Industrial production of chiral intermediate of cilastatin by nitrile hydratase and amidase catalyzed one-pot, two-step biotransformation

Article abstract of DOI:10.1016/j.molcatb.2014.02.005

An industrial one-pot, two-step bioprocess catalyzed by nitrile hydratase (NHase) and amidase was developed for (S)-2,2-dimethylcyclopropanecarboxamide (1), the key chiral intermediate of cilastatin. The key unit operations of the whole process including fermentative production of enzymes, biotransformation, isolation of product, and recycling of by-product were reported for the first time. The volumetric enzyme activities of NHase and amidase in 1000-L fermentor were enhanced to 351,000 and 5880 U/L, respectively. The two-step, one-pot biotransformation of rac-2,2-dimethylcyclopropanecarbonitrile (2) took full advantage of both enzymes, leading to accumulation of (S)-1 in 47% yield and 99.6% ee. (S)-1 and the by-product (R)-2,2-dimethylcyclopropanecarboxylic acid (3) were obtained with yields of 38% and 45%, respectively, by a novel macroporous resin adsorption chromatography. Moreover, the total yield of (S)-1 was further increased to 53% after one recycling of (R)-3. The new bioprocess dramatically improved process efficiency compared with the chemical route by elimination of six synthetic steps and proved to be a superior and more cost-effective approach towards (S)-1.

Full text of DOI:10.1016/j.molcatb.2014.02.005

Journal of Molecular Catalysis B: Enzymatic 102 (2014) 161–166
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Industrial production of chiral intermediate of cilastatin by nitrile
hydratase and amidase catalyzed one-pot, two-step
biotransformation
Ren-Chao Zhenga,b, Zhong-Yi Yangc,d, Cong-Cheng Lid, Yu-Guo Zhenga,b,∗
,
Yin-Chu Shena,b
a Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, People’s Republic of China
b Engineering Research Center of Bioconversion and Biopurification, Ministry of Education, Hangzhou, Zhejiang 310014, People’s Republic of China
c Taizhou University, Taizhou, Zhejiang 317000, People’s Republic of China
d Zhejiang Hisun Pharmaceutical Co. Ltd., Taizhou, Zhejiang 318000, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
An industrial one-pot, two-step bioprocess catalyzed by nitrile hydratase (NHase) and amidase was devel-
oped for (S)-2,2-dimethylcyclopropanecarboxamide (1), the key chiral intermediate of cilastatin. The key
unit operations of the whole process including fermentative production of enzymes, biotransformation,
isolation of product, and recycling of by-product were reported for the first time. The volumetric enzyme
activities of NHase and amidase in 1000-L fermentor were enhanced to 351,000 and 5880 U/L, respec-
tively. The two-step, one-pot biotransformation of rac-2,2-dimethylcyclopropanecarbonitrile (2) took
full advantage of both enzymes, leading to accumulation of (S)-1 in 47% yield and 99.6% ee. (S)-1 and
the by-product (R)-2,2-dimethylcyclopropanecarboxylic acid (3) were obtained with yields of 38% and
45%, respectively, by a novel macroporous resin adsorption chromatography. Moreover, the total yield of
(S)-1 was further increased to 53% after one recycling of (R)-3. The new bioprocess dramatically improved
process efficiency compared with the chemical route by elimination of six synthetic steps and proved to
be a superior and more cost-effective approach towards (S)-1.
Received 20 November 2013
Received in revised form 12 February 2014
Accepted 12 February 2014
Available online 20 February 2014
Keywords:
Nitrile hydratase
Amidase
One-pot
Two-step biotransformation
(S)-2,2-Dimethylcyclopropanecarboxamide
Cilastatin
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
(S)-2,2-Dimethylcyclopropanecarboxamide (1) is the chiral
intermediate of cilastatin. During the past years, considerable
Imipenem was the first carbapenem antibiotic selected for
development because of its highly potent, broad-spectrum antimi-
crobial activity and good safety profile [1]. As imipenem is rapidly
degraded by kidney dehydropeptidase I, it is administered in a
1:1 ratio with cilastatin, an inhibitor of dehydropeptidase I. Cilas-
tatin not only prevents the renal metabolism of imipenem but
also protects the kidneys against potential toxic effects exerted
by higher doses of imipenem [2]. During the past two decades,
imipenem/cilastatin has played an important role in both empirical
and targeted treatments of severe and difficult to treat infections
[3].
efforts have been devoted to the synthesis of this important
building block. The chemical resolution route usually began with
basic hydrolysis of rac-2,2-dimethylcyclopropanecarbonitrile (2),
followed by chloroformylation to give the corresponding acid chlo-
ride. Resolution of the racemate was achieved by several steps of
fractional crystallization using a chiral auxiliary, such as L-carnitine
oxalate, (S)-mandelic acid methyl ester, and L-menthol. The result-
ing (S)-2,2-dimethylcyclopropanecarboxylic acid (3) was finally
converted into optically pure (S)-1 via acylation and ammonolysis
(Scheme 1) [4–6]. The processes suffered from low overall yields
(∼20%) and the generation of large amounts of waste. Moreover, as
the resolution depended on the solubility difference of the diaste-
reomers, the optical purity of the product was not high enough to
satisfy the pharmaceutical quality standards.
Biocatalysis is rapidly evolving into a key technology for the
pharmaceutical industry owing to its high enantioselectivity, mild
reaction conditions, and environmental friendliness [7]. As a result,
several enzymatic routes have been exploited for the preparation
∗
Corresponding author at: Institute of Bioengineering, Zhejiang University of
Technology, Hangzhou, Zhejiang 310014, People’s Republic of China.
Tel.: +86 571 88320630; fax: +86 571 88320630.
E-mail address: zhengyg@zjut.edu.cn (Y.-G. Zheng).
http://dx.doi.org/10.1016/j.molcatb.2014.02.005
1381-1177/© 2014 Elsevier B.V. All rights reserved.

162
R.-C. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 161–166
OH
O
O
O
Me3N+
Cl-
(R)
OH
ion exchange
KOH
SOCl2
O
+
Reflux
Me3N
Cl-
OH
CN
COOH
COCl
(R)
O
O
O
O
O
(S)
O
O
O
O
Oxalic acid
2-3 Crystallizations
+
+
+
Me3N
HOOCCOO-
Me3N
Me3N
OH
OH
OH
(R)
HOOCCOO-
(R)
(R)
Basic hydrolysis
+SOCl2, NH3
OH
NH2
O
O
Scheme 1. Industrial chemical resolution of rac-1.
of enantiopure (S)-1 or (S)-3 [8–12]. Among these, the one-pot,
two-step biocatalytic route developed by Lonza was most competi-
tive [13]. This latter procedure involved a nitrile hydratase (NHase)
and R-amidase catalyzed sequential biotransformation, and started
from the existing, inexpensive racemic precursor 2.
We started industrial production of (S)-1 via an enzymatic sys-
tem of NHase and amidase using strains discovered in our lab in
2005 [14] (Scheme 2). The manufacturing process was continuously
improved over the past years, which made the bioprocess a superior
and more cost-effective approach toward (S)-1. Herein, we report
the key unit operations of the bioprocess, including fermentative
production of the enzymes, biotransformation, purification of the
product, and recycling of the by-product.
5 min, 800 ␮L reaction samples were withdrawn and the reaction
was quenched by 100 ␮L HCl (1.0 M), and then neutralized with
100 ␮L NaOH (1.0 M). After centrifugation at 12,000 × g for 3 min,
the supernatant was analyzed by gas chromatography. One unit of
NHase activity was defined as the amount of biocatalyst required
for the production of 1 ␮mol 1 per min under the above reaction
conditions.
Amidase activity was assayed using rac-1 as substrate. A 1.0-
mL aliquot of fermentation broth was centrifuged at 12,000 × g for
10 min. The cell pellet was suspended in 1.0 mL phosphate buffer
(50 mM, pH 7.0) and mixed with 3.0 mL of the same buffer con-
taining 8.0 g/L of 1. The bioconversion was carried out at 35 ◦C for
10 min with reciprocal shaking (200 rpm). Samples (800 ␮L each) of
the reaction mixture were withdrawn and the enzymatic reaction
was quenched by removing biomass through centrifugation. The
resulting supernatant was analyzed by gas chromatography. One
unit of amidase activity is represented by the release of 1.0 ␮mol
of 3 per minute under such conditions.
2. Materials and methods
2.1. Chemicals
The medium components were usual commercial products and
used without further purification. Rac-2 was synthesized according
to Nelson et al. [15] with modifications. Macroporous resins were
purchased from Shanghai Huazhen Sci. &Tech. Co. Ltd (Shanghai,
China). All the other chemicals were of reagent grade purity and
purchased from commercial sources.
2.4. Fermentation of R. boritolerans FW815 in 1000-L fermentor
To prepare liquid inocula, a loop of cells from 24-hour-old slant
cultures was initially inoculated into each 500 mL Erlenmeyer flask
containing 80 mL of seed medium: 10 g of glucose, 2 g of peptone,
5 g of yeast extract, 1 g of NaCl, 0.2 g of MgSO4, 0.1 g of K2HPO4, 0.1 g
of KH2PO4, 0.01 g of FeSO4·7H2O per liter of tap water, and culti-
vated at 30 ◦C for 24 h with shaking at 200 rpm. Thereafter, 500 mL
of the culture was transferred to a 15-L fermentor containing 10 L
of the fresh seed medium. Cells in the seed reactor were culti-
vated at 30 ◦C for 22 h with aeration at 0.5 vvm (air volume/culture
volume/minute) and agitation at 200 rpm. The culture broth was
subsequently inoculated to a 1000-L fermentor containing 600 L of
the same medium as that of seed culture with the exception that
1.0 g/L of ␧-caprolactam was supplemented as inducer. The fermen-
tations were carried out at 30 ◦C, agitated at 100 rpm and aerated
at 0.4 vvm.
2.2. Bacterial strains, media, and culture conditions
The NHase producing strain Rhodococcus boritolerans FW815
was screened using the enrichment culture technique [16]. It
was deposited at the China Center for Type Culture Collection
(CCTCC) under the accession number of CCTCC M208108. The
R-enantioselective amidase producing strain Delftia tsuruhaten-
sis ZJB-05174 was isolated employing a colorimetric method [17]
and deposited at CCTCC under the accession number of CCTCC
M205114. The heterologous expression of R-amidase gene (dam)
from D. tsuruhatensis ZJB-05174 in Escherichia coli BL21 (DE3) has
been previously described in detail [18].
2.5. Fermentation of E. coli BL21 (DE3)/pET-dam in 1000-L
fermentor
2.3. Enzyme activity assay
The NHase activity to rac-2 was determined according to the
production of 1. One milliliter of R. boritolerans FW815 fermen-
tation broth was centrifuged and the resultant cell paste was
resuspended in 50 mL phosphate buffer (50 mM, pH 7.0). Rac-2 was
added at a concentration of 12.2 g/L to initiate the reaction. After
Escherichia coli BL21 (DE3)/pET-dam was first cultured in 5 mL
LB medium and incubated for 10 h. It was then diluted into
500 mL flask cultures with 30 ␮g/mL kanamycin and grown at 35 ◦C
overnight. As the OD600 reached 0.8–1.0, 100 mL of the culture was
used to inoculate 10 L of 2 × YT medium composed of 10 g yeast

R.-C. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 161–166
163
R-amidase
NHase
+H2O
+
OH
NH2
NH2
CN
O
O
O
(S)-1
(R)-3
2
1
+SOCl2, NH3
Scheme 2. Production of (S)-1 via NHase and amidase-catalyzed one-pot, two-step biotransformation.
extract, 16 g tryptone and 5 g NaCl per liter of tap water. The seed
fermentation was performed at 35 ◦C, agitated at 300 rpm and aer-
ated at 1.0 vvm. When the OD600 reached 0.4–0.6, the culture was
supplemented with 10 g/L of lactose and induced by lowering the
temperature to 28 ◦C. After 15 h of induction, the main fermentation
cultures (600 L) were inoculated with these seed cultures. The oper-
ating conditions for the 1000-L fermentor were as follows: 35 ◦C,
100 rpm, 1.2 vvm. The DO concentration was maintained above 20%
of air saturation through automatic adjustment of the agitation
speed within a range of 200–600 rpm. Fermentation broth from
the 1000-L fermentor was harvested by centrifugation (continuous
flow) at 10,000 × g and flow rate of 50 L/h.
A suitable vessel was charged with 110 kg of (R)-3 as a n-
hexane solution, followed by slow addition of thionyl chloride (1.1
equiv). The mixture was refluxed for 2.5 h and the organic sol-
vent and excess thionyl chloride was distilled off. The residue was
heated to 135–140 ◦C and stirred under nitrogen atmosphere for
3 h until the optical rotation became zero. The obtained racemic
2,2-dimethylcyclopropanecarboxylic acid chloride (4) was slowly
added to 25% aqueous ammonia. The reaction mixture was stirred
at 0 ◦C for 2.5 h and then the pH of the solution was adjusted to
neutral with concentrated H2SO4. The resultant aqueous solution
was extracted with chloroform for two times and the organic layer
was evaporated in reduced pressure to give rac-1.
2.8. Analytical methods
2.6. Production of (S)-1 via NHase and amidase catalyzed
sequential biotransformations
The amounts of compound 1, 2, and 3 were assayed by a GC-
14 C instrument (Shimadzu, Japan) equipped with HP-5 capillary
column (Agilent, USA) and flame ionization detector (FID) using
N2 as carrier gas (1.0 mL/min). The column was conditioned and
operated at 140 ◦C; the detector and injector temperatures were set
at 220 ◦C. The retention times of 1, 2, and 3 were 6.4 min, 3.5 min,
and 5.2 min, respectively.
Enantiomeric excess (ee) of 1 and 3 was determined on an
Agilent 6890 GC system, equipped with a FID and a chiral capil-
lary column BGB-174 (50% 2,3-diacetyl-6-tert-butyldimethylsilyl-
alpha-cyclodextin dissolved in 14% cyanopropylphenyl- and 86%
methylpolysiloxane, BGB Analytik, Switerland). The column flow
rate was 1.6 mL/min (helium). An oven temperature program as fol-
lows was employed: the initial column temperature of 130 ◦C was
held for 5 min, then raised to 170 ◦C at a rate of 5 ◦C/min. Before
injection into the GC system, the samples were acidified by addi-
tion of 30 ␮L HCl (5.0 M) and extracted with ethyl acetate through
vigorous mixing. The ethyl acetate layer was collected and dried
over anhydrous Na2SO4. Retention times of the four enantiomers
were as follows: (R)-3, 5.8 min; (S)-3, 6.0 min; (S)-1, 10.3 min; (R)-1,
11.1 min.
Eight kilograms of R. boritolerans FW815 resting cells were sus-
pended in a 10,000-L reaction tank charged 8000 L of deionized
water and 32 L of acetonitrile. The resulting solution was main-
tained at 30 ◦C and stirred at 100 rpm. Rac-2 (203 kg) was fed to
the reactor continuously so that the substrate concentration was
always below 6 g/L in the reaction system. Samples were with-
drawn every 0.5 h and analyzed by GC. When the conversion of
2 was higher than 98%, the reaction mixture was heated to 35 ◦C
and 4 kg of E. coli BL21 (DE3)/pET-dam cells were charged. Samples
were withdrawn every 2 h to monitor the optical purity of 1. After
the bio-resolution, the pH of the reaction mixture was adjusted to
10.5 using 12% (w/v) NaOH solution.
2.7. Downstream processing of the reaction mixture
The resulting reaction mixture was centrifuged to remove res-
ting cells. The clarified supernatant (1000 L) was loaded onto a
column pre-packed with adsorbent HZ801 at a bed volume (BV)
of 1000 L. Thereafter, 4 BV of basic water (pH 12.0) was used to
wash the column until the concentration of 3 in eluates was below
0.2 g/L. The column was then washed with 1 BV of deionized water,
followed by elution with 2 BV of 85% (v/v) acetone aqueous solu-
tion. After 10–12 batches of purification, absorbent HZ801 was
regenerated by NaOH solution, 80% acetone solution and deion-
ized water. Eluates containing (S)-1 were combined, concentrated
under vacuum and cooled to 4 ◦C for crystallization overnight. The
final product was obtained after filtration and washing with water.
Meanwhile, eluates containing (R)-3 were acidified to pH 2.5 using
37% HCl and pumped into the regenerated chromatography col-
umn. After sample loading, 1 BV of deionized water was used to
wash the column, followed by elution with 1.3 BV of 16% sodium
hydroxide solution. The eluate was concentrated to a volume of
about 300 L by vacuum distillation. The pH of the remaining solu-
tion was again adjusted to 2.5 by 37% HCl and the solution extracted
two times with equal volume of n-heptane. The combined organic
layers were concentrated in vacuo to afford (R)-3.
3. Results and discussion
3.1. Production of NHase of R. boritolerans FW815 in 1000-L
fermentor
NHase catalyzes the single-step hydration of nitriles into amides
with excellent selectivity under environment-friendly reaction
conditions [19,20]. However, the pilot or large-scale fermentative
production of NHase has rarely been reported. Our previous study
revealed that R. boritolerans FW815 was a promising biocatalyst
for the industrial production of 2 [16]. Based on optimization of
the cultivation conditions in shake flask and 30-L fermentor, NHase
production of R. boritolerans FW815 was then carried out in a 1000-
L fermentor with a working volume of 600 L. The time course of
cell growth and NHase production is shown in Fig. 1. The cultures

164
R.-C. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 161–166
30
25
20
15
10
5
40
35
30
25
20
15
10
5
30
25
20
15
10
5
6000
5000
4000
3000
2000
1000
0
0
0
0
0
10
20
30
40
50
0
5
10
15
20
25
Culture time (h)
Culture time (h)
Fig. 1. Profiles of cell growth and NHase production in batch fermentation of R.
boritolerans FW815. Symbols: Growth (᭹); NHase activity (ꢀ).
Fig. 2. Time course of amidase production in batch fermentation of E. coli BL21
(DE3)/pET-dam in 3-L and 1000-L fermentors. Symbols: Growth in 3-L fermen-
tor (ꢀ); Growth in 1000-L fermentor (᭹); Amidase activity in 3-L fermentor (ꢀ);
Amidase activity in 1000-L fermentor (ꢁ).
grew exponentially soon after inoculation and the highest cell den-
sity (OD600) of 28.3 was observed after 46 h of cultivation. On the
other hand, the NHase production was found to be closely asso-
ciated with growth and the peak simultaneously occurred at 46 h
with total activity of 351,000 U/L, which was 2.1 times higher than
that of 167,000 U/L in the shake flask [16]. It has been reported that
agitation and aeration have significant effect on NHase production
[21]. In this study, the increased NHase production of R. boritolerans
FW815 in a stirred-tank reactor was considered to be dependent on
better agitation and aeration.
[25], recombinant expression [18] and optimization of cultivation
conditions, the efficient and low-cost production of biocatalysts
rendered the bioprocess more competitive. Moreover, as 0.1% (w/v)
R. boritolerans FW815 and 0.05% (w/v) E. coli BL21 (DE3)/pET-dam
resting cells were sufficient to catalyze the reaction, the low bio-
catalyst loading made the product recovery easier.
3.3. Production of (S)-1 in 10,000-L reactor by the one-pot,
two-step biotransformation
3.2. Production of R-enantioselective amidase of E. coli BL21
(DE3)/pET-dam in 1000-L fermentor
Our previous study showed that 1 formed in situ in the presence
of NHase had better miscibility properties than the chemically syn-
thesized counterpart (data not shown). Moreover, the solubility of
the amide could be further enhanced by applying cosolvents such
as acetonitrile or ethanol [26]. These two strategies were employed
to increase substrate loading of the bioprocess. The NHase and
amidase-catalyzed one-pot, two-step bioprocess took full advan-
tage of both enzymes, namely high activity of the NHase and strict
enantioselectivity of the amidase. In the first step, NHase-catalyzed
hydration of 2 proceeded rapidly and quantitatively. The amide 1
was accumulated in 30.1 g/L with 99.2% conversion within 2.5 h
(Fig. 3). Therefore, the concentration of substrate for subsequent
bio-resolution was notably increased compared to the bioprocess
catalyzed by amidase alone (6 g/L) [18]. As the NHases showed
low enantioselectivity [27], the optical purity of 1 was almost zero
during this period. After the enzymatic hydration, resting cells of
E. coli BL21 (DE3)/pET-dam were added to initiate kinetic resolution
of the resultant amide. The concentration of 3 increased linearly
until the substrate conversion reached approximately 40%. The
hydrolysis rate then slowed down and (S)-1 was produced in 47%
yield (14.3 g/L) and 99.6% ee, giving an E-value of 120.
Although amidases are often found to be coupled with NHase
in the microbial degradation pathway of nitriles, these NHase-
associated amidases are mostly S-stereospecific [9,10,22]. A novel
R-amidase producing strain D. tsuruhatensis ZJB-05174, which
exhibited strict enantioselectivity toward 1, was isolated in our
laboratory [23]. As the amidase-catalyzed resolution of 1 was the
rate-limiting step of the two-step biotransformation, efficient pro-
duction of amidase was of great importance to lower cost of the
whole bioprocess. However, the amidase activity in the wild strain
was low. Hence, the amidase gene was cloned and overexpressed
in a recombinant E. coli [18]. The expression of amidase in the engi-
neered E. coli was induced by lactose and the organism was grown
in 3-L and 1000-L fermentors (Fig. 2). E. coli BL21 (DE3)/pET-dam
exhibited a similar exponential mode of growth with a slight lag
timeinthe first 8 h. Thegrowthrate inthe 3-L fermentorcontinually
decreased until reached the stationary phase at 14 h. The cultures in
1000-L fermentor had a longer exponential growth phase and the
highest OD600 of 26.5 was observed after 19 h. Meanwhile, the yield
of amidase in 1000-L fermentor increased drastically to 5880 U/L,
which was 2.3 fold higher than that of 3-L process. The amidase
production improvement is summarized in Table 1, which demon-
strates that the volumetric amidase productivity is increased by
more than 10 times compared with that of wild strain in flask shake.
Therefore, the large-scale production of NHase and amidase was
reported for the first time. Through N+ ion implantation mutation
3.4. Isolation of (S)-1 from reaction mixture using macroporous
resin adsorption chromatography
When pH of the reaction mixture was adjusted to 12.0, the
huge polarity difference between 1 and 3 enabled them to be well
Table 1
Improvement of R-amidase production of D. tsuruhatensis ZJB-05174 and E. coli BL21 (DE)/pET-dam in different reactors.
Strain
Amidase activity (U/L)
Shake flask
3-L reactor
5-L reactor
100-L reactor
1000-L reactor
D. tsuruhatensis ZJB-05174
E. coli BL21(DE)/pET-dam
528 [24]
618
–
1758
358 [18]
2963 [18]
–
–
5880
5255 [18]

R.-C. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 161–166
165
Table 2
Yield of (S)-1 and (R)-3 during different steps of the bioprocess.
Steps
rac-2 (Kg)
(S)-1
(R)-3
Mass (Kg)
Yield
ee (%)
Mass (Kg)
Yield
ee (%)
Input
203
0
–
–
203
0
–
–
0
–
–
Coupled enzymatic reaction
Resin adsorption
Crystallization or extraction
Total
114.3
104.2
90.8
90.8
47
91
87
38
>99.5
>99.5
>99.5
>99.5
128.3
115.5
109.7
109.7
52
90
95
45
89.5
90.1
90.1
90.1
35
30
25
20
15
10
5
3.5. Isolation and recycling of by-product (R)-3
100
In order to establish a high-yield resolution process, isolation
and recycling of the by-product (R)-3 was explored. Based on
the polarity difference of 3 under basic and acidic conditions, the
by-product was conveniently isolated by a combination of macro-
porous resin adsorption and solvent extraction. 109.7 kg of 3 was
recovered in 90.1% ee and 45% overall yield (Table 2). However, it is
difficult to racemize the (R)-acid because the carboxylic acid group
is deprotonated first, which increases the pKa at the alpha-C. There-
fore, (R)-3 was first converted into the acid chloride using SOCl2,
and the resulting (R)-4 was racemized at 130∼135 ◦C, and subse-
quently converted into rac-1 by ammonolysis. Finally, 98.5 kg of 1
was afforded as white powder and recycled in R-amidase catalyzed
kinetic resolution. The yield of (S)-1 was increased to 53% after one
recycle of (R)-3. In regard to other biocatalytic strategies for (S)-1
[11], the present enzymatic process is the most economic route due
to its high yield and facile racemization of by-product.
80
60
40
20
0
S
e
0
0
5
10
15
20
25
Reaction time (h)
Fig. 3. Biocatalytic synthesis of (S)-1 from rac-2 via NHase and amidase catalyzed
sequential biotransformation. Symbols: concentration of 1 (ꢁ); concentration of 2
(ꢂ); concentration of 3 (ꢀ); ee of (S)-1 (ꢀ).
4. Conclusion
separated from each other. Therefore, a separation strategy involv-
ing macroporous resin adsorption chromatography combined with
crystallization was established for (S)-1 purification. Five differ-
ent macroporous resins HZ801, HZ806, HZ816, HZ820 and HZ841
were tested for their adsorption and desorption capacities of (S)-
1. Among these adsorbents, HZ801 had the best adsorption and
desorption performance. A stepwise elution protocol was applied
in this work (Fig. 4). In the first stage, the unbound 3 was quickly
eluted while (S)-1 was still bound to the resin. When the eluant
was changed to 85% (v/v) acetone aqueous solution, nearly all of
the bound 1 was desorbed and the elution peak was precipitous
and symmetric. The column chromatography recovered 91% of (S)-
1 and 90.8 kg of (S)-1 was obtained after crystallization with >99.5%
ee and a total yield (from rac-2) of 38% (Table 2), which was higher
than the 35% yield reported by Lonza [13].
The new biocatalytic route dramatically improved process effi-
ciency compared to the chemical one by elimination of six synthetic
steps. The bioprocess also avoided the use of expensive chiral aux-
iliary and remarkably reduced organic solvent usage. The reaction
was performed under mild conditions, reducing significant energy
consumption. This work demonstrates a successful example of an
environmentally benign pharmaceutical process by a combination
of efficient NHases and enantioselective amidases. Furthermore,
the bienzymatic cascade has been developed into a practical plat-
form technology for the production of valuable enantiomerically
pure amides and acids in our laboratory, which will find more and
more promising applications in pharmaceutical and agrochemical
industries.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (No. 21202150), National High Technology Research
and Development Program of China (No. 2012AA022201B) and
Major Basic Research Development Program of China (No.
2011CB710806).
12
10
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