JPS6274296A - Enzymic linking of n-acetyl-l-aspartic acid to l-phenylalanine alkyl ester - Google Patents

Abstract

PURPOSE:To efficiently carry out the titled linking without using an expensive protecting group or formic acid, by hydrolyzing N-acetyl-L-aspartyl-L- phenylalanine and a mino- and diesters thereof in an acid solution in a high concentration. CONSTITUTION:N-Acetyl-L-apartic acid and an L-phenylalanine alkyl ester are reacted in the presence of an enzyme, e.g. thermolysin, capable of serving as a medium for forming peptide bonds between the L-aspartic acid and the L-phenylalanine. The reaction mixture is acidified to liberate the N-acetyl-alpha-L- aspartyl-L-phenylalanine alkyl ester. The acetyl group is then removed therefrom with an aqueous solution of a strong acid, e.g. hydrochloric acid, in a high concentration or an aqueous solution of methanol.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention (d) involves reacting N-acetyl-L-aspartic acid and L-phenylalanine alkyl ester using protease in an aqueous solvent to obtain α-L-asnoξ The present invention relates to a method for producing rutile-monophenylalanine alkyl ester.

[Conventional technology]

α-L-7S glutyl-L-phenylalanine alkyl ester (especially methyl ester) is known as an excellent sweetener, and one of them? The method of production is also known between species.

For example, U.S. Pat. No. 3.786.039, or U.S. Pat.
933.721 describes a chemical synthesis method using N-protected mass and lagic acid anhydride, but in these methods unnecessary β-L-as celtyl-
It is not necessarily satisfactory for producing phenylalanine derivatives. On the other hand, in order to overcome the above-mentioned drawbacks, a method of generating Kf-1 bond using an enzyme is also being considered, for example, US Pat. No. 4,086,13
6 and JP-A-60-16-1.495, but the former method uses an expensive pennoluonyl group as a protecting group, and the latter method uses highly corrosive formic acid. In addition, from the viewpoint of yield, it was not necessarily necessary to add 6 (4: L: j).

[While the invention is trying to solve the problem: the origin]

The present invention addresses the problems encountered in the prior art*:' when enzymatically producing N-alpha-L-7/F-rutyl-L-phenylalanine alkyl ester using the above-mentioned N-sugaric acid, namely: The protecting group used is expensive, or 1/-1,
The purpose of the present invention is to solve the problem of strong corrosivity and to efficiently combine N-asnocragic acid and L-phenylalanine alkyl ester with an enzyme.

[Means for solving problems]

In order to solve the above-mentioned problems, the inventors worked diligently and surprisingly found that N-acetyl-L-mass 2-lachinic acid was efficiently combined with L-phenylalanine alkyl ester by an enzyme. On the other hand, the acetyl group is extremely inexpensive as a protecting group for the amine group in the synthesis of pezotide (although it is known that a large amount of noketopiveranone is produced as a by-product during removal (Protein Chemistry Vol. 1, Shiro Akahori). ), an efficient method is considered λ, is f, a −
Even in the process for producing L-mass and rutile-L-phenylalanine methyl ester, there is a description of it as a protecting group in the text of U.S. Patent No. 3.933.721, but there are no examples.
It has been considered that industrial implementation is practically impossible,
However, 1. What should I do in a highly concentrated acid solution? The present inventors have discovered that only the N-acetyl group can be selectively removed without cleaving the K peptide bond by total hydrolysis of rutile-L-7x nylalanine and its mono- and noesters, leading to the completion of the present invention. .

N-acetyl-L-asnoyaragic acid and L-phenyl-tynin alkyl ester used as raw materials in the method of the present invention can be easily synthesized (tζ) by a known method.

The enzyme used in the method of the present invention is not particularly limited as long as it is a proteolytic enzyme, and examples include thermolysin, trypsin, and proteinase, among which thermolysin is particularly preferably used. The pH of the reaction solution when carrying out is 5 to 85 for thermolysin, 7 to 8 for trypsin, and 6 to 7 for .
Enzyme reactions are usually carried out in aqueous solutions;
It goes without saying that organic solvents that do not adversely affect the reaction may coexist. The enzyme reaction of the present invention is carried out at a temperature of 10 to
90°C, preferred from the viewpoint of maintaining enzyme activity)
Carry out at 0-50°C. The reaction is usually completed in about 30 minutes to a few hours, but the reaction time is not limited to this. In the method of the present invention, there is no particular restriction on the concentration of both starting proboscis used, but 4, the N-acetyl-α-L-aspartyl-L-phenylalanine alkyl ester produced in the present enzyme reaction > i of the unreacted Since it forms an adduct with phenylalanine alkyl ester and precipitates, it is desirable that the ratio is higher. The ratio of the two starting materials used is also not particularly large, but in order to improve the yield of the reaction:
-The larger the ratio to phenylalanine alkyl ester, the better.

In the method of the present invention, the N-acetyl α-
Since L-7sno)0rutyl-L-phenylalanine alkyl ester is obtained as a 1:1 adduct with L-phenylalanine alkyl ester, free N-acetyl-α-L-asnosolutyl can be obtained by neutralization with acid. APM can be easily obtained by removing the acetyl group after forming L-phenylalanine alkyl ester.

7 In order to completely remove the cetyl group, it is better to contact the N-acetyl compound with a highly concentrated aqueous solution of a strong acid or an aqueous methanol solution.The concentration of the acid used in the method of the present invention is preferably 1.5 N or more, preferably 1. Or 15-6. ON is selected. Examples of strong acids include hydrochloric acid, sulfuric acid, and ρ-toluenesulfonic acid, among which hydrochloric acid is preferably used. In the case of N-acetyl-a-L-asnonrutile-L-phenylalanine methyl ester, a methanol aqueous solution is more preferable than a strong acid aqueous solution. or,
The ratio of methanol and water is not particularly limited and can be selected arbitrarily.

Examples will be described in detail below, but it goes without saying that the embodiments of the present invention are not limited thereto.

Example 1 N-acetyl-thi-ame/4'lagic acid 8.8.9 (5
0 mmole) was suspended in a small amount of water, and the pH was adjusted to 6 with an aqueous sodium hydroxide solution. L-phenylalanine methyl ester hydrochloride 10.67! (50mmole)
After adding I)l′! Wash all 6.5 times and add water to make the liquid volume 5Qml. After adding thermolysin 0.4.9 and reacting at 25° C. for 24 hours, the resulting slurry was separated. The white solid obtained is N-acetyl-α-L
It was confirmed that they were l=1 adducts of -as/glutyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester. When the total amount of N-acetyl-α-L-asnoculty-L-phenylalanine methyl ester in this solid was quantified by HPLC, 3,8I
It contained N-acetyl-a-L-7subarthyl-phenylalanine methyl ester. This indicates a yield of 35 winters.

Example 12 N-acetyl-L-as gragine e8.8g (50m
mole) was suspended in a small amount of water, and the pH was adjusted to 5.5K with an aqueous sodium hydroxide solution. After adding 2.19 (10 mmole) of L-phenylalanine methyl ester hydrochloride, the pH was adjusted to 58, and water was added to make a liquid layer of 25 m
I set it to 1. Thermolysin 0.5flk added L, 35゛C
After reacting for 2.5 hours, the resulting slurry was separated, and the resulting solid was washed with water and then dried. In this way, N-acetyl-α-L-asuno? l:1 of rutile-monophenylalanine methyl ester and L-phenylalanine methyl ester @ 2.3 g of main component (4,
6rnmole) was obtained.

This shows that the yield is 92%. ◎Example 3 N-acetyl-shi-asuno? lagic acid 8.8g (50m
mole)i was suspended in a small amount of water and adjusted to pH 5.5 with an aqueous sodium hydroxide solution. L-phenylalanine methyl ester hydrochloride 2], 2.9 (100mrn01e
) after adding 1. H was set to 6.6, and water was further processed to make the liquid volume 50ml. Thermolysin 1. After adding OI and reacting for 25 hours at 35°C, the resulting slurry was separated, and the solid was washed with water and then dried. In this way, a 1:1 addition product of N-acetyl-α-L-asno4rutile-L-phenylalanine methyl ester and L-phenylalanine methyl ester 11.51
(22,5rnmole) was obtained. This is a yield of 4
It shows that it is 5.

Example 4 44 g (250
mmole ) was suspended in a small amount of water and adjusted to p) 15.5 with an aqueous sodium hydroxide solution. L-phenylalanine methyl ester hydrochloride 10.5.9 (50 mmole
), the pH was adjusted to 58, and water was further added to make the liquid volume i25 Qml. After adding Thermolysin 2.5I and reacting at 35°C for 2.5 hours, the resulting slurry was separated, and the solid was washed with water and then dried. In this way, 11.5 g of a 1:1 addition product of N-acetyl-α-L-7-phenylalanine methyl ester and L-phenylalanine methyl ester (
22.5 mmole) was obtained. This corresponds to a yield of 92%.

This solid was dispersed in water, the pH was adjusted to 1.5, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure and N-
Acetyl-α-L-asA ruty-L-phenylalanine methyl ester 10.9t-1! Ta.

Add this to a mixed solvent of 5 ml of water and 3 ml of methanol, add 2 ml of concentrated hydrochloric acid, and heat at 60°C for 20 minutes. Then, add 5 ml of hydrochloric acid and heat at 25°C for 4 days.
Made it react. The precipitated α-L-as parent rutile-1-phenylalanine methyl ester hydrochloride was separated, and the α-L-asnoyalti-L-phenylalanine methyl ester in the crystals was quantified by HPLC. g (17,3 mmole) of a-L-as/J'rutile-L-phenylalanine methyl ester.

Example 5 N-acetyl-thias/gragic acid 8.8 F (5
0 mmole) was suspended in a small amount of water and hydroxylated.The pH was adjusted to 5.5 with an aqueous IJ solution. L-phenylalanine isopropyl ester hydrochloride 2.4 g (10 mrn
After adding ole), the PFL was brought to 58, and water was added to bring the liquid 1 to 50 ml. Thermolysin 0.5j
After adding j and reacting at 35°C for 25 hours, the resulting slurry was separated, and the resulting solid was washed with water and then dried. Thus, N-acetyl-α-L-77,
Nozorutyl-L-phenylalanine inglobil ester and L-phenylalanine inoglobil ester 1:
2.71 (4.7 mmole) of 1 addition product were obtained. This shows that the yield is 93%.

Comparative Example 1 (Compared to Example 3) N-formyl-L-as/P laginic acid 8.1. ! i'(
50 mmole) was suspended in a small amount of water and adjusted to pi-15.5 with an aqueous sodium hydroxide solution. L-phenylalanine methyl ester salt Shunshio 21.2/(100mm
After adding ole ), the pH was brought to 6.5 and water was added to bring the volume to 50 rnl. Thermolysin 1.
After adding OI and reacting at 35°C for 2.5 hours, the resulting slurry was separated, the solid was washed with water, and then dried. In this way, N-formyl-α-L-asnoyalti-L-phenylalanine methyl ester and L-
1:1 adduct of phenylalanine methyl ester9.
5E (19 mmole) was obtained. This indicates a yield of 38 mm.

Claims (7)

[Claims] (1) In the method for producing N-acetyl-α-L-aspartyl-L-phenylalanine alkyl ester, L-
In the presence of an enzyme known to mediate peptide bond formation between aspartic acid and L-phenylalanine, N
A process characterized in that acetyl-L-aspartic acid and L-phenylalanine alkyl ester are reacted and the mixture is then acidified to obtain a dipeptide product. (2) The method according to claim 1, wherein the L-phenylalanine ester is a lower alkyl ester. (3) The method according to claim 1, wherein the L-phenylalanine ester is a methyl ester. (4) The method according to claim 1, wherein the enzyme is thermolysin. (5) In the method for producing α-L-aspartyl-L-phenylalanine methyl ester, N
- α-L-aspartyl-L-phenylalanine, characterized in that acetyl-L-aspartic acid and L-phenylalanine methyl ester are reacted, and then the N-acetyl dipeptide ester is hydrolyzed to remove the acetyl group. Method for producing methyl ester. (6) The method according to claim 5, wherein the enzyme is thermolysin. (7) The method according to claim 6, wherein the condition for removing the acetyl group is an aqueous solution of a strong acid of 1.5N or more or an aqueous methanol solution.