Synthesis, characterization, X-ray crystal structure, DFT calculation, DNA binding, and antimicrobial assays of two new mixed-ligand copper(II) complexes

Article abstract of DOI:10.1016/j.saa.2015.01.088

Two new Cu(II) complexes, [Cu(L)(phen)] (1), [Cu(L)(bipy)] (2), where L^2- = (3-methoxy-2oxidobenzylidene)benzohydrazidato, phen = 1,10 phenanthroline, and bipy = 2,2′ bipyridine, were prepared and fully characterized using elemental analyses, F

Full text of DOI:10.1016/j.saa.2015.01.088

Accepted Manuscript
Synthesis, characterization, X-ray crystal structure, DFT calculation, DNA
binding and antimicrobial assays of two new mixed-ligand ternary copper (II)
complexes
S. Yousef Ebrahimipour, Iran Sheikhshoaie, Maryam Mohamadi, Sebastian
Suarez, Ricardo Baggio, Moj Khaleghi, Masoud Torkzadeh, Ali Mostafavi
PII:
S1386-1425(15)00109-2
http://dx.doi.org/10.1016/j.saa.2015.01.088
SAA 13256
DOI:
Reference:
To appear in:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
Received Date:
Revised Date:
Accepted Date:
17 September 2014
14 January 2015
29 January 2015
Please cite this article as: S. Yousef Ebrahimipour, I. Sheikhshoaie, M. Mohamadi, S. Suarez, R. Baggio, M.
Khaleghi, M. Torkzadeh, A. Mostafavi, Synthesis, characterization, X-ray crystal structure, DFT calculation, DNA
binding and antimicrobial assays of two new mixed-ligand ternary copper (II) complexes, Spectrochimica Acta Part
A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.088
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Synthesis, characterization, X-ray crystal structure, DFT calculation, DNA
binding and antimicrobial assays of two new mixed-ligand ternary copper (II)
complexes
a,
a
b
c
S. Yousef Ebrahimipour *, Iran Sheikhshoaie , Maryam Mohamadi , Sebastian Suarez ,
d
e
f
a
Ricardo Baggio , Moj Khaleghi , Masoud Torkzadeh , Ali Mostafavi
a
Department of Chemistry, Shahid Bahonar University of Kerman, Kerman, Iran.
b
Department of Chemistry, Payame Noor University (PNU), 19395–4697 Tehran, Iran
c
Departamento de Quımica Inorganica, Analıtica y Quımica, Fısica/INQUIMAE–CONICET, Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires,Buenos Aires, Argentina.
d
Gerencia de Investigacion y Aplicaciones, Centro Atomico Constituyentes, Comision Nacional de Energıa
Atomica, Buenos Aires, Argentina.
e
Department of Biology, Shahid Bahonar University of Kerman, 76169-14111, Kerman, Iran.
f
Department of Biotechnology, Institute of Science, High Technology and Environmental Science, Graduate
University of Advance Technology, Kerman, Iran
Corresponding author:
Dr. S. Yousef Ebrahimipour
Department of Chemistry
Shahid Bahonar University of Kerman
76169-14111
Kerman, Iran
Tel/fax: +98 34 3132 2143
E-mails adresses: Ebrahimipour@uk.ac.ir, Ebrahimipour@ymail.com
1

Abstract
Two new Cu(II) complexes, [Cu(L)(phen)] (1), [Cu(L)(bipy)] (2), [HL= (E)-N'-(2-hydroxy-3-
methoxybenzylidene)benzohydrazide , phen= 1,10 phenanthroline, bipy= 2,2’ bipyridine), were
prepared and fully characterized using elemental analyses, FT-IR, molar conductivity, and
electronic spectra. The structures of both complexes were also determined by X-ray diffraction.
It was found that, both complexes possessed square pyramidal coordination environment in
which, Cu(II) ions were coordinated by donor atoms of HL and two nitrogens of hetrocyclic
bases. Computational studies were performed using Density Functional Theory DFT, using
B3LYP exchange and correlation functionals. DNA binding activities of these complexes were
also investigated using electronic absorption, competitive fluorescence titration and cyclic
voltametry studies. The obtained results indicated that binding of the complexes to DNA was of
intercalative mode. Antimicrobial activities of these compounds were also screened against
microorganisms.
Keywords: Cu(II) complex, DFT calculation, Hydrazone Schiff base , DNA binding,
Antimicrobial activity, Crystal structure.
1
Introduction
In recent decades, hydrazone compounds have attracted lots of attention not only because of their
variety of structure, but also for their various biological and chemical applications [1, 2]. They
have the ability to form complexes with metal ions in different oxidation states [3]. Moreover,
recent researches show the presence of hydrazone moiety in these compounds can enhance their
biological activities [4]. Considerable attentions have been focused on Cu(II) complexes due to
2

significant properties in various fields [5, 6]. They have been used as models for metal-
containing sites in enzymes and several proteins of type 2 [9]. On the other hand, these
compounds can be appropriate candidates for the development of anticancer drugs as well as cis-
platin compounds [10].
Besides agents interacting with DNA covalently, a broad range of potentially genotoxic species
exhibit non-covalent DNA interactions. Intercalation and groove binding are two broad classes
of non-covalent DNA binding mode. Intercalators bind to DNA by inserting a planar aromatic
chromophore between adjacent DNA base pairs, whereas groove binders fit into the DNA
grooves causing a little perturbation of the DNA Structure [11].
Copper derivatives have been found capable to interact with DNA double helix non-covalently
rather than forming coordinated covalent adducts. Moreover, DNA-binding affinities of copper
complexes can be finely tuned by introducing intercalative ligand substituents capable of π−π
stacking [12]. However, DNA binding is dependent on the complex size, electron affinity and
geometry of the formed adduct, inducing an irreversible modification of the DNA
conformational structure. A large number of copper complexes has been and is still being tested
as DNA-targeting metallo-drugs [13].
Furthermore, with increasing of microbial resistance to current antibiotics, there is continues
demand for developing new antimicrobial agents. In this way, many Cu(II) complexes have been
reported as more effective antimicrobial agents [14].
Herein, we are going to focus on the preparation, structural characterization and DFT calculation
of two new mixed-ligand Cu(II) complexes, [Cu(L)(phen)](1), [Cu(L)(bipy)] (2), [HL= (E)-N'-
(
2-hydroxy-3-methoxybenzylidene)benzohydrazide , phen= 1,10 phenanthroline, bipy= 2,2’
bipyridine), as potent biological compounds. Computational studies were performed using
3

DFT/B3LYP. Furthermore the interaction between these complexes and Salmon sperm double
stranded DNA (dsDNA), were also studied by spectroscopic methods.
2
2
. Experimental
.1 Materials and instrumentation
All chemicals were of analytical grade and used as received. Elemental analyses were carried out
using a Thermo Finnigan Flash Elemental Analyzer 1112EA. IR spectra were recorded with a
FT-IR 8400-SHIMADZU spectrophotometer. Conductance measurements were made by means
of a Metrohm 712 Conductometer in DMSO. The electronic spectra were recorded in DMSO on
a SHIMADZU model 2550 UV–Vis spectrophotometer. [HL] have been prepared according to
our previous report [2].
2
.2. DNA binding studies
Salmon sperm double stranded DNA (dsDNA) was purchased from Sigma (St. Louis, USA). A
.0 mg/mL stock solution of the DNA was prepared in Tris–HCl (TE) buffer (pH 7.4) and kept
frozen. The concentration of DNA was determined according to the absorbance at 260 nm. The
1
−
1
−1
molar extinction coefficient (ε260) was found to be 6600 L mol cm (per P or nucleotide unit)
[
15]. Ethidium bromide (EB) was purchased from CinnaGen (Tehran, Iran).
-3
Stock solution of each synthesized complex with the concentration of 2.0×10 M was prepared
with dissolving appropriate amount of each complex in DMSO.
2
.3. Instrumentation
Electronic spectra were recorded on a double beam UV-Vis-NIR Varian Cary 500
spectrophotometer (Victoria, Australia). Fluorescence measurements were performed on a Cary
4

Eclipse fluorescence spectrophotometer. All voltammograms were obtained using an Autolab
PGSTAT 302 electrochemical system from Metrohm (Herisau, Switzerland) interfaced with a
personal computer for data acquisition and potential control. A three-electrode assembly was
employed composed of Ag/AgCl electrode as reference electrode, a platinum wire counter
electrode and a glassy carbon electrode as the working electrode.
2
.3.1. UV-Vis absorption studies
Absorption titrations of the complexes with salmon sperm DNA were carried out using fixed
-5
concentrations of the complexes (4.5×10 M) in 0.01 M PBS (pH=7.4) while the concentration
-4
-4
of the DNA was gradually increased (1.3×10 -4.0×10 M). The intrinsic binding constants (K )
b
of the complexes with the DNA were calculated using UV-Vis spectra of the compounds
recorded in the presence of different concentrations of the DNA.
For the titration of the DNA with the complexes, the electronic spectra of the salmon sperm
-4
DNA with the constant concentration of 4.4×10 M in 0.01 M PBS were recorded in the absence
and presence of different amounts of each complex. All experiments were carried out at room
temperature. In all experiments, the incubation time for the DNA and the complexes to
equilibrate was 10 min.
2
.3.2. Competitive fluorescence studies with DNA-EB adduct
In these experiments, different amounts of each copper complex were added to EB–DNA adduct
-5
-5
in PBS containing 1.0×10 M EB and 7.5×10 M DNA. Emission spectra of DNA-EB were
recorded in the range of 550–700 nm upon excitation at 525 nm while maintaining the
concentration of DNA-EB constant and varying those of the complexes.
5

2
.3.3. Thermal denaturation experiments
-5
In these studies, 8.0×10 M salmon sperm dsDNA was thermally denatured into single-stranded
-5
components in the absence and presence of each complex (5.5×10 M). At the same time, the
absorbance of the DNA at 259 nm was monitored at various temperatures. The temperature of
the sample was controlled with a thermocouple attached to the sample holder. The temperature
was varied from 25 to 95 °C with a rate of 2 °C/min and the melting temperature (T
was determined as the transition midpoint.
m
) of DNA
2
.3.4. Cyclic voltammetry measurements
Using cyclic voltammetry (CV), the electrochemical behavior of each complex was studied
before and after the addition of DNA by taking a fixed concentration of the complex in 0.1 M
acetate buffer at different scan rates (20-300 mV/s). The mixed complex–DNA solution was
allowed to equilibrate for 10 min at room temperature.
2
.4. Determination of antimicrobial activity
To study the antimicrobial effect of [HL], [Cu(L)(phen)] and[Cu(L)(bipy)], five standard
bacteria (Pseudomonas aeruginosa ATCC 27853; Staphylococcus aureus PTCC 1112;
Micrococcus luteus PTCC 1110, Bacillus cereus PTCC 1015 and Escherichia coli PTCC 1330),
three bacteria isolated from clinical samples (Pseudomonas sp; Klebsiella sp and Eterococcus
faecalis ) and one yeast (Candida albicans PTCC 5027) were used. The antimicrobial effects of
the compounds on the microorganisms were assayed by Agar well diffusion method. Brain Heart
Infusion medium (BHI-Merck) for Eterococcus faecalis, YGC medium (Merck) for yeast strain
6

and Mueller Hinton medium (Merck) for the others were used as test medium. Also, the
minimum inhibitory concentrations (MIC) and minimum lethal concentration (MBC) of the
compounds were determined by micro broth dilutions technique strictly following the National
Committee for Clinical Laboratory Standards (NCCLS) recommendations [16]. The inoculum
was prepared using an overnight culture of each bacterium and yeast strains (18-24h) adjusted to
a turbidity equivalent to a 0.5 McFarland standard [17].
Petriplates containing 20 ml Agar test plates were seeded with microorganism strains. Wells
were cut and 50 µl of the compounds (50 mg/ml; DMSO was used as solvent) were added. The
plates were then incubated at 37 °C for 24-48 h. Antibacterial activity was assayed by measuring
the diameter of the inhibition zone formed around the well. DMSO was used as a negative
control whereas media containing ciprofloxacin (standard antibiotic) and fluconazole (standard
antifungal drug) were used as the positive controls. The experiments were performed in
triplicate. Serial dilutions ranging from 100 mg/mL to 0.39 mg/mL were prepared in medium.
2
.5. The biofilm formation assessment
For biofilm formation, Microtiter plate adhesion assay was employed: Biofilm formation in this
assay was determined by the method reported earlier [18]. Staphylococcus aureus PTCC 1431
was used as positive control and the microbial medium without microorganism was used as
negative control.
2
.6. Synthesis of (2-hydroxy-3-methoxybenzylidene)benzoydrazide [HL]
7

A mixture of benzohydrazide (0.14 g, 1 mmol) and 3-methoxy-2-hydroxy benzaldehyde (0.15 g,
mmol) was refluxed in 10 ml methanol. After 1 h, the resulted precipitate was filtered, washed
with cold ethanol and dried in vacuum over anhydrous CaCl
Yield: 0.22 g, 83%. m.p.: 116 °C. Anal. Calc. for C15 O (270.28 g mol ): C, 66.66; H,
1
2
.
-1
H
14
N
2
3
-
1
5
3
1
.22; N, 10.36. Found: C, 66.18; H, 5.30; N, 10.53%. FT-IR (KBr), cm : ν(NH) 3571, ν(OH)
367, ν(CHar) 2839-3062, ν(C=O) 1647, ν(C=N) 1610, ν(C=Cring) 1470, ν(N-N) 1149, ν(C-O)
-
1
-1
296. UV/Vis (DMSO), λmax, nm (log(ε), L mol cm ): 307(4.61), 339(4.24), 423(2.90).
2
.7. Synthesis of (1,10-phenanthroline) (3-methoxy-2-oxidobenzylidene)benzohydrazonato
copper(II) [Cu(L)(phen)] (1)
Cu(OAc)
mmol) with constant stirring. Immediately after adding of 1, 10 phenanthroline (0.02 g,
.1mmol) to the reaction, the solution color turned back to dark green. The mixture was refluxed
on an oil bath for ca 1 h. After cooling, the resulted green precipitate was separated by filtration,
washed with cold methanol and dried in a desiccator over anhydrous CaCl . A suitable single
2
.4H
2
O (0.1 mmol, 0.02 g) was added dropwise to 5 ml solution of HL (0.02 g, 0.1
0
2
crystal for X-ray determination was obtained in mother liquor with slow evaporation after 6
days.
-1
2
-
Yield: 0.031 g, 61%. m.p.: >300 °C. Molar conductivity (1.0×10-3 M, DMSO): 31.7 Ω cm mol
1
-1
.
Anal. Calc. for C27
H20CuN
4
O (512.02 g mol ): C, 63.34; H, 3.94; N, 10.94. Found: C,63.05;
3
-1
H, 3.88; N 11.01, %. FT-IR (KBr), cm : ν(CH)ar 2824-2924, ν(C=N) 1595, ν(C=Cring) 1501,
-1
-1
ν(C-O) 1314, ν(N-N) 1163. UV/Vis (DMSO), λmax, nm (log(ε), L mol cm ): 324(4.64),
3
35(4.65), 348(4.58), 414(4.48),655(2.20).
8

2
.8. Synthesis of (2,2’-bipyridine) (3-methoxy-2-oxidobenzylidene)benzohydrazonato
copper(II) [Cu(L)(bipy)] (2)
Preparation of this compound is similar to that of 1. The difference is just in the use of 2,2’
bipyridine (0.1 mmol, 0.02 g) instead of 1,10 phenanthroline as heterocyclic aromatic base. X-
ray quality deep-brown single crystals were obtained by slow evaporation of the mother
liquor of this compound after 4 days.
-3
-1
2
-
Yield: 0.035 g, 72%. m.p.: >300 °C. Molar conductivity (1.0×10 M, DMSO): 30.2 Ω cm mol
1
-1
.
Anal. Calc. for C25
H20CuN
4
O (488.00 g mol ): C, 61.43; H, 4.13; N, 11.48. Found: C, 61.28;
3
-1
H, 4.30; N, 11.36%. FT-IR (KBr), cm : ν(CH)ar 2928-3099, ν(C=N) 1599, ν(C=Cring) 1501, ν(C-
-1
-1
O) 1300, ν(N-N) 1159. UV/Vis (DMSO), λmax, nm (log(ε), L mol cm ): 323(4.57), 334 (4.62),
3
47(4.56), 410(4.48), 623(2.28).
2
.9. Crystal structure determination
Single-crystal X-ray diffraction data for both complexes were collected at 294(2) K, on an
Oxford Diffraction CCD diffractometer using Mo-Kα radiation (λ = 0.71073 Å) with the ω scan
technique. An empirical absorption correction was applied to the raw intensities [19].
The structures were solved by direct methods (SHELXS-97) [20] and refined with full-matrix
2
least-squares technique on F using SHELXL-97 [20]. In structure (1) crystals were ill formed
and weakly diffracting. Even the crystal used for this report, the best of a large number of
samples analyzed, provided a poor data set (Rint = 11.5%), showing a generalized disorder
which refinement could only be accomplished with severe rigid body restraints, relaxed in the
final run in order to obtain the esd’s for all structural parameters. However, and in spite of the
9

large R value obtained, the final model appears to be satisfactory. In structure (2), the late
difference Fourier maps showed a peak very near the cation, which probably correspond to the
Cu cation of a small (0.03%), slightly miss-oriented chip. Its inclusion reduced the R factor from
−
.049 to 0.038 and the highest peak in the difference map, from 1.67 to 0.39 eÅ . Hydrogen
3
0
atoms were added at expected positions, riding in coordinates and thermal factors on their host
atoms.
2
.10. Computational
Full geometry optimization of the complexes in ground state (S ) was carried out using DFT-
0
B3LYP with 6-311+G(d,p) basis set for C, N, O, H and LANL2DZ for the Cu atom using the
G03 program [21]. Crystal structures used as starting point for DFT calculations. To validate the
optimization of the structures, frequency calculations were performed and the results showed no
negative (imaginary) frequency. Furthermore, determination of the origin of structural behaviors
was been done with Natural Band Orbital (NBO) analysis at the same level. Natural bond orbital
(
NBO) calculations were performed with the NBO code included in GAUSSIAN03 [22].
.
3 Results and discussion
Two new mixed-ligand copper(II) complexes were prepared. The complexes were stable in air
and were obtained as green crystals. They were soluble in DMSO and DMF and insoluble in n-
hexane and diethyl ether. These compounds possessed less solubility in other common solvents
like methanol, chloroform and acetonitrile. Molar conductivity values of complexes were in the
-1
2
-1
range 30.2-31.7 ohm cm mol in DMSO, indicating non-electrolyte behaviors of them.
10

Schematic diagram for the synthesis of both complexes are shown in Fig. 1. As can be seen, HL
has two main tatumeric forms that enol form contributes in the complexation.
3
3
.1 Spectral characterizations
.1.1. FT-IR
-1
Assignments of selected prominent IR bands in 400–4000 cm region for HL and its Cu(II)
complexes are listed in the experimental section. In the FT-IR spectrum of the ligand, the
-1
vibrations of NH and OH appear at 3571 and 3373 cm , respectively. Moreover, the observation
-1
of a sharp band with high intensity at 1678 cm indicates that HL is in the keto form [23]. In the
IR spectra of both complexes, the absence of these peaks is in agreement with the coordination
of HL to metal ion through deprotonated phenolic and aliphatic oxygen atoms in enolic form that
-1
-1
is also supported by the red shift of the C-O band from 1332 cm to 1314 and 1300 cm for 1
and 2, respectively [24]. The strong bond of CN moiety in the complexes assigns at 1595-1599
-1
cm which has been shifted towards lower wave numbers compared with related value in the
free ligand indicating the coordination of the azomethine nitrogen to central atom [25].
3
.1.2. Electronic spectra
The electronic spectra of title compounds were recorded in DMSO solution. In the UV-Vis
spectra of them, the bands in the region of 323-324 nm and 334-348 nm are assigned to π → π*
and n→ π* of the hydrazone and aromatic heterocyclic ligands (phen and bipy). For both
complexes, π→π* and n→π* transition showed hypsochromic shifts compared with
uncomplexed ONO ligand indicating the enolization followed with deprotonation of ligand
during the complaxation [26]. The bands appeared at about 410 nm may be due to ligand-to-
11

metal charge transfer (LMCT) transition. In a five coordinated Cu(II) complexes visible
spectrum can be used for predicting preferred geometry. SP or distorted SP geometries exhibit a
band in the 550-660 nm range that is assigned to d ,d → d
while TBP structures have a
xz
yz
2
2
x − y
characteristic band around >800 nm is related to d ,d
→ d . Both complexes showed d-d
z
xz
2
2
2
x − y
transition in the range of 623-655 nm consistent to with SP geometry [27].
3
.2 X-ray crystal structures
Full details of crystallographic data and structure refinement parameters can be found in Table 1,
while Table 2 provides coordination parameters for both structures.
Inspection of the monomeric units of (1) and (2) shown in Fig. 2 discloses the overall
resemblance of both structures, while superposition of both molecules through the L.S. fitting of
their cation environments (Fig. S1) suggests their differences, the most significant one residing
perhaps in the different bending of the two lateral “wings” of the L ligand, their L.S.
planes defining dihedral angles of 19.1(2)º in (1) and 13.0(1)º in (2). The more obvious similarity
resides in both (mostly planar) ligands binding almost perpendicular to each other (angles
2
between mean planes, 99.8(3) and 99.2(2)º, respectively) via two nitrogens (for the κ N,N’-
3
phen/bipy ) and two oxygens plus one nitrogen for (κ N,O,O’-L). The CuN
3
2
O cores thus
formed are better described as deformed square pyramids, as suggested by the τ parameters [28]
τ = 0.0 for a Square Pyramid (SP); 1.0 for Trigonal Bipyramid (TBP) ). This descriptor amounts
.36 for (1), suggesting a considerable TBP deformation, and 0.07 for (2), describing a more
clear SP geometry.
(
0
12

The basal plane in both coordination polyhedra are defined by all three L-donors and one of the
phen/bipy N’s. The Cu-O/N bond lengths for the L-donors are rather even, with spans of
1
.930(4)-1.966(5) Ǻ (1) and 1.925(2).1.960(2) Å (2). A larger difference, though still within
usual values, in found for the basal Cu-Nphen (1.972 (6) Å) as compared with the equivalent Cu-
bipy ( 2.052 (2) Å). As suggested by the τ parameters, the planarity of the basal group is sensibly
N
better in (2), with a RMS deviations from the L.S. plane of 0.043 Å as compared with 0.188 Å
for (1). The apical site is in turn occupied by the remaining Nphen/bipy donor, at a significant larger
distance (2.238(6) and 2.236(2) Å, respectively) consistent with the expected Jahn–Teller
deformation along the apical axis. The copper(II) ions depart from the L.S. basal plane by 0.180
Å in (1) and 0.204 Å in (2), towards the apical site.
As expected from the type of the available H-bonding donors, there are no strong intermolecular
interactions in none of the structures; the only ones present being either of π...π type (Table 3),
or C-H...O/N, C-H...π (Table 4).
For simplicity, we shall use in what follows the shorthand notation Tn-Em to mean Table n, mth
Entry. There is a single π...π interaction in each structure, and in both of them the heterocyclic
ligands are involved, though with different effects. The one in structure (1), (T3-E1) links
molecules related by a c glide plane, generating “chain-like” substructures oriented along "c"
(
Fig. 3 ).
In the case of (2) the π...π bond (T3-E2 ) connects centrosymmetrically related molecules to
generate isolated dimeric entities, reinforced by three intradimeric C-H...O contacts (T4-E4,5,6
and Fig. 4).
The remaining interactions (C-H..π bonds in (1) (T4-E1,2, 3) or C-H...N and C-H…π bonds in
(
2) (T4-E7,8) just serve to interconnect these elemental units into weakly bound 3D structures.
13

3
3
.3 Computational
.3.1. Geometry optimization
The optimizations of ground state of both complexes were performed with G03 using
DFT/B3LYP at 6-311+G(d,p) level of theory. Lack of imaginary vibration in frequency
calculation indicates that optimized structures are at global minima. Furthermore, the computed
global minimum energies are -3126.01 and -3049.78 a.u. for 1 and 2, respectively. Table 2 shows
the selected optimized bond length and angles for [Cu(L)(phen)] and [Cu(L)(bipy)] complexes
along with experimental data. The difference observed between calculated and experimental data
refers to this fact that theoretical calculations are performed for isolated molecule in gaseous
phase, while experimental results belong to solid state in which lattice interactions could be
effective in the structure formation.
3
.3.2. Electronic structure
Frontier molecular orbitals (FMO) have an important role for determination of chemical
reactivity of compounds. The HOMO and LUMO contours for [Cu(L)(phen)] and [Cu(L)(bipy)]
are presented in Fig. 5. HOMO largely acts as an electron donor while LUMO acts as an electron
acceptor. The gap between HOMO and LUMO determines molecular chemical stability.
Compounds with higher energy gap have a lower chemical reactivity. In both complexes,
HOMO is localized on Schiff base ligand while LUMO is centered on heterocyclic aromatic
bases (phen and bipy for 1 and 2, respectively). The molecular electrostatic potential is related to
electron density and has an important role for identifying sites for electrophilic attack and
nucleophilic reactions. It can also be used for describing many structural properties of the
molecule such as chemical reactivity and dipole moment. The MEP diagram for both complexes
14

were calculated by DFT/6-311+G(d,p) method. In Fig. 6, electrophilic sites (negative region) are
shown by red color while blue color indicates electron deficient regions (positive region).
Aromatic rings, especially heterocyclic bases, are suitable electrophilic sites while the oxygen of
methoxy moiety and nitrogen of hydrazine are potential negative sites. These regions confirm the
existence of molecular interactions in compounds.
3
.3.3. Thermodynamic properties
0
m
0
m
0
p,m
On the basis of vibrational analysis, enthalpy(H ), entropy (S ) and heat capacity (C ) as
standard thermodynamic functions for both complexes were obtained at different temperatures at
B3LYP/6-311+G(d,p) level of theory and are shown in Table S1. According to it, these
thermodynamic values increase with increasing temperature from 200 K to 700 K. This
phenomenon is in accordance with this fact that vibrational intensities increase with temperature.
The correlation equations between enthalpy, entropy, heat capacity and temperature for 1 and 2
are shown in the following equations [1-6].
2
H
m
° [1] = 0.0001T + 0.0318T + 258.71 (R² = 0.9999)
-5 2
S
m
°[1] = -9×10 T + 0.4385T + 72.834 (R² = 1)
2
C
p,m° [1]= -0.0002T + 0.485T - 10.651 (R² = 0.9999)
2
H
m
° [2] = 0.0001T + 0.0315T + 250.91 (R² = 0.9999)
-5 2
S °[2] = --9×10 T + 0.425T + 74.3 (R=1)
m
°
2
C
p,m [2]=-0.0002T +0.4583T-8.0694 (R² = 0.9998)
15

3
.4. Electronic absorption titrations
UV-Vis spectroscopic titration is an effective method that provides valuable information on the
modes of interaction. A hypochromism or hyperchromism and a hypsochrimism or
bathochromism often occur in the absorption spectrum of a metal complex when the complex is
bound to DNA.
3
.4.1. Titration of the complexes with salmon sperm DNA
-5
At first, the electronic spectra of (1) and (2) having the same concentration (4.5 × 10 M) have
been recorded separately in the range of 250–500 nm. Fig. 7 represents the spectra obtained for 2
(
A) and 1 (B) in the absence and presence of increasing amounts of the DNA [30-32].
4
As seen, the electronic spectrum of 2 shows a sharp absorption peak at 321.0 nm with ε=2.1×10
-1
-1
3
-1 -1
cm M (peak I) and a broad absorption peak at about 384.0 nm with ε=9.9×10 cm M (peak
4
-1 -1
II). For 1, a sharp absorption peak at 266.0 nm with ε=3.7×10 cm M (peak I), a peak at 320.0
4
-1 -1
nm with ε=2.0×10 cm M (peak II) and a broad absorption peak at about 389.0 nm with
3
-1 -1
ε=9.1×10 cm M (peak III) were observed. These observed intense absorption band scan be
attributed to the intra-ligand transitions of the coordinated ligands [33].
-4
-4
With the addition of increasing amounts of DNA (1.3×10 -6.7×10 M) on the fixed
concentration of 2, absorption peaks I and II show 2-nm shifts towards larger wavelength region
accompanied by hypochromism of ~18% and ~28%, respectively. Generally, hypochromism
accompanied by a red shift (bathochromism) is known to be a characteristic of the strong π– π
stacking interaction between the aromatic chromophore ligand of a metal complex and the
aromatic rings of DNA bases which is called intercalation interaction. The π* orbital of the
intercalated ligand can couple with the π orbital of the DNA base pairs, resulted in decrease of
16

the energy gap between the π and π* orbitals and subsequently, bathochromism is observed. The
coupling of π orbitals which are also partially filled by electrons reduces the transition
probability and thus results in hypochromism [34]. In the case of groove binding, sometimes a
hypochromism that is manifested in terms of a small decline in absorbance and either no or only
minor changes in λmax are observed [35, 36].
Upon titration with DNA, the spectral changes of [Cu(L)(bipy)] are characterized by one
isobestic point, at ~305 nm. Isobestic point indicates an equilibrium between DNA-bound and
free forms of the complex and suggests only one mode of binding (here intercalative mode) with
DNA [37, 38].
-4
-4
For (1) solution titrated with the DNA (1.3×10 -4.0×10 M), the band III presents a
hypochromism of up to 39% accompanied by a spectral change of 2-nm to higher energies,
suggesting tight binding probably by intercalation [39]. The same spectral shift is observed for
the band I but with ~14% decrease in its absorbance value (hypochromism). These
hypochromism effects suggest the intercalation of this compound into the DNA base pairs and
this may probably be due to the interaction of the electronic states of the intercalating
chromophore of the compound and those of the stacked base pairs of the DNA [40].
Binding constant values (K ) are used to assess the stability of compound–DNA adducts and can
b
be determined from the variation in the electronic spectra before and after the addition of DNA
by applying Benesi-Hildebrand equation [41, 42] given below:
Where A
presence of DNA, ε
respectively. From the plot of A
0
is the initial absorbance of free complex, A is the absorbance of the complex in the
and ε are molar extinction coefficients of the free and fully bound complex,
/(A-A ) versus 1/[DNA], the ratio of the intercept to the slope
f
b
0
0
17

4
4
gives the value of K . The binding constants of 2 and 1 are calculated to be 2.4×10 and 1.0×10
b
-1
M , respectively. [Cu(L)(bipy)] shows comparatively greater binding constant indicating
stronger affinity of 2 for DNA.
This may be attributed to its structural planarity or additional hydrophobic interactions with the
nucleotide bases of DNA or the presence of extended aromatic system which may bind strongly
with DNA.
3
.4.2. Titration of salmon sperm DNA with the complexes
As shown in Fig. 8, the absorption spectrum of salmon sperm DNA shows a broad band with a
maximum placed at about 259 nm due to the chromophoric groups in purine and pyrimidine
moieties of the DNA. The electronic spectra of the DNA obtained after titration with compound
(
2) is given in Fig. 8A. As it is evident, titration of the DNA solution with [Cu(L)(bipy)] slightly
increases the absorbance value of the DNA with a considerable red shift of 6 nm in its position
which indicates the interaction between this complex and the DNA. Furthermore, an isobestic
point at about 311 nm is observed in the spectroscopic titration curves.
The results of a similar experiment carried out for [Cu(L)(phen)] are given in Fig. 8B. As one
can see, addition of the complex significantly increases the absorption peak of the DNA (~17%
hyperchromisity) with a red shift of 5.0 nm.
From the obtained results, it can be concluded that both complexes may intercalate between two
strands of the DNA thereby increasing the absorption of DNA due to the unwinding of DNA
double helical. In the other words, interaction with the complexes causes conformational changes
in the DNA structure which made purine and pyrimidine bases be exposed [43].
18

Since the increase of DNA absorbance after interaction with [Cu(L)(phen)] is greater than that
observed after interaction with [Cu(L)(bipy)], one can conclude that (1) causes more
conformational changes in the DNA structure.
3
.5. Thermal denaturation studies
Fig. 9. reveals the plots of A /A (A and A are the absorbance of the DNA at 25˚C and applied
0
0
temperature, respectively) versus temperature (˚C) for salmon sperm DNA (a), the DNA
incubated with 2 (b) and the DNA incubated with 1 (c). As one can see, both complexes stabilize
the DNA helix and so increase its melting temperature (T
the values of T are obtained to be 67.5˚C for the DNA, 73.5˚C for the DNA in the presence of
2) and 74.5˚C for the DNA in the presence of (1).
It is believed that an intercalative mode of binding generally increases the stability of DNA helix
44], and so, increase T by about 5–8°C, while non-intercalative binding causes no obvious
increase in T [45]. Accordingly, the increases observed in T (6.0˚C and 7.0˚C respectively for
Cu(L)(bipy)] and [Cu(L)(phen)]) propose an intercalative mode for 2- and 1-DNA interaction.
m
). Under our experimental condition,
m
(
[
m
m
m
[
The structure stability of the DNA caused by [Cu(L)(phen)] is somewhat greater than that caused
by [Cu(L)(bipy)], which is consistent with the results of the DNA titration.
3
.6. Competitive binding experiments in the presence of ethidium bromide
The competitive studies of each complex with ethidium bromide (EB) have been investigated
with fluorescence spectroscopy in order to examine whether the compound can displace EB from
the DNA helix. EB is a cationic conjugated planar molecule which is well known as a DNA
intercalator. The fluorescence intensity of EB is usually weak. However, it increases upon the
19

addition of DNA. So, EB has been employed as a probe for the spectroscopic study of the
interaction between dsDNA and potent intercalating species because of 24-fold decrease in its
fluorescence when displaced from an intercalation site [46].
The Cu(II) complexes have been compared with EB using competitive binding studies. In these
studies, varying amounts of each complex are added to a DNA solution incubated with EB and
after a given time of equilibration, the fluorescence spectrum of the DNA-EB system is recorded.
The obtained results are presented in Fig. 10.
As can be seen, the fluorescence of DNA-EB adduct is quenched by both complexes and this
effect increases with increasing the concentration of the complex. This quenching indicates that
the complexes can drive out EB and take its place in the DNA double helix. Since EB
intercalates into DNA through interactions with the minor groove, an intercalative or minor
groove binding of the complexes is suggested [47, 48].
One can analyze the obtained quenching data and make a comparison between two complexes
using Stern-Volmer equation [49]:
Where F
0
and F are the fluorescence intensities in the absence and presence of the quencher or
complex (Q), respectively. KSV is Stern–Volmer constant or quenching constant which can be
obtained from the slope of the plot of F /F versus [Q] (Fig. 10, insets). Accordingly, the values of
0
4
4
-1
K
SV for 2 and 1 are 1.70×10 and 1.50×10 M , respectively indicating that [Cu(L)(bipy)] has a
higher quenching efficiency and a higher binding affinity to DNA.
The fluorescence quenching curves reaches a plateau, which allows determining the molar ratio
of each complex to EB needed to drive out maximum amount of EB from DNA helix. For both
complexes, this calculated ratio is less than 2 (about 1.8).
20

3
.7. Electrochemical characterization of the interaction between DNA and the complexes
The investigation of the interaction of DNA and electroactive compounds with electrochemical
techniques is a useful complement to spectroscopic methods and may yield information about
interactions with both reduced and oxidized form of the compound.
Cyclic voltammograms of fixed concentrations of the complexes were recorded in the absence
and presence of salmon sperm DNA. The cyclic voltammograms of both [Cu(L)(bipy)] and
[
Cu(L)(phen)] in the absence of DNA exhibit quasi-reversible single electron redox process
assignable to the Cu(II)/Cu(I) couple. The electrochemical characteristics of the complexes
including anodic and cathodic peak potentials (Epa and Epc, respectively), peak separation (∆E ),
formal potential ( and anodic and cathodic peak currents (Ipa and Ipc, respectively) extracted
from the voltammograms, are reported in Table 5. ∆E and are defined as the difference and
p
p
the average of Epa and Epc, respectively. As mentioned previously, the electrochemical behavior
of the copper complexes depends strongly on the structure of coordinated ligands [50]. So, these
values are different to some extent from 1 to 2.
After addition of the DNA, the shifts in the values of ∆E
the complexes with DNA. For both complexes Epa shows a positive shift while Epc shows a
negative shift resulted in the increase observed in ∆E (26% for 1 and 33% for 2). The increase
p
and
explain the binding ability of
p
of the formal potentials of the complexes accompanied by the decrease of the peak currents
observed in the presence of the DNA suggests that the complexes have interacted with the DNA
in an intercalating way. Generally, if the interaction mode is of an electrostatic binding nature,
the formal potential shifts to a more negative value, while intercalation binding results in a
formal potential shift to a more positive value [51-53].
21

Overall, the higher decrease of the peak currents observed for [Cu(L)(bipy)] compared to
Cu(L)(phen)] upon addition of DNA suggests that the binding affinity of the former to DNA is
[
stronger.
In addition, the net shift in the formal potential can be used to estimate the ratio of equilibrium
constants for the binding of the oxidized and reduced forms associated with a third species
(
DNA) in the solution using the following equation [54]:
where
and
are the formal potentials of Cu(II)/Cu(I) couple in the DNA-bound and free
complexes, respectively.
and
are the binding constants for the binding of the oxidized
and reduced forms to DNA.
The ratios of the constants for [Cu(L)(phen)] and [Cu(L)(bipy)] are found to be 1.9 and 1.6,
respectively. These values indicate that for both complexes the binding affinity of the Cu(II)-
containing complex is higher than that of the Cu(I)-containing complex which may be due to the
repulsion between the negative net charge of the reduced forms of the complexes and negative
charge of the phosphate backbone of the DNA.
Useful information can generally be acquired from the relationship between peak current and
scan rate. Therefore, the electrochemical behavior of each complex in the absence and presence
of salmon sperm DNA have been separately examined at scan rates varying from 20 to 300
mV/s. For example, the voltammograms obtained for (1) were depicted in Fig. S2. It can be seen
that with the increase of scan rate, the redox peak currents of (1) in the absence (Fig. S2A) and
presence (Fig. S2B) of the DNA increase simultaneously but the redox peak potentials show no
considerable shift.
22

As shown in Fig. 13, in the absence of DNA linear relationships between the square root of scan
rate (ν) and both anodic and cathodic peak currents of [Cu(L)(phen)] are observed indicating that
the electrode process is mainly controlled by diffusion mechanism [55]. The equations of Ipa
1
/2
−1
2
1/2
−1
(
µA)=0.0591ν
(mV s )+0.784; R =0.9978 and Ipc (µA)=-0.0534ν
(mV s )+0.1854;
2
R =0.9992, correspond to anodic and cathodic waves, respectively.
Addition of DNA decreases the redox peak current of [Cu(L)(phen)] at all scan rates, and at the
same time, shifts the anodic and cathodic peak potentials to more positive and negative values,
1
/2
respectively. The corresponding linear regression equations are Ipa (µA)=0.0230ν
(mV
−
1
2
1/2
−1
2
s )+0.0601; R =0.9910 and Ipc (µA)=-0.0225ν (mV s )+0.0746; R =0.9939.
As it is evident, the slopes of the lines obtained without DNA are greater than those obtained
with the DNA, suggesting that the diffusion coefficient of the free form of [Cu(L)(phen)] is
larger than that of the bound form of (1) to DNA. Accordingly, the binding of (1) to bulky,
slowly diffusing DNA which decreases the electron transport ability of (1), is suggested.
Moreover, this interaction decreases the apparent diffusion coefficient of (1) which, in turn,
decreases its concentration near the electrode surface and subsequent oxidation signal [56, 57].
3
.8. Antimicrobial activity
The antibacterial activities of [HL], [Cu(L)(phen)] and [Cu(L)(bipy)] against Pseudomonas sp;
Klebsiella sp, Eterococcus faecalis, isolated from clinical sample, Pseudomonas aeruginosa
ATCC 27853, Staphylococcus aureus PTCC 1112, Micrococcus luteus PTCC 1110, Bacillus
cereus PTCC 1015, Escherichia coli PTCC 1330, and Candida albicans PTCC 5027 were
assessed by evaluating the presence of inhibition zone (IZ), MIC and MBC values. Results
(
Table 6) showed that [Cu(L)(phen)] had a great potential of antibacterial activity; whereas,
23

[
Cu(L)(bipy)] and [HL] did not show any antimicrobial activities. It seems that the antibacterial
effect of [Cu(L)(phen)] is not only on Gram positive, but also on negative bacteria. In addition, it
was effective on Candida albicans PTCC 5027. However, according to the obtained results, the
bacteria which were isolated from clinical samples, were resistant to [Cu(L)(phen)] and
antibiotic (Ciprofloxacin).
The results corresponding to MIC and MBC are shown in Table 7. As can be seen, the MIC and
MBC values for [Cu(L)(phen)] were in the range of 100 mg/mL to 0.39 mg/mL. Antimicrobial
results showed that (1) is a potent antimicrobial agent which can inhibit the growth of
microorganisms as well as killing them.
3
.9. Biofilm formation
Biofilms are microbial communities attached to surfaces, and encased in an extracellular matrix
of microbial origin. In almost all instances, the biofilm lifestyle helps bacteria survive, and
persist within the environment. They are a major cause of human infections. The majority of
hospital-acquired infections are caused by biofilms, because they can be life-threatening
colonizers of biomedical devices. Indeed, a large proportion of nosocomial infections are related
to the colonization of pathogens on the surface of implanted medical devices, such as respirators,
catheters (central venous, urinary), prosthetic heart valves, and orthopedic devices. In industry,
biofilms cause food and drinking water contamination, metal surface corrosion, and clogging.
Due to the possibility of water contaminated with pathogens, biofilms present a serious hazard to
the drinking supply [58]. The development of a microbial biofilm can be described as a dynamic
process, involving successive steps. The first step is to attach the bacterial cells to the selected
abiotic or biotic surface. The second corresponds to the development of micro-colonies promoted
24

by the growth and division of the first attached cells (primary colonizers). The micro-colonies
progressively enlarge and coalesce to form the first layer of cells covering the surface. When
multiple layers of cells pile up on the surface, the third step of the formation is obtained,
indicated by the presence of a mature biofilm characterized by the presence of macro-colonies
surrounded by water channels that help distribute nutrients and signaling molecules. Finally, to
survive when lack of nutrients happens, or for simply spreading and colonizing to other niches,
some biofilm cells can detach individually or in clumps [59]. However, if biofilm formation
stops, we can control spread of infection. Therefore, in this study we have decided to investigate
the effect of the compounds on bacterial biofilm formation.
According to the biofilm formation results, we found that [Cu(L)(phen)] repressed biofilm
formation in P. aeruginosa ATCC 27853 (1.562 mg/mL), S. aureus PTCC 1112 (0.39 mg/mL)
and E. coli PTCC1330 (3.125 mg/mL) while it could not stop biofilm formation in M. luteus
PTCC 1110, B. cereus PTCC 1015 and C. albicans PTCC 5027. Also, the stop of biofilm
formation by [Cu(L)(phen)] occurred before minimum inhibitory concentration (Table 8).
4
. Conclusions
Two new mixed-ligand copper(II) complexes, [Cu(L)(phen)] and [Cu(L)(bipy)] have been
synthesized and fully characterized using various spectroscopic methods including single crystal
X-ray diffraction. Both complexes have SP geometry in which the positions around central atom
are occupied with donor atoms of hydrazone ligand, and nitrogens of heterocyclic aromatic
bases. For better definition, DFT calculations were done at B3LYP/6-311+G(d,p) level of theory
to predict the structural geometry and interpret the thermodynamic properties. DNA binding
activities of these complexes have been investigated with electronic absorption, competitive
25

fluorescence titration and cyclic voltammetry techniques. Accordingly, the interaction between
these complexes and DNA is of intercalative mode. Antimicrobial studies show that
[
Cu(L)(phen)] is effective on various bacteria and yeast. It can also inhibit the growth of
microorganisms or even in some cases kill them, while [Cu(L)(bipy)] and the parent ligand,
HL], haven't shown any antimicrobial activities. Furthermore, [Cu(L)(phen)] was used for
biofilm formation in some bacteria.
[
5
. Supplementary data
Crystallographic data for the structure reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre, CCDC Nos. 1003263 (2) and 1003264 (1). Copies of
this information may be obtained from the Director, CCDC, 12 Union Road, Cambridge, CB2
1
EZ,
UK
(fax:
+44-1223-336033;
e-mail:
deposit@ccdc.cam.ac.uk
or
http://www.ccdc.cam.ac.uk).
Acknowledgements
The authors gratefully acknowledge the financial support provided for this work by Kerman
Cancer Research Center. Also we express appreciation to the Shahid Bahonar University of
Kerman.
26

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