JP3316917B2 - New phenylalanine salt crystals and their production - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel optically active phenylalanine monomethyl sulfate crystal and a method for producing the same.

[0002]

2. Description of the Related Art Conventional L-phenylalanine (hereinafter referred to as L-phenylalanine).
In manufacturing, there are two impurities which are problematic in terms of quality. When one of them is manufactured by a fermentation method, L-tyrosine (hereinafter referred to as L-tyrosine) coexisting in the fermentation solution is used.
Tyr) and the other is LP
D-phenylalanine (hereinafter referred to as D)
-Phe).

With respect to the former impurity L-Tyr, some L-Phe derivative crystals which are considered to have good selectivity have been found (for example, Japanese Patent Application Laid-Open No. Sho 60-13746).
L-phenylalanine monosodium pentahydrate, L-phenylalanine 1/2 sulfate 1/2 hemihydrate disclosed in JP-A-56-79652). However, they are all L-Ph
Since the solubility of the e derivative crystal becomes extremely high, there is a problem that the loss of L-Phe to the crystallization mother liquor increases and the yield decreases. Therefore, in order to obtain a high yield, crystallization is performed by increasing the L-Phe concentration. As a result, the concentration of the slurry to be crystallized increases, the stirring power of the crystallization slurry increases, and the crystallization slurry adheres to the separated crystals. There is a problem that impurities in the mother liquor are mixed.

[0004] The other impurity D-Phe is L-Ph
L-Phe is generated by racemization under high temperature conditions and alkaline conditions at the stage of treating a fermentation solution containing e. Further, in the production of various substances using L-Phe as a raw material, the L-Phe derivative which could not be a target substance was produced by removing a substituent by a method such as hydrolysis.
-The method of recovering Phe and using it again as a raw material is economically advantageous, so that it is often carried out industrially,
Racemization also occurs during the manufacturing and recovery steps.

[0005] The D-Phe thus generated is:
Under normal crystallization conditions, it is difficult to efficiently remove L-Phe. When D-Phe in the solution precipitates as crystals, it forms a pair with the coexisting L-Phe to form a racemic DL-Phe crystal,
Its solubility is generally due to being much lower than that of L-Phe.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to find a salt of L-Phe which is easy to operate and has high selectivity for L-Tyr and D-Phe as impurities.

[0007]

As a result of intensive studies to solve the above problems, surprisingly, the crystallization operation from an aqueous solution containing monomethyl sulfate (hereinafter abbreviated as CH ₃ SO ₄ H) and L-Phe is performed. Is L-phenylalanine monomethyl sulfate (hereinafter, abbreviated as L-Phe.CH ₃ SO ₄ H salt), and L-phenylalanine having a low optical purity. Phe and CH ₃ SO ₄
From the crystallization system of H salt, L-Phe with extremely high optical purity
CH ₃ SO ₄ H salt is obtained, and furthermore, L-Ty
The inventors have also found that r has a very high selectivity, and have completed the present invention.

In addition, racemic DL-Phe and CH ₃ SO ₄ H
Crystals obtained by crystallization from an aqueous solution containing
e) the same powder X-ray diffraction spectrum as the crystals obtained from
Because of the infrared absorption spectrum, DL-Phe.
The CH ₃ SO ₄ H salt was found to be a racemic mixture.
This is an important finding in efficiently optically purifying optically impure L-Phe.

The amount of CH ₃ SO ₄ H to be subjected to the crystallization of the present invention may be at least equimolar to the corresponding L-Phe. When the amount of CH ₃ SO ₄ H is small, the crystallization yield is reduced, but when the amount of CH ₃ SO ₄ H is less than or equal to L-Phe, L-Phe · CH ₃ SO ₄ H salt crystals It may be obtained in a state where L-Phe crystals coexist. Again,
The L released from this mixed crystal can be easily obtained by the method described later.
-Phe can be obtained. Conversely, as the amount of CH ₃ SO ₄ H increases, the solubility of the formed salt tends to decrease significantly and the crystallization yield increases, but increasing the amount more than necessary is not economical.

The CH ₃ SO ₄ H to be used is easily prepared from methanol and sulfuric acid, but a salt of CH ₃ SO ₄ H and a base liberated with an acid such as sulfuric acid or hydrochloric acid can also be used. Examples of such salts include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium, and amine salts such as ammonia and triethylamine.

In the present invention, the crystallization is preferably carried out in an aqueous solvent, but there is no problem even if other organic solvents miscible with water are contained. Other organic solvents include alcohols such as methanol and ethanol, ketones such as acetone, carboxylic acids such as formic acid and acetic acid, and nitriles such as acetonitrile.

The crystallization method can be a conventional crystallization method such as concentration crystallization and cooling crystallization. However, since the difference in solubility depending on the temperature is large, cooling crystallization is efficient. Also, L-Phe.CH ₃
Since the solubility of the SO ₄ H salt is generally lower in organic solvents than in water, it can also be obtained by mixing an aqueous solution containing L-Phe and CH ₃ SO ₄ H with an organic solvent miscible with water. Further, when CH ₃ SO ₄ H is a salt with a base, neutralization crystallization with an acid is also possible.

In order to obtain the present crystal, CH ₃ SO ₄ H is added to the crystallization system.
And L-Phe need only be present, and are not affected by the mixing order of the two. For example, monomethyl sulfate sodium salt (hereinafter abbreviated as CH ₃ SO ₄ Na) is neutralized with sulfuric acid, and then L-Phe is added to and dissolved in a sulfuric acid aqueous solution even when crystalline L-Phe is added. And L ₃ -Phe.CH ₃ SO ₄ H salt can be obtained by adding CH ₃ SO ₄ H or sodium monomethyl sulfate to the mixture for crystallization.

At the time of crystallization, the concentration of L-Phe
When the amount of H ₃ SO ₄ H and an aqueous solvent containing an organic solvent miscible with water are used, the type and the content of the organic solvent greatly affect the solubility.
Crystallization is possible if the concentration is 0.1 g / dl or more.
When substantially no organic solvent is contained as a solvent, crystallization is preferably performed at 1 g / dl or more.

The temperature during crystallization is maintained at 60 ° C. or lower, preferably 30 ° C. or lower, because the lower the temperature, the lower the solubility and the higher the crystallization rate.

The thus obtained L-Phe.CH
_{The 3} SO ₄ H salt is prepared by removing CH ₃ SO ₄ H from the aqueous solution using an anion exchange resin, or using a commonly used base such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, ammonia and the like. Free L-Phe can be easily obtained by neutralizing and subjecting to crystallization.

The above description and the following embodiments are based on LP
Although he · CH ₃ SO ₄ H salt is described as the target substance, it goes without saying that the same applies when the enantiomer D-Phe · CH ₃ SO ₄ H salt is the target substance.

[0018]

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

[0019]

Example 1 13.4 g of CH ₃ SO ₄ Na (0.1 mol)
l), 4.13 g (25 mmol) of L-Phe
5 ml, and adjusted to pH = 0 with sulfuric acid. This was heated and melted at about 70 ° C., and then cooled. The precipitated crystals were separated by suction filtration, washed with a small amount of cold water, and dried under reduced pressure. 5.78 g. This crystal showed L-Phe as a result of various physical property measurements.
- it was CH ₃ SO ₄ H. The measurement results of the physical properties are shown in Table 1, FIG. 1, FIG. 2, and FIG.

[0020]

[Table 1]

[0021]

Example 2 4.13 g (25 mmo) of DL-Phe
Same as Example 1 except that l) was used. Crystal yield
4.57 g. The IR spectrum and powder X-ray diffraction of this crystal were the same as those of the crystal obtained in Example 1. or,
The melting point was 173-174.5 ° C.

[0022]

Example 3 3.72 g (22.5 mmo) of L-Phe
l), 0.41 g (2.5 mmol) of D-Phe (D
-Phe / L-Phe = 11%), except that Example 1 was used. Crystal yield 5.01 g. When the obtained crystals were analyzed by HPLC using an optically active column,
D-Phe was only 1% contained in L-Phe.

[0023]

Comparative Example 1 L-Phe 3.72 g (22.5 mmo
l), 0.41 g (2.5 mmol) of D-Phe (D
-Phe / L-Phe = 11%) was suspended in 50 ml of water, and sulfuric acid was added thereto to dissolve the suspension. 50% of this solution
The pH was adjusted to 6 with an aqueous sodium hydroxide solution, and the precipitated crystals were separated by filtration at 40 ° C. Crystal yield 2.98 g.
When the obtained crystals were analyzed by HPLC using an optically active column, D-Phe was 14.7 relative to L-Phe.
% Was included.

[0024]

Example 4 6.39 g (0.048 g) of CH ₃ SO ₄ Na
mol), 1.97 g (11.9 mmol) of L-Phe
l), L-Tyr 98mg (5wt% vs. L-Phe)
Was added to 15 ml of water, and the pH was adjusted to 0 with sulfuric acid. This was heated and melted at about 70 ° C., and then cooled. The precipitated crystals were separated by suction filtration, washed with a small amount of cold water, and dried under reduced pressure. Crystal yield 2.62 g. This crystal contains L-Phe
1.54 g (9.3 mmol), L-Tyr 28.
6 mg (1.9 wt% vs. L-Phe) was contained.

[0025]

Comparative Example 2 1.97 g of L-Phe (11.9 mmo)
l), L-Tyr 98 mg (5 wt% vs. Phe) was added to water 3
After adding to 0 ml and dissolving by heating at 80 ° C, the mixture was gradually cooled to 40 ° C. The precipitated crystals were separated by suction filtration, washed with a small amount of water, and dried under reduced pressure. Crystal yield 0.65 g. 0.59 g (3.6 mmol) of L-Phe and L-Ty
r was contained at 55 mg (9.4 wt% vs. L-Phe).

[0026]

Example 5 3.19 g (23.8 m) of CH ₃ SO ₄ Na
mol), 1.97 g (11.9 mmol) of L-Phe
l) was added to a mixture of 10 ml of water and 5 ml of acetic acid, and the pH was adjusted to 0.6 with sulfuric acid.
The precipitated crystals were separated by suction filtration. Crystal yield 1.78
g. The crystal contained 1.01 g of L-Phe.

[0027]

Example 6 2.38 g of L-Phe.CH ₃ SO ₄ H salt
Was added dropwise to 50 ml of acetone, and the mixture became cloudy. After the crystals were aged in a refrigerator, they were separated by suction filtration. Crystal weight after drying 1.8
6g.

[0028]

Example 7 95% sulfuric acid in 33 ml of methanol
8 ml was added, and the mixture was heated under reflux. Thereafter, methanol was distilled off under reduced pressure, and water was added until the total volume became 15 ml.
The H ₃ SO ₄ H solution was prepared. In addition, L-Phe 2.
7 g and D-Phe 0.3 g were added (D-Phe / L-
(Phe = 11.1%), and after heating to dissolve, the mixture was ice-cooled. The precipitated crystals were separated by suction filtration and washed with a small amount of cold water. 3.73 g of crystal yield. As a result of HPLC analysis using an optically active column, L-Phe
1.66 g and 0.049 g of D-Phe (D-Phe / L-Phe = 2.9%). Also, CH ₃ S
The O ₄ H solution contained 41 mg of L-phenylalanine methyl ester esterified with residual methanol.

[0029]

According to the method of the present invention, high-purity optically active Phe can be produced in a high yield using simple operations and inexpensive substances.

[Brief description of the drawings]

FIG. 1 is a powder X-ray diffraction pattern (C) of the crystals obtained in Example 1.
uKα ray)

FIG. 2 is an IR chart (KB) of the crystal obtained in Example 1.
r method)

FIG. 3 is an NMR chart of a crystal obtained in Example 1 (solvent: heavy water).

Continuation of the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07C 229/36 C07C 227/42 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. An optically active phenylalanine monomethyl sulfate crystal 2. A method for producing optically active phenylalanine monomethyl sulfate, comprising subjecting a solution containing monomethyl sulfate and phenylalanine to crystallization in an aqueous solvent in the presence or absence of an organic solvent.