A novel palladium(II) antitumor agent: Synthesis, characterization, DFT perspective, CT-DNA and BSA interaction studies via in-vitro and in-silico approaches

Article abstract of DOI:10.1016/j.saa.2020.119215

Since numerous people annually pass away due to cancer, research in this field is essential. Thus a newly made and water like palladium(II) complex of formula [Pd(phen)(acac)]NO₃, where phen is 1,10-phenanthroline and acac is acetylacetonato ligand, has been synthesized by the reaction between [Pd(phen)(H₂O)₂](NO₃)₂ and sodium salt of acetylacetone in the molar ratio of 1:1. It has been structurally characterized via the methods such as conductivity measurement, elemental analysis and spectroscopic methods (FT-IR, UV–Vis and ¹H NMR). The geometry optimization of this complex at the DFT level of theory reveals that Pd(II) atom is situated in a square-planar geometry. The complex has been screened for its antitumor activity against K562 cancer cells which demonstrated efficacious activity. The interaction of above palladium(II) complex with CT-DNA as a target molecule for antitumor agents and BSA as a transport protein was studies by a variety of techniques. The results of UV–Vis absorption and fluorescence emission indicated that the Pd(II) complex interacts with EB + CT-DNA through hydrophobic and with BSA by hydrogen bonding and van der Waals forces at very low concentrations. In these processes, the fluorescence quenching mechanism of both the macromolecules seems to be the combined dynamic and static. The interaction was further supported for CT-DNA by carrying out the gel electrophoresis and viscosity measurement and for BSA by the circular dichroism and F?rster resonance energy transfer experiments. Furthermore, results of partition coefficient determination showed that the [Pd(phen)(acac)]NO₃ complex is more lipophilic than that of cisplatin. Moreover, molecular docking simulation confirms the obtained results from experimental tests and reveals that the complex tends to be located at the intercalation site of DNA and Sudlow's site I of BSA.

Full text of DOI:10.1016/j.saa.2020.119215

Journal Pre-proofs
A novel palladium(II) antitumor agent: Synthesis, characterization, DFT per-
spective, CT-DNA and BSA interaction studies via in-vitro and in-silico ap-
proaches
Mehran Feizi-Dehnayebi, Effat Dehghanian, Hassan Mansouri-Torshizi
PII:
S1386-1425(20)31194-X
https://doi.org/10.1016/j.saa.2020.119215
SAA 119215
DOI:
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Spectrochimica Acta Part A: Molecular and Bio-
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6 November 2020
Please cite this article as: M. Feizi-Dehnayebi, E. Dehghanian, H. Mansouri-Torshizi, A novel palladium(II)
antitumor agent: Synthesis, characterization, DFT perspective, CT-DNA and BSA interaction studies via in-vitro
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A novel palladium(II) antitumor agent: Synthesis, characterization, DFT
perspective, CT-DNA and BSA interaction studies via in-vitro and in-silico
approaches
1
1*
1
Mehran Feizi-Dehnayebi , Effat Dehghanian , Hassan Mansouri-Torshizi
1Department of Chemistry, University of Sistan and Baluchestan, Zahedan, Iran
m.feizi@sutech.ac.ir
hmtorshizi@hamoon.usb.ac.ir
*
Corresponding author: dehghanian@chem.usb.ac.ir
1

Abstract
Since numerous people annually pass away due to cancer, research in this field is essential. Thus
a newly made and water like palladium(II) complex of formula [Pd(phen)(acac)]NO , where phen
3
is 1,10-phenanthroline and acac is acetylacetonato ligand, has been synthesized by the reaction
between [Pd(phen)(H O) ](NO ) and sodium salt of acetylacetone in the molar ratio of 1:1. It has
2
2
3 2
been structurally characterized via the methods such as conductivity measurement, elemental
1
analysis and spectroscopic methods (FT-IR, UV-Vis and H NMR). The geometry optimization of
this complex at the DFT level of theory reveals that Pd(II) atom is situated in a square-planar
geometry. The complex has been screened for its antitumor activity against K562 cancer cells
which demonstrated efficacious activity. The interaction of above palladium(II) complex with CT-
DNA as a target molecule for antitumor agents and BSA as a transport protein was studies by a
variety of techniques. The results of UV-Vis absorption and fluorescence emission indicated that
the Pd(II) complex interact with EB+CT-DNA through hydrophobic and with BSA by hydrogen
bonding and van der Waals forces at very low concentrations. In these processes, the fluorescence
quenching mechanism of both the macromolecules seems to be the combined dynamic and static.
The interaction was further supported for CT-DNA by carrying out the gel electrophoresis and
viscosity measurement and for BSA by the circular dichroism and Förster resonance energy
transfer experiments. Furthermore, results of partition coefficient determination showed that the
[
Pd(phen)(acac)]NO complex is more lipophilic than that of cisplatin. Moreover, molecular
3
docking simulation confirms the obtained results from experimental tests and reveals that the
complex tends to be located at the intercalation site of DNA and Sudlow's site I of BSA.
Keywords: cancer, palladium(II) complex, DNA-/BSA-binding, DFT, molecular docking
2

1
. Introduction
The last few decades have witnessed a remarkable interest in searching for antitumor drugs among
metal complexes which is the result of the successful development of cisplatin. The discovery of
cisplatin occurred during the period from 1960 to 1970. The platinum (Pt)-based antitumor agents
like cisplatin have some side effects such as nausea, nephrotoxicity, myelotocity and ototoxicity.
In addition, many tumor cells become resistant to platinum drugs over the time. So, the efforts
have been made to replace cisplatin with proper alternatives and different transition metal agents
have been tested for their anticancer activity [1-3] that can minimize the side effects and raise
therapeutic benefits.
Many researches show that palladium(II) complexes display a significant antitumor activity similar
to Pt-based drugs and illustrate fewer side effects compared to the other heavy metal anticancer
agents [4]. Palladium(II) complexes are efficacious in the treatment of tumors in the
gastrointestinal region and breast cancer where their platinum analogous fails [5]. An important
feature of palladium-containing chemotherapy drugs is that they have less kidney toxicity than
cisplatin [6]. Beside, therapeutic properties of transition metal based complexes always begin with
the hydrolysis. Interestingly, a good relationship is observed between the cytotoxicity behavior of
the Pd(II) complexes and their solubility [7]. The faster hydration rate of palladium (II) in compare
to Pt(II) has two consequences: i) it makes palladium based complexes better model for
investigating Pt(II) reactions [8], ii) they can interact with other molecules especially proteins,
preventing them to reach the DNA and show the therapeutics effect [9]. The latter is not desirable,
so this significantly greater activity of Pd(II) complexes signifies that if an anticancer Pd(II) drug
is to be developed then it must somehow be stabilized by a strongly coordinated nitrogen ligand
and proper ancillary ligands or substituting the coordinated monodentate ligands with these of
chelating ones.
3

1
,10-phenanthroline is frequently used ligand owing to its metal chelating properties, planar
structure, ability to interact with DNA through intercalation, and its chemotherapy effects [10, 11].
So it can act as an ancillary ligand. As a leaving group ligand, it is well known that acetylacetone
is an important compound because it forms stable complexes with various metal ions [12]. It exists
as a mixture of the keto and enol forms that are in equilibrium in solution and gaseous phases. The
enol form is dominant in both phases as it is stabilized by an internal hydrogen bond (Fig. 1) [13].
Fig.1. Keto (A) and enol (B) forms of acetylacetone.
It is worth mentioning that DNA has been a target for anticancer drugs in recent decades.
Interaction of chemotherapy drugs such as transition metal complexes with DNA is of interest in
order to identify the interaction sites and energy of DNA with complex, designing new anticancer
drugs, and understanding the mechanism of drug action. They can bind to the DNA through
covalent or non-covalent interactions. The latter can occur through the following ways i)
intercalation with base pairs ii) major and minor groove binding via dispersion force or hydrogen
bond iii) electrostatics force between negatively charged DNA and positively charged species [14,
1
5].
Serum albumin is the main soluble and circulatory protein constituents of the blood. Its most
important property is that it acts as a transport and storage protein, so it is crucial to examine the
interactions of metal complex drugs with this protein. Bovine serum albumin (BSA) is one the
most widely investigated protein due to its structural homology with human serum albumin (HSA)
[16]. The BSA protein is made up of three domains (I, II, and III). Each domain in turn constitutes
two subdomains A and B. Furthermore, BSA has two tryptophan which possess inherent
fluorescence emission [17]: Trp134 located in subdomains IA on the protein surface and Trp212
located in subdomains IIA within a hydrophobic binding pocket of the molecule.
4

In short, as mentioned in the above two paragraphs, DNA is the major target of an antitumor agent
while BSA acts as a carrier protein molecule in the blood stream for various compounds including
antitumor compounds. Thus interaction studies of antitumor agents with DNA and BSA will
complementary help pharmacological understanding of the use of these agents.
In order to obtain a new perspective into the interaction between a second raw transition metal
complex and DNA or BSA, a mononuclear palladium(II) complex which is analogous to
carboplatin (comprising N-N and O-O donor ligands) has been synthesized and characterized. The
second aim of this work is to disclose the interaction mode of [Pd(phen)(acac)]NO with DNA (as
3
the major target of anticancer agents) and BSA (as a carrier protein molecule in the blood stream
of mammals) through various spectroscopic methods. Also, the palladium(II) complex has been
docked into the three dimensional structure of DNA and BSA in order to envisage the binding
between them.
2
. Experimental
2
.1. Material
Type-1 CT-DNA (highly polymerized), BSA (with a molecular weight of approximately 66463
Da), Tris–HCl buffer, ethidium bromide (EB) and PdCl (palladium(II) chloride) were received
2
from Sigma Chemical Company and were used without further purification. Other reagents such
as silver(I) nitrate (AgNO ), 1,10-phenanthroline (phen), sodium chloride (NaCl), sodium
3
hydroxide (NaOH) and acetyl acetone (acac) were bought from Merck Chemical Company.
Commercial solvents (Merck) such as ethanol, methanol, and acetonitrile were of analytical grade
and were utilized as supplied. Doubly distilled water was applied all over experiments and
commercial acetone was distilled before use.
2
.2. Instrumentation and tools
UV–Vis absorption spectra of the metal complex and complex-DNA/-BSA were recorded on a
JASCO–V570 spectrophotometer. Utilizing the Varian fluorescence spectrometer (Cary Eclipse
model), the fluorescence spectra of the samples were recorded. All CD (circular dichroism) spectra
5

1
were taken via CD spectrometer (Aviv 215), USA in the far UV range (200–250 nm). The H
NMR spectrum was obtained on a 250 MHz Bruker spectrometer in presence of TMS as standard
reference. Infrared spectrum of the prepared Pd(II) complex was recorded in KBr pellets from 400
-1
to 4000 cm utilizing Perkin Elmer FT-IR spectrometer. Elemental analysis (C, H and N contents)
of the synthesized complex was determined by an elemental analysis of CHNO Rapid Heraus and
its molar conductivity was carried out on a HANA-EC215, using deionized water. Ostwald micro-
viscometer (SCHOT-geräte, Germany) was applied for viscosity experiments in a water-bath of
2
7±1 °C. Decomposition temperature was measured on an Electrothermal-9100 apparatus and was
reported uncorrected. Lipophilicity of the complex was determined by partition coefficient
experiment via shake-flask procedure. Gel electrophoresis was done on CCD-5 Gel
Documentation System utilizing Maxi HD 15 × 15 Tank, Ladder 100bp, Iran.
2
.3. Synthetic procedure of [Pd(phen)(acac)]NO3
Acetylacetonato-1,10-phenanthrolinepalladium(II) nitrate, C H N O Pd = 447 g/mol
1
7
15
3
5
1
mmol (0.357 g) of [Pd(phen)Cl ], which was prepared by Palocsay method [18], was suspended
2
in 50 mL double distilled water. In another container, 1mmol (0.04 g) of NaOH and 1 mmol (0.1
mL) of acetyl acetone was dissolved in 10 mL water-ethanol blend (1:1V/V) and stirred for 20 min
to get acetyl acetone sodium salt (pale yellow product). This solution was added slowly to
[
Pd(phen)Cl ] suspension and stirred at 50 °C for 2 h. Then it was cooled and 0.34 g (2 mmol)
2
AgNO was added and stirred at 55 °C for 7 h in dark and at room temperature for 10 h. The AgCl
3
precipitate was filtered off using 42 Wattman filter paper. The volume of yellowish filtrate was
decreased to 25 mL by evaporating at 45 °C and filtered again. Then Evaporation process was
continued to 5 mL and cooled to room temperature. The obtained orange precipitate was filtered,
washed by 5 mL chilled distilled water and 10 mL acetone and dried in an oven at 45-50 °C. The
precipitate was dissolved in 25 mL of acetonitrile-methanol mixture (1:1V/V) (for recrystallization).
On diffusing of diethyl ether to this mixture for 72 h, the yellow needle shaped microcrystals was
formed, which was filtered, washed with 10 mL of acetone and dried in an oven for 48 h at 50 °C.
The yield was 0.305 g (68%) and decomposed at 260-263 °C. Anal. Calcd. for the above complex:
C,25.64; H, 3.36; N, 9.40. Found: C, 25.73; H, 3.37; N, 9.39%. The proposed equations for the
synthesis of [Pd(phen)(acac)]NO are given in Fig. 2.
3
6

1
Fig.2. Proposed reaction and structure of [Pd(phen)(acac)]NO together with H NMR signing scheme.
3
2
.4. Geometry optimization and quantum analysis
DFT-based calculation methods have been capable of getting an insight into the electronic
structure attributes of inorganic and organic compounds [19]. The quantum chemical studies (DFT
calculations) of phen and acac ligands as well as that of the palladium(II) complex were carried
out by Gaussian 09W package [20], where their relaxed geometries were drawn by GaussView
5
.0. Geometry optimizations and frequency calculations (for ground state) were performed by
B3LYP method [21, 22] with effective core pseudo potentials (GENECP) [23] using 6-311G(d)
basis set for C, H, O and N atoms and aug-cc-pVTZ-PP [22, 24] for Pd in the gas phase. The
optimized structure corresponded to the configuration with true energy minimum (with no
imaginary vibrational frequency eigenvalues). Finally, to describe the electronic properties and
global reactivity parameters, the electrostatic potential surface and frontier molecular orbitals
(FMO) were plotted and analyzed at the same level of theory.
7

2
.5. Stability determination
Electronic absorption spectral patterns of the palladium(II) complex were recorded and checked
to evaluate its stability in water and Tris-HCl buffer (pH=7.2). These solutions (0.001 g/10 mL)
were incubated in thermostatic water bath at 37 ℃ for 0, 5, 10, and 20 h, respectively.
2
.6. Partition coefficients determination
UV-Vis spectroscopy method was conducted to determine partition coefficient (log P) of
Pd(phen)(acac)]NO complex via shake-flask procedure in a mixture of n-octanol and doubly
[
3
distilled water [25]. First, 0.001 g of the Pd(II) complex was added to n-octanol-water mixture (10
mL, 1:1V/V) and shaken continuously overnight. After we let it to equilibrate for 1 h, a phase
separation resulted and finally two layers (octanol and water) were carefully separated from each
other. The concentration of Pd(II) complex in each phase was evaluated from UV-visible
spectroscopy utilizing Beer-Lambert formula. The lipophilicity value of [Pd(phen)(acac)]NO3
complex in terms of log P (partition coefficient) was calculated from equation (1).
[
푃푑(퐼퐼) 푐표푚푝푙푒푥] 표푐푡푎푛표푙 푝ℎ푎푠푒
log 푃 = log
(1)
( [푃푑(퐼퐼) 푐표푚푝푙푒푥] 푤푎푡푒푟 푝ℎ푎푠푒
)
2
.7. In-vitro cytotoxicity assay
2
.7.1. Cell culture
In the present work, the cytotoxicity assay was carried out by utilizing leukemia cell line (K562),
obtained from Pasteur Institute cell bank (Iran). The cancer cells were cultured in Roswell Park
Memorial Institute (RPMI-1640) medium supplemented with 2 mM of glutamine, 10% of fetal
bovine serum and 5 µg/mL penicillin at 37 °C. The mentioned cells were grown in an incubator
under humidified condition of 5% CO /95% air.
2
8

2
.7.2. Determination of the cytotoxicity
In vitro cytotoxicity potential of the synthesized complex against K562 cell lines was assessed via
standard 3-(4,5-dymethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cancer
4
cultured cells were placed at a density of 3 × 10 cell/mL in a sterile 96-multiwell culture plate at
3
7˚C and grown overnight in incubator for 48 h. The Pd(II) complex was added to the wells in
diverse concentration range of 0 to 100 μM. Media in the presence of cisplatin were utilized as the
positive control cell. This assay (MTT assay) is based on the conversion and cleavage of yellow
MTT (tetrazolium salt) to formazan precipitate (purple colored) through viable cells. 25 μL of
MTT solution (5 mg/mL) in PBS was added to each well-plate before the end of incubations step.
The formazan precipitates so formed was dissolved in a solution of sodium dodecyl sulfate (SDS,
1
0%) and DMF (50%) at 37 °C for 3 h in dark. Subsequently, the well-cells were shaken for 5 min
before being read by multi well scanning spectrophotometer (ELISA Reader-Model Expert 96,
Austria) at λ = 570 nm. The viability of each group was reported as the percentage of control
absorbance, specified as IC (the 50% inhibition concentration) [26].
5
0
2
.8. CT-DNA and BSA interaction analysis
CT-DNA (1.5 mg/mL) and BSA (3 mg/mL) stock solutions were prepared in Tris-HCl buffer
containing NaCl 0.01 M, pH = 7.2), and stirred slowly till homogenous and stored at 4°C for all
experimental usage. The ratio of UV absorbance of CT-DNA solution at 260 nm and 280 nm
A /A ) was obtained between 1.8 – 1.9, illustrated that applied CT-DNA was sufficiently
(
(
2
60
280
devoid from protein [27, 28]. The actual concentrations of BSA and CT-DNA were measured
-1
-1
utilizing electronic absorbance at 280 nm (ε280, BSA= 44300 M cm ) and 260 nm (ε260, DNA= 6600
-1
-1
M cm ), respectively [29, 30]. Concentrated stock solution of metal complex was diluted in
doubly distilled water to attain the required concentration (10–4 M) for CT-DNA and BSA
interaction studies. The binding affinities of Pd(II) complex-CT-DNA/-BSA systems were
followed via diverse methods.
9

2
.8.1 Absorption spectroscopic experiments
Two titration experiments were conducted to evaluate the binding affinities between metal
complex with macromolecules (CT-DNA and BSA), utilizing electronic absorption spectroscopy
at 300 K in the range of 200-700 nm. In the first experiment, 30 μL of stock solution of CT-DNA
or 450 μL of stock solution of BSA was mixed with 1.97 and 1.05 mL of Tris-HCl buffer to get
the final volume of 2 (for CT-DNA solution) and 1.50 mL (for BSA solution), respectively and
were slowly homogenized. The resulted samples of CT-DNA or BSA were titrated by various
concentrations of Pd(II) complex which includes: 0.00 to 7.00 μM for the CT-DNA and 0.00 to
4
8.27 μM for BSA. After each injection, the resulting mixtures were kept for 3 min in 300 K to
equilibrate and then their electronic absorption patterns were recorded. Apparent association
constant (K ) for the CT-DNA/BSA-complex binding system can be obtained from the analysis
app
of absorption titration spectra utilizing the following equation [31, 32]:
1
1
1
1
=
+ 퐾
×
0
(2)
퐴 ― 퐴0
퐴∞ ― 퐴0
(퐴 ― 퐴 )
[푡푖푡푟푎푛푡]
푎푝푝
∞
where, A and A are the absorbance of CT-DNA (at 260 nm) or BSA (at 280 nm) in presence and
0
absence of the Pd(II) complex, [titrant] is the concentration of the Pd(II) complex after each
injection and A is the absorption pattern where CT-DNA or BSA was completely saturated by the
∞
complex. The value of Kapp was determined via plotting 1/[tirtant] in the x-axis and 1/A-A in the
0
y-axis [33]. The Kapp value for the CT-DNA or BSA binding system was utilized to determine the
change in the free energy (ΔG°) by equation ΔG° = - RTlnK , where R and T are the ideal gas
app
-1
-1
constant (1.987 cal.mol .K ) and the absolute temperature (300 K), respectively.
Second experiment which includes denaturation of the CT-DNA (30 μL of CT-DNA stock solution
in 1.97 mL Tris-HCl buffer) or BSA (450 μL of BSA stock solution in 1.05 mL of Tris-HCl buffer)
was carried out through monitoring the absorbance at 260 nm (for CT-DNA) and 280 nm (for
BSA). To the above mentioned samples, 20, 30, 45, …, 150 μL of the Pd(II) stock solution on CT-
DNA and 200, 250, 300, …, 1400 μL on BSA were added and their absorbance were recorded
after each injection. The absorbance at 640 nm (related to the turbidity absorption) for each
spectrum of CT-DNA or BSA solution was noted and then the absorbance values of A -A for
2
60 640
CT-DNA or A -A for BSA was plotted against total concentration of the metal complex. In
2
80 640
1
0

both experiments, an identical concentration of the metal complex was injected to the reference
cell in order to eliminate the complex effect.
2
.8.2 Fluorescence measurement
Fluorescence titration experiments were performed to get further support for the interaction
between [Pd(phen)(acac)]NO complex and CT-DNA or BSA. The fluorescence emission spectra
3
of CT-DNA and BSA in presence of various concentrations of Pd(II) complex were saved from
5
35 to 740 nm at excitation wavelength of 471 nm for CT-DNA and from 310 to 470 nm at
excitation wavelength of 295 nm for BSA at three temperatures of 293, 300, and 310. These
experiments were repeated three times. The excitation and emission slit were both kept at 5 nm
and the cuvette path length was 1 cm. For DNA binding, 90 µL of stock solution of CT-DNA and
1
5 µL of ethidium bromide (stock solution of 0.023 mg/mL) were added to 875 µL of Tris-HCl
buffer solution (pH = 7.2) and the final volume of 0.98 mL was achieved. Then for CT-DNA
titration, 0-125 µL of complex stock solution was added. For BSA experiment, 60 µL of stock
solution of BSA was added to 0.94 mL of Tris-HCl buffer to get the final volume of 1 mL. Then,
different amounts of stock solution of Pd(II) complex (0-95 µL) was added and titration was
accomplished. For both titration experiments, after each addition of Pd(II) solution, the mixture
was kept 2 minutes till equilibration and then its spectrum was saved. To evaluate the mechanism
of interaction Stern-Volmer equation was utilized as follows [34]:
퐹표
=
1 + 퐾 [푄]
(3)
푆푉
퐹
In this equation, F and F are the fluorescence intensities in the absence and presence of quencher,
0
K is Stern-Volmer quenching constant (K = k τ where k and τ are the rate constant and BSA
SV
SV
q
0
q
0
-8
or EB fluorescence lifetime,10 s, respectively) and [Q] is the concentration of quencher.
Furthermore, binding constant (K ) is a measure of bond strength and can be calculated according
b
to the following equation [35]:
퐹0 ― 퐹
log
= 푛log [푄] + log 퐾푏
(4)
퐹
1
1

here, n is the number of binding sites per molecule of BSA and/or per 1000 nucleotides of DNA.
In order to get thermodynamics parameters and the nature of binding mode between the metal
complex and CT-DNA/BSA, van’t Hoff equation was used as follows:
∆
퐻° ∆푆°
ln 퐾 = ―
+
(5)
푏
푅푇
푅
In equation (5), ΔH° and ΔS° are the changes in enthalpy and entropy, respectively. Finally, the
changes in Gibbs free energy (ΔG°) at three different temperatures were obtained from equation
(6):
∆ °
퐺 = ∆퐻 ― 푇∆푆
°
°
(6)
2
.8.3. Förster resonance energy transfer (FRET)
Using this method, one can calculate the distance between interacted moiety of the protein
molecule and bioactive metal complex which this distance is a measure of interaction affinity
between them. More detail about this method will be explained in section 3.6.3.
2
.8.4. Viscosity experiments
Ostwald micro-viscometer (Schott-geräte, Germany) was applied for performing viscosity
measurements by holding it in a water-bath of 27 °C. All samples were measured four times by
using digital stopwatch and an average value of flow time was used for calculation. The CT-DNA
concentration was kept 100 µM and the concentration of Pd(II) complex was diverse to give R (R=
[complex]/ [DNA]) from 0.00 to 0.40 with a spacing of 0.05. The obtained viscosity data were
1
/3
reported as (η/η ) versus ratio R, where η and η are the specific viscosity of CT-DNA alone and
0
0
CT-DNA-complex solution, respectively. The relative values of η and η were attained via the
0
following equation.
(
푡푃푑(퐼퐼) 푐표푚푝푙푒푥 ― 푡0 /푡0
)
η
=
(7)
η0
푡퐶푇 ― 퐷푁퐴 ― 푡0 /푡0
( )
1
2

here, 푡퐶푇 ― 퐷푁퐴 and 푡푃푑(퐼퐼) 푐표푚푝푙푒푥 are the average flow time of CT-DNA alone and CT-DNA in the
presence of different concentrations of the complex, respectively, and 푡 is related to the buffer
0
solution [36].
2
.8.5. CT-DNA binding by gel electrophoresis
CT-DNA interaction of [Pd(phen)(acac)]NO in Tris-HCl buffer under physiological pH (7.2) has
3
been monitored via the well-known method, agarose gel electrophoresis. Preparation of novel
complex stock solution was done by means of dissolving it in double distilled water and diluting
-4
with working buffer to the final concentration of 8 × 10 M. General process for performing
electrophoresis examination was as follows: initially, 20 µL CT-DNA (stock solution) was
exposed to various volumes of complex (0, 10, 15 and 20 µL). Afterward, dilution was done with
the working buffer to a total volume of 50 µL and the CT-DNA blend was left for overnight in
order to reaction be completed. Then, mixtures were incubated at 37 °C for 1 h. Finally, the mixture
of each sample (9 µL) and loading dye (1 µL) were added attentively into the wells on 3 % agarose
gel and electrophoresis mobility was accomplished at 70 V for 50 min in TAE buffer. Ethidium
-1
bromide (EB, 0.5 μg mL ) solution in water was utilized to stain the gel at room temperature for
4
5
5 min and then the gel was visualized and photographed under UV-illuminator utilizing a CCD-
Gel Documentation System.
2
.8.6. Circular dichroism (CD) of BSA
CD spectra (Far-UV, 200–250 nm) of BSA and BSA-Pd(II) complex system were recorded on a
circular dichroism spectrometer, AVIV model 215 at room temperature utilizing a cylindrical
cuvette cell with path length of 1 mm. For these experiments, the concentration of BSA in each
sample has been remained constant (5 μM) and concentration of prepared complex varied from 0
to 30 μM. All CD spectra have been collected via subtracting the background (working buffer)
signal from all measurements and converted to the molar ellipticity and then smoothed. CDNN
program was applied for the calculation of secondary structural elements [37].
1
3

2
.8.7. Docking method
Molecular docking method was applied as a supplementary path for prediction of binding site and
orientation of [Pd(phen)(acac)]NO complex during binding to DNA and BSA utilizing Auto Dock
3
4
.2 and Auto Dock Tools 1.5.6 programs. The optimized geometry of the complex was converted
to the PDB format with the help of GaussView 5.0 [38] and utilized as initial input for docking
process. The available crystal structure of DNA hexamer d(CGATCG) (ID: 1Z3F, complexed
2
with ellipticine through intercalation mode) [39] and BSA (ID: 4F5S) [40] were downloaded from
the Protein Data Bank (http://www.rcsb.org./pdb) and employed in the docking procedures. The
‘receptor’ (DNA or BSA) file was initially prepared via removing extra molecules such as water,
other ligands and monomers (for BSA) and adding polar hydrogens. Then, the partial atomic
charge (Gasteiger charge) was added to the complex. After that, the search space was assigned for
DNA or BSA, which was enclosed in a grid box of 52 × 56 × 56 points (for DNA), 80 × 88 × 76
points (for site I of BSA) and 76 × 56 × 54 points (for site II of BSA) in the x-, y- and z- axis,
respectively. Spacing of 0.375 Å with a grid center of x = -1.688, y = -0.012, z = -1.869 for DNA,
x = -3.053, y = 20.152, z = 97.594 for site I of BSA and x = -5.152, y = 20.404, z = 117.8 for site
II of BSA were chosen. After the generation of grid box, the Lamarckian genetic algorithm (LGA)
was employed to search for the stable pose of palladium(II) complex in the combination with DNA
or BSA. The LGA parameters were selected as follows: the number of runs, 100; fitness evaluation
energy, 2500000; maximum number of generations, 27000. Other parameters were remained as
their default values. During each docking process, the binding pose of palladium(II) complex with
the lowest free energy of binding (∆G°) was considered as the best binding mode. The obtained
poses from docking simulations were extracted by Chimera 1.11.2 software and analyzed through
Discovery Studio Visualizer 4.1 package.
3
. Results and discussion
On precipitating the chloride ions from [Pd(phen)Cl ] complex by AgNO in presence of acetyl
2
3
acetone-NaOH mixture, a new and bioactive palladium(II) complex of formula
Pd(phen)(acac)]NO was synthesized. In this complex, phen is 1,10-phenanthroline and acac is
[
3
deprotonated acetyl acetone that are both chelating ligands. Because it is well known that kinetic
1
4

lability of a metal complex could be lowered by coordinating the metal with chelating ligands
rather than monodentate ones [41]. The complex is yellow solid and soluble in water being the
first important phase for possible utilization in biological systems. Although the complex
preparation path is not so complicate, however, we have tried to get single crystal suitable for X-
Ray structural determination but have not yet being succeeded. However, satisfactory results were
obtained from other analytical methods like conductivity measurements, elemental analysis, FT-
1
IR, H NMR and UV-Vis studies. These results illustrated that the synthesized complex is of good
purity. Furthermore, partition coefficient test was performed to check the lipophilicity of the
complex. Antitumor activity of the compound was confirmed by testing it against K562 leukemia
cells. Theoretical studies including molecular orbital, electrostatic potential surface analysis and
molecular docking simulation were attempted. Rigorous interaction studies of the complex with
DNA as target and BSA as carrier protein molecules were carried out. The results of these above
studies are discussed herein below:
3
.1. Structural characterization of [Pd(phen)(acac)]NO3
-4
2
-1
The molar conductance of the complex solution (10 M) in deionized water was 130 cm ohm
-1
mol . This value suggests that the complex is 1:1 electrolyte [42]. The measured molar
conductance along with the results of CHN elemental analysis (given in the experimental section)
are in agreement with the formulation proposed for the above Pd(II) complex.
-1
The FT-IR spectrum of acetylacetone displayed two strong bands at 1729 and 1622 cm . These
are assigned to stretching frequencies of its ketonic and enolic forms of C=O group, respectively
-1
[
43]. In the [Pd(phen)(acac)]NO complex, the C=O stretching band is seen at 1518 cm ,
3
suggesting that the acetylacetone is present as bidentate chelate in this complex [44, 45]. Moreover,
-1
-
the sharp and intense band at 1383 cm in the spectrum of the Pd(II) complex shows that the NO
group is present as counter ion [46].
3
The UV-Vis spectrum of [Pd(phen)(acac)]NO complex was recorded in double distilled water.
3
Four broad electronic absorption bands with maxima at 205 nm (ε = 58760), 272 nm (ε = 22300),
a shoulder at 309 nm (ε = 7900) and a weak band at 360 nm (ε = 1760) were observed. These
bands are tentatively assigned to the first, second and higher internal transitions of phen and acac
1
5

-
ligands and NO as counter ion which have possibly showed overlapping [47-50]. Moreover, the
3
spectra of the complex in different solvents from less polar acetone to more polar water were
recorded and no blue- or red-shift in any of the peak positions were observed. This suggests that
either there is no charge transfer band or it has been hidden by the other absorption bands, leading
to no visible shift. Same explanation could be made for the weak d→d transition bands observed
for Pd(II) complexes having square planar geometry.
1
The H NMR spectrum of [Pd(phen)(acac)]NO was recorded in DMSO-d because of its low
3
6
solubility in D O. Tetramethylsilane was used as a standard internal reference. The protons of
2
1
,10-phenanthroline moieties of this complex were resonated between 8-9 ppm. These protons
showed upfiled shift in comparison to the corresponding 1,10-phenanthroline protons in
Pd(phen)Cl ] complex [51, 52]. A singlet (possibly triplet) signal in [Pd(phen)(acac)]NO appears
[
2
3
at 2.21 ppm is assigned to H (6H) protons and another singlet at 5.79 ppm is assigned to H (1H)
f
e
proton of coordinated acetylacetone moieties. These two signals in the corresponding free
acetylacetone resonates at 2.04 and 5.54 ppm, respectively. Thus, H and H protons showed
f
e
upfield shift of 0.17 and 0.15 ppm, respectively. Because the keto and enol forms of free
acetylacetone disappear on coordination [47].
On the bases of above characterization data, the suggested structure and NMR signing scheme for
the above Pd(II) complex was designed and shown in Fig. 2.
3
.2. Computational details
3
.2.1. Molecular geometry analysis
The optimized structure of palladium(II) complex obtained from Gaussian 09W and GaussView
.0 programs is demonstrated in Fig. 3 (a). The bond parameters (bond lengths and bond angles)
5
of the title complex were calculated utilizing DFT (B3LYP/ GENECP) method and aug-cc-pVTZ-
PP basis set for Pd atom and 6-311G(d) for other atoms. The bond angle between O-Pd-O, N-Pd-
o
N and N-Pd-O are 93.91, 81.24 and 92.42 , respectively. Also, the bond length of Pd-O and Pd-N
are 1.99 and 2.04 Å, respectively. The exact experimental results on the bond angles and lengths
of [Pd(phen)(acac)]NO are not available, so the x-ray diffraction geometrical parameters of
3
closely related complexes [53, 54] were utilized to compare the crystal data with that of the
1
6

investigated complex. From the calculated values, a good agreement is found between our
computational and experimental results. Slight difference between them could be attributed to the
fact that quantum mechanical calculations were accomplished in the gas phase while experimental
ones were carried out in the solid state.
Fig. 3. (a) Optimized geometry and (b) electrostatic potential surface plot of Pd(II) complex.
3
.2.2. Electrostatic potential surface
In order to determine the most reactive sites of Pd(II) complex for electrophilic and nucleophilic
attacks, electrostatic potential surface has been drawn using GaussView 5.0 program for the
optimized geometry of the title complex and is shown in Fig. 3 (b). Electrostatic potential surface
map simultaneously represents molecular shape, size, and electrostatic potential in terms of
different colors. In general, dark blue displays the regions of most positive electrostatic potential
and red shows the sites of most negative potential. For this complex, different values of the
electrostatic potential are shown by various shade of blue colors. From this figure, it can be
observed that the positive regions are mostly located on phen while negative regions are over acac
ligand. Positive (dark blue) and negative (light blue) regions of electrostatic potential are related
to nucleophilic and electrophilic reactivity, respectively. In the other word, the positive
electrostatic potential (dark blue) corresponds to the attraction of nucleophile and the repulsion of
1
7

the protons by the atomic nuclei and the negative molecular electrostatic potential (light blue) is
associated to the attraction of electrophile and proton by the aggregate electron density in the
compound. Electrostatic potential surface analysis illustrates that phen is more susceptible for
nucleophilic attack and acac is suitable for electrophilic substitutions of other species. In
conclusion, these active regions predict the ability of Pd(II) complex toward macromolecules such
as DNA and BSA and confirm its biological activity.
3
.2.3. HOMO-LUMO analysis
Investigation of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular
orbital (LUMO) is important for a compound as a ligand. LUMO can be thought as an orbital that
has a room to accept electron and its energy is related to electron affinity while HOMO contains
electrons that can donate and its energy is related to ionization energy. The energies of frontier
molecular orbitals, HOMO and LUMO, are important parameters to predict the chemical activity
and electrical property in the compound. The energy values of the Pd(II) complex and its ligands,
phen and acac, are EHOMO = -9.49, -6.47, -6.73 and ELUMO = -5.79, -1.65, -1.35 eV, respectively.
The atomic orbital components of the frontier molecular orbitals are demonstrated in Fig. 4. The
energy gap between their HOMO and LUMO are 3.70, 4.82 and 5.38 eV, respectively. As can be
observed in this figure the HOMO of Pd(II) complex is delocalized over acac while LUMO is
localized over phen in accordance with electrostatic potential surface results. This indicates π
nature charge transfer from acac to phen.
The molecule is called soft when the energy gap between HOMO and LUMO is small so that it
has low kinetic stability and high reactivity. The hardness implies the resistance toward
polarization under small perturbation during the chemical process. Absolute hardness (η), and
other parameters such as HOMO–LUMO energy gap (ΔE), chemical potentials (µ), absolute
electronegativitiy (χ) and global electrophilicity (ω) [55-57] are calculated using the following
equations:
퐸퐿푈푀푂 ― 퐸퐻푂푀푂
휂 =
(8)
(9)
2
Δ퐸 = 퐸퐿푈푀푂 ― 퐸퐻푂푀푂
1
8

퐸퐿푈푀푂 + 퐸퐻푂푀푂
휇 =
(10)
(11)
2
휒 = ― 휇
휇2
휔 =
(12)
2
휂
These quantities have been calculated for the title complex and its ligands and are summarized in
Table 1. It is observed from this table that the chemical potential of all compounds are negative
indicating that they are stable and do not decompose to their elements spontaneously. Note that
Pd(II) complex has the most negative chemical potential. From this table one can see that the value
of hardness for complex is lower than both free ligands that implies it has more polarizability and
reactivity. The calculated parameters describe the biological activity of the Pd(II) complex.
Fig. 4. HOMO and LUMO plots of Pd(II) complex and corresponding ligands.
1
9

Table 1. Absolute hardness (η), energy gap (ΔE), chemical potentials (µ), absolute electronegativitiy (χ) and global
electrophilicity (ω) of the complex and its ligands.
compounds
Pd(II) complex
phen
E
HOMO (eV)
E
LUMO (eV)
η (eV)
1.85
ΔE (eV)
3.70
µ (eV)
-7.64
-4.06
-4.04
χ (eV)
7.64
ω (eV)
15.78
3.42
-9.49
-5.79
-6.47
-1.65
2.41
4.82
4.06
acac
-6.73
-1.35
2.69
5.38
4.04
3.03
3
.3. Stability determination
In solution, a compound should be stable to cause specific biological activity. Double distilled
water or Tris-HCl buffer are commonly utilized to prepare sample solution for cytotoxic activity
and macromolecule interaction studies. Accordingly, the stability of [Pd(phen)(acac)]NO3
complex was determined in these solutions. The absorption spectra of this complex in these
solutions were remained unchanged with time intervals of 0, 5, 10 and 20h at 37 ℃ (supplementary
Fig. S1), demonstrating the stability of this complex in aqueous or Tris-HCl buffer solutions.
3
.4. Partition coefficient (log P)
The log P (water/n-octanol partition coefficient), a crucial pharmacokinetic parameter, is
frequently utilized to evaluate the lipophilicity of a compound. This is actually an index showing
liposolubility of a given compound which can cross through phospholipid part of cell membrane
[58]. The compounds with high lipophilicity can percolate the phospholipid part of cell membrane
without hardness and enter into the fatter body tissue. Here the resulting log P for the above Pd(II)
complex is -1.24 ± 0.04. On the other hand, the log P value of cisplatin listed in the literature is -
2
.28 ± 0.07 [59]. Thus the lipophilicity order of Pd(II) complex > cisplatin, exhibits that
[
Pd(phen)(acac)]NO is more lipophilic than that of cisplatin which facilitate its crossing from cell
3
membrane. This observation is in agreement with the results of cytotoxicity observed for this
newly made Pd(II) complex. It means that the complex can penetrate through phospholipid part of
cell membrane leading a rise in its concentration inside the cells and thus this may be the reason
that why it has higher cytotoxic character than cisplatin.
3
.5. In vitro cytotoxicity test
2
0

The in vitro anticancer activity of the novel Pd(II) complex against K562 cell lines was evaluated.
Following a 48 h exposure of the cancer cell lines to the newly synthesized palladium(II) complex,
the MTT assay was applied to determine the cytotoxicity activity of the compound. The effect of
the various concentrations (0-100 µM) of the above complex on the cancer cell lines and the IC50
(the 50% inhibition concentration) value are illustrated in Fig. 5. In this figure, the cytotoxicity
data was expressed as the percentage of cell viability versus complex concentration. According to
Fig. 5, the cell viability was reduced after adding the different concentrations of the complex and
the evaluated IC value related to the Pd(II) complex is 18 μM at incubation times of 48 h. On the
5
0
other hand, the IC value of the cisplatin, primary anti-cancer agent, against K562 cells was
5
0
detected 154 μM [60] in previous work at the identical condition, that is much higher in comparison
with the IC value of the above complex. Therefore, this preliminary researches reveal that this
5
0
complex inhibits tumor cell duplication through binding to DNA duplex and lead to block the cell
cycle. Thus guided us to carry on DNA-binding studies.
Fig. 5. Effect of the [Pd(phen)(acac)]NO complex against cell viability of the K562 cells utilizing MTT
3
assay. The K562 cells were treated with diverse concentrations of the complex (0-100 µM) for 48 hours.
3
.6. CT-DNA and BSA interaction analysis
2
1

3
.6.1. Electronic absorption titration
In order to probe the binding affinity of metal complex with DNA or protein, employing very
simple methods such as UV-Visible spectroscopy is useful, as the interaction with DNA or protein
leads to the electronic perturbations in their electronic absorption spectra [61-63]. On DNA-
complex binding system, this perturbation in the maximum wavelength of DNA absorption
spectrum is very important because from it the type of binding mode (i.e. intercalation or groove
binding) can be predicted. It is often in many works that π˗π stacking interaction between the
aromatic rings of the metal complex and DNA base pairs causes the hypochromism (reduction in
the intensity of spectrum at maximum wavelength) along with bathochromism (red shift) [64, 65].
The extent of the electronic perturbations (hypochromism and bathochromic) depends on the
potency of the interaction of the complex with DNA [66]. In the case of electrostatic attraction
between the compound and DNA, one can observe hyperchromism in the absorption patterns of
DNA [67]. The absorption measurements of the CT-DNA and BSA were performed in presence
of different concentration of [Pd(phen)(acac)]NO complex (0.00 to 7.00 μM from complex stock
3
solution for CT-DNA and 0.00 to 48.27 μM for BSA) and the spectra are demonstrated in Figs.
6
(a) and 6(c). From these figures, it is obvious that by supplying more volume of
[
Pd(phen)(acac)]NO complex, a significant case of hypochromism is observed in the electronic
3
absorption pattern of CT-DNA or BSA, along with 5 nm shift towards the larger values of
wavelengths (bathochromic shift) at λmax = 200 nm for CT-DNA. On the other hand, the percentage
of hypochromism was evaluated through the following equation H (%) = ((A –A )/A ) × 100,
0
M
0
where A and A are the absorbance of free and bounded CT-DNA or BSA, respectively [68]. The
0
M
obtained hypochromicity shifts for CT-DNA/BSA-complex system was 64.52 % and 49.96 %,
respectively. Also, during the CT-DNA titrations, a well-defined isosbestic point at 298 nm was
observed, proving the existence of, with strong possibility, one specific binding mode between this
complex and CT-DNA [69]. These evidences including a notable hypochromic followed by
bathochromic effect for CT-DNA binding, suggest that this complex may bind to CT-DNA via an
intercalating mode. This further illustrates a π˗π stacking interaction between the complex and CT-
DNA base pairs, as in the native structure of [Pd(phen)(acac)]NO complex, an aromatic fused
3
ring (phenanthroline ligand) is present which is capable of inserting between base pairs of DNA
by intercalation [34, 70].
2
2

Moreover, from the reduction in the absorption bands of CT-DNA and BSA in the presence of any
concentration of metal complex, one can conclude that Pd(II) complex interacts with CT-DNA or
BSA, thereby leading to the alteration in their native structures. The corresponding absorbing
groups in the primary structure of DNA and BSA, i.e. purine and pyrimidine bases in DNA and
phenylalanine, tryptophan and tyrosine residues in BSA, make above alteration by being hidden
from the UV waves, resulting a reduction in the intensity of absorption bands in the presence of
various concentrations of metal complex [71]. This type of conformational alteration can be related
to the refolding of CT-DNA and BSA, because the primary structures of DNA and BSA are folded
macromolecules.
Using UV titration spectra and the equations (2), apparent association constants (K ) was
app
calculated in terms of the interaction between the Pd(II) complex and CT-DNA/BSA to predict
their binding affinities. Figs. 6(a) and 6(d) illustrate the outline of 1/A-A vs. 1/[tirtant] . Utilizing
0
the intercept ratio (1/A -A ) acquired for the straight line to its corresponding slope (1/K (A -
∞
0
app
∞
5
-1
A0)), Kapp value was evaluated. The Kapp values of the complex are 2.87 × 10 M for CT-DNA
3
-1
binding and 1.37 × 10 M for BSA binding, indicating high binding affinity of Pd(II) complex to
CT‐DNA. The higher CT-DNA binding ability of [Pd(phen)(acac)]NO complex may be attributed
3
to the extended surface of aromatic ring of phenanthroline ligand which facilitates the π‐stacking
interaction of its structure with CT-DNA base pairs [72]. This value for CT-DNA-complex binding
system suggests a strong interaction between them and is a little bit higher than the binding affinity
5
-1
of classical intercalator (EB) to CT-DNA (K = 1.23 × 10 M ) [73], supporting the intercalative
b
site on CT-DNA for [Pd(phen)(acac)]NO . Using the K values, ΔGº (the free energy change)
3
app
for DNA/BSA–complex system is calculated as -7.49 (for CT-DNA binding) and -4.30 kcal mol-
1
(for BSA binding). The ΔG° specifies the spontaneity or non‐spontaneity of DNA/BSA-drug
binding. As a result, the negative values of free energy for both systems represent that the
interaction of [Pd(phen)(acac)]NO complex with CT-DNA or BSA is spontaneity.
3
2
3

Fig. 6. Absorption spectra of CT-DNA (a) and BSA (c) in the absence and presence of increasing
concentrations of [Pd(phen)(acac)]NO as titrant, [CT-DNA] = 120.75 μM, [BSA] = 12.50 μM and [titrant]
3
=
0.00 – 7.00 μM for CT-DNA and 0.00 – 48.27 μM for BSA at T = 300 K. (b) and (d) illustrate the outline
of 1/A-A vs. 1/[titrant] for CT-DNA and BSA, respectively to evaluate K values.
0
app
Second titration is related to the denaturation of CT-DNA or BSA which was performed by
analysis of their UV absorption spectra in the absence and presence of various volumes of
palladium(II) complex at 300 K. These titrations were continued till their absorption spectra did
not change with addition of further volumes of complex. The denaturation profiles of CT-DNA
and BSA are represented in Fig. 7. According to these figures, the concentration of
[
Pd(phen)(acac)]NO complex in the midpoint of transition, [L] , are 3.84 and 28.57 μM for CT-
3 1/2
DNA and BSA binding, respectively. As a results, this complex can denature CT-DNA or BSA at
very low concentration. This result indicates that if this Pd(II) complex is utilized as a potential
2
4

anticancer drug, low doses will be required for the medicinal usage, as a result its side effects is
probably low.
Fig. 7. The decrease in the absorbance of CT-DNA at 260 nm (a) and BSA at 280 nm (b) upon improving
the concentration of [Pd(phen)(acac)]NO at 300 K.
3
3
.6.2. Fluorescence titration experiments
Fluorescence emission titration tests were performed to monitor the binding of the new Pd(II)
complex with DNA/BSA. EB is phenanthridine dye that has low fluorescence emission due to its
quenching by solvent molecules. But in the presence of CT-DNA, it emits intense fluorescence
light around 600 nm because the planar phenanthridine intercalates with the base pairs of CT-DNA
and forms EB+CT-DNA complex. Fig. 8 (a) illustrates that the fluorescence intensity of EB+CT-
DNA at maximum wavelength of 600 nm decreases by increasing concentration of palladium(II)
complex due to the replacement of the EB molecule by the complex. In Fig. 8 (a), a weak intensity
spectrum near the base line appeared towards the end of titration which may be due to free EB
confirms that there is a competition between the EB and Pd(II) complex. This causes releasing of
EB molecules from CT-DNA and the appearance of free EB spectrum. For the case of BSA no EB
dye is required as it has intrinsically fluorescence activity due to the presence of wrapped
tryptophan in a hydrophobic environment. As can be seen in Fig. 8 (b), the emission spectrum of
BSA exhibits a notable decrease with increasing concentration of the new palladium(II) complex.
2
5

It shows that the secondary structure of BSA and therefore tryptophan environment could change
upon binding of BSA to the Pd(II) complex.
Fig. 8. Fluorescence emission spectra of EB+CT-DNA (a) and intrinsic fluorescence of BSA (b) in the
absence and presence of increasing concentration of [Pd(phen)(acac)]NO (a-i: 0.00, 19.60, 24.30, 28.80,
3
3
2
3.30, 37.70, 44.20, 50.40 and 56.50 µM of stock solution for CT-DNA and a-k: 0.00, 1.00, 1.47, 1.96,
.44, 3.38, 4.31, 5.21, 6.10, 7.40 and 8.67 µM for BSA). Stern-Volmer plots of fluorescence quenching of
EB+CT-DNA (c) and BSA (d) by the [Pd(phen)(acac)]NO at three different temperatures.
3
To characterize the mode of quenching mechanism of the palladium(II) complex with CT-DNA
and BSA, fluorescence emission titration was utilized. Fluorescence quenching mechanism are
generally categorized as dynamic and static quenching. Dynamic quenching corresponds to the
collision of metal complex with fluorophore in excited state and static one is due to the formation
2
6

of quencher-fluorophore complex in the ground state. These two mechanism can be distinguished
by following the behavior of Stern-Volmer constant upon raising temperature. In the other word,
if by increasing temperature the quenching constant and therefore Stern-Volmer constant are
enhanced then dynamic mechanism is predominant [71].
The Stern-Volmer plots of F /F versus [Q] are shown in the Fig. 8(c) for DNA and Fig. 8(d) for
0
BSA at three temperatures and the corresponding values of K and k are presented in Table 2. It
SV
q
is observed in this table that for both EB+CT-DNA and BSA interactions with
Pd(phen)(acac)]NO complex, the value of K increases with temperature, so the dynamic
[
3
SV
quenching mechanism is expected. On the other hand, the kq values of both systems are greater
1
0
-1 -1
than maximum dynamic quenching constant (2 × 10 M s ), so the static quenching may occur
too. Therefore, it is concluded that dynamic and static quenching happen concurrently. Although,
increment of Ksv with increasing temperature and the linear relation between F /F and [complex]
0
(
as shown in the Figs. 8(c) and 8(d)) implies that the dominant quenching mechanism is dynamic
74].
[
Table 2. Stern-Volmer quenching constants, intrinsic binding constant, number of binding sites and
thermodynamic parameters in the interaction of [Pd(phen)(acac)]NO with EB+CT-DNA and BSA.
3
DNA/BSA
binding
T
(K)
K
(M )
sv
k
q
K
(M )
b
ΔGº
(kcal/mol)
ΔHº
(kcal/mol)
ΔSº
(kcal/mol)
Log K
b
n
-1
-1 -1
-1
(M s )
2
3
3
2
93 (6.32±0.10)×103 (6.32±0.10)×1011
00 (7.60±0.10)×103 (7.60±0.10)×1011
10 (9.72±0.10)×103 (9.72±0.10)×1011
93 (5.11±0.05)×104 (5.11±0.05)×1012
4.30±0.16
5.10±0.14
6.86±0.18
3.93±0.10
3.82±0.07
3.40±0.07
1.98×104
1.29±0.03
-5.62±0.20
-7.27±0.22
-9.62±0.24
-5.33±0.12
-5.14±0.12
-4.88±0.10
DNA
binding
1.28×105
7.25×106
8.57×103
6.57×103
2.55×103
1.31±0.03
1.70±0.04
0.84±0.02
0.80±0.01
0.73±0.01
63.34±0.55
0.235±0.002
-0.026±0.001
300 (5.59±0.02)×104 (5.59±0.02)×1012
10 (5.64±0.05)×104 (5.64±0.05)×1012
BSA
binding
-13.11±0.48
3
All results were expressed as the mean ± standard deviation of three independent experiments.
2
7

Using equation (4) and interpolation of Fig. 9, the number of binding sites and the binding
constants between palladium(II) complex with CT-DNA/BSA were determined at three
temperatures and the results were gathered in Table 2. As evident from Table 2, the K values for
b
the interaction of [Pd(phen)(acac)]NO with CT-DNA at three temperatures fall in the range of
3
4
6
−1
1
0 -10 M , indicating the strong nature of the binding affinity between them. For BSA binding,
3
−1
K is about 10 M which implies low or intermediate binding affinity between complex and BSA
b
[
75]. On the basis of the K values, one may expect that the interaction of the Pd(II) complex with
b
BSA is weaker than that of CT-DNA. Thus we propose that this weak interaction may not bring
much structural change of BSA. Because a high structural change may disturb normal function of
BSA leading probable side effect [76]. While stronger interaction with CT-DNA may lead to
higher conformational change and prevents replication and transcription [77].
Fig. 9. The plot of log (F -F)/F vs. log [complex] for the interaction of CT-DNA (a) and BSA (b) with
0
[
Pd(phen)(acac)]NO at three temperatures of 293, 300 and 310 K.
3
By having binding constant at hand and equations (5) and (6), one can calculate the thermodynamic
parameters and estimate the type of interactions. The interaction of complex with CT-DNA/BSA
is (i) hydrogen bonds and van der Waals forces , if ΔHº < 0 and ΔSº < 0; (ii) hydrophobic
interactions, if ΔHº > 0 and ΔSº > 0 and; (iii) electrostatic forces, if ΔHº > 0 and ΔSº < 0 [78]. The
values of ΔHº and ΔSº were obtained in accordance with the van't Hoff equation (equation (6)) by
plotting lnK versus reciprocal absolute temperature. The linear plots for CT-DNA and BSA
b
binding are drawn in Fig. 10 and the obtained thermodynamic parameters are represented in Table
2
8

2
, respectively. According to the data presented in Table 2, the reaction of the Pd(II) complex with
CT-DNA is enthalpically unfavorable (positive ΔHº) and entropically favorable (positive ΔSº), so
the driving force for binding of complex to CT-DNA is hydrophobic. According to the data in
Table 2, the main interactions for BSA binding are van der Waals interactions and hydrogen bond
because both ΔHº and ΔSº are negative. Thereafter, the Gibbs free energy change (ΔGº) was
determined according to the equation (6). For both DNA/BSA binding the values of ΔGº are
negative indicating the processes are spontaneously [79].
Fig. 10. Van’t Hoff plot of ln K vs. 1/T for the interaction of CT-DNA (a) and BSA (b) with
b
[
Pd(phen)(acac)]NO in the temperature range of 293–310 K.
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3
.6.3. Energy transfer between [Pd(phen)(acac)]NO and BSA
3
Förster resonance energy transfer (FRET) is a basic method to calculate the distance between the
protein and the interacted bioactive molecules. Förster has proven that the yield of FRET between
acceptor and donor relies mainly on three agents: i) the donor molecule is expected to emit
fluorescence light, ii) the electronic absorbance spectrum of acceptor should overlap with
fluorescence emission spectrum of donor and iii) the donor-acceptor distance should be within the
range of 2-8 nm [80]. Therefore, referring to the extent of energy transfer, the distance between a
tryptophan amino acid residue in protein (as the donor) and [Pd(phen)(acac)]NO (as the acceptor)
3
can be evaluated. Accordingly, FRET is one of the most reliable techniques to detect binding
between the ligand and protein which are within the range of Förster distance [81]. Here, the
2
9