A Magnetic Heterogeneous Biocatalyst Composed of Immobilized Laccase and 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) for Green One-Pot Cascade Synthesis of 2-Substituted Benzimidazole and Benzoxazole Derivatives under Mild Reaction Conditions

Article abstract of DOI:10.1002/adsc.201800459

The design of reusable high-performance heterogeneous catalysts via the immobilization of chemical and biochemical species on magnetic nanoparticles increases the efficiency of catalytic systems by facilitating easy, fast, and clean separation processes. Laccase and 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl were separately immobilized on amine functionalized iron (II, III) oxide nanoparticles with covalent bonding using glutaraldehyde as a coupling reagent. The prepared catalyst was used to synthesize 12 benzoxazole and benzimidazole derivatives. The one-pot, two-step enzymatic aerobic oxidation reaction included the condensation of in situ-produced salicylaldehyde derivatives with aromatic amines, followed by an enzymatic dehydrogenation process. Optimal reaction conditions consisted of a citrate buffer (10 mM, pH 4.5) at 40 °C for an incubation time of 10 h and a heterogeneous catalyst containing immobilized laccase (80 mg, 100 U) and immobilized 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) (40 mg, 2 mol%). The catalyst retained more than 85% of its initial activity after 10 runs. In addition to the potential for reuse without significant losses in performance, eco-friendly attributes of this catalytic system include its high catalytic activity and the ease with which it can be recovered from the reaction mixture using an external magnet. (Figure presented.).

Full text of DOI:10.1002/adsc.201800459

Title: A Magnetic Heterogeneous Biocatalyst Composed of Immobilized
Laccase and 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO)
for Green One-Pot Cascade Synthesis of 2-Substituted
Benzimidazole and Benzoxazole Derivatives under Mild Reaction
Conditions
Authors: Mehdi Mogharabi-Manzari, Mahshid Kiani, Sima Arianejad,
Somaye Imanparast, Mohsen Amini, and Mohammad Ali
Faramarzi
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201800459
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10.1002/adsc.201800459
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
A Magnetic Heterogeneous Biocatalyst Composed of
Immobilized Laccase and 2,2,6,6-Tetramethylpiperidine-1-oxyl
(TEMPO) for Green One-Pot Cascade Synthesis of 2-
Substituted Benzimidazole and Benzoxazole Derivatives under
Mild Reaction Conditions
Mehdi Mogharabi-Manzaria,b, Mahshid Kiania, Sima Aryanejada, Somaye Imanparasta,
Mohsen Aminib, and Mohammad Ali Faramarzia*
a
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran
University of Medical Sciences, P.O. Box 14155–6451, Tehran 1417614411, Iran
Fax: (+98)-21-66954712; phone: (+98)-21-66954712; E-mail: faramarz@tums.ac.ir
Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 1417614411, Iran
b
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######
Abstract. The design of reusable high-performance Optimal reaction conditions consisted of a citrate buffer (10
heterogeneous catalysts via the immobilization of chemical mM, pH 4.5) at 40 C for an incubation time of 10 h and a
and biochemical species on magnetic nanoparticles increases heterogeneous catalyst containing immobilized laccase (80
the efficiency of catalytic systems by facilitating easy, fast, mg, 100 U) and immobilized 2,2,6,6-tetramethylpiperidine-
and clean separation processes. Laccase and 4-amino- 1-oxyl (TEMPO) (40 mg, 2 mol%). The catalyst retained
2,2,6,6-tetramethylpiperidine-1-oxyl
were
separately more than 85% of its initial activity after 10 runs. In
immobilized on amine functionalized iron (II, III) oxide addition to the potential for reuse without significant losses
nanoparticles with covalent bonding using glutaraldehyde as in performance, eco-friendly attributes of this catalytic
a coupling reagent. The prepared catalyst was used to system include its high catalytic activity and the ease with
synthesize 12 benzoxazole and benzimidazole derivatives. which it can be recovered from the reaction mixture using
The one-pot, two-step enzymatic aerobic oxidation reaction an external magnet.
included
the
condensation of
in
situ-produced
salicylaldehyde derivatives with aromatic amines, followed
by an enzymatic dehydrogenation process.
Keywords: Biocatalysis; Laccase; Magnetic nanoparticle;
Benzimidazole; Benzoxazole
application of enzymes in organic reactions is often
hampered by a lack of long-term stability under
process conditions and additional complications in
catalyst recovery and recycling.[4] Nevertheless, these
disadvantages can be overcome by performing
enzyme immobilization in a heterogeneous system to
enhance stability, facilitate easy separation from the
reaction mixture, and prevent protein contamination
of the product.[5] Inorganic materials, such as metal
oxides, minerals, and carbon-based nanomaterials,
have traditionally been used as support materials for
the immobilization of enzymes. These carriers are
remarkable due to their unique characteristics,
including good stability under harsh reaction
conditions and relatively simple synthesis procedures.
Consequentially, they are also readily available at
low costs.[5d]
Introduction
Homogeneous biocatalytic systems are widely used
in organic synthesis, and the growing demand for
green organic synthesis methods provides an
opportunity for the broad application of eco-friendly,
economical, and effective cascade processes.[1] In
addition, environmentally benign biocatalytic
processes are generally conducted under mild
conditions in terms of temperature and pH, allowing
for the integration of multiple transformations.[2]
However, the complicated nature of the processes
required to separate the catalyst from the desired
reaction products includes inherent drawbacks that
limit the use of biocatalysts in the food and
pharmaceutical industries, both of which require
exceptionally pure products.[3] The inability to reuse
enzymes, particularly those obtained via difficult and
costly expression, isolation, and purification
processes, can pose significant monetary and
With its potential to produce biologically active
compounds and pharmaceuticals via organic
synthesis, biocatalysis has thus emerged as one of the
best solutions to achieve green chemistry. Indeed, the
replacement of homogeneous biocatalytic processes
environmental
disadvantages.[3b]
Further, the
1
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10.1002/adsc.201800459
Advanced Synthesis & Catalysis
with efficient heterogeneous approaches is an
attractive strategy now under active investigation in
the field of catalysis studies.[6]
dicyano-l,4-benzoquinone
diacetoxyiodobenzene
(DDQ),
(PhI-(OAc)2),
pyridiniumchlorochromate (PCC), and BaMnO4.[15]
Green cascade reactions with oxygen as the oxidant
have also been used for aerobic catalytic synthesis of
benzoxazoles and benzoxazoles. Benzimidazol
derivatives, for example, have been synthesized via a
green approach using immobilized zinc oxide on the
surface of magnetic nanoparticles.[15b] Further,
ultrasound irradiation, in addition to being a green
process, alternative energy source, and convenient
procedure, has been investigated in the
environmentally benign synthesis of novel
benzoxazoles. This process offers such advantages as
reduced reaction time, mild reaction conditions, high
productivity and yield, ease of execution, and
economic viability.[15c]
Heterogeneous biocatalysts are prepared by
physical and chemical enzyme immobilization
techniques, which can include encapsulation,
crosslinking, and bonding to a carrier. Both porous
and non-porous supports are useful for enzyme
immobilization, with several reviews reporting
benefits from using a number of solid materials in
this process.[7] Magnetic nanoparticles (e.g., Fe3O4)
are used extensively for the immobilization of
enzymes because they show low toxicity and good
biocompatibility, have a large specific surface area,
and are easily separated from a reaction mixture via
the application of an external magnetic field.[8] These
magnetic nanoparticles can thus be readily
functionalized with appropriate surface modifications
that make them suitable for enzyme immobilization
via covalent attachment.[9]
In the present study, laccase and TEMPO were
separately immobilized on magnetic Fe3O4
nanoparticles and used in cascade enzymatic
oxidation of salicyl alcohols. This process was
followed by condensation of aromatic aldehydes with
o-phenylenediamine and o-aminophenol and further
biocatalytic aerobic dehydrogenation under mild
reaction conditions. Reaction conditions were
optimized: namely, pH, temperature, incubation time,
amount of each component, and choice of organic
solvents.
Laccases are one of the most important enzymes
applied in organic synthesis. They catalyze the
oxidation of a variety of organic compounds,
including methoxyphenols, ortho- and para-
diphenols, aminophenols, polyamines, and lignin
derivatives. Such oxidation is achieved via four-
electron reduction of molecular oxygen to water, with
molecular oxygen serving as an electron acceptor.
This results in concomitant one-electron oxidation of
a substrate.[10] The blue multi-copper oxidases,
another type of multi-copper oxidase, can also
catalyze the aerobic oxidation of a wide range of
substrates, leading to transformations of functional
groups and coupling reactions (e.g., dimerization,
polymerization, cyclization, amination).[11] The
laccase substrate range can be widened to include
selective oxidation of primary aromatic alcohols in
systems by the utilization of the commercially
Results and Discussion
Magnetic nanoparticle-supported catalytic systems
have emerged as promising catalysts that can be
easily separated from a reaction medium with the
simple application of an external permanent magnet.
In the present study, Fe3O4 nanoparticles of 10.7 nm
average diameter were prepared by co-precipitation
from FeCl3/FeCl2 solution with NH4OH, as
previously reported by Berger et al.[16] The
nanoparticles were characterized by scanning electron
microscopy (SEM), transmission electron microscopy
(TEM), X-ray diffraction (XRD), and Fourier
transform infrared (FT-IR) spectroscopy. Magnetic
measurements were taken by vibrating sample
magnetometry (VSM). The surface morphology and
size of the magnetic nanoparticles were determined
by SEM and TEM (Figure 1a-b). X-ray powder
diffraction of the prepared particles revealed a series
of characteristic peaks that agreed with the inverse
cubic spinel phase of Fe3O4 (Figure 1c). The obtained
results were similar to those reported in previous
literature.[17] Measurements of magnetic-field
dependence of magnetization of magnetic
nanoparticles at 300 K are presented in Figure 1d.
Superparamagnetic behavior is primarily generated
by a magnetic core. The saturation magnetization
available
redox
mediator
2,2,6,6-
tetramethylpiperidinyl-1-oxyl (TEMPO), which acts
as an electron shuttle to promote the laccase-
catalyzed oxidation of non-phenolic compounds.[12]
The oxidation of TEMPO produces a corresponding
oxoammonium cation that is capable of direct and
selective oxidation of primary alcohols, yielding
corresponding aldehydes.
Benzimidazole and benzoxazole derivatives are
five-member heterocyclic ring scaffolds found in a
range of biologically active and therapeutically
important natural and pharmaceutical products.[13]
These compounds have been extensively studied for
their biological and therapeutic properties, which
include anticancer, antifungal, antihistaminic,
antiviral, antiulcer, cathepsin S inhibitor, HIV reverse
transcriptase inhibitor, and orexin-1 receptor
antagonist activities.[14] Previous studies have
examined several synthetic methods for constructing
substituted benzimidazoles and benzoxazoles, with
the most common method being the heterocyclization
of o-diaminoarenes and o-aminophenoles with
aldehydes under acidic conditions and subsequent
oxidation with oxidants, such as 2,3-dichloro-5,6-
determined here indicated
a
saturation of
approximately 12 emu g-1 for Fe3O4 nanoparticles.
2
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10.1002/adsc.201800459
Advanced Synthesis & Catalysis
compounds by oxygen at room temperature. A time
course study of production 3a was performed using
immobilized TEMPO and immobilized laccase at
ratios of 1:1, 1:2, 1:3, 1:4, and 1:5. The optimal
amount of catalyst was observed at the ratio of 1:2,
which contained immobilized TEMPO (40 mg, 2
mol%) and immobilized laccase (80 mg, 100 U) after
10 h (Figure 2a-b).
The proposed mechanism for the conversion of
salicyl alcohol to a corresponding aldehyde, followed
by aerobic oxidative dehydrogenation, is shown in
Scheme 2. Laccase and TEMPO undergo oxidation to
form an oxoammonium ion. The oxidized TEMPO
then oxidizes the alcohol via simultaneous reduction
to hydroxyl amine. Oxoammonium cations as a
relatively strong oxidant are produced by the one-
electron oxidation of TEMPO. An oxoammonium
cation could be generated in situ by
disproportionation of TEMPO under acidic
conditions. Disproportionation takes place when
strongly acidic pH produces an oxoammonium cation
and TEMPOH. A reverse reaction occurs at pH ˃ 3:
TEMPOH undergoes one-electron oxidation by an
oxoammonium cation, affording two molecules of
TEMPO.[21a] Arends et al.[21b] studied laccase-
catalyzed reoxidation of TEMPOH to TEMPO and
proved that laccase was able to catalyze the
reoxidation of the reduced mediator. Optimal reaction
conditions for the synthesis of product 3a were also
investigated in the present study. Increasing the
temperature increased the yield of 2(2-
hydroxyphenyl)benzimidazole, with the maximum
yield achieved at 40 C. An acidic pH was more
suitable for cascade synthesis of benzimidazole (3a),
and a pH of 4.5 was found to be optimal (Figure 3a).
Figure 1. a) Scanning electron microscopy, b)
transmission electron microscopy images, c) X-ray
diffraction pattern, and d) vibrating sample magnetometry
of Fe3O4.
The surfaces of these magnetic nanoparticles were
functionalized using 3-aminopropyltriethoxysilane.
The infrared spectrum of the magnetic nanoparticles
showed peaks characteristic of a functionalized
TEMPO group, which clearly differed from the
spectra of the nonfunctionalized silica-coated Fe3O4
and the aminopropyl functionalized magnetic
nanoparticles. Laccase was immobilized on magnetic
mesoporous silica spheres by physical covalent
attachment methods. The immobilized laccase
retained its activity and exhibited greater resistance to
pH changes and thermal stability when compared to
the free enzyme. The kinetic parameters of the
immobilized laccase were also improved. The
oxidation of 2(2-hydroxyphenyl)benzimidazole (3)
using immobilized laccase and immobilized TEMPO
as the model reaction was then investigated as a
means of achieving environmentally friendly catalytic
synthesis of biologically important heterocyclic
compounds. Initially, the reaction was carried out in
the presence of laccase at room temperature under O2
in a citrate buffer (10 mM, pH 4.5). No reaction took
place, even after stirring the reaction mixture for 24 h.
Appropriate control reactions were performed to
investigate the probable catalytic activity of Fe3O4
nanoparticles. Fe3O4, SiO2-Fe3O4, and 4-amino-
TEMPO-Fe3O4 were separately used as catalysts. The
obtained results showed no significant catalytic
activity of magnetic nanoparticles in the reaction
conditions. Oxidation reactions using Fe3O4 as a
catalyst usually occurred in the presence of an
oxidizing agent, such as hydrogen peroxide[18] and
potassium carbonate.[19]
H2N
OH
Heterogeneous catalyst
N
+
Citrate buffer (10 mM, pH 4.5)
40 oC, 10 h
OH
N
H
H2N
1a
2a
3a
OH
Scheme 1. Preparation of 2-hydroxybenzimidazole (3a) by
heterogeneous laccase-catalyzed aerobic oxidation of 1a,
followed by an oxidative cyclization reaction and further
biocatalytic aerobic dehydrogenation. The catalyst
contained immobilized laccase (80 mg, 100 U) and
immobilized TEMPO (40 mg, 2 mol%).
Tromp et al.[21c] reported that the optimal
conversion conditions for primary alcohols (pH 4-5,
30 C) appeared to be strongly determined by the
general optimum acidic pH of laccases. However, in
the present study, the reaction between
oxoammonium and primary alcohols was slow at
such low pH values.[21c] The prepared catalyst was
recovered and washed with water to remove the
substrate and products for subsequent investigation of
the reusability of the catalytic system. As shown in
Figure 3b, the catalyst retained about 85% of its
initial activity after 10 runs, indicating that the
Synthesis of 2-hydroxybenzimidazole (3a) was
performed using heterogeneous laccase-catalyzed
aerobic oxidation of (1a), followed by oxidative
cyclization reaction and further biocatalytic aerobic
dehydrogenation (Scheme 1). Fabbrini et al.[20]
reported efficient oxidation of alcohols to carbonyl
3
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10.1002/adsc.201800459
Advanced Synthesis & Catalysis
heterogeneous catalyst had an appropriate stability
and reusability. The efficiency of the heterogeneous
catalyst separation process depends on a strong metal
as well as support interaction enhanced by
functionalizing the support surface with amino
groups.[22]
arylamine) exists in the reaction medium. In the
present study, three components existed in the
reaction: salicyl alcohol, aryl amine, and TEMPO as
redox mediator. Although laccase is able to oxidize
both TEMPO and aromatic amine, oxidation of
TEMPO (495 mV)[23] is preferable to oxidation of o-
phenylenediamine (650 mV)[24]
.
Therefore, o-
phenylenediamine acted as a nucleophile in this
reaction as a nucleophilic addition reaction is more
probable than dimerization in the presence of in situ-
generated aldehyde. Meanwhile, the yield of major
product ranged from 65-88%; 12-35% imines, or
dimer byproducts, were also produced. Arylamines
are one of the most applied nucleophiles in the
synthesis of nitrogen-containing organic compounds
catalyzed by laccase via an enzyme-catalyzed
oxidation reaction.[25] Hahn et al.[26] applied aniline
derivatives as nucleophiles in the synthesis of several
amine derivatives of benzoquinones using laccase of
Myceliophthora thermophila. Wellington et al.[27]
reported laccase-catalyzed nuclear diamination of p-
hydrobenzoquinones with arylamines, including
aniline,
4-isopropyleaniline,
4-aminophenethyl
alcohol, and 2-naphthylamine. An oxidative cascade
reaction was also carried out in the presence of
organic solvents in the present study. In a mixture of
citrate buffer and organic solvents (1:10, v/v), the
reaction proceeded with moderate yields, in the range
of 15-65%; the citrate buffer (10 mM, pH 4.5) was
thus deemed a suitable medium (Table 2). The results
from a Bradford assay also showed maximum
enzymatic loading was 10 mg of enzyme per 80 mg
of solid support. HPLC analysis of the washing
solution containing immobilized TEMPO indicated
that 3.4 mg of TEMPO were attached to every 40 mg
of solid support.
Colorcoded dot maps of copper and sulfur atoms
showed uniform distribution of laccase on the surface
of magnetic nanoparticles (Figure 4). The catalytic
efficiency of laccase was determined by the
Michaelis constant Km and Vmax. Kinetic parameters
for free and immobilized laccase were determined
using ABTS (0.1-10 mM) as a substrate and plotting
data to a double reciprocal Michaelis-Menten and
Lineweaver-Burk plot (Figure S1). The apparent Km
value of the immobilized laccase was 1.5 times
higher than that of the free laccase (Table 3). The
calculated Vmax of free laccase was 61 mM min-1,
while the corresponding value for the immobilized
laccase was 40 mM min-1. Laccase immobilized on
magnetic nanoparticles maintained its activity during
a 30-day storage period at 4 C. The result
demonstrated that immobilized laccase showed better
storage stability than free laccase at all three selected
temperatures (Figure 5). The free laccase was
deactivated after one and six days incubation at 40
and 25 C, respectively. The immobilized laccase
retained more than 80% of its initial activity at 4 C
after 30 days. Some studies have reported
cytotoxicity and mutagenicity in mammalian cells as
Figure 2. a) Time course study of product 3a by
ratios of each component of the catalyst, b)
optimization of the amount of immobilized laccase
and immobilized TEMPO.
In this study, the catalyst support contained a core
of magnetite that enabled its magnetic separation
from liquid systems. The magnetic nanoparticle
catalyst, which was readily prepared from
inexpensive starting materials, catalyzed the green
synthesis of benzimidazole and benzoxazole
derivatives under mild reaction conditions. The
efficiency of the prepared catalyst was studied for a
series of substituted salicyl alcohols with electron
donating and electron withdrawing substituents of o-
phenylenediamine and o-aminophenol under optimal
reaction conditions (Table 1). Aldehydes with
electron-withdrawing groups achieved relatively
higher yields when compared to aldehydes bearing
electron-donating
groups.
Laccase-catalyzed
a
result
of
TEMPO.
dimerization and polymerization reactions occur as
the main reaction only when one substrate (e.g.,
4
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10.1002/adsc.201800459
Advanced Synthesis & Catalysis
N
O
N
O
Laccase
Laccase
Laccase
Laccase
D
D
C
E
C
E
N
N
N
N
O
OH
O
OH
OH
NH
N
N
OH
N
H
O
O
O
O
1a
H
N
N
HO
OH
H
F
N
H
OH
B
N
H
H2N
H2N
A
HO
G
3a
2a
Scheme 2. Plausible mechanism for laccase/TEMPO catalyzed aerobic oxidation of salicyl alcohol (1a), followed by an
oxidative cyclization reaction and further biocatalytic aerobic dehydrogenation of intermediate F to produce 2-
hydroxybenzimidazole (3a).
N
R4
H2
N
OH
R4
Heterogeneous catalyst
+
Y
Citrate buffer
X
R3
R1
(10 mM, pH 4.5)
R5
X
R3
R1
R2
R2
Table 1. Synthesis of benzimidazole and benzoxazole
derivatives by the catalyst.
Entry
R1
R2
R3
R4
X
R5
Y
Time
(h)
12
21
20
10
10
18
19
19
16
15
13
Yield
(%)(a)
83
73
69
86
88
81
69
65
76
82
79
1
2
3
4
5
6
7
8
9
10
11
12
(a)
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
-
H
OMe
H
H
H
H
OMe
H
H
H
H
H
OMe
H
H
H
H
OMe
H
H
H
H
H
H
C
C
C
C
C
C
C
C
C
C
N
N
NH2
NH2
NH2
NH2
NH2
OH
OH
OH
OH
OH
N
N
N
N
N
O
O
O
O
O
N
O
Br
NO2
H
H
H
Br
NO2
H
H
H
NH2
OH
-
H
H
14
73
Reaction conditions: citrate buffer (10 mM, pH 4.5), 40
C, and a heterogeneous catalyst containing immobilized
laccase (80 mg, 100 U) and immobilized TEMPO (40 mg,
2 mol%). The yields were determined by HPLC.
However, the present study developed a green
approach for the synthesis of organic compounds in
which TEMPO was immobilized and reused to
reduce the environmental hazards associated with its
release into wastewater systems. Guo et al.[28]
investigated the cytotoxicity and genotoxicity of
TEMPO in mammalian cells using mouse lymphoma
and in vitro micronucleus assay. The obtained results
showed that TEMPO induced reactive oxygen species
and decreased glutathione levels in mouse lymphoma
cells.
Figure 3. a) optimization of laccase-catalyzed reaction
conditions (pH-temperature) for production of 3a, and d)
reusability of the catalyst in the aerobic enzymatic
oxidation of salicyl alcohol (1a) to salicylaldehyde (B)
(light gray) and synthesis of benzimidazole (3a) (dark
gray).
5
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10.1002/adsc.201800459
Advanced Synthesis & Catalysis
Table 2. Synthesis of benzimidazole (3a) in the presence
of organic solvent (1:10, v/v).
Entry
Solvent
log P
Time
(h)
10
8
Yield
(%)(a)
90
1
2
Water
Citrate buffer (10 mM, pH 4.5)
DMSO
-
-
95
3
-1.3
-1.1
-0.76
-0.23
0.68
1.3
1.8
2.0
2.5
2.9
3.5
4.0
4.5
15
15
15
15
15
12
12
12
10
10
10
10
10
65
4
Dioxane
68
5
Methanol
15
6
Acetone
15
7
Ethyl acetate
Hexanone
Hexanol
21
8
17
9
20
10
11
12
13
14
15
Chloroform
Toluene
23
50
Octanol
64
Hexane
68
Figure 5. Stability of the free and immobilized laccase at 4, 25,
and 40 C for 30 days.
Heptane
65
Octane
65
(a) Reaction conditions: 40 C and a heterogeneous catalyst
containing immobilized laccase (80 mg, 100 U) and
immobilized TEMPO (40 mg, 2 mol%). The yields were
determined by HPLC.
Conclusion
A heterogeneous catalyst was prepared by separately
immobilizing laccase and TEMPO on magnetic
nanoparticles for the catalysis of one-pot, two-step
enzymatic synthesis of benzimidazole and
benzoxazole derivatives. Immobilization of the
catalytic species enhanced catalyst stability and
simplified the catalyst separation process. This green
cascade procedure presented some attractive eco-
friendly characteristics, such as the simplicity of
catalyst recovery from the reaction mixture using an
external magnet, the use of molecular oxygen as a
non-toxic oxidant, and the production of water as the
only byproduct. Heterogeneous bio-catalysts can
therefore improve the performance of the catalytic
system in green organic syntheses, particularly in the
presence of organic solvents.
Experimental Section
Materials and instruments
Laccase from Trametes versicolor ( 10 U mg-1) and 4-
amino-TEMPO were purchased from Sigma-Aldrich (St.
Louis, MO, USA). All chemicals and solvents were
obtained commercially and used without further
purification. The 1H and 13C NMR spectra were recorded
on a Bruker DRX-500 Avance spectrometer at 500 and 125
MHz, respectively. The chemical shifts are given in 
relative to tetramethylsilane (TMS), and the coupling
constant (J) values are given in Hz. The splitting patterns
are designated as s (singlet), d (doublet), t (triplet), and m
(multiplet). Fourier Transform Infrared (FT-IR) spectra
Figure 4. a) Scanning electron microscopy image of the
catalyst, b) color coded dot maps of copper, and c) sulfur
atoms.
Table 3. Kinetic parameters of free and immobilized
laccase.
were recorded with
a
Shimadzu FTIR-8400S
spectrophotometer. Mass spectra were obtained using a
Shimadzu QP2010-PLUS instrument operating at 70 eV.
The yields of benzoxazole and benzimidazole derivatives
Entry
The Enzyme
Free laccase
Km
1.5
2.3
Vmax
61
1
2
Immobilized laccase
40
were determined by
a
high performance liquid
chromatography (HPLC) system equipped with a PDA
Detector 2800, a Smartline Pump 1000, and a degasser
5000, all from Knauer (Berlin, Germany). Samples were
filtered through 0.45 µm PTFE filters (Schleicher & Schull,
Germany) and injected (20 µL) by
Autosampler 3950 with a sample loop of 100 µL. The data
a
Smartline
6
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10.1002/adsc.201800459
Advanced Synthesis & Catalysis
were processed using ChromGate software (version 3.3.1)
from Knauer (Berlin, Germany). Chromatographic
separation was achieved on a Lichrospher 100 RP & C18
reverse phase column (C18, 250 × 4.6 cm id, 5 µm particle
size) from Knauer (Berlin, Germany). Isocratic mobile
phase was methanol/water at a ratio of 30:70. The retention
times were varied from 6.2 to 9.8 min.
30 °C. Absorption wavelength and the retention time for 4-
amino-TEMPO were 280 nm and 7.3 min, respectively.[16]
The leaching of TEMPO was investigated by incubation of
the immobilized TEMPO (40 mg) in citrate buffer (10 mM,
pH 4.5) at 40 C. After 10 h the suspension was
centrifuged and the supernatant was investigated for
detection of TEMPO using HPLC and the obtained results
showed no quantifiable amount of TEMPO.[31]
Laccase assay
Immobilization of laccase
The oxidative activity of laccase was assayed using ABTS
by the method previously reported by Forootanfar et al. [29]
In brief, enzyme (0.5 mL) was added to 0.5 mL freshly
prepared enzyme substrate (5 mM ABTS in 100 mM
citrate buffer pH 5) and was incubated at 37 °C and 120
rpm for 10 min. The absorbance was then recorded at 420
nm (ε420 = 36.000 M−1 cm−1) with a double beam PC
scanning UV–vis spectrophotometer (UVD 2950,
Labomed, Culver City, USA). One unit of laccase activity
was defined as the amount of enzyme that oxidizes 1 μmol
of ABTS per min.
Citrate buffer (0.1 M, pH 4.5) containing the magnetic
particle carriers was mixed with 1.6% glutaraldehyde at
25 °C for 3 h. The carriers were then washed with buffer
solution and distilled water. The glutaraldehyde-treated
particles (0.16 g) and laccase (20 mg) were dispersed in
citrate buffer and the mixture was incubated in a shaker at
150 rpm for 12 h at 25 °C. The particles were collected by
magnetic separation and washed with citrate buffer and
water to remove non-specifically bound enzyme. The
enzyme-immobilized particles were dispersed in water and
stored at 4 °C. A colorimetric procedure was used to
determine the enzyme activity. The uniform distribution of
sulfur and copper atoms of laccase was investigated by
scanning electron microscopy (SEM) image and color
coded dot maps.
Preparation of magnetic nanoparticles
Magnetic Fe3O4 nanoparticles were synthesized by
ultrasound-assisted reverse co-precipitation, as reported by
Berger et al.[16] A solution of FeCl2 (5.4 g) and FeCl3 (2 g)
in aqueous hydrochloride acid (2 M, 25 mL) was sonicated
at room temperature until the salts dissolved. Aqueous
ammonia (25%, 40 mL) was added slowly over 20 min to
the mixture under an argon atmosphere at room
temperature, followed by stirring for about 30 min with a
mechanical stirrer. The Fe3O4 nanoparticles were separated
with an external magnet and washed three times with
deionized water and three times with ethanol.
General procedure for synthesis of 2-substituted
benzoxazoles and benzimidazoles
A mixture was prepared in a two-necked flask at 40 °C by
combining salicyl alcohol (1 mmol), an o-phenylendiamine
(1 mmol), and the catalyst bearing immobilized laccase (80
mg, 100 U) and immobilized TEMPO (40 mg, 2 mol%) in
citrate buffer (0.1 M, pH 4.5). The resulting mixture was
stirred at 40 °C under air. The progress of the reaction was
monitored by TLC. The catalyst was separated with an
external magnet and the solution was dried with sodium
sulfate. The excess solvent was removed under reduced
pressure to give the corresponding products (Table 1). The
yields were determined by HPLC. The magnetic
heterogeneous biocatalyst was separated with a magnet,
washed with water, and used in the next oxidation process.
Three mixtures were separately prepared in two-necked
flasks at 40 °C by combining salicyl alcohol (1 mmol) and
o-phenylendiamine (1 mmol) in citrate buffer (0.1 M, pH
4.5). In each mixture, 2 mol% Fe3O4, SiO2-Fe3O4, and 4-
amino-TEMPO-Fe3O4 were separately added as catalysts.
The resulting mixture was stirred at 40 °C under air. The
progress of the reaction was monitored by TLC. TLC
analysis revealed that no reaction was took place. In
addition, separate control reactions were performed to
investigate the oxidation of alcohol and amine. No
detectable product was monitored by TLC.
Synthesis of silica-coated magnetite nanoparticles
The synthesized Fe3O4 nanoparticles were suspended in 35
mL ethanol and 6 mL deionized water and sonicated for 15
min. Tetraethyl orthosilicate (TEOS; 1.5 mL) was slowly
added to the mixture and sonicated for 10 min. Aqueous
ammonia (10%, 1.4 mL) was then slowly added over 10
min with mechanical stirring. The mixture was then heated
at 40 °C for 12 h. The silica-coated iron oxide
nanoparticles were separated with an external magnet,
washed three times with ethanol, and dried under
vacuum.[30]
Amination of the silica-coated magnetite nanoparticles
Dry silica-coated magnetite nanoparticle powder (10 g)
was mixed with 200 mL of toluene to produce a
homogeneously mixed solution, followed by sonication of
the mixture for 30 min. After addition of (3-
aminopropyl)triethoxysilane (APTES) (2.5 mL) with
mechanical stirring, the mixture was slowly heated to
105 °C and kept at this temperature for 20 h. The final
sample was separated with an external magnet, washed
three times with ethanol, and dried under vacuum.
Stability of the free and immobilized laccase
Thermal stability of the free and immobilized laccase was
evaluated by measuring the residual activity of the enzyme
that exposed to three different temperatures (4, 25, and 40
C) in acetate buffer (100 mM, pH 4.5) for 30 days. The
residual activity was defined as the fraction of the total
activity recovered after the immobilization of laccase on
the magnetic nanoparticles compared with the same
quantity of the free enzyme. An enzyme sample was
removed from each incubation solution with a subsequent
time interval and then assayed for enzymatic activity.
Immobilization of TEMPO on magnetite nanoparticles
4-Amino-TEMPO (1.5 mmol, 0.256) was added to a
mixture of aminated magnetic nanoparticles (0.3 g)
suspended in CH3OH (100 mL). NaBH4 (0.075 g, 2 mmol)
was divided into three portions: the first portion was added
after 3 h, the second after 12 h, and the third after 24 h.
After the final addition, the reaction mixture was
vigorously stirred at ambient temperature for a further 24 h.
The final product was separated with an external magnet,
washed three times with ethanol, and dried under vacuum.
Washing solution containing immobilized TEMPO was
investigated by HPLC analysis and it was found that 3.8
mg of TEMPO was attached per gram of solid support. In
this study, isocratic mobile phase methanol/water was used
at a ratio of 98:2 and the flow rate was 1.0 mL min-1 at
Determination of kinetic parameters for free and
immobilized laccase
The kinetic parameters, Michaelis constant (Km) and
maximum reaction velocity (Vmax), were calculated using
Lineweavere-Burk Plots plot from the intercepts at x and y
axes, respectively, by measuring initial rates of the reaction
with varying concentration of ABTS ranging 0.1–10 mM
in citrate buffer (100 mM, pH 4.5) at 45 C. The reaction
7
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10.1002/adsc.201800459
Advanced Synthesis & Catalysis
was shaked stirred for 5 min. Calculation of Km and Vmax
values were achieved by fitting the data to the Lineweaver-
Burk plot, resulting in the Michaelis-Menten curve by
plotting the obtained initial velocity against ABTS
concentrations using free laccase (0.1 mg) and
immobilized laccase (1 mg).
Bagherzadeh, M. A. Faramarzi, ChemCatChem 2018,
10, 1542-1546; d) S. Riva, Trends Biotechnol. 2006, 24,
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Catal. 2009, 351, 1187-1209.
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28, 694-705; b) L. Munk, A. M. Punt, M. A. Kabel, A.
S. Meyer, RSC Adv. 2017, 7, 3358-3368.
Acknowledgement
[13] J. Azizian, P. Torabi, J. Noei, Tetrahedron Lett. 2016,
57, 185-188.
This work was supported financially by the grant No. 97-01-90-
37228 from Biotechnology Research Center, Tehran University of
Medical Sciences, Tehran, Iran to M.A.F.
[14] a) X. -J. Fang, P. Jeyakkumar, S. R. Avula, Q. Zhou,
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2588; b) S. A. Galal, A. S. Abdelsamie, S. A. Shouman,
Y. M. Attia, H. I. Ali, A. Tabll, R. El-Shenawy, Y. S. E.
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10.1002/adsc.201800459
Advanced Synthesis & Catalysis
FULL PAPER
A Magnetic Heterogeneous Biocatalyst Composed
of Immobilized Laccase and 2,2,6,6-
Tetramethylpiperidine-1-oxyl (TEMPO) for Green
One-Pot Cascade Synthesis of 2-Substituted
Benzimidazole and Benzoxazole Derivatives under
Mild Reaction Conditions
Adv. Synth. Catal. Year, Volume, Page – Page
Mehdi Mogharabi-Manzari, Mahshid Kiani, Sima
Aryanejad, Somaye Imanparast, Mohsen Amini,
and Mohammad Ali Faramarzi
10
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