WO2011071058A1 - Procédé de préparation d'un ester d'acide 2-hydroxycycloalcane carboxylique optiquement actif - Google Patents

Procédé de préparation d'un ester d'acide 2-hydroxycycloalcane carboxylique optiquement actif Download PDF

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WO2011071058A1
WO2011071058A1 PCT/JP2010/071961 JP2010071961W WO2011071058A1 WO 2011071058 A1 WO2011071058 A1 WO 2011071058A1 JP 2010071961 W JP2010071961 W JP 2010071961W WO 2011071058 A1 WO2011071058 A1 WO 2011071058A1
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polypeptide
group
formula
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acid ester
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川野茂
田中辰佳
西山章
八十原良彦
喜多恵子
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株式会社カネカ
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
    • C07C209/54—Preparation of compounds containing amino groups bound to a carbon skeleton by rearrangement reactions
    • C07C209/56—Preparation of compounds containing amino groups bound to a carbon skeleton by rearrangement reactions from carboxylic acids involving a Hofmann, Curtius, Schmidt, or Lossen-type rearrangement
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B53/00—Asymmetric syntheses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
    • C07C209/54—Preparation of compounds containing amino groups bound to a carbon skeleton by rearrangement reactions
    • C07C209/58—Preparation of compounds containing amino groups bound to a carbon skeleton by rearrangement reactions from or via amides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/22—Separation; Purification; Stabilisation; Use of additives
    • C07C231/24—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to a process for producing an optically active 2-hydroxycycloalkanecarboxylic acid ester, particularly an optically active 2-hydroxycyclopentanecarboxylic acid ester.
  • 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.
  • 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 has been reported on a method for producing an optically active 2-hydroxycycloalkanecarboxylic acid ester, for example, 2-hydroxycyclopentanecarboxylic acid ester.
  • Patent Document 1 Non-Patent Document 1
  • Geotrichum candidum Geotrichum candidum
  • Mucor racemosus Geotrichum candidum
  • Mucor circinelloides Kningpomella guro
  • Non-patent document 2 Cunninghamella gloeosporoides
  • reductase KRED102, KRED103, and KRED106 Non-patent document 3
  • (1R, 2S) 2- Hydroxycyclopentanecarboxylic acid esters are reported to form.
  • Non-patent Document 3 reports that (1S, 2R) 2-hydroxycyclopentanecarboxylate is mainly produced. Has been.
  • 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.
  • the production method according to any one of [1] to [3], wherein R is a methyl group or an ethyl group.
  • the polypeptide is a genus Candida, genus Rhodotorula, genus Devosia, genus Ogataea, genus Brevundimonas, genus Lactobacillus, thermoanaerobium.
  • [1] to [4] derived from a microorganism selected from the group consisting of the genus (Themoanaerobium), the genus Rhodococcus, the genus Sporobolomyces, and the genus Sporidiobolus. The manufacturing method in any one.
  • the polypeptide is 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 above 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; A polypeptide having, The production method according to [6] or [7], which is a polypeptide selected from the group consisting of: [9] The polypeptide is the following (A1), (A2), (A3), and (A4); (A1) DNA set forth in SEQ ID NO:
  • an optically active 2-hydroxycycloalkanecarboxylic acid ester particularly an optically active 2-hydroxycyclopentanecarboxylic acid ester, can be produced efficiently and industrially.
  • FIG. 4 is a schematic diagram of a Curtius dislocation route.
  • the present invention relates to a general formula (9); Wherein the optically active 2-hydroxycycloalkanecarboxylic acid ester represented by the general formula (8):
  • the 2-oxocycloalkanecarboxylic acid ester represented by the above is reacted with a polypeptide, an organism that produces the polypeptide, or a processed product of the organism to reduce the 2-oxocycloalkanecarboxylic acid ester. It is characterized by that.
  • the specificity of the stereoselective reaction greatly differs depending on the number of asymmetric carbons.
  • the optical activity 2 represented by the above formula (8) having two asymmetric carbons It is possible to produce hydroxycycloalkane carboxylic acid esters.
  • n represents an integer of 0 to 8. Among them, it is preferably 0 to 4, more preferably 1 to 3, and most preferably 1.
  • n 1, the 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.
  • 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, a substituted or unsubstituted group having 7 to 11 carbon atoms.
  • the substituted or unsubstituted alkyl having 1 to 6 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 a phenyl group, p-methylphenyl group, o-methylphenyl group, m-methylbenzyl group, p-chlorophenyl group, p-nitrophenyl group, p -Trifluoromethylphenyl group, 1-naphthyl group, 2-naphthyl group and the like.
  • 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. .
  • a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms is preferable, and a methyl group and an ethyl group are more preferable.
  • * 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.
  • the optically active 2-hydroxycycloalkanecarboxylic acid ester (9) produced in the present invention means four kinds of stereoisomers of 2-hydroxycycloalkanecarboxylic acid ester (cis (1R, 2S)).
  • the ratio of the target stereoisomer in the (1S, 2R) isomer and the (1S, 2S) isomer and (1R, 2R) isomer of the trans isomer) is 50% or more, preferably 60% More preferably, it is 70% or more, more preferably 80% or more, still more preferably 85% or more, and most preferably 90% 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.
  • trans isomer ratio [Trans-body area] / ([Trans-body area] + [Cis-body area]) ⁇ 100
  • 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.
  • the ratio of the (1S, 2R) body and (1R, 2S) in the generated formula (9) can be determined as follows.
  • the proportion of (1S, 2R) or (1S, 2R) in the cis isomer is, for example, dinitrobenzoyl chloride under basic conditions when the 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.
  • any polypeptide may be used as long as it has an activity to act on 2-oxocycloalkanecarboxylic acid ester to produce optically active 2-hydroxycycloalkanecarboxylic acid ester. .
  • the production method of the present invention can be performed by allowing not only a polypeptide but also an organism producing the polypeptide or a processed product of the organism to act.
  • the organism may be a naturally occurring organism or a genetically modified organism.
  • the treated product of the organism is, for example, a crushed cell, a crude extract, a cultured cell, a lyophilized organism, an acetone-dried organism, or a ground product thereof, a mixture thereof, and a catalyst for the polypeptide. This means that the activity remains.
  • the origin of the polypeptide is not limited, but preferably the genus Candida, Rhodotorula, Devosia, Ogataea, Brevundimonas, Lactobacillus ( It is derived from a microorganism selected from the group consisting of the genus Lactobacillus, the genus Thermoanaerobium, the genus Rhodococcus, the genus Sporobolomyces, and the genus Sporidiobolus.
  • polypeptides that produce (1R, 2R) -2-hydroxycycloalkanecarboxylic acid esters are described below.
  • the polypeptide to be used is a microorganism selected from the group consisting of the genus Sporobolomyces and the genus Sporidiobolus The thing of origin is preferable.
  • Sporobolomyces yeast examples include, for example, Sporobolomyces albidus, Sporobolomyces alborubescens, Sporobolomyces albus, Sporobolomyces albus, Sporobolomyces albus Sporobolomyces antarcticus, Sporobolomyces bischofiae, Sporobolomyces blumeae, Sporobolomyces carnicolor, Sporobolomyces carnicolor Sprorobolomyces coprophilus, Sporobolomyces coprosmae, Sporobolomyces coprosmicola, Sporobolomyces coloriformis (Sporobolomyces coprophilus) s coralliformis, Sporobolomyces dimmenae, Sporobolomyces diospyroris, Sporobolomyces dracophyllus, Sporobolomyces elongatos, Sporobat Sporobolomyces falcatus, Sporobol
  • yeasts belonging to the genus Sporidiobolus include Sporidiobolus johnsonii, Sporidiobolus microsporus, Sporidiobolus pararoseus, and Sporidiobolus pararoseus.
  • Examples include Sporidiobolus ruineniae, Sporidiobolus ruineniae bar coprophilus, Sporidiobolus salmonicolor.
  • Sporoboromyces salmonicolor Sporobolomyces salmonicolor
  • Sporidiobolus salmonicolor etc.
  • Sporoboromyces salmonicolor is the name of the asexual generation (Anamorph) of Sporidiobolus salmonicolor.
  • new species of sporoboromyces and sporidiobolus newly isolated from nature are also included.
  • polypeptide used in the production method of 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 is exemplified.
  • 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)).
  • polypeptides can be prepared according to known methods described in Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1989), etc., and act on 2-oxocyclopentanecarboxylic acid ester and NADPH. Thus, as long as it has the activity of producing (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and NADP, it is included in the polypeptide.
  • 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.
  • the place where amino acids are substituted, inserted, deleted and / or added is not particularly limited, but it is preferable to avoid a highly conserved region.
  • 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.
  • amino acid sequence modified by substitution, insertion, deletion and / or addition may include only one type (for example, substitution) of modification, or two or more types of modification (for example, substitution and substitution). Insertion).
  • 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).
  • amino acids in the same group of the following groups are regarded as homologous amino acids.
  • Group 1 neutral nonpolar amino acids
  • Group 2 neutral polar amino acids
  • Ser, Thr, Gln, Asn, Trp, Tyr Group 3: acidic amino acids
  • Glu Asp
  • Group 4 basic amino acids
  • the polypeptide used in the production method of the present invention has a sequence identity of 85% or more with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and acts on 2-oxocyclopentanecarboxylic acid ester and NADPH.
  • it may be a polypeptide having the activity of producing (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and NADP (polypeptide of (a3)).
  • 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.
  • 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.
  • amino acid sequence shown in SEQ ID NO: 1 Amino acid sequences can be combined.
  • a tag sequence such as a histidine tag or HA tag may be added.
  • it can be a fusion protein with another protein.
  • it may be a peptide fragment as long as it has an activity of acting on 2-oxocyclopentanecarboxylic acid ester and NADPH to produce (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester and NADP.
  • any equivalent activity is encompassed by the polypeptide of the present invention.
  • “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, for example, by the method for measuring the reduction activity for 2-oxocyclopentanecarboxylic acid ester described later.
  • the polypeptide used in the production method of 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.
  • Microorganisms are cultured in an appropriate medium, and after collection, they are reacted with 2-oxocycloalkanecarboxylic acid ester in a buffer solution in the presence of nutrients such as glucose. 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.
  • 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 bacterial cells are crushed by an ultrasonic crusher or a physical method using glass beads or the like, and then the cell residue is removed by centrifugation to obtain a cell-free extract.
  • 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 reduction 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, 2-oxocycloalkanecarboxylic acid ester reducing activity, for example, 2-oxocyclopentanecarboxylic acid ester reducing activity can be evaluated by the following simple method.
  • the reduction activity for 2-oxocyclopentanecarboxylic acid ester is 30 mM 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 it is calculated from the rate of decrease in absorbance at a wavelength of 340 nm. Under this reaction condition, the enzyme activity that oxidizes 1 ⁇ mol of NADPH to NADP per minute is defined as 1 U. NADH may be used instead of NADPH.
  • the polypeptide used in the production method of the present invention may be expressed by a host cell introduced with a DNA encoding this polypeptide.
  • the DNA may be any DNA that can express the polypeptide in the introduced host cell, and may contain any untranslated region.
  • DNA obtained by synthesis is also included.
  • Examples of the DNA encoding the polypeptide include a 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.
  • a DNA complementary to the DNA shown in SEQ ID NO: 2 in the sequence listing and a complementary DNA are hybridized under stringent conditions and act on 2-oxocyclopentanecarboxylic acid ester and NADPH (1R, 2R)- It may be a DNA encoding a polypeptide having an activity to produce 2-hydroxycyclopentanecarboxylic acid ester and NADP (DNA of (A3)).
  • DNAs complementary to the DNA shown in SEQ ID NO: 2 in the Sequence Listing are hybridized under stringent conditions and act on 2-oxocyclopentanecarboxylic acid ester and NADPH (1R, 2R “) -DNA that encodes a polypeptide having an activity to produce 2-hydroxycyclopentanecarboxylic acid ester and NADP” is a probe of DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 2 in the sequence listing As a DNA obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, etc.
  • 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.
  • the hybridization conditions are not particularly limited to these conditions. A plurality of factors such as temperature and salt concentration are conceivable 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 produce (1R, 2R) -2-hydroxycyclopentanecarboxylic acid ester. As long as it has an activity to produce NADP, it is included in the DNA.
  • 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 using, for example, the following sequence analysis tools: GCG Wisconsin Package (Program Manual for The Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive Medicine, 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 and Spital University of Geneva, Geneva, Switzerland).
  • polypeptide can be obtained, a person skilled in the art can obtain DNA 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.
  • polypeptide isolated by the above-described method is digested with an appropriate endopeptidase, and the resulting peptide fragment is fractionated by reverse phase HPLC. Then, for example, part or all of the amino acid sequences of these peptide fragments are determined by an ABI492 type protein sequencer (manufactured by Applied Biosystems).
  • a PCR (Polymerase Chain Reaction) primer for amplifying a part of the DNA encoding the polypeptide is synthesized.
  • chromosomal DNA of the microorganism that is the origin of the polypeptide is prepared by a conventional DNA isolation method, for example, a method such as Visser (Appl. Microbiol. Biotechnol., 53, 415 (2000)).
  • cDNA is prepared from the mRNA of the microorganism that is the origin of the polypeptide.
  • 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, an inverse PCR method (Nucl. Acids Res., 16, 8186 (1988)). be able to.
  • the polypeptides to be used include the genus Candida, the genus Rhodotorula, and the genus Ogataea.
  • Preferred are those derived from microorganisms selected from the group consisting of the genus Thermoanaerobium. More preferably, Candida magnoliae, Candida boidinii, Rhodotorula glutinis var. Dairenensis, Ogataea minutavar. ), 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.
  • the CR enzyme is derived from International Publication WO01 / 040450
  • the RRG enzyme is derived from International Publication WO03 / 093477
  • Ogataea minuta var. Minuta NBRC0975 Reductases are described in International Publication WO06 / 013801.
  • the polypeptides to be used include Candida, Devosia, and Brevundimonas.
  • microorganisms selected from the group consisting of the genus Lactobacillus are preferred. More preferably, Candida maris, Devosia riboflavina, Brevundimonas diminuta, Lactobacillus brevis, Lactobacillus kefir polypeptide from 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 Bleundimonas diminuta. (Brevundimonas diminuta) NBRC13584-derived reductase (Example 11).
  • the FPDH enzyme is International Publication WO01 / 05996
  • the RRG enzyme is International Publication WO04 / 027055
  • the reductase derived from Brevundimonas diminuta NBRC13584 is the International Publication. WO07 / 114217.
  • the polypeptide to be used is selected from the group consisting of Candida genus and Rhodococcus genus. Those derived from microorganisms are preferred. More preferably, it is a polypeptide derived from Candida maltosa, Candida parapsilosis, Rhodococcus erythropolis. Particularly preferred polypeptides are the reductase derived from Candida maltosa NBRC1977 (Example 14), the reductase derived from Candida parapsilosis NBRC708 (Example 15), and the like.
  • the reductase derived from Candida maltosa NBRC1977 is the international publication WO08 / 066018, and the reductase derived from Candida parapsilosis NBRC708 is special. 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) (Marscheroder Weg 1b, D-38124 Brunschweig, Germany)
  • a genetically modified organism into which DNA encoding the polypeptide described above is introduced can be used.
  • a genetically modified organism can be obtained by inserting a DNA encoding a polypeptide into an expression vector to prepare a polypeptide expression vector, and introducing this vector into a host organism by transformation.
  • the polypeptide can be expressed by culturing using a medium in which the genetically modified organism can grow.
  • a genetically modified organism can also be obtained by introducing a DNA encoding a polypeptide into the chromosome of the host organism.
  • the expression vector is not particularly limited as long as it can express the polypeptide encoded by the DNA in a suitable host organism.
  • suitable host organisms include plasmid vectors, phage vectors, cosmid vectors, and shuttle vectors that can exchange genes with other host organisms can also be used.
  • such a vector when using E. coli as a host organism, 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. It can be suitably used as an expression vector comprising an expression unit operably linked to the DNA of the invention. Examples thereof include pUCN18 (see Example 2), pSTV28 (manufactured by Takara Bio Inc.), pUCNT (WO94 / 03613) and the like.
  • “Regulator” refers to a base sequence having a functional promoter and any associated transcription element (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).
  • “Operably linked” means that a gene is operably linked to various regulatory elements such as promoters, enhancers and the like that regulate the expression of the gene in a host organism. 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 organism. Vectors and promoters that can be used in various organisms are described in detail in "Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Publishing".
  • 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.
  • microorganisms include, for example, the genus Escherichia, the genus Bacillus, the genus Pseudomonas, the genus Serratia, the genus Brevibacterium, Bacteria such as the genus Corynebacterium, Streptococcus, and Lactobacillus; actinomycetes such as the genus Rhodococcus and Streptomyces; the genus Saccharomyces; The genus Kluyveromyces, the genus Schizosaccharomyces, the genus Zygosaccharomyces, the genus Yarrowia, the genus Trichosporon, the genus Rhodosporidium, the genus Rhodosporidium a And Candida Candida) yeasts such as; Neurospora (Neurospora) genus Aspergillus (Aspergillus)
  • a microorganism 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 large quantities in plants such as rapeseed, corn, and potatoes. Systems for expressing heterologous proteins have been developed and can be suitably used. Among these, from the viewpoint of introduction and expression efficiency, a microorganism is preferable, a bacterium is more preferable, and a genetically modified organism using Escherichia coli (commonly known as E. coli) as a host organism is particularly preferable.
  • Escherichia coli commonly known as E. coli
  • Polypeptide expression vectors can be introduced into host organisms by known methods.
  • the plasmid pUCAR2 Applied and Environmental Microbiology, 65 (12), 5207-5211, (1999)
  • the plasmid pUCAR2 prepared by introducing the DNA shown in SEQ ID NO: 2 into the vector pUC118
  • E. coli E. coli competent cell
  • E. coli competent cell manufactured by Takara Bio Inc.
  • a recombinant organism E. coli in which the vector is introduced into the host cell for example, E. coli HB101 ( pUCAR2) (see Example 1) is obtained.
  • NADH or NADPH is required as a coenzyme.
  • NADH or NADPH the ability to convert the oxidized coenzyme (NAD + or NADP + ) into a reduced form (NADH or NADPH) (hereinafter referred to as reduced complement)
  • reduced complement the ability to convert the oxidized coenzyme (NAD + or NADP + ) into a reduced form (NADH or NADPH)
  • reduced complement the amount of expensive coenzyme used can be greatly reduced by performing the reaction with an enzyme having an enzyme regeneration ability) together with its substrate, that is, a coenzyme regeneration system in combination with the polypeptide used in the present invention.
  • Such a reaction can be performed by adding a coenzyme regeneration system into the asymmetric reduction reaction system. It is also possible to breed a genetically modified organism in which both a polypeptide having asymmetric reduction activity and a polypeptide having reductive coenzyme regeneration ability are expressed in the same host organism. That is, it can be obtained by incorporating a DNA encoding a polypeptide having asymmetric reduction activity and a DNA encoding a polypeptide having reductive coenzyme regeneration ability into the same vector and introducing it into a host organism.
  • a DNA encoding a polypeptide having asymmetric reduction activity and a DNA encoding a polypeptide having reductive coenzyme regeneration ability are respectively incorporated into two different vectors of different incompatibility groups, and the same host is used.
  • a genetically modified organism obtained by introduction into an organism can also be used in the production method of the present invention.
  • 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.
  • 2-oxocycloalkanecarboxylic acid ester Using a polypeptide that reduces 2-oxocycloalkanecarboxylic acid ester or a recombinant organism that expresses the polypeptide, 2-oxocycloalkanecarboxylic acid ester (8); The optically active 2-hydroxycycloalkanecarboxylic acid ester (9); Can be carried out as follows. However, it is not necessarily limited to the following method.
  • the formula (8) When converting the formula (8) into the formula (9), the formula (8) is added to a suitable solvent such as 100 mM phosphate buffer (pH 6.5), and NADPH, NADP + , NADH Alternatively, a coenzyme such as NAD + and a polypeptide, an organism producing the polypeptide, or a processed product of the organism are added, and the mixture is reacted with stirring under pH adjustment.
  • a suitable solvent such as 100 mM phosphate buffer (pH 6.5), and NADPH, NADP + , NADH
  • a coenzyme such as NAD + and a polypeptide, an organism producing the polypeptide, or a processed product of the organism are added, and the mixture is reacted with stirring under pH adjustment.
  • 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.
  • the reaction substrate can be added at a feed 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.
  • 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.
  • 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.
  • 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. However, it is possible to collect 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
  • optically active 2-hydroxycycloalkanecarboxylic acid ester represented by the formula (9) produced by the method described above, a compound represented by the general formula (2); Or an optically active 2-aminocycloalkanol derivative particularly useful as a pharmaceutical intermediate represented by the general formula (2) ′;
  • the optically active 2-aminocycloalkanol represented by these can be manufactured.
  • 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.
  • the substituted or unsubstituted alkyl having 1 to 6 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 a phenyl group, p-methylphenyl group, o-methylphenyl group, m-methylbenzyl group, p-chlorophenyl group, p-nitrophenyl group, p -Trifluoromethylphenyl group, 1-naphthyl group, 2-naphthyl group and the like.
  • 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 6 carbon atoms, and more preferred are a methyl group and an ethyl group.
  • the formula (9) can be obtained by the reduction reaction of the corresponding ketoester using the microorganism or enzyme described above.
  • 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, a substituted or unsubstituted group having 7 to 11 carbon atoms. And substituted aralkyl groups.
  • the substituted or unsubstituted alkyl group having 1 to 6 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 a phenyl group, p-methylphenyl group, o-methylphenyl group, m-methylbenzyl group, p-chlorophenyl group, p-nitrophenyl group, p -Trifluoromethylphenyl group, 1-naphthyl group, 2-naphthyl group and the like.
  • 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. .
  • a substituted or unsubstituted alkyl group having 1 to 6 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.
  • the route for producing the formula (2) or the formula (2) ′ from the formula (9) is to react 2-hydroxycycloalkanecarboxylic acid amide (3) by reacting the formula (9) with ammonia.
  • n represents an integer of 0 to 8. Among them, it is preferably 0 to 4, more preferably 1 to 3, and most preferably 1.
  • * 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.
  • This route is a route for producing the above formula (3) by reacting the above formula (9) with ammonia, followed by rearrangement reaction to produce the above formula (2) or the above formula (2) ′. is there.
  • a method for producing the formula (3) by reacting the formula (9) with ammonia will be described.
  • R is the same as described above.
  • Ammonia used may be diluted in a solvent, or may be used without dilution.
  • 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.
  • ammonia 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.
  • the equivalent of ammonia to be used is not particularly limited, but 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).
  • the reaction temperature is usually in the range of ⁇ 50 to 120 ° C. Preferably it is in the range of 0 to 100 ° C, more preferably in the range of 20 to 100 ° C.
  • 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
  • alcohol solvents such as ethanol, ethanol, isopropanol, n-propanol, and water are more preferable.
  • the crude product can be obtained by concentrating the reaction solution. Moreover, you may acquire a composition by performing a general post-process.
  • 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.
  • a general extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane and the like.
  • 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 or the like to further increase the purity.
  • the diastereomer is not particularly limited.
  • the 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.
  • hydrocarbon solvents such as hexamethylbenzene, toluene, n-hexane, and cyclohexane
  • diethyl ether diethyl ether
  • tetrahydrofuran (THF) tetrahydrofuran
  • diisopropyl ether methyl tert-butyl ether
  • dimethoxyethane dimethoxyethane
  • Ether 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.
  • a combination of an ester solvent and an alcohol solvent is preferable, and a combination of ethyl acetate and methanol is more preferable, and a combination of ethyl acetate and isopropanol 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.
  • 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.
  • the crude product 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, the crude product 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 content of diastereomer contained in the formula (3) 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, even more preferably 1.0 mol% or less, and most preferably 0.5% 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.
  • crystallization for reaction of the following process it may be used as a dry crystal
  • the base used may be an inorganic base or an organic base.
  • 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.
  • Tertiary amine include, for example, trialkylamines having 1 to 12 carbon atoms such as trimethylamine, triethylamine, ethyldiisopropylamine, N, N-dimethylaniline, N, N-diethylaniline, N, N-dimethylaminopyridine and the like.
  • Tertiary amines consisting of alkyl groups having 1 to 4 carbon atoms and aryl groups or heteroaryl groups, nitrogen-containing organic bases such as pyridine, picoline, lutidine, N, N, N, N-tetramethyl-1,2-ethylenediamine N, N, N, N, N, N, N, N, N-tetramethyl-1,3-propanediamine, N, N, N, N-tetramethyl-1,6-hexanediamine, etc. -Tetramethyl- ⁇ , ⁇ -alkyldiamine and the like.
  • 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.
  • halogenating agent used in this reaction examples 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.
  • sodium hypochlorite and sodium hypobromite are preferable, and sodium hypochlorite is more preferable.
  • halogenating agents may be used as they are, or those diluted with water or an organic solvent may be used.
  • an aqueous solution is usually used.
  • the halogenating agent may be prepared in the system.
  • a methanol solution of sodium hypobromite can be prepared by reacting bromine and sodium methoxide in a methanol solvent.
  • the amount of the halogenating agent used in this reaction may be 1 equivalent or more, preferably 1 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the formula (3).
  • the reaction temperature is usually in the range of ⁇ 30 to 120 ° C., preferably ⁇ 20 to 80 ° C. More preferably, it is ⁇ 10 to 60 ° C.
  • 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-
  • ether solvents such as benzyl alcohols, and water are preferable, and tetrahydrofuran (THF), methanol, ethanol, isopropanol, n-propanol, and water are more preferable.
  • THF tetrahydrofuran
  • a general post-treatment may be performed.
  • the extraction operation is performed using a general extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane and the like.
  • a general extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane and the like.
  • the reaction solvent and the extraction solvent are distilled off from the obtained extract by an operation such as heating under reduced pressure, the target compound is obtained.
  • 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.
  • R 4 represents 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 substituted group having 2 to 12 carbon atoms.
  • an unsubstituted acyl group is represented.
  • the substituent include the same as those described for R.
  • the substituted or unsubstituted alkyloxycarbonyl group having 1 to 15 carbon atoms include t-butyloxycarbonyl group (Boc group), methoxycarbonyl group (Moc group), 9-fluorenylmethoxycarbonyl group (Fmoc group).
  • Examples of the substituted or unsubstituted aralkyloxycarbonyl group having 7 to 12 carbon atoms include benzyloxycarbonyl group (Cbz group), p-methoxybenzyloxycarbonyl group and the like.
  • Examples of the substituted or unsubstituted acyl group having 2 to 12 carbon atoms include an acetyl group and a benzoyl group.
  • 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).
  • * 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.
  • Theodora W. Greene, Peter G. M.M. It 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.
  • t-butyloxycarbonyl can be protected by reacting t-butyl dicarbonate in the presence of a base in an appropriate solvent, and benzyloxycarbonyl in the presence of a base in an appropriate solvent.
  • 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.
  • general formula (7) It may be induced to a hydrazide represented by
  • a general Curtius rearrangement reaction condition may be used.
  • it can be carried out according to the method described in Strategic Applications of Named Reaction in Organic Synthesis Elsevier Inc. Publishing.
  • 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.
  • 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.
  • the above formula (2) ′ can be produced.
  • the said Formula (2) can be manufactured by performing in the said alcohol (5) presence.
  • N- (benzyloxycarbonyl) -2-hydroxycyclopentanol can be produced by reacting 2-hydroxycyclopentanecarboxylic acid (1) with diphenylphosphoric acid azide in the presence of benzyl alcohol.
  • R is the same as described above.
  • R ′ is the same as described above.
  • n represents an integer of 0 to 8. Among these, 0 to 3 is preferable, 1 to 3 is more preferable, and 1 is most preferable.
  • * 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 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 above-described commonly used conditions for the Curtius rearrangement, by reacting the formula (9) with hydrazine, the general formula (7);
  • the hydrazide represented by the formula (1) may be produced and subsequently produced by reacting nitrite.
  • n, * 1, * 2 is the same as the above.
  • Example 1 Production of E. coli HB101 (pUCAR2) that produces a polypeptide (ARII) derived from Sporobolomyces salmonicolor
  • plasmid pUCAR2 Plasmid in which the structural gene of polypeptide ARII was introduced into vector plasmid pUC118
  • Escherichia coli E. coli HB101 pUCAR2
  • E. coli HB101 (pUC118) comparative containing E.
  • coli HB101 (pUCAR2) and vector plasmid pUC118 was mixed with 2 x YT medium (200% tryptone, 1.6% tryptone, yeast extract 1). 0.0%, NaCl 0.5%, pH 7.0) was inoculated into 5 ml, and cultured with shaking at 37 ° C. for 20 hours.
  • 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 Co., Ltd.), and the cell residue was removed by centrifugation to obtain a cell-free extract. The cell-free extracts were measured for 2-oxocyclopentanecarboxylic acid ester reducing activity.
  • the reduction activity for 2-oxocyclopentanecarboxylic acid ester is 30 mM by adding 5 mM methyl 2-oxocyclopentanecarboxylate, 0.25 mM coenzyme NADPH, 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 ⁇ mol of NADPH to NADP + per minute was defined as 1 U.
  • E. coli HB101 (pUC118) (comparative example) has a 2-oxocyclopentanecarboxylic acid ester reduction activity of 0.1 U / mg or less
  • E. coli HB101 (pUCAR2) in which ARII was expressed was used.
  • the reduction activity of 2-oxocyclopentanecarboxylic acid ester was 3.3 U / mg.
  • polypeptide ARII had a reducing activity against the target 2-oxocyclopentanecarboxylic acid ester.
  • Example 2 Production of ethyl (1R, 2R) -2-hydroxycyclopentanecarboxylate using 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, glucose dehydrogenase (trade name: GLUCDH “Amano” II, manufactured by Amano Enzyme) 2250 U, glucose 12.5 g, NADP + 22 mg, ethyl 2-oxocyclopentanecarboxylate 3.
  • glucose dehydrogenase trade name: GLUCDH “Amano” II, manufactured by Amano Enzyme
  • 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 by 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.
  • Example 3 Production of methyl (1R, 2R) -2-hydroxycyclopentanecarboxylate using 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.
  • glucose dehydrogenase (trade name: GLUCDH “Amano” II, manufactured by Amano Enzyme Co., Ltd.), 164 g of glucose, NADP + 115 mg, and 25 g of methyl 2-oxocyclopentanecarboxylate were added, It stirred at 30 degreeC, adjusting pH6.5 by dripping 5N sodium hydroxide aqueous solution. At 1.5, 3, and 6 hours after the start of the reaction, 25 g each was added, and 12.5 g of methyl 2-oxocyclopentanecarboxylate was added at 9.5 hours, and the reaction was stirred for 27 hours. I let you.
  • 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 analyzed under high performance liquid chromatography under the following conditions to analyze optical purity. As a result, the optical purity of the (1R, 2R) isomer was 98.8% e.e. e. Met.
  • Example 4 Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Candida magnolia 2 ml of 100 mM phosphate buffer (pH 6.5), 46 mg of glucose and candy CR enzyme prepared from Candida magnoliae NBRC0705 (see International Publication WO01 / 040450 pamphlet, Example 1) 100 U, glucose dehydrogenase (trade name: GLUCDH “Amano” II, manufactured by Amano Enzyme Co., Ltd.) 25 U, NADP + 1 mg and ethyl 2-oxocyclopentanecarboxylate 20 mg were added, and the mixture was stirred at 30 ° C.
  • glucose dehydrogenase trade name: GLUCDH “Amano” II, manufactured by Amano Enzyme Co., Ltd.
  • Example 5 Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Rhodola glutinis bar derlenensis Instead of CR enzyme, Rhodotorula glutinis bar derlenensis (Rhodotorula The reaction was carried out in the same manner as in Example 4 using 100 U of reductase RRG (see International Publication WO03 / 093477 pamphlet, Example 1) prepared from NBRC0415. 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 proportion of the (1S, 2S) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 97.9%.
  • Example 6 Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using polypeptide derived from Ogataea Minuta bar Minuta Instead of CR enzyme, Ogataea Minuta bar Minuta 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 (1S, 2S) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 70.0%.
  • Example 7 Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Candida boidini Alcohol dehydration derived from Candida boidinii instead of CR enzyme
  • the reaction was carried out in the same manner as in Example 4 using 20 mg of the enzyme enzyme CP (manufactured by Julich Fine chemicals).
  • the conversion rate 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) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 75.0%.
  • Example 8 Production of ethyl (1S, 2S) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Thermoanaerobium brocci Instead of CR enzyme, Thermoanaerobium brockii
  • the reaction was carried out in the same manner as in Example 4 using 50 mg of the alcohol dehydrogenase derived from Sigma (Aldrich Japan Co., Ltd.).
  • 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) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 80.4%.
  • Example 9 Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Candida maris Prepared from Candida maris NBRC10003 instead of CR enzyme The reaction was carried out in the same manner as in Example 4 using 100 U of reductase FPDH (see International Publication WO01 / 05996 pamphlet, Example 14). The conversion rate to ethyl 2-hydroxycyclopentanecarboxylate was 95%, and the cis-isomer ratio was 97.4%. The cis isomer ratio (%) was calculated by the following formula.
  • Example 10 Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Devosia riboflavina Reductase prepared from Devosia riboflavina NBRC13584 instead of CR enzyme
  • the reaction was carried out in the same manner as in Example 4 using 100 U of RDR (see International Publication WO 04/027055 pamphlet, Example 1).
  • the conversion 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 the (1S, 2R) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 95.5%.
  • Example 11 Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Brevundimonas diminuta Instead of CR enzyme, from Brevundimonas diminuta NBRC13584
  • the reaction was carried out in the same manner as in Example 4 using 100 U of the prepared reductase (International Publication WO07 / 114217 Pamphlet, see 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%.
  • Example 12 Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Lactobacillus brevis Alcohol dehydrogenase derived from Lactobacillus brevis instead of CR enzyme The reaction was carried out in the same manner as in Example 4 using 100 ⁇ l (manufactured by Julich Fine chemicals). 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%.
  • Example 13 Production of ethyl (1S, 2R) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Lactobacillus kefir Alcohol dehydrogenase derived from Lactobacillus kefir instead of CR enzyme The reaction was carried out in the same manner as in Example 4 using 50 mg (Fluka). 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 stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 97.0%.
  • Example 14 Production of ethyl (1R, 2S) -2-hydroxycyclopentanecarboxylate using polypeptide derived from Candida maltosa Prepared from Candida maltosa NBRC1977 instead of CR enzyme The reaction was carried out in the same manner as in Example 4 using 100 U of reductase (see International Publication WO08 / 0666018, 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 the (1R, 2S) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 97.7%.
  • Example 15 Production of ethyl (1R, 2S) -2-hydroxycyclopentanecarboxylate using a polypeptide derived from Candida parapsilosis Prepared from Candida parapsilosis NBRC708 instead of CR enzyme The reaction was carried out in the same manner as in Example 4 using 100 U of reductase (see JP 2003-289874 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 the (1R, 2S) isomer in the four stereoisomers of ethyl 2-hydroxycyclopentanecarboxylate was 97.7%.
  • Example 16 Production of ethyl (1R, 2S) -2-hydroxycyclopentanecarboxylate using polypeptide derived from Rhodococcus erythropolis Alcohol dehydration derived from Rhodococcus erythropolis instead of CR enzyme The reaction was carried out in the same manner as in Example 4 using 100 ⁇ l of elementary enzyme (manufactured by Julich Fine 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%.
  • Example 17 Production of (1R, 2R) -2-hydroxycyclopentanecarboxylic acid amide Obtained by the method described in Example 3 (1R, 2R) -2-hydroxycyclopentanecarboxylic acid methyl ester (73.34 g) 0.52 mol) and a 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.
  • Example 18 Production of (1R, 2R) -2-aminocyclopentanol (1R, 2R) -2-hydroxycyclopentanecarboxylic acid amide (34.00 g, 0) obtained by the method described in Example 17 .26 mol) was diluted in water (170 g), and a 30 wt% aqueous sodium hydroxide solution (70.20 g, 0.53 mol) was added dropwise at a bath temperature of 0 ° C. over 35 minutes. Subsequently, at the same temperature, a 13 wt% aqueous sodium hypochlorite solution (226.10 g, 0.40 mol) was added dropwise over 2.5 hours.
  • Example 19 Production of (1R, 2R) -2- (t-butyloxycarbonylamino) cyclopentanol (1R, 2R) -2-Aminocyclopentanol obtained by the method described in Example 18 A 30 wt% aqueous sodium hydroxide solution was added to the aqueous solution (net amount: 27.61 g, 0.21 mol) 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.
  • Example 20 Production of (1R, 2R) -2-aminocyclopentanol hydrochloride (1R, 2R) -2- (t-butyloxycarbonylamino) cyclopent obtained by the method described in Example 19 A 29.5 wt% hydrogen chloride / isopropanol solution (118.52 g, 0.96 mol) is added for 1.5 hours to a toluene solution of ethanol (net amount: 38.60 g, 0.19 mol) at a bath temperature of 50 ° C. It was dripped over.
  • Example 21 Production of (1R, 2R) -2-hydroxycyclopentanecarboxylic acid amide
  • (1R, 2R) -2-hydroxycyclopentanecarboxylic acid amide (62.02 g) obtained by the method described in Example 17 0.46 mol) was added ethyl acetate (223 g) and isopropanol (25 g), and the mixture was stirred for 18 hours under reflux conditions with a bath temperature of 80 ° C. Thereafter, the bath was cooled to 25 ° C. and stirred for 3 hours, and the precipitated solid was collected by filtration to obtain the title compound (yield 58.11 g, yield 94%).

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Abstract

La présente invention concerne un procédé industriel efficace de préparation d'un ester d'acide 2-hydroxycycloalcane carboxylique optiquement actif qui est utile comme intermédiaire dans la production de produits pharmaceutiques. L'invention concerne en particulier un procédé de préparation d'un ester d'acide 2-hydroxycycloalcane carboxylique optiquement actif au moyen d'une réduction par le biais de l'action d'un polypeptide, d'un organisme produisant ledit polypeptide, ou d'un produit transformé dudit organisme sur un ester d'acide 2-oxocylcoalcane carboxylique.
PCT/JP2010/071961 2009-12-08 2010-12-08 Procédé de préparation d'un ester d'acide 2-hydroxycycloalcane carboxylique optiquement actif WO2011071058A1 (fr)

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JP7021503B2 (ja) * 2017-11-02 2022-02-17 三菱瓦斯化学株式会社 脂肪族ジアミンの製造方法

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JP2020517284A (ja) * 2017-04-27 2020-06-18 コデクシス， インコーポレイテッド ケトレダクターゼポリペプチドおよびポリヌクレオチド
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CN112458142A (zh) * 2020-12-11 2021-03-09 上海合全药物研发有限公司 生物催化制备(1r,2r)-2-羟基环戊腈的方法
CN112458142B (zh) * 2020-12-11 2023-05-12 上海合全药物研发有限公司 生物催化制备(1r,2r)-2-羟基环戊腈的方法

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