The role of both intercalation and groove binding at AT-rich DNA regions in the interaction process of a dinuclear Cu(I) complex probed by spectroscopic and simulation analysis

Article abstract of DOI:10.1016/j.molliq.2021.116290

In this research paper, the synthesis process and characterization of a dinuclear Cu(I) phosphine complex, as well as spectroscopic and simulation analysis of its interaction with DNA helix, are presented. The monoclinic phase [Cu(PPh₃)(L_0.5)(I)]₂ with a tetrahedral molecular geometry was elucidated based on X-ray single-crystal diffraction data. The significant association constant (6.88 × 10⁵) and remarkable hyperchromism as obtained from UV–Visible spectra indicated a high binding affinity of the Cu(I) complex for DNA, which is in accordance with both intercalation and groove binding modes. The results of competitive fluorescence experiments along with molecular docking simulation proved that the planar part of the complex can insert into the core of adenine nucleobases and compete with methylene blue for the intercalative binding sites, while the bulky substituent binds in the minor groove of DNA via replacement of Hoechst molecules at A-T rich regions. According to thermodynamic parameters (the negative values of ΔH° and ΔS°), it is quite clear that the interaction process is enthalpy-favored while disfavored by entropy and van der Walls and hydrophobic forces play a major role in stabilization of right-handed B-DNA form during the interaction, as shown in the circular dichroism spectra. Based on the above findings partial intercalation mode at A-T rich region of DNA was proposed. The cytotoxicity and apoptosis results on MCF-7 cell line indicated positive effect of the Cu(I) complex in controlling growth and viability of breast cancer more than cisplatin.

Full text of DOI:10.1016/j.molliq.2021.116290

Journal of Molecular Liquids 335 (2021) 116290
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
The role of both intercalation and groove binding at AT-rich DNA regions
in the interaction process of a dinuclear Cu(I) complex probed by
spectroscopic and simulation analysis
a
a
b
c
Nahid Shahabadi a, , Farshad Shiri , Saba Hadidi , Kaveh Farshadfar , Maryam Darbemamieh ,
⇑
S. Mark Roe d
a Department of Inorganic Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Iran
b Department of Chemistry, Islamic Azad University, Central Tehran Branch, Poonak, Tehran 1469669191, Iran
c Department of Plant Protection, Faculty of Agriculture, Campus of Agriculture and Natural Resources, Razi University, Kermanshah, Iran
d Department of Chemistry, School of Life Sciences, University of Sussex, Brighton, BN1 9QJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
In this research paper, the synthesis process and characterization of a dinuclear Cu(I) phosphine complex,
as well as spectroscopic and simulation analysis of its interaction with DNA helix, are presented. The
monoclinic phase [Cu(PPh3)(L0.5)(I)]2 with a tetrahedral molecular geometry was elucidated based on
X-ray single-crystal diffraction data. The significant association constant (6.88 ꢀ 105) and remarkable
hyperchromism as obtained from UV–Visible spectra indicated a high binding affinity of the Cu(I) com-
plex for DNA, which is in accordance with both intercalation and groove binding modes. The results of
competitive fluorescence experiments along with molecular docking simulation proved that the planar
part of the complex can insert into the core of adenine nucleobases and compete with methylene blue
for the intercalative binding sites, while the bulky substituent binds in the minor groove of DNA via
replacement of Hoechst molecules at A-T rich regions. According to thermodynamic parameters (the neg-
Received 25 February 2021
Revised 17 April 2021
Accepted 21 April 2021
Available online 24 April 2021
Keywords:
Dinuclear Cu(I) complex
DNA binding
In vitro evaluation
Spectroscopic techniques
Docking simulation
Cytotoxicity study
ative values of
DH° and DS°), it is quite clear that the interaction process is enthalpy-favored while dis-
favored by entropy and van der Walls and hydrophobic forces play a major role in stabilization of right-
handed B-DNA form during the interaction, as shown in the circular dichroism spectra. Based on the
above findings partial intercalation mode at A-T rich region of DNA was proposed. The cytotoxicity
and apoptosis results on MCF-7 cell line indicated positive effect of the Cu(I) complex in controlling
growth and viability of breast cancer more than cisplatin.
Ó 2021 Elsevier B.V. All rights reserved.
1. Introduction
anti-angiogenic molecules in several types of cancers [6–9]. In this
regard, recent research studies have provided evidence that copper
Copper as a crucial trace element is essential for all living organ-
isms and cell metabolism [1]. It is needed for the proper function of
enzyme systems, most notably cytochrome oxidase, respiration,
DNA synthesis, ascorbate oxidase, energy production, and metabo-
lism [2,3]. Nevertheless, copper can also function as an essential
cofactor in tumor angiogenesis processes [4]. Consistently, major
levels of copper have been found in many human cancer types,
including breast, lung, brain, liver, colon, prostate, and bladder can-
cers [5]. On this base, the use of copper chelators is known as
complexes containing chelating phosphines act as a proteasome
inhibitor and apoptosis inducer in different human cancer cells
[10,11]. Hydrophilic phosphine ligands are also utilized as a strat-
egy to acquire easy membrane penetrating drugs [12]. The antipro-
liferative activity of a copper(I) phosphine complex [Cu(thp)4][PF6]
(CP) against human solid tumors with poor effects on non-tumor
cells is a promising point for considering these compounds as novel
anticancer agents [13,14]. CP demonstrated 40-fold more cytotoxic
potency than cisplatin and it was also able to overcome multi-drug
resistance problems [13]. The cytotoxic activities of copper(I) phos-
phine complexes may be correlated to their different mode of
action from that of cisplatin for damaging DNA as the main molec-
ular target for anticancer drugs [15–17]. Mechanistic studies
Abbreviations: CT-DNA, Calf thymus DNA; MB, Methylene blue; CD, Circular
dichroism.
⇑
Corresponding author.
E-mail address: n.shahabadi@razi.ac.ir (N. Shahabadi).
https://doi.org/10.1016/j.molliq.2021.116290
0167-7322/Ó 2021 Elsevier B.V. All rights reserved.

N. Shahabadi, F. Shiri, S. Hadidi et al.
Journal of Molecular Liquids 335 (2021) 116290
proved that cisplatin binds covalently to DNA [18,19] while copper
complexes generally interact with DNA via non-covalent interac-
tions and cause stronger damage to DNA [12,20–23]. Based on
the results obtained, DNA intercalators and groove binders show
promising anticancer effect with high binding constant on 105-
108 Mꢁ1 order of magnitude [24–26], while covalent DNA binders
such as cisplatin have lower binding constant in the order of 104
Mꢁ1 [19,27]. On the other hand, because of the low specificity of
DNA recognition and the omnipresence of its potential binding
sites, cisplatin is not a region-specific anticancer agent [28]. Dam-
age to DNA in various domains does not show a consistent rela-
tionship with cytotoxic activities of anticancer drugs contrary to
what has been observed in region-specific DNA damage. Thus,
specific DNA targeting offers the potential to improve antitumor
activities and treatment selectivity against tumors versus normal
cells. Understanding how drugs interact with DNA and how induce
DNA damage can be helpful to design more efficient and specifi-
cally targeted therapeutics, with lower side effects [24,29]. Hence,
with knowledge of the importance of the subject, we sought to
design a dinuclear Cu(I) complex containing chelating phosphine
ligand with the ability to interact with DNA in a remarkably speci-
fic way through non-covalent interactions.
2. Experimental
Scheme 1. Schematic view of the compounds and synthesis processes.
2.1. Materials
All materials including metal ion, buffers, nucleic acid, and
other chemicals for synthesis process and DNA interaction experi-
ments were of analytical reagent grade and used without purifica-
tion. Doubly distilled water was applied to prepare Tris-HCl buffer
and all aqueous stock solutions. A solution of 8.6 ꢀ 10ꢁ3 mol/L of
Calf thymus DNA (CT-DNA) in Tris–HCl buffer (pH 7.4) was used
as a stock solution of DNA and its protein purity was checked by
monitoring the ratio of A260/A280 > 1.80 [30]. All spectroscopic
studies were carried out in Tris-HCl buffer (50 mM, pH 7.4) includ-
ing 0.05% DMSO and 0.1% Ethanol.
2.3.2. Competitive fluorescence analysis
All fluorescence spectra were performed on JASCO spectrofluo-
rimeter Model FP-6200 equipped with a thermostated bath, using
a 1.0 cm quartz cell. 2 mL solution of DNA-Hoechst conjugate
(CDNA = 1.18 ꢀ 10ꢁ3 mol/L and CHoechst = 5 ꢀ 10ꢁ6 mol/L in Tris-
HCl pH = 7.4) was titrated by increasing concentration of the Cu
(I) complex to 1.12 ꢀ 10ꢁ4 mol/L at different temperatures (288,
293, 298, and 303 K) in the wavelength range of 350–550 nm with
exciting wavelength at 340 nm. The competitive interaction
between methylene blue (MB) and the Cu(I) complex was carried
out by the addition of increasing amounts of the Cu(I) complex
(from 0 to 2.33 ꢀ 10ꢁ4 mol/L) against a constant amount of the
probes (5 ꢀ 10ꢁ6 mol/L) and DNA (1.96 ꢀ 10ꢁ4 mol/L) at 288 K in
the wavelength range of 630–730 nm with exciting wavelength
at 630 nm. All fluorescence intensities were corrected for absorp-
tion of the exciting light and reabsorption of the emitted light to
decrease the inner filter effect using the following relationship
(Eq. (1)) [34]:
2.2. Synthesis of the Cu(I) complex
The synthesis of the Cu(I) complex [Cu(PPh3)(L0.5)(I)]2 was per-
formed according to previously published methods of our group
[31,32]. The ligand N,N’-Bis(2-pyridylmethylene)ethylenediamine1
(L) was prepared by mixing equivalent amounts of 1 mmol pyridine-
2-carbaldehyde and 1 mmol ethane-1,2-diamine in methanol solu-
tion under constant stirring for 24 h [33]. By adding 0.2 mmol of
the synthesized ligand solution to a mixture of 0.2 mmol of PPh3
and 0.2 mmol of Cu(I) iodide in acetonitrile under constant stirring
for 24 h, the formed orange-colored complex was filtered off (yield:
92%) (Scheme 1). Slow diffusion of diethyl ether into the filtered
solutions yielded orange needle crystal.
exþAem=2
Fcor ¼ Fobs ꢀ eA
ð1Þ
here Fcor and Fobs are the corrected and observed fluorescence inten-
sities, respectively and Aex and Aem are the absorption of the system
at the excitation and the emission wavelength, respectively.
2.3.3. Circular dichroism spectroscopy
Circular dichroism spectra of DNA (2.15 ꢀ 10ꢁ5 mol/L in Tris-
HCl pH = 7.4) in the presence of the Cu(I) complex (1.81 ꢀ 10ꢁ6
mol/L) were made on a JASCO spectropolarimeter Model J-810
using a 1.0 cm quartz cell. The CD spectra were recorded at room
temperature in a wavelength range of 200–300 nm.
2.3. DNA-binding procedures
2.3.1. UV–Visible absorption measurements
The UV–Visible absorption spectra of DNA (2 mL, 1.45 ꢀ 10ꢁ5
mol/L in Tris-HCl pH = 7.4) by increasing the concentration of the
Cu(I) complex from 0 to 4.69 ꢀ 10ꢁ6 mol/L were recorded in the
wavelength range of 200–400 nm on a Nordantec T80 UV–Visible
spectrophotometer.
2.3.4. Statistical analysis
All assays were executed in triplicate and the standard devia-
tion was calculated by the following equation (Eq. (2)) as the
square root of variance with determining each data point’s devia-
tion relative to the mean.
1
ChemSpider ID 92,443
2

N. Shahabadi, F. Shiri, S. Hadidi et al.
Journal of Molecular Liquids 335 (2021) 116290
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
X
displayed in Fig. 1. The X-ray analysis revealed that the Cu(I) com-
plex crystallized in the monoclinic space group P-21/n. The L ligand
binds to two copper centers in a bidentate chelating fashion
through N1 and N2 to Cu1 on side A and N3 and N4 to Cu2 on side
B. The two remaining coordination sites were occupied by PPh3
and iodide ligands. As evidence, the differences in similar angles
and bond lengths in two independent molecule parts are very
small. With no significant intermolecular interactions, the mole-
ðxi ꢁ ꢁxÞ2Þ=ðn ꢁ 1Þꢂ
ð2Þ
n
Standard Deviation ¼
½
i¼1
ꢁ
where xi is value of the ith point in the data set, x represents the
mean value of the data set and the number of data points in the data
set has been shown with n.
2.4. Molecular docking simulation
cules are further linked to each other as long as
pPPh3-pPPh3
The open-source AutoDock Vina (version 1.1.2) [35] and MGL
tools 1.5.6 [36,37] were used to perform docking simulations.
The partial charges of Gasteiger and polar hydrogens were added
to the Cu(I) complex and all rotatable bonds were defined. The B-
DNA sequence CGCGAATTCGCG/CGCGAATTCGCG was selected
from the Protein Data Bank (PDB ID: 1g3x) [38], because it has a
good structure resolution and closest sequence identity to the par-
ent AA/TT base for drug binding site in this work. Moreover, the
natural ligand present in the structure is considered as the possible
binding site for this structure. The DNA 3D structure and the resul-
tant docked were visualized by performing the visual molecular
dynamics, VMD. The DNA was enclosed in a box with the number
of points in x, y, and z dimensions of 20, 40, and 20 and center grid
box of 57.64, 49.00, and 58.63 with a grid spacing of 1.00 Å. The
docking calculation was performed using the Lamarckian genetic
algorithm (LGA) [39].
and PPh3- py to generate 3D network. Details of the crystallo-
p
p
graphic data and structure refinement parameters for the synthe-
sized complex are presented in Table 1.
3.2. DNA binding studies
3.2.1. UV–Visible absorption spectroscopy
The preliminary invitro evaluation of binding mode and binding
strength of the Cu(I) complex to DNA helix has been performed
through spectrophotometric titrations by following the changes
in absorbance values and the positions of the absorption band of
CT-DNA. Equal aliquots of the Cu(I) complex stock solution were
added to both DNA and reference solutions to eliminate the effect
of the Cu(I) complex absorbance (Fig. 2A). By increasing the Cu(I)
complex concentration, the maximum absorption of DNA helix at
260 nm represents a hyperchromism without any wavelength
shift, demonstrating that the Cu(I) complex has strong interaction
with DNA (Fig. 2B). The resulted hyperchromism might come from
2.5. Cell culture
p-p stacking interaction between the DNA-base pairs and the aro-
matic chromophores in the Cu(I) complex structure along with the
separation of DNA strands [44–46].
To determine the binding constant (Kb) the UV–Visible data
were analyzed based on the following equation (Eq. (3)) [47]:
The effects of the Cu(I) complex on human breast cancer cells
(MCF-7) and human umbilical venous endothelial cells (HUVEC)
were evaluated. The cell lines were routinely cultured on the
DMEM medium supplemented with fetal bovine serum and antibi-
otics. Proliferated cells were frozen for later use. The cells were
seeded in 96-well cell culture plate at a density of 1 ꢀ 104 cells
per well. The plate transferred to an incubator with 37 °C temper-
ature, 95% relative humidity and 5% CO2. After 24 h, the Cu(I) com-
plex and cisplatin with different concentrations (20, 40, 80, and
160 lg/mL), added on DMEM culture medium supplemented with
fetal bovine serum and penicillin/streptomycin as antibiotics. The
Cu(I) complex-free group considered as control. After 72 h of incu-
bation with the Cu(I) complex and cisplatin, about 20 lL of MTT
solution at a concentration of 5 mg/mL DPBS added to each well
and, finally, incubated in a 37 °C incubator for 4 h. At the end of
the incubation period, the medium was removed and 100 lL of
DMSO were added to each well then reading was performed at
570 nm with the ELISA reader. The results analyzed by SPSS soft-
ware using completely random design and then means compared.
1=ðA ꢁ A0Þ ¼ 1=ðA1 ꢁ A0Þ þ 1=KbðA1 ꢁ A0Þ ꢀ 1=½Complexꢂ
ð3Þ
here A0 represents the absorbance of DNA at 260 nm in the absence
of the Cu(I) complex, A1 shows the final absorbance of the Cu(I)
complex–DNA conjugate, and A is in accordance with the observed
absorbance at different complex concentrations. By the plotting of
1=ðA ꢁ A0Þversus 1=½Complexꢂ, the interaction binding constant (Kb)
of the Cu(I) complex-DNA system was determined to 6.88 ꢀ 105,
which is indicated that the Cu(I) complex has high DNA binding
affinity through insertion of the planer aromatic ring into the
DNA base pairs as observed for half sandwich Rh(III) and Ir(III) com-
plexes [24]. The resulted binding constant is also in the range of
both the intercalator copper complexes [48–50] and the groove bin-
der cases [51], while higher than anticancer metal complexes asuch
as cisplatin (5.51 ꢀ 104 L/mol) [27], and Cu(I) complexes with a
phenanthroline-phosphine set of ligands which have binding con-
stants in the range of 6.67 to 20 ꢀ 104 L/mol [12].
2.5.1. Investigation of apoptosis by acridin orange-ethidium bromide
dye
For this purpose, cells seeded on a 24-well plate and treated
with different concentrations of the Cu(I) complex and cisplatin
(20, 40, 80, and 160 lg/mL). Passing 72 h, supernatants removed
and each well was washed with DPBS solution that being removed
later. Then, paraformaldehyde solution was added to it for 15–
20 min. Fixing solution was removed and re-washed with DPBS.
Finally, Ethidium bromide and acridine orange (1:1) dye were
added to each well under dark condition. Cells were photographed
and evaluated under a fluorescent microscope.
3.2.2. Competitive fluorescence studies
The excitation scanning was performed to determine the fluo-
rescence properties of the Cu(I) complex (1 ꢀ 10ꢁ4 mol/L) but no
luminescence was observed for the Cu(I) complex upon excitation
either in aqueous solution or in the presence of DNA. Therefore, to
clarify the portion of the Cu(I) complex interacting with DNA, com-
petitive fluorescence experiments were used for further study of
the binding sites with both Hoechst 33,258 as a minor groove bin-
der probe at A-T rich regions [52] and methylene blue probe with
DNA intercalation ability [53–55]. In the Hoechst displacement
assay (Fig. 3A), it is observed that the fluorescence intensity of
the Hoechst-DNA conjugate decreases by nearly 100% upon subse-
quent titration of the Cu(I) complex, suggesting that the Cu(I) com-
plex is able to release the Hoechst molecules from DNA minor
groove into solvent solution after an exchange with them, which
supports the view that the Cu(I) complex is DNA minor groove bin-
3. Results and discussion
3.1. Single crystal X-ray structure determination
The molecular structure of the synthesized complex [Cu(PPh3)
(L0.5)(I)]2 (L = N,N’-Bis(2-pyridylmethylene)ethylenediamine) is
3

N. Shahabadi, F. Shiri, S. Hadidi et al.
Journal of Molecular Liquids 335 (2021) 116290
Fig. 1. The CPK and labeled licorice plots of the molecular structure of the Cu(I) complex [Cu(PPh3)(L0.5)(I)]2.
Table 1
adding the Cu(I) complex (Fig. 3B). Based on the results, the emis-
sion intensity of MB-DNA showed gradual enhancement up to
69.4% by increasing the Cu(I) complex concentration. So, it can
be concluded that an exchange between the Cu(I) complex and
methylene blue bonded to DNA took place, indicating an intercala-
tive kind of binding. Similarly, recovery of MB fluorescence was
attributed to an intercalation binding mode of curcumin Cu(II)
complex [57].
The crystal data and structure refinement parameters for the synthesized complex
and the selected bond lengths (Å) and angles (°) around the metal center.
Empirical formula
C50H44Cu2I2N4P2
Bond (Å) /angle (°)
Formula weight (g/mol)
Wavelength (Å)
T [K]
Crystal system
Space group
a [Å]
1143.71
1.54184
173.0
Monoclinic
P21/n
8.7424(2)
16.9780(6)
31.8558(10)
90
91.434(3)
90
4726.8(2)
4
1.607
12.297
2264.0
Cu1–I1
2.5803(9)
Cu1–P1
Cu1–N1
Cu1–N2
P1–Cu1–N1
P1–Cu1–N2
P1–Cu1–I1
N1–Cu1–N2
N1–Cu1–I1
N2–Cu1–I1
Cu2–I2
Cu2–P2
Cu2–N3
Cu2–N4
P2–Cu2–N3
P2–Cu2–N4
P2–Cu2–I2
N3–Cu2–N4
N3–Cu2–I2
N4–Cu2–I2
2.182(2)
2.103(5)
2.073(4)
115.0(1)
125.4(1)
124.02(5)
79.7(2)
b [Å]
c [Å]
3.2.3. Circular dichroism study
a
[°]
103.0(1)
99.8(1)
To obtain more information about the possible portion confor-
mational changes related to the binding of the Cu(I) complex to
specific DNA sites, the CD experiment was carried out. The CD
spectrum of DNA in the typical B conformation exhibits a positive
band at around 275 nm, which arises from base stacking and a neg-
ative band at around 245 nm due to right-handed helicity [58]. As
regards, any specific conversion of DNA morphology sensitizes the
CD signal, accordingly, the CD spectropolarimetry provides unique
possibilities to identify and interpret DNA binding modes of small
molecules with high sensitivity. Considering this, the CD spectra of
CT-DNA were monitored in the absence and presence of the Cu(I)
complex. It can be viewed that the presence of the Cu(I) complex
caused a considerable increase in the intensity of positive peak
than negative one without any significant change in the band posi-
tions (Fig. 4).
b [°]
c
[°]
2.6204(8)
2.193(1)
2.097(4)
2.092(4)
125.4(1)
122.1(1)
120.30(5)
79.1(2)
V [Å3]
Z
Density [g/cm3]
l
[mmꢁ1
]
F(0 0 0)
Crystal Size [mm]
Reflections collected
Rint [%]
0.3 ꢀ 0.03 ꢀ 0.03
26,539
0.0538
1.029
93.1(1)
107.4(1)
Goodness-of-fit on F2
Computer programs: CrysAlis PRO [40], SHELXT [41], SHELXL2014 [42] and OlexSys
[43].
der at A-T rich sequences, where the Hoechst molecule was
located, as similarly reported for groove binder macrocyclic Cu(II)
complex [56].
The enhancement of the positive CD band can offer typical
intercalation including
p-stacking and stabilization of the right-
Another kind of competitive assay based on the MB probe was
performed to prove the existence of intercalation mode. The emis-
sion spectra of MB–DNA conjugate were recorded before and after
handed B form of DNA [59,60]. The observed increase in the nega-
tive CD peak of DNA can be due to partial intercalation of planer
phenyl rings moieties between the base pairs in the minor groove
0.6
B
A
1.8
1.5
1.2
0.9
0.6
0.3
0.0
Free DNA
[DNA+Complex]-Free Complex
Free Complex
0.5
increasing
in complex
concentration*
0.4
0.3
0.2
0.1
0.0
DNA+Complex
DNA
240
Wavelength (nm)
220
240
260
280
300
320
220
260
280
300
320
Wavelength (nm)
Fig. 2. (A) UV–Visible absorption spectra of the Cu(I) complex (1.74 ꢀ 10ꢁ5 mol/L) in the absence and presence of DNA (3.02 ꢀ 10ꢁ5 mol/L), (B) UV–Visible absorption spectra
of 1.45 ꢀ 10ꢁ5 mol/L DNA by increasing the concentration of the complex from 0 to 4.69 ꢀ 10ꢁ6 mol/L up to 7 increments in 50 mmol/L Tris–HCl buffer (pH 7.4) at room
temperature. *All spectra are reported after removing the Cu(I) complex disturbance.
4

N. Shahabadi, F. Shiri, S. Hadidi et al.
Journal of Molecular Liquids 335 (2021) 116290
140
Hoechst+DNA
280
240
200
160
120
80
B
A
MB
120
Increasing
Increasing
in complex
concentration
100
80
60
40
20
in complex
concentration
40
MB+DNA
Hoechst
0
0
645 660 675 690 705 720
360 390 420 450 480 510 540
Wavelength (nm)
Wavelength (nm)
Fig. 3. (A) Competitive displacement analysis between the Cu(I) complex and Hoechst33258 in a pre-treatment Hoechst-DNA conjugate (CHoechst = 5 ꢀ 10ꢁ6 mol/L,
CDNA = 1.18 ꢀ 10ꢁ3 mol/L, Ccomplex = 0 to 1.12 ꢀ 10-4 mol/L) in 50 mmol/L Tris HCl buffer (pH 7.4) at 288 K (B) Competitive displacement analysis between the complex and
MB in a pre-treatment MB-DNA conjugate (CMB = 5 ꢀ 10ꢁ6 mol/L, CDNA = 1.96 ꢀ 10ꢁ4 mol/L, Ccomplex = 0 to 2.33 ꢀ 10ꢁ4 mol/L) in 50 mmol/L Tris-HCl buffer (pH 7.4) at 288 K.
centration at 288, 293, 298, and 303 K. Subsequent quenching of
the probe-DNA conjugate with the sequential addition of the Cu
(I) complex is considered as a direct approval for the interaction
of the Cu(I) complex with DNA helix [65]. The observed fluores-
cence quenching does not show any significant change in the peak
position. Stern–Volmer equation (Eq. (4)) was applied to evaluate
the fluorescence quenching mechanism at a quantitative level
[66,67]:
220
240
260
280
49.98%
300
2
75
50
1
0
0.01%
25
0
F0=F ¼ 1 þ kqs0½Qꢂ ¼ 1 þ Ks ½Qꢂ
ð4Þ
v
where the Stern–Volmer quenching constant is described by KSV
,
-25
-50
-75
the apparent bimolecular quenching rate constant is defining by
kq, [Q] is the concentration of the quencher complex and s0 the
unquenched fluorophore average lifetime which is about 1 ꢀ 10ꢁ8
S [68]. Applying different temperatures (288, 293, 298, and 303 K)
to eviscerate the Stern-Volmer plot, yields the KSV values at a fixed
y-axis intercept of 1 from the slop (Fig. 5E). The obtained Stern-
Volmer constants at applied temperatures found to be in the range
of 14.04–6.44 ꢀ 103 Mꢁ1 that is along with the groove binding
mode of the interaction [69]. The static or dynamic nature of the
quenching mechanism can be distinguished by their dependence
on temperature. Because an increase in temperature results in a lar-
ger diffusion coefficient and promotes electron transfer, the Stern–
Volmer quenching constant becomes larger as the temperature goes
up, implying a dynamic quenching mechanism. In contrast, in the
-1
-2
4.81%
29.37%
DNA
Complex
Solvent
200
220
240
260
280
300
Wavelength (nm)
Fig. 4. The CD spectra of the interaction of 1.81 ꢀ 10ꢁ6 mol/L complex with
2.15 ꢀ 10ꢁ5 mol/L DNA.
and cause the release of the water from the grooves consequently
the DNA helix can potentially tolerate more bending in the pres-
ence of the Cu(I) complex [61,62]. In overall, the changes in the
CD spectra indicated that the binding of the Cu(I) complex to
DNA caused stabilization of the right-handed B-DNA without any
transition in form (B-DNA to A or Z form) [63,64]. Similarly, in
the case of [Cu(Cur)(DIP)]+2, the increased intensity of the both
negative and positive bands indicated fixating the right-handed B
conformation of CT-DNA via intercalation binding mode [57]. The
effect of the solvent (DMSO 0.05%, Ethanol 0.1%) on the DNA struc-
ture was also investigated. As illustrated in Fig. 4, no significant
change in the intrinsic CD spectrum of the DNA was observed,
which indicated that the solvent does not disturb the DNA
structure.
case of
a static mechanism, the thermal stability of the
fluorophore-quencher complex decrease with increasing tempera-
ture, thus, the values of the Stern–Volmer quenching constants
are expected to be smaller. The interaction of the Cu(I) complex
with DNA leads to a significant decrease in KSV values to 54.09%
as temperature goes up to 303 K, supporting the involvement of sta-
tic mechanism in the quenching process, as reported for interaction
mechanism of a series of binary Cu(II) complexes containing inter-
calating ligand [70].
3.2.5. Binding equilibrium
The binding constant (Kb) and the Hill coefficient (n) for the
interaction of the Cu(I) complex with DNA were calculated via
the logarithmic Hill equation (Eq. (5)) (Fig. 5F) [71]:
3.2.4. Steady-state fluorescence studies
To provide a detailed analysis of the interaction mechanism,
thermodynamic parameters, and critical forces in the binding pro-
cess, a steady state fluorescence experiment was utilized. Regard-
ing the negligible fluorescence emission of both DNA helix and the
Cu(I) complex, a fixed amount of Hoechst 33,258 was used to probe
the interaction. Fig. 5A, B, C and D show the fluorescence emission
of Hoechst-labeled DNA with increasing of the Cu(I) complex con-
ðF0 ꢁ FÞ
Log
¼ LogKb þ nLog½Qꢂ
ð5Þ
F
where F0 and F represent the fluorescence intensity of the Hoechst-
labeled DNA in the absence and the presence of different the Cu(I)
complex concentrations [Q], n is the Hill cooperativity coefficient
to describe DNA binding sites cooperativity to put the Cu(I) complex
5

N. Shahabadi, F. Shiri, S. Hadidi et al.
Journal of Molecular Liquids 335 (2021) 116290
140
140
120
100
80
140
120
100
80
A, 288K
B, 293K
C, 298K
DNA+Hoechst
120
100
80
Increasing
in complex
concentration
60
60
60
40
40
40
20
20
20
Hoechst
0
0
0
360 390 420 450 480 510 540
360 390 420 450 480 510 540
360 390 420 450 480 510 540
Wavelength (nm)
D, 303K
2.4
140
0.5
E
288K
293K
298K
303K
F
288K
293K
298K
303K
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
120
100
80
60
40
20
0
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
-5.4 -5.1 -4.8 -4.5 -4.2 -3.9
360 390 420 450 480 510 540
Wavelength/nm
log [Complex]
0.00000 0.00002 0.00004 0.00006 0.00008 0.00010 0.00012
[Complex] (mol/L)
Fig. 5. A, B, C, and D) Fluorescence emission spectra of 5 ꢀ 10ꢁ6 mol/L and 1.18 ꢀ 10-3 mol/L of Hoechst and CT-DNA along with the Cu(I) complex concentration increasing
from 0.0 to 1.12 ꢀ 10ꢁ4 mol/L in 50 mmol/L Tris–HCl buffer (pH 7.4) at 288, 293, 298, and 303, respectively, (E) Stern–Volmer plot of the Cu(I) complex-DNA interaction by
applying temperatures 288, 293, 298, and 303 K, (F) logFo-F/F vs. log[Complex] plot for the determination of binding constants corresponding to 288, 293, 298, and 303 K.
Table 2
in these sites, and Kb (binding constant) utilizes DNA binding affin-
The binding parameters for the Cu(I) complex-DNA interaction.
ity of the Cu(I) complex. The calculated Kb and n are presented in
Kb (L molꢁ1
)
n
R2
Table 2. The downward trend of the Hill coefficient with tempera-
ture increasing, showing a negative cooperation effect. Additionally,
the Kb values decrease as temperature rises, suggesting that static
quenching occurs [34].
T (K)
288
293
298
303
8.06 ꢀ 105
4.85 ꢀ 105
1.59 ꢀ 105
4.62 ꢀ 104
1.45
1.43
1.34
1.21
0.9844
0.9953
0.9972
0.9915
3.2.6. Thermodynamic parameters
Intermolecular forces that exist between small ligands and bio-
molecules can be based on four types of non-covalent interactions
i.e. hydrogen bond, van der Waals force, electrostatic interaction,
and hydrophobic force [72]. According to the data of enthalpy
Table 3
The thermodynamic parameters for the Cu(I) complex-CT-DNA interaction.
T(K)
D
G (kcal molꢁ1
)
D
S (cal molꢁ1 Kꢁ1
ꢁ89.20
)
D
H (kcal molꢁ1
ꢁ33.59
)
288
293
298
303
ꢁ7.78
ꢁ7.62
ꢁ7.09
ꢁ6.47
changes (
between ligand and biomolecule can be concluded: (1)
and S > 0, hydrophobic forces; (2) H < 0 and S < 0, van der
Waals interactions and hydrogen bonds; (3) H < 0 and S > 0,
D
H) and entropy changes (
D
S), the model of interaction
D
H > 0
D
D
D
D
D
electrostatic interactions [73,74]. To identify the main interaction
force between DNA and the Cu(I) complex, the thermodynamic
parameters were derived using the Gibbs–Helmholtz equation
(Eq. (6)) [75]:
an exothermic reaction, which means that higher temperatures lead
to instability of the Cu(I) complex-DNA conjugate, confirmed by the
decrease in Kb values as temperature increases [76]. According to
Gꢃ ¼
D
Hꢃ ꢁ T
D
Sꢃ ¼ ꢁRTLnKb
ð6Þ
Ross and Subramanian studies [73], the negative values of
DH°
D
and S° are indicated of most probable the van der Waals interac-
D
tions as main driving forces in the Cu(I) complex binding to DNA
which is the main evidence for intercalation binding mode as
reported for the DNA interaction of pyrazoline based palladium(II)
complexes [27] and macrocyclic Cu(II) complex [56].
where Kb is the binding constant at the corresponding temperature,
G° shows the free energy change for the binding reaction, H°
represents the molar enthalpy change, the molar entropy change
is defined by
S°, R = 1.9872 cal molꢁ1 Kꢁ1 is the universal gas con-
stant, and T is the temperature in kelvins. The corresponding ther-
modynamic parameters, G°, H°, and S°, were reported in
Table 3. The negative binding free energies ( G°) evidenced favor-
able interactions between the Cu(I) complex and DNA [47]. The sig-
nificant volume reduction of the Cu(I) complex-DNA conjugate in
the intercalation binding mode or the changes in solvation during
D
D
D
D
D
D
D
3.2.7. Molecular docking studies
Based on the competitive fluorescence studies, the Cu(I) com-
plex is able to release the Hoechst molecules from DNA minor
groove at A-T rich region as well as MB from two DNA strands.
To confirm the experimental results, molecular docking simulation
was performed with DNA sequence CGCGAATTCGCG/
CGCGAATTCGCG (PDB ID: 1g3x), which contains A-T bases in the
the interaction process can be reflected in negative
DS° value. Based
on the negative value of H°, binding of the Cu(I) complex to DNA is
D
6

N. Shahabadi, F. Shiri, S. Hadidi et al.
Journal of Molecular Liquids 335 (2021) 116290
middle part (minor groove), where the Cu(I) complex is probably
bonded. From the docking analysis, the best conformer with the
lowest binding energy was picked from the 20 unique conforma-
tions among 500 runs (Fig. 6).
The selected docking pose showed that the Cu(I) complex can
bind to DNA model with high affinity (ꢁ7.4 kcal/mol). From
Fig. 6, it is clear that the planar aromatic rings of the Cu(I) complex
was inserted in the core of the adenine DA606 and adenine DA605
nucleobases at the middle part of DNA composed by AT base pairs
cisplatin showed a 39% reduction in cells viability at the same con-
centration, which proved more positive effect of the Cu(I) complex
on inhibition of cancer cell proliferation. Based on the published
data, cytotoxic mechanism of copper complexes is different than
that of platinum complexes [77–79], which is mainly related to
the copper affinity for binding to sites occupied by other metals,
and or their ability for DNA intercalation [13,21,80]. So, it can be
concluded that the more cytotoxicity effect of the complex is
attributed to DNA intercalation binding mode and lipophilic char-
acter, as reported for copper(I)-phosphine polypyridyl complexes
[20].
On the other hand, it can be clearly seen in Table 4 that the
HUVEC cell viability percent was higher than MCF-7 at all the Cu
(I) complex concentrations, that is indicated the Cu(I) complex
was more sensitive to inhibition of cancer cells than normal cells.
This is consistent with in vitro cell culture experiments that
demonstrated a significant antiproliferative activity for Cu(I) com-
pounds against several cancer cell lines with a preferential cell
growth inhibition towards tumour than nonmalignant cells [81].
Moreover, other studies showed that some binuclear copper-
based complexes can remarkably induce apoptosis and inhibit
angiogenesis to mediate tumour growth [82].
through
p-p stacking interactions in 4.54 Å and 5.19 Å, while the
remaining part of the Cu(I) complex lies in DNA minor groove
between DT619 to DA605. Dock results also show the presence
of weak hydrophobic forces in the interaction (Fig. 7). The obtained
results confirm the role of both intercalation (p-p interactions
between the bases) and groove binding at A-T rich region in the
interaction and the experiments results accuracy.
3.2.8. Cytotoxicity evaluation
The results of cytotoxicity evaluation of the Cu(I) complex and
cisplatin on MCF-7 cancer cells and HUVEC normal cells has been
shown in Table 4. As we can see the toxicity of the Cu(I) complex
toward MCF-7 cells was higher than cisplatin at all concentrations.
According to the findings, the highest descent in MCF-7 cells viabil-
ity reached to 21% by 160 mg/mL of the Cu(I) complex while
In addition, to determine apoptosis effects of the Cu(I) complex
and cisplan on MCF-7 and HUVEC cell lines, morphological assay of
Fig. 6. (A) Molecular modeling of the interaction between the Cu(I) complex and DNA dodecamer d(CGCGAATTCGCG/CGCGAATTCGCG) in a box with the number of grid
points in x ꢀ y ꢀ z directions, 20 ꢀ 40 ꢀ 20 and a grid spacing of 1.00 Å, (B) The stacking distance between the Cu(I) complex and DNA nucleobases.
p-p
Fig. 7. The involvement of hydrophobic and van der Waals forces in the interaction of the Cu(I) complex and DNA model.
7

N. Shahabadi, F. Shiri, S. Hadidi et al.
Journal of Molecular Liquids 335 (2021) 116290
Table 4
MCF-7 and HUVEC cell viability percent in the presence of the Cu(I) complex on 72 h incubation.
Concentration
% Viable MCF-7 cancer cells
Cu(I) complex
% Viable HUVEC cells
Cisplatin
Cu(I) complex
Cisplatin
20
40
80
l
l
l
g/mL
g/mL
g/mL
49.59 4.60
34.11 2.31
27.15 1.02
21.35 0.50
61.83 3.10
55.02 0.50
49.34 5.70
39.33 1.45
69.95 3.64
48.04 1.03
34.97 2.09
23.43 0.79
104.36 9.87
69.13 4.40
34.24 3.40
28.40 0.56
160 lg/mL
Fig. 8. Ethidium bromide-Acridine orange staining of MCF-7 (A, C) and HUVEC cells (B, D) treated 72 h with different concentrations of the Cu(I) complex and cisplatin.
cell death was investigated using acridin orange-ethidium bromide
dye staining. The cytotoxic activities of these compounds in their
induction of early and late apoptosis were analysed in a concentra-
tion dependent manner. 72 h after treatment different morpholog-
ical features like bright green early apoptotic cells with nuclear
margination and chromatin condensation were observed in all cell
lines, while nuclear morphology from control cells were seen uni-
formly green with normal morphology (Fig. 8). As the concentra-
tion of the Cu(I) complex and cisplatin increased, orange stained
cells which show high levels of apoptotic cell death became more
than bright green nucleus. The results clearly showed the Cu(I)
complex and cisplatin were able to induce apoptosis in MCF-7 can-
cer cells more than HUVEC normal cells.
aromatic chromophores of the Cu(I) complex results in DNA strand
separation which recognize by hyperchromism in UV–Visible spec-
tra. A decrease in the fluorescence intensity of Hoechst-DNA conju-
gate and an increase in the emission intensity of MB-DNA system,
confirmed that the Cu(I) complex was able to displace both
Hoechst molecules from the minor groove of DNA helix at A-T rich
region and MB dye from the intercalative binding sites. A gradual
decrease in KSV value by temperature increases indicated static flu-
orescence quenching involvement in the binding process. Based on
the thermodynamic data (negative
DH and DS values) and molec-
ular docking results van der Walls and hydrophobic forces play the
key role in the binding of the Cu(I) complex to DNA. The increased
intensity of the both negative and positive CD bands indicated
binding of the Cu(I) complex to DNA caused stabilization of the
right-handed B-DNA without any transition in form. The molecular
docking simulations confirmed the role of both intercalation and
groove binding in the interaction process and the accuracy of the
experimental results. The cytotoxicity results on human breast
cancer cells showed more anticancer effect of the Cu(I) complex
4. Conclusion
The monoclinic phase [Cu(PPh3)(L0.5)(I)]2 with a tetrahedral
geometry was identified by X-ray single-crystal diffraction data.
The p-p stacking interaction between the DNA base pairs and the
8

N. Shahabadi, F. Shiri, S. Hadidi et al.
Journal of Molecular Liquids 335 (2021) 116290
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than cisplatin against MCF-7 cells and also the Cu(I) complex was
more sensitive to inhibition of cancer cells than normal cells. The
present study not only enable us to have a new viewpoint of the
interaction mechanism of the Cu(I) complex with DNA, but also
helps us to design new molecular models of novel and more effi-
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(2020) 1.
Funding
[30] N. Shahabadi, S. Hadidi, F. Shiri, J. Biomol. Struct. Dyn. 38 (2020) 283.
[31] K. Gholivand, K. Farshadfar, S.M. Roe, M. Hosseini, A. Gholami, CrystEngComm
18 (2016) 7104.
There are no funders from any person or institution to report for
this submission.
[32] K. Gholivand, R. Salami, K. Farshadfar, R.J. Butcher, Polyhedron 119 (2016) 267.
[33] M. Abdoh, I. Warad, S. Naveen, N. Lokanath, R. Salghi, Acta Crystallographica
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Olson, J. Comput. Chem. 30 (2009) 2785.
CRediT authorship contribution statement
Nahid Shahabadi: Supervision. Farshad Shiri: Investigation.
Saba Hadidi: Investigation. Kaveh Farshadfar: Formal analysis.
Maryam Darbemamieh: Investigation. S. Mark Roe: Formal
analysis.
[37] R. Gaur, R.A. Khan, S. Tabassum, P. Shah, M.I. Siddiqi, L. Mishra, J. Photochem.
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Olson, J. Comput. Chem. 19 (1998) 1639.
Declaration of Competing Interest
[40] P. CrysAlis, Yarnton, Oxfordshire, England, 2014.
[41] G.M. Sheldrick, Acta Crystallogr., Sect. A: Found. Adv 70 (2014) C1437.
[42] G.M. Sheldrick, Acta Crystallogr. Sect. C: Struct. Chem. 71 (2015) 3.
[43] C.B. Hübschle, G.M. Sheldrick, B. Dittrich, J. Appl. Crystallogr. 44 (2011) 1281.
[44] M.M. Ackerman, C. Ricciardi, D. Weiss, A. Chant, C.M. Kraemer-Chant, J. Chem.
Educ. 93 (2016) 2089.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
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We thank Inorganic Research Lab. 2 of Razi University.
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Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molliq.2021.116290.
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