p-Toluate-bridged dinuclear Cu(II) complexes in combination with tridentate chelating ligand: Crystal structure, density functional theory calculation, DNA/protein binding and catecholase activity

Article abstract of DOI:10.1002/aoc.4506

The dinuclear Cu(II) complexes [Cu₂(L¹)₂(mb)]?ClO₄ (1) and [Cu₂(L²)₂(mb)]?ClO₄ (2) (HL¹?=?2-[(2-diethylaminoethylimino)methyl]phenol; HL²?=?2-[1-

Full text of DOI:10.1002/aoc.4506

Received: 27 February 2018
DOI: 10.1002/aoc.4506
Revised: 12 June 2018
Accepted: 17 June 2018
F U L L P A P E R
p‐Toluate‐bridged dinuclear Cu(II) complexes in
combination with tridentate chelating ligand: Crystal
structure, density functional theory calculation, DNA/
protein binding and catecholase activity
Samar Dolai1 | Kalipada Das1 | Apurba Bhunia1 | Valerio Bertolasi2 | Subal Chandra Manna1
1 Department of Chemistry and Chemical
The dinuclear Cu(II) complexes [Cu2(L1)2(mb)]⋅ClO4 (1) and [Cu2(L2)2(mb)]
Technology, Vidyasagar University,
⋅ClO4 (2) (HL1 = 2‐[(2‐diethylaminoethylimino)methyl]phenol; HL2 = 2‐[1‐
Midnapore 721102, West Bengal, India
2 Dipartimento di Scienze Chimiche e
(2‐diethylaminoethylimino)propyl]phenol; mb = 4‐methylbenzoate) were
Farmaceutiche, Centro di Strutturistica
synthesized and characterized using X‐ray crystal structure analysis and
Diffrattometrica, Università di Ferrara,
Via L. Borsari 46, 44100 Ferrara, Italy
spectroscopic methods. Complexes 1 and 2 are dinuclear with distorted square
pyramidal Cu (II) geometries, where Schiff base coordinates with tridentate
(N,N,O) chelating mode and mb bridges two metal centres. Optimized structures
and photophysical properties of ligands and complexes were calculated using
density functional theory and time‐dependent density functional theory methods
using B3LYP functional with 6‐31G (d,p) and LanL2MB basis sets. Interactions
of the complexes with bovine serum albumin (BSA) and human serum albumin
(HSA) were studied using UV–visible absorption and fluorescence spectroscopies
and the calculated values of association constants (M−1) are 1.7 × 105 (1–BSA),
5.7 × 105 (2–BSA), 1.6 × 105 (1–HSA) and 6.9 × 105 (2–HSA). Interactions of
the complexes with calf thymus DNA were also investigated and the binding
affinities are 1.4 × 105 and 1.6 × 105 M−1 for 1 and 2, respectively. Both complexes
catalytically oxidize 3,5‐di‐tert‐butylcatechol to 3,5‐di‐tert‐butylbenzoquinone in
the presence of molecular oxygen.
Correspondence
Subal Chandra Manna, Department of
Chemistry and Chemical Technology,
Vidyasagar University, Midnapore 721102,
West Bengal, India.
Email: scmanna@mail.vidyasagar.ac.in
KEYWORDS
catecholase activity, crystal structure, DFT calculation, dinuclear Cu (II) complex, DNA/protein
binding
1 | INTRODUCTION
salicylaldehyde and primary amines are studied since
these compounds have antiviral, anticancer and antibac-
Transition‐metal‐based coordination compounds are
important due to their potential application in the areas
of catalysis,[1] magnetism,[2] medicinal chemistry, etc.[3]
Schiff base complexes of 3d metals are important due to
their suitable biometric properties that can mimic active
site structures of biologically important molecules.[4]
Schiff base complexes derived from the condensation of
terial activities.[5] Copper is a bio‐essential element and,
due to its biological activity, copper complexes are well
studied by inorganic chemists in view of their medicinal
applications.[6] Moreover due to the strong Lewis acid
property of cupric ion, Cu (II) Schiff base complexes are
well studied in the area of DNA cleavage, and recently
many Cu(II) complexes of amino acid Schiff base ligands
Appl Organometal Chem. 2018;e4506.
wileyonlinelibrary.com/journal/aoc
© 2018 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4506

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DOLAI ET AL.
have shown artificial nuclease activity cleavage.[7,8]
Serum albumin is the major soluble protein in the blood
and plays an important role for the transportation of
many compounds like fatty acids, drugs, pharmaceuticals
etc.[9] and the study of the kinetics of interactions of
Cu(II) complexes with serum albumins is important
for the development of Cu(II) compound‐based metallo‐
pharmaceuticals. Literature survey reveals that Schiff‐base‐
coordinated Cu(II) complexes are promising candidates for
showing catecholase activities.[10]
of ligands were recorded with a Bruker 400 MHz instru-
ment in CDCl3. Electronic absorption spectra were
obtained with a Shimadzu UV‐1601 UV–visible spectro-
photometer at room temperature. Quartz cuvettes with
a 1 cm path length and a 3 cm3 volume were used for
all measurements. Emission spectra were recorded with
a Hitachi F‐7000 spectrofluorimeter. Room temperature
(300 K) spectra were obtained in methanol solution using
a quartz cell of 1 cm path length. The slit width was
2.5 nm for both excitation and emission.
In the work presented here, we used the Schiff base
The fluorescence quantum yield was determined
using phenol as a reference and water (refractive index
η= 1.333) medium for phenol. The solvent used for the
complexes was methanol (η= 1.329). Emission spectra
were recorded by exciting the complex and the reference
phenol at the same wavelength, maintaining nearly equal
absorbance (ca 0.1). The area of the emission spectrum
was integrated using the software available with the
instrument and the quantum yield was calculated accord-
ing to the following equation:
ligands 2‐[(2‐diethylaminoethylimino)methyl] phenol
(HL1)
and
2‐[1‐(2‐diethylaminoethylimino)propyl]
phenol (HL2) to synthesize the corresponding dinuclear
complexes [Cu2 (L1)2(mb)]⋅ClO4 (1) and [Cu2 (L2)2(mb)]
⋅ClO4 (2) (mb = 4‐methylbenzoate). In both 1 and 2, the
Schiff bases function as N,N,O donor tridentate chelat-
ing/bridging ligands with μ2‐η2:η1:η1 coordination mode
(Scheme 1). Electronic absorption spectral properties of
complexes and ligands were explained using time‐depen-
dent density functional theory (TD‐DFT) computation.
Using electronic absorption/fluorescence spectroscopic
technique, catecholase activities and calf thymus DNA
(CT‐DNA)/serum albumin interactions of the complexes
were studied.
As Ir η2s
Φs ¼ Φr
Ar Is η2r
where Φs and Φr are the fluorescence quantum yields of
sample and reference, respectively, As and Ar are the
respective optical densities at the wavelength of
excitation, Is and Ir correspond to the areas under the
fluorescence curve and ηs and ηr are the refractive index
values for sample and reference, respectively.
2 | EXPERIMENTAL
2.1 | Materials and Methods
High‐purity
N,N‐diethylethylenediammine,
2‐
2.2 | Synthesis of HL1 and HL2
hydroxypropiophenone, CT‐DNA, bovine serum albumin
(BSA), human serum albumin (HSA) and ethidium bro-
mide (EtBr) were purchased from Aldrich Chemical Co.
All other chemicals were of analytical grade. Solvents
used for spectroscopic studies were purified and dried
using standard procedures before use.[11]
The ligand HL1 was prepared by condensation reaction of
N,N‐diethylethylenediammine and 2‐hydroxybenzal-
dehyde in methanol at 70 °C. A methanolic solution
(10 ml) of N,N‐diethylethylenediammine (1 mmol,
0.116 g) was added dropwise to a methanolic solution
(10 ml) of 2‐hydroxybenzaldehyde (1 mmol, 0.122 g) with
constant stirring. The resulting yellow reaction mixture
was refluxed (70 °C) for 1 h. The yellow‐coloured
compound was separated out on evaporation of solvents
and the compound was recrystallized using a 1:1 mixture
of MeOH and EtOH solvents. Anal. Calcd for C13H20N2O
(220.31) (%): C, 70.87; H, 9.15; N, 12.72. Found (%): C,
Elemental analyses (carbon, hydrogen and nitrogen)
were performed using a PerkinElmer 240C elemental
analyser. Infrared (IR) spectra were recorded with a
Bruker Vector 22 FT‐IR spectrophotometer operating
from 400 to 4000 cm−1. H NMR and 13C NMR spectra
1
1
70.85; H, 9.12; N, 12.70. H NMR (CDCl3, 200 MHz, δ,
ppm): 1.01–1.03 (6H, m, ─ CH3), 2.35–2.43 (4H, m,
─ CH2─), 2.71–2.74 (2H, m, ─ CH2─ N), 3.60–3.70 (2H,
m, ─ CH2─ N), 4.86 (1H, s, Ar─ OH), 6.83–7.41 (4H, m,
Ar─ H), 8.46 (H, s, ─ CH═ N─). 13C NMR (400 MHz,
CDCl3, δ, ppm): 165.84 (─ CH═ N─), 158.12
(Ar─ C─ OH), 116.96–132.12 (Ar─ C), 44.6–56.90
(N─ CH2─ CH2─ N), 14.1 (─ CH3).
SCHEME 1 Structure of ligands and their coordination mode in
complexes

DOLAI ET AL.
3 of 16
The ligand HL2 was prepared by adopting the same pro-
Anal. Calcd for C34H45Cu2N4O8Cl (800.29) (%): C, 51.03;
H, 5.66; N, 7.00. Found (%): C, 51.05; H, 5.63; N, 7.05.
IR (KBr, selected bands, cm−1): 2970 (aromatic C─ H
stretching); 2925 (C (sp3)─ H stretching); 1739 (aromatic
cedure as for HL1 using 1‐(2‐hydroxyphenyl)propan‐1‐one
(1 mmol, 0.150 g) instead of 2‐hydroxybenzaldehyde. Anal.
Calcd for C15H24N2O (248.36) (%): C, 72.54; H, 9.74; N,
1
11.28. Found (%): C, 72.52; H, 9.71; N, 11.26. H NMR
ν
C═C); 1614 (OCO asymmetric stretching); 1472 (OCO
(CDCl3, 200 MHz, δ, ppm): 0.86–0.87 (3H, m, ─ CH3),
1.05–1.06 (6H, m, ─ CH3), 1.32–1.37 (2H, s, ─ CH2─),
2.35–2.43 (4H, m, ─ CH2─), 2.73–2.75 (2H, m, ─ CH2─ N),
3.60–3.70 (2H, m, ─ CH2─ N), 4.86 (1H, s, Ar─ OH), 6.83–
7.41 (4H, m, Ar─ H). 13C NMR (400 MHz, CDCl3, δ, ppm):
163.84 (─ CH═ N─), 156.12 (Ar─ C─ OH), 116.96–132.12
(Ar─ C), 44.6–56.90 (N─ CH2─ CH2─ N), 25.6 (aldehyde─
CH2─), 14.1 (amine─ CH3), 7.6 (aldehyde─ CH3).
symmetric stretching); 1217 (aromatic C═N); 1028
(ν(ClO4−) asymmetric stretching); 628 (ν(ClO4−) asymmetric
bending).
ν
2.3.2 | Synthesis of 2
Complex 2 was synthesized following the same procedure
as adopted for 1 using HL2 (1 mmol, 0.248 g) instead
of HL1. Green blocked‐shaped single crystals of suitable
2.3 | Synthesis of Complexes
TABLE 1 Crystal data and details of structure refinement for
complexes 1 and 2
Caution! Metal perchlorates in the presence of organic
ligands are potentially explosive. Only a small amount
of compound should be prepared and it should be
handled with care.
The complexes were synthesized by adopting the pro-
cedures shown schematically in Scheme 2.
1
2
Empirical formula
Formula mass
Crystal system
Space group
a (Å)
C34H45Cu2N4O8Cl
800.27
Monoclinic
P21
C38H53Cu2N4O8Cl
856.37
Orthorhombic
Pccn
2.3.1 | Synthesis of 1
8.0267(2)
21.3991(6)
10.6538(3)
90
8.4410(2)
13.8710(4)
34.2848 (10)
90
b (Å)
A methanolic solution (10 ml) of copper perchlorate
hexahydrate (1 mmol, 0.370 g) was added dropwise to
15 ml of a methanolic solution of a 1:1 mixture of
triethylamine (1 mmol, 0.10 g) and HL1 (1 mmol,
0.220 g) and stirred for 30 min. To this green‐coloured
reaction mixture was added an aqueous solution (5 ml)
of sodium 4‐methylbenzoate (Na (mb); 1 mmol) and the
resulting deep‐green‐coloured reaction mixture stirred
for 2 h and filtered. After a few days needle‐shaped
green crystals suitable for X‐ray structure determination
were obtained from the filtrate. Yield: 0.268 g (66%).
c (Å)
α (°)
β (°)
98.8210 (17)
90
90
γ (°)
90
Z
2
4
T (K)
295
295
V (Å3)
1808.30(9)
1.470
4014.24 (19)
1.417
Dcalcd (g cm−3
)
μ(mm−1
)
1.305
1.180
F (000)
832
1792
θrange (°)
2.7–30.1
19146
3.4–27.5
26343
No. of collected data
No. of unique data
Rint
9565
4575
0.044
0.055
h,k,lmax
11,30,14
7808
10,18,44
3419
Observed [I > 2σ(I)]
Goodness of fit ( F 2)
Parameters refined
R1[I > 2σ(I)]a
wR2[I > 2σ(I)]a
Δρ (e Å−3
1.087
1.015
447
243
0.0452
0.1179
−0.54, 0.50
0.0648
0.2041
)
−0.63, 0.64
2
aR1 ( F o) = Σ∣∣ F o∣–∣ F c∣∣/Σ∣ F o∣, wR2 ( F o2) = [Σw ( F o
–
F c 2)2/Σw
( F o2)2]1/2
.
SCHEME 2 Synthesis of 1 and 2

4 of 16
DOLAI ET AL.
X‐ray diffraction quality were obtained after a few days
on keeping the filtrate at room temperature. Yield:
solved by direct methods using the SIR97[14] system of
programs and refined using full‐matrix least‐squares with
all non‐hydrogen atoms anisotropically and hydrogens
included on calculated positions, riding on their carrier
0.269
g (63%). Anal. Calcd for C38H53Cu2N4O8Cl
(856.37) (%): C, 53.29; H, 6.23; N, 6.54. Found (%): C,
53.25; H, 6.21; N, 6.51. IR (KBr, selected bands, cm−1):
2973 (aromatic C─ H stretching); 2938 (C (sp3)─ H
stretching); 1739 (aromatic νC═C); 1589 (OCO asymmetric
stretching); 1412 (OCO symmetric stretching); 1321 (aro-
matic νC═N); 1079 (ν(ClO4−) asymmetric stretching); 621
(ν(ClO4−) asymmetric bending).
−
atoms. Both the complex cation and ClO4 anion of com-
plex 2 are situated on a two‐fold axis passing through the
C16, C17, C20 and C21 atoms of the mb ligand and the Cl
atom of the perchlorate anion. The atomic groups C9H2,
C12H2‐C13H3 and C14H2‐C15H3 of complex 2 were found
disordered and refined over two sites with occupancies of
0.6 and 0.4, respectively. All calculations were performed
using SHELXL‐97[15] and PARST[16] implemented in the
WINGX[17] system of programs. Details of crystallographic
data collection and refinement are given in Table 1.
2.4 | Crystallographic Data Collection and
Refinement
The crystal data of complexes 1 and 2 were collected at
room temperature (295 K) using a Nonius Kappa CCD
diffractometer with graphite monochromated Mo‐K
α radiation. The data sets were integrated with the Denzo‐
SMN package[12] and corrected for Lorentz, polarization
and absorption effects (SORTAV).[13] The structures were
2.5 | Theory and Computational Methods
Theoretical calculations for complexes and ligands were
carried out using Gaussian 09 (G09) software,[18] using
Becke's three‐parameter hybrid exchange functional and
FIGURE 1 1H NMR and 13C NMR spectra of HL1 (in CDCl3)

DOLAI ET AL.
5 of 16
the Lee–Yang–Parr non‐local correlation (B3LYP) func-
tional. The Los Alamos effective core potentials plus
MBS (LanL2MB)[19] (for 1 and 2) and 6‐31G (d, p) (for
HL1 and HL2) basis sets were used for calculation. In this
study the ligands were optimized in the ground state (sin-
glet) and structures of the complexes were fully optimized
in the ground state (triplet) at the B3LYP level and
vibrational frequency calculations were performed to
ensure that the optimized geometries represent local
minima associated with positive eigen values only.
TD‐DFT calculations were performed (in the gas
phase for 1 and 2, and using the conductor‐like polariz-
able continuum model (CPCM) in MeOH for HL1 and
HL2) to obtain possible vertical electronic excitations.[20]
For the calculation of fractional contribution of various
group to each molecular orbital, GaussSum[21] was
used.
2.6 | Albumin Binding Studies
Interactions of complexes with serum albumins were
investigated using the fluorescence spectroscopic tech-
nique. Stock solutions of HSA (4.24 μM) and BSA (3.13
μM) were prepared in HEPES buffer (pH = 7.2) and
solutions of 1 and 2 (0.1665 μM) were prepared in water.
The quenching of fluorescence intensity of serum
albumin upon addition of complexes is mainly due to
association of serum albumin with complex, serum
albumin denaturation or conformational change of serum
albumin.[22]
FIGURE 2 ORTEP view of cationic unit of complex 1 showing
thermal ellipsoids at 30% probability level
2.7 | DNA Binding Studies
2.7.1 | Electronic absorption spectral
studies
UV–visible absorption spectral titration is a useful tech-
nique for distinguishing different binding modes of com-
plexes with CT‐DNA. Hypochromism or hyperchromism
with red or blue shift is often observed in the absorption
spectrum of a metal complex when it is bound to DNA.
Generally intercalative binding mode between CT‐DNA
and
a
complex results in hypochromism and
bathochromism (red shift) in the UV–visible absorption
spectrum, whereas hyperchromism in the absorption
spectrum indicates electrostatic or non‐intercalative
binding mode. For both complexes 1 and 2 UV–visible
absorption spectral titrations were performed at a fixed
concentration of complexes (2 ml, 13.04 μM) with gradual
addition of 2 μl of 90.9 μM CT‐DNA solution. Intrinsic
binding constants (Kib) of the complexes with CT‐DNA
were determined using the equation[23]
FIGURE 3 ORTEP view of cationic unit of complex 2 showing
thermal ellipsoids at 30% probability level

6 of 16
DOLAI ET AL.
intercalation of the planar EtBr phenanthridium ring
between nearby base pairs of the CT‐DNA double helix.
Fluorescence spectral titrations were carried out with
gradual addition of 2 μl of 13.04 μM solution of
complexes to a solution of EtBr‐bound CT‐DNA (2 ml,
90.9 μM aqueous solution). The fluorescence intensity
at 607 nm (λex = 500 nm) gradually decreased keeping
emission wavelength fixed. The Stern–Volmer
equation[24] (I0/I = 1 + Ksv [quencher], where I0 and I
are the emission intensity in the absence and presence
of complex, Ksv is the Stern–Volmer constant and
[quencher] is the concentration of Cu (II) complex) was
used to calculate the quenching constant (Ksv).
½DNAꢀ ½DNAꢀ
1
¼
þ
εa−εf
εb−εf
Kibðεb−εf Þ
where [DNA] is the concentration of CT‐DNA, εa is the
extinction coefficient of the complex at a given CT‐DNA
concentration and εf and εb are the extinction coefficients
of the complex in free solution and when fully bound to
CT‐DNA, respectively. A plot of [DNA]/(εa − εf) versus
[DNA] gives a straight line with 1/(εb − εf) and 1/Kib
(εb − εf) as slope and intercept, respectively.
2.7.2 | Competitive binding fluorescence
measurement
3 | RESULTS AND DISCUSSION
3.1 | Synthetic Aspects
Multidentate coordinating ligands HL1 and HL2 were pre-
pared by a one‐pot synthesis employing condensation of
corresponding amine and aldehyde in methanol under
reflux condition. They were characterized using NMR
The competitive binding nature of the complexes with
CT‐DNA was investigated by adopting the fluorescence
spectroscopic method using aqueous solution of EtBr‐
bound CT‐DNA in HEPES buffer (pH = 7.2). EtBr shows
fluorescence and the intensity of such fluorescence
increases approximately 20‐fold in the presence of CT‐
DNA. The increase in fluorescence intensity is due to
TABLE 2 Experimental and calculateda coordination bond distances (Å) and angles (°) for complexes 1 and 2
Bond length
Bond angle
Exp
Calcd
Exp
Calcd
Complex 1
Cu(1)–O(1)
Cu(1)–O(2)
Cu(1)–O(3)
Cu(1)–N(1)
Cu(1)–N(2)
Cu(2)–O(1)
Cu(2)–O(2)
Cu(2)–O(4)
Cu(2)–N(3)
Cu(2)–N(4)
Cu(1) … Cu(2)
1.928(2)
2.449(2)
1.938(2)
1.936(4)
2.080(3)
2.455(2)
1.938(2)
1.938(2)
1.927(3)
2.106(3)
3.108(6)
2.037
2.398
1.929
1.974
2.300
2.228
2.043
1.939
1.974
2.284
3.317
O(1)–Cu(1)–O(2)
O(1)–Cu(1)–N(1)
O(1)–Cu(1)–N(2)
O(1)–Cu(1)–O(3)
O(2)–Cu(1)–O(3)
O(2)–Cu(1)–N(1)
O(2)–Cu(1)–N(2)
O(3)–Cu(1)–N(1)
N(1)–Cu(1)–N(2)
O(1)–Cu(2)–O(2)
O(4)–Cu(2)–N(3)
τ5 parameter
85.57(9)
75.00
91.00
92.71 (13)
163.82 (12)
89.87 (10)
94.62(9)
163.72
92.52
90.14
92.30 (11)
110.46 (10)
172.78 (13)
84.70 (14)
85.16(9)
101.96
120.81
167.87
82.24
77.47
173.12 (12)
0.0056
170.64
0.0461
Complex 2
Cu(1)–O(1)
Cu(1)–O(1′)
Cu(1)–O(2)
Cu(1)–N(1)
Cu(1)–N(2)
Cu(1) … Cu (1′)
1.888(3)
2.592(3)
1.956(3)
1.944(3)
2.070(3)
3.249(7)
2.008
2.213
2.279
2.026
2.185
3.235
O(1)–Cu(1)–O(2)
O(1)–Cu(1)–N(2)
O(1)–Cu(1)–O(1′)
O(1′)–Cu(1)–O(2)
O(1′)–Cu(1)–N(1)
O(2)–Cu(1)–N(1)
τ5 parameter[26]
91.3(1)
166.8(1)
82.6(1)
90.9(1)
93.2(1)
173.7(1)
0.115
89.23
150.81
75.34
88.76
89.45
131.78
0.317
aComplexes optimized in gas phase; LanL2MB basis set; B3LYP functional.

DOLAI ET AL.
7 of 16
spectroscopy (Figure 1 and Figures 1S and 2S). Using HL1
or HL2 in combination with mb, complexes 1 and 2 were
synthesized.
are crystallographically independent but chemically similar.
The geometries around Cu (II) atoms are distorted square
pyramidal, and the equatorial plane of which is occupied
by one oxygen atom and two nitrogen atoms of respective
N,N,O donor Schiff base ligands, while the fourth coordina-
tion site is occupied by the oxygen atom of mb. Selected
bond distances and angles are listed in Table 2.
3.2 | Crystal structures of 1 and 2
ORTEP views[25] of the cationic units of complexes 1 and
2 are shown in Figures 2 and 3, respectively. Both 1 and 2
consist of two metal centres, namely Cu1 and Cu2, which
Complex 1 crystallizes in the monoclinic crystal
system of P21 space group, while complex 2 crystallizes
FIGURE 4 Packing diagram of
complex 2
TABLE 3 Selected UV–visible energy transitions at the TD‐DFTa/B3LYP level for ligands and complexes
Excited
state
λcal (nm), εcal
Oscillator
strength ( f )
λexp (nm), εexp
(M−1 cm−1), (eV)
(M−1 cm−1), (eV)
Key transition
Characterb
HL1
HL2
S8
278.9,
0.0804
278,
HOMO‐1 → LUMO (30%)
π → π*
(0.030 × 105), 4.45
(10668.42), 4.44
S19
S8
213.06,
(16650.91), 5.81
276.13,
0.2095
0.1288
213,
HOMO‐3 → LUMO (10%)
HOMO‐1 → LUMO+1 (17%)
HOMO‐1 → LUMO (39%)
π → π*
π → π*
π → π*
(0.129 × 105), 5.82
276,
(9457.38), 4.49
(0.018 × 105), 4.45
1
2
T27
T26
372.19,
0.0080
0.0046
370,
HOMO (β) → LUMO+2 (β) (21%)
HOMO‐1 (β) → LUMO+2 (β) (17%)
ILCT
(0.130 × 106), 3.34
(26867.32), 3.33
369.92,
(27032.66), 3.35
369,
IELCT
(2.71 × 107), 3.36
aFor HL1 and HL2: using CPCM in methanol; basis set, 6‐31G (d‐p). For complexes 1 and 2: in gas phase; basis set, LanL2MB.
bILCT, intra‐ligand charge transfer from L to L; IELCT, inter‐ligand charge transfer from mb to L.

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DOLAI ET AL.
in orthorhombic crystals of space group Pccn. In both
complexes, the bond lengths between Cu atom and
nitrogen atoms are within the range 1.936(4) to
2.106(3) Å (Table 2). The average bond angle around
the Cu centre is ca 90°, whereas the lowest bond angle
is observed for N(1)–Cu(1)–N(2) at 84.70 (14)°. The
Schiff base ligand is coordinated in a tridentate fashion
to the metal centre (Cu1) through one phenoxy oxygen
(O1/O2) and two nitrogen (N1 and N2) atoms, and
forms an equatorial plane with one carboxylate oxygen
(O3/O4).
3.3 | Photophysical Study of Ligands
The electronic absorption spectrum of HL1 shows major
transitions at 215 nm (ε ~ 0.179 × 105 M−1 cm−1), 254 nm
(ε
~
0.087
×
105 M−1 cm−1), 278 nm (ε
~
0.030 × 105 M−1 cm−1), 316 nm (ε ~ 0.039 × 105 M−1 cm−1)
and 400 nm (ε ~ 0.012 × 105 M−1 cm−1). The spectrum
of HL2 shows significant transitions at 213 nm (ε ~
In both complexes, the equatorial Cu‐O bond length
(1.928(2) Å for 1, 1.888(3) Å for 2) is shorter than the
axial Cu‐O bond length (2.455(2) Å for 1, 2.592(3) for
2) due to Jahn–Teller distortion. The Cu‐O bond lengths
vary from 1.928(2) to 2.455(2) Å for 1 and 1.888(3) to
2.592(3) Å for 2. The Cu‐N bond lengths vary from
1.927 to 2.106 Å for 1 and 1.944 to 2.070 Å for 2
(Table 2). The distance between the two copper atoms
in 1 is 3.108 Å, and in 2 is 3.249 Å, indicating that
there is no bond between the two metal centres. Crystal
packing of 1 and 2 is shown in Figures 3S and 4,
respectively.
Calculated values of the trigonality parameter[26] τ are
0.0056 and 0.115 for 1 and 2, respectively, indicating that
both the complexes possess distorted square pyramidal
geometry.
FIGURE 6 Increase in 3,5‐DTBQ band at 400 nm after addition
of 10−4 M methanolic solution of complex 1 to 100‐fold
methanolic solution of 3,5‐DTBC. The spectra were recorded at
intervals of 5 min
FIGURE 5 Surface plots of frontier orbitals (β‐MOs) along with their energies and compositions of 1 using B3LYP functional in gas phase
(basis set: LanL2MB)

DOLAI ET AL.
9 of 16
0.129 × 105 M−1 cm−1), 250 nm (ε ~ 0.060 × 105 M−1 cm−1),
276 nm (ε ~ 0.018 × 105 M−1 cm−1), 323 nm (ε ~
0.023 × 105 M−1 cm−1) and 390 nm (ε ~ 0.012 × 105 M−1 cm−1).
The absorption bands appearing at around 200 nm to
400 nm are due to n → π*/π → π* (Table 3).
ν(C (sp3)─ H) stretching vibration appear at 2925 and
2938 cm−1 for 1 and 2, respectively (Figures 4S and 5S).
The νas (OCO) stretching vibrations for 1 and 2 appear
at 1614 and 1589 cm−1, respectively. Whereas νs (OCO)
stretching vibrations appear at 1472 and 1412 cm−1
for 1 and 2, respectively. The separation of stretching
frequencies Δν(= νas (OCO) − νs (OCO)) for 1 and 2 are
142 and 177 cm−1, respectively. But for divalent metal
3.4 | IR, Electronic Absorption and
Fluorescence Spectra of Complexes
carboxylates the observed trend is Δν(chelating) <Δν(bridging)
[27]
The most important IR spectral bands of 1 and 2 are
summarized in Section 2 and tabulated in Table 1S.
Aromatic ν(C═ C, C═ N) stretching vibrations of
complexes appear in the region 1217–1739 cm−1, and
the bands in the region 2970–2973 cm−1 correspond
to aromatic ν(C─ H) stretching vibrations. Aliphatic
<Δν(ionic) <Δν(monodentate)
.
It is noted that Δν for Na2
(mb) is 227 cm−1. Therefore Δνfor the complexes is less
than Δ νfor Na2 (mb), and the observed moderate
Δ νvalues of the complexes (142 and 177 cm−1
)
corroborate the crystallographically observed bridging of
carboxylate group in 1 and 2.
Electronic spectra of both complexes were recorded in
methanol solution. The spectrum of 1 shows four strong
transitions at 202 nm (ε ~ 1.21 × 106 M−1 cm−1), 222 nm
FIGURE 7 Change in absorption maxima at 400 nm with time
after addition of 10−4 M methanolic solution of complex to 100‐
fold methanolic solution of 3,5‐DTBC: (a) complex 1; (b) complex 2
FIGURE 8 (a) Rate versus substrate concentration and (b)
Lineweaver–Burk plot for complex 1

10 of 16
DOLAI ET AL.
(ε ~ 0.97 × 106 M−1 cm−1), 271 nm (ε ~ 0.51 × 106 M−1 cm−1
)
bands at 213 and 278 nm may be assigned as (HOMO
−1 → LUMO; HOMO −3 → LUMO +1) and HOMO
−1 → LUMO transitions, respectively. The spectral bands
at 213, 276 and 278 nm correspond to π → π* transition.
and 370 nm (ε ~ 0.13 × 106 M−1 cm−1) (Figure 6S). For 2,
the transitions appear at 202 nm (ε ~ 1.04 × 106 M−1 cm
−1), 225 nm (ε ~ 0.64 × 106 M−1 cm−1), 269 nm (ε
~ 0.3 × 106 M−1 cm−1) and 369 nm (ε ~ 0.09 × 106 M
−1 cm−1) (Figure 7S).
3.5.2 | DFT and TD‐DFT computations of
complexes
When excited at 271 nm (λex), a methanolic solution
of 1 shows fluorescence emission at 306 nm (λem) with
quantum yield of 0.35. On the other hand, when excited
at 269 nm (λex), 2 also shows emission at the same
wavelength of 306 nm (λem) with quantum yield of
0.41 (Table 2S).
Optimized structures of 1 and 2 along with their Mulliken
charge distributions are shown in Figures 11S and 12S.
Calculated energies of optimized geometries and other
physical parameters are presented in Table 7S. For 1,
the α‐state HOMO and LUMO energies are −6.57
and − 2.39 eV, respectively, and the β‐state HOMO and
LUMO energies are −6.34 and − 4.60 eV, respectively.
Whereas for 2, the α‐state HOMO and LUMO energies
are −5.01 and − 2.04 eV, respectively, and the β‐state
HOMO and LUMO energies are −5.55 and − 3.86 eV,
respectively. Surface plots of frontier molecular orbitals
of 1 and 2 are shown in Figures 5 and 13S.
3.5 | DFT and TD‐DFT Computations
3.5.1 | DFT and TD‐DFT computations of
HL1 and HL2
DFT and TD‐DFT calculation of HL1 and HL2 were
performed with B3LYP functional using 6‐31G (d, p) basis
set. Optimized structures of ligands are shown in Figures
8S and 9S, and physical parameters and energies of
molecular orbitals are presented in Table 3S. TD‐DFT
calculations were performed in methanol using CPCM
and theoretically possible spin‐allowed (singlet–singlet)
electronic transitions with their assignment are presented
in Table 3 and Tables 4S and 5S. For both HL1 and HL2,
HOMO → LUMO is the lowest energy transition, whereas
HOMO −2 → LUMO +1 and HOMO −1 → LUMO +1
transitions are the possible highest energy transitions in
HL1 and HL2, respectively. For HL1, the experimental
electronic spectral (Figure 10S) band at 276 nm may be
assigned as HOMO −1 → LUMO transition (Table 3).
On the other hand for HL2 the spectral (Figure 10S)
The contributions of Schiff base (L1/L2), copper and
carboxylate (mb) to the HOMO (α) of complexes are
95% L1, 3% Cu, 2% mb (for 1) and 76% L2, 24% Cu, 0%
mb (for 2), and for LUMO (α) the corresponding values
are 98% L1, 2% Cu, 0% mb (for 1) and 4% L2, 2% Cu,
94% mb (for 2). The contributions of Schiff base (L1/L2),
copper and carboxylate (mb) to the HOMO (β) of com-
plexes are 92% L1, 5% Cu, 3% mb (for 1) and 92% L2, 8%
Cu, 0% mb (for 2), and for LUMO (β) the values are
55% L1, 31% Cu, 14% mb (for 1) and 68% L2, 32% Cu,
0% mb (for 2). From the DFT/TD‐DFT calculations, the
values of electronic spectral transition wavelength (λcal
)
and oscillator strength ( f) were determined, and are pre-
sented in Tables 9S and 10S. The experimentally observed
TABLE 4 Kinetic parameters for oxidation of 3,5‐DTBC catalysed by complexes 1 and 2 and reported dinuclear Cu (II) complexes in
methanol solvent
Complex
Vmax
KM (M)
Kcat (h−1
)
Kcat/KM (M−1 h−1
)
Ref.
[Cu2 (L1)2(mb)]·ClO4 (1)
[Cu2 (L2)2(mb)]·ClO4 (2)
[Cu2 (L3)(OMe)][ClO4]2·2MeOH
[Cu2 (L4)(OMe)(MeOH)(ClO4)]ClO4
[Cu2 (L5)(OMe)(MeOH)(ClO4)]ClO4
[Cu2L6 (ClO4)2]
(2.31 0.31) × 10−7
(2.91 0.38) × 10−7
(1.90 0.23) × 10−3
(2.76 0.35) × 10−3
(2.3 0.2) × 10−3
(3.1 0.2) × 10−4
(1.4 0.2) × 10−3
(3.32 0.06) × 10−3
(4.60 0.2) × 10−3
42 0.09
(22.07 0.41) × 103
(19.11 0.32) × 103
This work
This work
[28a]
52.9 0.12
33
48
[28a]
43
[28a]
93.6
233.4
28.74
55
28.2 × 103
50.7 × 103
[10a]
[Cu2L6 (OH)]ClO4
[10a]
[Cu2 (H2L7)(μ‐OH)](ClO4)2
[Cu2 (LH,H‐O8)(OH)(MeCN)2][ClO4]2
[28b]
[28c]
HL3, 4‐bromo‐2‐(4‐methylpiperazin‐1‐ylmethyl)‐6‐[(2‐pyridylmethyl)aminomethyl]phenol; HL4, 4‐bromo‐2‐(4‐methylpiperazin‐1‐ylmethyl)‐6‐{[2‐(2‐pyridyl)
ethyl]aminomethyl}phenol;
HL5,
4‐bromo‐2‐(4‐methylpiperazin‐1‐ylmethyl)‐6‐{[2‐(1‐methyl‐2‐imidazolyl)ethyl]aminomethyl}phenol;
HL6,
2‐[[2‐
(diethylamino)ethylamino]methyl]phenol; H3L7, 2,6‐bis[{{(2‐hydroxybenzyl)(N′,N′‐(dimethylamino)ethyl)}amino}methyl]‐4‐methylphenol; LH,H‐O8, 1,3‐bis[(N,
N‐dimethylaminoethyl)aminomethyl]benzene.

DOLAI ET AL.
11 of 16
spectral transitions (Figures 14S and 15S) at 370 nm (for
1) and 369 nm (for 2) are due to HOMO (β) → LUMO
+2 (β) (21%) and HOMO −1(β) → LUMO +2 (β) (17%)
transitions, respectively. For 1, compositions of HOMO
(β) and LUMO +2(β) molecular orbitals are (92% L1, 5%
Cu, 3% mb) and (98% L1, 2% Cu, 0% mb), respectively.
For 2, compositions of HOMO −1 (β) and LUMO +2 (β)
molecular orbitals are (63% L2, 36% Cu, 1% mb) and (7%
L2, 2% Cu, 91% mb), respectively. Therefore electronic
spectral band at 370 nm for 1 is due to intra‐ligand charge
transfer in L, and that at 369 nm for 2 is due to inter‐
ligand charge transfer from mb to L.
geometry observed for 2 than 1, which corroborate the
observed order of catalytic activity. Kinetic parameters
of catecholase activities of reported dinuclear Cu (II)
28]
complexes[10a,
are presented in Table 4 and these
results indicate that 1 and 2 have catalytic activities
comparable to those of reported compounds.
3.7 | Protein Binding Studies
3.7.1 | Electronic absorption spectral
titration
Electronic absorption spectra were recorded in the range
200–500 nm using various concentrations of the Cu (II)
complexes with a constant concentration of BSA and
HSA. Electronic spectra of both BSA and HSA show an
3.6 | Catechol Oxidation Studies of
Complexes
Catecholase activities of complexes 1 and 2 were exam-
ined at room temperature (30 °C) by adding their
1 × 10−4 M methanolic solution to a 1 × 10−2 M methano-
lic solution of 3,5‐di‐tert‐butylcatechol (3,5‐DTBC) in the
presence of molecular oxygen, and the progress of the
reaction was studied by recording UV absorption spectra
of the mixture at five‐minute intervals. The changes of
the electronic absorption spectra of 3,5‐DTBC in the pres-
ence of the complexes are shown in Figures 6 and 16S.
A band at 400 nm corresponding to 3,5‐di‐tert‐
butylquinone (3,5‐DTBQ) gradually increases with time
after addition of complexes. The kinetics of oxidation of
3,5‐DTBC to 3,5‐DTBQ were determined by monitoring
the growth of the 400 nm band as function of time (t).
From the log[(A∞ − A0)/(A∞ − At)] (where A∞ and At
are the absorbance at infinite time and at t, respectively)
versus t plots (Figure 7), the rate constants for complex–
substrate mixtures were determined and the calculated
values were 8.21 × 10−3 and 8.94 × 10−3 min−1 for 1
and 2, respectively.
To determine the dependence of reaction rate on sub-
strate concentration, we added solutions of complex to
3,5‐DTBC solutions of various concentrations (1 × 10−3
to 10 × 10−3 M) and observed a first‐order dependence
of reaction rate at low concentration of substrate, and sat-
uration kinetics at higher concentration of 3,5‐DTBC
(Figures 8 and 17S).
As the complexes show saturation kinetics, we used
the Michaelis–Menten model for these systems. Using
the Lineweaver–Burk equation, 1/V = (KM/Vmax)(1/
[S]) + 1/Vmax, the kinetic parameters Vmax, KM and Kcat
were determined (Table 4) from a plot of 1/V versus 1/
[S]. Complex 2 shows higher turnover number (Kcat/KM)
than complex 1, indicating higher catalytic activity
(Table 4). The observed catalytic rate can be explained
considering the deviation from ideal geometry. Calcu-
lated τ values indicate more deviation from regular
FIGURE 9 Change of UV–visible absorption spectra of BSA
(2 ml, 3.13 μM aqueous solution) upon gradual addition of 10 μl
of 0.1665 μM aqueous solution of (a) complex 1 and (b) complex 2.
Insets: 1/(Aobs – A0) versus 1/[complex] plots of BSA absorption
titration

12 of 16
DOLAI ET AL.
absorption band at 280 nm. Gradual addition of 10 μl of
0.1665 μM aqueous solution of the complexes to 2 ml of
3.13 μM BSA solution results in an increase in absorbance
with blue shift for both complexes (280 to 265 nm for 1,
280 to 272 nm for 2) (Figure 9).
Similarly electronic absorption spectra of HSA also
show a blue shift with increase in absorbance (280 to
258 nm for 1, 280 to 272 nm for 2) (Figure 18S) upon
gradual addition of 10 μl of 0.1665 μM aqueous solution
of the complexes to 2 ml of 4.24 μM solution of HSA. This
hypsochromic shift of the spectral band corresponds to
ground‐state association between complex and serum
albumin. The apparent association constants (Kapp) were
calculated using the following equation:[29]
3.7.2 | Fluorometric protein binding
studies
To solutions of serum albumins (4.24 μM HSA; 3.13 μM
BSA) in HEPES buffer (pH = 7.2), Cu (II) complexes were
added at room temperature, and the quenching of emis-
sion intensities at 340 nm (λex = 280 nm) for BSA
(Figure 10) and 330 nm (λex = 280 nm) for HSA (Figure
19S) was recorded after gradual addition of 10 μl of
0.1665 μM aqueous solutions of 1 and 2.
Upon gradual increasing of complex concentration,
the fluorescence intensities of BSA and HSA were signifi-
cantly decreased. Moreover the electronic absorption spec-
tra of the serum albumins show significant change in the
presence of the complexes. These observations clearly
indicate that the fluorescence quenching occurs through
a ground‐state association process. The Stern–Volmer
equation[24] (I0/I=1+Ksv [quencher]=1+kqτ0[quencher],
where I0 and I are the emission intensity in the absence
and presence of complex, Ksv is the Stern–Volmer con-
stant, [quencher] is the concentration of Cu (II) complex,
kq is the quenching constant and τ0 is the lifetime (ca
10−8 s) of the fluorophore in absence of quencher) was
used to estimate the observed fluorescence quenching
behaviour, and the calculated values of Ksv are
4.4 × 105 M−1 (for 1–BSA) and 5.2 × 105 M−1 (for 2–BSA)
1
1
1
¼
þ
A
obs−A0 Ac−A0 KappðAc−A0Þ½complexꢀ
where Aobs is the observed absorbance at 280 nm of the
solution containing various concentrations of the com-
plex, A0 is the absorbance of serum albumin only and
Ac is the absorbance of serum albumin with complex. A
plot of 1/(Aobs – A0) versus 1/[complex] results in a
straight line (Figure 9 insets) with slope of 1/Kapp
(Ac – A0). From this slope the values of Kapp were
calculated, and the calculated values are 1.7 × 105 M−1
(for 1) and 5.7 × 105 M−1 (for 2) for BSA, and
1.6 × 105 M−1 (for 1) and 6.9 × 105 M−1 (for 2) for HSA
(Table 5). These results indicate that the strength of inter-
action varies in the order 1 <2.
(Figure 20S). The calculated values of kq are 4.4 × 1013
M
−1
s−1 (1–BSA) and 5.2 × 1013 M−1 s−1 (2–BSA). For
HSA, the calculated values of Ksv are 4.8 × 105 M−1 (for
1–HSA) and 4.9 × 105 M−1 (for 2–HSA) (Figure 21S), and
kq are 4.8 × 1013
M M
−1 s−1 (1–HSA) and 4.9 × 1013 −1 s−1
TABLE 5 Kinetic parameters of interaction of BSA/HSA with 1 and 2 and reported Cu (II) complexes
Compound
Kapp (M−1
)
n
Kb (M−1
)
Ksv (M−1
)
kq (M−1 s−1
)
Ref
BSA
[Cu2 (L1)2(mb)]·ClO4 (1)
[Cu2 (L2)2(mb)]·ClO4 (2)
[Cu2 (L9)2(N3)2]
1.7 × 105
5.7 × 105
0.70
0.76
1.06
4.7 × 105
5.8 × 105
2.92 × 105
8.35 × 104
4.4 × 105
5.2 × 105
4.4 × 1013
5.2 × 1013
5.15 × 1012
8.70 × 1012
1.18 × 1013
This work
This work
[30]
8.24 × 104
8.70 × 104
1.18 × 105
[Cu2 (L10) Cl (CH3OH)(dabt)]·CH3OH
[Cu2 (L10)(bpy)(H2O)](pic)·H2O
HSA
[31]
[31]
[Cu2 (L1)2(mb)]·ClO4 (1)
[Cu2 (L2)2(mb)]·ClO4 (2)
[Cu2 (L9)2(N3)2]
[Cu (L11)(OAc)]·H2O
[Cu (HL11)(C2O4) (EtOH)]·EtOH
[Cu (L11)(bza)]
1.6 × 105
6.9 × 105
0.70
1.02
1.39
0.70
1.15
1.11
1.18
4.9 × 105
5.7 × 105
1.28 × 105
0.14 × 105
1.67 × 105
1.52 × 105
3.38 × 105
4.8 × 105
4.9 × 105
7.57 × 104
4.8 × 1013
4.9 × 1013
4.73 × 1012
7.3 × 1012
5.3 × 1012
6.6 × 1012
6.8 × 1012
This work
This work
[30]
[32]
[32]
[32]
[32]
[Cu (L11)(sal)]
HL9, ligand derived from 2‐acetylpyridine and thiosemicarbazide; HL10, N‐phenolato‐N′‐[2‐(dimethylamino)ethyl]oxamide; HL11, 1‐(((2‐((2‐hydroxypropyl)
amino)ethyl)imino)methyl)naphthalene‐2‐ol.

DOLAI ET AL.
13 of 16
The binding constants and the number of binding
sites per albumin were calculated and the calculated
values are given in Table 5. A comparison of kinetic
parameters of BSA/HSA interaction of reported Cu (II)
complexes (Table 5) shows that 1 and 2 have relatively
strong interaction compared with the reported com-
pounds.[30–32]
3.8 | DNA Binding Studies of Complexes
3.8.1 | Electronic absorption spectral
study
Figure 11 shows the change in electronic absorption spec-
tra of complexes with gradual addition (2 μl, 90.9 μM) of
CT‐DNA solution to solutions of complexes (2 ml, 13.04
μM). The absorbance of the 268 nm band of complex 1
gradually increases with blue shift and finally appears at
265 nm. For 2, the absorbance of the 266 nm spectral
FIGURE 10 Change of fluorescence spectra of BSA (2 ml, 3.13
μM) upon gradual addition of 10 μl of 0.1665 μM aqueous
solution of (a) complex 1 and (b) complex 2. Insets: Scatchard plots
of the BSA fluorescence titration
(2–HSA). The values of Ksv and kq indicate that complex 2
has a good fluorescence quenching ability in comparison
to complex 1.
The equilibrium between free and protein‐bound
complexes is expressed by the Scatchard equation:[33]
ðF0− FÞ
log
¼ logkb þ n log½complexꢀ
F
where kb is the binding constant of complex with serum
albumin and n is the number of binding sites per serum
ðF0− FÞ
FIGURE 11 Change of electronic absorption spectra of (a)
complex 1 and (b) complex 2 (2 ml, 13.04 μM) in HEPES buffer
upon gradual addition of 2 μl of 90.9 μM aqueous solution of CT‐
DNA
albumin molecule. A plot of log
versus log
F
[complex] gives a straight line (insets of Figures 10 and
19S) with n and log kb as slope and intercept, respectively.

14 of 16
DOLAI ET AL.
TABLE 6 Kinetic parameters of interaction of CT‐DNA with complexes 1 and 2 and reported dinuclear Cu (II) complexes
Compound
Ksv (M−1
)
Kib (M−1
)
Ref.
[Cu2 (L1)2(mb)]·ClO4
[Cu2 (L2)2(mb)]·ClO4
2.8 × 104
2.9 × 104
1.4 × 105
1.6 × 105
This work
This work
[35]
{[Cu2 (L12)bipy)]·(ClO4)2·CH3CN}n
{[Cu2 (L13)(bipy)]·(ClO4)2·H2O}n
[Cu2 (L14)2(H2O)]n
0.72 × 105
2.1 × 105
0.049 × 105
0.008 × 105
[35]
[36]
[36]
{[Cu2 (L15)2(H2O)] H2O}n
H2L12, [2 + 2] condensation product of 1,3‐diaminopropane with 2,6‐diformyl‐4‐methylphenol; H2L13, [2 + 2] condensation product of 1,3‐diaminopropane with
2,6‐diformyl‐4‐fluorophenol; H2L14, Schiff base derived from condensation of N‐(2‐hydroxybenzaldehyde) with L‐methionine; H2L15, reduced form of H2L8.
band gradually increases with blue shift (up to 6 nm) and
finally the band appears at 260 nm.
Hyperchromism (Figure 11) with blue shift of spectral
band reveals non‐intercalative/electrostatic binding mode
of complexes with CT‐DNA.[34] The calculated values of
binding constant Kib are 1.4 × 105 and 1.6 × 105 M−1 for
1 and 2, respectively (Table 6).
3.8.2 | EtBr displacement studies
EtBr‐bound CT‐DNA shows emission at 607 nm on exci-
tation at 500 nm. Figure 12 shows the change of fluores-
cence spectra of EtBr‐bound CT‐DNA upon gradual
addition of 2 μl of a 13.043 μM solution of complexes.
Fluorescence intensity of EtBr‐bound CT‐DNA
gradually decreases with increasing concentration of
complexes. Hypochromism in the presence of the
complexes suggests that the complexes displace the EtBr
molecules from the DNA binding sites. From the Stern–
Volmer plot (Figure 22S) the binding constants (Ksv) were
calculated and the calculated values are 2.8 × 104 and
2.9 × 104 M−1 for 1 and 2, respectively. Table 6 presents
the kinetic parameters of the interaction of CT‐DNA with
reported dinuclear copper complexes. A comparison of
results shows that complexes 1 and 2 have stronger
interaction than the reported complexes.[35,36]
4 | CONCLUSIONS
In summary, we have presented here the synthesis,
crystal structure, DFT calculation, DNA/protein binding
and catecholase activities of two p‐toluate‐bridged
isostructural dinuclear Cu (II) complexes. Results of
DFT calculations for 1 and 2 are in good agreement with
their crystallographically determined structures and the
possible spin‐allowed triplet–triplet electronic transitions
of the complexes and singlet–singlet electronic transitions
of ligands have been assigned based on the results
obtained from TD‐DFT calculations. We have also
conducted TD‐DFT calculations for the complexes in
singlet state and the results are summarized in the
supporting information. A comparison of singlet‐ and
triplet‐state DFT results reveals that the triplet‐state
FIGURE 12 Change of fluorescence spectra of EtBr–CT‐DNA
upon gradual addition of (a) complex 1 and (b) complex 2 (2 μl,
13.043 μM) in HEPES buffer

DOLAI ET AL.
15 of 16
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Gao, Z. Li, J. Wei, C. Zheng, M. Sun, Bioorg. Med. Chem. Lett.
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Roth, H. H. Fiebig, W. Holzer, W. Jäger, M. Jenny, J. Hofmann,
J. Med. Chem. 2001, 44, 2164.
DFT results are closer to the experimental results. CT‐
DNA/protein binding of the complexes was investigated
using electronic absorption and fluorescence spectro-
scopic techniques and the results show that both
compounds effectively bind with CT‐DNA and serum
albumins. Complexes 1 and 2 are active for catalysing
oxidation of 3,5‐DTBC to 3,5‐DTBQ in the presence of
molecular oxygen in methanol, and the observed rate of
oxidation is higher where the complex deviates more
from its regular geometry.
[7] L. Li, J. Dong, J. Li, B. Jing, D. Wang, Transition Met. Chem.
2011, 36, 289.
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37, 175.
[9] a) B. X. Huang, H. Y. Kim, C. Das, J. Am. Soc. Mass Spectrom.
2004, 15, 1237. b) S. Shi, Y. Zhang, X. Chen, M. Peng, J. Agric.
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ACKNOWLEDGEMENTS
[10] a) A. Biswas, L. K. Das, M. G. B. Drew, C. Diaz, A. Ghosh,
Inorg. Chem. 2012, 51, 10111; b) S. Mistri, A. Paul, A. Bhunia,
R. Manne, M. K. Santra, H. Puschmann, S. C. Manna, Polyhe-
dron 2016, 104, 63; c) A. Bhunia, S. Manna, S. Mistri, A. Paul,
R. K. Manne, M. K. Santra, V. Bertolasi, S. C. Manna, RSC
Adv. 2015, 5, 67727.
The authors gratefully acknowledge the financial
assistance given by the CSIR, Government of India, to
S.C.M. (project no. 01 (2743)/13/EMR‐II). S.D. is grateful
to UGC, India, for providing a fellowship. S.C.M.
thanks UGC‐SAP, DST‐FIST New Delhi USIC (VU) and
Vidyasagar University for infrastructural facilities.
[11] D. D. Perrin, W. L. F. Armarego, D. R. Perrin, Purification of
Laboratory Chemicals, Pergamon Press, Oxford 1980.
[12] Z. Otwinowski, W. Minor, in Methods in Enzymology, (Eds: C.
W. Carter, R. M. Sweet) Vol. Part A, Vol. 276, Academic Press,
London 1997 307.
ORCID
Subal Chandra Manna
http://orcid.org/0000-0001-6823-
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How to cite this article: Dolai S, Das K, Bhunia
A, Bertolasi V, Manna SC. p‐Toluate‐bridged
dinuclear Cu(II) complexes in combination with
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density functional theory calculation, DNA/protein
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