WO2015141758A1 - Hydrolyse asymétrique de glutarimide substitué en position 3 - Google Patents

Hydrolyse asymétrique de glutarimide substitué en position 3 Download PDF

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WO2015141758A1
WO2015141758A1 PCT/JP2015/058155 JP2015058155W WO2015141758A1 WO 2015141758 A1 WO2015141758 A1 WO 2015141758A1 JP 2015058155 W JP2015058155 W JP 2015058155W WO 2015141758 A1 WO2015141758 A1 WO 2015141758A1
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substituted
group
formula
amino acid
acid sequence
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増俊 野尻
雅俊 大貫
伸行 堀之内
慎 日比
順 小川
八十原　良彦
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株式会社カネカ
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    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
    • C12Y305/02016—Maleimide hydrolase (3.5.2.16)

Definitions

  • the present invention relates to a method for producing an optically active 3-substituted glutaric acid monoamide useful as a pharmaceutical intermediate.
  • the following method is known as a method for producing an optically active 3-substituted glutaric monoamide.
  • a method for obtaining optically active 3-substituted glutaric acid monoamide by optical resolution of racemic 3-substituted glutaric acid monoamide by salt formation crystallization or the like Patent Document 1
  • An optically active 3-substituted glutaric acid monoamide obtained by obtaining an optically active 3-substituted glutaric acid monoester by asymmetric hydrolysis reaction of a 3-substituted glutaric acid diester using esterase or lipase and then amidating it.
  • Non-patent Document 2 An optically active 3-substituted glutaric acid monoamide obtained by obtaining an optically active 3-substituted glutaric acid monoester by asymmetric hydrolysis reaction of a 3-substituted glutaric acid diester using esterase or lipase and then amidating it.
  • the method 1) is an optical resolution method, a yield of 50% or more cannot be expected.
  • the method 2) requires high pressure and low temperature conditions in the amidation of the subsequent step of the asymmetric hydrolysis reaction. .
  • an object of the present invention is to provide a method suitable for industrial use for producing an optically active 3-substituted glutaric acid monoamide useful as a pharmaceutical intermediate.
  • the present inventors obtained a microorganism that asymmetrically hydrolyzes 3-substituted glutarimide.
  • optically active 3-substituted glutaric acid monoamide can be produced from 3-substituted glutarimide under mild reaction conditions, and the present invention has been completed.
  • R may have an optionally substituted alkyl group having 1 to 8 carbon atoms, an optionally substituted alkenyl group having 2 to 8 carbon atoms, or an optionally substituted group.
  • An enzyme source having asymmetric hydrolysis activity is allowed to act on the 3-substituted glutaric imide represented by the following formula (2); (Wherein, * represents an asymmetric carbon atom, R is the same as above), and relates to a process for producing an optically active 3-substituted glutaric acid monoamide compound.
  • an optically active 3-substituted glutaric acid monoamide can be produced by a reaction under mild conditions using a prochiral 3-substituted glutarimide as a raw material and an enzyme source.
  • R is an optionally substituted alkyl group having 1 to 8 carbon atoms, an optionally substituted alkenyl group having 2 to 8 carbon atoms, and a substituent.
  • An aralkyl group is shown.
  • the alkyl group having 1 to 8 carbon atoms may be linear or branched.
  • alkenyl group having 2 to 8 carbon atoms examples include ethenyl group, propenyl group, butenyl group, pentenyl group, hexenyl group, heptenyl group, octenyl group and the like.
  • alkynyl group having 2 to 8 carbon atoms examples include ethynyl group, propynyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group and the like.
  • the aryl group having 4 to 20 carbon atoms may have a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom.
  • the number of heteroatoms contained in one aryl group is not particularly limited, but is usually 1 to 2.
  • the heteroatom is particularly preferably a nitrogen atom.
  • Examples of the aryl group having 4 to 20 carbon atoms include phenyl group, naphthyl group, anthranyl group, pyridyl group, pyrimidyl group, indanyl group, and indenyl group.
  • the aralkyl group having 5 to 20 carbon atoms may have a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom.
  • the number of heteroatoms contained in one aralkyl group is not particularly limited, but is usually 1 to 2.
  • the heteroatom is particularly preferably a nitrogen atom.
  • Examples of the aralkyl group having 5 to 20 carbon atoms include benzyl group, naphthylmethyl group, anthranylmethyl group, pyridylmethyl group, pyrimidylmethyl group, indanylmethyl group, and indenylmethyl group.
  • the alkyl group, alkenyl group, alkynyl group, aryl group and aralkyl group may have a substituent, and examples of the substituent include a halogen atom, a hydroxyl group, an amino group, and a nitro group.
  • the number is not particularly limited, but is typically 1 to 2, preferably 1.
  • 3-substituted glutarimide represented by the formula (1) include 3-isobutyl glutarimide, 3-propyl glutarimide, 3-phenyl glutarimide, 3- (4-chlorophenyl) glutarimide and the like.
  • the optically active 3-substituted glutaric monoamide that is the product of the present invention has the following formula (2):
  • R is an optionally substituted alkyl group having 1 to 8 carbon atoms, an optionally substituted alkenyl group having 2 to 8 carbon atoms, and a substituent.
  • An aralkyl group is shown.
  • R in the optically active 3-substituted glutaric acid monoamide of the formula (2) as the product is the same as R in the 3-substituted glutarimide of the formula (1) as the starting material. Details of R in formula (2) are as described in detail for R in formula (1).
  • the absolute configuration of the optically active 3-substituted glutaric monoamide represented by the formula (2) may be R or S.
  • Specific examples of the optically active 3-substituted glutaric acid monoamide represented by the formula (2) include 3-isobutyl glutaric acid monoamide, 3-propyl glutaric acid monoamide, 3-phenylglutaric acid monoamide, whose absolute configuration is R, 3 -(4-chlorophenyl) glutaric acid monoamide, 3-isobutylglutaric acid monoamide having the absolute configuration S, 3-propylglutaric acid monoamide, 3-phenylglutaric acid monoamide, 3- (4-chlorophenyl) glutaric acid monoamide, etc. It is done.
  • the optically active 3-substituted glutaric monoamide represented by the formula (2) has an optical purity (% ee) exceeding 0% ee, preferably 5% ee or more, more preferably 10% ee or more. More preferably 20% ee or more, more preferably 30% ee or more, more preferably 40% ee or more, more preferably 50% ee or more, more preferably 60% ee or more, more preferably 65% ee or more, more The optical purity (preferably 70% ee or higher, more preferably 75% ee or higher, more preferably 80% ee or higher, more preferably 85% ee or higher, more preferably 90% ee or higher, more preferably 95% ee or higher.
  • optical purity is as described in Examples.
  • the three-dimensional structure of the optically active 3-substituted glutaric monoamide represented by the formula (2) whose absolute configuration is R is usually represented by the following formula.
  • the steric structure of the optically active 3-substituted glutaric monoamide represented by the formula (2) whose absolute configuration is S is usually represented by the following formula.
  • details of R are as described in detail for R in formula (1)).
  • the “enzyme source” includes an enzyme having an activity for asymmetric hydrolysis of the 3-substituted glutarimide of the formula (1) to the optically active 3-substituted glutaric acid monoamide of the formula (2). If it is, it will not specifically limit, The microorganisms which have the activity which asymmetrically hydrolyzes the said enzyme itself or the 3-substituted glutarimide of the said Formula (1) to the optically active 3-substituted glutaric acid monoamide of the said Formula (2) And the treated product thereof.
  • Microbial cells of microorganisms include, in addition to the cells themselves, a culture solution or cell suspension containing cells. In addition to wild-type strains, microbial cells include mutant strains that have gained advantageous properties, and further, DNA encoding the above-mentioned enzyme having asymmetric hydrolysis activity derived from the microorganism has been introduced. Transformed strains.
  • the “processed product” may be, for example, a crude extract, freeze-dried cells, acetone-dried cells, or a crushed product of these cells, and 3-substituted obtained from the cells. It may be an enzyme having an activity of asymmetric hydrolysis of glutaric imide.
  • the degree of purification of the enzyme obtained from the cells is not particularly limited, and may be a purified enzyme or a partially purified enzyme.
  • the enzyme source is the enzyme itself
  • the enzyme can be prepared by any means such as a cell-free protein synthesis system.
  • the degree of enzyme purification is not particularly limited.
  • an enzyme having an activity to asymmetrically hydrolyze 3-substituted glutarimide of the above formula (1) into an optically active 3-substituted glutaric acid monoamide of the above formula (2) contained in the above enzyme source or a processed product thereof
  • at least one selected from the group consisting of the following polypeptides (I) to (VI) can be preferably used.
  • amino acid substitution is, for example, 1 to 50, preferably 1 to 25, more preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • the amino acid substitution is preferably a conservative amino acid substitution.
  • Constant amino acid substitution refers to substitution between amino acids having similar physicochemical functions such as charge, side chain, polarity, and aromaticity.
  • Amino acids with similar physicochemical functions include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (asparagine, glutamine, serine, threonine, cysteine, tyrosine), Classified into nonpolar amino acids (glycine, leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched chain amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine) can do.
  • basic amino acids arginine, lysine, histidine
  • acidic amino acids aspartic acid, glutamic acid
  • uncharged polar amino acids asparagine, glutamine, serine, threonine, cysteine, tyrosine
  • amino acid sequence described in SEQ ID NO: 1 or 6 is preferably 1 in total to at least one of the N-terminal and C-terminal of the amino acid sequence described in SEQ ID NO: 1 or 6. Or addition of multiple amino acids.
  • the identity of the polypeptide of (III) or (VI) to the amino acid sequence shown in SEQ ID NO: 1 or 6 is preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably Is 95% or more, more preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.
  • the identity value of amino acid sequences indicates a value calculated with default settings using software (for example, FASTA, DANASYS, BLAST, Genetyx) that calculates identity between a plurality of amino acid sequences.
  • the amino acid sequence identity value is calculated by calculating the number of matching amino acid residues when aligning a pair of amino acid sequences so that the degree of matching is maximized, and comparing the number of matching amino acid residues. Calculated as a percentage of the total number of amino acid residues in the sequence. Here, when there is a gap, the total number of amino acid residues is the number of amino acid residues obtained by counting one gap as one amino acid residue.
  • the similarity of the polypeptide of (III) or (VI) to the amino acid sequence of SEQ ID NO: 1 or 6 is preferably 75% or more, more preferably 80% or more, more preferably 85% or more, More preferably, it is 90% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.
  • the similarity value of amino acid sequences is calculated by adding the number of amino acid residues that match when aligning a pair of amino acid sequences so that the degree of coincidence is maximized, and the amino acid residues that have similar physicochemical functions. Then, it is calculated as a ratio of the total to the total number of amino acid residues of the compared amino acid sequences.
  • amino acid sequence similarity can be calculated by a computer using the same software as described above with respect to amino acid sequence identity.
  • the method for calculating the total number of amino acid residues is as described above for amino acid identity.
  • the amino acid residues having similar physicochemical functions are as described above.
  • the polypeptide preferably has a specific activity per 1 mg of total protein of 0.1 mU / mg or more, more preferably 1.0 mU / mg or more, more preferably 5.0 mU / mg or more, more preferably 10 mU / mg or more, Most preferably, it is 100 mU / mg or more, and the upper limit is not particularly limited, but for example, it can be added to the reaction system in the form of a protein mixture or purified product of 3000 mU / mg or less, typically 2000 mU / mg or less.
  • the definition of the unit of enzyme activity and the measuring method are as described in Example 3 (1).
  • the optical purity range of the optically active 3-substituted glutaric monoamide of formula (2) obtained by the enzyme activity is as described above.
  • the enzyme source can be used in a state immobilized by known means. Immobilization can be performed by methods well known to those skilled in the art (for example, a crosslinking method, a physical adsorption method, a comprehensive method, etc.).
  • the carrier for immobilizing the enzyme source is not particularly limited and can be appropriately selected and used by those skilled in the art.
  • microorganisms described later in this specification can be obtained from a patent microorganism depositary or other research institution, and can also be separated from the natural world.
  • the microorganism identified by the NBRC number is the National Institute of Technology and Evaluation Biological Resource Center (Chiba, Japan)
  • the microorganism identified by the DSM number is the DSMZ-German microbial cell culture collection, AKU number.
  • the microorganisms identified are available from the Fermentation Physiology and Brewing Science Laboratory (Kyoto, Japan), Department of Applied Life Sciences, graduate School of Agriculture, Kyoto University.
  • the enzyme source having an asymmetric hydrolysis activity in the 3-substituted glutarimide is not particularly limited, and examples thereof include, for example, the genus Achromobacter, the genus Alcaligenes, the genus Burkholderia, the comamonas ( An enzyme source derived from a microorganism selected from the group consisting of the genus Comamonas, Delftia, and Pseudomonas.
  • examples of the enzyme source having the ability to produce 3-substituted glutaric monoamides whose absolute configuration is R include, for example, the genus Achromobacter, the genus Alcaligenes, and Burkholderia. ), An enzyme source derived from a microorganism selected from the group consisting of the genus Comamonas, the Delftia genus, and the Pseudomonas genus.
  • Achromobacter sp Preferably, Achromobacter sp.
  • microorganism having the ability to produce a hydrolase derived from the microorganism may be either a wild strain or a mutant strain.
  • microorganisms derived by genetic techniques such as cell fusion or gene manipulation can also be used.
  • a microorganism that produces the genetically engineered enzyme is, for example, a process described in WO 98/35025 for isolating and / or purifying these enzymes to determine part or all of the amino acid sequence of the enzyme, this amino acid sequence
  • a method comprising a step of obtaining a DNA sequence encoding an enzyme based on the above, a step of obtaining a recombinant microorganism by introducing this DNA into another microorganism, and a step of culturing the recombinant microorganism to obtain the enzyme, etc. Can be obtained.
  • Examples of the recombinant microorganism as described above include a transformed microorganism transformed with a plasmid having a DNA encoding the hydrolase.
  • the host microorganism is preferably Escherichia coli.
  • the culture medium for the microorganism used as the enzyme source is not particularly limited as long as the microorganism can grow.
  • a carbon source carbohydrates such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids such as oleic acid and stearic acid and esters thereof, oils such as rapeseed oil and soybean oil, and ammonium sulfate as a nitrogen source , Sodium nitrate, peptone, casamino acid, corn steep liquor, bran, yeast extract, etc.
  • Inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, potassium hydrogen phosphate, potassium dihydrogen phosphate and other malt sources, malt
  • malt A normal liquid medium containing an extract, meat extract or the like can be used.
  • an inducer may be added to the medium in order to induce microbial enzyme production.
  • the inducing agent include imide compounds.
  • the imide compound include succinimide, 2-methylsuccinimide, glutarimide, maleimide, phthalimide, 3-isobutylglutarimide, 3-propylglutarimide, 3-phenylglutarimide, 3- (4-chlorophenyl) glutarimide, and the like. Can be mentioned.
  • the amount of these inducers added to the medium is 0.05% to 2.0%, preferably 0.1% to 1.0%.
  • % is% by weight.
  • Cultivation is carried out aerobically. Usually, the cultivation time is about 1 to 5 days, the pH of the medium is 3 to 9, and the cultivation temperature is 10 to 50 ° C.
  • the stereoselective hydrolysis reaction of 3-substituted glutarimide (1) is carried out by adding 3-substituted glutarimide (1) as a raw material and the enzyme source to a suitable solvent, and adjusting the pH. This can be done by stirring.
  • Reaction conditions vary depending on the enzyme, microorganism or processed product, substrate concentration, etc. used. Usually, the substrate concentration is preferably about 0.1 to 99% by weight, more preferably 1 to 60% by weight.
  • the reaction temperature is preferably 10 to 60 ° C, more preferably 20 to 50 ° C.
  • the pH of the reaction is preferably 4-11, more preferably 6-9.
  • the reaction time is preferably 0.5 to 120 hours, more preferably 1 to 120 hours, and particularly preferably 1 to 72 hours.
  • the reaction can be carried out under atmospheric pressure conditions.
  • the substrate can be added in batches, divided or added continuously.
  • the reaction can be carried out batchwise or continuously.
  • the target optically active substance can be obtained only from the racemic starting material in a yield of 50% at the maximum.
  • the optically active 3-substituted glutaric acid monoamide (2) can be obtained in a yield exceeding 50% from the 3-substituted glutaric imide (1) as a starting material. Is very advantageous.
  • Each of the optically active 3-substituted glutaric acid monoamides produced by the reaction can be isolated and purified by a conventional method.
  • a reaction liquid containing an optically active 3-substituted glutaric acid monoamide generated by hydrolysis reaction is extracted with an organic solvent such as ethyl acetate and toluene, and the organic solvent is distilled off under reduced pressure. Then, distillation, recrystallization, Alternatively, it can be isolated and purified by performing a treatment such as chromatography. Further, the filtrate from which the enzyme source has been removed from the reaction solution can be isolated and purified by neutralizing and crystallization using sulfuric acid or the like, and filtering out the precipitated target product.
  • Example 1 Stereoselective hydrolysis of 3- (4-chlorophenyl) glutarimide Each microorganism shown in Table 1 was sterilized in a test tube in 5 ml of medium (trypton 0.5 w / v% in water, yeast extract 0.5 w / v %, Glucose 0.1 w / v%, dipotassium hydrogen phosphate 0.1 w / v%, pH 7.0), and cultured with shaking at 30 ° C. for 65 hours. After completion of the culture, the cells were collected by centrifugation and suspended in 0.2 ml of 100 mM phosphate buffer (pH 7.0).
  • Example 2 Stereoselective hydrolysis of 3-isobutylglutarimide Each microorganism shown in Table 2 was sterilized in a test tube in 5 ml of medium (trypton 0.5 w / v% in water, yeast extract 0.5 w / v%, glucose 0 0.1 w / v%, dipotassium hydrogen phosphate 0.1 w / v%, pH 7.0), and cultured with shaking at 30 ° C. for 22 hours. After completion of the culture, the cells were collected by centrifugation and suspended in 0.2 ml of 100 mM phosphate buffer (pH 7.0).
  • Example 3 Identification and production of imidase (1) Isolation and purification of imidase derived from Burkholderia phytofirmans strain DSM17436 Burkholderia phytofirmans strain DSM17436 was sterilized in vitro in 30 ml of TGY medium (5 g tryptone, 5 g yeast extract, 1 g glucose, deionized water). It was inoculated to 1 L, up to pH 7.0 before sterilization, and cultured with aerobic shaking at 28 ° C. for 22 hours. The whole amount of this culture solution was inoculated into 5 L of TGY medium sterilized in the flask, and cultured under aerobic shaking at 28 ° C. for 30 hours.
  • TGY medium 5 g tryptone, 5 g yeast extract, 1 g glucose, deionized water. It was inoculated to 1 L, up to pH 7.0 before sterilization, and cultured with aerobic shaking at 28 ° C. for 22 hours. The whole amount of this culture solution was inoculated into 5 L of TGY
  • the cells were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.5), disrupted by ultrasonic waves, and centrifuged. Ammonium sulfate was added to the obtained supernatant to a saturation concentration of 20%, and ammonium sulfate was further added to the supernatant obtained by centrifugation to a saturation concentration of 40%. The precipitate obtained by centrifuging this was suspended in 20 mM phosphate buffer (pH 7.5), and dialyzed with 20 mM Tris buffer (pH 7.5) using a cellulose tube as a dialysis membrane.
  • the obtained active fraction was concentrated using Amicon Ultra (centrifugal filter unit: manufactured by Merck Millipore), applied to Superdex 200 10/300 GL (manufactured by GE Healthcare), and subjected to gel filtration.
  • Ammonium sulfate was dissolved in the obtained active fraction to a final concentration of 2M, and then applied to RESOURCE PHE (manufactured by GE Healthcare) and subjected to column chromatography, and then with 20 mM Tris-HCl buffer (pH 7.5). Elution was performed with a gradient of 2M to 0M ammonium sulfate.
  • the obtained active fraction was buffer-exchanged with 20 mM Tris-HCl buffer (pH 7.5) using Amicon Ultra, applied to TSK gel DEAE-5PW (manufactured by Tosoh Corporation), and subjected to column chromatography.
  • the buffer was eluted with a gradient from 0M to 1M sodium chloride.
  • the obtained active fraction was buffer-exchanged with 20 mM Tris-HCl buffer (pH 7.5) using Amicon Ultra, and this was applied to MonoQ 5/50 GL (manufactured by GE Healthcare) and subjected to column chromatography.
  • the solution was eluted with a concentration gradient of 0 M to 1 M sodium chloride with the same buffer.
  • the obtained active fraction was subjected to SDS-polyacrylamide electrophoresis, and the N-terminal amino acid sequence of the obtained band was determined with a protein sequencer. This enzyme was named BpIH.
  • ⁇ Method for measuring enzyme activity during enzyme purification 5 ⁇ L of the active fraction was mixed with 55 ⁇ L of 100 mM phosphate buffer (pH 7.0) containing 0.1 w / v% of 3- (4-chlorophenyl) glutarimide, and reacted at 28 ° C. for 60 minutes. After the reaction, the reaction was stopped by diluting it twice with acetonitrile, the solid was removed by centrifugation, and the substrate and product in the reaction solution were analyzed by high performance liquid chromatography.
  • 1 U is defined as the amount of enzyme that produces 1 ⁇ mol of 3- (4-chlorophenyl) glutaric acid monoamide per minute.
  • the enzyme activity was performed in the same procedure not only in this experiment but also in the following experiment.
  • N-terminal amino acid sequence information and total amino acid sequence of BpIH The N-terminal amino acid sequence of imidase BpIH purified in (1) was examined using a protein sequencer (PPSQ-31B: manufactured by Shimadzu Corporation). The N-terminal sequence was determined to be PLDPNYPRDL. When this N-terminal sequence was searched from the genome information of Burkholderia phytofirmans strain DSM17436 using Protein BLAST, it was 100% identical to the protein of GenBank accession No .: ACD16728.1 (SEQ ID NO: 1), and this protein is urate catabolism It was annotated as protein.
  • the DNA obtained by PCR is confirmed by agarose gel electrophoresis, the size of the amplified fragment is confirmed, the target gene band is excised from the agarose gel, and the illustra GFX PCR DNA and Gel Band Purification Kit (manufactured by GE Healthcare) is used. And purified. PQE-60 treated with restriction enzymes HindIII and NcoI, and a DNA fragment encoding the full length of BpIH derived from Burkholderia phytofirmans DSM17436 strain obtained by the above procedure, Gibson Assembly Master Mix (manufactured by New England Biolabs) The recombinant vector pQE60-BpIH01 was constructed by ligating together.
  • E. coli JM109 competent cells were transformed.
  • E. coli JM109 / pQE60-BpIH01 was obtained.
  • the obtained transformant was inoculated into 2 mL of LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0) containing 50 ⁇ g / ml ampicillin and aerobic at 28 ° C. for 7 hours. After shaking culture, IPTG was added to the culture solution to a concentration of 1 mM, and shaking culture was further performed at 28 ° C. for 16 hours.
  • the cells were collected by centrifugation, suspended in 500 ⁇ L of 100 mM phosphate buffer (pH 7.0) containing 0.1 w / v% of 3- (4-chlorophenyl) glutarimide, and the activity was confirmed by resting cell reaction.
  • Faecalis NBRC 13111 Cloning of imidase gene derived from Alcaligenes faecalis subsp. Faecalis NBRC 13111 and production in E. coli From known genomic sequence data of Alcaligenes faecalis subsp. Faecalis NBRC 13111, genes and amino acid sequences highly homologous to the BpIH gene and amino acid sequence (SEQ ID NOs: 5 and 6) could be obtained.
  • the base sequence of SEQ ID NO: 5 has 71% identity to the base sequence of SEQ ID NO: 4, and the amino acid sequence of SEQ ID NO: 6 is It was calculated to have 75% identity to the amino acid sequence of SEQ ID NO: 1.
  • the AfIH gene using a DNA primer (SEQ ID NO: 7) having a sequence on the 5 ′ end side of the AfIH gene and a primer (SEQ ID NO: 8) having a sequence on the 3 ′ end side, the AfIH gene is used as in (3).
  • a gene fragment containing the full length of the gene was obtained, agarose gel electrophoresed, excised, and purified.
  • a recombinant vector pQE60-AfIH01 was constructed in the same manner as in (3).
  • E. coli JM109 / pQE60-AfIH01 was obtained, cultured, and confirmed for activity by resting cell reaction.
  • the cells were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.5), disrupted by ultrasonic waves, and centrifuged. The supernatant was dialyzed with 20 mM Tris buffer (pH 7.5) using a cellulose tube as a dialysis membrane. Subsequently, this was applied to MonoQ 10/100 GL (manufactured by GE Healthcare) and subjected to column chromatography, and eluted with a sodium chloride concentration gradient from 0 M to 1 M with the same buffer.
  • the obtained active fraction was concentrated using Amicon Ultra (centrifugal filter unit: manufactured by Merck Millipore), applied to Superdex 200 10/300 GL (manufactured by GE Healthcare), and subjected to gel filtration. Was eluted with 20 mM Tris-HCl buffer (pH 7.5) containing sodium chloride.
  • Amicon Ultra centrifugal filter unit: manufactured by Merck Millipore
  • Superdex 200 10/300 GL manufactured by GE Healthcare
  • gel filtration was eluted with 20 mM Tris-HCl buffer (pH 7.5) containing sodium chloride.
  • BpIH was detected as a single band, confirming the purity of the purified enzyme.
  • Recombinant AfIH was expressed and purified in the same procedure.
  • the purified enzymes BpIH and AfIH obtained in (5) were each used at an enzyme concentration of 1.3 mg / ml, and 28 in 250 ⁇ L of 100 mM phosphate buffer (pH 7.0) containing 0.5 w / v% of 3-isobutylglutarimide.
  • the reaction was carried out at 0 ° C. for 16 hours. After completion of the reaction, solids were removed by centrifugation, and the conversion rate (%) and optical purity (%) were determined by analyzing the substrate and product in the reaction solution by the high performance liquid chromatography described in Example 2. ee) was determined. The results are shown in Table 5.
  • the activity value (U) for each substrate calculated in% when the activity value (U) for 3- (4-chlorophenyl) glutarimide is 100 is shown as “relative activity (%)”.
  • the activity value (U) for each substrate is defined as the activity value (U) of the fixed amount of purified protein used in the reaction when 1 U is defined as the amount of enzyme that produces 1 ⁇ mol of each product per minute. Point to.
  • the method of the present invention can be used for the production of an optically active 3-substituted glutaric acid monoamide useful as a pharmaceutical intermediate.

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Abstract

La présente invention aborde le problème consistant à fournir un procédé de production d'un monoamide d'acide glutarique substitué en position 3 optiquement actif, qui est utile en tant qu'intermédiaire pharmaceutique, ledit procédé étant approprié pour des utilisations industrielles. La présente invention concerne, en tant que solution au problème décrit ci-dessus, un procédé de production d'un monoamide d'acide glutarique substitué en position 3 optiquement actif en soumettant un glutarimide substitué en position 3 facilement disponible à une hydrolyse asymétrique à l'aide d'une source d'enzyme.
PCT/JP2015/058155 2014-03-20 2015-03-19 Hydrolyse asymétrique de glutarimide substitué en position 3 WO2015141758A1 (fr)

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CN112368262A (zh) * 2018-06-06 2021-02-12 浙江华海药业股份有限公司 一种制备普瑞巴林中间体(r)-3-(氨甲酰甲基)-5-甲基己酸的方法
CN113502305A (zh) * 2021-07-16 2021-10-15 台州学院 一种利用重组酰亚胺酶合成(r)-异丁基戊二酸单酰胺的方法

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WO2011077463A1 (fr) * 2009-12-24 2011-06-30 Msn Laboratories Limited Procédé de préparation de la prégabaline et de son intermédiaire
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WO2011077463A1 (fr) * 2009-12-24 2011-06-30 Msn Laboratories Limited Procédé de préparation de la prégabaline et de son intermédiaire
CN102898320A (zh) * 2012-10-26 2013-01-30 浙江省天台县奥锐特药业有限公司 (3r)-(-)-3-(2-乙酰氨基)-5-甲基己酸的制备方法

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CLAUDIO DE OLIVEIRA, JOURNAL OF BACTERIOLOGY, vol. 194, no. 23, 2012, pages 6675 - 6676 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112368262A (zh) * 2018-06-06 2021-02-12 浙江华海药业股份有限公司 一种制备普瑞巴林中间体(r)-3-(氨甲酰甲基)-5-甲基己酸的方法
EP3805199A4 (fr) * 2018-06-06 2022-01-19 Zhejiang Huahai Pharmaceutical Co., Ltd Procédé de préparation d'acide (r)-3-(carbamoylméthyl)-5-méthylhexanoïque intermédiaire de la prégabaline
CN112368262B (zh) * 2018-06-06 2022-08-23 浙江华海药业股份有限公司 一种制备普瑞巴林中间体(r)-3-(氨甲酰甲基)-5-甲基己酸的方法
US11420932B2 (en) 2018-06-06 2022-08-23 Zheijiang Huahai Pharmaceutical Co., Ltd. Method for preparing pregabalin intermediate (R)-3-(carbamoylmethyl)-5-methylhexanoic acid
CN113502305A (zh) * 2021-07-16 2021-10-15 台州学院 一种利用重组酰亚胺酶合成(r)-异丁基戊二酸单酰胺的方法
CN113502305B (zh) * 2021-07-16 2024-01-30 台州学院 一种利用重组酰亚胺酶合成(r)-异丁基戊二酸单酰胺的方法

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