Synthesis, structure characterization, DNA binding, and cleavage properties of mononuclear and tetranuclear cluster of copper(II) complexes

Article abstract of DOI:10.17344/acsi.2014.797

Two copper(II) complexes, cluster 1, and mononuclear 2, have been synthesized by reacting acetylacetone and benzohydrazide (1:1 ratio for 1 and 1:2 ratio for 2) with CuCl₂ in a methanol solution. In 2, which is a new complex, the ligand acts as

Full text of DOI:10.17344/acsi.2014.797

DOI: 10.17344/acsi.2014.797
Acta Chim. Slov. 2015, 62, 122–129
122
Scientific paper
Synthesis, Structure Characterization, DNA Binding,
and Cleavage Properties of Mononuclear and Tetranuclear
Cluster of Copper(II) Complexes
Rasoul Vafazadeh,1,* Naime Hasanzade,1 Mohammad Mehdi Heidari2
and Anthony C. Willis3
1 Department of Chemistry, Yazd University, Yazd, Iran.
2 Department of Biology, Yazd University, Yazd, Iran.
3 Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
* Corresponding author: E-mail: rvafazadeh@yazd.ac.ir
Tel: +98 351 8214778 Fax: +98 351 7250110
Received: 09-07-2014
Abstract
Two copper(II) complexes, cluster 1, and mononuclear 2, have been synthesized by reacting acetylacetone and benzohy-
drazide (1:1 ratio for 1 and 1:2 ratio for 2) with CuCl2 in a methanol solution. In 2, which is a new complex, the ligand
acts as a tetradentate which binds the metal ion via two amide-O atoms and two imine-N atoms providing an N2O2 squa-
re-planar around the copper(II) ion. The absorption spectra data evidence strongly suggested that the two copper(II)
compounds could interact with CT-DNA (intrinsic binding constant, Kb = 0.45 × 104 M–1 for 1 and Kb = 2.39 × 104 M–1
for 2). The super coiled plasmid pBR322 DNA cleavage ability was studied with 1 and 2 in the presence and absence of
H2O2 as an oxidant. In both the absence and the presence of an oxidizing agent, complex 2 exhibited no nuclease acti-
vity. However, even in the absence of an oxidant, complex 1 exhibited significant DNA cleavage activity.
Keyword: copper complex; mononuclear; copper cluster; DNA binding; DNA cleavage
The former requires planar type structures while the latter
1. Introduction
needs coordination complexes with potential coordination
Recently, there has been considerable interest in the
interaction of transition metal complexes with DNA and
nuclease activity studies because of their various applica-
tions in nucleic acid chemistry. The use of such complexes
in foot-printing studies, as sequence specific DNA bin-
ding agents, as diagnostic agents in medicinal applica-
tions, and for genomic research, has generated the current
interest to further develop this chemistry.1–4 The first re-
port of a synthetic Cu(II) system capable of inducing
DNA cleavage was given by Sigman et al. in 1979.5 After-
wards, large numbers of copper(II) complexes and other
metal complexes, particularly of the first row transition
metals, have been synthesized, characterized, and evalua-
ted for their nuclease mimicking and anticancer activi-
ties.6–8 The two modes of binding with DNA residues ha-
ve been identified, i.e., intercalating and covalent binding.
sites.9, 10 There are three different types of DNA cleavage
mechanisms, viz oxidative cleavage, photochemical clea-
vage, and hydrolysis.11,12 Copper(II) complexes are ca-
pable of binding and cleaving double-stranded DNA un-
der physiological conditions.13,14
For these reasons, we have been systematically stud-
ying the DNA cleavage and nuclease activity of various
copper(II) complexes. Our group began to synthesize li-
gands derived from benzohydrazide and their copper
complexes and to evaluate their DNA binding and cleava-
ge abilities. In this paper, we describe the synthesis, cha-
racterization by elemental analysis, FT–IR, UV–Vis and
X-ray crystallography, and DNA binding and cleavage
abilities of a copper(II) complex with tetradentate ligand
derived from a benzohydrazide and tetranuclear cop-
per(II) cluster [Cu4Cl6O(C5H8N2)4].
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Acta Chim. Slov. 2015, 62, 122–129
The resulting somewhat viscous solution was clear and par-
123
2. Experimental
ticle-free. The CT-DNA in the buffer medium gave a ratio
of UV absorbance at 260 and 280 nm of 1.8:1, indicating
that the DNA was sufficiently free of protein.15 The DNA
concentration was measured from its absorption intensity at
260 nm using the molar absorption coefficient (ε) value of
6600 M–1 cm–1 as reported.16 The stock solutions of com-
plexes were freshly prepared by first dissolving complexes
in DMSO for 1 and in DMF for 2, then diluting them with
the buffer. The amount of DMSO or DMF was kept at 10%
(by volume) for each set of experiments and had no effect
on any experimental results. After equilibrium reached (ca.
5 min) the spectra were recorded against an analogous
blank solution containing the same concentration of DNA.
Absorption spectral titration experiments were performed
while maintaining a constant complex concentration and
varying the nucleic acid concentration. This was achieved
by dissolving an appropriate amount of the metal complex
(40 μM for 1 and 70 μM for 2) and DNA stock solutions
(0–60 μM) while maintaining the total volume constant (1
ml). The spectral bands exhibited hyperchromism for 1 and
hypochromism for 2 in each investigated complexes and
were recorded after the successive addition of CT-DNA.
The Tris–HCl buffer was used as a blank to make prelimi-
nary adjustments. The intrinsic binding constant (Kb) of the
complexes to CT-DNA were determined from the spectral
titration data using the following equation.17
2. 1. Reagents
All chemicals were used as supplied by Merck and
Fluka without further purification. Calf thymus DNA (CT-
DNA) and supercoiled pBR322 DNA were obtained from
Sigma–Aldrich and stored at 4°C. The Tris(hydroxy-
methyl)aminomethane–HCl (Tris–HCl) buffer was prepa-
red in doubly distilled water.
2. 2. Physical Measurements
Infrared spectra were taken with an Equinox 55
Bruker FT-IR spectrometer using KBr pellets in the
400–4000 cm–1 range. Absorption spectra were determi-
ned in the solvent methanol using a GBC UV-Visible Cin-
tra 101 spectrophotometer with 1 cm quartz, in the range
of 200–800 nm at 25 °C. Elemental analyses (C, H, N)
were performed using a CHNS-O 2400II PARKIN-
ELMER elemental analyzer.
2. 3. DNA Binding
The interaction of the complexes with calf thymus
CT-DNA was studied in the Tris–HCl buffer (5.0 mM
Tris–HCl, pH 7.2) containing 50mM NaCl at room tempe-
rature. Then, the solution was kept for over 24 h at 4 °C.
Table 1. Crystallographic data of the complexes 1 and 2
Compound
1
2
Chemical formula
Formula weight
Temperature (K)
Space group
C20H32Cl6Cu4N8O
867.43
200
C19H16CuN4O3
411.91
200
Monoclinic, p
Z=2
Orthorhombic, pbca, Z = 8
2/n,
Unit cell dimensions
a (Å)
b (Å)
c (Å)
α (°)
β (°)
13.3604 (3)
8.8530 (1)
16.6709 (4)
90
95.0682 (9)
90
19.8983 (10)
8.4055 (3)
20.8417 (10)
90
90
90
γ (°)
V (Å3)
1964.12 (7)
3485.9 (3)
F(000)
868
1.467
1688
1.570
DCalc (g cm–3)
Crystal size (mm)
μ (mm–1)
0.44 × 0.10 × 0.07 mm
2.57
0.54 × 0.04 × 0.01 mm
1.28
θ range (°)
2.6–27.5
2.8 – 25
Limiting indices
–17 ≤ h ≤ 17
–11 ≤ k ≤ 11
–21 ≤ l ≤ 20
0.043
–19 ≤ h ≤ 23
–10 ≤ k ≤ 9
–24 ≤ l ≤ 24
0.055
R[F2 > 2σ(F2)]
wR(F2) (all data)
0.108*
0.120**
*w = 1/[σ2(F2) + (0.04P)2 + 5.02P] , where P = (max(Fo2,0) + 2Fc2)/3
**w = 1/[σ2(F2) + (0.03P)2 + 9.61P] , where P = (max(Fo2,0) + 2Fc2)/3
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Acta Chim. Slov. 2015, 62, 122–129
lent of acetylacetone and 2 equivalents of benzohydrazide
124
[DNA]/(εa – εf) = [DNA]/( εb– εf ) +
+ 1/Kb(εb – εf)
(1)
in methanol by heating the mixture under reflux.
where [DNA] is the concentration of CT-DNA in base
pairs, the apparent absorption coefficients εa, εf and εb cor-
respond to Aobs/[DNA], the absorbance for the free-Cu(II)
complex (unbound), and the absorbance for the fully-
bound complex, respectively. A plot of [DNA]/(εa–εf) ver-
sus [DNA] gave a slope 1/(εb–εf) and a intercept 1/Kb(
εb–εf), so the value of Kb can be determined from the ratio
of the slope to the intercept.
2. 6. 1. Syntheses of Tetranuclear Copper(II)
[
]
Cluster Cu4Cl6O (C5H8N2)4 , 1
Acetylacetone (1.05 mL, 10 mmol) was added to a
methanol solution (25 mL) of benzohydrazide (1.36 g, 10
mmol), and the mixture was heated to reflux for 5 h. A so-
lution of CuCl2 · 2H2O (1.70 g, 10 mmol) in methanol was
added to the above-mentioned bright yellow solution. The
green solution was stirred at room temperature for 2 h. Af-
ter two days, green-brown precipitate was obtained upon
the slow evaporation of the solvents at room temperature.
The precipitate was recrystallized from acetone/2-propa-
nol/toluene (2:1:1 v/v). Brown-green rod-shaped crystals
appeared upon the slow evaporation of the solvents and
were washed with ethanol and dried in air. Yield 1.38 g
(40%). Anal. Calcd. for C20H32Cl6Cu4N8O (%): C, 27.69;
H, 3.72; N, 12.92. Found: C, 27.57; H, 4.01; N, 12.52. IR
(KBr, cm–1): υC=N = 1572, υN–H = 3335. Electronic
spectra in methanol: λmax(nm), (log ε): 813 (2.24), 311
(3.59).
2. 4. DNA Cleavage
Cleavage of plasmid DNA was monitored using aga-
rose gel electrophoresis. Supercoiled pBR322 DNA (0.1
mg/ml, 1.5 μL) in Tris-HCl buffer (5.0 mM, pH 7.2) with
50 mM NaCl was treated with the copper(II) complexes
(100–800 μM) in the presence and absence of additives.
The concentration of the complexes in DMF or the additi-
ves in the buffer corresponded to the quantity after the di-
lution of the complex stock to the 20 μL final volume us-
ing the Tris–HCl buffer. The oxidative DNA cleavage was
studied in the presence of H2O2 (3 mM, oxidizing agent),
DMSO (1.5 mM, hydroxyl scavenger), and NaN3 (1 mM,
singlet oxygen scavenger). The samples were incubated
for 2 h at 37 °C. The loading buffer (4 μL, 12.5% bromop-
henol blue and 25% xylene cyanol) was subsequently ad-
ded. The agarose gel (0.8%) containing 2 μL (10 mg/mL
stock) of ethidium bromide (EB) was prepared, and the
electrophoresis of the DNA cleavage products was perfor-
med on it. The gel was run at 60 V for 3 h in TAE
(Tris–acetate–EDTA) buffer, and the bands were identi-
fied by placing the stained gel under an illuminated UV
lamp.
2. 6. 2. Syntheses of Copper(II) Complex, 2
To a methanol solution, (30 ml) of bzpyzn (1.35 g, 4
mmol), CuCl2 · 2H2O (0.68 g, 4 mmol) was added, and the
green solution was stirred at room temperature for 2 h and
left in air at room temperature for slow evaporation. After
two days, the initial green color of the solution slowly be-
came brown, and a brown-green crystalline solid separa-
ted. The complex was collected by filtration and dried in
air. The solid was recrystallized from the DMF solution,
and the crystal appeared in about 4–5 days. Yield 0.33 g
(20%). Anal. Calcd. for C19H16CuN4O3 (%): C, 55.40; H,
3.92; N, 13.60. Found: C, 55.89; H, 4.03; N, 13.41. IR
(KBr, cm–1): υC=O = 1639, υC=N = 1587. Electronic
spectra in methanol: λmax(nm), (log ε): 587(2.21),
442(3.86), 344(4.65), 295 (4.95).
2. 5. X-ray Crystallography
Diffraction images were measured at 200 K on a
Nonius Kappa CCD diffractometer using Mo Kα, graphi-
te monochromator (λ = 0.71073 Å). Data was extracted
using the DENZO/SCALEPACK package.18 The structures
were solved by direct methods with the SIR92 and refined
on F2 by full matrix last-squares techniques using the
CRYSTALS program package, respectively.19,20 Atomic
coordinates, bond lengths and angles, and displacement
parameters were deposited at the Cambridge Crystallo-
graphic Data Centre. Crystallographic details for the two
crystals 1 and 2 are summarized in Table 1.
3. Result and Discussion
3. 1. Synthesis
The complex 1 was synthesized by a two-steps-
one-pot reaction with the initial formation of the pyra-
zol (without its isolation) and then adding the methano-
lic solution of CuCl2. The pyrazol was prepared in situ
from the reactions between acetylacetone and benzohy-
drazide in methanol under reflux. Initially, (3,5-di-
methyl-1H-pyrazol-1-yl)(phenyl)methanone (benz-
pyrazol) was obtained by the reaction of equimolar
amount of acetylacetone and benzohydrazide.22 The re-
sulting solution which was refluxed for 5 h, was used
for the synthesis of the complex without further purifi-
2. 6. Syntheses
The synthesis of 1-benzoyl-3,5-dimethyl-5-(1-ben-
zoylhydrazido) pyrazoline (bzpyzn) was prepared follo-
wing a published procedure.21 Briefly, bzpyzn ligand was
obtained by the condensation reaction between 1 equiva-
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Acta Chim. Slov. 2015, 62, 122–129
125
cation. From the reaction between CuCl2 and benz-
of the copper(II) cluster from the one-pot reaction of
3,5-dimethylpyrazole-1-carboxamide and CuCl2 in a
hot ethanol solvent.
Reaction between acetylacetone and benzohydrazi-
de in methanol under reflux will not lead to the formation
of the desired acetylacetone bis(benzoylhydrazone) li-
gand. However, as it is known, this type of the reaction
leads to cyclized 3,5-substituted pyrazolines (bzpyzn)
(Scheme 2).21,24
pyrazol at room temperature followed by its hydrolysis,
the brown-green rod-shaped crystalline of the cop-
per(II) cluster 1 was obtained (Scheme 1). The reaction
between the pyrazol and CuCl2 at room temperature
produced the brown-green rod-shaped crystalline of the
copper(II) cluster 1 (Scheme 1). Our search revealed
that the synthesis of the cluster was reported by Ja}imo-
vi} et al. in 2007.23 They synthesized the green crystals
Scheme 1 Synthesis of the cluster 1
Scheme 2 Synthesis of the complex 2
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Acta Chim. Slov. 2015, 62, 122–129
Complex 2 was synthesized by the reaction of
126
bzpyzn and CuCl2 · 2H2O in methanol under aerobic con-
ditions leads to the ring opening of bzpyzn and complexa-
tion of the copper by tetradentate ligand (Scheme 2). After
two days, the initial green color of the solution slowly
changed to brown, and a brown-green crystalline substan-
ce separated. The IR spectrum of bzpyzn showed bands
about 3277, 1666 and 1631 cm–1 attributable to N–H,
C=O and C=N groups, respectively. The IR spectrum of
the copper complex 2 did not display any band due to the
N–H group. The absence of an N–H group was consistent
with the ring opening of bzpyzn and complexation of the
copper by tetradentate ligand.21,24 The middle C-atom of
the acetylacetone residue in tetradentate ligand of com-
plex 2 underwent a four-electron-two-proton oxidation in
the presence of both water and oxygen and C=O group
formed.24 In complex 2, the bands observed at 1639 and
1587 cm–1 could be assigned to the C=O and C=N
groups.25 In the electronic spectrum, 2 showed a broad ab-
sorption band centered at 587 nm because of the spin-allo-
wed d–d transition of the copper(II) ion.26, 27
Fig. 1 The molecular structure of copper(II) complex 2 with label-
ing of selected atoms
Table 2. Selected bond lengths (Å) and angles (°) in complex 2
Cu1–O1
Cu1–O2
Cu1–N2
Cu1–N3
O1–C7
O2–C13
O3–C10
N1–N2
N1–C7
1.925 (4)
1.915 (4)
1.923 (4)
1.930 (6)
1.283 (6)
1.277 (6)
1.224 (6)
1.391 (6)
1.331 (6)
1.301 (6)
1.302 (6)
O1– Cu1–O2
O1– Cu1–N2
O2– Cu1–N2
O1– Cu1–N3
O2– Cu1–N3
N2– Cu1–N3
N2– N1–C7
N1– N2–Cu1
C8– C10–O3
C8– C10–C11
100.13 (15)
82.52 (17)
171.86 (18)
177.03 (17)
82.19 (17)
95.43 (19)
108.80 (5)
113.50 (3)
116.30 (5)
126.80 (4)
3. 2. Crystal Structures
Because a similar structure has been previously re-
ported,23 the structure and bond lengths and angles of clu-
ster 1 have been submitted as supplementary information.
N2–C8
N3–C11
3. 2. 1. Description of Complex 2
Single crystals of complex 2 suitable for X-ray were
obtained from the solution of complex 2 in DMF. The
complex crystallized in the orthorhombic space group
Pbca. The molecular structure of the complex with selec-
ted atoms labeled is shown in Figure 1, and selected bond
lengths and angles are listed in Table 2. In complex 2,
bzpyzn acted as a tetradentate ligand which bound the me-
tal ion via two amide-O atoms and two imine-N atoms
providing an N2O2 square-plane around the copper(II) ion.
The ligand formed two five-membered and one six-mem-
bered chelate rings (5-6-5) with a Cu(II) metal center. The
copper(II) ion was square planar with a slight tetrahedral
distortion. The deviation of the copper(II) center from the
N2O2 square plane is 0.048 Å. The dihedral angles bet-
ween the two planes [N(2)–Cu(1)–O(1) and N(3)–
Cu(1)–O(2)] were 8.32 Å, compared with 0 for a perfect-
ly square-planar arrangement and 90 for a perfect tetrahe-
dral arrangement. The angles at Cu(II) between two donor
atoms in the cis position were in the range of 82.19(17) –
95.43(19)° and between two donor atoms in the trans po-
sition were in the range of 171.86(18)° and 177.03(17)°
(Table 2).
the analogous compounds observed in the literature.21,24,28
Two Cu–N(imine) bond distances (1.923(4) and 1.930(4)
Å) were very similar. The same was also true for the two
Cu–O bond distances (1.925(4) and 1.915(4) Å). The di-
stances were comparable with distances observed for
square-planar copper(II) complexes having imine-N or
deprotonated amide-O coordinating atoms.26–29
3. 3. DNA Binding Studies
3. 3. 1. Absorption Spectral Studies of CT-DNA
with 1
UV–Visible spectroscopy was performed to study
the interaction of the complexes with DNA keeping the
complex concentration constant (40 μM) and varying the
concentration of CT-DNA (0–60 μM). The change in ab-
sorbance values at 260 nm was used to evaluate the intrin-
sic binding constant Kb. The absorption spectra of 1 in the
presence of CT-DNA are shown in Figure 2. With increa-
sing the concentrations of CT-DNA, the complex exhibited
hyperchromism without a shift in band position at 260 nm.
The hyperchromism indicated a strong interaction
between the complexes and CT-DNA mainly through
groove binding.30,31
The structure showed that the middle C-atom of the
acetylacetone residue had been oxidized with the forma-
tion of a C=O group (Figure 1). The C=O bond length in 2
was 1.224(6) Å, which distance is in good agreement with
The use of a tetranuclear copper(II) cluster with lar-
ge steric hindrance led to the poor binding affinity interac-
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Acta Chim. Slov. 2015, 62, 122–129
tion of the complex with the double stranded CT-DNA.
127
Since the complex cannot be intercalated well with DNA,
classical intercalative interaction was excluded. However,
since DNA possesses several hydrogen bonding sites
which are accessible both in the minor and major grooves,
a favorable hydrogen bonding may be formed between the
amine –NH– groups of the pyrazol ligand in cluster with
the base pairs in CT-DNA.32,33
Analysis of the spectrum data using Equation 1 in the
presence of DNA (Figure 2) gave a binding constant, Kb,
of 0.45 × 104 M–1 (r = 0.967 for seven points) for the copper
cluster. From the binding constant value, it was clear that
the cluster had moderate interaction with CT-DNA.
Fig. 3 Absorption spectra of complex 2 (70 μM) in Tris–HCl buffer
(pH = 7.2) with the increase in molar ratio of DNA to complex
(0–60 μM). Arrow shows the absorbance changing upon the increa-
se of DNA concentration. The inset shows plot of [DNA]/(εa–εf) vs.
[DNA] for titration of CT–DNA with the complex
intensity of the charge transfer spectral band of the complex
by increasing the concentration of CT-DNA (Figure 3). The
binding constant (Kb) for 2 is 2.39 × 104 M–1 (r = 0.986 for
six points), suggests that a strong interaction exists between
complex 2 and CT-DNA due to its square planar nature.36,37
The binding strength of the complex 2 is notably larger than
the free ligand, suggesting that the electronic configuration
of the metal center may be an important factor affecting the
DNA binding affinities of such as compound.
The intrinsic binding constants Kb obtained for 1 and 2
were lower than those observed for the typical classical in-
tercalator ethidium bromide (EB), Kb = 1.23 × 106 M–1.38
Therefore, interaction of the two complexes with CT-DNA
is considered to be weaker than with classical intercalator.
However, the Kb value for the complex 2 is higher than some
reported mononuclear copper(II) complexes, such as
[Cu(bpy)(Gly)Cl] · 2H2O, 1.84 × 103 M–1, [Cu(dpa)(Gly)Cl]
2H2O, 3.10 × 103 M–1,1 [Cu(L)(bpy)Cl] (HL = (E)-3-(2-
hydroxyphenylimino)-N-o-tolylbutanamide), 1.55 × 103
M–1,18 and is comparable to that observed for [CuL1](ClO4)2
(L1 = N,N’-bis-pyridin-2-ylmethyl-butane-1,4-diimine), 2.6
× 104 M–1,4 [Cu(nfH)2]Cl2 (nfH = norfloxacin), 4.08 × 104
M–1,10 and [Cu(o-vanile)(phen)], 2.13 × 104 M–1.37
Fig. 2 Absorption spectra of complex 1 (40 μM) in Tris–HCl buffer
(pH = 7.2) with the increase in molar ratio of DNA to complex
(0–60 μM). Arrow shows the absorbance changing upon the increa-
se of DNA concentration. The inset shows plot of [DNA]/(εa–εf) vs.
[DNA] for titration of CT–DNA with the complex
3. 3. 2. Absorption Spectral Studies of CT-DNA
with 2
Figure 3 illustrates the representative UV–Vis spec-
tra for the binding of complex 2 with DNA at a constant
complex concentration 70 μM and varying the concentra-
tion of CT-DNA (0–60 μM). Increasing the DNA concen-
tration showed hypochromicity in the absorption spectra.
The change in hyperchromic to hypochromic shift from
the copper(II) cluster 1 (Figure 3) to complex 2 indicated
that the type of binding mode was different. This can be at-
tributed to a difference in the structure of the two comple-
xes. The observable hypochromism is usually characteri-
zed by the non-covalently intercalative binding of the com-
pound to the DNA helix.31,34,35 Complex 2 bound to DNA
through intercalation and resulted in hypochromism due to
the strong stacking interaction between the aromatic chro-
mophore of the complex and the base pairs of DNA.35
The intrinsic binding constants Kb for complex 2 we-
re determined by monitoring the change of the absorption
3. 4. Supercoiled pBR322 Plasmid DNA
Cleavage Studies
Gel electrophoresis experiments using pBR322 cir-
cular plasmid DNA were performed with complexes 1 and
2 in the presence and absence of H2O2 as an oxidant. At
micro-molar concentrations, for 3 h incubation periods, in
the absence and presence of an oxidizing agent, complex
2 exhibited no nuclease activity. However, even in the ab-
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Acta Chim. Slov. 2015, 62, 122–129
sence of an oxidant, complex 1 exhibited significant DNA
128
per(II) compounds 1 and 2. Single crystal X-ray diffrac-
tion studies revealed that the two complexes have two dif-
ferent structures, namely a tetranuclear cluster and a mo-
nonuclear square-planar. The binding of the complexes
with CT-DNA was studied by UV-Vis spectroscopy, and
their strong binding ability was revealed. Complex 2 exhi-
bited no nuclease activity; however, complex 1 exhibited
significant DNA cleavage activity.
cleavage activity (Figure 4, lane 2). Control experiments
using DNA alone resulted in no significant cleavage of p-
BR322 circular plasmid DNA, even after longer exposure
times (Figure 4, lane 1). From the observed results, it was
concluded that complex 1 effectively cleaved the DNA as
compared to control DNA. The copper cluster at a higher
concentration (800 μM) showed more cleavage activity
than at the lower concentration (100 μM) of cluster 1 (Fi-
gure 4, lanes 3–5). This shows that the concentration of
optimal value led to extensive degradations, resulting in
the conversion of the supercoiled form (Form-I) into an
open-circular form (Form-II).
5. Supplementary material
The deposition numbers of the studied complexes, 1
and 2 are CCDC 963061 and 963060, respectively. These
data can be obtained free-of-charge via www.ccdc.cam.
ac.uk/data_request/cif, by emailing data-request@ccdc.
cam.ac.uk, or by contacting The Cambridge Crystallo-
graphic Data Centre, 12 Union Road, Cambridge CB2
1EZ, UK; fax +44 1223 336033.
6. Acknowledgments
The authors are grateful to the Yazd University and
the Australian National University for partial support of
this work.
Fig. 4 Agarose gel electrophoresis diagram showing the chemical
nuclease activity of the complexes 1 using Supercoiled pBR322
plasmid DNA (0.1 mg/ml, 1.5 μL): lane 1, DNA control; lane 2,
DNA + 1 (100 μM); lane 3, DNA + 1 (500 μM); lane 4, DNA + 1
(800 μM); lane 5, DNA + 1 (800 μM) + NaN3 (1 mM); lane 6, DNA
+ 1 (800 μM) + DMSO (1.4 mM)
7. References
1. M. S. Mohamed, A. A. Shoukry, A. G. Ali, Spectrochim. Ac-
ta A 2012, 86, 562–570.
2. K. Ghosh, V. Mohan, P. Kumar, U. P. Singh, Polyhedron
2013, 49, 167–176.
The nuclease activity of the complexes was also in-
vestigated in the presence of a free radical scavenger, di-
methylsulfoxide (DMSO), and a singlet oxygen quencher,
azide ion (NaN3). The presence of radical scavengers
(DMSO) and a singlet oxygen quencher (NaN3) did not
significantly reduce the efficiency of DNA cleavage (Fi-
gure 4, lanes 6, 7), ruling out the possibility of the invol-
vement of diffusible hydroxyl radicals and a singlet oxy-
gen in the cleavage.8,39
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Povzetek
Dva bakrova(II) kompleksa, klaster 1 in enojederni 2, sta bila sintetizirana pri reakciji acetilacetona in benzohidrazida
(v razmerju 1:1 za 1 in 1:2 za 2) z CuCl2 v metanolu. Pri 2, ki je nov kompleks, je ligand tetradentatni in vezan na ko-
vinski ion preko dveh amidnih O atomov in dveh iminskih N atomov in tvori N2O2 kvadratno-planarno geometrijo oko-
li bakrovega(II) ion. Podatki dobljeni iz absorpcijskih spektrov ka`ejo, da obe bakrovi(II) spojini lahko interagirata s
CT-DNA (Kb = 0.45 × 104 M–1 za 1 in Kb = 2.39 × 104 M–1 za 2). Na super coiled plasmidu pBR322 je bila testirana
zmo`nost 1 in 2 cepiti DNA v prisotnosti ali odsotnosti H2O2 kot oksidanta. V obeh primerih, tako v prisotnosti kot od-
sotnosti oksidanta, kompleks 2 nima nukleazne aktivnosti, medtem ko tudi v odsotnosti oksidanta kompleks 1 izkazuje
veliko aktivnost cepitve DNA.
Vafazadeh et al.: Synthesis, Structure Characterization, DNA Binding, ...

Acta Chim. Slov. 2015, 62, 122–130
S130
Supplement Material
Synthesis, Structure Characterization, DNA Binding,
and Cleavage Properties of Mononuclear and Tetranuclear
Cluster of Copper(II) Complexes
Rasoul Vafazadeh,* Naime Hasanzade, Mohammad Mehdi Heidari
and Anthony C. Willis
* Corresponding author: E-mail: rvafazadeh@yazd.ac.ir
Tel: +98 351 8214778 Fax: +98 351 7250110
Received: 09-07-2014
Table S. Selected bond lengths (Å) and angles (°) in complex 1a
Complex 1
Cu1–Cl1
Cu1–Cl4
Cu1–Cl2*
Cu1–N1
Cu1–O1
Cu2–Cl1
Cu2–Cl2
Cu2–Cl3
Cu2–N3
Cu2–O1
2.3580 (11)
2.4386 (19)
2.4069 (12)
1.9620 (3)
1.9072 (17)
2.3567 (10)
2.3810 (10)
2.4901 (11)
1.9530 (3)
1.9091 (16)
Cl1– Cu1–Cl2*
Cl1– Cu1–O1
Cl4– Cu1–O1
Cl1– Cu1–N1
Cl4– Cu1–N1
O1– Cu1–N1
Cl1– Cu2–Cl2
Cl1– Cu2–Cl3
O1– Cu2–N3
Cu1– O1–Cu2
116.83 (5)
85.46 (4)
82.59 (8)
96.12 (9)
92.75 (10)
174.33 (11)
125.74 (5)
117.96 (4)
175.97 (5)
107.53 (17)
a symmetry codes:–x+3/2, y, –z+3/2.
Fig. S The molecular structure of tetranuclear cluster of copper(II)
complex with labeling of selected atoms
Vafazadeh et al.: Synthesis, Structure Characterization, DNA Binding, ...

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