Synthesis of trimethoprim metal complexes: Spectral, electrochemical, thermal, DNA-binding and surface morphology studies

Article abstract of DOI:10.1016/j.saa.2012.03.055

Complexes of trimethoprim (TMP), with Cu(II), Zn(II), Pt(II), Ru(III) and Fe(III) have been synthesized. Then, these complexes have been characterized by spectroscopic techniques involving UV-vis, IR, mass and ¹H NMR. CHN elemental analysis, el

Full text of DOI:10.1016/j.saa.2012.03.055

Spectrochimica Acta Part A 94 (2012) 243–255
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis of trimethoprim metal complexes: Spectral, electrochemical, thermal,
DNA-binding and surface morphology studies
Nihat Demirezen, Derya Tarınc¸ , Duygu Polat, Mustafa C¸ es¸ me, Ays¸ egül Gölcü∗, Mehmet Tümer
Department of Chemistry, Faculty of Science and Letters, University of Kahramanmaras Sutcu Imam, Campuse of Avsar, 46100 Kahramanmaras, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Complexes of trimethoprim (TMP), with Cu(II), Zn(II), Pt(II), Ru(III) and Fe(III) have been synthesized.
Then, these complexes have been characterized by spectroscopic techniques involving UV–vis, IR, mass
and 1H NMR. CHN elemental analysis, electrochemical and thermal behavior of complexes have also been
investigated. The electrochemical properties of all complexes have been investigated by cyclic voltam-
metry (CV) using glassy carbon electrode. The biological activity of the complexes has been evaluated by
examining their ability to bind to calf-thymus DNA (CT DNA) with UV spectroscopy and cyclic voltam-
metry. UV studies of the interaction of the complexes with DNA have shown that these compounds can
bind to CT DNA. The binding constants of the complexes with CT DNA have also been calculated. The
cyclic voltammograms of the complexes in the presence of CT DNA have shown that the complexes can
bind to CT DNA by both the intercalative and the electrostatic binding mode. The antimicrobial activity
of these complexes has been evaluated against three Gram-positive and four Gram-negative bacteria.
Antifungal activity against two different fungi has been evaluated and compared with the reference drug
TMP. Almost all types of complexes show excellent activity against all type of bacteria and fungi. The
morphology of the CT DNA, TMP, metal ions and metal complexes has been investigated by scanning
electron microscopy (SEM). To get the SEM images, the interaction of compounds with CT DNA has been
studied by means of differential pulse voltammetry (DPV) at CT DNA modified pencil graphite electrode
(PGE). The decrease in intensity of the guanine oxidation signals has been used as an indicator for the
interaction mechanism.
Received 13 December 2011
Received in revised form 5 March 2012
Accepted 22 March 2012
Keywords:
Drug–metal complex
Trimethoprim
DNA binding
Voltammetry
PGE
SEM
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
toxicity is often associated with metal compounds. If they can be
delivered only to the tissues, cells and receptors where they are
TMP, chemically 5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-
diamine, the structure of which is shown in Fig. 1, belongs to the
class of chemotherapeutic agents known as dihydrofolate reduc-
tase inhibitors. It is used in prophylaxis treatment and urinary tract
infections [1].
The application of inorganic chemistry to medicine (“Elemental
Medicine”) is a rapidly developing field, and novel therapeutic and
diagnostic metal complexes have an impact on medical practice.
Advances in biocoordination chemistry are crucial for improv-
ing the design of compounds to reduce toxic side-effects and
understand their mechanisms of action. Most of the major classes
of pharmaceutical agents contain examples of metal compounds
which are in current clinical use [2], and new areas of applica-
tion are rapidly emerging. Some of these are discussed briefly
below with special emphasis on the targeting of metal complexes
and their bio-transformation. Targeting is important because the
required, the toxicity may be reduced. The ease with which many
metal complexes undergo ligand substitution and redox reactions
is likely to mean that the active species are bio-transformation
products of the administered complex. Identification of these active
species will lead to the more effective use of metal compounds as
drugs.
Compounds containing pyrimidine rings play a significant role
in many biological systems [3]. The pyrimidine ring system, present
in nucleic acids, several vitamins, coenzymes and antibiotics, pro-
vides potential binding sites for metal ions, and any information on
their coordinating properties is important as a means of under-
standing the role of the metal ions in biological systems. Many
compounds of therapeutic importance contain the pyrimidine
ring system. So, substituted 2,4-diaminopyrimidines are widely
employed as metabolic inhibitors of pathways leading to the
synthesis of proteins and nucleic acids. Among these kinds of lig-
ands, TMP is a well known biological agent, also employed as a
potent metabolic inhibitor of bacterial dihydrofolic acid reductase.
Trimethoprim has potential binding sites for metal ions. Several
authors have studied the interaction of this ligand with biological
∗
Corresponding author. Tel.: +90 344 219 12 85; fax: +90 344 219 10 42.
E-mail addresses: agolcu@ksu.edu.tr, ag518@ksu.edu.tr (A. Gölcü).
1386-1425/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.03.055

244
N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
complexes with CT DNA have been investigated with UV and CV
titration.
NH2
N
CH3
O
N
2. Experimental
2.1. General
CH3
H2N
O
TMP was kindly provided by Glaxo Smith Kline Pharmaceu-
tical Co. (Istanbul, Turkey). CT DNA was purchased from Sigma,
NaCl, metal salts (CuCl2·2H2O, ZnCl2, FeCl3·6H2O, K2PtCl4, and
RuCl2·3H2O), NaCl, 0.2 M Phosphate buffer at pH 2.0–12.0 and
Tris–HCl were purchased from Merck. All the chemicals and sol-
vents were reagent grade and were used as purchased. DNA stock
solution was prepared by dilution of CT-DNA to buffer solution
(containing 150 mM NaCl and 15 mM Tris–HCl at pH 7.0) followed
by exhaustive stirring at 4 ◦C for three days [28], and kept at 4 ◦C
for no longer than a week. The stock solution of CT-DNA gave a
ratio of UV absorbance at 260 and 280 nm (A260/A280) of 1.89, indi-
cating that the DNA was sufficiently free of protein contamination
[27]. The DNA concentration was determined by the UV absorbance
at 260 nm after 1:20 dilution using ε = 6600 M−1 cm−1 [29]. Ele-
mental analyses (C, H and N) were performed using a LECO CHNS
932 elemental analyser. Infrared spectra of the ligands and their
O
CH3
Fig. 1. The chemical structure of TMP.
metal ions and the coordination of TMP via a NH2 nitrogen atom
has been inferred on the basis of IR and visible measurements [4,5].
However, other authors have shown by X-ray diffraction methods
that the coordination site of the TMP molecule is the N1 of the
pyrimidine ring [6–13]. On the other hand, more recently, other
research groups [14–26] have prepared and characterized com-
plexes of TMP with metal(II)/(III) and the spectral and analytical
data show that the ligand acts as a monodentate or bidentate.
In this literature, the interactions of metal complexes of TMP
with DNA have not been studied. Whereas, the study of the inter-
actions of transition–metal complexes with DNA has been an active
field of research. Interest in this field stems from attempts to gain
some insight into the interaction model between DNA and the com-
plexes, and obtain some information about drug design and tools
of molecular biology [27]. Transition metal complexes can interact
with DNA through a non-covalent way, such as electrostatic inter-
action, groove binding and intercalation. Binding to DNA through
an intercalation mode with planar ligands intercalating into the
adjacent base pairs of DNA [27] correlates to the planarity of the
ligand, coordination geometry of the metal ion, and donor type of
the ligand [28]. Considerable useful applications of these complexes
need to be done to develop the complexes binding to DNA through
an intercalation mode with their structures containing fully planar
intercalating into the adjacent base pairs of DNA [27,28]. These give
valuable information to explore the potential chemotherapeutical
agents. Along this line, lots of copper(II) complexes, which pos-
sess biologically accessible redox potentials and demonstrate high
nucleobase affinity, have been synthesized and their interactions
with DNA extensively studied [29,30].
metal complexes were obtained using KBr discs (4000–400 cm−1
)
with a Perkin Elmer spectrum 400 FT-IR spectrophotometer. Far
spectra of the complexes were recorded using a Perkin Elmer spec-
trum 400 FT-IR/FT-FAR instrument. The electronic spectra were
obtained in the 200–900 nm range by a Perkin Elmer Lambda 45
spectrophotometer. Magnetic measurements were carried out by
the Gouy method using Hg[Co(SCN)4] as a calibrant. Mass spectra
of the ligands were recorded on a LC/MS APCI AGILENT 1100 MSD
spectrophotometer. 1H NMR spectra were recorded on a Bruker
400 MHz instrument. TMS was used as internal standard and DMSO
as solvent. The metal content of the complex was determined by an
Ati Unicam 929 Model AA Spectrometer in solutions prepared by
decomposing the compounds in aqua regia and then subsequently
digesting in concentrated HCl. The thermal analysis studies of the
complexes were performed on a Perkin Elmer STA 6000 simultane-
ous Thermal Analyzer under nitrogen atmosphere at a heating rate
of 10 ◦C/min. Scanning electron micrography associated with SEM
Neo Scope JSM-5000 was used for morphological evaluation.
2.2. Electrochemical measurements
Electrochemical investigations of nucleic acid binding
molecules–DNA interactions can provide a useful complement
to the spectroscopic techniques and yield information about
the mechanism at intercalation and the confirmation of anti
bacterial–DNA adduct. The immobilization of DNA onto an elec-
trode surface is crucial aspect in many ways of the developing DNA
biosensors for monitoring drug (or complex) because it dictates
the accessibility of the DNA to drug (or complex) in solution and
hence can influence the affinity of drug binding. Binding of drug (or
complex) to DNA and a general DNA damage has been described
through the variation of the electrochemical signal of guanine or
adenine [31,32]. Observing the electrochemical signal related to
DNA–drug (or DNA-complex) interaction can provide evidence for
the interaction mechanism and the nature of the complex formed,
binding constant, size of binding site and the role of free radicals
generated during interaction in drug (or complex) action. A good
sensitivity of oxidation signals for DNA structure would make
carbon electrodes extensively useful in DNA research especially
pencil graphite electrode [33], carbon paste electrode [34] and
glassy carbon electrode [35].
All voltammetric measurements at the glassy carbon working
electrode was performed using a BAS 100 W (Bioanalytical System,
USA) electrochemical analyzer. Glassy carbon working electrode
(BAS; ˚: 3 mm diameter), an Ag/AgCl reference electrode (BAS;
3 M KCl) and platinum wire counter electrode and a standard one-
compartment three electrode cell of 10 mL capacity were used in all
experiments. Glassy carbon working electrode was polished man-
ually with aqueous slurry of alumina powder (˚: 0.01 ␮m) on a
damp smooth polishing cloth (BAS velvet polishing pad), before
each measurement. All measurements were realized at room tem-
perature. Mettler Toledo MP 220 pH meters was used for the pH
measurements using a combined electrode (glass electrode refer-
ence electrode) with an accuracy of 0.05 pH.
2.3. Synthesis of a copper(II), zinc(II), platinum(II), iron(III) and
ruthenium(III) complex
All metal complexes have been obtained according to a general
procedure: a solution of a metal salt (1 mmol) dissolved in 5 mL
of MeOH have been added to a solution of TMP ligand (1 mmol)
in 5 mL of distilled water and finally 15 mL of MeOH have been
added to mixture and the mixture have been heated under reflux
In this context, mononuclear metal(II/III) complexes have been
synthesized and characterized. The complexes have been tested
for their ability to bind to CT DNA. The binding properties of the

N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
245
2.5. DNA-binding studies
H CO
3
H
N
2
OCH
3
N
The interaction of the complexes with CT DNA has been studied
with UV spectroscopy and CV in order to investigate the possible
binding modes to CT DNA and to calculate the binding constants to
CT DNA (Kb). In UV titration experiments, the spectra of CT DNA in
the presence of complex has been recorded for a constant CT-DNA
concentration in diverse [complex]/[CT DNA] mixing ratios (r). The
intrinsic binding constant, Kb, of the complex with CT DNA has been
determined through the UV spectra of the complex recorded for a
constant complex DNA concentration (4.2 × 10−8 M) in the absence
and presence of CT DNA for diverse r values [27]. The interaction of
the complex with CT DNA has also been investigated by monitoring
the changes observed in the cyclic voltammogram of a 3 × 10−4 M
buffer solution of the complex upon addition of CT DNA at diverse r
values. The buffer has also been used as the supporting electrolyte
and the cyclic voltammograms have been recorded at m = 100 mV/s.
Cl
N
M
OCH
3
Cl
N
H
2
n H O
2
Fig. 2. Proposed structure of Cu(II), Zn(II) and Pt(II) complexes (n = 0 for Cu(II), n = 3
for Pt(II) and n = 1 for Zn(II) complexes).
for one day. At the end of the reaction, determined by TLC, the pre-
cipitate have been filtered off, washed with distilled water, EtOH
and dried under vacuum. The proposed formulas of M(II)–TMP and
M(III)–TMP complexes have been given in Figs. 2 and 3, respec-
tively.
2.6. Morphology studies
2.4. Preparation of the microorganism cultures
Pencil graphite electrode (PGE) used for morphology studies.
So, DPV has been performed with an Autolab-PGSTAT 30 electro-
chemical analysis system with a General Purpose Electrochemical
Software (mlS) 4.9 software package (Eco Chemie, Utrecht, The
Netherlands). The three electrode system consisted of the PGE, as
the working electrode, a reference electrode (Ag/AgCl) and a plat-
inum wire as the auxiliary electrode. The renewable PGE process
has been described in literature 37 [37]. A Noki® pencil has been
used as a holder for Pentel® graphite leads. The pencil has been
hold vertically with 15 mm of the lead extruded outside (12 mm of
which was immersed in the solution) [38]. Each measurement has
been performed using a new graphite lead. PGE has been activated
at +1.40 V for 1 min in 0.50 M Tris–HCl buffer solution for electrode
surface pretreatment. The CT DNA has been immobilized onto the
pre-treatment PGE surface by applying a potential at +0.50 V during
240 s using 400 rpm stirring rate in 0.50 M Tris–HCl buffer contain-
ing 4.00 ␮g/mL CT DNA. Then, the electrode has been cleaned with
blank acetate buffer solution for 5 s for the removal of the unbound
CT DNA at the electrode surface. The CT DNA has been immobilized
and PGE has been immersed into Tris-buffer solution (30% ethanol)
containing same concentrations of compound (TMP or M(II)/(III)
complexes) during 180 s with 400 rpm stirring at open circuit sys-
tem. Then, the electrode has been rinsed with blank Tris–HCl buffer
solution for 5 s and SEM images have been recorded. All SEM images
have been taken on carbon strip.
The growth inhibitory activity of the chemical matter has been
tested against ten bacteria (Eschericha coli, Enterobacter cloaca,
Bacillus megaterium, Bacillus cereus, Pseudomonas sp., Brusella
melitensis, Saccharomyces cerevicia and Staphylococcus aureus)
and fungi (Candida albicans and Saccharomyces cerevicia). These
microorganisms have been provided from Microbiology Laboratory
Culture Collection, Department of Biology, Kahramanmaras Sutcu
Imam University, Kahramanmaras, Turkey. Antimicrobial activities
of the chemical matter have been determined using the agar-disc
diffusion method as described below. The bacteria have been first
incubated at 37 0.1 ◦C for 24 h in nutrient broth (Difco), and the
yeasts have been incubated in sabouraud dextrose broth (Difco)
at 25 0.1 ◦C for 24 h. The cultures of the bacteria and yeast have
been injected into the petri dishes (9 cm) in the amount of 0.1 mL.
And then, mueller hinton agar) and sabouraud dextrose agar (ster-
ilized in a flask and cooled to 45–50 ◦C) have been homogenously
distributed onto the sterilized petri dishes in the amount of 15 mL.
Subsequently, the sterilized blank paper discs of 6 mm diameter
have been saturated with 1200 ␮g of chemical matters per disc.
The discs have been placed onto the agar plates, which had previ-
ously been inoculated with the above organisms. In addition, blank
paper discs treated with TMP has been used as positive controls.
Afterwards the plates combined with the discs have been left at
4 ◦C for 2 h, the plates injected with yeast have been incubated at
25 0.1 ◦C for 24 h, and ones injected with bacteria have been incu-
bated at 37 0.1 ◦C for 24 h. After 24 h, inhibition zones appearing
around the discs have been measured and recorded in mm. The
initial number of microorganisms in the suspension has been deter-
mined for the total yeasts and bacterial count during 24 h at 37 ◦C
for bacteria and 48 h 25 ◦C for yeasts [36].
3. Results and discussion
In this study, metal(II/III) complexes of TMP have been prepared
and characterized by the analytical and spectroscopic methods. The
isolated complexes are stable in air, insoluble in water and common
organic solvents, but completely soluble in DMSO. The elemen-
tal analysis, color, and melting point together with the formula
weight obtained from the mass spectra for the complexes are given
in Table 1 and agree very well with molecular formula proposed.
The analytical data show the composition of the metal complexes
to be [Cu(Cl)2(TMP)], [Zn(Cl)2(TMP)]·(H2O), [Pt(Cl)2(TMP)]·3(H2O),
[Fe(Cl)3(TMP)(H2O)] and [Ru(Cl)3(TMP)(H2O)]·(H2O). The ratios of
the metal present in all complexes have been determined by atomic
absorption spectroscopy. The complexes have been decomposed in
HNO3/H2O2 (1/1) and then dissolved in 1.5 N HNO3. The amounts
of metals have been determined (Table 1). They support the struc-
tures given in Figs. 2 and 3. The molar conductivity measurements
have been done all compounds in DMSO (∼1.10−3 M solutions). The
conductivity data of the complexes are very low and they can be
H CO
3
H
N
2
OCH
3
N
Cl
N
Cl
M
OCH
3
Cl
N
H
OH
2
2
n H O
2
Fig. 3. Proposed structure of Ru(III) and Fe(III) complexes (n = 0 for Fe(III) and n = 1
for Ru(III) complexes).

246
N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
Table 1
Analytical and physical properties of trimethoprim complexes.
Compound
Formula weight (g/mol)
Color
Yield (%)
Freezing point (◦C)
Elemental analysis; calculated (found)
C
H
N
M
[Cu(Cl)2(TMP)]
424.76
551.79
470.53
444.64
610.35
Green
Black
Brown
White
Black
72
80
73
66
64
194
263
240
174
271
39.59 (41.14)
30.47 (30.21)
35.74 (35.09)
37.82 (38.09)
27.55 (27.50)
4.27 (4.86)
4.38 (4.11)
4.28 (4.36)
4.53 (4.46)
3.96 (3.12)
13.19 (13.34)
10.15 (9.50)
11.91 (11.29)
12.60 (12.34)
9.18 (8.62)
14.95 (14.13)
18.43 (19.03)
11.86 (11.69)
14.70 (14.54)
31.96 (30.98)
[Ru(Cl)3(TMP)(H2O)]·(H2O)
[Fe(Cl)3(TMP)(H2O)]
[Zn(Cl)2(TMP)]·(H2O)
[Pt(Cl)2(TMP)]·3(H2O)
regarded as non-electrolytes. Chloride ions in the all complexes
have been determined by titration with AgNO3.
Diverse crystallization techniques have been employed in order
to obtain a crystal suitable for the structure determination with
X-ray crystallography. Nevertheless, the complexes have been col-
lected as microcrystalline products.
The formation of the complexes also confirmed by UV–vis spec-
tra (Table 2). In the UV–vis measurements, acetonitrile/water (1/1,
v/v) have been used as a solvent. All complexes except Pt(II) and
Zn(II) complexes, shows d–d transitions between 409 and 603 nm
[39]. The spectra of the complexes show bands between 378 and
396 nm assigned to M→L and L→M (L: ligand, M: metal) charge
transfer transitions. There are n→(* and (→(* transitions between
325–374 and 233–296 nm, respectively.
The magnetic moments (as BM) of the complexes have been
measured at room temperature. The Zn(II) and Pt(II) complexes
have the diamagnetic character while the Zn(II) complex has tetra-
hedral geometry, the Pt(II) complex is square-planar around the
metal ion. The structures of the mononuclear Cu(II) complex are
supported by the magnetic moment data. The measured magnetic
moment value of the Cu(II) complex of the TMP is 1.80 BM, which
is very close to that of the spin only value (1.73 BM) expected for
a complex having one copper(II) ion with a single unpaired elec-
2
2
tron located in an essentially dx
orbital. This value suggests
–y
that the copper atom is in a square planar environment in its
chelates [40]. The observed magnetic moment of Fe(III) complex
is 5.20 BM. Thus, the complex formed has the octahedral geome-
try. Magnetic susceptibility measurement of the Ru(III) complex
at 298 K showed a value of e.m.u.g−1 with an effective magnetic
moment ꢀeff of 1.95 BM. The ꢀeff value is slightly smaller than the
spin-only moment of an unpaired electron. Thus, it can be con-
cluded that a change in the formal oxidation state of the ruthenium
atom from 0 to +1 has been achieved via oxidative addition of the
NH2 groups to ruthenium. This situation explores that a proton
displacement to give a low-spin d7 electronic configuration, Fig. 3.
An effective magnetic moment of 1.95 BM for this complex and the
assignment of the spectra are in conformity with the octahedral
geometry.
In order to clarify the mode of bonding and the effect of the
metal ion on the ligand, the FT-IR spectra of the TMP and metal
complexes have been compared and assigned on the basis of care-
ful comparison. The FT-IR spectra of TMP and its metal complexes
are listed in Table 3 and Fig. 4a and b. TMP possesses seven poten-
tial donor sites; two pyrimidinyl N atoms, two NH2 groups on the
pyrimidine rings and three methoxy groups. So, the ꢁas(N H) and
ꢁs(N H) modes of the pyrimidine-NH2 groups in the free TMP are
assigned to the strong and sharp bands at 3469 and 3317 cm−1
,
respectively, which are affected by the presence of hydrogen bonds
[40]. In all the complexes the bands ꢁ(N H), due to asymmetric and
symmetric vibrations, are present in the region 3482–3405 cm−1
;
Fig. 4. The infrared spectra of the [Zn(Cl)2(TMP)]·(H2O) (a) and [Fe(Cl)3(TMP)(H2O)]
these bands shifted significantly with respect to those of the ligand.
This confirms that the metal ion is coordinated to the trimetho-
prim through the two N atoms that belong to NH2 groups. ꢁ(C N)
of conjugated cyclic system of the ligand is lowered in complex
with respect to ligand. This is an indication of bonding of metal
(b) complexes.

N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
247
Table 2
Spectroscopic data of TMP metal complexes.
Compound
␲–␲* (nm)
n–␲* (nm)
Charge transfer (nm) (M→L or L→M)
d–d* (nm)
[Cu(Cl)2(TMP)]
274
252
264
233
296
334
341
374
338
325
381
396
379
378
379
603
409
559
–
[Ru(Cl)3(TMP)(H2O)]·(H2O)
[Fe(Cl)3(TMP)(H2O)]
[Zn(Cl)2(TMP)]·(H2O)
[Pt(Cl)2(TMP)]·3(H2O)
–
Table 3
FAR-and MID-IR results of TMP and its metal complexes.
Compound
ꢁas(N H)
ꢁs(N H)
ꢁ(C N)
ꢁ(M N)
ꢁ(M O)
ꢁ(M Cl)
TMP
[Cu(Cl)2(TMP)]
[Ru(Cl)3(TMP)(H2O)]·(H2O)
[Fe(Cl)3(TMP)(H2O)]
[Zn(Cl)2(TMP)]·(H2O)
[Pt(Cl)2(TMP)]·3(H2O)
3469
3451
3469
3478
3482
3547
3317
3405
3400
3353
3386
3323
1652
1640
1641
1629
1602
1632
–
605
473
605
645
471
–
–
366
472
–
–
264
274
263
274
261
–
ion through two–NH2 nitrogen of conjugated cyclic system. In
order to investigate the substituent effect upon both the ␦- and ␲-
contribution to the total metal–nitrogen and metal–chloride bond
strength, the FT-Far method have been used. Furthermore, the
effect of the substituents on the metal–halogen bond of the TMP
metal complexes has also been examined. The infrared spectra in
the region 700–30 cm−1 have been measured for the complexes,
and the maximum absorption frequencies are listed in Table 3. The
spectra of the [Zn(Cl)2(TMP)]·(H2O) and [Fe(Cl)3(TMP)(H2O)] com-
plexes are given in Fig. 4a and b, respectively. The far spectra of all
complexes of the TMP have similar features, they are characterized
by two or three strong metal–chlorine stretching bands and two
medium metal–nitrogen (ligand) stretching bands in this region.
Therefore, it can be reasonably assumed that the substituents
regions and this situation confirms that the nitrogen atom of the
amines group coordinates to the metal ions. Typical NMR spectra
of TMP and its zinc complex are given in Fig. 6a and b, respectively.
Thermal studies of all the complexes were performed
under nitrogen atmosphere in the temperature range of
20–1000 ◦C. The thermal curves of the [Pt(Cl)2(TMP)]·3(H2O)
and [Fe(Cl)3(TMP)(H2O)] complexes are given in Fig. 7a and b. All
complexes (except copper(II) complex) have the adsorbed and/or
hydrated water molecules. As seen from Fig. 7 and the figures of
the other complexes, the hydrated water molecules have been
eliminated at the first step in the 30–100 ◦C temperature range.
The intermediates are stable within the interval of 135–240 ◦C. The
loss of the hydrated water molecules has been seen as endothermic
peaks in the DTA curves. While the iron(III) and ruthenium(III)
complexes contain the coordinated water molecules and chloride
ions, zinc(II) and platinum(II) complexes contain only coordinated
chloride ions. Coordinated water molecules in the Fe(III) and Ru(III)
complexes eliminate within the temperature range of 100–200 ◦C.
The coordinated chloride ions in the complexes, loss from the
molecule in the 240–400 ◦C temperature range [41]. This process
is connected with the elimination of one and or two coordinated
chloride ions and the formation of [M(L)] specie which is thermally
stable up to the higher temperatures. Main mass losses are at the
higher temperatures. Then, the mass of the samples decreased
slightly up to 1000 ◦C. From the calculations, it follows that the
final decomposition product can be MO/M2O3 (MO is for the Cu(II),
Zn(II) and Pt(II) complexes; M2O3 is for the Ru(III) and Fe(III)
complexes).
(
NH2 groups) on the meta-position of the formyl ring change the
stereo-configuration of the metal complexes. On the whole, anal-
ogous to the case of the copper(II) complexes, the metal-chlorine
stretching (metal: Zn(II), Pt(II) Fe(III) and Ru(III)) frequencies due
to the effects of both electron-releasing and electron-attracting
substituents. This phenomenon is apparently due to the delocal-
ized nature of the coordination bonds in the complexes. In the
complexes, the ␯(M Cl) stretching frequency has been seen in the
274–261 cm−1 range. The ␯(M N) stretching frequencies have been
shown in the 471–645 cm−1 range. The ␯(M O) stretching fre-
quencies in [Fe(Cl)3(TMP)(H2O)] and [Ru(Cl)3(TMP)(H2O)]·(H2O)
complexes have been seen in 472 cm−1 and 366 cm−1, respectively.
The formulation of the complexes are deduced from analyti-
cal data, 1H NMR and further supported by mass spectroscopy.
The relatively low intensities of the molecular ion peaks, [M]+,
are indicative of the ease of fragmentation of the complexes, and
this may reflect the number of heteroatoms present in each struc-
ture. The spectra of the ruthenium complex shows peak at m/e
533. This peak can attribute to the molecular ion peaks [M]+. All
the compounds decompose in a similar way. In the mass spectra
of the ruthenium complex, the highest intensity peaks are at m/e
415 (100%) may be assigned to the [M CH3O2Cl2]+ ions which is
formed by the loss of other parts of the molecular ions. Typical mass
spectrum of [RuCl3(TMP)(H2O)]·(H2O) is given in Fig. 5.
The 1H NMR spectra of the TMP and its complexes have been
recorded using DMSO and obtained results are given in Table 4. In
the spectrum of the TMP, the–CH2 protons are seen at 3.40 ppm
as singlet. The singlets in the 3.72 and 3.52 ppm can be attributed
to the methoxy protons. The slightly broad bands at the 5.71 and
6.10 ppm can be attributed to the NH2 protons. The aromatic pro-
tons are shown at 6.55 and 7.51 ppm as singlets. In the spectra of
the complexes, the NH2 protons have been shifted to the lower
Fig. 5. Mass spectrum of the [RuCl3(TMP)(H2O)]· (H2O).

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N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
Table 4
The NMR results of TMP and its Zn(II) and Pt(II) complexes.
Compound
CH2
OCH3
NH2
Ar H
TMP
3.40 (s, 2H)
3.42 (s, 2H)
3.43 (s, 2H)
3.52, 3.72 (s, 6H)
3.72, 3.90 (s, 6H)
3.62, 3.74
5.71, 6.10 (s, 4H)
6.43, 6.74 (s, 4H)
6.43, 6.70
6.55, 7.51 (m, 2H)
6.99, 7.44 (m, 2H)
6.96, 7.46 (m, 2H)
[Zn(Cl)2(TMP)]·(H2O)]
[Pt(Cl)2(TMP)]·3(H2O)]
a
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
ppm
b
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
ppm
Fig. 6. NMR spectra of TMP (a) and [Zn(Cl)2(TMP)]·(H2O) complex (b).

N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
249
Fig. 7. The thermal curves of the [Fe(Cl)3(TMP)(H2O)] (a) and [Pt(Cl)2(TMP)]·3(H2O) (b) complexes.
[Pt(Cl)2(TMP)]·3(H2O), +0.10 V, 4 × 10−6 A for [Fe(Cl)3(TMP)(H2O)]
and (+0.13 V, 5.3 × 10−6 [Ru(Cl)3(TMP)(H2O)]·(H2O) and all com-
plexes are 3 × 10−4 M). Fig. 8 shows comparative CV of TMP in the
presence of copper(II) ions at cathodic directions for: (a) blank,
(b) TMP alone, (c) [Cu(Cl)2(TMP)]. In the figure (Fig. 8c) of the
[Cu(Cl)2(TMP)] complex as different from the CV curve (Fig. 8b)
of the TMP drug. The cathodic peak at +0.14 V shows the reduction
of the Cu(II) ion. This process is: Cu(II) + e− → Cu(I). In addition, the
anodic peak at +0.08 V is the oxidation peak of the [Cu(Cl)2(TMP)]
complex. This is Cu(I) → Cu(II) + e−. The oxidation and reduction
process are irreversible.
The pH of the supporting electrolyte has a significant effect on
the electro-reduction of the complexes at the GCE. Plots of pH vs.
Ep and Ip have been investigated using CV techniques. The peak
potential (Ep) at the redox process moved to less positive potential
values by raising the pH (Fig. 9).
In this research, TMP and its be [Cu(Cl)2(TMP)],
[Zn(Cl)2(TMP)]·(H2O), [Pt(Cl)2(TMP)]·3(H2O), [Fe(Cl)3(TMP)(H2O)]
and [Ru(Cl)3(TMP)(H2O)]·(H2O) complexes have been subjected
to a cyclic voltammetric study with the aim of the detailed char-
acterizing their electrochemical behavior, on the glassy carbon
electrode. Therefore, the electrochemical behaviors of compounds
have been studied over a wide pH range (2–12.0) with a glassy
carbon disc electrode in phosphate buffered aqueous media.
In earlier studies, Kotoucek et al. [42] evaluated the mecha-
nism of the polarographic reduction of TMP. In their study, in
concentrated sulfuric acid (c > 0.1 mol dm−3) the protonized TMP
has been reduced in a two-electron process. In this study, cyclic
voltammetric technique has been used. In CV studies, the scan-
ning has been started at +1.5 V in the positive direction, the
anodic oxidation of TMP does not occur until about 0.0 V for
GCE. After 0.0 V, TMP yields one well-defined peak and one ill-
defined peak has been observed on the cathodic branch for GCE
at −0.22 V (4.2 × 10−6 A for 3 × 10−4 M TMP solution) and −0.60 V
(8.8 × 10−7 A for 3 × 10−4 M TMP solution), respectively. When
examined of CV behavior of all metal complexes in same medium,
peak at −0.3 V is shifted more positive potential values and its cur-
rent increased (+0.14 V, 5.7 × 10−6 A for [Cu(Cl)2(TMP)], (+0.16 V,
5.9 × 10−6 A for [Zn(Cl)2(TMP)]·(H2O), +0.20 V, 6 × 10−6 A for
The plot of the peak potential vs. pH shows one straight line
between pH 2.0 and 12.0, which can be expressed by the following
equations in phosphate buffer:
Ep (mV) = −119.5–2.5 pH;
r = 0.94498528 for [Cu(Cl)2(TMP)] (pH = 2–4)

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N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
Fig. 8. Cyclic voltammogram of TMP in the presence of copper(II) ions at cathodic
directions for: (a) Supporting electrolyte, (b) 3 × 10−4
M
TMP, (c) 3 × 10−4
M
[Cu(Cl)2(TMP)]. Initial potential: 1500 mV, high potential: 1500 mV, low potential:
−1500 mV, scan rate: 100 mV/s, sensitivity: 10 ␮A/V, number of the segments: 2.
Ep (mV) = 1318.1–21.84 pH;
r = 0.9549 for [Zn(Cl)2(TMP)]·(H2O) (pH = 2–12)
Ep (mV) = 1409–29.38 pH;
r = 0.9720 for [Pt(Cl)2(TMP)]·3(H2O) (pH = 2–12)
Ep (mV) = 2807.3–152 pH;
r = 0.9858 for [Ru(Cl)3(TMP)(H2O)]·(H2O) (pH = 9–12)
Ep (mV) = 1330.2–23.15 pH;
r = 0.9304 for [Fe(Cl)3(TMP)(H2O)] (pH = 2–12)
The effects of pH on peak current of all complexes in the range
of pH 2.0–12.0 have also been evaluated. The best and sharpest
peak and reproducible results (except [Ru(Cl)3(TMP)(H2O)]·(H2O)
complex) have been obtained in pH 2.0 phosphate buffer. pH 9
is best media for [Ru(Cl)3(TMP)(H2O)]·(H2O) complex. Therefore
these mediums have been chosen in this study as the supporting
electrolyte for the electroanalytical investigations. Scan rate stud-
ies have been carried out to investigate whether the process at the
GCE is under diffusion or adsorption control. The effect of the poten-
tial scan rate between 5 mV/s and −500 mV/s on the peak current
and potential of all complexes have been evaluated in pH 2 or 9
phosphate buffer. The typical current–potential curve of scan rate
studies of [Cu(Cl)2(TMP)] complex has been given in Fig. 10. When
the scan rate has been varied from 5 to 500 mV/s in 1 × 10−4 M
complex solutions, a linear dependence of the peak current Ip (␮A)
upon the square root of the scan rate v1/2 (mV/s) was found by GCE,
demonstrating a diffusional behavior.
Fig. 9. Effects of pH on [Pt(Cl)2(TMP)]·3(H2O) (a), [Zn(Cl)2(TMP)]·(H2O) (b), and
[Fe(Cl)3(TMP)(H2O)] (c) anodic peak potential, complex concentration 1 × 10−4
M
and 0.2 M phosphate buffers.
The effect of scan rate on peak current has also been examined
under the above conditions with a plot of logarithm of peak current
vs. logarithm of scan rate giving a straight line within the same scan
rate range. These linear relationships have been obtained as follow
(n = 10 in all studies):
log Ip (ꢀA) = 0.6477 log v(mV/s) − 0.6491
r: 0.9969 for [Zn(Cl)2(TMP)]·(H2O)
log Ip (ꢀA) = 0.4821 log v(mV/s) − 0.0032
r: 0.9960 for [Pt(Cl)2(TMP)]·3(H2O)
log Ip (ꢀA) = 0.3650 log v(mV/s) − 0.315
r: 0.9900 for [Cu(Cl)2(TMP)]

N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
251
Fig. 11. UV spectra of [Cu(Cl)2(TMP)] in buffer solution in the presence of CT DNA
at increasing amounts. [Cu(Cl)2(TMP)] = 1 × 10−5 M. The arrow shows the intensity
changes upon increasing concentration of CT DNA.
Fig. 10. The scan rate (5–500 mV/s) effect on peak current of [Cu(Cl)2(TMP)] com-
plex.
[Zn(Cl)2(TMP)]·(H2O), from 296 to 272 for [Pt(Cl)2(TMP)]·3(H2O),
from 264 to 251 for [Fe(Cl)3(TMP)(H2O)] and from 252 to 240
for [Ru(Cl)3(TMP)(H2O)]·(H2O)) complex has been increased in the
presence of DNA up to r = 9 and a blue shift of 15 nm (259 nm) has
been observed for higher amounts of DNA. The hypsochromic effect
has been observed might be ascribed to external contact (electro-
static binding) [27,29] or that all complexes could uncoil the helix
structure of DNA and made more bases embedding in DNA exposed
[46–48]. The intrinsic binding constant Kb of [Cu(Cl)2(TMP)],
[Zn(Cl)2(TMP)]·(H2O), [Pt(Cl)2(TMP)]·3(H2O), [Fe(Cl)3(TMP)(H2O)]
and [Ru(Cl)3(TMP)(H2O)]·(H2O) complexes with CT DNA repre-
sents the binding constant per DNA base pair, can be obtained by
monitoring the changes in absorbances between 296 and 240 nm
with increasing concentrations of CT DNA from plots [DNA]/εa − εf
versus [DNA] and is given by the ratio of slope to the y intercept,
according to the following equation [47]:
log Ip (ꢀA) = 0.4774 log v(mV/s) − 0.2572
r: 0.9968 for [Fe(Cl)3(TMP)(H2O)]
log Ip (ꢀA) = 0.2750 log v(mV/s) + 0.4593
r: 0.9842 for [Ru(Cl)3(TMP)(H2O)]·(H2O)
The slopes (between 0.27 and 0.64) of the relationship are close
to the theoretically expected (0.5) for an ideal reaction of solu-
tion species, so in this case the process has a diffusive component
[43–45].
The mutual effect of the complexes with CT DNA has been
studied with UV spectroscopy and CV in order to investigate the
possible binding modes to CT DNA and to calculate the binding
constants to CT DNA (Kb). In UV titration experiments, the spectra
of CT DNA in the presence of each complexes (or TMP) have been
recorded for a constant CT DNA concentration in diverse [com-
plex (or TMP)]/[CT DNA] mixing ratios (r). The intrinsic binding
constants, Kb, of the complexes (or TMP) with CT DNA have been
determined through the UV spectra of the complexes recorded
for a constant complex (or TMP) DNA concentration (4 × 10−5 M)
in the absence and presence of CT DNA for diverse r values. The
interaction of the complexes (or TMP) with CT DNA has been
also investigated by monitoring the changes observed in the cyclic
voltammogram of a 1 × 10−5 M (1/9 methanol/buffer solution con-
taining) of the complexes upon addition of CT DNA at diverse r
values. The transition metal complexes are known to bind to DNA
via both covalent and/or noncovalent interactions [27,29,31,46]. In
covalent binding the labile ligand of the complexes is replaced by
a nitrogen base of DNA such as guanine N7. Moreover, the non-
covalent DNA interactions include intercalative, electrostatic and
groove (surface) binding of metal complexes along outside of DNA
helix, along major or minor groove. It has been reported that DNA
can provide three distinctive binding sites for all metal complexes;
namely, groove binding, electrostatic binding to phosphate group
and intercalation [47]. This behavior is of great importance with
regard to the relevant biological role of pyrimidine antibacterials
in the body [48]. Fig. 11 illustrates the spectral changes occurred
in 1 × 10−4 M methanolic solution of [Cu(Cl)2(TMP)] upon addi-
tion of increasing amounts of CT DNA. Even though no appreciable
change in the position of the intraligand band of metal complexes
have been observed by addition of CT-DNA, the intensity of the
band centered at 274 nm for [Cu(Cl)2(TMP)] (from 270 to 261 for
[DNA]
εa − εf
[DNA]
εb − εf
1
=
+
Kb(εb − εf)
where εa = Aobsd/[Complex], εf = extinction coefficient for the
free complex and εb = extinction coefficient for [Cu(Cl)2(TMP)],
[Zn(Cl)2(TMP)]·(H2O), [Pt(Cl)2(TMP)]·3(H2O), [Fe(Cl)3(TMP)(H2O)]
and [Ru(Cl)3(TMP)(H2O)]·(H2O) in the fully bound form. The high
value of Kb obtained for [Cu(Cl)2(TMP)], [Pt(Cl)2(TMP)]·3(H2O),
[Ru(Cl)3(TMP)(H2O)]·(H2O),
[Zn(Cl)2(TMP)]·(H2O)
and
[Fe(Cl)3(TMP)(H2O)], respectively, suggest
a
strong binding
of complexes to CT DNA (Table 5). Indeed, it is much higher
than Kb calculated for TMP (=1. 0.02 × 103 M−1), indicat-
ing that the coordination of TMP ligand to M(II)/(III) ion
enhance significantly the ability to bind to CT DNA. This is an
important point Kb of [Cu(Cl)2(TMP)], [Pt(Cl)2(TMP)]·3(H2O),
[Ru(Cl)3(TMP)(H2O)]·(H2O),
[Zn(Cl)2(TMP)]·(H2O)
and
[Fe(Cl)3(TMP)(H2O)] is higher than the EB binding affinity for
DNA (Kb = 1.23 0.07 × 105) suggesting that electrostatic and
Table 5
The intrinsic binding constants (Kb) of complexes with CT DNA.
Complexes
Kb ( 0.03)
TMP
[Cu(CI)2(TMP)]
[Pt(CI)2(TMP)]·3(H2O)
[Ru(Cl)3(TMP)(H2O)]·(H2O)
[Zn(CI)2(TMP)]·(H2O)
[Fe(CI)3(TMP)(H2O)]
2.50 × 104
6.66 × 107
6.57 × 107
6.48 × 107
6.25 × 107
6.14 × 107

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N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
Fig. 13. Biological diagram of TMP and metal complexes, 1: Candida albicans, 2: Sac-
charomyces cerevicia, 3: Eschericha coli, 4: Enterobacter cloaca, 5: Bacillus megaterium,
6: Bacillus cereus, 7: Pseudomonas sp, 8: Brusella melitensis, and 9: Staphylococcus
aureus.
Fig. 12. The cyclic voltammograms of the [Cu(Cl)2(TMP)] complex in the presence
of CT DNA in diverse r values. (a) 3 × 10−4 M [Cu(Cl)2(TMP)], (b) 100 ␮L DNA, and
(c) 500 ␮LDNA. Initial potential: 1500 mV, high potential: 1500 mV, low potential:
−1500 mV, scan rate: 100 mV/s, sensitivity: 10 ␮A/V, and number of the segments:
2.
CV study is that complexes [Cu(Cl)2(TMP)], [Pt(Cl)2(TMP)]·3(H2O),
[Ru(Cl)3(TMP)(H2O)]·(H2O),
[Zn(Cl)2(TMP)]·(H2O)
and
[Fe(Cl)3(TMP)(H2O)] can bind to DNA by both intercalation and
electrostatic interaction. Besides, the E1/2 values of all complexes
have been decreased after DNA addition.
The susceptibility of certain strains of bacterium towards TMP
and its complexes has been judged by measuring the size of
inhibition zone diameter. Antibacterial activities of TMP and its
complexes have been carried out with three Gram-positive (Bacil-
lus megaterium, Bacillus cereus, Staphylococcus aureus) and four
Gram-negative (Eschericha coli, Enterobacter cloaca, Pseudomonas
sp., Brusellamelitensis) bacteria. And antifungal screenings have
been tested against two fungi (Candida albicans, Saccharomyces
cerevicia). The test solutions have been prepared in DMSO. The
results of the biological activities are summarized in Fig. 13. The
synthesized compounds have been found to have remarkable bac-
tericidal and fungicidal properties. Surprisingly [Cu(Cl)2(TMP)] and
[Zn(Cl)2(TMP)]·(H2O) complexes show excellent activity against all
type of bacteria and fungi. But, [Ru(Cl)3(TMP)(H2O)]·(H2O) complex
show activity only against Saccharomyces cerevicia fungi.
Such an increased activity of the metal chelates as compared
to the TMP can be explained on the basis of chelation theory
[53]. Chelation considerably reduces the polarity of the metal ion
because of the partial sharing of its positive charge with the donor
groups and possible p-electron delocalization over the chelate ring.
Such chelation increases the lipophilic character of the central
metal ion, which subsequently favors the permeation through the
lipid layer of cell membrane. It is likely that the increased lipo-
solubility of the ligand up on metal chelation may contribute to
its facile transport into the bacterial cell which blocks the metal
binding sides in enzyme of microorganisms [54,55].
Fig. 14a–h presents the scanning electron microscopic (SEM)
images of the free PGE (a), activated PGE (b), CT DNA immo-
bilized onto PGE (c), TMP on CT DNA immobilized onto
PGE (d), [Cu(TMP)Cl2] on CT DNA immobilized onto PGE (e),
[Pt(Cl)2(TMP)]·3(H2O) CT DNA immobilized onto PGE (f), Cu(II)
ions on CT DNA immobilized onto PGE (g) and Pt(II) ions on CT
DNA immobilized onto PGE (h). The surface of the activated PGE
(Fig. 14b) is smoother than the surface of the free PGE (Fig. 14a).
When DNA is immobilized onto the surface of PGE (Fig. 14c), it
has been seen that the surface of PGE changes and some plats
occurs on the surface [56,57]. The SEM image of the TMP on CT
DNA immobilized onto PGE has been shown in Fig. 14d. The effec-
tive change on the surface and increase of the plats shows the
interaction between TMP and DNA. SEM photographs of the syn-
thesized copper and platinum complexes of TMP on CT DNA are
illustrated in Fig. 14e and f, respectively. The [Pt(Cl)2(TMP)]·3(H2O)
intercalative interaction may affect EB displacement [49]. Also, the
Kb value of free TMP has been found 2.5 × 104. This value is too
smaller than the complexes Kbs values. This result clearly shows
the importance of complexation.
The electrochemical investigations of metal–DNA interactions
can provide a useful complement to spectroscopic methods, e.g.,
for nonabsorbing species, and yield information about interactions
with both the reduced and oxidized form of the metal [50]. In
Fig. 12, the cyclic voltammograms of the [Cu(Cl)2(TMP)] in the
absence and presence of CT DNA, respectively, in diverse r values
in Tris–HCl (pH 7) buffer solution are shown. It has been pointed
out that the electrochemical potential of a small molecule will shift
positively when it intercalates into DNA double helix, and if it is
bound to DNA by electrostatic interaction, the potential would shift
in a negative direction [29,51]. Additionally, in the case of more
potentials than one, a positive shift of Ep1 and a negative shift of
Ep2 imply that the molecule can bind to DNA by both intercalation
and electrostatic interaction [20,52–54]. The quasi-reversible
redox couple for each complex in 1/9 methanol/buffer solution
that has been studied upon addition of CT-DNA and the shifts of
the cathodic Epc and anodic Epa potentials are given in Table 6. Any
new redox peaks have been appeared after the addition of CT DNA
to each complex, but the current intensity of all the peaks have
been increased significantly, suggesting the existence of an inter-
action between each complex and CT DNA. The increase in current
intensity can be explained in terms of an equilibrium mixture of
free and DNA-bound complex to the electrode surface [52]. It can
be observed that complex for [Cu(Cl)2(TMP)] exhibits the same
electrochemical behavior upon addition of CT DNA at diverse r
values. For increasing amounts of CT DNA, the anodic potential Epa1
does not change after DNA binding but the cathodic potential Epc1
shows a negative shift (ꢂEpc = −15 mV for [Cu(Cl)2(TMP)], while the
anodic potential Epa2 shift to more positive values (ꢂEpa = +56 mV
for [Cu(Cl)2(TMP)]. These shifts of the potentials show that all
complexes can bind to DNA by both intercalation and electro-
static interaction [54]. A positive shift of the cathodic potential
(ꢂEpc2 = +10 mV) of complex ([Cu(Cl)2(TMP)]) has been observed
while the first anodic potential remains unchanged (Fig. 13)
followed by a slight increase of current intensity. The existence
of intercalation between complex ([Cu(Cl)2(TMP)]) and CT DNA
bases can be suggested [27,50]. The conclusion derived from the

N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
253
Fig. 14. The SEM images of free PGE (a), activated PGE (b), CT DNA immobilized onto PGE (c), TMP CT DNA immobilized onto PGE (d), [Cu(TMP)Cl2] CT DNA immobilized
onto PGE (e), [Pt(Cl)2(TMP)]·3(H2O) CT DNA immobilized onto PGE (f), Cu(II) ions on CT DNA immobilized onto PGE (g) and Pt(II) ions CT DNA immobilized onto PGE (h).

254
N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
Table 6
Cathodic and anodic potentials and the shift of the potentials in the absence and presence of CT-DNA of [Cu(CI)2TMP] complex.
f
f
f
f
f
f
f
f
Complex
Redox couple
Cu(II)/Cu
Epc1
Epa1
ꢂE1
(E1/2
)
1
Epc2
Epa2
ꢂE2
(E1/2 )
2
[Cu(CI)2TMP]
+143
+8
−135
75.5
−603
+98
+701
−252.5
b
b
b
b
b
b
b
b
Complex
Epc1
Epa1
+6
ꢂE1
(E1/2
+67
)
1
Epc2
Epa2
ꢂE2
(E1/2
)
2
[Cu(CI)2TMP]
+128
−122
−593
+154
+747
−142.5
ꢂE = Epa − Epc; E1/2 = 1/2 × (Epa + Epc).
complex CT DNA immobilized onto PGE has changed the surface
much more than the [Cu(TMP)Cl2] complex CT DNA immobilized
onto PGE. In addition to all of these data, it has been recorded
that SEM images of Cu(II) ions on CT DNA immobilized onto
PGE (Fig. 14g) and Pt(II) ions on CT DNA immobilized onto PGE
(Fig. 14h). Similar to platinum complex, the platinum(II) ions have
caused big changes on the surface respectively to copper(II) ions.
This immobilization on the surface has also been recorded as
voltammetric signal [38,57]. The optimization of the immobiliza-
tion step of CT DNA on the PGE is shown in Fig. 15a as voltammetric
signal. The guanine oxidation peak has been used as an indicator.
Different concentrations of CT DNA have been immobilized and
the maximum surface coverage the PGE has been obtained before
interaction with TMP (or metal ions or metal complexes) by DPV
technique. The guanine has been oxidized at about +1.02 V. For
finding the optimum concentration of CT DNA, five different con-
centrations between 1 and 10.00 ppm have been studied. During
this step, CT DNA has been immobilized at +0.5 V. Guanine peak
current has been increased with increasing CT DNA concentra-
tion to 4 ppm and then leveled off. The effects of the experimental
parameters have been studied to find optimum analytical con-
ditions for interaction between TMP and CT DNA (Fig. 15b). The
binding of the TMP to the CT DNA modified PGE has been optimized
depending on the interaction concentration by DPV in Tris–HCl
buffer solution containing 30% ethanol. After the interaction with
TMP, the guanine signal has been decreased linearly. In this way,
different TMP concentrations have been studied between 0.00
and 12.00 ppm as seen in Fig. 15b. The best results have been
obtained at 10.00 g/mL TMP concentration. Similar calibration stud-
ies have been done for [Cu(TMP)Cl2] complex and the results have
been given in Fig. 15c. According to voltammetric results, voltam-
metric signal of TMP CT DNA immobilized onto PGE is greater
than the[Cu(TMP)Cl2] complex CT DNA immobilized onto PGE
(Fig. 15d).
Fig. 15. The effect of CT DNA concentration at oxidation signal at PGE for the optimization of immobilization of dsDNA (a). DP voltammograms for the interaction of 4 ppm CT
DNA modified PGE with several concentrations of TMP (b). DP voltammograms for the interaction of 4 ppm CT DNA modified PGE with several concentrations of [Cu(TMP)Cl2]
complex (c). DP voltammograms for the interaction of 4 ppm CT DNA modified PGE with 4 ppm TMP and 4 ppm [Cu(TMP)Cl2] complex (d).

N. Demirezen et al. / Spectrochimica Acta Part A 94 (2012) 243–255
255
4. Conclusion
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The synthesis and characterization of five new TMP to com-
plexes with Cu(II), Pt(II), Zn(II), Fe(III) and Ru(III) have been
synthesized with physicochemical and spectroscopic methods. The
study of the complexes interaction with CT DNA has been per-
formed with UV spectroscopy and cyclic voltammetry and it reveals
that the complexes can bind to DNA. UV spectroscopic titrations
have been used in order to calculate the binding strength of the
complexes with CT DNA which is mirrored in the intrinsic binding
constant Kb [Cu(TMP)Cl2] complex exhibits much higher intrinsic
binding constant to CT DNA than the other complexes. The results
have been described in this study show that changing the metal
environment can modulate the binding property of the complex
with DNA [42]. Information obtained from the present is helpful to
the development of nucleic acids molecular probes and therapeutic
agents. In addition, it would be of considerable interest if the novel
DNA adducts of [Cu(TMP)Cl2] complex led to a broader spectrum
of antibacterial activity. Cyclic voltammetric studies have shown
that all complexes bind to CT DNA by both intercalation and elec-
trostatic interaction. The most of bacterial infections now defy all
known antibiotics and the antibiotic resistance is a growing prob-
lem in our environment. There is a great need for new antibacterial
agents and metal complexes as novel derivatives of pyrimidines
can play an important role in this field. In most cases, it has been
evidenced that the antimicrobial activity of the complexes is com-
parable to free pyrimidine. According to our biological results,
because of the chelate effect, some complexes ([Cu(Cl)2(TMP)],
[Fe(Cl)3(TMP)(H2O)] and [Zn(Cl)2(TMP)]·(H2O)) exhibited higher
inhibitory activity than TMP against some microorganisms.
The investigation of drug–CT DNA interactions would provide
new compounds to be tested for an effect on a biochemical tar-
get, for the design of DNA biosensors, which will further become
DNA microchip systems [56]. In this paper, the interaction between
antibacterial drug TMP and its Cu(II), Zn(II), Pt(II), Fe(III), Ru(III) and
CT DNA has been investigated by voltammetry and spectrophotom-
etry for the first time. An electrochemical CT DNA biosensor has
been prepared by immobilizing CT DNA onto PGE surface. The oxi-
dation signal of guanine has been used as probe for the investigation
of the interaction between compounds and CT DNA. As a result of
the interaction of compounds in different concentrations with CT
DNA, a decrease has been observed in the response based on the
signal of guanine. This phenomenon can be explained by the dam-
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of the diffusion of compounds [57]. Also, interaction of CT DNA in
different concentrations with compounds, positive/negative peak
potentials shift may indicate that the compound binding to DNA by
both intercalation and electrostatic interaction. It is clear that, the
results of spectrophotometry and voltammetry used glassy carbon
electrode are in accordance with the results of voltammetry used
PGE. The utility of this electrochemical biosensor for the interaction
between CT DNA and compounds are cost effective and it provides
rapid detection.
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The authors wish to thank TUBITAK (Project No.: 109T020),
KSU (Project No.: 2009/4-12 and Project No.: 2010/5-19) for the
financial supports and Prof. Dr. Metin Dıgrak (Department of Biol-
ogy, Faculty of Science and Letters, University of Kahramanmaras
Sutcu Imam, Campuse of Avsar, 46100 Kahramanmaras, Turkey) for
antimicrobial studies.
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