SK280218B6 - Method of enantiomeric enrichment and stereoselective synthesis of chiral amines - Google Patents

Abstract

Amines in which the amino group is on a secondary carbon atom which is chirally substituted by R1 and R2 residuals, in which each of R1 and R2 is an alkyl or aryl group which is unsubstituted or substituted with an enzymatically non-inhibiting group and R1 is different from R2 in structure or chirality, said amines can be enantiomerically enriched by the action of an omega-amino acid transaminase which has the property of preferentially converting one of the two chiral forms to a ketone. The transformation is carried out in the presence of an amino acceptor. Since the reaction is in an equilibrium, it can be reversed, whilst in this case it can be used for the stereoselective synthesis of one chiral form of an amine in the presence of depicted transaminase to a ketone in the presence of an amino donor.

Description

Technical field

The invention relates to a process for enriching a mixture of two enantiomeric chiral amines with one of these enantiomers, as well as a process for the stereoselective synthesis of chiral amines.

BACKGROUND OF THE INVENTION

The carrier of the biological activity of chemical compounds, such as pharmaceutical and agricultural products that contain a chirality center, is often predominantly 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 carry out product enrichment 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 cleavage into enantiomers after which stereoselective conversion to chiral compounds can be performed. Up to now, the chemical production of chiral amines not containing the second enantiomer has relied mainly on the cleavage of a mixture of 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 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. Pat. No. 3,871,958 describes the enzymatic preparation of serine α-amino acid derivatives by coupling of aldehyde with glycine in the presence of E. coli-derived threonaldolase 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) disclose an omega-amino acid: pyruvate transaminase from Pseudomonas species for which pyruvate 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 low substrate specificity for omega-amino acids such as is hypothaurin, 3-aminopropanesulfonate, R-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 the Yonah omega-amino acid pyruvate transaminase and -aminobutyrate transaminase. The two transaminases can be distinguished by significantly different activity against B

-alanine (100: 3) -aminobutyrate (43: 100) and based on their different aminoacceptor requirements.

Burnett et al., J. C. Chem. Comm., 1979, 826-828, reported that the omega-amino acid: pyruvate transaminase and -aminobutyrate tramsaminase have different preferences over the two terminal hydrogen atoms in the third-labeled -aminobutyrate.

Tanizawa et al., Biochem. 21, 1104-1108 (1982) investigated bacterial L-lysine-e-aminotransferase and L-omitine-β-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 Bumette and others.

Yonaha et al., Agric. Biol. Chem., 47 (10), 2257-2265 (10g83) additionally characterized the omega-amino acid: pyruvate transaminase and -aminobutyrate transaminase (EC 2.6.1.18 and EC 2.6.1.19) and documented their distribution in different organisms.

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

Enzymatic methods are heretofore considered as methods for separating non-amino acid mixtures of chiral amines such as 2-aminobutanol. Most of these methods involve derivatization, especially of the amino group, and utilization of this protected group or other group in the molecule for self-separation. For example, EP-A 222 561 describes a process in which racemic 2-aminobutanol is converted to an 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 S-enantiomer of the H-carbamoyl derivative, which is then hydrolyzed. This process 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 to achieve stereospecificity in the enzymatic reaction.

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

Japanese Kokai JP 55-138 389 describes the preparation of vicinal 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 wherein d, 1 N-acyl-2-aminobutanol is treated with an aminoacylase derived from various Aspergillus, Penicillium and Streptomyces species, which hydrolyzes only d-N-acyl-2-aminobutanol.

Japanese Patent Publication Kokai 59-39,294 describes a process for resolution of 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 63-237 796 describes a process in which R, S-1-methyl-3-phenylpropylamine is treated under aerobic conditions by culturing specific microorganisms. The S-form is preferably metabolized. The highest yields and optical purity are obtained with Candida humicola and Trichosporon melibiosaceum. The enzymatic nature of S-form metabolism that occurs in these aerobic cultures, for example, oxidase, dehydrogenase, ammonia lysase, and the like, is not disclosed.

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

SUMMARY OF THE INVENTION

In the broadest sense, the invention encompasses the use of an omega amino acid transaminase in the presence of an amino acceptor for enantiomerically enriching a mixture of chiral amines with one of the enantiomers or for the stereoselective synthesis of chiral amines in which the amino group is bound to a nonterminal chiral substituted carbon. Thus, the invention is based on the discovery that omega amino acid transaminases act stereoselectively on non-omega amino groups and that this action can be used to enrich a mixture of chiral amines with one enantiomer and also to stereoselectively synthesize a chiral amine in only one configuration.

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

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

NHg omega-amino acid O | amino transaminase II

R ^X -CH- R ² + acceptor -----------------> R ¹ -c- r ² + amino 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 ² either in structure or chirality, or

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

The term amino acceptor as used herein refers to the various carbonyl compounds described in more detail below, which are capable of receiving the amino group of the amine shown by the action of an omega-amino acid transaminase. The term aminodonor is understood to mean the various amino compounds described in more detail below, which are capable of providing the amino group represented by the ketone, thereby becoming carbonyl compounds, also by treatment with the same omega-amino acid transaminase.

The enzymatic reaction shown above is characterized in particular by the omega-amino acid transaminase acting on the primary amine in which the amino group is not in the terminal (omega) position. Second, the transaminase acts on an amine, which may not be an amino acid. Third, the amine product consumed in the enzymatic transformation is not irreversibly metabolized, but can be stereoselectively converted again to a starting amine with uniform chirality.

In a first embodiment, the process of the invention relates to the enantiomeric enrichment of a mixture of chiral amines of formula (IA) and (IB) nh ₂ NH ₂

R ¹ * ·! * »? ² and R ² t »Č-« R ¹

HH (IA) (IB) wherein R ¹ and R ² are as defined herein, the action of an omega-amino acid transaminase in the presence of an amino acceptor. As can be seen, compounds of formula (IA) and (IB) are enantiomers (or diastereomers when either R ¹ or R ² contains a second chiral center) and are chiral due to the fact that R ¹ differs in structure or chirality from R 1 ² .

According to a second embodiment, the process of the invention relates to a process for the stereoselective synthesis of one chiral form of an amine of formula (IA) or (IB) in an amount substantially greater than that of the other chiral form wherein the ketone of formula (11),

R ¹ - C - R ² wherein R ¹ and R ² are as defined herein, are treated with an omega-amino acid transaminase in the presence of an aminodonor.

Both of these embodiments are based on the discovery that the omega-amino acid transaminase is not limited to omega-amino groups in its action and, moreover, is substantially or exclusively stereoselective with regard to amines belonging to a defined class and converts only one chiral form of amine to the corresponding. a ketone that is no longer chiral (at least with respect to the carbonyl carbon atom) and then converts the ketone to only one chiral form of the amine.

As used herein, enantiomeric enrichment refers to increasing the amount of one enantiomer relative to the amount of the other enantiomer. Enantiomeric enrichment may result in 1) a decrease in the amount of one chiral form compared to the other, 2) an increase in the amount of one chiral form compared to the other 3) a decrease in the amount of one chiral form and an increase in the amount of the other chiral form. An effective term for expressing enantiomeric enrichment is the concept of excess enantiomer (ee), which is defined by the equation ee = --------- x 100

E ¹ + E ² where

E ¹ represents the amount of the first chiral form of the amine and E ² represents the amount of the second chiral form of the same amine.

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

Substantially higher than is used herein to refer to the amount of one chiral form of the amine relative to the amount of the other chiral form of the amine in the stereoselective synthesis of the amine is understood to be an amount of at least about 3: 1 of the two chiral forms. at least about 50%.

The chiral amines of formula (IA) and (IB) used in the methods of the invention have several structural limitations. Firstly, the amino group contained therein is primary, but must be bound to a secondary carbon atom, i.e. a carbon atom. to a carbon bearing one hydrogen atom and two substituents which are other than hydrogen ^(R1 and ^R2). Secondly, R ¹ and R ² are selected from structures of the same type, but these groups must impart chirality to the molecule, i. R ¹ must necessarily be different from R ² in structure or chirality or R ¹ and R ² together must represent a chiral group. When R ^{@ 1} and R ^{@ 2 are} independent, they are generally alkyl, aralkyl or aryl groups, preferably straight or branched alkyl groups having 1 to 6 carbon atoms, straight or branched chain alkyl groups having 7 to 12 carbon atoms, or phenyl or naphthyl. Examples of such groups include methyl, ethyl, η-propyl, isopropyl, n-butyl, isobutyl, sec. butyl-, phenyl-, benzyl-, phenethyl-, 1-phenethyl-, 2-phenylpropyl, and the like. In addition, since the enzymatic reactions of the invention reach the depicted amino group and adjacent carbon atom, each of R ¹ and R ² may be optionally substituted with one or more groups, provided that they are not enzyme inhibiting groups, i. groups that would significantly affect or compete with the transaminase activity when the chiral amines or ketones bearing these groups are used in 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 chlorine, fluorine, bromine and iodine, hydroxy, lower alkyl, lower alkoxy, lower alkylthio, cycloalkyl, carbamoyl, mono- and di- di- (lower alkyl) substituted carbamoyl, trifluoromethyl-, phenyl-, nitro-, amino, and di- (lower alkyl) substituted amino, alkylsulfonyl-, arylsulfonyl-, alkylcarboxamido-, arylcarboxamido and the like.

Typical groups R ¹ and R ^{2 taken} together include methylbutane-1,4-diyl-, pentane-1,4-diyl-, hexane-1,4-diyl-, hexane-1,5-diyl- and 2-methylpentane-1,5-diyl.

Non-limiting examples of typical amines suitable for the process of the invention include 2-aminobutane, 2-amino-1-butanol, 1-amino-1-phenylethane, 1-amino-1- (2-methoxy-5- fluorophenyl) ethane, 1-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-phenylbutane, 1-phenyl-2-aminobutane, 1- (2,5- dimethoxy-4-methyl-phenyl) -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.

The method of the first embodiment of the invention in the broadest sense comprises a process wherein a mixture of chiral amines is treated with an omega-amino acid transaminase which is enzymatically active (with respect to the known amino group of at least one of said chiral amines) in the presence of an aminoacceptor.

NKj KHj

H (IA) (IB)

BQinO acceptor otnege-atuino acid transexclusive

0 NH ₂ R ^X -CR ² ' * R ^{3 -} C ⁴ R ⁴ H (II) (III}

wherein R ¹ and R ² are as defined in formula (III), R ^{3 is} either R ¹ while R ⁴ is R ² or R ³ is R ² while R ⁴ is R ¹ .

The enzymatic process usually proceeds with only one chiral form, or with a much larger extent than one with the chiral form. For example, in the case of R, S-amino-l-phenylethane (R ¹ = phenyl, R ² = methyl), only the S-form is converted to the corresponding nonchiral ketone, acetophenone, wherein R-1-amino-1-phenylethane unchanged . Similarly, the R, S-1-amino-l- (4-bromophenyl) ethane (R ¹ = 4-bromophenyl, R ² = methyl), the S-form is converted to the non-chiral ketone 4-bromoacetophenone, while R The amino-1- (4-bromophenyl) ethane remains unchanged. With R, S1-phenyl-3-aminobutane (R ¹ = phenethyl, R ² -methyl), the S-form is readily converted to the non-chiral 1-phenylbutan-3-one, while the R-form of 1-phenyl-3-aminobutane is converted to 1-phenylbutan-3-one in a range 0.05 times or less relative to the S-form.

In some cases, it is possible to assign chiral amines to the R- and S-configuration to identify which is converted to a ketone and which is not. However, the designation of the R- and S-configurations is carried out according to the Cahn-Ingold-Prelog method and depends on the pre-assigned values for R ¹ and R ² as determined by the sequence of substituents (sequence rule). Consequently, it is not always possible with a chiral amine, to which the enzyme acts, select a priori by the configuration of the chirality of R and S. The allocation of R and S configuration of the chiral amine of formula (III) will therefore be made conditional upon the primacy of the substituents R ^3, and R ⁴ according to the sequential rule, but the configuration of the chiral amine of formula III will be identical to one and only one of the enantiomers (IA) and (IB). For example, as mentioned above, the S-form of 1-amino-1-phenylethane is converted to the non-chiral ketone, acetophenone, leaving the R enantiomer unchanged. In the case of R, S-1-amino-1-phenyl-2-hydroxyethane (phenylglycinol), the enantiomer is converted with the same absolute configuration as 1-amino-1-phenylethane, but due to the substitution sequence rule, this isomer marked with the letter R.

Since the reaction is equilibrium, it can proceed in both directions. Stimulation of the reaction in the desired direction can be accomplished by adding additional starting materials or by removing the reaction products. For example, when a mixture of two chiral forms of amine is to be enriched with one of the enantiomers, an additional amount of amine acceptor (until saturation) may be added and / or the ketone formed may be continuously removed from the reaction mixture. When, on the other hand, one chiral form of an amine is stereoselectively synthesized, another ketone may be added (until saturation) and / or the amine formed may be removed.

When the undesired chiral amine form is converted to a ketone and the desired chiral form is not, the desired chiral form can be readily isolated by conventional procedures.

Partial separation can be carried out by acidifying the mixture, extracting with a hydrocarbon such as heptane to

The aqueous phase is rendered alkaline and re-extracted with a hydrocarbon such as heptane.

The by-products that are isolated are often valuable commodities themselves. For example, when the process is carried out to enantiomerically enrich a mixture of R-2-aminobutane and S-2-aminobutane (R ¹ = ethyl, R ² = methyl) with the R-chiral form, the S-chiral form is converted to methyl ethyl ketone, is itself a useful organic solvent.

On the other hand, when both forms of the amine are desired, the form that is converted to the ketone can be removed from the reaction mixture (or the aqueous phase in the biphasic mixture) and independently subjected to an omega-amino acid transaminase in the presence of an aminodonor to form the same chiral form. , which was originally converted to ketone. For example, starting from a mixture of R, S-amino-l-phenylethane (R ¹ = phenyl, R ² = methyl), the S-form is converted by the omega-amino acid transaminase to the corresponding nonchiral ketone, acetophenone, the R-amino- i-phenylethane remains unchanged. R1-amino-1-phenylethane is readily isolated from the reaction mixture as described, and the acetophenone byproduct is then treated with a transaminase in the presence of an aminodonor to produce S1-amino-1-phenylethane in a substantially higher percentage compared to the R-form.

The second embodiment of the invention may be carried out independently of the first. Stereoselective synthesis of one of the chiral polys of amines of general formula (IA) and (IB)

ΝΉ ₂ nh ₂ and R ^2b 'R - ** R ¹ H H

(IA) (13) in an amount substantially higher than the amount of the second chiral form may be carried out by treating the ketone of formula (II) with a

II

R ¹ - C - R ^{2 (II)} , wherein R ¹ and R ² are as defined above, treats the omega-amino acid with a transaminase in the presence of an aminodonor until a substantial amount of one of the chiral forms of the amine is formed. For example, when the above example is used, acetophenone can be treated with a transaminase in the presence of an aminodonor to produce S1-amino-1-phenylethane which does not contain R1-amino-1-phenylethane, or which 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 that can be converted to aminoacceptors by other methods using enzymes or whole cells may also be used. Examples of substances that can be converted to these aminoacceptors include fumaric acid (which is rapidly converted in situ to oxaleacetic acid), glucose (which is converted to pyruvate), lactate, maleic acid, and the like,

Aminodonors are amines comprising the non-chiral amino acid glycine and chiral amino acids having S

a configuration such as L-alanine or L-aspartic acid. Amines, both chiral and non-chiral such as S-2-aminobutane, propylamine, benzylamine, and the like can also be used.

Omega-amino acid transaminases useful in the methods of the invention are known pyridoxal phosphate-dependent enzymes, which are present in various microorganisms such as Pseudorrronas, 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. (See Agric. Biol. Chem., 47 (10), 2257). 2265 (1983).

Microorganisms having the desired activity can be readily isolated by means of a chemostat culture, i. cultivation in a constant but limited chemical environment with an aminoacceptor and an amine as the sole nitrogen source. The amine may be, but does not have to be, a chiral arine, since in the normal environment omega-amino acid transaminases metabolize primary amines. Among the non-chiral amines that have been successfully used to form omega amino acid transaminases are n-octylamine, cyclohexylamine, 1,4-butanediamine, 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, Z-omitine, β-alanine and taurine have also been used successfully.

By such methods, the culture is enriched for microorganisms producing the desired omega-amino acid transaminase. For example, one such chemostat culture using random soil samples that had no particular history of amine exposure was performed for about one month. As a dominant microorganism, it was then independently identified in the American Type Culture Collection Bacillus megaterium, which did not differ significantly from known strains and was similar to them in terms of phenotype.

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

Alternatively, the microorganism can be grown using an amine as the sole carbon source, thereby limiting the growth to those organisms capable of catalyzing the amine for carbon recovery.

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 onset of growth or during growth, with amine. such as 2-aminobutane, 1-phenyl 1-3-aminobutane, α-phenethylamine and the like, to induce the production of the desired transaminase activity.

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

The omega-amino acid transaminase may be in free form, either as a cell-free extract or said whole cell formulation, or in a form 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. Methods of immobilization are described in the literature (see, for example, Methods of Enzymology, 44, 1976). Methods using immobilized enzyme are preferred because, after immobilizing the enzyme, it is sufficient to pass the amino acceptor and the mixture of chiral amines only through the enzyme to obtain the desired enrichment and remove the ketone formed as described.

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 / pmol ketone per minute. To achieve uniformity, activity is measured as the number 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 transaminase activity values shown in the following examples.

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

sodium pyruvate 100 mM

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

A sample is taken and 20% by volume of 12% aqueous trichloroacetic acid is added. 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 100 x 8 mm 4 µm Novopak phenyl column. Elution is carried out in 40% isopropanol and 0.09% phosphoric acid in water. Under these conditions, 1-phenylbutan-3-one eluted in 5.3 minutes.

Purity of amines

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 the product is determined by reaction with (-) α- (trifluoromethylphenyl) methoxyacetyl chloride (see Gal,

J. Pharm. Sci., 66, 169, 1977 and Mosher et al., J. Org. Chem., 34, 25430, 1969) followed by capillary gas chromatography of the derivatized product on a fused silica Chrompack column.

Standard salt medium

Suitable salt medium for microbial transformations described in the following examples has the following composition: MgSO ₄ 1.00 g / l CaCl 0.021 g / l ZnSO ₄ .7H ₂ O 0.20 mg / l MnSO4.4H ₂ O 0.10 mg / l H3BO3 0.02 mg / l CuSO ₄ , 5H ₂ O 0.10 mg / l CoCl ₂ .6H ₂ O 0.05 mg / l NiCl ₂ .6H ₂ O 0.01 mg / l

FeSO ₄ 1.50 mg / L

NaMoO ₄ 2.00 mg / L

Fe EDTA 5.00 mg / L

KH ₂ PO 20.00 mM

NaOH to pH 7

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

microorganisms

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

Enrichment of microorganisms producing omega-amino acid transaminase

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 indicated, cells from the culture are centrifuged at 10,000 rpm for 10 minutes, resuspended in 10 mM phosphate buffer pH 7 and 0.5 mM pyridoxal phosphate, and crushed by two passes cooled with a French press operating at 103 MPa. . The crushed cells are separated by centrifugation at 10,000 rpm 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 definitions of the claims are decisive for the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

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

Bacillus megaterium is grown in a 3 liter shake flask (200 rpm) for 17 hours at 30 ° C using 1 liter of said saline, 60 mM sodium acetate, 30 mM phosphate buffer, 30 mM disodium 2ketoglutarate and 100 mM n. -propyl-amine as a nitrogen source. When the culture reaches a density of 0.6 g dry matter per liter, the cells are separated and the enzyme is recovered 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 a soil sample with no particular history regarding amine exposure, seeding the described chemostat and isolating dominant organisms (those capable of growing on R, S-1-phenyl-3-aminobutane). The strain was independently identified in the American Type Culturc Collcction as Bacillus megaterium, which does not differ significantly from the known ATCC strain No. 2. 14581 and which is phenotypically similar to ATCC 49097B.

Example 2

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

Pseudomonas aeuruginosa ATCC 15692 is grown on β-alanine as the sole carbon source as described by Way et al. In FEMS Micro. Lett., 34, 279 (1986). Cell extracts containing 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 is cultured as described in Example 1, and the extract is extracted from the cells. The specific activity of the omega-amino acid transaminase is 0.045 units / mg.

Example 4

The need for an amino acceptor is demonstrated in this example.

The enzyme extracts of Pseudomonas putida, Bacillus megaterium and Pseudomonas aeruginosa obtained as described above were tested at pH 9 in 50 mM TRIS / HCl using 30 mM R, S-1-phenyl-3-aminobutane in the presence or absence of 100 mM sodium pyruvate. The following relative conversion rates are achieved.

Relative speed of conversion /

p.putida E. megateriun P. aeruginosa

in the presence of pyruvate 100 100 100 in the absence of pyruvate 0 0 0 transaminase nature enzymatic effect

is evident from the action 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 of P.putida B.megaterium P.aeruginosa

no 100 100 100 gabakulín 0 13 0 hydroxylamine 3 10 0 The stereoselectivity of the omega-amino acid transaminase is evident from the results of the following assay using 15 mM

R-1-phenyl-3-aminobutane (with pyruvate).

relative conversion rate p.pu tida B. aega téri ujo P. aeruginosa R, S-l-phenyl-3aninobután 100 100 1OO R-l-phenyl-3anlnobután 3 15 4

Example 5

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

Bacillus megaterium is 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 inducer listed in the following table. After 30 to 40 hours, the enzyme is isolated and assayed as described.

specific activity (units / mg)

carbon source succinate acetate gluconate glucose R, S-l-phenyl-laninoetán 0.27 0.39 n. t. n. t. R-l-phenyl-1aminoetán 0.27 0.36 n. t. amp; R, S-i-phenyl-3aminobután 0.28 0.33 0.26 0.62 R-l-phenyl-3aninobután 0.21 0.26 n. t. n. t. R, S-2-aminobutane 0.13 0.14 n. t. amp; R-2-aminobutane 0.06 0.13 n. t. amp; tyranny n. t. 0.24 amp; amp;

Example 6

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

Bacillus megaterium is grown in a 12 L fermenter at pH 7 and 30 ° C and said salt medium supplemented with 10 g / L of 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. Its specific activity is 0.49 units / mg.

Example 7

This example illustrates the use of a soluble enzyme preparation for enantiomeric enrichment of a chiral amine racemate.

The omega-amino acid transaminase preparation is obtained as described in Example 1 from Bacillus megaterium. Its specific activity determined by the described assay is 0.375 units / mg. To 25 ml of a solution of 26.4 mg of this enzyme preparation, which additionally contains 0.4 mM pyri doxalphosphate and 40 mM sodium phosphate, is added 20 mM R, S-1-amino-1-phenylethane and 100 mM sodium pyruvate as aminoacceptor . The solution is incubated for 150 minutes at pH 7 and 30 ° C and then made alkaline (pH> 12) by the addition of

2.5 ml of 2N sodium hydroxide. The solution was extracted with nheptane and the extracts were evaporated. 30.8 mg (49% conversion) of R-1-amino-1-phenylethane with an ee of 96.4% is obtained. This example illustrates the use of a soluble enzyme preparation for enantiomeric enrichment of a chiral amine racemate.

The procedure of Example 7 is followed except that one of the following racemates is used in place of R, S-1-amino-1-phenylethane:

Starting substance

a) R, S-1-phenyl-1-aminobutane

b) R, S-1-amino-1- (4-bromophenyl) ethane

c) R, S-1-phenyl-2-aminopropane

d) R, S-1-amino-1-phenylethane

e) R, S-4- (4-methoxyphenyl) -2-aminobutane; R, S-5- (3-pyridyl) -2-aminopentane

Product ee% conversion

a) R-1-phenyl-3-aminobutane 98.4 60 b) R-1-amino-1- (4-bromophenyl) ethane 97.6 49 c) R-1-phenyl-2-aminopropane 98.6 49 d) R-1-amino-1-phenylethane 99.0 52 e) R-4- (4-Methoxyphenyl) -2- 99.0 58 aminobutane f) R-5- (3-pyridyl) -2- 99.0 49

-aminopentán

Example 9

In this example, the use of non-growing cells for enantiomeric enrichment of the chiral amine racemate is illustrated.

Cells from three 1 liter Bacillus megaterium cultures which were allowed to grow for 33 hours as described in Example 1 to 6 mM R, S1-phenyl-3-aminobutane as the sole nitrogen source were collected by centrifugation and washed by resuspending in 250 ml 10 mM phosphate buffer. (pH 6.8) and centrifugation.

The cell pellet is resuspended in 0.6 L of 10 mM phosphate buffer (pH 6.8) containing 10 mM R, S-1-phenyl-3-aminobutane and 50 mM oxaleacetic acid as aminoacceptor. After incubation in a circulating (orbital) incubator at 30 ° C for 4 hours, the solution is basified and extracted with heptane as described in Example 7. This gives R1-phenyl-3-aminobutane with an optical purity of 97.9% corresponding to ee 95 , the eighth

Example 10

The following example illustrates the use of growing cells for enantiomeric enrichment of chiral amine recemate and the use of an aminoacceptor precursor.

A 6 liter Bacillus megaterium inoculum produced essentially as described in Example 1, but using 10 mM R, S-1-phenyl-3-aminobutane as the sole nitrogen source, is cultured in 120 L of said salt medium supplemented with 3 mM fumarate as aminoacceptor precursor. An additional 30 mM fumarate is added 22 hours after inoculation, and after 6 hours the culture is harvested by removing the cells by ultrafiltration on a Romicon PM100 membrane. The solution was basified and extracted with heptane as described in Example 7. This gave R-1-phenyl-3-aminobutane in purity

99.5%, which corresponds to an ee of 96.4%.

Example 11

The following example illustrates the relative conversion rates determined directly or calculated from the kinetic data of various chiral amines using soluble enzyme preparations. The assay is performed as described above, but only changes the chiral amine. The results are shown in the following table.

anine (R, S) conc. (mM) relative conversion rate R-enantiomer R-enantiomer 1-phenyl-laminoetán 10 □ 100 l-phenyl-3aninobután 30 5 100 1- (4-brónfenyl) - 1-aroinoetán 30 0 100 l- (a-naphthyl) -laminoetán 10 100 0 phenylglycinol 10 100 0 2-aninooktán 5 0 100 5- (3-pyridyl) -2aniriopentďn 5 at LOQ 1- (4-nitrophenyl) - 2-aninopropane 5 0 100 3-phenyl-2-amincpropane 15 7 100 1-phenyl-laminopropán 20 11 100 3-phenylethoxy 2aminopropán 10 100 0

Example 12

This example illustrates the relative conversion rates of 1-phenyl-3-aminobutane using different amino acceptors instead of pyruvate in the assay performed as described. The results are shown in the following table:

acceptor concentration (MM) of relative conversion rate pyruvate 20 100 oxaloacetate 20 100 Heptaldehyde 25 80 glyoxylate 20 50 2-ketobutyrate 25 21 butane-2-one 20 20 acetaldehyde 20 50 propionaldehyde 20 100 butyraldehyde 20 90 benzaldehyde 25 17 2-pentanone 25 33 cyclopentanone 25 12 cyklchexanón 25 23 hydroxypyruvate 25 18

Acetophenone is also effective as an aminoacceptor, although its efficacy is significantly lower (relative rate <10).

SK 280218 Β6

Example 13

This example illustrates enantiomeric enrichment using a soluble enzyme preparation with continuous extraction of the enriched product.

A soluble enzyme preparation was obtained from Bacillus megaterium as described in Example 1. Its specific activity determined by said assay was 0.70 units / mg. An aqueous phase is prepared containing 450 mg of this extract, 0.12 M sodium pyruvate, 0.2 M R, S-1-phenyl-3-aminobutane, 1 mM pyridoxal phosphate and 0.5 M phosphate (pH 7.5). 500 ml of n-heptane are added and the biphasic mixture is stirred for 7 hours at 22 ° C. The value was adjusted to 4.5 by addition of hydrochloric acid and the aqueous layer was separated from the organic layer. The aqueous layer was basified by addition of sodium hydroxide and extracted with heptane. After removal of the heptane, analysis showed that the residue contained 96% R-1-phenyl-3-aminobutane.

Example 14

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 determined as described above is 0.58 units / mg. To 200 ml of an aqueous solution of 350 mg of this preparation, 0.4 mg of pyridoxal phosphate and 40 mM sodium phosphate were added 4.2 mM of 1-phenylbutan-3-one and 100 ml of 2-aminobutane as the aminodonora. The mixture is incubated at pH 7 and 30 ° C for 4 hours, after which time R-1-phenyl-3-aminobutane is present in the reaction mixture at a concentration of 3.35 mM corresponding to 80% conversion. The product was isolated by addition of 40 ml of 10 N sodium hydroxide and extraction of an alkaline aqueous solution of 250 ml of n-heptane. Evaporation of the heptane extracts yielded 100.5 g of the product which was analyzed by the described derivatization. The product contains 96.4% S-1-phenyl-3-aminobutane.

Similarly, S-1-phenyl-2-aminopropane was prepared from 1-phenylpropan-2-one at ee 96.4 in 94% yield. S-1-amino-1-phenylethane was prepared from acetophenone at ee 100 and in a yield of 44%.

Example 15

This example illustrates the enzymatic separation and isolation of the R- and S-enantiomers.

Using the incubation procedure, the procedure for preparing the R-enantiomer R, S-1-amino-1-phenylethane described in Example 7 is repeated. However, before alkalizing the incubation solution, this solution is extracted with n-heptane and the extracts stored. The aqueous phase is then treated as described in Example 7 to isolate R-1-amino-1-phenylethane.

Acetophenone is obtained by evaporation from the heptane extracts. When essentially reproducing the procedure of Example 14, except using 2.3 mM acetophenone in place of 1-phenylbutan-3-one, 56 mg of S-1-amino-1-phenylctane (100%) was obtained.

Example 16

This example illustrates the use of immobilized enzyme.

Immobilization is carried out as follows: The carrier matrix (0.4 g) in the form of a 47 mm diameter disc (Actidisk CFMC Corp.) is placed in a Millipore Sweenex equipped with an inlet and outlet pipe, a peristaltic pump and a reservoir. The matrix is washed successively at ambient temperature at a rate of 3 ml / min 1) 200 ml of a 50 mM phosphate buffer (pH 7) containing 0.5 mM pyridoxal phosphate for 20 minutes, 2) 11 ml of a 4.6 mg / ml enzyme solution obtained according to the method of Example 1, for 120 minutes, 3) 150 ml of 0.3 M sodium chloride in 50 mM phosphate buffer (pH 7) containing 0.5 mM pyridoxal phosphate, for 30 minutes and 4) 200 ml of 50 mM phosphate buffer (pH 7) containing 0.5 mM pyridoxal phosphate for 20 minutes.

The enrichment is carried out as follows: 140 ml of a solution of 10 mM R, S1-phenyl-3-aminobutane, 100 M sodium pyruvate, 0.1 mM pyridoxal phosphate and 25 mM potassium phosphate (pH 7) are circulated through the matrix at room temperature at 5 ml. / minute. After two hours, the circulating liquid is drained from the device. The concentration of the resulting 1-phenylbutan-3-one is 5.2 mM, while the concentration of R-1-phenyl-3-aminobutane is 4.8 mM. The pH is adjusted to 12.5 and the R-1-phenyl-3-aminobutane is quantitatively isolated by heptane extraction. After removal of the heptane by evaporation, the product is analyzed. The R-1-phenyl-3-aminobutane content of the product is 92.8%.

Claims (15)

PATENT CLAIMS 1. A process for the enantiomeric enrichment of a mixture of two enantiomeric chiral amines of the general formula “z”> 2 R ^ Ô — B ² sec • · • * HH where R ¹ and R ² are alkyl or aryl groups optionally substituted by an enzymatic non-inhibitory group, wherein R ¹ differs from R ^{2 in} structure or chirality, characterized in that the mixture of chiral amines is contacted with an omega-amino acid in an aqueous environment and in the presence of an aminoacceptor transaminase, which is enzymatically active against the illustrated amino group of one of said chiral amines, until a substantial amount of one of these chiral amines is converted to a ketone of formula ABOUT R ¹ - C - R ² wherein R ¹ and R ² have the same meaning as for the starting amine. The method of claim 1, wherein the contact is maintained for at least as long as there is no enantiomeric excess of the chiral amine that is converted to a ketone of at least 90% relative to the remaining chiral amine. The process according to claim 1, wherein the chiral amine, which is not converted to a ketone, is isolated from the reaction mixture. 4. The method of claim 1, wherein a substantial amount of ketone is recovered from the aqueous medium. The method of claim 4, wherein the aqueous isolated ketone is independently contacted with an omega-amino acid transaminase in the presence of an aminodonor for at least as long as the same chiral form as that at the beginning of the conversion to the ketone, in an amount substantially in excess of that of the second chiral form. The process according to claim 1, wherein the aminoacceptor is an α-ketocarboxylic acid, an aliphatic or cycloaliphatic ketone, an aliphatic or cycloaliphatic aldehyde or a substance which is biochemically converted to α-ketocarboxylic acid in situ in the reaction medium. The process according to claim 6, wherein the aminoacceptor is glyoxalic acid, pyruvic acid, oxaloacetic acid, salts thereof or heptaldchyd. A process according to claim 1, wherein each of R ¹ and R ^{2 is} independently straight or branched (C 1 -C 6) alkyl, straight or branched (C 7 -C 12) phenylalkyl or phenyl or naphthyl, wherein: each of these groups is unsubstituted or substituted by an enzymatically non-inhibitory group. The method of claim 8, wherein each of R ¹ and R ^{2 is} independently methyl, ethyl, η-propyl, isopropyl, η-butyl, isobutyl, sec. butyl, phenyl, benzyl or phenethyl. 10. The method of claim 1 wherein the mixture of chiral amines and aminoacceptor is contacted with whole cells of a microorganism that produces an omega-amino acid transaminase. 11. The method of claim 1, wherein the mixture of chiral amines and amino acceptor is contacted with an aqueous, omega-amino acid transaminase preparation that is free of cells. 12. The method of claim 1, wherein the mixture of chiral amines and aminosceptor is contacted with a carrier-immobilized omega-amino acid transaminase. 13. A process for the stereoselective synthesis of one chiral form of an amine of the general formula HH ₂ nh ₂ R ¹ * R ^{2 -} R ² or R ² - -Co R ¹ H H 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 the formula is straight or branched C 1 -C 6 alkyl, straight or branched C 7 -C 12 phenylalkyl or phenyl or naphthyl, each of which is unsubstituted or substituted by an enzymatically non-inhibitory group. 16. The method of claim 15, wherein characterized in that each of R ¹ and R ² each independently is methyl, ethyl, η-propyl, isopropyl, η-butyl, isobutyl, sec. butyl, phenyl, benzyl or phenethyl. 17. The method of claim 13, wherein the ketone and aminodonor are contacted with whole cells of a microorganism producing omega amino acid transaminase. 18. The method of claim 13, wherein the ketone and aminodonor are contacted with an aqueous cell-free omega-amino acid transaminase preparation. 19. The method of claim 13, wherein the ketone and the aminodonor are contacted with a carrier-immobilized omega-amino acid transaminase. 20. The process of claim 13, wherein a large molar excess of the aminodonor is used. End of document ABOUT R ¹ - C - R ^2, where R ¹ and R ² are the same as the prepared amine, contact 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. 14. The process of claim 13 wherein the aminodonor is 2-aminobutane, glycine, alanine or aspartic acid. 15. The method of claim 13, characterized in characterized in that each of R ¹ and R ² each independently pre-