DE2148953C3 - Process for the preparation of L-3,4-disubstituted phenylalanines - Google Patents

Description

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 - f ~ V - CH ₂ CHCOOH

NH,

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 process for the preparation of L-3,4-disubstituted phenylalanines of the general formula

R ¹ O

R ² O

CH ₂ CHCOOH
NH,

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 ₂ COCOOH (II)

in which the radicals R ¹ and R ² have the meanings stated above, with an aminating agent in the presence of a transaminase produced by microorganisms in the temperature range of 25 to 45 ^'C, a pH value of 6.5 to 10.5 and a reaction time from 20 to 60 hours.

wherein R ¹ and R ^{2 can be} a hydrogen atom or a methyl group, both being substituents

may be the same or different, and R ^{2 is} a methyl group when R ^{1 is} a hydrogen atom, so far produced by physical, chemical methods and with the use of 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 be treated (Charles J. S i h et al.

₄ or "Journal of the American Chemical Society", Vol. 91, p. 6204 [1969]). Another method involves condensing catechol with a special amino acid, such as tyrosine, serine or cysteine. For this purpose, tyrosine phenollyase is used, which is obtained from bacteria (Yamada, Ogata et al. "Biochemical and Biophysical Research Communications", Vol. 34, p. 266 [1969]).

From the DT-OS 2041418 are microorganisms known that have the property of 3,4-disubstituted phenylpyruvic acids in L-3,4-disubstituted To convert phenylalanines. These include the bacteria Pseudomonas, Bacillus, Micrococcus, Sarcina, Brevibacterium and the molds 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 (1) 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 of the genera Alcaligenes uses transaminase produced.

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

21

cultured in a culture medium that contains one or more of the following substances: glucose, sucrose, maltose, lactose or starch hydrolyzate as a carbon source, meat extract, peptone, polypeptone, casamino acid, corn water, ammonium salts or protein hydrolysates as a nitrogen source and sodium chloride, phosphates, metal ions, vitamins and growth substances. Thus, there is obtained an enzyme capable of converting the compounds represented by the general formula (II) into the compounds represented by the general formula (I), ₀ in the bacterial cell or in their fermentation broths. The process of the invention gives the compounds of formula (I) 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% by weight sodium chloride. It is cultured for 48 hours at a temperature of 30 "C., The compounds of the general formula (II) in the presence of the wet obtained ZEI brought len 1 hour with the amino acid to the reaction at a temperature of 37 'C ^1.

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

The L-3,4-dihydroxyphenylalanine is determined by the trihydroxyindole method (E u 1 e r, U. S. by: Hormones in Blood, Academic Press [1969]).

The L-3,4-dimethoxyphenyl-alanine and the L-3-methoxy-4-hydroxyphenyl-alanine are converted to the N-trifluoroacetyl-n-butyl ester 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 quantitatively determined by gas-liquid chromatography. The enzymatic activities of the microorganisms are determined from these analytical results. The unit of the enzyme activities is expressed by the amount of the enzyme which forms 1 mg of the compound represented by the general formula (I) at a temperature of 37 ^° C. over a period of 1 hour. As the enzymatic reaction system, a system was used in which the enzyme solution resulting from one ml of the culture solution is added with a solution of the following composition:

0.5 m phosphate buffer solution 0.5 ml

0.01% (w / v) pyridoxal

phosphate 0.5 ml

V ₁₆ In potassium L-glutamate 0.5 ml

V ₁₆ m potassium L-asparaginate 0.5 ml

V ₈ 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-dihydroxyphenylalanine, L-3-methoxy-4-hydroxyphenyl-alanine or L-S ^ -dimethoxyphenyl-alanine.

The amount of enzyme produced in 100 ml of fermentation for different bacteria 953

is expressed by the number of units defined above.

Amount of enzyme in the product obtained from bacteria

Bucclerium

Classic Enzyme (units / 100 ml)
fication amount

number connection connection connection connection

(A)

(B)

(C)

Alcaligenes 1015 98 53 69

I AM*)

faecalis

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

In the thus obtained transaminase which can convert the compounds represented by the general formula (II) in the compounds represented by the general formula (I) compounds by means of a transamination reaction, it is very advantageous that the transaminase ^r ÜR a method performed in an industrial scale Process for the preparation of the L-3,4-disubstituted phenylalanine compounds according to the formula (I) can be used. The ground cell fluid, the dried or wet cells or the fermentation broths of these microorganisms can be used as transaminase for the transamination.

As aminating agents in the process according to the invention, L-glutamic acid, 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 work is continued by precipitation at the isoelectric point, with ion exchange resins or with adsorbents.

Of the compounds of formula (I) obtained, L-3-methoxy-4-hydroxyphenylalanine or L-3,4-dimethoxyphenylalanine by demethylation 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 into 5 liters of a culture medium which consisted of 0.5% glucose, 0.5% casamic acid, 0.5% yeast extract, 0.1% sodium chloride, 0.1% monopotassium phosphate and 0.05% magnesium sulfate . Cultivation was carried out with movement and ventilation at an initial pH of 7.0 and at a temperature of 30 ^° C. for 64 hours. The bacterial cells were removed from the culture fluid by centrifugation. 35 g of the wet cells thus obtained were suspended in 300 ml of a solution obtained by dissolving

3 g of 3,4 - dihydroxyphenylpyruvic 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 cells and the protein removed from the reaction liquid and added the reaction liquid to activated carbon. The 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. Using IR analysis and thin layer chromatography the L-dopa was found to be a standard product.

Mi = -13.2 ° (c = 3.86 in In-HCl, 1 = 1).

B e i s ρ i e! 2

^r

Alcaligenes faecalis IAM 1015 was inoculated into 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. Cultivation was carried out with shaking at an initial pH of 7.0 at a temperature of 30 ^° C. for 48 hours. The culture liquid was combined with 2 liters of a solution 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 had been dissolved in a 1 -N aqueous potassium hydroxide solution Adjust the pH to 8.3. The solution thus obtained was kept at a temperature of 30 ° C. with agitation for 40 hours. The pH of the solution was then adjusted to a pH of 2.5 with 10% strength aqueous hydrochloric acid and centrifuged. The resulting supernatant liquid was concentrated under reduced pressure until the entire volume was concentrated to about 600 ml. The solution thus obtained was washed twice with about 300 ml of ethyl acetate solution to extract and separate the unreacted 3,4-dimethoxyphenylpyruvic acid. The aqueous solution was concentrated to about 300 ml and then the pH was adjusted to 4.5. It was allowed to stand overnight and 7.5 g of crude crystals of L-3,4-dimethoxyphenylalanine were obtained. 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 a 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-dimethoxyphenylalanine 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: M "= -5.75" ic = 3.30, in In-HClJ = 1).

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

The crystals were demethylated with hydrobromic acid to L-dopa, which is of optical purity owned by 100%.

[«]? = -12.7 "(c = 3.56, in In-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 cultivated 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 liters of a solution in which 15.0 g of 3-methoxy-4-hydroxyphenylpyruvic acid, 15.0 g of L-glutamic acid and 15.0 g of L-aspartic acid had been dissolved in 1N aqueous potassium hydroxide solution adjust the pH to 8.5. The thus prepared solution was held for 20 hours under agitation at 30 ⁰ C, and then the pH value of the solution with 10% aqueous hydrochloric acid solution was adjusted to 2.5 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: [«] !? = -5.60 ° (c = 3.05, 1 n-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.

Wed? = -12.3 ° (c = 3.56, In-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-, It is known that L-3,4-dihydroxyphenylalanine (hereinafter referred to as "L-Dopa") is a specific is a pharmaceutical agent for 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. As is known, L-3,4-disubstituted phenylalanine became the following general formula