Development of an Immobilized Ketoreductase for Enzymatic (R)-1-(3,5-Bis(trifluoromethyl)phenyl)ethanol Production

Article abstract of DOI:10.1021/op5003215

The development of an immobilized ketoreductase via covalent binding on resin EC-HFA has demonstrated that it is highly active and stable in organic solvents and can be recycled and reused many times in both batch mode and flow reactor mode. (R)-1-(3,5-Bi

Full text of DOI:10.1021/op5003215

Article
pubs.acs.org/OPRD
Development of an Immobilized Ketoreductase for Enzymatic
(R)‑1-(3,5-Bis(trifluoromethyl)phenyl)ethanol Production
Hongmei Li,* Johannah Moncecchi, and Matthew D. Truppo
Department of Process Chemistry, Merck Research Laboratories, Merck & Co., Inc., P.O. Box 2000, Rahway, New Jersey 07065,
United States
ABSTRACT: The development of an immobilized ketoreductase via covalent binding on resin EC-HFA has demonstrated that
it is highly active and stable in organic solvents and can be recycled and reused many times in both batch mode and flow reactor
mode. (R)-1-(3,5-Bis(trifluoromethyl)phenyl)ethanol, a key chiral intermediate in the synthesis of EMEND, was synthesized in
98% yield with >99% ee using the immobilized ketoreductase in a 50% hexanes/40% IPA/10% water solvent system. The
immobilized ketoreductase has also been applied to the synthesis of various chiral alcohols.
ketoreductases still remains challenging. To our knowledge,
only few examples of immobilization of ketoreductases have
been reported.15−17 One example uses a ketoreductase that
was immobilized by physical deposition on a nonporous glass
support, which was used in a continuous gas-phase reactor
without solvents.16 Yeast alcohol dehydrogenase was reported
to be immobilized covalently on Fe3O4 magnetic nanoparticles.17
The limitation of KRED immobilization work done to date is
that the immobilized ketoreductases did not tolerate operation
in organic solvents. Here we report the first demonstration of
an immobilized ketoreductase via covalent binding that is active
and stable in organic solvents and can be recycled and reused.
INTRODUCTION
■
Enzymes are naturally renewable, biodegradable, and sustain-
able catalysts, and biocatalysis has emerged as an important
technology for meeting the growing demand for green and
sustainable chemicals manufacture in the synthesis of flavours,
fragrances, and other fine chemicals.1Particularly, advances in
protein engineering technology have enabled biocatalysis to
become a preferred approach for the synthesis of enantiomeri-
cally pure compounds in the pharmaceutical industry because
of its excellent chemoselectivity, regioselectivity, and enantio-
selectivity.2 The reduction of prochiral ketones to chiral
alcohols is an excellent way to introduce enantioselectivity
into the synthesis of a molecule, as the enantioenriched alcohol
functionality can be easily transformed into other useful
functional groups without racemization.3 Many ketoreductases
(KREDs) are now commercially available for the synthesis of
chiral alcohols in good yields with excellent selectivity (often
>99% ee).4,5They generally operate under mild reaction condi-
tions and obviate the need for an ee upgrade, which is often
required with less selective metal-catalyzed transfer hydro-
genation approaches. Over the past few years the application of
KREDs to the synthesis of chiral alcohols has been
demonstrated on industrial scale.6,7 The ketoreductase reaction
is typically run in an aqueous buffer system and requires the
cofactor nicotinamide adenine dinucleotide phosphate
(NAD(P)H). The most efficient and cost-effective cofactor recycling
approach is the cosubstrate approach, in which isopropanol
(IPA) is used as a hydride source to recycle the nicotinamide
cofactor by the same KRED catalyst employed for the
reduction of the carbonyl substrate.8,9 Despite many advantages
and recent successes in biocatalysis, the use of enzymes is often
hampered by a lack of long-term operational stability due to
low substrate solubility in the aqueous reaction system coupled
with poor enzyme stability in commonly used organic solvents
and to difficulties in recovery and reuse of the enzyme.
Immobilization of enzymes provides one of the most attractive
approaches to overcome these drawbacks.10−12 We recently
reported the successful immobilization of a transaminase that
is capable of operating in organic solvent for many reaction
cycles.13 A variety of immobilized hydrolases are also widely
commercially available and used.14 However, immobilization of
RESULTS AND DISCUSSION
■
Both enantiomers of 1-(3,5-bis(trifluoromethyl)phenyl)ethanol
are pharmaceutically important alcohol intermediates for the
synthesis of NK-1 receptor antagonists. The enzymatic syn-
thesis of (S)-1-(3,5-bis(trifluoromethyl)phenyl)ethanol was
demonstrated with an enzyme isolated from Rhodococcus
erythropolis with excellent ee (>99%) and good conversion
(>98%).18 (R)-1-(3,5-Bis(trifluoromethyl)phenyl)ethanol is
currently incorporated into the orally active NK1 receptor
antagonist (EMEND) for chemotherapy-induced emesis and
has been synthesized by ruthenium-catalyzed transfer hydro-
genation or oxazaborolidine-catalyzed borane reduction with
91−93% ee, requiring an ee upgrade to >99% via a recrystalliza-
tion.19,20We were pleased to find that this important chiral
intermediate (R)-1-(3,5-bis(trifluoromethyl)phenyl)ethanol
can be synthesized with the commercially available ketoreductase
P1B2 from Codexis (Scheme 1). After thorough reaction op-
timization, the optimized aqueous reaction conditions for
the ketone reduction were found to be 150 g/L 3,5-
bis(trifluoromethyl)acetophenone, 1 wt % lyo KRED P1B2,
and 1 wt % NADP+ in 30% (v/v) IPA and 70% (v/v) 0.1 M
potassium phosphate buffer (pH 7) at 35 °C. The reaction
produces the desired (R)-alcohol product with 98−99%
Special Issue: Sustainable Chemistry
Received: October 13, 2014
© XXXX American Chemical Society
A
DOI: 10.1021/op5003215
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Article
Scheme 1. Enzymatic reduction of EMEND ketone with ketoreductase P1B2
conversion and >99% ee. A drawback of the aqueous reaction
system, which is common to many aqueous enzymatic reaction
systems, is the need for an extractive reaction workup to isolate
the product away from the enzyme catalyst. The extractive
workup results in denatured enzyme that cannot be reused and
that has the possibility to generate an emulsion layer that can
complicate the product isolation. The desire to eliminate these
issues led us to evaluate the immobilization of ketoreductase
P1B2 with several polymer-based resins.21 The resins selected
include three epoxide-functionalized supports for covalent
immobilization (EC-EP, EC-HFA, and EXE119) and two ad-
sorption supports for immobilization through hydrophobic
interactions (EXE120 and HP2MG).21
Figure 2. Illustration of enzyme immobilization through covalent
binding.22
organic solvent, and the reaction fails in 90% IPA. We further
optimized the immobilization conditions to provide for
maximum activity and stability. We found that increasing the
incubation temperature to room temperature provided for a
shortened incubation time of 6 h. Washing the resin with buffer
solution containing the NADP+ cofactor helped increase the activity
of the immobilized enzyme. The active enzyme loading on the
EC-HFA resin was estimated to be ∼5 wt % on the basis of an
activity comparison with the lyophilized enzyme. The ketoreductase
P1B2 immobilized on EC-HFA resin via covalent binding was
chosen and investigated further for activity and stability.
The lyophilized ketoreductase P1B2 and cofactor NADP+
were dissolved in buffer and incubated with each resin at 5 °C
for 48 h. The resins were then recovered, washed with buffer to
remove unbound enzyme, and dried under vacuum with a
nitrogen sweep at room temperature. Each resin was then
evaluated for activity on the 1-(3,5-bis(trifluoromethyl)phenyl)-
ethanone substrate in primarily organic solvent systems (90%
IPA and 10% buffer). All of the immobilized enzyme prepara-
tions showed activity in the 90% IPA system, with several
examples reaching >95% conversion within 24 h (Figure 1).
To study the long-term operational stability of the
immobilized enzyme, the activity and stability of immobilized
ketoreductase P1B2 in various solvents were investigated, using
IPA as a cosubstrate for cofactor regeneration. The use of buffer
was found to be unnecessary, with water providing similar or
better performance than buffer under the same reaction condi-
tions. This is an exciting result for immobilized ketoreductase
since aqueous buffer conditions are essential and required to
retain the activity and stability for general enzymatic reactions.
It was found that the level of water content in the organic
solvent system is crucial for the activity of the immobilized
ketoreductase. As shown in Figure 3, when no water was added,
the immobilized ketoreductase showed minimal reactivity, with
<10% conversion after 2 h. However, with the addition of only
2% water, the conversion jumped to 40% in 2 h. Good activity
(70% conversion in 2 h) was observed in IPA with a water
concentration of 10%. In addition to IPA used for cofactor
regeneration, additional cosolvents were studied. The immobi-
lized KRED was found to be active in a wide variety of organic
solvents. Hexanes, toluene, methyl tert-butyl ether (MTBE),
methyltetrahydrofuran (Me-THF), and cyclopentylmethyl
ether (CPME) as cosolvents (with a cosolvent/IPA/water
ratio of 50/40/10) all gave activities and conversions com-
parable to those under 90/10 IPA/water conditions (Figure 3).
A study of the effect of reaction temperature showed that 40 °C
is the optimal reaction temperature with the highest conver-
sion. The conversion slightly decreased when the tempera-
ture was increased to 60 °C (Figure 4). Reduction of 1-(3,5-
bis(trifluoromethyl)phenyl)ethanone using P1B2 immobilized
Figure 1. Conversion vs time for ketoreductase P1B2 immobilized on
each resin. Reaction conditions: 50 g/L ketone and 200 g/L
immobilized ketoreductase in 90% IPA and 10% buffer at 30 °C.
Immobilization on EC-HFA resin provided the highest activity,
with over 60% conversion in the first hour. EC-HFA is an
amino−epoxy-functionalized polymethacrylate resin, and the
enzyme is immobilized on it through covalent binding to
surface protein lysine residues (Figure 2). While the soluble
lyophilized KRED P1B2 can tolerate up to 60% IPA cosolvent,
the enzyme stability drops dramatically with increasing levels of
B
DOI: 10.1021/op5003215
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Article
Figure 3. Immobilized ketoreductase performance in different organic solvents and water concentrations. Reaction conditions: 50 g/L ketone and
100 g/L KRED immobilized on EC-HFA resin at 30 °C for 2 h.
three rounds of recycling, the reaction rate over the next
7 rounds remained consistent. In total, we demonstrated
10 consecutive recycles of the immobilized ketoreductase over a
200 h time period (Figure 5). Tight binding of the NADP+
cofactor to the immobilized KRED obviated the need for
cofactor addition at each round, further demonstrating the
efficiency of the reaction system.
The immobilized KRED P1B2 was also tested in a con-
tinuous reaction process. Immobilized ketoreductase (1 g) was
packed in a plug flow reactor (PFR) (6 cm length and 5 mL
volume). A 50 g/L ketone solution in 90/10 IPA/water was
flowed through the reactor via syringe pump, and the alcohol
Figure 4. Conversion as a function of reaction temperature. Reaction
conditions: 50 g/L ketone and 100 g/L KRED immobilized on EC-
HFA resin in 90% IPA and 10% water for 2 h.
product was collected in a flask from the other end. A plot of
reaction conversion versus residence time is shown in Figure 6.
A residence time of 4 h gave 93% conversion, and a residence
time of 10 h gave 98% conversion. After 7 days of holding the
PFR at 40 °C, we were pleased to find that the immobilized
ketoreductase P1B2 still retained 94% of its initial activity.23
Finally, we demonstrated the generality of this immobilized
ketoreductase in the synthesis of several chiral alcohols starting
from their prochiral ketones in batch mode, as shown in
Table 1.24 The ability to perform immobilized ketoreductase re-
actions in organic solvent (90% organic solvent and 10% water)
has significant advantages compared with running reactions
with soluble lyophilized enzyme in aqueous systems. Aqueous
reactions typically require the use of buffer to stabilize the re-
action pH in order to keep the enzymes active. Aqueous soluble
on EC-HFA resin was demonstrated on a 50 g scale in batch
mode under the following reaction conditions: 50 g/L ketone
and 100 g/L immobilized P1B2 in 50/40/10 hexanes/IPA/
water at 40 °C. The reaction gave the desired (R)-1-(3,5-
bis(trifluoromethyl)phenyl)ethanol with 99% conversion, 98%
yield and >99% ee.
With the optimized reaction conditions in hand, the stability
of the immobilized ketoreductase was evaluated through re-
cycling for multiple rounds. The enzyme was found to be quite
stable in the 90% IPA and 10% water system. Although the
initial rate of the reaction (1 h) decreased slightly after the first
Figure 5. Demonstration of reuse of immobilized ketoreductase for 10 rounds. Reaction conditions: 50 g/L ketone and 100 g/L KRED immobilized
on EC-HFA resin in 90% IPA and 10% water at 30 °C.
C
DOI: 10.1021/op5003215
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Article
most importantly, enables the enzyme catalyst to be reused
over multiple rounds, maximizing the utility of enzymes.
CONCLUSION
■
The first example of an immobilized ketoreductase capable of
operating in organic solvent has been developed and demon-
strated. Ketoreductase P1B2 was immobilized on a polymer-
based resin via covalent binding, and this catalytic system was
applied to the synthesis of (R)-1-(3,5-bis(trifluoromethyl)-
phenyl)ethanol, a key chiral intermediate in the synthesis of
EMEND. The immobilized enzyme exhibited excellent
selectivity and conversion and demonstrated that it could be
reused multiple times both in batch mode and in a continuous
plug flow reactor. Furthermore, this immobilized ketoreductase
was shown to be a generally useful tool for the rapid synthesis
of various chiral alcohols from ketones.
Figure 6. Test of immobilized KRED P1B2 in a continuous reaction
process. Conditions: plug flow reactor, 50 g/L ketone, 90% IPA and
10% water, 40 °C.
Table 1. Synthesis of chiral alcohols from ketones using
immobilized ketoreductase24
EXPERIMENTAL SECTION
■
Reagents and Enzymes. The ketoreductase enzyme P1B2
was purchased from Codexis. The resins used for immobiliza-
tion were purchased from Resindion or Chiralvision.
Analysis of the extent of conversion was performed by
reversed-phase HPLC using an Agilent HPLC system with an
Ascentis Express C18 column (2.7 μm, 100 mm × 4.6 mm)
with mobile phase A (water with 0.1 vol % H3PO4) and mobile
phase B (acetonitrile) in a gradient from 90/10 A/B to 10/90
A/B over 5 min and then holding at 10/90 A/B for 2 min. UV
absorbance was monitored at 210 nm. Both ketone substrates and
alcohol products were readily visible with this method, and the ee
was determined by chiral HPLC or supercritical fluid chromatog-
raphy (SFC) using the conditions listed below. Optical rotation
was measured on a PerkinElmer model 341 polarimeter.
Preparation of (R)-1-(3,5-Bis(trifluoromethyl)phenyl)-
ethanol from Lyo Ketoreductase P1B2. A mixture of 0.1 M
KH2PO4 (pH 7.0) buffer solution (140 mL), enzyme P1B2
(300 mg), and cofactor NADP (300 mg) was gently dissolved
to form a solution at rt, and the solution was heated to 30 °C.
1-(3,5-Bis(trifluoromethyl)phenyl)ethanone (30 g) in IPA
(60 mL) was added via an addition funnel over ∼60 min
while the temperature was kept at <35 °C. After the addition
was finished, the reaction mixture was aged at 35 °C for 24 h
until 99% conversion by HPLC was reached. The reaction
mixture was cooled to rt, and MTBE (150 mL, 5 vol) was
added. The mixture was allowed to settle for 30 min for layer
separation. The layers were cut, and the emulsion between the
layers was cut to the aqueous phase, after which the aqueous
layer was extracted with additional MTBE (90 mL, 3 vol). The
combined organic layers were washed with brine (150 mL,
5 vol). The organic layer was dried over MgSO4 and concen-
trated under reduced pressure (40 °C, 100 Torr), and the alcohol
product was obtained as an oil (29.2 g, 97% yield, >99% ee).
General Enzyme Immobilization Procedure for Resin
Screening. A 25 g/L solution of ketoreductase P1B2 from
Codexis was made in 100 mM potassium phosphate buffer
(pH 7.0) containing 2 g/L NADP. Immobilization resin (20 g)
was added to 10 mL of the enzyme solution, and the mixture
was placed on a shaking incubator at room temperature for
24 h. The resin was then filtered away from the enzyme solu-
tion and rinsed four times with 10 mL of 100 mM potassium
phosphate buffer (pH 7.0) containing 2 g/L NADP cofactor.
The immobilized enzyme resin was then dried under a nitrogen
sweep for 2 h.
Reaction conditions: 50 g/L ketone and 200 g/L immobilized
ketoreductase P1B2 in 90% IPA and 10% water at 30 °C for 20 h.
Reaction scale: 500 mg of ketone, reaction volume of 10 mL.
enzyme reaction systems typically require an extractive workup
in order to recover and isolate the desired product. Extractive
workups denature the enzyme, rendering it inactive, and can
potentially complicate the isolation because of the formation of
an emulsion layer. The immobilized enzyme reaction in organic
solvent greatly simplifies the workup to a simple filtration and,
D
DOI: 10.1021/op5003215
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Article
Optimized Enzyme Immobilization Procedure for
EC-HFA Resin. A 100 g/L solution of ketoreductase P1B2
from Codexis in 100 mM potassium phosphate buffer (pH 7.0)
containing 2 g/L NADP was made. Then 20 g of EC-HFA resin
(Chiralvision) was added to 10 mL of the enzyme solution, and
the mixture was placed on a shaking incubator at room
temperature for 6 h. The resin was then filtered away from the
enzyme solution and rinsed four times with 10 mL of 100 mM
potassium phosphate buffer (pH 7.0) containing 2 g/L NADP
cofactor. The immobilized enzyme resin was then dried under
a nitrogen sweep for 2 h. The typical water content of the
immobilized resin was 2−5 wt %. The immobilization yield
was ∼98% (immobilization yield = 100% × [enzyme added
(units/g of carrier) − unbound enzyme (units/g of carrier)]/
[enzyme added (units/g of carrier)]). The active enzyme
loading on the EC-HFA resin was estimated to be ∼5 wt % on
the basis of an activity comparison with the lyophilized enzyme.
General Immobilized Ketoreductase Reaction Protocol.
The ketone substrate (50 g/L) was dissolved in 90% 2-propanol
and 10% water, and 100 g/L immobilized ketoreductase P1B2
was added to the substrate solution. The reaction mixture was
heated to 30−40 °C and stirred with an orbital shaker for 20 h.
For immobilized ketoreductase reuse testing, the immobilized
enzyme was filtered away from the reaction solution and rinsed
with 3 volumes of 90/10 IPA/water to ensure removal of the
starting material and product. The recovered KRED was
then used directly in the subsequent reaction with no further
manipulation.
1H); 7.12−7.08 (m; 2H); 7.47−7.44 (m; 2H) ppm. 13C NMR
(100 MHz, CDCl3): δ 71.7−72.6 (q), 115.5−115.8 (d), 122.7−
25
589
129.3 (q), 129.3 (d), 129.8, 162.2−164.6 (d) ppm. [α] +51.1
(c 1.0, DMF).
Entry 3.
SFC with an AD-H column, iso 5% EtOH/25 mM isobutyl-
amine/CO2, 8 min run, 2 mL/min, 35 °C. 1H NMR (400 MHz,
CDCl3): δ 1.28 (t; J = 7.15 Hz; 3H); 2.81−2.69 (m; 2H); 3.31
(s; 1H); 4.20 (q; J = 7.14 Hz; 2H); 5.15 (dd; J = 8.70, 4.07 Hz;
1H); 7.32−7.28 (m; J = 7.70 Hz; 1H); 7.40−7.36 (m;
4H) ppm. 13C NMR (100 MHz, CDCl3): δ 14.1, 43.3, 60.9,
25
589
70.3, 125.7, 127.8, 128.5, 142.5, 172.4 ppm. [α]
(c 1.0, DMF).
−11.6
Entry 4.
Normal-phase HPLC with a Chiralpak OD-H column, 95%
1
Hex/5% EtOH, 40 °C, 1 mL/min, 210 nm, 15 min run. H
NMR (400 MHz, CDCl3): δ 1.48 (d; J = 6.50 Hz; 3H); 4.90
(q; J = 6.51 Hz; 1H); 7.44 (d; J = 8.24 Hz; 1H); 7.59 (dd; J =
8.21, 2.45 Hz; 1H); 8.32 (s; 1H) ppm. 13C NMR (100 MHz,
CDCl3): δ 25.3, 67.3, 127.9, 136.0, 140.5, 140.8, 147.8 ppm.
25
[α] +47.7 (c 1.0, DMF).
589
Preparation of (R)-1-(3,5-Bis(trifluoromethyl)phenyl)-
ethanol from Immobilized KRED P1B2. A mixture of P1B2
immobilized on EC-HFA resin (100 g), water (100 mL), IPA
(400 mL), hexanes (500 mL), and 1-(3,5-bis(trifluoromethyl)-
phenyl)ethanone (50 g) was stirred and heated to 40 °C and
aged for 15 h. The reaction gave >99% conversion by HPLC.
The immobilized enzyme was filtered off and rinsed with 95/5
IPA/water (200 mL). The combined fitrate was concentrated
and flashed with IPA to remove water under reduced pressure
(40 °C, 100 Torr), and finally the clean alcohol product was ob-
tained as an oil (32 g, 92 wt % by NMR, 98% yield, >99% ee).
The recovered immobilized enzyme could be reused several times.
NMR Data and Chiral Methods for Table 1.
Entry 1.
Entry 5.
SFC with an AD-H column, isocratic 5% EtOH with 25 μM
isobutylamine, 2 mL/min, CO2 200 psi, 40 °C, total 8 min run.
1H NMR (400 MHz, CDCl3): δ 4.41 (q; J = 7.99 Hz; 2H); 4.88
(q; J = 6.52 Hz; 1H); 7.31−7.26 (m; 2H); 8.28 (d; J = 2.58 Hz;
1H) ppm. 13C NMR (100 MHz, CDCl3): δ 24.2, 66.2−66.6
25
589
(q), 68.8, 120.3, 123.5, 136.1, 152.8, 157.5 ppm. [α] +40.5
(c 1.0, DMF).
Entry 6.
SFC with an AD-H column, gradient 4−40% EtOH with
25 μM isobutylamine in 6 min, hold 2 min, 2 mL/min, CO2
200 psi, 40 °C, total 8 min run. 1H NMR (400 MHz, CDCl3): δ
1.93 (1.60−2.05; m; 6H); 3.44−3.30 (m; 2H); 3.58−3.47 (m;
2H); 3.88−3.86 (m; 1H); 5.13 (d; J = 1.87 Hz; 2H); 7.34 (t;
J = 4.42 Hz; 5H) ppm. 13C NMR (100 MHz, CDCl3): δ 21.9−
Normal-phase HPLC with a Chiralcel OD-H column, 98%
hexanes/2% 2-propanol, 1 mL/min, 10 min run.18 1H NMR
(400 MHz, CDCl3): δ 1.53 (d; J = 6.51 Hz; 3H); 2.19 (s; 1H);
5.02 (q; J = 6.50 Hz; 1H); 7.78 (s; 1H); 7.83 (s; 2H) ppm. 13
C
NMR (100 MHz, CDCl3): δ 25.5, 69.2, 121.2 (d), 125.6 (d),
25
589
119.3−127.4 (q), 131.2−132.2 (q), 148.2 ppm. [α] +23.6
22.3, 35.1−35.3, 37.1−37.3, 41.0−41.4, 46.2−46.4, 67.0, 70.4−
25
589
(c 1.0, DMF).
Entry 2.
70.6, 127.8, 127.9, 128.5, 136.9, 156.2 ppm. [α] −10.1 (c 1.0,
DMF).
Entry 7.
Normal-phase HPLC with a Chiralcel OD-H column, 95%
1
heptane/5% EtOH, 40 °C, 1 mL/min, 12 min run. H NMR
Chiral GC with a Beta-Dex-SA column (30 m, 0.25 mm
(400 MHz, CDCl3): δ 2.78 (br s; 1H); 5.01 (q; J = 6.64 Hz;
ID, 0.25 um), heater 220 °C, H2 flow 40 mL/min, air flow
E
DOI: 10.1021/op5003215
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Article
(20) Xu, L.; Huang, Z.-H.; Sandoval, C. A.; Gu, L.-Q.; Huang, Z.-S.
Org. Process Res. Dev. 2014, 18, 1137−1141.
400 mL/min, makeup flow (He) 40 mL/min, constant pressure
12 psi, split 50:1, oven 170 °C hold 8 min, total 8 min run. 1H
NMR (400 MHz, CDCl3): δ 1.18 (s; 3H); 1.25 (s; 3H); 1.28−
1.31 (m; 1H); 1.40−1.52 (m; 1H); 1.88−1.82 (m; 2H); 3.61
(t; J = 12.11 Hz; 1H); 3.80 (dd; J = 12.29, 4.40 Hz; 1H); 4.00−
(21) (a) Cantone, S.; Ferrario, V.; Corici, L.; Ebert, C.; Fattor, D.;
Spizzo, P.; Gardossi, L. Chem. Soc. Rev. 2013, 42, 6262. The
Supporting Information for this article contains a variety of polymer
resin information. (b) Mateo, C.; Grazu, V.; Pessela, B. C. C.; Montes,
́
3.88 (m; 1H) ppm. 13C NMR (100 MHz, CDCl3): δ 23.1, 31.1,
́
́
T.; Palomo, J. M.; Torres, R.; Lopez-Gallego, F.; Fernandez-Lafuente,
25
589
35.6, 45.8, 60.0, 65.3, 72.8 ppm. [α] −20.1 (c 1.0, DMF).
R.; Guisan, J. M. Biochem. Soc. Trans. 2007, 35, 1593.
(22) Sheldon, R. A.; van Pelt, S. Chem. Soc. Rev. 2013, 42, 6223.
(23) The remaining activity of 94% for the immobilized enzyme
(stability study) was determined from the conversion in ketone
reduction with the immobilized enzyme after it was packed for 7 days
at 40 °C in the PFR versus the conversion with the immobilized
enzyme packed fresh in the PFR under the same reaction conditions
and residence time.
(24) In Table 1, the ee values of the alcohol products with
immobilized P1B2 are the same as or similar to those with the lyo
enzyme P1B2 reaction system. The ee values in entries 6 and 7 could
be lower or higher depending on the enzyme loading for both the
immobilized and lyo enzyme systems.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: hongmei06@gmail.com.
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Tufvesson, P.; Lima-Ramos, J.; Nordblad, M.; Woodley, J. M.
Org. Process Res. Dev. 2011, 15, 266−274.
(2) (a) Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz,
S.; Moore, J. C.; Robins, K. Nature 2012, 485, 185−194. (b) Turner,
N. J. Nat. Chem. Biol. 2009, 5, 567−573. (c) Hall, M.; Bommarius, A.
S. Chem. Rev. 2011, 111, 4088−4110.
(3) Truppo, M. In Comprehensive Organic Synthesis II, 2nd ed.;
Elsevier: Amsterdam, 2014; Vol. 8, pp 317−327.
(4) Moore, J. C.; Pollard, D. J.; Kosjek, B.; Devine, P. N. Acc. Chem.
Res. 2007, 40, 1412−1419.
(5) Huisman, G. W.; Liang, J.; Krebber, A. Curr. Opin. Chem. Biol.
2009, 14, 1−8.
(6) (a) Liang, J.; Lalonde, J.; Borup, B.; Mitchell, V.; Mundorff, E.;
Trinh, N.; Kochrekar, D. A.; Cherat, R. N.; Pai, G. G. Org. Process Res.
Dev. 2010, 14, 193−198. (b) Liang, J.; Mundorff, E.; Voldari, R.;
Jenne, S.; Gilson, L.; Conway, A.; Krebber, A.; Wong, J.; Huisman, G.;
Truesdell, S.; Lalonde, J. Org. Process Res. Dev. 2010, 14, 188−192.
(7) Magano, J.; Dunetz, J. R. Org. Process Res. Dev. 2012, 16, 1156−
1184.
(8) Ma, S. K.; Gruber, J.; Davis, C.; Newman, L.; Gray, D.; Wang, A.;
Grate, J.; Huisman, G. W.; Sheldon, R. A. Green Chem. 2010, 12, 81−
86.
(9) Calvin, S. J.; Mangan, D.; Miskelly, I.; Moody, T. S.; Stevenson, P.
J. Org. Process Res. Dev. 2012, 16, 82−86.
(10) (a) Hanefeld, U.; Cao, L.; Magner, E. Chem. Soc. Rev. 2013, 42,
6211−6212. (b) DiCosimo, R.; McAuliffe, J.; Poulose, A. J.;
Bohlmann, G. Chem. Soc. Rev. 2013, 42, 6437−6474.
(11) Gonzalo, G.; Lavandera, I.; Faber, K.; Kroutil, W. Org. Lett.
2007, 9, 2163−2166.
(12) Stepankova, V.; Bidmanova, S.; Koudelakova, T.; Prokop, Z.;
Chaloupkova, R.; Damborsky, J. ACS Catal. 2013, 3, 2823−2836.
(13) Truppo, M.; Strotman, H.; Hughes, G. ChemCatChem 2012, 4,
1071−1074.
(14) (a) Truppo, M. D.; Hughes, G. Org. Process Res. Dev. 2011, 15,
1033−1035. (b) Bruggink, A.; Roos, E. C.; de Vroom, E. Org. Process
Res. Dev. 1998, 2, 128−133. (c) Guo, Z.; Wong, M. K. Y.; Hickey, M.
R.; Patel, B. P.; Qian, X.; Goswami, A. Org. Process Res. Dev. 2014, 18,
774−780.
(15) Milagre, H. M. S.; Milagre, C. D. F.; Moran, P. J. S.; Santana, M.
H. A.; Rodrigues, J. A. R. Org. Process Res. Dev. 2006, 10, 611−617.
(16) Nagayama, K.; Spiess, A. C.; Buchs, J. Biochem. Eng. J. 2010, 52,
301−303.
(17) Chen, D.-H.; Liao, M.-H. Biotechnol. Lett. 2001, 23, 1723−1727.
(18) Pollard, D.; Truppo, M.; Pollard, J.; Chen, C.-y.; Moore, J.
Tetrahedron: Asymmetry 2006, 17, 554−559.
(19) Brands, K. M. J.; Payack, J. F.; Rosen, J. D.; Nelson, T. D.;
Candelario, A.; Huffman, M. A.; Zhao, M. M.; Li, J.; Craig, B.; Song, Z.
J.; Tschaen, D.; Hansen, K.; Devine, P. N.; Pye, P. J.; Rossen, K.;
Dormer, P. G.; Reamer, R. A.; Welch, C. J.; Mathre, D. J.; Tsou, N. N.;
McNamara, J. M.; Reider, P. J. J. Am. Chem. Soc. 2003, 125, 2129−
2135.
F
DOI: 10.1021/op5003215
Org. Process Res. Dev. XXXX, XXX, XXX−XXX