Microbial deracemization of α-substituted carboxylic acids: Substrate specificity and mechanistic investigation

Article abstract of DOI:10.1021/jo034253x

A new enzymatic method for the preparation of optically active α-substituted carboxylic acids is reported. This technique is called deracemization reaction, which provides us with a route to obtain the enantiomerically pure compounds, theoretically in 100% yield starting from the racemic mixture. This means that the synthesis of a racemate is almost equal to the synthesis of the optically active compound, and this concept is entirely different from the commonly accepted one in the asymmetric synthesis. Using the growing cell system of Nocardia diaphanozonaria JCM3208, racemates of 2-aryl- and 2-aryloxypropanoic acid are deracemized smoothly and (R)-form-enriched products are recovered in high chemical yield (>50%). In addition, using optically active starting compounds and deuterated derivatives as well as inhibitors, we have disclosed the fact that a new type of enzyme takes part in this biotransformation, and that the reaction proceeds probably via the same mechanism as that in rat liver.

Full text of DOI:10.1021/jo034253x

Micr obia l Der a cem iza tion of r-Su bstitu ted Ca r boxylic Acid s:
Su bstr a te Sp ecificity a n d Mech a n istic In vestiga tion
Dai-ichiro Kato,† Satoshi Mitsuda,‡ and Hiromichi Ohta*,†
Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama
223-8522, J apan, and Sumitomo Chemical Co. Ltd., 4-2-1, Takatsukasa, Takarazuka 665-8555, J apan
hohta@bio.keio.ac.jp
Received February 25, 2003
A new enzymatic method for the preparation of optically active R-substituted carboxylic acids is
reported. This technique is called deracemization reaction, which provides us with a route to obtain
the enantiomerically pure compounds, theoretically in 100% yield starting from the racemic mixture.
This means that the synthesis of a racemate is almost equal to the synthesis of the optically active
compound, and this concept is entirely different from the commonly accepted one in the asymmetric
synthesis. Using the growing cell system of Nocardia diaphanozonaria J CM3208, racemates of
2-aryl- and 2-aryloxypropanoic acid are deracemized smoothly and (R)-form-enriched products are
recovered in high chemical yield (>50%). In addition, using optically active starting compounds
and deuterated derivatives as well as inhibitors, we have disclosed the fact that a new type of
enzyme takes part in this biotransformation, and that the reaction proceeds probably via the same
mechanism as that in rat liver.
In tr od u ction
compounds (Scheme 1). There have been many reports
concerning this method and much effort has been devoted
to increase the enantioselectivity through the various
modifications of the reaction conditions and/or biocata-
lysts.2,4 Although kinetic resolution has already been a
well-established method for the preparation of optically
active compounds, this has a disadvantage in that the
maximum yield of the desired enantiomer is theoretically
limited to 50%. Also the separation of the product and
the recovered starting material is inevitable, which
sometimes is tedious.
To overcome this limitation, dynamic kinetic resolution
(DKR), which is a method of the combination of enzy-
matic kinetic resolution and in situ racemization of the
substrate, is attracting considerable attention.5 Theoreti-
cally, DKR is capable of giving the desired enantiomer
in 100% yield, which is a marked advantage for industrial
application. In this case, however, it is essential that the
starting material racemizes under the reaction condi-
tions, while the product does not. Accordingly, combina-
tions of sophisticated design of the substrate and incu-
bation conditions are the key to the successful procedure.
Biological activity of a compound often depends on the
absolute configuration of chiral centers in the molecule,
because the receptor to which the molecule binds is made
of an enantiomerically pure protein and its active site is
chiral. For example, (S)-enantiomers of 2-arylpropanoic
acids are widely used as nonsteroidal antiinflammatory
drugs (NSAIDs), while the (R)-antipodes are either
inactive or only weakly active in vivo.1 Furthermore, the
(R)-enantiomers of 2-aryloxypropanoic acids are utilized
as herbicide, while the (S)-antipodes are inactive.2 In
addition, (R)-2-(4-chlorophenoxy)propanoic acid lowers
the level of serum cholesterol and prevents platelet
aggregation. As the (S)-antipode of this compound inhib-
its the chloride channel in muscles, it causes side effects,
such as muscle irritability and spasms. Thus the prepa-
ration of optically pure enantiomers is extremely impor-
tant, and many kinds of approaches have been tried so
far.
The methods of obtaining the enantiomerically en-
riched compounds are classified into two broad catego-
ries: optical resolution of racemates and asymmetrization
of prochiral or meso compounds. Biocatalysts are widely
utilized in both cases.3 When the starting material is a
racemic mixture, the kinetic resolution process is a
standard procedure for the preparation of optically active
(3) (a) Faber, K. Biotransformations in Organic Chemistry, 4th ed.;
Spriger-Verlag: Berlin, Germany, 2000. (b) Drauz, K.; Waldmann, H.,
Eds. Enzyme Catalysis in Organic Synthesis; VCH: Weinheim, Ger-
many, 1995. (c) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic
Organic Chemistry; Pergamon: Oxford, UK, 1994.
(4) (a) Wu, S.-H.; Guo, Z.-W.; Sih, C. J . J . Am. Chem. Soc. 1990,
112, 1990-1995. (b) Cambou, B.; Klibanov, A. M. Biotechnol. Bioeng.
1984, 26, 1449-1454. (c) Liu, Y.-Y.; Xu, J .-H.; Hu, Y. J . Mol. Catal. B:
Enzymol. 2000, 10, 523-529.
(5) (a) Stecher, H.; Faber, K. Synthesis 1996, 1-16. (b) Caddick, S.;
J enkins, K. Chem. Soc. Rev. 1996, 25, 447-456. (c) Strauss, U. T.;
Felfer, U.; Faber, K. Tetrahedron: Asymmetry 1999, 10, 107-117. (d)
Ward, R. S. Tetrahedron: Asymmetry 1995, 6, 1475-1490. (e) Huerta,
F. F.; Minidis, A. B. E.; Backvall, J .-E. Chem. Soc. Rev. 2001, 30, 321-
331.
* To whom correspondence should be addressed. Phone: +81-45-
566-1703. Fax: +81-45-566-1551.
† Keio University.
‡ Sumitomo Chemical Co. Ltd.
(1) Rhys-Williams, W.; McCarthy, F.; Backer, J .; Hung, Y.-F.;
Thomason, M. J .; Lloyd, A. W.; Hanlon, G. W. Enzyme Microb. Technol.
1998, 22, 281-287 and reference cited therein.
(2) Colton, I. J .; Ahmed, S. N.; Kazlauskas, R. J . J . Org. Chem. 1995,
60, 212-217.
10.1021/jo034253x CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/22/2003
7234
J . Org. Chem. 2003, 68, 7234-7242

Deracemization of R-Substituted Carboxylic Acids
SCHEME 1. Com p a r ison of th e Con cep t of Tw o
Tech n iqu es for th e P r ep a r a tion of Op tica lly Active
Com p ou n d s
SCHEME 2. P r op osed Mech a n ism of
Der a cem iza tion Rea ction in Ra t Liver
Because of its structural limitation of the substrate, the
DKR method cannot be widely applied at present despite
its potential. In both resolution techniques, one enantio-
mer of the racemic compound is converted to a different
compound, such as the esterification of a carboxylic acid
(Scheme 1).
are capable of inverting the chirality of the (R)-enantio-
mer of 2-arylpropanoic acids to the (S)-antipode. There
is also an actinomycete, Rhodococcus sp.,12 which inverts
the configuration of 2-aryloxypropanoic acids from S to
R. In the case of rat liver, the reaction mechanism was
proposed based on various studies with enantiomerically
pure compounds and deuterated derivatives in which
three enzymes take part in this biotransformation system
(Scheme 2).7-9 The initial step of this biotransformation
is considered to be the enantioselective formation of the
Coenzyme A (CoA) thioester of (R)-acid. The thioester is
subsequently racemized by the aid of an epimerase and
cleaved by a hydrorase to release the free (S)-acid. Among
these three enzymes, only the acyl-CoA synthetase is
enantioselective. Thus, the enantiomeric ratio in the
reaction mixture shifts to the (S)-form with the repetion
of the reactions. These enzymes as well as genes were
purified and identified. In comparison to the rat liver
enzymes, the others have been little studied and many
reports on these biocatalysts concern only the pharma-
cological aspect of the system and not their synthetic
potentials. In addition, all of the previously reported
enzymes are only capable of inverting the chirality of
2-arylpropanoic acids from (R)- to (S)-configuration. Thus,
from the standpoint of synthetic chemistry, it is very
important to find new enzymes possessing the deracem-
ization activity for a wide range of compounds and to
invert the chirality of 2-arylpropanoic acid from (S)- to
(R)-configuration. Recently, we found a new biocatalyst,
Nocardia diaphanozonaria J CM3208, a kind of actino-
mycetes, affording the (R)-form of 2-aryl- and 2-aryloxy-
propanoic acid from their racemates.13 In addition, Mit-
sukura et al. also have reported the efficient preparative
On the other hand, if there is a method by which we
can convert a racemic compound to the corresponding
optically active one without changing its chemical struc-
ture, this will be the third option for obtaining an
optically active compound from a racemic mixture. In
such a case, we will be free from the fear of racemization
of the “product” and a tedious separation in the workup
procedure. We would like to call such a process “derace-
mization”, distinguishing it from DKR as well as kinetic
resolution. In this deracemization process, the chirality
of either enantiomer of a racemate is converted to the
other antipode via single or plural reactions, resulting
in an optically active compound starting from a racemic
mixture as a whole (Scheme 1). In total, successful
application of this process means that the synthesis of a
racemate is almost equal to the synthesis of the optically
active compound. This concept is entirely different from
the commonly accepted one in the asymmetric synthesis.
Most of the enzymatic deracemization reactions so far
reported utilize the oxidation-reduction process. A rep-
resentative example is the deracemization of secondary
alcohols, the key being the combination of two enzymes
that catalyze the oxidation of one enantiomer followed
by enantioselective reduction to the antipode.5c Quite
recently, Turner and co-workers reported the deracem-
ization of R-amino acids6 and R-methylbenzylamine7
combining the enantioselective enzyme-catalyzed oxida-
tion of the amine to imine and the nonenantioselective
chemical reduction. Here we present an enzymatic de-
racemization reaction of a quite different type of com-
pounds, 2-arylpropanoic acid and 2-aryloxypropanoic
acid. Not only are these important, physiologically active
compounds, but also it is interesting from the standpoint
of the reaction mechanism. That is because these com-
pounds are difficult to subject to the oxidation-reduction
process by either chemical or enzymatic reaction.
There are only a few reports on the enzymatic dera-
cemization of R-substituted carboxylic acids with bio-
catalysts, i.e., the use of rat liver8,9 and two fungi,
Cordyceps militaris1,10 and Verticillium lecanii,11 which
(8) (a) Knihinicki, R. D.; Day, R. O.; Williams, K. M. Biochem.
Pharmacol. 1991, 42, 1905-1911. (b) Knights, K.; Talbot, U. M.; Baillie,
T. A. Biochem. Pharmacol. 1992, 44, 2415-2417. (c) Menzel, S.; Waibel,
R.; Brune, K.; Geisslinger, G. Biochem. Pharmacol. 1994, 48, 1056-
1058. (d) Mu¨ller, S.; Mayer, J . M.; Etter, J .-C.; Testa, B. Biochem.
Pharmacol. 1992, 44, 1468-1470.
(9) (a) Shieh, W.-R.; Chen, C.-S. J . Biol. Chem. 1993, 268, 3487-
3493. (b) Brugger, R.; Al´ıa, B. G.; Reichel, C.; Waibel, R.; Menzel, S.;
Brune, K.; Geisslinger, G. Biochem. Pharmacol. 1996, 52, 1007-1013.
(c) Reichel, C.; Brugger, R.; Bang, H.; Geisslinger, G.; Brune, K. Mol.
Pharmacol. 1997, 51, 576-582.
(10) Rhys-Williams, W.; Thomason, M. J .; Hung, Y.-F.; Hanolon, G.
W.; Lloyd, A. W. Chirality 1998, 10, 528-534.
(6) (a) Beard, T. M.; Turner, N. J . Chem. Commun. 2002, 246-247.
(b) Alexandre, F.-R.; Pantaleone, D. P.; Taylor, P. P.; Fotheringham,
I. G.; Ager, D. J .; Turner, N. J . Tetrahedron Lett. 2002, 43, 707-710.
(7) Alexeeva, M.; Enright, A.; Dawson, M. J .; Mahmoudian, M.;
Turner, N. J . Angew. Chem., Int. Ed. 2002, 41, 3177-3180.
(11) Rhys-Williams, W.; Thomason, M. J .; Lloyd, A. W.; Hanlon, G.
W. Pharm. Sci. 1996, 2, 537-540.
(12) Bewick, D. W. E. Patent 133034, 1984.
(13) Kato, D.-i.; Mitsuda, S.; Ohta, H. Org. Lett. 2002, 4, 371-373.
J . Org. Chem, Vol. 68, No. 19, 2003 7235

Kato et al.
TABLE 1. Micr obia l Der a cem iza tion of 2-Ar ylp r op a n oic
Acid a
SCHEME 3. Op tim iza tion of th e Rea ction
Con d ition s w ith Use of th e Gr ow in g Cell System of
N. d ia ph a n ozon a r ia
time
(h)
yieldb
(%)
eec
(%)
entry
sub.
X
1
2
3
4
5
6
7
8
9
1b
1c
1d
1e
1f
1g
1h
1h
1h
4-isobutyl
3-fluoro-4-phenyl
4-methoxy
2-chloro
3-chloro
4-chloro
4-fluoro
4-fluoro
4-fluoro
48
48
48
48
48
48
6
85
99
60
59
62
61
83
58
35
9
6
4
9
4
11
racemic
6
16
48
10
a
The starting compounds were incubated with growing cells of
b
N. diaphanozonaria at 30 °C. Isolated yield after conversion to
the corresponding methyl ester (2b-h ). c Ee of the product was
determined by HPLC analysis after conversion to the correspond-
ing methyl ester.
method of (R)-2-phenylpropanoic acid using the resting
cells of this bacterium.14 In the present paper, we would
like to report the reaction mechanisms as well as the
substrate specificities using the whole cell system of N.
diaphanozonaria.
SCHEME 4. En ola te-Typ e In ter m ed ia te: P r op osed
In ter m ed ia te d u r in g th e Ch ir a l In ver sion P r ocess
Resu lts a n d Discu ssion
Su bstr a te Sp ecificity. (S)-2-Phenylpropanoic acid
((S)-1a ) was converted to the (R)-form (Scheme 3) by
incubation with the growing cells of N. diaphanozonaria.
To the best of our knowledge, this bacterium is the first
example that exhibits the deracemization activity toward
(S)-1a . Thus, 0.1% (w/v) of 1a was added to the suspen-
sion of 24-h incubated cells, and the mixture was shaken
at 30 °C (second incubation). When the reaction was
stopped after 48 h, enantiomerically enriched 1a was
recovered as the sole product, the yield and enantiomeric
excess (ee) of the product being 81% and 69% (R),
respectively. The period of the second incubation was
optimized based on two parameters, the yield and the ee
of the product. Although these two values consistently
increased over 48 h, the remarkable decrease was ob-
served thereafter. This is probably because of the oxida-
tive degradation of the substrate.
the hydrolase and was recovered intact (eq 2). These
results suggest that the free acid moiety is essential to
this enzyme system.
To expand the substrate specificity of the deracemiza-
tion reaction, several 2-phenylpropanoic acid deriva-
tives which have a substituent on the aromatic ring were
subjected to the reaction (Table 1). In the case of 2-(4-
isobutylphenyl)propanoic acid (ibuprofen, 1b ) and
2-[(3-fluoro-4-phenyl)phenyl]propanoic acid (flurbipro-
fen, 1c), N. diaphanozonaria did not show the deracem-
ization activity in contrast to the fact that both of these
compounds were deracemized by C. militalis.1,10 In ad-
dition, 2-(4-methoxyphenyl)propanoic acid (1d ) was not
accepted by the enzyme system of N. diaphanozonaria
(entry 3).
The methyl ester of 2-phenylpropanoic acid (2a ) was
also deracemized to the (R)-enantiomer although the
yield and ee of the product were lower (eq 1). In this case,
If the reaction proceeds via an enolate type intermedi-
ate as proposed by Shieh et al.9a (Scheme 4), the intro-
duction of an electron-withdrawing substituent on the
aromatic ring will stabilize the transition state and
accelerate the reaction. Unfortunately, however, 2-(chlo-
rophenyl)propanoic acid derivatives possessing a chlorine
atom on the ortho-, meta-, or para-position (1e-g) did
not undergo the deracemization reaction (entries 4-6).
Furthermore, the introduction of a fluorine atom on the
para-position (1h ) did not enhance the apparent chiral
the primary product was revealed to be free acid. Thus
the actual substrate would also be the free acid formed
in situ by the aid of hydrolase of N. diaphanozonaria.
On the other hand, 2-phenylpropanamide (3) could not
be recognized by either the deracemization enzymes or
(14) Mitsukura, K.; Yoshida, T.; Nagasawa, T. Biotechnol. Lett. 2002,
24, 1615-1621.
7236 J . Org. Chem., Vol. 68, No. 19, 2003

Deracemization of R-Substituted Carboxylic Acids
TABLE 2. Micr obia l Der a cem iza tion of Cyclic
An a logu es of 2-P h en ylp r op a n oic Acid a
TABLE 3. Micr obia l Der a cem iza tion of r-Su bstitu ted
P h en yla cetic Acid a
entry
sub.
n
yieldb (%)
eec (%)
entry
sub.
X
yieldb (%)
eec (%)
1
2
3
4a
4b
4c
1
2
3
65
70
58
racemic
12
9
1
2
3
4
6a
6b
6c
6d
F
74
77
85
76
55
C2H5
OMe
CH2OH
racemic
racemic
racemic
a
The starting compounds were incubated with growing cells of
b
N. diaphanozonaria at 30 °C. Isolated yield after conversion to
a
The starting compounds were incubated with growing cells of
the corresponding methyl ester (5a -c). c Ee of the product was
determined by HPLC analysis after conversion to the correspond-
ing methyl ester.
b
N. diaphanozonaria at 30 °C. Isolated yield after conversion to
the corresponding methyl ester (7a -d ). c Ee of the product was
determined by HPLC analysis after conversion to the correspond-
ing methyl ester.
inversion to any significant extent regardless of the small
size and the electronegativity of the fluorine atom (entries
7-9). In addition, after 48 h of incubation, the yield of
the product significantly decreased. This result was
estimated to be caused by the increase of the rate of
metabolic degradation by the introduction of a fluorine
atom on the aromatic ring. As expected, shortening the
incubation period raised the yield, although the ee of the
product was not improved (entry 7).
If the reaction proceeds via an enolate-type intermedi-
ate, which will be stabilized by the resonance effect of
the adjacent phenyl ring (Scheme 4). In this situation,
the orbital of the anion and the π electrons of the phenyl
ring should be parallel, and consequently the conforma-
tion of the intermediate in the active site of the enzyme
would be restricted. Accordingly, if the racemization step
is rate-determing, fixing the conformation of the sub-
strate in advance to the one that is favorable to the
delocalization of the carbanion will be expected to ac-
celerate the reaction because of the lowering of the
activation entropy. Thus we prepared some compounds
with a ring system (Table 2). However this attempt was
unsuccessful. The substrates with a four-, five-, or six-
membered ring (4a -c) were recovered as almost race-
mates.
It is also interesting how the R-substituent affects the
reactivity. R-Fluorophenylacetic acid (6a ), which has a
fluorine atom instead of a methyl group, was accepted
by the deracemization enzymes to give the (R)-form in a
moderate optical yield (Table 3, entry 1). On the other
hand, the enzyme system did not exhibit the deracem-
ization activity with 2-phenylbutanoic acid (6b), R-meth-
oxyphenylacetic acid (6c), and 3-hydroxy-2-phenylpro-
panoic acid (6d ), probably because of the steric bulkiness
of the R-substituents (entries 2-4).
Mandelic acid derivatives (8a -c), which have a hy-
droxyl group on the R-carbon, exhibited entirely different
reactivity, i.e., they underwent the enantioselective
degradation reaction (Table 4). Only the (S)-form was
converted to the corresponding benzoic acid derivatives
and (R)-mandelic acid derivatives were recovered in the
optically pure form.
TABLE 4. Micr obia l Der a cem iza tion of Ma n d elic Acid
Der iva tivesa
product
starting
compd yieldb eec
entry sub.
X
ee (%)
(%)
(%)
metabolic product
1
2
3
4
5
8a
8a
8a
H
H
H
racemic
R, >99
S, 83
45 >99 benzoic acid
84 >99
34 S, 23 benzoic acid
43 >99 4-chlorobenzoic acid
38 >99 4-methoxybenzoic acid
8b Cl
8c OMe racemic
racemic
a
The starting compounds were incubated with growing cells of
b
N. diaphanozonaria at 30 °C. Isolated yield after conversion to
the corresponding methyl ester (9a -c). c Ee of the product was
determined by HPLC analysis after conversion to the correspond-
ing methyl ester.
insertion of an oxygen atom will lower the acidity of the
R-methyne proton. To our surprise, however, the dera-
cemization reaction of 2-phenoxypropanoic acid (10a )
cleanly proceeded under the same reaction conditions as
those for 2-phenylpropanoic acid (1a ), both the yield and
the ee of the product being 75% after 48 h (Table 5, entry
2). Elongation of the second incubation period to 72 h
resulted in the improvement of the ee of the product,
being as high as 97% (entry 4). However, similar to the
case of 1a , incubation over 72 h had a negative effect on
the yield and the ee of the product (entry 5). Surprisingly
enough, the absolute configuration of the product was
revealed to be R judging from the sign of optical rota-
tion.15 This means that the spatial arrangement of the
ligands around the asymmetric center was opposite that
(15) Chordia, M. D.; Harman, W. D. J . Am. Chem. Soc 2000, 122,
2725-2736.
(16) Miyamoto, K.; Ohta, H. J . Am. Chem. Soc. 1990, 112, 4077-
4078.
(17) Fukuyama, Y.; Matoishi, K.; Iwasaki, M.; Takizawa, E.; Miyaza-
ki, M.; Ohta, H.; Hanzawa, S.; Kakidani, H.; Sugai, T. Biosci.,
Biotechnol., Biochem. 1999, 63, 1664-1666.
2-Aryloxypropanoic acid was selected as the next
substrate. These compounds have an oxygen atom be-
tween the aromatic ring and the asymmetric center. At
first, we presumed that these compounds would be
unfavorable for the deracemization reaction because the
(18) Miyazawa, T.; Kurita, S.; Ueji, S.; Yamada, T. Biocatal.
Biotransform. 2000, 17, 459-473.
(19) Ireland, R. E.; Thaisrivongs, S.; Dussault, P. H. J . Am. Chem.
Soc. 1988, 110, 5768-5779.
(20) Bach, T.; Ko¨rber, C. J . Org. Chem. 2000, 65, 2358-2367.
J . Org. Chem, Vol. 68, No. 19, 2003 7237

Kato et al.
TABLE 5. Micr obia l Der a cem iza tion of
2-P h en oxyp r op a n oic Acid a
TABLE 7. Micr obia l Der a cem iza tion of
2-(Ch lor op h en oxy)p r op a n oic Acid a
entry
sub.
position
yieldb (%)
eec (%)
reaction
time (h)
yieldb
(%)
eec
(%)
d
entry
[R]D
1
2
3
10l
10m
10c
2-Cl
3-Cl
4-Cl
61
73
95
17(S)
21(R)
97(R)
1
2
3
4
5
24
48
60
72
84
68
75
66
71
86
50
75
91
97
86
+41.0 (EtOH)
+41.2 (CHCl3)
a
The starting compounds were incubated with growing cells of
b
N. diaphanozonaria at 30 °C. Isolated yield after conversion to
the corresponding methyl ester (11c, l, and m ). c Ee of the product
was determined by HPLC analysis after conversion to the corre-
sponding methyl ester.
a
The starting compounds were incubated with growing cells of
b
N. diaphanozonaria at 30 °C. Isolated yield after conversion to
the corresponding methyl ester (11a ). c Ee of the product was
determined by HPLC analysis after conversion to the correspond-
ing methyl ester. Optical rotation was measured after conversion
to the corresponding methyl ester.
p-hydroxyl-substituted compounds, we observed only the
recovery of the racemate (entries 9 and 10). When the
aromatic ring was converted to the more bulky naphthyl
group, the deracemization reaction hardly proceeded (eq
3). Furthermore, while the enzyme system of N. diapha-
d
TABLE 6. Micr obia l Der a cem iza tion of
P a r a -Su bstitu ted 2-P h en oxyp r op a n oic Acid a
entry
sub.
X
yieldb (%)
eec (%)
[R]D(EtOH)d
1
2
3
4
5
6
7
8
9
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
F
Cl
Br
I
75
64
95
83
76
64
69
83
59
79
75
96
97
99
97
99
90
44
+41.0
+52.0
+44.7
+47.5
+43.4
+43.3
+53.3
+34.0
nozonaria could accept the p-chloro derivative, it could
not recognize the other chloro-substituted compounds,
such as 2-(2-chlorophenoxy)- and 2-(3-chlorophenoxy)-
propanoic acid (10l,m , Table 7, entries 1 and 2). The
introduction of one methylene spacer in 2-phenoxypro-
panoic acid between the oxygen atom and the aromatic
ring had considerable negative effect on the deracemiza-
tion reaction, as can be seen from the remarkable
decrease of the enantioselectivity for this type of com-
pound (eq 4).
CF3
CH3
OMe
COMe
OH
racemic
racemic
10
10j
a
The starting compounds were incubated with growing cells of
b
N. diaphanozonaria at 30 °C. Isolated yield after conversion to
the corresponding methyl ester (11a -j). c Ee of the product was
determined by HPLC analysis after conversion to the correspond-
d
ing methyl ester. Optical rotation was measured after conversion
to the corresponding methyl ester.
of 1a . To the best of our knowledge, this is the first
example of the inversion of the enantioselectivity between
these two substrates as we described in the previous
communication.13 It is very interesting that the insertion
of only one atom brought about a dramatic change in
enantioselectivity.
As seen from the results of compounds 10b-j, intro-
duction of a substituent on the para-position of the
aromatic ring had no negative effect on the deracemiza-
tion reaction in contrast to the case of 1a (Table 6). In
particular, this enzyme system was shown to be active
to a series of compounds that have an electronegative
substituent on the para-position. For example, 2-(4-
chlorophenoxy)propanoic acid (10c) was deracemized
smoothly, with the yield and the ee of the product being
as high as 95% and 97% (R) after 48 h of incubation
(entry 3). Moreover, the compounds which have an
electron-donating substituent also underwent a derace-
mization reaction, with a slight decrease of the ee of the
product after 48 h of incubation (entries 7 and 8).
Unfortunately, however, in the case of p-acetyl- or
Then we focused our attention on the effect of the
“spacer” atom between the R-carbon and the aromatic
ring. Thus we selected two compounds, 2-phenylthiopro-
panoic acid (14a ) and 2-methyl-3-phenylpropanoic acid
(15).
By the incubation with the growing cells of N. diapha-
nozonaria, however, degradation reactions proceeded
preferentially for these compounds, although the enzymes
concerning the oxidation of 14a and 15 will be different,
judging from the products. In the case of 14a , the (S)-
enantiomer was oxidized on the sulfur atom to give
2-phenylsulfinylpropanoic acid (14b, eq 5). The relative
1
configuration of this product could be determined by H
7238 J . Org. Chem., Vol. 68, No. 19, 2003

Deracemization of R-Substituted Carboxylic Acids
the configuration of (S)-1a was inverted to the other
antipode based on a calculation with the values of the
yield and ee of the product. It is impossible to explain
the result shown in Table 8 by supposing only the
enantioselective degradation via the â-oxidation process.
It is also interesting that the ee of (R)-1a was decreased
from 88% to 65% after 48 h of incubation (entry 2). These
results suggest that the deracemization process by this
bacterium consists of the combination of equilibrium
reactions and that a set of enzymes are participating.
To explain each reaction, the following experiments
were performed. When racemic 2-deuterio-2-phenylpro-
panoic acid (1i, D content 98%) was incubated with the
whole cells of N. diaphanozonaria, the D content of the
product decreased to 20% (entry 4). Because no decrease
of the D content was observed in the absence of the
microorganism, D-H exchange should be catalyzed by
enzymes and the reaction path should include C-D bond
fission. There are at least two possible paths that will
cause the loss of the R-methyne proton of 1a (Scheme 6).
One is the path via an enolate-type intermediate and the
other is an oxidation-reduction process via the exo
methylene-type intermediate; the former is proposed by
Shieh et al.9a for rat liver and the latter is the analogous
process for the deracemization of secondary alcohols. To
clarify which path was working, racemic 3,3,3-trideuterio-
2-phenylpropanoic acid (1j, D content >99%) was sub-
jected to the reaction. The substrate was recovered as
its optically active form without losing any deuterium
atom (entry 5). This fact strongly suggests that no C-D
bond fission occurred on the methyl group, although one
cannot totally neglect the possibility that the same
hydrogen atom comes back to the methyl group during
the oxidation-reduction process. However, considering
such a mechanism, it is still difficult to explain the fact
that only the hydrogen on C-2 exchanges, while the one
on C-3 does not. Thus, the most probable mechanism at
present is the path via an enolate-type intermediate
similar to the case of rat liver (Scheme 4).
NMR analysis, and equal amounts of syn and anti
isomers were detected. On the other hand, the (R)-
enantiomer was recovered intact in high optical yield. In
the case of 15, complete degradation proceeded without
enantioselectivity to give benzoic acid (16, eq 6).
If the enzyme system of N. diaphanozonaria also works
via the same reaction mechanism as that established for
the rat liver system (Scheme 2), the key intermediate will
be acyl-CoA, which is also the intermediate of the
â-oxidation process of carboxylic acids. Thus it is not
surprising that the degradation reaction took precedence
over the deracemization reaction for these compounds
under an aerobic condition (Scheme 5). Even if the
oxidative degradation process is a minor pass, the
substrate will be gradually metabolized because this
process is irreversible. In fact, 2-alkylpropanoic acid and
the analogous compounds, such as 2-methyldecanoic acid,
2-heptyloxypropanoic acid, and 2-chlorooctanoic acid,
were completely metabolized under the same conditions
by the growing cells of N. diaphanozonaria, and no
reaction product could be detected.
If the hypothesis that the â-oxidation process is
competing with the deracemization reaction is correct,
then it might be possible to obtain the optically active
form of 15 and related carboxylic acids by suppressing
the oxidation step by designing the reaction conditions
and/or by some additives. Thus we studied the reaction
mechanism aiming at obtaining more information.
In vestiga tion of th e Rea ction Mech a n ism . In
general, there are two possible reaction paths for the
formation of the optically active compounds starting from
the racemates. One is the deracemization of the substrate
via some mechanism and the other is the enantioselective
degradation. In case of the above-mentioned conversion
by the aid of N. diaphanozonaria, the former process is
considered to be the major path based on the yield of the
product although the degradation path is not negligible.
For the study of the strict reaction mechanism, the iso-
lation of the enzyme is essential. However, for the pur-
pose of designing the reaction conditions to obtain better
results, information that is available by using the whole
cell system is sometimes useful. To obtain such informa-
tion, we carried out some experiments. First, optically
active 2-phenylpropanoic acid (1a ) was subjected to the
reaction (Table 8). It was revealed that the configuration
of the product was R (entries 2 and 3). It is clear that
In case of rat liver, it is clarified that acyl-CoA
synthetase plays an important role in the recognition of
the configuration of the asymmetric center.8,9 Several
studies have confirmed that only the (R)-enantiomer of
2-arylpropanoic acid can yield acyl-CoA thioester. Forma-
tion of acyl-CoA may be essential not only in the chiral
inversion process but also in the â-oxidation of fatty acid.
It is of interest whether acyl-CoA synthetase also takes
part in the deracemization reaction in the case of N.
diaphanozonaria. Thus the deracemization procedure by
the growing cell system was carried out in the presence
of the typical substrate of acyl-CoA synthetase, i.e.,
benzoic acid and palmitic acid. These are known to be
the substrates of medium-chain acyl-CoA synthetase and
long-chain acyl-CoA synthetase, respectively. The influ-
ence of these carboxylic acids on the chiral inversion of
2-phenylpropanoic acid (1a ) and 2-(4-chlorophenoxy)-
propanoic acid (10c) was examined. The results are
summarized in Table 9. Racemic substrates were incu-
bated for 12 h with growing cells of N. diaphanozonaria
in the presence of 5 equiv of benzoic acid or palmitic acid.
When benzoic acid was added to the reaction mixture,
the deracemization reaction was completely inhibited. On
the other hand, palmitic acid had no effect on the chiral
inversion process. These experiments show that the
J . Org. Chem, Vol. 68, No. 19, 2003 7239

Kato et al.
SCHEME 5. Tw o Differ en t Kin d s of Rea ction P a th s in th e Meta bolic Rea ction of
2-Meth yl-3-p h en ylp r op a n oic Acid (15)
TABLE 8. Mech a n istic In vestiga tion of Der a cem iza tion
Rea ction w ith 2-P h en ylp r op a n oic Acid Der iva tivesa
both processes despite the fact that the enantioselectivity
between 1a and 10c was opposite (Scheme 3, Table 6).
Figure 1 shows the comparison of the inhibitory
activity of benzoic acid toward 1a and 10c. In this figure,
the relative activity is expressed by the ratio of ee values
of the product after 12 h of incubation in the presence
and absence of benzoic acid. Apparently, the effect of
benzoic acid on 1a is more serious compared to that on
10c. These results show that the affinity of 10c to acyl-
CoA synthetase is stronger than that of 1a , which is
reflected in the higher ee of 10c compared to that of 1a .
starting compd
D
product
D
ee
content yieldb eec contentd
entry sub.
X
Y
(%)
(%)
(%)
(%)
(%)
1
2
3
4
5
8a
1a
1a
1i
H
H
H
D
H
CH3 racemic
CH3 R, 88
CH3 S, 93
CH3 racemic
CD3 racemic
81
79
59
58
61
69
65
50
72
71
To obtain more information on the inhibitory activity
on the deracemization enzymes, the reaction of 10c was
carried out in the presence of 5 equiv of n-alkanoic acids
with various chain lengths (Table 10). It is known that
medium-chain acyl-CoA synthetase recognizes C4-C12
lengths of n-alkanoic acids and that long-chain acyl-CoA
synthetase is capable of catalyzing the reaction of
C14-C22 n-alkanoic acids.8c When n-alkanoic acids with
medium chain lengths (C4-C13) were present in the
reaction mixture, the chiral inversion process was com-
pletely inhibited (entries 2-9). On the other hand, long-
chain n-alkanoic acids, such as myristic acid, palmitic
acid, and stearic acid, affected the deracemization reac-
tion only a little and optically active 10c was obtained
(entries 10-12). Thus it was also shown that medium-
chain acyl-CoA synthetase takes part in the chiral
inversion process in N. diaphanozonaria.
98
>99
20
>99
1j
a
The starting compounds were incubated with growing cells of
b
N. diaphanozonaria at 30 °C. Isolated yield after conversion to
the corresponding methyl ester (2a ,i,j). c Ee of the product was
determined by HPLC analysis after conversion to the correspond-
ing methyl ester. D content was calculated from 1H NMR
d
analysis.
SCHEME 6. Tw o Typ es of In ter m ed ia te d u r in g
th e Der a cem iza tion Rea ction of
2-P h en ylp r op a n oic Acid (1a )
Con clu sion
We found a novel enzymatic method for the prepara-
tion of optically active R-substituted carboxylic acids. This
process can be performed under mild reaction conditions
via an extremely simple procedure. It gives optically
active products in good to high yields as well as enan-
tiomeric excess. This deracemization reaction is interest-
ing, in that a small modification of the substrate brings
about a drastic change in the enantioselectivity.
Though the detail of the reaction mechanism of this
biotransformation is not clear at present, plural enzymes
are likely to be related. The inhibition studies suggested
that the formation of acyl-CoA will be the key step as
reported in the case of rat liver, although the enantiose-
lectivity and reactivity are quite different. Low yields of
the product in some cases will be due to the oxidation of
these intermediates. Further investigation on the reac-
medium-chain acyl-CoA synthetase plays an important
role in the present deracemization reaction. This is in
marked contrast with the case of rat liver, in which the
chiral inversion process was inhibited by palmitic acid,
a typical substrate of long-chain acyl-CoA synthetase.8c
In addition, it is very interesting that the same compound
inhibited the deracemization of both 1a and 10c, which
means that the same acyl-CoA synthetase is involved in
7240 J . Org. Chem., Vol. 68, No. 19, 2003

Deracemization of R-Substituted Carboxylic Acids
TABLE 9. Rela tion of Acyl-CoA Syn th eta se to th e
Der a cem iza tion Rea ction a
Chemical Research (Riken), 2-1 Hirosawa, Wako 351-0106,
J apan.
2-P h en ylp r op ion a m id e (3). To a solution of 2-phenylpro-
ponitrile (0.52 g, 3.4 mmol) in MeOH (5 mL) was added a 35%
hydrogen peroxide solution (0.4 mL). The pH was adjusted at
8.0 by adding 2 M sodium hydroxide. After 1 h, 35% hydrogen
peroxide solution (0.4 mL) was added to the reaction. The
reaction mixture was stirred for an additional 1 h. The solvent
was removed in vacuo, and the residue was extracted with
EtOAc. 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
(hexane/EtOAc/EtOH 6/2/1) to give 2-phenylpropionamide
(0.50 g, 95% yield) as a colorless needle: mp 87-88 °C; IR
(KBr disk) 3361, 3185, 2983, 2801, 1657, 1451, 1405, 1287,
a
The starting compounds were incubated with growing cells of
1
1264, 1134, 1114, 696, 656 cm-1; H NMR (CDCl3) δ 7.33 (m,
N. diaphanozonaria for 12 h at 30 °C. Inhibitor was added (5 equiv)
to substrate 1a or 10c. Isolated yield was determined after
conversion to the corresponding methyl ester (2a and 11c). Ee of
the product was determined by HPLC analysis after conversion
to the corresponding methyl ester. Benzoic acid and palmitic acid
are typical substrates of medium-chain acyl-CoA synthetase and
long-chain acyl-CoA synthetase, respectively.
5H), 5.27 (br s, 2H), 3.60 (q, J ) 7.3 Hz, 1H), 1.55 (t, J ) 7.3
Hz, 3H).
Meth yl 2-(4-F lu or op h en yl)p r op a n oa te (2h ). To a solu-
tion of diisopropylamine (0.5 mL, 3.6 mmol) in tetrahydrofuran
(4 mL) was added n-butyllithium (1.52 M in hexane, 2.4 mL)
at -78 °C under Ar, and the mixture was stirred for 30 min.
Then a solution of methyl 4-fluorophenylacetate (0.50 g, 3.0
mmol) in tetrahydrofuran (5 mL) was added dropwise, and the
mixture was stirred for 20 min at the same temperature. The
mixture was then allowed to warm to 0 °C. Methyl iodide (0.3
mL, 4.8 mmol) was added and the mixture was stirred for 30
min. The reaction mixture was acidified by 2 M hydrochloric
acid and extracted with EtOAc. The organic layer was washed
with brine, dried over anhydrous sodium sulfate, and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane/EtOAc 19/1) to give methyl 2-(4-
fluorophenyl)propanoate (0.48 g, 89% yield) as a colorless oil;
IR (film) 2983, 2953, 1739, 1604, 1510, 1456, 1436, 1224, 1170,
1070, 838, 732, 551 cm-1; 1H NMR (CDCl3) δ 7.19 (dt, J ) 2.0,
8.8 Hz, 2H), 6.94 (tt, J ) 2.0, 8.8 Hz, 2H), 3.64 (q, J ) 6.8 Hz,
1H), 3.59 (s, 3H), 1.41 (d, J ) 6.8 Hz, 3H).
F IGURE 1. Comparison of the inhibitory activity of benzoic
acid toward 2-phenylpropanoic acid (1a ) and 2-(4-chlorophe-
noxy)propanoic acid (10c).
4-F lu or op n en ylp r op a n oic Acid (1h ). A solution of 2h
(0.25 g, 1.4 mmol) in EtOH (3 mL) was added to an aqueous
solution (3 mL) of potassium hydroxide (0.5 g, 9.0 mmol) at 0
°C. After being stirred for 8 h, the reaction mixture was
acidified by 2 M hydrochloric acid, and extracted with EtOAc.
The organic layer was washed with brine, dried over anhy-
drous sodium sulfate and concentrated in vacuo to give 2-(4-
fluorophenyl)propaoic acid (0.23 g, quant) as a colorless oil:
IR (film) 2985, 2626, 1705, 1603, 1506, 1457, 1410, 1225, 1161,
TABLE 10. Th e In flu en ce of Va r iou s Ch a in Len gth s of
n -Alk a n oic Acid on th e Der a cem iza tion P r ocess in N.
Dia ph a n ozon a r ia a
entry
inhibitorb
chain length
eed (%) yieldc (%)
1
2
3
4
5
6
7
8
9
none
n-butanoic acid
n-valeric acid
n-hexanoic acid
n-heptanoic acid
n-octanoic acid
n-decanoic acid
n-dodecanoic acid
n-tridecanoic acid
myristic acid
92
75
76
99
87
76
82
68
90
65
62
76
76
838, 722, 559 cm-1
.
4
5
6
7
8
10
12
13
14
16
18
racemic
racemic
racemic
racemic
racemic
racemic
racemic
racemic
52
Compounds 1d -g were prepared according to a similar
procedure.
In d a n -1-ca r boxylic Acid (4b). A solution of indan-1-one
(5.0 g, 37.8 mmol) in freshly distilled ethyl chloroacetate (6.9
g, 56.3 mmol) was added dropwise over 1 h to a solution of
potassium tert-butoxide (6.4 g, 57.0 mmol) in dry tert-butyl
alcohol (50 mL) at 0 °C. After completion of the addition, the
reaction mixture was allowed to warm to room temperature
and stirred for further 1.5 h. The excess tert-butoxide was
decomposed by passing CO2 through the reaction mixture and
the solvent was removed in vacuo at 35 °C. Ether (20 mL) was
added to the residue and the mixture was filtered through a
pad of Celite. The Celite was washed with ether and the
washing was combined with the filtrate and concentrated in
vacuo to give a dark-brownish residue. To a solution of
potassium hydroxide (3.5 g, 46.0 mmol) in ethanol (5 mL) was
added the solution of this residue in ethanol (5 mL) at 0 °C.
The reaction mixture was allowed to warm to room temper-
ature and stirred for an additional 3 h. The precipitate was
filtered and washed with ice-cold ethanol and ether. A vigorous
evolution of gas occurred when acetic acid (20 mL) was added
to this precipitate at 0 °C. After being stirred at room
temperature for 15 min, the reaction mixture was heated at
130 °C for 1 h. The reaction mixture was then cooled to room
temperature, diluted with water (10 mL), and extracted with
10
11
12
palmitic acid
stearic acid
91
52
a
The starting compounds were incubated with growing cells of
b
N. diaphanozonaria for 12 h at 30 °C. The inhibitor was added
(5 equiv) to substrate 10c. c Isolated yield after conversion to the
d
corresponding methyl ester (11c). Ee of the product was deter-
mined by HPLC analysis after conversion to the corresponding
methyl ester.
tion mechanism as well as the isolation of the enzymes
is now underway.
Exp er im en ta l Section
Micr oor ga n ism . The strain, Nocardia diaphanozonaria
J CM3208, used in this experiment is available from the J apan
Collection of Microorganism: The Institute of Physical and
J . Org. Chem, Vol. 68, No. 19, 2003 7241

Kato et al.
ether. The organic layer was washed with saturated sodium
hydrogen carbonate and brine, dried over anhydrous sodium
sulfate, and concentrated in vacuo to give a brownish oil. To a
solution of this oil in acetone (10 mL) was added dropwise
J ones’ reagent (12 mL) over 20 min at 0 °C and the reaction
mixture was stirred for an additional 1 h. The excess J ones’
reagent was quenched by the addition of MeOH, and the
reaction mixture was stirred for 15 min before additing 10%
NaCl solution. The reaction mixture was extracted with ether.
The organic layer was washed with brine and saturated
sodium hydrogen carbonate solution, dried over anhydrous
sodium sulfate, and concentrated in vacuo to give crude indan-
1-carboxylic acid (3.9 g, 64% yield) as a brownish oil. The crude
product was recrystallized from hexane/EtOAc to give indan-
1-carboxylic acid (2.0 g, 32% yield (4 steps starting from indan-
1-one)) as a yellowish crystal: IR (KBr disk) 2942, 2730, 1710,
1418, 1318, 1282, 1228, 940, 738 cm-1; 1H NMR (CDCl3) δ 7.42
(m, 1H), 7.20 (m, 3H), 4.08 (dd, J ) 6.3, 8.1 Hz, 1H), 3.12 (m,
1H), 2.93 (m, 1H), 2.40 (m, 2H).
2655, 2548, 1704, 1498, 1449, 1409, 1294, 1231, 1142, 943, 726,
1
697 cm-1; H NMR (CDCl3) δ 7.27 (m, 5H), 1.45 (s, 3H).
Meth yl 3,3,3-Tr ideu ter io-2-ph en ylpr opan oate (2j). Meth-
yl 3,3,3-trideuterio-2-phenylpropanoate (0.47 g, 96% yield) was
obtained as a colorless oil starting from methyl 2-phenylacetate
(0.44 g, 3.0 mmol) according to the same procedure as described
above for the synthesis of 2h except with trideuterio methyl
iodide instead of methyl iodide: IR (films) 3030, 2952, 2233,
1739, 1602, 1495, 1454, 1326, 1246, 1202, 1169, 1022, 943, 806,
1
768, 730, 698 cm-1; H NMR (CDCl3) δ 7.22 (m, 5H), 3.66 (s,
1H), 3.61 (s, 3H).
3,3,3-Tr id eu ter io-2-p h en ylp r op a n oic a cid (1j). 3,3,3-
Trideuterio-2-phenylpropanoic acid (0.20 g, 97% yield) was
obtained as a colorless oil starting from 2j (0.22 g, 1.3 mmol)
according to the same procedure as described above for the
synthesis of 1h : IR (film) 3032, 2929, 2722, 2233, 1705, 1601,
1497, 1455, 1416, 1290, 1226, 1186, 939, 731, 696 cm-1
.
Gen er a l P r oced u r e for th e Der a cem iza tion Rea ction
of R-Su b st it u t ed Ca r boxylic Acid s w it h t h e Aid of N.
d ia ph a n ozon a r ia J CM3208. The ingredients of the medium
were as follows: glycerol (10 g/L), peptone (2 g/L), beef extract
(3 g/L), yeast extract (3 g/L), KH2PO4 (1 g/L), K2HPO4 (1 g/L),
MgSO4‚7H2O (0.3 g/L), pH 7.0. To 90 mL of a nutrient medium
was added a suspension of 48-h incubated cells of N. diapha-
nozonaria in 10 mL of the broth and the incubation was carried
out at 30 °C for 24 h (first incubation). Then, 100 mg of (()-
R-substituted carboxylic acid was added to the suspension, and
the mixture was shaken for the appropriate time indicated in
the tables depending on the substrates (second incubation).
The reaction mixture was filtered through a pad of Celite to
remove the cells. The Celite was washed with EtOAc and the
washing was combined with the filtrate. After being acidified
by 2 M 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 treated with diazomethane and purified by pTLC
(hexane/EtOAc 9/1) to give the methyl ester of the starting
acid as a colorless oil. The incubation time, yield, and spectral
data of the esters are available as Supporting Information.
In h ibition Stu d y of Acyl-CoA Syn th eta se. To 90 mL of
a nutrient medium was added a suspension of 48-h incubated
cells of N. diaphanozonaria in 10 mL of the broth and the
incubation was carried out at 30 °C for 24 h (first incubation).
Then, the substrate (1a or 10c, 0.1 g) and appropriate amount
of the inhibitor (benzoic acid, palmitic acid, or n-alkanoic acid)
were added to the suspension, and the mixture was shaken
for 12 h (second incubation). The reaction mixture was filtered
through a pad of Celite to remove the cells. The Celite was
washed with EtOAc and the washing was combined with the
filtrate. After being acidified by 2 M hydrochloric acid, the mix-
ture was extracted with EtOAc. The organic layer was washed
with brine, dried over anhydrous sodium sulfate, and concen-
trated in vacuo. The residue was treated with diazomethane
and purified by pTLC (hexane/EtOAc 9/1) to give the methyl
ester of the starting acid as a colorless oil. The ee of the product
was determined by HPLC with a Daicel Chiralcel OJ column
(9/1 hexane/2-propanol; 0.5 mL/min; 254 nm).
Eth yl 2-(4-F lu or op h en oxy)p r op a n oa te (17b). Sodium
hydride (60% in paraffin, 80 mg, 2.0 mmol) was stirred for 5
min in hexane (5 mL) under Ar, then the solvent was removed
by a syringe followed by evaporation with a vacuum pump.
After the same operation was repeated three times, the residue
was suspended in tetrahydrofuran (5 mL) and the mixture was
cooled to 0 °C. To this mixture was added a solution of
4-fluorophenol (0.21 g, 1.9 mmol) in tetrahydrofuran (3 mL)
dropwise over 5 min, and the mixture was stirred for 5 min at
the same temperature. Then the mixture was allowed to warm
to room temperature and stirred for an additional 15 min. A
solution of ethyl 2-bromopropanoate (0.7 g, 3.9 mmol) in
tetrahydrofurane (2 mL) was added and the mixture was
stirred for 14 h. The reaction mixture was acidified by 2 M
hydrochloric acid and extracted with EtOAc. 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 (hexane/EtOAc 9/1) to give ethyl
2-(4-fluorophenoxy)propanoate (0.34 g, 86% yield) as a colorless
oil: IR (film) 2988, 2940, 1753, 1505, 1447, 1377, 1274, 1203,
1
1134, 1052, 829, 748 cm-1; H NMR (CDCl3) δ 6.96 (m, 2H),
6.83 (m, 2H), 4.67 (q, J ) 6.8 Hz, 1H), 4.22 (q, J ) 6.8 Hz,
2H), 1.60 (d, J ) 6.8 Hz, 2H), 1.25 (t, J ) 6.8 Hz, 3H).
2-(4-F lu or op h en oxy)p r op a n oic Acid (10b). A solution
of 17b (0.21 g, 1.0 mmol) in EtOH (3 mL) was added to an
aqueous solution (3 mL) of potassium hydroxide (0.62 g, 9.5
mmol) at 0 °C. After being stirred for 4 h, the reaction mixture
was acidified by 2 M hydrochloric acid and extracted with
EtOAc. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo to give
2-(4-fluorophenoxy)propanoic acid (0.18 g, quant) as a colorless
solid: IR (KBr disk) 2999, 2645, 1717, 1505, 1291, 1226, 1139,
922, 831, 757, 685, 516 cm-1
.
Compounds 10d -j, 12, 13, 14a , and 15 were prepared
according to a similar procedure. The spectral data of these
compounds are available as Supporting Information.
2-Deu ter io-2-p h en ylp r op a n oic Acid (1i). To a solution
of methyl 2-phenylpropanoate (1a , 2.0 g, 13.6 mmol) in
tetrahydrofuran (200 mL) was added dropwise n-butyllithium
(1.52 M in hexane, 8.8 mL) at -78 °C under Ar. After 1 h,
n-butyllithium (1.52 M in hexane, 17.5 mL) was added
dropwise. During this stage the colorless solution turned
yellow. The reaction mixture was stirred for an additional 100
min at -78 °C. Then the reaction mixture was allowed to warm
to 0 °C and deuterium chloride solution (30% in D2O, 5 mL)
was added. The yellow color immediately disappeared. After
60 min of stirring at room temperature, the solvent was
removed under reduced pressure and the residue was ex-
tracted with 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 chroma-
tography (hexane/EtOAc 3/1) to give 2-deuterio-2-phenylpro-
panoic acid (2.0 g, 98% yield) as a colorless oil: IR (film) 2982,
Ack n ow led gm en t. D.K. acknowledges support by
the Research Fellowships of the J apan Society for the
Promotion of Science for Young Scientists (J SPS Re-
search Fellowships for Young Scientists). This work was
also accomplished as a “Science and Technology Pro-
gram on Molecules, Supra-Molecules and Supra-Struc-
tured Materials” of an Academic Frontier Promotional
Project by the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT).
Su p p or tin g In for m a tion Ava ila ble: The spectral data
for 10d -j, 12, 13, 14a , and 15. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O034253X
7242 J . Org. Chem., Vol. 68, No. 19, 2003