JPH0416480B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to peptides.

Recent progress in research on bioactive peptides has been remarkable. One of them is the opioid peptide, endorphin, which has morphine activity.
Many studies have been conducted on enkephalins, etc. The present inventors have conducted studies on a series of such peptides, and as part of this research, they have arrived at the present invention.

That is, the gist of the present invention is that the general formula () Ser-Gln-Glu-Asp-Pro-Asn-Ala-Tyr-
AB-Glu-Leu-Phe-Asp-X
...() [In the formula, A is Ser or Tyr, B is Gly or GluX
Represents Ala or Val.

The configuration of the amino acid residue is D, L or DL. ] in the peptides shown.

The present invention will be explained in more detail below.

First, in the general formula (), Ser, Gln,
Glu, Asp, Pro, Asn, Ala, Tyr, Leu, Phe,
Val represents serine, glutamine, glutamic acid, aspartic acid, proline, asparagine, alanine, tyrosine, leucine, phenylalanine, and valine, respectively.

The peptides represented by the above general formula () include:
It also includes those in which the amino group at the N-terminus is protected with a protecting group commonly used in peptide chemistry. Examples include penzyloxycarbonyl, t-butyloxycarbonyl, acyl, p-toluenesulfonyl, and the like.

Furthermore, the C-terminal carboxyl group also includes those protected as commonly used benzyl esters, methyl esters, ethyl esters, etc. moreover,
It may form an amide (-CONR ¹ R ^{2 )} .
R ¹ and R ² represent H or an alkyl group).

Furthermore, the peptides of the present invention include acid addition salts of various commonly used inorganic acids and organic acids, as well as complex compounds with metal compounds, polyamic acids, and the like. Examples of these acids include hydrochloric acid, sulfuric acid, acetic acid, citric acid, and tartaric acid. Examples of the metal compounds include compounds of zinc, nickel, and cobalt, and examples of the polyamic acid include poly-L-glutamic acid.

The peptides of the present invention can be produced by appropriately selecting methods commonly used in peptide chemistry. That is, it can be obtained by either a liquid phase method or a solid phase method.

As protecting groups for amino groups that do not participate in the reaction,
Examples include p-toluenesulfonyl group, benzyloxycarbonyl group, t-butyloxycarbonyl group, and phthaloyl group.

Further, examples of carboxyl protecting groups that do not participate in the reaction include alkyl esters such as methyl and ethyl. Furthermore, the carboxyl group to be reacted with the amino group can be chloride, hydrazide,
It is desirable to activate it by changing to azide, mixed anhydride with organic acid, thioester, cyanomethyl ester, etc.

A method in which a predetermined peptide chain is sequentially extended to the N-terminal side of a starting material by a solid phase method is preferably employed.

In this method, the reaction of the repeated steps in each cycle (especially in the second and subsequent cycles) is carried out with N-hydroxysuccinimide, 1-
Preferably, the reaction is carried out in the presence of an active ester reagent such as oxybenzotriazole.

Removal of the protecting group from a peptide having a protecting group is carried out by a conventional method.

For example, trifluoroacetic acid treatment, hydrolysis,
Reduction etc.

Further, the obtained peptide can be purified using an ion exchange resin, various chromatography methods, and the like.

The peptides according to the present invention have the following general formula ()
It is shown by the general formula () and is useful as an intermediate for producing peptides having analgesic effect.
It is useful for preparing an antiserum that specifically acts on the peptide represented by , and is further useful for detecting and measuring prodynorphin.

H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-
Gln−Phe−Lys−Val−Val−Thr−Arg−
Ser−Gln−Glu−Asp−Pro−Asn−Ala−
Tyr-A-B-Glu-Leu-Phe-Asp-X-
Y...() [In the formula, A, B, and X have the same meanings as in the general formula (). Y represents OH or NR ¹ R ² (R ¹ and R ² represent H or an alkyl group)] When preparing antiserum, serum proteins () are treated with water-soluble carbodiimide, glutaraldehyde, etc. Hapten antigens can be obtained by employing methods such as binding a peptide to a carrier such as serum albumin (serum albumin), an amino acid polymer or copolymer, or adsorbing a peptide to a charcoal powder or inert polymer particles such as polyvinylpyrrolidone. can.

Furthermore, the peptide according to the present invention can be labeled with iodine ( ¹²⁵ I or ¹³¹ I) by a conventional method with the N-terminus protected or in its free form, to form a labeled antigen suitable for radioimmunoassay (RIA). can be provided.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples unless the gist thereof is exceeded.

The Boc-amino acid used in the Examples is L-amino acid and is a product of Peptide Institute Co., Ltd.
Furthermore, "Sephadex G-10" is a product of Pharmacia Fine Chemicals. moreover,
N,N'-cydichlorohexylcarbodiimide, chloromethylated polystyrene (divinylbenzene 1
%, 200-400 mesh, Cl: 0.71meq/g resin)
and N-hydroxysuccinimide are products of Peptide Institute Co., Ltd.

Example 1 H-Ser-Gln-Glu-Asp-Pro-Asn-Ala-
Tyr−Ser−Gly−Glu−Leu−Phe−Asp−Ala−
Boc-Ala esterification of OH synthetic resin is carried out as follows. To Boc-Ala (3.91 mequiv.) dissolved in dry Me ₂ SO (11 ml), add chloromethylated resin (2.50 g, 0.71 mequiv.Cl), and then add KO-t-Bu (3.73 mequiv.). . Shake the mixture vigorously and store the resulting slurry at 80°C.
Hold time. Then, the resin was treated with Me ₂ SO, EtOH
and CH ₂ Cl ₂ .

Peptide synthesis is carried out by a conventional solid phase method.

The synthesis is started by placing 2.50 g of Boc-Ala-OCH ₂ -resin into the reactor of a peptide synthesizer (Beckman 990B model), and amino acid sequences are sequentially synthesized. Removal of protecting groups was accomplished by treatment with 25% trifluoroacetic acid (TFA) in CH ₂ Cl ₂ for 30 min, followed by
10% triethanolamine _{(Et3N )} in _CH2Cl2
is neutralized by

Sequential coupling of each amino acid (3.26 mmol) is made with dicyclohexylcarbodiimide (3.26 mmol) in CH ₂ Cl ₂ for 2 hours. The amount of solvent is 6.5 dicyclohexylcarbodiimide.
Other than ml, it is 20ml.

One cycle of synthesis consists of the following operations.

(1) Washing with CH ₂ Cl ₂ (1.5 min, 3 times) (2) Deprotection with 25% TFA/CH ₂ Cl ₂ (prewash for 1.5 min, then treatment for 30 min) (3) Washing with CH ₂ Cl ₂ Washing (1.5 minutes, 6 times) (4) Neutralization with 10% Et ₃ N/CH ₂ Cl ₂ (1.5 minutes, 3 times) (5) Washing with CH ₂ Cl ₂ (1.5 minutes, 6 times) (6) Boc-amino acid (3.26mequiv., in _{CH2Cl2 ,}
5 min) Treatment (7) Without filtration, add in dicyclohexylcarbodiimide (3.26 mequiv.) and couple for 120 min.

(8) Washing with CH ₂ Cl ₂ (1.5 minutes, 6 times) (9) Repeat the steps from (4) onwards using 3.26 mequiv. of each Boc-amino acid and dicyclohexylcarbodiimide.

From the second cycle onwards, the reactions in the repeated steps are carried out in the presence of N-hydroxysuccinimide (6.52 mequiv.).

Boc-amino acids are dissolved _{in CH2Cl2} . However, Boc-Arg (TOS) is dissolved in dimethylformamide (DMF). Boc−Asn and Boc−
Gln is coupled as N-hydroxysuccinimide ester in DMF.

For this, Boc-Gln (3.26 mmol) and N-
Hydroxysuccinimide (6.52 mmol) in DMF
(20 ml) and the mixture is placed in a reaction vessel and then dicyclohexylcarbodiimide (3.26 mmol) in CH ₂ Cl ₂ (6.5 ml) is added. The reaction vessel is kept under a nitrogen atmosphere during the synthesis to minimize oxidation by air. A washing step is performed after each coupling reaction and the presence of unreacted free amino groups is monitored by the ninhydrin test.

The following protected amino acids are used as amino acids.

Boc-Ala, Boc-Asp (OBzl), Boc-Phe,
Boc−Leu−H ₂ O, Boc−Glu(OBzl), Boc−
Gly, Boc-Ser (Bzl), Boc-Tyr (2,6-Cl ₂
−Bzl), Boc−Asn, Boc−Pro, Boc−Gln.

After completing the entire cycle, 4.59 g of protected peptide -
Get resin.

One-third of the mixture (1.53 g) is treated with HF (20 ml) in the presence of anisole (4.0 ml) at 0° C. for 60 minutes in a conventional manner. HF is removed by a vacuum pump at 0°C. The resulting yellow resin is washed several times with ethyl acetate. Peptides are extracted with 1M acetic acid (100ml), concentrated and lyophilized. Crude peptide (518mg)
was applied to a "Sephadex" G-10 column (2.5 x 136 cm) and eluted with 1M acetic acid. The fractions (10 g each) are monitored spectrophotometrically at 280 nm. The fractions (tubes no. 26-27), (tubes no. 28-37) and (tubes no. 38-43) are collected, evaporated under reduced pressure and lyophilized.

Fraction and substance respectively
Contains 30mg, 325mg, and 60mg. The target substance is contained in the fraction. The resulting peptides are subjected to thin layer chromatography (TLC) and reversed phase high performance liquid chromatography (reversed phase HPLC).

Rf〓0.24, Rf〓0.38 (TLC) Solvent meter Rf〓: 1-BuoH-AcOH- _H2O (4:1:
5) Rf〓:1-BuOH-pyridine-AcOH-
H ₂ O (30:20:6:24) Amino acid analysis Peptide hydrolyzate (6NHCl, 24 hours, 110
℃) was carried out using a "Hitachi" 835 model amino acid analyzer. The amino acid composition is as follows.

Asp(3)3.08, Ser(2)1.85, Glu(3)3.13, Gly(1)
1.03, Ala(2)2.00, Leu(1)1.04, Tyr(1)1.02, Phe
(1)0.93, Pro(1)0.93 Example 2 H-Ser-Gln-Glu-Asp-Pro-Asn-Ala-
Tyr−Tyr−Glu−Glu−Leu−Phe−Asp−Val
Synthesis of -OH Boc-Ala esterification of the resin is carried out as follows. Boc dissolved in dry _Me2SO (11ml)
-Add chloromethylated resin (2.50g, 0.71mequiv.Cl) to -Val (4.45mequiv.), and then add KO-
Add t-Bu (3.73 mequiv.). The mixture is shaken vigorously and the resulting slurry is held at 80°C for 1 hour. The resin was then treated with Me ₂ SO, EtOH and
Wash _{with CH2Cl2} .

Synthesis is started by placing 2.50 g of Boc-Val-OCH ₂ -resin in a reaction vessel of a peptide synthesizer (Beckman 990B model), and the amino acid string is sequentially extended by a conventional solid phase method. Removal of protecting groups is achieved by treatment with 25% trifluoroacetic acid ( _TFA ) in _CH2Cl2 for 30 min, followed by neutralization with 10% ethanolamine ( _Et3N ) in _{CH2Cl2 .} .

Sequential coupling of each amino acid (0.06 mmol) is made with dicyclohexylcarbodiimide (4.06 mmol) in CH ₂ Cl ₂ for 2 hours. The amount of solvent is 8.0 dicyclohexylcarbodiimide.
Other than ml, it is 20ml.

One cycle of synthesis is as in Example 1 except that 4.06 mequiv. of Boc-amino acid and dicyclohexylcarbodiimide are used.

From the second cycle onwards, the reactions in the repeated steps are carried out in the presence of N-hydroxysuccinimide (8.12mequiv.).

Boc-amino acids are dissolved _{in CH2Cl2} .
However, Boc-Arg (Tos) is dissolved in dimethylformamide (DMF).

Boc-Asn and Boc-Gln are coupled as N-hydroxysuccinimide ester. For this, Boc-Gln (4.06 mmol) and N
-Hydroxysuccinimide (8.12mmnl)
Dissolve in DMF (20 ml) and place the mixture in a reaction vessel, then add cyclohexylcarbodiimide (4.06 mmol) in CH ₂ Cl ₂ (8.12 ml).

The reaction vessel is kept under a nitrogen atmosphere during the synthesis to minimize oxidation by air.

A washing step is performed after each coupling reaction and the presence of unreacted free amino groups is monitored by the ninhydrin test.

The protected amino acids used are the same as in Example 1, and Boc-Val is used as Val.

Complete the entire cycle and obtain 4.20 g of protected peptide resin.

One-third (1.40 g) thereof is treated in the same manner as in Example 1 to obtain a crude peptide (377 mg). This was applied to a "Sephadex" G-10 column (2.5 x 136 cm) and eluted with 50% AcOH. Fractions (tubes No. 24-25), (tubes No. 26-32) and (tubes No. 33-35) are obtained in the same manner as in Example 1. The substance content is 17mg, 313mg and
It is 23mg. The target substance is contained in the fraction. The resulting peptides are subjected to thin layer chromatography (TLC) and reversed phase high performance liquid chromatography (reversed phase HPLC).

Rf〓0.28, Rf〓0.50 (TLC) Solvent system: Same amino acid analysis as in Example 1 (carried out in the same manner as in Example 1) Asp(3)3.10, Ser(1)0.78, Glu(4)4.00, Ala (1)
1.03, Val(1)1.02, Leu(1)1.07, Tyr(2)1.97, Phe
(1)1.05, Pro(1)0.97

Claims (1)

[Claims] 1 General formula () Ser−Gln−Glu−Asp−Pro−Asn−Ala−Tyr−
AB-Glu-Leu-Phe-Asp-X
...() [In the formula, A is Ser or Tyr, B is Gly or Glu
represents Ala or Val. The configuration of the amino acid residue is D, L, or DL. ] Peptides indicated by 2 In the general formula (), A is Ser, B is Gly,
The peptides according to claim 1, wherein X is Ala.