JPH0143558B2 - - 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 peptide-splitting activity, the resulting peptide is degraded in parallel with the synthesis, presenting 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 gramicidin S, a decapeptide, and decapeptide (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. 10336/1983). However, in this method, the aminoacyl-tRNA synthetase used as the condensing agent is purified to a high degree of purity;
The procedure is complicated and it takes a long time to obtain the desired aminoacyl-tRNA synthetase.
There were cases in which tRNA synthetase activity was frequently lost during purification, which tended to result in a decrease in enzyme yield. In order to improve this problem, even if aminoacyl-tRNA synthetases were widely sought in the natural world, such as microorganisms, the yield of the obtained aminoacyl-tRNA synthetases tended to be low, and the above-mentioned problem could not be improved.
Furthermore, in order to improve the complexity of the operation, we considered using a crude extract obtained by crushing microbial cells with a homogenizer or Dynomill, but this method does not allow the use of other enzymes mixed during peptide synthesis. Perhaps due to these factors, side reactions were often observed, which tended to reduce the yield of the target peptide, and the target product was not sufficiently isolated and purified after the reaction.

Therefore, the present inventors conducted further intensive research to improve the above points, and surprisingly found that aminoacyl-tRNA synthetase obtained by treating the crude extract with a cation exchange resin having a phosphate group. The inventors have discovered that using a crude enzyme solution containing the above-mentioned solution solves all of the above problems and can synthesize peptides or peptide derivatives in high yields, and have completed the present invention.

That is, when synthesizing peptides or peptide derivatives from amino acids, the present invention involves treating a crude extract obtained by crushing biological cells with a cation exchange resin having a phosphate group to prepare a crude enzyme solution containing aminoacyl-tRNA synthetase. This is a method for synthesizing peptides or peptide derivatives, which is characterized by adding peptides or peptide derivatives to a reaction system.

The aminoacyl-tRNA synthetase used in the present invention belongs to enzyme classification 6.1.1 and has the following formula: amino acid + ATP + tRNA → aminoacyl-tRNA + AMP +
An enzyme that catalyzes the reaction of pyrophosphate, such as 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; 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 magateicus are preferred because of the stability of the enzyme. Aminoacyl-tRNA obtained from thermophilic bacteria such as
Synthetase is optimal.

These various aminoacyl-tRNA synthetases have specificity for various α-amino acids. For example, tyrosyl-tRNA synthetases have specificity for tyrosine, and tyrosyl-tRNA synthetases have specificity for leucine. is leucyl-tRNA synthetase, and those with specificity for valine include valyl-tRNA synthetase, other isoleucyl-tRNA synthetases,
Specific examples include phenylalanyl-tRNA synthetase, alanine-tRNA synthetase, glutamyl-tRNA synthetase, asparaginyl-tRNA synthetase, methionyl-tRNA synthetase, histidyl-tRNA synthetase, lysyl-tRNA synthetase, threonyl-tRNA synthetase, and seryl-tRNA synthetase. can give.

In the present invention, these aminoacyl-
To obtain a crude enzyme solution containing tRNA synthetase,
For example, it is necessary to crush the tissue or cells with a homogenizer, dyno mill, etc., and then treat the resulting crude extract with a cation exchange resin having a phosphate group.

For treatment with a cation exchange resin having a phosphate group, for example, PH3 to PH12, preferably
PH6 to PH9, optimally PH7 to PH8, concentration 1mM to 1M, preferably 20mM to PH8.
Add the above crude extract to a cation exchange resin containing phosphate groups equilibrated with 100mM buffer (batch method), or pack the resin into a column and pass the crude extract through the column (column method). ). The processing temperature at that time is aminoacyl-
It may be carried out at a temperature that maintains the activity of tRNA synthetase, but in general, a temperature between 0°C and 70°C is preferred;
In particular, a temperature between 0°C and 30°C is optimal. The buffer used at that time may be any buffer as long as it dissolves the aminoacyl-tRNA synthetase and provides the desired pH. Examples of such buffers include Tris-HCl buffer, Hepes buffer, triethanolamine buffer, imidazole buffer, and phosphate buffer. Furthermore, with the main purpose of preventing enzyme inactivation, treatment buffers include mercaptoethanol, sulfhydrylators such as dithiothreitol, protease inhibitors such as phenylmethylsulfonyl fluoride, and chelates such as sodium ethylenediaminetetraacetate. Agents may also be added. To process by the batch method, for example, the crude extract is added to a cation exchange resin having phosphate groups equilibrated with the above buffer solution, stirred for 5 minutes or more, preferably 30 minutes or more, and then left for a while. After that, the crude enzyme solution containing aminoacyl-tRNA synthetase is obtained by eluating the adsorbed enzyme with the same buffer solution, or with the above buffers with different pH values, with varying concentrations, or with buffers containing salt. Obtainable.
In addition, in order to perform treatment using the column method, for example, the desired enzyme may be adsorbed and desorbed within the column.
The principle is the same as the batch method. The treatment time is preferably as quick as possible, and the treatment is preferably carried out at a linear velocity of 1 cm·h ⁻¹ or more in the column. Further, in consideration of separation ability, it is more preferable that the linear velocity within the column is at most 60 cm·h ⁻¹ . Furthermore, as the salt used for elution in the batch method and column method, any salt can be used as long as it essentially completely dissociates; Salts that are the same as those of the liquid are preferred.

Aminoacyl-tRNA obtained by this treatment
A crude enzyme solution containing synthetase may be used as it is, or a solid solution obtained by further freeze-drying this may also be used.

Aminoacyl-tRNA obtained by this treatment
The crude enzyme solution containing synthetase may be further treated with DEAE resin and used for peptide synthesis.

As the cation exchange resin having a phosphoric acid group used in the present invention, any base material may be used as long as the exchange group is a phosphoric acid group. Among these, the use of cellulose phosphate resins based on cellulose (e.g., manufactured by Watmann, Bio-Rad, or Selva) allows for the rapid production of a crude enzyme solution containing the desired aminoacyl-tRNA synthetase with high yield. If the crude enzyme solution is used, peptides or peptide derivatives can be synthesized in high yield, which is preferable.

Next, a method for synthesizing a peptide or a peptide derivative using a crude enzyme solution containing aminoacyl-tRNA synthetase obtained by treatment with a cation exchange resin having a phosphate group will be specifically explained.

According to the present invention, an amino acid and an amino acid derivative derived from the amino acid are combined into an aminoacyl-tRNA.
Peptides or peptide derivatives can be synthesized by reacting in the presence of a crude enzyme solution containing synthetase. Furthermore, according to the present invention, a peptide or a peptide derivative is synthesized by mixing an amino acid and a crude enzyme solution containing aminoacyl-tRNA synthetase in advance to obtain a mixture, and then reacting the obtained mixture with an amino acid derivative. can do. Examples of amino acids preferably used for pre-mixing with the crude enzyme solution containing aminoacyl-tRNA synthetase include tyrosine, alanine, leucine,
isoleucine, phenylalanine, methionine,
Examples include α-amino acids such as lysine, serine, and valine, which may be either L-form or D-form. In addition, examples of amino acid derivatives preferably used in the above reaction include glycine, alanine, leucine,
α-amino acids such as isoleucine, phenylalanine, glutamic acid, glutamine, norleucine, cysteine, tyrosine, arginine, valine, lysine, histidine, aspartic acid, asparagine, methionine, tryptophan, threonine, β-alanine, β-aminoisobutyric acid, etc. Examples include esters, thioesters, amides, and hydroxamides of various amino acids such as β-amino acids, nitrogen-containing γ-amino acids such as creatine, γ-amino acids such as piperidic acid, and ε-amino acids such as ε-aminocaproic acid. is not limited to the above-mentioned exemplified compounds as long as it is an amino acid derivative in a free form. 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 mixture, e.g. PH5 to PH11
Preferably PH6 to PH10, optimally PH7 to PH10
This can be carried out by mixing amino acids and a crude enzyme solution containing aminoacyl-tRNA synthetase in the presence of adenosine triphosphate or deoxyadenosine triphosphate in a PH10 buffer. The mixing temperature at this time is generally preferably from 0°C to 70°C from the viewpoint of maintaining enzyme activity, and optimally from 0°C to 70°C.
Performed at 30°C. In addition, the buffer used at that time includes amino acids, adenosine triphosphate,
Deoxyadenosine triphosphate and aminoacyl-
Any solution may be used as long as it dissolves the crude enzyme solution containing tRNA synthetase and provides the desired pH. Examples include Tris-HCl buffer, Hepes buffer, triethanolamine buffer, malate buffer, and phosphate buffer. Furthermore, with the main purpose of making the reaction proceed smoothly and preventing enzyme deactivation, divalent cations such as magnesium and manganese, mercaptoethanol, etc.
sulfhydrylating agents such as dithiothreitol,
Pyrophosphatase may be added alone or in combination. Suitable concentrations of each additive include 0.01mM to 500mM of divalent cation, 0.001mM to 100mM of sulfhydrylating agent, and 0.001mM to 100mM of pyrophosphatase.
0.001 unit/ml to 100 units/ml, and the optimal concentration is 0.1 unit/ml for each divalent cation.
mM ~ 10mM, sulfhydrylation agent 0.01mM ~ 1
mM, pyrophosphatase 1 unit/ml to 10 units/ml. In addition, the amount of amino acid, crude enzyme solution containing aminoacyl-tRNA synthetase, and adenosine triphosphate or deoxyadenosine triphosphate used is not particularly limited, but in order to obtain a practical yield, it is necessary to use 1 part by weight of amino acid. Crude enzyme solution containing aminoacyl-tRNA synthetase 10 ³
~ ¹⁰⁶ parts by weight (the concentration of aminoacyl-tRNA synthetase is preferably 10 μM or more) The molar ratio of amino acids and adenosine triphosphate or deoxyadenosine triphosphate is 1:10.
It is preferable to carry out within the range of 1:100. When the reaction is carried out under the above conditions, the reaction proceeds smoothly,
It can be completed within a few seconds to 30 minutes.

Next, the mixture obtained as described above and an amino acid derivative are mixed and reacted to obtain the desired peptide or peptide derivative. (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 by performing gel chromatography such as G-25 (manufactured by Pharmacia) or G-75 (manufactured by Pharmacia),
It is also possible to remove adenosine triphosphate, adenosine monophosphate, pyrophosphate, etc. present after the reaction. In addition, the temperature of the peptidation reaction is preferably 0°C to 70°C, and from the viewpoint of preventing enzyme deactivation and obtaining an appropriate reaction rate, 10°C
It is preferable to carry out the reaction at a temperature between 20°C and 40°C, particularly between 20°C and 40°C. For pH, use various buffer solutions mentioned above,
5 to 11, preferably 6 to 10, optimally 7 to 9.

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 at low cost without using a protecting group, and can be produced in a higher yield, so that it is extremely useful industrially.

The present invention will be specifically explained below using examples.

Example 1, Comparative Example 1 Bacillus stearothermophilus cells 6Kg
of 100mM Tris-HCl buffer (PH
7.5), the cells were disrupted using Dynomill, and insoluble materials were removed by centrifugation to obtain a crude extract containing tyrosine-tRNA synthetase specific for tyrosine. The above solution was added to a column filled with phosphocellulose (manufactured by Watmann) equilibrated in advance with 50 mM phosphate buffer (PH7.0) containing 5 mM mercaptoethanol, 2 mM sodium ethylenediaminetetraacetate, and 0.1 mM phosphophenylsulfonyl fluoride. A solution of potassium chloride added to the above buffer was passed through the crude extract at a linear velocity of 60
When eluted at cm·h ⁻¹ , tyrosyl-tRNA synthetase was eluted. Collect and concentrate this division,
As a result of desalting, a crude enzyme solution containing tyrosine-specific tyrosyl-tRNA synthetase was obtained with a high yield of over 80%. All the above operations were performed at 4°C.

6 g of crude enzyme solution containing tyrosyl-tRNA synthetase (purity 8%) obtained by treatment with a cation exchange resin having a phosphate group, 0.4 g of magnesium chloride, and 0.1 g of adenosine triphosphate disodium salt.
g, 1 mg of L-tyrosine, 200 units of pyrophosphatase (manufactured by Boehringer Mannheim) and 0.01 mg of dithiothreitol were dissolved in 200 ml of 20 mM Hepes buffer pH 8.0 and mixed for 15 minutes at 4°C to obtain a mixture. Ta. 4 g of L-phenylalanine methyl ester was added to the resulting mixture, and the reaction system was adjusted 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, the precipitate was filtered off, and about 20 ml of the supernatant was collected 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 = 66.71 H = 6.37 N = 8.23.

Next, for comparison, a crude extract was obtained from 6 kg of Bacillus stearothermophilus in the same manner as above, and then anion exchange resin DEAE-cellulose column chromatography, hydroxyapatite column chromatography, DEAE -Tyrosyl-tRNA synthetase purified to a single form using Cephadex column chromatography, fractional precipitation with ammonium sulfate, hydroxyapatiteography, DEAE-Sephadex column chromatography, and Cephadex G-150 column chromatography. 0.1 mg of L-tyrosyl-L-phenylalanine methyl ester was obtained in the same manner as above.

Example 2, Comparative Example 2 Using 55 kg of Bacillus stearothermophilus cells, a crude extract obtained in the same manner as in Example 1 was mixed in advance with 5 mM mercaptoethanol, 2 mM sodium ethylenediaminetetraacetate, and 0.1 mM phosphophenylsulfonyl. 20mM containing fluoride
Add to Phosphate Selex (manufactured by Bio-Rad) equilibrated with phosphate buffer (PH7.5), stir for 30 minutes, leave for several minutes, remove the supernatant, and add sodium chloride to the above buffer. Tyrosyl-tRNA synthetase was eluted by elution with a diluted solution. All the above operations were performed at 30°C.

45 g of crude enzyme solution containing tyrosyl-tRNA synthetase (purity 11%) obtained by treatment with this cation exchange resin having a phosphate group, 150 g of magnesium chloride
mg, adenosine triphosphate disodium salt 300 mg,
9 mg of L-tyrosine, 200 units of pyrophosphatase (manufactured by Boehringer Mannheim) and 0.01 mg of dithiothreitol were dissolved in 200 ml of 25 mM phosphate buffer pH 8.5 and reacted at 4°C for 20 minutes.
The reaction mixture was applied to a G-75 (manufactured by Pharmacia) column, eluted with Hepes buffer, and 300 ml of void volume fractions were collected to isolate the reaction mixture. Add 1.0 g of D-valine methyl ester to the isolated mixture,
The reaction system was mixed well while maintaining the pH at 8.5, and the reaction was carried out for 30 minutes while maintaining the reaction temperature at 20°C.

Next, 1 part of acetone was added to the resulting reaction solution,
After filtering the precipitate, remove the supernatant using an evaporator to approximately 30ml.
The residue was concentrated, applied to a Bondapak _C18 column, and separated in the same manner as in Example 1 to obtain 14.6 mg of L-tyrosyl-D-valine methyl ester.

The elemental analysis (C ₁₅ H ₂₂ N ₂ O ₄ =294.36) was as follows: Calculated values (%): C=61.21 H=7.53 N=9.52 Measured values (%): C=61.41 H=7.50 N=9.39.

Next, for comparison, using the same amount of bacterial cells as above, a reaction was carried out with tyrosyl-tRNA synthetase obtained in the same manner as in Comparative Example 1 to obtain 3.6 mg of L-tyrosyl-D-valine methyl ester.

Example 3, Comparative Example 3 A crude extract obtained from baker's yeast in the same manner as in Example 1 was pretreated with 50mM phosphoric acid containing 10mM mercaptoethanol, 20mM sodium ethylenediaminetetraacetate, and 0.1mM phosphophenylsulfonyl fluoride. When passed through a column packed with cellulose phosphate (manufactured by Selva) equilibrated with a buffer solution (PH7.0) and eluted with a solution of potassium chloride added to the above buffer solution at a linear velocity of 10 cm h ^-1 , A crude enzyme solution containing methionyl-tRNA synthetase was eluted, and this was lyophilized to obtain a powdered crude enzyme preparation.

2 g of crude enzyme preparation containing methionyl-tRNA synthetase (purity 10%) obtained by treatment with this cation exchange resin having a phosphate group, magnesium chloride
20mg, adenosine triphosphate disodium salt 40mg,
1 mg of D-methionine, 10 units of pyrophosphatase (manufactured by Boehringer Mannheim) and 20μ of mercaptoethanol were mixed in 30ml of 50mM2,5-
After adding dimethylimidazole buffer solution PH 9.0 and reacting in the same manner as in Example 2 while maintaining the reaction system at PH 9.0, the reaction mixture was isolated in the same manner as in Example 2, and L-leucine ethyl 1 g of ester was added in solid form and reacted at 20°C for 5 hours. 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 1.8 mg of D-methionyl-L-leucine ethyl ester. .

The elemental analysis (C ₁₃ H ₂₆ N ₂ O ₃ S ₁ = 290.43) was as follows: Calculated value (%) C = 53.77 H = 9.02 N = 9.64 Measured value (%) C = 53.80 H = 8.90 N = 9.75 .

Next, for comparison, the crude extract obtained above was used as it was without being treated with a cation exchange resin having a phosphate group, and the reaction was carried out in the same manner as above to obtain D-methionyl-L-leucine ethyl ester. of
Obtained 0.2 mg.

Compared to Example 3, the yield tended to decrease, probably due to side reactions caused by other enzymes present in the crude extract.

Example 4, Comparative Example 4 A crude extract containing seryl-tRNA synthetase was obtained from rabbit liver in the same manner as in Example 1 using a homogenizer. The above crude extract was added to cellulose phosphate (manufactured by Watmann) which had been equilibrated in advance with 25mM imidazole buffer (PH7.5) containing 1mM dithiothreitol, 2mM sodium ethylenediaminetetraacetate and 0.1mM phosphophenylsulfonyl fluoride. The mixture was added, stirred for 1 hour, and left to stand, and then the supernatant was removed. When this is eluted with a solution of potassium chloride added to the above buffer,
Seryl-tRNA synthetase was eluted.

3 g of crude enzyme solution containing seryl-tRNA synthetase (purity 10%) obtained in this way, magnesium chloride
50 mg of deoxyadenosine triphosphate disodium salt, 1 mg of L-serine, 200 units of pyrophosphatase (manufactured by Boehringer Mannheim), and 0.01 mg of dithiothreitol. A reaction mixture was obtained using a 25 (manufactured by Pharmacia) column. Next, 4 g of β-alanylamide was added to this, and the reaction system was adjusted to pH 9.0.
The mixture was mixed at 50°C for 10 minutes. The obtained reaction solution was separated using a Bondapak _C18 column in the same manner as in Example 1, and L-seryl-β-
1.3 mg of alanilamide was obtained.

The elemental analysis (C ₆ H ₁₃ N ₃ O ₃ = 175.19) was as follows: Calculated values (%) C = 41.14 H = 7.48 N = 23.99 Measured values (%) C = 41.10 H = 7.43 N = 24.05.

Next, for comparison, the crude extract obtained above was used as it was without being treated with a cation exchange resin having a phosphoric acid group, and the reaction was carried out in the same manner as above to produce 0.2 L-seryl-β-alanylamide. I got mg.

Compared to Example 4, the yield tended to decrease.

Claims (1)

[Claims] 1. When synthesizing peptides or peptide derivatives from amino acids, a crude extract obtained by crushing biological cells is treated with a cation exchange resin having a phosphate group to obtain a crude enzyme solution containing aminoacyl-tRNA synthetase. A method for synthesizing a peptide or a peptide derivative, which comprises adding the obtained crude enzyme solution to a reaction system.