Mononuclear cobalt(III) and iron(II) complexes with diimine ligands: Synthesis, structure, DNA binding and cleavage activities, and oxidation of 2-aminophenol

Article abstract of DOI:10.1016/j.poly.2012.05.041

Mononuclear cobalt(III) and iron(II) complexes with diimine ligands were synthesized and characterized by elemental analyses, spectroscopic and electrochemical techniques. Single crystal X-ray diffraction studies revealed that the metal ions in both complexes are situated in distorted octahedral environments comprised of six nitrogen atoms from three diimine ligands. Interaction of the complexes with calf thymus DNA was investigated through UV-Vis and fluorescence spectroscopic methods and viscosity measurements. These results suggest that both complexes preferably interact with CT-DNA through the grove binding mode. Moreover, both complexes are capable of promoting DNA cleavage in the presence of suitable activating agent(s). The nuclease activity is strongly suggested to be oxidative, possibly due to the involvement of a diffusible hydroxyl radical mechanism. Furthermore, the cobalt(III) complex has been found to be an excellent oxidant for oxidative coupling of 2-aminophenol to the phenoxazinone chromophore under aerobic conditions.

Full text of DOI:10.1016/j.poly.2012.05.041

Polyhedron 43 (2012) 22–30
Contents lists available at SciVerse ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Mononuclear cobalt(III) and iron(II) complexes with diimine ligands: Synthesis,
structure, DNA binding and cleavage activities, and oxidation of 2-aminophenol
Anangamohan Panja
Department of Chemistry, Panskura Banamali College, Panskura RS, Purba Medinipur, West Bengal 721152, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Mononuclear cobalt(III) and iron(II) complexes with diimine ligands were synthesized and characterized
by elemental analyses, spectroscopic and electrochemical techniques. Single crystal X-ray diffraction
studies revealed that the metal ions in both complexes are situated in distorted octahedral environments
comprised of six nitrogen atoms from three diimine ligands. Interaction of the complexes with calf thy-
mus DNA was investigated through UV–Vis and fluorescence spectroscopic methods and viscosity mea-
surements. These results suggest that both complexes preferably interact with CT-DNA through the grove
binding mode. Moreover, both complexes are capable of promoting DNA cleavage in the presence of suit-
able activating agent(s). The nuclease activity is strongly suggested to be oxidative, possibly due to the
involvement of a diffusible hydroxyl radical mechanism. Furthermore, the cobalt(III) complex has been
found to be an excellent oxidant for oxidative coupling of 2-aminophenol to the phenoxazinone chromo-
phore under aerobic conditions.
Received 12 April 2012
Accepted 22 May 2012
Available online 15 June 2012
Keywords:
Cobalt(III) and iron(II) complexes
Crystal structure
DNA binding
Nuclease activity
Oxidation of 2-aminophenol
Ó 2012 Elsevier Ltd. All rights reserved.
1
. Introduction
The development of artificial nucleases is an important aspect
interaction of their complexes with various nitrogen donor ligands
with DNA has attracted much attention [15,16]. The DNA binding
and photo-cleavage ability of certain cobalt complexes have been
recently studied [17,18]. Some of the previous reports demonstrate
that hexamine cobalt complexes induce DNA condensation and can
be used to probe RNA hairpins [19]. Some of the cobalt(II) com-
plexes, such as [Co(tfa)(happ)], are known to function as specific
probes for DNA bulges due their ability to cleave DNA specifically
[20]. Most importantly, iron bleomycins (Fe-BLMs), naturally
occurring anti-tumor glycopeptide antibiotics, are used in chemo-
therapy [21]. However, the abilities of only a limited number of
functional models for iron(II)-BLMs have been studied because
Fe-BLMs are probably involved in the oxidative cleavage of DNA
in the areas of biotechnology and drug design, as well as molecular
biology [1–4]. Over the last few decades, transition metal com-
plexes have largely been employed for these purposes, not only be-
cause of their versatile electronic and structural features, but also
due to the fact that the DNA binding and cleavage ability of metal
complexes can be tuned by changing the coordination environ-
ment [5,6]. These studies are also important for the development
of new probes for nucleic acids and the determination of the mech-
anism of metal ion toxicity [7]. Cisplatin and related platinum
based drugs are some of the few most widely used antitumor
metallo-drugs for the treatment of certain human cancers with
incredible success, but they show severe toxic side effects, includ-
ing nephrotoxicity, and drug resistivity prevents their potential
efficiency [8,9]. Ruthenium(II) and bio-compatible copper(II) com-
pounds are considered as promising alternatives to platinum com-
plexes because of their remarkable anticancer activity and the fact
that they show general toxicity lower than platinum compounds
2
using O as the terminal oxidant [22–24]. The full therapeutic po-
tential of these complexes cannot be realized as they require acti-
vation by either light or an oxidant, and so the antitumor activity of
these complexes has not been well explored [25]. Redox-active
agents that damage DNA in vitro might show apoptotic activities
in living systems by inducing oxidative stress and/or damage of
DNA [26,27]. Thus, cobalt and iron polypyridyl complexes, which
may cleave DNA by generating Reactive Oxygen Species in vitro,
could be potential anticancer drugs by killing cancer cells [28,29].
The synthesis and reactivity studies of transition metal com-
plexes as structural and functional models for metalloenzymes
with oxidase (oxygenase) activity are of particular interest for
the development of bioinspired catalysts for oxidation reactions.
The enzyme phenoxazinone synthase [30,31], a multicopper oxi-
dase, is naturally found in the bacterium Streptomyces antibioticus
[
10–14]. Therefore, the design of new metal-based anticancer
drugs that exhibit enhanced selectivity and various non-covalent
DNA binding interaction modes, e.g. intercalation, groove binding
and external electrostatic binding, are of considerable interest.
Cobalt and iron are recognized as essential elements widely
spread in biological systems such as cells and organs, and the
E-mail address: ampanja@yahoo.co.in
0
277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.05.041

A. Panja / Polyhedron 43 (2012) 22–30
23
and catalyzes the oxidative coupling of two molecules of a substi-
2.3. Synthesis of complex 2
A mixture of FeSO
throline (180 mg, 1.0 mmol) in acetonitrile (40 mL) was heated to
boiling, with stirring. The solution was then filtered, and an aque-
tuted 2-aminophenol to the phenoxazinone chromophore in the fi-
nal step of the biosynthesis of actinomycin D [32], which is used
clinically for the treatment of Wilm’s tumor, gestational choriocar-
cinoma, and other tumors [33,34]. Only a limited numbers of func-
tional models based on cobalt and iron have been reported which
show phenoxazinone synthase activity [35,36].
A novel photoactive dinuclear zinc(II) complex with an azoben-
zene linker was recently reported by us in which the azobenzene
group could be photoisomerized between the trans and cis forms
4
ꢁ7H
2
O (93 mg, 0.33 mmol) and 1,10-phenan-
4
ous solution (5 mL) of NaBF (110 mg, 1.0 mmol) was added. Slow
evaporation of the resulting solution over 1 week afforded copious
quantities of purple needles that were suitable for structural stud-
ies. The remaining crystals were isolated via suction filtration,
washed with acetonitrile/ether, and air dried at room temperature.
[
37]. The two Zn(II) centers were positioned relatively far away
Yield: 0.096 g (64%). Anal. Calc. for C72
48 4 2
H B F16Fe N12O: C, 55.57;
from each other in the trans complex, where no appreciable DNA
cleavage activity was observed. In the cis complex, the Zn(II) cen-
ters were closely located, and the complex exhibited remarkable
DNA cleavage activity. We have also reported a study of the cat-
echolase activity of a mononuclear Cu(II) complex [38]. In this
work, two mononuclear complexes of cobalt(III) and iron(II) with
diimine ligands have been synthesized, and the crystal structures
are reported. DNA binding and chemical nuclease activities
through oxidative pathways have been studied. The cobalt(III)
complex also shows efficient oxidation of 2-aminophenol in the
presence of molecular dioxygen.
H, 3.11; N, 10.80. Found: C, 55.78; H, 3.32; N, 10.49%. IR (KBr,
ꢀ1
cm ): 3434 (br,
m
O–H); 3136–3086 (w,
B–F).
mC–H); 1629 (m, mC@N);
1427 (m, C–N); 1084 (br,
m
m
2.4. X-ray crystallography
Single crystals of 1 and 2 were coated with N-Paratone oil,
picked up with glass fibers and mounted on a Nonius Kappa CCD
diffractometer equipped with a cryogenic nitrogen cold stream.
Graphite-monochromated Mo K
a radiation (k = 0.71073 Å) was
used for data collection. Cell refinement, indexing and scaling of
the data sets were performed using the programs DENZO-SMN and
SCALEPACK [39]. The structures were solved by direct method and re-
fined by the full-matrix least-square technique based on F2 using
SHELXL97 [40] programs, using all the reflections. All the non-hydro-
gen atoms were refined anisotropically and all the hydrogen
atoms, except for the disordered water molecules, were placed in
calculated positions and refined isotropically using a riding model.
All the calculations were performed using the WinGX System, Ver.
2
. Experimental
2.1. Materials and physical measurements
0
0
4
,4 -Dimethyl-2,2 -bipyridine, 1,10-phenanthroline, cobalt(II)
nitrate hexahydrate, iron(II) sulfate heptahydrate, silver nitrate,
-aminophenol and tris(hydroxymethyl)aminomethane (Tris)
were purchased from Aldrich and Alfa Aesar. Calf thymus DNA
CT-DNA) and ethidium bromide (EB) were purchased from Sigma.
1
.70.01 [41]. The crystal parameters and basic information relating
2
to data collection and structure refinement for compounds 1 and 2
are summarized in Table 1.
(
All other chemicals and solvents were of reagent or analytical
grade and were used without further purification. Ultrapure MilliQ
water was used for all the experiments.
Table 1
Crystal data and structure refinement for complexes 1 and 2.
Elemental analyses for C, H and N were performed by the Pregl-
Dumas technique on a Thermo Fischer Flash EA1112. FT-IR spectra
of the complexes in KBr disks were recorded in the range 400–
Complex
1
2
Formula
Formula weight
T (K)
C
36
H
36CoF18
N
6
OP
3
C
72 48 4 2 12
H B F16Fe N O
1062.55
260(2)
0.71073
triclinic
P1ꢀ
10.082(3)
12.625(5)
17.561(6)
82.424(2)
83.500(3)
87.529(3)
2200.6(2)
2
1556.16
150(2)
0.71073
monoclinic
Cc
ꢀ
1
4
000 cm on a Nicolet 750 Magna-IR spectrometer. Electrochem-
k (Å)
ical data were collected on an EG&G Princeton Applied Research
potentiostat model 263A with a Pt working electrode, Pt counter
electrode and Ag/AgCl reference electrode. Absorption spectra
were measured using a Shimadzu UV-2450 spectrophotometer
with a 1 cm pathlength quartz-cell. Fluorescence experiments
were performed with a Hitachi F-4500 spectrofluorometer. Con-
ductivity measurements were made with a Systronics (model
Crystal system
Space group
a (Å)
b (Å)
c (Å)
35.768(8)
16.047(5)
12.030(4)
90
101.516(3)
90
6766(4)
4
1.528
a
(°)
b (°)
(°)
c
3
04) direct reading conductivity meter.
3
V (Å )
Z
D
l
calc (g cmꢀ3)
(mm
1.604
0.614
1072
ꢀ
1
)
0.529
3152
2.2. Synthesis of complex 1
F(000)
h Range (°)
3.18–27.49
15895
9734 (0.0379)
3.17–27.51
14254
14246 (0.0234)
0
A mixture of Co(NO
3
)
2
ꢁ6H
2
O (97 mg, 0.33 mmol) and 4,4 -di-
Reflections collected
Independent
reflections
0
methyl-2,2 -bipyridine (184 mg, 1.0 mmol) in acetonitrile (40 mL)
was heated to boiling, with stirring. The resulting red solution
int
(R )
was allowed to react with AgNO
5 mL) at room temperature with stirring for a period of 1 h, during
which time the mixture turned dark yellow. The resulting solution
was filtered, and an aqueous solution (5 mL) of KPF (184 mg,
.0 mmol) was added. Crops of dark yellow block-shaped crystals
3
(57 mg, 0.33 mmol) in water
Observed reflections
I > 2 (I)]
7490
9844
(
[
r
Data/restraints/
parameters
Goodness-of-fit (GOF)
on F2
Final R indices
9734/1/637
1.036
14246/2/965
1.022
6
1
of 1 were obtained by slow evaporation of the solution and these
were isolated via suction filtration, washed with acetonitrile/ether,
and air dried at room temperature. Yield: 290 mg (82%). Anal. Calc.
R
1
= 0.0975,
wR = 0.2626
= 0.1207,
wR = 0.2822
+1.697/ꢀ0.697
R
1
= 0.0780,
wR = 0.2127
= 0.1162,
wR = 0.2460
+1.032/ꢀ0.417
[I > 2r(I)]
2
2
R indices (all data)
R
1
R
1
for C36
H, 3.52; N, 7.61%. IR (KBr, cm ): 3443 (br,
C–H); 1622 (m, C@N); 1443 (m, C–N); 844 (s, m
H
6 3
38CoF18N OP : C, 40.62; H, 3.60; N, 7.89. Found: C, 40.78;
2
2
ꢀ
1
Largest difference in
peak/hole (e Å
m
O–H); 3132–2932 (w,
P–F); 558 (s, dP–F).
ꢀ
3
)
m
m
m

2
4
A. Panja / Polyhedron 43 (2012) 22–30
2.5. DNA binding and cleavage experiments
3. Results and discussion
The electronic absorption spectral method was employed for
the intrinsic binding of complexes 1 and 2 to CT-DNA in 2% DMF/
0 mM phosphate buffer at pH 7.5. A solution of CT-DNA gave a ra-
3.1. Synthesis and general characterizations
2
The cobalt(III) complex was synthesized by the reaction of co-
0
0
tio of UV absorbance at 260 and 280 nm of about 1.9, indicating
that the DNA was sufficiently free of proteins [42]. The concentra-
tion of CT-DNA in base pairs was measured using its molar absorp-
balt(II) nitrate with 4,4 -dimethyl-2,2 -bipyridine in a 1:3 molar ra-
tio, following the oxidation by silver nitrate in aqueous acetonitrile
medium. Dark yellow crystals of 1 were isolated by addition of
aqueous KPF6 in good yield. The iron(II) complex was prepared
by the treatment of iron(II) sulfate with phenanthroline in a 1:3
molar ratio, and it was isolated as the tetrafluoro borate salt. Both
the complexes were characterized by elemental analyses and var-
ious spectroscopic and physio-chemical methods. The molar con-
ꢀ
1
ꢀ1
tion coefficient of 6600 M cm
at 260 nm [43]. Both the
complexes at fixed concentrations were titrated with varying con-
centrations of CT-DNA, and the changes in the absorption spectra
were recorded after incubation for 5 min.
The relative binding affinity of the complexes to CT-DNA
was examined by fluorescence spectroscopy with EB-bound
ductivity measurements reveal that complex
1 is a 1:3
ꢀ
5
CT-DNA (2.5 ꢂ 10 M) in 2% DMF/20 mM phosphate buffer (pH
electrolytic type compound, while complex 2 behaves as a 1:2 elec-
trolyte. The infrared spectral data of the metal complexes are re-
ported in the experimental section with their tentative
7
.5). EB-bound CT-DNA was excited at 510 nm, and fluores-
cence intensities at 600 nm of EB-bound CT-DNA were recorded
for each incremental addition of the complexes after incubation
for 5 min.
ꢀ1
assignments. The bands at 843 and 558 cm for complex 1 are
attributed to the stretching and bending modes of vibrations of
ꢀ
ꢀ
Viscosity experiments were conducted on an Ostwald viscome-
ter operating at room temperature. The flow time was measured
with a digital stopwatch and an average flow time was calculated
for each sample based on three experiments. Data were presented
4
the PF6 counter ions, while those of BF for 2 were observed at
ꢀ1
1084 cm . These facts are in good agreement with the structural
characterization of the complexes.
1
/3
as (g/g
0
)
versus the ratio of the complexes to DNA, where g and
3.2. Description of the crystal structures
g
0
are the specific viscosities of CT DNA solution in the presence
and absence of the complexes, respectively. The viscosity values
were corrected taking into account the flow time of the buffer
The molecular structures of complexes 1 and 2 were deter-
mined by the single crystal X-ray diffraction technique. Crystallo-
graphic data and details of data collection and refinement for 1
and 2 are assembled in Table 1. Important bond lengths and angles
are summarized in Table 2. Complex 1 crystallizes in the triclinic
alone using the equation
flow time of the buffer itself and t is the flow time of DNA in the
presence or absence of the complexes [44].
g
= (t ꢀ t
0 0 0
)/t , where t represents the
Plasmid DNA (pUC19) was extracted by over expression in Esch-
erichia coli DH5a cells, and the isolated DNA was purified with a
QIAGEN kit (QIAprep spin miniprep kit-250). The purity of DNA
was checked by gel electrophoresis. The DNA cleavage activity of
the complexes was determined by monitoring the conversion of
supercoiled plasmid DNA (Form I) to nicked-circular DNA (Form
II) using agarose gel electrophoresis. Briefly, the cleavage reaction
ꢀ
P1 space group. The molecular structure of 1 consists of a discrete
0
3+
six-coordinate [Co(4,4 -dmbp)
3
]
cation, three non-coordinating
ꢀ
PF
6
anions and a disordered solvent water molecule. A perspec-
tive view of the complex cation of 1, with the selected atom num-
bering scheme, is depicted in Fig. 1. The coordination sphere
III
around the Co ion can be described as a distorted octahedral
0
geometry, having a CoN
6
chromophore comprised of three 4,4 -
was carried out by mixing plasmid DNA (0.005 lg/lL) with com-
dmbp ligands coordinated in a bidentate fashion. All the Co–N
bond distances are similar, and they vary in the narrow range from
.931(4) (Co1–N1) to 1.946(4) Å (Co1–N4). The Co–N bond lengths
plex 1 or 2 in 2% DMF/20 mM phosphate buffer (pH 7.5). The sam-
ples were incubated at 37 °C, followed by the addition of loading
buffer containing equal volumes of a 10ꢂ DNA loading buffer
1
are comparable to those reported for low spin cobalt(III) com-
plexes, and the bond lengths are much smaller than those of cobal-
t(II) complexes [46–49]. The average trans angle is close to
linearity, 175.98(17)°, while significant deviation of the bite angles
(
Takara-bromophenol blue) solution and a 10 mM aqueous
solution of ethylenediaminetetraacetic acid (EDTA). The reaction
products were subjected to electrophoresis on 1% agarose gel in
Tris–acetate–EDTA buffer (pH 8.0) for 40–60 min at 100 V. The
0
of 4,4 -dmbp [82.79(17)°, 83.36(17)° and 83.48(16)°] from the ideal
resulting gel was transferred to an EB solution (1 lg/1 lL) and
geometry are observed, which are mainly accountable for the geo-
stained. The bands of the supercoiled and nicked-circular DNA
forms were visualized, and the extent of cleavage of the SC DNA
was quantified by measuring the intensities of the bands using
the Gel Documentation System (Bio Rad). A correction factor of
III
metrical distortion around the Co ions. A similar deviation of the
Table 2
1
.3 was used for supercoiled DNA, considering the weaker interca-
Selected bond lengths (Å) and bond angles (°) for 1 and 2.
lating ability of ethidium bromide into supercoiled DNA relative to
the nicked-circular DNA [45].
1
Co1–N1
Co1–N2
Co1–N3
1.931(4)
1.943(4)
1.939(4)
Co1–N4
Co1–N5
Co1–N6
1.946(4)
1.933(4)
1.941(4)
2.6. Kinetics of the oxidation of 2-aminophenol
N1–Co1–N2
N3–Co1–N4
N5–Co1–N6
83.36(17)
82.79(17)
83.48(16)
N1–Co1–N5
N2–Co1–N4
N3–Co1–N6
175.01(17)
177.68(17)
175.24(17)
Kinetics of the aerobic oxidation of 2-aminophenol in the pres-
ence of the complexes were measured by monitoring the change in
3
2 (average value of two centers)
absorbance as
a
function of time at 433 nm
(
e
= 24 ꢂ 10
ꢀ1
ꢀ1
M
cm ), which is characteristic of 2-aminophenoxazin-3-one
Fe1–N1
Fe1–N2
Fe1–N3
1.986(6)
1.973(6)
1.983(6)
Fe1–N4
Fe1–N5
Fe1–N6
1.970(6)
1.988(6)
1.959(6)
in methanol. The reactions were studied under pseudo first-order
conditions, taking the complex as a minor component. The corre-
sponding rate constants were evaluated by means of a suitable
non-linear acquisition analysis system, with data taken for at least
three half-lives of the reactions.
N1–Fe1–N2
N3–Fe1–N4
N5–Fe1–N6
82.6(2)
82.9(2)
82.8(2)
N1–Fe1–N5
N2–Fe1–N4
N3–Fe1–N6
177.9(3)
174.5(2)
174.1(3)

A. Panja / Polyhedron 43 (2012) 22–30
25
complexes [50,51]. The molecular packing is primarily stabilized
by stacking interactions (3.903 Å) involving the phen ligands
p p
ꢁ ꢁ ꢁ
2
+
(
Fig. S2). In addition, [Fe(phen)
3
]
cations in 2 are associated in
pairs by C–Hꢁ ꢁ ꢁ
p interactions (3.515 Å) between the phen ligands.
3.3. Binding of the complexes to CT-DNA
Electronic absorption spectroscopy is one of the most useful
techniques for examining the binding modes of metal complexes
with DNA. A complex generally shows hypochromism and a red
shift (bathochromism) of the absorption band when it binds to
DNA through intercalation, resulting in a strong stacking interac-
tion between the aromatic chromophore of the complex and the
adjacent base pairs of DNA [52]. The extent of hypochromism gives
a measure of the strength of the intercalative binding. The cobal-
t(III) complex 1 exhibits intense absorption bands in the UV region,
⁄
which are attributed to an intraligand
p–p
transition of the coor-
0
dinated 4,4 -dmbp groups, while the absorption spectrum of 2 in
the visible region was characterized by a metal-to-ligand charge
transfer (MLCT) transition at a maximum wavelength (kmax) of
5
11 nm. The binding propensity of the complexes to CT-DNA was
Fig. 1. ORTEP diagram of complex cation
Hydrogen atoms are omitted for clarity.
1
with 50% probability ellipsoids.
determined by monitoring the change of the absorption intensities
of complexes 1 and 2 with the increasing concentration of CT-DNA
in 2% DMF/20 mM phosphate buffer at pH 7.5. The absorption
spectra of DNA in the absence and presence of 1 and 2 are given
in Figs. 3 and 4, respectively. The spectral changes show that upon
addition of an increasing amount of CT-DNA to the complexes only
moderate hypochromism was observed. In order to compare the
DNA binding strengths of complexes 1 and 2 quantitatively, the
bite angles has been observed for the analogous bipyridyl com-
plexes [46–49]. The molecular packing is primarily stabilized by
several edge-to-edge moderately strong (3.711 Å)
pꢁ ꢁ ꢁp stacking
0
interactions involving the 4,4 -dmbp groups (Fig. S1). In addition,
0
3+
the [Co(4,4 -dmbp)
3
]
cations in 1 are connected in pairs by C–
b
intrinsic binding constant (K ) was determined by non-linear
0
Hꢁ ꢁ ꢁ
p
interactions involving the 4,4 -dmbp ligands.
Single crystal analysis of complex 2 reveals that the compound
regression analysis using the following equations [53]:
2
2
b
1=2
crystallizes in the monoclinic Cc space group and the asymmetric
unit contains two independent Fe(phen)
gram of the complex cation of 2 is shown in Fig. 2. Each Fe ion is
coordinated by six N atoms of three phen ligands to form a dis-
torted octahedral environment. The Fe1–N1 bond length,
ðe
a
ꢀ
e
f
Þ=ð
e
b
ꢀ
e
f
Þ ¼ ðb ꢀ ðb ꢀ 2K C
t
½DNAꢃ=sÞ Þ=ð2K
b
C
t
Þ;
2+
3
centers. The ORTEP dia-
II
b ¼ 1 þ K
b
C
t
þ K ½DNAꢃ=2s
b
where ea is the extinction coefficient of the charge transfer band at a
given DNA concentration, ef is the extinction coefficient of the com-
plex free in solution, eb is the extinction coefficient of the complex
1
.988(6) Å, is slightly longer than the other Fe–N bonds; the aver-
age Fe–N bond length is 1.976 Å, in agreement with previously re-
ported results for analogous compounds [50,51]. The mean value of
the N–Fe–N bite-angle in the bidentate ligands is 82.77° and the
average trans angle for opposite nitrogen atoms in the coordination
polyhedra is 175.5°, which are consistent to those found in similar
when fully bound to DNA, C is the total metal complex concentra-
t
tion, [DNA] is the DNA concentration in nucleotides and s is the
binding site size in base pairs. The intrinsic binding constants (K )
b
3
of the complexes are found to be (1.1 ± 0.04) ꢂ 10 and
4
ꢀ1
(
1.9 ± 0.05) ꢂ 10 M for 1 and 2, respectively. The K
b
values are
Fig. 3. Absorption spectral changes upon the incremental addition of CT-DNA (0–
ꢀ
4
ꢀ5
Fig. 2. ORTEP drawing of complex cation
Hydrogen atoms are omitted for clarity.
2
with 50% probability ellipsoids.
1.0 ꢂ 10 M) to complex 1 (1.0 ꢂ 10 M) in a 2% DMF/20 mM phosphate buffer
(pH 7.5) at room temperature. Inset: Plot of ( )/( ) vs. [DNA].
e
a
ꢀ
e
f
b f
e – e

2
6
A. Panja / Polyhedron 43 (2012) 22–30
Fig. 5. Fluorescence emission spectral changes (kex = 510 nm) by addition of
Fig. 4. Absorption spectral changes upon the incremental addition of CT-DNA (0–
ꢀ
5
ꢀ
4
ꢀ5
increasing amount of complex
1
(0–12.5 ꢂ 10 M) to EB-bound CT-DNA
1
.2 ꢂ 10 M) to complex 2 (2.5 ꢂ 10 M) in 2% DMF/20 mM phosphate buffer (pH
5
(
2.5 ꢂ 10ꢀ M, 1:1). Inset: Plot of I
0
/I vs. [complex 1]/[DNA].
7
.5) at room temperature. Inset: Plot of ( )/( ) vs. [DNA].
e
a
ꢀ
e
f
b f
e ꢀ e
were 0.06 and 0.08, respectively, demonstrating a weaker affinity
of the complexes to CT-DNA than that of EB. The relative binding
constant (Kapp) of the complexes was calculated from the following
equation [57]:
similar to the values obtained for analogous complexes, but signif-
icantly lower than those reported for typical intercalators, indicat-
ing that the complexes do not bind to DNA through an
intercalation mode [54]. The results are also not consistent with
the charge of the complexes, and thus binding through electrostatic
force of attraction is also not the actual case. These results suggest
that intimate association of the complexes with CT-DNA is likely to
KEB ꢂ ½EBꢃ1=2 ¼ Kapp ꢂ ½complexꢃ1=2
be operative via the groove binding mode. Interestingly, the K
b
va-
ꢀ
5
where [EB]1/2 (2.5 ꢂ 10 M) and [complex]1/2 are the concentration
of EB and the complex, respectively, at 50% reduction of the fluores-
cence intensity. The [complex]1/2 value was obtained by an extrap-
lue obtained for complex 1 is slightly higher than the closely related
3
+
3
ꢀ1
complex [Co(bpy)
3
]
(K
b
, 9.4 ꢂ 10 M ), while it is significantly
3+
4
ꢀ1
lower that the reported value for [Co(phen)
53]. This difference in the extent of binding is consistent with
3
]
(K
b
,1.6 ꢂ 10 M
)
ꢀ3
olation of the plot of I
Fig. 5, inset) and 1.07 ꢂ 10 for 1 and 2, respectively. KEB repre-
sents the binding constant between EB and CT-DNA
0
/I versus [complex]/[DNA] as 1.66 ꢂ 10
M
[
ꢀ
3
(
the nature of the diimine ligands employed for those systems. The
phen ligand is a better planar ligand in this series, and thus
6
ꢀ1
(K
EB = 1.4 ꢂ 10 M ) [54]. Applying these values, Kapp for the com-
3
+
[
Co(phen)
3
]
shows the higher binding affinity compared to its
4
ꢀ1
plexes to EB bound CT-DNA were obtained as 2.1 ꢂ 10 M for 1
bipyridyl analogues, while the presence of two methyl substituents
4
ꢀ1
and 3.3 ꢂ 10 M for 2, which is in good agreement with the trend
in DNA binding affinities obtained from the absorption spectral and
viscosity studies (cf. below). These results support the hypothesis
that the compounds do not bind through an intercalation mode,
since the obtained binding constants of the complexes to DNA were
much smaller than that of EB [54]. However, the relative DNA bind-
ing constants (Kapp) of the complexes obtained from the EB dis-
placement assay (indirect method) are different from the values
0
in 4,4 -dmbp makes complex 1 a bit of a healthier candidate than
3
+
[
Co(bpy)
3
]
to interact in the grove region hydrophobically.
No luminescence was observed for complexes 1 and 2 at room
temperature in aqueous solution, or in the presence of CT-DNA.
So the binding of the complexes to CT-DNA cannot be directly
presented in the emission spectra. In order to further clarify the
interaction of the complexes with DNA, competitive binding exper-
iments were carried out. The emission intensity of EB decreases in
buffer because of the quenching effect by solvent molecules, but
increases significantly after intercalating into DNA [55]. Intercala-
tion of a complex to EB-bound DNA decreases the emission inten-
sity by release of EB from DNA, and the reduction of the emission
intensity is used as a measure of the DNA binding propensity of the
complex. However, the fluorescence intensity was not effectively
quenched by addition of five equivalents of complex 1 to EB-bound
DNA (Fig. 5). A similar observation was also noted for complex 2.
The quenching efficiency of the complexes was determined by
the classical Stern–Volmer equation [56],
b
(K ) obtained by the absorption spectral method. The same was re-
ported for several complexes, and the difference between the two
sets of binding constants may be due to the different spectroscopy
and different calculation methods employed [58].
Measurement of DNA viscosity, which is sensitive to DNA
length, is regarded as the least ambiguous and the most critical test
of binding in solution, especially in the absence of crystallographic
structural data [59]. Intercalating agents are expected to enlarge
the double helix to accommodate ligands between the base pairs,
leading to an increase in the viscosity of DNA. On the other hand,
a complex that binds exclusively in the DNA grooves by partial
and/or non-classical intercalation, under the same conditions, typ-
ically causes less pronounced (positive or negative) or no change of
I
0
=I ¼ 1 þ KSV ꢂ r
1
/3
where I and I are the fluorescence intensities in the absence and
0
viscosity in DNA solution. A plot of (
g/g
0
)
versus [complex]/
presence of the quencher, respectively. KSV is the linear Stern–Vol-
mer quenching constant, and r is the concentration ratio between
the quencher and DNA. The quenching constant KSV is a measure
of the quenching efficiency of the fluorescence intensity by the
complex to EB-bound CT-DNA, and is obtained from the slope of
[DNA] (Fig. 6) reveals that the viscosity of DNA remain almost un-
changed upon addition of the complexes. These results support
that the complexes do not intercalate with DNA, and possibly a
groove binding mode is operative. It is reflected from preceding
0
data that although both the 4,4 -dmbp and phen contain the same
0
the I /I versus r plot. The KSV values found for complexes 1 and 2
number of carbon atoms, the better planarity of the phen ligands

A. Panja / Polyhedron 43 (2012) 22–30
27
Fig. 6. Effect of an increasing amount of the complexes on the relative viscosity of
CT-DNA (100 M) at room temperature in 2% DMF/20 mM phosphate buffer (pH
.5).
l
7
Fig. 7. Cleavage of pUC19 DNA (0.005
presence of
phosphate buffer (pH 7.5) at 37 °C. Lane 1, DNA control; lane 2, DNA + 1; lane 3,
DNA + H + ascorbic acid; lane 4 DNA + 1 (10 mM) + H + ascorbic acid; lane 5,
DNA + 1 (20 mM) + H + ascorbic acid; lane 6, DNA + 1 (30 mM) + H + ascorbic
acid; lane 7, DNA + 1 (40 mM) + H + ascorbic acid; lane 8, DNA + 1
50 mM) + H + ascorbic acid.
l
g/
l
L) by complex 1 (10–50 mM) in the
0
offer a stronger interaction than that of 4,4 -dmbp in the grove
region.
2 2
H O (200 mM) and ascorbic acid (50 mM) in 2% DMF/20 mM
2
O
2
2 2
O
2
O
2
2 2
O
2
O
2
3.4. DNA cleavage activity
(
2 2
O
In order to assess any cleavage of DNA as a result of the binding
of complexes 1 and 2, agarose gel electrophoresis has been per-
formed with supercoiled pUC19 plasmid DNA. The DNA cleavage
ability of the complexes was analyzed by monitoring the conver-
sion of supercoiled DNA (Form I) to nicked-circular (Form II). Com-
plex 2 shows remarkable DNA cleavage activity in the presence of
hydrogen peroxide as an activating agent. On the other hand, no
significant cleavage of DNA was observed for complex 1 under
identical conditions. However, on incubation of DNA with complex
1
in the presence of both hydrogen peroxide and ascorbic acid, sig-
nificant cleavage was observed. Similar observations were also
noted for the nuclease activity by related metal complexes
[
16,60]. The cleavage reactions have been studied using different
complex concentrations and incubation times. Figs. 7 and 8 show
the results of gel electrophoretic separations of plasmid pUC19
DNA induced by an increasing concentration of complexes 1 and
2, respectively. It was observed that the cleavage efficiency of the
complexes gradually increases with the increase in concentration
of the complexes, which indicates that the DNA cleavage activity
of the complexes is obviously complex concentration-dependent.
Time courses of the gel electrophoresis pattern of pUC19 DNA
cleavage during the reactions in the presence of complexes 1 and
2
are shown in Figs. 9 and 10, respectively. With the increase in
Fig. 8. Cleavage of pUC19 DNA (0.005
presence of H
Lane 1, DNA control; lane 2, DNA + 2; lane 3, DNA + H
2 mM); lane 5, DNA + H + 2 (5 mM); lane 6, DNA + H
DNA + H + 2 (10 mM); lane 8, DNA + H + 2 (15 mM); lane 9, DNA + H
20 mM).
l
g/
l
L) by complex 2 (5–20 mM) in the
reaction time, the amount of Form II increased and Form I gradu-
ally disappeared. The results illustrate that the complexes can
effectively cleave DNA in the presence of suitable external agents,
and the cleavage of DNA by the complexes is dependent both on
the concentration of the complex and the reaction time. The time
course plots reveal that the disappearance of Form I and the
appearance of Form II with reaction time show the expected pseu-
do-first order kinetic profiles. On fitting the experimental data with
first-order consecutive kinetic equations, the pseudo-first order
rate constants for complexes
2
of supercoiled to nicked circular DNA.
In order to obtain information about the reactive oxygen species
which may be responsible for DNA damage, DNA cleavage has been
performed in the presence of hydroxyl radical scavengers (DMSO),
2 2
O (100 mM) in 2% DMF/20 mM phosphate buffer (pH 7.5) at 37 °C.
2
O
2
; lane 4, DNA + H
2
O
2
+ 2
(
2
O
2
2
O
2
+ 2 (8 mM); lane 7,
2
O
2
2 2
O
2
O
2
+ 2
(
singlet oxygen quenchers
(L-histidine), superoxide scavenger
(superoxide dismutase enzyme SOD) and hydrogen peroxide scav-
enger (catalase) (Table 3). From the data, it can be seen that signif-
icant inhibition is observed in the presence of DMSO, indicating
that the freely diffusible hydroxyl radical is responsible for the
DNA cleavage activity exhibited by the complexes. Therefore, the
mechanism of DNA cleavage by complex 2 involves the well known
Fenton type chemistry in which a DNA-bound iron(II) complex
reacts with hydrogen peroxide to generate the highly reactive
1
and
2 were obtained as
ꢀ4
ꢀ4 ꢀ1
.40 ꢂ 10 and 4.88 ꢂ 10
s , respectively, for the conversions

2
8
A. Panja / Polyhedron 43 (2012) 22–30
Table 3
Influence of common radical scavengers on the cleavage of pUC19 plasmid DNA from
Form I to Form II by the complexes in the presence of activating agent(s) at 37 °C.
Serial No.
Reaction condition
Form (%)
I
II
1
2
3
4
5
6
7
8
9
DNA control
96
87
18
15
12
95
88
16
12
15
4
DNA + 1 (10 M) + H
DNA + 1 (10 M) + H
DNA + 1 (10 M) + H
DNA + 1 (10 M) + H
DNA control
DNA + 2 (5 M) + H
DNA + 2 (5 M) + H
DNA + 2 (5 M) + H
DNA + 2 (5 M) + H
2
2
2
2
O
O
O
O
2
2
2
2
/ascorbic acid + DMSO
/ascorbic acid + -histidine
/ascorbic acid + SOD
13
82
85
88
5
12
84
88
85
L
/ascorbic acid + catalase
2
2
2
2
O
O
O
O
2
+ DMSO
2
id + -histidine
2
2
L
+ SOD
+ catalase
1
0
reducing and oxidizing agents are required in order to promote
oxidative DNA cleavage [60]. However, the chemical nuclease
activity of 2 is significantly higher than that of 1, probably due to
the higher binding affinity of 2 to DNA and/or the presence of dif-
ferent metal ions.
3.5. Electrochemical studies
Fig. 9. Time courses of cleavage of pUC19 DNA (0.005
lg/lL) by complex 1 (50 lM)
Since the electrochemical properties of the redox-active com-
in the presence of H (200 mM) and ascorbic acid (50 mM) in 2% DMF/20 mM
2 2
O
plexes are relevant to the oxidation process, these were studied
under the same experimental conditions as the kinetic studies.
The electrochemical data of complexes 1 and 2 have been recorded
in methanol containing 0.1 M tetrabutylammonium perchlorate as
supporting electrolyte at a platinum working electrode and using a
Ag/AgCl reference electrode. The cyclic voltammogram of complex
phosphate buffer (pH 7.5) at 37 °C. (A) Agarose gel electrophoresis patterns of
pUC19 DNA are shown at various reaction times: Lanes 1–9: reaction times of 0, 5,
1
0, 15, 20, 30, 40, 50 and 60 min, respectively. (B) Plots of percentages of
supercoiled (I) and nicked-circular (II) DNA vs. reaction time.
1
shows an electrochemically quasi-reversible one-electron reduc-
tion at +0.22 mV (E1/2 = 0.28 mV, = 120 mV) which can be as-
DE
p
III
II
signed to the Co /Co couple (Fig. 11). Similarly for complex 2,
at positive potentials a quasi-reversible process with an E1/2 value
p
of 950 mV (DE = 84 mV) is observed, which could be assigned to
II
III
the Fe /Fe redox couple. The electrochemical behaviors of the
complexes are comparable to those reported for analogous com-
plexes [16,61].
3.6. Oxidation of 2-aminophenol
The oxidation of 2-aminophenol (OAPH) by molecular dioxygen
in the presence of complex 1 was performed in methanol solution
Fig. 10. Time courses of cleavage of pUC19 DNA (0.005
20 M) in the presence of H (100 M) 2% DMF/20 mM phosphate buffer (pH
.5) at 37 °C. (A) Agarose gel electrophoresis patterns of pUC19 DNA are shown at
various reaction times: Lanes 1–8: reaction times of 0, 5, 10, 20, 30, 40, 50 and
0 min, respectively. (B) Plot of percentages of supercoiled (I) and nicked-circular
II) DNA vs. reaction time.
lg/lL) by complex 2
(
l
2
O
2
l
7
6
(
hydroxyl radical. On the other hand, the cobalt(III) complex bound
to DNA is first reduced to the cobalt(II) state by ascorbic acid,
which in turn produces reactive hydroxyl radicals on reaction with
hydrogen peroxide. It is worth noting that for all the reported co-
balt(III) complexes which show chemical nuclease activity, both
ꢀ3
Fig. 11. Cyclic voltammogram of complex 1 (1 ꢂ 10 M) in methanol at 25 °C
containing TEAP as the supporting electrolyte at a scan rate of 50 mV/s.

A. Panja / Polyhedron 43 (2012) 22–30
29
at 25 °C. Upon addition of 2-aminophenol to a methanol solution of
under anaerobic conditions, the absorption at 433 nm (kmax of a
first-order dependence was found when the kinetic experiments
were carried out by varying the complex concentration
1
1
typical band of 2-aminophenoxazine-3-one) increased until one
equivalent of 2-aminophenol to 1 was added, and further addition
of substrate did not increase the characteristic band of 2- amin-
ophenoxazine-3-one. The catalytic inefficiency is due to the failure
of the regeneration of the catalyst during the course of the reaction,
as the reduced form of complex 1 is stable in air and does not oxi-
dize by molecular oxygen in the timescale of the reaction. In order
to determine the rate dependence on various reactants, the oxida-
tion processes were performed with different substrates and cata-
lyst concentrations. The kinetic studies on the oxidation of OAPH
were monitored by UV–Vis spectroscopy, measuring the appear-
ance of the absorbance maximum of the reaction mixture at
(Fig. S3). In contrast, complex 2 is inactive towards the oxidation
of 2-aminophenol, which suggests that access of the higher oxida-
tion state of the metal ions is key for such oxidation processes.
4
. Conclusion
Mononuclear cobalt(III) and iron(II) complexes derived from
diimine ligands have been synthesized, and the molecular struc-
tures of the complexes were determined by X-ray crystallography.
The binding affinity of the complexes to CT-DNA was examined by
various techniques, and they support the preferable groove binding
mode of the complexes to DNA. The nuclease activity in the pres-
ence of activating agents shows that the cleavage efficiency was
depended both on the complex concentration and the incubation
period. Inhibition of nuclease activity was observed in the presence
of radical scavengers, and the possible role of reactive oxygen spe-
cies was speculated. Moreover, the cobalt(III) complex has been
found to be a good oxidant for the oxidative coupling of 2-amino-
phenol to the phenoxazinone chromophore.
4
33 nm. A typical time dependent formation of 2- aminophenox-
azine-3-one in the presence of 1 is depicted in Fig. 12. From the
variations of the reaction rates, saturation kinetics was found for
the observed rate constants versus the OAPH concentrations plot
(Fig. 13), which indicates that an intermediate complex–substrate
adduct forms, most probably through stacking interactions
p p
ꢁ ꢁ ꢁ
between the aromatic rings of 1 and the substrate in a pre-equilib-
rium process, followed by its redox decomposition in the rate-
determining step of the oxidation process. On the other hand,
Acknowledgements
The authors gratefully acknowledge the financial support of this
work by the University Grant Commission (UGC), India (Sanction
No. PSW-173/11-12).
Appendix A. Supplementary data
CCDC 873326 and 873327 contains the supplementary crystal-
lographic data for 1 and 2. These data can be obtained free of
charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
from the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: de-
posit@ccdc.cam.ac.uk. Supplementary data associated with this
article can be found, in the online version, at http://dx.doi.org/
1
0.1016/j.poly.2012.05.041.
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