NO854127L - PROCEDURE FOR THE PREPARATION OF AROMATIC SUBSTITUTED L-AMINO ACIDS. - Google Patents

Abstract

1. A process for the preparation of aromatically substituted L-amino acids by enzymatic cleavage of the racemate of the lower alkyl esters (except tert.-butylester) using alpha-chymotrypsin in the weakly acid pH range and extraction of the ester of the D form, which comprises the reaction mixture obtained in the enzymatic cleavage being made weakly alkaline, the ester of the D-amino acid being extracted from this alkaline solution using an organic solvent, and being racemized, whereupon the racemate is returned to the process, and the extracted alkaline solution being made weakly acid, whereupon the L-amino acid is isolated from it.

Description

The present invention relates to a method for producing aromatically substituted L-amino acids.

From the German specification no. 2 215 853 it is known to transfer racemic mono- and di-ring-substituted phenyl-alanines to the corresponding L-amino acids by first esterifying the racemate with an alkanol with up to 4 carbon atoms, subjecting the racemic ester to the influence of a chymotrypsin at an acidic pH value, isolates the L-amino acid by precipitation and optionally extracts and saponifies the ester of the D-amino acid from the filtrate. As an enzyme, α-chymotrypsin is preferred, the preferred pH range for the enzymatic cleavage is 5 to 6. The liquid that occurs after the separation of the L-amino acid is made alkaline before extraction of the ester of the D-amino acid.

It has now been found that aromatically substituted L-amino acids become particularly easily available by first making the mixture alkaline during the enzymatic cleavage, then extracting the ester of the D-amino acid, possibly racemizing the ester after drying the extract and returning the racemate to the process . From the alkaline residue after the extraction the ester of the D-amino acid is then isolated

The L-amino acid after acidification.

The invention therefore relates to a process for the production of aromatically substituted L-amino acids by cleavage of the racemic lower alkyl ester with α-chymotrypsin in a weakly acidic aqueous medium, which is characterized by the fact that the reaction mixture is made weakly alkaline, the ester of the D-amino acid is extracted with an organic solvent , the ester is racemized and fed back into the process, and the L-amino acid is isolated from the aqueous alkaline extract after acidification. Preferred embodiments of this method are described in more detail below, respectively defined in the patent claims.

As aromatically substituted amino acids, above all tryptophan, especially phenylalanine, tyrosine and 3,4-dihydroxy-phenylalanine in consideration.

The esterification of the racemic amino acids can be carried out as indicated in the German specification 2 215 853. Preferred are lower alkanols with up to 4 carbon atoms (except tert.-butanol), especially methanol, ethanol, n-propanol, isopropanol and n-butanol. .α-chymotrypsin is preferably used in fixed form, in this connection inorganic carriers are preferred. This fixed enzyme is then brought into contact with the aqueous substrate solution in a fixed or fluidized bed. The reaction conveniently takes place at 20 to 45°C, preferably at 30<0>C-40°C, and at a pH value in the range from 4.5 to 6.5, preferably at a constant pH value in the range from 5 .0 to 6.0, depending on the ester used. The substrate concentration in and of itself is not critical, but is suitably 5-15% by weight, especially 10-15% by weight.

The aqueous solution of the L-amino acid and the ester of the D-amino acid obtained after the enzymatic cleavage is made slightly alkaline, advantageously adjusted to a pH value in the range from 7.5 to 8.5, preferably 8.0, and extracted with a suitable organic solvent advantageously at a temperature of from 20 to 40°C, especially at room temperature.

As an organic solvent for the extraction, a solvent that is poorly miscible with water is advantageously used, since the racemization can particularly advantageously take place without isolation of the ester (the presence of water during the racemization can condition side reactions such as hydrolysis). Advantageous is therefore a solvent which forms an azeotope with water and thereby enables easy drying of the extract. However, it is also possible, if not generally advantageous, to completely separate the solvent used for the extraction and racemize the ester in solid form.

Suitable solvents for the extraction are aliphatic hydrocarbons such as hexane, in particular aromatic hydrocarbons such as toluene or xylol, ethers, in particular lower dialkyl ethers such as diethyl ether, di-isopropyl ether or di-n-butyl ether, ketones, in particular lower dialkyl ketones such as methyl ethyl ketone or methyl isobutyl ketone, halogenated hydrocarbons, in particular chlorinated lower aliphatics such as methylene chloride, chloroform or tetrachloromethane as well as esters, especially lower alkyl esters of lower aliphatic carboxylic acids such as ethyl acetate or butyl acetate.

The racemization can also be catalyzed in a manner known per se by means of keto compounds and/or acids. If the solvent is not a ketone, the addition of aldehydes or ketones can substantially accelerate the racemization. The racemization of optically active amino acid esters in ketones, optionally with the addition of an acid, is known from the published Japanese patent application 109 912/1979. In this known racemization reaction, a solvent such as toluene, tetrachloromethane or methanol can also be added. The racemization of free optically active amino acids with aldehydes and acids is known from published European patent application 57,092, as well as from S. Yamada et al., J. Org.Chem. 48 (1983) 843-846. Preferably, these keto compounds are used in catalytic amounts, preferably 0.001 to 0.1 mol per moles of ester. Preferred are acetaldehyde and salicylaldehyde.

The D, L mixture that occurs during the racemization is then extracted using slightly acidified water from the racemization mixture, thereby transferring the ester in salt form. This aqueous solution is adjusted by adding new substrate to the desired concentration, preferably 5 to 15, especially 10 to 15% by weight, and fed back into the process.

From the weakly alkaline, aqueous residue containing a large amount of the L-amino acid, this is isolated by acidification. Depending on the solubility of the acid, it can crystallize immediately or after concentration. The acid can also be isolated in a known manner by extraction with suitable solvents. The cleaning takes place in a known manner.

The method according to the invention allows the production of aromatically substituted L-amino acids in yields of over 90% and with an optical purity of over 98%. By the combination of enzymatic cleavage, extraction of the D-ester, racemization and its return, a particularly advantageous combination of these steps has been achieved, which allows a continuous execution of the process.

In the following examples, the invention is described in more detail. Percentages apply to % by weight unless otherwise stated.

Example 1

Immobilization of α-chymotrypsin

The fixation of the enzyme can take place on aluminum silicate according to Halwachs et al., "Biotechnology and Bioengineering XIX"

(1977) 1667-1677. The following working method is nevertheless particularly preferred: 138 g of dry silica gel (grain size 0.1 to 0.3 mm) is taken up in 1 1 3.5% acetonic 3-aminopropyl-triethoxysilane solution and stirred gently for 2 hours at room temperature. The acetone is then removed in a rotary evaporator and the silica gel is dried in a vacuum drying cabinet for 8 hours at 100°C. The product contains 0.89 mmol amino groups per g silica gel. This arained silica gel is taken up in 400 ml of 25% aqueous glutardialdehyde solution and kept for 2 hours at room temperature under vacuum (approx. 130 mbar). The carrier is then filtered off and washed thoroughly with desalted water.

138 g of this activated silica gel is introduced into a solution of 13.8 g of α-chymotrypsin (from the company Novo, "800 S Oral Grade") in 300 ml of 0.5 m phosphate buffer (pH 7.5) and stirred in 4 hours at room temperature. The catalyst is then filtered off and waxed successively with 1 1 of desalted water, saturated sodium chloride solution and desalted water. For storage, the immobilized α-chymotrypsin is taken up in 500 ml of 0.1 m phosphate buffer solution (pH 7.5, 0.1 mmol sodium azide).

For activity determination, 1 g of the immobilized α-chymotrypsin is added to 50 ml of a 10% aqueous solution of D,L-phenylalanine methyl ester hydrogen chloride, the pH is adjusted to 6.0 and the temperature is maintained using a thermostat at 30°C, and the solution is stirred. The pH value is kept constant by the addition of 0.1 N caustic soda using an automatic burette. The amount of caustic soda used is the measure and is directly proportional to the concentration of L-phenylalanine.

Activity: 338 U/g carrier (dry) = 3.35 kg L-phenylalanine/kg carrier

(U = international unit of enzyme activity,

1 U = 1 pinol/minute turnover.

Example 2

Enzymatic racemate resolution

In a reaction vessel with a thermostat, equipped with an inner tube, pH electrode and automatic burette, 100 g of D,L-phenylalanine methyl ester hydrogen chloride (residual content of D,L-phenylalanine below 0.3%, the detection limit by HPLC) is added and dissolved in 1 1 desalinated water. The pH value of the solution is adjusted to 6.0 with 5 N caustic soda (9.5 ml). The solution is set using the thermostat at 30°C and pumped at a flow rate of 5.0 1 per hour in circulation over a column filled with 150 ml of a fixed enzyme prepared as in Example 1 (5.0 cm cross-section, 8 cm height).

The pH value of the solution is maintained during the reaction at 6.0 by the addition of 5 N caustic soda (37.8 ml). After 25 minutes of reaction time, according to HPLC analysis, a conversion of 49.2% has been achieved, calculated on the basis of D ,L-phenylalanine methyl ester. At this point, the reaction is stopped and an enzyme bed is washed with 300 ml of desalted water.

Example 3

Extraction of D-phenylalanine methyl ester and subsequent racemization

The aqueous solution of D-phenylalanine methyl ester and L-phenylalanine prepared in example 2 is adjusted with stirring by the addition of 5 N caustic soda (36.5 ml) to pH 8.0, and decanted at room temperature three times with 300 ml of methyl isobutyl ketone. After the third extraction, in the aqueous phase, according to HPLC analysis, ester is no longer present (detection limit: below 0.2%). The combined extracts are mixed with 14.0 g of glacial acetic acid (0.5 mol equivalents, calculated on the basis of D-ester) and water present in the extract is azeotropically distilled off over a Vigreux column. The dry extract is heated for a further 2 hours under reflux, whereby the degree of racemisation is determined by determining the optical rotation value of the solution using a polarimeter. The solution is decanted, after cooling to room temperature, three times with 300 ml of 0.01 N hydrochloric acid. According to HPLC analysis, 98.5% of the ester used is found in the combined aqueous phase. This aqueous solution is adjusted by the addition of 10% D,L-phenylalanine methyl ester hydrogen chloride and used for a further enzymatic cleavage.

If n-hexane, di-n-butyl ether, toluene or xylol is used as the extraction agent, catalytic amounts of acetaldehyde, salicylaldehyde or benzaldehyde are added to increase the reaction rate.

Instead of glacial acetic acid, hydrochloric acid, benzoic acid, citric acid, p-toluenesulfonic acid or formic acid can also be used. Without acids, the racemization proceeds much more slowly and requires more than 10 hours. Acid concentrations above 0.5 mol equivalents do not cause any further increase in speed.

Example 4

Preparation and isolation of L-phenylalanine

The solution resulting after the extraction with methyl isobutyl ketone according to example 3, which contains approx. 3.5% L-phenylalanine, adjusted with concentrated hydrochloric acid to pH 5.5 (isoelectric point for phenylalanine) and evaporated in a rotary evaporator to a content of approx. 10% L-phenylalanine. The precipitated L-phenylalanine is brought into solution by heating to 90°C and the solution is filtered while hot. The clear solution is allowed to stand at room temperature, whereby the L-phenylalanine precipitates out in large crystals. The mother liquor is stored overnight at + 5°C, thereby white crystals precipitate out which are filtered off and washed with a little cold water. The crystals are dried for 3 hours at 105°C in a vacuum drying cabinet. 35.3 g of L-phenylalanine or 92% is obtained, calculated on the basis of 50 g of L-phenylalanine methyl ester hydrogen chloride used. The optical purity is 98.5 to 99.5%.

Example 5

Enzymatic racemate cleavage:

D,L-phenylalanine ethyl ester

Similarly to example 2, 150 g of D,L-phenylalanine ethyl ester hydrogen sulphate are dissolved in 1 1 of desalted water. The pH value of the solution is adjusted to 5.5 with 5 N caustic soda. The solution is set by means of a thermostat at 40°C and is pumped at a flow rate of 60 l/hour in a circuit over the column, while the pH value is kept at 5.5 during the entire reaction by the addition of 5 N caustic soda.

J) a further processing takes place corresponding to examples 3 and 4.

Example 6

Enzymatic racemate cleavage:

D,L-phenylalanine n-propyl ester

Analogous to example 5, D,L-phenylalanine-n-propyl ester hydrogen sulfate is reacted, except that the pH value is set and kept at 5.3. The further processing takes place analogously to examples 3 and 4.

With similarly favorable results, the isopropyl or n-butyl ester of D,L-phenylalanine can also be used.

Example 7

L-tryptophan

Analogously to example 2, 50 g of D,L-tryptophan methyl ester hydrogen chloride are dissolved in 1 1 of desalted water and split enzymatically as described. From the prepared aqueous solution, the D-tryptophan methyl ester is extracted by the method according to example 3, and the remaining aqueous phase is adjusted analogously to example 4 with concentrated hydrochloric acid to pH 5.9 (isoelectric point for tryptophan) and evaporated in a rotary evaporator to a content of approx. 5% L-tryptophan. The precipitated crystals are filtered off, washed with cold water and dried at 105°C for 3 hours. 19.4 g or 91% are obtained, calculated on the basis of 25 g of L-tryptophan methyl ester hydrogen chloride used. The optical purity is 98.7%.

Claims (9)

1. Process for the production of aromatically substituted L-amino acids by enzymatic cleavage of the racemate of the lower alkyl ester with α-chymotrypsin in a slightly acidic pH range and extraction of the ester of the D form, characterized by making the reaction mixture obtained by the enzymatic cleavage weak alkaline, from this alkaline solution is extracted and racemized .the ester of the D-amino acid with an organic solvent, after which the racemate is fed back into the process, and the extracted alkaline solution is made slightly acidic, whereupon the L-amino acid is isolated from it. 2. Method according to claim 1, characterized in that the α-chymotrypsin is used in fixed form. 3. Method according to claim 2. characterized in that the α-chymotrypsin is attached to an inorganic carrier. 4. Method according to one or more of the preceding claims, characterized in that an organic solvent is used which is poorly miscible with water for extraction of the D-ester. 5. Method according to claim 4, characterized in that a solvent is used which forms an azeotropic mixture with water. 6. Method according to one or more of the preceding claims, characterized in that the optionally dried extract of the D-ester is subjected to racemisation. 7. Method according to one or more of the preceding claims, characterized in that the racemized ester is extracted in an acidic state and, after amplification, is fed back into the method. 8. Method according to one or more of the preceding claims, characterized in that the ester is an alkyl ester with 1 to 4 carbon atoms, excluding tert-butyl. 9 . Method according to one or more of the preceding claims, characterized in that the ester is a methyl, ethyl, n-propyl, isopropyl or n-butyl ester of phenylalanine or tryptophan.