CZ283836B6 - Process of stereoselective synthesis of amine one chiral form - Google Patents

Abstract

Stereoselective synthesis of one chiral form of an amine of formula (IA) or (IB) in amt. greater than the other, where R1, R2 = different alkyl or aryl opt. substd. with an enzymatically non-inhibiting gp.; comprises contacting a ketone of formula R1-CO-R2 (II) with an omega-amino acid transaminase in the presence of an amine donor at least until a substantial amt. of one of the chiral amines is formed. Process for enantiomeric enrichment of a mixt. of enantiomeric chiral amines (IA) and (IB) comprises contacting the mixt., in aq. medium in presence of an amino acceptor, with an omega-amino acid transaminase which is enzymatically active w.r.t. the NH2 gp. of one of the chiral amines, at least until a substantial amt. of one of the amines is converted to ketone (II). (II) may be isolated from the medium and converted to chiral amine as above; and/or the chiral amine which is not converted to (II) may be recovered.

Description

Method for stereoselective synthesis of one chiral form of amine

Technical field

The invention relates to a process for the stereoselective synthesis of chiral amines.

BACKGROUND OF THE INVENTION

The biological activity of chemical compounds, such as pharmaceutical and agricultural products that contain a center of chirality, is often the predominant one of the possible chiral forms. Since most chemical syntheses are not inherently stereoselective, this phenomenon poses a serious problem in terms of chemical production. At some stage of the production, either after obtaining the resulting chiral compounds, or after preparing their chemical precursors with the same chiral center, it is necessary to enrich the product in favor of one chiral form. Whichever step of the reaction is chosen for enrichment, this process is inherently limited in that it can achieve a maximum theoretical yield of 50% of the desired enantiomer (unless a method for recycling the undesired enantiomer is available).

Many of the chiral compounds of this type are amines. In addition, due to the versatility of their reactions, they are good candidates for enantiomer resolution after which stereoselective conversion to chiral compounds can be performed. The chemical production of chiral amines not containing the second enantiomer has hitherto relied mainly on the cleavage of a mixture of the two chiral forms by the formation of diasteromeric derivatives such as salts with a chiral acid, stereoselective synthetic procedures and the use of chiral chromatography columns (see for example US Patent No. 3,944,608 and EP). A 36 265).

Some structural types of amines can be resolved enzymatically into enantiomers. Enzymatic reactions involving α-amino acids are well known and their use has been suggested for stereospecific preparations. For example, U.S. Patent No. 3,871,958 describes the enzymatic preparation of α-amino acid derivatives of serine by coupling an aldehyde with glycine in the presence of E. coli-derived threoninalolase and the related synthesis of seronil using ethanolamine.

Relatively little has been reported on enzymatic reactions of amino acids in which the amino group is not in the vicinal position to the carboxylic acid group. Yonaha et al., Agric. Biol. Chem. 42, (12). 2363-2367 (1978) discloses an omega-amino acid: pyruvate transaminase from Pseudomonas species for which puryvate is the exclusive aminoacceptor. This enzyme, which was recently produced in crystalline form and characterized (see Yonaha et al., Agric. Biol. Chem., 41 (9), 1701-1706 (1977)), had a low substrate specificity for omega-amino acids, such as hypothaurin, 3-aminopropanesulfonate, β-alanine, 4-aminobutyrate and 8-aminoctanoate, and catalyzed transamination between primary aminoalkanes and pyruvate.

Nakano et al., J. Biochem., 81, 1375-1381 (1977) identified two omega-amino acid transaminases in B. cereus: β-alanine transaminase, which corresponds to Yonah's omega-amino acid: pyruvate transaminase and -aminobutyrate transaminase. The two transaminases can be distinguished by their distinctly different activity towards β-alanine (100: 3) -aminobutyrate (43: 100) and their different requirements for aminoacceptors.

Bumett et al., J.C. Chem. Comm., 1979, 826-828, reported that omega-amino acid: pyruvate transaminase and -aminobutyrate transaminase show different preferences for the two terminal hydrogen atoms in tritium-labeled -aminobutyrate.

- 1 GB 283836 B6

Tanizawa et al., Biochem. 21, 1104-1108 (1982) investigated bacterial L-lysine-e-aminotransferase and L-omithine-d-aminotransferase and reported that although both of these enzymes are specific for L-amino acids, they act distally and with the same stereospecificity as - aminobutyrate transaminase studied by Bumett et al., supra.

Yonaha et al., Agric. Biol. Chem., 47 (10), 2257-2265 (1983) additionally characterized omega-amino acid pyruvate transaminase and -aminobutyrate transaminase (EC 2.6.1.18 and EC 2.6.1.19) and documented their distribution in various organisms.

Waters et al., FEMS Micro, Lett., 34 (1986) 279-282, in a report on the complete catabolism of βalanine and β-amino isobutyrate by β-aeruginose, reported that the first step involves transamination of β-alanine: pyruvate aminotransferase.

Enzymatic methods have hitherto been considered as methods for separating mixtures of non-amino acid chiral amines such as 2-aminobutanol. Most of these methods involve derivatization, especially the amino group, and use of the protected group or other group in the molecule for self-separation. For example, EP-A 222561 discloses a process in which racemic

The 2-aminobutanol is converted to the N-carbamoyl derivative, which is then contacted with an alkyl alkanoate in the presence of a lipase enzyme. The esterification of the free hydroxy group is apparently limited to the Senantiomer of the N-carbamoyl derivative, which is then hydrolyzed. This procedure is of course limited to amines bearing an esterifiable hydroxy group and, moreover, specifically requires prior protection of the amino group by forming a carbamoyl (-NH-CO-) group in order to achieve stereospecificity in the enzymatic reaction.

EP-A 239 122 describes a similar process applicable to a broader class of 2-amino-1-alkanols.

Japanese Kokai JP 55-138 389 discloses the preparation of primary amino alcohols by treating an alkyl or aralkyl substituted ethyleneimine with microorganisms of the genus Bacillus, Proteus, Erwinia or Klebsiella.

Japanese Patent Publication Kokai JP 58-198 296 discloses a process in which d, 1-N-acyl-2-aminobutanol is treated with an aminoacylase derived from various Asperigillus, Penicillium and Streptomyces species which hydrolyzes only d-N-acyl-2-aminobutanol.

Japanese Patent Publication Kokai JP 59-39,294 describes a process for resolving racemic 2-aminobutanol by converting it to an N-acetylderivative treated with Micrococcus acylase to give 1-2-aminobutanol and dN-acetyl-2-aminobutanol, wherein dN Acetyl-2-aminobutanol is chemically hydrolyzed to d-2-aminobutanol.

Japanese Patent Publication Kokai JP 63-237796 discloses a process in which R, S-1-methyl-

3-Phenylpropylamine is processed under aerobic conditions by culturing specific microorganisms. The S-form is preferably metabolized. The highest yields and optical purity are obtained using Candida humicola and Trichosporon melibiosaceum. The enzymatic nature of the S-form metabolism that occurs in these aerobic cultures, for example oxidase, dehydrogenase, ammonia lysase, etc., is not disclosed.

The abstract of Japanese Kokai Patent Publication JP 63-273486 discloses microbial synthesis of 1- (4-methoxyphenyl) -2-aminopropane with R-configuration at one of the two chiral centers of 1- (4-methoxyphenyl) -2-propane using Sarcino lutea.

SUMMARY OF THE INVENTION

In its broadest sense, the invention encompasses the use of an omega-amino acid transaminase in the presence of an aminodonor for the stereoselective synthesis of chiral amines in which the amino group is bound to a non-terminal chiral substituted carbon atom. The invention is therefore based on the discovery that the omega-amino acid transaminase acts stereoselectively on non-omega amino groups and that this action can be used for stereoselective synthesis of a chiral amine in only one configuration.

The term omega-amino acid transaminases means any enzymes capable of converting the terminal group of the formula -CH ₂ -NH ₂ omega-amino acids to the group of the formula -CH = O and vice versa.

The enzymatic equilibrium reaction employed in the process of the invention can be illustrated as follows:

omega-amino acid II II-C-R ² + transminase nh ₂ | amino-> donor <- 1 R'-CH-R ² + amino acceptor

where each of the symbols

R ¹ and R ² individually represent an alkyl or aryl group optionally substituted by one or more enzymatically non-inhibitory groups, and R ¹ differs from R 1 in either structure or chirality;

R ¹ and R ² together represent a hydrocarbon chain of 4 or more carbon atoms containing a chiral center.

The term "aminodonor" refers to the various amino compounds described in more detail below, which are capable of providing the amino group of the ketone shown, thereby becoming carbonyl compounds by the action of an omega-amino acid transaminase.

The term "amino acceptor" as used herein refers to various carbonyl compounds described in more detail below, which are capable of accepting the amino group of the depicted amine also by treatment with the same omega-amino acid transaminase.

In particular, the enzymatic reactions shown above are characterized in that the amino group in the primary amine obtained is not in the terminal (omega) position and that it need not be an amino acid.

The present invention provides a process for the stereoselective synthesis of one chiral form of an amine of formula IA or IB nh ₂

Ί · 2> R β

H nh _{2 E} ² 6m. C-AR ¹

H (ΙΑ) (IB) where

R ¹ and R ² are alkyl or aryl groups optionally substituted with an enzymatically non-inhibitory group, wherein R ¹ differs from R ^{2 in} structure or chirality in an amount substantially greater than the amount of the second chiral form, characterized in that the ketone of formula II

O

II (Π)

R ¹ - C - R ² wherein R ¹ and R ² have the same meaning as for the amine to be prepared; contacting the omega-amino acid with a transaminase in the presence of an aminodonor at least until a substantial amount of one of said chiral amines is formed.

The invention is based on the discovery that an omega-amino acid transaminase converts a non-chiral ketone (at least with respect to the carbonyl carbon atom) essentially to only a single chiral form of the amine.

As used herein, the term "enantiomeric enrichment" refers to increasing the amount of one enantiomer relative to the amount of the other enantiomer. In enantiomeric enrichment, 1) the amount of one chiral form may be reduced compared to the other, 2) the amount of one chiral form may be increased compared to the other, or 3) the amount of one chiral form may decrease and the amount of the other chiral form may be increased. An effective term for expressing enantiomeric enrichment is the term enantiomer excess (ee), which is defined by

E'-E ² ee = ---------- x 100

E ¹ + E ² where

E ¹ represents the amount of the first chiral form of the amine a

E ² represents the amount of the second chiral form of the same amine.

Thus, if the initial ratio of the two chiral forms is 50:50 and an enantiomeric enrichment giving a final ratio of 50:30 is achieved, the value of the excess enantiomer ee relative to the first chiral form is 25%, while when the final ratio of enantiomer is 70:30. ee relative to the first chiral form 40%. Usually, ee values of 90% or higher can be achieved using the process of the invention.

The term "substantially higher" as used in this specification to refer to the amount of one chiral form of an amine relative to the amount of the other chiral form of an amine in the stereoselective synthesis of that amine is an amount expressed by at least about 3: 1 corresponding to an ee of at least about 50%.

The chiral amines of formula IA and IB prepared by the process of the invention have several structural limitations. First, the amino group contained therein is primary, but must be bound to a secondary carbon atom, i.e. the carbon atom carrying one hydrogen atom and two substituents which are other than hydrogen (R ¹ and R ^2). Secondly, R ¹ and R ² are chosen from structures of the same type, but these groups must confer chirality on the molecule, ie R ¹ necessarily must be different from R ² in structure or chirality, or R ¹ and R ² must together represent a chiral group . When R ¹ and R ^{2 are} independent, they are usually alkyl, aralkyl or aryl groups, preferably straight or branched chain alkyl groups having 1 to 6 carbon atoms, straight or branched chain phenylalkyl groups having 7 to 12 carbon atoms, or phenyl or naphthyl. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, tert-butyl, isobutyl, sec-butyl, phenyl, benzyl, phenethyl, 1-phenethyl, 2- phenylpropyl, etc. In addition, since the enzymatic reaction of the invention extends to the shown amino group and the adjacent carbon atom, each of R ¹ and R ² may be optionally substituted by one or more groups, provided that they are not enzyme inhibiting groups, ie. groups that would significantly affect or compete with the transaminase effect when the chiral amines or ketones they carry are used at practical concentrations. This can be readily determined by a simple inhibition assay. When inhibition is detected, it can often be minimized by carrying out the reaction at low reagent concentrations. Typical substituents include, but are not limited to, halogens such as chloro, fluoro, bromo and iodo, hydroxy, lower alkyl, lower alkoxy, lower alkylthio, cycloalkyl, carbamoyl, mono- and di- (loweralkyl) substituted carbamoyl, trifluoromethyl-, phenyl-, nitro-, amino, and di- (lower alkyl) substituted amino, alkylsulfonyl-, arylsulfonyl-, alkylcarboxamido-, arylcarboxamido, etc.

Typical groups R @ ¹ and R @ ² taken together include methylbutene-1,4-diyl, pentane-1,4-diyl, hexane-1,4-diyl, hexane-1,5-diyl and 2. -methyl-pentane-1,5-diyl.

Non-limiting examples of typical amines which are suitable for the process of the present invention include 2-aminobutane, 2-amino-1-butanol, 1-amino-1-phenylethane, 1-amino-1- (2-methoxy-5-fluorophenyl) amine. ethane, 2-amino-1-phenylpropane, 1-amino-1- (4-hydroxyphenyl) propane, 1-amino-1- (4-bromophenyl) propane, 1-amino-1- (4- nitrophenyl) propane, 1-phenyl-2-aminopropane,

1- (3-Trifluoromethylphenyl) -2-aminopropane, 2-aminopropanol, 1-amino-1-phenylbutene, 1-phenyl-

2-aminobutene, 1- (2,5-dimethoxy-4-methylphenyl) -2-aminobutane, 1-phenyl-3-aminobutane, 1- (4-hydroxyphenyl) -3-aminobutane, 1-amino-2-methylcyclopentane, 1-amino-3-methylcyclopentane, 1-amino-2-methylcyclohexane and 1-amino-1- (2-naphthyl) ethane.

As an example of the process of the invention, acetophenone is treated with a transaminase in the presence of an aminodonor to form S-1-amino-1-phenylethane which does not contain R-1-amino-1-phenylethane or contains only a very small amount.

Aminoacceptors are ketocarboxylic acids, alkanones or substances from which these compounds are formed in situ. Typical examples of ketocarboxylic acids include α-ketocarboxylic acids such as glyoxalic acid, pyruvic acid, oxallacetic acid and the like, and salts thereof. A typical alkanone is butan-2-one.

Other substances which can be converted to aminoacceptors by other methods using enzymes or whole cells may also be used. As examples of substances that can be converted to these

Aminoacceptors include fumaric acid (which rapidly converts in situ to oxaloacetic acid), glucose (which converts to pyruvate), lactate, maleic acid, etc.

Aminodonors are amines including the non-chiral amino acid glycine and chiral amino acids having the S-configuration such as L-alanine or L-aspartic acid. It is also possible to use amines, both chiral and non-chiral, which are not amino acids, such as S-2 aminobutane, propylamine, benzylamine, etc.

The omega-amino acid transaminases useful in the methods of the invention are known 10 pyridoxal phosphate-dependent enzymes that are present in various microorganisms such as

Pseudomonas, Escherichia, Bacillus, Saccharomyces, Hansenula, Candida, Streptomyces, Aspergillus, and Neurospora. Two omega-amino acid transaminases that are particularly useful in the methods of the invention, EC 2.6.1.18 and EC 2.6.1.19, were prepared in a crystalline state and characterized by Yonaha et al., Agric. Biol. Chem., 47 (10), 2257-2265 (1983).

Microorganisms having the desired activity can be readily isolated by means of a chemostat culture, i.e. by cultivating in a constant but limited chemical environment with an aminoacceptor and an amine as the sole nitrogen source. The amine may be, but need not be, a chiral amine, since in the normal environment the omega-amino acid transaminases metabolize the primary 20 amines. Among the non-chiral amines which have been successfully used for the formation of omega-amino acid transaminases are n-octylamine, cyclohexylamine, 1,4-butanediemine, 1,6-hexanediamine, 6-aminohexanoic acid, 4-aminobutyric acid, tyramine and benzylamine . Chiral amines such as 2-aminobutane, α-phenethylamine and 2-amino-4-phenylbutane as well as amino acids such as L-lysine, L-omithine, β-alanine and taurine have also been used successfully.

Such procedures enrich the culture with microorganisms producing the desired omega-amino acid transminase. For example, one such chemostat culture using random soil samples having no particular history of amine exposure was performed for approximately one month. The dominant microorganism was then independently identified in the American Type Culture Collection Bacillus megaterium, which did not differ significantly from the known strains and was similar in phenotype.

The organisms thus isolated can be grown in various ways. First, a standard salt medium supplemented with phosphate buffer, sodium acetate, 35 carbon source, 2-ketoglutarate such as aminoacceptor, and a nitrogen containing compound such as npropylamine, n-octylamine, 2-aminobutane, 2-aminoheptane, cyclohexylamine, 1, 6-hexanediamine putrescine, 6-aminohexanoic acid, 4-aminobutyric acid, L-lysine, L-omithine, β-alanine, α-phenethylamine, 1-phenyl-3-aminobutane, benzylamine, tyramine, taurine, etc.

Alternatively, the microorganism may be grown using an amine as the sole carbon source, thereby limiting the growth to those organisms that are capable of catabolizing the amine to obtain carbon.

Third, the microorganism can be grown in an environment containing sodium succinate, sodium acetate or any other carbon source and ammonium salt or protein hydrolyzate, as the main nitrogen source to which it is then added, either at the beginning of or during growth, an amine such as 2-aminobutane, 1-phenyl-3-aminobutane, α-phenethylamine, etc., to induce the production of the desired transaminase activity.

True enzymatic conversion can be accomplished by conventional culture procedures in the presence of a ketone, using isolated but non-growing cells, or by contacting the ketone with a soluble omega-amino acid transaminase preparation.

-6GB 283836 B6

The omega-amino acid transaminase may be in free form, either as a cell-free extract or a whole cell preparation as described above, or immobilized on a suitable carrier or matrix such as cross-linked dextran or agarose, silica, polyamide or cellulose. It may also be encapsulated in polyacrylamide, alginates, fibers, and the like. Immobilization methods are described in the literature (see, for example, Mathods of Enzymology, 44, 1976). Methods using an immobilized enzyme are preferred, since after immobilizing the enzyme, it is sufficient to pass the aminodonor and the ketone through the enzyme to achieve the desired synthesis and to remove the chiral amine formed as described above.

While not necessarily necessary, it is often advantageous to increase the conversion rate by adding a pyridoxamine source such as pyridoxal phosphate to the reaction mixture.

The procedures and materials used are further illustrated by typical examples.

Enzyme activity: Enzyme activity is expressed in units / mg. An enzyme activity unit is defined as an activity that is capable of producing 1 μπιοί ketone per minute. To achieve uniformity, activity is measured as the number of pmoles of 1-phenylbutan-3-one made from R, S-1-phenyl-3-aminobutane. The following standardized assay is used to measure the omega-amino acid activity of the transaminases shown in the following examples.

A known volume of the enzyme preparation to be tested is incubated at 37 ° C and pH 7 in a solution of the following composition:

sodium pyruvate 100 mM

R, S-1-phenyl-3-aminobutane 30 mM pyridoxal phosphate 0.5 mM

Take a sample and add 20% of its volume to 12% aqueous trichloroacetic acid. The precipitated protein is collected by centrifugation and the concentration of 1-phenylbutan-3-one in the supernatant is determined by liquid chromatography on a 100x8 mm 4 µm Novopak phenyl column. Elution is carried out with 40% isopropanol and 0.09% phosphoric acid in water. Under these conditions, 1-phenylbuten-3-one eluted in 5.3 minutes.

Amine purity: The purity of the amines produced is determined by gas chromatography on a 1.83 x 2 mm chromium Q column of 10% SE-30 on a 100/120 mesh (125-150 µm) support at 210 ° C at a carrier gas flow rate of 10 mL / min.

Determination of enantiomeric enrichment: The ee of a given product is determined by reaction with (-) and (trifluoromethylphenyl) methoxyacetyl chloride (see Gal, J. Pharm. Sci., 66, 169 (1977)) and Mosher et al., J. Org. Chem. 25430 (1969) followed by capillary gas chromatography of the derivatized product on a fused silica Chrompack column.

Standard salt medium: A suitable salt medium for microbial transformations described in the following examples has the following composition:

MgSO ₄

CaCl ₂

ZnSO ₄ .7H ₂ O

MnSO ₄ .4H ₂ O

H ₃ BO ₃

CuSO ₄ .5H ₂ O

CoCl ₂ .6H ₂ O

NiCl ₂ .6H ₂ O

1.00 g / l

0.21 g / l

0.20 mg / l

0.10 mg / l

0.02 mg / l

0.10 mg / l

0.05 mg / l

0.01 mg / l

FeSO ₄

NaMoO ₄ Fe EDTA KH ₂ PO ₄

Naoh

1.50 mg / l 2.00 mg / l 5.00 mg / l 20.00 mM to pH 7

The composition of the salt medium is not critical, but has been standardized to eliminate it as a variable value.

Microorganisms: Cultures were either obtained from a labeled collection or isolated as described and then independently identified.

Enrichment of omega-amino acid transaminase producing microorganisms: A chemostat with 5 g / l R, S-2-aminobutane and 10 mM 2-ketoglutarate is maintained in a standard salt medium at a dilution rate of 0.03 / h. The chemostat is inoculated and kept in operation for about 1 month at 37 ° C and pH 6.8 to 7.0. The strains that develop are isolated and grown on minimal agar containing salt medium supplemented with 10 mM 2-ketoglutarate and 5 mM R, S-1-phenyl-3-aminobutane.

Enzyme Isolation: Unless otherwise noted, cells from the culture are centrifuged at 10,000 G for 10 minutes, resuspended in 10 mM phosphate buffer pH 7 and 0.5 mM pyridoxal phosphate, and crushed with two passages cooled by a French press operating at 103 MPa. The crushed cells are harvested by centrifugation at 10,000 G for 1 hour and the enzyme-containing supernatant is stored.

The invention is illustrated by the following examples. The examples are illustrative only and do not limit the scope of the invention in any way. Only the definition of the subject matter of the invention is decisive for the scope of the invention.

Example 1

The growth of omega-amino acid transaminase producing microorganisms using aminodonor as the sole nitrogen source is illustrated by the following example.

Bacillus megaterium was grown in a 3L shake flask (rotational speed of 200 min ^-1) for 17 hours at 30 ° C using 1 L of the above salt solution, 60 mM sodium acetate, 30 mM phosphate buffer, 30 mM 2-Metoglutarátu disodium and 100 mM n-propylamine as a nitrogen source. When the culture reaches a density of 0.6 g dry matter per liter of cell, the cells are separated and the enzyme isolated from them as described above. The specific activity of the omega-amino acid transaminase thus obtained is greater than 0.49 units / mg.

The Bacillus megaterium strain used in the previous procedure was obtained from soil samples with no particular history in terms of amine exposure, seeding of the chemostat described above and isolation of dominant organisms (those capable of growing on R, S-1-phenyl-3-aminobutane). The strain was independently identified in the American Type Culture Collection as Bacillus megaterium, which does not differ significantly from the known ATCC strain 14581 and which is phenotypically similar to ATCC 49097B.

Example 2

This example illustrates the growth of omega-amino acid transaminase producing microorganisms using aminodonor as the sole carbon source.

-8EN 283836 B6

Pseudomonas aeruginosa ATCC 15692 was grown to β-alanine as the sole carbon source, as described by Way et al. In FEMS Micro. Lett., 34, 279 (1986). Cell extracts containing the omega-amino acid transaminase are obtained as described above. When tested as described above, the specific activity of the omega amino acid transaminase was found to be 0.040 units / mg.

Example 3

Pseudomonas putida ATCC 39213 was cultured as described in Example 1, and the extract was extracted from the cells. The specific activity of the omega-amino acid transaminase is 0.045 units / mg.

Example 4

This example demonstrates the need for an aminoacceptor.

The enzyme extracts of Pseudomonas putida, Bacillus megaterium and Pseudomonas aeruginosa obtained above were assayed at pH9 in 50 mM Tris / HCl using 30 mM R, S-1-phenyl-3-aminobutane either in the presence or absence of 100 mM sodium pyruvate. The following relative conversion rates are achieved.

relative conversion rate

P. putida B. megaterium P. aeruginosa in the presence of pyruvate 100 100 100 in the absence of pyruvate 0 0 0

The transaminase nature of the enzymatic effect is evident in the causes of "suicide inactivators" known to be specific for transaminases (see, for example, Bumett et al., J. Bio. Chem. 225, 428-432, 1980). The inactivator (0.5 mM) is preincubated with assay medium before addition of R, S-1-phenyl-3-aminobutane.

relative conversion rate

Inactivator P. putida B. megaterium P. aeruginosa none 100 ALIGN! 100 ALIGN! 100 ALIGN! gabakulin 0 13 0 hydroxylamine 3 10 0

The stereoselectivity of the omega-amino acid transaminase is apparent from the results of the following assay using 15 mM R-1-phenyl-3-aminobutane (with pyruvate).

relative conversion rate P. putida B. megaterium of P. aeruginosa R, S-1-phenyl-3-aminobutane 100 ALIGN! 100 100 R-1-phenyl-3-aminobutane 3 15 4

Example 5

This example illustrates the growth of microorganisms using ammonium as the sole nitrogen source, wherein induction of omega-amino acid transaminase production is accomplished by the addition of an amine.

-9EN 283836 B6

Bacillus megaterium was grown in 1 liter cultures in standard salt medium supplemented with 40 mM carbon source listed in the following table, 5 mM ammonium chloride, 80 mM phosphate buffer and 2 mM amine as inducer listed in the following table. After 30 to 40 hours, the enzyme was isolated and assayed as described above.

specific activity (units / mg) carbon source succinate acetate gluconate glucose

R, S-1-phenyl-1-aminoethanol 0.27 0.39 m.p. m.p. R-1-Pheny1-1-aminoethane 0.27 0.36 m.p. m.p. R, S-1-phenyl-3-aminobutane 0.28 0.33 0.26 0.62 R-1-phenyl-3-aminobutane 0.21 0.26 m.p. m.p. R, S-2-amobutane 0.13 0.14 m.p. m.p. R-2-aminobutane 0.06 0.13 m.p. m.p. tyramine m.p. 0.24 m.p. m.p.

m.p. = not tested

Example 6

The following example illustrates the growth of microorganisms using a protein rich source and the subsequent induction of omega-amino acid transaminase production by addition of an amine.

Bacillus megaterium was grown in a 121 L fermenter at pH 7 and 30 ° C and the above salt medium supplemented with 10 g / l casamino acids. The contents of the fermenter are mixed and aerated. Sodium acetate was added sequentially to a final concentration of 120 mM.

At this point, the cell density is 3 g dry matter per liter. 1-Phenyl-3-aminobutane is added up to a total concentration of 10 mM. After 12 hours, the enzyme was separated and assayed as described above. Its specific activity is 0.49 units / mg.

Example 7

This example illustrates a typical synthesis of a chiral amine.

A soluble enzyme preparation is prepared from Bacillus megaterium by the method described in Example 1. Its specific activity as determined above is 0.58 units / mg. To 200 ml of an aqueous solution of 350 mg of this preparation, 0.4 mM pyridoxal phosphate and 40 mM sodium phosphate were added 4.2 mM 1-phenylbutan-3-one and 100 ml 2-aminobutane as aminodonor. The mixture was incubated at pH 7 and 30 ° C for 4 hours, after which time R1-phenyl- (3-aminobutane at 3.35 mM, corresponding to 80% conversion) was present in the reaction mixture. ml of 10N sodium hydroxide and extraction of an alkaline aqueous solution with 250 ml of n-heptane, evaporation of the heptane extracts gave 100.5 g of product, which was analyzed by the above derivatization, containing 96.4% S1-phenyl-3-aminobutane.

Similarly, S-1-phenyl-2-aminopropane was prepared from 1-phenylpropan-2-one at ee 96.4 in a yield of 94.8%. S-1-amino-1-phenylethane was prepared from acetophenone at ee 100 in a yield of 44%.

Claims (8)

PATENT CLAIMS A process for the stereoselective synthesis of one chiral form of an amine of formula IA or IB nk ₂ n · 2 Fz • H • • H (IA), (IB), where R ¹ and R ² are alkyl or aryl groups optionally substituted by an enzymatically non-inhibitory group, wherein R ¹ differs from R ^{2 in} structure or chirality in an amount substantially greater than the amount of the second chiral form, characterized in that the ketone of formula II O II (Π), R ¹ - C - R ² wherein R ¹ and R ² have the same meaning as the prepared amine are contacted with the omega-amino acid transaminase in the presence of an aminodonor at least until a substantial amount of one of said chiral amines is formed. The process according to claim 1, wherein the aminodonor is 2-aminobutane, glycine, alanine or aspartic acid. A process according to claim 1, wherein a ketone of formula II is used, wherein each of R ¹ and R ² independently represents a straight or branched (C 1 -C 6) alkyl group, a straight or branched (C 7 -C 12) phenylalkyl group. carbon or phenyl or naphthyl, each of which is unsubstituted or substituted by an enzymatically non-inhibitory group. A process according to claim 1, wherein a ketone of formula II is used, wherein each of R ¹ and R ^{2 is} independently methyl, ethyl, η-propyl, isopropyl, η-butyl, isobutyl. -, sec-butyl, phenyl, benzyl or phenethyl. The method of claim 1, wherein the ketone and the aminodonor are contacted with whole cells of an omega-amino acid transaminase producing transaminase. 6. The method of claim 1, wherein the ketone and the amidonor are contacted with the cell-free aqueous omega-amino acid transaminase. 7. The method of claim 1, wherein the ketone and the aminodonor are contacted with a carrier-immobilized omega-amino acid by a transaminase. -11EN 283836 B6 The process according to claim 1, wherein a large molar excess of the aminodonor is used.