Catalytic oxidation of polyphenol trihydroxybenzene by copper(II) β-alanylsulfadiazine complex

Article abstract of DOI:10.1016/j.molcata.2011.12.016

The copper(II) complex of sulfadiazine with β-alanine amino acid was synthesized and characterized using different tools such as IR, UV/Vis, ¹H NMR, elemental analysis, thermal analysis, TEM and EPR spectroscopy. The mode of binding of the meta

Full text of DOI:10.1016/j.molcata.2011.12.016

Journal of Molecular Catalysis A: Chemical 355 (2012) 192–200
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis A: Chemical
journal homepage: www.elsevier.com/locate/molcata
Catalytic oxidation of polyphenol trihydroxybenzene by copper(II)
␤-alanylsulfadiazine complex
Ahmed I. Hanafya,∗,1, Zeinhom M. El-Bahya,1, Ahmed A. El-Henawyb, Abeer A. Faheima,1
a Chemistry Department, Faculty of Science, Taif University, Taif, Saudi Arabia
b Al-Azhar University, Faculty of Science, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 August 2011
Received in revised form 7 December 2011
Accepted 9 December 2011
Available online 17 December 2011
The copper(II) complex of sulfadiazine with ␤-alanine amino acid was synthesized and characterized
using different tools such as IR, UV/Vis, 1H NMR, elemental analysis, thermal analysis, TEM and EPR
spectroscopy. The mode of binding of the metal shows that, the copper binds with the ligand through
nitrogen atom of the amino group and carbonyl oxygen atom. The transmission electron microscope
(TEM) demonstrated that the average particle size of the copper complex is found to be in the range of
35–40 nm. The complex, [CuL(OH)2] has been used as an effective catalyst for homogenous oxidation of
polyphenol 1,2,3-trihydroxybenzene in the presence of the green oxidant H2O2 to produce a first-order
rate constant kcat = 0.0043 s−1. The catalysis shows a catalytic proficiency of 1.7 × 103 times compared to
the uncatalyzed oxidation of THB under the same reaction conditions. The oxidation reaction is inhibited
by kojic acid with IC50 = 65 ␮M.
Keywords:
Sulfadiazine
␤-Alanine
Oxidation
Trihydroxybenzene
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
oxidation catalysis as copper-containing oxidases. These
biomimetic chemical systems may be better accessible, more
The oxidation of organic substrates with molecular oxygen
under mild conditions is of great interest for industrial and syn-
thetic processes, especially from economical and environmental
points of view [1,2]. Although the reaction of organic compounds
with dioxygen is thermodynamically favored it is kinetically hin-
dered due to the triplet ground state of O2. The synthesis and
investigation of functional model complexes for metalloenzymes
with oxidase or oxygenase activity is therefore of great interest
for the development of new and efficient catalysts for oxidation
reactions.
stable and more catalytically versatile than enzymes, thus may
have wider applications and provide chemical insight into the
mechanisms of enzymes [14–16].
Sulfadiazine compound has been chosen in this study due to its
relatively small molecular weight, its biological activity and medic-
inal uses. We have prepared a new simple and an environment
friendly copper(II) complex derived from the antibacterial sulfadi-
azine with ␤-alanine amino acid. This complex was characterized
and used as biomimetic of a copper-containing oxidase in the oxi-
dation of trihydroxybenzene.
Catechol oxidase, tyrosinase, and polyphenol oxidases are anal-
ogous metalloenzymes which oxidize phenolic compounds to the
corresponding quinones in the presence of oxygen. This kind of
reaction is of great importance in medical diagnosis for the deter-
mination of the hormonally active catecholamines adrenaline,
noradrenaline and dopa [3]. There have been great efforts to syn-
thesize model complexes as functional or structural models for
catechol oxidases or related copper containing enzymes [4–9].
Transition metal complexes have been widely studied in the
area of bio-inorganic, bio-organic, and catalytic chemistry [10–13].
Copper complexes are interesting compounds in the field of
2. Experimental
2.1. Materials
Sulfadiazine,
␤-alanine,
N,Nꢀ-dicyclohexylurea,
1,2,3-
trihydroxybenzene and Fmoc (9H-fluoren-9-ylmethoxycarbonyl)
were purchased from Sigma–Aldrich. Kojic acid and copper
chloride were purchased from Merck Company.
2.2. Synthesis of
3-amino-N-[4-(pyrimidine-2-ylsulfamoyl)-phenyl]-propionamide
(ˇ-alanylsulfadiazine)
∗
Corresponding author. Taif University, Faculty of Science, Taif, Saudi Arabia.
Tel.: +966 28322143; fax: +966 28322143.
A
mixture of sulfadiazine (0.01 mol) and Fmoc-␤-alanine
(0.01 mol) was dissolved in ∼30 ml tetrahydrofuran. The
E-mail address: ahmedih@yahoo.com (A.I. Hanafy).
Permanent address: Al-Azhar University, Faculty of Science, Cairo, Egypt.
mixture was cooled to 0 ◦C and then (2.06 g; 0.01 mol)
1
1381-1169/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcata.2011.12.016

A.I. Hanafy et al. / Journal of Molecular Catalysis A: Chemical 355 (2012) 192–200
193
H
∗
N
N
NH2
NH2
C
O
N
O
N
O
OH
S
O
S
N
N
N
N
O
H
H
Structure 1. Keto–enol tautomeric forms of ␤AS ligand.
N,Nꢀ-dicyclohexylcarbodiimide (DCCD) dissolved in ∼10 ml
tetrahydrofuran was added. The reaction mixture was stirred
for 3–5 h at 0 ◦C and allowed to stand for 24 h at room tempera-
ture. A few drops of acetic acid and water were added, then the
precipitate of N,Nꢀ-dicyclohexylurea was filtered off. The filtrate
was concentrated in vacuo to dryness. The residual material was
recrystallized from ethanol–water, and obtained in 80% yield,
Structure 1. The compound was chromatographically homoge-
nous when developed with iodine solution-benzidine and gave a
negative ninhydrin test.
Fig. 1. 1H NMR of ␤-alanylsulfadiazine in d6-DMSO.
2.3. Synthesis of CuII ˇAS complex
7.5). The resulting conformations were confirmed as minima by
vibrational analysis.
Copper(II) chloride (0.1 mol) was dissolved in ∼40 ml abso-
lute ethanol, then added to 0.1 mol of the prepared ligand
␤-alanylsulfadiazine (␤AS) dissolved in ∼40 ml absolute ethanol.
The mixture was heated under reflux for ∼2 h. The bluish precip-
itate was formed, filtered off and finally washed with hot ethanol
several times.
2.6. Kinetic reactions for trihydroxybenzene (THB) oxidation
The catalytic activity of the CuII ␤AS complex toward the
homogeneous oxidation of trihydroxybenzene (THB) in ethanol
solution at 25 ◦C was determined by measuring the initial rate
of (THB) oxidation. The increase of the absorption at 420 nm
(ε = 4583 M−1 cm−1) due to the oxidation product [17] with time
was obtained on a Varian Cary 3E spectrophotometer. A plot of
the formation of the product with respect to time gives the ini-
tial rate. To study the effect of the catalyst concentration on the
rate of the reaction, various amounts of the copper(II) complex
(10–300 ␮M) have been used with 200 ␮M H2O2 for oxidation of
1.0 mM THB at 25 ◦C. In the same time 30 ␮M of the catalyst has
been used in the oxidation of different concentrations of the sub-
strate (5–80 mM) in the presence of 200 ␮M H2O2 to study the
effect of THB concentration on the reaction. The rate laws were
determined and rate constants obtained. The dependence of H2O2
on THB oxidation by 30 ␮M CuII ␤AS was determined by mea-
suring the oxidation rate at different concentrations of hydrogen
peroxide (25–400 ␮M) in the presence of 1.0 mM THB in ethanol
at 25 ◦C. The auto-oxidation rate of THB was determined under the
same conditions in the absence of CuII ␤AS. Inhibitions were car-
ried out in a similar fashion as the kinetic measurements using
30 ␮M CuII ␤AS in the presence of 200 ␮M H2O2 and different
amounts of kojic acid.
2.4. Physical methods
Carbon, hydrogen and nitrogen contents were determined at
the Microanalytical Unit, Cairo University, Egypt. IR spectra of
the ligand and its solid complexes were measured in KBr on
a Mattson 5000 FTIR spectrometer. All electronic spectra and
kinetic measurement were performed using a Varian Cary 4 Bio
UV/Vis spectrophotometer. The 1H NMR spectrum of the ligand
was recorded on JEOL-90Q Fourier Transform (200 MHz) spec-
trometers in [d6] DMSO. Thermal analysis measurements (TGA,
DTA) were recorded on a Shimadzu thermo-gravimetric analyzer
model TGA-50H, using 20 mg samples. The flow rate of nitrogen
gas and heating rate were 20 cm3 min−1 and 10 ◦C min−1 respec-
tively. ESR spectra were obtained on a Bruker EMX spectrometer
working in the X-band (9.78 GHz) with 100 kHz modulation fre-
quency. The microwave power and modulation amplitudes were
set at 1 mW. The mass spectrum of the ligand was recorded on a
Shimadzu GC-S-QP 1000 EX spectrometer using a direct inlet sys-
tem. Thermal analysis, ESR and mass spectroscopy were recorded
at Cairo University, Egypt. The magnetic susceptibility measure-
ments for the complexes were determined using Gouy balance
with Hg[Co(NCS)4] as a calibrant at room temperature. Transmis-
sion electron microscope (TEM) micrographs were measured using
JEOL JEM-1010 transmission electron microscope, at an accelerat-
ing voltage of 60 kV. Suspensions of the samples were put on carbon
foil with a micro grid. TEM images were observed with minimum
electron irradiation to prevent damage to the sample structure.
3. Results and discussion
3.1. IR and 1H NMR spectra
The organic ligand (␤-alanylsulfadiazine) has been pre-
pared using (Fmoc) as
ally removed from the
a
N
protective group which is gener-
terminus of peptide chain by
a
2.5. Molecular modeling methods
acidolysis using trifluoroacetic acid (TFA) [18]. The IR spec-
trum of the 3-amino-N-[4-(pyrimidine-2-ylsulfamoyl)-phenyl]-
propionamide(␤-alanylsulfadiazine) shows bands at 3425, 3358
and 3259 cm−1 assigned to ꢀNH and ꢀNH2, respectively. The lig-
and also shows bands at 1649, 1586, 1494 and 1441 cm−1 assigned
2.5.1. Conformational analysis
Initial molecular structures of the ligands were built using the
HyperChem program 7.5. The conformational analysis has been
performed by use of MM+ (calculations in vacuo, bond dipole option
for electrostatics, Polake Ribiere algorithm, and RMS gradient of
0.01 kcal/mol). Minimum energy for compounds was performed
by a semi-empirical method PM3 (as implemented in HyperChem
to ꢀC O, ꢀC N, ␤-NH2 and ꢀ(O
S
O), respectively [19,20]. 1H
NMR spectrum of ␤-alanylsulfadiazine in dimethyl-sulfoxide-d6
(Fig. 1) exhibited signals at ı = 2.7 (d, 2H, CH2 CH2CH2NH2), 3.2 (b,
2H, CH2 CH2NH2), 5.9 (s, 1H, NH SO2NH), 6.5–7.4 (m, aromatic

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A.I. Hanafy et al. / Journal of Molecular Catalysis A: Chemical 355 (2012) 192–200
Fig. 2. Mass spectrum of the organic ligand ␤AS. Inset indicates the purity of the ligand ␤AS.
wavenumber in the complex spectrum (1610 cm−1) with a decrease
in its intensity. Moreover, this is also supported by shifting in
ꢀNH2 bands to higher wavelength (3953 and 3357 cm−1). The bands
protons), 7.8 (b, 2H, NH2), 8.4 (s, 1H, NH NHCO). All these data
together with the molecular weight determined from mass spec-
trometry [Fig. 2 and Scheme 1 (m/z = 322)] suggest the structure of
the ␤AS ligand as shown in Structure 1.
attributed to ꢀNH and ꢀ(O
S O) remain at the same position as in
By comparing the IR spectral data of the ligand with that of
the Cu(II) complex, it is found that the copper(II) binds to ␤AS
through the nitrogen atom of NH2 and carbonyl oxygen atom. This
suggestion was supported by shifting in carbonyl band to lower
the ligand spectrum, indicating no participation of NH or SO2 func-
tional groups in the coordination. The coordination of NH2 nitrogen
atom is also consistent with the presence of a new band at 464 cm−1
due to ꢀCu N. The IR spectrum shows a broad band observed at
H
H
N
H2N
N
NH2
C
O
C
O
C
O
N
N
O
N
O
O
S
S
S
N
N
N
N
N
N
NH2
O
O
O
H
H
H
235
277m/z
O
S
N
NH3
N
N
O
O
N
O
SH
O
N
NH2
S
N
N
N
N
N
H
95m/z
O
80m/z
H
158m/z
2
1
3
-CN
-CH
N
m
N
/
z
67 m/z
N
-CH2
O
N
O
S
S
N
N
N
N
O
O
H
H
185 m/z
1
9
9
m
/z
Scheme 1. Mass fragmentation pattern of ␤AS.

A.I. Hanafy et al. / Journal of Molecular Catalysis A: Chemical 355 (2012) 192–200
195
Fig. 3. EPR spectrum of CuII ␤AS complex.
5.0E-04
3
2.5
2
0.0E+00
-5.0E-04
-1.0E-03
-1.5E-03
-2.0E-03
-2.5E-03
TG
DTG
1.5
1
DTG
0.5
0
-0.5
0
200
400
600
800
1000
T(o C)
Fig. 4. TGA and DTG of the copper complex CuII ␤AS.
∼3420 cm−1 assigned to ꢀOH stretching vibration. The proposed
structure is confirmed by the presence of new band at 555 cm−1
attributed to Cu O.
complex. The elemental analysis calcd. (%) for CuII ␤AS: C, 37.40;
H, 4.07; N, 16.80; Cu, 15.20. Found: C, 38.01; H, 3.93; N, 15.97; Cu,
15.11, in addition to the IR, electronic and EPR spectral data suggest
that the structure of the CuII ␤AS complex is [CuL(OH)2], where
L = ␤AS, as shown in Structure 2.
3.2. Electronic spectral data
3.3. Thermal analysis
The magnetic moment (1.8 BM) of the Cu(II) complex CuII ␤-
AS at room temperature corresponds to one unpaired electron [21].
The electronic spectrum of CuII ␤AS complex recorded in ethanol
reveals an absorption band at 495 nm. This band can be attributed to
The TGA thermogram confirms the amount of solvent inside
and/or outside the coordination sphere and gives some infor-
mation about the stability of this compound. The thermogram
of CuII ␤AS (Fig. 4) shows three stages of mass loss over
the temperature range of 25–800 ◦C. The first stage at 140 ◦C
corresponds to removal of one water molecule inside the coor-
dination sphere with weight loss (calcd. = 4.3%, found = 5.0%). The
second peak in the temperature range of 240–350 ◦C corre-
sponds to removal of CH3CH2NH2, SO2 and CO2 molecules with
weight loss (calcd. = 36.5%, found = 37.0%). The third inflection point
2
d–d transition (2B1g → A1g), which support square-planar geome-
try around the metal ion [22]. The bands at 280 and 370 nm can be
assigned to a CT band from filled orbitals Cu(II) to the anti-bonding
␲* orbitals of the ligand [23].
The EPR spectrum of the CuII ␤AS complex was recorded as
a polycrystalline sample at room temperature. The spectrum of
the complex exhibits a single anisotropic broad signal. The anal-
ysis of the spectrum (Fig. 3) gives the g = 2.1212 and g = 2.0811.
||
⊥
These values indicate that the ground state of Cu(II) is predomi-
nately dx2 y2, which supports a square planar structure [24,25]. The
H
observed g value for CuII ␤AS is less than 2.3, thus, indicating
N
NH2
Cu
||
C
that the bonds between the organic ligand and copper ion have
a covalent character more than the ionic one. According to Hath-
N
O
O
S
OH
N
N
away and Billing [26,27], the G = (g − 2)/(g⊥ − 2), which measures
O
||
H
the exchange interaction between the copper centers in a poly-
crystalline solid has been calculated and found to be less than 4.0.
This value indicates to a considerable exchange interaction in solid
HO
Structure 2. Chemical structure of CuII ␤AS complex.

196
A.I. Hanafy et al. / Journal of Molecular Catalysis A: Chemical 355 (2012) 192–200
Table 1
Thermodynamic parameters for CuII ␤AS complex.
Decomposition temperature (K)
E (kJ mol−1
)
ꢁS (J K−1 mol−1
)
ꢁH (kJ mol−1
)
ꢁG (kJ mol−1
)
420–437
510–594
781–897
17.9
17.0
15.3
−290
−321
−345
14.4
12.4
8.1
136.2
189.6
307.9
Table 2
corresponds to C6H5NH2 (aniline) and C4H5N3 (pyridine-2-amine)
with weight loss (calcd. = 44.9%, found = 45%).
The thermodynamic activation parameters of the decomposi-
tion process were evaluated using the well known Coats–Redfern
equation [28] in the form:
Calculated energies of keto–enol tautomeric forms of ␤AS ligand and CuII ␤AS
complex.
Complex
Enol form
Keto form
Method PM3
−179.54
−4204.91
−4244.63
−9.00
−4.407
−3789.97
−3817.58
−9.63
−7.68
−3793.804
−3820.86
−9.68
Heat of formation (kcal/mol)
Total energy (kcal/mol)
Binding energy (kcal/mol)
HOMO (eV)
LUMO (eV)
Dipole (Deby)
ꢀ
ꢁ
− ln(1 − ˛)
E
AR
ˇE
ln
= −
+ ln
(1)
T2
RT
−9.16
−9.44
−9.65
where ˛ is the fraction of decomposition, R is the universal
gas constant, E is the activation energy, A is constant and ˇ
is the heating rate. Therefore, plotting ln[− ln(1 − ˛)/T2] against
1/T according to Eq. (1) gives a straight line whose slope is
directly proportional to the activation energy (−E/R). The activa-
tion entropy ꢁS, the activation enthalpy ꢁH, and the free energy
(Gibbs function ꢁG) were calculated (Table 1) using the following
equations [29]:
5.92
2.61
3.77
k
1
k
cat
CuII ␤AS + S ꢀ (CuII ␤AS) − S→CuII ␤AS + Prod.
(5)
k
−1
kcat [CuII ␤AS][S]
rate =
(6)
(7)
Kꢀ + [S]
ꢂ
ꢃ
1 + (KHꢀ
/[H2O2])
KTꢀ HB
Vmax
Ki(H
O
2
[THB]
Vo
O
)
2
2
2
=
[THB] +
1 +
Vmax
[H2O2]
ꢂ
ꢃ
Ah
kT
ꢁS = 2.303 log
R
(2)
ꢁH = E − RT
(3)
(4)
catalyst for further investigation of Cu-centered oxidation and oxy-
genation chemistry and medical diagnosis for determination of
some hormones [3]. Based on the concept that the attachment
of nanoparticles onto electrodes dramatically enhances the con-
ductivity and electron transfer from the redox analytes [30], this
complex can be used as electrochemical sensor [31] in bioelectro-
catalytic reactions.
ꢁG = ꢁH − TꢁS
where k and h are Boltzmann’s and Planck’s constants respectively,
T is the temperature involved in the calculation and selected as the
peak temperature of DTGA. The entropy ꢁS gives information about
the degree of disorder of the system, the enthalpy ꢁH gives infor-
mation about the total thermal motion and Gibbs or free energy
gives information about the stability of the system.
3.4. Molecular modeling
The CuII ␤AS complex was examined via transmission electron
microscope (TEM). Fig. 5 shows that the average particle size of the
copper(II) complex is in the range of 35–40 nm diameters. The sim-
plicity in the preparation of this Cu(II) complex and its high activity
suggest potential application of this synthetic model as oxidative
In trying to achieve better insight into the molecular structure
of the most preferentially tautomeric ligand forms and complexes,
the conformational analysis of the studied compounds were per-
formed. This was carried out by use of MM+ force field [32,33]
using Hyperchem. 7.5 program [34], (calculations in vacuo, bond
dipole option for electrostatics, Polak–Ribiere algorithm, RMS gra-
dient of 0.1 kcal/Å mol). Furthermore, the geometrical optimization
with semi-empirical (PM3) molecular orbital method was pre-
formed [35]. The computed molecular parameters, total energy,
binding energy, heat of formation, the lowest unoccupied molec-
ular orbital (LUMO) and the highest occupied molecular orbital
(HOMO) energies, and the dipole moment for studied compounds
were calculated (Table 2). It is obvious that there is a possibility of
existence the prepared ligand in both keto–enol forms. Moreover,
the experimental determination of the tautomeric ligand structure
is complicated and often unpredictable. The calculated molecular
parameters have been used to investigate the most stable isomer of
the keto–enol forms of the prepared ligand (␤AS) and showed that
the most stable tautomer is the keto form (Fig. 6). The conjugation
of the ␲-electrons of the carbonyl groups with the ␲-system of the
molecular skeleton probably reduces the energy of the keto form,
which leads to its predominance over the enol one.
The total energy, binding energy, heat of formation, LUMO and
HOMO energies were also, calculated for the copper(II) complex
(Table 2) and indicated that the CuII␤AS in the keto form is more
stable even when compared to the two ligand isomers. The bond
length between carbon and oxygen in CO in case of the keto form
of CuII␤AS was found to be shorter than that in case of COH in enol
form. The bond length and bond angle for the keto–enol forms of the
Fig. 5. TEM images of the CuII ␤AS complex.

A.I. Hanafy et al. / Journal of Molecular Catalysis A: Chemical 355 (2012) 192–200
197
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
0
1000
2000
3000
4000
5000
[ THB mM ]
Fig. 8. Oxidation of THB at 50 ␮M H2O2 (᭹), 100 ␮M H2O2 (ꢁ), 150 ␮M H2O2 (ꢁ),
200 ␮M H2O2
(
), 250 ␮M H2O2 (ꢂ), 300 ␮M H2O2 (ꢃ) using 40 ␮M CuII ␤AS at
25 ◦C.
concentrations which suggest an enzyme-like pre-equilibrium
kinetics. This kinetics can be described as the binding of THB with
the catalyst CuII ␤AS to form an intermediate THB CuII ␤AS com-
plex, followed by conversion of the bound substrate (THB) into
products (Eq. (5)). The rate law for this reaction can be obtained
with steady-state approximation similar to the Michaelis–Menten
kinetics in enzyme catalysis. The rate law for this reaction mecha-
Fig. 6. Ball and stick rendering for the most stable tautomer form of the ligand (keto
form of ␤AS) and complex (from above to below, respectively), as calculated by PM3
semi-empirical molecular orbital calculations.
nism can be expressed as in Eq. (6), wherein Kꢀ = (k + kcat)/k1 is the
ligand together with the most stable complex isomer are calculated
and listed in Tables S1 and S2 (supplementary data).
−1
dissociation constant of the THB CuII ␤AS complex. The reaction
in the presence of saturation amount of H2O2 (200 ␮M) pro-
duces a first-order rate constant kcat = 0.0043 s−1 (t1/2 = 161 s) and
dissociation constant Kꢀ = 13.5 mM. The CuII ␤AS affords a signif-
icant catalytic efficiency kcat/Kꢀ = 0.32 M−1 s−1 as the second order
rate constant. The catalysis shows 1.7 × 103 times rate enhance-
ment in terms of the first-order rate constant (kcat/ko, wherein
ko = 2.53 × 10−6 s−1 is the rate constant for the uncatalyzed reac-
tion “oxidation of 30 mM THB with 200 ␮M H2O2 under the same
reaction conditions”).
4. Oxidation of trihydroxybenzene
Since environmental and economic factors make the use of
harmful oxidants increasingly unacceptable except on a small scale,
hydrogen peroxide is used in the oxidation of polyphenol 1,2,3-
trihydroxybenzene (THB). In this study the CuII ␤AS complex was
used to activate the green oxidant H2O2 in the oxidation of THB
affording an effective catalyst.
The oxidation of the trihydroxybenzene as a function of H2O2
also shows a saturation pattern at high concentrations (Fig. 7,
inset), indicating direct binding of this oxidant to the active metal
center. Therefore, both THB and H2O2 are considered to be sub-
strates. For a bi-substrate binding mechanism, the binding of
two substrates (THB and H2O2) to the active center of CuII ␤AS
can be described in terms of the Hanes equation (Eq. (7)) [36].
In order to study the catalytic activity of the CuII ␤AS
and its interaction with H2O2 toward the oxidation of 1,2,3-
trihydroxybenzene, THB has been used as a substrate to provide
detailed kinetic information. The oxidation rates of THB by
30 ␮M CuII ␤AS at different concentrations of THB (Fig. 7) were
determined in the presence of 200 ␮M H2O2. The rate of THB oxi-
dation is found to be nonlinear, reaching saturation at high THB
6e+6
5e+6
4e+6
3e+6
2e+6
1e+6
0
0.00012
0.00010
0.0012
0.00008
0.0010
0.0008
0.00006
0.0006
0.0004
0.00004
0.0002
0.0000
0.00002
0
100 200 300 400 500 600
[H
-1e+6
-2e+6
O
2
] μM
2
0.00000
0
20
40
60
80
100
-2000
0
2000
4000
[THB] mM
[THB] mM
Fig. 7. Oxidation of THB using 30 ␮M of CuII ␤AS in the presence of 200 ␮M H2O2
at 25 ◦C. Inset shows the oxidation of 30 mM THB in the presence of different con-
centration of H2O2.
Fig. 9. Hanes plot of oxidation of THB using 40 ␮M CuII ␤AS in the presence of 50,
100, 150, 200, 250 and 300 ␮M H2O2 (from top).

198
A.I. Hanafy et al. / Journal of Molecular Catalysis A: Chemical 355 (2012) 192–200
Fig. 10. (a) Replotting the slope obtained from Fig. 9 versus 1/[H2O2]; (b) repotting the y-intercept obtained from Fig. 9 versus 1/[H2O2].
Hanes analysis was used to calculate the apparent and intrin-
sic dissociation constants (Fig. 8). It is important to determine
the rates at varying amounts of H2O2 with a fixed concentra-
tion of THB and vice versa. Fig. 10a and b shows the conversion
cooperative binding). To investigate the existence of free radical in
the reaction mechanism, various concentrations of (CH3)2SO have
been used in the oxidation of 1.0 mM THB in the presence of H2O2
using CuII ␤AS. The free radical scavenger (CH3)2SO [38] did not
inhibit the reaction under the experimental conditions, suggesting
the absence of free radical to induce the oxidation reaction (Fig. 11).
Because the oxidation of catechols is a two-electron transfer
process, the involvement of a dinuclear copper center is thus a pre-
ferred pathway as in the case of the enzyme [39,40]. Based on the
kinetic data described in this study and according to the catalytic
pathways of catechol oxidase, a random bi-substrate mechanism
is proposed (Fig. 12). In the absence of the green oxidant H2O2,
THB is bound to and oxidized by CuII ␤AS complex in form of a
dinuclear CuII-center by inner-sphere 2-electron transfer to yield
of the plot in Fig. 9 to a secondary plot of the slope (1/Vmax
)
and y-intercept (Kapp/Vmax) as a function of 1/[H O2], affording
K(ꢀTHB) = 404.39 mM, Ki(THB) = 560.88 mM, Kꢀ
=262.30 mM and
(H
O )
Ki(H
= 122.05 mM. The Kꢀ/Ki ratio is 0.72 2fo2r THB and 0.51 for
O
)
2
2
H2O2 which indicates that the binding of these two substrates are
not equally exclusive. At the same time it shows the presence of a
small cooperativity [37] among CuII centers for polyphenol oxida-
tion by CuII ␤AS. This cooperativity may be results from dinuclear
catalysis of the 2-electron oxidation of polyphenol. Cooperativity is
a phenomenon displayed by enzymes or receptors that have multi-
ple binding sites. When a substrate binds to one enzymatic subunit,
the rest of the subunits are stimulated and become active (positive
CuI and o-quinone product (steps A and B). The dinuclear cen-
2
ter can be formed from two CuII centers on two different copper
2H+
THB
+2
Cu
OH
OH
O
O
A
O
O
O
H
B
+1
Cu
u
Cu
+2
C
u
+2
C
O
H
+2
O
H
H2O2
Cu
+1
O2, H+
OH
H2O, H+
H2O2
D
C
F
O
O
G
H2O
2e-, 2H+
+ H2O
OH
3H+
+2
Cu
O
+2
Cu
O
O-
O
O
E
O-2
-O
-2O
u
C
u
+2
C
Cu
+2
2H+
THB
+2
Cu
+2
Fig. 11. Proposed mechanism for the oxidation of THB by CuII ␤AS in the absence (A–C and E–F) and presence (steps D–F) of H2O2.

A.I. Hanafy et al. / Journal of Molecular Catalysis A: Chemical 355 (2012) 192–200
199
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
0
100
200
300
400
500
600
[Kojic acid] µM
Fig. 13. Inhibition toward oxidation of THB using 40 ␮M CuII ␤AS by kojic acid in
the presence of 200 ␮M H2O2.
the dinuclear site of peroxide-bound CuII ␤AS without significant
distortion (Fig. 12a), in which the first hydroxy group of catechol
is bound to O center with an electrostatic bond and bound to
His109 with H-bond, while the second hydroxy group of catechol
forms a H-bond with Glu236 and electrostatic bond with His240
with energy (−450 kcal/mol). Also, CuII ␤AS forms H-bond with
(Asn119, His240 and His274) through (Cu atom, nitrogen atom of
NHSO2 and O atom of OSO) respectively (Fig. 12b), with lowest
energy (−570.12 kcal/mol).
In order to investigate the effect of the catalyst concentration on
the oxidation of THB with 200 ␮M H2O2, different concentrations
of the copper complex CuII ␤AS have been used in the oxidation
of 1.0 mM THB. The observed rate was found to be linear till 50 ␮M
of copper complex and then reaches saturation indicating that the
optimum concentration for the copper complex should be around
50 ␮M.
Inhibition of trihdyroxybenzene oxidation by kojic acid. Since kojic
acid is a well known compound for inhibition of polyphenol oxi-
dation by oxidases [46]. It was used to inhibit CuII ␤AS complex
toward oxidation of trihydroxybenzene. Fig. 13 shows that the kojic
acid significantly inhibits the oxidation of THB with IC50 ∼ 65 ␮M.
Fig. 12. Docking of catechol (stick mode) to the dinuclear CuII site reveals a favorable
binding configuration, green dot lines represented H-bonding with the bridging per-
oxide site, red dot lines represented electrostatic bonding with the bridging peroxide
site. Docking of CuII ␤AS (stick mode) to the dinuclear CuII site reveals a favorable
binding configuration, green dot lines represented H-bonding with the bridging per-
oxide site, red dot lines represented electrostatic bonding with the bridging peroxide
site. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of the article.)
5. Conclusion
complexes [15,41]. The inner-sphere electron transfer is followed
by dioxygen binding (C) (whereas the molecular oxygen serves as
a second substrate in this case) to form a dinuclear CuII-peroxo
center or its isoelectronic form [14,16] which can also be formed
upon peroxide binding (D). In the presence of THB and H2O2, the
dinuclear ␮-␩2:␩2-peroxo-Cu2II-THB transition state is eventually
formed by assembling two metal centers together via the bridging
peroxo (steps D and E) as in the case of many mononuclear CuII-
complexes [42,43]. Formation of the transition state is followed by
2-electron transfer from the bound catechol to the bound peroxide
(likely through the metal center) to yield Cu2II-␮-OH and o-quinone
to complete a catalytic cycle (step F). Under the reduction condi-
tions the CuII-peroxo intermediate is expected to undergo through
the pathway G to produce H2O2.
Molecular mechanical calculations have been applied to deter-
mine the structure of CuII ␤AS binding mode using (MOE version
2007). The kinetic studies results are in agreement with a dinuclear
catalysis model, the active oxygenated intermediate is modeled
with a dinuclear center analogous to that of hemocyanin with
a pseudo-C2h symmetry [44,45]. This oxy/peroxy dinuclear site
allows substrate binding to one of the metal ions from the top
or bottom of the Cu O2 Cu plane without distorting the over-
all coordination sphere. A catechol substrate can be docked into
The organic ligand ␤-alanylsulfadiazine (␤AS) was prepared by
reacting sulfadiazine with ␤-alanine amino acid. The ligand was
fully characterized by different techniques. The conformational
analysis showed that the keto form of ␤AS is more stable than the
enol one. The simple copper(II) complex of ␤-alanylsulfadiazine
was fully characterized by means of IR, UV/Vis, elemental anal-
ysis, EPR and thermal analysis. The geometry around the copper
ion is found to be square planar and the average diameter of the
complex is found to be in the range of 35–40 nm. The CuII ␤AS
complex has been used as a catalyst in the oxidation of polyphenol
1,2,3-trihydroxybenzene in the presence of H2O2 as a green oxi-
dant. CuII ␤AS affords a significant catalytic activity toward the
oxidation of THB compared to the uncatalyzed reaction. The oxi-
dation reaction mechanism proposed in this study demonstrated
two-electron transfer process involving dinuclear copper center as
in case of catechol oxidase. The oxidation reaction herein is inhib-
ited by kojic acid.
Acknowledgment
The authors acknowledge Taif University for supporting this
work by Taif University research program (1-430-473).

200
A.I. Hanafy et al. / Journal of Molecular Catalysis A: Chemical 355 (2012) 192–200
Appendix A. Supplementary data
[21] T. Ahamad, N. Nishat, J. Appl. Polym. Sci. 107 (2008) 2280–2288.
[22] J. Ribas, C. Diaz, R. Costa, Y. Journauv, C. Mathoniere, O. Kahn, A. Gleizes, Inorg.
Chem. 29 (1990) 2042–2047.
[23] C. Fraser, B. Bosnich, Inorg. Chem. 33 (1994) 338–346.
[24] T. Ahamad, V. Kumar, S. Parveen, N. Nishat, J. Coord. Chem. 61 (2008)
47–56.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcata.2011.12.016.
[25] N. Nishat, T. Ahamad, S.M. Alshehri, S. Parveen, Eur. J. Med. Chem. 45 (2010)
1287–1294.
References
[26] R.J. Dudley, B.J. Hathaway, J. Chem. Soc. (1970) 1725–1728.
[27] B.J. Hathaway, D.E. Billing, Coord. Chem. Rev. 5 (1970) 143–207.
[28] A.W. Coats, J.P. Redfern, Nature 201 (1964) 68–69.
[29] F. Yakuphanoglu, A.O. Gorgulu, A. Cukurovali, Physica B 353 (2004) 223–229.
[30] A.M. Yu, Z.J. Liang, J.H. Cho, F. Caruso, Nano Lett. 3 (2003) 1203–1207.
[31] Y. Xiao, F. Patolsky, E. Katz, J.F. Hainfeld, I. Willner, Science 299 (2003)
1877–1881.
[32] S. Profeta, N.L. Allinger, J. Am. Chem. Soc. 107 (1985) 1907–1918.
[33] N.L. Allinger, J. Am. Chem. Soc. 99 (1977) 8127–8134.
[34] HyperChem. Version 7.51, Hypercube Inc., FL, USA, 2003.
[35] J.J.P. Stewart, Applicat. J. Comput. Chem. 10 (1989) 221–264.
[36] Leskovac, Comperhensive Enzyme Kinetics, Kluwer/Plenum, Boston, 2002, p.
119.
[1] J. Haggin, Chem. Eng. News 71 (1993) 23–27.
[2] L.I. Simándi, Catalytic Activation of Dioxygen by Metal Complexes, Kluwer,
Dordrecht, 1992.
[3] R. Than, A.A. Feldman, B. Krebs, Coord. Chem. Rev. 182 (1999) 211–241.
[4] F. Zippel, F. Ahlers, R. Werner, W. Haase, H.F. Nolting, B. Krebs, Inorg. Chem. 35
(1996) 3409–3419.
[5] J. Reim, R. Werner, W. Haase, B. Krebs, Chem. Eur. J. 4 (1998) 289–298.
[6] B. Srinivas, N. Arulsamy, P.S. Zacharias, Polyhedron 10 (1991) 731–736.
[7] M.R. Malachowski, M.G. Davidson, Inorg. Chim. Acta 162 (1989) 199–204.
[8] K.D. Karlin, Y. Gultneh, T. Nicholson, J. Zubieta, Inorg. Chem. 24 (1985)
3725–3727.
[9] M.R. Malachowski, M.G. Davidson, J.N. Hoffman, Inorg. Chim. Acta 157 (1989)
91–94.
[10] E.V. Rybak-Akimova, D.H. Busch, P.K. Kahol, N. Pinto, N.W. Alcock, H.J. Clase,
Inorg. Chem. 36 (1997) 510–520.
[37] J.R. Florini, C.S. Vestling, Biochem. Biophys. Acta 25 (1957) 575–578.
[38] P.S. Rao, J.M. Luber, J. Milinowicz, P. Lalezari, H.S. Mueller, Biochem. Biophys.
Res. Commun. 150 (1988) 39–44.
[11] J.F. Larrow, E.N. Jacobsen, Y. Gao, Y.P. Hong, X.Y. Nie, C.M. Zepp, J. Org. Chem.
59 (1994) 1939–1942.
[39] E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Chem. Rev. 96 (1996) 2563–2605.
[40] C. Gerdemann, C. Eicken, B. Krebs, Acc. Chem. Res. 35 (2002) 183–191.
[41] W.B. Tolman, Acc. Chem. Res. 30 (1997) 227–237.
[42] N. Kitajima, T. Koda, Y. Iwata, Y. Morooka, J. Am. Chem. Soc. 112 (1990)
8833–8839.
[43] W.B. Tolman, Acc. Chem. Res. 30 (1997) 227–230.
[44] B. Hazes, K.A. Magnus, C. Bonaventura, J. Bonaventura, Z. Dauter, K.H. Kalk,
W.G.J. Hol, Protein Sci. 2 (1993) 597–619.
[45] K.A. Magnus, B. Hazes, H. Tonthat, C. Bonaventura, J. Bonaventura, W.G.J. Hol,
Proteins – Struct. Funct. Genet. 19 (1994) 302–309.
[46] G. Battaini, E. Monzani, L. Casella, L. Santagostini, R. Pagliarin, J. Biol. Inorg.
Chem. 5 (2000) 262–268.
[12] T. Punniyamurth, S. Velusamy, J. Igbal, Chem. Rev. 105 (2005) 2329–2364.
[13] H. Okawa, H. Sakiyama, Pure Appl. Chem. 67 (1995) 273–280.
[14] L.Q. Hatcher, K.D. Karlin, J. Biol. Inorg. Chem. 9 (2004) 669–683.
[15] E.A. Lewis, W.B. Tolman, Chem. Rev. 104 (2004) 1047–1076.
[16] N. Kitajima, Y. Moro-oka, Chem. Rev. 94 (1994) 737–757.
[17] V. Lykourinou, A.I. Hanafy, G.F.Z. da Silva, K.S. Bisht, R.W. Larsen, B.T. Livingston,
A. Angerhofer, L.J. Ming, Eur. J. Inorg. Chem. (2008) 2584–2592.
[18] E. Wünsch, E. Jaeger, L. Kisfaludy, M. Löw, Angew. Chem. 16 (1977) 317–318.
[19] H.Y. Mostafa, J. Pigment Resin. Res. Technol. 35 (2006) 71–75.
[20] P. Selvam, P. Rathore, S. Karthikumar, K. Velkumar, P. Palanisamy, S. Vijay-
alakhsmi, M. Witvrouw, Ind. J. Pharm. Sci. 71 (2009) 432–436.