WO2009107821A1 - Procédé de fabrication d'un acide carboxylique optiquement actif à l'aide d'une estérase issue d'une archaebactérie thermophile - Google Patents

Procédé de fabrication d'un acide carboxylique optiquement actif à l'aide d'une estérase issue d'une archaebactérie thermophile Download PDF

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WO2009107821A1
WO2009107821A1 PCT/JP2009/053783 JP2009053783W WO2009107821A1 WO 2009107821 A1 WO2009107821 A1 WO 2009107821A1 JP 2009053783 W JP2009053783 W JP 2009053783W WO 2009107821 A1 WO2009107821 A1 WO 2009107821A1
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optically active
esterase
ester
acid
reaction
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PCT/JP2009/053783
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Japanese (ja)
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宮本憲二
太田博道
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学校法人慶應義塾
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    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/004—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

Definitions

  • the present invention relates to a method for producing an optically active carboxylic acid used as an intermediate raw material for pharmaceuticals, agricultural chemicals, and the like, and more specifically, optically in one step using a domino reaction catalyzed by a thermophilic archaeal esterase.
  • the present invention relates to a method for producing an active carboxylic acid.
  • hydrolysis There are many types of enzymes that catalyze hydrolysis. Among them, lipases and esterases that act on ester bonds, and proteases that act on amide bonds, such as amidases, are the most studied. Research on substance conversion using hydrolases began about 40 years ago, and studies on esterases that catalyze the hydrolysis of steroid compounds have been made. At present, hydrolytic enzymes are widely used industrially, such as detergents and food additives. Among them, enzymes with high thermal stability are highly industrially useful because they exhibit high stability under high temperature conditions and in organic solvents.
  • archaea has attracted attention as a suitable screening target for such thermostable enzymes.
  • Archaea, Eubacteria (Eubacteria), a third group of organisms alongside eukaryotes (eukaryote), high temperature, high pressure, can grow in good UNA under extreme conditions of low P H and a high salt concentration known It has been. In this way, archaea are becoming increasingly important from the perspective of biotechnology applications that take advantage of the feature of thermal stability.
  • a prochiral compound is a compound that can be converted to a chiral compound with an asymmetric carbon by a one-step reaction ( Figure 1).
  • Figure 1 an optically pure compound can be obtained with a yield of 100%, which is an efficient reaction.
  • the substrate is a prochiral dicarboxylic acid ester
  • one of the equivalent esters is selectively hydrolyzed to obtain an optically active substance.
  • a reaction using a protic substrate is theoretically capable of obtaining an optically active form with a 100% enantiomeric excess in a yield of 100%, which is a very efficient reaction. Yes.
  • stereoselective hydrolysis of disubstituted malonic esters is a useful reaction that can theoretically yield optically pure malonic half esters in 100% yield.
  • PLE pig liver esterase
  • optically active substances are used in a wide range of fields such as agricultural chemicals, foods and fragrances, and recently liquid crystals not related to physiological activity.
  • pharmaceuticals for example, flurbiprofen, ibuprofen, ketoprofen, naproxen and the like shown in FIG. 2 are known as optically active substances.
  • optically active ⁇ -arylpropionic acids are known as non-steroidal anti-inflammatory drugs and have useful physiological activities for S-enantiomers. Therefore, it is required to efficiently synthesize only one enantiomer of optically active ⁇ -arylpropionic acid.
  • a domino-type reaction is a reaction in which multiple reactions occur in a single reaction solution.
  • the characteristic of the domino-type reaction is that when the yield of the elementary reaction is high, the product can be obtained efficiently because it eliminates the trouble of extracting and purifying the intermediate of the reaction.
  • This method is also used for the synthesis of complex compounds from simple compounds such as organic synthesis. (See Non-Patent Document 2).
  • Non-Patent Document 1 C. J. Sih, J. Am. Chera. Soc., 1975, 97, 4144
  • Non-Patent Document 2 D. Enders, C. Grondal, M.R.M.Huttl, Angew.Chem. Int.
  • the purpose of this study is to produce a novel domino-type reaction of hydrolysis and decarboxylation using esterase ST0071 as a catalyst, and to produce optically active carboxylic acids such as ⁇ -arylalkanoic acid.
  • the present inventors cloned the esterase ST0071 having high homology with a known esterase from the genome sequence of the thermoacidophilic archaeon Sulfolobus tokodai i strain 7, and revealed that the expressed enzyme has esterase activity as intended. (Y. Suzuki, K. Miyamoto, H. Ohta, FEMS Microbiol. Lett., 2004, 236, 97-102). Esterase ST0071 was found to have high thermal stability with an optimum reaction temperature of 70 ° C. and an optimum pH of 7.5 to 8.0. Furthermore, homology search confirmed that esterase ST0071 is closely related to thermostable esterase whose X-ray crystal structure has been solved.
  • the present inventors designed a novel domino-type reaction represented by Chemical Formula 1 using the isolated and purified thermophilic archaeal esterase ST0071 as a catalyst.
  • allylmalonic acid ethyl ester having prochiral and large steric hindrance was selected as the substrate.
  • half ester was first obtained by hydrolysis reaction using esterase ST0071 as a catalyst. This half ester was found to be stable at room temperature.
  • optically active aryl propionic acid can be obtained by decarboxylation within the active center of the enzyme by using a substrate that is easy to decarboxylate under high temperature conditions.
  • the stereoselective hydrolysis of the disubstituted malonic acid ester can be carried out with high optical purity, and the absolute configuration of the resulting compound is opposite to that obtained with PLE. Furthermore, by carrying out the reaction at a high temperature, the present inventors have succeeded in obtaining a product in which a decarboxylation reaction has proceeded subsequent to hydrolysis, thereby completing the present invention.
  • the present invention is as follows.
  • [4] A method for producing an optically active carboxylic acid from a dicarboxylic acid ester as a raw material, wherein the following (a) or (b) esterase is allowed to act on the dicarboxylic acid ester and optically active by a one-step domino reaction.
  • Method for producing carboxylic acid (a) an esterase consisting of the amino acid sequence represented by SEQ ID NO: 2
  • the starting dicarboxylic acid ester is a compound represented by the following chemical formula I,
  • R 1 and R 2 are different from each other, and are substituted or unsubstituted alkyl groups; substituted or unsubstituted aryl groups or aralkyl groups; substituted or unsubstituted cycloalkyl groups; A substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted sentence represents an unsubstituted alkoxy group, and R 3 and R 4 independently represent H or an alkyl group.
  • the starting dicarboxylic acid ester is a compound represented by the following chemical formula I:
  • R 1 and R 2 are different from each other, substituted or unsubstituted with a phenyl group
  • the starting dicarboxylic acid ester is 2-methyl-2-phenylmalonic acid diester or 2-methyl-2-benzylmalonic acid diester, and the resulting optically active rubonic acid is 2-phenylpropionic acid.
  • the method for producing an optically active carboxylic acid according to [4], which is 2-benzylpropionic acid is 2-benzylpropionic acid.
  • An optically active carboxylic acid is produced in one step from a dicarboxylic acid ester which is a raw material substrate by catalyzing a domino-type reaction with enantioselectivity, which contains esterase derived from Sulfolobus genus microorganisms as an active ingredient. Catalyst composition for carboxylic acid production.
  • [1 2] Catalyze a domino-type reaction having the following esterase (a) or (b) as an active ingredient and having an enantioselectivity, and in one step from dicarboxylic acid ester as a raw material substrate
  • Catalyst composition for producing an optically active carboxylic acid that generates an acid :
  • a method for producing an optically active malonic acid half ester by stereoselective hydrolysis using a dicarboxylic acid ester as a raw material which comprises reacting a dicarboxylic acid ester with an esterase derived from a microorganism belonging to the genus Sulfolobus.
  • PLE porcine liver esterase
  • the dicarboxylic acid ester as a raw material is a compound represented by the following chemical formula I,
  • R 1 and R 2 are different from each other, and are substituted or unsubstituted alkyl groups; substituted or unsubstituted aryl groups or aralkyl groups; substituted or unsubstituted cycloalkyl groups;
  • a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted alkoxy group is represented, and R 3 and R 4 independently represent H or an alkyl group.
  • RR 2 , R 3 and R 4 are the same as the compound represented by the above chemical formula I.
  • [1 7] A method for producing an optically active malonic acid half ester according to any one of [1 3] to [16], wherein the reaction between a dicarboxylic acid ester and a Sulfolobus microorganism-derived esterase is carried out at room temperature.
  • FIG. 1 shows an example of a prochiral compound.
  • FIG. 2 shows an example of an optically active pharmaceutical product.
  • FIG. 3 is a diagram showing an example of a substrate of Sulfolobus tokodaiii strain 7-derived estebuse ST0071.
  • FIG. 4 is a diagram showing a half ester produced from the substrate shown in FIG. 3 by hydrolysis with Sulfolobus tokodai i strain 7-derived esterase ST0071.
  • FIG. 5 is a diagram showing the procedure of the enzyme reaction.
  • FIG. 6 shows the absolute configuration of the product produced by porcine liver esterase (PLE).
  • FIG. 7 shows the results of HPLC analysis of 2-methyl-2-phenylmalonic acid ethyl ester.
  • FIG. 8 is a graph showing the yield and enantiomeric excess of half ester produced from the substrate shown in FIG. 3 by hydrolysis with Sulfolobus tokodai i strain 7-derived esterase ST0071.
  • FIG. 9 is a diagram showing a TLC analysis result of a reaction using 2-methyl-2- (p-diphenyl) maltyl ester malonate as a substrate.
  • FIG. 10 is a view showing a basic skeleton of a substrate of Sulfolobus tokodai i strain 7-derived esterase ST0071.
  • Figure 11 shows the electronic effects of substituents that affect the hydrolysis reaction.
  • FIG. 12 is a diagram showing the structure of 2-methylol-2-phenylmalonic acid jetyl ester.
  • FIG. 13 shows the structure of 2-methyl-2-benzylmalonic acid jetty / resester.
  • the esterase used in the present invention is an esterase derived from a microorganism belonging to the genus Sulfolobus, and includes esterase derived from Sulfolobus' tokodai i strain and esterase power S derived from Sulfolobus tokodai i strain 7. Esterase from Sulfolobus tokodai i strain 7
  • ST0071 is a thermoacidophilic archaeon Sulfolobus tokodai i strain 7 from Y Suzuki et al.
  • the esterase ST0071 can be purified from a culture by culturing Sulfolobus tokodai i strain 7.
  • Sulfolobus tokodai i strain 7 The gene to be transferred can be isolated and manufactured by genetic engineering techniques.
  • SUI IO IODUS tokodan strain is available from JCM u'apan collection of Microorgani sms (registration number JCM10545).
  • the base sequence and amino acid sequence of the esterase ST0071 are registered under the registration number BAB65028.1 in DDBJ (DNA Data Bank of Japan).
  • the base sequence and amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively.
  • the esterase of the present invention has a deletion or substitution of at least one, preferably one or several amino acids in the amino acid sequence as long as the protein comprising the amino acid sequence represented by SEQ ID NO: 2 has esterase activity. Even if a mutation such as addition occurs.
  • 1 or several means 1 to 10, more preferably 1 to 5.
  • amino acid sequence of SEQ ID NO: 2 and the basic amino acid search tool at the National Center are as amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2.
  • for Biological Information e.g., US National Biological Information Center Basic Local Alignment Search
  • etc. e.g., default or default parameters
  • a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 is substantially the same as a protein having the amino acid sequence of SEQ ID NO: 2.
  • a DNA that can hybridize with a DNA that is complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 under the following stringent conditions and that encodes a protein having esterase activity is also included in the esterase of the present invention. That is, using a filter with DNA immobilized, hybridization was performed at 68 ° C in the presence of 0 ⁇ 7 to 1.0M NaCl, and then a 0 ⁇ 1 to 2 fold concentration of SSC solution ( A 1-fold concentration of SSC is a condition that can be identified by washing at 68 ° C using 150raM NaCl and 15raM sodium citrate).
  • hybridization buffer 50% formamide, 4 X SSC, 50 mM HEPES (pH 7.0), 10 X Denhardf s solution, 100 / zg / ml salmon sperm DNA ] DNA that can form a hybrid by reaction at 42 ° C.
  • Sulfolobus-derived microorganism esterases such as Sulfolobus tokodai i strain 7 derived esterase ST0071, etc., use prochiral dicarboxylic acid ester (disubstituted malonic acid ester) as a substrate and preferentially hydrolyze one of the esters.
  • malonic acid half ester (monoester form). The half ester is optically pure and can be obtained in high yield. By this reaction, an optically active substance can be obtained.
  • Sulfolobus microorganism-derived esterases such as Sulfolobus tokodai i strain 7-derived esterase ST0071 catalyze a domino-type reaction with enantioselectivity.
  • the substrate used as a raw material for the above reaction is a dicarboxylic acid ester represented by the following chemical formula I.
  • R 1 and R 2 are different from each other, and are substituted or unsubstituted alkyl groups; substituted or unsubstituted aryl groups or aralkyl groups; substituted or unsubstituted cyclohexanes; It represents an alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted alkoxy group.
  • alkyl group examples include linear or molecular chain alkyl groups such as carbon number. ; ⁇ 20, preferably 1 to 10 carbon atoms, more preferably a linear or molecular alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, methylethyl, butyl, methylpropyl, Examples include pentyl, methylbutyl, ethylpropyl and the like.
  • alkenyl group include a straight chain or branched chain alkenyl group. For example, the straight chain or branched chain having 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms, and more preferably 2 to 5 carbon atoms.
  • Alkenyl groups such as bulle, propenyl and the like.
  • alkynyl group examples include a straight chain or branched chain alkynyl group.
  • the straight chain or branched chain having 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms, and more preferably 2 to 5 carbon atoms.
  • Alkynyl groups such as echel and probule.
  • alkoxy group examples include straight-chain or branched alkoxy groups, for example, carbon number:! To 20, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples include branched chain alkoxy groups, including methoxy, ethoxy, propoxy and the like.
  • aryl group examples include aryl groups having 6 to 20 carbon atoms, preferably 6 to 10 carbon atoms, and include phenyl, 1-naphthyl, 2-naphthyl and the like.
  • substituted alkyl group, cycloalkyl group, alkenyl group, alkynyl group, alkoxyl group, and aryl group include, for example, an aryl group, a cycloalkyl group, an alkyl group, an alkenyl group, an alkynyl group, and an analkoxy group. Amino group, nitro group, hydroxyl group, octalogen atom and the like. These substituents may be further substituted.
  • R 1 and R 2 are different from each other, and a phenyl group, 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 alkyl groups, 1 to 20 carbon atoms, preferably 1 to 10, more preferably 1 to 5 alkoxy or carboxy substituted or unsubstituted alkyl groups; 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms An alkyl group, an alkoxy group having 1 to 5 carbon atoms, a hydroxyl group, or a halogen-substituted or unsubstituted aryl group or aralkyl group; Preferably, R 1 and R 2 are different from each other and each represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group.
  • R 1 and R 2 are different from each other, and are substituted or unsubstituted alkyl groups substituted with a phenyl group, having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms.
  • a It represents an aryl group substituted or unsubstituted with an alkyl group.
  • One of R 1 and R 2 is preferably an alkyl group having 1 to 2 carbon atoms, more preferably a methyl group, and the other is an alkyl group substituted with a phenyl group or an alkyl group having 1 to 5 carbon atoms.
  • a substituted or unsubstituted aryl group is preferably an alkyl group having 1 to 2 carbon atoms, more preferably a methyl group, and the other is an alkyl group substituted with a phenyl group or an alkyl group having 1 to 5 carbon atoms.
  • R 3 and R 4 independently represent H or an alkyl group having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms.
  • Examples of the compound I include aryl carboxylic acid esters such as allyl malonic acid ester, and specifically, 2-methyl-2-phenylmalonic acid diester, particularly 2-methyl-2-phenylmalonic acid ester. Mention may be made of oleic acid ethyl ester or 2-methyl-2-benzylmalonic acid diester, in particular 2-methyl-2-benzylmalonic acid diethyl ester.
  • the first stereoselective hydrolysis reaction produces a half ester represented by the following chemical formula II.
  • R 2 COOH Chemical formula n R 2 COOH Chemical formula n
  • R 2 , R 3 and R 4 are the same as the compound represented by the chemical formula I.
  • the present invention also includes a method for producing a half ester represented by Formula II.
  • the half ester can be used as an intermediate for producing pharmaceuticals and agricultural chemicals.
  • the raw material compound I force S, aryl carboxylic acid ester such as aryl malonate,
  • the resulting compound is ⁇ -arylpropyl.
  • it is an ⁇ -arylalkanoic acid such as on-acid, and is a compound I force, 2-methyl-2-phenolmalonic acid diester or 2-methyl_2-benzylmalonic acid diester, -Phenylpropionic acid or 2-benzylpropionic acid.
  • Examples of other ⁇ -arylpropionic acid obtained by the method of the present invention include optically active flurbiprofen, ibuprofen, ketoprofen, naproxen and the like.
  • the enantiomeric excess of the half ester produced in the first hydrolysis reaction is 20ee (%) or more, preferably 80ee (%) or more, and the yield is 50% or more, preferably 90% or more.
  • the optically active carboxylic acid means that one optical isomer is contained more than the other optical isomer, and the enantiomeric excess of the carboxylic acid finally obtained is 20ee ( %) Or more, preferably 80ee (%) or more, and the yield is 20% or more, preferably 50% or more.
  • the reaction is performed at 0 to 100 ° C, preferably at room temperature to 80 ° C, such as 25 ° C to 80 ° C, more preferably 50 to 80 ° C.
  • room temperature to 80 ° C such as 25 ° C to 80 ° C, more preferably 50 to 80 ° C.
  • domino-type reactions are unlikely to occur at low temperatures below room temperature, and only reactions that produce half esters proceed. In this case, it is preferably carried out at 50 to 80 ° C.
  • the esterase concentration and substrate concentration during the reaction can be determined as appropriate according to the type of substrate.
  • the substrate concentration may be 10 mM
  • the enzyme is 500 U
  • the reaction volume is 20 mL.
  • the unit of esterase U (Unit) is 1 / 3 ⁇ 41 _nitrophenylbutanoate hydrolyzed at pH 8.0 and 25 ° C, 1 // ⁇ 1 _nitrophenol for 1 minute
  • the pH during the reaction is 5 to 10 and preferably 6 to 9.5.
  • the reaction may be performed in a buffer solution. .
  • the reaction time is:! ⁇ 300 hours.
  • the absolute configuration of optically active ruponic acid obtained by reaction using esterase derived from Sulfolobus genus microorganism is the porcine liver esterase (PLE) described in CJ Sih, J. Am. Chera. Soc., 1975, 97, 4144.
  • porcine liver esterase PLE
  • Sulfolobus tokodaii strain 7-derived esterase ST0071 may be used by immobilizing it on an insoluble carrier.
  • Sulfolobus tokodai i strain 7-derived esterase ST0071 is thermoacidophilic and stable to heat and acid. Therefore, it can be used repeatedly.
  • esterase derived from Sulfolobus genus microorganisms such as Sulfolobus tokodai i strain 7 derived esterase ST0071
  • the compound represented by the above chemical formula 1 is used as a raw material
  • the chemical formula 2 is used as an intermediate material for drugs such as anti-inflammatory agents and agricultural chemicals.
  • the optically active carboxylic acid represented can be produced in one step in a short time.
  • the present invention is a one-step optical process from a dicarboxylic acid ester as a raw material substrate that catalyzes a domino-type reaction with enantioselectivity, which contains an esterase derived from a genus Sulfolobus such as esterase ST0071 derived from Sulfolobus tokodai i strain 7 as an active ingredient.
  • generates active carboxylic acid is included.
  • E. coli BL21 Star (DE3) was transformed with plasmid pETstEst and cultured overnight at 37 ° C in 10 ml of LB medium (containing 50 ig / ral ampicillin). It was transferred to 1000 ml of LB medium and further cultured at 30 ° C for 5 hours. To induce enzyme expression, IPTG was added to a concentration of 0.1 mM, and the cells were further cultured for 3 hours.
  • JE0L JNM was used to measure the nuclear magnetic resonance spectrum.
  • EX-270 (270 MHz) Fourier transform nuclear magnetic resonance spectrum measuring instrument and JEOL A JNM AL-300 (300 MHz) Fourier transform nuclear magnetic resonance spectrum measuring device was used.
  • JASCO JASCO FT / IR-410 was used to measure the infrared absorption spectrum.
  • Sensyu Science SSC-3461 was used for high-speed liquid chromatography.
  • MERCK 1.005715 was used for analytical silica gel thin layer chromatography (TLC).
  • MERCK1.05744. was used for preparative Siri-Force gel thin layer chromatography (pTLC).
  • enzyme reaction and product analysis were performed by the following method.
  • the substrate and the crude enzyme solution were added to a 50 mL eggplant flask so that the substrate concentration was 10 mM, and the reaction was carried out by stirring at room temperature or 60 ° C for 1 to 10 days.
  • 2M hydrochloric acid was added to complete the reaction, the mixture was filtered through celite and extracted with ethyl acetate according to a conventional method.
  • the product was determined by nuclear magnetic resonance spectrum and infrared absorption spectrum.
  • the nuclear magnetic resonance spectrum and infrared absorption spectrum of the product were as follows.
  • 2-Methyl_2_phenylmalonic acid jetyl ester (5) was synthesized by methylation of 2-phenyl-2-remalonic acid jetyl ester with methyl fluoride using sodium methoxide as the base (Formula 2) .
  • 2-Ethyl-2-phenylmalonic acid jetyl ester (8) and 2-methyl-2-benzenolemalonic acid jetyl ester (9) use sodium hydride as a base and use 2-substituted malonic acid jetyl ester as a base. Synthesized by alkylation with alkyl (chemical formulas 3 and 4).
  • 2-Methyleno-2- (p-diphenyl) malonic acid jetyl ester (10) is obtained by arylating methylmalonic acid ethyl ester with sodium hydride as base and P-nitrochlorobenzene.
  • 2-Methyl-2- (P-methylphenyl) malonic acid ethyl ester (11) is obtained by esterifying 2- (p-methylphenyl) propionic acid, and using lithium diisopropylamine (LDA) as the base, And was synthesized by ethoxycarbonylation (Chemical Formula 6).
  • Sodium hydride (0.575 g, 14.4 mraol) was washed 3 times with 10 mL of anhydrous hexane. Hexane was removed, 15 mL of THF was added, and a THF solution (10 mL) of benzylmalonic acid jetyl ester (3.00 g, 12.0 olol) was added while maintaining the temperature at 0 ° C. After stirring for 30 minutes, a THF solution (10 mL) of methyl iodide (2.04 g, 14.4 ol) was added, and the mixture was heated to reflux at 80 ° C. for 6 hours.
  • the solution was washed three times with sodium hydride (1.12 g, 28.1 ol) in 10 mL of anhydrous hexane. Remove hexane, add DMFlO mL, and maintain the temperature at 0 ° C. A DMF solution (15 mL) of acid jetyl ester (3.00 g, 18.7 thigh ol) was added. After stirring for 30 minutes, a DMF solution (15 raL) of p-nitrochlorobenzene (4.43 g, 28.1 mraol) was added and heated to reflux at 100 ° C for 4 hours.
  • 2-Ethyl-2-phenylmalonic acid jetyl ester (0. 500 g, 1. 89 mmol) in ethanol (3 mL) was added to potassium hydroxide (0.159 g, 2.83 mmol) in water (3 mL). And stirred at room temperature. After 6 hours, the mixture was neutralized with hydrochloric acid and aqueous sodium hydroxide solution, the solvent was distilled off under reduced pressure, and water equivalent to the residue was added. After that, the aqueous layer was extracted using JETLE TERTEL and made basic with hydrochloric acid. Furthermore, extraction was performed using jetyl ether, and the organic layer was washed with saturated brine.
  • the absolute configuration was determined by comparing the product of the reaction with PLE and the product of the reaction with this enzyme using high-speed liquid chromatography.
  • esterase ST0071 catalyzes a domino-type reaction
  • an enzymatic reaction was first carried out using 2-methyl-2-phenylmalonic acid jetyl ester (5) as a substrate. At this time, the reaction temperature was examined at room temperature and 60 ° C.
  • this enzyme can hydrolyze 2-methyl-2-phenylmalonic acid jetyl ester (5) and catalyze a domino-type reaction under high temperature conditions.
  • the 2-methyl-2-phenylmalonic acid jetyl ester (5) used in the enzymatic reaction is prochiral and the resulting 2-methyl-2-phenylmalonic acid monoethyl ester (6) and 2-phenylpropionic acid (7) has an asymmetric carbon. Therefore, if these reactions occur within the active site of the enzyme, the product can be expected to be an optically active form.
  • the absolute configuration of product 6 cannot be determined because the retention time of high performance liquid chromatography (HPLC) is not known and there is no literature value of optical rotation. Therefore, it was determined by comparing the absolute configuration of the product with porcine liver esterase (PLE).
  • Table 1 shows that the product 6 at both temperatures is S-isomer with a high enantiomeric excess of 96-99% ee. Furthermore, the other product 7 in the reaction at 60 ° C was also found to be an optically active form of S form. Since two optically active products were obtained in this way, it was confirmed that enzymes were involved in this reaction.
  • reaction pathway in formula 1 0 60 The first route predicted from Chemical Formula 10 is that decarboxylation occurs, 2-phenylpropionic acid ethyl ester (16) is formed, and further ester hydrolysis occurs. The second is that ester hydrolysis takes place first, 2-methyl-2-phenylmalonic acid (17) is produced, and decarboxylation takes place.
  • the intermediates (16, 17) of these two predicted pathways have not been detected. However, since this enzyme does not have AMDase activity, it is presumed that it is a pathway via intermediate 16.
  • These reaction pathways can be determined by conducting an enzymatic reaction using each of the intermediates 16 and 17 and examining the progress of the reaction.
  • the product of the reaction to substrate 10 was carboxylic acid 14 instead of half ester.
  • the following results were obtained by following the reaction with TLC (Fig. 9). From Fig. 9, when all of the substrate 10 has been reacted (144 hours later), the product was only carboxylic acid 14. However, 12 hours after the start of the reaction, TLC analysis confirmed 2 Hafster 18 I was able to do it. Therefore, the reaction route of Chemical Formula 12 could be estimated.
  • Substrate 8 whose structure is shown in Fig. 12 has the methyl group of Substrate 5 substituted with an ethyl group. This suggests that the reason for the longer reaction time is that the electron donating property of the alkyl group is higher than that of the methyl group. Another possible factor is the steric hindrance of the ethyl group. The ethyl group is more sterically hindered than the methyl group, so it is presumed that it was less susceptible to hydrolysis.
  • the influence of the reaction time is due to the high electron donating property and steric hindrance of the ethyl group.
  • Substrate 9 whose structure is shown in Fig. 13 has the phenyl group of Substrate 5 substituted with a benzyl group.
  • a methylene group was inserted between the phenyl group and the ⁇ carbon. This has an effect on the carbonyl carbon inserted more than the phenyl group.
  • the electron donating property of the methylene group is larger (Fig. 13). Therefore, it is suggested that this reaction time is also caused by the electron donating property of the methylene group inserted next to the phenyl group.
  • Substrate 10 is a substrate in which a nitro group is added to the p-position of the phenyl group of substrate 5. Since the nitro and phenyl groups are electron withdrawing, the electron density of the carbonyl carbon of this substrate is significantly reduced. Therefore, the reaction time should be shortened. However, the reaction time was longer than that of substrate 5. As described above, since the substrate 10 is a domino type reaction, the reactivity of hydrolysis cannot be compared based on the reaction time. Therefore, we compare it with the reaction of substrate 5 at 60 ° C.
  • reaction time is the time until all of the substrate has reacted. Then, half-ester 6 was obtained in Chemical Formula 1, while half ester 18 could not be obtained in Chemical Formula 1-4. From this, it can be inferred that in the decarboxylation reaction, the reactivity of 18 was higher than that of Ifest 6 due to the electron withdrawing property of the nitro group.
  • Substrate 11 is a substrate in which a methyl group is added to the p-position of the phenyl group of substrate 5. This p-position methyl group is electron donating, so the electron density of carbonyl carbon of this substrate is higher than that of substrate 5. Therefore, it was predicted that the reaction time would be longer as with substrates 8 and 9, but the reaction time was shorter (Chemical Formula 15).
  • the reaction conditions for the substrate 10 are alkaline, which is a condition for promoting the decarboxylation reaction.
  • esterase ST0071 showed the same stereoselectivity with respect to all the substrates used in this example, and that stereoselectivity was reversed with PLE.
  • the size of the steric hindrance of the alkyl group was found to affect the enantiomeric excess.
  • Esterase ST0071 was found to hydrolyze ⁇ -arylmalic acid ketyl ester, which is highly sterically hindered and hardly hydrolyzed, with high stereoselectivity to produce a half ester.
  • carboxylic acid which was thought to have progressed through decarboxylation due to hydrolysis, was confirmed.
  • a domino-type reaction was successfully constructed under high temperature conditions (Chemical Formula 16).
  • E. coli BL21 Star a (DE3) and Development $ quality transformed with plasmid PETstEst, in 10 ml of LB medium (containing 50 ⁇ ⁇ / ⁇ 1 ampicillin), and cultured overnight at 37 ° C. It was transferred to 1000 ml of LB medium and further cultured at 30 ° C for 5 hours.
  • LB medium containing 50 ⁇ ⁇ / ⁇ 1 ampicillin
  • a substrate phenylmethylmalonic acid ester
  • enzyme (18 U) were added to a 50 mL eggplant flask so that the substrate concentration was 2 raM, and the mixture was stirred at 70 ° C. for 2 days to carry out the reaction.
  • an optically active carboxylic acid serving as an intermediate for pharmaceuticals and agricultural chemicals can be produced from a dicarboxylic acid ester.

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Abstract

L'invention vise à proposer une nouvelle réaction de type domino d'une réaction d'hydrolyse et d'une réaction de décarboxylation à l'aide d'une estérase ST0071 comme catalyseur pour obtenir de cette façon un acide carboxylique optiquement actif tel qu'un acide α-arylalcanoïque optiquement actif. L'invention porte sur un procédé de fabrication d'un acide carboxylique optiquement actif à l'aide d'un ester d'acide dicarboxylique comme matériau de départ, qui peut produire l'acide carboxylique optiquement actif par une réaction de domino à une étape comprenant la réaction de l'ester d'acide dicarboxylique avec une estérase issue de la souche 7 de Sulfolobus tokodaii.
PCT/JP2009/053783 2008-02-26 2009-02-23 Procédé de fabrication d'un acide carboxylique optiquement actif à l'aide d'une estérase issue d'une archaebactérie thermophile WO2009107821A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104447347A (zh) * 2013-09-17 2015-03-25 上海百灵医药科技有限公司 一种苹果酸苏尼替尼中间体的制备方法

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JP2003339391A (ja) * 1996-02-16 2003-12-02 Diversa Corp エステラーゼ

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JP2003339391A (ja) * 1996-02-16 2003-12-02 Diversa Corp エステラーゼ

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"87th Annual Meeting on Chemical Society of Japan in Spring Koen Yokoshu II, 2007", article SAWAZAKI M. ET AL.: "Hen'i Koso no Enantio Sentakusei ni Taisuru Ion Ekitai no Koka", pages: 1371 *
"88th Annual Meeting on Chemical Society of Japan in Spring Koen Yokoshu II, 12 March, 2008 (12.03.08)", article WADA R. ET AL.: "Konetsusei Kosaikin Yurai Esterase ST0071 o Mochiita Shinki Domino-gata Hanno no Kaihatsu", pages: 922 *
HERMANN M. ET AL.: "Alternative pig liver esterase (APLE) - cloning, identification and functional expression in Pichia pastoris of a versatile new biocatalyst.", J.BIOTECHNOL., vol. 133, no. 3, 1 February 2008 (2008-02-01), pages 301 - 310 *
MATOISHI K. ET AL.: "Mechanism of asymmetric decarboxylation of a-aryl-a-methylmalonate catalyzed by arylmalonate decarboxylase originated from Alcaligenes bronchisepticus", J.MOL.CATAL.B ENZYM., vol. 27, no. 4-6, 2004, pages 161 - 168 *
PARK Y.J. ET AL.: "A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression.", BIOCHIM.BIOPHYS.ACTA., vol. 1760, no. 5, 2006, pages 820 - 828 *
SUZUKI Y. ET AL.: "A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7.", FEMS MICROBIOL.LETT., vol. 236, no. 1, 2004, pages 97 - 102 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104447347A (zh) * 2013-09-17 2015-03-25 上海百灵医药科技有限公司 一种苹果酸苏尼替尼中间体的制备方法

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