The influence of the tertiary butyl group in the ligand frame on the catalytic activities, DNA cleavage ability and cytotoxicity of dinuclear nickel(II) complexes

Article abstract of DOI:10.1016/j.ica.2019.118993

Dinuclear phenoxo-bridged nickel complexes [Ni₂(L¹)H₂O(CH₃COO)](1) and [Ni₂(L²)H₂O(CH₃COO)](2) (where L¹H_3° = 2-(2′-hydroxyphenyl)-1,3-bis[4-(2-h

Full text of DOI:10.1016/j.ica.2019.118993

Accepted Manuscript
Research paper
The influence of the tertiary butyl group in the ligand frame on the catalytic
activities, DNA cleavage ability and cytotoxicity of dinuclear nickel(II) com-
plexes
Kapil Kumar, Ashish Kumar Dhara, Virendra Kumar Chaudhary, Nathani
Sandip, Partha Roy, Pankaj Verma, Kaushik Ghosh
PII:
S0020-1693(19)30697-8
https://doi.org/10.1016/j.ica.2019.118993
118993
DOI:
Article Number:
Reference:
ICA 118993
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
24 April 2019
28 June 2019
29 June 2019
Please cite this article as: K. Kumar, A. Kumar Dhara, V. Kumar Chaudhary, N. Sandip, P. Roy, P. Verma, K.
Ghosh, The influence of the tertiary butyl group in the ligand frame on the catalytic activities, DNA cleavage ability
and cytotoxicity of dinuclear nickel(II) complexes, Inorganica Chimica Acta (2019), doi: https://doi.org/10.1016/
j.ica.2019.118993
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The influence of the tertiary butyl group in the ligand frame on the
catalytic activities, DNA cleavage ability and cytotoxicity of
dinuclear nickel(II) complexes
a
a
a
b
Kapil Kumar, Ashish Kumar Dhara, Virendra Kumar Chaudhary, Nathani Sandip, Partha
b
a
a
Roy, Pankaj Verma, Kaushik Ghosh *
aDepartment of Chemistry, Indian Institute of Technology Roorkee,
Roorkee-247667 Uttarakhand INDIA.
bDepartment of Biotechnology, Indian Institute of Technology Roorkee,
Roorkee-247667 Uttarakhand INDIA.
E-mail: ghoshfcy@iitr.ernet.in
1

Abstract
1
Dinuclear
phenoxo-bridged
nickel
complexes
[Ni (L )H O(CH COO)](1)
and
2
2
3
2
1
[
Ni (L )H O(CH COO)](2) (where L H = 2-(2′-hydroxyphenyl)-1,3-bis[4-(2-hydroxyphenyl)-3-
2
2
3
3
2
azabutenyl]-3-enyl]-1,3-imidazolidine and L H = 2-(3,5-di-tert-butyl-2-hydroxyphenyl)-1,3-
3
bis[4-(3,5-di-tert-butyl-2-hydroxyphenyl)-3-azabutenyl]-1,3-idazolidine, and H stands for
dissociable protons) have been synthesized and characterized by different spectroscopic
methods. Molecular structure of 2 was determined by X-ray crystallography. Redox properties of
complexes 1 and 2 were investigated by cyclic voltammetric studies. As functional mimic of
active site of galactose oxidase enzyme, complexes 1 and 2 were utilized for the oxidation of
primary alcohol. Both the complexes exhibited catecholase activity. DNA interaction studies on
complexes 1 and 2 were also investigated. Self-activated DNA cleavage activity of complex 2
was observed.The cytotoxicity of the complexes 1 and 2 was measured using a standard MTT
assay and IC values for of 1 and 2 were found to be 21.92 µM and 20.69 µM on MCF-7 breast
5
0
cancer cell line respectively.
2

1
. Introduction
Galactose oxidase (GO) is fungal enzyme that contains mononuclear copper metal centre

in the active site [1]. However, this enzyme catalyzes 2e oxidation of substrate primary

alcohols to its corresponding aldehyde and this process of 2e reduction of oxygen
provides hydrogen peroxide (Scheme 1). Redox active phenolato function bound to
copper centre could produce phenoxyl radical. The phenoxyl radical is stabilized by
modified cysteine group present at ortho-position to the phenolato function of tyrosine
residue [1]. Keeping in mind the active site of galactose oxidase enzyme, several research
groups are involved in mimicking the active site of GO. Hence structural and functional
modelling of active site of GO are important areas of chemical research. Similar to
copper, nickel was also found to be redox active and nickel complexes also could
generate radical species during catalytic processes [2].
There are several reports of mononuclear nickel complexes and different research group explored
the chemistry of the phenoxyl radical complexes of nickel. Recently, Thomas and coworkers [2],
Benisvy and coworkers [3], Shimazaki et al. [4], Mukherjee and coworkers [5] have synthesized
mononuclear nickel complexes. However, reports on dinuclear complexes of nickel coordinated
to phenolato function are scares. Das and coworkers [6], Fondo and coworkers [7], Ray and
coworkers [8] Mitra and coworkers [9] are important to mention in this regard. Investigation of
literature clearly indicated that none of these reports described the activity of the complexes in
terms of functional modeling of active site GO. DNA interaction and cytotoxicity studies were
also not been investigated. This prompted us to explore the chemistry of dinuclear nickel
complexes, in the context of the functional modeling of GO enzyme. It has been found out that
such complexes can efficiently oxidize catechols (catecholase activity). Recently, Ghosh and
3

coworkers [10], Mukherjee and coworkers [11a] and Das and coworkers [6] have investigated
the dinuclear nickel complexes for the functional mimicking of catecholase activity. We have
reported [12] that the presence of tert-butyl group in the ligand frame could give rise to self
activated DNA cleavage and anticancer activity.
GOoxyactive
GOsemiinactive
O
II
.
O
NHis
NHis
Cu
O
1 e-
S
RCH OH
-
OH2
2
O
NHis
II
Cu
H2O2
NHis
OH2
S
RCHO
O2
OH
HO
Cu
I
NHis
NHis
S
OH2
GOred
Scheme 1. Schematic representation of the active site of galactose oxidase enzyme.
Keeping in mind the active site of galactose oxidase enzyme, we have synthesized tris-phenolato
based heptadentate ligand, which could stabilize phenoxyl radical complex after metallation.
Several groups are working using this heptadentate ligand (Scheme 2) with the different
substituents. Fondo et al. [7], Ray and co-workers [8], Batten et al. [13] have also worked with
this type of system but to the best of our knowledge no one has investigated the chemistry of
phenoxyl radical species and related biological activities.
Herein we report the synthesis and characterization of two dinuclear nickel complexes
1
2
[
Ni (CH COO)(L )(H O)](1) and [Ni (CH COO)(L )(H O)](2) derived from ligands
2 3 2 2 3 2
1
2
L H and L H (Scheme 2). Molecular structure of complex 2 was determined by X-ray
3
3
crystallography. We have investigated the oxidation of primary alcohol to aldehyde and
oxidation of catechol i.e. catecholase activity. DNA cleavage activity of complexes was
explored and cytotoxicity of the complexes was examined using MTT assay.
4

Scheme 2. Schematic diagram of ligands.
2
2
. Experimental
.1. Materials
All the solvents used were of reagent grade. Dichloromethane was purified by distillation over
P O and stored over molecular sieves. Analytical grade reagents salicylaldehyde (SRL,
2
5
Mumbai, India), 3,5 di-tert-butyl 2- hydroxyl benzaldehyde (Sigma Aldrich, Steinheim,
Germany), triethylenetetramine (Loba Chemie Pvt. Ltd. Mumbai, India) were used as obtained.
Analytical grade reagents, nickel acetate, hydrogen peroxide, cerric ammonium nitrate, (Himedia
Laboratories Pvt. Ltd., Mumbai, India), sodium hydride (Acros organics, USA), TEMPO
((2,2,6,6-Tetramethylpiperidin-1-yl)oxyl), TBHP (tert-Butyl hydroperoxide), 3,5-DTBC (3,5-Di-
tertbutylcatechol) were used as purchased. The supercoiled pBR322DNA was obtained from
Bangalore Genei (India) and stored at 4C. Ethidium bromide was purchased from Sigma-
Aldrich, Steinheim, Germany. Agarose was obtained from (Sisco Research Laboratories Pvt.
Ltd., Chennai, India). Tris-acetate-EDTA (TAE) buffer was prepared in deionised water. All
solvents purified by standard procedures and used for spectroscopic studies were HPLC grade
[17]. Human breast cancer cell line MCF-7 was procured from National Centre for Cell Science,
Pune, India. DMSO (Himedia, Mumbai, India), cells were cultured in DMEM high glucose
5

(HiMedia, India) and supplemented with 10% FBS and 10% antibiotic-antimycotic (GIBCO,
Invitrogen, USA) in a humidified incubator with 5% CO supply at 37 ᵒC. Cells were grown up
2
to 80-90% confluency andharvested with trypsin-EDTA (0.25%) (GIBCO, Invitrogen, USA) and
plated in 96-well plate at a desired concentration and allowed to grow for 24 h before any
treatment.
2
.2. Physical measurements
Micro analytical elemental analyses were performed on Elementar Vario MICRO (Germany).
Infrared spectra were recorded on Thermo Nicolet Nexus FTIR spectrophotometer and were
obtained in KBr pellets using 16 scans. Electronic absorption spectra of all the ligands and metal
1
complexes were obtained with Evolution 600, Thermo Scientific UV-Vis spectrophotometer. H
NMR spectra was taken in the deuterated solvents on Bruker AVANCE, 500.13 MHz
spectrometer. Mass spectra were obtained by using direct inlet system (70 eV) with detector (ES,
4
000 V) on a Perkin Elmer Gas chromatography-mass spectrometry (GC-MS). Cyclic
voltammetric study was performed on a CH-600 electroanalyzer with 0.1 M
–
3
tetrabutylammonium perchlorate (TBAP) as supporting electrolyte and 10 M concentration of
complexes in acetonitrile solution. A conventional three-electrode arrangement was made of
platinum wire as the auxiliary electrode, glassy carbon as the working electrode and Ag(s)/AgCl
as the reference electrode. The ferrocene/ferrocenium couple appeared at E /V(∆E /mV) =
1
/2
p
+
0.44(143) vs. Ag/AgCl (scan rate 0.1 V/s) in acetonitrile under the same experimental
conditions. All experiments were carried out at room temperature and solutions were thoroughly
degassed with nitrogen prior to beginning the experiments.
6

1
2
2
.3. Synthesis of ligand L H L H
3, 3
1
2
Two tris-phenolato based ligands L H and L H have been synthesized by the following
3
3
1
literature procedure [14] and satisfactorily characterized by IR, UV-Vis and H NMR
spectroscopic studies (Scheme S1 in ESI).
1
2
.4. Synthesis of [Ni (L )H O(CH COO)](1)
2
2
3
This nickel(II) complex 1 was synthesized by a literature preparation [7c] with some
1
modifications. A 8 ml acetonitrile solution of ligand L H (137.5 mg, 0.3 mmol) and sodium
3
hydride (7.2 mg, 0.3 mmol) in acetonitrile was stirred for 30 min. A solution of
Ni(CH COO) .4H O (148.8 mg, 0.6 mmol) in acetonitrile-methanol was added to the reaction
3
2
2
mixture with constant stirring. After 8 hrs stirring yellowish brown colored complex was
precipitated out. Evaporating the solvent and the solid was separated by filtration. Yellowish
brown colored compound came out in methanol when hexane was layered over it. Yield: 76%,
Anal. Calc. for C H N O Ni : C, 53.59; H, 4.96; N, 8.62. Found: C, 53.41; H, 4.65; N, 8.58.
2
9
32
4
6
2

1
Infrared spectrum (cm , KBr disk): 3429, 1630, 1579, 1402, 1265, 1111, 758. UV-Vis (CH Cl ;
2
2

1
1
λmax, nm (ε, M cm )): 231 (41056), 279 (27204), 398 (7698).
2
2
.5. Synthesis of [Ni (L )H O(CH COO)](2)
2
2
3
2
Similar to the procedure depicted above, 8 ml acetonitrile solution of ligand L H (238.2 mg, 0.3
3
mmol) was stirred with (7.2 mg, 0.3 mmol) sodium hydride for 30 min. A 8 ml acetonitrile-
methanol solution of Ni(CH COO) .4H O (148.8 mg, 0.6 mmol) was added into it with constant
3
2
2
stirring. After 6 h of stirring, yellowish brown colored complex was precipitated out.
Evaporating the solvent and the solid was separated by filtration. Yellowish brown colored
compound came out in methanol when hexane was layered over it. Yield: 72%, Anal. Calc. for
C H N O Ni : C, 64.52; H, 8.17; N, 5.6. Found: C, 64.32; H, 8.25; N, 5.80. Infrared spectrum
5
3
80
4
6
2
7


1
(
cm , KBr disk): 3438, 2941, 2810, 1633, 1490, 1458, 1406, 1273, 1159, 1111, 1015, 854, 834,

1
1
7
2
55. UV-Vis (CH Cl ; λ , nm (ε, M cm )): 228 (63500), 256 (34775), 396 (5325).
2 2 max
.6. X-ray crystallography
The yellowish brown colored crystals of complex 2 were grown in a solution of CH Cl -MeOH
2
2
solvents mixture by slow evaporation. Single crystal of suitable dimensions was used for data
collection. The X-ray diffraction data of nickel(II) complex 2 was obtained at 293K on a Bruker
Kappa Apex-II CCD diffractometer by the use of graphite monochromatic Mo-Kα radiation (λ =
0
.71073 Å) [15,16]. Direct methods were used to solve the crystal structure of metal complex.
Structure solution, refinement and data outputs were carried out with the help of SHELXTL
program [17]. Bond angle and bond distances were calculated using MERCURY software. To
refine hydrogen and non-hydrogen atoms, isotropic and anisotropic thermal parameters were
used respectively. DIAMOND program was used to get ORTEP images. Pertinent
crystallographic parameters are given in ESI (Table S1).
2
.7. Benzyl alcohol oxidation
-
As the galactose oxidase enzyme include 2e redox process and phenoxyl radical mechanism
during oxidation of primary alcohol, we carried out the reactivity of this enzyme towards aerial
oxidation of benzyl alcohol (primary alcohol). We have set up two different types of reactions
for investigated of this oxidation chemistry. The first TEMPO and TBHP method: Take 3 mmol
benzyl alcohol, 5 mol% of Et N, 0.2 mol% complexes as a catalyst, 5 mol% of TEMPO and 0.5
3
mmol of TBHP were added in 10 ml of different solvents and allowed to stir under oxygen at 70
°
C. For the second method, repeat the first procedure without adding the TEMPO. After 24 h to
an eluent, 0.5 ml standard solution of heptane was added and extracted the organic layer, washed
with water (pH = 4), then dried over anhydrous sodium sulphate, and the products were
8

quantified by GC-MS. Different yields were obtained with complex 1 and 2 under different
conditions given in ESI (Table S3). For complex 2, high % conversion of benzyl alcohol into
benzaldehyde was observed when aerial oxidation was carried out in CH CN solvent at 70˚C
3
temperature using TBHP and TEMPO as an oxidant. These results reflect the same function as
GO enzyme and both metal complexes are efficient to carry out the aerobic oxidation of primary
alcohol to aldehyde.
2
.8. Catecholase activity and H O detection
2 2
Catecholase activity was carried out by examining the oxidation of 3,5-DTBC (3,5-Di-
tertbutylcatechol) in acetonitrile solution. For this investigation, 10−4 M solutions of
−
2
−3
nickel(II) complexes 1 and 2 were added to 1 × 10 mol dm (100 equivalents) of 3,5-
DTBC in acetonitrile under aerobic conditions at 25 °C. The course of this reaction was
monitored by UV-Vis spectrophotometer.
The formation of H O during the catalytic reaction was investigated by following the
2
2
−
development of the characteristic band for I spectrophotometrically (λ = 353 nm; ε = 26 000
3
max

1
1

M cm ), upon reaction with I [18,19]. The oxidation reactions of 3, 5-DTBC in the presence

5
of different catalysts were carried out as in the kinetic experiments ([Complex] = 2.5 × 10 M;

5
[
3,5-DTBC] = 50 × 10 M). After 1 h of reaction an equal volume of water was added and the
quinine formed was extracted three times with dichloromethane. The aqueous layer was acidified
with H SO to pH = 2 to stop further oxidation, and 1 ml of a 10% solution of KI and three drops
2
4
of 3% solution of ammonium molybdate were added. In the presence of hydrogen peroxide,

+
occurs the reaction H O + 2I +2H → 2H O + I , and with an excess of iodide ions, the
2
2
2
2


triiodide ion is formed according to the reaction I (aq.) + I → I . The reaction rate is slow but
2
3
increases with increasing concentrations of acid, and the addition of an ammonium molybdate
9


solution renders the reaction almost instantaneously. The formation of I could be monitored
3
−
spectrophotometrically due to the development of the characteristic I band λ = 353 nm, ε = 26
3
−
1
1
0
00 M cm ) [20].
2
.9. DNA cleavage experiments
Cleavage of plasmid DNA was monitored by using agarose gel electrophoresis. Supercoiled
pBR322 plasmid DNA (100 ng) in Tris-acetate-EDTA (TAE) buffer (pH 8.20) was treated with
the nickel(II) complexes (5-50 μM) and 10% DMF (complexes 1 and 2) in the absence of
additives. The samples were incubated for 1 h at 37 ˚C. The agarose gel (1%) containing 2.5 µl
(10 mg/mL stock) of ethidium bromide (EB) was prepared and the electrophoresis of the DNA
cleavage products was performed on it. The gel was run at 60V for 2 h in TAE buffer and the
bands were identified by placing the stained gel in Gel doc system and illuminated UV lamp.
The fragments were photographed by using a gel documentation system (BIO RAD).
2
.10. Cell culture and cytotoxicity assay
To determine the cytotoxic effect of Complexes 1 and 2, MTT (3-(4,5-Dimethylthiazol-2-yl)-
2
7
,S-diphenyltetrazolium bromide) assay was carried out as described previously [21]. The MCF-
cells were cultured in DMEM media supplemented with 10% fetal bovine serum (heat
inactivated) and 1% antibiotic (100 U/ml of penicillin and 100 µg/ml streptomycin) mix at 37 ˚C
in a humidified atmosphere in a CO incubator. All the experiments were performed using cells
2
3
between 14 and 32 passages for MCF-7. In brief, 5×10 cells in 200 µl of medium were seeded in
9
6-well plates and grown under normal conditions. Serial dilutions of nickel(II) complexes 1 and
2
ranging from 5-100µM in DMSO were added to the monolayer in triplicates. Cultures were
assayed after 24 h by the addition of 20µl of 5 mg/ml MTT and incubating for 3-4 h at 37 ˚C.
The MTT-containing medium was aspirated and 200µl of DMSO (Himedia, Mumbai, India) was
added to solubilize the formazon crystals. Absorbance was determined on a Fluostar omega
1
0

(BMG Labtech, Germany) microplate reader at 570nm. The percentage inhibition was calculated
as:
Mean OD of vehicle treated cells Mean OD of treated cells
Mean OD of vehicle treated cells
×
100
3
3
. Result and discussion
.1. Synthesis
1
2
The ligands L H and L H were prepared (Scheme S1 in ESI) following a literature procedure
3
3
[14] and their reactions with Ni(II) salts were systematically investigated. Complexes 1 and 2
1
2
were prepared in one-pot synthesis, by direct interaction of L H and L H respectively. The
3
3
1
reaction of depronated ligand L H and Ni(CH COO) .4H O in acetonitrile-methanol (2:1)
3
3
2
2
1
solution afforded complex [Ni (L )H O(CH COO)](1). Nickel-salt to ligand ratio was 1:2.
2
2
3
2
Similarly, treatment of depronated liagand L H with Ni(CH COO) .4H O in a 1:2 molar ratio
3
3
2
2
2
yielded a yellowish brown colored complex [Ni (L )H O(CH COO)](2). The reaction was
2
2
3
carried out in aerobic condition at room temperature. The identities of complexes were
characterized by analytical and spectroscopic techniques. Molecular structure of complex 2 was
determined by single crystal X-ray diffraction technique.
The FTIR spectra of 1 and 2 were shown in supporting information (Fig. S2 and S3 in the ESI)
and these IR spectra proves that the coordination of the Schiff base ligands to Ni(II) ions. The IR
−
1
spectra of the two complexes show a strong band in the range 1630-1640 cm . This band was
assigned to νC=N, which suggested the coordination of the ligand to the metal ions through the
−
1
imine nitrogen atoms [6, 7b]. In addition, a broad band in the range 3425-3450 cm agrees with
the hydration of the compounds. The mode of binding of acetate to nickel(II) ions within the
framework of complex 2 has been established from the FTIR frequency ranges found for five
1
1

characteristic vibrational modes, the CH, CO, CC stretching modes (νCH, ν , νCC) and
CO
CH , COO deformation modes (νCH3, νCOO). All these appear near 2940, 1405, 1015, 1580 and
3
-1
7
50 cm respectively [22].
Electronic spectra of the complexes 1 and 2 in acetonitrile solvent provided two characteristic
bands near 300 and 400 nm (Fig. S4 and Fig. S5 in ESI). These two transitions are probably
assigned to intra-ligand charge transfer (ILCT) or metal to ligand charge transfer (MLCT)
[26,34a].
2
Fig. 1. ORTEP plot (40% probability level) of the complex [Ni (L )H O(CH COO)] (2).
2
2
3
Hydrogen atoms are omitted for clarity.
3
.2. Description of molecular structure
2
3-
To confirm the structure of the complex 2, and mode of coordination of the ligand (L ) , single
crystal X-ray structure determination of the complex 2 was carried out. The molecular structures
2
of dinuclear nickel(II) complex [Ni (L )H O(CH COO)](2) are shown in Fig. 1. The bond
2
2
3
lengths and bond angles are described in the ESI (Table S2) and the parameters of complex 2 are
1
2

depicted in the ESI (Table S1). The crystal structure revealed a dinuclear nickel(II) complex,
II
where one of the Ni ion is coordinated to water molecule and constituted hexacoordination. On
II
the other hand, another Ni ion was found to be pentacoordinated. One phenolato oxygen and
one accetato group acted as bridging ligands between two Ni(II) centers in complex 2. To
describe the geometry around the pentacoordinated nickel(II) centre we have calculated the
structural index parameter τ for the complex 2 [21,24]. The value of  = 0.346 clearly indicated a
distorted square pyramidal geometry around the pentacoordinated nickel(II) centre in 2. In
II
pentacoordinated Ni ion, phenolato oxygen (O4) was trans to amine nitrogen (N6) and another
imine nitrogen (N2) was trans to oxygen (O6) of acetato group. With coordinated water
molecule the second metal centre was coordinated to one amine nitrogen, one imine nitrogen and
II
a phenolato function. Two bridging ligands completed the hexacoordination of the Ni centre
II
complex 2. The geometry around the Ni was observed to be distorted octahedral. All Ni–N
imine
II
distances were found to be consistent with the reported Ni Schiff base complexes [6,7b].
Similarly Ni–O(phenolato) bond distances were found to be in the range from 1.933 Å to 1.978
Å and these distances are similar to the data reported by Benisvy and coworkers[3]. In the same
II
way, the bond distance (approx. 3.1 Å) between two Ni centers found to be consistent with
literature values [7a, 7c, 25].
3
.3. Generation of phenoxyl radical of 1 and 2
Phenoxyl radical complexes were generated by addition of ceric ammonium nitrate (CAN) in
acetonitrile solution,. The representative spectrum of complex 2 is shown in Fig. 2 and similar
spectra of complex 1 have shown in Supporting Information (Fig. S7 in the ESI). Generation of
1
3

phenoxyl radical in complexes 1 and 2 after addition of CAN is analyzed by UV-Vis
spectrophotometer.

5
Fig. 2. Generation of phenoxyl radical in 2 (10×10 M) after addition of CAN (1
equivalent, 2 equivalents) at 273K temperature.
After addition of CAN in acetonitrile solution of complex 2, a band at around 390 nm was
found to be disappeared and a new band at 410 nm and a broad band of low intensity at
around 730 nm were observed. However for complex 1 a broad band of very low intensity
around 700 nm was observed. This broad band observed around 700 nm and 730 nm in 1
and 2 respectively, expressed the formation of stable phenoxyl radical [26].
3
.4. Electrochemistry
We have studied the redox properties of nickel(II) complexes 1 and 2 at room temperature, cyclic
voltammetric experiments were performed under nitrogen atmosphere within the potential range
-1.2 V to +1.2V at a scan rate of 0.1 V/s by using Ag/AgCl as reference electrode, platinum wire
1
4

as auxiliary electrode, glassy carbon as working electrode. The data obtained from cylic
voltammetry at 298 K under inert atmosphere in acetonitrile are shown in Table 1. The cyclic
Table 1 Cyclic voltammetric data for complexes 1 and 2 at 298 K. Conditions: solvent,
acetonitrile; supporting electrolyte, TBAP (0.1 M); working electrode, glassy carbon;
-1
-3
reference electrode, Ag/AgCl; scan rate, 0.1 V s ; concentration, 10 M
Complex
Phenoxyl radical
E /V E /V E /V(∆E /mV)
pa
pc
1/2
p
2
1
0.761 0.714
.903 0.837
0.525 0.909
.661 1.030
0.737(47)
0.870(66)
0.593(136)
0.969(121)
0
0
E1/2 = 0.5 (E + E ) and ΔE = (E − E ), where E and E are anodic and cathodic peak
pa
pc
p
pa
pc
pa
pc
potentials respectively.

3
2
Fig. 3. Cyclic voltammograms of 10 M solutions of complex [Ni (L )H O(CH COO)](2)in the
2
2
3
presence of 0.1 M tetrabutylammonium perchlorate (TBAP), using a working electrode (glassy-
carbon), reference electrode (Ag/AgCl) and counter electrode (platinum wire), scan rate = 100

1
mVs .
1
5

-3
voltammograms of 10 M acetonitrile solution of the complex 2 in the potential range 1.20 V
to -1.2 V as shows in Fig. 3. As a substituent, tert-butyl groups also enhanced the reversibility of
2
complex, we have found quasireversible peaks at E1/2 = 0.737 V and 0.870 V vs. Ag/AgCl
These species were assigned as ligand-centered oxidation of phenoxyl radical species
successively [26]. Cyclic voltammogram for the complex 1 have shown in supporting
information (Fig. S8).
3
3
.5. Catalytic activity
.5.1. Benzyl alcohol oxidation
The catalytic conversion of benzyl alcohol (primary alcohol) into benzaldehyde was optimized
using the dinuclear nickel(II) complexes in the presence of TBHP and TEMPO/TBHP with a
small amount of base in different solvents shown in ESI (Table S3). We have optimized the
reaction condition at 70 °C temperature in presence of catalyst 0.2 mol% in air [34c]. The
conversion of benzyl alcohol to benzaldehyde increased with time and after 24 h no more
oxidation product was observed. In acetonitrile solvent, combination of TEMPO, TBHP and the
complex 2 afforded the conversion up to 68% of benzyl alcohol to benzaldehyde, however
combination of acetonitrile solvent, TBHP and complex 2 gave rise to benzaldehyde and benzoic
acid, with a conversion up to 53% for the aldehyde in these reactions.
In the oxidation of the primary alcohols, complex 2 was found to be more effective than complex
1
. These data clearly indicate the importance of complexes capable of generation of stable
phenoxyl radical in solution during the catalytic process. During the alcohol oxidation, the
addition of base played an important role in activation of the alcohol [27-28]. Here we have used
triethylamine as base for the activation of benzyl alcohol.
1
6

3
.5.2. Catecholase activity
In this study 3,5-Di-tertbutylcatechol (3,5-DTBC) was utilized as a substrate for the study of
catechol oxidase activity because its low reduction potential of 3,5-DTBC which makes it easy
to oxidize [29]. On the other hand, it is very important to note that the bulky tert-butyl groups
prevent further over oxidation reactions such as ring-opening and the oxidation product, 3,5-di-
tert-butylquinone (3,5-DTBQ), is more stable product. A characteristic absorption band maxima
at 402 nm gave rise to the formation of 3,5-di-tert-butylquinone (3,5-DTBQ) in pure methanol.
However it is clear that the spectra of 2 in acetonitrile solution showsed drastic changes
immediately after addition of 3,5-DTBC (100 equivalents). One band was observed near 402 nm
(Fig. 4) corresponds to 3,5-DTBQ in pure acetonitrile solution. Kinetics of oxidation of 3,5-
DTBC to 3,5-DTBQ mediated by 1 and 2 were determined by the
1.8
3,5DTBQ
1.2
Complex
0.6
3,5DTBC
0.0
300
400 500
Wavelength(nm)
600

4
Fig. 4. Changes observed in UV-Vis spectra of complex 2 (conc. 1 × 10 M) upon addition of

2
1
00-fold 3,5-DTBC (1 × 10 M) in acetonitrile solvent.
method of initial rates by monitoring the growth of the quinone band at 402 nm as a function of
time. The rate constant, for a particular complex and substrate mixture is determined from the
log [A /(A −A )] vs time plot. The substrate concentration dependence on the oxidation rates and
α
α
t
1
7

various kinetic parameters were determined by using 10−4 M solutions of 1-2 with different
concentration of 3,5-DTBC under aerobic conditions.
In all cases, a first-order kinetic dependence was observed at low concentrations of 3,5-DTBC,
whereas higher concentrations resulted in saturation kinetics. The observed rates versus
concentrations of substrate data were analyzed, on the basis of the Michaelis-Menten approach of
enzymatic kinetics Lineweaver-Burk plot and values of kinetic parameters Vmax, K , and K .
M
cat
Both the observed rate vs [substrate] and Lineweaver-Burk plots for 2 are shown in ESI (Fig. S9
and Fig. S10). A similar plot for 1 is given in the Supporting Information (Fig. S12 and S13 in
the ESI). The kinetic parameters for both complexes are deposited in Table 2. The k values can
cat
be calculated by dividing the Vmax values from the concentration of the corresponding nickel
3
1
1
complexes. This value for 2 [Kcat = 9.07×10 h ] is higher than that of 1 [K = 150 h ]. The
cat
oxidation of 3,5-DTBC to 3,5-DTBQ by nickel complexes being a two electron oxidation
process, for this two different path may be considered, (i) generation of ligand centred free
II
I
radicals with consequential oxidation of the substrate or (ii) reduction of Ni to Ni with
concurrent oxidation of 3,5-DTBC. In order to prove the possibility of radical participation we
have done the EPR study. The EPR spectra recorded immediately after mixing of nickel(II)
complexes with 3,5-DTBC exhibit broad signal with g value of 1.994 given in fig.5, Das and co-
workers [6] under same experimental conditions, verify the mixture of Ni(CH COO) .4H O and
3
2
2
3
,5-DTBC are EPR silent. The generation of EPR signal with g value of 1.994 clearly indicate of
the formation of ligand centred radical species, which is generated only when the Ni(II)
complexes are mixed with 3,5- DTBC and this radical formation is most likely responsible for
that oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylbenzoquinone (3,5-
1
8

DTBQ). In order to understand how inductive effect encumbrance on the ortho and para of the
phenolato oxygen can influence oxidation of 3,5-DTBC to 3,5-DTBQ.
Fig. 5. EPR spectra of the reaction mixture during the oxidation of 3,5-DTBC into 3,5-DTBQ at
2
98 K.
The catalytic activities of 1 and 2 are found to increase in the order H (1) < tert-butyl (2) where
3
1
complex 2 showing the highest activity [Kcat = 9.68×10 h ]. Complex 2 was also found to be
more efficient than reported nickel complexes in literature [31]. Casella and coworkers [29a] and
Karlin and coworkers [29b] previously suggested a mechanism for the oxidation of catechols to
o-quinones by dinuclear Cu(II) complexes, and they proposed that in the second step, two
electrons of the catecholate reduce the Cu(II) to Cu(I) after transference of two protons to the
bridging alkoxide groups [29]. It is postulated that an increase in steric encumbrance causes the
Cu O core to experience greater strain and become less planar, resulting in a faster rate of
2
2
proton transfer as well as reduction of the metal centers, and hence in higher reactivity towards
oxidation of 3,5-DTBC. This explains why increasing steric encumbrance from H (1) → tert-
butyl (2) brings about an increase in catecholase activity in the same order.
1
9

Table 2 Kinetic parameters for the oxidation of 3,5-DTBC to 3,5-DTBQ mediated by 1 and 2 in
acetonitrile.

1
Complex
V
(M
K (M)
K (h )
max
m
cat

1
s )


6
4
3
1
2
4.28×10
2.52×10
7.15×10
1.90×10
150
3
3
9.07×10
It was necessary to investigate the formation of either H O or water during the oxidation
2
2


process. UV-Vis spectral study clearly showed the generation of I by the oxidation of I to I
3
2
(Fig. S14 in the ESI). Reaction and proper work-up of the mixture of 3,5-DTBC, KI and complex
2
(see the Experimental section) indicated that oxygen was reduced to H O as previously
2 2
reported by other research groups [30-32].
3
.6. DNA Cleavage activity
The representative metal complexes 1 and 2 were studied for their DNA cleavage activity by the
agarose gel electrophoresis method against DNA of E. coli (pBR322 plasmid DNA). The
electrophoresis analysis clearly revealed that the DNA cleavage behavior of the complexes 1 and
2
under physiological conditions (37 °C), and observed by the transformation of the supercoiled
form (SC) to the nicked (NC) and linear (LC) forms of plasmid DNA in TAE (Tris-accetate-

1
EDTA) buffer. Test complexes 1 and 2 were prepared in 10% DMF (1ꢀmgꢀml ). Before
electrophoresis experiment, the reaction mixtures were incubated at 37˚C for 1 h. The samples of
reaction mixtures were electrophoresed for 30 min at 60 V on 1% agarose gel using TAE buffer
(pH = 8.20).
To estimate the DNA cleavage ability of both nickel(II) complexes and supercoiled pBR322
DNA was incubated with various concentrations of the complexes in TAE (pH = 8.20). Results
of the agarose gel electrophoretic separations of increasing concentrations of the Ni(II)
2
0

complexes with pBR322 plasmid DNA have been shown in Fig. 6 and Fig. 7. When the
concentrations of complex 1 (5-50 μM) was increased (Fig. 6, lanes: 3-7 for 1), it was observed
that complex 1 could cleave the supercoiled form (SC) of pBR322 plasmid DNA into linear
form(LC) and nicked form(NC) in a concentration-dependent fashion. However the amount of
the SC form of pBR322 DNA decreased gradually, whereas the amount of the NC form
increased. Hence complex 1 was able to induce significant cleavage of the plasmid at the
concentration of 5-50 μM.
However, we have observed a different type of nuclease activity of complex 2, in which the
disappearance of both bands (SC and NC DNA) was observed (Fig. 7). We observed similar
results in our previous study [33]. On increasing the concentration of 2 (∼50 μM) we found the
disappearance of DNA bands. Hence, in the absence of an external agent, 2 exhibited excellent
DNA cleavage activity i.e. self-activated DNA cleavage was observed [12]. This may be due to
•+
generation of phenoxyl radical complex [2] stabilized by tert-butyl group [33-35]. Therefore,
•+
•+
this radical complex [2] cleaved DNA effectively as compared to radical complex [1] .
Electrophoresis was done at 60V for 2 h in a Tris-acetate-EDTA (TAE) buffer; the bands were
visualized in a Gel doc system and under a UV illuminator take the photographing of
fluorescence of intercalated ethidium bromide. From these results, we can presuppose that in
DNA cleavage activity complex 2 may be important for studying anticancer activity.
Fig. 6. Gel electrophoresis separations showing the cleavage of pBR322 DNA by complex 1 in a
buffer containing 10% DMF and incubated at 37˚C for 1 h. (a) Lane 1: DNA control. Lane 2:
2
1

DNA + metal. Lane 3: DNA +1 (5 μM). Lane 4: DNA + 1 (10 μM).Lane 5: DNA + 1 (20 μM).
Lane 6: DNA + 1 ( 50 μM).
Fig. 7. Gel electrophoresis separations showing the cleavage of pBR322 DNA by complex 2 in a
buffer containing 10% DMF and incubated at 37˚C for 30 min. (a) Lane 1: DNA control. Lane 2:
DNA + metal. Lane 3: DNA +2 (5 μM). Lane 4: DNA + 2 (20 μM). Lane 5: DNA + 2 (30 μM).
Lane 6: DNA + 2 (40 μM). Lane 7: DNA + 2 (50 μM).
The generation of the reactive oxygen species (ROS) in the cleavage reaction was also
determined by the DPPH radical quenching experiment [36,37]. The degradation of the reactive
oxygen species was investigated by the absorption titration ∼60µM DPPH in the presence of
Fig. 8. UV-Vis spectra (A) and (B) of DPPH quenching assay in the presence of an increasing
concentration of 2 and 1 (0–200 μM) respectively.
complexes 1 and 2 (0–200 µM) in MeOH. The degradation of the DPPH band at 516 nm and 513
nm with addition of increasing concentration of the complex 1 and complex 2 (shown in Fig. 8),
complex 1 shows more quenching than complex 2. This also further indicates that phenoxyl
radical generation in 2 is more stable than 1 due to the tert-butyl groups present on the phenolato
ring.
2
2

3
.7. Cell cytotoxicity assay
DNA cleavage activity studies on complexes 1 and 2 afforded interesting data and these data
prompted us to determine the cytotoxiciy assays for these complexes. The cytotoxicity of the
complexes 1 and 2 were measured using a standard MTT assay, which measures mitochondrial
succinate dehydrogenase activity as an indication of cell viability against human-derived cell
lines (for example in this study the MCF-7 cell line: a breast adenocarcinoma cell line [37,38].
To determine the cytotoxic effect of Complexes 1 and 2 MTT (3-(4,5-Dimethylthiazol-2-yl)-2,S-
diphenyltetrazolium bromide) assay was carried out. The final DMSO concentration used for all
Fig. 9. (a) and (b) shows the Cytotoxic effect of 2 and 1 respectively on MCF-7 breast cancer
cell line. Cells were treated with10 µM dose of positive control 5-fluorouracil (PC) and 5, 10, 20,
4
0, 80, 100 µM doses of complexes 1 and 2 for 24 h. Data are expressed as mean ± SEM of
three independent experiments.
dilutions (0.5%) was used as a vehicle control. Cells were seeded in 96-well plate and treated
with different concentration (5, 10, 20, 40, 80 and 100 µM) of 1 and 2 for 24 hour. Cells were
also treated with 10 µM of positive control 5-fluorouracil (PC) for 24 hour. After treatment
media of each crystals formed inside cells were solubilized by adding 200 µL of DMSO into
each well. In proportion of viable cells violet crystals were visible and absorbance was taken at
5
70 nm in microplate reader. Values were quantified as the mean of three independent
2
3

experiments. The cell cytotoxicity was expressed as percentage cell viability in comparison to
1
2
control group. The ligands L H and L H afforded IC values of 67.85 µM and 60.54 µM
3
3
50
respectively(Fig. S15 in the ESI). Interestingly, both the complexes 1 and 2 exhibited more
1
2
cytotoxicity than ligands L H and L H for MCF-7 cells (shown in Fig. 9). Also the complex 2
3
3
exhibited better cytotoxicity as compared to 1. This indicated that Complex 2 has the higher
potency for MCF-7 cells. Cisplatin have showed IC values of 65.87 ± 5.97 μM towards MCF-7
5
0
cell line [21], however complexes 1 and 2 has shown IC50 values of 21.92 μM, 20.69 µM
respectively and approximately 3-4 times greater than cisplatin for MCF-7 cells.
4
. Conclusions
The following are the conclusions and principal findings of our study.
To investigate the influence of tert-butyl group in the ligand frame of dinuclear nickel(II)
complexes 1 and 2 were synthesized and molecular structure of 2 was determined by X-ray
crystallography. Electron donating tert-butyl substituent in the phenyl ring containing phenolato
function afforded a better stability of the phenoxyl radical complex in case of complex 2 as
compared to 1. These were supported by UV-Vis spectral studies and cyclic voltammetry.
Complexes 1 and 2 were utilized as functional model of galactose oxidase enzyme and oxidation
of benzyl alcohol to benzaldehyde was examined. Complex 2 was found to be more effective
catalyst than 1 for this mild oxidation. Complexes 1 and 2 exhibited catecholase activity and 2
was found to be more efficient than 1. Complex 2 exhibited excellent DNA cleavage activity.
The cytotoxicity of the complexes 1 and 2 were also measured using a MTT assay and IC50
values for of 1 and 2 were found to be 21.92 µM and 20.69 µM respectively. Cytotoxicity of
complexes 1 and 2 was found to be more as compared to Ni(CH COO) ·4H O towards MCF-7
3
2
2
2
4

cells. Complexes 1 and 2 exhibited approximately 3-4 times more potency than cisplatin for
MCF-7 cells. Detail mechanistic investigation on nuclease and biological activity studies are
currently under progress.
Acknowledgements
K. G. is thankful to SERB-DST (No. SR/S1/IC-47/2012) dated 13 May 2014, New Delhi, India
for financial assistance. KK and AKD are thankful to UGC and VKC is thankful to CSIR for
financial assistance. NS and PV are thankful to IIT Roorkee for their MRHD Fellowship. We are
also thankful to Central Instrumental Facility, IIT Roorkee.
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