Magnetic Attachment of Lipase Immobilized on Bacteriogenic Iron Oxide Inside a Microtube Reactor for the Kinetic Resolution of Secondary Alcohols

Article abstract of DOI:10.1055/s-0036-1589953

A PTFE microtube reactor was constructed with lipase immobilized on magnetized bacteriogenic iron oxide, which was retained inside of the tube by attraction to an external magnet. The reactor was used for the lipase-promoted kinetic resolution of secondary alcohols and gave sufficient catalytic activity, which was maintained during long-term flow over 14 days.

Full text of DOI:10.1055/s-0036-1589953

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Georg Thieme Verlag Stuttgart · New York
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letter
Synlett
K. Mandai et al.
Letter
Magnetic Attachment of Lipase Immobilized on Bacteriogenic
Iron Oxide Inside a Microtube Reactor for the Kinetic Resolution
of Secondary Alcohols
Kyoko Mandaia
Takehiro Fukudaa
Yuki Miyazakia
Hideki Hashimotoa
Hiroki Mandaia
Tadashi Emaa
Jun Takadaa,b
Seiji Suga*a
a
Division of Applied Chemistry, Graduate School of
Natural Science and Technology, Okayama University,
3
-1-1, Tsushima-naka, Kita-ku, Okayama, 700-8530,
Japan
b
CREST, Japan Science and Technology Agency (JST),
Okayama 700-8530, Japan
suga@cc.okayama-u.ac.jp
from the perspective of inorganic chemistry2 and micro-
a,4
Received: 17.11.2016
Accepted after revision: 27.12.2016
Published online: 24.01.2017
biology.5 BIOX produced by Leptothrix ochracea has been
DOI: 10.1055/s-0036-1589953; Art ID: st-2016-u0774-l
shown to be useful, environmentally benign, and ubiqui-
tous material that is suitable for use as an anode active ma-
Abstract A PTFE microtube reactor was constructed with lipase immo-
bilized on magnetized bacteriogenic iron oxide, which was retained in-
side of the tube by attraction to an external magnet. The reactor was
used for the lipase-promoted kinetic resolution of secondary alcohols
and gave sufficient catalytic activity, which was maintained during
long-term flow over 14 days.
3,6
terial for lithium-ion batteries, and as a precursor for pig-
7
ments, magnetic nanocomposites, and acidic silica. In the
field of organic chemistry, we have demonstrated that BIOX
8
can be used as an oxidation catalyst and as a solid support
for solid catalysts; i.e., immobilized enzymatic catalysts for
9
the kinetic resolution of secondary alcohols, immobilized
Key words iron oxide, microreactor, kinetic resolution, lipase, mag-
netic attachment
palladium catalysts for solvent-free Suzuki–Miyaura cross-
coupling reactions,10 and immobilized metal-porphyrin
catalysts for the synthesis of carbonates from epoxides and
carbon dioxide.11
Iron-based oxides produced by aquatic iron-oxidizing
bacteria are found ubiquitously as ocher deposits in aquatic
environments, and are considered to be generated during
1
bacterial vital activity. We refer to such iron oxides as bio-
genous iron oxides (BIOX). Currently, such bacteria are used
to purify drinking water at fresh-water-purification plants
2
by removing iron from the water. However, the resulting
large amount of BIOX causes problems and needs to be dis-
posed of as industrial waste.
Microscopic analyses of BIOX, and especially that pro-
duced by the bacteria Leptothrix ochracea, revealed that it
has unique features that cannot be achieved artificially;
BIOX is a unique tubular assemblage (Figure 1) of mild ag-
gregates of Fe -based amorphous iron oxide nanoparticles
with a diameter of ca. 3 nm, along with specific amounts of
silicon and phosphorus as minor elements in its structure
Figure 1 SEM image of BIOX produced by Leptothrix ochracea
3+
BIOX can be exposed to a range of heating and work-up
conditions for derivatization. One of the derivatives of BIOX
is magnetized iron oxide covered with silicate, hereafter re-
(ratio of Fe/Si/P = 73:22:5). The tubules have a complex po-
rous surface structure, which results in a relatively large
2
2a,3
12
surface area (280 m /g).
Further investigations of its
ferred to as m-BIOX, which is used as a support for mag-
structure, morphology and genesis have been conducted
netically recoverable and recyclable immobilized enzyme
©
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K. Mandai et al.
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catalyst.2b The catalyst (BCL/m-BIO-M) was prepared from
BIOX in three major steps, including heat treatment of BIOX
to give magnetized iron oxide (m-BIOX), surface modifica-
tion of m-BIOX to give m-BIO-M, and immobilization of li-
pase (Figure 2). The lipase, Burkholderia cepacia lipase
rials (BIOX and enzyme), in the microreactor could be even
more advantageous in terms of a green process, in addition
to reducing the need to separate the catalyst from the reac-
tion solution.19 The installation of magnetized immobilized
catalysts in microfluidic systems has received much atten-
20
(BCL), was anchored on the surface of m-BIOX through in-
tion. In previous reports on the application of enzymes in
microreactors,21 magnetized enzymes have also been in-
stalled in microchannels by using the interaction with a
magnetic field to fabricate readily replaceable and regener-
atable microreactors. Enzymes immobilized on magnetic
teraction with the organic cross-linkage, which was intro-
duced in the surface-modification step by the reaction of
m-BIOX with the silane coupling agent. We previously re-
ported that the catalyst, hereafter BCL/m-BIO-M, could be
used in a batch system for the kinetic resolution of second-
ary alcohols. It could be separated from the reaction mix-
ture by using an external magnet and showed sufficient
®
microparticles have been integrated into Teflon tubing for
22
flow injection analysis and into microchannels on micro-
chips for proteolysis23 and other enzymatic reactions.
Enzymes immobilized on magnetic beads or nanoparticles
have been embedded in capillaries for capillary electropho-
21d,21e
2b
catalytic activity in a recyclability test. However, BCL/m-
BIO-M was less active than the immobilized catalyst in
which BCL was grafted onto untreated BIOX in the same
manner as for the preparation of BCL/m-BIO-M. One reason
for this result could be the decrease in the surface area of
m-BIOX after heat treatment compared with that of un-
2
1f,21g
resis
and for the electrochemical detection of glu-
cose.2 Although a few enzyme microreactors have been
1h
24
used for organic reactions, including kinetic resolution, to
our knowledge, there has been no report on the application
of a microreactor that incorporated a magnetized immobi-
lized enzyme for organic synthesis. Herein, we describe the
construction of a microtube reactor that incorporates mag-
netic retention of an immobilized catalyst that is composed
of two bio-oriented materials (bacteriogenic iron oxide and
lipase) using an external magnet, and its application for or-
ganic synthesis. Our microtube reactor has several advan-
tages: (1) the catalyst used in the reactor is composed of
easily obtainable bio-oriented materials, bacteriogenic iron
oxide and lipase; (2) our microflow system is simple and
easy to construct by using a magnetic field; (3) the catalyst
in a microtube is easily attachable and detachable by on-off
application of the magnetic field; (4) no special packing
technique is required; (5) decreases in pressure during con-
tinuous flow operation can be prevented.
2
2
treated BIOX (280 m /g for BIOX vs 19 m /g for m-BIOX).
BIOX
Heat treatment
(i) heat treatment at 800 °C for 2 h under air
(ii) reduction at 550 °C for 45 min under 3% H2/Ar
(iii) heat treatment at 250 °C for 2 h under air
O
(
MeO)3Si
m-BIOX
Surface modification
O
BCL
m-BIO-M
BCL/m-BIO-M
toluene
Immobilization of lipase
BCL: Burkholderia cepacia lipase
Figure 2 Preparation of lipase immobilized on magnetized BIOX
(
BCL/m-BIO-M)
The microflow technique can make organic synthesis
quite powerful, especially from the perspective of reaction
A schematic diagram of the microtube reactor with
BCL/m-BIO-M, a photograph, and micrographs are shown in
Figure 3. BCL/m-BIO-M was placed inside a PTFE tube (0.75
mm inner diameter) by using a diaphragm pump as a suc-
tion device and retained by magnetic interaction with a lin-
ear assemblage of neodymium magnets placed immediately
outside of the tube. Aggregates of the catalyst spread out
under the magnetic field produced by the magnets. The
PTFE tube with BCL/m-BIO-M was then attached to a gas-
tight syringe filled with a solution of secondary alcohol and
vinyl acetate in diisopropyl ether via a PEEK ferrule and
adaptor, and the syringe was attached to a syringe pump.
Part of the PTFE tube and the magnet were kept in a water
bath at 30 °C.25 Micrographs of the magnetic retention of
BCL/m-BIO-M in the microtube, which was filled with the
reaction solvent (diisopropyl ether) were taken with a digi-
tal microscope (KEYENCE VHX-2000) (Figure 3, c and d).
The magnets clearly had a strong magnetic interaction with
BCL/m-BIO-M, which was densely packed into bundles with
a myriad of interspaces. The catalyst was not evenly dis-
persed, probably due to the non-uniformity of the magnetic
13,14
integration.
By taking advantage of a flow-microreactor,
short-lived, highly reactive intermediates such as organo-
lithium species can be controlled to promote the desired re-
15
actions. Integrated continuous-flow systems enable the
effective synthesis of even complex molecules such as bio-
16
17
active oligosaccharides, natural products and drugs. To
further promote the use of microflow systems in fine or-
ganic chemistry, modification of the reaction channel in
which the reaction actually takes place in a continuous-
flow system is of great importance. One attractive option to
accomplish this is through the introduction of a catalytic
polymer membrane between laminar aqueous and organic
layers inside a microchannel to achieve efficient catalytic
18
organic transformation. Inspired by this report, we envi-
sioned that our catalyst (BCL/m-BIO-M) could be attached
into a flow path by the magnetic attachment using the in-
teraction with an external magnet if we properly use the
magnetic property of BCL/m-BIO-M. Furthermore, the use
of our catalyst (BCL/m-BIO-M), which is composed of two
environmentally benign and ubiquitous bio-oriented mate-
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K. Mandai et al.
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field produced by the linear assemblage of tiny neodymium
magnets. The catalytic performance of BCL/m-BIO-M in the
reactor was evaluated in terms of the kinetic resolution of
filled with 4 mg of BCL/m-BIO-M from 1.4 to 37.8 μL/min by
changing the flow rate sequentially in descending order. A
sufficient amount of the steady-state product at each flow
rate was collected for H NMR spectroscopic and high-per-
formance liquid chromatography (HPLC) analyses.
1
1
-phenylethanol (rac-1a) at 30 °C, with reference to the re-
2b
action conditions previously reported for a batch system.
The reaction solution was injected from the syringe at a de-
fined flow rate until the system reached a steady state. We
used 2 equivalents of vinyl acetate to rac-1a and the con-
centration of the reaction solution was set at 0.2 M.26 First,
the optimal flow rate was examined by using a PTFE tube
As summarized in Table 1, as the flow rate decreased,
the conversion of alcohol (rac-1a) increased from 26 to 52%
and the enantiomeric excess of (S)-1a increased from 34 to
>99%. As the flow rate decreased, the enantiomeric excess
of ester 2a also decreased from 99 to 94%. The E values were
Figure 3 Schematic representation (a) and photograph (b) of the PTFE tube reactor system. Microscopic images of the PTFE tube with BCL/m-BIO-M
with a neodymium magnet (c) and a magnified view of the region highlighted in (c) (d)
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K. Mandai et al.
Letter
more than 200 at all of the flow rates tested. Overall, the ki-
netic resolution of rac-1a was found to proceed most effi-
ciently at a flow rate of 4.2 μL/min with good conversion,
good optical purities of the two products, (R)-2a and (S)-1a,
and high E values (Table 1, entry 5).
OAc
OH
OH
Me
BCL/m-BIO-M (4 mg)
CH2=CHOAc, i-Pr2O
+
Ph
Me
Ph
Me
Ph
30 °C, 4.2 µL/min
(R)-2a
(S)-1a
rac-1a
.0 g, 16.8 mmol
14 d
1.1 g, 7.0 mmol 0.9 g, 7.2 mmol
2
9
7% ee
89% ee
4
8% conversion, E = >200
Table 1 Effect of flow rate with BCL/m-BIO-M in the kinetic resolution
TTN = 2,079,000
a
of 1-phenylethanol
Scheme 1 Kinetic resolution of 1-phenylethanol under 14 days of con-
tinuous flow
OH
OAc
OH
Me
BCL/m-BIO-M
+
Ph
Me CH2=CHOAc, i-Pr2O
Ph
Me
Ph
3
0 °C, flow rate
(49,500).2b This indicates that the microreactor with
BCL/m-BIO-M ensures a more stable supply of the desired
products than a batch system, which often suffers from cat-
alyst disruption due to stirring or substrate inhibition.
The reactions with rac-1a and rac-1b were performed
in the reactor with a decreased amount of BCL/m-BIO-M (2
mg). The optimal flow rate with 2 mg of the catalyst was re-
examined by sending the reaction solution containing rac-
1a through the tube reactor, and was found to be 1.4
μL/min, at which the kinetic resolution of rac-1a was suffi-
ciently promoted (Table 2, entry 1). At this flow rate, rac-1b
also efficiently underwent transformation in the reactor
rac-1a
(R)-2a
(S)-1a
Entry
Flow rate (μL/min) Conv. (%)b ee (%)c
Ed
(
R)-2a
(S)-1a
1
2
3
4
5
6
37.8
12.6
7.0
26
41
46
46
49
49
52
99
99
98
98
98
96
94
34
68
>200
>200
>200
>200
>200
>200
>200
83
5.6
84
4.2
92
2.8
93
7
1.4
>99
(45% conversion, E value of >200, entry 2). Throughout the
a
BCL/m-BIO-M (4 mg) was placed inside a tube. The reaction was per-
formed with a 0.2 M solution of 1-phenylethanol, vinyl acetate (2 equiv) in
diisopropyl ether at 30 °C.
Determined by HPLC analysis using the equation: conversion
c) = ee(alcohol)/[ee(alcohol)+ee(ester)].
Determined by HPLC analysis.
Determined by HPLC analysis using the equation: E = ln{1–c[1+ee(es-
investigation, we found that the kinetic resolution of sec-
ondary alcohols proceeded efficiently in the microtube re-
actor with our immobilized catalyst. The catalyst took on
the jagged morphology spontaneously in the flow path un-
b
(
c
d
ter)]}/ln{1–c[1–ee(ester)]}.
Table 2 Kinetic resolution of secondary alcohols using less catalysta
Next, under the optimized flow rate (4.2 μL/min), a
solution of 1-phenylethanol (rac-1a) and vinyl acetate in di-
isopropyl ether (0.2 M) was continuously infused into the
PTFE tube containing BCL/m-BIO-M for 5 days. For continu-
ous monitoring of the performance of BCL/m-BIO-M (4 mg),
the eluent was collected for each 6 hour period and ana-
OH
OAc
OH
BCL/m-BIO-M
+
Ar
Me CH2=CHOAc, i-Pr2O Ar
Me
Ar
Me
3
0 °C, 1.4 µL/min
rac-1a,b
(R)-2a,b
(S)-1a,b
Entry
Alcohol
Conv. (%)b ee (%)c
Ed
1
(R)-2
(S)-1
lyzed by H NMR spectroscopy and HPLC (Figure 4). High
catalytic performance was maintained during the overall
experiment. In addition, we performed a longer-term con-
tinuous-flow experiment for 14 days (Scheme 1). In this
case, the syringe had to be periodically filled with the reac-
tion solution during the experiment. Overall, 2.0 g (16.8
mmol) of rac-1a was passed through the microtube reactor
during the total 14 days. Analyses and purification by silica
gel column chromatography of the collected eluent re-
vealed that the desired products were obtained in 48% con-
version with a high E value of >200 and sufficient enantio-
purities of (R)-2a (1.1 g, 7.0 mmol) and (S)-1a (0.9 g, 7.2
mmol): 97% ee and 89% ee, respectively. The total turnover
number (TTN), defined as the total number of moles of the
product formed per mole of the enzyme, during 14 days of
continuous flow was calculated to be 2,079,000, which is
much higher than that obtained in a four-fold recycling test
in the same reaction with BCL/m-BIO-M in a batch system
OH
Me
1
50
45
97
98
98
79
>200
rac-1a
OH
Me
2
>200
rac-1b
a
BCL/m-BIO-M (2 mg) was placed inside a tube. The reaction was per-
formed with a 0.2 M solution of 1-phenylethanol, vinyl acetate (2 equiv) in
diisopropyl ether at 30 °C.
Determined by HPLC analysis using the equation: conversion
c) = ee(alcohol)/[ee(alcohol)+ee(ester)].
Determined by HPLC analysis.
Determined by HPLC analysis using the equation: E = ln{1–c[1+ee(es-
ter)]}/ln{1–c[1–ee(ester)]}.
b
(
c
d
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K. Mandai et al.
Letter
Figure 4 Kinetic resolution of 1-phenylethanol during 5 days of continuous flow at the optimal flow rate
der application of the magnetic field without special ma-
nipulation (see Figure 3, c and d). We speculate that the
alignment would promote efficient mixing and turbulent
flow during passage of the reaction solution. Accordingly,
the catalyst effectively makes contact with the reactants at
a particular flow rate to promote the kinetic resolution re-
action with sufficient efficiency.
Acknowledgment
We thank Keyence Corp. for a detailed digital microscopic observation
of BCL/m-BIOX-M inside the PTFE tube. This study was conducted
with partial financial support from JST-CREST.
Supporting Information
In conclusion, we constructed a microtube reactor in
which lipase was immobilized on magnetized iron oxide
derived from bacteria-produced amorphous iron oxide. The
enzyme catalyst in the reactor showed sufficient catalytic
activity in the kinetic resolution of secondary alcohols at
the optimal flow rate of the reaction solution. Moreover,
the catalyst maintained its high catalytic activity in a long-
term flow experiment without flowing out. Our microtube
reactor is easily prepared without special care regarding the
introduction of the catalyst in a flow path to achieve effi-
cient organic reaction. The catalyst can be attached and de-
tached with technical ease by the on–off application of a
magnetic field. This is the first example of the use of a high-
ly environmentally benign catalyst made of readily avail-
able bio-oriented materials (bacteriogenic iron oxide and li-
pase). Further studies will be needed to determine whether
catalysts other than enzymes can be used in this type of mi-
crotube reactor.
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0036-1589953.
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References and Notes
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Letter
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(25) Typical procedure for construction of the microtube reactor:
The tube was placed in a narrow groove cut into a wooden block
(ca. 10 cm long) with almost the same width as the tube. One
side of the tube (40 cm) was connected to a diaphragm pump
via a PEEK connector and BCL/m-BIO-M (2 or 4 mg) was sucked
from the other side. BCL/m-BIO-M was retained by the mag-
netic interaction with an external assembly of neodymium
magnets placed at a middle part of the tube. The tube, wooden
stick, and the magnets were put on a wire-woven base in a
water bath filled with water (30 °C). The tube was then con-
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filled with alcohol and vinyl acetate in diisopropyl ether, and
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(
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were consistent with previous data.
1
(
18) Uozumi, Y.; Yamada, Y. M. A.; Beppu, T.; Fukuyama, N.; Ueno,
M.; Kitamori, T. J. Am. Chem. Soc. 2006, 128, 15994.
19) (a) Yoshida, J.; Kim, H.; Nagaki, A. ChemSusChem 2011, 4, 331.
(S)-1a: H NMR (CDCl , 600 MHz): δ = 7.39–7.26 (m, 5 H), 4.91
3
(q, J = 6.6 Hz, 1 H), 1.51 (d, J = 6.6 Hz, 3 H); HPLC [CHIRALCEL
OJ-H; hexane/i-PrOH, 99:1; flow rate 0.5 mL/min; UV detection
(
(
b) Mason, B. M.; Price, K. E.; Steinbacher, J. L.; Bogdan, A. R.;
λ = 254 nm]: R = 24.4 (S), 29.0 (R) min.
t
1
McQuade, D. T. Chem. Rev. 2007, 107, 2300.
(R)-2a: H NMR (CDCl , 600 MHz): δ = 7.35–7.27 (m, 5 H), 5.89
3
(20) (a) Park, C. P.; Kim, D.-P. Angew. Chem. Int. Ed. 2010, 49, 6825.
(q, J = 6.6 Hz, 1 H), 2.07 (s, 3 H), 1.54 (d, J = 6.6 Hz, 3 H); HPLC
(b) Jeong, G.-Y.; Singh, A. K.; Sharma, S.; Gyak, K. W.; Maurya, R.
[CHIRALCEL OJ-H; hexane/i-PrOH, 99:1; flow rate 0.5 mL/min;
A.; Kim, D.-P. NPG Asia Mater. 2015, 7, e173.
UV detection λ = 254 nm]: R = 60.0 (R), 64.9 (S) min.
t
(
21) (a) Li, Y.; Xu, X.; Yan, B.; Deng, C.; Yu, W.; Yang, P.; Zhang, X.
J. Proteome Res. 2007, 6, 2367. (b) Liu, J.; Lin, S.; Qi, D.; Deng, C.;
Yang, P.; Zhang, X. J. Chromatogr., A 2007, 1176, 169. (c) Li, Y.;
Yan, B.; Deng, C.; Yu, W.; Xu, X.; Yang, P.; Zhang, X. Proteomics
Kinetic resolution of 1b. The production of (S)-1b and (R)-2a
was confirmed by comparison of the H NMR spectrum of the
1
crude mixture with those of (S)-1b and (R)-2b reported previ-
11
ously.
2
2
007, 7, 2330. (d) Liu, X.; Lo, R. C.; Gomez, F. A. Electrophoresis
009, 30, 2129. (e) Riveros, T. A.; Lo, R.; Liu, X.; Valdez, A.;
HPLC [CHIRALCEL OJ-H; hexane/i-PrOH, 19:1; flow rate 0.6
mL/min, UV detection λ = 254 nm]: R = (S)-1b: 22.4 (S), 26.6 (R)
t
Lozano, M.; Gomez, F. A. Am. Lab. 2010, 42, 11. (f) Yu, D.;
Antwerpen, P. V.; Patris, S.; Blankert, B.; Kauffmann, J.-M. Comb.
Chem. High Throughput Screening 2010, 13, 455. (g) Yan, X.;
Gilman, S. D. Electrophoresis 2010, 31, 346. (h) Sheng, J.; Zhang,
L.; Lei, J.; Ju, H. Anal. Chim. Acta 2012, 709, 41.
min; (R)-2b: 42.3 (R), 56.5 (S) min.
(27) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc.
1982, 104, 7294.
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Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F