Synthesis, spectroscopic characterization, DNA interaction and antibacterial study of metal complexes of tetraazamacrocyclic Schiff base

Article abstract of DOI:10.1016/j.saa.2012.03.025

The template condensation reaction between benzil and 3,4-diaminotoulene resulted mononuclear 12-membered tetraimine macrocyclic complexes of the type, [MLCl₂] [M = Co(II), Ni(II), Cu(II) and Zn(II)]. The synthesized complexes have been characterized on the basis of the results of elemental analysis, molar conductance, magnetic susceptibility measurements and spectroscopic studies viz. FT-IR, ¹H and ¹³C NMR, FAB mass, UV-vis and EPR. An octahedral geometry has been envisaged for all these complexes, while a distorted octahedral geometry has been noticed for Cu(II) complex. Low conductivity data of all these complexes suggest their non-ionic nature. The interactive studies of these complexes with calf thymus DNA showed that the complexes are avid binders of calf thymus DNA. The in vitro antibacterial studies of these complexes screened against pathogenic bacteria proved them as growth inhibiting agents.

Full text of DOI:10.1016/j.saa.2012.03.025

Spectrochimica Acta Part A 93 (2012) 354–362
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, spectroscopic characterization, DNA interaction and antibacterial
study of metal complexes of tetraazamacrocyclic Schiff base
Mohammad Shakira,∗, Sadiqa Khanama, Farha Firdausa, Abdul Latifb, Mohammad Aatifc,
Saud I. Al-Resayesd
a Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
b Department of Ilmul Advia, Faculty of Unani Medicine, Aligarh Muslim University, Aligarh 202002, India
c Department of Biochemistry, Aligarh Muslim University, Aligarh 202002, India
d Department of Chemistry, Science College, King Saud University, Riyadh, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
The template condensation reaction between benzil and 3,4-diaminotoulene resulted mononuclear 12-
membered tetraimine macrocyclic complexes of the type, [MLCl2] [M = Co(II), Ni(II), Cu(II) and Zn(II)]. The
synthesized complexes have been characterized on the basis of the results of elemental analysis, molar
conductance, magnetic susceptibility measurements and spectroscopic studies viz. FT-IR, 1H and 13C NMR,
FAB mass, UV–vis and EPR. An octahedral geometry has been envisaged for all these complexes, while
a distorted octahedral geometry has been noticed for Cu(II) complex. Low conductivity data of all these
complexes suggest their non-ionic nature. The interactive studies of these complexes with calf thymus
DNA showed that the complexes are avid binders of calf thymus DNA. The in vitro antibacterial studies
of these complexes screened against pathogenic bacteria proved them as growth inhibiting agents.
© 2012 Elsevier B.V. All rights reserved.
Received 25 September 2011
Received in revised form 6 March 2012
Accepted 9 March 2012
Keywords:
Macrocycle
Schiff base
Spectroscopic studies
Antibacterial
1. Introduction
their biological activities i.e., toxicity against bacterial growth [13],
anticancerous [14] and other biochemical properties [15]. Polyaza
In recent years, macrocyclic chemistry has received great
attention for their unique properties such as biomimic functions
[1,2], ionic and molecular recognition [3,4], as building block in
supramolecular chemistry [5,6]. There is a continued interest in
synthesizing macrocyclic complexes because of their potential
applications in fundamental and applied sciences and importance
in the area of coordination chemistry [7]. The chemical proper-
ties of macrocyclic complexes can be tuned to force metal ions
to adopt unusual coordination geometry. The stability of macro-
cyclic metal complex depends upon a number of factors, including
the number and type of donor atoms present in the ligand and
their relative positions within the macrocyclic skeleton, as well as
the number and size of the chelate rings formed on complexation
[8]. The chemistry of Schiff base complexes has been an impor-
tant and popular area of research due to their simple synthesis,
versatility and diverse range of applications [9]. A considerable
number of Schiff-base complexes have potential biological inter-
est, being used as more or less successful models of biological
compounds [10]. Macrocyclic Schiff base nitrogen donor ligands
have received special attention because of their mixed hard–soft
donor character, versatile coordination behavior [11,12], and for
macrocyclic Schiff bases have been studied as potential inorganic
and organic cation receptors [16], electron transfer agents, homo-
geneous catalysts and DNA, RNA interacting agents [17]. Nitrogen
donor macrocycles are an important class of compounds due to
their prominent behavior of forming highly stable complexes with
a variety of transition metal ions [18,19]. Macrocyclic compounds
with basic nitrogen donors could also form protonated moieties,
which can interact with simple as well as with more complex
inorganic and organic anions [20]. The family of complexes with
aza-macrocyclic ligands has remained a focus of scientific atten-
tion for many decades [21]. Transition metal complexes with their
varied coordination environment and versatile redox and spectral
properties offer immense scope for designing species that are suit-
able to bind and cleave the DNA [22]. Recently, there has been
tremendous interest in studies related to the interaction of tran-
sition metal ions with nucleic acid because of their relevance in the
development of new reagents for biotechnology and medicine [23].
In view of aforesaid applications we found it worthwhile
to report a few 12-membered tetraazamacrocyclic complexes
of type, [MLCl2] formed by [2+2] template condensation reac-
tion of benzil and 3,4-diaminotoluene with Co(II), Ni(II), Cu(II)
and Zn(II) ions. These synthesized complexes were tested for
their in vitro antibacterial activity against some gram-positive
bacterial strains viz. Staphylococcus aureus, Streptococcus mutans,
Streptococcus pyogenes, Staphylococcus epidermidis, Bacillus cereus,
∗
Corresponding author. Tel.: +91 9837430035.
E-mail addresses: shakir078@yahoo.com, shakirnafees@yahoo.co.in (M. Shakir).
1386-1425/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2012.03.025

M. Shakir et al. / Spectrochimica Acta Part A 93 (2012) 354–362
355
Corynebacterium xerosis and gram-negative bacterial strains viz.
Escherichia coli, Klebsiella pneuomoniae, Proteus vulgaris and Pseu-
domonas aeruginosa. The results obtained were compared with
standard antibiotic: Ciprofloxacin (for gram-positive) and Gentam-
icin (for gram-negative) bacterial strains.
and benzil (2 mmol, 0.420 g) were added simultaneously with con-
stant stirring. The reaction mixture was then refluxed for 4–5 h,
resulting in a clear solution. The resultant solution was kept for
evaporation at room temperature leading to the isolation of a
microcrystalline product after a few days. The product was washed
with methanol and dried in vacuum.
2. Experimental
2.4. in vitro antibacterial activity
2.1. Materials and methods
2.4.1. Test microorganisms
The chemicals, 3,4-diaminotoluene, benzyl and ethidium bro-
mide (E. Merck) were used as received. The metal salts MCl2·6H2O,
M = Co(II) and Ni(II), CuCl2·2H2O and ZnCl2 (all E. Merck) were
commercially available pure samples. Methanol (AR) was used as
solvent. The solid media namely nutrient agar no. 2 (NA) (M 1269S-
500G, Himedia Labs Pvt. Ltd, Mumbai, India) was used for preparing
nutrient plates, while nutrient broth (NB) (M002-500G, Himedia
Labs Pvt. Ltd, Mumbai, India) was used for the liquid culture media.
Highly polymerized calf-thymus DNA sodium salt (7% Na content)
was purchased from Sigma. Other chemicals were of reagent grade
and used without further purification. Calf thymus DNA was dis-
solved to 0.5% (w/w), (12.5 mM DNA/phosphate) in 0.1 M sodium
phosphate buffer (pH 7.40) at 310 K for 24 h with occasional stir-
ring to ensure formation of homogeneous solution. The purity of
Ten bacterial strains (six gram positive and four gram negative)
were selected on the basis of their clinical importance in causing
diseases in humans. These were obtained from Hi media Labs Pvt.
Ltd., Mumbai, India and Microbial Type Culture Collection, Chandi-
garh, Punjab, India. The strains selected for the study are S. aureus
(ATCC 29213), S. mutans (ATCC 25175), Streptococcus pyrogenes
(MTCC 435), S. epidermidis (MTCC 435), B. cereus (MTCC 430), C.
xerosis (ATCC 373) (gram-positive bacterial strains), E. coli (ATCC
25922), K. pneuomoniae (MTCC 109), P. vulgaris (MTCC 426) and P.
aeruginosa (MTCC 424). These strains were screened for evaluation
of antibacterial activities of the synthesized complexes.
2.4.2. Primary screening
the DNA solution was checked from the absorbance ratio A260/A280
.
The antibacterial activity of the macrocyclic complexes was
evaluated by agar well diffusion method [26]. All the micro-
bial cultures were adjusted to 0.5 McFarland standards, which
is visually comparable to a microbial suspension of approxi-
mately 1.5 × 108 c.f.u./ml [27]. 20 ml of agar media was poured into
each petri plate these plates were then swabbed with a colony
from inoculum of the test microorganisms and kept for adsorp-
tion for 15 min. Using sterile cork borer of 6 mm diameter, wells
were bored into the seeded agar plates and these were loaded
with 50 ␮l volume with concentration of 10 mg/ml of each com-
pound reconstituted in dimethylsulphoxide (DMSO). All the plates
were incubated at 37 ◦C for 24 h. Antibacterial activity of all the
complexes was evaluated by measuring the diameter of zone of
inhibition in mm. The medium with dimethylsulphoxide (DMSO)
as solvent was used as a negative control whereas media with
ciprofloxacin (standard antibiotic for gram positive) and gentam-
icin (standard antibiotic for gram negative) were used as positive
control. The experiments were performed in triplicates.
Since the absorption ratio lies in the range 1.8 < A260/A280 < 1.9,
therefore no further deproteinization of DNA was needed.
2.2. Physical measurements
The elemental analyses were performed on Perkin-Elmer 2400
CHN Elemental Analyzer from the Micro-analytical Laboratory of
Central Drug Research Institute (CDRI), Lucknow, India. The FT-
IR spectra (4000–200 cm−1) of the complexes were recorded as
KBr/CsI discs on a Perkin-Elmer Spectrum RX-I spectrophotome-
ter. 1H and 13C NMR spectra were recorded in DMSO-d6 on Bruker
Avance II 400 NMR spectrometer with Me4Si as an internal stan-
dard from SAIF, Punjab University Chandigarh. FAB mass spectra
were recorded on a Joel SX-102 spectrometer. Metals and chlo-
ride were estimated volumetrically [24] and gravimetrically [25],
respectively. The electronic spectra of the complexes in DMSO
were recorded on a Cary 5E UV-VIS-NIR spectrophotometer at
room temperature. EPR spectrum was recorded at liquid nitro-
gen temperature on E-112 ESR spectrometer using TCNE as the
g-marker. Magnetic susceptibility measurements were carried out
using a Faraday balance at 25 ◦C. The molar conductivities of the
complexes (10−3 M solutions in DMSO) were obtained on a Sys-
tronic type 302 conductivity bridge equilibrated at 25.00 0.1 ◦C.
Fluorescence measurements were performed on spectrofluoropho-
tometer Model RF-5301PC (Shimadzu, Japan) equipped with a data
recorder DR-3. A fixed concentration of complex solution (30 ␮M)
was taken in a quartz cell and increasing amounts of calf thymus
DNA solution was titrated. The excitation wavelength and emis-
sion range for [CoLCl2], [NiLCl2], [CuLCl2], [ZnLCl2] were (350 nm,
355–520 nm), (365 nm, 370–430 nm), (250 nm, 340–490 nm), and
(250 nm, 375–480 nm), respectively. The path length of the sample
was 1 cm with 5 nm slit at 310 K.
2.4.3. Determination of minimum inhibitory concentration (MIC)
Minimum inhibitory concentration (MIC) is the lowest concen-
tration of an antimicrobial compound that will inhibit the visible
growth of microorganisms after overnight incubation. Minimum
inhibitory concentrations are important in diagnostic laboratories
to confirm resistance of microorganisms to antimicrobial agents
and also to monitor the activity of new antimicrobial agents.
The MIC of the macrocyclic complexes was tested against bacte-
rial strains through a broth dilution method. In this method, the
test concentrations of all the complexes were made from 2.5 to
0.01 mg/ml in the sterile wells of the micro-titer plates. In sterile
microtitre plates (96-u-shaped wells) 50 ␮l of the sterile nutrient
broth was poured in each well in three rows, then from fresh inocu-
lums so formed (108 c.f.u./ml diluted with 100 ␮l Nutrient broth to
have 106 c.f.u./ml) 50 ␮l of the suspension was poured in each well
in the first and third row, second row was again filled with 50 ␮l
of nutrient broth, finally the drug sample 50 ␮l was added in the
first row diluting uniformly from 2.5 to 0.01 mg/ml till the 8th well.
All the microtitre plates were incubated at 37 ◦C for 18–24 h. MIC
was expressed as the lowest dilution, which inhibited the growth
of bacteria observed by lack of turbidity in the well.
2.3. Synthesis of the complexes
2.3.1. Dichloro [2,3:8,9-tetraphenyl-5,6:11,12-tetrabenzo-
1,4,7,10-tetraazacyclododeca-1,3,7,9-tetraene] metal(II), [MLCl2]
[M = Co(II), Ni(II), Cu(II) and Zn(II)]
To a methanolic solution (20 ml) of metal salts (1 mmol), the
methanolic solutions of both 3,4-diaminotoluene (2 mmol, 0.244 g)

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M. Shakir et al. / Spectrochimica Acta Part A 93 (2012) 354–362
Fig. 1. Fluorescence emission spectrum of Co(II) complex in presence of increasing amount of CT-DNA. Inset shows the Stern–Volmer plot for the binding with CT-DNA.
2.5. DNA binding studies
the higher binding affinity of Cu(II) complex toward the DNA than
other complexes.
2.5.1. The fluorescence studies of ethidium bromide bound to
DNA in the presence of metal complexes
The state of the quenching due to molecular interaction between
luminophore and quencher can be purely static or purely dynamic.
However, these two states are the extremes and in most cases
the upward curvature of Stern–Volmer plot represents a com-
bination of both static and dynamic quenching as stated above
which occur simultaneously. The data plotted as emission intensity
against quencher concentration (Figs. 1–4) gives an upward curve
in our case as well, thus indicating the incidences of both static and
dynamic quenching.
The experiment was carried out at pH 7.0 in the buffer contain-
ing 50 mM NaCl and 5 mM Tris–HCl. DNA and ethidium bromide
(EB) were dissolved in buffer at the concentrations of 3 and 1 ␮g/ml,
respectively. The concentrations of the tested complex were 50 ␮M.
EB displays very weak fluorescence in aqueous solution. However,
in the presence of DNA, it exhibits intense fluorescence because
of the intercalation to base pairs in DNA. All the complexes were
added to EB bound with ct DNA and the intensity of fluorescence of
EB was measured. Fluorescence spectra were recorded using exci-
tation wavelength of 478 nm and the emission range set between
505 and 645 nm. Before examining the fluorescent properties of EB,
it was checked that the tested compounds did not quench the EB
fluorescence.
3. Results and discussion
The tetraaza Schiff base macrocyclic complexes of the type
[MLCl2] [M = Co(II), Ni(II), Cu(II), Zn(II)] have been synthesized by
[2+2] condensation reaction between 3,4-diaminotoulene and ben-
zil (Scheme 1). The purity of the complexes was checked by thin
layer chromatography (TLC) run in 1:1 benzene–methanol. How-
ever, inspite of all possible efforts a single crystal suitable for X-ray
crystallography could not be successfully isolated. All the com-
plexes were stable at room temperature and soluble in DMSO. The
molar conductance data of the 10−3 M solution of the complexes
measured in DMSO indicated the non-electrolytic nature of all the
complexes. The analytical data along with some physical properties
of the complexes are summarized in Table 1. The formation of Schiff
base complexes and bonding modes were inferred from positions
2.5.2. Binding data analysis of the complexes
To elaborate the fluorescence mechanism the Stern–Volmer
equation [28] was used for data analysis.
F0
= 1 + KSV[Q]
F
where, F and F0 are the fluorescence intensity with and without the
quencher (complex-DNA), KSV the Stern–Volmer quenching con-
stant, and [Q] the concentration of the quencher. The KSV value of
the complexes [CoLCl2], [NiLCl2], [CuLCl2] and [ZnLCl2] were cal-
of characteristic bands in FT-IR spectra and resonance signals in 1
H
and 13C NMR spectra. The overall geometry of all the complexes
was deduced from the observed values of magnetic moment and
the position of the bands in electronic spectra.
culated to be 1.74 × 104, 1.5 × 105, 3.83 × 105 and 3.6 × 105 M−1
,
respectively. A higher KSV value of Cu(II) complex suggest its
stronger quenching ability than other complexes. This implicates

M. Shakir et al. / Spectrochimica Acta Part A 93 (2012) 354–362
357
Table 1
Elemental analyses, color, yield, molar conductance, and melting point values of complexes.
Complexes
Color
Yield (%)
Found (calc.) %
Molar conductivity
(ohm−1 cm2 mol−1)/m.p. (◦C)
M
Cl
C
H
N
C42H32N4CoCl2
C42H32N4NiCl2
Dark brown
Brown
Light green
Pale white
42
48
50
51
8.22 (8.15)
8.25 (8.12)
8.50 (8.73)
8.75 (8.96)
9.62 (9.81)
9.55 (9.81)
9.52 (9.75)
9.40 (9.72)
69.40 (69.81)
69.60 (69.83)
69.19 (69.37)
69.08 (69.19)
4.22 (4.46)
4.15 (4.46)
4.20 (4.43)
4.18 (4.42)
7.55 (7.75)
7.48 (7.75)
7.41 (7.70)
7.36 (7.68)
30/>300
12/>300
33/>300
14/>300
C
C
42H32N4CuCl2
42H32N4ZnCl2
The 1H NMR spectrum of macrocyclic Zn(II) complex recorded in
DMSO-d6 shows a sharp signal at 2.08 ppm which may reason-
ably be assigned to the methyl protons ( CH3; 6H) [32]. Multiplets
observed in the region 7.23–7.96 ppm may be attributed to aro-
matic ring protons [33].
3.1. IR spectra
The preliminary identification regarding formation of the com-
plexes was obtained from the IR spectral findings (Table 2). The
IR spectra of all the complexes do not show bands correspond-
ing to free amino or carbonyl group rather a strong intensity band
appeared in the region 1618–1622 cm−1 characteristic of azome-
thine group ꢀ(C N) [29]. The presence of medium intensity band
in the region 440–460 cm−1 assignable to ꢀ(M N) vibration [30]
confirms the coordination of azomethine nitrogen to metal ions.
The bands characteristic of aromatic ring vibrations appeared in
the1440–1447, 1026–1061, 700–770 cm−1 regions in all the com-
plexes. A weak absorption band in the region 2906–2942 cm−1
may be assigned to CH3 stretching vibration. The coordination of
the chloro group has been ascertained by bands in 255–290 cm−1
region, which may be assigned to ꢀ(M Cl) vibration [31].
The 13C NMR spectrum of the Zn(II) complex shows a number
of sharp peaks corresponding to various carbon atoms in the pro-
posed structure, resulting due to the non-equivalence of the various
carbon atoms (Fig. 6). A sharp signal observed at 153 ppm may be
assigned to azomethine carbon [34]. The resonance signals for aro-
matic carbons appeared in the 127–141 ppm region [35]. A sharp
signal is observed at 21 ppm corresponding to methyl carbons [36].
3.3. Mass spectra
The mass spectra of macrocyclic compounds, showed molecular
ion peaks (M+1) at m/z 723, 723, 728 and 730 for [C42H32N4CoCl2],
[C42H32N4NiCl2], [C42H32N4CuCl2] and [C42H32N4ZnCl2], respec-
tively which are in good agreement with the respective molecular
formulae. The mass spectrum of Cu(II) complex has been depicted
in Fig. 7. The spectrum shows a molecular ion peak (M+1) at m/z
3.2. 1H and 13C NMR spectra
The 1H NMR spectrum gives some important information
regarding the formation of proposed macrocyclic moiety (Fig. 5).
Fig. 2. Fluorescence emission spectrum of Ni(II) complex in presence of increasing amount of CT-DNA. Inset shows the Stern–Volmer plot for the binding with CT-DNA.

358
M. Shakir et al. / Spectrochimica Acta Part A 93 (2012) 354–362
50
40
30
20
10
0
10
20
30
40
50
60
70
80
90
DNA(µM)
70
60
50
40
30
20
10
0
native
10µM
20µM
30µM
40µM
50µM
60µM
70µM
80µM
90µM
375
385
395
405
415
425
435
445
455
465
475
Wavelength (nm)
Fig. 3. Fluorescence emission spectrum of Cu(II) complex in presence of increasing amount of CT-DNA. Inset shows the Stern–Volmer plot for the binding with CT-DNA.
728, the fragment peaks observed at 712 and 658 are due to the
cleavage of CH3 and Cl, respectively.
3.5. Electronic spectra and magnetic susceptibility data
The electronic spectra of the macrocyclic Co(II) complex exhibit
three spin allowed bands at 8500, 14,260 and 21,500 cm−1
3.4. EPR spectrum
4
4
corresponding to 4T1g(F) → T1g(F), 4T1g(F) → A2g(F) and
4
4T1g(F) → T1g(P) transitions, respectively consistent with the
The EPR spectral studies of Cu(II) complex provides information
of the metal ion environment. The EPR spectrum of the Cu(II) com-
plex was recorded in DMSO at liquid nitrogen temperature (LNT).
The spectrum did not show any hyperfine splitting, and only a single
octahedral geometry around the cobalt(II) ion which is further
supported by a magnetic moment value of 4.49 B.M [40].
The electronic spectrum of Ni(II) complex exhibit three
bands at 9000, 15,400 and 22,550 cm−1 attributed to three spin
signal was observed (Fig. 8). The Cu(II) complex exhibits the g value
||
3
3
allowed transitions viz. 3A2g(F) → T2g(F), 3A2g(F) → T1g(F) and
of 2.12 and g value of 2.03. The trend g > g > 2.0023 observed for
⊥
ꢀ
⊥
3A2g(F) → T1g(P), respectively corresponding to an octahedral
3
the complex indicate that, the unpaired electron is localized in the
dx2–y2 orbital of the Cu(II) ion characteristic of the axial symmetry,
leading to a tetragonally elongated structure of the Cu(II) complex
[37]. The calculated gav value of 2.06 and its deviation from that
geometry for the Ni(II) complex. The observed magnetic moment
value of 3.12 B.M further complements the electronic spectral find-
ings [40,41].
The electronic spectrum of the copper(II) complex show bands
of the free electron (2.0023) may be due to the covalence in M
L
2
2
at 16,250 and 20,500 cm−1 which may be ascribed to B1g → Eg
bonding which is supported by Kivelson and Neiman [38]. However,
the calculated G parameter of 3 which is less than 4 indicates con-
siderable exchange interaction present between the Cu(II) centers
[39].
2
2
and B1g → B2g transitions, respectively corresponding to dis-
torted octahedral geometry. Magnetic moment of 1.83 B.M. further
confirms an octahedral geometry around the Cu(II) ion [40].
Table 2
IR vibrational frequencies (cm−1) of the complexes.
Complexes
ꢀ(C N)
ꢀ(M N)
ꢀ(M Cl)
ꢀ(C H)
Phenyl ring vibrations
[CoLCl2]
[NiLCl2]
[CuLCl2]
[ZnLCl2]
1621
1620
1618
1622
439
436
460
430
272
255
275
290
2925
2942
2919
2906
1442, 1060, 776
1447, 1062, 701
1440, 1026, 770
1445, 1061, 700

M. Shakir et al. / Spectrochimica Acta Part A 93 (2012) 354–362
359
40
30
20
10
0
10
20
30
40
50
60
70
80
90
DNA(µM)
60
50
40
30
20
10
0
native
10µM
20µM
30µM
40µM
50µM
60µM
70µM
80µM
90µM
360
380
400
420
440
460
480
Wavelength (nm)
Fig. 4. Fluorescence emission spectrum of Zn(II) complex in presence of increasing amount of CT-DNA. Inset shows the Stern–Volmer plot for the binding with CT-DNA.
3.6. Antibacterial activity
was performed at fixed concentration of 10 mg/ml. The results
obtained were compared with standard antibiotics: ciprofloxacin
(for gram-positive) and gentamicin (for gram-negative) bacterial
strains. All the complexes were found to be active on both types
of bacterial strains. On the basis of the data obtained for diame-
ter of zone of inhibition Cu(II) and Ni(II) complexes were found
to be very effective (Fig. 9a and b). In addition all the complexes
were found to be effective at different dilutions based on the activ-
ity. The minimum inhibitory concentration of these complexes
was determined by broth dilution method in which the effec-
tiveness was observed at lower concentrations (Table 3). It was
observed from the MIC values that Cu(II) complex was quite effec-
tive against S. mutans (0.156 mg/ml), S. pyogenes (0.312 mg/ml),
C. xerosis (0.312 mg/ml), K. pneuomoniae (0.312 mg/ml), E. coli
Antibacterial activity of the synthesized complexes was studied
against some bacterial strains viz. S. aureus, S. mutans, S. pyogenes,
S. epidermidis, B. cereus, C. xerosis, E. coli, K. pneuomoniae, P. vul-
garis and P. aeruginosa. Preliminary screening for all the complexes
Table 3
Minimum inhibitory concentration (MIC in mg/ml) of the macrocyclic complexes.
S.no
Bacterial Strains
Complexes
[CoLCl2]
[NiLCl2]
[CuLCl2]
[ZnLCl2]
1.
2.
3.
4
5.
6.
7.
8.
9.
S. aureus
S. mutans
S. epidermidis
S. pyrogenes
B. cereus
C. xerosis
E. coli
K. pneuomoniae
P. aeruginosa
P. vulgaris
0.312
0.625
1.250
0.625
2.500
1.250
2.500
0.312
2.500
1.250
0.625
0.312
0.625
0.312
0.625
1.250
2.500
0.156
1.250
0.625
2.500
0.156
2.500
0.312
1.250
0.312
0.625
0.312
1.250
0.625
1.250
2.500
1.250
1.250
2.500
1.250
0.625
1.250
0.625
1.250
10.
Fig. 5. 1H NMR spectrum of Zn(II) complex.

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M. Shakir et al. / Spectrochimica Acta Part A 93 (2012) 354–362
Fig. 6. 13C NMR spectrum of Zn(II) complex.
(0.625 mg/ml) and P. vulgaris (0.625 mg/ml). The MIC for Ni(II) com-
plex was found to be 0.156 mg/ml for K. pneuomoniae, 0.312 mg/ml
for S. mutans and S. pyogenes and 0.625 mg/ml for S. aureus, S.
epidermidis, B. cereus and P. vulgaris. For Co(II) complex MIC was
highest for S. aureus and K. neuomoniae (0.312 mg/ml) and then
0.625 mg/ml for S. mutans and S. pyogenes. The Zn(II) complex
showed comparatively low activity against most of the bacterial
strains.
3.7. Fluorescence measurements
3.7.1. DNA binding of the complexes
Fluorescence quenching is a useful method to monitor the
molecular interactions of chemical and biological systems because
of its high sensitivity [43,44]. Fluorescence intensity of
a
It is concluded that all the synthesized macrocyclic complexes
showed the antibacterial activity, but they were found to be more
potent inhibitors against gram positive bacterial strains (Table 3).
The Cu(II) complex showed comparatively more inhibition against
K. pneuomoniae as compared to the commercial antibiotic (Fig. 9b).
The reason for high antimicrobial activity of copper complex
can be explained in terms of the effect of copper metal ion on
the normal cell process. The complexation reaction reduces the
polarity of the metal ion by the partial sharing of metal ion pos-
itive charge with donor groups and electron delocalization over
the chelate ring [42]. Thus, the lipophilic character of the central
metal atom is enhanced which results in a higher capability to
penetrate the microorganisms through the lipid layer of the cell
membrane.
Fig. 7. FAB-mass spectrum of Cu(II) complex.
Fig. 8. EPR spectrum of Cu(II) complex.

M. Shakir et al. / Spectrochimica Acta Part A 93 (2012) 354–362
361
Fig. 9. (a) Comparison of zone of inhibition (in mm) of macrocyclic complexes with standard antibiotic against gram positive bacterial stains. Ciprofloxacin-standard antibiotic.
(b) Comparison of zone of inhibition (in mm) of macrocyclic complexes with standard antibiotic against gram negative bacterial stains. Gentamicin-standard antibiotic.
compound can quench as a result of variety of molecular inter-
actions such as excited state reactions, molecular rearrangements,
energy transfer, ground state complex formation and collisional
quenching. Dynamic quenching results from collision between
fluorophore and quencher whereas static quenching is due
to ground-state complex formation between fluorophore and
quencher [45]. In the present study, we investigated the interac-
tions of synthesized macrocyclic complexes with calf thymus DNA
by fluorescence spectroscopy. Fluorescence quenching is usually
classified as dynamic quenching and static quenching [46]. How-
ever, the characteristic Stern–Volmer plot of combined quenching
(both static and dynamic) is an upward curvature. The binding
of macrocyclic complexes with calf thymus DNA, was studied by
monitoring the changes in the intrinsic fluorescence of different
complexes at varying DNA concentrations. The representative flu-
orescence emission spectra of the complexes upon excitation at
different wavelengths are given in Figs. 1–4. The spectra illustrate
that excess of DNA caused a gradual decrease in the fluores-
cence emission intensity. The quenching of the fluorescence clearly
indicates that the binding of DNA to the macrocyclic complexes
changed the microenvironment of fluorophore residue. The spec-
tra illustrate that excess of DNA led to more effective quenching of
fluorophore molecule fluorescence (Fig. 10). There are three major
mode of DNA interaction relevant to the metal complexes depend-
ing on the presence of charged atoms, hydrophobicity and structure
of these complexes. As external binders the complexes with posi-
tive charges interact with the DNA backbone due to the electrostatic
interaction with negatively charged phosphates. Groove binders
interact with the DNA groove and hydrophobic interactions are
usually important components of this binding process. Such a mode
of interaction is also governed by geometric and steric factors such
as non-planer structures and the presence of methyl groups that
prevent intercalation. The third mode involves the insertion of a
planar fused aromatic ring system between the DNA base pairs
leading to intercalation [47–49]. Few studies on tetrazamacrocyclic
complexes have proposed intercalation as a possible mode of DNA
interaction [50,51]. In order to examine the possible mode of inter-
action of the complexes with DNA, ethidium bromide displacement
assay has been performed. Ethidium bromide, a polycyclic aromatic
dye, is the most widely used fluorescence probe for DNA struc-
ture. It binds to DNA by intercalation within the stacked bases
[52]. It has been reported that the enhanced fluorescence of the
EB-DNA complex can be quenched at least partially by the addi-
tion of a second molecule and this could be used to assess the
relative affinity of the molecule for DNA intercalation [53]. The
emission spectra of EB bound to DNA in the absence and presence
of complexes is given in Fig. 10. The addition of these complexes to
DNA being complexed with EB does not cause reduction in emis-
sion intensity, indicating that none of these complexes compete
with EB in binding to DNA. The external interaction with DNA
back bone was also ruled out as these complexes are non-cationic,
lacking in a nucleophilic attracting centre. These aromatic com-
plexes with methyl groups possibly interact with DNA within the
grooves via stabilization through hydrophobic cohesion. This is pre-
sumably explained due to steric hindrance encountered by methyl
groups, thus preventing the DNA base intercalation of these com-
plexes.
Fig. 10. Flourescence emission spectra of ethidium bromide bound to DNA in
absence and presence of Co(II), Ni(II), Cu(II) and Zn(II) complexes.

362
M. Shakir et al. / Spectrochimica Acta Part A 93 (2012) 354–362
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Acknowledgements
The chairman department of chemistry, Aligarh Muslim Uni-
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New Delhi for non-NET UGC fellowship. The authors are thankful to
the deanship of scientific research, King Saud University, Riyadh for
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