JPH06247913A - Recovery of l-phenylalanine - Google Patents

Abstract

PURPOSE:To recover L-phenylalanine from an aqueous layer left after the extraction and removal of L-phenylalanine ester with an organic solvent from a reactional product obtained by neutralizing an acid-catalyzed esterification reaction liquid of L-phenylalanine. CONSTITUTION:A reaction liquid obtained by esterifying L-phenylalanine with methanol in the presence of sulfuric acid is neutralized with a base in the presence of water and the produced L-phenylalanine methyl ester is extracted with an organic solvent. The aqueous layer left after the extraction is subjected to crystallization under acidic condition to recover the L-phenylalanine in the form of L-phenylalanine monomethylsulfuric acid salt.

Description

Detailed Description of the Invention

[0001]

[Industrial application] L-phenylalanine (hereinafter referred to as L-
(Abbreviated as Phe) and its methyl ester L
-Phenylalanine methyl ester (hereinafter abbreviated as L-PM) is an important substance as a raw material and intermediate for peptide synthesis. In particular, there is a great demand as a raw material for a dipeptide α-L-aspartyl-L-phenylalanine methyl ester (hereinafter abbreviated as α-APM) as a sweetener.

[0002]

2. Description of the Related Art Various methods for producing α-APM have been studied so far, but as an industrial method, L-Phe is methyl esterified to obtain L-phenylalanine methyl ester (hereinafter abbreviated as L-PM). A) is condensed with L-aspartic acid whose amino group is protected to give N-protected-L-aspartyl-L-phenylalanine methyl ester, and then the protecting group is eliminated to convert to α-APM.

To obtain this L-PM, L-Phe is esterified with methanol in the presence of an acid such as hydrochloric acid or sulfuric acid, and the obtained acidic reaction solution is used in the presence of water with a suitable base. Highly pure L-PM is the method of neutralizing and neutralizing liberated L-PM with an organic solvent immiscible with water such as toluene.
It is often used because in this way,
In the extracted water layer, L-Phe that did not react by esterification and LP generated by decomposition of L-PM in the neutralization operation and extraction operation
he is dissolved. In addition, L-PM that was not extracted in the organic solvent by the extraction operation is also dissolved.

By the way, since L-Phe is a relatively expensive raw material, industrially, L-Phe could not be a target substance.
A method is often adopted in which the Phe component is recovered and used again as a raw material. For example, in the α-APM manufacturing process,
A method for recovering from the α-APM crystallization mother liquor has been found (JP-A-63-159355, JP-A-57-1309).
58 etc.). However, the dissolved L-
No method for recovering Phe by an economical and simple operation has been found.

The reason for this is that, for example, when L-Phe is recovered by isoelectric focusing, the concentration of dissolved L-Phe is low, so that it is necessary to concentrate the extracted water layer. , The energy cost required for the concentration becomes enormous, and L-
This is because the economic meaning of recovering Phe is lost.

[0006]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
It is to find a method for efficiently recovering L-Phe from a water layer obtained by neutralizing a Phe methyl esterification reaction solution and extracting L-PM with an organic solvent.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, surprisingly, an acidic reaction solution obtained by esterification of L-Phe and methanol in the presence of sulfuric acid was neutralized with an aqueous sodium carbonate solution, By making the aqueous layer after extracting the produced L-PM with toluene acidic and crystallizing,
It was found that crystals were easily precipitated, and that L-Phe dissolved in the aqueous layer was accumulated in high yield in the present crystals. Further, it was revealed that the present crystal can be led to a free L-Phe crystal by dissolving the crystal in water and neutralizing with a base, and the present invention was completed.

L-Phe recovered from the aqueous layer of the present invention is L-phenylalanine monomethylsulfate (hereinafter referred to as L-Phe).
Since it is recovered as Phe.CH ₃ SO ₄ H salt), it is necessary to use sulfuric acid that produces monomethyl sulfuric acid in the reaction process in the esterification reaction.

As the base for neutralizing the esterification reaction liquid, a commonly used base such as sodium carbonate, sodium hydrogen carbonate, sodium hydroxide, potassium hydroxide, ammonia or the like can be used. The concentration of these bases is not particularly limited, but usually 5 to 50% is used.

The organic solvent used for extraction is not particularly limited as long as it is immiscible with water, but toluene,
Aromatic hydrocarbons such as benzene, carboxylic acid esters such as ethyl acetate and ethyl formate, ethers such as ethyl ether and isopropyl ether, and halogenated hydrocarbons such as chloroform and methylene chloride are preferable.

Although the organic solvent and methanol are dissolved in the aqueous layer from which L-PM has been extracted and removed, there is no great influence on crystallization. In particular, when performing concentrated crystallization for crystallization, the low boiling point solvent and the solvent azeotropic with water are distilled off during the concentration operation.

The extracted water layer is acidified to L-Phe.CH.
_The solution is concentrated until the concentration of ₃ SO ₄ H salt precipitates, but it may be acidified after the concentration. However, monomethylsulfate is
The former treatment procedure is preferable because it is more likely to decompose in the alkaline region than in the acidic region.

The degree of concentration depends on the concentration of dissolved L-Phe, but the esterification solution having an esterification reaction rate of 95% or more is neutralized with a 15% Na ₂ CO ₃ aqueous solution, and L is diluted with a suitable organic solvent. -1/2 in the water layer when PM is extracted
If concentrated to about _1/3 , the L-Phe.CH ₃ SO ₄ H salt will precipitate. Incidentally, it is necessary to concentrate the same aqueous layer to 1/10 or less in order to perform L-Phe isoelectric focusing and economically recover L-Phe.

Acids that make the extracted water layer acidic are hydrochloric acid,
Inexpensive ones such as mineral acids such as sulfuric acid and phosphoric acid, methanesulfonic acid, monomethyl sulfuric acid and the like can be used.

The pH of the extracted water layer is adjusted to 3 or less with these acids. The pH is preferably 2 or less, and more preferably 1 or less.

The crystallization method is not particularly limited, such as cooling crystallization and concentrated crystallization, but the solubility of the L-Phe.CH ₃ SO ₄ H salt changes greatly with temperature, and the solubility decreases as the temperature decreases. To do. Therefore, from the viewpoint of crystallization yield, it is preferable to crystallize at a low temperature. The temperature range is 60 ° C. or lower, preferably 30 ° C. or lower, and more preferably 15 ° C. or lower.

Crystals obtained by crystallization under acidic conditions are L-Phe
- Since is CH ₃ SO ₄ H salt, the monomethyl sulfate L-P
It suffices that the same amount is present with respect to he. However, when monomethylsulfuric acid is present in excess, L-Phe.C is produced due to the salting out effect.
The solubility of the H ₃ SO ₄ H salt is reduced and the crystallization yield is increased.
Desirably, two or more equivalent amounts should be present.

However, the above-mentioned esterification reaction rate is 95%.
In the extracted water layer generated from the above esterification reaction liquid,
Usually, 20 equivalents or more of monomethylsulfate is present with respect to L-Phe.

Although such a system may be crystallized as it is, a large amount of monomethyl sulfuric acid in the adhering mother liquor remains in the separated crystals. Since monomethylsulfuric acid has an extremely high solubility in water, it can be easily removed by washing with water. As described above, it is also possible to reduce the number to an equal amount and perform crystallization. In this way, crystallization is possible without concentrating the extracted water layer. Moreover, in this case, L-Phe added is D- which is its optical isomer.
There is no problem even if Phe is included, and added D
-Phe is removed on the side of the crystallization mother liquor, and the obtained crystals are optically pure.

Needless to say, L used for esterification
Even when D-Phe is mixed as an impurity in -Phe, this optical refining effect is similarly obtained.

L-Phe.CH thus obtained
_{The 3} SO ₄ H salt can be easily released by removing CH ₃ SO ₄ H from the aqueous solution using an anion exchange resin or by crystallization by neutralizing with a commonly used base such as sodium hydroxide. L-Phe can be obtained.

[0022]

EXAMPLES The present invention will be described in detail below with reference to examples.

(Example 1) 500 g of L-Phe (3.
03 mol) was suspended in 900 ml of methanol to give 98
% H ₂ SO ₄ 330 g (3.3 mol) was added. While keeping this mixture at 80 ℃ or higher, add methanol 400
0 ml was added continuously over 5 hours and approximately the same amount of methanol was distilled off. This reaction solution was mixed with 15% Na
Neutralized to pH = 8.3 with ₂ CO ₃ aqueous solution and formed LP
After M was extracted and separated with toluene, 900 ml of an aqueous layer was obtained. In this water layer, 7.71 g (46.
7 mmol), sodium monomethylsulfate 385 g
(2.87 mol) was included. This extracted water layer 3
After adjusting 00 ml to pH = 1 with hydrochloric acid, 100 ml
It concentrated under reduced pressure at 60 ° C. until it became. The resulting concentrate is 5
The mixture was stirred overnight at ° C, and the precipitated crystals were separated by suction filtration. Crystal yield 14.52g. In this crystal, 2.30 g (13.9 mmol: yield 89.1%) as L-Phe,
8.20 g (73.2 mmol) as monomethyl sulfuric acid
Was included. This crystal (13.2 g) was dissolved in water (30 ml), and the pH was adjusted to 5.8 with an aqueous sodium carbonate solution.
Crystals precipitated during the adjustment, but while stirring, 5
After crystallization at ℃ for 2 hours, it was separated by suction filtration, and the crystal was washed with a small amount of water. 1.36 g of L-Phe in this crystal
(8.2 mmol: Yield 64.5%) and contained 0.046 g (0.4 mmol) of monomethyl sulfuric acid.

(Example 2) 295 ml of the extracted aqueous layer obtained in Example 1 was concentrated under reduced pressure to 100 ml. The liquid was adjusted to pH = 2 with hydrochloric acid, cooled to 5 ° C., and the precipitated crystals were separated by suction filtration and washed with a small amount of cold water. Crystal yield 1
2.49 g. In this crystal, 1.73 as L-Phe was obtained.
g (10.5 mmol: yield 68.5%) was contained.

Comparative Example 1 300 ml of the aqueous extract layer obtained in Example 1 was adjusted to pH = 5.6 with hydrochloric acid and then concentrated under reduced pressure to 100 ml. The concentrated liquid was stirred overnight at 5 ° C., and the precipitated crystals were separated by suction filtration. Crystal yield 2
0.7 g. This crystal contained 0.91 g of L-Phe. Yield 35.5%.

(Example 3) 33.75 g (0.204 m) of L-Phe was added to 300 ml of the extracted aqueous layer obtained in Example 1.
ol), 3.75 g (22.7 mmol) of D-Phe.
(Total of L-Phe 36.32 g (0.2
2 mol), D-Phe / L-Phe = 10.3%),
While heating, sulfuric acid was added, and when the crystals were completely dissolved, the pH was adjusted to 1. The solution was stirred overnight at 8 ° C., the precipitated crystals were separated by suction filtration, and the crystals were washed with a small amount of cold water. Crystal yield 64.0 g. L- in this crystal
Phe is 32.39 g (0.196 mol: crystallization yield 8
9.1%), D-Phe is 1.04 g (D-Phe / L
-Phe = 3.2%), 27.6 as monomethylsulfate
1 g (0.246 mol) was contained.

Example 4 Using 165.2 g of L-Phe, the esterification reaction liquid obtained in the same manner as in Example 1 was neutralized with 28% ammonia water, and L-PM was extracted with toluene. The water layer after the layer separation contained 3.04 g of L-Phe. After adjusting the pH of this aqueous layer to 1 with hydrochloric acid,
It was concentrated under reduced pressure to 110 ml. This concentrate at 1 ℃ at 1
After stirring overnight, the precipitated crystals were separated by suction filtration. Crystal yield 15.3 g. L-Phe was 2.46 in this crystal.
g was included. Yield 80.9%.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Chiaki Mochizuki 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Central Research Laboratory

Claims (3)

[Claims] 1. A reaction solution obtained by esterification of L-phenylalanine and methanol in the presence of sulfuric acid in the presence of sulfuric acid is neutralized with a base in the presence of water, and the produced L-phenylalanine methyl ester is extracted with an organic solvent and separated into layers. A method for recovering L-phenylalanine, which comprises exposing the aqueous layer to crystallization conditions under acidic conditions to recover L-phenylalanine as L-phenylalanine monomethylsulfate. 2. The method according to claim 1, wherein the base used for neutralizing the esterification reaction liquid is ammonia or an inorganic base. 3. L-containing or not containing D-phenylalanine as at least an impurity in the extracted water layer according to claim 1.
A method for recovering L-phenylalanine, which comprises adding phenylalanine and exposing to crystallization conditions under acidic conditions to obtain precipitated L-phenylalanine monomethylsulfate.