Binuclear copper and zinc complexes possessing bio-potential ligands: Synthesis, characterization, antimicrobial, SOD mimetic, DNA binding, and cleavage studies

Article abstract of DOI:10.1080/00958971003699745

Schiff bases (L) viz, N,N′,N″,N′″-tetra-3,4-dimethoxybenzalidene-3,3′-diaminobenzidine (TDBD), N,N′,N″,N′″-tetra-4-hydroxy-3-methoxybenzalidene-3,3′-diaminobenzidine (THMBD), and N,N′,N″,N′″-tetra-3-hydroxy-4-nitrobenzalidene-3,3′-diaminobenzidine (THNBD) afford binuclear [M₂LCl₄] complexes where M = Cu(II) or Zn(II). These Schiff bases and their binuclear complexes have been characterized by analytical and spectral data showing square-planar geometry on metalation with Cu²⁺. Intercalative binding of these complexes with DNA has been investigated by electronic absorption spectroscopy, viscosity measurements, cyclic voltammetry, and differential pulse voltammetry. Control DNA cleavage experiments using pUC19 supercoiled (SC) DNA and minor groove binder distamycin suggest that these synthesized complexes bind to the major groove. In the presence of a reducing agent like 3-mercaptopropionic acid (MPA), they show chemical nuclease activity. They also show an efficient photo-induced DNA cleavage on irradiation with a monochromatic UV light of 360 nm in the presence of inhibitors. Control experiments indicate the inhibition of cleavage in the presence of singlet oxygen quencher like sodium azide and the enhancement of cleavage in D₂O show the formation of singlet oxygen as reactive species. The superoxide dismutase (SOD)-mimetic activity of the synthesized complexes has been assessed for their ability to inhibit the reduction of nitroblue tetrazolium chloride (NBT). The complexes have promising SOD-mimetic activity. The antimicrobial results indicate that the complexes inhibit the growth of bacteria and fungi more than free ligands.

Full text of DOI:10.1080/00958971003699745

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Binuclear copper and zinc complexes
possessing bio-potential ligands:
synthesis, characterization,
antimicrobial, SOD mimetic, DNA
binding, and cleavage studies
N. Raman a , A. Sakthivel a & R. Jeyamurugan a
a Research Department of Chemistry , VHNSN College, Madurai
Kamaraj University , Virudhunagar 626001, Tamil Nadu, India
Published online: 07 Apr 2010.
To cite this article: N. Raman , A. Sakthivel & R. Jeyamurugan (2010) Binuclear copper and
zinc complexes possessing bio-potential ligands: synthesis, characterization, antimicrobial, SOD
mimetic, DNA binding, and cleavage studies, Journal of Coordination Chemistry, 63:6, 1080-1096,
DOI: 10.1080/00958971003699745
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Journal of Coordination Chemistry
Vol. 63, No. 6, 20 March 2010, 1080–1096
Binuclear copper and zinc complexes possessing bio-potential
ligands: synthesis, characterization, antimicrobial, SOD
mimetic, DNA binding, and cleavage studies
N. RAMAN*, A. SAKTHIVEL and R. JEYAMURUGAN
Research Department of Chemistry, VHNSN College, Madurai Kamaraj University,
Virudhunagar 626001, Tamil Nadu, India
(Received 20 August 2009; in final form 29 October 2009)
Schiff bases (L) viz, N,N0,N00,N000-tetra-3,4-dimethoxybenzalidene-3,30-diaminobenzidine
(TDBD), N,N0,N00,N000-tetra-4-hydroxy-3-methoxybenzalidene-3,30-diaminobenzidine (THMBD),
and N,N0,N00,N000-tetra-3-hydroxy-4-nitrobenzalidene-3,30-diaminobenzidine (THNBD) afford
binuclear [M2LCl4] complexes where M ¼ Cu(II) or Zn(II). These Schiff bases and their
binuclear complexes have been characterized by analytical and spectral data showing
square-planar geometry on metalation with Cu2þ. Intercalative binding of these complexes
with DNA has been investigated by electronic absorption spectroscopy, viscosity measure-
ments, cyclic voltammetry, and differential pulse voltammetry. Control DNA cleavage
experiments using pUC19 supercoiled (SC) DNA and minor groove binder distamycin suggest
that these synthesized complexes bind to the major groove. In the presence of a reducing agent
like 3-mercaptopropionic acid (MPA), they show chemical nuclease activity. They also show an
efficient photo-induced DNA cleavage on irradiation with a monochromatic UV light of
360 nm in the presence of inhibitors. Control experiments indicate the inhibition of cleavage in
the presence of singlet oxygen quencher like sodium azide and the enhancement of cleavage in
D2O show the formation of singlet oxygen as reactive species. The superoxide dismutase
(SOD)-mimetic activity of the synthesized complexes has been assessed for their ability to
inhibit the reduction of nitroblue tetrazolium chloride (NBT). The complexes have promising
SOD-mimetic activity. The antimicrobial results indicate that the complexes inhibit the growth
of bacteria and fungi more than free ligands.
Keywords: Schiff base; Photo-cleavage; Binding constant; Antimicrobial; SOD
1. Introduction
The chemistry of multinuclear coordination metal complexes, especially of coupled
system, is of special interest in various fields of science due to the interaction between
metal centers lying at the crossover point of widely separated areas, physics of the
magnetic materials, and polynuclear reaction sites in biological processes [1].
Artificial nucleases have potential application in the fields of molecular biological
technology and drug development [2]. Transition metal complexes as synthetic
metallonuclease have been studied extensively due to their diversity in the structure
*Corresponding author. Email: drn_raman@yahoo.co.in
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2010 Taylor & Francis
DOI: 10.1080/00958971003699745

Binuclear copper and zinc complexes
1081
and reactivity [3], and some also exhibit antitumor or anti-HIV activities [4]. Binuclear
or polynuclear complexes have attracted attention as artificial nuclease [5, 6] since they
may demonstrate synergistic effects in the activation and recognition of active
phosphodiesters and nucleic acid [7]. In nature, there are many phosphoesterases that
are activated by two or more metal ions, such as 30,50-exonuclease from the Klenow
fragment of DNA polymerase I, Rnase H from HIV reverse tranciptase C, alkaline
phosphatase, P1 nuclease, and pherpholitase C. Tetrahymena ribozyme is also thought
to be activated by at least two metal ions. Binuclear Co(III) and Ln(III) complexes play
an important role because of their good nuclease activity [8, 9].
Binuclear Zn(II) complexes exhibit an excellent ability to cleave the phosphate ester
bond of active phosphodiesters and RNA model compounds. Breslow [10] first
prepared a binuclear Zn(II) complex and demonstrated that its cleaving activity toward
active phosphodiester BNP (bis-p-nitrophenol phosphate), RNA model HPNP
(2-hydroxypropyl-p-nitrophenyl phosphate), and UPV dinucleoside is five times more
than that of the corresponding mononuclear zinc(II) complex. Deal and Burstyn [11]
studied a series of macrocyclic copper(II) complexes and they have proposed
bis( p-nitrophenyl) phosphate and DNA hydrolysis. These works tempted us to
synthesize binuclear complexes and study their biological effects, such as superoxide
dismutase (SOD)-mimetic activity, DNA binding, chemical nuclease, photo-induced
DNA cleavage, and antimicrobial processes. We have reported a variety of mono-
nuclear complexes [12–14], but, in this article, we prepared the bimolecular copper
complexes for the first time and studied their DNA binding, redox-mediated DNA
cleavage, photo-induced DNA cleavage, SOD activity, and antimicrobial activities to
evaluate their biological efficiency with that of mononuclear complexes.
2. Experimental
2.1. Chemicals
All reagents
–
3,30-diaminobenzidene, 3,4-dimethoxybenzaldehyde, 4-hydroxy-
3-methoxybenzaldehyde, 3-hydroxy-4-nitrobenzaldehyde, and copper/zinc chlorides –
were from Merck and used as supplied. Anhydrous grade ethanol, DMF, and DMSO
were purified according to standard procedures. Solutions of plasmid pUC19 DNA in
5 mmol NaCl/50 mmol Tris-HCl (pH 7.2) gave a ratio of UV absorbance at 260 and
280 nm, A260/A280 of ca 1.8–1.9, indicating that the DNA was sufficiently free of protein
contamination [15]. The concentration of DNA was measured from the band intensity
at 260 nm with a known molar absorption coefficient (6600 molꢀ1 cmꢀ1) [16]. Stock
solutions were kept at 4ꢁC and used after not more than 4 days. Doubly distilled H2O
was used to prepare the buffer. The antimicrobial activities of the ligands and their
complexes were carried out by the well-diffusion method.
2.2. Physical measurements
1H-NMR spectra (300 MHz) of the samples were recorded in DMSO-d6 by employing
TMS as internal standard on a Bruker Avance DRX 300 FT-NMR spectrometer.
The fast atom bombardment mass spectra of the complexes were recorded on a JEOL

1082
N. Raman et al.
SX 102/DA-6000 mass spectrometer/data system using argon/xenon (6 kV, 10 mA) as
the FAB gas. The accelerating voltage was 10 kV and the spectra were recorded at room
temperature using m-nitrobenzylalcohol (NBA) as the matrix. The IR spectra were
recorded on a Shimadzu spectrophotometer from 4000 to 400 cmꢀ1 using KBr pellets.
The UV-Vis spectra were recorded on a Shimadzu UV-1601 spectrophotometer in
DMF. Magnetic susceptibility measurements of the complexes were carried out by
Gouy balance using copper sulfate as the calibrant. The purities of ligands and
complexes were evaluated by thin-layer chromatography. Molar conductance of the
complexes was measured using a Systronic conductivity bridge.
Electronic spectra of the complexes were recorded before and after the addition of
DNA in the presence of 5 mmol Tris-HCl/50 mmol NaCl buffer (pH 7.2). The intrinsic
binding constant for the interaction of complex with DNA was obtained from
absorption data. A fixed concentration of the complex (10 mmol) was titrated with
increasing amounts of DNA from 20 to 150 mmol. The equilibrium binding constants
(Kb) for the interaction of the complexes with DNA were obtained from the absorption
spectral titration data using the following equation:
½DNAꢂ=ð"a ꢀ "f Þ ¼ ½DNA=ð"b ꢀ "f Þꢂ þ 1=Kbð"b ꢀ "f Þ,
ð1Þ
where "a is the extinction coefficient observed for the charge transfer absorption at a
given DNA concentration, ꢀf the extinction coefficient of the complex free in solution,
"
the extinction coefficient of the complex when fully bound to DNA, Kb the
b
equilibrium binding constant, and [DNA] the concentration of nucleotides. A plot of
[DNA]/("aꢀ"f) versus [DNA] gives Kb as the ratio of the slope to the intercept.
Electrochemical studies were carried out using a CH Electrochemical analyzer,
Model CHI620C, controlled by Chi620c software. CV measurements were performed
using a glassy carbon working electrode, platinum wire auxiliary electrode, and
Ag/AgCl reference electrode. The supporting electrolyte was 50 mmol NaCl/10 mmol
Tris-HCl buffer (pH 7.2). Solutions were deoxygenated by purging with N2 prior to
measurements.
Viscometric experiments were carried out using an Oswald-type viscometer of 2 mL
capacity thermostated in a water bath at 27 ꢃ 1ꢁC. The flow rates of the buffer
(10 mmol), DNA (150 mmol), and DNA in the presence of all complexes at various
concentrations (5–40 mmol) were measured with a manually operated timer at least three
times to agree within 0.2 s. The relative specific viscosity was calculated according to the
relation ꢁ ¼ (tꢀt0)/t0, where t0 is the flow time for the buffer and t is the observed flow
time for DNA in the presence and absence of the complexes. A plot of (ꢁ/ꢁ0 )1/3 versus
1/R, R ¼ ([DNA]/[complex]), was constructed from viscosity measurements [17].
2.3. Synthesis of ligands (L)
N,N0,N00,N000-tetra-3,4-dimethoxybenzalidene-3,30-diaminobenzidine (TDBD) was syn-
thesized by refluxing an ethanolic solution of 3,30-diaminobenzidine (0.01 mol, 2.14 g)
and 3,4-dimethoxybenzaldehyde (0.04 mol, 6.64 g) for 2 h. Upon cooling, the yellow
microcrystalline compound obtained was filtered and washed with ethanol followed
by diethyl ether and recrystallized from DMF and dried in vacuo. Yield: 65%. IR (KBr):
1
2840 ((methyl)–CH), 1624 (–CH¼N) cmꢀ1. H-NMR (DMSO-d6): (phenyl multiplet)
6.4–7.2 ꢂ(m), ꢂ(OCH3) 3.9(s), (–CH¼N) 8.1(s): (807): m/z: 807; Anal. Calcd for

Binuclear copper and zinc complexes
1083
[C48H46N4O8]: C 71.4, H 5.7, N, 6.9; Found: C 70.8, H 5.7, N. 6.8 (%); ꢃmax in DMF,
255 and 383 nm (scheme 1).
N,N0,N00,N000-tetra-4-hydroxy-3-methoxybenzalidene-3,30-diaminobenzidine (THMBD)
and N,N0,N00,N000-tetra-3-hydroxy-4-nitrobenzalidene-3,30-diaminobenzidine (THNBD)
were synthesized according to the above-described procedure by the replacement of
3,4-dimethoxybenzaldehyde by 4-hydroxy-3-methoxybenzaldehyde (6.08 g) and
3-hydroxy-4-nitrobenzaldehyde (6.68 g), respectively. THMBD: Yield: 48%. IR (KBr):
1
3250–3500 (–OH), 2865 ((methyl)–CH), 1632 (–CH¼N) cmꢀ1. H-NMR (DMSO-d6):
(phenyl multiplet) 6.5–7.2 ꢂ(m), (OCH3) 3.8 ꢂ(s), (–CH¼N) 7.9 ꢂ(s) and (–OH) 11.9 ꢂ(s):
m/z: 751: Anal. Calcd for [C44H38N4O8] (%): C 70.3, H 5.1, N, 7.4. Found (%): C 69.8, H
5.0, N. 7.3; ꢃmax in DMF, 265 and 390 nm. THNBD: Yield: 62%. IR (KBr): 3225–3500
(–OH), 1338 (aromatic-NO2), 1628 (–CH¼N) cmꢀ1
.
1H-NMR (DMSO-d6):
(phenyl multiplet) 6.5–7.2 ꢂ(m), (–CH¼N) 8.5(s) and (–OH) 12.5(s); m/z: 811; Anal.
Calcd for [C40H26N8O12] (%): C 59.2, H 3.2, N, 13.8; Found (%): C 59.1, H 3.1,
N. 12.9; ꢃmax in DMF, 256 and 395 nm.
2.4. Synthesis of [Cu2(TDBD)Cl4]
To a solution of TDBD (0.005 mol 4.03 g) in DMF, a solution of copper chloride
(0.01 mol, 1.72 g) in ethanol was added, the mixture refluxed for 2 h, concentrated to
one-third volume and kept at 0ꢁC for 10 h. The solid product formed was filtered,
washed several times with small amounts of ethanol and diethyl ether, and dried
in vacuo. Yield: 74%. IR (KBr): 2839 ((methyl)-CH), 1602 (–CH¼N) cmꢀ1. m/z: 1076,
Anal. Calcd for [Cu2C48H46N4O8Cl4] (%): Cu 11.8, C 53.5, H 4.3, N 5.2; Found (%):
Scheme 1. Outline of the synthesis of ligands.

1084
N. Raman et al.
Cu 11.1, C 52.9, H 4.3, N 5.1. ꢃM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1) 7.8; ꢄeff (B.M.), 2.85. ꢃmax in
DMF, 257, 383, 525, and 914 nm, IC50, 36 mmol dmꢀ1
.
Similarly, [Cu2(THMBD)Cl4] and [Cu2(THNBD)Cl4] were synthesized according to
the above-described procedure. [Cu2(THMBD)Cl4]: Yield: 54%. IR (KBr): 3250–3500
(–OH), 2865 ((methyl)–CH), 1608 (–CH¼N) cmꢀ1. m/z: 1020, Anal. Calcd for
[Cu2C44H38N4O8Cl4] (%): Cu 12.4, C 51.8, H 3.7, N 5.4; Found (%): Cu 11.9,
C 51.2, H 3.7, N 5.4. ꢃM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1), 12.4; ꢄeff (B.M.), 2.74. ꢃmax in DMF,
262, 392, 544, and 915 nm IC50, 15 mmol dmꢀ1. [Cu2(THNBD)Cl4]: Yield: 47%. IR
(KBr): 3225–3500 (–OH), 1338 (aromatic-NO2), 1604 (–CH¼N) cmꢀ1. m/z: 1079, Anal.
Calcd for [Cu2C40H26N8O12Cl4] (%): Cu 11.7, C 44.5, H 2.4, N 10.3; Found (%):
Cu 11.2, C 44.1, H 2.4, N 10.1. m/z: 1010; ꢃM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1), 9.8; ꢄeff
(B.M.), 2.77. ꢃmax in DMF, 252, 394, 547, and 911 nm, IC50, 12 mmol dmꢀ1
.
2.5. Synthesis of [Zn2(TDBD)Cl4]
A solution of TDBD (0.005 mol, 4.03 g) in DMF was added to an ethanolic solution of
ZnCl2 (0.01 mol, 1.36 g), the mixture was refluxed for 2 h, concentrated to one-third
volume, and kept at 0ꢁC for 10 h. The solid product formed was filtered, washed several
times with small amounts of ethanol and diethyl ether, and dried in vacuo. Yield: 42%.
1
IR (KBr): 2842 ((methyl)–CH), 1604 (–CH¼N) cmꢀ1. H-NMR (DMSO-d6): (phenyl
multiplet) 6.4–7.2 ꢂ(m), ꢂ(OCH3) 3.9(s), (–CH¼N) 7.9(s): m/z: 1079. Anal. Calcd for
[Zn2C48H46N4O8Cl4] (%): Zn 11.1, C 53.4, H 4.3, N 5.1; Found (%): Zn 10.9, C 52.8,
H 4.3, N 5.1. ꢃM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1), 4.8; ꢃmax in DMF, 265 and 372 nm,
IC50, 56 mmol dmꢀ1
.
Similarly, [Zn2(THMBD)Cl4] and [Zn2(THNBD)Cl4] were synthesized according to
the above-described procedure. [Zn(THMBD)Cl4]: Yield: 28%. IR (KBr): 3250–3500
(–OH), 2838 ((methyl)–CH), 1610 (–CH¼N) cmꢀ1
.
1H-NMR (DMSO-d6): (phenyl
multiplet) 6.5–7.2 ꢂ(m), (OCH3) 3.8 ꢂ(s), (–CH¼N) 7.7 ꢂ(s) and (–OH) 11.9 ꢂ(s): m/z:
1023. Anal. Calcd for [Zn2C44H38N4O8Cl4] (%): Zn 12.7, C 51.6, H 3.7, N 5.4; Found
(%): Zn 12.1, C 50.7, H 3.7, N 5.4. ꢃM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1), 11.9; ꢃmax in DMF, 279
and 368 nm, IC50, 65 mmol dmꢀ1: [Zn2(THNBD)Cl4]: Yield: 32%. IR (KBr): 3225–3500
(–OH), 1338 (aromatic-NO2), 1601 (–CH¼N) cmꢀ1
.
1H-NMR (DMSO-d6): (phenyl
multiplet) 6.5–7.2 ꢂ(m), (–CH¼N) 8.3(s) and (–OH) 12.5(s) m/z: 1083. Anal. Calcd for
[Zn2C40H26N8O12Cl4] (%): Zn 12.0, C 44.3, H 2.4, N 10.3; Found (%): Cu 11.8, C 44.1,
H 2.4, N 10.3. ꢃM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1), 15.4; ꢃmax in DMF, 248, 385 nm,
IC50, 49 mmol dmꢀ1
.
2.6. DNA cleavage study
The extent of the cleavage of supercoiled (SC) pUC19 DNA (33.3 mmol, 0.2 mg) to its
nicked circular (NC) form was determined by an agarose gel electrophoresis in 5 mmol
Tris-HCl buffer (pH 7.2) containing 50 mmol NaCl. For the photo-induced DNA
cleavage, the reactions were carried out irradiated at 360 nm. After exposure to light,
each sample was incubated for 1 h at 37ꢁC and analyzed for photocleaved products
using gel electrophoresis as discussed below. Inhibition reactions for the ‘‘chemical
nuclease’’ reactions were carried out in the dark by adding reagents (distamycin,
50 mmol; DMSO, 4 mL) prior to the addition of the complexes and the reducing agent

Binuclear copper and zinc complexes
1085
3-mercaptopropionic acid (MPA). The inhibition reactions for the photo-induced DNA
were carried out at 360 nm using NaN3 (100 mmol) and DMSO (4 mL) prior to the
addition of the complexes. For the D2O experiment, this solvent was used for the
dilution of the sample to 18 mL; samples after incubation for 1 h at 37ꢁC in a dark
chamber were added to the loading buffer containing 25% bromophenol blue, 0.25%
xylene cyanol, 30% glycerol (3 mL), and the solution was finally loaded on 0.8% agarose
gel containing 1 mg mLꢀ1 ethidium bromide. Electrophoresis was carried out in a dark
chamber for 3 h at 50 V in Tris-HCl–EDTA buffer. Bands were visualized by UV light
and photographed.
2.7. Antimicrobial activity
The in vitro biological screening of the investigated compounds were tested against the
bacteria Salmonella typhi, Staphylococcus aureus, Escherichia coli, and Bacillus subtilis
by the well-diffusion method [18] using agar nutrient as the medium and streptomycin
as the standard. Antifungal activities were evaluated by the well-diffusion method
against the fungi Aspergillus niger, Aspergillus flavus, Candida Albicans, and Rhizoctonia
bataticola cultured on potato dextrose agar as medium and nystatin as the standard.
The stock solution (10ꢀ2 mol) was prepared by dissolving the compounds in DMSO and
the solutions were serially diluted in order to find the minimum inhibitory
concentrations (MICs). In a typical procedure [19], a well was made on the agar
medium inoculated with microorganisms. The well was filled with the test solution using
a micropipette and the plate was incubated, 24 h for bacteria and 72 h for fungi at 35ꢁC.
During this period, the test solution diffused and the growth of the inoculated
microorganisms was affected. The inhibition zone was developed, at which the
concentration was noted.
2.8. SOD activity
The SOD activity of the complexes was assayed using nitroblue tetrazolium chloride
(NBT) as reported previously [20]. The concentration of complex required to yield 50%
inhibition of the reduction of NBT (named IC50) was determined.
3. Results and discussion
The ligands and their complexes are stable in air and soluble only in DMF and DMSO.
Elemental analysis data of all ligands and complexes are in agreement with the
presented formulae. The physico-chemical and spectroscopic studies presented here
indicate the formation of bimetallic complexes of stoichiometry [M2LCl4] where L is a
ligand possessing four azomethine group (CH¼N–) endocyclic substituents, a mimic to
cisplatin [21].

1086
N. Raman et al.
3.1. Mass spectra
The FAB-mass spectra of synthesized ligands and their complexes were recorded and
the obtained molecular ion peaks confirm the proposed formulae. The mass spectrum
of ligand shows a peak at m/z 807 (12.5%) corresponding to [C48H46N4O8]þ ion. Also,
the spectrum exhibited the fragments at m/z 404, 260, 75, and 31 corresponding to
(C24H23N2O4), [C9H10O2]þ, [C6H3]þ, and [OCH3]þ, respectively. The mass spectrum of
[C48H46N4O8Cu2Cl4] shows peaks at 1076, 934, 807, and 404 with 9.2%, 3.5%, 7.4%,
and 15.7% abundances, respectively. The one at 1076 may represent the molecular ion
peak of the complex and the other peaks are isotopic species. The strongest peak (base
peak) at m/z 404 represents the stable species (C24H23N2O4). Also, the spectrum
exhibited fragments at m/z 260, 75, and 31 corresponding to [C9H10O2]þ, [C6H3]þ, and
[OCH3]þ, respectively. Thus, the mass spectral data confirm the stoichiometry of the
complexes as being of [M2LCl4], further supported by the FAB-mass study of other
complexes. This stoichiometry is further supported by the elemental analyses, which are
in close agreement with the values calculated from molecular formulae assigned to the
complexes.
3.2. Infrared spectra
IR spectra provide valuable information regarding the nature of the functional group
attached to the metal. In the IR spectra of the Schiff bases, a sharp band at
1620–1640 cmꢀ1 is assigned to ꢅ(C¼N) of azomethine. This band shifts to lower wave
numbers (ꢄ25 cmꢀ1) in all the complexes, suggesting the coordination of the
azomethine nitrogen to the metal, further substantiated by the presence of a new
band at 410–480 cmꢀ1, assigned to ꢅ(M–N) [22]. THMBD and THNBD show a broad
band for –OH at ca 3250–3500 cmꢀ1. The appearance of this peak in copper and zinc
complexes indicates that chelation does not take place via –OH. Weak bands were
observed in the free ligand spectrum at 2350 cmꢀ1, attributed to C–H vibrations.
Further, an intense band at 750 cmꢀ1 is typical of aromatic ring vibrations. As expected,
these bands are not affected by chelation.
3.3. Electronic absorption spectra
The electronic absorption spectra of the complexes were recorded in DMF.
The Cu(II) complexes show two bands in the visible region, 10,940–10,976 and
2
2
2
18,281–19,047 cmꢀ1, which are assigned to B1g ! A1g and B1g ! Eg transitions,
respectively. The electronic spectral data suggest a square-planar geometry around
Cu(II) ion. The electronic absorption spectra of the Zn(II) complexes show bands
(25,974–27,173 and 35,842–40,322 cmꢀ1) assigned to intraligand charge transfer
transitions [23].
3.4. Magnetic susceptibility
The majority of Cu(II) complexes [24] are square-planar or distorted-octahedral. In the
case of square-planar, tetrahedral, or distorted-octahedral Cu(II) complexes, the room
temperature magnetic moment values are usually observed as 1.8–2.2 B.M. [25] and are

Binuclear copper and zinc complexes
1087
not affected appreciably by the temperature and magnetic field. In practice, compounds
whose geometry approaches octahedral usually exhibit magnetic moments at the lower
end of the range, while those with square-planer or tetrahedral geometry are at the
higher end. For square-planar dimeric or polynuclear species, the complexes display
subnormal magnetic moments [25]. The observed room temperature magnetic moment
of Cu(II) complexes in the present case is 2.74–2.85 B.M. The spacer [26] nature of the
ligand, being two azomethine nitrogens of benzidine moiety far apart, and the analytical
data coupled with the magnetic moment suggest that the complex is binuclear [27] with
square-planar geometry at each metal center.
3.5. Nuclear magnetic resonance spectra
1H-NMR spectra of ligands and their zinc complexes were recorded in DMSO-d6.
TDBD gives the following signals: phenyl as multiplet at 6.4–7.2, –OCH3 at 3.9, and
–CH¼N– at 8.1 ꢂ. THMBD and THNBD give phenyl as multiplet at 6.5–7.2, –OCH3 at
3.8, and –CH¼N– at 7.9 and 8.5, and 12.5 and 11.9 ꢂ, which are attributable to the
phenolic –OH present in 3-hydroxy-4-nitro benzaldehyde and 4-hydroxy-3-methoxy
benzaldehyde. The presence of the phenolic –OH proton for the zinc complex confirms
the –OH free from complexation. The azomethine (–N¼CH–) proton signal in the zinc
complex is shifted downfield compared to the free ligand, suggesting the deshielding of
the azomethine due to the coordination with metal. There was no appreciable change in
the other signals of the complex.
The decoupled 13C NMR spectra of TDBD and its zinc complex (in DMSO-d6)
confirm the presence of azomethine (–C¼N–) and –OCH3. In zinc complex, the imine
carbon was deshielded compared to the free ligand, suggesting the coordination of
azomethine nitrogen. Comparison of all other macrocyclic carbon peaks of the ligand
with those of the zinc complex [Zn2LCl4] shows some upfield and downfield shifts,
which are not significant. The data for the free ligand and its zinc complex given in
‘‘Supplementary material’’ are in good agreement with the assigned structure of the
ligand and its complexes. Based on the above spectral and analytical data, the structure
of the Schiff-base complexes is represented in figure 1.
3.6. DNA binding studies
3.6.1. Absorption titration. Electronic absorption spectroscopy is a powerful tech-
nique for probing metal ion DNA interactions. Binding of the macromolecule leads to
Figure 1. The proposed structure of the Schiff-base complexes.

1088
N. Raman et al.
changes in the electronic spectrum of the metal complex. Base binding is expected to
perturb the ligand field transition of the metal complex. Intercalative mode of binding
usually results in hypochromism and bathochromism due to the strong stacking
interaction between an aromatic chromophore and the base pairs of DNA. The extent
of hypochromism parallels the intercalative binding strength. On the other hand, metal
complexes which bind non-intercalatively or electrostatically with DNA may result in
hyperchromism or hypochromism.
Electronic absorption spectra of [Cu2(TDBD)Cl4] in the presence of increasing
amounts of CT-DNA in 5 mmol Tris–HCl, 50 mmol NaCl, pH 7.2 buffer is shown in
figure 2. In the UV region, the intense absorption bands with a maxima of 383.5 nm for
[Cu2(TDBD)Cl4], 392 nm for [Cu2(THMBD)Cl4], 394 nm for [Cu2(THNBD)Cl4],
372 nm for [Zn2(TDBD)Cl4], 368 nm for [Zn2(THMBD)Cl4], and 385 nm for
[Zn2(THNBD)Cl4] complexes were attributed to intraligand ꢆ–ꢆ* transition.
Increasing the concentration of CT-DNA resulted in hypochromism and blueshift in
the UV spectrum of the copper complex, suggesting that copper complex binds to DNA
by an intercalative mode. After intercalating the base pairs of DNA, the ꢆ* orbit of the
intercalated ligand could couple with the ꢆ orbital of base pairs, decreasing the ꢆ–ꢆ*
transition energy and resulting in blueshift. The coupling of ꢆ orbit was partially filled
by electrons, thus decreasing the transition probabilities and concomitantly resulting in
hypochromism. A similar type of binding mode was observed for other complexes with
CT-DNA (table 1).
In order to compare the binding strength of the complexes with CT-DNA, the
intrinsic binding constants (Kb) are obtained by monitoring the changes in
the absorbance for the complexes with increasing the concentration of DNA. The Kb
values are shown in table 1.
Figure 2. Electronic absorption spectrum of [Cu2TDBDCl4] in the absence (dash line) and presence
(dark line) of increasing amounts of DNA.

Binuclear copper and zinc complexes
1089
3.6.2. Electrochemical studies. Electrochemical methods to study the metallointerca-
lation and coordination of transitional metal complexes to DNA provide a useful
complement to UV-Vis spectroscopy [28]. Typical cyclic voltammograms of
[Cu2(TDBD)Cl4] in the absence and in presence of varying amounts of [DNA] are
shown in figure 3.
In the absence of DNA, the first redox couple cathodic peak appears at ꢀ0.412 V for
Cu(III) ! Cu(II) (Epa ¼ ꢀ0.393 V, Epc ¼ ꢀ0.412 V, DEp ¼ 0.019 V, and E1/2
¼
ꢀ0.403 V) and second redox couple cathodic peak appears at ꢀ0.997 V for
Cu(II) ! Cu(I) (Epa ¼ ꢀ0.982 V, Epc ¼ ꢀ0.97 V, DEp ¼ 0.012 V, and E1/2 ¼ ꢀ0.976 V).
These two redox couples ratio of ipc/ipa is approximately unity. This indicates that the
reaction of the complex on the glassy carbon electrode surface is quasi-reversible.
Incremental addition of DNA to the complex for the second redox couple causes a
negative shift in E1/2 of 36 mV and a decrease in DEp of 23 mV. The ipc/ipa values also
decreases in the presence of DNA in first couple (Cu(III) ! Cu(II)), but in second
couple (Cu(II) ! Cu(I)) it increases. Peak potentials, Epc and Epa, as well as E1/2 shift
to more negative potential, indicating a binding interaction between the complex and
DNA that makes the complexes less readily reducible. Changes of the voltammetric
currents in the presence of CT-DNA can be attributed to the diffusion of the metal
complex bound to the large, slowly diffusing DNA molecule. The changes of the peak
currents observed for the complexes upon the addition of CT-DNA may indicate that
[THNBD] and [THMBD] complexes possess higher DNA-binding affinity than the
other complexes. The results parallel the above spectroscopic and viscosity data of the
complexes in the presence of DNA.
Zn(II) complexes show only the oxidation peak from ꢀ1.2 to ꢀ1.12 V (Ep) and no
reduction peak in the absence of DNA. Incremental addition of DNA on Zn(II)
complexes shows a decrease in the current intensity and negative shift of the oxidation
peak potential. The resulting changes in the current and potential demonstrate the
interaction between Zn(II) and DNA. The electrochemical parameters of the Cu(II) and
Zn(II) complexes are shown in tables 2 and 3, respectively. According to these data, all
the Cu(II) and Zn(II) complexes interact with DNA through intercalation.
Differential pulse voltammograms of [Cu2(TDBD)Cl4] in the absence and presence of
varying amount of [DNA] are given in figure 4. Increase in the concentration of DNA
causes a negative potential shift along with a significant increase of current intensity.
The shift in potential is related to the ratio of binding constant
Ebo
0
ꢀ Eo ¼ 0:0591 logðK =K2þÞ,
ð2Þ
þ
0
f
Table 1. Absorption spectral properties of complexes with DNA.
ꢃmax
Complexes
Free
Bound
Dꢃ (nm)
Hypochromicity H (%)
Kb ꢅ 105 (Mꢀ1
)
[Cu2(TDBD)Cl4]
[Cu2(THMBD)Cl4]
[Cu2(THNBD)Cl4]
[Zn2(TDBD)Cl4]
[Zn2(THMBD)Cl4]
[Zn2(THNBD)Cl4]
383.5
392
394
372
368
385
376.0
384.0
381.5
367.5
361.0
373.0
7.5
8.0
12.5
5.5
7.0
10.0
5.8
7.1
9.3
5.2
6.4
8.1
3.2
4.7
5.8
2.6
3.9
4.3

1090
N. Raman et al.
where Ebo0 and Efo0 are formal potentials of the Cu(II)/Cu(I) complex couple in the
bound and free form, respectively. The ratio of the binding constants (K /K2þ) for the
þ
DNA binding of Cu(II)/Cu(I) complexes were calculated and found to be less than
unity (table 2). This indicates that the binding of Cu(I) complex to DNA is small
compared to that of the Cu(II) complex. The above electrochemical results indicate the
preferential stabilization of Cu(II) form over Cu(I) form on binding to DNA. The
possible mechanism is shown below.
0
f
–
–
E
e
e
Cu(II)–Cu(I) +
Cu(I)–Cu(I)
'
+
Cu(II)–Cu(II)
K
+
K
2+
0
b
E
Cu(I)–Cu(I)–DNA
Cu(II)–Cu(I)–DNA
'
Cu(II)–Cu(II)–DNA
Figure 3. Cyclic voltammogram of [Cu2TDBDCl4] both in the absence (dash line) and presence (dark line)
of different concentrations of DNA.
Table 2. Electrochemical parameters for the interaction of DNA with Cu(II) complexes.
E1/2 (V)
DEp (V)
Complexes
Redox couple
Free
Bound
Free
Bound
K [red]/K [oxd]
ipc/ipa
[Cu2(TDBD)Cl4]
[Cu2(THMBD)Cl4]
[Cu2(THNBD)Cl4]
Cu(III)/Cu(II)
Cu(II)/Cu(I)
Cu(III)/Cu(II)
Cu(II)/Cu(I)
Cu(III)/Cu(II)
Cu(II)/Cu(I)
ꢀ0.403
ꢀ0.976
ꢀ0.394
ꢀ0.970
ꢀ0.399
ꢀ0.958
ꢀ0.375
ꢀ0.940
ꢀ0.313
ꢀ0.932
ꢀ0.308
ꢀ0.907
0.019
0.012
0.025
0.012
0.024
0.013
0.030
0.035
0.044
0.040
0.039
0.028
4.4
0.6
1.9
0.7
2.3
0.8
0.90
0.75
0.87
0.85
0.82
0.92

Binuclear copper and zinc complexes
1091
Differential pulse voltammograms of the Zn(II) complexes have a negative potential
shift along with a significant decrease of current intensity during the addition of
increasing amounts of DNA, indicating that zinc ions stabilize the duplex (GC pairs) by
intercalation. Hence, for the complexes of Zn(II) with DNA, the electrochemical
reduction reaction can be divided into two steps:
2+
2+
–
Zn
DNA
Zn + DNA
–
2+
0
+
Zn
2e
Zn
The dissociation constant (Kd) of the Zn(II)–DNA complex was obtained using the
following equation:
kd
i2p ¼
ði2po ꢀ i2pÞ þ ip2o ꢀ ½DNAꢂ
ð3Þ
½DNAꢂ
Table 3. Electrochemical parameters for the interaction of DNA with Zn(II) complexes.
Ep (V) ip (A)
Complexes
Free
Bound
Free
Bound
Kd ꢅ 10ꢀ10 (mol Lꢀ1
)
[Zn2(TDBD)Cl4]
[Zn2(THMBD)Cl4]
[Zn2(THNBD)Cl4]
ꢀ0.65
ꢀ0.71
ꢀ0.87
ꢀ0.58
ꢀ0.63
ꢀ0.75
0.42
0.15
0.54
0.34
0.09
0.43
6.7
8.6
0.9
Figure 4. Differential pulse voltammogram of [Cu2TDBDCl4] in the absence and presence of different
concentrations of DNA.

1092
N. Raman et al.
where Kd is dissociation constant of the complex Zn(II)–DNA and i2po and ip2 are the
reduction current of Zn(II) in the absence and presence of DNA, respectively. Using
equation (3), the dissociation constant was determined. The low dissociation constant
values (table 3) of Zn(II) ions were indispensable for the catalytic function and
structural stability of zinc enzymes which participate in the replication, degradation,
and translation of genetic material of all species. Moreover, Zn(II) ions were probably
interacting not only with the active site of the enzyme during these processes, as already
well-known in the literature [29], but also with DNA.
3.6.3. Viscosity measurements. To further clarify the interaction between the
complexes and DNA, viscosity measurements were carried out. Hydrodynamic
measurements that are sensitive to the length change (i.e., viscosity and sedimentation)
are regarded as the least ambiguous and the most critical tests of a binding model in
solution in the absence of crystallographic structural data. A classical intercalation
model demands that the DNA helix lengthen as base pairs are separated to
accommodate the binding ligand, leading to an increase in the DNA viscosity. In
contrast, a partial and/or non-classical intercalation ligand could bend (or kink) the
DNA helix, reduce its effective length, and concomitantly its viscosity. Figure 5 shows
the relative viscosity of DNA in the presence of varying amounts of copper and zinc
complexes. The relative viscosity steadily increases upon increasing the concentration of
zinc or copper complexes. These results suggest that both complexes bind to CT-DNA
intercalatively. However, the large increases in the relative viscosity values for THNBD
complexes reveal that THNBD complexes are better intercalators than other complexes.
Figure 5. The effect of [Cu2THNBDCl4] (m), [Zn2THNBDCl4] (ꢆ), [Cu2THMBDCl4] (^),
[Zn2THMBDCl4] (g), [Cu2TDBDCl4] (ꢅ), and [Zn2TDBDCl4] (*) on the viscosity of DNA; relative specific
viscosity vs. R ¼ [complex]/[DNA].

Binuclear copper and zinc complexes
1093
3.7. SOD activity
The SOD-mimetic activity values (IC50) of synthesized complexes are given in Section 2.
On comparing the SOD activities of the present complexes with the reported metal
complexes of EDTA and related chelators, our synthesized complexes exhibit
significant SOD-mimetic activities (using a modified NBT assay) [30]. In a variety of
complexes which act as a SOD enzyme [31], either there is one electron oxidation
followed by the reduction of metal ion or the formation of a superoxide complex which
then gets reduced to peroxide by another superoxide ion. To explore the mechanism of
action, the absorption spectrum of complexes were recorded in the presence and
absence of alkaline DMSO (alkaline DMSO acts as a source of O2ꢀ). The spectrum got
suppressed in alkaline DMSO containing buffer (pH 8.6). However, upon the addition
of NBT, which act as O2ꢀ scavenger, these peaks were reverted to their original
position. Thus, this experiment indicates that O2ꢀ is initially attached to the metal
complexes, which later get reduced by another O2ꢀ ion.
The lower SOD-like activity (IC50) of the copper complexes is due to the weak
resistance of these chelates toward Oꢀ2 or the product O2. The most active metal
complexes compare favorably with a number of synthetic SOD mimics developed for
therapeutic purposes [32].
3.8. Chemical nuclease activity
Chemical nuclease activity is controlled by the relaxation of the SC circular
conformation of pUC19 DNA to NC and/or linear conformations. When electrophor-
esis is applied to a circular plasmid DNA, fastest migration will be observed for the
DNA of closed circular conformations (Form I). If one strand is cleaved, the SC will
relax to produce a slower-moving nicked conformation (Form II). If both strands
are cleaved, a linear conformation (Form III) will be generated that migrates in
between [33].
The ligand alone is inactive for DNA cleavage in the presence and absence of any
external agents. The oxidative cleavage of pUC19 DNA (Supplementary material) in
the presence of an external reducing agent like MPA (5 mmol) (type-I) has been
studied by gel electrophoresis using SC pUC19 DNA (0.2 mg, 33.3 mmol) in 5 mmol
Tris-HCl/50 mmol NaCl buffer (14 mL, pH 7.2) and the complexes (50 mmol). Control
experiments using MPA or the synthesized complexes alone show an apparent cleavage
of SC DNA in the dark, suggesting that the complexes bind to DNA. To determine the
groove selectivity of the complexes, control experiments are performed using minor
groove binder distamycin. The addition of distamycin does not inhibit the cleavage
for all the complexes, indicating major groove binding for the synthesized complexes
with DNA. Control experiments show that DMSO inhibits DNA cleavage, suggesting
the possibility of hydroxyl radical and/or ‘‘copper-oxo’’ intermediate as the reactive
species [34]. SOD addition does not have any apparent effect on the cleavage activity,
indicating the non-involvement of superoxide radical in the cleavage reaction.
The mechanism involved in the DNA cleavage reactions is believed to be similar
to that proposed by Sigman et al. [35] for the chemical nuclease activity of metal
complexes.

1094
N. Raman et al.
3.9. DNA photo-cleavage study
Photo-induced DNA cleavage experiments have been carried out in UV light using the
Cu(II) and Zn(II) complexes (50 and 100 mmol) and SC pUC19 DNA (0.2 mg,
33.3 mmol) in the absence of any external agent.
The ligand alone is inactive for DNA cleavage. All complexes cleave the pUC19
DNA from its SC to NC form even in the absence of inhibitors on irradiation with UV
light at 360 nm. Singlet oxygen quencher, such as sodium azide, inhibits the cleavage.
1
Enhancement of the photo-cleavage of DNA is observed in D2O in which O2 has
longer lifetime [36]. Hydroxyl radical scavengers DMSO or KI do not show any
significant inhibition in the DNA cleavage activity, indicative of the presence of type-II
in which the photo-excited complexes activate molecular oxygen from its stable triplet
to the cytotoxic singlet state. Moreover, it suggests the formation of singlet oxygen as
the respective species in a type-II process in the metal assisted photo-excitation process
involving ligand n–ꢆ* and ꢆ–ꢆ* transitions (Supplementary material). The proposed
mechanistic pathway for photo-induced cleavage of SC DNA is shown below:
3
O
h
v=360 nm
1
*
2
[Complex]
S
O
2
Type-II
Type-I
SC DNA
MPA
O
.
2
Reduced form of
complexes
OH
Cleaved DNA
SC DNA
3.10. Antimicrobial activity
For in vitro antimicrobial activity, the compounds were tested against bacteria and
fungi. The MICs are summarized in tables 4 and 5. A comparative study of the ligands
and their complexes (MIC values) indicates that complexes exhibit higher antimicrobial
activity than the free ligand. From the MIC values, THNBD complexes are more potent
than other complexes. Such increased activity of the complexes can be explained on the
basis of Overtone’s concept [37] and Tweedy’s chelation theory [38]. These complexes
also disturb the respiration process of the cell and thus block the synthesis of the
proteins that restricts further growth of the organism. Furthermore, the mode of action
Table 4. MIC of the compounds against the growth of four fungi (mg mLꢀ1).
Compound
A. niger
A. flavus
C. albicans
R. bataticola
TDBD
THMBD
THNBD
[Cu2(TDBD)Cl4]
[Cu2(THMBD)Cl4]
[Cu2(THNBD)Cl4]
[Zn2(TDBD)Cl4]
[Zn2(THMBD)Cl4]
[Zn2(THNBD)Cl4]
Nystatin
78
65
75
42
36
35
38
33
27
10
69
52
68
38
32
27
24
28
30
8
63
58
62
24
28
43
45
47
42
12
71
74
67
36
44
28
37
38
31
14

Binuclear copper and zinc complexes
1095
Table 5. MIC of the compounds against the growth of four bacteria
(mg mLꢀ1).
Compound
E. coli
S. typhi
S. aureus
B. subtilis
TDBD
THMBD
THNBD
[Cu2(TDBD)Cl4]
[Cu2(THMBD)Cl4]
[Cu2(THNBD)Cl4]
[Zn2(TDBD)Cl4]
[Zn2(THMBD)Cl4]
[Zn2(THNBD)Cl4]
Streptomycin
60
57
47
28
24
14
20
29
18
14
71
68
57
24
32
28
33
32
27
18
74
71
63
32
29
18
22
30
25
12
82
77
73
21
22
22
18
26
23
10
of the compound may involve the formation of a hydrogen bond through the
azomethine group with the active center of cell resulting in interference with the normal
cell process and in general metal complexes are more active than the ligands because
metal complexes may serve as a vehicle for activation of ligands as the principle
cyctotoxic species [39].
4. Conclusions
Three new Schiff bases and their Cu(II) and Zn(II) complexes were synthesized
and characterized. They are bimetallic and square-planar geometry around metal(II)
ions. The planarity and extended conjugation effect the DNA binding and cleavage
activity of the complexes. Mechanistic investigations show a major groove binding for
the synthesized macrocyclic complexes with DNA. The synthesized complexes exhibit
chemical nuclease activity in the dark in the presence of a reducing agent (MPA) via
mechanistic pathway involving the formation of hydroxyl radical as the reactive species.
Pathways involving singlet oxygen in the DNA photo-cleavage reactions are proposed
from the observation of the complete inhibition of the cleavage in the presence of
sodium azide and the enhancement of cleavage in D2O. Hydroxyl radical scavengers
like DMSO do not show any significant effect on the DNA cleavage activity, suggesting
the formation of singlet oxygen as the reactive species in a type-II process.
The synthesized complexes exhibit significant SOD-mimetic activities and have
higher antimicrobial activity than the ligands. These binuclear complexes exhibit
more SOD, DNA binding (from the DNA binding constants), and cleaving ability
(from redox behavior) compared to mononuclear complexes.
Acknowledgments
The authors express their heartfelt thanks to the College Managing Board, VHNSN
College, Virudhunagar, for providing research facilities. N. Raman and A. Sakthivel
express their gratitude to the UGC, New Delhi, for financial assistance.

1096
N. Raman et al.
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