JPS62259597A - Enzymic synthesis of n-protected peptide - Google Patents

Abstract

PURPOSE:To efficiently obtain the titled compound, by reacting an N-protected amino acid with an amino acid in an aqueous layer in a two-phase system reaction medium of an organic solvent containing a quaternary ammonium salt, etc., and water using a proteolytic enzyme and migrating the resultant product to an organic solvent phase. CONSTITUTION:An N-protected amino acid, e.g. N-acetyl-L-aspartic acid, etc., or a derivative thereof is reacted with an amino acid, e.g., L-phenylalanine, etc., or a derivative thereof in an aqueous layer in a two-phase system reaction medium of a water-immiscible organic solvent containing a dissolved quaternary ammonium salt, e.g. trioctylmethylammonium chloride, etc., or quaternary phosphonium salt and water using a proteolytic enzyme to form an N-protected peptide containing one or more carboxyl groups. The resultant N-protected peptide is then converted into a lipophilic quaternary ammonium salt or phosphonium salt and migrated to the organic solvent phase to afford the aimed compound.

Description

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

In recent years, methods using enzymes in peptide synthesis have attracted attention because they frequently cause ceramization, and attempts have been made to utilize them in various peptide syntheses. By the way, since this method uses proteolytic enzymes as enzymes, it is common to use proteolytic enzymes once they have been generated. Accompanied by hydrolysis reaction of notide. By the way,
As is well known, this reaction using a protease is an equilibrium reaction.

Therefore, in order to efficiently proceed with the peptide bond formation reaction, it is important to suppress the hydrolysis reaction.Currently, there are methods to prevent the hydrolysis reaction by precipitating the product and removing it from the reaction system, Mainly used is a method in which the reaction is carried out in a two-phase reaction medium in which an organic solvent that is immiscible with water is added to water, and the product is extracted into the organic solvent layer and removed from the reaction system. However, in these methods, products with carboxyl groups have high solubility in water under normal enzyme reaction conditions, so they do not precipitate or are extracted into the organic solvent layer, and the reaction is not efficient. It doesn't progress well. In particular, highly hydrophilic formyl group (hereinafter abbreviated as For) and acetyl group (hereinafter A
It is abbreviated as e. ) is particularly noticeable. By the way, there are two types of proteolytic enzymes: endo-type and exo-type. When Cal, an exo-type enzyme, uses xipezotidase, an amino acid is used as the substrate on the amino group side, so the Cal-d of the product peptide is ? The xyl terminal becomes a free carboxyl group, and the above example applies exactly.

Therefore, the present inventors have conducted intensive studies on 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, when this enzyme reaction is carried out in the presence of a quaternary ammonium salt or quaternary phosphonium salt in a two-phase reaction medium of organic solvent and water, no migration occurs in the organic solvent layer. The present inventors have discovered that the peptide production reaction can be efficiently progressed by transferring the product that does not react as a lipophilic quaternary ammonium salt or quaternary phosphonium salt to an organic solvent layer, and have completed the present invention.

The method of the present invention utilizes N-protected-α-L-aspartyl-L-phenylalanine (α-L-aspartyl-L-phenylalanine methyl ester), which is an important intermediate in the production of aspartame (α-L-aspartyl-L-phenylalanine methyl ester), which has recently attracted attention as an excellent sweetener. It is particularly effective when applied to the synthesis of N-protected-α-AP (hereinafter abbreviated as N-protected-α-AP). That is, N-protected-α-
AP is easily converted to aspartame (Special Publications Act 1986)
-50.200), is the only method for producing N-protected-α-AP that uses N-protected-L-asnocarboxylic anhydride and L-protected α-AP.
- A method of reacting phenylalanine in acetic acid is known (Japanese Patent Publication No. 55-26, 133), but this method produces by-products of HN-holding-β-L-aspartyl-L-phenylalanine and is corrosive. It is hard to say that it is a good method, such as using high-quality acetic acid. By applying the method of the present invention, the above drawbacks can be overcome and only N-protected-α-AP can be efficiently synthesized.

As shown in the Examples, when the method of the present invention is used, the yield of the N-protected peptide produced is significantly improved compared to an enzymatic reaction in water. For example, when the protecting group is an acetyl group, in the reaction in an aqueous solution, N-acetyl-α-L-asno-rutile-L-phenylalanine (hereinafter referred to as Ac-α-A
It is abbreviated as P. ) is 5.2% (Comparative Example 2), but when the method of the present invention is used, the yield of Ae-α-AP is improved to 20% (Example 7). Also, when the benzyloxycar protecting group is a nyl group, in the reaction in an aqueous solution, N
- The yield of α-L-aspartyl-L-phenylalanine is 2.4% (Comparative Example 6)
However, when the method of the present invention was used, it improved to 8 inches (Example 15).

According to the method of the present invention, firstly, a two-phase reaction medium is used to
-Perform the protected peptide production reaction and generate LAN-protection-
(The peptide production reaction can proceed efficiently by transferring 'zotide as a lipophilic quaternary ammonium salt or quaternary phosphonium salt to the organic solvent layer, but
Second, after reacting the N-protected amino acid or its derivative with the amino acid or its derivative in water using a protease, an organic solvent and a quaternary ammonium salt or
Using the difference in organic solvent/water partition coefficient between the N-protected peptide, the raw material N-protected amino acid or its derivative, and the amino acid or its derivative, the N-protected peptide is converted into an organic It is also possible to extract into a 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 added to the aqueous layer again to perform a peptide production reaction in a two-layer reaction medium. It goes without saying that if this is carried out repeatedly, the peptide production reaction can proceed efficiently.

Of course, the peptide production reaction may be repeated by the second method.

The N-protecting groups of the N-protected amino acids and their derivatives used in the method of the present invention include protecting groups used in ordinary peptide synthesis, such as formyl group, acetyl group, benzyloxycarboxyl group, t-butyloxycarboxyl group, and -rudenyl group, phenoxyacetyl group, l-methyl-
In addition to 2-acetylvinyl group and acetoacetyl group, amino acid residues are also used, and among them, formyl group, acetyl group, pennoloxycar, and nyl group are preferably used. N where the amino acid residue is an N-protecting group
-Protected amino acids include, for example, N-formyl-L-
Alanyl-L-aspartic acid. Further, as the N-protected amino acid derivative, for example, N-formyl-L-
Aspartic acid-α-methyl ester.

Among the amino acids or derivatives thereof to be reacted with the N-protected amino acid or derivative thereof, the last amino acid derivative is, 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 with it is such that the resulting N-protected-e7''tide or its derivative is at least 1
It must have one free carboxyl group. As mentioned above, the generated 9N-homitomi peptide or its derivatives are combined with the cation moiety of a quaternary ammonium salt or a quaternary phosphonium salt dissolved in an organic solvent to create a new N-protected peptide or This is to obtain a lipophilic quaternary ammonium salt or quaternary phosphonium salt of the derivative.

Further, as the organic solvent, any solvent can be used as long as it can dissolve the quaternary ammonium salt or the quaternary phosphonium salt, is not uniformly miscible with water, and is not particularly active for the starting material and the target product. I can do it. Hydrocarbons such as toluene, xylene, and hexane; esters such as ethyl acetate and butyl acetate; halodanized 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 include ethers such as diethyl ether, diinzolovyl ether, etc., and any two of these
It is also possible to use a mixed solvent consisting of more than one type of solvent.

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

In the method of the present invention, there are no particular limitations on the concentrations of both starting materials used, but higher concentrations are desirable in order to allow the peptide production reaction to proceed more efficiently.

From the viewpoint of advancing the peptide production reaction, the concentration of the starting materials is important, and there is no particular restriction on the ratio of the two used.

There are no particular limitations on the quaternary ammonium salt or phosphonium salt used in the method of the present invention, including trioctylmethylammonium chloride (Henkel A1.
1quat 336, etc.), cetyldimethylbenzylammonium chloride, tetra-butylphosphonium bromide, and the like are preferably used. In addition, the larger the amount relative to 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, so the total amount of both starting materials should be adjusted. Against 0.
It can be used at a ratio of 5 to 5.0 times by weight.

In order to separate and recover the N-protected peptide or its derivative transferred to the organic solvent layer in the form of a quaternary ammonium salt or phosphonium salt, the following may be performed, for example. 4
N in the form of ammonium or phosphonium salts
- The protected peptide or its derivative can be released, for example, by mixing with saline or the like. That is,
The separated organic solvent layer may be extracted with saline. The concentration of the saline solution is preferably between 2 and 20 (by weight).

The present invention will be further explained below with reference to Examples and Comparative Examples.

Example I N-acetyl-L-aspartic acid (hereinafter referred to as Ac-As
It is abbreviated as p. ) 0.54 g (3.1 mmot)
and L-phenylalanine (hereinafter abbreviated as Phe)
Dissolve 0.1 g (0.6 mmot) in an appropriate amount of water,
After adjusting the pH to 4.5 by adding an aqueous sodium hydroxide solution, the total volume was made up to 5 ml. 50 Da of Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) was added to this aqueous solution. After complete dissolution, trioctylmethylammonium chloride (A1
1quat 336) 1.25 g (3.1mm
ot) was added to the mixture, and the mixture was shaken at 40°C for 24 hours.

When Aa-α-AP in the reaction solution (aqueous layer and toluene layer) was quantified using high-performance liquid chromatography (hereinafter abbreviated as HPLC), it was found that it was produced at a yield of 4.2% relative to Pb. (aqueous layer 1.2%,) luene layer 3.0%
).

Example 2 Ac-Aspo, 54 g (3,1 mmot) and P
1 g (0.6 mmot) of heO was dissolved in an appropriate amount of water, the pH was adjusted to 4.5 by adding an aqueous sodium hydroxide solution, and the total volume was made up to 51 nl. Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) 501n9 was added to this water bath solution. After complete dissolution, add 1.25 g of trioctylmethylammonium chloride (A11quat336) (3,
1mmot) of carbon tetrachloride solution lQmA! Add 40
Shake at ℃ for 24 hours.

When Ae-α-AP in the reaction solution (aqueous layer and carbon tetrachloride layer) was quantified by HPLC, it was 4.3% relative to phe.
It was produced at a yield of 1.1% (1 m of water, 3.2% carbon tetrachloride layer).

Example 3 Ac-Asp 0.54 g (3.1 mmot) and P
1 g (0.6 mmol) of heO was dissolved in an appropriate amount of water, the pH was adjusted to 4.5 by adding an aqueous sodium hydroxide solution, and the total was brought to 5 mA'. 50 m9 of Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) was added to this aqueous solution. After complete dissolution, 1.23 g (3.1 mmot) of cetyldimethylbenzyl ammonium chloride was added to 10 ml of a homogeneous solution, and the mixture was shaken at 40° C. for 24 hours.

Ae-α-AP in the reaction solution (aqueous layer and chloroform layer)
When quantified by HPLC, l'? It was produced at a yield of 4.2 parts based on he (aqueous layer 1.2%, chloroform layer 3.0 parts).

Example 4 Ae-Asp 0.54 g (3,1rrrnot
) and PBS 0.1 g (0.6 mmot) were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pH to 4.
After adjusting the volume to 5 ml, the total volume was adjusted to 5 ml. 50 doses of Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) was added to this aqueous solution. After complete dissolution, 10 m of a solution of 1.05 g (3,1 mmot) of tetra n-butylphosphonium bromide in 1,2-dichloroethane was added, and the mixture was shaken at 40°C for 24 hours.

Ae in the reaction solution (aqueous layer and 1,2-dichloroethane layer)
-α-AP was quantified by PLC.
The product was produced at a yield of 3.1 compared to that of the aqueous layer (1.6 qb of aqueous layer, 1.5 qb of l,2-dichloroethane layer).

Comparative Example 1 (Comparative Example for Examples 1 to 5) Ac-Asp
0.54 g (3,1 mmot) and Phe 0.
1 g (0.6 mmol) was dissolved in an appropriate amount of water, and after adjusting the pH to 4.5 K by adding an aqueous sodium hydroxide solution, the total pH was adjusted to 5 d. Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) 50rn9 was added to this aqueous solution. After complete dissolution, it was shaken at 40°C for 24 hours.

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

Example 5 2.17 g (12.4 mmot) of Ae-Asp and 0.1 g (0.6 mmot) of Phe were dissolved in an appropriate amount of water, and after adjusting to -4.5 by adding an aqueous sodium hydroxide solution, The whole thing was 54 busy. 50 μm of Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) was added to this aqueous solution. After complete dissolution, trioctylmethylammonium chloride (A
11quat336) 1.25g (3.1mr
10 g of a toluene solution of (not) was added, and the mixture was shaken at 40°C for 44 hours.

Ae-α-AP in the reaction solution (aqueous layer and toluene layer),
I (quantified by PLC, 11.
It was produced at a yield of 1.1% (aqueous layer: 3.3%, luene: 47.8%).

Example 6 Ac-Asp 2.17 g (12.4 mmot) and Phe O, 1 g (0.6 mmol) were dissolved in an appropriate amount of water, and a sodium hydroxide water bath solution was added to give a Pll of 4.5
After adjusting to 5 ml, the total volume was adjusted to 5 ml. 50m9 of Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) was added to this aqueous solution. After completely dissolving, add 1.25 g of trioctylmethylammonium chloride (A11quat336) (
A toluene/hexane (1/1) solution 104 of 3.1 mmot) was added, and the mixture was shaken at 40° C. for 44 hours.

Ac-α in the reaction solution (aqueous layer and toluene/hexane layer)
-AP was quantified by PLC, and it was found that it was produced at a yield of 11.0% relative to Rho (aqueous layer 3.1 section,
toluene/hexane layer 7.9%).

Example 7 Ac-Asp 2.17 g (12.4 mmot) and phe 0.1 g (0.6 mmot) were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the concentration to 4.5 mm. , the total volume was 5 ml. 50 μm of Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) was added to this aqueous solution. After complete dissolution, 20 rnl of a chloroform solution of 2.46 g (6.2 mmot) of cetyldimethylbenzyl ammonium chloride was added, and the mixture was shaken at 40° C. for 44 hours.

Ae-α-AP in the reaction solution (aqueous layer and chloroform layer)
was quantified by HPLC [20
, with a yield of 11 (aqueous layer 5.1%, chloroform layer 15.0%).

Comparative Example 2 (Comparative Example for Examples 5 to 7) Ae-Asp
2.17 g (12,4 mmot) and Ph@0.1
g (0.6 mmot) was dissolved in an appropriate amount of water, the pH was adjusted to 4.5 by adding sodium hydroxide water bath solution, and the total volume was made up to 5 ml. Add 50% of Protease M (manufactured by Ohno Pharmaceutical) to this aqueous solution. n9 was added. After complete dissolution, it was shaken at 40°C for 44 hours.

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

Example 8 Ac-Asp 0.54 g (3,1rr1rrIo
t) and Ph s O, 1 g (0.6 mmot) were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to prepare -I4.
．． After adjusting to 5, the total volume was 5 ml KL.

200 Da of Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) was added to this aqueous solution. After completely dissolving, trioctylmethylammonium chloride (A11quat336) 1
．． 25 g (3,1 mmot) of toluene solution 10
mA' was added and shaken at 40°C for 48 hours.

AC-α-AP in the reaction solution (aqueous layer and toluene layer),
When quantified by HPLC, it was found that the toluene layer was produced at a yield of 5.0% based on Rho (aqueous layer: 1.5%).
5chi).

Example 9 Aa-Asp 0.54 g (3.1 mmot) and Ph
Dissolve 0.1 g (0.6 rrvnol) in an appropriate amount of water and hydroxylate)
, After adjusting to 0, the whole was set to 5.

50 μm of Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) was added to this aqueous solution. After complete dissolution, trioctylmethylammonium chloride (A11quat336) 1.25
Add 10 m/g (3,1 mmot) of toluene solution,
It was shaken at 40°C for 22 hours.

Ae-α-AP in the reaction solution (aqueous layer and toluene layer),
When fixed in HPLC, 3.
It was produced with a yield of 4% (aqueous layer 09%, toluene layer 2%).
5chi).

Example 10 Aa-Asp O, 54g (3.1 mmot) and Ph
0.1 g (0.6 mmot) was dissolved in an appropriate amount of water and hydroxylated. After adjusting to -4.5 by adding an aqueous solution of IJ, the total volume was made up to 5 ml. Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) 5o+y was added to this aqueous solution. After complete dissolution, 1.25 g trioctylmethylammonium chloride (Aliquat 336) (3,
1 mmot) of toluene solution IQm/ was added, and the mixture was shaken at 25° C. for 96 hours.

Ae-α-AP in the reaction solution (aqueous layer and toluene layer),
When quantified using HPLIC, it was found that phe was produced at a yield of 4.0% (aqueous layer: 1.2%, toluene layer: 2.0%).
8chi).

Example 11 N-formyl-L-asno9ragic acid (hereinafter referred to as For-
It is abbreviated as Asp. ) 0.5 g (3.1mm
P
After adjusting to H4 and 5, the whole was set to 5H. Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) 507Q was added to this aqueous solution. After completely dissolved, trioctylmethylammonium chloride (A11quat 336) 1.25
A toluene solution of Ion/g (3,1rrmot) was added and the mixture was shaken at 40°C for 72 hours.

N-formyl-α- in the reaction solution (aqueous layer and toluene layer)
L-asuno9rutile-L-7enylalanine (hereinafter referred to as F
It is abbreviated as or-α-AP. ) was quantified by HPLC, and it was found that it was produced in a yield of 2.9 yen based on Ph. (0.7% in water layer, 2.2% in toluene layer).

Comparative Example 3 (Comparative Example with respect to Example 11) For-Asp
0.5 g (3,1 mmot) and Phe 0.1 g
(0.6 mmot) was dissolved in an appropriate amount of water, the pH was adjusted to 4.5 by adding an aqueous sodium hydroxide solution, and the whole was adjusted to 5-. Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) 501n9 was added to this aqueous solution. After completely dissolving, 40
Shake for 72 hours at °C.

When F'or-α-AP in the aqueous solution was quantified using HPI, C, it was found that it was produced at a yield of 0.8% based on phe.

Example 12 For-Asp 2. Og (12,4 mmol)
and phaQ, 1 g (0.6 mmot) were dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the p) to 14.5, and the total volume was made up to 5 ml. 50 μm of Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) was added to this aqueous solution. After complete dissolution, add 1.25 g of trioctylmethylammonium chloride (A11quat 336) (3,
IQml of a toluene solution of 1 mmot) was added, and the mixture was shaken at 40°C for 76 hours.

When For-α-AP in the reaction solution (aqueous layer and toluene layer) was quantified by HPLC, it was found to be 6.3% relative to Phe.
The toluene layer was produced at a yield of 2.0m (aqueous layer 2.0m).
3chi).

Example 13 For-Asp 0.5 g (3,1rnmot)
and P.he 0.1 g (0.6 mmot) 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 whole was set to 5d. Add orientase 5A (manufactured by Oriental Yeast Co., Ltd.) 50rII9 to this aqueous solution.
was added. After complete dissolution, trioctylmethylammonium chloride (A11quat 336)
1.25 g (3.1 mmot) of ethyl acetate solution 1
01117 was added and shaken at 40°C for 71 hours.

For-α-AP in the reaction solution (aqueous layer and ethyl acetate layer)
When quantified by HPLC, it was found to be 1.1 with respect to Phe.
% yield (aqueous layer 0.3%, ethyl acetate layer 0.8%).

Comparative Example 4 (Comparative Example to Example 13) For-Asp
0.5 g (3,1 mmot) and phe o,
After dissolving 1 g (0.6 mmot) in an appropriate amount of water and hydrating it, the pH was adjusted to 4.5 by adding an aqueous IJ solution, and the whole was adjusted to 5-. Add orientase 5A to this aqueous solution.
(Oriental Yeast Co., Ltd. #) 50μ was added. After complete dissolution, it 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 at a yield of 0.4% based on Pha.

Example 14 N-formyl-L-aspartic acid-α-methyl ester (hereinafter abbreviated as For-A@p-OMe) 0,
54 g (3,1 mmot) and P+h* 0.1 g
(0.6 mmot) was dissolved in an appropriate amount of water, the pH was adjusted to 4.5 by adding an aqueous sodium hydroxide solution, and the total pH was adjusted to 51111.

Add Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) 50Tn to this aqueous solution.
9 was added. After complete dissolution, trioctylmethylammonium chloride (A11quat336)
1.25 g (3,1 ramot) of toluene solution I
Qml was added and shaken at 40°C for 65 hours.

For-α-AP in the reaction solution (aqueous layer and toluene layer) was quantified by HPLC and found to be 7.2% relative to Phe.
The toluene layer was produced at a yield of 5. (aqueous layer 1.9%).
Section 3).

Comparative Example 5 (Comparative Example to Example 14) For-Asp
-OMe O, 54g (3,1 mmot) and Phe
Dissolve O.1g (0.6mmot) in an appropriate amount of water,
After adjusting the pH to 4.5K by adding an aqueous sodium hydroxide solution, the entire solution was brought to 5H. Protease M (
50 μm (manufactured by Amano Pharmaceutical Co., Ltd.) was added. After complete dissolution, it 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 at a yield of 2.1% based on Pbe.

Example 15 11J, benzyloxycarboxylic acid (hereinafter abbreviated as Z-Asp) 0.83
g (3,1 mmot) and Phs 0.1 g (0,6
After adjusting the temperature to -14.5 K by adding an aqueous IJ solution, the whole solution was heated to 5 K.
I changed it to al. Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) 50Tn9 was added to this aqueous solution. After complete dissolution, trioctylmethylammonium chloride (A11quat
336) 10 ml of a toluene solution of 1.25 g (3.1 mmot) was added and shaken at 40° C. for 44 hours.

N-benzyloxycarbonyl-α-L-as/4'rutile-L-7enylalanine (hereinafter abbreviated as 2-α-AP) in the reaction solution (aqueous layer and toluene layer) was analyzed by HPL.
When quantified at C, it was found that it was produced at a yield of 8.1% based on Phe (aqueous layer 1.1%, toluene layer 7.0%)
.

Comparative Example 6 (Comparative Example to Example 15) Z-Asp 0
．． 83 g (3,1 mmot) and Phe 0.1 g
(0.6 mmot) was dissolved in an appropriate amount of water, and an aqueous sodium hydroxide solution was added to adjust the pl (4.5), and the total volume was then adjusted to 5 rnl. Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) 50rn9 was added to this aqueous solution. After complete dissolution, it was shaken at 49°C for 44 hours.

When 2-α-AP in aqueous solution was quantified by HPLC,
It was produced at a yield of 2.4% based on Pbs.

Example 16 Z-Asp 0.67 g (2.5 mmot) and L-
Dissolve 0.45 g (2.5 mmot) of Fz-''lualanine methyl ester (hereinafter abbreviated as PM) in an appropriate amount of water, and adjust the pH to 6.2 by adding an aqueous sodium hydroxide solution.
After adjusting to 18mA overall! I made it. Samoase (manufactured by Daiwa Kasei Co., Ltd.) 120~ was added to this aqueous solution. After complete dissolution, 2.06 g of trioctylmethylammonium chloride (A11quat 336) (
Add 207 d of toluene solution of 5.1 mmot),
It was shaken at ℃ for 3 hours.

When N-benzyloxycarbonyl-α-L-phenylalanine methyl ester (hereinafter abbreviated as 2-α-APM) in the reaction solution (aqueous layer and toluene layer) was quantified by HPLC, it was found to be 54% relative to Phe. The yield of .6 was produced (aqueous layer 18.0%), toluene layer 36.6%
).

Comparative Example 7 (Comparative Example to Example 16) Z-Asp 0
．． 67 g (2°5 mmot) and PMo, 45 g (
Dissolve 2.5 mmot) in an appropriate amount of water and hydroxylate) I
After adjusting the pH to 6.2 by adding a Jium aqueous solution, the total volume was adjusted to 181 m. Samoase (manufactured by Daiwa Kasei Co., Ltd.) 120Tn9 was added to this aqueous solution. After completely dissolving, 4
It was shaken at 0°C for 3 hours.

When 2-α-APM in the aqueous solution was quantified by HPI, C, it was found to be produced at a yield of 19.44 based on Phe.

Example 17 N-acetyl-L-alanine (hereinafter abbreviated as Aa-Ata) 0.4 g (3.0 mmot) and I,
-Leucine (hereinafter abbreviated as L@u* 0.08 g
(0.6 mmot) is dissolved in an appropriate amount of water and hydroxylated)
After adjusting the pH to 44.5 by adding IJum aqueous solution,
Overall 5. Q: I changed it to ml. 50 rv of Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) was added to this aqueous solution. After complete dissolution, add trioctylmethylammonium chloride (
A11quat336) 1,22 g (3,0 mmo
t) toluene solution 10ml! was added and shaken at 40°C for 49 hours.

Ac-Ala-Le in the reaction solution (aqueous layer and toluene layer)
Assuming that u was quantified by HPLC, 2 for Le+1
．． It was produced at a yield of 3% (aqueous layer 0.6%, toluene layer 1.7%).

Comparative example 8 (comparative example to Example 17) Aa-A, la
0.4 g (3,0 mmot) and Lsu 0.08
g (0.6 mmot) was dissolved in an appropriate amount of water, the pT was adjusted to 4.5 by adding an aqueous sodium hydroxide solution, and the total volume was made up to 5 tnl.Protease M (manufactured by Ohno Pharmaceutical Co., Ltd.) 50 μm was added to this aqueous solution. After complete dissolution, 4
Shake at 0°C for 49 hours.

When Ae-Ara-Lsu in the aqueous solution was quantified by HPLC, it was found to be produced at a yield of 0.71 relative to Lea.

Example 18 For-Asp2 obtained by reacting in the same manner as Comparative Example 3
．． 5 g, For-α-AP 0.009 g and L
50 tnl of a toluene solution containing 6.25 g of trioctylmethylammonium chloride was added to 25 rnl of the enzyme reaction solution containing 05 g of -PheO, the pH was adjusted to 6.0, and the mixture was shaken at 37°C for 15 minutes.

Separate the toluene layer, For-Asp, For-α
-AP and L-Ph. were quantified by HPI and C, and it was found that 76% of F'or-α-APFi was extracted in the toluene layer.

On the other hand, For-Asp and Phe were 9% and 7%, respectively.
Only % was extracted. By the way, F'or-α-
A.P.

The water-toluene partition coefficients of For-Asp and Phe were 1.35, 0.04, and 0.03, respectively.

Comparative Example 9 (Comparative Example to Example 18) An experiment similar to Example 18 was conducted except that trioctylmethylammonium chloride was not added.
was not extracted into the toluene layer at all, and its partition coefficient was 0.

Example 19 Ac-Asp obtained by reacting in the same manner as Comparative Example 1 2.
6g + Ae-α-AP0.015g and Phe O,
25ml of enzyme reaction solution containing 5g VC trioctylmethylammonium chloride 6.25g of toluene solution 50ml
After adding A and adjusting -■ to 4.4, the mixture was shaken at 37°C for 15 minutes.

Separate the toluene layer and prepare Ac-Asp*Ac-α-A
When P and (J L -Pbe were quantified by T (PLC), 63% of Ac-α-AP was extracted in the toluene layer. On the other hand, Ac-Asp and Phe were
Only 9 sections and 2 sections were extracted. By the way, AC-
α-AP.

The water-toluene partition coefficients of Ac-Asp and L-Phe were 1.03, 0.07, and 0.01, respectively.

Comparative Example 10 (Comparative Example to Example 19) An experiment similar to Example 19 was conducted except that trioctylmethylammonium chloride was not added.
was not extracted into the toluene layer at all, and its partition coefficient was O.

Claims (2)

[Claims] (1) N-protected amino acid or its derivative and amino acid or its derivative are mixed with water and a water-immiscible organic solvent in which a quaternary ammonium salt or quaternary phosphonium salt is dissolved.
The reaction is carried out using a proteolytic enzyme in the aqueous phase in a phase-based reaction medium, and the resulting N-protected peptide having at least one carboxyl group is transferred to the organic solvent phase as a lipophilic quaternary ammonium salt or quaternary phosphonium salt. 1. A method for enzymatic synthesis of N-protected peptides or derivatives thereof, which is characterized by translocation. (2) The N-protected amino acid or its derivative is reacted with the amino acid or its derivative in water using a protease, and the resulting N-
1. A method for enzymatically synthesizing an N-protected peptide or a derivative thereof, which comprises extracting and recovering the protected peptide with a water-immiscible organic solvent in which a quaternary ammonium salt or a quaternary phosphonium salt is dissolved.