Synthesis, structure, redox properties and DNA interaction studies on mononuclear iron(III) complexes with amidate ligand

Article abstract of DOI:10.1016/j.ica.2013.11.034

A new family of mononuclear Fe(III) complexes [Fe(Pamp)(MeOH)Cl ₂], 1 and [Fe(Pamp)₂](ClO₄), 2 were synthesized using designed tridentate ligand PampH having pyridine and amide nitrogen donors (PampH is N′-phenyl-N′-(pyridin-2-yl)picolinohydrazide) and H stands for dissociable proton). Both the complexes (1 and 2) were characterized by different spectroscopic studies and molecular structure of [Fe(Pamp) ₂](ClO₄), 2 was determined by single crystal X-ray diffraction. Geometry around metal centre was described as distorted octahedral with two meridionally oriented Pamp^- ligands. Electrochemical studies afforded E_1/2 values of Fe(III)/Fe(II) couple +0.065 V (for 1) and -0.077 V (for 2) versus Ag/AgCl electrode. DNA binding properties of these complexes were investigated and complex 1 exhibited nuclease activity. Mechanistic investigation revealed the possible participation of hydroxyl radical in nuclease activity which was supported by rhodamine B assay.

Full text of DOI:10.1016/j.ica.2013.11.034

Inorganica Chimica Acta 412 (2014) 20–26
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, structure, redox properties and DNA interaction studies
on mononuclear iron(III) complexes with amidate ligand
⇑
Kaushik Ghosh , Nidhi Tyagi, Pramod Kumar, Udai P. Singh
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2013
Received in revised form 22 November 2013
Accepted 26 November 2013
Available online 10 December 2013
A new family of mononuclear Fe(III) complexes [Fe(Pamp)(MeOH)Cl2], 1 and [Fe(Pamp)2](ClO4), 2 were
synthesized using designed tridentate ligand PampH having pyridine and amide nitrogen donors (PampH
is N0-phenyl-N0-(pyridin-2-yl)picolinohydrazide) and
H stands for dissociable proton). Both the
complexes (1 and 2) were characterized by different spectroscopic studies and molecular structure of
[Fe(Pamp)2](ClO4), 2 was determined by single crystal X-ray diffraction. Geometry around metal centre
was described as distorted octahedral with two meridionally oriented Pampꢀ ligands. Electrochemical
studies afforded E1/2 values of Fe(III)/Fe(II) couple +0.065 V (for 1) and ꢀ0.077 V (for 2) versus Ag/AgCl
electrode. DNA binding properties of these complexes were investigated and complex 1 exhibited
nuclease activity. Mechanistic investigation revealed the possible participation of hydroxyl radical in
nuclease activity which was supported by rhodamine B assay.
Keywords:
Amidate ligand
Iron complexes
Crystal structure
DNA interaction
Nuclease activity
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
abilities [33]. Coordination chemistry of iron-amidate group partic-
ipate important role in biomolecules such as anti-tumor drug ble-
There has been considerable current interest for the synthesis of
metal based synthetic nuclease because of their applications in dis-
covery of artificial DNA cleaving agents, DNA foot-printing and in
medicinal chemistry [1–6]. Cis-platin and Fe-bleomycin are the
established metal based anticancer drugs [7–9]. Cis-platin is hav-
ing lot many side effects and researchers are interested to discover
metal based anti cancer agents with biologically benign metal
rather than toxic metal like platinum [10]. Among first row transi-
tion elements iron exhibit flexible range of coordination geome-
tries, oxidation states, spin states and redox potential and hence,
iron became one of the suitable metals for the synthesis of artificial
nuclease [11–13]. Several iron complexes were reported as artifi-
cial nucleases, however, most of them are multinuclear [3,14–23]
and very few of them are mononuclear [24–26]. Among mononu-
clear complexes most of them were reported with salen and salen
related ligands [27–29]. On the other hand, Roelfes et al. and Muk-
herjee et al. reported synthetic DNA cleavage agent of mononuclear
iron complex in which ligand was covalently attached to a DNA-
binder arm or intercalator as imidazopyridine ring and 9-aminoac-
ridine (also acts as photosensitizer) respectively [30–31]. Wong
et al. also reported nuclease activity of mononuclear iron
complexes with tripodal ligands [32]. It is now well known in
the literature that the ligation of deprotonated amidate nitrogen(s)
omycin [34] and enzyme nitrile hydratase [35–36]. Fe(III)
complexes derived from such ligand(s) have been exploited now
as DNA cleaving agents [37]. Mascharak and co-workers reported
mononuclear iron complex mimicking the iron binding site of bel-
omycine (by designing the ligand having amidate group) however
DNA binding and nuclease activity of these complexes were not
reported [38].
Recently, we have communicated the role of carboxamido
nitrogen in superoxide scavenging activity and nuclease activity
[39–40]. Herein, we report the synthesis and characterization of
two mononuclear iron complexes [Fe(Pamp)(MeOH)Cl2], 1 and
[Fe(Pamp)2](ClO4), 2, derived from the amidate ligand PampH
(shown in Scheme 1). These complexes were characterized by
spectroscopic and electrochemical studies. Molecular structure of
complex 2 was determined by X-ray crystallography. DNA interac-
tion and nuclease activity were examined. We also investigated the
mechanism of nuclease activity.
2. Experimental
2.1. Materials
All the solvents used were reagent grade. Analytical grade
reagents picolinic acid (Wilson Laboratories, Mumbai, India), dicy-
clohexylcarbodiimide (SRL, Mumbai, India), 1-hydroxybenzotriazol
(Himedia Laboratories Pvt. Ltd., Mumbai, India), phenylhydrazine,
(S. D. Fine, Mumbai, India) and 2-chloropyridine (Acros organics,
stabilizes the iron(III) centres due to their strong
r-donating
⇑
Corresponding author. Tel.: +91 1332285547; fax: +91 1332 273560.
E-mail address: ghoshfcy@iitr.ernet.in (K. Ghosh).
0020-1693/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2013.11.034

K. Ghosh et al. / Inorganica Chimica Acta 412 (2014) 20–26
21
N
O
N
H
N
1:1
C
N
2:1
[Fe(Pamp)(MeOH)Cl2]
[Fe(Pamp)2](ClO4)
anhydrous FeCl3
Fe(ClO4)3.xH2O
1
2
PampH
Scheme 1. Synthetic scheme of complex 1 and 2.
USA) were used as obtained. Anhydrous FeCl3 was purchased from
Rankem, Delhi, India and Fe(ClO4)3ꢁxH2O, Fe(ClO4)2ꢁxH2O, rhoda-
mine B dye and 1,3-diphenylisobenzofuran (DPBF) were purchased
from Sigma Aldrich, Steinheim, Germany. Solvent used for
spectroscopic studies were HPLC grade and purified by standard
procedure before use [41]. Supercoiled pBR322 DNA and CT DNA
were purchased from Bangalore Genei (India). Agarose was
purchased from (Himedia Laboratories Pvt. Ltd., Mumbai, India).
Tris(hydroxymethyl)aminomethane–HCl (Tris–HCl) buffer was
prepared in deionised water.
brownish-green solid was separated out. This green solid was
filtered and washed with methanol and diethylether. Yield:
45 mg, (51%).
2.3. Physical measurements
Elemental analyses were carried microanalytically at Elemenlar
Vario EL III. IR spectra were obtained as KBr pellets with Thermo
Nikolet Nexus FT-IR spectrometer, using 16 scans and were
reported in cmꢀ1. Electronic absorption spectra were recorded
with an Evolution 600, Thermo Scientific UV–Vis spectrophotome-
ter. Emission quenching titrations were carried out on Varian fluo-
rescence spectrophotometer. Circular dichroism (CD) spectra of
complexes (1 and 2) were recorded on Chirascan circular dichro-
ism spectrometer, Applied photophysics, UK. Magnetic susceptibil-
ities were determined at 296 K with Vibrating Sample
Magnetometer model 155, using nickel as a standard. Diamagnetic
corrections were carried out with Pascal’s increments [42]. Molar
conductivities were determined in dimethylformamide (DMF) at
10ꢀ3 M at 25 °C with a Systronics 304 conductometer. Cyclic vol-
tammetry measurements were carried out using a CH-600 electro-
analyzer. A conventional three-electrode arrangement was using
consisting a platinum wire as auxiliary electrode, glassy carbon
as working electrode and the Ag(s)/AgCl as reference electrode.
These measurements were performed in the presence of 0.1 M tet-
rabutylammonium perchlorate (TBAP) as the supporting electro-
lyte, using complex concentration 10ꢀ3 M in dichloromethane
and acetonitrile. The ferrocene/ferrocenium couple occurs at E1/
2 = +0.40 (75) V versus Ag/AgCl under the same experimental con-
ditions. All experiments were performed at room temperature and
solutions were thoroughly degassed with nitrogen prior to begin-
ning the experiments.
Caution! Perchlorate salts of metal complexes with organic
ligands are potentially explosive. Only small amount of material
should be prepared and handled with caution.
2.2. Synthesis of complexes
2.2.1. [Fe(Pamp)(MeOH)Cl2], 1
A batch of (34 mg, 0.27 mmol) anhydrous FeCl3 in 5 mL of
methanol was added dropwise to stirred solution of ligand PampH
(79.7 mg, 0.27 mmol) in 15 mL of methanol. The colour of solution
was changed to dark green. The green solution was filtered to re-
move dirty suspension. Filtrate was again stirred for 3 h and the
solution was kept in freeze for slow evaporation. Green colour
semi-crystalline precipitate was obtained which was filtered and
washed with small amount of methanol and diethylether. Though,
complex 1 is precipitated as nice crystalline solid but all attempts
to get single crystals were unsuccessful. Yield: 52.2 mg, (42%). Se-
lected IR data (KBr,
kmax/nm (
/Mꢀ1cmꢀ1)]: 626 (1450), 321 (9750), 248 (15,000). leff
(296 K): 5.19 BM. KM
ꢀ1 cm2 molꢀ1 (in DMF): 12. Anal. Calc. for
18H17N4O2Cl2Fe: C, 48.25; H, 3.82; N, 12.50. Found: C, 49.01; H,
3.67; N, 12.53%.
m m-C@O. UV–Vis [CH2Cl2,
max/cmꢀ1): 1658, 1603
e
/X
C
2.2.2. [Fe(Pamp)2](ClO4)
2.4. X-ray crystal structure determination of complex 2
2.2.2.1. Method A. A batch of (50 mg, 0.14 mmol) Fe(ClO4)3ꢁxH2O in
5 mL of methanol was added dropwise, to the stirred solution of
(80.7 mg, 0.28 mmol) ligand (PampH) in 10 mL of methanol. The
colour of solution changed to red and then to brown. After 5 min,
it turns to brownish-green solution which was stirred for 4 h. Dark
green solid was separated out which was filtered and washed with
methanol and small amount of diethylether. Single crystals of the
complex for X-ray crystallography were obtained within a week
on slow diffusion of acetonitrile/ethylacetate-diethyl ether mix-
Crystal and refinement data is given in Table 1. The X-ray data
collection and processing for complex 2 was performed on Bruker
Kappa Apex-II CCD diffractometer by using graphite monochro-
mated Mo Ka radiation (k = 0.71070 Å) at 296 K. Crystal structure
was solved by direct methods. Structure solution, refinement and
data output was carried out with the SHELXTL program [43–44].
All non-hydrogen atoms were refined anisotropically. Hydrogen
atoms were placed in geometrically calculated positions and re-
fined using a riding model. Images were created with the DIA-
MOND program [45].
ture in freezer. Yield: 75.0 mg, (73%). Selected IR data (KBr, mmax
cmꢀ1): 1602,
-C@O, 1090, 622 mClO4ꢀ. UV–Vis [CH3CN, kmax/nm
/Mꢀ1 cmꢀ1)]: 801 (2000), 437 (3100), 315 (14,300), 251
(23,150). leff (296 K): 2.75 BM, KM
ꢀ1 cm2 molꢀ1 (in DMF): 58.
/
m
(e
/X
2.5. DNA binding experiments
Anal. Calc. for C34H26N8O6ClFe: C, 55.64; H, 3.57; N, 15.27. Found:
C, 55.60; H, 3.72; N, 15.23%.
Fluorescence quenching experiments were carried out by the
successive addition of complexes 1 and 2 to the DNA (25
lM)
2.2.2.2. Method B. A batch of (30 mg, 0.12 mmol) Fe(ClO4)2ꢁxH2O in
5 mL of methanol was added dropwise, to the stirred solution of
(72.5 mg, 0.25 mmol) ligand (PampH) in 10 mL of methanol. The
colour of solution was changed to purple and within 1 min
solutions containing 5 M ethidium bromide (EB) in 100 mM
l
phosphate buffer (pH 7.2). For better solubility of complex 1
and 2, we use 5% DMF. These samples were excited at 250 nm
and emissions were observed between 500 and 700 nm. Stern–Vol-

22
K. Ghosh et al. / Inorganica Chimica Acta 412 (2014) 20–26
Table 1
The degradation of the dye was monitored by the UV absorbance
of rhodamine B at 544 nm. The fluorescent probes DPBF were used
to detect the probable formation of 1O2 during the nuclease reac-
Summary of crystal data and data collection parameters for complex 2.
Empirical formula
C34H26N8O6ClFe
tion. Complexes 1 and 2 (0–100 lM) with DPBF (0.05 lM) in a
Formula weight (g molꢀ1
)
733.93
methanol solution were used to monitor the changes in the fluo-
rescence kex = 405 nm, kem = 479 nm.
T (K)
k (Å) (Mo K
Crystal system
Space group
a (Å)
296(2)
0.71073
monoclinic
P21/n
11.1466(8)
9.2634(7)
a
)
3. Results and discussion
b (Å)
c (Å)
30.935(2)
91.306(3)
3193.4(4)
3.1. Synthesis
b (°)
V (Å3)
The tridentate ligand PampH was synthesized and character-
ized by the reported procedure [39]. Ligand PampH when reacted
with FeCl3 in 1:1 equivalent ligand to metal ratio yielded green so-
lid of [Fe(Pamp)(MeOH)Cl2], 1. On the other hand, reaction of
PampH with Fe(ClO4)3ꢁxH2O or Fe(ClO4)2ꢁxH2O in 2:1 (ligand to
metal ratio) gave rise to [Fe(Pamp)2](ClO4), 2, in case of Fe(II) start-
ing material we ended up with complex 2 due to aerial oxidation.
Interestingly, during synthesis of 1 and 2 base was not needed for
the deprotonation of PampH, the Lewis acidity of Fe(III) centre was
efficient to deprotonate the ligand (PampH) [37]. The synthetic
procedures of complexes 1 and 2 were described in Scheme 1.
Z
4
qcalc (g cmꢀ3
)
1.527
Crystal size (mm)
F(000)
h Range for data collection
Index ranges
0.25 ꢂ 0.17 ꢂ 0.11
3280
1.32–26.32
ꢀ13 < h < 9, ꢀ9 < k < 11, ꢀ37 < l < 38
Refinement method
Data/restraints/parameters
full matrix least-squares on F2
6475/0/451
1.812
0.0722
0.0892
0.2554
0.2641
Goodness-of-fit (GOF) on F2a
b
R1 [I > 2
R1[all data]
wR2 [I > 2r(I)]
wR2 [all data]
r
(I)]
c
P
GOF = [ [w(Fo ꢀ Fc2)2]/M–N)]1/2 (M = number of reflections, N = number of
a
2
parameters refined).
3.2. Structure of complex 2
P
P
b
R1
=
||Fo| ꢀ |Fc||/ |Fo|.
P
P
wR2 = [ [w(Fo ꢀ Fc2)2]/ [w(Fo2)2]]1/2
.
c
2
The ORTEP diagram for cation of complex [Fe(Pamp)2]+, 2 is
shown in Fig. 1. Matrix parameters, selected bond distances and
bond angles are described in Table 1 and Table 2. Crystals of com-
plex 2 were obtained from diethyl ether diffusion to a solution of 2
in acetonitrile-ethyl acetate (1:1) mixture at room temperature
within a week. Complex 2 was crystallized in monoclinic space
group P21/n consisting of mononuclear iron(III) complex with six
nitrogen atoms from two Pampꢀ ligands in distorted octahedral
geometry. Complex 2 was isolated as the mer isomer with two car-
boxamido nitrogen (N2, N5) atoms occupying positions trans to
each other however, all pyridine nitrogen donors (N1, N3, N4 and
N6) were cis to each other. In complex 2, the Fe–Nam bond lengths
(1.884(4) Å and 1.890(4) Å) were consistent with reported values
for complexes [Fe(pmp)2]+ (1.887(4) and 1.890(4) Å) [49] and
[Fe(bpc)(1-MeIm)2]+ (1.886 (4) Å, bpc = 4,5-dichloro-1,2-bis(pyri-
dine-2-carboxamido)-benzene) [50] however, longer than in com-
plex [Fe(Pypep)2](ClO4) (1.957(2) and 1.958(2) Å) [38]. The average
Fe–Npy distance (1.958 Å) in 2 was shorter than reported low-spin
Fe(III) complexes 1.978 Å [37], 1.982 Å [38], 1.968 (3) Å [51] and
1.953(3) Å [52]. All the six Fe–N bonds have different bond lengths,
the average Fe–N distance follows the order Fe–Nam (1.89 Å) < Fe–
mer quenching constants were calculated using the given
equation,
I0=I ¼ 1 þ KsvQ;
Where I0 and I are the fluorescence intensities in the absence
and presence of complex and Q is the concentration of quencher
(complexes 1 and 2). Ksv is a linear Stern–Volmer constant and gi-
ven by the ratio of slope to intercept in the plot of I0/I versus Q [46].
Circular dichroism (CD) spectra of CT-DNA in absence and pres-
ence of the iron complexes were recorded with a 0.1 cm path-
length cuvette after 10 min incubation at 25 °C. The concentration
of the complexes and CT-DNA were 50 and 200 lM respectively.
2.6. Nuclease activity
DNA cleavage was measured by the conversion of supercoiled
pBR322 plasmid DNA to nicked circular and linear DNA forms.
Supercoiled pBR322 DNA in (TBE) Trisboric acid-EDTA buffer (pH
8.2) was treated with complexes 1 and 2 taken in dimethylform-
amide (10%) in the presence or absence of additives. The oxidative
DNA cleavage by the complexes were studies in the presence of
H2O2 (oxidizing agent) and BME (reducing agent). The samples
were incubated at 37 °C, added loading buffer (25% bromophenol
blue and 30% glycerol). The agarose gel (0.8%) containing 0.4 ng/
mL of ethidium bromide (EB) was prepared and the electrophoresis
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 plac-
ing the stained gel under an illuminated UV lamp. The fragments
were visualized by using a UV illuminator (BIO RAD).
NPy (1.95 Å). The appreciably shorter Fe–Nam bond was due to good
donating properties of the amide nitrogen as well as the normal
2.7. Detection of reactive oxygen species (ROS)
The generation of reactive oxygen species (ROS) in the cleavage
reaction was detected by dye degradation (rhodamine B) [47] or by
using sensitive radical sensor (DPBF) [48]. Hydroxyl radical forma-
Fig. 1. ORTEP diagram (50% probability level) of complex [Fe(Pamp)2]+, 2, hydrogen
tion was quantified by 5
lM of rhodamine B in the presence of
complexes 1–2 (0–300 M) in MeOH under aerobic conditions.
l
atoms were omitted for clarity.

K. Ghosh et al. / Inorganica Chimica Acta 412 (2014) 20–26
23
Table 2
the solid state at 296 K, suggesting a high-spin (S = 5/2) electronic
configuration of iron(III). The effective magnetic moment value for
complex 2 was 2.75 BM at 296 K supports the low-spin (S = ½) con-
figuration in complex 2 [38].
Selected bond lengths (Å) and angles (°) of complex 2.
Bond distances (Å)
Bond angles (°)
Fe1–N5
Fe1–N2
Fe1–N4
Fe1–N6
Fe1–N1
Fe1–N3
N2–N7
N5–N8
1.884(4)
1.890(4)
1.961(4)
1.946(4)
1.950(4)
1.975(4)
1.408(5)
1.396(5)
N1–Fe1–N2
N2–Fe1–N3
N4–Fe1–N5
N5–Fe1–N6
N2–Fe1–N5
N4–Fe1–N6
N1–Fe1–N3
C13–C14–N3
C30–C29–N5
81.29(15)
81.62(16)
81.57(16)
81.33(16)
176.63(17)
162.79(16)
162.62(16)
114.0(4)
3.4. Electrochemistry
Both complexes were electrochemically examined at a glassy
carbon working electrode in acetonitrile (for 1) and dichlorometh-
ane (for 2) solutions. Representative cyclic voltammograms are
displayed in Fig. 2 and reduction potentials are listed in Table 3.
115.8(4)
The redox processes were quasi-reversible (
D
Epꢃ125 mV) for both
complexes (1 and 2) and exhibit a clean one electron-redox couple
(iPa/iPcꢃ1). The half-wave potential (E1/2) of the Fe(III)/Fe(II) couple
of 1 was + 0.065 V (vs Ag/AgCl electrode) while 2 exhibit E1/2 value
of ꢀ0.077 V (vs Ag/AgCl electrode). The E1/2 value of complex 1 was
more positive side than reported literature (ꢀ0.040 V vresus Ag/
AgCl) with one amidate ligand [59]. The E1/2 value of complex 2
close to low-spin [Fe(tacn)2]3+ (ꢀ0.065 V versus Ag/AgCl) [60]
however, very small as compared to [Fe(Pypep)2]+ (ꢀ0.235 V ver-
sus Ag/AgCl) [38]. Interestingly, the redox potential for Fe(III)/Fe(II)
couple of 2 was observed at a more negative potential than that of
1 indicating stabilization of the Fe(III) species. The E1/2 values of
both complexes clearly designated that coordination of two amide
tendency of tridentate ligands to have longer metal–ligand dis-
tances for the terminal bonds. The N–Fe–N angles showed maxi-
mum deviation from octahedral geometry with angles N2-Fe-N3
(81.62 (16)°), N1–Fe–N2 (81.29 (15)°), N4–Fe–N5 (81.57 (16)°)
and N5–Fe–N6 (81.33(16)°). This was due to the short bite of the
rigid pyridine-2-carboxamido moieties (five-membered chelate
rings) all four Npy–Fe–Nam angles were ꢃ81°, similar value was ob-
served in [M(bpca)2]+ complexes.[53–54]. Further support of strain
in the five-membered chelate rings came from the C13–C14–N3
(114.0 (4)°) and C29–C30–N4 (115.82 (4)°) angles, which was away
from the normal sp2 angle of 120°.
nitrogens provide extra stability (due to strong
r donating capabil-
ity) to the +3 oxidation state of low-spin Fe(III) in complex 2 as
compared to high-spin Fe(III) complex 1.
3.3. Spectroscopic properties
Coordination of the deprotonated amido nitrogen to Fe(III) cen-
tre in 1 and 2 was expressed by the shift of mC
free ligand to 1657 and 1602 cmꢀ1 for complexes 1 and 2 respec-
tively. The decrease in mC frequency in both complexes clearly
indicate the binding of deprotonated ligand (Nam) to the metal cen-
tre because partial double bond character in carbonyl moiety
(shown in Scheme 2) is generated in C@O due to metal coordina-
@
O from 1694 cmꢀ1 in
3.5. DNA interaction studies
@
O
We have investigated the DNA binding properties of complexes
1 and 2 by EtBr displacement assay via fluorescence spectropho-
tometer and circular dichroism spectral studies. We would like to
mention here that complexes 1 and 2 in buffer did not show any
characteristic peak by which we could study the titration with
CT-DNA for the determination of binding constant to CT DNA.
The Stern–Volmer quenching constant KSV values (Fig. 3) for com-
plexes 1 and 2 were 5.30 ꢂ 104 and 7.88 ꢂ 104 Mꢀ1 respectively,
displayed poor binding propensity probably via external or surface
binding to CT-DNA. Circular dichroic spectra of CT DNA exhibits a
positive band at 275 nm due to base stacking, and a negative band
at 245 nm due to the right handed helicity (Fig. S3 in Supporting
information) [61]. On addition of complexes, the CD spectrum of
tion. Similar, red shift of mC
@ has been noted with other Fe(III)
O
complexes having coordinated carboxamido nitrogens [38].
Deprotonation of amido nitrogens were further supported by
disappearance of the band m
N–H (3353 cmꢀ1) in IR spectra of metal
complexes 1 and 2. The presence of counter ion (ClO4ꢀ) in complex
2 was also confirmed by 1090 and 622 cmꢀ1 bands. The IR spectra
of both complexes were dominated by vibrations of the pyridyl
rings as evidenced by the absorptions ꢃ1465 and ꢃ1555 cmꢀ1
[55]. The UV–Vis spectra of complexes 1 and 2 were recorded in
dichloromethane and acetonitrile respectively. For complex 1 the
peak near 321 nm was assigned as chloro-to-Fe(III) charge transfer
transition [56] and 248 nm was assigned as p–
p⁄, n–p⁄ transitions
Complex 1
Complex 2
of ligand. Complex 1 also showed a broad band near 626 nm due to
ligand-to-metal charge transfer transition (LMCT). Interestingly,
for complex 2, an absorption band near 800 nm assigned as ligand
to metal charge transfer transition which was different than other
low-spin Fe(III) complexes reported in literature [57]. The molar
conductivities were determined in dimethylformamide at ca.
10
0
10ꢀ3 M at 25 °C. The values for 1 and 2 were 12.0 and 58.0 Xꢀ1
-
cm2 molꢀ1 respectively. These values for 1 and 2 were designated
neutral and uni-univalent (1:1) electrolyte behaviour respectively
[41,58]. Complex 1 showed a magnetic moment of 5.91 BM in
-10
O
O
Fe+3
N
H
N
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
Potential (V)
Fe+3
Fig. 2. Cyclic voltammograms of a 10-3 M solution of complexes 1 and 2: using
working electrode: glassy-carbon, reference electrode: Ag/AgCl; auxiliary electrode:
platinum wire, scan rate 0.1 V/s.
Scheme 2. Partial double bond character at carbonyl moiety.

24
K. Ghosh et al. / Inorganica Chimica Acta 412 (2014) 20–26
Table 3
Tris–boric acid–EDTA buffer. Nuclease activities of both complexes
Cyclic voltammogram of complexes 1 and 2.
were examined by gel electrophoresis, which allowed quantitative
evaluation of different forms of the DNA. The percentage of total
DNA cleavage was calculated by the following equation: DNA
cleavage = (DII + 2 ꢂ DIII)/(DI + DII + 2 ꢂ DIII), where DI, DII, DIII: inte-
grated density of SC-form, NC-form and LC-form respectively and
shown in Tables S1–S3 in Supporting information [23]. It is clear
from Fig. 4 that complex 1 alone exhibits a significant cleavage
and single strand cleavage was enhanced as the complex concen-
d
Complexa
Fe(III)/Fe(II)
n = iPa/iPc
Epa/V
Epc/V
0.052
ꢀ0.137
E1/2b, V ( Epc, mV)
D
1
2
0.180
ꢀ0.016
0.065 (128)
ꢀ0.077 (121)
0.77
1.00
a
Measured in acetonitrile for 1 and in dichloromethane for 2 with 0.1 M tetra-
butylammonium perchlorate (TBAP).
b
Data from cyclic voltammetric measurements; E1/2 is calculated as average of
tration was increased (15–100
lM) (Fig. 4(A), lane 2–8) (50.4%, to-
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.
tal DNA cleavage with 100 M complex concentration, Table S1 in
l
d
Constant-potential coulometric data n = ipa/ipc calculated for 1eꢀ transfer.
Supporting information). On the other hand, anhydrous FeCl3 did
not cleave plasmid DNA (Fig. 4(A), lane 2) at all. The oxidative
DNA cleavage behaviour of the complex 1 was shown by combina-
tion of 1 (50 lM) and H2O2 (150–200 lM) resulted single strand
12
10
scission (Fig. 4(B), lane 6) and double strand scission (Fig. 4(B),
lanes 7–8) (ꢃ75%, total DNA cleavage, Table S2 in Supporting infor-
mation) however, Kurosaki and co-workers [59] reported double
strand cleavage of Fe(III)-BLM (0.1 mM) analogues with very high
concentration of H2O2 (3 mM). Similar behaviour was observed
30
8
6
4
2
0
20
-2
1.0 1.1 1.2 1.3 1.4 1.5
-6
for BME (10–100 lM) (Fig. 4(C), lane 3–6) with increase in BME
R x 10
concentration strand scission increased (SC form to NC and LC
form) (ꢃ80%, total DNA cleavage, Table S3 in Supporting
information).
10
0
Complex 2 was cleavage inactive in the presence oxidising
(H2O2) or reducing agents (BME). No nuclease was appeared even
in high concentration of BME (3 mM) (Fig. S4 in Supporting infor-
mation). Mechanistic aspects of activity of nuclease of complex 1
was studied using different quenchers: singlet oxygen scavengers
like NaN3 or L-histidine and hydroxyl radical scavengers like
DMSO, KI, urea, ethanol, D2O. To check the mechanism of DNA
cleavage activity via hydrolytic pathway we perform the experi-
ment in presence of complex 1 and radical scavengers (Fig. S5 in
Supporting information) nuclease activity was not inhibited by
radical scavengers showing that only complex 1 could not gener-
ated reactive oxygen species (ROS) which were responsible for
nuclease activity and exclude the possibility through hydrolytic
pathway [46]. In case of oxidative cleavage, efficient inhibition
was observed in presence of KI (Fig. 5, lane: 3) and small inhibitory
effect was also found in presence of DMSO (Fig. 5, lane: 4). Inhibi-
tion in presence of KI and DMSO suggested the possibility of the
formation of ÅOH radicals as the reactive species from probable
hydroperoxo intermediate: [FeIII–OOH] ? [FeIV = O] + ÅOH. Similar
result was reported by Chakravarty et al. with synthetic model
for the bleomycins [Fe(L0)(L00)](PF6)3 [31].
550
600
650
700
Wavelength (nm)
(A)
14
12
10
8
25
20
15
10
5
6
4
2
0
-2
1.0 1.2 1.4 1.6 1.8 2.0
-6
R x 10
0
550
600
650
700
Wavelength (nm)
3.7. Detection of reactive oxygen species (ROS)
(B)
Reactive oxygen species in the cleavage reaction was detected
by rhodamine B dye degradation [47] or by using sensitive radical
sensor 1,3-diphenylisobenzofuran (DPBF) [48]. Participation of OHÅ
radicals or singlet oxygen (1O2) generation was also evaluated by
rhodamine B dye and DPBF respectively, in presence of complexes
1 and 2.
Fig. 3. EtBr–DNA fluorescence quenching titrations of using (A) complex 1 (0–
38 M), (B) complex 2 (0–34
M), SternꢀVolmer plots of Fo/F vs. [R] for both
complexes shown in insets of respective graphs. Tests were performed in the
conditions of 50 mM phosphate buffer (pH 7.2) at 298 K. CDNA = 25 M, CEtBr = 0.5 -
M; kex = 250 nm, kem = 602 nm.
l
l
l
l
We have observed the decrease in absorbance of dye at 544 nm
with increasing concentration of complex 1 (Fig. 6) however, there
is no or slight change in absorbance of dye at 544 nm with increas-
ing concentration of complex 2 (Fig. 6). These observations sug-
gested the formation of OH_ radical in presence of 1, which is
probably responsible for degradation of dye. Generally, rhodamine
B was oxidized by OHÅ radical to yield a photo inactive rhodamine
B adducts [63]. 1,3-diphenylisobenzofuran (DPBF) is the most well
known 1O2 acceptor and its emission is quenched in presence of
1O2. In our experiment, DPBF was incubated with both complexes
1 and 2 and fluorescence at 479 nm (kem = 405 nm) is monitored.
We observed slight quenching in fluorescence spectra of DPBF in
DNA undergoes changes in both the positive and negative bands
but no shift in the band positions. Considering the data obtained
by fluorescence and circular dichroism spectral studies external
interaction with CT-DNA was speculated [62].
3.6. Nuclease activity
The DNA cleavage behaviour of the complexes 1 and 2 were
studied under physiological conditions (37 °C) and observed by
the transformation of supercoiled form (SC or DI) to the nicked
(NC or DII) and linear (LC or DIII) forms of pBR322 DNA plasmid in

K. Ghosh et al. / Inorganica Chimica Acta 412 (2014) 20–26
25
Fig. 4. Gel electrophoresis separations showing cleavage of supercoiled pBR322 DNA (50 ng) by complex 1 in 10% dimethylformamide incubated at 37 °C for 3 h. (A) lane 1:
DNA, lane 2: DNA + FeCl3 (100 M), lane 3: DNA + 1 (15 M), lane 4: DNA + 1 (25 M), lane 5: DNA + 1 (35 M), lane 6: DNA + 1 (50 M), lane 7: DNA + 1 (75 M), lane 8:
DNA + 1 (100 M). (B) lane 1: DNA, lane 2: DNA + H2O2 (200 M), lane 3: DNA + 1 (25 M), Lane 4: DNA + 1 (25 M) + H2O2 (25 M), lane 5: DNA + 1 (25 M) + H2O2 (50
M), lane 7: DNA + 1 (50 M) + H2O2 (150 M), lane 8: DNA + 1 (50 M) + H2O2 (200 M). (C) lane 1: DNA, lane 2: DNA + BME (100
M), lane 4: DNA + 1 (25 M) + BME (25 M), lane 5: DNA + 1 (25 M) + BME (50 M), lane 6: DNA + 1 (25 M) + BME (100 M).
l
l
l
l
l
l
l
l
l
l
l
l
l
l
M),
M),
lane 6: DNA + 1 (25
lane 3: DNA + 1 (25
l
l
M) + H2O2 (100
M) + BME (10
l
l
l
l
l
l
l
l
l
l
l
l
Complex 2
Complex 1
0.6
0.4
0.2
0.0
Fig. 5. Gel electrophoresis separations showing cleavage of supercoiled pBR322
DNA (60 ng) by complex 1 in 10% dimethylformamide incubated at 37 °C for 3 h.
lane 1: DNA, lane 2: DNA + 1 (15
(15 M) + H2O2 (100 M) + KI (50 mM), lane 4: DNA + 1 (15
(100 M) + DMSO (100 mM), lane 5: DNA + 1 (15 M) + H2O2 (100
(100 mM), lane 6: DNA + 1 (15 M) + H2O2 (100 M) + NaN3 (100 mM), lane 7:
DNA + 1 (15 M) + H2O2 (100 M) + L-his (100 mM), lane 8: DNA + 1 (15 M) + H2-
O2 (100 M) + D2O (100 mM).
l
M) + H2O2 (100
l
M), lane 3: DNA + 1
M) + H2O2
M) + Urea
l
l
l
l
l
l
l
l
l
l
l
l
presence of complexes 1 and 2 (Fig. S6 in Supporting information).
These data indicated that 1O2 was not produced the reaction
mixture in presence of 1 or 2 [47].
0
100
200
300
Concentration (µM)
Fig. 6. Plot of decrease in absorbance of rhodamine B dye (5
increasing concentration of metal complexes 1, 2 (0–300 M).
lM) at 544 nm vs
l
4. Conclusion
[Fe(Pamp)(MeOH)Cl2], 1 and [Fe(Pamp)2](ClO4), 2. Complexes 1
and 2 were characterized by conductivity measurements, IR and
In the present work, we synthesised two mononuclear iron(III)
complexes derived from a tridentate amidate ligand PampH,

26
K. Ghosh et al. / Inorganica Chimica Acta 412 (2014) 20–26
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ent spin states namely high-spin state for 1 and low-spin state for
2. Molecular structure of complex [Fe(Pamp)2](ClO4), 2 was deter-
mined by single crystal X-ray crystallography. The stereochemistry
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K.G. is thankful to DST, New Delhi, India, for financial support
(File No. SR/S1/IC-47/2012). N.T. and P.K. are thankful to CSIR, In-
dia, for financial assistance. UPS is thankful to IIT Roorkee for single
crystal X-ray facility. We are thankful to Rajan Kumar for his help.
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.ica.2013.11.034.
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