JPH029800B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for synthesizing peptides or peptide derivatives.

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 peptide synthesis methods known to date include, for example, Pharmacia, Review, No. 3, pp. 27-47 (1980).
As summarized in , it can be roughly divided into two types: chemical synthesis methods and enzymatic methods. 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. The fragment condensation method has a serious drawback in that racemization is particularly likely to occur.

On the other hand, an enzymatic method using protease has been proposed as a method to avoid the occurrence of racemization as much as possible, but this method also requires a long reaction time and the need to protect the terminal amino group with a protecting group. The complexity of the process could not be improved.
Furthermore, in the enzymatic method using this protease,
Since the enzyme used inherently has peptidolytic activity, the resulting peptide is degraded in parallel with synthesis, resulting in a serious drawback in that the desired peptide is often not obtained. In particular, when applied to the synthesis of oligopeptides, a serious drawback has been pointed out: unintended peptides lacking some amino acids can be obtained (Journal of Biological Chemistry, Vol. 256, 1301). Page (1981). In addition to the protease method, methods for synthesizing peptides using enzymatic methods include methods that use special enzymes that control only the synthesis of a single peptide with a specific amino acid sequence. Examples of seed enzymes include glutamic acid/cysteine/
Glutathione synthetase that synthesizes a tripeptide having the sequence of glycine (Japanese Patent Application Laid-Open No. 122793/1983).
gramicidin S synthase, which synthesizes the decapeptide gramicidin S (Gendai Kagaku December 1974 issue)
(p. 12) have been reported. However, these enzymes are special enzymes, and only a limited number of peptides can be synthesized by these enzymes, and it is not possible to synthesize any desired peptide. Therefore, at present, this method cannot be used as a general peptide synthesis method.

In view of the usefulness of peptides, the present inventors have solved the above-mentioned drawbacks, particularly the need for protective groups that cause racemization, occurrence of side reactions, and complexity of the reaction, and at the same time impair economic efficiency. As a result of extensive research aimed at providing a new and versatile peptide synthesis method, we discovered that amino acids are a type of nucleic acid.
Surprisingly, we discovered that aminoacyl-tRNA synthetase, an enzyme that binds to tRNA and had no known ability to form peptide bonds, has the ability to synthesize peptides, and used this enzyme as a condensing agent. We discovered that all of the above objectives could be achieved, and filed a patent application (Japanese Patent Application No. 10336/1982). However, this method is necessary to obtain the desired product in good yield as a raw material for peptides or peptide derivatives. Expensive amino acid derivatives were added to the reaction system at high concentrations, which tended to increase costs. Further, after the reaction, it may be complicated to separate and purify the peptide or peptide derivative synthesized from the reaction solution, and improvements have been desired.

Therefore, the present inventors conducted further intensive research in order to improve the above points, and surprisingly, when acetonitrile was added to the reaction system, good yields were obtained even if the concentration of the raw amino acid derivative was lowered. The inventors discovered that it is possible to synthesize peptides or peptide derivatives, and completed the present invention.

That is, the present invention is a method for synthesizing a peptide or peptide derivative from an amino acid, which is characterized by the presence of acetonitrile in the reaction system and the use of aminoacyl-tRNA synthetase as a condensing agent.

The present invention is characterized by the presence of acetonitrile in the reaction system and the use of aminoacyl as a condensing agent in the enzymatic peptide synthesis method.
The purpose of this invention is to synthesize peptides or peptide derivatives in high yield without protecting amino groups by using tRNA synthetase.

The aminoacyl-tRNA synthetase used in the present invention belongs to enzyme classification 6.1.1 and is an enzyme that catalyzes the reaction of the following formula: amino acid + ATP + tRNA → aminoacyl -tRNA + AMP + pyrophosphate.
Those obtained from animal tissues such as horses, cows, rats, chickens, and snakes; those obtained from plant tissues such as rice, potatoes, and tomatoes; those obtained from microorganisms such as molds, yeasts, mushrooms, bacteria, actinomycetes, and algae. Things can be given. Among them, enzymes obtained from microorganisms are preferred because they are easy to obtain, and Bacillus stearothermophilus, Thermus, thermophilus, Thermus flavus, Clostridium thermoaceticum, and Thermus magna are preferred because of the stability of the enzyme. Aminoacyl obtained from thermophilic bacteria such as Atticus
tRNA synthetase is optimal.

These various aminoacyl-tRNA synthetases can be obtained by disrupting the above-mentioned tissues or cells using a homogenizer, dyno mill, etc., and then subjecting them to DEAE-cellulose column chromatography as described in, for example, Biochemistry, Vol. 13, p. 2307 (1974). It can be obtained by purification using conventional enzyme purification methods such as chromatography such as E, hydroxyapatite column chromatography, and fractional precipitation using ammonium sulfate. Aminoacyl-tRNA synthetases are used that have specificity for various α-amino acids,
For example, those specific for tyrosine include tyrosyl-tRNA synthetase, those specific for leucine include leucyl-tRNA synthetase, and those specific for valine include valyl-tRNA synthetase, Other isoleucyl-tRNA synthetase, phenylalanyl-tRNA synthetase, alanyl-tRNA
Synthetase, glutamyl-tRNA synthetase, asparaginyl-tRNA synthetase, methionyl-tRNA synthetase, histidyl-tRNA synthetase
tRNA synthetase, lysyl-tRNA synthetase, threonyl-tRNA synthetase, seryl-
A specific example is tRNA synthetase.

The acetonitrile used in the present invention provides good results for the stability of the enzyme, and the concentration of acetonitrile in the entire reaction solution by volume is 0.5 to 85%, preferably 5 to 60%, optimally 10%.
It ranges from 50% to 50%. Furthermore, acetonitrile only needs to be present at the time of the peptidation reaction, and it does not matter when it is added.

Hereinafter, the method of the present invention for synthesizing peptides or peptide derivatives from amino acids will be specifically explained.
According to the present invention, a peptide or a peptide derivative can be synthesized by reacting an amino acid and an amino acid derivative derived from the amino acid in the presence of aminoacyl-tRNA synthetase and acetonitrile. Furthermore, according to the present invention,
A peptide or peptide derivative can be synthesized by reacting an amino acid with an aminoacyl-tRNA synthetase in advance to obtain a reaction mixture, and then reacting the obtained reaction mixture with an amino acid derivative and acetonitrile. Examples of amino acids preferably used to react with this aminoacyl-tRNA synthetase include α-amino acids such as tyrosine, alanine, leucine, isoleucine, phenylalanine, methionine, lysine, serine, and valine; Any D-configuration may be used. Further, amino acid derivatives preferably used in the above reaction include, for example, glycine, alanine, leucine, isoleucine, phenylalanine, glutamic acid,
Glutamine, illleucine, cysteine, tyrosine, arginine, valine, lysine, histidine,
α-amino acids such as aspartic acid, asparagine, methionine, tryptophan, and threonine;
β-amino acids such as β-alanine and β-aminoisobutyric acid, nitrogen-containing γ-amino acids such as creatine,
Examples include esters, thioesters, amides, and hydroxamides of various amino acids such as γ-amino acids such as piperidic acid, and ε-amino acids such as ε-aminocaproic acid. It is not limited to the exemplified compounds. As the ester, various esters can be used, from simple hydrocarbon esters such as methyl, ethyl, propyl, cyclohexyl, phenyl, and benzyl to those in which the above amino acids are esterified with the 3'-OH of tRNA. Can be done. Furthermore, as the amide, in addition to free amide, for example, oligopeptides or polypeptides in which different or the same type of amino acids are amide-bonded can also be used. It is also possible to use esters, thioesters, hydroxamides, and etherified oligopeptides and polypeptides. Furthermore, the above amino acid derivatives may be used in the form of an aqueous solution or in a solid state.

Then, to obtain a reaction mixture, e.g.
This may be carried out by mixing the amino acid and aminoacyl-tRNA synthetase in the presence of adenosine triphosphate or deoxyadenosine triphosphate in a buffer solution of PH11, preferably PH6 to PH10, optimally PH7 to PH10. The reaction temperature at that time is generally 0°C from the viewpoint of maintaining enzyme activity.
to 70°C, optimally carried out at 0°C to 30°C. In addition, any buffer can be used as long as the amino acid, adenosine triphosphate, deoxyadenosine triphosphate, and aminoacyl-tRNA synthetase can be dissolved and the desired pH can be obtained. Good too. for example,
Examples include Tris-HCl buffer, Hepes buffer, triethanolamine buffer, malate buffer, and phosphate buffer. In addition, divalent cations such as magnesium and manganese, sulfhydrylating agents such as mercaptoethanol and dithiothreitol, and pyrophosphatase were added to the reaction system to facilitate the reaction and prevent enzyme deactivation. Alternatively, they may be mixed and added. The preferred concentration of each additive is 0.01mM to 0.01mM for divalent cations.
500mM, sulfhydrylating agent 0.001mM to 100m
M, pyrophosphatase 0.001 unit/ml ~ 100
The optimal concentrations are 0.1 to 10 mM for divalent cation, 0.01 to 1 mM for sulfhydrylating agent, and 1 to 10 units/ml for pyrophosphatase. The amounts of amino acids, aminoacyl-tRNA synthetase, and adenosine triphosphate or deoxyadenosine triphosphate used are not particularly limited, but in order to obtain a practical yield, the molar ratio of amino acids and aminoacyl-tRNA synthetase should be 1:1. 1-1:
10. The molar ratio of amino acid and adenosine triphosphate or deoxyadenosine triphosphate is 1:10 to 1:
It is preferable to do this within a range of 100. When the reaction is carried out under the above conditions, the reaction proceeds smoothly and is completed within a few seconds to 30 minutes.

Next, the desired peptide or peptide derivative can be obtained by mixing and reacting the reaction mixture obtained as described above with an amino acid derivative and acetonitrile. (This step is hereinafter referred to as peptideization.) The reaction mixture used at this time can be used as it is for the peptidation reaction, but G-25 (manufactured by Pharmacia) G-75
By performing gel chromatography such as (manufactured by Pharmacia), adenosine triphosphate, adenosine monophosphate, pyrophosphate, etc. mixed in after the reaction can be removed and used. In addition, the temperature of the peptidation reaction ranges from 0°C to 70°C.
℃ is preferable, and from the viewpoint of preventing enzyme deactivation and obtaining an appropriate reaction rate, the temperature is 10℃ to 50℃, especially 20℃.
Preferably, the temperature is between 40°C and 40°C. The pH may be 5 to 11, preferably 6 to 10, optimally 7 to 9, using various buffer solutions as described above.

The mixing ratio of the reaction mixture and the amino acid derivative may be, for example, in the range of 1:0.1 to 1:100 in terms of volume. Further, the concentration of the amino acid derivative used at this time is in the range of 10mM to 10M, but it can also be used at a lower concentration.

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

The peptide derivatives obtained by the present invention have various hormones such as bradykinin, which has a hypotensive effect, somatostatin, which has an endocrine and exocrine suppressive effect, and other biologically active substances, such as antibiotic peptides and taste peptides. Useful as a substance.

According to the present invention, the above-mentioned useful peptide or peptide derivative can be produced without using a protecting group and using raw materials at a low concentration, so that the production cost is also low.

The present invention will be specifically explained below using examples.

Example 1, Comparative Examples 1 and 2 0.5 g of tyrosine-specific tyrosyl-tRNA synthetase purified from Bacillus stearothermophilus according to the method described in Biochemistry, Vol. 13, p. 2307 (1974), Magnesium chloride 0.4g, adenosine triphosphate disodium salt 0.1
1 mg of L-tyrosine, 200 units of pyrophosphatase (manufactured by Boehringer Mannheim), and 0.01 mg of dithiothreitol were dissolved in 140 ml of 20 mM Hepes buffer pH 8.0 and reacted at 4°C for 15 minutes to form a reaction mixture. I got it. The resulting reaction mixture was
Add 0.4 g of phenylalanine methyl ester and 60 ml (30 volume%) of acetonitrile, and adjust the reaction system to pH.
Mix well while maintaining the temperature at 8.0, and reduce the reaction temperature to 30.
The mixture was kept at ℃ and allowed to react for one day.

Next, 200 ml of acetone was added to the resulting reaction solution, and after filtering out the precipitate, about 20 ml of the supersoak was added to the reaction solution using an evaporator.
Concentrated _to Obtained 0.4 mg.

The elemental analysis (C ₁₉ H ₂₂ N ₂ O ₄ = 342.39) was as follows: Calculated values (%) C = 66.65 H = 6.48 N = 8.18 Measured values (%) C = 66.48 H = 6.52 N = 8.52.

For comparison (Comparative Example 1), the same procedure as in Example 1 was carried out except that 200 ml of 20 mM Hepes buffer and 4 g of L-phenylalanine methyl ester were used, and acetonitrile was not present.

As a result, the yield of L-tyrosyl-L-phenylalanine methyl ester was 0.4 mg, which was the same as the yield in Example 1. While the amount of L-phenylalanine methyl ester used in Comparative Example 1 was 4 g, in Example 1, 0.4 g, which was 1/10 of that amount, was enough.

Furthermore, for comparison (Comparative Example 2), the same procedure as in Example 1 was carried out except that 200 ml of 20 mM Hepes buffer was used and glycerin was not present.

As a result, the yield of L-tyrosyl-L-phenylalanine methyl ester was 0.1 mg, which was 1/4 of the yield in Example 1.

Example 2, Comparative Examples 3 and 4 Tyrosyl-tRNA synthetase used in Example 1 5g, magnesium chloride 50mg, adenosine triphosphate disodium salt 300mg, L-tyrosine 9mg,
Pyrophosphatase (Boehringer Mannheim) 200 units and dithiothreitol 0.01mg
Dissolve in 20ml of 10mM Hepes buffer PH8.5,
After reacting at ℃ for 20 minutes, the reaction mixture was heated to G-75.
The reaction mixture was applied to a column (manufactured by Pharmacia) and eluted with the same Hepes buffer as above, and 30 ml of void volume fractions were collected to isolate the reaction mixture. D- to the isolated reaction mixture
0.5 g of leucine ethyl ester and 5 ml (14% by volume) of acetonitrile were added, mixed well while maintaining the reaction system at pH 8.5, and reacted for 30 minutes while maintaining the reaction temperature at 20°C.

Next, apply the resulting reaction solution as it is to Bonda Pack.
C ₁₈ column and separated in the same manner as in Example 1.
L-tyrosyl-D-leucine ethyl ester 15mg
I got it.

Its elemental analysis (C ₁₇ H ₂₃ N ₂ O ₄ = 322.39) was as follows: Calculated values (%) C = 63.33 H = 8.13 N = 8.69 Measured values (%) C = 63.30 H = 8.08 N = 8.75.

Next, for comparison (Comparative Example 3), the same procedure as in Example 2 was carried out except that 4 g of D-leucine ethyl ester was used and acetonitrile was not present.

As a result, the yield of L-tyrosyl-D-leucine ethyl ester was 16 mg.

Further, for comparison (Comparative Example 4), the same procedure as in Example 2 was carried out except that acetonitrile was not allowed to coexist.

As a result, the yield of L-tyrosyl-D-leucine ethyl ester was 5 mg.

Examples 3 and 4, Comparative Example 5 Tyrosyl-tRNA synthetase 20 prepared from baker's yeast according to the method described in Journal of Biochemistry, Vol. 63, p. 434 (1968)
mg, magnesium chloride 60 mg, adenosine triphosphate disodium salt 5 mg, D-tyrosine 0.1 mg, pyrophosphatase (Boehringer Mannheim)
10 units and 20μ of mercaptoethanol
ml 30mM 2,5-dimethylimidazole buffer
In addition to PH9, after reacting in the same manner as in Example 2,
The reaction mixture was isolated as in Example 2 and added to L
-0.1 g of leucine ethyl ester and 10 ml (25% by volume) of acetonitrile were added, and the reaction system was reacted at 20° C. for 5 hours while maintaining the pH of the reaction system at 9.0. 20 ml of acetone was added to the resulting reaction product, the resulting precipitate was filtered, concentrated to about 10 ml using an evaporator, and separated in the same manner as in Example 1 to obtain D-tyrosyl-L-leucine ethyl ester (Example 3).

Next, 10 ml of dimethyl sulfoxide was added in place of 10 ml of acetonitrile, and the same procedure as above was carried out (Example 4).

Furthermore, for comparison (Comparative Example 5), the same procedure as in Example 3 was carried out except that 10 ml of acetonitrile was not present.

As a result, the yield of D-tyrosyl-L-leucine ethyl ester was as shown below.

Yield (mg) Example 3 0.16 Example 4 0.15 Comparative example 5 0.08

Claims (1)

[Claims] 1. A method for synthesizing a peptide or peptide derivative from an amino acid, the method comprising making acetonitrile exist in the reaction system and using aminoacyl-tRNA synthetase as a condensing agent.