US20130005002A1 - Amidase and use thereof for producing 3-aminocarboxylic acid esters - Google Patents

Amidase and use thereof for producing 3-aminocarboxylic acid esters Download PDF

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US20130005002A1
US20130005002A1 US13/497,985 US201013497985A US2013005002A1 US 20130005002 A1 US20130005002 A1 US 20130005002A1 US 201013497985 A US201013497985 A US 201013497985A US 2013005002 A1 US2013005002 A1 US 2013005002A1
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aminocarboxylic acid
alkyl
acid ester
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Bernhard Hauer
Thomas Friedrich
Rainer Stürmer
Nina Schneider
Susanne Krauser
Wolf-Rüdiger Krahnert
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BASF SE
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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 new amidase and use thereof for producing optically active 3-aminocarboxylic acid ester compounds, and derivatives thereof.
  • Asymmetric synthesis i.e. reactions in which a chiral group is produced from a prochiral group, so that the stereoisomeric products (enantiomers or diastereomers) are formed in unequal amounts, has become tremendously important chiefly in the pharmaceutical industry, as often only a particular optically active isomer is therapeutically active.
  • optically active intermediates of the active compounds are also becoming increasingly important. This also applies to 3-aminocarboxylic acid esters (Formula I), and derivatives thereof.
  • WO 97/41214 describes biocatalysts with aminoacylase activity, which do not have lipase or esterase activity.
  • WO 2008/003761 describes a process for producing optically active 3-aminocarboxylic acid esters in which an enantiomeric mixture of a simply N-acylated 3-aminocarboxylic acid ester, enriched in one enantiomer, is submitted, by adding an acidic salt-forming substance, to a deacylation and a subsequent further enantiomeric enrichment by crystallization.
  • the problem to be solved by the present invention is therefore to provide a simple and therefore economical process for producing optically active 3-aminocarboxylic acid esters and derivatives thereof.
  • R 1 and R 2 have the meanings given above and R 3 stands for hydrogen, alkyl, cycloalkyl or aryl, is submitted, by adding a polypeptide according to claim 1 or 2 , to an enantioselective deacylation.
  • the invention further relates to a process for producing optically active 3-aminocarboxylic acid ester compounds of general Formula I′, and derivatives thereof,
  • the invention further relates to a polypeptide with amidase activity, selected from
  • the invention further relates to a polypeptide with amidase activity, selected from
  • Chiral compounds are, in the context of the present invention, compounds with at least one chiral centre (i.e. at least one asymmetric atom, e.g. at least one asymmetric carbon atom or phosphorus atom), with chiral axis, chiral plane or helical shape.
  • chiral catalyst comprises catalysts that have at least one chiral ligand.
  • Achiral compounds are compounds that are not chiral.
  • Prochiral compound means a compound with at least one prochiral centre.
  • Asymmetric synthesis denotes a reaction in which, from a compound with at least one prochiral centre, a compound is produced with at least one chiral centre, a chiral axis, chiral plane or helical shape, wherein the stereoisomeric products form in unequal amounts.
  • Steps are compounds with the same constitution but with different atomic arrangement in three-dimensional space.
  • Enantiomers are stereoisomers that relate to one another as object to mirror image.
  • R and S are the descriptors of the CIP system for the two enantiomers and represent the absolute configuration on the asymmetric atom.
  • the process according to the invention leads to products that are enriched with respect to a particular stereoisomer.
  • the “enantiomeric excess” (ee) achieved is as a rule at least 95%, preferably at least 98% and especially preferably at least 99%.
  • “Diastereomers” are stereoisomers that are not enantiomeric to one another.
  • stereochemical concepts presented herein refer, unless expressly stated otherwise, to the carbon atom of the respective compounds corresponding to the asymmetric ⁇ -carbon atom in compound I or I′. If further stereocentres are present, they are ignored in the naming in the context of the present invention.
  • alkyl comprises linear and branched alkyl groups.
  • they are linear or branched C 1 -C 20 -alkyl, preferably C 1 -C 12 -alkyl, especially preferably C 1 -C 8 -alkyl and quite especially 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
  • alkyl also comprises substituted alkyl groups, which can generally carry 1, 2, 3, 4 or 5, preferably 1, 2 or 3 and especially preferably 1 substituents, selected from the groups cycloalkyl, aryl, hetaryl, halogen, COOR f , COO ⁇ M + and NE 1 E 2 , wherein R f stands for hydrogen, alkyl, cycloalkyl or aryl, M + stands for a cation equivalent and E 1 and E 2 , independently of one another, stand for hydrogen, alkyl, cycloalkyl or aryl.
  • alkoxyalkyl comprises linear and branched alkyl groups that are linked to an alkoxy residue.
  • the alkoxy residue can also be linear or branched.
  • they are linear or branched C 1 -C 20 -alkyl, preferably C 1 -C 12 -alkyl, especially preferably C 1 -C 8 -alkyl and quite especially preferably C 1 -C 6 -alkyl groups, which are linked C 1 -C 12 -alkoxy, especially preferably C 1 -C 6 -alkoxy residues.
  • alkyl groups are mentioned above; examples of alkoxy groups are in particular methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, sec.-butoxy. Examples of alkoxyalkyls are in particular methoxymethyl, ethoxymethyl, ethoxyethyl, ethoxypropyl.
  • alkenyl comprises linear and branched alkyl groups, which still bear at least one C ⁇ C double bond. Preferably they are linear C 1 -C 20 -alkyl groups, bearing a C ⁇ C double bond. Examples of alkenyl groups are in particular 1-propenyl, 1-butenyl, 1-pentenyl, 1-hexenyl.
  • cycloalkyl comprises, in the sense of the present invention, 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 can generally bear 1, 2, 3, 4 or 5, preferably 1, 2 or 3 and especially preferably 1 substituents, preferably selected from alkyl and the substituents mentioned for alkyl.
  • heterocycloalkyl comprises, in the sense of the present invention, saturated cycloaliphatic groups generally with 4 to 7, preferably 5 or 6 ring atoms, in which 1 or 2 of the ring carbon atoms are replaced with heteroatoms, preferably selected from the elements oxygen, nitrogen and sulphur, and which optionally can be substituted, wherein in the case of a substitution, these heterocycloaliphatic groups can bear 1, 2 or 3, preferably 1 or 2, especially preferably 1 substituents, selected from alkyl, cycloalkyl, aryl, COOR f , COO ⁇ M + and NE 1 E 2 , preferably alkyl, wherein R f stands for hydrogen, alkyl, cycloalkyl or aryl, M + stands for a cation equivalent and E 1 and E 2 independently of one another stand for hydrogen, alkyl, cycloalkyl or aryl.
  • heterocycloaliphatic groups we may mention pyrrolidinyl, piperidinyl, 2,2,6,6-tetramethylpiperidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, morpholidinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, piperazinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, dioxanyl.
  • aryl comprises, in the sense of the present invention, unsubstituted and substituted aryl groups, and preferably stands for phenyl, tolyl, xylyl, mesityl, naphthyl, fluorenyl, anthracenyl, phenanthrenyl or naphthacenyl, especially preferably for phenyl or naphthyl, wherein these aryl groups in the case of a substitution can generally bear 1, 2, 3, 4 or 5, preferably 1, 2 or 3 and especially preferably 1 substituents, selected from the groups alkyl, alkoxy, nitro, cyano or halogen.
  • 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, wherein these heterocycloaromatic groups can, in the case of a substitution, generally bear 1, 2 or 3 substituents, selected from the groups alkyl, alkoxy, acyl, carboxyl, carboxylate, —SO 3 H, sulphonate, NE 1 E 2 , alkylene-NE 1 E 2 or halogen, wherein E 1 and E 2 have the meanings given above.
  • acyl stands, in the sense of the present invention, for alkanoyl or aroyl groups generally with 2 to 11, preferably 2 to 8 carbon atoms, for example for the acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, 2-ethylhexanoyl, 2-propylheptanoyl, benzoyl, naphthoyl or trifluoroacetyl group.
  • Halogen stands for fluorine, chlorine, bromine and iodine, preferably for fluorine, chlorine and bromine.
  • M + stands for a cation equivalent, i.e. a monovalent cation or the unipositive component of the charge of a multiple cation. This includes e.g. Li, Na, K, Ca and Mg.
  • R 1 preferably stands for C 1 -C 6 -alkyl, 1-C 3 -C 6 -alkenyl, or C 6 -C 14 -aryl, which can optionally be substituted, as mentioned at the beginning.
  • R 1 stands for methyl, ethyl, n-propyl, isopropyl, n-butyl, tert.-butyl, 1-propenyl, 1-heptenyl, or phenyl, especially for methyl and phenyl.
  • R 2 preferably stands for unsubstituted or substituted C 1 -C 6 -alkyl, C 3 -C 7 -cycloalkyl or C 6 -C 14 -aryl.
  • R 2 residues are methyl, ethyl, n-propyl, isopropyl, n-butyl, tert.-butyl, trifluoromethyl, cyclohexyl, phenyl and benzyl.
  • R 2′ stands for hydrogen, M + , and for the meanings stated for R 2 .
  • R 3 stands for hydrogen, alkyl, cycloalkyl or aryl, in particular for hydrogen, methyl, ethyl, trifluoromethyl, benzyl and phenyl.
  • an enantiomeric mixture of compounds I.b is submitted, by adding an amidase, to an enantioselective deacylation and the resultant ammonium salt of a 3-aminocarboxylic acid ester, enriched with respect to a stereoisomer, is isolated.
  • the isomeric mixture of compounds of general Formula I.b used for the deacylation also comprises the corresponding enantiomer, or starting from chiral ⁇ -ketoesters also diastereomers in non-negligible amounts.
  • the process therefore makes possible the production of optically active compounds of general Formula I, starting from isomeric mixtures of compounds of general Formula I.b, such as are obtainable for example from the precursor compounds by usual asymmetric hydrogenation of enamides.
  • enantiomeric mixtures are used that comprise the enantiomers in the same molar ratio or else are already enriched in one enantiomer.
  • the ee value of these mixtures is preferably above 75% and especially preferably above 90%.
  • racemates or mixtures already enriched in one enantiomer are produced.
  • enantioselective hydrogenation processes are chosen, for example such as are mentioned in WO 2008/003761, whose description is expressly included here by reference.
  • the deacylation is preferably carried out at a temperature of 20-40° C., especially preferably between 20 and 30° C.
  • the reaction is usually carried out in an aqueous buffer.
  • the invention further relates to a process comprising the reaction stages a) to c) and optionally d) and e) described below.
  • stage a) of the process according to the invention a ⁇ -ketoester of Formula I.1 is reacted with at least one carboxylic acid amide of formula R 3 —C(O)NH 2 , in the presence of an amidation catalyst with removal of the reaction water, to a 3-aminocarboxylic acid ester of Formula I.a (step a.1).
  • the carboxylic acid amides of formula R 3 —C(O)NH 2 are acetamide, propionic acid amide, benzoic acid amide, formamide or trifluoroacetamide, in particular benzoic acid amide or acetamide.
  • Solvents suitable for step a.1 are those that form a low-boiling azeotrope with water, from which the reaction water can be removed by separation techniques (e.g. azeotropic distillation) known by a person skilled in the art.
  • they 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-toluenesulphonic acid, methanesulphonic acid, sulphuric acid or the like. p-Toluenesulphonic acid is preferably used.
  • the reaction in process step a.1 takes place at a temperature in the range from 20 to 110° C., especially preferably 60 to 90° C. Especially preferably, the temperature is above the boiling point of the solvent used under S.T.P.
  • Process step a.1 is usually carried out at a pressure from 0.01 to 1.5 bar, in particular 0.1 to 0.5 bar.
  • the aminocarboxylic acid ester obtained in step a.1 can be submitted to a purification by usual methods known by a person skilled in the art, e.g. by distillation.
  • a ⁇ -ketoester of Formula I.1 is reacted with aqueous ammonia and then with a carboxylic acid derivative of formula R 3 —C(O)X to the N-acylated, ⁇ -unsaturated (Z)-3-aminocarboxylic acid ester (I.a), in which X stands for halogen or a residue of 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 stands for chlorine and R 3 has the meaning given above, or carboxylic acid anhydrides, wherein X stands for OC(O)R 4 and R 4 preferably has the same meaning as R 3 , especially preferably the carboxylic acid derivatives are acetyl chloride, benzoyl chloride or acetic anhydride.
  • the acylation in step a.2 is carried out at a temperature in the range from 20° C. to 120° C., especially 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 nonpolar solvent, preferably the polar solvent is a carboxylic acid of formula R 3 COOH or a tertiary amine, haloalkanes and aromatics are suitable in particular as nonpolar solvent, especially preferably acetic acid or triethylamine is used as solvent.
  • the polar solvent is a carboxylic acid of formula R 3 COOH or a tertiary amine, haloalkanes and aromatics are suitable in particular as nonpolar solvent, especially preferably acetic acid or triethylamine is used as 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 solvent, non-nucleophilic bases are preferred, such as tertiary amines, especially preferably these are triethylamine and/or dimethylaminopyridine (DMAP).
  • a catalyst this can be used both in catalytic amounts and stoichiometrically or as solvent
  • non-nucleophilic bases are preferred, such as tertiary amines, especially preferably these are triethylamine and/or dimethylaminopyridine (DMAP).
  • the (Z)-3-aminocarboxylic acid ester will be obtained as a mixture with the (E)-3-aminocarboxylic acid ester and optionally further acylation products.
  • the (Z)-3-aminocarboxylic acid ester of Formula I.a will be isolated by methods known by a person skilled in the art. A preferred method is separation by distillation.
  • the ⁇ -unsaturated (Z)-3-aminocarboxylic acid ester compounds of Formula I.a obtained in stage a) can then be submitted to a hydrogenation, optionally an enantioselective hydrogenation, in the presence of an optionally chiral hydrogenation catalyst, obtaining a racemate or an enantiomeric mixture of simply N-acylated ⁇ -aminocarboxylic acid esters of general formula (I.b) enriched in one enantiomer.
  • At least one complex of a transition metal of groups 8 to 11 of the periodic table of the elements, which comprises at least one chiral, phosphorus atom-containing compound as ligand, is used as hydrogenation catalyst in stage b).
  • a chiral hydrogenation catalyst is used, which is capable of hydrogenating the ⁇ -unsaturated, N-acylated 3-aminocarboxylic acid ester (I.a) used preferentially for the desired isomer.
  • the compound of Formula I.b obtained in stage b) has, after the asymmetric hydrogenation, an ee value of at least 75%, especially preferably at least 90%.
  • an ee value of compound Lb is at least 75%.
  • the process according to the invention makes enantioselective hydrogenation possible at substrate/catalyst ratios (s/c) of at least 1000:1, especially preferably at least 5000:1 and in particular at least 15000:1.
  • a complex of a metal of group 8, 9 or 10 with at least one of the ligands stated hereunder is used for the asymmetric hydrogenation.
  • the transition metal is selected from Ru, Rh, Ir, Pd or Pt. Catalysts based on Rh and Ru are especially preferred. Rh catalysts are preferred in particular.
  • the phosphorus-containing compound used as ligand is preferably selected from bidentate and multidentate phosphine, phosphinite, phosphonite, phosphoramidite and phosphite compounds.
  • Catalysts are preferred for hydrogenation that have at least one ligand selected from the compounds of the following formulae,
  • Ar stands for optionally substituted phenyl, preferably for tolyl or xylyl.
  • P-chiral compounds such as DuanPhos, TangPhos or Binapine are preferred in particular.
  • Suitable chiral ligands coordinating to the transition metal via at least one phosphorus atom are known by a person skilled in the art and for example are commercially available from Chiral Quest ((Princeton) Inc., Monmouth Junction, N.J.). The nomenclature of the examples of chiral ligands given above corresponds to their commercial designation.
  • Chiral transition-metal complexes can be obtained in a manner known by a person skilled in the art (e.g. Uson, Inorg. Chim. Acta 73, 275 1983, EP-A-0 158 875, EP-A-437 690) by reaction of suitable ligands with complexes of the metals that comprise labile or hemilabile ligands.
  • X can be any anion known by a person skilled in the art, generally unstable in asymmetric synthesis.
  • Examples of X are halogens such as Cl ⁇ , Br ⁇ or I ⁇ , BF 4 ⁇ , ClO 4 ⁇ , SbF 6 ⁇ , PF 6 ⁇ , CF 3 SO 3 ⁇ , BAr 4 ⁇ .
  • BF 4 ⁇ , PF 6 ⁇ , CF 3 SO 3 ⁇ , SbF 6 ⁇ are preferred for X.
  • the chiral transition-metal complexes can either be produced in situ in the reaction vessel before the actual hydrogenation reaction or can be produced separately, isolated and then used. It may happen that at least one solvent molecule adds onto the transition-metal complex.
  • the common solvents e.g. methanol, diethyl ether, tetrahydrofuran (THF), dichloromethane, etc.
  • THF tetrahydrofuran
  • dichloromethane dichloromethane
  • the hydrogenation step (step b) of the process according to the invention is as a rule carried out at a temperature from ⁇ 10 to 150° C., preferably at 0 to 120° C. and especially preferably at 10 to 70° C.
  • the hydrogen pressure can be varied in a range between 0.1 bar and 600 bar. Preferably it is in a pressure range from 0.5 to 20 bar, especially preferably between 1 and 10 bar.
  • solvents for asymmetric hydrogenation are suitable as solvents for the hydrogenation reaction of the enamides I.a.
  • Preferred solvents are lower alkyl alcohols such as methanol, ethanol, isopropanol, and toluene, THF, ethyl acetate.
  • ethyl acetate or THF is used as solvent in the process according to the invention.
  • the hydrogenation catalysts (or hydrogenation precatalysts) described above can also be immobilized in a suitable way, e.g. by attachment via functional groups suitable as anchor groups, adsorption, grafting, etc., on a suitable support, e.g. of glass, silica gel, synthetic resins, polymer supports, etc. They are then also suitable for use as solid-phase catalysts.
  • catalyst consumption can be lowered further by these methods.
  • the catalysts described above are also suitable for a continuous reaction, e.g. after immobilization, as described above, in the form of solid-phase catalysts.
  • the hydrogenation in stage b is carried out continuously. Continuous hydrogenation can take place in one or preferably in several reaction zones.
  • Several reaction zones can be formed by several reactors or by spatially different regions within one reactor. When several reactors are used, they can be identical or different. They can in each case have identical or different mixing characteristics and/or can be subdivided once or more by internal fittings.
  • the reactors can be connected together in any way, e.g. in parallel or in series.
  • Suitable pressure-proof reactors for hydrogenation are known by a person skilled in the art. These include the generally usual reactors for gas-liquid reactions, for example tubular reactors, shell-and-tube reactors, stirred reactors, gas circulating reactors, bubble columns, etc., which can optionally be filled or subdivided by internal fittings.
  • process step c) the enantiomeric mixture of compounds I.b obtained in the hydrogenation is submitted to an enantioselective deacylation by adding a polypeptide with amidase activity and the resultant ammonium salt of a 3-aminocarboxylic acid ester, enriched with respect to a stereoisomer, is isolated.
  • the polypeptide with amidase activity can be used as purified enzyme, as partially purified raw extract or in the form of a living or killed microorganism, which contains the amidase.
  • Preferred amidases are those with the primary structure SEQ ID NO:2 or NO:4 or variants of SEQ ID NO:2 or NO:4, which are obtained by insertion, deletion or substitution of a few amino acids, preferably 1-20, especially preferably 1-10 amino acids.
  • the reaction usually takes place in aqueous buffer.
  • the resultant reaction product can be purified and isolated by usual methods.
  • the ammonium salts isolated in the enantiomer-enriching deacylation by amidase reaction can be submitted to further processing.
  • a suitable base preferably NaHCO 3 , NaOH, KOH.
  • the product of deacylation is dissolved or suspended in water and then the pH is adjusted by addition of base to about 8 to 12, preferably about 10.
  • a suitable organic solvent e.g.
  • an ether such as methyl butyl ether, a hydrocarbon or hydrocarbon mixture, e.g. an alkane, such as pentane, hexane, heptane, or an alkane mixture, naphtha or petroleum ether, or aromatics, such as toluene.
  • a preferred extractant is toluene.
  • 3-aminocarboxylic acid esters can be derivatized using methods known by a person skilled in the art. Possible derivatizations comprise for example saponification of the ester or stereoselective reduction of the carboxyl carbon atom to an optically active alcohol.
  • Derivatives of compounds of Formula I′ according to the invention therefore comprise 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.
  • the invention further relates to polypeptides that can catalyse an amidase reaction, and comprise the following primary structure (amino acid sequence):
  • polypeptide sequence that has at least 96%, preferably 98%, especially preferably 99% identity with SEQ ID NO:2.
  • polypeptide sequence that has at least 80%, preferably at least 85%, especially preferably at least 95% identity with SEQ ID NO:4.
  • R1 and R3 in each case stand for methyl and R2 stands for ethyl.
  • amidase with SEQ ID NO:2 can for example be isolated by cloning from Rhodococcus equi DSM 19590.
  • the coding region of the S-selective amidase from Rhodococcus equi was amplified by PCR with the following oligonucleotide primers:
  • Rhodococcus equi is a soil isolate, which was isolated from screening for 3-acetylamino-3-phenyl-propionic-acid ethyl esters. The strain was determined at the DSMZ. The strain was deposited at the DSM under No. 19590.
  • the genomic DNA was obtained by means of a Qiagen kit:
  • chromosomal DNA For isolation of chromosomal DNA from Rhodococcus equi, a bacterial culture was inoculated in 30 ml FP medium and incubated overnight at 30° C.
  • the culture was centrifuged at 5000 ⁇ g and 22 ⁇ l RNase A solution was added to an 11 ml aliquot of B1 buffer.
  • the cell pellet was resuspended in each case with 11 ml RNase-containing B1 buffer.
  • 300 ml of lysozyme stock solution (100 mg/ml) and 500 ⁇ l of proteinase-K stock solution (20 mg/ml) were added and, for lysis of the cells, incubated at 37° C. for 30 min. Meanwhile, a QIAGEN Genomic-tip 500/G was equilibrated with 10 ml QBT buffer. The clear lysate was applied to the column and allowed to pass through.
  • the amplified gene was cut with the restriction enzymes Ndel and HindIII and ligated into the multiple cloning site of the vector pDHE-vector, which possesses a rhamnose-inducible promoter. This vector was expressed in TG1 cells (DSMZ 6056).
  • This strain was fermented as fed-batch at 37° C. in a minimal medium.
  • the cells were used in the tests as bio-moist-matter with a bio-dry-matter of 150 g/l.
  • the specific enzyme activity was 50 U/g bio-dry-matter (BDM).
  • Inoculate FP medium with cells.
  • the cells are incubated at 28° C. and 180 rpm. After 20 h of growth, the wild type-strain is induced with a solution of 1 g/l 3-acetylamino-3-phenyl-propionic-acid ethyl ester and incubated for a further 7 h.
  • the cells are lysed and the raw extract is used in the activity test.
  • the formation of the amine or the degradation of the amide is measured by HPLC.
  • the 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
  • the concentration of 3-amino-3-phenyl-propionic acid ethyl ester reaches a maximum after about 24 hours. After that, the amine that formed is also degraded. The reactions at 40° C. go faster at the beginning, but collapse earlier than at 28° C.
  • Phase A 20 mM KH 2 PO 4 pH2.5
  • Test conditions 500 mM AAPPEE (rac./enriched)
  • FIG. 3 shows a comparison of the reaction with racemic or enantiomer-enriched substrate
  • FIG. 4 shows the reaction of enriched S-AAPEE
  • the wild-type strain Rhodococcus erythropolis was used as amidase (SEQ ID NO:4).
  • This amidase can be produced by genetic engineering methods that are familiar to a person skilled in the art, for example by expression of the nucleic acid according to SEQ ID NO:3 in a suitable host system, e.g. E. coli.
  • Phase A 10 mM KH 2 PO 4 pH2.5
  • Temp. progr. 90° C., 15 min, 10° C., 10 min, 160° C., 15 min
  • FIG. 6 shows the variation 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 erythropolis wild-type strain.

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