Synthesis, structure, DFT study and catechol oxidase activity of Cu(II) complex with sterically constrained phenol based ligand

Article abstract of DOI:10.1016/j.molstruc.2019.04.110

A mononuclear Cu(II) complex [Cu^II(L)](1) [H₂L=N,N′-bis(2-hydroxy-3,5-di tertiarybutylbenzyl)-homopiperazine] has been synthesized by using sterically constrained tetradentate phenol-based ligand. Characterization of this compound ha

Full text of DOI:10.1016/j.molstruc.2019.04.110

Accepted Manuscript
Synthesis, structure, DFT study and catechol oxidase activity of Cu(II) complex with
sterically constrained phenol based ligand
Bikramaditya Mandal, Mithun Chandra Majee, Trilochan Rakshit, Snehasis Banerjee,
Partha Mitra, Debdas Mandal
PII:
S0022-2860(19)30521-6
DOI:
https://doi.org/10.1016/j.molstruc.2019.04.110
MOLSTR 26482
Reference:
To appear in: Journal of Molecular Structure
Received Date: 17 December 2018
Revised Date: 22 April 2019
Accepted Date: 25 April 2019
Please cite this article as: B. Mandal, M.C. Majee, T. Rakshit, S. Banerjee, P. Mitra, D. Mandal,
Synthesis, structure, DFT study and catechol oxidase activity of Cu(II) complex with sterically
constrained phenol based ligand, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.04.110.
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Synthesis, structure, DFT study and catechol oxidase activity of Cu(II) complex
with sterically constrained phenol based ligand
c
Bikramaditya Mandal a, Mithun Chandra Majee b, Trilochan Rakshit a, Snehasis Banerjee , Partha
Mitra b, Debdas Mandal a,*
a Department of Chemistry, Sidho-Kanho-Birsha University, Purulia - 723104, West Bengal, India.
b
Department of Inorganic Chemistry, Indian association for the cultivation of science, Kolkata –
700032, India.
c Department of Chemistry, Govt. College Of Engineering and Leather Technology,
Salt Lake Sector-III, Block-LB, Kolkata- 700106, India
* Corresponding author E mail – id: deb_mandal@yahoo.co.in (Debdas Mandal)
Abstract
A
mononuclear
Cu(II)
complex
[CuII(L)](1)
[H2L=N,N’-bis(2-hydroxy-3,5-di
tertiarybutylbenzyl)-homopiperazine] has been synthesized by using sterically constrained
tetradentate phenol-based ligand. Characterization of this compound has been carried out by various
spectroscopic tools. Compound 1 crystallizes in orthorhombic space group P212121 with a = 9.9794
Å, b = 11.944 Å, c = 28.719 Å. Single crystal X-ray diffraction study reveals that in mono nuclear
copper(II) complex [CuII(L) ] 1, the metal center adopts a square planar environment. The geometry
of the Cu(II) complex has been optimized using density functional theory (DFT) calculations.
Electronic excitation energies of the above mentioned complex are calculated by TDDFT/UB3LYP
method combined with the SMD solvation in methanol medium. Electrochemical nature of the bis
phenol based copper complex confirms that both the oxidation process (E1/2 = 0.56 V and E1/2 = 0.89
V) arise due to ligand based oxidation of the phenol moiety to phenoxyl radical. Compound 1 acts as
an effective catalyst towards the oxidation of 3,5-di-tert-butylcatechol in methanol solvent to its
corresponding quinone derivative in aerial oxygen. The kinetic parameters have been determined
using Michaelis–Menten approach
.
The Kcat value for 1 in methanol is 722 h-1.
Keywords: Syntheses, Characterization, X-ray structure. DFT study, Catecholase activity.

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1. Introduction
Metalloenzyme containing copper ions at their active site are usually involved in a range of
biological processes, such as electron transfer or oxidation of organic substrates [1]. The ability of
copper proteins to reversibly bind dioxygen at ambient conditions has inspired numerous research
groups to study their structural, spectroscopic and catalytic properties [2]. Catechol oxidase is an
enzyme with a type-3 di-copper active site that catalyzes the oxidation of a range of ortho-diphenol
(catechol) substrates to the corresponding ortho-quinones. The generated o-quinones are auto
polymerized producing a brown polyphenolic pigment, i.e., melanin, a process which is considered to
protect damage tissues against pathogens or insects [3]. In 1998, X-ray crystallographic
characterization of catechol oxidase, isolated from sweet potatoes, was reported. The reported results
reveals that the active center consists of a hydroxo-bridged dicopper(II) center in which each
copper(II) center is coordinated to three histidine nitrogens and adopts a trigonal pyramidal
environment with one nitrogen at the apical site [4]. The ability of di-copper complexes to oxidize
phenols and catechols is well established from the report of various research groups [5]. Several
monocopper (II) complexes are also known to exhibit catecholase activity [6]. There are different
approaches used in the literature to study the mechanism of the catalytic oxidation of the substrate
(catechol) by these compounds. There are several model system follow the pathway which produces
two molecules of quinone and water [7]. At the same time many complexes oxidise catechol through
an alternate pathway which involves the production of quinone along with H2O2 rather than water
[8].
Herein, we prepare mononuclear square planar Cu(II) complex for [CuL] (1) with redox active di-
tertiary butyl substituted phenol based tetradentate N2O2 ligand. X- ray crystallography, electronic
spectroscopy and ESI- MS have been carried out to characterize this complex. The electrochemical
studies of 1 show two reversible oxidation peaks in the positive potential range due to the ligand
centered redox processes in which phenolate group yields phenoxyl radical in this complex.
Furthermore, the copper complex 1 is found to be an effective functional model of the enzyme as it
catalyzes the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylbenzoquinone
(3,5-DTBQ) with molecular oxygen in methanol at 25°C.

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2. Experimental section
2.1. Materials
Homopiperazine, 2,4-di-tert-butylphenol and 3,5-di-tert-butylcatechol were purchased from
Aldrich. Solvents were reagent grade, purified using appropriate drying agents and distilled under
nitrogen prior to their use [9]. All other chemicals were commercially available and were of reagent
grade and used as received without further purification.
2.2. Synthesis
2.2.1. Ligand
N,N’-bis(2-hydroxy-3,5-di tertiarybutylbenzyl)-homopiperazine (H2L) Homopiperazine (1.0
g,10 mmol) was taken in methanol (50 mL). To the methanolic solution about 0.60 g (20 mmol) of
paraformaldehyde was added. The resulting Solution was refluxed for 3 h. To this 2,4-di-tert-
butylphenol (4.12 g, 20 mmol) was added slowly with constant stirring. The resulting mixture was
refluxed further for 12 h to obtain a white crystalline precipitate which was filtered off, washed with
methanol (2 × 10 mL) and diethyl ether (2 ×10 mL) and finally dried in vacuum. The compound was
recrystallized from acetone-pet ether (60-80oC) mixture. Yield: 4.28 g (80 %). Anal. Calcd. for
C35H56N2O2: C, 78.31; H, 10.51; N, 5.22. Found: C, 77.92; H, 10.20; N, 5.02.
1H NMR (300 MHz, CDCl3, 25oC), δ/ppm: 1.32 (s, 18H, CH3), 1.38 (s, 18H, CH3), 1.97
(s, 2H, -CH2-), 2.77 (s, 4H, N-CH2 -), 2.83 (s, 4H, CH2-CH2), 3.77 (s, 4H, benzylic), 6.82 (s, 2H,
aryl), 7.21 (s, 2H, aryl).
IR (KBr disk, cm-1) : 2956, 2904, 1481, 1434, 1360, 1236, 1164, 1093, 877, 812, 723.
Scheme 1. Schematic presentation of preparation of ligand.

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2.2.2. Preparation of complex
[CuL] (1) Cu(OAc)2.2H2O (0.1 g , 0.5 mmol ) was added to the methanol solution of H2L
(0.27g, 0.5 mmol) and the resulting mixture was stirred. After 1h stirring, solution was turned into
dark color. Resulting solution was filtered. The filtrate was kept undisturbed in open air at room
temperature for crystallization. After 1 day, brown crystalline compound along with single crystals
was obtained. IR (KBr disk, cm-1) : 2952, 2855, 1464, 1436, 1366, 1306, 1257, 1167, 1146, 827, 736,
526. Anal. Calcd. for C35H54CuN2O2: C, 70.25; H, 9.10; N, 4.68. Found: C, 70.10; H, 8.88; N, 4.21.
UV-Vis (CH2Cl2), [λmax /nm (ε
/mol-1cm2) : 422 (1351), 550(848). ESI-MS in CH2Cl2: m/z (M+H+):
598.
2.3. Physical measurements
UV-visible spectra in solution were recorded on a Perkin-Elmer 950 UV/VIS/NIR
spectrophotometer, while for infrared spectra were employed a Nicolet Magna 750 FT-IR
spectrometer, series II with samples prepared as KBr pellets. Mass spectra (ESI-MS in positive ion
mode) were recorded on a QTOF Model YA263 Micro Mass Spectrometer. The electrochemical
measurements were carried out with a BAS epsilon electrochemistry system. A three–electrode
assembly comprising a Pt (for oxidation) or glassy carbon (for reduction) working electrode, Pt
auxiliary electrode and Ag/AgCl reference electrode were used. The cyclic voltammetric study (CV)
was carried out at 25°C in CH3CN solution of the complex (ca.1 mM) and the concentration of the
supporting electrolyte tetraethylammonium perchlorate (TEAP) was maintained at 0.1 M. All of the
potentials reported in this study were referenced against the Ag/AgCl electrode, which under the
given experimental conditions gave a value of 0.36 V for the ferrocene/ferrocenium couple.
2.3.1. X-ray crystallography
Diffraction quality crystals of 1 were grown at room temperature by slow diffusion of
methanol solution of the compound. Intensity data for the compound was measured on a Bruker
SMART 1000 CCD diffractometer using a graphite monochromatic Mo Kα radiation (λ = 0.71073 Å)
at 293 K. Intensity data were collected with θmax of 25 deg. No crystal decay was observed during the
data collections. Relevant crystal data and refinement details are given in Table 1. The structures

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were solved by direct methods [10] and refined on F2 by a full-matrix least-squares procedure [11]
using the program SHELXL 97.
Table 1 Summary of the Crystallographic Data for the Complex 1.
Empirical formula
Formula weight
T (K)
C35 H54 Cu N2 O2
598.34
293 (2)
0.71073
P212121
orthorhombic
9.9794
λ
(Mo Kα), Å
Space group
Crystal system
a (Å)
b (Å)
11.944
c (Å)
28.719
V (Å3)
3423
Z
4
DCalcd. (g cm-3)
1.161
ꢀ
(mm_1)
F (000)
ranges (º)
0.668
1292
θ
1.42 – 25.00
-11
-13
-34
≤
h
≤ 9
≤
k
≤
14
Index ranges
≤
l
≤
33
Reflections collected
Rint
25114
0.0949
1.032
373
Goodness of fit
No. of parameters
R1a (Fo), wR2b(Fo) (all data)
Largest diff. peak, deepest hole (eÅ-3)
0.0599, 0.1306
0.621, -0.347
2
2 2
aR =
∑
||Fo| - |Fc||/
∑
|Fo|. bwR = [
∑
[w((Fo - Fc2)2]/
∑
w(Fo ) ]

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2.3.2. Theoretical methods
All of the molecular parameters needed in this paper are calculated by unrestricted
density functional theory (DFT) using Gaussian 09 program [12]. The geometries are
optimized by B3LYP functional starting from the X-ray crystallographic data [13]. This
functional was combined with the 6-31G(d,p) atomic basis set for all first and second-row
atoms. Relativistic effects were included for Cu atom using LANL2DZ pseudopotential [14].
The nature of the stationary points was confirmed by computing the Hessian at the same level
of theory. To explore the absorption spectral properties for the investigated complex, the
time-dependent density functional theory (TD-DFT) was adopted on the basis of the
optimized ground state of doublet state.
2.3.3.Catalytic oxidation of 3,5-DTBC
The catecholase activity of complex 1 was monitored using most familiar 3,5-di-
tertbutylcatechol (3,5-DTBC) as the substrate in a methanol solution under aerobic conditions
at room temperature [15]. In order to check the catechol oxidase activity of the complex, a
10-4 M solution of 1 in methanol solvent was mixed with 100 equiv. of 3,5-ditert-
butylcatechol (3,5-DTBC) under aerobic conditions at room temperature. The reaction was
carried out spectrophotometrically by observing the increase in the maximum absorbance of
the quinone band around 400 nm as a function of time [16]. Absorbance vs. wavelength of
the solution was recorded at a regular time intervals of 5 min. It may be noted here that a
blank experiment without catalyst does not show formation of the quinone up to 6 h in
MeOH. To determine the dependence of rate on substrate concentration and upon various
kinetic parameters,
1.75 x 10–4 M solution of complex 1 was mixed with increasing amounts
of 3,5-DTBC from 1.4 x 10-3 M to 5 x 10-3 M. In each case, the reaction was monitored
spectrophotometrically by watching the increase in the absorbance at 400 nm up to 6 hr. The
rate constant versus substrate concentration data were then analyzed on the basis of the
Michaelis–Menten approach of enzymatic kinetics to get the Lineweaver–Burk plot as well as
the values of the parameters Vmax, KM, and Kcat .

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2.3.4. Detection of Hydrogen Peroxide in the Catalytic Reactions
Detection of H2O2 was observed by modified iodometric method during catalytic
reaction [17]. The reaction mixture of catalyst and 3,5 DTBC was prepared as in the kinetic
experiments. After 1 h of reaction, equal volume of water was added to the reaction mixture.
The quinone was extracted from the reaction mixture by using dichloromethane. Few drops
of H2SO4 is mixed with aqueous extract to stop further oxidation. 1 mL of a 10% solution of
KI and three drops of 3% solution of ammonium molybdate were added to the reaction
mixture. Hydrogen peroxide obtained from catalytic reaction oxidizes I- to I2. In presence of
-
excess iodide ions, the tri- iodide ion is produced according to the reaction I2(aq) + I -
→
I3 .
The reaction rate increases with increasing concentrations of acid and the addition of an
ammonium molybdate solution condenses the reaction almost immediately. The formation of
-
-
I3 was detected by UV – vis spectroscopy due to the development of the characteristic I3
band.

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3. Results and Discussion
3.1. Syntheses
The copper complex has been prepared readily by mixing the copper acetate with the
methanolic solution of sterically hindered phenol based N2O2 ligand in stoichiometric amounts (1:1
mole ratio). The detailed strategy is described in scheme 2. Mononuclear compound [CuII L] (1) was
obtained with nearly square planar geometry and the compound is stable in air at room temperature.
The compound was characterized using IR, UV–VIS spectroscopy, mass spectroscopy and single
crystal X-ray crystallography. The complex was initially characterized by IR spectroscopy. IR
spectra of this complex display all the characteristic bands of the coordinated L2- ligand [18]. One
such prominent band appears at 1257 cm-1 due to υ(c-o/phenolate) stretching vibrations [19].
Scheme 2. Schematic presentation of preparation of complex 1.
3.2. Description of crystal structure
The molecular structure of complex 1 is shown in Fig.1. Its important inter atomic parameters are
listed in Table 2. The complex crystallizes in orthorhombic space group P212121. The Cu(II) center in
this mononuclear complex is four-coordinated using a doubly deprotonated tetradentate ligand L2-,
providing O(1), N(1), N(2) and O(2) donor sites. The bond distances of Cu–N (2.020(5) – 2.041(4)
Å) and Cu–O (1.883(4) – 1.898(3) Å) are all in the expected ranges [20]. There is no hydrogen
bonding interaction present within the molecule, as well as no ion dipole interaction is present. This
will also be supported from the theoretically obtained results as discussed later. Addison and
Reedijk’s define the geometric parameter (τ) as an index of the degree of trigonality, within the
structural continuum between trigonal bipyramidal and rectangular pyramidal which is applicable to
five-co-ordinate structures [21]. Yang et. al propose a very simple geometry index for four-

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coordinate complexes
,
τ4, which can be used to quantify the geometry of four-coordinate species
[22]. The equation is as follows
ꢄ
ꢈ
ꢀꢁꢂ°ꢃ ꢅꢆꢇ
τ4
=
ꢉꢊꢉ°
Where α and β are the two largest θ angles in the four-coordinate species. Using the above mentioned
equation the magnitude τ4 value for this copper compound is 0.143.
Fig. 1. Molecular structure of the complex 1 showing the atom-numbering scheme.
Table 2 Selected Bond Distances (Ǻ) and Angles (deg) for 1.
Bond distances (
Ǻ
)
Bond angles (deg)
O(1)-Cu-O(2)
O(1)-Cu-N(1)
O(2)-Cu-N(1)
O(1)-Cu-N(2)
O(1)-Cu-N(2)
N(1)-Cu-N(2)
Cu-O(1)
Cu-O(2)
Cu-N(1)
Cu-N(2)
1.883(4)
1.898(3)
2.020(5)
2.041(4)
93.35(16)
170.27(18)
94.82(18)
93.99(18)
169.54(18)
78.6(2)

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3.3. Electronic spectra
The electronic spectra of the complex 1 have been observed in methanol solution of the
compound at room temperature and the data are represented in the experimental section. The
electronic spectrum of the copper complex shows bands at 422 nm and 550 nm which can be
2
assigned to 2B1g → 2A1g and 2B1g → E1g transitions as displayed in Fig. 2 [ 23-24]. Remaining band
maxima appearing around at UV region are due to ligand internal transition.
Fig. 2. Electronic absorption spectrum of complex 1 in MeOH
3.4. Electrochemical studies
The redox behavior of the complex has been examined by cyclic voltammetry in acetonitrile
solution (0.1 M TBAP). Compound display main features in the potential range 0.2 V to +1.4 V vs
Ag/AgCl reference as shown in Fig.3. The electrochemical studies reveal that there are two

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reversible oxidation peaks for [CuL] 1 complex in the positive potential range. The first reversible
2
electrochemical process appeared at E1/21 = 0.56 V and the second reversible process at E 1/2 = 0.89
V. These redox processes are probably attributed to the ligand centered redox processes in which
phenolate group yields phenoxyl radical in the complex [25]. Such type of observation has been
reported previously in other complexes containing tertiary butyl substituted phenol based ligand [26].
0.0
0.2
0.4
0.6
0.8
E (V)
1.0
1.2
1.4
Fig. 3. Cyclic voltammograms of complex 1 in CH3CN /0.1 M tetraethylammonium perchlorate
(TEAP) with platinum as the working electrode and saturated Ag/AgCl as the reference electrode.
Comparison of the electrochemical nature of complex 1 with related copper complexes suggests that
both the oxidation processes arise due to ligand based oxidation of the phenol. For example, {[(3-
tert-butyl5methyl2hydoxybenzyl)(3’tertbutyl5’methyl2’oxobenzyl)(2pyridylmethyl)]amine}Cu(OAc)
exhibited two electrochemical processes in the anodic region on the CV timescale. The first quasi-
reversible electrochemical process appeared at E1/2 = 0.72 V [0.21 V vs Fc/Fc+ ] and the second
quasi-reversible process at E1/2 = 1.10 V [ 0.60 V vs. Fc/Fc+ ] [25a]. Apart from these structural
analogues, copper bis-phenolate complex namely (N-benzyl-bis-N’,N’’-salicylidene)-cis- 1,3,5-

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triaminocyclohexane copper(II) showed two reversible one electron oxidations at E1/2 = 0.89 V (0.34
V vs Fc/Fc+. ) and E1/2 = 1.13 V (0.58 V vs. Fc/Fc+ ) [27].
In our cases, redox couples are ascribed to the formation of mono- and di-radical species according
to following pathway.
Oxidation of one phenolate moeity into a phenoxyl radical does influence significantly the redox
potential of second oxidation of the another phenolate moiety to phenoxyl radical as reported
analogues, copper bis phenolate complex [28].
3.5. Theoretical study
In this study, the geometries of ground state for the title complex were optimized by means of
unrestricted density functional theory (DFT) without any symmetry constrains. The best agreement
with experimental results were observed for the title complex at UB3LYP/6-31G (d,p) levels. These
results confirm that the complex has distorted square planar geometry as obtained in the X –ray data.
In this geometry, two Cu
−N bond distances are 2.11 Å and Cu
−O bond distances are about 1.92 Å.
The well agreement with the X-ray data (Cu
the theoretical methods used here.
−N
2.04 Å and Cu−N 1.89 Å) verified the reliability
The title complex has a spin doublet ground state. The data of frontier molecular orbital
(FMO) analysis in terms of energies are shown in Table S1 and Fig. 4. A larger HOMO-LUMO gap
of 4.46 eV indicates a very stable nature of the investigated complex. In order to reduce the
computational cost for this large molecule, some methyl groups were excluded from the complex in
the excitation process as shown in Fig. S1. On the basis of optimized ground state geometry, the
TDDFT/UB3LYP method combined with the SMD solvation in methanol medium was used to
calculate the absorption properties of the investigated complexes. The most important excited states,
their oscillator strengths, dominant orbital excitations, and their assignments are shown in Table 3. In
Fig.5 the normalized absorption spectra obtained theoretically along with the experimental spectra
are shown. The simulated curve is in good agreement with that of experimentally obtained curve,
however, some transitions that are found theoretically are not present in the simulated results.

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A larger HOMO-LUMO energy gap (4.46 eV) for the
(2.1 eV) obviously indicate the low energy electronic excitations are from
(Table 3) shows that, for 1, the lowest-lying absorption at 557 nm mainly originates from
LUMO.This absorption band is close to the experimentally obtained band at 570 nm. The other
important strong bands at 466 nm come mainly from -H-5 to -LUMO with the other contributions
-LUMO. Slightly higher energy transition at 344 nm mainly originates
-H-2 to the -LUMO. This band can be correlated with the assigned
α
-MOs compared to energy
-MOs. FMO analysis
-H-2→β
β-MOs
β
β
-
β
β
from
from
β
-H-10 and
β-H-6 to β
α
-HOMO and
α
α
experimentally obtained band at 330 nm which is slightly red shifted by about 14 nm. The strong
absorption at 286 nm is in excellent agreement with the experimentally obtained band at 288 nm.
This band is contributed from α-HOMO to α-LUMO.
Table 3 Selected calculated wavelength (nm)/energies (eV), oscillator strength (ƒ), major
contribution and the experimental wavelength (nm) for the investigated complexes. H indicates
HOMO, L indicates LUMO.
Expt.
Complex
1
f
configuration
λ
/E
(nm)
β
-H-2→β-LUMO (63%),
557
2.22
0.004
570
β
-HOMO→
LUMO (11%)
β
-H-10→β-LUMO (10%), β-H-
466
418
2.66
2.96
0.019
0.005
450
6
→β-LUMO (10%), -H-5→β
β
-
LUMO (36%)
β
-H-4→β-LUMO (92%)
α
1
-H-1→α-LUMO (12%),
L+4 (10%), -HOMO→α
LUMO (11%), -H-1→β-L+1 (11%)
α-H-
344
286
3.6
0.082
0.032
330
288
→
α
-
β
4.33
α
-HOMO→α-LUMO (65%)

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Fig. 4. Energy level energy gaps (in eV) of the FMOs for the complex 1.

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expt.
theo.
0.6
0.4
0.2
0.0
200
300
400
500
600
700
wavelength (nm)
Fig. 5. Normalized simulated and experimental absorption spectra of the investigated complex.

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3.6. Catecholase activity
The catechol oxidase activity of mononuclear copper complex [CuIIL] (1) was monitored
using 3,5-di-tert-butyl catechol (3,5-DTBC) as a model substrate in methanol under aerobic condition
at room temperature. With addition of 3,5- di-tert-butyl catechol in methanolic solution of the
complex in presence of air, there was a gradual increase in absorbance at 400 nm as shown in Fig.6.
characteristic to the formation of 3,5-di-tert-butyl benzoquinone. Most widely used 3,5-di-tert-
butylcatechol (3,5 - DTBC) has been chosen as the substrate as it can easily be oxidised to 3,5-di-
tert-butyl benzoquinone (3,5 - DTBQ). Two bulky substituents on the catechol ring makes it easily
oxidizable to the corresponding o-quinone 3,5-DTBQ and shows a maximum absorption at 400 nm
in methanol [15-16]. Kinetic experiments were performed spectrophotometrically with complex 1
and the substrate 3,5-DTBC in methanol at 25°C. The conversion of 3,5–DTBC to 3,5-DTBQ
(Quinone band maxima) was observed with time at a wave length of 400 nm for [CuIIL] (1) in
methanol. The rate constant for a particular complex–substrate concentration ratio was obtained by
change in absorbance versus time plot by choosing initial rate method.
The substrate concentration dependence of the oxidation rate was watched under aerobic
conditions using 1.75 x 10–4 M solution of complex 1 and increasing amounts of 3,5-DTBC from 1.4
x 10-3 M to 5 x 10-3 M. The rate constant versus substrate concentration data were explained on the
basis of the Michaelis–Menten approach of enzymatic kinetics to get the Line weaver–Burk plot as
well as the values of the parameters Vmax, KM, and Kcat. The observed rate vs. [substrate] plot in
methanol solution, as well as Line weaver–Burk plot, is given in Fig.7. The Vmax value is 5.798 x 10-5
M s-1 (Std. error 1.195 x 10-4) whereas KM value is 11.83 x10-3 M (Std. error 7.546 x 10-3) .
The mononuclear Cu(II) complex showed high turnover number ( 722 h-1) for the catalytic oxidation
of 3,5-DTBC to 3,5-DTBQ under mild conditions by molecular oxygen and the existence of 3,5-
DTBQ was identified from ESI-MS+ (m/z) study (Fig. 8).
Table 4 Kinetic parameters for the oxidation of 3,5-DTBC catalyzed by 1.
Solvent
MeOH
Vmax (M min-1)
6.74 × 10-3
Std. error
KM (M)
Std. error
kcat (h-1)
722
12.55 × 10-3 1.17 × 10-2
2.55 × 10-3

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Fig.6. Increase of quinone band at 400 nm after addition of 100 equivalents of 3,5-DTBC to a
solution containing complex 1 (10-4 M) in methanol at 25 °C.
Fig. 7. Plot of rate vs. [substrate] in the presence of 1 in MeOH; inset: Lineweaver–Burk plot.

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Table 5 Comparison of Kcat value for the oxidation of 3,5-DTBC to 3,5-DTBQ by different copper
complexes
Complex
[Cu(H2La)(HL)]ClO4
Solvent
Vmax(M min-1)
151.8 ×10-4
KM(M)
Kcat (h-1)
Ref.
29
methanol
2.62 ×10-3 9.11 ×103
1.20 ×10-3 2.08 ×104
[Cu(H2La)(HL)Cu(L)]+
[Cu(L2a)(NO3)2]
methanol
methanol
methanol
methanol
346.2 ×10-4
2.83 ×10-5
3.59 ×10-5
2.35 ×10-4
29
30
30
31
1.2 ×10-3
2.7 ×10-2
6.91 ×10-6
3
6
[Cu(L2b)(NO3)2]
[Cu(dicl)2(2-pic)2]
140.04
[Cu(dicl)2(4-pic)2]
methanol
methanol
1.69 ×10-4
1.59 ×10-4
144 ×10-7
9.84 ×10-5
3.44 ×10-5
7.9 ×10-4
101.16
95.04
28.44
31
31
32
[Cu(dicl)2(2-ampy)2]
.
[Cu2(H2bbppnol)(µOAc)(H2O)2]Cl2 2H2O methanol
[CuII2(L3−O)(OH)(OClO3)2]·1.5H2O
methanol
methanol
methanol
15 ×10-6
1.56 ×10-4
6.74 × 10-3
0.40 ×10-3
3.32 ×10-3
1.17 × 10-2
36
33
34
[Cu2L4 (ClO4)2]
93.6
722
2
Complex 1
Present
work
Where H2La
=
Pyridine-2,6-dimethanol,
L2a
=
N-(2,6-diethylphenyl)-2-[(pyridin-2-
ylmethyl)amino]acetamide, L2b = N-(mesityl)-2-[(pyridin-2-ylmethyl)amino]acetamide, Diclofenac =
Hdicl = 2-(2,6-dicholoroanilino)phenylacetic acid), 2-pic = 2-picolin,4-pic = 4-picoline, 2-
ampy = 2-aminopyridine, H3-bbppnol = N,N′-bis(2-hydroxybenzyl)-N,N′-bis-(pyridylmethyl)]-2-
hydroxy-1,3-propanediamine, L3−OH=1,3-bis[(2-dimethylaminoethyl)iminomethyl]phenol, HL4 = 2-
[[2-(diethylamino)-ethylamino]methyl]phenol.

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Table 5 shows the catalytic activities of some mononuclear Cu(II) as well as some multi nuclear
Cu(II) complexes. It is worth mentioning that our complex showed quite high TON (Kcat) for the
catalytic oxidation of 3,5 DTBC to 3,5 DTBQ by comparing with other reported work [29 -34].
O
O
+
Na
Fig. 8. ESI-MS+ (m/z) spectrum for the ultimate catecholase product during the reaction of 1 with
3,5-DTBC.
Herein, we may suggest a plausible mechanism for the catalytic oxidation of 3,5 DTBC to 3,5
DTBQ in presence of air. In the first step of catalytic cycle molecular oxygen take part in the ligand
based oxidation reaction of neutral copper complex 1 with redox active di-tertiary butyl substituted
phenol based ligand to generate active phenoxyl radical complex species 1a and simultaneously
molecular oxygen is reduced to the H2O2. A ligand–centered radical species is most likely
responsible for catechol oxidation. Phenoxyl radical complex 1a (O• – CuII – O• ) oxidizes 3,5 DTBC
to 3,5 DTBQ by capturing two electron from the substrate and itself is transformed to previous

ACCEPTED MANUSCRIPT
neutral copper complex 1. If, this oxidation reaction is performed in the presence of a radical
scavenger, TEMPO ( TEMPO = 2,2,6,6 –tetra methyl piperidinoyl), the formation of quinone is
prevented. When catecholase activity of copper complex is monitored in presence of TEMPO,
quinone band at 400 nm is not observed spectroscopic ally. This result suggests that the catechol
oxidation catalyzed by Cu (II) complexes is occurred via radical generation [35]. Cyclic
voltammetric study supplies additional information of these phenomenons.
The di-oxygen of atmosphere is reduced to H2O2 during the oxidation process. To detect the
formation of hydrogen peroxide during the catalytic reaction we followed the modified iodometric
-
method as indicated in reported paper [17]. Qualitative as well as quantitative detection of I3 band (
353 nm) by UV- vis spectroscopy is the indication of formation of H2O2 during catalytic reaction.
The estimation of H2O2 clearly indicates that nearly 1 mol of H2O2 was shown to be produced per
mole of 3,5 DTBC along with 1 mol of 3,5 DTBQ. Such incident clearly suggests that in the catalytic
cycle two electron reduction of molecular oxygen is occurred as reported earlier [36].
Scheme 3. A plausible mechanistic pathway for the aerobic catechol oxidation catalyzed by
complex 1.

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4. Conclusion
In summary, we have reported a square planar copper(II) complex with a sterically
constrained redox active tetradentate phenol based ligand with N2O2 donor site. Structure of this
compound is characterized by X-ray single crystal diffractometer. A very good agreement between
the X-ray and DFT calculated data was obtained. Electronic spectrum calculations of the complex
were made by TDDFT/UB3LYP method combined with the SMD salvation in methanol.
Comparison of the electrochemical nature of complex 1 with related copper bis phenolate complexes
confirms that both the oxidation processes arise due to ligand based oxidation of the phenol moiety to
phenoxyl radicals. In addition the copper complex 1 efficiently catalysed the oxidation of 3,5 DTBC
to 3,5 DTBQ in presence of air. The turnover number of this reaction is 722 h-1.
Supplementary data
CCDC1861935 contains the supplementary crystallographic data for compound 1. These data
can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223-
336-033; or e-mail: deposit@ ccdc.cam.ac.uk.
.
Acknowledgment
Financial support received from Department of Science and Technology, Government of
West Bengal, Kolkata (Project memo no. 1071(Sanc)/ST/P/S&T/15G-4/2015 dated 23.02.2016) is
gratefully acknowledged. We are grateful for the instrumental support from the Department of
Inorganic Chemistry, Indian Association for the Cultivation of Science.

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A square planar copper(II) complex with a sterically constrained redox active tetradentate
phenol based ligand with N2O2 donor site have been prepared. Structure of this compound is
characterized by X-ray single crystal diffract meter. Comparison of the electrochemical
nature of complex 1 with related copper bis phenolate complexes confirms that both the
oxidation processes arise due to ligand based oxidation of the phenol moiety to phenoxyl
radicals. The copper complex 1 efficiently catalysed the oxidation of 3,5 DTBC to 3,5
DTBQ in presence of air. The turnover number of this reaction is 722 h-1.

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Highlights
•
•
The mononuclear complex of general formula [CuIIL] was prepared.
The structure of the complex was characterized by single crystal X-ray
crystallography and various spectroscopic tools.
•
•
Geometries of ground state for the complex were optimized by density functional
theory.
Catechol oxidase activity of the complex was performed.