Efficient chemoenzymatic synthesis of (S)-α-amino-4-fluorobenzeneacetic acid using immobilized penicillin amidase

Article abstract of DOI:10.1016/j.bioorg.2018.06.020

An efficient chemoenzymatic route was developed for synthesis of (S)-α-amino-4-fluorobenzeneacetic acid, a valuable chiral intermediate of Aprepitant, using immobilized penicillin amidase catalyzed kinetic resolution of racemic N-phenylacetyl-4-fluorophen

Full text of DOI:10.1016/j.bioorg.2018.06.020

Bioorganic Chemistry 80 (2018) 174–179
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Efficient chemoenzymatic synthesis of (S)-α-amino-4-fluorobenzeneacetic
acid using immobilized penicillin amidase
⁎
Chao-Ping Lin, Xiao-Ling Tang, Ren-Chao Zheng , Yu-Guo Zheng
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People’s
Republic of China
Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of
China
A R T I C L E I N F O
A B S T R A C T
Keywords:
An efficient chemoenzymatic route was developed for synthesis of (S)-α-amino-4-fluorobenzeneacetic acid, a
(
S)-α-amino-4-fluorobenzeneacetic acid
valuable chiral intermediate of Aprepitant, using immobilized penicillin amidase catalyzed kinetic resolution of
Immobilized penicillin amidase
Kinetic resolution
racemic N-phenylacetyl-4-fluorophenylglycine. The optimum temperature, pH and agitation rate of the reaction
−1
were determined to be 40 °C, 9.5 and 300 rpm, respectively. Kinetic resolution of 80 g L
fluorophenylglycine by immobilized amidase 20 g L
N-phenylacetyl-4-
Chemoenzymatic approach
−1
resulted in 49.9% conversion and > 99.9% e.e. within
3
h. The unreacted N-phenylacetyl-4-fluorophenylglycine can be easily racemized and then recycled as substrate.
The production of (S)-α-amino-4-fluorobenzeneacetic acid was further amplified in 1 L reaction system, af-
fording excellent conversion (49.9%) and enantioselectivity (99.9%). This chemoenzymatic approach was de-
monstrated to be promising for industrial production of (S)-α-amino-4-fluorobenzeneacetic acid.
1. Introduction
procedure, but also avoided using environmental unfriendly reagents.
Asymmetric synthesis and crystallisation-induced resolution are two
Aprepitant is a potent and orally active antagonist of human neu-
main types of routes reported for synthesis of 4 [11,16,17]. However,
those traditional chemical processes exhibited a lengthy procedure and
required the used of toxic organic reagents. By comparison, the enzy-
matic process has become an appealing approach for producing 4, be-
cause of its shorter process, greener solvent, and environmental
friendliness [18–23]. The transaminase mediated synthesis of 4 using
prochiral keto-acid as substrate has been reported [24] (Scheme 7a).
Although with excellent enantioselectivity (98% e.e.), substrate loading
of the enzymatic reaction was low (2.3 g L ) and reaction time was
long (71 h). In addition, the reversible enzymatic process requires
pyridoxal 5-phosphate as co-factor and aspartate as amino donor, re-
sulting in increased cost and incomplete conversion. Biocatalytic de-
racemization in the preparation of 4 from 1 using whole cells (Nocardia
Corallina CGMCC 4.1037) containing R-amino acid oxidase and S-
aminotransferase has been reported [25] (Scheme 7b). The low sub-
strate concentration (10 mM) and relatively inferior catalytic efficiency
(63% yield) restrict its industrial applications. Therefore, developing a
robust biocatalysts and an efficient synthesis strategy for producing 4 is
urgent. A penicillin amidase which exhibited a high hydrolytic activity
and excellent enantioselectivity toward N-phenylacetyl-4-fluor-
ophenylglycine, was employed as biocatalyst for enzymatic synthesis of
rokinin-1 (NK-1) receptor in the treatment of chemotherapy-induced
emesis, and also has good effect to release severe major depression,
pain and migraine [1–3]. The compound 6 is a critical precursor of
Aprepitant (Fig. 1), and its synthesis has aroused great interest. Several
synthetic routes of 6 have been reported [4–8] (Fig. 2), and most of
them introduced the chiral building block via direct addition reaction,
or by asymmetric synthetic methodologies. One of the most econom-
ically attractive route for synthesis of 6 starts from (R)-α-methylben-
zylamine [1] (Scheme 1). However, this method involved a reduction
−1
4
step where hazardous reagents like DIBALH and NaBH are used. Be-
sides, the poor optical purity of intermediate compound oxazinone and
the redundant procedure restricted its industrial applications. Other
potential synthetic routes using N-benzyl ethanolamine, p-fluor-
obenzaldehyde, 3, 5-bis (trifluoromethyl)-1-vinylben or 4-fluor-
ophenylacetic acid as raw material also have been reported [7,9–15]
Schemes 2–5). However, the dangerous reagents and precious metal
catalyst used in the synthesis of key chiral block resulted in security
risks and cost pressures. Comparatively, an additional approach
(
(
Scheme 6) based on the improvement of Scheme 5 with (S)-α-amino-4-
fluorobenzeneacetic acid 4 as raw material not only reduced the
⁎
Corresponding author at: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou
3
10014, People’s Republic of China.
E-mail address: zhengrc@zjut.edu.cn (R.-C. Zheng).
https://doi.org/10.1016/j.bioorg.2018.06.020
Received 26 April 2018; Received in revised form 11 June 2018; Accepted 11 June 2018
Available online 15 June 2018
0045-2068/ © 2018 Published by Elsevier Inc.

C.-P. Lin et al.
Bioorganic Chemistry 80 (2018) 174–179
Fig. 1. Structures of Aprepitant and its chiral precursor 6.
Fig. 2. Synthetic routes of 6. The synthetic routes (Schemes 1–5) have been reported.
(
immobilized amidase) was prepared by covalent immobilization of the
recombinant penicillin amidase from Bacillus megaterium onto epoxy
resin. After optimization of the reaction parameters, the chemoenzy-
matic reaction was carried out with high substrate concentration, high
conversion and excellent enantioselectivity. This highly efficient che-
moenzymatic route appears to be industrial promising in the production
of (S)-α-amino-4-fluorobenzeneacetic acid.
2. Materials and methods
Scheme 7. Enzymatic synthesis of 4.
2.1. General
4
. However, free enzyme has some drawbacks such as poor stability,
Recombinant penicillin amidase was constructed by our laboratory
difficulty in separation and recycling. Fortunately, these problems
could be overcome by immobilized enzyme.
In this work, we aim to develop a practical chemoenzymatic ap-
proach for efficient synthesis of 4 via kinetic resolution combined with
flexible chemical racemization (Scheme 8). The biocatalyst
[26]. 4-Fluorophenylglycine was purchased from Shanghai Rongli
Chemical Co. Ltd. (Shanghai, China). Phenylacetyl chloride was pur-
chased from Shanghai Mayao Chemicals Co. Ltd. (Shanghai, China).
Epoxy resin (LX-1000EP) was purchased from Xian Lanxiao Technology
Co. Ltd. (Xian, China). Analytical reagent grade solvents were pur-
chased from TEDIA, USA. All other reagents were obtained from
175

C.-P. Lin et al.
Bioorganic Chemistry 80 (2018) 174–179
Scheme 8. Immobilized amidase mediated chemoenzymatic synthesis of 4.
commercial sources and used without further purification.
2.5. Optimization of substrate concentration
The effects of substrate concentration on enzymatic reaction were
evaluated at different concentrations of 2 (40–100 g L ). The enzy-
matic reactions were performed at 40 °C and pH 9.5 with 0.1 g of im-
mobilized amidase in 10 mL reaction mixture.
High-pressure liquid chromatography (HPLC) analysis was con-
1
13
ducted on Model LC-20AT HPLC (Shimadu, Japan). H and C NMR
were recorded on a Bruker AVANCE III (Bruker, Switzerland). High-
resolution mass spectra (MS) were analyzed on an Agilent 6210 TOF
LC/MS (Agilent, USA). Optical rotator dispersion was carried out by
Rudolph Autopol IV polarimeter (Rudolph, USA). Concentrations of
substrate and product were monitored on a C18 column (Welch
Materials, Inc., Shanghai, China) by HPLC.
−1
2.6. Optimization of agitation rate
The effects of agitation rate on the enzymatic reaction were also
investigated. The reaction was performed with different agitation rate
2.2. Preparation of 2
(
100–500 rpm) at 40 °C and pH 9.5 in 20 mL reaction mixture con-
Rac-1 (0.4 mol, 67.61 g) was dissolved in 4.0 M sodium hydroxide
500 mL) at ambient temperature with stirring for 20 min. Phenylacetyl
−1
−1
taining 80 g L
substrate and 0.3 g L
immobilized amidase. The
(
sample was centrifuged at 12,000g for 1 min and diluted with ultrapure
water (1:50) for HPLC analysis.
chloride was added dropwise while the mixture was vigorously stirred
at 0 °C. After 10 h, the reaction mixture was extracted using di-
chloromethane to remove small amount of phenylacetic acid.
Subsequently, the aqueous layer was acidified to pH 2.0 with hydro-
chloric acid (6.0 M). The precipitate was filtered, washed with ultra-
pure water three times. The filtered precipitate dried at 70 °C for 12 h to
afford the product 2. Yield: 94.5%, 108.54 g white powder. 1H NMR
2.7. Optimization of immobilized amidase loading
The influence of immobilized amidase dose (5–30 g L−1) on the
enzymatic reaction was examined. The reaction was performed at 40 °C
with loading 80 g L substrate concentration for 3 h. The supernatant
was diluted with ultrapure water (1:50) and analyzed by HPLC.
−
1
(
4
(
500 MHz, D2O) δ 7.30 (dd, J = 7.2, 4.5 Hz, 2H), 7.10 – 7.00 (m, 2H),
1
3
.28 (s, 1H); C NMR (126 MHz, D2O) δ 181.00 (s), 162.87 (s), 160.94
s), 138.15 (s), 128.61 (d, J = 8.4 Hz), 115.47 (s), 115.30 (s), 59.86 (s);
+
MS (ESI): m/z = 285.7 [M + H]
.
2
.8. Kinetic resolution of 2 in 1L reaction system
2.3. Activity assay of immobilized amidase
The time course for catalyzed hydrolysis of 2 at the optimum re-
action condition was evaluated. The reaction was carried out in a 1 L
system with reaction mixture containing 80 g 2 dissolved in water (pH
was adjusted to 9.5 with 5% anmmonia solution) and 20 g immobilized
amidase at 40 °C. Samples were taken periodically and centrifuged,
then assayed by HPLC.
The immobilized amidase activity was assayed in a 10 mL reaction
mixture containing 50 mM substrate 2 and 0.1 g immobilized amidase
the pH was adjusted to 9.5 with 5% anmmonia solution). The reaction
was performed at 40 °C for 3 min and then terminated by centrifuging at
2,000g for 1 min. The concentration of 4 was determined by HPLC.
(
1
One unit (U) of immobilized amidase activity was defined as the
amount of immobilized amidase that produced 1.0 μmol 4 per minute
under the described conditions.
2.9. Racemization of 3
The unreacted (R)-enantiomer 3 (28.71 g, 0.1 mol) and phenylacetic
2
.4. Optimization of temperature and pH
acid (16.34 g, 0.12 mol) were added in three-necked flask. The reaction
mixture was heated in an oil bath at 160 °C for 20 min, and added into
100 mL 5% anmmonia solution before cooling to room temperature.
The mixture was extracted with dichloromethane to remove phenyla-
cetic acid and the residue 2 was reused as substrate. Then, the mixture
solution was used for optical rotation determination by polarmeter and
concentration analysis by HPLC.
The effect of temperature on immobilized amidase activity was in-
vestigated at different temperatures ranging from 30 to 50 °C under the
assay conditions mentioned above, and the effect of pH on immobilized
amidase activity was examined at 40 °C in buffers with different pHs
(
7.5–11.5), respectively.
176

C.-P. Lin et al.
Bioorganic Chemistry 80 (2018) 174–179
2
.10. Analytical method
100
1
00
90
The concentrations of 2, 4 and 5 were determined by HPLC
8
0
(
(
Shimadzu
Co.,
Kyoto,
Japan)
with
a
C18
column
8
7
0
0
5 μm × 250 mm × 4.6 mm, Welch Materials, Inc., Shanghai, China)
using acetonitrile-0.1% perchloric acid (50:50, v/v) as the mobile phase
60
40
20
0
−
1
at a flow rate of 0.7 mL min . The separated peaks were monitored
using a UV detector (SPD-10A, Shimadzu Co., Japan) at 220 nm. The
retention times of 2, 4 and 5 were 8.20 min, 3.74 min and 6.56 min,
respectively.
The ee value of 4 was determined on a chiral column (Chirobiotic™
R 250 × 4.6 mm, particle size 5 μm, Sigma, USA) using acetonitrile-
0
60
5
4
3
2
0
0
0
0
Relative activity
ee
p
.5% acetic acid (80:20, v/v) as the mobile phase at a flow rate of
−1
1
.0 mL min . The separated peaks were monitored using a UV detector
25
30
35
40
45
50
55
at 220 nm. The retention times of (S)-4 and (R)-4 were 13.02 min and
o
C
Temperature
(
)
14.84 min, respectively.
Fig. 3. Effect of temperature on the activity of immobilized amidase. The im-
mobilized amidase activities were assayed at different temperatures (30–50 °C)
in a 10 mL reaction mixture (pH 9.5) containing 50 mM substrate 2, 0.1 g im-
mobilized amidase.
2
.11. Synthesis of (S)-α-amino-4-fluorobenzeneacetic acid 4
Rac-2 (80 g L−1) was dissolved in water and the pH was adjusted to
9
.5 with 5% anmmonia solution. Subsequently, the enzymatic reaction
−1
in the temperature range from 25 to 65 °C. In the light of this, 40 °C was
chosen as the optimal temperature in the subsequent reactions.
To evaluate the effect of pH on the immobilized amidase-catalyzed
hydrolysis, we investigated different pH and found that the activity of
immobilized amidase gradually increased with the increase of pH from
was carried out by adding 20 g L immobilized amidase with 300 rpm
at 40 °C in 100 mL reaction system. When the enzymatic reaction was
finished after 3 h, the mixture was filtered to separate immobilized
amidase. The supernatant was acidified to pH 4.0 with concentrated
hydrochloric acid and then extracted with dichloromethane
7.5 to 11.5 and the maximum activity was achieved at pH 9.5 (Fig. 4).
(
3 × 100 mL) to remove by-product 5. The aqueous layer was further
However, the immobilized amidase activity decreased ramarkably with
further increase of pH from 9.5 to 11.5. The enantioselectivity was not
affected noticeably by pH in the enzymatic reaction. Generally, the
amidase-catalyzed hydrolysis reaction were carried out in buffers or by
adding extra alkaline solution to ensure the stable activity of biocatalyst
[28,29]. Interestingly, the immobilized amidase mediated hydrolysis
for the synthesis of 4 could work efficiently without pH regulation by
additional alkali.
acidified with concentrated hydrochloric acid to pH 2.0 at 0 °C with
stirring. Then the unreacted 3 in the solution was slowly precipitated.
The aqueous solution was collected and the pH was adjusted to 7.0. (S)-
4
was obtained via the evaporation of water, and then redissolved in
hot methanol. The product (S)-α-amino-4-fluorobenzeneacetic acid was
2
5
−1
finally crystallized from methanol. [α] = +85.7° (c = 20 mg mL
,
D
1
methanol); H NMR (500 MHz, Acetone) δ 7.49 (dd, J = 8.5, 5.4 Hz,
2
H), 7.34 (d, J = 7.3 Hz, 2H), 7.29 (t, J = 7.4 Hz, 2H), 7.22 (t,
13
J = 7.2 Hz, 1H), 7.13 (t, J = 8.8 Hz, 2H), 5.57 (s, 1H), 3.65 (s, 2H);
C
NMR (126 MHz, Acetone) δ 181.62 (s), 180.71 (d, J = 9.3 Hz), 173.97
3
.3. The effect of substrate concentration on activity of immobilized
(
(
s), 172.03 (s), 146.50 (s), 144.17 (t, J = 3.5 Hz), 140.10 (s), 139.73
s), 138.81 (s), 137.07 (s), 125.84 (d, J = 21.8 Hz), 66.26 (d,
amidase
The effects of substrate concentration (40–100 g L−1) on immobilized
J = 11.6 Hz), 52.75 (d, J = 5.5 Hz); MS (ESI): m/z = 167.9 [M + H]+.
amidase-catalyzed hydrolysis were investigated. As shown in Fig. 5, the
activity of immobilized amidase gradually increased with the increase of
3
. Results and discussion
−1
the substrate concentration (40–60 g L ) and then maintained high value
−1
3.1. Synthesis of substrate 2
in the range of 60–80 g L . Mild inhibition was observed when the
−1
substrate concentration beyond 60 g L . However, the activity sharply
Racemic 2 was prepared in one simple step by mixing 4-fluor-
ophenylglycine 1 with phenylacetyl chloride under alkaline condition.
To investigate the substrate scope of the immobilized amidase, we
synthesized acetyl, benzoyl and N-phenylalanyl derivatives of the 4-
fluorophenylglycine. Interestingly, only N-phenylacetyl-4-fluor-
ophenylglycine 2 was able to be recognized as a substrate of the im-
mobilized amidase. Meanwhile, only (S)-2 was hydrolyzed by im-
mobilized amidase, indicating strict stereoselectivity of the enzymatic
process.
100
1
00
8
6
4
0
0
0
8
6
4
2
0
0
0
0
0
Relative activity
3.2. Effects of temperature and pH on activity of immobilized amidase
ee
p
20
0
The effect of temperature on the kinetic resolution of 2 was studied
at a temperature range from 25 to 65 °C. The results of the activity and
enantioselectivity at different temperatures were shown in Fig. 3. The
immobilized amidase activity increased with the increase of reaction
temperature and the maximum activity was observed at 40 °C. How-
ever, with further increase of temperature, the activity decreased sig-
nificantly, which might ascribe to the partial inactivation of the im-
mobilized amidase by conformation change under high temperature
7
8
9
10
11
12
pH
Fig. 4. Effect of pH on the activity of immobilized amidase. The reactions were
carried out at 40 °C and the immobilized amidase activities were assayed with
different pHs (7.5–11.5) in a 10 mL reaction mixture containing 50 mM sub-
strate 2, 0.1 g immobilized amidase.
[
27]. Interestingly, temperature has less effect on the enantioselectivity
177

C.-P. Lin et al.
Bioorganic Chemistry 80 (2018) 174–179
1
9
7
6
05
0
1
8
6
4
00
5
4
3
0
0
0
1
000
0
8
6
4
2
00
00
00
00
0
5
0
0
0
45
Conversion
Immobilized amidase activity
20
3
1
0
5
20
0
ee
p
ee
p
1
0
4
0
50
60
70
80
90
100
5
10
15
20
25
30
-1
Concentration(g L )
-1
Immobilized amidase loading (g L )
Fig. 5. Effect of substrate concentration on the activity of immobilized amidase.
The reactions were performed at 40 °C in reaction mixtures (pH 9.5) consisted
of 0.1 g immobilized amidase with and various substrate concentration
Fig. 7. Effect of immobilized amidase loading on the kinetic resolution of 2.
The reactions were performed at different immobilized amidase loading (5–30 g
−1
−1
L
) with 300 rpm agitation rate in reaction mixture containing 80g L
sub-
−
1
(40–100 g L ).
strate.
decreased with further increase of substrate concentration (80–100 g L−1).
The increase of substrate concentration can accelerate the reaction rate
from the perspective of chemical equilibrium. However, higher substrate
concentration could cause inhibition of enzyme activity. Considering the
practical industrial application, 80 g L was chosen as the optimal sub-
strate concentration in further study.
immobilized amidase (5–30 g L−1) on the enzymatic reaction and ob-
served that the conversion steadily increased with the increase of im-
−1
mobilized amidase loading from 5 to 20 g L and leveled out at values
for further increasing the amount of immobilized amidase (Fig. 7).
−1
−1
Thus, 20 g L
was chosen as the optimal dosage of immobilized ami-
dase in further study.
3.4. Effect of agitation rate
3.6. Time course of enzymatic kinetic resolution process
It was observed that the reaction mixture gradually became viscous
The enzymatic reaction was scaled up to further evaluate the fea-
sibility of the route. The reaction was performed with 80 g substrate
loading in a 1 L thermostated stirred tank reactor under the optimized
reaction parameters. As shown in Fig. 8, the desired 4 was obtained
with high conversion (49.9%) and excellent enantioselectivity (99.9%
e.e.) after 3 h.
because of precipitation of compound 4 during the reaction course at a
−
1
substrate loading of 80 g L . Thus, the effect of the agitation rate on
the enzymatic reaction was investigated. As shown in Fig. 6, it was
found that the conversion was significantly improved with the increase
of the agitation rate (100–300 rpm), and then was basically flat with the
agitation rate increased (300–500 rpm), indicating that it was sufficient
to afford satisfied mass transfer with 300 rpm in this reaction. Thus,
3.7. Recycling of 3 by racemization
3
00 rpm was chosen as the optimal agitation rate in further study.
The recycling of remaining enantiomer 3 was explored by chemical
3
.5. Effect of immobilized amidase loading
racemization after the enzymatic reaction was accomplished.
Compound 3 was completely racemized to obtain racemic 2 with 95.3%
yield. The racemization makes the enzymatic resolution process over-
come the limitation of the theoretical yield of 50%.
The dosage of biocatalyst was an importance factor in terms of in-
dustrial application. We evaluated the effect of different doses of
To assess the feasibility of the chemoenzymatic strategy, enzymatic
1
8
6
4
00
0
5
4
3
2
1
0
0
0
0
0
1
00
5
4
3
2
1
0
0
0
0
0
80
60
40
0
0
Conversion
Conversion
ee
p
20
0
ee
p
2
0
0
1
00
200
300
400
500
0
30
60
90 120 150 180 210 240 270
Time (min)
Rotation rate (rpm)
Fig. 6. Effect of agitation rate on the kinetic resolution of 2 using the im-
mobilized amidase. The conversions of the reactions were investigated at 40 °C
with different agitation rates (100–500 rpm). The reaction mixture (pH 9.5) was
Fig. 8. Time course of kinetic resolution of 2 using immobilized amidase. The
reaction was performed at 40 °C with 300 rpm agitation rate in reaction mixture
−
1
−1
−1
composed of 80 g L substrate 2 and 0.3 g immobilized amidase.
(1 L, pH 9.5) consisted of 80 g L substrate and 20 g L immobilized amidase.
178

C.-P. Lin et al.
Bioorganic Chemistry 80 (2018) 174–179
resolution and the subsequent separation steps were carried out, af-
fording 4 in 46.2% isolated yield. The unreacted enantiomer of the
substrate was completely racemized to produce substrate 2, which was
again subjected to the enzymatic resolution, giving 4 in 21.5% yield.
Thus, the total yield of 67.7% was achieved after one recycle of (R)-2.
to the synthesis of aprepitant: a potent human NK-1 receptor antagonist,
Tetrahedron Lett. 48 (2007) 8001–8004.
[
7] P.J. Pye, K. Rossen, S.A. Weissman, A. Maliakal, R.A. Reamer, R. Ball, N.N. Tsou,
R.P. Volante, P.J. Reider, Crystallization-induced diastereoselection: asymmetric
synthesis of substance P inhibitors, Chem. Eur. J. 8 (2002) 1372–1376.
8] B.H. Hoff, E. Sundby, Preparation of pharmaceutical important fluorinated 1-ar-
ylethanols using isolated enzymes, Bioorg. Chem. 51 (2013) 31–47.
9] R.M. Satyanarayana, S. Eswaraiah, M. Venkatesh, M. Venkazesh, An improved
process for the preparation of aprepitant. USA. Patent. US 14379948. 16.02.04.
[10] E. Vedejs, R.W. Chapman, S. Lin, M. Müller, D.R. Powell, Crystallization-induced
asymmetric transformation vs quasi-racemate formation in tetravalent boron
complexes, J. Am. Chem. Soc. 122 (2000) 3047–3052.
[
[
−
1
−1
The space time yield of the bioprocess reached 10.6 g L
h
, which
was 963.6 and 240.9 times higher than that reported by Cameron et al.
−
2
−1 −1
−2
−1 −1
(
1.1 × 10
g L
h
) [24] and Xia et al. (4.4 × 10
g L
h
)
[
25], respectively. Thus, this chemoenzymatic approach using im-
mobilized amidase as the biocatalyst exhibited higher efficiency and
feasibility compared with other enzymatic method ever reported.
[
11] N. Kolla, C.R. Elati, M. Arunagiri, S. Gangula, P.J. Vnjaneyulu, Y. Anjaneyulu,
A. Bhattacharya, A.S. Venkatraman, An alternative approach to achieve en-
antiopure (3S)-4-benzyl-3-(4-fluorophenyl)morpholin-2-one: a key intermediate of
1
aprepitant, an NK receptor antagonist, Org. Process Res. Dev. 11 (2007) 455–457.
4
. Conclusions
[
12] J. Ji, Q. Zhang, F. Du, Y. Jin, T. Zhang, N. Guo, X. Yan, Y. Yang, B. Li, Preparation
method of 5-[[2(R)-[1(R)-[3,5-bis(trifluoromethyl) phenyl]ethoxy]-3(S)-4-fluor-
ophenyl-4-morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazole-3-one. USA. Patent.
US 8940890. 15.01.27.
In summary, we have developed a simple, high efficient che-
moenzymatic route for the synthesis of (S)-α-amino-4-fluor-
obenzeneacetic acid using immobilized amidase as biocatalyst. The
enzymatic reaction was carried out using water as reaction medium
without pH regulation during the reaction process. The reaction para-
meters such as pH, temperature effect, catalyst loading, and agitation
rate were optimized. The hydrolysis reaction was demonstrated to be a
robust catalytic process for preparation of (S)-α-amino-4-fluor-
obenzeneacetic acid with a high efficiency (49.9% conversion) and
excellent enantioselectivity (99.9% e.e.) on a scale of 80 g L substrate
concentration in 1 L reaction system. Moreover, the unreacted en-
antiomer of substrate could be easily racemized and reused. The overall
process has an excellent industrial potential, and further study on re-
cycle of immobilized amidase and pilot scale are in progress.
[
[
[
13] B.V.S. Reddy, G. Narasimhulu, P.S. Lakshumma, Y.V. Reddy, J.S. Yadav,
Phosphomolybdic acid: a highly efficient solid acid catalyst for the synthesis of
trans-4,5-disubstituted cyclopentenones, Tetrahedron Lett. 43 (2012) 1776–1779.
14] C.P. Dorn, J.J. Hale, M. Maccoss, S.G. Mills, S.K. Shah, T. Ladduwahetty,
Morpholine and thiomorpholine tachykinin receptor antagonists. USA. Patent. US
6048859. 01.04.11.
15] M. Van der Mey, C.G. Janssen, F.E. Janssens, M. Jurzak, X. Langlois, F.M. Sommen,
B. Verreet, A.D. Windhorst, J.E. Leysen, J.D. Herscheid, Synthesis and biodis-
11
1
tribution of [ C]R116301, a promising PET ligand for central NK receptors,
Bioorg. Med. Chem. 13 (2005) 1579–1586.
−
1
[16] D.A. Evans, T.C. Britton, J.A. Ellman, R.L. Dorow, The asymmetric synthesis of α-
amino acids. Electrophilic azidation of chiral imide enolates, a practical approach to
the synthesis of (R)- and (S)-α-azido carboxylic acids, J. Am. Chem. Soc. 21 (1990)
1
2601–12605.
[17] K.M. Brands, A.J. Davies, Crystallization-induced diastereomer transformations,
Chem. Rev. 106 (2006) 2711–2733.
18] Y.P. Xue, C.H. Cao, Y.G. Zheng, Enzymatic asymmetric synthesis of chiral amino
[
acids, Chem. Soc. Rev. 46 (2018) 1516–1561.
Acknowledgement
[19] X. Chen, Z.Q. Liu, C.P. Lin, Y.G. Zheng, Chemoenzymatic synthesis of (S)-duloxetine
using carbonyl reductase from Rhodosporidium toruloides, Bioorg. Chem. 65 (2016)
8
2–89.
This work was financially supported by the National High
Technology Research and Development Program of China (No.
012AA022201).
[
[
[
20] Y.P. Xue, T. Jiang, X. Liu, Y.G. Zheng, Efficient production of S-(+)-2-chlor-
ophenylglycine by immobilized penicillin G acylase in a recirculating packed bed
reactor, Biochem. Eng. J. 74 (2013) 88–94.
21] K. Buchholz, A breakthrough in enzyme technology to fight penicillin re-
sistance—industrial application of penicillin amidase, Appl. Microbiol. Biotechnol.
100 (2016) 3825–3839.
22] P.C. Pereira, I.W.C.E. Arends, R.A. Sheldon, A green and expedient synthesis of
enantiopure diketopiperazines via enzymatic resolution of unnatural amino acids,
Tetrahedron Lett. 55 (2014) 4991–4993.
23] R.A. Sheldon, J.M. Woodley, Role of biocatalysis in sustainable chemistry, Chem.
Rev. 118 (2017) 801–838.
24] M. Cameron, D. Cohen, I.F. Cottrell, D.J. Kennedy, C. Roberge, M. Chartrain, The
highly stereospecific enzyme catalysed transamination of 4-fluorophenylglyoxylic
acid to 4-(S)-fluorophenylglycine, J. Mol. Cata. B: Enzym. 14 (2001) 1–5.
25] S.W. Xia, W.J. Xiong, Y.C. Wei, H.E. Cong-Lin, X.U. Hong-Mei, Biocatalytic dera-
cemization of (R S)-4-fluorophenylglycine to (S)-4-fluorophenylglycine by whole
cells of Nocardia Corallina CGMCC 4.1037, J. Mol. Catal. 29 (2015) 307–314.
26] Y.P. Xue, Y.G. Zheng, Z.Q. Liu, X. Liu, J.F. Huang, Y.C. Shen, Efficient synthesis of
non-natural l-2-aryl-amino acids by a chemoenzymatic route, ACS Catal. 4 (2014)
2
References
[
1] M.M. Zhao, J.M. Mcnamara, G.J. Ho, K.M. Emerson, Z.J. Song, D.M. Tschaen,
K.M. Brands, U.H. Dolling, E.J. Grabowski, P.J. Reider, Practical asymmetric
synthesis of aprepitant, a potent human NK-1 receptor antagonist, via a stereo-
selective Lewis acid-catalyzed trans acetalization reaction, J. Org. Chem. 67 (2002)
[
[
6
743–6747.
2] K.M.J. Brands, J.F. Payack, J.D. Rosen, T.D. Nelson, A. Candelario, M.A. Huffman,
M.M. Zhao, J. Li, B. Craig, Z.J. Song, Efficient synthesis of NK receptor antagonist
[
1
[
[
aprepitant using a crystallization-induced diastereoselective transformation, J. Am.
Chem. Soc. 125 (2003) 2129–2135.
3] Gregory A. Reichard, Carmine Stengone, Sunil Paliwal, Ingrid Mergelsberg,
Sapna Majmundar, Cheng Wang, Robert Tiberi, Andrew T. McPhail, John
J. Piwinski, NengYang Shih, Asymmetric synthesis of 4,4-disubstituted-2-imidazoli-
[
3
051–3058.
1
dinones: potent NK antagonists, Org. Lett. 5 (2003) 4249–4251.
[
27] R.S. Phillips, Temperature modulation of the stereochemistry of enzymatic cata-
[
[
[
4] B.H. Landis, P.B. Mullins, K.E. Mullins, P.T. Wang, Kinetic resolution of β-amino
esters by acylation using immobilized penicillin amidohydrolase, Org. Process Res.
Dev. 6 (2002) 539–546.
5] S. Servi, D. Tessaro, G. Pedrocchi-Fantoni, Chemo-enzymatic deracemization
methods for the preparation of enantiopure non-natural α-amino acids, Coord.
Chem. Rev. 252 (2008) 715–726.
lysis: prospects for exploitation, Trends. Biotechnol. 14 (1996) 13–16.
28] J. Wang, N. Liu, X. Cheng, L. Chen, An efficient continuous kinetic resolution of
racemic 2-aminobutanol over immobilized penicillin G acylase, Synth. Comm. 46
[
(2016) 956–962.
[
29] S. Liu, Q. Song, D. Wei, Y. Zhang, X. Wang, Preparation of optically pure tert-
leucine by penicillin G acylase-catalyzed resolution, Prep. Biochem. Biotechnol. 36
6] C.R. Elati, N. Kolla, S. Gangula, A. Naredla, P.J. Vankawala, M.L. Avinigiri,
S. Chalamala, V. Sundaram, V.T. Mathad, A. Bhattacharya, A convergent approach
(2006) 235–241.
179