DE2148953B2 - PROCESS FOR THE PREPARATION OF L-3,4 DISUBSTITUTED PHENYLALANINES - Google Patents

Description

IO R ¹ O

in which the radicals R ¹ and R ^{2 can be the} same or different from one another and each represent a hydrogen atom or a methyl group with the proviso that R ^{2 is} not a methyl group when R ^{1 is} a hydrogen atom by reacting a 3,4-disubstituted phenylpyruvic acid the general formula

(I)

NH ₂

20th

R ¹ O

R ² O

CH ₂ COCOOH (II)

in which the radicals R ¹ and R ^{2 have} the meaning given above, with an aminating agent in the presence of a transaminase generated by microorganisms in the temperature range from 25 to 45 ° C, a pH of 6.5 to 10.5 and a reaction time of 20 to 60 hours, characterized in that a transaminase produced by bacteria of the genera Alcaligenes is used.

The invention relates to a method of manufacture of L-3,4-disubstituted phenylalanines of the general formula

40

R ¹ O

45

R ² O

CH ₂ CHCOOH

NH ₂

(D

in which the radicals R ¹ and R ^{2 can be the} same or different from one another and each represent a hydrogen atom or a methyl group with the proviso that R ^{2 is} not a methyl group when R ^{1 is} a hydrogen atom by reacting a 3,4-disubstituted phenylpyruvic acid the general formula

R ¹ O

R ² O

CH, COCOOH (II)

in which the radicals R ¹ and R ^{2 have} the meaning given above, with an aminating agent in the presence of a transaminant generated by microorganisms in the temperature range from 25 to 45 ° C., a pI value of 6.5 to 10.5 and a reaction time of 20 until (SO hours.

wherein R ¹ and R ^{2 can be} a hydrogen atom or a methyl group, where both substituents can be the same or different, and R ^{2 is} a methyl group when R ^{1 is} a hydrogen atom, previously produced by physical, chemical methods and using enzymes. A mixture of the D- and L-form of a 3,4-disubstituted phenylalanine (hereinafter referred to as "D- or L-compound"), as it is chemically synthesized, can be broken down into the optically pure compounds of the L- However, the yield higher than 50% of the compound cannot be obtained unless the D-compound obtained as a by-product is rearranged to the L-compound. Therefore, the decomposition method for separation into the optical components has great disadvantages when carried out on an industrial scale.

For the production of L-dopa by fermentation, a method has been proposed in which L-tyrosine derivatives such as L-N-formyl tyrosine with a fungus-derived polyphenol oxidase (Charles J. S i h et al. "Journal of the American Chemical Society", Vol. 91, P. 6204 [1969]). Another method involves condensing catechol with a special amino acid, such as with tyrosine, serine or cysteine. For this purpose, tyrosine phenollyase is used, obtained from bacteria (Ya mad a, Ogata et al. »Biochemical and Biophysical Research Communications ", Vol. 34, p. 266 [1969]).

From DT-OS 20 41418 are microorganisms known that have the property of 3,4-disubstituted Convert phenylpyruvic acids into L-3,4-disubstituted phenylalanines. This includes the bacteria Pseudomonas, Bacillus, Micrococcus. Sarcina. Brevibacterium and the types of mold Aspergillus, Penicillium and Mucor, as well as the yeasts Candida and Saccharomyces.

It has now become an advantageous and industrially feasible process for the production of L-3,4-disubstituted phenylalanine compounds (I) found from compounds of the general formula (II).

The invention thus relates to a process for the preparation of L-3,4-disubstituted phenylalanines of the type described at the outset, which is characterized in that one of bacteria the transaminase generated by the gations Alcaligenes uses.

In the method according to the invention, Alcaligenes bacteria are thus under the stated conditions

^ 2148

cultured in a culture medium that contains one or more of the following substances: glucose, Sucrose, maltose, lactose or starch hydrolyzate is carbon source, meat extract, peptone, polypeptone, Casamino acid, corn water, ammonium salts or protein hydrolysates as a nitrogen source as well as sodium chloride, phosphates, metal ions, vitamins and growth substances. So an enzyme is obtained that shown in the general formula (II) Can convert compounds into the compounds represented by the general formula (I), and in the bacterial cell or in their fermentation broths. The method of the invention yields the compounds of formula (1) in very high yields.

Although these optimal fermentation conditions according to the selected microorganism can be varied, the following productivity results for the Products of these microorganisms receive:

The bacteria are inoculated into the culture medium, which consists of 0.5% by weight meat extract, 1.0% by weight Peptone and 0.5 wt% sodium chloride. It will be at a temperature of 30 C for 48 hours cultivated. In the presence of the wet cells obtained the compounds corresponding to the general formula (II) at a temperature of 37 ° C Reacted with the amino acid for 1 hour.

The compounds thus prepared and represented by the general formula (1) become quantitative determined by the following methods.

The L-3,4-dihydroxyphenylalanine is determined by the trihydroxyindole method (Euler, U.S. by: Hormones in Blood, Academic Press [1969]).

The L-3,4-dimethoxyphenyl-alanine and the L-3-methoxy-4-hydroxyphenyl-alanine are in the N-trifluoroacetyl-n-butyl ester converted according to the procedure described in the following reference: Charles W. Grehek et al., “Analytical Chemistry ", Vol. 37, p. 383 (1965). The N-trifluoroacetyl-n-butyl ester thus prepared is subjected to gas-liquid chromatography quantified. The enzymatic results are derived from these analytical results Determines the activities of the microorganisms. The unit of enzyme activities is expressed by the amount of the enzyme containing 1 mg of the compound represented by the general formula (I) forms at a temperature of 37 C over a period of 1 hour. As an enzymatic reaction system a system was used in which the enzyme solution obtained from one ml of the culture solution is mixed with a solution of the following composition:

0.5 m phosphate buffer solution 0.5 ml

0.01% (wt.; Vol.) Pyridoxal

phosphate 0.5 ml

¹ _lh m potassium L-glutamate 0.5 ml

^l : _ih m potassium L-asparaginate 0.5 ml

'/ "M connections of general

Formula (II), 1.0 ml ^

The total amount was made up to 5 ml.

In the following overview, the compounds (A), (B) and (C) show the results for L-3,4-dihydroxyphenyl-alaniii, LO-Methoxy ^ -hydroxy-phenyl-alanine and L-S ^ -dimethoxyphenylalanine, respectively.

The amount of enzyme produced in 100 ml of the fermentation broth for various bacteria.

953

is expressed by the number of units defined above

Amount of enzyme in the product obtained from bacteria

bacterium Classic Enzyme- (Units / 100 ml) Ver fiction mcnge L bond number Ver Ver (C) binding binding 69 (Aj (B) Alcaligenes 1015 98 53 I AM*) faecalis

*) Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan.

In the thus obtained transaminase containing the compounds represented by the general formula (II) into the compounds represented by the general formula (I) by means of a transamination reaction can convert, it is very advantageous that the transaminase for an industrial scale Process for the preparation of the L-3.4-disubstituted phenylalanine compounds according to the form! (I) can be used. It can be the ground cell fluid, the dried or wet Cells or the fermentation broths of these microorganisms for transamination as transaminase can be used.

L-glutamic acid can be used as the aminating agent in the process according to the invention. L-alanine. L-aspartic acid, glycine or mixtures thereof, peptone or protein hydrolysates such as, for example Wheat protein hydrolyzates can be used.

The compounds represented by the general formula (I) can be easily isolated by using the reaction solution acidified with a mineral acid, the cells or the mycelium and the soluble protein of the Separating the reaction solution, the unreacted keto acid with organic solvents, such as Ethyl acetate, and then the purified solution thus prepared is concentrated. Then by precipitation at the isoelectric point, continued working with ion exchange resins or with adsorbents.

Of the compounds according to formula (11 can L-S-Methoxy ^ -hydroxyphenyl-alanine or L- 3,4-dimeihoxyphenylalanine by enamelylation can be converted into L-dopa obtained in a high yield and an optical 100% purity.

The method according to the invention is illustrated in the following examples.

example 1

Alcaligenes faecalis IAM 1015 was inoculated in 5 l of a culture medium which was composed of 0.5% glucose, 0.5% casamirt acid, 0.5% yeast extract, 0.1% sodium chloride, Consisted of 0.1% monopotassium phosphate and 0.05% magnesium sulfate. With exercise and ventilation was at an initial pH of 7.0 and at a temperature of 30 ° C for 64 hours cultivated. The bacterial cells were removed from the culture fluid by centrifugation. 35 g of the wet cells obtained in this way were in 300 m! a solution suspended by dissolving

3 g of 3,4 - dihydroxyphenylpyric acid, 3 g of L-glutamic acid and 3 g of L-aspartic acid in aqueous Ammonia had been adjusted to a pH of 7.5. This liquid was taking Movement or stirring kept at a temperature of 30 ° C.

After 18 hours, the tent and the protein removed from the reaction liquid and added the reaction liquid to activated carbon. That adsorbed L-dopa was eluted with water, and the eluate was then concentrated and in the cold ditched. 1.4 g of crude crystals of L-3,4-dihydroxyphenylalanine were obtained. 1.0 g of pure L-dopa was obtained by recrystallizing the crude L-dopa from water. By means of IR analysis and Dünr ^ Layer chromatography found that the L-Dopa was a standard product.

[«] '/ = -13.2 ° (c = 3.86 in In-HCL 1 = 1).

Example 2 _m

Alcaligenes faecalis IAM 101 *> was in 400 ml of a nutrient medium consisting of 1.0% glucose. 3.0% polypeptone, 0.3% sodium chloride, 0.15% potassium phosphate and 0.01% magnesium sulfate. While shaking was at a starting pH of 7.0 cultivated at a temperature of 30 ° C for 48 hours. The culture liquid wedge: was with 2 1 one Solution combined in which 12.0 g of 3,4-dimethoxyphenylpyruvic acid, 6.5 g of L-glutamic acid and 6.5 g of L-aspartic acid in a 1-N aqueous Potassium hydroxide solution had been dissolved to adjust the pH to 8.3. The st preserved Solution was kept at a temperature of 30 ° C. with agitation for 40 hours. Subsequently was the pH of the solution is adjusted to a pH of 2.5 with 10% aqueous hydrochloric acid and centrifuged. The obtained supernatant liquid was concentrated under reduced pressure until the total volume had been reduced to about 600 ml. The solution thus obtained was used twice Washed with about 300 ml of ethyl acetate solution to remove the unreacted 3,4-dimethoxyphenylpyruvic acid to extract and separate. The aqueous solution was concentrated to about 300 ml, and then the pH was adjusted to 4.5. It was left to stand overnight and 7.5 g was obtained crude crystals of L-3,4-dimethoxyphenyl-alanine. The L-3,4-dimethoxyphenylalanine remaining in the mother liquor was adsorbed on activated carbon, and the adsorbate was eluted with water. The eluate was concentrated and left in one cold room. This gave 1.8 g of crude crystals.

The purities of these crude crystals were 89 and 94% as determined by gas chromatography could. The crude crystals were dissolved in 100 ml of an about 2% aqueous solution of hydrochloric acid and washed with ethyl acetate to obtain white crystals. Those white crystals were recrystallized twice by precipitation at the isoelectric point. The white ones obtained afterwards Crystals were further recrystallized from water, and 5.5 g of pure L-3,4-dimethoxyphenyl-alanine crystals were obtained, which were examined by means of thin-layer and gas chromatography.

The physical and chemical properties of these crystals were as follows:

Melting point: 203.0 to 209.9 C (decomposition).

Optical rotation: | Vj? - -5.75 " {c = 3.30. In In-HClJ = 1).

IR Absorption Spectrum (Nujol Mull) 3230 (NH ₃ ⁺ ), 2150-2800, 1610 (COCT) [cm " ¹ ].

The crystals were blocked with hydrobromic acid L-dopa demethylated, which had an optical purity of 100%.

[α] «= -12.7 (f = 3.5α in 1N-HCl, 1 = 1).

Example 3

Alcaligenes faecalis IAM 1015 was inoculated into 500 ml of a nutrient medium consisting of 1.0% glucose, 3.0% polypeptone, 0.3% sodium chloride, 0.15% potassium phosphate and 0.01% magnesium sulfate. It was cultured with agitation at an initial pH of 7.0 and at a temperature of 30 ° C. for 48 hours. The culture liquid was combined with 2 1 of a solution in which 15.0 g 3 - methoxy hydroxyphenylpyruvic -A-. 15.0 g of L-glutamic acid and 15.0 g of L-aspartic acid were dissolved in 1N aqueous potassium hydroxide solution to adjust the pH to 8.5. The solution thus prepared was kept under agitation at 30 ° C. for 20 hours, and then the pH of the solution was adjusted to 2.5 with a 10% aqueous hydrochloric acid solution and centrifuged. The resulting supernatant liquid was concentrated under reduced pressure until the total volume became about 300 ml. This solution was filtered off, and the filtrate was washed twice with the same amount of ethyl acetate as in the extraction and recovery of 3-methoxy-4-hydroxyphenylpyruvic acid from the filtrate. The pH of the aqueous layer was adjusted to 4.5, and the aqueous layer was left to stand in a cold room overnight. 5.2 g of crude crystals of L-3-methoxy-4-hydroxyphenylalanine were obtained. The purity of the crystals was 90% and was determined by gas chromatography. The crystals were dissolved in 100 ml of an about 2% aqueous solution of hydrochloric acid and then washed with ethyl acetate. The crystals thus treated were purified twice by precipitation at the isoelectric point, and pure white crystals were obtained. These crystals were recrystallized again from water, and 3.6 g of pure crystals of L-3-methoxy-4-hydroxyphenylalanine were obtained. The product was examined by means of thin layer and gas chromatography.

The physical and chemical properties of these crystals were as follows:

Melting point: 208 to 212 "C (decomposition).

Optical rotation: [a] i ⁵ = -5.60 (c = 3.05, In-HCl, 1 = 1).

IR absorption spectrum (Nujol Mull) 3280 (NH ₃ ⁺ ), 1627 (COO-) [cm ' ¹ ].

These crystals were demethylated with hydrobromic acid. L-dopa was obtained with an optical 100% purity.

[*]? = -12.3 ° (c = 3.56, 1N-HCl, 1 = 1).

Claims (1)

Claim: Process for the preparation of L-3,4-disubstituted Phenylalanines of the general formula R ¹ O R ² O CH ₂ CHCOOH
NH ₂ It is known that L-3,4-dihydroxyphenylalanine (hereinafter referred to as "L-Dopa") is a specific pharmaceutical agent against Parkinson's disease L-3-methoxy-4-hydroxyphenylalanine and L-3 , 4-Dimethoxyphenyl-alanine are important industrial starting materials for the production of L-Dopa. ,.,., .. It is known that L-3,4-disubstituted phenylalanine became of the following general formula