KR20010039357A - Preparation of optically active secondary alcohol by enzymatic asymmetric reduction - Google Patents

Abstract

The present invention relates to a method for preparing an optically active secondary alcohol by enzymatic asymmetric reduction using microorganisms.

The present invention provides the preparation of (3S, 1'R) -3- (1'-t-Butyldimethylsilyloxyethyl) -4-acetoxyazetidin-2-one (4-AA) and carbapenem intermediates by using an enzymatic asymmetric reduction reaction using microorganisms. The invention can be used to prepare an optically active secondary alcohol which is useful as an intermediate of the city, and can be easily separated and purified from the optically active secondary alcohol of the desired (2S, 3R).

Description

Preparation method of optically active secondary alcohol by enzymatic asymmetric reduction {Preparation of optically active secondary alcohol by enzymatic asymmetric reduction}

The present invention relates to a method for preparing an optically active secondary alcohol by enzymatic asymmetric reduction using microorganisms.

A number of studies have been published to produce optically active secondary alcohols by asymmetrically reducing carbonyl compounds using enzymes or microorganisms containing the enzymes. Research in this field has been actively studied since the 1980s, and has been developed with the attempt to use biocatalysts such as enzymes and microorganisms in pure organic chemical reactions. Among them, asymmetric methods of carbonyl compounds using baker's yeast have been widely used, and most of them are known to produce (S) body alcohols according to the prelog law. However, the discovery of enzymes or microorganisms that produce optically active secondary alcohols of the (R) form is of great importance in that it can provide a useful building block for the preparation of intermediates in the development of optically active pharmaceuticals. As a result, several reports were published. Among them, Yarrowia lipolytica has been reported to produce (R) alcohols (Tetrahedron, 52 (10), 3547 (1996)) based on linear ketone compounds or compounds such as acetophenone. Bradshaw et al. Also studied the asymmetric reduction of various ketone compounds using alcohol dehydrogenases from Pseudomonas sp. And Lactobacillus kefir (JOC, 57, 1526). 1992, ibid, 57, 1532 (1992)). However, examples of these are not mentioned for compounds having two chiral centers. In addition, examples of asymmetric reduction of a compound having two chiral centers include (2S, 3R) secondary alcohols obtained by using α-alkylated β-keto ester compounds as substrates (Tetrahedron Asymmetry 4, 1259 (1993)). However, the side chain at position 2 is limited to a compound to which an alkyl group of 1 to 2 carbon atoms is linked, and is particularly useful as an intermediate of carbapenem antibiotics (3S, 1'R) -3- (1'-t-Butyldimethylsilyloxyethyl Substrate used for the synthesis of 4-acetoxyazetidin-2-one (4-AA) is a compound in which R ¹ is a methyl group in the following general formula (II) and nitrogen atoms must be included in the side chain, and the general formula (II) In the same case, there is an example (J. Chem. Soc. Perkin Trans. 1, 2247 (1993)) that causes a reduction reaction using a biocatalyst, but only secondary alcohols of (2R, 3S) and (2S, 3S) are produced. Only, examples of generating optically active secondary alcohols of the desired (2S, 3R) bodies have not been reported. No.

The present invention has been made on the basis of the above facts, and an object of the present invention is an optically active secondary alcohol such as general formula (Ia), which is useful as an intermediate of carbapenem antibiotics by enzymatic asymmetric reduction using microorganisms and generally It is to provide a strain for producing an enzyme that can be used for the production of secondary alcohols of the (R) body, such as the general formula (I) and a manufacturing method using the same.

The present invention is a microorganism capable of catalyzing the asymmetric reduction reaction of the compound of the general formula (II) or salts thereof in order to obtain a compound of the general formula (I), wherein the microorganism is a genus of Lactobacillus and Kluyveromyces The asymmetric reduction reaction was carried out by contacting with an enzyme derived from the microorganism or an enzyme having the same structure as the enzyme derived from the microorganism to prepare a compound of the following general formula (I).

Wherein R ¹ is alkyl, substituted alkyl or aryl and R ² is hydrogen, alkyl, aryl or an alkali metal, Z is an ammonium, amide, imide or substituted amine group with substituents as alkyl, aryl and anhydride There is a ride group.

The enzymatic asymmetric reduction of the present invention provides an effective means for obtaining compounds of formula (I) which can be used as intermediates in the preparation of 4-AA and other carbapenem intermediates. In addition, by using the asymmetric reduction method according to the present invention that can be carried out under mild reaction conditions, the optically active secondary alcohol of the desired (2S, 3R) can be easily purified by general chemical synthesis.

Hereinafter, the specific configuration and operation of the present invention will be described in detail.

As used herein, the term "enzymatic method" means a method using an enzyme or microorganism. The term "alkyl" as used herein, alone or as part of another group, refers to an optionally substituted straight or branched chain hydrocarbon containing from 1 to 12, preferably 1 to 6, carbon atoms in the normal chain. For example methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, Nonyl, decyl, undecyl, dodecyl, and various branched chain isomers thereof. Representative substituents that may be substituted for these alkyls include aryl such as halo (particularly chloro), trihalomethyl, alkoxy, unsubstituted aryl (eg phenyl), alkyl-aryl or haloaryl, unsubstituted cycloalkyl and Cycloalkyl, hydroxy or protected hydroxy, carboxyl, alkyloxycarbonyl, alkylamino, dialkylamino such as dimethylamino, alkylcarbonylamino such as acetylamino, amino, arylcarbonylamino, nitro, cya And at least one group selected from furnaces, thiols, or alkylthios. As used herein, the term "aryl" refers to a monocyclic or bicyclic substituted or unsubstituted aromatic group containing 6 to 12 carbon atoms in the ring moiety, for example phenyl, biphenyl, naphthyl, substituted phenyl Substituted biphenyl or substituted naphthyl.

Representative substituents (preferably up to 3) include alkyl such as unsubstituted alkyl, haloalkyl or cycloalkyl, alkoxy such as halogen, unsubstituted alkoxy or haloalkoxy, hydroxy such as hydroxy, phenyl or halophenyl, phenoxy and Such as aryloxy, alkylcarbonyloxy or aroyloxy, allyl, cycloalkyl, alkylamino, dialkylamino, alkylcarbonylamino or arylcarbonylamino, amino, nitro, cyano, alyl, thiol, Alkylcarbonyl, or arylcarbonyl, or methylenedioxy, wherein the methylene group is a lower alkyl group (s) (ie, said alkyl group having 1 to 6 carbon atoms), arylalkenyl group (s) and / or alkylthio group (s) Can be substituted by one or more groups.

The term "halo" or "halogen" as used herein means chlorine, fluorine, bromine or iodine. The term "salt" as used herein refers to acidic and / or basic salts formed with inorganic and / or organic bases. Non-toxic pharmaceutically acceptable salts are preferred. Representative pharmaceutically acceptable salts include salts formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium and ammonia, ethylenediamine, chloroprocaine, diethanolamine, procaine, N Salts formed from amines such as -benzylethylamine.

As used herein, the term "ATCC" refers to the accession number of the American Type Culture Collection, Rockville Parkron Drive 12301, United States 20852, Microbial Depositary.

Compounds of formula (II) for use in the asymmetric reduction of the present invention may be provided by methods known to those skilled in the art.

As a preferable compound of this invention, the compound of general formula (I) which has the following general formula (Ia), the homolog in which one part or all of R <_1> and R <_2> were halogenated, or these alkali metal salts are mentioned.

Wherein R ¹ is alkyl and R ² is the same as defined in general formula (I).

Particularly preferred examples are (2S, 3R) -ethyl 2- (phthalimidomethyl) -3-hydroxy butyrate of the general formula (Ib), 4-AA as an intermediate in the preparation of carbapenems useful as antibiotics, for example. This includes.

Compounds of the general formula (II) having the general formula (IIa) below, homologs in which some or all of R ₁ and R ₂ are halogenated, or alkali metal salts thereof are preferred for use as starting materials.

Wherein R ¹ is alkyl and R ² is the same as defined above in formula (II).

Particularly preferred examples are ethyl 2- (phthalimidomethyl) acetoacetate of the general formula (IIb).

All products according to the invention can be separated and purified by known methods such as filtration and separation of cells and cellular or chemicals by appropriate extraction, filtration, centrifugation, crystallization, thin film or column chromatography, high performance liquid chromatography, and the like. Can be.

The enzymes and microorganisms used in the method of the present invention may be all enzymes and microorganisms having the catalytic function of the reduction reaction as described herein regardless of their origin and purity. Suitable microbial genera as enzyme catalyst sources include the genus Lactobacillus and Kluyveromyces. Representative species suitable for use in the present invention are particularly preferred Kluyveromyces marxianus ATCC 46537, Lactobacillus kefir ATCC 35411.

The method of the present invention in the use of microorganisms can be carried out using any microbial cell material having a reducing catalytic function as described herein. The cells can be used in the form of intact wet cells or dry cells such as lyophilized, spray dried or heat dried cells. The cells can also be used in the form of treated cellular material such as ruptured cells or cell extracts. Alternatively, it can be used in a form fixed to the support by physical adsorption, collection. One or more microorganisms may be used in carrying out the present invention.

The process of the invention can be carried out by washing the microbial material used with water or buffer and then contacting the obtained microbial material with a starting material compound of the general formula (II). The process of the invention may also be carried out in situ fermentation and reaction, ie in the presence of actively growing microorganisms.

The reaction can be carried out under inert (static) conditions or with stirring. Agitation, such as vibration-flask cultivation, or aeration agitation, is preferably used when the starting compound of formula (II) is added to a suspension solution of inactive cells obtained by centrifugation or to an actively growing culture.

Cultivation of microorganisms can be carried out by those skilled in the art, for example by using nutrients and trace elements such as carbon and nitrogen sources. Representative anabolic carbon sources include glucose, glycerol, maltose, dextrin, starch, lactose, sucrose, molasses, soybean oil, cottonseed oil and the like. Representative sources of assimilation nitrogen include soybean meal, peanut flour, cottonseed flour, fish flour, corn steep liquor, peptone, rice bran, meat extract, yeast, yeast extract, sodium nitrate and nitrate Ammonium, ammonium sulfate, etc. are mentioned. Inorganic salts such as sodium chloride, phosphate, calcium carbonate and the like can be added to the culture medium. Small amounts of metal salts or heavy metals may also be added.

The same or different media can be used depending on the various stages of growth of the microorganism. Preferred microbial growth media are the media described in the Examples herein, and these media can be used for the growth of all microorganisms used in the methods of the present invention.

If an enzyme is used, it is preferably an enzyme derived from the microorganisms described above, and may also be prepared synthetically or by other methods. For example, enzymes can be derived from genetically modified host cells. Also contemplated are the use of genetically modified host cells themselves, or otherwise modified cells, capable of producing enzymes having structures of enzymes derived from the above-mentioned microorganisms.

The process of the invention can be carried out in a buffered or aqueous solution. Aqueous is conveniently water, preferably deionized water and a suitable aqueous buffer solution is preferably a phosphate buffer solution. In the asymmetric reduction method of the present invention, the use of a buffer solution medium is preferred.

In addition, the reaction of the present invention may be carried out in an organic solvent or a mixed solvent of an organic solvent and an aqueous solvent. The use of organic, or organic / aqueous solvents may increase the solubility of less water-soluble starting material compounds of general formula (II), such as compounds in which Z is an amine residue. Less water soluble starting materials are dissolved in organic solvents, for example methyl or ethyl alcohol, and this solution can be added to the aqueous solvent for conversion. The liquid forming the organic solvent may be water immiscible but is preferably water miscible. Representative organic solvents include toluene, hexane, benzene, acetone, dimethyl sulfoxide, cyclohexane, xylene, trichlorotrifluoroethane, alkanols (e.g. methyl, ethyl, propyl or isopropyl alcohol, or butanol). Can be mentioned.

The starting material is preferably dissolved in, for example, water, alcohols, acetone and polar organic solvents before addition to buffer or aqueous solutions for the reaction.

The reaction solution preferably contains about 0.5 to about 3 mg of the starting material compound of formula (II) per ml of solution. The pH of the reaction solution is preferably about 6.0 to about 7.5.

In order to carry out the asymmetric reduction reaction of the invention, water or an organic solvent, for example an alkanol such as methyl or ethyl alcohol, can be added. The materials are preferably used in molar excess, preferably in significant molar excess, based on the starting material compound of general formula (II).

When microbial cells are used in the process of the invention, the amount of microbial cells added is preferably in an amount of about 10 to about 1,000 mg per mg of starting material compound of general formula (II). When enzymes are used in the process of the invention, the amount of enzyme added is preferably in an amount of about 1 to 100 units per mg of compound of general formula (II) which is a starting material.

On the other hand, the enzyme titer was measured by using a 0.2mM NADPH, an appropriate amount of the purified purified enzyme solution, 10μl ethanol solution of 0.5M β-ketoester. One unit of this enzyme activity was defined by measuring at 340 nm as the amount of enzyme that oxidizes 1 mmol of NADPH per minute at 23 ° C.

The reaction temperature is preferably maintained at about 20 ° C to 40 ° C, most preferably about 25 ° C to about 34 ° C. The reaction time may be appropriately changed depending on the amount of enzyme produced or used by the microbial cell and its specific activity. Typical reaction times are from about 2 hours 30 minutes to about 72 hours. The reaction time can be reduced by increasing the reaction temperature and / or the amount of enzyme added to the reaction solution.

Hereinafter, the specific configuration and operation of the present invention will be described in detail based on the following examples. The following examples illustrate preferred forms of the invention, but the invention is not limited thereto.

Preparation example. Preparation of Purifying Enzyme

The inoculated strains of Kluyveromyces martianus were inoculated in YM medium for 24 hours at 30 ° C., and then centrifuged (1 ° C., 2100 g × 15 min) from 1 L of the culture medium to obtain 10 g of cells. The cells were washed with 100 ml of 0.1 M phosphate buffer (pH 6.5), suspended again in 100 ml of buffer and disrupted with a cell sonicator (Sonics & Materials). The glass beads used were 0.1-0.2 mm in diameter and a flow rate of 60 ml / min. The lysate was centrifuged to obtain supernatant and used as a crude enzyme source (protein amount 750 mg).

Ammonium sulfate (40-80% (W / V) fraction) was added to the supernatant over 2 hours, followed by centrifugation (4 ° C., 14,000 g × 30 min) to obtain 0.53 g of active protein as a precipitate to obtain partially purified enzyme. Used as a circle (inactive 0.05 unit / mg).

The protein precipitate to be used as a partially purified enzyme source was completely dissolved by adding 100 ml of 10 mM Tris hydrochloric acid buffer solution (pH 8.0) and dialyzed using a cellulose bag for 18 hours to remove salt.

The protein solution thus obtained was passed through a Q-sepharose column (mono-Q, 130 mL) and a phenyl sepharose column (30 mL) to obtain an enzyme active fraction, which was then concentrated by conventional dialysis to be used as a purified enzyme source. (Protein amount = 2.8 mg / ml).

Protein concentrations were measured at UV 270 nm or by Bobford serum albumin as standard (inactive 3.8 units / mg).

The method for obtaining the cells, the partially purified enzyme source and the purified enzyme source used in the following examples is in accordance with the preparation example unless otherwise specified.

Example 1

Frozen stock Kluyveromyces marxianus (ATCC 46537) was thawed and used to inoculate a separate 500 ml flask containing 100 ml LB medium. Each flask was placed on a stirrer at about 300 rpm at 25 ° C. and stirred for 24 hours. After stirring for 24 hours, the culture solution was centrifuged, the supernatant was discarded, and the cell pellet was taken (cell weight 11 g). 100 ml of phosphate buffer (pH 7.5) was added to the obtained cell pellet, and 10 ml of 40 mg / ml ethanol solution of general formula (II) was added thereto, followed by stirring and reduction at 25 ° C. at a speed of about 300 rpm for 24 hours. The reaction proceeded. After stirring, the reaction solution was extracted with 100 ml of ethyl acetate and used for analysis. The ethyl acetate extract was dried over a sufficient amount of anhydrous magnesium sulfate, filtered under reduced pressure, and the filtrate was concentrated under vacuum to give 380 mg of concentrate.

Quantification of the resulting (3R) -ethyl 2- (phthalimidomethyl) -3-hydroxy butyrate was carried out by dissolving the concentrate obtained above at 1% concentration in a high performance liquid chromatography mobile phase solution followed by high performance liquid chromatography. It was. Analysis of the reactants in the net phase using a Chiralcel OD column yielded (2S, 3R)-and (2R, 3R) -ethyl 2-phthalimidomethyl-3-hydroxy butyrate in yields 28% and 57%, respectively. It became.

The concentrate was fractionated by silica gel chromatography using n-hexane / ethyl acetate solvent (40: 1 to 4: 1), and each fraction was concentrated under reduced pressure, and recrystallized in ethyl acetate solution to obtain pure ( 100 mg and 212 mg of 2S, 3R)-and (2R, 3R) bodies were obtained, respectively.

(2S, 3R): mp 44.5-45 ° C., [α] _D ²⁵ + 38 ° (c 0.1, EtOH)

¹ H NMR (CDCl ₃ ): 1.19 (t, 3H), 1.26 (d, 3H), 2.70 (m, 1H), 3.58

(d, 1H), 3.94-4.20 (complex, 5H), 7.71-7.78,

7.84-7.88 (dm, 4 H)

(2R, 3R) sieve: mp 73-74 ° C., [α] _D ²⁵ + 27 ° (c 1, EtOH)

¹ H NMR (CDCl ₃ ): 1.13 (t, 3H), 1.32 (d, 3H), 2.79-2.89 (m, 1H),

3.02 (s, 1H), 3.91-4.20 (complex, 5H),

7.70-7.76, 7.82-7.91 (dm, 4H)

Column: Chiralcel OD (0.46 × 25 cm); Column temperature: 40 ° C .; Mobile phase: n-hexane / ethanol = 30: 1; Flow rate: 1 ml / min, detection: 220 nm; Elution time (Rt): (2S, 3R) sieve -20.4 minutes, (2R, 3R) sieve-26.1 minutes

Example 2

Cells were disrupted by sonicating 1 g of Kluyveromyces marcianus strain cultured in Example 1 in 10 ml of phosphate buffer (pH 6.0). The crushed cells were centrifuged at 2000 g for 10 minutes to obtain a supernatant portion, which was then used as a crude enzyme source. The protein amount of the solution used as the crude enzyme source was 5 mg / ml.

2 ml of this crude enzyme solution and 200 µl of a 2-% ethanol solution of ethyl 2- (phthalimidomethyl) acetoacetate were added thereto, followed by stirring at 30 ° C for 24 hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate in the same manner as in Example 1, and the extract obtained by distillation of the solvent was analyzed under the conditions of Example 1, and the yields were 32% and 53% (2S, 3R)-and (2R, 3R) -ethyl 2-phthalimidomethyl-3-hydroxy butyrate was produced.

Example 3

500 mg of the cell pellet of the strain obtained in Example 1 (wet state) was suspended in 50 ml of phosphate buffer solution (pH 6.0), and 50 mg of ethyl 2- (benzamidomethyl) acetoacetate was dissolved in a small amount of acetone. After the addition, the mixture was stirred at 28 ° C. for 24 hours to carry out a reduction reaction. After completion of the reaction, the reaction solution was extracted with ethyl acetate in the same manner as in Example 1, the solvent was distilled off under reduced pressure, and the extract was analyzed under the conditions of Example 1, and the yield was 31% and 58%, respectively (2S, 3R)-and (2R, 3R) -ethyl 2-benzamidomethyl-3-hydroxy butyrate were produced.

Example 4

50 mg of glucose, 1 mg of NADPH, and 100 mg of ethyl 2- (phthalimidomethyl) acetoacetate were added to 50 ml of the enzyme solution obtained as in Example 2, partially purified by ammonium sulfate precipitation. Was adjusted with sodium hydroxide (pH 6.0) and stirred for 20 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate in the same manner as in Example 1, the solvent was distilled off under reduced pressure, and the extract was analyzed under the conditions of Example 1. The yield was 40% and 51%, respectively (2S, 3R). )-And (2R, 3R) -ethyl 2-phthalimidomethyl-3-hydroxy butyrate were produced.

Example 5

As a result of protein purification using a Q-sepharose column and a phenyl sepharose column, the enzyme solution obtained by partially purifying the crude enzyme source obtained in Example 2 by ammonium sulfate precipitation was analyzed. An active fraction was obtained. This enzyme active fraction was dialyzed as in Preparation Example to remove salts, and asymmetric reduction was carried out with the purified enzyme.

2.5 mg of 0.1 M phosphate buffer (pH 6.5) containing 10 units of purified enzyme, 25 mg of ethyl 2- (phthalimidomethyl) acetoacetate, and 0.25 mg of NADPH was stirred at 30 ° C. for 24 hours. After the completion of the reaction, the reaction solution was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered under reduced pressure, and the extract obtained by distillation under reduced pressure and then concentrated under the conditions of Example 1 was analyzed to yield 42% and 38% ( 2S, 3R)-and (2R, 3R) -ethyl 2-phthalimidomethyl-3-hydroxy butyrate were obtained.

Example 6

Cryoprotected Kluyveromyces marxianus (ATCC 46537) was thawed and inoculated into a separate 50 ml flask containing 10 ml of YM medium (cell concentration; about 100 mg (wet) / ml, inoculation) 100 μl) each flask was stirred at 30 ° C. on a stirrer of about 300 rpm for 24 hours. After stirring for 24 hours, the culture was adjusted to pH 6.0 with 1N sodium hydroxide, and then 500 mg of ethyl 2- (phthalimidomethyl) acetoacetate was added to the culture solution, followed by reaction for 48 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered under reduced pressure, and the extract obtained by distillation under reduced pressure was analyzed under the conditions of Example 1, and the yield was 27% and 41%, respectively. (2S, 3R)-and (2R, 3R) -ethyl 2-phthalimidomethyl-3-hydroxy butyrate were obtained.

Example 7

2 g of the cell pellet of the strain obtained in Example 6 (wet state) was suspended in 10 ml of phosphate buffer (pH 7.0), and 50 mg of ethyl 2- (benzamidomethyl) acetoacetate was added in a small amount of dimethylformamide ( DMF) was added and dissolved, followed by stirring at 30 ° C. for 72 hours to effect reduction. After completion of the reaction, the reaction solution was extracted with toluene, dried over anhydrous magnesium sulfate, filtered under reduced pressure, and the extract obtained by distillation under reduced pressure and concentrated under the conditions of Example 1 was analyzed. The yield was 31% and 57%, respectively (2S , 3R)-and (2R, 3R) -ethyl 2-benzamidomethyl-3-hydroxy butyrate were produced.

Example 8

Asymmetric reduction was carried out with the purified enzyme obtained in the preparation example. 5 ml of 0.1 M phosphate buffer (pH 6.0) containing 20 units of purified enzyme, 50 mg of ethyl 2- (phthalimidomethyl) acetoacetate, and 0.5 mg of NADPH was stirred at 25 ° C. for 24 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate, distilled under reduced pressure, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting solution was analyzed under the conditions of Example 1, and the yield was 75% (2S, 3R). -Ethyl 2-phthalimidomethyl-3-hydroxy butyrate was obtained.

The optical isomer yield for each embodiment of the present invention is as shown in Table 1 below.

Optical isomer yield by Example Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Remarks (Reduction method by NaBH ₄ ) (2S, 3R) 28 32 31 40 42 27 31 75 25% (2R, 3S) n.d n.d n.d n.d n.d n.d n.d n.d 25% (2R, 3R) 57 53 58 51 38 41 57 15 25% (2S, 3S) 8 7 5 5 n.d 15 8 n.d 25%

As shown in Table 1, when using the enzymatic asymmetric reduction reaction using the microorganism of the present invention (2R, 3S) body is not produced, the second optical activity of the target (2S, 3R) body compared to the chemical synthesis The alcohol can be easily separated and purified by the method of column chromatography and recrystallization.

The present invention provides the preparation of (3S, 1'R) -3- (1'-t-Butyldimethylsilyloxyethyl) -4-acetoxyazetidin-2-one (4-AA) and carbapenem intermediates by using an enzymatic asymmetric reduction reaction using microorganisms. The invention can be used to prepare an optically active secondary alcohol which is useful as an intermediate of the city, and can be easily separated and purified from the optically active secondary alcohol of the desired (2S, 3R).

Claims (8)

A microorganism selected from the group of Kluyveromyces capable of catalyzing the asymmetric reduction reaction of the general formula (II) or a salt thereof using the compound of the general formula (II) as a substrate, a cell crush obtained by pulverizing the microorganism, Or contacting an enzyme derived from the microorganism to carry out the asymmetric reduction reaction, to prepare a compound of formula (I) or a salt thereof. Wherein R ¹ is alkyl or aryl, R ² is alkyl, aryl and hydrogen, an alkali metal, Z is an ammonium, amide, imide or substituted amine group with substituents as alkyl, aryl and anhydride groups. The method of claim 1, wherein the compound of formula (lb) is prepared. The method of claim 1, wherein the microorganism is Kluyveromyces marxianus ATCC 46537, wherein the compound of formula (I) or a salt thereof is prepared. 4. The process according to claim 3, wherein the compound of formula (II) is carried out in an aqueous solution or a buffer solution in the step of contacting the compound of formula (II) with a microorganism or an enzyme. 5. A process according to claim 4, wherein the pH of the aqueous or buffered solution is between 6.0 and 7.0. The process according to claim 4, wherein the reaction temperature is 20 ° C to 35 ° C. The method of preparing a compound of formula (I) or a salt thereof according to claim 4, wherein the buffer solution is a phosphate buffer solution. An enzyme derived from a microorganism selected from Kluyveromyces according to claim 1, which can catalyze the asymmetric reduction reaction of Formula (II) or its salt using the compound of Formula (II) as a substrate. Contacting to perform the asymmetric reduction reaction.