Efficient preparation of both enantiomers of 3,3,3-trifluoro-2-hydroxy-2- methylpropanoic acid catalyzed by Shinella sp. R-6 and Arthrobacter sp. S-2

Article abstract of DOI:10.1016/j.molcatb.2014.01.024

Several microorganisms that can enantioselectively hydrolyze 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide have been isolated from soil samples. These strains were capable of growing in a medium containing 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide as the sole nitrogen source. Among them, Shinella sp. R-6 was identified as a strain capable of exhibiting R-selective hydrolysis activity, while Arthrobacter sp. S-2 was capable of exhibiting S-selective hydrolysis activity. The preparation of both enantiomers of 3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid via the two-step whole-cell reaction was investigated using these two strains.

Full text of DOI:10.1016/j.molcatb.2014.01.024

Journal of Molecular Catalysis B: Enzymatic 102 (2014) 115–119
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Efficient preparation of both enantiomers of
3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid catalyzed by
Shinella sp. R-6 and Arthrobacter sp. S-2
Ken-ichi Fuhshukua, Shunsuke Watanabea, Tetsuro Nishiic, Akihiro Ishiic,
Yasuhisa Asanoa,b,∗
a Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
b Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
c Chemical Research Center, Central Glass Co., Ltd., 2-17-5 Nakadai, Kawagoe, Saitama 350-1159, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Several microorganisms that can enantioselectively hydrolyze 3,3,3-trifluoro-2-hydroxy-2-
methylpropanamide have been isolated from soil samples. These strains were capable of growing
in a medium containing 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide as the sole nitrogen source.
Among them, Shinella sp. R-6 was identified as a strain capable of exhibiting R-selective hydrolysis activ-
ity, while Arthrobacter sp. S-2 was capable of exhibiting S-selective hydrolysis activity. The preparation
of both enantiomers of 3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid via the two-step whole-cell
reaction was investigated using these two strains.
Received 18 December 2013
Received in revised form 25 January 2014
Accepted 29 January 2014
Available online 14 February 2014
Keywords:
Amidase
Enrichment culture technique
Kinetic resolution
Screening
© 2014 Elsevier B.V. All rights reserved.
Trifluoromethyl
1. Introduction
there still remains a drawback to this method. To obtain (S)-acid 1,
the recovered (S)-amide 2 has to be hydrolyzed under harsh con-
Trifluoromethyl-substituted compounds, especially optically
active ones, have received much attention of scientists and
industry [1]. Both enantiomers of 3,3,3-trifluoro-2-hydroxy-2-
methylpropanoic acid 1, containing hydroxyl and trifluoromethyl
groups attached to the same carbon atom, were previously shown
to be intermediates for the synthesis of a number of fine chemi-
cals and pharmaceuticals, like the pyruvate dehydrogenase kinase
inhibitor for the treatment of diabetes, and bradykinin antagonist
in anti-inflammatory and painkiller medicines (Fig. 1) [2,3].
Many microbial amidases have been characterized and applied
to the synthesis of useful compounds for the preparation of car-
boxylic acids [4]. Shaw has reported a process for the large-scale
synthesis of enantiomerically pure acid 1, which employed an
enantioselective amidase from Klebsiella oxytoca PRS1 and used it
in the kinetic resolution of ( )-amide 2 to obtain (R)-acid 1 and (S)-
amide 2 [5]. While this enzymatic procedure is a very efficient way,
ditions. Konigsberger has reported the process that employed the
enzymatic resolution of 1,1,1-trifluoroacetone cyanohydrin acyl
derivatives followed by the hydrolysis to yield (R)-acid 1 [6]. How-
ever, this method showed low enantioselectivity for (S)-acid 1.
Therefore, the efficient preparation of both enantiomers of this
valuable molecule is still desired.
In this study, we focused on enantioselective hydrolysis activity
from microbial sources that acted on amide 2 to develop kinetic
resolution for the preparation of both enantiomers of acid 1 under
mild conditions.
2. Materials and methods
2.1. Materials
(
)-Amide 2 was prepared by the hydrolysis of the correspond-
ing cyanohydrin prepared from 1,1,1-trifluoroacetone and NaCN
according to a previously reported procedure [5,7]. All other chem-
icals were purchased from commercial sources and were used
without further purification.
∗
Corresponding author at: Toyama Prefectural University, Biotechnology
Research Center and Department of Biotechnology, 5180 Kurokawa, Imizu, Toyama
939-0398, Japan. Tel.: +81 766 56 7500; fax: +81 766 56 2498.
E-mail address: asano@pu-toyama.ac.jp (Y. Asano).
http://dx.doi.org/10.1016/j.molcatb.2014.01.024
1381-1177/© 2014 Elsevier B.V. All rights reserved.

116
K. Fuhshuku et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 115–119
temperature: 40 ◦C; detection: 254 nm; tR = 13.6 min for (R)-1 and
16.0 min for (S)-1] for enantiomeric excess.
2.4. Examination of substrate concentration
Shinella sp. R-6 or Arthrobacter sp. S-2 cells were inoculated
into 5.0 mL of the medium as described above, and incubated with
shaking at reciprocal shaking (300 strokes/min) for 24 h at 30 ◦C.
Cells were harvested by centrifugation (20,000 × g, 10 min, 4 ◦C)
and washed with 20 mM KPB (pH 7.0). Wet cells from 10 mL of
the culture were suspended in 20 mM KPB (pH 7.0) containing
10–1000 mg of ( )-2 (sub. conc. 0.20–20%) in a total volume of
5.0 mL. A 1000 ␮L aliquot was withdrawn after incubation with
reciprocal shaking (300 strokes/min) for an appropriate period
at 30 ◦C. After appropriate dilution and centrifugation (20,000 × g,
10 min, 4 ◦C), an 800 ␮L of supernatant was then withdrawn. After
further centrifugation (20,000 × g, 10 min, 4 ◦C), the supernatant
was analyzed by HPLC, as described above.
Fig. 1. 3,3,3-Trifluoro-2-hydroxy-2-methylpropanoic acid 1 and the corresponding
amide 2, as intermediates for the synthesis of pharmaceuticals.
2.2. Isolation of 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide
(2) degrading microorganisms using the enrichment culture
technique
2.5. Kinetic resolution of
3,3,3-trifluoro-2-hydroxy-2-methylpropanamide (2) by the
two-step reaction using Shinella sp. R-6 and Arthrobacter sp. S-2
A screening medium consisting of 2.0 g of KH2PO4, 2.0 g of
K2HPO4, 1.0 g of NaCl, 0.50 g of yeast extract, 0.20 g of MgSO4, 1.0 mL
of the vitamin mixture solution, 1.0 mL of the trace element solu-
tion, and 2.0 g of ( )-amide 2 in 1000 mL of water was used to
isolate amide 2 degrading microorganisms. The vitamin mixture
solution was composed of 20 mg of inositol, 4.0 mg of nicotinic
acid, 4.0 mg of Ca·pantothenate, 4.0 mg pyridoxine·HCl, 4.0 mg of
thiamine·HCl, 2.0 g of p-aminobenzoic acid, 2.0 mg of riboflavin,
0.10 mg of folic acid, and 20 ␮g of biotin in 100 mL of water. The
trace element solution was composed of 500 mg of CyDTA, 200 mg
of FeSO4·7H2O, 30 mg of H3BO4, 20 mg of CoCl2·6H2O, 10 mg of
ZnSO4·7H2O, 3.0 mg of MnCl2·4H2O, 3.0 mg of Na2MoO4, 2.0 mg of
NiCl2·6H2O, and 1.0 mg of CuCl2·2H2O in 100 mL of water. Amide
2 degrading microorganisms were isolated as follows. Soil samples
were taken from different locations in Toyama, Japan. A spoonful of
the soil sample was added to 2.5 mL of screening medium, and incu-
bated with reciprocal shaking (300 strokes/min) at 30 ◦C. A 50 ␮L
aliquot was transferred to fresh medium every 3 or 4 days. After
three transfers, each of the culture broths was spread onto agar
plates containing the same medium and 1.5% agar, respectively.
Arthrobacter sp. S-2 cells were inoculated into 5.0 mL of the
medium as described above, and incubated with reciprocal shak-
ing (300 strokes/min) for 24 h at 30 ◦C. Grown cells were added
into 500 mL of the same medium, and incubated with shaking
at 150 rpm for 24 h at 30 ◦C. A 200 mL aliquot was withdrawn,
and cells were harvested by centrifugation (9000 × g, 10 min, 4 ◦C)
and washed with 20 mM KPB (pH 7.0). Wet cells from 200 mL of
the culture were suspended in 20 mM KPB (pH 7.0) containing
2.00 g of ( )-2 (sub. conc. 2.0%) in a total volume of 100 mL. A
1000 ␮L aliquot was withdrawn after incubation for an appropri-
ate period at 30 ◦C with shaking at 150 rpm. After centrifugation
(20,000 × g, 10 min, 4 ◦C), an 800 ␮L of supernatant was withdrawn.
After further centrifugation (20,000 × g, 10 min, 4 ◦C), the super-
natant was analyzed by HPLC with the Cosmosil® C18-MS-II column
as described above. After incubation for 22 h at 30 ◦C with shaking
at 150 rpm, the reaction mixture was centrifuged (9000 × g, 10 min,
4 ◦C). The supernatant was collected, and wet cells were washed
with water (10 mL) twice. The combined supernatant was extracted
with EtOAc five times. The combined organic extract was dried over
Na2SO4, and concentrated in vacuo. The residue was the recovered
(R)-2 (991 mg, 50%). The residual aqueous layer was acidified to pH
4 by the addition of 2 N HCl, and extracted with Et2O five times. The
combined organic extract was dried over MgSO4, and concentrated
in vacuo. The residue was (S)-1 (921 mg, 46%). Based on HPLC anal-
ysis with the Sumichiral OA-5000 column as described above, the
enantiomeric excess of (S)-1 was determined to be 96.7%.
Shinella sp. R-6 cells were inoculated into 5.0 mL of the medium
as described above, and incubated with reciprocal shaking (300
strokes/min) for 24 h at 30 ◦C. Grown cells were added into 500 mL
of the same medium, and incubated with shaking at 150 rpm for
48 h at 30 ◦C. A 400 mL aliquot was withdrawn, and cells were
harvested by centrifugation (9000 × g, 10 min, 4 ◦C) and washed
with 20 mM KPB (pH 7.0). Wet cells from 400 mL of the culture
were suspended in 20 mM KPB (pH 7.0) containing the recovered
(R)-2 described above (991 mg) in a total volume of 200 mL. A
1000 ␮L aliquot was withdrawn after incubation for an appropri-
ate period at 30 ◦C with shaking at 150 rpm. After centrifugation
(20,000 × g, 10 min, 4 ◦C), an 800 ␮L of supernatant was withdrawn.
After further centrifugation (20,000 × g, 10 min, 4 ◦C), the super-
natant was analyzed by HPLC with the Cosmosil® C18-MS-II column
as described above. After incubation for 34 h at 30 ◦C with shaking
at 150 rpm, the reaction mixture was centrifuged (9000 × g, 10 min,
4 ◦C). The supernatant was collected, and wet cells were washed
2.3. Screening and analytical method
The medium used for screening was the same as that described
above. Microorganisms isolated from soil samples described above
or stored as stock cultures were inoculated into 2.5 mL of medium,
and then incubated with reciprocal shaking (300 strokes/min) for
48 h at 30 ◦C. Cells were harvested by centrifugation (20,000 × g,
5 min, 4 ◦C) and washed with 20 mM potassium phosphate buffer
(KPB, pH 7.0). Wet cells from 2.0 mL of the culture were sus-
pended in 980 ␮L of 20 mM KPB (pH 7.0). A total of 20 ␮L of 10%
(
)-2 aqueous solution was added to this suspension as a sub-
strate and the mixture was incubated with shaking at 1000 rpm
for 18 h at 30 ◦C. A 600 ␮L aliquot was withdrawn after centrifu-
gation (20,000 × g, 10 min, 4 ◦C) and mixed with 400 ␮L of water.
After further centrifugation (20,000 × g, 10 min, 4 ◦C), the super-
natant was analyzed using HPLC with the Cosmosil® C18-MS-II
column (5 ␮m, 4.6 mm × 150 mm, Nacalai Tesque, Inc.) (solvent:
0.1% H3PO4 aq./MeCN = 9/1; flow rate: 0.80 mL/min; temperature:
40 ◦C; detection: 210 nm; tR = 4.7 min for amide 2 and 7.6 min for
acid 1) for conversion and with the Sumichiral OA-5000 column
(5 ␮m, 4.6 mm × 150 mm, Sumika Chemical Analysis Service, Ltd.)
[solvent: 2 mM CuSO4 aq./MeCN = 85/15; flow rate: 2.0 mL/min;

K. Fuhshuku et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 115–119
117
Table 1
3,3,3-Trifluoro-2-hydroxy-2-methylpropanamide 2 hydrolysis activity from stock cultures.
Number of strains
Genus
Stereoselectivity toward2 (>50% ee of1)
Microorganisms tested
Hydrolysis of 2 (>10%)
R
S
Pseudomonas
Arthrobacter
Alcaligenes
Brevibacterium
Variovorax
Corynebacteirum
Yersinia
Microbacterium
Klebsiella
Rhodococcus
Serratia
78
51
34
10
8
18
3
9
14
7
6
3
3
2
2
2
1
1
1
1
–
3
–
–
2
1
–
1
–
–
–
–
1
–
8
2
5
4
–
–
–
–
1
1
–
1
–
–
14
8
16
10
4
193
442
Acinetobacter
Others
Total
43
with water (10 mL) twice. The combined supernatant was washed
with EtOAc five times, acidified to pH 4 by the addition of 2 N HCl,
and extracted with Et2O five times. The combined organic extract
was dried over MgSO4, and concentrated in vacuo. The residue was
(R)-1 (902 mg, 90%, and 45% for two steps). Based on HPLC analy-
sis with the Sumichiral OA-5000 column as described above, the
enantiomeric excess of (R)-1 was determined to be 99.5%.
were 83, 4, and 68, respectively. Strains 6-1 and 23-2 were then
chosen as candidates R-6 and S-2 for further studies, respectively.
The taxonomical characteristics of strain R-6 were as follows:
rod cells (0.7–0.8 × 1.5–2.0 ␮m), Gram negative, non-spore-
forming, and motile. The most variable region of the 16S rDNA
sequence revealed 99.9% identity to Shinella zoogloeoides IAM
12669 [9]. These characteristics have indicated that strain R-6
belongs to the genus Shinella sp. because it is rod-shaped, Gram-
negative, non-spore-forming, motile, glucose oxidation-negative,
catalase-positive, and oxidase-positive [9]. Shinella sp. R-6 was
deposited in National Institute of Technology and Evaluation (NITE)
under the accession number NITE P-1527.
3. Results
3.1. Isolation and identification of microorganisms
The taxonomical characteristics of strain S-2 were as fol-
lows: rod cells (rod-coccus cycle, 24 h: 0.7–0.8 × 1.5–2.5 ␮m,
72 h: 0.7–0.8 × 1.0–1.2 ␮m), Gram positive, non-spore-forming,
and non-motile. The most variable region of the 16S rDNA sequence
revealed 98.2% identity to Arthrobacter nitroguajacolicus G2-1 [10].
These characteristics have indicated that strain S-2 belongs to the
genus Arthrobacter sp. because it is rod-shaped and rod-coccus
cycle, Gram-positive, non-spore-forming, non-motile, glucose
oxidation-negative, catalase-positive, and oxidase-negative [10].
Arthrobacter sp. S-2 was deposited in National Institute of Tech-
nology and Evaluation (NITE) under the accession number NITE
P-1526.
We have screened microorganisms that can enantioselectively
hydrolyze ( )-amide 2 from stock cultures. After growing on the
screening medium containing 0.20% ( )-amide 2 as the sole nitro-
gen source, more than 400 microorganisms from 19 genera were
assayed for hydrolytic activity on ( )-amide 2 (Table 1). Although
some microorganisms were obtained that could show hydrolytic
activity on amide 2, most of them showed no or scarce enantiose-
lectivity.
We have also screened microorganisms that can assimilate ( )-
amide 2 as the sole nitrogen source. After growing on the screening
medium containing 0.20% ( )-amide 2 from 50 soil samples, the
188 isolated microorganisms were assayed for hydrolytic activity
on ( )-amide 2 (Table 2). Although some microorganisms were
obtained that exhibited hydrolytic activity on amide 2, most had
low enantioselectivity. Of these isolates, strains 6-1 and 13-1 were
found actively hydrolyze amide 2 R-selectively, while strain 23-2
showed high S-selective hydrolysis activity toward amide 2. E val-
ues [Vmax(fast)/Km(fast)]/[Vmax(slow)/Km(slow)], developed by Sih and
co-workers [8] as an index for the efficiency of a kinetic resolution,
3.2. Effect of substrate concentration
The influence of the substrate concentration on hydrolysis
activity toward amide 2 was investigated at substrate concen-
trations between 0.20% and 20% (Table 3). When the reaction in
the 5.0 mL scale was carried out using wet cells from 10 mL of
the culture, the reaction using Arthrobacter sp. S-2 was completed
Table 2
3,3,3-Trifluoro-2-hydroxy-2-methylpropanamide 2 hydrolysis activity from soil samples.
Number of strains
Soil sample
Isolated microorganisms
Degradation of 2 (>10%)
Stereoselectivitytoward 2 (>50% ee of1)
R
S
50
188
63
2
1
Strain no.
Recovery of 2 (%)
Yield of 1 (%)
Ee of 1 (%)
E value
6-1
13-1
23-2
44.8
29.9
49.1
41.6
33.5
47.8
95.4 (R)
51.2 (R)
89.8 (S)
83
4
68

118
K. Fuhshuku et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 115–119
Table 3
Effect of substrate concentration of 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide 2 on the hydrolysis activity of Shinella sp. R-6 and Arthrobacter sp. S-2.
Species
Sub. conc. (%)
Time (h)
Yield of 1 (%)
Ee of 1 (%)
0.20
0.50
1.0
2.0
5.0
24
24
24
24
24
24
4
4
8
24
24
24
24
54.7
47.0
33.0
18.8
7.3
90.6 (R)
93.3 (R)
94.6 (R)
91.1 (R)
84.0 (R)
79.6 (R)
90.9 (S)
95.0 (S)
96.4 (S)
96.0 (S)
96.7 (S)
91.8 (S)
80.1 (S)
Shinella sp. R-6
10
2.6
0.20
0.50
1.0
2.0
5.0
53.7
47.3
47.3
50.1
19.8
3.2
Arthrobacter sp. S-2
10
20
1.2
smoothly within 24 h using less than 2.0% of the substrate con-
centration. On the other hand, the reaction using Shinella sp.
R-6 only proceeded with a substrate concentration of less than
0.5%. Although the high enantioselectivity of the reaction using
Arthrobacter sp. S-2 was maintained up to a substrate concentra-
tion of 10%, that using Shinella sp. R-6 decreased to over 2.0%. These
results indicated that Arthrobacter sp. S-2 can be tolerant and show
higher enantioselectivity than Shinella sp. R-6 at high substrate
concentrations.
pH 4, and its yield and enantiomeric excess were 46% and 96.7%,
respectively. The recovered (R)-amide 2 was applied to the second
reaction using Shinella sp. R-6 at a substrate concentration of 0.50%,
converted to (R)-acid 1 after 34 h, and its yield and enantiomeric
excess achieved 45% for two steps and 99.5%, respectively.
4. Discussion
The screening and isolation of two microorganisms that can
enantioselectively hydrolyze amide 2 have been described in
this study. Although the intramolecular amide bond was gen-
erally resistant to hydrolysis and could only be cleaved under
harsh conditions, Shinella sp. R-6 and Arthrobacter sp. S-2, newly
isolated from soil samples, exhibited enantioselective hydroly-
sis activity toward amide 2 under mild conditions. Interestingly,
Shinella sp. R-6 and Arthrobacter sp. S-2 showed opposite enantio-
preferences from each other, and could be used in the preparation
of both enantiomers of acid 1. A previously reported amidase
from K. oxytoca PRS1 showed R-selective hydrolysis activity toward
amide 2 [5], and the amidase from Mycobacterium neoaurum ATCC
25795 also exhibited R-selectivity toward 2-amino-3,3,3-trifluoro-
2-methylpropanamide containing an amino group not a hydroxyl
group [11]. On the other hand, no microorganism or its enzymes
exhibiting S-selective hydrolysis activity toward amide 2 have been
reported to date. Arthrobacter sp. S-2 is the first example of a
microorganism that exhibits S-selective hydrolysis activity toward
amide 2.
The incorporation of fluoride can have marked effects on
the properties of fine chemicals and pharmaceuticals. Consid-
ering the divergent biological activities of both enantiomers of
trifluoromethyl-substituted compounds, the availability of these
compounds in their enantiomerically pure form is highly desirable.
Although many routes toward enantiomerically pure compounds
have been developed using chemical and enzymatic methods,
kinetic resolution is still useful for the preparation of both enan-
tiomers. Among them, the enzymatic enantioselective hydrolysis
3.3. Preparation of both enantiomers of
3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid (1)
To demonstrate the applicability of the biocatalytic enantiose-
lective hydrolysis of amide 2 to acid 1 catalyzed by Shinella sp. R-6
and Arthrobacter sp. S-2, we also performed the conversion on a
preparative scale using wet cells from an appropriate volume of
culture (Scheme 1). Encouraged by the complementary enantiose-
lectivity of the two strains Shinella sp. R-6 and Arthrobacter sp. S-2,
we applied them to the two-step reaction. The first reaction using
Arthrobacter sp. S-2, selected to increase the productivity, should
have produced (S)-acid 1 at a substrate concentration of 2.0%. The
recovered (R)-amide 2 should then have been applied to the reac-
tion using Shinella sp. R-6 at a substrate concentration of 0.50%, and
converted to (R)-acid 1 with increased enantiomeric excess under
mild conditions.
The reaction mixture using Arthrobacter sp. S-2 in 100 mL
consisted of 2.00 g of ( )-amide 2, 20 mM KPB (pH 7.0), and wet
cells from 200 mL of the culture. This was incubated at 30 ◦C with
shaking. Progress was monitored by HPLC analysis. The hydroly-
sis of ( )-amide 2 had proceeded by approximately 50% after 22 h.
After centrifugation, the supernatant was extracted with EtOAc. In
this step, only recovered (R)-amide 2 could be extracted, and the
(S)-acid 1 produced remained in the aqueous layer. (R)-Amide 2 and
(S)-acid 1 could be separated easily using only an extraction step.
(S)-Acid 1 could be extracted from the aqueous layer with Et2O at
Scheme 1. The preparation of both enantiomers of 3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid 1 via the two-step whole-cell reaction.

K. Fuhshuku et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 115–119
119
of amides has been highly established; however, only a few reports
Acknowledgment
have been investigated the preparation of enantiomerically pure
trifluoromethyl-substituted aliphatic acids [4,11]. In addition, it
has been reported that the enzymatic hydrolysis of ␣-hydroxy
amide, like lactamide, proceeds at a nearly equal rate toward both
enantiomers [12]. We also used a set of incubated microorganisms
involving microorganisms such as Pseudomonas sp. and Rhodococ-
cus sp., which have been used for the hydrolysis of many amides;
however, none exhibited high enantioselectivity toward amide
2, although some showed high hydrolysis activity, as shown in
Table 1. Furthermore, although the recovered enantiomer had to
be converted to an acid for the preparation of both enantiomers,
the reaction proceeded only under harsh conditions such as strong
acidic pH and high temperature. Under mild conditions, the two
newly isolated microorganisms, Shinella sp. R-6 and Arthrobacter
sp. S-2, could easily prepare both enantiomers of acid 1 as enan-
tiomerically pure forms.
This research was partly supported by a Grant-in-Aid for Scien-
tific Research (A) (23248015) from Japan Society for the Promotion
of Science (JSPS) to Y. Asano.
References
[1] J. Nie, H.-C. Guo, D. Cahard, J.-A. Ma, Chem. Rev. 111 (2011) 455–529.
[2] J.S. Parker, J.F. Bower, P.M. Murray, B. Patel, P. Talavera, Org. Process Res. Dev.
12 (2008) 1060–1077.
[3] K. Menzel, F. Machrouhi, M. Bodenstein, A. Alorati, C. Cowden, A.W. Gibson, B.
Bishop, N. Ikemoto, T.D. Nelson, M.H. Kress, D.E. Frantz, Org. Process Res. Dev.
13 (2009) 519–524.
[4] T. Sonke, B. Kaptein, in: K. Drauz, H. Gröger, O. May (Eds.), Enzyme Catalysis in
Organic Synthesis, 2, third ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim,
Germany, 2011, pp. 561–650.
[5] N.M. Shaw, A. Naughton, K. Robins, A. Tinschert, E. Schmid, M.-L. Hischier, V.
Venetz, J. Werlen, T. Zimmermann, W. Brieden, P. de Riedmatten, J.-P. Roduit,
B. Zimmermann, R. Neumueller, Org. Process Res. Dev. 6 (2002) 497–504.
[6] K. Konigsberger, K. Prasad, O. Repicˇ, Tetrahedron: Asymmetry 10 (1999)
679–687.
[7] R.A. Darrall, F. Smith, M. Stacey, J.C. Tatlow, J. Chem. Soc. (1951) 2329–2332.
[8] C.S. Chen, Y. Fujimoto, G. Girdaukas, C.J. Sih, J. Am. Chem. Soc. 104 (1982)
7294–7299.
[9] D.-S. An, W.-T. Im, H.-C. Yang, S.-T. Lee, Int. J. Syst. Evol. Microbiol. 56 (2006)
443–448.
[10] L. Kotouckova, P. Schumann, E. Durnova, C. Sproer, I. Sedlacek, J. Neca, Z. Zdrahal,
M. Nemec, Int. J. Syst. Evol. Microbiol. 54 (2004) 773–777.
[11] B. Koksch, P.J.L.M. Quaedflieg, T. Michel, K. Burger, Q.B. Broxterman, H.E. Schoe-
maker, Tetrahedron: Asymmetry 15 (2004) 1401–1407.
[12] L. Krieg, M.B. Ansorge-Schumacher, M.-R. Kula, Adv. Synth. Catal. 344 (2002)
965–973.
5. Conclusions
This study has described the screening and isolation of microor-
ganisms that enantioselectively hydrolyze amide 2. The two newly
isolated microorganisms, Shinella sp. R-6 and Arthrobacter sp. S-
2 exhibited R- and S-selective hydrolysis activity toward amide
2, respectively. The preparation of both enantiomers of acid 1 via
the two-step whole-cell reaction was investigated using these two
strains.