Macrocyclic nickel(II) complexes: Synthesis, characterization, superoxide scavenging activity and DNA-binding

Article abstract of DOI:10.1016/j.molstruc.2012.01.048

A new series of nickel(II) complexes with the tetraaza macrocyclic ligand have been synthesized as possible functional models for nickel-superoxide dismutase enzyme. The reaction of 5-amino-3-methyl-1-phenylpyrazole-4- carbaldehyde (AMPC) with itself in the presence of nickel(II) ion yields, the new macrocyclic cationic complex, [NiL(NO₃)₂], containing a ligand composed of the self-condensed AMPC (4 mol) bound to a single nickel(II) ion. A series of metathetical reactions have led to the isolation of a number of newly complexes of the types [NiL]X₂; X = ClO₄ and BF₄, [NiLX₂], X = Cl and Br (Scheme 1). Structures and characterizations of these complexes were achieved by several physicochemical methods namely, elemental analysis, magnetic moment, conductivity, and spectral (IR and UV-Vis) measurements. The electrochemical properties and thermal behaviors of these chelates were investigated by using cyclic voltammetry and thermogravimetric analysis (TGA and DTG) techniques. A distorted octahedral stereochemistry has been proposed for the six-coordinate nitrato, and halogeno complexes. For the four-coordinate, perchlorate and fluoroborate, complex species a square-planar geometry is proposed. The measured superoxide dismutase mimetic activities of the complexes indicated that they are potent NiSOD mimics and their activities are compared with those obtained previously for nickel(II) complexes. The probable mechanistic implications of the catalytic dismutation of O2- by the synthesized nickel(II) complexes are discussed. The DNA-binding properties of representative complexes [NiLCl ₂] and [NiL](PF₄)₂ have been investigated by the electronic absorption and fluorescence measurements. The results obtained suggest that these complexes bind to DNA via an intercalation binding mode and the binding affinity for DNA follows the order: [NiLCl₂] □ [NiL](PF₄)₂.

Full text of DOI:10.1016/j.molstruc.2012.01.048

Journal of Molecular Structure 1015 (2012) 56–66
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Macrocyclic nickel(II) complexes: Synthesis, characterization, superoxide
scavenging activity and DNA-binding
⇑
Abd El-Motaleb M. Ramadan
Chemistry Department, Faculty of Science, Kafr El-Sheikh University, Kafr El-Sheikh 33516, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of nickel(II) complexes with the tetraaza macrocyclic ligand have been synthesized as
possible functional models for nickel–superoxide dismutase enzyme. The reaction of 5-amino-3-
methyl-1-phenylpyrazole-4-carbaldehyde (AMPC) with itself in the presence of nickel(II) ion yields,
the new macrocyclic cationic complex, [NiL(NO3)2], containing a ligand composed of the self-condensed
AMPC (4 mol) bound to a single nickel(II) ion. A series of metathetical reactions have led to the isolation
of a number of newly complexes of the types [NiL]X2; X = ClO4 and BF4, [NiLX2], X = Cl and Br (Scheme 1).
Structures and characterizations of these complexes were achieved by several physicochemical methods
namely, elemental analysis, magnetic moment, conductivity, and spectral (IR and UV–Vis) measure-
ments. The electrochemical properties and thermal behaviors of these chelates were investigated by
using cyclic voltammetry and thermogravimetric analysis (TGA and DTG) techniques. A distorted octahe-
dral stereochemistry has been proposed for the six-coordinate nitrato, and halogeno complexes. For the
four-coordinate, perchlorate and fluoroborate, complex species a square-planar geometry is proposed.
The measured superoxide dismutase mimetic activities of the complexes indicated that they are potent
NiSOD mimics and their activities are compared with those obtained previously for nickel(II) complexes.
The probable mechanistic implications of the catalytic dismutation of OÅ2ꢀ by the synthesized nickel(II)
complexes are discussed. The DNA-binding properties of representative complexes [NiLCl2] and
[NiL](PF4)2 have been investigated by the electronic absorption and fluorescence measurements. The
results obtained suggest that these complexes bind to DNA via an intercalation binding mode and the
binding affinity for DNA follows the order: [NiLCl2] h [NiL](PF4)2.
Received 12 December 2011
Received in revised form 27 January 2012
Accepted 28 January 2012
Available online 4 February 2012
Keywords:
Synthesis
Characterization
SOD
Nickel(II)
DNA-binding
Biomimetic
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
resent the first line of defense against OÅ2ꢀ. Preclinical studies have
revealed that SOD enzymes play a protective effect in animal mod-
Superoxide anion (OÅ2ꢀ), the one-electron reduction product of
dioxygen (O2), is a toxic reactive oxygen species [1]. In eukaryotes,
its principal source is the mitochondrial inner membrane, both
sides of which release superoxide as a byproduct of aerobic metab-
olism [1]. Superoxide anion and the reactive oxygen species (ROS)
that be derived from it is the respiratory chain within the mito-
chondria [2]. ROS include hydroxyl radicals, superoxide anion,
hydrogen peroxide and nitric oxide. They are very transient species
due to their high chemical reactivity that leads to broad array of
diseases [3] including autoimmune, inflammatory disorders such
as rheumatoid arthritis (RA) and ischemic diseases as well as organ
failure from species or trauma. There is much evidence implicating
oxidative stress and mitochondrial dysfunction in Alzheimer’s and
Parkinson’s diseases, amyotrophic lateral sclerosis (ALS), and, in-
deed, in aging and age-associated decline [4]. By catalyzing the
conversion of OÅ2ꢀ to H2O2 and O2, superoxide dismutase (SOD) rep-
els of several diseases [5]. However, the therapeutic use of the na-
tive SOD has several limitations related with low cell permeability
and short half-life. In addition, bovine SOD was tested in clinical
trials but immunological problems lead to its withdrawal from
the market [6]. To overcome this problem, synthetic SOD mimetic
compounds have emerged as a potential novel class of drugs. A list
of diseases that may be treatable with SOD mimics now includes
heart attacks, stroke, autoimmune diseases (such as osteoarthritis),
neurodegenerative diseases (including Alzheimer’s and Parkin-
son’s) and aging [7]. It is the first time that a molecule is a totally
selective SOD catalyst. And by eliminating superoxide, the produc-
tion of pro-inflammatory cytokines can be also eliminated. There-
fore selective non-toxic superoxide substituents for SODs are
desirable for medical purposes [8]. Artificial scavengers with vari-
ous metal ions (Mn, Fe and Cu) have been prepared and tested. Out
of these manganese is the most suitable transition metal for phar-
maceutical applications due to its relatively low toxicity [9]. How-
ever other transition metal complexes are also studied to help
elucidating structure/activity relationships and other properties
⇑
Tel.: +20 403317838.
E-mail address: ramadanss@hotmail.com
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2012.01.048

A.E.-M. M. Ramadan / Journal of Molecular Structure 1015 (2012) 56–66
57
influencing SOD activity. Among these SOD-functional models, me-
tal complexes with macrocyclic ligands constitute an important
group [10].
favorable enthalpy for the formation of metal ligand bonds over-
comes the unfavorable entropy of the ordering of the multidentate
ligand around metal ion thus promotes the cyclization reaction
[26]. This work describes the synthesizes and characterization of
a series of new tetraaza macrocyclic nickel(II) complexes. It has
been documented in the literature that small molecule SOD mimics
exhibit DNA binding ability and nuclease activity [27]. Hence, be-
fore studying the DNA interaction of the complexes, we examined
the superoxide scavenging activity by phenazine methosulphate
(PMS) and nitroblue tetrazolium (NBT) assay. The DNA binding
activity was also investigated and is described in this study.
Two classes of superoxide detoxification enzymes are known:
superoxide dismutase (SOD) enzymes and superoxide reductase
(SOR) enzymes [1]. The most recently identified superoxide dismu-
tases is nickel superoxide dismutase (NiSOD). This enzyme has been
isolated from a number of Streptomyces bacteria and from cyano-
bacteria. The crystal structure has been published by two groups
[11,12] and show a hexameric structure with a molecular weight
of approximately 80,000 Daltons, where each monomeric unit con-
tains what has been described as a ‘‘nickel-hook.’’ Each nickel is
bound by a small peptide chain with binding ligands that consist
of two amides, one from the N-terminal and one from the backbone
of cysteine 2, two thiolates, one from cysteine 2 and one from cys-
teine 6, and an imidazole from histidine 3. This is the first SOD in
which the binding ligands are anything other than histidines, aspar-
tates and water/hydroxide. The nickel is redox active but the details
of the mechanism are still to be unraveled, in large part because of
the complexity of making nickel redox active with superoxide.
One significant difference between NiSOD and the other SODs is
the apparent lack of ionic strength effect on the catalytic rate con-
stant [13], suggesting there is limited need to generate an electro-
static funneling mechanism. Thus far, preparation of a short chain
peptide to mimic the nickel hook region has failed to produce a
functional NiSOD mimic although it has produced spectroscopic
mimics [14]. The role of the protein is clearly crucial but the details
remain to be elucidated. The enzymatic activity of NiSOD is as high
as that of Cu–ZnSOD at about 109 Mꢀ1 Sꢀl per metal center. Re-
cently some low molecular weight nickel(II) complexes mimicking
superoxide dismutase (SOD) activity with high catalytic activity
have been reported [15].
2. Experimental
2.1. Materials
All chemicals used were of analytical grade. 5-amino-3-methyl-
l-phenylpyrazole-4-carbaldehyde (AMPC) was prepared according
to the method described elsewhere [28a].
2.2. Synthesis of the tetraaza macrocyclic [Ni(II)L(NO3)2] complex
A solution of freshly prepared 5-amino-3-methyl-l-phenylpyra-
zole-4-carbaldehyde (0.024 mol) in absolute ethanol (50 ml) was
heated and stirred. While the 5-amino-3-methyl-l-phenylpyra-
zole-4-carbaldehyde solution was under reflux, a solution of the
appropriate amount of nickel nitrate Ni(NO3)2 6H2O in 50 ml of
absolute ethanol was added. The reaction mixture was boiled un-
der reflux and stirred for 24 h, during which time colored products
precipitated. After cooling, the solution was filtered and the iso-
lated solid was washed with ethanol and ether and finally dried
in vacuum over CaO at room temperature for 2 weeks.
The design of small molecules that bind and react at specific se-
quences of DNA under physiological conditions via oxidative and
hydrolytic mechanisms has been attracted great interest in the field
of bioinorganic chemistry [16–18]. Intercalators are small molecules
that contain a planar aromatic heterocyclic functionality which can
insert and stack between the base pairs of double helical DNA [19].
Recently interaction of transition metal complexes with DNA has at-
tracted much attention [20]a–e. In general, transition metal com-
plexes can interact non-covalently with DNA by intercalation, via
groove binding or external electrostatic binding. Among the factors
governing the binding modes, it appears that the most significant is
likely to be that of molecular shape. Studies reveal that the ligand
modifications in geometry, size, hydrophobicity, planarity, the
charge and hydrogen-bonding ability of the complexes, may lead
to subtle or to spectacular changes in the binding modes, location,
affinity, and to a different cleavage effect [21]. These properties have
turned chemical nucleases into useful tools as adjuvant in PCR diag-
nostics [22], nucleic acid attacking and cleaving agents [23]. There-
fore, extensive studies using different structural transition metal
complexes to evaluate and understand the factors that determine
the mode of binging interactions with DNA and the cleavage mech-
anisms are necessary. A broad variety of transition metal complexes
have been widely exploited for these purposes not only because of
their unique spectral and electrochemical signatures but also due
to the fact that by changing the ligand environment one can tune
the DNA binding and cleaving ability of a metal complex [24]. Sev-
eral efficient cleaving agents have been developed in course of time.
However, most of the studies are dealing about non-cyclized mono-
nuclear metal complexes. In that few of them are reported as macro-
cyclic mononuclear metal complexes which show greater cleaving
efficiency or DNA interaction than the non-cyclized complexes [25].
Many synthetic routes to macrocyclic ligand involve the use of
the metal ion template to orient the reacting groups of the reacting
substrates in the desired conformation for the ring closure. The
2.3. Synthesis of [NiLX2] and [NiL]X2 complexes
Metathetical displacement of the nitrate ion was accomplished
by dissolving [NiL(NO3)2] in hot MeOH, and adding an excess of
NaX (X = Cl, Br, ClO4 or BF4) aqueous solution. The reaction mixture
was stirred for 60 min at room temperature. In most cases the
complex salts precipitated after slow evaporation of MeOH. The
colored precipitates were isolated by filtration, washed with water,
ethanol and finally dried in vacuum at room temperature over CaO.
2.4. Physical measurements
The I. R. spectra were recorded using KBr disks in the 4000–
200 cmꢀ1 range on a Unicam SP200 spectrophotometer. The elec-
tronic absorption spectra were obtained in DMF solution with a Shi-
madzu UV-240 spectrophotometer. Magnetic moments were
measured by Gouy’s method at room temperature. ESR measure-
ments of the polycrystalline samples at room temperature were
made on a Varian E9 X-band spectrometer using a quartz Dewar ves-
sel. All spectra were calibrated with DPPH (g = 2.0027). The specific
conductance of the complexes was measured using freshly prepared
10ꢀ3 M solutions in electrochemically pure MeOH or DMF at room
temperature, using an YSI Model 32 conductance meter. The ther-
mogravimetric measurements were performed using a Shimadzu
TG 50-Thermogravimetric analyzer in the 25–800 °C range and un-
der an N2 atmosphere. Elemental analyses (C, H, and N) were carried
out at the Micro analytical Unit of Cairo University.
2.5. Electrochemical measurements
Cyclic voltammetric measurements were performed by a com-
puterized Electrochemical Trace Analyzer Model 273A-PAR

58
A.E.-M. M. Ramadan / Journal of Molecular Structure 1015 (2012) 56–66
(Princeton Applied Research, Oak Ridge, TN, USA) controlled via
270/250 PAR software was used for the cyclic voltammetry mea-
surements. The electrode assembly (Model 303A-PAR) incorporat-
3. Results and discussion
3.1. General
ing of
a micro-electrolysis cell of a three electrode system
comprising of a hanging mercury drop electrode as a working elec-
trode (area: 0.026 cm2), an Ag/AgCl/KCls reference electrode and a
platinum wire counter electrode, was used. Stirring of the solution
in the micro-electrolysis cell was performed using a magnetic stir-
rer (305-PAR) to provide the convective transport during the pre-
concentration step. The whole measurements were automated
and controlled through the programming capacity of the appara-
tus. The supporting electrolyte used was 0.1 M n-Bu4NClO4. The
solvents used were sufficiently pure and the concentration of the
complexes was 0.001 M.
The reaction of 5-amino-3-methyl-1-phenylpyrazole-4-carbalde-
hyde (AMPC) with itself in the presence of nickel(II) ions yields, new
macrocyclic cationic complex, [NiL(NO3)2], containing a ligand com-
posed of the self-condensed AMPC (four mol) bound to a single nicke-
l(II) ion. A series of metathetical reactions have led to the isolation of a
number of newly complexes of the types [NiL]X2; X = ClO4 and BF4,
[NiLX2], X = Cl and Br (Scheme 1). These isolated complexes have been
identified and characterized, principally, based on the analytical data
as complexes containing the fully cyclized ligand (L). However, at-
tempts to synthesize the corresponding metal-free macrocyclic ligand
did not prove successful. All the complexes are microcrystalline in nat-
ure, stable to atmosphere and are soluble in the polar solvents such as
DMF and DMSO at room temperature. The results of elemental analysis
(Table 1) agree well with the proposed mononuclear structure of these
Schiff-base macrocyclic complexes. The formation of the Schiff base
macrocyclic frame work was further confirmed by IR-spectra and
TGA data. The halide or nitrate ions were found to be coordinated to
the metal ion as confirmed by thermal analysis data, values of magnetic
moments and conductivity measurements. The molar conductivity
data for ca. 10ꢀ3 M of [NiLX2] (X = NO3, Cl or Br), in DMF solutions at
room temperature are in accordance with those expected for a non-
electrolyte, while the measured value for [NiL]X2 (X = ClO4 or BF4), un-
der the same conditions is as expected for a 1:2 electrolyte [29]. These
observations imply that the counter anions in the nitrato and halogeno
complex species are coordinated to the nickel(II) ion, while ClO4 or BF4
in [NiL]X2 solution do not participate in coordination. Several at-
tempts failed to obtain a single crystal suitable for X-ray crystallog-
raphy. However, the analytical, spectroscopic and magnetic data
enable us to predict the possible structure of these newly synthe-
sized complexes (Scheme 1).
2.6. Superoxide dismutase assay
SOD mimetic catalytic activity of the investigated copper(II)
complexes was assayed by using phenazine methosulphate (PMS)
to photogenerate a reproducible and constant flux of OÅ2ꢀ at
pH = 8.3 (phosphate buffer). Reduction of nitroblue tetrazolium
to blue formazan was used as an indicator of OÅ2ꢀ production and
followed spectrophotometrically at 560 nm. The addition of PMS
(9.3 ꢁ 10ꢀ5 M), and phosphate buffer (final volume of 2 ml) caused
a OD(D1 560/min change of 0.035. The reaction blank samples and
)
in the presence of nickel(II) complexes was measured for 5 min.
Each experiment was performed in duplicate and the SOD activity
has been defined as the concentration of the tested compound for
the 50% inhibition of the NBT (1.7 ꢁ 10ꢀ4 M) reduction (IC50 value)
by the superoxide anion radical (OÅ2ꢀ) produced.
2.7. DNA-binding studies
2.7.1. Absorption spectra method
DNA binding experiments were carried out in 0.1 M phosphate
buffer (pH 7.2) using a solution of calf thymus (CT) DNA which
3.2. Infrared spectra
gave a ratio of UV–Vis absorbance at 260 and 280 nm (A260/A280
)
The IR spectra in the (4000–200 cmꢀl) region provide informa-
tion regarding the coordination mode in the complexes and were
analyzed by comparison with data for the free AMPC and the pure
isolated complexes. The most relevant bands and proposed assign-
ments for all the complexes are mentioned throughout this section.
In the IR spectra of the complexes, the absence of band in the region
of ca. 1.8 indicating that the CT-DNA was sufficiently protein free.
The concentration of DNA solution was determined by the absor-
bance at 260 nm using e260 value of 6600 Mꢀ1 cmꢀ1 at 260 nm as
reported in the literature [28b]. Absorption titration experiments
of nickel(II) complexes [NiLCl2] and [NiL](PF4)2 were performed
using a fixed complex concentration to which increments of the
DNA stock solution were added. Complex solutions employed were
3400 cmꢀ1 corresponding to free primary
t(NH2) and the absence of
free carbonyl function C@O at 1700 cmꢀ1 suggests, that complete
20 lM in concentration and calf thymus DNA was added to give a
ratio of 8:1 [DNA]/[complex]. Complex-DNA solutions were al-
lowed to incubate for 10 min before the absorption spectra were
recorded.
self condensation of amino group by carbonyl group has taken place.
A strong intensity band at 1620 cmꢀ1 attributable to
t(C@N) [30]
provide strong evidence for the presence of cyclic product. These re-
sults strongly suggest that the proposed ligand framework is
formed. This has been further corroborated by the appearance of a
sharp band in the 450–440 cmꢀ1 region, which may reasonably be
2.7.2. Fluorescence spectra measurements
The fluorescence spectra was employed to determine the rela-
tive DNA binding properties of the complexes [NiLCl2] and
[NiL](PF4)2 to CT-DNA in 50 mM Tris–HCl/1 mM NaCl, buffer, pH
7.5. Fluorescence intensities of the examined complexes at
305 nm with an excitation wavelength of 285 nm were measured
at different CT-DNA concentrations. Reduction in the emission
intensity was observed with addition of CT-DNA. The relative bind-
ing tendency of the complexes to CT-DNA was determined from
Stern–Volmer plot. Plotting the relative emission intensities (I0/I)
against [DNA] yields a linear Stern–Volmer plot that describes
the static quenching process. I0 and I are the fluorescence intensi-
ties of complex in the absence and presence of DNA (quencher)
respectively. The slope of this plotted line yields ksv, the static
quenching constant or associative equilibrium constant which
measure the binding ability of the complexes to CT-DNA.
assigned to
t
(MAN) vibration. Bands observed in the region 1160–
1200 cmꢀ1 may reasonably be assigned to
t
(CAN) stretching vibra-
tions. The spectral features of these nickel(II) macrocyclic complexes
are similar to that reported for the structurally analogous
[Pt(TAAP)]2+, [Pd(TAAP)]2+ [31] and [Ni(TAAB)]2+ [32]. Bands attrib-
uted to functional groups in the macrocycles are observed in the fol-
lowing regions (cmꢀ1): 3000–3150, aromatic
aliphatic (CAH), 1610–1620;
t
(CAH), 2900–3000;
t
t(C@C), 1560–1575 and the out of
plan bending of phenyl, at ca. 710–745.
The infrared absorption bands of the anions in these complexes
show that the perchlorate and fluoroborate are not coordinated to
nickel(II) center. This fact was inferred from the strong broad
bands occur at 1085 and 621 cmꢀ1 and a medium broad band at
412 cmꢀ1 for the perchlorate and at 1050, 620, and 412 cmꢀ1 for

A.E.-M. M. Ramadan / Journal of Molecular Structure 1015 (2012) 56–66
59
Table 1
Molecular formulae, elemental analyses, and physical properties of the tetraaza macrocyclic nickel(II) complexes.
Complex
Color
K
(X
ꢀ1 cm2 molꢀ1
)
Found (cacld.)
C
H
N
M
1. [NiLCl2]
2. [NiLBr2]
3. [NiL(NO3)2] 2H2O
4. [NiL](PF4)2
5. [NiL](ClO4)2
Red
Brown
Dark red
Pale brown
Greenish yellow
21.10
15.26
27.16
145.78
148.65
60.77(61.26)
55.33(55.54)
55.21(55.53)
56.97(56.99)
53.12(53.34)
4.13(4.17)
3.67(3.79)
4.45(4.21)
4.17(3.88)
3.57(3.63)
19.22(19.51)
17.44(17.68)
17.65(17.68)
18.11(18.14)
16.86(16.98)
6.79(6.81)
5.59(6.17)
6.64(6.17)
6.51(6.34)
5.39(5.93)
the fluoroborate. The spectrum of the nitrato complex displays
strong bands at about 1410 and 1310 cmꢀ1 and a band of medium
intensity at 1065 cmꢀ1. These features indicate that the nitrate an-
ions are coordinated to nickel(II) ion in the monodentate fashion
[31]. The coordination of the nitrato group is further supported
by appearance of new band at 260 cmꢀ1, which may reasonably
room temperature magnetic moments which confirmed the pro-
posed stereochemistries for these macrocyclic nickel(II) chelates.
In addition to the ligand field d–d bands, the spectra exhibit
strong absorption bands in the high energy region (27,027–
27,777 cmꢀ1), arising from the charge-transfer transitions, e.g.
M ? L(pꢂ) and L ? M. The spectrum of the free AMPC shows a
medium band at 26,715 cmꢀ1 owing to the pyrazole moiety or
the aromatic benzene ring bound to the nitrogen of the pyrazole
be assigned to
t(MAO) of the OANO2 group [33]. The absorptions
due to the lattice water molecules are evident in the spectrum of
nitrato complex, occurring as
a
broad absorption at about
ring, and is assignable to p ?
pꢂ. This band is shifted to slightly
3450 cmꢀ1 and a weak shoulder at 1655 cmꢀ1 [33]. For the halo-
geno complexes, [CuLX2]; X = Cl or Br, the nonelectrolyte halogens
exhibit far IR absorptions at 310–340 cmꢀ1, indicating an axial
coordinated halide ions [33].
higher energies in the spectra of complexes and the extinction
coefficient is approximately up to four times as large as the coeffi-
cient for AMPC itself. This is a reasonable result since each complex
molecule is composed of four AMPC moieties.
3.3. Electronic absorption spectra
3.4. Magnetic moment measurements
The electronic spectra of the synthesized mononuclear nickel(II)
macrocyclic complexes are measured in DMF solutions at room
temperature and the results obtained are tabulated in Table 2.
The electronic absorption spectra of the six-coordinate complexes
show three absorption bands in the regions of 13,460–14,126,
17,564–18,321 and 19,598–20,032 cmꢀ1. These absorption bands
The effect of a series of ligands of increasing strength on the
spin state of certain transition metal ions is expected to produce
high-spin compounds for weak field ligands and low-spin com-
pounds for strong field ligands. Consequently, the magnetic mo-
ments of such metal complexes are expected to lie in the region
of the two extremes, this indeed being the case with the reported
nickel(II) macrocyclic complexes.
may be assigned to the 3A2g ? 3T2g
(t t2) and
1), 3A2g ? 3T1g (F), (
3A2g ? 3T1g (P), (
t
3) transitions, respectively, and reflect that nick-
The results of room temperature magnetic measurements are
recorded in Table 2. These data show an increase of the magnetic
moment from diamagnetism to complete paramagnetic as the
strength of the axial perturbation is increased. With anions of poor
coordinating ability, viz., ClOꢀ4 , and BFꢀ4 , the resultant complexes
are diamagnetic and therefore have singletꢀground states. With an-
ions of greater coordinating ability, viz., NO3 , Clꢀ and Brꢀ the resul-
tant complex exhibits moments consistent with triplet ground
state. However, with anions, viz., Clꢀ and Brꢀ, the observed mag-
netic moments are lower than that for nitrato complex. During this
change from singlet to triplet ground states, it appears reasonable
to assume that the in-plane ligand field remains essentially con-
stant. Visible reflectance spectra on solid samples are consistent
with the aꢀssociated changes in spin of the ground state. The spectra
of the ClO4 , and BFꢀ4 complexes are consistent with the presence of
a planar nickel(II) species, while that of NOꢀ3 , Clꢀ and Brꢀ suggest
octahedral or tetragonal nickel(II) structure. These anomalous
room temperature magnetic moments of the chloro and bromo
el(II) d8 ions is in a distorted octahedral geometrical environment
[34,35]. The d–d absorption bands for these complexes increase to-
ward higher energies in the order Br < Cl < NO3, indicating that the
presence of these coordinated axial ligands affects the ligand field
strength of the complexes. The Dq values of [NiL(NO3)2], [NiLCl2]
and [NiLBr2] are 1412, 1400 and 1346 cmꢀ1, respectively.
The electronic absorption spectra of the four-coordinate perchlo-
rate and fluoroborate complexes display three d–d transitions in the
low energy region at, 13,699–14,285, 16,666–16,806 and 19,607–
20,000 cmꢀ1. These bands are assignable to the spin-allowed d–d
bands: dxy (b2g) ? dx2–y2 (b1g), d2 (a1g) ? dx2–y2 (b1g) and dxz,yz
Z
(eg) ? dx2–y2 (b1g) corresponding to: 1A1g ? 1A2g
,
1A1g ? 1B1g and
1A1g ? 1Eg transitions, respectively. These spectral features of
[NiL](ClO4)2 and [NiL](BF4)2 are similar to each other and consis-
tence with the low-spin d8-metal ion in the square planer environ-
ment [34,35]. The geometry of the reported nickel(II) tetraaza
macrocyclic complexes is further supported by measuring their
Table 2
Electronic absorption spectra (cmꢀ1) of the reported nickel(II) complexes.
Complex
kmax (cmꢀ1
3A2g 3T2g
)
?
3A2g
?
3T1g (F)
3A2g
?
3T1g (P)
p
?
pꢂ
Dq
leff BM
[NiLCl2]
[NiLBr2]
[NiL(NO3)2] 2H2O
13,460
14,000
14,126
17,564
17,986
18,321
19,598
19,805
20,032
32,786
32,258
33,898
1346
1400
1412
3.59
3.56
3.93
1A1g
?
1A2g
1A1g
?
1B1g
1A1g 1Eg
?
[NiL](PF4)2
[NiL](ClO4)2
14,285
13,966
16,806
16,666
20,000
19,607
34,482
33,003
Diamagnetic
Diamagnetic

60
A.E.-M. M. Ramadan / Journal of Molecular Structure 1015 (2012) 56–66
complex species were interpreted on the assumption of the exis-
tence of equilibrium between high and low spin isomers.
3.6. Electrochemical properties
It has been reported that for nickel(II) complexes, the relative
energies of the singlet and triplet states be very sensitive to the
nature of two ligands located axially, above and below the metal
atom which is otherwise bound to a planar array of donor atoms
[35,36]. Most simply, so long as the axial ligands compare closely
in their binding strength to the in-plane ligands, the structure is
grossly octahedral and the triplet state lies lowest. As the axial li-
gand-field strength is decreased, the singlet state drops in energy
relative to the triplet. Thus, at some particular point in relative ax-
ial-in-plane ligand field strengths, the singlet and triplet states
should coexist. On the other hand, an examination of the axial per-
turbation by anions X, X = Cl, Br and NO3, in NiL4X2, where L4 rep-
The electrochemical behavior of the synthesized nickel(II) mac-
rocyclic complexes was investigated, as the redox potential is an
important parameter in electron transfer processes in general
and also in our catalytic systems. The redox potentials of the cata-
lysts and species involved in the redox cycles have to match some
criteria. The oxidation of Ni(II) center to Ni(III) by the substrate
(OÅ2ꢀ) and the subsequent reduction of Ni(III) to Ni(II) by another
molecule of OÅ2ꢀ are significant steps in the catalytic cycle. The re-
dox properties of the complexes reported in the present work were
studied by cyclic voltammetry in the potential-range ꢀ1.0 to 1.5 V
in dimethylformamide containing 10ꢀ1 M tetra (n-butyl) ammo-
nium perchlorate. (Supplementary material S2), depicts the cyclic
voltammogram of the nitrato complex [NiL (NO3)2]. All the com-
plexes undergo reduction and oxidation potentials and their cyclic
voltamrnetric behavior is found to be similar and displayed two
well-defined electrode couples. The complexes showed two suc-
cessive one electron processes and the first electrode couple is
safely assigned to the reversible couple NiII/NiI with E1/2 of ꢀ417
resents
a planar field produced by four neutral donors,
demonstrated that: complexes with anions of negligible or low-
coordinating ability are diamagnetic while those of medium to
good coordinating power exhibit room temperature magnetic mo-
ments consistent with octahedral or weakly tetragonal nickel(II)
complexes. No examples were obtained with intermediate room
temperature magnetic moments, the data merely dividing the
complexes into two groups, those with singlet ground states and
those with triplet ground states as reported in the present study.
to ꢀ526 mV (
DEp = 58–70 mV) and represented as follows:
½NiLꢃ2þ þ e ꢀ ½NiLꢃþ
ð1Þ
The second electrode couple with E1/2 = 420–523 mV is assigned
to the reversible electrode couple NiIII/NiII by comparison with
analogous Ni(II) complexes [37]. The pertinent redox couple is rep-
resented in the following electron transfer series (2):
3.5. Thermal studies
The structure characterization of the synthesized nickel(II) mac-
rocyclic complexes, was further elucidated by employing the ther-
mogravimetric technique and the proposed structures of the
reported nickel(II) chelates are confirmed. The thermogravimetric
analysis (TGA/DTG), and curves were obtained at a heating rate
of 10 °C/min in a nitrogen atmosphere over a temperature range
of the ambient temperature to 850 °C. The weight loss correspond-
ing to different stages of pyrolysis were calculated and compared
with those of the expelled groups (Table 3).
2þ
½NiLꢃ3þ þ e ꢀ ½NiLꢃ
ð2Þ
The complexes that undergo reduction are completely regener-
ated following electrochemical oxidation (the ratio of the peak cur-
rents ia/ic is about 1.0), suggesting
Ninþ ꢀ Niðnþ1Þ one-electron transfer processes (1) and (2). How-
ever, the value of the limiting peak-to-peak separation ( Ep),
a chemically reversible
D
which lies within the normal values for a reversible one-electron
redox process suggests that the heterogeneous electron-transfer
The thermogram of the two halogeno and the fluoroborate com-
plex species are similar and consist of two well defined stages. The
first decomposition stage starts at 170–200 °C and continues until
250–300 °C is corresponding to the removal of the two axially
coordinated halogeno ions and the electrolytic BF4 counter ions
constituting about 8.11%, 14.59% and 16.55% of the total weight
loss (calcd. 8.23%, 14.63% and 16.83%) for the chloro, bromo and
fluoroborate complexes respectively. Since the decomposition
started above 170 °C, the presence of any solvent/water molecule
may be ruled out which are also evident from the elemental anal-
ysis. The second stage runs over 300 °C and is consistent with the
degradation of the organic part of the complex molecule in succes-
sive unresolved steps. Above 800 °C the TGA curve shows no
change even when the temperature was raised to 850 °C. This hor-
izontal constant curve may be due to the presence of metal oxides
(NiO) residue in the remaining part. The total loss of weight agrees
with the corresponding calculated data.
process in these complexes is easily reversible (DEp, 60 mV for a
reversible one-electron redox process) [38] and not accompanied
by stereochemical reorganization [39]. The impact of the electro-
chemical properties on the catalytic reactivity of these complexes
will be discussed below. Table 4, summarizes the half-wave poten-
tial (DEp) data calculated as the arithmetic mean of the Epc and Epa
values. The EI/2 values lie between the potentials of the reduction of
O2 to OÅ2ꢀ (ꢀ0.16 V vs. NHE at pH 7) and OÅ2ꢀ to H2O2 (+0.89 V vs.
NHE) as it is expected for compounds with a SOD-like activity in
dependence on the OÅ2ꢀ concentration [40]. However, the similarity
in the E1/2 values for the reported nickel(II) complexes show that
the axially coordinating and electrolytic anions, have a negligible
contribution on the redox potentials.
3.7. Superoxide dismutase (SOD) biomimetic catalytic activity
The TG curve of the nitrato complex [NiL(NO3)2].2H2O is shown
in (Supplementary material S1), which exhibits three decomposi-
tion steps within the temperature range from 75 to 850 °C. The first
two steps of decompositions within the temperature range 75–
300 °C corresponds to the loss of water molecules of hydration,
and the coordinated nitrate ions with a mass loss of 3.33% (calcd.
3.78%) and 12.34% (calcd. 13.03%). The subsequent step (300–
850 °C) with the maximum decomposition peaks DTGmax at
650 °C, corresponds to the removal of the organic part of the ligand
leaving metal oxide as a residue. The overall weight loss amounts
to 91.55% (calcd. 92.65%). The mass losses in this stage are in a
good agreement with calculated mass losses and the final product
is quantitatively proved to be anhydrous metallic oxide (NiO).
The superoxide dismutase mimetic activity of the reported
nickel(II) complexes was assayed by the use of phenazine metho-
sulphate (PMS) as a photogenerator of the superoxide anion radical
in association with nitroblue tetrazolium (NBT) chloride as scaven-
ger of the superoxide [41]. Inhibition of the reduction of NBT to for-
mazan (F) by the synthesized nickel(II) complexes was used for
detection of the SOD like activity of theses macrocyclic chelates
in the phosphate buffer under similar biological conditions as pre-
viously described [42]. The chromophore concentration required to
yield 50% inhibition of the reduction of NBT (IC50) was determined
by following the literature method [15b]. A 50% inhibition reduc-
tion of NBT (the IC50 value) for complexes 1–5 was obtained from
the plot of percentage of inhibition vs the complex concentration

A.E.-M. M. Ramadan / Journal of Molecular Structure 1015 (2012) 56–66
61
Table 3
Thermogravimetric analysis data of the tetraaza macrocyclic nickel(II) complexes 1–4.
Complex
[NiLCl2]
Step
DTG (°C)
Temp. range (°C)
Mass loss (%) found (calc.)
Species formed
1
2
180
635
170–250
250–720
08.11(08.23)
83.25(83.52)
NiL
NiO
[NiLBr2]
1
2
215
680
190–250
250–720
16.55(16.83)
74.87(75.85)
NiL
NiO
[NiL(NO3)2] 2H2O
1
2
3
90
280
650
75–100
100–300
300–750
03.33 (03.78)
12.34(13.03)
75.88(75.84)
[NiL(NO3)2]
NiLNiO
[NiL](PF4)2
1
2
260
500
200–300
300–740
14.59(14.63)
77.12(77.79)
NiL
NiO
as shown in Fig. 1 for complex [NiL(NO3)2]. The IC50 values are
summarized in Table 5 along with other IC50 values obtained pre-
viously for nickel(II) complexes. The data in Table 5 reveal that the
IC50 values of reported nickel(II) complexes are in the same level of
the other nickel(II) SOD-mimics [15a,15c].
structural features of these complexes. The overall mechanism has
been called a ping–pong mechanism proposed for the dismutation
of superoxide anion radical by both the native M-SOD and metal
complexes (SOD-mimics) is thought to involve redox cycling of
Mn+ ion as follows:
The SOD-like activity of some metal complexes is a function of
several factors, among them is the exchange ability of the axial
coordinated ligand, the steric hindrance and flexibility of the metal
ion to the geometrical changes. The fast exchange of ligand coordi-
nated axially to the central metal ion and limited steric hindrance
to the approach of the OÅ2ꢀ anion are considered essential require-
ments for the successful binding of the OÅ2ꢀ. The flexibility of the
metal complex to geometrical arrangement changes, during the re-
dox cycling of Mn+/M(nꢀ1), which facilitates the interaction of the
OÅ2ꢀ is also important. In addition, the nature of coordinated ligands
to the metal ion is also playing an important role in enhancing the
SOD like activity of the SOD mimics [43]. The macrocycle frame-
work provides stable and flexible environment similar to that in
the active site of the native enzyme, ensuring the stable existence
of the complexes at the investigated pH value in aqueous solution.
In addition our spectral and electrochemical investigations dis-
played that this tetraaza macrocyclic ligand system provides these
mentioned requirements for its nickel(II) complexes as SOD mim-
ics (see Table 5).
Mðnþ1Þþ-SOD þ O2Åꢀ ! Mnþ-SOD þ O2
ð3Þ
Mnþ-SOD þ O2Åꢀ þ 2Hþ ! Mðnþ1Þþ-SOD þ H2O2
ð4Þ
where M = Cu (n = 1); Mn (n = 2); Fe (n = 2); Ni (n = 2). In this reac-
tion the oxidation state of the metal cation oscillates between n and
n + 1.
Self-dismutation of OÅ2ꢀ at pH 7.4 occurs with a rate constant,
k ꢄ 5 ꢁ 105 Mꢀ1 sꢀ1, and is increased more than three orders of
magnitude in the presence of SOD [1]. All SOD enzymes, regardless
of the type of metal (Mn, Fe, Cu, Ni), have metal-centered reduction
potential around +300 mV vs NHE, which is midway between the
potential for the reduction of O2 (+890 mV vs. NHE) and oxidation
of OÅ2ꢀ (ꢀ160 mV vs. NHE). Thus, both processes are thermodynam-
ically equally favored at ꢄ+300 mV vs NHE. The data in Table 4 dis-
play that the redox potential (E1/2
) values of the reported
macrocyclic nickel(II) complexes are in the allowed ranges of an
SOD mimic, and follow the order [NiL](PF4)2 h [NiL](ClO4)2 h [NiL-
Cl2] h [NiLBr2] h [NiL(NO3)2] and the SOD mimetic activity of the
complexes follows the inverse of this order.
The data in Table 5 show that, all the synthesized nickel(II)
complexes exhibit promising SOD mimetic catalytic activity which
can be related to the redox potential of the couple NiIII/NiII and the
The remarkable appreciable SOD-like activity observed for the
coordinately saturated octahedral complexes [NiLCl2], [NiLBr2]
and [NiL(NO3)2] is explained in term of the exchange ability of
one of the axially coordinate ligands with the approaching super-
oxide anion. A ligand exchange interaction is expected to occur be-
tween the highly reactive and more basic OÅ2ꢀ and one of the
counter anions occupies the axial position of the octahedral com-
plexes [NiLCl2], [NiLBr2] and [NiL(NO3)2]. The little higher reactiv-
ity of complex [NiL(NO3)2] compared to [NiLBr2], which has E1/2
almost equal to [NiL(NO3)2] may be attributed to the fact that:
the relatively weak axial coordination of nitrato ligands are readily
dissociated in solution to provide axial sites on Ni(II) for OÅ2ꢀ bond-
ing. The dissociation would also facilitate any necessary geometri-
cal changes induced by OÅ2ꢀ bonding during catalysis as in the
native SOD.
Remarkably although the square planar complex [NiL](ClO4)2
possess very similar redox potential value compared to the hexa-
coordinate octahedral complex [NiLCl2] the later exhibits greater
activity. As well as the square-planar complex [NiL](ClO4)2 is coor-
dinately unsaturated and already has vacant coordination sites
available for OÅ2ꢀ bonding. Similar to the previously reported stud-
ies of copper(II) SOD mimics, the difference in IC50 values for these
complexes should be ascribed to the evident discrepancy in the
structures between them, especially in geometry of nickel(II) cen-
ter. A possible correlation between the strength of the equatorial
Fig. 1. Plot of percentage of NBT inhibition reduction with an increase in the
concentration of complex [NiL(NO3)2].

62
A.E.-M. M. Ramadan / Journal of Molecular Structure 1015 (2012) 56–66
Table 4
Electrochemical data (mv) for the reported nickel(II) complexes.
Complex
First electrode couple
Second electrode couple
Epc
Epa
E1/2
D
Ep
Epc
Epa
E1/2
DEp
[NiLCl2]
[NiLBr2]
[NiL(NO3)2] 2H2O
[NiL](PF4)2
[NiL](ClO4)2
ꢀ485
ꢀ480
ꢀ385
ꢀ495
ꢀ490
ꢀ553
ꢀ550
ꢀ450
ꢀ557
ꢀ548
ꢀ519
ꢀ515
ꢀ417
ꢀ526
ꢀ519
68
70
65
62
58
553
457
450
556
545
490
390
390
490
485
512
423
420
523
515
63
67
66
60
60
peroxide molecules to be released in the reductive and oxidative
phases, respectively.
Table 5
Comparison of the superoxide dismutase biomimetic catalytic activity of the reported
nickel(II) complexes with analogous known nickel(II) complexes.
The proposed mechanism for catalysis of OÅ2ꢀ disproportionation
to molecular oxygen and hydrogen peroxide by the reported nick-
el(II) macrocyclic SOD functional models may proceed similarly.
For the six coordinated octahedral complexes an exchange interac-
tions between the highly reactive superoxide anion radical with
the good leaving group X, X = Cl, Br or NO3), will lead to the forma-
tion of a [superoxo-nickel(II)LX] adduct. As a consequence of this
interaction, and due to the excessive build up of the electron density
on nickel(II) center and according to the electro neutrality principal,
nickel(II) undergoes rapid oxidation to nickel(III) with the concom-
Complex
IC50 (M)
kc (Mꢀ1 Sꢀ1
)
Reference
[NiLCl2]
[NiLBr2]
(38.8 0.15) ꢁ 10ꢀ6
(30.5 0.81) ꢁ 10ꢀ6
(21.4 0.19) ꢁ 10ꢀ6
(55.38 1.33) ꢁ 10ꢀ6
(49.50 1.10) ꢁ 10ꢀ6
ꢅ4.00 ꢁ 10ꢀ9
2.60 ꢁ 105
3.33 ꢁ 105
4.72 ꢁ 105
1.82 ꢁ 105
2.04 ꢁ 105
2.52 ꢁ 109
2.10 ꢁ 105
1.55 ꢁ 105
2.52 ꢁ 105
2.10 ꢁ 105
2.34 ꢁ 105
1.74 ꢁ 105
This work
This work
This work
This work
This work
[15a]
[15c]
[15c]
[15a]
[15a]
[NiL(NO3)2] 2H2O
[NiL](PF4)2
[NiL](ClO4)2
Native NiSOD
[NiL](BF4)
48.00 ꢁ 10ꢀ6
[NiL](ClO4)
65.00 ꢁ 10ꢀ6
[NiL1](ClO4)2 (1)
[NiL2](ClO4)2 (2)
[NiL3L](ClO4)2 (3)
[NiL3L0] (4)
40 00 ꢁ 10ꢀ6
48.00 ꢁ 10ꢀ6
itant formation of the peroxide anion O22ꢀ. The protonation of O22ꢀ
-
43.00 ꢁ 10ꢀ6
[15a]
[15a]
prior to its release from the complex is required, because the perox-
ide anion is highly basic and thus too unstable to be released in its
unprotonated form. Owing to the high basicity of O22ꢀ, the six-coor-
dinate [peroxo-nickel(III)-X] adduct becomes highly unstable be-
cause of the excessive build-up of the electron density on the
metal ion. At this stage, probably, the axially coordinated X could
be forced to dissociate from the complex in the solution as HX. Dis-
sociation of the axial X offers the possibility of hydrogen ion (H+)
bonding. The hydrogen proton can then coordinate to the coordi-
nately unsaturated [peroxo-nickel(III)-X] adduct which rearranges
to give the perhydroxyl radical HOꢀ2 . The basic perhydroxyl (hydro-
peroxide) formed is further protonated as a result of electrophilic at-
tack by a proton on the oxygen-bonded nickel(III) with release of
H2O2 and generation of the catalyst in its oxidized inactive form.
In a fast reaction a second superoxide anion radical (OÅ2ꢀ) will coordi-
nate to nickel(III) center in the five coordinate [Ni(III)LX] intermedi-
ate. As a consequence of this interaction, nickel(III) undergoes rapid
reduction to nickel(II) with the release of O2, and regeneration of the
catalyst in its original reduced active form. This assumption mecha-
nism finds supports from the following facts:
58.00 ꢁ 10ꢀ6
field and the SOD activity was suggested [44]. It has been previ-
ously reported that a strong equatorial ligand field opposes the
interaction of the complexed copper with OÅ2ꢀ, disfavoring the
probable formation of the intermediate copper superoxide adduct
[45]. More over, a strong equatorial ligand field is a factor which
disfavors the reduction process from copper(II) to copper(I) during
the catalytic cycle. Coordination of the fifth ligand to copper(II) ion
in the five coordinate square pyramidal or trigonal bipyramidal
complex may reduce the strength of the equatorial field experi-
enced by the copper(II) centers as compared to the four coordi-
nated square planar complexes. Another important factor in the
catalytic process is the ability of the five-coordinate complex to
change its geometry as the geometry of copper in the CuSOD en-
zyme also changes during the catalytic dismutation process [46].
This hypothesis finds support from Patel and co-workers, they re-
ported that the higher SOD activity of the five coordinate copper(II)
complexes is due to the presence of the fifth ligand in the axial po-
sition [47]. A great interaction between the superoxide anion rad-
ical and copper(II) ion in the five coordinate geometry is induced
due to the strong axial bond which results in an increased catalytic
activity [48]. This augment may clarify the difference in the SOD
like activity between the reported octahedral complex [NiLCl2]
and the square-planar complex [NiL](ClO4)2.
(1) The superoxide anion radical (OÅ2ꢀ) has strong reducing and
oxidizing power, and consequently can behave as a good
oxidizing and as well reducing agent.
(2) The fact that OÅ2ꢀ can reduce and subsequently reoxidize the
nickel center requires this center to possess a redox poten-
tial between those of the couples (OÅ2ꢀ/O2) and (OÅꢀ/H2O2)
2
The reaction mechanism for the disproportionation of the toxic
superoxide radical to molecular oxygen and hydrogen peroxide by
the nickel-dependent superoxide dismutase (NiSOD) has been
studied using the B3LYP hybrid DFT method [49]. The obtained re-
sults demonstrated that: Ni-SOD is believed to act like other SODs
according to the formal Eqs. (3) and (4), of the dismutation reac-
tion, where Ni(III)-SOD and Ni(II)-SOD represented the oxidized
and reduced metal center. The catalytic cycle consists of two
half-reactions, each initiated by the successive substrate approach
to the metal center. The first (reductive) phase involves Ni(III)
reduction to Ni(II), and the second (oxidative) phase involves the
metal reoxidation back to its resting state. The Cys2 thiolate sulfur
serves as a transient protonation site in the interim between
the two half-reactions, allowing for the dioxygen and hydrogen
ꢅ 300 mV vs NHE [1]. Our electrochemical results demon-
strated that the macrocyclic framework provides the ability
of the nickel(II/III) redox couple to reach this desire poten-
tial. The probable reaction sequence for the catalytic dispro-
portionation of OÅ2ꢀ can be represented as shown in
Scheme 2.
3.8. DNA-binding
3.8.1. Electronic spectral studies
Electronic absorption spectroscopy has been widely employed to
determine the binding ability of metal complexes with DNA [50a–d].
The binding ability of the synthesized nickel(II) complexes [NiLCl2]

A.E.-M. M. Ramadan / Journal of Molecular Structure 1015 (2012) 56–66
63
Scheme 1. Structure of nickel(II) tetraaza macrocyclic complexes 1–5.
Scheme 2. The proposed mechanism of the catalytic dismutation of OÅ2ꢀ by nickel(II) tetraaza macrocyclic complexes. X = Cl, Br and NO3 in the hexa-coordinate octahedral
Ni(II) complex species (Scheme 1).
and [NiL](PF4)2 with calf thymus (CT) DNA were assayed by measur-
ing the spectral changes of their electronic spectra during the inter-
action with DNA. Complex binding with DNA through intercalation
usually results in hypochromism and bathochromism, due to inter-
calative mode involving a strong stacking interaction between an
aromatic chromophore of the bound ligand and the base pairs of
DNA [51,52]. In the presentinvestigation the interaction of macrocy-
clic nickel(II) complexes [NiLCl2] and [NiL](PF4)2 in DMF solutions
with CT DNA have been investigated. Absorption titration experi-
ments of nickel(II) complexes in buffer were performed by using a
fixed concentration of nickel(II) complex to which increments of
the DNA stock solution were added. The binding of nickel(II) com-
plexes [NiLCl2] and [NiL](PF4)2 to duplexes DNA led to decrease in
the absorption intensities (hypochromism) with a small amount of
red shifts in the UV–Vis absorption spectra (bathochromism). After
intercalating the base pairs of DNA, the pꢂ orbital of the intercalated
the changes of absorbance at 305 nm for complex [NiLCl2] with
increasing concentration of DNA (Supplementary material S3).
The appreciable decrease in absorption intensity and red shift of
the kmax band at 305 nm of [NiLCl2] is suggesting that the complex
binds to DNA strongly [20]. From the absorption titration data, the
binding constant (kb) was determined using the following equation
[54]:
½DNAꢃ
½DNAꢃ
1
¼
þ
ð5Þ
ea
ꢀ
ef eb
ꢀ
ef kbðeb
ꢀ
ef
Þ
where [DNA] is the concentration of CT-DNA in base pairs, ea corre-
sponds to the extinction coefficient observed (Aobs/[complex]), ef is
the extinction coefficient of the free complex, eb is extinction coef-
ficient of the complex fully bound to CT-DNA, and kb is the intrinsic
binding constant. The ratio of slope to intercept in the plot of [DNA]/
(ea–ef) vs [DNA] (Fig. 2) gave the value of kb.
The binding constants (kb) of complexes [NiLCl2] and [NiL](PF4)2
are calculated as 10.83 ꢁ 104 and, 6.50 ꢁ 104 Mꢀ1, respectively.
The obtained results display that both complexes exhibited hypo-
chromism but of varied degree. Slight bathochromic shift was ob-
served with complex [NiLCl2], which was negligible with complex
[NiL](PF4)2. Comparing the intrinsic binding constant of the two
ligand can couple with the
porbital of the base pairs, thus decreasing
the p–
pꢂ transition energy and resulting in the bathochromism [53].
On the other hand, the coupling pꢂ orbital is partially filled by
electrons, thus decreasing the transition probabilities and con-
comitantly resulting in hypochromism. To compare quantitatively
the affinity of the two complexes toward DNA, the binding constants
kb of the two complexes to CT DNA were determined by monitoring

64
A.E.-M. M. Ramadan / Journal of Molecular Structure 1015 (2012) 56–66
Fig. 2. Plot of the ratio [DNA]/(ea–ef) vs. DNA concentration for the reaction between
DNA and complexes [NiLCl2] and[NiL](PF4)2.
Fig. 4. Plot the ratio of I0/I vs. DNA concentration for the reaction between DNA and
complexes [NiLCl2] and [NiL](PF4)2 at 296 K.
complexes with those of DNA-intercalative macrocyclic binuclear
copper(II) complex, such as [Cu2(L)](ClO4) (1.72 ꢁ 104) [55], we
can deduce that complex [NiLCl2] and [NiL](PF4)2 bind strongly to
DNA by intercalation. These spectral characteristics obviously sug-
gest that the two complexes in the present study interact with
DNA most likely through a mode that involves a stacking interac-
tion between the aromatic chromophore and the base pairs of DNA.
DNA led to more effective quenching of the f1uorophore molecule
fluorescence. The reduction of the emission intensity gives a mea-
sure of the DNA binding propensity of the complexes and stacking
interaction (intercalation) between the adjacent DNA base pairs
[56]. The fluorescence quenching curve of DNA-complexes [NiLCl2]
and [NiL](PF4)2 Fig. 4, illustrates that the quenching of complexes
[NiLCl2] and [NiL](PF4)2 is in a good agreement with the linear
Stern–Volmer Eq. (6), which provides further evidence that the
complexes [NiLCl2] and [NiL](PF4)2 bind to DNA [57]:
3.8.2. Fluorescence spectral studies
The fluorescence spectroscopy provides insight of the changes
taken place in the microenvironment of DNA molecule on ligand
binding. To further clarify the interaction of the complexes with
DNA, the fluorescence measurements were performed to deter-
mine the binding ability of the metal complexes with DNA. The
binding of these compounds with calf thymus DNA, were studied
by monitoring the changes in the intrinsic fluorescence of these
complexes at varying DNA concentration. Fig. 3 shows the repre-
sentative fluorescence emission spectra of complex [NiLCl2] upon
excitation at 285 nm. Increment additions of DNA at constant con-
centration of complex solution caused a gradual decrease in the
fluorescence emission intensity with a conspicuous change in the
emission spectra (Fig. 3). The spectra illustrate that an excess of
I0=I ¼ 1 þ ks ½Qꢃ
ð6Þ
v
where I0 and I are the fluorescence intensities of complex in the ab-
sence and presence of DNA (quencher) respectively. ksv is the Stern–
Volmer constant and [Q] is the concentration of DNA. Plotting the
relative emission intensities (I0/I) against [Q] for static process
should yield a linear Stern–Volmer plot that describes the static
quenching process. The slope of this plotted line yields ksv, the static
quenching constant or associative equilibrium constant. The ksv val-
ues for complexes [NiLCl2] and [NiL](PF4)2 are 35.0 ꢁ 103 Mꢀ1
(R = 0.998) and 23.75 ꢁ 103 Mꢀ1 (R = 0.9898), respectively. This data
suggests that the interaction of complex [NiLCl2] with CT-DNA is
stronger than that of complex [NiL](PF4)2, which is consistent with
the above absorption spectral results.
The quenching of the complexes fluorescence clearly indicated
that the binding of the DNA to complexes molecule changed the
microenvironment of fluorophore residue. The shift in emission
peak further depicts effective interaction at higher DNA concentra-
tion, which is more prominent in case of [NiLCl2]. The reduction in
the intrinsic fluorescence of these synthesized complex molecules
upon interaction with DNA could be due to masking or burial of
compound fluorophore upon interaction between the stacked
bases within the helix and/or surface binding at the reactive nucle-
ophilic sites on the heterocyclic nitrogenous bases of DNA
molecule.
4. Conclusion
The reaction of 5-amino-3-methyl-1-phenylpyrazole-4-carbal-
dehyde (AMPC) with itself in the presence of nickel(II) ions yields,
new macrocyclic complex, [NiL(NO3)2], containing a ligand com-
posed of the self-condensed AMPC (four mol) bound to a single
nickel(II) ion. A number of newly complexes of the types [NiL]X2;
X = ClO4 and BF4, [NiLX2], X = Cl and Br (Scheme 1) have been
Fig. 3. Fluorescence emission spectra of [NiLCl2] complex in the absence and
presence of increasing amount of DNA.

A.E.-M. M. Ramadan / Journal of Molecular Structure 1015 (2012) 56–66
65
Inorg. Biochem. 103 (2009) 401;
prepared via series of metathetical reactions. A square planar
geometry was deduced for the four-coordinate complex species
[NiL](PF4)2 and[NiL](ClO4)2, while an octahedral environment with
slight distortion in nitrato and halogeno complexes was inferred on
the bases of electronic and magnetic moment data. The catalytic
activity for the SOD reaction of the title complexes has been dem-
onstrated by in vitro measurements. Taking into account the
reduction potentials of the couples OÅ2ꢀ/O2 and O2Åꢀ/H2O2 (ꢀ0.16 V
and 0.89 V, vs. NHE at pH 7, respectively) any redox pair with an
intermediate potential value between these limits can act as a cat-
alyst for the SOD reaction. The observed redox processes of the re-
ported nickel centers lie in the middle of this range. Therefore, the
electrochemical behavior observed for these complexes is in agree-
ment with their SOD mimetic activity. The absorption and fluores-
cence studies demonstrated a considerable interaction between
complexes [NiLCl2] and [NiL](PF4)2 with calf thymus DNA.
(d) Kaushik Chosh, Pramod Kumar, Nidhi Tyagi, Udai P. Singh, Nidhi Coel,
Ajanta Chakraborty, Partha Roy, Maria Camilla Baratto, Polyhedron 30 (2011)
2667;
(e) X.L. Wang, H. Chao, X.L. Hong, Y.J. Liu, L.N. Ji, Transition Met. Chem. 30
(2005) 305–311.
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