Stabilization of Mn(II) and Mn(III) in mononuclear complexes derived from tridentate ligands with N2O donors: Synthesis, crystal structure, superoxide dismutase activity and DNA interaction studies

Article abstract of DOI:10.1016/j.jinorgbio.2009.09.014

A new family of tridentate ligands PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydazono)methyl)phenol), N-PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydrazono)methyl)napthalen-1-ol), Me-PhimpH (2-(1-(2-phenyl-2-(pyridine-2-yl)hydrazono)ethyl)phenol) have been synthesized and characterized. The ligands PhimpH and N-PhimpH after deprotonation react with manganese(II) and manganese(III) starting materials affording [Mn(Phimp)₂] (1), [Mn(Phimp)₂](ClO₄) (2), [Mn(N-Phimp)₂] (3), [Mn(N-Phimp)₂](ClO₄) (4). Complexes [Mn(Phimp)₂] (1) and [Mn(N-Phimp)₂] (3) convert to [Mn(Phimp)₂]⁺ (cation of 2) and [Mn(N-Phimp)₂]⁺ (cation of 4) respectively upon oxidation. Ligand Me-PhimpH stabilized only manganese(III) centre resulting [Mn(Me-Phimp)₂](ClO₄) (5). The molecular structures of [Mn(Phimp)₂], 1 and [Mn(Phimp)₂](ClO₄), 2 were determined by single crystal X-ray diffraction. X-ray crystal structures of 1 and 2 have revealed the presence of distorted octahedral MnN₄O₂ coordination sphere having meridionally spanning ligands. Electrochemical studies for the complexes showed Mn(II)/Mn(III), (E_1/2 = 0.14-0.40 V) and Mn(III)/Mn(IV), (E_1/2 = 0.80-1.06 V) couples vs. Ag/AgCl. The redox properties were exploited to examine superoxide dismutase (SOD) activity using Mn(II)/Mn(III) couple. The complexes 1, 2, 4 and 5 have been revealed to catalyze effectively the dismutation of superoxide (O₂^{· -}) in xanthine-xanthine oxidase-nitro blue tetrazolium assay and IC₅₀ values were found to be 0.29, 0.39, 1.12 and 0.76 μM respectively. DNA interaction studies with complex 2 showed binding of DNA in a non-intercalative pathway. Complexes 1, 2 and 4 exhibited nuclease activity in presence of H₂O₂ and inhibition of activity was noted in presence of KI.

Full text of DOI:10.1016/j.jinorgbio.2009.09.014

Journal of Inorganic Biochemistry 104 (2010) 9–18
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Stabilization of Mn(II) and Mn(III) in mononuclear complexes derived
from tridentate ligands with N2O donors: Synthesis, crystal structure,
superoxide dismutase activity and DNA interaction studies
*
Kaushik Ghosh , Nidhi Tyagi, Pramod Kumar, Udai P. Singh, Nidhi Goel
Department of Chemistry, Indian Institute of Technology, Roorkee, Roorkee 247 667, Uttarakhand, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 May 2009
Received in revised form 17 September
2009
Accepted 21 September 2009
Available online 26 September 2009
A new family of tridentate ligands PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydazono)methyl)phenol), N–
PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydrazono)methyl)napthalen-1-ol), Me–PhimpH (2-(1-(2-phe-
nyl-2-(pyridine-2-yl)hydrazono)ethyl)phenol) have been synthesized and characterized. The ligands
PhimpH and N–PhimpH after deprotonation react with manganese(II) and manganese(III) starting mate-
rials affording [Mn(Phimp)2] (1), [Mn(Phimp)2](ClO4) (2), [Mn(N–Phimp)2] (3), [Mn(N–Phimp)2](ClO4)
(4). Complexes [Mn(Phimp)2] (1) and [Mn(N–Phimp)2] (3) convert to [Mn(Phimp)2]+ (cation of 2) and
[Mn(N–Phimp)2]+ (cation of 4) respectively upon oxidation. Ligand Me–PhimpH stabilized only manga-
nese(III) centre resulting [Mn(Me–Phimp)2](ClO4) (5). The molecular structures of [Mn(Phimp)2], 1 and
[Mn(Phimp)2](ClO4), 2 were determined by single crystal X-ray diffraction. X-ray crystal structures of 1
and 2 have revealed the presence of distorted octahedral MnN4O2 coordination sphere having meridio-
nally spanning ligands. Electrochemical studies for the complexes showed Mn(II)/Mn(III), (E1/2 = 0.14–
0.40 V) and Mn(III)/Mn(IV), (E1/2 = 0.80–1.06 V) couples vs. Ag/AgCl. The redox properties were exploited
to examine superoxide dismutase (SOD) activity using Mn(II)/Mn(III) couple. The complexes 1, 2, 4 and 5
have been revealed to catalyze effectively the dismutation of superoxide (Oꢀ2ꢁ) in xanthine–xanthine oxi-
Keywords:
Manganese complexes
Crystal structure
Electrochemistry
Superoxide dismutase
DNA binding
Nuclease activity
dase–nitro blue tetrazolium assay and IC50 values were found to be 0.29, 0.39, 1.12 and 0.76 lM respec-
tively. DNA interaction studies with complex 2 showed binding of DNA in a non-intercalative pathway.
Complexes 1, 2 and 4 exhibited nuclease activity in presence of H2O2 and inhibition of activity was noted
in presence of KI.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
nitrosyl complexes [9]. Stabilization of manganese oxidation states
has been implicated in manganese toxicity [10]. However, our curi-
Manganese, an essential element for human, is widely distrib-
uted in nature and is second in terms of its terrestrial abundance
among the first row transition metals [1]. In biosystem several en-
zymes namely superoxide dismutase (SOD) [2], oxalate oxidase [3],
lipoxygenase (LO) [4], catalase [5] etc. require manganese as cofac-
tor for their catalytic activities. In biological reactions manganese
sometime acts as a Lewis acid, on the other hand, it could partici-
pate in redox reactions by flipping its +2, +3 and +4 oxidation states
[6]. Hence it has plural roles in biosystem. However, the role of
manganese in oxygen evolving complex (OEC) of photosystem-II
(PSII) is unique [2]. Coordination chemistry of manganese has been
exploited for the structural and functional modelling of metallo-
protein e.g. mimicking of OEC in PSII [7]. Other applications are
catalytic activity studies [8] and synthesis of photolabile metal
osity originated from the great demand of manganese chemistry
with ligands having phenolato donors for the mimicking of SOD
activity. Moreover, it has been documented in the literature that
native SOD as well as small molecule SOD mimics exhibit nuclease
activity [11,12]. Hence we were also interested to study DNA inter-
action as well as nuclease activity studies.
Superoxide dismutases are important class of redox enzymes
which are responsible for the disproportionation of superoxide
ion (Oꢀ2ꢁ) produced by one electron reduction of oxygen. The redox
reaction is shown below:
2O2ꢀꢁ þ 2Hþ ) O2 þ H2O2
Superoxide ion is known to be involved in oxidative stress [13]
and this reactive species is responsible for several diseases like
reperfusion injury, arthritis, neuronal apoptosis, cancer and ac-
quired immunodeficiency syndrome [14]. These metalloenzymes
can perform this catalytic redox cycle by several metal ions namely
* Corresponding author. Tel.: +91 1332 285365; fax: +91 1332 2735601.
E-mail address: ghoshfcy@iitr.ernet.in (K. Ghosh).
0162-0134/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2009.09.014

10
K. Ghosh et al. / Journal of Inorganic Biochemistry 104 (2010) 9–18
copper/zinc or manganese or iron [15] or nickel [16]. Manganese
complexes, which mimic the activity of superoxide dismutase
enzyme, are used for the destruction of detrimental superoxide
ions [14,17,18] and in several cases as therapeutic agents [14,17].
Salen complexes of manganese and its derivatives were exten-
sively used for the mimicking of SOD activity [17,19]. However,
in very few instances other than salen, ligands with phenolato do-
nor were used [20,21].
Xanthine, nitro blue tetrazolium (NBT) and catalase were ob-
tained from Himedia and xantine oxidase (XO) from bovine milk,
agarose (molecular biology grade) and ethidium bromide (EB)
were purchased from Sigma. The supercoiled pBR322 DNA and calf
thymus DNA (CT-DNA) were purchased from Bangalore Genei
(India). Tris buffer was prepared in deionised water.
2.2. Methods
Interaction of DNA with transition metal complexes has gained
considerable current interest due to its various applications in can-
cer research and nucleic acid chemistry [22,23]. Although cisplatin
and carboplatin are in use, there are several side effects of these che-
motherapeutic drugs [24]. Among other transition metal complexes,
copper [25] and ruthenium [26,27] complexes are used now-a-days
extensively to study metal complex–DNA interactions. However, re-
port of biologically relevant and lesser toxic manganese complexes
[17] are fewer [28–35] compared to copper and ruthenium. Only
manganese-salen related [28,29] complexes were used for DNA
interaction and nuclease activity studies, however, other than salen,
to the best of our knowledge Zhang et al. [32] and Bochu and
co-workers [34] used ligands affording phenolato donor.
Hence, we designed three tridentate ligands (shown in
Scheme 1) containing single phenolato donor along with pyridine
and imine donors. Synthesis and spectroscopic characterization of
the new family of ligands and their mononuclear manganese com-
plexes are described here. Molecular structures of two representa-
tive complexes were determined by X-ray crystallography.
Investigation of electrochemical studies prompted us to examine
superoxide dismutase activity of these complexes. We have deter-
mined IC50 values for these complexes using xanthine–xanthine
oxidase-nitrobluetetrazolium (NBT) assay. We also report the inter-
action studies of this family of manganese complexes with DNA and
their nuclease activity.
2.2.1. Physical measurements
Elemental analyses were carried out microanalytically at Ele-
menlar Vario EL III. Melting points were obtained using Perfit (In-
dia) melting point apparatus. IR spectra were obtained as KBr
pellets with Thermo Nikolet Nexus FT-IR spectrometer, using 50
scans and were reported in cmꢁ1. GC-MS data were obtained on
a quadrupole Perkin Elemer Clarus 500 MS coupled to a Perkin Ele-
mer Clarus 500 GC fitted with an Elite-1 column and mass detector
was operated at 70 eV. Electronic absorption spectra were re-
corded in dichloromethane, acetonitrile, DMF or DMSO solvents
with an Evolution 600, Thermo Scientific UV–visible spectropho-
tometer. Emission quenching titrations were carried out on Varian
fluorescence spectrophotometer. 1H and 13C NMR were recorded
on Bruker AVANCE, 500.13 MHz spectrometer, chemical shift for
1H NMR spectra are related to internal standard Me4Si for all resid-
ual protium in the deutrated solvents. Magnetic susceptibilities
were determined at 297 K with Vibrating Sample Magnetometer
model 155, using nickel as a standard. Diamagnetic corrections
were carried out with Pascal’s increments [37]. Molar conductivi-
ties were determined in DMF at 10ꢁ3 M at 25 °C with a Systronics
304 conductometer. Cyclic voltammetry measurements were car-
ried out using a CHI-600C electroanalyzer in dichloromethane
and acetonitrile. A conventional three-electrode arrangement con-
sisting of platinum wire as auxiliary electrode, glassy-carbon as
working electrode and Ag(s)/AgCl electrode as reference electrode,
was used. These measurements were performed in the presence of
0.1 M tetrabutylammonium perchlorate (TBAP) as the supporting
electrolyte, using complexes concentration 10ꢁ3 M in dichloro-
methane and acetonitrile. The ferrocene/ferrocenium couple was
found at E1/2 = +0.42 (72) V vs. Ag/AgCl under the same experimen-
tal conditions. All experiments were performed at room tempera-
ture, and solutions were thoroughly degassed with nitrogen prior
to beginning the experiments, and during the measurements nitro-
gen atmosphere was maintained.
2. Experimental
2.1. Materials
All the solvents used were reagent grade. Removal of all solvents
was carried out under reduced pressure. Toluene, diethylether,
dimethylsulfoxide (DMSO), dimethylformamide (DMF), chloroform
and dichloromethane were purified by distillation over 4 Å molecu-
lar sieves and stored over sieves. Methanol was purified by distilla-
tion over 3 Å molecular sieves and stored over sieves. Acetonitrile,
acetone were dried by refluxing and distillation over P2O5 (0.5%,
w/v) [36]. Analytical grade reagents salicylaldehyde, o-hydroxyace-
tophenone (SRL, Mumbai, India), 2-hydroxy-1-napthaldehyde (Sig-
ma Aldrich, Steinheim, Germany), triethylamine (Thomas Baker,
India), sodium perchlorate and tert-butylhydroperoxide (tBuOOH)
(Himedia Laboratories Pvt. Ltd, Mumbai, India), sodium hydride
(Acros organics, USA), MnCl2ꢀ4H2O, Mn(CH3COO)2ꢀ4H2O (Merck
Limited, Mumbai, India) Mn(ClO4)2ꢀ6H2O, Mn(CH3COO)3ꢀ2H2O
(Sigma Aldrich, Steinheim, Germany) were used as obtained.
[Mn(DMF)6](ClO4)3 was prepared according to the method reported
in the literature [17].
2.3. Synthesis of the ligands
2-(1-phenylhydrazinyl)pyridine was prepared according to the
method reported [38] in the literature. Ligands PhimpH (2-((2-
phenyl-2-(pyridin-2-yl)hydazono)methyl)phenol), N–PhimpH (2-
((2-phenyl-2-(pyridin-2-yl)hydrazono)methyl)napthalen-1-ol),
Me–PhimpH (2-(1-(2-phenyl-2-(pyridine-2-yl)hydrazono)ethyl)-
phenol) were synthesized by reacting 2-(1-phenylhydrazinyl)-
pyridine with salicylaldehyde, 2-hydroxy-1-napthaldehyde and
o-hydroxyacetophenone respectively in methanol. Details of their
synthesis are described in the Supporting Information.
Scheme 1. Schematic drawing of tridentate Schiff’s base ligands and abbreviations.

K. Ghosh et al. / Journal of Inorganic Biochemistry 104 (2010) 9–18
11
2.4. Synthesis of metal complexes
2.4.4. Synthesis of [Mn(N–Phimp)2](ClO4) (4)
A batch of triethylamine (Et3N) (17.2 mg, 0.17 mmol) was added
to stirred solution of ligand (N–PhimpH) (57.6 mg, 0.17 mmol) in
10 mL of dichloromethane/methanol (1:1). After stirring for
30 min, a batch of Mn(ClO4)2ꢀ6H2O (61.5 mg, 0.17 mmol) in 5 mL
methanol was added dropwise, the color of solution changed from
yellow to orange and after 2–3 h the color of solution changes to
dark brown. After 12 h of stirring 5 mL of diethylether was added
to above reaction mixture, brown solid separated out which was fil-
tered washed with small amount of methanol and dried in vacuo.
Perchlorate salts of metal complexes with organic ligands are
potentially explosive. Only small quantities of these compounds
should be prepared and handled with proper protection. All the
complexes were prepared by more than one procedure. One meth-
od is described here and the rest are described in the Supporting
Information.
2.4.1. Synthesis of [Mn(Phimp)2] (1)
Yield: 85.2 mg, (60.2%). IR data (KBr,
1331, C–Ophenol, 1090, 622, ClO4–. UV–visible [CH3CN, kmax/nm
/Mꢁ1 cmꢁ1)]: 510 (4920), 448 (21,307), 415 (13,460), 384
(35,307), 363 (35,307), 331 (35,384), 317 (33,384), 248 (84,307).
leff (297 K): 4.97 lB
ꢁ1 cm2 molꢁ1 (in DMF): 61 (1:1). Anal.
m ,
max/cmꢁ1): 1611, mC@Nimine
Complex 1 was synthesized using three different manganese(II)
salts separately, however overall procedure was the same. A batch
of triethylamine (Et3N) (133.3 mg, 1.32 mmol) was added to stirred
solution of ligand (PhimpH) (381.0 mg, 1.32 mmol) in 10 mL dichlo-
romethane. After stirring for 30 min, a batch of Mn(ClO4)2ꢀ6H2O
(238.0 mg, 0.66 mmol) in 10 mL methanol was added dropwise.
The color of solution changed to orange. After 3 h of stirring, solvent
was evaporated to give orange solid which was washed with small
amount of methanol, diethylether and dried in vacuo. Orange block
shaped crystals appeared by diethylether diffusion in dichloro-
methane/methanol (1:1) solution of the complex within 24 h. Yield:
m
m
(e
.
KM/X
Calcd for C44H32N6O6ClMn: C, 63.57; H, 3.85; N, 10.11, Found: C,
63.45; H, 3.50; N, 10.08.
2.4.5. Synthesis of [Mn(Me–Phimp)2](ClO4) (5)
A batch of sodium hydride (NaH) (24.0 mg, 1.00 mmol) was
added to stirred solution of ligand (Me–PhimpH) (303.0 mg,
1.00 mmol) in dichloromethane (5 mL). After stirring for 15 min,
a batch of [Mn(DMF)6](ClO4)3 (395.0 mg, 0.50 mmol) in 10 mL
acetonitrile was added dropwise; the color of solution changed
from yellow to dark brown. After 3 h of stirring 5 mL of diethyl-
ether was added to above reaction mixture and a brown solid
separated out which was filtered, washed with small amount of
methanol and dried in vacuo. Yield: 482.3 mg, (63.6%). IR data
545.6 mg, (65.6%). IR data (KBr,
UV–visible [CH2Cl2, kmax/nm
(10,780), 334 (20,520), 310 (20,260), 240 (27,800). leff (297 K):
5.87 lB
ꢁ1 cm2 molꢁ1 (in DMF): 6 (neutral and nonelectro-
m
max/cmꢁ1): 1598,
/Mꢁ1cmꢁ1)]: 399
mC@Nimine, 1301,
mC–Ophenol
.
(e
.
KM/X
lyte). Anal. Calcd for C36H28N6O2Mn: C, 68.46; H, 4.43; N, 13.31,
Found: C, 68.40; H, 4.50; N, 13.49.
(KBr,
ClO4–. UV–visible [CH3CN, kmax/nm (
348 (16,980), 233 (52,670), 217 (59,070). leff (297 K): 4.77 lB
ꢁ1 cm2 molꢁ1 (in DMF): 54 (1:1). Anal. Calcd for
m
max/cmꢁ1): 1603,
m
C@Nimine, 1345,
mC–Ophenol, 1095, 622,
/Mꢁ1 cmꢁ1)]: 421 (7830),
2.4.2. Synthesis of [Mn(Phimp)2](ClO4) (2)
m
e
A batch of ligand (PhimpH) (578.0 mg, 2.00 mmol) was dissolved
in 7 mL of dichloromethane, methanol and water (2.0:4.5:0.5). After
stirring for 10 min, a batch of Mn(CH3COO)3ꢀ2H2O (268.0 mg,
1.00 mmol) was added dropwise, the color of solution changed to
dark brown. After 15 min stirring sodium perchlorate (140.0 mg,
1.00 mmol) was added to the above reaction mixture. Brown solid
precipitated out within 30 min, reaction mixture was further stirred
for 3 h. After 3 h of stirring, reaction mixture was filtered; dark
brown solid obtained was washed with excess of methanol and
dried in vacuo. Dark brown block shaped crystals appeared by slow
evaporation of a solution of complex 2 in acetonitrile/diethylether
(5 mL/3 drop) after a week at room temperature. Yield: 680.0 mg,
.
KM/X
C38H32N6O6ClMn: C, 60.11; H, 4.21; N, 11.07, Found: C, 60.13; H,
4.58; N, 10.88.
2.5. X-ray structure determination
The X-ray data collection and processing for 1 and 2 were per-
formed on Bruker Kappa Apex-II CCD diffractometer by using
graphite monochromated Mo-K
a radiation (k = 0.71070 Å) at
293 K and 296 K respectively. Crystal structures were solved by di-
rect methods. Structure solution, refinement and data output were
carried out with the SHELXTL program [39,40]. All non-hydrogen
atoms were refined anisotropically. Hydrogen atoms were placed
in geometrically calculated positions and refined using a riding
model. Images were created with the DIAMOND program [41].
(46.6%). IR data (KBr,
1096, 622, ClO4–. UV–visible [CH3CN, kmax/nm (
421 (4798), 341 (17,791), 306 (18,640), 245 (31,034). leff (297 K):
4.69 lB
ꢁ1 cm2 molꢁ1 (in DMF): 59 (1:1). Anal. Calcd for
m
max/cmꢁ1): 1608,
m
C@Nimine, 1344, mC–Ophenol
,
m
e
/Mꢁ1 cmꢁ1)]:
.
KM/X
C37H32N6O7ClMn: C, 58.19; H, 4.19; N, 11.01, Found: C, 58.11; H,
4.12; N, 11.51.
2.6. Measurement of superoxide dismutase activity
2.4.3. Synthesis of [Mn(N–Phimp)2] (3)
Superoxide dismutase activity of complexes 1, 2, 4 and 5 were
determined by indirect method using nitro blue tetrazolium
(NBT) assay. In this method superoxide anion is generated in situ
enzymetically by xanthine/xantine oxidase system and detected
spectrophotometrically by reduction of NBT which produced a
formazan dye of blue color. Absorbance at 560 nm got increased
because of production of formazan dye [42]. However, the increase
rate of the absorbance was reduced with the increase of complexes
concentration. The assay was carried out in phosphate buffer
(50 mM) at pH 7.8 using 0.2 mM xanthine, 0.12 mM NBT, 0.07
U/mL xanthine oxidase and catalase 1000 U/mL (final volume =
A batch of sodium hydride (NaH) (7.7 mg, 0.32 mmol) was
added to stirred solution of ligand (N–PhimpH) (101.7 mg,
0.30 mmol) in toluene (15 mL) under the continuous flow of
nitrogen atmosphere. After stirring for 30 min,
a batch of
Mn(ClO4)2ꢀ6H2O (54.3 mg, 0.15 mmol) in 5 mL methanol was
added dropwise, the color of solution changed from yellow to or-
ange. After 3 h of stirring in nitrogen atmosphere, reaction mixture
was concentrated to 5 mL, orange solid separated out which was
filtered. This orange solid was washed with excess of toluene and
small amount of methanol and dried in vacuo. Yield: 128.6 mg,
(58.5%). IR data (KBr,
m
max/cmꢁ1): 1605,
/Mꢁ1 cmꢁ1)]: 448 (24,063), 429
(24,900), 385 (33,479), 332 (38,502), 253 (78,887). leff (297 K):
5.49 lB
ꢁ1 cm2 molꢁ1 (in DMF): 8.2 (neutral and nonelectro-
m
C@Nimine, 1325, mC–Ophenol
.
750 lL). The tested compounds were dissolving in DMSO/DMF
UV–visible [CH Cl2, kmax/nm (
e
(for 1 and 5 in DMSO and 2 and 4 in DMF) and the final concentra-
tion of DMSO/DMF in reaction mixture was 0.1% in phosphate buf-
fer at pH 7.8. The reaction was started by adding 0.07 U/mL
xanthine oxidase and measurement was started after 15 min for
each experiment.
2
.
KM/X
lyte). Anal. Calcd for C44H32N6O2Mn: C, 72.22; H, 4.37; N, 11.49,
Found: C, 72.32; H, 4.39; N, 11.55.

12
K. Ghosh et al. / Journal of Inorganic Biochemistry 104 (2010) 9–18
2.7. DNA-binding studies by spectroscopic titration and cleavage
experiments
together with a band at 623 cmꢁ1 were found in all manganese(III)
complexes (2, 4 and 5). The lack of splitting of these two bands sug-
gests the presence of non-coordinated perchlorate ion to the metal
centre [47]. A high intensity band at (ꢂ1297–1324 cmꢁ1) in the
Schiff bases can be assigned as phenol C–O stretching. Shift of
The experiments were carried out in 0.1 M phosphate buffer at
pH 7.2 using a solution of calf thymus DNA (CT-DNA) which gave a
ratio of absorbance at 260 nm and 280 nm (A260/A280) of ca. 1.8,
indicating that the CT-DNA was sufficiently protein free [43]. The
concentration of DNA solution was determined by UV–visible
absorbance at 260 nm. The extinction coefficient e260 was taken
as 6600 cmꢁ1 as reported in literature [44]. Absorbance titration
experiments were carried out with a complex concentration of
m
C–O to higher frequency supports deprotonation and the formation
of metal oxygen bond [48]. It is further supported by disappearance
of the broad O–H band in IR spectra of all metal complexes. The mo-
lar conductivity measurements in DMF at ca. 10ꢁ3 M determined at
25 °C for complexes and were found to be 6.0 and
8.0
ꢁ1 cm2 molꢁ1 respectively whereas the same for 2, 4 and 5
were 59.0, 61.0 and 54.0
ꢁ1 cm2 molꢁ1 respectively. These values
m
1
3
X
23
l
M varying the CT-DNA concentration from 0 to 68
Fluorescence quenching experiments were carried out by the
successive addition of 0–7 M of the manganese complex to the
DNA (25 M) solutions containing 5 M EB in 0.1 M phosphate
l
M.
X
for 1 and 3 confirm the neutral electrolyte behaviour whereas val-
ues for 2, 4 and 5 clearly indicate uni-univalent (1:1) electrolyte
behaviour in solution [49]. For complexes 1 and 3, room tempera-
l
l
l
buffer (pH 7.2) these sample were excited at 510 nm and emission
were observed between 530 and 550 nm.
Cleavage of plasmid DNA was monitored by using agarose gel
electrophoresis. Supercoiled pBR32 DNA (200 ng) in TBE (pH 8.2)
was treated with manganese complexes (100 lM) dissolved in
DMF (10%) in the presence or absence of additives. The oxidative
DNA cleavage by the complexes were studied in the presence of
H2O2 (200–400 lM, oxidizing agent) and KI (400 lM, radical scav-
enger). The samples were incubated for 1.5 h at 37 °C, added load-
ing buffer (25% bromophenol blue and 30% glycerol). The agarose
gel (0.8%) containing 0.4 lg/mL of EB was prepared and the elec-
trophoresis of the DNA cleavage products was performed on it.
The gel was run at 60 V for 2 h in TBE buffer and the bands were
identified by placing the stained gel under an illuminated UV lamp.
The fragments were photographed by using gel documentation
system (BIO RAD).
ture (297 K) magnetic moments were 5.87 and 5.49 lB respectively,
predicting the stabilization of high-spin d5 manganese(II) in the
above two complexes. The magnetic moment values for 2, 4 and 5
were 4.69, 4.97 and 4.77 lB respectively, which were expected for
a high-spin magnetically dilute d4 manganese(III) ion, indicating lit-
tle or no antiferromagnetic interaction [50]. The absorption maxima
at 400 nm for complex 1 and 430, 450 nm for complex 3 were as-
signed to be ligand-to-metal charge transfer (LMCT) transitions
[51]. The bands near ꢃ420 nm respectively for 2, 4 and 5 were as-
signed to phenolato oxygen to manganese(III) ligand-to-metal
charge transfer (LMCT) transition [52]. However, 4 possesses an-
other two absorption maxima at ꢃ450 nm (
e
= 21307 Mꢁ1 cmꢁ1
e
= 4920 Mꢁ1 cmꢁ1) which were also of charge trans-
)
and ꢃ510 nm (
fer type [53].
3.2. Interconversions
Characteristic UV–visible spectra of the synthesized manganese
complexes in this report prompted us to monitor their interconver-
sion (shown in Scheme 2) through UV–visible spectral studies.
Interconversion of 1 to cation of 2 is shown in Fig. 1 whereas that
of 3 to cation of 4 is shown in Fig. 2. Disappearance of the peak at
399 nm confirms the formation of [MnIII(Phimp)2]+ as shown in
Fig. 1. The decrease in intensity of peaks at 429 nm and 448 nm
and increase in intensity of peaks at 384 nm in Fig. 2 indicate the
formation of cation of 4 from 3. The time dependent plots (inset
of Fig. 2a and b) clearly show higher rate of conversion of oxidised
3. Results and discussion
3.1. Syntheses and characterization of ligands and their metal
complexes
The tridentate ligands PhimpH, N–PhimpH and Me–PhimpH
were synthesized and their characterizations were established by
elemental analysis, UV–visible, IR, GC-MS spectrometry, and NMR
(1H and 13C) spectroscopic studies (shown in Supporting Informa-
tion). [Mn(Phimp)2] (1) was synthesized by the reaction of the
deprotonated ligand with manganese(II) starting materials.
[Mn(Phimp)2](ClO4) (2) was prepared by manganese(III) starting
t
product in presence of BuOOH as compared to the conversion in
presence of oxygen. Hence complexes 2 and 4 could be synthesized
by the oxidation of 1 and 3 respectively. Detailed experimental
procedure is described in Supporting Information. Characterization
t
materials as well as via the oxidation of 1 by BuOOH.
t
[Mn(N–Phimp)2] (3) was prepared by manganese(II) starting
materials in inert atmosphere. In presence of oxygen, 3 undergoes
oxidation to [Mn(N–Phimp)2]+. The manganese(III) derivative 4
was independently prepared by the reaction of deprotonated li-
gand with [Mn(DMF)6](ClO4)3. In case of Me–PhimpH the deproto-
nated ligand does not stabilize manganese(II) centre and in all of
our synthetic attempts we ended up with [Mn(Me–Phimp)2](ClO4)
(5). This may be due to the presence of electron donating methyl
(–Me) group attached to the ligand [45]. The synthetic procedures
of 1, 2, 3, 4, 5 and their conversions (vide infra) have been summa-
rized in Scheme 2.
of 2 and 4 derived from the oxidation of 1 and 2 by BuOOH gave
rise to similar data in IR spectra, conductivity measurement, mag-
netic moment measurement and electrochemical investigation.
We were unable to find the reason for the deviation of the UV–vis-
ible data.
3.3. Description of the structures
In order to get the structure of the new family of ligands, single
crystals of representative ligand PhimpH were grown by slow
evaporation of a dichloromethane solution. Molecular structure
of PhimpH was solved by single crystal X-ray diffraction and de-
scribed in the Supporting Information. The crystal structure gave
rise to C@N bond distance of 1.283(3) Å which is similar to the re-
ported value of 1.282(3) Å [46].
The azomethine (–HC@N–) characteristic band in IR for the free
ligand was observed at ꢂ1607–1620 cmꢁ1. Coordination of the
nitrogen to the metal centre reduced the electron density in the azo-
. . .
methine moiety and thus lowered the (–HC N–) frequency [46].
•
Decrease in stretching frequencies for
m
–HC@N in [Mn(Phimp)2] and
Molecular structures of manganese complexes [Mn(Phimp)2]
(1) and [Mn(Phimp)2](ClO4) (2) are depicted in Figs. 3 and 4 respec-
tively. All the crystallographic parameters are tabulated in Table 1
and selected bond distances and bond angles are listed in Table 2.
Crystals of complex 1 were obtained from diethylether diffusion to
[Mn(N–Phimp)2] clearly indicates the ligation of azomethine nitro-
. . .
gen (–HC N–) to metal centre. The m–HC
for [Mn(Phimp)2]-
. . .
•
N
•
(ClO4), [Mn(N–Phimp)2](ClO4) and [Mn(Me–Phimp)2](ClO4) are
1608, 1611 and 1603 cmꢁ1 respectively. IR bands near 1090 cmꢁ1

K. Ghosh et al. / Journal of Inorganic Biochemistry 104 (2010) 9–18
13
Scheme 2.
In complex 1 the molecule lies on a twofold axis of symmetry
and complete structure of the molecule was obtained after grow-
ing. The deprotonated ligand PhimpH is coordinated meridionally
to the central manganese ion in both complexes 1 and 2. In both
complexes 1 and 2 the metal centre is coordinated by two trans
imine nitrogens, two cis pyridine nitrogen and two cis phenolato
donors in distorted octahedral fashion. In complex 1, Mn–Npy dis-
tance is 2.255(2) Å which is consistent with the reported manga-
nese(II) Schiff base complexes [46,54,55]. Similarly Mn–Ophenolato
,
(2.0405(18) Å) bond distance is shorter than that of structurally
characterized [MnIIL](ClO4), (Mn–Ophenolato distance, 2.078(5) Å,
where
L
is heptadentate ligand) and [Mn(pyo3tren)]2+ ion
(Mn–Ophenolato distance, 2.192(3) Å, where pyo3tren is also a hep-
tadentate ligand) [56,57]. Mn–Nimine distance 2.2984(17) Å is also
closely related with reported data [56,58,59]. The MnN4O2 coordi-
nation shows a considerable distortion because of ligand’s rigidity.
Thus the angles at the metal centre [N2–Mn–N2; 153.94(9)°, O1–
Mn–N1; 100.9(3)°, O1–Mn–N1; 151.24(6)° (shown in Table 2)]
show large deviation from ideal octahedron.
Complex [Mn(Phimp)2](ClO4), (2) was crystallized after slow
evaporation of a solution of 2 in acetonitrile-diethylether. It was
crystallized in trigonal space group P3121. The distortion of perfect
octahedral geometry was manifested by N2–Mn1–N1 angle of
74.96(12)°, N1–Mn1–N1; 166.57(16)° (shown in Table 2) which
differ significantly from ideal 90° and 180°. In complex 2, Mn–Npy
Fig. 1. Titration of [Mn(Phimp)2] (1) with tBuOOH: spectra were recorded after
successive addition of 5
l
L of (0.1 M) tBuOOH to 10
l
L of 5.5 ꢄ 10ꢁ6 M solution of
[Mn(Phimp)2] (1) in DMF (1 mL) at 25 °C.
a solution of 1 in dichloromethane–methanol (1:1) mixture. Com-
pound 1 crystallized in monoclinic space group C2/c consisting of
neutral mononuclear manganese(II) complex in N4O2 donor set.

14
K. Ghosh et al. / Journal of Inorganic Biochemistry 104 (2010) 9–18
Fig. 4. Ball-and-stick representation of the crystal structure of [Mn(Phimp)2](ClO4)
(2), atoms are shown as sphere of arbitrary diameter.
and Mn–Ophenolato distances are consistent with the reported data
[21,60,61]. Mn–Nimine bond distances were found to be
2.176(3) Å which is longer than the usual reported values of
2.001(6) Å in [MnIII(L)Cl]+ where L is Schiff base ligand [58] and
other structurally characterized manganese(III) complexes
[21,48,61,62]. A d4 system of manganese(III) is susceptible to un-
dergo Jahn–Teller distortion and is expected to show elongation
of trans bonds. In our complex the trans imine bond lengths are
longer than the usual distances, may be due to Jahn–Teller distor-
tion [51].
Examination of packing diagram of complexes 1 and 2 revealed
non-covalent interactions. These types of non-covalent and
hydrogen bonding interactions are very important in the crystal
engineering and supramolecular frameworks [63]. The data for
non-covalent interaction are described in Table S3 and Fig. S23 in
Supporting Information.
Fig. 2. (a) Aerial oxidation for conversion of 3 to cation of 4 by repetitive scan of
10
lL
of 2.87 ꢄ 10ꢁ3 mM of [Mn(N–Phimp)2] in dichloromethane (1 mL) and
There is no conjugation between the pyridine ring and the phe-
nolato ring; however they remain in the same plane in the ligand
as well as in metal complexes. The angle between the phenyl ring
attached to the nitrogen and plane containing phenolato ring and
pyridine ring is 88.14° in ligand, 86.38° in complex 1 and 87.84°
in complex 2 (phenyl ring vs. mer plane). Comparing the structure
of free ligand and the ligand bound to the metal centre, the follow-
ing facts were observed. A very little change in N1–C8–N2 and
O1–C1–C6 angles of the ligand was observed in the complexes 1
and 2. On the other hand due to the coordination of imine nitrogen
to the metal centre in both complexes, N3–C7–C6 angle of the
ligand was decreased by ꢃ7o. The Mn–Npy, Mn–Ophenolato and
Mn–Nimine bond lengths in case of 2 are lower than that of 1 which
confirms stabilization of manganese(II) centre for 1 and manga-
nese(III) centre for 2. This is consistent with our magnetic moment
data.
changes in spectral patterns were recorded after each 0.6 min interval. (b) Titration
of [Mn(N–Phimp)2] by 5
the changes in spectral patterns were recorded after 1 min interval in DMF (1 mL) at
l
L of 3.25 ꢄ 10ꢁ3 mM with 0.2 L of tBuOOH (0.01 M) and
l
25 °C.
3.4. Redox properties and SOD activity
In order to examine the influence of the newly synthesized
ligands on metal centre, we investigated the electrochemical prop-
erties of the five (1–5) complexes isolated and characterized in this
report. The representative voltammograms of complexes 1 and 5
are shown in Fig. 5 (figures for 2, 3 and 4 are shown in Supporting
Information (Fig. S21)), and the redox potentials for all the
Fig. 3. Ball-and-stick representation of the crystal structure of [Mn(Phimp)2] (1),
atoms are shown as sphere of arbitrary diameter, and the molecule lies on a twofold
axis of symmetry.

K. Ghosh et al. / Journal of Inorganic Biochemistry 104 (2010) 9–18
15
Table 1
Crystallographic data for complexes [Mn(Phimp)2] (1) and [Mn(Phimp)2](ClO4) (2).
Empirical formula C36H28MnN6O2 (1)
C36H28ClMnN6O6 (2)
Color
Orange
631.58
Brown
731.03
Formula weight
(g molꢁ1
)
Temperature (K)
k (Å) (Mo-K
Crystal system
Space group
a (Å)
293(2)
296(2)
a)
0.71073
Monoclinic
C2/c
18.599(5)
9.786(5)
17.368(5)
90.00
0.71073
Trigonal
P3121
10.775(2)
10.775(2)
26.197(5)
90.00
b (Å)
c (Å)
a
(°)
b (°)
96.407(5)
90.00
3141(2)
0.24 ꢄ 0.19 ꢄ 0.13
4
90.00
120.00
2633.9(10)
0.27 ꢄ 0.23 ꢄ 0.17
3
c
(°)
V (Å3)
Crystal size (mm)
Z
qcalcd (g cmꢁ3
F (0 0 0)
)
1.336
1308
1.383
1600
h range for data
collection
Index ranges
2.20–28.27
2.18–28.19
Fig. 5. Cyclic voltammograms of a 10ꢁ3 M solution of 1 in dichloromethane and 5 in
acetonitrile, in presence of 0.1 M tetrabutylammonium perchlorate (TBAP), using
working electrode: glassy-carbon, reference electrode: Ag/AgCl; auxiliary electrode:
platinum wire, scan rate 0.1 V/s.
ꢁ24 < h < 24, ꢁ12 < k < 13, ꢁ14 < h < 14, ꢁ14 < k < 14,
ꢁ22 < l < 23
ꢁ30 < l < 34
Refinement
method
Full matrix least-squares
on F2
Full matrix least-squares
on F2
Data/restraints/
parameters
GOFaon F2
3835/0/204
4331/0/227
Table 3
1.017
1.775
Electrochemical data for the redox couples Mn(II)/Mn(III) and Mn(III)/Mn(IV) at
R1b (I > 2
r
(I))
R1 (all data)
wR2c (I > 2
(I))
0.0405
0.0812
0.1193
0.1577
0.0613
0.0696
0.2054
0.2163
298 Ka vs. Ag/AgCl.
r
Complex Mn(II)/Mn(III)
Epa(V) Epc(V) E1/2
Mn(III)/Mn(IV)
wR2 (all data)
b
b
(D
Epc)(V) Epa(V) Epc(V) E1/2 Epc)(V)
(D
P
2
1=2
GOF ¼ ½ ½wðF2o ꢁ F2c Þ ꢅ=M ꢁ Nꢅ
(M = number of reflections, N = number of
a
1
2
3
4
5
0.410 0.243 0.327 (167)
0.323 0.247 0.285 (76)
0.388 0.293 0.341 (95)
0.454 0.366 0.410 (88)
0.184 0.101 0.143 (83)
1.133 0.979 1.056 (154)
1.014 0.930 0.927 (84)
1.020 0.916 0.968 (104)
0.949 0.843 0.896 (106)
0.838 0.766 0.802 (72)
parameters refined).
P
P
b
R1 ¼ kFo j ꢁ j Fck= Fo j.
P
P
2
2
1=2
wR2 ¼ ½ ½wðF2o ꢁ Fc2Þ ꢅ= ½wðF2o Þ ꢅꢅ
.
c
a
Measured in dichloromethane for 1, 3 and in acetonitrile for 2, 4 and 5 with
Table 2
0.1 M tetrabutylammonium perchlorate (TBAP).
Selected bond distances (Å) and bond angles (°) at the manganese centre for
[Mn(Phimp)2] (1) and [Mn(Phimp)2](ClO4) (2). In complex 1 the molecule lies on a
twofold axis of symmetry. In complexes 1 and 2 the complete molecular structure
were available after growing.
b
Data from cyclic voltammetric measurements; E1/2 is calculated as average of
anodic (Epa) and cathodic (Epc) peak potentials E1/2 = 1/2(Epa + Epc).
c
D
Ep = Epa ꢁ Epc at scan rate 0.1 V/s.
[Mn(Phimp)2] (1)
[Mn(Phimp)2](ClO4) (2)
Bond distances (Å)
Mn–O1
Mn–N1
process attributed to the oxidation of [MnIIL2] to [MnIIIL2]+ (where
L = Phimpꢁ, N–Phimpꢁ, Me–Phimpꢁ) indicating Mn(II)/Mn(III)
redox couple. However, another redox wave detected at
E1/2 =0.80–1.06 V vs. Ag/AgCl is described as Mn(III)/Mn(IV) redox
couple. These data match with the values reported [20,58,64] for
the manganese complexes with similar ligand frame. E1/2 values
for Mn(II)/Mn(III) and Mn(III)/Mn(IV) couples in 5 are 0.143 V
and 0.802 V respectively. These data for 5 are comparatively smal-
ler than that other complexes (Table 3) which is probably due to
electron donating (–Me) group attached to the ligand [45].
Superoxide dismutation is a redox process and hence electro-
chemical properties of manganese complexes are very important
for SOD activity studies. Several reports [20,60,65–71] are there
by which one can select a manganese complex that could mimic
SOD activity considering the electrochemical properties. Our elec-
trochemical data showed the Mn(II)/Mn(III) couple, responsible for
the catalytic activity, was within the proper range [60,69]. These
prompted us to investigate the SOD activity of the complexes.
IC50 value for SOD activity has been defined as the concentration
of a particular tested compound for 50% inhibition of NBT reduc-
tion by superoxide produced in the xanthine/xanthine oxidase sys-
tem. The IC50 data of the SOD activity assay are described in Table 4
and inhibition curve for complex 1 is shown in Fig. 6 (curves for
complexes 2, 4 and 5 are shown in Supporting Information
(Fig. S22)). According to our data complex 2 shows the lowest
2.0405(18)
2.255(2)
2.2985(17)
Mn1–O1
Mn1–N2
Mn1–N1
1.879(3)
2.099(3)
2.176(3)
Mn–N2
Bond angles (°)
O1–Mn–O1
O1–Mn–N2
O1–Mn–N2
N2–Mn–N2
N1–Mn–N2
N1–Mn–N2
N1–Mn–N1
O1–Mn–N1
O1–Mn–N1
98.08(13)
81.31(6)
116.53(6)
153.94(9)
91.45(7)
70.08(6)
91.21(11)
92.33(8)
151.24(6)
O1–Mn1–O1
O1–Mn1–N1
O1–Mn1–N1
N1–Mn1–N1
N2–Mn1–N1
N2–Mn1–N1
N2–Mn1–N2
O1–Mn1–N2
O1–Mn1–N2
95.8(3)
87.23(11)
101.84(13)
166.57(16)
74.96(12)
85.74(18)
95.05(12)
91.89(15)
161.72(12)
complexes are described in Table 3. The neutral uncomplexed
ligands do not show any cyclic voltammogram over the range from
ꢁ1.0 to 1.4 V; hence all the curves were attributed to the redox
activity of our complexes. The shapes of the cyclic voltammograms
for all the complexes are similar with two redox peaks. For com-
plex 1 both the peaks are quasi-reversible whereas complex 2
showed reversible peaks. For complexes 3 and 4 both the redox
peaks are approximately reversible, however, both redox couples
are reversible in 5. From the data (Table 3), it has been shown that
one electron is involved in this redox process. The wave detected at
0.14–0.40 V vs. Ag/AgCl has been considered as one-electron-redox

16
K. Ghosh et al. / Journal of Inorganic Biochemistry 104 (2010) 9–18
Table 4
IC50 values and kinetic catalytic constant of 1, 2, 4 and 5 complexes.
kMcCF (Mꢁ1 sꢁ1
)
ꢄ 107
a
b
Complex
IC50
(lM)
1
2
4
5
0.29
0.39
1.12
0.76
4.09
3.04
1.06
1.56
a
IC50 values are the average of two replicate experiments. kMcCF were calculated
by K = kNBT ꢄ [NBT]/IC50, kNBT (pH 7.8) = 5.94 ꢄ 104 Mꢁ1 sꢁ1 [73,74].
Fig. 6. SOD activity of complex 1 (in DMF) in the xanthine oxidase–nitro blue
tetrazolium (NBT) assay.
IC50 value, on the other hand complex 4 shows the highest IC50
value. Comparing the IC50 data for all Mn(III) complexes, 2 showed
the lowest and 4 showed the highest IC50 value. Complexes 1 and 2
utilized the same Mn(II)/Mn(III) redox couple to destroy superox-
ide ions however, complex 1 (for its +2 oxidation state) converted
Oꢀ2ꢁ to O22ꢁ whereas other Mn(III) complexes converted O2ꢀꢁ to O2
using same redox couple. Due to the instability of 3 in air we were
unable to examine its activity. The IC50 value of complex 5 is con-
sistent with the data reported by Kitajima et al. [68], Xiang et al.
[69] and Cisnetti et al. [20]. Complex 4 gave rise to IC50 value of
Fig. 7. (a) Absorption spectra of [Mn(Phimp)2](ClO4), 2 in 0.1 mM phosphate buffer
(pH 7.2) in presence of increasing amounts of DNA ([DNA] = 0–68 M). (b) Plot of
[DNA]/(eA F) vs. [DNA].
l
ꢁ
e
1.12
[70], Lin et al. [60], Deroche et al. [71], Ouyang et al. [67] and Pol-
icar et al. [65]. However the IC50 values (0.29 M and 0.39
lM which is better than the data reported by Faulkner et al.
l
lM
The binding strength of complex 2 with CT-DNA could be ex-
pressed in terms of intrinsic binding constant Kb, which represents
the binding constant per DNA base pair. This constant could be
obtained by monitoring the changes in the absorbance at a kmax
with increase in concentration of CT-DNA by following the
equation [30].
respectively) for complexes 1 and 2 are not only the best among
the new family of manganese complexes we report here but also
among the very good SOD mimics known in the literature [66,72].
3.5. DNA-binding studies
½DNAꢅ=ðeA
ꢁ
eFÞ ¼ ½DNAꢅ=ðeB
ꢁ
eFÞ þ 1=KbðeB
ꢁ
e
9mÞ
To examine the nuclease activity of this family of complexes we
extended our investigation by starting DNA interaction with these
small molecule SOD mimics [12]. Among all the complexes, 2 was
selected for DNA interaction studies due to its solubility in biolog-
ical buffer solution. Other complexes were not soluble in the con-
dition set for the DNA-binding studies. In order to understand the
DNA binding properties of 2, we followed two techniques namely
UV–visible spectral studies and EB fluorescence quenching studies.
The experimental results are shown in Figs. 7 and 8 respectively.
The intensity of the peak at 307 nm in UV–visible spectra got
decreased due to the addition of CT-DNA. A considerable hypo-
chromicity of 4.41% without change in wavelength implies that
some interaction occurred between complex 2 and the surface of
DNA molecule and the interaction is not an intercalation [75,76].
where [DNA] is the concentration of DNA in base pairs and eA, eF and
eB correspond to the ratio of Aabs/[complex], the molar absorptivity
for the free manganese complex, and the molar absorptivity for the
manganese complex in the fully bound form respectively. In the
plot of [DNA]/(eA
ꢁ
eF) vs. [DNA], Kb is given by the ratio of the slope
to the Y-intercept. The binding constant Kb was found to be
7.87 ꢄ 104 Mꢁ1 which is lower than the values reported for the
intercalation of manganese complexes [31,77]. The examinations
of fluorescence spectral studies were carried out to further exclude
the possibility of intercalative binding mode. EB is an intercalator
that gives a significant increase in fluorescence emission when
bound to DNA, and its displacement from DNA results in a decrease
in fluorescence intensity. Addition of 2 to EB-DNA system shows

K. Ghosh et al. / Journal of Inorganic Biochemistry 104 (2010) 9–18
17
complex with CT-DNA. We further extended our DNA interaction
studies by examining the nuclease activity of the complexes. The
cleavage of supercoiled pBR322 DNA by the complexes were
studied by gel electrophoresis in tris buffer. We performed the
nuclease activity in 10% DMF for complexes 1, 2 and 4. The
strand scissions of plasmid pBR322 were assayed in the presence
of H2O2. Examining the data obtained from DNA gel electropho-
resis, it is clear that in presence of H2O2 the complexes can
cleave plasmid pBR322 DNA to a mixture of supercoiled and
nicked DNA (shown in Fig. 9). In search of the mechanism of
nuclease activity of this family of complexes we investigated
the activity in presence of NaN3 and KI which are known to be
singlet oxygen and hydroxyl radical scavengers respectively
[76]. No change in nuclease activity in presence of NaN3 and
inhibition of activity in presence of KI were observed. These data
indicate that possible role of reactive oxygen species (ROS) is
involved in nuclease activity.
4. Conclusions
Fig. 8. Fluorescence emission spectra of the EB-CT-DNA ([DNA] = 25
lM) system in
absence (dashed line) and presence (solid line) of the complex 2 (0–7
l
M).
In this study we prepared tridentate PhimpH, N–PhimpH and
Me–PhimpH ligands and characterized the ligands with UV–visi-
ble, IR, GC-MS and NMR studies. These ligands after deprotonation
were reacted with Mn(II) and Mn(III) starting materials and in all
synthetic attempts we ended up with bis complexes. Both the
Mn(II) and Mn(III) centre were stabilized by the ligand PhimpH
and N–PhimpH whereas only Mn(III) complex was isolated and
characterized for ligand Me–PhimpH. UV–visible and IR spectral
studies, magnetic moment data and conductivity measurements
confirm the formation of [Mn(Phimp)2] (1), [Mn(Phimp)2](ClO4)
(2), [Mn(N–Phimp)2] (3), [Mn(N–Phimp)2](ClO4) (4), [Mn(Me–
Phimp)2](ClO4) (5). X-ray crystal structures of complexes 1 and 2
revealed the formation of manganese(II) and manganese(III) com-
plexes respectively and for both complexes ligand was bound to
the metal centre in meridional fashion. Bond distances support
the stabilization of high-spin manganese(II) in complex 1 and man-
ganese(III) in complex 2. Investigation of electrochemical studies
showed Mn(II)/Mn(III) and Mn(III)/Mn(IV) couples. Our complexes
satisfy the properties required for a small molecule SOD mimic.
Among them, complexes 1 and 2 with IC50 values 0.29 and
0.39 lM respectively provide a novel example of low molecular
Fig. 9. Gel electrophoresis separations showing the oxidative cleavage of super-
coiled pBR322 DNA (200 ng) by complexes (100 M) (A), (B) and (C)
respectively. Complexes 1, 2 and 4 in 10% DMF incubated at 37 °C for 1.5 h. (A) DNA
(Lane 1); DNA + H2O2 (150 M) (Lane 2); DNA + 1 (Lane 3); DNA + 1 + H2O2
(150 M) (Lane 4); DNA + 1 + H2O2 (300 M) (Lane 5); DNA + 1 + H2O2 (400 M)
(Lane 6); DNA + 1 + H2O2 (150 M) + KI (300 M) (Lane 7). (B) DNA (Lane 1);
DNA + DMF (2 L) (Lane 2); DNA + H2O2 (150 M) (Lane 3); DNA + 2 (Lane 4);
DNA + 2 + H2O2 (150 M) (Lane 5); DNA + 2 + H2O2 (400 M) (Lane 6);
DNA + 2 + H2O2 (150 M) (Lane 7). (C) DNA (Lane 1); DNA + H2O2
(150 M) (Lane 4);
DNA + 4 + H2O2 (300 M) (Lane 6);
DNA + 4 + H2O2 (150
weight SOD mimics on the basis of activity relationship. DNA-bind-
ing studies of complex 2 indicated a non-intercalative interaction
and a probable groove binding or external binding with CT-DNA
has been suggested. The phenyl ring, which is approximately per-
pendicular to the ligand binding plane, probably inhibits the inter-
calation with DNA. Another major finding was that complexes 1, 2
and 4 exhibited nuclease activity. Hence our small molecule SOD
mimics set another example to support the data reported in the lit-
erature [12]. Inhibition of the nuclease activity in presence of KI
indicated the probable participation of reactive oxygen species in
DNA cleavage.
l
1
2
4
l
l
l
l
l
l
l
l
l
l
l
M) + KI (300 l
l
M) (Lane 2); DNA + 4 (Lane 3); DNA + 4 + H2O2 (150
M) (Lane 5); DNA + 4 + H2O2 (400
M) + KI (300 M) (Lane 7).
l
l
l
l
l
very little change in fluorescence (shown in Fig. 8). The lower Kb va-
lue and small change in fluorescence spectral studies indicate the
non-intercalative binding interaction with DNA and probable
groove binding or external binding is suggested [75,76]. Structure
solution for 2 also revealed that the phenyl ring attached to the
nitrogen is roughly perpendicular (87.84o) to the ligand binding
plane having phenolato ring and pyridine ring. This phenyl ring
probably inhibits the intercalation with DNA.
Acknowledgements
KG is thankful to DST, New Delhi, India for SERC FAST Track pro-
ject support. NT and PK are thankful to CSIR, India for financial
assistance. We are thankful to IITR for Faculty Initiation Grant
and instrumentation facilities. UPS is thankful to Head, Instrumen-
tation Centre, IIT Roorkee for single crystal X-ray facility.
3.6. Nuclease activity
Appendix A. Supplementary material
The absorption and fluorescence quenching studies with com-
plex 2 indicated non-intercalative interaction of the manganese
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jinorgbio.2009.09.014.

18
K. Ghosh et al. / Journal of Inorganic Biochemistry 104 (2010) 9–18
[41] B. Klaus, University of Bonn, Germany DIAMOND, Version 1.2c, 1999.
References
[42] C. Beauchmp, I. Fridovich, Anal. Biochem. 44 (1971) 276–287 (and references
therein).
[43] J. Marmur, J. Mol. Biol. 3 (1961) 208–218.
[44] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954)
3047–3053.
[45] M. Maneiro, M.R. Bermejo, M.I. Fernández, E. Gómez-Fórneas, A.M. González-
Noya, A.M. Tyryshkin, New J. Chem. 27 (2003) 727–733.
[46] O. Pouralimardan, A.-C. Chamayou, C. Janiak, H. Hosseini-Monfared, Inorg.
Chim. Acta 360 (2007) 1599–1608.
[47] M.R. Rosenthal, J. Chem. Educ. 50 (1973) 331–335.
[48] M. Maneiro, M.R. Bermejo, A. Sousa, M. Fondo, A.M. González, A. Sousa-
Pedrares, C.A. McAuliffe, Polyhedron 19 (2000) 47–54.
[49] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81–122.
[50] O.L. Hoyos, M.R. Bermejo, M. Fondo, A. García-Deibe, A.M. González, M.
Maneiro, R. Pedrido, J. Chem. Soc. Dalton Trans. (2000) 3122–3127.
[51] S. Biswas, K. Mitra, S.K. Chattopadhyay, B. Adhikary, Transition Met. Chem. 30
(2005) 393–398.
[52] J.A. Bonadies, M.J. Maroney, V.L. Pecoraro, Inorg. Chem. 28 (1989) 2044–2051.
[53] A. Neves, S.M.D. Erthal, I. Vencato, A.S. Ceccato, Y.P. Mascarenhas, O.R.
Nascimento, M. Horner, A.A. Batista, Inorg. Chem. 31 (1992) 4749–4755.
[54] M.E.D. Vries, R.M.L. Crois, G. Roelfes, H. Kooijman, A.L. Spek, R. Hage, B.L.
Feringa, Chem. Commun. (1997) 1549–1550.
[55] J. Limburg, J.S. Vrettos, R.H. Crabtree, G.W. Brudivg, J.C.D. Paula, A. Hassan, A.-L.
Barra, C. Duboc-Toia, M.-N. Collomb, Inorg. Chem. 40 (2001) 1698–1703.
[56] O. Horner, J.-J. Girerd, C. Philouze, L. Tchertanov, Inorg. Chim. Acta 290 (1999)
139–144.
[57] S. Gou, X. You, K. Yu, J. Lu, Inorg. Chem. 32 (1993) 1883–1887.
[58] L. Sabater, C. Hureau, R. Guillot, A. Aukauloo, Inorg. Chem. 45 (2006) 2373–
2375.
[59] S. Naskar, D. Mishra, S.K. Chattopadhyay, M. Corbella, A.J. Blake, Dalton Trans.
(2005) 2428–2435.
[60] J. Lin, C. Tu, H. Lin, P. Jiang, J. Ding, Z. Guo, Inorg. Chem. Commun. 6 (2003)
262–265.
[61] J.-P. Costes, F. Dahan, B. Donnadieu, M.-J.R. Douton, M.-I.F. Garcia, A.
Bousseksou, J.-P. Tuchagues, Inorg. Chem. 43 (2004) 2736–2744.
[62] A. Panja, N. Shaikh, S. Gupta, R.J. Butcher, P. Banerjee, Eur. J. Inorg. Chem.
(2003) 1540–1547.
[1] D.W. Christianson, Prog. Biophys. Molec. Biol. 67 (1997) 217–252.
[2] J.J.R. Frausto da Silva, R.J.P. Williams, The Biological Chemistry of the Elements,
Clarendon Press, Oxford, 1993. p. 4.
[3] E.-J. Woo, J.M. Dunwell, P.W. Goodenough, A.C. Marvier, R.W. Pickersgill, Nat.
Struct. Biol. 7 (2000) 1036–1040.
[4] C. Su, E.H. Oliw, J. Biol. Chem. 273 (1998) 13072–13079.
[5] T.L. Stemmler, B.E. Sturgeon, D.W. Randall, R.D. Britt, J.E. Penner-Hahn, J. Am.
Chem. Soc. 119 (1997) 9215–9225.
[6] V.L. Pecoraro (Ed.), Manganese Redox Enzymes, VCH, New York, 1992.
[7] S. Mukhopadhyay, S.K. Mandal, S. Bhaduri, W.H. Armstrong, Chem. Rev. 104
(2004) 3981–4026.
[8] G. Yin, A.M. Danby, D. Kitko, J.D. Carter, W.M. Scheper, D.H. Busch, Inorg. Chem.
46 (2007) 2173–2180 (and references therein).
[9] K. Ghosh, A.A. Eroy-Reveles, B. Avila, T.R. Holman, M.M. Olmstead, P.K.
Mascharak, Inorg. Chem. 43 (2004) 2988–2997.
[10] L. Quintanar, Inorg. Chim. Acta 361 (2008) 875–884.
[11] W. Jiang, Y. Han, Q. Pan, T. Shen, C. Liu, J. Inorg. Biochem. 101 (2007) 667–677.
[12] M. Devereux, D.O. Shea, A. Kellett, M. McCann, M. Walsh, D. Egan, C. Deegan, K.
Kedziora, G. Rosair, H. Muller-Bunz, J. Inorg. Biochem. 101 (2007) 881–892.
[13] D. Salvemini, C. Muscoli, D.P. Riley, S. Cuzzocrea, Pulm. Pharmacol. Therapeut.
15 (2002) 439–447.
[14] D. Salvemini, Z.-Q. Wang, J.L. Zweier, A. Samouilov, H. Macarthur, T.P. Misko,
M.G. Currie, S. Cuzzocrea, J.A. Sikorski, D.P. Riley, Science 286 (1999) 304–306.
[15] I. Bertini, S.J. Gray, S.J. Lippard, J.S. Valentine, Bioinorganic Chemistry,
University Science Book, Mill Valley, California, 1994.
[16] D.P. Barondeau, C.J. Kassmann, C.K. Bruns, J.A. Tainer, E.D. Getzoff,
Biochemistry 43 (2004) 8038–8047.
[17] D.P. Reley, Chem. Rev. 99 (1999) 2573–2588.
´
´
[18] I. Batinic-Haberle, I. Spasojevic, R.D. Stevens, P. Hambright, P. Neta, A. Okado-
Matsumoto, I. Fridovich, Dalton Trans. (2004) 1696–1702.
[19] F. Fucassi, J.E. Lowe, K.D. Pavey, S. Shah, R.G.A. Faragher, M.H.L. Green, F. Paul,
D.Ó. Hare, P.J. Cragg, J. Inorg. Biochem. 101 (2007) 225–232.
[20] F. Cisnetti, G. Pelosi, C. Policar, Inorg. Chim. Acta 360 (2007) 557–562.
[21] S. Durot, C. Policar, F. Cisnetti, F. Lambert, J.-P. Renault, G. Pelosi, G. Blain, H.
Korri-Youssoufi, J.-P. Mahy, Eur. J. Inorg. Chem. (2005) 3513–3523.
[22] N. Farrell, Coord. Chem. Rev. 232 (2002) 1–4.
[23] H. Ali, J.E.V. Lier, Chem. Rev. 99 (1999) 2379–2450.
[24] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451–2466.
[25] S. Mahadevan, M. Palaniandavar, Inorg. Chem. 37 (1998) 693–700.
[26] M.J. Clarke, Coord. Chem. Rev. 232 (2002) 69–93.
[27] Y.K. Yan, M. Melchart, A. Habtemariam, P.J. Sadler, Chem. Commun. (2005)
4764–4776.
[28] D.J. Gravert, J.H. Griffin, Inorg. Chem. 35 (1996) 4837–4847 (and references
therein).
[29] S.S. Mandal, N.V. Kumar, U. Varshney, S. Bhattacharya, J. Inorg. Biochem. 63
(1996) 265–272.
[30] A. Dimitrakopoulou, C. Dendrinou-Samara, A.A. Pantazaki, M. Alexiou, E.
Nordlander, D.P. Kessissoglou, J. Inorg. Biochem. 102 (2008) 618–628 (and
references therein).
[31] Z.-D. Xu, H. Liu, M. Wang, S.-L. Xiao, M. Yang, X.-H. Bu, J. Inorg. Biochem. 92
(2002) 149–155.
[32] S.-S. Zhang, S.-Y. Niu, B. Qu, G.-F. Jie, H. Xu, C.-F. Ding, J. Inorg. Biochem. 99
(2005) 2340–2347.
[33] S. Niu, M. Zhao, R. Ren, S. Zhang, J. Inorg. Biochem. 103 (2009) 43–49.
[34] T. Jun, W. Bochu, Z. Liancai, Colloids Surf. B 55 (2007) 149–152.
[35] S.C.M. Gandini, V.E. Yushmanov, J.R. Perussi, M. Tabak, I.E. Borissevitch, J. Inorg.
Biochem. 73 (1999) 35–40.
[36] W.L.F. Armaergo, C.L.L. Chai, Purification of Laboratory Chemicals, fifth ed.,
Butter Heinemann, Elsevier Science, USA, 2002. pp. 85–86.
[37] R.L. Dutta, A. Syamal, Elements of Magnetochemistry, second ed., Affiliated
East-West Press, New Delhi, 1993. p. 8.
[38] A. Kotschy, J. Faragó, A. Csámpai, D.M. Smith, Tetrahedron 60 (2004) 3421–
3425.
G.R. Dasiraju, T. Steiner, The weak hydrogen bond, in: J. John (Ed.), Structural
[63]
Chemistry and Biology, Oxford University Press, New York, 1999.
[64] M.U. Triller, D. Pursche, W.-Y. Hsieh, V.L. Pecoraro, A. Rompel, B. Krebs, Inorg.
Chem. 42 (2003) 6274–6283.
[65] C. Policar, S. Durot, F. Lambert, M. Cesario, F. Ramiandrasoa, I. Morgenstern-
Badarau, Eur. J. Inorg. Chem. (2001) 1807–1818.
[66] R. Kachadourian, I. Batinic´-Haberle, I. Fridovich, Inorg. Chem. 38 (1999) 391–
396.
[67] X.-M. Ouyang, B.-L. Fei, T.-A. Okamura, W.-Y. Sun, W.-X. Tang, N. Ueyama,
Chem. Lett. 3 (2002) 362–363.
[68] N. Kitajima, M. Osawa, N. Tamura, Y. Moro-oka, T. Hirano, M. Hirobe, T.
Nagano, Inorg. Chem. 32 (1993) 1879–1880.
[69] D.F. Xiang, C.Y. Duan, X.S. Tan, Q.W. Hang, W.X. Tang, J. Chem. Soc. Dalton
Trans. (1998) 1201–1204.
[70] K.M. Faulkner, R.D. Stevens, I. Fridovich, Arch. Biochem. Biophys. 310 (1994)
341–346.
[71] A. Deroche, I. Morgenstern-Badarau, M. Cesario, J. Guilhem, B. Keita, L. Nadjo,
C. Houe-Levin, J. Am. Chem. Soc. 118 (1996) 4567–4573.
[72] I. Batinic-Haberle, I. Spasojevic, P. Hambright, L. Benov, A.L. Crumbliss, I.
Fridovich, Inorg. Chem. 38 (1999) 4011–4022.
[73] R.F. Pasternack, B. Halliwell, J. Am. Chem. Soc. 101 (1979) 1026–1031.
[74] Z.-R. Liao, X.-F. Zheng, B.-S. Luo, L.-R. Shen, D.-F. Li, H.-L. Liu, W. Zhao,
Polyhedron 20 (2001) 2813–2821.
[75] S. Sarkar, A. Mondal, D. Chopra, J. Ribas, K.K. Rajak, Eur. J. Inorg. Chem. (2006)
3510–3516.
[76] J. Chen, X. Wang, Y. Shao, J. Zhu, Y. Zhu, Y. Li, Q. Xu, Z. Guo, Inorg. Chem. 46
(2007) 3306–3312.
[77] Z.-D. Xu, H. Liu, S.-L. Xiao, M. Yang, X.-H. Bu, J. Inorg. Biochem. 90 (2002) 79–
84.
[39] G.M. Sheldrick, Acta Cryst. A 46 (1990) 467–473.
[40] G.M. Sheldrick, SHELXTL-NT 2000 Version 6.12, Reference Manual. University
of Gottingen, Pergamon, New York, 1980.