DNA-binding, spectroscopic and antimicrobial studies of palladium(II) complexes containing 2,2′-bipyridine and 1-phenylpiperazine

Article abstract of DOI:10.1016/j.saa.2012.07.012

With the purpose of evaluating the ability of Pd(II) complex to interact with DNA molecule as the main biological target, two new complexes [Pd(bpy)(OH₂)₂] (1) and [Pd(Phenpip)(OH₂) ₂] (2), where (bpy = 2,2′-bip

Full text of DOI:10.1016/j.saa.2012.07.012

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 586–593
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
DNA-binding, spectroscopic and antimicrobial studies of palladium(II)
complexes containing 2,20-bipyridine and 1-phenylpiperazine
Azza A. Shoukry a, Mervat S. Mohamed b,
⇑
a Department of Chemistry, Inorganic Chemistry Speciality, Faculty of Science, Cairo University, Egypt
b Department of Chemistry, Biochemistry Speciality, Faculty of Science, Cairo University, Egypt
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
[Pd(bpy)(OH2] (1) and [Pd(Phenpip)(OH2) (2) where, (bpy = 2,20-bipyridine; Phenpip = 1-phenylpiper-
azine) have been synthesized and characterized on the basis of elemental analysis. The interaction of
these complexes with calf thymus DNA was extensively investigated by a variety of techniques, viz. spec-
troscopic, electrochemical, ethanol precipitation and gel electrophoresis. All studies showed that both
complexes presumably intercalate in DNA by electrostatic and/or groove binding mode. The calculated
binding strength (Kb) of the two complexes to CT-DNA was estimated to be of lower magnitude than that
of the classical intercalator EB (Ethidium bromide) (Kb = 1.23( 0.07) ꢀ 105 Mꢁ1) supporting the electro-
static and/or groove binding mode. The antimicrobial tests showed that both complexes exhibited anti-
microbial properties, and they were found to be more active against Gram-negative than Gram-positive
bacteria.
"
Two new complexes [Pd(bpy)(OH2]
(1) and [Pd(Phenpip)(OH2)] (2) have
been synthesized.
The binding properties of the two
complexes with CT-DNA were
investigated.
The intrinsic binding constants Kb
have been calculated.
The mode of binding is an
electrostatic and/or groove one.
Our complexes may act as model
anticancer agents.
"
"
"
"
30
a
20
10
b
0
-10
-20
-30
-40
-50
Complex 1 alone (100 uM)
Complex 1 (100 uM) + DNA (37 uM)
-550
-500
-450
-350
-300
-250
-200
-150
Potent-i4a0l0/mV vs Ag/AgCl (KCl)
a r t i c l e i n f o
a b s t r a c t
Article history:
With the purpose of evaluating the ability of Pd(II) complex to interact with DNA molecule as the main
biological target, two new complexes [Pd(bpy)(OH2)2] (1) and [Pd(Phenpip)(OH2)2] (2), where (bpy = 2,20-
bipyridine; Phenpip = 1-phenylpiperazine), have been synthesized and the binding properties of these
complexes with CT-DNA were investigated. The intrinsic binding constants (Kb) calculated from UV–
Vis absorption studies were 3.78 ꢀ 103 Mꢁ1 and 4.14 ꢀ 103 Mꢁ1 for complexes 1 and 2 respectively. Ther-
Received 23 April 2012
Received in revised form 13 June 2012
Accepted 5 July 2012
Available online 16 July 2012
mal denaturation has been systematically studied by spectrophotometric method and the calculated
DTm
Keywords:
was nearly 5 °C for each complex. All the results suggest an electrostatic and/or groove binding mode for
the interaction between the complexes and CT-DNA. The redox behavior of the two complexes in the
absence and in the presence of calf thymus DNA has been investigated by cyclic voltammetry. The cyclic
Pd(II) amine complexes
Calf thymus DNA
UV–Vis spectroscopy
Gel electrophoreses
Cyclic voltammetry
Antimicrobial activity
voltammogram exhibits one quasi-reversible redox wave. The change in E1/2, DEp and Ipc/Ipa supports that
the two complexes exhibit strong binding to calf thymus DNA. Further insight into the binding of com-
plexes with CT-DNA has been made by gel electrophoresis, where the binding of complexes is confirmed
through decreasing the intensity of DNA bands. The two complexes have been screened for their antimi-
crobial activities using the disc diffusion method against some selected Gram-positive and Gram-negative
⇑
Corresponding author. Tel.: +20 2 100 1964580; fax: +20 2 5727213.
E-mail addresses: mervat_sayed@yahoo.com, mervat@sci.cu.edu.eg (M.S. Mohamed).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.07.012

A.A. Shoukry, M.S. Mohamed / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 586–593
587
bacteria. The activity data showed that both complexes were more active against Gram-negative than
Gram-positive bacteria. It may be concluded that the antimicrobial activity of the compounds is related
to cell wall structure of bacteria.
Ó 2012 Elsevier B.V. All rights reserved.
Introduction
intramolecular hydrogen bonding [23,24] with the Pt-DNA adduct,
favoring interaction with DNA and (2) the piperazine ring may un-
dergo stacking interactions with the sugar group of DNA, again
The interaction and reaction of metal complexes with DNA has
long been the subject of intense investigation in relation to the
development of new reagents for biotechnology and medicine
[1]. A number of metal complexes have been used as probes of
DNA structure in solution, as agents for mediation of strand scis-
sion of duplex DNA and as chemotherapeutic agents [2–4]. The
landmark discovery of cisplatin by Rosenberg in 1965 heralded a
new era of anticancer drug research based on metallo-pharmaceu-
ticals [5]. To date, cisplatin and its analogs are some of the most
effective chemotherapeutic agents in clinical use as the first line
of treatment in testicular and ovarian cancers. Unfortunately, they
have several major drawbacks. Common problems include cumula-
tive toxicities of nephrotoxicity and ototoxicity [6–9]. In addition
to the serious side effects, the therapeutic efficacy is also limited
by inherent or treatment-induced resistant tumor cells. These
drawbacks have provided the motivation for alternative chemo-
therapeutic strategies. Naturally, every therapeutic drug interacts
with its bio-targets to remediate undesirable disease condition
associated with various side effects and drug resistances. Enhanc-
ing the quality of life of patients is generally a prerequisite for
proper chemotherapy. Therefore, there is particular interest in
the search of anticancer agents that can minimize side effects
and enhance the therapeutic effects. Mechanistic investigations
of the mechanism of action of Pt(II) anticancer drugs, their Pd(II)
analogues are suitable model compounds since they exhibit ca.
104–105 times higher reactivities, whereas their structural and
equilibrium behavior are very similar [10].The similarity between
the coordination chemistry of platinum(II) and palladium(II) com-
pounds supports the theory that palladium complexes can act suc-
cessfully as antitumor drugs. Several studies demonstrate that
palladium derivates exhibit a noticeable cytotoxic activity, simi-
larly to standard platinum-based drugs (e.g. cisplatin, carboplatin
and oxaliplatin) used as reference, and show fewer side effects rel-
ative to other heavy metal anticancer compounds [11]. Moreover,
palladium(II) complexes can serve as good models for the under-
standing of more inert platinum(II) anti-cancer drugs [12]. Also,
it has been suggested by Saddler et al. [13] that the palladium com-
plexes may be useful for the treatment of tumors of the gastroin-
testinal region where cisplatin fails.
favoring interaction with DNA. The latter effect is similar to that
reported for carboplatin, where the stacking interaction between
the cyclobutane ring and the sugar group is part of the increased
antitumor activity [27]. Also, piperazine is an important pharmic
intermediate, its rings are capable of resting in the minor groove
of GC base pairs [28,29].
In continuation of our previous work on equilibria of complex
formation reactions of cis-(diamine) palladium(II) complexes with
DNA, the major target in chemotherapy of tumors [30], and amino
acids, peptides, dicarboxylic acids and esters [31,32], in this project
we study the mode of binding of the two complexes 1 and 2 with
Calf thymus DNA. Their interaction studies are attempted by
UV–Vis spectroscopy, electrochemical technique and gel electro-
phoresis as well as ethanol precipitation. The two complexes have
been screened for their antimicrobial activities against some
selected Gram-positive and Gram-negative bacteria.
Experimental
Materials and reagents
Palladium(II) chloride was purchased from Acros organics. The
ligands 2,20-bipyridine and 1-phenylpiperazine were from Aldrich
and Fluka. Calf thymus DNA was provided from Sigma. All solvents
used were of the analar grade. All DNA-binding experiments were
carried out in Tris–HCl buffer solution (50 mM NaCl, 5 mM
Tris–HCl, pH 7.1). Tris–HCl buffer was prepared using deionized
triple distilled water. Solutions of CT-DNA in buffer gave a ratio of
UV–Vis absorbance of 1.8–1.9:1 at 260 and 280 nm, indicating that
the DNA was sufficiently free of protein [33]. The concentration of
DNA was determined spectrophotometrically
(e
260 = 6600 Mꢁ1
cmꢁ1) [34]. Stock solution of DNA was stored at ꢁ20 °C. Concen-
trated stock solution of the palladium complexes (1.0 ꢀ 10ꢁ3 M)
was prepared by dissolving an appropriate amount of the complex
into 50 ml of deionized doubly distilled water and diluted suitably
with Tris–HCl buffer to the required concentrations for all the
experiments.
Apparatus and measuring techniques
In this paper we have synthesized two model Pd(II) amine com-
plexes, [Pd(bpy)(OH2)2] (1) and [Pd(phenpip)(OH2)2] (2), where
bpy: is 2,2’-bipyridine and phenpip: is 1-phenylpiperazine. In gen-
eral, the pyridyl compounds operate as bidentate chelating ligands
for metal binding. The UV–Vis absorption properties of this class of
bipyridyl metal complexes have been utilized to monitor the inter-
action processes with DNA. In addition, the molecular nature of
bipyridyl metal complex provides a square-planar geometry to
intercalate with DNA. These evidences suggest that DNA does
represent the potent target for many bipyridyl metal complexes.
Recent progresses have emphasized the antiproliferative effects
and molecular mechanisms from diverse metals, gold(III) [14,15],
copper(II/I) [16,17], palladium(II) [18,19], rhodium(III) [20],
ruthenium(II) [21,22], zinc(II) [23], and platinum(II) [24,25] with
bipyridyl ligands.
The microchemical analysis of the separated solid complexes
for C, H and N was performed in the microanalytical center, Cairo
University. The analyses were performed twice to check the accu-
racy of the analytical data. Absorption titration experiments were
made using TB-85-thermobath Shimadzu model UV spectropho-
tometer. Cyclic voltammetric measurements were made on an
EG&G PAR model 253 VersaStat potentiostat/galvanostat with
electrochemical analysis software 273 using a three electrode
set-up comprising a glassy carbon working, platinum wire auxil-
iary and a saturated calomel reference electrode (SCE).
Synthesis of the complexes
The study of Phenyl piperazine complexes were performed be-
cause (1) piperazine and many of its derivatives are notable succes-
full drugs, piperazine has O6–Nꢂ ꢂ ꢂH [26] and/or phosphate-Nꢂ ꢂ ꢂH
[Pd(bpy)Cl2] and [Pd(phenpip)Cl2] were prepared by heating
PdCl2 (0.177 g, 1.0 mM) in 40 ml H2O and KCl (0.149 g, 2.0 mM)
in 10 mL water to 70 °C with stirring. The clear solution of

588
A.A. Shoukry, M.S. Mohamed / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 586–593
[PdCl4]2ꢁ was filtered and the ligands (0.156 g, 1.0 mM) of 2,20-
bipyridine and (0.162 g, 1.0 mM) of 1-phenyl piperazine were
dissolved in 10 ml H2O and added dropwise to the stirred [PdCl4]2ꢁ
solution. The pH value was adjusted to 2–3 by the addition of HCl
and/or NaOH. A yellowish-brown precipitate of [Pd(bpy)Cl2] or
[Pd(phenpip)Cl2] is formed. The precipitates were stirred for an
additional 30 min at 50 °C. After filtering off the precipitates, they
were thoroughly washed with H2O, ethanol, and diethyl ether. Yel-
low powders were obtained. For the [Pd(bpy)Cl2], C10H8N2PdCl2:
Calc. (%): C, 36.0; H, 2.40; N, 8.40. Found: C, 35.78; H, 2.02; N,
8.1% and for [Pd(phenpip)Cl2], C10H14N2PdCl2: Calc.: C, 35.3; H,
4.12; N, 8.2%. Found: C, 35.1; H, 4.02; N, 8.1%. Aqueous solutions
of the diaqua analogues of [Pd(bpy)Cl2] or [Pd(phenpip)Cl2]
complexes were prepared in situ by the addition of slightly less
than two mole equivalents of AgNO3 to a solution of a known
amount of the dichloro complexes and stirred over night. The
white precipitate of AgCl that formed was filtered off using a
0.1 lm pore membrane filter. Great care was taken to ensure that
the resulting solution was free of Ag+ ion and that the dichloro
complexes have been converted completely into the diaqua
species.
Ethanol precipitation
Palladium(II) complexes (175 lM of [Pd(bpy)(OH2)2] and
247
lM
of [Pd(phenpip)(OH2)2] were interacted with DNA
(50
l
m) in 5–10 mL of the above-mentioned Tris–HCl buffer of
pH 7.1. To this interacted solution, an excess of absolute ethanol
was added. The precipitated DNA was separated and washed with
alcohol. This precipitate was redissolved in the same buffer and the
solution was monitored spectrophotometrically for DNA at 258 nm
and Pd(II) complexes at their kmax (nm).
DNA gel electrophoresis
The binding of palladium complexes with CT-DNA was moni-
tored using agarose gel electrophoresis. The reaction mixture con-
taining (16
(5, 10, 15, 20 and 25
90 min at 37 °C followed by the addition of (3
l
M) CT-DNA and different concentrations of complexes
M) in tris-buffer solution was incubated for
l) loading buffer
l
l
containing (bromophenol blue 0.25%, SDS 20%, 0.5 M EDTA
(PH:8) and glycerol 30%). Electrophoresis was performed at
(50 V) for 2 h. in TBE buffer (10.8% tris, (Tris (hydroxymethyl) ami-
nomethane), 5.5% boric acid, (0.5 M) EDTA (pH 8)) using 0.8% aga-
rose gel containing (2 ll) 1.0 mg/ml ethidium bromide. Bands were
visualized using UV light and photographed. The binding ability
2+
2+
N
OH2
Pd
was observed from the intensity of bands.
OH2
N
N
N
Cyclic voltammetric measurements
Pd
All the electrochemical measurements were carried out at
room temperature in a 15 ml electrolytic cell by using (5 mM
Tris–HCl/50 mM NaCl buffer (pH 7.1) as the supporting electrolyte.
The GCE surface was freshly polished to a mirror prior to each
experiment with 0.05 mm a-Al2O3-paste and then cleaned in
water. The working electrode was cleaned after every electrochem-
ical assay. The voltammogram of 15 ml of the solution of the
OH2
H2O
Complex 1
Complex 2
complexes, [complex] = 100 lM, was recorded in absence of DNA.
Absorption titration
The procedure was then repeated for systems of 15 ml of a mixture
containing constant concentration of the complexes ([com-
Absorption titration experiments were carried out by varying
the DNA concentration in the range of (0, 29.2, 43.6, 59.36, 77.28,
plex] = 100
complex 1, 100
80 and 125 M) in the molar ratios (1:0.4, 1:0.8 and 1:1.25) respec-
tively. For Complex 2 the concentration of the DNA used was
lM) and varying the concentration of DNA. For
lM of the complex was mixed with ([DNA] = 37,
89.8, 96 and 128.8
tion constant at (20
l
l
M) and maintaining the complex concentra-
M). Upon measuring the absorption spectra,
l
equal amount of DNA was added to both the palladium complex
solution and the reference solution to eliminate the absorbance
of DNA itself. The reference solution was the corresponding buffer
solution. Absorbance values were recorded after each successive
addition of DNA solution and equilibration (ca. 10 min). The sam-
ple solution was scanned in the range of 200–400 nm. Each sample
was measured three times and an average value was calculated.
The absorption data were analyzed for an evaluation of the intrin-
sic binding constant, Kb, of the complexes with CT-DNA.
Thermal denaturation experiments were carried out by moni-
toring the change in the absorption of CT-DNA at 260 nm at various
temperatures to evaluate the melting temperature (Tm). Whereas,
Tm is defined as the temperature at which 50% of double stranded
DNA becomes single stranded. Tm was measured in absence and in
presence of the complexes in Tris–HCl buffer pH 7.1 containing a
([DNA] = 100 and 150 lM); in the molar ratios (1:1 and 1:1.5).
Antimicrobial activity
The Pd(II) diaqua complexes 1 and 2 were evaluated for their
antibacterial activity against Bacillus subtilis and Staphylococcus
aureus (as Gram-positive bacteria) and Escherichia coli and Neisseria
gonorrhoeae (as Gram-negative bacteria) using the disc diffusion
technique [35] as described in British Pharmacopoeia (2000). Paper
discs of Whatman filter paper (No. 42) of uniform diameter (2 cm)
were sterilized in an autoclave. The paper discs, soaked in the de-
sired concentration of the complex solutions, were placed asepti-
cally in the petri dishes containing nutrient agar media (agar
15 g + beef extract 3 g + peptone 5 g) seeded with S. aureus and B.
subtilis bacteria separately. The petri dishes were incubated at
37 °C and the inhibition zones were recorded after 24 h of incuba-
tion. The antibacterial activities are calculated as a mean of three
replicates. The antibacterial activity of a common standard antibi-
otic Tetracycline was also recorded using the same protocol as
above and at the same concentration and solvent. The antibacterial
results of the compounds were compared with the standard and
the% activity index for the complexes was calculated by using
the formula:
mixture of 60 lM CT-DNA and 20 lM of the complex. The mixture
was stirred continuously and the temperature was elevated gradu-
ally from 30 to 90 °C with a reading of absorbance taken every 5 °C.
All experiments were made using TB-85-thermobath Shimadzu
model UV spectrophotometer equipped with cell-temperature
controller. The denaturation temperature (Tm) was taken as the
midpoint of the hyperchromic transition. All measurements of Tm
were repeated three times and the data presented are the average
values.

A.A. Shoukry, M.S. Mohamed / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 586–593
589
Zone of inhibition by test compoundðdiameterÞ
are capable of resting in the minor groove of GC base pairs
[28,29,38]. As far as the pd(II) is concerned, it is likely that the li-
gands facilitate the formation of the van der Waal’s contacts within
the walls of groove or hydrogen bonds in DNA grooves when inter-
act with DNA in Tris–HCl buffer.
% Activity index ¼
Zone of inhibition by standardðdiameterÞ
ꢀ100
In order to further illustrate the binding strength of the palla-
dium(II) complexes with CT-DNA, the intrinsic binding constant
Kb was determined from the spectral titration data. It can be calcu-
lated by monitoring the changes in absorbance at the correspond-
ing kmax with increasing concentrations of CT-DNA, using the
following Eq. (1) [39]:
Results and discussion
DNA-binding studies
Absorbance titration experiments
One of the most common techniques in DNA-binding studies of
metal complexes is electronic absorption spectroscopy. The magni-
tude of spectral perturbation is an evidence for DNA-binding [36].
The absorption titrations were carried out by using a fixed amount
of each metal complex (20
DNA in the range from (0–160
the corresponding buffer solution. While measuring the absorption
spectra, equal amount of DNA was added to both the compound
solution and the reference solution to eliminate the absorbance
of DNA itself. Each sample solution was scanned in the range of
200–400 nm. The absorption spectra of the title complexes with
increasing concentrations of CT-DNA are given in Figs. 1 and 2
respectively. On increasing the concentration of CT-DNA, the
absorption bands of the complexes at 253 nm for complex 1 and
at 251 nm for complex 2 were affected, resulting in hyperchromic-
ity and slight red shift. The absorption intensity is increased due to
the fact that the purine and pyrimidine DNA-bases are exposed be-
cause of binding of the complexes to DNA.
½DNAꢃ
½DNAꢃ
1
¼
þ
ð1Þ
ð
ea
ꢁ
ef
Þ
ð
eb
ꢁ
ef
Þ
Kbðeb
ꢁ
ef
Þ
Where [DNA] is the concentration of DNA in base pairs, ef
,
e
a, and eb
l
M) with increasing concentrations of
M). The reference solution was
correspond to the extinction coefficient, respectively, for the free
palladium diaqua complexes (II), for each addition of DNA to the
palladium(II) complex and for the palladium(II) complex in fully
l
bound form.
½DNAꢃ
A plot of ð
Þ versus [DNA] gives Kb, insets in Figs. 1 and 2, as the
eaꢁef
½DNAꢃ
ratio of the slope to the intercept. From the ð
Þ versus [DNA] plots,
ea ef
the intrinsic binding constant Kb for complexꢁ1 was 3.78 ꢀ 103 Mꢁ1
(R2 = 0.98 for eight .points) and that for complex
2 was
4.15 ꢀ 103 Mꢁ1 (R2 = 0.99 for eight points). The higher Kb value ob-
tained for complex 2 may plausibly be due to more base pairs avail-
able for binding than in complex 1. The calculated Kb values for
complexes 1 and 2 are of lower magnitude than that of the classical
intercalator EB (Ethidium bromide) (Kb = 1.23 ( 0.07) ꢀ 105 Mꢁ1
)
[40] and both reveal a strong binding to CT-DNA.
Metal complexes may bind to DNA with several different bind-
ing modes, namely intercalation or non intercalation, such as
groove binding and binding to the phosphate group [37]. In the
present study it is assumed that the positively charged diaqua
complexes 1 and 2 would electrostatically interact with the nega-
tively charged phosphate backbone at the periphery of the double
helix CT-DNA. This electrostatic binding mode of the two com-
plexes to CT-DNA is also evidenced by the strong hyperchromism
obtained for both complexes suggesting tight binding to CT-DNA
and stabilization. The observed strong hyperchromism is most pro-
Thermal denaturation experiments
The consequences of adduct formation on the stability of the
double helix in CT-DNA were assayed by recording the DNA melt-
ing profiles. Thermal behavior of DNA in the presence of complexes
can give insight into their conformational changes when tempera-
ture is raised, and offers information about the interaction strength
of complexes with DNA.
Previous studies have revealed that when a cationic species
interact with double helix the stability increases and so does the
DNA melting temperature. In general, groove binding or electro-
static binding along the phosphate backbone of DNA gives rise to
nounced in the p–
p⁄ transition, indicating that the complexes are
actively in associating with the DNA. This agrees with the previous
study that mentioned that piperazine rings and bipyridine rings
1
0.8
0.6
0.4
0.2
30
Kb = 3.16 x 104 M-1
25
R2 = 0.9866
20
15
10
5
[10]
[1]
0
0
50
100
M
150
[10]
[DNA]x10-6
[1]
0
230.20,
250.20,
270.20,
290.20,
310.20,
330.20,
Wavelength, nm
Fig. 1. Absorption spectra of [Pd(bpy)(OH2)2](1) in Tris–HCl buffer upon addition of CT-DNA. [complex] = 20
[4],(60.3)[5],(79.8) [6], (82.9) [7],(89.8) [8], (96) [9],(128.9) [10] M. Arrow shows the absorbance changing upon the increase of DNA concentration. Inst: Pot of [DNA]/
f) versus [DNA] for the titration of CT-DNA with the complex.
lM, [DNA] = (0) [1], (43.6) [2],(45.6) [3],(59.3)
l
(ea–e

590
A.A. Shoukry, M.S. Mohamed / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 586–593
0.8
60
Kb = 4.25 x 105M-1
R2 = 0.9992
48
36
24
12
0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
[7]
[1]
0
20 40 60 80 10 12 14
[DNA]x10-6
M
0
0
0
[1]
220.00,
240.00,
260.00,
280.00,
300.00,
320.00,
340.00,
Wavelength
Fig. 2. Absorption spectra of [Pd(phenpip)(OH2)2]2+(2) in Tris–HCl buffer upon addition of CT-DNA. [complex] = 20
l
M, [DNA] = (0) [1], (29.2) [2],(43.6) [3],(59.3) [4],(59.3)
[5],(77.8) [6], 160 [7]
with the complex.
l
M. Arrow shows the absorbance changing upon the increase of DNA concentration. Inst: plot of [DNA]/(ea f) versus [DNA] for the titration of CT-DNA
– e
only a small change in thermal denaturation temperature, while
intercalation binding mode leads to a significant rise in thermal
denaturation temperature of DNA due to the stabilization of the
Watson–Crick base paired duplex [41,42]. The DNA melting studies
for the present complexes show a moderate positive shift in the
such as UV spectra [49]. The redox behavior of the two studied
complexes 1 and 2 in absence and presence of CT-DNA was studied
at room temperature within potential range of ꢁ550 to ꢁ150 mV
for complex 1 and ꢁ400 to 400 mV for complex 2 at a scan rate
of 200 mVsꢁ1. The results indicate that, the two complexes are
redox active and show quasi-reversible cyclic voltammetric
response. The cyclic voltammogram of complex 1 exhibits a
quasi-reversible redox wave with Epc and Epa values of ꢁ344 and
ꢁ454 mV, respectively. The ratio of cathodic to anodic peak cur-
melting temperature (DTm) of nearly 5 °C for each complex,
Fig. 3. Here
D
Tm ¼ Tm ꢁ T0 , where, Tm and T0 refer to the melting
m
m
temperature of DNA in presence and absence of complexes respec-
tively. The extent of
DTm is in the interface between the value in-
duced by electrostatic and intercalative binding and it is relatively
lower as compared to those observed for common organic interca-
lators such as ethidium (13 °C) [43] and some derivatives of por-
phyrins (ꢄ15 °C) [44–46]. This indicates primarily electrostatic
and/or groove binding nature of the complexes in preference to
an intercalative mode of binding to DNA [47], which is in accor-
dance with the results obtained from the absorption titration
experiments and from the calculation of Kb.
rents Ipc/Ipa was 0.413. The formal electrode potentials E1/2, DEp
(difference in cathodic Epc and anodic Epa peak potentials) were
ꢁ399.4 and 109.3 mV respectively. At constant temperature, the
addition of CT-DNA resulted in the shift in E1/2 = ꢁ407 mV and
D
Ep = 106 mV, Fig. 4, respectively. The ratio of Ipc/Ipa is 0.25 for
CT-DNA bound metal complex 1. The shift in potentials and de-
crease in current ratio suggest the binding of complex 1 to CT-
DNA [50]. The cyclic voltammogram of complex 2 at scan rate
200 mVsꢁ1 features a quasi-reversible redox wave with E1/2
, DEp
Cyclic voltammetry
Cyclic voltammetry is one of the most important electroanalyt-
ical techniques employed for interaction of metal complexes with
biomolecules due to the similarity between various redox chemical
and biological processes [48]. It is extremely useful in probing the
nature and mode of DNA binding of metal complexes and it pro-
vides a useful complement to the above method of investigation
30
a
20
b
10
0
-10
-20
-30
0.9
CT-DNA alone
0.8
CT-DNA + Complex 1
0.7
CT-DNA + Complex 2
0.6
0.5
0.4
-40
-50
Complex 1 alone (100 uM)
Complex 1 (100 uM) + DNA (37 uM)
0.3
25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
-550
-500
-450
-400
-350
-300
-250
-200
-150
Temperature / oC
Potential /mV vs Ag/AgCl (KCl)
Fig. 3. Melting curves of CT-DNA in Tris–HCl buffer in the absence and presence of
complexes, [DNA] = 60 uM, [complex] = 20 uM, showing an increase in Tm due to
binding.
Fig. 4. Cyclic voltammograms of complex (1) in Tris–HCl buffer in the absence (a)
and presence (b) of CT-DNA. M; [DNA]: (a) 0, (b)
= 200 mV sꢁ1, [complex] = 100
37 M.
m
l
l

A.A. Shoukry, M.S. Mohamed / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 586–593
591
10
8
10
8
a
c
a
6
6
b
4
4
2
2
0
0
-2
-4
-6
-8
-2
(a) Complex2 alone (100 uM)
-4
-6
-8
(b) Complex2 (100 uM) + DNA (150 uM)
(a) Complex 2 alone (100 uM)
(b) Complex 2 (100 uM) + DNA (100 uM)
(c) Complex 2 (100 uM) + DNA (150 uM)
-400
-300
-200
-100
0
100
200
300
400
Potential/mV vs Ag/AgCl (KCl)
-400
-300
-200
-100
0
100
200
300
400
Fig. 5. Cyclic voltammograms of complex (2) in Tris–HCl buffer in the absence (a)
and presence (b) of CT-DNA. M; [DNA]: (a) 0, (b)
= 200 mV sꢁ1, [complex] = 100
150 M.
Potential/mV vs Ag/AgCl (KCl)
m
l
l
Fig. 7. Cyclic voltammograms of 100
absence (a) and presence of 100 M DNA (b) and 150
l
M complex (2) in Tris–HCl buffer in the
l
lM DNA (c).
m
= 200 mVsꢁ1
.
and Ipc/Ipa values of 122 mV, 168 mV and 0.43 respectively. Upon
addition of CT-DNA under the same recording conditions complex
the peak currents due to variation of the binding state and slowing
the mass transfer of the complex after binding to CT-DNA frag-
ments as well as a shift in the E1/2. The decrease of the anodic
and cathodic peak currents of the complexes in presence of DNA
is due to formation of slowly diffusing complex-DNA supramolec-
ular complex, which in turn lowers the concentration of the free
complex (mainly responsible for the transfer of current).
2 experience a shift in E1/2 (103 mV),
DEp (182 mV) and ratio of
anodic to cathodic peak currents Ipa/Ipc is 0.38 (Fig. 5).
The mode of the drug vs DNA interaction can be judged from the
variation in formal potential. In general, it was reported that the
positive shift (anodic shift) in formal potential is caused by the
intercalation of the cationic drug into the double helical structure
of DNA [51], while negative shift is observed for the electrostatic
interaction of the cationic drug with the anionic phosphate of
DNA backbone [52,53]. So the obvious negative peak potential shift
(cathodic shift) in the CV behavior of both complexes 1 and 2 by
the addition of DNA supports our previous assumption for an elec-
trostatic interaction of the positively charged diaqua complexes
with the polyanionic DNA. The effect of concentration of CT-DNA
on potential and current peaks of the complexes 1 and 2 was also
The shift in the value of the formal potential (D
E0) can be used
to estimate the ration of equilibrium binding constants (KR/K0)
according to the model of interaction described by Bard and Carter
[54]. From this model one can obtain that:
D
E0 ¼ E0 ꢁ E0 ¼ 59:15 logðKR=KOÞ
ð2Þ
b
f
Where E0b and E0f are the formal potentials of the bound and free
complex forms, KR and K0 are the corresponding binding constants
for the binding of reduction and oxidation species to CT-DNA,
respectively. The KR/KO values for complex 1 and 2 are 0.9 and
0.48 respectively, suggesting the stronger binding affinity of the re-
duced form of complex 1 compared to the oxidized form. The lower
value obtained for complex 2 (0.48) suggests that complex 2 is
strongly bounded to CT-DNA in its oxidized form. This is further
confirmed by the higher cathodic peak potential shift observed for
complex 2, which indicates that the reduced state of complex 2 is
easier to oxidize in presence of DNA because its oxidized form is
more strongly bound to DNA than its reduced form. Complex 1
shows only small cathodic peak shift indicating that the more
strongly bound species is the reduced form.
studied using a constant concentration of the complex (100
and varying the concentration of DNA. With complex 1:
([DNA] = 37, 80 and 125 M) and with complex 2: ([DNA] = 100
and 150 M), Figs. 6 and 7. The results showed that the incremen-
lM)
l
l
tal addition of CT-DNA to both complexes causes a diminution of
40
a
20
0
d
Ethanol precipitation
The binding between DNA and metal complexes was also stud-
ied by precipitating the DNA from the interacted DNA–metal com-
plexes with absolute ethanol. If the interaction between DNA and
metal complex is very weak, the DNA should be precipitated sepa-
rately and all free metal complexes will remain in the supernatant
[55]. In this experiment, the precipitated DNA was separated out
and washed several times with alcohol. This precipitate was re-dis-
solved in Tris–HCl buffer, and the solution was monitored spectro-
photometrically for DNA at 258 nm, for complex 1 at 253 nm and
for complex 2 at 251 nm. The spectral data indicated that the band
corresponding to free DNA was not detected at 258 nm, and the
bands observed for complexes 1 and 2 are shifted from their previ-
ously recorded values at 253 and 251 respectively. This supports
-20
-40
-60
(a) Complex 1 alone (100 uM)
(b) Complex 1 (100 uM) + DNA (37 uM)
(c) Complex 1 (100 uM) + DNA (80 uM)
(d) Complex 1 (100 uM) + DNA ( 125 uM)
-550
-500
-450
-400
-350
-300
-250
-200
-150
Potential/ mV vs Ag/AgCl (KCl)
Fig. 6. Cyclic voltammograms of 100
absence (a) and presence of 37 M DNA (b), 80
= 200 mV sꢁ1
l
M complex (1) in Tris–HCl buffer in the
l
l
M DNA (c) and 125 M DNA (d).
l
m
.

592
A.A. Shoukry, M.S. Mohamed / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 586–593
biological potential of the synthesized diaqua complexes in vivo
against different species of bacteria as well. In testing the antimi-
crobial activity of these compounds, we used more than one test
organism to increase the chance of detecting antibiotic principles
in tested materials. The tested complexes show a remarkable bio-
logical activity against Gram-positive (G+) and Gram-negative (Gꢁ)
bacteria. The in vitro antimicrobial activity of the complexes was
tested against B. subtilis and S. aureus (as Gram-positive bacteria),
E. coli and N. gonorrhoeae (as Gram-negative bacteria) and then
compared with the Standard tetracycline antibacterial agent. The
results are listed in Table 1. The results obtained show the
following:
1
2
3
4
5
C
1' 2' 3' 4' 5'
1- [Pd(phenpip)(OH2)2] was found to have higher reactivity
against the different strains of bacteria more than
[Pd(bpy)(OH2)2] which was found to be moderately active
against them.
Fig. 8. Agarose gel electrophoresis of CT-DNA samples showing the interaction
between the two complexes 1 and 2 and CT-DNA in TBE buffer. Lane C: untreated
2- [Pd(phenpip)(OH2)2] exhibits relatively higher reactivity
against Gram-negative bacteria than Gram-positive ones.
CT-DNA. Lanes 1–5: 16
lM
CT-DNA + (5, 10, 15, 20 and 25 M) complex
l
1
respectively. Lanes 10–50: 16
respectively.
lM CT-DNA + (5, 10, 15, 20 and 25 lM) complex 2
It was reported that Gram-negative bacteria have a thin cell
wall consisting of few layers of peptidoglycan surrounded by a sec-
ond lipid membrane containing lipopolysaccharides and lipopro-
teins. Whereas, Gram-positive bacteria possess a thick cell wall
containing many layers of peptidoglycan and teichoic acids. These
differences in cell wall structure can produce differences in anti-
bacterial susceptibility and some antibiotics can kill only Gram-po-
sitive bacteria and are ineffective against Gram-negative pathogens
[60]. Therefore the interest in these results is that the two studied
complexes 1 and 2 were found to be effective against both Gram-
negative and Gram-positive bacteria, in addition increasing interests
are now directed to the line of antibiotics affecting Gram-negative
bacteria, since there are certain organisms which have proved dif-
ficult to treat and most of them are Gram negative rods [61–63]. It
may be concluded that the antibacterial activity of the compounds
is related to cell wall structure of the bacteria. This is possible be-
cause the cell wall is essential to the survival of bacteria and some
antibiotics are able to kill bacteria by inhibiting a step in the syn-
thesis of peptidoglycan [64].
the strong binding ability of both complexes to CT-DNA, and the
inability of ethanol to separate between them. Similar results were
obtained for another series of Pt(II) and Pd(II) complexes of 2,20-
bipyridine and amino acids [56] .
Gel electrophoresis
The ability of binding of the synthesized complexes to CT-DNA
was investigated by gel electrophoresis through examination of the
effect of different concentrations of the Pd(II) complexes on
CT- DNA. The results are presented in Fig. 8, it is clear that the
intensity of the bands recorded for both complexes after binding
to CT-DNA were decreased, as compared to the band of the free
CT-DNA. The decrease in the intensity of bands observed after
binding of the complexes to CT-DNA is believed to be due to deg-
radation of the DNA double helix and /or quenching of fluorescence
from DNA-intercalated ethidium bromide. Previous work hypothe-
sized that DNA degradation may rather have occurred via back-
bone cleavage due to a nucleophilic attack of basic residues [57].
It was reported that the intensity of bands in gel electrophoresis
visualized by the fluorescence of ethidium bromide intercalate to
DNA base pairs depends not only on the number of molecules,
but also on DNA length [58]. Previous report mentioned that the
fluorescent light could be quenched by the addition of a second
molecule [59]. Therefore, the reduction in the intensity of electro-
phoretic bands of CT-DNA upon interaction with Pd(II) complexes
could be due to masking of complex upon interaction between the
stacked bases within the helix and/or surface binding at the reac-
tive nucleophilic sites on DNA double helix.
Conclusion
The study of the interaction of Pd(II) amine complexes with
CT-DNA is of special interest for pharmaceutical and biomedical re-
search. The present study describes the interaction of two Pd(II)
complexes; [Pd(bpy)(OH2)2]2+ (1) and [Pd(phenpip)(OH2)2]2+ (2)
with CT-DNA. The introduction of heteroaromatic nitrogen base
such as bipyridine and /or phenylpiperazine rings into the Pd(II)
coordination sphere results in decreasing the electron density on
the metal center, making it more electrophilic. As a consequence,
the acidity of the coordinated water molecule in the diaqua com-
plexes would increase and this would favor the binding with CT-
DNA. The binding of two complexes with CT-DNA has been studied
by UV–Vis spectroscopy and electrochemical techniques, revealing
their strong binding ability. The calculated binding strength (Kb) of
Antimicrobial activity
Pd(II) amine complexes are well known to have enhanced
antitumor activity. Therefore it seems interesting to screen the
Table 1
Antimicrobial and antifungal activities of [Pd(bpy)(OH2] and [Pd(phenpip)(OH2)].
Microorganism
Gram reaction
Inhibition zone diameter (mm/mg sample)
[Pd(bpy)(OH2]
[Pd(phenpip)(OH2)]
Standard
Bacillus subtilis
(G+)
(G+)
(G+)
(Gꢁ)
(Gꢁ)
(Gꢁ)
15
14
13
13
14
14
23
21
18
19
25
25
28
29
31
30
31
31
Staphylococcus aureus
Streptococcus faecalis
Escherichia coli
Neisseria gonorrhoeae
Seudomonas aeruginosa

A.A. Shoukry, M.S. Mohamed / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 586–593
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