Synthesis, characterization and DNA interaction of new copper(II) complexes of Schiff base-aroylhydrazones bearing naphthalene ring

Article abstract of DOI:10.1016/j.jphotobiol.2013.02.014

Two new copper(II) complexes with the condensation products of methyl 2-naphthyl ketone with 4-hydroxybenzohydrazide, 4-hydroxy-N′-[(1Z)-1- (naphthalen-2-yl)ethylidene]benzohydrazide [HL¹] and (Z)-ethyl 2-(4-(2-(1-(naphthalen-2-yl)ethylidene)hydrazinecarbonyl)phenoxy)acetate (HL²) were synthesized and characterized by elemental analysis, infrared spectra, UV-Vis electronic absorption spectra, magnetic susceptibility measurements, TGA, powder XRD and SEM-EDS. The binding properties of the copper(II) complexes with calf thymus DNA were studied by using the absorption titration method. DNA cleavage activities of the synthesized copper complexes were examined by using agarose gel electrophoresis. The effect of complex concentration on the DNA cleavage reactions in the absence and presence of H₂O₂ was also investigated. The experimental results suggest that the copper complexes bind significantly to calf thymus DNA by both groove binding and intercalation modes and cleavage effectively pBR322 DNA. The mechanistic studies demonstrate that a hydrogen peroxide-derived species and singlet oxygen (¹O₂) are the active oxidative species for DNA cleavage.

Full text of DOI:10.1016/j.jphotobiol.2013.02.014

Journal of Photochemistry and Photobiology B: Biology 122 (2013) 15–23
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis, characterization and DNA interaction of new copper(II) complexes
of Schiff base-aroylhydrazones bearing naphthalene ring
⇑
Cansu Gökçe, Ramazan Gup
Mugla Sıtkı Koçman University, Department of Chemistry, 48000 Mugla, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new copper(II) complexes with the condensation products of methyl 2-naphthyl ketone with 4-
hydroxybenzohydrazide, 4-hydroxy-N0-[(1Z)-1-(naphthalen-2-yl)ethylidene]benzohydrazide [HL1] and
(Z)-ethyl 2-(4-(2-(1-(naphthalen-2-yl)ethylidene)hydrazinecarbonyl)phenoxy)acetate (HL2) were syn-
thesized and characterized by elemental analysis, infrared spectra, UV–Vis electronic absorption spectra,
magnetic susceptibility measurements, TGA, powder XRD and SEM–EDS. The binding properties of the
copper(II) complexes with calf thymus DNA were studied by using the absorption titration method.
DNA cleavage activities of the synthesized copper complexes were examined by using agarose gel elec-
trophoresis. The effect of complex concentration on the DNA cleavage reactions in the absence and pres-
ence of H2O2 was also investigated. The experimental results suggest that the copper complexes bind
significantly to calf thymus DNA by both groove binding and intercalation modes and cleavage effectively
pBR322 DNA. The mechanistic studies demonstrate that a hydrogen peroxide-derived species and singlet
oxygen (1O2) are the active oxidative species for DNA cleavage.
Received 20 December 2012
Received in revised form 21 February 2013
Accepted 25 February 2013
Available online 14 March 2013
Keywords:
Aroylhydrazone
Naphthalene ring
Copper(II) complexes DNA binding
DNA cleavage
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
synthesis and characterization of new Cu(II) complexes of Schiff
base-hydrazone ligands and their DNA binding and cleavage
The interaction of coordination compounds with DNA has been
of interest due to their possible applications in cancer therapy [1–
5] and molecular biology [6,7]. They show unique spectral and
electrochemical properties, as well as the ability of their ligands
to be adjusted to DNA interaction abilities. Schiff base complexes
have been attracted much attention due to their significances in
the development of new therapeutic agents and novel nucleic acid
structural probes [8–11]. Copper is an important trace element for
plants and animals and is involved in complex formation in a num-
ber of biological processes [12–14]. Copper(II) complexes have
found possible medical uses in the treatment of many diseases
including cancer [15,16]. Therefore, investigations on copper com-
plexes are becoming more prominent in the research area of bioin-
organic chemistry [17–23]. Copper complexes have been known to
cleave DNA by different mechanisms viz. hydrolytic [24] and oxi-
dative pathways [25]. Artificial metallonucleases require ligands,
delivering the metal ion to the proximity of DNA. Aroylhydrazone
type ligands have a large number of potential donor atoms and
hence display versatile behavior in metal coordination. The mode
of coordination depends on the nature of the central metal atom,
type of the ligand as well as on the presence of other species capa-
ble to compete for coordination pockets. Herein, we described the
activities.
2. Experimental
2.1. Material
All chemicals used were analytical reagent grade. Copper(II)
acetate, ethyl 4-hydroxybenzoate, methyl 2-naphthyl ketone, ethyl
bromoacetate, K2CO3, acetone and hydrazine monohydrate were
purchased from Fluka and Sigma–Aldrich and used without further
purification. Calf thymus DNA (CT-DNA) was purchased from Sig-
ma–Aldrich. pBR322 DNA was purchased from Fermantas. 1H and
13C NMR spectra were recorded on a Bruker 400 MHz spectrometer
in DMSO_d6 with TMS as the internal standard. IR spectra were re-
corded on a Perkin Elmer 1605 FTIR spectrometer as KBr pellets.
The electronic spectra of the ligands and complexes were recorded
on a UV-1601 Shimadzu spectrophotometer. Carbon, hydrogen and
nitrogen analyses were carried out on a LECO 932 CHNS analyzer
and copper content was determined by atomic absorption spec-
troscopy using the DV 2000 Perkin Elber ICP-AES. Room tempera-
ture magnetic susceptibility measurements were carried out on
powdered samples using a Sherwood Scientific MK1 Model Gouy
Magnetic Susceptibility Balance. Melting points were determined
on an Electrothermal IA 9100 digital melting point apparatus.
The thermogravimetric analysis was carried out in dynamic
⇑
Corresponding author. Tel.: +90 2522111496; fax: +90 2522111472.
E-mail address: rgup@mu.edu.tr (R. Gup).
1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2013.02.014

16
C. Gökçe, R. Gup / Journal of Photochemistry and Photobiology B: Biology 122 (2013) 15–23
nitrogen atmosphere (20 mL minꢀ1
)
with
a
heating rate of
(DMSO_d6, ppm) d 1.24 (t, J = 7.1, CH3), 2.48 (s, 3H, N@CACH3),
4.17 (q, J = 7.1, OCH2CH3), 4.87 (s, ArAOCH2), 7.03 (d, 2H, J = 8.3,
ArHd), 7.23 (d, 2H, J = 8.3, ArHe), 7.52–7.90 (m, 5H, ArH), 10.59 (s,
1H, NH); 13C NMR (DMSO_d6, ppm) 169.2 (C19), 162.0 (C1),
156.4 (C2), 138.6 (C17), 128.2 (C14), 126.0 (C15), 114.7 (C16),
136.8, 133.3, 133.7, 129.4, 128.0, 127.9, 127.3, 126.8, 125.2, 124.4
(C3AC12), 61.4 (C18), 65.3 (C20), 21.5 (C13), 14.7 (C21). Analysis
(%Calculated/found) for C23H22N2O4 C: 70.75/71.05, H: 5.68/5.49,
N: 7.17/7.42.
20 °C minꢀ1 using a Perkin Elmer Pyris 1 TGA thermal analyzer in
the Central Laboratory at METU. SEM investigations were per-
formed using a JEOL SEM 7700F, equipped with an EDS system in
the Central Laboratory at Mugla Sıtkı Koçman University. The
XRD analyses of the powder samples were recorded with a Rigaku
Corporation X-ray diffractometer (Model smartlab) analyzer in the
Central Laboratory at Mugla Sıtkı Koçman University. All the dif-
fraction patterns were obtained by using Cu Ka1 radiation, with a
graphite monochromator at 5j/min scanning rate. The Schiff
base-hydrazone ligands were prepared according to Scheme 1.
2.4. Synthesis of Cu(II) complexes
2.2. Synthesis of 4-hydroxy-N0-[(1Z)-1-(naphthalen-2-yl)
ethylidene]benzohydrazide (HL1)
A solution of 1 mmol copper(II) acetate dihydrate in EtOH
(10 mL) was added to a hot solution containing triethylamine
(2 mmol, 0.202 g) and 2 mmol HL1 and HL2 in absolute ethanol
(25 mL) with stirring. The reaction mixture was refluxed for 3 h.
On standing over night, the complexes separated, were collected
by filtration, and finally washed with water.
Methyl 2-naphthyl ketone (1 mmol, 0.170 g) dissolved in etha-
nol (10 mL) was added drop wise to a suspension of 4-hydrox-
ybenzohydrazide (I) (4 mmol, 0.152 g) with two drops of glacial
acetic acid in ethanol (15 mL) in room temperature. The reaction
mixture was refluxed for further 8 h and the colorless product
was filtered. The pure aroylhydrazone was collected by crystalliza-
tion from acetone.
For [Cu(L1)2]: brown complex; yield: 83%; m.p.: 260 °C.
l
eff = 1.75 B.M.; UV (DMF, nm) 268, 315, 408, 432; FT-IR (KBr,
cmꢀ1): 3275 b (OAH), 1605 s (C@NAN@C), 1365 (CAN), 1228 s
and 1166 m (CAOAC). Analysis (%Calculated/found) for
Yield 77%; Mp 249–251 °C; UV (EtOH, nm) 267, 302, 389; IR
C
38H30CuN4O4 C: 68.06/67.89, H: 4.48/4.25, N: 8.36/8.73, Cu:
9.48/9.71.
For [Cu(L2)2]: dark brown complex; yield: 80%; m.p.: 177 °C.
eff = 1.69 B.M.; UV (DMF, nm) 266, 302, 400; FT-IR (KBr, cmꢀ1):
(KBr) (m
, cmꢀ1) 3290 (OH), 1634 (C@O)amide, 1606 (C@N), 1371
(CAN), 1234 and 1175 (CAOAC); 1H NMR (DMSO_d6, ppm) d
2.48 (s, 3H, N@CACH3), 6.89 (d, 2H, J = 8.3, ArHd), 7.52 (d, 2H,
J = 8.3, ArHe), 7.83–8–29 (m, 7H, ArH), 10.15 (s, 1H, OH), 10.64 (s,
1H, NH); 13C NMR (DMSO_d6, ppm) 161.3 (C1), 155.1 (C2), 139.3
(C17), 128.9 (C14), 125.2 (C15), 115.6 (C16), 136.3, 133.9, 133.6,
129.2, 128.4, 127.5, 127.1, 127.0, 125.2, 124.3 (C3AC12), 21.2
l
3450 (OAH), 1758 (C@@O)ester, 1601 (C@NAN@C), 1255 and
1167 (CAOAC). Analysis (%Calculated/found) for C46H44CuN4O9 C:
64.52/64.89, H: 5.11/4.95, N: 6.51/6.81, Cu: 7.56/7.23.
(C13). Analysis (%Calculated/found) for
75.37, H: 5.26/5.09, N: 9.21/9.12.
C19H16N2O2 C: 75.00/
2.5. DNA binding
2.3. Synthesis of (Z)-ethyl 2-(4-(2-(1-(naphthalene-2-yl)
ethylidene)hydrazinecarbonyl)-phenoxy)acetate (HL2)
2.5.1. Electronic absorption titrations
All the experiments involving the interaction of the complexes
with CT-DNA were carried out in water buffer containing 5 mM tris
[tris(hydroxymethyl)aminomethane] and 50 mM NaCl, and ad-
justed to pH 7.3 with HCl. The solution of CT-DNA in the buffer gave
a ratio of UV absorbance of 1.8–1.9:1 at 260 and 280 nm, indicating
that the CT-DNA was sufficiently free of protein [26]. The CT-DNA
concentration per nucleotide was determined spectrophotometri-
cally by employing an extinction coefficient of 6600 Mꢀ1 cmꢀ1 at
260 nm [27]. An appropriate amount of the copper complex was
dissolved in a solvent mixture of 1% DMF and 99% tris–HCl buffer.
Absorption titration experiments were performed by maintaining
A
mixture of 4-hydroxy-N0-[(1Z)-1-(aphthalene-2-yl)ethyli-
dene]benzohydrazide (HL1) (1 mmol, 0.304 g), ethyl bromoacetate
(10 mmol, 1.670 g) and dry K2CO3 (10 mmol, 1.380 g) in 40 mL ace-
tone was refluxed with stirring for 24 h and poured to 200 mL of
cold water. The white precipitate formed was filtered and washed
with water and finally recrystallized from ethanol.
Yield 66%; Mp 153–155 °C; UV (EtOH, nm) 272, 307sh, 366; IR
(KBr) (m ,
, cmꢀ1) 3373 (NH), 1760 (C@O)ester, 1645 (C@O)amide
1603 (C@N), 1386 (CAN), 1244 and 1077 (CAOAC); 1H NMR
OH
O
O
ii
i
HO
HO
OC2H5
HN NH2
HN
I
II
N
O
H3C
HL1
iii
6
10
11
O
1
5
3
8
7
'
15
N
12
'16 14
N
H
2
4
9
21
O
18 17
15
16
13
20 19
O
HL2
O
Scheme 1. Schematic diagram showing the synthesis of the acylhydrazone ligands. (i) NH2NH2ꢁH2O, EtOH, reflux 4 h. (ii) methyl 2-naphthyl ketone, EtOH, AcOH, reflux 4 h;
and (iii) ethyl bromoacetate, K2CO3, acetone, reflux 24 h.

C. Gökçe, R. Gup / Journal of Photochemistry and Photobiology B: Biology 122 (2013) 15–23
17
the metal complex concentration as constant while gradually
increasing the concentration of the CT-DNA within 0–100 lM.
and 162.0 ppm and 155.1 and 156.4 ppm for HL1 and HL2 ligands,
respectively. The signal at 169.2 ppm is assignable to the ester car-
bonyl group (C14) of the ligand HL2. The chemical shifts for the car-
bon atoms of the aromatic rings were recorded between 114.7 and
139.3 ppm. The signals observed between 14.7 and 65.3 ppm are
attributed to the aliphatic carbon atoms. These assignments are
in good agreement with those previously reported for similar com-
pounds [28–37]. These results strongly suggest that the proposed
acylhydrazone compound has been formed. In the 1H and 13C
NMR spectra of the HL1 and HL2 only one signal was observed for
all hydrogens and carbons indicating that both acylhydrazones
are the E configuration [31,32] (Fig. 2). Due to the paramagnetic
nature of the copper(II) complexes, their 1H and 13C NMR spectra
could not be obtained.
2.6. DNA Cleavage
pBR322 plasmid DNA was used for all cleavage activities. In a
typical experiment, 7 l plasmid DNA (50 ng/ l) was mixed with
different concentrations of complexes (25, 50, 75 and 100 M dis-
solved in DMF) to determine optimum activation concentration.
l H2O2 (5 mM) was added to mixture to oxidize the reactant. Fi-
nally the reaction mixture was diluted with the Tris buffer
(100 mM Tris, pH: 8) to a total volume of 30 l. After that reaction
mixtures were incubated at 37 °C for 2 h. Samples (20 l) were
then incubated at 37 °C and loaded with 4 l loading dye (0.25%
bromophenol blue, 0.25% xylene cyanol, 30% glycerol, 10 mmol
EDTA) on a 1% agarose gel containing 1 g/ml of EtBr. The gel
l
l
l
5
l
l
l
l
l
3.2. IR spectroscopy
was run at 100 V for 3 h in TBE buffer and photographed under
UV light. To test for the presence of reactive oxygen species gener-
ated during strand scission, reactive oxygen intermediate scaveng-
ers were added alternately to the reaction mixture. These
The IR spectrum of HL1 shows a broad OAH stretching vibration
at 3290 cmꢀ1. However, in the IR spectrum of HL2 this band disap-
pears indicating that condensation takes place. The characteristic
amide I bands are observed at 1634 and 1645 cmꢀ1 for HL1 and
HL2, respectively. On the other hand, the band observed at
1760 cmꢀ1 is assigned to the ester carbonyl stretching vibration
which is also characteristic for carboxylic esters. The amide NH
stretching band of the HL1 was not observed in the IR spectra prob-
ably due to overlapping with the broad OH stretching frequency.
However, this band is observed clearly at 3373 cmꢀ1 in IR spectra
of the HL2 ligand which is hydroxyl group free. The other charac-
teristic IR peaks of the new compounds synthesized in this work
are given in the experimental section. These values are in accord
with those previously reported such compounds [28–34,37–42].
To determine the modes of the acylhydrazone ligands coordina-
tion with Cu(II) ion, we compared the IR spectra of the metal free li-
gands with those of the complexes. The IR spectra of the copper(II)
scavengers were DMSO (100
lM), KI (100 lM), NaN3 (100 lM)
and EDTA (100 M). Samples were treated as described above.
l
3. Results and discussion
Acylhydrazones such as synthesized in this work may exit in
the keto or in the enol tautomeric form in the solid state (Fig. 1).
The observation of strong m
(C@O) peaks for HL1 and HL2 in the
infrared spectra of the aroylhydrazones suggests that the ligands
are in the keto form in the solid state [28–30]. The tautomeric keto
forms of the compounds were also indicated by observation of the
amide NH and carbonyl (C@O) signals of keto forms in the 1H NMR
and 13C NMR spectroscopies, respectively.
complexes lack absorptions due to amide I [
and
(NAH), but show a new band between 1605 and 1601 cmꢀ1
probably due to (C@@NAN@@C) stretching suggesting that the
m(C@@O)], m(C@@N)imine
3.1. 1H and 13C NMR spectroscopy
m
m
The main 1H NMR signals for each of the acylhydrazone com-
pounds are given in the experimental section. Formation of the
HL2 was confirmed by the absence of the OH proton signal at
10.15 ppm assigned to the starting material HL1. The D2O
exchangeable singlet resonances appeared at 10.64 and
10.59 ppm for HL1 and HL2 ligands are assigned to NH protons.
The singlet and triplet signals observed at 4.87 ppm and at
4.17 ppm are attributed to methylene protons of ACH2OPh and
AOCH2CH3 groups, respectively, in the 1H NMR spectra of HL2.
The other obtained values for 1H NMR chemical shifts of the acyl-
hydrazones are given in the experimental section. These data are in
agreement with those previously reported for similar compounds
[28–38].
NH proton is likely lost via deprotonation induced by the metal
and the resulting enolic oxygen and the azomethine nitrogen coor-
dinate to the copper [28–34,36–39]. Therefore, from the IR spectra,
it is concluded that acylhydrazones act as mono anionic bidentate
ligands coordinating through the azomethine nitrogen and the eno-
lic oxygen (Fig. 3). In the IR spectra of the copper complexes, the phe-
nolic hydroxyl and (C@@O)ester stretching vibrations are observed
almost at same positions as in the metal-free ligands indicating
non-participation of these groups in coordination. On the other
hand, a new broad band at around 3450 cmꢀ1 for [Cu(L2)2] complex
is assignable to the OH vibration of water molecule.
The 1H NMR spectral assignments of the new acylhydrazone
compound are also supported by the 13C NMR spectrum. The char-
acteristic chemical shifts of the amide carbonyl (C1) and azome-
thine (C2) groups of the aroylhydrazones are observed at 161.3
3.3. X-ray diffraction analysis
Single crystal XRD could not be employed to confirm the struc-
tures of the complexes since attempts to isolate crystals suitable
HO
CH
3
CH
3
N
O
N
N
NH
OR
OR
Enol form
Keto form
Fig. 1. Keto–enol forms of the aroylhydrazones.

18
C. Gökçe, R. Gup / Journal of Photochemistry and Photobiology B: Biology 122 (2013) 15–23
concluded from Fig. 6 that each complex contains only C, N, O
and copper ions and existence of no other elemental peak assures
the highly purity of prepared copper(II) complex powders. The EDS
spectrum was appeared identical for both complexes confirming
the formation of the copper complexes.
CH
3
CH
NH
3
N
O
3.5. Thermal analyses
N
O
HN
According to TGA curves, thermal degradation of [Cu(L1)2] com-
plex occurs in one step and it is stable under 300 °C (see Fig. S2).
The degradation of this copper complex occurred within tempera-
ture range 300–480 °C corresponds the removal of the organic part
of the aroylhydrazone side of the copper complexes and leaving
copper oxide as a residue. On the other hand, the thermal degrada-
tion of [Cu(L2)2] occurred in two steps. This complex showed the
loss of one mole water molecule within the temperature range
185–200 °C. This high temperature loss confirms that the water
molecule participate in coordination. The other step of decomposi-
tion of this complex appeared within temperature range 275–
480 °C involves the removal of the organic part of ligand leaving
copper oxide as a residue.
RO
OR
configuration
E
configuration
Z
Fig. 2. Structural configurations of the acylhydrazones.
for single X-ray diffraction were unsuccessful. To obtain further
evidence about the structure of the metal complexes, powder X-
ray diffraction was performed (see Fig. S1). Both copper complexes
show sharp crystalline peaks due to their crystalline nature. The
XRD patterns for complexes are quite similar and suggest that
the complexes have similar structure. The line broadening of the
crystalline diffraction peaks in the [Cu(L1)2] complexes show high-
er crystallinity than that of the [Cu(L2)2] complex.
3.6. Electronic absorption spectra
The UV–Vis spectra of the aroylhydrazones and their transition
metal complexes in DMF are given in the experimental section. The
ligands exhibit a band around 270 nm assignable to the aromatic
3.4. SEM and EDS analyses
ring transition p ?
pꢄ, which remains almost unchanged in the
metal complexes. In addition, in the spectrums of the aroylhydraz-
ones there are two transition bands at 302 and 389 for HL1 and 307
and 366 for HL2 corresponding to the n ? pꢄ type electronic tran-
sitions. By comparison of the electronic absorption spectra of the
free hydrazone ligands and their copper complexes, it is observed
that the maxima bands of the free ligands showed a red shift,
which may be the result of the enolization of the hydrazone ligands
as well as the coordination to the copper center. The electronic
spectrums of copper complexes show the metal-to-ligand charge
transfer band at 432 nm and 400 nm for [Cu(L1)2] and [Cu(L2)2]
complexes, respectively, which is comparable to those of previ-
ously reported complexes [28–34]. Unfortunately the expected
weak d–d transition in the visible region for the paramagnetic cop-
per complexes cannot be detected even with concentrated solu-
tions. It may be lost in the low energy tail of the charge transfer
transition [28–31]. The observed magnetic moment values for
[Cu(L1)2] and [Cu(L2)2] complexes are 1.69 and 1.75 BM, respec-
tively, which are somewhat lower and higher than the expected
spin-only magnetic moment of an S = 1/2 (1.73 BM), Cu(II) d9
The scanning electron micrographs revealed the morphology of
the aroylhydrazones and their copper(II) complexes (Figs. 4 and 5).
The scanning electron micrographs were taken at 15 kV accelerat-
ing voltage and magnification was fixed according to ꢂ500. The
SEM picture of the HL1 ligand in Fig. 4a is aligned with rod-shaped
blocks vertically and horizontally while its complex [Cu(L1)2] dis-
plays agglomerated particles in non-uniform shape. The SEM im-
age of the HL2 (Fig. 5a) exhibits the crystalline objects are
needles and rods with diagonal dimension in micrometer size.
The [Cu(L2)2] shows square shapes with irregular boundary
(Fig. 5b). The average particle sizes for ligands and their Cu(II) com-
plexes were found ꢃ100
lm, 20 lm, 10 lm and 15 l
m for HL1,
HL2, Cu(L1)2 and Cu(L2)2, respectively. From SEM images, it is clear
that there is a strong change in morphology of the hydrazone li-
gands after coordination to copper ion.
Energy dispersive spectroscopy (EDS) allows determining the
chemical composition of a sample. Therefore, the composition of
the copper complexes was defined by EDS analysis (Fig. 6). It is
O
O
OH
C2H5
O
CH3
OH2
CH3
N
O
O
N
N
Cu
N
Cu
N
N
O
N
N
O
H3C
H3C
O
HO
O
O
C2H5
Fig. 3. Suggested structures of the copper complexes.

C. Gökçe, R. Gup / Journal of Photochemistry and Photobiology B: Biology 122 (2013) 15–23
19
Fig. 4. SEM images of (a) HL1 and (b) [Cu(L1)2].
Fig. 5. SEM images of (a) HL2 and (b) [Cu(L2)2].
Fig. 6. Energy dispersive spectrometer of (a) [Cu(L1)2] and (b) [Cu(L2)2].
system, probably due to monomeric nature of the copper com-
plexes and there is no possibility of an exchange interaction.
interactions between aromatic chromophore of molecule and the
base pairs of DNA. On the other hand, the absorption intensities
of drugs are increased (hyperchromism) upon increasing the con-
centration of CT DNA due to a damage of the CT-DNA double-helix
structure. The extent of the hyperchromism is indicative of the par-
tial or non-intercalative binding modes, such as electrostatic
forces, vander Waals interaction, hydrogen bonds and hydrophobic
interaction.
In the present study, the interaction of the copper complexes in
DMF solutions with calf thymus DNA was investigated by the
changes of absorbance at 267 nm and 314 nm for [Cu(L1)2]
(Fig. 7A) and 267 nm 304 nm for [Cu(L1)2] with increasing concen-
tration of CT-DNA (Fig. 7B). The spectra show clearly that the bands
at 314 nm and 304 nm exhibit hypochromism of 10.13% and 9.32%
3.7. DNA binding studies
3.7.1. Electronic absorption titrations
It is well known that metal complexes can bind to DNA via both
covalent and non-covalent interactions [43]. Non-covalent interac-
tions include intercalation, and binding to minor groove, major
groove, sugar–phosphate backbone and electrostatic binding mode
[44]. Absorption titration is an effective method to examine the
binding mode of DNA with metal complexes [45–47]. Drugs bind-
ing with DNA via intercalation usually result in hypochromism and
bathochromism of the absorption bands due to strong stacking

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with a blue-shift of ꢃ2 nm for the [Cu(L1)2] and [Cu(L2)2] com-
plexes, respectively. On the other hand, upon addition of DNA
the absorption band at 267 nm in both copper complexes is shifted
to ꢃ273 nm as well as hyperchromism of 40.85% and 31.08% in the
spectra of CT-DNA/[Cu(L1)2] and CT-DNA/[Cu(L2)2] complexes,
respectively. These changes are typical of the complexes bound
to DNA through non-covalent interaction [48]. These red shifts in
absorbance are accompanied by an increase in molar absorptivity,
so that isosbestic points were formed at 298 nm for [Cu(L1)2] and
at 296 nm for [Cu(L2)2] indicating the existence of single mode of
binding. These spectral characteristics suggest that the binding
nature of the copper complexes with DNA is similar and they might
bind to DNA by an intercalative mode as well as by groove binding.
The observed hypochromism could be attributed to as results of
the contraction of DNA helix axes as well as the conformational
changes on molecule of DNA. The two complexes in our paper
interact with DNA, most likely through a mode that involves a
stacking interaction between the aromatic naphthalene ring and
the base pairs of DNA. On the other hand, the hyperchromism
could be a result of the secondary damage of DNA double helix
structure [45,49]. The carbonyl and hydroxyl groups in the copper
complexes could form hydrogen bonds with suitable donors like
N7 and O6 of adjacent guanine base of DNA, supported by the
favorable hydrophobic interaction of naphthalene ring on the sur-
face of DNA, contributing to the overall hyperchromism. It can be
also concluded from Fig. 7 that the electronic absorption spectra
of the copper complexes are not significantly changed upon addi-
tion of DNA, suggesting that the architectures of the copper com-
plexes are not significantly modified by binding.
The intrinsic binding constant, Kb, was determined by using the
equation, [DNA]/(ea
[DNA] is the concentration of DNA in base pairs, ea
ꢀ
e
f) = [DNA]/(eo
ꢀ
e
f) + 1/Kb(eo
ꢀ
e
f), where
e
,
e
f and o corre-
spond to Aobsd/[M], the extinction coefficient of the complexes and
the extinction coefficient of the complex in the fully bound form,
respectively, and Kb is the intrinsic binding constant. The ratio of
the slope to intercept in the plot of [DNA]/(ea
ꢀ
ef) versus [DNA]
gives the value of Kb and for complex. The calculated binding con-
stants Kb of the copper(II) complexes [Cu(L1)2] and [Cu(L2)2] are
1.16 ꢂ 105 and 8.00 ꢂ 104 Mꢀ1, respectively. The results indicate
that the binding strength of the [Cu(L1)2] is stronger than that of
[Cu(L2)2], probably due to strong hydrogen bond of the hydroxyl
group on the ligand of the [Cu(L1)2] with suitable DNA nucleobases.
However, the binding constants of these complexes are lower in
comparison to those of the classical intercalators (for ethidium
bromide and [Ru(phen)DPPZ]) [43,44].
3.8. DNA cleavage studies
The ability of the copper complexes to cleave DNA in the pres-
ence and absence of H2O2 was studied by gel electrophoresis using
the supercoiled form of pBR322 at 37 °C in TBE buffer [21,50–53].
We prepared four different concentrations of the complexes with
the range from 25 to 100
lM. The progress of the cleavage reaction
with increasing concentrations of each copper complex in the
Fig. 7. Electronic spectra of Cu(II) complexes in the presence of increasing amounts of CT-DNA. [CT-DNA] = 0–100 l
M. (A): [Cu(L1)2] and (B): [Cu(L2)2]. (—) presence of T-DNA,
(---) absence of CT-DNA.

C. Gökçe, R. Gup / Journal of Photochemistry and Photobiology B: Biology 122 (2013) 15–23
21
Fig. 8. Agarose gel electrophoresis of oxidative cleavage of pBR322 plasmid DNA by different concentrations of copper(II) complexes in the presence of H2O2. Lane 1: Cu(L1)2
(25
(25
l
l
M) + DNA + H2O2, lane 2: Cu(L1)2 (50
l
M) + DNA + H2O2, lane 3: Cu(L1)2 (75
l
M) + DNA + H2O2, lane 4: Cu(L1)2 (100
l
M) + DNA + H2O2, lane 5: Cu(L2)2
M) + DNA + H2O2, lane 6: Cu(L2)2 (50 M) + DNA + H2O2, lane 7: Cu(L2)2 (75
l
l
M) + DNA + H2O2, lane 8: Cu(L2)2 (100
lM) + DNA + H2O2, lane 9: DNA control, lane 10:
1 kB marker.
Fig. 9. Agarose gel electrophoresis of oxidative cleavage of pBR322 plasmid DNA by different concentrations of copper(II) complexes in the absence of any oxidative agent.
Lane 1: Cu(L1)2 (25 M) + DNA, lane 2: Cu(L1)2 (50 M) + DNA, lane 3: Cu(L1)2 (75 M) + DNA, lane 4: Cu(L1)2 (100 M) + DNA, lane 5: Cu(L2)2 (25 M) + DNA, lane 6: Cu(L2)2
(50 M) + DNA, lane 9: DNA control.
M) + DNA, lane 7: Cu(L2)2 (75 M) + DNA, lane 8: Cu(L2)2 (100
l
l
l
l
l
l
l
l
Fig. 10. Agarose gel electrophoresis of pBR322 plasmid DNA treated with the copper(II) complexes and potential inhibitors agents in the presence of hydrogen peroxide. Lane
1: Cu(L1)2 (100 M) + DNA + H2O2 + NaN3, lane 2: Cu(L1)2 (100 M) + DNA + H2O2 + EDTA, lane 3: Cu(L1)2 (100 M) + DNA + H2O2 + KI, lane 4: Cu(L1)2 (100
M) + DNA + H2-
O2 + DMSO, lane 5: Cu(L2)2 (100 M) + DNA + H2O2 + NaN3, lane 6: Cu(L2)2 (100 M) + DNA + H2O2 + EDTA, lane 7: Cu(L2)2 (100 M) + DNA + H2O + KI, lane 8: Cu(L2)2
(100 M) + DNA + H2O2 + DMSO, lane 9: DNA control.
l
l
l
l
l
l
l
l
presence of H2O2 is given in Fig. 8. From the results shown in Fig. 8,
it can be deduced that both copper complexes exhibit effective
nuclease activity in the presence of hydrogen peroxide. An increase
of complex concentration gives rise to an increase in the DNA
cleavage. In lanes 1 and 2 for the complex [Cu(L1)2], the circular
supercoiled DNA band is completely lost and forms II and III are
present. The cleavage percentage of nicked form is much higher
than that of linear form. When the concentration of the [Cu(L1)2]
Interestingly, both copper complexes exhibit slight nuclease
activity in the absence of hydrogen peroxide as an oxidant agent.
They slightly converted the form I to form II, but the supercoiled
form is still seen (Fig. 9), implying that they probably undergo a
mainly single-strand cleavage pathway in the absence of an oxi-
dant agent. It is also clearly seen that as the concentration of
the complex [Cu(L1)2] increases, the concentration of the nicked
DNA increases. It is reasonably expected in the absence of an oxi-
dant agent the DNA cleavage is a hydrolytic. On the other hand, it
is also reported that the DNA cleavage mediated by the some cop-
per complexes occur via an oxidative mechanism without any
reducing agent [20,21,49,53]. In this case, the oxidative DNA
cleavage can be ligand-based, possibly due to the involvement
of a non-diffusible organic radical mechanism which causes oxi-
dative DNA cleavage. The other way is that the hydrogen abstrac-
tion from the deoxyribose sugar in the presence of dioxygen
produces dihydrogen peroxide. Then, dihydrogen peroxide cou-
ples with Cu(II) in a Fenton-type reaction to produce reactive dif-
fusible oxygen species, which makes the DNA cleavage oxidative
is increased to 75
pletely into small pieces.
With 25, 50 and 75
M concentrations, the [Cu(L2)2] is found to
lM, the circular supercoiled DNA degrades com-
l
cleave the supercoiled DNA to nicked and linear DNA forms and no
band is observed for form I (lanes 5–7) as a result of double-strand
breaks over the plasmid molecule. It is also seen that the intensity
of nicked DNA is much higher than that of form III. With increasing
concentration of the copper complex, the intensity of form III also
decreases. With a complex concentration of 100 lM, no band is ob-
served, lane 8, probably due to complete degradation of DNA into
small pieces.

22
C. Gökçe, R. Gup / Journal of Photochemistry and Photobiology B: Biology 122 (2013) 15–23
and catalytic. Further studies are undergoing to elucidate the
cleavage mechanism.
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