Synthesis, characterization, DNA interaction and antimicrobial screening of isatin-based polypyridyl mixed-ligand Cu(II) and Zn(II) complexes

Article abstract of DOI:10.2298/JSC091020054R

Several mixed ligand Cu(II)/Zn(II) complexes using 3-(phenylimino)1,3- dihydro-2H-indol-2-one (obtained by the condensation of isatin and aniline) as the primary ligand and 1,10-phenanthroline (phen)/2,2'-bipyridine (bpy) as an additional ligand were synt

Full text of DOI:10.2298/JSC091020054R

J. Serb. Chem. Soc. 75 (6) 773–788 (2010)
JSCS–4006
UDC 546.562’472+542.913+
547.477.2+547.963.32:615.28
Original scientific paper
Synthesis, characterization, DNA interaction and antimicrobial
screening of isatin-based polypyridyl mixed-ligand
Cu(II) and Zn(II) complexes
NATARAJAN RAMAN* and SIVASANGU SOBHA
Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, India
(
Received 20 October 2009, revised 17 March 2010)
Abstract: Several mixed ligand Cu(II)/Zn(II) complexes using 3-(phenylimi-
no)-1,3-dihydro-2H-indol-2-one (obtained by the condensation of isatin and
aniline) as the primary ligand and 1,10-phenanthroline (phen)/2,2’-bipyridine
(
bpy) as an additional ligand were synthesized and characterized analytically and
spectroscopically by elemental analyses, magnetic susceptibility and molar
conductance measurements, as well as by UV–Vis, IR, NMR and FAB mass
spectroscopy. The interaction of the complexes with calf thymus (CT) DNA
was studied using absorption spectra, cyclic voltammetric and viscosity mea-
surements. They exhibit absorption hypochromicity, and the specific viscosity
increased during the binding of the complexes to calf thymus DNA. The shifts in
the oxidation–reduction potential and changes in peak current on addition of
DNA were shown by CV measurements. The Cu(II)/Zn(II) complexes were
found to promote cleavage of pUC19 DNA from the supercoiled form I to the
open circular form II and linear form III. The complexes show enhanced
antifungal and antibacterial activities compared with the free ligand.
Keywords: 1,10-phenanthroline/2,2’-bipyridine; Cu(II) and Zn(II) complexes; an-
timicrobial activity; DNA binding and cleavage.
INTRODUCTION
The therapeutic and diagnostic properties of transition metal complexes have
attracted considerable attention leading to their application in many areas of mo-
1
dern medicine. Many coordination compounds of transition metal ions accom-
2
plish nucleolytic cleavage. In this regard, mixed-ligand metal complexes were
found to be particularly useful because of their potential to bind DNA via a
multitude of interactions and to cleave the duplex by virtue of their intrinsic che-
3
–8
mical, electrochemical, and photochemical reactivities.
A recent study on the
incorporation of good intercalators, such as phen (1,10-phenanthroline), bpy
*
Corresponding author. E-mail: drn_raman@yahoo.co.in
doi: 10.2298/JSC091020054R
7
73
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RAMAN and SOBHA
(
2,2’-bipyridine), etc. found high affinity between DNA base pairs and their pla-
9
nar structure through stacking interaction. A singular advantage in the use of
these metallo-intercalators for such studies is that the ligands or the metal ion in
them can be varied in an easily controlled manner to facilitate individual appli-
1
0,11
cations.
Although DNA interactions of number of mixed-ligand complexes
previously appeared in the literature, there is still scope to design and study
isatin-based Schiff base containing 1,10-phenantroline/2,2’-bipyridine with the
Cu(II) and Zn(II) as new chemical nucleases. Bearing these facts in mind, the
nuclease activity of mixed-ligand complexes of Cu(II) and Zn(II) containing
1
,10-phenantroline/2,2’-bipyridine is reported herein. DNA binding was also re-
searched. Hence, the present study is very valuable in understanding the mode of
complex binding to DNA, as well as laying the foundation for the rational design
of DNA structure probes and antitumor drugs.
EXPERIMENTAL
All chemicals used in the present work, viz., isatin, aniline, 1,10-phenanthroline, 2,2’-bi-
pyridine and copper and zinc chlorides were of analytical reagent grade (produced by Merck,
Germany). Commercial solvents were distilled and then used for the preparation of the ligand
and its complexes. CT DNA and pUC19 DNA was purchased from Bangalore Genei (India).
Microanalyses (C, H and N) were performed using a Carlo Erba 1108 analyzer at the sophis-
ticated analytical instrument facility (SAIF), Central Drug Research Institute (CDRI), Luck-
now, India. Molar conductivities in DMSO (10- mol/dm ) at room temperature were mea-
sured using a Systronic model-304 digital conductivity meter. Magnetic susceptibility measu-
rements of the complexes were realized by a Gouy balance using copper sulfate pentahydrate
as the calibrant. The IR spectra were recorded on a Perkin–Elmer 783 spectrophotometer in
the 4000–400 cm-1 range using KBr pellets. The NMR spectra were recorded on a Bruker
2
3
Avance Dry 300 FT-NMR spectrometer in DMSO-d with TMS as the internal reference. The
6
FAB-MS spectra were recorded using a VGZAB-HS spectrometer at room temperature in a
3
-nitrobenzyl alcohol matrix. Electron paramagnetic resonance spectra (EPR) of the mixed
ligand complexes of copper(II) were recorded on a Varian E 112 EPR spectrometer in DMSO
solution both at room temperature and liquid nitrogen temperature (77 K) using TCNE (tet-
racyanoethylene) as the g-marker. The absorption spectra were recorded using a Shimadzu
model UV-1601 spectrophotometer at room temperature. Electrochemical studies were rea-
lized using a CHI Electrochemical analyzer, controlled by CHI620C software. The CV mea-
surements were performed using a glassy carbon working electrode and an Ag/AgCl reference
electrode (all potentials refer to this reference electrode) and the supporting electrolyte was 50
mmol/L NaCl–5 mmol/L Tris-HCl buffer (pH 7.2). All solutions were deoxygenated by purging
with N for 30 min prior to the measurements.
2
Preparation of ligand (L)
The Schiff base was synthesized according to a literature procedure.12 A methanolic so-
lution of aniline (0.040 mol, 3.65 mL) was added to a methanolic solution (25 mL) of isatin
(
0.040 mol, 5.8 g) and the reaction mixture was refluxed for ca. 6 h. The end of the reaction
was monitored by TLC. In this technique, a small quantity of a solution of the Schiff base to
be analyzed was deposited as a small spot on a TLC plate which had a thin layer of silica gel
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BIOLOGICALLY ACTIVE Cu(II) AND Zn(II) COMPLEXES
775
(
SiO ) coated on a glass plate as the absorbent. It was used as the stationary phase and the mo-
2
bile phase was toluene:ethanol (9:1). The observed R value was 0.68.
f
The formed yellowish orange product (L) was filtered and washed with methanol and
dried in vacuo. Yield: 86 %. The preparation of the Schiff base is schematically presented in
Fig. 1.
Fig. 1. Synthesis of the Schiff base.
Synthesis of the [ML(Phen) ]Cl /[ML(bpy) ]Cl complexes
2
2
2
2
The complexes were prepared by mixing the appropriate molar quantities of the ligand
and the metal salts using the following procedure. A methanolic solution of Schiff base
(
0.0030 mol) was stirred with a methanolic solution (5 mL) of the required anhydrous me-
tal(II) chloride (0.0030 mol) for ca.1 h. To the above mixture, a methanolic solution (5 mL) of
,10-phenanthroline (phen)/2,2’-bipyridine (bpy) (0.0060 mol) was added in a 1:1:2 molar
1
ratio and the stirring was continued for ca. 1 h. The obtained solid product was filtered and
washed with methanol.
DNA binding experiments
The interactions between the metal complexes and DNA were studied using electroche-
mical and electronic absorption methods. The disodium salt of calf thymus DNA was stored at
4
°C. A solution of DNA in the buffer 50 mmol/L NaCl–5 mmol/L Tris-HCl (pH 7.2) in water
gave a ratio 1.9 of the UV absorbance at 260 and 280 nm, A260/A280, indicating that the DNA
1
3
was sufficiently free from protein. The concentration of DNA was measured using its ex-
tinction coefficient at 260 nm (6600 mol- L cm ) after a 1:100 dilution. Stock solutions were
stored at 4 °C and used no more than 4 days. Doubly distilled water was used to prepare the
solutions. Concentrated stock solutions of the complexes were prepared by dissolving the
complexes in DMSO and diluting suitably with the corresponding buffer to the required con-
centration for all the experiments. The data were then fitted to Eq. (1) to obtain the intrinsic
1
-1
binding constant (K ) values for the interaction of the complexes with DNA:
b
[
DNA]/(ε – ε ) = [DNA]/(ε – ε ) + 1/K (ε – ε )
(1)
a
f
b
f
b
b
f
where ε , ε , and ε are the apparent, free and bound metal complex extinction coefficients,
a
f
b
respectively. A plot of [DNA]/(ε – ε ) vs. [DNA] gave a slope of 1/(ε – ε ) and a y-intercept
b
f
b
f
–
1
equal to [K /(ε – ε )] ; thus, K is the ratio of the slope to the y-intercept. Viscosity mea-
b
b
f
b
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RAMAN and SOBHA
surements at room temperature were performed using a semi-micro dilution capillary visco-
meter. Each experiment was performed three times and an average flow time was calculated.
The data are presented as (η/η0)1 vs. the binding ratio, where η is the viscosity of a DNA
/3
solution in the presence of a complex and η is the viscosity of DNA in solution alone.
0
pUC19 DNA cleavage study
The cleavage of pUC19 DNA was determined by agarose gel electrophoresis. The gel-
-electrophoresis experiments were performed by incubation of the samples containing 30
μmol/L pUC19 DNA, 50 μmol/L copper complex and 50 μmol/L hydrogen peroxide (H O )
2
2
in Tris-HCl/NaCl buffer (pH 7.2) at 37 °C for 2 h. After incubation, the samples were elec-
trophoresed for 2 h at 50 V on 1 % agarose gel using Tris-acetic acid–EDTA buffer (pH 7.2).
-
3
The gel was then stained using 1 μg cm ethidium bromide (EB) and photographed under 360
nm ultraviolet light. All experiments were performed at room temperature unless otherwise
stated.
Antimicrobial activity studies
The in vitro antibacterial activity of the ligand and its complexes were tested against the
bacteria Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus
epidermidis, Klebsiella pneumoniae by the paper disc method using nutrient agar as the me-
dium. The antifungal activity was evaluated by the paper disc method against the fungi As-
pergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataticola and Candida albi-
cans cultured on potato dextrose agar medium. Streptomycin and nystatin were used as stan-
-
2
-1
dards for bacteria and fungi, respectively. A stock solution (10 mol L ) was prepared by
dissolving the compound in DMSO and the solution was serially diluted in order to find the
minimum inhibitory concentration (MIC) value. Test extract loaded discs inoculated with mic-
roorganisms were incubated at 30 °C for 24 h for the bacteria and 72 h for fungi. During the
incubation period, the test solution diffused and the growth of the inoculated microorganisms
was affected. The concentration at which an inhibition zone developed was noted.
RESULTS AND DISCUSSION
The ligand (L) and the mixed ligand complexes containing L and phen/bpy
were stable in air. L is soluble in common organic solvents but the complexes are
soluble only in DMF and DMSO. Elemental analyses of L and the complexes
were in agreement with the assigned formula. The higher molar conductance va-
lues of the complexes in DMSO show their electrolytic nature. The elemental
analysis and FAB-mass data, together with other properties, are given in Table I.
Mass spectra
The FAB-mass spectra of L and its complexes having phen were recorded
and the obtained molecular ion peaks confirmed the proposed formulae. The mass
+
spectrum of L (C H N O) exhibited peaks at 222 (M ), 223 (M+1) and 224
1
4 10 2
(
M+2) with 68, 100 and 49 % abundances, respectively. The most abundant peak
at m/z 223 represents the molecular ion peak of L. The mass spectrum of the
+
copper complex [CuC H N O]Cl showed peaks at 717 (M ), 718 (M+1), 719
3
8
26
6
2
(
M+2 ) with 8.7, 9.2 and 9.9 % abundances, respectively. The most abundant
peak at m/z 719 represents the molecular ion peak of the complex and the other
peaks are isotopic species. In addition, the spectrum exhibited fragments at m/z
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BIOLOGICALLY ACTIVE Cu(II) AND Zn(II) COMPLEXES
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1
80 and 77, which represent phen and phenyl moieties, respectively. The m/z of
all the fragments of L and the mixed ligand complexes, together with the relative
intensities confirmed the stoichiometry of the complexes to be of the type
[
ML(phen) /(bpy) ]Cl (where L = isatin-based Schiff base).The structural for-
2 2 2
mulas of the complexes are shown in Fig. 2. Thus, the mass spectral data rein-
forced the conclusion drawn from the analytical and conductance values.
TABLE I. Physical and analytical data of the synthesized Schiff base and its complexes
Compound
Yield Color
%
Found (Calcd.), %
Λ
M
2
MW
-1
µ
eff / µ
B
M
–
C
H
N
S cm mol
–
L
86 Yellowish
orange
72.7 5.0 14.1 222.0
(72.9) (5.0) (14.4)
–
[
[
[
[
CuL(phen)
CuL(bpy) ]Cl
ZnL(phen)
2
]Cl
2
71
67
79
83
Pale
green (8.5) (63.1) (3.5) (11.2)
Pale 9.5 61.0 3.9 12.5 669.0
green (8.9) (59.2) (3.3) (12.1)
Pale 9.0 63.4 3.6 11.6 718.9
brown (8.7) (59.7) (3.1) (11.4)
Pale 9.7 60.8 3.9 12.5 670.9
brown (9.3) (60.6) (3.8) (12.0)
8.8 63.6 3.6 11.7 717.1
104.5
115.2
102.9
112.6
1.89
1.86
2
2
2
]Cl
2
Diamag-
netic
Diamag-
netic
ZnL(bpy) ]Cl
2
2
(
a)
(b)
Fig. 2. The structural formulas of the complexes a) [ML(phen) ]Cl and
2
2
b) [ML(bpy) ]Cl ; M = Cu(II) or Zn(II).
2
2
IR Spectra
The IR spectrum of the free ligand (L) showed a broad band around 3257
–
1
cm , which can be attributed to NH stretching vibration of the isatin moiety. The
position of this band remained at nearly the same frequency in spectra of the me-
–
1
tal complexes, suggesting the uncoordination of this group. The band at 1763 cm
of isatin moiety, shifted to-
in the spectrum of the free ligand, assigned to ν
C=O
–
1
wards lower values, around 1719–1710 cm , in the complexes, indicating the
coordination of the carbonyl oxygen atom of the isatin residue. The C=N stretch-
–1
ing frequency of the Schiff base ligand appears in the region 1613–1608 cm ,
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RAMAN and SOBHA
which was shifted towards lower values in all the complexes, indicating the in-
volvement of the –C=N nitrogen in coordination to the central metal ion. All the
–
1
complexes showed bands in the regions 1090–1100 and 700–750 cm . These
can be assigned to phen/bpy ring –C–H and –C=N stretching vibrations, respecti-
–
1
vely. The appearance of two new bands in the regions 486–472 and 547–533 cm
in the spectra of the complexes, due to νM–N and νM–O stretching vibrations, res-
pectively, also confirmed the formation of metal complexes. The characteristic IR
data are presented in Table II.
-
1
TABLE II. IR spectral data (cm ) of the ligand and its complexes
Compound
L
ν
N–H
ν
C=O
ν
C=N
ν
M–N
ν
M–O
3257
3262
3267
3268
3265
1763
1711
1710
1717
1719
1645
1613
1608
1611
1619
–
–
[
[
[
[
CuL(phen)
2
]Cl
2
472
481
476
486
533
536
537
547
CuL(bpy) ]Cl
2
2
ZnL(phen)
2
]Cl
2
ZnL(bpy) ]Cl
2
2
1H-NMR Spectra
1
H-NMR (300 MHz, DMSO-d , δ / ppm) spectrum of the Schiff base ex-
6
hibited the following signals: the signal at 10.92 (1H, s) was assigned to the NH
proton of isatin and the multiplet signal around 6.43–7.10 (4H, m) to aromatic
protons. In addition to these, one singlet peak observed at 7.21 was ascribed to
1
aniline ring protons. In the H-NMR spectra of the Zn(II) complexes, the protons
of L are shifted downfield due to coordination to the metal ions. The resonance
peaks observed in the spectrum of the [ZnL(phen) ]Cl complex at 7.34–7.40 and
2
2
7
–
.81–8.37 were assigned to phen protons and the signals at 7.54–7.73 and 8.23–
8.37 in the spectrum of [ZnL(bpy) ]Cl were assigned to bpy protons.
2
2
EPR Spectra
The EPR spectra of the copper complexes exhibited an auxiliary symmetric
g-tensor parameters with g > g >2.0023, indicating that the copper site had a
dx –dy ground state characteristic of octahedral geometry. According to Hath-
║
┴
1
4
2
2
,
1
5
away, if the value of G is greater than four, the exchange interaction between
the copper(II) centers in the solid state is negligible, whereas when G is less than
four, there is considerable exchange interaction from the polypyridyl nitrogen
atom. The G values calculated for the present copper complexes lie within the
range 2.936–3.277, which are consistent with a dx
2
–dy ground state.
2
2
Apart from this, the covalency parameters α (covalent in-plane σ-bonding)
2
and β (covalent in-plane π-bonding) were calculated using a simplified MO theory
and the results are summarized in Table III.
2
2
The α and β values indicated a greater interaction in the in-plane π-bond-
ing than in the covalent in-plane σ-bonding, whereas the in-plane π-bonding is
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BIOLOGICALLY ACTIVE Cu(II) AND Zn(II) COMPLEXES
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2
2
almost ionic. The higher value of α compared to β indicates that the in-plane
σ-bonding is more covalent than the in-plane π-bonding. Based on these observa-
tions, a distorted octahedral geometry is proposed for both the copper complexes.
TABLE III. The spin Hamiltonian parameters of the copper complexes in DMSO solution at
300 and 77 K
4
-1
4
-1
2
2
Compound
g║
g┴
A ×10 / cm A ×10 / cm
α
β
G
║
┴
[
[
CuL(phen)
2
]Cl
2
2.232 2.071
2.351 2.072
169.0
171.3
66.0
64.3
0.767 0.615
0.724 0.518
3.2
2.9
CuL(bpy) ]Cl
2
2
Electronic absorption spectra
The UV–Vis spectra of the copper complexes were recorded in DMSO solu-
tion. They show a d–d band at around 734–737 nm. This band may be assigned
2
2
to E → T transitions, characteristics for a distorted octahedral structure. In
g
2g
addition, the complexes exhibited intense bands in the 395–398 nm region, which
are attributed to charge transfer transitions. The intense higher energy bands at
around 272 and 285 nm can be attributed to intra-ligand π–π* transitions. The
copper complexes showed magnetic moment values in the range of 1.87–1.89 µB
which indicate the monomeric nature of the complexes. The zinc(II) complexes
showed bands at 375 and 378.5 nm, assigned to intra-ligand charge transfer tran-
sitions. They are diamagnetic.
Based on the elemental analysis, magnetic moments, mass, EPR spectra,
1
electronic, IR and H-NMR data, the proposed structures of the complexes are
given in Fig. 2.
CT-DNA binding studies
Electronic absorption titration. The binding interaction of the metal com-
plexes with DNA was monitored by UV–Vis spectroscopy. The absorption spec-
tra of the complexes in the presence or without DNA were mutually compared,
which is shown in Fig. 3. In the UV region of the spectra, all the copper com-
plexes exhibited an intense absorption around 395–398 nm and zinc complexes
showed a band at 375–378.5 nm, which are attributed to n–π* transitions. With
increasing concentration of DNA, both the copper and zinc complexes showed
hypochromicity and a red-shifted charge transfer peak maxima in the absorption
spectra.
The hypochromicity values of the complexes [CuL(phen) ]Cl ,
2
2
[
CuL(bpy) ]Cl , [ZnL(phen) ]Cl and [ZnL(bpy) ]Cl observed in the presence
2 2 2 2 2 2
of DNA were 2.16, 1.08, 1.07 and 1.05 respectively, and their hypsochromic
shifts were 8.6, 4.2 and 4.0, 5.6 nm, respectively. The change in the absorbance
values with increasing amount of DNA was used to evaluate the intrinsic binding
constant K for the present complexes, the values of which are given in Table IV.
b
The change in hypochromicity may be attributed to the nature of the binding of
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RAMAN and SOBHA
the mixed ligand complexes with DNA, which is significant due to π-stacking or
hydrophobic interactions of the aromatic phenyl rings. However, the metal ions
play crucial role in DNA binding by these complexes. In general, the DNA bind-
ing abilities seem to follow the order: [CuL(phen) ]Cl > [CuL(bpy) ]Cl >
2
2
2
2
>
[ZnL(bpy) ]Cl > [ZnL(phen) ]Cl . The high binding nature of the metal com-
2 2 2 2
plexes may be due to additional π-π interaction through the aromatic phenyl rings.
Fig. 3. Electronic absorption spec-
trum of [CuL(phen) ] in the absence
2
(dash line) and presence (dark line)
of different concentrations (0 to 400
μM) of DNA.
TABLE IV. Electronic absorption spectral properties of the Cu(II) and Zn(II) complexes
λmax / nm
-5
-1
3
Compound
∆λmax / nm
H / %
K ×10 / mol dm
b
Free
397.8
393.2
375.0
378.4
Bound
389.2
389.0
371.0
372.8
[
[
[
[
CuL(phen)
2
]Cl
2
8.6
4.2
4.0
5.6
2.16
1.08
1.07
1.50
5.84
5.36
2.74
3.34
CuL(bpy) ]Cl
2
2
ZnL(phen) ]Cl
2
2
ZnL(bpy) ]Cl
2
2
Viscosity studies. A useful technique to prove intercalation is viscosity mea-
surements, which are sensitive to change in length of the DNA and are regarded
as the least ambiguous and the most critical tests of binding mode in solution in
the absence of crystallographic structural data or NMR spectra.16 Under appro-
priate conditions, intercalation of drugs, such as ethidium bromide [EB], causes a
significant increase in the viscosity of a DNA solution due to the increase in the
separation of the base pairs of the intercalation sites and hence, result in an in-
crease in the overall DNA contour length, as shown in Fig. 4. On the other hand,
drug molecular binding exclusively in the DNA grooves causes a less pronoun-
1
7
ced or no changes in the viscosity of a DNA solution. The viscosity of the
DNA solution increased with increasing ratio of both the copper and zinc com-
plexes to DNA. This result further suggests an intercalating binding mode of the
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BIOLOGICALLY ACTIVE Cu(II) AND Zn(II) COMPLEXES
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complexes with DNA and also parallels the above spectroscopic results, such as
hypochromism and bathochromism of the complexes in the presence of DNA.
Fig.4. Change in the relative viscosity
1
/3
(
η/40) of CT DNA as a function of r,
the molar ratio of the compound to the
DNA base pairs. The effect of increasing
amounts of:
[
[
[
[
CuL(phen) ]Cl (▲),
2 2
CuL(bpy) ]Cl (■),
2
2
ZnL(phen)2]Cl2 (♦) and
ZnL(bpy) ]Cl (●)
2
2
on the relative viscosity of DNA.
Redox studies. The application of cyclic voltammetry to the studies of the
complexes bound to DNA provides a useful complement to the previously used
investigation methods, i.e., UV–Vis spectroscopy and viscosity measurements.
The cyclic voltammograms of the glassy carbon electrode in solutions containing
[
CuL(phen) ]Cl in the absence and in the presence of varying amounts of DNA
2 2
are shown in Fig. 5.
Fig.5. Cyclic voltammograms of the glassy carbon electrode in solutions containing
[
CuL(phen)2]Cl2 in the absence (dash line) and presence (dark lines) of different
concentrations (0 to 400 μM) of DNA.
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In the absence of DNA, the first redox couple cathodic peak appeared at 0.29
V for Cu(III) → Cu(II) (E = 0.49 V, Epc = 0.29 V, ∆E = 0.20 V, and E =
pa
p
1/2
=
0.39 V) and the second redox couple cathodic peak appeared at –0.16 V for
Cu(II) → Cu(I), (Epa = 0.17 V, Epc = –0.16 V, ∆E = 0.33 V, and E = 0.17 V).
p
1/2
The ratio of cathodic to anodic peak currents, I /I = 0.95, indicated a quasi-re-
pa pc
versible redox process. The formal potential (E1/2), taken as the average of Epc
and E , was 0.39 V for the first redox couple in the absence of DNA. The
pa
presence of DNA in the solution at the same concentration as [CuL(phen) ]Cl
2
2
caused a negative shift in the potential ∆E by 0.160 V and a decrease in E by
p
1/2
0
.35 V, Table V. The value of I /I also decreased with increasing DNA con-
pa pc
centration. The decrease of the anodic and cathodic peak currents of the complex
in the presence DNA is due to a decrease in the apparent diffusion coefficient of
the Cu(II) complex upon complexation with the DNA macromolecules. These re-
sults show that [CuL(phen) ]Cl stabilizes the duplex (GC pairs) by intercalation.
2
2
TABLE V. Electrochemical parameters for the interaction of DNA with the Cu(II) complexes
E
1/2 / V
∆E
p
/ V
Compound
Redox couple
K /K
pc pa
I /I
+
2+
Free Bound Free Bound
[
[
CuL(phen)
2
]Cl
2
Cu(III) → Cu(II) 0.39
0.35
0.20
0.16
0.28
0.14
0.12
0.0965
0.4081
0.0826
0.7042
0.95
0.93
0.84
0.75
Cu(II) → Cu(I) 0.005 0.014 0.33
CuL(bpy) ]Cl
2
2
Cu(III) → Cu(II) 0.42
Cu(II) → Cu(I) 0.02
0.27
0.07
0.16
0.06
Considering the CV behavior of the glassy carbon electrode in solutions
containing [CuL(bpy) ]Cl in the absence of DNA, the first redox couple ca-
2
2
thodic peak appeared at 0.338 V for Cu(III) → Cu(II), (Epa = 0.50 V, Epc = 0.34
V, ∆E = 0.16 V and E = 0.42 V), while the second cathodic peak appeared at
p
1/2
0
=
.17 V for Cu(II) → Cu(I) (E = 0.23 V, Epc = 0.17 V, ∆E = 0.06 V and E
=
pa
p
1/2
0.20 V). The I /I ratios of these two redox couples were approximately
pa pc
unity. This indicates that the reaction of the complex on the working electrode
surface was a quasi-reversible redox process. The incremental addition of DNA
to the complex caused a negative shift in the potential of the second cathodic
peak and a decrease in the current intensity. The changes in the voltammetric
currents in the presence of CT DNA can be attributed to the slow diffusion of the
metal complex bound to the large, slowly diffusing DNA molecule. The changes
of the peak currents observed for the complexes upon addition of CT DNA may
indicate that the complexes possess a higher DNA binding affinity. The results
lead conclusions similar to those deduced from the spectroscopic and viscosity
data of the complexes in the presence of DNA.
The Zn(II) complexes showed only a reduction peak from –0.99 to –0.80 V
(Epc) and no oxidation peak in the absence of DNA. The incremental addition of
DNA to the Zn(II) complexes increased the current intensity and there was a po-
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BIOLOGICALLY ACTIVE Cu(II) AND Zn(II) COMPLEXES
783
sitive shift of the reduction peak potential. This result changes the reduction cur-
rent which indicates interaction of Zn(II) with CT DNA.
Differential pulse voltammogram study. The differential pulse voltammo-
grams of the glassy carbon electrode in solutions containing [CuL(phen) ]Cl2
2
both in the absence and presence of varying amounts of DNA are shown in Fig.
6
.
Fig.6. Differential pulse voltammograms of the glassy carbon electrode in solutions
containing [CuL(phen) ]Cl in the absence (dash line) and presence (dark lines)
2
2
of different concentrations (0 to 400 μM) of DNA.
The differential pulse voltammograms of the the glassy carbon electrode in
solutions containing [CuL(phen) ]Cl showed that increasing the concentration
2
2
of DNA resulted in a negative potential shift together with a significant decrease
of the current intensity. The shift in potential is related to the ratio of the binding
constants:
E – E = 0.0591log (K /K )
(2)
b
f
+
2+
where E and E are the formal potential of the Cu(II)/Cu(I) complex couple in
b
f
the bound and free forms, respectively. The ratio of the binding constants
K /K ) for DNA binding of the Cu(II)/Cu(I) couple of the complex was cal-
(
+
2+
culated and found to be less than unity. This indicates that the binding of the
Cu(I) complex to DNA is small compared to that of the Cu(II) complex. The
above electrochemical experimental results indicate that the Cu(II) complex binds
to DNA molecules. The possible mechanism is:
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RAMAN and SOBHA
2
+
For a complex of the electro-active substance Zn with DNA, the electro-
chemical reduction reactions can be divided into two steps:
2
+
The dissociation constant (K ) of the Zn –CT-DNA complex was obtained
d
using the following equation:
2
2
K (I − I )
2
d
po
p
2
I =
+ I −[DNA]
(3)
p
po
[
DNA]
2
where K is the dissociation constant of the complex Zn(II)–DNA; I is the re-
d
po
2
duction current of Zn(II) in the absence of CT-DNA; Ip is the reduction current
of Zn(II) in the presence of CT DNA; [DNA] is the concentration of added DNA
in the solution. Using the above equation, the dissociation constant was deter-
mined (Table VI).
TABLE VI. Electrochemical parameters for the interaction of DNA with the Zn(II) complexes
Ep / V
Ip / A
9
-1
Compound
K ×10 / M
d
Free
–0.989
–0.801
Bound
–0.931
–0.789
Free
0.83
0.89
bound
0.71
0.78
[
[
ZnL(phen)
2
]Cl
2
5.41
5.44
ZnL(bpy) ]Cl
2
2
The low values of the dissociation constant (Table VI) of Zn(II) ions are
indispensable for the catalytic function and structural stability of zinc enzymes
which participate in the replication, degradation, and translation of genetics of all
species. Moreover, the Zn(II) ion probably not only interacted with the active site
1
8
of the enzyme during these processes, as is already known in the literature, but
also with DNA.
Chemical nuclease activity
The cleavage efficiency of the complexes compared to that of the control is
due to their efficient DNA-binding ability. DNA cleavage was controlled by re-
laxation of the supercoiled form of pUC19 DNA into the nicked circular form
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BIOLOGICALLY ACTIVE Cu(II) AND Zn(II) COMPLEXES
785
and linear form. When pUC19 DNA is subjected to electrophoresis, the fastest
migration will be observed for the supercoiled form (form I). If one strand is
cleaved, the supercoils relax to produce the slower-moving open circular form
(
form II). If both strands are cleaved, the linear form (form III) will be generated
that migrates between the other two forms.
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 pre-
sence of the oxidant H O . The electrophoresis results are shown in Fig. 7.
2
2
From Fig. 7, it is evident that the complexes cleave DNA more efficiently in
the presence of an oxidant (H O ). This may be attributed to the formation of
2
2
hydroxyl free radicals. The production of a hydroxyl radical due to the reaction
between the metal complex and oxidant may be explained as shown below:
Ligand)Cu2+ + H O → (Ligand)Cu + OH· + OH–
3+
(
2 2
Fig. 7. Changes in the agarose gel electrophoretic pattern of pUC19 DNA induced by H2O2
and the Cu(II)/Zn(II) complexes. Lane 1, DNA alone; Lane 2, DNA + Ligand+ H O ;
2
2
Lane 3, DNA + [CuL(phen) ]Cl complex + H O ; Lane 4, DNA + [CuL(bpy) ]Cl
2
2
2
2
2
2
complex + H2O2; Lane 5, DNA + [ZnL(phen)2]Cl2 complex + H2O2;
Lane 6, DNA + [ZnL(bpy) ]Cl complex + H O .
2
2
2
2
The OH· free radicals participate in the oxidation of the deoxyribose moiety,
followed by hydrolytic cleavage of a sugar phosphate backbone. All the com-
plexes showed pronounced nuclease activity in the presence of the oxidant H O ,
2
2
which may be due to the increased production of hydroxyl radicals. Control ex-
periments using DNA alone resulted in no significant cleavage of pUC19 DNA,
even after longer exposure times. From the observed results, it was concluded
that all the complexes effectively cleaved the DNA as compared to control DNA.
Furthermore, the presence of a smear in the gel diagram indicates the presence of
1
9
radical cleavage.
Antimicrobial screening
The synthesized ligand and its complexes were tested for their in vitro anti-
microbial activity. They were tested against the bacteria Staphylococcus aureus,
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RAMAN and SOBHA
Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Kleb-
siella pneumoniae and the fungi Aspergillus niger, Fusarium solani, Culvularia
lunata, Rhizoctonia bataticola and Candida albicans. The minimum inhibitory
concentration (MIC) values of the investigated compounds are summarized in
Tables VII and VIII. A comparison of the MIC value of L with those of the com-
plexes indicates that the metal complexes exhibited higher antimicrobial activity
than L and the control sample. Such increased activity of the complexes can be
2
0
21
explained based on the Overtone’s concept and the Tweedy chelation theory.
According to the Overtone’s concept of cell permeability, the lipid membrane
that surrounds the cell favors the passage of only lipid-soluble materials, due to
which liposolubility is an important factor that controls antimicrobial activity. On
chelation, the polarity of the metal ion will be reduced due to the partial sharing
of positive charges with donor groups. Furthermore, it increases the
delocalization of π-electrons over the whole chelate ring and enhances the
lipophilicity of the complexes. This increased lipophilicity enhances the
penetration of the complexes into lipid membranes and the blocking of the metal
binding sites in the enzymes of microorganisms. The results obtained from the
antifungal and antibacterial tests showed that all the tested complexes were more
active towards bacteria than fungi. Moreover, the copper complexes were more
active than the zinc complexes against the tested microorganisms.
TABLE VII. Antibacterial studies of the investigated compounds (minimum inhibitory con-
4
centration×10 , µmol/L)
Compound
L
S. aureus P. aeruginosa E. coli
S. epidermidis
K. pneumoniae
16.3
1.4
2.1
2.7
3.6
1.7
14.8
1.6
2.5
3.4
4.8
1.9
16.6
1.2
2.0
2.4
2.5
1.8
16.2
1.0
1.7
3.1
3.9
1.3
14.5
1.9
2.2
4.2
4.4
2.6
[
[
[
[
CuL(phen)
CuL(bpy) ]Cl2
ZnL(phen) ]Cl
2
]Cl
2
2
2
2
ZnL(bpy)2]Cl2
Streptomycin
TABLE VIII. Antifungal studies of the investigated compounds (minimum inhibitory con-
4
centration×10 , µmol/L)
Compound
L
A. niger
F. solani
C. lunata
R. bataticola
C. albicans
12.8
2.0
3.2
5.1
6.3
1.1
13.1
2.6
3.4
5.3
6.7
1.6
14.3
3.9
4.3
5.9
6.1
1.2
11.5
3.8
4.1
6.1
6.5
1.0
15.7
4.9
5.3
6.4
6.9
1.5
[
[
[
[
CuL(phen)
2
]Cl
2
CuL(bpy) ]Cl
2
2
ZnL(phen)
CuL(phen) ]Cl
2
]Cl
2
2
2
Nystatin
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BIOLOGICALLY ACTIVE Cu(II) AND Zn(II) COMPLEXES
787
CONCLUSIONS
Four mixed ligand Cu(II) and Zn(II) complexes of an isatin-based Schiff
base (L) and 1,10-phenanthroline/2,2’-bipyridine were synthesized and characte-
rized. Elemental analyses, conductivity measurements and FAB-Mass spectro-
metry revealed the stoichiometry and composition of the complexes to be
1
[
ML(bpy) /(phen) ]Cl . The FTIR, UV–Vis, H-NMR, EPR spectra and magne-
2
2
2
tic measurements data confirmed the bonding features of the studied mixed li-
gand complexes. Electronic absorption spectroscopy, cyclic voltammetry, diffe-
rential pulse voltammetry and viscometric studies demonstrated a considerable
interaction between the complexes and calf-thymus DNA. The results of clea-
vage studies using pUC19 DNA showed that the complexes had higher nuclease
activities than the isatin-based ligand. The MIC values against the growth of mic-
roorganisms are much larger for the complexes than those of the ligand and the
control sample.
Acknowledgements. The authors express their sincere thanks to the College Managing
Board, Principal and Head of the Department of Chemistry, VHNSN College, and Virud-
hunagar, India, for providing the necessary research facilities.
И З В О Д
СИНТЕЗА, КАРАКТЕРИЗАЦИЈА, ИНТЕРАКЦИЈА СА ДНК И АНТИМИКРОБНО
ИЗУЧАВАЊЕ Cu(II) И Zn(II) КОМПЛЕКСА СА МЕШОВИТИМ ЛИГАНДИМА НА БАЗИ
ИЗАТИНСКЕ И ПОЛИПИРИДИЛНЕ СТРУКТУРЕ
NATARAJAN RAMAN и SIVASANGU SOBHA
Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, India
Синтетисано је неколико мешовито лигандних Cu(II)/Zn(II) комплекса коришћењем
3
-(фенилимино)-1,3-дихидроиндол-2Н-она (добијеног кондензацијом изатина и анилина) као
примарног лиганда и 1,10-фенантролина (phen)/2,2’-бипиридина (bpy) као додатног лиганда
и окарактерисано аналитички и спектроскопски елементалном анализом, мерењима магнет-
не сусцептибилности и моларне проводљивости, UV–Vis, IR, NMR и FAB масеним спек-
трима. Интеракција комплекса са ДНК телећег тимуса (CT) је изучавана помоћу апсорпцио-
них спектара, мерењима цикличне волтаметрије, вискозности и гел-електрофорезом. Они
имају апсорпциони хипохромизам и специфична вискозност расте у току везивања
комплекса за ДНК тeлeћег тимуса. Померања оксидо-редукционог потенцијала и промене у
пиковима струје додатком ДНК су показане CV мерењима. Cu(II)/Zn(II) комплекси изазивају
раскидање pUC19 ДНК из суперувијеног облика I у отворени кружни облик II и линеарни
облик III. Комплекси су показали повећану антифунгалну и антибактеријску активност у
односу на слободни лиганд.
(Примљено 20. октобра 2009, ревидирано 17. марта 2010)
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