A Laccase Heterogeneous Magnetic Fibrous Silica-Based Biocatalyst for Green and One-Pot Cascade Synthesis of Chromene Derivatives

Article abstract of DOI:10.1002/ejoc.201801784

Green cascade approaches, which utilize sustainable and recyclable heterogeneous biocatalysts, can be used in the catalysis of multicomponent organic reactions to synthesize biologically important substances. Three magnetic nanoparticles, iron (II, III) oxide, cobalt ferrite, and nickel ferrite, were prepared by co-precipitation and functionalized with fibrous silica (KCC-1), and characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and vibrating sample magnetometry (VSM). Laccase immobilized on CoFe₂O₄-KCC-1 was used as a heterogeneous biocatalyst for green and one-pot cascade synthesis of 2-amino-5-oxo-4-aryl-4H,5H-pyrano[3,2-c]chromene-3-carbonitriles. The reaction was performed in the presence of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) as a synthetic redox mediator. The optimum reaction conditions were found to be as immobilized laccase (100 mg, 95 U) and TEMPO (2 mol-%) in a 100 mm sodium citrate buffer (pH 4.5), 40 °C, and incubation time 17 h. About 80 % of initial activity of the immobilized laccase was retained after 15 independent runs.

Full text of DOI:10.1002/ejoc.201801784

A Journal of
Accepted Article
Title: A laccase heterogeneous magnetic fibrous silica-based
biocatalyst for green and one-pot cascade synthesis of chromene
derivatives
Authors: Mehdi Mogharabi-Manzari, Mohammad Hossein Ghahremani,
Tabassom Sedaghat, Fatemeh Shayan, and Mohammad Ali
Faramarzi
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201801784
Link to VoR: http://dx.doi.org/10.1002/ejoc.201801784
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10.1002/ejoc.201801784
European Journal of Organic Chemistry
FULL PAPER
A laccase heterogeneous magnetic fibrous silica-based
biocatalyst for green and one-pot cascade synthesis of chromene
derivatives
Mehdi Mogharabi-Manzari,[a,b] Mohammad Hossein Ghahremani,[c] Tabassom Sedaghat,[a] Fatemeh
Shayan,[a] and Mohammad Ali Faramarzi*[a]
Abstract: Green cascade approaches, which utilize sustainable and
recyclable heterogeneous biocatalysts, can be used in the catalysis
of multicomponent organic reactions to synthesize biologically
important substances. Three magnetic nanoparticles, iron (II, III)
oxide, cobalt ferrite, and nickel ferrite, were prepared by co-
precipitation and functionalized with fibrous silica (KCC-1), and
characterized by scanning electron microscopy (SEM), X-ray
diffraction (XRD), and vibrating sample magnetometry (VSM).
heterogeneous biocatalyst composed of immobilized enzymes
provides a broader range of operating temperature and pH than
that of free enzymes.[5] In the last decade, the use of
nanoparticles in enzyme stabilization has attracted increased
attention based on their very small size of particles and large
surface area.[6] Magnetic nanoparticles show a high binding
capacity to conjugated enzymes and can be used for enzyme
immobilization.[7] The potential of magnetic nanoparticles as
carriers for immobilized enzymes can be increased by the use of
nanoparticles containing silica with an ordered structure, such as
KCC-1, on the surface of magnetic supports. This has unique
properties, including fibrous morphology, high surface-to-volume
ratio, and good thermal and mechanical stability.[8] The potential
application of heterogeneous biocatalysts in organic synthesis
has increased in recent decades due to major advancements in
enzyme immobilization techniques and efforts to imitate the
production of organic compounds. Among the enzymes applied
in organic synthesis, oxidoredactases are widely used as
biocatalysts for oxyfunctionalization and oxidation reactions.[9]
Laccases, copper-containing oxidases, can catalyze the
Laccase immobilized on CoFe2O4-KCC-1 was used as
heterogeneous biocatalyst for green and one-pot cascade synthesis
of 2-amino-5-oxo-4-aryl-4H,5H-pyrano[3,2-c]chromene-3-
a
carbonitriles. The reaction was performed in the presence of 2,2,6,6-
tetramethylpiperidine-1-oxyl (TEMPO) as a synthetic redox mediator.
The optimum reaction conditions were found to be as immobilized
laccase (100 mg, 95 U) and TEMPO (2 mol%) in a 100 mM sodium
citrate buffer (pH 4.5), 40 °C, and incubation time 17 h. About 80%
of initial activity of the immobilized laccase was retained after 15
independent runs.
oxidation of
substrates.
a wide range of phenolic and nonphenolic
Introduction
Laccase-catalyzed oxidation of various phenolic compounds,
such as diphenols, diamines, and benzenethiols, occurs by four-
electron reduction of O2 to HO2.[10] The scope of laccase
substrates can be extended to compounds with high redox
potential using natural or synthetic mediators. The ability of
laccase-mediator systems to catalyze multicomponent reactions
provides a powerful and effective strategy for synthesis of
complicated organic compounds. These reactions are
considered pivotal in the production of several important natural
products, such as coumarin and chromene derivatives.
Chromenes belong to a main category of biologically active and
natural oxygen-containing heterocycles, which are commonly
found in flavonoids and alkaloids.[11] These compounds possess
a range of biological activity and pharmacological properties,
such as anti-inflammatory, antioxidant, antimalarial, antimicrobial,
anticoagulant, antiallergenic, and anticancer activities.[11a,12]
Chromene derivatives are also applied in laser dyes, liquid
crystal display, optical brighteners, optical chemo-sensors, and
fluorescence markers.[13] Amino-4H-chromene derivatives was
previously prepared by Michael addition of an aromatic aldehyde
to malononitrile, followed by cyclization with hydroxycoumarins.
Although several synthetic approaches have been proposed for
production of chromenes, these methods suffer from some
limitations, including the usage of highly toxic and chlorinated
solvents, transition metal catalysts, lengthy multistep work-up
procedures, and low yields.[14] Elimination of these restrictions is
essential for the development of an efficient, simple, and
sustainable method for the synthesis of 2-amino-4H-
chromenes.[15]
Heterogeneous biocatalytic processes are considered an
important route to green and sustainable organic synthesis.[1]
Heterogeneous catalysts have attracted substantial attentions
because of their various advantages, such as improved atom
efficiency of reactions, enhanced turnover number, facile
catalyst separation, recovery, and reusability.[2] Moreover, the
simplicity of catalyst recovery in heterogeneous biocatalytic
processes provides economic and green approaches for the
production of biological substances.[3] Several types of
nanocarriers have been developed to progress traditional
immobilization methods leading to enhancement of enzyme
loading, activity, and stability, thereby aiding the development of
environmentally
friendly
synthetic
applications.[4]
A
[a]
M. Mogharabi-Manzari, T. Sedaghat, F. Shayan, and Prof. Dr. M.A.
Faramarzi
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy,
and Biotechnology Research Center
Tehran University of Medical Sciences
P.O. Box 14155–6451, Tehran 1417614411, Iran
E-mail: faramarz@tums.ac.ir
Homepage: http://www.tums.ac.ir/faculties/faramarz
M. Mogharabi-Manzari
The Institute of Pharmaceutical Sciences
Tehran University of Medical Sciences
Tehran 1417614411, Iran
[b]
[c]
Prof. Dr. M.H. Ghahremani
Department of Pharmacology and Toxicology, Faculty of Pharmacy
Tehran University of Medical Sciences
Tehran 1417614411, Iran
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.201801784
European Journal of Organic Chemistry
FULL PAPER
The present study describes an approach for the preparation of
a heterogeneous biocatalyst by laccase immobilization on the
surface of KCC-1 functionalized magnetic nanoparticles. The
immobilized laccase was applied in green and one-pot cascade
synthesis of chromenes in the presence of TEMPO as a shuttle
redox mediator.
nanoparticles were decreased (ca. 10 emu g-1) which was
expected and could be explained by diamagnetic contribution of
coated silica layer. However, the decrease showed no dramatic
effect on the efficiency of the prepared catalyst.
The wide-angle X-ray diffraction (XRD) patterns of the prepared
magnetic nanoparticles revealed that the nanoparticles
embedded in the core had not changed, despite being fabricated
with fibrous silica (Figure 1h). Elemental mapping of sulfur and
copper atoms investigated by SEM image and color-coded maps
revealed uniform distribution of the enzyme on the surface of the
fibrous silica coated magnetic support (Figure 2).
Results and Discussion
Magnetic nanoparticles (Fe3O4, CoFe2O4, and NiFe2O4) were
prepared via co-precipitation as described in the literature
(Supporting Information) and then coated by a fibrous silica layer
using sol-gel method. Morphology of all magnetic nanoparticles,
KCC-1 coated nanoparticles, and the enzyme immobilized
nanoparticles were studied and the porous surface of the silica-
coated nanoparticles and the core-shell structure of the
magnetic KCC-1 were observed in the scanning electron
microscopy (SEM) and transmision electron microscopy (TEM)
images, respectively (Figure 1a-f). The Fe3O4-KCC-1
nanoparticles had a uniform size, with an average diameter of
100 nm and good dispersity. The magnetization curves of the
Fe3O4-KCC-1, CoFe2O4-KCC-1, and NiFe2O4-KCC-1 magnetic
samples (Figure 1g) indicated saturation magnetization values of
76, 59, and 19 emu g−1, respectively, which are in the range of
reported values for amorphous silica coated magnetic
nanoparticles.[16] Three nanoparticles, Fe3O4, CoFe2O4, and
NiFe2O4, were examined for immobilization of laccase. The
obtained results showed that CoFe2O4-KCC-1 nanoparticles
were able to immobilize a higher amount of the enzymes than
other two nanoparticles (see Supporting Information). Although
iron oxide nanoparticles showed better magnetic saturation
values than CoFe2O4 nanoparticles (Figure 1e), the obtained
results from the kinetic parameters showed that CoFe2O4-KCC-1
was a suitable support for the enzyme immobilization (see
Supporting Information).
Figure 1. SEM images of a) CoFe2O4-KCC-1, b) Fe3O4-KCC-1, c) NiFe2O4-
KCC-1, TEM images of d) CoFe2O4-KCC-1, e) Fe3O4-KCC-1, f) NiFe2O4-KCC-
1, g) VSM of magnetic nanoparticles, and h) XRD pattern of magnetic
nanoparticles.
The magnetic properties of the KCC-1 coated magnetic particles
were measured and it was found that the saturation
magnetization values of the fibrous silica-coated magnetic
Figure 2. Color coded dot maps of copper atoms of immobilized laccase on a) Fe3O4, b) CoFe2O4, c) NiFe2O4, and sulfur atoms on d) Fe3O4, e) CoFe2O4, and f)
NiFe2O4.
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10.1002/ejoc.201801784
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Radial-like morphology and large surface area helped the target
enzymes attach to the fibrous structure quickly and efficiently,
thereby decreasing the time of attachment. The enzyme was
immobilized on the modified surface of the prepared magnetic
fibrous silica KCC-1 activated by (3-aminopropyl)triethoxysilane
and glutaraldehyde. The obtained results showed that laccase
immobilization increased enzyme storage stability at 4 and 25 °C
(Figure 3). The study of enzyme reusability showed that
immobilized laccase on the surface of the CoFe2O4-KCC-1
magnetic nanoparticles could retain around 85% of its initial
activity after 15 independent runs (Figure 4).
The activated nanoparticles and the immobilized laccase were
investigated by Fourier transform infrared (FTIR) spectroscopy
and it was found that carbonyl adsorption band (1578 cm-1) of
the attached glutaraldehyde was disappeared after the enzyme
immobilization. Disappearing of the carbonyl band proves the
reaction between carbonyl groups in the surface of nanoparticles
with amine groups of the enzyme leading to conformational
changes of laccase. Wang et al.[16b] showed that the carbonyl
stretching vibration of carboxyl groups of modified mesoporous
carbon was disappeared after immobilization and amide peaks
were used to determine the conformational change of protein.
The yield and efficiency of immobilization were calculated as the
percentage of total immobilized enzyme activity and the
percentage of the bound enzyme, respectively. The yield and
efficiency of immobilization were found to be 78% and 86%,
respectively. Therefore, laccase immobilized on CoFe2O4-KCC-1
was selected as a heterogeneous catalyst for aerobic oxidation
of some aromatic alcohols containing one, two, and three
aromatic rings. The biocatalyst effectively catalyzed the
oxidation reaction, with a yield up to 95% (Figure 5 & Table 1).
An ionic mechanism has been proposed for laccase-induced
oxidation of aromatic alcohols using a synthetic redox mediator.
In this mechanism, the oxygen lone pair electrones of the
aromatic alcohol attacks onto the laccase-catalyzed generated
oxoammonium, which leads to a transient complex formation.
Fainally, carbonyl-containing product is achieved by
deprotonation
of
this
adduct.[17]
Figure 3. Residual activity of a) free enzyme, and immobilized laccase on b) CoFe2O4, c) Fe3O4, and d) NiFe2O4 at 4, and 25 °C.
Proper control experiments were carried out in the absence of
laccase to explore the possible catalytic activity of the
nanoparticles. The magnetic nanoparticles did not exhibit
significant catalytic activity in the reaction conditions. Although
laccase is used extensively as a biocatalyst in simple organic
reactions, such as oxidation, dimerization, and polymerization,
only a few studies have applied this enzyme in multicomponent
organic reactions for the synthesis of heteroatom-containing
polycyclic compounds.[18]
Therefore, in the present study, to investigate the capability of
this heterogeneous biocatalytic system in the catalysis of
multicomponent organic reactions in aqueous solution and under
slight reaction conditions, the system was applied in the green
synthesis
of
2-amino-5-oxo-4-aryl-4H,5H-pyrano[3,2-
c]chromene-3-carbonitriles. Aromatic alcohols were oxidized to
corresponding aldehyde via an enzyme-catalyzed reaction.
Chromenes were obtained by the addition of malononitrile and
4-hydroxycoumarin to the in situ generated aldehydes. Alcohol
oxidation was completed after 10 h. The optimum amounts of
the heterogeneous catalyst and TEMPO were 100 mg (95 U)
Figure 4. Relative activity of laccase after 15 independent cycles (pH 4.5, and
40 °C).
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10.1002/ejoc.201801784
European Journal of Organic Chemistry
FULL PAPER
and 2 mol%, respectively (Figure 6). Study of the effect of pH
(3.5-7.5) and temperature (25-50 °C) on the yield of 2-amino-5-
oxo-4-phenyl-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile
demonstrated that the optimal reaction conditions were pH 4.5 at
40 °C (Figure 7). The biocatalyst effectively catalyzed the
synthesis of chromenes, with a yield up to 88% (Table 2).
Figure 6. Effect of immobilized laccase and TEMPO on the yield of oxidation
reaction in citrate buffer (100 mM, pH 4.5) under air at 40 °C.
Although, rising the temperature up to 40 °C improved the yield
by more than 90%, the yield was decreased with further
increases in reaction temperature that caused by enzyme
deactivation. The efficiency of laccase in organic synthesis
depends on its tendency toward time-dependent inactivation
under reaction conditions. There is a belief that thermal
inactivation is mainly caused by denaturation of the tertiary
structure of protein which leads to unfolding and disruption of the
Figure 5. Time-course study of consumption of benzyl alcohol and production
of benzaldehyde in citrate buffer (100 mM, pH 4.5) under air at 40 °C.
Table 1. Aerobic oxidation of aromatic alcohols using immobilized
laccase in the presence of TEMPO (2 mol%) at pH 4.5 and 40 °C.
active site.[19] As shown by
a literature survey, several
Immobilized laccase
homogeneous or heterogeneous catalysts have been applied for
the production of chromene derivatives, such as piperidine,
cetyltrimethylammonium chloride (CTAB), triethyl amine,
tetrabutylammonium bromide (TBAB), triethylbenzylammonium
chloride, N,N-dimethyl aminoethyl benzyl dimethyl ammonium
chloride, hydrotalcite, chitosan, heteropolyacid, ionic liquids, and
methane sulfonic acid.[20, 21]
TEMPO
Ar
Ar
OH
O
Citrate buffer (100 mM, pH 4.5)
2a-m
1a-m
Products (reaction time, yield)
O
O
O
F
Cl
2a (10 h, 95%)
2d (9 h, 85%)
2b (8 h, 93%)
2c (9 h, 88%)
O
O
O
O
HO
2e (10 h, 78%)
Br
2f (10 h, 80%)
O
O
O
O
O
O
NO2
2g (9 h, 80%)
2h (8 h, 75%)
2i (8 h, 78%)
O
O
O2N
2j (8 h, 85%)
2k (10 h, 88%)
2l (10 h, 79%)
O
Figure 7. Effect of reaction conditions (pH and temperature) on the yield of
laccase-catalyzed oxidation reaction.
2m (12 h, 68%)
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10.1002/ejoc.201801784
European Journal of Organic Chemistry
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Table 2. One-pot synthesis of 2-amino-4-(aryl)-5-oxo-4H,5H-pyrano[3,2-
c]chromene-3-carbonitrilein aqueous medium using immobilized laccase.
H14[NaP5W30O110] as a heteropolyacid catalyst under reflux
conditions for 3‒5 h.[21]
O
Ar
In the present study, the cascade one-pot reaction was
performed in various aqueous organic media. Some ratios of
organic and aqueous solution including 1:10, 2:10, and 3:10
were examined. The obtained results showed that activity of the
enzyme and the yield of the product decreased by increasing the
ratio of organic solvents. Therefore, the ratio of 1:10 was
selected as the optimal ratio. The reaction proceeded in a
mixture of both organic and aqueous solutions (1:10, v/v) and
the product was achieved with moderate yields (55–85%).
CN
Immobilized laccase
TEMPO
O
Ar
OH
Malononitrile
O
NH2
4-Hydroxycoumarin
Citrate buffer (100 mM, pH 4.5)
1a-m
3a-m
Products (reaction time, yield)(a)
Cl
F
The obtained results showed that the citrate buffer (100 mM, pH
4.5) was the appropriate reaction medium (Table 3). Water
molecules are bonded to the surface of enzymes and act as a
lubricant or plasticizer, thereby enabling these enzymes to
exhibit the conformational mobility required for optimum enzyme
structure and activity. Hydrophobic solvents are more suitable
than hydrophilic ones for promoting enzyme activity due to their
reduced ability to strip essential water from enzymes and to
restrict conformational flexibility. The most important driving
force for enzyme activity is the bonding energy between the
substrate and active site.
O
O
O
CN
O
CN
CN
O
O
O
NH2
O
NH2
O
NH2
3a (17 h, 85%)
3b (15 h, 88%)
3c (15 h, 88%)
OH
Br
Hydrophobic active site of some enzymes provides a large
energetic barrier that restricts movement of hydrophilic
substrates from water into the active site and decreases rate of
catalytic reaction. On the other hand, when the reaction is
performed in the presence of organic solvent, the energetic
advantages of partitioning decrease, and the ground state
energy of the hydrophobic molecule is more stabilized in organic
solvents than water.[22] Therefore, the increased activation
barrier slows down the enzymatic reaction.[22] Kinetic parameters
(Vmax and Km) were calculated using the Lineweaver–Burk curve
and applying ABTS as substrate. Calculated values of Vmax for
free and immobilized laccase was found to be 61 and 56 mM
min‒1, respectively.
O
O
O
CN
CN
CN
O
O
O
O
NH2
O
NH2
O
NH2
3d (16 h, 85%)
3e (18 h, 78%)
NO2
3f (18 h, 75%)
NO2
O
O
O
O
O
CN
CN
O
CN
O
O
O
NH2
O
NH2
O
NH2
Table 3. Synthesis of 2-Amino-5-oxo-4-phenyl-4H,5H-pyrano[3,2-
c]chromene-3-carbonitrile (5a) in the presence of organic solvent (1:10,
v/v) after 17 h incubation at pH 4.5 and 40 °C.
3g (15 h, 85%)
3h (15 h, 78%)
3i (17 h, 75%)
Entry
Solvent
water
Citrate buffer
DMSO
Dioxane
Hexanone
Toluene
Octanol
hexane
log P
-
-
Time (h)
17
15
17
18
20
17
20
20
Yield (%)
85
N
1
2
3
4
5
6
7
8
9
85
60
75
78
80
75
55
80
O
O
-1.3
-1.1
1.3
2.5
2.9
3.5
4.0
4.5
O
CN
CN
O
O
CN
O
O
NH2
O
NH2
O
NH2
Heptane
Octane
17
17
10
80
3j (18 h, 80%)
3k (20 h, 75%)
3l (20 h, 75%)
(a)Reaction condition: benzyl alcohol (1 mmol), 4-hydroxy coumarin (1.2
mmol), malononitrile (0.5 mmol) immobilized laccase (100 mg, 95 U), and
TEMPO (2 mol%) in a citrate buffer (100 mM, pH 4.5) at 40 °C.
A slight declining trend of Vmax occurred due to conformational
changes, which reduced the availability of the active site of
laccase after immobilization (Supporting Information). The Km
values of the immobilized and free laccase were estimated as
1.4 and 2.2 µM, respectively. Kinetic parameters of an enzyme
usually change significantly after immobilization on a solid
support. These parameters can be improved by applying a
suitable support and an appropriate immobilization method.
Some conformational changes may be occurred on the tertiary
Kaur et al.[20] reported a series of 4-aryl/heteroaryl-4H-fused
pyrans that were produced in a microwave synthesizer via a
multicomponent reaction and showed remarkable in vitro
inhibition of xanthine oxidase.[20] Another study reported the
synthesis
of
2-amino-4H-chromene
derivatives
using
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10.1002/ejoc.201801784
European Journal of Organic Chemistry
FULL PAPER
conducted using silica gel (SiO2 60, 230–400 mesh, Fluka, Buchs,
Switzerland).
structure of the enzyme during the immobilization procedures
which affect the kinetic behavior of the immobilized enzyme.[22a]
Covalent bonds between the enzyme and the support may
increase accessibility of the active site and lead to improved
kinetic constants. In addition, catalytic properties are also
influenced by diffusion of substrate and product from porous
matrix of the support. The found values showed an increase in
affinity of the immobilized enzyme toward the substrate.[22b] The
most key effecting factors on the affinity of an immobilized
enzyme toward a substrate are the loss of required flexibility of
enzyme that is necessary for substrate.[23]
Laccase assay
The activity of laccase was measured using a method previously
reported by Johannes
&
Majcherczyk.,[24] using 2,2′-azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS). Briefly, 0.5 ml laccase
solution was added to equal volume of freshly prepared ABTS (2 mM) in
100 mM citrate buffer pH 4.5. Then the reaction mixture was incubated
for 10 min at 37 °C and 120 rpm and the absorbance was recorded at
420 nm (ε420 = 36,000 M−1 cm−1). One unit of laccase activity was
considered as the amount of enzyme that oxidized 1 μmol of ABTS per
one minute.
Conclusions
Synthesis of Fe3O4 nanoparticles
In summary, laccase was immobilized on CoFe2O4-KCC-1, and
the prepared catalyst was characterized. This heterogeneous
catalytic system was used to yield 2-amino-5-oxo-4-aryl-4H,5H-
pyrano[3,2-c]chromene-3-carbonitriles via a one-pot cascade
enzymatic reaction. The optimal reaction conditions, including
pH, temperature, incubation time, and amounts of
heterogeneous biocatalyst and mediator, were investigated.
The prepared catalytic system described herein can be used to
enable improvements in future biocatalytic systems that utilize
heterogeneous biocatalysts in cascade organic reactions.
Moreover, the cascade and enzymatic approach described in
the present study can be considered as an effective and
straightforward method for advancement of applying simple
biotransformations in the targeted synthesis of valuable
compounds via green multicomponent reactions.
The magnetic Fe3O4 nanoparticles were prepared by ultrasound-assisted
reverse co-precipitation method, as previously reported by Wei et al.[25] In
brief, an aqueous solution of iron (II) chloride (5.4 g) and iron (III) chloride
(2 g) in 25 ml hydrochloric acid (2 M) was sonicated at 25 °C until the
salts dissolved. A solution of aqueous ammonia (25%, 40 ml) was slowly
added over 20 min to the above mixture under a N2 atmosphere at at 25
°C, followed by 30 min stirring with a mechanical stirrer. The prepared
Fe3O4 nanoparticles were separated using an external magnetic field.
Then the magnetic nanoparticles were washed three times with distilled
water and three times with ethanol.
Preparation of magnetic CoFe2O4 nanoparticles
Cobalt ferrite (CoFe2O4) nanoparticles were prepared by co-precipitating
a mixtures of cobalt (II) chloride and iron (III) chloride aqueous solutions
in an alkaline medium.3 Mixed solutions of 100 ml CoCl2.6H2O (1.0 M)
and 100 ml FeCl3.6H2O (2.0 M) were prepared and kept at 60 °C for half
an hour. The mixture was abruptly added to a boiling solution of NaOH
(1,200 ml, 0.63 M). Then the solution was stirred vigorously and
maintained at 85 °C for one hour. At this stage, fine particles were
collected using an external magnetic field. The prepared nanoparticles
were washed several times with distilled water and dried at room
temperature.[26]
Experimental Section
Materials and instruments
Laccase from Trametes versicolor (≥ 10 U mg-1), 2,2′-azino-bis(3-
ethylbenzthiazoline-6-sulfonic
acid)
(ABTS),
and
2,2,6,6-
Preparation of magnetic NiFe2O4 nanoparticles
tetramethylpiperidine-1-oxyl (TEMPO) were obtained from Sigma-Aldrich
(St. Louis, MO, USA). All other chemicals and solvents were purchased
from Merck (Darmstadt, Germany) and applied without further purification.
Nickel ferrite (NiFe2O4) nanoparticles were produced via the
autocombustion assisted sol-gel approach, as previously reported by
Feng et al.[6] Briefly, Fe(NO3)3.9H2O (2 mol) and Ni(NO3)2.6H2O (1 mol)
were dissolved in deionized water and in the presence of citric acid (3
mol) as a chelating agent. Ammonia solution (28 %) was added dropwise
to control the pH value at 7. Then the solution was evaporated at 60 °C
to form a sticky gel and followed by increasing the temperature up to 80
°C to form a thick gel. The gel was combusted by hold on a hot plate
(200 °C) to release large amounts of gases, such as CO2, H2O, and N2. A
dark brown ferrite powder was washed three times with distilled water
and collected using an external magnetic field.[27]
A
high-performance liquid chromatography (HPLC) apparatus was
employed from Knauer (Berlin, Germany). The apparatus was equipped
with a smart line pump 1000, PDA detector 2800, and degasser 5000.
Activity of the enzyme was measured using a double-beam PC scanning
UV-vis spectrophotometer (UVD 2950, Labomed, Culver City, USA).
Scanning electron microscopy was performed on a Hitachi S-4700
(Tokyo, Japan) instrument, with beam energy of 4 kV. The 1H and 13C
nuclear magnetic resonance (NMR) spectra were recorded on a Bruker
DRX-500 Avance instrument (Bruker Biospin AG, Faellanden,
Switzerland) at 500 and 125 MHz, respectively. The spectra were
measured in deuterated dimethyl sulfoxide (DMSO-d6) relative to
tetramethylsilane (TMS). The chemical shifts and coupling constant (J)
are given in δ and Hz, respectively. The splitting patterns of spectra are
presented as singlet (s), doublet (d), triplet (t), and multiplet (m). Mass
spectra were recorded by electrospray ionization technique using a
micromass LCT spectrometer (Waters, Milford, MA, USA) using. Thin-
layer chromatography (TLC) was carried out using precoated aluminum-
backed plates (Kieselgel 60 F254, Merck, Darm-stadt, Germany) and
visualized by ultraviolet irradiation at 254 nm. Flash chromatography was
Preparation of silica-coated nanoparticles
The prepared magnetic nanoparticles were suspended in a mixture of
ethanol (35 ml) and distilled water (6 ml). the mixture was sonicated for
15 min. Then 1.5 ml tetraethyl orthosilicate (TEOS) was dropwise added
to the solution and placed in an ultrasonic water bath for 15 min.
Subsequently, 1.4 ml aqueous ammonia (10%) was dropwise added over
10 min, while the mixture is stirred mechanically. The mixture was kept at
40 °C for an overnight. The iron oxide nanoparticles were washed three
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10.1002/ejoc.201801784
European Journal of Organic Chemistry
FULL PAPER
times with ethanol. Finally, the silica coated nanoparticles were collected
by an external magnetic field and dried over vacuum condition.[28]
up and the catalyst was separated by an external magnet. Excess
solvent was removed under reduced pressure to yield the product.
Preparation of Amin functionalized magnetite nanoparticles
Acknowledgements
A mixture containing 10 g of silica-coated magnetite nanoparticle was
dispended in dry toluene (200 ml) to form a uniform suspension. The
mixture was sonicated for 30 min. After the dropwise addition of 2.5 ml
(3-aminopropyl) triethoxysilane (APTES), the mixture was stirred
mechanically and then the temperature was slowly increased up to
105 °C and remained for 20 h. Finally, the amine functionalized magnetic
nanoparticles were washed three times with ethanol and collected by an
external magnet. The prepared particles were dried under vacuum.[29]
This work was financially supported by the grant No. 97-02-90-
38618 from Biotechnology Research Center, Tehran University
of Medical Sciences, Tehran, Iran to M.A.F.
Keywords: Laccase • Immobilization • Cascade reaction •
Green synthesis • Biocatalysis
Immobilization of laccase
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Magnetic carrier (10 mg) was suspended in citrate buffer (100 mM, pH
4.5), 1.6% glutaraldehyde was added and the mixture slightly stirred at
room temperature for 2 h. After attachment of glutaraldehyde, the
nanoparticles were washed with the same buffer solution. The activated
particles (0.16 g) were dispersed in a solution of the enzyme (20 mg) and
the prepared mixture was slightly shacked at room temperature for 12 h
(Supporting Information). The immobilized enzymes were collected by an
external magnetic field and non-covalently-bounded enzymes were
removed by washing with citrate buffer. The immobilized biocatalysts
were dispersed in buffer and stored at 4 °C. The enzyme activity was
measured using a colorimetric procedure. The yield and efficiency of
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COMMUNICATION
Biocatalysis
Mehdi Mogharabi-Manzari, Mohammad
Hossein Ghahremani, Tabassom
Sedaghat, Fatemeh Shayan, and
Mohammad Ali Faramarzi*
Page No. – Page No.
Text for Table of Contents
A laccase heterogeneous magnetic
fibrous silica-based biocatalyst for
green and one-pot cascade synthesis
of chromene derivatives
A heterogeneous biocatalyst was prepared by laccase immobilization on the surface of KCC-1 functionalized magnetic
nanoparticles. The catalyst was applied in green and one-pot cascade synthesis of chromenes in the presence of TEMPO as
a shuttle redox mediator.
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