WO2005005648A1

WO2005005648A1 - Novel process for producing optically active carboxylic acid

Info

Publication number: WO2005005648A1
Application number: PCT/JP2004/009932
Authority: WO
Inventors: Hiroshi Kawabata; Makoto Ueda
Original assignee: Mitsubishi Pharma Corporation
Priority date: 2003-07-11
Filing date: 2004-07-12
Publication date: 2005-01-20
Also published as: JPWO2005005648A1

Abstract

An enoate reductase, cells containing the same, a preparation comprising the cells, or a culture obtained by culturing the cells is caused to act on a 4-halocrotonic acid derivative represented by the following general formula (I): to produce an optically active 4-halobutyric acid derivative represented by the following general formula (II). (In the formulae (I) and (II), R represents halogeno, nitro, hydroxy, optionally substituted alkyl, optionally substituted aryl, or optionally substituted alkoxy; X represents halogeno; and A1 and A2 each represents hydrogen or halogeno.)

Description

Specification

Production method of new optically active carboxylic acid

Technical field

[0001] The present invention relates to a carbon-carbon double bond of a 4,18-mouth crotonic acid derivative using enoate reductase, a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell. To an optically active 4-halobutyric acid derivative which is a compound useful as an intermediate material for pharmaceuticals, agricultural chemicals and the like. The present invention also provides (R) -N- (4,4,4_trifluoro-2_methinolebutyl) _3_ [2-methoxy_4_ (o_tolylsulfonylcarbamoy) utilizing the method for producing the 4_halobutyric acid derivative. Nore) benzyl] _1-methinoleindol-5-carboxamide.

Background art

[0002] Optically active 4-halobutyric acid derivatives are industrially useful compounds as intermediate raw materials for pharmaceuticals, agricultural chemicals and the like. For example, (R) —4,4,4_trifluoromethyl-2-methylbutyric acid (2R—methyl-4,4,4—trifluorobutanoic acid) is useful as a leukotriene antagonist (R) —4,4 It is known to be an intermediate for the synthesis of 4-methyl-2-butyrylamine, 4-trifluorene (EP-A-489548). As a method for obtaining an optically active 4-octabutyric acid derivative, there has been known a method for reducing a 4,8-octacrotonic acid derivative using a complex catalyst comprising an optically active phosphine conjugate and a ruthenium compound (European Patent Application Publication No. 673911). However, such a reaction must be performed under high pressure, and the catalyst used is expensive.

[0003] Enoate reductase is an enzyme that catalyzes the reaction of reducing the carbon-carbon double bond of enoate (Henoate), and its presence has been reported in microorganisms such as Clostridia (Journal). of Biological Chemistry, vol. 276, No. 8,

P5779-5787, 2001). Asymmetric reduction of various enoates using microorganisms having enoate reductase activity has been reported (Studies in Natural Products Chemistry, vol. 20, 817, 1998), but carbon at position 4 of enoates is halogenated. As for the 4-halocrotonic acid derivative, there was no report of asymmetric reduction using enoate reductase. Disclosure of the invention It is an object of the present invention to provide a novel method for producing an optically active 4-halobutyric acid derivative with higher optical purity at a low cost and simply. The present invention also relates to (R) -N- (4,4,4-trifluoro-2-methylbutyl) _3_ [2-methoxy-14- (o-tolylsulfonylcarbamoinole) benzyl] -11-methylindole —To provide a new method for producing carboxamide.

The present inventors have conducted intensive studies on a method for producing an optically active 4-halobutyric acid derivative in order to solve the above-mentioned problems. As a result, the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 was determined. Has been found to catalyze the asymmetric reduction reaction of the 4-halocrotonic acid derivative used as a raw material. Further, a transformed cell into which a gene encoding the protein has been introduced is prepared, and the cell, a preparation of the cell, or a culture solution obtained by culturing the cell is used as a starting material for the production of 418-croton. It has been found that by acting on an acid derivative, it is possible to obtain an optically active 4-halobutyric acid derivative as a target substance with high optical purity and high concentration. Furthermore, by using (R) -4,4,4-trifluo-2-methylbutyric acid obtained by this method, (R) —N— (4,4,4-trifluoro-2-methylbutyl) -3 — It was found that [2-methoxy-14- (o-tolylsulfonylcarbamoinole) benzyl] _1-methylindole-5-carboxamide can be efficiently produced. Thus, the present invention has been completed.

[0006] That is, the present invention is as follows.

(1) The following general formula (I)

[Chemical 1]

Is reacted with a 4-halocrotonic acid derivative represented by the following formula, or a cell containing the same, a preparation of the same cell, or a culture solution obtained by culturing the same cell. [Chemical 2]

A method for producing an optically active 4-halobutyric acid derivative represented by the general formula (II), which comprises producing an optically active 4-halobutyric acid derivative represented by (Formulas (1) and (II), R represents a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted alkoxy group, X represents a halogen atom, A and

1 and A represent a hydrogen atom or a halogen atom).

2

(2) The method for producing (1), wherein the enoate reductase is a protein represented by the following (A), (B) or (C):

(A) a protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or

(B) an amino acid sequence having at least 50% homology with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and having a 4-octacrotonic acid derivative represented by the general formula (I); A protein having an enzymatic activity for converting into an optically active 4-halobutyric acid derivative represented by the general formula (Π).

(C) In the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, one or several amino acids are substituted, deleted or added, and are represented by the general formula (I). A protein having an enzymatic activity for converting a halocrotonic acid derivative into an optically active 4-halobutyric acid derivative represented by the general formula (II).

(3) The method according to (1), wherein the cell containing enoate reductase is a cell transformed with a DNA encoding enoate reductase.

(4) The method according to (3), wherein the cell force S is a cell transformed with a DNA encoding enoate reductase, and the cell is a cell transformed by integrating the DNA on a chromosome.

(5) Cell force transformed with DNA encoding enoate reductase DN The method according to (3), which is a cell transformed with a vector containing A.

(6) The method for producing any one of (3) and (5), wherein the DNA encoding enoate reductase is a DNA encoding a protein represented by the following (A), (B) or (C): :

(7) The method according to any one of (3) to (5), wherein the DNA encoding enoate reductase is a DNA represented by the following (D), (E) or (F):

(D) DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or

(E) a 4-halocrotonic acid derivative represented by the general formula (I), which has a base sequence that hybridizes under stringent conditions with the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a complementary sequence thereof, DNA encoding a protein having an enzymatic activity for converting into an optically active 4-halobutyric acid derivative represented by the formula (Π).

(F) In the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, one or several nucleotides are substituted, deleted or added, and comprise a complementary nucleotide sequence and represented by the general formula (I) DNA encoding a protein having an enzymatic activity that converts a 4-halocrotonic acid derivative into an optically active 4-halobutyric acid derivative represented by the general formula (II).

(8) In the compounds of the general formulas (I) and (II), R is an S methyl group, and X, A and A

(1) The method according to any one of (1) to (7), wherein 1 2 is a fluorine atom.

(9) According to the method of (8), 4,4,4_trifluorofluoric acid represented by the general formula (ΠΙ) is converted to (R) -4,4,4_trifluorofluoride represented by the general formula (IV) Converting to 2-methylbutyric acid, the (R) Converting -4,4,4_trifluoromethyl-2-methylbutyric acid to (R) -4,4,4_trifluoromethyl-2-methylbutylamine represented by the general formula (V), And reacting the compound represented by the general formula (VI) with the above (R) -4,4,4_trifluoro1-2-methylbutylamine to obtain the (R) _N_ ( (R) _N_, including the step of producing 4,4,4_trifluoro- 2_methylbutyl) _3_ [2-methoxy-4_ (o_tolylsulfonylcarbamoinole) benzyl] _1_methylindolinole_5_carboxamide (4,4,4_Trifluoro-2-methylbutyl) _3_ [2-methoxy_4_ (o_tolylsulfonylcarbamoyl) benzyl] _1_methylindoleno-5_carboxamide.

[Formula 3]

(VII) Best mode for carrying out the invention

The present invention is described in detail below.

<1> Method for producing optically active 4-halobutyric acid derivative In the present invention, enoate reductase or a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell is used as a reaction substrate for the following general formula (I):

By acting on a 4-halocrotonic acid derivative represented by the following formula, the carbon 'carbon: heavy bond of the compound is asymmetrically reduced to give the following general formula (II)

[Formula 5]

To produce an optically active 4-halobutyric acid derivative represented by the formula:

In the above general formulas (I) and (II), R represents a halogen atom, a nitro group, a hydroxy group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted X is a halogen atom; A and A are a hydrogen atom or a halo group; an aryl group or an optionally substituted alkoxy group;

1 2

Indicates a gen atom.

Here, examples of the alkyl group include a methynole group, an ethyl group, an n-propyl group, an isopropyline group, a cyclopropyl group, an n_butyl group, an isobutyl group, a tertiary butyl group, an n-pentyl group, Examples include straight-chain, branched-chain and cyclic alkyl groups having 118 carbon atoms such as isopentyl group, neopentyl group, tertiary pentyl group, isoamyl group, n-hexyl group and the like. Of these, a methyl group or an ethyl group is particularly preferable, and an alkyl group having 14 to 14 carbon atoms is particularly preferable. Examples of aryl groups include phenyl, mesityl, and naphthyl. I can get lost. Examples of the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a tertiary butoxy group, and the like. Among them, an alkoxy group having 14 to 14 carbon atoms is preferable.

[0010] The alkyl group, aryl group and alkoxy group may be substituted. The substituent is not particularly limited as long as it does not adversely affect the asymmetric reduction reaction, but specifically, an alkyl group, an aryl group, an alkoxy group, a halogen group, a cyano group, an amino group, Examples include a nitro group and a hydroxy group. Therefore, in the general formulas (I) and (Π), specific examples of the substituted alkyl group include a benzyl group, a phenethyl group, a trifluoromethyl group, a cyanomethyl group, an aminomethyl group, a hydroxymethyl group, a nitromethyl group, and a methoxymethyl group. A xymethyl group and the like. Specific examples of the substituted aryl group include a chlorophenyl group, an aminophenyl group, a hydroxyphenyl group, a nitrophenyl group, and a methoxyphenyl group. Specific examples of the substituted alkoxy group include a benzyloxy group, a phenoxy group, and a trifluoromethoxy group.

[0011] X represents a halogen atom, and A and A represent a hydrogen atom or a halogen atom.

1 2

Is more preferably a fluorine atom, preferably a fluorine atom, a chlorine atom or a bromine atom. It is particularly preferred that all of X, A and A are fluorine atoms.

1 2

R in the above general formulas (I) and (II) is preferably an alkyl group having 14 to 14 carbon atoms, a benzyl group or a phenyl group, and more preferably an alkyl group having 14 to 14 carbon atoms. And particularly preferably a methyl group.

The compound represented by the general formula (I) has a molecular weight of 1,000 or less, preferably 500 or less, more preferably 300 or less. Specifically, for example, the compound represented by the general formula (III) 4,4,4_Trifluorotiglic acid and the like. The compound represented by the general formula (I) is, for example, an aldehyde represented by the following general formula (VIII) (X, A

1, A is the same substituent as above 2

The Wittig reaction with the desired stabilized ylide is carried out according to the method described in the 4th Edition Experimental Chemistry Course (Vol. 19, _p. 62, 1992), and the obtained unsaturated ester is easily hydrolyzed. Can be synthesized. [0014] [Formula 6]

Further, the compound represented by the general formula (Π) has a molecular weight of 1000 or less, preferably 500 or less, more preferably 300 or less. Specifically, the compound represented by the general formula (IV) (R) _4,4,4-trifluro mouth-2-methylbutyric acid and the like.

[Formula 7]

[0016] Enoate reductase generally refers to an enzyme that catalyzes the reduction reaction of the carbon'carbon double bond of the enoate (Henoate) (Studies in Natural Products Chemistry, vol. 20, p817, 1998). A protein capable of asymmetrically reducing the carbon'carbon double bond of a 4-halocrotonic acid derivative to produce an optically active 4-halobutyric acid derivative. Here, the optical purity of the generated 4-halobutyric acid derivative is preferably at least 60% ee, more preferably at least 90% ee, and at least 98% ee. It is particularly preferred that the ee is 99.5% ee. Such an activity can be measured by measuring the initial rate of decrease of NADH in a reaction system containing a 4-nitrocrotonic acid derivative as a substrate and further containing NADH as a coenzyme.

[0017] The enoate reductase that can be used in the production method of the present invention is not particularly limited as long as it has the above activity, and examples thereof include those having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. These are enoate reductases derived from Moorella thermoautotrophica and Clostridium acetobutylicum, respectively. Further, in the present invention, homologs of these homologs having the above-mentioned enzyme activity may be used. Homologs include, for example, those having an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 within a range that does not impair the activity. be able to. Here, the term “several” specifically means 20 or less, preferably 10 or less, and more preferably 5 or less.

[0019] The homolog has 35% or more, preferably 50% or more, more preferably 80%, particularly preferably 95% or more homology with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. It may be protein. Incidentally, the homology search of the above-mentioned protein can be performed using, for example, a program such as FASTA or BLAST for GenBank or DNA Databank of JAPAN (DDBJ). A homology search was performed using the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 with the BLAST program against GenBank, and as a result, the sequence of SEQ ID NO: 2 was 2-enoate reductase derived from Clostridium tyrobutyricum (Accession No. CAA71086) and 59% showed homology, eye ti歹¹ of SEQ ID NO: 4 J is Clostridium Tyrobutyricum from 2- enoate reductase (Accession No.

CAA71086) and 49% homology. In addition, the sequences of SEQ ID NO: 2 and SEQ ID NO: 4 showed 50% homology with each other. In this specification, Accession No. indicates that of GenBank.

The enoate reductase used in the production method of the present invention may be any microorganism having enoate reductase activity using a probe prepared based on the nucleotide sequence of a gene encoding a part or all of enoate reductase. Can be obtained by isolating a DNA encoding enoate reductase from E. coli and expressing the DNA in a host such as E. coli. It can also be obtained by purifying from a microorganism having enoate reductase activity, for example, a bacterium of the genus Clostridium or Moorella. As a method for obtaining enoate reductase from bacterial cells, for example, the method described in Eur. J. Biochem. Vol. 97, pl03 (1979) can be referred to.

[0021] Examples of Clostridium bacteria include, for example, Clostridium acetobutylicum ATCC824 strain, which has enoate reductase that can be suitably used in the present invention. For example, as a bacterium belonging to the genus Moorella, for example, Moorella thermoautotrophica

The strain DSM1974 has enoate reductase that can be suitably used in the present invention. The former is an ATCC (American Type Culture Collection) online catalog (

http://www.atcc.org/) and the ATCC power is available. Also, the latter is an online catalog of DSZ (Deutsche ^ ammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures)).

http://www.dsmz.de/) and the DSMZ force is also available.

In the production method of the present invention, the 4_nodrocrotonic acid derivative may be allowed to react with a purified enzyme of enoate reductase, but a cell containing enoate reductase, a preparation of the cell, or the cell is used. The culture solution obtained by culturing may be reacted with a 4,18-mouth crotonic acid derivative to produce an optically active 4,18-mouth butyric acid derivative. The cell containing enoate reductase is preferably a cell transformed with DNA encoding enoate reductase.

In this case, the DNA encoding enoate reductase includes a DNA encoding enoate reductase having the amino acid sequence of SEQ ID NO: 2 or 4. Further, a 4-halocrotonic acid derivative having an amino acid sequence IJ having 50% or more homology with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and represented by the general formula (I) is represented by the general formula (Π) )) May be a DNA encoding a protein having an enzymatic activity for converting into an optically active 4-halobutyric acid derivative. Specifically, a DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 can be mentioned. Such DNA can be obtained by PCR using a primer designed based on the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Furthermore, in the production method of the present invention, a DNA homolog of a DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein having an enoate reductase activity may be used.

Here, a homolog is defined as a protein having an enoate reductase activity, in which one or several bases are deleted, substituted, or added to the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3. Includes DNA consisting of the base sequence and the complementary strand of the elementary sequence. Here, the term "several" specifically refers to 60 or less, preferably 30 or less, and more preferably 10 or less. It is.

[0025] The homolog may be a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 1 or 3 or a complementary strand thereof under stringent conditions and encodes a protein having an enoate reductase activity. Les ,. Here, “DNA that hybridizes under stringent conditions” refers to a method using a probe DNA and a colony hybridization method, a plaque hybridization method, or a Southern blot hybridization method under stringent conditions. The term "stringent conditions" refers to DNA obtained by performing the hybridization method or the like.For example, in the colony hybridization method and the plaque hybridization method, After performing hybridization at 65 ° C in the presence of 0.7-1.0 M sodium chloride using a filter on which the DNA or a fragment of the DNA has been immobilized, a 0.1 X 2 SSC solution (The composition of 1 X SSC is 150 mM sodium chloride, 15 mM sodium citrate). Conditions for washing the filter under C conditions can be mentioned.

[0026] DNA encoding enoate reductase can be isolated, for example, by the following method. First, enoate reductase is purified from microbial cells or the like by the above-mentioned method or the like, and then the N-terminal amino acid sequence is analyzed. N-terminal amino acid sequence analysis is performed by cleaving the purified protein with enzymes such as lysyl endopeptidase and V8 protease, purifying the peptide fragment by reverse-phase liquid chromatography, etc., and then analyzing the amino acid sequence with a protein sequencer. By determining the amino acid sequence of Using primers designed based on the determined amino acid sequence, and performing PCR using the chromosomal DNA or cDNA library of the enoate reductase-producing microbial strain as type III, the DNA encoding the enoate reductase is obtained. Able to obtain a part (DNA fragment). Furthermore, the above DNA fragment is probed from a library or cDNA library obtained by introducing a restriction enzyme digest of chromosomal DNA of an enoate reductase-producing microorganism strain into a phage, a plasmid, etc., and transforming E. coli. By performing colony hybridization, plaque hybridization, and the like, DNA encoding enoate reductase can be obtained.

[0027] In addition, the base sequence of the DNA fragment obtained by the PCR is analyzed, and Then, PCR primers were designed to extend outside the sequenced region, and the chromosomal DNA of the enoate reductase-producing microbial strain was digested with appropriate restriction enzymes, and the DNA cyclized by the autocyclization reaction was used. DNA encoding enoate reductase can be obtained by performing invese PCR (Genetics vol. 120, p621-623 (1988)) as type 铸.

[0028] The DNA encoding enoate reductase can also be obtained by chemically synthesizing DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

[0029] A person skilled in the art would be able to introduce a site-specific mutation into the DNA of SEQ ID NO: 1 or 3 (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 Aroach, IRL Press, p200 (1991)), etc. By introducing an additional mutation, DNA encoding enoate reductase that can be used in the production method of the present invention can be obtained.

[0030] Further, based on all or a part of the amino acid sequence of SEQ ID NO: 2 or 4, or all or a part of the nucleotide sequence of SEQ ID NO: 1 or 3, based on a database such as GenBank or DDBJ. It is also possible to obtain a base sequence information of a DNA homologue encoding enoate reductase by conducting a homology search. A person skilled in the art can obtain a DNA encoding enone reductase by PCR or the like from a deposited strain (available from ATCC, DSMZ, etc.) based on this base sequence information.

[0031] Further, DNA encoding enoate reductase was prepared from any microorganism having enoate reductase activity using a DNA having all or a part of the base sequence of SEQ ID NO: 1 or 3 as a probe. The DNA is subjected to hybridization under stringent engineering conditions by colony hybridization, plaque hybridization, Southern blot hybridization, or the like, and hybridized. It can also be obtained by obtaining DNA. Here, the term “part” refers to a 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.

[0032] Each hybridization was performed by Molecular Cloning 2nd Edt., Cold Spring Harbor. It can be carried out according to the method described in Laboratory Press (1989) and the like.

The enoate reductase expression vector is provided by inserting the DNA encoding enoate reductase isolated as described above into a known expression vector so that it can be expressed. By culturing cells transformed with this expression vector, enoate reductase can be obtained from the cells. Transformed cells can also be obtained by incorporating DNA encoding enoate reductase into a chromosomal DNA of a known host cell so that it can be expressed.

[0034] As a method for producing a transformed cell, specifically, an expression vector constructed by incorporating DNA encoding enoate reductase into a plasmid vector or a phage vector stably existing in a microorganism is used. Or the DNA encoding enoate reductase must be introduced directly into the host genome and the genetic information must be transcribed and translated.

[0035] At this time, if the DNA encoding the enoate reductase does not contain a promoter that can be expressed in the host microorganism, an appropriate promoter is placed 5 'upstream of the DNA strand encoding the enoate reductase. Must be incorporated. Further, 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 a microorganism used as a host, provided that they are promoters and terminators. Regarding promoters and terminators, for example, "Basic Lectures on Microbiology 8Genetic Engineering 'Kyoritsu Shuppan'," especially for yeast, Adv. Biochem. Eng. Vol. 43, P75-102 (1990), Yeast vol. 8, It is described in detail in p423_488 (1992).

[0036] The host microorganism to be transformed for expressing the enoate reductase of the present invention is not particularly limited as long as the host itself does not adversely affect the present reaction. For example, the following microorganisms can be mentioned.

[0037] The genus Escherichia, the genus Bacillus, the genus Pseudomonas, the genus Serratia, the genus Brevibacterium, the genus Corynebacterium, the genus Streptococcus (Streptococcus) An established host vector system belonging to the genus (Lactobacillus). [0038] Actinomycetes having an established host vector system belonging to the genera Rhodococcus, Streptomyces, and the like.

[0039] The genus Saccharomyces, the genus Kluyveromyces, the genus Schizosaccharomyces, and the genus Saccharomyces (

Zygosaccharomyces) j¾ _¾ Yarowizo (Yarrowia Bruno Chen, Torikosuhoron (Tnchosporon) / Woo, Rodosuporijiumu (Rhodosporidium) genus Hansenula (Hansenula) spp, Pichia (Pichia) sp., Are established in the host-vector system belonging to such Candida (Candida) spp Yeast.

[0040] An established mold of a host vector belonging to the genus Neurospora, the genus Aspergillus, the genus Cephalosporium (halosporium C), the genus Trichoderma, or the like.

[0041] Among the above microorganisms, the host is preferably a genus Escherichia, a genus Bacillus, a genus Brevibacterium, or a corynebacterium (

Corynebacterium), and particularly preferably, Escherichia and Corynebacterium j.

[0042] The procedure for producing transformed cells, the construction of a recombinant vector suitable for a host, and the culture method of the host are in accordance with techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering. (See, eg, "Molecular Cloning Second Edition," Cold Spring Harbor Laooratory Press (Re, 1989)).

Hereinafter, specific examples of preferred host microorganisms, preferred transformation methods for each microorganism, vectors, promoters, terminators, and the like will be given, but the present invention is not limited to these examples.

[0044] In the genus Escherichia, particularly in Escherichia coli, plasmid vectors include pBR and pUC-type plasmids, and promoters include lac (i3-galatatosidase) and trp (tryptophan operon). , Tac, trc (fusion of lac and trp), λ phage PL, PR and the like. Examples of the terminator include a terminator derived from trpA, phage, and rrnB ribosomal RNA.

[0045] In the genus Bacillus, vectors include pUBlO-based plasmid and pC194-based plasmid. And chromosomes. As the promoter and the terminator, promoters and terminators of enzyme genes such as alkaline protease, neutral protease and α-amylase can be used.

In the genus Pseudomonas, vectors include Pseudomonas' putida (

Pseudomonas putida), Nyu ¹ Pseudomonas - Se Nono Na (Pseudomonas cepacia nona and ⁵ common host vector system to finalize A or plasmids involved in the degradation of Torueni匕合was wide was basically the TOL plasmid A host range vector (including a gene necessary for autonomous replication derived from RSF1010 or the like) pKT240 (Gene, vol. 26, p273_82 (1983)) can be mentioned.

[0047] In the genus Brevibacterium, particularly in Brevibacterium lactofermentum, examples of the vector include a plasmid vector such as pAJ43 (Gene vol. 39, p281 (1985)). Various promoters and terminators used in Escherichia coli can be used as the promoter and terminator.

[0048] The genus Corynebacterium, particularly Corynebacterium 'daltamicum (Corynebacterium

In glutamicum), examples of the vector include a plasmid vector such as pCS11 (Japanese Patent Publication No. 57-183799, pCB101 (Mol. Gen. Genet, vol. 196, pl 75 (1984))).

[0049] The genus Saccharomyces, especially Saccharomyces cerevisiae (

In Saccharomyces cerevisiae), examples of the vector include YRp, YEp, YCp, and Yip plasmids. In addition, promoters and terminators of various enzyme genes such as alcohol dehydrogenase, dalyselanoledehydrido 3-phosphate dehydrogenase, acid phosphatase, / 3_galactosidase, phosphodarycelate kinase, and enolase can be used.

[0050] In the genus Schizosaccharomyces, examples of the vector include a plasmid vector derived from Schizosaccharomyces bomb described in Mol. Cell. Biol. Vol. 6, p80 (1986). . In particular, _pAUR224 is commercially available from Takara Shuzo and can be easily used. [0051] In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae, etc. are the most studied among molds, and integration into plasmids and chromosomes is available. Yes, and a promoter derived from extracellular protease ゃ amylase is available (Trends in

Biotechnology vol. 7, p283_287 (1989)).

[0052] 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 systems have been established in plants and animals, especially in animals such as insects using silkworms (Nature vol. 315, p592-594 (1985)), rapeseed, A system for expressing a large amount of a heterologous protein in plants such as corn and potato and a system using a cell-free protein synthesis system such as a cell-free extract of Escherichia coli or wheat germ have been established and can be suitably used.

[0053] In the production method of the present invention, a cell transformed with a DNA encoding enoate reductase may be allowed to act on a 4,18-mouth crotonic acid derivative as a reaction substrate. The transformed cells may be allowed to act as they are in the reaction solution. However, a preparation of the transformed cells, for example, the transformed cells may be treated with an organic solvent such as acetone, dimethyl sulfoxide (DMS〇), toluene, or a surfactant. , Freeze-dried, physically or enzymatically disrupted cell cell preparations, and the enzyme fraction of the present invention extracted from the transformed cells as a crude or purified product Further, those immobilized on a carrier represented by polyacrylamide gel, carrageenan gel or the like may be used. In the production method of the present invention, a culture solution obtained by culturing the cells, that is, a culture solution containing the cells may be directly used.

[0054] In the production method of the present invention, it is preferable to add coenzyme NAD ⁺ or NADH to the reaction solution. The concentration of the additive is 0.001 mM, preferably 100 mM. When these coenzymes are added, it is preferable to regenerate NAD ⁺ generated from NADH into NADH as a preferable regeneration method for improving production efficiency.

(The method using the NAD ⁺ reducing ability of the host microorganism itself,

(ii) Microorganisms or their preparations capable of producing NADH from NAD ⁺ , or glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase A method of adding an enzyme (regenerating enzyme) that can be used to regenerate NADH, such as elemental or organic acid dehydrogenase (malate dehydrogenase, etc.), or

(iii) A method for producing a transformed cell, wherein a gene of the above-mentioned regenerative enzymes, which is an enzyme available for the regeneration of NADH, is introduced into a host simultaneously with DNA encoding enoate reductase.

[0055] Among them, in the above method (i), it is preferable to add glucose, ethanol, 2-propanol, formic acid or the like to the reaction system.

[0056] Further, in the method (ii), microorganisms containing the above-mentioned regenerated enzymes, those obtained by treating the microorganism cells with acetone, those subjected to freeze-drying, those physically or enzymatically crushed, and the like. A cell preparation, a product obtained by extracting the enzyme fraction as a crude product or a purified product, or a product obtained by immobilizing these on a carrier represented by polyacrylamide gel, carrageenan gel, etc. may be used. Alternatively, a commercially available regenerating enzyme may be used. In this case, the amount of the regenerating enzyme used is, specifically, about 0.01 to 100 times, preferably about 0.5 to 20 times the enzyme activity as compared with the enoate reductase of the present invention. Added. Compounds that serve as substrates for the above-mentioned regenerating enzymes, for example, glucose when using glucose dehydrogenase, formic acid when using formate dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, etc. Force required to be added The amount of addition is 0.1 to 20 times molar equivalent, preferably 115 to 5 times molar equivalent, to the 4-halocrotonic acid derivative as a reaction raw material.

[0057] In the above method (iii), a method of integrating DNA encoding enoate reductase and DNA of the above-mentioned regenerating enzymes into a chromosome, and transforming a host by introducing both DNAs into a single vector Alternatively, a method in which both DNAs are separately introduced into a vector and then a host is transformed can be used.In the case of a method in which both DNAs are separately introduced into a vector and the host is transformed, It is necessary to select a vector in consideration of the incompatibility between both vectors. When multiple genes are introduced into a single vector, a method involving ligating regions related to expression control, such as a promoter and a terminator, to each gene, or using multiple cistrons such as ratatosoperon. It can also be expressed as an operon that contains it. [0058] The production method of the present invention contains, for example, a reaction substrate, the transformed cell of the present invention and / or the transformed cell preparation, various coenzymes added as necessary, and a regeneration system thereof. The reaction can be carried out in an aqueous medium or in a mixture of the aqueous medium and an organic solvent.

The compound represented by the general formula (I) serving as a reaction substrate in the production method of the present invention generally has a substrate concentration of 0.01% 90% w / v, preferably 0.1% to 30% w / v. It can be used within the range. The reaction substrate may be added all at once at the beginning of the reaction, but from the viewpoint of reducing the effects of enzyme substrate inhibition and improving the accumulated concentration of the product, continuous or It is desirable to add intermittently.

[0060] The aqueous medium includes water or a buffer, and the organic solvent includes a reaction substrate such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, and dimethyl sulfoxide. The one with high solubility can be used.

[0061] The method of the present invention can be carried out, for example, at a reaction temperature of 4 to 60 ° C, preferably 10 to 45 ° C, and at pH 3 to 11, preferably pH 5 to 8. It is also possible to carry out the reaction using a membrane reactor or the like. It is also effective to remove sodium by adding sodium sulfite to the reaction solution or sealing the reaction solution with nitrogen or argon gas to prevent inactivation of enoate reductase by oxygen. It is.

[0062] The optically active 4-halobutyric acid derivative produced by the method of the present invention is obtained by separating the bacterial cells in the reaction solution by centrifugation or membrane treatment after completion of the reaction, and then removing the organic compound such as ethyl acetate or toluene. Purification can be performed by appropriately combining extraction with a solvent, distillation, column chromatography, crystallization, and the like.

[0063] <2> (R) -N- (4,4,4_trifluoro-2-methylbutyl) _3_ [2-methoxy_4_ (o-tolylsulfonylcarbamoinole) benzyl] _1_methylindoleno_5_carboxamide book (R) -N- (4,4,4-trifluoro-2-methylbutyl) -3- [2-methoxy-4-((o_tolylsulfonylcarbamoinole) benzyl] of the invention _1_methylindoleno_5_carboxami The method for producing the enzyme is as follows: enoate reductase, a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell, are treated with the general formula (4). A trifle Reacting with orotiglic acid to form (R) -4,4,4-monotrifluoro-2-methylbutyric acid represented by the general formula (IV) (a), wherein (R) -4,4 Conversion of 2,4-trifluoro-2-methylbutyric acid into (R) -4,4,4,4-trifluoro-2-methylbutylamine represented by the general formula (V) (b), And reacting the above-mentioned (R) -4,4,4_trifluoro-2-methylbutylamine with the compound represented by the general formula (VI) to produce the (R) _N_ (4 , 4,4_trifluoro-2-methylbutynole) _3_ [2-methoxy-4_ (o_tolylsulfonylcarbamoinole) benzyl] _1 monomethylindole-5-carboxamide.

[0064] [Formula 8]

[0065] Step (a) can be performed in the same manner as described above. In the step (b), a usual method for converting a carboxylic acid to an amine can be used. For example, a method of reacting with ammonia to amidate and then reducing (reaction formula (IX) below), reacting with a pendinoleamine or the like or an ammonia equivalent (eg, HN (SiMe), etc.), and then reducing the reactant And more contact

3 2 A deprotection method such as hydrogenolysis (reaction formula (X)) can be used.

[0066] [Formula 9]

(IX)

(X)

In step (c), (R) _4,4,4-monotrifluoro-2-methylbutynoleamine of formula (V) obtained in step (b) is reacted with a compound of formula (VI) to give a compound of formula (VI) (VII) (R) -N- (4,4,4-Trifluoro 2-methylbutyl) _3_ [2-Methoxy-1- (o-tolylsulfonylcarbamoyl) benzyl] -1-1-methylindole-5_carboxamide Get. This step can be carried out in the same manner as in the usual reaction between amine and carboxylic acid. Specifically, for example, the method is performed in the same manner as disclosed in European Patent Application Publication No. 489547 (Especially Example 5) and European Patent Application Publication No. 489548 (Especially Example 2). be able to. The compound of the general formula (VI) used in this step may be a compound obtained by the above-mentioned production method. For example, as disclosed in the above-mentioned patent gazette, 4_ (5-methoxycarbonyl) 1-Methylindole-3-ylmethyl) _A compound obtained by reacting 3-methoxybenzoic acid (general formula XI) with 2-methylbenzenesulfonamide (general formula ΧΠ) and hydrolyzing the resulting compound Can be used. [0068] [Formula 10]

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

(1) Preparation of Transformed Cells Transformed with Moenoella thermoacetica-derived Enoate Reductase Gene

Based on the DNA sequence (Accession No. Y16136, REGION: 47..2050, SEQ ID NO: 1) encoding 2_enoate reductase (Accession No. CAA76082, SEQ ID NO: 2) derived from Moorella thermoacetica (Moorella thermoacetica) 5 and 6 primers were synthesized. 15 pmol of each of these primers, 10 nmol of dNTP each, 25 ng of genomic DNA of Moorella thermoacetica DSM1974, 10 X buffer for KOD-plus- (Toyobo Co., Ltd.) 5 μ KOD-plus-1 unit (Toyobo Co., Ltd.) , Denaturation (94 ° C, 15 seconds), anneal (57 ° C, 30 seconds), elongation (68.C, 2 minutes) in 30 cycles The 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 was detected.

[0071] The above reaction solution was purified using MinElute PCR Purification kit (manufactured by Qiagen). Purification The DNA fragment thus obtained was digested with restriction enzymes EcoRI and Xbal, subjected to agarose gel electrophoresis, excised from the band of interest, purified and recovered using a Qiagen Gel Extraction kit (manufactured by Qiagen). The obtained DNA fragment was ligated to pUCl18 digested with EcoRI and Xbal using Ligation high (manufactured by Toyobo Co., Ltd.), and Escherichia coli JM109 strain was transformed by a heat shock method.

[0072] The transformed cells were grown on an LB agar medium containing ampicillin (50 ag / mL), and colony direct PCR was performed on the obtained colonies to confirm the size of the imported fragment. The transformed cells containing the DNA fragment of interest are cultured in an LB medium containing 50 μg / mL ampicillin, and the plasmid is purified using the QIAPrepSpin Mini Prep kit (Qiagen). , PUCMtERl. 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 nucleotide sequence of SEQ ID NO: 1.

(2) Preparation of Transformed Cell Transformed with Clonotridium acetobutylicum-derived Enoate Reductase Gene

DNA sequence encoding 2-enoate reductase (Accession No. AAK81302, SEQ ID NO: 4) derived from Clostridium acetobutylicum

Based on Accession No. AE007834, REGION: 859.2853, SEQ ID NO: 3), primers described in SEQ ID NOs: 7 and 8 were synthesized. These primers were used in an amount of 15 pmol each, dNTP each 10 nmol, genomic DNA of Clostridium acetobutylicum ATCC824 25 ng, 10X buffer for KOD-plus— (Toyobo) 5 μL, KOD-plus-1 unit ( with 50's reaction solution containing Toyobo Co., Ltd.), modified (94 ° C, 15禾少), Aninore (57.G, 30禾少), I monkey length (68 ⁰ C, 2 min) 30 A PCR reaction was performed using PTC-200 (manufactured by MJ Research) in a cycle. As a result of analyzing a part of the PCR reaction solution by agarose gel electrophoresis, a band considered to be specific was detected.

[0074] The above reaction solution was purified using MinElute PCR Purification kit (manufactured by Qiagen). The purified DNA fragment was digested with restriction enzymes EcoRI and Xbal, subjected to agarose gel electrophoresis, excised a band of interest, purified using a Qiagen Gel Extraction kit (manufactured by Qiagen), and collected. The obtained DNA fragment was digested with EcoRI and Xbal. In FIG. 8, ligation was performed using Ligation high (manufactured by Toyobo Co., Ltd.), and Escherichia coli JM109 strain was transformed. The transformed cells were grown on LB agar medium containing ampicillin (50 μg / mL), and the resulting colonies were subjected to colony direct PCR to confirm the size of the inserted fragment. The transformed cells containing the DNA fragment of interest are cultured in an LB medium containing 50 xg / mL ampicillin, and the plasmid is purified using the QIAPrepSpin Mini Prep kit (Qiagen). pUCCaERl. When the nucleotide sequence of the DNA inserted into the plasmid was analyzed by a dye terminator method, the inserted DNA fragment was identical to the nucleotide sequence of SEQ ID NO: 3.

(3) Synthesis of (R) _2_methyl_4,4,4_trifluorobutyric acid using E. coli transformed with DNA encoding enoate reductase

The transformed cells obtained in the above (1) and (2) were subjected to Aneropaque's culture in an LB medium containing ampicillin (50 x gZmL) and ImM isopropyl β-D-thiogalatatopyranoside (IPTG). (Mitsubishi Gas Chemical Company) under anaerobic conditions at 37 ° C for 30 hours. After 5 ml of the obtained cell broth was collected by centrifugation to obtain cells, the enoate reductase activity of the cells was determined using 4,4,4 trifluorotiglic acid as a substrate by the method described below. Sure

5, then /

[0076] The substrate 4,4,4 trifluorotiglic acid was synthesized as follows. That is, 50% water-containing methanol (20 mL) and 8 mol / L NaOH (3 mL) were added to 4,4,4_trifluoroethyltignate ethyl ester (3.64 g, 20 mmol), and the obtained mixture was added to about 4 mL. Stir for 2 hours. The solvent was distilled off from the reaction mixture under reduced pressure, the pH was adjusted to 2 or less by adding concentrated hydrochloric acid, methylene chloride extraction was performed, and the obtained organic layer was concentrated under reduced pressure to obtain a transparent oily product as a transparent oil. Trifluorotiglic acid (2.9 g, yield 94%) was obtained.

[0077] A 200 μL reaction solution (0.6 g / L NAD ⁺ (manufactured by Oriental Yeast), 50 mM potassium phosphate buffer (pH 7.0), 100 mM glucose, 0.2 g ZL glucose dehydrogenase (Amano) was added to the cells. After addition of 25 mM 4,44_triphnorelotiglic acid (manufactured by Pharmaceutical Co., Ltd.), the mixture was reacted at 37 ° C. for 20 hours under anaerobic conditions using Anellopak'Kenki (manufactured by Mitsubishi Gas Chemical Company). The reaction was terminated by adding 10 μL of 6N HC1, and the mixture was extracted with ethyl acetate, and 2-methyl-4,4,4_trifluorobutyric acid was quantified. Determination is ethyl acetate The solution was measured using gas chromatography (GC).

[0078] GC conditions are as follows.

Column: i3-DEX225 (SUPELCO, 30m X O. 25mmID, 0.25 ^ m film

)

Carrier: He 1.5 ml / min, split 1/50

Column temperature: 110 ° C

Injection temperature: 250 ° C

Detection: FID 250 ° C

GC: Shimadzu GC-14A (Shimadzu Corporation)

[0079] As a result, when E. coli transformed with pUCMtERl was used, the yield of (R) _2-methyl-4,4,4_triphnolebutyric acid was f up to 625 zg, and optical purity f up to 67.2. It was% ee. When using E. coli transformed with pUCCaERl, the yield of (R) -2-methyl-4,4,4-trifluorobutyric acid was 757 / ig, and the optical purity was 99.8% ee or more. .

Industrial potential

According to the present invention, it has become possible to obtain an optically active 418-butyric acid derivative having high optical purity, which is an industrially useful compound as an intermediate material for pharmaceuticals, agricultural chemicals and the like. In addition, (R) _N_ (4,4,4_trifluoro-2-ethylmethyl) _3_ [2-methoxy_4_ (o_tolylsulfonylcarbamoyl) benzyl] _1_methylindolinole-5—carboxamide useful as a leukotriene antagonist Can be manufactured efficiently.

Claims

Claims [1] The following general formula (I)

[Chemical 1]

Is reacted with a 4-halocrotonic acid derivative represented by the following formula, a cell containing the same, a preparation of the same cell, or a culture solution obtained by culturing the same cell.

[Formula 2]

A method for producing an optically active 4-octabutyric acid derivative represented by the general formula (II), characterized by producing an optically active 4-halobutyric acid derivative represented by the general formula (1), (II) Wherein R is a halogen atom, a nitro group, a hydroxyl group, an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted alkoxy group, X is a halogen atom, A

1 and A represent a hydrogen atom or a halogen atom).

2

[2] The production method according to claim 1, wherein the enoate reductase is a protein represented by the following (A), (B) or (C):

(B) having at least 50% homology with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 A protein comprising an amino acid sequence represented by the general formula (I) and having an enzymatic activity for converting a 4-halocrotonic acid derivative represented by the general formula (I) into an optically active 4-halobutyric acid derivative represented by the general formula (Π).

[3] The production method according to claim 1, wherein the cell is a cell transformed with DNA encoding enoate reductase.

[4] The production method according to claim 3, wherein the cell force S is a cell transformed with a DNA encoding enoate reductase, and the cell is a cell transformed by incorporating the DNA on a chromosome.

[5] The production method according to claim 3, wherein the cell is a cell transformed with a vector containing the DNA, the cell being transformed with a DNA encoding enoate reductase.

[6] The production according to any one of claims 3 to 5, wherein the DNA encoding enoate reductase is a DNA encoding a protein represented by the following (A), (B) or (C): Method:

(B) a 4-halocrotonic acid derivative comprising an amino acid sequence having 50% or more homology with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and represented by the general formula (I); A protein having an enzymatic activity for converting into an optically active 4-halobutyric acid derivative represented by Π).

[7] The production method according to any one of claims 3 to 5, wherein the DNA encoding enoate reductase is a DNA represented by the following (D), (E) or (F): (D) DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or

(E) a 4-halocrotonic tonic acid derivative which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or its complementary sequence under stringent conditions and is represented by the general formula (I); DNA encoding a protein having an enzymatic activity for converting into an optically active 4-halobutyric acid derivative represented by the formula (II).

(F) In the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, most of the nucleotide sequence is composed of a substituted, deleted or added nucleotide sequence and its complementary strand, and represented by the general formula (I) DNA encoding a protein having an enzymatic activity that converts a 4-halocrotonic acid derivative into an optically active 4-halobutyric acid derivative represented by the general formula (II).

[8] In the compounds of the general formulas (I) and (Π), R is a methyl group, and X, A and A are

The production method according to any one of claims 11 to 17, wherein each of the two is a fluorine atom.

[9] The method according to claim 8, wherein 4,4,4-trifluorotiglic acid represented by the general formula (III) is converted to (R) -4,4,4 represented by the general formula (IV). (4) a step of converting into monotrifluoro-2-methylbutyric acid, wherein the (R) —4,4,4-trifluoro-2-methylbutyric acid is converted to (R) —4,4 represented by the general formula (V) Converting to 4,4-trifluoro-2-methylbutylamine, and reacting the compound represented by the general formula (VI) with the (R) -4,4,4-trifluoro-2-methylbutylamine (R) _N_ (4,4,4_trifluoro-2-methylbutyl) _3_ [2-methoxy-1- 4- (o_tolylsulfonylcarbamoyl) benzyl] _1_methylindole represented by the general formula (VII) (R) _N_ (4,4,4-trifluoro-2-methylbutyl) -1_3 [[2-methoxy-14_ (o_tolylsulfonylcarbamoyl) benzyl] _1 monomethyl including the step of producing _carboxamide Method for producing Indore No_5_carboxamide.

[Formula 3] ) n