SODs, DNA binding and cleavage studies of new Mn(III) complexes with 2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol

Article abstract of DOI:10.1016/j.saa.2013.01.025

Newly synthesized ligand [2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol] (Bpmp) react with manganese(II) to form mononuclear complexes [Mn(phen)(Bpmp)(CH₃COO)(H₂O)]·4H₂O (1), (phen = 1,10-phenanthroline) and [Mn(Bpmp)

Full text of DOI:10.1016/j.saa.2013.01.025

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 203–212
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
SODs, DNA binding and cleavage studies of new Mn(III) complexes
with 2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol
⇑
L. Shivakumar, K. Shivaprasad, Hosakere D. Revanasiddappa
Department of Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
"
Synthesis of new Schiff base and its
Manganese(III) complexes.
Spectral and thermal
"
characterization of the synthesized
compounds.
"
"
SODs activity.
Antimicrobial activity of the
synthesized complexes.
"
DNA binding properties and
oxidative induced supercoiled DNA
cleavages of the complexes.
a r t i c l e i n f o
a b s t r a c t
Article history:
Newly synthesized ligand [2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol] (Bpmp) react with man-
Received 16 October 2012
Received in revised form 8 January 2013
Accepted 10 January 2013
Available online 31 January 2013
ganese(II) to form mononuclear complexes [Mn(phen)(Bpmp)(CH
3
COO)(H
2
O)]ꢀ4H
2
O (1), (phen = 1,
1
0-phenanthroline) and [Mn(Bpmp) (CH COO)(H O (2). These complexes were characterized
2
3
2
O)]ꢀ5H
2
1
by elemental analysis, IR, H NMR, Mass, UV–vis spectral studies. Molar conductance and thermogravi-
metric analysis of these complexes were also recorded. The in vitro SOD mimic activity of Mn(III) com-
plexes were carried out and obtained with good result. The DNA-binding properties of the complexes
1 and 2 were investigated by UV-spectroscopy, fluorescence spectroscopy and viscosity measurements.
The spectral results suggest that the complexes 1 and 2 can bind to Calf thymus DNA by intercalation
mode. The cleavage properties of these complexes with super coiled pUC19 have been studied using
the gel electrophoresis method, wherein both complexes 1 and 2 displayed chemical nuclease activity
This work is dedicated to Dr. P.G. Ramappa,
Former Professor of Inorganic Chemistry,
University of Mysore, on his 75th birth
anniversary.
Keywords:
2 2
in the absence and presence of H O via an oxidative mechanism. All the complexes inhibit the growth
of both Gram positive and Gram negative bacteria to competent level. The MIC was determined by micro-
titer method.
Mn(III) complexes
SOD mimic activity
DNA binding
Oxidative DNA cleavage
Antimicrobial activity
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
been investigated as an area of considerable interest in inorganic
biochemistry [2]. Schiff bases derived from salicylaldehyde and
Schiff bases belong to a class of organic compounds which have
potential as anticancer drugs, indeed the activity of these com-
pounds increase when they complex with metal ions [1]. Coordina-
tion chemistry of manganese in various oxidation states has long
various amines have been extensively used to synthesize many
complexes of manganese(III) [3–5]. Mn-Salen complexes have been
exploited as a catalyst for reactions [6,7], models for catalytic scav-
engers (hydrogen peroxide and superoxide) [8], protective activity
in various experimental disease models like EUK-8 (in vivo) [9,10].
Similar to Fe(III), the high spin Mn(III) is a trivalent cation and is
classified as a hard Lewis acid by virtue of its high charge density.
⇑
Corresponding author. Tel.: +91 821 2419669; fax: +91 821 2419363.
E-mail address: hdrevanasiddappa@yahoo.com (H.D. Revanasiddappa).
1
386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.01.025

2
04
L. Shivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 203–212
In contrast to Mn(II), Mn(III) forms stable complexes with hard do-
nor atoms, such as oxygen donors [11].
buffer solution was prepared using deionized, sonicated triply-
distilled water.
The investigation of biological properties of Schiff base com-
plexes of Mn(III) have received less attention in comparison to
the corresponding Mn(II) complexes. A large number of Schiff base
complexes of manganese(III) find important roles in metalloen-
zymes, redox and non-redox proteins [12]. A few reports on the
antimicrobial activity of Mn(III) complexes have been found in
the literature [13,14]. The DNA interaction of a number of manga-
nese complexes with different functionalized Salen derivatives has
been investigated in literature [15–17]. These studies have indi-
cated that while some complexes bind DNA avidly, others induce
Physical measurement
Elemental analysis was carried out on an Elemental Vario EL
1
elemental analyzer. H NMR spectra were recorded on a Bruker
3
AV-400 MHz FT NMR spectrometer in CDCl . The electro-spray
ion mass spectra (ESIMS) were recorded on API 2000 Applied
Bio-system triple quadrupole mass spectrometer. The solution
was introduced into the ESI source through a syringe pump at
the rate 5
l
L/min. The molar conductance data were recorded in
effective DNA scission via a redox process.
ꢁ3
1
0
M DMF solution at room temperature using an Elico CM-
ꢁ
Reactive oxygen species (ROS) like the superoxide radical (O ),
2
180 conductometer. The cell constant of the conductivity cell used
formed following a one-electron reduction of molecular oxygen.
ꢁ1
was 0.5 cm . Infrared spectra were recorded in the range 4000–
Superoxide dismutases (SODs) are metalloenzymes that catalyze
ꢁ1
4
00 cm on a Jasco FT/IR-4100 Series FT-IR spectrometer by Nujol
ꢁ
the conversion of superoxide radical (O ) to oxygen (O
2
) and
2
mull method. Electronic spectra were obtained with a Hitachi
U-2000, recording spectrophotometer in DMF solution at concen-
2 2
hydrogen peroxide (H O ) at rates approaching the diffusion con-
trolled limit [18]. Therefore, they play a crucial role in protecting
biological systems against the damage mediated by this deleteri-
ous radical. SODs, is in fact one of the first line ROS-defense en-
zymes in cells whose role is becoming increasingly important as
more pathological and disease states are discovered to be linked
with oxidative damage [19]. Many studies on the reactivity of
the native SODs enzymes and low molecular weight manga-
nese(II,III) based SOD mimetic (SODm) towards superoxide have
been reported [20]. Manganese has been the preferred metal for
the development of SODm complexes, due to its low toxicity com-
pared to the other metals [21]. Additionally, it has been shown that
high intracellular manganese concentrations can provide protec-
tion against oxidative stress [22].
Although some important classes of SODm have proven to be
effective in clinical trials in manipulating a variety of disease states
related to reactive oxygen and nitrogen species, the key mecha-
nisms behind their bioactivity are still unclear [23]. It has been
documented in the literature that native SODs enzyme also inter-
act with DNA and exhibit nuclease activity [24,25]. This prompted
us to study the DNA interaction as well as nuclease activity of man-
ganese complexes having SODs activity.
ꢁ3
tration of 10 M. Magnetic measurement were carried out by
the Gouy method at room temperature (28 ± 2 °C) using
Hg[Co(SCN) ] as the calibrant. Thermogravimetric and Differential
4
thermal analysis were performed by Universal V4.3A TA Instru-
ments. Fluorescence spectra were recorded on a Shimadzu RF-
5
301pc spectrofluorimeter. The metal contents in the complexes
were determined by literature method [27].
Synthesis of the ligand (Bpmp)
A solution of salicylaldehyde (0.245 g, 2 mmol) in methanol
(
20 mL) was added to a methanolic solution (30 mL) of 3-(benzyl-
oxy)pyridin-2-amine (0.400 g, 2 mmol). The reaction mixture was
heated under reflux for 8 h. The solution was cooled and left to
stand for the evaporation, after few days reddish brown colored
crystals of Bpmp were obtained in a good yield (Yield = 0.522 g,
8
1%). m.p: 90 °C; Anal. Calcd. for C19
16 2 2
H N O : C, 74.98; H, 5.30;
N, 9.20; O, 10.51; Found: C, 74.92; H, 5.28; N, 9.12; O, 10.46%;
ꢁ1
FT-IR (Nujol) (
m/z: 305 [M + 1] ; H NMR (CDCl
AO), 10.54 (s, PhAOH), 9.18 (s, HC@N), 7.26–6.56 (m, ArAH).
mmax/cm ): 1670 (HC@N), 3613 (PhAOH); ESI-MS,
+
1
3
, 300 MHz, d ppm): 5.14 (ACH2-
Therefore, in this paper the authors have tried to present man-
ganese complexes represents manganese-SODs for a treatment of
the oxidative stress like disease, using indirect spectrophotometric
detection of superoxide decomposition method in aqueous media.
The structure of newly synthesized Schiff base and its manga-
nese(III) complexes were accomplished by spectral characteriza-
tion viz., IR, UV–vis, mass, and elemental analysis and
thermogravimetric analysis. Also DNA interactions of the two com-
plexes with calf-thymus DNA (CT-DNA) were investigated using
UV–vis absorption titration, fluorescence spectra and viscosity
measurements. In addition, their oxidative-cleavage reactions with
pUC19 supercoiled plasmid DNA were elucidated by gel electro-
phoresis. The complexes were also tested for their in vitro antimi-
crobial activity against some Gram positive and Gram negative
strains using the paper disc diffusion method. Microtiter method
was used to determine MIC values.
Synthesis of the complex (1) [Mn(phen)(Bpmp)(CH
3
COO)(H
2
O)]ꢀ4H
2
O
A methanolic solution (10 mL) containing 1.0 mmol (0.245 g)
quantity of Mn(CH COO) ꢀ4H was reacted with 1.0 mmol
3
2
2
O
(
0.198 g) of the heterocyclic base (phen) in 10 mL MeOH under
stirring at 40 °C for 0.5 h. The resulting solution was then reacted
with 1.0 mmol (0.305 g) of Bpmp taken in 10 mL hot MeOH. The
mixture was refluxed with stirring for 10 h and cooled. After slow
evaporation at room temperature, a light yellowish brown micro-
crystalline product was deposited, collected with filtration, washed
with methanol and dried.
Complex 1: Yield: (73%). m.p; >300 °C. Anal. Calc. for [C33
MnN ] (%); C, 57.64; H, 5.28; N, 8.15; O, 20.94; Found: C,
7.04; H, 5.20; N, 8.10; O, 20.83; FT-IR (Nujol) (
H
36-
4 9
O
ꢁ
1
5
m
max/cm ): 3395,
ꢁ
ꢁ
(
H
2
O); 1282,
m(COO )sy; 1580 m(COO )asy; 561, 543 (MAN); 442
+
ꢁ1
2
(
MAO). ESI-MS, m/z: 688 [M + 1] . Conductance (
K
, ohm cm -
ꢁ1
mol ), 19.2;
leff (BM), 4.73.
Experimental
Chemicals
Synthesis of the complex (2) [Mn(Bpmp)
2
(CH
3 2 2
COO)(H O)]ꢀ5H O
All reagents were procured from Merck and Sigma Aldrich Co.
and used without further purification unless specially noted. The
solvents used for spectroscopic studies were purified by standard
procedures [26]. Calf Thymus (CT) and Supercoiled (SC) pUC19
DNA were purchased from Genei, Bangalore (India). Tris–HCl
The complex was prepared by stirring a MeOH solution (25 mL)
of Bpmp (2 mmol, 0.61 g) and adding successively a MeOH solution
(25 mL) of Mn(CH
3
COO)
2
2
ꢀ4H O (1 mmol, 0.245 g) under reflux. The
initial yellow color of the solution rapidly changed to brown. After
24 h of stirring at room temperature slow evaporation of solvent

L. Shivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 203–212
205
lead to deposition brown compounds. The products were collected
by filtration and washed with ethanol–diethyl ether mixture (1:1).
complex concentrations. DNA was pretreated with EB in the ratio
[DNA/EB] = 1 for 30 min at 27 °C. The metal complexes with the
Complex 2: Yield: (64%). m.p >300 °C. Anal. Calc. for [C40
MnN 12] (%); C, 57.97; H, 5.47; N, 6.76; O, 23.17; Found: C,
6.88; H, 5.51; N, 6.28; O, 22.93; FT-IR (Nujol) (
H
45-
increasing concentration (0–50
lM) were then added to the
4
O
DNA-EB (50 M) mixture and their effect on the emission intensity
l
ꢁ1
5
m
max/cm ): 3412,
was measured.
ꢁ
ꢁ
(
(
H
2
O); 1285,
m(COO )sy; 1585 m(COO )asy; 554, 541 (MAN); 436
+
ꢁ1
2
MAO). ESI-MS, m/z: 829 [M + 1] . Conductance (
K
, ohm cm -
Viscosity measurements
ꢁ1
mol ), 15.9;
leff (BM), 4.95.
Viscosity measurements were carried out with an Ubbelodhe
viscometer maintained at a constant temperature of 28.0 ± 0.1 °C
in a thermostated bath. DNA samples of ca. 200-bp average length
were prepared by sonication in order to minimize complexities
arising from DNA flexibility [32]. The concentration of DNA was
Superoxide dismutase activity
SOD activity of the complexes 1 and 2 were evaluated according
to the reported method [28]. This in vitro method is based on the
ability of tested compounds (1 mg/mL, 4% DMSO solution) to inhi-
2
00
to 200
EB and each sample were tested three times to get an average cal-
l
M, the EB and complex concentrations were varied from 0
lM and the flow times were measured with a digital timer.
bit the reduction of nitro blue tetrazolium (NBT) (0.3 mL, 300
by superoxide radical O2 generated by the phenazine metho sul-
l
M)
ꢁ
1/3
culated flow time. Data were presented as (
ratio [33], where is the viscosity of DNA in the presence of com-
plex, being the viscosity of free DNA solution alone. The viscos-
g/g0
)
versus binding
fate (PMT) (0.1 mL, 186 lM) with NADH (0.2 mL, 780 lM) in phos-
g
phate buffer pH 7.8, of total volume 3 mL. The extent of NBT
reduction was followed spectrophotometrically by measuring the
absorbance at 560 nm. Each experiment was performed in dupli-
cate and the SOD activity has been defined as the concentration
of the tested compound for the 50% inhibition of the NBT reduction
g
0
ity values were calculated from the observed flow time of CT-DNA
containing solutions (t) duly corrected for that of the buffer alone
(t ),
0
g
0 0
= (t ꢁ t )/t .
(
IC50 value) by superoxide produced.
Oxidative DNA cleavage efficiency
The DNA cleavage activity of the metal complexes was studied
by agarose gel electrophoresis. Supercoiled plasmid pUC19 DNA
DNA binding and cleavage activity
(
30
taining 50 mM NaCl and added to the different concentration of
complexes (100, 200, 300 and 400 M) prepared in less than 4%
DMSO:H O. The mixtures were incubated at 37 °C for 24 h and
then mixed with the loading buffer (2 L) containing 25% bromo-
phenol blue, 0.25% xylene cyanol and 30% glycerol. Each sample
L) was loaded into 0.8% w/v agarose gel. Electrophoresis was
lM) was dissolved in a 50 mM (Tris–HCl) buffer (pH 7.2) con-
Electronic absorption titration
All spectroscopic titrations were carried out in 5 mmol L
Tris–HCl buffer (pH 7.1) containing 50 mmol L
temperature. A solution of CTꢁDNA in the buffer gave a ratio of
UV absorbance at 260 and at 280 nm of 1.84:1, indicating that
the DNA was sufficiently free of protein [29]. The concentration
of DNA was estimated from its absorption intensity at 260 nm with
a known molar absorption coefficient value of 6600 M cm [30].
Stock solutions were stored at 4 °C and used within 4 days. Before
adding CT-DNA to complexes 1 and 2, complexes were dissolved in
ꢁ
1
ꢁ1
l
NaCl at room
2
l
(5 l
ꢁ
1
ꢁ1
undertaken for 1 h at 50 V in Tris–acetate-EDTA (TAE) buffer. The
gel was stained with EB for 5 min after electrophoresis, and then
photographed under UV light. The proportion of DNA in each frac-
tion was quantitatively estimated from the intensity of each band
with the Alpha Innotech Gel documentation system (AlphaImager
less than 4% DMSO: H
a fixed complex concentration (50
tration of the CT DNA from 0 to 300
2
O. Titration experiments were performed by
M) while varying the concen-
M. While measuring the
l
2
200). To enhance the DNA cleaving ability by the complexes,
hydrogen peroxide (100 M) was added into each complex
400 M). Moreover, the cleavage mechanism was further investi-
gated by using scavengers for the hydroxyl radical species (4 L,
DMSO) and the singlet oxygen species (100 M, NaN ). All experi-
l
l
absorption spectra, equal quantity of CT DNA was added to both
the complex solution and the reference solution to eliminate the
absorbance of CT DNA itself. The complex-DNA solutions were al-
lowed to equilibrate for 10 min before spectra were recorded. The
(
l
l
l
3
ments were carried out in triplicate, under the same conditions.
Assays in the presence of the minor groove binder distamycin
(
0
intrinsic binding constant (K
with CT DNA was determined from a plot of [DNA]/(
DNA] using absorption spectral titration data [31] and the follow-
ing equation,
b
) for the interaction of the complexes
e
a
ꢁ
e
f
) versus
8
lM) and the major groove binder methyl green (2.5
lL of a
[
.01 mg/mL solution) were also performed.
½
DNAꢂ=ð
e
a
ꢁ
e
f
Þ ¼ ½DNAꢂ=ð
e
b
ꢁ
e
f
Þ þ 1=K
b
ðe
b
ꢁ
e
f
Þ
Antimicrobial activities
where [DNA] is the concentration of DNA, the apparent absorption
coefficients and correspond to Aobsd/[complex], the extinc-
tion coefficient for the free metal complex and the extinction coef-
ficient for the metal complex in the fully bound form, respectively.
b
The K value is given by the ratio of the slope to the intercept.
e
a
,
e
f
e
b
The complexes and ligand were tested for their in vitro antibac-
terial activity against Escherichiacoli, Staphylococcusaureus, Xantho-
monas vesicatoria and Ralstonia solanacearum strains. Initial
screening of compounds was performed by following disc diffusion
method. Suspensions in sterile distilled water from 24 h cultures of
microorganisms were adjusted to 0.5 McFarland. Muller–Hinton
Petri dishes of 90 mm were inoculated using these suspensions. Pa-
Fluorescence spectral study
The relative binding of the ternary complexes to CT DNA was
studied by fluorescence spectral method using EB (Ethidium Bro-
mide) bound CT DNA solution in Tris–HCl/NaCl buffer (pH 7.2).
Emission intensity measurements were carried out on a Shimadzu
RF-5301pc spectrofluorometer with a 1 cm quartz cell. Tris buffer
was used as the blank to make preliminary adjustments. For all
fluorescence measurements, the entrance and exit slits were main-
tained at 10 nm each. All the experiments were measured after
per disks (5 mm in diameter) containing 100 lL of the substance to
be tested (at a concentration of 2 mg/mL in DMF) were placed at
the edge of the petri plates containing nutrient agar (NA). One
5 mm diameter agar disc of test organism from a 1 week old NA
culture was placed in the center of the plate. Incubation of the
plates was done at 37 °C for 24 h. Reading of the results was done
by measuring the diameters of the inhibition zones generated by
the tested substances, using a ruler. Chloramphenicol was used
as a reference substance.
5
min at a constant room temperature, 302 K. Fluorescence inten-
sities at 545 nm (376 nm excitation) were measured at different

2
06
L. Shivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 203–212
The in vitro anti-fungal assay was performed by the disc diffu-
NMR spectra
1H NMR spectral data of the ligand in CDCl
posed structure of the ligand (Fig. 2). The sharp signal at d
5.14 ppm in the spectra of Bpmp is assigned to the (ACH AO)
sion method. The complexes and ligand were tested against the
fungi Aspergillus niger and Aspergillus flavus, cultured on potato
dextrose agar as medium. In a typical procedure, a well was made
at the center on the agar medium inoculated with the fungi. The
3
confirms the pro-
2
well was filled with the test solutions (100
l
L) using a micropi-
methylene group, allied with the benzene ring. The free ligand
pette and the plate was incubated at 37 °C for 72 h. During this
period, the test solution diffused and the growth of the inoculated
fungi was affected. The inhibition zone developed on the plate was
measured. Fluconazole was used as a reference compound.
The prepared compounds were further used to determine
minimum inhibitory concentration (MIC) in 96 well, sterile, flat
bottom microtiter plates, based on broth microdilution assay,
which is an automated colorimetric method, uses the absorbance
spectrum shows a sharp signal at d 9.18 ppm is assigned to the azo-
methine (ACH@NA) moiety [36]. The H NMR spectrum of ligand
shows a broad singlet at d 10.54 ppm due to phenolic AOH proton
[37]. The spectrum also shows the multiple bands in the region d
7.26–6.56 ppm, assigned for the aromatic ring protons.
1
IR spectra
(
optical density) of cultures in a microtiter plates [34]. Each well
of microtiter plates was filled with 200 L of nutrient broth/po-
tato dextrose broth, 1 L of test organism and 15 L of different
The IR spectra of Bpmp and its complexes were presented in
l
Figs. S1–S3. The IR spectral data of the Bpmp show a medium
l
l
ꢁ1
intensity absorption band at 3613 cm
which is attributed to
concentration of selected compounds. For bacteria and fungi the
microtiter plates were incubated at 35 ± 2 °C for 24 h. After the
incubation period, the plates were read at 610 nm using ELISA
reader (ELX 800 MS, Biotek instruments, Inc. USA). MIC, which
was determined as the lowest concentration of compound inhib-
iting the growth of the organism, was determined based on the
optical density.
the phenolic
m
(OH) stretching vibration. The phenolic CAO stretch-
ꢁ1
ing can also be observed at 1286 cm range. These bands are dis-
appeared in complexes indicating that the ligand is coordinated to
metal through deprotonated hydroxyl group [38]. The high inten-
sity band at 1670 cm
ꢁ1
appeared which is attributed to the
m(CH@N) vibration of the ligand, and it disappeared upon coordina-
tion with metal ion. The presence of medium intensity bands in
ꢁ1
2
600–2700 cm region indicates the existence of CAH group of
ꢁ1
the ligand. The bands in the range 3370–3420 cm
and
Results and discussion
ꢁ1
1
620 cm are attributable to OAH stretching and bending of coor-
dinated water ligands or water of crystallization [39,40]. Ligand
Chemistry
coordination to the manganese center in 1 and 2 is confirmed by
ꢁ
1
two bands appearing about 576–543 and 468–436 cm , attribut-
able to (MnAN) and (MnAO) [41], respectively. The IR spectra of
the acetate complexes 1 and 2 show an absorption band in the re-
The synthesis of Bpmp involves the addition of a solution of sal-
icylaldehyde in methanol to a stirred methanolic solution of 3-ben-
zyloxypyridin-2-ylamine in a 1:1 ratio. The level of the purity of
Bpmp has been checked by running TLC on a silica gel coated plate
m
m
ꢁ1
ꢁ
gion 1574–1590 cm which is assigned to
m(COO ) asymmetric
stretching of acetate ion and another in the region 1274–
using EtOAc–EtOH (6:4, v/v) as the mobile phase. The structure of
ꢁ
1
ꢁ
ligand is characterized by IR, 1H NMR, mass and UV spectra. The
1295 cm
and which can be assigned to m(COO ) symmetric
stretching vibration of acetate ion. A difference between (
m
as
ꢁ
s
m )
reaction of the ligands Bpmp/o-phen and Mn(CH
3
COO)
2
2
ꢀ4H O in
ꢁ1
ꢁ1
is around 300–295 cm which is greater than 144 cm indicates
the unidentate coordination of the acetate ion with the central me-
tal ion [42].
different ratio produced the mononuclear complexes 1 and 2.
Bpmp and its complexes 1 and 2 are insoluble in water, dichloro-
methane, ethyl ether and soluble in common polar organic sol-
vents. The structures of complexes were characterized by
elemental analysis, molar conductivities, IR, UV spectra, mass and
thermogravimetric analysis. The elemental analysis data of the
Bpmp and its metal complexes agree well with the proposed struc-
ture (Fig. 1). The synthesis, spectroscopic and analytical data of
prepared manganese complexes are presented in the Experimental
Section. The molar conductivities of complexes 1 and 2 in DMF are
Electronic spectra and magnetic measurements
The UV–vis spectra of the ligand and its manganese(III) com-
plexes in DMF were recorded in the region of 800–200 nm. All
the complexes show similar electronic spectra to those previously
reported for related other manganese(III) complexes [43]. The
ꢁ1
2
ꢁ1
4
found to be 19.2 and 15.9 ohm cm mol , respectively, which
indicate that these complexes are neutral and non-electrolytes
spectrum of all the d manganese(III) complexes in the present
study are similar and consist of three regions of absorption. The
[
35]. The relative low molar conductivities show that these com-
electronic spectrum of Bpmp shows two absorption bands
ꢃ
plexes are stable in solution. Both the ligand and its complexes
are very stable at room temperature in the solid state.
257 nm and 284 nm corresponding to
p
?
p
transition within
the structure. The absorption bands of the complex 1 and 2 are
OOCCH3
OOCCH3
2
2
2
2
(
1)
(2)
Fig. 1. Proposed structure of the complexes 1 and 2.

L. Shivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 203–212
207
Fig. 2. 1H NMR spectrum of the of the Bpmp.
shifted to longer wavelength region 278–283 nm and 298–304 nm
of starting amine compound. Other fragments are in good agree-
ment with the ligand structure. The FAB mass peak corresponds
to the Schiff base complex (1) was appeared at 688 m/z (M + 1) .
compared to the ligand. The bands appearing later at the low en-
ꢃ
+
ergy side are attributable to n ?
p
transitions associated with
the azomethine chromophores. The band obtained at ca. 412–
Mass spectrum of the complex 1 show molecular ion peak at
556 m/z, for the ligands attached to metal center with one water
molecule after elimination of acetate moiety [48]. Similarly, for
complex 2 the peak at 680 m/z appeared corresponds to the ligand
attached to metal center with one water molecule. The peak ap-
peared at 688 and 829 m/z for the complexes 1 and 2 represents
ꢃ
4
26 nm region may be attributed to phenolate O(p
p
) ? Mn(d
p
) li-
gand-to-metal charge transfer by analogy with some similar man-
ganese(III) complexes [44]. Two bands with shoulders have been
found around 520–626 nm suggesting an octahedral geometry,
6
4
4
6
4
4
with the
A
1g ? T1g ( G),
A1g ? T2g ( G) transition for com-
plexes. These bands are reasonably assignable to d–d transition
on the basis of their low extinction coefficients [45].
the structural formula {[Mn(phen)(Bpmp)(CH
and {[Mn(Bpmp) (CH COO)(H O}, respectively. These
O)]ꢀ5H
peaks match the weakly present lattice water molecules with me-
tal ion, which is not unlikely. To confirm the presence of lattice H
molecules in the complexes, we carried out thermogravimetric
analyses of complexes. However the coordinated ion acetate to
metal was also supported by IR spectra. Other fragments are in
good agreement with the complexes structure.
3
COO)(H
2
O)]ꢀ4H
2
O}
2
3
2
2
2
O
Magnetic measurements
The magnetic susceptibility data for manganese(III) complexes
1
and 2 found to be 4.73 and 4.95 BM, respectively, indicating high
4
3
1
spin magnetically dilute d configurations (t2 , e ). This value is ex-
g
g
pected for four unpaired electrons and lack of any kind of exchange
interaction [46]. Mn(II) has undergone aerial oxidation to Mn(III) in
both the complexes. This is probably due to the formation of more
stable complexes by harder Mn(III) ion with ligands containing
harder donor atoms [47]. The elemental analysis, the molar con-
ductivity, the electronic spectra and the magnetic moments sug-
gest an octahedral geometry for complexes 1 and 2, with a
coordinated water molecule and acetate ion in the axial positions.
Thermal studies
Room temperature stability for the complexes 1 and 2 was re-
vealed by its thermogravimetric study (Figs. S7 and S8) carried
out from ambient temperature to 800 °C in nitrogen atmosphere,
at a heating rate of 10 °C/min. The TGA indicated that complexes
1
and 2 lose ꢄ20% and ꢄ19% of the total weight in the 35–209 °C
temperature range, respectively. This weight loss corresponds to
the release of lattice, as well as coordinated water molecules and
one coordinated acetate ion (calcd., 21.68% and 20.16%, for 1 and
2, respectively) [49]. When the temperature hold on rising, the
complex 1 loses ꢄ70% of the total weight in the 220–800 °C tem-
perature range, corresponding to the removal of phenanthroline
moiety followed by Bpmp (calcd. 70.20%) and finally the formation
of the residue. In complex 2, the second stage of weight loss (calcd.,
73.1%; found, ꢄ72%) occurs between 215–600 °C, corresponding to
Mass spectra
Mass spectra of all the compounds stand in good agreement
with proposed structure, the representative mass spectra shown
in Figs. S4–S6. A moderate peak at m/z 305 is due to the formation
of Schiff base (Bpmp + 1) . A distinct but less abundant peak ob-
served at (m/z = 275) due to elimination of CO from the parent
compound. One intense peak at m/z 201 is due to the formation
+

2
08
L. Shivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 203–212
the removal of two Bpmp thereby leading to the formation of metal
oxides.
Superoxide dismutase activity
Different methods (nonenzymatic and enzymatic methods) can
be used to ascertain the superoxide radical scavenging activity of
various compounds. The SOD-like activity for the reaction of differ-
ent mimics was determined studying the effect of the complexes
on superoxide generated by the NADH/PMS system as described
in experimental section. This method is very sensitive for evalua-
tion of superoxide radical scavenging activity and was used to cal-
culate the IC50 of the active compounds (Table 1). As the reaction
proceeds, the farmazan color is developed and a color change from
yellow to blue, which was associated with an increase in the absor-
bance at 560 nm.
Fig. 3. Comparison of SODs activity of complexes 1 and 2 with standard SOD
mimetic in IC50 value.
5
(a) 0.45
4
Complexes 1 and 2 show good SODs activity (Fig. 3) with re-
3
0
0
0
.40
.35
.30
spect to the standard complexes [50]. Under the reaction condi-
2
1
0
ꢁ
tions used, the
O
radicals activity was almost completely
M (IC50 = 4.2 M). Com-
plex 2 inhibited at the concentration of 7.5 M (IC50 = 3.4 M).
2
inhibited by 1 at a concentration of 9.6
l
l
l
l
0.5
1
1.5
2
-
2.5
M
3
The SODs activity of the present complexes 1 and 2 may be consis-
tent with the replacement of only one inner-sphere water mole-
cule and phosphate binding in a monodentate manner. From this
observation one can presume that the SODs activity of 1 and 2
are not so strongly affected by phosphate, because there is still
one labile acetate ion present in its sixth coordinate environment,
which can be easily replaced by superoxide [51]. It cannot be ruled
out that, the reason for the observed SODs activity of 1 and 2 is
may be the presence of Mn(III)salen entity itself, not just the re-
sults of free manganese species potentially present in the solution
4
[
DNA] x 10
0.25
0
0
.20
.15
2
00
300
400
500
nm
600
700
b) 0.7
(
5
4
3
2
1
0
[
20]. The IC50 value exhibited by SODs 1 and 2 is lower than those
0
0
0
.6
.5
.4
reported for the standard Mn(III)–SODm, nevertheless, further sys-
tematic studies like kinetic effect of complexes on catalytic cycle of
superoxide dismutation are in progress.
0
.5
1
1.5
2
2.5
M
3
DNA binding study
-
4
[
DNA] x 10
Electronic absorption titration
0.3
Electronic absorption spectroscopy is widely employed to
determine the propensity for binding of the complexes to calf thy-
mus DNA (CT-DNA). Hypochromism happens when the DNA-bind-
0
.2
200
300
400
nm
500
600
700
ing mode of
a complex has an electrostatic effect or an
intercalation which stabilizes the DNA duplex [52]. The absorption
spectra of the complexes 1 and 2 in the absence and presence of
CT-DNA are shown in Fig. 4a and b. With increasing CT-DNA con-
centration, for complex 1, the hypochromism in the band at
Fig. 4. Variation in the absorption spectra of complexes 1 (a) and 2 (b) in Tris–HCl
buffer upon addition of increasing concentration of CT-DNA [0–300 M].
Complex] = 50 M.
l
[
l
3
4
2
04 nm reaches as high as 34.7% with a small bathochromism of
nm. Complex 2 also shows an evident hypochromism of about
9.2% at 298 nm and bathochromism of about 2 nm. These spectro-
ꢃ
base pairs, thus, decreasing the
p ? p transition energy and
resulting in the bathochromism. To compare quantitatively the
affinity of the two complexes toward DNA, the intrinsic binding
scopic characteristics suggest that these two complexes interact
with DNA most likely through a mode that involves a stacking
interaction between the aromatic chromophore and the base pairs
of DNA [53]. If the binding mode is intercalation, the
the intercalated ligand can couple with the
constants K
by monitoring the changes of absorbance, with increasing concen-
tration of DNA. The intrinsic binding constant K of complexes 1
b
of the two complexes to CT-DNA were determined
ꢃ
p
orbital of
p
orbital of the DNA
b
4
ꢁ1
4
ꢁ1
and 2 obtained were 6.06 ꢅ 10 M and 1.09 ꢅ 10 M , respec-
tively, which is lower than that reported for classical intercalator
(for ethidium bromide and [Ru(phen)DPPZ] whose binding con-
stants have been found to be in the order of 10 –10 M) [54,55]
and comparable with those reported for manganese complexes
Table 1
SODs activity of complexes 1 and 2 in IC50
(
lM).
6
7
Compounds
Suppression ratio, IC50
(l
M)
Reference
4
ꢁ1
[
{[MnCl(bpma)]
2
[Mn(
l-Cl)
(H
4 2
O)
2
]}ꢀCH
3
CN (1.37 ꢅ 10 M )] [56]
EUK-189
MnL7
Complex 1
Complex 2
1.4
2.1
4.2
3.4
[45]
[45]
This work
This work
This work
4
ꢁ1
and [[MnL(pic)
2
]ꢀH
2
O (4.35 ꢅ 10 M )] [57]. Comparing the intrin-
sic binding constant of the two complexes, we can deduce that
both complexes bind to DNA by partial intercalation. However,
the binding strength of complex 1 is greater than that of complex
2. The additional planar aromatic moiety (phen) in the manga-
a
Mn(CH
3
COO)
2
ꢀ4H
2
O
No activity
a
Checked up to 200
lM.

L. Shivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 203–212
209
nese(III) complex 1, may facilitate its potential intercalative DNA
binding.
quenching efficiency of complexes 1 and 2 may be due to the inter-
action of former with DNA through additional planar aromatic
phen moiety, so releasing some free EB from the EB–DNA complex.
Competitive studies with EB
In general, measurement of the ability of a complex to affect the
EB fluorescence intensity in the EB-DNA adducts allows determina-
tion of the affinity of the complex for DNA, whatever the binding
mode may be. If a complex can replace EB from DNA-bound EB,
the fluorescence of the solution will be quenched due to the fact
that free EB molecules are readily quenched by the surrounding
water molecules [58]. Two mechanisms have been proposed to ac-
count for this reduction in the emission intensity: the replacement
of molecular fluorophores (EB in this case) and/or electron transfer
Viscosity measurements
Furthermore, the interactions between the complexes and DNA
were investigated by viscosity measurements. In the absence of X-
ray structural data, viscosity measurement is regarded as the most
effective means to study intercalative binding mode of DNA in
solution [60]. A classical intercalative mode causes significant in-
crease in viscosity of the DNA solution due to an increase in sepa-
ration of base pairs at the intercalation sites and hence an increase
in overall DNA length. In contrast, a partial, non-classical intercala-
tion of ligand could bend (or kink) the DNA helix, reduce its effec-
tive length and, concomitantly, its viscosity [61]. Fig. 6 shows the
viscometric titration results of complexes 1 and 2 towards CT
DNA. The viscosity of CT DNA increases with increase in the ratio
of 1 and 2 to CT DNA, also this result resembles the binding mode
of EB to CT DNA. These results also suggested that both of the com-
pounds could bind to DNA in an intercalative mode. Notably, 1
caused a greater extent of viscosity changes, indicating the stron-
ger binding ability than that of 2. This result was also consistent
with those obtained in fluorescent and electronic absorption titra-
tion measurements.
[
59]. For all the synthesized compounds, no emission was observed
either alone or in the presence of CT-DNA in the buffer. A compet-
itive binding of the Mn(III) complexes to CT DNA resulted in the
reduction of the emission intensity due to displacement of bound
EB. The quenching of the emission spectra of EB bound to DNA
by the complexes 1 and 2 are shown in Fig. 5a and b. According
0 0
to the classical Stern–Volmer equation [29], I /I = 1 + KSV [Q]; I
and I are the fluorescence intensities in absence and in presence
of the quencher, respectively. KSV is a linear Stern–Volmer quench-
ing constant, [Q] is concentration of the quencher. The quenching
of EB bound to CT-DNA by both complexes is in good agreement
with the linear Stern–Volmer equation, which provides further evi-
dence that the complexes bind to DNA. The Ksv values for com-
DNA cleavage efficiency studies
4
ꢁ1
3
ꢁ1
plexes
1
and
2
are 1.02 ꢅ 10 M
and 6.6 ꢅ 10 M
,
It is known that DNA cleavage activity is proceeds by relaxation
of supercoiled circular conformation of plasmid DNA to nicked cir-
cular and/or linear conformation. When electrophoresis is applied
to circular plasmid DNA, fastest migration will be observed for
DNA of closed circular conformations (Form I). If one strand is
cleaved, the supercoiled will relax to produce a slower moving
nicked conformation (Form II). If both strands are cleaved, a linear
conformation (Form III) will be generated and migrates in between
respectively. The data suggest that the interaction of complex 1
with CT-DNA is stronger than that of complex 2, which is consis-
tent with the above absorption spectral results. The difference in
Complex 1
(
(
a) 80
4
3
2
1
0
[
62]. Incubating plasmid pUC19 DNA with the increasing amounts
of the complex concentration of 100, 200, 300 and 400 M, in pres-
ence of oxidizing agent H under physiological conditions pro-
6
4
2
0
0
0
0
l
2 2
O
1
2
3
4
5
duced the outcome shown in Fig. 7. The results show that both
complexes 1 and 2 behave as a chemical nucleases by nicking
the DNA Form I into Form II, at the concentration of 100 lM. The
increasing concentrations of complexes led to a gradual diminish
in the band intensity of Form I, while increasing Form II progres-
sively suggesting single strand DNA cleavage. In contrast, the start-
ing material Mn(II) acetate salt does not seemed to cleave the DNA
at 400 lM (data not showed). Poor cleavage of DNA was observed
on incubation of complexes 1 and 2 in the absence of hydrogen
-5
[
M] x 10
5
20
540
nm
560
580
Complex 2
b)100
5
4
3
2
1
8
6
4
2
0
0
0
0
0
0
1
2
3
4
5
-
5
[
M] x 10
5
00
520
540
560
580
nm
Fig. 5. Emission spectra of EB bound to DNA in the presence of increasing (a)
complex 1 and (b) complex 2 concentration ([EB] = 10 M, [DNA] = 10 M, [com-
plex] = 0–50 M, kex = 376 nm; kem = 545 nm for complexes 1 and 2 respectively);
Inset: Stern Volmer plot of I /I vs. [complex] for the titration of the complexes to
0
DNA-bound EB system.
l
l
l
Fig. 6. Effect of increasing the concentration of the complexes 1 (j), 2 (N), and EB
(ꢀ) on the relative viscosities of CT-DNA at 28.0 ± 0.1 °C in 5 mM Tris–HCl buffer
(pH 7.2).

2
10
L. Shivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 203–212
3
viz. DMSO as hydroxyl radical scavengers, NaN as singlet oxygen
quencher under identical conditions (Fig. 8a and b). This experi-
ment reveals that DMSO inhibits the DNA cleavage to the maxi-
mum caused by 1 and 2, suggesting the involvement of hydroxyl
radicals in the cleavage process. On the other hand the complexes
show no inhibition in the DNA cleavage activity in the presence of
3
singlet oxygen quencher NaN . Therefore, the presence of singlet
Fig. 7. Cleavage picture of SC pUC19 DNA (30
lM) by different concentration of
oxygen should be ruled out of the DNA cleavage process. Thus,
the catalytic pathway for the DNA cleavage mechanism by com-
plexes 1 and 2 is a hydrolytic cleavage process [65].
complexes 1 and 2 (100–400 M) in 10 mM Tris–HCl/1 mM EDTA buffer (pH 8.0).
l
In order to study the binding of the complexes to pUC19 DNA,
the major groove binder methyl green and the minor groove binder
distamycin were added prior to the addition of the complexes to
DNA [66]. Methyl green was found to inhibit the DNA breakage
mediated by both complexes (lane 8, Fig. 8a and b). In contrast,
distamycin had no effect on the cleavage produced by both com-
plexes (Fig. 8a and b, lane 7). These findings suggest that the com-
plexes prefer to bind through the major groove only.
Antimicrobial activities
Antimicrobial drugs are designed to inhibit/kill the infecting
organisms and to have no minimal effect on the recipient. Manga-
nese is better against antimicrobial activity with low toxicity and
high selectivity than nickel, copper, titanium, zirconium, silver,
cadmium and zinc. Maneb, the coined name for manganese ethyl-
ene bis-dithiocarbamate has been used as commercial fungicide,
against a wide variety of diseases particularly of vegetable and
fruits [67]. The results of the antimicrobial activity have been com-
pared with the conventional fungicide Fluconazole and the bacte-
ricide Chloramphenicol used as the standards. The results
achieved from these studies have been enlisted in Tables 2 and 3.
The data of the anti-fungal and antibacterial activity indicated that
the metal chelates are more active than the ligands. However, the
increased biocidal properties after complexation can be very well
explained by chelation theory [68]. The mechanism of the toxicity
of the complexes with the ligands may be ascribed to the increase
of the lipophilic nature of the complexes arising from chelation.
Due to the complexity of biological system, it is rather difficult to
stipulate the exact mechanism for such activities.
Fig. 8. Cleavage of SC pUC19 DNA (30
lM) by complexes 1 (a) and 2 (b) (400 lM) in
the presence of H (100 M), in 10 mM Tris–HCl/1 mM EDTA buffer (pH 8.0). The
2
O
2
l
strong inhibitions of DNA cleavage to complexes was observed in the presence of
the hydroxyl radical scavenger DMSO and their ability of groove binding.
peroxide (data not showed). This indicates that hydrogen peroxide
plays a role to aid the complexes in DNA degradation by oxidative
cleavage, and engages in an oxidation reaction on the DNA strand.
This result is comparable with previous studies of DNA cleavage by
other Manganese complexes where hydrogen peroxide is needed
and reactive hydroxyl radicals are involved [63,64].
Furthermore, in order to investigate the nature of reactive spe-
cies that is responsible for the cleavage of the plasmid DNA by 1
and 2, reaction was carried out with standard radical scavengers,
Table 2
Antimicrobial results of Hbid and its metal complexes.
Compounds
% Inhibition against bacteriaa
% Inhibition against fungia
S. aureus
X. vesicatoria
R. solanacearum
E. coli
A. niger
A. flavus
Bpmp
32
36
34
100
–
26
33
30
100
–
36
46
42
100
–
25
30
30
100
–
34
45
44
–
36
48
46
–
Complex 1
Complex 2
Chloramphenicol
Fluconazole
100
100
a
The results obtained were the average of three replicates.
Table 3
Minimum inhibitory concentration results in (lg/mL).
Compound
IC50 (l
g/mL)a
S. aureus
X. vesicatoria
R. solanacearum
E. coli
A. niger
A. flavus
Bpmp
>100
75
70
<20
–
>100
65
80
20
–
>100
60
55
<20
–
>100
80
85
20
–
>100
55
60
–
>100
50
55
–
Complex 1
Complex 2
Chloramphenicol
Fluconazole
20
20
a
The results obtained were the average of three replicates.

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The author L. Shiva Kumar is thankful to the UGC, New Delhi for
the award of Rajiv Gandhi National Fellowship. We also thank
Head of the Department of Biotechnology, University of Mysore,
for providing the help in carrying out antimicrobial and nuclease
activities.
[
[
[
[
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2013.01.025.
[
[
3
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