Synthesis and characterization of Pd(II) antitumor complex, DFT calculation and DNA/BSA binding insight through the combined experimental and theoretical aspects

Article abstract of DOI:10.1016/j.molstruc.2021.130535

A novel palladium(II) complex, [Pd(bpy)(acac)]NO₃ (where bpy and acac are 2,2′-bipyridine and acetylacetonato), has been synthesized and fully characterized through ¹H NMR, UV-Vis and FT-IR spectra, conductivity measurement and elemental analysis. Geometry optimization using DFT calculation demonstrated that a square-planar geometry is built around Pd(II) atom. The MEP and FMO analysis were also applied. Partition coefficient evaluation was conducted to determine the lipophilicity of the compound which follows the order complex > cisplatin. The antitumor activity of the complex has been evaluated on K562 cancer cells by MTT method, which exhibited an appropriate activity in compare to cisplatin. The interaction of [Pd(bpy)(acac)]NO₃ towards biomolecules (CT-DNA and BSA) was investigated through in-detail fluorescence, UV–Vis and other techniques. The findings of fluorescence titration demonstrated that the Pd(II) complex binds to CT-DNA via hydrophobic and with BSA through van der Waals forces and hydrogen bond. UV–Vis experiment revealed a reduction in the absorption spectra of biomolecules in presence of the complex. The binding affinity was further endorsed for CT-DNA utilizing viscosity measurement and gel electrophoresis and for BSA via CD spectroscopy and FRET calculation. In addition, molecular docking supports the findings of experiments and indicates that the Pd(II) complex appears to be situated at DNA intercalation position and BSA Sudlow's site I. All results have shown that this compound may be a useful potential antitumor candidate for further medical purposes.

Full text of DOI:10.1016/j.molstruc.2021.130535

Journal of Molecular Structure 1240 (2021) 130535
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis and characterization of Pd(II) antitumor complex, DFT
calculation and DNA/BSA binding insight through the combined
experimental and theoretical aspects
∗
Mehran Feizi-Dehnayebi, Effat Dehghanian , Hassan Mansouri-Torshizi
Department of Chemistry, University of Sistan and Baluchestan, Zahedan, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
ꢀ
A novel palladium(II) complex, [Pd(bpy)(acac)]NO3 (where bpy and acac are 2,2 -bipyridine and acetylace-
tonato), has been synthesized and fully characterized through 1H NMR, UV-Vis and FT-IR spectra, conduc-
Received 16 January 2021
Revised 12 April 2021
Accepted 19 April 2021
Available online 24 April 2021
tivity measurement and elemental analysis. Geometry optimization using DFT calculation demonstrated
that a square-planar geometry is built around Pd(II) atom. The MEP and FMO analysis were also applied.
Partition coefficient evaluation was conducted to determine the lipophilicity of the compound which fol-
lows the order complex > cisplatin. The antitumor activity of the complex has been evaluated on K562
cancer cells by MTT method, which exhibited an appropriate activity in compare to cisplatin. The inter-
action of [Pd(bpy)(acac)]NO3 towards biomolecules (CT-DNA and BSA) was investigated through in-detail
fluorescence, UV–Vis and other techniques. The findings of fluorescence titration demonstrated that the
Pd(II) complex binds to CT-DNA via hydrophobic and with BSA through van der Waals forces and hydro-
gen bond. UV–Vis experiment revealed a reduction in the absorption spectra of biomolecules in presence
of the complex. The binding affinity was further endorsed for CT-DNA utilizing viscosity measurement
and gel electrophoresis and for BSA via CD spectroscopy and FRET calculation. In addition, molecular
docking supports the findings of experiments and indicates that the Pd(II) complex appears to be situ-
ated at DNA intercalation position and BSA Sudlow’s site I. All results have shown that this compound
may be a useful potential antitumor candidate for further medical purposes.
Keywords:
K562
DFT calculation
FMO
Biomolecules
Molecular docking
©
2021 Elsevier B.V. All rights reserved.
1
. Introduction
of proteins and peptides. This leads to inactivation of platinum
complexes, severe side effects, and cellular resistance [4]. These
drawbacks of platinum based complexes are a motivation for syn-
thesis and testing new anticancer agents with different transition
metals.
Cisplatin is a chemotropic drug discovered by Rosenberg [1] and
currently administered in several types of cancers such as colorec-
tal, testicular, ovarian, and lymphoma [2,3]. One accepted hypothe-
sis about the mechanism of action for cisplatin (cis-[PtCl (NH ) ])
Since both platinum(II) and palladium(II) ions have d8 elec-
tron configuration and nearly the same size, so they exhibit sim-
ilar chemical properties and form square planner complexes [5].
As a result, therapeutics effects and antitumor activity of palla-
dium is of interest for further studies. However, Pd-complexes are
more labile and reactive than Pt-analogs where by choosing ap-
propriate chelate ligand, one can keep this reactivity at the rea-
sonable limit and in accordance with biological system conditions.
Some researchers have demonstrated that many palladium com-
plexes show anticancer activity at pH = 6.8 that belongs to cancer
cell lines where healthy cells live in pH = 7.4 [6]. Another interest-
ing point about palladium complexes is that some of trans com-
plexes have more cytotoxicity activity than cis ones [7].
2
3 2
as an anticancer compound is that when it enters a cell, hy-
drolysis happens and two chloride ligands are replaced by wa-
ter molecules. As a result, the ionic complex with positive charge
is produced which has a tendency to interact with nucleophilic
species like DNA. This action distorts DNA and causes cytotox-
icity property of platinum based complexes including cisplatin.
Since amine groups (NH ) are the same in cisplatin, carboplatin,
3
and nedaplatin (Fig. 1), therefore they may demonstrate equivalent
DNA binding mode. On the other hand, platinum based complexes
have drastic potency for interacting with sulfur in the amino acids
∗
ꢀ
Corresponding author.
2,2 -bipyridine ligand (bpy) consists of two aromatic nitrogen-
E-mail
addresses:
m.feizi@sutech.ac.ir
(M.
Feizi-Dehnayebi),
containing pyridine (C H N) rings. Such a pyridine ring interacts
5
4
dehghanian@chem.usb.ac.ir (E. Dehghanian), hmtorshizi@hamoon.usb.ac.ir (H.
Mansouri-Torshizi).
with metals mostly as a σ-donor/π-acceptor agent. Since the dis-
https://doi.org/10.1016/j.molstruc.2021.130535
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Fig. 1. Molecular structure of platinum based complexes with two NH3 ligands (A: cisplatin, B: carboplatin, and C: nedaplatin).
covery of bpy at the end of nineteenth century [8] it has been
largely used in the synthesis of complexes with metal ions. As it
is a neutral compound it forms charged complexes with transition
metal ions. Acetylacetone, an organic molecule with the general
company. CT-DNA (Calf thymus DNA, highly polymerized-type1),
Tris–HCl buffer and BSA were obtained from Sigma-Aldrich and
commercial solvents (Merck) including acetonitrile, ethanol, ace-
tone and methanol were reagent grade and were used as re-
ceived. Deionized water was utilized all over measurements. Simi-
lar synthetic procedure [13] was carried out for the preparation of
formula CH COCH COCH , is commonly abbreviated to acac, is an
3
2
3
appropriate chelating compound as i) it is available at proper cost
ii) it forms stable chelating complexes with over 60 metals even at
low concentration of metals and iii) it is soluble in organic as well
as some inorganic solvents [9].
[Pd(bpy)Cl ].
2
Fourier Transfer Infrared (FT-IR) spectra of the complex and
ligands were measured in KBr pellets on a Perkin Elmer FT-
IR spectrometer (400–4000 cm 1). UV–Vis spectra of free metal
complex and interacted metal complex with biomolecules were
taken utilizing a JASCO, V-570. The lipophilicity value of the com-
pound was evaluated utilizing partition coefficient experiment
by shake-flask method. Molar conductivity of the Pd(II) com-
−
DNA or deoxyribonucleic acid [10] is the hereditary substance
that carries genetic information in almost all organisms. Each DNA
includes a backbone of sugar and phosphate along with some at-
tached bases to it. The name is taken from its combination, in the
other word, "deoxyribo" refers to the sugar and the nucleic acid
refers to the bases and phosphate. The bases are adenine, cytosine,
guanine, and thymine, where their pattern encodes huge complex-
ity. DNA is assumed to be the main target for anticancer drugs, be-
cause it is involved in important mechanisms in the cells. So, the
definitive target for palladium based complexes is widely accepted
to be DNA. But the exact mechanism by which the palladium based
compounds interact with DNA and lead to the cell death is un-
known.
plex was measured on HANA-EC215, using deionized water. 1
H
NMR spectrum of the synthesized metal complex was recorded
in the range of 0–12 ppm by a Brucker-250 NMR spectrome-
ter utilizing DMSO–d6 as solvent and TMS as standard reference.
Elemental analysis for CHN was conducted on a Heraus CHNO-
RAPID elemental analyzer. Using the fluorescence spectrometer,
Cary Eclipse, the emission patterns of the samples were mea-
sured. The circular dichroism (CD) measurement of BSA in the
presence and absence of Pd(II) complex were recorded through
Aviv-215 CD spectrometer, USA. Using Maxi HD 15 × 15 Tank, Lad-
der 100 bp, electrophoresis assay was performed on CCD-5 Gel
Doc System. Decomposition temperature of complex was measured
through electrothermal model 9100 using glass capillary and are
uncorrected. Viscosity experiments were performed utilizing Ost-
wald micro-viscometer (SCHOT-geräte) at ambient temperature of
27 ± 1 °C.
Serum albumin (SA) is the most abundant protein in the blood-
stream of many organisms and some tissues. Investigating the in-
teraction between albumin and drug is essential and of vital im-
portance since the distribution and action of many drugs are re-
lated with their binding affinities to SA. Bovine serum albumin
(
BSA) is often chosen as protein model for human serum albu-
min (HSA) because of structural similarity to HSA, unusual bind-
ing properties, water solubility, availability and its low cost [11].
It is composed of 3 homologous domains i.e., I, II, and III and 2
subdomains (A and B). BSA has 2 tryptophan residues (Trp134 and
Trp212) that have intrinsic fluorescence emission property [12].
Keeping the above subject matters in mind, we intend to syn-
thesize and characterize the new water-soluble palladium(II) com-
plex. Partition coefficient value of the complex was determined to
check the lipophilicity of the palladium(II) complex in compari-
son to cisplatin. The antitumor activity of the complex was de-
termined for cancer cells by MTT assay. Density functional the-
ory (DFT) calculations i.e. geometry optimization, frontier molec-
ular orbital (FMO) analysis and molecular electrostatic potential
2
.2. Synthesis process of [Pd(bpy)(acac)]NO3
ꢀ
Acetylacetonato-2,2 -bipyridinepalladium(II) nitrate, C15H15N O
3
5
Pd = 423 g/mol
.1 mL (1 mmol) of acetyl acetone and 0.04 g (1 mmol) of
NaOH were dissolved in a blend of water and ethanol (20 mL,
:1 V/V) and stirred at 40 °C for 20 min for obtaining sodium salt
of acetyl acetonate. This mixture was poured on a suspension of
Pd(bpy)Cl ] (0.333 g, 1 mmol) in 50 mL double distilled water and
0
1
[
2
(MEP) surface were conducted in the gas phase. The in-detail in-
stirred for 2 h at 50 °C. It was then cooled to 28 °C and 2 mmol
0.335 g) of AgNO3 in 10 mL water was added and stirred at 50 °C
for 7 h in dark. Stirring continued at room temperature for an-
other 12 h. The AgCl precipitate was filtered off through Wattman
teraction of [Pd(bpy)(acac)]NO3 with DNA as the principal target
of anticancer agents and BSA as a transport protein in the blood
circulation was investigated by different experimental methods. In
addition, in order to predict the correlation between computational
and experimental results, the compound has been interacted with
DNA and BSA via in-silico molecular modeling.
(
4
2 filter paper. The volume of yellowish filtrate was reduced by
evaporation at 45 °C to 35 mL and filtered again. Evaporation was
continued to 7 mL and left a side overnight. The obtained yellow
precipitate was filtered, washed with acetone and dried in an oven
at 50 °C. Recrystallization was carried out via dissolving the precip-
itate in a 5 mL mixture of acetonitrile methanol (1:1 V/V). On dif-
fusing diethyl ether to this blend for 72 h, the cauliflower shaped
precipitate was formed which was filtered, washed with acetone
and dried in an oven at 50 °C. Yield: 0.44 g (70%) and decomposed
at 226–228 °C. The suggested equations are illustrated in Fig. 2.
2. Experimental
2
.1. Material and method
Acetyl acetone, NaCl, ethidium bromide (EB), PdCl , NaOH,
2
ꢀ
AgNO3 and 2,2 -bipyridine were bought from Merck chemical
2

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Fig. 2. Structure and reactions suggested for [Pd(bpy)(acac)]NO3 complex involving 1H NMR signing scheme.
2
.3. Computational details
The cells were cultivated in humidified conditions (5% CO2 and
9
5% air atmosphere). The MTT (3-(4,5-dymethylthiazol-yl)−2,5-
The initial 3D structure of Pd(II) complex and its related lig-
diphenyltetrazolium bromide) assay was applied for the measure-
ment of cytotoxicity effect of this complex against the cells [21].
This experiment is dependent on the conversion and cleavage of
MTT to formazan via active mitochondrial dehydrogenase of liv-
ing cells. Harvested sample cells were seeded into a 96-well cul-
ands were built using Gauss view 5.0 software. DFT method has
been applied to achieve an insight into the palladium(II) complex
and ligands electronic structure in the gas phase through Gaussian
09 software. The B3LYP (the Becke’s exchange functional [14] and
4
the Lee, Yang and Parr’s [15] correlation functional)/GENECP (with
effective core pseudo potentials) level of theory were applied for
geometry optimization and frequency calculations of ground state
ture plate (1.5 × 10 cell/mL) with diverse concentrations of the
[Pd(bpy)(acac)]NO3 from 0 to 100 μM and incubated for 24 h. Be-
fore the end of incubations step, 25 μL of MTT solution (5 mg/mL)
in PBS was poured into each well-plate. The resulted formazan pre-
cipitates were dissolved in a 50% DMF solution and sodium dode-
cyl sulfate (SDS, 10%) and incubated for 3 h at 37 °C in dark. Opti-
cal density (OD) was read to reagent blank utilizing ELISA Reader-
Model Expert 96 (Austria) at 570 nm. In this assay, absorbance is a
function of converted dye concentration. The viability of each study
group was reported as percentage control absorbance and specified
as 50% inhibition concentration (IC50) [22].
[
16]. Aug-cc-pVTZ-PP, and 6–311 g(d) basis sets were used for Pd
and nonmetal atoms (H, C, N and O), respectively [17-19]. The cal-
culated structure was confirmed to be at true minimum (lack of
imaginary vibrational frequencies) on global PES (potential energy
surface). Also, molecular orbital studies (electronic properties and
global reactivity parameters) and MEP analysis were carried out by
using Gauss View 5.0 software package.
2
.4. Stability test
2
.7. Macromolecules-metal complex binding studies
In general, electronic absorption spectrum of a given compound
is recorded in order to determine its stability in aqueous media. A
solution of [Pd(bpy)(acac)]NO3 (0.001 g/10 mL) in double distilled
water or Tris–HCl buffer was prepared and stirred in a thermo-
static water bath of 37 °C. Then, the electronic absorption spec-
trum of this sample was measured from 0 to 20 h to check if there
is disappearance or any change in the peak position with the time.
All solutions in this work were freshly prepared before any
usage. BSA (3 mg/mL) and CT-DNA (1.5 mg/mL) stock solutions
were prepared in Tris–HCl buffer, pH = 7.2, and stirred at 4 °C
till homogenous and kept for no more than 10 days. Concentrated
stock solution of the complex was dissolved in deionized water
to get the concentration of 10–4 M for biomolecules binding stud-
ies. The concentration of biomolecules was obtained spectrophoto-
metrically utilizing the values of molar extinction coefficients (ε
2
.5. Partition coefficients determination
−
1cm
−1
2
60 nm = 6600 M
and ε BSA, 280 nm = 44,300
DNA,
− 1
The lipophilicity value of the Pd (II) complex was determined
−1
M
cm ) [23,24]. The binding affinities between the metal com-
utilizing “shake flask” method in a blend of deionized water and n-
octanol media [20]. At first, [Pd(bpy)(acac)]NO3 (0.001 g) was dis-
solved in a blend of water-octanol (10 mL, 1:1v/v) via shaking for
plex and biomolecules were described through variety of following
methods.
12 h. Then, this blend was left a side at ambient temperature for
one h to achieve phase separation. The concentration of the Pd(II)
complex in each layer (octanol and water) was determined from
their UV–Vis spectra. Finally, the lipophilicity value of the complex
was obtained in terms of partition coefficient (log P) via the fol-
lowing formula:
2.7.1. UV–Vis experiments
Two titration measurements were performed using UV–
Vis spectroscopy in the range of 200–700 nm at 300 K. In the first
measurement, 1.97 and 1.55 mL of Tris–HCl buffer was added sep-
arately to 30 μL of CT-DNA stock solution and 450 μL of BSA stock
solution to attain the overall volume of 2 mL and were mixed until
homogenized. The resulted solutions were titrated via metal com-
plex stock solution (20 to 300 μL for DNA and 250 to 1250 μL
for BSA). After each step of addition, the resulting solution was re-
mained for 3 min to equilibrate, then the UV pattern was mea-
sured. In both measurements, an equivalent volume of Pd(II) com-
plex was added to the reference cell in order to delete the com-
pound absorbance at 260 and 280 nm for CT-DNA and BSA solu-
tion, respectively. From the absorption data, we can evaluate the
apparent binding constant (Kapp) for the biomolecule-metal com-
ꢀ
ꢁ
[
Pd(II) complex]in octanol
log P = log
(1)
[
Pd(II) complex]in water
2
.6. In-vitro cytotoxicity experiment
The leukemia cell line, K562, was received from Pasteur In-
stitute cell bank (Iran), which was kept in RPMI (Roswell Park
Memorial Institute, 1640-medium) filled with fetal calf serum (FCS,
1
0%), 5 μg/mL penicillin and 2 × 10−3 M l-glutamine at 37 °C.
3

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
quencher. The τ0 value is 10 8 s for EB+CT-DNA and BSA [29,30].
−
plex according to the Eq. (2) [25,26]:
[
Q] is the Pd(II) complex concentration and K is Stern-Volmer
SV
1
1
1
1
=
+
×
(2)
quenching constant. The KSV values for the case of DNA and BSA
A − A0
A∞ − A0
Kapp(A∞ − A0
)
[complex]
were calculated from the slope of F /F plot versus [Q].
0
where, A∞ is the absorption when biomolecule was completely
saturated via the metal complex solution, [complex] is the concen-
tration of the Pd(II) complex and A0 and A are the absorbance of
CT-DNA (at 260 nm) or BSA (at 280 nm) in absence and presence
of metal complex. A linear relationship can be acquired between
Then, the binding constant (K ) and number of binding sites per
b
1000 nucleotides of DNA and/or per molecule of BSA (n) were eval-
uated via plotting log(F -F)/F vs. log[Q] according to the following
0
formula (5) [31]:
F0 − F
1
/(A-A ) and the reverse of the complex concentration, then Kapp
log
= n log [Q] + log Kb
(5)
0
F
value is obtained from the ratio of the intercept to the slope of
this chart [27]. The Kapp values for CT-DNA-/BSA-complex systems
were used to evaluate the change in the free energy (ꢀG°) through
the following formula (3):
Also, thermodynamics parameters (ꢀH° and ꢀS°) were evalu-
ated using van’t Hoff equation as follows (6) [32]:
◦
◦
ꢀH
ꢀS
ln K = −
+
(6)
b
◦
RT
R
ꢀ
G = −RTln Kapp
(3)
in Eq. (6), T is the temperature in kelvin. Finally, the ꢀG° values
were obtained from Eq. (7):
−1
−
1) and T is
where R is the ideal gas constant (1.987 cal mol
K
absolute temperature (300 K).
◦
◦
◦
ꢀ
G = ꢀH − TꢀS
(7)
Second measurement was conducted to follow the biomolecule
denaturation via adding different volumes of the Pd(II) complex so-
lution. In this step, 30 μL of CT-DNA stock solution or 450 μL BSA
stock solution was reached to the final volume of 2 mL using Tris–
HCl buffer. To these blends, 20, 30, 45, …, 300 μL of metal com-
plex stock solution were added on CT-DNA and 250, 300, 400, …,
These thermodynamic parameters were utilized to estimate the
binding modes of Pd(II) complex and biomolecules [33].
2.7.3. BSA-FRET
This technique can be utilized to quantify the distance of the
bioactive metal complex to the interacted protein molecule, which
is a criterion for the interaction between the two. Section 3.5.3 will
provide more information regarding this process.
1250 μL on BSA and their patterns were noted at 260 for CT-DNA
and at 280 nm for BSA after each injection. For each biomolecule,
the absorbance at 640 nm was recorded and then the absorbance
values (A280-A640 for BSA and A260-A640 for CT-DNA) were plotted
versus the total concentration of the complex.
2.7.4. CT-DNA gel electrophoresis
Agarose gel electrophoresis was applied for monitoring the ef-
fect of [Pd(bpy)(acac)]NO3 on the CT-DNA structure. At the begin-
ning of electrophoresis experiment, four solutions were prepared
in 4 different clean tubes, that include separately: tube 1 (30 μL
of CT-DNA stock solution and 30 μL Tris–HCl buffer), tube 2 (30 μL
2
.7.2. Fluorescence emission titrations
Fluorescence emission experiments were conducted to assess
the binding mode of the novel metal complex on CT-DNA and BSA
and obtain the thermodynamic parameters. Fluorescence emission
of this new complex was checked and observed to be negligible.
For the case of DNA, since the amount of fluorescence emission
is small, ethidium bromide was added as a fluorescent probe. But,
BSA has intrinsically fluorescence because of the presence of ty-
rosine, tryptophan and phenylalanine residues. To perform titra-
tion, the solutions of EB+CT-DNA and BSA were prepared as fol-
lows: (i) 90 μL of stock solution of CT-DNA along with 20 μL of EB
−
4
CT-DNA, 10 μL complex (8 × 10 M) and 20 μL buffer), tube 3 (30
μL CT-DNA, 15 μL complex and 15 μL buffer) and tube 4 (30 μL CT-
DNA, 20 μL complex and 10 μL buffer). All the tubes were blended
and left for one night in the refrigerator till the reactions were
completed. Afterwards, the solutions were remained for 60 min at
37 °C. 1 μL of loading dye and 9 μL of each sample were mixed
and loaded onto a 3% agarose gel. The electrophoresis process was
performed in TAE buffer (contains a blend of EDTA, acetic acid and
Tris base) at the voltage of 70 V for 60 min. Finally, the gel came
out from the buffer tank and was visualized by CCD-5 Gel Docu-
mentation System, Iran, using UV light and photographed.
(
0.023 mg/mL) were dissolved in 0.87 mL of Tris–HCl buffer to get
the final volume of 0.98 mL, and (ii) 60 μL of stock solution of BSA
and 0.94 mL of Tris–HCl buffer were mixed to achieve the final vol-
ume of 1 mL. Then for CT-DNA binding, various amounts of Pd(II)
complex stock solution (30–160 μL) was added and titration was
performed at 3 temperatures of 293, 300, and 310 K. For BSA, 15–
2
.7.5. Hydrodynamic measurement
Viscosity experiments were performed for different solutions
125 μL of Pd(II) stock solution was used to perform titration at the
of CT-DNA and Pd(II) complex utilizing Ostwald micro-viscometer
SCHOT-geräte, Germany) at ambient temperature of 27 ± 1 °C.
In each sample, the concentration of CT-DNA was kept unchanged
60 μM) while varying the concentration of Pd(II) complex (0–
above mentioned temperatures. For both cases, after each addition,
the mixture was slowly mixed and maintained 3 min until equi-
libration and then its spectrum was recorded. These experiments
were repeated three times. In these experiments, excitation wave-
length for EB+CT-DNA and BSA were taken as 471 and 295 nm,
respectively. The fluorescence spectra were measured at the range
of 530 to 740 nm for DNA and 310 to 470 nm for BSA. The path
length of fluorescence cell was 1 cm and excitation and emission
slits were selected both 5 nm. The binding of the metal complex
to CT-DNA as well as BSA and subsequent fluorescence quenching
mechanism were investigated via Stern-Volmer Eq. (4) [28]:
(
(
2
4 μM) to attain the ratios of r = [Pd(II) complex]/[CT-DNA] = 0.00,
i
0
.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40. Then, the viscosity mea-
surements were carried out and the flow time was noted four
times for each sample with a digital stopwatch and the average
value was used for the calculation. The findings were illustrated
as (η/η )1 against r , where η and η0 are the viscosity of CT-
/3
0
i
DNA/Pd(II) complex solution and CT-DNA alone, respectively. The
relative values of η /η0 were achieved by Eq. (8):
Fo
ꢂ
ꢃ
=
1 + kq
τ
0[Q] = 1 + KSV [Q]
(4)
tPd(II) complex − t0 /t0
η
F
=
(8)
η
0
(t
− t0)/t0
CT−DNA
where, F and F0 are the fluorescence intensities of biomolecule in
the presence and absence of metal complex, respectively, kq is ap-
parent bimolecular quenching constant, τ0 is the average fluores-
cence lifetime of the fluorophore (EB or BSA) in the absence of
where tPd(II) complex and tCT-DNA are the average flow time of CT-
DNA in presence of palladium(II) complex and CT-DNA alone, re-
spectively. t0 is related to the buffer [34].
4

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
2
.7.6. BSA-CD spectra
ration or incubation time to be the three minutes. The finding of
these above approaches are discussed below:
BSA CD spectra (5.5 μM) with and without [Pd(bpy)(acac)]NO3
complex (different concentration of 0 to 65 μM) were collected on
an AVIV CD spectrometer-model 215, USA. A cylindrical cuvette cell
3
.1. Structural characterization of [Pd(bpy)(acac)]NO3
(
path length of 1 mm) was applied to perform the measurements
in far-UV region (200–250 nm) in a nitrogen atmosphere at 298 K.
The CD spectrum of background (working buffer) was subtracted
from all CD signals and they were changed to the molar ellipticity
and then smoothed via CD software [35,36].
[
Pd(bpy)(acac)]NO3 is yellow color, while both of its ligands
are colorless. It melts/decomposes at higher temperature (226–
2
27 °C) than that of respective ligands (bpy = 70–73 °C and
acac = −23 °C). These are two indications of the formation
of the complex. The theoretical results calculated for palladium
(25.06), carbon (42.55), hydrogen (3.55) and nitrogen (9.93) con-
tents (%) were found to be in favor to experimental values ob-
tained (Pd = 25.00, C = 42.38, H = 3.50 and N = 9.94%). The molar
conductance value of the above Pd(II) complex in double distilled
2
.7.7. Docking protocol
Molecular docking has an extensive diversity of usage in
drug discovery and development, including chemical mechanism,
structure-activity relationship (SAR), lead optimization, and provid-
ing hypotheses to facilitate predictions for studies based on mu-
tagenesis [37]. In order to determine ࢞G0 and predict the appro-
priate binding modes and non-covalent molecular binding of new
Pd(II) complex with two main biomolecules (DNA and BSA), the
molecular docking method was employed. Herein, Auto Dock 4.2
and Auto Dock Tools 1.5.6 programs were utilized for in-silico dock-
ing procedure. For the representation of the schematic 3D inter-
actions and energy values of each residue, Discovery Studio Vi-
sualizer 4.1 was applied. The crystal structures of DNA (PDB ID:
water (10 4 M) was 144 ꢁ mol cm2 illustrating the complex
−1
−
−1
is 1:1 electrolyte [40] and a rough support that both ligands are
bidentate chelates. Further support that Pd(II) ion is coordinated to
both oxygen atoms of acetylacetonato and two nitrogen atoms of
ꢀ
2,2 -bipyridine comes from the findings obtained from UV–Vis, FT-
IR and 1H NMR spectra of the metal complex.
In the FT-IR pattern of free acetylacetone ligand, 2 characteristic
bands have been reported [41] at 1729 cm 1 and 1622 cm . These
bands are attributed to the ketonic and enolic stretching frequen-
cies of the C=O groups, respectively. In the Pd(II) complex, only
−
−1
ꢀ ꢀ ꢀ ꢀ
1
XRW ) octamer 5 -CCTCGTCC-3 /3 -GGAGCAGG-5 and BSA (PDB
−1
ID: 4F5S) were obtained from https://www.rcsb.org/ (Protein Data
Bank site). The optimized geometry of the Pd(II) complex was used
for docking simulation. The macromolecules structures were ini-
tially prepared via eliminating the ligands, water molecules and
another monomer (available in BSA structure) and adding polar hy-
drogens. Then, the Gasteiger and Kollman partial atomic charges
were added to the PDB files of the macromolecules and complex
structure, respectively. Following the creation of PDBQT files of
mentioned structures, they were used as inputs for setting grid
boxes. The grid maps along X-, Y- and Z-axis were set to 56, 70
one band at 1547 cm
was observed which shows coordination
of acac ligand as bidentate chelate [42,43]. This is because of coor-
dination, the negative charge on acac is delocalized on both C=O
groups present in the chelate ring and thus spectrum shows a sin-
gle stretching frequency due to both resonated CO groups. The in-
−
1
tense and sharp stretching peak that appeared at 1383 cm in the
−
spectrum of [Pd(bpy)(acac)]NO3 is assigned to NO3 ion, showing
that this group is present as counter ion and not coordinated one
[44].
1H NMR pattern of [Pd(bpy)(acac)]NO3 complex was measured
in DMSO–d6 using TMS as standard reference. In this pattern, two
sets of resonance signals were observed. In the first set, two sharp
signal resonating at 2.1 and 5.7 ppm are assigned to HF (6H) and
HE (1H) of acac moiety (see Fig. 2). The second set signals are
and 50 A3 (0.375 A grid spacing) for DNA and 70, 70 and 70 A
with the same grid spacing for both drug binding sites of BSA, uti-
lizing Auto Grid program. LGA (Lamarckian genetic algorithm) was
implemented for 10 number of runs [38,39]. Maximum numbers
of generations and fitness evaluations were fixed to 27,000 and
˚
˚
˚ 3
ꢀ
attributed to the protons of coordinated 2,2 -bipyridine moiety.
2
,500,000, respectively. Default values were used for other param-
These doublet signals resonate at 7.8 (2H), 8.31 (2H), 8.37 (2H) and
eters. Chimera 1.11.2 software was used for visualizing the output
of docked poses.
8.60 ppm (2H), which are assigned to HB, H , H and H , respec-
C D A
tively (Fig. 2). The integration area under these signals are in the
ratio of 6: 1: 2: 2: 2 (HF: HE: H : H H : H ), support the protons
B
C:
D
A
of coordinated acac and bpy moieties to the Pd(II) center.
3. Results and discussion
In the electronic absorption spectrum of [Pd(bpy)(acac)]NO3
in H O, there are three broad absorption bands at 308 nm
2
A
novel and bioactive Pd(II) complex of general formula
(
ε = 17,354), 234 (ε = 29,580) and 205 (ε = 58,540) which are
[
Pd(bpy)(acac)]NO3 was prepared through precipitating the chlo-
∗
∗
assigned to π→π and n→π transitions within organic ligands
ride ions from [Pd(bpy)Cl ] complex via silver(I) nitrate in pres-
2
−
as well as NO3 ion that show overlapping and thus could not be
ence of acac-NaOH blend. This compound is yellow solid and wa-
ter or Tris–HCl buffer-soluble, being the first serious step towards
future usage in biological systems. We have attempted to obtain
a single crystal ideal for X-Ray experiment, but have not yet been
successful. However, satisfactory structural findings were acquired
from various spectroscopic (FT-IR, 1H NMR and UV–Vis) and non-
spectroscopic procedures including elemental analysis and conduc-
tivity measurement (complex is of good purity) as well as DFT cal-
culations. The results of experimental and computational methods
are aligned with those of the suggested structure. On the other
hand, the lipophilicity determination of the novel metal complex
was checked in term of log P. In-vitro cytotoxicity of the com-
plex was checked by investigating it against K562 cells. In-detailed
biomolecular interaction studies of the complex were investigated.
In all interaction experiments, we found no any spectral changes
occur after three minutes of injecting the Pd(II) complex solution
to CT-DNA or BSA solutions. In this way we found out time du-
separated.
The above characterization results of conductivity measure-
ment, elemental analysis, 1H NMR, UV–Vis and FT-IR are in accor-
dance with the proposed structure given in Fig. 2.
3.2. Theoretical approaches
3.2.1. Geometry optimization
The optimized molecular geometry of the Pd(II) complex was
evaluated via Gaussian09 program and is demonstrated in Fig. 3(a)
along with some selected bond angles and lengths demonstrated
in the figure. Since, the X-ray diffraction (XRD) of this complex is
not yet reported so we checked the validity of our quantum me-
chanical results with XRD data of similar complexes [45,46]. We
found a good agreement between our computational results with
the reported XRD data.
5

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Fig. 4. HOMO (lower) and LUMO (upper) plots of Pd(II) complex, bpy and acac.
accept an electron. The frontier molecular orbitals, HOMO and
LUMO, ascertain how a compound interacts with other molecules
and specify its stability and reactivity. For the purpose of drug de-
sign, different new chemical reactivity descriptors including elec-
tron affinity, global hardness, ionization energy, electrophilicity
and chemical potential were introduced. By having FMO ener-
gies, the first ionization energy (I) and first electron affinity (A)
can be determined as: I = -E
, A = -E
[48]. The hard-
LUMO
HOMO
ness (η), chemical potential (μ) and global electrophilicity (ω)
are expressed via the following relations: η = (E – EHOMO)/2,
LUMO
2
μ = (E
+ E
)/2 and ω = μ /2η, respectively [49–51].
LUMO
HOMO
The hardness signifies the resistance of compound for polariza-
tion during the chemical process and the deformation of electron
cloud. Hard molecules are small with low polarizability and soft
molecules are large and polarizable. The negative value of chem-
ical potential implies that the compound is stable. Electrophilic-
ity (ω) descriptor represents the stabilization in energy when the
compound obtains an electron from other species.
Fig. 3. (a) Optimized geometry and (b) Cut from MEP plot of Pd(II) complex.
3
.2.2. MEP analysis
The HOMO and LUMO diagrams of the compounds are repre-
sented in Fig. 4. As can be seen in this figure, HOMO is localized
over transition metal and acac ligand, while the LUMO is localized
over transition metal and bpy. Consequently, the HOMO→LUMO
transition involves the transfer of electron density from acac to
bpy ligand. This demonstrates that charge transfer happens within
the complex through the conjugation. For the newly made complex
and its related ligands, EHOMO, ELUMO, energy gap (ꢀE = ELUMO-
EHOMO), I, A, global hardness (η), global electrophilicity (ω) and
chemical potential (μ) are listed in Table 1. It is clear that
the chemical potential of Pd(II) complex is negative and lower
than that of both ligands, so it is stable and does not decom-
pose spontaneously to its initial elements. The hardness value of
[Pd(bpy)(acac)]NO3 is higher than that of bpy and lower than acac.
Since it has a moderate value of hardness and kinetic stability so
it is reactive and has enough polarizability to interact with another
species like macromolecules.
MEP is relevant to the electronic density and illustrates the
charge distribution within the surface of chemical system. The
electrostatic potential is an appropriate approach for analyzing
processes like drug-receptor interactions which are based on the
recognition of the drug compound by the macromolecule because
it is via their electrostatic potentials that they first feel each other
[
47]. In order to investigate the reactive regions of nucleophilic and
electrophilic attacks along with hydrogen bonding for Pd(II) com-
plex, MEP calculation was performed for the optimized geometry.
Fig. 3(b) demonstrates the electrostatic potential contour map
of the investigated complex. In this figure, the colors grading is
used to show different values of the electrostatic potential. Red and
blue color represents the most electronegative (rich in electron)
and electropositive (poor in electron) sites of compound, respec-
tively. For this compound the light blue shade corresponds to the
negative electrostatic potential and dark blue is related to the posi-
tive electrostatic potential. Negative potential sites generally corre-
spond to the free electron pair of electronegative atoms. The MEP
map illustrates that the negative charge covers acac ligand and the
positive area is over the bpy rings. The more electronegativity in
the C=O groups of acac ligand makes it the most reactive part of
the molecule for electrophilic attacks and can also act as an elec-
tron donner in hydrogen bond interactions.
3
.3. Solution stability and partition coefficient (log P)
The stability of molecules being a momentous feature affecting
their clinical usages in medicine. Therefore, the stability of Pd(II)
complex in aqueous solutions (water and Tris–HCl buffer) was in-
vestigated at 37 °C through electronic absorption measurements
before performing biological tests. According to the obtained re-
sults, no alteration in the absorption spectra of the complex was
observed (supplementary Fig. S1). This complex remained stable in
aqueous solutions over the period of time, which illustrates that
3
.2.3. FMO analysis
The HOMO (highest occupied molecular orbital) energy shows
the tendency to donate an electron while the LUMO (lowest un-
occupied molecular orbital) energy demonstrates the capability to
6

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Table 1
Quantum chemical descriptors of Pd(II) complex, bpy and acac (the values are given in eV).
Compound
EHOMO
ELUMO
ꢀE
I
A
η
μ
ꢁ
Pd(II) complex
−9.54
−3.64
−6.73
−5.90
−0.76
−1.35
3.64
2.88
5.38
9.54
3.64
6.73
5.90
0.76
1.35
1.82
1.44
2.69
−7.72
−2.20
−4.04
16.37
1.68
3.03
bpy
acac
only shows antitumor potency of the agent, but also indicates its
probable interaction with K562 cells DNA and thus encouraged us
to prove the later by detailed DNA-binding studies.
3
3
.5. Macromolecules binding studies
.5.1. UV–Vis titrations
UV–Vis spectroscopy is a critical and fundamental technique
to investigate the binding tendency of metal based drugs during
drug-macromolecule (DNA or protein) interaction, as the affinity
to macromolecule causes the electronic perturbations in their UV
patterns [56,57]. These perturbations affect the location and max-
imum intensity of related peak and includes the following types:
hypsochromic (blue shift), bathochromic (red shift), hypochromic
Fig. 5. Diagram representing the effect of [Pd(bpy)(acac)]NO3 complex on K562
(
decreasing intensity) and hyperchromic (increasing intensity) [58].
leukemia cell line.
Generally, for the case of DNA, hypochromic along with the red
shift is related to the intercalation of metal complex through
strong π-π interaction with the base pairs of DNA, while hyper-
chromism along with the blue shift is universally attributed to the
groove binding. Moreover, partial intercalation for complexes with
large functional groups in their structures and without complete
intercalation capability leads to hypochromism without any con-
siderable red shift. Surface interaction was demonstrated to cause
a small absorbance decrease with no notable shift in maximum
wavelength [59–61]. Fig. 6 illustrates the absorption spectra of CT-
DNA and BSA titrated with different volumes of [Pd(bpy)(acac)]NO3
complex (20 to 300 μL from complex stock solution for CT-DNA
and 250 to 1250 μL for BSA, respectively). From Fig. 6, it is clear
that the peak intensity of CT-DNA or BSA decreases with raising
complex concentration, while, λmax in Fig. 6(a) shifts towards the
larger wavelengths. These results represent the binding affinities of
[Pd(bpy)(acac)]NO3 to CT-DNA or BSA [62,63]. On the other hand,
the calculated hypochromicity for CT-DNA and BSA-complex sys-
tems were 32.17% and 38.67% respectively with a red shift of 4 nm
for CT-DNA binding system. As a result, intercalation binding mode
is recommended relating to the binding of synthesized Pd(II) com-
plex with duplex of DNA. Generally, compounds containing fused
aromatic rings are proper to intercalate into the duplex of DNA
through inserting their planar functional groups between the base
pairs of DNA, while those possess unfused aromatic rings can dis-
cover desired orientation for binding to the groove sites of DNA
[64,65]. The aromatic fused ring of [Pd(bpy)(acac)]NO3 complex i.e.
bpy ligand is able to be inserted between the bases pairs of DNA
via intercalation binding mode.
no dissociation happens when utilizing doubly distilled water or
Tris–HCl buffer to dissolve the complex.
Lipophilicity is a prominent factor which is used in drug de-
sign and discovery process. It is generally accepted to be the desire
of nonpolar compounds to form aggregates to decrease their con-
tact surface with the polar compounds such as water. Hydropho-
bic interactions, the initial driving force in many phenomena, are
some of the main interactions in the nature [52]. High lipophilic
compounds can easily penetrate to the cell membrane and access
the tissue without difficulty. As described in the experimental sec-
tion the lipophilic behavior of the Pd(II) complex is evaluated in
terms of log P utilizing n-octanol/water system. Here, the obtained
value of log P for the newly made Pd(II) complex is −0.85 ± 0.03.
Furthermore, the known value of log P for cisplatin reported in
the literature is −2.28 ± 0.07 [53]. Also, the lipophilicity values
of most anticancer compounds are between −0.4 to 5.6 (in aver-
age 2.52) [54]. However, the lipophilicity of the complex reported
in this article and the well-known anticancer drug, cisplatin, fol-
lows the order complex > cisplatin. This higher lipophilicity char-
acter of the [Pd(bpy)(acac)]NO3 in compare to that of cisplatin sup-
ports its cytotoxicity potency (see Section 3.4.) and vis versa. In the
other word, higher lipophilicity suggests that the title compound
can pass through the cell membrane more easily which leads to an
increase in its concentration within the cells. Therefore, the com-
plex may have a better cytotoxic character than cisplatin.
3
.4. Cytotoxicity experiment
For quantitative comparison, Kapp for CT-DNA/BSA-complex sys-
tems were also calculated utilizing Eq. (2) as summarized in
Table 2 (Fig. 6, insets). The calculated Kapp of [Pd(bpy)(acac)]NO3
complex to CT-DNA was found to be a bit higher than that of EB
The novel [Pd(bpy)(acac)]NO3 complex has been measured for
its in-vitro cytotoxicity versus K562 cell line via MTT measurement.
The cells were incubated for 24 h with various complex concen-
trations (0–100 μM). The result of cytotoxicity was plotted as the
percentage of cell viability against concentration of the complex
5
−1
(Kb = 1.23 × 10
M
) [66]. These findings demonstrate that prob-
ably interaction mode of [Pd(bpy)(acac)]NO3 complex to CT-DNA
should be intercalation similar to that of classical intercalator (EB).
On the other hand, the binding affinities of [Pd(bpy)(acac)]NO3 to
(
μM), which is shown in Fig. 5. As demonstrated in Fig. 5, the cell
viability was decreased after addition of diverse concentrations of
Pd(bpy)(acac)]NO3 and IC50 was determined. According to Fig. 5,
5
−1
[
CT-DNA (Kapp, CT-DNA = 2.35 × 10
M
) is much higher than that
−1
) indicating that this complex
3
the evaluated IC50 value of the newly made complex is 13 μM at
incubation time of 24 h. So, IC50 value of the anticancer agent,
cisplatin, under the similar experimental conditions was detected
of BSA (Kapp, BSA = 1.73 × 10
M
has less interaction affinity in compare to CT-DNA and thus no
abrupt structural damage of BSA is expected [67]. The ꢀG° val-
ues of the complex-CT-DNA and -BSA binding systems at 300 K
were obtained to be - 7.37 and - 4.45 Kcal/mol, respectively, which
1
54 μM [55], which is much higher in compared with the IC50
value of [Pd(bpy)(acac)]NO3 complex. This growth inhibition not
7

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Fig. 6. Absorption patterns of CT-DNA (a) and BSA (b) in the absence and presence of increasing concentrations of [Pd(bpy)(acac)]NO3, [CT-DNA] = 127 μM, [BSA] = 8.2 μM
and [complex] = 0.00 – 13.00 μM for CT-DNA and 0.00 – 38.50 μM for BSA at T = 300 K. insets illustrate the outline of 1/A-A0 vs. 1/[complex] for CT-DNA and BSA
respectively to calculate Kapp values.
Table 2
The evaluated binding parameters from UV–Vis spectroscopic method for the interaction of [Pd(bpy)(acac)]NO3
complex with CT-DNA and BSA.
Kapp (M 1)
−
[L]1/2 (mM)
ꢀG° (Kcal/mol)
H
∗
(%)
T (K)
5
CT-DNA binding
BSA binding
300
300
2.35 × 10
1.73 × 10
0.0048
0.023
−7.37
−4.45
32.17
38.67
3
∗
H = percentage hypochromicity.
demonstrate spontaneous binding between this complex and both
biomolecules.
in the native structure of CT-DNA and BSA at very low concentra-
tion, 2) the [L]1/2 values for the above metal complex in CT-DNA
binding is lower than BSA binding, indicating that if this complex
is applied as a potential antitumor agent, its antitumor attributes
is high (due to potent binding to CT-DNA) and its side effects is
probably low (due to weak binding to BSA).
In addition, the reduction in the absorption patterns of CT-DNA
and BSA upon increasing concentration of Pd(II) complex demon-
strates that a type of conformational alteration in the primitive
structure of biomolecules occur. The absorbing components in the
native structure of DNA (pyrimidine and purine bases) and BSA
The above discussion is complemented by the two following
paragraphs:
(
R groups attached to amino acid residues) make above changes
via hiding from the UV waves, causing a decrement in the inten-
sity of electronic absorption patterns with increasing concentration
of [Pd(bpy)(acac)]NO3 [68]. These classes of conformational alter-
ations may be attributed to CT-DNA or BSA refolding.
Binding constant in the interaction of the complex with DNA
5
3
(2.35 × 10 ) and BSA (1.73 × 10 ) (Table 2) tells us that interac-
tion affinity with DNA is more than that of BSA. From the other
side, since we have not done in-vivo studies, one can just say:
High interaction affinity may cause halting DNA- duplication lead-
ing to cancer cell death. While low interaction affinity with BSA
possibly bring about the reversibility binding behavior of BSA with
the complex which is necessary for uploading in BSA and releasing
near the target cells.
In the second experiment, the profile of alterations in the pat-
terns of absorbance at 260 nm for CT-DNA and 280 nm for BSA
were plotted versus the concentrations of the complex (Fig. 7).
From the analysis of this graph, we are able to obtain a crucial
parameter ([L]1/2). This parameter denotes the complex concentra-
tion at the midpoint of the transition of biomolecule from primary
structure to deformed conformation. As Fig. 7 illustrates, the ob-
tained values of [L]1/2 in the binding between [Pd(bpy)(acac)]NO3
and CT-DNA and BSA are 0.0048 and 0.023 mM, respectively.
The obtained results from absorption experiments are: 1)
In the cell (cuvette), where interaction is carried out, the inter-
fering molecules may be NaCl or Tris- HCl buffer only. Thus, UV–
Vis spectra of the Pd(II) complex, CT-DNA and BSA were recorded
in i) deionized water ii) in 10 mM Tris- HCl buffer solution of
pH = 7.2 and iii) in 10 mM NaCl solution (i.e. about the same
concentration in the blood stream). All the three showed exactly
[
Pd(bpy)(acac)]NO3 complex leads to the conformation alterations
Fig. 7. The decrease in the absorbance of CT-DNA at 260 nm (a) and BSA at 280 nm (b) upon increasing the concentration of [Pd(bpy)(acac)]NO3 at 300 K.
8

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Table 3
Comparison of Kapp, L1/2 and IC50 values found for [Pd(bpy)(acac)]NO3 with its structurally related compounds found from the
literature.
Kapp, DNA (M 1)
−
Kapp, BSA (M
−1
)
L1/2, DNA (μM)
L1/2, BSA (μM)
IC50 (μM)
Reference
compound
5
3
[
[
[
[
Pd(bpy)(acac)]NO3
Pd(phen)(acac)]NO3
Pd(bpy)(SA)]
2.35 × 10
1.73 × 10
4.8
3.84
18
23
13
18
36
27
5
3
2.87 × 10
1.37 × 10
28.57
[26]
[68]
[68]
4
3.90 × 10
–
–
–
–
5
Pd(bpy)(SSA)]
1.09 × 10
13
Fig. 8. The emission spectra of interacted EB+CT-DNA (a) and intrinsic fluorescence of BSA (b) in the absence and presence of the [Pd(bpy)(acac)]NO3 complex (0.00, 2.97,
3.92, 5.31, 6.67, 7.98, 9.26, 10.90, 12.50, 14.00 and 65.57 μM of stock solution for CT-DNA and 0.00, 1.48, 1.96, 3.38, 4.30, 5.21, 6.10, 7.41, 8.67, 9.91 and 11.11 μM for BSA) at
300 K. Insets show Stern-Volmer plots for binding of EB+CT-DNA (a, inset) and BSA (b, inset) to [Pd(bpy)(acac)]NO3 complex.
the same spectral pattern, indicating that NaCl and Tris- HCl buffer
structurally do not interfere with the Pd(II) complex, CT-DNA and
BSA solutions. However, we have not checked the interfering effect
of other molecules or ions.
Fig. 8(b) illustrates that the BSA emission spectra reduce in the
presence of complex without sudden alteration in the location of
maximum emission peak. In the other word, the interaction of
the [Pd(bpy)(acac)]NO3 to the BSA diminishes the inherent fluo-
rescence ascribed primarily to the Tyr, Phe-and particularly Trp-
residues of BSA. This means the interaction of Pd(II) complex with
BSA causes some conformational changes in hydrophobic environ-
ment around tryptophan residues of BSA and leads to the reduc-
tion in emission intensity of BSA.
It is noteworthy here to compare UV–Vis and cytotoxicity stud-
ies of the Pd(II) complex under investigation with structurally re-
lated compounds available in the literature: [Pd(bpy)(acac)]NO3
bears N–N and O–O donor ligands, similar to that of well-known
anticancer drug, carboplatin (Fig. 1). In these type of compounds,
the N–N donor ligands are ancillary and the O–O donor are leav-
ing groups, which can get substituted with biomolecules like DNA.
There are many O–O bonded metal complexes in the literature.
However, for few of them the same experiments had been carried
out as we did in present paper. These finding results for struc-
turally related metal complexes include: Kapp, L1/2 and IC50 and are
gathered in Table 3.
Quenching is a procedure in which the emission intensity of a
fluorophore substance reduces due to one of the following rea-
sons: formation of non-fluorescent complex in the ground state
(static quenching), the collision of given substance and fluorophore
in the excited state which leads to the energy transfer between
them (dynamic quenching). In static quenching, increasing temper-
ature leads to the decrease in complex formation and therefore de-
creasing the Stern-Volmer constants. While in dynamic quenching
raising temperature causes more collision between fluorophore and
quencher and increases quenching and Stern-Volmer constants. Us-
ing fluorescence emission data, the KSV and kq values were de-
termined and summarized in Table 4 for biomolecules interaction.
The results in this table for DNA shows that with increasing tem-
perature, KSV reduces and the kq values are greater than maximal
As the data in Table 3 shows, there are no proper trends in the
values of Kapp and L1/2. However, IC50 value of [Pd(bpy)(acac)]NO3
is the lowest, indicating higher in-vitro activity of this compound.
3
.5.2. Fluorescence titration studies
Fluorescence titration technique was utilized to understand the
type of the complex interactions with EB+CT-DNA and BSA at
temperatures of 293, 300, and 310 K. The fluorescence spectra of
EB+CT-DNA and BSA in presence of Pd(II) complex are illustrated
in Fig. 8(a and b). Fig. 8(a) shows by addition of more amount of
complex, a smooth reduction in the emission intensity of EB+CT-
DNA is observed without even any noticeable change in the peak
position. It has been confirmed that if the florescence intensity
of the EB+DNA in presence of a given compound decreases more
than 50% and the ratio of complex concentration to DNA is less
than 100, then it is assumed that they interact via intercalation
dynamic quenching constant (2 × 10 M–1s–1). It could be con-
cluded that the fluorescence quenching mechanism in the binding
of [Pd(bpy)(acac)]NO3 to CT-DNA is likely to be static quenching.
In addition, the obtained results for BSA demonstrate that with in-
creasing temperature, KSV increases which implies the quenching
mechanism is dynamic. Furthermore, the values of kq are greater
10
than 2 × 10 M–1s–1 which confirms static quenching. Therefore,
the fluorescence quenching mechanism of interaction between BSA
and [Pd(bpy)(acac)]NO3 is a combination of static and dynamic.
Such a quenching mechanism was observed for the reported Pd(II)
10
[
69]. In this work, the addition of [Pd(bpy)(acac)]NO3 caused a
complexes [26,68]. Although, the linear relation between F /F and
0
considerable reduction in the emission intensity of the EB+CT-DNA
system at the end of titration (50.0%, Fig. 8(a)), which indicates
that this complex is probably bound to CT-DNA in a similar mode
to EB, i.e., intercalation mode.
[
complex] (as illustrated in the inset of Fig. 8) and the increase
in KSV value with rising temperature shows that the dominant
quenching mechanism is dynamic [70,71].
9

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Table 4
Stern-Volmer constants, bimolecular quenching rate constants, binding constants, number of binding sites and thermodynamic parameters of [Pd(bpy)(acac)]NO3 with
CT-DNA and BSA at different temperatures.
4
−1
12
−1 −1
4
−1
)
T (K)
Ksv × 10 (M
)
kq × 10 (M
s
)
Kb × 10 (M
n
ꢀG° (kcal/mol)
ꢀH° (kcal/mol)
15.37 ± 0.07
ꢀS° (kcal/K.mol)
0.07 ± 0.002
2
93
1.41 ± 0.02
1.30 ± 0.02
1.04 ± 0.03
5.17 ± 0.03
5.26 ± 0.02
5.87 ± 0.03
1.41 ± 0.02
1.30 ± 0.02
1.04 ± 0.03
5.17 ± 0.03
5.26 ± 0.02
5.87 ± 0.03
1.20 ± 0.04
2.14 ± 0.05
5.09 ± 0.04
0.99 ± 0.02
1.05 ± 0.01
1.15 ± 0.03
1.09 ± 0.02
0.90 ± 0.02
0.63 ± 0.01
−5.46 ± 0.09
−5.96 ± 0.07
−6.67 ± 0.07
−6.90 ± 0.05
−5.83 ± 0.05
−4.30 ± 0.04
CT-DNA binding
BSA binding
300
3
2
10
93
14.00 ± 0.04
1.60 ± 0.04
0.10 ± 0.04
300
10
−51.73 ± 0.06
−0.15 ± 0.001
3
All results were expressed as the mean ± standard deviation of three independent experiments.
Fig. 9. The plot of log(F0-F)/F vs. log[complex] for the CT-DNA-[Pd(bpy)(acac)]NO3 system (a) and BSA-[Pd(bpy)(acac)]NO3 system (b) at three temperatures (293, 300, and
10 K). Van’t Hoff plot for the interaction of CT-DNA (c) and BSA (d) with [Pd(bpy)(acac)]NO3 complex at three various temperatures.
3
Utilizing Eq. (5) and titration data, the binding constants (Kb)
and number of binding sites on biomolecules were evaluated.
Fig. 9(a and b) demonstrate the plots of log (F0 – F)/F against log[Q]
for CT-DNA and BSA binding, respectively. Then, the Kb and n val-
ues were calculated from the intercept and slope of these plots, re-
spectively. The values of Kb and n for [Pd(bpy)(acac)]NO3 at three
temperatures are specified in Table 4 for biomolecules interaction.
As can be seen in this table, the binding constant of complex
with CT-DNA increases with raising temperature which confirms
that the formation of complex-CT-DNA system is more favorable at
higher temperatures. In contrast, for BSA interaction with increas-
ing temperature, the Kb values decreases. This shows the interac-
tion between metal complex and BSA is exothermic. Furthermore,
the n value is ~1, demonstrating that there is only one interaction
site on BSA and CT-DNA for [Pd(bpy)(acac)]NO3 complex.
thermodynamic quantities and type of interactions are correlated
[72] as follows: ꢀH° < 0 and ꢀS° < 0 show hydrogen bonds and
van der Waals forces, ꢀH° > 0 and ꢀS° > 0 refer to hydrophobic
interactions and ꢀH° < 0 and ꢀS° > 0 indicate electrostatic inter-
actions. In this study, ꢀH° < 0 and ꢀS° < 0 for BSA binding indi-
cate that the forces holding [Pd(bpy)(acac)]NO3 complex with BSA
are hydrogen bond and van der Waals forces. Moreover, ꢀH° > 0
and ꢀS° > 0 for DNA binding illustrates that the obtained CT-DNA-
Pd(II) complex adduct is stabilized mainly by hydrophobic interac-
tions. Same results have been observed for the structurally analo-
gous Pd(II) complexes [26,68]. As shown in Table 4 the ꢀG° values
are negative. Hence, the formation of CT-DNA-/BSA-Pd(II) complex
are spontaneous process [73,74].
3.5.3. FRET calculation
Utilizing Eqs. (6) and (7) and the values of K , the thermody-
distance-dependent binding between the various electronic ex-
cited states of compounds is called fluorescence resonance energy
transfer, which is one of the most widely used techniques for mea-
suring the distance between drug and protein in a complex. This
procedure permits the excitation energy to be transferred from a
donor compound to an acceptor compound without emission of a
photon from donor molecular system [75]. The procedure is also
known as Förster’s resonance energy transfer. FRET expresses the
energy transfer efficiency (E) as follows [76]:
b
namic parameters were determined. They can be utilized to es-
timate the essence of driving forces of the complex and EB+CT-
DNA/BSA. These forces are electrostatic, hydrophobic, van der
Waals interactions and hydrogen bond. Fig. 9 (c and d) show the
plot of LnKb against 1/T based on the formula (6) for EB+CT-DNA
and BSA, respectively. The ꢀS° and ꢀH° values were determined
using the intercept and slope of these plots. The changes in binding
free energy, ꢀG°, were obtained using Eq. (7) for both systems. The
values of thermodynamic parameters are mentioned in Table 4 for
biomolecules interaction. Yang et al. concluded that the sign of the
6
0
F
R
E = 1 −
=
(9)
6
0
6
0
F0
R + r
10

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Fig. 10. Overlap of the fluorescence emission spectrum of BSA (a) with electronic
absorption spectrum of [Pd(bpy)(acac)]NO3 (b). The concentrations of BSA and
metal complex were equal to 8 μM.
Fig. 11. Agarose gel electrophoresis pattern of: Marker (marker is circular DNA
for checking the instrument), lane 1, untreated CT-DNA (4.23 mM) and lanes 2–4,
Table 5
4.23 mM CT-DNA + (0.13, 0.20 and 0.26 mM) of [Pd(bpy)(acac)]NO3 complex.
The calculated energy transfer parameters in the Pd(II) complex-BSA system.
3
−1
)
J(cm .L.mol
E
r (nm)
2.75
R0 (nm)
2.56
Fig. 11. Free CT-DNA demonstrates a long band in lane 1. Lanes 2–
obviously demonstrate that the complex induces mobility dis-
14
Pd(II) complex-BSA system
1.085 × 10−
0.394
4
placement of CT-DNA at different concentration without changing
in the initial profile pattern. In the current work, no CT-DNA cleav-
age activity of [Pd(bpy)(acac)]NO3 has been observed. The retarda-
tion of CT-DNA band in the gel could be due to an increment in
its mass or reduction of CT-DNA negative charge after interaction
with Pd(II) complex.
where F0 and F are the emission intensity of BSA without and with
Pd(II) complex. Also, r0 and R0 are the binding distance and crit-
ical distance between donor and acceptor when the efficiency of
transfer is 50%. The magnitude of R0 depends on the spectral at-
tributes of the donor and acceptor and is obtained from the fol-
lowing Eq. (10):
3
.5.5. Hydrodynamic measurement
ꢄ
ꢅ
6
0
−25
K N−4∅J(λ)
2
R = 8.8 × 10
(10)
Viscosity experiments were conducted to confirm the nature of
binding mode of novel Pd(II) complex to CT-DNA. Viscosity mea-
surements, a component of hydrodynamic methods, are sensitive
to increase or decrease of DNA length and in the absence of spec-
troscopic and crystallographic data are regarded as the most effi-
cient paths of probing the binding of small compounds and DNA
where, N is the refractive index of a medium, ø is the quantum
2
yield of the donor without the acceptor and K is the spatial ori-
entation factor of the dipole. The overlap integral of the donor flu-
orescence emission spectrum with the acceptor UV spectrum, J (λ),
was determined via Fig. 10 and numerical integration with the fol-
lowing Eq. (11):
[
82]. A prevalent intercalative mode such as intercalation of ethid-
ium bromide under proper conditions leads to a serious increment
in the DNA viscosity because of an increment in the dissociation
of base pairs at the intercalation positions and therefore an in-
creasing in the length of the overall DNA. In groove binding (mi-
nor and major) or electrostatic mode, the DNA helix length is un-
changed and leads to no apparent alteration in viscosity of DNA so-
lution [83,84]. Besides, a non-classical and/or partial intercalation
of molecule could screw (or bend) the DNA helix, decreasing its
effectual length and viscosity [85,86]. The effects of increasing the
synthesized Pd(II) complex and EB concentration on the CT-DNA
viscosity are illustrated in Fig. 12. As can be observed in Fig. 12,
∞
4
∫
F(λ)ε(λ)λ dλ
∞
0
J(λ) =
(11)
∫
F(λ)dλ
0
here, ε (λ) is the molar extinction coefficient of the acceptor at
wavelength of λ and F (λ) is the corrected emission intensity of the
donor. For Pd(II) complex-BSA system taking K2 = 2/3, N = 1.336
and ø = 0.15 [77], the obtained values of J (λ), R , r and E are
0
listed in Table 5. The r value demonstrates that both acceptor
(
[Pd(bpy)(acac)]NO ) and donor (BSA) lie close together and can
3
possess striking interaction with each other [78]. Moreover, the
donor to acceptor distance (r < 8 nm) illustrates that the quench-
ing mechanism in Pd(II) complex-BSA system is static [79,80] at
high concentration, which supports the results obtained from fluo-
rescence titration.
3
.5.4. CT-DNA electrophoresis assay
The interaction ability of the Pd(II) complex with CT-DNA was
studied via examining the effect of various concentrations of
Pd(bpy)(acac)]NO3 on CT-DNA, utilizing gel electrophoresis. Fluo-
[
rescent dye EB that is inserted between nucleic acids bases (inter-
calation site) was utilized to visualize DNA in agarose gel. The DNA
phosphate backbone is negatively charged and when located in an
electric field, fragments of DNA move to the positively charged an-
ode [81]. Since DNA has identical ratio of mass/charge, the DNA
molecules can be distinguished according to their size. The CT-
DNA electrophoretic mobility patterns in the absence and presence
of diverse concentrations of [Pd(bpy)(acac)]NO3 are illustrated in
Fig. 12. The relative viscosities of CT-DNA with increasing concentration of Pd(II)
complex at 27 ± 1 °C. [CT-DNA] = 60 μM and [Pd(II) complex] = [EB] = 0–24 μM.
11

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
with raising the value of r = [Pd(II) complex]/[CT-DNA] (0 to 0.40),
CD studies, the decrease in percentage of β-turn and random coil
with α-helix rise can be attributed to the occurrence of a kind of
contraction in the native structure of BSA. Thus, the CD and UV–
Vis studies display the same conformational alterations in the BSA
structure and endorse each other.
i
the relative viscosity of DNA solution increases significantly. This
result may be due to the insertion of the aromatic rings (bpy and
acac ligands) of [Pd(bpy)(acac)]NO3 into the CT-DNA base pairs re-
sulting an increase in molecular length of CT-DNA at the interca-
lation site. As viscosity experiment result showed the mentioned
complex binds with CT-DNA via intercalation mode, which is in
agreement with the spectroscopic findings [87].
3
.5.7. Docking on macromolecules
In-silico molecular docking, which identifies the binding sites
of small molecules on macromolecules, was also utilized to gain
unique insights into the interaction of Pd(II) complex with DNA
and BSA. The optimized structure of the complex was docked into
the DNA and BSA fragments. The best pose of the complex with
DNA (with more negative ࢞G°), is demonstrated in Fig. 14. This
figure demonstrates the nucleotides and the complex structures in-
volved in the interactions along with the distance between them.
It is evident from this figure that the entire docked complex is lo-
cated within the intercalation site of DNA between cytosine and
guanine on both strands, leads to the existence of hydrophobic in-
teractions. In the other word, there are π-alkyl interactions be-
tween acac ligand of Pd(II) complex and Cyt4 and Gua5 of DNA.
The π-π interactions are also found between the planar rings of
bpy ligand and Gua5 and Cyt4 of DNA strand. ࢞G° of the com-
3
.5.6. BSA-CD spectral
Circular Dichroism (CD), an intuitive spectroscopic technique to
elucidate conformational changes of protein in protein-molecules
interactions [88], was utilized in the current study. In Fig. 13(a),
CD spectra of the free BSA and BSA with various concentrations
of [Pd(bpy)(acac)]NO3 (0–65 μM) are shown. All CD spectra con-
taining BSA and BSA-Pd(II) complex demonstrated two minima at
around 208 and 222 nm, particular peaks for α-helix structure that
occur because of the π-π and n-π transitions in α-helix. The in-
tensity of peaks at the minima reflects the α-helical amount of the
protein. The related helical values of native BSA and interacted BSA
were determined via the mean residual ellipticity values at 208 nm
∗
∗
(MRE208) through the following relations (12) and (13) [89]:
−1
plex with DNA was evaluated to be −7.22 kcal. mol , which is
ꢂ
ꢃ
2
−1
−1
Observed CD deg.cm .dmol
Cpnl × 10
consistent with the UV–Vis result (࢞G°
–
= −7.37 kcal. mol ).
UV Vis
[
MRE]λ
=
(12)
(13)
Therefore, taking into account the findings of the docking simula-
tion and the experimental procedures, it could be concluded that
the Pd(II) complex binds to DNA by intercalation.
−
MRE208 − 4000
α − Helix (%) =
× 100
Two potential interaction sites on BSA (site I and II) were in-
vestigated in the case of the BSA-docking simulation (Fig. 15). The
obtained ࢞G° of the complex to BSA site I (࢞G° = −6.19 kcal.
3
3000 − 4000
where Cp, n and l represent BSA molar concentration, number of
−1
amino acid residues (583) and the cell path length, respectively.
mol ) was more negative than that of site II (࢞G° = −5.58 kcal.
−
4
000 is related to the MRE of the random coil and β-form confor-
mol 1), suggesting the complex primarily is located in BSA site I.
mation at λ = 208 nm while 33,000 is the magnitude of a pure
α-helix MRE at the same wavelength.
The calculated percentage values of α-helix, parallel, β-turn,
anti-parallel and random coil are summarized in Fig. 13(b). The re-
sults demonstrate an enhancement in negative ellipticity and α-
helix content from 72.6 to 79.7 and 83.4% at two concentrations
So, for further analysis of docking result, we concentrate on BSA
site I findings. The obtained ꢀG°UV
–
(−4.45 kcal/mol) for BSA in-
Vis
teraction, varies marginally from the computational value, due to
the fact that the experimental method was conducted in solution
environment (body physiological condition) while the docking sim-
ulation results has been obtained in the gas phase and the effect of
water and working buffer have been neglected [90]. The values of
vdW+Hbond+desolve and electrostatic energies were evaluated to
(
45 and 65 μM) . All CD spectra were observed to be resembling in
appearance shape without any notable move in the peak position
Fig. 13(a)), exhibiting stabilization of the secondary structure of
−1
(
be −6.19 and 0.00 kcal. mol , respectively, demonstrating that hy-
drogen bond and van der Waals forces play a vital function, while
electrostatic attraction has no particular role in the interaction pro-
cess. Docking simulation illustrated that the newly complex in-
teracts with the following BSA residues: Glu100, Leu103, Ser104,
Lys106, Tyr147, Ile202, Gln203, Lys242, Cys245 and His246. A hy-
drogen bond is observed between His246 and the oxygen atom of
BSA and interacted BSA with [Pd(bpy)(acac)]NO . It also suggests
3
that the BSA structure was mainly α-helix even after interaction
with the complex. Here, it is interesting to look at the coincidence
of CD results with that of UV–Vis discussed in Section 3.5.1. We
saw in Section 3.5.1 that when BSA comes in contact with increas-
ing amounts of Pd(II) complex, its absorption decreases at 280 nm.
This decrease is due to the hiding of the absorbing BSA groups
from UV light and the contraction of the native BSA happens. In
˚
the Pd(II) complex with the distance of 3.1 A. Other residues are
involved in the formation of van der Waals force between the BSA
Fig. 13. (a) Circular dichroism spectra of native BSA (5.5 μM) with increasing concentration of [Pd(bpy)(acac)]NO3 complex (0–65 μM). (b) the values and relative percentages
of secondary structure elements of BSA in the absence (black column) and presence (blue and red column) of [Pd(bpy)(acac)]NO3 complex.
12

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Fig. 14. Docking pose of Pd(II) complex with DNA.
Fig. 15. Docking pose of Pd(II) complex with BSA.
and the complex. The distance between CH3 group of acac ligand
tually supporting. In addition, DNA-viscosity measurements, DNA-
gel electrophoresis assay, BSA-FRET calculation provided more in-
sights into the interaction mechanisms of the complex with DNA
and BSA. MEP map revealed the electrophilic and nucleophilic cen-
ters of the complex. FMO analysis showed the charge transfer oc-
curs from acac to bpy ligand within the complex. In-silico molecu-
lar docking results exhibited an excellent agreement with the ex-
perimental finding in the case of binding mechanism and energy of
the synthesized compound with macromolecules. Thus, the struc-
tural variation of N–N and O–O donor set of palladium(II) complex
towards nucleic acid and albumin provide us more detail about
the binding affinities of drug-macromolecules and are of vital rel-
evance in biochemistry and pharmacy.
in [Pd(bpy)(acac)]NO3 and the O atom in C = O group of Trp134
˚
residue is 21.60 A (2.16 nm) evidently lower than 8 nm, which
is absolutely in line with the result of the FRET calculation. The
obtained results from computational (࢞G°DNA, docking = −7.22 and
1
࢞
G°BSA, docking = −6.19 kcal. mol− ) and experimental approaches
imply the well binding affinities of the complex with CT-DNA via
intercalation site and moderate interaction with BSA via van der
Waals forces and hydrogen bonding.
4. Conclusion
Novel mononuclear Pd(II) complex with cytotoxicity activity
much better than cisplatin was synthesized and fully character-
ized via various physicochemical techniques. The findings of spec-
Credit author statement
troscopic methods along with DFT calculation indicated that the
ꢀ
Mehran Feizi-Dehnayebi: Ph.D. student,
Effat Dehghanian: Guide,
PdN O lies in the aromatic plane of 2,2 -bipyridine which may
2
2
improve its binding behavior. The lipophilicity determination of the
new complex was performed in terms of log P and the obtained
value was higher than that of cisplatin. In-vitro biomolecules inter-
action profiles of the complex utilizing in-detail fluorescence and
UV–Vis methods supported the fact that it binds to CT-DNA by
intercalation mode and demonstrates a weaker binding affinity to
BSA. UV–Vis as well as CD studies displayed the same conforma-
tional alterations in the native structure of the BSA and are mu-
Hassan Mansouri-Torshizi: Co-Guide.
Declaration of Competing Interest
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.
13

M. Feizi-Dehnayebi, E. Dehghanian and H. Mansouri-Torshizi
Journal of Molecular Structure 1240 (2021) 130535
Acknowledgements
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II) antitumor agent: synthesis, characterization, DFT perspective, CT-DNA and
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The authors gratefully acknowledge the University of Sistan and
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