Microbial deracemization of α-substituted carboxylic acids: Control of the reaction path

Article abstract of DOI:10.1016/j.tetasy.2004.06.049

A novel approach to preparing optically active α-substituted carboxylic acids using the whole cells of Nocardia diaphanozonaria JCM 3208 is described. When 2-phenylthiopropanoic acid and 2-methyl-3-phenylpropanoic acid were subjected to the reaction under aerobic conditions, the oxidation reaction proceeded preferentially rather than deracemization of these substrates. Herein, we report the design of reaction conditions to increase the deracemization activity in preference to oxidation reactions. In addition, we have successfully detected a metabolic intermediate in the reaction mixture of 2-methyl-3-phenylpropanoic acid, which indicates that the deracemization is a competitive reaction against the α-oxidation pathway of fatty acid metabolism.

Full text of DOI:10.1016/j.tetasy.2004.06.049

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2965–2973
Microbial deracemization of a-substituted carboxylic acids:
control of the reaction path
Dai-ichiro Kato, Kenji Miyamoto and Hiromichi Ohta*
Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
Received 23 April 2004; accepted 28June 2004
Available online 12 September 2004
Abstract—A novel approach to preparing optically active a-substituted carboxylic acids using the whole cells of Nocardia diaphano-
zonaria JCM 3208is described. When 2-phenylthiopropanoic acid and 2-methyl-3-phenylpropanoic acid were subjected to the reac-
tion under aerobic conditions, the oxidation reaction proceeded preferentially rather than deracemization of these substrates.
Herein, we report the design of reaction conditions to increase the deracemization activity in preference to oxidation reactions.
In addition, we have successfully detected a metabolic intermediate in the reaction mixture of 2-methyl-3-phenylpropanoic acid,
which indicates that the deracemization is a competitive reaction against the b-oxidation pathway of fatty acid metabolism.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
CH3
CO2H
CH3
CO2H
inversion
R
R
R
R
What is the best way for the preparation of optically
active compounds starting from racemates? Although
many scientists have made the effort to solve this prob-
lem, we still do not have a perfect answer at present.
Recently, we and another group have introduced a
new approach to preparing optically active a-substituted
propanoic acids using a type of actinomycete.1,2 This
method is known as a deracemization reaction, which
is a third option for obtaining optically active com-
pounds after the following two techniques, that is,
kinetic resolution and dynamic kinetic resolution.3
Deracemization is a reaction, which inverts the configu-
ration of either enantiomer of a racemate to the other
antipode, resulting in an optically active compound
starting from a racemic mixture (Scheme 1). Theoreti-
cally, this method is capable of giving the desired enantio-
mer in 100% yield and it means that the synthesis of
racemates is almost equal to the synthesis of optically
active compounds. This process can be realized by
ꢀenantioselective racemizationꢁ, because repetition of
racemization of one enantiomer finally results in the
accumulation of the other enantiomer.
Enantioselective Racemization
CH3
CO2H
CH3
CO2H
retention
racemate
enantiomeric excess : 100%
chemical yield : 100%
Scheme 1.
cells of Nocardia. diaphanozonaria JCM 3208.1 The en-
zyme system of N. diaphanozonaria catalyzes the inver-
sion of the configuration of 2-phenylpropanoic acid 1
from (S)- to (R)-configuration (Scheme 2). In addition,
the deracemization reaction also proceeds with 2-(4-
chlorophenoxy)propanoic acid 2 to give the (R)-enantio-
mer in high enantiomeric excess. Surprisingly enough,
the spacial arrangement of the ligands around the stereo-
genic center was opposite to that of 1. Although the
Cl
CH3
R=p-ClPhO
CH3
CO2H
R=Ph
CH3
CO2H
We have already reported the deracemization reactions
of 2-aryl- and 2-aryloxypropanoic acids using growing
R
CO2H
O
1
racemate
2
81%, 69% ee
95%, 97% ee
*
Corresponding author. Tel.: +81 45 566 1703; fax: +81 45 566
1551; e-mail: hohta@bio.keio.ac.jp
Scheme 2.
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.06.049

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D. Kato et al. / Tetrahedron: Asymmetry 15 (2004) 2965–2973
CH3
CO2H
resting cells
48 h
CH3
COSCoA
CH3
CO2H
LACS
S
hydrolase
iBu
iBu
(R)-Ibuprofen
( )-3
(R)-Ibuprofenyl-CoA
2-Arylpropionyl-CoA
epimerase
CH3
CO2H
CH3
CO2H
S
S
O
CH3
CO2H
CH3
(R)-3
49%, 94% ee
4
COSCoA
47%, syn/anti = 1/1
hydrolase
iBu
iBu
(S)-Ibuprofenyl-CoA
(S)-Ibuprofen
Scheme 4.
Scheme 3.
able for 2-aryl- and 2-aryloxypropanoic acid, degrada-
tion reactions proceeded preferentially in the case of
other acids, which have sulfur, methylene, or amine at
b-position.1 In the case of 2-phenylthiopropanoic acid
3, enantiomer-differentiating oxidation occurred on the
sulfur atom (Scheme 4). While the (R)-enantiomer was
recovered intact in good enantiomeric excess, the (S)-
enantiomer was converted to 2-phenylsulfinylpropanoic
acid 4, which consisted of equal amounts of syn- and
anti-isomers. To determine whether the oxidation reac-
tion on the sulfur atom was enantioselective or not,
the resulting 2-phenylsulfinylpropanoic acid 4 was con-
verted to methyl 2-methyl-2-phenylsulfinylpropanoate
5 and analyzed by chiral phase HPLC (Scheme 5). It be-
came clear that the sulfoxide was racemic thus indicating
that the oxygenase did not differentiate the prochirality
of the sulfide. This is surprising when compared to the
fact that the reaction of 2-phenylthioacetic acid 6 pro-
ceeded in an enantioselective manner and (S)-7 was
obtained in 73% ee (Scheme 6). Although the yield
was low, the rest of the recovered material is the starting
compound, and the material balance is nearly
quantitative.
reason is not clear at present, it is very interesting to
note that the insertion of only one atom brought about
a dramatic change in enantioselectivity.
The deracemization reaction of 2-arylpropanoic acid has
also been carried out using rat liver with the mechanistic
studies already performed.4 It was proposed that three
enzymes related in this case and the (R)-2-arylpropanoic
acid inverted to the (S)-form (Scheme 3). The initial step
of this reaction is considered to involve the enantioselec-
tive formation of the coenzyme A (CoA) thioester of the
(R)-acid with the aid of long-chain acyl-CoA synthetase
(LACS). The thioester is subsequently epimerized and
cleaved by a hydrolase to release the racemic acid. In
these three steps, only the LACS-catalyzed reaction pro-
ceeded in an enantioselective manner. Thus, the enantio-
meric ratio of the acid shifted to the (S)-form over the
course of the reaction. These enzymes as well as the
corresponding genes were purified and identified.
We supposed that deracemization using N. diaphanozo-
naria was likely to proceed via essentially the same reac-
tion mechanism as that of rat liver. Based on the
deuterium exchange experiments using 2-deuterio-2-
phenylpropanoic acid, we have proposed that enolate-
type intermediate was formed during the deracemization
reaction.1 On the other hand, inhibitory effect experi-
ments using the typical substrate of acyl-CoA synthe-
tase, that is, benzoic acid and n-alkanoic acids with
various chain length suggested that medium-chain
acyl-CoA synthetase (MACS) took part in this chiral
inversion process. This result is in marked contrast with
the case of rat liver. Herein we report the improvement
in reaction conditions to suppress the metabolic reac-
tions and to lead the reaction to the direction of the
deracemization using the resting cells of N. diaphanozo-
naria. In addition, we will show some supporting evi-
dences that the deracemization process is a competitive
reaction against the fatty acid metabolism and proceeds
via a part of b-oxidation pathway.
In the case of 2-methyl-3-phenylpropanoic acid 8, a
complete degradation reaction proceeded without enan-
tioselectivity to give benzoic acid 9 (Scheme 7). The IR
and NMR spectra, and melting point agreed with those
1) CH3I, NaHCO3
/DMF
CH3
CO2H
H3C CH3
S
S
CO2Me
2) CH3I, K2CO3
/DMF
O
O
4
5
77%, 2 steps
Scheme 5.
resting cells
24 h
S
CO2H
S
CO2H
O
2. Results and discussion
(S)-7
6
17%, 73% ee
Although the previously reported reaction conditions
using the growing cells of N. diaphanozonaria are suit-
Scheme 6.

D. Kato et al. / Tetrahedron: Asymmetry 15 (2004) 2965–2973
2967
Table 1. Deracemization reaction of 2 under the inert gas conditionsa
resting cells
48 h
CH3
CO2H
Cl
Cl
CO2H
CH3
CO2H
under Ar
CH3
CO2H
9
( )-8
resting cells
O
O
78%
(R)-2
( )-2
Scheme 7.
Entry
Reaction time (h)
24
Yield (%)b
91
Ee (%)c
65
1
2
3
of an authentic sample. 2-Methyl-3-(2-thienyl)propanoic
acid 10 was oxidized in an enantioselective manner to
give a,b-unsaturated carboxylic acid 11 and the (S)-form
enriched starting material (Scheme 8). Any other meta-
bolic product, such as thiophenecarboxylic acid, could
not be detected in the reaction mixture. 2-Aminophenyl-
propanoic acid 12 was converted to aniline 13, also in an
enantioselective manner, leaving behind the (R)-isomer
in high ee (Scheme 9). These four substrates can be clas-
sified into two groups by the type of oxidation. The first
group consists of 2-phenylthiopropanoic acid 3, which is
oxidized by oxygenase. The second group includes 8, 10,
and 12, which are thought to be oxidized by dehydro-
genase in the first step.
48
96
6
8
79
87
>99
a The starting compounds were incubated with the resting cells of
N. diaphanozonaria under Ar at 30ꢁC.
b Isolated yield after conversion to the corresponding methyl ester.
c Ee of the product was determined by HPLC analysis after conversion
to the corresponding methyl ester.
Table 2. Suppression of the oxidation reaction on the sulfur atoma
X
X
CH3
CH3
under Ar
S
CO2H
S
CO2H
resting cells
( )-3 (X=H), 14 (X=Cl)
(R)-3 or 14
At first, we tried to suppress the oxidation on the sulfur
atom, because the oxidation was thought to be catalyzed
by monooxygenase. This side reaction was suppressed
by performing it under an inert gas. We also tested to
see if the deracemization reaction itself proceeded under
oxygen-free conditions using 2-(4-chlorophenoxy)prop-
anoic acid 2, which is one of the best substrates for this
biocatalyst. When 2 was incubated with the resting cells
of this bacterium under Ar, it took 96h to complete the
deracemization reaction (Table 1, entry 3), while only
24h were needed to complete the reaction under the aero-
bic conditions. Thus, deracemization was confirmed to
proceed in the absence of oxygen although the reaction
Entry
X
Reaction time (h)
Yield (%)b
6828
Ee (%)c
1
2
3
4
5
6
H
H
H
H
Cl
Cl
Cl
24
4879
42
21
96
240
8
688
9
92
31
48 3
96
8
77
574
8
1688
8Cl
9
240
Cl
8
90 8
336
77
91
a The starting compounds were incubated with the resting cells of
N. diaphanozonaria under Ar at 30ꢁC.
b Isolated yield after conversion to the corresponding methyl ester.
c Ee of the product was determined by HPLC analysis after conversion
to the corresponding methyl ester.
CH3
CO2H
resting cells
48 h
rate was greatly reduced. Also in the case of 2-phenyl-
thiopropanoic acid 3 and 2-(4-chlorophenylthio)propa-
noic acid 14, the enzyme system of N. diaphanozonaria
showed the deracemization activity under Ar as
expected (Table 2). The chiral inversion proceeded
smoothly without the formation of other metabolic
products. After 96-h incubation, the yield and the ee
of 3 were 68% and 88% (R), respectively (Table 2, entry
3). In the case of 14, the product was recovered in high
enantiomeric excess (90% ee) after a 240-h incubation
(Table 2, entry 8).
S
( )-10
CH3
CH3
CO2H
CO2H
S
S
(S)-10
42%, 93% ee
11
38%
Scheme 8.
CH3
CO2H
resting cells
24 h
We then proceeded to the next stage to suppress the met-
abolic process of the second group. The key for directing
the reaction toward deracemization was to take into
consideration the possibility that the reaction proceeded
via a common intermediate, an a,b-unsaturated carb-
oxylic acid (Fig. 1). If we supposed that the enzyme
N
H
( )-12
CH3
CO2H
N
H
NH2
CH3
Ar
(R)-12
13
46%
X
CO2H
20%, >99% ee
Scheme 9.
Figure 1. The common metabolic intermediate for 8, 10, and 12.

2968
D. Kato et al. / Tetrahedron: Asymmetry 15 (2004) 2965–2973
hydrolysis
activation
CH3
CO2H
epimerization
CH3
X CO2H
CH3
COSCoA
CH3
COSCoA
Ar
Ar
Ar
Ar
X
X
X
deracemization
ACDH oxidation
CH3
COSCoA
Ar
β
-oxidation pathway
X
Scheme 10.
Table 3. Reaction of 8 under Ara
system of N. diaphanozonaria acted via the same reaction
mechanism as in the case of rat liver, the first step of the
deracemization reaction would be the activation of the
carboxylic acid by the formation of a thioester linkage
with CoA (Scheme 3). The acyl-CoA would be dehydro-
genated by acyl-CoA dehydrogenase (ACDH) and fur-
ther metabolized via b-oxidation pathway. It is not
surprising that this undesired reaction took precedence
over the deracemization reaction under aerobic condi-
tions. In other words, the deracemization process may
be a competitive reaction against the fatty acid meta-
bolism (Scheme 10).
CH3
under Ar
CO2H
resting cells
( )-8
CH3
CO2H
(S)-8
CH3
CO2H
15
Entry
Reaction
time (h)
(S)-8
15
Yield (%)b
Ee (%)c
Yield (%)d
Thus, we focused our attention on an ACDH-catalyzed
oxidation as it would be the initial step towards the b-
oxidation pathway. If this oxidation step can be inhib-
ited by some method, the following downstream will
be directed to deracemization process. ACDH is a flavo-
enzyme and utilizes molecular oxygen for the coenzyme
regeneration. Accordingly, the blocking of this coen-
zyme regeneration system was considered to be one of
the effective options.
1
2
3
4860
96
33
20
91
53
42
40
49
240
>99
a The starting compounds were added to the resting cells of N.
diaphanozonaria under Ar at 30ꢁC.
b Isolated yield after conversion to the corresponding methyl ester and
selective removal of a-cinnamic acid under the condition of double
bond cleavage oxidation.
c Ee of the product was determined by HPLC analysis after conversion
to the corresponding methyl ester.
d Yields are calculated in comparison with the value of integration in
1H NMR spectrum of the mixture of 8 and 15.
Thus, as the first trial, the reaction of 2-methyl-3-phen-
ylpopanoic acid 8 with the resting cells of N. diaphanozo-
naria was carried out under Ar (Table 3). The ee of the
starting material gradually increased and finally enantio-
merically pure (S)-8 and a-methylcinnamic acid 15 were
recovered in the ratio of 50:50 (Table 3, entry 3). How-
ever, these two compounds could not be separated by
column chromatography or chiral phase HPLC. Thus,
the ee of the product was determined after selective re-
moval of 15 as a benzaldehyde via the oxidative cleavage
of the double bond with the aid of osmium tetroxide–
sodium metaperiodite system. As 15 is considered as
the metabolic intermediate of the b-oxidation pathway,
formation of this compound indicates that the deracemi-
zation process is a competitive reaction against b-oxida-
tion pathway.
itor under aerobic conditions. 2-Methyl-3-phenyl-
propanoic acid 8 was recovered in its optically active
form with a minimum amount of by-product (Table
4). The best result was obtained under the conditions
in entry 1. In the presence of 1.2mM of 2-bromohexa-
noic acid, 8 was actually deracemized and recovered in
its (S)-form in 88% ee after 48h incubation. As this
result could not be realized via simple enantioselective
degradation, it clearly shows the presence of chiral
inversion process. In this case, only 15 was formed as
by-product while 9 could not be detected in the reaction
mixture. It is interesting that the deracemization activity
was affected dramatically by the chain length of the
inhibitors and their concentration (entry 1 vs 4, 6 vs 8).
Thus to inhibit the b-oxidation path more efficiently, the
reaction of 8 was carried out in the presence of an
ACDH inhibitor, such as 2-bromohexanoic acid and
2-bromooctanoic acid.5 However, when compound 8
was incubated with the resting cells under Ar in the pres-
ence of 3.0mM of these inhibitors, deracemization activ-
ity was lost completely, and only the racemic starting
material was recovered. After several trials, we found
that the deracemization reaction proceeded preferen-
tially in the presence of an appropriate amount of inhib-
In the case of 2-methyl-3-(2-thienyl)propanoic acid 10,
however, 2-bromohexanoic acid could not suppress the
unsaturation reaction by ACDH (Table 5). a,b-Unsatu-
rated product 11 was formed regardless of the presence
or absence of 2-bromohexanoic acid (2-BH) while the
concentration of the inhibitor had no effect. In the case
of 2-aminophenylpropanoic acid 12, the deracemization
reaction did not become the major path even under an

D. Kato et al. / Tetrahedron: Asymmetry 15 (2004) 2965–2973
2969
Table 4. Suppression of the dehydrogenase reaction of 8 ACDH
inhibitora
Table 6. Metabolic reaction of 12 under the various conditions
Entry
Condition
Reaction
time (h)
(S)-12
13
Entry Inhibitorb Concn
(mM)
(S)-8
15
Yield
(%)a
Ee
(%)b
Yield
(%)c
Yield (%)c Ee (%)d Yield (%)e
1
2-BH
2-BH
2-BH
2-BH
2-BO
2-BO
2-BO
1.2
3.0
71
74
88
82
19
34
35
42
26
22
1
2
3
4
Aerobicd
Are
24
20
>99
46
2
4837
4869
4842
65
ND
3
6.1
7865
77
2-BHf
2-BOg
0
19
0
ND
4
12.2
0.63
1.2
4
12
12
5
73
75
a Isolated yield after conversion to the corresponding methyl ester.
b Ee of the product was determined by HPLC analysis after conversion
to the corresponding methyl ester.
6
7
82-BO
3.0
71
71
83
4.1
3
1
c Isolated yield after conversion to acetanilide.
a The starting compounds were added to the resting cells of N.
diaphanozonaria after the incubation with appropriate amount of
ACDH inhibitor for 10min at 30ꢁC.
d The starting compounds were incubated with resting cells of N.
diaphanozonaria under aerobic conditions at 30ꢁC.
e The starting compounds were incubated with resting cells of N.
diaphanozonaria under Ar at 30ꢁC.
b 2-BH: 2-bromohexanoic acid, 2-BO: 2-bromooctanoic acid.
c Isolated yield after conversion to the corresponding methyl ester and
selective removal of a-cinnamic acid under the condition of double
bond cleavage oxidation.
f The starting compounds were added to the resting cells of N. diapha-
nozonaria after the incubation with 1.2mM 2-bromohexanoic acid
for 10min at 30ꢁC.
d Ee of the product was determined by HPLC analysis after conversion
to the corresponding methyl ester.
e Yields are calculated in comparison with the value of integration in
1H NMR spectrum of the mixture of 8 and 15.
g The starting compounds were added to the resting cells of N.
diaphanozonaria after the incubation with 1.2mM 2-bromooctanoic
acid for 10min at 30ꢁC.
diate, an a,b-unsaturated thioester. Second, ACDH will
catalyze the metabolic reaction after the chiral inversion
step because of the following reason: it has already
been reported that ACDH recognizes the chirality of a-
methylacyl-CoA,6 and works on the (S)-form. As
the (R)-enantiomer of 8 is degraded or converted to
the (S)-enantiomer, it is reasonable to suppose that the
oxidative metabolism occurs after the racemization step,
which is considered to be also playing an important role
in the deracemization reaction.
Table 5. Effect of ACDH inhibitor on the reaction of 10a
Entry Concn of 2-BH Reaction time
(mM)
(S)-10
11
(h)
Yield Ee Yield
(%)b (%)c (%)b
1
0
3
6
ND
70
74
73
38
75
66
ND ND
2
0
51
93
93
10
24
25
3
0
12
24
4842
3
4
0
5
0
93
6
1.2
1.2
185
30
3. Conclusion
7
6
7
81.2
9
12
ND
ND ND
70 93
We have succeeded in establishing the reaction condi-
tions necessary to deracemize 2-phenylthiopropanoic
acid 3 and 2-methyl-3-phenylpropanoic acid 8, and thus
expand the applicability of the biotransformation sys-
tem of N. diaphanozonaria. While, the degradation reac-
tions proceeded preferentially under aerobic conditions
for these substrates, when 3 and its derivative 14 were
subjected to the resting cells of N. diaphanozonaria
under Ar, the deracemization reaction became the major
pathway. In the case of 8, the enzyme system of the
microorganism showed the deracemization activity in
the presence of an ACDH inhibitor. In addition, a key
intermediate of the fatty acid metabolism, a-methylcin-
namic acid 15, was also detected in the reaction mixture,
which suggests that the deracemization process is a com-
petitive reaction against the b-oxidation pathway. Fur-
ther investigation on the reaction path and enzyme
purification is now underway.
1.2
1.2
3.0
6.0
24
93 484384
4852
27
10
11
12
93
54
36
16
4879
a The starting compounds were added to the resting cells of N.
diaphanozonaria after the incubation with 2-bromohexanoic acid (2-
BH) for 10min at 30ꢁC and incubated under aerobic conditions at
30ꢁC.
b Yields are calculated in comparison with the value of integration in
1H NMR spectrum.
c Ee of the product was determined by HPLC analysis after conversion
to the corresponding methyl ester.
atmosphere of an inert gas or in the presence of ACDH
inhibitor (Table 6). When 2-BH was present, however,
aniline 13 could not be detected in the reaction mixture
although the ee of recovered 12 was 0%. This result indi-
cates that under aerobic conditions, the metabolic reac-
tion of 12 catalyzed by ACDH would give an imine
intermediate, followed by hydrolysis to produce 13.
4. Experimental
4.1. General
Although, we could not find the reaction conditions to
make the deracemization reaction major path for 10
and 12, the investigation gave us some precious hints
for the elucidation of reaction mechanism. First, the
metabolic reaction will proceed via a common interme-
Starting materials and reagents were purchased and
used without purification unless otherwise noted. Ana-
lytical thin layer chromatography (TLC) was developed

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D. Kato et al. / Tetrahedron: Asymmetry 15 (2004) 2965–2973
on E. Merck Silica Gel 60 F256 plates (0.25mm thick-
ness). Column chromatography was performed on Kat-
ayama Chemical Co., Inc. Silica Gel 60 K070-WH (70–
230 mesh). NMR spectra were obtained on JEOL AL-
300, GX-400 spectrometers (1H at 300, 400MHz and
(8.3g, 0.10mol), and ethyl 2-bromopropanoate (18g,
0.10mol) in EtOH (3mL) was heated under reflux for
25h. After being cooled to room temperature, the mix-
ture was diluted with water (40mL) and extracted with
ether. The organic layer was washed with brine, dried
over anhydrous sodium sulfate, and concentrated in
vacuo. The oily residue was then heated under reflux
in 10% aqueous NaOH (70mL) for 2h. The mixture
was then cooled to room temperature and washed with
ether. The aqueous layer was cooled in an ice-water bath
and the solution acidified to pH2 with concentrated
HCl. The resulting slurry suspension was allowed to
stand in an ice-water bath for 12h, filtered, washed
sequentially with water (200mL) and hexane (200mL),
and dried under high vacuum. This crude precipitate
was recrystallized from chloroform/acetone to give 2-
phenylaminopropanoic acid8 (11.8g, 71% yield) as a col-
orless crystal: IR (KBr disc) 2979, 2775, 1572, 1495,
1
13C at 100MHz) at ambient temperature. H chemical
shifts were referenced with CDCl3 at 7.26ppm and 13C
chemical shifts with CHCl3 at 77.0ppm or acetone-d6
at 29.8ppm. IR spectra were measured as films for oils
and as KBr discs for solids with a JASCO FT/IR-410
instrument. Optical rotations were measured on a JAS-
CO DIP 360 polarimeter. The strain, N. diaphanozonaria
JCM3208, used in this experiment is available from the
Japan Collection of Microorganism: The Institute of
Physical and Chemical Research (Riken), 2-1 Hirosawa,
Wako 351-0106, Japan.
4.2. Synthesis of substrates
1394, 1358, 1090, 958, 850, 756, 696, 553cmꢀ1 1H
;
2-(4-Chlorophenoxy)propanoic acid 2, 2-phenylthio-
propanoic acid 3, 2-methyl-3-phenylpropanoic acid 8,
and 2-(4-chlorophenylthio)propanoic acid 14 were syn-
thesized according to the same procedure described.1
NMR (CDCl3): d 7.09 (m, 2H), 6.61 (m, 3H), 4.09 (q,
J = 6.8Hz, 1H), 1.45 (d, J = 6.8Hz, 3H); 13C NMR (ace-
tone): d 175.7 (CO), 148.4 (C), 129.6 (CH · 2), 117.8
(CH), 113.6 (CH · 2), 52.2 (CH), 18.8 (CH3).
4.2.1. 2-Methyl-3-(2-thienyl)propanoic acid 10. Sodium
ethoxide (1.10g, 16.2mmol) was dissolved in EtOH
(6mL) at 0ꢁC. After stirring for 5min, the mixture
was allowed to warm to room temperature. A solution
of diethyl methylmalonate (2.00g, 11.5mmol) in EtOH
(2mL) was added dropwise over a period of 5min and
the mixture stirred for 30min at the same temperature.
Then, an ethanol solution (2mL) of 2-chloromethylthio-
phene (3.30g, 24.8mmol), which was freshly prepared
from 2-thiophenemethanol and thionyl chloride, was
added. The mixture was heated under reflux for 15h.
The reaction mixture was cooled in an ice bath, acidified
with 2M hydrochloric acid and extracted with EtOAc.
The organic layer was concentrated in vacuo. To a solu-
tion of KOH (2.6g, 9.5mmol) in water (15mL), was
added this oily residue dissolved in EtOH (15mL). The
mixture was stirred at room temperature for 11h. The
reaction mixture was cooled in an ice-water bath, acidi-
fied with 2M hydrochloric acid and extracted with
EtOAc. The organic layer was concentrated in vacuo.
The resulting solid was dissolved in a mixture of water
(50mL) and concentrated sulfuric acid (5mL), and
heated under reflux for 30h. The mixture was then
cooled to room temperature and extracted with EtOAc.
The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo.
The residue was distilled (140ꢁC/200Pa) to give 2-
methyl-3-(2-thienyl)propanoic acid7 (1.54g, 81% yield)
as colorless oil; IR (film) 2978, 2659, 1707, 1462, 1292,
4.3. General procedure for the deracemization reaction of
a-substituted carboxylic acids by the aid of N. diaphano-
zonaria JCM3208
4.3.1. General procedure for the deracemization reaction
under aerobic conditions. The ingredients of the med-
ium were as follows: glycerol (10g/L), peptone (2g/L),
beef extract (3g/L), yeast extract (3g/L), KH2PO4 (1g/
L), K2HPO4 (1g/L), MgSO4Æ7H2O (0.3g/L), pH7.0. To
90mL of a nutrient medium, was added a suspension
of 48-h incubated cells of N. diaphanozonaria in 10mL
of the broth and the incubation carried out at 30ꢁC
for 24h. The wet cells were harvested by centrifugation
(5000rpm, 10min) and washed with phosphate buffer
(0.1M, pH7.0). The wet cells, together with the sub-
strate (100mg, 0.1% w/v), were re-suspended in 100mL
of a phosphate buffer (0.1M, pH7.0) in a 500-mL shak-
ing culture (Sakaguchi) flask. The flask was shaken at
30ꢁC on a reciprocal shaker for the appropriate time
as indicated in Tables 1–6. The reaction mixture was fil-
tered through a pad of Celite to remove the cells. The
Celite was washed with EtOAc and the washing com-
bined with the filtrate. After being acidified by 2M
hydrochloric acid, the mixture was extracted with
EtOAc. The organic layer was washed with brine, dried
over anhydrous sodium sulfate, and concentrated
in vacuo. The residue was converted to the correspond-
ing methyl ester with diazomethane, and purified by sil-
ica gel column chromatography to give the ester as a
colorless oil.
1234, 937, 850, 698cmꢀ1 1H NMR (CDCl3): d 7.15
;
(dd, J = 1.3, 5.1Hz, 1H), 6.93 (dd, J = 3.5, 5.1Hz,
1H), 6.83 (d, J = 3.5Hz, 1H), 3.26 (dd, J = 6.6,
14.7Hz, 1H), 2.94 (dd, J = 7.3, 14.7Hz, 1H), 2.80 (m,
1H), 1.24 (d, J = 7.0Hz, 3H); 13C NMR (CDCl3): d
182.0 (CO), 141.2 (C), 126.8 (CH), 125.7 (CH), 123.9
(CH), 41.6 (CH2), 33.2 (CH), 16.5 (CH3).
4.3.2. General procedure for the deracemization reaction
under inert gas condition. The wet cells harvested as de-
scribed in Section 4.3.1 and the substrate (100mg, 0.1%
w/v), re-suspended in 100mL of a phosphate buffer
(0.1M, pH7.0) in a 500-mL shaking culture (Sakaguchi)
flask. The flask was purged with argon, equipped with a
balloon filled with argon, and shaken at 30ꢁC on a
4.2.2. 2-Phenylaminopropanoic acid 12. A solution of
aniline (9.3g, 0.10mol), anhydrous sodium acetate

D. Kato et al. / Tetrahedron: Asymmetry 15 (2004) 2965–2973
2971
reciprocal shaker for the appropriate time as indicated
in Tables 1–6. The work-up procedure was the same as
Section 4.3.1.
(200lL, 3.60mmol). The mixture was stirred at room
temperature for 3h. The saturated ammonium chloride
(10mL) was then added and extracted with hexane.
The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo.
The residue was purified by silica gel column chroma-
tography (hexane/EtOAc = 9/1 then 1/1) to give methyl
2-(phenylsulfinyl)propanoate as a colorless oil.
4.3.3. General procedure for the deracemization reaction
in the presence of acyl-CoA dehydrogenase inhibi-
tor. The wet cells harvested as described above and
the appropriate amount of acyl-CoA dehydrogenase
inhibitor were suspended in 100mL of a phosphate buf-
fer (0.1M, pH7.0) in a 500-mL shaking culture (Saka-
guchi) flask. The flask was shaken for 10min. To this cell
suspension, the substrate (100mg, 0.1% w/v) was added
and shaken at 30ꢁC on a reciprocal shaker for the
appropriate time as indicated in Tables 1–6. The work-
up procedure was the same as Section 4.3.1.
A solution of methyl 2-(phenylsulfinyl)propanoate,
potassium carbonate (0.11g, 0.80mmol), and methyl
iodide (60lL, 0.93mmol) in DMF (4mL) was stirred at
room temperature. After 16h, saturated ammonium
chloride (10mL) was added and extracted with hexane.
The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo.
The residue was purified by silica gel column chroma-
tography (hexane/EtOAc = 1/1) to give methyl 2-
methyl-2-(phenylsulfinyl)propanoate (47.5mg, 77%
yield) as a colorless oil: IR (film) 3059, 2925, 1734,
4.4. Recovery and analysis of the products
4.4.1. Methyl (R)-(+)-2-(4-chlorophenoxy)propanoate,
methyl ester of 2. The extract of the reaction mixture
was treated with diazomethane to give the methyl ester
as a colorless oil: IR (film) 2954, 1758, 1596, 1492,
1577, 1475, 1441, 1327, 1456, 1076, 744, 687, 594cmꢀ1
;
1H NMR (CDCl3): d 7.50 (m, 5H), 3.65 (s, 3H), 1.58
1
1283, 1240, 1135, 1090, 979, 825, 667cmꢀ1; H NMR
(s, 3H), 1.28(s, 3H); 13C NMR (CDCl3): d 171.0
(CDCl3): d 7.25 (dd, J = 3.4, 9.9Hz, 2H), 6.83 (dd,
J = 3.4, 9.9Hz, 2H), 5.50 (q, J = 6.8Hz, 1H), 3.78 (s,
3H), 1.64 (d, J = 6.8Hz, 3H); 13C NMR (CDCl3): d
172.2 (CO), 156.0 (C), 129.4 (CH · 2), 126.5 (C), 116.3
(CH · 2), 72.9 (CH), 52.4 (CH3), 18.6 (CH3). Ee 97%
(Daicel Chiralcel OJ column (9/1 hexane/2-propanol;
(CO), 139.8(C), 131.6 (C), 12.86 (CH
(CH · 2), 66.3 (C), 52.5 (CH3), 20.5 (CH3), 16.0
(CH3). Ee racemate (Daicel Chiralcel OD column (9/1
· 2), 125.4
hexane/2-propanol; 0.5mL/min;
tR = 22.2min, 25.4min.
254nm,
37ꢁC):
0.5mL/min; 254nm): (R)-(major) tR = 18.5min, (S)-
4.4.5. Methyl (S)-phenylsulfinylacetate, methyl ester of
7. The extract of the reaction mixture was treated with
diazomethane to give the methyl ester as a colorless oil:
IR (film) 2981, 1736, 1583, 1481, 1441, 1367, 1282, 1151,
1028, 742, 690cmꢀ1; 1H NMR (CDCl3): d 7.69 (m, 2H),
7.55 (m, 3H), 3.85 (d, J = 13.7Hz, 1H), 3.71 (s, 3H), 3.67
(d, J = 13.7Hz, 1H); 13C NMR (CDCl3): d 169.5 (CO),
134.9 (C), 129.9 (CH · 2), 128.9 (CH · 2), 126.8(C),
61.5 (CH2), 45.2 (CH3). Ee 73% (Daicel Chiralcel OB
column (9/1 hexane/2-propanol; 0.5mL/min; 254nm,
37ꢁC): (S)-(major) tR = 73.1min, (R)-(minor) tR =
82.6min). The absolute configuration was determined
by comparison of the retention time with that described
in a literature.12
20
D
(minor) tR = 25.0min), ½aŠ ¼ þ44:7 (c 0.94, EtOH),
lit.,9 (S)-form, 96% ee, [a]D = ꢀ41.1 (c 50, EtOH).
4.4.2. Methyl (R)-(+)-2-phenylthiopropanoate, methyl
ester of 3. The extract of the reaction mixture was
treated with diazomethane to give the methyl ester as
a colorless oil: IR (film) 3059, 2989, 2952, 1733, 1437,
1330, 1260, 1227, 1191, 1161, 1067, 854, 749, 691cmꢀ1
;
1H NMR (CDCl3): d 7.38(m, 2H), 7.23 (m, 2H), 3.72
(q, J = 6.8Hz, 1H), 3.60 (s, 3H), 1.41 (d, J = 6.8Hz,
3H); 13C NMR (CDCl3): d 172.9 (CO), 136.6 (C),
133.0 (CH · 2), 128.8 (CH · 2), 128.0 (C), 52.3 (CH),
45.2 (CH3), 17.5 (CH3). Ee 93% (Daicel Chiralcel OJ
column (9/1 hexane/2-propanol; 0.5mL/min; 254nm):
(R)-(major) tR = 30.1min, (S)-(minor) tR = 39.7min),
4.4.6. Methyl (S)-(+)-2-methyl-3-phenylpropanoate,
methyl ester of 8. The reaction mixture was treated
with diazomethane. To a mixture of the methyl esters
of 8 and 15, sodium metaperiodate (0.3g, 1.40mmol),
water (3mL), and diethyl ether (3mL) were added
20mM osumium tetroxide in tert-BuOH (1.5mL,
0.03mmol). The mixture was stirred at room tempera-
ture for 12h. Saturated sodium thiosulfate (2mL) was
then added and extracted with diethyl ether. The organic
layer was washed with brine, dried over anhydrous
sodium sulfate, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (hex-
ane/EtOAc = 19/1) to give methyl 2-methyl-3-phenyl-
propanoate as colorless oil: IR (film) 3028, 2975, 2951,
23
22
½aŠ ¼ þ145:7 (c 1.02, EtOH), ½aŠ ¼ þ141:2 (c 0.68,
D
D
acetone), lit.,10 (R)-form, [a]D ¼ þ65.5 (c 1.0, acetone).
4.4.3. Methyl 2-(phenylsulfinyl)propanoate, methyl ester
of 4. The extract of the reaction mixture was treated
with diazomethane to give the methyl ester11 as a color-
less oil: IR (film) 3058, 2982, 2939, 2359, 1731, 1318,
1253, 1206, 1167, 1050, 858, 693cmꢀ1
;
1H NMR
(CDCl3): d 7.59 (m, 5H), 3.84 (q, J = 7.1 Hz, 0.5H, dia-
stereomer), 3.51 (q, J = 7.1Hz, 0.5H, diastereomer), 3.67
(s, 1.5H, diastereomer), 3.66 (s, 1.5H, diastereomer),
1.47 (d, J = 7.1Hz, 1.5H, diastereomer), 1.32 (d,
J = 7.1Hz, 1.5H, diastereomer).
1739, 1496, 1455, 1166, 986, 832, 745, 701cmꢀ1 1H
;
4.4.4. Methyl 2-methyl-2-(phenylsulfinyl)propanoate
5. To the extract from 100mL reaction mixture dis-
solved in DMF (1.5mL) was added sodium hydrogen
carbonate (0.3g, 3.57mmol) and methyl iodide
NMR (CDCl3): d 7.33 (m, 5H), 3.73 (s, 3H), 3.12 (m,
1H), 2.79 (m, 2H), 1.25 (d, J = 6.8Hz, 3H); 13C NMR
(CDCl3): d 176.5 (CO), 139.3 (C), 128.9 (CH · 2),
128.3 (CH · 2), 126.3 (CH), 51.2 (CH3), 41.4 (CH2),

2972
D. Kato et al. / Tetrahedron: Asymmetry 15 (2004) 2965–2973
22
enantiomer could be detected, ½aŠ ¼ þ84:6 (c 0.53,
39.7 (CH), 16.7 (CH3). Ee 88% (Daicel Chiralcel OJ col-
umn (9/1 hexane/2-propanol; 0.5mL/min; 254nm): (R)-
(minor) tR = 24.0min, (S)-(major) tR = 28.6min),
D
MeOH).
25
20
D
½aŠ ¼ þ39:3 (c 0.95, MeOH), lit.,13 (S)-form, ½aŠ
¼
4.4.11. Acetoanilide, N-acetyl protection of 13. To the
extract of the reaction mixture from 100mL medium
in CH2Cl2 (1mL) were added pyridine (200lL,
2.47mmol) and acetic anhydride (114lL, 1.22mmol).
The mixture was stirred at room temperature for 2h.
Water (5mL) was then added and acidified by 2M
hydrochloric acid. The mixture was extracted with
EtOAc. The organic layer was washed with 0.2M hydro-
chloric acid, an aqueous solution of saturated sodium
hydrogen carbonate, and brine, and then dried over
anhydrous sodium sulfate, and concentrated in vacuo.
The residue was purified by silica gel column chroma-
tography (hexane/EtOAc = 1/1) to give acetoanilide as
colorless needles, mp 113ꢁC: IR (KBr disc) 3294, 3136,
1664, 1599, 1556, 1434, 1323, 1263, 1041, 756, 694, 509
D
þ49:8 (c 2.042, MeOH).
4.4.7. Benzoic acid 9. The product was analyzed by
HPLC with a Shenshu Pak PEGASIL Silica 60-5
(98%/2%/0.05% hexane/2-propanol/TFA; 1.0mL/min;
254nm): tR = 5.84min.
4.4.8. Methyl 2-methyl-3-(2-thienyl)propanoate, methyl
ester of 10. The extract of the reaction mixture was
treated with diazomethane to give the methyl ester as
a colorless oil: IR (film) 2956, 2860, 1738, 1460, 1275,
1072, 850, 696cmꢀ1 1H NMR (CDCl3): d 7.11 (dd,
;
J = 1.0, 5.1Hz, 1H), 6.91 (dd, J = 3.3, 5.1Hz, 1H),
6.80 (dd, J = 1.0, 3.3Hz, 1H), 3.22 (dd, J = 7.0,
14.7Hz, 1H), 2.93 (dd, J = 7.2, 14.7Hz, 1H), 2.77 (m,
1H), 1.20 (d, J = 7.0Hz, 3H); 13C NMR (CDCl3): d
165.5 (CO), 146.0 (C), 126.8(CH), 125.6 (CH), 123.8
(CH), 51.7 (CH3), 41.8(CH ), 33.6 (CH), 16.8(CH ).
1
cmꢀ1; H NMR (CDCl3): d 7.49 (d, J = 7.9Hz, 2H),
7.31 (t, J = 7.9Hz, 2H), 7.10 (d, J = 7.2Hz, 1H), 2.17
(s, 3H); 13C NMR (CDCl3): d 168.4 (CO), 137.8 (C),
129.0 (CH · 2), 124.3 (CH), 119.9 (CH · 2), 24.6
(CH3).
2
3
Ee 93% (Daicel Chiralcel OJ column (9/1 hexane/2-pro-
panol; 0.5mL/min; 254nm): (R)-(minor) tR = 9.90min,
(S)-(major) tR = 10.9min).
4.4.12. Methyl (R)-(+)-2-(4-chlorophenylthio)propanoate,
methyl ester of 14. The extract of the reaction mixture
was treated with diazomethane to give methyl ester as a
colorless oil: IR (film) 2952, 1738, 1574, 1477, 1329,
4.4.9. Methyl 2-methyl-3-(2-thienyl)propenoate, methyl
ester of 11. The extract of the reaction mixture was
treated with diazomethane to give the methyl ester as
a colorless oil: IR (film) 3020, 1716, 1653, 1522, 1473,
1
1263, 1163, 1095, 1012, 823, 733, 499cmꢀ1; H NMR
(CDCl3): d 7.31 (m, 2H), 7.21 (m, 2H), 3.69 (q,
J = 7.3Hz, 1H), 3.61 (s, 3H), 1.40 (d, J = 7.3Hz, 3H);
13C NMR (CDCl3): d 172.7 (CO), 134.4 (C), 134.4
(CH · 2), 131.4 (C), 129.0 (CH · 2), 52.4 (CH),
45.3 (CH3), 17.4 (CH3). Ee 90% (Daicel Chiralcel OJ
1
1421, 1215, 928, 762, 699cmꢀ1; H NMR (CDCl3): d
7.86 (s, 1H), 7.49 (d, J = 5.1Hz, 1H), 7.28(d,
J = 3.3Hz, 1H), 7.12 (dd, J = 3.7, 5.1Hz, 1H), 3.81 (s,
3H), 2.22 (s, 3H); 13C NMR (CDCl3): d 169.0 (CO),
139.2 (C), 131.7 (C), 131.7 (CH), 129.1 (CH), 127.3
(CH), 124.7 (CH), 52.1 (CH3), 14.3 (CH3). The product
was analyzed by HPLC with a Daicel Chiralcel OJ col-
umn (9/1 hexane/2-propanol; 0.5mL/min; 254nm):
tR = 12.8min.
column (9/1 hexane/2-propanol; 0.5mL/min; 254nm):
(R)-(major) tR = 15.4min, (S)-(minor) tR = 17.3min), ½aŠ
21
D
¼
þ140:2 (c 1.0, MeOH), lit.,9 (S)-form, ½aŠ ¼ ꢀ144:6
D
(c 1.0, MeOH).
4.4.13. Methyl a-methylcinnamate, methyl ester of
15. The extract of the reaction mixture was treated
with diazomethane to give the methyl ester as a colorless
oil: IR (film) 2991, 1709, 1259, 1120, 933, 768, 710,
4.4.10. Methyl (R)-(+)-2-phenylaminopropanoate, methyl
ester of 12. To the extract of the reaction mixture from
100mL medium in methanol (6mL) was added trimeth-
ylsilyl chloride (600lL, 4.72mmol). The mixture was
stirred at room temperature for 24h. Water (5mL)
and an aqueous solution of saturated sodium hydrogen
carbonate (5mL) were then added and extracted with
EtOAc. The organic layer was washed with an aqueous
solution of saturated sodium hydrogen carbonate and
brine, dried over anhydrous sodium sulfate, and concen-
trated in vacuo. The residue was purified by silica gel
column chromatography (hexane/EtOAc = 5/1) to give
methyl 2-phenylaminopropanoate as a colorless oil: IR
(film) 3392, 2952, 1739, 1604, 1508, 1315, 1207, 1163,
1
511cmꢀ1; H NMR (CDCl3): d 7.39 (s, 1H), 7.25 (m,
5H), 3.82 (s, 3H), 2.13 (s, 3H); 13C NMR (CDCl3): d
169.2 (CO), 138.9 (C), 135.9 (CH), 129.6 (CH · 3),
128.4 (CH), 128.3 (C), 52.1 (CH3), 14.1 (CH3). The
product was analyzed by HPLC with a Daicel Chiral-
cel OJ column (9/1 hexane/2-propanol; 0.5mL/min;
254nm): tR = 29.7min.
Acknowledgements
1055, 750, 694cmꢀ1
;
1H NMR (CDCl3): d 7.19 (m,
D.K. acknowledges the financial support by Research
Fellowships of the Japan Society for the Promotion of
Science for Young Scientists (JSPS Research Fellow-
ships for Young Scientists). This study was performed
through Special Coordination Funds for Promoting Sci-
ence and Technology from the Ministry of Education,
Culture, Sports, Science, and Technology, the Japanese
Government.
2H), 6.76 (m, 1H), 6.63 (m, 2H), 4.16 (q, J = 6.8Hz,
1H), 3.73 (s, 3H), 1.49 (d, J = 6.8Hz, 3H); 13C NMR
(CDCl3): d 174.8(CO), 146.0 (C), 129.3 (CH · 2),
11.88(CH), 113.8(CH
· 2), 52.2 (CH), 52.2 (CH3),
18.8 (CH3). Ee >99% (Daicel Chiralcel OJ column (9/1
hexane/2-propanol; 0.5mL/min; 254nm): (R)-(major)
tR = 28.2min, (S)-(minor) tR = 44.1min), Only the (R)-

D. Kato et al. / Tetrahedron: Asymmetry 15 (2004) 2965–2973
2973
Geisslinger, K.; Brune, K. Mol. Pharmacol. 1997, 51,
576–582.
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