Asymmetric reduction of 2-chloroacrylic acid to (S)-2-chloropropionic acid by a novel reductase from Burkholderia sp. WS

Article abstract of DOI:10.1016/j.tetasy.2004.06.035

Burkholderia sp. WS has a novel enzyme that catalyzes the asymmetric reduction of 2-chloroacrylic acid to yield (S)-2-chloropropionic acid, which is used as a building block for the synthesis of aryloxyphenoxypropionic acid herbicides. NADPH is required as a co-substrate for this reaction.

Full text of DOI:10.1016/j.tetasy.2004.06.035

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2837–2839
Asymmetric reduction of 2-chloroacrylic acid to
(S)-2-chloropropionic acid by a novel reductase from
Burkholderia sp. WS
Atsushi Kurata,a Tatsuo Kurihara,a Harumi Kamachib and Nobuyoshi Esakia,*
aInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
bCorporate R&D Center, Showa Denko KK, Chiba 267-0056, Japan
Received 1 June 2004;accepted 18 June 2004
Available online 3 August 2004
Abstract—Burkholderia sp. WS has a novel enzyme that catalyzes the asymmetric reduction of 2-chloroacrylic acid to yield (S)-2-
chloropropionic acid, which is used as a building block for the synthesis of aryloxyphenoxypropionic acid herbicides. NADPH is
required as a co-substrate for this reaction.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Biologically active compounds, including pharmaceuti-
cal and agrochemical products, often contain stereogenic
centers. In most cases, one of the enantiomers has the
desired biological activity, whereas the other does not
or is even hazardous. Thus, there exists a continuing
demand for the development of technology to produce
optically active compounds efficiently.
We have previously isolated bacterial strains that use 2-
CAA as their sole carbon and energy source.2 One of
these strains, formerly called Pseudomonas sp. WS,
was used herein. We re-characterized this strain by 16S
rRNA sequencing and found that the sequence had
the highest similarity to those of bacteria belonging to
the genus Burkholderia. Thus, we have renamed this
strain Burkholderia sp. WS.
(S)-2-Chloropropionic acid (CPA) is used as a building
block for the synthesis of aryloxyphenoxypropionic
acid, the (R)-isomer of which has herbicidal activity.
In the industrial synthesis of this herbicide, (S)-2-CPA
is produced by resolution, in which the (R)-2-CPA com-
ponent of a racemic 2-CPA mixture is selectively de-
graded using (R)-2-haloacid dehalogenase.1 Since the
theoretical maximum yield of (S)-2-CPA from a racemic
mixture of 2-CPA by this procedure is only 50%, it
would be advantageous to establish an asymmetric syn-
thesis procedure that could produce (S)-2-CPA directly.
Herein, we have developed an enzymatic process in
which (S)-2-CPA is produced by asymmetric reduction
of the carbon–carbon double bond in 2-chloroacrylic
acid (2-CAA).
Burkholderia sp. WS exhibits 2-CPA dehalogenase activ-
ity when grown in media containing 2-CPA as the sole
carbon source.2 We also found that this bacterium has
a gene homologous to the (S)-2-haloacid dehalogenase
[(S)-DEX] gene (data not shown). The deduced amino
acid sequence of (S)-DEX of Burkholderia sp. WS [(S)-
DEX WS] is highly similar to that of Pseudomonas sp.
YL (76% identity),3 which catalyzes the hydrolysis of
(S)-2-CPA with inversion of the C2 configuration to pro-
duce (R)-lactate.4–6 Although 2-CPA dehalogenase
activity was too low to be detected in a crude extract
of cells grown on 2-CAA,2 the above-mentioned obser-
vations raised the possibility that 2-CAA is first con-
verted into (S)-2-CPA, and that (S)-2-CPA is
subsequently hydrolyzed to produce (R)-lactate by (S)-
DEX WS (Scheme 1).
In order to verify this scheme, we first examined
whether intact Burkholderia sp. WS cells can metabolize
2-CAA to release chloride ions. Cells were grown in the
*
Corresponding author. Tel.: +81-774-38-3240;fax: +81-774-38-
3248;e-mail: esaki@scl.kyoto-u.ac.jp
0957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.06.035

2838
A. Kurata et al. / Tetrahedron: Asymmetry 15 (2004) 2837–2839
following medium: 0.2% 2-CAA, 0.2% (NH4)2SO4, 0.05%
yeast extract (DIFCO), 0.1% K2HPO4, 0.2% NaH2PO4,
and 0.01% MgSO4Æ7H2O, pH7.1. The reaction was car-
ried out at 30°C in a mixture consisting of 60mM potas-
sium phosphate buffer (pH7.1), 0.2% 2-CAA, and 1%
(w/v) cells. We monitored the reaction by measuring
the concentrations of 2-CAA and the chloride ions. 2-
CAA was analyzed by high-performance liquid chroma-
tography (HPLC) using an Asahipak ODP-50 column
(4.6mm by 25m, Showa Denko) eluted using 20% aceto-
nitrile containing 0.1% trifluoroacetic acid at a flow
rate of 0.5mL/min and temperature of 25°C. Eluted
products were detected at 246nm. Chloride ions were
quantified spectrophotometrically using mercuric
thiocyanate and ferric ammonium sulfate.7 As shown
in Figure 1, the concentration of chloride ions increased
as the 2-CAA concentration decreased. This result is
consistent with Scheme 1, although it does not exclude
the possibility that the chloride ion was released directly
from 2-CAA.
20
15
10
5
20
15
10
5
0
0
0
5
10 15 20 25
Time (h)
Figure 1. Degradation of 2-CAA and production of chloride ions
using Burkholderia sp. WS cells. The concentrations of 2-CAA (d) and
chloride ion (j) in the reaction mixture were monitored.
metry was conducted in the EI+ ionization mode with a
mass range of 29–130 m/z. The culture medium was
shown to contain a metabolite eluting at a retention time
of 12.6min (Fig. 2c), which is identical to that of authen-
tic 2-CPA (Fig. 2a). The mass spectrum of the metabolite
shows peaks at m/z values of 35 and 45, which were as-
signed to the chloride atom and the carboxyl group,
respectively (Fig. 2d). The fragment ion at an m/z of
64 was probably produced by the release of COO
(m/z=44) from [35Cl]-2-CPA (m/z=108). All of these
peaks were found in the spectrum of authentic 2-CPA
(Fig. 2b). Thus, although the unstable molecular ion of
[35Cl]-2-CPA (m/z=108) was not detected in the mass
spectrum (Fig. 2d), the metabolite in the culture medium
could be identified as 2-CPA.
Cl
H
Cl
(S)-DEX WS
2-CAA reductase
OH
H
HOOC
COOH
COOH
Scheme 1. Putative pathway of 2-CAA metabolism in Burkholderia sp.
WS.
In order to see whether 2-CPA was produced as a me-
tabolite of 2-CAA, the culture medium was analyzed
by gas chromatography–mass spectrometry (GC–MS)
(Fig. 2). The culture supernatant (1mL) was dried and
the residue was solubilized in 0.2mL of methanol. The
sample was injected into a GC–MS system equipped with
a capillary column (DB-WAX, 0.32mm by 30m, J & W).
The temperature was kept at 50°C for 1min and then
raised to 180°C at a rate of 15°C/min. Mass spectro-
Next, GC using a chiral column was used to determine
the configuration of the 2-CPA, that was generated.
First, the 2-CPA produced by the Burkholderia sp. WS
cells was treated with a boron trifluoride methanol solu-
Figure 2. GC–MS analysis of the metabolite of 2-CAA. (a) Chromatogram of authentic 2-CPA. (b) Mass spectrum of authentic 2-CPA. (c)
Chromatogram of the metabolite. (d) Mass spectrum of the compound indicated by the arrow in (c).

A. Kurata et al. / Tetrahedron: Asymmetry 15 (2004) 2837–2839
2-CAA
2839
tion of 2-CAA only in the presence of NADPH (Fig.
3). Over the course of the reaction, the peak correspond-
ing to [35Cl]-2-CAA at m/z=105 decreased, while the
peaks corresponding to [37Cl]-2-CPA (m/z=109) and
lactate (m/z=89) increased in size. These trends support
the metabolic pathway shown in Scheme 1. In this
experiment, an increase in size of the peak at m/z=87
was also observed, suggesting that lactate is further con-
verted into pyruvate.
0 min
100
50
0
86
90
95
100
105
110
20 min
100
pyruvate
lactate
2-CAA 2-CPA
3. Conclusion
50
In summary, we found that 2-CAA-grown Burkholderia
sp. WS produces a novel enzyme that catalyzes the
asymmetric reduction of 2-CAA to (S)-2-CPA. This en-
zyme required NADPH as a co-substrate. Since (S)-2-
CPA is probably further metabolized by (S)-DEX, dele-
tion of the (S)-DEX gene would be essential in order to
accumulate (S)-2-CPA in Burkholderia sp. WS. Cloning
of the gene encoding this novel enzyme, as well as the
overproduction and further characterization of the gene
product, are currently in progress. It may be possible to
produce a large amount of (S)-2-CPA by coupling this
2-CAA reducing enzyme with a system for regenerating
NADPH.
0
86
90
95
100
105
110
80 min
100
pyruvate
2-CAA 2-CPA
lactate
50
0
86
90
95
100
105
110
m/z
Figure 3. Mass spectrometric monitoring of the degradation of 2-CAA
using a cell-free extract in the presence of NADPH. Spectra were
obtained for the range between 86 and 110 atomic mass units. [35Cl]-2-
CAA, [37Cl]-(S)-2-CPA, lactate, and pyruvate were detected at m/z
values of 105, 109, 89, and 87, respectively.
Acknowledgements
This work was supported in part by a Grant-in-Aid for
Scientific Research on Priority Areas 13125203 (to
tion to convert it into a methyl ester, extracted with
ethyl acetate, and concentrated. GC analysis of the 2-
CPA methyl ester was performed on a GC-14A chroma-
tograph (Shimadzu) equipped with a flame ionization
detector and a chiral chromatographic column (CP-
Chirasil-DEX CB, 0.32mm by 25m, Varian). The col-
umn temperature was kept at 70°C. The 2-CPA methyl
ester sample derived from the product of the Burkholde-
ria sp. WS cells exhibited a single peak and co-eluted
with authentic (S)-2-CPA methyl ester, and not with
authentic (R)-2-CPA methyl ester (data not shown).
These results demonstrate that Burkholderia sp. WS cells
produce the (S)-enantiomer of 2-CPA, using an enzyme
that catalyzes the asymmetric reduction of 2-CAA to
(S)-2-CPA.
N.E.),
a Grant-in-Aid for Young Scientists (A)
14703021 (to T.K.) from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan, the
National Project on Protein Structural and Functional
Analyses, and Grant-in-Aid from the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan
(21st Century COE on Kyoto University Alliance for
Chemistry).
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