Evaluation of DNA/Protein interactions and cytotoxic studies of copper(II) complexes incorporated with N, N donor ligands and terpyridine ligand

Article abstract of DOI:10.1016/j.ijbiomac.2016.11.022

A series of four new copper(II) heteroleptic complexes, [Cu(2?-pytpy) (L)] (NO₃)₂·2H₂O (1–4), where 2?-pytpy?=?4′-(2′′′-Pyridyl)-2, 2′:6′, 2′′-terpyridine, L?=?bipyridyl (bpy), 1, 10 phenanthroline(phen), dipyridoquinoxaline(dpq) and dipyridophenazine (dppz) were synthesized and characterized by spectroscopic techniques. Further, the molecular structure of the complex (2) was confirmed by single crystal X-ray diffraction technique and the data revealed a penta coordinated, distorted square-pyramidal geometry with triclinic system. The interactions of four complexes with calf thymus DNA and bovine serum albumin (BSA) were investigated by electronic absorption, fluorescence and circular dichroism spectroscopy techniques. Spectral studies substantiated an intercalative binding mode of metal complexes with ct-DNA. Significant binding interactions of the complexes with protein have been further revealed from fluorescence studies. Furthermore, all the four complexes show potential cytotoxicity towards the human liver carcinoma cell line (HepG-2).

Full text of DOI:10.1016/j.ijbiomac.2016.11.022

Accepted Manuscript
Title: Evaluation of DNA/Protein interactions and cytotoxic
studies of copper(II) complexes incorporated with N, N donor
ligands and terpyridine ligand
Author: Tummalapalli Kiran C.S. Vasavi Punnagai Munusami
Madhvesh Pathak M.M. Balamurali
PII:
S0141-8130(16)30969-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.11.022
BIOMAC 6709
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
23-7-2016
2-11-2016
3-11-2016
Please cite this article as: Tummalapalli Kiran, C.S.Vasavi, Punnagai Munusami,
Madhvesh Pathak, M.M.Balamurali, Evaluation of DNA/Protein interactions and
cytotoxic studies of copper(II) complexes incorporated with N, N donor ligands
and terpyridine ligand, International Journal of Biological Macromolecules
http://dx.doi.org/10.1016/j.ijbiomac.2016.11.022
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Evaluation of DNA/Protein interactions and cytotoxic studies of copper(II)
complexes incorporated with N, N donor ligands and terpyridine ligand
Tummalapalli Kirana, C. S Vasavib, Punnagai Munusamic, Madhvesh Pathak*a, Balamurali
M M †a
a
Department of Chemistry, School of Advanced Sciences, VIT University, Vellore, Tamilnadu 632014, India.
Bioinformatics Division, School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu 632 014,
b
India.
c
Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology,
Hyderabad 500 032, India.
* Corresponding author. Tel.: +91-416-2202553; E-mail: madhveshpathak@vit.ac.in (M. Pathak)
Co-corresponding author. Tel.: +91-416-2202321. E-mail: balamurali.mm@vit.ac.in (Balamurali M M)
†
Graphical abstract
A series of four new heteroleptic derivatives of copper(II) synthesized and structurally
characterized as a distorted square-pyramidal geometry around central metal. Significant
biological applications e.g. DNA interactions, protein binding, molecular docking, Density
Functional Theory (DFT) calculations and cytotoxicity of these complexes exhibited their noted
worth.

Highlights
 A series of four metallo-organic complexes of copper(II) have been synthesized.
 Physico-chemical characterization techniques established very well and penta coordinated,
distorted square-pyramidal geometry with triclinic system was confirmed by single crystal
X-ray diffraction technique.
 DNA and protein binding studies displayed the mode of binding in an elaborative fashion.

Molecular docking and Density Functional Theory (DFT) calculations are providing
additional essential features of these new derivatives

All the complexes have been carried out for cytotoxic studies with fruitful results
Abstract
A series of four new copper(II) heteroleptic complexes, [Cu(2‴-pytpy) (L)] (NO3)2.2H2O (1-4),
where 2‴-pytpy= 4′-(2′′′-Pyridyl)-2, 2′:6′, 2′′-terpyridine, L=bipyridyl (bpy), 1, 10
phenanthroline(phen), dipyridoquinoxaline(dpq) and dipyridophenazine (dppz) were synthesized
and characterized by spectroscopic techniques. Further, the molecular structure of the complex (2)
was confirmed by single crystal X-ray diffraction technique and the data revealed a penta
coordinated, distorted square-pyramidal geometry with triclinic system. The interactions of four
complexes with calf thymus DNA and bovine serum albumin (BSA) were investigated by
electronic absorption, fluorescence and circular dichroism spectroscopy techniques. Spectral
studies substantiated an intercalative binding mode of metal complexes with ct-DNA. Significant
binding interactions of the complexes with protein have been further revealed from fluorescence
studies. Furthermore, all the four complexes show potential cytotoxicity towards the human liver
carcinoma cell line (HepG-2).
Keywords: Copper(II) complexes, DNA and protein binding studies, Molecular docking, Density
Functional Theory (DFT) calculations and Cytotoxicity studies.

1. Introduction
Along the path towards innovative progress for the treatment of cancer, the metal-based organic
compounds are being intensively explored for the past several decades. Though cis-platin is
popularly known as an anti-cancer drug since 1978 [1-3], the antitumor activity of second
generation platinum complexes have been identified and thoroughly studied for their structure
activity relationships while designing drug molecules [4, 5]. An in-depth investigation on the
structure and activity of new organic probes have gained their significance despite the fact that
DNA and proteins are the preliminary targets for anticancer drugs. The molecule which acts as
target selective agent for DNA/protein mainly depends on the structure of the ligand, nature of
functional groups and metal centers thereby mimicking the biological enzymes or endonucleases
[6]. Recent days metal complex based anticancer agents, with potential in vitro toxicity have
gained immense importance [7, 8]. As it is proven that platinum-based drugs show cellular
resistance and undesirable side effects [9-11], much efforts are made to develop non-platinum
based anticancer agents with bio-essential metal ions such as zinc(II), copper(II) and iron(III) were
found to be successful [12-15]. These new metal complexes are known to have strong influence
on the structural and conformational changes of DNA and protein inside the cell.
Copper is the third most abundant transition metal present in the cellular body after iron and zinc
[16-18]. Enzymes like dismutase (containing copper) have their key roles in maintaining cellular
metabolic wastes [19, 20]. Recently, derivatives of above-said metals incorporated with
terpyridine were reported as potent chemotherapeutic drugs [21, 22]. Fine tuning and selectivity
of the drug molecule depends on substitutions of the ligand that coordinates with the metal center.
Moreover, it is quite obvious that the nature of the ligand plays an important role in the binding of
metal complexes to the bio molecules.
Metal complexes having extended planarity tend to bind with DNA intercalatively [23]. However,
metal complexes with ligands lacking planar structure are also reported to bind with DNA through
non-intercalative mode [24]. The central metal ion is also expected to play a role in determining
the efficacy of the metal complexes e. g. a redox active metal ion such as Cu(II) may promote
transformation of a biomolecule through an oxidative pathway [25]. On account of these
descriptions and expandable potentials, copper based complexes have been investigated in the
present work and the new observations make additional contributions to the field concerned. In

this work an attempts have been made to understand the effect of metal ions with heteroleptic
ligands like terpyridine and phenanthroline with extended planarity. The DNA binding ability and
the cytotoxicity of these complexes have also been investigated.
2. Experimental
2.1. Materials
Deoxyribonucleic acid sodium salt from calf thymus, 2-acetyl pyridine, 2-pyridine-
carboxaldehyde, bipyridyl and 1, 10 phenanthroline monohydrate were obtained from
Sigma Aldrich. All the solvents and copper(II) nitrate trihydrate were procured
commercially (SDFCL) and used without further purification. All the buffer solutions were
prepared by using Millipore Water and pH was maintained in the range of 7.2-7.4. The
ancillary ligands dipyrido [3, 2-d: 2′, 3′-f]-quinoxaline (dpq), and dipyrido [3, 2-a: 2′, 3′-c]
phenazine (dppz) were prepared as reported elsewhere [26].
2.2. Instrumentation
1
13
The H and C magnetic resonance spectral data were recorded on a Bruker Avance III-
400 MHz spectrometer, using CDCl3 as solvent and Trimethylsilane (TMS) as an internal
standard. UV-Vis absorption spectra were obtained using Jasco v-670 UV-VIS-NIR
spectrophotometer. FT-IR spectra of ligands and new complexes were recorded as KBr
pellets on a Shimadzu IR affinity-1 spectrophotometer in the range of 4000-400 cm-1. The
elemental analysis (C, H, N, O) of metal complexes were performed on Elementar Vario
EL III analyzer. The molecular weight of ligand and complexes were ensured from Agilent
Technologies 6310 Ion Trap LC/MS with 1200 Series. Fluorescence emission spectra were
obtained from Hitachi F-7000FL spectrometer. Circular dichroism studies for ct-DNA and
protein (BSA) with metal complexes were measured on a circular dichroism
spectropolarimeter JASCO-J717 under nitrogen flush and 1 cm Quartz cell at 0.4 nm
intervals in the wavelength range of 200-300 nm.
2.3. Preparation of 4′-(2′′′-Pyridyl)-2, 2′:6′, 2′′-terpyridine ligand

The ligand 4′-(2′′′-pyridyl)-2, 2′:6′, 2′′-terpyridine was prepared as reported in the literature
[27]. 2-Acetyl pyridine (20.0 mmol, 2.24 ml) was added drop wise to an ethanolic solution
of 2-pyridinecarboxaldehyde (10.0 mmol, 0.95 ml) containing potassium hydroxide (10.0
mmol, 0.77 g) and 30 % aqueous ammonia (80.0 mmol, 5 ml) under stirring conditions.
The resultant dark brownish-yellow colored solution was stirred vigorously for 8h in
ambient condition to get precipitated. The precipitate was collected, filtered and washed
several times with distilled water followed by cold ethanol. An off white solid obtained was
dried under vacuum and recrystallized using chloroform-methanol system.
Yield: 53% (1.62g, 6.4mmol) off white solid, m.p. 231-233 °C. IR (KBr cm−1): 3053 w,
3010 w, 1581 s, 1546 m, 1467 m, 1390 m, 1259 w, 1070 w, 991 m, 777 s. UV–vis [(λmax
(nm), ε (L mol−1 cm−1)]: 206 (35,160), 242 (41,565), 280 (42,745), 316 (11,945). 1H NMR
(CDCl3, 400MHz): δ (ppm): 7.36-7.33 (m, 3H, CHAr), 7.89-7.81(m, 3H, CHAr), 8.08-8.06
(d, J= 8Hz, 1H, CHAr), 8.67-8.65 (d, J=8Hz, 2H, CHAr ), 8.75-8.73 (m, 2H, CHAr ), 8.81-
13
8.79 (m, 1H, CHAr), 9.10 (s, 2H, CHAr). C NMR (CDCl3, 100MHz): δ (ppm): 118.63,
121.28, 121.32, 123.72, 123.83, 136.83, 136.87, 148.64, 149.20, 150.04, 155.11, 156.21,
156. 25. ESI-MS: found: 311.4 (M+1), calcd: 310.12.
2.4. Synthesis of [Cu(2‴-pytpy) (B)] (NO3)2.2H2O (B = bpy, 1; phen, 2; dpq, 3; dppz, 4)
All the complexes were prepared by stirring a methanolic solution of Cu(NO3)2.3H2O (1.0
mmol, 241mg) with 2‴-pytpy (1.0 mmol, 310mg) at room temperature for 30 min.
Subsequently, methanolic solution of ancillary ligand B (1.0 mmol of bpy (156mg)/ phen
(180mg)/ dpq (232mg)/ dppz (282mg)) was added drop wise into the former solution of
copper salt and continued stirring for 3h. The obtained reaction mixture was filtered and
kept aside for slow evaporation. Later solid products (accordingly) were isolated as blue
(1, 2 and 3), green (4) and were washed with diethyl ether followed by drying under
vacuum. The crystallization of complex
acetonitrile.
2 for X-ray diffraction was processed in
[Cu(2‴-pytpy) (bpy)] (NO3)2.2H2O (1). Yield: 82%. Light blue crystalline solid. M.p.:217-
219 °C Anal. Calc. for C30H26CuN8O8: C, 52.91; H, 3.80; N, 16.24; O, 18.55. Found: C,
52.84; H, 3.97; N, 16.37; O, 18.14. UV-Vis (methanol): λmax, nm (ε, dm3mol-1cm-1) 220

(135150), 288 (34695), 334 (9240), 348 (8484). FT-IR (KBr phase, ν cm-1): 3387 br, 3057
w, 1616 m, 1560 m, 1498 m, 1473 m, 1384 vs, 1274 w, 1022 w, 898 w, 839 w, 785 s, 729
w, 657 w, 493 w, 416 w (br, broad; vs, very strong; s, strong; m, medium; w, weak). LC-
MS: found: 531(M+2), calcd: 529.12.
[Cu(2‴-pytpy) (phen)] (NO3)2.2H2O (2). Yield: 78%. Blue crystalline solid. M.p.:221-223
°C Anal. Calc. for C32H26CuN8O8: C, 53.82; H, 3.67; N, 15.69; O, 17.92. Found: C, 53.73;
H, 3.61; N, 16.07; O, 17.86. UV-Vis (methanol): λmax, nm (ε, dm3mol-1cm-1) 222
(141850), 266 (44900), 288 (38000), 344 (11465), 348 (9910). FT-IR (KBr phase, ν cm-1):
3371 br, 3059 w, 1616 m, 1516 m, 1475 m, 1384 vs, 1244 w, 1018 m, 893 w, 848 m, 783
s, 725 s, 655 w, 497 w. LC-MS: found:554 (M+1), calcd: 553.12.
[Cu(2‴-pytpy) (dpq)] (NO3)2.2H2O (3). Yield: 82%. Dark blue crystalline solid. M.p.:266-
268 °C Anal. Calc. for C34H26CuN10O8: C, 53.30; H, 3.42; N, 18.28; O, 16.71. Found: C,
53.27; H, 3.49; N, 18.36; O, 16.67. UV-Vis (methanol): λmax, nm (ε, dm3mol-1cm-1) 222
(144230), 252 (49600), 288 (46390), 334 (15860), 348 (12745). FT-IR (KBr phase, ν cm-
1): 3327 br, 3057 w, 1614 m, 1554 w, 1471 m, 1384 vs, 1244 w, 1159 w, 1083 w, 1018 w,
898 w, 827 w, 783 s, 727 w, 655 m, 497 w, 437 w. LC-MS: found:606 (M+1), calcd: 605.13.
[Cu(2‴-pytpy) (dppz)] (NO3)2.2H2O (4). Yield: 82%. Green crystalline solid. M.p.: 273-275
°C Anal. Calc. for C38H28CuN10O8: C, 55.92; H, 3.46; N, 17.16; O, 15.68. Found: C, 55.84;
H, 3.51; N, 17.23; O, 15.71. UV-Vis (methanol): λmax, nm (ε, dm3mol-1cm-1) 226
(140350), 288 (27920), 334 (9320), 348 (8825). FT-IR (KBr phase, ν cm-1): 3439 br, 3062
w, 1604 m, 1560 m, 1473 m, 1384 vs, 1251 w, 1157 m, 785 s, 725 m, 655 m, 617 w, 497
w, 418 w. LC-MS: found:657 (M+2), calcd: 655.14.
2.5 Single crystal X-ray crystallographic data collection and refinement of the structure
All the complexes were subjected for crystallization by slow evaporation technique. The
single crystal X-ray diffraction analysis of complex
APEXII diffractometer using graphite filter monochromatic MoKα radiation (λ=0.71073
A°) at 23°C with crystal dimension of 0.250×0.200×0.100 mm3. The complex structure (
2 was performed on Bruker X8 Kappa
2
)
was resolved by using SHELX-97 program [28] and all non-hydrogen atoms were solved
with anisotropic thermal parameters by full matrix least squares refinement on F2 using

SHELX-97 software. The structure was partially solved by using direct method and
converged to reliable structure using full matrix least square method. The final reliability
index is 0.055. The hydrogen atoms were added geometrically and refined (using riding
model) with fixed isotropic thermal parameters. The copper(II) complexes crystallize in
triclinic with Centro-symmetric space group (P1).
2.6 Theoretical calculations
Gaussian 09 program [29] was used for the Density Functional Theory (DFT) calculations
for all the copper complexes 1-4. The complexes were fully optimized in the electronic
ground state (S0) using the three-parameter hybrid functional B3LYP. The standard basis
sets LANL2DZ were used for Cu, 6-31+G** for N and 6-31G* for C and H. The optimized
geometries of copper complexes 1-4 were verified for the absence of negative frequencies
by performing frequency calculations. Table 1 summarizes the optimized molecular
structure parameters in the ground state (S0) for the complex 1-4. In order to obtain the
estimates of the vertical electron excitation energies, time-dependent density functional
theory (TD-DFT; includes electron correlation) was performed on the calculated S0
geometries using same B3LYP method and basis set [30, 31]. The solvent (methanol) effect
was simulated using the polarizable continuum model with the integral equation formalism
(IEF-PCM). Natural bond orbital (NBO) calculations were carried out with the NBO code
included in GAUSSIAN-09. All the excitation energies, oscillator strength and the
corresponding HOMO and LUMO contributions from all the electronic transitions are
given in Table S3†.
2.7 DNA binding studies
In order to determine the binding interactions of newly synthesized metal complexes with
ct-DNA, absorption spectral titration was carried out in 5 mM tris-HCl buffer (pH-7.2). The
protein free form of ct-DNA was confirmed by the ratio of UV absorbance at 260 and 280
nm (A260/A280) of ca. 1.8 [32]. The concentration of ct-DNA per nucleotide was determined
by its molar absorption coefficient (6600 m-1) at 260 nm [33]. Stock solutions of metal
complexes were prepared in DMF and further dilutions were made in 5 mM Tris-HCl
buffer. The binding experiments were carried out with fixed concentration of the complex

(20 µM) and increasing concentrations of ct-DNA (0–26 µM) in both reference and sample
solutions to nullify the absorbance peak of ct-DNA.
The competitive binding of metal complexes with ethidium bromide bound ct-DNA is
explored by fluorescence spectroscopic technique. Solutions of EtBr and ct-DNA each at 5
µM concentration were prepared in 5 mM Tris-HCl buffer (pH-7.2), incubated for 1 hour
at room temperature before being used for fluorescence experiments. The ethidium bromide
bound ct-DNA was used as the reference solution. 515 nm and 597 nm were used as
excitation and emission wavelengths, respectively. The reference solution (EtBr-DNA) is
titrated against different concentrations of metal complexes (0–20 µM).
The stock solution of BSA (10 µM) was prepared in 50 mM phosphate buffer (pH-7.2) and
stored at 4 °C until used. The binding mechanism of newly synthesized metal complexes
with BSA (10 µM) were determined by UV-Visible absorption titration in the range of 200-
500 nm. The quenching interactions of metal complexes with BSA were monitored by
change in their fluorescence intensities at a fixed excitation wave length of 285 nm and
emission value at 345 nm, respectively. The same emission and excitation scan rates and
slit widths were maintained throughout the experiments. Fixed protein concentration (1
µM) was used to titrate against different concentrations of the metal complexes (0-24 µM).
The CD spectral studies were also carried out with ct-DNA (100 µM) and BSA (10 µM)
with varying concentrations of metal complexes (0-12 µM) in the range of 200-300 nm,
using buffer alone as a blank control. All the solutions for ct-DNA and protein studies were
prepared in 5 mM Tris-HCl buffer (pH-7.2) and phosphate buffer (pH-7.2), respectively.
Each experiment was performed in triplicates and the average value was taken to minimize
the signal to noise ratio.
2.8 Computational studies to reveal the nature of interaction of copper complexes with DNA and
protein
2.8.1 Receptor and Ligand preparation
The interactions of the four copper complexes 1-4 towards the DNA and BSA (protein)
were studied using molecular docking analysis. The X-ray crystal structures of Bovine

serum albumin protein in complex with 3, 5-diiodosalicylic acid (PDB ID: 4JK4) and ct-
DNA (PDB ID: 355D) obtained from Brookhaven Protein Data Bank were considered for
docking studies [34, 35]. The crystal structures were refined by removing the water
molecules and hetero atoms. Hydrogen atoms were added to the protein and ct-DNA
molecule. The kollman charges [36] were assigned to the protein atoms using Auto
DockTools-1.5.6. The 3D coordinates of the synthesized complexes 1-4 were built and
optimized using molecular mechanics. Gasteiger partial atomic charges [37] were added to
the optimized complexes and ct-DNA. The possible flexibility and torsion of the complexes
were defined by using Auto Tors. The copper metal complexes were treated to be flexible
using AutoDock 4.2. The most active binding site for 3, was identified [35] by defining a
grid box around the active site. In case of ct-DNA, a blind docking was performed by
covering the entire DNA to identify the binding site. Grid maps of interaction energies
between protein/DNA and various types of atoms present in the complexes were pre-
calculated using Auto Grid program.
2.8.2 Docking studies
Docking calculations were performed with default parameters implemented in AutoDock
4.2 [38]. It evaluates the conformational changes obtained after the binding of ligand to the
receptor and also their structural integrity. Scoring functions elucidate the intermolecular
energy difference between the bound and unbound states of the protein. It also calculates
the intermolecular energy due to receptor-ligand interactions. Prior to docking, the
geometries of the complexes were optimized with LANL2DZ, 6–31+G** and 6–31G*
basis and a B3LYP functional in Gaussian 09 program. The probable bound conformations
and the energies of the flexible complexes were explored using Lamarckian genetic
algorithm (LGA) and AMBER force field, respectively. The binding energy was evaluated
using the following scoring function:
∆G= ∆Gvdw+∆Ghbond+∆Gelec+ ∆Gtor+∆Gdesolv
The free energy upon binding of the flexible ligand to the rigid target can be calculated
using the equation that includes parameters like ∆Gvdw (dispersion/repulsion), ∆Gelec

(electrostatic interaction), ∆Ghbond (hydrogen bonding), ∆Gtor (torsional constraints) and
∆Gsol (desolvation effects).
2.9 Evolution of cytotoxicity by MTT assay
Cytotoxic studies of copper(II) complexes (1-4) were carried out on human liver carcinoma
cell line (HepG-2), procured from the American Type Culture Collection (ATCC HB-
8065), USA. The viability of cells was screened by MTT assay [39]. The obtained cells
were cultured in a sterile T75 flask containing Dulbecco's Modified Eagle's medium
(DMEM) supplemented with fetal bovine serum (10% v/v) at 37 °C in an incubator with
humidified atmosphere of 95% air and 5% CO2.
The complexes were dissolved in 5 mM phosphate buffered saline (PBS, pH 7.2) containing
10% DMSO. Concentration of the DMSO was maintained below 0.5% for cell survival.
The HepG-2 cells were plated separately in 96 well plates at a concentration of 1×105
cells/well. After 24h, the cells were washed twice with 100 µl of serum-free medium and
starved for an hour at 37 °C. After starvation, the cells were treated with different
concentrations of complexes (50-150 µg/ml) for 24h. At the end of the treatment period,
the medium was aspirated and serum free medium containing MTT (0.5 mg/ml) was added
and incubated for 4h at 37 °C in a CO2 incubator. The MTT containing medium was then
discarded and the cells were washed with PBS (200 µl). Only the live cells can take in MTT
and convert it in to formazan crystals. Afterwards, the cells were lysed by adding DMSO
and further dissolved formazan imparted purple blue colour to the buffer solution. This
conversion was monitored by change in absorbance at 579 nm in a micro titer plate reader
(Biorad 680). Cytotoxicity was determined using Microsoft excel. The percentage of cell
viability was calculated using the formula;
% viability = {OD of the experiment samples/OD of the control} × 100

3. Results and Discussion
3.1 Synthesis
2.0 Moles of 2-acetyl pyridine and 1.0 mole of 2-pyridinecarboxaldehyde were used for the
preparation of 4′-(2′′′-Pyridyl)-2, 2′:6′, 2′′-terpyridine ligand in presence of KOH as a base
(Scheme 1) and the ligand structure was confirmed by spectroscopic techniques. Copper(II)
nitrate tri hydrate [Cu (NO3)2.3H2O] was used as precursor for the synthesis of new
copper(II) derivatives (Scheme 2). All the newly synthesized metal complexes were
characterized by elemental analysis and various spectroscopic techniques. The molecular
structure of copper complex
2 was confirmed by single crystal X-ray diffraction studies.
3.2 Characterization
The electronic absorption spectra of ligand (2‴-pytpy) and its metal complexes (1-4) show
intense bands in the range of 200-400 nm (Fig. S1, ESI†). All the new copper(II) complexes
exhibit four bands. The bands at 242 and 280 nm can be assigned to n-* and π-π*
transitions of aromatic rings. The bands appearing in the range of 252-288 nm correspond
to the intra-ligand transitions (π-π*, n-π*) of 4′-(2′′′-Pyridyl)-2, 2′:6′, 2′′-terpyridine ligand.
The bands appearing at ~334 nm and ~ 349 nm, corresponds to the ligand to metal charge
transfer band (LMCT). The weak d-d transitions which are supposed to be in the visible
region (~600 nm) of the spectrum were not observed for copper(II) complexes due to the
dominant tail arising from the charge transfer transition [40, 41].
The FT-IR spectrum of 4′-(2′′′-Pyridyl)-2, 2′:6′, 2′′-terpyridine shows strong band at 1581
cm-1 which corresponds to the C=N stretching (Fig. S2, ESI†). In case of complexes (1-4),
the medium C=N stretch band is shifted towards higher values and are observed in the range
of 1604-1616 cm-1 (Fig. S3-S6, ESI†). This indicates the binding of nitrogen atom to copper
metal is through coordination fashion. The uncoordinated ancillary ligands show the same
medium C=N stretching band in the frequency range of 1579-1589 cm-1. In all complexes
a strong intense band at 1384 cm-1 confirms the presence of nitrate as counter ion. The
stretching band corresponding to the coordinated water molecule was found between 783-
785 cm-1.

4′-(2′′′-Pyridyl)-2, 2′:6′, 2′′-terpyridine structure was confirmed by both NMR and mass
1
spectrometry techniques (Fig. S7-S9, ESI†). The H NMR spectrum of the ligand shows
expected chemical shift values for aromatic protons in the region of 7.26 - 9.10 δ ppm and
13C NMR spectrum reveals chemical shift values for all identical carbon entities of the
ligand in the range of 118.63 - 156.25 δ ppm. The molecular weight of the ligand was
confirmed by ESI mass spectroscopy. The molecular ion peak M+1 was observed at 311.40
m/z (310.12). The molecular mass of all the synthesized complexes were confirmed by LC-
MS (Fig. S10-S13, ESI†). Complexes
1
and
4 show M+2 peaks at 531 (529.12), 657
(655.14) and complex
(605.13), respectively.
2, 3 display molecular ion peak (M+1) at 554 (553.12) and 606
3.3 Crystal Structure
Single crystal X-ray diffraction analysis was carried out for the extensive structural
characterization of complex . The perspective view of the complex, ORTEP diagram is
given in Fig. 1. A keen investigation on the structure revealed that it belongs to the P1 space
group of the triclinic system and detailed crystallographic data of complex is summarized
in Table S1†. The penta-coordinated crystal structure of complex exhibited a distorted
2
2
2
square-pyramidal geometrical (Cu-N5) system in tridentate and bidentate binding fashion
from 4′-(2′′′-Pyridyl)-2, 2′:6′, 2′′-terpyridine (2‴-pytpy) ligand and the phenanthroline base,
respectively. The 2‴-pytpy ligand binds to the bansal plane of the complex
2 and an axial-
equatorial mode of binding was observed with the phenanthroline base. In the bansal plane
of 2‴-pytpy ligand, nitrogen {N(2)} is positioned at a distance of 1.932 ꢀ and the axial
position of phenanthroline base nitrogen {N(5)} was at a bond distance of 2.278 ꢀ from
the central copper ion. The other three nitrogen’s {N (1), N (3) and N (4)} are linked with
central metal ion at a distance of 2.055 ꢀ, 2.060 ꢀ and 1.999 ꢀ, respectively. Selected bond
distances and bond angles are tabulated in Table S2†.
3.4 Computation methods
The optimized structures of copper(II) complexes (1-4) are depicted in Fig. S14, ESI†.
Calculated and experimental geometrical parameters of all the derivatives are shown in
Table 1. The results reveal that all the calculated geometrical parameters are in good
agreement with the experimental values except for the bond length of Cu–N (the difference

in the bond length is ~ 0.50 Å). The reason for this deviation could be due to constraints
arising from the crystal packing arrangement. The theoretically optimized geometry data
were compared with the crystallographic data and the validation of the method was
authenticated from the structural similarity observed between both the methods. Further,
similar computational procedures were employed for other complexes
crystal structure data are not available. The calculated geometrical parameters of the
complexes ( and ) are also given in Table 1 along with the calculated electronic
1, 3 and 4 for which
1
,
3
4
transition bands of the complexes (1-4) (Table S3†). Theoretical absorption peaks of all the
complexes were compared with the experimental (electronic absorption) values. All the
theoretical and experimental absorption values are found to be in good agreement. The
molecular orbitals (Fig. 2) involved in the transitions and their major contributions for the
transitions are tabulated in Table S3† (Fig. S15, ESI†). Theoretical and experimental values
are supporting each other in the frame work of TD-DFT.
3.5.1 UV-Visible absorption titration
UV-Visible absorption spectroscopy was used to calculate the binding propensity of metal
complexes with ct-DNA. Metal complexes (20 µM) were titrated with varying
concentrations of ct-DNA (0-26 µM) and is depicted in Fig. 3. The increasing concentration
of ct-DNA to metal complexes shows a significant hypochromism (48.71-67.19 %) with
small red shift (~1 nm) due to the strong stacking interaction between the DNA base pairs
and aromatic chromophore of the ligands [42]. The results indicate that complexes bind to
ct-DNA via intercalative mode. The intrinsic binding constant (Kb) values of complexes
with ct-DNA were calculated using the following equation:
[DNA]/ (εa- εf) = [DNA]/ (εb- εf) +1/ Kb (εb- εf)
Here [DNA] is the concentration of the DNA in base pairs, the apparent absorption
coefficients εa, εf and εb correspond to Aobsd / [complex], extinction coefficient for complex
in free form and extinction coefficient for DNA bound complex, respectively. The intrinsic
binding constant (Kb) is obtained from the ratio of slope to intercept [43]. The binding
constant values (Kb) of metal complexes (1-4) were found to be in the range of 2.75×105 to
7.85×105 M-1. The mode of binding of the metal complexes with DNA might have been

influenced by the planarity of 4′-(2′′′-Pyridyl)-2, 2′:6′, 2′′-terpyridine and ancillary ligands
(bpy, phen, dpq and dppz). The dpq and dppz containing complexes ( ) show high
3
& 4
binding constants when compared with bpy and phen containing metal complexes (
which could be due to the extended aromatic moiety [44].
1 & 2)
3.5.2 Competitive binding interaction between Ethidium Bromide (EtBr) and metal complexes with
ct-DNA
Neither metal complexes nor the ct-DNA displayed intrinsic fluorescence emission either
alone or in combination. Therefore, the binding interaction of complexes with ct-DNA
could not be identified directly through fluorescence emission. A competitive binding
experiment was carried out to evaluate the mode of interaction, wherein EtBr was not only
used as a fluorescent probe but also to compete with the binding of ct-DNA to the metal
complexes. EtBr a planar cationic dye with phenanthridine ring is used as a fluorescence
probe for native DNA. The intense fluorescence emission observed from EtBr in the
presence of ct-DNA is due to strong interaction between adjacent DNA base pairs via
intercalative binding [45]. The remarkable decrease in fluorescence intensity of EtBr-ct-
DNA with increasing concentration (0-20 µM) of quencher is shown in Fig. 4. The emission
band at 598 nm displayed hypochromism up to 79.50 %, 85.71 %, 85.83 % and 87.80 % of
the initial fluorescence intensity for 1-4, respectively. The EtBr bound ct-DNA enhanced
fluorescence intensity values were quenched by metal complexes due to its replacement for
EtBr in ct-DNA: EtBr complex. The quenching constants of metal complexes were
analyzed by following Stern-Volmer equation:
I0 / I = 1+Ksv [Q]
Here I0 and I are the emission intensities in the absence and presence of quencher,
respectively. Ksv is the linear Stern-Volmer quenching constant, and [Q] is the concentration
of quencher. The Ksv value was obtained from slope of I0 / I vs. [Q]. The quenching plot
depicts that the quenching of EtBr bound to ct-DNA by metal complexes are in good
agreement with the Stern-Volmer equation. The quenching constant values (Ksv) for
complexes (1-4) were found to be 1.023×105 M-1, 1.09×105 M-1, 1.183×105 M-1 and

1.264×105 M-1, respectively. The apparent binding constant (Kapp) values for metal
complexes were obtained from the following equation:
KEB [EB] = Kapp [complex]
Here KEB = 1.0 × 107 M-1, [EB] = 5 µM and [complex] is the complex concentration at 50
% reduction in the fluorescence intensity of EB. The quenching constant Kapp values of
metal complexes (1-4) are found to be 5.11×106 M-1, 5.45×106 M-1, 5.91×106 M-1 and
6.37×106 M-1, respectively. These results suggest that all the metal complexes bind with ct-
DNA via intercalation mode and binding constant values are less while comparing with
other classical intercalators [46].
3.5.3 In-silico analysis
In chemotherapy treatments, the biological processes inside the cell are influenced by the
structure and conformations of protein / DNA while binding partners interact. In order to
establish this, docking calculations were performed to observe the interaction and binding
affinity of all the four copper(II) complexes with ct-DNA and BSA protein (Fig. 5 and Fig.
6). In case of ct-DNA, initially a blind docking was executed to identify the binding
pockets. Similar procedure was then repeated with grid boxes covering only the major and
minor grooves independently. The binding (∆GBE) and intermolecular energies (∆Gintermol
)
of the complexes 1-4 with ct-DNA and protein are given in Table 2 and Table 3,
respectively. Docking calculations of complexes 1-4 with ct-DNA revealed similar
binding energies (~ −10.00 kcal/mol). The interaction for complexes 1-3 mainly depends
upon hydrogen bonding. The pyrimidine (C21 OP1) of complexes 1, 2 and
3 formed
hydrogen bonds with the nitrogen atom of terpyridine ring. ct-DNA docking studies
revealed that the complexes 1-4 interact with ct-DNA via major groove with high binding
affinities. The interactions reveal that the complexes 1-4 can serve as potent therapeutic
candidates and chemotherapeutic agents for the treatment of cancers.
Molecular docking was also carried out to investigate the protein-ligand interactions and
hence to display all structural features of the protein and the stereo-chemical properties of
all the binding complexes. In this study the interaction of the four synthesized copper(II)

complexes were analyzed for their binding affinity and their interactions towards the BSA
protein. Fig. 6 depicts the residues involved in the interaction with copper complexes 1-4
and their binding energy, torsional energy, intermolecular, electrostatic energy for each
protein–complex are provided in Table 3.
In case of Bovine serum albumin binding studies, the interaction of complex
4 is
predominantly stabilized by hydrophobic interactions with the residues Lys116, Pro117,
Leu178, Arg185, Thr514 and Pro516. The intermolecular energy contribution of the
complex
4 to the total energy was ~ −6.00 kcal/mol for BSA protein and the contribution
of complex 1-4 to the total energy was ~ −11.00 kcal/mol for ct-DNA. This shows that
there exists a strong intermolecular van der Waals, hydrogen bonding, desolvation and
electrostatic interactions. The best docked conformation of complexes with ct-DNA and
BSA obtained by docking calculations is exhibited in Fig. 5 and Fig. 6, respectively. Based
on docking calculations, it is observed that complex
4 shows better binding affinity with
BSA compared to other complexes. These results are agree well with the experimental data.
3.6 BSA interaction studies
In order to study the nature of interaction between BSA and the metal complexes, electronic
spectrum was recorded by titrating a fixed amount of protein against varying concentration
of metal complexes (1-4). Quenching effect was observed with (Fig .7). The decrease in
the emission intensity of BSA with metal complexes indicates the type of interaction
between protein and metal complex and induces static quenching rather than dynamic
quenching as a shift in the absorption spectra was not observed [47].
Mainly the presence of amino acids phenylalanine, tyrosine and tryptophan are responsible
for the fluorescence of BSA [48-50]. Out of these three, tryptophan plays a major role in
the intrinsic fluorescence of BSA. Fluorescence spectral titrations were carried out in the
wave length range of 285-500 nm upon excitation at 280 nm. The effect of copper(II)
complexes on fluorescence spectra of protein is given in Fig. 7. The increasing
concentration of metal complexes (0 - 24 µM) shows a significant quenching of
fluorescence intensity at 345 nm, up to 84.07 %, 89.78 %, 93.07 % and 82.56 %,
respectively of the initial fluorescence intensity of BSA accompanied by a hypsochromic

shift of ~ 2 - 4 nm for the metal complexes (1-4). The blue shift observed is mainly due to
the binding of metal complexes with the active site of protein in a hydrophobic
environment.
The fluorescence quenching constant values are described by the Stern-Volmer equation
I0 / I = 1+Ksv [Q]
Here I0 and I indicate the fluorescence intensities in the absence and presence of metal
complex (quencher), respectively. Ksv is a linear Stern-Volmer quenching constant, and [Q]
is the quencher concentration. The quenching constant (Kq) can be calculated using the plot
of log (I0 / I) versus log [Q].
If small molecules bind independently to a set of equivalent sites on a macromolecule, the
equilibrium between free and bound molecules is represented by the Scatchard equation:
[5, 52]
log [(I0- I)/ I] = log Kb + n log [Q]
Here Kb is the binding constant of the complex with BSA and n is the number of binding
sites. Employing the plot of log [(I0- I)/ I] versus log [Q], the number of binding sites (n)
and the binding constant (Kb) values could be obtained. The quenching constant (Kq),
binding constant (Kb) and number of binding sites (n) for the interaction of the Cu(II)
complexes with BSA are shown in Table 4. The results indicate that in all the complexes,
only one binding site is available for interaction with BSA.
3.7 Circular Dichroism spectral analysis of metal complexes (1-4) with ct-DNA and BSA
Circular Dichroism spectral analysis was carried out to investigate conformational changes
induced in the secondary structure of ct-DNA by the metal complexes [53]. The
characteristic CD spectrum of ct-DNA in its right handed B-form, exhibits a positive band
at 275 nm and a negative band at 245 nm corresponding to π-π base stacking and helical
structure, respectively [54]. The interaction studies revealed a slight increase in the intensity
of negative band and a slight decrease in intensity of positive band with a blue shift of 10
– 17 nm and 2 – 11 nm, respectively with hypsochromic shift for all the complexes as

provided in Fig. 8. Surprisingly, in case of complexes 2-4 with increase in concentrations
(0-12 µM), there is a huge blue shift in the long wave length band from 275 nm to ~269
nm. These results suggest that complexes bind with DNA via intercalative mode with strong
conformational changes at higher concentrations of metal complexes [55, 56].
The conformational change caused by the metal complexes upon binding with protein is
also investigated from the CD spectra. Metal complexes may affect the secondary or tertiary
structure of proteins. The typical CD spectrum of BSA shows two negative bands at 208
nm and 222 nm. The red shift observed in the band around 222 nm corresponds to the n-
π* transition from the peptide bond of α-helix [57, 58]. Fig. 9 reveals the influence of
copper(II) complexes (1-4) on the secondary structure of the protein. At lower
concentration of metal complexes, there is a slight increase in right handed helical content
of BSA. These results suggest that there is a conformational change in the secondary
structure of protein in the presence of metal complexes.
3.8 In vitro cytotoxicity studies
MTT assay was followed to assess the influence of complexes (1-4) on the percentage cell
viability of HepG-2 cell lines. From the cell viability (%) vs complex concentration plot
(Fig. 10), a dose dependent activity was observed towards HepG-2 cell lines. The half
maximal inhibitory concentration (IC50) values of complexes 1-4 were found to be 76.9,
51.4, 39.2 and 23.8 µg/ml, respectively. Among the four metal complexes,
cytotoxicity towards the HepG-2 cell line.
4 exhibits better
4. Conclusion
Herein, new copper(II) heteroleptic mononuclear complexes were synthesized, structurally
characterized and assessed for their biological activities. The single crystal X-ray
diffraction studies of complex
2 reveals a penta-coordination around copper ion with
distorted square-pyramidal geometry. An intercalative binding mode of metal complexes
with DNA was observed when their binding properties with DNA were getting examined
by UV-Visible absorption, fluorescence and CD spectral titrations. The binding efficacy of

DNA with complexes follows the order 1 < 2 < 3 < 4. The interaction of complexes with
protein was revealed from the observation of static quenching in electronic spectrum of
BSA. In addition, in vitro cytotoxicity of copper(II) complexes against HepG-2 cell lines
provides satisfactory IC50 values. Thus, based on the above investigations followed by
description, it could be concluded that these new copper(II) complexes enrich the library
of potent anticancer drugs and could open up avenues for further exploringtheir applications
towards next level of biological studies such as genomic and topoisomerase activities.
Acknowledgement
Authors extend their heartfelt thanks to the VIT University, Vellore, India for expediting
essential requirements and financial support. Author T.K. gratefully acknowledge
administration of VIT University for awarding him research associateship.

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Fig. 1. ORTEP diagram of complex 2 (CCDC: 1431231).

Fig. 2. Contour plots of HOMOs and LUMOs with the orbital energies of complex 1-4.
3.5 DNA interaction studies

Fig. 3. Electronic absorption spectra depicting the titration of complexes 1-4 vs DNA. The arrows
indicate the decrease in the absorbance with increasing concentration of DNA concentration (0 –
26 µM). The intrinsic binding constant can be calculated from the intercept of the inset graph.

Fig. 4. Fluorescence quenching spectra depicting the titration of Ethidium Bromide bound DNA
(Equimolar concentrations) vs complexes 1-4 (0-20 µM). The inset figure shows the Stern-Volmer
plot and slope represents the quenching constant for the corresponding complex.