The enzymatic resolution of 1-(4-chlorophenyl)ethylamine by Novozym 435 to prepare a novel triazolopyrimidine herbicide

Article abstract of DOI:10.1002/chir.23016

The kinetic resolution of (R,S)-1-(4-chlorophenyl)ethylamine was accomplished using a commercial lipase from Candida antarctica (Novozym 435). The performance of this lipase was investigated for the enantioselective amidation of (R,S)-1-(4-chlorophenyl)ethylamine, leaving the target product (S)-1-(4-chlorophenyl)ethylamine in its unreacted form. The effects of various types of solvents and an acyl donor, the molar ratio of the substrate to the acyl donor, and the reaction temperature were studied. The optimum reaction conditions were found to result in amidation with methyl 2-tetrahydrofuroate at 40°C in methyl tert-butyl ether, with a substrate/acyl donor molar ratio of 1:2.4. The conversion rate of (R,S)-1-(4-chlorophenyl)ethylamine was 52%, with an enantiomeric excess of 99% towards the unreacted substrate in a reaction time of 22?hours. Finally, using optically pure (S)-1-(4-chlorophenyl)ethylamine as the raw material, the chemical synthesis of (S)-N-(1-(4-chlorphenyl)ethyl)-2-(5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylthio)acetamide, a novel triazolopyrimidine herbicide, was achieved, and the total yield and purity were 83.5% and 95.3%, respectively.

Full text of DOI:10.1002/chir.23016

Received: 2 April 2018
DOI: 10.1002/chir.23016
Revised: 24 July 2018
Accepted: 6 August 2018
R E G U L A R A R T I C L E
The enzymatic resolution of 1‐(4‐chlorophenyl)ethylamine
by Novozym 435 to prepare a novel triazolopyrimidine
herbicide
Yinjun Zhang
| Feifei Cheng | Hongde Yan | Jianyong Zheng
| Zhao Wang
Key Laboratory of Bioorganic Synthesis of
Zhejiang Province, College of
Biotechnology and Bioengineering,
Zhejiang University of Technology,
Hangzhou, China
Abstract
The kinetic resolution of (R,S)‐1‐(4‐chlorophenyl)ethylamine was accom-
plished using a commercial lipase from Candida antarctica (Novozym 435).
The performance of this lipase was investigated for the enantioselective
amidation of (R,S)‐1‐(4‐chlorophenyl)ethylamine, leaving the target product
(S)‐1‐(4‐chlorophenyl)ethylamine in its unreacted form. The effects of various
types of solvents and an acyl donor, the molar ratio of the substrate to the
acyl donor, and the reaction temperature were studied. The optimum
reaction conditions were found to result in amidation with methyl 2‐
tetrahydrofuroate at 40°C in methyl tert‐butyl ether, with a substrate/acyl
donor molar ratio of 1:2.4. The conversion rate of (R,S)‐1‐(4‐chlorophenyl)
ethylamine was 52%, with an enantiomeric excess of 99% towards the
unreacted substrate in a reaction time of 22 hours. Finally, using optically
pure (S)‐1‐(4‐chlorophenyl)ethylamine as the raw material, the chemical syn-
thesis of (S)‐N‐(1‐(4‐chlorphenyl)ethyl)‐2‐(5,7‐dimethyl‐[1,2,4]triazolo[1,5‐a]
pyrimidin‐2‐ylthio)acetamide, a novel triazolopyrimidine herbicide, was
achieved, and the total yield and purity were 83.5% and 95.3%, respectively.
Correspondence
Zhao Wang, Key Laboratory of Bioorganic
Synthesis of Zhejiang Province, College of
Biotechnology and Bioengineering,
Zhejiang University of Technology,
Address No. 18, Chaowang Road,
Hangzhou, China.
Email: hzwangzhao@163.com
Funding information
Scientific and Technological Innovation
Activity Plan for Students in Zhejiang
Province, Grant/Award Number:
2017R403090; Zhejiang Provincial Natural
Science Foundation, Grant/Award Num-
ber: LY18B020021
KEYWORDS
1‐(4‐chlorophenyl)ethylamine, amidation, lipase, Novozym 435, resolution
1 | INTRODUCTION
acetolactate synthase inhibitor, which is a new type of
aromatic amide compound containing triazolopyrimidine
Chiral 1‐phenylethylamine and its derivatives are impor-
tant intermediates for the synthesis of chiral pesticides
and pharmaceuticals.1,2 Also, these compounds play crit-
ical roles during the process of chiral synthesis, such as in
the form of chiral auxiliaries,3,4 chiral resolving agents,5
and base materials.6 Therefore, the synthesis of optically
active isomers of 1‐phenylethylamine compounds has
been a focus in recent years. (S)‐1‐(4‐chlorophenyl)
ethylamine ((S)‐1) is a key intermediate for the synthesis
of (S)‐N‐(1‐(4‐chlorphenyl)ethyl)‐2‐(5,7‐dimethyl‐[1,2,4]
triazolo[1,5‐a]pyrimidin‐2‐ylthio)‐acetamide ((S)‐4), an
structure developed by Zheng.7 According to the prelimi-
nary study, this compound has high herbicidal activity,
low residue, and a wide herbicidal spectrum, but is safe
to gramineae.8
Preparation of chiral 1‐phenylethylamine and its
derivatives by enzymatic resolution has been proven a
practicable universal method owing to its high catalytic
efficiency, excellent stereoselectivity, and environmental
friendliness.1,9-11 For example, lipases from Candida ant-
arctica and Pseudomonas cepacia were demonstrated to
possess high enantioselectivity and catalytic efficiency
Chirality. 2018;1–8.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

2
ZHANG ET AL.
for the enantiomeric kinetic resolution of 1‐phenylethyl-
amine.1,11 A new amidohydrolase from Arthrobacter
aurescens, AcR5b, was found for deacetylating several
N‐acetyl‐1‐phenylethylamine derivatives with (R)‐
stereopreference.12 Enzymatic enantioselective amidation
of p‐methoxy‐1‐phenylethylamine was achieved using
immobilized lipases from C. antarctica.13 In this study,
the lipase from C. antarctica (Novozym 435) was screened
for the stereoselective acylation of (R,S)‐1 to obtain (S)‐1
(Scheme 1A). Some factors affecting the resolution of
(R,S)‐1 were investigated, such as its organic solvent, acyl
donor, molar ratio, and reaction temperature. Then, high
optical purity of (S)‐1 was utilized for the further synthe-
sis of (S)‐4 (Scheme 1B).
2.3 | Synthesis of (S)‐4
Chloroacetyl chloride (0.012 mmol) was slowly added to a
solution of (S)‐1 (0.01 mol) in 20 mL of acetone at 5°C for
30 minutes (Scheme 1B). The crystal of (S)‐1‐chloro‐N‐[1‐
(4‐chlorophenyl) ethyl]acetamide ((S)‐3) was precipitated
while continuously stirring deionized water into the
mixture. The filtered precipitate was washed with 18%
hydrochloric acid to remove pyridine, and then washed
with water until the pH became neutral.
A total of 2.05 g of the 2‐mercapto‐5,7‐dimethyl‐1,2,4‐
triazolo [1,5‐a] pyrimidine dissolved in 30 mL of 0.43 M
sodium hydroxide reacted with 2.28 g (S)‐3 dissolved in
100 mL of 95% ethanol for 30 minutes at a temperature
of less than 25°C to prepare (S)‐4. The product was pre-
cipitated through the addition of deionized water, and
was washed successively with 3% hydrochloric acid, 5%
sodium bicarbonate, and deionized water until the wash-
ing liquid became neutral. Following this, the product
was dried at 50°C.
2 | MATERIALS AND METHODS
2.1 | Materials
Novozym 435 was purchased from the China Headquar-
ters of Novozymes (Beijing, China). (R,S)‐4 (purity-
99.8%) was provided by the pesticide institute of the
Zhejiang University of Technology (Hangzhou, China).
The standard sample of (R,S)‐1 was purchased from
Aladdin Chemistry Co., Ltd. (Shanghai, China). All other
chemicals used were of analytical grade.
2.4 | Analytical methods
The conversion rate and enantiomeric excesses of (R,S)‐1
(e.e.s) and (R,S)‐2 (e.e.p) were measured by HPLC with a
chiral CD‐Ph column (250 mm × 4.6 mm, 5 μm; Daicel,
Hyogo, Japan), the mobile phase was composed of
methanol/0.5 M sodium perchlorate solution at a ratio
of 90/10. The flow rate was 1 mL/minute; UV wavelength
detection was performed at 254 nm. The enantiomeric
excess was calculated according to the peak areas of (S)‐
enantiomer and (R)‐enantiomer. The enantioselectivity
(E) was calculated according to the following equation14:
2.2 | Enzymatic resolution of (R,S)‐1
In a typical experiment, the reaction mixtures were com-
posed of (R,S)‐1 (0.7 mmol), acyl donor (200 μL), organic
solvent (10 mL), and lipase (20 mg). The mixtures were
incubated on a shaker at 200 rpm and 30°C for 24 hours.
Reaction mixtures without lipase were also run to exclude
any possible spontaneous nonenzymatic reactions. At
appropriate intervals, the samples were withdrawn and
filtered, then analyzed by high‐performance liquid chro-
matography (HPLC).
½S1ꢀ − ½R1ꢀ
e:e:s ¼
e:e:p ¼
× 100%
× 100%
(1)
(2)
½S1ꢀ þ ½R1ꢀ
½R2ꢀ − ½S2ꢀ
½S2ꢀ þ ½R2ꢀ
SCHEME 1 A, The lipase‐catalyzed
resolution of (R,S)‐1‐(4‐chlorophenyl)
ethylamine ((R,S)‐1). B, The synthesis of
(S)‐N‐(1‐(4‐chlorphenyl)ethyl)‐2‐(5,7‐
dimethyl‐[1,2,4]triazolo[1,5‐a]pyrimidin‐2‐
ylthio)acetamide ((S)‐4).

ZHANG ET AL.
3
e:e:s
e:e:s þ e:e:p
TABLE 1 The screening of commercial lipases
e.e.s e.e.p Conversion
C ¼
E ¼
× 100%
(3)
(4)
Enzyme
Source
(%)
(%)
(%)
E
ln½ð1 − CÞð1 − e:e:sÞꢀ
ln½ð1 − CÞð1 þ e:e:sÞꢀ
Novozym
435
Candida
antarctica
68.6 57.3
54.5
7.4
Lipozyme Thermomyces
1.6
0.5
3.5
1.7
31.4
22.6
1.1
1.0
where C represents the conversion ratio of (R,S)‐1. e.e.s
represents the enantiomeric excess of the residual (R,S)‐
1. e.e.p represents the enantiomeric excess of the product
(R,S)‐2. [R1] and [S1] are the peak areas corresponding
to (R)‐1 and (S)‐1. [R2] and [S2] are the peak areas corre-
sponding to (R)‐2 and (S)‐2, respectively.
The optical purity (S)‐4 was determined by measuring
the optical rotation at 589 nm through a polarimeter
(NJ 07840, Rudolph, USA). The structural identification
of (S)‐4 was measured by ESI‐MS (Waters, USA) and
NMR (500 MHz). The yield was calculated at the practical
and theoretical weight of (S)‐4 subsequent to its drying.
Finally, the purity of (S)‐4 was detected by HPLC with
Hypersil ODS2 (250 mm × 4.6 mm, 5 μm; Thermo Fisher
Scientific, Germany). The mobile phase was composed of
ethanol/water at a ratio of 60/40.
TLIM
languginosa
Lipase P
Penicillium
camemberti
Lipase R
Rhizopus niveus
Aspergillus niger
2.3
2.2
6.4
6.7
26.4
24.6
1.2
1.2
Amano
lipase A
Amano
lipase
AK
Pseudomonas
fluorescens
1.6
4.7
25.2
1.1
Lipase VII Candida rugosa
1.4
4.2
26.2
1.1
Reaction were carried out at 30°C, 200 rpm for 24 hours, including 100 μL
(R,S)‐1 (0.7 mmol), 200 μL isopropenyl acetate, 10 mL MTBE, and 20 mg
lipase.
TABLE 2 The effects of organic solvents on the enantioselective
amidation of (R,S)‐1
Organic
solvents
Log e.e.s
(%)
e.e.p
(%)
Conversion
(%)
3 | RESULTS AND DISCUSSION
3.1 | Screening of the lipases
P
E
Tetrahydrofuran 0.46 18.3
25.8
9.3
41.5
56.4
53.9
18.6
2.0
1.3
7.8
1.8
Ethyl acetate
MTBE
0.68 12.0
1.29 68.9
Screening is the key step for selection of suitable
biocalysts.15 Fifty commercial lipases were selected for
selective amidation of (R,S)‐1. Seven lipases displayed
amidation activity and displayed a (R)‐stereopreference,
leaving the target product an (S)‐enantiomer. The results
are summarized in Table 1. Novozym 435 afforded high
conversion and e.e.s among the screened lipases, and the
enantioselectivity was calculated with E = 7.4 from a con-
version of 54.5%. Therefore, Novozym 435 was chosen for
further study.
58.9
26.3
Methylene
chloride
1.41
6.0
Isopropyl ether
Chloroform
Toluene
1.9
2.0
33.4
5.4
5.7
15.2
34.3
3.2
85.5
26.2
50.4
77.8
1.4
1.4
2.8
1.2
2.74 34.9
3.76 11.1
n‐Hexane
Reaction were carried out at 30°C, 200 rpm for 24 hours, including 100 μL
(R,S)‐1 (0.7 mmol), 200 μL isopropenyl acetate, 10 mL different organic sol-
vents, and 20 mg Novozym 435.
3.2 | The effects of organic solvents
in methyl tert‐butyl ether (MTBE), with the highest
E value being 7.8 and the conversion being 53.9%. Similar
results were obtain in the enantioselective acylation of
2‐phenylcycloalkanamines17 and 1‐(3′‐bromophenyl)‐
ethylamine,18 and MTBE seemed to be an excellent
choice as a solvent in the lipase‐catalyzed kinetic resolu-
tion of chiral amines. Finally, MTBE was selected as the
optimum solvent for the following tests.
It is well known that organic solvents can affect the solu-
bility of substrates and the activity and enantioselectivity
of enzymes16. Different organic solvents with log P values
of from .46 to 3.76 were used to investigate the effects
of solvents on the kinetic resolution of (R,S)‐1. The
results in Table 2 revealed that enzyme activity and
enantioselectivity were not correlated with the log P
values. Low conversions and low E values were observed
in methylene chloride and chloroform. The high conver-
sions of 77.8% and 85.5% were obtained in n‐hexane and
isopropyl ether, but the E values were both low. The
other tested solvents afforded moderate conversions
(41.5% to 56.4%), and the best performance was obtained
3.3 | The effects of the acyl donor
The use of diverse acyl donors with differing chemical
structures has been proven to have cause different results

4
ZHANG ET AL.
in enzymes' acylation activity. The effects of the appropri-
ate acyl donor can resemble very closely the “pocket”
structures of enzymes that can fully expose enzymes'
active centres.19,20 As demonstrated in Table 3, methyl
2‐tetrahydrofuroate had an obviously positive effect on
the activity and enatioselectivity of Novozym 435,
affording the highest e.e.s (96.8%) and enantioselectivity
(E = 44.6), with a conversion of 53.8%. Isopropenyl ace-
tate also displayed good results, with a conversion of
54% and an E value of 7.8, but the isopropenyl acetate
reaction had poor reproducibility. According to Gill's
observation, extensive (up to 60%) by‐product was formed
with certain batches of isopropenyl acetate.18 Miranda
et al also found that an imine derivative formed between
the free amine and propanone (derived from the
isopropenyl ester).21 Ethyl methoxyacetate, which is less
reactive than isopropenyl acetate but much more reactive
than simple esters, afforded a high conversion (52.9%)
and moderate enantioselectivity (E = 5.6). The results
were in agreement with the reported of Schmid et al.
They assumed that the methoxy residue in the acyl part
of the amide greatly enhances the reaction rate, com-
pared to the acetate or the butyric residue, which is
sterically almost equivalent.22 Another enol ester, vinyl
acetate, a widely used acyl donor for resolving chiral
alcohols,23 gave a low conversion rate (19.1%) and low E
value (2.1). The results were similar to those reported
for the lipase‐catalyzed acylation of (R,S)‐phenylethyl-
amine and1‐(3′‐bromophenyl)ethylamine,17,24 where
vinyl acetate also had low E values. This may be due
to the fact that the use of vinyl acetate resulted in nonse-
lective nonenzymatic acetylation, as well as the formation
of several side products.17 Dimethyl succinate and
diethyl succinate displayed low conversion and low
enantioselectivity. But in the report of Gröger et al, an
analog, diethyl malonates, served as a highly efficient acyl
donor in the resolution of 1‐phenylethyl amine and (R,S)‐
[1‐(4‐bromophenyl)]ethyl amine with E value >100.25
This may be due to the differences of the nature of
amines. Ethyl acetate and ethyl 3‐hydroxybutyrate also
gave low conversion and low enantioselectivity. Thus,
methyl 2‐tetrahydrofuroate was chosen as the acyl donor
to employ in further experiments.
3.4 | The effects of the substrate
concentration
Substrate concentration is a crucial factor that may
affect enzyme activity or even result in substrate inhibi-
tion and affect the enantioselectivity of the reaction.
Figure 1 displays the performance of Novozym 435 on
the resolution of (R,S)‐1 using different substrate con-
centrations. The results showed that high conversion
(>50%) and high e.e.s (>96%) were obtained when the
substrate concentration was below 70 mM, and signifi-
cant decreases of conversion and e.e.s were observed
when the substrate concentration was above 70 mM.
When the substrate concentration was 70 mM, the e.e.s
and e.e.p could reach 96.8% and 86.7%, with an E value
of 54.6. Similar results were reported in the kinetic res-
olution of (R,S)‐1‐phenylethylamine, where an increase
in substrate concentration also led to a less selective
reaction.26
TABLE 3 The effects of acyl donors on the enantioselective
amidation of (R,S)‐1
e.e.s
(%)
e.e.p
(%)
Conversion
(%)
Acyl Donor
E
Dimethyl succinate
Diethyl succinate
Isopropenyl acetate
11.6
4.5
41.1
10.6
58.1
71.6
22.0
29.8
54.0
53.8
2.7
1.3
7.8
68.2
96.8
Methyl 2‐
44.6
tetrahydrofuroate
Ethyl methoxyacetate
Ethyl acetate
58.6
17.0
5.2
52.2
30.2
14.7
52.9
36.0
26.1
5.6
2.2
1.4
Ethyl 3‐
hydroxybutyrate
FIGURE 1 The effects of substrate concentrations on the
enantioselective amidation of (R,S)‐1. The reactions were carried
out at 30°C, 200 rpm for 24 hours, and utilized different
concentrations of (R,S)‐1, 200 μL methyl 2‐tetrahydrofuroate, 10 mL
MTBE, and 20 mg Novozym 435
Vinyl acetate
7.7
32.6
19.1
2.1
Reaction were carried out at 30°C, 200 rpm for 24 hours, including 100 μL
(R,S)‐1 (0.7 mmol), 200 μL different acyl donor, 10 mL MTBE, and 20 mg
Novozym 435.

ZHANG ET AL.
5
ratios resulted in a decrease in enantioselectivity.24
Thus, considering the optical purity of the target prod-
uct, a molar ratio of 1:2.4 was selected for the following
experiments.
3.5 | The effects of the molar ratio of the
substrate to the acyl donor
As shown in Figure 2, the conversion rate increased
with an increase in the molar ratio of the substrate to
the acyl donor. The e.e.s also increased with an increase
in the molar ratio of the substrate to the acyl donor,
reaching a maximum value of 96.8% (ratio 1:2.4), but
decreased subsequently with further increases in the
molar ratio. The results were similar to the reports
concerning the enantioselective acylation of (R,S)‐
phenylethylamine by Novozym 435, where the molar
ratio of 1:4 gave the highest E value, and higher molar
3.6 | The effects of temperature
The effects of temperature on reaction conversion
rates and e.e.s are described in Figure 3. There was a con-
tinuous increase in the conversion rate from 20°C to
40°C, and then a slight decrease from 40°C to 45°C. The
e.e.s showed an obvious increase from 20°C (85%) to
30°C (96.8%), a smooth increase from 30°C (96.8%)
FIGURE 2 The effects of molar ratios of substrate to acyl donor
on the enantioselective amidation of (R,S)‐1. The reactions were
carried out at 30°C, 200 rpm for 24 hours, and employed 100 μL (R,
S)‐1 (0.7 mmol), different amounts of methyl 2‐tetrahydrofuroate,
10 mL MTBE, and 20 mg Novozym 435
FIGURE 4 The time course for the enantioselective amidation
of (R,S)‐1. The reactions were carried out at 40°C and 200 rpm
and entailed the use of 100 μL (R,S)‐1 (0.7 mmol), 200 μL methyl
2‐tetrahydrofuroate (1.7 mmol), 10 mL MTBE, and 20 mg
Novozym 435
FIGURE 3 The effects of temperature on the enantioselective
amidation of (R,S)‐1. The reactions were carried out at 200 rpm at
different temperatures for 24 hours; the experiment used 100 μL (R,
S)‐1 (0.7 mmol), 200 μL methyl 2‐tetrahydrofuroate (1.7 mmol),
10 mL MTBE, and 20 mg Novozym 435
FIGURE 5 The reuse cycles of immobilized Novozym 435 on the
preparation of (S)‐1. The reactions were carried out at 40°C at
200 rpm for 22 hours, and entailed 100 μL (R,S)‐1 (0.7 mmol),
200 μL methyl 2‐tetrahydrofuroate (1.7 mmol), 10 mL MTBE, and
20 mg Novozym 435

6
ZHANG ET AL.
to 40°C (98%), and then an obvious decrease from 40°C to
45°C (90%). The highest e.e.s was found at approximately
98% at 40°C, with a conversion rate of 57%. Thus, a reac-
tion temperature of 40°C was selected for the subsequent
experiments.
the e.e.s to rise as the reaction time increased to 22 hours.
At this reaction time, the final e.e.s reached about 99%
with a conversion rate of 52%.
3.8 | Reusability of immobilized lipase in
the preparation of (S)‐1
3.7 | The time course for the
enantioselective amidation of (R,S)‐1
Compared with free enzymes, there are several advan-
tages for using immobilized enzymes, which include their
stability of conformation, easy separation, and reusabil-
ity.27,28 As shown in Figure 5, the e.e.s was maintained
at a high level (>95%) following the first 12 batches.
Although the conversion rate slightly decreased as the
The time course for the enantioselective amidation of
(R, S)‐1 at the optimized reaction conditions was next
investigated. The results are presented in Figure 4. There
was a marked tendency for the conversion, together with
FIGURE 6 1H NMR spectra of (S)‐4
FIGURE 7 Mass spectrogram of the
product (S)‐4

ZHANG ET AL.
7
2. Yun H, Yun H, Cho BK, Kim BG. Kinetic resolution of (R,S)‐sec‐
butylamine using omega‐transaminase from Vibrio fluvialis JS17
under reduced pressure. Biotechnol Bioeng. 2004;87(6):772‐778.
batches progressed, the residual activity was greater than
90% following 12 reuse cycles. Therefore, the immobilized
Novozym 435 had an improved application in the prepa-
ration of (S)‐1.
3. Wang WG, Ma FN, Shen XM, Zhang C. New chiral auxiliaries
derived from (S)‐α‐phenylethylamine as chiral solvating agents
for carboxylic acids. Tetrahedron: Asymmetry. 2007;18:832‐837.
3.9 | Synthesis of (S)‐4
4. Juaristi E, Escalante J, León‐Romo JL, Reyes A. Recent applica-
tions of α‐phenylethylamine (α‐PEA) in the preparation of
enantiopure compounds. Part 1: incorporation in chiral cata-
lysts. Part 2: α‐PEA and derivatives as resolving agent.
Tetrahedron: Asymmetry. 1998;29:715‐740.
The chemical synthesis of (S)‐4 was developed from (S)‐
1. First, (S)‐3 was synthesized through the chemical
acylation of (S)‐1. Then, (S)‐3 was used to obtain (S)‐4
through its reacting with 2‐mercapto‐5,7‐dimethyl‐1,2,4‐
triazolo[1,5‐a]pyrimidine. Under the appropriate condi-
tions, the target, (S)‐4, was acquired, and the total yield
and e.e. values were 83.5% and 95.3%, respectively.
(S)‐4 was white solid. Formula: C17H18ClN5OS1H.
Melting point: 179~181°C. 1HNMR (500 MHz, CDCl3)
(Figure 6): δ: 1.41~1.42 (d,3H), 2.65 (s,3H), 2.67 (s,3H),
3.79~3.91 (m,2H), 5.01~5.05 (m,1H), 6.79 (s,1H),
7.07~7.13 (m,4H), and 7.78~7.79 (d,1H). MS (ESI) m/z
(Figure 7): 375 [M + H]+.
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ethyl)‐2‐(5,7‐dimethyl‐[1,2,4]triazolo[1,5‐a]pyrimidin‐2‐ylthio)
acetamideand raw material 1‐(4‐chorophenyl)ethanone. China:
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4 | CONCLUSION
The asymmetric amidation of (R,S)‐1 by Novozym 435
was achieved with MTBE. Methyl 2‐tetrahydrofuroate
was proven an ideal acyl donor, with the molar ratio of
substrate to acyl donor being 1:2.4. When the reaction
was carried out at 40°C for 22 hours, the enantiomeric
excess of (S)‐1 was able to reach as high as 99%, with a
conversion rate of 52%. Therefore, a green and highly effi-
cient protocol for the asymmetric resolution of (R,S)‐1
was developed. Using the obtained (S)‐1 as the raw mate-
rial, (S)‐4 was successfully synthesized with an optical
purity of 95.3%.
9. Bea HS, Seo YM, Cha MH, Kim BG, Yun H. Kinetic resolution
of α‐methylbenzylamine by recombinant Pichia pastoris express-
ing ω‐transaminase. Biotechnol Bioprocess Eng. 2010;15:429‐434.
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ACKNOWLEDGEMENTS
phenylethylamine amidohydrolase,
a new enzyme from
Arthrobacter aurescens AcR5b. Appl Microbiol Biotechnol. 1997;
47(6):650‐657.
This study was funded by the Zhejiang Provincial Natural
Science Foundation (LY18B020021) and the Scientific
and Technological Innovation Activity Plan for Students
in Zhejiang Province (Emerging Artists Talent Plan,
2017R403090).
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ORCID
Yinjun Zhang http://orcid.org/0000-0001-8504-1036
Jianyong Zheng http://orcid.org/0000-0002-9821-1062
15. Cheng F, Tang XL, Kardashliev T. Transcription factor‐based
biosensors in high‐throughput screening: advances and applica-
tions. Biotechnol J. 2018;13:1700648.
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