A novel magnetically separable laccase-mediator catalyst system for the aerobic oxidation of alcohols and 2-substituted-2,3-dihydroquinazolin-4(1H)-ones under mild conditions

Article abstract of DOI:10.1002/aoc.5899

In this study, a magnetically reusable artificial metalloenzyme has been constructed by co-immobilization of palladium nanoparticles as a strong oxidizing catalyst and laccase as an oxygen-activating enzyme into the cavities of magnetic mesocellular foams

Full text of DOI:10.1002/aoc.5899

Received: 17 January 2020
DOI: 10.1002/aoc.5899
Revised: 29 May 2020
Accepted: 30 May 2020
F U L L P A P E R
A novel magnetically separable laccase-mediator
catalyst system for the aerobic oxidation of alcohols
and 2-substituted-2,3-dihydroquinazolin-4(1H)-ones
under mild conditions
Zahra Shokri | Nahid Azimi | Sirvan Moradi | Amin Rostami
Department of Chemistry, Faculty of
In this study, a magnetically reusable artificial metalloenzyme has been con-
structed by co-immobilization of palladium nanoparticles as a strong oxidizing
catalyst and laccase as an oxygen-activating enzyme into the cavities of mag-
netic mesocellular foams silica (Pd-Laccase@MMCF). The combination of Pd-
Laccase@MMCF and hydroquinone (HQ) act as electron-transfer mediator
system and make stepwise electron transfer from substrate to molecular oxy-
gen. This catalyst system was used for the aerobic (i) oxidation of alcohols to
the corresponding carbonyl compounds and (ii) dehydrogenation of
Science, University of Kurdistan, Zip Code
6177-15175, Sanandaj, Iran
6
Correspondence
Amin Rostami, Department of Chemistry,
Faculty of Science, University of
Kurdistan, Zip Code 66177-15175,
Sanandaj, Iran.
Email: a.rostami@uok.ac.ir
2-substituted-2,3-dihydroquinazolin-4(1H)-ones in phosphate buffer (0.1 M,
pH 4.5, 4 mL)/THF (4%, 1 mL) as solvent under mild conditions. The co-
immobilization of both laccase and Pd onto high surface area mesoporous sup-
port, high catalytic activity and magnetically separable and reusable make the
present catalyst system superior to other currently available electron-transfer
mediator systems.
K E Y W O R D S
aerobic oxidation, laccase, magnetic mesocellular foam silica, Metalloenzyme, palladium
1
| INTRODUCTION
oxidase usually found in plants and fungi, is able to cata-
lyze the oxidation of a wide variety of phenolic com-
In recent decades, the use of enzymes as safe catalysts in
organic reactions has attracted considerable attention
because enzyme-catalyzed reactions meet most of the
pounds and aromatic amines in the presence of O as
2
the oxidant under mild conditions and produce H O as
2
[
4]
the only by-product.
Notwithstanding these advan-
[1]
criteria of green chemistry. This holds particularly true
tages, the utilization of laccase in organic reactions is
for enzyme-catalyzed oxidations in the presence of O as
limited owing to low stability, high production costs and
2
[2]
[5,6]
the oxidant. O is not only one of the cheapest oxi-
the difficult recovery and reuse of it.
It has been
2
dants available, but also regarded as an environmentally
shown that the immobilization of laccase on heteroge-
neous system can overcome the aforementioned
disadvantages by improving the stability of the enzyme,
facilitating its separation from the reaction mixture, and
reducing the cost factor because immobilized enzyme
friendly oxidant. Another potential advantage of O is
2
the formation of non-toxic H O as the only byproduct
2
during the course of oxidase-catalyzed oxidations with
O . Within this context, laccase-catalyzed oxidations are
2
[
6–9]
receiving particular attention among synthetic chemists
can be easily reused.
Morever, another major
[3]
in recent years.
Laccase, a multicopper-containing
obstacle using free laccase as the catalyst in organic
Appl Organomet Chem. 2020;e5899.
wileyonlinelibrary.com/journal/aoc
© 2020 John Wiley & Sons, Ltd.
1 of 15
https://doi.org/10.1002/aoc.5899

2
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SHOKRI ET AL.
reactions is the low redox potential. It is well-known
that the redox potential of laccase can be broadened by
natural mediators such as 4-hydroxybenzoic acid and
transfer mediator (ETM) and palladium as substrate-
selective catalyst. They obtain a stepwise electron transfer
from substrate to molecular oxygen to perform desired
transformation under mild conditions (Scheme 1).
3
-hydroxyanthranilic acid or by artificial mediators
including 2,2-Azino-bis-(3-ethylbenzothiazoline)-6-sul-
fon0ic acid (ABTS), 1-hydroxybenzo-triazole (HBT), and
Although these systems presented an interesting
breakthrough in the field of aerobic oxidation, these
methods require transition metal complexes and expen-
sive ligands which are intrinsically difficult to separate
from the product and recycle. Therefore, there is an
increasing demand for the development of heterogeneous
cooperative catalytic systems with simple separation and
recycling in the ligand free aerobic oxidation of organic
compounds under mild conditions.
2
,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). Although,
the use of laccase-mediator systems (LMS) proceeds the
[
10]
oxidation of substrates with higher redox potentials
,
the mentioned mediators are quite expensive and difficult
to recover from reaction mixture, which hampers the
applications of LMS. In order to overcome these limita-
tions, co-immobilization of laccase and mediators on dif-
[
4]
ferent support materials such as cellulose beads,
Based on the mentioned background and our
systematic studies about the application of laccase and
[6]
[11]
magnetic Fe O
and mesoporous silica
have been
3
4
[19]
made. Despite these developments, the co-immobilization
of laccase and new more effective mediator on to high spe-
cific surface areas support is still demand.
laccase/quinone catalytic system in organic reactions,
our main objectives in this study are: (1) the synthesis of
a magnetically separable cooperative catalyst system via
the immobilization of Pd nanoparticles as a strong
oxidizing catalyst and laccase enzyme as an oxygen-
activating and sustainable alternative to transition metal
complex on MMCF (Pd-Laccase@MMCF); and (2) the
investigation of the catalytic ability of this biohybrid
catalyst for the aerobic oxidation of alcohols and
2-substituted-2,3-dihydroquinazolin-4(1H)-ones in the
presence of hydroquinone instead of benzoquinone as
electron transfer mediator (Scheme 2). One of the main
novelty and advantage of this cooperative catalytic sys-
tem is that redox catalysts are immobilized on magneti-
cally reusable support.
Mesoporous silica materials have been widely
explored for their unique properties such as high surface
areas, large pore volumes, tunable pore sizes, and a con-
[12]
trollable framework composition.
In recent years,
magnetic silica materials have attracted a lot of attention
as support for enzyme immobilization because they pos-
sess not only the unique features of mesoporous mate-
rials, but also magnetic properties make these materials
can be simply separated by an external magnetic field
[13]
and recycled.
The selective oxidation of alcohols to carbonyl com-
pounds plays a key role in organic chemistry and in indus-
try owing to the wide application of the resulting carbonyl
compounds as precursors for many drugs, agricultural
[14]
chemicals and fine chemicals.
Although a variety of
2 | EXPERIMENTAL
stoichiometric reagents have been traditionally used to
achieve this transformation, their use causes environmen-
tal problems owing to generation of a large amount of
2.1 | Material and physical measurements
[15]
waste.
To overcome this shortcoming, catalytic meth-
All substances were bought from the Merck and Aldrich
Chemical Companies and utilized without additional puri-
fication. The 2-substituted-2,3-dihydroquinazolin-4(1H)-
ones were prepared by previous procedures in our
odologies employing molecular oxygen as the sacrificial
[16]
oxidant have been emerged.
Thus, much attention was
given to palladium catalysts for the aerobic oxidation of
[
17]
alcohols.
However, because of the unfavored electron
0
transfer between Pd and O palladium-catalyzed oxida-
tions with direct reoxidation of Pd by O require high
2,
0
2
reaction temperatures, which are not suitable for ther-
mally unstable substrates. Therefore, it seems that there is
still an increasing need for the development of systems
that can operate under mild conditions. In order to over-
come this problem, Bäckvall and coworkers have reported
several mild palladium-catalyzed aerobic oxidations that
proceed through a multistep electron transfer containing a
[18]
triple catalytic system.
These catalytic systems consist
of macrocyclic transition metal complex that acts as the
oxygen-activating component, benzoquinone as electron
SCHEME 1 Cooperative catalytic aerobic oxidation systems

SHOKRI ET AL.
3 of 15
FIGURE 1 XRD pattern of magnetic mesocellular foam silica
the Fe (NO ) -impregnated foam was reacted with
3
3
ꢀ
propionic acid (4.6 ml) at 85 C for 3 hr to form the iron
propionate complex. Subsequently, the sample was
ꢀ
ꢀ
heated to 300 C in air (heating rate of 1 C/min) and
maintained at this temperature for 30 min. The resulting
material was named as MMCF.
SCHEME 2 Pd-Laccase@MMCF/HQ as a cooperative
catalytic aerobic oxidation system
The synthesized MMCF was characterized by X-ray
diffraction (XRD). As is shown in Figure 1, the XRD
spectrum of the MMCF exhibited a broad reflection
o
(
2θ = 11-29 ), approving the presence of amorphous sil-
[
23]
ica.
This spectrum also indicated multiple peaks in the
[20]
o
o
research lab.
The particle size was obtained by
2θ range 30 -70 , which revealed the presence of γ-Fe O
2
3
[
22]
SEM using FESEM-TESCAN MIRA3. The chemical com-
position of the prepared nanocatalyst was determined by
EDX (Energy Dispersive X-ray Spectroscopy). Pd, Cu
and Fe percentages of the prepared nanocatalyst were
obtained by inductively coupled plasma-optical emission
spectrum (ICP-OES) on Optima 7300D spectrophotome-
ter. Magnetic measurements were carried out using
a vibration sample magnetometer (VSM, Meghnatis
Daghigh Kavir Co., Iran) under magnetic fields up to
nanoparticles inside foam spheres.
2.3 | Preparation of amine functionalized
MMCF
The synthesized MMCF (1 g) suspended in dry toluene
(20 ml) was added to 10 ml of toluene solution containing
2.7 ml of 3-aminopropyltrimetoxysilane. The conten-
ts were heated to reflux under argon atmosphere for
24 hr. Then, the carrier was magnetically collected,
washed with toluene, ethanol, and dichloromethane
2
0 kOe. X-ray diffraction (XRD) measurement was done
on a X'PertPro Panalytical.
[
24]
and dried.
AmP-MMCF.
The resulting material was named as
2.2 | Preparation of magnetic
mesocellular foam silica (MMCF)
Spherical mesocellular foam silica was prepared follow-
2.4 | Immobilization of Pd nanoparticles
onto AmP-MMCF
[21]
ing a previously reported procedure.
The characteris-
tics of the obtained MCFs are as follows: BET surface
2
−1
3
−1
area = 503 m g , specific pore volume = 1.1 cm g ,
window size = 7 nm and Cell size = 24 nm.
First, 500 mg of the amine functionalized MMCF was
suspended in pH-adjusted deionized water solution
(pH 8) by adding LiOH (15 ml, 0.1 N) and stirring for
Next, the magnetic particles were introduced into the
[
22]
MCF as follows
: Fe (NO ) Á9H O (2.68 g) was dis-
5 min. Thereafter, Li PdCl4 was prepared by mixing
3
3
2
2
solved in methanol (5 ml), followed by addition of 1 g of
foam. After evaporation of methanol at 80 C in an oven,
PdCl2 (145.41 mg, 0.82 mmol) and LiCl (69.52 mg,
1.64 mmol) in 10 ml distilled water, the suspension was
ꢀ

4
of 15
SHOKRI ET AL.
ꢀ
stirred at 80 C until a homogeneous solution was
achieved. The resulting solution was filtered, pH-adjusted
(
1 mmol of ABTS per minute. The findings of this proce-
dure revealed that the amount of immobilized laccase
was 22 U per 0.5 g of support.
pH 8), and then added to the mixture of AmP-MMCF in
water. After being stirred at room temperature for 24 hr,
the resultant Pd (II)-precatalyst was collected by applying
an external magnetic field and washed with distilled
water. Then, the Pd (II)-precatalyst was re-suspended in
water (15 ml), and a solution of distilled water (5 ml) con-
2.7 | General procedure for the aerobic
oxidation of alcohols
taining NaBH (310.2 mg, 8.2 mmol) was added to reduce
4
Pd (II) to Pd(0). After 30 min, the final fabricated parti-
cles were magnetically separated, washed with water,
To a mixture of alcohol (1 mmol), Pd-Laccase@MMCF
(0.25 g) and hydroquinone (HQ, 0.27 mmol) in an open-
air round-bottom flask, NaPBS/THF (5 ml, 4/1 v/v) was
[
24]
acetone, and dried.
as Pd-AmP-MMCF.
The resulting material was named
ꢀ
added. Then, the resulting mixture was stirred at 40 C
for an appropriate time. The progress of reaction was
monitored by TLC. After completion of the reaction, the
catalyst was separated using an external magnet and the
2
.5 | Immobilization of laccase onto Pd-
AmP-MMCF
product was extracted with Et O. The organic phase was
2
dried over anhydrous MgSO . Finally, the purity of the
4
5
00 mg of the Pd-AmP-MMCF suspended in potassium
products was evaluated by GC without any chromato-
graphic purification.
phosphate buffer (30 ml, 100 mM, pH 8.0) was mixed
with glutaraldehyde solution (50% in H O, 230 mg,
2
0
.99 mmol) at room ambient for 24 hr. After magnetic
separation, the GA-modified particles were collected,
washed with the phosphate buffer and acetone and
applied to react with the amino groups of the enzyme.
For this purpose, 500 mg of the Glutaraldehyde-
functionalized Pd(0)-AmP-MMCF particles was re-
dispersed in the enzyme solution prepared by dissolving
laccase enzyme (30 U) in potassium phosphate buffer
2.8 | General procedure for the aerobic
dehydrogenation of 2-substuted-
2,3-dihydroquinazolin-4(1H)-ones
To a magnetically stirred solution of Pd-Laccase@MMCF
(0.25 g), and HQ (0.34 mmol) in NaPBS/THF (4/1 ml)
under air, substrate (1 mmol) was added. Then, the reac-
(3 ml, 100 mM, pH 7.2). The reaction mixture was then
ꢀ
stirred at room temperature for 12 hr. Finally, the enzyme-
bound magnetic particles, named as Pd-Laccase@MMCF,
were collected, washed with phosphate buffer and dried
under reduced pressure.
tion mixture was stirred at 40 C for an appropriate
time. After the reaction, the catalyst was easily separated
using an external and the product was extracted with
Et O (3 × 5 ml). The organic layer was dried with anhy-
2
drous sodium sulfate, the excess solvent was removed
under reduced pressure. The crude product was purified
by recrystallization from ethanol or chromatography on
silica gel using n-hexane/ethyl acetate (3:1).
2
.6 | Activity assay of immobilized laccase
in Pd-laccase@MMCF
In order to assessment the amount of immobilized
[25]
laccase onto support, the Bradford method
was used
as follows: The activity of laccase in Pd-Laccase@MMCF
was assayed spectrophotometrically with 2,2-azino-bis-
3 | RESULTS AND DISCUSSIONS
3
-ethylbenzothiazoline-6-sulfonic acid (ABTS) as sub-
3.1 | Preparation of Pd-laccase@MMCF as
strate (5 mM) in Na-acetate buffer (100 mM, pH 5)
by measuring absorbance increase at 420 nm at room
temperature. Suitable amount of Pd-Laccase@MMCF
in Na-acetate buffer (100 ml) was added to the mixture
and the initial rate was immediately measured as
an artificial metalloenzyme
An artificial metalloenzyme (ArM) is a metalloprotein
made in the laboratory which cannot be found in the
nature and can catalyze certain desired chemical reac-
tions. Three different approaches have been developed to
[25]
increase in optical density at 420 nm.
The molar
extinction coefficient for the oxidation of ABTS at
anchor transition metals to native enzymes to form
−
1
−1
[26]
4
20 nm is 3.6 × 104 M cm . One unit of activity is
hybrid artificial metalloenzymes.
A limitation with the
defined as the amount of enzyme required to oxidize
approach of binding a metal catalyst into the active site

SHOKRI ET AL.
5 of 15
of the enzyme is that the natural enzyme activity is
inhibited. Therefore, the search for new successful strate-
gies in the construction of artificial metalloenzymes
that maintain the activity and stereoselectivity of enzyme
with the original function of the active site has become
a very active area of research. In this context,
several hybrid catalysts with metalloenzyme-like proper-
3.2 | Characterization of Pd-
laccase@MMCF
Scanning electron microscopy (SEM) was performed to
elucidate the surface morphology and size of the Pd-
Laccase@MMCF particles. As shown in SEM image in
Figure 2, the catalyst is composed of aggregated particles
with a size range of 21–62 nm, confirming the nanostruc-
ture of the prepared catalyst.
[27]
ties have been devolved.
Backvall and co-workers
designed heterogenuse hybrid catalyst consisting of
palladium nanoparticles and lipase enzyme that has the
properties of a metalloenzyme for cooperative tandem
In order to show the elemental composition of the
catalyst, the EDX analysis of Pd-Laccase@MMCF
nanocomposite was carried out. As is shown in Figure 3,
EDX spectrum reveals the existence of C, N, O, Fe, Si, Pd
and as well as Cu species in catalyst structure. To be
noted that the attendance of Cu element in this analysis
a good indication for the immobilization of laccase. In
addition, the elemental mapping images also confirmed
the presence of C, N, O, Fe, Si, Pd and Cu in the
synthesized nanocomposite (Figure 4). On the other
hand, we have determined the Pd, Cu and Fe contents of
the synthesized nanocomposite by ICP-OES analysis.
This analysis displayed that the Pd-Laccase@MMCF
nanocomposite contained 16.5 wt % Pd, 0.025 wt % Cu
and 10.4 wt % Fe.
[28]
catalysis.
This research prompted us to design a mag-
netically reusable cooperative catalyst system by the
stepwise immobilization of palladium nanoparticles
and laccase enzyme into the cavities of magnetic
mesocellular foam (MMCF) (Scheme 3). As shown in
scheme 3, initially magnetic nanoparticles were grafted
onto the pore surface of the mesocellular foam silica.
Subsequently, the MMCF was modified with 3-amino-
propyltriethoxysilane to introduce amine groups onto
their surfaces. Then, palladium nanoparticles were
immobilized in the amino-functionalized magnetic mate-
rial. Finally, laccase was immobilized by covalent attach-
ment on the functionalized magnetic nanocomposite
to prepare Pd-Laccase@MMCF by glutaraldehyde
crosslinking. In Pd-Laccase@MMCF, each cavity might
act as an artificial metalloenzyme, containing both amino
acid constituents in the form of the immobilized laccase
and a metal cofactor in the form of Pd nanoparticles. This
hybrid catalyst was characterized using SEM, EDX, ICP,
FT-IR and VSM techniques.
FT-IR spectra of succeeding steps in the synthesis of
the hybrid catalyst are presented in Figure 5.
The FT-IR spectrum of MMCF (Figure 5a) indicated
the following peaks: Fe-O stretching vibration at 587 cm ,
Si-O-Si symmetric stretching, asymmetric stretching and
−1
−
1
binding vibrations at 1096, 810 and 466 cm and O-H
−
1
stretching vibration at 3420 cm .
SCHEME 3 The process of Pd-Laccase@MMCF synthesis

6
of 15
SHOKRI ET AL.
FIGURE 2 SEM image of Pd-
Laccase@MMCF
quite alike and no indicative peaks for laccase
were not distinguished in the FT-IR spectrum of Pd-
Laccase@MMCF (Figure 5d).
Figure 6 displays the magnetic hysteresis loops of
MMCF and Pd-Laccase@MMCF nanoparticles. These cur-
ves display zero coercivity and no remanence, suggesting
the synthesized samples are superparamagnetic. Mean-
while, the decrease of saturation magnetization (Ms)
from 19.79 emu.g− of MMCF to 9.10 emu.g of Pd-
Laccase@MMCF was owing to the coating of the MMCF
by non-magnetic materials.
1
−1
3
.3 | Catalytic activity of the Pd-
laccase@MMCF nanoparticles
FIGURE 3 EDX spectrum of Pd-Laccase@MMCF
We examined the aerobic oxidation of alcohols to the
corresponding carbonyl compounds in the presence of
Pd-Laccase@MMCF under mild conditions. To deter-
mine the most appropriate conditions for the oxidation
of alcohols, our investigations focused on the oxidation
of 1-phenyl ethanol (1a) as a model reaction. The oxida-
tion of 1-phenyl ethanol in the presence of Pd-
Laccase@MMCF (0.25 g) in phosphate buffer (0.1 M,
In the FT-IR spectrum of AmP-MMCF (Figure 5b), in
addition to the above vibrations, the characteristic peaks
of –CH groups of the aminopropyl chain were detected
2
−
1
−1 [24]
at 2931 cm , 2,862 cm .
In the case of FT-IR spectrum of GA-Pd-AmP-MMCF
(
Figure 5c), the existence of the C=N stretching
-l
ꢀ
vibration at 1662 cm clearly proved that free –NH2
group of Pd-AmP-MMCF reacted with –CHO group
glutaraldehyde.
pH 4.5, 4 ml)/THF (4%, 1 ml) at 40 C afforded the
corresponding ketone in low yield (Table 1, entry 1). We
wondered whether it is probable to increase the yield of
the reaction by using a mediator. For this purpose, the
model reaction was conducted in the existence of two
[29]
Comparing the FT-IR spectra of Pd-Laccase@MMCF
and GA-Pd-AmP-MMCF demonstrated that they are

SHOKRI ET AL.
7 of 15
FIGURE 4 Elemental mapping of the Pd-Laccase@MMCF
different mediators including hydroquinone (HQ) and
p-benzoquinone (BQ). Notably, the yield of 2a amounted
to 99% in the existence of any of the mediators (Table 1,
entries 2–3). The reaction time in the presence of BQ was
shorter but HQ was selected as the mediator for the sub-
sequent experiments owing to its low toxicity. Next, to
determine the appropriate solvent, the model reaction
was carried using different solvents such as THF/NaPBS,
CH CN/NaPBS, THF and CH CN (Table 1, entries
either THF/NaPBS or CH CN/NaPBS afforded the
3
corresponding product in high yield, the less toxicity of
THF resulted in us choosing THF/NaPBS as the reaction
medium. The next set of experiments was dedicated to
find out the effect of the amount of the mediator and cat-
alyst on the model reaction. The results clearly indicated
that reduction in the amount of HQ or catalyst led to the
decrease in the yield of product (Table 1, entries 7–8).
The effect of the temperature was screened upon the
model reaction. When the reaction temperature was
3
3
2
, 4–6). Even though the oxidation of 1-phenyl ethanol in

8
of 15
SHOKRI ET AL.
FIGURE 5 FT-IR spectra of a) MMCF, b) AmP-MMCF, c) GA-Pd-AmP-MMCF, d) Pd-Laccase@MMCF
In summary, the most appropriate conditions for oxi-
dation of 1-phenyl ethanol (1a) was achieved in the pres-
ence of Pd-Lacasse@MMCF(0.25 g), HQ (0.27 mmol) in
phosphate buffer (0.1 M, pH 4.5, 4 ml)/THF (4%, 1 ml)
ꢀ
under air at 40 C (Table 1, entry 2).
The generality of the developed protocol has been
demonstrated by oxidation of a representative range of
alcohols under the optimized conditions (Table 2).
As can be seen from Table 2, Pd-Laccase@MMCF
works fine in the selective oxidation of a variety of pri-
mary benzylic alcohols to the corresponding benzalde-
hydes in excellent yields, over-oxidation to carboxylic
acids was not detected (Table 2, entries 1–5). Notably, the
catalytic performance of this hybrid catalyst was
influenced by the nature of the substituents in the aro-
matic ring and it was observed that oxidation of primary
benzylic alcohols with electron-donating groups (Table 2,
entry 2) occur faster than for electron-withdrawing ones
(Table 2, entries 3–5). Similarly, the present catalyst sys-
tem also presented high activity and selectivity for the
aerobic oxidation of secondary benzylic alcohols to the
expected ketones (Table 2, entries 6–7). It should be
noted that heterocyclic alcohols were also smoothly
converted into the respective products in excellent yield
and selectivity (Table 2, entry 8). To our delight, both pri-
mary and secondary aliphatic alcohols could be success-
fully oxidized to the respective carbonyl compounds
using the present method in good to high yields (Table 2,
entries 9–11).
FIGURE 6 Magnetization curves of (a) MMCF (b) Pd-
Laccase@MMCF
down to room temperature, the reaction didn't go to
completion and only 50% yield was obtained (Table 1,
entry 9).
Moreover, we surveyed other catalysts including mag-
netic MCF (MMCF), Pd@MMCF, and Laccase@MMCF
instead of Pd-Laccase@MCF in the presence of HQ for
the aerobic oxidation of 1-phenyl ethanol (Table 1,
entries 10–12). MMCF failed to catalyze the desired oxi-
dative reaction (Table 1, entry 10). Using Pd@MMCF as
catalyst led to 30% conversion into the desired product
(Table 1, entry 11). The model reaction was also con-
ducted in the presence of Laccase@MMCF as catalyst. It
was observed that the desired product was obtained in
only 15% GC yield (Table 1, entry 12). Furthermore, we
also examined the aerobic oxidation of 1-phenyl ethanol
with HQ in the absence of Pd-Laccase@MMCF and
found that the desired product was not formed (Table 1,
entry 13). The above results clearly indicate that palla-
dium nanoparticles and laccase immobilized into the cav-
ities of magnetic MCF (heterogeneous catalyst) and HQ
obtain a stepwise electron transfer from substrate to
molecular oxygen and act cooperatively to facilitate the
aerobic oxidation under mild conditions.
In sustainable organic synthesis, the recovery and
reusability of the catalyst are the two most important
properties of catalytic processes. Therefore, to survey
this issue, we carried out the oxidation of 1-phenyl
ethanol (3 mmol) using Pd-Laccase@MCF (0.75 g) and
HQ (0.81 mmol) in phosphate buffer (0.1 M, pH 4.5,
ꢀ
12 ml)/THF (4%, 3 ml) at 40 C. After the completion
of the reaction, the magnetic heterogeneous biocatalyst
was separated with external magnet and washed with
Et O (3 × 5 ml), dried and used in the next oxidation
2

SHOKRI ET AL.
9 of 15
a
TABLE 1 Optimizbic oxidation of 1-phenylethanol
ꢀ
Entry
Catalyst (g)
Solvent
Mediator (mmol)
-
Temperature ( C)
Time (hr)
GC Yield (%)
1
Pd-Laccase@MMCF (0.25)
Pd-Laccase@MMCF (0.25)
Pd-Laccase@MMCF (0.25)
Pd-Laccase@MMCF (0.25)
Pd-Laccase@MMCF (0.25)
Pd-Laccase@MMCF (0.25)
Pd-Laccase@MMCF (0.25)
Pd-Laccase@MMCF (0.2)
Pd-Laccase@MMCF (0.25)
MMCF (0.25)
THF/NaPBS
THF/NaPBS
THF/NaPBS
40
40
40
40
40
40
40
40
r.t.
40
40
40
40
16
16
15
16
16
16
16
16
16
16
16
16
16
40
99
99
99
80
80
80
75
50
-
a
2
HQ (0.27)
BQ (0.27)
HQ (0.27)
HQ (0.27)
HQ (0.27)
HQ (0.15)
HQ (0.27)
HQ (0.27)
HQ (0.27)
HQ (0.27)
HQ (0.27)
HQ (0.27)
3
4
5
6
7
8
9
CH
THF
CH CN
3
CN/NaPBS
3
THF/NaPBS
THF/NaPBS
THF/NaPBS
THF/NaPBS
THF/NaPBS
THF/NaPBS
THF/NaPBS
1
1
1
1
0
1
2
3
Pd@MMCF (0.25)
30
15
-
Laccase@MMCF (0.25)
-
a
ꢀ
Reaction conditions unless stated otherwise: 1-phenylethanol (1 mmol), phosphate buffer (0.1 M, pH 4.5, 4 ml)/THF (4%, 1 ml), air, 40 C.
reaction run. The recovered catalyst was added to fresh
model reaction mixture (1-phenyl ethanol, HQ and
phosphate buffer/THF) under same reaction conditions.
When the reaction time was set to 16 hr, which
proved to be sufficient for conversion of 1-phenyl
ethanol, comparably excellent yields of acetophenone
were obtained in 9 subsequent runs (Figure 7) to give
a total turnover number (TON) of 90,830 for the
laccase catalyst.
Due to the high catalytic efficiency of the Pd-
Laccase@MMCF for the oxidation of alcohols, we
became interested to test the activity of the prepared
catalyst for the aerobic dehydrogenation of 2-substituted-
experimental conditions for the model reaction. The fur-
ther experiments presented in Table 3 revealed that
larger amount of HQ resulted in complete substrate con-
version to the corresponding product.
With the reaction conditions explained above
(Table 3, entry 3), dehydrogenation of a number of
2-substituted-2,3-dihydroquinazolin-4(1H)-ones was eval-
uated. The results summarized in Table 4 assert that
2-substituted −2,3-dihydroquinazolin-4(1H)-ones with
electron-donating groups are more appropriate substrates
than for electron-withdrawing ones. The oxidation of
2-substituted-2,3-dihydroquinazolin-4(1H)-ones carrying
the electron-withdrawing groups also afforded the respec-
tive products in high yields but in these conditions longer
reaction times were required (Table 4, entries 8–10).
2
,3-dihydroquinazolin-4(1H)-ones to Quinazolin-4(3H)-
ones. Quinazolin-4(3H)-ones are important nitrogen-
containing heterocycles which have various biological
[30,31]
and medicinal activities.
-aminobenzamides with aldehydes followed by subse-
quent oxidation is common method for the synthesis of
The cyclization of
2
3.4 | Selected spectral data of
2-substituted-2,3-dihydroquinazolin-4(1H)-
ones
[32,33]
quinazolin-4(3H)-ones.
Under the conditions developed for the oxidation
of alcohols, the aerobic oxidation of 2-phenyl-2,-
[
34]
2-phenylquinazolin-4(3H)-one (Table 4, entry 1):
1
3
-dihydroquinazolin-4(1H)-one was conducted as
model reaction, which gave the corresponding product in
5% yield. This result managed us to acquire the optimal
a
H NMR (DMSO-d , 250 MHz): δ (ppm) = 12.50
6
(s, 1H), 8.16–8.14 (m, 3H), 7.80–7.69 (m, 2H), 7.54–7.49
(m, 4H).
8

10 of 15
SHOKRI ET AL.
a
TABLE 2 Aerobic oxidation of various alcohols
b
Entry
substrate
product
Time (hr)
GC Yield (%)
1
18
99
2
3
4
5
17
20
19
20
99
97
97
97
6
7
16
19
19
99
99
99
8
9
24
20
97
97
1
0

SHOKRI ET AL.
11 of 15
TABLE 2 (Continued)
b
Entry
substrate
product
Time (hr)
GC Yield (%)
1
1
20
96
a
General procedure: Substrate (1 mmol), Pd-Laccase@MMCF (0.25 g), HQ (0.27 mmol), phosphate buffer (0.1 M, pH 4.5, 4 ml)/THF (1 ml),
ꢀ
air, 40 C.
b
GC yields were determined by internal standard method.
2
-(4-Methylphenyl)quinazolin-4(3H)-one (Table 4,
[
34]
1
entry 2):
H NMR (DMSO-d , 250 MHz): δ
6
(
ppm) = 12.61 (s, 1H), 8.19–8.09 (m, 3H), 7.79 (t,
J = 7.25 Hz, 1H), 7.67 (d, J = 8 Hz, 1H), 7.45 (t,
J = 7.25 Hz, 1H), 7.05 (d, J = 8.5 Hz, 2H), 2.47 (s, 3H).
2
-(4-Methoxyphenyl)quinazolin-4(3H)-one (Table
[
34] 1
4
, entry 3):
H NMR (DMSO-d , 250 MHz): δ
6
(
ppm) = 12.38 (s, 1H), 7.04–8.18 (m, 8H), 3.81 (s, 3H).
2
-(3-Fluorophenyl)quinazolin-4(3H)-one (Table 4,
1
entry 8): H NMR (DMSO-d , 250 MHz): δ (ppm) = 12.59
6
(
s, 1H), 8.15 (d, J = 7.75 Hz, 1H), 8.057–7.970 (m, 2H),
7.86–7.27 (d, J = 7.5 Hz, 2H), 7.63–7.40 (m,3H).
FIGURE 7 The recyclability of Pd-Laccase@MMCF as a
catalyst in the aerobic oxidation of 1-phenyl ethanol
2-(3-Bromophenyl)quinazolin-4(3H)-one (Table 4,
[
35]
1
entry 9):
H
NMR (DMSO-d6, 250 MHz): δ
a
TABLE 3 Optimization of reaction conditions for oxidative dehydrogenation of 2-phenyl-2,3-dihydroquinazolin-4(1H)-one
ꢀ
Entry
Catalyst amount (g)
HQ (mmol)
0.27
Temperature ( C)
GC Yield (%)
1
2
3
Pd-Laccase@MMCF (0.25)
Pd-Laccase@MMCF (0.3)
Pd-Laccase@MMCF (0.25)
40
40
40
85
85
99
ꢀ
0.27
0.34
a
Reaction conditions: Pd-Laccase@MMCF (0.25 g), HQ (0.34 mmol), air, phosphate buffer (0.1 M,pH 4.5, 4 ml)/THF (4%, 1 ml), 20 hr, 40 C.

12 of 15
SHOKRI ET AL.
a
TABLE 4 Aerobic dehydrogenation of 2-substituted −2,3-dihydroquinazolin-4(1H)-ones
ꢀ
Entry
Substrate
Product
Time (hr)
Isolated Yield (%)
M.P. ( C) [Ref.]
[
[
[
34]
34]
34]
1
20
92
236–238
238–240
244–247
2
3
18
16
95
94
[
34]
4
5
6
16
16
16
95
92
95
181–182
242–243
257–260
[
35]
[
34]
[
36]
7
18
91
235–238
8
30
90
288–289

SHOKRI ET AL.
13 of 15
TABLE 4 (Continued)
ꢀ
M.P. ( C) [Ref.]
Entry
Substrate
Product
Time (hr)
Isolated Yield (%)
[
35]
9
30
32
87
87
295–296
363–364
[
37]
1
0
a
Reaction conditions: Substrate (1 mmol), Pd-Laccase@MMCF (0.25 g), HQ (0.34 mmol), air, phosphate buffer (0.1 M, pH 4.5, 4 ml)/THF
1 ml), 40 C.
ꢀ
(
that Pd (II) atoms are actual oxidizing agents. After
treatment of Laccase with HQ, the active Pd (II) atoms
required for the oxidation reaction are generated
through adsorption of BQ to the Pd(0) nanoparticles in
[
38]
Pd-Laccase@MMCF
(Scheme 4). Laccase can be
reoxidized by molecular oxygen, thus completing the
catalytic cycle (Scheme 2).
4
| CONCLUSIONS
To summarize, we have prepared a magnetically
recoverable cooperative catalyst oxidation system by
immobilizing palladium nanoparticles and laccase on
MMCF, which were then applied an excellent catalyst for
aerobic oxidative dehydrogenation of C–O and C–N
bonds using an ideal oxidant under mild conditions. The
advantages of these protocols are the use of air as the
cheapest and greenest oxidant available, the mild reac-
tion conditions and good to excellent yield of the prod-
ucts. It is also noteworthy that the immobilization of
both of laccase and Pd onto magnetic foam provides a
heterogeneous catalyst system that can be easily sepa-
rated using an external magnetic field and reused several
times without a significant loss of activity.
SCHEME 4 Pd (II) atoms are generated after treatment of BQ
with Pd(0) atoms on Pd-Laccase@MMCF
(ppm) = 12.62 (s, br, 1H), 8.37 (s, 1H), 8.20 (d, J = 7.5 Hz,
1
7
H), 8.17 (d, J = 7.5 Hz, 1H), 7.88–7.76 (m, 3H),
.57–7.50 (m, 2H).
Although, the actual role of present cooperative
catalyst system is not exactly clear now, however on the
basis of previously reported mechanisms for the
application of Pd@MCF/BQ catalyst system in the
[
24,38]
oxidation reactions
and aerobic oxidation of HQ to
[19]
BQ in the presence of laccase,
it is hypothesized

14 of 15
SHOKRI ET AL.
ACKNOWLEDGEMENTS
We gratefully acknowledge financial support of this
research by University of Kurdistan.
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1990, 112, 5160.
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