Synthesis and structure elucidation of new open cubane tetranuclear [CuII4] Cluster: Evaluation of the DNA/HSA interaction and pBR322 DNA cleavage pathway and cytotoxicity

Article abstract of DOI:10.1016/j.ica.2017.04.031

New open–cubane tetranuclear copper(II) complex of formulation {[Cu₄(HL)₄(H₂O)₂]·(CH₃OH)₂·(H₂O)₅}(1) derived from Schiff base ligand 2–((E)–(1,3–dihydroxy–2–methylpropan–2–ylimino)methyl)–6–methoxyphenol (HL) was synthesized. The characterization of both ligand (HL) and tetranuclear Cu complex was carried out by analytical, spectroscopic and single crystal X–ray diffraction. The structure analyses revealed that in the tetranuclear core, the metal centers are mutually interconnected via two phenoxo oxygen and two alkoxo oxygen atoms of four H₂L^2? ligand to generate a distorted single–open [Cu₄(μ₃–O)₂]⁶⁺ open cubane core with the four Cu atoms disposed in an extended “butterfly–like” arrangement. Binding of complex 1 with CT DNA was investigated by using vivid spectroscopic techniques to study the mode of the interaction of 1 towards CT DNA. The results obtained indicated that 1 avidly binds to CT DNA via electrostatic mode with an intrinsic binding constant, K_b value 4.63?×?10⁴?M^–1. Complex 1 showed an impressive nuclease activity generating single–strand breaks and the mechanistic investigation confirms the contribution of the reactive oxygen species (ROS). Likewise, the interaction of 1 with human serum album (HSA) revealed the change in intrinsic fluorescence intensity of HSA and was supposed to be associated with the microenvironment of Trp residue. Furthermore, the interaction of complex 1 with DNA and HSA was further investigated by the molecular docking studies which corroborated well with above findings. We have also tested 1 for cytotoxicity against the PC3 (Prostate) and K562 (Leukemia) cancer cell lines. The results exhibited were moderate when compared to standard drug viz, adriamycin (ADR).

Full text of DOI:10.1016/j.ica.2017.04.031

Accepted Manuscript
Research paper
Synthesis and Structure Elucidation of New Open Cubane Tetranuclear [CuII
4] Clusters: Evaluation of the DNA/HSA Interaction and pBR322 DNA Cleav-
age Pathway and Cytotoxicity
Sartaj Tabassum, Mohd. Afzal, Hamad Al-Lohedan, Mehvash Zaki, Rais
Ahmad Khan, Musheer Ahmad
PII:
S0020-1693(17)30158-5
http://dx.doi.org/10.1016/j.ica.2017.04.031
ICA 17536
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 February 2017
28 March 2017
18 April 2017
Please cite this article as: S. Tabassum, Mohd. Afzal, H. Al-Lohedan, M. Zaki, R.A. Khan, M. Ahmad, Synthesis
and Structure Elucidation of New Open Cubane Tetranuclear [CuII 4] Clusters: Evaluation of the DNA/HSA
Interaction and pBR322 DNA Cleavage Pathway and Cytotoxicity, Inorganica Chimica Acta (2017), doi: http://
dx.doi.org/10.1016/j.ica.2017.04.031
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Synthesis and Structure Elucidation of New Open Cubane Tetranuclear
[CuII ] Clusters: Evaluation of the DNA/HSA Interaction and pBR322 DNA
4
Cleavage Pathway and Cytotoxicity
Sartaj Tabassum,a,b* Mohd. Afzal,b Hamad Al–Lohedan,a Mehvash Zaki,b Rais Ahmad Khan,a
and Musheer Ahmadc
aSurfactant Research Chair, Department of Chemistry, College of Science, King Saud
University, P.O. Box 2454, Riyadh –11451, Saudi Arabia.
bDepartment of Chemistry, Aligarh Muslim University, Aligarh, UP–202002, India.
cDepartment of Applied Chemistry, Aligarh Muslim University, Aligarh, UP–202002, India
E–mail address: tsartaj62@yahoo.com (S. Tabassum)
Abstract
New open–cubane tetranuclear copper(II) complex of formulation
{[Cu4(HL)4(H2O)2]·(CH3OH)2·(H2O)5}(1) derived from Schiff base ligand 2–((E)–(1,3–
dihydroxy–2–methylpropan–2–ylimino)methyl)–6–methoxyphenol (HL) was synthesized. The
characterization of both ligand (HL) and tetranuclear Cu complex was carried out by analytical,
spectroscopic and single crystal X–ray diffraction. The structure analyses revealed that in the
tetranuclear core, the metal centers are mutually interconnected via two phenoxo oxygen and two
alkoxo oxygen atoms of four H2L2– ligand to generate a distorted single–open [Cu4(µ3–O)2]6+
open cubane core with the four Cu atoms disposed in an extended “butterfly–like” arrangement.
Binding of complex 1 with calf thymus DNA (CT–DNA) was investigated by using vivid
spectroscopic techniques to study the mode of the interaction of 1 towards CT DNA. The results
obtained indicated that 1 avidly binds to CT DNA via electrostatic mode with an intrinsic
binding constant, Kb value 4.63 x 104 M–1. Complex 1 showed an impressive nuclease activity
generating single–strand breaks and the mechanistic investigation confirms the contribution of
the reactive oxygen species (ROS). Likewise, the interaction of 1 with human serum album
1

(HSA) revealed the change in intrinsic fluorescence intensity of HSA and was supposed to be
associated with the microenvironment of Trp residue. Furthermore, the interaction of complex 1
with DNA and HSA was further investigated by the molecular docking studies which
corroborated well with above findings. We have also tested 1 for cytotoxicity against the PC3
(Prostate) and K562 (Leukemia) cancer cell lines. The results exhibited were moderate when
compared to standard drug viz, adriamycin (ADR).
Keywords: Tetranuclear Cu complex, DNA/HSA binding, Nuclease activity
Abbreviations
UV–vis
CT DNA
Tris
UV–visible
Calf thymus DNA
Tris(hydroxymethyl)aminomethane
Human serum albumin
HSA
1. Introduction
In recent year, tetranuclear copper(II) clusters are of great interest to researchers should not only
because of their interesting topologies in bioinorganic chemistry, multi–electron transfer,
catalytic and new extended material investigations but could also shed light on biological
processes that involve copper biomolecules in relation to metalloproteins [1–5]. The successful
and facile approach for the synthesis of such complexes is based upon the appropriate choice of
well–designed organic moiety as building blocks and metal ions [6,7]. A mononuclear complex
can be transformed into a multinuclear complex by the judicial choice of the bridging ligand,
metal ion, and reaction conditions [8]. Schiff–base ligands are well known in the syntheses of
multinuclear complexes because it can stabilize various metal ions in different oxidation states
[9,10].
2

Poly alcoholic ligand containing metal cluster that can interact to DNA are gaining huge
attention due to their various applications in the area of bioinorganic chemistry viz. diagnostic
agents for medical applications, development of cleavage agents for probing nucleic acid
structure.[11,12] For anticancer drug design, DNA has been identified as a primary intracellular
target and lies amongst the ultimately promising biological receptors.[13] The interaction
between transition metal complexes and DNA are essential for the design of efficacious
molecular entities which exhibit different properties than the mainstream protocol drugs viz.
cisplatin and its derivatives, etc. which are currently in use.[14,15] On the other hand, drug
interactions at the protein binding level significantly affect the apparent distribution volume and
their elimination rate. Therefore, the interaction of metal complexes with serum albumins is of
particular interest for a researcher by studying metallopharmaceuticals. HSA is the most
multifunctional transport and distribution proteins present in plasma that can reversibly bound
and carries several endogenous and exogenous substances including proteins and fatty acids to
specific target [16].
To date, many kinds of metal complexes were synthesized for these studies. Among all,
copper complexes showed encouraging perspectives based on the assumption that endogenous
metals may be less toxic to normal cells with respect to cancer cells.[17] The altered metabolism
of cancer cells and differential response between normal and tumor cells to copper are the basis
for the development of copper complexes capable with antineoplastic characteristics.[18] Also,
copper complexes which possess biologically accessible redox potentials and demonstrate high
nucleobase affinity are potential reagents for the cleavage of DNA. Meanwhile, the interaction of
these copper(II) complexes of Schiff base with DNA has been extensively studied in the past
decades.[19,20] Due to the site–specific binding properties and gaining prominence application
3

in medicinal chemistry, these coordination compounds were suitable candidates as DNA
secondary structure probes. Moreover, copper(II) Schiff–base complex was found to be effective
in reducing tumor size, delay in metastasis, and considerably increasing the survival of the
hosts.[21] Compared to the number of studies dealing with mono– and binuclear complexes,
relatively few studies on tetranuclear complexes binding to DNA have been reported, encourage
us to synthesize new tetranuclear complex and its interaction towards molecular target DNA and
serum albumin protein HSA.
In quest of our ongoing research on DNA binding and cleavage activities of transition
metal complexes to obtain more insight into the selectivity and efficiency towards DNA, herein
we report the synthesis, crystal structure, DNA binding and cleavage activity of a new open–
cubane tetra–nuclear copper(II) complex 1 having the formula
{[Cu4(HL)4(H2O)2]·(CH3OH)2·(H2O)5} (1). The asymmetric unit contains four Cu(II) ions, four
HL2– ligands, two coordinated water molecules and two methanol and five aqua molecules in the
lattice. It possesses a distorted [Cu4(µ3–O)2]6+ open core with the four Cu atoms disposed of in an
extended “butterfly–like” arrangement. The complex binds avidly to CT DNA via electrostatic
mode and efficiently cleaves supercoiled plasmid DNA in the presence reactive oxygen species.
The cleavage of DNA takes place via the oxidative pathway. Furthermore, the interaction of
tetranuclear complex 1 and HSA was monitored by employing fluorescence quenching
mechanism which revealed the changes of intrinsic fluorescence intensity of HSA was induced
by the microenvironment of Trp 214 residue. The mode of interaction of 1 with DNA and HSA
was studied via molecular docking techniques. Furthermore, the complex 1 was tested for
cytotoxicity against the PC3 (Prostate) and K562 (Leukemia) cancer cell lines by SRB Assay.
4

2. Experimental Section
2.1. Materials and Methods
Reagents available commercially were used as supplied without further purification.
CuClO4·6H2O, o–Vanillin, 2–amino–2–methylpropane–1,3–diol, HSA (fatty acid–free, 99%)
and Calf thymus DNA (CT DNA) (Sigma–Aldrich). 6X loading dye (Fermental Life Science)
and Supercoiled plasmid DNA pBR322 (Genei) was utilized as received. Human serum albumin
of 1.0×10–3 M was prepared by dissolving protein in Tris–HCl buffer solution at pH 7.4.
Microanalyses for the compounds were performed using CE–440 elemental analyzers (Exeter
Analytical Inc.).Infrared spectra were obtained (KBr disk, 400–4000 cm–1) on a Perkin–Elmer
Molar conductance was measured at room temperature on CON 510 Bench conductivity TDS
Meter. Model 1320 spectrometer. ESI mass spectra were recorded on a WATERS Q–TOF
Premier mass spectrometer. The EPR spectrum of the Cu(II) complex was acquired on a Varian
E 112 ESR spectrometer using X–band frequency (9.5 GHz) at liquid nitrogen temperature in a
liquid state. Electronic spectrum was recorded on UV–1700 PharmaSpec UV–vis
spectrophotometer (Shimadzu) in DMSO cuvettes of 1 cm path length. Data were reported in
λmax/nm. Fluorescence measurements were determined on RF–5301 PC spectrofluorophotometer
(Shimadzu). DNA cleavage experiments were performed with the help of Axygen made
electrophoresis supported by a Genie power supply with a potential range of 50–500 volts,
visualized and photographed by a Vilber–INFINITY gel documentation system.
5

2.2 Synthesis
2.2.1. Synthesis of ligand 2–((E)–(1,3–dihydroxy–2–methylpropan–2–ylimino)methyl)–6–
methoxyphenol (HL)
The proligand HL was synthesized from the reaction of o–vanillin (0.65 mmol, 100 mg) and 2–
amino–2–methylpropane–1,3–diol (0.65 mmol, 70 mg) according to the procedure reported
earlier.[22]
2.2.2. Synthesis of the complex [Cu4(HL)4(H2O)2]·(CH3OH)2·(H2O)5 (1)
To a stirred methanolic solution (15 ml) of Schiff base ligand LH (1 mmol, 0.239g) was added a
solution of CuClO4·6H2O (1 mmol, 0.370g) dissolved in 15 mL methanol dropwise. The
resulting solution was refluxed for ca. 4 h to obtain a clear green color solution and then the
excess of methanol was reduced to 5 ml by the rotatory evaporator. Block shaped green crystals
of 1 were collected in ~54% yield. The crystals were washed with hexane and then dried in
vacuo. Melting point 248 °C. Anal. calcd. for C50H82Cu4N4O25: C, 41.37; H, 5.93; N, 4.02 %.
Found: C, 40.79; H, 5.81; N, 3.92 %. IR (cm–1): 3384(s), 1607(s), 1422(s), 1376(s), 1340(m),
1205(m), 940(m), 802(m), 776(s), 720(s). 538 (w), 497 (w), 467 (w), 453 (w), 424 (w). ESI–MS:
m/z (100%) 1394.03 [M+1]+.
2.3. X–ray crystallography
Single crystal X–ray data of complex 1 was collected on a Bruker SMART APEX CCD
diffractometer at 100 K using graphite monochromated MoKα radiation (λ = 0.71073 Å). The
linear absorption coefficients, scattering factors for the atoms and the anomalous dispersion
corrections were referred from the International Tables for X–ray Crystallography.[23] The data
integration and reduction were worked out with SAINT software.[24] Empirical absorption
correction was applied to the collected reflections with SADABS,[25] and the space group was
6

determined using XPREP.[26] The structure was solved by the direct methods using SHELXTL–
97 [27] and refined on F2 by full–matrix least–squares using the SHELXTL–97 programme
package.[28] Only a few H atoms could be located in the difference Fourier maps in the
structure. The rest were placed in calculated positions using idealized geometries (riding model)
and assigned fixed isotropic displacement parameters. All non–H atoms were refined
anisotropically. Several DFIX commands were used for fixing some bond distances in complex
1. The unit cell contains a large number of lattice water and methanol molecules with mutually
overlapping electron densities which could not be modeled as discrete atomic sites. However, we
employed PLATON/SQUEEZE [29] refinements to calculate the diffraction contribution of
solvent molecules and thus gets a set of solvent–free diffraction intensities. For complex 1,
squeeze refinement results show two methanol molecule and five water molecules in per formula
weight. The contribution of all the solvent atoms has been incorporated in both the empirical
formula and formula weight of the complex. The crystal and refinement data are presented in
Table 1, and selective bond distances and angles are given in Table S1.
2.4. DNA Binding and cleavage experiments
DNA binding experiments include absorption spectral traces, and emission spectroscopy
conformed to the standard methods and practices previously adopted by our laboratory whereas
DNA cleavage experiment has been performed by the standard protocol.[30–32] Upon
measuring the absorption spectra an equimolar DNA was added to both the compound as well as
the reference solution to eliminate the absorbance of the CT–DNA itself, and Tris buffer was
subtracted through base line correction.
7

2.5. HSA binding studies
Human Serum Albumin (HSA) of 1 x 10–3 M was prepared in Tris–HCl buffer solution (pH 7.4).
The HSA concentration was calculated spectrophotometrically using an extinction coefficient of
35219 M–1cm–1 at 280 nm.[33] A stock solution of the complex (1 x 10–3 M) was prepared by
dissolving compound in double distilled water. 1 M NaCl (analytical grade) solution was used to
for maintaining the ionic strength of buffer 0.1 M at pH 7.4 by using HCl. Working standard
solution was obtained by appropriate dilution of the stock solution.
2.6. Molecular docking method
The molecular docking studies (rigid) were performed by using HEX 8.0.0 software,[34] which
is an interactive molecular graphics program for computing and exposing possible docking
modes of protein. The Hex 8.0.0 performs protein docking using Spherical Polar Fourier
Correlations (Ritche and Venkataraman, 2010). Hex 8.0.0 necessitates the ligand and the
receptor as input in PDB format. The parameters used for docking include correlation type –
shape only, FFT mode – 3D, grid dimension – 0.6, receptor range – 180, ligand range – 180,
twist range – 360, distance range – 40. The coordinates of complex 1 were taken from its crystal
structure as a .cif file and were converted to the PDB format using Mercury software. The crystal
structure of the DNA (PDB ID: 1BNA) human serum albumin (PDB ID: 1h9z) was obtained
from the protein data bank (http://www.rcsb.org./pdb). Visualization of minimum energy
favorable docked poses has been performed using Discovery Studio 4.1 and PyMol.[35]
2.7. Antitumor activity assays
The PC3 (Prostate) and K562 (Leukemia) cancer cell lines were used for in vitro antitumor
activity. In RPMI–1640 medium along with 10% Fetal Bovine Serum (FBS) and antibiotics to
study growth pattern of these cells, cell lines were procured and grown. The Sulphorhodamine–B
8

(SR–B) semi–automated assay was carried out to check the proliferation of the cells upon
treatment with the complex. In 96 well plates, cell lines were seeded and then the number of cells
counted, and by titration readings, the cell count was adjusted, to get the linear range (0.5 to 1.8)
of optical density and was incubated at 37 ºC in a CO2 incubator for 24 h. The stock solution of
the complex (100 mg/mL in DMSO) and four concentrations viz, 10, 20, 40, 80 µL, (in
triplicates) was tested, each well receiving 90 µL of cell suspension. The plates were labeled and
incubated for 48 h. Termination of the experiment was done by gently layering the cells with 50
µL of chilled 30% TCA (for adherent cells) and 50% TCA (for suspension cell lines) to fix cells
and preserved at 4 ºC for 60 min. 50 µL of 0.4 % SRB were used to stain the plates for 20 min.
All experiments were done in triplicate.
3. Results and discussions
The tetranuclear Cu(II) complex {[Cu4(HL)4(H2O)2]·(CH3OH)2·(H2O)5}(1) was synthesized by
mixing stoichiometric amounts of CuClO4·6H2O with Schiff base HL in methanol (Scheme 1),
which was characterized by analytical and spectral studies. The synthesized complex is stable to
air and soluble in DMF and DMSO. Furthermore, the molecular structure of the complex was
further confirmed by determination of the X–ray crystal structure which revealed that complex 1
exists in two different coordination a square pyramidal environment.
3.1. Spectroscopic Characterization
The IR spectrum of the complex in the region of 4000–400 cm–1 was recorded by using the KBr
pellet technique and analyzed in a careful comparison with that of the free HL ligand which
provides evidence for coordination of ligand to the metal ions. The IR spectrum of ligand LH
exhibited characteristic azomethine band ν(C=N) at 1637 cm–1 which was shifted to lower
wavenumber at 1607 cm–1 in the complex 1 indicating coordination via azomethane nitrogen to
9

the metal cores.[36] It was worth noticing that a sharp band at 1466 cm–1 assigned to the skeletal
vibration related to the phenolic oxygen of the free ligand was found at a lower frequency at
1422 cm–1 in the complex, ascribed to the phenoxo–O bonding in the complex and the
disappearance of the ν(OH) band at 3413 cm–1, indicates the complexation occurs via proton
displacement through a covalent bond with oxygen of the phenolic group [37]. Furthermore,
complex 1 shows a medium intensity bands at 424–467 cm–1 and 497–575 cm–1 were assigned to
stretching frequencies of ν(Cu–N) and ν(Cu–O), respectively which were absent in the spectrum
of the free ligand.
The electronic spectra of complex exhibited an intense transition at 295 nm, attributed to
the π–π* transition of the Schiff base ligand while the absorption band at 415 nm characteristic
of the complex appeared at higher complex concentration ascribed to the ligand to metal charge–
transfer. The complex 1 displayed d–d band in the range of low energy region with a maximum
appearing at 628 nm exhibiting different coordination environment around Cu(II) ion.[38]
The X–band EPR spectrum (Fig. S1) of the complex 1 was acquired at a frequency of 9.1 GHz
under the magnetic field strength 3,000 1,000 Gauss using tetracyanoethylene (TCNE) as field
marker (g = 2.0027) at LNT. The spectrum displayed a well resolved anisotropic signal with g|| =
2
2.24 and g⊥ = 2.09 and gav = 2.14 computed from the formula gav2 = g||2+2g⊥ /3. The trend of g
values (g||>g⊥>2.0023) observed for the complex 1 indicates that the unpaired electrons lie
predominantly in the dx2–y2 orbital of Cu(II) ion. For a Cu(II) complex, g|| is a moderately
sensitive parameter to indicate covalence. In case of covalent environment g|| < 2.3 and for ionic
environment g|| < 2.3 or more. In the Cu(II) complex, g|| < 2.3 indicates an appreciable metal–
ligand covalent character [39]. The spectrum displayed hyperfine splitting due to the coupling of
one unpaired electron with 63,65Cu nuclei. The geometric parameter “G,” which is an extent of
10

the exchange interaction between copper centers in the polycrystalline compound, is calculated
using Hathway expression (G = g||–2.0023/g⊥–2.0023).[40] According to Hathway, if the value
of G > 4, the exchange interaction is negligible in solid complexes but G < 4 indicates
considerable interaction in solid complexes. The observed ‘G’ value for complex 1 was 2.71,
which indicative of substantial exchange interaction among the Cu(II) centers in the solid phase
due to intermolecular spin exchange which is caused by the strong spin coupling during the
collision of paramagnetic centers.
3.2. Structure Description
Once isolated, complex 1 was found to be air–stable and soluble in common organic solvents
like ethanol, methanol, DMF and DMSO. Single crystals of tetranuclear complex 1, suitable for
X–ray crystallographic analysis, was achieved by slow evaporation of water and methanol (1:1)
solution at room temperature. The tetranuclear copper cluster of Complex 1 crystallizes in the
triclinic system and successfully converged and solved in P–1 space group (Fig.1a and b) The
asymmetric unit contains four Cu(II) ions, four HL2– ligands, two coordinated water molecules
and two methanol and five aqua molecules in the lattice. It possesses a distorted [Cu4(µ3–O)2]6+
open core with the four Cu atoms disposed of in an extended “butterfly–like” arrangement
(Fig.2). Selected bond distances and bond angles are given in Supporting Information, Table. S1.
The tetranuclear cluster consists of four symmetry–nonequivalent Cu(II) atoms, Cu1, and Cu4
centers exhibit CuN1O4 coordination environment arranged in distorted square pyramidal
geometries (τ=0.14). The Cu1 atom is essentially similar to Cu4, and the coordination
environment was ligated with planar O2, O3, O4 and N1atoms [Cu1–O= 1.935(5)–2.002(5)Å
and Cu1–N1= 1.952(6)Å] while the axial position is occupied by O1W atom [Cu1–O1W=
1.944(5) Å]. The Cu4 atom has identical coordination fashion as described for Cu1 atom and the
11

planar positions are satisfied by O8, O15, O16, N4 atoms [Cu4–O=1.945(5)–2.005(6)Å and
Cu4–N4=1.955(6)Å] and axial sites completed with O2W [Cu4–O2W=2.278(5)Å]. The six–
coordinated Cu2 represent distorted square pyramidal CuN1O4 environment, coordinated
through N3, O2, O7 and O8 atoms [Cu2–N3=1.950(7)Å and Cu2–O=2.001(5)–1.955(5)Å] in
equatorial positions of the H2L2– ligand moieties, whereas an axial site is occupied by O6 [Cu2–
O6= 2.356(5)Å] of H2L2– ligand. Interestingly, Cu3 atom also exhibited distorted square
pyramidal CuN1O5 geometry with τ = 0.12, the equatorial position coordinated from N2, O3,
O11 and O15 [Cu3–N2=1.926(6) Å and Cu3–O=1.964(5)–2.006(5)Å] and O14 [Cu3–
O14=2.314(5) Å] of H2L2– ligand moieties. In the tetranuclear core, the metal centers are
mutually interconnected via two phenoxo oxygen and two alkoxo oxygen atoms of four H2L2–
ligand to generate a distorted single–open [Cu4(µ3–O)2]6+ open cubane core with idealized S4–
symmetry. The molecules in the unit cell exhibited intricate π···π stacking and –CH···π
interactions which lead to the stabilization of the molecules and hence generated 2D
supramolecular architecture (Figs S2 and S3)
3.3. DNA binding studies
3.3.1. Absorption spectral studies
Absorption spectroscopy is an imperative method to examine the binding modes and propensity
for binding of the metal complexes with DNA. In general, complex binding with DNA via
intercalation results in hypochromism with a red shift (bathochromism) of the absorption band
due to stacking interaction between planar aromatic moieties present in the ligand and DNA base
pairs. On the other hand, hyperchromism is observed in the absorption spectra of a compound
with increasing concentration of DNA evidence the non–intercalative interaction between the
complex and DNA.[41] The absorption spectra of the tetranuclear copper(II) complex 1 in the
12

absence and presence of ctDNA was given in Fig.3. With the incremental amount of ctDNA (0–
5.25 x 10–5 M) to complex 1 of fixed concentration (6.67 x 10–6 M), an increase in the molar
absorptivity, hyperchromism of the π–π* absorption band was observed which reflects the
binding propensity of the complex 1 for DNA. The observed hyperchromic effect suggests that
the binding of complex 1 towards CT DNA most likely through external contact (electrostatic
binding) with the phosphate backbone of DNA double helix owing to the degradation of the
DNA double–helix structure [42]. Nonexistence of the isosbestic point in the absorption spectra
of the complex 1 upon addition of DNA implies that 1:1 drug: DNA stoichiometry is probably
not maintained during the binding process and hence, more than one type of binding of the
complex 1 with DNA cannot be ruled out.
To quantify the binding affinity of complex 1 to CT–DNA, the intrinsic binding constant,
Kb was calculated by monitoring the changes in the absorbance at the corresponding λmax with
increasing concentrations of CT–DNA and is given by the ratio of the slope to the intercept in
plots from the Wolfe–Shimer Eq. (1), via relationship plot of [DNA]/εa–εf vs. [DNA], where
[DNA] represents the concentration of DNA, and εa, εf and εb the apparent extinction coefficient
(Aobs/[M]), the extinction coefficient for free metal complex (M), and the extinction coefficient
for the metal complex (M) in the fully bound form, respectively. In plots of [DNA]/εa–εf vs.
[DNA], Kb is given by the ratio of slope to intercept [43].
[DNA] [DNA]
εa −ε f εb −ε f Kb (εa −ε f )
1
(1)
=
+
The Kb value of 1 was found to 4.63 x 104, indicating that 1 avidly binds to CT DNA through
hydrogen–bonding interactions between coordinated mutihydroxy –OH and –C=N groups of
Schiff base ligand with DNA nucleobases in addition to the cationic core of metal complex to the
13

oxygen phosphate backbone of DNA. We have adopted other experimental tools as described in
the forthcoming sections to further establish the mode of binding of complex 1 with DNA.
3.3.2. Fluorescence spectral studies
To further investigate the mode of binding of the complex 1 with DNA, fluorometric titration
studies were carried out. Complex 1 displayed maximum emission wavelength at 354 nm in
0.01Tris–HCl/50 mM NaCl buffer when excited at 270 nm. With incremental addition of ctDNA
concentration (0–5.25 x 10–5 M), to fixed amount of 1, the emission intensity of intensity
gradually increases without any apparent change in the shape and position of the emission
maximum (Fig. 4), indicating that complex 1 penetrates into the hydrophobic environment inside
the DNA, thereby avoiding the quenching effect of solvent water molecules. Thus, because of
the hydrophobic environment inside the DNA helix reduces accessibility of the solvent
molecules and restricts its mobility at the binding site which causes reduction of the vibrational
modes leading to higher emission intensity.[44] Hydrophobic interactions between the complex 1
and polyelectrolyte DNA may induce changes in the excited state properties either due to the
electrostatic association or intercalation.[45] The intercalative mode of binding will be sensitive
to ligand characteristics such as planarity of ligand, the extent of aromatic π system available for
stacking and depth of ligand which can penetrate into the double helix. On the other hand, the
electrostatic interaction would be more sensitive to the charge of the metal ion, ligand
hydrophobicity, and size of the complex. An observed increase in emission intensity is
associated with the electrostatic interaction. The binding constant K was calculated by the
Scatchard equations.[46]
CF = CT (I / Io − P)(1− P)
(2)
14

r / CF = K(n − r)
(3)
where, CF is the free probe concentration, CT is the total concentration of the probe added, I and
I0 are fluorescence intensities in presence and absence of CT–DNA, respectively and P is the
ratio of the fluorescence quantum yield exhibited by the bound probe to that of the free probe.
The value P was obtained as the intercept by extrapolating from a plot of I/Io vs. 1/[DNA], r
denotes ratio of CB (= CT–CF) to the DNA concentration, i.e., the bound probe concentration to
the DNA concentration, K is the binding constant and CF, is the free metal complex
concentration and “n” is the binding site number. The measured binding constant (K) value of
complex 1 with CT–DNA was found to be 5.26 x 104 M–1, consistent in magnitude with the value
obtained from electronic absorption studies. Three−dimensional (3D) fluorescence spectroscopy
was recorded from 200 to 700 nm with 1 in the absence and presence of CT−DNA to confirm the
interaction of 1 with the DNA. The 3D fluorescence spectrum and contour ones of 1 consisted of
two prominent peaks, peak A (λex = 270 nm) and peak B (λem = 354 nm) while Peak C comes
due to the second–ordered scattering peak (λem = 2λex) (Fig. 5). In the presence of DNA, the
fluorescence intensity of excitation peak A and peak B increases significantly which clearly
indicated that complex binds to DNA and could be protected efficiently from the hydrophobic
environment inside the DNA helix consistent with other DNA binding studies.
To further examine the interaction mode of complex 1 with DNA, steady–state emission
quenching experiments of ctDNA bound complex 1 was studied in relation to the quenching of
complex in in 0.01Tris–HCl/50 mM NaCl buffer using [Fe(CN)6]4– as a quencher. [Fe(CN)6]4– is
a dynamic fluorescence quencher and affords a sensitive tool to examine the nature of the
interacting probe molecule with DNA.[47] [Fe(CN)6]4– poorly quench the fluorescence of
complexes which are strongly bound to the DNA whereas the complexes which are free in
15

solution are quenched efficiently due to ion pairing.[48] The quenching efficiency is evaluated
by the Sterne–Volmer equation. The emission intensity of the complex 1 was greatly affected by
the addition of anionic quencher. The decrease in emission intensity of the complex was due to
the repulsion of highly negative charged [Fe(CN)6]4– from the DNA polyanion backbone, which
hinders access of [Fe(CN)6]4– to the DNA–bound complexes.[49] The Ksv value obtained for free
complex 1 was 1.34 x 102 M–1. In the presence of complex bound to DNA at saturation level of
interactions, the quenching slope was depressed reflecting the protection of complex by the DNA
helix and the Ksv value of 1 decreased to 85 M–1 (Fig. S4). From the Ksv value, it was
undoubtedly perceived that no appreciable reduction in the quenching efficiency thus rules out
the intercalation binding and indicates that complex 1 resides at the DNA exterior phosphate
backbone which is directly accessible to the quencher that resides in the aqueous phase.
3.3.3. Viscosity measurement
To decipher the binding mode of the complex 1 towards DNA, viscosity measurements was
carried out on CT DNA solutions.[50] Hydrodynamic measurements that are sensitive to length
change are regarded as the least ambiguous most critical tests of a binding model in a solution in
the absence of any crystallographic data. The addition of complexes results in a decrease in the
relative viscosity of DNA indicating that the complexes could bend and kink the DNA helix,
reducing its effective length and consequently its viscosity. Since DNA is a polyanion, the
repulsion between the negative charges makes the DNA molecule more extended in solution.
The negative charges of DNA are partially neutralized when the complexes bind to DNA through
electrostatic interaction to the phosphate group of DNA backbone leading to contraction of the
DNA helix, shortening of DNA molecule and a decrease in viscosity of DNA. On contrary,
lengthening of DNA helix occurs on intercalation as base pairs are separated to accommodate the
16

binding ligand leading to swelling in DNA viscosity a classical intercalative mode causes a
significant increase in viscosity of DNA solution due to its increase in separation of base pairs at
intercalation sites and increases in overall DNA length.[51] The relative specific viscosity of
DNA decreases steadily with increasing concentration of the complex 1, indicating that the DNA
becomes more compact because of its interaction with 1 via electrostatic mode which leads to the
bending of the DNA chain resulting in a decrease in the DNA viscosity (Fig 6). Thus, the results
obtained from viscosity studies validate those obtained from UV–Vis and fluorescence titrations.
3.3.4. Circular dichroic studies
Circular dichroic studies are useful for monitoring changes in the morphology of DNA during
complex–DNA interactions.[52] The observed CD spectrum of CT DNA consists of a positive
band at 276 nm due to base stacking and a negative band at 242 nm due to helicity which is
characteristic of DNA in the right–handed B–form, in the UV region.[53] The CD spectra of
tetranuclear complex 1 on the addition of CT DNA does not reveal any significant change in the
intrinsic CD spectrum of the DNA indicating that the binding of the probe with ctDNA does not
disturb the stacking of the bases (Fig. 7). Both the bands show a slight change in the intensities.
Groove binding and electrostatic interaction of the complexes with DNA reveals slight changes
in the intensity of both negative as well as a positive band of DNA.[54] Therefore, this spectral
changes categorically rules out intercalation of 1 within the DNA helix and thereby implies that
the complex 1 binds to the host DNA through groove binding that stabilizes the right–handed B–
form of DNA. By all of the spectroscopic studies, we concluded that the complex 1 binds to CT
DNA preferentially in a non–covalent groove binding mode.
17

3.3.5. Thermal Melting
The binding mode of interaction between complex 1 and CT–DNA has also been corroborated
from the DNA helix melting experiment. The temperature at which 50% of double–stranded
DNA becomes single stranded is melting temperature (Tm). The breakage of ds–DNA into single
strand DNA can be detected by the hyperchromic effect in the UV absorption because the molar
extinction coefficient at 260 nm of the DNA base pairs of ss–DNA is much higher than in ds–
DNA. In general, groove binding and electrostatic binding along the phosphate backbone of
DNA gives rise to only a small change in thermal denaturation temperature, while intercalation
leads to a significant increase in thermal denaturation temperature of DNA due to the
stabilization of the DNA double helix.[55] The DNA melting curves in the absence and presence
of complex 1 were presented in Fig. 8. The estimated Tm value of ctDNA in the absence of 1
(unbound DNA) was 65.69 ºC. However, it was increased by only 1 ºC in the presence of
complex 1 in our experimental conditions. Thus, we conclude that the ∆Tm value of DNA in the
presence of complex 1 does not lead to an appreciable change, which was characteristic of non–
intercalative binding behavior.
3.4. HSA binding studies
3.4.1. Fluorescence quenching studies with HSA
Investigation of the interaction of drugs with human serum albumin can elucidate the properties
of drug–protein complex, as it may provide useful information on the structural features that
determine the therapeutic effectiveness of drugs. In particular, fluorescence spectroscopy is an
effective method to explore the interaction between the drug molecule and the receptor (serum
albumins). Fluorescence of HSA is associated with the tryptophan and tyrosine residues. When
the HSA molecule is excited at 280 nm, both tryptophan and tyrosine amino acid residues give
18

fluorescence emissions, however, at an excitation wavelength of 295 nm only tryptophan residue
has strong fluorescence emission while the emission from tyrosine get quenched totally, if it is
ionized or having a carboxyl, an amino group, or a tryptophan in its close proximity. Thus, the
fluorescence of HSA is dominated by the Trp–214 residue in subdomain IIA.[56,57] When a
small molecule binds to HSA, the microenvironment of Trp residues gets disturbed that gives
rise to small changes of intrinsic fluorescence intensity of HSA. The emission spectra of HSA in
the absence and presence of complex 1 as a quencher in Tris–HCl buffer (pH 7.4) were
monitored at 370 nm (Fig.9). On addition of increasing concentration of 1 (0.67 x 10–5 to 4.6 x
10–5 M) to fixed amount of HSA, the intrinsic fluorescence intensity of HSA decreases gradually
with a red shift in the emission maxima. The observed quenching of HSA emission clearly
implicates the existence of an interaction between complex 1 and HSA which changes the local
environment around Trp–214 residues and the tertiary structure of HSA.[58]
The fluorescence intensity of a molecule can be quenched by several mechanisms of
interaction, viz, molecular rearrangements, excited–state reactions, complex formation, energy
transfer, and collision quenching.[59] Collisional or dynamic quenching refers to a process that
the fluorophore and the quencher come into contact during the excited–state, whereas static
quenching refers to fluorophore–quencher complex formation.
To determine the fluorescence quenching mechanism of HSA in the presence of 1,
classical Stern–Volmer equation is applied:
Fo
=1+ Kqτo[Q] =1+ Ksv[Q]
(1)
F
where, Fo and F are the fluorescence intensities in the absence and presence of quencher,
respectively, Kq, Ksv, τo and [Q] are the quenching rate constant of the biomolecules, the Stern–
19

Volmer quenching constant, the average life–time of the molecule without quencher (τo =10–8 s)
and the concentration of the quencher, respectively. The Stern–Volmer plots of Fo/F versus [Q]
for the quenching of HSA fluorescence by 1 was depicted in Fig. S5 and the calculated KSV and
Kq values are found to be 2.24 × 103 M–1 and 2.24 × 1012 M–1s–1 respectively. The observed Kq
value is larger than the limiting diffusion constant Kdif of the biomolecules (Kdif = 2.0×1010 M–1s–
1), indicating that the fluorescence quenching is mainly due to the specific interaction of 1 with
HSA, consistent with the static quenching mechanism.[60] For static quenching, the Scatchard
equation is employed to calculate the binding constant and number of binding sites [61]:
F − F




o
log
= log K + nlog[Q]
(2)


F
where, Fo and F are the fluorescence intensities of HSA in the absence and presence of quencher,
K and n are the binding constant and the number of binding sites, respectively. Thus, a plot of
log[(Fo–F)/F] versus log[Q] is used to determine K (binding constant) from the intercept on Y–
axis and n (binding sites) from the slope (Fig. S6). From the corresponding Scatchard plot, the K
and n values for 1 is calculated to be 1.6 x 104 M−1 and 1.1 respectively. The calculated K value
of 1 is lower than the association constant of one of the strongest known non–covalent bonds of
avidin–ligands interaction (K ≈ 1015 M−1) suggesting a possible release from the serum albumin
to the targeted cells.
To gain an understanding of the conformational changes of proteins, 3D fluorescence
spectral studies were carried out in absence and presence of the complex 1. The 3D fluorescence
spectra and contour ones of HSA alone and in the presence of complex 1 were shown in Fig. 10.
Peak A is the Rayleigh scattering peak (λex = λem). Peak B (λex = 295, λem = 370 nm)
dominatingly displays spectral behavior of the Trp residue, and the fluorescence intensity of this
20

residue is associated with the micro–environment’s polarity of HSA. The results reveal that
fluorescence intensity of peak A increases and that of peak B (295, 370 nm, λex, λem) decreases
significantly indicating quenching of fluorescence induced by Trp residue of HSA. However, the
fluorescence intensity of peak B is strongly quenched revealing that 1 binds to HSA near the
tryptophan residues.[62] The results show that the interaction of 1 with HSA induced micro–
environmental changes in the structure of HSA, corroborated well with our spectroscopic results
obtained from UV–vis, and fluorescence measurements.
3.4.2. Investigations on the circular dichroism of HSA
Circular dichroism is sensitive to the variation of the structure of HSA. To determine the
potential impact of complex 1 binding on the secondary structure of HSA, CD spectra of HSA in
absence and presence of complex 1 was measured in the UV range of 200–400 nm (Fig. 11). The
CD spectra of free HSA exhibit two negative bands in the ultraviolet region at 208 and 222 nm
attributed to n–π* transfer for the peptide bond of α–helix.[63] It was observed that in the
presence of complex 1, the CD signal of HSA increased which indicated that the interaction of
complex 1 to HSA induces a significant conformational change in HSA which increases the
helical secondary structure content. However, the CD spectra of HSA in the presence or absence
of complex 1 was similar in shape, indicative of the structure of HSA as predominantly α–
helical. The CD results were expressed in terms of MRE (mean residue ellipticity) in deg cm2 d
mol–1 as the following equation:




Observed CD(mdeg)
C pnl ×10
(15)
MRE =




21

Where, Cp molar concentration of the protein, n the number of amino acid residues (585) and l
the path length (0.2 cm). By the MRE value at 208 nm, the α–helical contents of free and
combined HSA were calculated adopting the following equation as described earlier [64]:
− (MRE) − 4000


208
(16)
α − helix(%) =
×100




33000− 4000
Where, the value of MRE at 208nm is MRE208, at 208 nm, the MRE of the β–form and random
coil conformation cross is 4000, and of a pure α–helix is 33000.
From Eq.16, the quantitative analysis results of the α–helix in the secondary structure of HSA
were obtained. They differed from that of 57.51% in free HSA to 41.32% after interaction with
complex 1 at our experimental conditions. The decrease in α–helix character in the presence of
complex 1 suggested that interaction between complex 1 and HSA leads to a change in
secondary structure of HSA. From the above results, it was apparent that the effects of complex 1
on HSA cause a conformational change of the protein, with the loss of helical stability.[65]
3.5. Nuclease Activity
3.5.1. DNA cleavage without added reductant
To assess the chemical nuclease activities of the tetranuclear copper(II) complex 1 for DNA
strand scission, pBR322 DNA was incubated with the complex 1 in 5 mM Tris–HCl/50 mM
NaCl buffer (pH 7.3) in the absence of any external additives (Fig.12). The evaluation of the
DNA cleavage activity can be monitored by gel electrophoresis. When circular pBR322 DNA is
subjected to electrophoresis, relatively fast migration will be observed for the intact supercoiled
form (Form I). If one strand scission (nicking), the supercoiled form will relax to generate a
slower–moving nicked form(Form II). However, if both strands get cleaved, a linear form (Form
III) that migrates between Form I and Form II will be generated.[66] The pBR322 DNA
22

exhibited two clear bands in the absence of the complex 1(Lane 1), as expected. The intense
band corresponded to Form I, and the other band corresponded to Form–II. DNA cleavage
activity was clearly observed in the presence of complex 1 (Lanes 2–7). At the lowest complex 1
concentration, the two bands exhibited similar intensities, suggesting that nicked form. As the
complex 1 concentration increased (Lanes 4 and 5), the intensity of these two bands decreased,
which was accompanied by the appearance of Form III DNA before the disappearance of Form I
DNA (Lanes 6 and 7). Hence, the complex 1 exhibited DNA cleavage activity in the absence of
an external agent.
3.5.2. DNA cleavage in the presence of activators
To gain some insight into the DNA cleavage pathway of the presented complex 1, furthermore,
we studied the effect of some activating agents such as H2O2, 3–mercaptopropionic acid (MPA)
and ascorbic acid (Asc) (Fig. 13a). The nuclease efficiency of the copper(II) complexes is known
to depend on the activators used for initiating the DNA cleavage.[67] The cleavage activity of 1
was significantly enhanced by these activators. Their activating efficacy follows the order Asc
>MPA >H2O2 (Lane 2–4). Thus, complex 1 exhibited a significant DNA cleavage activity in the
presence of ascorbic acid. Several authors have studied the influence of different activators on
the cleavage of DNA by copper(II) complexes.[68–71] Chiou et al. have shown that ascorbate is
more efficient in cleaving DNA than MPA due to its ability to generate hydrogen peroxide in the
presence of oxygen and metal ions.[72] The addition of H2O2 to the system also shows some
increase in the DNA cleavage efficiency of the complex 1. This can be a result of the reduction
of complex 1 to Cu+ followed by the oxidation to Cu2+, again with the formation of ROS species.
In both instances (addition of ascorbic acid or H2O2), the redox cycling of Cu2+/Cu+ results in the
production of ROS that are responsible for the nuclease activity exhibited by the 1.
23

3.5.3. DNA cleavage in the presence of reactive oxygen species (ROS)
To apprehend evidence about the active oxygen species which was responsible for the DNA
damage, we investigate the mechanism of DNA cleavage mediated by complex 1 in presence of
some standard radical scavengers viz; DMSO, tert–butyl alcohol as hydroxyl radical scavengers,
sodium azide as singlet oxygen scavenger and SOD as superoxide oxygen scavenger under
identical conditions (Fig. 13a). On adding DMSO and tert–butyl alcohol (Lanes 5 and 6), DNA
cleavage were inhibited suggestive of the involvement of diffusible (●OH) hydroxyl radicals as
one of the ROS responsible for DNA breakage. On the other hand, the addition of sodium azide
(singlet oxygen scavenger) and superoxide dismutase (superoxide scavenger) to the reaction
mixture did not show any effect on the cleavage reaction revealing which obviously ruled out the
possibility of singlet oxygen and superoxide anion responsible for the cleavage (Lanes 7 and 8).
Thus, freely diffusible oxygen intermediate or hydroxyl radical is involved in the strand scission,
and hence a simple diffusible radical mechanism is applicable.
The mechanistic pathway proposed for the interaction of complex 1 with DNA was given
below: The first step is the interaction of Cu(II) complex with DNA through outer sphere, and
then in the second step, reduction of the Cu(II) to Cu(I) complex by the reaction with the
¯
reducing agent. The reaction of the cuprous state with dioxygen leads to the generation of O2 .
¯
Cu(I)+ O2 → Cu(II) + O2
(1)
In this system, it is thought that superoxide dismutase to H2O2,
¯
2O2 + 2H+ → O2 + H2O2
(2)
The ROS formed are responsible for initiating DNA strand scission chemistry.
ROS species + DNA → DNA cleavage. (3)
24

3.5.4. DNA cleavage in presence groove binders
The DNA recognition (groove binding) propensity of the complex 1 was studied by using minor
groove binding agent, DAPI and major groove binding agent, methyl green to probe the potential
interacting site of complex 1 with supercoiled plasmid pBR322 DNA. The supercoiled DNA was
treated with DAPI or methyl green earlier to the addition of complex 1. The patterns presented in
the (Fig.13b), demonstrated that neither major nor minor groove grooves are the favored
responding sites for the complex. Z. Guo et al. have attributed such pattern of gel electrophoresis
to the interaction of 1 directly with outside phosphates of DNA via electrostatic attraction [73].
3.6. Molecular docking study of 1 with DNA
The docked model (Fig.14) revealed that complex 1 was tailored tightly into the outside edge
curved contour of the targeted DNA within G–C rich region, anchored in such a way that
successfully formed Hydrogen bonding with the sugar phosphate back bone and planar part of
the aromatic rings made favorable stacking interactions between DNA base pairs and produced
electrostatic and hydrophobic contacts with the DNA functional groups which would define the
stability of the docked complex, detail description of which is given in Table 2. The resulting
relative binding energy of docked structure was found to be –325.49 eV, indicating the strong
binding affinity between DNA and complex 1, correlating well with the experimental DNA
binding studies and minor groove binder, gel mobility assay.
3.7. Molecular docking study of 1 with HSA
To provide considerable insight into the interactions of HSA with complex 1, a molecular
docking technique was employed to find out the exact binding sites inside the molecular target
HSA. From the 3–D structure of crystalline albumin, it is known that HSA encompasses three
homologous domains (I, II, and III): I (1–195), II (196–383) and III (384–585) residues that
25

assemble to form a heart–shaped molecule. The principal region of complex 1 binding sites of
HSA is located in hydrophobic cavities in subdomains IIA and IIIA, corresponding to sites I and
II, respectively, and the tryptophan residue (Trp–214) of HSA in subdomain IIA. A large
hydrophobic cavity is present in subdomain IIA (a binding site I) to accommodate complex 1.
The minimum energy docked pattern (Fig.15) indicates that complex 1 is located within the
subdomain IIA of HSA close to the TRP214 residue, forming numerous hydrophobic contacts
(Pi–Pi T–Shaped, Pi–Alkyl, and Alkyl) with HIS242, LYS195, CYS200, CYS245, CYS246,
ALA291 residues of hydrophobic binding site IIA. Furthermore, there are also a number of
hydrogen bonds and specific electrostatic interaction formed by the complex 1 (Table 3). The
docked structures, binding energy was found to be –436.17 eV indicating efficient binding
affinity. The molecular docking studies results revealed that the interaction of complex 1 with
the HSA was dominated by the hydrophobic forces, correlating well with the experimental
fluorescence quenching studies.
4. Antitumor activity assays
In vitro anticancer activity of tetranuclear complex 1 has been evaluated in terms of GI50, TGI
and LC50 (Table 2) values against two different human carcinoma cell lines of histological origin:
PC3 (Prostate), HL60 (Leukemia). The Sulforhodamine–B (SRB) assay was used to assess the
cellular proliferation [74]. A close inspection of cytotoxic data revealed that complex 1 exhibited
selectivity for both phenotypes of cancer cell lines PC3 (Prostate) and HL60 (Leukemia) with
pronounced GI50 values 35.3 and 32.2 µg/ml, respectively which was near to the GI50 value <10
µg/ml in case of well–known drug adriamycin (Fig. 14a and b). The results showed it has the
potential to act as antitumor drug candidate as expected from in vitro DNA and HSA binding
studies. However, at this stage, we are not committing any better prospects than adriamycin,
26

further in vivo investigations are in progress for a better understanding of the mechanism of the
antiproliferative activity.
5. Conclusion
Herein, we have described the synthesis and characterization of new tetranuclear copper(II)
complex. The complex has distorted open–cubane Cu4O4 core in which copper and oxygen
atoms are present at alternative vertices. The central core of the complex has two types of chelate
rings; (i) a five–membered Cu2NO2 chelate ring and (ii) a six–membered Cu2N2O2 chelate ring
with a half–chair conformation. The in vitro DNA binding studies of complex 1 was carried out
by using various biophysical techniques which revealed the electrostatic mode of binding with
DNA with Kb value 4.5 x 104 M–1. The gel electrophoresis assay demonstrated that the complex
1 cleaves supercoiled plasmid DNA in a concentration dependent manner and mechanistic
investigation showed that the reactive oxygen species was responsible for cleavage activity.
Furthermore, affinity of complex 1 for HSA was investigated in order to appreciate the carrier
role of serum albumin for complex 1 towards molecular target DNA, and experimental results
established that complex 1 binds to HSA with low affinity as compared to DNA, suggesting a
possible release from the serum albumin to the DNA in the targeted cells. These results were
further validated by molecular docking studies. The in vitro cytotoxicity of the complex 1 against
the PC3 (Prostate) and K562 (Leukemia) cancer were low that does not mean that this molecule
does not have potential. There are several drugs that have shown low activity in vitro analysis
but found to be very active on in–vivo results. These findings should be valuable in
understanding the relationship of DNA–binding behaviors of tetranuclear complexes as well as
laying a foundation for the rational design of novel, powerful agents for probing and targeting
27

nucleic acids and proteins. Furthermore, the in vivo studies are needed to warrant its application
as a potential drug candidate.
Acknowledgements
This project was financially supported by King Saud University, Vice Deanship of Scientific
Research, Research Chairs. We express our gratitude to Department of Chemistry, Aligarh
Muslim University, India for instrumentation. We gratefully acknowledge the IIT Kanpur for X–
Ray crystallography.
Supporting Information
Tables presented for selected bonds lengths and bond distances, EPR, Stern–Volmer plots plot of
the fluorescence quenching of complex 1 with DNA and HSA and Logarithmic plot of the
fluorescence quenching of HSA in the presence of complex 1. Crystallographic data in CIF
format have been deposited with the Cambridge Structural Database (CCDC 1523085).
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