JPH0634745B2 - Enzymatic synthesis of N-protected peptides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for enzymatically synthesizing N-protected peptides, more particularly N-having at least one carboxyl group.
The present invention relates to a method for enzymatically synthesizing a protected peptide.

In recent years, in peptide synthesis, attention has been paid to the fact that the method using an enzyme does not cause ceramization, and its use in various peptide synthesis has been attempted. By the way, since a proteolytic enzyme is used as an enzyme in this method, it is usually accompanied by a hydrolysis reaction of a peptide once formed. Incidentally, as is well known, the reaction using this proteolytic enzyme is an equilibrium reaction.

Therefore, it is an important point to suppress the hydrolysis reaction in order to efficiently proceed the peptide bond formation reaction, and at present, a method of precipitating the product to remove it from the reaction system to prevent the hydrolysis reaction or water A method in which a reaction is carried out in a two-phase reaction medium in which an organic solvent immiscible with is added to water and the product is extracted to the organic solvent layer side and removed from the reaction system is mainly used. However, in these methods, products having a carboxyl group in the product have high solubility in water under normal enzymatic reaction conditions, and therefore do not precipitate and are not extracted into the organic solvent layer, and the reaction is efficient. Does not progress well. In particular, a highly hydrophilic formyl group (hereinafter abbreviated as For) or an acetyl group (hereinafter Ac
Is abbreviated. ) Is particularly remarkable. By the way, there are endo-type and exo-type proteolytic enzymes.When carboxypeptidase which is an exo-type enzyme is used, an amino acid is used as a substrate on the amino group side. It becomes a free carboxyl group, which is exactly the case in the above example.

Therefore, the present inventors have earnestly studied a method for efficiently synthesizing an N-protected peptide having at least one carboxyl group by reacting an N-protected amino acid or its derivative with an amino acid or its derivative using a protease. As a result, surprisingly, by carrying out the present enzymatic reaction in a two-phase reaction medium of an organic solvent and water in the presence of a quaternary ammonium salt or a quaternary phosphonium salt, the enzyme does not originally move to the organic solvent layer. The product is lipophilic 4
As a quaternary ammonium salt or quaternary phosphonium salt,
The present invention has been completed by finding that it can be transferred to an organic solvent layer and the peptide production reaction can proceed efficiently.

The method of the present invention is an important intermediate for the production of aspartame (α-L-aspartyl-L-phenylalanine methyl ester), which has been attracting attention as an excellent sweetener in recent years, and is N-protected-α-L-aspartyl-L-. It is particularly effective when applied to the synthesis of phenylalanine (hereinafter abbreviated as N-protected-α-AP). That is, N-protected-α-AP
Is easily converted to aspartame (Japanese Patent Publication No. 60-5)
0,200) is the only method for producing N-protected-α-AP,
There is a method of reacting -protected-L-aspartic anhydride and L-phenylalanine in acetic acid (JP-B-55-26,133), but in this method, N-protected-β-L-aspartyl-L-phenylalanine is used. Is a by-product,
It is difficult to say that it is an excellent method such as using highly corrosive acetic acid. By applying the method of the present invention, the above-mentioned drawbacks can be overcome and only N-protected-α-AP can be efficiently synthesized.

As shown in the examples, the use of the method of the present invention significantly improves the yield of the produced N-protected peptide as compared with the enzymatic reaction in water. For example, when the protecting group is an acetyl group, the yield of N-acetyl-α-L-aspartyl-L-phenylalanine (hereinafter abbreviated as Ac-α-AP) is 5.2% in the reaction in an aqueous solution (comparative). Example 2), the yield of Ac-α-AP is improved to 20% (Example 7) by using the method of the present invention. Even when the protecting group is a benzyloxycarbonyl group, the yield of N-benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine is 2.4% (Comparative Example 6) in the reaction in an aqueous solution. The method improved to 8% (Example 15).

According to the method of the present invention, firstly, N
-By carrying out a protected peptide synthesis reaction and transferring the produced N-protected peptide as a lipophilic quaternary ammonium salt or quaternary phosphonium salt to the organic solvent layer, the peptide production reaction can proceed efficiently. Two
The N-protected peptide produced by reacting an N-protected amino acid or its derivative with an amino acid or its derivative in water using a protease and then adding an organic solvent and a quaternary ammonium salt or a quaternary phosphonium salt. It is also possible to extract the N-protected peptide produced by utilizing the difference in the organic solvent / water partition coefficient between the N-protected amino acid or its derivative and the organic solvent / water derivative of the starting material into the organic solvent layer.

After separating the organic solvent layer and the aqueous layer, by the first method,
An organic solvent containing a quaternary ammonium salt or a quaternary phosphonium salt is again added to the aqueous layer to carry out a peptide formation reaction in a two-layer reaction medium, and if this is repeated, the peptide formation reaction can proceed efficiently. It goes without saying that you can do it. Of course, the peptide production reaction may be repeated by the second method.

As the N-protecting group of N-protected-L-aspartic acid and its derivative used in the method of the present invention, a protecting group used in usual peptide synthesis, for example, formyl group, acetyl group, benzyloxycarbonyl group, t
-Butyloxycarbonyl group, phenoxyacetyl group,
A 1-methyl-2-acetylvinyl group and an acetoacetyl group are used, and an amino acid residue is also used. Among them, a formyl group, an acetyl group and a benzyloxycarbonyl group are preferably used. Examples of N-protected amino acids in which an amino acid residue is an N-protecting group include N-formyl-L-alanyl-L-aspartic acid. Examples of N-protected amino acid derivatives include N-formyl-L-aspartic acid-α-methyl ester.

The amino acid which is reacted with N-protected-L-aspartic acid or its derivative is L-phenylalanine or its derivative, for example L-phenylalanine methyl ester.

In addition, the combination of the N-protected amino acid or its derivative and the amino acid or its derivative to be reacted therewith must be such that the N-protected peptide or its derivative produced has at least one free carboxyl group.
As described above, the N-protected peptide or its derivative is newly bonded by binding the produced N-protected peptide or its derivative with the cation portion of the quaternary ammonium salt or quaternary phosphonium salt dissolved in an organic solvent. This is because it is a lipophilic quaternary ammonium salt or quaternary phosphonium salt.

As the organic solvent, any solvent can be used as long as it can dissolve the quaternary ammonium salt or the quaternary phosphonium salt and is not uniformly miscible with water and is not particularly active in the starting material and the intended product. You can Hydrocarbons such as toluene, xylene and hexane, esters such as ethyl acetate and butyl acetate, halogenated hydrocarbons such as chloroform, carbon tetrachloride and ethylene dichloride, alcohols such as butanol and amyl alcohol, and ketones such as methyl ethyl ketone. Typical examples thereof include ethers such as diethyl ether and diisopropyl ether, and a mixed solvent composed of any two or more of these may be used.

The enzyme used in the method of the present invention is not particularly limited as long as it is a protease. Further, the pH of the reaction solution when carrying out the enzyme reaction varies depending on the type of the enzyme used, and is 3 to 7 in the case of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) and Orientase 5A (manufactured by Oriental Yeast Co., Ltd.),
In the case of Samoaze (manufactured by Daiwa Kasei Co., Ltd.), it is 6 to 8. The enzymatic reaction of the present invention is preferably carried out at a temperature of 10 to 90 ° C, and 20 to 50 ° C from the viewpoint of maintaining the enzymatic activity.

In the method of the present invention, the concentrations of both starting materials used are not particularly limited, but are preferably higher in order to allow the peptide-forming reaction to proceed more efficiently. From the viewpoint of promoting the peptide-forming reaction, the concentration of the starting material is important, and the ratio of use of both is not particularly limited.

The quaternary ammonium salt or phosphonium salt used in the method of the present invention is not particularly limited, and trioctylmethylammonium chloride (Aliqua manufactured by Henkel) is used.
t336), cetyl dimethyl benzyl ammonium chloride, tetra n-butyl phosphonium bromide and the like are preferably used. Also, the larger the amount of both starting materials, the more the target product can be transferred to the organic solvent layer, but if it is too large, the enzyme activity may be reduced. It can be used in a ratio of 0.5 to 5.0 times by weight.

In order to separate and collect the N-protected peptide or its derivative transferred to the organic solvent layer in the form of a quaternary ammonium salt or a phosphonium salt, for example, the following may be performed. Four
N- in the form of a primary annium salt or phosphonium salt
The protected peptide or its derivative can be released by mixing with, for example, saline. That is, the separated organic solvent layer may be extracted with saline. The concentration of saline is preferably 2 to 20 (weight)%.

Hereinafter, the present invention will be further described with reference to Examples and Comparative Examples.

Example 1 0.54 g (3.1 mmol) of N-acetyl-L-aspartic acid (hereinafter abbreviated as Ac-Asp) and 0.1 g (0.6 mmol) of L-phenylalanine (hereinafter abbreviated as Phe) in appropriate amounts. After dissolving in water and adding sodium hydroxide aqueous solution to adjust the pH to 4.5, the total volume was adjusted to 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolved, 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat 336) in toluene solution 1
0 ml was added, and the mixture was shaken at 40 ° C. for 24 hours.

When Ac-α-AP in the reaction solution (aqueous layer and toluene layer) was quantified by high performance liquid chromatography (hereinafter abbreviated as HPLC), it was produced in a yield of 4.2% with respect to Phe. (Water layer 1.2%, toluene layer 3.0%).

Example 2 Ac-Asp (0.54 g, 3.1 mmol) and Phe (0.1 g, 0.6 mmol) were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH to 4.
After adjusting to 5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, 10 ml of a carbon tetrachloride solution containing 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat 336) was added, and the mixture was shaken at 40 ° C. for 24 hours.

Ac-α-AP in the reaction solution (water layer and carbon tetrachloride layer)
When quantified by LC, it was found to be produced in a yield of 4.3% based on Phe (water layer 1.1%, carbon tetrachloride layer 3.2%).

Example 3 Ac-Asp (0.54 g, 3.1 mmol) and Phe (0.1 g, 0.6 mmol) were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH to 4.
After adjusting to 5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, 10 ml of a chloroform solution containing 1.23 g (3.1 mmol) of cetyldimethylbenzylammonium chloride was added, and the mixture was shaken at 40 ° C. for 24 hours.

Ac-α-AP in the reaction solution (water layer and chloroform layer),
When quantified by HPLC, it was produced in a yield of 4.2% based on Phe (water layer 1.2%, chloroform layer 3.0%).

Example 4 Ac-Asp (0.54 g, 3.1 mmol) and Phe (0.1 g, 0.6 mmol) were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH to 4.
After adjusting to 5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After being completely dissolved, a solution of tetra-n-butylphosphonium bromide 1.05 g (3.1 mmol) in 1,2-dichloroethane 1
0 ml was added, and the mixture was shaken at 40 ° C. for 24 hours.

Ac- in the reaction solution (water layer and 1,2-dichloroethane layer)
When α-AP was quantified by HPLC, it was 3.1% with respect to Phe.
Was produced in a yield of (water layer 1.6%, 1,2-dichloroethane layer 1.5%).

Comparative Example 1 (Comparative Example for Examples 1 to 5) 0.54 g (3.1 mmol) of Ac-Asp and 0.1 g (0.6 mmol) of Phe were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH to 4.
After adjusting to 5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, the mixture was shaken at 40 ° C. for 24 hours.

When Ac-α-AP in the aqueous solution was quantified by HPLC, it was produced in a yield of 1.6% based on Phe.

Example 5 2.17 g (12.4 mmol) of Ac-Asp and 0.1 g (0.6 mmol) of Phe were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH.
After adjusting to 4.5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, 10 ml of a toluene solution containing 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat 336) was added, and the mixture was shaken at 40 ° C. for 44 hours.

Ac-α-AP in the reaction solution (water layer and toluene layer) was analyzed by HPLC.
The yield was 11.1% based on Phe (water layer 3.3%, toluene layer 7.8%).

Example 6 2.17 g (12.4 mmol) of Ac-Asp and 0.1 g (0.6 mmol) of Phe were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH.
After adjusting to 4.5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After complete dissolution, 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat 336) in toluene /
10 ml of a hexane (1/1) solution was added, and the mixture was shaken at 40 ° C. for 44 hours.

Ac-α- in the reaction solution (water layer and toluene / hexane layer)
When AP was quantified by HPLC, it was found to be produced in a yield of 11.0% with respect to Phe (water layer 3.1%, toluene / hexane layer 7.9%).

Example 7 2.17 g (12.4 mmol) of Ac-Asp and 0.1 g (0.6 mmol) of Phe were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH.
After adjusting to 4.5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, 20 ml of a chloroform solution of 2.46 g (6.2 mmol) of cetyldimethylbenzylammonium chloride
Was added and shaken at 40 ° C. for 44 hours.

Ac-α-AP in the reaction solution (water layer and chloroform layer),
When quantified by HPLC, it was found to be produced in a yield of 20.1% with respect to Phe (water layer 5.1%, chloroform layer 15.0%).

Comparative Example 2 (Comparative Example to Examples 5 to 7) 2.17 g (12.4 mmol) of Ac-Asp and 0.1 g (0.6 mmol) of Phe were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH.
After adjusting to 4.5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, the mixture was shaken at 40 ° C. for 44 hours.

When Ac-α-AP in the water solution was quantified by HPLC, it was produced in a yield of 5.2% based on Phe.

Example 8 Ac-Asp (0.54 g, 3.1 mmol) and Phe (0.1 g, 0.6 mmol) were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH to 4.
After adjusting to 5, the total volume was 5 ml. To this aqueous solution, 200 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) was added. After completely dissolving, 10 ml of a toluene solution containing 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat336) was added, and the mixture was shaken at 40 ° C. for 48 hours.

Ac-α-AP in the reaction solution (water layer and toluene layer) was analyzed by HPLC.
The yield was 5.0% based on Phe (water layer 1.5%, toluene layer 3.5%).

Example 9 0.54 g (3.1 mmol) of Ac-Asp and 0.1 g (0.6 mmol) of Phe were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH to 3.
After adjusting to 0, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, 10 ml of a toluene solution containing 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat336) was added, and the mixture was shaken at 40 ° C. for 22 hours.

Ac-α-AP in the reaction solution (water layer and toluene layer) was analyzed by HPLC.
The yield was 3.4% based on Phe (water layer 0.9%, toluene layer 25%).

Example 10 Ac-Asp (0.54 g, 3.1 mmol) and Phe (0.1 g, 0.6 mmol) were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH to 4.
After adjusting to 5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, 10 ml of a toluene solution containing 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat336) was added, and the mixture was shaken at 25 ° C. for 96 hours.

Ac-α-AP in the reaction solution (water layer and toluene layer) was analyzed by HPLC.
The yield was 4.0% based on Phe (water layer 1.2%, toluene layer 2.8%).

Example 11 N-formyl-L-aspartic acid (hereinafter abbreviated as For-Asp) 0.5 g (3.1 mmol) and Phe 0.1 g (0.6 mmol)
Is dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution is added.
After adjusting to pH 4.5, the whole was brought to 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolved, 10 ml of a toluene solution containing 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat 336) was added, and the mixture was shaken at 40 ° C. for 72 hours.

N-formyl-α-in the reaction liquid (water layer and toluene layer)
L-aspartyl-L-phenylalanine (hereinafter, For
Abbreviated as -α-AP. ) Was determined by HPLC.
The yield was 2.9% based on e (water layer 0.7%, toluene layer 2.2%).

Comparative Example 3 (Comparative Example to Example 11) 0.5 g (3.1 mmol) of For-Asp and 0.1 g (0.6 mmol) of Phe were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH to 4.
After adjusting to 5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, the mixture was shaken at 40 ° C. for 72 hours.

When For-α-AP in the aqueous solution was quantified by HPLC, it was found to be produced in a yield of 0.8% with respect to Phe.

Example 12 2.0 g (12.4 mmol) of For-Asp and 0.1 g (0.6 mmol) of Phe were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH.
After adjusting to 4.5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, 10 ml of a toluene solution containing 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat336) was added, and the mixture was shaken at 40 ° C. for 76 hours.

HPLC of For-α-AP in the reaction solution (water layer and toluene layer)
The yield was 6.3% based on Phe (water layer 2.0%, toluene layer 4.3%).

Example 13 For-Asp 0.5 g (3.1 mmol) and Phe 0.1 g (0.6 mmol) were dissolved in an appropriate amount of water, and a sodium hydroxide aqueous solution was added to adjust the pH to 4.
After adjusting to 5, the total volume was 5 ml. To this aqueous solution, 50 mg of Orientase 5A (manufactured by Oriental Yeast Co., Ltd.) was added. After completely dissolving, 10 ml of an ethyl acetate solution containing 1.25 g (3.1 mmol) of trioctylmethylammonium chloride (Aliquat 336) was added, and the mixture was shaken at 40 ° C. for 71 hours.

HPL of For-α-AP in the reaction solution (water layer and ethyl acetate layer)
When quantified with C, the yield was 1.1% based on Phe (water layer 0.3%, ethyl acetate layer 0.8%).

Comparative Example 4 (Comparative Example to Example 13) For-Asp 0.5 g (3.1 mmol) and Phe 0.1 g (0.6 mmol) were dissolved in an appropriate amount of water, and a sodium hydroxide aqueous solution was added to adjust the pH to 4.
After adjusting to 5, the total volume was 5 ml. To this aqueous solution, 50 mg of Orientase 5A (manufactured by Oriental Yeast Co., Ltd.) was added. After completely dissolving, the mixture was shaken at 40 ° C. for 71 hours.

When For-α-AP in the aqueous solution was quantified by HPLC, it was found to be produced in a yield of 0.4% based on Phe.

Example 14 0.54 g (3.1 mmo) of N-formyl-L-aspartic acid-α-methyl ester (hereinafter abbreviated as For-Asp-OMe)
l) and Phe 0.1 g (0.6 mmol) were dissolved in an appropriate amount of water, and a sodium hydroxide aqueous solution was added to adjust the pH to 4.5, and then the whole was adjusted to 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After complete dissolution, trioctylmethyl ammonium chloride (Aliquat 336)
Add 10 ml of a 1.25 g (3.1 mmol) toluene solution, and add 40
Shake at 65 ° C. for 65 hours.

HPLC of For-α-AP in the reaction solution (water layer and toluene layer)
The yield was 7.2% based on Phe (water layer 1.9%, toluene layer 5.3%).

Comparative Example 5 (Comparative Example to Example 14) For-Asp-OMe 0.54 g (3.1 mmol) and Phe 0.1 g (0.6 mmo
l) was dissolved in an appropriate amount of water, an aqueous sodium hydroxide solution was added to adjust the pH to 4.5, and then the whole was adjusted to 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, the mixture was shaken at 40 ° C. for 65 hours.

When For-α-AP in the aqueous solution was quantified by HPLC, it was found to be produced in a yield of 2.1% based on Phe.

Example 15 N-benzyloxycarbonyl-L-aspartic acid (hereinafter abbreviated as Z-Asp) 0.83 g (3.1 mmol) and Ph
0.1 g (0.6 mmol) of e was dissolved in an appropriate amount of water, an aqueous sodium hydroxide solution was added to adjust the pH to 4.5, and then the whole was adjusted to 5 ml. Protease M (Amano Pharmaceutical Co., Ltd.) 5
0 mg was added. After completely dissolving, 1.25 g of trioctylmethyl ammonium chloride (Aliquat 336)
Add 10 ml of a toluene solution of (3.1 mmol), and add 4 at 40 ° C.
Shake for 4 hours.

N-benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine (hereinafter abbreviated as Z-α-AP) in the reaction solution (water layer and toluene layer) was quantified by HPLC, and it was determined to be Phe. Was produced in a yield of 8.1% (water layer 1.1%, toluene layer 7.0%).

Comparative Example 6 (Comparative Example to Example 15) 0.83 g (3.1 mmol) of Z-Asp and 0.1 g (0.6 mmol) of Phe were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH.
After adjusting to 4.5, the total volume was 5 ml. To this aqueous solution was added 50 mg of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.). After completely dissolving, the mixture was shaken at 40 ° C. for 44 hours.

When Z-α-AP in the aqueous solution was quantified by HPLC, it was produced in a yield of 2.4% based on Phe.

Example 16 Z-Asp 0.67 g (2.5 mmol) and L-phenylalanine methyl ester (hereinafter abbreviated as PM) 0.45 g (2.5 m)
(mol) was dissolved in an appropriate amount of water, sodium hydroxide aqueous solution was added to adjust the pH to 6.2, and then the whole was adjusted to 18 ml. To this aqueous solution, 120 mg of Samoases (manufactured by Daiwa Kasei Co., Ltd.) was added. After completely dissolving, 20 ml of a toluene solution containing 2.06 g (5.1 mmol) of trioctylmethylammonium chloride (Aliquat 336) was added, and the mixture was shaken at 40 ° C. for 3 hours.

N-benzyloxycarbonyl-α-L-phenylalanine methyl ester (hereinafter abbreviated as Z-α-APM) in the reaction solution (water layer and toluene layer) was quantified by HPLC, and it was 54.6 relative to Phe. % (Water layer 18.0%, toluene layer 36.6%).

Comparative Example 7 (Comparative Example to Example 16) 0.67 g (2.5 mmol) of Z-Asp and 0.45 g (2.5 mmol) of PM were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH.
After adjusting to 6.2, the total volume was 18 ml. To this aqueous solution, 120 mg of Samoases (manufactured by Daiwa Kasei Co., Ltd.) was added. After completely dissolving, the mixture was shaken at 40 ° C. for 3 hours.

When Z-α-APM in the aqueous solution was quantified by HPLC, it was found to be produced in a yield of 19.4% with respect to Phe.

Example 17 For-Asp 2.5 g, F obtained by reacting in the same manner as in Comparative Example 3
Trioctylmethylammonium chloride 6. was added to 25 ml of an enzyme reaction solution containing 0.009 g of or-α-AP and 0.5 g of L-Phe.
After adding 50 g of 25 g toluene solution to adjust the pH to 6.0, the mixture was shaken at 37 ° C. for 15 minutes.

The toluene layer is separated, and For-Asp, For-α-AP and L-Phe are separated.
Was quantified by HPLC. As a result, For-α-A was found in the toluene layer.
76% of P was extracted. On the other hand, For-Asp and Phe were only extracted by 9% and 7%, respectively. By the way,
The water-toluene distribution coefficients of For-α-AP, For-Asp and Phe were 1.35, 0.04 and 0.03, respectively.

Comparative Example 8 (Comparative Example to Example 17) The same experiment as in Example 18 was conducted except that trioctylmethylammonium chloride was not added.
No r-α-AP was extracted into the toluene layer, and its partition coefficient was 0.

Example 18 Ac-Asp 2.6 g, Ac-obtained by the same reaction as in Comparative Example 1
25 ml of enzyme reaction solution containing 0.015 g of α-AP and 0.5 g of Phe
50 ml of a toluene solution of 6.25 g of trioctylmethylammonium chloride was added to the mixture to adjust the pH to 4.4, and then 37
Shake at 15 ° C for 15 minutes.

The toluene layer was separated, and Ac-Asp, Ac-α-AP and L-Phe were separated.
As a result of quantification by HPLC, 63% of Ac-α-AP was extracted in the toluene layer. On the other hand, Ac-Asp and Phe were only extracted by 9% and 2%, respectively. By the way, Ac
The distribution coefficients of -α-AP, Ac-Asp and L-Phe between water and toluene were 1.03, 0.07 and 0.01, respectively.

Comparative Example 9 (Comparative Example to Example 18) The same experiment as in Example 19 was carried out except that trioctylmethylammonium chloride was not added.
-Α-AP was not extracted in the toluene layer at all, and its partition coefficient was 0.

Claims (2)

[Claims] 1. N-protected-L-aspartic acid or a derivative thereof and L-phenylalanine or a derivative thereof,
N- produced by reacting with a protease in a water layer in a two-phase reaction medium of a water-immiscible organic solvent in which a quaternary ammonium salt or a quaternary phosphonium salt is dissolved and water
Protecting -α-L-aspartyl-L-phenylalanine or its derivative with a lipophilic quaternary ammonium salt or 4
A method for enzymatically synthesizing N-protected-α-L-aspartyl-L-phenylalanine or a derivative thereof, which comprises migrating as a primary phosphonium salt to an organic solvent phase. 2. N-protected-α-L-aspartyl-L-produced by reacting N-protected-L-aspartic acid or its derivative with L-phenylalanine or its derivative in water using a protease. An enzyme of N-protected-α-L-aspartyl-L-phenylalanine or a derivative thereof, which comprises extracting and recovering phenylalanine or a derivative thereof with a water-immiscible organic solvent in which a quaternary ammonium salt or a quaternary phosphonium salt is dissolved. Synthetic method.