U.S. patent application number 14/618900 was filed with the patent office on 2015-08-06 for process for producing optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane.
The applicant listed for this patent is GENENTECH, INC.. Invention is credited to Norihiko HIRATA, Kazuhiro YAMAUCHI.
Application Number | 20150218602 14/618900 |
Document ID | / |
Family ID | 40321442 |
Filed Date | 2015-08-06 |
United States Patent
Application |
20150218602 |
Kind Code |
A1 |
HIRATA; Norihiko ; et
al. |
August 6, 2015 |
PROCESS FOR PRODUCING OPTICALLY ACTIVE
2-ALKYL-1,1,3-TRIALKOXYCARBONYLPROPANE
Abstract
A process for producing an optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane (2), comprising a step of
asymmetric hydrolysis of 2-alkyl-1,1,3-trialokoxycarbonylpropane
(1) by using an enzyme capable of selectively hydrolyzing an ester
moiety of either one enantiomer of
2-alkyl-1,1,3-trialkoxycarbonylpropane (1), or by using a culture
of a microorganism capable of producing the enzyme or a treated
object thereof.
Inventors: |
HIRATA; Norihiko; (Osaka,
JP) ; YAMAUCHI; Kazuhiro; (Osaka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC. |
South San Francisco |
CA |
US |
|
|
Family ID: |
40321442 |
Appl. No.: |
14/618900 |
Filed: |
February 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12599338 |
Nov 9, 2009 |
8969051 |
|
|
PCT/JP2008/058991 |
May 9, 2008 |
|
|
|
14618900 |
|
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Current U.S.
Class: |
435/135 |
Current CPC
Class: |
Y02P 20/52 20151101;
C12N 9/18 20130101; C12P 7/62 20130101; C12P 41/005 20130101 |
International
Class: |
C12P 7/62 20060101
C12P007/62 |
Foreign Application Data
Date |
Code |
Application Number |
May 14, 2007 |
JP |
2007-127704 |
May 29, 2007 |
JP |
2007-141542 |
Mar 27, 2008 |
JP |
2008-083302 |
Claims
1-11. (canceled)
12. A process for producing an optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane of Formula (2): ##STR00003##
wherein: R.sub.1, R.sub.2 and R.sub.3, which may be the same or
different, are a C1-C4 alkyl group; and * represents an
asymmetrical center; comprising hydrolyzing a
2-alkyl-1,1,3-trialokoxycarbonylpropane of Formula (1):
##STR00004## wherein R.sub.1, R.sub.2 and R.sub.3 are as defined
above, to provide the optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane of Formula (2).
13. The process according to claim 12, wherein R.sub.1 is
methyl.
14. The process according to claim 12, wherein R.sub.2 is
methyl.
15. The process according to claim 12, wherein R.sub.3 is
methyl.
16. The process according to claim 12, wherein both R.sub.1 and
R.sub.2 are methyl.
17. The process according to claim 12, wherein both R.sub.2 and
R.sub.3 are methyl.
18. The process according to claim 12, wherein R.sub.1, R.sub.2 and
R.sub.3 are methyl.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/599,338, filed Nov. 9, 2009, now allowed,
which is a 371 of International Patent Application No.
PCT/JP2008/058991, filed May 9, 2008, which was published on Nov.
20, 2008 under International Publication No. WO 2008/140127 A1. The
entire content of the applications referenced above are hereby
incorporated by reference herein. This application also claims
priority to Japanese Patent Application No. 2007-127704, filed May
14, 2007, Japanese Patent Application No. 2007-141542, filed May
29, 2007, and Japanese Patent Application No. 2008-083302, filed
Mar. 27, 2008.
TECHNICAL FIELD
[0002] The present invention relates to a process for producing an
optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane.
BACKGROUND ART
[0003] An optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane
is a compound useful as a material for formation of an asymmetric
carbon atom in synthesis of natural products, pharmaceuticals and
so on.
[0004] Heretofore, as a method for producing such an optically
active 2-alkyl-1,1,3-trialkoxycarbonylpropane, for example, a
method of addition of a silyl enolate to an alkylidenemalonate in
the presence of an asymmetric copper catalyst (Non-patent document
1) is known. By ester-exchange of the thioester obtained in this
production method, an optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane can be obtained. In this
method, however, a halogenated solvent is used, and a low
temperature condition is required for improving the optical purity
of the objective substance. Additionally, even when the reaction is
conducted at -78.degree. C., the optical purity is at most 93% ee
(43% ee when the alkyl group that binds to the carbon atom serving
as optically-active center is a methyl group), so that this method
is not necessarily satisfactory from the industrial view.
[0005] [Non-patent document 1] J. Am. Chem. Soc., 122, 9134
(2000)
DISCLOSURE OF THE INVENTION
[0006] In view of the above, the present inventors have studied
intensely about a process for producing an optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane by hydrolyzing a racemic
form of 2-alkyl-1,1,3-trialkoxycarbonylpropane as a material in an
optically selective manner using various kinds of enzymes under an
ordinary enzymatic reaction condition, and have found that both
isomers of the optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane can be produced easily with
excellent efficiency.
[0007] Specifically, the present invention provides the following
[1]-[11].
[1] A process for producing an optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane represented by Formula
(2):
##STR00001##
(wherein, R.sub.1, R.sub.2 and R.sub.3, which may be the same or
different, represent a C1-C4 alkyl group, and * represents that the
carbon atom is an asymmetrical center) comprising a step of
asymmetric hydrolysis of an 2-alkyl-1,1,3-trialokoxycarbonylpropane
represented by Formula (1):
##STR00002##
(wherein R.sub.1, R.sub.2 and R.sub.3 are as defined above) by
using an enzyme capable of selectively hydrolyzing an ester moiety
of either one enantiomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane represented by Formula (1),
or a culture of a microorganism capable of producing the enzyme or
a treated object thereof. [2] The process according to [1], wherein
R.sub.1 in the 2-alkyl-1,1,3-trialkoxycarbonylpropane represented
by Formula (1) is a methyl group. [3] The process according to [1],
wherein R.sub.2 in the 2-alkyl-1,1,3-trialkoxycarbonylpropane
represented by Formula (1) is a methyl group. [4] The process
according to [1], wherein R.sub.3 in the
2-alkyl-1,1,3-trialkoxycarbonylpropane represented by Formula (1)
is a methyl group. [5] The process according to [1], wherein both
R.sub.1 and R.sub.2 in the 2-alkyl-1,1,3-trialkoxycarbonylpropane
represented by Formula (1) are methyl groups. [6] The process
according to [1], wherein both R.sub.2 and R.sub.3 in the
2-alkyl-1,1,3-trialkoxycarbonylpropane represented by Formula (1)
are methyl groups. [7] The process according to [1], wherein all of
R.sub.1, R.sub.2 and R.sub.3 in the
2-alkyl-1,1,3-trialkoxycarbonylpropane represented by Formula (1)
are methyl groups. [8] The process according to any one of [1] to
[7], wherein the enzyme is a hydrolase originated from a
microorganism of Candida or Bacillus. [9] The process according to
any one of [1] to [7], wherein the enzyme is a hydrolase originated
from a microorganism of Arthrobacter globiformis, Candida
cylindracea, Candida rugosa, Candida antactica, Bacillus
licheniformis, Bacillus subtilis or Chromobacterium chocolatum or a
thermophilic microorganism. [10] The process according to any one
of [1] to [7], wherein the enzyme is an esterase or a lipase
originated from Arthrobacter strain SC-6-98-28 (FERM BP-3658) or
Chromobacterium strain SC-YM-1 (FERM BP-6703). [11] The process
according to any one of [1] to [7], wherein the enzyme is a protein
containing an amino acid sequence selected from the following a) to
e):
[0008] a) an amino acid sequence represented by SEQ ID NO: 1 or
3;
[0009] b) an amino acid sequence i) which is encoded by a
nucleotide sequence of DNA having at least 90% homology to DNA
having a nucleotide sequence represented by SEQ ID NO: 2 or 4, and
ii) which is an amino acid sequence of a protein capable of
selectively hydrolyzing an ester moiety of either one enantiomer of
the 2-alkyl-1,1,3-trialkoxycarbonylpropane;
[0010] c) an amino acid sequence i) which is encoded by a
nucleotide sequence of DNA that hybridizes with DNA having a
nucleotide sequence represented by SEQ ID NO: 2 or 4 under a
stringent condition, and ii) which is an amino acid sequence of a
protein capable of selectively hydrolyzing an ester moiety of
either one enantiomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane;
[0011] d) an amino acid sequence i) in which one or plural amino
acids are deleted, replaced or added in the amino acid sequence
represented by SEQ ID NO: 1 or 3, and ii) which is an amino acid
sequence of a protein capable of selectively hydrolyzing an ester
moiety of either one enantiomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane; and
[0012] e) an amino acid sequence i) having at least 90% homology to
the amino acid sequence represented by SEQ ID NO: 1 or 3, and ii)
which is an amino acid sequence of a protein capable of selectively
hydrolyzing an ester moiety of either one enantiomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane.
MODE FOR CARRYING OUT THE INVENTION
[0013] In the following, the present invention will be explained in
detail.
[0014] The 2-alkyl-1,1,3-trialkoxycarbonylpropane represented by
Formula (1) (hereinafter, simply denoted by
2-alkyl-1,1,3-trialkoxycarbonylpropane (1)) which is a material
used in the process of the present invention can be produced in any
known method such as a method of a reaction of dialkyl malonate and
.beta.-alkyl-.alpha.,.beta.-unsaturated alkyl ester in the presence
of a base (for example, see Tetrahedron, 44, 119 (1988)), and used
in the present invention.
[0015] Typical examples of C1-C4 alkyl group represented by
R.sub.1, R.sub.2 and R.sub.3 in the compounds represented by the
formulas (1) and (2) of the present invention include a methyl
group, an ethyl group, an n-propyl group, an isopropyl group, an
n-butyl group, an isobutyl group, a sec-butyl group, and a
tert-butyl group.
[0016] Typical examples of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1) include
2-methyl-1,1,3-trimethoxycarbonylpropane,
2-methyl-1,1,3-triethoxycarbonylpropane,
2-methyl-1,1,3-tri-n-propoxycarbonylpropane,
2-ethyl-1,1,3-trimethoxycarbonylpropane,
2-ethyl-1,1,3-triethoxycarbonylpropane,
2-n-propyl-1,1,3-trimethoxycarbonylpropane,
2-n-propyl-1,1,3-triethoxycarbonylpropane,
2-methyl-1,1-diethoxycarbonyl-3-methoxycarbonylpropane,
2-methyl-1,1-di-n-propoxycarbonyl-3-methoxycarbonylpropane,
2-ethyl-1,1-diethoxycarbonyl-3-methoxycarbonylpropane,
2-ethyl-1,1-di-n-propoxycarbonyl-3-methoxycarbonylpropane,
2-n-propyl-1,1-diethoxycarbonyl-3-methoxycarbonylpropane,
2-methyl-1,1-dimethoxycarbonyl-3-ethoxycarbonylpropane,
2-methyl-1,1-di-n-propoxycarbonyl-3-ethoxycarbonylpropane,
2-ethyl-1,1-dimethoxycarbonyl-3-ethoxycarbonylpropane and
2-n-propyl-1,1-dimethoxycarbonyl-3-ethoxycarbonylpropane.
[0017] Examples of the enzyme capable of selectively hydrolyzing an
ester moiety of an S isomer of
2-alkyl-1,1,3-trialkoxycarbonylpropane (1) include an enzyme
originated from a microorganism of Candida such as Candida
cylindracea and Candida rugosa, a microorganism of Chromobacterium
chocolatum, pig liver and a thermophilic microorganism. The enzyme
from a microorganism of Candida such as Candida cylindracea and
Candida rugosa, a microorganism of Chromobacterium chocolatum and a
thermophilic microorganism are preferred.
[0018] More specific examples of the enzyme capable of selectively
hydrolyzing an ester moiety of an S isomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1) include an enzyme
originated from Chromobacterium strain SC-YM-1 (FERM BP-6703) and
commercially available enzymes CHIRAZYME (registered trademark) E-3
(originated from thermophilic microorganism), lipase CHIRAZYME
(registered trademark) L-3 (originated from Candida rugosa),
cholesterol esterase (originated from Candida cylindracea) (the
above-mentioned are produced by Roche Diagnostics), lipase
ChiroCLEC-CR (produced by Altus Biologics), lipase Lipase-MY
(produced by Candida cylindracea) (produced by Meito Sangyo Co.,
Ltd.), and lipase Lipase OF (produced by Meito Sangyo Co., Ltd.)
and PLE-A (produced by Amano Enzyme Inc.). The enzyme originated
from Chromobacterium strain SC-YM-1 (FERM BP-6703) is more
preferred.
[0019] Examples of the enzyme capable of selectively hydrolyzing an
ester moiety of an R isomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1) include an enzyme
originated from a microorganism of Bacillus such as Bacillus
licheniformis and Bacillus subtilis, a microorganism of
Arthrobacter globiformis, a microorganism of Candida antactica,
bovine pancreas and a thermophilic microorganism. The enzyme
originated from a microorganism of Bacillus such as Bacillus
licheniformis and Bacillus subtilis, a microorganism of
Arthrobacter globiformis, and a thermophilic microorganism are
preferred.
[0020] More specific examples of the enzyme capable of selectively
hydrolyzing an ester moiety of an R isomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1) include an enzyme
originated from Arthrobacter strain SC-6-98-28 (FERM BP-3658), and
commercially available enzymes such as esterase CHIRAZYME
(registered trademark) E-4 (originated from thermophilic
microorganism), protease CHIRAZYME (registered trademark) P-1
(originated from Bacillus licheniformis) (the above-mentioned are
produced by Roche Diagnostics), protease Purafect (registered
trademark) 4000E (produced by GENENCOR), protease
.alpha.-Chymotrypsin (produced by SIGMA), and lipase SP-525
(produced by Novozymes Japan). The enzyme originated from
Arthrobacter strain SC-6-98-28 (FERM BP-3658) is more
preferred.
[0021] The enzyme capable of selectively hydrolyzing an ester
moiety of either one of enantiomers of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1) (hereinafter, referred
to as the present enzyme) may be enzymes originated from mutants
induced by a treatment of these microorganisms with a mutagenic
agent, ultraviolet rays or the like, enzymes produced by a
recombinant microorganism transformed with a gene encoding the
present enzyme possessed by these microorganisms, or mutated
enzymes in which one or several amino acids in the present enzyme
mentioned above are deleted, added, or replaced by genetic
engineering technique in the process of the present invention as
far as they are capable of selectively hydrolyzing an ester moiety
of either one enantiomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1).
[0022] A recombinant microorganism transformed with a gene encoding
the present enzyme can be prepared, for example, by an ordinary
genetic engineering technique described, for example, in Molecular
Cloning 2nd edition (written by J. Sambrook, E. F. Fritsch, and T.
Maniatis, published by Cold Spring Harbor Laboratory, 1989) or
similar methods. In addition, it can be prepared according to the
methods described in JP2001-46084A, JP3855329, JP3875283, or
JP3151893, or similar methods. Examples of the present enzyme
produced by a recombinant microorganism that can be prepared in
such a manner as described above include an esterase originated
from Chromobacterium strain SC-YM-1 (FERM BP-6703) (JP3875283) or
an esterase originated from Arthrobacter strain SC-6-98-28 (FERM
BP-3658) (JP3151893).
[0023] A typical example of the esterase originated from
Chromobacterium strain SC-YM-1 (FERM BP-6703) is an enzyme having
an amino acid sequence represented by SEQ ID NO: 1, and a typical
example of the esterase originated from Arthrobacter strain
SC-6-98-28 (FERN BP-3658) is an enzyme having an amino acid
sequence represented by SEQ ID NO: 3.
[0024] In the gene that encodes the present enzyme, for example,
DNA having a nucleotide sequence represented by SEQ ID NO: 2 is a
gene encoding the esterase originated from Chromobacterium strain
SC-YM-1 (FERM BP-6703), and for example, DNA having a nucleotide
sequence represented by SEQ ID NO: 4 is a gene encoding the
esterase originated from Arthrobacter strain SC-6-98-28 (FERM
BP-3658).
[0025] In the present invention, it is preferred to use an enzyme
made of a protein having an amino acid sequence of either one of
the following a) to e):
a) an amino acid sequence represented by SEQ ID NO: 1 or 3; b) an
amino acid sequence i) which is encoded by a nucleotide sequence of
DNA having at least 90% homology to DNA having a nucleotide
sequence represented by SEQ ID NO: 2 or 4, and ii) which is an
amino acid sequence of a protein capable of selectively hydrolyzing
an ester moiety of either one enantiomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane; c) an amino acid sequence
i) which is encoded by a nucleotide sequence of DNA that hybridizes
with DNA having a nucleotide sequence represented by SEQ ID NO: 2
or 4 under a stringent condition, and ii) which is an amino acid
sequence of a protein capable of selectively hydrolyzing an ester
moiety of either one enantiomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane; d) an amino acid sequence
i) in which one or plural amino acids are deleted, replaced or
added in the amino acid sequence represented by SEQ ID NO: 1 or 3,
and ii) which is an amino acid sequence of a protein capable of
selectively hydrolyzing an ester moiety of either one enantiomer of
the 2-alkyl-1,1,3-trialkoxycarbonylpropane; and e) an amino acid
sequence i) having at least 90% homology to the amino acid sequence
represented by SEQ ID NO: 1 or 3, and ii) which is an amino acid
sequence of a protein capable of selectively hydrolyzing an ester
moiety of either one enantiomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane.
[0026] Among them, it is more preferred to use an enzyme made of a
protein having an amino acid sequence represented by at least 1st
to 362nd amino acids in the amino acid sequence represented by SEQ
ID NO: 1 in which the 43rd N (asparagine) is replaced by S (serine)
(N43SA363term described in JP2000-78988A), or having an amino acid
sequence represented by at least 1st to the 362nd amino acids in
the amino acid sequence represented by SEQ ID NO: 1 in which the
160th G (glycine) is replaced by S (serine) and the 189th G
(glycine) is replaced by F (phenylalanine) (160S189F363term
described in JP3486942).
[0027] "DNA that hybridizes with DNA having a nucleotide sequence
represented by SEQ ID NO: 2 or 4 under a stringent condition"
refers to such DNA that (1) can form a DNA-DNA hybrid with DNA
having a nucleotide sequence encoding an amino acid sequence
represented by SEQ ID NO: 1 by allowing hybridization at 65.degree.
C. under a high ion concentration [for example, 6.times.SSC (900 mM
of sodium chloride, 90 mM of sodium citrate) can be mentioned], and
(2) can keep the hybrid even after 30-minute incubation at
65.degree. C. under a low ion concentration [for example,
0.1.times.SSC (15 mM of sodium chloride, 1.5 mM of sodium citrate)
can be mentioned], in Southern hybridization method described, for
example, in "Cloning and Sequencing" (supervised by Itaru Watanabe,
edited by Masahiro Sugiura, 1989, published by Nosonbunka-sha).
Specifically, for example, DNA having a nucleotide sequence
encoding an amino acid sequence represented by SEQ ID NO: 1 or 3,
DNA having a nucleotide sequence in which a certain base is
deleted, replaced or added in a nucleotide sequence encoding an
amino acid sequence represented by SEQ ID NO: 1 or 3, DNA having at
least 90%, preferably at least 95% homology to DNA having a
nucleotide sequence encoding an amino acid sequence represented by
SEQ ID NO: 1 or 3, and the like are recited.
[0028] The above-mentioned DNA may be DNA cloned from naturally
occurring DNA, or DNA in which a certain base is artificially
deleted, replaced or added in a nucleotide sequence of such cloned
DNA, or artificially synthesized DNA. The homology can be
calculated by using a sequence analyzing tool such as BESTFIT
program supplied, for example, from UWGCG Package (Devereux et al
(1984) Nucleic Acids Research 12, p 387-395), PILEUP and BLAST
algorism (Altschul S. F. (1993) J. Mol Evol 36: 290-300; Altschul
S. F. (1990) J. Mol Biol 215: 403-10).
[0029] "The amino acid sequence in which one or plural amino acids
are deleted, replaced or added in an amino acid sequence
represented by SEQ ID NO: 1 or 3" is preferably an amino acid
sequence in which one amino acid or two amino acids is/are deleted,
replaced or added in an amino acid sequence represented by SEQ ID
NO: 1 or 3. In addition, examples of the present enzyme include an
amino acid sequence having at least 90%, preferably at least 95%
homology to an amino acid sequence represented by SEQ ID NO: 1 or
2.
[0030] Microorganisms which produce the present enzyme can be
liquid-cultured in any ordinary method. Various culture media
appropriately containing a carbon source, a nitrogen source, an
inorganic substance and the like used in ordinary microorganism
culture can be used for the culture medium. For example, glucose,
glycerin, organic acid and molasses can be used for the carbon
source; peptone, yeast extract, malt extract, soy powder, corn
steep liquor, cotton seed powder, dry yeast, casamino acid,
ammonium chloride, ammonium nitrate, ammonium sulfate and urea can
be used for the nitrogen source; and salts, sulfates, and
phosphates of potassium, sodium, magnesium, iron, manganese,
cobalt, zinc and the like, specifically, potassium chloride, sodium
chloride, magnesium sulfate, ferrous sulfate, manganese sulfate,
cobalt chloride, zinc sulfate, potassium phosphate, sodium
phosphate and the like can be used for the inorganic substance. For
improving the asymmetric hydrolyzing ability of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1) possessed by the
aforementioned microorganism, triglyceride such as olive oil or
tributyrin or the 2-alkyl-1,1,3-trialkoxycarbonylpropane (1) may be
added appropriately to the culture medium.
[0031] In the case of a transformant formed by introducing a
plasmid in which DNA encoding the present enzyme is downstream
connected with a promoter of the type that is induced by
allolactose such as tac promoter, trc promoter and lac promoter,
for example, a small amount of isopropyl thio-.beta.-D-galactoside
(IPTG) can be added into a culture medium as an inducer for
inducing production of the protein of the present invention.
[0032] In general, culture is preferably conducted aerobically, and
shaking culture or culture under aeration and stirring is
appropriate. Culture temperature is about 20 to 40.degree. C., and
preferably about 25 to 35.degree. C., and pH is preferably about 6
to 8. Culture time is preferably about 1 to 7 days, though it
varies depending on various conditions.
[0033] A solid culture method may also be appropriately employed,
if required, as far as bacterial cells capable of asymmetrically
hydrolyzing the 2-alkyl-1,1,3-trialkoxycarbonylpropane (1) can be
obtained.
[0034] Purification of the present enzyme from a microbial culture
obtained in the above manner may be conducted in a method
ordinarily used in purification of enzyme. For example, bacterial
cells in the microbial culture are disrupted by a method such as an
ultrasonic treatment, a Dyno mill treatment, or a French press
treatment at first. After removing insoluble materials from the
obtained disrupted solution by centrifugation or the like, an
objective enzyme can be purified by either one or combination of
cation-exchange column chromatography, anion-exchange column
chromatography, hydrophobic column chromatography, gel filtration
column chromatography and the like that are ordinarily used in
purification of enzyme. Examples of carriers usable in these column
chromatographies include DEAE-Sepharose (registered trademark),
fastflow (produced by GE Healthcare Bioscience) and Butyl-Toyopearl
(registered trademark) 650S (produced by TOSOH Corporation).
[0035] The present enzyme can be used in various forms including a
purified enzyme, a crude enzyme, a microbial culture, a bacterial
cell, and a treated object thereof. Examples of the treated object
used herein include a lyophilized bacterial cell, an acetone-dried
bacterial cell, a ground bacterial cell, an autodigested substance
of bacterial cell, an ultrasonic-treated object of bacterial cell,
bacterial cell extract, or an alkaline-treated object of bacterial
cell. Further, enzymes in various purities or forms as described
above may be immobilized for use, for example, by known methods
including an adsorption method to an inorganic carrier such as
silica gel and ceramics, cellulose, ion-exchange resin and so on, a
polyacrylamide method, a sulfur-containing polysaccharide gel
method (for example, a carrageenan gel method), an alginic acid gel
method, an agar gel method and so on.
[0036] A used amount of the present enzyme is appropriately
determined so that delay of reaction time or decrease in
selectivity will not occur, and for example, when a purified
enzyme, a crude enzyme or a commercially available enzyme is used,
the amount is usually 0.001 to 2 times by weight, preferably 0.002
to 0.5 time by weight, relative to that of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1), and when a microbial
culture, a bacterial cell or a treated object thereof is used, the
amount is usually 0.01 to 200 times by weight, preferably 0.1 to 50
times by weight, relative to that of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1).
[0037] Water used in the asymmetrical hydrolysis reaction may be a
buffered aqueous solution. Examples of the buffered aqueous
solution include buffered aqueous solutions of inorganic acid salt
such as aqueous alkali phosphate solutions such as an aqueous
sodium phosphate solution and an aqueous potassium phosphate
solution, and buffered aqueous solutions of organic acid salt such
as aqueous alkali acetate solutions such as an aqueous sodium
acetate solution and an aqueous potassium acetate solution. A used
amount of such water may be usually 0.5 time by mol or more,
occasionally a solvent amount, and usually 200 times by weight or
less, relative to that of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1).
[0038] Asymmetric hydrolysis reaction may be conducted in the
presence of an organic solvent such as a hydrophobic organic
solvent or a hydrophilic organic solvent. Examples of the
hydrophobic organic solvent include ethers such as tert-butyl
methyl ether and isopropyl ether, hydrocarbons such as toluene,
hexane, cyclohexane, heptane, octane and isooctane, and examples of
the hydrophilic organic solvent alcohols such as tert-butanol,
methanol, ethanol, isopropanol, isobutanol and n-butanol, ethers
such as tetrahydrofuran, sulfoxides such as dimethyl sulfoxide,
ketones such as acetone, nitriles such as acetonitrile, amides such
as N,N-dimethylformamide. These hydrophobic organic solvents and
hydrophilic organic solvents are respectively used alone or in
combination of two or more kinds, and a combination of hydrophobic
organic solvent and hydrophilic organic solvent may be used.
[0039] When the organic solvent is used, a used amount thereof is
usually 200 times by weight or less, preferably in the range of
about 0.1 to 100 times by weight, relative to that of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1).
[0040] Asymmetric hydrolysis reaction is conducted, for example, by
mixing water, the 2-alkyl-1,1,3-trialkoxycarbonylpropane (1) and
the present enzyme, and when an organic solvent is used, the
organic solvent, water, the 2-alkyl-1,1,3-trialkoxylcarbonylpropane
(1) and the present enzyme may be mixed.
[0041] The pH value of the reaction system, at which asymmetric
hydrolysis by the present enzyme proceeds with high selectivity, is
appropriately selected, and is usually about pH 4 to 10, and
preferably about pH 6 to 8, though it is not limited. The pH may be
adjusted to a value in the selected range appropriately by adding a
base during the reaction. Examples of the base include alkali
hydroxides such as sodium hydroxide and potassium hydroxide, of
alkali and alkaline earth carbonates such as sodium carbonate,
potassium carbonate and calcium carbonate, alkali bicarbonates such
as sodium bicarbonate and potassium bicarbonate, phosphates such as
sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium
dihydrogen phosphate, and dipotassium hydrogen phosphate, organic
bases such as triethyl amine and pyridine, and ammonia. The bases
may be used alone or in combination of two or more kinds. While the
base is usually used in an aqueous solution, it may be used in an
organic solvent or a mixed solution of organic solvent and water
when an organic solvent is used in the reaction. The organic
solvent may be the same as that used in the reaction. Further, the
base may be used in solid or in the state of being suspended in a
solution.
[0042] A reaction temperature at which asymmetric hydrolysis by the
present enzyme proceeds with high selectivity is appropriately
selected. Though the temperature is not limited, too high
temperature tends to reduce the stability of the enzyme and too low
temperature tends to decrease the reaction speed. On the other
hand, the lower the temperature the more the selectivity increases.
It is usually in the range of about -10 to 65.degree. C.,
preferably about -5 to 50.degree. C.
[0043] Thus a solution of an optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane represented by the formula
(2) (hereinafter, simply referred to as optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane (2)) is obtained, and
usually, a post treatment operation is further conducted for
separation from the enzyme and the buffer used in the reaction, and
the carboxylic acid generated by hydrolysis reaction.
[0044] Examples of the post treatment include a method of
conducting separation and purification using silica gel
chromatography after distilling off the solvent in the reaction
solution, and a method of conducting separation and purification by
distillation after distilling off the solvent, and a method of
conducting separation and purification by liquid separation
operation.
[0045] Separation and purification by liquid separation operation
may be conducted after removing an organic solvent soluble in both
water and a hydrophobic organic solvent may be removed by
distillation when such an organic solvent is used in the reaction.
When there are an insoluble enzyme, an immobilizing carrier and so
on in the solution, these may be removed by filtration.
[0046] For separating the optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane (2) which is an objective
substance from the enzyme, the buffer and other water-soluble
components, a hydrophobic organic solvent may be used for
extracting the optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane (2) with an organic phase,
and the organic phase may be separated from an aqueous phase.
[0047] Examples of the hydrophobic organic solvent include ethers
such as tert-butyl methyl ether and isopropyl ether, hydrocarbons
such as toluene, hexane, cyclohexane, heptane, octane and
isooctane, halogenated hydrocarbons such as dichloromethane,
dichloroethane, chloroform, chlorobenzene and orthodichlorobenzene,
and esters such as ethyl acetate, methyl acetate and butyl acetate.
When the hydrophobic organic solvent is used in the reaction, the
liquid separation operation may be conducted directly.
Alternatively, when the hydrophobic organic solvent is not used in
the reaction, or when the liquid separation can not be achieved due
to the small amount used, or when the liquid separation cannot be
readily achieved due to the small amount of the used water, the
hydrophobic organic solvent, water and the like may be
appropriately added before the liquid separation. A used amount of
the hydrophobic organic solvent is usually 0.1 to 200 times by
weight, and preferably in the range of about 0.2 to 100 times by
weight, relative to that of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1), though it is not
limited.
[0048] The pH in the extraction of the objective substance is
usually in the range of about 6 to 10, and preferably in the range
of about 7 to 9.
[0049] For adjusting the pH of the solution into the range, an acid
or a base may be appropriately used. Examples of the acid include
inorganic acids such as hydrogen chloride, hydrogen bromide,
sulfuric acid and phosphoric acid and salts thereof, and bases
thereof, and organic acids such as acetic acid, citric acid and
methanesulfonic acid, and salts thereof. A base similar to that
used for adjusting pH in the reaction may be used.
[0050] When extraction of the objective substance from the aqueous
phase is insufficient, the same extraction and liquid separation
operation may be repeated plural times. Likewise, when removal of
the water-soluble components from the organic phase is
insufficient, the same extraction and liquid separation operation
may be repeated plural times
[0051] Next, by distilling off the organic solvent in the obtained
organic phase, the optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane (2) which is an objective
substance can be isolated.
[0052] The obtained optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane (2) may further be purified
by column chromatography or distillation.
[0053] In the present invention, when an enzyme capable of
selectively hydrolyzing an ester moiety of an S isomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1) is used for the present
enzyme, the optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane
(2) obtained from the organic phase in the liquid separation
treatment is rich in R isomer, while when an enzyme capable of
selectively hydrolyzing an ester moiety of an R isomer of the
2-alkyl-1,1,3-trialkoxycarbonylpropane (1) is used for the present
enzyme, the optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane
(2) obtained from the organic phase in the liquid separation
treatment is rich in S isomer.
[0054] The aqueous phase after the liquid separation operation
usually contains a hydrolysis product having opposite
stereoisomerism to that of the objective substance obtained from
the organic phase. In other words, when an enzyme capable of
selectively hydrolyzing an ester moiety of an S isomer is used, the
aqueous phase contains a hydrolysis product of the optically active
(S)-2-alkyl-1,1,3-trialkoxycarbonylpropane, and when an enzyme
capable of selectively hydrolyzing an ester moiety of an R isomer
is used, the aqueous phase contains a hydrolysis product of the
optically active (R)-2-alkyl-1,1,3-trialkoxycarbonylpropane. These
hydrolysis products may be collected by distilling off water from
the aqueous phase, or by extracting the aqueous phase with an
organic solvent after adjusting the aqueous phase to acidic, and
concentrating the obtained organic phase. By esterifying the
obtained hydrolysis product, the corresponding optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane (2) can be obtained. Such
esterification may be conducted by conventional methods, for
example, a method of reacting the hydrolysis product with alcohol
in the presence of sulfuric acid; a method of reacting the
hydrolysis product with alcohol in the presence of trimethylsilyl
azide or trimethylsilyl chloride.
[0055] Examples of the typical optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane (2) thus obtained include
(R)-2-methyl-1,1,3-trimethoxycarbonylpropane,
(R)-2-methyl-1,1,3-triethoxycarbonylpropane,
(R)-2-methyl-1,1,3-tri-n-propoxycarbonylpropane,
(R)-2-ethyl-1,1,3-trimethoxycarbonylpropane,
(R)-2-ethyl-1,1,3-triethoxycarbonylpropane,
(R)-2-n-propyl-1,1,3-trimethoxycarbonylpropane,
(R)-2-n-propyl-1,1,3-triethoxycarbonylpropane,
(R)-2-methyl-1,1-diethoxycarbonyl-3-methoxcarbonylpropane,
(R)-2-methyl-1,1-di-n-propoxycarbonyl-3-methoxycarbonylpropane,
(R)-2-ethyl-1,1-diethoxycarbonyl-3-methoxycarbonylpropane,
(R)-2-ethyl-1,1-di-n-propoxycarbonyl-3-methoxycarbonylpropane,
(R)-2-n-propyl-1,1-diethoxycarbonyl-3-methoxycarbonylpropane,
(R)-2-methyl-1,1-dimethoxycarbonyl-3-ethoxycarbonylpropane,
(R)-2-methyl-1,1-di-n-propoxycarbonyl-3-ethoxycarbonylpropane,
(R)-2-ethyl-1,1-dimethoxycarbonyl-3-ethoxycarbonylpropane,
(R)-2-n-propyl-1,1-dimethoxycarbonyl-3-ethoxycarbonylpropane, and
compounds wherein (R) is replaced by (S).
EXAMPLES
[0056] In the following, the present invention will be explained
more specifically by way of examples; however, the present
invention is not limited to these examples.
Examples 1 to 14
[0057] To 35 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane and
each of various enzymes shown in Table 1 respectively weighed in
the amount shown in Table 2, 2 mL of 100 mM potassium phosphate
buffer (pH 7.0) was added. The resultant solution was stirred at
25.degree. C. for 20 hours, and then 2.5 mL of acetonitrile was
added thereto and the mixture was drawn through a membrane filter.
The optical purity of the resultant filtrate was analyzed by high
performance liquid chromatography [column: CHIRALCEL (registered
trademark) OB-H, 4.6 mm.phi..times.15 cm, 5 .mu.m (produced by
Daicel Chemical Industries, Ltd.)], and the chemical purity was
analyzed by high performance liquid chromatography [column: SUMIPAX
ODS D-210FF, 4.6 mm.phi..times.15 cm, 3 .mu.m (available from
Sumika Chemical Analysis Service, Ltd.)]; and thus, yield and
enantiomer excess of the obtained optically active
2-methyl-1,1,3-trimethoxycarbonylpropane were determined. The
results are shown in Table 2.
Examples 15, 16
[0058] To 70 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane and an
enzyme shown in Table 1 weighed in the amount shown in Table 2, 4
mL of 100 mM potassium phosphate buffer (pH 7.0) was added. The
resultant solution was stirred at 25.degree. C. for 3.5 hours, and
then 5 mL of acetonitrile was added thereto and the mixture was
drawn through a membrane filter. The filtrate was analyzed in the
same method as in Examples 1 to 14, and yield and enantiomer excess
of obtained optically active
2-methyl-1,1,3-trimethoxycarbonylpropane were determined. The
results are shown in Table 2.
TABLE-US-00001 TABLE 1 Enzyme manufacturer Preparation
(commercially method of Example Name of enzyme Origin of enzyme
available enzyme) enzyme 1 Cholesterol Candida Roche Diagnostics
Esterase cylindracea 2 CHIRAZYME E-3, lyo Thermophilic Roche
Diagnostics micro-organism 3 Cholesterol esterase, lyo Candida
Roche Diagnostics cylindracea 4 PLE-A Pig liver Amano Enzyme 5
ChiroCLEC-CR Candida rugosa Altus Biologics 6 CHIRAZYME L-3 Candida
rugosa Roche Diagnostics 7 Lipase OF Candida Meito Sangyo
cylindracea 8 Lipase-MY Candida Meito Sangyo cylindracea 9 Esterase
originated from Arthrobacter Prepared Arthrobacter strain
globiformis according to the SC-6-98-28 method described in JP
3151893 10 CHIRAZYME E-4, Lyo. Thermophilic Roche Diagnostics
micro-organism 11 CHIRAZYME P-1, Bacillus Roche Diagnostics
Lyo.(Subtilisin) licheniformis 12 Purafect 4000E Bacillus subtilis
GENENCOR 13 SP-525 Candida antactica Novozymes Japan 14
.alpha.-Chymotrypsin Bovine pancreas SIGMA 15 Esterase
Chromobacterium Prepared 160S189F363term chocolatum according to
the originated from method described Chromobacterium strain in JP
3486942 SC-YM-1 16 Esterase Chromobacterium Prepared
160A189Y363term chocolatum according to the originated from method
described Chromobacterium strain in JP 3486942 SC-YM-1
TABLE-US-00002 TABLE 2 Amount of Enantiomer Excess enzyme Yield
excess optical Example (mg) (%) (% ee) isomer 1 2.2 9.6 100
(R)-isomer 2 2.1 9.6 100 (R)-isomer 3 2.1 5.6 100 (R)-isomer 4 2.1
2.8 100 (R)-isomer 5 2.0 24.4 51.5 (R)-isomer 6 2.0 77.0 7.3
(R)-isomer 7 2.2 61.3 18.7 (R)-isomer 8 2.4 77.5 8.2 (R)-isomer 9
74.8 14.4 100 (S)-isomer 10 2.0 46.9 46.5 (S)-isomer 11 2.1 53.0
36.3 (S)-isomer 12 10.2 62.6 31.3 (S)-isomer 13 1.4 53.8 14.0
(S)-isomer 14 2.1 71.2 6.6 (S)-isomer 15 7.0 37.5 95.0 (R)-isomer
16 7.0 36.5 96.5 (R)-isomer
Examples 17, 18
[0059] To 70 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane and 7.0
mg of esterase derived from Chromobacterium strain SC-YM-1
(160S189F363term), 4 mL of 100 mM potassium phosphate buffer either
at pH 5 or pH 9 was added. The resultant solution was stirred at
25.degree. C. for 3.5 hours, and then 5 mL of acetonitrile was
added thereto and the mixture was drawn through a membrane filter.
The filtrate was analyzed in the same method as in Examples 1 to
14, and yield and enantiomer excess of the obtained optically
active 2-methyl-1,1,3-trimethoxycarbonylpropane were determined.
The results compared with Example 15 (pH7) are shown in Table
3.
TABLE-US-00003 TABLE 3 Enantiomer excess Excess optical Example pH
Yield (%) (% ee) isomer 17 9 36.2 100.0 (R)-isomer 15 7 37.5 95.0
(R)-isomer 18 5 78.0 21.0 (R)-isomer
Examples 19, 20
[0060] To 70 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane and 7.0
mg of esterase derived from Chromobacterium strain SC-YM-1
(160S189F363term), 4 mL of 100 mM potassium phosphate buffer
(pH7.0) was added. The resultant solution was stirred at 10.degree.
C. or 0.degree. C. for a time described in Table 4, and then 5 mL
of acetonitrile was added thereto and the mixture was drawn through
a membrane filter. The filtrate was analyzed in the same method as
in Examples 1 to 14, and yield and enantiomer excess of the
obtained optically active 2-methyl-1,1,3-trimethoxycarbonylpropane
were determined. The results compared with Example 15 (25.degree.
C.) are shown in Table 4.
TABLE-US-00004 TABLE 4 Excess Temperature Time Enantiomer optical
Example (.degree. C.) (hr) Yield (%) excess (% ee) isomer 15 25 3.5
37.5 95.2 (R)-isomer 19 10 8.0 47.3 91.8 (R)-isomer 20 0 21.0 42.3
100.0 (R)-isomer
Examples 21-29
[0061] E. coli strain JM105 was transformed with a plasmid
including each esterase gene shown in Table 5. The obtained
transformant was inoculated onto sterile LB (1% Bacto-Triptone,
0.5% Bacto-Yeast extract, 1% sodium chloride) culture medium (100
ml) containing 0.1 mM IPTG and 50 .mu.g/ml of ampicillin, and
cultured under shaking (37.degree. C., 24 hours). The obtained
culture liquid was centrifuged, to obtain about 0.6 g of wet
bacterial cells. About 0.6 g of wet bacterial cells were suspended
in 5 mL of 0.1 M potassium phosphate buffer (pH 7.0), 5 g of glass
beads of 0.1 mm in diameter was added, and then disrupted by a
Multi-beads shocker (produced by Yasui Kikai Corporation, 2500 rpm,
20 minutes). The obtained disrupted liquid was centrifuged (10000
rpm, 4.degree. C., 10 minutes), and the supernatant was provided as
a crude enzyme liquid.
[0062] To 70 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane, the
crude enzyme liquid was added in the enzyme amount shown in Table
6, and further 4 mL of 170 mM potassium phosphate buffer (pH 7.0)
was added. The solution was stirred at 25.degree. C. for 5 hours, 2
mL of acetonitrile containing biphenyl (internal standard
substance) was added thereto, and the mixture was drawn through a
membrane filter. The filtrate was analyzed in the same method as in
Examples 1 to 14, and yield and enantiomer excess of the obtained
optically active 2-methyl-1,1,3-trimethoxycarbonylpropane were
determined. The results are shown in Table 6.
TABLE-US-00005 TABLE 5 Example Name of enzyme Origin of enzyme
Plasmid 21 Esterase N43SA363term Chromobacterium pCCN43SA363term
originated from chocolatum (see JP2000-78988A) Chromobacterium
strain SC-YM-1 22 Esterase Chromobacterium pCC160S189Y363term
160S189Y363term chocolatum (see JP3486942) originated from
Chromobacterium strain SC-YM-1 23 Esterase Chromobacterium
pCC160A189H363term 160A189H363term chocolatum (see JP3486942)
originated from Chromobacterium strain SC-YM-1 24 Esterase
Chromobacterium pCC160S189H363term 160S189H363term chocolatum (see
JP3486942) originated from Chromobacterium strain SC-YM-1 25
Esterase Chromobacterium pCC160A189F363term 160A189F363term
chocolatum (see JP3486942) originated from Chromobacterium strain
SC-YM-1 26 Esterase V325I originated Chromobacterium pCCV325I from
Chromobacterium chocolatum (see JP2000-78988A) strain SC-YM-1 27
Esterase originated from Chromobacterium pCC363term Chromobacterium
strain chocolatum (see JP3486942) SC-YM-1 28 Esterase
Chromobacterium pCC160A189Y363term 160A189Y363term chocolatum (see
JP3486942) originated from Chromobacterium strain SC-YM-1 29
Esterase T240AV288A Chromobacterium pCCT240AV288A originated from
chocolatum (see JP2000-78988A) Chromobacterium strain SC-YM-1
TABLE-US-00006 TABLE 6 Amount of Enantiomer Excess enzyme Yield
excess optical Example (mg) (%) (% ee) isomer 21 34.6 40.9 100
(R)-isomer 22 34.9 18.1 100 (R)-isomer 23 35.3 23.8 100 (R)-isomer
24 35.3 10.9 100 (R)-isomer 25 35.2 6.2 100 (R)-isomer 26 34.6 9.5
100 (R)-isomer 27 34.7 20.9 100 (R)-isomer 28 34.9 12.2 100
(R)-isomer 29 35.1 6.3 100 (R)-isomer
Example 30-1
Enzymatic Hydrolysis
[0063] To 11.37 g of 2-methyl-1,1,3-trimethoxycarbonylpropane and
68.4 g of 100 mM potassium phosphate buffer (pH 7.0), 1.0 g of a
crude enzyme liquid prepared in the same method as in Examples 21
to 29 from a transformant of E. coli strain JM105 containing
plasmid (pCCN43SA363term) was added. The resultant solution was
stirred at 0.degree. C. for 23 hours. During stirring, pH of the
solution was kept at 7 by dropping 10 wt % of aqueous sodium
hydroxide solution. After stirring the solution, 20 g of tert-butyl
methyl ether was added, and the obtained mixture was drawn through
a glass filter. The filtrate was separated into an organic phase
and an aqueous phase. To the aqueous phase, 20 g of tert-butyl
methyl ether was added and a phase separation gave 81.8 g of an
aqueous phase. The obtained organic phases were combined and washed
with 5.1 g of 5 wt % of aqueous sodium bicarbonate solution. The
washed organic phase was concentrated under reduced pressure, to
obtain 4.49 g of (R)-2-methyl-1,1,3-trimethoxycarbonylpropane as a
yellow oily substance. Yield: 47.7%, Enantiomer excess: 100%
ee.
Example 30-2
Collection of Hydrolysate
[0064] To 79.8 g of the aqueous phase obtained in Example 30-1,
3.12 g of 35 wt % of hydrochloric acid was added to adjust pH at
2.0. After adding 20.0 g of ethyl acetate and 25.0 g of sodium
chloride and stirring the same, the obtained mixture was subjected
to liquid separation. 25.0 g of ethyl acetate was added to the
obtained aqueous phase, and extracted. The obtained organic phases
were combined and washed with 25.0 g of 25 wt % of aqueous sodium
chloride solution. The washed organic phase was concentrated under
reduced pressure to obtain 5.1 g of a yellow-brown oily
substance.
Example 30-3
Esterification
[0065] 1.0 g of the yellow-brown oily substance obtained in Example
30-2 was dissolved in 20.0 g of methanol. The obtained solution was
cooled to 0.degree. C., and 1.0 g of trimethylsilyl chloride was
added dropwise over about 10 minutes. After dropping, temperature
of the obtained reaction solution was raised to 20 to 25.degree.
C., and kept at this temperature for 45.5 hours. The obtained
reaction solution was diluted with 32 g of tert-butyl methyl ether.
After adding 40 g of 5 wt % of aqueous sodium bicarbonate solution
and stirring the same, liquid separation was conducted to obtain
36.2 g of an organic phase and 57.8 g of an aqueous phase. For each
of the obtained organic phase and aqueous phase, the chemical
purity was analyzed by high performance liquid chromatography, and
for the organic phase, the optical purity was analyzed by high
performance liquid chromatography, and the yield and enantiomer
excess of the obtained optically active
2-methyl1,1,3-trimethoxycarbonylpropane were determined. The
chemical purity and the optical purity were analyzed in the same
method as in Examples 1 to 14. Yield of
2-methyl-1,1,3-trimethoxycarbonylpropane contained in the organic
phase and aqueous phase was 46.8%, and enantiomer excess of
2-methyl-1,1,3-trimethoxycarbonylpropane contained in the organic
phase was 86.8% ee (S isomer).
Example 31-1
Enzymatic Hydrolysis
[0066] To 150 g of 2-methyl-1,1,3-trimethoxycarbonyl propane and
1035 g of 100 mM potassium phosphate buffer (pH 7.0), 17.0 g of a
crude enzyme liquid prepared in the same method as in Examples 21
to 29 from a transformant of E. coli strain JM105 containing
plasmid (pCC160S189F363term) was added. The resultant solution was
stirred at 0.degree. C. for 42 hours. During stirring, the pH of
the solution was kept at 7 by dropping 10 wt % of aqueous sodium
hydroxide solution. The solution after end of the stirring was
added with 613 g of ethyl acetate, and the obtained mixture was
drawn through a glass filter. The filtrate was separated into an
organic phase and an aqueous phase, and the aqueous phase was
separated by adding 750 g of ethyl acetate. The obtained organic
phases were combined, to obtain 1500 g of an organic phase and 1321
g of an aqueous phase. The organic phase was further washed with
150 g of 5 wt % of aqueous sodium bicarbonate solution. The washed
organic phase was concentrated under reduced pressure, to obtain
67.6 g of
[0067] (R)-2-methyl-1,1,3-trimethoxycarbonylpropane as a yellow
oily substance. Yield: 39.1%, Enantiomer excess: 100% ee.
Example 31-2
Collection of Hydrolysate
[0068] To 1315 g of the aqueous phase obtained in Example 31-1, 61
g of 35 wt % hydrochloric acid was added to adjust pH at 2.0. After
adding 300 g of ethyl acetate and 415 g of sodium chloride and
stirring the same, the obtained mixture was subjected to liquid
separation. To the obtained aqueous phase, 375 g of ethyl acetate
was added and extracted. The obtained organic phases were combined
and washed with 375 g of 25 wt % of aqueous sodium chloride
solution. The washed organic phase was concentrated under reduced
pressure to obtain 105 g of a yellow-brown oily substance.
Example 31-3
Esterification
[0069] 1.0 g of the yellow-brown oily substance obtained in Example
31-2 was dissolved in 20.1 g of methanol. The obtained solution was
cooled to 0.degree. C., and 1.0 g of trimethylsilyl chloride was
added dropwise over about 10 minutes. After dropping, temperature
of the obtained reaction solution was raised to 20 to 25.degree.
C., and kept at this temperature for 45 hours. The obtained
reaction solution was diluted with 32 g of tert-butyl methyl ether.
After adding 40 g of 5 wt % of aqueous sodium bicarbonate solution
and stirring the same, liquid separation was conducted to obtain
37.0 g of an organic phase and 57.3 g of an aqueous phase. When
analyzing in the same method as in Example 30-3, yield of
2-methyl-1,1,3-trimethoxycarbonylpropane contained in the organic
phase and aqueous phase was 51.1%, and enantiomer excess of
2-methyl-1,1,3-trimethoxycarbonylpropane contained in the organic
phase was 70.6% ee (S isomer).
INDUSTRIAL APPLICABILITY
[0070] According to the method of the present invention, it is
possible to produce an optically active
2-alkyl-1,1,3-trialkoxycarbonylpropane with high optical purity
without using a low temperature condition.
Sequence CWU 1
1
41370PRTChromobacterium chocolatum 1Met Thr Leu Phe Asp Gly Ile Thr
Ser Arg Ile Val Asp Thr Asp Arg1 5 10 15 Leu Thr Val Asn Ile Leu
Glu Arg Ala Ala Asp Asp Pro Gln Thr Pro 20 25 30 Pro Asp Arg Thr
Val Val Phe Val His Gly Asn Val Ser Ser Ala Leu 35 40 45 Phe Trp
Gln Glu Ile Met Gln Asp Leu Pro Ser Asp Leu Arg Ala Ile 50 55 60
Ala Val Asp Leu Arg Gly Phe Gly Gly Ser Glu His Ala Pro Val Asp65
70 75 80Ala Thr Arg Gly Val Arg Asp Phe Ser Asp Asp Leu His Ala Thr
Leu 85 90 95 Glu Ala Leu Asp Ile Pro Val Ala His Leu Val Gly Trp
Ser Met Gly 100 105 110 Gly Gly Val Val Met Gln Tyr Ala Leu Asp His
Pro Val Leu Ser Leu 115 120 125 Thr Leu Gln Ser Pro Val Ser Pro Tyr
Gly Phe Gly Gly Thr Arg Arg 130 135 140 Asp Gly Ser Arg Leu Thr Asp
Asp Asp Ala Gly Cys Gly Gly Gly Gly145 150 155 160Ala Asn Pro Asp
Phe Ile Gln Arg Leu Ile Asp His Asp Thr Ser Asp 165 170 175 Asp Ala
Gln Thr Ser Pro Arg Ser Val Phe Arg Ala Gly Tyr Val Ala 180 185 190
Ser Asp Tyr Thr Thr Asp His Glu Asp Val Trp Val Glu Ser Met Leu 195
200 205 Thr Thr Ser Thr Ala Asp Gly Asn Tyr Pro Gly Asp Ala Val Pro
Ser 210 215 220 Asp Asn Trp Pro Gly Phe Ala Ala Gly Arg His Gly Val
Leu Asn Thr225 230 235 240Met Ala Pro Gln Tyr Phe Asp Val Ser Gly
Ile Val Asp Leu Ala Glu 245 250 255 Lys Pro Pro Ile Leu Trp Ile His
Gly Thr Ala Asp Ala Ile Val Ser 260 265 270 Asp Ala Ser Phe Tyr Asp
Leu Asn Tyr Leu Gly Gln Leu Gly Ile Val 275 280 285 Pro Gly Trp Pro
Gly Glu Asp Val Ala Pro Ala Gln Glu Met Val Ser 290 295 300 Gln Thr
Arg Asp Val Leu Gly Arg Tyr Ala Ala Gly Gly Gly Thr Val305 310 315
320Thr Glu Val Ala Val Glu Gly Ala Gly His Ser Ala His Leu Glu Arg
325 330 335 Pro Ala Val Phe Arg His Ala Leu Leu Glu Ile Ile Gly Tyr
Val Gly 340 345 350 Ala Ala Ala Asp Pro Ala Pro Pro Thr Glu Ala Ile
Ile Ile Arg Ser 355 360 365 Ala Asp 37021110DNAChromobacterium
chocolatumCDS(1)..(1110) 2atg acc ctg ttc gac ggc atc acg tct cgc
atc gtc gac acc gac cgc 48Met Thr Leu Phe Asp Gly Ile Thr Ser Arg
Ile Val Asp Thr Asp Arg 1 5 10 15 ctg acc gtg aac atc ctg gag cgc
gcg gcc gac gac ccg cag acc ccg 96Leu Thr Val Asn Ile Leu Glu Arg
Ala Ala Asp Asp Pro Gln Thr Pro 20 25 30 ccc gac cgc acg gtc gtg
ttc gtc cac ggg aat gtg tcc tcc gcg ctg 144Pro Asp Arg Thr Val Val
Phe Val His Gly Asn Val Ser Ser Ala Leu 35 40 45 ttc tgg cag gag
atc atg cag gac ctg ccg agc gac ctg cgc gcc atc 192Phe Trp Gln Glu
Ile Met Gln Asp Leu Pro Ser Asp Leu Arg Ala Ile 50 55 60 gcg gtc
gac ctg cgc ggc ttc ggc ggc tcg gag cac gcg ccg gtc gac 240Ala Val
Asp Leu Arg Gly Phe Gly Gly Ser Glu His Ala Pro Val Asp 65 70 75 80
gcc acc cgc ggc gtc cgc gac ttc agc gac gat ctg cac gcg acc ctc
288Ala Thr Arg Gly Val Arg Asp Phe Ser Asp Asp Leu His Ala Thr Leu
85 90 95 gag gcg ctc gac atc ccg gtc gcg cat ctg gtc ggc tgg tcg
atg ggc 336Glu Ala Leu Asp Ile Pro Val Ala His Leu Val Gly Trp Ser
Met Gly 100 105 110 ggc ggc gtc gtc atg cag tat gcc ctc gac cac ccg
gtg ctg agc ctg 384Gly Gly Val Val Met Gln Tyr Ala Leu Asp His Pro
Val Leu Ser Leu 115 120 125 acc ctg cag tcg ccg gtg tcg ccc tac ggc
ttc ggc ggc acc cgc cgt 432Thr Leu Gln Ser Pro Val Ser Pro Tyr Gly
Phe Gly Gly Thr Arg Arg 130 135 140 gac ggc tca cgc ctc acc gac gac
gat gcc ggc tgc ggt ggc ggc ggt 480Asp Gly Ser Arg Leu Thr Asp Asp
Asp Ala Gly Cys Gly Gly Gly Gly 145 150 155 160 gcg aac ccc gac ttc
atc cag cgc ctc atc gac cac gac acc tcc gac 528Ala Asn Pro Asp Phe
Ile Gln Arg Leu Ile Asp His Asp Thr Ser Asp 165 170 175 gat gcg cag
acc tcg ccc cgg agc gtc ttc cgc gcc ggc tac gtc gcc 576Asp Ala Gln
Thr Ser Pro Arg Ser Val Phe Arg Ala Gly Tyr Val Ala 180 185 190 tcg
gac tac acc acc gac cac gag gac gtg tgg gtc gaa tcg atg ctc 624Ser
Asp Tyr Thr Thr Asp His Glu Asp Val Trp Val Glu Ser Met Leu 195 200
205 acc acg tcc acc gcc gac gga aac tac ccc ggc gat gcg gtg ccg agc
672Thr Thr Ser Thr Ala Asp Gly Asn Tyr Pro Gly Asp Ala Val Pro Ser
210 215 220 gac aac tgg ccg ggc ttc gcc gcc ggc cgc cac ggc gtg ctg
aac acc 720Asp Asn Trp Pro Gly Phe Ala Ala Gly Arg His Gly Val Leu
Asn Thr 225 230 235 240 atg gcc ccg cag tac ttc gat gtg tcg ggg att
gtc gac ctg gcc gag 768Met Ala Pro Gln Tyr Phe Asp Val Ser Gly Ile
Val Asp Leu Ala Glu 245 250 255 aag cct ccg atc ctg tgg atc cac ggc
acc gcg gac gcg atc gtc tcc 816Lys Pro Pro Ile Leu Trp Ile His Gly
Thr Ala Asp Ala Ile Val Ser 260 265 270 gac gcg tcg ttc tac gac ctc
aac tac ctc ggc cag ctg ggc atc gtc 864Asp Ala Ser Phe Tyr Asp Leu
Asn Tyr Leu Gly Gln Leu Gly Ile Val 275 280 285 ccc ggc tgg ccc ggc
gaa gac gtc gcg ccc gcg cag gag atg gtg tcg 912Pro Gly Trp Pro Gly
Glu Asp Val Ala Pro Ala Gln Glu Met Val Ser 290 295 300 cag acc cgc
gat gtc ctc ggc cgc tac gct gcg ggc ggc gga acg gtc 960Gln Thr Arg
Asp Val Leu Gly Arg Tyr Ala Ala Gly Gly Gly Thr Val 305 310 315 320
acc gag gtc gcc gtc gag ggc gcg ggc cac tcc gcg cac ctg gag cgt
1008Thr Glu Val Ala Val Glu Gly Ala Gly His Ser Ala His Leu Glu Arg
325 330 335 ccc gcg gtg ttc cgc cac gcg ctg ctc gag atc atc ggc tac
gtc ggc 1056Pro Ala Val Phe Arg His Ala Leu Leu Glu Ile Ile Gly Tyr
Val Gly 340 345 350 gcg gcg gcc gac ccc gcc ccg ccg acc gag gcg atc
atc atc cgc tcc 1104Ala Ala Ala Asp Pro Ala Pro Pro Thr Glu Ala Ile
Ile Ile Arg Ser 355 360 365 gcc gac 1110Ala Asp 370
3375PRTArthrobacter globiformis 3Met Asp Ala Gln Thr Ile Ala Pro
Gly Phe Glu Ser Val Ala Glu Leu1 5 10 15 Phe Gly Arg Phe Leu Ser
Glu Asp Arg Glu Tyr Ser Ala Gln Leu Ala 20 25 30 Ala Tyr His Arg
Gly Val Lys Val Leu Asp Ile Ser Gly Gly Pro His 35 40 45 Arg Arg
Pro Asp Ser Val Thr Gly Val Phe Ser Cys Ser Lys Gly Val 50 55 60
Ser Gly Leu Val Ile Ala Leu Leu Val Gln Asp Gly Phe Leu Asp Leu65
70 75 80Asp Ala Glu Val Val Lys Tyr Trp Pro Glu Phe Gly Ala Glu Gly
Lys 85 90 95 Ala Thr Ile Thr Val Ala Gln Leu Leu Ser His Gln Ala
Gly Leu Leu 100 105 110 Gly Val Glu Gly Gly Leu Thr Leu Ala Glu Tyr
Asn Asn Ser Glu Leu 115 120 125 Ala Ala Ala Lys Leu Ala Gln Met Arg
Pro Leu Trp Lys Pro Gly Thr 130 135 140 Ala Phe Gly Tyr His Ala Leu
Thr Ile Gly Val Phe Met Glu Glu Leu145 150 155 160Cys Arg Arg Ile
Thr Gly Ser Thr Leu Gln Glu Ile Tyr Glu Gln Arg 165 170 175 Ile Arg
Ser Val Thr Gly Ala His Phe Phe Leu Gly Leu Pro Glu Ser 180 185 190
Glu Glu Pro Arg Tyr Ala Thr Leu Arg Trp Ala Ala Asp Pro Ser Gln 195
200 205 Pro Trp Ile Asp Pro Ala Ser His Phe Gly Leu Ser Ala Asn Ser
Ala 210 215 220 Val Gly Asp Ile Leu Asp Leu Pro Asn Leu Arg Glu Val
Arg Ala Ala225 230 235 240Gly Leu Ser Ser Ala Ala Gly Val Ala Ser
Ala Glu Gly Met Ala Arg 245 250 255 Val Tyr Ala Ala Ala Leu Thr Gly
Leu Ala Ala Asn Gly Asp Arg Ala 260 265 270 Ala Val Ala Pro Leu Leu
Ser Glu Glu Thr Ile Gln Thr Val Thr Ala 275 280 285 Glu Gln Val Phe
Gly Ile Asp Arg Val Phe Gly Glu Thr Ser Cys Phe 290 295 300 Gly Thr
Val Phe Met Lys Ser His Ala Arg Ser Pro Tyr Gly Ser Tyr305 310 315
320Arg Ala Phe Gly His Asp Gly Ala Ser Ala Ser Leu Gly Phe Ala Asp
325 330 335 Pro Val Tyr Glu Leu Ala Phe Gly Tyr Val Pro Gln Gln Ala
Glu Pro 340 345 350 Gly Gly Ala Gly Cys Arg Asn Leu Glu Leu Ser Ala
Ala Val Arg Lys 355 360 365 Ala Val Thr Glu Leu Ala Gln 370
37541125DNAArthrobacter globiformisCDS(1)..(1125) 4gtg gat gca cag
acg att gcc cct gga ttc gaa tca gtc gcc gaa ctc 48Val Asp Ala Gln
Thr Ile Ala Pro Gly Phe Glu Ser Val Ala Glu Leu 1 5 10 15 ttt ggc
cgt ttc ctg agc gaa gac cgg gaa tat tca gcc cag ctc gcg 96Phe Gly
Arg Phe Leu Ser Glu Asp Arg Glu Tyr Ser Ala Gln Leu Ala 20 25 30
gcc tac cac cgc gga gtc aag gta ttg gac atc agc ggt ggg ccg cac
144Ala Tyr His Arg Gly Val Lys Val Leu Asp Ile Ser Gly Gly Pro His
35 40 45 cgc cgc ccg gat tcc gtg acc ggt gtt ttc tcc tgc tcc aag
gga gta 192Arg Arg Pro Asp Ser Val Thr Gly Val Phe Ser Cys Ser Lys
Gly Val 50 55 60 tcc ggg ctg gtc atc gca ctt ttg gtc cag gac ggc
ttc ctc gac ctc 240Ser Gly Leu Val Ile Ala Leu Leu Val Gln Asp Gly
Phe Leu Asp Leu 65 70 75 80 gac gcc gaa gtg gtc aag tac tgg ccg gaa
ttc ggc gcc gaa gga aag 288Asp Ala Glu Val Val Lys Tyr Trp Pro Glu
Phe Gly Ala Glu Gly Lys 85 90 95 gcc acg att acc gtg gcc cag ctg
ctc tcc cac cag gcc ggg ctt ctg 336Ala Thr Ile Thr Val Ala Gln Leu
Leu Ser His Gln Ala Gly Leu Leu 100 105 110 gga gtc gaa ggc gga ctc
acc ctc gcg gaa tac aac aac tcc gaa ctg 384Gly Val Glu Gly Gly Leu
Thr Leu Ala Glu Tyr Asn Asn Ser Glu Leu 115 120 125 gcc gcc gcc aag
ctc gcg cag atg cgg ccg ctg tgg aag ccc ggg acc 432Ala Ala Ala Lys
Leu Ala Gln Met Arg Pro Leu Trp Lys Pro Gly Thr 130 135 140 gcc ttc
ggg tac cac gcc ctg acc atc ggc gtc ttc atg gag gag ctt 480Ala Phe
Gly Tyr His Ala Leu Thr Ile Gly Val Phe Met Glu Glu Leu 145 150 155
160 tgc cgc cgg atc acc ggg tcc acg ctc cag gaa atc tac gaa cag cgg
528Cys Arg Arg Ile Thr Gly Ser Thr Leu Gln Glu Ile Tyr Glu Gln Arg
165 170 175 atc cgc tcg gtc acg ggc gcc cac ttc ttc ctg gga ctg cct
gag tcc 576Ile Arg Ser Val Thr Gly Ala His Phe Phe Leu Gly Leu Pro
Glu Ser 180 185 190 gag gaa ccc cgc tat gcc acc ctc cgt tgg gct gca
gac ccc tcc cag 624Glu Glu Pro Arg Tyr Ala Thr Leu Arg Trp Ala Ala
Asp Pro Ser Gln 195 200 205 ccg tgg att gat ccc gcc agc cat ttc ggc
ctt tcc gca aac tcg gcc 672Pro Trp Ile Asp Pro Ala Ser His Phe Gly
Leu Ser Ala Asn Ser Ala 210 215 220 gtg ggg gac atc ctt gac ctg ccc
aac ctc cgc gag gtc cgc gca gcc 720Val Gly Asp Ile Leu Asp Leu Pro
Asn Leu Arg Glu Val Arg Ala Ala 225 230 235 240 ggc ctg agt tca gcc
gcc gga gtc gcc agc gcg gaa ggc atg gcc cgc 768Gly Leu Ser Ser Ala
Ala Gly Val Ala Ser Ala Glu Gly Met Ala Arg 245 250 255 gtc tac gct
gcg gca ctc acc gga ctt gcc gcc aac ggc gac cga gcc 816Val Tyr Ala
Ala Ala Leu Thr Gly Leu Ala Ala Asn Gly Asp Arg Ala 260 265 270 gcc
gtc gcg ccc ctc ctc agc gaa gag acc atc caa acc gtc acg gcc 864Ala
Val Ala Pro Leu Leu Ser Glu Glu Thr Ile Gln Thr Val Thr Ala 275 280
285 gag cag gtc ttc ggc atc gac cgg gtg ttc ggc gag acg agc tgc ttt
912Glu Gln Val Phe Gly Ile Asp Arg Val Phe Gly Glu Thr Ser Cys Phe
290 295 300 ggg aca gtg ttc atg aaa tcg cat gca cgc tcg cct tat ggc
agc tac 960Gly Thr Val Phe Met Lys Ser His Ala Arg Ser Pro Tyr Gly
Ser Tyr 305 310 315 320 cgg gcg ttc ggg cac gac ggc gcc agc gca tct
ttg ggg ttc gct gac 1008Arg Ala Phe Gly His Asp Gly Ala Ser Ala Ser
Leu Gly Phe Ala Asp 325 330 335 cct gtg tat gaa ctc gcc ttc ggg tac
gtg ccg caa cag gcc gag ccg 1056Pro Val Tyr Glu Leu Ala Phe Gly Tyr
Val Pro Gln Gln Ala Glu Pro 340 345 350 ggc gga gcg gga tgc cgc aac
ctt gag ctg agc gcc gcc gtg cgg aag 1104Gly Gly Ala Gly Cys Arg Asn
Leu Glu Leu Ser Ala Ala Val Arg Lys 355 360 365 gca gtc acc gaa ctg
gct cag 1125Ala Val Thr Glu Leu Ala Gln 370 375
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