Continuous Flow Chemo-Enzymatic Baeyer-Villiger Oxidation with Superactive and Extra-Stable Enzyme/Carbon Nanotube Catalyst: An Efficient Upgrade from Batch to Flow

Article abstract of DOI:10.1021/acs.oprd.9b00132

Continuous flow chemo-enzymatic Baeyer-Villiger oxidation in the presence of exceptionally active Candida antarctica lipase B immobilized via simple physical adsorption on multiwalled carbon nanotubes has been investigated. The nanobiocatalyst was used to generate peracid in situ from ethyl acetate and 30 wt % aq. hydrogen peroxide as the primary oxidant. Application of the highly stable and active nanobiocatalyst in the Baeyer-Villiger oxidation of 2-methylcyclohexanone to 6-methyl-?-caprolactone after 8 h at 40 °C led to a high product yield (87%) and selectivity (>99%). Environmentally friendly ethyl acetate was applied as both solvent and the peracid precursor. To determine the most favorable reaction conditions, a series of experiments using various parameters was performed. The main contribution of this work is that it describes the first application of the nanobiocatalyst in a chemo-enzymatic Baeyer-Villiger oxidation in a flow system. Since the process was performed in a flow reactor, many improvements were achieved. First of all, substantially shorter reaction times as well as a significant increase in the product yield were obtained as compared to the batch process. Since peracids are unstable, a large increase in the safety of the process was demonstrated under mild conditions in this work. In summary, this work shows a particularly efficient upgrade in the studied processes by transfer from a batch to a flow system.

Full text of DOI:10.1021/acs.oprd.9b00132

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Full Paper
Continuous Flow Chemo-Enzymatic Baeyer-Villiger
Oxidation with Superactive and Extra-Stable Enzyme/Carbon
Nanotube Catalyst: An Efficient Upgrade from Batch to Flow
Anna Szelwicka, Przemyslaw Zawadzki, Magdalena Sitko,
Slawomir Boncel, Wojciech Czardybon, and Anna Chrobok
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00132 • Publication Date (Web): 20 Jun 2019
Downloaded from http://pubs.acs.org on June 20, 2019
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Continuous Flow Chemo-Enzymatic Baeyer-Villiger
Oxidation with Superactive and Extra-Stable
Enzyme/Carbon Nanotube Catalyst: An Efficient
Upgrade from Batch to Flow
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Anna Szelwickaa, Przemysław Zawadzkib, Magdalena Sitkoa, Sławomir Boncelc, Wojciech
Czardybonb, Anna Chroboka*
a Department of Chemical Organic Technology and Petrochemistry, Silesian University of
Technology, Krzywoustego 4, 44-100 Gliwice, Poland.
bSelvita S.A., Bobrzynskiego 14, 30-348 Cracow, Poland
cDepartment of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian
University of Technology, Krzywoustego 4, 44-100 Gliwice, Poland.
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GRAPHICAL ABSTRACT
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ABSTRACT: Continuous flow chemo-enzymatic Baeyer-Villiger oxidation in the presence of
exceptionally active Candida antarctica lipase B immobilized via simple physical adsorption
on multi-walled carbon nanotubes has been investigated. The nanobiocatalyst was used to
generate peracid in situ from ethyl acetate and 30 wt.% aq. hydrogen peroxide as the primary
oxidant. Application of the highly-stable and active nanobiocatalyst in the Baeyer-Villiger
oxidation of 2-methylcyclohexanone to 6-methyl-ε-caprolactone after 8 h at 40 °C led to a
high product yield (87%) and selectivity (>99%). Environmentally-friendly ethyl acetate was
applied as both solvent and the peracid precursor. To determine the most favorable reaction
conditions, a series of experiments using various parameters was performed. The main
contribution of this work is that it describes the first application of the nanobiocatalyst in a
chemo-enzymatic Baeyer-Villiger oxidation in a flow system. Since the process was
performed in a flow reactor, many improvements were achieved. First of all, substantially
shorter reaction times as well as a significant increase in the product yield were obtained as
compared to the batch process. Since peracids are unstable, a large increase in the safety of
the process was demonstrated under mild conditions in this work. In summary, this work
shows a particularly efficient upgrade in the studied processes by transfer from a batch to a
flow system.
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KEYWORDS: (nano)biocatalyst, carbon nanotubes, flow chemistry, lipase, chemo-enzymatic
Baeyer-Villiger oxidation
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INTRODUCTION
Environmental regulations concerning the safety and waste disposal of industrial processes
have caused a considerable acceleration in research involving the design of sustainable
processes. A continuous flow reactor strategy is one of the modern tools that offers greener
approaches to organic synthesis. Flow systems using microreactors have several advantages
over the traditional batch processes, such as lower waste generation and safer experimental
conditions. Additionally, more efficient mass and heat transfer, as well as precise
temperature control to avoid hazardous exothermic side reactions are among the other
benefits. Another advantage is a rapid early stage reaction optimization and a direct scale-up.
Three different approaches in flow chemistry to produce larger amounts of product can be
applied: running the process longer (scaling-out), reactors-in-parallel (numbering up), or
larger continuous reactors (scaling-up).1-3
Heterogeneous catalysts for flow systems
Catalytic continuous flow systems with homogenous or heterogeneous catalysts have been
reported in the literature.4 When the homogeneous catalyst is employed, it flows through
the reactor together with the reactants, and the additional step to separate the product from
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the catalyst is necessary. In the case of heterogeneous catalyst, the catalyst is confined in the
reactor, the reagents pass through the system, and no separation of the product from the
catalyst is required. However, catalyst leaching is the main limitation of the flow systems.
And so, transition metal catalysts such as palladium, copper, ruthenium, and nickel have
been immobilized on silica, monoliths, magnetic nanoparticles and polymer supports. These
catalytic systems have been applied in a range of reactions including Heck, Sonogashira,
Suzuki, Kumada, olefin metathesis, hydrogenation and benzannulation reactions.1-4
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Apart from the metal catalysts, enzymes have been used as biocatalysts in micro enzyme-
reactors.5-7Among others, lipases, Novozyme-435 (which is commercially available Candida
antarctica lipase B (CALB) physically immobilized on a mesoporous poly(methyl
methacrylate) support), protease and dehydrogenases have been used to catalyze
oligosaccharide synthesis, polymerization, dehalogenation reactions, esterification and
hydrolysis or transesterification reactions.8 Enzyme micro-reactors have been constructed
either in the solution phase or by immobilization of enzymes. Enzyme immobilization
reduces the overall costs of the process since the biocatalyst can be easily reused, often
multiple times.9,10 Organic, inorganic or hybrid materials – based mainly on polymers and
silica – can be used as solid supports for enzyme immobilization. The selection of the support
may be a critical step in the design of the biocatalyst.11 An interesting example is shown in
the application of a micro-reactor for the polymerization of ε-caprolactone catalyzed by
Novozyme-435. Using a flow system enabled the reaction to be performed at elevated
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temperatures in organic media; additionally, faster polymerization and higher molecular
weight polymers were achieved compared to the batch reactors. Therefore, continuous
biotransformations using immobilized enzymes are becoming more important.12 Another
important example is flow chemo-enzymatic epoxidation of olefins mediated by Novozyme-
435. After the recently discovered hydrolase-catalyzed synthesis of peroxycarboxylic acids,
the chemo-enzymatic methods for oxidation were developed.13 For example, Novozyme-435
was very active and stable in flow chemo-enzymatic epoxidation in the packed-bed flow
reactor over 24 h.14
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Nevertheless, the critical limitation of enzyme catalyzed reactions is leaching of the
enzymes from solid beds into the products which reduces the life-time of the biocatalyst. In
general, however, enzyme leaching is lower in flow systems than in batch reactors.15
Chemo-enzymatic Baeyer-Villiger oxidation
A one-pot chemo-enzymatic approach for Baeyer-Villiger reaction involves the oxidation
of long-/medium-chain carboxylic acids or ethyl acetate with H2O2 to generate peracids.
Then, peracids oxidize cyclic ketones to lactones. In this method, organic peracids are
generated in situ hence one avoids direct handling of those hazardous (shock sensitive)
materials16.
To date, the former reactions have demonstrated that enzymes could serve as efficient
catalysts in a batch system in various configurations: CALB (Novozyme-435),16-21 CALB
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immobilized as cross-linked enzyme aggregates (CLEA),22 perhydrolase immobilized as
CLEA,23 CALB immobilized on multi-walled carbon nanotubes (MWCNTs),24 CALB
incorporated into mesoporous silica materials25 or in the native form of acyltransferase and
CALB26 The stability of the above heterogeneous biocatalysts in the recycle tests was studied
in our group only in a batch system. By this, we intended to illustrate the practical
applications, using mesoporous silica materials25 and MWCNTs24 as CALB carriers.
Additionally, we have applied ionic liquids in order to improve the stability of lipase.27
The aim of this paper is to investigate the chemo-enzymatic oxidation of a model ketone
using a microreactor in the continuous flow mode. Lipase has been non-covalently
immobilized on MWCNTs and used as the innovative, highly-active and stable
nanobiocatalyst. The nanobiocatalysis has been indeed recently emerged as the critical
innovation involving synergy of nanotechnology and biotechnology. By definition,
nanobiocatalysis embraces amalgamation of enzyme molecules and nanocarriers toward
significantly enhanced and desirable kinetics and selectivity, including stereoselectivity, for
the substrates.24
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To the best of our knowledge, this is the first attempt to apply flow system to perform
Baeyer-Villiger oxidation of ketones to lactones. It is worth to emphasize that the reaction
system is complex and characterized by many issues to overcome. One of them is biphasic
system and the application of aqueous solution of hydrogen peroxide which can deactivate
the enzyme. The hereby proposed environmentally benign, chemo-enzymatic process avoids
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the direct use of unstable peracid which is generated in situ during the enzymatic stage in
the reaction system of the proposed method. To determine the most promising recycling
strategy, catalytic reactions were carried out with lipase immobilized on MWCNTs. The tests
performed using the batch system have produced a recyclable catalytic system. Upgrading to
the flow system resulted in the enhanced activity. This advantage constitutes the basis for
the future design of continuous flow fixed-bed reactors for industrial applications.
Importantly, lactones belong to the group of key substances in the fine chemical sector26
applicable in pharmaceutical, food, cosmetic, perfume and polymer industry. The fine
chemical market is projected to grow at a CAGR of 5.76%, and to reach $201.57 billion by
2023.28 And here, by developing the chemo-enzymatic continuous process we demonstrate
that production of those compounds via alternative, green technology using the flow reactor
is conveniently scalable.
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EXPERIMENTAL
Materials and methods
Ketones, n-octanoic acid, potassium phosphate buffer (pH=7), Novozyme-435 and an aqueous
solution of Candida antarctica lipase B were purchased from Sigma-Aldrich. Nanocyl
NC7000 MWCNTs were purchased from Nanocyl (Belgium). Industrial grade CheapTubes
MWCNTs are available from Cheap Tubes Inc. (United States of America). Toluene, ethyl
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acetate, dichloromethane and chloroform were obtained from Chempur and were used
without additional purification.
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NMR spectra of the products were recorded using a Varian 500 spectrometer at the following
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operating frequencies: H 600 MHz and C 150 MHz. Chemical shifts are reported as parts
per million (ppm) in reference to tetramethylsilane (TMS) for 0.020 g of the sample.
Gas Chromatography analyses were performed using SHIMADZU 2010 chromatograph
equipped with SPB (30 m × 0.25 mm × 0.25 mm) column. The parameters of analysis were set
as follows: injection temperature 250 °C, a FID detector temperature of 280 °C, the initial
temperature of 40 °C (hold for 2 min) with an increasing temperature rate of 20 °C/min and
the final temperature of 280 °C (hold for 10 min), with a linear flow set as 30 mL/min, a split
ratio of 25:1 and a run time of 24 min. The qualitative analyses were performed using Agilent
Technologies 7890C connected to a mass spectrometer Agilent Technologies 5975C detector.
The GC-MS chromatograph was equipped with an HP 5MS column (30 m × 0.25 mm × 0.25
μm film), and electron impact (EI) ionization at 70 eV was used. (for retention times see SI,
Table S1).
Lipase loadings on the surface of the carbon materials were determined by
thermogravimetric analysis (TGA) using a Mettler Toledo TGA851e thermobalance. A
sample of approximately 0.010 g was heated from 25 °C to 800 °C at a rate of 10 °C/min in
standard 70 µL Al2O3 crucibles under a dynamic nitrogen flow of 60 mL/min.
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Nitrogen adsorption/desorption isotherms for MWCNTs were obtained using a Micrometrics
ASAP 2420M instrument at −196 °C to calculate their specific surface area (SBET) and pore
volume. The size of the pores was obtained using the Barrett–Joyner–Halenda (BJH) method
with the Kruk–Jaroniec–Sayari correction. Prior to the experiments, the carbon nanocarrier
samples were outgassed at 200 °C and 1.33×10-3 Pa for 5 h.
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Synthetic procedures
Synthesis of pristine MWCNTs: Pristine MWCNTs were synthesized via catalytic chemical
vapor deposition (c-CVD) using a slightly modified protocol from the literature.29
Immobilization of Candida antarctica lipase B on nanocarbon solid supports: The
immobilization step was carried out according to a literature procedure.30 Into a 100 mL
round bottom flask, a solution of CALB (4 mL), the adequate nanocarbon solid support (1.000
g), and a potassium phosphate buffer at pH = 7 (30 mL) were added. The immobilization was
carried out for 3 h at 20 °C in a thermostatic shaker (180 rpm). After this time, the mixture
was filtered under vacuum and the nanobiocatalyst was washed with 200 mL of potassium
phosphate buffer at pH = 7. Then, the nanobiocatalyst was dried over anhydrous P2O5 for 3
days in a desiccator at 5 °C.
Batch Baeyer-Villiger process: Into a 25 mL round-bottom flask, biocatalyst (0.080 g / 1
mmol of ketone), 2-methylcyclohexanone (0.25 mmol, 0.028 g), n-octanoic acid (0.5 ml), n-
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decane (an internal standard, 20% wt. of ketone) and solvent (0.5 mL) were added. Next,
30% aqueous solution of hydrogen peroxide (0.50 mmol, 0.057 g) was added dropwise at 40
°C. The reaction mixture was stirred in a thermostatic shaker (40 °C, 250 rpm) for 6 h, and
samples were taken for GC-FID analysis (10 μL of sample diluted in 0.5 mL of
dichloromethane). After completion of the reaction, the nanobiocatalyst was filtered off and
solvent (10 mL) and water were added (5 mL). The organic phase was washed using a
saturated aq. solution of sodium bicarbonate (35 mL). Next, the collected water phases were
extracted using methylene chloride (35 mL) and the organic phase was dried over
anhydrous MgSO4 and concentrated under reduced pressure (8 mbar, 25 °C, 1 h). The residue
was purified via column chromatography using SiO2 as the stationary phase and n-
hexane:ethyl acetate 8:2 v/v as the eluent yielding 90% of the lactone.
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Flow Baeyer-Villiger process: To carry out the Baeyer-Villiger reaction, a Vapourtec R2
series two-module flow reactor with two pumps was used. The apparatus was equipped with
a back-pressure regulator located in between the reactor and the collection tube, and was set
to 8 bar. Pump A pumped an organic phase (ketone concentration: 4.50 mmol (0.504 g)/10
mL ethyl acetate). Pump B pumped 30 wt.% aqueous solution of hydrogen peroxide (20-80
molar excess). The total flow was set to 0.040-0.133 mL/min. The process was carried out for
2-24 h at 25-55 °C using various residence times (12-38 min) and the constant amount of
(nano)biocatalyst (0.5 g). During the process the samples were taken for GC-FID analysis
(100 μL of sample diluted in 0.5 mL of dichloromethane).
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For the synthesis of 6-methyl-ε-caprolactone, a dedicated isolation method was developed.
The post-reaction mixture – after the oxidation of 2-metylocyclohexanone – was collected
(25 mL), and then water was added thereto (25 mL). The organic phase was washed using a
saturated aq. solution of sodium bicarbonate (325 mL). Next, the collected aqueous phases
were extracted using methylene chloride (325 mL), the organic phase was dried over
anhydrous MgSO4 and concentrated under reduced pressure (8 mbar, 25 °C, 1 h). The residue
was purified via column chromatography using SiO2 as the stationary phase and n-
hexane:ethyl acetate 8:2 v/v as the eluent, yielding 90% of the lactone.
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6-methyl-ε-caprolactone: H NMR (600 MHz, CDCl3, TMS): δ/ppm 1.36 (d, 3H, J=5.9 Hz),
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1.58-1.71 (m, 4H), 1.88-1.93 (m, 2H), 2.59-2.70 (m, 2H), 4.44-4.63 (m, 1H). C NMR (150
MHz, CDCl3): δ/ppm 22.58, 22.89, 28.28, 35.01, 36.22, 68.38, 175.57.
6-methyl-ε-caprolactone: GC-MS (EI) m/z (%): 128 (1), 113 (1), 84 (61), 67 (10), 55 (100), 47
(43), 21 (14).
ε-caprolactone: GC-MS (EI) m/z (%): 114 (22), 84 (28), 70 (20), 67 (5), 55 (92), 43 (100), 28
(28).
-valerolactone: GC-MS (EI) m/z (%): 100 (30), 81 (1), 70 (17), 56 (54), 42 (100), 28 (42), 15
(3).
4-tert-butyl-ε-caprolactone: GC-MS (EI) m/z (%): 171 (4), 155 (4), 137 (5), 114 (56), 95 (8),
86 (54), 68 (18), 57 (100), 41 (48), 29 (24).
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4-phenyl-ε-caprolactone: GC-MS (EI) m/z (%): 190 (90), 162 (20), 148 (99), 117 (100), 106
(95), 91 (53), 77 (30), 63 (18), 51 (22), 45 (19) 39 (24), 24 (12).
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4-oxatricyclo[4.3.1.13.8]undecan-5-one: GC-MS (EI) m/z (%): 166 (2), 122 (5), 107 (18), 93
(18), 80 (100), 67 (16), 53 (28), 41 (17).
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RESULTS AND DISCUSSION
MWCNTs have been described as versatile supports for enzyme immobilization due to
their small size, large surface area, mechanical/thermal stability and other unique properties.
These characteristics enable, among others, higher enzyme loadings and, more importantly,
increased enzyme stability in the presence of hydrogen peroxide without the enzyme
leakage.31-36 Both non-covalent and covalent methods to immobilize various enzymes on
MWCNTs have been reported.37 The simple, direct physical adsorption method is based on
hydrophobic, van der Waals and hydrogen bonding interactions between MWCNTs and the
enzyme macromolecules.38
To demonstrate the catalytic potential of non-covalently immobilized lipase on MWCNTs
in a flow reactor, chemo-enzymatic Baeyer-Villiger oxidation of 2-methylcyclohexanone
was carried out as the model reaction (Scheme 1).
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O
O
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O
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O
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OH
OH
O
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H2O2
H2O
nanobiocatalyst
Scheme 1. Chemo-enzymatic Baeyer-Villiger oxidation of 2-methylcyclohexanone.
Chemo-enzymatic Baeyer-Villiger oxidation of 2-methylcyclohexanone in the batch system
The activity of CALB non-covalently immobilized on MWCNTs of various morphologies
and surface chemistry has been previously studied in the chemo-enzymatic oxidation of
cyclic ketones to lactones in a batch reactor at 20 °C.24 The most promising results were
achieved, when unmodified, pristine MWCNTs – synthesized according to the literature29 –
were used as the CALB support.
In order to apply this nanobiocatalyst in a continuous flow chemo-enzymatic Baeyer-
Villiger process, which in fact can be dedicated for the industry, it was necessary to choose
commercially available support to increase stability of the enzyme. Therefore, two
inexpensive, unmodified, commercially available supports of CALB: MWCNTs from Nanocyl
(100 EUR/1 kg) and MWCNTs from CheapTubes (100 USD/1 kg) were tested under pre-
optimized reaction conditions at 40 °C. The results were compared with pristine (in-house)
MWCNTs as a CALB support synthesized via catalytic chemical vapor deposition (c-CVD) in
our laboratories as well as with the native form of CALB and Novozyme-435.
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CALB was immobilized on carbon nanomaterials using a physical adsorption technique in
accordance with the procedure described in the literature.24 All carbon nanomaterials were
characterized (Table 1) and the amount of immobilized CALB was determined using TGA.
Based on previous studies,24,38 the model reaction system of the chemo-enzymatic
oxidation of 2-methylcyclohexanone in toluene consisted of: (1) heterogeneous
nanobiocatalyst to form the peracid, (b) 30 wt.% aq. H2O2 as the primary oxidant, and (3) n-
octanoic acid as the peracid precursor. The conversion of ketone after 3 and 4 h was
measured by GC/FID and compared with the model reaction carried out in presence of
CALB-MWCNT nanobiocatalyst (Table 2).
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Table 1. Characterization of carbon nanotube-based supports and CALB loadings.
Support
SBET,
m2/g
Pore
Length,
Outer
diameter, diameter,
nm
9.5
Inner
CALB loading,
wt.%
volume, µm
cm3/g
nm
7
Nanocyl
NC7000
253
89
1.06
0.49
0.11
1.5
19.8
15.7
6.0
CheapTubes
MWCNTs
10-30
130-370
20-40
35-85
5-10
In-house
34
15-40
MWCNTs
Table 2. The influence of the carbon-based support on the biocatalyst activity in chemo-
enzymatic Baeyer-Villiger oxidation curried out in the batch reactor.
Biocatalyst
CALB-MWCNTs
Time, h
α, %
98
99
3
4
3
CALB-Nanocyl NC7000 MWCNTs
90
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68
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CALB-CheapTubes MWCNTs
Novozyme-435
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CALB native
Reaction conditions: 40 °C; 250 rpm, 2-methylcyclohexanone (0.25 mmol, 0.029 g);
(nano)biocatalyst 0.080 g/ 1 mmol of ketone; toluene 0.5 mL; n-octanoic acid 0.5 mL; 30% aq.
H2O2 (0.50 mmol, 0.057 g), the conversion of ketone () was determined by GC with 95%
selectivity toward the lactone. All experiments were performed in triplicate and the results
for each of the three measurements differed by less than 1%.
The activity of nanocarbon-supported lipase was higher than the native one. Among the
commercially available CALB supports, Nanocyl NC7000 MWCNTs emerged as the support
the most capacious one. This support was characterized by the highest specific surface area
and the lowest aspect ratio (length-to-diameter ratio) (Table 1). Hence, the highest amount
of CALB was immobilized on the surface of Nanocyl 7000 MWCNTs and, at the same time, it
was prone to catalyze the peracid formation. High conversion of the ketone 93% after 4 h
was reached, which was only slightly lower than in the case of in-house MWCNTs. CALB
anchored on the surface of CheapTubes MWCNTs was also comparatively active to the
relatively expensive Novozym-435 (30,000 EUR/1 kg). The lowest activity demonstrated the
native CALB (the reaction courses are presented in SI, Fig. S7) which was used for the
reaction in the same amount as for immobilized on the support.
Chemo-enzymatic Baeyer-Villiger oxidation of 2-methylcyclohexanone in the flow system
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Based on the most promising results from the batch reactor (including economy aspects),
we selected [CALB Nanocyl NC7000 MWCNTs] system to generate peracid in situ and
further oxidize ketones24 in a series of continuous system experiments.
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Vapourtec R2 (SI, Scheme S1) flow reactor equipped with two HPLC pumps (one pumping
the organic phase: a mixture of n-octanoic acid with substrates and solvent and the second
one pumping aqueous solution of H2O2). A column reactor with a 4 mL-working volume
charged with 0.500 g of [CALB-Nanocyl NC7000 MWCNTs] was used (Scheme 2). The
apparatus was equipped with a back-pressure regulator set to 8 bar.
Pump A
Organic phase
Column reactor
Product
Mixer
Pump B
30% aq. H2O2
Scheme 2. Diagram of the flow system used in this study. Organic phase: a mixture of n-
octanoic acid with substrates and solvent.
The influence of residence time on the flow rate was calculated from the exact volume of
the reactor which is the difference of the volume of the empty reactor and the volume of
catalyst. In the case of the porous catalysts, it was necessary to consider the volume of the
pores in the calculations. The application of a solvent with a known density was helpful here.
To determine the volume, the reactor was densely packed with 0.500 g of CALB-Nanocyl
NC7000 and weighted. Ethyl acetate was pumped through the reactor for 10 minutes to
ensure evacuation of air from the catalyst pores. The amount of solvent was established with
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the front of the catalytic bed and weighted once again. The exact volume of the reactor was
determined to be 1.56 mL (SI).
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Influence of solvent on the conversion of the 2-methylcyclohexanone
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In the beginning, the possibility of using different solvents instead of toluene was
investigated. To this aim, chloroform as solvent of low viscosity was also tested. In these two
cases, n-octanoic acid as peracid precursor was used. A different approach was the use of
ethyl acetate which can be used as solvent and the acid precursor at one.
The initial parameters of the reactor operation were determined. The total flow rate was
maintained at 0.133 mL/min with a residence time of 12 min. The flow rate ratio of pump A
to pump B was maintained at the level 1:3. Pump A pumped the organic phase (2-
methylcyclohexanone and solvent (and when necessary n-octanoic acid), while
a 30 wt.% aq. solution of hydrogen peroxide was introduced through pump B (Figure 1).
The investigations concerning application of various solvent/precursor systems in flow
chemo-enzymatic oxidation of 2-methylcyclohexanone indicated the use of ethyl acetate as
the most efficient solvent. In the experiment with ethyl acetate, the highest conversion of
ketone (60% after 30 min) was observed with 100% selectivity to the corresponding lactone
(in the batch reactor the lower selectivity 95% was caused by a partial hydrolysis of lactone
to the appropriate hydroxyacid). At the same time, the conversion of ketone in the system
with toluene/n-octanoic acid was only 39%.
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ethyl acetate
chloroform
toluene
10
30
60
120
Time (min)
Figure 1. The influence of the solvent type on the conversion of ketone in chemo-
enzymatic Baeyer-Villiger oxidation carried out in the flow reactor. All
experiments were performed in triplicate and the results for each of the three
measurements differed by less than 1%.
Reaction conditions: 40 °C; 2-methylcyclohexanone (0.05 mol/100 mL, 5.040 g/100 mL);
CALB-Nanocyl™ NC7000 MWCNTs (0.500 g); solvent (n-octanoic acid was applied as
peracid precursor when toluene and chloroform were used as solvents; ethyl acetate was
applied as both: solvent and peracid precursor, 2.00 mol/1 mol of ketone, i.e. 288 g/1 mol of
ketone); 30% aq. H2O2 (0.91 mol/100 mL), flow rate ratio: organic phase:oxidant 1:3; total
flow 0.133 mL/min; residence time 12 min.; the conversion of ketone () was determined by
GC with 100% selectivity to the lactone. Reaction time = sampling time after reaching the
residence time.
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This approach allowed this process to be performed in a non-chlorinated solvent without
any additional peracid precursor. Moreover, ethyl acetate can be called environmentally
‘preferred solvent’ due to non-toxicity and safety reasons as well as the low price.39
Additionally, the application of ethyl acetate resulted in a lower total pressure in the reactor
during the process. Those characteristics limited the potentially detrimental effect of the
reaction system on the nanobiocatalyst. Importantly, a use of chloroform is not advisable
from the point of view of green chemistry and scaling-up. Nevertheless, further
improvements in the overall performance of flow system were necessary as the 60% yield of
lactone was not satisfying.
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Influence of the residence time on the conversion of the 2-methylcyclohexanone
The residence time is an important parameter in the continuous processes as it corresponds
to the time at which substances are in contact in the reactor. It describes the number
of molecules leaving the reactor per unit of time and the contact time of the reagents with
the nanobiocatalyst which, in turn, affect the conversion and stability of the enzyme. Hence,
the next step was to investigate the influence of the residence time on the substrate
conversion (Table 3).
As shown in Table 3, the optimal residence time for this process was found to be 20 min,
since this is the time where the highest conversion of 2-methylcyclohexanone as well as the
highest stability of nanobiocatalyst were achieved. Under these conditions, 87% conversion
of the ketone was obtained after only 5 min, and this conversion was maintained
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approximately for 2 h. In contrast, nearly twice lower conversions were observed in the
other cases. Moreover, a drop in the conversion of ketone was obtained after only 2 h in both
processes which were carried out at the longer residence times. The reason for these
phenomena is likely the generation of ethanol and acidic acid via ethyl acetate hydrolysis,
which are detrimental to the activated enzyme structure and hence its performance.
Additionally, prolonged contact of the nanobiocatalyst with the oxidant and the non-polar,
hydrolysable solvent, such as ethyl acetate, are other possible reasons for the observed
decrease in the enzyme activity.
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Table 3. The influence of the total flow rate on the conversion of ketone in chemo-
enzymatic Baeyer-Villiger oxidation carried out in the flow reactor.
Time, min
α, %
Flow rate
A, Pump B, mL/ Total, mL/
Residence
time, min
Pump
mL/ min
min
min, (flow
rate ratio)
0.133 (1:3)
10
30
60
120
5
15
60
120
5
15
60
120
5
55
60
59
60
87
87
90
93
37
76
93
76
27
0.033
0.019
0.013
0.010
0.100
12
20
30
38
0.057
0.039
0.030
0.076 (1:3)
0.052 (1:3)
0.040 (1:3)
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15
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45
84
78
7
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Reaction conditions: 40 °C; 2-methylcyclohexanone (0.05 mol/100 mL, 5.040 g/100 mL);
CALB-Nanocyl NC7000 MWCNTs (0.500 g); ethyl acetate as solvent; 30% aq. H2O2 (0.91
mol/100 mL), the conversion of ketone () was determined by GC with 100% selectivity to
the lactone. All experiments were performed in triplicate and the results for each of the
three measurements differed by less than 1 %. Reaction time = sampling time after reaching
the residence time.
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Influence of the flow rate ratio on the conversion of the 2-methylcyclohexanone
In the next step, the influence of the flow ratio on the conversion of ketone was
investigated. The total flow rate was maintained at 0.075-0.076 mL/min with a residence
time of 20 min. The influence of the flow rate ratio of pump A to pump B within the range of
1:1 to 1:4 was investigated. Pump A pumped the organic phase (2-methylcyclohexanone and
ethyl acetate), while a 30 wt.% aq. solution of hydrogen peroxide was introduced through
pump B (Table 4).
Table 4. The influence of the flow rate ratio on the conversion of ketone in chemo-
enzymatic Baeyer-Villiger oxidation carried out in the flow reactor.
Time, min
α, %
Flow rate
Pump A, mL/ Pump B, mL/ Flow rate ratio
min
min
5
15
29
60
0.038
0.038
1:1
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60
120
5
15
60
120
5
15
60
120
5
15
60
120
40
36
35
38
48
40
87
87
90
93
43
45
50
57
0.025
0.019
0.015
0.050
0.057
0.060
1:2
1:3
1:4
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Reaction conditions: 40 °C; 2-methylcyclohexanone (0.05 mol/100 mL, 5.040 g/100 mL);
CALB-Nanocyl NC7000 MWCNTs (0.500 g); ethyl acetate as solvent; 30% aq. H2O2 (0.91
mol/100 mL); total flow 0.075-0.076 mL/min; residence time 20 min; the conversion of
ketone () was determined by GC with 100% selectivity to the lactone. All experiments were
performed in triplicate and the results for each of the three measurements differed by less
than 1 %. Reaction time = sampling time after reaching the residence time.
The highest conversion of the model ketone was achieved when the flow rate ratio of the
organic to the aqueous phase of 1:3 was used. Below this value, a twice lower conversion of
the substrate (at the comparable reaction time) was obtained due to an insufficient amount of
the oxidant to generate the necessary concentration of the peracid. On the other hand, a
higher amount of oxidant led to deactivation of the enzyme causing a lower conversion
of the ketone.
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Influence of temperature on the conversion of 2-methylcyclohexanone
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In order to determine the influence of temperature on the continuous flow chemo-
enzymatic Baeyer-Villiger oxidation of 2-methylcyclohexanone, a series of experiments at
three various temperatures: 25, 40 and 55 °C were performed. CALB is sensitive to higher
temperatures, especially in the presence of hydrogen peroxide. Unfortunately, proceeding at
lower temperature slowed down the reaction and, even if the residence time was relatively
long (20 min), the conversion of ketone reached only 20%. The increase of temperature to 55
°C have accelerated the reaction (with almost a full conversion of ketone after 15 min) but,
on the other hand, the conversion decrease was visible after 1 h, probably caused by a partial
deactivation of lipase.
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Influence of the primary oxidant concentration on the conversion of 2-methylcyclohexanone
Another crucial factor influencing the nanobiocatalyst stability is concentration of
hydrogen peroxide in the aqueous phase. In the preliminary studies in the batch system,24 the
possibility of application in chemo-enzymatic continuous process urea hydrogen peroxide
UHP – anhydrous form of hydrogen peroxide – was eliminated due to the solid state of this
complex and practical insolubility in the reaction system. For comparison, the experiment
involving application of 30% aq. H2O2 as the primary oxidant was performed (Figure 3).
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Figure 2. The influence of the temperature on the conversion of ketone in chemo-
enzymatic Baeyer-Villiger oxidation carried out in the flow reactor. All
experiments were performed in triplicate and the results for each of the three
measurements differed by less than 1%.
Reaction conditions: 2-methylcyclohexanone (0.05 mol/100 mL, 5.040 g/100 mL); CALB-
Nanocyl NC7000 MWCNTs (0.500 g); ethyl acetate as solvent; 30% aq. H2O2 (0.91 mol/100
mL), flow rate ratio organic phase:oxidant 1:3; total flow 0.076 mL/min; residence time 20
min; the conversion of ketone () was determined by GC with 100% selectivity to the
lactone. Reaction time = sampling time after reaching the residence time.
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60
30% aq. H2O2
60% aq. H2O2
5
15
60
120
Time (min)
Figure 3. The influence of the H2O2 concentration on the conversion of ketone in chemo-
enzymatic Baeyer-Villiger oxidation carried out in the flow reactor. all
experiments were performed in triplicate and the results for each of the three
measurements differed by less than 1 %.
Reaction conditions: 40 °C; 2-methylcyclohexanone (0.05 mol/100 mL, 5.040 g/100 mL);
CALB-Nanocyl NC7000 MWCNTs (0.500 g); ethyl acetate as solvent; flow rate ratio organic
phase:oxidant 1:3; total flow 0.075 mL/min; residence time 20 min; the conversion of ketone
() was determined by GC with 100% selectivity to the lactone. Reaction time = sampling
time after reaching the residence time.
As expected, higher concentration of hydrogen peroxide proved a negative effect on the
lipase lifetime. Although, in the beginning, both processes were carried out with the same
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reaction rate (conversion of ketone 87% after 5 and 15 min), after 1 h a significant drop in
the conversion of ketone was observed while using 60% aq. H2O2.
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Substrate scope
In order to broaden the scope of possible substrates and hence to confirm the practical
potential of the proposed approach, a selection of various substrates was subjected to the
reaction under so-optimized conditions (Table 5). The experiments were carried out for
seven various ketones: 2-methylcyclohexanone, cyclohexanone, 4-phenylcyclohexanone, 4-
tert-butylcyclohexanone, cyclopentanone and 2-adamantanone.
Table 5. The influence of the type of the substrate on the conversion of ketone in chemo-
enzymatic Baeyer-Villiger oxidation carried out in the flow reactor.
Ketone
Lactone
α, %
O
O
O
85
O
O
90
O
O
O
O
83
O
O
O
97
O
C6H5
O
95
99
O
C6H5
O
O
O
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Reaction conditions: 40 °C; ketone (0.05 mol/100 mL); CALB-Nanocyl NC7000 MWCNTs
(0.500 g); ethyl acetate as solvent; 30% aq. H2O2 (0.91 mol/100 mL); total flow rate 0.076
mL/min; flow rate ratio organic phase:oxidant 1:3; residence time 20 min; reaction time 60
min, the conversion of ketones () was determined by GC with 100% selectivity to the
lactone. All experiments were performed in triplicate and the results for each of the three
measurements differed by less than 1 %. Reaction time: time of sample collection after
crossing of residence time.
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60
All ketones were obtained in high to excellent yields (83-99%) and excellent selectivities
(100%). We have therefore clearly demonstrated that the developed method is
comprehensive and competitive to other non-enzymatic methods of oxidation.
Stability of the (nano)biocatalysts in the flow system
Finally, the studies concerning the stability of nanobiocatalyst in the long-term
experiments were performed. Two (nano)biocatalysts were taken into consideration: the one
proposed in this work, i.e. immobilized via simple, one-step physical adsorption – [CALB-
Nanocyl NC7000 MWCNTs] nanobiocatalyst and commercially available Novozyme-435 for
comparison (Table 6).
Table 6. The stability of the CALB-Nanocyl NC7000 MWCNTs in comparison with
Novozyme-435 in chemo-enzymatic Baeyer-Villiger oxidation carried out in the
flow reactor.
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Page 29 of 38
Organic Process Research & Development
1
2
3
4
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7
8
9
(nano)biocatalys Time, min
t
α, %
Flow rate
mL/ min
0.048
Pump
mL/ min
A, Pump
B, Total, mL/
min, (flow
rate ratio)
Novozyme-435
5
82
96
99
95
90
74
68
0
87
87
90
93
95
88
87
16
0.016
0.064 (1:3)
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30
31
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36
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40
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44
45
46
47
48
49
50
51
52
53
54
55
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58
59
60
15
60
120
240
360
480
1440
5
CALB-NanocylT
NC7000
0.019
0.057
0.076 (1:3)
15
MWCNTs
60
120
240
360
480
1440
Reaction conditions: 40 °C; 2-methylcyclohexanone (0.05 mol/100 mL, 5.040 g/100 mL);
CALB-Nanocyl NC7000 MWCNTs or Novozyme-435 (0.500 g); ethyl acetate as solvent; 30%
aq. H2O2 (0.91 mol/100 mL); flow rate ratio organic phase:oxidant 1:3; residence time 20 min;
the conversion of ketone () was determined by GC with 100% selectivity to the lactone. All
experiments were performed in triplicate and the results for each of the three measurements
differed by less than 1 %. Reaction time = sampling time after reaching the residence time.
Due to different morphology, specific surface, grain size and other properties, it was necessary
to determine the exact volume of the reactor (SI) filled with 0.500 g of Novozym-435. Here,
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