Synthesis and characterization of copper–based anticancer compound; in?vitro interaction studies with DNA/HSA, SOD mimetic, cytotoxic activity and molecular docking investigation

Article abstract of DOI:10.1080/07391102.2020.1733663

A new copper(II) complex derived from the ligand ethylene [O,O′–bis{5–{(1E)–(2 hydroxyphenyl)methylene]amino} ? 2–hydroxybenzoic acid] was designed, synthesized and characterized by spectroscopic (IR, UV–Vis, ESI–MS, X–ray diffraction, EPR and elemental a

Full text of DOI:10.1080/07391102.2020.1733663

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Synthesis and characterization of Copper–based
anticancer compound; in vitro interaction studies
with DNA/HSA, SOD mimetic, cytotoxic activity and
molecular docking investigation
Waddhaah M. Al–asbahy & Manal Shamsi
To cite this article: Waddhaah M. Al–asbahy & Manal Shamsi (2020): Synthesis and
characterization of Copper–based anticancer compound; inꢀvitro interaction studies with DNA/
HSA, SOD mimetic, cytotoxic activity and molecular docking investigation, Journal of Biomolecular
Structure and Dynamics, DOI: 10.1080/07391102.2020.1733663
To link to this article: https://doi.org/10.1080/07391102.2020.1733663
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Synthesis and characterization of Copper–based anticancer compound; in
vitro interaction studies with DNA/HSA, SOD mimetic, cytotoxic activity and
molecular docking investigation
*
Waddhaah M. Al–asbahy and Manal Shamsi
Department of Chemistry, Taiz University, Taiz, Yemen
*
Correspondence: Waddhaah M. Al–asbahy, Department of Chemistry, Faculty of Science,
Taiz University, Taiz, Yemen. E–mail address: wadahm2007@yahoo.com (W.M. Al–asbahy).
Tel.: +967–777439393
ABSTRACT
A new copper(II) complex derived from the ligand ethylene [O,O′–bis{5–{(1E)–(2
hydroxyphenyl)methylene]amino}–2–hydroxybenzoic acid] was designed, synthesized and
characterized by spectroscopic (IR, UV–Vis, ESI–MS, X–ray diffraction, EPR and elemental
analytical methods. In vitro interaction studies were carried out with CT DNA, HSA and SOD
targets by employing various biophysical methods viz., UV–Vis, fluorescence, FTIR (in case of
HSA only), circular dichroism and gel electrophoresis. Notably, the copper(II) complex
exhibited a high propensity for DNA binding via electrostatic mode; The results of fluorescence
titration revealed that the copper(II) complex strongly quench the intrinsic fluorescence of HSA
through a static quenching procedure. Copper(II) complex cleaves pBR322 DNA via hydrolytic
mechanism and strongly binds to the DNA minor groove. Furthermore, Copper(II) complex
cleaves pBR322 DNA via hydrolytic pathway. Our results showed that copper(II) complex is

capable of inhibiting the growth of MCF7, A2780 and K562 tumor cell lines revealed
–
1
remarkable cytotoxicity of Cu(II) complex with GI values <10 μg mL for most of the cell
5
0
lines tested, which accompanied by systemic toxicity as was evident from the slightly significant
histopathologic alterations in liver of mice. In addition, Cu(II) complex exhibit excellent
superoxide dismutase (SOD) mimetic activity with an IC50 of 0.109 μM. Molecular docking
studies of the Cu(II) complex were carried out with DNA, HSA and SOD targets to ascertain the
specific binding mode thereby substantiated the spectroscopic results.
KEY WORDS: Cu(II) complex, DNA binding, SOD mimic, Albumin, Aminosalicylic acid,
Molecular docking.
1
. Introduction
Cancer is a multifaceted class of disease in which cells divides wildly away from the normal
limits, consequently, intruding to the near or/distant tissues (metastasis), and ultimately leads to
death (Waddhaah, et al., 2016). Cancer is the second leading cause of death globally and is a rise
in cancer incidence which is predicted to increase from 12.8 million new cases in 2008 to 22.2
million in 2030 (Bray, Jemal, Grey, Ferlay, & Forman, 2012). In a recent report in Nature,
cancer surpassed all other causes of mortality, outweighing all other health concerns viz.
cardiovascular diseases, cerebrovascular diseases, HIV/AIDS, lower respiratory infection
including pneumonia, malaria, and cirrhosis, as well as road accidents (Brody, 2011). Therefore,
an important challenge for chemist today is in the area of medicinal chemistry, to develop and
seek new drugs for treating chronic diseases which show promise i) good intrinsic properties,
including saline solubility and enough stability to arrive intact at the cellular target; ii) efficient

transport properties in blood and through membranes; iii) efficient DNA–binding properties but
slow reactivity with proteins; iv) the ability to differentiate between cancerous and normal cells;
and (v) activity against tumors that are, or have become, resistant to cisplatin and derivatives.
(
Imtiyaz, et al., 2019). Metal complexes have been extensively explored for anticancer activities
after the protocol success of cisplatin for treating most aggressive solid tumors. The strategies for
rational drug design can be envisaged by studying the interactions of small molecules with DNA
and also their reaction at specific sites along a DNA strand (Ananya, & Santanu, 2012).
Copper complexes have been studied on the supposition that endogenous metals may be less
toxic for normal cells in comparison to cancer cells (Fatemeh, & Hassan, 2018). The altered
metabolism of cancer cells and reverent response to Cu are the root for progress of copper
complexes able to have antineoplastic characteristic (Carlo, et al., 2014). It has been shown that
copper accumulates in tumors due to the selective permeability of cancer cell membranes to
copper compounds. Because of this, a number of copper complexes have been screened for anti–
cancer activity and some of them were found active both in vivo and in vitro (Niladri, et al.,
2
019). Much attention has been directed towards the design of metal‐based cancer
chemotherapeutics, the design of new metal‐based pharmaceuticals depending on the ligand
framework and the choice of metal ion. Ligands can modify the reactivity, stabilization of the
oxidation state and cytotoxicity of metal ions in metal‐based medicinal agents depending on the
requirements for chemotherapy (Waddhaah, Sabah, and Manal, 2018). In this regard,
Aminosalicylic acid (5–ASA) has been utilized to combat many inflammatory diseases such as
Crohns disease and ulcerative colities. Anti‒inflammatory drugs have been shown to be active
for the treatment or prevention of cancer in particular; colorectal cancer (Farukh, and Mohd,
2
010). On the other hand, human serum albumin (HSA) is the principal plasma protein in the

bloodstream, that greatly augments the reversible binding and delivering of a variety of diverse
endo– and exogenous drug entities. In particular, human serum albumin (HSA) is known to
accumulate in tumors, being taken up by tumor cells at increased levels compared to normal
cells, and has been exploited as the carrier conjugate of various organic anticancer drugs such as
chlorambucil, doxorubicin, and paclitaxel (Sabah, Manal, and Waddhaah, 2018). Therefore,
drug–HSA interaction studies provide a deeper understanding of the absorption, transportation,
distribution, metabolism and excretion properties of drug and hence, are recognized as a crucial
step in assessing the therapeutic effectiveness as well as pharmacological response of the drug in
the body (Shipra, et al, 2015). Moreover, The small molecular weight SOD mimics transition
metal complexes which are able to catalyze the dismutation of superoxide anion and therefore
provide a suitable alternative to SOD in clinical application. These studies provide an important
rationale for design of new lead anticancer drugs and their specific delivery at the active site of
action, besides providing the pharmacological profile in vivo and in vitro.
We report herein, the synthesis, spectroscopic characterization and in vitro DNA/HSA binding
studies of the Cu(II) complex. We also demonstrated the in vitro antitumor activity of Cu (II)
complex against a panel of different human cancer cell lines (U373MG, PC3, MCF7, K562,
HCT15, Hop–62, and A2780) and SOD activity at the molecular level. Additionally, molecular
dockingNstudies have been employed to explore the orientation, specific binding sites and non–
covalent interaction of Cu(II) complex within the binding cavity of targeted biological
macromolecule (DNA, HSA and SOD).

2
. Experimental
2
.1. Reagents and materials
Commercially available materials were used as received. Salicylaldhyde (alfaaster), 1,2
dibromoethane (merck), 5−aminosalcylic acid disodium salt of calf thymus (CT DNA)
Tris(hydroxymethyl)aminomethane, agarose gel, ascorbic acid, sodium azide (NaN3),
dimethylsulfoxide (DMSO), superoxide dismutase (SOD), methyl green DAPI,
mercaptopropionic acid (MPA) (Sigma Aldrich), Cu(NO ) .3H O (Fisher Scientific) and
3
2
2
supercoiledpBR322 DNA (Genei), 6× loading dye (Fermental Life Science). and Supercoiled
plasmid DNA pBR322 (Genei) were utilized as received. Disodium salt of calf−thymus DNA
(CT‒DNA), Human serum albumin (HSA) with a molecular mass of 66 kDa were purchased
○
from Sigma Chem. Co. and stored at 4 C. Doubly distilled water was used as the solvent
throughout the experiments. All solvents were analytical grade.
2
.2. Methods and instrumentation
Microanalysis carbon, hydrogen, and nitrogen contents were determined using Carlo Erba
Analyzer Model 1108. Molar conductance was performed at room temperature on a Digisun
Electronic conductivity Bridge. Fourier−transform IR (FTIR) spectra were recorded on an
Interspec 2020 FTIR spectrometer. Electronic spectra were recorded on UV−1700 PharmaSpec
spectrophotometer (Shimadzu). Data were reported in λmax/nm. The EPR spectrum of the copper
complex was acquired on a Varian E 112 spectrometer using Xb and frequency (9.1 GHz) at
liquid nitrogen temperature in the solid state using tetracyanoethylene (TCNE) as field marker.
TGA was performed with a universal V3.8 B TA SDT Q600 Build 51 Thermal Analyzer under
nitrogen atmosphere using alumina powder as reference material. X−ray powder diffraction

(XRPD) was recorded on Rikagu mini Flex II Instrument. Electrospray mass spectra were
recorded on Micromass Quattro II triple quadrupole mass spectrometer. Fluorescence spectra
were recorded on Hitachi F−2500 fluorescence spectrophotometer equipped with quartz cells
(1.0 cm), setting the widths of both excitation and emission slits to 10 nm. CD spectra were
measured on Jasco J−815 CD spectropolarimeter at room temperature using a 1 cm quartz
cuvette. 3D fluorescence spectra were recorded on Hitachi FL–4500 fluorescence
spectrophotometer. Cleavage experiments were performed with the help of Axygen
electrophoresis supported by a Genei power supply with a potential range of 50–500 V,
visualized and photographed by Vilber INFINITY gel documentation system.
2
.3. Synthesis
2
.3.1. Synthesis of ligand ethylene [O,O′–bis{5–{(1E)–(2 hydroxyphenyl) methylene] amino}–2–
hydroxybenzoic acid] (L)
The salicylaldehyde derivative was synthesized according to the procedure reported earlier
Salih, & Hamdi, 2008) which is described below (scheme 1).
(
To a stirred methanolic (30 mL) solution of 5–aminosalicylic acid (0.612g, 4mmol) was added
dropwise salicylaldehyde derivative (0.54g, 2mmol) in methanol (20 mL). The reaction mixture
was reflux for 4 hours to ensure the completion of reaction which yielded yellow colored solid
product, washed with methanol (15 mL) and diethyl ether (30 mL). The yellow solid was then
dried to afford ligand L. (Yield: 0.309g, 57.2%). m.p. 240 °C. Anal. Calc. for C H N O (%): C,
3
0
24
2
8
–
1
6
6.66; H, 4.48; N, 5.18. Found: C, 66.69; H, 4.50; N, 5.21. Selected IR data on KBr/nujol, cm :
3
081 ν(Ar−CH); 1606 ν(C=N); 1677 ν(C=O); 1483 ν(C=C); 1265; 1206 ν(Ar−O); 755
1
ν(Substituted benzene). H NMR (DMSO–d , 400 MHz) δ (ppm): 8.78 (a) (s, 2H, C(H)=N);
6

13
6
.75−7.97 (b&c) (ov,m, 14H Ar); 5.58 (e) (s, 2H, –OH); 3.20 (d) (s, 4H, C H ). C NMR
2 5
(
DMSO–d , 100 MHz) δ(ppm): 171.7 (c) (C=O); 159.6 (a) (C=N); 158.4 (d) (CO); 112.9–142.8
6
(b&c) (ArC) (scheme 2).
2
.3.2. Synthesis of Cu (II) complex
To a stirred methanolic (15 mL) solution of Cu(NO ) .3H O (0.241g, 1mmol) was added slowly
3
2
2
ligand L (0.54 g, 1mmol) in methanol (25 mL). The resulting mixture was refluxed for ca4 hours
until a dark brown product was obtained and washed thoroughly with methanol and diethyl ether
then dried in vacuo. (Yield: 0.453g, 74%). Decomposition temperature ˃300 °C. Anal. Calc. for
C H N O Cu (%): C, 59.45; H, 4.00; N, 4.64. Found: C, 59.65; H, 3.98; N, 4.61. Selected IR
30
24
2
8
–
1
data on KBr/nujol, cm : 3081 ν(Ar−CH); 1602 ν(C=N); 1677 ν(C=O); 1486 ν(C=C); 1260,
1
204 ν(Ar−O); 747 ν(Substituted benzene); 564 ν(Cu−O), 475 (Cu–N). Molar Conductance, ᴧM
–
3
–1
2
–1
–3
(
1×10 M, DMSO): 51 Ω cm mol (non–electrolyte). UV–Vis absorption: (10 M, DMSO),
3
−1
−1
λmax/nm (ε/10 M cm ): 270 (4.81); 375 (3.25); 538 (1.73).
.4. In vitro DNA binding and cleavage studies
2
DNA binding experiments include absorption spectral traces, emission spectroscopy and circular
dichroism conformed to the standard methods and practices previously described (Waddhaah,
Sabah, & Manal, 2018).
−1
The DNA concentration was determined spectrophotometrically by assuming ε260 nm= 6600 M
−
1
cm . EB, 3,8–diamino–5–ethyl–6–phenylphenanthridinium bromide, is a planar aromatic
heterocyclic intercalator that gives a significant increase in fluorescence emission when bound
to DNA and its displacement from DNA results in a decrease in fluorescence intensity. In the
presence of DNA, it emits intense fluorescence due to intercalation of planar phenanthridinium

ring between adjacent base pairs on the DNA double helix. EB was non–emissive in Tris–HCl
buffer medium due to fluorescence quenching of free EB by solvent molecules. Addition of
second DNA binding molecule results in a decrease in emission intensity of EB–DNA adduct by
either replacing the EB and/or by accepting the excited–state electron of the EB through a
photoelectron transfer mechanism. The CD contribution by the CT DNA and Tris buffer was
subtracted through base line correction. The cleavage experiments of supercoiled pBR322 DNA
(300 ng) by copper complex (10–30 µM) in Tris–HCl/NaCl (5:50 mM) buffer at pH 7.2 was
carried out using agarose gel electrophoresis. The samples were incubated for 45 min at 310 K.
A loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol was
added and electrophoresis was carried out at 50 V for 1 h in Tris–HCl buffer using 1% agarose
gel containing 1.0 mg/ml ethidium bromide. The DNA cleavage with added reductant was
monitored as in case of cleavage experiment without added reductant using agarose gel
electrophoresis.
2
.5. In vitro HSA binding and photo–induced cleavage studies
−
3
Human serum albumin of 1×10 M was prepared by dissolving protein in Tris−HCl buffer
solution at pH 7.4. The protein concentration was determined spectrophotometrically using an
−
1
−1
−3
extinction coefficient of 35219 M cm at 280 nm. Stock solution of complex (1×10 M) was
prepared by dissolving complex in DMSO. NaCl (analytical grade, 1M) solution was used to
maintain the ionic strength of buffer at 0.1M pH was adjusted to 7.4 by using HCl. Working
standard solution was obtained by appropriate dilution of the stock solution.
The fluorescence spectra were recorded under the following conditions: λ =280 nm and λ =346
ex
em
nm. The excitation and emission slits were set at 10 nm. The CD spectra of HSA were collected
−1
in the presence of complex at molar ratio of 1:1 in the range of 200 to 260 nm, with 20 nm min

scan speed and a response time of 2s. FT IR spectra of HSA in the presence and absence of
−
1
complex was recorded in the range of 1700–1600 cm at room temperature. Photo–induced
protein cleavage experiments were performed following to the standard methods according to the
literature (Huilun, et al., 2016). The photo–induced protein cleavage studies were carried out
using incubating HSA in 50 mM Tris–HCl buffer (pH 7.4). The protein solutions (15 µM)
containing complex with increasing concentrations 100 to 200 µM were photo–irradiated at 365
○
nm for 4h incubation at 37 C followed by SDS–PAGE analysis.
2
.6. Antitumor activity assays
The positive results obtained from the previous biological studies, namely, DNA binding and
cleavage, encouraged us to test their in vitro cytotoxic activity. The activity of complex was
evaluated in terms of GI , TGI and LC values against nine different human carcinoma cell
5
0
50
lines of different histological origin: U373MG (CNS), PC3 (Prostate), MCF7 (Breast), K562
Leukemia), HCT15 (Colon), Hop–62 (Lung), A2780 (Ovarian). The in vitro anti−tumor
(
screening of complex was evaluated as a consequence of their binding affinity towards HSA by
applying microculture Sulforhodamine B test (SRB) (Lammers, et al., 2007).
2
.7. In vivo biochemical studies
Animals were cared for at the Department of biology, Taiz University, Taiz, Yemen. All animal
experiments were performed as per Institutes animal ethics guidelines under standard laboratory
○
conditions (25 ± 2 C, 12 h light/dark cycle) and had access to standard rat chow and clean
drinking water provided ad libitum. The mice were divided into three treatment groups of five
mice each for evaluation of the systemic toxicity of Cu (II) complex. Group I of rats received
single dose of DMSO intra−peritoneally (ip) which served as a control while Group II and Group

–
1
III received 200 and 400 µg kg body weight of single ip doses of Cu (II) complex, respectively.
Liver tissues was removed after 24 h treatment of rats for histopathologic studies.
2
.8. Histopathologic examination of tissues
After treatment of liver tissue by Cu (II) complex, the liver tissues were carefully taken. They
were sliced and fixed in Bouins fluid for 24 h. The tissues were dehydrated with alcohol of
graded concentrations and then cleaned in xylene for 15 min. Further, they were treated with
xylene plus paraffin wax (1 : 1) for 15 min, and then complete infiltration was occurred with
their treatment with only paraffin wax for further 15 min. They were cast into blocks; sections of
tissues were then cut on a microtome to 5 mm by using microtome (Rotary type). They were
later attached to slide which was pre−smeared with glycerine and albumin (1 : 1) and allowed to
○
stick to glass slide by keeping them over hot plate (40–45 C). These slides were then dipped in
xylene for 5 min and then rehydrated in alcohol grades. The prepared slides were subsequently
stained in haematoxylin−eosin and examined under light microscope. The photographs of the
samples were taken and recorded.
2
.9. Determination of the superoxide dismutase activity
Superoxide dismutase activity of the copper (II) complex was assayed by using its ability to
inhibit the reduction of nitroblue tetrazolium, NBT, at 560 nm by superoxide ions produced by
the xanthine–xanthine oxidase system (Tabassum, et al., 2013). The assay was carried out in the
assay buffer containing 50mM Tris HCl, pH 8.0, 0.1 mM diethylene triamine penta acetic acid
(DTPA) and 0.1 mM hypoxanthine. Radical detector consists of a tetrazolium salt and is diluted

by assay buffer. Similarly, the solutions of SOD standards and xanthine oxidase were prepared in
sample buffer consisting of 50mM Tris–HCl, pH 8.0. The SOD mimetic activity of tested copper
complex in aqueous solution at 25 °C was evaluated from the absorbance decrease at 560 nm
comparing to the blank (the reaction mixture without the copper complex). The concentration of
complex required to yield 50% inhibition of NBT reduction (the IC value) was determined
5
0
from a plot of percentage inhibition versus copper complex concentration (µM).
.10. Molecular docking
2
Molecular docking studies were performed by using HEX 8.0 software, which is an interactive
molecular graphics program for calculating and displaying feasible docking modes of DNA,
protein and SOD enzyme. Structure of the Cu(II) complex was converted it into PDB format
from mol format by OPENBABEL (http://www.vcclab.org/lab/babel/) (Ali et al., 2018). The
crystal structure of the B–DNA (PDB ID: 1BNA), HSA (PDB ID: 1h9z) and native SOD (PDB
ID: 1SXA) were downloaded from the protein data bank (http://www.rcsb.org./pdb). All
calculations were carried out on an Intel Pentium 4,2.4 GHz based machine running MS
Windows XP SP2 as operating system. Visualization of the docked pose has been done by using
PyMol (http://pymol.sourceforge.net/) molecular graphic program.
3
. Results and discussion
The copper (II) complex was synthesized as depicted in scheme 1 a & b. Empirical formulae and
proposed structure were ascertained by elemental analysis, molar conductivity measurements,
UV–Vis, FTIR spectroscopy and XRPD studies. The complex is soluble in organic polar
solvents, DMSO and DMF. On the basis of UV–Vis and EPR data, the proposed geometry of the
copper (II) was assigned to be square planar (four–coordinated environment).

3
.1. IR spectroscopy
‒
1
The IR spectrum of free ligand L displayed a strong characteristic band at 1606 cm due to
‒
1
v(C=N) which shifted slightly to 1602 cm in the copper (II) complex implying the coordination
of the azomethine nitrogen of N atom of the ligand to the metal center. The bands around 1677
‒
1
‒1
cm and 1260 cm observed in the copper (II) complex attributed to antisymmetric and
symmetric ν(COOH) stretching vibrations (Farukh, and Mohd, 2010). also, the broad bands at ~
–1
3
350 cm in the spectra of copper (II) complex attributed to the presence of free ν(OH) group of
–1
phenolic moiety. The spectra of the complex is dominated by bands at 3081 cm
due to
–
1
ν(Ar−CH) groups, other medium intensity bands at 747cm were attributed to the characteristic
signatures of aromatic ring vibration. The complex formation of copper (II) complex was also
revealed by the presence of medium intensity (Cu–O) and (Cu–N) bands appear at 564 and 475
–
1
cm , respectively in the far IR region (Jarzȩcki, Anbar, and Spiro, 2004). rzȩcki, Anbar, and
Spiro, 2004).
3
.2. EPR studies
The solid state X–band EPR spectrum (Figure S1) of polycrystalline Cu(II) complex was
recorded at room temperature with tetracyanoethylene (TCNE) as field marker (g = 2.0027). The
spectrum displayed an isotropic signal and exhibits axial symmetrical line shape with
2
av
2
||
2
┴
g =2.133 and g =2.055 and g =2.08 computed from the formula g =g +2g /3. These
||
┴
av
parameters are consistent with the square planar geometry of the copper(II) metal ion and are
quite similar to the values reported for other related square planar copper(II) systems. The trend
of g values (g >g >2.0023) observed for the copper(II) indicates that the unpaired electrons
||
┴

2
2
2
lies predominantly in the dx –y orbital of Cu(II) ion (spectroscopic state B ). The exchange
1
g
interaction parameter G = 2.42, suggesting presence of exchange coupling between the copper
centers (Géraldine, et al., 2007).
3
.3 Electronic Spectra and
The absorption spectra of the complex was recorded in DMSO solution. The Cu(II) complex
3
−1
−1
reveals bands at 270 nm and a broad band at 538 nm (ε/10 M cm : 4.81 and 1.73,
2
2
respectively), which were assigned to (π→π*) and ( B – A ) transitions, respectively. These
1
g
1g
results are consistent with the square planar environment around Cu(II) center (Duward, et al.,
2
014).
3
.4. X–ray diffraction analysis
To obtain further evidence about the structure of the metal complex, X–ray powder diffraction
studies of the Cu(II) complex was performed as it was difficult to isolate single crystals suitable
for X–ray crystallography could not be obtained. The crystalline nature of the complex was
authenticated by XRPD measurements. Figure 1 showed the XRPD patterns indicate crystalline
nature for the Cu(II) complex. The X–ray diffractogram of the Cu(II) complex shows reflecting
peaks at 2θ scattering angles of 26.18, 43.44 and 78.28 assigned to (112), (014), (024), and (118)
crystal planes respectively, characteristic of a well ordered hexagonal arrangement of Cu(II)
atoms (JCPDF:89–2531). A summary of the refined XRPD parameters of complex is given in
Table 1.

3
.5. Interaction studies with CT DNA
.5.1. Absorption titrations
3
The absorption spectra of the complex in the absence and presence of calf thymus DNA (CT‐
DNA) at constant concentration of copper complex are shown in Figure 2. Upon addition of
–
5
–5
increasing amount of CT–DNA (0.00–1.66×10 M) to the fixed amount of complex (0.01×10
M), a significant “hyperchromic” effect was observed. Thus, from these spectral changes, we
deduce that the complex bind to the CT–DNA by a strong electrostatic interaction mode. These
changes are typical for complex bounded to DNA through noncovalent interaction
(Karunganathan, et al. 2018). Hypochromism results from the contraction of DNA helix as well
as the conformational changes in DNA, while hyperchromism results from the damage of
secondary structure of DNA double helix.
To study quantitatively, The intrinsic binding constant K of the complex to CT DNA was
b
determined from Equation (1), through a plot of [DNA]/ – vs [DNA], where [DNA] represents
a
f
the concentration of DNA, and  ,  and  the apparent extinction coefficient (A /[M]), the
a
f
b
obs
extinction coefficient for free metal complex (M), and the extinction coefficient for the free
metal complex (M) in the fully bound form, respectively. In plots of [DNA]/ – vs. [DNA], K
a
f
b
is given by the ratio of slope to intercept. The binding ability of Cu(II) complex with CT–DNA,
4
–1
the intrinsic binding constant K value was determined and found to be 3.38 ×10 M .
b

[
DNA]
[DNA]
1


(1)
a  f b  f
K (  )
b
a
f
An explanation for higher K value of copper complex is apparently that copper complexes are
b
‘borderline’ metals which show high affinity for both nucleobase as well as phosphate ends of
the DNA helix. In particular, Cu(II) complex binds specifically to N7 of guanine nucleobase of
DNA helix.
3
.5.2. Fluorescence spectroscopic studies
The Cu(II) complex exhibited luminescence either in DMSO or in presence of CT‒DNA. Hence,
binding studies of metal complex to CT‒DNA was carried out by fluorescence spectral titration
–
5
in absence of any probe. Fixed volumes (1.0 ×10 M) of the studied complex was titrated,
–
5
respectively with increasing concentration of DNA in the range from (0.00–1.66 ×10 M). As
seen from the Figure 3. When Cu(II) complex is excited at 280 nm, the intensity of emission
increased appreciably in the presence of DNA. The observed enhancement could be due to
relatively non–polar environment of the bound metal complex in the presence of DNA, such that
the complex was less deeply inserted inside the hydrophobic pockets or grooves of CT–DNA.
Cationic complex usually bind to DNA non–covalently as the cationic core of the complexes
exerts a strong electrostatic attraction to the anionic phosphate backbone of DNA thus precluding
substantial overlap with the base pairs leading to higher emission intensity indicative of
electrostatic binding of the probe to the DNA.
Binding constant K of the Cu(II) complex was also determined from Scatchard Equations (2)
b
and (3) by employing emission titration (Eamonn, 2007).
C = C (I / I – P) (1–P)
(2)
(3)
F
T
o
r /C = K (n–r)
F
b

Where C is the free probe concentration, C is the total concentration of the probe added I and
F
T
,
I0 are fluorescence intensities in presence and absence of CT DNA, respectively and P is the ratio
of the observed fluorescence quantum yield of the bound probe to that of the free probe. The
value P was obtained as the intercept by extrapolating from a plot of I/I vs 1/[DNA], r denotes
o
ratio of C (=C –C ) to the [DNA] concentration i.e., the bound probe concentration to the DNA
B
T
F
concentration, K is the binding constant and C , is the free metal complex concentration and “n”
b
F
is the binding site number.
4
–1
The binding constant (K ) estimated for Cu(II) complex by equation 3 was 3.06 ×10 M . These
b
results are consistent with the findings obtained from UV–Vis spectral studies.
3
.5.3. Circular dichroic studies
CD spectroscopy is a useful technique in diagnosing changes in DNA morphology during drug–
DNA interactions. The CD spectrum of CT DNA exhibits a positive band at 275 nm (UV: λmax
60 nm) due to base stacking and a negative band at 245 nm due to the right–handed helicity of
,
2
B–DNA form which are quite sensitive to the mode of DNA interactions with small molecules.
Simple groove binding and electrostatic interaction of the Cu (II) complex with DNA shows less
or no perturbation on the base stacking and helicity bands while intercalator enhances the
intensities of both bands (Lu, Zhu, Zhang, Liu, Kang, 2002). The interaction of the Cu (II)
complex with DNA induces a change in the CD spectrum of B DNA (Figure 4). The intensities
of both the negative and positive bands decrease significantly (shifting to zero levels). This
suggests that the DNA binding of the complex induces certain conformational changes, such as
the conversion from a more B–like to a more C–like structure within the DNA molecule. From

the results of UV absorption, fluorescence and CD spectroscopic studies, we conclude that the
Cu (II) complex avidly binds to CT DNA.
3
.5.4. Gel electrophoresis
The Cu (II) complex showed maximum binding propensity with CT–DNA, therefore, the
cleavage activity has been evaluated for Cu (II) complex. It has been well established in
literature that copper(II) complexes are selective for the cleavage of DNA either through
oxidative or hydrolytic mechanism. The nuclease activity of complex Cu (II) complex was
studied using supercoiled pBR322 DNA as a substrate (Figure 5). To ascertain the DNA
cleavage ability of complex, supercoiled pBR322 DNA was incubated with different
concentration of complex in aqueous buffer solution (5mM Tris–HCl/40 mM NaCl, pH 7.4)
without addition of any reductant. As shown in Figure 5, there was a gradual decrease in the
amount of Form I with a simultaneous increase in Form II (Lanes 2 and 4) on increasing the
concentration of the complex. A notable cleavage feature observed for this complex is the
appearance of Form III before the disappearance of Form I (Lanes 5 and 6).
The reduction in the intensity of Form I and appearance of more intense Form II of pBR322
DNA on the gel shows that the Cu (II) complex is involved in an efficient double strand DNA
cleavage pathway. In the presence of activators viz; glutathione (GSH), ascorbic acid (Asc),
hydrogen peroxide (H O ) and mercaptopropionic acid (MPA), the Cu(II) complex showed
2
2
significant increase in the cleavage of the DNA and follows the order H O > MPA >GSH ≈Asc
2
2
(Figure 6).

DNA recognition elements (groove binding) minor groove binding agent, DAPI and major
groove binding agent, methyl green (Farukh, & Jamsheera, 2011) were used to probe the
potential interacting site of Cu(II) complex with supercoiled plasmid pBR322 DNA. As shown in
Figure 7, DNA cleavage is inhibited in the presence of minor groove binder (lane 2), However,
no apparent inhibition of DNA damage was observed in presence of major groove binding agent
(lane 3), suggesting that minor grooves are the preferred interacting sites of Cu(II) complex
which is also in accordance with the conclusion drawn from molecular docking studies.
In order to identify the reactive oxygen species that are responsible for the DNA cleavage reaction,
experiments in the presence different common scavengers such as DMSO and Ethyl alcohol
(
hydroxyl radical scavengers), NaN (singlet oxygen scavenger) and SOD (superoxide oxygen
3
scavenger) were carried out, respectively with Cu (II) complex. As shown in Figure 8. The cleavage
activity of Cu (II) complex is reduced dramatically by the presence of hydroxyl radical scavengers
ethyl alcohol and DMSO (Figure 8, lanes 2,3), indicating that the freely diffusible hydroxyl radical is
one of the intermediates involved in the DNA scission process. On the other hand, addition of
1
sodium azide (singlet oxygen scavenger) decreases the cleavage efficiencies, this reveals that O is
2
also the activated oxygen intermediate responsible for the cleavage (Figure 8, Lane 5). Addition of
superoxide dismutase (superoxide scavenger) to the reaction mixture reveals no significant
quenching of the cleavage reaction indicating that superoxide anion is not the active species
responsible for the cleavage (Figure 8, Lane 4).
Thus, freely diffusible oxygen intermediate or hydroxyl radical may be involved in the strand
scission and hence a simple diffusible radical mechanisms applicable. Since the Cu (II) complex are

able to cleave DNA in the absence of any reducing agent, it may be assumed that DNA might be
cleaved by a discernible hydrolytic path.
3
.6. Antitumor activity assays
The positive results obtained from the previous biological studies, namely, DNA binding and
cleavage, and anti–oxidative studies for Cu (II) complex, encouraged us to test their in vitro
cytotoxic activity. The activity of Cu (II) complex was evaluated in terms of GI , TGI and LC
50
5
0
values against nine different human carcinoma cell lines of different histological origin:
U373MG (CNS), PC3 (Prostate), MCF7 (Breast), K562 (Leukemia), HCT15 (Colon), Hop–62
(
Lung), A2780 (Ovarian). The in vitro anti−tumor screening of Cu (II) complex was evaluated as
a consequence of their binding affinity towards HSA by applying microculture Sulforhodamine
B test (SRB). The initial cytotoxic screening data (Table 2) shows that the Cu (II) complex act as
a potential selective anticancer agent with a significant GI values specifically towards MCF7,
5
0
A2780 and K562 tumor cell lines.
3
.7. Histopathologic examination of liver tissue
Light microscopic images of liver tissue showed that the control, large polygonal cells with
prominent nuclei with eosinophilic cytoplasm and few spaced sinusoids arrangement. In contrast,
–
1
the group receiving Cu (II) complex at a single dose of 40 µg kg showed hepatotoxicity,
particularly degeneration of hepatic cords, inflammation, and congestion of portal vein whereas,
–
1
slightly significant histopathologic alterations in liver of mice treated at a dose of 20 µg kg of
Cu (II) complex were observed (Figure 9 (a–c)).
3
.8. Superoxide Dismutase Activity

Superoxide dismutase (SOD) are metalloenzyme that catalyze the dismutation of the superoxide
anion to molecular oxygen and hydrogen peroxide and thus form a crucial part of the cellular
antioxidant defense mechanism of organisms for its ability to protect cells from the damage of
the toxic superoxide by catalyzing the dismutation of superoxide radicals effectively.
Therefore, it was interesting to investigate the SOD–like activity of this Cu(II)–complex by using
the xanthine–xanthine oxidase–nitroblue tetrazolium (NBT) assay. A plot (Figure 10) of NBT
percent inhibition with an increase in concentration of Cu (II) complex, necessary to produce
5
0% inhibition (IC ) of the reaction are presented Table S1†, together with the values found for
50
the previously investigated copper complexes and the one obtained with native SOD. The IC50
value of Cu (II) complex is 0.109 µM, which is considered to be an excellent SOD mimic among
the most active Schiff base complexes but it is somewhat less active than the IC value obtained
5
0
for native enzyme (IC = 0.04 µM). In the enzyme, the flexible 5−aminosalcylic ligand can
5
0
accommodate and stabilize the space structure of the protein and provide needed protons to the
peroxide anion during the catalytic cycle. Since, the proton exchange between substrates and
buffer is a rapid process in the small model systems; the buffer can directly transfer protons to
peroxide anion in the mechanism.
•‾
Cu(II) + O2
Cu(I) + O2
•‾
+
Cu(I) + O + 2H
Cu(II) + H O2
2
2
Furthermore, the high superoxide dismutase activity of Cu(II) complex may be possibly
due to cooperation of Cu(II) center in close proximity, acting in concord in free radical binding
and electron transfer.
3
.9. In vitro binding studies with HSA
.9.1. Electronic absorption studies.
3

Electronic absorption spectroscopy is a reliable tool to understand the morphological changes in
secondary structure of HSA via complex formation between drug and HSA. On addition of
−
5
Cu(II) complex with incremental increase of concentration (1.00–3.33 × 10 M) to HSA of
−
6
constant concentration (1.00 × 10 M), there was a sharp increase in absorption intensity,
hyperchromism (Supplementary information, Figure S4†), of the intraligand band at 280 nm,
(Figure 2) with a slight red shift to 282 nm toward long wavelengths, which implicated that
interacted strong binding affinity of Cu(II) complex involving electrostatic interaction and could
be facilitated by hydrogen–bond formation due to the molecular topology of peptide ligand
scaffold of Cu(II) complex.
In order to evaluate the binding propensity quantitatively with HSA, the intrinsic binding
constant (K ) of the Cu(II) complex with HSA was calculated using equation 4 which was
b
described earlier, (Stephanos, 1996) as follows:
A
HSA HSA
1



(4)
A A
B  K C
B
complex
where A and A are the absorbance of HSA at 280 nm, in the absence and presence of complex,
o
respectively. εHSA and ε are the molar extinction coefficient of HSA and the HSA bound
B
complex. Thus, the double reciprocal plot of 1/A–A vs. 1/Ccomplex is linear and binding constant
o
can be estimated from the ratio of intercept to the slope. The binding constant, K value was
b
4
−1
found to be 1.48 × 10 M . The K value of complex suggested of moderate binding propensity
b
to HSA followed by conformational transition. Since moderate affinity of a drug towards a
protein helps in the diffusion of the drug from the circulatory system to reach its target site, thus
obtained K value for complex fulfill this criterion for their transport in the blood circulation and
b
diffusion at the target site.

3
.9.2. Fluorescence interaction studies
Fluorescent quenching techniques involves a variety of molecular interactions such as excited
state reactions, molecular rearrangements, energy transfer, the formation of ground–state
complex, collision, etc. Therefore, fluorescence quenching experiments were undertaken to
investigate the interaction between Cu(II) complex and HSA. When HSA molecule is excited at
2
80 nm, both tryptophan and tyrosine amino acid residues give fluorescence emissions at an
excitation wavelength of 340nm in which tryptophan has strong fluorescence emissions while
tyrosine does not absorb in this region. The fluorescence spectra of HSA in the absence and
presence of Cu(II) complex as a quencher in Tris–HC1 buffer (pH 7.4) were monitored with an
excitation wavelength of 295 nm (Figure S5†). HSA exhibits a strong fluorescence emission with
a peak at 370 nm due to the emission from the single tryptophan residue (Trp–214). However,
intrinsic fluorescence intensity of HSA decreased regularly and no shift was observed in the
emission wavelength with the increase in concentration of Cu(II) complex. The strong quenching
of the HSA fluorescence implicated the existence of interaction between Cu(II) complex and
HSA which changes the local environment around Trp–214 residues.
In order to determine the fluorescence quenching mechanism, the fluorescence quenching data at
different temperatures (298, 308 and 318 K) were analyzed using the classical Stern–Volmer
Equation 5: (Joseph, & Gregorio, 1973).
F
(5)

1 k  [Q] 1 K [Q]
q  SV
F
where F and F are the fluorescence intensities in absence and presence of quencher,
0
respectively, k , K , τ and [Q] are the quenching rate constant of the biomolecule, the Stern–
q
SV
o
Volmer dynamic quenching constant, the average lifetime of the molecule without quencher
−
8
(
τo=10 s) and the concentration of the quencher, respectively. Figure S3† shows the Stern–

Volmer plots of F /F versus [Q] at three different temperatures and the calculated K and k
q
0
SV
values are summarized in Table S2. The results revealed that the Stern–Volmer quenching
constant K is inversely correlated with temperature and k is larger than the limiting diffusion
SV
q
1
0
−1 −1
constant Kdif of the biomolecule (Kdif = 2.0 × 10
M
s ), indicating that fluorescence
quenching was caused by a specific interaction between HSA and Cu(II) complex, consistent
with the static quenching mechanism.
For static quenching, the following equation was employed to calculate the binding constant and
number of binding sites:

F  F 

(6)
log
 log K  nlog[Q]


b
F


where F and F are the fluorescence intensities of HSA in the absence and presence of quencher,
0
K and n are the binding constant and the number of binding sites, respectively. Thus, a plot of
b
log[(F −F)/F] versus log [Q] was used to determine K as well as n. The binding data (K and n)
0
b
b
at different temperatures were presented in Figure S6† and ESI Table 3. It was found that the
binding constant (K ) decreased with the increase of temperature, resulting in a reduction of the
b
stability between Cu(II) complex–HSA binding reaction. Literature reveals that Cu(II) metal ion
most likely binds to the hydrophobic pocket located within subdomain IIA; Trp–214 which is
near or within the binding site. These results were further validated by docking simulation
studies.
3
.9.3. Binding mode
The acting forces between a small molecule and bio–macromolecule are mainly include weak
interactions such as hydrogen bonds, van der Waals forces, electrostatic and hydrophobic
interactions. The thermodynamic parameters, enthalpy change (∆H), entropy change (∆S) and

free energy change (∆G) are the main evidence to determine the binding mode. From the
thermodynamic standpoint, ∆H > 0 and ∆S > 0 implies a hydrophobic interaction; ∆H< 0 and ∆S
<
0 reflects the van der Waals force or hydrogen bond formation; and ∆H≈0 and ∆S > 0 suggests
an electrostatic force (Hamid, et al. 2018). The thermodynamic temperatures chosen for
measurements were 298, 308 and 318 K, so that HSA does not undergo any structural
degradation. The thermodynamic parameters can be calculated from the Van’t Hoff equation:

RT
H S
ln K 

(7)
(8)
R

G  H TS  RT ln K
where K is the Lineweaver–Burk static quenching constant at corresponding temperature and R
is the gas constant, in which ∆H and ∆S of reaction could be determined from the linear
relationship between ln K and the reciprocal absolute temperature (Figure S7†). The free energy
change (∆G) could be calculated by the Equation 8. As shown in Table S2, ∆G and ∆H were
negative, and ∆S was positive. Therefore, the formation of Cu(II) complex–HSA was
spontaneous and exothermic process. The positive ∆S value is frequently taken as evidence for a
−
1
hydrophobic interaction. Furthermore, the negative ∆H value (−25.30 kJ mol ) observed cannot
be mainly attributed to electrostatic interactions since for electrostatic interactions ∆H is very
small, almost zero. Therefore, the negative ∆H and positive ∆S values suggest that hydrophobic
and hydrogen bond interactions play major roles in the Cu(II) complex–HSA binding reaction
and contributed to the stability of the complex.
3
.9.4. Energy transfer and Binding distance between Cu(II) complex and HSA
Fluorescence energy transfer occurs via overlapping of a fluorophore (donor) with the absorption
spectrum of molecule (acceptor). The overlap of the absorption spectrum of the Cu(II) complex

with the fluorescence emission spectra of HSA is shown in Figure 11. According to fluorescence
resonance energy transfer theory, the rate of energy transfer depends on the extent of overlapping
between fluorescence emission spectrum of donor with the absorption spectrum of acceptor, the
relative orientation of the donor and acceptor transition dipoles, and the distance between donor
and acceptor (Zahra, et al. 2019).
A F ster resonance energy transfer (FRET) mechanism is involved in the quenching of Trp
Ö
fluorescence by Cu(II) complex, the efficiency of energy transfer (E) is given by:
6
F
R
(9)
E 1

6
6
F R  r
where F and F are the fluorescence intensities of HSA in the presence and absence of quencher,
0
r is the distance between acceptor and donor and R is the critical distance when the transfer
ο
efficiency is 50%. The value of R can be calculated using the equation 10:
ο
6
25
2
4
R  8.7810 K N J
(10)

2
where K is spatial orientation factor between the emission dipole of the donor and the
absorption dipole of acceptor, N is the refractive index of the medium, ф is the fluorescence
quantum yield of the donor and J is the overlap integral of the fluorescence emission spectrum of
the donor and the absorption spectrum of the acceptor and is given by:

4
0
F()() 
0
J 
(11)

F()
where F(λ) is the corrected fluorescence intensity of the donor at wavelength λ and ε(λ) is the
molar absorption coefficient of the acceptor at wavelength λ. Under the experimental condition,
2
K =2/3, N =1.336, ф= 0.188, according to equations. (9)–(11), the value of E, R , r and J were
0
−
14
−1
3
calculated and found to be 0.99, 2.5 nm, 3.7 nm and 1.6 × 10 M cm , respectively. The donor

to acceptor distance r < 7 nm indicated that the energy transfer from tryptophan residue in HSA
to Cu(II) complex occur with high probability.
3
.9.5. 3D fluorescence measurements
3
D fluorescence measurements have been getting more important in protein conformational
analysis as they provide detailed information about the conformational and structural changes of
proteins by changing excitation and emission wavelengths simultaneously. As shown in Figure
1
2 and Table 4 , it was observed that the fluorescence intensities of both peaks a and b decreased
significantly and the maximum emission wavelength of the peak changed in the presence of
Cu(II) complex indicating the quenching of fluorescence of HSA. Peak a was the Rayleigh
scattering peak (λ = λ ). Peak b (280, 370 nm, λ , λ ) mainly reflected the spectral behavior
ex
em
ex em
of the Trp residue, and the maximum emission wavelength and the fluorescence intensity of the
residue associated with its microenvironment’s polarity. Peak c was the second–ordered
scattering peak (λ =2 λ ) (Rodríguez–Cuesta, et al. 2003). We can conclude that the
ex
em
interaction of Cu(II) complex with HSA induced some conformational and micro–environmental
changes in HSA, corroborated well our spectroscopic results obtained from UV–Vis, FT–IR and
CD measurements.
3
.9.6. Investigations on the circular dichroism of HSA
To ascertain the possible influence of Cu(II) complex binding on the secondary structure of
HSA, CD measurement was also performed in the presence of Cu(II) complex at different
concentrations. As shown in Figure 13, CD spectra of free HSA (line a) exhibits two negative
bands in the ultraviolet region at 208 and 222 nm attributed to n→π* transfer for the peptide
bond of α–helix (Nida, Mohammad, Gulam, Ejaz, & Rizwan, 2013). It was observed that with

the increase in Cu(II) complex concentration, the CD signal of HSA increased, suggestive of the
binding of Cu(II) complex to HSA induces a significant conformational change in HSA (line b
and c). However, the CD spectra of HSA in the presence or absence of Cu(II) complex is similar
in shape, indicating that the structure of HSA is also predominantly α–helical. The CD results
2
−1
were expressed in terms of MRE (mean residue ellipsticity) in deg cm d mol as the following
equation 12:



Observed CD(mdeg)
(12)
MRE  
C nl 10


p


where C is the molar concentration of the protein, n the number of amino acid residues (585)
p
and l the path length (0.2 cm). The α–helical contents of free and combined HSA were calculated
from the MRE value at 208 nm using the following equation as described earlier:



(MRE)208 4000

 helix(%) 
100
(13)

33000 4000

where MRE208 is the observed MRE value at 208 nm, 4000 is the MRE of the β–form and
random coil conformation cross at 208 nm, and 33000 is the MRE value of a pure α–helix at 208
nm.
From Equation 13, the quantitative analysis results of the α–helix in the secondary structure of
HSA were obtained. They differed from that of 59.79% in free HSA to 49.14% and 46.33% in
the Cu(II) complex–HSA system at pH 7.4 and temperature 25 °C (curves a–b in Figure 13). The
α–helix gradually decreases with the increase in concentration of Cu(II) complex, which reveals
that interaction between Cu(II) complex and HSA leads to a change in secondary structure of
HSA.
3
.9.7. Fourier transform infrared (FTIR) measurements

To further investigate the conformational change of HSA induced by Cu(II) complex, FT–IR
experiment was performed. The spectrum in Figure S8a† was obtained by subtracting the
absorption of the Tris–HCl from the spectrum of the HSA solution. The spectrum in Figure S8b†
was obtained by subtracting the absorption of the Cu(II) complex–free form from that of the
Cu(II) complex–bound form. Infrared spectra of HSA exhibit a number of the amide bands,
which represent different vibrations of the peptide moiety. The amide I peak observed in the
–
1
–1
range 1600–1700 cm (mainly C=O stretch) and amide II band in the region 1500–1600 cm
(C–N stretch coupled with N–H bending mode). It is well known that the amide bands have a
relationship with the secondary structure of HSA, and amide I is more sensitive than amide II for
change of secondary structure of HSA (Huilun et al., 2015). As shown in Figure S8†, the peak
–
1
–1
position of amide I bands were shifted from 1637.49 cm to 1645.35 cm , indicating the
secondary structure of the HSA was changed in presence of Cu(II) complex.
3
.9.8. HSA photocleavage studies.
There is a considerable interest in the development of synthetic chemical proteases that are
activated by radical–generating metal complex. Such chemical proteases can be utilized for
breaking large proteins into smaller fragments that are more amenable to sequencing. The ability
of Cu(II) complexes to mediate protein cleavage is well established in literature (Sarbadhikary,
&
Dube, 2017) owing to the fact that these complex show their own selectivity for a cleavage
mechanism, it is therefore imperative to study the effect of Cu(II) complex on HSA in presence
of different concentration and reactive oxygen species.
In order to ascertain the ability of the Cu(II) complex to serve as a synthetic
metalloproteases, the HSA photocleavage activity of the Cu(II) complex was studied using 4 μM

HSA in 50 mM Tris–HCl buffer on exposure to UV–A1 light of 365 nm with different complex
concentrations for 20 min and the extent of cleavage was compared with the untreated HSA
band. The photoinduced protease activity of Cu(II) complex was assayed by SDS–PAGE (12%)
analysis. As shown in Figure 14, it was observed that HSA alone (Lane 1) did not show any
apparent cleavage under these conditions. However, upon increasing the concentration of Cu(II)
complex (100–250 µM), HSA showed significant smearing or fading of the band (Lane 2–5),
indicating photocleavage of HSA under physiological reaction conditions. At the same time,
when the concentration of Cu(II) complex reached to 250 μM, HSA cleaved into smaller
fragment that could not be observable by SDS–PAGE. The smearing of the band results from
non–specific cleavage of the HSA by the highly diffusible HO∙ radicals generated by the
complex upon photoexcitation, leading to the cleavage of HSA into very small fragments. The
Cu(II) complex did not show any cleavage activity in the dark, thus ruling out any hydrolytic
cleavage of HSA (Lane 5).
3
.10. Molecular docking
3
.10.1. Molecular docking with DNA
Molecular docking techniques are an attractive scaffold to understand the drug–DNA
interactions in rational drug design, as well as in the mechanistic study by placing a small
molecule into the binding site of the target specific region of the DNA mainly in a non−covalent
fashion. Molecular docking simulations of the synthesized Cu(II) complex and DNA were
performed to explore in more detail the complex conformation, to get more insight into the mode
of binding and their orientations in the active site of the receptor. Cu(II) complex was
successively docked with DNA duplex of sequence d(CGCGAATTCGCG) dodecamer (PDB
2