Synthesis, characterization, biological studies (DNA binding, cleavage, antibacterial and topoisomerase I) and molecular docking of copper(II) benzimidazole complexes

Article abstract of DOI:10.1016/j.jphotobiol.2012.05.003

To explore the therapeutic potential of copper-based benzimidazole complexes, tetranuclear Cu(II) complex 1 and dinuclear ternary amino acid complexes 2 and 3 {L-trp and L-val, respectively} were synthesized and thoroughly characterized. In vitro DNA binding studies of complexes 1-3 were carried out employing UV-vis titrations, fluorescence, circular dichroic and viscosity measurements which revealed that the complexes 1-3 bind to CT DNA preferably via groove binding. Complex 1 cleaved pBR322 DNA via hydrolytic pathway (validated by T4 DNA ligase assay), accessible to major groove while 2 followed oxidative mechanism, binding to minor groove of DNA double helix; binding events were further validated by molecular docking studies. Additionally, the complexes 1 and 2 exhibit high Topo-I inhibitory activity at different concentrations. The complexes 1-3 were evaluated for antibacterial activity against Escherichia coli and Staphylococcus aureus, and 2 was found to be most effective against Gram-positive bacteria.

Full text of DOI:10.1016/j.jphotobiol.2012.05.003

Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis, characterization, biological studies (DNA binding, cleavage,
antibacterial and topoisomerase I) and molecular docking of copper(II)
benzimidazole complexes
a
a
b
Farukh Arjmand a, , Shazia Parveen , Mohd. Afzal , Mohd. Shahid
⇑
a Department of Chemistry, Aligarh Muslim University, Aligarh 202 002, India
b Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh 202 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
To explore the therapeutic potential of copper-based benzimidazole complexes, tetranuclear Cu(II)
complex 1 and dinuclear ternary amino acid complexes 2 and 3 {L-trp and L-val, respectively} were syn-
thesized and thoroughly characterized. In vitro DNA binding studies of complexes 1–3 were carried out
employing UV–vis titrations, fluorescence, circular dichroic and viscosity measurements which revealed
that the complexes 1–3 bind to CT DNA preferably via groove binding. Complex 1 cleaved pBR322 DNA
via hydrolytic pathway (validated by T4 DNA ligase assay), accessible to major groove while 2 followed
oxidative mechanism, binding to minor groove of DNA double helix; binding events were further vali-
dated by molecular docking studies. Additionally, the complexes 1 and 2 exhibit high Topo-I inhibitory
activity at different concentrations. The complexes 1–3 were evaluated for antibacterial activity against
Escherichia coli and Staphylococcus aureus, and 2 was found to be most effective against Gram-positive
bacteria.
Received 16 January 2012
Received in revised form 24 April 2012
Accepted 3 May 2012
Available online 24 May 2012
Keywords:
Cu(II)-benzimidazole
In vitro DNA binding
Cleavage activity
Topo-I inhibitors
Groove binding
Molecular docking
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
ligand capable of forming many geometric topological variations,
which include polynuclear to dinuclear species. Control over the
Benzimidazoles (both natural and synthetic) are the key compo-
nents of many bioactive compounds. The benzimidazole scaffold is
a useful structural motif for the development of therapeutic
agents/pharmaceutical preparations {such as omeprazole (proton
pump inhibitor), pimobendan (ionodilator) and mebendazole
(anthelmintic) (Fig. 1a–c)} [1]. Furthermore, benzimidazole residue
is a constituent of vitamin B12 which supports their potential use as
therapeutics [2]. In lieu of broad spectrum of biological activities
exhibited by benzimidazole and their derivatives viz. antimicrobial
[3], H2 receptor blocker, antioxidant [4], anti-allergic [5], antihista-
mine [6] and antitumor [7]; they are highly in demand for use as
precursors in metal-based drug regime. Benzimidazole sometimes
called 1,2-dideazapurine and its derivatives can serve as model
compounds for purine due to structural similarity [8].
Previously, we have shown interest in chiral tridentate facially
coordinating ligand, H2bzimed, 1,2-bis(1H-benzimidazol-2-yl)eth-
ane-1,2-diol, for the synthesis of metal-based cancer chemothera-
peutics [9,10]. Since DNA has been identified as primary target for
many small therapeutic molecules [11–13], therefore, DNA-H2bzi-
med complex interactions were carried out. H2bzimed is versatile
formation of one or other species has been attributed to the part
of ligand which supplies the terminal ligands [14]. For example,
Cu(II) forms many dinuclear structures with benzimidazole with
basically square planar coordination linking, the two metals via
alkoxy (RAOH) or hydroxide groups [15]. Since, Cu(II) can readily
accept fifth ligand about the plane and offers association of two di-
meric units to give so called ‘cubane tetranuclear complexes’. A
number of structures have been reported exhibiting cubane struc-
tures and unsupported complexes with bidentate diamine ligands
(such as bipyridine), amino acids and diketones which act as termi-
nal ligands have also been reported [16]. Nevertheless, biological
properties of these complexes have not been intensely investigated
so far. Although, most of the bis-benzimidazole derived complexes
are typically specific groove binders, with Hoechst 33258 binding
in a specific manner to the A–T sequence at preorganised site of
the minor groove of the DNA duplex [17]. There is considerable
interest in developing novel antimicrobials due to growing concern
over rise in rate of infection by antibiotic-resistant microorgan-
isms. The antimicrobial activity of benzimidazole derivatives has
been attributed to bioactive benzimidazole pharmacophore;
although literature supports that the efficacy of such heterocyclic
compounds gets enhanced after metal complexation, in particular
if they are combined with other recognition elements. Such
compounds exhibit tendency to form chelates with metals causing
⇑
Corresponding author. Tel.: +91 5712703893.
E-mail address: farukh_arjmand@yahoo.co.in (F. Arjmand).
1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2012.05.003

16
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
Fluorescence measurements were determined on a Hitachi F-2500
fluorescence spectrophotometer. EPR spectrum of copper(II) com-
plexes was recorded on Varian E 112 spectrometer at the X-band fre-
quency (9.1 GHz) at room temperature and LNT (liquid nitrogen
temperature). Electrospray mass spectra were recorded on Micro-
mass Quattro II triple quadrupol mass spectrometer. The viscosity
measurements were carried out using Oswald capillary viscometer
maintained at 25 °C. Circular Dichroism was performed on Applied
Photophysics Chirascan Circular Dichroism Spectrometer with Stop
Flow. While measuring the absorption spectra an equal amount of
DNA was added to both the complex solution and the reference solu-
tion to eliminatethe absorbanceof the CT DNA itself, and the CD con-
tribution by the CT DNA and Tris buffer was subtracted through base
line correction.
(a)
(b)
(c)
Fig. 1. Pharmacologically active benzimidazole drugs (a) omeprazole, (b) pimob-
endan and (c) mebendazole.
2.3. Synthesis
2.3.1. Synthesis of ligand, 1,2-bis(1H-benzimidazol-2-yl)ethane-1,2-
diol (L)
The ligand was synthesized from L(+)tartaric acid (3.0 g,
20 mmol) and 1,2-diaminobenzene (4.32 g, 40 mmol) in ethanol–
water mixture according to the procedure reported earlier [19]
(Scheme 1).
drastic change in their biological properties [18]. Chelation re-
moves the undesirable metal ions and can deactivate either the
carcinogenic metal or the enzyme which is necessary for the rapid
growth of both the healthy and malignant cells.
Herein, we report the differences in the attributes of tetranu-
clear benzimidazole Cu(II) complex and dinuclear Cu(II) complexes
for DNA interaction and also evaluate the inhibitory effect of these
complexes (Topo-I poisoning). Molecular docking studies validate
the binding events suggestive of major groove in the case of tetra-
nuclear complex 1 while minor groove in the case of dinuclear ter-
nary complexes 2 and 3. The complexes 1–3 were evaluated for
in vitro antibacterial activity against Escherichia coli and Staphylo-
coccus aureus. Among the complexes screened, the ternary amino
acid complex 2 was found most effective against Gram-positive
bacteria (S. aureus).
2.3.2. Synthesis of tetrakis (S,S-1,2-bis(benzimidazol-2-yl)-1,2-
ethanediolato) tetracopper diperchlorate) nitrate, [Cu4L4(ClO4)2]
(NO3)2ꢀ4H2O, 1
The complex 1 was synthesized by the slight modification of
the method described previously [15] in which a mixture of
Cu(ClO4)2ꢀ6H2O (0.741 g, 2 mmol), Cu(NO3)2ꢀ3H2O (0.483 g,
2 mmol) and L (1.170 g, 4 mmol) was dissolved in 10 mL of EtOH
and 10 mL of H2O, which on slow evaporation yielded green crys-
talline solid, washed in hexane and dried in vacuo.
Yield, 42%, m.p. 236 °C (d), Anal. Calc. for C64H60N18O26Cl2Cu4
(%) C, 42.18; H, 3.32; N, 13.83. Found C, 42.97; H, 3.98; N, 13.97.
IR (KBr, cmꢁ1) 3198 b
t
(N–H), 1532
(ClOꢁ4 ), 743 s (Ar). UV–vis (kmax, nm, 10ꢁ4 M,
(MeOH),+166.00.
t(C@N), 1280 (imidazole
2. Materials and methods
NH), 1093 vs.
t
t
2.1. Materials
MeOH), 243, 273, 281, 349(sh), 682 (d–d). [a]
D
Molar conductance, Km, S m2 molꢁ1 (10ꢁ3 M, MeOH, 25 °C) 219
(1:2 electrolyte). ESI-MS (m/z) 875 (35%) [C64H60N18O26Cl2Cu4–
4H2O/2].
Reagent grade chemicals were used without further purifica-
tion. 1,2-diaminobenzene, L-(+)-Tartaric acid (Merck), Cu(-
NO3)2ꢀ3H2O, Sodium hydroxide (Merck) were used as received.
All amino acids were purchased from Sigma. 6X loading dye (Fer-
mental Life Science), T4 DNA ligase enzyme, Topo-I (CalBioChem)
and Supercoiled plasmid DNA pBR322 (Genei) were utilized as re-
ceived. Calf thymus DNA (CT DNA) was purchased from Sigma
Chemical Co.
2.3.3. Synthesis of [Cu2(L-trp)L(H2O)](NO3)2 and [Cu2(L-val)L(H2O)]
(NO3)2 (2 and 3)
The complexes 2 and 3 were prepared from a general synthetic
procedure in which L-trp (0.204 g, 1 mmol)/L-val (0.234 g,
2.0 mmol) pretreated with aqueous NaOH (0.04 g, 1 mmol) were
added to a 10 mL aqueous solution of Cu(NO3)2ꢀ3H2O (0.483 g,
2 mmol) and stirred for 30 min. A 10 mL methanolic solution of li-
gand L (0.294 g, 1 mmol) was added to the resulting solution and
refluxed at 60 °C for 4 h. A green precipitate formed was filtered
off. The volume of the resulting solution was reduced which on
slow evaporation at room temperature yielded green product
which was isolated by filtration, washed with hexane and dried
in vacuo (Scheme 2).
2.2. Methods and instrumentation
Elemental analyses of the complexes were performed on a Carlo
Erba Analyzer Model 1108. Molar conductance was measured at
room temperature on conductivity meter CM 180. IR spectra were
recorded on Interspec 2020 FTIR spectrometer in KBr pellets from
400 to 4000 cmꢁ1. Electronic spectra were recorded on UV-1700
PharmaSpec UV–vis spectrophotometer (Shimadzu) in methanol
using cuvettes of 1 cm path length. Data were reported in kmax/nm.
[Cu2(L-trp)L(H2O)](NO3)2, 2: Yield, 60%, m.p. 230–233 °C (d),
Anal. Calc. for C27H26N8O11Cu2 (%) C, 42.41; H, 3.43; N, 14.66.
Scheme 1. Synthesis of ligand, L.

F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
17
Scheme 2. Schematic representation of synthesis of complexes 1–3.
Found C, 42.22; H, 3.98; N, 14.89. IR (KBr, cmꢁ1) 3390 b
t
(H2O),
(C@N), 1457 s d(CH2),
s(COO), 1279 (imidazole NH), 832 (rocking
t
(Ar). UV–vis (kmax, nm, 10ꢁ4 M, MeOH), 244, 274,
samples were loaded on 1% agarose gel and visualized by staining
with an ethidium bromide solution.
3196 b
1386 vs.
t
t
(N–H), 1623 s
tas(COO), 1530 t
(NOꢁ3 ), 1353
t
OH)water, 744 s
2.5. Topoisomerase I inhibition assay
280, 348 (sh), 523 (d–d). [a]D (MeOH),+139.92. Molar conductance,
Km, Sm2 molꢁ1 (10ꢁ3 M, MeOH, 25 °C) 182 (1:2 electrolyte). ESI-
Human DNA topoisomerase I (Topo-I) was purchased from CAL-
BIOCHEM and was used without further purification. One unit of
MS (m/z) 650 (24%) [C27H26N8O11Cu2—2NOꢁ þ 0:5H2O].
3
[Cu2(L-val)L(H2O)](NO3)2, 3: Yield, 60%, m.p. 235 °C (d). Anal.
the enzyme was defined as completely relax 1
supercoiled pBR322 DNA in 30 min at 310 K under the standard as-
say conditions. The reaction mixture (30 L) contained 35 mM
Tris–HCl (pH 8.0), 72 mM KCl, 5 mM MgCl2, 5 mM DTT, 2 mM sper-
midine, 0.1 mg/mL BSA, 0.25 g pBR322 DNA, 2 Unit Topo-I and
lg of negatively
Calc. for C21H25N7O11Cu2 (%); C, 37.22; H, 3.72; N,14.48. Found C,
37.86; H, 3.98; N, 14.92. IR (KBr, cmꢁ1) 3482 b
t
(H2O), 3228 b
(C@N), 1457 s d(CH2), 1383 vs.
s(COO), 1282 (imidazole NH), 830 (rocking OH)water
t
(Ar). UV–vis (kmax, nm, 10ꢁ4 M, MeOH), 247, 273, 280, 343
l
t
t
(NH), 1622 s
t(C@O), 1526 t
(NOꢁ3 ), 1351
t
,
l
747 s
complexes 1 and 2. These reaction mixtures were incubated at
310 K for 30 min, and the reaction was terminated by addition of
4 lL of 5X buffer solution consisting of 0.25% bromophenol blue,
4.5% SDS and 45% glycerol. The samples were electrophoresed
(sh), 528 (d–d). [
a
]
(MeOH),+51.88. Molar conductance, Km,
D
Sm2 molꢁ1 (10ꢁ3 M, MeOH, 25 °C) 198 (1:2 electrolyte). ESI-MS
(m/z), 565 (68%) [C21H25N7O11Cu2—2NOꢁ þ 0:5H2O].
3
through 1% agarose in TBE at 30 V for 8 h.
2.4. DNA binding and cleavage experiments
2.6. Antibacterial activity
DNA binding experiments include absorption spectral traces,
emission spectroscopy and viscosity conformed to the standard
methods and practices previously adopted by our laboratory [20–
23]. DNA cleavage experiment has been performed by the standard
protocol as described in [24]. While measuring the absorption
spectra an equal amount of DNA was added to both the complex
solution and the reference solution to eliminate the absorbance
of the CT DNA itself, and Tris–HCl buffer was subtracted through
base line correction.
2.6.1. Screening of the drugs for antibacterial activity
The test organisms chosen for antimicrobial activity were one
Gram positive (E. coli, ATCC 25922) and one Gram negative (S. aur-
eus, ATCC 25923) bacteria. A 9 cm nutrient agar plate was swabbed
with the strains of S. aureus and E. coli. Wells of 3–4 mm diameter
were punched in the agar plates. 20
complexes 1–3 (in DMSO) was inoculated in the respective wells
and 20 L of DMSO was put as a control in a separate well in order
lL suspension of each of the
l
to look for any antibacterial activity of DMSO itself. The plates were
then incubated at 37 °C for 16–18 h, after which the zones (if pres-
ent) around the wells were measured, to assess susceptibility to
the particular complex.
2.4.1. Religation experiment with T4 DNA ligase enzyme
The DNA religation experiments were performed using T4 DNA
ligase enzyme to support the hydrolytic mechanism of DNA cleav-
age by following the standard DNA religation protocol [25]. The
complex 1 treated with pBR322 plasmid DNA (2 mg), ligation buf-
fer of 1.5 mL in 10 X, T4 DNA ligase 1 mL (2 units) and 2.5 mL of
H2O were mixed and incubated at 4 °C for 1 h. Subsequently, the
2.6.2. Bacterial strains used for analyzing the antibacterial potential of
the complexes
For initial screening, standard strains of S. aureus (ATCC 25923)
and E. coli (ATCC 25922) were used. Subsequently the complexes

18
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
were tested for minimum inhibitory concentrations (MICs) against
genetically characterized isolates harboring antibiotics resistance
genes (bla genes). E. coli (ATCC 25922) was used as a control. The de-
tails of the genetically characterized isolates are shown in Table S1.
m
(CAO) frequency at 1317 and 1267 cmꢁ1, respectively [29,30].
The IR spectra of complexes 1–3 exhibited a broad band at ꢂ3390–
3482 cmꢁ1 corresponding to the coordinated amino group of the
1,2-diaminobenzene and the amino acid moiety (in 2 and 3), sug-
gesting the coordination of NH2 group to copper [31]. The character-
istic
m(C@N) undergoes a negative shift at 1532, 1530 and
2.6.3. Measurement of minimum inhibitory concentrations (MICs)
1526 cmꢁ1 in the complexes 1–3, respectively indicative of the
involvement of N atoms in the formation of complexes. The presence
of bands in the region 1279–1282 cmꢁ1 were attributed to the imid-
azole ring NH [32]. Furthermore, in the amino acid complexes 2 and
3, the bands around 1660 and 1430 cmꢁ1 observed in free amino
acid assigned to antisymmetric and symmetric OAC@O stretching
vibrations were shifted to lower frequencies suggesting the terminal
coordination mode of the amino acids to the copper ion via deproto-
nation. The presence of bands in the region of 1622–1626 cmꢁ1 and
1353–1383 cmꢁ1 in the complexes 2 and 3 corresponds to the anti-
symmetric and symmetric OAC@O stretching vibrations confirms
Doubling dilutions of the drugs were prepared in sterile nutrient
broth, and 100
each well of a sterile microtitre plate. 100
l
L of each drug dilution was inoculated serially in
L of the bacterial sus-
l
pension in sterile nutrient broth (turbidity adjusted to 0.5 McFar-
land standards) was inoculated in each well. Appropriate complex
and bacterial controls were put up on the same plate. The plates
were then incubated at 37 °C for 16–18 h. MIC was taken as the con-
centration of the drug in the last well in which no visible bacterial
growth was observed. Confirmation of the MIC value was done by
spot inoculation of the drug-bacterial suspension on a plate of
blood agar, two wells on either side of the observed MIC value.
the presence of the amino acid moiety. In complexes 2 and 3, Dt va-
lue is >243 cmꢁ1 which reflects the monodendate nature of the car-
boxylate moiety [33]. Coordinated water in complexes 2 and 3 was
confirmed by the presence of rocking mode of water at ꢂ830 cmꢁ1
[34]. The uncoordinated NOꢁ3 in complexes 2 and 3 was clearly de-
tected at ꢂ1383 cmꢁ1, suggesting their presence as counter ions.
The X-band EPR spectra of complexes 1 and 2 were recorded
both at room temperature and at LNT at a frequency of 9.1 GHz un-
der the magnetic field strength 3000 1000G, with tetracyanoeth-
ylene (TCNE) as field marker (g = 0.0027) (Fig. S1). In the spectra of
complex 1 at room temperature, a very broad axial symmetrical sig-
nal with g|| at 2.31, g\ at 2.093 and gav at 2.16 computed from the
formula gav = 1/3 (g||+2g\) was observed. The EPR spectra of binu-
clear Cu(II) complex 2 show a single broad band centered at g|| at
2.14, g\ at 2.08 and gav at 2.10. The calculated values g|| and g\
for the complexes show the order as g|| > g\ > 2.0023 which is
2.7. Molecular docking
The rigid molecular docking studies were performed by using
HEX 6.1 software [26], is an interactive molecular graphics program
for calculating and displaying feasible docking modes of pairs of pro-
tein, enzymes and DNA molecule. The coordinates of metal complex
was taken from its crystal structure as a CIF file and was converted to
the PDB format using Mercury software [27]. The crystal structure of
the B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) and hu-
man-DNA-Topo-I complex (PDB ID: 1SC7) were downloaded from
the protein data bank (http://www.rcsb.org./pdb). The human-
DNA- Topo-I complex was prepared using a 50-bridging phosphoro-
thiolate duplex oligonucleotide [28]. The oligonucleotide sequence
of the cleavable strand of the duplex oligomer was 50-AAAAA-
GACTTsX-GAAAATTTTT-30, where ‘s’ represents the 50-bridging
phosphorothiolate of the cleaved strand and ‘X’ represents any of
the four bases A, G, C or T. All calculations were carried out on an In-
tel pentium4, 2.4 GHz based machine running MS Windows XP SP2
as operating system. Visualization of the docked pose has been done
by using CHIMERA (www.cgl.ucsf.edu/chimera) and PyMol (http://
pymol.sourceforget.net/) molecular graphics program.
indicative of the presence of an unpaired electron in the dx2
orbi-
ꢁy2
tal [35]. At LNT both complexes show hyperfine splitting due to
coupling of one unpaired electron with 63,65Cu nuclei [36]. The geo-
metric parameter G, which is a measure of the exchange interaction
between copper centers in the polycrystalline compound, is calcu-
lated using Hathway expression (G = g||ꢁ2.0023/g\ꢁ2.0023) [37].
According to Hathway, if the value of G > 4, the exchange interac-
tion is negligible in solid complexes but G < 4 indicates considerable
interaction in solid complexes. In the complexes 1 and 2 the ‘G’ val-
ues are 3.50 and 1.77, respectively, indicating considerable ex-
change interaction between the Cu(II) centers in the solid phase.
The absorption spectra of complexes 1–3, showed three ligand
centered electronic bands in the UV region at 244 nm, high energy
bands at 274 nm and 280 nm which could be attributed to intrali-
3. Results and discussion
Benzimidazole-derived Cu(II) complexes 1–3, where 1 is a tetra-
nuclear cluster while 2 and 3 are dinuclear ternary amino acid
complexes (viz. L-tryptophan and L-valine, respectively) were syn-
thesized and thoroughly characterized by elemental analysis and
various spectroscopic techniques. In vitro DNA binding, cleavage
and the biological activities (Topo-I and antibacterial) were carried
out to validate their potential to act as efficient therapeutic agents.
All of these complexes 1–3 are air stable for extended periods, 1:2
electrolytic and remarkably soluble in methanol, DMF and DMSO.
The complex 1 displayed d–d band in the range of 682 nm exhib-
iting five and six coordination environment while complexes 2
and 3 exhibited bands at 523 and 528 nm, respectively suggestive
of Cu(II) in a four-coordinate environment. All the complexes 1–3
gand (IL)
p
–
pꢃ transitions [38]. One charge transfer spectral band
near 350 nm (shoulder) was assignable to the
p
–
pꢃ electronic tran-
sitions of the coordinated benzimidazolyl ligand. Complex 1 exhib-
ited one low energy band at 682 nm confirming the dissociation of
complex 1 in solution [15]. Complexes 2 and 3 exhibited low en-
ergy bands at 523 and 528 nm, respectively, which is in the range
for expected d–d transitions (dxy; dyz ! dx2 ) suggesting that Cu(II)
y2
ions exhibit four-coordinate square planar environment [39].
Since, only one d–d transition was observed which indicates that
both the Cu(II) atoms were in the same environment.
exhibited optical rotation, [
+51.88, respectively, indicative of chiral nature of these complexes.
a
]
D
values of +166.00, +139.92 and
3.2. In vitro DNA binding studies
3.1. Synthesis and spectroscopic characterization
It is a well-known fact that DNA is the primary pharmacological
target of many antitumor compounds, and hence, the interaction
between DNA and metal complexes is of paramount importance
in understanding the mechanism of binding. Thus, the mode of
binding of complexes 1–3 to CT DNA were studied with the aid
of different techniques.
The IR spectra of complexes 1–3 were recorded in the region of
4000–400 cmꢁ1. The free ligand, L displayed
m
(OAH) bands at
3497 cmꢁ1, a medium intensity band at 1538 cmꢁ1 assigned to
(C@N) of the imidazole moiety and a characteristic (NAH)
stretching band at 3180 cmꢁ1 in addition to other
(CAN) and
m
m
m

F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
19
3.2.1. Electronic absorption spectroscopy
tions, the observed quenching is due to the photoelectron transfer
mechanism [43]. Relative binding of these complexes to CT DNA is
measured from the extent of reduction in emission intensity. The
Stern–Volmer quenching constant value, Ksv, obtained as a slope
of I0/I vs. r ([complex]/[DNA]) were evaluated for complexes 1–3
and were found to be 1.92, 0.72 and 0.36, respectively. A large de-
crease in emission intensity suggests stronger binding propensity
to CT DNA [34].
Electronic absorption spectroscopy is an efficient method to
examine the binding mode of DNA with metal complexes. The
absorption spectra of complexes 1–3 were recorded for a fixed con-
centration of free metal complex [6.66 ꢄ 10ꢁ6 M] with increasing
concentrations of CT DNA [0–4.0 ꢄ 10ꢁ5 M] (Fig. 2). A strong
‘‘hyperchromic effect’’ was observed for increasing concentrations
of CT DNA, although no appreciable change in the position of the
intraligand band in the complexes 1–3 was observed. Hyperchro-
mism has been attributed to the presence of synergic non-covalent
interactions: external contact (electrostatic binding), hydrogen
bonding and groove surface binding (major or minor) along outside
of DNA helix. As there is no change in position of absorption bands
(bathochromic or hypsochromic shift), it can be inferred that com-
plexes exhibit groove binding interactions to DNA. The intrinsic
binding constant, Kb for complexes 1–3 were calculated from
3.2.3. Viscosity measurements
To investigate further the binding nature of the complexes to
DNA, viscosity measurements on the solutions of CT DNA incu-
bated with the complexes have been carried out. Hydrodynamic
measurements that are sensitive to length change (i.e. viscosity)
are regarded as the least ambiguous most critical tests of a binding
model in a solution in the absence of any crystallographic data
[44]. Lengthening of DNA helix occurs on intercalation as base
pairs are separated to accommodate the binding ligand leading
to increase in DNA viscosity. Partial or non-classical intercalation
of ligand may bend or kink the DNA helix, thereby decreasing its
effective length and subsequently viscosity. The relative specific
viscosity of the DNA reduces steadily upon addition of complexes
1–3 indicating the complexes bind to DNA via non-covalent inter-
actions [45] (Fig. S2). The decreased relative viscosity of DNA may
be explained by a binding mode which produced bends or kinks in
the DNA and thus reduced its effective length and concomitantly
its viscosity.
Wolfe–Shimmer Eq. (1), through a plot of [DNA]/ea
where [DNA] represents the concentration of DNA, and ea
–
ef vs. [DNA],
,
ef and
eb the apparent extinction coefficient (Aobs/[M]), the extinction
coefficient for free metal complex (M) and the extinction coeffi-
cient for the free metal complex (M) in the fully bound form,
respectively, and were found to be 1.38 ꢄ 105, 2.4 ꢄ 104 and
1.11 ꢄ 104 Mꢁ1, respectively.
½DNAꢅ
½DNAꢅ
1
¼
þ
ð1Þ
j
ea
ꢁ
ef
j
j
eb
ꢁ
ef
j
Kbðeb
ꢁ
ef
Þ
From the observed Kb values, it is evident that the complex 1
binds more strongly to DNA in comparison to 2 and 3 which show
more or less similar binding strength. This can be attributed to the
different binding modes of Cu(II) complexes due to their structure,
charge and type of ligands [40]. The complex 1 is a tetranuclear
molecular entity possessing four copper centers in cubane arrange-
ment, two copper centers in five coordinate and other two in six
coordinate arrangements. On the other hand, the complexes 2
and 3 are dinuclear ternary Cu(II) complexes showing four coordi-
nate arrangement, the geometrical topology being restricted by
amino acid scaffold, though all the complexes possess same charge.
Evidently, complex 1 is structurally different from 2 and 3 owing to
its tetranuclearity and maximum number of AOH groups due to
four benzimidazole moieties in comparison to complexes 2 and
3, and the DNA double helix possess many hydrogen binding sites
which are accessible both in the minor and major grooves, it is
likely that the AOH group of complex 1 could form extensive
hydrogen bonding with DNA [41]. The complexes with ANH/
AOH groups (benzimidazole) can form hydrogen bonds with A–T
base pairs (N-3 of adenine or O-2 of thymine) of the DNA [42].
Though the intercalation of the benzimidazole moiety between a
set of adjacent base pairs of DNA is sterically impossible, but some
type of partial intercalation can be envisioned in such complexes.
3.2.4. Circular dichroic studies
Circular dichroic studies are useful in diagnosing changes in the
morphology of DNA during complex–DNA interactions. The ob-
served CD spectrum of CT DNA consists of a positive band at
275 nm due to base stacking and a negative band at 245 nm due
to helicity which is characteristic of DNA in right-handed B-form,
in the UV region. The CD spectra of complexes 1–3 on addition of
CT DNA showed conformational changes upon the addition of the
complexes without any changes in the wavelength (Fig. 4). Both
the bands show a slight change in the intensities. Groove binding
and electrostatic interaction of the complexes with DNA reveals
slight changes in the intensity of both negative as well as positive
band of DNA. Therefore, CD spectral changes suggest groove bind-
ing interaction that stabilizes the right-handed B-form of DNA [46].
On the basis of all of the spectroscopic studies, we concluded
that the complexes 1–3 bind to CT DNA preferentially in a non-
covalent groove binding mode.
3.3. DNA cleavage activity
DNA cleavage was controlled by relaxation of supercoiled circu-
lar form of pBR322 DNA into the nicked and linear form. When a
circular plasmid DNA is subjected to agarose gel electrophoresis,
the fastest migration will be observed for supercoiled form (Form
I). If one strand is cleaved, the supercoils will relax to produce a
slower moving open circular form (Form II). If both strands are
cleaved, a linear form (Form III) will be generated that migrates
in between. Fig. S3 shows agarose gel electrophoresis patterns of
pBR322 DNA after incubation with complexes 1 and 2 for 45 min
in Tris–HCl buffer (pH 7.2) at 37 °C. Compared with DNA control
(Lane 1), the patterns of Lanes 2–6, demonstrate that the DNA
cleavage occurs only when incubated with complexes 1 and 2.
The conversion of supercoiled form (Form I) to nicked form (Form
II) becomes more efficient with increasing concentration of com-
plexes 1 and 2 and a third form (linear form) migrated in between
Forms I and II suggesting double strand DNA cleavage.
3.2.2. Emission spectral studies
To further confirm the interaction between the complexes 1–3
and CT DNA, emission titrations were performed. Since, no lumi-
nescence was observed for complexes 1–3 at room temperature
in solution or in the presence of CT DNA, competitive binding stud-
ies using Ethidium Bromide, (a planar intercalating non-emissive
molecule, emitting intense fluorescence when bound to DNA) were
carried out. The emission intensity of EB is used as a structural
probe as EB shows reduced emission intensity in buffer solution
because of solvent quenching or due to photoelectron transfer
mechanism [43] and an enhancement of emission intensity when
bound intercalatively to DNA. When complexes 1–3 were added
to DNA pretreated with EB, the DNA induced emission intensity
at 560 nm (270 nm excitation) was decreased (Fig. 3). As these
complexes bind to DNA via non-covalent groove binding interac-
When inhibitors of oxygen-based radicals, ascorbic acid, gluta-
thione (GSH), H2O2 and 3-mercaptopropionic acid (MPA) were

20
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
Fig. 2. Absorption spectral traces of complexes 1–3, in 0.1 M Tris–HCl/10 M NaCl buffer at pH 7.2 upon the addition of CT DNA. Inset: plots of [DNA]/ea–e
f (m2 cm) vs. [DNA]
for the titration of CT DNA with complexes j, experimental data points; full lines, linear fitting of the data. [Complex] 6.66 ꢄ 10ꢁ6 M, [DNA] 0–4.0 ꢄ 10ꢁ5 M.

F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
21
Fig. 3. Emission spectra of EB bound to DNA in the presence of complexes 1–3 in Tris–HCl buffer (pH 7.2). Arrows indicate the intensity changes upon increasing
concentration of the complexes. Inset: fluorescence quenching curve of DNA bound EB with complexes j, experimental data points; full lines, linear fitting of the data.
[Complex] 6.66–4 ꢄ 10ꢁ6 M, [EB–DNA] 6.66 ꢄ 10ꢁ7 M.

22
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
ment of hydroxyl radical in the cleavage process. In the case of SOD
(Lane 9) the Form II of plasmid DNA was converted to linear Form
III indicating two subsequent and proximate single strand breaks
of DNA non-randomly. This result demonstrates that there is no
involvement of superoxide radical in DNA scission. Since complex
1 is able to cleave DNA in the absence of any reducing agent, it may
be assumed that DNA might be cleaved by a discernible hydrolytic
path. The commonly accepted mechanism of hydrolysis of deoxy-
nucleotide phosphates involves nucleophilic attack of hydroxyl
oxygen on the phosphorus to give a five-coordinate phosphate
intermediate [47]. The complex 1 lacks coordinated water mole-
cule but has CuAN groups, and it has been reported previously that
a phosphate ester bond can be also hydrolytically cleaved by Cu(II)
complexes with N-containing ligands [48], and this eventuality
cannot therefore, be ruled out. Direct evidence in support of hydro-
lytic cleavage mechanism of exhibited by complex 1 was obtained
by the religation assays in which supercoiled pBR322 DNA was
treated with T4 DNA ligase enzyme and subjected to gel
electrophoresis.
In the case of complex 2, the addition of DMSO (Lane 6), EtOH
(Lane 7) did not show any inhibition of the nuclease activity. Addi-
tion of singlet oxygen quencher NaN3 (Lane 8) did not show any
appreciable inhibitory effect on the chemical nuclease activity by
the complex, so there is no involvement of singlet oxygen of DNA
cleavage process. No obvious inhibitions are observed for the Cu(II)
complex in the presence of SOD (Lane 9), the results rule out the
possibility of DNA cleavage by superoxide. These results are indic-
ative of the involvement of hydroxyl radical and/or ‘‘metal-oxo’’
intermediate. Furthermore, there was no DNA-cleavage (complete
inhibition) observed (Lane 3, Fig. 5). The DNA cleavage reaction of
the complex 2 in the presence of MPA probably proceeds through
the oxidative hydroxyl radical pathway in a similar way as pro-
posed by Sigman and co-workers as shown below [49].
Fig. 4. CD spectra of CT DNA alone (——dashed line), in presence of complex (a) j, 1
(b) I, 2 (c) d, 3. [Complex] 1 ꢄ 10ꢁ4 M, [DNA] 1 ꢄ 10ꢁ4 M.
added to the complexes 1 and 2 to establish the reactive species
responsible for DNA cleavage, the cleavage ability of 1 was signif-
icantly enhanced by these activators following the order
H2O2 ꢆ MPA > GSH ꢇ Asc (Fig. 5). The conversion to Form III (lin-
ear DNA) was observed in the presence of MPA and GSH. Similarly,
complex 2 exhibited DNA cleavage in the presence of these activa-
tors showing complete inhibition in the case of H2O2 and MPA, fol-
lowing the order H2O2 ꢆ MPA > GSH > Asc.
To determine the potential interacting site of 1 and 2 with
pBR322 DNA, control experiments was performed using DNA rec-
ognition elements DAPI (minor groove binding) and methyl green
(major groove binding) prior to the addition of complexes 1 and
2. The cleavage reaction mediated by complex 1 was quenched in
presence of methyl green (Lane 10, Fig. 5a) However, complex 2
inhibited DNA cleavage in presence of DAPI (Lane 11 Fig. 5b), sug-
gesting that major grooves are the preferred interacting sites of
complex 1, whereas complex 2 prefers minor grooves which was
further validated by molecular docking studies.
Reactive oxygen species generated during the interaction be-
tween metal complexes and dioxygen or redox reagents are major
cause of DNA damage. To postulate a mechanism responsible for
DNA cleavage mediated by complexes 1 and 2, electrophoretic
study with some standard radical scavengers was also carried
out, viz., ethanol, DMSO as hydroxyl radical scavengers, sodium
azide as singlet oxygen scavenger and SOD as superoxide radical
scavenger under identical conditions (Fig. 5). Copper complexes
are able to cleave DNA through both oxidative and hydrolytic pro-
cesses. The addition of DMSO (Lane 6), EtOH (Lane 7) to complex 1
diminishes the cleavage activity which is indicative of the involve-
CuðIIÞ complex þ DNA ! CuðIIÞ ꢁ DNA
CuðIIÞ ꢁ DNA þ MPA ! CuðIÞ ꢁ DNA
3
CuðIÞ ꢁ DNA þ O2 ! ꢀOH þ SC DNA ! Cleaved DNA:
3.4. T4 DNA ligase assay
The hydrolytic mechanism of pBR322 DNA cleavage by Cu(II)
complex 1 was further supported by DNA religation experiments
in which supercoiled pBR322 DNA was treated with T4 DNA ligase
enzyme and subjected to gel electrophoresis. The complex 1
yielded linearized DNA which was relegated by using T4 DNA li-
gase enzyme (Fig. S4). In DNA hydrolytic cleavage, 30-OH and 50-
OPO3 (50-OH and 30-phosphate) fragments must exclusively afford
and that these fragments can be enzymatically ligated [50]. In
some cases, the hydrolytic products either did not end at the re-
quired 50-phosphate and 30-OH (ribose) termini or the complex
sometimes bound to the termini of cleaved DNA which would re-
sult in the religation being incomplete or even failing completely
[51]. The complex 1 yielded linearized DNA which was religated
by using T4 DNA ligase enzyme. In our case, DNA religation was
complete and after ligation there was complete repair which was
quantitatively ascertained by comparison of complex treated
DNA and with the control DNA alone in SC form.
Fig. 5. Agarose gel electrophoresis pattern for the cleavage of pBR322 supercoiled
DNA (300 ng) by complex (a) 1 and (b) 2 in presence of different activating agents at
3.5. Topoisomerase inhibition
310 K after incubation for 45 min. Lane 1: DNA control; Lane 2: 25
complex + H2O2 (0.4 M)+DNA; Lane 3: 25 M of complex + MPA (0.4 M) + DNA;
Lane 4: 25 M of complex + GSH (0.4 M)+DNA; Lane 5: 25 M of complex + ASc
(0.4 M) + DNA; Lane 6: 25 M of complex + DMSO (0.4 M) + DNA; Lane 7: 25 M of
complex + EtOH (0.4 M) + DNA; Lane 8: 25 of complex + sodium azide
(0.4 M) + DNA; Lane 9: 25 M of complex + SOD (15 units)+DNA; Lane 10: 25
of complex + DNA + DAPI (8 M); Lane 11: 25 M of complex + DNA + methyl green
(2.5 L of a 0.01 mg/mL solution).
lM of
l
l
l
Topo-I is one of the DNA-targeting enzymes that control the
topology of the DNA by a single-strand cut, allowing the DNA helix
to swivel and release torsional strain which results in relaxation of
supercoiled DNA, which is required during the transcription or rep-
lication. Blocking the binding of transcription factors to DNA by
l
l
lM
l
lM
l
l
l

F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
23
DNA-binding drugs and in turn modulating gene expression have
become an attractive strategy in cancer therapy [52].
A plasmid DNA cleavage assay of the complexes 1 and 2 were
used to investigate the influence of the complexes on the activity
of human Topo-I by agarose gel electrophoresis. This assay pro-
vides direct means of determining if the drug affects the unwind-
ing of a supercoiled (SC) duplex DNA substrate, because the
enzyme converts SC DNA to relaxed (R) DNA and open circular
(OC) DNA. When catalytic activity of Topo-I was assayed, com-
plexes 1 and 2 inhibited this process in a concentration-dependent
manner [53]. As shown in Fig. 6, supercoiled DNA was fully con-
verted into open circular form (Lane 2, Topo-I) in the absence of
complex. However, upon increasing concentrations of the com-
Fig. 6. The cleavage patterns of the agarose gel electrophoresis diagram showing
effect of different concentration of complexes 1 and 2 on the activity of DNA Topo-I;
Lane 1: DNA control; Lane 2: Topo-I + DNA; Lane 3: 10
Lane 4: 15 M of 1 + DNA + Topo-I; Lane 5: 20 M of 1 + DNA + Topo-I; Lane 6:
25 M of 1 + DNA + Topo-I; Lane 7: 10 M of 2 + DNA + Topo-I; Lane 8: 15 M of
of 2 + DNA + Topo-I; Lane 10: 25
lM of 1 + DNA + Topo-I;
l
l
l
l
l
2 + DNA + Topo-I; Lane 9: 20
lM
lM
of
2 + DNA + Topo-I.
plexes 1 and 2 (10–25
diminished (Lanes 3–10). The DNA relaxation effects caused by
Topo-I were totally inhibited by complexes 1 and 2 at 20
(IC50 ꢂ20 M) and 25 M (IC50 ꢂ25 M), respectively, comparing
lM), the levels of open circular form were
lM
l
l
l
with some classical Topo-I inhibitors (Table 1) [54–58]. These find-
ings imply that both complexes may block the DNA strand passage
event of the enzyme, and serve as catalytic inhibitor of Topo-I, due
to (i) relatively strong DNA binding ability of complexes 1 and 2
with DNA at higher concentration which expels the Topo-I from
approaching plasmid DNA and in turn leads to a lower level of
DNA relaxation or (ii) direct interference with the enzyme (i.e., sta-
bilization of topoisomerase-DNA cleavable complexes) and inacti-
vating its proper functioning or (iii) ligand that occupies the
topoisomerase binding site may suppress the association of topoi-
somerase with DNA, thus influencing the topoisomerase activity.
3.6. Molecular docking
Fig. 7. In vitro antibacterial activities of complexes 1–3 indicated by MIC (
against genetically characterized bacterial isolates.
l
g/mL)
3.6.1. Molecular docking studies with DNA
The design of molecules that can recognize specific sequences
and structures of nucleic acids plays an important role both for
understanding nucleic acid molecular recognition as well as for
the development of new chemotherapeutic drugs. Molecular dock-
ing technique is an attractive scaffold to understand the Drug–DNA
interactions for the rational drug design and discovery, as well as in
the mechanistic study by placing a small molecule into the binding
site of the target specific region of the DNA mainly in a non-cova-
lent fashion [59], which can substantiate the spectroscopic results.
In our experiment, molecular docking studies of complexes 1–3
with DNA duplex of sequence d(CGCGAATTCGCG)2 dodecamer
(PDB ID: 1BNA) were performed in order to predict the chosen
binding site along with preferred orientation of the molecules in-
side the DNA groove. The energetically most favorable conforma-
Fig. 8. Graph showing zone inhibition diameter vs. complexes 1–3.
tion of the docked pose, Fig.
9 revealed that complex 1
approaches towards the gap between DNA major groove mainly
through benzimidazole ring while complexes 2 and 3 fits snugly
into the curved contour of the targeted DNA in the minor groove
(Supplementary Fig. S5a and b and is situated within narrower
A–T (ꢂ10.8 Å) regions compared to G–C (ꢂ13.2 Å) ones, due to
preferential binding of complexes 1–3 bearing benzimidazole moi-
ety to A–T regions compared to the G–C region and leads to van der
Waals interaction and hydrophobic contacts with DNA functional
groups which define the stability of groove [60]. It is well known
that the interactions of chemical species with the minor groove
of B-DNA differ from those occurring in the major groove, both in
terms of electrostatic potential and steric effects, because of the
narrow shape of the former. Small molecules interact with the
minor groove, while large molecules tend to recognize the major
groove binding site [61]. Changes in accessible surface area of
interacting residues show a preferential binding of complexes 1–
3 between A–T base pairs, and bends the DNA slightly in such a
way that a part of the molecule comes between the two base pairs
of the DNA helix which makes favorable stacking interactions be-
Table 1
IC50 (lM) values of the complexes 1 and 2 on topoisomerase I activities, some of the
classical Topo-I inhibitors.
Complexes
IC50
(
lM)
Ref.
Etoposide
>1000
17
>100
>100
17
[54]
[55]
[55]
[55]
[55]
[56]
[57]
[57]
Camptothecin
Doxorubicin
Novobiocin
Topostatin
Hoechst 33258
30
D
K
-[Ru(bpy)2(uip)]2+
ꢂ40
ꢂ40
ꢂ25
20
-[Ru(bpy)2(uip)]2+
[Ru(bpy)2(appo)]2+
Complex 1
[58]
This work
This work
Complex 2
25
tween the ring systems of the DNA bases and the benzimidazole
ring of complexes. Hydrogen bonding from the C-2 carbonyl oxy-
gen of T or the N-3 nitrogen of A to the minor groove binders is

24
F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
Fig. 9. Molecular docked model of complex 1 with DNA dodecamer duplex of
sequence d(CGCGAATTCGCG)2 (PDB ID: 1BNA) and the brown solid line showing
hydrogen bond interaction between them. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
important [62]. In addition, there are H-bonds (2.8–3.0 Å) between
the AOH groups of benzimidazole and C-2 carbonyl oxygen of T8 at
a distance of 3.71 Å. The resulting binding energy of docked metal
complexes 1–3 were found to be ꢁ321.3, ꢁ294.6 and
ꢁ262.4 KJ molꢁ1, respectively, correlating well with the experi-
mental DNA binding values. The more negative the relative bind-
ing, the more potent the binding as between DNA and target
molecules. Thus, we can conclude that there is a mutual comple-
ment between spectroscopic techniques and molecular docked
model, which can be substantiate our spectroscopic results and
at the same time provides further evidence of groove binding.
Fig. 10. Molecular docked model showing intercalation of complex (a) 1 and (b) 2
in the cleavage site of human DNA topoisomerase I (PDB ID: 1SC7).
of Topo-I docked complexes 1 and 2 were found to be ꢁ361.6,
ꢁ347.1 KJ molꢁ1 respectively, which revealed that complex 1 binds
the cleavable complex more efficiently suggesting that it can be a
potential scaffold to be used for therapeutic purpose. Furthermore,
DNA intercalating forces was much more important than hydrogen
bonding of the ligand to the surrounding amino acids residues of
the protein, or to the base pairs [65]. So, we can conclude that
the complex occupies the topoisomerase binding site may suppress
the association of topoisomerase with DNA, thus influencing the
topoisomerase inhibition activity or form stable complex with
DNA and have a high potential to act as DNA-targeting anticancer
drugs.
3.6.2. Docking study of complexes 1 and 2 with DNA-Topo-I
In order to further rationalize the observed Topo-I inhibitory as-
say, molecular docking studies of complexes 1 and 2 with human-
DNA-Topo-I (PDB ID: 1SC7) were performed to understand the
putative binding site. The X-ray crystallographic structure of the
human-DNA-Topo-I complex was retrieved from Protein Data Bank
in which Topo-I is bound to the oligonucleotide sequence 50-
AAAAAGACTTsX-GAAAATTTTT-30, where‘s’ is 50-bridging phosp-
horothiolate of the cleaved strand and ‘X’ represents any of the four
bases A, G, C or T. The phosphoester bond of G12 in 1SC7 was re-
built and SH of G11 on the scissile strand was changed to OH
[63]. The analysis of best docked conformation (Fig. 10a and b) re-
vealed that both complexes 1 and 2 approaches towards the DNA
cleavage site in the Topo-I-DNA complex and forming a stable
4. Antimicrobial activity
The diameter of the zone is measured to the nearest millimeter.
Test substances which produce the zone of inhibition with diame-
ters 9 mm or more are regarded as positive, i.e. having antimicro-
bial activity; while those cases where the diameter is below 9 mm,
the bacteria are resistant to the sample tested and the sample is
said to have no antimicrobial activity. In our experimental sets,
all the complexes were found to exhibit significant antimicrobial
activity. The MIC (minimum inhibitory concentration) expressed
in micrograms, of the complexes showing positive antimicrobial
property is given in Table S2 (Fig. 7). The results for the zones of
inhibition of the respective complexes against S. aureus (ATCC
25923) and E. coli (ATCC 25922) are shown in Fig. 8. Among the
complexes screened, the complex 2 was found as the most effec-
tive complex against Gram-positive bacteria (S. aureus) whereas
complex through p–p stacking interactions between the G11 (+1)
and pyrimidine ring of T10 (ꢁ1) in the minor groove on the scissile
strand and C112 and A113, on the non-scissile strand, parallel to
the plane of base pairs without having hydrogen bonds with the
enzyme, subsequently leading to inhibitory effect on Topo-I [64].
The intercalation of complexes between the DNA base pairs at
the cleavage site results in increased physical distance between
the cleaved DNA termini and consequently inhibition of the religa-
tion step catalyzed by Topo-I. The resulting relative binding energy

F. Arjmand et al. / Journal of Photochemistry and Photobiology B: Biology 114 (2012) 15–26
25
the complexes 1 and 3 were found to demonstrate substantial
Appendix A. Supplementary material
activity against Gram-negative bacteria (E. coli).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2012.
05.003.
4.1. Activity against resistant bacterial isolates harboring blaCTX-M
blaampC, blaSHV, and blaTEM
,
The tetranuclear Cu(II) complex 1 demonstrated marked activi-
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