Synthesis and characterization of transition metal 2,6-pyridinedicarboxylic acid derivatives, interactions of Cu(II) and Ni(II) complexes with DNA in vitro

Article abstract of DOI:10.1016/j.saa.2008.10.001

Mononuclear complexes M(L)Cl₂ where M = Mn(II), Fe(II), Co(II), Ni(II) and Cu(II) and (L = N,N-diethylpiperazinyl,2,6-pyridinedicarboxylate), have been synthesized and characterized by elemental analysis, FT-IR, ¹H NMR spectroscopy,

Full text of DOI:10.1016/j.saa.2008.10.001

Spectrochimica Acta Part A 72 (2009) 421–428
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis and characterization of transition metal 2,6-pyridinedicarboxylic acid
derivatives, interactions of Cu(II) and Ni(II) complexes with DNA in vitro
Sadaf Khan , Shahab A.A. Nami , K.S. Siddiqi , Eram Husain , Imrana Naseemb
a
a
a,∗
b
a
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
Department of Biochemistry, Aligarh Muslim University, Aligarh 202002, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 June 2008
Accepted 10 October 2008
Mononuclear complexes M(L)Cl2 where M = Mn(II), Fe(II), Co(II), Ni(II) and Cu(II) and (L = N,N-
diethylpiperazinyl,2,6-pyridinedicarboxylate), have been synthesized and characterized by elemental
analysis, FT-IR, H NMR spectroscopy, UV–vis, magnetic moment, TGA/DSC, cyclic voltammetry and con-
1
ductivity measurement data. The spectral data suggests that the dipicolinic acid acts as a bidentate ligand
and is coordinated to the metal ion through the carboxylate oxygen. The cyclic voltammogram for Cu(L)Cl2
complex was found to display two reversible Cu(II)/Cu(I) and Cu(II)/Cu(III) redox couple. The ligand
exhibits a two-step thermolytic pattern while the complexes decompose in three stages respectively.
An octahedral geometry has been proposed for both the complexes. The investigation of the interaction of
the complexes with calf thymus DNA has been performed with absorption spectroscopy and fluorescence
quenching experiments, which showed that the complexes are avid binders of calf thymus DNA. Also
the interaction of the Cu(II) and Ni(II) complexes with plasmid DNA (pUC 19) was studied using agarose
gel electrophoresis. The results revealed that these complexes can act as effective DNA cleaving agents
resulting in the nicked form of DNA (pUC 19) under physiological conditions. The gel was run both in the
absence and presence of an oxidizing agent (H2O2). The ligand and its complexes have also been screened
against microbes in order to study their antibacterial action. The results revealed that the Cu(II) complex
has activity comparable with the reference drugs gentamycin and flucanzole.
Keywords:
Mononuclear
Dipicolinic acid
Piperazine
DNA cleavage
Electrophoresis
Antibacterial activity
©
2008 Elsevier B.V. All rights reserved.
1
. Introduction
as an oxidative degradation product of vitamins, coenzymes and
alkaloids and are also an important constituent of fulvic acids. DPA
complexes of iron are well-known electron carriers in various bio-
logical models and are recognized as specific molecular tools for
DNA cleavage [7]. DPA is also an intermediate in the tryptophan
degradation pathway and is a precursor for NAD [8].
Pyridine-2,6-dicarboxylic acid or dipicolinic acid (DPA) is known
to form stable chelates with metal ions and oxometal cations and
can display widely varying coordination demeanour functioning as
a bidentate, tridentate, meridian or bridging ligand [1,2]. DPA can
stabilize unusual oxidation states and can form bridging hydrogen
bonds due to its functional groups [3]. It is a versatile N,O-chelating
agent with limited stearic hindrance and can further provide possi-
bility to form polymeric complexes through bridging coordination
of carboxylates under suitable conditions [4]. DPA can act as an
interesting ligand due to its ability to form strong covalent bonds.
Also the spatial separation of two carboxylate groups attached
to the same aromatic ring leads to either a polymeric chain or a
cyclo-oligomeric ring structure and there is a potential influence of
nitrogen atom on the coordination mode [5].
Transition metal complexes have been the subject of thorough
investigation because of their extensive applications in wide rang-
ing areas from material sciences to biological sciences [9]. Metal
complexes are well-known to accelerate the drug action and the
efficacy of a therapeutic agent can often be enhanced upon coor-
dination with a metal ion [10]. The pharmacological activity has
also been found to be highly dependent on the nature of the
metal ion and the donor sequence of the ligands as different lig-
ands exhibit different biological properties [11]. In recent years,
the binding studies of transition metal complexes have become an
important field in the development of DNA molecular probes and
chemotherapeutics [12]. There is a substantial literature supporting
the application of artificial DNA cleaving agents in biotechnology;
structural studies of nucleic acids or development of new drugs.
Compounds showing the properties of effective binding as well as
cleaving double stranded DNA under physiological conditions are
A very important characteristic of these ligands is their diverse
biological activity [6]. They are present in many natural products,
∗ Corresponding author.
E-mail address: kssiddiqi@yahoo.co.in (K.S. Siddiqi).
1386-1425/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2008.10.001

4
22
S. Khan et al. / Spectrochimica Acta Part A 72 (2009) 421–428
the 600–200 cm 1 range was scanned with CsI on a Nexus FT-IR
Thermo Nicolet (Wisconsin). The Electronic spectra were recorded
on a Cintra 5GBC spectrophotometer in DMSO. The NMR spectra
were recorded on a DPX-300 spectrometer in DMSO at room
temperature. The conductivity measurements were carried out
on a CM-82T Elico conductivity bridge in DMSO. Magnetic sus-
ceptibility measurements were done with a 155 Allied Research
vibrating sample magnetometer at room temperature. TGA was
performed with a PerkinElmer (Pyris Diamond) thermal analyzer
under nitrogen atmosphere using alumina powder as reference.
The weight of the sample was kept between 8 and 12 mg and the
−
of much importance since these could be used as diagnostic agents
in medicinal and genomic research. With the recognition of cis-
platin as the anticancer drug the introduction of metal-based drugs
has gained importance. However, the greatest disadvantage of cis-
platin and other heavy metal-based drugs is their toxicity [13]. It
will be more appropriate to use biologically soft metal ions and
their complexes, reducing the toxic effect and enabling faster and
efficient removal of the drug from the body. Transition metal com-
plexes interact with DNA through covalent binding, electrostatic
interactions, groove binding or intercalation [14,15]. Studies have
proved that macrocyclic complexes as chemical nucleases are the
efficient DNA cleavage agents. Most of the enzymes that partici-
pate in the biochemistry of nucleic acids require divalent metal ion
cofactors to promote their activity for the cleavage of phosphodi-
ester bond of DNA [16].
◦
heating rate was maintained at 10 C/min. Cyclic voltammetry
was performed on a 0.1 mM DMSO solution. The potential was
scanned at variable rates from 25–100 mV/s. The potentiometer
was a Princeton Applied Research model 263A attached to a
P.C. using Electrochemistry Powersuite Software. The working
electrode was a glassy carbon electrode, the counter electrode
was a platinum wire and the reference electrode consisted of Ag,
AgCl/KCl (saturated).
Metal complexes of pyridine carboxylic acids and some of their
derivatives have also been used as model systems for the design
of new metallopharmaceutical compounds [17]. Lately DPA has
become one of the more suitable ligands for modelling potential
pharmacologically active compounds because of its low toxicity and
amphophilic nature [18].
2.2. Synthesis
Copper being a bio-essential element its complexes find more
applications in nucleic acid chemistry as compared to other heav-
ier transition metal elements [19]. Over a dozen of enzymes that
depend on copper, for their activity have been identified; the
metabolic conversions catalyzed by all these enzymes are oxida-
tive in nature [20]. Due to their importance in biological processes,
Cu(II) complexes synthesis and activity studies have been the
focus from different perspectives. Recently, the studies have proved
that various macrocycles of copper as chemical nucleases are effi-
cient DNA cleaving agents [21]. Due to the versatile biological and
chemical properties of DPA we are reporting the synthesis and
characterization of a ligand L where L = N,N-diethylpiperazinyl,2,6-
pyridinedicarboxylate and its mononuclear complexes with Mn(II),
Fe(II), Co(II), Ni(II) and Cu(II) ions. Since Cu(II) and Ni(II) are bio-
chemically active their interaction with pUC 19 plasmid DNA has
been investigated in order to ascertain the probable mechanism of
their cleavage under physiological conditions.
2.2.1. Synthesis of the ligand L
(L = N,N-diethylpiperazinyl,2,6-pyridinedicarboxylate)
DPA (2 mmol, 0.33 g) was dissolved in 15 mL of methanol.
Dichloroethane (4 mmol, 0.35 mL) was dissolved in 5 mL of the
same solvent. The dissolved dichloromethane was then carefully
added drop by drop into the methanolic solution of DPA under con-
tinuous stirring. This reaction mixture was then refluxed on a hot
plate for 3 h. A white precipitate appears at this time which dis-
solves and becomes a clear solution on further refluxing of 4 h. This
clear solution was then brought to room temperature. Piperazine
hexahydrate (2 mmol, 0.38 g) was dissolved in 15 mL methanol and
then it was added to the clear solution form of reaction mixture
slowly accompanied by continuous stirring on a magnetic stirrer.
The addition of piperazine hexahydrate gives an immediate pre-
cipitation. The reaction mixture in the form of a white precipitate
was further refluxed for 4 h in order to ascertain complete precip-
itation. Finally after refluxing, the white precipitate obtained was
filtered using a Whatman filter paper (no. 100). It was then washed
with methanol and dried in vacuo (Scheme 1). The progress of the
reaction was monitored by thin layer chromatography (TLC).
2. Experimental
2.1. Chemicals and methods
2.2.2. Synthesis of complexes
Hydrated metal chlorides, 2,6-pyridinedicarboxylic acid
Aldrich), dichloroethane (Merck), piperazine hexahydrate (Loba
2.2.2.1. Synthesis of the complex Mn(L)Cl2 where L = N,N-
diethylpiperazinyl,2,6-pyridine dicarboxylate. The ligand (2 mmol,
0.60 g) was dissolved in 15 mL of hot methanol in a round bottom
flask. Metal halide (2 mmol, 0.39 g) was dissolved in minimum
quantity of the same solvent. It was then gradually added to the
ligand solution with stirring, to obtain an immediate precipitation
accompanied by a visible colour change. The reaction mixture was
then refluxed for 6 h on a hot plate, which affords the precipitate.
(
Chemie) were used as received. CT-DNA and pUC 19 plasmid
DNA were purchased from Sigma. Methanol was distilled prior to
use. Elemental analyses (C, H, N and S) were carried out with a
Carlo Erba EA-1108 analyzer. The metal contents were estimated
−
1
by complexometric titration [22]. IR spectra (4000–400 cm
)
were recorded on a RXI FT-IR spectrometer as KBr disc while
Scheme 1. Schematic diagram for the preparation of the ligand.

S. Khan et al. / Spectrochimica Acta Part A 72 (2009) 421–428
423
Scheme 2. Schematic diagram for the preparation of the metal complexes where M = Mn(II), Fe(II), Co(II), Ni(II), and Cu(II).
The precipitation of the complex ensures the completion of the
reaction. The complex was then carefully filtered by using What-
man filter paper (no. 100), washed with methanol and dried in
vacuo (Scheme 2).
ligand solution with stirring, to obtain an immediate precipitation
accompanied by a visible colour change. The reaction mixture was
then refluxed for 6 h on a hot plate, which affords the precipitate.
The precipitation of the complex ensures the completion of the
reaction. The complex was then carefully filtered by using What-
man filter paper (no. 100), washed with methanol and dried in
vacuo (Scheme 2).
2
.2.2.2. Synthesis of the complex Fe(L)Cl2 where L = N,N-
diethylpiperazinyl,2,6-pyridinedicarboxylate.. The ligand (2 mmol,
.60 g) was dissolved in 15 mL of hot methanol in a round bottom
0
flask. Metal halide (2 mmol, 0.39 g) was dissolved in minimum
quantity of the same solvent. It was then gradually added to the
ligand solution with stirring, to obtain an immediate precipitation
accompanied by a visible colour change. The reaction mixture was
then refluxed for 6 h on a hot plate, which affords the precipitate.
The precipitation of the complex ensures the completion of the
reaction. The complex was then carefully filtered by using What-
man filter paper (no. 100), washed with methanol and dried in
vacuo (Scheme 2).
2.3. Interaction of the complexes with DNA–fluorescence
spectroscopy
The DNA binding studies were carried out in 0.02 mol/L of phos-
phate buffer containing 60 mM NaCl at pH 7.0 at room temperature.
Calf thymus DNA as sodium salt (CT-DNA) was purchased from
Sigma and the purity was checked by the absorbance A260/A280
which was found to be 1.86 in phosphate buffer [23] at pH 7.0.
The concentration of ethidium bromide (EB), which binds to DNA
by interacting and enhancing the fluorescence intensity, was kept
at 4 ␮M. The concentration of DNA (25 ␮M) and EB was kept con-
stant. The fluorescence studies were carried out with increasing
concentration of the complexes ranging from 5 to 25 ␮M. Since the
metal complexes do not fluoresce themselves a fluorophore like EB
was utilized. All the samples were excited at 340 nm. Emission was
recorded at 500–600 nm. The emission slit was 10 nm.
2
.2.2.3. Synthesis of the complex Co(L)Cl2 where L = N,N-
diethylpiperazinyl,2,6-pyridinedicarboxylate.. The ligand (2 mmol,
.60 g) was dissolved in 15 mL of hot methanol in a round bottom
0
flask. Metal halide (2 mmol, 0.47 g) was dissolved in minimum
quantity of the same solvent. It was then gradually added to the
ligand solution with stirring, to obtain an immediate precipitation
accompanied by a visible colour change. The reaction mixture was
then refluxed for 6 h on a hot plate, which affords the precipitate.
The precipitation of the complex ensures the completion of the
reaction. The complex was then carefully filtered by using What-
man filter paper (no. 100), washed with methanol and dried in
vacuo (Scheme 2).
2.4. Interaction of the complexes with CT-DNA-absorption
spectroscopy
DNA stock solutions (5 mM) were prepared by dilution of CT-
DNA with buffer (containing 150 mM NaCl and 15 mM trisodium
◦
citrate at pH 7.0) followed by exhaustive stirring at 4 C for 3 days
◦
2
.2.2.4. Synthesis of the complex Ni(L)Cl2 where L= N,N-
diethylpiperazinyl,2,6-pyridinedicarboxylate.. The ligand (2 mmol,
.60 g) was dissolved in 15 mL of hot methanol in a round bottom
[24] and the solutions were kept at 4 C for a week. The nucleotide
concentrations were determined by the absorption at 260 nm using
− −1
1
0
ε = 6600 M cm (expressed as phosphate).
flask. Metal halide (2 mmol, 0.47 g) was dissolved in minimum
quantity of the same solvent. It was then gradually added to the
ligand solution with stirring, to obtain an immediate precipitation
accompanied by a visible colour change. The reaction mixture was
then refluxed for 6 h on a hot plate, which affords the precipitate.
The precipitation of the complex ensures the completion of the
reaction. The complex was then carefully filtered by using What-
man filter paper (no. 100), washed with methanol and dried in
vacuo (Scheme 2).
2
.5. Assay of nuclease activity
The DNA cleavage by the complexes was probed using pUC 19
supercoiled plasmid DNA employing agarose gel electrophoresis.
The metal complexes were dissolved in 50 mM Tris–HCl/18 mM
NaCl buffer (pH 7.2). Increasing concentrations of the metal com-
plexes 1 ␮L from different concentrations (5–25 ␮M) were taken in
clean eppendorf tubes and plasmid DNA (1 ␮L of 0.10 ␮g/mL) was
◦
added. The contents were incubated at 37 C for 12 h and loaded on
2
.2.2.5. Synthesis of the complex Cu(L)Cl2 where L = N,N-
diethylpiperazinyl,2,6-pyridinedicarboxylate.. The ligand (2 mmol,
.60 g) was dissolved in 15 mL of hot methanol in a round bottom
1% agarose gel after mixing 5 ␮L of loading buffer (25% bromophe-
nol, 30% glycerol (3 ␮L) and 0.25% xylene cyanol). Electrophoresis
was performed at a constant voltage (80 V) till the bromophenol
blue reached to three-fourth of the gel. The gel was stained for
10 min. by immersing it in ethidium bromide solution. It was then
0
flask. Metal halide (2 mmol, 0.34 g) was dissolved in minimum
quantity of the same solvent. It was then gradually added to the

4
24
S. Khan et al. / Spectrochimica Acta Part A 72 (2009) 421–428
de-stained by keeping in sterile distilled water for 10 min. Plasmid
band was visualized by viewing the gel under transilluminator and
photographed. The efficiency of the DNA cleavage was measured
by determining the ability of the complex to form open circular or
nicked forms, respectively.
3.1. Infrared spectroscopy
Various coordinating modes exists in the pyridine carboxylic
acid complexes which can be distinguished from the difference
in asymmetric and symmetric carboxylate [26] stretching bands,
−
−
ꢀ
ꢁ = ꢁasCOO − ꢁsymCOO . If there is a marked difference of over
−
1
2.6. Pharmacology
200 cm it implies monodentate coordination while a difference
of about 100 cm confirms a bidentate coordination. The carboxy-
late ion may coordinate to a metal ion in one of the following ways.
−
1
The antibacterial and antifungal activity of a solution of freshly
synthesized complexes in dichloromethane was tested against
some gram positive and gram negative bacteria such as, E. coli, S.
aureus, C. albicans and A. flavus. The activity was then compared
with some reference antibiotics that were purchased from the mar-
ket. All the complexes and the parent ligand were screened for their
activity against the test organisms. The hole plate diffusion method
was adopted for the activity measurements. The bacterial strains
were grown in nutrient agar slants and the fungal strains were
grown in Sabouraud dextrose agar slants. The viable bacterial cells
were swabbed onto Nutrient agar plates and the fungal spores onto
Sabouraud dextrose agar plates. The compounds were dissolved in
dichloromethane to a final concentration of 0.1%. A 0.5 cm diameter
well was cut in a medium inoculated with the respective cultures,
and the test solutions of the compounds in different concentrations
In the IR spectra of the complexes the ꢁ(C–O) bands decrease in
intensity significantly as compared to free ligand, L and new bands
−
1
appear at 1623, 1625 and 1415, 1412 cm corresponding to ꢁas and
ꢁ
ꢁ
s frequencies, respectively. The large difference between ꢁas and
−
1
s (ꢀꢁ > 200 cm ) frequencies indicates monodentate coordina-
−
1
tion [27]. The absence of any band in the 3000–3450 cm region
implies deprotonation of carboxylate groups leading to a ring like
structure (Scheme 1) [28]. Furthermore, the absence of any band in
(
5 and 1 ␮g) for bacterial cultures and (15 and 50 ␮g) for fungal
cultures were allowed to stay in the wells. The petri plates were
incubated for 36 h for bacterial cultures and 72 h for fungal cultures.
All the compounds were screened against flucanazole as reference
for fungal cultures and gentamycin for bacterial cultures in their
standard concentration of 200 ␮g/well. The activity of the com-
pounds was evaluated by measuring the diameter of the inhibition
zone around the respective wells.
−
1
3
000–3450 cm region can also be corroborated with the depro-
tonation of piperazine. Piperazine exists in two conformations i.e.
chair and boat form although the chair form has a higher stability.
However, the boat form is predominant [29] in case of bidentate
chelation with the same metal ion such as [PtCl (Me ppz)]. In our
2
2
case too, the piperazinato moiety is constrained to form a ring like
structure thus existing in a boat conformation (Table 2) [30].
3. Results and discussion
3.2. NMR spectroscopy
The complexes were obtained in moderate yield by the reaction
of metal chloride, MX .nH O with ligand, L in methanolic media
The 1H NMR spectrum of the free DPA in DMSO exhibits a sharp
2
2
(
Scheme 2).
singlet at ı 10.14 ppm due to the carboxylic acid protons, which is
found to be absent in metal complexes indicating the formation of
C–O–C bonds [31]. The eight piperazinium protons were found to
resonate at 3.15 ppm as a singlet in case of ligand and do not alter
after complexation [32]. The methylene protons were observed at
2.64 ppm in case of ligand and are shifted slightly when complexed
with metal ions (2.66–2.69 ppm) [33].
MX ·nH O + L → M(L)X
2
2
2
where M = Mn(II), Fe(II), Co(II), Ni(II), Cu(II), X = Cl and L = N,N-
diethylpiperazinyl,2,6-pyridinedicarboxylate. Elemental analysis,
FT-IR, UV–vis spectroscopy, 1H NMR, cyclic voltammetry and
TGA/DSC were used to characterize the complexes. The com-
plexes are soluble in H O and common organic solvents. The
2
−3
conductivity measurement (10 M) in DMSO indicated them to
be non-electrolytes [25] (Table 1).
3.3. Electronic spectroscopy and magnetic moment
The interaction of Ni(L)Cl2 and Cu(L)Cl2 complexes with pUC
9 was studied by monitoring the conversion of circular super-
The electronic spectral bands and the magnetic moments of
1
the complexes are listed in Table 3. In octahedral environment,
Mn(II) complex gives spin forbidden as well as parity-forbidden
bands. In addition to the band due to n–␲* transition, the elec-
tronic spectrum of Mn(II) complex in DMSO exhibits three more
coiled DNA (Form I) to nicked DNA (Form II). The amount of strand
scission was assessed by agarose gel electrophoresis. The interca-
lating ability of the complexes was probed by DNA–EB system using
fluorescence quenching spectroscopic experiments which suggests
that the Cu(II) complex is a better intercalator than the Ni(L)Cl2.
−1
−1
and
bands in the region 31104–30350 cm ; 22542–20533 cm
17513–16890 cm
−1
which have been assigned to 4T1g(P) ← A1g;
6
Table 1
Physicochemical properties of the ligand and its complexes.
Molar conductance (ꢂ 1 mol cm2)
−
−1
Analysis, (%) Found (calculated)
Compounds
Colour
Yield (%)
C
H
N
O
M
L
White
Pale yellow
Brown
Grey
Green
Blue
80
62
67
70
68
55
17
27
42
17
11
29
58.78 (59.0)
41.93 (41.74)
41.48 (41.69)
41.63 (41.40)
41.46 (41.42)
40.87 (40.96)
6.19 (6.27)
4.53 (4.43)
4.57 (4.43)
4.28 (4.40)
4.35 (4.40)
4.22 (4.35)
13.79 (13.76)
9.48 (9.74)
9.66 (9.72)
9.43 (9.65)
9.44 (9.66)
9.41 (9.55)
20.74 (20.95)
14.55 (14.84)
15.08 (14.81)
14.81 (14.70)
14.86 (14.71)
14.75 (14.55)
–
Mn(L)Cl2
Fe(L)Cl2
Co(L)Cl2
Ni(L)Cl2
Cu(L)Cl2
12.82 (12.74)
12.85 (12.92)
13.48 (13.54)
13.68 (13.49)
14.67 (14.44)

S. Khan et al. / Spectrochimica Acta Part A 72 (2009) 421–428
425
Table 2
Characteristic IR bands (in cm−1) of the ligand and its complexes.
Complex
ꢁasym
ꢁsym
Ring vibration
ꢁ(C–N)
ꢁ(M–X)
L
1724
1623
1625
1629
1631
1635
–
1300 w, 1270 m
1288 w, 1262 m
1285 w, 1264 m
1291 w, 1266 m
1284 w, 1254 m
1278 w, 1244 m
1634
1598
1606
1604
1588
1586
–
Mn(L)Cl2
Fe(L)Cl2
Co(L)Cl2
Ni(L)Cl2
Cu(L)Cl2
1415
1412
1425
1431
1418
380 w
367 w
355 w
340 w
327 w
Table 3
Magnetic susceptibility, electronic spectra and ligand field parameters of the complexes.
−
Log ε (l mol cm1)
−1
−1
−1
)
Complex
Mn(L)Cl2
Magnetic moment (B.M.)
5.77
Electronic bands (cm 1
)
Possible assignments
10 Dq (cm
20075
)
B (cm
944
ˇ
4
A1(G) ← 6A1
22430
3.2
2.7
4.1
3.0
2.1
3.2
2.8
1.8
1.2
0.75
4
5
4
4
1
1
2
2
6
20640
T1(G) ← A1
5
Fe(L)Cl2
Co(L)Cl2
5.32
4.52
23529
17460
E ← T2
–
–
–
–
–
–
T1(P) ← 4A2(F)
4
1
1380
23696
5503
18529
4814
T1(F) ← A2(F)
1
Ni(L)Cl2
Cu(L)Cl2
Diamagnetic
2.13
B1g ← A1g
15560
–
1613
–
0.64
–
1
1
A2g ← A1g
2
A1g ← B1g
1
Eg ← 2B1g
4T2g(G) ← A and 4T1g(G) ← A transitions, respectively. The
6
6
B.M. regardless of the stereochemistry and is temperature indepen-
dent. However, the room temperature magnetic moment of 2.11
B.M. in our case is in agreement with that found for octahedral
Cu(RPO)py2 complex [42].
1g
5
1g
high spin d configuration gives an essentially spin-only mag-
netic moment of ∼5.9 B.M., which is temperature independent.
The Mn(II) complex under consideration has the value of 5.84
B.M. which is close to the calculated value. Thus the ligand field
bands and magnetic moment value support an octahedral geometry
around the metal ion [34].
3.4. Cyclic voltammetry
For the octahedral spin-free Fe(II) complexes the magnetic
moment values lies at about 5.5 B.M. and is nearly independent
of temperature. In the present case the magnetic moment values
are found to be 5.30 B.M. for Fe(L)Cl2 complex. The deviation in the
magnetic moment value may be ascribed to a slight distortion from
the regular octahedral geometry [35] (Table 3).
The complexes are soluble in common organic solvents and
the electron transfer behavior of the Cu-complex was studied
in CH CN by two-vertex cyclic voltammetry in −2.0 to +2.0 V
3
range with variable scan rate (25–100 mV/s). The cyclic voltammo-
gram at Pt disk electrode for this complex display two reversible
Cu(II)/Cu(I) and Cu(II)/Cu(III) redox couple (Fig. 1). It is known
that the redox potential of Cu(II)/Cu(I) process is shifted towards
more negative potential as the electron-donating ability of the
substituents on the ligand framework becomes higher [43]. The
ligand is electrochemically active due to the presence of the
Three intense bands exhibited by the Co(L)Cl at 15480; 20781
2
−1
4
and 24272 cm are attributed to the three transitions from T1g(F)
ground state to 4T2g(F), A2g(F) and 4T1g(P) states, respectively
which is characteristic of the high-spin octahedral Co(II) complexes
4
−1
−1
[
36]. Dq = 1580 cm and B = 753 cm are also in accordance with
pyridinium moiety [44]. The copper(II) ion yields two quasi-
reversible redox couple, located at E1 = 0.445 V and E
2
=
the octahedral environment of Co(II) complexes [37]. The room
temperature magnetic moment of 4.39 B.M. further supports an
octahedral arrangement around Co(II) ion in Co(L)Cl2.
1/2
1/2
0.805 V which are assignable to Cu(II)/Cu(I) and Cu(II)/Cu(III),
respectively (E1/2 = (Epa + Epc)/2). The criterion of reversibility was
Generally, bivalent octahedral Ni(II) complexes exhibit three
spin allowed d–d bands which shift to a higher wave num-
ber depending upon the nature of the coordinating ligand [38].
The electronic spectrum of the Ni(L)Cl2 complex in aqueous
medium exhibits two absorption bands in the region 9560–9090
verified by the ratio, i
pa/ pc
= 0.89–0.92 ␮A with variation of scan
i
rates. Any redox behaviour of the ligand in the range −1.25 to 1.50 V
is ruled out because dipicolinic acid shows quasi-reversible and
irreversible process at −1.58 V [45].
−1
corresponding to the 4T2g ← A
4
and 16722–16129 cm
and
2g
4
4
T1g(F) ← A transitions, respectively, which is characteristic of
2g
−
1
an octahedral Ni(II) ion [39]. A broad hump observed at 12500 cm
is also present and is probably due to the spin forbidden transi-
tion similar to that observed by Prushan et al. for the Ni(L)Cl2 and
Ni(L)(NO3)2 where L = trithiatridecane-2,12–dionedioxime [40].
Generally the magnetic moment for octahedral Ni(II) complex, lies
between 2.9 and 3.4 B.M. Our value (3.26 B.M) is well within the
specified limit therefore, an octahedral geometry is proposed for
the Ni(II) ion.
The blue colored Cu(L)Cl2 complex in the present case exhibits
a broad band centered at 730 nm encompassing several tran-
sitions characteristic of an octahedral geometry [41]. However,
several attempts to resolve this band were unsuccessful due to
the absence of cubic field symmetry. The magnetic moment of
mononuclear Cu(II) complexes generally lies between 1.75 and 2.20
Fig. 1. Cyclic voltammogram of Cu(L)Cl2.

4
26
S. Khan et al. / Spectrochimica Acta Part A 72 (2009) 421–428
Table 4
Thermal degradation of ligand and its complexes.
Complex
First decomposition stage
Second decomposition stage
Third decomposition stage
Residue
Fragments
Temperature
range ( C)
Mass loss
data, found
Fragments
Temperature
range ( C)
Mass loss
data, found
(calculated)
(%)
Fragments
Temperature
range ( C)
Mass loss
data, found
(calculated)
(%)
◦
◦
◦
(
(
calculated)
%)
L
C8H16 N2
2HCl
2HCl
2HCl
2HCl
162–255
182–220
180–224
182–220
180–224
182–220
46.31 (45.93)
16.91 (16.76)
17.02 (16.58)
16.91 (16.76)
17.02 (16.58)
16.91 (16.76)
C7H3NO4
C8H14 N2
C8H14 N2
C8H14 N2
C8H14 N2
C8H14 N2
260–400
225–330
225–310
225–330
225–310
225–330
52.44 (54.07)
31.11 (31.77)
31.25 (31.43)
31.11 (31.77)
31.25 (31.43)
31.11 (31.77)
–
–
–
–
Mn(L)Cl2
Fe(L)Cl2
Co(L)Cl2
Ni(L)Cl2
Cu(L)Cl2
C7H3NO3
C7H3NO3
C7H3NO3
C7H3NO3
C7H3NO3
360–470
360–470
360–470
360–470
360–470
33.87 (34.28)
33.57 (33.90)
33.87 (34.28)
33.57 (33.90)
33.87 (34.28)
MnO
FeO
CoO
NiO
CuO
2HCl
3.5. Thermal studies (TGA/DSC)
The thermal analysis of the ligand and its mononuclear com-
plexes was carried out under an inert atmosphere (N ). The
2
ligand exhibits a two-step thermolytic pattern while the com-
plexes decompose in three stages, respectively (Table 4). The
◦
decomposition of the free ligand begins at 162 C and exhibits a
◦
sharp endotherm (absent in the complexes) at 195 C. However, in
◦
the case of complexes the first decomposition spans from 180 C
◦
through 220 C corresponding to the liberation of chloride ion as
HCl (found ∼17%). The results show a significant increase in the
decomposition temperature when compared with the ligand, indi-
cating a higher thermal stability of the complexes [46]. The absence
◦
of any thermal change before 150 C indicates that the restructuring
of the ligand and its complexes does not take place before the ini-
tiation of the degradation process and also rules out the possibility
Fig. 2. Emission spectra of Cu(L)Cl2 complex (excited at 550 nm, 5 ml solution):
a) control (4 ␮M EB + 25 ␮DNA); (b) 4 ␮M EB + 25 ␮DNA + 5 ␮M Cu(L)Cl2; (c) 4 ␮M
of any water or solvent molecule in the ligand and its complexes
(
◦
[
47]. The second thermolytic step starts at 220 C and terminates
EB + 25 ␮DNA + 10 ␮M Cu(L)Cl2; (d) 4 ␮M EB + 25 ␮DNA + 15 ␮M Cu(L)Cl2; (e) 4 ␮M
EB + 25 ␮DNA + 20 ␮M Cu(L)Cl ; (f) 4 ␮M EB + 25 ␮DNA + 25 ␮M Cu(L)Cl .
◦
at about 360 C, corresponding to the decomposition of piperazine
and ethane linkage. The third stage of pyrolysis corresponds to
2
2
3
4% weight loss consistent with the expulsion of dipicolinic moiety
pretreated with EB causes appreciable reduction in the emission
intensity indicating that the DNA bound EB fluorophore is partially
replaced by the Cu(II) complex (Fig. 2). A similar treatment with
Ni(II) complex showed a less pronounced effect indicating that
the Cu(II) complex is a better intercalator than the Ni(II) analogue
leaving behind metal oxide as the end product [48].
The DSC plots are consistent with the decomposition pattern of
the ligand and its complexes. Two broad peaks observed at 195 and
3
A sharp exothermic peak obtained for the liberation of chloride
reflects fast decomposition process, implying volatilization of the
complex upon heating. However there is no well-defined peak for
the formation of metal oxide as the end product.
◦
65 C indicate the pyrolysis of the ligand as an exothermic process.
(Fig. 3).
3.6. Interaction of the complexes with Calf thymus
DNA-fluorescence spectroscopy
EB is one of the most sensitive fluorescent probes that can bind
with DNA. There is an increase in the fluorescence of EB due to
intercalation with DNA. If the metal complex intercalates with
DNA it leads to a decrease in the binding sites of DNA available
for EB, which is reflected, from a decrease in the fluorescence of
the EB-DNA system [49]. Generally metal complexes interact with
DNA via intercalation or through covalent binding of the nitrogen
bases. However, due the presence of benzene ring, an intercala-
tive binding mode may be more plausible. The metal complex
might have penetrated between the double helical structure of DNA
through hydrogen bonding leading to a decrease in the binding
sites of DNA–EB system. Consequently a decrease in fluorescence
is observed on addition of metal complex in the DNA–EB system
and the effect is enhanced with the increase in the concentra-
tion of the complex. Accordingly, the titration of DNA–EB with
Ni(II) and Cu(II) complexes decreased the intensity of fluorescence
spectra in our system. The addition of Cu(L)Cl2 complex to DNA
Fig. 3. Emission spectra of Ni(L)Cl2 complex (excited at 550 nm, 5 ml solution): (a)
Control (4 ␮M EB + 25 ␮DNA); (b) 4 ␮M EB + 25 ␮DNA + 10 ␮M Ni(L)Cl2; (c) 4 ␮M
EB + 25 ␮DNA + 15 ␮M Ni(L)Cl2; (d) 4 ␮M EB + 25 ␮DNA + 20 ␮M Ni(L)Cl2; (e) 4 ␮M
EB + 25 ␮DNA + 25 ␮M Ni(L)Cl2.

S. Khan et al. / Spectrochimica Acta Part A 72 (2009) 421–428
427
Fig. 5. Agarose gel electrophoresis diagram showing the cleavage of pUC 19 plasmid
DNA by Cu(L)Cl2 in various concentrations in TAE buffer at pH 7.2. lane (a) DNA con-
trol showing pUC 19 plasmid DNA in supercoiled form, form I; lane (b) DNA + Cu(L)Cl2
(
5 ␮M); lane (c) DNA + Cu(L)Cl2 (5 ␮M) + H2O2; lane (d) DNA + Cu(L)Cl2 (10 ␮M);
lane (e) DNA + Cu(L)Cl2 (10 ␮M) + H2O2 lane (f) DNA + Cu(L)Cl2 (20 ␮M); lane (g)
DNA + Cu(L)Cl2 (20 ␮M) + H2O2.
Fig. 4. Agarose gel electrophoresis diagram showing the cleavage of pUC 19 plasmid
DNA by Ni(L)Cl2 in various concentrations in TAE buffer at pH 7.2. lane (a) DNA con-
trol showing pUC 19 plasmid DNA in supercoiled form, form I; lane (b) DNA + Ni(L)Cl2
(
5 ␮M); lane (c) DNA + Ni(L)Cl2 (10 ␮M); lane (d) DNA + Ni(L)Cl2 (15 ␮M); lane (e)
in the intensity of supercoiled DNA band although the intensity of
the nicked DNA increases apparently. These observations confirm
that the Cu(II) complex acts as a better nuclease at a physiologically
relevant concentration.
DNA + Ni(L)Cl2 (20 ␮M) lane (f) DNA + Cu(L)Cl2 (20 ␮M).
3
.7. Interaction of complexes with pUC 19 plasmid DNA
The DNA cleavage efficiency of metal complexes was examined
3.8. Study of the complexes with CT-DNA-absorption
spectroscopy
using supercoiled pUC 19 plasmid DNA as the target. Circular plas-
mid DNA is ideally suited to probe the cleavage as the DNA exists
in a supercoiled state in its native form and converts to a relaxed
form upon single strand scission. This is exhibited by altered migra-
tion rate during agarose gel electrophoresis, the fastest migration
will be observed for the supercoiled form (Form I). If one strand is
cleaved, the supercoils will relax to produce a slower moving open
circular form (Form II) [50]. If both the strands are cleaved a linear
form will be generated that migrates in between. In the quest to
find out whether the Ni(II) and Cu(II) complexes would exhibit DNA
cleavage activity in vitro their effects at various concentrations were
studied using pUC 19 plasmid DNA and the results are summarized
in Figs. 4 and 5. It was found that both the Ni(II) and Cu(II) complexes
can effectively cleave the supercoiled DNA, but the nuclease activity
of the later complex was pronounced. This behavior is verified from
the gel electrophoresis experiments (Fig. 4) where copper shows a
better nuclease activity as compared to nickel at the highest equiv-
alent concentration (20 ␮M) leading to a pronounced DNA damage.
The greater nuclease activity of Cu(II) complex may be attributed
to its redox nature i.e. the ease of reduction of Cu(II) to Cu(I).
It was also observed that the DNA cleaving ability of Cu(L)Cl2
complex was further enhanced in the presence of an oxidant like
H O . It is evident from Fig. 5 that the copper complex cleaves DNA
The absorption spectra of the interaction of the Ni(L)Cl2 and
Cu(L)Cl2 complexes with CT-DNA have been recorded for a constant
−
4
CT-DNA concentration of 3.125 × 10 M in presence and absence
of an oxidant, H2O2. Representative spectra of Ni(L)Cl2 and Cu(L)Cl2
−
6
complexes having a concentration of 4 × 10 M along with CT-DNA
alone is given in Fig. 6. The spectra clearly shows that Cu(L)Cl2 com-
plex is a better intercalator than its Ni analogue which might be due
to the redox nature of Cu(L)Cl2 complex. The DNA cleavage activity
of Cu(L)Cl2 complex is further enhanced in presence of H2O2 due
to the increase in hydroxyl ion concentration by Fenton reaction
(Fig. 6).
3.9. Antibacterial activity
The results of the antimicrobial activity are presented in the
Table 5. The complexes display stable inhibition zones and are more
2
2
more efficiently in the presence of an oxidant, which is attributed to
the formation of hydroxyl radicals. The hydroxyl radical may have
been produced due to the reaction between the metal complex and
H O as shown below
2
2
Cu + H O → Cu + OH + OH•
+
2+
2
2
•
These OH free radicals participate in the oxidation of the
deoxyribose moiety, followed by hydrolytic cleavage of the sugar
phosphate backbone. The more pronounced nuclease activity in
case of Cu(L)Cl2 complex may be due to the greater ease of its oxi-
dation. The increase in concentration in each well led to a decrease
Fig. 6. Absorption spectra of Ni(L)Cl2 and Cu(L)Cl2 complexes in the presence of CT-
◦
DNA. The r value is 1:100. The spectra were recorded at 25 C after complexes had
◦
been incubated with CT- DNA for 24 h at 37 C.

4
28
S. Khan et al. / Spectrochimica Acta Part A 72 (2009) 421–428
Table 5
Inhibition zone in mm.
Compounds
E. coli
␮g
S. aureus
A. flavus
C. albicans
5
1 ␮g
5 ␮g
1 ␮g
5 ␮g
1 ␮g
5 ␮g
1 ␮g
L
14.2
15.6
11.2
21.9
23.5
25.0
26.0
11.2
13.6
10.2
20.2
20.3
22.6
26.0
15.7
21.5
17.5
21.9
27.3
28.7
30.0
13.2
20.3
15.8
20.5
23.4
25.5
30.0
15.4
11.0
9.5
16.4
18.2
19.8
20.0
13.7
10.0
9.0
15.9
17.0
18.6
22.0
10.4
16.9
10.2
15.3
18.3
20.3
22.0
10.2
14.8
10.0
14.8
18.1
20.1
22.0
Mn(L)Cl2
Fe(L)Cl2
Co(L)Cl2
Ni(L)Cl2
Cu(L)Cl2
Gentamycin/flucanazole
active than the ligand. They possess potential inhibitory activity in
amounts as low as 1 ␮g/well for bacterial cultures and 15 ␮g/well
for fungal cultures. The activity of the complexes is measured in
terms of inhibition of the replication of DNA by interacting with the
enzyme prosthetic group. The antibacterial results evidently show
that the activity of the ligand became more pronounced and signifi-
cant when coordinated to the metal ion (Table 5). This enhancement
in the activity may be due to an efficient diffusion of the metal com-
plexes in to the bacterial cell or interaction with the bacterial cell
walls. Also it has been observed that the activity of Mn(II), Fe(II)
and Co(II) complexes is lower than that of Cu(II) complex, while
that of Cu(II) and Ni(II) analogues is quite comparable. The antimi-
crobial activity of the metal complexes generally depends on the
following factors: the chelation ability of the ligand, the nature of
nitrogen donor ligands, the total charge of the complex, the exis-
tence and the nature of the metal ion neutralizing the ionic complex
and the nuclearity of the metal center in the complex [51]. Higher
activity of Cu(II) complex is probably due to the effective chelation.
This activity is quite comparable with the reference drugs gen-
tamycin/flucanzole. However, the reduced activities in some cases
can be attributed to the inability of the complexes to form hydrogen
bonds with the cell constituents [52].
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