EP2480664A1 - Amidase et son utilisation pour la production d'esters d'acide 3-aminocarboxilique - Google Patents

Amidase et son utilisation pour la production d'esters d'acide 3-aminocarboxilique

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Publication number
EP2480664A1
EP2480664A1 EP10757596A EP10757596A EP2480664A1 EP 2480664 A1 EP2480664 A1 EP 2480664A1 EP 10757596 A EP10757596 A EP 10757596A EP 10757596 A EP10757596 A EP 10757596A EP 2480664 A1 EP2480664 A1 EP 2480664A1
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EP
European Patent Office
Prior art keywords
alkyl
formula
aryl
acid ester
cycloalkyl
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EP10757596A
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German (de)
English (en)
Inventor
Bernhard Hauer
Thomas Friedrich
Rainer STÜRMER
Nina Schneider
Susanne Krauser
Wolf-Rüdiger KRAHNERT
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BASF SE
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BASF SE
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Priority to EP10757596A priority Critical patent/EP2480664A1/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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • 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)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • 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
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C12P41/007—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures by reactions involving acyl derivatives of racemic amines

Definitions

  • the present invention relates to a novel amidase and its use for the preparation of optically active 3-aminocarboxylic acid ester compounds, and their derivatives.
  • WO 97/41214 describes biocatalysts with aminacylase activity which have no lipase or esterase activity.
  • WO 2008/003761 describes a process for the preparation of optically active 3-aminocarboxylic acid esters in which an enantiomerically enriched enantiomer mixture of a singly N-acylated 3-aminocarboxylic acid ester is added by addition of an acidic salt former of a deacylation followed by further enantiomeric enrichment by crystallization subjects.
  • the present invention is therefore based on the object to provide a simple and therefore economical process for the preparation of optically active 3-aminocarboxylic acid esters and derivatives thereof.
  • R 1 is alkyl, alkoxyalkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, or hetaryl, and
  • R 2 is alkyl, cycloalkyl or aryl, in which one enantiomeric mixture of a simple N-acylated 3-aminocarboxylic ester of the general wherein R 1 and R 2 have the meanings given above and R 3 is hydrogen, alkyl, cycloalkyl or aryl, by addition of a polypeptide according to claim 1 or 2 undergoes an enantioselective deacylation, is dissolved.
  • Another object of the invention is a process for the preparation of optically active 3-amino carboxylic acid ester compounds of the general formula ⁇ , and their derivatives,
  • R 1 is alkyl, alkoxyalkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, or hetaryl, and
  • R 2 is hydrogen, a cation equivalent M + , alkyl, cycloalkyl or aryl, in which a) a ⁇ -keto ester of the general formula 1.1 wherein R 1 and R 2 have the meanings given above, a 1) with at least one carboxylic acid amide of the formula R 3 -C (O) NH 2, in which R 3 has the abovementioned meaning, in the presence of an amidation catalyst, or
  • R 1 , R 2 and R 3 have the meanings given above, c) subjecting the enantiomeric mixture of compounds Ib obtained in the hydrogenation to an enantioselective deacylation by addition of a polypeptide having amidase activity and the ammonium salt enriched in a stereoisomer of a 3 -Aminocarboxylic acid ester is isolated, and d) optionally the isolated ammonium salt is converted into the 3-aminocarboxylic acid ester, and e) optionally the 3-aminocarboxylic acid ester in the free 3-aminocarboxylic acid or a salt thereof.
  • Another object of the invention is a polypeptide having amidase activity selected from
  • polypeptide containing an amino acid sequence according to SEQ ID NO: 2 a) polypeptide containing an amino acid sequence according to SEQ ID NO: 2, and b) polypeptide containing an amino acid sequence which has at least 96%, preferably 98%, particularly preferably 99% identity with SEQ ID NO: 2.
  • Another object of the invention is a polypeptide having amidase activity selected from
  • polypeptide containing an amino acid sequence according to SEQ ID NO: 4 and d) polypeptide containing an amino acid sequence which is at least 80%, preferably 85, 88%, 90%, more preferably 92%, 94%, 96%, 98%, 99% % Identity with SEQ ID NO: 4.
  • Chiral compounds in the context of the present invention are compounds having at least one chiral center (that is to say at least one asymmetric atom, eg at least one asymmetric C atom or P atom), with chirality axis, chirality plane or helical turn.
  • chiral catalyst includes catalysts having at least one chiral ligand.
  • prochiral compound a compound having at least one prochiral center.
  • Asymmetric synthesis refers to a reaction in which, from a compound having at least one prochiral center, a compound having at least one chiral center, a chiral axis, a plane of chirality, or a helical coil is generated, whereby the stereoisomeric products are formed in unequal amounts.
  • Steps are compounds of the same constitution but of different atomic order in three-dimensional space.
  • Enantiomers are stereoisomers that behave as image to mirror image to each other.
  • ee [%] (RS) / (R + S) * 100.
  • R and S are the descriptors of the ClP system for the two enantiomers and represent the absolute configuration at the asymmetric atom.
  • the process according to the invention leads to products enriched in a particular stereoisomer.
  • the achieved "enantiomeric excess" (ee) is usually at least 95%, preferably at least 98%, and particularly preferably at least 99%.
  • Diastereomers are stereoisomers that are not enantiomeric to one another.
  • stereochemical terms listed herein refer to the carbon atom of the respective compounds corresponding to the asymmetric ⁇ -carbon atom in compound I or ⁇ . If further stereocenters are present, they are neglected in the context of the present invention in the designation.
  • alkyl includes straight-chain and branched alkyl groups. These are preferably straight-chain or branched C 1 -C 20 -alkyl, preferably C 1 -C 12 -alkyl, particularly preferably C 1 -C 6 -alkyl and very particularly preferably C 1 -C 6 -alkyl groups.
  • alkyl groups are, in particular, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 2-methylbutyl, 3-methylbutyl, 1, 2 Dimethylpropyl, 1, 1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 2-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl, 1 - Ethyl 2-methyl
  • alkyl also includes substituted alkyl groups which are generally 1, 2, 3, 4 or 5, preferably 1, 2 or 3 and particularly preferably 1 substituent selected from the groups cycloalkyl, aryl, hetaryl, halogen, COOR f , CO " M + and NE can carry 1 E 2 , wherein R f is hydrogen, alkyl, cycloalkyl or aryl, M + is a cation equivalent and E 1 and E 2 are independently hydrogen, alkyl, cycloalkyl or aryl.
  • alkoxyalkyl includes straight-chain and branched alkyl groups linked to an alkoxy group.
  • the alkoxy radical can likewise be straight-chain or branched. These are preferably straight-chain or branched C 1 -C 20 -alkyl, preferably C 1 -C 12 -alkyl, particularly preferably C 1 -C 6 -alkyl and very particularly preferably C 1 -C 6 -alkyl groups which are denoted by C 1 -C 12 Alkoxy, particularly preferably C 1 -C 6 alkoxy linked.
  • alkyl groups are mentioned above;
  • alkoxy groups are in particular methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutene toxy, sec-butoxy.
  • alkoxyalkyls are in particular methoxymethyl, ethoxymethyl, ethoxyethyl, ethoxypropyl.
  • cycloalkyl in the context of the present invention comprises both unsubstituted and substituted cycloalkyl groups, preferably C 3 -C 8 -cycloalkyl groups, such as cyclopentyl, cyclohexyl or cycloheptyl, which in the case of a substitution, generally 1, 2, 3, 4 or 5, preferably 1, 2 or 3 and particularly preferably 1 substituent, preferably selected from alkyl and the substituents mentioned for alkyl, can carry.
  • heterocycloalkyl in the context of the present invention comprises saturated, cycloaliphatic groups having generally 4 to 7, preferably 5 or 6, ring atoms in which 1 or 2 of the ring carbon atoms are replaced by heteroatoms, preferably selected from the elements oxygen, nitrogen and sulfur and which may be optionally substituted, wherein in case of substitution, these heterocycloaliphatic groups are 1, 2 or 3, preferably 1 or 2, more preferably 1 substituent selected from alkyl, cycloalkyl, aryl, COOR f , COO-M + and NE 1 E 2 , preferably alkyl, where R f is hydrogen, alkyl, cycloalkyl or aryl, M + is a cation equivalent and E 1 and E 2 are independently hydrogen, alkyl, cycloalkyl or aryl.
  • heterocycloaliphatic groups are pyrrolidinyl, piperidinyl, 2,2,6,6-tetramethylpiperidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, morpholidinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, piperazinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, dioxanyl called.
  • aryl in the context of the present invention comprises unsubstituted and substituted aryl groups, and is preferably phenyl, tolyl, xylyl, mesityl, naphthyl, fluorenyl, anthracenyl, phenanthrenyl or naphthacenyl, particularly preferably phenyl or naphthyl, these aryl groups in the In case of a substitution in general 1, 2, 3, 4 or 5, preferably 1, 2 or 3 and particularly preferably 1 substituent selected from the groups alkyl, alkoxy, nitro, cyano or halogen, can carry.
  • heterocycloaromatic groups preferably the groups pyridyl, quinolinyl, acridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, imidazolyl, pyrazolyl, indolyl, purinyl, indazolyl, benzotriazolyl , 1, 2,3-triazolyl, 1, 3,4-triazolyl and carbazolyl, these heterocycloaromatic groups in the case of a substitution generally 1, 2 or 3 substituents selected from the groups alkyl, alkoxy, acyl, carboxyl, carboxylate, - SO 3 H, sulfonate, NE 1 E 2 , alkylene-NE 1 E 2 or halogen, where E 1 and E 2 have the meanings given above.
  • acyl in the context of the present invention for alkanoyl or
  • Aroyl groups having generally 2 to 11, preferably 2 to 8, carbon atoms for example the acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, 2-ethylhexanoyl, 2-propylheptanoyl, benzoyl, naphthoyl or trifluoroacetyl group.
  • Halogen is fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine.
  • M + represents a cation equivalent, ie a monovalent cation or the single positive charge fraction of a multiple cation. These include z. Li, Na, K, Ca and Mg.
  • the processes according to the invention enable the preparation of optically active compounds of the general formula I, as well as the preparation of their derivatives.
  • R 1 is preferably C 1 -C 6 -alkyl, C 1 -C 3 -alkenyl, or C 6 -C 4 -aryl, which may optionally be substituted as described above.
  • R 1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, 1-propenyl, 1 -heptenyl, or phenyl, especially methyl and phenyl.
  • R 2 is preferably unsubstituted or substituted C 1 -C 6 -alkyl, C 3 -C 7 -cycloalkyl or C 6 -C 14 -aryl.
  • Particularly preferred radicals R 2 are methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, trifluoromethyl, cyclohexyl, phenyl and benzyl.
  • R 2 ' is hydrogen, M + , as well as the meanings given for R 2 .
  • R 3 is hydrogen, alkyl, cycloalkyl or aryl, in particular hydrogen
  • Methyl, ethyl, trifluoromethyl, benzyl and phenyl Methyl, ethyl, trifluoromethyl, benzyl and phenyl.
  • an enantiomeric mixture of the compounds 1b is subjected to enantioselective deacylation by addition of an amidase, and the ammonium salt of a 3-aminocarboxylic acid ester which has been enriched in respect of a stereoisomer is isolated.
  • the process according to the invention that in the isomer mixture of compounds of the general formula Ib used for the deacylation, the corresponding enantiomer or, starting from chiral ⁇ -ketoesters, also diastereomers are present in non-negligible amounts.
  • the process thus enables the preparation of optically active compounds of general formula I, starting from mixtures of isomers of compounds of general formula Ib, as obtainable for example from the precursor compounds by conventional asymmetric hydrogenation of enamides.
  • enantiomeric mixtures which contain the enantiomers in the same molar ratio or are already enriched in an enantiomer.
  • the ee value of these mixtures is preferably greater than 75% and particularly preferably greater than 90%.
  • racemates or mixtures already enriched in one enantiomer are produced.
  • enantioselective hydrogenation processes for example those as mentioned in WO 2008/003761, the description of which is hereby expressly referred to.
  • the deacylation is carried out at a temperature of 20-40 ° C, more preferably between 20 and 30 ° C.
  • the reaction is usually carried out in aqueous buffer.
  • Another object of the invention relates to a process comprising the reaction steps a) to c) described below and optionally d) and e).
  • step a) of the process according to the invention a ⁇ -keto ester of the formula I.1 having at least one carboxamide of the formula
  • step a.1 in the case of the carboxylic acid amides of the formula
  • R 3 -C (O) NH 2 to acetamide, propionamide, benzoic acid amide, formamide or trifluoroacetamide, in particular benzoic acid amide or acetamide.
  • Suitable solvents for step a.1 are those which form a low-boiling azeotrope with water, from which the water of reaction can be removed by separation methods known to those skilled in the art (such as, for example, azeotropic distillation).
  • these are aromatics, such as toluene, benzene, etc., ketones such as methyl isobutyl ketone or methyl ethyl ketone, etc., and haloalkanes such as chloroform.
  • toluene is used.
  • Suitable amidation catalysts are, for example, acids such as p-toluenesulfonic acid, methanesulfonic acid, sulfuric acid or the like. Preferably, p-toluenesulfonic acid is used.
  • the reaction in process step a.1 preferably takes place at a temperature in the range from 20 to 110 ° C., particularly preferably 60 to 90 ° C. Particularly preferably, the temperature is above the boiling point of the solvent used under normal conditions.
  • Process step a.1 is usually carried out at a pressure of 0.01 to 1.5 bar, in particular 0.1 to 0.5 bar. If appropriate, the aminocarboxylic acid ester obtained in step a.1 can be purified by customary methods known to the person skilled in the art, eg. B. be subjected to distillation.
  • a ⁇ -ketoester of the formula 1.1 is reacted with aqueous ammonia and then with a carboxylic acid derivative of the formula R 3 -C (O) X to give the N-acylated, ⁇ -unsaturated (Z) -3-aminocarboxylic acid ester (La), wherein X is halogen or a radical of the formula OC (O) R 4 , in which R 4 has the meaning given above for R 3 (step a.2).
  • the carboxylic acid derivative is preferably selected from carboxylic acid chlorides, wherein X is chlorine and R 3 has the meaning given above, or carboxylic anhydrides, wherein X is OC (O) R 4 and R 4 preferably has the same meaning as R 3 , is particularly preferred the carboxylic acid derivatives are acetyl chloride, benzoyl chloride or acetic anhydride.
  • the acylation in step a.2 is preferably carried out at a temperature in the range from 20 ° C. to 120 ° C., more preferably at a temperature in the range from 60 ° C. to 90 ° C.
  • the acylation in step a.2 is carried out in a polar solvent or a mixture of a polar solvent with a non-polar solvent, it is preferable that the polar solvent is a carboxylic acid of the formula R 3 COOH or a tertiary amine, as a non-polar solvent in particular haloalkanes and aromatics are suitable, with particular preference being given to using acetic acid or triethylamine as the solvent.
  • the polar solvent is a carboxylic acid of the formula R 3 COOH or a tertiary amine, as a non-polar solvent in particular haloalkanes and aromatics are suitable, with particular preference being given to using acetic acid or triethylamine as the solvent.
  • the acylation in step a.2 can be carried out using a catalyst, this can be used both in catalytic amounts and stoichiometrically or as a solvent, preferably non-nucleophilic bases, such as tertiary amines, more preferably these are triethylamine and / or dimethylaminopyri - din (DMAP).
  • a catalyst this can be used both in catalytic amounts and stoichiometrically or as a solvent, preferably non-nucleophilic bases, such as tertiary amines, more preferably these are triethylamine and / or dimethylaminopyri - din (DMAP).
  • the (Z) -3-aminocarboxylic acid ester is obtained as a mixture with the (E) -3-aminocarboxylic acid ester and optionally further acylation products.
  • the (Z) -3-aminocarboxylic acid ester of the formula I.a will be isolated by methods known to those skilled in the art. A preferred method is separation by distillation.
  • the ⁇ -unsaturated (Z) -3-aminocarboxylic acid ester compounds of the formula Ia obtained in stage a can subsequently undergo hydrogenation, optionally an enantioselective hydrogenation, in the presence of an optionally chiral hydrogenation catalyst to give a racemate or an enantiomeric mixture enriched in an enantiomer simply N-acylated ß-aminocarboxylic acid ester of the general formula (lb) are subjected.
  • step b) as the hydrogenation catalyst at least one complex of a transition metal of groups 8 to 1 1 of the Periodic Table of the Elements is used, which comprises as ligand at least one chiral, phosphorus atom-containing compound.
  • a transition metal of groups 8 to 1 1 of the Periodic Table of the Elements which comprises as ligand at least one chiral, phosphorus atom-containing compound.
  • a chiral hydrogenation catalyst which is capable of hydrogenating the ⁇ -unsaturated, N-acylated 3-aminocarboxylic acid ester (Ia) used with preference to the desired isomer.
  • the compound of the formula I.b obtained in step b) preferably has an ee value of at least 75%, particularly preferably at least 90%, after the asymmetric hydrogenation.
  • the ee value of the compound 1b is preferably at least 75%.
  • the process according to the invention preferably allows the enantioselective hydrogenation at substrate / catalyst ratios (s / c) of at least 1000: 1, particularly preferably at least 5000: 1 and in particular at least 15000: 1.
  • the transition metal is selected from Ru, Rh, Ir, Pd or Pt.
  • catalysts based on Rh and Ru Especially preferred are Rh catalysts.
  • the phosphorus-containing compound used as ligand is preferably selected from bidentate and polydentate phosphine, phosphinite, phosphonite, phosphoramidite and phosphite compounds.
  • catalysts which have at least one ligand selected from compounds of the following formulas,
  • TrichickenfootPhos MiniPhos or their enantiomers wherein Ar is optionally substituted phenyl, preferably tolyl or xylyl.
  • bidentate compounds of the aforementioned classes of compounds are particularly preferred.
  • P-chiral compounds such as DuanPhos, TangPhos or Binapine are preferred.
  • Suitable chiral ligands which coordinate to the transition metal via at least one phosphorus atom are known to the person skilled in the art and are commercially available, for example, from Chiral Quest ((Princeton) Inc., Monmouth Junction, NJ). The naming of the previously exemplified chiral ligands corresponds to their commercial name.
  • Chiral transition metal complexes can be prepared in a manner known to the person skilled in the art (for example Uson, Inorg. Chim. Acta 73, 275 1983, EP-A-0 158 875, EP-A-437 690) by reacting suitable ligands with complexes of Metals containing labile or hemilabile ligands obtained.
  • suitable ligands for example Uson, Inorg. Chim. Acta 73, 275 1983, EP-A-0 158 875, EP-A-437 690
  • precatalysts complexes such as
  • X can be any anion known to those skilled in the art and generally useful in asymmetric synthesis.
  • Examples of X are halogens such as Ch, Br or I, BF 4 -, CI0 4 -, SbF6 -, PF 6 -, CF 3 S0 3 -, BAr 4 -.
  • Preferred for X are BF 4 -, PF 6 -, CF 3 S0 3 -, SbF 6 -.
  • the chiral transition metal complexes can either be generated in situ before the actual hydrogenation reaction in the reaction vessel or else be generated separately, isolated and then used. It may happen that at least one solvent molecule attaches to the transition metal complex.
  • the common solutions for example, methanol, diethyl ether, tetrahydrofuran (THF), dichloromethane, etc.
  • THF tetrahydrofuran
  • dichloromethane dichloromethane
  • the hydrogenation step (step b) of the process according to the invention is generally carried out at a temperature of from -10 to 150.degree. C., preferably from 0 to 120.degree. C. and more preferably from 10 to 70.degree.
  • the hydrogen pressure can be varied within a range between 0.1 bar and 600 bar. This is preferably in a pressure range of 0.5 to 20 bar, more preferably between 1 to 10 bar.
  • Suitable solvents for the hydrogenation reaction of the enamides Ia are all solvents known to the person skilled in the art for asymmetric hydrogenation.
  • Preferred solvents are lower alkyl alcohols such as methanol, ethanol, isopropanol, and toluene, THF, ethyl acetate.
  • ethyl acetate or THF is particularly preferably used as the solvent.
  • the hydrogenation catalysts (or pre-catalysts) described above can also be suitably, for. B. by attachment via suitable as anchor groups functional groups, adsorption, grafting, etc. to a suitable carrier, eg. Example of glass, silica gel, resins, polymer carriers, etc., are immobilized. They are then also suitable for use as solid phase catalysts.
  • a suitable carrier eg. Example of glass, silica gel, resins, polymer carriers, etc.
  • the catalyst consumption can be further reduced by these methods.
  • the catalysts described above are also suitable for a continuous reaction, z. B. after immobilization, as described above, in the form of solid phase catalysts.
  • the hydrogenation in stage b is carried out continuously.
  • the continuous hydrogenation can be carried out in one or preferably in several reaction zones. Multiple reaction zones may be formed by multiple reactors or by spatially distinct regions within a reactor. When using multiple reactors may each be the same or different reactors. These may each have the same or different mixing characteristics and / or be subdivided by internals one or more times.
  • the reactors can be interconnected as desired, z. B. parallel or in series. Suitable pressure-resistant reactors for the hydrogenation are known to the person skilled in the art. These include the commonly used reactors for gas-liquid reactions, such. B. tubular reactors, tube bundle reactors, stirred tank, gas circulation reactors, bubble columns, etc., which may be filled or divided by internals.
  • process step c) the mixture of enantiomers of the compounds I.b obtained in the hydrogenation is subjected to an enantioselective deacylation by addition of a polypeptide having amidase activity and the resulting stereoisomer-enriched ammonium salt of a 3-aminocarboxylic acid ester is isolated.
  • the polypeptide having amidase activity can be used as a purified enzyme, as a partially purified crude extract or in the form of a living or killed microorganism containing the amidase.
  • Preferred amidases are those having the primary structure SEQ ID NO: 2 or NO: 4 or variants of SEQ ID NO: 2 or NO: 4, which by insertion, deletion or substitution of fewer amino acids, preferably 1 -20, particularly preferably 1 - 10 amino acids, can be obtained.
  • the reaction is usually carried out in aqueous buffer.
  • the resulting reaction product can be purified by conventional methods and isolated.
  • the ammonium salts isolated in the enantiomerically enriching deacylation by amidase reaction may be subjected to further work-up.
  • a suitable base preferably NaHCO 3, NaOH, KOH.
  • the product of the deacylation is dissolved or suspended in water and then the pH is adjusted by base addition to about 8 to 12, preferably about 10.
  • a suitable organic solvent e.g. Example, an ether such as methyl butyl ether, a hydrocarbon or hydrocarbon mixture, for.
  • alkane such as pentane, hexane, heptane, or an alkane mixture, ligroin or petroleum ether, or aromatics, such as toluene to extract.
  • a preferred extractant is toluene.
  • the 3-aminocarboxylic acid esters may be derivatized using methods known to those skilled in the art. Possible derivatizations include, for example, saponification of the ester or stereoselective reduction of the carboxyl carbon to an optically active alcohol.
  • Derivatives of the invention of compounds of the formula ⁇ thus include, for example, ammonium salts of the 3-aminocarboxylic acid esters, the free carboxylic acid in which R 2 'is hydrogen, salts of the free carboxylic acid in which R 2' is M + , and optically active 3-aminoalcohols.
  • a further subject of the invention are polypeptides which can catalyze an amidase reaction and have the following primary structure (amino acid sequence):
  • polypeptide sequence having at least 96%, preferably 98%, most preferably 99% identity with SEQ ID NO: 2.
  • polypeptide sequence having at least 80%, preferably at least 85%, most preferably at least 95% identity with SEQ ID NO: 4.
  • R1 and R3 are each methyl and R2 is ethyl.
  • amidase with the SEQ ID NO: 2 can be, for example, Rhodococcus eq
  • Example 1 Cloning of an amidase from Rhodococcus equi The coding region of the S-selective amidase from Rhodococcus equi was amplified by means of a PCR with the following oligonucleotide primers:
  • Rhodococcus equi is a soil isolate isolated from a 3-acetylamino-3-phenyi-propionic acid ethyl ester screening. The strain was determined at the DSMZ. Strain was deposited with the DSM under No. 19590.
  • the genomic DNA was obtained using a Qiagen kit:
  • the culture was centrifuged at 5000 xg and 22 ⁇ RNase A solution was added to an 11 ml aliquot of B1 buffer.
  • the cell pellet was resuspended with 11 ml each of RNase-containing Bl buffer.
  • 300 ml of lysozyme (100 mg / ml) and 500 ⁇ proteinase K stock solution (20 mg / ml) were added and for lysis of the cells at 37 ° C for 30 min. incubated.
  • a QIAGEN Genomic-tip 500 / G was equilibrated with 10 ml of QBT buffer. The clear lysate was added to the column and allowed to run through.
  • the column was washed twice with 15 ml of QC buffer.
  • the genomic DNA was eluted with 5 ml of QF buffer.
  • the chromosomal DNA could then be precipitated with isopropanol and transferred with a glass rod in TE buffer.
  • the amplified gene was cut with the restriction enzymes Ndel and HindIII and ligated into the multiple cloning site of the vector pDHE vector having a rhamnose-inducible promoter. This vector was expressed in TG 1 cells (DSMZ 6056).
  • This strain was fermented at 37 ° C in a minimal medium as a fed-batch.
  • the cells were used as biomass with a dry biomass of 150 g / l in the experiments.
  • the specific enzyme activity was 50 U / g dry biomass (BTM).
  • Inoculate FP medium with cells.
  • the cells are incubated at 28 ° C and 180 rpm.
  • the wild-type strain is induced with a solution of 1 g / l of 3-acetylamino-3-phenyl-propionic acid ethyl ester and incubated for a further 7 h.
  • the formation of the amine or the degradation of the amide is measured by HPLC.
  • the course samples are measured by chiral GC.
  • Fig. 1 shows the formation of 3-acetylamino-3-phenyl-propionic acid ethyl ester as a function of reaction time and temperature
  • Phase A 20 mM KH 2 P0 4 pH 2.5
  • Phase B acetonitrile
  • Rhodococcus erythropolis Rhodococcus erythropolis
  • FIG. 3 shows a comparison of the reaction with racemic or enantiomerically enriched substrate
  • SEQ ID NO: 4 This amidase can be determined by genetic engineering methods familiar to the skilled worker, for example by expression of the nucleic acid according to SEQ ID NO: 3 in a suitable host system, e.g. E. coli, manufacture.
  • Phase A 10 mM KH 2 PQ 4 pH 2.5
  • Phase B acetonitrile
  • Temp.Progr . 90 ° C, 15 ', 10 ° C, 10', 160 ° C, 15 '
  • Fig. 5 shows the course of the concentrations of 3-acetylamino-butyric acid methyl ester, 3-amino-butyric acid methyl ester, and a control without enzyme
  • LU8676 denotes the Rhodococcus erythropolls wild-type strain.

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Abstract

L'invention concerne un procédé de production de composés d'ester d'acide 3-aminocarboxilique optiquement actifs de la formule générale I, et ses sels d'ammonium. Dans la formule générale I, R1 représente alkyle, alcoxyalkyle, alcényle, cycloalkyle, hétérocycloalkyle, aryle ou hétaryle, et R2 représente alkyle, cycloalkyle ou aryle. Selon ce procédé, on soumet un mélange d'énantiomères d'un ester N-acylé simple d'acide 3-aminocarboxilique de la formule générale (l.b), dans laquelle R1 et R2 sont tels que définis précédemment et R3 représente un hydrogène, alkyle, cycloalkyle ou aryle, à une désacylation énantiosélective par apport d'un polypeptide conformément à la revendication 1.
EP10757596A 2009-09-25 2010-09-24 Amidase et son utilisation pour la production d'esters d'acide 3-aminocarboxilique Withdrawn EP2480664A1 (fr)

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PCT/EP2010/064098 WO2011036233A1 (fr) 2009-09-25 2010-09-24 Amidase et son utilisation pour la production d'esters d'acide 3-aminocarboxilique

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EP2438179B1 (fr) 2009-06-04 2016-12-21 Basf Se Procédé de réduction enzymatique d'énoates
US20130273619A1 (en) 2012-04-16 2013-10-17 Basf Se Process for the Preparation of (3E, 7E)-Homofarnesol
CN108070605B (zh) * 2018-01-05 2021-06-01 南京农业大学 多菌灵降解酶CbmA及其编码基因和应用

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DE3574704D1 (de) 1984-04-19 1990-01-18 Hoffmann La Roche Chirale rhodium-diphosphinkomplexe fuer asymmetrische hydrierungen.
DE4001019A1 (de) 1990-01-16 1991-07-18 Degussa Verfahren zur asymmetrischen hydrierung von (alpha)-ketocarbonylverbindungen zu optisch aktiven (alpha)-hydroxycarbonylverbindungen
EP1889903A1 (fr) 1996-04-25 2008-02-20 Novartis AG Biocatalysateurs possédant une activité aminé acylase
JP3899080B2 (ja) * 2004-03-03 2007-03-28 独立行政法人科学技術振興機構 新規ウレタナーゼ
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US20090299089A1 (en) 2006-07-06 2009-12-03 Basf Se Method for producing optically active 3-aminocarboxylic acid esters
JP5119783B2 (ja) * 2006-07-26 2013-01-16 味の素株式会社 Ｎ−アセチル−（Ｒ，Ｓ）−β−アミノ酸アシラーゼ遺伝子
DE602007005880D1 (de) * 2006-07-26 2010-05-27 Ajinomoto Kk N-Acetyl-(R,S)-B-Aminosäuren-Acylase-Gene
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CN102549151A (zh) 2012-07-04
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US8932837B2 (en) 2015-01-13
US20130005002A1 (en) 2013-01-03

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