Probing the DNA-binding behavior of tryptophan incorporating mixed-ligand complexes

Article abstract of DOI:10.1007/s00706-012-0718-4

Mixed-ligand Cu(II), Ni(II), Co(II), and Zn(II) complexes using a tryptophan-derived Schiff base (obtained by the condensation of tryptophan and benzaldehyde) as the primary ligand and 1,10-phenanthroline as the co-ligand were synthesized and characterized analytically and spectroscopically by performing elemental analyses, magnetic susceptibility and molar conductance measurements, UV-Vis, IR, NMR, and FAB-MS. The binding properties of metal complexes with DNA were investigated by electronic absorption spectroscopy, cyclic voltammetry, and by performing viscosity measurements, and the results showed that these complexes have the ability to interact with DNA via an intercalative mode. The DNA cleavage efficiencies of these complexes with pUC19 DNA were investigated by gel electrophoresis. The complexes were found to promote the cleavage of pUC19 DNA from the supercoiled form I to the open circular form II and the linear form III in the presence of ascorbic acid. Finally, the in vitro antibacterial activities of the Schiff base and its mixed-ligand metal complexes were screened against the bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhi. The antibacterial screening data revealed that the complexes show growth inhibitory activity against bacteria. Springer-Verlag 2012.

Full text of DOI:10.1007/s00706-012-0718-4

Monatsh Chem
DOI 10.1007/s00706-012-0718-4
ORIGINAL PAPER
Probing the DNA-binding behavior of tryptophan incorporating
mixed-ligand complexes
•
Natarajan Raman Sivasangu Sobha
•
Muthusamy Selvaganapathy
Received: 1 November 2011 / Accepted: 6 January 2012
Ó Springer-Verlag 2012
Abstract Mixed-ligand Cu(II), Ni(II), Co(II), and Zn(II)
complexes using
Introduction
a
tryptophan-derived Schiff base
(obtained by the condensation of tryptophan and benzal-
dehyde) as the primary ligand and 1,10-phenanthroline as
the co-ligand were synthesized and characterized analyti-
cally and spectroscopically by performing elemental
analyses, magnetic susceptibility and molar conductance
measurements, UV-Vis, IR, NMR, and FAB-MS. The
binding properties of metal complexes with DNA were
investigated by electronic absorption spectroscopy, cyclic
voltammetry, and by performing viscosity measurements,
and the results showed that these complexes have the
ability to interact with DNA via an intercalative mode. The
DNA cleavage efficiencies of these complexes with pUC19
DNA were investigated by gel electrophoresis. The com-
plexes were found to promote the cleavage of pUC19 DNA
from the supercoiled form I to the open circular form II and
the linear form III in the presence of ascorbic acid. Finally,
the in vitro antibacterial activities of the Schiff base and its
mixed-ligand metal complexes were screened against the
bacteria Staphylococcus aureus, Bacillus subtilis, Pseudo-
monas aeruginosa, Escherichia coli, and Salmonella typhi.
The antibacterial screening data revealed that the com-
plexes show growth inhibitory activity against bacteria.
The medicinal uses and applications of metals and metal
complexes are of increasing clinical and commercial
importance. Recent advances in chelation research have
paved the way for the development of ‘‘magic bullets’’ for
chemotherapy using different strategies and pharmacolog-
ical manipulation, thus demonstrating the significant
potential of the utilization of metal complexes as drugs,
and highlighting that medical inorganic biochemistry is a
flourishing field [1]. Physiologically interesting mixed-
ligand complexes of amino acids with heavy metal ions
play an important role in biological systems, and have
proven to be a subject of great interest to researchers [2–8].
Complexes of amino acids are involved in the exchange
and transport of trace metal ions in the human body [9].
Binding studies of small molecules to DNA are very
important in the development of DNA molecular probes
and new therapeutic reagents. Over the past few decades,
DNA-binding metal complexes have been extensively
studied as DNA structural probes, DNA-dependent electron
transfer probes, DNA footprinting and sequence-specific
cleaving agents, and potential anticancer drugs [10–13].
Many compounds exert their antitumor activity by binding
to DNA, and can damage DNA in cancer cells, blocking
the division of cancer cells and causing cell death [14].
The interaction of metal complexes with DNA is an area
of intense interest to both inorganic chemists and bio-
chemists. Transition metal complexes have been widely
exploited in probes of nucleic acid structure, and they show
nuclease activity. The important criterion for the develop-
ment of metallodrugs as chemotherapeutic agents is the
ability of the metallodrug to cause DNA cleavage [15].
Bacterial resistance is a major global health problem,
along with multidrug resistance in pathogenic species.
Keywords Tryptophan-derived Schiff base ꢀ
Mixed-ligand complexes ꢀ DNA binding ꢀ
DNA cleavage ꢀ Antibacterial activity
N. Raman (&) ꢀ S. Sobha ꢀ M. Selvaganapathy
Research Department of Chemistry, VHNSN College,
Virudhunagar 626 001, India
e-mail: drn_raman@yahoo.co.in
123

N. Raman et al.
UV spectra measurements
There is an urgent need to develop new antibacterials with
novel mechanisms of action to overcome the problem of
resistance [16]. Therefore, researchers are increasingly
turning their attention to folk medicine, looking for new
leads to develop better drugs for treating microbial
infections.
The geometries of the metal complexes were deduced from
the electronic spectra and magnetic data for the complexes.
The electronic spectra for the complexes were recorded in
DMSO solution. All of the complexes show a high-energy
absorption band in the region 27,248–33,112 cm-1. This
transition can be attributed to a charge-transfer band. The
electronic spectrum of the copper(II) complex 2 displays a
d–d transition band in the region 13,586 cm-1, which is
Bearing these facts in mind, in this paper we describe
the synthesis, structures, and antibacterial and cleavage
studies of a tryptophan-derived Schiff base and its mixed-
ligand transition metal complexes. To the best of our
knowledge, this is the first report on the chemical nuclease
activity of Schiff base mixed-ligand metal complexes of
tryptophan derivatives containing 1,10-phenanthroline and
various metal ions. The electron-transfer mechanism of the
mixed-ligand metal complexes was investigated using
cyclic voltammetry. The results enhance our understanding
of novel agents for targeting nucleic acids, as well as aid in
the synthesis of novel antibacterial drugs for bacterial
infections.
2
2
due to the Eg ? T2g transition. This d–d transition band
strongly favors a distorted octahedral geometry around the
metal ion. The absorption spectrum of the nickel(II) com-
plex 3 displays three d–d bands at 14,749, 17,168, and
23,392 cm-1. These bands correspond to A2g(F) ?
3
3
3
3
3T2g(F) (t1), A2g(F) ? T1g(F) (t2), and A2g(F) ?
3T1g(P) (t3) transitions, which are characteristic of an
octahedral geometry. This geometry is further supported by
its magnetic susceptibility value (3.21 BM). The electronic
spectrum of the cobalt(II) complex 4 displays three
d–d transition bands in the regions 14,992, 17,513, and
22,652 cm-1, which can be assigned to T1g(F) ?
4
Results and discussion
4
4
4
4T2g(F) (t1), T1g(F) ? A2g(F) (t2), and T1g(F) ?
4T2g(P) (t3) transitions. This indicates that the complex of
Co(II) is a six-coordinate complex, and probably has an
octahedral geometry, which is also supported by its mag-
netic susceptibility value (4.88 BM). The Zn(II) complex 5
is diamagnetic. According to the empirical formula, an
octahedral geometry is proposed for this complex.
The Schiff base ligand and its Cu(II), Ni(II), Co(II), and
Zn(II) mixed-ligand complexes were synthesized and
characterized based on spectral and elemental analysis
data. The complexes were found to be air stable. The ligand
was soluble in common organic solvents, and all of its
complexes were freely soluble in CHCl3, DMF, and
DMSO. The preparation of the Schiff base is presented
schematically in Fig. 1.
Mass spectra
The mass spectrum of the Schiff base ligand showed a peak
at m/z = 330 corresponding to the [C18H15KN2O2] ion.
The spectrum also exhibited peaks for the fragments at
m/z = 238, 115, 90, and 77, corresponding to [C11H7KN2O2],
[C8H5N], [C7H6], and [C6H5]. The spectra of the Cu(II),
Co(II), Ni(II), and Zn(II) complexes showed molecular ion
peaks at m/z = 715 (M?), 711 (M?), 710 (M?), and 717
([M?1]?), respectively. The Cu(II) complex gave a frag-
ment ion peak with the loss of one fragment of
phenanthroline at m/z = 534. All these fragments lead to
Elemental analysis and molar conductivity
measurements
Elemental analyses of the Schiff base ligand (KL, 1) and its
complexes were in agreement with the assigned formulae.
The metal complexes were dissolved in DMSO and the
molar conductivities of 10-3 mol/dm3 solutions were
measured at 25 °C. The high molar conductance values
(45.37–58.75 X-1 cm2 mol-1) of the complexes support
their electrolytic nature.
Fig. 1 Synthesis of the Schiff
base ligand 1
O
KOH, EtOH
HN
Reflux, 5 h
O
O
K
+
N
HN
OH
O
H2N
123

Probing the DNA-binding behavior of tryptophan incorporating mixed-ligand complexes
the formation of the species [ML], which further undergoes
demetallation to yield the species [L], which gives the
fragment ion peak at m/z = 291.
7.01–7.43 ppm due to the presence of aromatic protons.
The resonance peaks observed in the spectrum of the
[ZnL(phen)2]Cl complex at d = 7.77–8.26 ppm were
assigned to the protons of 1,10-phenanthroline (phen). The
zinc(II) complex also showed a multiplet signal at 6.63–
6.94 ppm due to indole protons present in the Schiff base
ligand. The azomethine proton (–CH=N) signal in the
spectrum of the zinc complex was shifted downfield
compared to the free ligand, suggesting deshielding of the
azomethine group due to coordination with the metal ion.
The 13C NMR spectrum of the ligand showed aromatic
carbons at 105.62–117.95 ppm and indole carbons at
118.74–136.62 ppm. The ligand also showed the COO-
carbon at 171.56 ppm and the HC=N carbon at
167.11 ppm, which were shifted downfield (to 165.47 and
157.25 ppm, respectively) upon coordination, indicating
that the COO- and HC=N groups participate in complex
formation. There were no appreciable changes in other
peaks.
IR spectra
The modes and sites of the coordination of the ligand to the
metal ions were investigated by comparing the infrared
spectra of the free ligand with those of its metal complexes.
The IR spectra of the complexes were very similar to each
other, except for some slight shifts and changes in intensity
by a few vibration bands with different metal ions, which
indicate that the complexes have similar structures. The
characteristic t(NH) mode due to the NH group of the
tryptophan moiety present in the Schiff base ligand was
observed at 3,252 cm-1. The appearance of this band in all
of the complexes indicates that the NH group is free from
complexation. In the IR spectrum of the Schiff base ligand,
the band observed at 1,614 cm-1 was shifted to a lower
frequency by 10–33 cm-1 on complexation, suggesting the
coordination of the azomethine nitrogen [17]. Moreover,
the coordination of the ligand to the metal center via the
carboxylic group is quite apparent based on the difference
in the positions of the maxima observed for tsy(COO-) and
Electron paramagnetic resonance spectra
The EPR spectrum of the copper complex 2 provides
important information relating to the metal ion environ-
ment. EPR spectra were recorded in DMSO at liquid
nitrogen temperature (LNT) and at room temperature (RT).
The spectrum of the copper complex at RT showed one
intense absorption band at high field, and is isotropic due to
the tumbling motion of the molecules. However, at LNT,
this complex showed well-resolved peaks in the low-field
t
asy(COO-). The bands were detected at 1,375 and
1,456 cm-1 for the free ligand, so Dvꢀ = 81 cm-1. For
comparison, tsy(COO-) and tasy(COO-) were recorded at
1,315–1,319 and 1,444–1,427 cm-1, respectively, for all
of the metal complexes. These results reveal that the
organic ligand is involved in coordination through the
carboxyl group. The new bands in the region of 503–516
and 470–484 cm-1 in the spectra of the complexes can be
assigned to the stretching frequencies of (M–O) and (M–N)
bonds, respectively.
region. The copper complex had A = 156.4 [ A\ = 34.1;
k
g = 2.259 [ g\ = 2.066, which suggests that the com-
k
plex is present in an axially elongated octahedral
geometry, and that the unpaired electron lies predomi-
nantly in the dx2ꢁy2 orbital. The axial symmetry parameter
G is defined as
Nuclear magnetic resonance spectra
1
The H NMR spectra of the Schiff base and its zinc(II)
g ꢁ 2:0023
?
k
G ¼
:
complex were recorded at room temperature in CDCl3. The
signal at d = 10.30 ppm (s, 1H) in the spectrum for the
Schiff base, assigned to the –NH proton of tryptophan,
remained unchanged in the zinc(II) complex. This shows
that the –NH group does not take part in complexation. The
ligand also showed one singlet at 8.90 ppm due to the
azomethine (CH=N) proton, and a multiplet signal at
around 6.81–7.39 ppm due to indole protons of the tryp-
tophan moiety. Moreover, it showed a phenyl multiplet
signal at 7.49–7.77 ppm due to aromatic protons of the
benzaldehyde moiety present in the tryptophan-derived
g ꢁ 2:0023
If G [ 4.0, the local tetragonal axes are aligned
parallel to each other, or are only slightly misaligned. If
G \ 4.0, significant exchange coupling is present and
the misalignment is appreciable. The observed value for
the exchange interaction parameter for the copper
complex (G = 4.04) suggested that the local tetragonal
axes are aligned parallel to each other or are slightly
misaligned, and the unpaired electron is present in the
dx2ꢁy2 orbital. This result also indicates that exchange
coupling effects do not operate in the present complex
[18]. Based on the above spectral and analytical data, the
proposed geometry of the metal(II) complexes is shown
in Fig. 2.
1
Schiff base. In the H NMR spectrum of the Zn(II) com-
plex 5, the protons of the Schiff base ligand were shifted
downfield due to the coordination with the metal ion.
Further, a set of multiplets was observed in the range
123

N. Raman et al.
structure. This spectral change highlighted the changes in
the conformation and structure of DNA after complexation
with DNA. Hypochromism results from a contraction of
the DNA along the axis of the helix, as well as from a
change in the conformation of the DNA, while hyper-
chromism results from damage to the double-helix
structure of the DNA [19]. The absorption spectrum of
[CuL(phen)2]Cl (2) showed an intense absorption band at
354.0 nm, [CoL(phen)2]Cl (4) showed such a band at
343 nm, [NiL(phen)2]Cl (3) did so at 342.6 nm, and
[ZnL(phen)2]Cl (5) at 351.9 nm in 5 mM Tris–HCl and
50 mM NaCl buffer solution (pH 7.2). These bands are due
to intraligand p–p* transitions. Increasing the concentra-
tion of CT-DNA resulted in a slight bathochromic shift of
2.8–1.9 nm and significant hypochromicity (31–23%).
These spectral characteristics clearly suggest that the metal
complexes most likely interact with DNA through a mode
that involves a stacking interaction between the aromatic
chromophore and the base pairs of DNA.
N
M
N
N
N
O
N
O
HN
Cl
Fig. 2 The proposed structure of the metal complexes. M = Cu(II),
Ni(II), Co(II), or Zn(II)
Investigating the interactions of the metal complexes
with DNA using electronic absorption spectroscopy
Electronic absorption spectroscopy has proven to be one of
the most useful techniques applied in DNA-binding stud-
ies. Absorption spectra of [CuL(phen)2]Cl (2) in the
absence and presence of DNA are given in Fig. 3.
The intrinsic binding constants (Kb) of the metal com-
plexes with CT-DNA were obtained by monitoring the
changes in the intraligand band as the concentration of
DNA was increased, and applying the following equation:
As the concentration of CT-DNA was increased, the
absorption bands of the complexes were affected, resulting
in a tendency for hypochromism, and a slight redshift was
observed in all of the complexes due to the intercalative
binding between DNA and the metal complexes. The
‘‘hyperchromic effect’’ and ‘‘hypochromic effect’’ are
spectral features of DNA relating to its double-helix
½DNAꢂ
½DNAꢂ
1
¼
þ
;
ðea ꢁ efÞ ðeb ꢁ efÞ Kbðeb ꢁ efÞ
where [DNA] is the concentration of DNA in base pairs, and
the absorption coefficients ea, ef, and eb are the apparent, free,
and bound metal complex extinction coefficients, respec-
tively. A plot of [DNA]/(eb - ef) versus [DNA] gives a slope
of 1/(eb - ef) and a Y intercept equal to [Kb/(eb - ef)]-1, where
Kb is the ratio of the slope to the Y intercept. Intrinsic binding
constants of 3.91 9 105, 2.20 9 105, 1.78 9 105, and
1.37 9 105 M-1 were determined for [CuL(phen)2]Cl,
[CoL(phen)2]Cl, [NiL(phen)2]Cl, and [ZnL(phen)2]Cl,
respectively. These values (Table 1) are comparable to that
of the classical DNA intercalator ethidium bromide
(1.4 3 106 M-1) [20]. These results suggest that the four
metal complexes interact with DNA via an intercalative
mode.
Table 1 Electronic absorption spectral properties of Cu(II), Ni(II),
Co(II), and Zn(II) complexes
Complex
k
max/nm
Dk/nm
H%
Kb 9 105/M-1
Free
Bound
Wavelength/nm
2
3
4
5
356.8
342.5
343.0
351.5
354.0
344.8
345.5
353.4
2.8
2.3
2.5
1.9
31
28
29
24
3.9
1.7
2.2
1.3
Fig. 3 Absorption spectral changes upon the addition of CT-DNA to
a solution of [CuL(phen)2]Cl in buffer (pH 7.2 at 25 °C) and in the
presence of increasing amounts of DNA. The arrow indicates the
change in absorbance upon increasing the DNA concentration
123

Probing the DNA-binding behavior of tryptophan incorporating mixed-ligand complexes
Viscosity measurements
The application of an optical photophysical technique to
investigate the interactions of DNA with metal complexes
generally provides clues that are needed—but not sufficient
by themselves—to support an intercalative binding model.
Therefore, viscosity measurements were introduced to
provide further support for this type of interaction between
the complexes and DNA. In the absence of crystallographic
structural data, hydrodynamic methods—which are sensi-
tive to the length of the DNA—are known to be among the
definitive and critical indicators of binding strength.
Intercalation is an effect of increasing the viscosity of DNA
[21]. The significant increase in the viscosity of DNA that
occurred upon the addition of a complex was due to
intercalation, which caused the DNA bases to separate in
order to the increase the effective size of the DNA, which
could be the reason for the increase in the viscosity. A plot
Potential/V
Fig. 5 Cyclic voltammogram of [CuL(phen)2]Cl in the presence of
increasing amounts of DNA in buffer (pH 7.2 at 25 °C). The arrow
indicates the change in voltammetric currents upon increasing the
DNA concentration
of (g/g0)1/3 versus [complex]/[DNA] (R, Fig. 4) gives a
measure of the change in viscosity. A gradual increase in
the relative viscosity was observed upon the addition of the
metal complexes to the DNA solution, suggesting that the
complexes mainly bind via an intercalation mode.
(EPa1 = -0.060 V, EPa2 = -0.097 V, EPa3 = -0.364 V)
and three cathodic peaks (EPc1 = -0.617 V, EPc2
=
Electrochemical properties of metal complexes
-0.399 V, EPc3 = -0.089 V). The oxidation of peak EPa1
refers to Cu(0)/Cu(I) and the reduction of Cu(I) occurred
at -0.617 V upon reversing the scan. The anodic
and cathodic peak potentials were separated by 0.557 V.
The ratio of the anodic to cathodic peak currents,
IPa/IPc = 1.35, indicated a quasi-reversible redox process.
The oxidation of peak EPa2 refers to Cu(I)/Cu(II) and the
reduction of Cu(II) occurred at -0.399 V upon reversing
the scan direction. The anodic and cathodic peak potentials
were separated by 0.496 V, and the ratio of the anodic to
cathodic peak currents, IPa/IPc = 1.40, indicated a quasi-
reversible redox process. The oxidation of peak EPa3 refers
to Cu(II)/Cu(III), and the reduction of Cu(III) occurred at
-0.089 V upon reversing the scan direction. The anodic
and cathodic peak potentials were separated by 0.453 V,
and the ratio of the anodic to cathodic peak currents,
IPa/IPc = 1.14, indicated a quasi-reversible redox process.
The formal potential E1/2, calculated as the average of EPa
and EPc, was -0.338, 0.116, and 0.137 V, respectively.
When CT-DNA was added to a solution of the complex,
both the anodic and cathodic peak current heights for the
complex decreased in the same way with increasing con-
centrations of DNA (Fig. 5). Also, during DNA addition,
the anodic peak potential (EPa), cathodic peak potential
(EPc), and E1/2 (calculated as the average of EPc and EPa) all
showed positive shifts. These positive shifts are considered
to be evidence for intercalation of the complex into the
DNA, because this kind of interaction is due to hydro-
phobic interactions. On the other hand, if a molecule binds
The cyclic voltammogram of [CuL(phen)2]Cl in buffer (pH
7.2 at 25 °C) is given in Fig. 5. The cyclic voltammogram
of 2 in the absence of CT-DNA featured three anodic peaks
R
Fig. 4 Effect of increasing amounts of [CuL(phen)2]Cl (filled
squares), [CoL(phen)2]Cl (filled diamonds), [NiL(phen)2]Cl] (filled
circles), and [ZnL(phen)2]Cl] (filled triangles) on the viscosity of
DNA. R = [complex]/[DNA]
123

N. Raman et al.
electrostatically to the negatively charged deoxyribose–
phosphate backbone of DNA, negative peak potential shifts
should be detected. Therefore, the positive shifts in the CV
peak potentials of the complex are indicative of an inter-
calative binding mode of the complex with DNA [22].
The cyclic voltammogram of [NiL(phen)2]Cl in the
absence of CT-DNA featured two anodic peaks
(EPa1 = 0.007 V, EPa2 = 0.272 V) and two cathodic peaks
(EPc1 = 0.411 V, EPc2 = -0.178 V). The oxidation of peak
EPa1 refers to Ni(I)/Ni(II), and the reduction of Ni(II)
occurred at -0.411 V upon reversing the scan direction. The
anodic and cathodic peak potentials were separated by
0.418 V, and the ratio of the anodic to cathodic peak cur-
rents, IPa/IPc = 1.38, indicated a quasi-reversible redox
process. The oxidation of peak EPa2 refers to Ni(II)/Ni(III),
and reduction of Ni(II) occurred at -0.178 V upon reversing
the scan direction. The anodic and cathodic peak potentials
were separated by 0.450 V, and the ratio of the anodic to
cathodic peak currents, IPa/IPc = 1.05, indicated a quasi-
reversible redox process. The formal potentials E1/2, taken as
the average of EPa and EPc, were -0.202 and 0.094 V.
For Co(III) ? Co(II), the redox couple cathodic peak
appeared at 0.105 in the absence of CT-DNA
(EPa = 0.365 V, EPc = 0.123 V, DEp = 0.242 V, and
E1/2 = -0.244 V). Note that the ratio IPa/IPc is approxi-
mately unity. This indicates that the reaction of the complex
on the glassy carbon electrode surface is a quasi-reversible
redox process. The incremental addition of CT-DNA to the
complex caused a positive shift in the formal potential
(E1/2), indicating that [CoL(phen)2]Cl had bonded favorably
with DNA via intercalation. Finally, the Zn(II) complex
exhibited a quasi-reversible transfer process with the redox
couple Zn(II) ? Zn(0), and the cathodic peak appeared at
-0.096 V in the absence of DNA (EPa = 0.418 V,
EPc = -0.096 V, DEp = 0.514 V, and E1/2 = -0.161 V).
The ratio of IPa/IPc is 0.92, which indicates a quasi-
reversible redox process for the metal complex. The
incremental addition of CT-DNA to the complex also
caused a positive shift in the formal potential (E1/2), indi-
cating that [ZnL(phen)2]Cl stabilizes the duplex (GC pairs)
by intercalation. The electrochemical parameters of the
Cu(II), Ni(II), Co(II), and Zn(II) complexes are shown in
Table 2. From these data, it is clear that all of the syn-
thesized complexes interact with DNA via intercalation.
Table 2 Electrochemical parameters for the interactions of DNA
with the Cu(II), Ni(II), Co(II), and Zn(II) complexes
Complex Redox couple
E
1/2/V
DEp/V
IPa/IPc
Free
Bound Free Bound
2
3
Cu(III) ? Cu(II) 0.137 0.128 0.453 0.435 1.35
Cu(II) ? Cu(I) -0.151 -0.116 0.496 0.503 1.40
Cu(I) ? Cu(0)
Ni(III) ? Ni(II)
Ni(II) ? Ni(I)
-0.338 -0.332 0.557 0.554 1.14
0.094 0.079 0.450 0.469 1.05
-0.202 -0.191 0.418 0.402 1.38
4
5
Co(III) ? Co(II) 0.244 0.260 0.242 0.245 1.18
Zn(II) ? Zn(0) 0.161 0.178 0.514 0.529 0.92
and DNA footprinting agents. Some chiral complexes have
the ability to display enantioselective DNA binding
between right- and left-handed DNA. Observations indi-
cate that DNA changes its conformation due to the binding
of metal complexes to DNA. DNA cleavage was analyzed
by monitoring the conversion of supercoiled DNA (form I)
to nicked DNA (form II) and linear DNA (form III) in the
presence of the oxidant ascorbic acid.
Figure 6 shows the results of gel electrophoretic sepa-
rations of plasmid pUC19 DNA induced by the addition of
metal(II) complexes in the presence of ascorbic acid.
Control experiments using DNA alone did not show any
significant cleavage of pUC19 DNA, even with long
exposure times (lane 1). Under the same conditions, free
ascorbic acid produced no cleavage of pUC19 DNA (lane
2), and the free metal complex alone did not show any
apparent cleavage of pUC19 DNA (lane 3). When pUC19
DNA was allowed to interact with metal complexes in the
presence of ascorbic acid, the mobility of the band was
found to increase slightly, as shown in Fig. 6. These phe-
nomena imply that Cu(II), Co(II), and Ni(II) complexes do
more damage to plasmid pUC19 DNA than the Zn(II)
complex does in the presence of ascorbic acid.
Antibacterial activity
Multi-drug-resistant Gram-positive and Gram-negative
pathogens have become a serious problem in hospitals and
the community [23]. To overcome the alarming problem of
microbial resistance to antibiotics, the discovery of novel
compounds that are active against new targets is a matter of
urgency. Hence, nowadays, researchers are focusing their
attention to folk medicine, looking for new leads to
develop better drugs against bacterial infections. So, at
present, pharmaceutical industries are looking for synthe-
sizing the alternative compounds which act as drugs. There
is a continuous search for more potent and cheaper raw
materials to feed the industry, so pharmaceutical industries
are now looking at synthesizing alternative compounds that
DNA cleavage efficiencies of the metal complexes
Great efforts have been made to develop DNA cleavage
compounds due to their potential application in biotech-
nology and as therapeutic agents. Studies pertaining to
DNA cleavage by synthetic reagents are of considerable
interest because of their utility as tools in molecular biol-
ogy. This is true of both sequence-specific DNA cleavers
123

Probing the DNA-binding behavior of tryptophan incorporating mixed-ligand complexes
1
2
3
4
5
6
7
8
Form I
Form III
Form II
Fig. 6 Changes in the agarose gel electrophoretic pattern of pUC19
DNA induced by ascorbic acid and Cu(II), Ni(II), Co(II), and Zn(II)
complexes: lane 1, DNA alone; lane 2, DNA ? ascorbic acid; lane 3,
DNA ? ligand ? ascorbic acid; lane 4, DNA ? [CuL(phen)2]Cl;
lane 5, DNA ? [CuL(phen)2]Cl ? ascorbic acid; lane 6, DNA ?
[NiL(phen)2]Cl ? ascorbic acid; lane 7, DNA ? [CoL(phen)2]
Cl ? ascorbic acid; lane 8, DNA ? [ZnL(phen)2]Cl ? ascorbic acid
act as drugs. Much attention is currently focused on the
synthesis of new metal complexes and the evaluation of
these agents for antibacterial activity. In the present work,
we tested the activity of the ligand and its metal(II) com-
plexes against bacteria. Ampicillin was used as a standard
drug for comparison. The microorganisms used in the
present investigations included Staphylococcus aureus and
Bacillus subtilis (Gram-positive bacteria) and Pseudomo-
nas aeruginosa, Escherichia coli, and Salmonella typhi
(Gram-negative bacteria). The diffusion agar technique
was used to evaluate the antibacterial activities of the
synthesized mixed-ligand complexes. Biocidal activity data
for the investigated compounds are summarized in Table 3.
From this table, we can infer that all of the metal
complexes have greater inhibitory effects than the free
ligand. The higher antimicrobial activities of the metal
complexes compared to the Schiff base may be due to
changes in structure that occur due to coordination and
chelation that cause the metal complexes to act as more
powerful antibacterial agents.
also contains many phosphates, carbonyls, and cysteinyl
ligands that maintain the integrity of the membrane by acting
as a barrier to diffusion and also provide suitable sites for
binding. Furthermore, increased lipophilicity enhances the
penetration of the complexes into the lipid membrane and the
blocking of metal-binding sites in the enzymes of microor-
ganisms [24]. While chelation is not the only criterion for
antibacterial activity, it is an intricate blend of several factors
such as the nature of the metal ion and the ligand, the
geometry of the metal complex, the lipophilicity, the pres-
ence of co-ligands, and steric and pharmacokinetic factors.
Conclusion
Four mixed-ligand Cu(II), Ni(II), Co(II), and Zn(II) com-
plexes of the tryptophan-containing Schiff base 1 and 1,10-
phenanthroline have been synthesized and characterized.
Elemental, conductivity, and FAB-MS analyses revealed the
stoichiometry and composition of the complexes of
[ML(phen)2]Cl. FT-IR, UV-Vis, 1H NMR, and EPR spectral
data, as well as magnetic measurements, confirmed the
bonding features of the above mixed-ligand complexes. The
binding of these complexes to CT-DNA was investigated in
detail via electronic absorbance titration, viscosity, and cyclic
voltammetry. The experimental results reveal that all of the
complexes can interact with DNA through an intercalative
binding mode. The results of agarose gel electrophoresis
indicate that the complexes exhibit the ability to cleave
pUC19 DNA in the presence of ascorbic acid. A study of the
antibacterial activities of the complexes showed that they all
exhibit good biological activity against different organisms.
Chelation reduces the polarity of the metal ion consider-
ably because of the partial sharing of its positive charge with
donor groups, and also due to p-electron delocalization
across the whole chelating ring. Lipids and polysaccharides
are important constituents of the cell wall and membranes
that are preferred for metal ion interactions. The cell wall
Table 3 Minimum inhibitory concentrations (in mg cm-3) of the
synthesized compounds with respect to the growth of five bacteria
species
Compound S. aureus P. aeruginosa E. coli B. subtilis S. typhi
1
2
3
4
5
8
11
23
18
21
14
16
6
10
27
19
21
23
16
13
31
24
19
21
19
19
15
17
17
25
21
18
20
14
Experimental
All reagents, tryptophan, benzaldehyde, and metal(II)
chlorides were Merck products and used as supplied.
Ampicillin 12
123

N. Raman et al.
Commercial solvents were distilled and then used to pre-
pare the ligand and its complexes. DNA was purchased
from Bangalore Genei (India). Microanalyses (C, H, N)
were performed on a Carlo Erba 1108 analyzer at the
Sophisticated Analytical Instrument Facility (SAIF), Cen-
tral Drug Research Institute (CDRI), Lucknow, India.
Molar conductivities were measured via 10-3 mol/dm3
solutions in DMSO at room temperature using a Systronic
model 304 digital conductivity meter. Magnetic suscepti-
bility measurements of the complexes were carried out
using a Gouy balance with copper sulfate pentahydrate
employed as the calibrant. IR spectra were recorded with a
Shimadzu spectrophotometer in the 4,000-400 cm-1
range using KBr pellets. NMR spectra were recorded on a
Bruker Avance 300 FT-NMR spectrometer in CDCl3 using
TMS as the internal reference. FAB-MS spectra were
recorded with a VGZAB-HS spectrometer at room tem-
perature in a 3-nitrobenzylalcohol matrix. EPR spectra
were recorded on a Varian E 112 EPR spectrometer in
DMSO solution at both room temperature (300 K) and
LNT (77 K) using diphenylpicrylhydrazyl as the g-marker.
The absorption spectra were recorded using a Shimadzu
model UV-1601 spectrophotometer at room temperature.
anhydrous metal(II) chloride (0.003 mol) for ca. 1 h. A
methanolic solution (5 cm3) of 1,10-phenanthroline (0.006
mol) was added to this mixture, and the stirring was con-
tinued for 1 h. The solid product obtained was filtered and
washed with ethanol.
2-(Benzylideneamino)-3-(indol-3-yl)propionatebis-
(1,10-phenanthroline)copper(II) chloride
(2, C42H31ClCuN6O2)
Yield 86%; FT-IR (KBr): vꢀ = 3,252 (NH), 1,581 (HC=N),
1,427 tasy(COO), 1,315 tsy(COO), 511 (M=O), 478 (M=N)
cm-1
; ; leff = 1.76 BM;
Km = 45.37 X-1 cm2 mol-1
UV-Vis (DMSO): kmax = 31,496, 27,248, 13,586 cm-1
MS: m/z = 715.
;
2-(Benzylideneamino)-3-(indol-3-yl)propionatebis-
(1,10-phenanthroline)nickel(II) chloride
(3, C42H31ClN6NiO2)
Yield 76%; FT-IR (KBr): vꢀ = 3,252 (NH), 1,571 (HC=N),
1,431 tasy(COO), 1,315 tsy(COO), 516 (M=O), 473 (M=N)
cm-1
; ; leff = 3.21 BM;
Km = 51.23 X-1 cm2 mol-1
UV-Vis (DMSO): kmax = 23,392, 17,168, 14,749 cm-1
MS: m/z = 710.
;
2-(Benzylideneamino)-3-(indol-3-yl)propionatebis-
(1,10-phenanthroline)cobalt(II) chloride
(4, C42H31ClCoN6O2)
Potassium 2-(benzylideneamino)-3-(indol-3-yl)propionate
(1, C18H15KN2O2)
Tryptophan (2.04 g, 0.01 mol) was dissolved in 40 cm3 of
a water–ethanol mixture (1:1) and added to a hot ethanolic
solution (30 cm3) of KOH. The resulting solution was
stirred to obtain a homogeneous solution. Then, an
ethanolic solution of 1.06 g benzaldehyde (0.01 mol) was
added to this solution dropwise, and the resultant mixture
was refluxed for ca. 5 h. A pale yellow colored solution
was obtained, which was reduced to one-third on a water
bath. The solid complex that precipitated was filtered off,
washed thoroughly with ethanol, and dried in vacuo. Yield
77%; FT-IR (KBr): vꢀ = 3,252 (NH), 1,581 (HC=N), 1,456
Yield 71%; FT-IR (KBr):vꢀ = 3,256 (NH), 1,593 (HC=N),
1,436 tasy(COO), 1,317 tsy(COO), 507 (M = O), 481
(M = N) cm-1; Km = 55.40 X-1 cm2 mol-1; leff = 4.88
BM; UV-Vis (DMSO): kmax = 22,652, 17,513,
14,992 cm-1; MS: m/z = 710.
2-(Benzylideneamino)-3-(indol-3-yl)propionatebis-
(1,10-phenanthroline)zinc(II) chloride
(5, C42H31ClN6O2Zn)
Yield 73%; FT-IR (KBr):vꢀ = 3,256 (NH), 1,604 (HC=N),
1,444 tasy(COO), 1,319 tsy(COO), 503 (M=O), 478 (M=N)
1
cm-1; H NMR (CDCl3): d = 10.61 (s, 1H, NH), 8.60 (s,
t
asy(COO), 1,375 tsy(COO) cm-1 1H NMR (CDCl3):
;
1H, CH=N), 7.77–8.26 (m, 16H, phen), 7.01–7.43 (m, 5H,
phenyl), 6.63–6.94 (m, 5H, indole), 3.20 (q, 1H, CH), 2.60
(t, 2H, CH2) ppm; 13C NMR (CDCl3): d = 52.60 (CH),
54.02 (CH2), 105.43, 108.15, 113.79, 112.85, 118.45
(aromatic C), 120.25, 125.97, 127.57, 129.18, 139.94,
147.54 (indole C), 157.25 (CH=N), 165.47 (COO-) ppm;
d = 10.30 (s, 1H, NH), 8.90 (s, 1H, CH=N), 7.49–7.77 (m,
5H, phenyl), 6.81–7.39 (m, 5H, indole), 2.70 (q, 1H, CH),
2.40 (t, 2H, CH2) ppm; 13C NMR (CDCl3): d = 53.90
(CH2), 55.40 (CH), 105.62, 108.15, 111.82, 112.37, 117.95
(aromatic C), 118.74, 121.40, 125.11, 126.33, 128.53,
136.62 (indole C), 167.11 (CH=N), 171.56 (COO-) ppm;
UV-Vis (DMSO): kmax = 42,564, 36,978 cm-1; MS:
m/z = 330.
Km = 58.75 X-1 cm2 mol-1
;
UV-Vis (DMSO):
kmax = 33,112, 29,492 cm-1; MS: m/z = 717.
DNA-binding experiments
Synthesis of [ML(phen)2]Cl complexes
The interactions between the metal complexes and DNA
were studied using electronic absorption spectroscopic,
viscosity, and electrochemical methods. Disodium salt of
calf thymus DNA was stored at 4 °C. A solution of DNA in
the buffer 50 mM NaCl, 5 mM Tris HCl (pH 7.2) in water
The complexes were prepared by mixing the appropriate
molar quantity of the ligand 1 with the metal salt using the
following procedure: an ethanolic solution of 1 (0.003 mol)
was stirred with 5 cm3 of an ethanolic solution of the
123

Probing the DNA-binding behavior of tryptophan incorporating mixed-ligand complexes
gave a UV absorbance ratio at 260 and 280 nm, A260/A280
,
B. subtilis, P. aeruginosa, E. coli, and S. typhi by the paper
disc method using nutrient agar as the medium. The solu-
tion was prepared by dissolving the compound in DMSO,
and the entire blank discs were moistened with the solvent.
To perform disc assays, paper (6 mm) containing the
compound was placed on nutrient agar plates onto which
0.1 cm3 of overnight cultures of microorganisms had
already been spread. The diameter of the inhibition zone
was measured after 36 h of incubation at 37 °C.
of 1.9:1, indicating that the DNA was sufficiently free from
protein [25]. The concentration of DNA was measured
using its extinction coefficient at 260 nm (6,600 M-1) after
1:100 dilution. Stock solutions were stored at 4 °C and
used for no more than 4 days. Doubly distilled water was
used to prepare solutions. Concentrated stock solutions of
the complexes were prepared by dissolving the complexes
in DMF and diluting suitably with the corresponding buffer
to the required concentration in all experiments. Absorp-
tion titration experiments were carried out by varying the
DNA concentration and keeping the complex concentration
constant. Absorbance values were recorded after each
successive addition of DNA solution and equilibration
(ca. 10 min). The absorption data were analyzed to eval-
uate the intrinsic binding constant Kb using the reported
procedure [26].
Acknowledgments The authors express their sincere thanks to the
College Managing Board, the Principal, and the Head of the
Department of Chemistry, VHNSN College, Virudhunagar, India for
providing the necessary research facilities. NR thanks the University
Grants Commission (UGC), New Delhi for financial assistance.
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Electrochemical studies were carried out using a CHI
electrochemical analyzer controlled by CHI620C software.
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123