JP2013046572A - Method for producing optically active 2-hydroxycycloalkanecarboxylic acid ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an efficient industrial production method of an optically active 2-hydroxycycloalkanecarboxylic acid ester useful as an intermediate for a pharmaceutical product.SOLUTION: The optically active 2-hydroxycycloalkanecarboxylic acid ester is produced by treating a 2-oxocycloalkanecarboxylic acid ester with a polypeptide, an organism that produces the polypeptide or a processed product of the organism to cause reduction. The polypeptide is derived from a microorganism selected from the group consisting of genera Candida, Rhodotorula, Devosia, Ogataea, Brevundimonas, Lactobacillus, Thermoanaerobium, Rhodococcus, Sporobolomyces and Sporidiobolus.

Description

  The present invention relates to a method for producing an optically active 2-hydroxycycloalkanecarboxylic acid ester, particularly an optically active 2-hydroxycyclopentanecarboxylic acid ester. The optically active 2-hydroxycycloalkanecarboxylic acid ester is a useful compound as a synthetic raw material and intermediate for pharmaceuticals, agricultural chemicals and the like.

  Optically active 2-hydroxycycloalkanecarboxylic acid esters, especially optically active 2-hydroxycyclopentanecarboxylic acid esters, are useful compounds as synthetic raw materials and intermediates for pharmaceuticals, agricultural chemicals and the like.

  Reduction of the carbonyl group at the 2-position of 2-oxocycloalkanecarboxylic acid ester with sodium borohydride or the like generates two asymmetric carbons, and therefore, there are four stereoisomers (cis (1R, 2S) 2-hydroxycycloalkanecarboxylic acid ester in which (form) and (1S, 2R) form, and (1S, 2S) form and (1R, 2R) form of trans form are mixed.

  What is useful as a synthetic raw material for pharmaceuticals, agricultural chemicals, and the like is a specific steric optically active 2-hydroxycycloalkanecarboxylic acid ester. If other stereoisomers are contained, it becomes an impurity. Therefore, there is a need for an efficient production technique for a desired optically active 2-hydroxycycloalkanecarboxylic acid ester that does not contain unnecessary stereoisomers as much as possible.

  One method for producing an optically active 2-hydroxycycloalkanecarboxylic acid ester is an asymmetric reduction method of 2-oxocycloalkanecarboxylic acid ester using a microbial cell or enzyme as a catalyst.

  The following is reported about the manufacturing method of optically active 2-hydroxycycloalkanecarboxylic acid ester, for example, 2-hydroxycyclopentanecarboxylic acid ester.

  Klebsiella pneumoniae (patent document 1, non-patent document 1), Geotrichum candidum, Mucor racemosus, Mucor circinelloides, Kuninggames grooeporoid In the asymmetric reduction of 2-oxocyclopentanecarboxylic acid ester using Cunninghamella gloeosporoides (Non-patent Document 2), reductases KRED102, KRED103, and KRED106 (Non-patent Document 3), (1R, 2S) 2- Hydroxycyclopentanecarboxylic acid esters are reported to form.

  In addition, asymmetric reduction of 2-oxocyclopentanecarboxylic acid ester using reductases KRED107 and KRED131 (Non-patent Document 3) reports that (1S, 2R) isomer of 2-hydroxycyclopentanecarboxylic acid ester is mainly produced. Has been.

  In addition, 2-oxocyclopentanecarboxylic acid using Absidia glauca, Rhizopus arrhizus, Penicillium chrysogenum (Non-patent document 2), and reductase KRED113 (Non-patent document 3). In the asymmetric reduction of acid ester, it is reported that (1S, 2S) -form 2-hydroxycyclopentanecarboxylic acid ester is mainly produced.

  As described above, although there are examples of asymmetric reduction of 2-oxocyclopentanecarboxylic acid esters using microorganisms and enzymes, they are limited to reduction reactions using specific microorganisms and enzymes. There has never been a report that the main product is a (1R, 2R) 2-hydroxycyclopentanecarboxylic acid ester.

JP 5-192187

Biosci. Biotech. Biochem., 60 (5), 760-764, (1996) Tetrahedron Letters, 27 (23), 2631-2634, (1986) Tetrahedron, 62, 901-905, (2006)

  An object of the present invention is to efficiently and industrially produce an optically active 2-hydroxycycloalkanecarboxylic acid ester, particularly an optically active 2-hydroxycyclopentanecarboxylic acid ester, useful as a pharmaceutical intermediate.

  As a result of repeated studies to develop a simple and efficient method for producing an optically active 2-hydroxycycloalkanecarboxylic acid ester, the present inventors have caused a specific polypeptide to act on 2-oxocyclopentanecarboxylic acid ester. As a result, it was found that a specific stereoisomer of 2-hydroxycyclopentanecarboxylic acid ester can be efficiently produced by asymmetric reduction, and the present invention has been completed.

  That is, the present invention has the following one or more features.

  The present invention relates to a process for producing an optically active 2-hydroxycycloalkane carboxylic acid ester, preferably 2-hydroxycyclopentane carboxylic acid ester, wherein the 2-oxocycloalkane carboxylic acid ester produces a polypeptide and the polypeptide. The 2-oxocycloalkanecarboxylic acid ester is reduced by the action of a living organism or a processed product of the organism. Polypeptides include Candida, Rhodotorula, Devosia, Ogataea, Brevundimonas, Lactobacillus, Thermoanaerobium, and Thermoanaerobium. And a polypeptide derived from a microorganism selected from the group consisting of genus, genus Rhodococcus, genus Sporobolomyces and genus Sporidiobolus.

  The present invention also relates to a method for producing (1R, 2R) -2-hydroxycycloalkanecarboxylic acid ester, wherein 2-oxocycloalkanecarboxylic acid ester is added to a polypeptide, preferably a genus Sporobolomyces. It is a method characterized by allowing a polypeptide derived from a microorganism selected from the group consisting of the genus Sporidiobolus to act.

The present invention also relates to a method for producing (1R, 2R) -2-hydroxycycloalkanecarboxylic acid ester, wherein 2-oxocycloalkanecarboxylic acid ester is a polypeptide, preferably the following (a1), (a2) And reducing a 2-oxocycloalkanecarboxylic acid ester by acting a polypeptide selected from the group consisting of: and (a3), an organism that produces the polypeptide, or a processed product of the organism It is a featured method:
(A1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing;
(A2) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, and 2-oxocyclopentanecarboxylic acid ester and NADPH A polypeptide having an activity of acting on (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and NADP,
(A3) It has a sequence identity of 85% or more with the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, and acts on 2-oxocyclopentanecarboxylic acid ester and NADPH to (1R, 2R) -2-hydroxy A polypeptide having an activity to produce cyclopentanecarboxylic acid ester and NADP.

In addition, an organism producing a polypeptide selected from the group consisting of (a1), (a2), and (a3) above is from (A1), (A2), (A3), and (A4) below. A recombinant organism into which a DNA selected from the group is introduced is preferred.
(A1) DNA set forth in SEQ ID NO: 2 in the Sequence Listing,
(A2) DNA encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing,
(A3) hybridizes with a DNA complementary to the DNA of SEQ ID NO: 2 in the sequence listing under stringent conditions, and acts on 2-oxocyclopentanecarboxylic acid ester and NADPH (1R, 2R)- DNA encoding a polypeptide having activity to produce 2-hydroxycyclopentanecarboxylic acid ester and NADP,
(A4), having a sequence identity of 85% or more with the base sequence set forth in SEQ ID NO: 2 in the sequence listing and acting on 2-oxocyclopentanecarboxylic acid ester and NADPH (1R, 2R) -2- A DNA encoding a polypeptide having an activity to produce hydroxycyclopentanecarboxylic acid ester and NADP.

  In the above production method, the recombinant organism is preferably Escherichia coli.

  The present invention also relates to a method for producing (1S, 2S) -2-hydroxycycloalkanecarboxylic acid ester, wherein 2-oxocycloalkanecarboxylic acid ester includes Candida genus, Rhodotorula genus, Ogataair. It is a method characterized in that a polypeptide derived from a microorganism selected from the group consisting of the genus (Ogataea) and the genus Thermoanaerobium is allowed to act.

  The present invention also relates to a method for producing (1S, 2R) -2-hydroxycycloalkanecarboxylic acid ester, wherein 2-oxocycloalkanecarboxylic acid ester includes Candida genus, Devosia genus, It is a method characterized by allowing a polypeptide derived from a microorganism selected from the group consisting of the genera Brevundimonas and Lactobacillus to act.

  The present invention also relates to a method for producing (1R, 2S) -2-hydroxycycloalkane carboxylic acid ester, and the 2-oxocycloalkane carboxylic acid ester comprises a genus Candida and a genus Rhodococcus. It is a method characterized by allowing a polypeptide derived from a microorganism selected from the group to act.

  In any of the above production methods, it is preferable to further act a polypeptide having the ability to regenerate reduced coenzyme.

  The present invention also relates to a method for producing 2-hydroxycycloalkanecarboxylic acid amide, which comprises reacting 2-hydroxycycloalkanecarboxylic acid ester obtained by the above method with ammonia.

  The present invention also relates to a process for producing 2-aminocycloalkanol, characterized in that the 2-hydroxycycloalkanecarboxylic acid amide obtained above is reacted with a halogenating agent under basic conditions.

  According to the method of the present invention, an optically active 2-hydroxycycloalkanecarboxylic acid ester, particularly an optically active 2-hydroxycyclopentanecarboxylic acid ester, can be produced efficiently and industrially.

  Hereinafter, the present invention will be described in detail using embodiments. In addition, this invention is not limited by these.

  The optically active 2-hydroxycycloalkanecarboxylic acid ester produced in the present invention has a ratio of the desired stereoisomer in the four stereoisomers of 2-hydroxycycloalkanecarboxylic acid ester of 50% or more, preferably Is 60% or more, more preferably 70% or more, and still more preferably 80% or more of 2-hydroxycycloalkanecarboxylic acid ester. This will be described in detail later.

Polypeptide used in the present invention Any polypeptide can be used as long as it has an activity to act on 2-oxocycloalkanecarboxylic acid ester to produce optically active 2-hydroxycycloalkanecarboxylic acid ester. May be. The method for obtaining this polypeptide will be described in detail later.

  Hereinafter, the polypeptide used in the present invention will be described in detail using the polypeptide used for the production of (1R, 2R) 2-hydroxycycloalkanecarboxylic acid ester as an example. The following contents can also be applied to polypeptides used for the production of other stereoisomeric 2-hydroxycycloalkanecarboxylic acid esters.

  As a polypeptide used in the present invention, for example, when producing a (1R, 2R) 2-hydroxycycloalkanecarboxylic acid ester, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing can be mentioned. (Polypeptide of (a1)). The polypeptide used in the present invention is a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. (Polypeptide of (a2)). These polypeptides can be prepared according to known methods described in Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1989) and the like, and act on 2-oxocyclopentanecarboxylic acid ester and NADPH. As long as it has an activity to produce (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and NADP, it is included in the above polypeptide.

  Here, the “plural amino acids” are, for example, 50, preferably 30, more preferably 15, more preferably 10, 9, 8, 7, 6, 5, 4 Means 3 or less amino acids.

  In the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, the place where an amino acid is substituted, inserted, deleted and / or added is not particularly limited, but it is preferable to avoid a highly conserved region. Here, the highly conserved region represents a region in which amino acids are identical between a plurality of sequences when the amino acid sequences are optimally aligned and compared for a plurality of enzymes having different origins. The highly conserved region can be confirmed by comparing the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequence of a known microorganism-derived alcohol dehydrogenase using a tool such as GENETYX.

  The amino acid sequence modified by substitution, insertion, deletion and / or addition may include only one type of modification (for example, substitution), or two or more modifications (for example, substitution and substitution). Insertion).

In the case of substitution, the amino acid to be substituted is preferably an amino acid having a property similar to that of the amino acid before substitution (cognate amino acid). Here, amino acids in the same group of the following groups are regarded as homologous amino acids.
(Group 1: neutral nonpolar amino acids) Gly, Ala, Val, Leu, Ile, Met, Cys, Pro, Phe
(Group 2: neutral polar amino acids) Ser, Thr, Gln, Asn, Trp, Tyr
(Group 3: acidic amino acids) Glu, Asp
(Group 4: basic amino acids) His, Lys, Arg.

  The polypeptide used in the present invention has 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and acts on 2-oxocyclopentanecarboxylic acid ester and NADPH ( 1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and a polypeptide having an activity of producing NADP may be used (polypeptide of (a3)).

  The sequence identity to the amino acid sequence of SEQ ID NO: 1 in the sequence listing is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more or 99% or more.

  The sequence identity of the amino acid sequences is determined by comparing the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing with the amino acid sequence to be evaluated, dividing the number of amino acid positions in both sequences by the total number of amino acids, Furthermore, it is represented by a value multiplied by 100.

  As long as it has the activity of acting on 2-oxocyclopentanecarboxylic acid ester and NADPH to produce (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and NADP, it is added to the amino acid sequence of SEQ ID NO: 1. Amino acid sequences can be combined. For example, a tag sequence such as a histidine tag or HA tag may be added. Alternatively, it can be a fusion protein with another protein. Moreover, as long as it has the activity which acts on 2-oxocyclopentanecarboxylic acid ester and NADPH and produces | generates (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and NADP, a peptide fragment may be sufficient.

  Even when the level of activity of acting on 2-oxocyclopentanecarboxylic acid ester and NADPH to produce (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and NADP is reduced, Any equivalent activity is encompassed by the polypeptide of the present invention. Here, “substantially equivalent activity” refers to an activity of, for example, 50% or more, usually 70% or more, preferably 80% or more, more preferably 90% or more or 95% or more with respect to the original activity. . The activity can be measured by the method described later.

Method for Obtaining Polypeptide Used in the Present Invention The polypeptide used in the present invention can be obtained from a microorganism having the ability to reduce 2-oxocycloalkanecarboxylic acid ester to produce optically active 2-hydroxycycloalkanecarboxylic acid ester. . A microorganism having the polypeptide can be found, for example, by the following method.

  The microorganism is cultured in an appropriate medium, and after collection, the microorganism is reacted with 2-oxocycloalkanecarboxylic acid ester in the presence of nutrients such as glucose in a buffer solution. After the reaction, extraction with a solvent, etc., and analysis by gas chromatography, high performance liquid chromatography, etc. confirm the amount of 2-hydroxycycloalkanecarboxylic acid ester produced and the composition ratio of the four stereoisomers. do it.

  As a medium for culturing a microorganism, a normal liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients and the like can be used as long as the microorganism grows. The culture can be performed, for example, by shaking or aeration at a temperature of 25 ° C. to 37 ° C. and a pH of 4 to 8.

  Isolation of the polypeptide used in the present invention from a microorganism can be carried out by using an appropriate combination of known protein purification methods. For example, it can be implemented as follows. First, microorganisms are cultured in an appropriate medium, and the cells are collected from the culture solution by centrifugation or filtration. The obtained microbial cells are crushed by an ultrasonic crusher or a physical method using glass beads and the like, and the microbial cell residue is removed by centrifugation to obtain a cell-free extract. And heat treatment, salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fraction precipitation with acetone or ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, limiting The polypeptide used in the present invention is isolated from the cell-free extract by using a technique such as external filtration alone or in combination.

  The reduced activity of the isolated polypeptide against 2-oxocycloalkanecarboxylic acid ester is determined by reacting the polypeptide to be evaluated with 2-oxocycloalkanecarboxylic acid ester and then generating 2-hydroxycycloalkanecarboxylic acid ester. It can be evaluated by analyzing with gas chromatography or the like. Further, the 2-oxocycloalkanecarboxylic acid ester reducing activity, for example, the 2-oxocyclopentanecarboxylic acid ester reducing activity can be evaluated by the following simple method.

[Method for measuring reduction activity for 2-oxocyclopentanecarboxylic acid ester]
The reduction activity for 2-oxocyclopentanecarboxylic acid ester was increased by adding 10 mM 2-oxocyclopentanecarboxylic acid ester, 0.25 mM coenzyme NADPH, and crude enzyme solution to 100 mM phosphate buffer (pH 6.5). The reaction is carried out at 1 ° C. for 1 minute, and the rate of decrease in absorbance at a wavelength of 340 nm is calculated. Under this reaction condition, the enzyme activity that oxidizes 1 μmol of NADPH to NADP per minute is defined as 1 U. NADH may be used instead of NADPH.

  The origin of the polypeptide used in the present invention is not limited, but preferably the genus Candida, the genus Rhodotorula, the genus Devosia, the genus Ogataea, the Brevundimonas Derived from a microorganism selected from the group consisting of genus, Lactobacillus genus, Thermoanaerobium genus, Rhodococcus genus, Sporobolomyces genus, Sporidiobolus genus Is.

  When producing (1R, 2R) -2-hydroxycycloalkanecarboxylic acid ester, the polypeptide used is a microorganism selected from the group consisting of the genus Sporoboromyces and the genus Sporidiobolus The thing of origin is preferable.

  Examples of yeasts belonging to the genus Sporobolomyces include Sporobolomyces albidus, Sporobolomyces alborubescens, Sporobolomyces albus, Sporobolomyces albus, Sporobolomyces albus Sporobolomyces antarcticus, Sporobolomyces bischofiae, Sporobolomyces blumeae, Sporobolomyces carnicolor, Sporoboromyces carnicolor Sprorobolomyces coprophilus, Sporobolomyces coprosmae, Sporobolomyces coprosmicola, Sporobolomyces coralliformis Sporobolomyces dimmenae, Sporobolomyces diospyroris, Sporobolomyces dracophyllus, Sporobolomyces elongatus, Sporoboromyces elongatus・ Falcatus (Sporobolomyces falcatus), Sporobolomyces foliicola (Sporobolomyces foliicola), Sporobolomyces gracilis, Sporobolomyces griseoflavus (Sporobolomyces griseoflavus), Sporoboromyces porobus ces , Sporobolomyces inositophilus, Sporobolomyces intermedius, Sporobolomyces intermedius (Sporobolo myces kluyveri-nielii), Sporobolomyces lactophilus, Sporobolomyces lactosus, Sporobolomyces linderae, Sporobolomyces lophatheri, Sporobolomyces lophatheri Sporobolomyces magnisporus, Sporobolomyces marcillae, Sporobolomyces miscanthi, Sporobolomyces naganoensis, Sporobolomyces naganoensis Novadia randicas (Sporobolomyces novalandicus), Sporobolomyces nylandii, Sporobolomyces odoratus, Sporobolomyces odorusus, Sporobolomyces ogasawarensis, Sporobolomyces oryzicola, Sporobolomyces pararoseus, Sporobolomyces phaffii, Sporoboromyces phaffii Sporobolomyces phylladus), Sporobolomyces phyllomatis, Sporobolomyces pollaccii, Sporobolomyces poonsookiae, Sporobolomyces poonsookiae, Spoolomyces ponyus ces Sporobolomyces pyrrosiae, Sporobolomyces roseus, Sporobolomyces ruber, Sporobolomyces ruber bar・ Albus (Sporobolomyces ruberrimus var. Albus), Sporobolomyces ruberrimus ・ Bar luberrimus (Sporobolomyces ruberrimus var. Ruberrimus), Sporobolomyces salicinus, Sporobolomyces salmoneus salmon (Sporobolomyces salmoneus) Sporobolomyces salmonicolor, Sporobolomyces sasicola, Sporobolomyces shibatanus, Sporobolomyces singularis, Sporobolomyces singularis (Sporobolomyces subbrunneus), Sporobolomyces subroseus, Sporobolomyces syzygii, Sporobolomyces taupoensis, spo Sporobolomyces tenuis, Sporobolomyces tsugae, Sporobolomyces vermiculatus, Sporobolomyces weijmanii, Sporobolomyces weijmanii xanthus), Sporobolomyces yamatoanus, Sporobolomyces yuccicola, Sporobolomyces yunnanensis, and the like.

  Examples of the yeast belonging to the genus Sporidiobolus include Sporidiobolus johnsonii, Sporidiobolus microsporus, Sporidiobolus pararoseus, and Sporidiobolus pararoseus. Examples include Sporidiobolus ruineniae, Sporidiobolus ruineniae var. Coprophilus, and Sporidiobolus salmonicolor.

  In addition, new species of sporoboromyces and sporidiobolus newly isolated from nature are also included.

  Preferably, Sporoboromyces salmonicolor (Sporobolomyces salmonicolor), Sporidiobolus salmonicolor (Sporidiobolus salmonicolor), etc. are mentioned.

  In the case of producing (1S, 2S) -2-hydroxycycloalkanecarboxylic acid ester, the polypeptides to be used include Candida genus, Rhodotorula genus, Ogataea genus, Thermoanaerobium (Themoanaerobium genus). And) derived from a microorganism selected from the group consisting of genus. More preferably, Candida magnoliae, Candida boidinii, Rhodotorula glutinis var. Dairenensis, Ogataea minuta var. Minuta ), A polypeptide derived from Thermoanaerobium brockii. Particularly preferred polypeptides are the reductase CR enzyme derived from Candida magnoliae (Example 4), the reductase RRG derived from Rhodotorula glutinis var. Dairenensis NBRC0415 (Example 5). ), A reductase derived from Ogataea minuta var. Minuta NBRC0975 (Example 6), and the like. Regarding the physicochemical properties, amino acid sequences, and obtaining methods of these enzymes, CR enzyme is derived from International Publication WO01 / 040450, RRG enzyme is derived from International Publication WO03 / 093477, Ogataea minuta var. Minuta NBRC0975 Reductases are described in International Publication WO06 / 013801.

  In the case of producing (1S, 2R) -2-hydroxycycloalkanecarboxylic acid ester, the polypeptides to be used include Candida genus, Devosia genus, Brevundimonas genus, Lactobacillus. Those derived from microorganisms selected from the group consisting of genera are preferred. More preferably, a polypeptide derived from Candida maris, Devosia riboflavina, Brevundimonas diminuta, Lactobacillus brevis, Lactobacillus kefir It is. Particularly preferred polypeptides are the reductase FPDH derived from Candida maris NBRC10003 (Example 9), the RDR reductase derived from Devosia riboflavina NBRC13584 (Example 10), and Brevundimonas diminuta. (Brevundimonas diminuta) NBRC13584-derived reductase (Example 11). Regarding the physicochemical properties, amino acid sequences, and obtaining methods of these enzymes, the FPDH enzyme is the international publication WO01 / 05996, the RRG enzyme is the international publication WO04 / 027055, and the reductase derived from Brevundimonas diminuta NBRC13584 is the international publication. WO07 / 114217.

  In the case of producing (1R, 2S) -2-hydroxycycloalkanecarboxylic acid ester, the polypeptide to be used is preferably derived from a microorganism selected from the group consisting of the genus Candida and the genus Rhodococcus. . More preferably, it is a polypeptide derived from Candida maltosa, Candida parapsilosis, Rhodococcus erythropolis. Particularly preferred polypeptides include reductase derived from Candida maltosa NBRC1977 (Example 14), reductase derived from Candida parapsilosis NBRC708 (Example 15), and the like. Regarding the physicochemical properties, amino acid sequences, and obtaining methods of these enzymes, the reductase derived from Candida maltosa NBRC1977 is a special reductase derived from International Publication WO08 / 066018 and the reductase derived from Candida parapsilosis NBRC708. No. 2003-289874.

The above microorganisms can be obtained from, for example, the following culture collection.
・ National Biotechnology Headquarters, Biotechnology Headquarters, National Institute of Technology and Evaluation (NBRC) (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818).
・ RIKEN BioResource Center Microbial Materials Development Office (JCM) (2-1 Hirosawa, Wako, Saitama 351-0198)
German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (Marschroder Weg 1b, D-38124 Brunswick, Germany).

DNA encoding the polypeptide used in the present invention
The DNA encoding the polypeptide used in the present invention may be any DNA that can express the polypeptide in a host cell introduced according to the method described below, and may contain any untranslated region. . In addition to DNA and cDNA cloned from nature, DNA obtained by synthesis is also included.

  If the polypeptide can be obtained, such DNA can be obtained by a person skilled in the art from a microorganism that is the origin of the polypeptide by a known method. For example, it can be acquired by the method shown below.

  Unless otherwise specified, genetic manipulations such as DNA cloning, vector preparation and transformation described later in this specification are described in documents such as Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989). Can be implemented by any method. Further,% used in the description of the present specification means% (w / v) unless otherwise specified.

  First, the polypeptide used in the present invention isolated by the method described in “Method for obtaining polypeptide used in the present invention” is digested with an appropriate endopeptidase, and the resulting peptide fragment is subjected to reverse phase HPLC. Sort by. Then, for example, part or all of the amino acid sequences of these peptide fragments are determined by an ABI492 type protein sequencer (Applied Biosystems).

  Based on the amino acid sequence information thus obtained, a PCR (Polymerase Chain Reaction) primer for amplifying a part of the DNA encoding the polypeptide is synthesized. Next, chromosomal DNA of the microorganism that is the origin of the polypeptide is prepared by a conventional DNA isolation method, for example, the method of Visser et al. (Appl. Microbiol. Biotechnol., 53, 415 (2000)). Alternatively, cDNA is prepared from the mRNA of the microorganism that is the origin of the polypeptide. Using these DNAs as templates, PCR is performed using the PCR primers described above, a part of the DNA encoding the polypeptide is amplified, and the base sequence is determined. The base sequence can be determined using, for example, Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems).

  If the partial base sequence of DNA encoding the polypeptide is clarified, the entire sequence is determined by, for example, the inverse PCR method (Nucl. Acids Res., 16, 8186 (1988)). be able to.

  Examples of the DNA encoding the polypeptide of the present invention thus obtained include DNA encoding the polypeptide shown in SEQ ID NO: 1 in the sequence listing (DNA of (A2)). Specifically, the DNA (DNA of (A1)) shown in SEQ ID NO: 2 in the Sequence Listing can be mentioned.

  The DNA used in the present invention hybridizes with a DNA complementary to the DNA shown in SEQ ID NO: 2 in the sequence listing under stringent conditions, and acts on 2-oxocyclopentanecarboxylic acid ester and NADPH. (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and a DNA encoding a polypeptide having an activity to produce NADP (DNA of (A3)).

  The DNA used in the present invention has a sequence identity of 85% or more with the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing, and acts on 2-oxocyclopentanecarboxylic acid ester and NADPH (1R , 2R) -2-hydroxycyclopentanecarboxylic acid ester and a DNA encoding a polypeptide having an activity to produce NADP (DNA of (A4)).

  Here, “hybridizes with DNA complementary to the DNA shown in SEQ ID NO: 2 in the sequence listing under stringent conditions and acts on 2-oxocyclopentanecarboxylic acid ester and NADPH (1R, 2R) “2-hydroxycyclopentanecarboxylic acid ester and a DNA encoding a polypeptide having an activity to produce NADP” means that a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 2 in the sequence listing is used as a probe. , DNA obtained by using a colony hybridization method, a plaque hybridization method, or a Southern hybridization method under stringent conditions, and acts on 2-oxocyclopentanecarboxylic acid ester and NADPH ( 1R, 2R) -2-hydroxycyclo It means a DNA encoding a polypeptide having the activity to form Ntankarubon ester and NADP.

  Hybridization can be performed according to the method described in Molecular Cloning, A laboratory manual, second edition (Cold Spring Harbor Laboratory Press, 1989). Here, “DNA that hybridizes under stringent conditions” means, for example, complementation at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA is immobilized. After strand formation, the filter is washed under conditions of 65 ° C. using a 2 × concentration SSC solution (composition of 1 × concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate). The DNA which can be acquired can be mentioned. It is preferably washed at 65 ° C. with a 0.3-fold concentrated SSC solution, more preferably at 65 ° C. with a 0.1-fold concentrated SSC solution, and even more preferably at 65 ° C. with a 0.05-fold concentrated SSC solution. DNA that can be obtained by

  Although the hybridization conditions have been described as described above, the conditions are not particularly limited. A plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.

  The DNA hybridizable under the above conditions is 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, with the DNA shown in SEQ ID NO: 2. Most preferably, 99% or more of DNA can be mentioned, and the encoded polypeptide acts on 2-oxocyclopentanecarboxylic acid ester and NADPH to give (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester. As long as it has an activity to produce NADP, it is included in the DNA.

Here, “sequence identity (%)” means that the two DNAs to be compared are optimally aligned, and the nucleobases (eg, A, T, C, G, U, or I) match in both sequences. The number of positions is divided by the total number of comparison bases, and the result is expressed by a value obtained by multiplying by 100.
Sequence identity can be calculated, for example, using the following sequence analysis tool: GCG Wisconsin Package (Program Manual for The Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive Medison, Wisconsin, USA) 53711; Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 1RQ, England) and the ExPASy World Wide Web molecular biology server (Geneva University Hospital and University of Geneva, Geneva, Switzerland).

Recombinant organisms producing polypeptides used in the present invention Recombinant organisms producing polypeptides used in the present invention include, for example, (A1), (A2), (A3) or (A4) described above. A recombinant organism into which the DNA described in any one of the above has been introduced can be mentioned.

  If a host organism is transformed with a polypeptide expression vector prepared by inserting a DNA encoding the polypeptide used in the present invention into an expression vector, a recombinant organism producing the polypeptide used in the present invention can be obtained. The polypeptide of the present invention can be expressed by culturing using a medium in which this organism can grow. Furthermore, a method of introducing a polynucleotide encoding the polypeptide used in the present invention into a chromosome can also be used.

  The expression vector used above is not particularly limited as long as it can express the polypeptide encoded by the DNA in a suitable host organism. Examples of such vectors include plasmid vectors, phage vectors, cosmid vectors, and shuttle vectors that can exchange genes with other host strains can also be used.

  For example, in the case of Escherichia coli, such a vector usually contains regulatory elements such as lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, etc., and operates with the DNA of the present invention. It can be suitably used as an expression vector comprising expression units that are ligated together. For example, pUCN18 (see Example 2), pSTV28 (manufactured by Takara Bio Inc.), pUCNT (WO94 / 03613) and the like can be mentioned.

  As used herein, the term “regulatory element” refers to a base sequence having a functional promoter and any associated transcription element (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).

  As used herein, the term “operably linked” means that a gene is operably linked to various regulatory elements such as promoters and enhancers that regulate the expression of the gene. It is well known to those skilled in the art that the type and kind of the control factor can vary depending on the host.

  Vectors and promoters that can be used in various organisms are described in detail in "Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan".

  The host organism used to express the polypeptide used in the present invention is an organism that is transformed with a polypeptide expression vector containing DNA encoding each polypeptide and can express the polypeptide encoded by the introduced DNA. If there is no particular limitation, organisms for which host vector systems have been developed are preferred.

  Examples of usable microorganisms include, for example, the genus Escherichia, the genus Bacillus, the genus Pseudomonas, the genus Serratia, the genus Brevibacterium, the genus Corynebacterium, Streptococcus genus and bacteria such as Lactobacillus genus; Rhodococcus genus and Streptomyces genus actinomycetes; Saccharomyces genus, Kluyveromyces genus, The genus of Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Pichia, and Candida Yeast; Neurospora (N Examples include molds such as the genus eurospora, the genus Aspergillus, the genus Cephalosporium, and the genus Trichoderma.

  In addition to microorganisms, various host and vector systems have been developed for plants and animals, especially in insects such as moths (Nature 315, 592-594 (1985)) and in plants such as rapeseed, corn and potatoes. Systems for expressing heterologous proteins have been developed and can be suitably used. Among these, microorganisms are preferable, bacteria are more preferable, and Escherichia coli (commonly known as E. coli) is particularly preferable in terms of introduction and expression efficiency.

  A polypeptide expression vector containing a DNA encoding a polypeptide used in the present invention can be introduced into a host organism by a known method.

  For example, when the plasmid pUCAR2 (Applied and Environmental Microbiology, 65 (12), 5207-5211, (1999)) prepared by introducing the DNA shown in SEQ ID NO: 2 into the vector pUC118 is introduced into E. coli as the host microorganism. By using a commercially available E. coli competent cell (manufactured by Takara Bio Inc.) according to the protocol, a recombinant organism E. coli introduced with the vector into a host cell, such as E. coli JM109 (pUCAR2 (See Example 1).

  In addition, a recombinant organism in which both the polypeptide used in the present invention and the polypeptide having the ability to regenerate reduced coenzyme described below are expressed in the same cell can also be bred. That is, it can be obtained by incorporating the DNA encoding the polypeptide used in the present invention and the DNA encoding the polypeptide having the ability to regenerate reduced coenzyme into the same vector and introducing it into a host cell. In addition, the DNA encoding the polypeptide used in the present invention and the DNA encoding the polypeptide having the ability to regenerate reduced coenzyme are respectively incorporated into two types of vectors of different incompatibility groups, and these are incorporated into the same host cell. Also by introduction, a recombinant organism that produces both the polypeptide used in the present invention and the polypeptide having the ability to regenerate reduced coenzyme in the same cell can be bred.

Production of optically active 2-hydroxycycloalkanecarboxylic acid ester using polypeptide or recombinant organism 2-oxocycloalkanecarboxylic acid ester (8) using the above-mentioned polypeptide or a recombinant organism expressing the polypeptide ;

The optically active 2-hydroxycycloalkanecarboxylic acid ester (9);

Can be carried out as follows. However, it is not necessarily limited to the following method.

  In the formulas (8) and (9), n represents an integer of 0 to 8. Among these, preferably 0 to 4, more preferably 1 to 3, and most preferably 1. When n = 1, 2-oxocycloalkanecarboxylic acid ester is 2-oxocyclopentanecarboxylic acid ester, and the product optically active 2-hydroxycycloalkanecarboxylic acid ester is 2-hydroxycyclopentanecarboxylic acid ester. It is.

  In the formulas (8) and (9), R is a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, and a substituted or unsubstituted group having 7 to 11 carbon atoms. Represents a substituted aralkyl group. Examples of the substituted or unsubstituted alkyl having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group. Examples of the substituted or unsubstituted aryl group having 6 to 10 carbon atoms include phenyl group, p-methylphenyl group, o-methylphenyl group, m-methylbenzyl group, p-chlorophenyl group, p-nitrophenyl group, p -A trifluoromethylphenyl group, 1-naphthyl group, 2-naphthyl group, etc. are mentioned. Examples of the aralkyl group having 7 to 11 carbon atoms include benzyl group, p-methylbenzyl group, o-methylbenzyl group, m-methylbenzyl group, p-nitrobenzyl group, p-trifluoromethylbenzyl group and the like. . Among them, preferred is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and more preferred are a methyl group and an ethyl group.

  In the formula (9), * 1 and * 2 represent asymmetric carbon. The (S) configuration may be independent of * 1 and * 2, respectively, or the (R) configuration may be used. Among these, * 1 and * 2 are preferably in the (R) configuration.

  In addition, the optically active 2-hydroxycycloalkanecarboxylic acid ester (9) produced in the present invention is four kinds of stereoisomers (cis (1R, 2S) of 2-hydroxycycloalkanecarboxylic acid ester. ) And (1S, 2R) isomers, and (1S, 2S) isomers and (1R, 2R) isomers in the trans isomers) are 50% or more, preferably 60% More preferably, it is 70% or more, more preferably 80% or more of 2-hydroxycycloalkanecarboxylic acid ester.

  The ratio of (1R, 2R) body and (1S, 2S) in the formula (9) can be determined as follows, for example.

When the formula (9) to be analyzed is, for example, ethyl 2-hydroxycyclopentanecarboxylate, by analyzing under the following gas chromatography conditions, the trans isomer ratio (trans isomers in the four stereoisomers ((1S , 2S) and (1R, 2R) isomers) can be determined by the following formula.
[Trans-body ratio (%)] = [Trans-body area] / ([Trans-body area] + [Cis-body area]) × 100

[Analysis conditions by gas chromatography]
Column: ULBON HR-20M (0.25 mm (inner diameter) × 25 m; manufactured by Shinwa Kako), detection: FID, column temperature: 130 ° C., injection temperature: 200 ° C., detection temperature: 200 ° C., carrier gas: helium (130 kPa) ), Elution time: ethyl 2-hydroxycyclopentanecarboxylate (trans isomer) approximately 15.7 minutes, ethyl 2-hydroxycyclopentanecarboxylate (cis isomer) approximately 8.1 minutes, ethyl 2-oxocyclopentanecarboxylate approximately 8.8 minutes.

  The proportion of (1R, 2R) or (1S, 2S) in the trans isomer was determined by derivatizing ethyl 2-hydroxycyclopentanecarboxylate with dinitrobenzoyl chloride under basic conditions. It can be easily calculated by analyzing under the conditions of high performance liquid chromatography.

[Analysis conditions by high performance liquid chromatography]
Column: Chiralcel AD-H (4.6 μm (inner diameter) × 250 mm; manufactured by Daicel Chemical Industries), column temperature: 30 ° C., detection wavelength: 245 nm, mobile phase: n-hexane / ethanol = 70/30, retention time: ( 1R, 2S) -2-hydroxycyclopentanecarboxylic acid ethyl about 7.2 minutes, (1S, 2R) -2-hydroxycyclopentanecarboxylic acid ethyl about 9.2 minutes, (1S, 2S) -2-hydroxycyclopentane Ethyl carboxylate about 15.3 minutes, ethyl (1R, 2R) -2-hydroxycyclopentanecarboxylate about 17.0 minutes.

  From the above two analysis results, the ratio of (1R, 2R) isomer or (1S, 2S) isomer to 2-hydroxycyclopentanecarboxylic acid ester can be easily calculated.

  The ratio of the (1S, 2R) body and (1R, 2S) in the generated formula (9) can be determined as follows.

When the formula (9) to be analyzed is, for example, ethyl 2-hydroxycyclopentanecarboxylate, the cis-isomer ratio (trans isomers in the four stereoisomers ((1S , 2R) and (1R, 2S) isomers) can be determined by the following formula:
[Cis body ratio (%)] = [Cis body area] / ([Trans body area] + [Cis body area]) × 100

  The ratio of (1S, 2R) or (1S, 2R) to the cis isomer is, for example, when dinitrobenzoyl chloride is used under basic conditions when formula (9) is ethyl 2-hydroxycyclopentanecarboxylate. It can be easily calculated by derivatizing and analyzing under the conditions of the above-mentioned high performance liquid chromatography.

  From the above two analysis results, the ratio of the (1S, 2R) body or (1S, 2R) body in the formula (9) can be easily calculated.

When converting the formula (8) into the formula (9), the formula (8) is added to an appropriate solvent such as 100 mM phosphate buffer (pH 6.5), and NADPH, NADP ⁺ , NADH And a coenzyme such as NAD ^+, a culture of the recombinant organism and / or a processed product thereof, and the like, and the mixture is reacted with stirring under pH adjustment.

  Here, the treated product is, for example, a crushed cell, a crude extract, a cultured cell, a freeze-dried organism, an acetone-dried organism, or a ground product thereof, or a mixture thereof, and the polypeptide catalyst. This means that the activity remains.

  The reaction is carried out at a temperature of 5 to 80 ° C., preferably 10 to 60 ° C., more preferably 20 to 40 ° C., and the pH of the reaction solution during the reaction is 3 to 10, preferably 4 to 9, more preferably 5 to 8. To maintain. The reaction can be carried out batchwise or continuously. In the case of a batch system, the reaction substrate may be added at a charge concentration of 0.01 to 100% (w / v), preferably 0.1 to 70%, more preferably 0.5 to 50%. Further, a substrate may be newly added during the reaction.

  Furthermore, it is also effective to add a surfactant such as Triton (manufactured by Nacalai Tesque), Span (manufactured by Kanto Chemical Co., Ltd.), Tween (manufactured by Nacalai Tesque) to the reaction solution. In addition, an organic solvent insoluble in water such as ethyl acetate, butyl acetate, isopropyl ether, toluene, hexane, or the like is added to the reaction solution in order to avoid inhibition of the reaction by the substrate and / or the alcohol that is the product of the reduction reaction. May be. Furthermore, for the purpose of increasing the solubility of the substrate, an organic solvent soluble in water such as methanol, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide and the like can be added.

  The collection of the optically active 2-hydroxycycloalkanecarboxylic acid ester produced by the reduction reaction is not particularly limited, but after separating the cells or the like directly from the reaction solution, ethyl acetate, toluene, t-butyl methyl ether, hexane, Extraction with a solvent such as n-butanol or dichloromethane, and the solvent may be distilled off by an operation such as heating under reduced pressure. Furthermore, if it refine | purifies by distillation, silica gel column chromatography, etc., the said highly purified Formula (9) can be obtained easily.

Polypeptide having reducible coenzyme regeneration ability Optically active 2-hydroxycycloalkane by reducing 2-oxocycloalkanecarboxylic acid ester (8) using a recombinant organism capable of producing the polypeptide used in the present invention When synthesizing the carboxylic acid ester (9), NADH or NADPH is required as a coenzyme. As described above, the present invention can also be carried out by adding NADH or NADPH in a required amount to the reaction system. However, an enzyme having the ability to convert the oxidized coenzyme (NAD ⁺ or NADP ⁺ ) into reduced form (NADH or NADPH) (hereinafter referred to as reduced coenzyme regeneration ability) together with its substrate, that is, coenzyme regeneration By using the system in combination with the polypeptide used in the present invention, the amount of expensive coenzyme used can be greatly reduced. Examples of the enzyme having the ability to regenerate reduced coenzyme include hydrogenase, formate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, and glucose dehydrogenase. Preferably, glucose dehydrogenase is used.

  Such a reaction can also be carried out by adding a coenzyme regeneration system into the asymmetric reduction reaction system, but it encodes a DNA encoding the enzyme of the present invention and a polypeptide having a reduced coenzyme regeneration ability. When a recombinant organism transformed with both DNAs is used as a catalyst, the reaction can be efficiently performed without separately preparing an enzyme having the ability to regenerate reduced coenzyme and adding it to the reaction system. . Such a recombinant organism can be obtained by the method described above.

Conversion of optically active 2-hydroxycycloalkanecarboxylic acid ester to other compound Optically active 2-hydroxycycloalkanecarboxylic acid ester (9) produced by the method described above;

2-aminocycloalkanol derivative (2) particularly useful as a pharmaceutical intermediate;

Or optically active-2-aminocycloalkanol (2) ';

Can be manufactured.

  In the formula (9), R is a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted aralkyl group having 7 to 11 carbon atoms. Represents. Examples of the substituted or unsubstituted alkyl having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group. Examples of the substituted or unsubstituted aryl group having 6 to 10 carbon atoms include phenyl group, p-methylphenyl group, o-methylphenyl group, m-methylbenzyl group, p-chlorophenyl group, p-nitrophenyl group, p -A trifluoromethylphenyl group, 1-naphthyl group, 2-naphthyl group, etc. are mentioned. Examples of the aralkyl group having 7 to 11 carbon atoms include benzyl group, p-methylbenzyl group, o-methylbenzyl group, m-methylbenzyl group, p-nitrobenzyl group, p-trifluoromethylbenzyl group and the like. . Among these, a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms is preferable, and a methyl group or an ethyl group is more preferable.

  The formula (9) can be obtained by the reduction reaction of the corresponding ketoester using the microorganism or enzyme described above.

  In the formula (2), R ′ is, for example, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted group having 7 to 11 carbon atoms. And substituted aralkyl groups. Examples of the substituted or unsubstituted alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group. Examples of the substituted or unsubstituted aryl group having 6 to 10 carbon atoms include phenyl group, p-methylphenyl group, o-methylphenyl group, m-methylbenzyl group, p-chlorophenyl group, p-nitrophenyl group, p -A trifluoromethylphenyl group, 1-naphthyl group, 2-naphthyl group, etc. are mentioned. Examples of the aralkyl group having 7 to 11 carbon atoms include benzyl group, p-methylbenzyl group, o-methylbenzyl group, m-methylbenzyl group, p-nitrobenzyl group, p-trifluoromethylbenzyl group and the like. . Among these, a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms and an aralkyl group having 7 to 11 carbon atoms are preferable, and a methyl group, an ethyl group, a t-butyl group, and a benzyl group are more preferable.

  As a route for producing the formula (2) or the formula (2) ′ from the formula (9), by reacting the formula (9) with ammonia, the 2-hydroxycycloalkanecarboxylic acid amide (3) is obtained. The Hofmann rearrangement route for producing and subsequently carrying out the rearrangement reaction, or the Curtius rearrangement route for carrying out the rearrangement reaction via 2-hydroxycycloalkanecarboxylic acid azide (4) from the above formula (1) (see FIG. 1) . Since the Curtius rearrangement uses an azide compound with explosion risk, it is preferably a route for performing the Hofmann rearrangement.

  In said Formula (9), (2), (2) '(3), (4), n represents the integer of 0-8. Among these, preferably 0 to 4, more preferably 1 to 3, and most preferably 1.

  In the formulas (9), (2), (2) ′ (3) and (4), * 1, * 2 represents an asymmetric carbon. The (S) configuration may be independent of * 1 and * 2, respectively, or the (R) configuration may be used. Among these, * 1 and * 2 are preferably in the (R) configuration.

  Hereinafter, these two routes will be described in detail.

The route using the Hofmann rearrangement reaction is that the above formula (9) is reacted with ammonia to produce the above formula (3), and then the rearrangement reaction is carried out to formula (2) or the above formula (2). 'The route to manufacture.

  First, a method for producing the formula (3) by reacting the formula (9) with ammonia will be described.

  In the formula (9), R is the same as described above.

  Ammonia used may be diluted in a solvent, or may be used without dilution. Examples of the solvent to be diluted include alcohol solvent solutions such as methanol, ethanol, n-propanol, isopropanol, and n-butanol, and aqueous solutions. Among these, a methanol solution or an aqueous solution is preferable.

  When ammonia is used after being diluted, its concentration is not particularly limited, but is preferably a 5 to 40% by weight solution, more preferably a 10 to 30% by weight solution, and even more preferably 15 to 28% by weight.

  Although the equivalent of ammonia to be used is not particularly limited, it is preferably 1 to 30 equivalents, more preferably 5 to 25 equivalents, and further preferably 10 to 20 equivalents with respect to the formula (9).

  What is necessary is just to perform reaction temperature within the range of -50-120 degreeC normally. Preferably it exists in the range of 0-100 degreeC, More preferably, it exists in the range of 20-100 degreeC.

  The reaction can be carried out under normal pressure (1 atm), but can be carried out under pressure by heating using a sealed reaction can.

  The solvent to be used is not particularly limited, and examples thereof include aprotic polar solvents such as N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone, hexamethylphosphoric triamide, hexamethylbenzene, Hydrocarbon solvents such as toluene, n-hexane, cyclohexane, ether solvents such as diethyl ether, tetrahydrofuran (THF), diisopropyl ether, methyl tert-butyl ether, dimethoxyethane, chlorobenzene, methylene chloride, chloroform, 1,1,1 -Halogen solvents such as trichloroethane, ester solvents such as ethyl acetate and butyl acetate, nitrile solvents such as acetonitrile and butyronitrile, methanol, ethanol, isopropanol, n-propanol, butanol , Alcohol solvents octanol and water. These may be used alone or in combination of two or more. Among these, alcohol solvents, hydrocarbon solvents, and water are preferable, and toluene, methanol, ethanol, isopropanol, n-propanol, and water are more preferable.

  After completion of the reaction, the crude product can be obtained by concentrating the reaction solution. Moreover, you may acquire a composition by performing a general post-process. As a general post-treatment, for example, an extraction operation is performed using water and a general extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane and the like. The target compound is obtained by distilling off the reaction solvent and the extraction solvent from the resulting extract by an operation such as heating under reduced pressure.

  The target product thus obtained may be used as it is in the next step, but may be further purified by a general method such as crystallization purification, fractional distillation, column chromatography, etc. to further increase the purity.

  When the diastereomer of the formula (3) is contained in the crude product, a crystallization operation is performed to obtain the formula (2) or the formula (2) ′ having sufficient quality as a pharmaceutical intermediate. Is preferred. That is, diastereomers can be removed by crystallization of a crude product containing the formula (3).

  Although it does not specifically limit as a diastereomer, For example, when the configuration which is preferable in the said Formula (3), that is, when * 1 and * 2 are both (R), a diastereomer is (1R, 2S) -2-hydroxycyclo Alkaneamide and / or (1S, 2R) -2-hydroxycycloalkanamide.

  The crystallization solvent is not particularly limited, and examples thereof include hydrocarbon solvents such as hexamethylbenzene, toluene, n-hexane, and cyclohexane, diethyl ether, tetrahydrofuran (THF), diisopropyl ether, methyl tert-butyl ether, and dimethoxyethane. Ether solvents, halogen solvents such as chlorobenzene, methylene chloride, chloroform, 1,1,1-trichloroethane, ester solvents such as ethyl acetate and butyl acetate, nitrile solvents such as acetonitrile and butyronitrile, methanol, ethanol, isopropanol, Examples thereof include alcohol solvents such as n-propanol, butanol and octanol, and water. These may be used alone or in combination of two or more. Among these, a combination of an ester solvent and an alcohol solvent is preferable, and a combination of ethyl acetate and methanol is more preferable.

  The amount of the solvent used for crystallization is 1 to 200 times by weight, preferably 1 to 50 times by weight with respect to the compound represented by the formula (9).

  The temperature at the time of crystallization is in the range of -10 ° C to 120 ° C, preferably in the range of -10 ° C to 70 ° C, and most preferably in the range of -10 ° C to 50 ° C.

  The crystallization method is not particularly limited, and any method may be used. For example, a method may be used in which a crystallization solvent is added to a crude product containing the compound represented by the formula (9), heated to a uniform solution, and then cooled and crystallized. After dissolving in a rich solvent, crystallization may be performed by adding a poor solvent. Moreover, after dissolving the crude product containing the compound represented by the formula (9) in a mixed solution of a rich solvent and a poor solvent, it may be crystallized by removing the good solvent by concentration distillation or the like. Furthermore, operations such as seed crystal addition can be appropriately combined with the above-described method.

  The diastereomer content contained in the formula (9) obtained by crystallization is 10.0 mol% or less, preferably 5.0 mol% or less, more preferably 2.0 mol%, in terms of molar ratio. % Or less.

  The crystals of the compound represented by the formula (3) thus obtained are subjected to solid-liquid separation by, for example, centrifugal separation, pressure filtration, reduced pressure filtration, etc., and cake washing is performed as necessary. Can be obtained as Further, dry crystals can be obtained by further drying under reduced pressure.

  When using the acquired crystal | crystallization for reaction of the following process, it may be used as a dry crystal | crystallization and may be used with a wet body.

  Next, a method for producing Formula (2) or Compound (2) ′ by reacting Formula (3) with a halogenating agent in the presence of a base will be described.

  The base used may be an inorganic base or an organic base. Examples of the inorganic base include metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, magnesium hydroxide, and barium hydroxide, and carbonates such as sodium carbonate, potassium carbonate, and sodium bicarbonate.

  Although it does not specifically limit as an organic base, A tertiary amine is preferable. Examples of tertiary amines include trialkylamines having 1 to 12 carbon atoms such as trimethylamine, triethylamine, ethyldiisopropylamine, N, N-dimethylaniline, N, N-diethylaniline, N.I. Tertiary amines composed of alkyl groups having 1 to 4 carbon atoms such as N-dimethylaminopyridine and aryl groups or heteroaryl groups, nitrogen-containing organic bases such as pyridine, picoline, and lutidine, N, N, N, N-tetra 1 to 10 carbon atoms such as methyl-1,2-ethylenediamine, N, N, N, N-tetramethyl-1,3-propanediamine, N, N, N, N-tetramethyl-1,6-hexanediamine, etc. N, N, N, N-tetramethyl-α, ω-alkyldiamine and the like can be mentioned.

  These bases may be used alone or in combination. Among these, lithium hydroxide, sodium hydroxide, and potassium hydroxide are preferable because they are inexpensive and easily available.

  The amount of the base used may be 1 equivalent or more with respect to the formula (3), preferably 1 to 10 equivalents, and more preferably 1 to 5 equivalents.

  The base used in this reaction may be used without being diluted in a solvent, or may be used after being diluted.

  Examples of the halogenating agent used in this reaction include sodium hypochlorite, potassium hypochlorite, sodium hypobromite, chlorine, bromine, iodine, N-chlorosuccinimide (NCS), N-bromosuccinimide ( NBS), N-iodosuccinimide (NIS), N-chloroisocyanuric acid and the like. Of these, sodium hypochlorite and sodium hypobromite are preferable, and sodium hypochlorite is more preferable.

  These halogenating agents may be used as they are, or those diluted with water or an organic solvent may be used. In particular, when sodium hypochlorite is used, an aqueous solution is usually used.

  The halogenating agent may be prepared in the system. For example, a methanol solution of sodium hypobromite can be prepared by reacting bromine and sodium methoxide in a methanol solvent.

  What is necessary is just to use 1 equivalent or more with respect to the said Formula (3) as a quantity of the halogenating agent used for this reaction, Preferably it is 1-10 equivalent, More preferably, it is 1-5 equivalent.

  The reaction temperature is usually within the range of -30 to 120 ° C, preferably -20 to 80 ° C. More preferably, it is -10-60 degreeC.

  The solvent used in this step is not particularly limited, and examples thereof include aprotic polar solvents such as N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone, hexamethylphosphoric triamide, hexa Hydrocarbon solvents such as methylbenzene, toluene, n-hexane, cyclohexane, ether solvents such as diethyl ether, tetrahydrofuran (THF), diisopropyl ether, methyl tert-butyl ether, dimethoxyethane, chlorobenzene, methylene chloride, chloroform, 1, Halogen solvents such as 1,1-trichloroethane, ester solvents such as ethyl acetate and butyl acetate, nitrile solvents such as acetonitrile and butyronitrile, methanol, ethanol, isopropanol, n-propanol, Ethanol, alcohol solvents octanol and water. These may be used alone or in combination of two or more. Of these, ether solvents, alcohol solvents, and water are preferable, and tetrahydrofuran (THF), methanol, ethanol, isopropanol, n-propanol, and water are more preferable.

  By performing this reaction in the presence of water, the above formula (2) ′ can be obtained. Alcohol (5);

By performing in the presence, the formula (2) can be obtained.

  In the formulas (2) and (5), R ′ is the same as described above.

  In order to obtain a crude product from the reaction solution after completion of the reaction, a general post-treatment may be performed. For example, the extraction operation is performed using a general extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane and the like. The target compound is obtained by distilling off the reaction solvent and the extraction solvent from the resulting extract by an operation such as heating under reduced pressure. Furthermore, after completion of the reaction, the reaction solvent may be distilled off or replaced by an operation such as heating under reduced pressure, and then the same operation as described above may be performed.

  2-aminocyclopentanol (2) 'synthesized as described above;

From general formula (6)

N-protected-2-aminocyclopentanol derivative represented by the following formula can be obtained.

In the formula (6), R ⁴ is a substituted or unsubstituted alkyloxycarbonyl group having 1 to 15 carbon atoms, a substituted or unsubstituted aralkyloxycarbonyl group having 7 to 12 carbon atoms, or a substitution having 2 to 12 carbon atoms. Or an unsubstituted acyl group is represented. Examples of the substituent include the same as those described for R. Examples of the substituted or unsubstituted alkyloxycarbonyl group having 1 to 15 carbon atoms include t-butyloxycarbonyl group (Boc group), methoxycarbonyl group (Moc group), and 9-fluorenylmethoxycarbonyl group (Fmoc group). Etc. Examples of the substituted or unsubstituted aralkyloxycarbonyl group having 7 to 12 carbon atoms include benzyloxycarbonyl group (Cbz group) and p-methoxybenzyloxycarbonyl group. Examples of the substituted or unsubstituted acyl group having 2 to 12 carbon atoms include an acetyl group and a benzoyl group. Among them, t-butyloxycarbonyl group (Boc group), methoxycarbonyl group (Moc group), 9-fluorenylmethoxycarbonyl group (Fmoc group), benzyloxycarbonyl group (Cbz group), and acetyl group are preferable. More preferred are t-butyloxycarbonyl group (Boc group) and benzyloxycarbonyl group (Cbz group).

  In the formula (6), * 1 and * 2 represent asymmetric carbons. The (S) configuration may be independent of * 1 and * 2, respectively, or the (R) configuration may be used. Among these, * 1 and * 2 are preferably in the (R) configuration.

  The method of deriving from the formula (2) ′ to the formula (6) may be performed under general protection conditions. For example, Theodora W. Greene, Peter G. M.M. This can be carried out according to the deprotection method described in Wuts Protective Groups in Organic Chemistry (3rd edition) published by JOHN WILEY & SONS, INC.

  For example, t-butyloxycarbonyl (Boc) can be protected by allowing t-butyl dicarbonate to act in a suitable solvent in the presence of a base, and benzyloxycarbonyl can be protected in a suitable solvent in the presence of a base. By reacting with chloride, benzyloxycarbonyl (Cbz) protection can be performed.

  The formula (2) ′ isolated by the method described above may be used to derive the formula (6), or the reaction solution containing the formula (2) ′ may be used as it is without performing an isolation operation. May be used to derive the equation (7).

Route Using Curtius Rearrangement Next, the Curtius rearrangement route for producing Formula (2) or Formula (2) ′ from Formula (9) via Formula (4) will be described.

  In order to produce the formula (2) from the formula (9), a general Curtius rearrangement reaction condition may be used. For example, it can be performed according to the method described in Strategic Applications of Named Reaction in Organic Synthesis Elsevier Inc.

  In this reaction, 2-hydroxyacycloalkanecarboxylic acid (1) that can be produced by the hydrolysis reaction of the formula (9);

Can also be used.

  The formula (1) can be obtained by general hydrolysis using an acid or base of 2-hydroxycycloalkanecarboxylic acid ester produced by the above production method. For example, 2-hydroxycyclopentanecarboxylic acid methyl ester produced by a reduction reaction using an enzyme can be produced by allowing sodium hydroxide to act in a water-methanol mixed solvent.

  By carrying out this reaction in the presence of water, the above formula (2) ′ can be produced. Moreover, the said Formula (2) can be manufactured by performing in the said alcohol (5) presence.

  For example, N- (benzyloxycarbonyl) -2-hydroxycyclopentanol can be produced by reacting 2-hydroxycyclopentanecarboxylic acid (1) with diphenylphosphoric acid azide in the presence of benzyl alcohol.

  In the formula (9), R is the same as described above.

  In the formulas (2) and (5), R ′ is the same as described above.

  In said Formula (1), (2), (2) ', (4), (9), n represents the integer of 0-8. Among these, preferably 0 to 3, more preferably 1 to 3, and most preferably 1.

  In the formulas (1), (2), (2) ′, (4) and (9), * 1 and * 2 represent asymmetric carbons. The (S) configuration may be independent of * 1 and * 2, respectively, or the (R) configuration may be used. Among these, * 1 and * 2 are preferably in the (R) configuration.

  Usually, the said Formula (4) is used for reaction, without isolating. The production method of the formula (4) is not particularly limited, and in addition to the conditions generally used for the Curtius rearrangement described above, the hydrazide (7) can be obtained by reacting the formula (9) with hydrazine.

May be produced by the subsequent action of nitrite.

  In said formula (7), n, * 1, * 2 is the same as the above.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by these. Detailed operation methods relating to the recombinant DNA technology used in the following examples are described in the following documents:
Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience).

E. coli JM109 (pUCAR2) that produces a polypeptide (ARII) derived from Sporobolomyces salmonicolor
E. coli J having plasmid pUCAR2 (plasmid obtained by introducing the structural gene of polypeptide ARII into vector plasmid pUC118) by the method described in Applied and Environmental Microbiology, 65 (12), 5207-5211, (1999)
M109 (pUCAR2) was bred. Recombinant E. coli E. coli HB101 (pUC118) (comparative example) with E. coli JM109 (pUCAR2) and vector plasmid pUC118 was compared with 2 × YT medium (Tripton 1.G) containing 0.1 mM IPTG, 200 μg / ml ampicillin. 6%, yeast extract 1.0%, NaCl 0.5%, pH 7.0) was inoculated into 5 ml, and cultured with shaking at 37 ° C. for 20 hours.

  About each culture solution obtained by said culture | cultivation, the microbial cell was collected by centrifugation and suspended in 5 ml of 100 mM phosphate buffer (pH 6.5). This was crushed using a UH-50 type ultrasonic homogenizer (manufactured by SMT), and then the cell residue was removed by centrifugation to obtain a cell-free extract. The cell-free extract was measured for 2-oxocyclopentanecarboxylic acid ester reducing activity.

The reduction activity for 2-oxocyclopentanecarboxylic acid ester is 30 mM by adding methyl 2-oxocyclopentanecarboxylate 5 mM, coenzyme NADPH 0.25 mM, and crude enzyme solution to 100 mM phosphate buffer (pH 6.5). The reaction was carried out at 1 ° C. for 1 minute, and it was calculated from the rate of decrease in absorbance at a wavelength of 340 nm. Under these reaction conditions, the enzyme activity that oxidizes 1 μmol of NADPH to NADP ⁺ per minute was defined as 1 U.

The 2-oxocyclopentanecarboxylic acid ester reducing activity of E. coli HB101 (pUC118) (comparative example) is 0.1 U / mg or less, whereas that of E. coli HB101 (pUCAR2) expressing ARII The reduction activity of 2-oxocyclopentanecarboxylic acid ester was 3.3 U / mg.

As described above, the polypeptide ARII had a reducing activity on the target 2-oxocyclopentanecarboxylic acid ester.

Production of ethyl (1R, 2R) -2-hydroxycyclopentanecarboxylate using recombinant organism E. coli HB101 (pUCAR2)
After culturing E. coli HB101 (pUCAR2) in the same manner as in Example 1, cell disruption with an ultrasonic homogenizer was performed to obtain 180 ml of a cell-free extract. To 180 ml of this cell-free extract, 2250 U of glucose dehydrogenase (trade name: GLUCDH “Amano” II, Amano Enzyme), 12.5 g of glucose, 22 mg of NADP ⁺ , 3.6 g of ethyl 2-oxocyclopentanecarboxylate The mixture was added and stirred at 30 ° C. while adjusting to pH 6.5 by dropwise addition of 5N aqueous sodium hydroxide solution. At 2 hours after the start of the reaction, 3.6 g and an additional 1.8 g of ethyl 2-oxocyclopentanecarboxylate were added at 4.75 hours and stirred for 27 hours to react. After the reaction, a part of the reaction solution was sampled and extracted with ethyl acetate. The extract was analyzed under the following gas chromatography conditions, and the conversion rate to ethyl 2-hydroxycyclopentanecarboxylate and the trans isomer ratio were measured. As a result, the conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 100%, and the trans isomer ratio was 76.4%. The trans isomer ratio (%) was calculated by the following formula.
[Trans-body ratio (%)] = [Trans-body area] / ([Trans-body area] + [Cis-body area]) × 100.

[Analysis conditions by gas chromatography]
Column: ULBON HR-20M (0.25 mm (inner diameter) × 25 m; manufactured by Shinwa Kako), detection: FID, column temperature: 130 ° C., injection temperature: 200 ° C., detection temperature: 200 ° C., carrier gas: helium (130 kPa) ), Elution time: ethyl 2-hydroxycyclopentanecarboxylate (trans isomer) approximately 15.7 minutes, ethyl 2-hydroxycyclopentanecarboxylate (cis isomer) approximately 8.1 minutes, ethyl 2-oxocyclopentanecarboxylate approximately 8.8 minutes.

  The product was extracted from the reaction solution with toluene, and the solvent was distilled off to obtain 8.35 g of ethyl 2-hydroxycyclopentanecarboxylate. The obtained ethyl 2-hydroxycyclopentanecarboxylate was derivatized with the action of dinitrobenzoyl chloride under basic conditions, and analyzed under the conditions of high performance liquid chromatography under the following conditions to analyze the optical purity. As a result, the optical purity of the (1R, 2R) isomer was 99.3% e.e. e. Met.

[Analysis conditions by high performance liquid chromatography]
Column: Chiralcel AD-H (4.6 μm (inner diameter) × 250 mm; manufactured by Daicel Chemical Industries), column temperature: 30 ° C., detection wavelength: 245 nm, mobile phase: n-hexane / ethanol = 70/30, retention time: ( 1R, 2S) -2-hydroxycyclopentanecarboxylic acid ethyl about 7.2 minutes, (1S, 2R) -2-hydroxycyclopentanecarboxylic acid ethyl about 9.2 minutes, (1S, 2S) -2-hydroxycyclopentane Ethyl carboxylate about 15.3 minutes, ethyl (1R, 2R) -2-hydroxycyclopentanecarboxylate about 17.0 minutes.

  From the analysis results of the trans ratio and optical purity of ethyl 2-hydroxycyclopentanecarboxylate, the proportion of (1R, 2R) isomers in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 76.1%. there were.

Production of methyl (1R, 2R) -2-hydroxycyclopentanecarboxylate using recombinant organism E. coli HB101 (pUCAR2)
After culturing E. coli HB101 (pUCAR2) in the same manner as in Example 1, cell disruption with an ultrasonic homogenizer was performed to obtain 1200 ml of a cell-free extract. To 1200 ml of the cell-free extract, glucose dehydrogenase (trade name: GLUCDH “Amano” II, manufactured by Amano Enzyme) 15000 U, glucose 164 g, NADP ⁺ 115 mg, and 25 g of methyl 2-oxocyclopentanecarboxylate were added to 5N. The solution was stirred at 30 ° C. while adjusting the pH to 6.5 by dropwise addition of an aqueous sodium hydroxide solution. At 1.5 hours, 3 hours and 6 hours after the start of the reaction, 25 g of each was added, and 12.5 g of methyl 2-oxocyclopentanecarboxylate was added at 9.5 hours, and the mixture was stirred for 27 hours to react. It was. After the reaction, a part of the reaction solution was sampled and extracted with ethyl acetate. The extract was analyzed under the conditions of gas chromatography described below, and the conversion rate to methyl 2-hydroxycyclopentanecarboxylate and the trans isomer ratio were measured. As a result, the conversion rate to methyl 2-hydroxycyclopentanecarboxylate was 98%, and the trans isomer ratio was 83.1%.

[Analysis conditions by gas chromatography]
Column: ULBON HR-20M (0.25 mm (inner diameter) × 25 m; manufactured by Shinwa Kako), detection: FID, column temperature: 130 ° C., injection temperature: 200 ° C., detection temperature: 200 ° C., carrier gas: helium (130 kPa) ), Elution time: methyl 2-hydroxycyclopentanecarboxylate (trans isomer) approximately 14.3 minutes, methyl 2-hydroxycyclopentanecarboxylate (cis isomer) approximately 7.6 minutes, methyl 2-oxocyclopentanecarboxylate approximately 8.0 minutes.

  The product was extracted from the reaction solution with toluene, and the solvent was distilled off to obtain 95.8 g of methyl 2-hydroxycyclopentanecarboxylate. The obtained methyl 2-hydroxycyclopentanecarboxylate was derivatized with the action of dinitrobenzoyl chloride under basic conditions, and the optical purity was analyzed by analyzing under the conditions of high performance liquid chromatography under the following conditions. As a result, the optical purity of the (1R, 2R) isomer was 98.8% e.e. e. Met.

[Analysis conditions by high performance liquid chromatography]
Column: Chiralcel AD-H (4.6 μm (inner diameter) × 250 mm; manufactured by Daicel Chemical Industries), column temperature: 30 ° C., detection wavelength: 245 nm, mobile phase: n-hexane / ethanol = 70/30, retention time: ( 1R, 2S) -2-hydroxycyclopentanecarboxylate methyl about 9.5 minutes, (1S, 2R) -2-hydroxycyclopentanecarboxylate methyl about 13.4 minutes, (1S, 2S) -2-hydroxycyclopentane Methyl carboxylate about 19.1 minutes, methyl (1R, 2R) -2-hydroxycyclopentanecarboxylate about 22.4 minutes.

  From the analysis results of the trans ratio and optical purity of methyl 2-hydroxycyclopentanecarboxylate, the proportion of (1R, 2R) isomer in the four stereoisomers of methyl 2-hydroxycyclopentanecarboxylate was 82.6%. there were.

Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using polypeptide derived from Candida magnolie In 2 ml of 100 mM phosphate buffer (pH 6.5), 46 mg of glucose, Candida magnolie (Candida magnoliae) CR enzyme prepared from NBRC0705 (International publication WO01 / 040450 pamphlet, Example 1) 100 U, glucose dehydrogenase (trade name: GLUCDH "Amano" II, manufactured by Amano Enzyme) 25 U, NADP ⁺ 1 mg, 2- 20 mg of ethyl oxocyclopentanecarboxylate was added and stirred at 30 ° C. for 24 hours. The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate after the reaction, the trans isomer ratio, and the optical purity thereof were measured by the method described in Example 2. As a result, the conversion rate was 45% and the trans isomer ratio was 57.6%. The optical purity is 93.6% e.e. in (1S, 2S) form. e. Therefore, the proportion of the (1S, 2S) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 55.8%.

Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Rhodotorula glutinis bar dyrenensis Instead of CR enzyme, Rhodotorula glutinis var. Dairenensis The reaction was carried out in the same manner as in Example 4 using 100 U of reductase RRG prepared from NBRC0415 (see International Publication WO03 / 093477 pamphlet, Example 1). The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 99%, and the trans isomer ratio was 97.9%. The optical purity is (1S, 2S) and 100% e.e. e. Therefore, the ratio of the (1S, 2S) form in the four types of stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 97.9%.

Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using polypeptide derived from Ogataea Minuta bar Minuta Instead of CR enzyme, Ogataea minuta var. Minuta The reaction was carried out in the same manner as in Example 4 using 100 U of reductase prepared from NBRC0975 (see International Publication WO06 / 013801 pamphlet, Example 1). The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 98%, and the trans isomer ratio was 70.0%. The optical purity is (1S, 2S) and 100% e.e. e. Therefore, the proportion of the (1S, 2S) form in the four types of stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 70.0%.

Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using polypeptide derived from Candida boidini Instead of CR enzyme, alcohol dehydrogenase CP (Julich derived from Candida boidinii) The reaction was carried out in the same manner as in Example 4 using 20 mg of Fine Chemicals. The conversion to ethyl 2-hydroxycyclopentanecarboxylate was 60%, and the trans isomer ratio was 75.0%. The optical purity is (1S, 2S) and 100% e.e. e. Therefore, the proportion of the (1S, 2S) form in the four types of stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 75.0%.

Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Thermoanaerobium brocci Alcohol dehydrogenation from Thermoanaerobium brockii instead of CR enzyme The reaction was carried out in the same manner as in Example 4 using 50 mg of the enzyme (manufactured by Sigma Aldrich). The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 100%, and the trans isomer ratio was 80.4%. The optical purity is (1S, 2S) and 100% e.e. e. Therefore, the proportion of the (1S, 2S) form in the four types of stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 80.4%.

Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using polypeptide derived from Candida maris Reductase FPDH prepared from Candida maris NBRC10003 (International) The reaction was carried out in the same manner as in Example 4 using 100 U) (see publication WO01 / 05996 pamphlet, Example 14). The conversion to ethyl 2-hydroxycyclopentanecarboxylate was 95%, and the cis-isomer ratio was 97.4%. The cis isomer ratio (%) was calculated by the following formula.
[Cis body ratio (%)] = [Cis body area] / ([Trans body area] + [Cis body area]) × 100

  The optical purity is 99.1% e.e. in (1S, 2R) isomer. e. Therefore, the proportion of the (1S, 2R) isomer in the four types of stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 97.0%.

Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Debosia riboflavina Reductase RDR prepared from Devosia riboflavina NBRC13584 instead of CR enzyme (International Publication WO04) The reaction was carried out in the same manner as in Example 4 using 100U. The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 96%, and the cis-isomer ratio was 95.7%. The optical purity is 99.5% e.e. (1S, 2R) isomer. e. Therefore, the proportion of (1S, 2R) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 95.5%.

Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Brevundimonas diminuta Instead of CR enzyme, a reductase prepared from Brevundimonas diminuta NBRC13584 ( The reaction was carried out in the same manner as in Example 4 using 100 U) (see International Publication WO07 / 114217 Pamphlet, Example 4). The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 80%, and the cis-isomer ratio was 93.4%. The optical purity is (1S, 2R) isomer, 98.6% e.e. e. Therefore, the proportion of the (1S, 2R) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 92.7%.

Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Lactobacillus brevis Alcohol dehydrogenase derived from Lactobacillus brevis (Julich Fine chemicals) instead of CR enzyme The reaction was carried out in the same manner as in Example 4 using 100 μl. The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 98%, and the cis-isomer ratio was 98.7%. The optical purity is 99.5% e.e. (1S, 2R) isomer. e. Therefore, the proportion of the (1S, 2R) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 98.5%.

Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Lactobacillus kefir Alcohol dehydrogenase derived from Lactobacillus kefir (Fluka) instead of CR enzyme Reaction was carried out in the same manner as in Example 4 using 50 mg. The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 98%, and the cis-isomer ratio was 97.7%. The optical purity is 98.5% e.e. in (1S, 2R) isomer. e. Therefore, the proportion of the (1S, 2R) isomer in the four types of stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 97.0%.

Production of ethyl (1R, 2S) -2-hydroxycyclopentanecarboxylate using a Candida maltosa-derived polypeptide Instead of CR enzyme, a reductase prepared from Candida maltosa NBRC1977 (International Publication) The reaction was carried out in the same manner as in Example 4 using 100 U (see WO08 / 0666018 pamphlet, Example 1). The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 40%, and the cis-isomer ratio was 98.2%. The optical purity was 99.0% e.e. in (1R, 2S) form. e. Therefore, the proportion of (1R, 2S) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 97.7%.

Production of ethyl (1R, 2S) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Candida parapsilosis Reductase prepared from Candida parapsilosis NBRC708 instead of CR enzyme 2003-289874 pamphlet, Example 1) The reaction was carried out in the same manner as Example 4 using 100 U. The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 40%, and the cis-isomer ratio was 98.2%. The optical purity was 99.0% e.e. in (1R, 2S) form. e. Therefore, the proportion of (1R, 2S) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 97.7%.

Production of ethyl (1R, 2S) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Rhodococcus erythropolis Instead of CR enzyme, alcohol dehydrogenase derived from Rhodococcus erythropolis (Julich Fine) The reaction was carried out in the same manner as in Example 4 using 100 μl of Chemicals). The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 40%, and the cis-isomer ratio was 99.8%. The optical purity was 97.5% e.e. (1R, 2S). e. Therefore, the proportion of the (1R, 2S) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 98.6%.

Preparation of (1R, 2R) -2-hydroxycyclopentanecarboxylic acid amide Methyl (1R, 2R) -2-hydroxycyclopentanecarboxylic acid (73.34 g, 0.52 mol) obtained by the method described in Example 3 And 20 wt% NH 3 / MeOH solution (658.20 g, 7.74 mol) were stirred in an autoclave for 48 hours at a bath temperature of 80 ° C. in a sealed state. After completion of the reaction, the autoclave was opened at room temperature, and the inner solution was concentrated using a rotary evaporator until the total amount became 110.14 g. After adding ethyl acetate (490 g) to the concentrate, the precipitated solid was obtained by filtration to obtain the title compound (yield 35.17 g, yield 53%). From 1H-NMR, the diastereomer present in (1R, 2R) -2-hydroxycyclopentanecarboxylic amide was 2.0% in terms of molar ratio.
¹ H-NMR (CDCl ₃ ) 1.54-1.80 (m, 4H), 1.90-2.04 (m, 2H), 2.51-2.57 (m, 1H), 4.26 (Dt, 1H)

Production of (1R, 2R) -2-aminocyclopentanol (1R, 2R) -2-hydroxycyclopentanecarboxylic acid amide (34.00 g, 0.26 mol) obtained by the method described in Example 17 was The solution was diluted to (170 g), and a 30 wt% aqueous sodium hydroxide solution (70.20 g, 0.53 mol) was added dropwise over 35 minutes at a bath temperature of 0 ° C. Subsequently, at the same temperature, a 13 wt% sodium hypochlorite aqueous solution (226.10 g, 0.40 mol) was added dropwise over 2.5 hours. After stirring for 30 minutes at the same temperature, stirring was further performed for 1 hour at a bath temperature of 40 ° C. After cooling the reaction solution to 20 ° C., the pH in the system was adjusted to 6.8 by adding 35 wt% hydrochloric acid. As a result of quantitative analysis by gas chromatography compared with a standard product of the reaction solution ((1R, 2R) -2-aminocyclopentanol hydrochloride), it was confirmed that the title compound was contained (yield 27.61 g, yield). 80%).

(1R, 2R) -2- (t-butyloxycarbonylamino) cyclopentanol An aqueous solution of (1R, 2R) -2-aminocyclopentanol obtained by the method described in Example 17 (net amount: 27. 61 g, 0.21 mol) was added with a 30 wt% aqueous sodium hydroxide solution to adjust the pH in the system to 10.4. Under a bath temperature of 20 ° C., t-butyl dicarbonate (68.94 g, 0.32 mol) was added. During the reaction, since the pH in the system was lowered, 30 wt% NaOH was appropriately added to adjust the pH to 10 ± 0.5. After stirring at the same temperature for 14 hours, concentrated hydrochloric acid was added to adjust the pH of the system to 4.0. Extraction was performed twice using toluene (70 g × 2), and the obtained organic layer was washed with water (70 g). As a result of gas chromatography quantitative analysis comparing with the standard product of the reaction solution, it was confirmed that the title compound was contained (yield 39.04 g, yield 93%). Using a rotary evaporator, the organic layer was concentrated to a total amount of 145.55 g.
¹ H-NMR (CDCl ₃ ) 1.33-1.39 (m, 1H), 1.45 (s, 9H), 1.61-1.70 (m, 2H), 1.75-1.83 (M, 1H), 1.97-2.14 (m, 2H), 3.59-3.66 (m, 1H), 3.96-4.00 (1H, m), 4.06 (br , 1H), 4.71 (br, 1H)

(1R, 2R) -2-aminocyclopentanol hydrochloride obtained in the manner described in Example 19) (1R, 2R) -2- (t-butyloxycarbonylamino) cyclopentanol in toluene solution ( 29.5 wt% hydrogen chloride / isopropanol solution (118.52 g, 0.96 mol) was added dropwise over 1.5 hours at a bath temperature of 50 ° C. with respect to the net amount: 38.60 g, 0.19 mol). After stirring for 1.0 hour at the same temperature, 1.0 hour at a bath temperature of 20 ° C., and 3.0 hours at a bath temperature of 10 ° C., a precipitated solid was obtained by filtration. The obtained solid was washed with a mixed solution of toluene (21.70 g) and isopropanol (38.20 g) and then dried under reduced pressure to obtain the title compound (yield 20.50 g, yield 78%).
¹ H-NMR (DMSO) 1.43-1.57 (m, 2H), 1.60-1.71 (m, 2H), 1.85-2.03 (m, 2H), 3.09- 3.14 (m, 1H), 3.96-4.02 (m, 1H), 5.20-5.21 (d, 1H), 8.08 (br, 3H)

Claims (22)

General formula (9);

(In the formula, R represents a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted aralkyl group having 7 to 11 carbon atoms. n represents an integer of 0 to 8. * 1, * 2 represents an asymmetric carbon.) represented by general formula (8);

(Wherein R and n are the same as above), a polypeptide, an organism producing the polypeptide, or a processed product of the organism is allowed to act on the 2-oxocycloalkanecarboxylic acid ester represented by Then, the 2-oxocycloalkanecarboxylic acid ester is reduced. The manufacturing method according to claim 1, wherein n is 1. Polypeptides are Candida, Rhodotorula, Devosia, Ogataea, Brevundimonas, Lactobacillus, Thermoanaerobium The production according to claim 1 or 2, which is derived from a microorganism selected from the group consisting of a genus, a genus Rhodococcus, a genus Sporobolomyces, and a genus Sporidiobolus. Method. The manufacturing method according to claim 1 or 2, wherein the optically active 2-hydroxycycloalkanecarboxylic acid ester represented by the formula (9) is (1R, 2R) -2-hydroxycycloalkanecarboxylic acid ester. . The production method according to claim 4, wherein the polypeptide is derived from a microorganism selected from the group consisting of the genus Sporobolomyces and the genus Sporidiobolus. The production method according to claim 4, wherein the polypeptide is a polypeptide selected from the group consisting of the following (a1), (a2), and (a3):
(A1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing;
(A2) consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, and acts on the formula (8) and NADPH A polypeptide having the activity to produce NADP with the formula (9),
(A3) an activity having 85% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing and acting on the above formula (8) and NADPH to produce the above formula (9) and NADP; Having a polypeptide. The organism producing a polypeptide selected from the group consisting of (a1), (a2) and (a3) is selected from the group consisting of the following (A1), (A2), (A3) and (A4) The production method according to claim 4, which is a recombinant organism into which the DNA to be introduced is introduced:
(A1) DNA set forth in SEQ ID NO: 2 in the Sequence Listing,
(A2) DNA encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing,
(A3) Hybridizes with a DNA complementary to the DNA of SEQ ID NO: 2 in the sequence listing under stringent conditions, and acts on the formula (8) and NADPH to generate the formula (9) and NADP. DNA encoding a polypeptide having the activity of:
(A4), having a sequence identity of 85% or more with the base sequence set forth in SEQ ID NO: 2 in the sequence listing and acting on 2-oxocyclopentanecarboxylic acid ester and NADPH (1R, 2R) -2- A DNA encoding a polypeptide having an activity to produce hydroxycyclopentanecarboxylic acid ester and NADP. The production method according to claim 7, wherein the recombinant organism is Escherichia coli. In the formula (9), * 1 is the absolute configuration of (S), * 2 is (S), and the polypeptide is a genus Candida, a genus Rhodotorula, a genus Ogataea, The production method according to claim 1 or 2, which is derived from a microorganism selected from the group consisting of the genus Thermoanaerobium. In the above formula (9), * 1 is the absolute configuration of (S), * 2 is (R), and the polypeptide is a genus Candida, a genus Devosia, or a Brevundimonas. The production method according to claim 1 or 2, which is derived from a microorganism selected from the group consisting of a genus and a genus Lactobacillus. In the formula (9), * 1 is the absolute configuration of (R), * 2 is (S), and the polypeptide is selected from the group consisting of the genus Candida and the genus Rhodococcus. The production method according to claim 1 or 2, which is derived from a microorganism. The production method according to any one of claims 1 to 11, wherein a polypeptide having the ability to regenerate reduced coenzyme is further acted. 13. The production method according to claim 12, wherein a recombinant organism having the ability to produce a polypeptide having a reduced coenzyme regeneration ability is used in addition to the polypeptide of (a1), (a2), or (a3). . The production method according to claim 12 or 13, wherein the polypeptide having reductive coenzyme regeneration ability is glucose dehydrogenase. The production method according to claim 1, wherein R is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms. The production method according to claim 1, wherein R is a methyl group or an ethyl group. A general formula (9) produced by the method according to any one of claims 1 to 16;

(In the formula, R represents a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted aralkyl group having 7 to 11 carbon atoms. n represents an integer of 0 to 8. * 1, * 2 represents an asymmetric carbon.) 2-hydroxycycloalkanecarboxylic acid ester represented by formula (3);

(Wherein, * 1 and * 2 are the same as above) to obtain a 2-hydroxycycloalkanecarboxylic acid amide compound. The production method according to claim 17, wherein the 2-hydroxycycloalkanecarboxylic acid amide represented by the formula (3) is purified by crystallization. The production method according to claim 18, wherein the diastereomeric content present in the formula (3) is 5.0 mol% or less in terms of a molar ratio. The production method according to claim 17 or 19, wherein the diastereomer content present in the formula (3) is 2.0 mol% or less in terms of molar ratio. Said formula (3) manufactured by the method in any one of Claims 17-20;

(Wherein, * 1 and * 2 are the same as those described above) 2-hydroxycycloalkanecarboxylic acid amide represented by general formula (2);

(In the formula, R ′ represents a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, and a substituted or unsubstituted aralkyl group having 7 to 11 carbon atoms. * 1, * 2 are the same as above) or a 2-aminocycloalkanol derivative represented by formula (2) ′;

(Wherein, * 1 and * 2 are the same as described above). 2-Production method characterized by obtaining 2-aminocycloalkanol. The production method according to any one of claims 17 to 21, wherein * 1 and * 2 are both in the (R) configuration.