JPS6296097A - Production of peptide or peptide derivative - Google Patents

Abstract

PURPOSE:To produce the titled peptide useful as pharmaceuticals, etc., in high yield at a low cost without necessitating protective group, by reacting an alpha-amino acid with other amino acid, etc., in the presence of a specific enzyme, nucleotide-3-phosphate and a chelating agent. CONSTITUTION:A peptide (derivative) can be produced by mixing (A) >=0.1mM of an alpha-amino acid such as alanine, (B) 1-10<-9> equivalent of an aminoacyl- tRNA synthetase derived from thermophilic bacteria, etc., based on 1 equivalent of the amino acid A, (C) 10mM-10M of an amino acid (derivative) such as creatine, (D) 0.01mM-1M of a chelating agent such as ethylene diaminetetraacetic acid and, if necessary, (E) 0.01-500mM of a water-soluble organic catalyst such as methanol or a bivalent cation such as Mg and (F) <=100mM of Hepes buffer solution, etc. dissolving the mixture in an aqueous medium and reacting the components with each other at 7-9pH and 0-70 deg.C for several sec. - several days.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a novel method for producing peptides or peptide derivatives.

(Prior Art) In recent years, it has become increasingly known that peptides have various physiological activities, and their importance as pharmaceuticals for treatment and diagnosis as well as as taste substances is increasing. Along with this, development of peptide synthesis methods is also active. The main methods of peptide synthesis known to date are the chemical synthesis method and the enzymatic method, as summarized in Pharmacia, Review, No. 3, pp. 27-47 (1980). It can be roughly divided into Typical chemical synthesis methods include the azide method, mixed acid anhydride method, active ester method, and carbodiimide method in which amino acids are condensed sequentially and fragments. The synthetic method also has various problems such as racemization and side reactions are likely to occur, the reaction time is long, and the terminal amino group must be protected with a protecting group before the reaction. For the fragment condensation method.

It has a serious drawback that racemization is particularly likely to occur.

On the other hand, as a way to avoid racemization as much as possible.

An enzymatic method using protease has been proposed.

Even in this method, the reaction time was long, and the terminal amino group had to be protected with a protecting group, so that the complexity of the operation could not be improved.

Furthermore, in the enzymatic method using this protease, since the enzyme used originally has peptidolytic activity, the resulting peptide is degraded while being synthesized.

A serious drawback was that the desired peptide was often not obtained. In particular, it has been pointed out that when oligopeptide synthesis is applied, a serious drawback is that unintended peptides lacking some amino acids can be obtained [Journal of Biological Chemistry, 256
Volume, 1301 pages (1981)]. In addition to the protease method, methods for synthesizing peptides using enzymatic methods include methods using special enzymes that control only the synthesis of a single peptide having a specific amino acid sequence. Examples of this type of enzyme include glutathione synthase (Japanese Unexamined Patent Publication No. 122793/1983), which synthesizes a tripeptide with the sequence glutamic acid/cystine/glycine, and gramicidin S synthase, which synthesizes gramicidin S, a decapeptide. (Gendai Kagaku, December 1974 issue, p. 12) have been reported. However, these enzymes are special enzymes.

The peptides that can be synthesized by this enzyme are limited to only one type, and it is not possible to synthesize any desired peptide. For this reason.

At present, this method cannot be used as a general peptide synthesis method.

In view of the usefulness of peptides, the present inventors sought to solve the above-mentioned drawbacks, particularly the need for protective groups that cause racemization, side reactions, and complexity of two reactions, and at the same time impair economic efficiency. As a result of extensive research with the aim of providing a new and versatile peptide synthesis method, we have discovered that an enzyme that has the ability to bind amino acids to tRNA, a type of nucleic acid, has not previously had the ability to form peptide bonds at all. They surprisingly discovered that a previously unknown aminoacyl-t RNA synthetase has the ability to synthesize peptides, and discovered that all of the above objectives could be achieved by using this enzyme as a condensing agent, and filed a patent application earlier. (Refer to Japanese Patent Application Laid-Open No. 58-146539).

(Problems to be Solved by the Invention) In the method described in JP-A-58-146539, nucleoside triphosphate is added when producing a peptide or a peptide derivative.

To obtain peptides or peptide derivatives in good yield,
It was necessary to add it in a large excess of 10 to 100 times the molar ratio to the α-amino acid.

(Means for Solving the Problems) Therefore, the present inventors conducted further intensive research to improve the above points, and surprisingly found that when the reaction is carried out in the presence of a chelating agent, nucleoside The inventors have discovered that peptides or peptide derivatives can be synthesized in good yields even when a small amount of phosphoric acid is used relative to the α-amino acid, and the present invention has been completed.

That is, the present invention provides a method for producing a peptide or a peptide derivative by reacting an α-amino acid with an amino acid or an amino acid derivative derived from the amino acid in the presence of an aminoacyl-t RNA synthetase and a nucleoside triphosphate. This is a method for producing a peptide or a peptide derivative, characterized in that the step is carried out in the presence of a chelating agent.

The aminoacyl-t RNA synthetase used in the present invention belongs to enzyme classification 6.1.1 and has the primary formula amino acid+A
Tp+tRNA- is an enzyme that catalyzes the reaction of aminoacyl-tRNA+AMP+birophosphoric acid.9 For example, rabbit.

Those obtained from animal tissues such as horses, cows, rats, chickens, and snakes, those obtained from plant tissues such as rice, potatoes, and tomatoes, and those obtained from microorganisms such as molds, yeasts, mushrooms, bacteria, and actinobacteria, and algae. There are things that can be done. Among these, enzymes obtained from microorganisms are preferred because they are easy to obtain, and Bacillus stearothermophilus, Thermus thermophilus, and Thermus warabas are preferred because of the stability of the enzyme. Clostridium
Aminoacyl-tRNA synthetases obtained from thermostable bacteria such as Thermoaceticum and Thermus aquaticus are most suitable.

These various aminoacyl-tRNA synthetases are 1
Those with specificity for various α-amino acids are used. For example, one with specificity for tyrosine is tyrosyl-amino acid.
tRNA synthetase.

Leucine-specific tRNA synthetase is specific to valine, and valine-specific tRNA synthetase is valyl-tRNA synthetase, as well as isolosyl-tRNA synthetase, phenylalanyl-t RNA synthetase, and alanyl-t RNA. synthetase, glutaminyl-tRNA synthetase, asparaginyl-tRNA synthetase, methionyl-tRNA synthetase
tRNA synthetase, histidyl-tRNA synthetase.

lysyl-t RNA synthetase, threonyl-tRN
A synthetase, seryl-tRNA synthetase, asparatyl-t RNA synthetase, glutamyl-t
RNA synthetase, cysteinyl-tRNA synthetase, prolyl-tRNA synthetase, glycyl-t
RNA synthetase.

Specific examples include arginyl-t RNA synthetase and tryptophanyl-t RNA synthetase.

These various aminoacyl-t RNA synthetases can be obtained by disrupting the above-mentioned tissues or cells using a homogenizer, Dynomill, etc. 2 For example, Biochemistry Magazine, 13
Vol., p. 2307 (1974), chromatography such as DEAE-cellulose column chromatography, hydroxyapatite column chromatography, and fractional precipitation with ammonium sulfate.

It can be obtained by purification using a conventional enzyme purification method.

Examples of α-amino acids used in the present invention include tyrosine, alanine, leucine, isoleucine, phenylalanine, methionine, and lysine.

Serine, valine, asparagine, aspartic acid.

Glycine, glutamine, glutamic acid, cysteine, threonine, tryptophan, histidine.

Examples include amino acids such as proline and arginine.

Either L-form or D-form may be used. Examples of the amino acid or amino acid derivative to be reacted with the α-amino acid include glycine, alanine, and leucine.

Isoleucine, phenylalanine, glutamine.

cysteine, tyrosine, arginine, valine, lysine,
histidine, aspartate, methionine.

α-amino acids such as tryptophan and threonine.

β-amino acids such as β-alanine and β-aminoisobutyric acid, and nitrogen-containing γ-amino acids such as creatine.

Examples include various amino acids such as γ-amino acids such as piperidic acid, ε-amino acids such as ε-amic acid and pronic acid, and esters, thioesters, amides, and hydroxamides of these various amino acids.

The compounds are not limited to the above-mentioned exemplified compounds as long as they are amino acid derivatives in which the amino group is in a free form.

Examples of the ester include methyl and ethyl.

From simple hydrocarbon esters such as propyl, cyclohexyl, phenyl, and benzyl, the 3'-
Various esters can be used, including those obtained by esterifying the above amino acids with OH.

Further, as the amide, in addition to a free amide, it is also possible to use an oligopeptide or a polypeptide in which two different or the same amino acids are amide-bonded. These oligopeptides and polypeptides are further processed into esters, thioesters, and hydroxamides.

It is also possible to use an etherified one.

The nucleoside triphosphate used in the present invention is a compound that serves as an energy source for one reaction,
Specific examples of such include adenosine trisonic acid, 3'
- Deoxyadenosine triphosphate, β or γ-thio analogs of adenosine triphosphate.

Another example is adenosine triphosphate, which has a substituent on the adenine ring.

The chelating agent used in the present invention may be any agent as long as it can form a chelate with a divalent metal cation, including Co'', Zn'', and Cu''.

Those that can form a chelate with Ni2'' or Ca2+ are preferred. Examples of such compounds include 2.

Ethylenediaminetetraacetic acid, trans-1,2-diaminocyclohexanetetraacetic acid, ethylene glycol-bis-(
β-aminoethyl ether)-tetraacetic acid.

Ortho-phenanthroline, 8-hydroxyquinoline-
5-sulfonic acid is preferred.

According to the present invention, a peptide or a peptide derivative can be produced by mixing an α-amino acid, an amino acid or an amino acid derivative, an aminoacyl-tRNA synthetase, a nucleoside triphosphate, and a chelating agent and reacting the mixture in a liquid phase medium. . At this time, the chelating agent may be added in any order, and the chelating agent may be added to an aqueous medium containing an α-amino acid, an amino acid or an amino acid derivative, an aminoacyl-tRNA synthetase, and a nucleoside triphosphate; Or aminoacyl-
Add a chelating agent to the solution containing tRNA synthase in advance and mix well.

After equilibration, the α-amino acid, amino acid or amino acid derivative and nucleoside triphosphate may be added.

Furthermore, if a chelating agent is added at this time, the amount of nucleoside triphosphate used will be 1 to 10 equivalents relative to the α-amino acid, so there is no need to add a large excess. Furthermore, the concentration of α-amino acid is suitably 0.1 mM or more, preferably 1 mM or more. and 1/1 to 1/10 g of 1-aminoacyl-tRNA synthetase to α-amino acid.
The amino acid or amino acid derivative at a concentration of equivalents, preferably 1/10" to 1/10' equivalent, of the amino acid or amino acid derivative, usually in the range of 10 mM to IOM, and the chelating agent.

0.01mM to LM, preferably 1mM to 25
It is preferable to add in a range of 0mM.

As the medium used for the reaction at this time, a solvent containing water as a main component is selected since the nine-method is a reaction using an enzyme as a catalyst. Furthermore, a water-soluble organic catalyst may be added to the extent that enzyme activity can be maintained. Examples of water-soluble organic catalysts include methanol, ethanol, acetonitrile, dioxane, tetrahydrofuran, N,N-dimethylacetamide, N-methylpyrrolidone, and dimethylsulfoxide. Addition of such an organic catalyst is particularly effective when some of the raw materials are poorly soluble in water. At this time, divalent cations such as magnesium and manganese, and mercaptoethanol are added to the reaction system for the main purpose of making the 9 reactions proceed smoothly or preventing enzyme deactivation.

Sulfhydryl agents such as dithiothreitol and pyrophosphatase may be added alone or in combination. The preferred concentration of each additive is 0.01 mM divalent cation.
~500mM, S)L, 7hydryl agent 0, OO1
mM to 100 mM, pyrophosphatase 0.001
1 nit/m (1 to 1002 nift/ml).

The optimal concentration is 0.1mM of each divalent cation.
~10mM, sulfhydryl agent 0.01mM to 1mM,
Pyrophosphatase 1]nift/ml 1 to 10 nift/ml. Further, in order to maintain the activity of the enzyme, it is preferable to add a buffer to the solvent.

The concentration of the buffer solution is preferably 100 mM or less.

This buffer solution dissolves α-amino acids, amino acids or amino acid derivatives, aminoacyl-tRNA synthetase, nucleoside triphosphates, and chelating agents, maintains enzyme activity, obtains the desired pH, and prevents side reactions. You may use any material as long as it does not cause any irritation. Specific examples of such buffers include Hepes buffer, triethanolamine buffer, malate buffer, phosphate buffer, Bicine buffer, Esopus buffer, and the like.

In addition, the pH of the reaction solution when producing the peptide or peptide derivative is
Since NA synthetase 1 normally has an optimum pH for its reaction around 7 to 9,
Preferably, it is kept at 6 to 10. Also 1
The reaction temperature is not particularly limited as long as the catalytic activity of aminoacyl-tRNA synthetase can be maintained.

The temperature is usually preferably 0 to 70°C, most preferably 10 to 40°C.

Under the above conditions, the peptidation reaction is completed in a few seconds to several days, and the desired peptide or peptide derivative can be obtained.

(Example) The present invention will be specifically explained below using examples.

Reference Example 1 6 kg of bacterial cells of Bacillus stearothermophilus UK788 (Feikoken Kyori No. 5141) were added to 100
Suspended in mM Tris-Salt B2”D buffer (pH 7.5),
After disrupting the cells using Dynomill, insoluble materials were removed by centrifugation to obtain a crude extract containing tyrosine-specific tyrosyl-tRNA synthetase. 5mM in advance
50mM Tris buffer (pH 7) containing mercaptoethanol and 0.1mM phosphophenylsulfonyl fluoride.
．． 5) Pass the above crude extract through a column filled with Martresox Gel Blue A (manufactured by Amicon) equilibrated with 5).

A solution of potassium chloride added to the above buffer solution at a linear velocity of 60
When eluted with .h-1, tyrosyl-LRNA synthetase was eluted. Collect this division.

As a result of concentration 1 and desalting, a crude enzyme solution containing tyrosine-specific tyrosyl-t RNA synthetase was obtained with a yield of about 70%. All the above operations were performed at 4°C.

Reference example 2 Satucharomyces cerevisiae αS288C1 0 0
After disrupting 0 kg of cells with Dynomill, the resulting crude extract was subjected to ammonium sulfate fractionation, DEAE-cellulose chromatography, and cellulose phosphate (Whatman).
Chromatography, DEAE-Sephacel (manufactured by Pharmacia) Chromatography, Ultrogel ACA3
4 chromatography and CM-cellulose (Whatman) chromatography, 3.2 g of leucine-specific leucyl-tRNA synthetase was obtained.

Example 1, Comparative Examples 1 and 2 Tyrosyl-tRNA synthetase 0.0 obtained in Reference Example 1.
6 mg. 20mM Heves buffer (pH 8.0) 1m
A and 200rr+M disodium ethylenediaminetetraacetate (pH 8.0) 3mj+ were mixed and left at 30° C. for 1 hour. Thereafter, this mixed solution was mixed with 0.3 g of magnesium chloride and 10 μg of L-tyrosine.

20mM Hepes containing 100 μm of disodium adenosine triphosphate (equivalent to 3.3 equivalents to tyrosine), 5g of L-phenylalanine methyl ester, pyrophosphatase (manufactured by Boehringer Mannheim) 2001 Nift, and 0.01mg of dithiothreitol. Buffer solution (pH 8
．． 0) 46mj! were added to the solution, mixed well while maintaining the pH at 8.0, and allowed to react for 18 hours while maintaining the reaction temperature at 30°C.

Next. The resulting reaction solution was applied to a µponda pack Clll column (manufactured by Waters) and treated with acetonitrile for 10.
0 IN Hydrochloric acid aqueous solution 85/15 pH 4 was used as a developing solvent to separate L-tyrosyl-L-phenylalanine methyl ester hydrochloride. I got 1.

Its elemental analysis (C,9H23C I N20.=378
．． 89).

Calculated value (%) C=60.23 H=6.13
N=7.40 Measured value (%>c-6o.2'y H=6.2
ON=7.35.

For comparison (Comparative Example 1), the same procedure as in Example 1 was conducted except that disodium ethylenediaminetetraacetate was not added.

As a result, the yield of L-tyrosyl-d-phenylalanine methyl ester hydrochloride was 6 ml, which was 35% of the yield in Example 1.

Furthermore, for comparison (Comparative Example 2), disodium adenosine triphosphate was added from 100 ■ to 300 ■ (17
Corresponds to equivalent weight. ) was carried out in exactly the same manner as in Example 1, except that

As a result, the yield of L-tyrosine-phenylalanine methyl ester hydrochloride was 16.5 [1 g].

The yield was almost the same as that of Example 1.

Example 2.3 Instead of disodium ethylenediaminetetraacetate used in Example 1, 50 mM ethylene glycol-bis-(
β-aminoethyl ether)-tetraacetic acid disodium (p
H8.0) using 3 mJ (Example 2), and 20 m
Using 6 ml of M 1,10-ortho-phenanthroline (Example 3), the same procedure as in Example 1 was carried out.

As a result, the yield of L-tyrosyl-phenylalanine methyl ester hydrochloride was as shown below.

Yield (■) Example 2 17.8 Example 3 16.6 Example 4. Comparative Example 3.4 Leucyl-t RNA synthetase 6 obtained in Reference Example 2
, 40 μm of magnesium chloride, 20 μm of L-leucine, and 130 μm of disodium adenosine triphosphate (corresponding to 1.54 equivalents to leucine).

1.5 g of L-phenylalanine amide, pyrophosphatase (manufactured by Boehringer Mannheim) 201 Nift.

Mercaptoethanol 40μ! 50mM Ebbs buffer (pH 8,
5) 30 m I! 20 mg of L-leucine was added to the mixture, and the reaction was carried out at 30° C. for 5 hours while maintaining p) (of 3 reactions at 8.5).

Next, the obtained reaction product was applied to a μ Bondapak column, and the product was separated using an acetonitrile 150 mM potassium phosphate aqueous solution as a developing solvent.

The product was L-isoyl-L-phenylalanine amide, and the yield was 36 ml.

Its elemental analysis (C+ s H23N :l O□=27
7.41).

Calculated value (%) C=64.94 8=8.37N=15
．． 15 Measured value (%) C=64.96 H=8.47
N=15.09.

In addition, for comparison (comparative example 3), ethylene glycol-
The procedure was exactly as in Example 4, except that disodium bis-(β-aminoethyl ether)-tetraacetate was not added.

Furthermore, for comparison (Comparative Example 4), disodium adenosine triphosphate was added without adding disodium ethylene glycol-bis-(β-aminoethyl ether)-tetraacetate.
The same procedure as in Example 4 was carried out except that 30 nv was replaced with Ig (corresponding to 12 equivalents to leucine).

As a result, the yield of L-leucyl-phenylalanine amide was as shown below.

Yield (■) Comparative Example 3 19.3 Comparative Example 4 35.1 Example 5 Instead of disodium ethylene glycol-bis-(β-aminoethyl ether)-tetraacetate used in Example 4, 5-nitro- The same procedure as in Example 4 was carried out except that 1,10-phenanthroline 31 was added.

The yield of the condensed L L-leucyl-phenylalaninamide was 33.2 .

(Effects of the Invention) The peptide derivatives obtained by the present invention are useful for various hormones such as bradykinin, which has antihypertensive effects, softstatin, which has endocrine and exocrine suppressive effects, antibiotic peptides, and taste peptides. Useful as other biologically active substances.

According to the present invention, the above-mentioned useful peptides or peptide derivatives can be produced in good yields without using protective groups and even with a reduced amount of nucleoside triphosphates, resulting in low production costs. It is.

Claims (1)

[Claims] (1) When producing a peptide or peptide derivative by reacting an α-amino acid with an amino acid or an amino acid derivative derived from the amino acid in the presence of an aminoacyl-tRNA synthetase and a nucleoside triphosphate, the reaction is carried out using a chelating agent. A method for producing a peptide or a peptide derivative, characterized in that the method is carried out in the presence of a peptide or a peptide derivative.