JP2971667B2 - Process for producing optically active α- or β-hydroxycarboxylic acid esters or hydroxyacetals - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing optically active α- or β-hydroxycarboxylic acid esters or hydroxyacetals using an enzyme immobilized on a water-absorbing polymer.

[0002] Optically active α- or β-hydroxycarboxylic acid esters or hydroxyacetals are important compounds as intermediates for various organic chemical products such as medicines and agricultural chemicals, fragrances and the like, or as raw materials for production.

[0003] Examples of such raw materials and intermediates for pharmaceuticals and fragrances include salazabril (Roche, Switzerland) and enalapril (Merck, USA), which are antihypertensive agents that inhibit angiotensin converting enzyme. , Benzapril (Ciba-Geigy (Switzerland)) and Delapril (Takeda Pharmaceutical Co., Ltd.)
(R) -3-hydroxybutanoic acid, which is a raw material for producing phenyl-2-hydroxybutanoic acid and its ester (JP-A-2-16987, WO890717), 4-acetoxyazetidinone which is a synthetic intermediate of penem antibiotics, Its esters (Tetrahedron Lett.)
26 , 4647 (1985)), (R) -3-chlorolactic acid and its ester, which are raw materials for the production of levocarnitine chloride, a metabolic drug (Journal of the Society of Synthetic Organic Chemistry, 45 , 331 (1987)),
Grahamimimycin A ₁ (g
(S) -1- which is a synthetic intermediate of rahamiycin A ₁ )
(1,3-dithian-2-yl) -2-hydroxypropane and (S) -3-hydroxybutyraldehyde dimethyl acetal (tetrahedron letters, 24 , 28
7 (1983)), (S)-(+)-3-hydroxy-2-methylpropanoic acid and its ester (Herpetica kimika), which are the raw materials of vitamin E and muscone as a fragrance
Acta (Helv. Chim. Acta), 60 , 925 (1977) and Journal of Organic Chemistry (J. Org. C)
hem), 41 , 3505 (1976)).

[0004]

2. Description of the Related Art Conventionally, as a method for producing optically active α- or β-hydroxycarboxylic acid esters or hydroxyacetals, there is a method using a dehydrogenase. ) Glycerol dehydrogenase derived from Geotricum candium and nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD ⁺ ) as a coenzyme (tetrahedron letters, 29 , 2453 (1988)) and 2) horse liver-derived Method using alcohol dehydrogenase and NAD ⁺ (Journal of American Chemical Society (J. Am. Chem. Soc.), 104 , 4458 (198
2)) has been reported. However, these methods are disadvantageous in that the compounds of the general formulas (I) and (II) are accompanied by hydrolysis and polymerization of the ester group because the method is carried out in an aqueous solution and the substrate is hardly dissolved. A reduction reaction of 4-chloro-3-oxobutanoate using a mixture of an organic solvent and an aqueous solution in consideration of the solubility of the substrate (Appl. Environ. Microbiol.), 56 , 2374
(1990) has been reported.

However, in this method, since the dehydrogenase is inactivated by the use of the organic solvent, the activity of the dehydrogenase in the aqueous solution is reduced after the reaction, and it is difficult to reuse the dehydrogenase industrially. Dehydrogenases and coenzymes are expensive and are desired to be reused by treatment such as immobilization. The general method of immobilization is as follows: 4) Horse liver by polymerization of acrylamide and N-acryloxysuccinimide Immobilization method of alcohol dehydrogenase of origin (Journal of American Chemical Society, 102 , 6234 (1980)), 5) introducing an aminoethyl group into nitrogen at position 6 of the adenine ring of NAD ⁺ , and using this as a spacer, Method of bonding to polyethylene glycol having a carboxyl group by dehydration amidation (Journal of Applied Biochemistry (J. Appl. Biochem.), 3 , 301 (1981))
And 6) a method in which a monomer side chain is introduced into the nitrogen at the 6-position of the adenine ring of NAD ⁺ and copolymerized with acrylamides (Biochem., 62 , 629 (1980)). The immobilized enzyme obtained by the method of 4) is used for asymmetric reduction of pyruvaldehyde dimethyl acetal which is an α-ketoacetal represented by the general formula (III) (Journal of American Chemical Society, 107 , 4028 (1985)).

However, the methods 4) to 6) involve inactivation of enzymes and coenzymes and complicated operations.
A simple method for immobilizing dehydrogenase and coenzyme has been desired.

In view of the above circumstances, the present invention provides an optically active α, which is an important compound as an intermediate or a raw material for producing various organic chemical products such as medical and agricultural chemicals and fragrances.
An object of the present invention is to provide a simple and economical process for producing-or β-hydroxycarboxylic acid esters and acetals.

The inventors of the present invention have conducted intensive studies to achieve the above object, and as a result, using α- or β-ketoesters or ketoacetals as raw materials, dissolve dehydrogenase and coenzyme in water or a buffer solution. Contact with an immobilized catalyst immobilized on a water-absorbing polymer and stereoselectively reducing the carbonyl group at the α or β position in an organic solvent to obtain optically active α- or β-hydroxycarboxylic acid esters. Alternatively, the present inventors have found that hydroxyacetals can be obtained, and that the immobilized enzyme and coenzyme can be reused, and that the continuous reaction in a column system using the immobilized enzyme is also possible, thereby completing the present invention.

[0009]

The present invention provides a compound of the general formula (I):

[0010]

Embedded image

(Wherein R, R 'and R "are the same or different and each is a linear or branched alkyl group having 1 to 18 carbon atoms, a linear or branched alkenyl group having 1 to 18 carbon atoms) Group, aromatic group, halogen atom or hydrogen atom
However, R ″ is not a halogen atom, a represents 0, 1 or 2, b represents 0 or 1, and the general formula (II):

[0012]

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(Wherein R is the same as above ,
Not n, but n represents 3 or 4) or the general formula (III):

[0014]

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(Wherein R, R ', a and b are the same as above, R ¹ and R ² are the same or different, and are a linear or branched C1-C18 alkyl group, or turned by _{- (CH 2) k - (} k in the formula 2 or 3) to form a ring represented by, X is α- or β- ketoesters or keto acetals represented by an oxygen atom or a sulfur atom) and the primary or secondary alcohol in an organic solvent and a reducing agent, dissolved in water or a buffer solution de
By contacting the hydrogenase and the coenzyme with an immobilized enzyme obtained by immobilizing the coenzyme on a water-absorbing polymer, stereoselectively reducing the carbonyl group at the α- or β-position; The present invention relates to a method for producing a hydroxycarboxylic acid ester or a hydroxyacetal.

[0016]

In the present invention, general formula (I):

[0017]

Embedded image

(Wherein R, R ′ and R ″ are the same or different and are linear or branched alkyl groups having 1 to 18 carbon atoms, linear or branched alkenyl groups having 1 to 18 carbon atoms) Group, aromatic group, halogen atom or hydrogen atom
However, R ″ is not a halogen atom, a represents 0, 1 or 2, b represents 0 or 1, and the general formula (II):

[0019]

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(Wherein R is the same as above ,
Not n, but n represents 3 or 4) or the general formula (III):

[0021]

Embedded image

(Wherein, R, R ', a and b are the same as above, R ¹ and R ² are the same or different, and are a linear or branched C1-C18 alkyl group, or turned by _{- (CH 2) k - (} k in the formula 2 or 3) to form a ring represented by, X is α- or β- ketoesters or keto acetals represented by an oxygen atom or a sulfur atom) Is reacted with an enzyme and a coenzyme immobilized on a water-absorbing polymer to convert the carbonyl group at the α- or β-position to a hydroxyl group in a stereoselective manner, thereby obtaining an optically active α- or β-hydroxycarboxylic acid ester or Hydroxyacetals (hereinafter also including hydroxythioacetals) are obtained.

In the present invention, examples of the substituent represented by R, R 'and R "include a hydrogen atom, a methyl group,
Ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, alkyl group such as stearyl group, vinyl group, allyl group, alkenyl group such as isopropenyl group,
Examples include aromatics such as phenyl, 4-methoxyphenyl and 4-chlorophenyl, and halogens such as chlorine and bromine. Also, R ¹ and R
Examples of the substituent represented by ² include an alkyl group such as a methyl group, an ethyl group, and a propyl group, and an ethylene group and a propylene group that form a cyclic structure.

The dehydrogenase that can be used in the present invention includes sporoboromycetes, Enterobacter, and Cryptococcus (C).
ryptococcus), Sporotrichum, Thermoanaerobium, Lactobacillus, Apiotrichu
m) genus, Candida genus, tolulopsis (Toru
lopsis), Trichosporon, Saccharomyces, Rhodosporidium (R
hodosporidium), Pichia, Klebsiella (K
lebsiella), Rhodococcus, Rhodotorula, Paracoccus,
Nocardioides, Geotricum
Genus (Geotrichum), genus Kloeckera, genus Nocardia, Staphylococcus
s), Thermus, Aerobacter
ter), Bacillus, Cellulomonas
monas), enterobacter, genus Leuconostoc, dehydrogenase produced by microorganisms belonging to the genus Proteus, pig heart, pig muscle, rabbit muscle, horse liver, bovine heart or bird Heart-derived dehydrogenase or dehydrogenase produced by baker's yeast, etc., which can stereoselectively reduce the carbonyl group at the α-position or β-position of α- or β-ketoesters or ketoacetals. Any one may be used as long as it is an excellent dehydrogenase, and preferable examples thereof are glycerol dehydrogenase derived from Geotricum candium (manufactured by Amano Pharmaceutical Co., Ltd.) and cholesterol derived from Nocardia Dehydrogenase (eg, CHD
H "Amano" manufactured by Amano Pharmaceutical Co., Ltd., lactate dehydrogenase derived from Staphylococcus sp.
DH “Amano” Amano Pharmaceutical Co., Ltd., trade name), Thermus derived malate dehydrogenase (eg, MDH “Amano” Amano Pharmaceutical Co., Ltd., trade name), Bacillus derived glucose dehydrogenase ( Examples thereof include GLUCDH "Amano" (manufactured by Amano Pharmaceutical Co., Ltd., trade name), D-butyric acid dehydrogenase derived from Lactobacillus reichmannii (manufactured by Sigma), and alcohol dehydrogenase derived from horse liver (manufactured by Sigma).

These enzymes may be used alone or, if necessary, glycerol dehydrogenase derived from Geotricum candium, cholesterol dehydrogenase derived from the genus Norcadia, lactate dehydrogenase derived from the genus Staphylococcus, or genus Thermus. An enzyme for coenzyme regeneration such as malate dehydrogenase from horse liver alcohol dehydrogenase from horse liver, L-lactate dehydrogenase from rabbit muscle,
A mixture with a substrate reducing enzyme such as D-hydrogen lactate derived from Lactobacillus can also be used. These enzymes can be obtained by culturing the microorganisms that produce them, and may be used as they are or as crude enzymes or purified enzymes when immobilized on the water-absorbing polymer.

The coenzymes that can be used in the present invention include nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and the like.

The water-absorbing polymer used for immobilization is
Any material may be used as long as it has water retention and retains the activity of enzymes and coenzymes. Examples thereof include anionic and cationic polymer water-absorbing resins. Examples of the anionic water-absorbing resin include acrylic acid polymer, methacrylic acid polymer, polysaccharide-acrylic acid or methacrylic acid graft copolymer, acrylic acid or methacrylic-
Acrylamide-sulfonated acrylamide terpolymer or an alkali metal or alkaline earth metal salt thereof, such as acrylic acid or acrylate polymer, acrylic acid or acrylate-methacrylic acid or methacrylate copolymer , Starch-acrylic acid or acrylate graft copolymer, polysaccharide-acrylic acid or methacrylic acid alkyl ester graft copolymer, saponified polysaccharide-acrylonitrile graft copolymer, polysaccharide-acrylamide copolymer Saponified polymer, alkyl acrylate or alkyl methacrylate-vinyl acetate copolymer, starch-acrylonitrile-acrylamide-2
-Saponified methylpropane sulfonic acid graft copolymer, starch-acrylonitrile-saponified vinyl sulfonic acid graft copolymer, sodium carboxymethyl cellulose and the like, acrylic acid, methacrylic acid, alkali metal salts thereof or Instead of alkaline earth metal salts or their esters, the general formula
(IV):

[0028]

Embedded image

(Wherein the substitution position of the —SO ₃ R ³ group is 2
-, 3-or 4-position, R ³ represents straight-chain or branched alkyl group having 1 to 18 carbon atoms, a hydrogen atom, or an alkali metal or alkaline earth metal, a is 0
Or 1)) or a derivative thereof. These may be used alone or in combination of two or more, and may be used as a crosslinked product.

The cationic water-absorbing resin is represented by the following general formula (V):

[0031]

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[0032] (wherein, R ⁴ is hydrogen or methyl, R ⁵
Represents a linear or branched alkyl group having 1 to 18 carbon atoms or a hydrogen atom, and j represents 1, 2, 3, 4 or 5
Wherein Y represents an oxygen atom or a nitrogen atom, and Z represents a sulfonate ion or a halogen ion), or a copolymer with N-alkyl-acrylamide or N-alkyl-methacrylamide. And so on. Commercial products include Aquaric (trade name, manufactured by Nippon Shokubai Co., Ltd.), Poise and Wonder Gel (trade name, manufactured by Kao Corporation), Aqua Keep (trade name, manufactured by Sumitomo Seika Co., Ltd.), Arasorp (Trade name, manufactured by Arakawa Chemical Co., Ltd.), Diamond Wet (Mitsubishi Yuka Corporation)
Sumikagel (Sumitomo Chemical Co., Ltd., trade name), Sunwet (Sanyo Chemical Co., Ltd., trade name), P
Q Polymer (manufactured by Osaka Organic Chemical Industry Co., Ltd., trade name) and the like are preferable. PQ polymer (grade: BL-100) (manufactured by Osaka Organic Chemical Industry Co., Ltd.) -56512), Aqua Keep and Wonder Gel.

The alcohol as the reducing agent may be any alcohol as long as it is oxidized by the dehydrogenase and does not inhibit the reduction of the carbonyl group of the substrate by the coenzyme, for example, the general formula (VI):

[0034]

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(In the formula, R ⁶ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a linear or branched alkenyl group having 1 to 18 carbon atoms, an aromatic group or a hydrogen atom. , R ⁷ represents R ⁶ or —COOR ⁶ , a represents 0 or 1) or a general formula (VII):

[0036]

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(Where a is the same as above, j is 1, 2,
A primary or secondary alcohol represented by 3, 4 or 5), and preferred are ethanol,
Examples include propanol, butanol, benzyl alcohol, cyclopentanol, cyclohexanol, and lactate.

Water or a buffer is used for the immobilization. Examples of the buffer include commonly used buffers of inorganic acid salts such as sodium phosphate and potassium phosphate, and buffers of sodium acetate and sodium citrate. Such a buffer as an organic acid salt may be used, and the pH is preferably adjusted to the optimum pH of the enzyme to be used.

The immobilized enzyme is obtained by permeating water or a buffer in which dehydrogenase and coenzyme are dissolved into a water-absorbing polymer. The concentration of dehydrogenase and coenzyme in water or buffer, and the amount of water-absorbing polymer used can facilitate the reduction of the substrate represented by the general formula (I), (II) or (III) and regeneration of the coenzyme. If it progresses to, it is not particularly limited. The concentration of dehydrogenase is generally 1 × 10 ^-4
To 0.1 unit / l, preferably 1 × 10 ^{−3 to} 0.1 unit / l, and the concentration of coenzyme is generally 1 × 10 ⁻³ to 10 mol / l.
and preferably 0.05 to 5 mol / l, and the amount of water-absorbing polymer used is 0.1 to 100 wt.
%, Preferably 1 to 10% by weight.

The organic solvent used in the method of the present invention is obtained by dissolving an ester, acetal or thioacetal as a substrate represented by the general formula (I), (II) or (III), dehydrogenase or coenzyme. The activity is not particularly limited as long as the requirement that the activity is not inhibited is satisfied. Such organic solvents include hydrocarbons, halogenated hydrocarbons, aromatic hydrocarbons,
Halogenated aromatic hydrocarbons, alkyl or aromatic ethers, alkyl or aromatic nitriles, alkyl or aromatic esters, and the like, may be used alone or as a mixture, and examples thereof include n- Pentane, cyclopentane, n-hexane, cyclohexane, carbon tetrachloride, dichloromethane, toluene, benzene, chlorobenzene, isopropyl ether, propionitrile, butyl acetate, and the like. Preferred examples thereof include n-pentane and cyclopentane. , N-hexane, cyclohexane, benzene and toluene.

The ratio of the alcohol as a reducing agent to the substrate represented by the general formula (I), (II) or (III),
The amount of the immobilized catalyst and the amount of the organic solvent used are not particularly limited as long as the reduction of the substrate and the regeneration of the coenzyme proceed smoothly. The usage ratio of the alcohol as the reducing agent is 1.0 to 1.5 molar equivalents, and the usage amount of the immobilized catalyst is generally 1 to 1000 g / mol, preferably 100 to 500 g / mol. The organic solvent has a concentration of the above-mentioned substrate generally of 0.1 to 0.1.
It is desirable to use 50 wt%, preferably 1 to 15 wt%.

The reaction temperature may be any temperature as long as the activity of the enzyme is maintained. In the case of an enzyme obtained from a thermostable bacterium, the reaction temperature is 0 to 90 ° C. In this case, it is 0 to 60 ° C. The reaction time may be several days, and is not particularly limited.
~ 30 hours.

The time and end point of the reaction can be confirmed by high performance liquid chromatography or gas chromatography.

The enzyme and coenzyme immobilized by the water-absorbing polymer have activity even after the reaction, and the recovered immobilized enzyme can be reused in a batch system, and the immobilized enzyme can be used as a filler even in a column system. The reaction can be performed continuously.

In the case of the batch method, after the reaction is completed, the reaction solution and the immobilized enzyme are separated, concentrated, and the optically active α- or β-hydroxyl is obtained by applying a conventional method such as distillation or column chromatography. Ester or hydroxyacetal can be purified and obtained.

In the case of the column system, the above-mentioned organic solvent in which the raw material represented by the general formula (I), (II) or (III) is dissolved and the alcohol represented by the general formula (VI) or (VII) are By injecting the immobilized enzyme alone or under pressure into a column packed with another packing material, concentrating the reaction solution that has completed the reaction flowing out of the column, and applying a conventional method such as distillation or column chromatography, The optically active α- or β-hydroxyester or hydroxyacetal can be purified and obtained. In order for the reaction to proceed efficiently and the reaction solution to flow out, it is desirable to use the immobilized enzyme in a mixture with other fillers, and the other fillers used inhibit the activity of dehydrogenase and coenzyme. It is not particularly limited as long as it satisfies the requirement of not. Examples of such packing materials include silica gel for column separation, acidic, basic and neutral alumina, cation and anion exchange resins, celite, quartz sand and sea sand, and these may be used alone or as a mixture. Preferred examples thereof include silica gel (200 to 300 mesh) (manufactured by Wako Pure Chemical Industries, Ltd.) and Celite (521) (manufactured by Aldrich (USA)).

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

Example 1 (Preparation of immobilized enzyme) Tris buffer (pH 8.0) (100 ml) containing dehydrogenase (1040 units) and nicotinamide adenine dinucleotide (NAD ⁺ ) (predetermined amount)
A water-absorbing polymer (6.00 g) was added to obtain an immobilized enzyme.

(Reaction) The above-prepared immobilized enzyme was added to an organic solvent (400 ml) in which butyl pyruvate (2.88 g, 0.02 mol) and cyclopentanol (1.89 g, 0.022 mol) were dissolved. Shake at ℃. After confirming the completion of the reaction by gas chromatography, the immobilized enzyme was removed by filtration, the organic solvent was distilled off, and the obtained oil was purified by silica gel column chromatography to obtain (R) -butyl lactate.

The optical purity of (R) -butyl lactate was determined by gas chromatography analysis after esterifying the hydroxyl group with optically active 2-methoxy-2-trifluoromethylphenylacetic acid chloride. The yield and optical purity (% ee) of the obtained compound were as shown in Table 1.

Further, the boiling point, optical rotation, and ¹
The H-NMR spectrum data is shown below.

Boiling point: 71-72 ° C. (10 Torr)

[0053]

(Equation 1)

¹ H-NMR (200 MHz, CDCl
₃ , δ value (ppm)): 0.93 (t, 3H), 1.20 to 2.14
(M, 7H), 3.02 to 3.28 (bs, 1H), 4.03 to 4.53
(M, 3H)

[0055]

[Table 1]

Example 2 An organic solvent (400 g) in which ethyl pyruvate (2.32 g, 0.02 mol) and 2-propanol (1.32 g, 0.022 mol) were dissolved.
ml), the immobilized enzyme prepared in the same manner as in Example 1 was added, and shaken at 35 ° C. After confirming the completion of the reaction by gas chromatography, the immobilized enzyme was removed by filtration, the organic solvent was distilled off, and the obtained oil was purified by silica gel column chromatography to obtain (R) -ethyl lactate.

The optical purity of (R) -ethyl lactate was determined by gas chromatography analysis after esterifying the hydroxyl group with optically active 2-methoxy-2-trifluoromethylphenylacetic acid chloride. The yield and optical purity (% ee) of the obtained compound were as shown in Table 2.

Also, the optical rotation of the obtained compound, ¹ H-N
The MR spectrum data is shown below.

Boiling point: 69-70 ° C (36 Torr)

[0060]

(Equation 2)

¹ H-NMR (200 MHz, CDCl
₃ , δ value (ppm)): 1.30 (t, 3H), 1.39 (d, 3
H), 3.00 to 3.26 (bs, 1H), 4.06 to 4.55 (m, 3
H)

[0062]

[Table 2]

Example 3 (Preparation of immobilized enzyme) D-butyrate dehydrogenase (manufactured by Sigma)
(1040 units), glycerol dehydrogenase derived from Geotricum candium (Amano Pharmaceutical Co., Ltd.) (1040
Units) and nicotinamide adenine dinucleotide (NAD ⁺ ) (predetermined amount) (pH 8.
0) (100 ml) was added with a water-absorbing polymer (6.00 g) to obtain an immobilized enzyme.

(Reaction) Benzene (400 ml) in which ethyl 2-oxo-4-phenylbutanoate (4.12 g, 0.02 mol) and cyclopentanol (1.89 g, 0.022 mol) were dissolved.
)), The immobilized enzyme prepared above was added, and the mixture was shaken at 35 ° C. After confirming the completion of the reaction by gas chromatography, the immobilized enzyme was removed by filtration, the organic solvent was distilled off, and the obtained oil was purified by silica gel column chromatography to give (R) -2-hydroxy-4- Ethyl phenylbutyrate was obtained.

The optical purity of ethyl (R) -2-hydroxy-4-phenylbutyrate was determined by gas chromatography analysis after esterifying the hydroxyl group with optically active 2-methoxy-2-trifluoromethylphenylacetic acid chloride. . Yield, optical purity (% ee) of the obtained compound
Was as shown in Table 3.

[0066]

[Table 3]

Example 4 Ethyl 4-chloro-3-oxybutanoate (3.29 g is 0.1%
02 mol) and 2-propanol (1.32 g, 0.022 mol) dissolved in benzene (400 ml) were added with the immobilized enzyme prepared in the same manner as in Example 1 and shaken at 35 ° C.
After confirming the completion of the reaction by gas chromatography, the immobilized enzyme was removed by filtration, the organic solvent was distilled off, and the obtained oil was purified by silica gel column chromatography to give (S) -4-chloro-3- Ethyl hydroxybutanoate was obtained.

The optical purity of ethyl (S) -4-chloro-3-hydroxybutanoate is determined based on the optical activity of 2-methoxy-2-ethyl-2-methoxy-2-hydroxybutanoate.
The hydroxyl group was esterified with trifluoromethylphenylacetic acid chloride and determined by gas chromatography analysis. The yield and optical purity (% e.
e.) was as shown in Table 4.

[0069]

[Table 4]

Example 5 (Purification of immobilized enzyme) Alcohol dehydrogenase derived from horse liver (1040 units) (Sigma), alcohol dehydrogenase derived from Geotricum candium (1040 units) (Amano Pharmaceutical Co., Ltd.) Nicotinamide adenine dinucleotide (NAD ⁺ ) (predetermined amount) and NaCl
(3.51 g) containing Tris buffer (pH 8.0) (100 ml)
Then, a water-absorbing polymer (6.00 g) was added to obtain an immobilized enzyme.

(Reaction) Ethyl 4-chloro-3-oxybutanoate (3.29 g, 0.02 mol) and 2-propanol (1.32 g)
g, 0.022 mol) in benzene (400 ml)
The immobilized enzyme prepared above was added, and shaken at 35 ° C.
After confirming the completion of the reaction by gas chromatography, the immobilized enzyme was removed by filtration, the organic solvent was distilled off, and the obtained oil was purified by silica gel column chromatography to give (R) -4-chloro-3- Ethyl hydroxybutanoate was obtained.

The optical purity of ethyl (R) -4-chloro-3-hydroxybutanoate is determined based on the optically active 2-methoxy-2-ethyl-2-methoxy-2-hydroxybutanoate.
The hydroxyl group was esterified with trifluoromethylphenylacetic acid chloride and determined by gas chromatography analysis. The yield and optical purity (% e.
e.) was as shown in Table 5.

[0073]

[Table 5]

Example 6 Pyruvaldehyde dimethyl acetal (2.36 g, 0.02 g
Mol) and 2-propanol (1.32 g, 0.022 mol) were dissolved in benzene (400 ml), and the immobilized enzyme prepared in the same manner as in Example 5 was added and shaken at 35 ° C. After confirming the completion of the reaction by gas chromatography, the immobilized enzyme was removed by filtration, the organic solvent was distilled off, and the obtained oil was purified by silica gel column chromatography.
(S) -2-Hydroxypropionaldehyde dimethyl acetal was obtained.

The optical purity of (S) -2-hydroxypropionaldehyde dimethyl acetal was determined by gas chromatography analysis after esterifying the hydroxyl group with optically active 2-methoxy-2-trifluoromethylphenylacetic acid chloride. The yield and optical purity (% ee) of the obtained compound were as shown in Table 6.

[0076]

[Table 6]

Example 7 Pyruvaldehyde dimethyl acetal (2.64 g, 0.02 g
Mol) and 2-propanol (1.32 g, 0.022 mol) were dissolved in benzene (400 ml), and the immobilized enzyme prepared in the same manner as in Example 5 was added thereto, followed by shaking at 35 ° C. After confirming the completion of the reaction by gas chromatography, the immobilized enzyme was removed by filtration, the organic solvent was distilled off, and the obtained oil was purified by silica gel column chromatography (S)-.
3-Hydroxybutyraldehyde dimethyl acetal was obtained.

The optical purity of (S) -3-hydroxybutyraldehyde dimethyl acetal was determined by using optically active 2-methoxy-2-trifluoromethylphenyl acetic acid chloride.
The hydroxyl group was esterified and determined by gas chromatography analysis. The yield and optical purity (% ee) of the obtained compound were as shown in Table 7.

[0079]

[Table 7]

Example 8 2-ethoxycarbonylcyclopentanone (3.12 g, 0.02 g
Mol) and 2-propanol (1.32 g, 0.022 mol) were dissolved in n-hexane (400 ml), and the immobilized enzyme prepared in the same manner as in Example 1 was added thereto, followed by shaking at 35 ° C. After confirming the completion of the reaction by gas chromatography, the immobilized enzyme was removed by filtration, the organic solvent was distilled off, and the obtained oil was purified by silica gel column chromatography.
(1S, 2R) -2-ethoxycarbonylcyclopentanol was obtained.

The optical purity of (1S, 2R) -2-ethoxycarbonylcyclopentanol was determined by using optically active 2-methoxy-2-trifluoromethylphenylacetic acid chloride.
The hydroxyl group was esterified and determined by gas chromatography analysis. The yield and optical purity (% ee) of the obtained compound were as shown in Table 8.

[0082]

[Table 8]

Example 9 (When the immobilized enzyme is reused in a batch system) (Preparation of the immobilized enzyme) Glycerol dehydrogenase derived from Geotricum candium (manufactured by Amano Pharmaceutical Co., Ltd.)
BL-100 (manufactured by Osaka Organic Chemical Industry Co., Ltd.) (6.00 g) was added to a Tris buffer solution (pH 8.0) (100 ml) containing NAD ⁺ (8.0 × 10 ⁻³ mol) and NAD ⁺ (8.0 × 10 ⁻³ mol). ) Was added to obtain an immobilized enzyme.

(Reaction) The immobilized enzyme prepared above was added to n-hexane (400 ml) in which butyl pyruvate (2.88 g, 0.02 mol) and cyclopentanol (1.89 g, 0.022 mol) were dissolved. Shake at 35 ° C. After confirming the completion of the reaction by gas chromatography, the immobilized enzyme and the reaction solution were filtered, the reaction solution was concentrated, and the obtained oil was purified by silica gel column chromatography to obtain (R) -butyl lactate. Using the recovered immobilized enzyme as it was, a reduction reaction was further performed twice by the above method.

The optical purity of (R) -butyl lactate was determined by gas chromatography analysis after esterifying the hydroxyl group with optically active 2-methoxy-2-trifluoromethylphenylacetic acid chloride. The yield and optical purity (% ee) of the compound obtained by the three reduction reactions were as shown in Table 9.

[0086]

[Table 9]

Example 10 (When the immobilized enzyme is used in a continuous reaction in a column system) (Packing of the immobilized enzyme in a column) Glycerol dehydrogenase derived from Geotricum cadmium (Amano Pharmaceutical Co., Ltd.)
(2080 units) and NAD ⁺ (1.6 × 10 ^-3 mol) in Tris buffer (pH 8.0) (200 ml), and water-absorbing polymer BL-100 (manufactured by Osaka Organic Chemical Industry Co., Ltd.) (12.
0g) and silica gel (200-300 mesh) (24.0 g) (Wako Pure Chemical Industries, Ltd.) for column preparative separation were mixed, and a glass column (diameter 1.6 cm, length
50 cm).

(Reaction) n-Hexane (8 L) in which butyl pyruvate (57.6 g, 0.40 mol) and cyclopentanol (37.8 g, 0.44 mol) were dissolved was added to the column under pressure using a pump at room temperature. And the flow rate was maintained at 15 ml / hr. The reaction solution flowing out was analyzed by gas chromatography to confirm the completion of the reaction. After the completion of the injection, the inside of the glass column was washed with n-hexane (100 ml), and the eluted reaction solution and the washing solution were combined and concentrated. The obtained oil was purified by silica gel column chromatography to give (R) -butyl lactate. I got

The optical purity of (R) -butyl lactate was determined by gas chromatography analysis after esterifying the hydroxyl group with optically active 2-methoxy-2-trifluoromethylphenylacetic acid chloride. The yield of (R) -butyl lactate obtained was 99%, the optical purity was 99% ee or more, and the turnover of NAD ⁺ was 25,000.

[0090]

According to the present invention, expensive dehydrogenases and coenzymes are immobilized on a water-absorbing polymer using a catalytic amount without being deactivated, and reused in a batch system or continuous reaction in a column system By using the compound, an important compound as an intermediate or a raw material for production of various organic chemicals such as medicines and agricultural chemicals, flavors, and the like, optically active α- or β-hydroxycarboxylic acid esters or hydroxyacetals. It can be manufactured simply and economically.

────────────────────────────────────────────────── ─── Continued on the front page (72) Kumi Terada, Inventor 12-7, Kamitano Kitadacho, Sakyo-ku, Kyoto (56) References JP-A-3-285689 (JP, A) (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12P 7/62 C12P 7/02 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] [Claim 1] General formula (I): (Wherein R, R ′ and R ″ are the same or different and each is a linear or branched alkyl group having 1 to 18 carbon atoms, a linear or branched alkenyl group having 1 to 18 carbon atoms, groups, but I Table halogen atom or a hydrogen atom, R "is
Is not a halogen atom, a is 0, 1 or 2, b is 0
Or 1), general formula (II): (In the formula, R is the same as above, except that it is not a halogen atom.
Wherein n represents 3 or 4) or the general formula (III): (Wherein R, R ′, a and b are the same as above, R ¹ and R ² are the same or different, and are a linear or branched alkyl group having 1 to 18 carbon atoms, or
(CH _{2) k} - _(k in the formula 2 or 3) to form a ring represented by, X is a α- or β- ketoesters or keto acetals represented by an oxygen atom or a sulfur atom), an organic In a solvent, a primary or secondary alcohol is used as a reducing agent, and a dehydrogenase and a coenzyme dissolved in water or a buffer solution are brought into contact with an immobilized enzyme obtained by immobilizing the coenzyme on a water-absorbing polymer. Or a method for producing optically active α- or β-hydroxycarboxylic acid esters or hydroxyacetals, which comprises reducing the β-carbonyl group. 2. The method according to claim 1, wherein after the reaction, the immobilized enzyme is recovered by a separation operation and reused. 3. The method according to claim 1, wherein an immobilized enzyme obtained by immobilizing a dehydrogenase and a coenzyme dissolved in water or a buffer solution on a water-absorbing polymer is continuously used as a filler in a column system for the reaction. Manufacturing method.