CS258132B2 - Method of l-phenylalanine and l-tryphtofan production - 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 invention relates to a process for the preparation of L-phenylalanine and L-tryptophan by enzymatic resolution of the racemate of lower alkyl esters.

German Offenlegungsschrift 2,215,853 discloses the conversion of racemic phenylalanines which are mono- and disubstituted into the corresponding L-amino acids by first esterifying the racemate with an alkanol of up to 4 carbon atoms and treating the racemic ester with chymotrypsin in the acidic region. The pH, L-amino acid is isolated by precipitation, and optionally the D-amino acid ester is extracted from the filtrate and saponified. Preferred as the enzyme is α-chymotrypsin, whose preferred pH range in enzymatic cleavage ranges from 5 to 6. The liquid remaining after separation of the L-amino acid is basified prior to extraction of the D-amino acid ester.

It has now been found that L-phenylalanine and L-tryptophan are particularly advantageously obtained by first alkalinizing the mixture obtained by enzymatic cleavage, then extracting the D-amino acid ester, optionally after drying the extract, the ester is racemized and the racemate is returned to the process . The L-amino acid is then isolated from the alkaline residue after extraction of the D-amino acid ester after acidification.

The present invention provides a process for the preparation of L-phenylalanine and L-tryptophan by enzymatic resolution of the racemate of C 1 -C 4 alkyl esters, except tert-butyl ester, with α-chymotrypsin in the pH range of 4.5 to 6.5 and ester extraction The D-form, which comprises adjusting the reaction mixture obtained by enzymatic cleavage to a pH in the range of 7.5 to 8.5, extracts the D-amino acid ester from this alkaline solution with an organic solvent which is poorly miscible with water, such as hydrocarbons that are optionally chlorinated, lower dialkyl ethers, lower dialkyl ketones as well as lower alkyl esters and racemized, after which the racemate is returned to the process and the extracted alkaline solution is slightly acidified and then the L-amino acid is isolated therefrom.

The esterification of the racemic amino acids can be carried out according to the data disclosed in German Offenlegungsschrift 2,215,853. Preferred are lower alkanols of up to 4 carbon atoms, with the exception of tert-butyl alcohol, especially methanol, ethanol, n-propyl alcohol, isopropyl alcohol and n-butyl alcohol.

The .alpha.-Chymotrypsin is preferably used in fixed form, with inorganic carriers being the preferred carriers. The fixed enzyme is then contacted with an aqueous substrate solution in a solid or liquid bed.

The reaction is conveniently carried out at temperatures of from 20 to 45 ° C, preferably at temperatures of from 30 to 40 ° C, and at a pH in the range of from 4.5 to 6.5, preferably at a constant pH in the range of from 5.0 to 6, 0, depending on the ester used. The substrate concentration is not in itself a critical condition and is suitably 5 to 15% by weight, in particular 10 to 15% by weight.

The aqueous solution of the L-amino acid and the D-amino acid ester obtained after the enzymatic cleavage is slightly alkalinized, preferably to a pH in the range of 7.5 to 8.5, preferably to a pH of 8, and then conveniently at temperatures of 20 to 40 °. C, especially at room temperature, is extracted with a suitable organic solvent.

Suitably, the organic solvent for the extraction is a solvent which is poorly miscible with water, since the racemization can then be carried out particularly advantageously without ester isolation (the presence of water during racemization may be conditional on side reactions, such as hydrolysis). However, it is also possible, although generally not preferred, to completely separate the solvent used for the extraction and then to racemise the ester in the mass.

Suitable solvents for the extraction are aliphatic hydrocarbons such as hexane, especially aromatic hydrocarbons such as toluene or xylene, ethers, especially lower dialkyl ethers such as diethyl ether, diisopropyl ether or di-n-butyl ether, ketones, especially lower dialkyl ketones such as methyl ethyl ketone or methylisobutyl ketone, in particular chlorinated lower aliphatic compounds, such as methylene chloride, chloroform or carbon tetrachloride, and esters, in particular lower alkyl esters of lower aliphatic carboxylic acids, such as ethyl acetate or butyl acetate.

Racemization can be carried out in a manner known per se under catalysis with keto compounds and / or acids. Thus, if the solvent is not a ketone, racemization can be substantially accelerated by the addition of aldehydes or ketones. Racemization of optically active amino acid esters in ketones, optionally with the addition of acid, is known from Japanese Patent Application Publication No. 109 912/1979. In this known racemization reaction, a solvent such as toluene, carbon tetrachloride or methanol may also be added. Racemization of free optically active amino acids by aldehydes and acids is known from European Patent Publication No. 57,092, as well as from S. Yamada et al., J. Org. Chem. 48 (1983) 843-846. Suitably these keto compounds are used in catalytic amounts, preferably in an amount of 0.001 to 0.1 mol per 1 mol of ester. Acetaldehyde and salicylaldehyde are preferred.

The racemization D-, L-mixture is then easily extracted from the racemization mixture with acidified water, converting the ester to a salt. This aqueous solution is brought to the desired concentration by adding a new substrate, preferably to a concentration of 5 to 15% by weight, in particular to 10 to 15% by weight, and returned to the process.

β

The amino acid is isolated from the weakly alkaline aqueous residue containing the amino acid by acidification. Depending on the solubility of the acid, it can be crystallized immediately or after concentration. The acid can also be isolated in a known manner by extraction with suitable solvents. The purification is carried out in a known manner.

The process according to the invention makes it possible to obtain aromatically substituted L-amino acids in yields above 90% and in optical purity above 98%. The combination of enzymatic cleavage, extraction of the D-ester, its racemization and returning to the process results in a particularly expedient combination of these working steps, which also permits continuous operation of the process.

In the following examples, the invention is explained in more detail. The percentages are by weight unless otherwise indicated.

Example 1

T-mobilization of α-chymotrypsin

The enzyme fixation can be carried out on aluminum silicate according to I-Ialwachs et al., Biotechnology and Bioengineering XIX (1977) 1667-1677. However, the following process is particularly preferred:

138 g of dry silica gel (particle size 0.1 0,3 0.3 mm) were suspended in 1 liter of 3.5% acetonic separation of 3-aminopropyltricthoxysilane and the suspension was stirred at room temperature for 2 hours. The acetone was then evaporated on a rotary evaporator and the silica gel was dried in a vacuum oven at 100 ° C for 8 hours. The product contains 0.89 mmol of amino groups per g of silica gel. This aminated silica gel is taken up in 400 ml of a 25% aqueous solution of glutardialdehyde and held for 2 hours at room temperature under vacuum (about 1300 Pa). The carrier is then filtered off and washed thoroughly with demineralized water.

138 g of the activated silica gel are introduced into a solution of 13.8 g of α-chymotrypsin (manufactured by Novo, 800 S Oral Grade) in 300 ml of 0.5 M phosphate buffer (pH 7.5) and the mixture is stirred at room temperature for 4 hours. . The catalyst is then filtered off and washed with 1 liter of demineralized water, saturated sodium chloride solution and again with 1 liter of demineralized water. For storage, the immobilized α-chymotrypsin is taken up in 500 ml of a 0.1 M phosphate buffer solution (pH 7.5, 0.1 mmol sodium azide).

To determine activity, add 1 g of immobilized α-chymotrypsin to 50 ml of a 10% aqueous solution of methyl ester D, L-phenylalanine, adjust the pH to 6.0 and store in a thermostat at 30 ° C, after which the solution spinal cord. The pH is kept constant by the addition of 0.1 N sodium hydroxide solution using an automatic burette. The amount of sodium hydroxide consumed is a measured quantity and is directly proportional to the concentration of L-phenylalanine.

Activity: 338 U / g carrier (dry) = 3.35 g L-phenylalanine / kg carrier and h (U = International Unit for Enzyme Activity, 1 U = 1 µmol / min Kouverse).

Example 2

Enzymatic resolution of the racemate

A thermostatically heated reaction vessel equipped with an internal stirrer, pH electrode and automatic burette was charged with 100 g of D, L-phenylalanine methyl ester hydrochloride (residual D, L-phenylalanine content below 0.3 why), by high performance liquid chromatography) and dissolved in 1 liter of demineralised water. The pH of the solution was adjusted to 6.0 with 5N sodium hydroxide solution (9.5 ml). The solution was thermostatized to 30 ° C and circulated through the pump at a flow rate of 5.0 liters / hour through a column filled with 150 ml of the enzyme fixed according to Example 1 (column diameter 5.0 cm, bed height 8 cm).

The pH of the solution was maintained at 6.0 by adding 5N sodium hydroxide solution (37.8 mL) during the reaction. After a reaction time of 25 minutes, the high-performance liquid chromatography conversion was 49.2% based on D, L-phenylalanine methyl ester. The reaction is then stopped and the enzyme bed is washed with 300 ml of demineralized water.

Example 3

Extraction of D-phenylalanine methyl ester followed by racemization

The aqueous solution of D-phenylalanine methyl ester and L-phenylalanine methyl ester obtained according to Example 2 was adjusted to pH 8.0 by addition of 5N sodium hydroxide solution (36.5 ml) with stirring and then extracted three times with 300 ml of methylisobutyl ketone each. After the third extraction, no ester is present in the aqueous phase according to HPLC analysis (yield limit: below 0.2%). To the combined extracts was added 14.0 g of glacial acetic acid (0.5 mol equivalents, based on D-ester) and the water contained in the extract was azeotropically distilled through a Vigreux column. The dried extract was heated under reflux for a further 2 hours to determine the degree of racemization by determining the optical rotation of the solution using a polarimeter. After cooling to room temperature, the solution is extracted three times with 300 ml of 0.05 N hydrochloric acid each. In the combined aqueous phases, 98.5% of the este258132 r used was found by high performance liquid chromatography analysis. This aqueous solution is adjusted to 10% by addition of methyl ester D, L-phenylalanine hydrochloride and used for further enzymatic cleavage.

When n-hexane, di-n-butyl ether, toluene or xylene is used as the extracting agent, a catalytic amount of acetaldehyde, salicylaldehyde or benzaldehyde is added to accelerate the reaction.

Hydrochloric acid, benzoic acid, citric acid, p-toluenesulfonic acid or formic acid may also be used instead of glacial acetic acid. In the absence of acid, racemization proceeds much slower and requires more than 10 hours. An acid concentration above 0.5 mol equivalents does not cause any further increase in velocity.

Example 4

Processing and isolation of L-phenylalanine

The solution obtained after extraction with methylisobutyl ketone according to Example 3 containing about 3.5% L-phenylalanine is adjusted to pH 5.5 (isoelectric point of phenylalanine) by adding concentrated hydrochloric acid and concentrated on a rotary evaporator to about 10% L-phenylalanine . The precipitated L-phenylalanine is dissolved by heating to 90 ° C and the solution is filtered while hot. The clear solution is allowed to stand at room temperature, whereby L-phenylalanine precipitates as large crystals. The mother liquor was left at + 5 ° C overnight, leaving white crystals, which were filtered off and washed with a little cold water. The crystals are then dried for 3 hours at 105 ° C in a vacuum oven. 35.3 g of L-phenylalanine or 92% yield based on 50 g of L-phenylalanine methyl ester hydrochloride used are obtained.

The optical purity is between 98.5 and 99.5%.

Example 5

Enzymatic resolution of the racemate:

Ethyl ester D, L-phenylalanine

In a manner analogous to Example 2, 150 g of D, L-phenylalanine ethyl ester hydrogen sulfate are dissolved in 1 liter of demineralized water. The pH of the solution was adjusted to 5.5 with 5N sodium hydroxide solution. The solution was maintained at 40 ^DEG C. with a thermostat and circulated through the pump at a flow rate of 60 liters / hour through the column, maintaining the pH at 5.5 throughout the reaction time by adding 5N sodium hydroxide solution.

Further processing is carried out in an analogous manner to Examples 3 and 4.

Example 6

Enzymatic resolution of the racemate:

n-propyl ester of D, L-phenylalanine

In an analogous manner to that described in Example 5, n-propyl ester D, L-phenylalanine hydrogen sulphate was reacted, but the pH was adjusted to 5.3 and maintained at that value. Further processing is carried out analogously to Examples 3 and 4.

The isopropyl ester or the n-butyl ester D, L-phenylalanine can also be used with the same success.

Example 7

L-tryptophan

In a manner analogous to Example 2, 50 g of D, L-tryptophan methyl ester hydrochloride is dissolved in 1 liter of demineralized water and then cleaved as described above. D-tryptophan methyl ester was extracted from the aqueous solution thus obtained as described in Example 3, and the remaining aqueous solution was adjusted to pH 5.9 (tryptophan isoelectric point) by addition of concentrated hydrochloric acid analogously to Example 4 and concentrated to about 5 on a rotary evaporator. % L-tryptophan. The precipitated crystals are filtered, washed with cold water and dried at 105 ^DEG C. for 3 hours. 19.4 g (97% yield) based on 25 g of L-tryptophan methyl ester hydrochloride used are obtained. The optical purity was 98.7%.

Claims (5)

CLAIMS 1. A process for the preparation of L-phenylalanine and L-tryptophan by enzymatic resolution of the racemate of a C1 -C4 alkyl ester, with the exception of the tert-butyl ester, by α-chymotrypsin in the pH range of 4.5 to 6.5 and extraction of the D- ester. characterized in that the reaction mixture obtained by enzymatic digestion is adjusted to a pH in the range from 7.5 to 8.5, from this alkaline solution the D-amino acid ester is extracted with an organic solvent which is poorly miscible with water , such as hydrocarbons that are optionally chlorinated, lower dialkyl ethers, lower dialkyl ketones as well as lower alkyl esters and racemized, after which the racemate is returned to the process and the extracted alkaline solution is slightly acidified and then the L-amino acid is isolated therefrom. 2. A process according to claim 1, characterized in that solvents which form an azeotropic mixture with water, such as methyl isobutyl ketone, are used. 3. The process according to claim 1, wherein the optionally dried D-ester extract is racemized. 4. The process of claims 1 to 3, wherein the racemized ester is extracted under acidic conditions and returned to the process after concentration. 5. A process according to any one of claims 1 to 4, wherein the ester is methyl, ethyl, n-propyl, isopropyl or phenylalanine n-butyl ester or tryptophan.