JPH05503005A - Method for producing optically active 3-hydroxy-2-methylbutyric acid esters - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Technical field of production method of optically active 3-hydroxy-2-methylbutyric acid esters The present invention provides an optically active 3-hydrocarbon compound useful as a raw material for synthesis of ferroelectric liquid crystal compounds, etc. The present invention relates to a method for producing xy-2-methylbutyric acid esters.

That is, the expression 2-methyl-3-oxobutyric acid represented by [wherein R represents an ester-forming group] When esters are reduced using conventional chemical methods, e.g. , the following four stereoisomers are generated due to the asymmetric carbons at the 2nd and 3rd positions (21?, 3S) syn 2 (2S, 3S) anti 3 (2S, 3R) syn 4 (2R, 3R) anti 5 [In each formula, R has the same meaning as above, and in parentheses The symbols indicate the absolute coordination of the asymmetric carbon atoms at the 2nd and 3rd positions].

Of these four stereoisomers, the present invention provides (2R,3S) represented by formula (2). Related to a method for selectively producing syn2 bodies by asymmetric reduction using microorganisms . The isomer of formula (2) is extremely applicable as a raw material for the synthesis of ferroelectric liquid crystal compounds, etc. It has a high value.

Conventional technology Optically active 3- Production of 2-methylbutyric acid esters by asymmetric reduction using microorganisms A known method for this is the method using baker's yeast [Ei Furuichi et al. Natural Biological Chemistry (K, F uruichi etc. al, : Agric, B iol, Chem, ), ±day, 9.2563 ~2570 (1985); Kei Nakamura et al., Tetrahedron Letters (Na kamura, K, et al: TetrahedronLette rs), 27.3155 (1986); Kei Nakamura et al., Bulletin of Kei Michal Society of Japan (Naka+aura.

K, et al, : Bull, Chem, Soc, J pn, ), l 7.1179 (1989)] and Candid albicans (Candid A method using yeast such as A. albicans [H. Akita et al., Chemical ・And Pharma’/Neatical Bulletin (Akita, H, et al , :Chew, Pharm.

Bull, ), Main ±, 4376 (1983)]. However, this These methods suffer from poor diastereoselectivity and/or low enantioselectivity. This is not a commercially advantageous method. In addition, 2-methyl-3-oxo-butyric acid ester Microbial asymmetric reduction of Stellaceae by bacteria is not known.

Purpose of invention The main object of the present invention is to provide ferroelectric liquid crystal compounds, polyoxo antibiotics, and various Optical activity 3 represented by formula (2) is a useful synthetic raw material for natural physiologically active substances, etc. -Hydroxy-2-methylbutyric acid esters, the corresponding 3-ol of formula (1) By using microorganisms, especially bacteria, from the xoester, selectively and efficiently An object of the present invention is to provide a manufacturing method.

This and other objects and advantages of the invention will be apparent to those skilled in the art from the following description. Will.

Summary of the invention The present inventors discovered that the 2-methyl-3-oxobutyric acid ester represented by formula (1) It is efficiently carried out by asymmetric reduction by living organisms or by using bacteria, and the target light is It was discovered that a chemically active 3-hydroxy compound can be produced.

That is, according to the present invention, 2-methyl-3-oxobutyric acid ester represented by formula (1) A formula ( 2) Production of (2R,3S)-3-hydroxy-2-methylbutyric acid ester A manufacturing method is provided.

Disclosure of invention In the production method of the present invention, 2-methyl represented by formula (1) used as a starting material The ester-forming group represented by R of -3-oxobutyric acid ester is particularly limited. For example, lower alkyl, alkoxyalkyl or aral Kills are mentioned. Examples of lower alkyl include methyl, ethyl, propyl , isopropyl, butyl, isobutyl, 5ec-butyl, tert-butyl, Examples include alkyl having 1 to 6 carbon atoms such as pentyl, neopentyl, and hexyl.

As alkoxyalkyl, methoxyethyl, ethoxyethyl, butquinethyl Examples include alkoxyalkyl having 3 to 6 carbon atoms such as ru. Also, Aralquil and are benzyl, phenethyl, 1-methylbenzyl, 1-ethylbenzyl, 1 - Phenethyl such as methylphenethyl, 2-methylphenethyl, 2-ethylphenethyl Examples include nyl (lower) alkyl. In particular, 2-methyl-3-oxobutyric acid ester Tyl ester, 2-methyl-3-oxobutyric acid isobutyl ester, 2-methyl 3 -Oxobutyric acid butoxyethyl ester and 2-methyl-3-oxobutyric acid benzene esters are preferred. In particular, from a practical standpoint, ethyl 2-methyl-3-oxobutyrate preferable.

Although formula (1) shows the keto form, the compound can also exist in the dinolic form.

As used herein, the term "2-methyl-3-oxobutyric acid" refers to each of these including variants and mixtures thereof.

Bacteria used in the asymmetric reduction reaction of the present invention may be those having the ability of asymmetric reduction. There are no particular limitations. For example, Enterobacter spp., Erwinia spp., Enosia spp. Rhinochia, Crabsiella, Proteus, Salmo, Serratia, Drumstick The genus Rus or Pedeioco.

Examples include bacteria belonging to the genus Casas. Newly isolated from soil, food, animals and plants, etc. Even if it is a bacterial strain, the compound represented by formula (1) to the compound represented by formula (2) Any can be used as long as it has the ability to produce by asymmetric reduction. . In addition, these strains were obtained through artificial mutation through ultraviolet irradiation or treatment with mutagens. It may be a mutant that exhibits the reducing activity, or it may be an artificially isolated mutant that is necessary for the expression of the reducing activity. Other thin mm bodies incorporating gene fragments may also be used.

Used in the asymmetric reduction of 2-methyl-3-oxobutyric acid esters represented by formula (1) A specific example of a bacterial strain that can be used is Enterobacter aerogenes (Enterobacter aerogenes). obacter aerogenes) IF O13534, Enterobacter E nterobacter cloacae IF O 13535, Elk C Nia herbico la) I　　　　　　　12686, Enosialinohi 7” Cori (E 5cheri chia coli) I F O3549, Club Sierra niumoniae (K) 1ebsiella pneumoniae) IF 03319, Clubsi K1ebsiella terrigena A TCC3 3257 (IF○ 14941, FERM BP-2710), Proteus Bu P roteus vulgaris rF O3045, monkey Monella typhimurium (S almonella typhimurium) IF O14194, S erratia gr. ia + essii) I F O13537, Bacillus pumilus (Bacillus lus pua+1lus) IF O12090 and Pediococcus Pediococcus pentosaeus I F O3892 is mentioned. Among these strains, Clubsiella terrigena (K Iebsiella terrigena) AT CC33257 is American Can Type Culture Collection (AIlerican Type C) "Catalog, O-Catalog" published by "A.T.C. Culture Collection (ATCC)" Bu.

Bacteria and Bacteriophages (Catalogue of BACT Published in ERIA & BACTERIOPHAGES) Volume 17, 19B9J It is a known strain and can be easily obtained from ATCC. Also, this strain is Since September 14, 1989, the Fermentation Research Institute (Osaka) (IFO) has received the accession number IF. Furthermore, as O14941, the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microbial Technology Research Institute (F RI) from December 26, 1989 under the Budapest Treaty with accession number FER. It has been deposited as M BP-2710. In addition, other strains are published by IF○. [LIST OF CULTURES Volume 8, 19 It is a known strain listed in 88j and can be easily obtained from IFO.

To carry out the production method of the present invention, these bacterial strains are first cultured.

These strains can be cultured using conventional static culture, shaking culture, aerated agitation culture, or solid-state culture. It can be carried out continuously or intermittently by culturing or the like. The medium used is It may be of a normal composition on which bacteria can grow. Carbohydrates and fats are carbon sources. You can select assimilated materials as appropriate from acids, organic acids, alcohols, etc. Can be used alone or in combination. Nitrogen sources are e.g. peptone, yeast Extract, meat extract, malt extract, corn steep liquor, soy flour, urea, etc. In addition to which organic nitrogen sources, there are also ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphorus, etc.! Also contains a nitrogen source.

They may be used alone or in an appropriate mixture, if necessary. The medium contains these carbon sources and nitrogen. In addition to raw materials, the growth of minerals, amino acids, vitamins, etc. necessary for the growth of bacterial strains It is advisable to add essential factors and growth-promoting substances.

Caustic alkaline solution, sodium carbonate aqueous solution, calcium for the purpose of pH control during culture Salts can be added as appropriate. Also, use antifoaming agents for foam control purposes. is also valid. Furthermore, depending on the microorganisms used, the purpose of making the environment anaerobic is It is also effective to aerate nitrogen gas or carbon dioxide gas.

Then, the cultured strain or its treated product is brought into contact with the compound represented by formula (1). The asymmetric reduction reaction is then carried out.

Bacterial strains can be obtained from the resulting culture itself or by suitable isolation means, e.g. Heart separation, sedimentation separation, flocculation separation, or porous membrane, polymer membrane, ceramic membrane, etc. The bacterial cells separated by filtration or the like can be used in the asymmetric reduction reaction.

The "processed product" used in the present invention refers to the bacterial cells or culture obtained in the above Physical and chemical methods, such as ultrasound, French press, osmotic pressure, freeze-thaw, freeze-drying , treated with alumina grinding, Meien enzymes, surfactants or organic solvents, etc. It refers to a crushed bacterial cell material that has the ability to asymmetrically reduce the compound represented by formula (1). Also , the treated product is obtained by purifying these bacterial cells and crushed bacterial cells by the method described below. It also includes crude or purified enzymes. The enzyme freezes the bacterial cells obtained above, for example. Physicochemical methods such as melting, grinding, ultrasound, osmotic pressure, cell wall membrane dissolution, surfactants, etc. It can be solubilized by subjecting it to treatment. The solubilized enzyme is further purified. Rotamine treatment, salting out, organic solvent treatment, isoelectric precipitation, ion exchange chromatography -, gel filtration, affinity chromatography, crystallization, etc. It can be purified by subjecting it to more than one conventional enzyme purification operation. instructor The enzyme obtained by Comprehensive immobilization in synthetic polymers such as polyamide and urethane resins, or activated carbon and ceramics. Support materials such as dextran, agarose-based materials, porous glass, ion exchange resins, etc. It can be attached to the body and immobilized.

Contacting the compound represented by formula (1) with a culture of a bacterial strain or a treated product thereof In order to carry out the asymmetric reduction reaction by It is used at a degree of 5% by weight, more preferably from 0.2 to 3% by weight. Furthermore, the formula ( Together with the compound shown in 1), it serves as a hydrogen donor as a carbon source ( For example, carbohydrates, fatty acids, organic acids, alcohols) are preferably added. Also , minerals and vitamins can also be added to accelerate the reaction. .

The reaction temperature is not particularly limited as long as the asymmetric reduction can proceed, but is preferably The temperature is preferably in the range of 20 to 60°C, more preferably in the range of 25 to 50°C. of pH The range varies depending on the strain used.

When using strains belonging to the genus Bedioco and Kas, the pH should be between 3 and 7, but for other strains. When using, the pH is preferably 5 to 9. To make the reduction reaction proceed efficiently It is desirable to stir the reaction solution appropriately, but when using strains of the genus Vedioco and Kas In such cases, it is preferable to maintain anaerobic conditions. For this purpose, nitrogen gas or carbon dioxide gas is used. It is also good to ventilate. On the other hand, when using other bacterial strains, under sufficient aerobic conditions Preferably, the reaction is carried out.

The reaction continues until the asymmetric reduction of the compound represented by formula (1) is completed. usually, Approximately 8 to 96 hours.

After the reaction, the optically active 3-hydroxy compound produced is subjected to one or more methods such as extraction and concentration. It can be easily recovered and collected by the above-mentioned known purification method.

The compound represented by formula (2) obtained by the method of the present invention has a hydroxyl group known per se. The corresponding 3-alcokyne-2- Can be converted to methylbutyrate. Next, this 3-alcoquine-2 - Methyl butyrate and side chain components that do not contain chiral centers (or contain chiral centers) A ferroelectric liquid crystal compound with excellent liquid crystal properties. can be easily synthesized (e.g. European Patent Application Publication No. 3228 No. 62). As described above, according to the method of the present invention, ferroelectric liquid crystal compounds, etc. Which useful synthetic raw material is (2R,3S)-3-hydroxy-2-methylbutyric acid ester? Steles can be easily and efficiently produced.

The present invention will be explained in detail below by giving examples and reference examples, but these are It does not limit the scope of the invention.

Example 1 2-Methyl-3-oxobutyric acid ethyl ester was used as a substrate and shown in Table 1. By using the strain, the following reduction reaction was carried out.

Broth slant cultures of each strain were mixed with 1% Bacto-tryptone, Bacto-tryptone 1% - Sterilized consisting of 05% yeast extract, 0.5% NaCρ and 1% glucose Inoculate a 200 mρ Erlenmeyer flask containing 20 m (l) of seed medium, and inoculate at 200 rpm. The cells were cultured at 28°C for 16 hours with rotational shaking. This seed culture solution (0.3 mf2 ), meat extract 1%, polypeptone 1%, NaC & 0.5%, CaCO 34% and 30 ml (l) of sterile main medium (pH 7) consisting of 2% glucose (separately sterilized). Transfer it to a 200 m 12-volume pleated Erlenmeyer flask, rotate at 20 rpm, and at a temperature of 3. Culture was performed with shaking at 0°C. 24 hours after the start of culture, 120 u of substrate and 1.0 g of glucose were added. Add 2g to each flask and react for 24 hours under the same conditions as the main culture. I made it happen. After the reaction was completed, the reaction solution was extracted twice with an equal volume (30 °C) of ethyl acetate, The collected ethyl acetate layer was washed with water, dehydrated, and then brought to a constant volume. A part of the extract is used as a sample. The number of reductants and syn/an I determined the ti ratio. Meanwhile, the remaining extract was concentrated under reduced pressure. A small amount of the resulting concentrate After dissolving in ether, it was subjected to Kugel distillation to obtain a colorless oily reduced product. . The optical purity (enantiomeric excess) of this reduced product was measured using a high-speed liquid Determined by romatography.

The results are shown in Table 1. For both strains, diastereomers and enantiomers Mer selectivity is that obtained in the known microbial asymmetric reduction using baker's yeast. taller than.

The analytical method and conditions used are as follows.

(1) Gas chromatography Using a Hitachi G-3000 gas chromatograph, /Nyl alcohol was used as an internal standard. Chemically synthesized (2R,35) and (2S,3S)-3-hydroxy-2- Analysis was conducted under the following conditions using methyl butyric acid ethyl ester as a standard.

Column: ULBON-20M (0.25mm 11x25m) Column temperature = 110°C Carrier gas: He Detection: FID (2) High performance liquid chromatography Using a Waters 600-E liquid chromatograph, chemically synthesized 3-hydrocarbon Four stereoisomers of Dorogyu/-2-methylbutyric acid ethyl ester were used as standards. The analysis was performed under the following conditions.

Column: Chiral Cell OB < 4.6tnm 8250in) 2 columns Column temperature 25°C Eluent: Hegyu San: 2-propatool (99,5:0.5) Flow rate: 1, O mQ1 minute Detection UV220nm *) ce enantiomer efses (enantiomer excess) Example 2 Bediofunoccus grown by puncture culture in a commercially available GAM agar medium (Tamizu Pharmaceutical Co., Ltd.) Bentosaceus (Pediococcus pentosaceus) I F03892 strain, glucose 2%, polypeptone 1%, yeast extract 05%, Meat extract 1%, Tween 800.1%, sodium acetate 03%, trianium citrate Monium 0.2%, Mg5O, 78200.02% and Mn5O, 4-6 200 m containing 30 mQ of sterile seed medium (pH 6,6) consisting of H, OO, 005% (It was inoculated into an Erlenmeyer flask and cultured stationary at 30°C for 24 hours. This seed culture solution (1 , 5n++2 each) with the same composition as the seed medium above, except that 1% CaCO was added. Sterilized main medium (pH 6,6) 200 m containing 30 ml (12 volume conical flask with folds) The cells were transplanted to a plant and cultured statically at 30°C. 24 hours after the start of culture, 2-methyl- Add 60 mg of 3-oxobutyric acid ethyl ester and 1.2 g of glucose to each flask. added. After replacing the gas phase with nitrogen gas, the reaction system was tightly stoppered and the rotation speed was 200 rpm. The reaction was carried out at 30°C for 24 hours with shaking.

After the reaction was completed, the reduction product was analyzed in the same manner as in Example 1, and the reduction yield was 30%, syn/anti ratio 97.3, optical purity of two syn bodies is 99%ee or higher It was above.

Example 3 K. 1ebsiella pneumoniae ) IF O3319 was cultured in the same manner as in Example 1. 24 hours after starting main culture In between, 120 mg of each of the five types of substrates shown in Table 2 and 1.2 g of glucose were added to each It was added to a flask and allowed to react for 24 hours under the same conditions as the main culture. After the reaction , each reaction solution was extracted twice with an equal volume (30 m) of ethyl acetate. The ethyl layer was washed with water, dehydrated, and then brought to a constant volume.

A portion of the extract was subjected to gas chromatography as described in Example 1. obtained and a chromatogram obtained by separately reducing the same substrate using the baker's yeast method. The peaks of the syn and anti-isomers were identified by comparing the two. difference Furthermore, the syn/anti ratio was calculated from the area ratio of both peaks. Meanwhile, the remaining extract was concentrated under reduced pressure. After dissolving the concentrate in a small amount of ether, it is subjected to Kugel distillation. A colorless oily reduced product was obtained. This reduced product is converted into ethanol 811I2. Dissolve and add strongly basic anion exchange resin Amberlite IRA402 (ROHM And Haas (USA)) was added. The mixture was stirred at room temperature for 48 hours. A transesterification reaction was carried out. The resulting ethyl ester was processed using the high speed method described in Example 1. Separate and quantify using liquid chromatography and calculate the enantiomeric excess did. The optical purity was expressed by this rate. The results are shown in Table 2.

Example 4 In the same manner as in Example 1, K. levsiellap A seed culture of IF O3319 was prepared. This culture solution 20-, meat extract 1%, polypeptone 1%, NaCQ O, 5%, antifoaming agent 0.0 Contains sterile main medium (pH 7) 2Q consisting of 5% and glucose 2% (separately sterilized) Transplanted to a 5Q capacity jar fermenter, stirring number 80 Orpm, aeration rate 0.6 Culture was carried out with aeration and stirring at Q/min and temperature of 30'C.

When the pH of the culture solution drops below 6.5 during culture, add 15% (w/v) sodium hydroxide. The pH was maintained at 6.5 by adding an aqueous solution of um. 18 hours after the start of culture, 2- 35 g of methyl-3-oxobutyric acid ethyl ester and 40% (w/v) glucose water A solution of 200 mσ was added and the reaction was carried out for 24 hours under the same conditions as the main culture. Before The pH was adjusted in the same manner as described above. After the reaction was completed, the reaction solution (2,1812) was diluted with acetic acid. Extracted once with ethyl 2Q and then twice with dithyl acetate IT2. combined ethyl acetate The filtrate layer was filtered to remove insoluble materials, and the filtrate was washed with water and dehydrated. One of this extract sample and analyzed it by gas chromatography as described in Example 1. As a result, the amount of remaining substrate was 0.5 g, and the amount of reduced product produced was 34.0 g (reduction yield 97%). , the gyn/anti ratio was 99/l. On the other hand, the extract was extracted with acetic acid under reduced pressure. When the chill was distilled off, 35.1 g of concentrate was obtained. Add the concentrate to a minimum amount of After dissolving in tel, it was subjected to Kugel distillation to obtain 335 g of a colorless oily reduced product. Ta. This reduced product was separated and determined by high performance liquid chromatography as described in Example 1. The amount of syn2, anti3 and anti5 produced was 3 each. 3.0g, 0.25g and 0610g, and syn 4 was not detected. In the same manner as in 4, Klebsiella lapne umoniae) IF O3319 was cultured to obtain a main culture solution of about 2a.

1) Extraction of reductase The IQ of the main culture solution was 10. The cells were collected by centrifugation at OOOxg for 20 minutes. Applicable The bacterial cells were treated with 2-mercaptoethanol (5mM) and phenylmethylsulfonate fluoride. 10mM phosphate buffer (pH 6.0, hereinafter simply buffer) containing 2μg/m The cells were centrifugally washed once with about 200 mg of the microorganisms, to obtain about 30 g (wet weight) of washed bacterial cells.

All washed bacterial cells were suspended in a buffer solution to make a total volume of 150 mJ. That 50m Each Q was subjected to ultrasonic treatment at an output of 180 W for 20 minutes. Combined sonication suspension A supernatant (145 mf2) was obtained by centrifugation at 35,500 xg for 20 minutes. child The protein concentration of the supernatant was determined by protein assay using bovine serum albumin as a standard. Say Kit (Protein As5ay Kit) (Bio-Rad (Bi o-Rad (manufactured in the United States), it was 33.5 mg/rM. Ma In addition, the 2-methyl-3-oxobutyric acid ethyl ester reducing activity determined by the measurement method described below was 0. ．． It was 30U/mQ.

Enzyme activity was measured as follows.

Phosphate buffer (pH 7.0) 40 g mol, NADPH 0.2 μ mol, substrate (2- Total volume 1 consisting of 2 μmol of methyl-3-oxobutyric acid ethyl ester) and enzyme solution The mQ reaction mixture was kept at 30°C and the decrease in absorbance at 340 nm was continuously monitored. did. Enzyme activity is an enzyme that oxidizes 1 g mole of NADPH per minute under the above conditions. The amount was defined as 1 unit (U).

ii) Purification of the enzyme The supernatant was diluted with a buffer solution so that the protein concentration was 10 mg/m& After diluting the solution, solid ammonium sulfate was gradually added to make a 40% saturated solution. Discard the formed precipitate It was removed by heart separation and solid ammonium sulfate was added to make an 80% saturated solution. occured Collect the precipitate by centrifugation, dissolve it in buffer and adjust the volume to 55 m& did. This solution was mixed with buffer solution (pH 8,0) 2j+ at 4°C, and buffer solution (p H8,0) was changed and dialyzed for a total of 20 hours. The insoluble matter generated during this dialysis It was removed by centrifugation to obtain a clear supernatant.

The total volume of this supernatant was equilibrated with 10mM' phosphate buffer (pH 8,0). Add to TSK-Gel DEAE-5PW column (20x150mm) and The ram was washed with 200 mQ of the same buffer. In the same buffer, the salt concentration was O-0,1 The enzyme was eluted in a linear manner up to 5M. Collect the active fraction and add ammonium sulfate to it. In addition, a 90% saturated solution was obtained. The resulting precipitate was collected by centrifugation.

This precipitate was dissolved in a small amount of 50mM'J acid buffer (pH 7.0), and the solution was , AF-Red Toyopearl column equilibrated with the same buffer (2 5x 50mm). Linearly increase the salt concentration from 0 to 1M in the same buffer. The enzyme was eluted. The active fractions were collected and subjected to ammonium sulfate salting out in the same manner as above. A precipitate was collected.

This precipitate was dissolved in a minimum amount of 50mM'J acid buffer (pH 7, 0). TSK-GelG3000 SW equilibrated with the same buffer solution. column (20x300 mm) and eluted with the same buffer. Active fraction (13m The protein concentration of C) is 0.52 mg/mQ, and the enzyme activity is 1.170/m12. It was. Therefore, when using the ultrasonic extraction liquid as a standard, the recovery rate of the target enzyme is approximately 35%. %, the degree of purification was approximately 250 times higher.

The homogeneity of the purified enzyme was confirmed by polyacryloamide gel electrophoresis.

1ii) Reduction of 2-methyl-3-oxobutyric acid ethyl ester by mm enzyme Phosphate buffer (pH 7.0) 400 μmol, ethyl 2-methyl-3-oxobutyrate Ester 200μmol, glucose 200μmol, NADPHO, 8μmol, glucose Lucose dehydrogenase (Bacillus sp. ), manufactured by Sigma (USA)) and the purified enzyme IU, total volume 4mQ The reaction mixture was kept at 30'C and reacted for 6 hours with gentle shaking. . After the reaction was completed, the reaction solution was extracted twice with an equal volume (4 m) of ethyl acetate. The ethyl acid layer was dehydrated and its volume was adjusted to a predetermined volume. In the same manner as in Example 1, When the reduction products in this sample were analyzed, the amount of syn and anti forms produced was were 197 and 0.6 μmol, respectively.

Furthermore, all syn bodies were 5yn2, and 5yn4 was not detected. In addition, The rotational speed of NADPH under these reaction conditions was about 250.

Reference example 4'-octyloxy-4-biphenylcarbon[4-[(2R,,3S)-3 -Production of methoxy-2-methylbutyryl oxy/yuphenyl ester 1) (2R,3S) Production of 3-methoxy-2-methylbutyric acid (2R,3S)-3 -Hydroxy-2-methylbutyric acid ethyl ester 8.20 g, methyl iodide 78 ．． 0 g was dissolved in 150 ml of N,N-dimethylformamide. water in this solution While cooling, add 2.46 g of oily hydrogenated oil (approximately 60%) little by little, and add water. The mixture was stirred for 1 hour while cooling. The reaction solution was poured into 300 mQ of ethyl acetate, and the mixture was diluted with 1 Stir for 0 minutes. Insoluble materials were removed by filtration, and the filtrate was washed with water (3 times). Dry the organic layer was concentrated and distilled under reduced pressure (b, p, 80°C/30 Hg) to give (2R,3S)-3 6.87 g of -methoxy-2-methylbutyric acid ethyl ester was obtained.

2.55 g of this methyl ether was dissolved in 50 mff of dioxane, and concentrated hydrochloric acid (3.5 g) was dissolved in 50 mff of dioxane. 6%) and 50 ml of water were added and the solution was heated at 90'C for 5 hours.

The reaction solution was poured into 100 mQ of water, and the mixture was extracted with ethyl acetate (4 times). If The combined extract was dehydrated, and 1141 L was distilled under reduced pressure (b, p, 105°C/1 m o + Hg) 1.49 g of (2R,3S)-3-methoxy-2-methylbutyric acid Obtained.

The 'H-NMR and IR spectra of this compound are shown below.

'HNMR (90MHz, CDC+23) δ: 1.15 (3H, s), 1.23 (3H, s), 2.5-2.8 (IH, n).

3.39 (3H, s), 3.47-3.74 (LH, m), 6.0 (IH, b) IR shneatc+n-': 3450, 1710.1100ax ii) Esterification 0.79 g of (2R,3S)-3-methoxy-2-methylbutyric acid produced in i), 4 ’-Octyl-ox-4-biphenylcarboxylic acid 4-hydroxyphenyl ester Dissolve 2.5 g of alcohol and 266 g of tributylamine in 40 mQ of dry tetrahydro-7ran. 1.84 g of 2-chloro-1-methylpyridinium iodide was added to The mixture was heated to reflux for an hour. Insoluble materials were removed by filtration, and the filtrate was concentrated under reduced pressure. Column the residue. ROMATOGRAPHY "Silica gel, developing solvent/chloroform, n-hexane (4: 1) Separate and purify with l and recrystallize from ethanol to obtain 0.36 g of the title compound. I got something.

The 'H-NMR and IR spectrum data of this compound are as follows.

'HNMR (90MHz, CDCQs) δ: 0.90 (3H, t), 1.05 ~ 1.6 (16H, m), 1.8 (2H, b).

2.65-2.9 (IH, m), 3.40 (3H, s), 3.6-3.82 (I H, m), 4.01 (2H, t), 6.94-8.25 (12H, n+) IRv KBrcm-': 1760, 1730, 1610, 1510゜1ax 1100.830 Elemental analysis value Calculated value (C,, H,, O, included): C, 74,41; H, 7,57 Actual value: C, 74, 69: H, 7, 52 summary 2-Methyl-3-oxobutyric acid ester was treated with bacteria or (2R,3S)-3-hydroxy-2-methylbutyric acid ester by reduction efficiently and selectively.

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Claims (12)

[Claims] 1. Asymmetric reduction of 2-methyl-3-oxobutyric acid ester by bacteria or its treated product (2R,3S)-3-hydroxy-2-methylbutyric acid ester How to make stel. 2. The bacterium is of the genus Enterobacter, Erwinii. Genus Erwinia, Genus Escherichia, Kiebsiella spp., Proteus spp. Salmonella spp., Serratia spp., drumstick The genus Bacillus or Pediococcus 2. The method according to claim 1, wherein the method is selected from bacteria belonging to the genus .). 3. The bacterium is Enterobacter aerogenes. erogenes), Enterobacter crocae (Enterobacter crocae) cloacae), Erwinia herb icola), Escherichia coli , Klebsiella pneumoniae , Klebsiella terrigena, professional Proteus vulgaris, Salmonella typhoid Salmonella typhimurium, Serratia gu Serratia grimessii, Bacillus pumilus ( Bacillus pumilus) and Pediococcus pentosaceus (Pediococcus pentosaceus). 3. The method according to claim 2. 4. The bacterium is Enterobacter aerogenes. erogenes) IFO13534, Enterobacter crocae (Ente robacter cloacae) IFO13535, Erwinia helvi Cola (Erwinia herbicola) IFO12686, Esshari Escherichia coli IFO3549, Crabzi Klebsie11a pneumoniae IFO33 19. Klebsiella terrigena ATCC33257 (IFO14941, FERMBP-2710), Proteu Proteus vulgaris IFO3045, Salmo Salmonella typhimurium IF O14194, Serratia grimessi i) IFO13537, Bacillus pumilu s) IFO12090 and Pediococcus pentosaceus (Pedioc occus pentosaceus) IFO3892. 4. The method according to claim 3. 5. The bacterium is Klebsiella pneumoniae. 5. The method according to claim 4, wherein the method is iae) IFO3319. 6. The bacterium is Klebsiella terrigena. 5. The method according to claim 4, which is na) ATCC33257. 7. 2. The method according to claim 1, wherein the treated product is a crushed bacterial cell product. 8. 2. The method according to claim 1, wherein the treated product is a reductase contained in the bacteria. 9. Use 2-methyl-3-oxobutyric acid ester at a concentration of 0.1-5% by weight The method according to claim 1. 10. 2. The method according to claim 1, wherein the asymmetric reduction is carried out at 20 to 60<0>C. 11. 2. The method according to claim 1, wherein the asymmetric reduction is carried out at a pH of 3 to 9. 12. 2. The method according to claim 1, wherein the asymmetric reduction is carried out for 8 to 96 hours.