RU2112805C1 - Method for enantiomeric enrichment of mixture of d- and l-treo-2-amino-3-hydroxy-3-phenyl propionic acids, or derivatives thereof, or salts thereof - Google Patents

Abstract

FIELD: organic chemistry. SUBSTANCE: mixture of enantiomeric D- and L-treo-2-amino-3-hydroxy-3-phenyl propionic acids or derivatives thereof or salts thereof are affected by stereoisomeric enrichment. Method is carried out contacting mixture with D-treonine aldolase. D- and L-treo-2-amino-3-hydroxy-3-(4-methylsulfonylphenyl) propionic acid is treated with D-treonine aldolase while L-treo-2-amino-3-hydroxy-3-(4-methylsulfonylphenyl)pronionic acid is prepared at high yield and at high selectivity. Benzaldehyde and amino acid which are obtained of D-treoisomer may be recycled at condensation stage. EFFECT: improved efficiency . 10 cl, 1 tbl

Description

 This application is a private continuation of Series N 07 / 689.300, recorded April 22, 1991 and series N 07 / 676.102, registered March 27, 1991

 The present invention relates to the stereoisomeric enrichment of 2-amino-3-hydroxy-3-phenylpropionic acids.

The biological activity of many chemical compounds having a chiral center, in particular pharmaceutical and agricultural preparations, has been found to be mainly found in one of the chiral forms. Since most synthesis reactions are not stereoselective, serious chemical processing problems arise as a result. Therefore, enrichment of predominantly one of the chiral forms should be carried out at any stage of the process either for the final chiral compounds or for their chemical precursors, which have the same chiral center as the final product. Regardless of the choice of the stage at which enrichment is carried out, and in the absence of a method for recycling the undesirable one or more stereoisomers, the method is essentially limited to a maximum yield corresponding to 1 / (2 ⁿ ) • 100% of the desired stereoisomer, where n is the total number of chiral centers with the molecule.

 2-amino-3-hydroxy-3-phenylpropionic acids and their derivatives are used as intermediates in the synthesis of a number of biologically useful chemical compounds. L-threo-2-amino-3- (4-methylsulfonylphenyl) propionic acid is used as an intermediate in the synthesis of antibiotics D-threo-1-fluoro-2- (dichloroacetamido) -3- (4-methylsulfonyl-phenyl) propan-1- ol (also known as florfenicol) and D-threo-2- (dichloroacetamido) -3 (4-methylsulfonylphenyl) propan-1,3-diol, known as thiamphenicol. In addition, L-threo-2-amino-3- (4-methylthiophenyl) -3-hydroxypropionic acid is an intermediate in the synthesis of antibiotics. Similarly, L-threo-2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid, also known, is used as an intermediate of the synthesis of 1-2-amino-2-methyl-3- (3,4-dihydroxyphenyl) propionic acid called L-methyldopa.

 Since 2-amino-3-hydroxy-3-phenylpropionic acid and its derivatives possess two chiral centers, they exist in the form of four stereoisomers. These include two pairs of enantiomers - these are D, L-erythro- and D, L-threo-enantiomers. In cases where the final product contains two chiral centers, then, as a rule, only one stereoisomer, usually the L-threo stereoisomer, is suitable for the preparation of biologically active compounds. In the preparation of L-methyldopa, the distinction between erythro and threo is not important, since hydrogenation eliminates the chiral center associated with the 3-hydroxy group of 2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid, i.e., the target the product has only one chiral center. And in this case, however, only one enantiomer, its L-form, is suitable. If there are two chiral centers in the molecule, the desired stereoisomer should be isolated from the mixture or enriched in a mixture of D, L-erythro- and D, L-threo-stereoisomers in order to more effectively obtain the final compound of optical purity.

 British Patent No. 1,268,867, Akiyama et al, to which the applicants refer, discloses a process for the preparation of thiamphenicol, according to which an alcohol solution of a glycine alkali metal salt is contacted with two moles of para-methylsulfonylbenzaldehyde per mole of glycine in the presence of alkali metal carbonate. As a result, mixtures are formed that are enriched specifically with D, L-threo-enantiomeric forms of β- (4-methylsulfonylphenyl) serine, and not with D, L-erythro-forms. The separation of the L-threo-enantiomer from the D-threo-enantiomer can then be carried out using conventional chemical separation. The yields, however, are low, not more than 50% of the desired threo isomer from the threo mixture.

 The present invention is a significant improvement in the process for producing L-threo-2-amino-3-hydroxy-3-phenylpropionic acid, 2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid and their derivatives, unsubstituted or containing substituents in phenyl ring. When processing a mixture of D-threo-and L-threo-form-2-amino-3-hydroxy-3-phenylpropionic acid, which, as a rule, but not necessarily racemic, D-threo selectively cleaves with D-threonine aldolase -2-amino-3-hydroxy-3-phenylpropionic acid to glycine and the corresponding benzaldehyde without significant splitting of L-threo-2-amino-3-hydroxy-3-phenylpropionic acid. Similarly, when processing a mixture of the enantiomers of D, L-threo-2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid with D-threonine-aldolase by the method proposed in the present invention, selective cleavage of D-threo-2-amino occurs -2-methyl-3-hydroxy-3-phenylpropionic acid into alanine and the corresponding benzaldehyde, and no significant decomposition of L-threo-2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid occurs. In each case, the corresponding benzaldehyde and the corresponding amino acid can be easily isolated from the mixture for recycling.

 The basis of the present invention was the fact that D-threonine aldolase selectively cleaves the bond between the carbon atom in position 2 (a carbon atom bearing an amino group) and that which is in position 3 (a carbon atom carrying a hydroxy group) in D-threo-2 amino-3-hydroxy-3-phenylpropionic acid or in D-threo-2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid, leaving the corresponding L-threo-isomer predominantly unaffected. Thus, the present invention is particularly suitable for the preparation of the biologically active compounds described above, for example florfenicol, thiamphenicol and L-methyldopa.

в которой каждый
R¹ и R², независимо друг от друга представляют собой водород, гидроксигруппу, галоген, нитрогруппу, трифторметил, низший алкил, низший алкоксил, низший алкилсульфонил, низший алкилсульфинил, или низшую алкилтиогруппу; R³ - водород или метил; R⁴ - водород или карбоксизащитная группа.In a broader sense, the present invention includes enantiomeric enrichment of a mixture of D, L-threo-2-amino-3-hydroxy-3-phenylpropionic acids or D, L-threo-2-amino-2-methyl-3-hydroxy-3-phenylpropionic acids of the formula

in which every
R ¹ and R ² are independently hydrogen, hydroxy, halogen, nitro, trifluoromethyl, lower alkyl, lower alkoxyl, lower alkylsulfonyl, lower alkylsulfinyl, or lower alkylthio; R ³ is hydrogen or methyl; R ⁴ is hydrogen or a carboxy protecting group.

 Also included are the corresponding salts of the aforementioned 2-amino-2-methyl-3-hydroxy-3-phenylpropionic acids.

 By the term lower alkyl is meant a monovalent saturated branched or unbranched hydrocarbon radical containing from 1 to 6 carbon atoms. These include alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, isohexyl and the like.

 By the term “lower alkoxyl” is meant lower alkyl connected to the remainder of the molecule via ether oxygen, for example methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentoxy, hexoxy and the like. groups.

 Halogen is chlorine, bromine, fluorine and iodine.

The carboxy group can be protected with an ester group R ⁴ , which is selectively removed under mild conditions without destroying the desired structure of the molecule, in particular it can be an alkyl ether containing from 1 to 12 carbon atoms in the alkyl part, for example methyl or ethyl ether, and in particular alkyl branched ester at position 1, for example tert-butyl ether; lower alkyl ester which is substituted at position 1 or 2: (1) a lower alkoxy group, for example, it is methoxymethyl, 1-methoxyethyl and ethoxyethyl, (2) a lower alkylthio group, for example, methylthiomethyl and 1-ethylthioethyl; (3) halogen, for example, 2,2,2-trichloroethyl, 2-bromoethyl and 2-iodoethoxycarbonyl; (4) one or two phenyl groups, each of which may be unsubstituted or mono-, di- or trisubstituted, for example with lower alkyl, such as tert-butyl, lower alkoxyl, such as methoxy, hydroxy, halogen, for example chlorine and a nitro group, for example, such as benzyl, 4-nitrobenzyl, diphenylmethyl, di- (4-methoxy) phenylmethyl; or (5) aroyl, such as phenylacyl. The carboxy group may also be protected by an organosilyl group, such as a tri-lower alkylsilyl, for example trimethylsilyloxycarbonyl.

 As indicated, the present invention also extends to salts of the above compounds, in particular salts of alkali metals, alkaline earth metals, ammonium salts and organic amines, for example, salts in which cations are sodium, potassium, magnesium, calcium cations or protonated cations obtained from ethylamine, tyretylamine, ethanolamine, diethylaminoethanol, ethylenediamine, piperidine, morpholine, 2-piperidinoethanol, benzylamine, procaine and the like. Since salts are used as intermediates of a chemical reaction, there is no need to make physiological acceptability requirements for them.

в которой каждый R¹ и R² независимо друг от друга представляет собой водород, гидрокси-, метокси-, метилсульфонил- или метилтио-группу; R³ - водород или метил; R⁴ - водород, C₁-C₃ алкил, или катион (т.е. их соли).In particular, the method is well suited for the preparation of compounds of the formula

in which each R ¹ and R ² independently from each other represents hydrogen, hydroxy, methoxy, methylsulfonyl or methylthio group; R ³ is hydrogen or methyl; R ⁴ is hydrogen, C ₁ -C ₃ alkyl, or a cation (i.e. their salts).

в которой
E¹ - количество одной хиральной формы (такой, как L-трео-форма) и E² - количество другой хиральной формы (такой, как D-трео-форма). Таким образом, если исходное отношение двух хиральных форм равно 50 : 50 (1 : 1), а достигается такое энантиомерное обогащение, при котором обеспечивается конечное отношение L-трео-формы к D-треоформе, равное 50 : 30 (5 : 3), то "и.э." по отношению к L-трео-форме составляет 25%. Если же конечное отношение L-трео-формы к D-трео-форме равно 70 : 30 (7 : 3), то "и.э." по отношению к L-трео-форме составляет 40%. Как правило, в соответствии с предлагаемым способом могут быть достигнуты значения "и.э.", равные 85% и выше.The term "enantiomeric enrichment," as used herein, refers to an increase in the amount of one enantiomer compared to another. A convenient method for expressing the achieved enantiomeric enrichment is to use the concept of excess enantiomer, "ie," expressed as

wherein
E ¹ is the amount of one chiral form (such as the L-threo form) and E ² is the amount of another chiral form (such as the D-threo form). Thus, if the initial ratio of two chiral forms is 50:50 (1: 1), and enantiomeric enrichment is achieved, which ensures a final ratio of the L-threo form to the D-threoform equal to 50: 30 (5: 3), then "IE" in relation to the L-threo form is 25%. If the final ratio of the L-threo-form to the D-threo-form is 70: 30 (7: 3), then "IE" in relation to the L-threo form is 40%. As a rule, in accordance with the proposed method can be achieved values "IE" equal to 85% and above.

 Mixtures of the stereoisomeric forms of 2-amino-3-hydroxy-3-phenylpropionic acid or 2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid are usually obtained by simple condensation of glycine or alanine, respectively, with a suitable benzaldehyde using well known procedures. Since this synthesis is not stereospecific, the result is a racemic mixture of D-threo and L-threo isomers of the corresponding 2-amino-3-hydroxy-3-phenylpropionic acid or 2-amino-2-methyl-3-hydroxy-3- phenylpropionic acid.

 According to the proposed method, a mixture of D, L-threo-2-amino-3-hydroxy-3-phenylpropionic acid or D, L-threo-2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid is treated with D-threonine - aldolase. The enzymatic process affects only one chiral form (or affects one of the chiral forms to a much greater extent than the other). As a result, D-threonine aldolase not only cleaves the undesired D-isomer, thereby facilitating the enrichment of the desired enantiomer, but also affects the bond between the 2-carbon atom and the 3-carbon atom of propionic acid, which results in the formation of the corresponding benzaldehyde and amino acids - glycine or alanine. Benzaldehyde and the amino acid, which are the products of the enzymatic reaction, are at the same time the starting products in the synthesis of D, L-threo-forms of 2-amino-3-hydroxy-3-phenylpropionic acid and 2-amino-2-methyl-3-hydroxy-3 -phenylpropionic acid. The reaction products as such can be isolated and recycled back to the condensation step for further synthesis into 2-amino-3-hydroxy-3-phenylpropionic acid or, respectively, into 2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid.

в которой
R¹, R², R³ и R⁴ - каждый имеет значение, определенное выше.For example, D, L-threo-2-amino-3-hydroxy-3-phenylpropionic acids of the formula (I) are treated with D-threonine aldolase according to the following scheme:

wherein
R ¹ , R ² , R ³ and R ⁴ - each has the meaning defined above.

 D-threonine aldolase enzymes are known and can be obtained, for example, by methods described by Kato et al. and Yamada et al., infra. This enzyme can also be isolated from microorganisms that can adapt to various environmental conditions, ubiquists, living in the soil and not exposed to threonine. In this latter method, microorganisms are grown and incubated in a suitable nutrient broth and D-threonine, and then the culture is re-cultured and incubated in a chemostat from the nutrient broth and D-threonine. After further incubation, a single colony can be isolated that produces the enzyme D-threonine aldolase.

 An enzyme can be isolated from cells using known techniques, for example, those associated with cell disruption and the isolation of an enzyme-containing supernatant. The target enzyme can be used in an unbound form, either in a cell-free extract or in the form of a whole cell preparation, or can be immobilized on a suitable support or matrix such as crosslinked dextran, agarose, silica gel, polyamide or cellulose. The enzyme may also be encapsulated in polyacrylamide, alginates, fibers, etc. Methods of this kind of immobilization are described in the literature (see, for example, Methods of Enzymology, 44, 1976).

In a practical embodiment of the method, the enzyme is added to a mixture of D- and L-threo-2-amino-3-hydroxy-3-phenylpropionic acid or D- and L-threo-2-amino-2-methyl-3-hydroxy-3- phenylpropionic acid and the reaction mixture are then kept at an enzyme activity temperature (at about 40 ^° C.) until the required excess of the L-form enantiomer is achieved. This point can be easily determined using, for example, the HPLC method.

 It is often convenient to remove one reagent from the reaction mixture in order to obtain a higher "IE" according to the law of the masses. There is no need to remove the reagent physically, which is just enough to exclude (isolate) from the zone of the enzymatic reaction. For example, the use of a co-solvent in which one of the reaction products has a preferred solubility contributes to an increase in "IE". Benzaldehydes are readily soluble in halogenated alkanes, such as methylene chloride and chloroform, in lower alkanones containing from 3 to 10 carbon atoms, such as pentan-2-one or methyl isobutyl ketone, and aromatic hydrocarbons, for example benzene. The inclusion of these solvents in the reaction mixture will have a beneficial effect. In another case, the addition of a non-ionic resin of an adsorbing type, in particular Amberlite or Dowex (Amberlite, Dowex), on which the aldehyde is adsorbed, will also lead to an increase in "ue" in accordance with the same principles.

 Following or during the enzymatic reaction, the resulting benzaldehyde and amino acid can be recycled in accordance with the procedure described above. The unsplit L-threo form of 2-amino-3-hydroxy-3-phenylpropionic acid is isolated and treated according to known procedures. For example, it can be esterified and then reduced according to the previously described procedure to form the corresponding 2-amino-3-phenylpropane-1,3-diol. If the phenyl group in position 4 has a methylsulfonyl group as a substituent, then such a diol can simply be dichloroacetylated to give thiamphenicol. If the phenyl group at position 4 is substituted with a methylthio group, then the diol can be dichloroacetylated and then oxidized with peracetic acid, whereby thiamphenicol will also be obtained.

 To obtain florfenicol, thiamphenicol is directly fluorinated using standard conditions again and D-threo-2- (dichloroacetamido) -3- (3-methylsulfonylphenyl) -3-fluoropropan-1-ol is obtained. Alternatively, L-threo-2-amino-3-hydroxy-3-phenylpropionic acid is first esterified and reduced as described above, and the amino group is then protected, for example, by the formation of a phthalimide derivative, which is then fluorinated, and after the removal of the amino protecting group D- threo-2-amino-3- (3-methylsulfonylphenyl) - 3-fluoropropan-1-ol is dichloroacetylated as described above.

 The following examples will serve as illustrative material that is characteristic of the present invention, but does not limit its scope, while the scope of the claims is determined solely by the claims.

Example 1
Racemic D, L-threo-2-amino-3-hydroxy-3- (4-methylthiophenyl) propionic acid (0.7 g) is added to a mixture containing 20 ml of 50 mM sodium borate (pH 8.4), 1 ml 8 mM pyridoxal 5-phosphate and 4 ml of D-threonine aldolase extract. The reaction mixture was kept at 40 ^° C. until an excess of the enantiomer was achieved (determined by HPLC) in an amount of at least 98% L-threo-2-amino-3-hydroxy-3- (4-methylthiophenyl) propionic acid; usually under these conditions the enrichment process lasts approximately 60 - 70 minutes

 Example 2

A mixture of 2.5 g of D-threo-2-amino-3- (4-methylthiophenyl) propane-1,3-diol obtained by the method described in example 1 and 3.6 ml of ethyl dichloroacetate are heated at about 100 ^° C about 3 hours. The reaction product is dissolved in ethylene chloride and filtered through activated carbon. The filtrate was cooled and recrystallized from nitroethane to give D-threo-2-dichloroacetamido-3- (4-methylentiophenyl) propan-1,3-diol, mp. 111.6 - 112.6 ^o C, [α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = +12 ^° (1% in ethanol).

 Example 3

L-threo-2-amino-3-hydroxy-3- (4-methylsulfonylphenyl) propionic acid is obtained from D, L-threo-2-amino-3-hydroxy-3- (4-methylsulfonylphenyl) propionic acid, following the procedure described in Example 1. Then, L-threo-2-amino-3-hydroxy-3- (4-methylsulfonylphenyl) propionic acid is esterified and reduced as described above to give D-threo-2-amino-3- (4-Methylsulfonylphenyl) propane-1,3-diol; mp. 201 - 202 ^o C, [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ = -25.5 ^° .
D-threo-2-amino-3- (4-methylsulfonylphenyl) propane-1,3-diol is then dichloroacetylated in a manner similar to that described in example 2, and as a result, thiamphenicol is obtained, so pl. 164.3 - 166.3 ^o C, [α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = + 12.9 ^° (1% in ethanol).

 Alternatively, thiamphenicol can be prepared from D-threo-2-dichloroacetamido-3- (4-methylthiophenyl) propan-1,3-diol from Example 2 by oxidation with peracetic acid under conventional conditions.

 Example 4

6 g (150 mmol) of sodium hydroxide and 8.9 g (100 mmol) of D, L-alanine are dissolved in 25 ml of water and the solution is cooled to about 5.5 ^° C. with stirring under a nitrogen atmosphere. To the solution was added 21.2 g (200 mmol) of benzaldehyde and the mixture was stirred at 5.5 ^° C. for about 1 hour. Then the mixture was warmed to room temperature and held for about 20 hours. Then, concentrated hydrochloric acid was added and the pH was adjusted to 2. After stirring The acidified solution is separated in 2 hours, the aqueous phase is extracted with ethyl acetate and evaporated in vacuo. The resulting material was extracted twice with 80 ml of absolute ethanol, and then ethanol was evaporated. The resulting product was again extracted with 40 ml of absolute ethanol, followed by removal of ethanol. The extract is then dissolved in an aqueous methanol solution with a ratio of at least 1: 1 and absorbed on a column filled with polymethyl methacrylate. The contents of the column are then eluted therein with a methanol-aqueous solution (1: 1), 30 ml fractions are combined and evaporated. Then, the purification was repeated again, passing through a column filled with polymethyl methacrylate using 90% ethanol. The result is a racemic 2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid.

 Example 5

 The following procedure is confirmed by the example of the use of D-threonine aldolase as a means that allows the efficient isolation of the L-isomer from the racemate of D, L-threo-2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid.

 D, L-threo-2-amino-2-methyl-3-hydroxy-3-phenylpropionic acid (1.4 mM) is maintained at pH 8.75 in 50 mM borate buffer and 0.8 mM pyridoxalphosphate in the presence of disrupted cells, containing about 300 μl of enzyme.

 After certain periods of time, comprising about 30, 120, and 210 minutes, 200 μl of the incubation mixture is taken and diluted with 800 μl of 1% perchloric acid. After centrifugation of the mixture, the upper layer was analyzed by HPLC. The data in the table demonstrate the effectiveness of the enzyme on the process of separation of the racemic mixture.

L-threo-2-amino-3-hydroxy-3- (4-methylthiophenyl) propionic acid obtained in this way can be esterified and reduced with sodium borohydrate according to known methods; the result is D-threo-2-amino-3- (4-methylthiophenyl) propan-1,3-diol, mp. 151.9 - 152.9 ^o C, [α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = -21 ^° (1% in ethanol).

 Example 6

 D-threonine aldolase is produced by various microorganisms, such as Alcaligenes faecalis (deposited at the Osaka Institute of Enzymes, Japan, under IFO-12.669), Pseudomonas DK-2 (deposited at the Institute of Enzyme Research, Agency of Industrial Science and Technology, Ministry of Internacional Trade and Industry, Japan, under the number FERM-P N 6200) and Arthrobacter DK-19, FERM-P N 6201. See, for example, US Pat. No. 4,492,757, Kato et al., And Japan Patent 56-209983, Yamada et al. . 1985.

 In addition, microorganisms producing D-threonine aldolase can be isolated from soil samples as follows.

A soil sample previously treated with threonines is inoculated in a flask shaken with 50 ml of an aqueous medium (hereinafter referred to as “medium A”), which typically contains the following components:
NH ₄ Cl - 1 g / l
MgCl ₂ - 1 g / l
CaCl ₂ - 0.015 g / l
KH ₂ PO ₄ - 2.7 g / l
NaOH - 0.5 g / l
and standard solutions of trace elements - 1 ml / l
MgSO ₄ - 1 g / l
CaCl ₂ - 0.21 g / l
ZnSO ₄ • 7H ₂ O - 0.2 mg / l
MnSO ₄ • 4H ₂ O - 0.1 mg / L
H ₃ BO ₃ - 0.02 mg / l
CuSO ₄ • 5H ₂ O - 0.10 mg / l
CuCl ₂ • 6H ₂ O - 0.05 mg / l
NiCl ₂ • 6H ₂ O - 0.01 mg / l
FeSO ₄ - 1.5 mg / L
NaMoO ₄ - 2.0 mg / L
Fe EDTA - 5.0 mg / L
KH ₂ PO ₄ - 20.0 mm
NaOH - Up to pH 7
The composition of a standard solution of trace elements is not a critical parameter that determines the course of the process, but in all procedures it is standardized in order to eliminate its effect as a variable.

Medium A and soil containing the microorganism were inoculated with 3 g / L D-threonine and incubated at 30 ^° C. with shaking (200 rpm) for 5 days. 1 ml of the sample is then subcultured into the same shaken flask and incubated again, as described above until turbidity. 5 ml of clouded culture is then inoculated in a chemostat with medium A and 3.0 g / l of D-threonine and incubated with constant dilution at a rate of 0.03 / min for two weeks. The liquid medium of their chemostat is plated on a plate or Petri dish with medium A and 15 g / l of noble agar. After an incubation period of 5 days at 30 ^° C., one colony is isolated. An isolated strain is a rod-negative gram-negative microorganism that has been identified as Alcaligenes dentrificans xylosoxydans by analysis of the fatty acids that make up the cell wall.

50 ml of medium A containing 6.0 g / l D-threonine are inoculated with Alcaligenes dentrificans xylosoxydans. After the mixture was incubated at 37 ^° C. for 48 hours, 10 ml of the mixture were transferred to 250 ml of medium A containing 6.0 g / l D-threonine. After 40 h incubation at 37 ^o C the cells by centrifugation at 10000 G was concentrated to a paste and washed with 50 ml phosphate buffer pH 7 and again concentrated to a paste by centrifugation at 10000 G. The washed paste was placed under a French Press at a pressure of 17,000 pounds / ⁱⁿ² (1195 kg / cm ² ) to destroy cells and obtain a cell extract. Cell debris was removed by centrifugation for 1 h at 100,000 G, collecting the top layer containing the enzyme.

 Example 7

 After enrichment of the racemic mixture of D, L-threo 2-amino-3-hydroxy-3- (4-methylthiophenyl) propionic acid or D, L-threo-2-amino-3-hydroxy-3- (4-methylsulfonylphenyl) -propionic acids in the L-threo form regenerate the corresponding 4-methylthiobenzaldehyde or 4-methylsulfonylbenzaldehyde, respectively, from an aqueous solution, lowering its pH to 0.5 by adding hydrochloric acid in order to split Schiff base. The benzaldehyde thus obtained is filtered off and recycled back to the condensation step.

 Example 8

 A mixture of D- and L-threo-2-amino-3-hydroxy-3- (4-methylsulfonyl) propionic acid (0.8 g) is combined with 10 ml of a 50 mM sodium phosphate solution (pH 8.0), 0.5 mM pyridoxalphosphate and 20 ml of methylene chloride. 10 ml of crude D-threonine aldolase is added to initiate the reaction. After 90 minutes "I.E." amounted to 94%.

 Example 9

A mixture of D- and L-threo-2-amino-3-hydroxy-3- (4-methylsulfonyl) propionic acid (2.0 g) is added to 10 ml of a solution of 50 mM sodium phosphate (pH 8.0), 0.8 mM pyridoxal 5-phosphate and 100 mM sodium chloride. Add 20 g of Amberlit XAD-16 brand wetted resin together with 10 ml of crude D-threonine aldolase. The reaction mixture was vigorously stirred at 42 ^° C. for 120 minutes, and as a result, L-threo-2-amino-3-hydroxy-3- (4-methylsulfonylphenyl) propionic acid and c. 96%

Claims (10)

1. The method of enantiomeric enrichment of a mixture of D- and L-threo-2-amino-3-hydroxy-3-phenylpropionic acids or their derivatives of the formula

where R ¹ and R ^{2 are} independently hydrogen, a hydroxyl group, a halogen, trifluoromethyl, lower alkyl, lower alkoxy group, lower alkylsulfonyl, lower alkylsulfinyl or lower alkylthio group;
R ³ is hydrogen or lower alkyl;
R ⁴ is hydrogen or a carboxy protecting group,
or their salts with a base when R ⁴ is hydrogen, characterized in that the said mixture is treated in an aqueous medium with a D-threonine aldolase active with respect to the D-threo enantiomer and practically not active with respect to the L-threo enantiomer until a significant amount of the D-threo enantiomer is converted to benzaldehyde and glycine or alanine. 2. The method according to claim 1, characterized in that a mixture of D- and L-threo-enantiomers of the above compounds is used, in which R ^{1 is} methylthio at position 4 of the phenyl group, R ² is hydrogen. 3. The method according to claim 1, characterized in that a mixture of D- and L-threo enantiomers of the above compounds is used, in which R ¹ is a methylsulfonyl group at position 4 of the phenyl group and R ² is hydrogen. 4. The method according to claim 1, characterized in that a mixture of D- and L-threo enantiomers of the above compounds is used, in which R ³ is methyl.  5. The method according to claim 1, characterized in that the L-threo-enantiomer is isolated from the reaction mixture.  6. The method according to claim 1, characterized in that a substantial amount of at least one of said benzaldehyde and said amino acid derivatives is isolated from the reaction mixture.  7. The method according to claim 1, characterized in that the D-threonine-aldolase treatment process is conducted in the presence of at least one substance or agent capable of removing both benzaldehyde and a glycine or alanine derivative from the reaction mixture.  8. The method according to claim 7, characterized in that as a substance capable of removing one of the reaction products, an organic solvent is used in which one of the reaction products is preferably soluble.  9. The method according to claim 8, characterized in that halogenated alkanes, mainly methylene chloride, are used as the organic solvent.  10. The method according to claim 7, characterized in that as a means capable of removing one of the reaction products, a non-ionic absorbent based on resin is used.

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