HK1068151B - Enzymatic method for the enantiomeric resolution of amino acids - Google Patents

Description

Enzymatic process for enantiomeric resolution of amino acids

The present invention relates to a novel enzymatic process which enables the enantiomeric resolution of amino acids in the form of racemic mixtures.

These amino acids can be used in a wide variety of industries, for example, often as biologically active compounds, or as synthetic intermediates for the preparation of compounds which are primarily of pharmaceutical, chemical or agricultural interest. Thus, it was soon discovered that there was a problem that it was often necessary to be able to obtain one or the other optically active enantiomer from these amino acids. Thus, separation methods of the enantiomers of pro-chiral amino acids have been developed. In particular, a number of enzymatic methods for enantiomeric resolution of these amino acids have been disclosed, which are interesting alternatives to asymmetric synthesis methods.

Thus, Solosochok et al [ Tetrahedron (Tetrahedron): assymetry, Vol.6 (7), 1995, p.1601-1610, proposed an enzymatic method for enantiomeric resolution of β -amino acids according to the following reaction scheme:

similarly, Topgi et al (Bioorg. Med. chem., 1999, Vol.7, p. 2221-2229) describe a process for the resolution of the enantiomers of ethyl (R) -and (S) -3-amino-4-pentynoate in the form of pure enantiomers, the reaction scheme of which is as follows:

the starting phenylacetamide is obtained by acylation of the corresponding amine by the action of phenylacetic acid, or:

patent application WO 98/50575 more generally describes a process for the preparation of a chiral β -amino acid which comprises contacting a racemic mixture of said amino acid with an acyl donor and a penicillin G acylase (or amidohydrolase) under suitable conditions so as to stereoselectively acylate one enantiomer of the racemic mixture of β -amino acid to its corresponding N-acylated derivative, while obtaining the opposite enantiomer of the β -amino acid, in enantiomerically enriched form, which has the following reaction scheme:

the "acyl donor" described above has the following general formula:

in the formula R₃Different derivatives selected from the group consisting of phenyl, phenoxy, amino, phenyl and pyridyl, R₄Selected from hydroxy, alkoxy, alkyl, alkenyl, alkynyl, haloalkyl, aryl (aryl), arylalkyl, saccharide or steroid.

Patent application WO 98/50575 also describes an alternative process for the preparation of a chiral β -amino acid which comprises contacting an amide in racemic form with penicillin G acylase under suitable conditions to selectively deacylate one of the enantiomers of the amide in racemic form to its corresponding β -amino acid while obtaining the opposite enantiomer of the amide in enriched enantiomeric form, the reaction scheme of which is as follows:

however, the previously discussed processes suffer from the major drawback of passing an amide intermediate prior to or simultaneously with the enzymatic step, the chemical formula of which can be summarised as follows:

aromatic rings, preferably phenyl

Such an amide is insoluble in an aqueous medium because it contains an aromatic ring in its structure, thereby causing inconvenience. For example, it is known that certain enzymes are soluble in aqueous media and tend to be sensitive to the presence of organic solvents. However, in order to optimize the yield of the enzymatic reaction, it is important that the solubility of the substrate be better than that of the enzyme so that they can be brought into intimate contact.

This is the main drawback to be overcome by the present invention. In fact, the present invention relates firstly to a new process for the separation of the enantiomers of an amino acid by treating a racemic mixture of said amino acid with glutaric anhydride and then with glutaryl 7-ACA acylase, in order to recover one enantiomer of said amino acid, the other enantiomer remaining in the form of the corresponding glutarylamide derivative.

This process is particularly advantageous because the use of glutaric anhydride enables the reaction to proceed through the glutarylamide derivative intermediate corresponding to the starting amino acid, the glutaryl function making the component soluble in aqueous medium. Thus, the process of the invention can be carried out in an aqueous medium under mild reaction conditions, in particular without the use of organic cosolvents.

The process of the invention also has the advantage of being applicable to any form of amino acid (α, β, γ, etc.). In the context of the present invention, the term "amino acid" includes not only the amino acid itself (i.e. the compound having an amino function and an acid function-COOH) but also the corresponding ester derivative thereof (i.e. the compound in which the acid function is replaced by an ester function COOR). Preferably, the process of the invention applies more precisely to amino acids of the following general formula (I):

in the formula:

-n is an integer selected from 0, 1, 2, 3, 4, 5 and 6,

-R represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a condensed polycyclic hydrocarbon group, or a heterocyclic group, all of which may be optionally substituted,

-and R' represent alkyl, alkenyl, alkynyl, cycloalkyl, aryl, condensed polycyclic hydrocarbon radicals, heterocyclic radicals, or oxy, thio, sulfoxide or sulfonyl substituted by alkyl, aryl, cycloalkyl or heterocyclic radicals, all of which are also optionally substituted.

Thus, in the case where these amino acids are of the general formula (I), the process of the invention can be represented by the reaction scheme of FIG. 1. This scheme shows a first step of treatment with glutaric anhydride and a second step of treatment with glutaryl 7-ACA acylase. The configuration of each resulting product, i.e. the amino acid on the one hand and the glutarylamide derivative on the other, depends on the nature of the R' group.

In the present invention, the alkyl group, the alkenyl group and the alkynyl group are generally a straight chain or a branched chain having 1 to 30 carbon atoms, but this is not limitative. The same applies to the case where these radicals are substituents of other radicals. Preferably, these groups are straight or branched chains containing from 1 to 20 carbon atoms, more preferably, from 1 to 10 carbon atoms. These alkyl groups may be selected, for example, from: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, isopropyl, isobutyl, isopentyl, isohexyl, 3-methylpentyl, neopentyl, octylhexyl, 2, 3, 5-trimethylhexyl, sec-butyl, tert-pentyl. Preferred alkyl groups are, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl and isohexyl. The alkenyl group may be selected from, for example, vinyl, 1-propenyl, allyl, butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, and 3-methyl-2-butenyl. Alkynyl groups may be selected, for example, from ethynyl, 1-propynyl and propargyl.

In the present invention, the cycloalkyl group generally has 3 to 12 carbon atoms. Preferably, the cycloalkyl group is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl. According to another aspect of the invention, the cycloalkyl group may be polycyclic. Preferably, these groups are selected from bicycloalkyl and tricycloalkyl.

According to the invention, "aryl" means a monovalent aromatic hydrocarbon radical. Among these aryl groups, an optionally substituted phenyl group is preferred.

In the present invention, the term "condensed polycyclic hydrocarbon" means a group preferably selected from: pentacene, indene, naphthalene, azulene, heptalene, biphenylene, asymmetric indacene, symmetric indacene, acenaphthylene, fluorene, chrysene, phenanthrene, anthracene, fluoranthene, acephenanthrene, aceanthrylene, triphenylene, pyrene, chrysene, naphtalene, pleiadene, dinaphthylene, perylene, pentaphene, pentacene, tetraphenylene, hexaphene, hexacene, rubicene, coronene, terphenyl, heptophene, enanthocene, or ovalene.

In the present invention, the term "heterocycle" denotes monocyclic or fused polycyclic compounds containing one or more heteroatoms, each ring being composed of 3 to 10 segments. Preferably, the heterocyclic ring of the present invention contains 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen in a ring composed of 3 to 10 segments. The heterocycles of the invention are preferably selected from thiophene, benzo [ b ] thiophene, naphtho [2, 3-b ] thiophene, thianthrene, furan, 2H-pyran, isobenzofuran, 2H-chromene, xanthene, phenoxathiine, 2H-pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, 3H-indole, 1H-indazole, purine, 4H-quinolizine, isoquinoline, quinoline, phthalazine, 1, 8-naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, 4a H-carbazole, beta-carboline, phenanthridine, acridine, perimidine, 1, 7-phenanthroline, phenazine, isothiazole, phenothiazine, isoxazole, furazan, phenoxazine, isochroman, pyrrolidine, Δ 2-pyrroline, thianthrene, furan, 2H-pyran, xanthene, phenoxane, phenoxathiine, phenoxathiin, phenanthroline, quinoxaline, and quinoxaline, Imidazoline, Δ 2-imidazoline, pyrazolidine, Δ 3-pyrazoline, piperidine, piperazine, 2, 3-dihydroindole, iso 2, 3-dihydroindole, quinuclidine and morpholine.

In the present invention, when these various groups defined above are substituted, said substituent or substituents may be generally selected from the group consisting of halogen atoms, aryl groups, heterocyclic groups, hydroxyl groups, alkoxy groups, aryloxy groups, thio groups, alkylthio groups, arylthio groups, alkylsulfoxide groups, arylsulfoxide groups, alkylsulfonyl groups, arylsulfonyl groups, cyano groups, nitro groups, sulfonamido groups, alkylsulfonylamino groups and arylsulfonylamino groups, depending on the nature of these groups. Preferably, these groups as defined above are substituted 1, 2 or 3 times. According to a preferred aspect, the halogen atom may be selected from chlorine, fluorine, bromine and iodine. In the case where these alkyl groups are substituted with halogen, the halogen atom is preferably a fluorine atom. Preferably, the number of fluorine substituents is 1, 2, 3, 4, 5, 6 or 7. For example, trifluoromethyl.

Preferably, the amino acids of the invention are selected from compounds of general formula (I) wherein n is an integer selected from 0, 1, 2 and 3, R represents a hydrogen atom, an alkyl group or an aryl group, R' is as defined above.

More preferably, the amino acid of the invention is selected from compounds of general formula (I) wherein n is an integer selected from 0, 1 and 2, R represents a hydrogen atom or an alkyl group, R' is selected from optionally substituted aryl or heterocyclic ring. In the latter case, these aryl and/or heterocyclic rings are preferably substituted 1, 2 or 3 times.

Glutaryl 7-ACA acylases have been used as catalysts in many industrial processes, especially in the hydrolysis of beta-lactams such as N-glutaryl 7-aminoacetoacetoxycephalosporin). The enzymes can be obtained from a number of microorganisms, such as the genera Acinetobacter (Acinetobacter), Arthrobacter (Arthrobacter), Bacillus (Bacillus), Pseudomonas (Pseudomonas), Stenotrophomonas (Stenotrophomonas) or Xanthomonas (Xanthomonas), according to techniques well known to those skilled in the art, i.e., to experts in the field of enzymes. Glutaryl 7-ACA acylases are also commercially available, for example from Roche Diagnostic GmbH (Roche Molecular Biochemicals, Standard variety 116, D-68305 Mannheim) or Recordatio S.p.A. (Stabilimento di opera, Via Lambro 38, 1-20090 opera (MI)).

Different forms of glutaryl 7-ACA acylase can be used, but this does not alter the stereospecificity and stereoselectivity. For example, glutaryl 7-ACA acylase can be used in soluble form or in immobilized form. In the second case, the enzyme is generally immobilized by techniques well known to those skilled in the art. For example, the enzyme may be embedded in a polymer gel or immobilized on a solid support by covalent bonding, cross-linking, adsorption or encapsulation. Suitable supports which are generally used are, for example, porous glass, porous ceramics, synthetic polymers (for example polystyrene, polyvinyl alcohol, polyethylene, polyamide or polyacrylamide), or polymers of natural origin (for example cellulose).

By using glutaryl 7-ACA acylase it is possible to obtain an enantiomer with a high enantiomeric excess (ee), in particular higher than or equal to 90%, even higher than or equal to 95%, preferably higher than or equal to 99%. In the present invention, "enantiomeric excess" refers to the excess of one of the enantiomers in% based on the racemic mixture. More precisely, the enantiomeric excess is calculated according to the following formula:

[ R ] and [ S ] represent the concentrations of the (R) -enantiomer and the (S) -enantiomer, respectively

In general, the amount of enzyme used is from 1 to 100 units (U) per millimole of substrate, preferably from 10 to 40 units (U) per millimole of substrate, based on the total amount of starting amino acids (substrate). 1 unit of enzyme is the amount of enzyme required to hydrolyze 1 micromole of N-glutaryl 7-aminoacetoacetoxycephalosporanic acid per minute under standard pH and temperature conditions known to those skilled in the art.

According to the invention, the reaction is carried out in an optionally buffered aqueous medium. In this case, the aqueous buffer solution having a concentration of 10 to 200mM may be selected from an acetate buffer solution which can be used at pH5 to 6.5, or a phosphate buffer solution which can be used at pH6.5 to 8, or a pyrophosphate buffer solution which can be used at pH8 to 9.

Thus, the process of the invention can be carried out in a medium whose pH can be controlled and adjusted to 6 to 9. Preferably, the pH of the reaction medium can be precisely controlled and adjusted to 7.5-8.5, more preferably, 8-8.5. The pH homeostasis can be controlled using a pH-regulator by adding an acid (such as, for example, hydrochloric acid, sulfuric acid or phosphoric acid) and adding a base (such as, for example, sodium hydroxide, potassium hydroxide or ammonium hydroxide).

The amino acids of the invention are treated with glutaric anhydride at a temperature of 20-40 c, preferably 25-35 c. Alternatively, the second step is carried out at a temperature of 10-50 deg.C, preferably 25-35 deg.C, using glutaryl 7ACA acylase.

Finally, the reaction time varies widely and may be from 1 to 100 hours, depending mainly on the concentration of the amino acids and enzymes involved. Generally, the reaction is carried out for the time required to obtain the desired enantiomer with a satisfactory enantiomeric excess. The amount and enantiomeric excess of the resulting chiral amino acid can be controlled by employing classical techniques known to those skilled in the art. Preferably, HPLC (high performance liquid chromatography) is used for the control.

According to the present invention, since one of the (R) -enantiomer and the (S) -enantiomer obtained by the aforementioned method is in the form of a soluble amine in an aqueous medium and the other is in the form of a solid amide, they can be easily separated. Therefore, another object of the present invention relates to the process as previously disclosed, which further comprises a step of separating the (R) -enantiomer from the (S) -enantiomer.

The separation of the (R) -and (S) -enantiomers can be easily carried out by means of classical techniques known to the person skilled in the art. For example by filtration, extraction, chromatography or crystallization.

In case the other enantiomer which is desired to be separated is in the form of an amino acid instead of in the form of a glutarylamide derivative, the enantiomer of a glutarylamide derivative which has been separated according to the previous process can be subjected to hydrolysis in order to recover the corresponding amino acid in enantiomeric form. It should be noted that this method is advantageous because this hydrolysis allows to preserve the stereochemistry of the compound used, without causing racemization of the chiral glutarylamide derivative. In the case where the amino acid is of formula (I), this additional step can be represented by the reaction scheme of FIG. 2.

The hydrolysis may be carried out according to classical techniques known to the person skilled in the art. The invention relates in particular to acidic or alkaline hydrolysis. In the latter case, for example, in the presence of a base such as sodium hydroxide, at a temperature of 50-90 ℃ and at atmospheric pressure. However, other suitable operating conditions known to those skilled in the art may also be used.

The process of the invention can be used to isolate an amino acid in racemic form, thus making it possible to obtain one or the other enantiomer of said amino acid, said enantiomer being particularly useful as a synthesis intermediate.

For example, the present invention enables, for example, 3-amino-3-phenylpropionic acid of the (S) form, which is an intermediate for the synthesis of compounds of the general formula:

it is an antagonist of the VLA4 receptor which is particularly implicated in asthma.

In addition to the foregoing, the present invention also includes features and advantages of the invention that may be derived from the following examples, which should be considered illustrative of the invention and not limiting its scope.

Drawings

FIG. 1:the scheme shows the first step of treating the amino acid of formula (I) according to the invention with glutaric anhydride, with glutaryl 7-CAC acylThe second step of the transferase treatment. The configuration of each product obtained, i.e. the amino acid on the one hand and the glutarylamide derivative on the other, depends on the nature of R'.

FIG. 2:the scheme shows the step of converting a chiral glutarylamide derivative into its corresponding chiral amino acid by hydrolysis.

Examples

Example 1: resolution of 3-amino-3- (4' -nitrophenyl) propionic acid in the form of a racemic mixture

a) Acylation of racemic 3-amino-3- (4' -nitrophenyl) propionic acid

40.6 g of racemic 3-amino-3- (4' -nitrophenyl) propionic acid are dissolved in 200 ml of distilled water and 55 ml of triethylamine. 29.4 g of glutaric anhydride were added in small portions and the reaction mixture was stirred for 1 hour. The reaction mixture was then sulfated with 8.2 ml of 95% (w/v). The precipitate thus obtained is filtered, washed 3 times with 15 ml of distilled water each time and then dried under vacuum at 55 ℃ until a constant weight is reached. This gives 52.84 g of racemic 3- (glutarylamide) -3- (4' -nitrophenyl) propionic acid.

The properties of the resulting product were determined by HPLC (high Performance liquid chromatography).

b) Enzymatic deacylation using crystalline glutaryl 7-CAC acyltransferase in suspension (100) Milliliter reactor)

20 g of the acid obtained in the previous step were dissolved in 75 ml of distilled water. 11.2 ml of 30% (w/v) sodium hydroxide solution were added to adjust the pH of the suspension to 8.2. To this solution was added 826 mg (626 units) of a crystalline glutaryl 7-CAC acylase suspension. The reaction mixture was stirred at 35 ℃ for 51 hours with control and adjustment of the pH to 7.9-8.1. After the reaction was completed, the reaction mixture was cooled to 20 ℃ and 5N hydrochloric acid was added to adjust the pH to 7.0. At this point 20 ml of ethanol was added. This gives a precipitate of (R) -3-amino- (4' -nitrophenyl) propionic acid which is filtered off and washed three times with 30 ml of ethanol each time and then dried under vacuum at 45 ℃ until a constant weight is reached. This gives 5.65 g of (R) -3-amino-3- (4' -nitrophenyl) propionic acid in an enantiomeric excess of more than 99%.

The mother liquor was acidified with 17.5 ml of 5N hydrochloric acid. The precipitate formed is filtered and washed 2 times with 10 ml of distilled water each time. The resulting filter cake was dried under vacuum at 45 ℃ until constant weight was reached. This gives 8.25 g of (S) -3- (glutarylamide) - (4 '-nitrophenyl) propionic acid and (R) -3- (glutarylamide) -3- (4' -nitrophenyl) propionic acid in a ratio of 91: 9.

The properties of the resulting product were determined by HPLC.

c) Use of immobilized glutaryl 7-CAC Acytransfer from Roche Diagnostic GmbH Enzymatic deacylation of enzymes (100 ml reactor)

20.5 g of the acid obtained in step a) were dissolved in 75 ml of distilled water. 11.9 ml of 30% (w/v) sodium hydroxide solution were added to adjust the pH of the suspension to 8.2. To this solution 6.2 g (632.4 units) of wet immobilized glutaryl 7-CAC acylase (Roche Diagnostic GmbH) were added. The reaction mixture was stirred at 35 ℃ for 18 hours with control and adjustment of the pH to 7.9-8.1. After the completion of the reaction, 30% (w/v) sodium hydroxide solution was added to adjust the pH of the reaction mixture, and the volume of the reaction mixture was adjusted to 200 ml, so as to dissolve the produced (R) -3-amino-3- (4' -nitrophenyl) propionic acid. The immobilized enzyme was removed by filtration, and 5N hydrochloric acid was added to adjust the pH of the mother liquor to 7.0. At this point 50 ml of ethanol was added to the reaction mixture. The (R) -3-amino-3- (4' -nitrophenyl) propionic acid precipitate was filtered, washed with 10 ml of ethanol and then dried under vacuum at 45 ℃ until constant weight was reached. This gave 5.375 g of (R) -3-amino-3- (4' -nitrophenyl) propionic acid, the enantiomeric excess amounting to 98%.

The mother liquor was acidified with 20 ml of 5N hydrochloric acid. The precipitate formed is filtered and washed 2 times with 15 ml of distilled water each time. The filter cake was dried under vacuum at 45 ℃ until constant weight was reached. This gives 6 g of (S) -3- (glutarylamide) -3- (4' -nitrophenyl) propionic acid in a ratio of 94: 6.

The properties of the resulting product were determined by HPLC.

d) Enzymatic deacylation using crystalline glutaryl 7-CAC acyltransferase in suspension (500) Milliliter reactor)

100 g of racemic 3- (glutarylamide) -3- (4' -nitrophenyl) propionic acid, obtained in step a), are dissolved in 400 ml of distilled water. 60 ml of 30% (w/v) sodium hydroxide solution are added to adjust the pH of the suspension to 8.2. To this solution was added 6.4 g (4653 units) of a crystalline glutaryl 7-CAC acyltransferase suspension. The reaction mixture was stirred at 35 ℃ for 30 hours with the pH being controlled and adjusted to 7.9-8.1. After the reaction was completed, the reaction mixture was cooled to 20 ℃ and 25 ml of 30% (w/v) sodium hydroxide solution was added to adjust the pH to 13, thereby dissolving the resulting (R) -3-amino-3- (4' -nitrophenyl) propionic acid. The insoluble particles were filtered and 27 ml of 36% hydrochloric acid was added to adjust the pH of the mother liquor to 7.0. At this point 100 ml of ethanol was added to the reaction mixture. A precipitate of (R) -3-amino-3- (4' -nitrophenyl) propionic acid is obtained, which is filtered and washed 2 times with 100 ml of ethanol each time and then dried under vacuum at 45 ℃ until a constant weight is reached. This gives 28.87 g of (R) -3-amino-3- (4' -nitrophenyl) propionic acid in an enantiomeric excess equal to 97%.

The properties of the resulting product were determined by HPLC.

Example 2: resolution of 3-amino-3-phenylpropionic acid in form of racemic mixture

a) Acylation of racemic 3-amino-3-phenylpropionic acid

488 g of racemic 3-amino-3-phenylpropionic acid, containing 236 g of sodium hydroxide flakes, was dissolved in 2 l of distilled water. 437.5 g of glutaric anhydride were added in small portions to the reaction mixture at 20 ℃ over one hour with stirring. After 1 hour, the reaction mixture was acidified with 236 ml of 95% (w/v) sulfuric acid and cooled to 10 ℃. The precipitate thus formed was filtered and washed 3 times with 600 ml portions of distilled water. 1610 g of the wet filter cake thus obtained contain 602 g of racemic 3-glutarylamide-3-phenylpropionic acid. The properties of the resulting product were determined by HPLC.

b) Enzymatic deacylation Using crystalline glutaryl 7-CAC acylase in suspension (5 liters) Reactor)

1575 g of the wet cake from the previous step, containing 589 g of racemic 3-glutarylamide-3-phenylpropionic acid, was dissolved in 1610 ml of distilled water. 440 ml of 30% (w/v) sodium hydroxide solution are added to adjust the pH of the suspension to 8.0. To this solution was added 52.6 mg (3200 units) of a suspension of crystalline glutaryl 7-CAC acylase. The reaction mixture was stirred at 35 ℃ for 41 hours with the pH being controlled and adjusted to 7.9-8.1. After the reaction was completed, the reaction mixture was cooled to 20 ℃ and 25 ml of 30% (w/v) sodium hydroxide solution was added to adjust the pH to 13, thereby dissolving the resulting (R) -3-amino-3- (4' -nitrophenyl) propionic acid. The insoluble particles were filtered and 27 ml of 36% hydrochloric acid solution was added to adjust the pH of the mother liquor to 7.0.

The (R) -3-amino-3-phenylpropionic acid thus obtained was precipitated, filtered and washed with 150 ml of distilled water, and then dried under vacuum at 45 ℃ until a constant weight was reached. This gives 96.9 g of (R) -3-amino-3-phenylpropionic acid, the enantiomeric excess equaling 98%.

The mother liquor was acidified with 180 ml of 95% (w/v) sulfuric acid. The precipitate thus formed was filtered and washed 2 times with 400 ml of cold distilled water each time. The filter cake was dried under vacuum at 45 ℃ until constant weight was reached. This gives 314.6 g of (S) -3- (glutarylamide) -3-phenylpropionic acid and (R) -3- (glutarylamide) -3-phenylpropionic acid in a ratio of 90: 10.

The properties of the resulting product were determined by HPLC.

c) Deacylation of (S) -3-glutarylamide-3-phenylpropionic acid by alkaline hydrolysis

557.2 g of the mixture of (S) -3- (glutarylamide) -3-phenylpropionic acid and (R) -3- (glutarylamide) -3-phenylpropionic acid obtained in the preceding step b) (in a ratio of 90: 10) were dissolved in 3.91 l of distilled water and 1.65 l of 30% (w/v) sodium hydroxide. The reaction mixture was stirred at 70 ℃ for 4 days.

The reaction mixture was then cooled to 15 ℃. 3.21 l of 37% (w/v) hydrochloric acid were added and the resulting product was precipitated, filtered and dried under vacuum at 45 ℃ until constant weight was reached. This gives 202.4 g of (S) -3-amino-3-phenylpropionic acid and (R) -3-amino-3-phenylpropionic acid in an enantiomeric excess of more than 98%.

The properties of the resulting product were determined by HPLC.

Claims (16)

1. A process for the separation of enantiomers of an amino acid, which process comprises treating a racemic mixture of said amino acid with glutaric anhydride and then with glutaryl 7-ACA acylase, so as to recover one enantiomer of said amino acid, the other enantiomer remaining in the form of the corresponding glutarylamide derivative.

2. The method according to claim 1, characterized in that the amino acid has the following general formula (I):

in the formula:

-n is an integer selected from 0, 1, 2, 3, 4, 5 and 6,

-R represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a condensed polycyclic hydrocarbon or a heterocycle, all of which may be optionally substituted, and

-R' represents alkyl, alkenyl, alkynyl, cycloalkyl, aryl, condensed polycyclic hydrocarbon, heterocycle, or oxy, thio, sulfoxide or sulfonyl substituted by alkyl, aryl, cycloalkyl or heterocycle, all of which are also optionally substituted.

3. The method according to claim 2, characterized in that said amino acid is selected from the group consisting of the compounds of the following general formula (I):

wherein n is an integer selected from 0, 1, 2 and 3, R represents a hydrogen atom, an alkyl group or an aryl group, R' represents an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a condensed polycyclic hydrocarbon, a heterocyclic ring, or an oxy group, thio group, sulfoxide group or sulfonyl group substituted with an alkyl group, an aryl group, a cycloalkyl group or a heterocyclic ring, all of which are optionally substituted.

4. A method according to claim 2 or 3, characterized in that said amino acid is selected from the group consisting of compounds of the following general formula (I):

wherein n is an integer selected from 0, 1 and 2, R represents a hydrogen atom or an alkyl group, and R' is selected from an optionally substituted aryl or heterocyclic ring.

5. A method according to any of the preceding claims 1-3, characterized in that glutaryl 7-ACA acylase is used in soluble or immobilized form.

6. Process according to any one of claims 1 to 3, characterized in that the amount of enzyme used is 1 to 100 units per millimole of substrate, based on the total amount of starting amino acids.

7. A process according to any one of the preceding claims 1 to 3, characterised in that the reaction is carried out in a buffered aqueous medium.

8. The method according to claim 7, characterized in that the concentration of the aqueous buffer is 10-200mM, said aqueous buffer being selected from the group consisting of acetate buffer at pH5-6.5, phosphate buffer at pH6.5-8, and pyrophosphate buffer at pH 8-9.

9. A method according to any of the preceding claims 1-3, characterized in that the pH is controlled and adjusted to 6-9.

10. A method according to any of the preceding claims 1-3, characterized in that the amino acid is treated with glutaric anhydride at a temperature of 20-40 ℃.

11. A process according to any one of the preceding claims 1 to 3, characterized in that the treatment with glutaryl 7ACA acylase is carried out at a temperature of 10 to 50 ℃.

12. A process according to any one of the preceding claims 1 to 3, characterised in that the reaction time is from 1 to 100 hours.

13. A process according to any one of the preceding claims 1 to 3, characterized in that the (R) -enantiomer and the (S) -enantiomer are also separated.

14. The process according to claim 13, characterized in that the separation of the (R) -and (S) -enantiomers is carried out by filtration, extraction, chromatography or crystallization.

15. A process according to claim 13, characterized in that the separated glutarylamide-derived enantiomer is also subjected to hydrolysis in order to recover the corresponding amino acid in enantiomeric form.

16. The process according to claim 14, characterized in that the separated glutarylamide-derived enantiomer is also subjected to hydrolysis in order to recover the corresponding amino acid in enantiomeric form.