JP2007029089A - Method for producing optically active hydroxyamino acid derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for easily producing an optically active 3-hydroxyamino acid derivative in higher optical purity at a low cost. <P>SOLUTION: The method for producing an optically active 3-hydroxyamino acid derivative comprises the formation of a 3-oxoamino acid derivative of formula (2) (R<SP>1</SP>is an alkyl, an aryl or a heterocyclic group; R<SP>2</SP>is a ≥4C alkyl, a ≥4C alkoxy, an aryl or a heterocyclic group; R<SP>3</SP>is an alkyl or an aryl; the alkyl, alkoxy, aryl or heterocyclic group in R<SP>1</SP>to R<SP>3</SP>may have substituents) with a microbial cell and/or treated cell of a microorganism capable of stereospecifically reducing the β-carbonyl group of the 3-oxoamino acid derivative. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention uses a microorganism containing carbonyl reductase, a preparation of the microorganism, or a culture solution obtained by culturing the microorganism, and three-dimensionally recognizing the amino group at the 2-position of a 3-oxoamino acid derivative. The present invention relates to a method for producing an optically active 3-hydroxyamino acid derivative which is an industrially useful compound as an intermediate material for pharmaceuticals, agricultural chemicals and the like by stereoselectively reducing the carbonyl group.

  3-Hydroxyamino acid derivatives are industrially useful compounds as intermediate materials for pharmaceuticals, agricultural chemicals and the like. For example, (2R, 3R) -3-cyclohexyl-3-hydroxyamino acid derivatives are used as pharmaceutical intermediates. As a method for producing this, an optically active epoxide is formed to produce 3-cyclohexyl-3. It has been reported that a method leading to a hydroxyamino acid derivative (Patent Document 1) or a 3-cyclohexyl-3-oxoamino acid derivative reduced by an asymmetric catalyst (Non-Patent Document 1), The former has a high unit cost of raw material compounds and has industrial problems, and the latter requires a special reaction apparatus for high-pressure hydrogenation, and the optical purity is not satisfactory, making industrialization difficult.

  Carbonyl reductase is an enzyme that catalyzes a reaction that reduces the carbon-oxygen double bond of a carbonyl group, and its presence has been reported in various microorganisms such as yeast, bacteria, and mold. For example, production of a threonine derivative by reduction of an N-acetylated 3-oxoamino acid derivative as in the following reaction formula is described (Patent Documents 2, 3, and 4).

  However, in this method, selective synthesis of diastereomers and optical isomers has not been performed.

JP 2003-55358 A JP-A-2-60592 Japanese Patent Laid-Open No. 2-60593 Japanese Patent Laid-Open No. 2-60594 Angew Chem Int Ed (2004) vol43 pp882-884

  An object of the present invention is to provide a novel method for producing an optically active 3-hydroxyamino acid derivative with higher optical purity at a low cost and in a simple manner.

  In order to solve the above-mentioned problems, the present inventors have intensively studied a method for producing an optically active 3-hydroxyamino acid derivative, and as a result, a microbial cell and / or a processed product of the microbial cell is used as a raw material. It has been found that it catalyzes an asymmetric reduction reaction of an amino acid derivative. Furthermore, a transformed cell into which a gene encoding a protein that catalyzes the asymmetric reduction reaction is prepared, and the cell, a preparation of the cell, or a culture solution obtained by culturing the cell is used as a raw material. It was found that the target optically active 3-hydroxyamino acid derivative can be obtained with high optical purity and high concentration by acting on the resulting 3-oxoamino acid derivative. The present invention has been completed based on these findings.

（式中、Ｒ¹はアルキル基、アリール基、又はヘテロ環基を示し、Ｒ²は炭素数４以上のアルキル基、炭素数４以上のアルコキシ基、アリール基、又はヘテロ環基を示し、Ｒ³はアルキル基またはアリール基を示し、Ｒ¹からＲ³におけるアルキル基、アルコキシ基、アリール基及びヘテロ環基は置換されていてもよい）
で表される３−オキソアミノ酸誘導体のβ位のカルボニル基を立体特異的に還元する能力を有する微生物の菌体及び／または該菌体処理物を、前記一般式（１）で表される３−オキソアミノ酸誘導体に作用させて、下記一般式（２）

（式中、Ｒ¹、Ｒ²、及びＲ³は前記と同義である）
で表される光学活性３−ヒドロキシアミノ酸誘導体を生成させることを特徴とする、光学活性３−ヒドロキシアミノ酸誘導体の製造方法。 That is, according to the present invention, the following inventions are provided.
(1) The following general formula (1)

(In the formula, R ¹ represents an alkyl group, an aryl group, or a heterocyclic group; R ² represents an alkyl group having 4 or more carbon atoms, an alkoxy group having 4 or more carbon atoms, an aryl group, or a heterocyclic group; ³ represents an alkyl group or an aryl group, and the alkyl group, alkoxy group, aryl group and heterocyclic group in R ¹ to R ³ may be substituted.
A microbial cell and / or a treated product thereof having the ability to stereospecifically reduce the carbonyl group at the β-position of the 3-oxoamino acid derivative represented by the formula (1) is represented by 3 -By reacting with an oxo amino acid derivative, the following general formula (2)

(Wherein R ¹ , R ² and R ³ are as defined above)
An optically active 3-hydroxyamino acid derivative represented by the formula:

(2) The microorganisms include the genus Absidia, the genus Acremonium, the genus Agrobacterium, the genus Alcaligenes, the genus Arthrobacter, the genus Bacillus, the Brevibacterium ( Brevibacterium genus, Brevundimonas genus, Cellulomonas genus, Citrobacter genus, Comamonas genus, Delftia genus, Mycobacterium genus, Nocardioides (Nocardioides) ) Genus, Ochrobactrum genus, Paracoccus genus, Pseudomonas genus, Rhizobium genus, Rhodococccus genus, Serratia genus, Sinorhizobces genus ) Genus, valid The method for producing an optically active 3-hydroxyamino acid derivative according to (1), which is a microorganism belonging to the genus Variovorax or the genus Exiguobacterium.

(3) The method for producing an optically active 3-hydroxyamino acid derivative according to (1), wherein the microorganism is a transformant in which any one of the following DNAs (A) to (F) is expressed:
(A) DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A 3-oxoamino acid derivative represented by the general formula (2) having an amino acid sequence having 50% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and represented by the general formula (1) A DNA encoding a protein having an enzymatic activity to be converted into an optically active 3-hydroxyamino acid derivative.
(C) a 3-oxo amino acid derivative consisting of an amino acid sequence represented by general formula (1) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 2 A DNA encoding a protein having an enzyme activity which is converted into an optically active 3-hydroxyamino acid derivative represented by the general formula (2).
(D) DNA having the base sequence set forth in SEQ ID NO: 1.
(E) A 3-oxoamino acid derivative represented by the general formula (2) is hybridized with a DNA comprising the base sequence set forth in SEQ ID NO: 1 or a complementary sequence thereof under stringent conditions and represented by the general formula (1) A DNA encoding a protein having an enzymatic activity to be converted into the optically active 3-hydroxyamino acid derivative represented.
(F) a 3-oxo amino acid comprising a base sequence in which one or several bases are substituted, deleted or added in the base sequence set forth in SEQ ID NO: 1 and its complementary strand, and represented by the general formula (1) A DNA encoding a protein having an enzyme activity for converting a derivative into an optically active 3-hydroxyamino acid derivative represented by the general formula (2).

(4) The method for producing an optically active 3-hydroxyamino acid derivative according to (3), wherein the transformant is transformed by integrating the DNA on a chromosome.
(5) The method for producing an optically active 3-hydroxyamino acid derivative according to (3), wherein the transformant is a transformant transformed with a vector containing the DNA.

(6) The optically active 3-hydroxyamino acid according to any one of (1) to (5), wherein R ² is an aryl group in the compounds of the general formulas (1) and (2) A method for producing a derivative.
(7) The method for producing an optically active 3-hydroxyamino acid derivative according to any one of (1) to (6), wherein the optical purity of the general formula (2) is 98% ee.
(8) The absolute configuration of the general formula (2) is 2R or 3R as represented by the following general formula (3), according to any one of (1) to (7) A method for producing an optically active 3-hydroxyamino acid derivative.

  The present invention makes it possible to obtain an optically active 3-hydroxyamino acid derivative, which is an industrially useful compound as an intermediate material for pharmaceuticals, agricultural chemicals, and the like, with high optical purity.

  The present invention will be described in detail below, but the present invention is not limited to the following description.

(3-oxoamino acid derivative, optically active 3-hydroxyamino acid derivative)
In the present invention, the following general formula (1), in which a microbial cell and / or a treated product thereof is a reaction substrate:

(In the formula, R ¹ represents an alkyl group, an aryl group, or a heterocyclic group; R ² represents an alkyl group having 4 or more carbon atoms, an alkoxy group having 4 or more carbon atoms, an aryl group, or a heterocyclic group; ³ represents an alkyl group or an aryl group, and the alkyl group, alkoxy group, aryl group and heterocyclic group in R ¹ to R ³ may be substituted.
In the following general formula (2):

(Wherein R ¹ , R ² and R ³ are as defined above)
To produce an optically active 3-hydroxyamino acid derivative represented by:

R ¹ represents an alkyl group, an aryl group, or a heterocyclic ring. Here, examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tertiary butyl, n-pentyl, isopentyl, and neopentyl. Group, tertiary pentyl group, isoamyl group, n-hexyl group, n-decyl group, etc., 1 to 30 carbon atoms, preferably 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms straight chain or branched chain Or a cyclic alkyl group.

  As an aryl group, C6-C20 aryl groups, such as a phenyl group, a naphthyl group, a benzoyl group, a thiophenyl group, can be mentioned, for example.

  Heterocyclic groups include 3- to 10-membered saturated or unsaturated heterocyclic groups containing at least one of N, O or S atoms, which may be monocyclic, A condensed ring may be formed with other rings. The heterocyclic group is preferably a 5- to 6-membered unsaturated heterocyclic group optionally having a condensed ring, more preferably a 5- to 6-membered aromatic heterocyclic ring optionally having a condensed ring. It is a group. Specific examples of the heterocyclic ring in the heterocyclic group include, for example, pyrrolidine, piperidine, piperazine, morpholine, thiophene, furan, pyrrole, imidazole, pyrazole, pyridine, pyrimidine, pyrazine, pyridazine, triazole, triazine, indole, indazole, purine, Thiadiazole, oxadiazole, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, acridine, phenanthroline, phenazine, tetrazole, thiazole, oxazole, benzimidazole, benzoxazole, benzthiazole, benzselenazole, indolenine, tetrazain Den and the like.

  The above alkyl group, aryl group and heterocyclic group may be substituted. The substituent that the alkyl group, aryl group, and heterocyclic group may have is not particularly limited as long as it does not adversely affect the reaction. Specifically, the alkyl group, aryl group, alkoxy group , Halogen group, cyano group, amino group, nitro group, hydroxyl group and the like.

  Specific examples of the substituted alkyl group include benzyl group, phenethyl group, trifluoromethyl group, cyanomethyl group, aminomethyl group, hydroxymethyl group, nitromethyl group, methoxymethyl group and the like. Specific examples of the substituted aryl group include a tolyl group, a mesityl group, a chlorophenyl group, an aminophenyl group, a hydroxyphenyl group, a nitrophenyl group, and a methoxyphenyl group.

Moreover, the carbon atom in the said alkyl group and aryl group may be substituted by hetero atoms, such as a nitrogen atom, a sulfur atom, and an oxygen atom. For example, the alkyl _{group, CH 3 (CH 2) n} S (CH 2) n -, CH 3 (CH 2) n NH (CH 2) n -, CH 3 (CH 2) n O (CH 2) n (Wherein n represents an integer of 1 or more, preferably 1 to 10, more preferably 1 to 5).

R ¹ is preferably an alkyl group having 1 to 30 carbon atoms, an aryl group, or a hetero group. More preferably, it is a linear, cyclic or branched alkyl group having 1 to 20 carbon atoms, phenyl group, chlorophenyl group, naphthyl group, thiophenyl group, more preferably methyl group, ethyl group, n-propyl group, isopropyl group. , A cyclohexyl group, an n-decyl group, a phenyl group, a chlorophenyl group, a naphthyl group, or a thiophene group.

R ² represents an alkyl group having 4 or more carbon atoms, an alkoxy group having 4 or more carbon atoms, an aryl group, or a heterocyclic group. Examples of the alkyl group having 4 or more carbon atoms include those having 4 or more carbon atoms among the alkyl groups mentioned in the above R ¹ . Examples of the alkoxy group having 4 or more carbon atoms include an alkoxy group obtained by substituting one hydrogen atom of the alkyl group having 4 or more carbon atoms with a hydroxyl group. The alkoxy group has 4 to 20 carbon atoms, preferably 4 to 4 carbon atoms. 10, more preferably a linear, branched, or cyclic alkoxy group having 4 to 6 carbon atoms, specifically, t-butoxy group, —O—CH ₂ —C ₆ H _{5 and the} like. Can be mentioned. Examples of the aryl group and heterocyclic group represented by R ² include the aryl group and heterocyclic group mentioned in the above R ¹ .

The alkyl group having 4 or more carbon atoms, the alkoxy group having 4 or more carbon atoms, the aryl group, or the heterocyclic group represented by R ² may be substituted. The substituent that the alkyl group having 4 or more carbon atoms, the alkoxy group having 4 or more carbon atoms, the aryl group, or the heterocyclic group may have is not particularly limited as long as it does not adversely affect the reaction. Specific examples include an alkyl group, an aryl group, an alkoxy group, a halogen group, a cyano group, an amino group, a nitro group, and a hydroxyl group.

R ² is preferably a phenyl group or a tert-butyl group.

R ³ represents an alkyl group or an aryl group. Examples of the alkyl group and aryl group represented by R ³ include those exemplified above for R ¹ .

The alkyl group or aryl group represented by R ³ may be substituted. The substituent that the alkyl group or aryl group may have is not particularly limited as long as it does not adversely affect the reaction. Specifically, the alkyl group, aryl group, alkoxy group, halogen group, Examples include a cyano group, an amino group, a nitro group, and a hydroxyl group.

R ³ is preferably a linear or branched alkyl group having 1 to 6 carbon atoms or a benzyl group. Particularly preferred as R ³ is a methyl group, an ethyl group, an n-propyl group or an isopropyl group.

Specific compounds of the general formula (1) can include the following compounds, but are not limited thereto.
Methyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate,
Ethyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate,
N-propyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate,
Isopropyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate,
Phenyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate,
Benzyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate,
Methyl 2-benzoylamino-4-methyl-3-oxopentanoate,
Ethyl 2-benzoylamino-4-methyl-3-oxopentanoate,
N-propyl 2-benzoylamino-4-methyl-3-oxopentanoate,
Isopropyl 2-benzoylamino-4-methyl-3-oxopentanoate,
Benzyl 2-benzoylamino-4-methyl-3-oxopentanoate,
Isopropyl 2-benzoylamino-3-oxobutanoate,
Isopropyl 2-benzoylamino-3-oxopentanoate,
Isopropyl 2-benzoylamino-3-oxohexanoate,
Isopropyl 2-benzoylamino-3-oxotridecanoate,
Isopropyl 2-benzoylamino-4,4-dimethyl-3-oxopentanoate,
Isopropyl 2-benzoylamino-3-phenyl-3-oxopropionate,
Isopropyl 2-benzoylamino-3- (4-chlorophenyl) -3-oxopropionate,
Isopropyl 2-benzoylamino-3- (thiophen-2-yl) -3-oxopropionate,
2- (thiophene-2-carboxamido) -3- (thiophen-2-yl) -3-oxopropionate isopropyl,
Isopropyl 2-benzoylamino-3- (naphthalen-2-yl) -3-oxopropionate,
Isopropyl 3-oxo-3-phenyl-2-pivaloylaminopropionate,
Isopropyl 3-oxo-2-pivaloylaminobutanoate,
Isopropyl 3-oxo-2-pivaloylaminopentanoate,
Isopropyl 3-oxo-2-pivaloylaminohexanoate,
Isopropyl 4,4-dimethyl-3-oxo-2-pivaloylaminopentanoate,
Isopropyl 3- (4-chlorophenyl) -3-oxo-2-pivaloylaminopropionate,
Isopropyl 3-oxo-2-pivaloylamino-3- (thiophen-2-yl) propionate,
Ethyl 2-t-butoxycarbonylamino-3-cyclohexyl-3-oxopropionate,

  The specific compound of the general formula (2) is an optically active 3-hydroxyamino acid derivative obtained by stereospecific reduction of the carbonyl group at the β-position of the specific compound of the general formula (1).

  Since the compound of the general formula (2) has asymmetric carbons at the 2-position and the 3-position, it has four isomers: (2R, 3R), (2R, 3S), (2S, 3R) , (2S, 3S) isomers, but in the production using the microbial cells and / or the treated microbial cells in the present invention, one of them is preferentially produced. In addition, it is preferable that it is a (2R, 3R) body as a compound which has a demand as an intermediate body of a medicine. The (2R, 3R) and (2S, 3S) isomers are referred to as anti (anti) (2R, 3S) and (2S, 3R) isomers.

(Cells of microorganisms used in the present invention and / or treated cells thereof)
The microorganism used in the present invention and / or the treated product thereof acts on the 3-oxoamino acid derivative represented by the general formula (1) to stereospecifically reduce the carbonyl group at the β-position. Any compound may be used as long as it generates an optically active 3-hydroxyamino acid derivative represented by the general formula (2).

  As a method for confirming the production of the optically active 3-hydroxyamino acid derivative, usually, the target microorganism and its treated cell are allowed to act on an aqueous solution containing the 3-oxoamino acid derivative of the general formula (1), and the reaction solution is used. The optically active 3-hydroxyamino acid derivative represented by the general formula (2), which is a substrate and a product, may be qualitatively and quantified using thin layer chromatography, gas chromatography, or liquid chromatography.

  The optically active 3-hydroxyamino acid derivative represented by the general formula (2) in the present invention preferably has an optical purity of 96% ee or higher, more preferably 98% ee or higher, more preferably 99% ee or higher. . Low optical purity necessitates further purification for use as a pharmaceutical intermediate. In general, separation and purification of optical isomers is not an easy task such as using a special column or crystallization with another optically active substance. It is important to provide a method for manufacturing without purification.

  The microbial cells and / or the processed microbial cells that can be used in the present invention are subjected to the above production confirmation method, and when the reaction is carried out for several minutes or more, preferably one day or more, a decrease in the raw material substrate can be confirmed. More specifically, when the reaction is carried out for several hours or more, preferably one day or more, the raw material substrate is reduced by 1% or more, preferably 5% or more.

  Examples of the microorganism that acts on the 3-oxoamino acid derivative represented by the general formula (1) to produce the optically active 3-hydroxyamino acid derivative represented by the general formula (2) include microorganisms belonging to the genus Absidia, Microorganisms belonging to the genus Acremonium, microorganisms belonging to the genus Agrobacterium, microorganisms belonging to the genus Alcaligenes, microorganisms belonging to the genus Arthrobacter, microorganisms belonging to the genus Bacillus, Microorganisms belonging to the genus Brevibacterium, microorganisms belonging to the genus Brevundimonas, microorganisms belonging to the genus Cellulomonas, microorganisms belonging to the genus Citrobacter, microorganisms belonging to the genus Comamonas, Mycobacte, a microorganism belonging to the genus Delftia Microorganisms belonging to the genus Mycobacterium, microorganisms belonging to the genus Nocardioides, microorganisms belonging to the genus Ochrobactrum, microorganisms belonging to the genus Paracoccus, microorganisms belonging to the genus Pseudomonas, Rhizobium ) Microorganisms belonging to the genus, microorganisms belonging to the genus Rhodococccus, microorganisms belonging to the genus Serratia, microorganisms belonging to the genus Sinorhizobium, microorganisms belonging to the genus Streptomyces (Variovorax) Examples include microorganisms belonging to the genus, microorganisms belonging to the genus Exibacterium, and recombinant microorganisms having a gene encoding carbonyl reductase.

Specific examples of the microorganism belonging to the genus Absidia include Absidia lichthemi OUT1010.
Specific examples of microorganisms belonging to the genus Acremonium include Acremonium chrysogenum ATCC14615.
Examples of microorganisms belonging to the genus Agrobacterium include Agrobacterium rhizogenes MAFF03-01724, MAFF07-20001, MAFF301724, IAM13570 Agrobacterium tumefaciens MAFF301001, MAFF301222, IAM12048, IAM13129. It is done.

Specific examples of microorganisms belonging to the genus Alcaligenes include Alcaligenes faecalis JCM10169.
As microorganisms belonging to the genus Arthrobacter, specifically, Arthrobacter crystallopoietes JCM2522 Arthrobacter sulfureus JCM1338 Arthrobacter sp JCM1336, IFO14 Can be mentioned.
Specific examples of microorganisms belonging to the genus Bacillus include Bacillus lentus IAM11055.

As microorganisms belonging to the genus Brevibacterium, specifically, Brevibacterium sulfureum
(JCM1485).
Specific examples of microorganisms belonging to the genus Brevundimonas include Brevundimonas diminuta IFO12697.
Specific examples of microorganisms belonging to the genus Cellulomonas include Cellulomonas biazotea ATCC486 Cellulomonas flavigena ATCC482.

Specific examples of the microorganism belonging to the genus Citrobacter include Citrobacter youngae ATCC11102.
Specific examples of microorganisms belonging to the genus Comamonas include Comamonas terrigena IFO13299, IFO12685, and NBRC14918.
Specific examples of microorganisms belonging to the genus Delftia include Delftia acidovorans JCM6218.

Specific examples of the microorganism belonging to the genus Mycobacterium include Mycobacterium sp. NCIMB11678 and NCIMB11677.
Specific examples of microorganisms belonging to the genus Nocardioides include Nocardioides simplex IAM1398.
Specific examples of microorganisms belonging to the genus Ochrobactrum include Ochrobactrum anthropi ATCC49237 Ochrobactrum sp IFO12950.

Specific examples of microorganisms belonging to the genus Paracoccus include Paracoccus denitrificans IFO12442 and IFO13301.
Specific examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas alkaligenes JCM5967 (Pseudomonas alcaligenes) Pseudomonas putida NCIMB11924 Pseudomonas taetrolens IFO3460, Pseudomonas sp.
Specific examples of microorganisms belonging to the genus Rhizobium include Rhizobium haukuii MAFF 210250.

Specific examples of the microorganism belonging to the genus Rhodococccus include Rhodococccus rhodochrous ATCC21291.
Specific examples of microorganisms belonging to the genus Serratia include Serratia marcescens IFO3046 Serratia plymuthica NCIMB8285.
Specific examples of microorganisms belonging to the genus Sinorhizobium include Sinorhizobium meliloti IAM12611 and MAFF 303042.

Examples of microorganisms belonging to the genus Streptomyces include Streptomyces achromogenes subsp. Achromogenes IFO12735.
Specific examples of microorganisms belonging to the genus Variodorax include Variodorax paradoxus DSM30034 and DSM30162.
Specific examples of microorganisms belonging to the genus Exiguobacterium include Exiguobacterium sp MCI4322 strain (FERM-P-19239 strain). Available from the Deposit Center.

These microorganisms can be obtained from each research institution.
IAM Culture Collection (http://www.iam.u-tokyo.ac.jp/misist/ColleBOX/jp/IAMcollection.html)
JCM (RIKEN BioResource Center) (http://www.jcm.riken.jp/JCM/JCM_Home_J.html)
NBRC (including former IFO) Independent Administrative Institution Biotechnology Headquarters Biotechnology Resource Division (NBRC) Genetic Resource Conservation Section (http://www.nbrc.nite.go.jp/gene.html)
IAM (Cellular Information Research Center IAM Culture Collection, University of Tokyo) (http://www.iam.u-tokyo.ac.jp/misyst/ColleBOX/jp/IAMcollection.html)
NCIMB (Japan Agency, NMB Japan) (http://www.ncimb.co.jp/index.html)
ATCC (American Type Culture Collection (http://www.atcc.org/Home.cfm)) (Japanese agency: Sumisho Pharma International ATCC Business Group)
(Http://www.summitpharma.co.jp/japanese/index_j.html)
DSM (http://www.dsmz.de/species/strains.htm)
CBS (Centraalbureau voor Schimmelcultures Uppsalalaan) http://www.cbs.knaw.nl/databases/index.htm
NCYC (National Collection of Yeast Cultures) http://www.ifrn.bbsrc.ac.uk/NCYC/)
OUT: Department of Fermentation Technology, Faculty of Engineering, Osaka University, Osaka, Japan
MAFF: Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan
(http://www.gene.affrc.go.jp/micro/index_j.html)
NRRL: Agricultural Research Service Culture Collection, National Center for Agricultural Utilization
FERM-P; National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center

  A carbonyl reductase generally refers to an enzyme that catalyzes the reduction reaction of a carbon-oxygen double bond of carbonyl. In the present invention, the carbon-oxygen double bond of a 3-oxoamino acid derivative is asymmetrically reduced. An enzyme having an activity to produce an optically active 3-hydroxyamino acid derivative.

  Examples of the carbonyl reductase that can be used in the production method of the present invention include those having the amino acid sequence set forth in SEQ ID NO: 2. These are carbonyl reductases derived from Exigubacterium.

  Moreover, in this invention, it is these homologs, Comprising: The enzyme activity which converts the 3-oxo amino acid derivative represented by General formula (1) into the optically active 3-hydroxy amino acid derivative represented by General formula (2) You may use what has. Examples of homologs include those having an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence set forth in SEQ ID NO: 2 within a range that does not impair the activity. Here, the term “several” specifically means 20 or less, preferably 10 or less, more preferably 5 or less.

  The homologue may be a protein having a homology of 35% or more, preferably 50% or more, more preferably 80% or more with the amino acid sequence shown in SEQ ID NO: 2. Incidentally, the homology search of the above protein can be performed using programs such as FASTA and BLAST, for example, for GenBank and DNA Database of JAPAN (DDBJ). Using the amino acid sequence described in SEQ ID NO: 2 as a homology search by BLAST program for GenBank, the sequence of SEQ ID NO: 2 is 57% of oxidoreductase (Accession No. AAK76748) of Clostridium acetobutylicum. Showed homology.

  The carbonyl reductase used in the production method of the present invention encodes carbonyl reductase from any microorganism having carbonyl reductase activity using a probe prepared based on the base sequence of a gene encoding part or all of carbonyl reductase. After DNA is isolated, it can be obtained by expressing the DNA in a host such as E. coli. Moreover, it can also obtain by refine | purifying from the microorganisms which have carbonyl reductase activity, for example, the microbial cell of the bacterium of the genus Exigobacteria. As a method for obtaining carbonyl reductase from bacterial cells, for example, Eur. J. et al. Biochem. Vol. 97, p103 (1979).

  Examples of microorganisms belonging to the genus Exiguobacterium include carbonyl reductase that can be suitably used in the present invention, for example, Exibacterium sp. FERM-P-19239 (MCI4322). It is available from the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary.

  In the production method of the present invention, a 3-oxoamino acid derivative may be reacted with a purified enzyme of carbonyl reductase, but a cell containing carbonyl reductase, a preparation of the same cell, or a culture obtained by culturing the same cell The solution may be reacted with a 3-oxoamino acid derivative to produce a 3-hydroxyamino acid derivative. The cell containing carbonyl reductase is preferably a cell transformed with DNA encoding carbonyl reductase.

  In this case, the DNA encoding carbonyl reductase includes DNA encoding carbonyl reductase having the amino acid sequence of SEQ ID NO: 2. In addition, a 3-oxo amino acid derivative having an amino acid sequence having 50% or more homology with the amino acid sequence of SEQ ID NO: 2 and represented by the general formula (1) is represented by the optical formula represented by the general formula (2). It may be a DNA encoding a protein having an enzymatic activity to be converted into an active 3-hydroxyamino acid derivative. Specific examples include DNA having the base sequence of SEQ ID NO: 1. In the production method of the present invention, a DNA homologue having the base sequence described in SEQ ID NO: 1 and encoding a protein having carbonyl reductase activity may be used.

  Here, as long as it encodes a protein having carbonyl reductase activity, a homolog is a base sequence in which one or several bases are deleted, substituted, or added to the base sequence described in SEQ ID NO: 1 and its complementary strand DNA consisting of Here, the term “several” means specifically 60 or less, preferably 30 or less, and more preferably 10 or less.

  In addition, the homologue may be a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 1 or 3 or its complementary strand under stringent conditions and encodes a protein having a carbonyl reductase activity. Here, “DNA that hybridizes under stringent conditions” refers to a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like using a probe DNA under stringent conditions. For example, in the colony hybridization method and the plaque hybridization method, a colony or plaque-derived DNA or a filter on which the DNA fragment is immobilized is used as the “stringent conditions”. After hybridization at 65 ° C. in the presence of 0.7 to 1.0 M sodium chloride, 0.1 to 2 × SSC solution (1 × SSC composition is 150 mM sodium chloride, 15 mM sodium citrate) The conditions for washing the filter at 65 ° C. can be mentioned.

  DNA encoding carbonyl reductase can be isolated by the following method, for example. First, carbonyl reductase is purified from microbial cells by the above method and the like, and then the N-terminal amino acid sequence is analyzed. N-terminal amino acid sequence analysis is performed by cleaving a purified protein with an enzyme such as lysyl endopeptidase or V8 protease, purifying a peptide fragment by reverse phase liquid chromatography, etc., and then analyzing the amino acid sequence with a protein sequencer. It is done by deciding. Using a primer designed based on the determined amino acid sequence and performing PCR using a chromosomal DNA or cDNA library of a carbonyl reductase-producing microbial strain as a template, to obtain a part of the DNA encoding the carbonyl reductase (DNA fragment) Can do. Furthermore, from the library or cDNA library obtained by introducing a restriction enzyme digest of carbonyl reductase producing microorganism strain into phage, plasmid, etc., and transforming E. coli, using the above DNA fragment as a probe DNA encoding carbonyl reductase can be obtained by performing colony hybridization, plaque hybridization, and the like.

  In addition, the base sequence of the DNA fragment obtained by the PCR is analyzed, and a PCR primer is designed to extend from the obtained sequence to the outside of the sequence-determined region, and the chromosome of the carbonyl reductase-producing microorganism strain The DNA encoding carbonyl reductase is obtained by performing inves PCR (Genetics vol. 120, p621-623 (1988)) using DNA that has been cyclized by a self-cyclization reaction as a template after digesting the DNA with an appropriate restriction enzyme. It is also possible to obtain.

  The DNA encoding carbonyl reductase can also be obtained by chemically synthesizing DNA having the base sequence of SEQ ID NO: 1.

  Those skilled in the art will be able to introduce site-directed mutagenesis into the DNA described in SEQ ID NO: 1 (Nucleic Acid Res. Vol. 10, p6487 (1982), Methods in Enzymol. Vol. 100, p448 (1983), Molecular Cloning 2nd. Edt., Cold Spring Harbor Laboratory Press (1989), PCR: A Practical Approach, IRL Press, p200 (1991)) and the like, by appropriately introducing substitutions, deletions, insertions and / or addition mutations. It is possible to obtain a DNA encoding a carbonyl reductase that can be used in the production method.

  Further, based on the whole or part of the amino acid sequence of SEQ ID NO: 2 or the whole or part of the base sequence of SEQ ID NO: 1, a homology search is performed on a database such as GenBank or DDBJ, and carbonyl reductase is obtained. It is also possible to obtain base sequence information of the encoded DNA homolog. A person skilled in the art can obtain DNA encoding carbonyl reductase by PCR or the like from the deposited strain (available from ATCC, DSMZ, etc.) based on this nucleotide sequence information.

  Furthermore, the DNA encoding carbonyl reductase is obtained by using a DNA having all or part of the nucleotide sequence of SEQ ID NO: 1 as a probe, and using a colony hybridization method for DNA prepared from any microorganism having carbonyl reductase activity, It can also be obtained by performing hybridization under stringent conditions by a plaque hybridization method, a Southern blot hybridization method, or the like to obtain hybridized DNA. Here, “part” refers to DNA having a length sufficient to be used as a probe, specifically 15 bp or more, preferably 50 bp or more, more preferably 100 bp or more.

  Each hybridization was performed using Molecular Cloning 2nd Edt. , Cold Spring Harbor Laboratory Press (1989) and the like.

  A carbonyl reductase expression vector is provided by inserting the DNA encoding carbonyl reductase isolated as described above into a known expression vector so as to allow expression. By culturing cells transformed with this expression vector, carbonyl reductase can be obtained from the cells. A transformed cell can also be obtained by integrating a carbonyl reductase-encoding DNA into a chromosomal DNA of a known host cell so that it can be expressed.

  As a method for producing transformed cells, specifically, a DNA encoding carbonyl reductase is incorporated into a plasmid vector or phage vector that is stably present in the microorganism, and the constructed expression vector is introduced into the microorganism. Alternatively, it is necessary to introduce DNA encoding carbonyl reductase directly into the host genome, and to transcribe and translate the genetic information.

  At this time, if the DNA encoding carbonyl reductase does not contain a promoter that can be expressed in the host microorganism, it is necessary to incorporate an appropriate promoter upstream of the DNA strand encoding carbonyl reductase. Furthermore, it is preferable to incorporate a terminator downstream of the 3 'side. The promoter and terminator are not particularly limited as long as they are known to function in microorganisms used as hosts, and vectors, promoters and terminators that can be used in these various microorganisms are, for example, “ Microbiology Basic Course 8, Genetic Engineering, Kyoritsu Shuppan ", especially regarding yeast, Adv. Biochem. Eng. vol. 43, p75-102 (1990), Yeast vol. 8, p423-488 (1992).

  The host microorganism to be transformed for expressing the carbonyl reductase of the present invention is not particularly limited as long as the host itself does not adversely affect this reaction, and specifically, the following microorganisms: Can be mentioned.

  Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacter, Streptococcus b Bacteria with established host vector systems belonging to the genus.

  Actinomycetes with established host vector systems belonging to the genus Rhodococcus, the genus Streptomyces, and the like.

  Saccharomyces (genus), Kluyveromyces (genus), Schizosaccharomyces (genus), Zygosaccharomyd (genus) Yeast with established host vector systems belonging to the genus Hansenula, Pichia, Candida and the like.

  Molds with established host vector systems belonging to the genera Neurospora, Aspergillus, Cephalosporia, Trichoderma, and the like.

  Among the microorganisms, the host is preferably the genus Escherichia, the genus Bacillus, the genus Brevibacterium, the genus Corynebacterium, particularly preferably the genus Escherichia, It belongs to the genus Corynebacterium.

  The procedure for preparing transformed cells, the construction of a recombinant vector suitable for the host, and the method for culturing the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering ( See, for example, “Molecular Cloning 2nd Edition”, Cold Spring Harbor Laboratory Press (1989)).

  Specific examples of preferred host microorganisms, preferred transformation techniques in each microorganism, vectors, promoters, terminators and the like are given below, but the present invention is not limited to these examples.

  In the genus Escherichia, especially Escherichia coli, plasmid vectors include pBR and pUC plasmids, and promoters include lac (β-galactosidase), trp (tryptophan operon), tac, trc ( lac, trp fusion), promoters derived from λ phage PL, PR, and the like. Examples of the terminator include trpA-derived, phage-derived, and rrnB ribosomal RNA-derived terminators.

  In the genus Bacillus, examples of the vector include a pUB110-type plasmid and a pC194-type plasmid, and can also be integrated into a chromosome. As the promoter and terminator, promoters and terminators of enzyme genes such as alkaline protease, neutral protease, α-amylase and the like can be used.

In the genus Pseudomonas, vectors include general host vector systems established in Pseudomonas putida, Pseudomonas cepacia, etc., plasmids involved in the degradation of toluene compounds, and TOL plasmids. Basic broad host range vector (including genes necessary for autonomous replication derived from RSF1010 etc.) pKT240 (Gene, vol.
26, p273-82 (1983)).

  In the genus Brevibacterium, in particular, Brevibacterium lactofermentum, examples of the vector include plasmid vectors such as pAJ43 (Gene vol. 39, p281 (1985)). As the promoter and terminator, various promoters and terminators used in E. coli can be used.

  In the genus Corynebacterium, in particular, Corynebacterium glutamicum, as vectors, pCS11 (Japanese Patent Laid-Open No. 57-183799), pCB101 (Mol. Gen. Genet. Vol. 196, p175 (1984) ) And the like.

  In the genus Saccharomyces, particularly in Saccharomyces cerevisiae, vectors include YRp, YEp, YCp, and YIp plasmids. In addition, promoters and terminators of various enzyme genes such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase, β-galactosidase, phosphoglycerate kinase, and enolase can be used.

  In the genus Schizosaccharomyces, the vector is Mol. Cell. Biol. vol. 6, p80 (1986), and a plasmid vector derived from Schizosaccharomyces pombe. In particular, pAUR224 is commercially available from Takara Shuzo and can be easily used.

  In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae, etc. are the most studied among molds, and integration into plasmids and chromosomes is available. Promoters derived from external proteases or amylases can be used (Trends in Biotechnology vol. 7, p283-287 (1989)).

  In addition to the above, host vector systems corresponding to various microorganisms have been established, and these can be used as appropriate. In addition to microorganisms, various host / vector systems have been established in plants and animals, especially in animals such as insects using moths (Nature vol. 315, p592-594 (1985)), rapeseed, corn, A system for expressing a heterogeneous protein in a large amount in a plant such as potato and a system using a cell-free protein synthesis system such as an E. coli cell-free extract or wheat germ have been established and can be suitably used.

(Production method of the present invention)
In the production method of the present invention, a microbial cell and / or a treated product thereof is allowed to act on a 3-oxoamino acid derivative which is a reaction substrate. The treated microbial cells include, for example, those obtained by treating the microbial cells with an organic solvent or surfactant such as acetone, dimethyl sulfoxide (DMSO), toluene, lyophilized, physically or enzymatically disrupted. Cellular cell preparations such as those obtained, and those obtained by removing the carbonyl reductase fraction of the present invention in the microbial cells as a crude product or purified product, and these are represented by polyacrylamide gel, carrageenan gel, etc. It indicates what is immobilized on the carrier.

In the production method of the present invention, it is preferable to add coenzyme NADP ⁺ (nicotinamide adenine dinucleotide phosphate oxidation type) or NADPH (nicotinamide adenine dinucleotide phosphate reduction type) to the reaction solution. The addition concentration is 0.001 mM to 100 mM, preferably 0.01 to 10 mM. When these coenzymes are added, it is preferable to regenerate NADP + produced from NADPH to NADPH in order to improve production efficiency. As a regeneration method, (i) a method using the NADP + reducing ability of the host microorganism itself, (ii) a microorganism having the ability to produce NADPH from NADP + or a preparation thereof, or glucose dehydrogenase, alcohol dehydrogenase, A method of adding an enzyme (regenerative enzyme) that can be used for NADPH regeneration such as amino acid dehydrogenase into the reaction system, or (iii) an enzyme that can be used for NADPH regeneration when producing transformed cells. And a method for introducing the gene for the regenerative enzyme and a DNA encoding carbonyl reductase into the host at the same time.

  Among these, in the method (i), it is preferable to add glucose or 2-propanol to the reaction system.

  In the above method (ii), a microorganism preparation containing a regenerative enzyme, a microorganism prepared by treating the microorganism with acetone, freeze-dried, physically or enzymatically disrupted, etc. In addition, a product obtained by removing the enzyme fraction as a crude product or a purified product, a product obtained by immobilizing the enzyme fraction on a carrier represented by polyacrylamide gel, carrageenan gel, or the like may be used. May be used. In this case, the amount of the regenerative enzyme used is specifically added so that the enzyme activity is 0.01 to 100 times, preferably about 0.5 to 20 times that of the carbonyl reductase of the present invention. To do. In addition, it is necessary to add a compound that becomes a substrate for the regenerative enzyme, for example, glucose when using glucose dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, In addition, 0.1 to 100-fold molar equivalent, preferably 1 to 20-fold molar equivalent, is added to the 3-oxoamino acid derivative that is a reaction raw material.

  In the method (iii), a method for incorporating a DNA encoding a carbonyl reductase and a DNA for the regenerating enzyme into a chromosome, a method for transforming a host by introducing both DNAs into a single vector, and The method of transforming the host after introducing both DNAs separately into the vector can be used, but in the case of the method of transforming the host after separately introducing both DNAs into the vector, the two vectors are incompatible. It is necessary to select a vector in consideration of sex. When introducing multiple genes into a single vector, methods such as linking promoter-terminator-related regions such as promoters and terminators to each gene, or expressing them as an operon containing multiple cistrons such as the lactose operon. Is possible.

  The production method of the present invention can be carried out in an aqueous medium or a mixture of the aqueous medium and an organic solvent. Examples of the aqueous medium include water or a buffer, and examples of the organic solvent that can be used include ethyl acetate, butyl acetate, toluene, dimethyl sulfoxide, isopropanol, and the like that have high solubility of the reaction substrate. What can increase the solubility of reaction substrates, such as various surfactant, can also be used for said aqueous medium.

  The compound represented by the general formula (1) serving as a reaction substrate in the production method of the present invention can be produced by combining existing chemical synthesis.

  For example, it can be manufactured by the following route.

  Specifically, an oxazolone ring compound represented by the general formula (5) is obtained by a condensation reaction of glycine derivatives such as hippuric acid and acetylglycine represented by the general formula (4). An acylating agent is allowed to act on the oxazolone ring compound, or O-acylated to form an oxazole compound, and then the acyl group is further rearranged to obtain a diacyloxazolone ring compound represented by the general formula (6). By reacting this diacyloxazolone ring compound with alcohols, a 3-oxoamino acid derivative represented by the general formula (1) can be obtained. This reaction can also be performed in the same reactor without isolating the oxazolone ring compound represented by the general formula (5) and / or the diacyloxazolone ring compound represented by the general formula (6).

  It is also possible to manufacture by the following route.

  Specifically, an oxazole compound represented by the general formula (7) is obtained from a glycine derivative such as hippuric acid and acetylglycine represented by the general formula (4), and an acid chloride is added thereto in the presence of a Lewis acid. By making it act, the acyl oxazolone compound represented by General formula (8) is obtained. A 3-oxoamino acid derivative represented by the general formula (1) can be obtained by allowing an alcohol to act on the acyloxazolone compound.

  In the production method of the present invention, the compound represented by the general formula (1) serving as a reaction substrate usually has a substrate concentration of 0.001 to 90% w / v, preferably 0.01 to 30% w / v. Can be used. The reaction substrate may be added all at once at the start of the reaction, but it is desirable to add it continuously or intermittently from the viewpoint of reducing the decomposition of the substrate and the solubility of the substrate.

  In the production method of the present invention, the amount of the microorganism used and / or the processed product thereof is usually 0.01 to the compound represented by the general formula (1) serving as a reaction substrate. It can be used in an amount of 500 times (W / W), preferably 1 to 200 times. The microbial cells and / or the processed microbial cells may be added all at once at the start of the reaction. However, if a decrease in the reaction is observed, it is also effective to add them intermittently.

  The production method of the present invention can be carried out, for example, at a reaction temperature of 4 to 60 ° C., preferably 20 to 50 ° C., and pH 3 to 12, preferably pH 5 to 10. In addition, in order to prevent decomposition of the carboxylic acid ester portion of the substrate, a compound that prevents the decomposition of the ester portion, specifically an esterase inhibitor, or a salt such as ammonium sulfate or sodium sulfate may be added to the reaction solution. Is valid. Similarly, an additive may be added to prevent decomposition of the amide bond. Specific examples include acylase inhibitors.

  In the production method of the present invention, the reaction time is not particularly limited as long as the compound represented by the general formula (1) is converted to produce the optically active 3-hydroxyamino acid derivative represented by the general formula (2). Usually, it is performed for 1 minute to 7 days, preferably 1 hour to 4 days.

  The reaction form may be either a batch method or a continuous method, but in the case of the continuous method, the 3-oxoamino acid derivative of the formula (1), the culture solution of the microorganism, the microbial cell and / or the processed microbial cell product, If necessary, it may be added as appropriate, and the cells isolated from the target product may be recycled.

  After completion of the reaction, the optically active 3-hydroxyamino acid derivative produced by the production method of the present invention is separated from cells and proteins in the reaction solution by centrifugation, membrane treatment, etc., and then with an organic solvent such as ethyl acetate or toluene. Purification can be performed by appropriately combining extraction, distillation, column chromatography, crystallization, and the like.

(Use of optically active 3-hydroxyamino acid derivative)
The optically active 3-hydroxyamino acid derivative represented by the general formula (2) is derivatized by itself or by various techniques and is useful as an intermediate for various pharmaceuticals and agricultural chemicals. In particular, the following general formula (9)

Is industrially useful as an intermediate of a drug having anti-HIV activity (WO01 / 40227). As a method for converting this (9) and its analogs, the compound represented by the general formula (2) is represented by the following general formula (10).

It is converted into a compound represented by As a method for this conversion, there is a method in which hydrolysis or hydrolysis with a hydrolase is performed in the presence of acid or base.

  The compound represented by the general formula (10) can be converted to the compound represented by the general formula (9) by performing t-butoxycarbonylation under basic conditions.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited to this.

(Synthesis Example 1)
(Synthesis of 4- (1-cyclohexyl-1-hydroxymethylidene) -2-phenyl-5-oxazolone)
A 100 ml flask was charged with 43 ml of methylene chloride and 4.28 g (21.1 mmol) of 5-acetoxy-2-phenyloxazole, 3.10 ml (23.2 mmol) of cyclohexanecarboxylic acid chloride was added, and after stirring for a while, tin tetrachloride was added. 3.87 ml (73.2 mmol) was added dropwise over 10 minutes. After 24 hours, the disappearance of the raw materials was confirmed by TLC, 10 ml of 4N hydrochloric acid aqueous solution and 150 ml of ethyl acetate were added, the organic layer was separated, and further washed with 50 ml of water twice and with 50 ml of saturated brine, and sulfuric acid. After drying with magnesium, the solvent was distilled off. The obtained residue was purified by silica gel column chromatography (silica gel 150 g, hexane: ethyl acetate = 100: 2 to 100: 4), and the desired product 4- (1-cyclohexyl-1-hydroxymethylidene)-was obtained as a yellow solid. 2.20 g of 2-phenyl-5-oxazolone was obtained (yield after purity conversion: 52%).

¹ H-NMR (400 MHz, DMSO-d): δ 1.14-1.51 (5H, m), 1.68-1.79 (5H, m), 3.31-3.37 (1H, m) 7.53-7.57 (3H, m), 7.96-8.01 (2H, m)

(Synthesis Example 2)
(Synthesis of isopropyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate)
After charging 47.5 g (175 mmol) of 4- (1-cyclohexyl-1-hydroxymethylidene) -2-phenyl-5-oxazolone prepared in Synthesis Example 1 and 238 ml of 2-propanol, the mixture was stirred for 2 hours with heating under reflux. After concentration under reduced pressure, 66.1 g of isopropyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate (53.9 g as a pure component, yield 93%) was obtained as a white solid.

¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.20-1.38 (9H, m), 1.45-1.56 (2H, m), 1.69-1.85 ((4H, m) 2.08-2.10 (1H, br-s), 2.86-2.93 (1H, m), 5.10-5.17 (1H, m), 5.52 (1H, d, J = 6.6 Hz), 7.29 (1H, d, J = 6.1 Hz), 7.43-7.55 (3H, m), 7.83-7.85 (m, 2H)

(Synthesis Example 3)
(Synthesis of ethyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate)
After charging 100 mg (0.37 mmol) of 4- (1-cyclohexyl-1-hydroxymethylidene) -2-phenyl-5-oxazolone prepared in Synthesis Example 1 and ethanol (2.0 ml), the mixture was stirred for 2 hours with heating under reflux. The solution was concentrated under reduced pressure to obtain 112 mg of ethyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate as a white solid (108 mg as a pure component, yield 96%).

¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.20-1.55 (8H, m), 1.68-1.84 (4H, m), 2.06-2.10 (1H, br-s) ), 2.86-2.90 (1H, m), 4.25-4.33 (2H, m), 5.56 (1H, d, J = 6.7 Hz), 7.30-7.32. (1H, d, J = 6.7 Hz), 7.43-7.48 (2H, m), 7.52-7.56 (1H, m), 7.83-7.86 (m, 2H)

(Synthesis Example 4)
(Production of isopropyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate)
A 200 mL flask was charged with 5.00 g (27.9 mmol) of N-benzoylglycine, 16 mL (170 mmol) of 4-picoline and 50 mL of acetone, and 15.2 mL (112 mmol) of cyclohexanecarboxylic acid chloride was added dropwise over 30 minutes under ice cooling. The mixture was allowed to react for 30 minutes at room temperature under ice-cooling for 20 hours. 25 mL of isopropanol was added to the reaction solution and heated under reflux for 1 hour. Then, 0.68 g (5.6 mmol) of 4-dimethylaminopyridine was dissolved in 2.5 mL of 2-propanol, and the mixture was further heated under reflux for 7 hours. After completion of the reaction, the reaction mixture was concentrated, and the resulting concentrate was extracted with 100 mL of toluene, washed with 40 mL of 2N hydrochloric acid, and then the organic layer was concentrated. The residue was crystallized with 5 mL of ethyl acetate and 90 mL of heptane to obtain 6.16 g of isopropyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate (chemical purity 98%, yield 67%) as white crystals.

(Synthesis Example 5)
(Production of 4- (1-cyclohexylcarbonyloxy-1-cyclohexylmethylidene) -2-phenyl-5-oxazolone)
A 500 mL flask was charged with 20.0 g (112 mmol) of N-benzoylglycine, 65 mL (670 mmol) of 4-picoline and 300 mL of acetonitrile, and 61 mL (450 mmol) of cyclohexanecarboxylic acid chloride was added dropwise over 30 minutes under ice cooling. After reacting for 5 hours under ice cooling, 100 mL of 1N hydrochloric acid was added, and the crystals were collected by filtration. The crystals were dried and 37.9 g of 4- (1-cyclohexylcarbonyloxy-1-cyclohexylmethylidene) -2-phenyl-5-oxazolone as white crystals (a mixture of two geometric isomers containing a small amount of impurities, approximately 85 : 15, yield 89%).

major-isomer: ¹ H-NMR (400 MHz, CDCl ₃ ): δ1.15-1.50 (8H, m), 1.62-1.93 (10H, m), 2.10-2.16 (2H M), 2.66 (1H, tt, J = 11.1, 3.8 Hz), 3.58-3.63 (1H, m), 7.46-7.50 (2H, m), 7 .54-7.56 (1H, m), 7.97-7.99 (2H, m).

minor-isomer: ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.20-1.47 (8H, m), 1.59-1.85 (10H, m), 2.11-2.15 (2H M), 2.65 (1H, tt, J = 11.4, 3.7 Hz), 3.26-3.31 (1H, m), 7.46-7.50 (2H, m), 7 .54-7.56 (1H, m), 8.04-8.07 (2H, m).

(Synthesis Example 6)
(Production of methyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate)
0.99 g (26.0 mmol) of 4- (1-cyclohexylcarbonyloxy-1-cyclohexylmethylidene) -2-phenyl-5-oxazolone obtained by the method of Synthesis Example 5 in a 30 mL flask, 0.25 mL of 4-picoline (2.6 mmol) and 5 mL of methanol were charged, and after heating and refluxing for 1 hour, 68 mg (0.56 mmol) of 4-dimethylaminopyridine was added, and the mixture was further heated to reflux for 4 hours. After completion of the reaction, the reaction mixture was concentrated, and the resulting concentrate was extracted with 10 mL of ethyl acetate, washed with 2 mL of 2N hydrochloric acid and 2 mL of saturated brine, and the organic layer was concentrated. The residue was purified by column chromatography to obtain 0.58 g of methyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate (including a small amount of impurities, yield of about 60%) as a white solid.

¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.18-1.55 (5H, m), 1.65-1.87 (4H, m), 2.04-2.11 (1H, m), 2.83-2.91 (1H, m), 3.83 (3H, s), 5.59 (1H, d, J = 6.8 Hz), 7.33 (1H, d, J = 6.3 Hz) ), 7.43-7.49 (2H, m), 7.51-7.56 (1H, m), 7.82-7.87 (2H, m).

(Synthesis Example 7)
(Production of isopropyl 2-benzoylamino-4-methyl-3-oxopentanoate)
A 200 mL flask was charged with 2.5 g (13.7 mmol) of N-benzoylglycine, 7.66 g (80.6 mmol) of 4-picoline, and 25 mL of acetonitrile, and 6.07 g (112 mmol) of isobutyric chloride over 30 minutes under ice cooling. The mixture was added dropwise and reacted at room temperature for 5 hours under ice-cooling. 12.5 mL of isopropanol was added to the reaction solution, heated and refluxed for 2 and a half hours, and then 0.34 g (2.8 mmol) of 4-dimethylaminopyridine was dissolved in 1.25 mL of 2-propanol and further heated to reflux for 4 hours. I let you. After completion of the reaction, the reaction mixture was concentrated, and the resulting concentrate was extracted with 100 mL of toluene, washed with 40 mL of 2N hydrochloric acid, and then the organic layer was concentrated. The residue was purified by column chromatography to obtain 2.63 g of isopropyl 2-benzoylamino-4-methyl-3-oxopentanoate (yield 66%) as a pale yellow solid.

¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.16 (3H, d, J = 6.4 Hz), 1.26 (3H, d, J = 6.4 Hz), 1.27 (3H, d, J = 6.4 Hz), 1.32 (3 H, d, J = 6.6 Hz), 3.16 (1 H, sept, J = 6.4 Hz), 5.13 (1 H, sept, J = 6.6 Hz) 5.56 (1H, d, J = 6.8 Hz), 7.30 (1H, d, J = 6.8 Hz), 7.44-7.47 (2H, m), 7.52-7. 55 (1H, m), 7.84-7.85 (2H, m).

(Synthesis Example 8)
(Production of isopropyl 2-benzoylamino-4,4-dimethyl-3-oxopentanoate)
A 300 mL flask was charged with 2.01 g (11.0 mmol) of N-benzoylglycine, 6.28 g (67.4 mmol) of 4-picoline, and 20 mL of acetone, and 5.17 g (42.3 mmol) of pivalic acid chloride was added under ice cooling. The mixture was added dropwise over a period of 30 minutes under ice-cooling, and heated at reflux for 6 hours at room temperature for 14 hours. Further, 30 mL of acetonitrile was added, and the mixture was heated to reflux for 4 hours. After adding 17 mL of isopropanol to the reaction solution and heating under reflux for 1 hour, 0.28 g (2.3 mmol) of 4-dimethylaminopyridine was dissolved in 1.7 mL of 2-propanol and added, and further 17 mL of 2-propanol was added for 1 hour. The mixture was further heated to reflux for 9 and a half hours. The reaction solution was concentrated, diluted with 75 mL of toluene, washed with 30 mL of 2N hydrochloric acid, and the organic layer was concentrated. The residue was purified by column chromatography to obtain 0.36 g of isopropyl 2-benzoylamino-4,4-dimethyl-3-oxopentanoate as an orange-brown solid (including a small amount of impurities, yield about 10%). .

¹ H-NMR (400 MHz, CDCl ₃ ): δ1.25 (3H, d, J = 6.0 Hz), 1.30 (3H, d, J = 6.0 Hz), 1.31 (9H, s), 5.09 (1H, sept, J = 6.0 Hz), 5.93 (1H, d, J = 8.0 Hz), 7.12 (1H, d, J = 8.0 Hz), 7.43-7 .47 (2H, m), 7.51-7.82 (1H, m), 7.83-7.84 (2H, m).

(Synthesis Example 9)
(Production of isopropyl 2-benzoylamino-3-oxotridecanoate)
A 100 mL flask was charged with 2.00 g (10.9 mmol) of N-benzoylglycine, 6.5 mL (67.0 mmol) of 4-picoline and 20 mL of acetonitrile, and 8.99 g (42.6 mmol) of undecanoyl chloride was cooled with ice. The solution was added dropwise over 25 minutes, and stirred at room temperature for 2 hours under ice-cooling for 45 minutes. 10 mL of isopropanol was added to the reaction solution, and the mixture was heated under reflux for 2 hours. Then, 0.27 g (2.2 mmol) of 4-dimethylaminopyridine was added, and the mixture was further heated under reflux for 3 hours. The reaction solution was concentrated, diluted with 45 mL of ethyl acetate, washed with 15 mL of 2N hydrochloric acid and 15 mL of saturated brine, and the organic layer was concentrated. The residue was purified by column chromatography, and the oily substance obtained was dissolved in 120 mL of chloroform and washed with 30 mL of saturated aqueous sodium hydrogen carbonate solution. The organic layer was concentrated to obtain 2.38 g of isopropyl 2-benzoylamino-3-oxotridecanoate (including a small amount of impurities, yield of about 45%) as a white solid. Further, the obtained solid was purified by hexane suspension washing to obtain 0.59 g (yield 14%) of isopropyl 2-benzoylamino-3-oxotridecanoate as a white solid.

¹ H-NMR (400 MHz, CDCl ₃ ): δ 0.88 (3H, t, J = 6.8 Hz), 1.26-1.30 (14H, m), 1.28 (3H, d, J = 6) .3 Hz), 1.33 (3 H, d, J = 6.3 Hz), 1.62-1.67 (2 H, m), 2.78 (2 H, dt, J = 17.4, 7.5 Hz) 5.14 (1H, sept, J = 6.3 Hz), 5.38 (1H, d, J = 6.3 Hz), 7.30 (1H, d, J = 6.3 Hz), 7.43− 7.47 (2H, m), 7.51-7.55 (1H, m), 7.82-7.85 (2H, m).

(Synthesis Example 10)
(Production of phenyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate)
In a 50 mL flask was obtained 2.00 g (5.14 mmol) of 4- (1-cyclohexylcarbonyloxy-1-cyclohexylmethylidene) -2-phenyl-5-oxazolone obtained by the method of Example 1, and 0.54 g of 4-picoline. (5.72 mmol), 10 mL of phenol and 10 mL of acetonitrile were added and heated at 45 ° C. for 6 hours. Then, 0.13 g (1.0 mmol) of 4-dimethylaminopyridine was added and the mixture was heated at the same temperature for 12 hours and at 55 ° C. for 6 hours and a half. Heated. After completion of the reaction, the reaction mixture was diluted with 110 mL of ethyl acetate, washed twice with 10 mL of 2N hydrochloric acid and once with 20 mL of saturated brine, and then the organic layer was dried and concentrated. The residue was purified by column chromatography to obtain 2.7 g of phenyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate (including phenol, 47% yield).

¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.20-1.50 (4H, m), 1.50-1.63 (1 H, m), 1.68-1.76 (1 H, m), 1.80-1.95 (3H, m), 2.11-2.19 (1H, m), 2.97-3.06 (1H, m), 5.79 (1H, d, J = 6) .6 Hz), 7.11-7.25 (2H, m), 7.26-7.29 (1H, m), 7.37-7.42 (3H, m), 7.44-7.48. (2H, m), 7.52-7.56 (1H, m), 7.86-7.89 (2H, m).

(Synthesis Example 11)
(Production of N-benzoylglycine isopropyl ester)
A 500 mL flask was charged with 24 g (0.134 mol) of N-benzoylglycine, 50 mL of 2-propanol, 60 mL of toluene, and 0.2 g of p-toluenesulfonic acid, and refluxed for 4 hours with a Dean stark apparatus. After cooling, 60 mL of ethyl acetate was added, and the reaction mixture was washed successively with 30 mL of saturated sodium bicarbonate solution and 30 mL of brine and dehydrated with sodium sulfate. The colorless solid produced by concentrating the solvent was collected by filtration and air-dried to obtain 27 g of the desired product (yield 93%).

(Synthesis Example 12)
(Production of N-pivaloylglycine isopropyl ester)
Add 4.8 g (31.3 mmol) of glycine isopropyl ester, 50 mL of dichloromethane, and 6.3 g (62.6 mmol) of triethylamine to a 200 mL flask and cool to about 15 ° C., and add 10 mL of dichloromethane solution containing 3.8 g of pivaloyl chloride. Slowly dropped. At this time, the reaction temperature was kept at 15 ° C. or lower. After completion of dropping, the mixture was further stirred at room temperature for 30 minutes. The reaction mixture was washed with 1N hydrochloric acid 20 mL × 2 saturated brine 20 mL × 2 and dehydrated with sodium sulfate. A colorless solid produced by concentrating the solvent was collected by filtration and air-dried to obtain 5.5 g of the desired product (yield 87%).

(Synthesis Example 13)
(Production of isopropyl 2-benzoylamino-3-oxobutanoate)
With reference to a report by Tanabe et al. (J. Am. Chem. Soc., 2005, 127, 2854), isopropyl 2-benzoylamino-3-oxobutanoate was produced. A 100 mL flask was charged with 16 mL of dichloromethane, 1.77 g (8 mmol) of N-benzoylglycine isopropyl ester, 0.79 g (9.6 mmol) of 1-methylimidazole, 0.63 g (8 mmol) of acetyl chloride, and −45 ° C. under a nitrogen atmosphere. Then, the mixture was stirred for 10 minutes, and at that temperature, 5.3 g (28 mmol) of titanium tetrachloride and 5.9 g (32 mmol) of tri-n-butylamine were gradually added dropwise. After completion of the dropping, the reaction solution was further stirred for 1 hour, poured into 50 mL of water, the organic phase was taken, washed successively with water and brine, and dehydrated with sodium sulfate. The organic phase was concentrated and purified by silica gel column chromatography to obtain 0.5 g of the desired product (containing a small amount of impurities, yield 35%).

¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.27 (3H, d, J = 8 Hz), 1.29 (3H, d, J = 8 Hz), 2.45 (3H, s), 5.15 ( 1H, septet, J = 8 Hz), 5.40 (1H, d, J = 4 Hz), 7.30 (1H, brs), 7.40-7.55 (3H, m), 7.80-7. 90 (2H, m).

(Synthesis Example 14)
The following compounds were obtained in the same manner as above.
(Isopropyl 2-benzoylamino-3-oxopentanoate: 35% yield)
¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.14 (3H, t, J = 8 Hz), 1.27 (3H, d, J = 8 Hz), 1.29 (3H, d, J = 8 Hz), 2.74-2.90 (2H, m), 5.15 (1H, septet, J = 8 Hz), 5.40 (1H, d, J = 4 Hz), 7.32 (1H, brs), 7. 40-7.55 (3H, m), 7.80-7.90 (2H, m).

(Isopropyl 2-benzoylamino-3-oxohexanoate: yield 39%)
¹ H-NMR (400 MHz, CDCl ₃ ): δ 0.98 (3H, t, J = 8 Hz), 1.27 (3H, d, J = 8 Hz), 1.29 (3H, d, J = 8 Hz), 1.65-1.74 (2H, m), 2.71-2.82 (2H, m), 5.14 (1H, septet, J = 8 Hz), 5.39 (1H, d, J = 4 Hz) ), 7.32 (1H, brs),
7.40-7.55 (3H, m), 7.84-7.86 (2H, m).

(Isopropyl 2-benzoylamino-3- (naphthalen-2-yl) -3-oxopropionate: yield 11%)
¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.04 (3H, d, J = 8 Hz), 1.23 (3H, d, J = 8 Hz), 5.05 (1H, septet, J = 8 Hz), 6.50 (1H, d, J = 8 Hz), 7.4-7.7 (6H, m), 7.80-8.20 (6H, m), 8.84 (1H, s).

(Isopropyl 2-benzoylamino-3-oxo-3- (thiophen-2-yl) propionate: 17% yield)
¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.15 (3H, d, J = 8 Hz), 1.27 (3H, d, J = 8 Hz), 5.08 (1H, septet, J = 8 Hz), 6.17 (1H, d, J = 4 Hz), 7.2-7.5 (5H, m), 7.8-7.9 (3H, m), 8.19 (1H, s).

(Isopropyl 2-benzoylamino-3- (4-chlorophenyl) -3-oxopropionate: yield 11%)
¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.10 (3H, d, J = 8 Hz), 1.26 (3H, d, J = 8 Hz), 5.05 (1H, septet, J = 8 Hz), 6.29 (1H, d, J = 8 Hz), 7.4-7.6 (6H, m), 7.85-7.88 (2H, m), 8.12-8.14 (2H, m ).

(Isopropyl 3-oxo-3-phenyl-2-pivaloylaminopropionate: 53% yield)
¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.04 (3H, d, J = 8 Hz), 1.19 (3H, d, J = 8 Hz), 1.26 (9H, s), 4.98 ( 1H, septet, J = 8 Hz), 6.10 (1H, d, J = 4 Hz), 7.00 (1H, brs), 7.45-7.65 (3H, m), 8.10-8. 13 (2H, m).

(Isopropyl 3- (4-chlorophenyl) -3-oxo-2-pivaloylaminopropionate: yield 20%)
¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.08 (3H, d, J = 8 Hz), 1.23 (3H, d, J = 8 Hz), 1.25 (9H, s), 5.02 ( 1H, septet, J = 8 Hz), 6.05 (1H, d, J = 4 Hz), 6.98 (1H, brs), 7.46-7.50 (2H, m), 8.05-8. 08 (2H, m).

(Isopropyl 3-oxo-2-pivaloylamino-3- (thiophen-2-yl) propionate: yield 35%)
¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.13 (3H, d, J = 8 Hz), 1.24 (3H, d, J = 8 Hz), 1.26 (9H, s), 4.98 ( 1H, septet, J = 8 Hz), 5.92 (1H, d, J = 4 Hz), 6.95 (1H, brs), 7.15-7.25 (1H, m), 7.70-7. 80 (1H, m), 8.05-8.15 (1H, m).

Example 1
(Preparation of transformed cells transformed with a carbonyl reductase gene derived from Exiguobacterium sp. MCI4322)
Plasmid pExSDR1 and transformed Escherichia coli JM109 strain were obtained in the same manner as in Japanese Patent Application Laid-Open No. 2004-267130. That is, Exibubacterium sp. Primers described in SEQ ID NOs: 3 and 4 were synthesized based on a DNA sequence (genbank Accession No. gp: AB154409, SEQ ID NO: 1) encoding a carbonyl reductase derived from MCI4322 (Accession No. 3016330A, SEQ ID NO: 2).

SEQ ID NO: 1:
atgaaatata cggtgattac tggagcaagt tcaggaattg gatatgagac ggcaaaacta 60
ctcgcaggaa aaggaaaatc actcgttctc gtcgcgcgac ggacgtctga gctcgaaaaa 120
cttcgggatg aagtcaaaca aatctcacca gatagtgatg tcatcctcaa gtcggtcgat 180
ctcgcagata accaaaatgt ccatgattta tatgagggat taaaggaact cgacatcgaa 240
acgtggatca acaatgctgg attcggcgat tttgatctcg tccaggacat tgagctcggg 300
aaaatcgaga aaatgcttcg cttgaacatc gaggcgctaa cgattctatc gagtctgttc 360
gtccgcgatc atcatgacat cgaaggaacg acgctcgtca atatctcgtc agcaggtggc 420
taccggatcg tgccgaacgc ggtcacgtat tgcgcgacga agttctatgt cagtgcttat 480
acagaagggc tcgcgcaaga actgcaaaaa ggtggggcaa aacttcgggc gaaagtactg 540
gcaccagctg cgactgagac agagtttgcg gatcgttcgc gcggcgaagc aggtttcgac 600
tacagcaaga acgtcaaaaa gtaccatacg gcggctgaaa tggcaggctt cctgcatcag 660
ttgattgaaa gcgacgcgat cgtcggcatc gtcgacggtg agacgtatga gttcgaattg 720
cgtggtccat tgttcaacta cgcaggataa 750

SEQ ID NO: 2:
Met Lys Tyr Thr Val Ile Thr Gly Ala Ser Ser Gly Ile Gly Tyr Glu
Thr Ala Lys Leu Leu Ala Gly Lys Gly Lys Ser Leu Val Leu Val Ala
Arg Arg Thr Ser Glu Leu Glu Lys Leu Arg Asp Glu Val Lys Gln Ile
Ser Pro Asp Ser Asp Val Ile Leu Lys Ser Val Asp Leu Ala Asp Asn
Gln Asn Val His Asp Leu Tyr Glu Gly Leu Lys Glu Leu Asp Ile Glu
Thr Trp Ile Asn Asn Ala Gly Phe Gly Asp Phe Asp Leu Val Gln Asp
Ile Glu Leu Gly Lys Ile Glu Lys Met Leu Arg Leu Asn Ile Glu Ala
Leu Thr Ile Leu Ser Ser Leu Phe Val Arg Asp His His Asp Ile Glu
Gly Thr Thr Leu Val Asn Ile Ser Ser Ala Gly Gly Tyr Arg Ile Val
Pro Asn Ala Val Thr Tyr Cys Ala Thr Lys Phe Tyr Val Ser Ala Tyr
Thr Glu Gly Leu Ala Gln Glu Leu Gln Lys Gly Gly Ala Lys Leu Arg
Ala Lys Val Leu Ala Pro Ala Ala Thr Glu Thr Glu Phe Ala Asp Arg
Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys Lys Tyr
His Thr Ala Ala Glu Met Ala Gly Phe Leu His Gln Leu Ile Glu Ser
Asp Ala Ile Val Gly Ile Val Asp Gly Glu Thr Tyr Glu Phe Glu Leu
Arg Gly Pro Leu Phe Asn Tyr Ala Gly

Sequence number 3: ggaggcgaat tcatgaaata tacggtgatt
Sequence number 4: taatcactgc agttatcctg cgtagttgaa

  These primers were used at 15 pmol each, 10 nmol each dNTP, Exibacterium, sp. Using a reaction solution of 50 μL containing 25 ng of genomic DNA of MCI4322, 5 μL of 10 × buffer for KOD-plus- (Toyobo Co., Ltd.) and 1 unit of KOD-plus-1 (manufactured by Toyobo Co., Ltd.) Second), annealing (57 ° C., 30 seconds), extension (68 ° C., 2 minutes) in 30 cycles, and PCR reaction was performed using PTC-200 (manufactured by MJ Research). As a result of analyzing a part of the PCR reaction solution by agarose gel electrophoresis, a band considered to be specific could be detected. The reaction solution was purified with MinElute PCR Purification kit (Qiagen). The purified DNA fragment was digested with restriction enzymes EcoRI and PstI, and subjected to agarose gel electrophoresis. The target band portion was excised and collected after purification by Qiagen Gel Extraction kit (manufactured by Qiagen). The obtained DNA fragment was ligated using pKK223-3 (manufactured by Amersham-Pharmacia) digested with EcoRI and PstI and Takara Ligation Kit to transform Escherichia coli JM109 strain.

  The transformed cells were grown on an LB agar medium containing ampicillin (50 μg / mL), and colony direct PCR was performed to confirm the size of the inserted fragment. Transformed cells that are considered to have the target DNA fragment inserted are cultured in LB medium containing 50 μg / mL ampicillin, and the plasmid was purified using QIAPrepSpin Mini Prep kit (manufactured by Qiagen) to obtain pExSDR1. . When the base sequence of the DNA inserted into the plasmid was analyzed by the dye terminator method, the inserted DNA fragment was identical to the base sequence of SEQ ID NO: 1. )

(Example 2)
(Synthesis of isopropyl (2R, 3R) -2-benzoylamino-3-cyclohexyl lu 3-hydroxypropionate using E. coli transformed with DNA encoding carbonyl reductase)
The transformant having pExSDR1 obtained in Example 1 was grown for 1.7 hours at 30 ° C. in CircleGrow medium (domestic chemistry) containing ampicillin (50 μg / mL), and isopropyl β-D to 0.1 mM. -Thiogalactopyranoside (IPTG) was added and further cultured at 30 ° C for 20 hours. 2 mL of the obtained bacterial cell broth was collected by centrifugation, and 100 μL of a buffer solution (100 mM Tris containing 10% sodium sulfate and 10% glucose (final pH 9.9)) was added and mixed well. 30 μL of 10 g / L NADP + (manufactured by Oriental Yeast Co., Ltd.), 30 μL of glucose dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd., 1 unit / ml), 20 μL of 10 g / L 2-benzoylamino-3-cyclohexyl obtained in Synthesis Example 2 After adding isopropyl oxo-3-oxopropionate (DMSO solution), the mixture was reacted with shaking at 40 ° C. for 18 hours. The reaction solution after completion of the reaction was extracted with ethyl acetate, concentrated, dissolved in 200 μL of 2-propanol, and isopropyl 2-benzoylamino-3-cyclohexyl-3-hydroxypropionate was quantified.

Quantification was performed using high performance liquid chromatography (HPLC). The conditions of HPLC are as follows.
Column: ODS column Acentis Amide (manufactured by Spelco, 4.6 mm × 10 cm) 40 ° C.
Eluent: 10 mM ammonium acetate / acetonitrile = 40/60
Flow rate: 1 ml / min
Detection: UV254nm

  As a result, the yield of isopropyl 2-benzoylamino-3-cyclohexyl-3-hydroxypropionate was 14.1 μg.

  Further, when E. coli having the plasmid pKK223-3 not containing the gene was cultured and reacted in the same manner as above, the product was not observed.

Next, the optical purity was measured using HPLC.
The conditions of HPLC are as follows.
Column: OD-RH (manufactured by Daicel Corporation 4.6 mm × 15 cm) 30 ° C.
Eluent: 40% acetonitrile Flow rate: 0.5 ml / min
Detection: UV254nm
The optical purity is 100% e.e. e. The solid was R, R.

(Example 3)
(Synthesis of ethyl (2R, 3R) -2-benzoylamino-3-cyclohexyl-3-hydroxypropionate using Escherichia coli transformed with DNA encoding carbonyl reductase)
Transformants were cultured in the same manner as in Example 2. 2 mL of the obtained bacterial cell broth was collected by centrifugation, 50 μL of 40% ammonium sulfate, 50 μL of 50% glucose, and 20 μL of 1M Tris were added and mixed well. 30 μL of 10 g / L NADP + (manufactured by Oriental Yeast Co., Ltd.), 30 μL of glucose dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd., 1 unit / ml), 20 μL of 10 g / L 2-benzoylamino-3-cyclohexyl obtained in Synthesis Example 2 After addition of ethyl -3-oxopropionate (DMSO solution), the mixture was shaken at 40 ° C. for 18 hours. The reaction solution after completion of the reaction was extracted with ethyl acetate, concentrated, dissolved in 200 μL of 2-propanol, and ethyl 2-benzoylamino-3-cyclohexyl-3-hydroxypropionate was quantified in the same manner as in Example 1.

  As a result, the yield of ethyl 2-benzoylamino-3-cyclohexyl-3-hydroxypropionate was 48.0 μg. Further, when E. coli having the plasmid pKK223-3 not containing the gene was cultured and reacted in the same manner as above, the product was not observed.

  Next, when the optical purity was measured in the same manner as in Example 2, the optical purity was 100% e.e. e. The solid was 2R, 3R.

Example 4
(Synthesis of isopropyl (2R, 3R) -2-benzoylamino-3-cyclohexyl-3-hydroxypropionate by Agrobacterium tumefaciens (IAM12048))
A medium containing 10 g / L of yeast extract, 1 g / L of glucose, 1 g / L of potassium phosphate, 3 g / L of dipotassium phosphate, 0.5 g / L of magnesium sulfate, and 0.01 g / L of manganese chloride was prepared. The flask was placed in a 200 ml fluted Erlenmeyer flask and sterilized at 120 ° C. for 20 minutes. Agrobacterium tumefaciens (IAM12048) was inoculated into this medium and cultured at 30 degrees for 24 hours. The culture broth was centrifuged for 1 ml to obtain bacterial cells. To this, 50 μl of 20 mM Tris-HCl buffer (pH 7.5) and 10 μl of 50% glucose solution were added and vigorously stirred. To this suspension was further added 10 μl of 1% NAD ⁺ (manufactured by Oriental Yeast), 10 μl of 1% NADP ⁺ , 10 μl of glucose dehydrogenase (1 unit / ml from Amano Pharmaceutical), 1 g / g obtained in Synthesis Example 2 10 μl of L isopropyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate (DMSO solution) was added and reacted at 30 ° C. for 18 hours. 2-Propanol was added to the reaction solution to make the total volume 1 ml, and quantified in the same manner as in Example 2. As a result, isopropyl 2R, 3R′-2-benzoylamino-3-cyclohexyl-3-hydroxypropionate was 1.7 mg / L. Concentration. The optical purity is 100% e.e. e. The solid was 2R, 3R.

(Example 5)
(Synthesis of ethyl 2-benzoylamino-3-cyclohexyl-3-hydroxypropionate by various bacteria)
As in Example 4, various bacteria described in Table 1 were inoculated and cultured at 30 ° C. for 24 hours. However, Variovorax paradoxus (DSM66) was cultured for 1 week. The culture broth was centrifuged for 30 ml to obtain bacterial cells. 5 ml of distilled water was added and the mixture was sonicated and the crude enzyme solution was recovered by centrifugation. To 200 μl of this crude enzyme solution, 30 μl of 1M Tris-HCl buffer (pH 7.5) and 10 μl of 50% glucose solution were added and vigorously stirred. Further, 10 μl of 1% NAD ⁺ (manufactured by Oriental Yeast), 10 μl of 1% NADP ⁺ , 10 μl of glucose dehydrogenase (1 unit / ml manufactured by Amano Pharmaceutical), 10 g / 10 μl of L 2-benzoylamino-3-cyclohexyl-3-oxopropionate (DMSO solution) was added and reacted at 30 ° C. for 18 hours. 2-Propanol was added to the reaction solution to determine the total volume to 1 ml. Quantification was performed using high performance liquid chromatography (HPLC). The conditions of HPLC are as follows.

Column: ODS column Acentis Amide (manufactured by Spelco, 4.6 mm × 10 cm) 40 ° C.
Eluent: 10 mM ammonium acetate / acetonitrile = 55/45
Flow rate: 1 ml / min
Detection: UV254nm

  Table 1 shows the amount of ethyl 2-benzoylamino-3-cyclohexyl-3-hydroxypropionate, which is a reduced product. All were anti bodies and no syn bodies were detected.

(Example 6)
(Synthesis of isopropyl 2-benzoylamino-3-cyclohexyl-3-hydroxypropionate by various microorganisms)
In the same manner as in Example 4, various microorganisms listed in Table 2 were inoculated and cultured at 30 ° C. until they grew well for 1 to 7 days. The culture broth was centrifuged for 1 ml to obtain bacterial cells. To this, 100 μl of 0.1 M Tris-HCl buffer (pH 10), 20 μl of 50% glucose solution, 20 μl of 1% NAD ⁺ (manufactured by Oriental Yeast), 20 μl of 1% NADP ⁺ were added and vigorously stirred. To this suspension, 10 μl of glucose dehydrogenase (Amano Pharmaceutical 1 unit / ml) and 2.5 g / L of 2-benzoylamino-3-cyclohexyl-3-oxopropionate (DMSO solution) obtained in Synthesis Example 3 were added. 20 μl was added and reacted at 30 ° C. for 1 day. 0.3 ml of 2-propanol was added to the reaction solution and quantified by HPLC. Table 2 shows the amount of isopropyl 2-benzoylamino-3-cyclohexyl-3-hydroxypropionate that is a reduced product. The conditions of HPLC are as follows.

Column: ODS column Acentis Amide (manufactured by Spelco, 4.6 mm × 10 cm) 40 ° C.
Eluent: 10 mM ammonium acetate / acetonitrile = 40/60
Flow rate: 1 ml / min
Detection: UV254nm

(Example 7)
18 ml of 10 mM potassium phosphate buffer (final pH 7.0) containing 0.1 mM dithiothreitol was added to 5.66 g of the transformant obtained by culturing in the same manner as in Example 2 and mixed well. This bacterial cell suspension was sonicated and the crude enzyme solution was recovered by centrifugation. This crude enzyme solution had a protein concentration of 49 g / L.

(1) Measurement of carbonyl reductase activity Measurement of carbonyl reductase activity was performed using a reaction solution containing 100 mM Tris-HCl buffer (pH 7.5), 0.32 mM NADPH, and 10 mM 3-oxo-3- (2′-thienyl) propionic acid ethyl ester. Was carried out at 37 ° C. 1 U was defined as the amount of enzyme that oxidizes 1 μmol of NADPH per minute under the above reaction conditions. As a result, it was 5 U / ml.

  The methyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate obtained in Synthesis Example 6 was dissolved in DMSO or 2-propanol so as to be 10 g / L.

  Crude enzyme solution 180 μL or crude enzyme solution 20 μL with distilled water 160 μL, or distilled water 180 μL with 25 μL 1M potassium phosphate buffer (final pH 7.0), 10 μL 50 mM NADP + (produced by Oriental Yeast), 10 μL glucose Dehydrogenase (Amano Pharmaceutical Co., Ltd., 10 unit / ml), 10 μL of 50% glucose solution, and 10 μL of 10 g / L substrate solution were added, and the mixture was shaken at 30 ° C. for 90 minutes. After completion of the reaction, 500 μL of ethyl acetate was added to the reaction solution, and after stirring well, the organic layer was transferred to another container and dried. After drying, it was dissolved in 20 μL of isopropanol and 2 μL of the sample was analyzed by TLC. (Silica gel plate developing solution hexane / ethyl acetate = 1/1 detected by UV irradiation) The amount of reductant produced was larger when DMSO was the solvent in which the substrate was dissolved. Samples reacted with DMSO solution were analyzed by LC-Mass.

LC-Mass device: Hewlett-Packard 1100MSD
HPLC column: MCI-GEL ODS-1HU (150 x 4.6 mm)
Eluent A: 10 mM ammonium acetate solution Eluent B: 100% acetonitrile A: B = 50: 50
Flow rate: 0.5 ml / min
Temperature: 40 ° C
UV220nm
Ionization voltage: 20V
Ionization method: API-ES method positive ion measurement mode

Under the condition of 180 μL of the crude enzyme solution, the conversion rate was 84%, and the reduced product yield was 30%. The diastereoselectivity is 86% d. e. And the anti form was the main. In addition, 47% of the decarboxylated compound was produced ([M + H] ⁺ = 248.1).

Under the condition of 20 μL of the crude enzyme solution, the conversion rate was 22% and the yield of the reduced product was 7%. The diastereoselectivity is 91% d. e. And the anti form was the main. The decarboxylated compound ([M + H] ⁺ = 248.1) was 9% produced.

The substrate was not converted at all under the condition of 180 μL of distilled water.
When the optical purity was measured in the same manner as in Example 4, 100% e.e. e. The solid was 2R, 3R.

(Example 8)
The crude enzyme solution obtained in Example 7 was purified by anion exchange chromatography. A carbonyl reductase activity peaked in the vicinity of 0.15 M sodium chloride with a gradient elution of sodium chloride from 0 to 0.6 M. This was used as a crudely purified enzyme. The carbonyl reductase activity was 1 U / ml.

  25 μL of 1 M potassium phosphate buffer (final pH 7.0), 10 μL of 50 mM NADP + (manufactured by Oriental Yeast), 10 μL of glucose dehydrogenase (manufactured by Amano Pharmaceutical, 10 unit / ml), 5 μL of 1 M glucose solution When 5 μL of 10 g / L substrate solution was added and reacted at 30 ° C. for 3 and a half hours, the conversion rate was 100% and the yield of reduced product was 99%. The diastereoselectivity is 90% d. e. And the anti form was the main.

Example 9
(Synthesis of various (2R, 3R) -2-benzoylamino-3-cyclohexyl-3-hydroxypropionic acid esters)
10 mM potassium phosphate buffer (final pH 7.0)) was added to 25 g of the transformant obtained by culturing in the same manner as in Example 2, and mixed well to 150 ml. This bacterial cell suspension was sonicated and 30 g of ammonium sulfate was added and dissolved, and then the crude enzyme solution was recovered by centrifugation. This crude enzyme solution had a carbonyl reductase activity of 2.6 U / ml. Glucose dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd.) was further added to this so as to be 2 units / ml.

  25 μL of 1 M potassium phosphate buffer (final pH 7.0), 10 μL of 50 mM NADP + (manufactured by Oriental Yeast), 10 μL of 50% glucose solution, 10 μL of 10 g / L substrate solution were added to 88 μL of this crude purified enzyme at 30 ° C. The reaction was carried out for 19 hours. Substrate is ethyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate, isopropyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate 2-benzoylamino-3-cyclohexyl-3-oxopropionate n-propyl Was used. The ethyl ester had a conversion rate of 97%, a reduced product yield of 97%, the isopropyl ester had a conversion rate of 77%, a reduced product yield of 76%, and the n-propyl ester had a conversion rate of 87% and a reduced product yield of 82%. .

(Example 10)
(Generation of various optically active 3-hydroxyamino acid derivatives represented by Chemical Formula 2 from various 3-oxoamino acid derivatives represented by Chemical Formula 1)
A buffer solution (10% sodium sulfate, 100 mM sodium hydrogen carbonate-10 mM sodium carbonate (final pH 8.5)) was added to 10 g of the transformant obtained by culturing in the same manner as in Example 2, and the total amount was 40 ml and mixed well. This bacterial cell suspension was sonicated and the crude enzyme solution was recovered by centrifugation. This crude enzyme solution had a protein concentration of 80 g / L. This crude enzyme solution had a carbonyl reductase activity of 5 U / ml. Glucose dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd.) was further added to this so as to be 100 units / ml.

  10 μL of 50 mM NADP + (produced by Oriental Yeast), 5 μL of 50% glucose solution, 10 μL of 10 g / L to 100 μL of this crude enzyme solution, various 3-oxo amino acid derivatives (DMSO solution) represented by Chemical Formula 1 shown in Table 3 were added After that, the reaction was shaken at 37 ° C. for 1 to 2 days. 1000 μL of 2-propanol was added to the reaction solution after completion of the reaction, and the mixture was stirred well and analyzed by LC-Mass.

The conditions of LC-Mass are as follows.
LC-Mass device: Hewlett-Packard 1100MSD
HPLC column: L-column ODS-1HU (250 x 4.6 mm)
Eluent A: 10 mM ammonium acetate solution Eluent B: 100% acetonitrile gradient Method 1 A: B = 50: 50 (0 min) → A: B = 0: 100 (10 min)
A: B = 0: 100 (10-15 minutes)
Gradient method 2 A: B = 40: 60 (0 min) → A: B = 0: 100 (10 min)
A: B = 0: 100 (10-15 minutes)
Flow rate: 1 ml / min
Temperature: 40 ° C
UV254nm
Ionization voltage: 20V
Ionization method: API-ES method Positive ion measurement mode Analysis with gradient method 1 is analysis condition 1 and analysis with gradient method 2 is analysis condition 2.
The results of the analysis are shown in Table 3.

  The area% in Table 3 represents the ratio to the total area value of the reduced substance and substrate peaks obtained by the mass chromatogram. Both the mass ion and the retention time of the reduced form coincided with the 3-hydroxyamino acid derivative obtained by chemically reducing the 3-oxoamino acid derivative.

(Example 11)
For the substrate having high reactivity and high diasteromer excess in Example 10, the reaction was carried out at a large reaction scale in order to examine the three-dimensional structure of the obtained reduced product and 3-hydroxyamino acid derivative, and the reduced product was scraped off by preparative TLC. Collected with ethyl acetate and concentrated. The obtained crude purified product was examined for diastereomer excess (de%) and optical purity (enantiomer excess; ee%).

The diasteromer excess was measured using HPLC under the following conditions.
Column: Inertsil-CN (GL Science Co., Ltd., 4.6 mm × 15 cm) 40 ° C.
Eluent A: 80% 10 mM ammonium acetate, 20% acetonitrile eluent B: 100% acetonitrile gradient Method 3 A: B = 90: 10 (0 min to 20 min) → A: B = 0: 100 (30 min )
A: B = 0: 100 (30 to 35 minutes)
Gradient method 4 A: B = 80: 20 (0 to 20 minutes) → A: B = 0: 100 (30 minutes)
A: B = 0: 100 (30 to 35 minutes)
Flow rate: 1 ml / min
Detection: UV230nm
Analysis by gradient method 3 is analysis condition 3, and analysis by gradient method 4 is analysis condition 4.

The optical purity was measured using HPLC under the following conditions.
Column: OD-H (manufactured by Daicel Corporation 4.6 mm × 15 cm) 30 ° C.
Eluent: Eluent 5 Hex / IPA = 95/5 Eluent 6 Hex / IPA = 90/10
Flow rate: 0.5 ml / min
Detection: UV230nm
The analysis with the eluent 5 is the analysis condition 5 and the analysis with the eluent 6 is the analysis condition 6. The results are shown in Table 4.

  These were then subjected to NMR measurements. The results are shown below.

Isopropyl 2-benzoylamino-3-hydroxybutanoate,

1H NMR (400MHz, CDCl3): δ 1.14 (3H, d, J = 6.3Hz), 1.24 (3H, d, J = 6.3Hz), 1.25 (3H, d, J = 6.3Hz),
3.66 (1H, m), 4.25 (1H, m), 4.78 (1H, dd, J = 6.6,3.0Hz), 5.06 (1H, sept, J = 6.3Hz),
7.08 (1H, brd, J = 5.6Hz), 7.38-7.50 (3H, m), 7.76-7.78 (2H, m)
13C NMR (100 MHz, CDCl 3): δ 18.48, 21.73, 59.14, 69.53,
70.23, 127.21,128.69,132.11, 133.28, 168.43, 169.71 ppm

Isopropyl 2-benzoylamino-3-hydroxypentanoate,

1H NMR (400MHz, CDCl3): δ 0.95 (3H, t, J = 7.5Hz), 1.24 (3H, d, J = 6.3Hz), 1.25 (d, J = 6.3Hz),
1.47 (2H, m), 3.38 (1H, brd, J = 7.3Hz), 3.92 (1H, m), 4.77 (1H, dd, J = 6.8,3.0Hz),
5.07 (1H, sept, J = 6.3Hz), 7.09 (1H, brd, J = 6.0Hz), 7.36-7.49 (3H, m), 7.73-7.78 (2H, m)

Isopropyl 2-benzoylamino-3-hydroxy-4-methylpentanoate

1H NMR (400 MHz, CDCl3): δ1.00 (3H, d, J = 6.6 Hz), 1.05 (3H d, J = 6.6 Hz), 1.31 (3H, d, J = 6.0 Hz), 1.31 (3H, d , J = 6.0Hz), 1.78 (1H, sept, J = 6.6Hz), 3.22 (1H, brs), 3.64 (1H, d, J = 7.3Hz), 4.90 (1H, dd, J = 6.3, 3.0Hz ), 5.13 (1H, sept, J = 6.0Hz), 7.19 (1H, d, J = 6.3Hz), 7.43-7.55 (3H, m), 7.82-7.84 (2H, m) ppm
13C NMR (100 MHz, CDCl3): δ 18.04, 18.12, 20.71, 20.76, 30.67, 55.63, 68.99, 77.92, 126.14, 127.64, 130.94, 132.53, 166.56, 169.42 ppm

Isopropyl 2-benzoylamino-3-hydroxyhexanoate,

1H NMR (400MHz, CDCl3): δ0.86 (3H, t, J = 7.3Hz), 1.24 (3H, d, J = 6.3Hz), 1.25 (3H, d, J = 6.3Hz),
1.28-1.55 (4H.m), 3.45 (1H, brd, J = 7.1Hz), 4.02 (1H.m), 4.77 (1H, dd, J = 6.6,3.0Hz),
5.07 (1H, sept, J = 6.3Hz), 7.09 (1H, brd, J = 6.1Hz), 7.37-7.49 (3H, m), 7.76-7.78 (2H, m)

2-Benzoylamino-3-hydroxy-4,4-dimethylpentanoic acid isopropyl ester

1H NMR (400MHz, CDCl3): δ1.00 (9H, s), 1.31 (6H, d, J = 6.3Hz), 3.58-3.67 (1H, brs), 3.67 (1H, d, J = 2.8Hz), 4.92 (1H, dd, J = 7.6,2.8Hz), 5.07 (1H, sept, J = 6.3Hz), 7.14 (1H, d, J = 7.6Hz), 7.42-7.46 (2H, m), 7.50-7.55 (1H, m), 7.80-7.82 (2H, m) ppm
13C NMR (100 MHz, CDCl3): δ20.45, 20.74, 25.15, 34.63, 54.04, 69.08, 80.15, 126.13, 127.66, 130.94, 132.55, 166.39, 170.39 ppm

Isopropyl 2-benzoylamino-3-hydroxy-3-phenylpropionate,

1H NMR (400MHz, CDCl3): δ 1.27 (6H, d, J = 6.3Hz), 4.73 (1H, d, J = 6.1Hz), 5.07 (1H, sept, J = 6.3Hz), 5.17 (1H, dd, J = 6.6, 3.0Hz), 5.40 (1H, m), 6.91 (1H, d, J = 6.1Hz), 7.26-7.34 (5H, m), 7.42-7.46 (2H, m), 7.51-7.55 (1H, m), 7.73-7.76 (2H, m) ppm
13C NMR (100 MHz, CDCl3): δ 20.65, 20.71, 58.88, 69.41, 74.52, 125.03, 126.15, 127.02, 127.23, 127.70, 131.14, 132.15, 138.17, 167.81, 167.88 ppm

Isopropyl 2-benzoylamino-3-cyclohexyl-3-hydroxypropionate

¹ H-NMR (400 MHz, CD3OD): δ 0.80-1.27 (5H, m), 1.58-1.79 (5H, m), 1.96-
1.99 (1H, m), 3.42 (1H, m), 4.00 (1H, d, J = 2.3Hz).

Isopropyl 2-benzoylamino-3- (4-chlorophenyl) -3-hydroxypropionate,

1H NMR (400MHz, CDCl3): δ1.20 (3H, d, J = 6.4Hz), 1.18 (3H, d, J = 6.4Hz), 5.00 (1H, sept, J = 6.4Hz), 5.05 (1H, dd, J = 6.3, 2.8Hz), 5.29 (1H, m), 6.89 (1H, brs), 7.14-7.23 (4H, m), 7.34
7.50 (3H, m), 7.68 (2H, d, J = 8.1Hz)
13C NMR (100 MHz, CDCl3) δ 20.69, 20.72, 58.88, 69.65, 73.93, 126.16, 126.48, 127.37, 127.76, 131.31, 131.88, 132.75, 136.84, 167.65, 167.89

Isopropyl 2-benzoylamino-3-hydroxy-3- (thiophen-2-yl) propionate

1H NMR (400MHz, CDCl3): δ1.22 (3H, d, J = 6.3Hz), 1.24 (1H, d, J = 6.0Hz), 5.03 (1H, sept, J = 6.0Hz), 5.16 (1H, dd, J = 7.4,3.3Hz), 5.60 (1H, m), 6.83-6.89 (2H, m), 6.98 (1H, brd, J = 6.1Hz), 7.17 (1H, m), 7.37-7.72 (3H , m), 7.74 (2H, m)
13C NMR (100 MHz, CDCl3) δ 20.69, 20.72, 58.71, 6975, 71.60, 123.49, 124.24, 125.55, 126.26, 127.73, 131.28, 131.99, 141.53, 167.31, 168.34

Isopropyl 2-benzoylamino-3-hydroxy-3- (naphthalen-2-yl) propionate,

1HNMR (400MHz, CDCl3): δ 1.16 (6H, d, J = 6.3Hz), 5.00 (1H, sept, J = 6.3Hz) 5.18 (1H, dd, J = 6.8, 3.0Hz), 5.50 (1H, d, J = 3.0Hz), 6.56 (1H, brd, J = 6.6Hz), 7.33-7.47 (6H, m), 7.67-7.76 (6H, m)
13C NMR (100 MHz, CDCl 3) δ 20.64, 20.70, 58.95, 69.46, 74.64, 122.97, 124.13,
124.99, 125.17, 126.16, 126.66, 126.94, 126.98, 127.70, 131.16, 132.03, 132.11,
132.14, 135.76, 167.84, 167.89

Isopropyl 2-benzoylamino-3-hydroxytridecanoate,

1H-NMR (400 MHz, CDCl3): δ 0.82 (3H, t, J = 6.9 Hz), 1.12-1.21 (14H, m), 1.23 (3H, d, J = 6.2 Hz), 1.25 (3H, d, J = 6.2Hz), 1.26-1.57 (4H, m), 3.50 (1H, brs),
3.97-4.04 (1H, m), 4.77 (1H, dd, J = 6.8,3.0Hz), 5.01-5.12 (1H, m), 7.10 (1H, d, J = 6.8Hz),
7.36-7.42 (2H, m), 7.44-7.49 (1H, m), 7.75-7.78 (2H, m) ppm.
13C-NMR (100 MHz, CDCl 3): δ 13.09, 20.75, 21.66, 24.69, 28.30,
28.47, 28.48, 28.55, 28.58, 30.88, 32.27, 57.53, 69.08, 72.40, 126.21, 127.66,
131.03, 132.39, 167.09, 168.93 ppm.

Isopropyl 3-hydroxy-3-phenyl-2-pivaloylaminopropionate,

Isopropyl 3- (4-chlorophenyl) -3-hydroxy-2-pivaloylaminopropionate,

MeCN / 50mMAcONH4 = 60/40 230nm L-column ODS (4.6 x 250mm), 1 mL / min, 40 ° C 4.84min
anti: rt 7.45 min, syn: rt8.01 min

Isopropyl 3-hydroxy-2-pivaloylamino-3- (thiophen-2-yl) propionate,

MeCN / 50mMAcONH4 = 60/40 230nm L-column ODS (4.6 × 250mm), 1 mL / min, 40 ℃
anti: rt 5.58 min ,, syn: rt5.91 min

2-Benzoylamino-3-cyclohexyl-3-hydroxypropionic acid phenyl

MeCN / 50mMAcONH4 = 2 / 8- (15min) -1/0 (5minHOLD), 254nm L-column ODS (4.6 × 250mm), 1 mL / min, 40 ℃ anti ： rt14.10 min, syn ： rt14.29 min

Isopropyl 3-cyclohexyl-3-hydroxy-2-pivaloylaminopropionate

MeCN / 50mMAcONH4 = 55/45, 230nm
L-column ODS (4.6 × 250mm), 1 mL / min, 40 ℃ anti ： rt11.42 min, syn ： rt10.96 min

Benzyl 2-benzoylamino-3-hydroxy-4-methylpentanoate,

1H-NMR (400 MHz, CDCl3): δ0.88 (3H, d, J = 6.6 Hz), 0.94 (3H, d, J = 6.6 Hz), 1.60-1.67 (1H, m), 3.56 (1H, dd, J = 8.6,3.0Hz), 4.93 (1H, dd, J = 7.1,3.0Hz),
5.16 (1H, d, J = 12.4Hz), 5.22 (1H, d, J = 12.4Hz), 7.07 (1H, d, J = 7.1Hz), 7.24-7.34 (5H, m),
7.35-7.41 (2H, m), 7.44-7.49 (1H, m), 7.72-7.77 (2H, m) ppm.
13C-NMR (100 MHz, CDCl 3): δ 17.92, 18.03, 30.55, 55.27, 66.58,
77.91, 126.15, 127.36, 127.59, 127.65, 127.66, 130.99, 132.46, 133.94, 166.50,
169.81 ppm.

(Example 12)
(Escherichia coli co-expressing mutant carbonyl reductase was used with isopropyl (2R, 3R) -2-benzoylamino-3-cyclohexyl-3-hydroxypropionate and (2R, 3R) -2-benzoylamino-3-cyclohexyl Synthesis of ethyl 3-hydroxypropionate)
The procedure was the same as in Examples 1 and 2, except that a gene encoding a mutant carbonyl reductase in which phenylalanine at the 88th amino acid of SEQ ID NO: 2 was converted to isoleucine was used. 1 mL of the obtained bacterial cell broth was collected by centrifugation, and 70 μL of a buffer solution (100 mM Tris containing 10% ammonium sulfate and 3% glucose (final pH 7.5)) was added and mixed well. 10 μL of 1 g / L NADP + (manufactured by Oriental Yeast), 10 μL of glucose dehydrogenase (manufactured by Amano Pharmaceutical, 10 unit / ml), 10 μL of 50% glucose, 10 μL of 2.5 g / L 2-benzoylamino-3- After adding isopropyl cyclohexyl-3-oxopropionate (DMSO solution) or 10 g / L ethyl 2-benzoylamino-3-cyclohexyl-3-oxopropionate (DMSO solution), the mixture was shaken at 33 ° C. for 24 hours. After completion of the reaction, 750 μL of 2-propanol was added to the reaction solution, and the reduced product was quantified. The amount of isopropyl (2R, 3R) -2-benzoylamino-3-cyclohexyl-3-hydroxypropionate and ethyl (2R, 3R) -2-benzoylamino-3-cyclohexyl-3-hydroxypropionate was 6.2 μg and 29, respectively. 3 μg was produced. The diastereoselectivity is 100% d. e. It was anti. The optical purity is 100% e.e. e. The solid was 2R, 3R.

FIG. 1 shows an outline of the reaction in the synthesis example.

[Sequence Listing]
SEQUENCE LISTING
<110> API Corporation
<120> A method for producing an optically active hydroxyl amino acid derivative
<130> A61442A
<160> 4
<210> 1
<211> 750
<212> DNA
<213> Exiguobacterium sp.
<400> 1
atgaaatata cggtgattac tggagcaagt tcaggaattg gatatgagac ggcaaaacta 60
ctcgcaggaa aaggaaaatc actcgttctc gtcgcgcgac ggacgtctga gctcgaaaaa 120
cttcgggatg aagtcaaaca aatctcacca gatagtgatg tcatcctcaa gtcggtcgat 180
ctcgcagata accaaaatgt ccatgattta tatgagggat taaaggaact cgacatcgaa 240
acgtggatca acaatgctgg attcggcgat tttgatctcg tccaggacat tgagctcggg 300
aaaatcgaga aaatgcttcg cttgaacatc gaggcgctaa cgattctatc gagtctgttc 360
gtccgcgatc atcatgacat cgaaggaacg acgctcgtca atatctcgtc agcaggtggc 420
taccggatcg tgccgaacgc ggtcacgtat tgcgcgacga agttctatgt cagtgcttat 480
acagaagggc tcgcgcaaga actgcaaaaa ggtggggcaa aacttcgggc gaaagtactg 540
gcaccagctg cgactgagac agagtttgcg gatcgttcgc gcggcgaagc aggtttcgac 600
tacagcaaga acgtcaaaaa gtaccatacg gcggctgaaa tggcaggctt cctgcatcag 660
ttgattgaaa gcgacgcgat cgtcggcatc gtcgacggtg agacgtatga gttcgaattg 720
cgtggtccat tgttcaacta cgcaggataa 750
<210> 2
<211> 249
<212> PRT
<213> Exiguobacterium sp.
<400> 2
Met Lys Tyr Thr Val Ile Thr Gly Ala Ser Ser Gly Ile Gly Tyr Glu
1 5 10 15
Thr Ala Lys Leu Leu Ala Gly Lys Gly Lys Ser Leu Val Leu Val Ala
20 25 30
Arg Arg Thr Ser Glu Leu Glu Lys Leu Arg Asp Glu Val Lys Gln Ile
35 40 45
Ser Pro Asp Ser Asp Val Ile Leu Lys Ser Val Asp Leu Ala Asp Asn
50 55 60
Gln Asn Val His Asp Leu Tyr Glu Gly Leu Lys Glu Leu Asp Ile Glu
65 70 75 80
Thr Trp Ile Asn Asn Ala Gly Phe Gly Asp Phe Asp Leu Val Gln Asp
85 90 95
Ile Glu Leu Gly Lys Ile Glu Lys Met Leu Arg Leu Asn Ile Glu Ala
100 105 110
Leu Thr Ile Leu Ser Ser Leu Phe Val Arg Asp His His Asp Ile Glu
115 120 125
Gly Thr Thr Leu Val Asn Ile Ser Ser Ala Gly Gly Tyr Arg Ile Val
130 135 140
Pro Asn Ala Val Thr Tyr Cys Ala Thr Lys Phe Tyr Val Ser Ala Tyr
145 150 155 160
Thr Glu Gly Leu Ala Gln Glu Leu Gln Lys Gly Gly Ala Lys Leu Arg
165 170 175
Ala Lys Val Leu Ala Pro Ala Ala Thr Glu Thr Glu Phe Ala Asp Arg
180 185 190
Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys Lys Tyr
195 200 205
His Thr Ala Ala Glu Met Ala Gly Phe Leu His Gln Leu Ile Glu Ser
210 215 220
Asp Ala Ile Val Gly Ile Val Asp Gly Glu Thr Tyr Glu Phe Glu Leu
225 230 235 240
Arg Gly Pro Leu Phe Asn Tyr Ala Gly
245
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 3
ggaggcgaat tcatgaaata tacggtgatt 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 4
taatcactgc agttatcctg cgtagttgaa 30

Claims (8)

The following general formula (1)

(In the formula, R ¹ represents an alkyl group, an aryl group, or a heterocyclic group; R ² represents an alkyl group having 4 or more carbon atoms, an alkoxy group having 4 or more carbon atoms, an aryl group, or a heterocyclic group; ³ represents an alkyl group or an aryl group, and the alkyl group, alkoxy group, aryl group and heterocyclic group in R ¹ to R ³ may be substituted.
A microbial cell and / or a treated product thereof having the ability to stereospecifically reduce the carbonyl group at the β-position of the 3-oxoamino acid derivative represented by the formula (1) is represented by 3 -By reacting with an oxo amino acid derivative, the following general formula (2)

(Wherein R ¹ , R ² and R ³ are as defined above)
An optically active 3-hydroxyamino acid derivative represented by the formula: Absidia genus, Acremonium genus, Agrobacterium genus, Alcaligenes genus, Arthrobacter genus, Bacillus genus, Brevibacterium genus Brevundimonas genus, Cellulomonas genus, Citrobacter genus, Comamonas genus, Delftia genus, Mycobacterium genus, Nocardioides genus, Ochrobactrum genus, Paracoccus genus, Pseudomonas genus, Rhizobium genus, Rhodococccus genus, Serratia genus, Sinorhizobium genus, Streptomyces genus, Streptomyces genus, Streptomyces Valid Borax (Va The method for producing an optically active 3-hydroxyamino acid derivative according to claim 1, which is a microorganism belonging to the genus riovorax) or the genus Exiguobacterium. The method for producing an optically active 3-hydroxyamino acid derivative according to claim 1, wherein the microorganism is a transformant expressing any one of the following DNAs (A) to (F):
(A) DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A 3-oxoamino acid derivative represented by the general formula (2) having an amino acid sequence having 50% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and represented by the general formula (1) A DNA encoding a protein having an enzymatic activity to be converted into an optically active 3-hydroxyamino acid derivative.
(C) a 3-oxo amino acid derivative consisting of an amino acid sequence represented by general formula (1) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 2 A DNA encoding a protein having an enzyme activity which is converted into an optically active 3-hydroxyamino acid derivative represented by the general formula (2).
(D) DNA having the base sequence set forth in SEQ ID NO: 1.
(E) A 3-oxoamino acid derivative represented by the general formula (2) is hybridized with a DNA comprising the base sequence set forth in SEQ ID NO: 1 or a complementary sequence thereof under stringent conditions and represented by the general formula (1) A DNA encoding a protein having an enzymatic activity to be converted into the optically active 3-hydroxyamino acid derivative represented.
(F) a 3-oxo amino acid comprising a base sequence in which one or several bases are substituted, deleted or added in the base sequence set forth in SEQ ID NO: 1 and its complementary strand, and represented by the general formula (1) A DNA encoding a protein having an enzyme activity for converting a derivative into an optically active 3-hydroxyamino acid derivative represented by the general formula (2). The method for producing an optically active 3-hydroxyamino acid derivative according to claim 3, wherein the transformant is transformed by integrating the DNA on a chromosome. The method for producing an optically active 3-hydroxyamino acid derivative according to claim 3, wherein the transformant is a transformant transformed with a vector containing the DNA. In the compound of General formula (1) and (2), R < ² > is an aryl group, The manufacturing method of the optically active 3-hydroxyamino acid derivative as described in any one of Claims 1-5 characterized by the above-mentioned. The method for producing an optically active 3-hydroxyamino acid derivative according to any one of claims 1 to 6, wherein the optical purity of the general formula (2) is 98% ee. The optically active 3-hydroxy according to any one of claims 1 to 7, wherein the absolute configuration of the general formula (2) is 2R or 3R as represented by the following general formula (3). A method for producing an amino acid derivative.

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