Synthesis, spectral characterization, DNA binding ability and antibacterial screening of copper(II) complexes of symmetrical NOON tetradentate Schiff bases bearing different bridges

Article abstract of DOI:10.1016/j.molstruc.2012.04.017

A novel series of four copper(II) complexes were synthesized by thermal reaction of copper acetate salt with symmetrical tetradentate Schiff bases, N,N′bis(o-vanillin)4,5-dimethyl-l,2-phenylenediamine (H₂L ¹), N,N′bis(salicylaldehyde

Full text of DOI:10.1016/j.molstruc.2012.04.017

Journal of Molecular Structure 1020 (2012) 188–196
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, spectral characterization, DNA binding ability and antibacterial
screening of copper(II) complexes of symmetrical NOON tetradentate Schiff
bases bearing different bridges
a
a,b,
⇑
, Maher M. El-Naggar a
Saleh O. Bahaffi , Ayman A. Abdel Aziz
a
Department of Chemistry, Faculty of Science, University of Tabuk, Tabuk 71421, Saudi Arabia
Chemistry Department, Faculty of Science, Ain Shams University, 11566 Abbassia, Cairo, Egypt
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of four copper(II) complexes were synthesized by thermal reaction of copper acetate salt
with symmetrical tetradentate Schiff bases, N,N bis(o-vanillin)4,5-dimethyl-l,2-phenylenediamine
Received 14 March 2012
Received in revised form 6 April 2012
Accepted 7 April 2012
0
1
0
2
0
(
H
2
L ), N,N bis(salicylaldehyde)4,5-dimethyl-1,2-phenylenediamine (H
2
L ), N,N bis(o-vanillin)4,5-
L ) and N,N bis(salicylaldehyde)4,5-dichloro-1,2-phenylenediamine
L ), respectively. All the new synthesized complexes were characterized by using of microanalysis,
FT-IR, UV–Vis, magnetic measurements, ESR, and conductance measurements, respectively. The data
3
0
dichloro-1,2-phenylenediamine (H
2
Available online 16 April 2012
4
(H
2
Keywords:
Synthesis
1–4
revealed that all the Schiff bases (H
2
L
) coordinate in their deprotonated forms and behave as tetraden-
tate NOON coordinated ligands. Moreover, their copper(II) complexes have square planar geometry with
general formula [CuL1 ]. The binding of the complexes with calf thymus DNA (CT-DNA) was investigated
by UV–Vis spectrophotometry, fluorescence quenching and viscosity measurements. The results indi-
cated that the complexes bind to CT-DNA through an intercalative mode. From the biological activity
view, the copper(II) complexes and their parent ligands were screened for their in vitro antibacterial
activity against the bacterial species Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli
and Pseudomonas aeruginosai by well diffusion method. The complexes showed an increased activity in
comparison to some standard drugs.
Cu(II) complexes
NOON donors
Characterization
CT-DNA binding
Antibacterial screening
–4
Ó 2012 Elsevier B.V. All rights reserved.
1
. Introduction
Deoxyribonucleic acid (DNA) plays a significant role in the life
It is known that copper is a bioessential element with relevant
oxidation states [8]. A diverse variety of copper-containing metal-
loenzymes occur in nature. It is revealed that Azurins [9], plastocy-
anins [10] and laccases [11] are involved in electron transfer
reactions. Moreover, Tyrosinases [12] and ascorbate oxidases [13]
are used in oxygenation reactions. Also, Hemocyanins [14] are cop-
per-containing oxygen transport metalloproteins found in arthro-
pods and mollusks. The role played by copper ions in the active
sites of a large number of metalloproteins droved many research-
ers to design and characterize copper complexes as models for a
better understanding of biological systems [15–17].
process. This implies carrying the inheritance information, the bio-
logical synthesis of proteins and enzymes through the replication
and transcription of genetic information in living cells. Also DNA
is considered the primary target molecule for most anticancer
and antiviral therapies according to cell biologists. Since DNA is
an important cellular receptor, many chemicals exert their antitu-
mor effects through binding to DNA thereby changing the replica-
tion of DNA and inhibiting the growth of the tumor cell [1]. Thus
new and more efficient antitumor drugs were designed [2,3].
The interaction between DNA and transition metal complexes is
an important fundamental issue in life sciences [4]. Consequently
the design and synthesis of new complexes that interact with
DNA are of considerable interest nowadays [5–7].
The aim of the present work is to investigate the interaction of
calf thymus DNA (CT-DNA) with novel copper(II) complexes syn-
thesized via thermal reaction with symmetrical tetradentate
2 2
N O Schiff base ligands derived from condensation reactions of
4,5-dimethyl-1,2-phenylendiammine and 4,5-dichloro-1,2-pheny-
lendiammine with o-vanillin and salicylaldehyde. The new synthe-
sized complexes have been fully characterized by different
spectroscopic techniques. Moreover, from biological activity view
the in vitro antibacterial activity of the new copper(II) complexes
were screened against the bacterial species Staphylococcus aureus,
⇑
Corresponding author. Permanent address: Chemistry Department, Faculty of
Science, Ain Shams University, 11566 Abbassia, Cairo, Egypt. Tel.: +20 1001300155;
fax: +20 22483631.
E-mail addresses: aymanaziz31@gmail.com, aabdelaziz@ut.edu.sa (A.A. Abdel
Aziz).
0
022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.04.017

S.O. Bahaffi et al. / Journal of Molecular Structure 1020 (2012) 188–196
189
Staphylococcus epidermidis, Escherichia coli and Pseudomonas aerug-
inosai by well diffusion method.
R
R
NH2
NH2
OHC
HO
+
2
. Experimental
R/
Microanalyses (C, H and N) of all the synthesized compounds
were performed on a JEOL JMS-AX500 MASS SPECTROMETER. FT-
ꢀ1
IR spectra (4000–400 cm , KBr discs) of the samples were re-
corded on a Unicam-Mattson 1000 FT-IR. The electronic spectra
were recorded on Shimadzu UV 1800 Spectrophotometer,
equipped with a PC, using UVProbe software, version 2. Fluores-
cence spectra were recorded on a Jenway 6270 Fluorimeter. The
molar conductivities of the complexes in dimethyl sulfoxide
R
R
ꢀ3
(
DMSO) solution (1 ꢁ 10 M) were measured at room tempera-
ture by using Jenway 4010 conductivity meter. Magnetic moments
were determined on a Sherwood Scientific magnetic moment bal-
ance (Model N0: MK1) at room temperature (25 °C), using
4
Hg[Co(SCN) ] as a calibrant. Corrections for diamagnetism of the
constituents were made using Pascal constants [18]. Electron spin
resonance (ESR) measurements of solid complexes were recorded
at room temperature on Bruker EPR spectrometer at 9.706 GHz
N
N
HO
OH
(
X-band), the microwave power was (1.0 mW) with 4.0 G modula-
R/
R/
tion amplitude, using 2,2-diphenylpyridylhydrazone (DPPH) as
standard (g = 2.0037). Copper content of the complexes were esti-
mated by atomic absorption spectroscopy after decomposing the
/
1
R= CH
R= CH
and R = OCH
H
H L
2
L
3
3
2
/
2
3
4
complexes with concentrated HNO
AAS [19].
3
using Buck Scientific 205
3
and R = H
/
R= Cl and R = OCH H L
3
2
/
R= Cl and R = H
H2L
2.1. Materials
4
,5-Dimethyl-1,2-phenylendiammine, 4,5-dichloro-1,2-pheny-
lendiammine, o-vanillin, salicylaldehyde and Cu(CH COO)
were supplied from Aldrich. calf thymus DNA (CT-DNA) and ethi-
dium bromide (EB) were purchased from Sigma Chemicals Co.
3
2
ꢂH
2
O
(
USA). All experiments of the interaction of the complexes with
CT-DNA were carried out at room temperature in triply distilled
water buffer containing 5 mM Tris–HCl/50 mM NaCl and adjusted
to pH 7.2 with hydrochloric acid. The stock solution of CT-DNA
R
R
(
4
1 mM) was prepared in Tris–HCl/NaCl buffer, pH 7.2 (stored at
°C and used within 4 days). Solution of CT-DNA in the buffer gave
a ratio of UV absorbance at 260 and 280 nm (A260/A280) of 1.87,
indicating that the DNA was sufficiently pure and free of protein
N
N
[
20]. The CT-DNA concentration per nucleotide was determined
Cu
by absorption spectroscopy using the molar absorption coefficient
ꢀ
1
ꢀ1
(
6600 M cm ) at 260 nm [21]. All solvents were of analytical
O
O
grade and were used without further purification.
R/
R/
2.2. Syntheses
Scheme 1. Synthetic route of Schiff base ligands (H
2
L1–4) and their corresponding
copper(II) complexes.
Synthetic route of Schiff base ligands (H
2
L1–4) and their corre-
sponding copper(II) complexes (1–4) is shown in Scheme 1.
2
.2.2. Synthesis of copper(II) complexes (1–4)
The copper(II) complexes were prepared by using the following
general procedure. A methanolic solution (10 mL) of Cu(OAc)
ꢂH
1 mmol) was added drop wisely to a hot methanolic solution
10 mL) of the corresponding Schiff bases. After complete addition,
1
–4
2.2.1. Synthesis of the Schiff base ligand (H
2
L
)
2
2
O
The ligands were synthesized as described in literature [22] by
(
(
the following general procedure. 20 mmol of aldehyde was added
drop wisely to a hot methanolic solution containing 10 mmol of
the amine. The yellow solution obtained was refluxed for 2 h. After
cooling, the precipitated product (Schiff base) was collected by fil-
tration and recrystallized from methanol. The ligands were dried in
a desiccator over anhydrous CaCl under vacuum. The resultant
2
product was washed with ethanol and the purity of the ligands
was checked by TLC.
1
2
2 2
the mixture was refluxed at 80 °C for 3 h in case of H L and H L
and 8 h for H L and H L . Thereafter, the microcrystalline complex
2 2
3
4
was separated on cooling and filtered under suction. The complex
crystals were washed with hot petroleum ether 40–60 °C, recrys-
tallized from chloroform and finally kept in a desiccator over anhy-
2
drous CaCl .

1
90
S.O. Bahaffi et al. / Journal of Molecular Structure 1020 (2012) 188–196
2
2
.3. DNA-binding experiments
g
¼ ðt ꢀ t
o
Þ=t
o
ð3Þ
where t is the observed flow time of the CT-DNA-containing solu-
tion and t is the flow time of buffer alone [26]. The data were pre-
sented as (
.3.1. Absorption study
Absorption studies were performed with fixed complex concen-
o
1
/3
1–4
g/g
o
)
vs mole ratio of [CuL ]/[DNA] [27].
trations (50
lM) while varying the CT-DNA concentration within
0
–50 M. Because of their low solubility in the buffer solution,
l
2
.4. Anti bacterial screening
the copper(II) complexes was dissolved in ethanol first to get a
stock solution and then a small amount of the stock solution was
added to the DNA solutions to get the desired concentration. While
measuring the absorption spectra, equal increments of CT-DNA
were added to both the complex solution and the reference solu-
tion to eliminate the absorbance of CT-DNA itself. From the absorp-
The in vitro biological activity of the investigated Schiff bases
and their copper(II) complexes was tested against the bacteria
strains: (i) S. aureus, (ii) S. epidermidis (iii) E. coli, and (iv) P. aerug-
inosai by well diffusion method using nutrient agar as medium. The
stock solution was prepared by dissolving the compounds in
DMSO. In a typical procedure, a well was made on agar medium
inoculated with microorganism. The well was filled with the test
solution using a micropipette and the plate was incubated for
24 h at 37 °C. During this period, the test solution diffused and
the growth of the inoculated microorganism was affected. The
inhibition zone was developed, at which the concentration was
noted. Preliminary screening was performed using two concentra-
b
tion data, the intrinsic binding constant K was determined from a
plot of [DNA]/(
e
a
ꢀe
f
) versus [DNA] using the equation [23]:
½
DNAꢃ ½DNAꢃ
1
¼
þ
ð1Þ
ð
e
a
ꢀ
e
f
Þ
e
b
ꢀ
e
f
½K
b
ð
e
b
ꢀ
e
f
Þꢃ
where [DNA] is the concentration of DNA in base pairs, the apparent
a, b f
extinctions coefficient e e and e
correspond to Aobs./[CuL1 ], the
–4
extinction coefficient of the copper(II) complex in the free solution
and the extinction coefficient of the copper(II) complex in the fully
bound form, respectively. The intrinsic binding constant (K ) is gi-
b
ven by the ratio of the slope to the intercept.
tions, 100 lg/1.0 mL and 200 lg/1.0 mL of the tested compounds.
DMSO was used as a control under the same conditions for each
organism and no activity was found. Finally the activity results
are calculated as a mean of triplicates. The standard drugs strepto-
mycin and amoxycillin were also tested for their antibacterial
activity at the same concentration under similar conditions to that
of the test compounds.
2.3.2. Fluorescence study
The relative binding of the copper(II) complexes to CT-DNA was
studied with an EB-bound CT-DNA solution in 5 mM Tris–HCl/NaCl
buffer (pH 7.2) using fluorescence spectrophotometry. The fluores-
3
. Results and discussion
cence intensities of EB (10
measured at 602 nm (524 nm excitation) after addition of different
complex concentrations within range 0–50 M. The relative bind-
lM) bound to CT-DNA (10 lM) were
The physical properties of the ligands and the complexes are
l
grouped in Table 1. The elemental analysis data (Table 1) was
found to agree well with the proposed formulae of the complexes
and confirmed the [CuL ] composition with 1:1 [M:L] ratio. Gen-
ing of the copper(II) complexes to CT-DNA was determined by cal-
culating the quenching constant (K) from the slopes of straight
lines of Stern–Volmer equation [24]:
1–4
erally all the complexes were colored, quite stable towards air,
insoluble in water and soluble in acetone, ethanol, CH
2 2
Cl , DMF
F
o
=F ¼ 1 þ K r
ð2Þ
CH CN and DMSO, respectively. The Molar conductance values
3
ꢀ
3
(K
m
) of the complexes in DMSO (1 ꢁ 10 M) solution at 25 °C
where F
o
and F are the fluorescence intensities in the absence and
ꢀ1 ꢀ1 2
1–4
were found in the range 8.45–15.7
X
mol cm . These low val-
presence of the quencher, respectively and r = [CuL ]/[DNA].
ues indicated that all of the complexes have non-electrolytic nat-
ure [28].
2
.3.3. Viscosity measurements
Viscosities of CT-DNA at different complexes concentrations (0–
6
0
l
M) in buffer solution were measured using Ubbelodhe viscom-
3.1. Characterization of the complexes
3.1.1. Infrared spectra
eter at the temperature of 25 ± 0.1 °C in a thermostatic water-bath.
CT-DNA samples, averaging approximately 200 base pairs (bp)
were prepared using sonication in order to minimize the complex-
In order to study the binding of Schiff bases ligands (H
2
L1–4) to
1
–4
ities arising from DNA flexibility [25]. Different ratios of [CuL ]/
DNA] were prepared before use, using [CT-DNA] = 100 M. Aver-
Cu(II) ion, a comparison between the IR spectra of the complexes
and the free ligands were made. The selected IR spectra of the li-
gands and their copper(II) complexes along with their tentative
[
l
age flow time was recorded after three time measurements for a
sample with a digital stopwatch. Relative viscosities for CT-DNA
in the presence and absence of complexes were calculated from
the relation:
1
assignments are presented in Table 2. The IR spectra of [CuL ]
2
and [CuL ] complexes as a representative example are shown in
Fig. 1. It is illustrated that all the IR spectra of the complexes are
Table 1
Yield, molecular weight, micro analysis and conductivity data of the Schiff bases ligands (H
2
L1–4) and their copper(II) complexes.
ꢀ
1
cm2 molꢀ1
)
Complexes
Yield (%) M (Wt) Color
(Calculated) Found (%)
K
m
(X
C
H
N
Cl
Cu
–
H
2
L1
C
24
H
24
N
2
O
H
4
89
85
63
79
88
404.46
465.99
344.41
405.94
445.30
506.83
385.25
466.78
Orange
Brown
Yellow
(71.27) 71.01 (5.98) 5.77 (6.92) 6.78
61.86 (61.79) 4.75 (4.68) 6.01 (5.86)
(76.72) 76.21 (5.85) 5.72 (8.13) 7.98
–
–
–
–
–
1
[
CuL ] (1) (C24
22
N
2
2
2
O
O
O
O
4
2
4
2
)Cu
)Cu
(13.63) 13.56 14.9
–
15.65 (15.61) 15.70
2
H
2
L
C
22
H
20
N
2
O
2
–
2
[
CuL ] (2) (C22
H
18
N
Dark green 65.09 (64.98) 4.46 (4.37) 6.90 (6.88)
3
H
2
L
C
22
H
18
N
2
O
4
Cl
2
Yellow
Brown
Yellow
Brown
(59.34) 59.21 (4.07) 3.92 (6.29) 6.11 (15.92) 15.75
(52.13) 52.03 (3.18) 3.13 (5.52) 5.46 (13.99) 13.87 (12.53) 12.49 9.74
(62.35) 62.29 (3.66) 3.60 (7.27) 7.25 (18.40) 18.38
(51.46) 51.38 (2.59) 2.53 (6.00) 5.93 (15.19) 15.02 (13.61) 13.58 8.45
–
–
3
[
CuL ] (3) (C22
H
16
N
Cl
2
)Cu 75
70
)Cu 63
4
H
2
L
C
20
H
14
N
2
O
2
Cl
2
–
–
4
[
CuL ] (4) (C20
H
12
N
2
Cl
2

S.O. Bahaffi et al. / Journal of Molecular Structure 1020 (2012) 188–196
191
Table 2
The infrared (cmꢀ1), UV–Vis spectral data of the Schiff bases ligands (H
L1–4) and their copper(II) complexes.
(CAO) (CuAO)
2
Compound
L1
(OH)
m
(C@N)
m
m
m(CuAN)
UV–Vis kmax (nm) (Ethanol)
⁄
a
b
b
H
2
3389(br.)
1610(s)
1612(s)
1617(s)
1615(s)
1608(s)
1606(s)
1616(s)
1608(s)
1276(m)
1334(m)
1278(m)
1329(m)
1274(m)
1299(m)
1279(m)
1332(m)
–
–
224 , 308
1
a
c
c
c
c
[
CuL ] (1)
–
488(w)
–
497(w)
–
466(w)
–
554(w)
436(w)
–
441(w)
–
435(w)
–
475(w)
228 , 311 , 400
2
a
b
b
H
2
L
3428(br.)
–
223 , 301
2
a
[
CuL ] (2)
224 , 305 , 397
3
a
b
b
H
2
L
3394(br.)
–
216 , 297
3
a
[
CuL ] (3)
219 , 299 , 388
4
a
b
b
H
2
L
3444(br.)
–
214 , 295
4
a
CuL ] (4)
218 , 288 , 386
⁄
IR peak intensity; s, strong; m, medium; w, weak; br., broad.
a
b
c
ꢄ
p–p .
ꢄ
n–
p .
MLCT.
ꢄ
ꢄ
very similar. This finding would indicate that, the four complexes
have the same IR coordination structures. Generally, the position
and/or the intensities of IR peaks are expected to change upon che-
lation. Moreover, the appearance of new peaks are also guiding for
chelation. The discussion is confined to the most important vibra-
p–p
and n ? p transitions within the Schiff base ligands. These
absorptions also present in the spectra of copper(II) complexes
but they undergone bathochromic shift. This shift in the spectra
of the complexes supported the coordination of the ligands to
Cu(II) ion. Furthermore, the UV–Vis spectra of the complexes
showed an intense band in the region 386–400 nm which can be
considered as ligand to metal charge transfer (LMCT) transitions
as those were absent in the spectra of the respective free ligands.
ꢀ1
tions of the 4000–400 cm region in relation to structure. It can be
clearly seen that the absence of the characteristic aldehydic car-
bonyl stretching bands, and the appearance of the azomethine
ꢀ1
AC@NA bands at 1608–1617 cm . This behavior gives an evi-
dence for formation of the Schiff bases. Also, the broad band of
3.1.3. Electron spin resonance spectra
ꢀ1
medium intensity which appeared in the region 3389–3444 cm
was assigned to the intra-molecular hydrogen-bonded OAH group
29]. As the hydrogen bond becomes stronger, the bandwidth in-
The ESR spectra of copper(II) complexes provide valuable infor-
mation in studying the Cu(II) ion environment. ESR spectra of the
polycrystalline copper (II) complexes exhibited an isotropic signal,
without any hyperfine splitting. Representative ESR spectra of
[
creases, and this band sometimes cannot be detected [30]. The
strong band attributed to (C@N) stretching vibrations of imine
nitrogen, present in the free ligands was found to be shifted to low-
1
2
[CuL ] and CuL complexes are given in Fig. 2. The g tensor values
of the copper (II) complexes could be used to obtain the ground
state [37]. For ionic environment g|| is normally 2.3 or larger, but
for covalent environment g is less than 2.3 [38]. The trend in the
ꢀ1
er wave numbers in the range 1606–1615 cm in accordance with
the coordination of the azomethine to the metal ion for all the
complexes [31]. Coordination of the Schiff base to the metal
through the nitrogen atom is expected to reduce electron density
in the azomethine as a result of the withdrawal of electron density
from the nitrogen atom and lower the C@N absorption frequency.
On the other hand, the involvement of the deprotonated phenolic-
OH group in chelation is confirmed by clarifying the effect of che-
||
observed ‘‘g’’ values of copper (II) complexes at room temperature
(Table 3), was g > g > g (2.0023). This trend provide an evidence
||
\
e
of localization of the unpaired electron in dx2–y2 orbital [39,40]. The
‘gaver’ values of the complexes were found to be in the range
2.09–2.12. Moreover, for the reported copper(II) complexes,
g < 2.3 value, indicating the covalent character of the metal-ligand
||
lation on the
m
(CAO). The medium intensity band in the region
bond [40]. This behavior is further supported by calculating the
axial symmetry parameter (G) using Kneubuhl’s method G =
(g ꢀ 2.0023)/(g ꢀ 2.0023) [41]. According to Hathaway [42], if
ꢀ1
1
274–1279 cm
was ascribed to the phenolic CAO stretching
vibration in the case of salicylideneanilines [32]. On complexation
this band was shifted to higher frequency in the range 1299–
|
|
\
the G value >4, the exchange interaction is negligible, while a value
of <4 give an indication for considerable exchange interaction in
the complex. The axial symmetry parameter (G) of the reported
copper(II) complexes were <4 suggesting exchange interaction in
the solid state.
ꢀ1
1
334 cm . The co-ordination of the azomethine nitrogen and phe-
nolic oxygen were further supported by the appearance of two
non-ligand bands at 488–554 cm
ꢀ1
ꢀ1
and 435–475 cm
due to
m
(CuAO) and (CuAN) [33,34], respectively. From the above argu-
m
ments together with the elemental analyses, it was concluded that
Schiff base ligands behave as a dianionic tetradentate Schiff base
ligand with NOON donor sites.
3.2. DNA binding studies
DNA binding is the critical step for DNA cleavage in most cases.
Many workers [43,44] suggested two major binding modes of
interaction of substrate with DNA. The first suggestion is that the
metal complexes squeeze in between the double helix through
hydrogen bonding. The second suggestion based on covalent bind-
ing, where two major sites are available for the metal ion to inter-
act with the DNA. In the later case, one being the electron donor
groups of the bases, more preferably at the guanine N-7 and the
other is the phosphate moieties of the ribose-phosphate backbone.
3
.1.2. Electronic spectra and magnetic susceptibility
Magnetic measurements and electronic spectra were conducted
in order to obtain information about the geometry of the com-
plexes. The values of magnetic susceptibility for the copper(II)
complexes are given in Table 3. The magnetic susceptibility values
(leff) of all these complexes were in 1.74–1.81 BM range, which
was consistent with presence of a single unpaired electron [35].
This behavior suggest square-planar geometry for the copper(II)
complexes [36]. On the other hand, the electronic spectra of the
free ligands and the complexes were recorded in ethanol
3.2.1. UV–Vis spectroscopic studies
ꢀ5
(
1 ꢁ 10 M). The absorption region and the assignment of the
The application of electronic absorption spectroscopy in DNA-
binding studies is one of the useful techniques. The binding of cop-
per(II) complexes with DNA helix have been characterized through
absorption spectral studies by following changes in absorbance
absorption bands are listed in Table 2. Evidently the electronic
spectra of the free ligands exhibit two absorption bands at the
range 214–224 nm and 295–308 nm which were assigned to

1
92
S.O. Bahaffi et al. / Journal of Molecular Structure 1020 (2012) 188–196
1
2
Fig. 1. FT-IR spectra of [CuL ] and [CuL ] complexes.
and shift in wavelength. In general, the absorption spectra of metal
Table 3
The
complex bound to DNA through intercalation exhibit significant
l
eff and g parameter values of the reported copper(II) complexes (1–4).
ꢄ
hypochromism and bathochromism shift due to the strong
p–p
⁄
Complex
leff. (BM)
g
||
g
\
g
aver.
G
stacking interaction between the aromatic chromophore ligand of
metal complexes and the base pairs of DNA [44]. The absorption
spectra of copper(II) complexes in the absence and presence of
DNA, using a constant concentration of the copper(II) complex
are given in Fig. 3. The results showed that as the DNA concentra-
tion increased, the transition bands of the complexes exhibited
1
[
[
[
[
CuL ] (1)
1.78
1.80
1.74
1.81
2.15
2.14
2.11
2.11
2.06
2.07
2.05
2.04
2.12
2.11
2.09
2.08
2.56
2.03
2.26
2.85
2
CuL ] (2)
3
CuL ] (3)
4
CuL ] (4)
⁄
=
1/3(g
\
+ 2 g||); G = (g||-2.0023)/(g
\
-2.0023).

S.O. Bahaffi et al. / Journal of Molecular Structure 1020 (2012) 188–196
193
2
000
000
0
The affinity of the complexes toward DNA was determined
quantitatively by calculating the intrinsic binding constant (K
from the observed spectroscopic changes by regression analysis
1
1
[
CuL ] (1)
b
)
2
1
2
[CuL ] (2)
4
ꢀ1
using Eq. (1). The K
b
values were 4.754 ꢁ 10 M for complex 1,
4
ꢀ1
4
ꢀ1
4.257 ꢁ 10 M for complex 2, 3.637 ꢁ 10 M for complex 3,
4
ꢀ1
and 2.144 ꢁ 10 M
for complex 4, respectively revealing that
-
-
-
-
1000
2000
3000
1 > 2 > 3 > 4 in binding to CT-DNA.
3.2.2. Fluorescence quenching studies
Fluorescence measurements were carried out to get further
proof for the binding of the copper(II) complexes to DNA. The bind-
ing of the copper(II) complexes to CT-DNA was evaluated by mea-
suring the fluorescence emission intensity of EB bound to CT-DNA
as a probe. EB (3,8-diamino-5-ethyl-6-phenylphenanthridinium
bromide) is a large, flat basic conjugate planar molecule that
resembles a DNA base pair. EB shows weak reduced fluorescence
intensity in buffer due to quenching by solvent molecules. When
EB bound to DNA, EB fluorescence is enhanced due to its intercala-
tion into the helix and again it is quenched by the addition of an-
other molecule that displaces EB from DNA [47]. The fluorescence
measurement for the copper(II) complexes showed that no emis-
sion band was observed either with or without CT-DNA at ambient
4000
2
500
3000
3500
4000
4500
Magnetic Induction (G)
1
2
Fig. 2. ESR spectra of [CuL ] and [CuL ] complexes at 298 K.
hypochromism with a red shift of 6 nm, 4 nm, 2 nm and 1 nm for
1
–4
complexes [CuL ] (1–4) respectively. This behavior gives an indi-
cation for binding of the four complexes to DNA. Moreover, it is ob-
served that the extent of hypochromism was 45.60% for complex 1,
3
2.31% for complex 2, 21.67% for complex 3 and 17.67% for com-
temperature. The fluorescence intensity of EB (10
lM) bound to
plex 4, respectively. The extent of hypochromism is consistent with
intercalative interaction [45]. Generally, the extent of the hypo-
chromism is commonly consistent with the strength of intercala-
tive interaction [46].
DNA (10 M) at 602 nm in the absence and the presence of cop-
l
per(II) complexes (1–4), using variable complex concentrations
are shown in Fig. 4. In case of the addition of complexes showed
a significant decrease in the fluorescence intensity of the EB-DNA
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
1.2
5
5
4
4
.5
.0
.5
.0
4.0
3.5
3.0
2.5
2.0
1.5
1
[
CuL ]
3.5
.0
2.5
[
CuL2
]
3
1.0
0.8
0.6
0.4
0.2
0.0
2.0
1.5
1.0
0.5
0.0
1.0
0.5
0.0
0
0
5 10 15 20 25 30 35 40 45 50 55
0
5
10 15 20 25 30 35 40 45 50 55
[
DNA] x 10-6
M
[DNA] x 10-6
M
2
50
300
350
400
450
500
250
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
4
.0
3
.0
2.5
.0
1.5
.0
.5
.0
3
CuL4
3.5
3.0
2.5
2.0
[
CuL ]
[
]
2
1
1
.5
.0
1
0
0.5
.0
0
0
0 5 10 15 20 25 30 35 40 45 50 55
0
5
10 15 20 25 30 35 40 45 50 55
DNA] x 10-6
[DNA] x 10-6
M
[
M
250
300
350
400
450
500
250
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
Fig. 3. Absorption spectra of copper(II) complexes (50
lM) in the absence (dashed line) and presence of CT-DNA (5,10, 15, 20, 25, 30, 35, 40 and 50
lM) at 25 ± 0.1 °C. Arrows
show the changes in absorbance with respect to an increase of DNA concentration (Inset: plot of [DNA] vs [DNA]/(e
a
f
ꢀe )).

1
94
S.O. Bahaffi et al. / Journal of Molecular Structure 1020 (2012) 188–196
system. The decrease in fluorescence intensity give an indication of
the binding of the copper(II) complexes to DNA. The extent of the
quenching gives a measure of the DNA binding propensity of the
complexes. These results indicate that the copper(II) complexes
could partially displace EB from the EB-DNA system, as often ob-
served in intercalative complex-DNA modes [48].
small molecule intercalate between the DNA base pairs, it unwinds
the DNA helix and hence lengthen it, resulting in significant in-
crease in the viscosity of DNA solution. However, a partial and/or
non-classical intercalation of ligand may bend (or kink) the DNA he-
lix, resulting in the decrease of its effective length and concomi-
tantly its viscosity [49]. The effect of the investigated copper(II)
complexes on the viscosity of CT-DNA solution was studied in order
to assess the binding mode and strength of these complexes with
CT-DNA. The effects of copper(II) complexes (1–4) on the viscosity
of rod-like CT-DNA at 25 ± 0.1 °C are shown in Fig. 5. Viscosity
experimental results clearly showed that the relative viscosity of
CT-DNA increased steadily on addition of increasing concentration
of the copper (II) complexes in the following order 1 > 2 > 3 > 4. Sev-
eral factors which include the shape and hydrophobicity of the
complex as well as extension of planarity and the presence of addi-
tional donor functionalities on the ligand have been cited to be an
importance in rendering these complexes to be strong intercalative
binding agents. The complexes 1 and 2 intercalate more strongly
and deeply than the complexes 3 and 4, leading to the greater in-
crease in viscosity of the CT-DNA with an increasing concentration
of complex. The viscosity increase of CT-DNA is ascribed to the
intercalative binding mode of the copper(II) complexes which cause
the effective length of the DNA to increase [51].
The fluorescence quenching of EB bound to DNA by the cop-
per(II) complexes (1–4) is plotted against r values (r = [complex]/
[
DNA]) and it was found to be in agreement with the Stern–Volmer
equation (Fig. 4 inset). The Stern–Volmer constant (K) value give an
indication about the degree of interaction the copper(II) complexes
to CT-DNA. The K values corresponding to copper(II) complexes (1–
4
o
) were obtained from the slope of F /F–r plot using Eq. (2), were
found to be 1.95, 1.57, 1.05, and 0.96 respectively. These values
give an indication for the binding ability of the copper(II) com-
plexes with CT-DNA follow the order 1 > 2 > 3 > 4 which in consis-
tent with absorption study.
3.2.3. Viscosity studies
The binding of the copper(II) complexes (1–4) with CT-DNA was
further elucidated by measuring the relative specific viscosity of
DNA after the addition of varying concentration of complexes. Vis-
cosity studies give valuable information regarding mode of binding
metal complexes with DNA in the absence of crystallographic struc-
tural data [49]. Also, viscosity measurements are proved to be least
ambiguous to support a complex-DNA binding model [50] as these
measurements are very much sensitive to length change. When a
In conclusion, it is suggested that, the copper(II) complexes (1–
4
) may bind with DNA through intercalative mode. The binding
molecular mode for copper(II) complexes via an intercalative mode
is represented in Scheme 2.
1
000
1000
2
.2
2.0
1
2.0
1.8
2
9
8
7
6
5
4
3
2
1
00
00
00
00
00
00
00
00
00
0
[CuL ]
900
800
700
600
500
400
300
200
100
0
1
1
.8
.6
[CuL ]
1.6
1
1
1
.4
.2
.0
1.4
1
1
0
.2
.0
.8
0
0.8
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
r
r
5
00
550
600
650
700
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
1
000
1000
900
800
700
600
500
400
300
200
100
0
1
.8
.6
1
.6
1.4
.2
1
9
8
7
6
5
4
3
2
1
00
00
00
00
00
00
00
00
00
0
3
4
[
CuL ]
1.4
1.2
[
CuL ]
1
1
.0
1.0
0.8
0
.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
0.8
r
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
r
5
00
550
600
650
700
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
Fig. 4. Effect of increasing amount of copper(II) complexes (0–50
lM) on emission spectra EB-DNA (10
lM) at 25 ± 0.1 °C (dashed line in absence of Cu(II) complex). Arrows
show the changes in emission intensity with respect to an increase of complex concentration (Inset: plot of Stern–Volmer plot of F
o
/F vs [CuL]/[DNA]).

S.O. Bahaffi et al. / Journal of Molecular Structure 1020 (2012) 188–196
195
2
2
1
1
1
1
1
0
0
.2
.0
.8
.6
.4
.2
.0
.8
.6
1
CuL ]
2
[
[
[
CuL ]
3
CuL ]
4
CuL ]
0
.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
[Complex]/[DNA]
Fig. 5. Effect of increasing amount of copper(II) complexes on the relative viscosity
of CT-DNA at 25 ± 0.1 °C. [DNA] = 100
l
M and [complex] = 0–60 lM.
Fig. 6. Zone of inhibitions of reported compounds (200
against gram (+) and gram (ꢀ) bacteria strains.
lg/mL) and antibiotics
R
R
3.3. Antibacterial screening
The Schiff base ligands H
2
L1–4 and their copper(II) complexes
1–4) were tested for their inhibitory effects on the growth of bac-
N
N
(
Cu
teria strains: S. aureus, S. epidermidis, E. coli, and P. aeruginosai. The
antibacterial activities of the prepared compounds are listed in Ta-
ble 4. The results indicated that the tested complexes displayed
more activity against the same microorganisms compared to the
parent ligands under identical experimental conditions. The anti-
O
O
R/
R/
bacterial activity of the reported compounds (200 lg/mL) is repre-
sented graphically in Fig. 6. The data showed that, the activity
increase with increasing the concentration of the test solution.
Such increase in the activity of the complexes compared to that
of ligands can be explained on the basis of Overtone’s concept
[
52] and Tweedy’s Chelation theory [53]. According to Overtone’s
concept of cell permeability, the lipid membrane that surrounds
the cell favors the passage of only the lipid-soluble materials which
makes liposolubility as an important factor that controls the anti-
bacterial activity. On chelation, the polarity of cation will be re-
duced to a greater extent due to the overlap of the ligand orbital
and partial sharing of the positive charge of the metal ion with
Scheme 2. Molecular mode for copper(II) complexes interaction with CT-DNA.
Table 4
Antibacterial activities of the Schiff bases ligands (H
2
L1–4) and their copper(II) complexes.
Compound
Diameter of inhibition zone (mm)
Gram +ve)
S. aureus
(
(Gram ꢀve)
S. epidermidis
E. coli
P. aeruginosai
100 (ppm)
200 (ppm)
100 (ppm)
200 (ppm)
100 (ppm)
200 (ppm)
100 (ppm)
200 (ppm)
1
H
H
H
H
[
[
[
[
2
2
2
2
L
L
L
L
7
6
10
9
19
17
23
20
25
28
0
10
8
6
5
10
9
17
15
23
19
21
27
0
9
8
6
5
9
8
7
6
5
10
9
15
12
19
15
22
24
0
9
8
2
3
4
15
12
22
20
27
25
28
30
0
14
11
21
19
25
21
27
31
0
11
10
19
17
20
19
24
26
0
12
11
16
15
19
17
25
26
0
8
1
CuL ] (1)
16
14
17
15
21
23
0
2
CuL ] (2)
3
CuL ] (3)
4
CuL ] (4)
Streptomycin
Amoxycillin
DMSO

1
96
S.O. Bahaffi et al. / Journal of Molecular Structure 1020 (2012) 188–196
donor groups. Furthermore, the chelation increases the delocaliza-
tion of -electrons over the whole chelate ring and enhances the
(2010) 1421;
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(
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lipophilicity of the complexes. This increase in lipophilicity en-
hances the penetration of the complexes into lipid membranes,
and blocks the metal binding sites of the enzymes of the microor-
ganism. Metal complexes also disturb the respiration process of the
cell and thus block the synthesis of the proteins that restricts fur-
ther growth of the organism. Generally, the electronic nature (elec-
tron withdrawing and electron releasing) and position of
substituents of the phenyl ring dictates the antimicrobial activities.
The inhibitory action gets enhanced with the introduction of elec-
tron-withdrawing chloro groups in the phenyl ring, whereas, elec-
tron-releasing substituents such as methyl groups are less active
compared to un-substituted phenyl ring [54].
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(
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(
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by direct thermal reaction between Cu(OAc)
neutral N
vanillin)4,5-dimethy-l,2-phenylenediamine (H
aldehyde)4,5-dimethyl-1,2-phenylenediamine (H
2
ꢂH
2
O and symmetrical
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2
O
2
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1
0
2
L ), N,N bis(salicyl-
[
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[
tively. The new complexes were fully characterized by
physicochemical and spectroscopic methods. Characterizations of
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complexes with 1:1 (metal:ligand) stoichiometry. The study of
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