JPH0453515B2 - - 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 2010), it can be broadly divided into 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 it is necessary to protect the terminal amino group with a protecting group before the reaction. For the fragment condensation method,
In particular, it has a serious drawback that racemization easily occurs.

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 acid 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 the synthesis, resulting in a serious drawback in that the desired peptide is often not obtained. It was hot. 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
Journal of Biological Chemistry, Volume 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. Species of enzymes include, for example, glutathione synthase (Japanese Unexamined Patent Publication No. 122793/1983) which synthesizes a tripeptide with the sequence glutamic acid/cysteine/glycine, and gramicidin S synthase (which synthesizes gramicidin S, which is a decapeptide). modern chemistry 1974
(December 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 sought to solve the above-mentioned drawbacks, particularly the need for protective groups that cause racemization, occurrence of side reactions, and complexity of 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. Surprisingly, they discovered that a previously unknown aminoacyl-tRNA 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. 146539/1983). However, in order to obtain the desired product in good yield, this method requires
Expensive aminoacyl-tRNA synthetase is added to the reaction system at a high concentration, which tends to increase costs.

In addition, unexpected side reactions often occur during reaction operations, and this tendency was particularly observed when tryptophan, methionine, cysteine, etc. were included as constituents of the peptide or peptide derivative.
Furthermore, there was a tendency for aminoacyl-tRNA synthetase activity to be lost during the reaction procedure.

Therefore, the present inventors conducted further intensive research to improve the above points, and found that, surprisingly, when the reaction was carried out under an inert gas atmosphere, side reactions could be suppressed and expensive aminoacyl-tRNA synthetase could be used. The present invention was completed based on the discovery that peptides or peptide derivatives can be synthesized in high yields even when deactivation is prevented and the concentration of raw materials, especially enzymes, is lowered.

That is, the present invention combines amino acids and amino acid derivatives derived from the amino acids into aminoacyl-
This is a method for synthesizing a peptide or peptide derivative, which is characterized in that the reaction is carried out under an inert gas atmosphere when the peptide or peptide derivative is synthesized by reaction in the presence of 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, and actinobacteria; and algae. Things can be given. Among these, enzymes obtained from microorganisms are preferred because they are easy to obtain, and in addition, enzymes obtained from microorganisms such as Bacillus stearothermophilus, Thermus thermophilus, Thermus flavus, Clostridium thermoaceticum, Thermus Aminoacyl obtained from heat-resistant bacteria such as Macateicus
tRNA synthetase is optimal.

Among these various aminoacyl-tRNA synthetases, tyrosyl-tRNA synthetase has specificity for various α-amino acids, leucyl-tRNA synthetase has specificity for leucine, and further has specificity for valine. Examples of valyl-tRNA synthetase, other isorosyl-tRNA synthetase, phenylalanyl-tRNA synthetase, phenylalanyl-tRNA synthetase, and alanyl-tRNA synthetase.
tRNA synthetase, glutamyl-tRNA synthetase, asparaginyl-tRNA synthetase,
Methionyl-tRNA synthetase, histidyl-
tRNA synthetase, lysyl-tRNA synthetase, threonyl-tRNA synthetase, seryl-
tRNA synthetase, asparatyl-tRNA synthetase, glutamyl-tRNA synthetase, cysteinyl-tRNA synthetase, prolyl-
Specific examples include tRNA synthetase, glycyl-tRNA synthetase, arginyl-tRNA synthetase, and tryptophanyl-tRNA synthetase.

Examples of amino acids preferably used in the present invention include tyrosine, alanine, leucine, isoleucine, phenylalanine, methionine, lysine, serine, valine, asparagine, aspartic acid, glycine, glutamine, glutamic acid, cysteine, threonine, tryptophan, histidine, Examples include α-amino acids such as proline and arginine, which may be in either the L-form or the D-form. Preferably used amino acid derivatives include, for example, glycine, alanine, leucine, isoleucine, phenylalanine, glutamine, isoleucine, cysteine, tyrosine, arginine,
valine, lysine, histidine, aspartic acid,
α-amino acids such as methionine, tryptophan, and threonine; β-amino acids such as β-alanine and β-aminoisobutyric acid; nitrogen-containing γ-amino acids such as creatine; γ-amino acids such as piperidic acid; and ε-amino acids such as ε-aminocaproic acid. Examples include esters, thioesters, amides, hydroxamides, etc. of various amino acids such as amino acids, but as long as they are amino acid derivatives in which the amino group is in a free form,
It is not limited to the above-mentioned exemplified compounds. Examples of esters include simple hydrocarbon esters such as methyl, ethyl, propyl, cyclohexyl, phenyl, and benzyl;
Various esters can be used, including those obtained by esterifying the above amino acids with 3'-OH. 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.

The inert gas used in the present invention may be any non-oxidizing gas. Preferred specific examples include carbon dioxide, nitrogen, helium, argon, and neon. To carry this out, for example, the reactor must be degassed using a vacuum pump or the like and then returned to normal pressure with an inert gas, or it can be carried out under conditions where an inert gas is passed through the reactor. Alternatively, the reaction may be carried out in a closed reactor with an inert gas atmosphere.

In order to synthesize a peptide or a peptide derivative in the present invention, first a mixture of an amino acid and an amino acid derivative is obtained under an inert gas atmosphere, or a mixture of an amino acid derivative and an aminoacyl derivative is obtained under an inert gas atmosphere.
Preferably, a mixture with tRNA synthetase is obtained. For this purpose, the amino acid and the amino acid derivative are mixed under an inert gas atmosphere in the presence of water or, for example, adenosine triphosphate or deoxyadenosine triphosphate in a buffer solution, or the amino acid and the amino acid derivative are mixed in the presence of water or, for example, adenosine triphosphate or deoxyadenosine triphosphate in a buffer solution. This may be carried out by mixing the amino acid derivative and aminoacyl-tRNA synthetase under an inert gas atmosphere in the presence of triphosphate. At this time, the main purpose of making the next reaction proceed smoothly and preventing enzyme deactivation is to add divalent cations such as magnesium and manganese, sulfhydrylating agents such as mercaptoethanol and dithiothreitol, and pyrolytic cations to the reaction system. Phofatase may be added alone or in combination. Suitable concentrations of each additive include 0.01mM to 500mM of divalent cation, sulfhydrylating agent
0.001mM to 100mM, pyrophosphatase 0.001 unit/ml to 100 units/ml, and the optimal concentration is 0.1mM to divalent cation, respectively.
10mM, sulfhydrylation agent 0.01mM to 1mM, pyrophosphatase 1 unit/ml to 10 units/ml
ml. Also, amino acids, aminoacyl-
The amounts of 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 acid and aminoacyl-tRNA synthetase should be 10 ⁹ :1 to 1:1. The molar ratio of amino acid to adenosine triphosphate or deoxyadenosine triphosphate is preferably within the range of 1:1 to 1:100.
Furthermore, the concentration of the amino acid derivative was 10mM.
A range of 10M to 10M is appropriate, and a range of 100mM to 2M is preferred. Further, it can be used even lower than this.

The buffer used at this time includes amino acids,
amino derivatives, aminoacyl-tRNA synthetase and adenosine triphosphate or deoxyadenosine triphosphate are dissolved and maintain enzyme activity;
Any material may be used as long as the desired pH can be obtained. Specific examples of such buffers include, for example, Tris-hydrochloride buffer, Hepes buffer, triethanolamino buffer, malate buffer, and phosphate buffer. A mixed medium in which a hydrophilic organic solvent is added to these buffers can also be used as long as the above conditions are satisfied. As the hydrophilic organic solvent, ethers such as dioxane and tetrahydrofuran, as well as dimethyl sulfoxide, dimethyl formamide, acetonitrile, acetone, and the like are preferably used. These organic solvents may be used alone or in combination of two or more. The volume concentration of the organic solvent in the entire mixed liquid at this time is in the range of 0.5 to 85%, preferably 5 to 60%, and optimally 10 to 50%.

Next, the mixture of the amino acid and amino acid derivative obtained above and aminoacyl-tRNA synthetase are mixed in an inert gas atmosphere, or the amino acid derivative obtained above and aminoacyl-tRNA synthetase are mixed together in an inert gas atmosphere.
The mixture with tRNA synthetase and the amino acid are mixed under an inert gas atmosphere, and the amino acid and the amino acid derivative are reacted in the presence of the aminoacyl-tRNA synthetase. The pH when carrying out this reaction is 5-11, preferably 6-10, optimally 7-10.
The range is 9. The reaction temperature is generally 0°C to 70°C, preferably 10°C to 50°C, and optimally 20°C to 40°C from the viewpoint of maintaining enzyme activity.

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 are useful for various hormones and antibiotic peptides, such as Met-enkephalin, which has a morphine effect, bradykinin, which has a hypotensive effect, and somatostatin, which has an endocrine- and exocrine-suppressing effect. It is useful as other biologically active substances such as

According to the present invention, it is possible to suppress side reactions, prevent the deactivation of expensive aminoacyl-tRNA synthetase, and furthermore, it is possible to produce peptide derivatives even with low concentrations of raw materials, especially enzymes, thereby reducing production costs. It is also cheap.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Reference example 1 6 kg of Bacillus stearothermophilus UK788 (Feikoken Bacterial Serial No. 5141) was doubled in amount.
Suspend in 100mM Tris-HCl buffer (PH7.5),
After disrupting the cells using Dynomill, insoluble materials were removed by centrifugation to obtain a crude extract containing tyrosine-specific tyrosyl-tRNA synthetase. A column packed with Matrex Gel Blue-A (manufactured by Amicon) equilibrated in advance with 50 mM Tris buffer (PH7.5) containing 5 mM mercaptoethanol, 2 mM sodium ethylenediaminetetraacetate, and 0.1 mM phosphophenylsulfonyl fluoride. Then, the above crude extract was eluted with a solution prepared by adding potassium chloride to the above buffer solution at a linear velocity of 60 cm·h ^-1 , and tyrosyl-tRNA synthetase was eluted. The fractions were collected, concentrated, and desalted to obtain a crude enzyme solution containing tyrosine-specific tyrosyl-tRNA synthetase with a yield of about 70%. All the above operations were performed at 4°C.

Reference example 2 Satucharomyces cerevisiae αS288C 1000Kg
After disrupting the cells with Dynomill, the resulting crude extract was subjected to ammonium sulfate fractionation, DEAE-cellulose chromatography, cellulose phosphate (Watmann) chromatography, DEAE-Sefacel (Pharmacia) chromatography, and Ultrogel. 3.2 g of leucine-specific leucyl-tRNA synthetase was obtained by ACA34 chromatography and CM-cellulose (Watmann) chromatography.

Reference Example 3 After crushing 1,000 kg of rabbit liver with a Waring blender, centrifugation (15,000 g), and further ultracentrifugation (105,000 g) of the supernatant were performed to obtain a soluble protein fraction, which was purified using ammonium sulfate fractionation Sephadex G-
200 (manufactured by Pharmacia) gel chromatography, DEAE Cephacel chromatography, and hydroxyapatite chromatography to obtain 4.5 g of methionyl-tRNA synthetase.

Reference Example 4 1.5 g of asparatyl-tRNA synthetase specific for aspartic acid was obtained from 150 kg of Escherichia coli K-12 strain by the same column operation as in Reference Example 1.

Reference example 5 Bacillus stearothermophilus NCA1503
Using the same column operation as in Reference Example 1, 1.1 g of arginyl-tRNA synthetase specific for arginine was obtained from 100 kg of the stock.

Example 1, Comparative Example 1 To 45 ml of 20 mM Pipes buffer, pH 7.0, containing 8 g of L-phenylalanine ethyl ester, 0.15 g of magnesium chloride, 0.3 g of disodium adenosine triphosphate, 10 mg of L-tyrosine, and pyrophosph. 200 units of Actase (manufactured by Boehringer Mannheim) and 0.01 mg of dithiothreitol were added, and the volume of the mixed solution was adjusted to 50 ml. While replacing the air in the reactor with nitrogen gas and maintaining the pH of this mixture at 7.0, the tyrosyl-
Add 0.2 mg of tRNA synthetase under nitrogen atmosphere,
The mixture was thoroughly mixed, the reaction temperature was maintained at 30°C, and the mixture was allowed to react for one day.

The resulting reaction solution was then applied to a Pondapak _C18 column (manufactured by Waters) and eluted with a linear gradient of acetonitrile/0.1% trifluoroacetic acid aqueous solution to separate L-tyrosyl-L-phenylalanine ethyl ester. I got 18 mg.

The elemental analysis (C ₂₀ H ₂₄ N ₂ O ₂ = 356.42) was as follows: Calculated value (%) C = 67.39 H = 6.80 N = 7.86 Measured value (%) C = 67.28 H = 6.93 N = 7.79.

In addition, for comparison (Comparative Example 1), the reaction was not performed in a nitrogen atmosphere, and the amount of tyrosyl-tRNA synthetase used in Example 1 was 30,000 times that of the amount of tyrosyl-tRNA synthetase used in Example 1.
Using 6 g, add to 50 ml of 20 mM Pipes buffer, pH 7.0, containing the same amount of magnesium chloride, sodium adenosine triphosphate, L-tyrosine, pyrophosphatase and dithiothreitol as in Example 1,
The reaction mixture was reacted at 4°C for 15 hours.
-75 (manufactured by Pharmacia) column, eluted with the same Pipes buffer, and collected the void volume fraction.
The reaction mixture was isolated, 8 g of L-phenylalanine ethyl ester was added thereto, and the mixture was kept at 30° C. in the atmosphere and allowed to react overnight.

As a result, the yield of L-tyrosyl-L-phenylalanine ethyl ester was 4 mg, and Example 1
It was about 1/4 of the yield. Further, while the amount of tyrosyl-tRNA synthetase used in Comparative Example 1 was 6 g, in Example 1 only 0.2 mg was required, which was 1/30000 of that amount.

Example 2, Comparative Example 2 50mM Hepes buffer containing 1.5g of glycyl-glycyl-phenylalanyl-methionine, PH7.9, 50
ml contains 100 mg of magnesium chloride, 200 mg of disodium adenosine triphosphate, and ¹⁴ C-radiolabeled L-
Tyrosine 25mg, dithiothreitol 0.005mg,
The tyrosyl obtained in Reference Example 1 was added to a 100 ml three-necked flask sealed with helium gas, and stirred with a magnetic stirrer.
Add 0.1 mg of tRNA synthetase and mix well.
The reaction temperature was maintained at 40°C and the reaction was carried out for 7 hours.

A portion of the resulting reaction product was collected and analyzed and identified using cellulose thin layer chromatography (developing solvent: n-butanol/pyridine/acetic acid/water = 15/10/3/12). Radioactivity was also detected at the position of -glucyl-phenylalanyl-methionine (hereinafter referred to as enkephalin), confirming the synthesis of the desired oligopeptide, enkephalin. This substance was further digested with carboxypeptidase, and the digested material was analyzed over time using the cellulose thin layer chromatography described above.

As a result, the radioactivity was found to be enkephalin [1-4],
Enkephalin [1-3], Enkephalin [1]
-2] with time, and it was confirmed that the substance obtained in this reaction was the desired enkephalin. The yield was determined to be 75% based on the tyrosine used based on the measured radioactivity.

Next, for comparison, a helium gas seal was not performed and the same amounts of magnesium chloride, disodium adenosine triphosphate, L-tyrosine radioactively labeled with ¹⁴ C, and dithiothreitol were included as in Example 2.
Add 0.1 g equivalent to 1000 times the amount of the same tyrosyl tRNA synthetase as in Example 2 to 50 ml of 50 mM Hepes buffer, pH 7.9, mix well, and react at 4°C for 15 hours. It was isolated in the same manner as in Comparative Example 1, and 1.5 g of glycyl-glycyl-phenylalanyl-methionine was added thereto in the atmosphere, and while the pH was maintained at 7.9 with a small amount of sodium hydroxide, 40 g was added in the same manner as in Example 2. When reacted at ℃ for 7 hours, despite using a large amount of enzyme,
The reaction product was analyzed by thin layer chromatography in the same manner as in Example 2. As a result, the production of concephalin was very low, perhaps due to side reactions, and was barely detectable.

Example 3, Comparative Example 3 30mM 2,5-dimethylimidazole buffer PH containing 2g of L-phenylalanine methyl ester
7.2, 13ml contains 20mg of magnesium chloride, 50mg of disodium adenosine triphosphate, 10 units of pyrophosphatase (manufactured by Boehringer Mannheim),
Add 20μ of mercaptoethanol and 3mg of leucyl-tRNA synthetase obtained in Reference Example 2, and increase the volume while maintaining the pH of the mixture at 7.2 under a nitrogen stream.
The volume was adjusted to 15ml. Add 1 mg of L-leucine to this, mix well under a nitrogen stream, and adjust the pH to 7.2 and reaction temperature.
The mixture was kept at 20°C and reacted for 5 hours.

The resulting reaction mixture was then packed into a bonder pack.
Separated using a _C18 column in the same manner as in Example 1, L-
Leucyl-L-phenylalanine methyl ester
Obtained 2.2 mg.

Its elemental analysis (C ₁₆ H ₂₄ N ₂ O ₃ = 292.36) was as follows: Calculated value (%) C = 65.73 H = 8.27 N = 9.58 Measured value (%) C = 65.80 H = 8.17 N = 9.49.

Next, for comparison (Comparative Example 3), 15 ml of the same buffer as in Example 3 containing the same amounts of magnesium chloride, disodium adenosine triphosphate, L-leucine, pyrophosphatase, and mercaptoethanol. 3 g equivalent to 1000 times the amount of the same leucyl-tRNA synthetase as in Example 3 was added to
Mix well and react at 4°C for 20 minutes. The resulting reaction mixture was isolated in the same manner as in Comparative Example 1. 2 g of L-phenylalanine methyl ester was added thereto, and the reaction was carried out in the same manner as in Example 3. Ivy. At this time, all operations were not performed under a nitrogen stream.

As a result, the yield of L-leucyl-L-phenylalanine methyl ester was 1.8 mg, which is almost the same yield as in Example 3, but the amount of leucyl-tRNA synthetase was 1000 times larger.

Example 4, Comparative Example 4 Magnesium chloride 100 mg, L-
A mixed solution was obtained by adding 2 mg of methionine, 100 units of pyrophosphatase (manufactured by Boehringer Mannheim), and 0.01 mg of dithiothreitol. Add 1 mg of the methionyl-tRNA synthetase obtained in Reference Example 3 to this mixture, adjust the volume to 40 ml while maintaining the pH of the mixture at 7.5, and add 100 mg of disodium deoxyadenosine triphosphate to the mixture to adjust the pH to 7.5. 5, and once the pressure of the reactor was reduced to about 1 to 0.1 mmHg using a vacuum pump, argon gas was introduced to return the reactor to normal pressure, the reactor was immediately plugged, and the reaction was carried out in the same manner as in Example 3. Separate to L-methionyl-β-
2.5 mg of alanilamide was obtained.

The elemental analysis (C ₈ H ₁₇ N ₃ O ₂ S = 219.30) was as follows: Calculated value (%) C = 43.81 H = 7.82 N = 19.16 Measured value (%) C = 43.90 H = 7.70 N = 19.13.

Next, for comparison (Comparative Example 4), the same amounts of magnesium chloride, disodium deoxyadenosine triphosphate, L-methionine, pyrophosphatase, and dithiochloride as in Example 4 were used without degassing and subsequent argon substitution. The same methionyl solution as in Example 4 was added to 40 ml of the same buffer as in Example 4 containing threitol.
1 g corresponding to 1000 times the amount of tRNA synthetase was added, mixed well, 5 g of β-alanylamide was added in the same manner as in Comparative Example 1, and the reaction was carried out in the same manner as in Example 4.

As a result, the yield of L-methionyl-β-alanylamide was 1.3 mg, which was a low yield even though 1000 times more enzyme was used.

Example 5, Comparative Example 5 20mM Pipes buffer containing 8g of L-phenylalanine ethyl ester, pH 7.1, 45ml of magnesium chloride 150mg, adenosine triphosphate disodium 300mg, L-aspartic acid 2mg, pyrophosphatase (manufactured by Boehringer Mannheim) 200
Add unit and dithiothreitol 0.01mg under helium atmosphere, and make the mixture PH7.1, volume 50ml.
Adjusted to. Add 0.1 mg of asparatyl-tRNA synthetase obtained in Reference Example 4 to this, mix well,
The mixture was left to react for one day under a helium atmosphere at a pH of 7.1 and a reaction temperature of 30°C. This reaction solution was treated in the same manner as in Example 1 to obtain 3.7 mg of L-asparatyl-L-phenylalanine ethyl ester.

Next, for comparison (Comparative Example 5), the same buffer as in Example 5 containing the same amounts of magnesium chloride, disodium adenosine triphosphate, L-aspartic acid, pyrophosphatase and dithiothreitol as in Example 5 was prepared. Add the same asparatyl as in Example 5 to 50 ml.
2g, equivalent to 20000 times the amount of tRNA synthetase
was added, mixed well, and reacted at 4°C for 20 minutes. The resulting reaction mixture was isolated in the same manner as in Comparative Example 1, and L-phenylalanine ethyl ester was added to it.
8 g was added and the reaction was carried out in the same manner as in Example 5. At this time, all operations were not performed under a helium atmosphere.

As a result, 3.0 mg of L-asparatyl-L-phenylalanine ethyl ester was obtained.

Examples 6 and 7, Comparative Example 6 Contains 4g of L-tyrosine t-butyl ester
20mM Hepes buffer PH8.0, 45ml magnesium chloride 300mg, adenosine triphosphate disodium 350
1 mg of L-arginine, 200 units of pyrophosphatase (manufactured by Boehringer Mannheim) and 0.01 mg of dithiothreitol were added under a helium atmosphere to adjust the pH of the mixture to 8.0 and the volume to 50 ml.
Add 0.2 mg of arginyl-tRNA synthetase obtained in Reference Example 5 to this, mix well, keep the pH at 8.0, and perform the same operation as in Example 5 to obtain L-arginyl-L-tyrosine t-butyl ester. 0.5mg
I got it.

Next, for comparison (Comparative Example 6), arginyl-
L-arginyl-L was prepared in the same manner as in Comparative Example 5 using the same amount as in Example 6, except that 0.2 g of tRNA synthetase was used and the procedure was not carried out in a helium atmosphere.
-Tyrosine t-butyl ester (0.3 mg) was obtained.

Next, a reaction was carried out in exactly the same manner as in Example 6 using D-tyrosine t-butyl ester instead of L-tyrosine t-butyl ester (Example 7).

As a result, L-arginyl-D-tyrosine t-
0.4 mg of butyl ester was obtained.

Claims (1)

[Claims] 1. A peptide or peptide characterized in that when a peptide or peptide derivative is synthesized by reacting an amino acid and an amino acid derivative derived from the amino acid in the presence of aminoacyl-tRNA synthetase, the reaction is carried out under an inert gas atmosphere. Synthesis method of derivatives.