Co-immobilization of Laccase and TEMPO onto Glycidyloxypropyl Functionalized Fibrous Phosphosilicate Nanoparticles for Fixing CO2 into β-Oxopropylcarbamatesin

Article abstract of DOI:10.1007/s10562-019-02894-5

Abstract: TEMPO or Anchoring 2,2,6,6-tetra-methylpiperidine-1oxyl radical into the nanospaces a fibre of phosphosilicate with laccase compound causes an unheard potent to be producing which called bifunctional nanocatalyst (TEMPO@FPS-laccase). TEMPO@FPS-l

Full text of DOI:10.1007/s10562-019-02894-5

Catalysis Letters
https://doi.org/10.1007/s10562-019-02894-5
Co‑immobilization of Laccase and TEMPO onto Glycidyloxypropyl
Functionalized Fibrous Phosphosilicate Nanoparticles for Fixing CO2
into β‑Oxopropylcarbamatesin
Liqun Fan1 · Jinhu Wang1 · Xianman Zhang1 · Seyed Mohsen Sadeghzadeh2,3 · Rahele Zhiani2,3 · Mina Shahroudi2,3
Fatemeh Amarloo2,3
·
Received: 15 April 2019 / Accepted: 8 July 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
TEMPO or Anchoring 2,2,6,6-tetra-methylpiperidine-1oxyl radical into the nanospaces a fbre of phosphosilicate with lac-
case compound causes an unheard potent to be producing which called bifunctional nanocatalyst (TEMPO@FPS-laccase).
TEMPO@FPS-laccase indicated proper catalytic activity for synthesis of β-oxopropylcarbamates in aqueous medium with-
out any pollutants through a multi component coupling of CO2, amines and propargyl alcohols in moderate condition. Free
laccases may not be recovered but can be easily disabled in diferent environmental conditions. Enzyme immobilization is
known as an expanding way to enhance resistor to extreme conditions and stability as well as recycled of laccase.
Graphic Abstract
O
O
R1
OH
R2
H
N
R2
R3
CO2
N
R4
O
+
+
R3
R4
R1
TEMPO
laccase
laccase
TEMPO
TEMPO
laccase
FPS
Keywords Nanocatalyst · Green chemistry · Kinetic · One-pot synthesis · Nanoparticle · Laccase
2
* Jinhu Wang
wangjinhu@uzz.edu.cn
Young Researchers and Elite Club, Neyshabur Branch,
Islamic Azad University, Neyshabur, P.O. Box 97175-613,
Iran
* Seyed Mohsen Sadeghzadeh
3
seyedmohsen.sadeghzadeh@gmail.com
New Materials Technology and Processing Research Center,
Department of Chemistry, Neyshabur Branch, Islamic Azad
University, Neyshabur, Iran
1
College of Chemistry, Chemical Engineering and Materials
Science, Zaozhuang University, Zaozhuang 277160,
Shandong, China
Vol.:(0123456789)
1 3

L. Fan et al.
Various scholars have investigated the application of
immobilized laccase in the production of organic materials,
nevertheless, some laccase-catalysed reactions inevitably
require the use of intermediate reagents with high costs, such
as 2,2,6,6-tetramethyl-1-piperidinyloxy, creating recycling of
both highly favourable enzyme and mediator, particularly for
large-scale reactions [18]. Co-immobilization of a laccase or a
mediator in materials that have some porosity should hence be
proper, because each cavity of the support will have deferent
active species [19].
1 Introduction
In the recent years, many studies have focused on the
chemical fxation of CO2 due to its low toxicity, cheap,
unfamable, ubiquitous and reproducible attributes, that
shows great facilities for the making of C–N and C–C
bonds in organic production. These properties of CO2 may
be ascribed to its kinetic inertness and great stability in
term of thermodynamic properties. Recently, signifcant
approach has been obtained in this subject. Using CO2 to
produce diferent organic compound, like amides, esters,
alcohols, carbonates and carboxylic acids as well as car-
bamates. It has been favored with diferent efcient cata-
lytic approaches. Considerable progress has been built,
and several approaches have been developing in the case
of the reaction of CO2 to products like cyclic carbonates
and urethanes as well as salicylic acid and so on. One of
these composite, carbamates are one of important diverse
composites. Thus, utilizing carbon dioxide to synthesis
an eco-friendly approach that could efciently admit car-
bamates is attractive [1–4]. Among them, carbamates like
β-Oxopropylcarbamates have been vastly utilized in agron-
omy, organic production, and pharmaceutics. Most newly,
for production of boxopropylcarbamates were applied from
CO2, secondary amines and propargylic alcohols. In addi-
tion, diferent catalytic approaches like Ruthenium [5, 6],
Fe [7], Cu [8, 9], Ag [10], and bicyclic guanidine [11] have
been expanded to the multicomponent reaction. Cu, and
Ag have been illustrated to be the most impressive cata-
lysts to this reaction. Silver compounds have been showed
to be the huge transition metal catalysts with particular
selectivity, and they have consider able benefts over other
catalysts.
Inspired by the high accessibility and low difusion limita-
tions of fbrous silica nanoparticles (KCC-1), fbrous phospho-
silicate (FPS) was engineered using a microemulsion system
[20, 21]. In the present study, an approach for the making of
an impressive bifunctional hybrid catalyst through co-immo-
bilization of TEMPO as well as laccase in the same cavities
into fbres of FPS and utilized to the one-pot production of
β-oxopropylcarbamates via the three-component reaction of
propargylic alcohols and amines and CO2 at moderate condi-
tions. Exclusively, this catalytic apparatus might be quickly
recycled about ten times by the lowermost catalyst loading
ever reported. These great catalytic performances are ascribed
to the usage of the enzyme that might do as the dual activators
to both substrates and CO2 (Scheme 1).
2 Experimental Section
2.1 Materials and Methods
All the chemical substances that were utilized in the present
paper had high purity and were purchased from Fluka and
Merck. Melting points of materials were obtained in open
capillaries by taking advantage of an Electrothermal 9100
system. For all the purchased powders, FTIR spectra were
mapped on a spectrometer of VERTEX 70 with the mode of
transmission in spectroscopic grade KBr spherical pellets. The
size and structure of nano particles was considered utilizing
a microscope (Philips CM10 transmission electron that acti-
vated at 100 kV). Powder X-ray difraction information were
used by Bruker D8 Advance model and considering Cu ka
radiation. The thermogravimetric analysis (TGA) was done
on a NETZSCH STA449F3 under a constant heating rate of
10 °C min−1 and in the present of inert gas (nitrogen). Elemen-
tal analyses for atoms N, H and C were doneutilizing a Heraeus
CHN–O-Rapid analyser. The purity defnition of the products
as well as reaction monitoring were carried out using TLC on
Laccase can be obtained from many sources such as
plants, fungi and insects. Laccase is a multicopper oxidase
[12]. In the catalytic center of laccase, four copper atoms
exist. It catalyses the oxidation of polyamines, polyphe-
nols, benzenethiols and phenols by and reducing oxygen to
H2O [13–15]. In the recent years, they have attracted great
attention and has been utilized in several usages, like pro-
duction of the biofuel, pulp bleaching, bioremediation, and
organic synthesis [16, 17]. However, it is hard to obtain
free Laccasses and their efciency in the environment of
reaction is severely reduced that decrease their application
in the industry.
Scheme 1 Synthesis of
O
R2
R1
R1
β-oxopropylcarbamates in the
CO2
H
O
TEMPO@FPS-laccase
N
+
+
OH
R2
presence of TEMPO@FPS-
R3
R4
R3
laccase NPs
N
R
O
1 3

Co-immobilization of Laccase and TEMPO onto Glycidyloxypropyl Functionalized Fibrous…
silica gel polygram SILG/UV 254 plates. Mass spectra were
obtained on Shimadzu GCMS-QP5050 Mass Spectrometer.
chromatography onto silica gel by petroleum ether/ethyl
acetate (100:1–20:1).
2.2 General Procedure for the Preparation of FPS
3 Results and Discussion
In a stirred solution with a specifc volume (1.5 mL) of
1-pentanol and 30 mL of cyclohexane and, 3.7 g of trip-
olyphosphate as well as 2.0 g of tetraethyl orthosilicate
(TEOS) were dissolved. A solution contained of 1 g of CPB
and also 0.5 g of urea in 30 mL of water was enhanced to the
introduced mixture. The produced mixture was then stirred,
continually, for 2700 s at r.t. and after that putted into a
reactor and heated at a temperature of 120 °C for 5 h. FPS
was isolated for energy saving by centrifugation and washed
utilizing deionized water and acetone as well as dried by a
drying oven.
In the present work, FPS solution was produced with regards
to reported methods and then became applicable by using
(3-glycidyloxypropyl)trimethoxysilane, followed by reduc-
tive amination to make the corresponding co-immobilized
TEMPO as well as enzyme. This process can be observed
in Scheme 2.
The morphology and the physical structure of TEMPO@
FPS-laccase NPs and FPS were analysed with FESEM and
TEM analyses (Fig. 1). As seen in Fig. 1a and c, the FPS
solution sample includes of wall-like domains with stable
sizes of wall. Scrutiny of TEM and FESEM scheme indi-
cated that the TEMPO@FPS-laccase is consist of dendri-
meric fbers with thicknesses of 8–10 nm ordered in three
dimensions to create walls, which can allow straight for-
ward access to the high level available. From FESEM and
TEM analyses of TEMPO@FPS-laccase NPs, it can be seen
that this solution is not change after changing the morphol-
ogy of NPs (Fig. 1b and d). Figure 2 demonstrates TGA
analysis of TEMPO@FPS-laccase NPs. The deletion of the
solvent of chemisorbed and physisorbed on the surface of
the TEMPO@FPS-laccase material leading the weight loss.
In addition, weight loss in range of 250–450 °C is almost
26.5 wt%, that is relevant to the organic group derivatives.
Reducing the weight at this range of temperature can be
rationalized using oxidation of TEMPO and laccase by about
180–350 °C. In fact, the residual mass after the decomposi-
tion of TEMPO@FPS-laccase NPs is because of exiting the
FPS NPs.
2.3 General Procedure for the Preparation of FPS/
GMSI NPs
Twenty milliliter of THF was added to 200 mg of FPS. Then,
0.002 mol of NaH was difused by ultrasonication. 0.022 mol
of (3-glycidyloxypropyl)trimethoxysilane was added in r.t.
and stirred for 16 h under a constant temperature of 50 °C.
The product was washed by deionized water and an alcohol,
then under vacuum dried for 3 h at a temperature of 50 °C.
2.4 General Procedure for the Preparation
of TEMPO@FPS‑laccase NPs
Some FPS, about 100 mg, activated by glycidyloxypropyl
was released in 10 mL of acetate bufer (0.1 M and pH 4.5)
comprising a constant amount of 4-hydroxy-TEMPO and
laccase. The suspension was shaken under speed of 120 rpm
for a few hours to produce nanoparticles containing TEMPO
and co-immobilized laccase. By using acetate bufer, the
resultant nanoparticles were washed several times and stored
under a temperature of 4 °C.
O
O
O
O
2.5 General Procedure for Catalytic Synthesis
of β‑Oxopropylcarbamates
TEMPO
Si
Si
O
O
O
O
O
O
laccase
Catalyst by about 5 mg, propargylic alcohols 4.95 mmol in
water 0.5–1 mL and secondary amines by around 5 mmol
were mixed with a Schlenk tube that equipped to a stir bar.
After that the apparatus was clean with carbon dioxide for
more than two times, the blend was stirred under the tem-
perature of 50 °C and pressure of 1.5 bar for carbon dioxide
for the desired time. When the considered reaction fnished,
the mixture was adapted by diethyl ether (3–15 mL). The
upper layers were gathered and dried by vacuuming to obtain
the crude yields that might be purifed using more column
EtO
+
EtO Si
EtO
O
FPS
O
Scheme 2 The process of co-immobilization of laccase and TEMPO
onto glycidyloxypropyl-functionalized FPS nanoparticles
1 3

L. Fan et al.
Fig. 1 TEM images of FPS NPs
(a); TEMPO@FPS-laccase NPs
(b); FESEM images of FPS NPs
(c); and TEMPO@FPS-laccase
NPs (d)
100
95
90
85
80
75
70
indicated using the brighter yellowish white color increased
with reducing T/W, ofering the enhance in the catalyst sur-
face roughness.
FTIR spectra indicated the existence of surface hydroxyl
and silanols as well as phosphate groups. Figure 4 showed
(a): FPS, (b): amino functionalized FPS nanoparticles.
The pure FPS indicated a common broad peak by about
3399 cm−1 afliated with the existence of hydroxyl groups.
As seen, the intensity of this peak enhances that could be
because of the existence of immobilized phosphate group on
the structure of the FPS. The FPS substance sindicated addi-
tional peak about 1483 cm−1 that is attributed to the phos-
phate moiety achieving from TPP [22]. The basic peak in
the spectrum of FPS is the extra peak emerged at 1232 cm−1,
that can be related to the –P=O stretching vibration showing
the existence of phosphate group [23, 24]. The band around
963 cm−1 and the shoulder around 1108 cm−1 are because
of exiting the TO and LO modes of asymmetric stretching
of Si–O–P bonds [25, 26]. The band at around 724 as well
as 795 cm−1 were related to the asymmetric expanding of
the bridging oxygen atoms to a phosphor atom [27]. As seen
in Fig. 5a, these peaks ofered proper reaction between both
0
100
200
300
400
T/oC
500
600
700
800
Fig. 2 TGA diagram of TEMPO@FPS-laccase NPs
Figure 3 shows the XRD pattern of FPS and TEMPO@
FPS-laccase NPs. It displays a number of crystalline peaks,
frm with a same reports (Fig. 3a). Figure 3b indicates a
common XRD pattern of the TEMPO@FPS-laccase NPs.
There was no variation. The roughness of TEMPO@
FPS-laccase NPs surface was determined by atomic force
microscopy analysis (AFM). Figure 4 shows the topographic
images of it. As seen in Fig. 4, the greater height region
1 3

Co-immobilization of Laccase and TEMPO onto Glycidyloxypropyl Functionalized Fibrous…
Fig. 3 XRD analysis of a FPS
NPs; b TEMPO@FPS-laccase
NPs
FPS-laccase NPs due to low bicontinuous concentric
TEMPO and also laccase morphology of the nanocatalyst.
The nitrogen adsorption–desorption isotherms of FPS sup-
ported catalysts can be seen in Fig. 6. The FPS indicated a
type IV isotherm, with a H1-type hysteresis loop, proposing
the existence of mesopores. The related pore size section
predicted by the desorption branch of the nitrogen isotherm
by the BJH procedure displayed a narrow pore measure
repartition peaked at 9 nm (Table 1). The large mesopore
size of FPS with high capacity may load TEMPO and lac-
case which have comparative large molecular size.
We began investigation by the synthesis of
β-oxopropylcarbamate as model substrates in existence of
TEMPO@FPS-laccase NPs by 5 mg that is a nanocatalyst
substance. At the beginning, we examined the infuence of
various solvents on fnal product. Table 2 shows the results
of our investigation. Trace amount of β-oxopropylcarbamate
was obtained for n-Hexane, Toluene and Cyclohexane (refer
to Table 2, Entries 15–17). It is determined that the reaction
performed proper in CHCl3, i-PrOH CH2Cl2, CH3CN, EtOH,
MeOH and CCl4, (Table 2, Entries 1–7). The best product
(yield 97%) was obtained by adding water as the reaction
environment for 12 h at a temperature of 60 °C (Table 2,
Entry 14). According to the obtained results, the water is
proper solvent due to its eco-friendly property synthesis of
β-oxopropylcarbamate. As seen in Table 2 (Entry 20), the
minimum essential temperature to carry out the reaction
was studied. After investigating various temperatures, it was
determined that temperature of 50 °C is the best temperature
for this reaction. The temperature more than 50 °C did not
Fig. 4 Three-dimensional of AFM images of TEMPO@FPS-laccase
NPs
the TEOS and the TPP. These obviously demonstrate the
grafting of GMSI onto the FPS surface. The GMSI-FPS
composite indicates bands at about 1091 and 793 as well
as 462 cm−1. A strong and broad absorption band at around
3000–3550 cm−1 is associated to the -OH and -NH stretch-
ing vibrancies. Figure 5b indicates that peak emerged at
around 2930 cm−1 are because of the stretching of the C–H
aliphatic group.
The analysis of Nitrogen physisorption indicated that the
specifc surface of BET area of FPS as well as TEMPO@
FPS-laccase were 679, and 378 m2/g. The decrease surface
area of TEMPO@FPS-laccase in comparison with FPS
can be due to the specifying TEMPO in the FPS. In addi-
tion, the decrease of surface area was clearer in TEMPO@
1 3

L. Fan et al.
Fig. 5 FTIR spectra of a FPS
NPs, and b GMSI-FPS NPs
Fig. 6 Adsorption–Desorption isotherms of the FPS NPs (a); TEMPO@FPS-laccase NPs (b); and BJH pore size distributions of the FPS NPs
(c); TEMPO@FPS-laccase NPs (d)
1 3

Co-immobilization of Laccase and TEMPO onto Glycidyloxypropyl Functionalized Fibrous…
Table 1 Structural parameters of FPS, and TEMPO@FPS-laccase
product was obtained for that spreading about 3 mg cata-
lyst after 12 h persistence. Increasing the catalyst amount
to 5 mg enhanced the product of β-oxopropylcarbamate to
97%. Adding more than 5 mg of TEMPO@FPS-laccase NPs
is not proper to the fnal yield amount (Table 2, Entry 22).
Entry 26 and 27 showed the infuences of time. Product yield
enhanced by increasing the time from 8 to 10 h. More time
to 10 h did not afect the fnal yield.
NPs
Catalysts
SBET (m2 g−1
)
Va (cm3 g−1
)
DBJH (nm)
FPS
679
3.3
2.1
9
4
TEMPO@FPS- 378
laccase
In addition, to enhance production of β-oxopropylcarbamate,
carbon dioxide pressure was studied. The proper pressure of
CO2 for yielding the best production of TEMPO@FPS-lac-
case NPs would be specifed whereas the kinetics of the mass
transfer reactions might be changed using the difusion and
the reaction between CO2, and 2-methylbut-3-yn-2-ol, pyr-
rolidine. The infuence of pressure was experimentally deter-
mined ranging from 0.5 to 3.5 Mpa pressure. The function of
TEMPO@FPS-laccase NPs increased forcefully while the CO2
pressure enhanced from 0.5 to 1.5 Mpa pressure, after that it
supportable in the pressure ranging from 1.5 to 3.5 MPa that
can be observed in Fig. 7. These contrary facts determined the
requirement for an optimum pressure around 1.5 bar for the
best products of β-oxopropylcarbamate.
Table 2 Synthesis of β-oxopropylcarbamate by TEMPO@FPS-lac-
case NPs in diferent solvents, temperature, amount catalyst, and time
Entry Solvent
Tem-
Catalyst (mg) Time (h) Yield (%)a
perature
(°C)
1
2
3
4
5
6
7
8
EtOH
60
60
60
60
60
60
60
60
60
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
5
3
1
–
5
5
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
10
8
23
34
31
31
28
38
36
28
82
26
53
76
82
97
7
11
–
16
–
97
85
97
63
21
–
MeOH
i-PrOH
CH2Cl2
CH3CN
CHCl3
CCl4
DMF
DMA
1,4-Dioxane 60
EtOAc
THF
DMSO
H2O
Toluene
n-Hexane
Cyclohexane 60
Anisole 60
Solvent-free 60
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
A time period investigation for production of
β-oxopropylcarbamate was performed by immobilized
TEMPO and immobilized laccase at diferent ratios (1:1,
1:2, 1:3, 1:4, and 1:5). As seen in Fig. 8, the best propor-
tion of catalyst was achieved at the ratio of 1:1 and after
10 h. CO2 cannot fxing into β-oxopropylcarbamatesin using
the immobilized laccase and immobilized TEMPO singly
(Fig. 9). Nevertheless, it can be observed that the co-immo-
bilized TEMPO and laccase displaying a large capacity for
synthesis of β-oxopropylcarbamate, which determined a
97% product after 10 h. The obtained results also confrmed
that the TEMPO and laccase were properly supporting on
the FPS. This is for the fact that the immobilized laccase
and immobilized TEMPO cannot oxidase benzyl alcohol,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
60
60
60
60
60
60
50
40
50
50
50
50
50
50
100
80
60
40
20
0
97
85
aIsolated yield
enhance yield. Another parameter which should be tested for
the introduced reaction is amount of TEMPO@FPS-laccase
NPs. The reaction was done for 12 h and under temperature
of 50 °C, in the existence catalyst (1 mg, 3 mg, 5 mg, and
7 mg). As seen in Table 2 (Entry 25), without using a cata-
lyst, the reaction failed to yield the β-oxopropylcarbamate.
As predicted, selecting the TEMPO@FPS-laccase NPs as
catalyst made a distinct change in the product amount. The
reaction which using 1 mg catalyst made 21% yield and 63%
0
1
2
3
4
CO2 Pressure (MPa)
Fig. 7 The efect of CO2 pressure on the synthesis of
β-oxopropylcarbamate
1 3

L. Fan et al.
Table 3 Scope of the reaction of propargylic alcohols, secondary
amines and CO2
Entry Amine
Alcohol
Product
Yielda (%)
97
O
1
O
OH
NH
N
O
O
O
N
O
2
82
94
OH
NH
O
OH
3
O
N
O
NH
O
OH
4
80
O
N
O
NH
Fig. 8 Time course study of product by ratios of each component of
O
the catalyst
OH
5
56
77
O
NH
N
O
O
OH
6
NH
O
N
O
O
OH
Ph
7
65
86
O
NH
N
Ph
O
O
OH
8
NH
O
O
N
O
9
O
91
95
OH
OH
NH
N
O
O
10
O
O
O
NH
O
N
Fig. 9 Synthesis of β-oxopropylcarbamate by diferent FPS nanopar-
O
ticles
11
92
O
OH
NH
N
O
properly, however, the of the co-immobilized laccase and
TEMPO, this limitation was dominate with the adding
mediator of TEMPO. Notably, there was not much diference
in the reaction yields when reaction was carried out using
TEMPO@FPS-laccase NPs and TEMPO/laccase catalyst,
however, TEMPO/laccase is not recoverable and reusable
for the next runs. These observations show that the reaction
cycle is mainly catalyzed by TEMPO/laccase on the FPS
nanostructure.
O
O
n-Bu
n-Bu
n-Bu
O
O
OH
OH
NH
N
12
13
88
65
n-Bu
O
O
NH
N
O
OH
OH
NH
O
14
15
74
48
N
O
O
NH
Since the conditions of reaction are improved, we then
tested the substrate range using examination of the reac-
tivity of aryl or alkyl substituents by diferent secondary
amines. The reaction of pyrrolidine with various terminal
propargylic alcohols directing alkyl substituents in the
propargylic position underwent at the same condition and
admitted the corresponding carbamates for yielding great
O
O
N
HO
O
NH
OH
HO
O
O
16
59
N
aGC yields (%)
1 3

Co-immobilization of Laccase and TEMPO onto Glycidyloxypropyl Functionalized Fibrous…
Scheme 3 Proposed catalytic
mechanism of the TEMPO@
FPS-laccase system
TEMPO
O H
R1
R2
TEMPO
laccase
CO2
laccase
O H
R1
R2
O
O
-O
O
R1
O
R2
O
laccase
O
O
O
laccase
R1
R2
R1
R2
TEMPO
R3
NH
R4
O
O
O
NR3R4
OH
NR3R4
O
O
R1
R1
R2
R2
100
90
80
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
Reuse
Fig. 10 Stability of the free and immobilized laccase at 5, and 25 °C
for 30 days
Fig. 11 The
reusability
of
catalysts
for
synthesis
of
β-oxopropylcarbamate
yield (as observed in Table 3, Entries 1–4). Commonly,
tertiary alcohols are further active compared to primary
alcohols and secondary alcohols and simultaneously indi-
cated distinct reactivity with various substituent groups.
Propargylic carbonate intermediate was quickly produced
via the reaction of tertiary alcohols and carbon dioxide
1 3

L. Fan et al.
It important to know that the heterogeneous attribute of
TEMPO@FPS-laccase NPs reduce its recovery property
from the environment of reaction. Some power of catalyst
of the recycled TEMPO@FPS-laccase NPs was investi-
gated at the proper conditions. At the end of the reaction,
TEMPO@FPS-laccase NPs was separated by taking advan-
tage of fltration process and it’s washed by alcohols and
dried by using the pump. The laccase activity was reduced
only about 3.9% after 10 processes and no considerable
deactivation of the hybrid catalyst was seen after 6 pro-
cesses (can be observed in Fig. 11). This reusability also
demonstrates the high stability of this new heterogeneous
system.
Eventually, we ran a leaching study to check whether
this catalytic apparatus is really heterogeneous or whether
this catalysis is promoted homogeneously using some of
laccase and TEMPO substances leaching in the solution.
The reaction was primarily mount using optimized condi-
tions, in the existence of a ten-times reused batch of the
catalyst. After a few hour ago, catalyst was deleted using
hot fltration and the remained solution was stirred for six
more hours. Figure 12 indicates the reaction in contrast to
time with a ten-times reused catalyst batch (can be seen as
a green curve) and how no more reagent use was discov-
ered after the catalyst was deleted from the mixture (can
be seen as a blue curve).
Fig. 12 Leaching
test
for
catalyst
for
synthesis
of
β-oxopropylcarbamate
by the support of proper catalysts. Propargylic alcohols
by cycloalkyl substitution done lower reactivity (can be
observed in Table 3, Entries 5 and 6), and the reason
would be the steric hindrance infuence on the carbon-
ate intermediate formation. In addition, other propargylic
alcohols by unsaturated groups such as phenyl-substitution
propose good products after adding the certain value of
co-catalyst. Moreover, secondary amines represented good
reactivity. Nevertheless, for the Entries 16, some lower
reactivity was determined because of the steric hindrance
in methyl or cyclohexyl within amines pending the nucleo-
philic add to a-methylene carbonate.
4 Conclusions
In summary, a very efective hybrid catalyst was syn-
thesized on basis of heterogeneous substances including
fundamentally various catalytic species. In this multi-
functional catalyst, the laccase and mediator collaborate
in an unprecedented procedure. The hybrid catalyst
was well characterized, and the co-immobilization of
both TEMPO and enzyme on the fbres of the FPS was
observed. TEMPO@FPS-laccase indicated proper cata-
lytic activity for production of β-oxopropylcarbamates
via the three-component coupling reaction of CO2, pro-
pargylic alcohols and amines. This green method showed
some attractive environmentally friendly characteristics,
like the simplicity of catalyst improvement from the reac-
tion mixture by fltration. Heterogeneous bio-catalysts can
enhance the efciency of the catalytic apparatus in green
organic production, exclusively in the presence of eco-
friendly solvents.
Relying on the above yields, a proper mechanism of
TEMPO@FPS-laccase NPs is showed and it has been deter-
mined in Scheme 3. Firstly, TEMPO provide the –OH of the
2-methylbut-3-yn-2-ol and enhance the nucleophilicity of the
–OH to the CO2 that is snared and puted using the synergistic
infuences of TEMPO. Next, the species of laccase activates a
triple bond to provide the composition of the charged oxygen
by the carbon in the triple bond, causing the production of
the fve-membered ring. Moreover, the catalyst is dropped via
fve-membered ring and the main free intermediate is made.
Finally, the amine ofensives the carbonyl the intermediate
group, followed with the tautomerism of enol to ketone as well
as the production of the corresponding β-oxopropylcarbamates.
Laccase based on FPS NPs hold its activity for a thirty-
day storage term under a temperature of 5 °C. Study indi-
cated that immobilized laccase demonstrated better storage
stability compared to free laccase at both selected tempera-
tures (can be observed in Fig. 10). The free laccase was
undo after few days’ incubation at under temperatures of 5
and 25 °C, respectively. Some studies have presented cyto-
toxicity and mutagenicity in mammalian cells due to having
TEMPO.
Acknowledgements The study was supported by “Natural Science
Foundation of Shandong Province (Grant Nos. ZR201702200464,
ZR2019QB013)” and “Science and Technology Development Plan
Project of Zaozhuang City (Grant No. 2019GX07)”.
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Co-immobilization of Laccase and TEMPO onto Glycidyloxypropyl Functionalized Fibrous…
15. Mate DM, Alcalde M (2015) Biotechnol Adv 33:25–40
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