of 1 and 2 with salmon sperm DNA have been investigated by viscosity measurements, UV, fluorescence and circular dichroism (CD) spectroscopic techniques. Absorption spectral (K_b = 3.30 × 10⁵ M^-1 (1), 6.63 × 10⁵ M^{- 1}(2)), emission spectral (K_sv = 7.58 × 10 ³ M^-1 (1), 1.52 × 10⁴ M ^-1 (2)), and viscosity measurements reveal that 1 and 2 interact with DNA through intercalation and 2 exhibits a higher DNA binding ability. In addition, CD study indicates 2 cause a more evident perturbation on the base stacking and helicity of B-DNA upon binding to it. In fluorimetric studies, the enthalpy (ΔH > 0) and entropy (ΔS > 0) changes of the reactions between the compounds with DNA demonstrate hydrophobic interactions. 1 and 2 were also screened for their cytotoxic ability and 2 demonstrates higher growth inhibition of the selected cancer cells at concentration of 50 μM, this result is identical with their DNA binding ability order. All the experimental results show that the involvement of Cu (II) centers has some interesting effect on DNA binding ability and cytotoxicity of the chiral Schiff base.

Full text of DOI:10.1016/j.jphotobiol.2014.01.018

Journal of Photochemistry and Photobiology B: Biology 132 (2014) 36–44
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Effects of copper ions on DNA binding and cytotoxic activity of a chiral
salicylidene Schiff base
a
a
a
a
c
c
Bao-Li Fei a,b, , Wu-Shuang Xu , Hui-Wen Tao , Wen Li , Yu Zhang , Jian-Ying Long , Qing-Bo Liu ,
⇑
Bing Xia c, Wei-Yin Sun b,
⇑
a College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
b Coordination Chemistry Institute, State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, China
c College of Science, Nanjing Forestry University, Nanjing 210037, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A chiral Schiff base HL N-(5-bromo-salicylaldehyde)dehydroabietylamine (1) and its chiral dinuclear cop-
per complex [Cu2L4]ꢀ4DMF (2) have been synthesized and fully characterized. The interactions of 1 and 2
with salmon sperm DNA have been investigated by viscosity measurements, UV, fluorescence and circu-
lar dichroism (CD) spectroscopic techniques. Absorption spectral (Kb = 3.30 ꢁ 105 Mꢂ1 (1),
6.63 ꢁ 105 Mꢂ1(2)), emission spectral (Ksv = 7.58 ꢁ 103 Mꢂ1 (1), 1.52 ꢁ 104 Mꢂ1 (2)), and viscosity mea-
surements reveal that 1 and 2 interact with DNA through intercalation and 2 exhibits a higher DNA bind-
ing ability. In addition, CD study indicates 2 cause a more evident perturbation on the base stacking and
helicity of B-DNA upon binding to it. In fluorimetric studies, the enthalpy (DH > 0) and entropy (DS > 0)
changes of the reactions between the compounds with DNA demonstrate hydrophobic interactions. 1 and
2 were also screened for their cytotoxic ability and 2 demonstrates higher growth inhibition of the
Received 4 September 2013
Received in revised form 5 January 2014
Accepted 28 January 2014
Available online 12 February 2014
Keywords:
Cytotoxicity
Dehydroabietylamine Schiff base
Chiral
Dinuclear copper complex
DNA
selected cancer cells at concentration of 50 lM, this result is identical with their DNA binding ability
order. All the experimental results show that the involvement of Cu (II) centers has some interesting
effect on DNA binding ability and cytotoxicity of the chiral Schiff base.
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
chiral metal complexes has attracted the attention of many re-
search groups [4]. The DNA molecule is chiral itself and enantiome-
DNA is the primary target molecule for most anticancer and
antiviral therapies; the DNA-binding studies of small metal com-
plexes have received considerable attention during the past dec-
ades [1]. Revealing the features that contribute to enhance DNA
binding ability by small ligands or metal complexes is crucial for
designing effective chemotherapeutic agents and better DNA
targeted anticancer drugs [2].
Cisplatin targeted to DNA is an effective anticancer drug widely
used in the treatment of several human carcinomas [3]. However,
side effects or toxicity of cisplatin and its second generation ana-
logs made it is an urgent need to develop new drugs with minimal
side effects and maximal curative potential [3]. Chirality is usually
a significant factor in the field of pharmaceuticals, agrochemicals,
flavors and fragrances, and chirality is known to improve the phar-
macological behavior of metal complexes [3]. Bio-manifestation of
rically pure complexes may therefore lead to different
diastereomeric interactions with DNA [5]. Consequently, investi-
gating the interactions of chiral metal complexes with biomole-
cules, especially with DNA is likely to provide useful clues for
designing targeted bioactive molecules [4].
The choice of metal ion is the most important factor in the de-
sign of metal-based chemotherapeutic agent [3]. Copper is a bio-
essential and bio-relevant element. Many copper(II) complexes
with biological activities such as antibacterial, anti-cancer and can-
cer-inhibiting properties have appeared in the previous literature
[6,7]. Palaniandavar et al. pointed out that copper(II) complexes
are the best alternatives to cisplatin [7]. Schiff base transition me-
tal complexes have been paid much attention for their diverse bio-
logical and pharmaceutical activities [6,8–10]. Therefore, the
investigation on the interactions of novel copper(II) Schiff base
complexes with DNA has a great significance for disease defense,
new medicine design and filtration and clinical application of
drugs.
⇑
Corresponding authors. Address: College of Chemical Engineering, Nanjing
Forestry University, Nanjing 210037, China. Tel.: +86 0 159 962 308 59; fax: +86 25
85418873 (B.-L. Fei).
Salicylaldehyde derived Schiff bases have anticancer, antivirial,
antibacterial, anti-inflammatory and antifungal activities, and their
E-mail address: hgfbl@njfu.edu.cn (B.-L. Fei).
http://dx.doi.org/10.1016/j.jphotobiol.2014.01.018
1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

B.-L. Fei et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 36–44
37
transition metal complexes exhibiting enhanced pharmacological
properties [11]. Dehydroabietylamine derived from abietic acid
which is the main component of rosin, has gained much attention
for its special tricyclic hydrophenanthrene structure, three chiral
carbon atoms and extensive use. Its derivatives maybe develop
into new drugs, for the reason of their broad-spectrum biological
properties, such as antibacterial, antifungal, antipenetrant and
anti-inflammatory activities [12]. Lin et al. reported that dehydro-
abietylamine-substituted salicylidene Schiff bases had anti-cancer
activities, but they did not give any mechanism to explain their re-
sults [13]. According to the superposition principle of activity, the
Schiff base will have much more stronger bioactivity when it
contains two bioactive sub-groups.
Rational design and synthesis of novel metal complexes with
chirality, new composition, structure and promising properties is
still interesting targets for the future. In addition, understanding
and reasonably explaining the mechanism of the reaction are also
urgent topics. Compared to normal transition metal complexes,
very few researches have been done on DNA binding and biological
properties of chiral ones and understanding related structure–
activity relationship [3].
by dissolution of calculated amounts of compounds in a corre-
sponding amount of solvent and were diluted suitably with the
corresponding buffer to the required concentrations for all the
experiments. All the measurements about interactions of the com-
pounds with salmon sperm DNA were conducted using solutions of
the corresponding compound in Tris–HCl buffer (pH = 7.32)
containing 50 mM NaCl and 5 mM Tris–HCl at room temperature.
Elemental analysis (C, H and N) was performed on Elementar
Vario Micro analyzer. IR spectra were taken in the range of 400–
4000 cmꢂ1 on a Mattson Alpha-Centauri spectrometer with KBr
pellets. Electrospray mass spectra were determined using an LCQ
electron spray mass spectrometer (ES-MS, Finnigan). UV–visible
(UV–Vis) spectral measurements for the compounds and DNA-
binding studies were recorded on a TU-1900 UV–visible spectro-
photometer (Beijing Purkinje General Instrument Co., Ltd.). Fluore-
scene measurements were analyzed with a Perkin–Elmer LS-55
fluorescence spectrophotometer. Viscosity measurements were
carried out using Ubbelodhe viscometer with a temperature con-
troller in the thermostatic bath. The circular dichroism (CD) spec-
tra were measured with JASCO J-810 spectropolarimeter at room
temperature.
Based on the above mentioned, we describe a comparative
study of the DNA binding and biological properties of a chiral Schiff
base HL [N-(5-bromo-salicylaldehyde)dehydroabietylamine] (1)
(Scheme 1) and its chiral dinuclear copper complex (2) here. The
DNA interaction capacity and cytotoxic activity of the two com-
pounds were evaluated. The structure difference of 1 and 2 is the
copper(II) ion involvement, which permit us to make a better
understanding of transition metal effect on the binding affinity
and biological properties. The results demonstrate that 1 and 2
interact with DNA through intercalation. In addition, 2 exhibit bet-
ter cytotoxic activity against HeLa, NCI-H460, MCF-7 and HepG-2
cancer cell lines compared with 1. The results should be valuable
in understanding the DNA-binding mode and cytotoxic activity of
chiral compounds based on rosin-derivative and similar natural ac-
tive products, as well as laying a foundation for the rational design
of novel and powerful DNA targeted agents.
2.2. Synthesis
2.2.1. Synthesis of Schiff base N-(5-bromo-
salicylaldehyde)dehydroabietylamine (1)
An absolute ethanolic solution (40 mL) of 5-bromo-salicylalde-
hyde (6.031 g, 30 mmol) was added dropwise to a vigorously stir-
red solution of dehydroabietylamine (8.564 g, 30 mmol) in 30 mL
absolute ethanol. The resulting solution was refluxed for 6 h before
cooling to room temperature. The crude compound of 1 was depos-
ited as light yellow solid. Following recrystallization from DMF,
needle-like single crystals suitable for X-ray analysis were ob-
tained by slow evaporation of the filtrate at room temperature.
Yield: 69%. Anal. Calcd for C27H34BrNO (%) C 69.22, H 7.32, N
2.99. found: C 69.37, H 7.42, N 2.84. [
CHCl3). FT-IR (KBr (OH), 2929 m
a
]20 D = ꢂ30.5 (C = 0.02,
m
/cmꢂ1): 3431
m
(CH2), 1633
m
(C@N). CD (nm): 325 (negative), 291 (negative), 273 (negative),
263 (positive). 1H NMR (500 MHz, CDCl3): d 8.26 (s, 1H, CH@N),
6.90–7.40 (m, 6H, Ph), 3.46–3.54 (m, 2H, N-CH2), 1.07–2.84 (m,
24H, aliphatic cyclo). ESI-MS (m/z): 469 [C27H34BrNO+H]+.
2. Experimental
2.1. Materials and physical measurements
2.2.2. Synthesis of the copper(II) complex (2)
All chemicals were commercial available and used without
further purification unless otherwise noted.
A solution of copper(II) acetate (0.200 g, 1 mmol) in 10 mL DMF
was added slowly to a vigorously stirred solution of Schiff base 1
(0.469 g, 1 mmol) in 25 mL DMF. The brown reaction mixture
was refluxed for 1.5 h before filtration. Brown needle-like single
crystals suitable for X-ray analysis were obtained by slow evapora-
tion of the filtrate after three weeks. Yield: 64%. Anal. Calcd for
Solutions of DNA in the buffer 50 mM NaCl/5 mM Tris–HCl in
water (pH = 7.32) gave a ratio of UV absorbance at 260 and
280 nm (A260/A280) of about 1.8–1.9 [10], indicating that the
DNA was sufficiently free of protein. The concentration of DNA
was determined by measuring the UV absorption at 260 nm, taking
the molar absorption coefficient (e .
260) of DNA as 6600 Mꢂ1 cmꢂ1
C
108H132Br4Cu2N4O4 (%) C 64.96, H 6.66, N 2.81. found: C 65.05 H
6.78, N 2.65. [ ]20 D = +700 (C = 0.00005, CHCl3). FT-IR (KBr
/cmꢂ1): 2924
(CH2), 1620 (C@N), 699 (Cu@O), 473 (Cu@N).
a
m
Stock solutions were stored at 4 °C and used after no more than
4 days. Concentrated stock solutions of compounds were prepared
m
m
m
m
CD (nm): 385 (positive), 375 (negative), 363 (positive), 350 (nega-
tive), 334 (positive), 302 (negative), 288 (positive), 280 (negative),
261 (positive).
2.3. X-ray crystallography
OH
The intensity data for suitable single crystal for
1
(0.20 mm ꢁ 0.20 mm ꢁ 0.16 mm) and
2
(0.20 mm ꢁ 0.10 mm ꢁ
0.50 mm) were collected on Bruker SMART APEX II CCD X-ray dif-
H
N
fractometer at 296(2) K (1) and 153(2) K (2) with graphite–mono-
chromatized Mo K
a radiation (k = 0.71073 Å) by u–x scans. An
Br
empirical absorption correction was employed. Both structures of
1 and 2 were solved by direct methods and refined by full-matrix
least-squares against F2 using SHELXTL software (G.M. Sheldrick,
Scheme 1. Chiral Schiff base HL [N-(5-bromo-salicylaldehyde)dehydroabietyl-
amine] (1).

38
B.-L. Fei et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 36–44
where Kb and n are the binding constant and the number of the
binding sites, respectively. [Q] is the concentration of quenching
reagent (compound) and F0 is the fluorescence intensity of the
EB-DNA system alone, while F is the fluorescence intensity of the
system in the presence of compound.
The determination of quenching process type was carried out
by the Stern–Volmer quenching method. The plots from the fluo-
rescence titration data under two different temperatures (300,
312 K) were investigated according to the Stern–Volmer equation.
Bruker AXS, Madison, WI, 2008). The positions and anisotropic
thermal parameters of all non-hydrogen atoms were refined on
F2 by full-matrix least-squares techniques with the SHELX-97 pro-
gram package (G.M. Sheldrick, Bruker AXS, Madison, WI, 2008). All
H atoms were positioned geometrically and treated as riding on
their parent atoms. CCDC-867771 (for 1) and ꢂ867770 (for 2) con-
tain the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. De-
tails of the crystallographic data and structure refinement
parameters for compounds 1 and 2 are summarized in Table S1
(Supporting Information).
2.4.3. CD spectroscopic studies
CD spectra of DNA were recorded on a Jasco J-810 spectropolar-
imeter with a scanning speed of 100 nm/min with scope of 200–
400 nm at room temperature in the absence or presence of
2.4. DNA-binding experiments
1.0 ꢁ 10ꢂ4
M
compound. The concentration of DNA was
2.4.1. Absorption titration
1.0 ꢁ 10ꢂ4 M and the buffer solution was 1% CH3CN/50 mM NaCl/
5 mM Tris–HCl. The sample was completely mixed and standed
5 min before scanning. The buffer background was subtracted
automatically. Each CD spectrum has been subtracted with that
of buffer solution and the compounds, thus the spectrum purely re-
flect the changes of DNA structure upon binding with the
compounds.
Absorption titration experiment was performed with fixed con-
centrations of the two compounds, while gradually increasing the
concentration of DNA. The compounds were dissolved in a mixed
solvent of 20% DMF and 80% Tris–HCl buffer. The reference solution
was the corresponding Tris–HCl buffer solution. While measuring
the absorption spectra, equal amount of DNA was added to both
the compound solution and the reference solution to eliminate
the absorbance of DNA itself. After addition of DNA to the com-
pounds in Tris–HCl buffer, the resulting solution was allowed to
equilibrate at room temperature for 5 min. Then, the sample solu-
tion was scanned in the range 200–600 nm. For compounds 1 and
2, the binding constant (Kb) was determined from the spectro-
scopic titration data using the following equation (Eq. (1)) [14]:
2.4.4. Viscosity experiments
Viscosity experiments were carried on an Ubbelodhe viscome-
ter, immersed in
a thermostated water-bath maintained at
28.0 0.5 °C. Flow time was measured with a digital stopwatch
for different concentrations of the compounds, maintaining the ini-
tial DNA concentration. Each sample was measured three times
½DNAꢃ=ðea
ꢂ
ef Þ ¼ ½DNAꢃ=ðeb
ꢂ
ef Þ þ 1=Kbðeb
ꢂ
ef
Þ
ð1Þ
where [DNA] is the concentration of DNA in base pairs, the apparent
and an average flow time was calculated. Data were presented as
absorption coefficient ( a) was obtained by calculating Aobsd/[com-
pound]. The terms f and b correspond to the extinction coefficient
of free (unbound) and the fully bound compound, respectively. A
plot of [DNA]/(ea f) versus [DNA] will give a slope 1/(eb
e
1/3
(g/g0
)
versus binding ratio r (r = [compound]/[DNA] [16], where
0 is the
viscosity of DNA alone. The relative viscosity values for DNA in the
presence ( ) and absence ( 0) of the compounds were calculated
= (t ꢂ t0)/t0, where t is the observed flow time
e
e
g
is the viscosity of DNA in the presence of compound, and g
ꢂ
e
ꢂ
ef)
g
g
and an intercept 1/Kb(eb
ꢂ
ef). Kb is given by the ratio of the slope
using the relation
g
to the intercept.
in seconds.
2.4.2. Fluorimetric studies
Emission intensity measurements were carried out using a 20%
DMF/50 mM NaCl/5 mM Tris–HCl buffer solution as a blank to
make preliminary adjustments. For fluorescence quenching exper-
iments, DNA was pretreated with EB in the ratio of [DNA]/
[EB] = 10:1 for 10 h at room temperature. The two compounds
(1.0 ꢁ 10ꢂ5 M, 40 uL per scan, respectively) were then added to
this mixture and their effect on the emission intensity was mea-
sured. The extent of fluorescence quenching of EB bound to DNA
can be used to determine the extent of binding between the second
molecule and DNA. The competitive binding experiments were car-
ried out in the buffer by keeping [DNA]/[EB] and varying the con-
centrations of the compounds. The fluorescence spectra of EB
were measured using an excitation wavelength of 520 nm and
the emission range was set between 550 and 700 nm.
2.5. Cytotoxicity assay
Cell lines: human cervical cancer (HeLa) cells, breast cancer
(MCF-7) cells, non small cell lung cancer (NCI-H460) cells and
human liver carcinoma (HepG-2) cells were obtained from
Nanjing KeyGen Biotech. Co. Ltd., Nanjing, China. Tumour cell
lines were grown in RPMI-1640 medium supplemented with
10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 U/mL
streptomycin at 37 °C, in a highly humidified atmosphere of 95%
air/5% CO2. The cytotoxicity of MT and title compounds against
HeLa, NCI-H460, MCF-7 and HepG-2 cell lines were evaluated by
the microculture tetrozolium (MTT) assay [17]. The experiments
were carried out with reported procedure [17]. The growth
inhibitory rate of treated cells was calculated using the data from
three replicate tests by (Mean ODcontrol – Mean ODtest)/Mean
ODcontrol ꢁ 100%. The complexes incubated with cell lines for
The binding constant Kb at different temperature given in Tables
1 and 2 was determined with the following equation (Eq. (2)) [15]:
logðF0 ꢂ F=FÞ ¼ log Kb þ n log½Qꢃ
ð2Þ
48 h with concentration 50 lM.
Table 1
Binding constants (Kb), n, the quenching constants (KSV) of EB-DNA system by 1 and thermodynamic parameters for the binding of 1 to DNA at different temperatures.
T (K)
Kb (Mꢂ1
)
n
KSV (Mꢂ1
)
D
G0 (KJ molꢂ1
)
D
H0 (KJ molꢂ1
)
D )
S0 (J molꢂ1 Kꢂ1
301
304
311
3.37 ꢁ 104
3.90 ꢁ 103
1.86 ꢁ 104
1.14
0.93
0.98
7.58 ꢁ 103
8.96 ꢁ 103
2.20 ꢁ 104
ꢂ22.26
ꢂ23.10
ꢂ25.97
64.13
64.13
64.13
287.40
287.40
287.40

B.-L. Fei et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 36–44
39
Table 2
Binding constants (Kb), n, the quenching constants (KSV) of EB–DNA system by 2 and thermodynamic parameters for the binding of 2 to DNA at different temperatures.
T (K)
Kb (Mꢂ1
)
n
KSV (Mꢂ1
)
D
G0 (KJ molꢂ1
)
D
H0 (KJ molꢂ1
)
D )
S0 (J molꢂ1 Kꢂ1
300
305
312
3.64 ꢁ 104
3.27 ꢁ 103
2.09 ꢁ 104
1.09
0.84
1.01
1.52 ꢁ 104
1.63 ꢁ 104
1.88 ꢁ 104
ꢂ24.00
ꢂ27.03
ꢂ25.56
10.09
10.09
10.09
113.74
113.74
113.74
almost planar. The two methyl groups are in the axis position of
the cyclohexane ring.
3. Results and discussion
3.1. Crystal structures of the compounds
3.2. DNA binding studies
The compounds 1 and 2 crystallized in monoclinic system with
space group P21 and triclinic system with space group P1, respec-
tively. The crystal structure of 1 shown in Fig. S1 (Supporting Infor-
mation) has appeared in literature [18], and the crystal structure of
2 is discussed in detail here.
3.2.1. Absorption titrations
To monitor the changes in absorption spectra of small mole-
cules upon addition of increasing amounts of DNA is one of the
most widely used techniques for studying the binding characteris-
tics [21]. Small molecules binding with DNA through intercalation
usually result in the changes in the absorbance and shift in wave-
length [22]. As given in Fig. 2, the two compounds displayed in-
tense absorption bands around 262 nm assigned to intraligand
transition. Upon addition of increasing amounts of DNA
([DNA] = 7.70 ꢁ 10ꢂ5 M) to 1 and 2 solution with fixed concentra-
tion, their absorption spectra both show ‘‘hypochromic’’ effect. For
1, hypochromism is about 13.95% (262 nm), and for 2 it is 15.49%
(262 nm) with 2 nm (1) and 6 nm (2) blue-shift. These changes
are characteristic of the compounds bound to DNA through non-
covalent interactions or the compound could uncoil the helix and
made more bases embedding in DNA exposed [22]. Hypochromism
is suggested to be generated through an intercalatative mode of
binding involving a strong stacking interaction between an aro-
matic chromophore and the base pairs of DNA [23]. The extent of
the hypochromism commonly parallels the intercalative binding
strength. The spectroscopic changes suggest that the two com-
pounds bound with DNA and it is also likely that these compounds
bound to DNA via intercalation [24]. 2 showed more hypochromic-
ity than 1, indicating that the binding strength of the copper(II)
complex is much stronger than that of the free ligand.
Crystals of 2 consists of [Cu2L4] and four DMF molecules, the
molecular structure of 2 without DMF and hydrogen atoms is
shown in Fig. 1. Selected distances and angles are listed in
Table S2 (Supporting Information).
It is interesting that the two copper centers have different coor-
dination geometry. As we can see from Fig. 1, both the central
Cu(II) are coordinated with two imine nitrogen atoms. However,
Cu1 is four-coordinated with two phenoxide oxygen O1 and O2,
and the coordination geometry around Cu1 is an intermediate
structure between square-planar and tetrahedral for which the
four-coordinate geometry index s4 of 0.48 lies almost midway be-
tween the ideal values of zero (square-planar) and 1 (tetrahedral)
[19]. Whereas, the Cu2 is five-coordinated with three phenoxide
oxygen O1, O3 and O4, displaying a slightly distorted square pyra-
midal geometry with the value of trigonality index
s = 0.05 (s = 1
for idealized trigonal bipyramidal and = 0 for idealized square
s
pyramidal) [20]. The N3, N4, O3 and O4 are located in the basal
positions, whereas the O1 occupies the axial position. The apical
bond length Cu2–O1 is 2.765(8) Å and is longer than the other
coordination bonds in the equatorial plane. The two phenoxo-
bridged copper centers have a Cu1–O1–Cu2 angle of 99.3(3)° and
the Cuꢀ ꢀ ꢀCu separation in the dinuclear unit is 3.584 Å.
The extent of hypochromism gives a measure of the strength of
the intercalatative binding [25]. In order to compare the DNA-
binding affinities of the compounds quantitatively, their intrinsic
binding constants (Kb) with DNA were obtained using Eq. (1) and
In the complex, L contains four rings, the two cyclohexane rings
form a trans ring junction with classic chair and half-chair confor-
mations, respectively. The benzene ring and the pyridine ring are
Fig. 1. Ball-and-stick representation of the molecular structure of 2 showing the coordination environment of copper(II) centers. Hydrogen atoms and the four DMF
molecules are omitted for clarity.

40
B.-L. Fei et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 36–44
cence intensity is highly enhanced upon addition of DNA, due to its
strong intercalation with DNA base pairs [27]. Addition of a second
molecule binding to DNA can quench the DNA-induced EB emis-
sion, at least partially, which proved the intercalation of the mole-
cule to the base pairs of DNA [28].
The competitive DNA binding of the compounds 1 and 2 has
been studied by monitoring changes in emission intensity of EB
bound to DNA as a function of added compound concentration.
The emission spectra of EB bound to DNA in the absence/presence
of compounds 1 and 2 are shown in Fig. 3. Upon addition of the
compounds (0–28 lM) to DNA pretreated with EB, an appreciable
reduction in the DNA-induced emission intensity of EB is caused,
indicating the compound bind to DNA at the sites occupied by
EB. The data were analyzed by means of the following Stern–Vol-
mer equation [29].
F0=F ¼ 1 þ Ksv½Qꢃ
ð3Þ
The quenching plots (insets of the resp. Figs.) illustrate that the
fluorescence quenching of EB bound to DNA by 1 and 2 is in linear
agreement with the Stern–Volmer equation, which confirms that
the compounds bound to DNA. In the plot of F0/F vs. [Q], F0 is the
emission intensity of EB-DNA in the absence of compound; F is
the emission intensity of EB-DNA in the presence of compound;
Ksv is a linear Stern–Volmer quenching constant, which is given
by the slope to the intercept. The Ksv values for 1 and 2 are
7.58 ꢁ 103 Mꢂ1 and 1.52 ꢁ 104 Mꢂ1, respectively. These values
prove that the partial replacements of EB bound to DNA by the
two compounds result in a decrease of the fluorescence intensity,
and also suggest that the DNA binding affinities of the compounds
follow the order of 2 > 1. The result is consistent with that of
absorption spectroscopic studies. Anyway, it maybe conclude that
1 and 2 bound to DNA via the similar mode and the quenching con-
stant of 2 suggest that the interaction of the compound with DNA
should be intercalation [30]. Thus, the spectroscopic studies lead
us to a conclusion that the two compounds can bind with DNA.
3.2.3. Fluorescence quenching studies
Fluorescence quenching refers to the process in which the fluo-
rescence intensity of EB-DNA decreases upon adding the two com-
pounds as quenchers. Stern–Volmer constant (Ksv) is used to
evaluate the fluorescence quenching efficiency. To distinguish be-
tween both static and dynamic mechanisms of the quenching pro-
cesses, we addressed here the Ksv dependence on temperature.
Fig. 4 shows the Stern–Volmer plots for quenching of EB-DNA fluo-
rescence by 1 and 2 at two different temperatures. It is evident that
the Stern–Volmer plots are linear. It means that only one type of
quenching process occurs, either static or dynamic quenching
[31]. In our experiments, the Stern–Volmer constant (Ksv) in-
creased from 7.58 ꢁ 103 Mꢂ1 to 2.20 ꢁ 104 Mꢂ1 for 1, and
1.52 ꢁ 104 Mꢂ1 to 1.88 ꢁ 104 Mꢂ1 for 2, with the temperature
increasing from 300 K to 312 K. It means that the dynamic quench-
ing is responsible for fluorescence quenching in this investigation.
Since dynamic quenching depends upon diffusion, higher temper-
atures lead to larger diffusion coefficients, the Ksv can be increased
by rising the temperature. In contrast, increased temperature de-
creases compounds stability, and thus lower values of the static
quenching constants were resulted [32].
Fig. 2. Absorption spectra of 1 (1.0 ꢁ 10ꢂ4 M) and 2 (2.0 ꢁ 10ꢂ5 M) in the absence
and presence of increasing amounts of DNA (7.70 ꢁ 10ꢂ5 M) at room temperature in
20% DMF/50 mM NaCl/5 mM Tris–HCl buffer (pH = 7.32). [DNA]/[Compound] for 1:
(a) 0, (b) 0.08, (c) 0.15, (d) 0.23, (e) 0.31, (f) 0.38, (g) 0.46, (h) 0.54, (i) 0.62. [DNA]/
[Compound] for 2: (a) 0, (b) 0.04, (c) 0.08, (d) 0.12, (e) 0.16, (f) 0.20, (g) 0.24, (h)
0.28, (i) 0.32. The arrows show the absorbance change upon increasing the DNA
concentration. Inset shows the plot of [DNA]/(ea
the two compounds with DNA.
ꢂ ef) vs. [DNA] for the titration of
were found to be 3.30 ꢁ 105 Mꢂ1 (1);6.63 ꢁ 105 Mꢂ1 (2), respec-
tively. The data are comparable to that observed for typical classi-
cal intercalator, ethidium bromide, acridine orange and methylene
blue with binding constants of KEB = 6.58 ꢁ 104 Mꢂ1, KAO = 2.69
ꢁ 104 Mꢂ1 and KMB = 2.13 ꢁ 104 Mꢂ1
, respectively [26]. The
observed value of Kb reveals that it is possible that 1 and 2 bound
to DNA via intercalative mode. The higher Kb value for 2 compared
to 1 illustrates that the binding strength of 2 is stronger than that
of 1 and the binding affinity is 1 < 2. Although the electronic
absorption studies have confirmed that the two compounds can
bind to DNA by intercalation, it is necessary to carry out other
experiments to prove the binding mode.
3.2.4. CD spectral studies
CD spectroscopy is a powerful technique to give useful informa-
tion on changes in DNA morphology during drug–DNA interac-
tions, since CD signals are quite sensitive to the mode of DNA
interactions with small molecules [30]. The CD spectrum of salmon
sperm DNA exhibits a positive band at 275 nm due to the base
stacking and a negative band at 245 nm due to the right-handed
helicity of B-type DNA [30]. Observed changes in these CD signals
3.2.2. Fluorescence spectral studies
The ethidium bromide (EB) fluorescence displacement experi-
ments were employed to further investigate the interaction mode
between the two compounds and DNA. EB does not show any
appreciable emission in buffer solution due to fluorescence
quenching of the free EB by the solvent molecules and the fluores-

B.-L. Fei et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 36–44
41
Fig. 3. Emission spectra of EB bound to DNA in the presence of 1 and 2. ([DNA] = 1.0 ꢁ 10ꢂ4 M, [EB] = 1.0 ꢁ 10-5 M, [Quencher] (
l
M) for 1 and 2: (a) 0, (b) 4, (c) 8, (d) 12, (e) 16,
(f) 20, (g) 24, (h) 28. The arrow shows the intensity changes upon increasing the complex concentration. Inset: plot of F0/F vs. [Quencher].
of DNA are usually assigned to corresponding changes in its struc-
ture. The simple groove binding or electrostatic interaction be-
tween small molecules and DNA causes less or no perturbation
on the base stacking and helicity bands, whereas a classical inter-
calation can stabilize the helix conformation of B-DNA, and en-
hances the intensities of both the CD bands, as observed for the
classical intercalator methylene blue [30].
As shown in Fig. 5, with the addition of the compounds 1 and 2
into DNA solutions, the intensities of both the negative and posi-
tive bands change in some degree. In the case of 1, a decrease in
intensity of the positive band with 2 nm red shift and a larger de-
crease (shifting to zero levels) in intensity of the negative band
with 3 nm red shift are observed. In the case of 2, a decrease in
intensity of the positive band with 1 nm blue shift and a larger in-
crease in intensity of the negative band with 2 nm blue shift are
shown. Observed changes in those CD signals of DNA suggest that
the stacking mode and the orientation of base pairs in DNA were
disturbed with the binding of the compounds. More significant
changes for the negative band indicate that the compounds may
have a more evident effect on the helicity of B-DNA than the base
stacking. On the other hand, the CD spectral changes caused by the
2 are more pronounced.
The observed decrease in intensity of the positive band is likely
due to a transition from the extended nucleic acid double helix to
the more denatured structure; the decrease or increase in the
intensity of negative band indicates a decrease or increase in the
right-handed helicity [33]. The above mentioned data suggest that
the binding of compounds to DNA could induce certain conforma-
tional changes, a more B- to more A- conformational change of
DNA could be induced upon the binding of the copper complex
[32,34]; in the case of the free ligand, the conformational change
should be a more B- to more C-DNA transition [35].
In addition, for 2, a significant broad induced CD signal with
negative ellipticity at ca. 320 nm was observed, which points out

42
B.-L. Fei et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 36–44
From the results of UV/Vis absorption, fluorescence and CD
spectroscopic studies, we conclude that the copper(II) complex
and the Schiff base can effectively bind to DNA, and the Cu(II)
complex exhibits little stronger binding ability.
3.2.5. Viscosity measurements
Although spectral methods are necessary, it is not sufficient to
support a binding mode. In the absence of crystallographic struc-
tural data, hydrodynamic measurement is considered as least
ambiguous and the most critical test to find binding mode of
DNA with small molecules [38]. Viscosity measurement is sensitive
to the changes in the length of DNA molecule, the sensitivity of this
method is largely depend on the changes in the length of DNA that
occur as result of its different binding modes with guest molecules.
In classical intercalation, the DNA helix lengthens as base pairs are
separated to accommodate the bound guest leading to increased
DNA viscosity [39], whereas in groove binding or electrostatic
mode, the length of the helix unchanged results in not apparent
alteration in DNA viscosity [40]. In contrast, compounds that bind
exclusively in the DNA groove by partial and/or non classical inter-
calation, under the same conditions, typically cause less pro-
nounced (positive or negative) or no change in the DNA solution
viscosity [41]. A classical intercalation mode causes a significant
increase in viscosity of DNA due to an increase in separation of
base pairs at intercalation sites and hence an increase in overall
DNA length.
To further clarify the interaction modes of the two compounds
and DNA, the viscosity measurement was carried out. The plots of
1/3
relative viscosity (
g/g0
)
versus [compound]/[DNA] ratio (0–0.6)
(Fig. 6) illustrated a significant increase in the relative viscosity
of DNA on increasing the concentration of the compounds, which
is similar to that of the proven intercalator EB [42]. This may be ex-
plained by the insertion of the compounds between the DNA base
pairs, leading to an increase in the separation of base pairs at inter-
calation sites and, thus, an increase in overall DNA length. On the
basis of the viscosity results, the compounds bind with DNA
through the intercalation mode. The increased degree of viscosity,
which may depend on its affinity to DNA follows the order of 2 > 1.
Thus, the viscosity measurement is consistent with the results of
the electronic absorption titrations and EB fluorescence displace-
ment experiments.
Fig. 4. Plots of F0/F versus the concentration of the two compounds for the binding
of them with DNA at different temperature.
From the experiment evidences observed in the electronic
absorption titrations, EB fluorescence displacement experiments,
CD spectral studies and viscosity measurements, we speculate that
Fig. 5. Circular dichroism spectra of DNA in 1% CH3CN/50 mM NaCl/5 mM Tris–HCl
buffer (pH = 7.32) at 25 °C in the absence and presence of 1 and 2 at 1:1 ratio of DNA
(1.0 ꢁ 10ꢂ4 M) and compounds (1.0 ꢁ 10ꢂ4 M).
that the compound tightly bound (such as intercalation) to the chi-
ral scaffold of the DNA double helix, acting as a new CD chromo-
phore [36–38]. The changing intensity follow the order of 2 > 1,
which means the coordination with metal can strength the interac-
tion of a organic compound with DNA.
Fig. 6. Effect of increasing amount of 1 and 2 on the relative viscosities of DNA at
28.0 ( 0.5)°C ([DNA] = 1.31 ꢁ 10ꢂ4 M, r = [Compound]/[DNA], respectively).

B.-L. Fei et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 36–44
43
the compounds 1 and 2 could interact with DNA through interca-
lation and 2 has little stronger binding affinity than 1.
3.2.6. Thermodynamic studies
In order to further elucidate the interaction between the com-
pounds and DNA, the thermodynamic parameters with varied tem-
perature were analyzed. The thermodynamic parameters, enthalpy
change (
main evidence for verifying binding modes. From the thermody-
namic standpoint, H > 0 and S > 0 indicates a hydrophobic inter-
action; H < 0 and S < 0 implies the Van der Waals force or
DH) and entropy change (DS) of binding reaction are the
D
D
D
D
hydrogen bond formation; and
trostatic force [43].
D
H ꢄ 0 and
DS > 0 suggests an elec-
The temperature-dependence of the binding constants for the
two compounds was studied at three different temperatures
(300, 305, and 312 K) using the following thermodynamic
equations:
Fig. 7. Cytotoxic activity of 1 and 2 against four human tumor cell lines incubated
with 1 and 2 at concentration of 50 lM for 48 h. Results are mean values obtained
from three independent experiments corresponding DMSO control, and bars
represent the standard deviations.
ln K ¼
G ¼
D
H=RT þ
D
S=R
ð4Þ
ð5Þ
D
D
H ꢂ T S ¼ ꢂRT ln K
D
4. Conclusion
where K is the Stern–Volmer quenching constant at corresponding
temperature and R is the gas constant. If the temperature does
not vary significantly, the enthalpy change can be regarded as a
The synthesis and characterization of two chiral compounds,
Schiff base N-(5-bromo-salicylaldehyde)dehydroabietylamine (1)
and its dinuclear copper(II) complex (2) have been achieved. The
crystal structures of the compounds 1 and 2 have been determined
by X-ray crystallography, and 2 represents the first dinuclear transi-
tion metal complex based on dehydroabityl Schiff base. The interac-
tion studies of the compounds 1 and 2 with DNA suggest that they
were involved in intercalative interaction, and 2 had stronger affinity
to DNA. CD spectrum shows that the two compounds could perturb
the base stacking and helicity of B-DNA on binding to it. The in vitro
antitumor activity of the compounds is consistent with their DNA-
binding abilities and follow the order of 2 > 1. These results have at-
tested that the coordination with transition metal of a chiral Schiff
base can result in some differences in its DNA binding and cytotoxic-
ity properties. In other words, the affinity magnitudes of a organic
compound toward DNA and anticancer activity may be controlled
and tuned to some extent by exploring the nature of the transition
metal ions in its coordination complex, and this strategy should be
valuable for the rational design of novel, powerful agents based on
bioactive biomass for targeting DNA, and may further broaden the
medical application fields of plant-derived natural products.
constant.
ear relationship between lnK and the reciprocal absolute tempera-
ture. The free energy (
G0) was calculated by Eq. (5). When we
apply this analysis to the binding of the compounds with DNA,
we find that H > 0 and S > 0 in the both cases (Tables 1 and 2).
DH and DS of the reactions were determined from the lin-
D
D
D
The positive changes in enthalpy and entropy determined by the
Stern–Volmer fluorescence quenching method at three tempera-
tures (300, 305, and 312 K), indicate that the binding processes
are dominated by hydrophobic interaction [15]. The negative value
of
DG reveals that the interaction process is spontaneous.
3.3. Evaluation of the in vitro anticancer activity
The positive results obtained from DNA binding of the two com-
pounds encouraged us to test its cytotoxicity against four human
cancer cell lines (HepG-2, HeLa, NCI-H460 and MCF-7) by the
MTT assay method. Compounds were dissolved in DMSO and blank
samples containing the same volume of DMSO were taken as con-
trols to identify the activity of the solvent in this cytotoxicity
experiment. After incubation of tumor cells and each compound
Acknowledgements
at concentration of 50 lM for 48 h under identical experimental
The authors gratefully acknowledge the financial support from
State Key Laboratory of Coordination Chemistry of Nanjing Univer-
sity, Nanjing Forestry University, the Priority Academic Program
Development of Jiangsu Higher Education Institutions, College
and University Graduate Research Innovation Project of Jiangsu
Province (No. CXLX13_517), and University Science Research
Project of Jiangsu Province (No. 13KJB220006).
conditions, as shown in Fig. 7, it is evident that compound 2 dem-
onstrates better cytotoxicity than compound 1 against HepG-2,
HeLa and NCI-H460 cell lines, the result for MCF-7 is on the versa.
It is noticed that 2 and cisplatin exhibit similar cytotoxicity against
HepG-2, HeLa and NCI-H460 cell lines.
In contrast to the low inhibition rates (less than 70%) of 1 to-
ward HepG-2, HeLa and NCI-H460, the data for 2 are all higher
than 82%. The different cytotoxic activities of 1 and 2 against differ-
ent cancer cells under identical experimental conditions illustrate
the diversities of different cancer cell lines and indicate that they
exhibit certain selectivity against different tumors simultaneously.
The better cytotoxic activity of 2 corroborates with its stronger
DNA binding ability, which further suggests the binding of the
compounds to DNA and thus consequently leads to cell death.
The results demonstrate that 2 has high potential to act as effective
metal-based chiral anticancer drug, as expected from the DNA
binding studies. Accordingly, 2 may provide useful scientific evi-
dence for designing novel chiral metal-based anticancer drugs.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2014.
01.018.
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