JP2003299495A - Method for producing optically active monoester of 3- methylglutaric acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an optically active monoester of 3-methylgulutaric acid. <P>SOLUTION: The method for producing the optically active monoester of the 3-methylgulutaric acid represented by general formula (2) [wherein R is the same or different, and a 1-4C lower alkyl group; and (*) represents an asymmetric carbon atom] comprises asymmetrically hydrolyzing a diester of the 3-methylgulutaric acid represented by general formula (1) (wherein, R is the same or different and a 1-4C lower alkyl) by using an enzyme having an activity for preferentially hydrolyzing one ester site of the diester, or a cultured product of microorganisms having productivity of the enzyme or a treated product thereof. An isomer of the optically active monoester of the 3- methylgulutaric acid can easily be produced by the method. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an optically active 3-methylglutarate monoester.

[0002]

2. Description of the Related Art Optically active 3-methylglutarate monoester is a compound useful as a raw material for introducing asymmetric carbon in the synthesis of natural products and pharmaceuticals. Conventionally, as methods for producing such optically active 3-methylglutarate monoester, chemical asymmetric synthesis, optical resolution, and enzymatic synthesis methods are known. As a chemical asymmetric synthesis method, for example, a method of adding a silyl enolate and ethylidene malonate using a bisoxazoline-based asymmetric catalyst and then obtaining the target compound through two steps (J. Am. Chem. Soc. (2000), 12
2 (38), 9134-9142.), A method of ring-opening 3-methylglutaric anhydride in the presence of alcohol using a chemically modified cinchona alkaloid (J. Am. Chem. Soc. (2000),
122 (39), 9542-9543.), A method of ring-opening 3-methylglutaric anhydride using an optically active Lewis acid (J. O.
Chem. (1998), 63 (4), 1190-1197.), a method of obtaining an objective compound through two steps after reacting optically active 1-naphthylethanol with 3-methylglutaric anhydride (J. Org. Chem. (1993), 58 (1), 142-6.), A method for obtaining a target compound through multiple steps using an asymmetric Michael addition reaction using an optically active sulfoxide (Tetrahedron (19
86), 42 (11), 2919-29.) And the like. These chemical asymmetric synthesis methods require expensive reagents that are complicated in the synthesis method, require a long number of steps, and have a low yield, and the obtained optical purity is not always sufficient.
As an optical resolution method, a method of obtaining an R-form from racemic 3-methylglutarate monoester using cinchonine and an S-form using keinine (Tetrahedron (1985),
41 (3), 627-33., J. Chem. Soc., (1950), 3333-
5.) is known. These optical resolution methods have a problem that they require recrystallization a plurality of times in order to obtain sufficient optical purity. As a method using an enzyme, a 3-methylglutaric anhydride is used as a raw material, and lipolysis is performed with lipase P (manufactured by Amano enzyme) or lipase PS (manufactured by Amano enzyme). Method of carrying out reaction to obtain target compound (JP 01091789 A
2, Agric. Biol. Chem. (1988), 52 (12), 3087-9
2., Liebigs Ann. (1996), (9), 1443-1448.) Or pig liver lipase or lipase P (Amano Enzyme) using 3-methylglutarate diester as a raw material.
(Manufactured by Amano enzyme) to obtain the target compound by hydrolysis (US 5262313 A, Tetrahedron (1988), 44.
(5), 1477-87., J. Am. Chem. Soc. (1982), 104 (2
5), 7294-9., Agric. Biol. Chem. (1988), 52 (12),
3087-92.) Is known. In any of these reactions by the enzymatic method, the optical purity of the target compound was not always sufficient under ordinary enzymatic reaction conditions. As a special method for improving the optical purity, a method of obtaining a target compound having a high ee by causing a porcine liver lipase to act on 3-methylglutarate diester in a 20% aqueous methanol solution at a low temperature of -10 ° C (J. Org. Chem.
(1986), 51 (11), 2047-50.), Or a method for obtaining a target product with high ee using porcine liver lipase immobilized on a filter element having a fibrous layer (JP 09501565).
T2) is known. Of the above special methods, the former requires a low temperature in order to achieve the purpose, so that the activity of the enzyme is low and the reaction time is long and the amount of the enzyme is large compared with the usual reaction temperature. Further, in order to separate the target substance from the enzyme after the completion of the reaction, it is necessary to first distill off methanol and then to extract it into an organic solvent, which is more disadvantageous than the usual reaction not using methanol. The latter had a problem that it was necessary to use an enzyme immobilized by a complicated method.

[0003]

Therefore, the present inventors have found that
As a result of extensive studies on a method for producing an optically active 3-methylglutarate monoester by optically selective hydrolysis using various enzymes using a 3-methylglutarate diester as a raw material under normal enzymatic reaction conditions, It was found that both isomers of optically active 3-methylglutaric acid monoester can be easily and efficiently produced, respectively, and the present invention has been completed.

[0004]

That is, the present invention is based on the general formula (1) (In the formula, Rs are the same or different and each represents a lower alkyl group having 1 to 4 carbon atoms.) An enzyme having the ability to preferentially hydrolyze one ester site of 3-methylglutarate diester Alternatively, the general formula (2) is characterized by asymmetric hydrolysis using a culture of a microorganism capable of producing the enzyme or a treated product thereof. (In the formula, R has the same meaning as described above, and * represents an asymmetric carbon atom.) The present invention provides a method for producing an optically active 3-methylglutarate monoester.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The 3-methylglutarate diester represented by the general formula (1), which is a raw material used in the method of the present invention, is
For example, it can be obtained according to the method described in Tetrahedron (1988), 44 (1), 119-26. However, it is not limited to this method, and those obtained by other methods can also be used. be able to.

Specific examples of the lower alkyl group having 1 to 4 carbon atoms represented by R in the compound represented by the general formula of the present invention include methyl group, ethyl group, n-propyl group, isopropyl group and n-butyl group. , Isobutyl group, sec-butyl group and tert-butyl group are exemplified.

Specific examples of the 3-methylglutarate diester represented by the general formula (1) include dimethyl 3-methylglutarate, diethyl 3-methylglutarate, and 3
-Di-n-propyl methyl glutarate, diisopropyl 3-methyl glutarate, di-n-butyl 3-methyl glutarate, diisobutyl 3-methyl glutarate, di sec-butyl 3-methyl glutarate, di-t-methyl glutarate
ert-butyl, ethyl 4-methoxycarbonyl-3-methylbutanoate, n-propyl 4-methoxycarbonyl-3-methylbutanoate, 4-methoxycarbonyl-3-
Isopropyl methylbutanoate, n-butyl 4-methoxycarbonyl-3-methylbutanoate, isobutyl 4-methoxycarbonyl-3-methylbutanoate, sec-butyl 4-methoxycarbonyl-3-methylbutanoate, 4-methoxycarbonyl-3-methylbutanoic acid tert-butyl, 4-ethoxycarbonyl-3-methylbutanoic acid n-
Propyl, isopropyl 4-ethoxycarbonyl-3-methylbutanoate, n-butyl 4-ethoxycarbonyl-3-methylbutanoate, isobutyl 4-ethoxycarbonyl-3-methylbutanoate, 4-ethoxycarbonyl-3
-Sec-butyl methylbutanoate, tert-butyl 4-ethoxycarbonyl-3-methylbutanoate, 4-n-
Isopropyl propyloxycarbonyl-3-methylbutanoate, n-butyl 4-n-propyloxycarbonyl-3-methylbutanoate, isobutyl 4-n-propyloxycarbonyl-3-methylbutanoate, 4-n-propyloxycarbonyl-3- Sec-Butyl methylbutanoate, tert-butyl 4-n-propyloxycarbonyl-3-methylbutanoate, n-butyl 4-isopropyloxycarbonyl-3-methylbutanoate, isobutyl 4-isopropyloxycarbonyl-3-methylbutanoate, 4- Sec-Butyl isopropyloxycarbonyl-3-methylbutanoate, tert-butyl 4-isopropyloxycarbonyl-3-methylbutanoate, 4-n-
Isobutyl butyloxycarbonyl-3-methylbutanoate, sec-butyl 4-n-butyloxycarbonyl-3-methylbutanoate, tert-butyl 4-n-butyloxycarbonyl-3-methylbutanoate, 4-isobutyloxycarbonyl-3- Examples include sec-butyl methylbutanoate, tert-butyl 4-isobutyloxycarbonyl-3-methylbutanoate, and tert-butyl 4-sec-butyloxycarbonyl-3-methylbutanoate.

It has asymmetric hydrolytic ability to 3-methylglutaric acid diester represented by the general formula (1) which can be used in the present invention, and selectively (R) -3-methylglutaric acid monoester. The enzyme produced is Burkholderia cepacia,
Or Chromobacter
Enzymes derived from microorganisms such as ium viscosum).

More specifically, the (R) -3-methylglutarate monoester-selective enzyme is, more specifically, Burkholderia cepacia SC-20 (FERM P-1740).
6) strain, or Chromobacterium SC-YM-1 (F
ERM BP-6703) -derived enzyme, or as a commercially available enzyme, CHIRAZYME E-3 (derived from a thermophilic microorganism) (Roche Diagnostics
stics)).

The compound has an asymmetric hydrolysis ability with respect to the 3-methylglutaric acid diester represented by the general formula (1) which can be used in the present invention, and selectively selects (S) -3-methylglutaric acid monoester. The enzyme produced is Arthrobacter globiformis
biformis), Aspergillus f
lavus), Aspergillus ory
zae), Bacillus lichenif
ormis), Burkholderia c
epacia), Candida a
ntarctica), or Pseudmonas sp.
omonas sp.) microorganism-derived enzymes.

More specifically, the (S) -3-methylglutarate monoester-selective enzyme is more specifically Arthrobacter SC-6-98-28 (FERM BP-365).
8) Stock or Aspergillus flavus ATCC11
492 strain, or as a commercially available enzyme, Kirazyme L-2 (C
andida antarctica), Kirazyme L-5 (Candida a
(from ntarctica), Kirazyme L-6 (Burkholderia cepa
cia), Kirazyme P-1 (Bacillus licheniformis
Origin) (above, Roche Diagnostics (Roche
Diagnostics, Lipase PS (Burkholderia cepaci)
a)), LPL Amano 3 (Pseudomonas sp.) (above, Amano enzyme (Amano enzyme)), lipase 6
2285 (from Aspergillus oryzae) (manufactured by Fluka) can be mentioned.

These (R) -3-methylglutarate monoester selective enzymes or (S) -3-methylglutarate monoester selective enzymes (hereinafter referred to as the present enzyme)
For example, even if an enzyme derived from a mutant derived from these microorganisms by treatment with a mutagen or ultraviolet rays is used, the gene encoding the enzyme of these microorganisms is introduced and transformed. An enzyme produced by a microorganism or a mutant enzyme in which one or several specific amino acids in the amino acid sequence of the present enzyme are deleted, added or substituted by a genetic engineering method, Of course, it can be used in the production method of the present invention as long as it has an asymmetric hydrolysis ability with respect to the 3-methylglutarate diester represented by the general formula (1).

As a method for producing a transformed recombinant microorganism into which a gene encoding the present enzyme has been introduced, for example, J. Sambrook, EFFritsch, T. Maniatis; Molecular Cloning 2nd edi.
tion), Cold Spring Harbor Laboratory (Co
ld Spring Harbor Laboratory), 1989, etc. More specifically, Japanese Patent Laid-Open No. 2001-460
No. 84 (Esterase AF), No. 10-210975 (Repose A), No. 7-163364 (Esterase CC-WT (KOS)), or No. 5-56787 (Esterase AR). A method similar to the described method can be mentioned. Examples of the present enzyme produced by a recombinant microorganism that can be produced in this manner include esterases derived from Aspergillus flavus ATCC 11492 strain (JP-A 2001-46084), Burkholderia cepacia SC- 20 strains (FERM P-17
406) -derived lipase (JP-A-10-210975), Chromobacterium SC-YM-1 strain (FER)
Esterase derived from MBP-6703 (JP-A-7-
No. 163364) or Arthrobacter SC-6
Esterase derived from the -98-28 strain (FERM BP-3658) (Japanese Patent Laid-Open No. 5-56787) and the like can be mentioned.

Further, as a method for producing a mutant enzyme by a genetic engineering method, for example, Olfert Landt et al.
96 125-128 1990). More specifically, JP 2000-78988 A (Esterase CC-I, AA, S) or JP 7-213280 A (Esterase CC-SY, SF, AH, SH, AF, AY (RKOS)) An example of the mutant enzyme that can be prepared in this manner is a mutant esterase prepared from an esterase derived from Chromobacterium SC-YM-1 strain. Alternatively, lipase and the like can be mentioned.

Any of the microorganisms producing the present enzyme can be liquid-cultured by an ordinary method. As the culture medium, various culture media that are appropriately used for culturing microorganisms, including carbon sources, nitrogen sources, inorganic substances and the like can be used.
For example, as a carbon source, glucose, glycerin, organic acids, molasses, etc., as a nitrogen source, peptone, yeast extract, malt extract, soybean flour, corn steep liquor, cottonseed flour, dry yeast, casamino acid, ammonium chloride, Ammonium nitrate, ammonium sulfate, urea and the like, as inorganic substances, salts of potassium, sodium, magnesium, iron, manganese, cobalt, zinc and the like, sulfates, and phosphates, 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. Further, in order to enhance the asymmetric water-dissolving ability of the 3-methylglutarate diester represented by the general formula (1) possessed by the microorganism, triglycerides such as olive oil or tributyrin or 3-methylglutaric acid represented by the general formula (1) The diester may be appropriately added to the medium.

Culturing is usually carried out aerobically, and shaking culture or aeration-agitation culture is suitable. The culture temperature is about 20 to 40 ° C, preferably about 25 to 35 ° C, and the pH is preferably about 6 to 8. The culturing time varies depending on various conditions, but is preferably about 1 to 7 days. Further, if necessary, the solid culture method can also be appropriately adopted as long as it is a method capable of obtaining a microbial cell having an asymmetric water-dissolving ability of 3-methylglutarate diester represented by the general formula (1).

The enzyme of the present invention can be purified from the microbial culture cultivated as described above by a method generally used in the purification of enzymes. For example, first, the cells in the microbial culture are disrupted by a method such as ultrasonic treatment, dynomill treatment, or French press treatment. After removing insoluble matter from the obtained disrupted solution by centrifugation, etc., cation exchange column chromatography, anion exchange column chromatography, hydrophobic column chromatography, gel filtration column chromatography, etc., which are usually used for enzyme purification The desired enzyme can be purified by appropriately combining one or more of the above. As an example of a carrier used for these column chromatography, DEAE-Sepharose fas is used.
Examples thereof include tflow (manufactured by Amersham Pharmacia Biotech) and Butyl-Toyopearl 650S (manufactured by Toyo Soda Kogyo Co., Ltd.).

The present enzyme can be used in various forms such as purified enzyme, crude enzyme, microbial culture, bacterial cells, and processed products thereof. Here, the treated product means, for example, freeze-dried bacterial cells, acetone-dried bacterial cells, bacterial cell grinds, autolyzed bacterial cells, ultrasonically treated bacterial cells, bacterial cell extract, or alkaline treatment of bacterial cells. Refers to things etc. Furthermore, the enzyme having various purities or forms as described above is adsorbed onto, for example, an inorganic carrier such as silica gel or ceramics, cellulose, an ion exchange resin, etc., a polyacrylamide method, a sulfur-containing polysaccharide gel method (eg, carrageenan gel method). ), An alginic acid gel method, an agar gel method and the like may be used after being immobilized.

The amount of the present enzyme used is appropriately selected so that the reaction time is not delayed or the selectivity is not lowered. For example, when a purified enzyme, a crude enzyme, or a commercially available enzyme is used, the amount used is represented by the general formula ( It is usually 0.001 to 2 times by weight, preferably 0.002 to 0.5 times by weight, relative to 3-methylglutaric acid diester represented by 1), and is a microbial culture, microbial cell, or a treated product thereof. When used, the amount used is usually about 0.01 to 200 times by weight with respect to the 3-methylglutarate diester represented by the general formula (1),
It is preferably about 0.1 to 50 times by weight.

The water used in the asymmetric hydrolysis reaction may be a buffered aqueous solution. Examples of the buffer aqueous solution include a buffer aqueous solution of an inorganic acid salt such as an aqueous solution of an alkali metal salt of phosphate such as an aqueous solution of sodium phosphate and an aqueous solution of potassium phosphate, an organic acid salt such as an aqueous solution of sodium acetate and an aqueous solution of potassium acetate. Buffered aqueous solution and the like. The amount of such water used is usually 0.5 mol times or more with respect to the 3-methylglutarate diester represented by the general formula (1), and in some cases, it is used as a solvent amount, and usually 200 times by weight or less. is there.

The asymmetric hydrolysis reaction may be carried out 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, toluene, hexane, cyclohexane, heptane,
Hydrocarbons such as octane and isooctane include hydrophilic organic solvents such as tert-butanol, alcohols such as methanol, ethanol, isopropanol, isobutanol, n-butanol, ethers such as tetrahydrofuran, and dimethyl sulfoxide. And the like, ketones such as acetone, nitriles such as acetonitrile, and amides such as N, N-dimethylformamide. These hydrophobic organic solvents and hydrophilic organic solvents may be used alone or in combination of two or more, and the hydrophobic organic solvent and the hydrophilic organic solvent may be used in combination.

When such an organic solvent is used, the amount thereof used is usually not more than 200 times by weight, preferably 0.1 times the weight of the 3-methylglutaric acid diester represented by the general formula (1).
It is in the range of about 100 times by weight.

The asymmetric hydrolysis reaction is carried out, for example, by a method of mixing water, 3-methylglutarate diester represented by the general formula (1) and the present enzyme. When an organic solvent is used, the organic solvent and water are used. , 3 represented by the general formula (1)
-Methylglutarate diester and the present enzyme may be mixed.

The pH of the reaction system is appropriately selected so that the asymmetric hydrolysis by the present enzyme proceeds with good selectivity and is not particularly limited, but is usually about pH 4 to 10, preferably pH 6 to.
The range is about 8. During the reaction, the pH may be adjusted within an appropriately selected range by adding a base. Examples of the base include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal and alkaline earth metal carbonates such as sodium carbonate, potassium carbonate and calcium carbonate, sodium hydrogen carbonate and potassium hydrogen carbonate. Alkali metal bicarbonate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and other phosphates, triethylamine, organic bases such as pyridine, and ammonia are used. . You may use such a base individually or in mixture of 2 or more types. Such a base is usually used as an aqueous solution, but when an organic solvent is used for the reaction, it may be used as an organic solvent or a mixed solution of an organic solvent and water. The same organic solvent as that used in the reaction can be used. Further, the base may be used as a solid or in a state of being suspended in a solution.

If the reaction temperature is too high, the stability of the enzyme tends to decrease, and if it is too low, the reaction rate tends to decrease. Therefore, the reaction temperature is usually about 5 to 65 ° C, preferably 20 to 50 ° C. It is a range of degrees.

Thus, a solution of the optically active 3-methylglutaric acid monoester represented by the general formula (2) is obtained, which is separated from the enzyme or buffer used in the reaction or the raw material when the reaction is incomplete. Further post-treatment operations may be carried out to achieve this. Examples of such post-treatment include a method of distilling off the solvent in the reaction solution and then separating and purifying using silica gel chromatography, a method of distilling off the solvent and then separating and purifying by distillation, and a method of separating and purifying by a liquid separation operation. And so on.

When separating and purifying by a liquid separation operation, when an organic solvent which is soluble in both water and a hydrophobic organic solvent is used during the reaction, it may be removed by distillation before use. When the solution contains insoluble enzymes, immobilized carriers, etc., these may be removed by filtration.

After completion of the reaction, if the reaction is incomplete and the 3-methylglutarate diester represented by the general formula (1) as a raw material remains or a neutral impurity is produced, a hydrophobic organic solvent is used. Optical activity 3 represented by the general formula (2), which is a target substance, is obtained by extracting into an organic layer and separating from an aqueous layer.
It can be separated from the methyl glutaric acid monoester. 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, dichloromethane, dichloroethane, chloroform, chlorobenzene and orthodichlorobenzene. And halogenated hydrocarbons, esters such as ethyl acetate, methyl acetate and butyl acetate. When these hydrophobic organic solvents are used during the reaction, the liquid separation operation can be performed as it is. In addition, when a hydrophobic organic solvent is not used during the reaction, or when the amount used is too small to allow easy liquid separation, or when the amount of water used is too small to allow easy liquid separation, a hydrophobic The organic layer may be separated after adding an organic solvent or water as appropriate. The amount of the hydrophobic organic solvent used is not particularly limited, but is usually 0. 0 with respect to the 3-methylglutarate diester represented by the general formula (1).
It is in the range of 1 to 200 times by weight, preferably 0.2 to 100 times by weight. The pH during the liquid separation operation for removing such neutral components is usually in the range of about 6 to 10, preferably 7 to 9.
It is a range of degrees. Acids and bases may be used as appropriate to adjust the solution to such a pH. Examples of such an acid include inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid and phosphoric acid and salts thereof, organic acids such as acetic acid, citric acid and methanesulfonic acid and salts thereof, and the like. As the base, the same base as that used for pH adjustment during the reaction can be used. When the neutral component is insufficiently removed from the aqueous layer, the same extraction and liquid separation operations may be repeated multiple times.

In order to separate the objective optically active 3-methylglutarate monoester represented by the general formula (2) from water-soluble components such as enzymes and buffers, a hydrophobic organic solvent is generally used for the organic layer. The optically active 3-methylglutarate monoester represented by the formula (2) may be extracted and separated from the aqueous layer. As such a hydrophobic organic solvent, the same one as used in the liquid separation operation for removing the neutral component can be used. When these hydrophobic organic solvents are used during the reaction, the liquid separation operation can be performed as it is. In addition, when a hydrophobic organic solvent is not used during the reaction, or when the amount used is too small to allow easy liquid separation, or when the amount of water used is too small to allow easy liquid separation, a hydrophobic The organic layer may be separated after adding an organic solvent or water as appropriate. The amount of the hydrophobic organic solvent used is usually 0.1 to 200 times by weight, preferably 0.2 to 100 times the weight of the 3-methylglutaric acid diester represented by the general formula (1).
The range is about twice the weight. PH when extracting the target substance
Is usually in the range of about 1 to 7, preferably in the range of about 2 to 5. Acids and bases may be used as appropriate to adjust the solution to such a pH. As the acid and base, the same ones as those used in the liquid separation operation for removing the neutral component can be used. When extraction of the desired product from the aqueous layer is insufficient, the same extraction and liquid separation operations may be repeated multiple times.

Then, the organic solvent in the obtained oil layer is distilled off, whereby the optically active 3-methylglutaric acid monoester represented by the general formula (2) can be isolated. The obtained optically active 3-methylglutaric acid monoester represented by the general formula (2) may be further purified by column chromatography, distillation or the like.

The optically active 3-methylglutarate monoester represented by the general formula (2) thus obtained is specifically (R) -3-methylglutarate methyl, (R)
Ethyl-3-methyl glutarate, n-propyl (R) -3-methyl glutarate, isopropyl 3-methyl glutarate, n-butyl (R) -3-methyl glutarate,
(R) -3-Methyl glutarate isobutyl, (R) -3
-Sec-butyl methyl glutarate, tert-butyl (R) -3-methyl glutarate, methyl (S) -3-methyl glutarate, ethyl (S) -3-methyl glutarate, (S) -3-methyl N-Propyl glutarate, 3-
Isopropyl methyl glutarate, n-butyl (S) -3-methyl glutarate, isobutyl (S) -3-methyl glutarate, sec-butyl (S) -3-methyl glutarate, (S) -3-methyl glutarate Tert-Butyl acid may be mentioned.

[0032]

INDUSTRIAL APPLICABILITY According to the method of the present invention, the optical activity 3 represented by the general formula (2) can be obtained by using a specific enzyme.
-Methylglutarate monoester can be easily and efficiently produced.

[0033]

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

Examples 1-19 A solution prepared by dissolving 25 mg of dimethyl 3-methylglutarate in 5 ml of 100 mM potassium phosphate buffer (pH 7.0) is shown in Table 2 for each of the various enzymes shown in Table 1. The amount was added to the place where it was weighed out. This solution at 25 ℃
After stirring for 20 hours, 1% of 3.4% phosphoric acid aqueous solution
1, 2.1 g of sodium chloride and 10 ml of tert-butyl methyl ether were added and mixed. The oil layer was subjected to HPLC [column: CHIRALCEL OB-H, 4.6 mmφ ×
15 cm (manufactured by Daicel)], and the yield and enantiomeric excess of the obtained optically active monomethyl 3-methylglutarate were determined. The results are shown in Table 2.

[0035]

[Table 1]

[Table 2]

[Table 3]

[0036]

[Table 4]

Comparative Example 1 The yield of the optically active 3-methylglutarate monoester obtained in the same manner as in Examples 1 to 27 except that 1.0 mg of PLE-A (manufactured by Amano Enzyme) was used as the enzyme, and When the enantiomer excess was determined, the yield was 91%, and the enantiomer excess was 68% ee.

Example 20 1.16 g of dipotassium hydrogen phosphate was dissolved in 66.6 g of water, and 0.23 g of phosphoric acid was added to adjust the pH to 6.9. A culture containing 0.50 g of dimethyl 3-methylglutarate and 0.50 of the esterase 160A189Y363term derived from the strain Chromobacterium SC-YM-1 prepared according to the method described in JP-A-7-213280.
g was added and the mixture was stirred at 25 ° C. for 26 hours. Furthermore, a culture containing the same esterase 160A189Y363term from Chromobacterium SC-YM-1 as described above 9.
50 g was added, and the mixture was stirred at 25 ° C. for 13 hours. During this reaction, 38.4 g of a 10% sodium carbonate aqueous solution was continuously added dropwise so that the pH of the reaction solution would be 7.0. Sodium chloride (370.7 g) and ethyl acetate (500.0 ml) were added to the obtained reaction mixture, and 35% hydrochloric acid (49.9 g) was added.
Was added to bring the pH to 4.0. Furthermore, Celite was added to this solution, and the mixture was stirred for 10 minutes, the solid matter was removed by filtration, and then an oil layer and an aqueous layer were separated by liquid separation. Ethyl acetate (500.0 ml) was added to the aqueous layer, extraction and liquid separation operations were performed again to remove the aqueous layer, and then the oil layer obtained previously was combined. The combined oil layer was washed with 200 g of saturated saline, and then the organic solvent of the oil layer was distilled off under reduced pressure to obtain 54.6 g of monomethyl (R) -3-methylglutarate as an oily substance. The obtained oily substance was subjected to gas chromatography and HPLC [column: CHIRALCEL OB-H, 4.6 mmφ ×
15 cm (manufactured by Daicel)] and the yield and enantiomeric excess of the obtained optically active 3-methylglutaric acid monoester were determined. The yield was 91.0%, the enantiomeric excess Was 100% ee.

Comparative Example 2 6.86 g of dipotassium hydrogen phosphate was dissolved in 393.6 g of water, and 1.15 g of phosphoric acid was added to adjust the pH to 7.0. 59.1 g of dimethyl 3-methylglutarate and PL were added to this solution.
Add 1.00 g of EA (manufactured by Amano Enzyme) to add 25
The mixture was stirred at ° C for 22 hours. During this reaction, 632.6 g of a 7% aqueous sodium hydrogen carbonate solution was continuously added dropwise so that the pH of the reaction solution would be 7.0. After adding 30.0 g of sodium chloride and 50.0 g of ethyl acetate to the obtained reaction mixture, 6.53 g of 35% hydrochloric acid was added to adjust the pH to 4.0. Furthermore, 5.0 g of Celite was added to this solution, and the mixture was stirred for 10 minutes. After removing the solid matter by filtration, an oil layer and an aqueous layer were separated by liquid separation. Ethyl acetate (50.0 g) was added to the aqueous layer, extraction and liquid separation operations were performed again to remove the aqueous layer, and then the oil layer obtained above was combined. After washing the combined oil layer with 50 g of saturated saline, the organic solvent of the oil layer was distilled off under reduced pressure,
9.2 g of monomethyl (R) -3-methylglutarate was obtained as an oily substance. The oily substance obtained was subjected to gas chromatography and HPLC [column: CHIRALCEL
OB-H, 4.6 mmφ × 15 cm (manufactured by Daicel)], and the yield and enantiomeric excess of the obtained optically active 3-methylglutarate monoester were determined. The yield was 98. 7%, enantiomeric excess 81.1
% Ee.

Claims (7)

[Claims] 1. A general formula (1) (In the formula, Rs are the same or different and each represents a lower alkyl group having 1 to 4 carbon atoms.) An enzyme having the ability to preferentially hydrolyze one ester site of 3-methylglutarate diester Alternatively, the general formula (2) is characterized by asymmetric hydrolysis using a culture of a microorganism capable of producing the enzyme or a treated product thereof. (In the formula, R represents the same meaning as described above, and * represents an asymmetric carbon atom.) A method for producing an optically active 3-methylglutaric acid monoester. 2. The production method according to claim 1, wherein in the 3-methylglutarate diester represented by the general formula (1), R is methyl. 3. The enzyme is Arthrobacter.
Genus, Aspergillus, Bacillus
us), Burkholderia, Candida, Chromobacteriu
The method according to claim 1 or 2, wherein the hydrolase is derived from a microorganism of the genus m) or the genus Pseudomonas. 4. The enzyme is Arthrobacter globiformis, Aspergillus flavus, Aspergillus oryzae, Bacillus licheniformis, and Burkholderia cepacia. , Or Candida
The method according to claim 1, 2 or 3, wherein the hydrolase is derived from a microorganism of Candida antarctica. 5. The enzyme is Arthrobacter SC-6-98.
-28 strain (FERM BP-3658) (esterage AR),
Aspergillus flavus ATCC 11492 strain (Esterer AF), Burkholderia cepacia SC-20 strain (F
ERM P-17406) (repose A, esterase S), or chromobacterium SC-YM-1 strain (FERM BP
-6703) (esterase CC) -derived esterase or lipase. 6. The enzyme is a commercially available enzyme, and the kirazyme (CHI
RAZYME) E-3 (derived from thermophilic microorganism), Kirazyme L-2
(From Candida antarctica), Kirazyme L-5 (Candida antarctica)
from antarctica), Kirazyme L-6 (Pseudomonas sp.
Origin), Kirazyme P-1 (derived from Bacillus licheniformis) (above, Roche Diagnostics
agnostics), Lipase PS (Burkholderia cepaci
a), LPL Amano 3 (derived from Pseudomonas sp.), (above, manufactured by Amano enzyme), lipase 6
2285 (from Aspergillus oryzae) (manufactured by Fluka), The production method according to claim 1, 2, 3 or 4. 7. An enzyme, which is a mutant enzyme having an enzymatic activity in which one or more specific amino acids of the enzyme according to claim 3, 4, 5 or 6 is deleted, added or substituted. The manufacturing method according to claim 1, 2, 3, or 4.