Preparing β-blocker (R)-Nifenalol based on enantioconvergent synthesis of (R)-p-nitrophenylglycols in continuous packed bed reactor with epoxide hydrolase

Article abstract of DOI:10.1016/j.tet.2019.01.034

An engineered epoxide hydrolase from Vigna radiate (VrEH2_M263N) shows near-perfect enantioconvergence in single enzyme mediated hydrolysis of racemic p-nitrostyrene oxide (pNSO). To explore industrial potential of the promising biocatalyst, we tried to immobilize the VrEH2 variant by covalently linking onto a commercially available amino resin ECR8405F. Then a 5-mL packed bed reactor filled with the immobilized VrEH2_M263N was connected with macroporous resin NKA-11 for in situ product adsorption, and the product (R)-p-nitrophenyl glycol (pNPG) was harvested by methanol elution, with 91% isolated yield and 97% ee. The continuous reactor was operated stably for more than 100 h with a space time yield of 20 g?L^?1?h^?1. Subsequently, the β-blocker (R)-Nifenalol was prepared by chemically synthesized from (R)-pNPG, affording the product in an overall yield of 61.3% (1.5 g) and an enantiopurity of 99.9% ee after recrystallization.

Full text of DOI:10.1016/j.tet.2019.01.034

Accepted Manuscript
Preparing β-blocker (R)-Nifenalol based on enantioconvergent synthesis of (R)-p-
nitrophenylglycols in continuous packed bed reactor with epoxide hydrolase
Fu-Long Li, Yu-Cong Zheng, Hao Li, Fei-Fei Chen, Hui-Lei Yu, Jian-He Xu
PII:
S0040-4020(19)30061-4
https://doi.org/10.1016/j.tet.2019.01.034
TET 30086
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 12 November 2018
Revised Date: 9 January 2019
Accepted Date: 15 January 2019
Please cite this article as: Li F-L, Zheng Y-C, Li H, Chen F-F, Yu H-L, Xu J-H, Preparing β-blocker (R)-
Nifenalol based on enantioconvergent synthesis of (R)-p-nitrophenylglycols in continuous packed bed
reactor with epoxide hydrolase, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.01.034.
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Preparing β-Blocker (R)-Nifenalol Based on Enantio-
convergent Synthesis of (R)-p-Nitrophenylglycols in
Continuous Packed Bed Reactor with Epoxide Hydrolase
Fu-Long Li, Yu-Cong Zheng, Hao Li, Fei-Fei Chen, Hui-Lei Yu and Jian-He Xu
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of
Science and Technology, Shanghai 200237, P. R. China.

1
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Tetrahedron
journal homepage: www.elsevier.com
Preparing β-Blocker (R)-Nifenalol Based on Enantioconvergent Synthesis of (R)-
p-Nitrophenylglycols in Continuous Packed Bed Reactor with Epoxide
Hydrolase
Fu-Long Li, Yu-Cong Zheng, Hao Li, Fei-Fei Chen, Hui-Lei Yu and Jian-He Xu
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and
Technology, Shanghai 200237, P. R. China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
An engineered epoxide hydrolase from Vigna radiate (VrEH2M263N) shows near-perfect
enantioconvergence in single enzyme mediated hydrolysis of racemic p-nitrostyrene oxide
(pNSO). To explore industrial potential of the promising biocatalyst, we tried to immobilize the
VrEH2 variant by covalently linking onto a commercially available amino resin ECR8405F.
Then a 5-mL packed bed reactor filled with the immobilized VrEH2M263N was connected with
macroporous resin NKA-11 for in situ product adsorption, and the product (R)-p-nitrophenyl
glycol (pNPG) was harvested by methanol elution, with 91% isolated yield and 97% ee. The
continuous reactor was operated stably for more than 100 h with a space time yield of 20 g L-1 h-
Available online
In Memory of Professor Wei-Shan Zhou
for his legacy in natural product synthesis.
Keywords:
1. Subsequently, the
β-blocker (R)-Nifenalol was prepared by chemically synthesized from (R)-
Vigna radiate epoxide hydrolase
Enantioconvergent hydrolysis
Immobilized enzyme
Packed bed reactor
(R)-Nifenalol.
pNPG, affording the product in an overall yield of 61.3% (1.5 g) and an enantiopurity of 99.9%
ee after recrystallization.
2018 Elsevier Ltd. All rights reserved
.
———
Corresponding authors: e-mail: huileiyu@ecust.edu.cn; jianhexu@ecust.edu.cn.

2
Tetrahedron
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1. Introduction
(R)-Nifenalol is a β-adrenergic blocker drug endowed with
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development of commercial resins provided new carriers for
immobilization of EHs. They were more easily used by the
covalently bonding the enzyme than the other carriers and have
been successfully applied in recent study.23,24 Continuous process
by biocatalyst has been regarded as a green strategy for
sustainable industrial production.25 The design of continuous
reactor based on the immobilized enzyme have attracted much
attention for large-scale preparation of fine chemicals.26 Packed
bed reactor (PBR) is one of common continuous-flow reactors,
which can improve enzyme loading per unit volume, reduce the
mechanical damage to the enzyme and avoid the experimental
loss of enzyme.27,28 Herein, to develop the industrial application
potential of VrEH2, a number of commercial resins were used to
prepare immobilized the epoxide hydrolase. A packed bed reactor
with immobilized enzyme was designed for continuous
preparation of (R)-pNPG from rac-pNSO. To collect the product,
an adsorbed column with macroporous resin NKA-11 was
connected with PBR, and thus can construct a continuously
prepared apparatus. Finally, the synthesis of β-blocker (R)-
Nifenalol was performed based on enzymatic enantioconvergent
preparation of (R)-p-nitrophenyl glycol, with good yield and high
enantiopurity.
anti-anginal and anti-arhythmic properties.1,2 One of the tempting
ways to this drug was to use (R)-p-nitrophenyl glycol [(R)-
pNPG] as intermediate combining with chemical routes (Scheme
1). In the past two decades, several chemical and biological
methods have been explored to obtain the intermediate (R)-
pNPG. For example, the carbonyl reductases (RCR, 0.223 U/mg
cell free extract) or organic catalysts was used to produce the
chiral vicinal diols from hydroxyketone compound,3,4 as well as
oxidation of olefins by chromium Jacobsen's catalysts I or P450s
(P450pyrTM, 12 U g-1 cdw-1).5,6 However, these enzymes
suffered low catalytic activity and poor stability. Although
chemical routes can performed in good yield, the expensive
organic catalysts and harsh reaction conditions would face the
environmental and safety problems.4,7 As an alternative green
route, epoxide hydrolase mediated enantioconvergent hydrolysis
of styrene oxides can highly produce the corresponding vicinal
diols.8,9 Furstoss group first performed the availability to
synthesize chiral (R)-pNPG using step-wise hydrolysis of p-
nitrostyrene oxide (pNSO) with AnEH and H2SO4, which led to
satisfied yield (94%), but the ee value was only 80%.10
Similarly, we utilized the native VrEH2 and H2SO4 to step-wisely
synthesize (R)-pNPG with 82% ee and 84% yield.11 Although the
preparation of (R)-pNPG can be performed by chemo-enzymatic
routes, the degree of enantioconvergence was still unsatisfactory.
2. Results and Discussion
2.1. Preparation of covalently immobilized VrEH2M263N
OH
OH
O
OH
NH
c
: iPrNH2
a
: 33%HBr-HAc
To improve the stability of the VrEH2M263N, we selected two
kinds of commercial epoxy and amino resins for immobilization.
Free enzyme can be covalently bonded with the functional groups
of these resins, and seven immobilized enzymes were
successfully prepared. The loading and activity recovery rate
(Table 1) indicate that the immobilized efficiency of amino resins
was better than that of epoxy resins, and VrEH2M263N-ECR8405F
exhibited the highest loading (13.2 U/gresin) and activity recovery
rate (57.4%). Subsequently, to investigate the wear resistant
performance of these resins, the immobilized enzymes ESR-1,
ESQ-1, ECR8405F and ECR8205F was selected. The
immobilized resins’ shapes were observed after magnetic stirring
in Fig. 1. The resins ECR8405F and ECR8205F maintained the
relatively overall spherical morphology, while the shapes of
ESR-1 and ESQ-1 was severely destroyed. Hence, the
ECR8405F and ECR8205F were selected for subsequent
b: K2CO3
O2N
O2N
O2N
Scheme 1. Chemical synthesis of β-blocker (R)-Nifenalol based on (R)-p-
nitrophenyl glycol which can be produced by enzymatic hydrolysis of
epoxide.
Compared to the chemo-enzymatic combination, the
enantioconvergent hydrolysis by a single and special enzyme is
very attractive for preparation the product (R)-diols with high
yield
and
enantiopurity.
However,
the
perfect
enantioconvergence depends on the highly specific and opposite
regioselectivity of an epoxide hydrolase towards both
enantiomers of the epoxide substrate, thus caused a challenge for
enantioconvergent hydrolysis of epoxide hydrolase.12-15
Fortunately, we recently obtained
a single site mutant
optimization
of
immobilized
conditions
(Supporting
VrEH2M263N which allowed the near-perfect enantioconvergence
(>99% analytical yield, 98% ee) in hydrolysis of rac-pNSO
information).
through
structure-guided
regioselectivity
engineering.16
Nevertheless, the unsatisfactory operation stability of epoxide
hydrolases and the spontaneous hydrolysis of racemic epoxides
cannot be ignored. Under mechanical shaking condition, the
stability of VrEH2M263N was very poor, it showed no activity after
24 h. Besides, due to the spontaneous hydrolysis of rac-pNSO in
aqueous solution, the eep value was decreased from 98% to 92%
under high concentration of substrate (100 mM), which also
limited the wide application of VrEH2M263N
.
Immobilization technology is a powerful strategy for elevating
enzymes’ stability and recycling efficiency.17-20 Faber group
prepared the immobilized Nocardia EH1 through DEAE-
cellulose anion adsorption, it can keep a residual activity of 55%
after five batch reactions.21 Furstoss group synthesized a stable
immobilized StEH by the using dextran and glyoxal-agarose,
which showed a 300-fold increase in thermal stability under 60
oC and retained high selectivity.22 In recent years, the
Fig. 1. The shape insights of immobilized resins after magnetic shaking
(24 h). A) ECR8405F; B) ECR8205F; C) ESR-1; D) ESQ-1.

4
Tetrahedron
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Table 1
The loading and activity recovery rates of the immobilized enzymes prepared with commercial resins.
Activity
recovery (%)
Functional group
Resins
Diameter (Å)
Enzyme loading (U/gresin)
ECR8205F
ECR4204F
ECR8215F
ES-103
150~300
150~300
150~300
100~300
4.3 ± 0.1
2.4 ± 0.2
0.8 ± 0.0
2.0 ± 0.1
18.5
10.5
3.5
Epoxy
8.8
ESR-1
ESQ-1
100~300
100~300
150~300
9.9 ± 0.0
5.5 ± 0.1
13.2 ± 0.1
45.2
25.1
57.4
Amino
ECR8405F
2.2. Thermal and operational stability of immobilized enzyme
VrEH2M263N-ECR8405F
In order to utilize the immobilized enzyme for the
preparation of (R)-pNPG, Synthesis of (R)-pNPG by
VrEH2M263N-ECR8405F in a stirred reactor was first explored.
The substrate loading can reach to 100 mM (10% DMSO, v/v) in
a 40-mL scale, the immobilized enzyme was recycled by a
filtration step. After 10 batches, (R)-pNPG was obtained in 91%
yield and 92% ee, with a space time yield (STY) was up to 16.7
g L-1 h-1. However, the deterioration of the optical purity of the
product was observed, which is possibly caused by the
spontaneous hydrolysis of substrate under the high concentration
(100 mM). Mechanical operation and physical loss also led to the
irreversible inactivation, so the residual activity of immobilized
enzyme was only 43.5 % after 10 batches.
By measuring the inactivation curves of the immobilized
enzyme VrEH2M263N-ECR8405F, we found that the thermal
stability of the immobilized enzyme was obviously altered
comparing with the free enzyme. The residual activity of the free
enzyme was only 16.3% after 96 h’s incubation and the half-life
(t1/2) was 55.1 h at 30 °C, while the immobilized enzyme retained
42.3% residual activity after 200 h’s incubation and its t1/2 was
improved to 193.2 h (Fig. 2A). After incubation for 20 h at 40
°C, the residual activity of free enzyme was only 18.0% with a
t1/2 of only 11.1 h, but the immobilized enzyme retained 23.8%
residual activity after 150 h of incubation with an increased t1/2 of
101.9 h, (Fig. 2B). The free enzyme showed no activity after
incubation for 15 min at 50 °C, while the 21.9% residual activity
was retained after 45 min for immobilized enzyme (Fig. S1).
These results indicate that the immobilization of the enzyme by
covalently bonding of the resin carrier can significantly improve
its thermal stability.
Fig. 3. Residual activity of the immobilized enzyme and ee value of
product was determined after per batch.
Fig. 2. The inactivation curves of immobilized enzymes and free
enzymes at 30 °C (A) and 40 °C (B).
2.4. Construction of PBR for continuous synthesis of (R)-pNPG
As the batch reaction cannot provide the satisfactory result,
we tried to address the problem with continuous synthesis mode.
A packed bed reactor (PBR) was designed and optimized in
several aspects including the flow direction, space velocity and
the height to diameter ratio (H-D) of the PBR (Supporting
information). The performance of PBR was subsequently
determined, including the stability and space time yield (STY). In
a first trial, the conversion of PBR (H-D: 2:1) was only 74.7%
under the equal biocatalyst loading and space velocity (SV),
resulting in the STY of 41.0 g L-1 h-1. Using the same enzyme
amount and SV, when the H-D ratio improved by 4:1 or 8:1, a
higher STY (54.9 g L-1 h-1) were achieved (Table 2), which was
much better than that of the stirred reactor (16.7 g L−1 h−1). By
measuring the stability of reactor, we found that the lower H-D
The operational stabilities of free enzyme and immobilized
enzyme were also investigated. Consequently, the free enzyme
lost its activity after 24 h, while the immobilized enzyme still
maintained 73% of initial activity after 150 h (Fig. S2), indicating
the operational stability of the epoxide hydrolase was also
improved. To verify the reaction performance, a number of
batches reaction for immobilized enzyme was performed, it
retained a residual activity of 77% after 20 batches and the ee
value of product can be reached to 97% (Fig. 3). The loss of
activity was mainly attributed to the mechanical damage and
physical recovery loss.
2.3. Synthesis of (R)-pNPG from rac-pNSO in a stirred reactor

5
ratio of PBR, the higher residual activity of the immobilized
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enzyme. It was mainly because the height of the reactor increased

6
Tetrahedron
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Table 2
Key reaction parameters of PBR with different H/D ratios.
Type (H-D)a
Residual activityb
S.T.Y. (g L−1 h−1)c
Conversion (%)
2:1
4:1
>99%
97.4%
95.5%
41.0
54.9
54.9
74
99
99
8:1
a
H-D: height to diameter ratio of the PBR
b Specific activity of immobilized enzyme after continuous operation;
c Space time yield normalized by the reactor volume.
3. Conclusion
the pressure at the bottom of the packed bed, resulting in the loss
of enzyme activity. Compromising the productivity with the
stability of biocatalyst, the H-D ratio of PBR was set to 4:1 in the
following research.
In this study, the key intermediate of (R)-Nifenalol, (R)-
pNPG, was successfully prepared by VrEH2 in an
enantioconvergent manner. The immobilized VrEHs were
prepared by covalently cross-linking with a commercially
2.5. Screening macroporous resins for product adsorption
available amino resin ECR8405F.
A simple continuous
Due to the poor solubility of the epoxides, the substrate
concentration was limited at 5 mM with 10% DMSO in reaction.
For improving the efficiency of the product recovery from large
volume reactant with low concentration, an additional product
adsorption column was connected with the PBR for product
enrichment. Three kinds of polar macroporous adsorption resins
were compared and the adsorption capacity of these resins under
different space velocities were investigated (Fig. S3). It indicates
that the largest adsorption capacity of resin NKA-11 was 165.5
mg/g. the adsorption efficiency of resin NKA-11 showed more
than 99% at space velocity of 0.4 min-1, so NKA-11 was selected
for recovery of product.
manufacturing module was subsequently established to prepare
the valuable intermediate, the STY of the reactor can reach 20.0
g L-1 h-1 with high enantiopurity (99% ee) after recrystallization.
This allowed the preparation of enantiopure (R)-Nifenalol
combination of chemical method with a 61.3% overall yield.
4. Experimental section
4.1 General
Substrate rac-pNSO was prepared according to the reported
method.29 All the biological and chemical reagents were
commercial available. Immobilized resins were provided from
Tianjin Nankai Hecheng Science and Technology Co., Ltd and
Based on this mini scale continuous preparation apparatus
(Fig. 4), a gram-scale preparation of (R)-pNPG was carried out.
As expected, the PBR still maintained more than 99% conversion
After 100 h continuous production and showed good operational
stability (Fig. S4). Then product of adsorbed column could be
readily eluted by methanol and the resin was recycled, 3.00 g (R)-
pNPG was obtained with a yield of 91% and 99% ee. The STY of
the continuous apparatus reached 20.0 g L-1 h-1, which was
higher than that of the stirred batch reaction. Subsequent
recrystallization in chloroform gave 2.66 g highly pure (R)-pNPG
with 87% yield and 99.9% ee.
1
Purolite (China) Co., Ltd. The NMR spectra H and 13C NMR
were recorded in CDCl3 solution on Brucker-400 MHz. The
1
chemical shift (δ) of H NMR and 13C NMR was given in ppm
relative to solvent residual peak according to the reference 30.
The optical rotation of (R)-Nifenalol was measured by Autopol I
(Rudolph, American). The ee value of product was determined by
chiral OD-H column as previously described.16 High resolution
mass spectra were determined on Waters GCT Premier (EI-OA-
TOF).
4.2. Enzyme Immobilization
Genes of VrEH2M263N were constructed with pET-28a(+)
and expressed in Escherichia coli BL21 (DE3). The methods for
cells cultivation, enzyme expression and purification were
described previously.16 The collected recombinant cells were
disrupted by a high-pressure homogenizer and centrifuged, clear
lysate was lyophilized by a freeze dryer. The lyophilized enzyme
powder (50 mg) was dissolved in 10 mL of phosphate buffer (pH
7.0, 100 mM) and mixed with 1.0 g amino resins (activated by 2%
(v/v) glutaraldehyde) or epoxy resins. The mixture was reacted in
a shaker (16 °C, 180 rpm) for 12 h. The immobilized enzyme was
obtained by filtration and washed with buffer carefully to avoid
the remaining of glutaraldehyde. The activity recovery rate was
calculated by the activity of immobilized enzyme and the initial
activity of free enzyme.
Fig. 4. Scheme of continuous synthesis apparatus, including PBR (5 mL)
and a product adsorbed column (20 mL).
2.6. Synthesis of (R)-Nifenalol from (R)-pNPG
4.3. Thermostability and operational stability of immobilized
enzyme
According to the reported method of Furstoss group,10 the
gram-scale synthesis of (R)-Nifenalol was performed, which led
to the (R)-Nifenalol in 61.3% overall yield after recrystallization.
Investigation the wear resistance of different resins. In 10-mL
reactor (KPB buffer, pH 7.0, 100 mM), 1 g epoxy resins or amino
resins was stirred (400 rpm, 30 °C) for 24 h. Then morphological

7
shapes of the immobilized carrier were observed by using a
Cryo-SEM (Scanning Electron Microscope).
sodium hydrogen carbonate, and then extracted with ethyl
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acetate. The organic phase was washed with saturated NaCl,
dried over anhydrous sodium sulfate and evaporated. The
resulting residual was dissolved in methanol (20 mL) and
anhydrous K2CO3 (2.0 g, was added. The mixture was reacted at
room temperature for 1 h, the reaction mixture was concentrated
in vacuum after filtration and affording (R)-pNSO (1.6 g, 89%
yield).
Thermostability of the immobilized enzyme. In 1 mL KPB
buffer (pH 7.0, 100 mM), 0.2 g immobilized enzyme was
incubated at different temperatures (30 °C, 40 °C and 50 °C). The
residual activities were measured at different time intervals and
the inactivation curves were then made.
Operational stability of the immobilized enzyme. In 1 mL
KPB buffer (pH 7.0, 100 mM), 0.2 g immobilized enzyme was
shaken in a constant temperature mixer (1000 rpm. 30 °C). The
residual activities were measured under different time intervals.
(R)-pNSO (1.6 g, 9.8 mmol) was dissolved in absolute
ethanol (20 mL), and then 6 mL isopropylamine (4 g) were added
drop-wise. The reaction was reacted and refluxed under 50 °C for
24 h. When the reaction was completed, the ethanol and the
remaining isopropylamine were evaporated to obtain a crude
product. The final crude product was recrystallized in n-
hexane/ethyl acetate to afford (R)-Nifenalol (61.3% yield, 99.9%
ee).
Immobilized enzyme for batch reaction. In 10 mL KPB buffer
(pH 7.0, 100 mM), substrate rac-pNSO was dissolved with 10%
DMSO (v/v) as a final concentration of 10 mM, and 1 g
immobilized enzyme was used. The reaction was performed in a
shaker (200 rpm, 30 °C) until the substrate was converted
completely. Then the immobilized enzyme was recycled for the
next batch reaction. After each batch, the ee value of product and
the residual activity of immobilized enzyme were measured.
(R)-Nifenalol 13C NMR (101 MHz, CDCl3) δ 150.59 (s),
147.25 (s), 126.50 (s), 123.60 (s), 71.02 (s), 54.27 (s), 48.75 (s),
23.05 (d, J = 16.9 Hz). 1H NMR (400 MHz, CDCl3) δ 8.30 – 8.12
(m, 2H), 7.55 (dd, J = 6.9, 1.9 Hz, 2H), 4.72 (dd, J = 8.8, 3.8 Hz,
1H), 3.01 (ddd, J = 12.3, 3.8, 1.4 Hz, 1H), 2.83 (dt, J = 12.6, 6.3
Hz, 1H), 2.57 (dd, J = 12.2, 8.8 Hz, 1H), 1.67 (s, 2H), 1.09 (t, J =
4.4 Synthesis of (R)-pNPG from rac-pNSO in a stirred
reactor
30
5.7 Hz, 6H). [α]D20= -11.3 (c 1.0, EtOH) lit 31. [α]D = -11.0 (c
1.0, EtOH). HRMS: [M]+, found 223.1057. C11H16 N2O3 requires
223.1088.
In 36 mL KPB buffer (pH 7.0, 100 mM), substrate rac-
pNSO was dissolved with 10% DMSO (v/v) as a final
concentration of 100 mM, and 4 g immobilized enzyme was
added. The reaction was performed in a 100-mL reactor (400
r/min, 30 °C) until the substrate was converted completely, then
the immobilized enzyme was recycled for the next batch reaction.
Acknowledgments
The work was financially supported by the National Natural
Science Foundation of China (Nos. 21536004 and 21871085) and
the Fundamental Research Funds for the Central Universities
(No. 22221818014).
4.5 Resin screening for the product adsorption
30 mg macroporous resin (NKA-11, NKA-9, AS-17) and
500 ꢀL product solution (100 mM) was added in 2 mL EP tube,
then the adsorption was performed in a shaker (500 rpm, 30 °C)
for 5 h. When the adsorption reached saturation, the largest
adsorption capacity of resin was calculated. In a packed bed
column (H: 60 mm; D: 15mm), 2 g macroporous resins was
washed and equilibriumed with KP buffer. The product solution
(5 mM) was continuously fed into the reactor from top to bottom
by peristaltic pump with different space velocities (0.1 min-1, 0.2
min-1, 0.4 min-1 and 0.5 min-1), and the eluent solution was
detected for calculating the adsorption efficiency.
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The substrate solution (5 mM) was continuously fed into the
PBR (H: 60 mm, D: 15 mm) from top to bottom by peristaltic
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4.7 Preparation of (R)-Nifenalol from (R)-pNPG in a gram-
scale
In a 50-mL reaction flask, enantiopure (R)-pNPG (2.0 g, 10.9
mmol) was slowly dropped by 33% hydrobromide acetic acid
solution on ice-bath. After the diols was completely dissolved,
the system was reacted at 50 °C for 1 h. After cooling to room
temperature, the reaction mixture was poured into ice water, the
reaction pH was adjusted to neutral condition with saturated

8
Tetrahedron
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