Synthesis, photophysical behavior and biomolecular reactivity of new triphenylphosphonium-based Pd(II)salphens as new anticancer candidates

Article abstract of DOI:10.1016/j.jphotochem.2019.112083

The synthesis of new bis-triphenylphosphonium-based dimethlysalphens (salphen = N,N`-bis-(salicylidene)-o-phenylenediimine) and their complexes (Pd(II)-salphens) for chemotherapeutic applications were reported. The in vitro anticancer efficacy of new comp

Full text of DOI:10.1016/j.jphotochem.2019.112083

JournalofPhotochemistry&PhotobiologyA:Chemistry385(2019)112083
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Synthesis, photophysical behavior and biomolecular reactivity of new
triphenylphosphonium-based Pd(II)salphens as new anticancer candidates
Mohammad Y. Alfaifia, Mohamed A.-E. Zeinb,c, Ali A. Shatia, Mohammed A. Alshehria,
⁎
Serag Eldin I. Elbehairia,d, Hani S. Hafeze, Reda F.M. Elshaarawyf,g,
a Biology Department, Faculty of Science, King Khalid University, 9004 Abha, Saudi Arabia
b Chemistry Department, Faculty of Science, Damanhour University, Egypt
c Chemistry department, College of Alwajh, Tabuk University, Saudi Arabia
d Cell Culture Lab, Egyptian Organization for Biological Products and Vaccines (VACSERA Holding Company), Giza 12311, Egypt
e Faculty of Science, Zoology Department, Suez University, 43533 Suez, Egypt
f Faculty of Science, Chemistry Department, Suez University, 43533 Suez, Egypt
g Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine Universität Düsseldorf, 40204 Düsseldorf, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
The synthesis of new bis-triphenylphosphonium-based dimethlysalphens (salphen = N,N`-bis-(salicylidene)-o-
phenylenediimine) and their complexes (Pd(II)-salphens) for chemotherapeutic applications were reported. The
in vitro anticancer efficacy of new compounds against human hepatocellular carcinoma (HepG2) cell lines de-
monstrated higher activity for Pd-complexes as compared with their parent ligands. Among tested compounds,
Pd(II)-salphen3 was found to be the most potent one in the suppression of HepG2 proliferation (IC50 = 3.01 μM),
˜3-fold lower than the clinical drug (cisplatin, CDDP) (IC50 = 8.28 μM). The Pd(II)-salphens can effectively bind
to CT-DNA via a non-covalent interaction (intercalation/ electrostatic) as revealed from the hyperchromism of
28–53% along with 3–6 nm bathochromism observed during the electronic absorption titration of Pd(II)-sal-
phens with CT-DNA. On the other hand, the H-bonding and hydrophobic interactions play crucial roles in the
binding of Pd(II)-salphens to BSA as indicating from the BSA fluorescence quenching by Pd(II) complexes.
Noteworthy, Pd(II)-salphen3 displayed the highest reactivity towards DNA/ BSA with intrinsic binding constants
(Kb/ KF) (3.01 × 105/ 2.95 × 106 M–1).
TPP-based Pd(II) salphens
Cytotoxicity
CT-DNA binding
Spectrophotometry
Fluorescence quenching of BSA
1. Introduction
classes of pharmacological candidates that have the capacity to effec-
tively interacted with the nucleic acids and consequently induced their
The inability of Pt-based chemotherapy [1] to treat a wide range of
cancers along with their intrinsic toxicity and their resistance [2], as
well, have motivated many medicinal researchers for exploring in-
novative and safe non-Pt anticancer agents with broad-spectrum an-
ticancer action [3]. Meanwhile, great attention has been devoted to Pd
(II) complexes due to their remarkable efficacies in suppressing the
proliferation of a wide range of cancers [4] along with minimal toxic
effects [5]. The wide range anticancer activity of Pd-based che-
motherapeutic agents in comparison to Pt-based ones could be attrib-
uted to the preferable aqueous solubility and consequently higher
bioavailability of Pd complexes, moreover, the rates of ligand-exchange
and aquation reactions (and thus biomolecular reactivity) for Pd(II)
complexes is ˜105 fold faster than that of Pt(II) analogous [6].
degradation [7,8]. Recently, it was reported that Pd(II) complexes can
suppress the proliferation and induce the apoptosis of liver cancer cells
through different mechanisms such as DNA cleavage [6,9], probe-pro-
tein interaction [10], DNA photocleavage [11], inhibition of human
ribonucleotide reductase [12], and blocking topoisomerase IIa [11].
The amazing pharmacological aspects of triphenylphosphonium
(TPP) salts such as their ability to preferentially transfer from an aqu-
eous to the hydrophobic environment and selectively accumulation in
the mitochondria of cancer cells inducing their apoptosis [13,14] makes
them important pharmacophores for biological molecules to achieve
ultimate pharmacological effects [15,16]. Meanwhile, several studies
revealed that the cytotoxicity of TPP salts was significantly enhanced as
the electron density on TPP increased [17,18]. Therefore, TPP could act
as prospective motifs for clinical therapy of cancer.
Noteworthy, metallosalens and metallosalphens provide attractive
⁎ Corresponding author at: Faculty of Science, Chemistry Department, Suez University, 43533 Suez, Egypt.
E-mail addresses: reda.elshaarawy@suezuniv.edu.eg, reel-001@hhu.de (R.F.M. Elshaarawy).
https://doi.org/10.1016/j.jphotochem.2019.112083
Received 10 June 2019; Received in revised form 28 August 2019; Accepted 6 September 2019
Availableonline08September2019
1010-6030/©2019ElsevierB.V.Allrightsreserved.

M.Y. Alfaifi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry385(2019)112083
By taking the previous facts into consideration and in continuation
2.4. Anticancer survey
of our ongoing efforts for exploring of novel, more effective and se-
lective chemotherapeutic candidates for cancer therapy along with
minimum side effects [19], we reported in this work the prepration and
the anticancer actvity of new Pd(II)-salphen complexes anchored TPP
motifs against cultured HepG2 cells. Futhermore, the biomolecular re-
activity of the new compounds toward DNA and BSA was addressed
based on the electronic absorption and fluorescence studies.
2.4.1. Cell cultures
Human hepatocellular carcinoma (HepG2) cells were provided by
the tissue culture unit in VACSERA, and cultured in RPMI-1640 media
(10% FBS, 1% L-glutamine, HEPES buffer, and gentamycin (50 μg/
mL)). HepG2 cells were sustained at 37 °C in 5% CO2. The HepG2 cells
viability was recorded as indicative of the cytotoxicity of the tested
compound.
2. Experimental
2.4.2. Cytotoxic effect assay
The cytotoxic action of the salphen ligands and their Pd(II)-com-
plexes on HepG2 cells were assayed according to the experimental
protocol adopted by the Regional Center for Mycology & Biotechnology
(RCMB), Egypt. Briefly, HepG2 cells with a density of (104 cells per
well) were cultivated in 100 μL RPMI-1640 using 96-well plates. Then,
the tested compounds solutions of final concentrations (50, 25, 12.5,
6.25, 3.125, 1.56 μg/mL) were added to the cell culture media and kept
in a humidified incubator at 37 °C under 5% CO2 atmosphere for 48 h.
Each concentration was applied to consecutive three wells. Control cells
were cultivated without target compounds and treated with 0.5%
DMSOaq. After 48 h, the viable HepG2 cells were investigated by a
colorimetric technique where the culture media were stained with the
1% crystal violet (CV) solution and maintained at 37 °C for 4 h. Then
the CV was excluded, and the plates were washed by distilled water.
Thereafter, the content of each well was dissolved in 30% acetic acid
and the absorbance of each plate was determined after gentle shake on
a microplate reader (TECAN, Inc.), at 490 nm. All experiments were
done in triplicate and the average was calculated. The cytotoxic efficacy
of each sample was calculated according to the reported equations [20].
2.1. Materials and methods
The commercial reagents along with their chemical suppliers, ana-
lytical techniques applied for characterization of new compounds, and
some experimental protocols for preparation of the key starting mate-
rials (4-Chloropentoxy-2-hydroxyacetophenone (1) and TPP salts (2a-c)
and salphen ligands (H2salphen1-3) are provided in supplementary
materials.
2.2. Preparation of Pd(II)-salphens
PdCl2 (0.139 g, 1.1 mmol/ 5 ml EtOH) was gradually added to a
solution of salphen ligands (1 mmol/ 10 ml EtOH) containg few drops of
conc HCl under stirring at room temprature. Then, the reaction contents
was heated under reflux for 6 h. Thereafter, the solvent was partially
evaporated under reduced pressure and the solution was left aside al-
lowing the deposition of solid products. The isolated solids were filtered
off followed by washing with a chilly mixture of methanol/ diethyl
ether (1 : 2) and finally diethyl ether to give Pd(II)-salphens as solid
products.
2.5. DNA-binding study
The DNA binding study was performed using commmon experi-
ments [21], including absorption, fluorescence spectral studies and
viscosity measurements.
2.2.1. [Pd(salphen1)]
Pale brown powder (63%)), mp > 300 °C. FTIR (KBr, cm−1): 1642
(s, sh), 1268 (s, sh), 616 (m, sh), 493 (m, sh). ESI MS: m/z 1206.9 and
585.5 ([C70H70ClN2O4P2Pd]+ and [C70H70N2O4P2Pd]2+ corresponding
to [M – Cl–]+ and [M – 2 Cl–]2+, respectively). Anal. Calcd. for
C70H70Cl2N2O4P2Pd (M = 1242.59): C, 67.66; H, 5.68; N, 2.25%;
Found: C, 67.42; H, 5.69; N, 2.22%.
2.5.1. UV–vis spectrophotometric titration
Stock calf thymus DNA (CT-DNA) solutions were prepared in an
aqueous 10 mM Tris−HCl buffer/ 50 mM NaCl at pH 7.2. The molar
absorptivity of CT-DNA at 260 nm (6600 M−1 cm−1) was used for es-
timation of the [CT-DNA] solution [22]. Moreover, the purity of CT-
DNA was checked by recording the relative absorbance of its solution in
a respective buffer at 260 and 280 nm, which was found to be > 1.8
confirming that the DNA sample is free of any proteinaceous con-
taminants. CT-DNA stock solutions were kept at 4 °C and used for
various experiments within four days. UV-Visible spectrophotometric
titration experiments were carried out by changing [CT-DNA]
(0–5 mM) whereas the parent ligand or Pd-complex concentration was
kept constant at 0.06 and 0.1 mM for free ligand and Pd-complex, re-
spectively. All spectra were recorded after the equilibration of titration
mixture for 25 min after each addition.
2.2.2. [Pd(salphen2)]
Brown powder (61%), mp > 300 °C. FTIR (KBr, cm−1): 1644 (vs, sh),
1267(s, sh), 839 (vs, sh), 610 (m, sh), 558 (m, sh), 489 (w, br). ESI MS: m/
z 1316.5 and 585.5 ([C70H70F6N2O4P3Pd]+ and [C70H70N2O4P2Pd]2+
corresponding to [M – PF6–]+ and [M – 2 PF6–]2+, respectively). Anal.
Calcd. for C70H70F12N2O4P4Pd (M = 1461.61): C, 57.52%; H, 4.83; N,
1.92; Found: C, 57.33; H, 4.87; N, 1.78%.
2.2.3. [Pd(salphen3)]
Reddish-brown powder (59%), mp > 300 °C. FTIR (KBr, cm−1): 1643
(vs, sh), 1269 (s, sh), 1056 (vs, sh), 614 (m, sh), 490 (w, br). ESI-MS:
1258.3 and 585.5 ([C70H70BF4N2O4P2Pd]+ and [C70H70N2O4P2Pd]2+
corresponding to [M – BF4–]+ and [M – 2 BF4–]2+, respectively). Anal.
Calcd. for C70H70B2F8N2O4P2Pd (M = 1345.29): C, 62.50; H, 5.24; N,
2.08%; Found: C, 62.28; H, 5.31; N, 1.96%..
2.5.2. Emission spectral studies
The relative bindings of the Pd(II)-salphens with CT-DNA were also
surveyed using emission spectroscopy in a saline phosphate buffer
(pH = 7.4) at room temperature. Each experiment was performed by
titration of a constant concentration (30 μM) of Pd(II)-salphen com-
plexes with growing concentrations of DNA.
2.3. Stability of Pd(II)-salphens under physiological parameters
2.5.3. Viscosity measurements
The stability profile of Pd(II)-salphens under physiological condi-
tions could be investigated using UV–vis spectral survey. Where the
absorption spectra of a complex solution (1 mM) in a phosphate-DMSO
buffer of pH 7.4 were measured through time intervals (t =0 h - 3
weeks) under physiological temperature.
Viscosity measurements were performed using a thermostatically
controlled semimicro-viscometer held in a water bath at 27 °C. The flow
rate for pure buffer (pH = 7.2), free CT-DNA (200 μM) and CT-DNA
blended with various concentrations of salphen ligands or their Pd(II)
complexes (0 to 0.25 mM) were recorded in triplicate for each sample,
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M.Y. Alfaifi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry385(2019)112083
and the mean values of flow times were calculated and used to calculate
the relative viscosities (η/η0) (where η and η0 are the viscosities of CT-
DNA with and without sample, respectively). Then, the cubical root of
the relative viscosities (η/η0)1/3 were plotted versus the molar con-
centration ratio of the sample to CT-DNA (1/R) [23]. Viscosity values
based on the observed flow time of CT-DNA solutions (t) were corrected
by eliminating the buffer (Tris−HCl) interference (t0) using η = (t – t0).
AreOH vibration band coupled with the changes in intensities and/ or
positions of HeC]N and AreO peaks such as their blue -/ red-shifted
by 15
2 cm−1 and 9
1 cm−1, respectively (Fig. 1), are indicative
markers for the deprotonation of AreOH and the participation pheno-
late oxygen with azomethinic nitrogen in building up the coordination
sphere of Pd(II) ion.
3.4. The geometry of Pd(II)-salphens and their stability under physiological
parameters
2.6. BSA-binding experiments
A 100 μM stock solution of bovine serum albumin (BSA) was pre-
pared in the Tris−HCl buffer (50 mM, pH 7.2), and stored in a dark
place at 4 °C for further usage. For UV–Vis absorption study, the ab-
sorption spectra of BSA solution were measured in the absence and
presence of 4 μM of salphen ligands or their Pd(II) complexes. In the
fluorescence quenching study, the emission spectra of diluted BSA so-
lution (1.0 μM) were recorded also in the absence and presence of
variable concentrations of Pd(II)-salphen complexes (0–20 μM)
UV-Vis spectroscopy could be used to investigate the geometry of Pd
(II)-salphens and their stability of Pd(II)-salphens under physiological
conditions, as well. The electronic spectra of Pd(II)-salphens complexes
exhibit a broad peak around 450 nm Fig S6 (ESI†) attributable for the D-
1
1
d electronic transition (particularly, A1g→ B1g), typical for the square-
planar geometry of these complexes [24]. The stability Pd(II)-salphens
under physiological conditions can be investigated by storing 1 mM
solution of Pd(II) complex in a PBS-DMSO buffer (pH = 7.4) and re-
cording its electronic spectra at time intervals (0–3 weeks) at room
temperature. As shown in Fig S6 (ESI†) there are negligible changes in
the UV–vis spectra of Pd(II)-salphen2 complex with time (over 3
weeks), confirming that this complex exhibited high stability under
physiological conditions.
3. Results and discussion
3.1. Synthesis protocol
The parent salphen ligands bearing bis-TPP compartments were
synthesized by the Schiff-base condensation of ionic TPP salts (2a-c)
with 4,5-dimethyl-1,2-phenylenediamine (dmphen) in a 2 : 1 M ratio
using Schlenck technique. Our synthesis protocol starts with the pre-
paration of TPP salts using 2,4-dihydroxyacetophenone (1) as a starting
substrate through diverse consecutive chemical reactions (alkylation,
quarternization and anion exchange) (Scheme 1) following our earlier
work [19]. Thereafter, the reaction of ionic TPP salts with dmphen via
Schiff-base condensation yielded the target salphen ligands
(H2salphen1-3) which were applied as Pd(II) chelators to fabricate TPP-
based Pd(II)-salphens. The isolated salphens and their Pd(II) complexes
were acquired in acceptable yields and structurally characterized using
elemental analysis, and spectral methods (FTIR, UV-Vis, NMR (1H, 13C,
19F, 31P) and ESI-MS, as well).
3.5. Anticancer activity study
All synthesized TPP-based salphens (H2salphen1-3) and their complexes
were surveyed for their capacity to suppress the proliferation of HepG2
cells in comparison to a standard anticancer drug, cisplatin (Cl2H6N2Pt
(CDDP), 300.05 g/mol). All tested compounds were found to be cytotoxic
to HepG2 cells (Fig. 2). All the tested samples have significantly dimin-
ished the cell viability of HepG2 in a concentration- and microstructure-
dependent manner (Fig. 2A). Generally, Pd(II)-salphen1-3 are more cyto-
toxic than the parent salphen ligands (H2salphen1-3). This could be as-
cribed to the square-planar geometry of Pd(II)-complexes enables them to
facilely interact with the nucleotides of DNA and thus barring its re-
plication [25]. Our findings agrees with Quiroga et al. who concluded that
the anticancer effect of Pd(II)-complexes could be attributed to their
ability to suppress the ribonucleotide reductase and induce genetic ma-
terial deterioration [12a]. According to the IC50 values of the tested
3.2. Elemental analyses and mass spectral data
The quantitative elemental analysis of the parent salphens and their
Pd(II)-complexes is in complete consistency with their suggested
structural forms (Experimental section).
samples (Fig. 2B), complexes Pd(II)-salphen1 (IC50 = 11.33
1.12 μM)
0.72 μM) showed promising antic-
and Pd(II)-salphen3 (IC50 = 3.01
ancer efficacy. Interestingly that complex Pd(II)-salphen3 showed higher
anticancer activity than the standard drug, CDDP. Meanwhile, the effect of
counteranions of the cytotoxicity of target compounds revealed that
complex Pd(II)-salphen3 with tetrafluoroborate counteranions was more
cytotoxic than other complexes (Pd(II)-salphen1 with Cl–; Pd(II)-salphen2
with PF6–.
Moreover, the common features of mass spectrometry with positive
electrospray ionization mode (ESI-MS) for new salphen ligands and
their complexes (Figs S3-S5, ESI†) are the notice of dominant peaks
characteristic for the monocationic and dicationic fragments produced
from the sequential exodus of counteranions from the parent com-
pounds (i.e [M – X–]+ and [M – 2 X–]2+, X = Cl−, PF6 and BF4−).
This may be due to the ease of hydrolysis of harmful tetra-
fluoroborate anions, even at room temperature, liberating very toxic
fluoride ions which easily penetrate the cancer cells and inducing their
death [26]. Moreover, previous theoretical calculations revealed that
the small hydrophilic anions tetrafluoroborate (BF4−) and chloride
(Cl−) have highly interacted with H2O molecules than bulky ions such
−
Additional peaks were observed at m/z corresponding to the departure
of one or both TPP compartments leaving the following fragments [M –
+
X– – Ph3P]+, [M – 2 X–– Ph3PH+
]
+ and [M – 2 X–– Ph3P – Ph3PH+
]
.
3.3. FTIR spectra
as hexafluorophosphate (PF6−) [27]. Nevertheless, BF4 can form
−
FTIR spectroscopy offers a preliminary tool to confirm the successful
preparation of salphens and to investigate the coordination mode of the
salphen ligands to Pd(II) ions in their complexes, as well. Where the
stronger H-bonds than did Cl− or PF6−, suggesting that anion-H2O
−
clusters formed by BF4 are more stable than those formed by Cl− or
PF6−. Accordingly, the tetrafluoroborate-based complex can form
stable clusters in aqueous solutions resulting in a higher bioavailability,
enhanced interactions with membrane components, and sturdy cyto-
toxic effect [28]. In conclusion, the remarkable activity of Pd(II)-sal-
phen3 may be attributed to the presence of multiple active cytotoxic
sites in the same molecule (Fig. 3) may be attributed to the existence of
multiple cytotoxic active sites in a single molecule which can exert
diverse synergistic cytotoxicity mechanisms. For instance, Pd(II), BF4–,
salphen, and TPP fragments which may induce oxidative stress,
notice IR peaks at the regions of 3425
14, 1643
2 and
1276
2 cm−1 characteristic for the stretching vibrations of phenolic
hydroxyl (AreOH), azomethinic (HeC]N) and phenolic oxygen
(Ar–O) fragments, respectively, in the spectra of salphen ligands was
considered as strong spectral markers for their successful preparation.
The fluctuations or missing of these spectral markers in the FTIR spectra
of Pd(II) salphen complexes can provide informative spectral markers to
recognize the Pd(II)-salphen binding sites. For instance, loss of the
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M.Y. Alfaifi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry385(2019)112083
Scheme 1. Synthesis protocol for TPP salts (2a-c), H2salphens and their Pd(II)-complexes.
mitochondrial failure, DNA damage, and eventually apoptosis of cancer
cells [29]. More studies on the structure-activity relationship (SAR) and
gene profiling, in future, will provide further insight for the anticancer
mechanism of our complexes against HepG2 cells.
3.6. DNA-binding studies
Generally, metal complexes can bind to DNA by covalent and/or
non-covalent interactions. During covalent interactions, the labile
atoms or groups of complexes may be exchanged by active N-atoms
from the DNA nucleotide, while non-covalent interactions take place
through the intercalative, or electrostatic binding [30].
3.6.1. Electronic absorption titration
The biomolecular interaction of Pd(II)-salphen1-3 with CT-DNA was
preliminarily investigated using a spectrophotometric titration tech-
nique. Electronic absorption titration was used to evaluate the binding
profile and binding constant (Kb) between the complexes and CT-DNA
[31]. The UV–vis spectra of the most anticancer active Pd(II) complex
(Pd(II)-salphen1) in the absence and presence of serial concentrations of
Fig. 1. FTIR spectral segment (1700-1200 cm−1) representing the fluctuations
in the azomethine and phenolate stretches due to their involvement in the
construction of the coordination sphere of Pd(II) ion.
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M.Y. Alfaifi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry385(2019)112083
Fig. 2. (A) Concentration- cytotoxicity correlation and (B) IC50 (μM) of new TPP-based compounds against HepG2 cells.
CT-DNA are given in Fig. 4A. The hyperchromism with or without
bathochromism of transition peaks was used as a spectral marker for
quantization the extent of complex-DNA interaction under physiolo-
gical conditions. Notable that the non-covalent intercalation of bio-
molecules to DNA results in hypochromism due to the π→π stacking
interaction between the aromatic chromophores from biomolecule with
nucleotides in DNA [32]. Upon titration of the Pd(II) complex solution
with CT-DNA, a hyperchromism of 28–53% along with 3–6 nm bath-
ochromism occurred in the transition peak confirming considerable
binding of the complex with CT-DNA through non-covalent interactions
[33]. The value of intrinsic binding constant (Kb), quantifying the
complex–DNA binding affinities, can be obtained by plotting of
{[DNA]/ (εa - εf)} versus [DNA] according to Eq. (1) [34];
concentration of complex, (Aobs/[complex]). The ratio of the slope {1/
(εb – εf)} to intercept {1/ Kb(εb – εf)} in the plot of [DNA]/(εa – εf) vs.
[DNA] (Fig. 4B) gives the Kb values. The calculated Kb values were
found to be 2.39 × 105, 1.99 × 105, and 3.01 × 105 M–1 for Pd(II)-
salphen3, Pd(II)-salphen1 and Pd(II)-salphen2 respectively. Pd(II)-sal-
phen3 has the highest Kb and consequently highest DNA binding affi-
nity. This may be due to the following facts; (i) The liberation of the
strongly hydrophilic anions, [BF4]−, from the Pd(II) complex to form a
dicationic species, [Pd(Me)2salphen(TPP+)2]2+, which can electro-
statically interact with the phosphate moieties distributed on the sur-
face of CT-DNA. (ii) Intramolecular hydrogen bonding among different
HBA/ HBD sites (cf. Fig. 4) of the target compound and nucleobases in
DNA, particularly the tetrafluoroborate group which offers significant
HBA scaffold for nucleobases.
[DNA]
[DNA]
1
b
=
+
(
)
(
)
Kb (
)
f
a
f
b
f
(1)
3.6.2. Fluorescence titration
Where [DNA] is the concentration of DNA in base pairs, εa, εb, and εf
correspond to the extinction coefficients of the apparent, bound and
free complex, respectively. The apparent extinction coefficient, εa, was
estimated as the ratio between the observed absorbance (Aobs) and the
The fluorescence measurements provide further information for the
binding modes of the Pd-salphen complexes with CT-DNA. Where the
fluorescence titration of Pd(II)-salphen3 with different concentrations of
CT- DNA in a Tris−HCL buffer (pH = 7.4) at the emission wavelength
Fig. 3. All possible active cytotoxic sites: H-bonding acceptor (HBA) sites (azomethinic N, tetrafluoroborate F, and phenolic O), active metal, hydrophobic and ionic
sites characteristic for the most potent anticancer compound (Pd(II)-salphen3).
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M.Y. Alfaifi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry385(2019)112083
Fig. 4. (A) Electronic absorption titration of complex (Pd(II)-salphen3), (10 mM) with CT-DNA of serial concentrations 0–28 mM. The arrows show peak hyper-
chromism with increasing [CT-DNA]. (B) A plot of [DNA]/(εa – εf) versus [DNA]. (C) Changes in the fluorescence spectra of Pd(II)-salphen3 (30 μM) in Tris−HCl
buffer (pH = 7.4) in upon addition of CT-DNA (0–4.5 × 10−4 M). Arrow shows the intensity change upon increasing [CT-DNA]. (D) Effect of Pd-salphen complexes
(Pd(II)-salphen1-3) on the viscosity of CT-DNA solution (1/R = [Complex]/ [CT-DNA]).
of this complex (459 nm) resulted in a significant enhancement in the
fluorescence intensity by a maximum of 16 folds (Fig. 4C) indicating
that complex Pd(II)-salphen3 strongly interacts with CT-DNA via in-
tercalative mode [35]. Thus our new Pd-salphen complexes may offer
promising CT-DNA fluorescence probes in future use.
3.7. BSA interaction studies
Interaction of the most abundant protein, bovine serum albumin
(BSA), with complexes has attracted huge interest because of its
structural similarity with the human serum albumin (HSA) [36]. To
understand the mode of interaction between pharmacological agent and
BSA, UV–vis absorption and fluorescence quenching studies were per-
formed.
3.6.3. Viscosity measurements
Ultrasensitivity of the relative viscosity (η/η0) of DNA (where η and
η0 are the specific viscosities of DNA in the presence and absence of
complexes) toward any alterations in the DNA double helix as a result
of interaction with foreign molecules, maks it a powerful tool to give an
insightful view regarding the degree and mode of interaction of dif-
ferent substance with DNA. The effects of Pd-salphen complexes on the
relative viscosity of CT-DNA solutions are depicted in Fig. 4D. It is
noted that the relative viscosity of CT-DNA solutions steadily increases
upon addition of increasing amounts of complexes, as a consequence of
elongation of the CT-DNA double helix. This lengthening of DNA helix
is due to an intercalative interaction of the complex with DNA strand
and, as a result, the gap between DNA nucleobases increases [22],
whereas a groove‐bounded molecule causes lesser or no alteration in
DNA length. The viscosity values changes are fully consistent with the
trends of complex-DNA binding affinities as investigated from absorp-
tion and emission studies. For example, the ability of complexes to
elongate DNA's helix and consequently increases the CT-DNA's viscosity
follows the trend of Pd(II)-salphen3 > Pd(II)-salphen1 > Pd(II)-sal-
phen2.
3.7.1. UV–vis spectrophotometric titrations
The BSA-binding activity of the TPP-based Pd-salphen complexes
(Pd(II)-salphen1-3) was preliminarily investigated by means of UV–Vis
absorption spectroscopy. As shown in Fig. 5A, the UV–Vis spectrum of
pure BSA exhibited characteristic maxima at ˜ 280 nm ascribed to the
microenvironment of aromatic amino acid (AAA) segment in the BSA
backbone (Trp, Tyr and Phe) [37]. As the absorption maximum of BSA
is significantly affected by the ambient microenvironment and the
changes in BSA conformation, the UV–Vis spectrum of BSA solution has
to be recorded in the absence and presence of target compounds to
check the reactivity of our compounds toward BSA. The UV–Vis spectral
features of BSA have remarkably fluctuated upon addition our com-
pounds as noticed in Fig. 5A, where the intensity of absorption maxima
at 280 nm was boosted up. This hyperchromic effect provided strong
evidence for alteration of ambient environment for AAA and the ex-
istence of a static quenching mechanism as a result of the formation of a
non-fluorescent ground-state complex between the Pd(II)salphens and
BSA and thus the fluorescence intensity of BSA was significantly
6

M.Y. Alfaifi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry385(2019)112083
Fig. 5. (A) UV–Vis absorption spectra of BSA (10 × 10–6 M) in the absence and presence of (4 × 10–6 M) Pd(II)-salphen1-3. (B) Quenching fluorescence plateaus of
BSA in the absence and presence of the most potent Pd(II) complex (Pd(II)-salphen3). [BSA] = 10–6 M and [Complex] = 0–20 × 10–6 M. (C, D) Stern–Volmer and
Scatchard plot graphs of the fluorescence titrations of the Pd(II) complexes (Pd(II)-salphen1-3) with BSA.
diminished upon addition of increasing amount Pd(II)-salphen due the
formulation of higher population of Pd(II)salphen-BSA pairs allowing
the Pd(II)salphen to be close enough to BSA to instantaneously quench
its excited state as reported in earlier similar work [38,39,40].
The Pd(II)salphen-BSA binding constant (Kb) can be calculated
based on the UV–vis absorption titration of Pd(II)-salphen complexes
(1.5 × 10−4 M) with increasing concentrations of BSA at 280 nm (Fig.
S7, ESI†), according to the following equation [24].
3.7.2. Fluorescence quenching titration
The fluorescence features of BSA arises from intrinsic characteristics
of the AAA residues (Trp, Tyr and Phe with relative fluorescence in-
tensity of 100:9:0.5). Thus, the fluorescence performance of BSA as-
cribed mainly to the presence of two Trp fragments, namely Trp-134
belonged to the surface of sub-domain IB and Trp-213 centred in the
hydrophobic pocket of sub-domain IIA [41]. Thus, the fluctuation of the
fluorescence profile of BSA arises primarily from the interaction of its
Trp components as a consequence of BSA substrate binding, subunit
association, conformational changes, or denaturation. Therefore, the
fluorescence behaviour of BSA can provide significant information
about the interaction between the Pd(II) complexes and BSA (binding
mechanism, binding constants and sites). The changes in the fluores-
cence behaviour of BSA upon addition of different concentrations of the
Pd(II) complexes to its solution are depicted in Fig. 5B and Figs. S8, S9
(ESI†). As shown in these Figs., the addition of Pd(II)-salphen1-3 to BSA
solution is resulted in synergistic effects of fluorescence quenching at
340 nm by 83.5, 74.9 and 85.2% along with the hypsochromic shifts of
2, 2 and 3 nm for Pd(II)-salphen1-3, respectively. These data confirm the
interaction of BSA with the Pd(II) complexes to form new BSA-quencher
ground-state complex inducing a conformational alteration in the pro-
tein structure and changes in the Trp microenvironment, as well [42].
To get a quantitative insight into the quenching fluorescence perfor-
mance, the quenching constant (Kq) and equilibrium binding constant
(KF) were estimated based upon the following equations (Stern–Volmer
(Eq. 3), Eq. 4),.
A0
G
G
1
=
+
x
[BSA]
Kb
(A
A0)
(2)
TC
G
TC
G
Where A0 and A are the absorbances of Pd(II)-salphen in the absence
and presence of BSA, εG and εTC are the absorption coefficients of Pd(II)-
salphen and its transition complex with BSA, respectively. The ratio of
the slope to intercept of a A0/(A- A0) vs. 1/ [BSA] graph yielded the Kb
values which were collected in Table 1.
Table 1
Quenching constant (Kq), BSA binding constants (Kb/ KF)‡, and number of
binding sites (n) for Pd(II) complexes (Pd(II)-salphen1-3).
Complex
Kq [M−1
]
Kb [M−1
]
KF [M−1
]
n
Pd(II)-salphen1
Pd(II)-salphen2
Pd(II)-salphen3
1.97 × 105
1.68 × 105
2.46 × 105
1.23 × 106
1.11 × 106
1.65 × 106
2.31 × 106
2.01 × 106
2.95 × 106
1.15
1.06
1.19
F0/ F = 1 + KSV [Q] = 1 + τ0 Kq [Q]
(3)
‡
Kb and KF are Pd(II)salphen-BSA binding constants calculated based on the
UV–vis absorption titration and fluorescence quenching study, respectively.
7

M.Y. Alfaifi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry385(2019)112083
log [(F0 – F)/ F] = log KF + n log [Q]
(4)
Acknowledgement
where F and F0 are the fluorescence intensities in the presence and
absence of the Pd(II) complex, respectively, KSV is the dynamic
Stern–Volmer quenching constant, τ0 is the average lifetime of BSA in
the absence of any quencher and is generally equal to 2 × 10−8 [s]
[41], [Q] is the concentration of quencher and n is the number of
binding sites. The Kq values were derived from the graph between F0/F
and [Q], while, KF values were from plotting log[(F0 – F)/F] against log
[Q] (Fig. 5C). A good linear correlation between the relative fluores-
cence intensities (F0/F) and quencher concentration [Q] (Fig. 6D) re-
vealed a single mode of quenching mechanism [41], a static quenching
mechanism. Moreover, the values of KF and Kq (Table 1) are within the
optimum range (104–106 M−1) [43] revealing the enhanced binding
ability of our complexes to BSA. Among the tested Pd(II) complexes, Pd
(II)-salphen3 exhibited the highest affinity to interact with BSA. Even-
tually, the obtained n values are ≈ 1.0, indicating that there is only a
single binding site available for BSA- Pd(II) complexes interaction.
Noteworthy, the UV–vis absorption spectrum of BSA fluorophore
offers a more simple and facile tool to explore the mode of fluorescence
quenching. A static quenching usually leads to perturbation of the
fluorophore and as a result changes in its UV–vis spectrum [36],
however, in case of dynamic quenching a perceivable change in UV–vis
spectrum of the fluorophore does not prospect. Thus the results of
fluorescence quenching and UV − Vis titrations experiments are in
good agreement.
The authors extend their appreciation to the Dean of Scientific
Research at King Khaled University for supporting this work through
general research project fund under grant number (G.R.P-118-39).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2019.
112083.
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and show no conflict of interest.
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