EP2480664A1

EP2480664A1 - Amidase and use thereof for producing 3-amino carboxylic acid esters

Info

Publication number: EP2480664A1
Application number: EP10757596A
Authority: EP
Inventors: Bernhard Hauer; Thomas Friedrich; Rainer STÜRMER; Nina Schneider; Susanne Krauser; Wolf-Rüdiger KRAHNERT
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2009-09-25
Filing date: 2010-09-24
Publication date: 2012-08-01
Also published as: WO2011036233A1; CN102549151A; US8932837B2; US20130005002A1; JP2013505710A; US20130337512A1

Abstract

The invention relates to a method for producing optically active 3-amino carboxylic acid ester compounds of general formula I, and the ammonium salts thereof, where R¹ is alkyl, alkoxyalkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, or hetaryl, and R² is alkyl, cycloalkyl or aryl, wherein an enantiomer mixture of a simple N-acylized 3-amino carboxylic acid ester of general formula (1.b), where R¹ and R² are as defined above and R³ is hydrogen, alkyl, cycloalkyl, or aryl, is subjected to an enantioselective deacylation by adding a polypeptide according to claim 1.

Description

AMIDASE AND 1HRE USE FOR THE PREPARATION OF 3-AMINOCARBOXIC ACID ESTERS

description

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.

The asymmetric synthesis, ie reactions in which a chiral moiety is generated from a prochiral moiety, so that the stereoisomeric products (enantiomers or diastereomers) are formed in unequal amounts, has gained immense importance, above all in the pharmaceutical industry often only a certain optically active isomer is therapeutically active. In this context, optically active intermediates of the active ingredients are becoming increasingly important. This also applies to 3-aminocarboxylic acid esters (formula I), and their derivatives.

(Formula I)

Thus, there is a great need for effective synthetic routes for the preparation of optically active compounds of the general formulas I.

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.

Surprisingly, it has now been found that the stated object is achieved by a process for the preparation of optically active 3-aminocarboxylic acid ester compounds of the general formula I, and also their ammonium salts,

wherein

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 Γ, and their derivatives,

wherein

R ^{2 is} hydrogen, a cation equivalent M ⁺ , alkyl, cycloalkyl or aryl, in which a) a β-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

a 2) with ammonia and then with a carboxylic acid derivative of the formula R ³ -C (O) X, where X is halogen or a radical of the formula OC (O) R ⁴ , where R ^{4 has} the meaning given above for R ³ , to give the corresponding N-acylated, a-unsaturated

(Z) -3-aminocarboxylic acid ester, the general formula (l.a) reacts,

in which R ¹ , R ² and R ^{3 have} the meanings indicated above, b) subjecting the enamide (Ia) obtained in this reaction to hydrogenation to give a mixture of enantiomers of N-acylated β-aminocarboxylic acid ester of the general formula (Ib),

in which 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

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

c) 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. The term "chiral catalyst" includes catalysts having at least one chiral ligand.

"Achiral connections" are compounds that are not chiral.

By a "prochiral compound" is meant 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.

"Stereoisomers" 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. The "enantiomeric excess" achieved in an asymmetric synthesis

(enantiomeric excess, ee) results from the following formula:

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 enantiomerically pure compound (ee = 100%) is also called "homochiral compound". 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.

Although other asymmetric atoms may be present in the compounds covered by the present invention, the stereochemical terms listed herein, unless expressly stated otherwise, refer to the carbon atom of the respective compounds corresponding to the asymmetric β-carbon atom in compound I or Γ. If further stereocenters are present, they are neglected in the context of the present invention in the designation.

In the following, the term "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. Examples of 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-methylpropyl, n-heptyl, 2-heptyl, 3-heptyl, 2-ethylpentyl, 1-propylbutyl, n-octyl, 2-ethylhexyl, 2-methylheptyl, nonyl, decyl, 2-propylheptyl.

The term "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.

The term "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. Examples of alkyl groups are mentioned above; Examples of alkoxy groups are in particular methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutene toxy, sec-butoxy. Examples of alkoxyalkyls are in particular methoxymethyl, ethoxymethyl, ethoxyethyl, ethoxypropyl.

The term "alkenyl" encompasses straight-chain and branched alkyl groups which still carry at least one C =C double bond. Preferably, these are straight-chain C ₁ -C 2o-alkyl groups which carry a C = C double bond. Examples of alkenyl groups are in particular 1-propenyl, 1-butenyl, 1-pentenyl, 1-hexenyl.

The term "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.

The term "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. Examples of such heterocycloaliphatic groups are pyrrolidinyl, piperidinyl, 2,2,6,6-tetramethylpiperidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, morpholidinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, piperazinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, dioxanyl called.

The term "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.

The term "hetaryl" for the purposes of the present invention comprises unsubstituted or substituted 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.

The above explanations for the terms "alkyl", "cycloalkyl", "aryl", "heterocycloalkyl" and "hetaryl" apply correspondingly to the terms "alkoxy", "cycloalkoxy", "aryloxy", "heterocycloalkoxy" and "hetaryloxy".

The term "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.

As described above, 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. In particular, 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.

According to the invention, 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. It is a characteristic feature of 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 β-ketoesters, also diastereomers are present in non-negligible amounts. Advantageously, 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.

Usually, in this process step enantiomeric mixtures are used 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%. Depending on the choice of hydrogenation of the enamide (Ia), racemates or mixtures already enriched in one enantiomer are produced. In order to obtain mixtures which are already enriched in one enantiomer, one generally selects enantioselective hydrogenation processes, for example those as mentioned in WO 2008/003761, the description of which is hereby expressly referred to.

Preferably, 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).

Stage a)

In one embodiment of step a) of the process according to the invention, a β-keto ester of the formula I.1 having at least one carboxamide of the formula

R ³ -C (O) NH 2, reacted in the presence of an amidation catalyst with removal of the water of reaction to a 3-amino carboxylic acid ester of the formula Ia (step a.1).

Preferably, in 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). In particular, these are aromatics, such as toluene, benzene, etc., ketones such as methyl isobutyl ketone or methyl ethyl ketone, etc., and haloalkanes such as chloroform. Preferably, 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.

In an alternative embodiment, a β-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, β-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 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).

Optionally, in steps a.1 and a.2, the (Z) -3-aminocarboxylic acid ester is obtained as a mixture with the (E) -3-aminocarboxylic acid ester and optionally further acylation products. In this case, 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.

Stage b)

The α-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.

Preferably, in 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.

For the hydrogenation, preference is given to using a chiral hydrogenation catalyst which is capable of hydrogenating the α-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. However, such high enantiomeric purity is often not necessary in the process according to the invention since, according to the process of the invention, a further enantiomeric enrichment takes place in the subsequent deacylation step. However, 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.

For the asymmetric hydrogenation, preference is given to using a complex of a group 8, 9 or 10 metal with at least one of the ligands mentioned below. Preferably, the transition metal is selected from Ru, Rh, Ir, Pd or Pt. Especially preferred are 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.

For hydrogenation, preference is given to catalysts which have at least one ligand selected from compounds of the following formulas,

Tang Pho Pho Phane Phos

TrichickenfootPhos MiniPhos or their enantiomers, wherein Ar is optionally substituted phenyl, preferably tolyl or xylyl.

Particularly preferred are bidentate compounds of the aforementioned classes of compounds. In particular, 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. Here, as precatalysts complexes such as

Pd ₂ (dibenzylideneacetone) ₃ , Pd (OAc) ₂ (Ac = acetyl), RhCl ₃ , Rh (OAc) ₃ , [Rh (COD) Cl] ₂ ,

[Rh (COD) OH] ₂ , [Rh (COD) OMe] ₂ (Me = methyl), Rh (COD) acac, Rh ₄ (CO) i ₂ , Rh ₆ (CO) i ₆ ,

[Rh (COD) ₂ )] X, Rh (acac) (CO) ₂ (acac = acetylacetonato), RuCl ₃ , Ru (acac) ₃ , RuCl ₂ (COD), Ru (COD) (methallyl) ₂ , Ru ( Ar) l ₂ and Ru (Ar) Cl ₂ , Ar = aryl, both unsubstituted and substituted, [Ir (COD) Cl] ₂ , [Ir (COD) ₂ ] X, Ni (allyl) X can be used. Instead of COD (= 1, 5-cyclooctadiene), NBD (= norbornadiene) can also be used. Preferred are

[Rh (COD) Cl] ₂ , [Rh (COD) ₂ )] X, Rh (acac) (CO) ₂ , RuCl ₂ (COD), Ru (COD) (methallyl) ₂ , Ru (Ar) Cl ₂ , Ar = aryl, both unsubstituted and substituted, and the corresponding systems with NBD instead of COD. Particularly preferred are [Rh (COD) ₂ )] X and [Rh (NBD) ₂ )] X.

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.) for the complex preparation are known in the art.

Phosphine, phosphinite, phosphonite, phosphoramidite and phosphite metal or metal-LM complexes (LM = solvent) with at least one labile or hemilabile ligand are suitable precatalysts from which the actual hydrogenation under the conditions of hydrogenation Catalyst is generated.

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. In the process according to the invention, 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. Advantageously, 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.

In a further embodiment, 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.

Step c)

In 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.

Step d)

If desired, the ammonium salts isolated in the enantiomerically enriching deacylation by amidase reaction may be subjected to further work-up. For example, to release the optically active compound of the formula I, it is possible to bring the product of the crystallization into contact with a suitable base, preferably NaHCO 3, NaOH, KOH. In a suitable procedure, 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. To isolate the free 3-aminocarboxylic acid ester, it is possible to add the basic solution or suspension with a suitable organic solvent, e.g. Example, an ether such as methyl butyl ether, a hydrocarbon or hydrocarbon mixture, for. As an 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. In this procedure, the 3-amino acid ester can be obtained almost quantitatively, whereby the ee value is retained.

Steps) Optionally, 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 Γ 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):

SEQ ID NO: 2

or a polypeptide sequence having at least 96%, preferably 98%, most preferably 99% identity with SEQ ID NO: 2.

SEQ ID NO: 4

or a polypeptide sequence having at least 80%, preferably at least 85%, most preferably at least 95% identity with SEQ ID NO: 4.

As amidase reaction in the context of this invention, the following model reaction is understood:

wherein R1 and R3 are each methyl and R2 is ethyl.

The following reaction conditions were chosen:

200 μΙ cells

50 μM 1 M KH ₂ P0 ₄ buffer pH 7.0

1 -10 g / L substrate racemic or S-enantiomer enriched

740 μΙ H ₂ 0.

Cultivation of the cells see example 2.

The amidase with the SEQ ID NO: 2 can be, for example, Rhodococcus eq

Isolate by cloning in 19590.

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:

The sequence obtained in this way was, in order to introduce the corresponding cloning sites, amplified in a further PCR with the following primers: 5 '-GGGATACTCATATGAGTACATCGGATCCGGG-3'

3'-GAGTCTCAAGCTTACGCCACCGGTCGACGATCC-5 '

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:

To isolate chromosomal DNA from Rhodococcus equi, a bacterial culture was inoculated into 30 ml of FP medium and transferred to N.N. incubated at 30 * C.

The culture was centrifuged at 5000 xg and 22 μΙ 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. Subsequently, 300 ml of lysozyme (100 mg / ml) and 500 μΙ proteinase K stock solution (20 mg / ml) were added and for lysis of the cells at 37 ° C for 30 min. incubated. Meanwhile, 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. Subsequently, the column was washed twice with 15 ml of QC buffer. Finally, 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).

Example 2: Preparation of 3-amino-3-phenyl-propionic acid ethyl ester with a wild-type strain Rhodococcus equi

a) Preparation of the cells:

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 of 3-acetylamino-3-phenyl-propionic acid ethyl ester and incubated for a further 7 h.

The cells are disrupted and the crude extract used in the activity test. b) Reaction of 3-acetylamino-3-phenyl-propionic acid ethyl ester:

To a buffer (100 mM KH ₂ P0 ₄ pH 7) are 1 g / l of ethyl 3-acetylamino-3-phenylpropionate (AAPEE) and x μΙ (see Table) cell-free crude extract (see Table 1) overnight at 28 ° C or 40 ° C incubated.

The formation of the amine or the degradation of the amide is measured by HPLC. For the determination of the enantioselectivity the course samples are measured by chiral GC.

Approach:

Tab. 1 Approaches for the reaction of ethyl 3-acetylamino-3-phenylpropionate Results:

Fig. 1 shows the formation of 3-acetylamino-3-phenyl-propionic acid ethyl ester as a function of reaction time and temperature

As can be seen in Figure 2, the concentration of ethyl 3-amino-3-phenylpropionate reaches a maximum after about 24 hours. Thereafter, the resulting amine is also degraded. Reactions at 40 ° C are faster at the start, but break earlier than at 28 ° C.

Analytics: achiral HPLC

Column: Onyx Monolith C18, 50 ^* 4.6 mm, Fa. Phenomenex

Mob. Phase A: 20 mM KH ₂ P0 ₄ pH 2.5

Mob. Phase B: acetonitrile

Flow: 1, 5ml / min

Oven temp .: 45 ° C

Inj. Vol .: 2μΙ

Gradient: 0.0 min 20% B

0.5 min. 20% B

0.6 min 80% B

1.2 min 80% B

1.3 min 20% B

2.0 min 20% B

Detection: UV 210 nm

Retention time: educt 1, 49 min

Product 0.74 min

Chiral GC:

Solvent: acetonitrile

Derivatization ~ 100μΙ solution

+ 300μΙ TFAA (trifluoroacetic anhydride)

~ Leave for 30 minutes at 100 ° C GC conditions

Column 25m Lipodex G 0,25mm Inside 0,25μιη FD

Oven program 80/10/2/180/10/700

Injection 1 -5 μΙ depending on concentration at 250 ° C

Detector FID at 250 ° C

Carrier gas helium 16,7 PSI, flow 1, 6 ml / min, split 100: 1

Comparison: implementation with racemic vs. enriched substrate

Testbed: 500 mM AAPPEE (rac. / Enriched)

100 mM KH ₂ P0 ₄ pH 7.0

25 g / l (dry biomass) cells from fermenter discharge (cloned enzyme from

Rhodococcus erythropolis)

30 ° C

FIG. 3 shows a comparison of the reaction with racemic or enantiomerically enriched substrate

Up to 20 g / l of methyl 3-acetylamino-3-phenylpropionate (AAPEE) were reacted. Substituting racemic substrate, one obtains an enrichment of the S enantiomer (ee ~ 94%). On the other hand, if enriched substrate (ee ~ 80%) is used, one can obtain> 99% ee.

Preparative 4-l approach

4I approach:

130 mM AAPPEE

100 mM KH ₂ P0 ₄ pH 7.0

34 g / L BTM cells (cloned enzyme from Rhododcoccus erythropolis)

30 ° C, 5 h

Work-up:

Adjust the reactor to pH 3.0 with 60 ml / 102 g H ₃ P0 (85%). The weight was 4113 g / 4150 ml. The mixture was centrifuged off (5000 g, 20 min) and the pellet was washed with 200 ml. One got a clear, slightly yellow

Supernatant (weight 3804 g).

This was extracted with 3 × 1400 ml of 2-butanol in order to first separate off any starting material still present and by-products from the reactant synthesis. The mixture was then adjusted to pH 10 at 10 ° C. with 20% NaOH and the amino acid ester was isolated by extraction with 1500 ml of 2-butanol and subsequent removal of the solvent in vacuo. Obtained were 23.0 g of almost enantiomerically pure (99.3% ee) amino esters as a pale yellow oil. The chemical purity is> 98% (GC). Fig. 4 shows the conversion of enriched S-AAPEE

Example 3: Preparation of methyl 3-aminobutyric acid

As amidase, the wild-type strain Rhodococcus erythropolis was used

(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.

Procedure analogous to Example 2.

Analytics: achiral HPLC

Column: Luna C8 (2), 150 ^* 3.0 mm, Fa. Phenomenex

Mob. Phase A: 10 mM KH ₂ PQ ₄ pH 2.5

Mob. Phase B: acetonitrile

Flow: 1, 0 ml / min

Oven temperature: 40 ° C

Inj. Vol .:

Gradient: 0% B

7.0 min 30% B

10 min 30% B

1.2 min 80% B

1.3 min 20% B

2.0 min 20% B

Detection: UV 200 nm

Retention time: educt 4.35 min

Product 1, 13 min chiral GC

Column: Hydrodex-β-6-TBDM, 25 * 0.25 mm, film thickness 16pm M & N

Temp.Progr .: 90 ° C, 15 ', 10 ° C, 10', 160 ° C, 15 '

Detector: FID

Retention time: starting material enant. 1 21, 46 min

(only starting material) Approach:

Tab. 2 Approaches for the reaction of 3-acetylamino-Buttersäurernethylester

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.

Claims

Claims 1. Polypeptide having amidase activity selected from

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% identity with SEQ ID NO: 2.

2. Polypeptide with amidase activity selected from

a) polypeptide containing an amino acid sequence according to SEQ ID NO: 4, and b) polypeptide containing an amino acid sequence which has at least 80% identity with SEQ ID NO: 4.

3. Process for the preparation of optically active 3-aminocarboxylic ester compounds of the general formula I, and also their ammonium salts,

wherein

R ^{2 is} alkyl, cycloalkyl or aryl, in which an enantiomer mixture of a singly N-acylated 3-aminocarboxylic acid ester of the general formula (Ib),

wherein R ¹ and R ^{2 have} the meanings given above and R ^{3 is} hydrogen, alkyl, cycloalkyl or aryl, by adding a polypeptide according to claim 1 or 2 of an enantioselective deacylation.

4. A process for preparing optically active 3-aminocarboxylic ester compounds of the general formula I ', and derivatives thereof,

wherein

R ^{2 is} hydrogen, a cation equivalent M ⁺ , alkyl, cycloalkyl or aryl, in which a) a β-keto ester of the general formula 1.1 wherein R ¹ and R ^{2 have} the meanings given above, a 1) having at least one carboxamide of the formula R ³ -C (O) NH 2, wherein R ^{3 has} the abovementioned meaning, in the presence of an amidation tion catalyst, or

a 2) with ammonia and then with a carboxylic acid derivative of the formula R ³ -C (O) X, where X is halogen or a radical of the formula OC (O) R ⁴ , where R ^{4 has} the meaning given above for R ³ , to give the corresponding N-acylated, α - ß -unsaturated

(Z) -3-aminocarboxylic acid ester, the general formula (l.a) reacts,

in which R ¹ , R ² and R ^{3 have} the meanings given above, b) subjecting the enamide (Ia) obtained in this reaction to hydrogenation to give an enantiomeric mixture of N-acylated β-aminocarboxylic acid ester of the general formula (Ib), in which 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 enantioselective deacylation by addition of a polypeptide according to claim 1 and the ammonium salt of a stereoisomer enriched therewith Isolated 3-aminocarboxylic acid ester, and d) optionally converting the isolated ammonium salt into the 3-aminocarboxylic acid ester, and e) optionally the 3-aminocarboxylic acid ester in the free

3-aminocarboxylic acid or a salt thereof.

5. The method of claim 3, wherein a ß-ketoester of formula 1.1 with at least one carboxamide of the formula R ³ -C (0) NH 2, in the presence of an amidation catalyst under removal of the water of reaction to a 3-aminocarboxylic acid ester of the formula la is reacted ,

6. The method according to any one of the preceding claims, wherein the deacylation is carried out in aqueous buffer as the reaction medium.

7. The process according to claim 4, wherein the hydrogenation b) is carried out in the presence of a hydrogenation catalyst which comprises at least one transition metal complex of groups 8 to 11 of the Periodic Table of the Elements and comprises as ligand at least one chiral phosphorus atom-containing compound.

8. The method according to claim 3 or 4, wherein R ^{1 is} phenyl and R ² and R ^{3 have} the meanings mentioned in claim 2.