Microbial deracemization of alpha-substituted carboxylic acids.

Article abstract of DOI:10.1021/ol0170556

An enzyme system of Nocardia diaphanozonaria JCM 3208 catalyzes the inversion of the chirality of various alpha-substituted carboxylic acids, such as 2-phenylpropanoic acid and 2-phenoxypropanoic acid derivatives, via a novel deracemization reaction.

Full text of DOI:10.1021/ol0170556

ORGANIC
LETTERS
2002
Vol. 4, No. 3
371-373
Microbial Deracemization of
r-Substituted Carboxylic Acids
,†
Dai-ichiro Kato,† Satoshi Mitsuda,‡ and Hiromichi Ohta*
Center for Life Science and Technology, Keio UniVersity, 3-14-1 Hiyoshi,
Kohoku-ku, Yokohama 223-8522, Japan, and Sumitomo Chemical Co. Ltd.,
4-2-1 Takatsukasa, Takarazuka 665-8555, Japan
hohta@chem.keio.ac.jp
Received November 15, 2001
ABSTRACT
An enzyme system of Nocardia diaphanozonaria JCM 3208 catalyzes the inversion of the chirality of various r-substituted carboxylic acids,
such as 2-phenylpropanoic acid and 2-phenoxypropanoic acid derivatives, via a novel deracemization reaction.
The methods of preparation of optically active compounds
are classified into two broad categories: optical resolution
of racemic compounds and asymmetrization of prochiral
compounds. Biocatalysts are widely utilized in both cases.1
When the starting material is a racemic mixture, the most
popular enzymatic approach to obtain the optically active
compounds is kinetic resolution. However, the maximum
theoretical yield is limited to 50% and the tedious procedures
for the separation of the recovered starting material and the
product are inevitable.
combination of sophisticated design of the substrate and
incubation conditions is the key to the successful procedure.
The most straightforward method is the synthesis of the target
molecule in racemic form and its conversion to the optically
active form.2c
In this Letter, we report a preparation of optically active
R-substituted carboxylic acids via a novel enzymatic dera-
cemization reaction using whole cells. This deracemization
reaction inverts the chirality of one enantiomer of a racemate
to the other antipode, resulting in an optically active
compound starting from a racemic mixture. Thus this process
is entirely different from the above-mentioned dynamic
resolution,2 because in this reaction the starting material and
the product are the same compound except for their chirality.
The most common enzymatic deracemization processes
previously reported are oxidation of sec-alcohol followed by
enantioselective reduction to the antipode.2c Here we present
a different option.
To overcome this drawback, there is a fascinating method
known as dynamic resolution.2 In this case, it is essential
that the starting material racemizes under the reaction
conditions, while the product does not. Accordingly, a
† Keio University
‡ Sumitomo Chemical Co. Ltd.
(1) (a) Faber, K. Biotransformations in Organic Chemistry, 4th ed.;
Spriger-Verlag: Berlin, 2000. (b) Drauz, K., Waldmann, H., Ed. Enzyme
Catalysis in Organic Synthesis; VCH: Weinheim, 1995. (c) Wong, C.-H.;
Whitesides, G. M. Enzymes in Synthetic Organic Chemistry; Pergamon:
Oxford, 1994.
(2) (a) Stecher, H.; Faber, K. Synthesis 1996, 1-16. (b) Caddick, S.;
Jenkins, 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.
The (S)-enantiomers of 2-arylpropanoic acid derivatives
constitute a subgroup of the widely used nonsteroidal
antiinflammatory drugs,3 while the (R)-enantiomer is either
inactive or only weakly active in vivo. The preparation of
an optically pure enaniomer is extremely important for these
10.1021/ol0170556 CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/09/2002

Table 1. Deracemization of R-Substituted Phenylacetic Acids by the Aid of the Enzyme System of N. diaphanozonaria
c
entry
starting compd
(()-1a
(R)-1a (88% ee)
(S)-1a (93% ee)
(()-1be (D content: 98%)
(()-1ce (D content: 100%)
(()-1d
reactn time (h)
producta
yieldb (%)
[R]D
eed (%)
1
2
3
4
5
6
7
48
48
48
48
48
16
48
(R)-1a
(R)-1a
(R)-1a
81
58
48
66
61
58
74
-71.3
69
72
52
78
71
6
(R)-1a e (D content: 20%)
(R)-1a e (D content: 100%)
(R)-1c
-5.6
(()-1e
(R)-1d
55
a The corresponding starting compounds were incubated with grown cells of N. diaphanozonaria at 30 °C. b Isolated yield after conversion to the
corresponding methyl ester by treatment with diazomethane. c Optical rotation was measured in EtOH, c ) 1.00-1.03 after conversion to the corresponding
methyl ester. d ee (%) of the product was determined by HPLC analysis after conversion to the corresponding methyl ester. e D content was calculated from
1H NMR analysis.
compounds, and some of these approaches have been
previously reported.4,5
the inversion of the chirality of 2-phenylpropanoic acid from
(S)- to the (R)-configuration. To the best of our knowledge,
this is the first example of inverting the chirality of
2-arylpropanoic acid derivatives from S to R.
Only a few reports are known for the enzymatic derace-
mization of 2-arylpropanoic acid, i.e., the use of rat liver6,7
and two fungi, Cordyceps militaris3,8 and Verticillium leca-
nii,9 which are capable of inverting the chirality of 2-aryl-
propanoic acid derivatives from the (R)-enantiomer to its (S)-
antipode. In this case, the mechanism of the biotransformation
system was proposed based on various studies using enan-
tiomerically pure compounds and deuterated derivatives.3,6,8,9
These studies, however, have been carried out from the
perspective of the pharmacological effect of racemic com-
pounds and were intended to clarify the destiny of two
enantiomers in vivo. As described above, this type of reaction
is also interesting from the standpoint of synthetic chemistry.
Thus, we tried to examine the applicability of such a novel
type of reaction as well as the substrate specificities and
mechanism of the reaction.
The best incubation conditions so far obtained for 2-
phenylpropanoic acid are as follows. To 90 mL of a nutrient
medium was added a suspension of 48 h incubated cells of
N. diaphanozonaria in 10 mL of broth, and the incubation
was carried out at 30 °C for 24 h (first incubation). Then,
0.1% w/v of (()-2-phenylpropanoic acid was added to the
suspension of the grown cells, and the mixture was shaken
for another 48 h (second incubation). Extraction of the broth,
followed by treatment with diazomethane and subsequent
purification of the product, gave the methyl ester of the
starting acid. The yield and enantiomeric excess were
determined to be 81% and 69% (R), respectively. Elongation
of the time of the second incubation resulted in the
remarkable decrease of the yield and enantiomeric excess
of the product.
There are two possible paths for the asymmetrization of
the substrates. One is deracemization of the substrate via
some mechanism and the other is the enantioselective
degradation of the (S)-enantiomer. The latter is supposed to
be a minor path, if at all, based on the yield and the ee
mentioned above. To clarify, if the enantioselective degrada-
tion path was actually working, optically active 2-phenyl-
propanoic acid was subjected to the reaction (Table 1).
Regardless of the configuration of the starting material (88%
R or 93% S), the recovery of the product was around 70%
and the configuration was R (72% ee from R and 52% ee
from S-starting acid). These results indicate that the enan-
tioselective degradation process is very unlikely in this
system. In addition, when (()-2-deuterio-2-phenylpropanoic
acid (D content 98%) was incubated with the whole cells of
After some screening, we found a deracemization activity
in a type of actinomycetes, Nocardia diaphanozonaria JCM
3208.10 The enzyme system of N. diaphanozonaria catalyzes
(3) Rhys-Williams, W.; McCarthy, F.; Backer, J.; Hung, Y.-F.; Thoma-
son, M. J.; Lloyd, A. W.; Hanlon, G. W. Enzyme Microb. Technol. 1998,
22, 281-287 and reference cited therein.
(4) (a) Wu, S.-H.; Guo, Z.-W.; Sih, C. J. J. Am. Chem. Soc. 1990, 112,
2, 1990-1995. (b) Cambou, B.; Klibanov, A. M. Biotechnol. Bioeng. 1984,
XXVI, 1449-1454. (c) Liu, Y.-Y.; Xu, J.-H.; Hu, Y. J. Mol. Catal. B:
Enzymol. 2000, 10, 523-529.
(5) Colton, I. J.; Ahmed, S. N.; Kazlauskas, R. J. J. Org. Chem. 1995,
60, 212-217.
(6) (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.
(7) (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.
(8) Rhys-Williams, W.; Thomason, M. J.; Hung, Y.-F.; Hanolon, G. W.;
Lloyd, A. W. Chirality 1998, 10, 528-534.
(9) Rhys-Williams, W.; Thomason, M. J.; Lloyd, A. W.; Hanlon, G. W.
Pharm. Sci. 1996, 2, 537-540.
(10) The strain 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.
372
Org. Lett., Vol. 4, No. 3, 2002

N. diaphanozonaria, the D content of the product decreased
to 20%. Clearly the reaction path includes C-D bond fission.
On the other hand, (()-3,3,3-trideuteriophenylpropanoic acid
was recovered as its optically active form without losing any
deuterium atom. This fact indicates that no C-D bond fission
occurred on the methyl group, such as formation of meth-
ylene followed by reduction to the methyl group. Thus, the
most probable mechanism is the formation of enolate-type
intermediate as proposed by W-.R. Shieh et al.7a The direct
binding of the phenyl group to the R-position will facilitate
the formation of such an intermediate.
Table 2. Deracemization of 2-Phenoxypropanoic Acids by the
Aid of the Enzyme System of N. diaphanozonaria
entry producta
X
reactn time (h) yieldb (%) [R]D eed (%)
c
Introduction of a fluorine atom on the p-position is
expected to stabilize the enolate-type intermediate by its
electronegative effect and accelerate the reaction. However,
with 2-(4-fluorophenyl)propanoic acid, the apparent chiral
inversion process did not occur to any significant extent
(entry 6, Table 1). This fact shows that this enzyme system
is affected dramatically by the electronic effect of the
substituent on the aromatic ring. Unfortunately, other p-
substituted derivatives that have a chlorine atom or a methoxy
group did not appear to undergo the deracemization reaction.
The enzyme system accepted a compound that has a
fluorine atom on the R-carbon instead of a methyl group.
R-Fluorophenylacetic acid underwent the deracemization
reaction to give the corresponding optically active form (entry
7, Table 1). On the other hand, the enzyme system was
shown to be inactive to 2-phenylbutanoic acid, probably
because of the steric bulkiness of the R-substituent.
In the second group of substrates, we used 2-phenoxypro-
panoic acid derivatives.11 (R)-2-Phenoxypropanoic acid
derivatives are utilized as herbicides, while the (S)-antipodes
are inactive. In addition, (R)-2-(4-chlorophenoxy)propanoic
acid lowers the level of serum cholesterol and prevents
platelet aggregation.5 As the (S)-antipode inhibits the chloride
channel in muscles, it causes side effects, such as muscle
irritability and spasms.
1
2
3
4
5
6
7
8
9
2a
2a
2a
2a
2b
2c
2c
2d
2e
2f
H
H
H
H
36
48
60
72
48
12
48
48
48
48
72
75
66
71
64
75
95
83
76
64
68
75
91
97
96
92
97
99
97
99
+41.0
+41.2
+52.0
F
Cl
Cl
Br
I
+44.5
+47.5
+43.4
+43.3
10
CF3
a The corresponding racemic substrates were incubated with grown cells
of N. diaphanozonaria at 30 °C. b Isolated yield after conversion to the
corresponding methyl ester by treatment with diazomethane. c Optical
rotation was measured in EtOH, c ) 0.71-1.05, except for entry 4 in which
CHCl3 was used: c ) 0.58, after conversion to the corresponding methyl
ester. d ee (%) of the product was determined by HPLC analysis after
conversion to the corresponding methyl ester.
to that of 2-phenylpropanoic acid. The mode of the inter-
action between the enzyme and the substrates is not clear at
present, but it is very interesting that insertion of only one
atom brought about a dramatic change in enantioselectivity.
In this case, introduction of an electronegative substituent
on the aromatic ring had no negative effect on the reaction,
in contrast to the case of 2-phenylpropanoic acid (entries
5-10, Table 2). As seen in entry 7, 2-(4-chlorophenoxy)-
propanoic acid deracemized smoothly, with the yield and
ee of the product being as high as 95% and 97% (R),
respectively.
In conclusion, we found an enzyme system that gives (R)-
2-phenoxypropanoic acid, which is the pharmacologically
active enantiomer, via a novel deracemization reaction. This
process can be performed under mild reaction conditions
using an extremely simple procedure and gives good to high
yield and enantiomeric excess. Further investigation on the
scope and limitation of the reaction, as well as the mecha-
nistic study, is now underway.
As these type of compounds have an oxygen atom between
the aromatic ring and the asymmetric center, we presumed
that it was unfavorable for the reaction. To our surprise,
however, the deracemization reaction of 2-phenoxypropanoic
acid (entry 1 to 4, Table 2) cleanly proceeded, and enantio-
meric excess of the product reached up to 97% after a 72 h
incubation. Moreover, judging from the sign of optical
rotation, the absolute configuration of the product was
revealed to be R.12 This means that the spacial arrangement
of the ligands around the asymmetric center was opposite
(11) (a) Guo, Z.-W.; Sih, C. J. J. Am. Chem. Soc. 1989, 111, 6836-
6841. (b) Bewick, D. W. Eur. Pat. Appl. EP 133,033 (Chem. Abstr. 1985,
102, 165249a). (c) Bewick, D. W. Eur. Pat. Appl. EP 133,034 (Chem. Abstr.
1985, 102, 165251v).
(12) Chordia, M. D.; Harman, W. D. J. Am. Chem. Soc 2000, 122, 2725-
2736.
Supporting Information Available: Synthetic details and
spectroscopic data for a representative product. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL0170556
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