Structural, electrochemical, phosphate-hydrolysis, DNA binding and cleavage studies of new macrocyclic binuclear nickel(ii) complexes

Article abstract of DOI:10.1039/b923078e

New macrocyclic binuclear nickel(ii) complexes have been synthesized by using the bicompartmental mononuclear complex [NiL] [3,30-((1E,7E)-3,6-dioxa-2, 7-diazaocta-1,7-diene-1,8-diyl)bis(3-formyl-5-methyl-2-diolato)nickel(ii)] with various diamines like 1

Full text of DOI:10.1039/b923078e

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PAPER
www.rsc.org/dalton | Dalton Transactions
Structural, electrochemical, phosphate-hydrolysis, DNA binding and cleavage
studies of new macrocyclic binuclear nickel(II) complexes†
Sellamuthu Anbu,a Muthusamy Kandaswamy*a and Babu Vargheseb
Received 4th November 2009, Accepted 11th February 2010
First published as an Advance Article on the web 11th March 2010
DOI: 10.1039/b923078e
New macrocyclic binuclear nickel(II) complexes have been synthesized by using the bicompartmental
mononuclear complex [NiL] [3,30-((1E,7E)-3,6-dioxa-2,7-diazaocta-1,7-diene-1,8-diyl)bis(3-
formyl-5-methyl-2-diolato)nickel(II)] with various diamines like 1,2-bis(aminooxy)ethane (L1),
1,2-diamino ethane (L2), 1,3-diamino propane (L3), 1,4-diamino butane (L4), 1,2-diamino benzene (L5),
and 1,8-diamino naphthalene (L6). The complexes were characterized by elemental analysis and
spectroscopic methods. The molecular structures of the symmetrical binuclear complex [Ni2L1(H2O)4]-
(ClO4)2 (1) and unsymmetrical binuclear complex [Ni2L3(H2O)4](ClO4)2·(H2O)4 (3) were determined by
single-crystal X-ray diffraction. The geometry around both the nickel(II) ions in each molecule is a
˚
slightly distorted octahedral. The distance between the Ni ◊ ◊ ◊ Ni centers for complex 1 is 3.039 A and for
˚
complex 3 is 3.059 A. The influence of the coordination geometry and the ring size of the binucleating
ligands on the electronic, redox, phosphate hydrolysis, DNA binding and cleavage properties have been
studied. Electrochemical studies of the complexes show two quasi-reversible one electron reduction
processes between -0.49 to -1.69 V. The reduction potential of the binuclear Ni(II) complexes shifts
towards anodically upon increasing the macrocyclic ring size. The observed first order rate constant
values for the hydrolysis of 4-nitrophenyl phosphate reaction are in the range from 8.69 ¥ 10-3 to 1.85 ¥
10-2 s-1. The complexes show good binding propensity to calf thymus DNA giving binding constant
values in the range from 1.4 ¥ 104 to 17.5 ¥ 104 M-1. The absorption, fluorescence and CD spectral data
suggests that the complexes are strongly interacting with DNA. These complexes display hydrolytic
cleavage of supercoiled pBR322DNA in the presence of H2O2 at pH 7.2 and 37 ◦C. The hydrolytic
cleavage of DNA by the complexes is supported by the evidence from free radical quenching and T4
ligase ligation. The pseudo-Michaelis–Menten kinetic parameters kcat = 1.27 0.4 h-1 and KM = 7.7 ¥
10-2 M for naphthalene diimine containing macrocyclic binuclear nickel(II) complex, (6) were obtained.
dinuclear Ni(II) complexes are more active catalysts for phosphate
Introduction
hydrolysis.6 Along this line, lots of nickel(II) complexes synthesized
and their interactions with DNA have been studied.7–11 Compared
with the number of studies dealing with mononuclear complexes,
relatively few studies on binuclear complexes12 have been re-
ported to date. So, the enhancement of DNA cleavage activity
for binuclear complexes13,14 stimulates to design and synthesize
binuclear Ni(II) complexes to evaluate and understand the factors
on the DNA-binding properties. Very recently, we have reported
the catalytic, nuclease activities of macrocyclic binuclear Cu(II)
analogues.15 With this in view, the new symmetrical and a series
of unsymmetrical macrocyclic binuclear Ni(II) complexes have
been prepared (Scheme 1) and crystal structure reported.16 One
compartment of the macrocyclic ligand size is fixed by O-alkyl
oxime moiety and other compartment of the macrocyclic ligand
ring size is modified by using various alkyl and aromatic diamines.
Nickel(II) complexes of a macrocyclic ligand containing mixed
donors have attracted much attention because they are used
as a model for nickel centred enzymes1–4 such as bifunctional
carbon monoxide dehydrogenase/acetyl-CoA synthase, nickel
containing superoxide dismutase, urease and phosphatase. Metal
ion mediated hydrolysis of phosphate esters by metallonuclease
enzymes is therefore a common catalytic pathway in nucleic
acid biochemistry.5 It was reported that the hexacoordinate
aDepartment of Inorganic Chemistry, University of Madras, Guindy Campus,
Chennai, 600 025, India. E-mail: mkands@yahoo.com; Fax: +91-44-
22300488
bSophisticated Analytical Instruments Facility, Indian Institute of Technol-
ogy, Chennai, 600 036, India
† Electronic supplementary information (ESI) available: The crystal
packing of the macrocyclic binuclear Ni(II) complex 1 (Fig. S1), cyclic
voltammograms of complexes 1, 5 and 2, 3 and 4 (at anodic potentials)
(Fig. S2, S3 and S4, respectively), the pH dependence plot for the binuclear
Ni(II) complexes on phosphate hydrolysis reaction (Fig. S5), The DNA
binding plots of complexes 1, 3 and 6 (Fig. S6), the effect of addition
of complexes 1, 3 and 6 on the emission intensity of CT DNA-bound
ethidium bromide (Fig. S7), gel electrophoresis diagrams (Fig. S8, S9 and
S10). CCDC reference number 710629. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/b923078e
Result and discussion
General properties
The positive ion FAB mass spectrum of the symmetrical binuclear
nickel(II) complex [Ni2L1(H2O)4](ClO4)2 (1) showed a peak at
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Scheme 1 Synthesis of mono and binuclear Ni(II) complexes.
m/z = 767.39, due to the formation of [L1 + 2Ni + ClO4]+ ion.
Similarly a series of unsymmetrical binuclear nickel(II) complexes
[Ni2L2-6(H2O)4](ClO4)2.(H2O)4 showed a peak at m/z = 866.24 (2),
781.42 (3), 795.41 (4), 815.17 (5) and 865.12 (6) corresponding to
the [L2–6 + 2Ni + ClO4]+ ion. The positive ion FAB mass spectral
data and elemental analysis are consistent with the proposed
formula of all the binuclear Ni(II) complexes. The absorption
spectra of all the complexes in CH3CN, displayed three bands
over the range 500–900 nm. These are the characteristic of Ni2+
in the six coordination environment.17,18 These are assigned to the
3
3A2g → T1g(P), 3T1g(F) and 3T2g(F) transitions, respectively.19
Description of the complex molecular structure
Complex 1 (Fig. 1) crystallizes in a monoclinic system with space
group P21/n. Half of the molecule forms the asymmetric unit.
The two halves of the molecule are related through center of
inversion. The nickel atom(s) have distorted octahedral geometry
with approximate square planar base (N1 N2 O3 O3_1, symm_1:
-x, -y, -z) and fifth and sixth coordination sites are occupied by
two water molecules. The ligand bite angles with metal are 88.53◦
and 88.19◦. The distance between Ni ◊ ◊ ◊ Ni centres was found to be
Fig. 1 ORTEP diagram of [Ni2L1(H2O)4](ClO4)2 (1) Only asymmet-
ric unit is labelled. Displacement ellipsoids are drawn at the 50%
probability level. The perchlorate anions are omitted for the sake of clarity.
˚
3.039 A. Crystallographic data, the list of selected bond lengths,
(ESI Fig. S1).† The trans angles at the Ni(II) centres are deviated
from 180◦, ranging from 170.04(6) to 174.18(6). All other angles
subtended at Ni(II) centres are deviated from 90◦, ranging from
86.78 (6) to 100.91 (6), which indicates geometry around Ni(II)
angles and hydrogen bonds are given in the Tables 1 and 2. The
packing of the molecule is stabilized through three dimensional
OH ◊ ◊ ◊ O hydrogen bonds mediated through perchlorate anions
3824 | Dalton Trans., 2010, 39, 3823–3832
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Table 1 Crystallographic data for Ni(II) complex 1
Parameters
Ni2L1 (1)
Empirical formula
Formula weight
T/K
C22H30Cl2N4Ni2O18
826.82
293(2)
0.71069
Monoclinic
P21/n
˚
l/A
Crystal system
Space group
˚
a/A
8.978(2)
˚
b/A
12.042(2)
14.186(2)
90
90.981
90
1533.5(5)
2
1.791
˚
c/A
a/◦
b/◦
g /◦
3
˚
V/A
Z
D
calcd/Mg m-3
m/mm-1
Crystal size/mm
1.491
0.25 ¥ 0.20 ¥ 0.16
2.22 to 32.28
0.754 and 0.652
6545/7/254
R1 = 0.0415
wR2 = 0.1120
1.322 and -1.039
q range for data collection/◦
Max. and min. transmission
Data/restraints/parameters
Final R indices [I > 2s(I)]
R indices (all data)
Fig. 2 ORTEP diagram of [Ni2L3(H2O)4](ClO4)2·(H2O)4 (3) The unit cell
content of the title compound, showing the atomic numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level. The
perchlorate anions are omitted for the sake of clarity.
Largest diffraction peak and hole/e A-3
the electrochemical data are summarized in Table 3. The complex
◦
˚
Table 2 Selected bond lengths (A) and angles ( ) for the Ni(II) ion
environment of 1
1 showed two quasi-reversible reduction waves (E1 = -0.49 V
1/2
and E2 = -1.30 V vs. Ag/AgCl) in the cathodic region (ESI,
1/2
Fig. S2).† The complexes 2 to 6 also show two quasi-reversible
reduction waves (Fig. 3 and ESI, Fig. S3)† in the potential ranges
E1pc = -1.01 to -1.26 V and E2pc = -1.55 to -1.67 V. Coulometric
experiments by potentiostatic exhaustive electrolysis performed
at 100 mV more negative to the first reduction wave consumed
approximately one electron (n = 0.95) per complex allowing to
N1–Ni1 2.0370(16)
N2–Ni1 1.9849(15)
O3–Ni1 2.0058(12)
O3¢–Ni1 2.0328(13)
O4–Ni1 2.0951(15)
O5–Ni1 2.1682(15)
Ni1–Ni1¢ 3.039)
Ni1–O3–Ni1¢ 97.62(5)
Ni1–O3–Ni1¢ 97.62(5)
N2–Ni1–O3 88.19(6)
N2–Ni1–N1 100.91(6)
O3–Ni1–O4 86.78(6)
O3–Ni1–N1 170.04(6)
O4–Ni1–O5 174.18(6)
O4–H4A–O6 0.850(9) 1.976(12) 2.818(3) 171(3)
O4–H4B–O5 0.853(10) 2.010(14) 2.834(2) 162(3)
O5–H5B–O1 0.846(10) 2.215(17) 3.009(2) 156(3)
are more distorted than the geometry around Ni(II) centres in
unsymmetrical macrocyclic Ni(II) analogue17 (3). Fig. 2 shows the
molecular structure of the unsymmetrical macrocyclic binuclear
Ni(II) complex [Ni2L3(H2O)4](ClO4)2·(H2O)4 (3). The molecule
is discrete binuclear species in which each nickel(II) ions are in
slightly distorted octahedral. The Ni ◊ ◊ ◊ Ni distance was found to
˚
be 3.059 A.
Electrochemistry
The cyclic voltammogram of binuclear Ni(II) complexes 1–6 were
measured in CH3CN from the potential region +1.0 to -1.67 and
Fig. 3 Cyclic voltammograms of Ni(II) complexes 2, 3 and 4.
Table 3 Electrochemical data of binuclear Ni(II) complexes
Reduction process
Oxidation process
E1pc/V
E1pa/V
E11/2/V DE1/mV
E2pc/V
E2pa/V
E21/2/V DE2/mV
E1pa/V
E2pa/V
E2pc/V
E21/2/V DE2/V
1
2
3
4
5
6
-0.56
-1.05
-1.03
-1.01
-1.26
-1.19
-0.42
-0.83
-0.81
-0.81
-1.16
-1.10
-0.49
-0.94
-0.92
-0.91
-1.21
-1.14
140
220
210
200
100
090
-1.40
-1.67
-1.60
-1.55
-1.63
-1.58
-1.2
-1.30
-1.61
-1.53
-1.50
-1.63
-1.53
200
120
100
090
110
090
+0.68
+0.24
+0.22
+0.21
+0.26
+0.23
+1.02
+0.58
+0.59
+0.62
+0.56
+0.54
—
—
—
-1.55
-1.50
-1.46
-1.57
-1.49
+0.38
+0.40
+0.44
+0.43
+0.41
+0.48
+0.50
+0.53
+0.49
+0.47
200
190
180
140
130
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II,I
attribute this quasi reversible signal to the Ni2II,II/Ni2 redox
It must be noted that the symmetrical macrocyclic ligand
containing electronegative oxygen atoms of the oxyime group
causes a destabilization of the oxidized form of the complex and
therefore the irreversibility of the anodic electrochemical responses
were observed. There is slight anodic shift was observed as the
number of methylene groups (chain length) increases in the imine
compartment of unsymmetrical macrocyclic Ni(II) analogues 2–
4. This can be due to the presence of the azomethine group in
one compartment affects electron-transfer potentials25 leading to
a strong negative shift in the cathodic waves and slight positive
shift is observed for the oxidation waves.
couple. The second quasi-reversible wave can be attributed to the
formation of the Ni2 species from the reduction of Ni2II,I. Both
the mixed-valence Ni2 and Ni2 species are stable at the time
scale of the CV (0.05 V s-1). Based on these observations, it is
reasonable to suggest that the reduction process may involve the
stepwise redox processes depicted.
I,I
II,I
I,I
NiIINiII ꢀ NiIINiI ꢀ NiINiI
(1)
The effect of scan rate (u) on the cyclic voltammetric response
was examined in a wide scan rate range from 50 to 300 mV s-1.
DE values are greater than 59/n mV (of the order of 100 mV) and
increases with scan rate (ESI, Fig. S2 and S3).† The cathodic and
anodic peak current ratio is greater than unity. The nature of the
wave shape broadens as this scan rate increases. This indicates that
the redox processes are quasi-reversible in nature. Two different
reduction and oxidation waves of all the Ni(II) complexes may
be due to Ni(II) ions present in two different compartments as
well as electrostatic effects arises during redox processes. When
the first Ni(II) ion is getting reduced then the charge of the
complex decreases from +2 to +1. Therefore, the second Ni(II)
ion reduced at higher negative potentials. It was reported20 that
the metal ion in oxime moiety reduces at less negative potential
than the metal ion in imine21a moiety. We have reported15 that the
influence of more electronegative oxygen atoms in the alkyl oxime
moiety makes the Cu(II) ion to reduce at less negative reduction
potentials. The donor nature of alkyl group in the alkyl imine21b
moiety increases the electron density around the Cu(II) ion and
makes to reduce at higher negative potentials. It is noticed that
geometry around both the Cu(II) ions in unsymmetrical Cu(II)
analogues is square pyramid. Presently, geometry around both
the Ni(II) ions in alkyl oxime and imine moiety is octahedral.
Like Cu(II) complexes, the Ni(II) ion in oxime moiety may reduces
first than the Ni(II) ion in imine moiety. As the chain length
of the alkylimine compartment increases, the entire macrocyclic
ring becomes more flexible. The distortion around the geometry
also increases. It has been suggested22–24 that reduction of electron
density on the metal ions and distortion in geometry favours the
reduction process (NiII/NiI) at less negative potentials, as observed
in the complex of ligand L4 relative to ligand L2. As the size of the
macrocycle is increased, shifting of both first and second reduction
potentials towards anodic is observed for the binuclear nickel(II)
complexes. Complexes containing aromatic group (5 and 6) in the
imine nitrogen compartment get reduced at higher potential than
the other complexes containing alkyl group in the imine nitrogen
compartment. The higher reduction potential can be attributed
due to the greater planarity and electronic properties those are
associated with aromatic rings.24
Phosphate hydrolysis
Many hydrolytic processes in enzyme-catalysis involve metal ions
that are assumed to activate a water molecule which is more
easily to form hydroxyl group as a nucleophilic group in reaction
system.26 Presently, all the binuclear Ni(II) complexes possesses
in its structure a potential nucleophile constituted by the metal
coordinated water molecule, and their catalytic activities on
hydrolysis of 4-nitrophenyl phosphate (4-NPP) was investigated
spectrophotometrically by following the absorption increase at
400 nm due to the formation of 4-nitrophenolate ion over time. The
effect of pH on the rate of reaction was determined and correlated
with the pKa of coordinated water in 1. The pH dependence plot for
the binuclear Ni(II) complexes on phosphate hydrolysis reaction
showed a pH-independent rate above 8.5 and a range below this
pH where the initial rate of hydrolysis increases with pH (ESI,
Fig. S5).† The derived sigmoidal pH-rate profiles are characteristic
of a kinetic process controlled by an acid–base equilibrium and
exhibit inflection points corresponding to the pKa value is 7.89
for one of the coordinated water molecule. This indicates that the
[Ni2(L)(H2O)3(OH)] complex is the reactive species. The Ni(II)-
bound OH- acts as a nucleophile to attack the phosphate atom
of the 4-NPP and hydrolysis takes place. Since the substrate
concentration was essentially constant during the measurement,
the initial first order rate constant (kobs) was measured at different
concentrations of the catalyst at pH 7.6 and 25 0.1 ◦C. Plots
of rate constant (kobs) vs. complex concentration are presented
in Fig. 4. As can be seen, for all complexes, the rate of 4-NPP
cleavage initially increases linearly with the increase of complex
concentration but gradually the reaction order in the catalyst con-
centration deviates from unity. Inother words, the reactionexhibits
a first order dependence only at low Ni(II) complex concentrations.
The kobs value for phosphate hydrolysis reaction by nickel(II)
perchlorate hexahydrate salt was found as 2 ¥ 10-13 s-1. This is
negligibly small when compare to the kobs value (108 times more
faster) for binuclear Ni(II) complexes. The first order rate constants
k were obtained for the Ni(II) complexes, from Lineweaver–
Burk plot, i.e., 1/V0 vs. 1/[4-NPP] by changing concentration
of substrate (Fig. 5) and the results of calculation are summarized
in Table 4. These values are comparable to the constants reported
by Yamaguchi et al.6 for the hydrolytic cleavage of 4-NPP by
binuclear Ni(II) complexes. From Table 4, it can be seen that the
catalytic activity of the complexes (2, 3 and 4) are found to increase
as the macrocyclic ring size increases, because of the intrinsic
flexibility of the ligand makes the geometry around metal ion
is more distorted. The aromatic diimines containing complexes (5
and 6) are also shows remarkable catalytic activity. It is noted that
In the positive potential region complex (1) showed two
irreversible oxidation waves (E1pa = +0.68 V and E2pa = +1.02 V).
Whereas, complexes 2–6 showed an irreversible (E1 = +0.21 V
pa
to +0.26 V) followed by a quasi-reversible (E2 = +0.48 V to
1/2
+0.53 V vs. Ag/AgCl) oxidation waves (ESI, Fig. S4).† Controlled
potential electrolysis experiment indicates that the two oxidation
peaks are associated with stepwise oxidation process at metal
center.
NiIINiII → NiIINiIII ꢀ NiIIINiIII
(2)
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Table 4 Rate constants for phosphate hydrolysis and DNA binding
of metal coordinated water molecule. It has been assumed that the
geometry around the Ni(II) ions and the intermetallic distance
are the two key factors that determine the catalytic activity
of the complexes. The distance between the Ni ◊ ◊ ◊ Ni centres is
parameters of the binuclear Ni(II) complexes
4-NPP
a
b
Complexes Hydrolysis k/s-1 104 Kb
105Kapp
CD Dlmax + Dec
˚
smaller (3.039 A (1)) than the unsymmetrical Ni(II) analogues. The
1
2
3
4
5
6
1.85 ¥ 10-2
8.69 ¥ 10-3
1.20 ¥ 10-2
1.73 ¥ 10-2
1.68 ¥ 10-2
1.70 ¥ 10-2
3.1
—
1.4
—
—
17.5
4.2
—
3.7
—
—
12.2
-3 (11)
-2 (1)
Ni ◊ ◊ ◊ Ni distance also influence the rate of phosphate hydrolysis
reaction. The first order rate values of the unsymmetrical binuclear
Ni(II) complexes were found to increase as the macrocyclic ring
size increases. It is evident from the literature,24,27–29 that the first
order rate values for the more distorted complexes are higher than
those of the less-distorted complexes.
-2 (3)
-3 (4)
-3 (10)
-4 (11)
a Binding constants (M-1) were determined by absorption spectrophoto-
metric titration. b Apparent binding constants (M-1) were determined by
fluorescence spectrophotometric method. c Dlmax is the shift in nm of the
positive DNA CD band at 274 nm. De (the value in parentheses) is the
difference between the maximum ellipticity (in ◦) observed for the positive
CD band in the spectrum of a 2 : 1 reaction mixture, and the ellipticity
observed at the same wavelength in the spectrum of free CT DNA.
DNA binding and cleavage properties
Absorption spectral studies. Absorption spectral titration ex-
periment is used to monitor the interaction of Ni(II) complexes
1, 3 and 6 with CT DNA (Fig. 6, Table 4). A complex generally
shows hypochromism and a red shift30 (bathochromism) of the
absorption band when it binds to DNA through intercalation,
resulting in a strong stacking interaction between the aromatic
chromophore of the ligand and the base pairs of the DNA. The
extent of hypochromism31 gives a measure of the strength of an
intercalative binding. The observed trend in hypochromism among
the present complexes follows the order complexes 6 > 1 > 3.
The binding constants KB of the Ni(II) complexes to CT DNA
were determined by monitoring the changes of absorbance with
increasing concentration of DNA. The binding constant KB (ESI,
Fig. S6)† of complexes 1, 3 and 6 are calculated as 3.1 ¥ 104,
1.4 ¥ 104 and 17.5 ¥ 104 M-1, respectively. The better binding of
the complex 6 than the complexes 1 and 3 may be due to the
co-planarity of the naphthalene ring system in the macrocyclic
ring. It is expected to be stacked between the base pairs upon
the interaction of the complex with DNA.32 Structurally, the
ligand 6 should provide aromatic moiety to overlap with the
stacking base pairs of the DNA helix by intercalation which results
in hypochromism and bathochromism. The increasing aromatic
moiety in macrocyclic ligands of the nickel(II) complexes, that
facilitates its potential intercalative binding, while complexes 1
and 3 may prefer electrostatic interaction.
Fig. 4 Dependence of the reaction rate on the concentration of 1–4 for
the 4-NPP hydrolysis at pH 7.60 and 25 0.1 ◦C. Conditions: [4-NPP] =
5.0 ¥ 10-5 M, [Ni2 complex] = 5.0 ¥ 10-5 to 5.0 ¥ 10-4, [buffers] = 50 mM,
I = 0.1 M (NaClO4). Inset shows the hydrolysis of 4-NPP by the binuclear
nickel(II) complex 4.
Fig. 5 Lineweaver–Burk plot for the 4-NPP hydrolysis by complexes 1,
4, 5 and 6.
Fig. 6 Absorption spectra of complex 1 (1 ¥ 10-5 M) in the absence (line-a)
and presence of increasing amounts of CT-DNA (0-2.5 ¥ 10-3 M) at room
temperature in 50 mM Tris-HCl/NaCl buffer (pH = 7.5). Arrow shows the
absorbance changing upon increasing DNA concentrations. Insets shows
the saturation in absorption intensity hypochromism is indicated by the
plot of A0/A vs. [DNA].
the oxime containing symmetrical Ni(II) complex 1 display higher
catalytic activity than the unsymmetrical Ni(II) analogues 2–6.
This may be due to more electronegative oxygen atom of the oxime
nitrogen atoms of both the compartments, which reduces the
electron density around metal ion and favors easy deprotonation
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Fluorescence spectral studies. The fluorescence spectral
method is used to study the relative binding of these complexes
to CT-DNA. The emission intensity of ethidium bromide (EB)
is used as a spectral probe.33 The fluorescence of EB increases
after intercalating into DNA. If the metal complex intercalates
into DNA, it leads to a decrease in the binding sites of DNA
available for EB resulting in decrease in the fluorescence intensity
of the EB-DNA system.34 Nickel(II) perchlorate hexahydrate salt
and complexes 1, 3 and 6 were added to DNA pretreated with
EB. No significant quenching was observed in fluorescence of
EB bound DNA while adding increasing amounts of nickel(II)
perchlorate hexahydrate (ESI, Fig S7).† Complexes 1, 3 and 6
cause an appreciable reduction in fluorescence intensity, (Fig. 7)
indicating that complexes competes with EB to bind with DNA.35
The fluorescence quenching curve of DNA-bound EB by complex
6 is in good agreement with the linear Stern–Volmer equation.
In the linear fit plot of I0/I vs. [complex]/[DNA], K is given by
the ratio of the slope to intercept. (I0 is the emission intensity of
EB-DNA in the absence of complex; I is the emission intensity
of EB-DNA in the presence of complex) The K value of the
complexes 1, 3 and 6 were calculated as 6.61(R = 0.985), 5.01(R =
0.991) and 12.6(R = 0.989), respectively. The concentrations of
the complexes are taken for observing 50% reduction of emission
intensity of EB.22 From the data in the ESI, Fig. S7,† it is also
know that 50% of EB molecules were replaced from DNA-bound
EB at a concentration ratio of [Ni2complex]/[EB] = K1. The K1
value of the complexes 1, 3 and 6 were found as 23.8, 27.0 and 8.19,
respectively. By taking a DNA binding constant 1.0 ¥ 107 M-1 for
EB36 an apparent DNA binding constant Kapp of the complexes
(4.2 ¥ 105 (1), 3.7 ¥ 105 (3) and 1.22 ¥ 106 (6) M-1) were derived
from the equation (Kb(EB)/K1). The Kapp values imply that the
complex 6 can strongly interact with DNA and protected by DNA
efficiently. The intercalation of 4-methylphenol group and also
the hydrophobic property of the rigid macrocyclic ligands also
facilitate the DNA binding.36 The greater Kapp value for complex
6 than the complexes 1 and 3 may be due to the presence of
more aromatic moiety, which enhances the binding propensity
of the molecule to DNA. The DNA binding constants Kapp of
the complexes were obtained from EB displacement (indirect
method) are different from those obtained (KB) from absorption
spectral method (direct method). The same was reported for Co3+
and Ru2+ complexes.31,37 This difference between the two sets of
binding constants may be caused by the different spectroscopy
and different calculation method.
Viscosity measurements. In order to clarifying the binding
mode of Ni(II) complexes 1, 3, 6 with DNA, viscosity of DNA
solutions containing varying amount of added complexes were
measured. A classical intercalation model demands that the DNA
helix lengthens as base pairs are separated to accommodate the
binding ligand, leading to the increase of DNA viscosity.38 The
effects of complexes 1, 3, 6 and EB on the viscosity of rod-like
DNA, are shown in Fig. 8. EB, a well known DNA intercalator,
gave rise to a strong change in DNA viscosity upon complexation.
Complexes 1 and 3 binds by electrostatic intercalations only,
exerted essentially no such effect. After increasing the amounts
of 6 the relative viscosity of DNA increases steadily, similar to
EB. The increase in relative viscosity, expected to correlate with
the compound’s DNA-intercalating potential, followed the order
EB > 6 > 1 > 3. These results suggest that complex 6 can bind to
DNA through intercalation, due to the presence of naphthalene
ring system in one compartment of the ligand.
Fig. 8 Effect of increasing amounts of EB (a), Ni(II) complexes 1, 3 and
6 (b, c and d, respectively) on the relative viscosity of calf thymus DNA at
25 ( 0.1) ◦C. The total concentration of DNA is 0.5 mM.
CD spectral studies. Circular dichorism (CD) is a useful
method to access whether nucleic acids undergo conformational
changes as a result of complex formation or changes in the
environment.39 The UV circular dichoric spectrum of CT DNA
exhibits positive band at 272 nm (UV: lmax 260 nm) due to base
stacking and negative band at 239 nm due to helicity of B-DNA.40
It was reported30 that the change in ellipticity and shifting to
higher energy of the positive CD signals are due to intercalative
mode of binding. Incubation of the DNA with the complexes
1–6, induced considerable changes in CD spectrum (Fig. 9). At
a [Ni2complex] : CT DNA ratio of 2 : 1 all nickel(II) complexes
produced shifts to higher energy for the positive CD signal as well
as an enhancement of CD ellipticity at 272 nm. Examination of
Table 4 shows that the magnitude of the increases in elipticity at
272 nm increase in the following order. 2 < 3 < 4 < 1 < 5 < 6.
The result reveals that the changes induced by 5 and 6 are more
significant than those by 1–4, which suggests that 6 have higher
affinity for CT DNA than the complexes 1–4 does. It is reasonable
to suggest that the higher affinity of complexes 5 and 6 with CT
DNA due to presence of aromatic moiety in the one compartment
of the macrocyclic ligand, which enhances the interaction, while
the complexes 1–4 induced only slight conformational changes
in DNA may be due to presence of aliphatic moiety in both the
compartments.
Fig. 7 Emission spectrum of EB bound to DNA in the presence of
([EB] = 3.3 mM, [DNA] = 40 mM, [complex] = 0-25 mM, lex = 430 nm).
Arrow shows the absorbance changing upon increasing Ni(II) complex
(6) concentrations. Inset shows the plots of emission intensity Io/I vs.
[DNA]/[Ni(II) complex].
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activity, these rules out the possibility of cleavage by hydroxyl
radical and superoxide, respectively. The EDTA (lanes 6 and 7)
efficiently inhibit the DNA cleavage activity of the Ni(II) complexes
in a similar way to that for nuclease.36 In order to ascertain the
hydrolytic cleavage mechanism, the cleavage studies were carried
out under anaerobic conditions as shown in the ESI, Fig. S9.†
Under anaerobic conditions, the binuclear Ni(II) complex display
considerable cleavage (lanes 1–5). This fact implies that the DNA
cleavage reaction by the binuclear Ni(II)/H2O2 system should be
due to hydrolytic mechanism. To confirm the hydrolytic cleavage,
the linear form obtained from the cleavage of SC DNA was
reacted with T4 ligase enzyme (ESI, Fig. S10)† and observed the
complete conversion of the linear DNA to its original form.42
It is well known that in DNA hydrolytic cleavage 3¢-OH and 5¢-
OPO3 (5¢-OH and 3¢-OPO3) fragments remain intact and that these
fragments can be enzymatically ligated.43 The linear DNA was
recoveredfrom low meltingpoint gelbycuttingoff the gel fragment
and subjecting to overnight ligation reaction with T4 ligase. The
electrophoretic results (ESI, Fig. S10)† show that the linear DNA
fragments cleaved by 5 and 6 can be re-ligated by T4 ligase just
like the linear DNA mediated by the natural enzyme EcoRI.44 The
kinetic aspects of the hydrolytic DNA cleavage (Fig. 11) by 6 is
found to vary exponentially with incubation time and it follows
pseudo-first order kinetics. Kinetic plots showing the formation
of nicked circular (NC) DNA, linear DNA and the degradation
of SC DNA vs. time follow pseudo-first order kinetics and they fit
well to a single exponential curve. Under true Michaelis–Menten
conditions in which the complex concentration is kept constant
at 55 mM and the DNA concentration is varied from 41–180 mM,
we are able to obtain the rate constant of 1.27 0.4 h-1 using
150 mM SC pBR322 DNA. The linear plot of log (%SC-DNA)
vs. time (Fig. 12a) from which we have determined the hydrolytic
rate constant is shown in Fig. 12b. The pseudo Michaelis–Menten
Fig. 9 CD spectra recorded over the wavelength range 230–320 nm for
solutions containing 2 : 1 ratio of CT-DNA (200 mM) and binuclear Ni(II)
complexes 1–6 (100 mM). (a = CT DNA, b = 2 + DNA, c = 3 + DNA,
d = 4 + DNA, e = 1 +DNA, f = 5 + DNA and g = 6 + DNA).
DNA cleavage activity. The interaction of complexes 2, 5 and
6 with pBR322 DNA was studied by monitoring the conversion of
circular supercoiled DNA (Form I) to nicked (Form II) and linear
(Form III) DNA. The amount of strand scission was assessed
by agarose gel electrophoresis. Fig. 10 shows the electrophoretic
pattern of plasmid DNA treated with complexes 2, 5 and 6 (30–
50 mM) in the presence of H2O2 (40 mM). Control experiments
suggest that untreated DNA and DNA incubated with either
complex or peroxide alone did not show any significant DNA
cleavage (lanes 1–5). At 50 mM concentration of the binuclear
complexes, the cleavage is found to be significant, as is seen
from the formation of nicked circular (form II) and linear form
(form III) in lanes 6–8. The cleavage mechanism of pBR322
DNA induced by complexes 3 and 6 were investigated (ESI, Fig.
S8)† and clarified in the presence of hydroxyl radical scavenger
0.4 M DMSO (lanes 2 and 4), superoxide quencher SOD (4
units) (lanes 3 and 5) and EDTA as a chelating agent under
aerobic conditions.41 As shown in the ESI, Fig. S8,† the DNA
cleavage mechanism by complexes 3 and 6 are shown as follows:
both DMSO and SOD (lanes 2–5) do not alter DNA cleavage
kinetic parameters kcat = 1.27 0.4 h-1 and KM = 7.7 ¥ 10-2
M
Fig. 11 Cleavage activity of 6 monitored by 0.8% agarose gel elec-
trophoresis, where [DNA] (0.2 mg, 33 mM) by (6) 50 mM, and [H2O2] 40 mM.
Time course measured in 10 mM Tris buffer, pH 7.4, 37 ◦C, showing the
disappearance of supercoiled DNA (I) at (1) 0 min, (2) 5 min, (3) 10 min,
(4) 15 min, (5) 20 min, (6) 25 min, (7) 30 min, (8) 35 min (9) 40 min. (Gel
image showing supercoiled (Form I), circular relaxed (Form II), linear
(Form III) DNA.)
Fig. 10 Cleavage of SC pBR322 DNA (0.2 mg, 33 mM) by Ni(II) complexes
1, 5 and 6 (30–50 mM) in the presence of H2O2 (40 mM) in 50 mM
Tris-HCl/NaCl buffer (pH 7.2). Lane 1, DNA control; lane 2, DNA +
H2O2; lane 3, DNA + 1 (30 mM); lane 4, DNA + H2O2 + 5(30 mM); lane
5, DNA + H2O2 + 6 (30 mM); lane 6, DNA + H2O2 + 1 (50 mM); lane 7,
DNA + H2O2 + 5 (50 mM); lane 8, DNA + H2O2 + 6 (50 mM).
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NiL (0.5 g, 1.13 mmol) in methanol (25 mL), a methanolic solution
of Ni(ClO4)2·6H2O (0.41 g, 1.13 mmol) was added slowly and
the mixture was stirred for 15 min to obtain a clear solution.
Then the methanolic solution (5 mL) of 1,2-bis(aminooxy)ethane
(0.10 g, 1.13 mmol) was added drop wise to the above solution and
refluxed for 3 h. A resulting solid was separated on evaporating
the solution at room temperature. Green crystals suitable for X-ray
analysis were obtained after several days by slow evaporation of
acetonitrile solution (0.62 g 84%). C22H30Cl2N4Ni2O18 (627.88): C
42.05, H 4.78, N 8.92. Found: C 42.12, H 4.74, N 8.87. FAB-MS in
NBA: m/z: 767.39 [L1 + 2Ni + ClO4]+. FT-IR (KBr, n/cm-1: 3445
br, 2925 s, 1638 s, 1107 vs, 625 s, 502 w. UV-vis: lmax (CH3CN)/nm:
952, 742, 560, 328 and 265 (e/dm3 mol-1 cm-1: 30, 40, 62, 54 000
and 170 000).
Fig. 12 (a) Plot of log (%SC DNA) vs. time for a complex concentration
of 50 mM. (b) Saturation kinetics of the cleavage of pBR322 DNA using
50 mM complex 6 with different concentrations of SC DNA (33–183 mM)
at 37 ◦C in 50 mM Tris-HCl/NaCl buffer (pH 7.2).
Synthesis of [Ni2L2(H2O)4]4H2O·2ClO4 (2). This complex was
prepared by the method used for 1, using 1,2-diamino ethane in
place of compound I, offered orange coloured solid (0.73 g, 74%).
Elemental analysis data: calcd (%) for C22H38Cl2N4Ni2O20 (866.84):
C 30.46, H 4.38, N 6.46. Found (%): C 30.41, H 4.44, N 6.38. FAB-
MS in NBA: m/z: 866.24 [L2 + 2Ni + ClO4]+. FT-IR (KBr, n/cm-1:
3428 br, 2921 m, 1625 s, 1343 m, 1087 s, and 623 s. UV-vis: lmax
(CH3CN)/nm: 970, 560, 426, 375 and 263 (e/dm3 mol-1 cm-1: 30,
180, 2820, 58 000 and 33 000).
for complex 6 was calculated. This kcat value of complex 6 is
comparable to the DNA hydrolytic rate constant (kcat) value of
reported12,45 Ni(II) complexes.
Experimental
Materials and measurements
2,6-Diformyl-4-methylphenol46 and 1,2-bis(aminooxy)ethane47
were prepared by following the literature methods. Tetra(n-
butyl)ammonium perchlorate (TBAP) was purchased from Fluka
and recrystallized from hot methanol. (Caution! TBAP is po-
tentially explosive; hence care should be taken in handling
the compound.) All the solvents were purified by reported
procedures.48 CT-DNA and pBR322DNA were purchased from
Bangalore Genie (India). All other chemicals and solvents were
of analytical grade and used as received. Elemental analysis was
conducted on a Carlo Erba model 1106 elemental analyzer. FT-
IR spectra were obtained on a Perkin Elmer FTIR spectrometer
with samples prepared as KBr pellets. UV-vis spectra were
recorded using a Perkin Elmer Lambda 35 spectrophotometer
operating in the range of 200–1000 nm with quartz cells and e
are given in M-1 cm-1. CH11008 Electrochemical analyzer using
a three- electrode cell setup comprised of glassy carbon working,
platinum wire auxiliary, and saturated Ag/AgCl electrodes. The
concentration of the complexes was 10-3 M and TBAP (10-1 M)
was used as the supporting electrolyte.
Synthesis of [Ni2L3(H2O)4]4H2O·2ClO4 (3). This complex was
prepared by the method used for 1, using 1,3-diamino propane in
place of compound I, offered green coloured crystals (0.75 g, 75%).
Elemental analysis data: calcd (%) for C23H40Cl2N4Ni2O20 (880.87):
C 31.33, H 4.54, N 6.36. Found (%): C 31.41, H 4.62, N 6.41. FAB-
MS in NBA: m/z: 781.42 [L3 + 2Ni + ClO4]+. FT-IR (KBr, n/cm-1:
3431 br, 2922 m, 1628 s, 1342 m, 1088 s, and 624 s. UV-vis: lmax
(CH3CN)/nm: 975, 566, 432, 380 and 265 (e/dm3 mol-1 cm-1: 33,
182, 2830, 58 500 and 35 000).
Synthesis of [Ni2L4(H2O)4]4H2O·2ClO4 (4). This complex was
prepared by the method used for 1, using 1,4-diamino butane in
place of compound I offered pale green coloured solid (0.76 g,
75%). Elemental analysis data: calcd (%) for C24H42Cl2N4Ni2O20
(894.9): C 32.18, H 4.69, N 6.26. Found (%): C 32.24, H 4.72,
N 6.32. FAB-MS in NBA: m/z: 795.41 [L4 + 2Ni + ClO4]+. FT-
IR (KBr,n/cm-1: 3432 br, 2925 m, 1630 s, 1343 m, 1089 s, and
625 s. UV-vis: lmax (CH3CN)/nm: 976, 569, 440, 383 and 269
(e/dm3 mol-1 cm-1: 35, 186, 2910, 58 900 and 38 000).
Synthesis of mononuclear nickel(II) complex
Synthesis of [Ni2L5(H2O)4]4H2O·2ClO4 (5). This complex was
prepared by the method used for 1, using 1,2-diamino benzene in
place of compound I offered orange coloured solid (0.70 g, 70%).
Elemental analysis data: calcd (%) for C26H34Cl2N4Ni2O18 (914.88):
C 34.10, H 3.71, N 6.12. Found (%): C 34.21, H 3.68, N 6.18. FAB-
MS in NBA: m/z: 815.17 [L5 + 2Ni + ClO4]+. FT-IR (KBr, n/cm-1:
3435 br, 2921 w, 1635 s, 1342 m, 1087 s, and 625 s. UV-vis: lmax
(CH3CN)/nm: 975, 572, 442, 380 and 266 (e/dm3 mol-1 cm-1: 32,
182, 2830, 60 500 and 40 000).
Synthesis of [NiL]. To a solution of 2,6-diformyl-4-methyl phe-
nol (3.0 g; 1.8 mmol) in warm dimethyl formamide (30 mL), 1,2-
bis(aminooxy)ethane (0.84 g. 0.9 mmol) (I) was added dropwise
under constant stirring. Solid Ni(OAc)2·4H2O (1.8 g; 0.9 mmol)
was added and the solution was stirred at 60 ◦C for 2 h. A greenish
yellow colored mononuclear complex [NiL] precipitated. The solid
was separated by filtration and washed with isopropyl alcohol and
diethyl ether (3.6 g, 44%). C20H18N2NiO6 (441.32): C 54.46, H 4.11,
N 6.35. Found: C 54.42, H 4.06, N 6.32.
Synthesis of [Ni2L6(H2O)4]4H2O·2ClO4 (6). This complex was
prepared by the method used for 1, using 1,8-diamino naphthalene
in place of compound I offered green colored solid (0.73 g, 69%).
Elemental analysis data: calcd (%) for C30H36Cl2N4Ni2O18 (964.94):
C 37.31, H 3.73, N 5.80. Found (%): C 37.43, H 3.79, N 5.95. FAB-
MS in NBA: m/z: 865.12 [L6 + 2Ni + ClO4]+. FT-IR (KBr, n/cm-1:
Synthesis of binuclear nickel(II) complexes
Synthesis of [Ni2L1(H2O)4]2ClO4 (1). The binuclear Ni(II)
complex 1 was prepared from a general synthetic procedure in
which the vigorously stirred suspension of mononuclear complex
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3441 br, 2920 w, 1636 s, 1345 m, 1088 s, and 625 s. UV-vis: lmax
(CH3CN)/nm: 980, 576, 448, 386 and 267 (e/dm3 mol-1 cm-1: 35,
190, 2780, 61 100 and 39 000).
The fluorescence spectral method using EB as a reference
was used to determine the relative DNA binding properties
of the complexes 1, 3 and 6 to CT DNA in 50 mM Tris
HCl/1 mM NaCl buffer, pH 7.5). Fluorescence intensities of EB
at 610 nm with an excitation wavelength of 430 nm were measured
at different complex concentrations. Reduction in the emission
intensity was observed with addition of the complexes. The
apparent binding constant (Kapp) was calculated using the equation
X-Ray crystallography
Crystals of complexes 1 and 3 were translucent green and cut to
suitable size and mounted on Kappa Apex2 CCD diffractometer
equipped with graphite monochromated Mo Ka radiation (l =
K
EB[EB]/Kapp[complex], where the complex concentration was the
˚
0.71073 A). The intensity data were collected using w and j
value at a 50% reduction of the fluorescence intensity of EB and
KEB = 1.0 ¥ 107 M-1 ([EB] = 3.3 mM).54
scans with frame width of 0.5◦. The frame integration and data
reduction were performed using Bruker SAINT-Plus (version
7.06a) software.49 The multi-scan absorption corrections were
applied to the data using SADABS (Bruker 1999)50 program. Both
samples were stable at room temperature. The structures were
solved using SIR92 (Altornare et al., 1993).51 Full-matrix least-
squares refinement was performed using SHELXL-97 (Sheldrick,
1997) programs. All the non-hydrogen atoms were refined with
anisotropic displacement parameters. All the hydrogen atoms
could be located in a difference Fourier map. However, they were
relocated at chemically meaningful positions and were given riding
model refinement. The refinement of water hydrogen atoms were
restrained such that they remain in the vicinity of the respective
difference peak.
Viscosity measurements were carried out using an Ostwald-
type viscometer of 2 mL capacity, maintained at a constant
temperature of 25.0 0.1 ◦C in a thermostatic bath. DNA samples
approximately 200 bp in length were prepared by sonication in
order to minimize complexities arising from DNA flexibility.55
The flow time was measured with a digital stopwatch, and each
sample was tested three times to get an average calculated time.
Data were presented as (h/h0)1/3 vs. binding ratio,56 where h is the
viscosity of DNA in the presence of complex, h0 is the viscosity of
free DNA.
Cyclic dichroic (CD) spectra of the CT DNA were measured
using a JASCO J-715 spectropolarimeter equipped with a Peltier
◦
temperature control device at 25 0.1 C. All experiments were
done using a quartz cell of 1 cm path length. Each CD spectrum
was recorded after averaging over at least 5 accumulations using a
scan speed of 100 nm min-1.
Phosphate hydrolysis
Kinetic experiments for the hydrolysis of 4-nitrophenyl phosphate
were followed spectrophotometrically on a Perkin Elmer UV-
spectrophotometer. The effect of pH on the reaction rate for the
hydrolysis of 4-NPP promoted by complexes 1–6 was determined
over the pH range 3.9–10.5. Reactions were performed using the
following conditions: 3 mL of freshly prepared buffer aqueous
solution (50 mM, 0.1 mM KCl, buffer: acetato (pH 3.9 and 4.9),
MES (pH 6), Bis-Tris propano (pH 7.0, 7.6 and 8.0), CHES
(pH 8.94 and 10.52) and 1 mL of 4 ¥ 10-3 M complex solution
[acetonitrile–water (2.5% (v/v))] were added to a 1 cm path length
at 25 ◦C.
DNA cleavage experiments
The cleavage of plasmid DNA was monitored by agarose gel
electrophoresis. Supercoiled pBR322DNA (0.020 mg mL-1) in
50 mM Tris-HCl/NaCl buffer (pH 7.2) was treated with the
complexes 2, 5 and 6 (30–50 mM) and H2O2 (40 mM). All the
samples were incubated for 30 min at 37 ◦C followed by its
addition to the loading buffer containing 25% bromophenol blue,
0.25% xylene cyanol, 30% glycerol (3 mL). All the samples were
finally loaded on 0.8% agarose gel containing EB (1 mg mL-1).
Electrophoresis was carried out at 50 V for 1 h in TBE buffer
(45 mM Tris, 45 mM H3BO3, 1 mM EDTA, pH 8.3). Resulting
bands were visualized by UV light and photographed. DNA
ligation experiments43 as follows: after incubation of pBR322
DNA with 5 and 6 (50 mM) in the presence of hydrogen peroxide
for 1 h at 37 ◦C, the cleavage product, i.e. linear form, was purified
by DNA gel extraction kit. The linear DNA (2 mL) (nicked by
Ni(II) complexes 5 and 6) was incubated for 12 h at 16 ◦C with
1mL of T4 ligase (4 units) and 1 mM ATP containing ligation buffer
(10 mL). Afterwards, the ligation products were electrophoresed,
stained and imaged. Quantification was performed by fluorescence
imaging by use of a Gel-Doc 1000 (BioRad) and data analysis with
Multianalysis software (version 1.1).
DNA binding experiments
The DNA binding experiments were performed in Tris-HCl/NaCl
buffer (50 mM Tris-HCl/1 mM NaCl buffer, pH 7.5) using
dimethyl formamide (DMF) (10%) solution of the complexes
1, 3 and 6. The concentration of CT DNA was determined
from the absorption intensity at 260 nm with a e value52 of
6600 M-1 cm-1. Absorption titration experiments were made using
different concentration of CT DNA, while keeping the complex
concentration as constant. Due correction was made for the
absorbance of the CT DNA itself. Samples were equilibrated
before recording each spectrum. The intrinsic binding constant,
Kb for the complexes 1, 3 and 6 has been determined from the
spectral titration data using the following equation.53
Conclusions
[DNA]/(ea - ef) = [DNA]/(eb - ef) + 1/Kb(eb - ef)
(3)
Here, ea, ef, and eb, correspond to Aobsd/[Ni(II) complex], the
extinction coefficient for the free complex, and the extinction
coefficient for the complex in the fully bound form, respectively.
The non-linear least-squares analysis was done using Origin Lab,
version 6.1.
Based on the electrochemical studies, the Ni(II) ion located in
the alkyl oxime moiety, reduces at less negative potential than
the Ni(II) ion present in the imine moiety. Phosphate hydrolysis
studies show that the complex 1 has a higher catalytic activity
than the other corresponding unsymmetrical binuclear Ni(II)
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analogues. The DNA binding experiments results shows that
the aromatic diimine containing Ni(II) complex 6 display better
binding propensity with DNA than aliphatic diimine containing
Ni(II) analogues 1 and 3. All the complexes can cleave the DNA
through hydrolytically, because a classical radical scavenger, such
as dimethyl sulfoxide (DMSO), was completely ineffective in the
cleavage activity.
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Abbreviations
DMF
MES
CHES
EB
N,N-dimethylformamide
2-(N-morpholino)ethanesulfonic acid
2-(Cyclohexylamino)ethanesulfonic acid
Ethidium bromide
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dron, 2007, 26, 3825.
CT-DNA
Tris
TBE
Calf thymus DNA
Tris(hydroxymethyl)aminomethane
Tris-boric acid-EDTA
EDTA
SOD
Ethylenediaminetetraacetic acid
Superoxide dismutase
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Acknowledgements
The authors thank the Department of Science and Technology
(DST-FIST), New Delhi, Government of India, for financial
support. Dr M. R. N. Murthy and Mr. M. Govindaraju, Molecular
Biophysics Unit, Indian Institute of Science, Bangalore, India,
are gratefully acknowledged for providing the Circular Dichroism
Spectral facility. S. A. is grateful to CSIR (SRF), New Delhi,
Government of India, for a fellowship.
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