JPH0458318B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-phenylalanine.

L-phenylalanine is one of the essential amino acids and is a useful compound that is used in nutritional fortifiers, pharmaceutical amino acid infusions, combined amino acid preparations, etc., as well as as a food additive. In recent years, it has also attracted attention as a raw material for the sweetener aspartame.

Conventional methods for producing L-phenylalanine can be roughly divided into two methods: a method by fermentation and a method by splitting a chemically synthesized racemate. According to the fermentation method, it is possible to produce only the L-form, but L-phenylalanine accumulates in large amounts in the culture to the extent that it is seen in the production of other amino acids. No method has been found to prevent it. On the other hand, the mainstream method for producing racemates through chemical synthesis is to use enzymes, and these can be divided into methods using esterase and methods using acylase. . An example of the esterase method can be found in, for example, Japanese Patent Publication No. 57-35956. This method uses N-acyl-
Using DL-phenylalanine methyl ester as a raw material, it is treated with an enzyme having esterase activity to form N-acyl-L-phenylalanine and N-acyl-L-phenylalanine.
-Acyl-D-phenylalanine methyl ester, but since these two compounds have very similar properties, it is difficult to completely separate them. Also, through this method, L-
Regarding the method to obtain phenylalanine, it is necessary to remove the acyl group.

The acylase method, which is currently the most commonly used method, uses N-acyl-DL-phenylalanine as a raw material, and the products L-phenylalanine and N-acyl-D- When extracting and separating phenylalanine using an organic solvent,
N-acyl-D-phenylalanine in the organic layer,
Furthermore, when L-phenylalanine is transferred to the aqueous layer, the partition between the two is not completed completely, so it is necessary to repeat extraction and salting-out extraction, and N-acyl-D-phenylalanine is transferred to the aqueous layer. Various drawbacks existed, such as racemization having to be carried out using acetic anhydride at a high temperature (~100°C) when reusing it.

The present inventors conducted various studies on the production method of optically active phenylalanine, and found that N-acyl-
When acylase is applied to DL-phenylalanine alkyl ester, the enzyme performs a two-step action of first eliminating the alkyl group at the ester site and then eliminating the acyl group at the N-acyl site; It has been found that this effect occurs only in the L form. In addition, in this enzymatic reaction, when the reaction is carried out in a two-phase solvent of an organic solvent and water, the L-phenylalanine produced is dissolved in the water layer, and the N-
It has been found that since the acyl-D-phenylalanine alkyl ester is dissolved in the organic solvent layer, these two compounds can be separated by an extremely simple operation of separating them into two layers.

The present invention has been made based on this knowledge. Therefore, the present invention uses a two-phase solvent of water and an organic solvent that is immiscible with water and forms a two-phase system, and in the solvent, the general formula: N ^{- acyl-}
By allowing acylase to act on DL-phenylalanine alkyl ester, N-acyl-L
- By allowing acylase to act on the phenylalanine alkyl ester, the acyl and alkyl groups of the N-acyl-L-phenylalanine alkyl ester are eliminated, and then the produced L-phenyl group dissolved in the aqueous phase is removed. The present invention provides a method for producing L-phenylalanine characterized by obtaining enylalanine, and also provides a method for producing D-phenylalanine at the same time.

The present invention will be explained in detail below.

In the method of the present invention, N-acyl-DL-phenylalanine alkyl ester represented by the general formula () is used as a raw material, but in typical embodiments, this is miscible with water. It dissolves in organic solvents forming a two-phase system. Meanwhile, dissolve or suspend acylase in water and adjust the pH to 6.5.
-8.5, preferably 7.0-7.5. Next, the above organic solvent solution and the above aqueous solution (or aqueous suspension) are combined. Both solutions are immiscible and form a two-phase system, and the entire reaction system is adjusted to a temperature of 20-40°C and stirred vigorously. At this time, depending on the type of enzyme used, Co, Ca,
It is desirable to add metal ions such as Mg, Mn, Zn, etc. to the reaction system. The reaction time is usually 3 hours to 2 days, during which time stirring is continued.

During this stirring, the acylase has the general formula ()
It specifically acts only on the L form of the N-acyl-DL-phenylalanine alkyl ester represented by the formula, first eliminating the alkyl group at the alkyl ester site, and then eliminating the acyl group at the N-acyl site. Let go. Eventually, L-phenylalanine is produced, and N-acyl-D-phenylalanine alkyl ester that does not participate in the enzyme reaction remains. After the stirring is complete, the reaction solution is allowed to stand still for a while and is separated into two layers: an aqueous layer and an organic solvent layer. The produced L-phenylalanine is dissolved in the aqueous layer, and the N-acyl-D-phenylalanine alkyl ester is dissolved in the organic solvent layer.

This aqueous layer is separated, its pH is adjusted to about 5.8, and it is allowed to stand, or the separated aqueous layer is concentrated and then cooled to crystallize L-phenylalanine.
This L-phenylalanine has such high optical purity that it does not require recrystallization treatment.

N-acyl-D-phenylalanine alkyl ester present in the remaining organic solvent layer after separating the aqueous layer is racemized to N-acyl-DL-
It can be converted into a phenylalanine alkyl ester and used cyclically as a raw material for the process of the invention. The racemization of the N-acyl-D-phenylalanine alkyl ester was carried out by using the organic solvent layer as it was without subjecting it to any special treatment, and adding 0.1 to 0.5 of the alkali metal alcoholate to the organic solvent layer.
This can be achieved by adding % (weight ratio) amount and heating to a temperature of about 30 to 50°C.

In addition, the N-acyl-D-phenylalanine alkyl ester present in the organic solvent layer can also be used for the purpose of producing D-phenylalanine. In that case, the separated organic solvent layer is washed with water, dehydrated with a dehydrating agent such as anhydrous metal salt of an inorganic acid such as anhydrous magnesium sulfate, and the organic solvent is distilled off. D-phenylalanine can be crystallized by adding an inorganic acid such as an acid, boiling it, distilling off the inorganic acid, and adjusting the pH of the residue as appropriate.

In the method of the present invention, buffer salts such as sodium hydrogen carbonate, sodium carbonate, potassium hydrogen carbonate, potassium carbonate, sodium phosphate, potassium phosphate, Tris-(oxymethyl)-
It is possible to contain aminomethane or the like.

Examples of organic solvents used in the method of the invention include hexane, pentane, chloroform, ethylene dichloride, tricrine, benzene, toluene, ethyl acetate, methyl acetate,
Examples include propionic acid methyl ester.

The acylase used in the method of the present invention is particularly
It is not specified and may be derived from microorganisms or animal tissues. Acylase and aminoacylase of the enzyme system (EC3, 5, 1, 14) can be used. Specific examples include acylase "Amano" 15000 and acylase.

In the method of the present invention, the N-acyl-DL-phenylalanine alkyl ester used as a raw material can be easily produced by a conventionally known method. Examples of such methods include, for example, a method in which DL-phenylalanine is converted to N-acyl-DL-phenylalanine using an acyl chloride and then esterified, and a method in which DL-phenylalanine is esterified and then, There is a way to obtain this by acylation. In addition, as a method other than these, the compound represented by the general formula () is the most common N-acetyl-DL-
Regarding the case of phenylalanine methyl ester, 2-methyl-4-(phenylmethylene)-5(4H)-oxazolone is obtained from N-acetylglycine and benzaldehyde [Org.
Synthesis, Coll. Vol., 1 (1943)] and treated with sodium methoxide in methanol to give α-N-acetamidocinnamic acid methyl ester (AHCook, et al., J.
Chem.Soc., 1056 (1948)), followed by catalytic reduction using Raney nickel as a catalyst [J.
M. Roper et al., Synthesis 1041 (1983)].

The method of the present invention is an industrial method as follows:
It has various advantages such as:

The raw material N-acyl-DL-phenylalanine alkyl ester is a substance that is almost insoluble in water, and the L-phenylalanine produced is water-soluble, and the N-acyl-D that did not participate in the reaction - Since the phenylalanine alkyl ester is dissolved in the organic solvent layer and the distribution ratio of both substances to the aqueous layer and the organic solvent layer is extremely high, the separation of both substances can be carried out extremely efficiently.

When aiming for D-phenylalanine,
D-phenylalanine can be obtained by treating the separated N-acyl-D-phenylalanine alkyl ester with a dilute acid.

The N-acyl-D-phenylalanine alkyl ester can be racemized by a simple method of treatment with an alcoholate and recycled as a raw material for the process of the present invention.

EXAMPLES The present invention will be specifically explained below with reference to Examples.

Example 1 Acylase 14mg (SIGMA0.23units/mg
solid) and cobalt chloride hexahydrate 10.8mg at 2%
Dissolve NaHCO ₃ in 15 ml of water to make an aqueous solution. N-
2.0 g of acetyl-DL-phenylalanine methyl ester is dissolved in 15 ml of toluene and added to the above aqueous solution. Stir vigorously for 5 hours at 37°C. Leave to stand still to separate the aqueous layer and toluene layer.
Extract in ml. Both toluene layers were combined, dehydrated with anhydrous magnesium sulfate, and then distilled off to yield 1.0 g of N-acetyl-D-phenylalanine methyl ester.
([α] _D = -84.4° (toluene, c = 2) Optical purity 96
%)was gotten. On the other hand, the aqueous layer was aged at 37° for 24 hours after adjusting the pH to 7.0 using 5N-NaOH. When about 2/3 of the water was distilled off, 627 mg of L-phenylalanine [Y: 84%, [α] _D = -32.7° (water, c
=2) optical purity of 99%] was obtained.

Example 2 200 mg of acylase (Amano 15000) and 10.8 mg of cobalt chloride hexahydrate are dissolved in 15 ml of 2% NaHCO ₃ water to form an aqueous solution. 4.0 g of N-acetyl-DL-phenylalanine methyl ester was added to 18 g of toluene.
ml and add this toluene solution to the above aqueous solution. Add N-NaOH dropwise to this mixture,
Stir vigorously at 37°C for 3 hours while adjusting the pH to 7.0-7.3. After stirring, the mixture is allowed to stand still and separated into an aqueous layer and a toluene layer. A toluene solution of 4.0 g of N-acetyl-DL-phenylalanine methyl ester is further added to this aqueous layer, and the mixture is vigorously stirred at 37° C. for 3 hours while adjusting the pH to 7.0 to 7.3. After separating the toluene layer in the same manner as above, repeat the above operation again, add the above toluene solution, and heat at 37°C.
Stir vigorously for 6 hours. After separating the toluene layer, the aqueous layer was extracted with 18 ml of toluene. After combining the toluene layers and washing the entire toluene layer with water,
It was dehydrated with anhydrous magnesium sulfate and evaporated. 15 ml of 4N-HCI was added to the residue and boiled for 2 hours. After removing hydrochloric acid gas under reduced pressure, the pH was adjusted to 5.6 with 5N-NaOH.
When adjusted to 3.7g of D-phenylalanine
(Y: 82.6%, [α] _D = +32.3° optical purity 98%) was obtained. Meanwhile, add 17 ml of concentrated hydrochloric acid to the aqueous layer and add 1.5
boiled for an hour. Distill about half of the water and 5N−
When the pH was adjusted to 5.6 using NaOH, L-phenylalanine was precipitated. After taking it, I washed it with a small amount of ice water and then dried it, and it weighed 3.6g.
A yield of (Y: 80.4%, [α] _D = -32.1° (water, c = 2) optical purity 97.3%) was obtained.

Reference Example 1 Racemization of N-acetyl-D-phenylalanine methyl ester 1.5 g of N-acetyl-D-phenylalanine methyl ester obtained in Example 1 was added to 30 ml of toluene.
38mg of sodium methoxide dissolved in this
was added and stirred vigorously at 40°C for 1 hour. The sodium methoxide is removed by decantation and the toluene is distilled off. N-acetyl-DL-
Phenylalanine methyl ester 1.41g (Y:94
%, racemization rate 100%).

Reference Example 2 (1) Synthesis of α-N-acetamidocinnamic acid methyl ester [AHCook, G.Harris, Ian Heilbron
and G. Shaw, J. Chem. Soc., 1056−1060
(1948)] Dissolve 0.54 g (0.01 mol) of sodium methoxide in 25 ml of methanol, and add 2-methyl-4
Add 18.7 g (0.1 mol) of -(phenylmethylene)-5(4H)-oxazolone (Org.Synth.Coll.Vol., (1943)). After stirring for 15 minutes at room temperature, methanol is distilled off. The residue was dissolved in 120 ml of methylene chloride and washed with 40 ml of N-HCl and 100 ml of water in this order. The methylene chloride layer was removed, dehydrated with anhydrous magnesium sulfate, and then distilled off to give α-N-acetamidocinnamic acid methyl ester (mp. 122-123.5℃).
21.9g (Y: quantitative) was obtained.

(2) Synthesis of N-acetyl-DL-phenylalanine methyl ester [JMRoper, DPBauer,
Synthesis 1041 (1983)] 21.9 g (0.1 mol) of α-N-acetamidocinnamic acid methyl ester obtained in (1) was added to 70 ml of methanol.
dissolve. To this, 10% Pd-C (50% by weight) 2.2
g was added thereto, and catalytic reduction was carried out at room temperature under a hydrogen atmosphere for 2 hours. When the catalyst was removed and methanol was distilled off, 22.1 g (Y: quantitative) of N-acetyl-DL-phenylalanine methyl ester (m.p 60-61°C) was obtained.

Claims (1)

[Claims] 1. Using a two-phase solvent of water and an organic solvent that is immiscible with water and forming a two-phase system, in the solvent, the general formula, N ^{- acyl-}
By allowing acylase to act on DL-phenylalanine alkyl ester, N-acyl-L
- A method for producing L-phenylalanine, which comprises removing the acyl group and alkyl group of a phenylalanine alkyl ester, and then obtaining the produced L-phenylalanine dissolved in an aqueous phase. .