WO2005108592A1 - Procédé pour la production d'alcool propargylique optiquement actif - Google Patents

Procédé pour la production d'alcool propargylique optiquement actif Download PDF

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WO2005108592A1
WO2005108592A1 PCT/JP2005/009016 JP2005009016W WO2005108592A1 WO 2005108592 A1 WO2005108592 A1 WO 2005108592A1 JP 2005009016 W JP2005009016 W JP 2005009016W WO 2005108592 A1 WO2005108592 A1 WO 2005108592A1
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formula
represented
optically active
protein
propargyl
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PCT/JP2005/009016
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English (en)
Japanese (ja)
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Hiroaki Yamamoto
Masatake Kudoh
Motoko Hayashi
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Taisho Pharmaceutical Co., Ltd.
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Publication of WO2005108592A1 publication Critical patent/WO2005108592A1/fr

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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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic

Definitions

  • the present invention relates to a method for producing optically active propargyl alcohol, which is useful as an optically active raw material for various pharmaceuticals, particularly as a raw material for an ⁇ side chain essential for a prostaglandin compound.
  • the method for producing the optically active propargyl alcohol represented by Formula 2 or Formula 5 as defined herein includes the following:
  • the method (1) requires a low yield and an expensive resolving agent
  • the method (2) has a low optical purity
  • the method (3) requires an expensive optical resolution column
  • the method (4) requires an expensive asymmetric reduction catalyst, cryogenic temperature
  • the method (5) has low yield and low optical purity.
  • the method (6) has low productivity
  • the method (7) requires an expensive resolving agent and requires complicated steps, which is problematic as an industrial production method. Disclosure of the invention
  • the present invention relates to an optically active propargyl alcohol represented by the formula (2) or (5), in particular, (S) -1-cyclohexyl-2-propyne-11-ole represented by the formula (4) or (R) represented by the formula (6)
  • An object of the present invention is to provide a method for producing 1-cyclohexyl-2-propyne-11-ol, which can provide a product with high optical purity in good yield.
  • the present inventors did not use a resolution method in which the theoretical yield does not exceed 50%, but instead used a 100% raw material available, and obtained propargyl ketone by an asymmetric reduction reaction using a highly stereoselective enzyme.
  • a method for producing optically active propargyl alcohol with high optical purity was studied.
  • CHPN (1-cyclohexylpropynone, hereafter abbreviated as CHPN) as a substrate, using an enzyme that asymmetrically reduces a large number of phenols, 1-cyclohexylone 2-propyne-1 1-onole (1-cyclohexyl- 2 -propyn- 1- ol, hereafter abbreviated as CHP0).
  • TdCRl protein consisting of the amino acid sequence of SEQ ID NO: 8
  • (S) -specific secondary alcohol dehydrogenase derived from Candida parapsilosis can asymmetrically reduce ethyl acetate acetate to give (S) -3 — Has been reported to produce ethyl ethyl butyrate, but has virtually no activity against CHPN.
  • ScYDRl having a homology of 61% to TdCRl Japanese Patent Application No. 2003-113402 had substantially no activity against CHPN.
  • NAD + generated from NADH to NADH accompanying the above reaction and NADPH
  • the regeneration of NADPH produced from methane to NADP + can be carried out by, for example, dalkose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase, etc.
  • dalkose dehydrogenase formate dehydrogenase
  • alcohol dehydrogenase amino acid dehydrogenase
  • organic acid dehydrogenase etc.
  • (c) a protein consisting of the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been substituted, deleted, inserted and / or added, and reduced by reducing a propargyl ketone derivative represented by the formula 1.
  • R represents a C 1 -C 10 substituted or unsubstituted linear, branched or cyclic alkyl group, alkenyl group or alkynyl group, or C 6 -C 20 aryl group.
  • a method for producing an optically active propargyl alcohol derivative comprising producing an optically active propargyl alcohol derivative represented by the formula:
  • a protein encoded by the polynucleotide according to any of the above, a microorganism or a transformant functionally expressing the protein, or a processed product thereof is expressed by the following formula 1:
  • R represents a C 11 -C 10 substituted or unsubstituted linear, branched or cyclic alkyl group, alkenyl group or alkynyl group, or a C 6 -C 20 aryl group.
  • a method for producing an optically active propargyl alcohol derivative comprising producing an optically active propargyl alcohol derivative represented by the formula:
  • a propargyl ketone derivative represented by the formula 1 is represented by the formula 3:
  • An optically active propargyl alcohol derivative represented by the formula 5 is 1-cyclohexyl 2-propyne 11-one represented by the formula 3,
  • Figure 1 shows the construction of ScGRE2 expression plasmid P SE- Y0L1.
  • FIG. 2 shows the construction of the co-expressed plasmid pSG-SCR1 with ScGRE2 and glucose dehydrogenase from Bacillus subtilis.
  • the present invention provides the following) to (e);
  • one or more amino acids are A protein consisting of substituted, deleted, inserted and / or added amino acids, which has the activity of reducing a propargyl ketone derivative represented by the formula 1 to produce an optically active propargyl alcohol derivative represented by the formula 2
  • the present invention relates to a method for producing an optically active polyester pargylic alcohol derivative represented by Formula 2.
  • the protein described in SEQ ID NO: 2 includes, for example, (R) -2-octanol dehydrogenase derived from Pichia Finlandandi force.
  • the enzyme can be prepared from Pichia-Finlandi-powered cells by the method described in WO 01Z61014.
  • Pichia Finlandia DSM 70280 strain can be suitably used as the Pichia Finlandia force.
  • DNA encoding the protein described in SEQ ID NO: 2 which is the amino acid sequence of the (R) -2-octanol dehydrogenase derived from Pichia's Finlandi force DSM 70280 strain, and Pichia's Finlandy force DSM 70280 strain, etc.
  • (R) -2-octanol using a recombinant cloned from E. coli or chemically synthesized from a DNA encoding the amino acid sequence of SEQ ID NO: 2 and expressed in a heterologous host such as Escherichia coli.
  • the enzyme can also be obtained from a recombinant by expressing a dehydrogenase.
  • the measurement of the enzyme activity of the (R) -2-octanol dehydrogenase used in the present invention can be confirmed as follows.
  • the present invention provides the following (a) to (e);
  • the present invention relates to a method for producing an oral pargylic alcohol derivative.
  • the carbohydrate reductase ScGRE2 used in the present invention can be prepared from baker's yeast by the method described in J. Org. Chem. 63, 4996-5000 (1998).
  • the fragility described in SEQ ID NO: 3 encoding ScGRE2 derived from Saccharomyces cerevisiae was cloned from Saccharomyces cerevisiae (for example, strain X2180-1B) (Yeast Genetic Stock Center) by PCR or the like, and transferred to a heterologous host such as Escherichia coli.
  • the enzyme can also be obtained from the recombinant by expressing ScGRE2 using the recombinant introduced in an expressible form.
  • the measurement of the enzyme activity of SGGRE2 used in the present invention can be confirmed as follows.
  • NADPH reduced-cotinamide adenidine dinucleotide phosphate
  • 1 U was defined as the amount of enzyme that catalyzes the decrease of NADPH by ⁇ per minute.
  • the carbonyl reductase ScYGD9 used in the present invention is a DNA described in SEQ ID NO: 5 encoding ScYGD9 derived from Saccharomyces cerevisiae, for example, as described in Japanese Patent Application No. 2003-113304.
  • Saccharomyces' Celevige By way of Saccharomyces' Celevige
  • the carbonyl reductase TdCRl used in the present invention can be obtained by the method described in Japanese Patent Application No. 2003-113134.
  • TdCRl derived from Torulaspora delpreckii was cloned from Torulaspora delprezkie (eg, strain IF0381) by PCR or the like, and used for TdCRl can be expressed using a recombinant introduced in a form that can be expressed in a heterologous host, and the enzyme can be obtained from the recombinant.
  • the measurement of the enzymatic activity of TdCRl according to the present invention can be confirmed as follows. Reaction at 30 ° C in a reaction mixture containing 100 mM potassium phosphate buffer (pH 6.5), 0.2 mM NADPH, 5 mM 3,4-dimethoxyphenylacetone, and enzyme. Measure the decrease in absorbance at 340 nm. 1 U was the amount of enzyme that catalyzes the reduction of l / z mol of NADPH per minute.
  • the yeast Sacharomyces cerevisiae, Torrasbora del Delpreky
  • a common medium used for culturing yeast such as a YM medium.
  • the cells are collected, crushed in a buffer solution containing a reducing agent such as 2-mercaptoethanol / phenylmethanemethanefluonyl fluoride or a protease inhibitor, and combined with a cell-free extract. I do.
  • Protein-free fractionation from cell-free extracts eg, precipitation with organic solvents or salting out with ammonium sulfate, etc.
  • cation exchange anion exchange
  • gel filtration hydrophobic chromatography
  • chelate dye
  • roasting using such antibodies - for example c can be purified by combining tee one chromatography scratch as appropriate, Hue - Lou Sepharose hydrophobic chromatography using anion-exchange chromatography using M ono Q, butyl- It can be purified to a single band electrophoretically through hydrophobic chromatography using Sepharose, adsorption chromatography using hydroxyapatite, and the like.
  • amino acid sequence in which one or more (preferably 1 to 20, more preferably 1 to 10, and still more preferably about 1 to 5) amino acids are deleted, substituted, inserted and / or added in the present invention is a method for site-directed mutagenesis of the polynucleotide of SEQ ID NO: 1, 3, 5, or 7 (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Using mutations such as substitution, deletion, insertion, and Z or addition, as appropriate, using Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989), PGR A Practical Approach IRL Press pp. 200 (1991)) It is possible to obtain by doing.
  • the polynucleotide homolog of the present invention is a protein having at least 70%, preferably at least 80%, more preferably 90% or more homology with the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8.
  • the homology search of proteins can be performed, for example, on the database of amino acid sequences of proteins such as SWISS-PR0T, PIR, and DAD, on the database of DNA sequences such as DDBJ, EMBL, or Gene-Bank, and on amino acid sequences predicted based on DNA sequences. It can be performed on a database or the like by using a program such as BLAST or FASTA, for example, through the Internet.
  • the homology of the present invention refers to, for example, a numerical value represented by identity using the BLAST program.
  • a polynucleotide that can hybridize under stringent conditions with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7 is any polynucleotide described in any of the polynucleotide SEQ ID NOs.
  • DNA which is one or more selected from 0, preferably at least 30, for example, 40, 60 or 100 continuous sequences, is used as a probe DNA, for example, ECL direct nucleic acid labeling and detection system (Amersham The polynucleotides that hybridize under the conditions described in the manual (for example, wash: 42 ° C, primary wash buffer containing 0.5x SSC) using Pharmaica Biotech. More specific “stringent conditions” are, for example, usually 42. C, 2X SSC, 0.1% SDS, preferably 50 ° C, 2X SSC, 0.1% SDS, more preferably 65 ° C, 0.1X The conditions are SSC and 0.1% SDS, but are not particularly limited to these conditions. Factors that affect the stringency of the hybridization can be considered as multiple factors such as temperature and salt concentration, and those skilled in the art will realize the optimal stringency by appropriately selecting these factors. It is possible.
  • an expression vector for each enzyme is provided.
  • the transformation transformed with this expression vector By culturing the recombinant, an alcohol dehydrogenase and a carbohydrate reductase usable in the present invention can be obtained from the recombinant.
  • the microorganism to be transformed is transformed with a recombinant vector containing a polynucleotide encoding each enzyme, and the activity of each enzyme is reduced. There is no particular limitation as long as it can express the organism. Examples of usable microorganisms include the following microorganisms.
  • Genus Escherichia Genus Bacillus, Genus Pseudomonas, Genus Serratia, Genus Brevibacterium, Genus Corynebacterium, Streptococcus
  • Bacteria for which a host vector system has been developed such as the genus (Streptococcus) and the genus Lactobacillus;
  • Actinomycetes whose host vector system has been developed, such as the genus Rhodococcus and the genus Streptomyces;
  • Yeasts that have been developed as a host vector system such as genus (Trichosporon), genus Rhodosporidium, genus Pichia, and genus Candida; genus Neurospora, genus Aspergillus Fungi that have been developed in a host vector system such as genus, Cephalosporium, and Trichoderma genus:
  • Procedures for producing a transformant and construction of a recombinant vector suitable for a host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, see Sambrook et al.). , Molecular Cloning, Cold Spring Harbor Laboratories).
  • NADPH NADPH
  • a promoter corresponding to a unit for controlling transcription and translation may be incorporated upstream of the 5′-side of the DNA strand of the present invention, and more preferably, a terminator may be incorporated downstream of the 3′-side.
  • a promoter and terminator it is necessary to use a promoter and terminator known to function in a microorganism used as a host.
  • promoters, and terminators that can be used in these various microorganisms, see “Basic Lectures on Microbiology, 8 Genetic Engineering, Kyoritsu Shuppan”. Especially for yeast, see Adv. Biochem. Eng. 43, 75-102 (1990). ), Yeast 8, 423-488 (1992), and so on.
  • An optically active propargyl alcohol derivative can be produced by bringing a transformant into contact with a reaction solution to carry out a desired enzyme reaction.
  • the contact form between the enzyme and the reaction solution is not limited to these specific examples.
  • the processed products of microorganisms containing alcohol dehydrogenase or carbonyl reductase according to the present invention include microorganisms whose cell membrane permeability has been changed by treatment with an organic solvent such as a surfactant or toluene.
  • an organic solvent such as a surfactant or toluene.
  • a cell-free extract obtained by disrupting cells by glass beads or enzyme treatment, or a partially purified extract thereof is included.
  • R represents a C 1 -C 10 substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl group, or C 6- Represents an aryl group of C 20.
  • R methinole, ethinole, propinole, petitinole, pentinole, hexinole, heptinole, octyl, noel, decyl, isopropyl, isoptyl, t-butyl, 2-methylpentinole, 3-methinolehexinole, cyclopentinole, cyclohexinole
  • substituents may be further substituted with chlorine, bromine, iodine, trialkylsilyl, trialkyltin and the like.
  • a protein having (R) -2-octanol dehydrogenase activity, a microorganism producing the enzyme or protein, or a processed product of the microorganism is treated with 1-cyclohexyl represented by the formula (3).
  • (S) -propynone (CHPN) to produce (S) 1-1-cyclohexyl-2-propyne-1-1-ol ((S) -CHP0) represented by Formula 4; 1)
  • a method for producing 1-cyclohexyl 2-l-open pin is provided.
  • a protein having a potent luponyl reductase activity selected from ScGRE2, ScYGD9, TdCRl or a mutant thereof, a microorganism producing the enzyme or protein, or a processed product of the microorganism is converted into 1-cyclo represented by Formula 3.
  • Regeneration of NAD + or NADP + generated from NADH or NADPH in association with the above reduction reaction to NADH or NADPH depends on the ability of microorganisms to reduce NAD +, NADP + (glycolysis system, methylotroph C 1 compound utilization pathway, etc.) Can be performed. These NAD + and NADP + reducing ability can be enhanced by adding glucose, ethanol, etc. to the reaction system. Alternatively, the reaction can be carried out by adding a microorganism capable of producing NADH and NADPH from NAD + and NADP +, a processed product thereof, and an enzyme to the reaction system.
  • NADPH can be regenerated.
  • the components constituting the reaction necessary for the regeneration of NADH and NADPH can be added to the reaction system for producing the optically active alcohol according to the present invention, immobilized components can be added, or exchange of NADH and NADPH is possible. Contact can be made through a membrane.
  • the present invention can include a step of culturing a transformant transformed with a recombinant vector containing a polynucleotide encoding a polypeptide usable in the present invention.
  • a step of culturing a transformant transformed with a recombinant vector containing a polynucleotide encoding a polypeptide usable in the present invention when live cells of a microorganism transformed with a recombinant vector containing the polynucleotide of the present invention are used in the method for producing an optically active alcohol, an additional method for regenerating NADH and NADPH is required. In some cases, unnecessary reaction systems can be eliminated.
  • an efficient reaction can be performed in a reduction reaction using a transformant without adding an enzyme for regenerating NADH and NADPH.
  • genes such as glucose dehydrogenase, alcohol dehydrogenase, formate dehydrogenase, amino acid dehydrogenase, and organic acid dehydrogenase (malate dehydrogenase) that can be used for NADH and NADPH regeneration,
  • glucose dehydrogenase alcohol dehydrogenase
  • formate dehydrogenase amino acid dehydrogenase
  • organic acid dehydrogenase malate dehydrogenase
  • two or more of these genes can be introduced into a host by transforming the host with a recombinant vector in which the genes have been separately introduced into multiple vectors of different origins of replication.
  • a method of introducing both genes into a single vector, a method of introducing both genes, or a method of introducing one gene into a chromosome can be used.
  • the expression control region such as promoter and terminator must be linked to each gene, or expressed as an operon containing multiple cistrons such as the lactose operon. It is also possible.
  • enzymes for regenerating NADH and NADPH Bacillus subtilis
  • glucose dehydrogenase derived from Thermoplasma acidophilum are available.
  • formate dehydrogenase derived from Mycopacterium 'pakje and its mutants can be suitably used.
  • the reduction reaction using the enzyme of the present invention is carried out in an organic solvent that is hardly soluble in water or water, for example, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, methyl isobutyl ketone, methyl tertiary peptide.
  • an organic solvent soluble in water such as methanol, ethanol, isopropyl alcohol, acetonitrile, acetone, and dimethyl sulfoxide.
  • the reaction of the present invention can be carried out using an immobilized enzyme, a membrane reactor, or the like.
  • the reaction of the present invention is carried out at a reaction temperature of 4 to 60 ° C, preferably 10 to 40 ° C, pH 3 to 9, preferably pH 5 to 7, and a substrate concentration of 0.01 to 50%, preferably 0.1 to 10%. %, More preferably 0.1-5%.
  • a reaction temperature 4 to 60 ° C, preferably 10 to 40 ° C, pH 3 to 9, preferably pH 5 to 7, and a substrate concentration of 0.01 to 50%, preferably 0.1 to 10%. %, More preferably 0.1-5%.
  • propargyl ketone represented by the formula 1 as a substrate for the reaction of the present invention is generally unstable in water, particularly on the alkali side, it is desirable to carry out the reaction at a low temperature in a weakly acidic region.
  • the reaction system may contain coenzyme NAD +, NADH, NADP + or NADPH at 0.001 mM 0 mM, preferably 0.101 to 10 mM can be added.
  • the substrate can be added all at once at
  • NADH and NADPH for regeneration of NADH and NADPH, for example, glucose when using glucose dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, formic acid and amino acid dehydrogenation when using formate dehydrogenase
  • Amino acids when using enzymes, malic acid when using malate dehydrogenase, and glycerol when using glycerol dehydrogenase are added to the reaction system. These compounds can be added in a molar ratio of 0.1 to 20 and preferably 1 to 5 times in excess with respect to the substrate ketone.
  • enzymes for regenerating NADH and NADPH such as glucose dehydrogenase, alcohol dehydrogenase, formate dehydrogenase, amino acid dehydrogenase, malate dehydrogenase, and glycerol dehydrogenase, are used in the present invention. It can be added 0.1 to 100 times, preferably 0.5 to 20 times as much as the enzyme activity compared to NADH and NADPH-dependent enzymes.
  • Propargyl ketone as a substrate can be synthesized, for example, by the method described in Literature. Org. Chem., 52, 2860 (1987).
  • optically active propargyl alcohol produced by the reduction of propargyl ketone of the present invention can be purified by appropriately combining bacterial cells, protein, centrifugation, separation by membrane treatment, solvent extraction, distillation and the like.
  • an organic solvent such as ethyl acetate, butyl acetate, tonolen, hexane, benzene, methyl isobuty / leketone, methino-lethalate butynooleate is added directly to the reaction solution containing the microbial cells.
  • Butanol, hexane, heptane and the like are added and extracted, and then concentrated under reduced pressure to obtain optically active propargyl alcohol.
  • silica gel column chromatography, distillation, crystallization, etc. can be used to further purify the product.
  • the cell-free extract obtained from recombinant Escherichia coli exhibited (R) -2-octanol oxidation activity in a NAD + -dependent manner, with a specific activity of 35.9 U / mg-protein.
  • the measurement of CHPN reduction activity was performed at 30 ° C in a reaction solution containing 100 mM potassium phosphate buffer (pH 6.5), 0.2 mM NADH, 1% dimethyl sulfoxide, 2 mM CHPN and enzyme. The decrease in absorbance at 340 nm due to the decrease in NADH was measured. 1 U was the amount of enzyme that catalyzes the production of 1 ⁇ mol NADH per minute.
  • the cell-free extract obtained from the recombinant Escherichia coli showed NADH-dependent CHPN reduction activity, and the specific activity was 8.01 U / mg protein.
  • Glucose dehydrogenase activity was measured by reacting at 30 ° C in a reaction solution containing 100 mM potassium phosphate buffer (pH 6.5), 2.5 mM NAD +, 100 mM D-glucose and enzyme. The increase in absorbance at 340 nm due to the increase in the absorbance was measured. 1 U was the amount of enzyme that catalyzes the production of 1 mol NADH per minute.
  • the specific activity of the cell-free extract obtained from recombinant E. coli was 1.18 U / mg protein.
  • the transformed E. coli obtained in Example 1 was cultured in 50 mL of 2X YT medium (Dco-Tryptone 20 g / L, Difco-Yeast extract 10 g / L, NaCl 10 g / L, pH 7.2). After induction of gene expression with ImM IPTG, cells were collected. One half of the obtained cells was diluted with 50 mL of 400 mM phosphate buffer (pH 6.5), 1% CHPN (73 mM), 1% dimethyl sulfoxide, 1.6% glucose (88 mM). In addition to the reaction solution, the reaction was started at 20 ° C with shaking. One hour after the start of the reaction, half of the collected cells was further added, and the reaction was continued overnight.
  • 2X YT medium Dco-Tryptone 20 g / L, Difco-Yeast extract 10 g / L, NaCl 10 g / L, pH 7.2. After induction of gene expression with ImM IPTG, cells were collected. One half
  • the reaction solution prepared in Example 3 was extracted three times with 150 mL of hexane. A portion of this hexane extract was taken, and the conversion was measured by gas chromatography.
  • FFAP 20% Chromosorb W (AW), 80-100 mesh (0.32 x 210 cm in diameter, Shinwa Kako) was used.
  • the column chamber temperature and inlet temperature were 200 ° C
  • the detector temperature was 230 ° C
  • nitrogen was used as carrier gas
  • a flame ionization detector was used for detection.
  • the retention time of each compound was 5.0 minutes for CHPN and 8.5 minutes for CHP0. As a result of the quantification, the conversion was 100%.
  • the organic layer was filtered to remove insoluble components, the solvent was distilled off under reduced pressure.
  • the optical purity was measured by high performance liquid chromatography using an optical resolution column.
  • CHIRALCEL 0D ⁇ 0.46 ⁇ 25 cm, Daicel Chemical Industries
  • Column chamber temperature is 40.
  • elution was hexane 2-propanol (97/3, flow rate 1 mL / min), and detection wavelength was 220 nm.
  • the retention time of each enantiomer was 13.0 minutes for the (S) form and 14.9 minutes for the (R) form.
  • the optical purity of the product was 100% ee (S).
  • the cell-free extract obtained in Example 6 was measured.
  • the cell-free extract obtained from recombinant Escherichia coli showed (R) -2-octanol oxidation activity in a NAD + -dependent manner, with a specific activity of 18.2 U / mg-protein.
  • the formate dehydrogenase activity was measured at 30 ° C. in a reaction solution containing 100 mM potassium phosphate buffer (pH 7.0), 2.5 mM to 100 mM sodium formate, and the enzyme. 1 U was the amount of enzyme that catalyzes the production of 1 / Z mol of NADH per minute.
  • the specific activity of the cell-free extract obtained from the recombinant E. coli was 1.11 U / mg protein.
  • Example 8 Synthesis of (S) -CHP0 by Pichia-Finlandi-derived (R) -2-octanol dehydrogenase and formate dehydrogenase from Mycobacterium pakae Transformation obtained in Example 6 Cultured Escherichia coli in 50 mL of 2X YT medium (Dif Co-Tryptone 20 g / L, Difco-Yeast extract 10 g / NaCl 10 g / pH 7.2), and induced gene expression with 0.1 mM IPTG After that, the cells were collected.
  • 2X YT medium Dif Co-Tryptone 20 g / L, Difco-Yeast extract 10 g / NaCl 10 g / pH 7.2
  • One half of the obtained cells was diluted with 400 mM phosphate buffer (pH 6.5), 1% CHPN (73 mM), 1% dimethyl sulfoxide, 1.6% glucose (88 mM), 1 mM NAD + was added to 25 mL of the reaction solution, and the reaction was continued at 20 ° C with shaking.
  • the reaction solution was extracted twice with 75 mL of ethyl acetate, and quantitatively determined by gas chromatography. As a result, the conversion was 84%.
  • Saccharomyces cerevisiae X2180-IB (Yeast Genetic Stock Center) was cultured in YM medium to prepare cells. Purification of chromosomal DNA from the cells was performed by the method described in Meth. Cell Biol. 29, 39-44 (1975).
  • Y0L-ATG1 GACACATGTCAGTTTTTGTTTCAGGTGCTAAC
  • Y0L-XbaIF CTGTCTAGAGGAGAATCGTGACTCTGTAAAATTC SEQ ID NO: 12
  • Y0L-TAA1 GTCGAATTCCTCTATTAAATACGGCCCTCAAATTTTAAAATTTGGGAG
  • the amplification product was treated with phenol, it was double-digested with restriction enzymes BspLUllI and Xbal, and the obtained fragment was purified.
  • primers Y0L-XbalF and Y0L-TAA1 were added to each of 25 pmo1, dNTP10 nmo1, Saccharomyces cerevisiae-derived chromosomal DNA 50 ng, Pfu DNA polymerase buffer (manufactured by STRATAGENE), Pfu DNA polymerase 2 U Using a 50 reaction mixture containing (STRATAGENE), denaturation (95 ° C, 45 seconds), anneal (55 ° C, 30 seconds), elongation (72 ° C, 1 minute 20 seconds) ) For 30 cycles using GeneAmp PCR System 2400 (manufactured by PerkinElmer), and specific amplification products were obtained.
  • the obtained two PCR-amplified DNA fragments digested with the restriction enzymes were ligated with a vector pSE420D (Japanese Unexamined Patent Publication No. 2000-189170) that was double-digested with restriction enzymes Ncol and EcoRI and a TAKARA Ligation Kit.
  • Escherichia coli JM109 strain was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the plasmid was purified from the resulting transformant by FlexiPrep.
  • FlexiPrep As a result of analyzing the nucleotide sequence of the inserted DNA portion of the obtained plasmid, it was completely identical to the nucleotide sequence registered in DDBJ except for the primer portion.
  • plasmid was designated as pSE-Y0L1.
  • Figure 1 shows the process of plasmid construction. Construction of Example 1 1] Plasmid P SG- SCR1 coexpressing glucose dehydrogenase gene derived from Karuponiru reductase ScGRE2 and Bacillus subtilis
  • PSE-Y0L1 was double-digested with Nhel and Csp45I, and a DNA fragment consisting of 719 bp was purified. Further, the plasmid was double digested with Csp45I and EcoRI to purify a 469 bp DNA fragment. Plasmid pSE-BSGl containing the glucose dehydrogenase gene from Bacillus subtilis
  • E. coli HB101 E. coli HB101 (pSG-SCRl)
  • pSG-SCR1 obtained in Example 11
  • IPTG IPTG
  • the cells were crushed with a closed ultrasonic crusher UCD-200TM (manufactured by Cosmo Bio), and the supernatant was centrifuged to obtain a cell-free extract.
  • Example 12 Using the cell-free extract obtained in Example 12, the enzyme activity was measured. The measurement of CHPN reduction activity was performed at 25 ° C in a reaction solution containing 100 mM potassium phosphate buffer (pH 6.5), 0.2 mM NADPH, 2 mM CHPN, l ° / o dimethyl sulfoxide and enzyme. Reacted. 1 U was defined as the amount of enzyme that catalyzes the production of 1 ⁇ MDH per minute.
  • the cell-free extract obtained from recombinant Escherichia coli showed CHDP reduction activity in a NADPH-dependent manner, and its specific activity was 10.8 U / mg-extract. W
  • the reaction solution prepared in Example 14 was extracted with twice the volume of ethyl acetate.
  • the solvent was distilled off under reduced pressure, and the residue was dissolved in 1-fold volume of hexane / 2-propanol (97/3).
  • the optical purity of this sample was measured using high performance liquid chromatography using an optical resolution column. The analysis was performed by the method described in Example 5. As a result, the optical purity of the product was 99.8% ee (R).
  • Example 14 Using the sample prepared in Example 14, the conversion rate was measured by gas chromatography according to the method described in Example 4. The conversion rate calculated from the peak area was 28.4%.
  • the amplified product was double-digested with the restriction enzymes BspHI and Nhel, and ligated with the vector pSE420D, which was double-digested with the restriction enzymes Ncol and Xbal, using the TAKARA Ligation Kit.
  • Escherichia coli strain J1109 was transformed with the ligated DNA, grown in an LB medium containing ampicillin (50 mg / L), and plasmid was purified from the resulting transformant by FlexiPrep.
  • the nucleotide sequence of the inserted DNA portion of the plasmid was analyzed, and the results are shown in SEQ ID NO: 15. The obtained nucleotide sequence completely matched the nucleotide sequence registered in DDBJ.
  • the obtained plasmid was designated as pSE-YGD9.
  • PSE-YGD9 is double-digested with two restriction enzymes, EcoRI and Hindlll, and glucose dehydrated from the plasmid pSE-BSG1 (JP-A-2000-189170) containing the glucose dehydrogenase gene derived from Bacillus subtilis.
  • the DNA fragment containing the gene was ligated with Takara Ligation Kit.
  • Escherichia coli JM109 strain was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the plasmid was purified from the resulting transformant by FlexiPrep, and glucose dehydrogenase and YGL039w were simultaneously purified. to obtain a P SG-YGD9 is expressible plasmids.
  • E. coli HB101 strain E. coli HB101 (pSG-YGD9) transformed with pSG-YGD9 obtained in Example 17 was cultured overnight in an LB medium containing 50 mg / L of ampicillin. It was added to induce gene expression, and the cells were further cultured for 4 hours. After collecting the obtained cells, the cells were broken with a closed ultrasonic crusher UCD-200TM (Cosmo Bio) and centrifuged. Clarified as cell-free extract
  • Example 18 Using the cell-free extract obtained in Example 18, the enzyme activity was measured according to the method of Example 13.
  • the cell-free extract obtained from recombinant Escherichia coli showed CHDP reduction activity in a NADPH-dependent manner, and its specific activity was 22.7 U / mg-extract.
  • reaction solution containing 1 U of the enzyme solution was reacted at 25 ° C. with shaking.
  • the optical purity of the reaction solution prepared in Example 20 was measured in the same manner as in Example 5. As a result, the optical purity of the product was 99.3% ee (R).
  • Example 20 Using the sample prepared in Example 20, the conversion was measured in the same manner as in Example 4. As a result, the conversion rate calculated from the peak area was 11.6%.
  • the obtained PCR product was recovered as ethanol precipitate after phenol-chloroform extraction. After digestion of Td-PCR2 with Xbal, agarose gel electrophoresis was performed to cut out the target band, which was then purified using Sephaglas BandPrep Kit (Pharmacia).
  • the restriction enzyme-digested Td-PCR2 was ligated with pUC18 (manufactured by Takara Shuzo) digested with Xbal using Takara Ligation Kit to transform Escherichia coli JM109 strain.
  • the transformed strain was grown on LB medium containing ampicillin, and the nucleotide sequence of the inserted fragment was analyzed.
  • the plasmid obtained here was designated as pUC-TDX.
  • the plasmid pUC-TDX is digested with the restriction enzyme Xbal, precipitated with ethanol, and subjected to agarose gel electrophoresis to cut out a band of about 0.8 bp containing a part of the TdCRl gene. Purification and recovery by SephaglasBandPrep (Amersham Pharmacia Biotech) did. The obtained DNA fragment was digested with the same restriction enzymes, treated with alkaline phosphatase, and then subjected to phenol extraction, phenol-chloroform extraction, chloroform extraction, ethanol-precipitated plasmid pSG-TDX, and TaKaRa Ligation Kit. And ligated.
  • Escherichia coli JM109 strain was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and plasmid was purified from the resulting transformant by FlexiPrep.
  • pSG-TDRl a plasmid capable of simultaneously expressing glucose dehydrogenase and TdCRl, was obtained.
  • E. coli HB101 strain E. coli HB101 (pSG-TDRl) transformed with pSG-TDRl obtained in Example 23 was cultured overnight in an LB medium containing 50 mg / L of ampicillin. It was added to induce gene expression, and the cells were further cultured for 4 hours. After collecting the obtained cells, the cells were cleaved with a closed ultrasonic crusher UCD-200TM (manufactured by Cosmo Bio), and the supernatant was centrifuged to obtain a cell-free extract.
  • UCD-200TM manufactured by Cosmo Bio
  • Example 24 the enzyme activity was measured according to the method of Example 13.
  • the cell-free extract obtained from the recombinant Escherichia coli showed NADPH-dependent CHPN reduction activity, and the specific activity was 5.7 U / mg-protein.
  • reaction solution containing 0.5 U of the TdCRl enzyme solution was reacted at 25 ° C. with shaking.
  • the optical purity of the reaction solution prepared in Example 26 was measured in the same manner as in Example 5. As a result, the optical purity of the product was 99.7% ee (R).
  • Example 26 Using the sample prepared in Example 26, the conversion was measured in the same manner as in Example 4. As a result, the conversion rate calculated from the peak area was 9.0%. Industrial applicability

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Abstract

Cette invention fournit un procédé pour la production d'alcool propargylique optiquement actif utile comme intermédiaire dans la synthèse de médicaments. Selon l'invention, de l'alcool propargylique optiquement actif est produit par réduction asymétrique de propargylcétone avec une alcool secondaire déshydrogénase ou une carbonyle réductase.
PCT/JP2005/009016 2004-05-12 2005-05-11 Procédé pour la production d'alcool propargylique optiquement actif WO2005108592A1 (fr)

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US8404461B2 (en) 2009-10-15 2013-03-26 SK Biopharmaceutical Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
US8501436B2 (en) 2009-06-22 2013-08-06 Sk Biopharmaceuticals Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
CN107607635A (zh) * 2017-08-15 2018-01-19 东北制药集团股份有限公司 一种采用顶空气相色谱检测左磷右胺盐中丙炔醇含量的方法

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8501436B2 (en) 2009-06-22 2013-08-06 Sk Biopharmaceuticals Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
US8404461B2 (en) 2009-10-15 2013-03-26 SK Biopharmaceutical Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
US9068207B2 (en) 2009-10-15 2015-06-30 Sk Biopharmaceuticals Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
US9434970B2 (en) 2009-10-15 2016-09-06 Sk Biopharmaceuticals Co., Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
CN107607635A (zh) * 2017-08-15 2018-01-19 东北制药集团股份有限公司 一种采用顶空气相色谱检测左磷右胺盐中丙炔醇含量的方法
CN107607635B (zh) * 2017-08-15 2020-07-31 东北制药集团股份有限公司 一种采用顶空气相色谱检测左磷右胺盐中丙炔醇含量的方法

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