1
SPECIFICATION
PROCESS FOR PREPARING CARBOXYLIC ACID USING SURFACTANT-
MODIFIED ENZYME
TECHNICAL FIELD
[0001]
The present invention relates to a process for
preparing a carboxylic acid by hydrolyzing a carboxylic
acid ester in the presence of a surfactant-modified enzyme
in a uniform solvent comprising water, and an organic
solvent which forms a uniform phase with water, in parti-
cular, it relates to a process for preparing an optically
active carboxylic acid (for example, an a or -amino acid)
by hydrolyzing a carboxylic acid ester (racemic mixture,
for example, an a or p-amino acid ester (racemic mixture))
having an asymmetric carbon atom at other than an ester
portion. Incidentally, the surfactant-modified enzyme of
the present invention means a material in which an enzyme
surface is covered by a surfactant, and, in view of a
chemical aspect, the surface of the enzyme and a hydro-
phiiic group(s) of the surfactant are binding with an
interaction such as a hydrogen bonding, etc. The carbo-
xylic acid, in particular, an optically active carboxylic
acid (for example, an optically active a or p-amino acid)
to be obtained according to the present invention is useful
as a starting material or a synthetic intermediate for
medicinal and agricultural chemicals, or for physiological-
ly active substances such as physiologically active
peptides or lactam series antibiotics, etc. (for example,
see Patent Literatures 1-3).
BACKGROUND ART
[0002]
Heretofore, as a method for preparing a carboxylic
acid by hydrolyzing a carboxylic acid ester in the presence

2
of a surfactant-modified enzyme, it has been disclosed a
method in which triglyceride is hydrolyzed in the presence
of a surfactant-coated lipase in isooctane to obtain a
mixture of triolein, diolein and monoolein (for example,
see Non-Patent Literature 1). However, according to this
method, there is no description other than hydrolysis of
triglyceride.
[0003]
On the other hand, as a method of preparing an
optically active a or -amino acid ester by hydrolyzing a
carboxylic acid ester which is an a or p-amino acid ester
(racemic mixture), it has been disclosed a method in which,
for example, one of enantiomers of ethyl 3-amino-3-aryl-
propionate (racemic mixture) is selectively hydrolyzed in
water in the presence of a lipase (Trade name: Amano PS)
originated from Burkholderia cepacia {Pseudomonas cepacia)
to obtain an optically active (S)-3-amino-3-arylpropionic
acid and an optically active ethyl (R)-3-amino-3-aryl-
propionate (for example, see Non-Patent Literature 2.) .
[0004]
However, according to this method, an E value which
is an index of selectivity between enantiomers by an enzyme
is low, and when the produced optically active carboxylic
acid is water-soluble, it is difficult to recover the
product with a yielded amount of 100% from an aqueous
solution after completion of the reaction. In addition,
there is a problem that, in the presence of a large amount
of water, optical purity is lowered due to self-hydrolysis
of the substrate. Incidentally, the E value is widely
utilized as an index of selectivity of kinetic optical
resolution (for example, see Non-Patent Literature 3.).
[0005]
Also, as a method of obtaining an optically active
3-amino-3-arylpropionic acid, it has been known a method in
which good yield and good optical purity can be accom-
plished by making an ester portion propyl ester (for

3
example, see Patent Literature 4).
[0006]
However, according to this method, a large amount of
water must be used, so that there is a problem that reac-
tion operation is complicated, for example, a pH adjustment
of an aqueous phase is indispensable, etc. Incidentally,
as an optically active p-amino acid ester, a methyl ester
or an ethyl ester is desired to be prepared in many cases,
so that the resulting optically active -amino acid propyl
ester must be led to a desired methyl ester or an ethyl
ester by interesterification, etc., whereby it is not an
efficient method.
[0007]
Moreover, as a method of obtaining an optically
active 3-amino-3-arylpropionic acid, it has been known a
method which can realize high enantio-selectivity by
carrying out enzymatic hydrolysis of a 3-amino-3-aryl-
propionic acid ester (racemic compound) in a two-phase
system comprising water and an organic solvent (for
example, see Patent Literature 5).
[0008]
However, according to this method, when the produced
optically active carboxylic acid is water-soluble, it is
difficult to recover 100% of the product from an aqueous
solution after completion of the reaction, and there is a
problem that optical purity of the product is lowered due
to self-hydrolysis of the substrate in the presence of a
large amount of water.
Non-Patent Literature 1: Chemical Engineering theses,
11(3) , 424 (1993)
Non-Patent Literature 2: Tetrahedron Lett., 41, 2679(2000)
Non-Patent Literature 3: J. Am. Chem. Soc., 104, 7294(1982)
Non-Patent Literature 4: J. Org. Chem., .60., 2244(1995)
Non-Patent Literature 5: "Chemical Dictionary", published
by Tokyo Kagaku Dozin Co., Ltd., p.948 (2000)

4
Non-Patent Literature 6: J. Member. Sci., 19, 237(1984)
Patent Literature 1: WO2001/042192
Patent Literature 2: WO2004/092116
Patent Literature 3: US2003/0199692
Patent Literature 4: JP 2003-325195 A
Patent Literature 5: JP 2003-325197 A
Patent Literature 6: JP 64-80282 A
Patent Literature 7: JP 4-23981 A
Patent Literature 8: JP 6-269285 A
Patent Literature 9: JP 9-118844 A
Patent Literature 10: JP 10-248558 A
Patent Literature 11: Japanese Patent No. 3218794
DISCLOSURE OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0009]
An object of the present invention is to solve the
above-mentioned problems, and to provide a process for
preparing a carboxylic acid by hydrolyzing a carboxyiic
acid ester with a simple and easy method, in particular, to
provide a process for preparing an optically active (S or
R) - a or p-substituted carboxylic acid and an optically
active (R or S)-a or p-substituted carboxylic acid ester
simultaneously by hydrolyzing a carboxylic acid ester
(racemic mixture) having an asymmetric carbon atom at a
portion other than an ester portion, for example, an a or
p-substituted carboxylic acid ester (racemic mixture) with
high E value.
MEANS TO SOLVE THE PROBLEMS
[0010]
The problems of the present invention can be solved
by a process for preparing a carboxylic acid which
comprises selectively reacting water and a carboxylic acid
ester, provided that triglyceride is excluded, in an
organic solvent in the presence of a surfactant-modified

5
enzyme.
BEST MODE FOR CARRYING OUT THE INVENTION
[0011]
The carboxylic acid ester to be used in the hydroly-
sis of the present invention is not specifically limited,
and is preferably a carboxylic acid ester (racemic mixture)
having an asymmetric carbon atom at other than an ester
portion, more preferably an a or p-substituted carboxylic
acid ester (racemic mixture; hereinafter referred to as
Compound (I)) represented by the formula (I):
[0012]

[0013]
wherein R represents an alkyl group, alkenyl group,
alkynyl group, cycloalkyl group, aralkyl group, aryl
group or heteroaryl group, each of which may have a
substituent(s), R1 represents an alkyl group which
may have a substituent(s), Z represents an amino
group which may have a protective group, a hydroxyl
group which may have a protective group or an alkyl
group, * represents an asymmetric carbon atom, and n
is 0 or 1.
[0014]
R in Compound (I) represents an alkyl group, alkenyl
group, alkynyl group, cycloaikyi group, araikyl group, aryl
group or heteroaryl group, each of which may have a substi-
tuent (s) .
[0015]
As the alkyl group of an alkyl group which may have
a substituent(s) in the above-mentioned R, there may be
mentioned a straight or branched alkyl group having 1 to 10
carbon atoms, for example, an alkyl group such as a methyl

6
group, ethyl group, propyl group, butyl group, pentyl
group, hexyl group, heptyl group, octyl group, nonyl group,
decyl group, etc., preferably an alkyl group having 1 to 8
carbon atoms such as a methyl group, ethyl group, n-propyl
group, isopropyl group, n-butyl group, n-octyl group, etc.,
more preferably a methyl group, or ethyl group. Incident-
ally, these groups contain various kinds of isomers.
[0016]
As the substituent(s) of the alkyl group which may
have a substituent(s), there may be mentioned, for example,
a halogen atom such as a fluorine atom, chlorine atom,
bromine atom, iodine atom, etc.; a hydroxyl group; an
alkoxy group having 1 to 4 carbon atoms such as a methoxy
group, ethoxy group, propoxy group, butoxy group, etc.; an
amino group; a dialkylamino group such as a dimethylamino
group, diethylamino group, etc.; a cyano group; or a nitro
group, preferably a fluorine atom, chlorine atom, hydroxyl
group, amino group, or dialkylamino group.
[0017]
As the alkyl group having such a substituent(s),
there may be mentioned, for example, a fluoromethyl group,
chloromethyl group, hydroxymethyl group, methoxymethyl
group, aminomethyl group, dimethylaminomethyl group, 2-
chloroethyl group, 2,2-dichloroethyl group, 2-hydroxyethyl
group, and 2-cyanoethyl group, etc., preferably a fluoro-
methyl group, chloromethyl group, hydroxymethyl group,
aminomethyl group, dimethylaminomethyl group, 2-chloroethyl
group, or 2-cyanoethyl group.
[0018]
As the alkenyl group of an alkenyl group which may
have a substituent(s) in the above-mentioned R, there may
be mentioned, for example, an alkenyl group having 2 to 10
carbon atoms such as a vinyl group, propenyl group, butenyl
group, pentenyl group, hexenyl group, heptenyl group,
octenyl group, nonenyl group, decenyl group, etc., prefer-
ably an alkenyl group having 2 to 7 carbon atoms such as a

7
vinyl group, propenyl group, butenyl group, pentenyl group,
etc., more preferably an alkenyl group having 2 to 3 carbon
atoms such as a vinyl group, 1-propenyl group, 2-propenyl
group, etc. Incidentally, these groups contain various
kinds of isomers.
[0019]
As the substituent(s) of the alkenyl group which may
have a substituent(s), there may be mentioned, for example,
a halogen atom such as a fluorine atom, chlorine atom,
bromine atom, iodine atom, etc.,- a hydroxyl group; an
alkoxyl group having 1 to 4 carbon atoms such as a methoxyl
group, ethoxyl group, propoxyl group, butoxyl group, etc.;
an amino group; a dialkylamino group such as a dimethyl-
amino group, diethylamino group, etc.; a cyano group; or a
nitro group, preferably a fluorine atom, chlorine atom,
hydroxyl group, amino group, or dialkylamino group.
[0020]
As the alkenyl group having such a substituent(s),
there may be mentioned, for example, a 1-fiuoroethenyi
group, 1-chloroethenyl group, 1-hydroxyethenyl group, 1-
methoxyethenyl group, 1-aminoethenyl group, 1-cyanoethenyl
group, 2-fIuoroethenyi group, 2-chloroethenyl group, 2-
hydroxyethenyl group, 2-methoxyethenyl group, 2-amino-
ethenyl group, 2-cyanoethenyl group, 1,2-dimethylamino-
ethenyl group, 1-fluoro-2-propenyl group, l-chloro-2-
propenyl group, 1-hydroxy-2-propenyl group, l-methoxy-2-
propenyl group, l-amino-2-propenyl group, i-cyano-2-
propenyl group, 3-fluoro-1-propenyl group, 3-chloro-l-
propenyl group, 3-hydroxy-2-propenyl group, 3-methoxy-2-
propenyl group, 3-amino-2-propenyl group, 2-cyano-2-
propenyl group, 3,3-dimethylamino-2-propenyl group, 3,3-
dichloro-2-propenyl group, etc., preferably 1-fIuoroethenyi
group, 1-chloroethenyl group, 1-hydroxyethenyl group, 1-
aminoethenyl group, 1-cyanoethenyl group, 1-fluoro-2-
propenyl group, 1-chloro-2-propenyl group, and 1-cyano-2-
propenyl group.

8
[0021]
As the alkynyl group of an alkynyl group which may
have a substituent(s) in the above-mentioned R, there may
be mentioned, for example, an alkynyl group having 2 to 10
carbon atoms such as an ethynyl group, propynyl group,
butynyl group, pentynyl group, hexynyl group, heptynyl
group, octynyl group, nonynyl group, decynyl group, etc.,
preferably an alkynyl group having 2 to 7 carbon atoms such
as an ethynyl group, propynyl group, butynyl group,
pentynyl group, etc., more preferably an alkynyl group
having 2 to 3 carbon atoms such as an ethynyl, 1-propynyl
group, 2-propynyl group, etc. Incidentally, these groups
contain various kinds of isomers.
[0022]
As the substituent(s) of the alkynyl group which may
have a substituent(s), there may be mentioned, for example,
a halogen atom such as a fluorine atom, chlorine atom,
bromine atom, iodine atom, etc.; a hydroxyl group; an
alkoxyl group having 1 to 4 carbon atoms such as a methoxyl
group, ethoxyl group, propoxyl group, butoxyl group, etc.;
an amino group; a dialkylamino group such as a dimethyl-
amino group, diethylamino group, etc.; a cyano group; and a
nitro group, preferably a fluorine atom, chlorine atom,
hydroxyl group, amino group, and dialkylamino group.
[0023]
As the alkynyl group having such a substituent(s),
there may be mentioned, for example, a 2-fiuoroethynyi
group, 2-chloroethynyl group, 2-hydroxyethynyl group, 2-
methoxyethynyl group, 2-aminoethynyl group, 2-cyanoethynyl
group, 1-fluoro-2-propynyl group, 1-chloro-2-propynyl
group, 1-hydroxy-2-propynyl group, 1-methoxy-2-propynyl
group, l-amino-2-propynyl group, 1-cyano-2-propynyl group,
1,l-dichloro-2-propynyl group, and 1, 1-diamino-2-propynyl
group, etc., preferably a 2-fIuoroethynyi group, 2-chloro-
ethynyl group, 2-hydroxyethynyl group, 2-aminoethynyl
group, 1-fluoro-2-propynyl group, and 1,l-dichloro-2-

9
propynyl group.
[0024]
The cycloalkyl group of a cycloalkyl group which may
have a substituent(s) in the above-mentioned R is a
cycloalkyl group having 3 to 10 carbon atoms, and there may
be mentioned, for example, a cycloalkyl group such as a
cyclopropyl group, cyclobutyl group, cyclopentyl group,
cyclohexyl group, cycloheptyl group, cyclooctyl group,
cyclononyl group, and cyclodecyl group (Incidentally, these
groups contain various kinds of isomers), preferably a
cycloalkyl group having 3 to 8 carbon atoms such as a
cyclopropyl group, cyclobutyl group, cyclopentyl group,
cyclohexyl group, cycloheptyl group, and cyclooctyl group,
etc., more preferably a cycloalkyl group having 3 to 6
carbon atoms such as a cyclopropyl group, cyclobutyl group,
cyclopentyl group, and cyclohexyl group, etc.
[0025]
As the substituent(s) of the cycloalkyl group which
may have a substituent(s), there may be mentioned an alkyl
group having 1 to 6 carbon atoms, a halogen atom such as a
fluorine atom, chlorine atom, bromine atom, iodine atom,
etc.; a hydroxyl group; an alkoxyl group having 1 to 4
carbon atoms such as a methoxyl group, ethoxyl group,
propoxyl group, butoxyl group, etc.; an amino group; a
dialkylamino group having 2 to 12 carbon atoms such as a
dimethylamino group, diethylamino group, etc.; a cyano
group; and a nitro group, preferably a fluorine atom,
chlorine atom, hydroxyl group, amino group, and dialkyl-
amino group.
[0026]
As the cycloalkyl group which may have such a sub-
stituent (s) , there may be mentioned, for example, a 1-
fluorocyclopropyl group, 2-chlorocyclopropyl group, 3-
fluorocyclobutyl group, methoxycyclopropyl group, amino-
cyclopentyl group, dimethylaminocyclohexyl group, 2-chloro-
cyclopropyl group, 2,2-dichlorocyclohexyl group, 2-hydroxy-

10
cyclobutyl group, and 2-cyanocyclohexyl group, etc.,
preferably a fluorocyclopropyl group, and chlorocyclobutyl
group.
[0027]
The aralkyl group of an aralkyl group which may have
a substituent(s) in the above-mentioned R, there may be
mentioned, for example, an aralkyl group such as a benzyl
group, phenethyl group, phenylpropyl group, and phenylbutyl
group, etc., preferably a benzyl group, 1-phenethyl group,
2-phenethyl group, 3-phenylpropyl group, and 3-phenylbutyl
group. Incidentally, these groups contain various kinds of
isomers.
[0028]
As the substituent(s) of the aralkyl group which may
have a substituent(s), there may be mentioned, for example,
an alkyl group having 1 to 10 carbon atoms such as a methyl
group, ethyl group, propyl group, butyl group, pentyl
group, hexyl group, heptyl group, octyl group, nonyl group,
decyl group, etc. (Incidentally, these groups contain
various kinds of isomers.); a hydroxyl group; a nitro
group; a halogen atom such as a fluorine atom, chlorine
atom, bromine atom, iodine atom, etc.; an alkoxyl group
having 1 to 10 carbon atoms such as a methoxyl group,
ethoxyl group, propoxyl group, butoxyl group, pentyloxyl
group, hexyloxyl group, heptyloxyl group, octyloxyl group,
nonyloxyl group, decyloxyl group, etc. (Incidentally, these
groups contain various kinds of isomers.); an aralkyloxyi
group having 7 to 10 carbon atoms such as a benzyloxyl
group, phenethyloxyl group, phenylpropoxyl group, etc.
(Incidentally, these groups contain various kinds of
isomers.); an aryloxyl group such as a phenyloxyl group,
nephthyloxyl group, etc. (Incidentally, these groups
contain various kinds of isomers.); an alkoxyalkoxyl group
having 2 to 12 carbon atoms such as a methoxymethoxyl
group, methoxyethoxyl group, etc. (Incidentally, these
groups contain various kinds of isomers.); a monoalkylamino

11
group having 1 to 6 carbon atoms such as a raethylamino
group, ethylamino group, etc. (Incidentally, these groups
contain various kinds of isomers.); a dialkylamino group
having 2 to 12 carbon atoms such as a dimethylamino group,
diethylamino group, etc. (Incidentally, these groups
contain various kinds of isomers.); an acylamino group
having 1 to 6 carbon atoms such as a formylamino group,
acetylamino group, benzoylamino group, etc. (Incidentally,
these groups contain various kinds of isomers.); a nitro
group; a cyano group; a halogenated alkyl group having i to
6 carbon atoms such as a trifluoromethyl group, etc.
[0029]
As the araikyl group having such a substituent (s) ,
there may be specifically mentioned, for example, a 2-
fluorobenzyl group, 3-fluorobenzyl group, 4-fluorobenzyl
group, 3,4-difluorobenzyl group, 2,4-difluorobenzyl group,
2-chlorobenzyl group, 3-chlorobenzyl group, 4-chlorobenzyl
group, 2,4-dichlorobenzyl group, 3,4-dichlorobenzyl group,
2-bromobenzyl group, 3-bromobenzyl group, 4-bromobenzyl
group, 2,4-dibromobenzyl group, 3,4-dibromobenzyl group, 2-
iodobenzyl group, 3-iodobenzyl group, 4-iodobenzyl group,
2,3-diiodobenzyl group, 3,4-diiodobenzyl group, 2-methyl-
benzyl group, 3-methylbenzyl group, 4-methylbenzyl group,
2-ethylbenzyl group, 3-ethylbenzyl group, 4-ethylbenzyl
group, 2-hydroxybenzyl group, 3-hydroxybenzyl group, 4-
hydroxybenzyl group, 2-methoxybenzyl group, 3-methoxybenzyl
group, 4-methoxybenzyl group, 2,4-dimethoxybenzyi group,
3,4-dimethoxybenzyl group, 2-ethoxybenzyl group, 4-ethoxy-
benzyl group, 2-trifluoromethylbenzyl group, 4-trifluoro-
methylbenzyl group, 4-benzyloxybenzyl group, 2-nitrobenzyl
group, 3-nitrobenzyl group, 4-nitrobenzyl group, 2-cyano-
benzyl group, 3-cyanobenzyl group, 4-cyanobenzyl group, 4-
dimethylaminobenzyl group, 4-formylaminobenzyl group, 2-
acetylaminobenzyl group, 3-acetylaminobenzyl group, 4-
acetylaminobenzyl group, 4-benzoylaminobenzyl group, 2-(2-
fluorophenyl)ethyl group, 2-(3-fluorophenyl)ethyl group, 2-

12
(4-fluorophenyl)ethyl group, 2-(3,4-difluorophenyl)ethyl
group, 2-(2,4-difluorophenyl)ethyl group, 2-(2-chloro-
phenyl)ethyl group, 2-(3-chlorophenyl)ethyl group, 2-(4-
chlorophenyl)ethyl group, 2-(2,4-dichlorophenyl)ethyl
group, 2-(3,4-dichlorophenyl)ethyl group, 2-(2-bromo-
phenyl)ethyl group, 2-(3-bromophenyl)ethyl group, 2-(4-
bromophenyl)ethyl group, 2-(2,4-dibromophenyl)ethyl group,
2-(3,4-dibromophenyl)ethyl group, 2-(2-iodophenyl)ethyl
group, 2-(3-iodophenyl)ethyl group, 2-(4-iodophenyl)ethyl
group, 2-(2,3-diiodophenyl)ethyl group, 2-(3,4-diiodo-
phenyl)ethyl group, 2-(2-tolyl)ethyl group, 2-(3-tolyl)-
ethyl group, 2-(4-tolyl)ethyl group, 2-(2-ethylphenyl)ethyl
group, 2-(3-ethylphenyl)ethyl group, 2-(4-ethylphenyl)ethyl
group, 2-(2-hydroxyphenyl)ethyl group, 2-(4-hydroxyphenyl)-
ethyl group, 2-(2-methoxyphenyl)ethyl group, 2-(3-methoxy-
phenyl)ethyl group, 2-(4-methoxyphenyl)ethyl group, 2-(2, 4-
dimethoxyphenyl)ethyl group, 2-(3,4-dimethoxyphenyl)ethyl
group, 2-(2-ethoxyphenyl)ethyl group, 2-(4-ethoxyphenyl)-
ethyl group, 2-(2-trifluoromethylphenyl)ethyl group, 2-(4-
trifluoromethylphenyl)ethyl group, 2-(4-benzyloxyphenyl)-
ethyl group, 2-(2-nitrophenyl)ethyl group, 2-(3-nitro-
phenyl)ethyl group, 2-(4-nitrophenyl)ethyl group, 2-(2-
cyanophenyl)ethyl group, 2-(3-cyanophenyl)ethyl group, 2-
(4-cyanophenyl)ethyl group, 2-(4-dimethylaminophenyl)ethyl
group, 2-(4-formylaminophenyl)ethyl group, 2-(2-acetyl-
aminophenyl)ethyl group, 2-(3-acetylaminophenyl)ethyl
group, 2-(4-acetylaminophenyl)ethyl group, 2-(4-benzoyl-
aminophenyl)ethyl group, 3-(2-fluorophenyl)propyl group, 3-
(4 -fluorophenyl)propyl group, 3 -(4 -chlorophenyl)propyl
group, 3 -(4-bromophenyl)propyl group, 3-(4-iodophenyl)-
propyl group, 3-(2-chlorophenyl)propyl group, 3 -(2-methoxy-
phenyl) propyl group, 3-(4-methoxyphenyl)propyl group, 3-
(3 , 4-dimethoxyphenyl)propyl group, 3-(4-trifluoromethyl-
phenyl)propyl group, 3-(2-trifluoromethylphenyl)propyl
group, 3-(4-nitrophenyl)propyl group, 3-(4-cyanopheny1)-
propyl group, 3-(4-acetylaminophenyl)propyl group, etc.,

13
preferably 2-fluorobenzyl group, 3-fluorobenzyl group, 4-
fluorobenzyl group, 2-chlorobenzyl group, 3-chlorobenzyl
group, 4-chlorobenzyl group, 2-bromobenzyl group, 3-bromo-
benzyl group, 4-bromobenzyl group, 2-iodobenzyl group, 3-
iodobenzyl group, 4-iodobenzyl group, 2-methylbenzyl group,
3-methylbenzyl group, 4-methylbenzyl group, 2-hydroxybenzyl
group, 4-hydroxybenzyl group, 2-methoxybenzyl group, 3-
methoxybenzyl group, 4-methoxybenzyl group, 3,4-dimethoxy-
benzyl group, 2-trifluoromethylbenzyl group, 4-trifluoro-
methylbenzyl group, 4-benzyioxybenzyl group, 2-nitrobenzyl
group, 3-nitrobenzyl group, 4-nitrobenzyl group, 2-cyano-
benzyl group, 3-cyanobenzyl group, 4-cyanobenzyl group, 4-
formylaminobenzyl group, 3-acetylaminobenzyl group, 4-
acetylaminobenzyl group, 4-benzoylaminobenzyl group, 2-(2-
fluorophenyl)ethyl group, 2-(3-fluorophenyl)ethyl group, 2-
(4-fluorophenyl)ethyl group, 2-(2-chlorophenyl)ethyl group,
2 -(3 -chlorophenyl)ethyl group, 2 -(4 -chlorophenyl)ethyl
group, 2-(2-bromophenyl)ethyl group, 2-(3-bromophenyl)ethyl
group, 2-(4-bromophenyl)ethyl group, 2-(2-iodophenyl)ethyl
group, 2-(3-iodophenyl)ethyl group, 2-(4-iodophenyl)ethyl
group, 2-(2-tolyl)ethyl group, 2-(3-tolyl)ethyl group, 2-
(4-tolyl)ethyl group, 2-(2-ethylphenyl)ethyl group, 2-(2-
hydroxyphenyl)ethyl group, 2-(4-hydroxyphenyl)ethyl group,
2 -(2-methoxyphenyl)ethyl group, 2 -(3-methoxyphenyl)ethyl
group, 2-(4-methoxyphenyl)ethyl group, 2-(2,4-dimethoxy-
phenyl)ethyl group, 2-(3,4-dimethoxyphenyl)ethyl group, 2-
(2-trifluoromethylphenyl)ethyl group, 2-(4-trifluoromethyl-
phenyl)ethyl group, 2-(4-benzyloxyphenyl)ethyl group, 2-(2-
nitrophenyl)ethyl group, 2-(3-nitrophenyl)ethyl group, 2-
(4-nitrophenyl)ethyl group, 2-(2-cyanophenyl)ethyl group,
2-(3-cyanophenyl)ethyl group, 2-(4-cyanophenyl)ethyl group,
2-(2-acetylaminophenyl)ethyl group, 2-(3-acetylamino-
phenyl)ethyl group, 2-(4-acetylaminophenyl)ethyl group, 2-
(4-benzoylaminophenyl)ethyl group, 3-(2-fluorophenyl)propyl
group, 3-(4-fluorophenyl)propyl group, 3 -(4-chlorophenyl)-
propyl group, 3-(4-bromophenyl)propyl group, 3-(4-iodo-

14
phenyDpropyl group, 3-(2-chlorophenyl)propyl group, 3-(2-
methoxyphenyDpropyl group, 3-(4-methoxyphenyl)propyl
group, 3-(3,4-dimethoxyphenyl)propyl group, 3-(4-trifluoro-
methylphenyl)propyl group, 3-(2-trifluoromethylphenyl)-
propyl group, 3-(4-nitrophenyl)propyl group, 3-(4-cyano-
phenyl)propyl group, 3-(4-acetylaminophenyl)propyl group,
more preferably (a 2-fluorobenzyl group, 4-fluorobenzyl
group, 2-chlorobenzyl group, 4-chlorobenzyl group, 2-
bromobenzyl group, 4-bromobenzyl group, 2-iodobenzyl group,
4-iodobenzyl group, 2-methylbenzyl group, 4-methylbenzyl
group, 4-hydroxybenzyl group, 2-methoxybenzyl group, 4-
methoxybenzyl group, 3,4-dimethoxybenzyl group, 2-tri-
fluoromethylbenzyl group, 4-trifluoromethylbenzyl group, 4-
benzyloxybenzyl group, 2-nitrobenzyl group, 4-nitrobenzyl
group, 2-cyanobenzyl group, 3-cyanobenzyl group, 4-cyano-
benzyl group, 3-acetylaminobenzyl group, 4-acetylamino-
benzyl group, 2-(2-fluorophenyl)ethyl group, 2-(4-fluoro-
phenyl)ethyl group, 2-(2-chlorophenyl)ethyl group, 2-(4-
chlorophenyl)ethyl group, 2-(2-bromophenyl)ethyl group, 2-
(4-bromophenyl)ethyl group, 2-(2-iodophenyl)ethyl group, 2-
(4-iodophenyl)ethyl group, 2-(2-tolyl)ethyl group, 2-(4-
tolyl)ethyl group, 2-(4-hydroxyphenyl)ethyl group, 2-(2-
methoxyphenyl)ethyl group, 2-(4-methoxyphenyl)ethyl group,
2-(3,4-dimethoxyphenyl)ethyl group, 2-(2-trifluoromethyl-
phenyl) ethyl group, 2-(4-trifluoromethylphenyl)ethyl group,
2-(4-benzyloxyphenyl)ethyl group, 2-(2-nitrophenyl)ethyl
group, 2-(4-nitrophenyl)ethyl group, 2-(2-cyanophenyl)ethyl
group, 2-(4-cyanophenyl)ethyl group, 2-(2-acetylamino-
phenyl) ethyl group, and 2-(4-acetylaminophenyl)ethyl group.
[0030]
As the aryl group of an aryl group which may have a
substituent(s) in the above-mentioned R, there may be
mentioned a phenyl group, naphthyl group, anthranyl group,
phenanthryl group, biphenyl group, and binaphthyl group.
[0031]
As the substituent(s) of the aryl group which may

15
have a substituent (s), there may be mentioned an alkyl
group having 1 to 4 carbon atoms such as a methyl group,
ethyl group, propyl group, butyl group, etc. (Incidentally,
these groups contain various kinds of isomers.); a hydroxyl
group; a halogen atom such as a chlorine atom, bromine
atom, iodine atom, fluorine atom, etc.; an alkoxyl group
having 2 to 4 carbon atoms such as an ethoxyl group, etc.
(Incidentally, these groups contain various kinds of
isomers.); an alkylenedioxyl group having 1 to 4 carbon
atoms such as a methylenedioxyl group, etc.; a nitro group;
a cyano group; a halogenated alkyl group such as a tri-
fluoromethyl group, etc.
[0032]
As the aryl group which may have such a substitu-
ent (s), there may be mentioned, for example, a 2-tolyl
group, 3-tolyl group, 4-tolyl group, 2,3-xylyl group, 2,6-
xylyl group, 2,4-xylyl group, 3,4-xylyl group, mesityl
group, 2-hydroxyphenyl group, 3-hydroxyphenyl group, 4-
hydroxyphenyl group, 2,3-dihydroxyphenyl group, 2,4-
dihydroxyphenyl group, 3,4-dihydroxyphenyl group, 2-
chlorophenyl group, 3-chlorophenyl group, 4-chlorophenyl
group, 2,3-dichlorophenyl group, 2,4-dichlorophenyl group,
3,4-dichlorophenyl group, 3,5-dichlorophenyl group, 2-
bromophenyl group, 3-bromophenyl group, 4-bromophenyl
group, 2-iodophenyl group, 3-iodophenyl group, 4-iodophenyl
group, 2-fluorophenyl group, 3-fluorophenyl group, 4-
fluorophenyl group, 2,3-difluorophenyl group, 2,4-difluoro-
phenyl group, 3,4-difluorophenyl group, 3-bromo-5-chloro-2-
hydroxyphenyl group, 2-methoxyphenyl group, 3-methoxyphenyl
group, 4-methoxyphenyl group, 2,3-dimethoxyphenyl group,
2,4-dimethoxyphenyl group, 3,4-dimethoxyphenyl group, 3,5-
dimethoxyphenyl group, 3,4-methylenedioxyphenyl group, 4-
ethoxyphenyl group, 4-butoxyphenyl group, 4-isopropoxy-
phenyl group, 1-phenoxyphenyl group, 4-benzyloxyphenyl
group, 4-trifluoromethylphenyl group, 2-nitrophenyl group,
3-nitrophenyl group, 4-nitrophenyl group, 4-cyanophenyl

16
group, 4-methoxycarbonylphenyl group, 1-naphthyl group, and
2-naphthyl group, etc., preferably a phenyl group, 2-tolyl
group, 3-tolyl group, 4-tolyl group, 2,3-xylyl group, 2-
hydroxyphenyl group, 3-hydroxyphenyl group, 4-hydroxyphenyl
group, 2,3-dihydroxypheny1 group, 2,4-dihydroxypheny1
group, 3,4-dihydroxypheny1 group, 2-chlorophenyl group, 3-
chlorophenyl group, 4-chlorophenyl group, 2,3-dichloro-
phenyl group, 2,4-dichlorophenyl group, 3,4-dichlorophenyl
group, 3,5-dichlorophenyl group, 2-bromophenyl group, 3-
bromophenyl group, 4-bromophenyi group, 2-iodophenyi group,
3-iodophenyl group, 4-iodophenyl group, 2-fluorophenyl
group, 3-fluorophenyl group, 4-fluorophenyl group, 3,4-
difluorophenyl group, 3-bromo-5-chloro-2-hydroxyphenyl
group, 2-methoxyphenyl group, 3-methoxyphenyl group, 4-
methoxyphenyl group, 2,3-dimethoxyphenyl group, 2,4-
dimethoxyphenyl group, 3,4-dimethoxyphenyl group, 3,5-
dimethoxyphenyl group, 3,4-methylenedioxyphenyl group, 4-
ethoxyphenyl group, 4-trifluoromethylphenyl group, 4-
nitrophenyl group, 4-cyanophenyl group, 1-naphthyl group,
and 2-naphthyl group, more preferably a phenyl group, 2-
tolyl group, 3-tolyl group, 4-tolyl group, 2,3-xylyl group,
4-hydroxyphenyl group, 3,4-dihydroxypheny1 group, 2-chloro-
phenyl group, 3-chlorophenyl group, 4-chlorophenyl group,
2,3-dichlorophenyl group, 2,4-dichlorophenyl group, 3,4-
dichlorophenyl group, 3,5-dichlorophenyl group, 2-bromo-
phenyl group, 3-bromophenyl group, 4-bromophenyl group, 4-
iodophenyl group, 2-fluorophenyl group, 3-fluorophenyl
group, 4-fluorophenyl group, 3,4-difluorophenyl group, 2-
iodophenyl group, 3-iodophenyl group, 4-iodophenyl group,
2-methoxyphenyl group, 3-methoxyphenyl group, 4-methoxy-
phenyl group, 2,3-dimethoxyphenyl group, 2,4-dimethoxy-
phenyl group, 3,4-dimethoxyphenyl group, 3,5-dimethoxy-
phenyl group, 3,4-methylenedioxyphenyl group, 4-trifluoro-
methylphenyl group, 4-nitrophenyl group, 1-naphthyl group,
2-naphthyl group, 3-pyridyl group, particularly preferably
phenyl group, 2-tolyl group, 3-tolyl group, 4-tolyl group,

17
2,3-xylyl group, 2-chlorophenyl group, 3-chlorophenyl
group, 4-chlorophenyl group, 2,3-dichlorophenyl group, 2,4-
dichlorophenyl group, 3,4-dichlorophenyl group, 3,5-di-
chlorophenyl group, 2-bromophenyl group, 3-bromophenyl
group, 4-bromophenyl group, 2-fluorophenyl group, 3-
fluorophenyl group, 4-fluorophenyl group, 3,4-difluoro-
phenyl group, 2-iodophenyl group, 3-iodophenyl group, 4-
iodophenyl group, 2-methoxyphenyl group, 3-methoxyphenyl
group, 4-methoxyphenyl group, 2,3-dimethoxyphenyl group,
3 , 4-dimethoxyphenyl group, 3,5-dimethoxyphenyl group and
3,4-methylenedioxyphenyl group.
[0033]
As the heteroaryl group of a heteroaryl group which
may have a substituent(s) in the above-mentioned R, there
may be mentioned for example, a 2-furyl group, 3-fury1
group, 2-pyridyl group, 3-pyridyl group, 4-pyridyl group,
2-pyrrolyl group, 3-pyrrolyl group, 2-thienyl group, 3-
thienyl group, 2-indolyl group, 3-indolyl group, 2-
imidazolyl group, 4-imidazolyl group, 3-pyrazolyl group, 2-
pyrimidyl group, 4-pyrimidyl group, and quinolyl group.
[0034]
As the substituent(s) of the heteroaryl group which
may have a substituent(s), there may be mentioned an alkyl
group having 1 to 4 carbon atoms such as a methyl group,
ethyl group, propyl group, butyl group, etc. (Incidentally,
these groups contain various kinds of isomers.); a hydroxyl
group; a halogen atom such as a chlorine atom, bromine
atom, iodine atom, fluorine atom, etc.; an alkoxyl group
having 2 to 4 carbon atoms such as an ethoxyl group, etc.
(Incidentally, these groups contain various kinds of
isomers.); an amino group; a nitro group; a cyano group; a
halogenated alkyl group such as a trifluoromethyl group,
etc.
[0035]
As the heteroaryl group which may have a substitu-
ent (s), there may be mentioned, for example, a 2-(3-

18
methyl)fury1 group, 2-(4-methyl)fury1 group, 2-(3-ethyl)-
furyl group, 2-(4-ethyl)fury1 group, 2-(3-fluoro)furyl
group, 2-(3-chloro)furyl group, 2-(3-hydroxy)furyl group,
2-(3-methoxy)furyl group, 2-(3-amino)furyl group, 2-(3-
nitro)furyl group, 2-(3-cyano)furyl group, 2-(3-methyl)-
pyridyl group, 2-(4-methyl)pyridyl group, 2-(3-ethyl)-
pyridyl group, 2-(4-ethyl)pyridyl group, 2-(3-fluoro)-
pyridyl group, 2-(4-chloro)pyridyl group, 2-(3-hydroxy)-
pyridyl group, 2-(3-methoxy)pyridyl group, 2-(3-amino)-
pyridyl group, 2-(3-nitro)pyridyl group, 2-(3-cyano)pyridyl
group, 2-(3,5-dichloro)pyridyl group, 3-(2-chloro)pyridyl
group, 2-(3-methyl)pyrrolyl group, and 2-(3-methyl)thienyl
group, etc., preferably a 2-(3-methyl)furyl group, 2-(3-
fluoro)furyl group, 2-(3-methyl)pyridyl group, 2-(3-
fluoro)pyridyl group, 2-(3-nitro)pyridyl group, 2-(3-
cyano)pyridyl group, and 2-(3,5-dichloro)pyridyl group.
[0036]
Z in Compound (I) represents an amino group which
may have a protective group, a hydroxyl group which may
have a protective group or an alkyl group.
[0037]
As the above-mentioned protective group, there may
be mentioned, for example, a benzyl group, 4-methoxyphenyl-
methyl group, methoxymethyl group, trimethylsilyl group,
triethylsilyl group, t-butyldimethylsilyl group, acetyl
group, benzoyl group, isopropylidene acetal group, 9-
fluorenylmethoxycarbonyl group, t-butoxycarbonyl group, and
benzyloxycarbonyl group, etc.
[0038]
As the above-mentioned alkyl group, there may be
mentioned a straight or branched alkyl group having 1 to 10
carbon atoms, and there may be specifically mentioned, for
example, an alkyl group such as a methyl group, ethyl
group, propyl group, butyl group, pentyl group, hexyl
group, heptyl group, octyl group, nonyl group, decyl group,
etc. Incidentally, these groups contain various kinds of

19
isomers.
[0039]
R1 in Compound (I) represents an alkyl group which
may have a substituent(s).
[0040]
As the alkyl group of the alkyl group which may have
a substituent(s) in the above-mentioned R1, there may be
mentioned a straight or branched alkyl group having 1 to 10
carbon atoms, and there may be specifically mentioned, for
example, an alkyl group such as a methyl group, ethyl
group, propyl group, butyl group, pentyl group, hexyl
group, heptyl group, octyl group, nonyl group, decyl group,
etc., preferably an alkyl group having 1 to 6 carbon atoms
such as a methyl group, ethyl group, n-propyl group,
isopropyl group, n-butyl group, isobutyl group, sec-butyl
group, n-pentyl group, n-hexyl group, etc., more preferably
an alkyl group having 1 to 4 carbon atoms such as a methyl
group, ethyl group, n-propyl group, n-butyl group, isobutyl
group, etc. Incidentally, these groups contain various
kinds of isomers.
[0041]
As the substituent(s) of the alkyl group which may
have a substituent(s), there may be mentioned a halogen
atom such as a fluorine atom, chlorine atom, bromine atom,
iodine atom, etc.; a hydroxyl group; an alkoxyl group
having 1 to 4 carbon atoms such as a methoxyl group,
ethoxyl group, propoxyl group, butoxyl group, etc.; a
dialkylamino group such as a dimethylamino group, diethyl-
amino group, etc.; a cyano group, etc., preferably a
fluorine atom, chlorine atom, methoxyl group, ethoxyl
group, hydroxyl group, and cyano group, more preferably a
fluorine atom, chlorine atom, methoxyl group, and ethoxyl
group.
[0042]
As the alkyl group which may have such a substitu-
ent (s), there may be mentioned, for example, a 2-fluoro-

20
ethyl group, 2-chloroethyl group, 2,2-difluoroethyl group,
2,2-dichloroethyl group, 2,2,2-trichloroethyl group, 2,2,2-
trifluoroethyl group, 2-methoxyethyl group, 2-ethoxyethyl
group, methoxymethyl group, 2-hydroxyethyl group, 2-cyano-
ethyl group, 2-bromoethyl group, 2-dimethylamino group, 2-
chloropropyl group, 3-chloropropyl group, etc., preferably
a 2-chloroethyl group, 2,2,2-trichloroethyl group, 2,2,2-
trifluoroethyl group, methoxymethyl group, 2-methoxyethyl
group, and 2-ethoxyethyl group.
[0043]
Incidentally, n is 1 or 2.
[0044]
As the surfactant-modified enzyme to be used in the
hydrolysis of the present invention, for example, those
enzymes in which a hydrolase is subjected to covering
treatment with a surfactant and solubilized in an organic
solvent are suitably used.
[0045]
With regard to the above-mentioned surfactant-modi-
fied enzyme, as a hydrolase to be used as a base material,
there may be mentioned, for example, a protease, esterase,
lipase, etc., preferably a lipase obtained from microorg-
anisms isolatable from yeast or bacteria, more preferably
at least one kind selected from a lipase (for example,
Amano PS (available from AMANO ENZYME CO.), etc.)
originated from Burkholderia cepacia (Pseudomonas cepacia)
and a lipase (for example, NOVOZYM 435 (available from
NOVOZYMS COM.), etc.) originated from Candida Antarctica is
used. Incidentally, as these hydrolases, commercially
available product can be used as such as a natural form or
an immobilized enzyme, and may by used alone or in admix-
ture of two or more kinds. Also, an enzyme-immobilizing
agent contained in the commercially available product is
previously removed and then the treated product may be
used.
[0046]

21
The above-mentioned hydrolases are desirably used
after subjecting the commercially available product sold as
a natural form or an immobilized enzyme to chemical treat-
ment or physical treatment.
[0047]
The synthetic method of the surfactant-modified
enzyme having the above-mentioned characteristics is not
specifically limited, and it may be a preparation method in
which the product shows properties wherein it has high
solubility in an organic solvent, and the dissolved enzyme
fraction shows high activity in an organic solvent. There
may be mentioned a method in which, for example, a surfac-
tant dissolved in a small amount of methanol, ethanol,
propanol, acetone, methyl ethyl ketone, or other hydro-
philic organic solvents is mixed under cooling with a
buffer in which a hydrolase had been dissolved therein, to
precipitate a surfactant-modified enzyme. This precipitate
is separated by ultracentrifugation or filtration, etc.,
washed with a buffer and distilled water, and lyophilized
as such to obtain a powder state surfactant-modified enzyme
(for example, see the above-mentioned Patent Literatures 6
to 10 and Non-Patent Literature 4). Or else, a surfactant
dissolved in a small amount of methanol, ethanol, propanol,
acetone, methyl ethyl ketone, or other hydrophilic organic
solvents is mixed under cooling with a buffer in which a
hydrolase had been dissolved therein, and the resulting
mixed solution is lyophilized to obtain a powder state
surfactant-modified enzyme (described in the following
Reference example 2). Also, it may be obtained by vigor-
ously stirring an aqueous hydrolase solution and a water-
insoluble organic solvent in the presence of a surfactant
which can form a reverse phase emulsion to form an emulsion
and then drying the same (for example, see the above-
mentioned Patent Literature 11).
[0048]
Incidentally, lyophilization is a method in which an

22
aqueous solution and a substance containing water are
frozen rapidly at a temperature not more than the freezing
point, and a pressure is reduced to the water vapor
pressure or lower of the frozen product to remove water by
sublimation whereby drying the substance (for example, see
Non-Patent literature 5).
[0049]
As the above-mentioned buffer, there may be mention-
ed, for example, an aqueous solution of an inorganic acid
salt such as an aqueous sodium phosphate solution, an
aqueous potassium phosphate solution, etc.; an aqueous
solution of an organic acid salt such as an aqueous sodium
acetate solution, an aqueous ammonium acetate solution, an
aqueous sodium citrate solution, etc., preferably an
aqueous sodium phosphate solution, an aqueous potassium
phosphate solution, an aqueous ammonium acetate solution
is/are used. Incidentally, these buffers may be used
singly or in admixture of two or more kinds.
[0050]
A concentration of the above-mentioned buffer is
preferably 0.005 to 2 mol/L, more preferably 0.01 to 0.5
mol/L, and a pH of the buffer is preferably 4 to 9, more
preferably 7 to 8.5.
[0051]
An amount of the buffer to be used at the time of
lyophilization is not particularly limited so long as it is
a concentration that the surfactant-modified enzyme is
completely dissolved, and preferably 10 ml to 1000 ml, more
preferably 10 ml to 100 ml based on 1 g of the hydrolase.
[0052]
As the hydrolase to be used for the preparation of
the above-mentioned surfactant-modified enzyme, a commer-
cially available product which is available as a natural
form or an immobilized enzyme may be used as such.
[0053]
An amount of the above-mentioned hydrolase to be used

23
is preferably 0.1 to 1000 mg, more preferably 1 to 200 mg
based on 1 g of Compound (I).
[0054]
As the above-mentioned surfactant, a surfactant
having both of a hydrophobic group such as a long-chain
alkyl group, and a hydrophilic group is suitably used, and
it may be either a natural surfactant or a synthetic
surfactant. Depending on a kind of the hydrophilic group,
the surfactant is classified into four groups such as an
anionic surfactant, a cationic surfactant, a nonionic
surfactant, and an amphoteric surfactant. Incidentally,
these surfactants may be used alone or in admixture of two
or more kinds.
[0055]
As the above-mentioned anionic surfactant, there may
be mentioned, for example, soap, a base material for soap,
metal soap, triethanol amine N-acyl-L-glutamate, sodium N-
acyl-L-glutamate, sodium alkyl sulfate, polyoxyethylene
alkyl ether sodium sulfate, polyoxyethylene alkyl ether
phosphate, polyoxyethylene alkylphenyl ether phosphate,
sodium palm oil fatty acid methyl taurine, lauryl sulfate
triethanol amine, lauryl sodium lauryl sulfate, sodium
lauroyl sarcosinate, sodium lauroylmethyl a-alanine
solution or sodium lauroylmethyl taurine, etc.
[0056]
As the above-mentioned cationic surfactant, there
may be mentioned, for example, invert soap, ethyl sulfate
lanolin fatty acid aminopropylethyl dimethyl ammonium,
alkyltrimethyl ammonium chloride, dialkyldimethyl ammonium
chloride, distearyldimethyl ammonium chloride, stearyl
dimethylbenzyl ammonium chloride, benzalkonium chloride or
benzethonium chloride, etc.
[0057]
As the above-mentioned nonionic surfactant, there
may be mentioned, for example, polyethylene glycol,
sorbitan alkyl ester, polyoxyethylene glycol alkyl ester,

24
polyoxyethylene glycol alkyl ether, dialkyl glutamate
gluconamide, dimethicone copolyol, sucrose aliphatic ester,
sucrose stearic ester, polyoxyethylene sorbit tetraoleate,
poly(oxyethylene-oxypropylene)methylpolysiloxane copolymer,
polyoxyethylene octylphenyl ether, polyoxyethylene hardened
castor oil, polyoxyethylene stearyl ether, polyoxyethylene
stearyl amide, polyoxyethylene cetyl ether, polyoxyethylene
polyoxypropyleneglycol, polyglycerin aliphatic acid ester,
polyoxyethylene sorbitan monooleate, ethylene glycol
monostearate, glycerin monostearate, sorbitan monostearate,
propylene glycol monostearate, polyoxyethylene glycerin
monostearate, polyoxyethylene sorbitan monostearate, poly-
oxyethylene sorbitan monopalmitate, polyethylene glycol
monolaurate, polyoxyethylene sorbitan monolaurate, polyoxy-
ethylene sorbit monolaurate or diethanol amide laurate,
etc.
[0058]
As the above-mentioned amphoteric surfactant, there
may be mentioned, for example, 2-alkyl-N-carboxymethyl-N-
hydroxyethyl imidazolium betaine, alkyldiaminoethyl glycine
chloride solution or lauryl dimethylamino acetic acid
betaine, etc.
[0059]
As the synthetic example of the above-mentioned
synthetic surfactant, it can be easily synthesize by, for
example, refluxing L-glutamic acid, and a long chain
alcohol such as dodecanol, oleyl alcohol, etc. in toluene
in the presence of p-toluenesulfonic acid to prepare L-
glutamic acid di-long chain ester, and further, refluxing
the resulting ester product with 8-gluconolactone in
methanol (for example, see the above-mentioned Non-Patent
Literature 6).
[0060]
As the above-mentioned synthetic surfactant, N-D-
glucono-L-glutamic acid diesters are most suitably used.
[0061]

25
The hydrolysis of the present invention is usually
carried out in a uniform phase solvent containing water,
and an organic solvent which forms a uniform phase with
water. Incidentally, there is substantially no bad effect
in the hydrolysis of the present invention even when water
is not completely dissolved in the organic solvent to form
a suspended state.
[0062]
As the above-mentioned water, purified water such as
deionized water and distilled water, etc. is usually used,
and water may contain an inorganic salt such as sodium
phosphate, potassium phosphate, etc., or an organic salt
such as sodium acetate, ammonium acetate, sodium citrate,
etc.
[0063]
An amount of the above-mentioned water to be used is
not specifically limited so long as the reaction solution
forms a uniform phase, and preferably 0.5 to 10 mol, more
preferably 0.5 to 5.0 mol, particularly preferably 1.0 to
3.0 mol based on 1 mol of Compound (I).
[0064]
As the above-mentioned organic solvent, there may be
mentioned, for example, an aliphatic hydrocarbon such as n-
pentane, n-hexane, n-heptane, n-octane, cyclopentane,
cyclohexane, cyclopentane, etc.; an aromatic hydrocarbon
such as benzene, toluene, xylene, etc.; an ether such as
diethyl ether, t-butyl methyl ether, diisopropyl ether,
cyclopentyl methyl ether, tetrahydrofuran, 1,4-dioxane,
etc.; a ketone such as acetone, methyl ethyl ketone, etc.;
and an ester such as ethyl acetate, butyl acetate, etc.,
preferably n-hexane, n-heptane, cyclopentane, cyclohexane,
toluene, diisopropyl ether, t-butyl methyl ether, cyclo-
pentyl methyl ether, tetrahydrofuran, more preferably n-
hexane, cyclohexane, toluene, diisopropyl ether, t-butyl
methyl ether, cyclopentyl methyl ether, particularly
preferably cyclohexane, toluene, or t-butyl methyl ether is

26
used. Incidentally, these organic solvents may be used
alone or in admixture of two or more kinds.
[0065]
An amount of the above-mentioned organic solvent to
be used is preferably 2 to 200 mL, more preferably 5 to 80
mL based on 1 g of Compound (I).
[0066]
The hydrolysis of the present invention can be
carried out by a method, for example, where Compound (I), a
surfactant-modified enzyme, water (if necessary, it may
contain an inorganic salt or an organic salt) and an
organic solvent which can form a uniform phase with water
are mixed and reacted under stirring, etc. A reaction
temperature at that time is preferably 0 to 8 0°C, more
preferably 10 to 50°C, particularly preferably 30 to 45°C,
and a reaction pressure is not particularly limited.
[0067]
In the hydrolysis of the present invention, when an a
or p-substituted carboxylic acid ester (racemic mixture)
represented by the formula (I):
[0068]

[0069]
wherein R, R1, Z, n and * have the same meanings as
defined above,
is used, an optically active a or p-substituted carboxylic
acid (hereinafter referred to as Compound (II)) represented
by the formula (II):
[0069]


27
[0070]
wherein R, Z, n and * have the same meanings as
defined above,
and an optically active a or p-substituted carboxylic acid
ester (hereinafter referred to as Compound (III))
represented by the formula (III):
[0071]

[0072]
wherein R, R1, Z, n and * have the same meanings as
defined above,
which is remained and not hydrolyzed can be obtained
simultaneously.
[0073]
Compound (II) and Compound (III) obtained by the
hydrolysis of the present invention can be separated by,
for example, after completion of the reaction, filtering
the precipitated Compound (II) as such, or adding a
suitable organic solvent (for example, acetonitrile,
acetone, etc.) to the reaction mixture and filtering to
obtain Compound (II), and concentrating the organic layer
after adjusting a pH of the filtrate to obtain Compound
(III) . Also, sodium chloride may be added to the mixture
before separation to improve the recovery rate of Compound
(II) . Incidentally, the resulting Compound (II) and
Compound (III) may be further purified according to the
usual method such as crystallization, recrystallization,
distillation, column chromatography, etc.
EXAMPLES
[0074]
Next, the present invention is explained more spe-
cifically by referring to Examples, but the scope of the
present invention is not limited by these.

28
[0075]
Reference example 1 (Preparation of surfactant-modified
enzyme A)
To 50 mL of 0.1 mol/L phosphate buffer (a phosphate
buffer prepared by adding 0.1 mol/L aqueous disodium
hydrogen phosphate solution to 0.1 mol/L aqueous sodium
dihydrogen phosphate solution and adjusted to pH 7.0) was
added 200 mg of a lipase (Amano Lipase PS (trade name);
available from Aldrich) originated from Burkholderia
cepacia {Pseudomonas cepacia), and the mixture was stirred
at room temperature. After 3 0 minutes, insoluble materials
were removed by ultracentrifugation, and cooled to 4°C. To
the obtained enzyme solution was added dropwise a solution
in which 400 mg of didodecyl N-D-glucono-L-glutamate was
dissolved in 2.5 mL of ethanol, and the mixture was stirred
at 4°C. After 4 hours, stirring was stopped and the mix-
ture was allowed to stand for 5 8 hours. The supernatant
was removed by ultracentrifugation, and the remaining pre-
cipitate was washed twice with 0.1 mol/L phosphate buffer
(pH 7.0), and once with distilled water. The solid after
washing was lyophilized to obtain 180 mg of a surfactant-
modified enzyme A as white powder.
[0076]
Reference example 2 (Preparation of surfactant-modified
enzyme B)
To 200 mL of 0.01 mol/L phosphate buffer (a phos-
phate buffer prepared by adding 0.01 mol/L aqueous disodium
hydrogen phosphate solution to 0.01 mol/L aqueous sodium
dihydrogen phosphate solution and adjusted to pH 7.0) was
added 1.60 g of a lipase (Amano Lipase PS (trade name);
available from Aldrich) originated from Burkholderia
cepacia {Pseudomonas cepacia), and the mixture was stirred
at room temperature. After 3 0 minutes, insoluble materials
were removed by ultracentrifugation, and cooled to 4°C. To
the obtained enzyme solution was added dropwise a solution
in which 1.60 g of didodecyl N-D-glucono-L-glutamate was

29
dissolved in 20.0 mL of ethanol, and the mixture was
stirred at 4°C. After 4 hours, stirring was stopped and
the mixture was allowed to stand for 66 hours. The mixture
was lyophilized to obtain 1.87 g of a surfactant-modified
enzyme B as white powder.
[0077]
Reference example 3 (Synthesis of 3-amino-3-phenylpropionic
acid (racemic mixture))
To 250 mL of isopropyl alcohol were added 17.7 g
(0.17 mol) of benzaldehyde, 18.2 g (0.17 mol) of malonic
acid and 2 5.6 g (0.33 mol) of ammonium acetate, and the
materials were reacted under reflux (80 to 90°C) for 7
hours while stirring. After completion of the reaction,
the resulting reaction solution was stirred at 0 to 5°C for
1 hour and filtered to obtain 19.2 g (Isolation yield based
on benzaldehyde: 70.0%) of 3-amino-3-phenylpropionic acid
(racemic mixture) as white powder.
Incidentally, physical properties of the 3-amino-3-
phenylpropionic acid (racemic mixture) are as follows.
[0078]
1H-NMR (5 (ppm) , D2O+DC1) : 3.06 (dd, 1H, J=17.1, 6.8Hz),
3.17 (dd, 1H, J=17.1, 7.3Hz), 4.76 (dd, 1H, J=7.3, 6.8Hz),
3.77 (s, 2H), 7.45 (m, 5H)
13C-NMR (5 (ppm), D2O+DC1): 40.5, 54.4, 130.0, 132.3, 132.6,
138.0, 176.3
MS (El) m/z: 165 (M+)
MS (CI, i-C4H10) m/z: 166 (MH+)
Elemental analysis; Calcd: C, 65.44%; H, 6.71%; N, 8.48%
Found: C, 65.18%; H, 6.78%; N, 8.34%
[0079]
Reference example 4 (Synthesis of ethyl 3-amino-3-phenyl-
propionate (racemic mixture))
To 6.00 mL (103 mmol) of ethanol were added 2.00 g
(12.1 mmol) of 3-amino-3-phenylpropionic acid (racemic
mixture) synthesized in Reference example 3 and 1.78 g
(18.2 mmol) of cone, sulfuric acid, and the materials were

30
reacted under stirring at 60°C for 4 hours. After comple-
tion of the reaction, the obtained reaction mixture was
concentrated under reduced pressure, 6 mol/L aqueous sodium
hydroxide solution was added to the residue, and a pH of
the reaction mixture was adjusted to 8.5. Then, the
mixture was extracted with 10 mL of ethyl acetate and 4 mL
of water, and the organic layer was dried over anhydrous
magnesium sulfate. After filtration, the filtrate was
concentrated under reduced pressure to obtain 1.98 g
(Isolation yield based on 3-amino-3-phenylpropionic acid
(racemic mixture): 84.5%) of -3-amino-3-phenylpropionic acid
ethyl ester (racemic mixture) as colorless liquid.
Incidentally, physical properties of the 3-amino-3-
phenylpropionic acid ethyl ester (racemic mixture) are as
follows.
[0080]
1H-NMR (6 (ppm) , CDC13) : 1.19 (t, 3H, J=7.3Hz), 3.15 (dd,
1H, J=7.3, 16.6Hz), 3.25 (dd, 1H, J=7.3, 16.6Hz), 4.15 (q,
2H, J=7.3Hz), 4.85 (dd, 1H, J=7.3, 7.3Hz), 7.50-7.55 (m,
5H)
13C-NMR (5 (ppm), CDCl3) : 16.0, 40.9, 54.3, 65.2, 129.9,
132.2, 132.5, 137.8, 174.3
MS (El) m/z: 193 (M+)
MS (CI, i-C4H10) m/z: 194 (MH+)
[0081]
Example 1 (Synthesis of (S)-3-amino-3-phenylpropionic acid
and ethyl (R)-3-amino-3-phenylpropionate)
To 1.0 mL of t-butyl methyl ether saturated with
water were added 100 mg (0.517 mmol) of ethyl 3-amino-3-
phenylpropionate (racemic mixture) and 10.0 mg of the
surfactant-modified enzyme A prepared in Reference example
1, and the materials were reacted under stirring at 30°C.
After 4 6 hours, 0.5 mL of acetone was added to the reaction
mixture and the mixture was filtered to obtain 34.2 mg
(Isolation yield based on ethyl 3-amino-3-phenylpropionate
(racemic mixture)=40.0%) of (S)-3-amino-3-phenylpropionic

31
acid.
The (S)-3-atnino-3-phenylpropionic acid was led to n-
propyl (S)-3-amino-3-phenylpropionate according to the
conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 99.9%ee.
The ethyl (R)-3-amino-3-phenylpropionate was led to
ethyl (R)-3-(2-furoylamino)-3-phenylpropionate according to
the conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 96.6%ee.
Incidentally, the E value in the present reaction
was 5522.
[0082]
Analytical conditions of high performance liquid chromato-
graphy;
Optically active n-propyl 3-amino-3-phenylpropionate
Column: CHIRAL CD-Ph (0.46 cmdx25 cm, available from
SHISEIDO CO., LTD.)
Solvent: acetonitrile/water (=1/9 (volume ratio))
Potassium dihydrogen phosphate 40 mmol/L
Adjusted to pH 3.5 with phosphoric acid
Flow rate: 0.5 mL/min
Temperature: 3 0 °C
Wavelength: 22 0 nm
[0083]
Optically active ethyl 3-(2-furoylamino)-3-phenylpropionate
Column: CHIRAL CEL OJ-H (0.4 6cmΦx 2 5cm, available from
DAICEL CHEMICAL INDUSTRIES, LTD.)
Solvent: hexane/isopropyl alcohol (=9/1 (volume ratio))
Flow rate: 0.5 mL/min
Temperature: 3 0°C
Wavelength: 220 nm
[0084]
Also, physical properties of the (S)-3-amino-3-
phenylpropionic acid were the same as those shown in

32
Reference example 3.
Physical properties of the ethyl (R)-3-amino-3-
phenylpropionate were the same as those shown in Reference
example 4.
[0085]
Example 2 (Syntheses of (S)-3-amino-3-phenylpropionic acid
and ethyl (R)-3-amino-3-phenylpropionate)
To 1.0 mL of t-butyl methyl ether saturated with
water were added 100 mg (0.517 mmol) of ethyl 3-amino-3-
phenylpropionate (racemic mixture) and 5.0 mg of the
surfactant-modified enzyme B prepared in Reference example
2, and the materials were reacted under stirring at 30°C.
After 90 hours, 0.5 mL of acetone was added to the reaction
mixture and the mixture was filtered to obtain 34.4 mg
(Isolation yield based on ethyl 3-amino-3-phenylpropionate
(racemic mixture)=40.2%) of (S)-3-amino-3-phenylpropionic
acid.
The (S)-3-amino-3-phenylpropionic acid was led to n-
propyl (S)-3-amino-3-phenylpropionate according to the
conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 99.9%ee.
The ethyl (R)-3-amino-3-phenylpropionate was led to
ethyl (R)-3-(2-furoylamino)-3-phenylpropionate according to
the conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 96.3%ee.
Incidentally, the E value in the present reaction
was 8106.
[0086]
Analytical conditions of high performance liquid chromato-
graphy;
Optically active n-propyl 3-amino-3-phenylpropionate
Column: CHIRAL CD-Ph (0.46 cmΦx25 cm, available from
SHISEIDO CO., LTD.)
Solvent: acetonitrile/water (=1/9 (volume ratio))

33
Potassium dihydrogen phosphate 4 0 mmol/L
Adjusted to pH 3.5 with phosphoric acid
Flow rate: 0.5 mL/min
Temperature: 3 0°C
Wavelength: 22 0 nm
[0087]
Optically active ethyl 3-(2-furoylamino)-3-phenylpropionate
Column: CHIRAL CEL OJ-H (0.46 cmOx 25cm, available from
DAICEL CHEMICAL INDUSTRIES, LTD.)
Solvent: hexane/isopropyl alcohol (=9/1 (volume ratio))
Flow rate: 0.5 mL/min
Temperature: 30°C
Wavelength: 220 nm
[0088]
Also, physical properties of the (S)-3-amino-3-
phenylpropionic acid were the same as those shown in
Reference example 3.
Physical properties of the ethyl (R)-3-amino-3-
phenylpropionate were the same as those shown in Reference
example 4.
[0089]
Reference example 5 (Synthesis of ethyl 2-amino-4-phenyl-
butyrate (racemic mixture))
To 6.00 mL (103 mmol) of ethanol were added 2.00 g
(11.2 mmol) of 2-amino-4-phenylbutyric acid (racemic
mixture) and 1.78 g (16.8 mmol) of cone, sulfuric acid, and
the materials were reacted under stirring at 60°C for 4
hours. After completion of the reaction, the obtained
reaction mixture was concentrated under reduced pressure, 6
mol/L aqueous sodium hydroxide solution was added to the
residue, and a pH of the mixture was adjusted to 8.5.
Then, the mixture was extracted with 10 mL of ethyl acetate
and 4 mL of water, and the organic layer was dried over
anhydrous magnesium sulfate. After filtration, the
filtrate was concentrated under reduced pressure to obtain
1.89 g (Isolation yield based on 2-amino-4-phenylbutyric

34
acid (racemic mixture): 82.0%) of ethyl 2-amino-4-phenyl-
butyrate (racemic mixture) as colorless liquid.
Incidentally, physical properties of the ethyl 2-
amino-4-phenyl butyrate (racemic mixture) are as follows.
[0090]
1H-NMR (8 (ppm) , CDC13) : 1.28 (t, 3H, J=7.3Hz), 1.81-1.90
(m, 2H), 2.06 (ddd, 1H, J=5.4, 6.4, 13.7Hz), 2.08 (ddd, 1H,
J=5.4, 6.4, 13.7Hz), 3.44 (dd, 1H, J=5.4, 7.8Hz), 4.18 (q,
2H, J=7.3Hz), 7.17-7.30 (m, 5H)
13C-NMR (S (ppm), CDCI3) : 14.3, 32.0, 36.5, 54.1, 60.9,
126.1, 128.4, 128.5, 141.4, 176.0
MS (El) m/z: 207 (M+)
MS (CI, i-C4H10) m/z: 208 (MH+)
[0091]
Example 3 (Syntheses of (S)-2-amino-4-phenylbutyric acid
and ethyl (R)-2-amino-4-phenylbutyrate)
To 1.0 mL of t-butyl methyl ether saturated with
water were added 100 mg (0.482 mmol) of ethyl 2-amino-4-
phenylbutyrate (racemic mixture) and 10.0 mg of the
surfactant-modified enzyme A prepared in Reference example
1, and the materials were reacted under stirring at 3 0°C.
After 66 hours, 0.5 mL of acetone was added to the reaction
mixture and the mixture was filtered to obtain 35.5 mg
(Isolation yield based on ethyl 2-amino-4-phenylbutyrate
(racemic mixture)=41.0%) of (S)-2-amino-4-phenylbutyric
acid.
The (S)-2-amino-4-phenylbutyric acid was led to
ethyl (S)-2-amino-4-phenylbutyrate according to the
conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 61.5%ee.
The ethyl (R)-2-amino-4-phenybutyrate was led to
ethyl (R)-2-(2-furoylamino)-4-phenylbutyrate according to
the conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 93.5%ee.

35
Incidentally, the E value in the present reaction
was 14.
[0092]
Analytical conditions of high performance liquid chromato-
graphy;
Optically active ethyl 2-amino-4-phenylbutyrate
Column: CHIRAL CD-Ph (0.46 cmOx25 cm, available from
SHISEIDO CO., LTD.)
Solvent: acetonitrile/water (=1/9 (volume ratio))
Potassium dihydrogen phosphate 4 0 mmol/L
Adjusted to pH 3.5 with phosphoric acid
Flow rate: 0.5 mL/min
Temperature: 3 0°C
Wavelength: 22 0 nm
[0093]
Optically active ethyl 2 -(2-furoylamino)-4-phenylbutyrate
Column: CHIRAL CEL OJ-H (0.46cmΦx 25cm, available from
DAICEL CHEMICAL INDUSTRIES, LTD.)
Solvent: hexane/isopropyl alcohol (=9/1(volume ratio))
Flow rate: 0.5 mL/min
Temperature: 3 0°C
Wavelength: 22 0 nm
[0094]
Also, physical properties of the (S)-2-amino-4-
phenylbutyric acid are as follows.
1H-NMR (5 (ppm) , CDC13) : 1.97-2.19 (m, 2H) , 2.54-2.69 (m,
2H), 3.91 (t, 1H, J=6.4Hz), 7.09-7.22 (m, 5H)
13C-NMR (5 (ppm), CDC13) : 33.2, 34.3, 55.3, 129.6, 131.4,
131.8, 143.0, 174.6
MS (El) m/z: 179 (M+)
MS (CI, i-C4H10) m/z: 180 (MH+)
[0095]
Physical properties of the ethyl (R)-2-amino-4-
phenybutyrate were the same as those shown in Reference
example 5.
[0096]

36
Example 4 (Syntheses of (S)-2-amino-4-phenylbutyric acid
and ethyl (R)-2-amino-4-phenybutyrate)
To 1.0 mL of t-butyl methyl ether saturated with
water were added 100 mg (0.482 mmol) of ethyl 2-amino-4-
phenylbutyrate (racemic mixture) and 10.0 mg of the
surfactant-modified enzyme B prepared in Reference example
2, and the materials were reacted under stirring at 30°C.
After 70 hours, 0.5 mL of acetone was added to the reaction
mixture and the mixture was filtered to obtain 34.9 mg
(Isolation yield based on ethyl 2-amino-4-phenyibutyrate
(racemic mixture)=40.3%) of (S)-2-amino-4-phenylbutyric
acid.
The (S)-2-amino-4-phenylbutyric acid was led to
ethyl (S)-2-amino-4-phenylbutyrate according to the
conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 75.1%ee.
The ethyl (R)-2-amino-4-phenybutyrate was led to
ethyl (R)-2-(2-furoylamino)-4-phenylbutyrate according to
the conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 84.6%ee.
Incidentally, the E value in the present reaction
was 19.
[0097]
Analytical conditions of high performance liquid chromato-
graphy;
Optically active ethyl 2-amino-4-phenylbutyrate
Column: CHIRAL CD-Ph (0.46 cmOx25 cm, available from
SHISEIDO CO., LTD.)
Solvent: acetonitrile/water (=1/9 (volume ratio))
Potassium dihydrogen phosphate 4 0 mmol/L
Adjusted to pH 3.5 with phosphoric acid
Flow rate: 0.5 mL/min
Temperature: 3 0 ° C
Wavelength: 220 nm

37
[0098]
Optically active ethyl 2-(2-furoylamino)-4-phenylbutyrate
Column: CHIRAL CEL OJ-H (0.46 cmΦx25 cm, available from
DAICEL CHEMICAL INDUSTRIES, LTD.)
Solvent: hexane/isopropyl alcohol (=9/1 (volume ratio))
Flow rate: 0.5 mL/min
Temperature: 3 0°C
Wavelength: 220 nm
[0099]
Also, physical properties of the (S)-2-amino-4-
phenylbutyric acid were the same as those shown in Example
3.
Physical properties of the ethyl (R)-2-amino-4-
phenylbutyrate were the same as those shown in Reference
example 5.
[0100]
Reference example 6 (Synthesis of ethyl 2-amino-3-phenyl-
propionate (racemic mixture))
To 10.0 mL (171 mmol) of ethanol were added 2.00 g
(12.1 mmol) of 2-amino 3-phenylpropionic acid (racemic
mixture) and 1.42 g (14.5 mmol) of cone, sulfuric acid, and
the materials were reacted under stirring at 60°C for 4
hours. After completion of the reaction, the obtained
reaction mixture was concentrated under reduced pressure, 6
mol/L aqueous sodium hydroxide solution was added to the
mixture, and a pH of the reaction mixture was adjusted to
8.5. Then, the mixture was extracted with 10 mL of t-butyl
methyl ether and 4 mL of water, and the organic layer was
dried over anhydrous magnesium sulfate. After filtration,
the filtrate was concentrated under reduced pressure to
obtain 2.34 g (Isolation yield based on 2-amino 3-phenyl-
propionic acid (racemic mixture): 89.0%) of ethyl 2-amino
3-phenylpropionate (racemic mixture) as colorless liquid.
Incidentally, physical properties of the ethyl 2-
amino 3-phenylpropionate (racemic mixture) are as follows.
[0101]

38
1H-NMR (8 (ppm) , CDCI3) : 1.22 (t, 3H, J=7.1Hz), 2.85 (dd,
1H, J=7.8, 13.5Hz), 3.06 (dd, 1H, J=5.4, 13.5Hz), 3.69 (dd,
1H, J=5.4, 7.8Hz), 7.17-7.30 (in, 5H)
13C-NMR (5 (ppm), CDC13) : 14.2, 41.2, 55.9, 60.8, 126.7,
128.5, 129.3.137.4, 175.0
MS (CI, i-C4H10) m/z: 194 (MH+)
Elemental analysis; Calcd: C, 68.37%; H, 7.82%; N, 7.25%
Found: C, 66.29%; H, 7.69%; N, 7.02%
[0102]
Example 5 (Syntheses of (S)-2-amino-3-phenylpropionic acid
and ethyl (R)-2-amino-3-phenylpropionate)
To 1.00 mL of t-butyl methyl ether saturated with
water were added 100 mg (0.517 mmol) of ethyl 2-amino 3-
phenylpropionate (racemic mixture) and 5.0 mg of the
surfactant-modified enzyme A prepared in Reference example
2, and the materials were reacted under stirring at 30°C.
After 24 hours, 0.5 mL of acetone was added to the reaction
mixture and the mixture was filtered to obtain 35.8 mg
(Isolation yield based on 2-amino-3-phenylpropionic acid
ethyl ester (racemic mixture)=41.8%) of (S)-2-amino-3-
phenylpropionic acid and a lipase as a mixture.
The (S)-2-amino-3-phenylpropionic acid was led to
ethyl (S)-2-(2-furoylamino)-3-phenylpropionate according to
the conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 95.9%ee.
The ethyl (R)-2-amino-3-phenyipropionate was led to
ethyl (R)-2-(2-furoylamino)-3-phenylpropionate according to
the conventional manner, and an optical purity thereof was
measured by using high performance liquid chromatography
which uses an optically active column, then it was 91.7%ee.
Incidentally, the E value in the present reaction
was 155.
[0103]
Analytical conditions of high performance liquid chromato-
graphy;

39
Optically active ethyl 2-(2-furoylamino)-3-phenylpropionate
Column: CHIRAL CEL OJ-H (0.46 cmΦ25 cm, available from
DAICEL CHEMICAL INDUSTRIES, LTD.)
Solvent: hexane/isopropyl alcohol (=8/2(volume ratio))
Flow rate: 0.5 mL/min
Temperature: 30°C
Wavelength: 220 nm
[0104]
Also, physical properties of the (S)-2-amino-3-
phenyLpropionic acid are as follows.
[0105]
1H-NMR (5 (ppm) , CD3OD) : 3.12 (dd, 1H, J=8.0, 14.5Hz), 3.29
(dd, 1H, J=5.2, 14.5HZ), 3.99 (dd, 1H, J=5.2, 8.0Hz), 7.32-
7.45 (m, 5H)
13C-NMR (8 (ppm), CD3OD) : 39.2, 58.9, 130.5, 132.0, 132.2,
138.0, 176.8
MS (CI, i-C4H10) m/z: 166 (MH+)
Elemental analysis; Calcd: C, 65.44%; H, 6.71%; N, 8.48%
Found: C, 61.36%; H, 6.69%; N, 7.94%
[0106]
Physical properties of the ethyl (R)-2-amino 3-
phenylpropionate were the same as those shown in Reference
example 1.
UTILIZABILITY IN INDUSTRY
[0107]
The present invention relates to a process for
preparing a carboxylic acid by hydrolyzing a carboxylic
acid ester in a uniform solvent comprising water and an
organic solvent which can form a uniform phase with water,
in the presence of a surfactant-modified enzyme, in parti-
cular, it relates to a process for preparing an optically
active carboxylic acid (for example, a or p-amino acid) by
hydrolyzing a carboxylic acid ester (racemic mixture; for
example, a or [3-amino acid ester (racemic mixture)) having
an asymmetric carbon atom at a portion other than an ester
portion. The carboxylic acid obtainable by the present

40
invention, in particular, the optically active carboxylic
acid (for example, optically active a or (3-amino acid) is
useful as a starting material or a synthetic intermediate
for medicinal and agricultural chemicals, or for physio-
logically active substances such as physiologically active
peptide or lactam series antibiotics, etc.

41
CLAIMS
1. A process for preparing a carboxylic acid using a
surfactant-modified enzyme which comprises selectively
reacting water and a carboxylic acid ester, provided that
triglyceride is excluded, in an organic solvent in the
presence of a surfactant-modified enzyme.
2. The preparation process according to Claim 1, wherein
the carboxylic acid ester is a carboxylic acid ester which
is a racemic mixture and having an asymmetric carbon atom
at the portion other than an ester portion.
3. The preparation process according to Claim 2, wherein
the carboxylic acid ester which is a racemic mixture and
having an asymmetric carbon atom at the portion other than
an ester portion is an a or (3-substituted carboxylic acid
ester which is a racemic mixture and represented by the
formula (I):

wherein R represents an alkyl group, alkenyl group,
alkynyl group, cycloalkyl group, aralkyl group, aryl
group or heteroaryl group, each of which may have a
substituent(s), R1 represents an alkyl group which
may have a substituent(s), Z represents an amino
group which may have a protective group, a hydroxyl
group which may have a protective group or an alkyl
group, * represents an asymmetric carbon atom, and n
is 0 or 1,
and the carboxylic acid is an optically active a or p-
substituted carboxylic acid represented by the formula
(II) :

42

wherein R, Z, n and * have the same meanings as
defined above.
4. The preparation process according to Claim 2 or 3,
wherein the carboxylic acid ester (racemic mixture) having
an asymmetric carbon atom at the portion other than an
ester portion is an a or p-substituted carboxylic acid
ester represented by the formula (I):

wherein R, R1, Z, n and * have the same meanings as
defined above,
the optically active carboxylic acid is an optically active
a or p-substituted carboxylic acid represented by the
formula (II):

wherein R, Z, n and * have the same meanings as
defined above,
and the carboxylic acid ester which is not hydrolyzed and
remained is an optically active a or p-substituted
carboxylic acid ester represented by the formula (III) :

wherein R, R1, Z, n and * have the same meanings as
defined above,

43
provided that it has a reverse absolute configuration to
that of the compound of the formula (II).
5. The preparation process according to Claim 1, wherein
the surfactant-modified enzyme is an enzyme in which a
hydrolase is subjected to coating treatment with a
surfactant to be solubilized in an organic solvent.
6. The preparation process according to Claim 1 or 5,
wherein the hydrolase is a protease, an esterase or a
lipase.
7. The preparation process according to Claim 6, wherein
the hydrolase is at least one hydrolase selected from the
group consisting of a lipase originated from Burkholderia
cepacia {Pseudomonas cepacia) and a lipase originated from
Candida Antarctica.
8. The preparation process according to Claim 5, wherein
the surfactant is N-D-glucono-L-glutamic acid diester.
9. The preparation process according to Claim 1, wherein
an amount of water to be used is 0.5 to 10 mol based on 1
mol of the carboxylic acid ester.

10. The preparation process according to Claim 1, wherein
the organic solvent is at least one organic solvent
selected from the group consisting of an ether, a ketone,
an ester, an aliphatic hydrocarbon and an aromatic hydro-
carbon .
11. The preparation process according to Claim 3 or 4,
wherein R1 is a methyl group or an ethyl group each of
which may have a substituent(s).
12. The preparation process according to Claim 11, wherein

44
the substituent (s) of R1 is a halogen atom or an alkoxy
group.
13. The preparation process according to Claim 3 or 4,
wherein Z is an amino group substituted by an amino group
or an aralkyl group.
14. The preparation process according to Claim 4, wherein
each of the optically active a or p-substituted carboxylic
acid represented by the formula (II):

wherein R, Z, n and * have the same meanings as
defined above,
which had formed by the hydrolysis, and the remaining a or
p-substituted carboxylic acid ester represented by the
formula (III):

wherein R, R1, Z, n and * have the same meanings as
defined above,
which had not been hydrolyzed, provided that it has a
reverse absolute configuration to that of the compound of
the formula (II),
is isolated from a mixture thereof.

The present invention relates to a process for
preparing a carboxylic acid using a surfactant-modified
enzyme which comprises selectively reacting water and a
carboxylic acid ester, provided that triglyceride is
excluded, in an organic solvent in the presence of a
surfactant-modified enzyme.