JPS58146539A - Synthesis of peptide or peptide derivative - Google Patents

Abstract

PURPOSE:To prepare a peptide or a peptide derivative from amino acids, economically, without protecting the amino group, by using aminoacyl t-RNA synthetase as a condensation agent. CONSTITUTION:1mol of an amino acid is reacted with 1-10mol of aminoacyl t-RNA synthetase in a buffer solution of 7-10pH in the presence of 10-100mol of adenosine triphosphate, etc. at 0-30 deg.C, and 1pt.vol. of the reaction mixture is made to react with 0.1-100pts.vol. of an amino acid derivative at 20-40 deg.C to obtain a peptide or a peptide derivative (e.g. L-tyrosine-L-phenylalanine methyl etster). USE:Various hormones such as bradikinin (having hypotensive activity) somastatin (having external secretion suppressing activity), antibiotic peptide, and peptide for seasoning.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for the synthesis of -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. At the same time, the development of peptide synthesis methods is also active. The main methods of peptide synthesis known to date are 1, for example, Fanima V7. As summarized in V View, No. 3, pp. 27-47 (1980), methods 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 spreading time is long, and the terminal amino group must be protected with a protective film before the reaction. For the fragment condensation method.

In particular, it has a serious drawback that racemization is 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 takes a long reaction time and requires operations such as protecting the terminal amino group with a protecting group. However, it was not possible to improve the complexity of the system.

Furthermore, in the enzymatic method using this protease.

Because the enzyme used originally has peptidolytic activity,
This method presented a serious drawback in that the resulting peptide was degraded during synthesis, often making it impossible to obtain the desired peptide. In particular, it has been pointed out that when applied to the synthesis of oligopegated compounds, a serious drawback is that unintended peptides lacking some amino acids can be obtained (Journal-Og.
Biological e-chemistry, volume 256, 15
01 page (1981), in addition to the globase method, there is also a known enzymatic peptide synthesis method that uses a special enzyme that controls only the synthesis of a single peptide having a constant N sequence of amino acids. There is. This type of enzyme has 9
For example, glutathione synthase (Japanese Patent Application Laid-open No. 122793/1983) synthesizes a tripeptide with the sequence glutamic acid/cysteine/glycine, and gutumicidin S synthase (Modern Chemical (December 1974 issue, p. 12) have been reported. However, these enzymes are special enzymes, and the peptides that can be synthesized by these enzymes are only one type of peptide, and it is not possible to synthesize any desired peptide. 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 need for protective groups, which cause the above-mentioned drawbacks, especially fusemization, the occurrence of multiple reactions, and the complexity of 9 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 t-RNA, a type of nucleic acid, is an enzyme that has the ability to C-bond amino acids, and has never previously formed a peptide bond. Aminoa V/I/l-RNA V-n?, whose effect was unknown.
#-SeE*<< Especially, it was discovered that the enzyme has the ability to synthesize peptides, and when this enzyme is used as a condensing agent, all of the above objects can be achieved. 1. The present invention has been made.

That is, the present invention uses aminoacyl t as a condensing agent when synthesizing peptides or peptide derivatives from amino acids.
- A method for synthesizing peptides or peptide derivatives, characterized by using RNA synthetase.

The feature of the present invention is that in the enzymatic peptide synthesis method, aminoa Vs/l-RJ is used as a condensing agent.
By using A syndetase, peptides or peptide derivatives are synthesized without protecting amino groups. This aminoacyl t-RNA synthetase belongs to enzyme classification A, 1.1 and has the following formula: amino acid + ATP + t-RNA → aminoacyl-t
- An enzyme that catalyzes the reaction of RNA + AMP + birophosphate 9 For example, rabbit.

Items obtained from animal tissues such as horses, cows, rats, chickens, and snakes; Items obtained from plant tissues such as rice, potatoes, and tomatoes; molds, yeast, and mushrooms; 11!1#I
, and those obtained from microorganisms and algae such as Hoban Moe. Among these, enzymes obtained from microorganisms are preferred because they are easy to obtain, and in addition, enzymes obtained from microorganisms such as Bacillus stearonermophilus, Thermus thermophilus, Thermus huopas, Clostridium t-moaceticum, and T. -Aminoacyl t-RNA obtained from heat-resistant microorganisms such as trout φ aquaticus
Synthetase is optimal.

These various aminoamine A/l-RNA synthetases can be prepared by crushing the Imperial tissues or cells using a homogenizer, Dyno-μ, etc. 1 For example, Biochemistry Magazine Vol.
DEA as described on page 307 (1974)
It can be obtained by purification using ordinary p4Jf and elementary purification methods such as chromatography such as E-cellulose chromatography and human tetraxiapatite chromatography and fractional precipitation with ammonium sulfate. Aminoacyl'vt-RNA synthetase is used that has specificity for nine different a-amino acids; for example, one that has specificity for tilcin.

Tiroshi vt-R? iA synthetase is specific for leucine, and leucyl t-RNA synthetase is specific for valine.
vt-RNA synthetase, alanylj-RNA
Synthetase, Gutami 7%/l-1 RNA synthetase.

Asvafugi k t-RNA synthetase, methionine t-RNA synthetase histidinyl t-RNA synthetase 5 detase, rigid pv t-RNA synthetase-4, threonyl t-RNA synthetase, seryl t-RNA
Specific examples include V.

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 with a Salel amino acid derivative derived from the amino acid in the presence of aminoacyt-RNA synthetase. Furthermore, according to the present invention, the amino acid and the aminoacyl t-
React with RNA synthetase and dilute the reaction mixture to 1% 1
Next, a peptide or peptide derivative can be synthesized by reacting the obtained reaction mixture with an amino acid derivative. Examples of amino acids preferably used for the pre-reaction with this amino acid 1vt-RNA synthetase include thyrovinine and alanine.

Examples include a-amino acids such as leucine, isoleucine, phenylawonine, methionine, 9-zine, serine, and valine, and may be in either the L-form or the D-form.

In addition, examples of amino acid derivatives preferably used in the above reaction include glycine and alanine.

Leucine, inleucine, phenylafnine, glutamic acid, glutamine,) fv serine, cysteine, tyrosine, arginine, valine, lysine.

α-amino acids such as histidine, asbawogic acid, 'asbawogin, methion, trygtophan, threonine, β-amino acids such as β-afnin, β-aminoisoaN, nitrogen-containing γ-amino acids such as creatine, and r-amino acids such as piperidic acid. amino acid.

Ephthyl, thioester, and amide of various amino acids such as ε-aminocaproic acid and C-amino acids.

Examples include hydroxamide, but the compound is not limited to the above-mentioned exemplified compounds as long as it is an amino acid derivative in which the amino group is in a free form. Examples of the ester V include t-R
Nine different types of Este V can be used, including those in which the above amino acid is converted to Este V at the 3'OH of NA. Furthermore, as the amide, in addition to a free amide, an oligopeptide or a polypeptide in which 9 amino acids, for example, 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. Further, the above amino acid derivatives are used in the form of an aqueous solution, but they may also be used in a solid state.

Then to obtain a reaction mixture, for example pH 5 to pl
i11 preferably pH 6 to pH 10° most INIK-
may be carried out by mixing the amino acid and aminoacyl t-RNA synthetase in the presence of adenosine myphosphate or deoxyadenosine myphosphate in a p)17 to pFlo buffer.

The reaction temperature at this time is preferably from Co°C to 70°C from the viewpoint of maintaining enzyme activity.

Optimally it is carried out at 0°C to 30°C. Also.

The mild solution used at that time is amino acids.

Adenosine diphosphate, deoxyadenosine myphosphate and aminoacyl A/t-RNA sy synthetase/f
Any solution may be used as long as the desired pH can be obtained. For example, Tris-HCl buffer, Hepes buffer, triethanolamine Il stock solution, malate buffer, phosphate buffer. etc. Furthermore, with the main purpose of promoting smooth activation and preventing enzyme deactivation, divalent cations such as magnesium and manganese, sulfur hydrylating agents such as captoethanol, dithiothreate-V, and pyrrolidine are added to the reaction system. Phosphatases may be added alone or in combination. Suitable temperatures for each additive include 0.01 MM to 500 ml of divalent cation, 0.001 ml to 100 ml of sulfhydrylating agent, and 0.001 ml to 100 ml of pyrophosphatase, and the optimum concentration. are 0.1 ymM to 10 11 M of divalent cation, 0.01 mM to 1 mM of sulfhydrylation agent, and 1 unit/d to 10 units/d of pyrophosphatase, respectively. In addition, although there are no particular restrictions on the amount of amino acids, aminoacyl A/1-RNA S/synthetase, and adenosine diphosphate or deoxyadenosine myphosphate, in order to obtain a practical yield, it is necessary to The molar ratio is 1 to 1:10. It is preferable to carry out the molar ratio of amino acid and adenosine diphosphoric acid or deoxyadenosine diphosphoric acid within the range of 1:10 to 1:10G. When the reaction is carried out under the conditions described below, the reaction proceeds smoothly and the reaction takes place within a few seconds. Completed within 5 minutes.

Next, the reaction mixture obtained as described above and the amino acid derivative are mixed and reacted to obtain the desired peptide or peptide derivative. (This step is hereinafter referred to as peptidation.) 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 (manufactured by PharmaVa), etc. By performing gel chromatography of
Adenosine-phosphoric acid, birophosphoric acid, etc. can also be removed and used. In addition, the temperature of the peptidation reaction is preferably from 0°C to 7DC, and Tsunaten Kagame et al.
It is preferable to carry out the reaction at a temperature of 20°C to 40'°C.

As for PH.

5 to 11, preferably 6 to 10, using the various buffer solutions mentioned above. For maximum m, use 7 to 9.

The mixing ratio of the reaction mixture and the amino acid derivative is 12, for example, the volume may be 1:Q, and the production amount may be 1 to 1:100. Further, the concentration of the amino acid M1 form used at this time is within the range of 10111M Carr et al. IOM.

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 peptides or peptide derivatives obtained by the present invention are 1) various hormones such as gudgekinin which has antihypertensive effects, somatostatin which has endocrine and exocrine suppressive effects, and other biological substances such as antibiotic peptides and taste peptides. It is useful as a clinically active substance.

According to the present invention, the useful peptide or peptide derivative described above can be produced at low cost without using a protecting group.

The present invention will be specifically explained below using examples.

Example 1 Thiovs/1-R specific for tyrosine purified from Bacillus suderothermophii V according to the method described in Biochemistry, Vol. 13, p. 2307 (1974)
Nk synthetase 0.4f, magnesium chloride 0.41
F, Adenosine jade phosphate 0.1f, L-tyrosine 1
qPyrophosphater-V (Peringer Mannheim?
tll) 200 units and ditheothlate-AI 0
．． 01'F -200s/2011fM Hepes buffer p l'! 8.0 and reacted at 4° C. for 15 minutes to obtain a reaction mixture. L-phenylaphnine methyl ester tv4f was added to the resulting reaction mixture, mixed well, and allowed to react for one day while maintaining the reaction temperature at 30°C.

Next, 200 d of acetone was added to the resulting reaction solution to remove the sediment, and the supernatant was concentrated in an evaporator to a concentration of about 20.
Bondapack cis Kago (manufactured by Waters) was applied to acetone triy15Q aqueous potassium phosphate solution,
85/15. The mixture was separated using pH 7 as a developing solvent to obtain 0.4q of L-tyrosine-L-phenylated methane ester.

Its elemental analysis (Cse H It Oa = 342-
59).

Calculated value (%) C=66.65 H=6.48N=
8.18 Measured value (%) C=66.55 H=6.4ON=
It was 8.27.

Example 2 Tyro$'A/t-RNA synthetase 41. used in Example 1. Magnesium chloride 50III, adenosine myphosphate 20 da, L-thyrokune 9 cape, pyrophosphatase (manufactured by Pehringamannheim) 200 units, and dithiothreitol 0.0111 F at 20 g/1
Dissolved in 011M Hepes buffer pH 8.5 and incubated at 4°C for 2
After reacting for 00 minutes, the reaction mixture was applied to 075 (Pharmacia) Kago, eluted with the same Hehes buffer as above, and 50 d of the void volume was collected to isolate the reaction mixture.

Isolated reaction mixture 1;.

14/4'L of D-leucine ethyl ester was added, mixed well, and reacted for 30 minutes while maintaining the 1/vS temperature at 20°C.

Next, the obtained reaction solution was directly applied to a bonderback CI column and separated in the same manner as in Example "1".

L-thi psil-D-leucine ethyl ester 16 capes were obtained.

Its elemental analysis (Csma H1・Nl 04 = 322-
59).

Calculated value (excellent) C depth 63.35 H=8.13N=8
．． 69 Measured value (excellent) Cm65.42 11=8.1ON=
It was 8.57.

Example 3 Tyrosyl t-RM mucintetase 20 capes, prepared from baker's yeast according to the method described in Journal Op, 11 Biochemistry, Vol. 63, p. 434 (1968);
Magnesium chloride 20q, adenosine 9sq, D
- Tyrosine 0.11 II, Hyrophosphatase (Boehringer Mannheim II) 102 nits and MeV Cub)
Utah, -pv 20 pi 20m 50111M2
．． After adding 5-dimethylimidazole buffer pH 9 and reacting in the same manner as in Example 2, the reaction mixture was isolated in the same manner as in Example 2, and L-leucine ethyl ester A/1
g was added as a solid and subjected to Flflff reaction at 2DC for 5 hours. 20 d of acetone was added to the obtained reaction product, the resulting precipitate was filtered, concentrated to about 10 gg using an evaporator, and separated in the same manner as in Example 1. 15 q of D-tylovtv-L-leucine ethyl ester. I got it.

Its elemental analysis (CKy If 曾* Nl 04 = 3
22.39) is.

Calculated value (*) Cm6L53 H=8.15N=8
．． 69 Measured value (j) Cm63.15 H=8.24N=
It was 8.50.

Example 4 From Pig Pancreas Jhana J4/II Journal of Biological Chemistry, Vol. 237, p. 3698 (1962
) was prepared according to the method described in ). J pv t −
RWA synthetase 0.4f, magnesium chloride 50q
, 20q of deoxyadenosine phosphoric acid, 1 drop of L-serine, 200 units of pyrophosphater 41 (manufactured by Boehringer Inheim), and 10.0 units of dithiothreate-A.
Using 1, *, m corresponds to F-z as in Example 2, and G25 (
A reaction mixture was obtained using a column (manufactured by Tulmacia).

Next, 4 g of β-AwojiV amide was added to this.

The mixture was mixed and reacted at 50°C for 10 minutes. The resulting reaction solution was separated using a Bondabafuku C column in the same manner as in Example 1.
One portion of L-seryl-β-alanylaminocyamide was obtained.

Its elemental analysis (Cm Fats Ha Oh = 17
5-19).

Calculated value Cm41.14 11=7.48N m2
3.99 Measurement [I Cm, 41.20 1'l=7.52N
= 24.10.

Agent Yu Kodama 3 Procedural amendments (spontaneous) April 7, 1980 Director-General of the Patent Office April 80, 1980 Submission 1, Indication of the case Patent application No. 10336/1982 2, Title of the invention: Peptide Or Synthesis Method of Pepti F Derivatives 3, Relationship with the Amendment Case Patent Applicant 4, Agent 5? # Column 6 of Detailed Description of the Invention of the Positive Subject Specification, Contents of Amendment (11 Specification Box, page 2, bottom 1st line, change “Oligopepte” to “Oligopepte”)
"Oligopepti," he corrected.

(2) "Double reaction" in the second line of page 4 of the same book is corrected to "side reaction."

(3) [Aminoamyl LRNA syntera] in the first line of page 6 of the same book is corrected to [aminoacyl t-RN^synteter].

(4) "Nitrogen-containing" in the same book, page 8, line 1 O, is "nitrogen-containing"
I am corrected.

(5) "Probilge" in the fourth line of the same page of the same book is corrected to "propyl, shi".

(6) "Chlohexylphenyl" in the third line directly below the same document is corrected to "chlorhexyl, phenyl." (7) "Thio" in line 5 of page 9 of the same book is corrected to "thio".

(8) "Activation" in line 6 of page 10 of the same book is corrected to "reaction."

(9) "Sulfhydryl" in the fifth line of the same document is corrected to "sulfhydryl."

In the same book, page 11, line 3, "1:1~l:, 10J is corrected to rlo:1~1:IOJ.

01) “Adenosine triphosphate” in the same book, page 13, line 8
is corrected to "adenosine triphosphate disodium."

0no Same book, same page, line 1θ [Jiteosure J is corrected as ``Dichiosure''.

03 Same book, same page, line 11 r -200*120m
Correct MJ to 20mMJ of r200m#.

(14) “Eva Preku” in the third line just below the same book as “
"Evaporator", corrected.

OS "Acetontrile" in the first line just below the same book as "
Acetonitrile,” he corrected.

as　EW 1st tribute Correct it to r　250　mgJ.

αη "Phosphoric acid" in the 8th line of the same document is corrected to "disodium phosphate".

oIl "Phosphoric acid" in line 6 of page 10 of the same book is corrected to [disodium phosphate].

0911 "Phosphoric acid" in the second line of page 17 of the same book is corrected to "disodium phosphate".

(To) Add the following example after "was." on the 18th line of the same page in the same book.

"Example 5. 6.7 0.4 g of aspartic acid-specific asparatyl-TRNA synthetase purified from Bacillus stearothermophile according to the method described in Nucleus Acid Research, Vol. 4, p. 2233 (1977), chloride. Magnesium 0.4g, adenosine triphosphate disodium salt 0
．． 1 g, L-aspartic acid 1 tag. Pyrophosphatase (manufactured by Boehringer Mannheim)
200 units and 9.01 mg of dithiothreitol were dissolved in 140 ml of 20 mM Heves buffer pH 8.0.

A reaction mixture was obtained by reacting at 4°C for 15 minutes. 4 g of L-phenylalanine t-butyl ester was added to the obtained reaction mixture, mixed well, and kept at a reaction temperature of 30° C. for one day to react.

Next, 200 ml of acetone was added to the resulting reaction solution, and the precipitate was filtered off. The supernatant was concentrated to about 204-1 in an evaporator, and then applied to a Bondapak CI8 column (manufactured by Waters), and evaporated with acetonitrile/50 mM. It was separated using a potassium phosphate aqueous solution, 85/15, PH 7 as a developing solvent to obtain 0.4 mg of L-asparatyl-phenylalanine t-butyl ester (Example 5).
).

Its elemental analysis value (C17 N25 N205 = 33
7, 40).

Calculated value (%) C=60.52 H=7.47
N=8.30 Measured value (%) C=60.50 H=
7.38 N=8.35.

Next, instead of L-phenylalanine t-butyl ester, L-phenylalanine ethyl ester (Example 6) or L-phenylalanine methyl ester (
The reaction was carried out in exactly the same manner as in Example 5, except that Example 7) was used. the result.

L-Asparatyl-L-phenylalanine ethyl ester 0.2a+gG. Also, L-asparatyl-L-
0.3 mg of phenylalanine methyl ester was obtained in each case.

-5~ Example 8 From Satucharomyces, Biohim Biophys Acta Magazine,
Asparatyl-t RNA synthetase 2OB prepared according to the method described in Vol. 249, p. 263 (1973)
, magnesium chloride 20 mg, adenosine triphosphate disodium salt 5B, L-aspartic acid 0.1 mg,
Pyrophosphatase (Boehringer Mannheim) 1
0 unit and mercaptoethanol 20μm for 20m
+1 in 30 mM 2.5-dimethylimidazole buffer pH 8,0 and reacted in the same manner as in Example 1, the reaction mixture was isolated in the same manner as in Example 1, and L-methionine ethyl ester 1. 2g was added and reacted at 20°C for 5 hours. 20 ml of acetone was added to the obtained reaction product, the resulting precipitate was filtered off, and the mixture was concentrated to about 10 ml using an evaporator.

Separation was carried out in the same manner as in Example 1 to obtain 0.13 mg of L-asparatyl-L-methionine ethyl ester.

Example 9.10.11 4 g of tyrosyl-tRN^ synthetase used in Example 1
, magnesium chloride 50 mg, adenosine triphosphate disodium 200 mg, I, tyrosine 9 mg, pyrophosphatase (Boehringer Mannheim) 20
0 units and 0.01 mg of dithiothreitol in 2
Dissolve in 0ml of 10mM Hepes buffer PH8.5.

After reacting at 4°C for 200 minutes, the 9 reaction mixture was transferred to G-75.
The reaction mixture was applied to a column (manufactured by Pharmacia) and melted using the same Hepes buffer as above, and both void volumes 30a+1 were collected to isolate the reaction mixture. 5 g (870 mM) of D-arginine ethyl ester was added to the isolated reaction mixture, mixed well, and allowed to stand to react for 300 minutes while maintaining the reaction temperature at 20°C (Example 9).

Next, the obtained reaction solution was separated in the same manner as in Example 1, and 1. -Tyrosyl-D-arginine ethyl ester 14
．． 5 mg was obtained.

Its elemental analysis value (C17H27N504 = 365.4
3).

Calculated value (%) C=55.88 H=7.45 N=
19.17 Measured value (%) C= 55.80 H=
7.52 N = 19.0.

-6 = Next, a reaction was carried out in exactly the same manner as in Example 9 except that 3 g of L-arginine t-butyl ester was added instead of D-arginine ethyl ester (Example 10). Its result J
10 mg of IL, L-tyrosine-L-arginine t-butyl ester was obtained.

Furthermore, instead of the D-arginine ethyl ester of Example 9, 4 g of L-arginine ethyl ester was added to Hehes w.
The reaction was carried out in exactly the same manner as in Example 9, except that d,2,5-dimethylimigsol 81% solution pH 8.0 was used instead of k solution pH 8.5 (7) (Example 11).

As a result, 12 mg of L-tyrosine-arginine ethyl ester was obtained.

Examples 12 and 13 0.4 g of arginine-specific argyl-tRNA synthetase purified from Bacillus stearothermophilus according to the method described in February 1976, Vol. 62, p. 190, 0.4 g of magnesium chloride,
Adenosine triphosphate disodium salt 0.1 g.

L-alkyrine 111g + pyrophosphase (Bage 8
-7- 200 units (manufactured by Nger Mannheim) and 0.01 mg of dithiothreitol in 140ml of 20mM
Hepes! The mixture was dissolved to pH 8.0 and reacted at 4° C. for 15 minutes to obtain a reaction mixture. The resulting reaction mixture was
After adding 4 g of tyrosine t-butyl ester and mixing well, the reaction temperature was maintained at 30° C. and allowed to react for one day (Example 12).

Thereafter, in the same manner as in Example 1, 1.6 mg of L-arginine-1,-tyrosine t-butyl ester was obtained.

Its elemental analysis value (C19) 131 N504 = 36
5.43) is.

Calculated value (%) C=58.00 H=7.94
N=17.80 Measured value (%) C=57.83 H
=7.90 N=17.78.

Next, L-tyrosine [- D- instead of butyl ester]
The reaction was carried out in exactly the same manner as in Example 12, except that 5 g of tyrosine ethyl ester was added, and 1.3 mg of L-arginine-D--tyrosine ethyl ester was obtained. "9-

Claims (1)

[Claims] (1) When synthesizing peptides or peptide derivatives from amino acids, aminoacyl t-RNA is used as mixture a.
A method for synthesizing a peptide or a peptide derivative, characterized by using a synthetase.