JPS60161999A - Peptide - Google Patents

Abstract

NEW MATERIAL:A compound (salt) expressed by formulas I -VII (R<4> is H or protecting group of mercapto). USE:An antigen for analyzing hepatitis B viruses and antigen for hepatitis B vaccines. PREPARATION:For example, serine having amino group protected with tert- butoxycarbonyl group is linked to a chloromethylated styrene-divinylbenzene copolymer resin as a carrier to give N-tert-butoxycarbonyl-O-benzylserine resin. The amino group of the resultant resin is then eliminated, and amino acids having protected amino groups are condensed with the resin according to the amino acid sequence of the aimed peptide in the presence of a condensing agent. The operation to eliminate protecting groups is repeated successively to afford a peptide resin, which is then treated with hydrogen fluoride in the presence of a solvent to separate the peptide from the carrier and give the aimed peptide expressed by formulas I -VII.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel peptide, and more specifically, a novel peptide and a novel peptide useful for the analysis of hepatitis B virus antigens, and also useful as antigen compositions for hepatitis B vaccines, etc. It relates to pharmaceutically acceptable salts thereof.

The novel peptides of this invention have the following formulas [I] to [■
] is indicated.

[I] [+1] [ml [■ko[V] H-A la -P r o-T h r-Cys-
P r o -G Iy-G In-Asn-9e r
-G In-9e r-Pro-Thr-Se r
-Asn-Hi 5-Ser-OHH-Ala-Pr
o-Dinghr-Cys-Pro-Gly-Gin-Asn
-Ser-GIn-9er-Pro-Thr-Ser-
Asn-His-Ser-OH[■co[■] (However, in the above formula, R4 means hydrogen or a mercapto protecting group) In this specification, regarding amino acids, protecting groups, active groups, solvents, etc., IUPAC-IUB co
Missionon Biological Nom
It may be indicated by an abbreviation based on "enclature" or a common abbreviation in the field, examples of which are as follows. Met: Methionine, Gysnidistine, ) Iis: Histidine, Pro/Proline, I
le: isoleucine, Asp: aspartic acid, Asn
: Asparagine, Thr: Threonine, Ala: Alanine, Gln: Glutamine, Glu: Glutamine,
Gly nigericin, Lys: lysine, His: histidine, Ser: serine, Boast-butoxycarbonyl, cl-z: 2-chlorohensyloxycarbonyl,
Bzl: pencil, Acm: acetamidomethyl, To
s-P-1 luenesulfonyl, 0-cHex: cyclohexyl ester.

Compounds [I] to [■] of this invention and pharmaceutically acceptable salts thereof can be produced by various methods, and these methods will be explained below.

(1) Method-1 (Peptide synthesis by solid phase method): 1) Method 1-1 R'-9er (R3)-resin [Ial"2) Method 1-2 R'-C:ys(Ra')- Resin [11a] - [11
b) Method 1-3 R'-5et (R3)-resin [Ial-resin (m'b) (2) Method 2 (protecting group removal method): l) Method 2-1 2) Method 2 -2 [Hb] → 3) Method? -3 [Ill bl → (3) Method 3 (elimination of mercapto M-retaining group and cyclization reaction).

l) Method 3-1 [IV] 2) Method 3-2 [Vl] and (or) [■] 3) Method 3-3 [V] (blank below) In the above formula, R1 is an amino protecting group, and R2 is Carboxy protection i
ix, R3 means a hydroxy protecting group, and Ra4 means a mercapto protecting group. These definitions are explained below.

(1) About the amine/protecting group in R Diamino protecting groups include common amino protecting groups commonly used in the fields of amino acid and peptide chemistry, and preferred examples of such amino protecting groups include alkoxycarbonyl or cycloalkoxycarbonyl (e.g. t-butoxycarbonyl, t-pentoxycarbonyl, cyclohexylcarbonyl, etc.), substituted or unsubstituted phenyl lower alkoxycarbonyl (e.g. benzyloxycarbonyl, 2
-chlorobenzyloxycarbonyl, etc.), substituted or unsubstituted allenesulfonyl (eg, benzenesulfonyl, p-)luenesulfonyl, etc.), and the like.

(2) Carboxy protecting group in R2: Carboxy protecting groups include common carboxy protecting groups commonly used in the fields of amino acid and peptide chemistry, and preferred examples of such protecting groups include lower alkyl (e.g. alkyl such as methyl, ethyl, etc.), cycloalkyl (e.g. cyclopentyl, cyclohexyl, etc.), mono- or diphenyl lower alkyl (e.g. benzyl,
Examples include aralkyl such as diphenylmethyl, etc., and aroylalkyl (eg, phenacyl, toluoyl ethyl, etc.).

(3) Regarding the hydroxy protecting group in R8: Hydroxy protecting groups include common hydroxy protecting groups commonly used in the fields of amino acid and peptide chemistry, and suitable examples of such hydroxy protecting groups include, for example, alkanoyl ( Examples include acyl such as acetyl, substituted or unsubstituted aralkyl (such as benzyl, 2,6-dichlorobenzyl, etc.), and the like.

(4) Regarding mercapto protecting groups in R4 and Ra': Preferred examples of mercapto protecting groups include protecting groups that do not leave off under the elimination conditions of carboxy protecting groups, amine protecting groups and hydroxy protecting groups; Preferred examples of protective groups include acylaminoalkyl (e.g., acetamidomethyl, benzamidomethyl, etc.), arylmercaptan (e.g., 3-nitropyridine-2-thiol, etc.), and substituted or unsubstituted lower alkyl (e.g., tert-butyl), substituted or unsubstituted aralkyl (e.g. benzyl, P-methoxybenzyl, P-
methylbenzyl, 3,4-dimethylbenzyl, diphenylbenzyl, trityl, etc.), substituted or unsubstituted lower alkyl mercapto (e.g. ethyl mercapto, t-bunal mercapto, etc.), etc., by appropriately selecting the removal conditions for each protecting group. It can be used by (5) Regarding pharmaceutically acceptable salts: Pharmaceutically acceptable salts in compounds [I] to [■] include, for example, alkali metal salts (
Salts with inorganic or organic bases, such as sodium salts, potassium salts, alkaline earth metal salts (e.g. calcium salts), ammonium salts, organic amine salts (e.g. ethanolamine salts, triethylamine salts, dicyclohexylamine salts, etc.) and addition salts of organic or inorganic acids such as trifluoroacetic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid. The above various methods will be explained below. (1) Regarding method 1: This method is based on the compound [Ia
] and [IIa], each protected constituent amino acid was coupled sequentially to the compounds [Ib], [IIb] and [mb
].

Compounds [Ia] and [
IIa] respectively include known substances (for example, Biopolymer Vol. 12, p. 2513, 1873) and new substances, and the new substances can be produced in the same manner as the method described in the literature. In addition, as for the resin to be used, if it is a resin used in peptide synthesis by solid phase method,
Any can be used; examples of such resins include, for example, chloromethylated styrene-divinylbenzene copolymer, hydroxymethylated styrene-divinylbenzene copolymer, and aminomethylated styrene. -Polystyrene type resins such as divinylbenzene copolymers, polyamide type resins such as polydimethylacrylamide resins, and the like.

This method is generally carried out as one cycle of steps 1 to 11 as follows for each protected constituent amino acid.

1) Step 1: This step is carried out to wash and swell the starting material, the protected amino buty-resin, and a suitable example of the solvent used for this purpose is methylene chloride. , chloroform, dimethylformamide, or a mixed solvent thereof.

2) Step 2: This step is a step of removing the amine protecting group in the protected amino acid-resin, and the reaction in this step is carried out by a conventional method such as a method using an acid or a method using a base. be exposed.

Among the above desorption methods, the method using an acid is most often used, so the acid method will be explained below.

This reaction is carried out in a solvent such as methylene chloride, chloroform, acetic acid, or water in the presence of an inorganic or organic acid such as trifluoroacetic acid, @acid, p-)luenesulfonic acid, hydrochloric acid, or hydrobromic acid, preferably ethane. This is done by adding dithionyl or anisole.

Among the acids exemplified above, trifluoroacetic acid and vi acid are also used as solvents.

This reaction is usually carried out under cooling (eg -78°C) to room temperature.

3) Step 3: This step is carried out to remove impurities and swell the resin, and is carried out in substantially the same manner as Step 1 above.

4) Step 4: This step is performed to shrink the resin and increase the cleaning effect, and the amino acid-resin is treated with alcohol (methanol, ethanol, propatool, 2-propanol, butanol, etc.), dioxane, etc. Preferably, it is treated.

5) Step 5: This step is carried out to remove impurities and swell the resin, and is carried out in substantially the same manner as in Step 1 above.

6) Step 6: This step is a step carried out for desalting when the amino acid in the amino acid-resin obtained by the treatments in Steps 1 to 5 above exists as an acid addition salt at the α-amino group. This is carried out, for example, by treatment with a base such as triethylamine.

7) Step 7: This step is carried out to remove impurities and swell the resin, and is carried out in substantially the same manner as Step 1 above.

8) Step 8: This step involves coupling each protected constituent amino acid and is carried out in a solvent such as methylene chloride, chloroform, dimethylformamide in the presence of a conventional condensing agent such as dicyclohexylcarbodiimide. It is also possible to convert the carboxy group of each protected amino acid into
It is also possible to activate it into an acid anhydride, active ester, etc. by a conventional method, and then carry out the reaction in the above-mentioned solvent.

8) Step 9: This step is carried out to remove impurities and swell the resin, and is carried out in substantially the same manner as Step 1 above.

10) Step 10: This step is carried out to shrink the resin and improve the cleaning effect, and is carried out by substantially the same method as step 4) above.

11) Step 11: This step is carried out to wash and swell the resin, and is carried out in substantially the same manner as Step 1 above.

The marking process is generally carried out at room temperature.

It is preferable to repeat each step (except step 8) about 2 to 3 times.

(2) Regarding method 2: This method is applicable to compounds [I b] , [II b] and [
mb] is subjected to an elimination reaction of an amine protecting group, a carboxy protecting group and a hydroxy protecting group to obtain compounds [Ic], [lIc1 and [mc], respectively.

The method for removing the amine protecting group is carried out by substantially the same method as Step 2 of the above method (I), catalytic reduction method, liquid ammonia-alkali metal method, zinc acid method, hydrazine method, etc. The protecting group and hydroxy protecting group are removed by conventional methods such as hydrolysis and reduction.

1) Hydrolysis: Hydrolysis is preferably carried out in the presence of an acid or a base.

Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid,
Sulfuric acid, etc.) organic acids (eg, formic acid, acetic acid, trifluoroacetic acid, propionic acid, penzenesulfonic acid, P-)luenesulfonic acid, etc.), acidic ion exchange resins, and the like.

Suitable bases also include alkali or alkaline earth metal hydroxides, carbonates or bicarbonates, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, sodium bicarbonate, calcium hydroxide. , magnesium hydroxide, etc.), and inorganic bases such as ammonium hydroxide.

Hydrolysis is carried out under relatively mild conditions such as cooling or heating, and using solvents that do not adversely affect the reaction [such as water, alcohols (such as methanol, ethanol, propatool, etc.)]. ! The process is carried out in an aqueous solution, acetone, N,N-dimethylformamide, tetrahydrofuran, dioxane, dimethyl sulfoxide, etc., or a mixed solvent thereof.

2) Reduction: Reduction methods including chemical reduction and catalytic reduction methods are carried out by conventional methods.

Preferred reducing agents used in chemical reduction include, for example, metals (e.g. tin, zinc, iron, etc.) or metal compounds (e.g. chromium chloride, chromium acetate, etc.) and organic or inorganic acids (e.g. formic acid, acetic acid, propionic acid, trichloride, etc.). Fluoroacetic acid, p
-toluenesulfonic acid, hydrochloric acid, hydrobromic acid, etc.).

Preferred catalysts used for catalytic reduction include platinum catalysts (e.g. platinum plate, platinum sponge, platinum black, platinum colloid, platinum oxide, platinum wire, etc.), palladium catalysts (palladium sponge, palladium black, palladium oxide, palladium carbon, etc.). , palladium colloid, palladium-barium sulfate, palladium-barium carbonate, etc.), nickel catalyst (reduced nickel, nickel oxide, Raney nickel, etc.), cobalt catalyst (e.g. reduced cobalt, Raney cobalt, etc.),
Examples include iron catalysts (for example, reduced iron, Raney iron, etc.), copper catalysts (for example, reduced copper, Raney copper, Ullmann copper, etc.). Reduction is usually carried out in a solvent. Preferred solvents include:
For example, water, alcohol (e.g. methanol, ethanol,
propatool, etc.), acetic acid and other common organic solvents or mixtures thereof. Furthermore, the above-mentioned liquid acid used in the chemical reduction can also be used as a solvent. In addition to the above-mentioned solvents, preferred solvents used in the catalytic reduction include diethyl ether, dioxane, tetrahydrofuran, etc., or mixtures thereof.

The reaction is rapidly carried out under relatively mild conditions such as cooling or heating.

Depending on the type of amine protecting group, carboxy protecting group, and hydroxy protecting group, they may be eliminated at the same time by the above elimination reaction. acid,
Examples include a method of treatment with an acid such as trifluoromethanesulfonic acid, and this reaction can be carried out in the presence or absence of a conventional solvent that does not affect these acids.

In this method, good results are often obtained when the reaction is carried out in the presence of methionine, anisole, etc.

Further, the above acid can also be used as a solvent. This reaction is usually preferably carried out at a temperature of around 0°C.

(3) Regarding method 3: This method uses compounds [Ic], [IIc] and [m
c] is subjected to a mercapto-protecting group elimination reaction to produce a compound from which the corresponding mercapto-protecting group has been eliminated and S- in the molecule.
5 bonds and/or intermolecular S-5 bonds (i.e. compounds [I d] , [II dl
.

[mdl, [IV], [V], [VI] and [
■]).

As a method for obtaining compounds with these intramolecular and/or intermolecular S-5 bonds, compounds having a free mercapto group (i.e., compounds [I'd], [IIdl
, l'l jump [mdl] and there are methods that do not go through it. These will be explained below.

■) Method via a compound having free mercapto x: 1)-1 (Compound [Ic] → Compound [Idco, Compound [11cl → Compound [11d1. Compound [mc] → Compound [md]): This method is the compound [Icl, [11c or [m
c] metal salts such as mercury acetate, mercury trifluoroacetate, and silver acetate, organic phosphorus compounds such as triphenylphosphine and tributylphosphine, mercaptoethanol, mercaptoacetate, ethanedithiol, and thiophenol.

By treatment with reagents such as alkyl or arylthiols such as dithiothreitol and dithioerythritol, the corresponding compounds [Idl, compounds [11
d] or compound [md].

This reaction is usually carried out in a solvent, and preferred solvents include, for example, water, alcohol (eg, methanol, ethanol, propatool, etc.), dioxane, dimethylformamide, and mixed solvents thereof.

This reaction is rapidly carried out under relatively mild conditions such as cooling to heating.

1)-2 (Compound [I dl→ Compound [VI] and (
or) [■], compound [11d] → compound [■
, compound [mdl→compound [VI): This method involves treating compound [Idl, compound [IId] or compound [111d] under relatively mild oxidative conditions.
Compound [VI] and/or compound [■
], compound [IV] corresponding to compound [11d], compound [md
], respectively, to obtain the compound [VI].

Preferred examples of the oxidizing agent used here include oxygen in the air, potassium ferricyanide, and the like.

2) Method not via a compound having a free mercapto group: (Compound [Icl→Compound [VI] and/or)
[VI[], Compound [IIc] → Compound [■], Compound [mc] → Compound [VI): This method
By treating cl, compound [IIC] or compound [mc] with an electronic reagent such as iodine or dithiocyanogen, compound [VI] and/or compound [■] corresponding to compound [Icl] or compound [ IIc] corresponding to the compound [I
This is a method to directly obtain compound [VI] corresponding to compound [mc].

This reaction is usually carried out in a solvent, and examples of preferred solvents include water, alcohol (eg, methanol, ethanol, propatool, etc.), dioxane, dimethylformamide, and the like.

Further, this reaction is carried out under relatively mild conditions such as cooling to heating.

Compounds [I] to [■] of this invention include one or more isomers due to asymmetric carbon atoms in the molecule, and all such isomers are included within the scope of this invention.

Peptides of this invention, that is, compounds [I] to [■]
and pharmaceutically acceptable salts thereof are useful for the analysis of hepatitis B virus antigens, as mentioned above, and are also useful as antigen compositions for hepatitis B vaccines, and furthermore, these compounds are useful for preventing infection. It also has activity and is useful as an antibacterial agent.

Test examples showing the infection-preventing effect of the peptides [I] to [■] of the present invention will be shown below.

Test example (protective effect against bactericidal infection) (a) Method: -Test animals: ICR mice, male, 4 withdrawals, 10/group -Method of drug administration: 4 days before infection, each mouse was given a predetermined concentration of the present invention. Peptide (aqueous solution) was administered intraperitoneally.

- Infecting bacteria and infection method = E. coli No. cultured at 37°C for 20 hours.

22 (concentration: 2.OX 108 cfu/ml) was suspended in sterile physiological saline, and 0.2 ml of this was intraperitoneally infected. The survival of mice was observed for 4 days after infection, and the survival rate (%) was calculated.

(B) Results: The pharmaceutical compositions of this invention can be used in the form of preparations, for example in solid, semi-solid or liquid form, which contain the active ingredients of this invention for external, oral or parenteral administration. Contained in admixture with suitable organic or inorganic carriers or excipients. The active ingredient can be, for example, tablets, pellets,
They can be formulated with conventional non-toxic pharmaceutically acceptable carriers for capsules, halves, solutions, emulsions, suspensions, or any other dosage form suitable for use. Possible carriers are water,
Glucose, lactose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate,
Talc, corn starch, keratin, finely divided (colloidal) silica, potato starch, urea and other carriers suitable for preparing solid, semi-solid or liquid form preparations. In addition to the carrier, auxiliary agents, stabilizers, thickeners and coloring agents, fragrances, and the like may be used.

The pharmaceutical composition may also contain protective or bacteriostatic agents for the purpose of preserving the activity of the active ingredient. The active ingredient compound is present in the pharmaceutical composition in an amount sufficient to produce the desired therapeutic effect on the treatment or medical condition.

When administering this composition to humans, it is preferably administered intravenously, intramuscularly or orally.

The dosage or therapeutically effective amount of the active compounds of this invention will vary depending on the type of compound and the age and condition of each patient being treated, but will generally range from about 0.1 to 1000 mg per kg of animal body weight per day. Administer the ingredients for therapeutic purposes. The average dose per dose is approximately 50mg.
, 100mg, 250+ng and 500mg are used.

Next, examples of the present invention will be shown, and each amino acid in each example refers to the L-configuration.

Example 1-1 N-t-butoxycarbonyl-〇-benzylserine resin (2.15 g) [Serine content: 0.82 mmol/
The reaction order of the amino acids used in the reaction was Set, Pro, IIs.

Pro, Ile, l:ys, Thr, Cy
s, Asn, Gly, Asp, Set, P
ro, Lys.

A peptide having 16 amino acid residues was synthesized by repeating the schedule shown in the table for 15 cycles. Note that the α-amino group of these amino acids is t
-butoxycarbonyl group, and other side chain functional groups are protected by Set and Thr by benzyl group, Cys by acetamidomethyls (, Asp by cyclohexyl ester, and Lys by 2-chlorobenzyloxycarbonyl group). I kept it.

That is, starting materials (
Alternatively, the peptide chain can be extended by sequentially reacting the solvents and reagents listed in the same table with an intermediate substance obtained by extending the peptide chain by coupling an amino acid to the N-terminal amine group of the starting material. It was. The eighth step (coupling reaction) in the same procedure was generally carried out in a methylene chloride solution, but only when reacting Cys protected with an S-acetamidomethyl group, methylene chloride and dimethylformamide were used. (2:1)
A mixed solution was used. In addition, in the Asn coupling reaction, N-t-butoxycarbonyl asparagine is
Hydroxybenzotriazole (1:1 equivalent) and dicyclohexylcarbodiimide (1 equivalent) were added to a dimethylformamide-methylene chloride (1:1) mixture and heated to 0°C.
The mixture was activated by stirring for 10 minutes and used in the 8th step. Introducing the seventh and subsequent amino acids (
After the completion of the first reaction using Cys),
The second step shown in the table was carried out by adding 5% by volume of ethanedithiol to the methylene chloride-trifluoroacetic acid mixture. In addition, when introducing the 12th and subsequent amino spirits after the coupling reaction of one Asp, the neutralization in the sixth step is performed using a water-cooled solution of triethylamine, and the treatment time is also 1. Shortened to 5 minutes.

The completion of each coupling reaction was determined by Kaiser's ninhydrin test. When it was detected that the coupling was insufficient, steps 8.9, 10.11 and 1 in the table were repeated in the order listed.

In the experiment, Cys (6th), Gln, Asp
, Set were subjected to the above-mentioned repeating process. After coupling the last Thr, the resulting resinous material was washed with methanol and further dried under reduced pressure, resulting in a peptide resin (1-1) (5, 438g).

Boc-Thr(Bzl)-Lys(CI-z)-Pr
o-9et(Bzl)-As p (0)1eri-G
Iy-Asn-Cys(Acm)-Thr(Bz I
)-Cys (Acm)-I 1e-Pro-11e-
Pro-Set (Bzl)-5et (Bzl)-Resin Example 1-2 The peptide resin (1-1) (2,67 g) obtained above was treated with hydrogen fluoride ( 50m
The mixture was stirred at −6 to −5° C. for 1 hour in 1). Thereafter, hydrogen fluoride was distilled off under reduced pressure at the same temperature for 2 hours and at room temperature for 1 hour. IM vinegar (100 ml) and ether (50+nl) were added to the residue, and the mixture was stirred for 1 hour while cooling with water. The resin was filtered off and washed with 1M acetic acid. The filtrate and washing liquid were combined, the ether layer was separated, and the aqueous layer was passed through a DOWEX 1×2 (acetic acid form, 20'0 ml) column and freeze-dried to obtain crude H-Thr-Lys-Pro-3et-Asp. −
Gly-Asn-Cys(Acm)-Thr-Cys(
Acm)-11e-Pro-11e-Pro-Ser-
5er-OH (1,242 g) was obtained [Starting material C
Yield 81.5% in terms of I)].

This crude product (1.14 g) was added to carboxymethyl cellulose [War/l・77eM-52, 2.8×5Bcm
, averaged with 0.1M pyridine-acetic acid buffer (p) 15.4)
The same buffer solution (each with 10
Elution at 0100O, fractions 240-264n+l were collected and lyophilized to yield purified product (447+ig). From the next fraction (265-312ml), the purity level is slightly lower, but it can be considered a purified product (495mg)
was gotten.

The above purified product (43 mg) was added to Sephadex G-15 (
3.2X 80cm: equilibrated with 1% acetic acid)
Desalting was carried out by passing it through a column. Elute with 1% vinegar and butter, 1
Fractions from 44 to 208 ml were collected and lyophilized to obtain desalted target substance (1-2: 38 mg).

(1) Acid hydrolyzate (110'C with 6N hydrochloric acid, 2
Amino acid analysis of 4-hour processed product: Thr: 0.82X
2 Lys:1.00Pro+1.0? X 3 As
p:1.03X 2Gly:1. +3 11e:1. O
IX 2Ser: 1.02X 3 (2) Thin layer chromatography Rf = 0.40 [cellulose or Merck No. 5552, n-butanol-
Acetic acid-water-pyridine (15:3+12:10) detected by ninhydrin] (3) High performance liquid chromatography Retention time = 5.8 minutes Column: Nucleosyl 5CIB (150X 4mm) Eluent: 0.1M phosphoric acid-phosphoric acid at pH 4,8 84:16 mixture flow rate of secondary potassium buffer and acetonitrile: 1. Oml 7 minutes detection: UV210nm Example 2-1 Nt-butoxycarbonyl-5-acetamidomethylcystine resin (4,02g Nidistine content:
0.48 mmol/g) as a starting material, a peptide bond-forming reaction was carried out according to Example 1-1.

However, the reaction order of amino acids is Set, Pro, Ph
e, Met.

Ssr, Thr, Gly, Gin, Ala,
Pro j Ie, Thr, Cys, Thr, L
It was ys. The liquid mixture used in the second step in the table contained 5% by weight of ethanediol.

Further, in the coupling reaction of the eighth step, the reaction of S-acetamide Cys was carried out in a methylene chloride solution containing 30% by volume of dimethylformamide. The coupling of glutamine was carried out in accordance with the asparagine coupling of Example 1-1. It should be noted that the repetition of steps 8.9, 10.11, and 1 was necessary due to insufficient coupling when the 1st Set and 10th Pro were subjected to the coupling reaction. The last Ly
At the stage where the coupling of s was completed, the obtained resinous material was treated in the same manner as in Example 1-1, and a peptide resin (2-1) (8.32 g) represented by the following formula was obtained. .

Boc-Lys(CI-z)-Thr(Bzl)-Cy
s(Aca+)-Thr(Bzl)-11e-Pro-
Ala-Gln-Gly-Thr(Bzl)-3er(
Bzl)-Met-Phe-Pro-9et(Bzl)
-Cys(Acm)-Resin Example 2-2 The peptide resin (3.0 g) obtained above was treated with 50% triple oloacetic acid containing 5% ethanedithiol for 30 minutes in a solid-phase reaction vessel, and then chlorinated. Methylene (3 minutes x 3
), 2-propanol (3 minutes x 2), methylene chloride (3
minutes x 3), 5% triethylamine in methylene chloride (
3 minutes x 2) and methylene chloride (3 minutes x 3). After washing this resin with methanol, it was dried over phosphorus pentachloride under reduced pressure. The product from which the protecting group was partially removed by the above treatment was combined with anisole (4.5ml) in Example 1.
The mixture was stirred at 0°C for 1 hour in hydrogen fluoride (45 ml) in the same manner as in -2. Hydrogen fluoride was distilled off under reduced pressure at the same temperature for 1 hour and 40 minutes, and 1M acetic acid (100 ml) and ether (40 ml) were added to the residue. After freeze-drying in the same manner as in Example 1-2, crude H-Lys-Thr-Cys
(Acm)-Thr-11e-Pro-Ala-Gln
-Gly-Thr-9er-Met-Phe-Pro-
9er-Cys(Acm)-0H (1,02 f(g)) was obtained.

This crude product (100 mg) was added to carboxymethyl cellulose [Whatman CM-52, 2.4 x 32 cm, 0
．． 1 equilibrated with pyridine-acetate buffer (pH 5,4)] and eluted with the same buffer (300 ml each) whose concentration was changed linearly from 0.1 M to 0.4 M. The ~2681 fractions were collected and lyophilized to yield the purified product (31 mg). Next, this material was coated with Sephadex L H-20 (3.2 x 80 cm:
The mixture was passed through a column (equilibrated with 1% acetic acid) and eluted with the same solvent. The fractions of 144 to 168 ml were collected and lyophilized to obtain the purified target substance (2-2: 24.9 ml
g) was obtained.

(1) Acid hydrolyzate (110'C12 with 6N hydrochloric acid)
Amino acid analysis of 4-hour processed product): Thr: 0.82X3 Lys: 1.00Pro:
1.12X2Ala: 1.19Gly: 1.0
2 11e: 0.98Glu: 1.16 Met:
1.04Ser: 0.92X2 Phe: 1.0
0(2) Thin layer four-layered graphography Rf=0.59 [Cellulose Merck Nn5552, n-butanol-vinegar-
Water-Pyridine (15:3:12:10) Ninhydrin Detection Column (3) High Performance Liquid Chromadog 2 Filter Retention Time: 4.8 minutes Column: Nucleoge #10 CIs (250x4
mm) Eluate: 81:19 mixture of 0.1 M phosphoric acid-potassium phosphate buffer at pH 4,8 and acetonitrile Flow rate: 1.5 ml/G Detection: UV 210 nm (4) Elemental analysis: C2eHusN2o02sSs ・
9HtOCH,C00H Calculated value C46,16H6,60N 13.85 Experimental value C46,21H6,81N 13.82 Example 3-1 N-t-butoxycarbonyl-O-benzylserine resin (z, 5g: Serine content: 0 Using .82 mmol/g) as a starting material, a peptide bond forming reaction was performed according to Example 1-1 and Example 2-1, and the reaction was repeated for 16 cycles).
By recycling, a peptide consisting of 17 amino acids was produced. The reaction order of amino acids is His+Asnt
Ser+Thr+Pro, Ser+Gin, Ser,
AsntG1n+Gly+Pro +Cys +Thr
+Pro +A1a, protection of α-amine group and side chain functional group was carried out in accordance with Example 1-1. However, His is p-
) protected with a luenesulfonyl group.

Whether the coupling reaction is completed or not is determined by Kaiser et al.'s ninhydrin test. Each step of the process was repeated. After the last Ala coupling reaction, the product was dissolved in methanol (50ml
) and drying, the peptide resin (
3-1) (aasg) could be obtained.

Boc-Ala-Pro-Thr (Bz 1 )-
Cys (Acm)-Pro-Gly-Gin-As
n-5er(Bzl)-Gin-5er(Bzl)-P
ro-Thr(Bzl)-5er(Bzl)-Asn
-His(Tos)-5er(Bzl)-Resin Example 3-2 The peptide resin (3.0 g) obtained above was mixed with anisole (3.0 g).
hydrogen fluoride (4 ml) for 1 hour at 0°C.
0 ml). After the reaction was completed, the peptide from which the protecting groups (except for the S-acetamidomethyl group) had been removed was extracted with IN acetic acid (50 ml). The extract was diluted with ethyl acetate (20
ml), then DOWEX 1×2 (acetic acid form, 3 ml) was washed.
00ml) column and eluted with IN acetic acid. Collect the fractions (300 ml) of the ninhydrin test (positive),
After concentrating to about 50ml and freeze-drying, 1.43g of the crude peptide of the following formula (N-t-butoxycarbonyl-〇-
A yield of 83.5% calculated from benzylserine resin was obtained.

H-Ala-Pro-Thr-Cys(Acm)-Pr
o-Gly-Gin-Asn-Ser-Gin-5er
-Pro-Thr-8er-Asn-His -8e
r -OH crystal (aoomg) is converted into carboxymethyl cellulose [
Whatman CH-52, 2, 3x32cm+0.05
It was passed through a column equilibrated with M acetate-pyridine buffer (pH 4,8) and eluted with the same buffer.

Collect fractions of 6.5 g each and collect Folin qv-(Fo
lin-Lowry) method at 9750 nm.

Fractions showing peaks (positive numbers 22 to 36) were collected and lyophilized. The residue was dissolved in 1% acetic acid (3 ml) and passed through Sephadex G-15 (3 x 63 cm, equilibrated with 1% acetic acid) to collect fractions of 6.5 g each. 23
The fraction with a peak UV absorption at 0 nm (N12
5-34) are collected and freeze-dried, the purified product (zs2m
g) was obtained.

(1) Amino acid analysis of hydrolyzate with 6N hydrochloric acid: A
1 a: 1.0 G 1 y: 1.02 (1)
His: 0.86 (1) Asp: 1.9
5 (2) G 1 u : 1.87 (2) Th r
: 1.28 (2) P r o : 2.80 (3)
Ser: 3.39 (4) (2) Amino acid analysis of AP-M digest: G1y: 1. OHis:
1.22 (1) A 1 a: 0.74 (11As
p: 1.83 (2) G 1 u: 1.75 (
2) Thr: 1.60 (2) Pro: 2.78
(3) Ser: 3.2 (4) (3) Thin layer chromatography Rf = 0.34 [Seru p-su, n-butanol (2): acetic acid (2): water (2): pyridine (1) (4) High performance liquid chromatography retention time: 23.9 minutes Column: RP-18 (4X250mm) Eluent: 0.1
M phosphoric acid - dibasic potassium phosphate (1) H2,7) (10:
1. Volume ratio) Flow rate: 1 ml/min Detection: UV 210 nm Example 4 H-Ala-Pro-Thr-Cy obtained in Example 3-2
s(Acm)-Pro-Gly-Gin-Asn-5e
r-Gin-8er-Pro-Thr-8er-As
Iodine (74.9 mg) in acetic acid (8,
5ml 1) solution was added. After stirring the reaction solution at room temperature for 1 hour, 0.5M sodium thiosulfate is added to decompose excess iodine. The solution was evaporated to dryness, the residue was dissolved in 1% acetic acid (3 ml), and then Sephadex LH-20
(3 x 60 cm: equilibrated with monoacetic acid), 4 ml fractions were collected and measured by UV absorption (230 nm). The fractions that showed a peak (Nn34-39) were collected and lyophilized to obtain (40 mg).

(1) Amino acid analysis of hydrolyzate with 6N hydrochloric acid: Gl
y:1. OHis: 1.22 Ala: 0.74 (1) Asp: 1.83 (2) Gl
u:1.75f2) Thr:1.60(2)P r
o: 2.78 (3) Ser: 3.20
(4) (2) High performance liquid chromatography Retention time: 5.2 minutes Column: RP-18 (4X250) Eluent: 0.1M phosphoric acid - dipotassium phosphate (1) H2,
7) Niacetonitrile (9:1, volume ratio) Rate: 0.5 ml/G Detection: UV210 nm Example 5 H-Lys-Thr-Cys (Ac
m)-Thr-Ile-Pro-At a-Gin-G
ly-Thr-9er-Met-Phe-Pro-
8er-Cys(Acm)-0H (160mg) at 80%
It was dissolved in acetic acid (xc+2ml), and to this solution was added a solution of 85% iodine (227mg) in acetic acid (32ml) at room temperature.

The reaction mixture was stirred at the same temperature for 1 hour, and a dilute aqueous solution of sodium thiosulfate was added until the brown color disappeared. The reaction mixture was concentrated and the residue was filtered onto a Sephadex G-15 column (
3.2 x 65 cm) and eluted with 1% acetic acid.

When the eluate is lyophilized, the peptide (i
42 mg) of crude product was obtained.

This crude product (140 mg) was added to Sephadex G-25 (
Fine: 2.6 x 44 cm) column to adsorb it, and
-Butano- was purified by fractional chromatography using a mixed solvent system consisting of 0.6 M ammonium acetate solution in 0.1% acetic acid - pyridine (5:1 to 3). 7g of each fraction was collected using the Foley-Lowry method (wavelength 750).
nm). The main fractions (fractions 19 to 24) were collected and adsorbed onto a column of Sephadex G-15 (2.8 x 55 cm), and eluted with 1% acetic acid.
0.3 mg) was obtained.

(1) Acid hydrolyzate (110°C with 6N hydrochloric acid.

Amino acid analysis of 24-hour treated product): Lys: 0.87 Thr: 1.0OX3Pro: 1.
0OX2Ala:1.00Gly:0.96 Ser
:0.83X2Phe:1.10 Cys 20.35
I 1 e: 1.03 G 1 u: 1.23
Met: 1.04 (2) Enzyme decomposition product (1.75 mg of substrate dissolved in 300 ml of 1M ammonium bicarbonate aqueous solution, added with 1.2 units of aminopeptidase and treated at 37°C for 24 hours) Amino acid analysis: Lys: 0.77 Thr + Gin: 0.92X4
11e:1.05 Pro:0.78X2Gly:Q,
98 Ser: 0.95X2Phe: 1.06 Cy
s: 0.84 AJa: 1.00 Met: 1.25 (3) [α] D = -94,67° (C = 0.477
, H2O) (4) Thin layer chromatography Rf=0
．． 64 [Cellulose Merck tm5552. n-Butanol-acetic acid-water-pyridine (15:3:12:10) Detection using ninhydrin (5) High performance liquid chromatography Retention time 2 6.2 minutes Column: Nucleosil 10C+a (250 x 4 mm)
Eluent: 0.1 M phosphoric acid-dibasic potassium phosphate buffer niacetonitrile (80:20).

pH 4,8 Flow rate: 1. Oml/min Detection: UV210nm (4) Elemental analysis: c? OH+tzN+aOzsS1
・10H20・CH,C0OH Calculated value C45,27H7,18N 13.20 Experimental value C44,71H6,27N 13.13 Example 6 The same crude raw material peptide (7,34
mg: 4.5 fimol) was dissolved in water (6 ml),
The pH was adjusted to 4 by adding monothiacetic acid. Silver acetate (284m
A solution of g) in water (2.8 ml) was added and stirred at room temperature for 1 hour and 45 minutes. Next, hydrogen sulfide gas was blown into this solution (for 15 minutes), and nitrogen gas was further blown into the solution for 10 minutes to purge the hydrogen sulfide. The reaction solution was filtered through a pad of cellulose powder, and a portion (o
, 2 oml) and 0.1M phosphate buffer (pH 8.0).
) to dilute to 5 ml, and then add 0.1 M of 5,5'-dithiobis(2-nitrobenzoic acid): Ellman's reagent.
Two drops of phosphate buffer (pH 7,0) solution were added and left to stand for 1 hour. The absorbance at 412 nm was 0.39, which means that the intermediate peptide from which the SH protecting group of cysteine was removed was in the reaction mixture at 3,42 μmol (theoretical yield: 84.4
%) indicates that it exists.

After adding dilute ammonium water to the solution to adjust the pH to 8,
Stirred at room temperature for 24 hours. The reaction mixture was concentrated and adsorbed onto 2 μm of Sephadex G-15 (14×35 cm), eluted with 0.1% acetic acid, and lyophilized to obtain the crude peptide. It was confirmed by high performance liquid chromatography that it was the same as the compound obtained in Example 5.

Example 7 1H-Thr-Lys-Pro-3e obtained in Example 1-2
r-Asp-Gly-Asn-Cys (Acm)
-Thr-Cys (Acm)-I 1 e-Pro
-11e-Pro-8er-5er-OH (200mg
+ e), 114 mol) was dissolved in water (100 ml), and the pH was adjusted to 4 by adding 0.1% acetic acid (20 ml). Add a solution of silver acetate (80 mg) in water (60 ml),
The reaction mixture was stirred at room temperature for 2 hours. Next, hydrogen sulfide gas was blown in for 15 minutes, and then nitrogen gas was blown in for 30 minutes to purge excess hydrogen sulfide. The reaction mixture was filtered through a pad of cellulose powder, and the filtrate and washings were combined.
Diluted to 50ml. Diluted anthonia water was added to this to adjust the pH to 8.

A portion (o, 2 oml) was diluted with 5 ml of 0.1M phosphate buffer (pH 8,0), and further diluted with 5,5'-dithiobi,',[:2-=)benzoin M (Ellman's Ei
0.1 M phosphate buffer (pH 7,0)
Two drops of the solution were added and left for 1 hour. The absorbance at 412 nm was 0.226, which indicates that 0.0937 mmole of the peptide represented by the following formula (in which Cys is free) was present in the reaction mixture.

H-Thr-Lys-Pro-5er-Asp-Gly
-Asn-Cys-Thr-Cys-IIe-Pro-
8er-8er-OH The reaction mixture was then left at room temperature for 3 days and further concentrated. Sephadex G-1
The crude peptide was obtained by adsorption onto a column of No. 5 (3.2×65 cm), elution with 1% acetic acid, and lyophilization.

Quartz is a mixture of isomers represented by the formula:

and (1) Thin layer chromatography Rf = 0.30 and 0.12 [Cellulose plate/Merck N15552, detected by n-butanol-acetic acid-water-pyridine (3:1:2:1) ninhydrin] (2) High performance liquid Chromatography retention time: 5.2 minutes and 6.8 minutes Column: Nucleosil 10C+a (250X4mm
) Eluent: 0.1% trifluoroacetic acid-acetonitrile (75:25) Flow rate: tomt/min Detection: UV 210 nm Applicant: Japan Institute of Chemotherapy and Serum Therapy Fujisawa Pharmaceutical Co., Ltd.

Claims (1)

[Claims] H-Ala-Pro-Dhr-Cys-Pro-GIy
-Gln-Asn-9er-Gjn-Set-Pro-
Thr-9er-Asn-His-Ser-0)1H-
Ala-Pro-Thr-Cys-Pro-Gly-G
In-Asn-9er-GIn-9er-Pro-Th
A peptide selected from r-9er-ASn-His-Ssr-OH and (in each of the above formulas, R'1 means hydrogen or a mercapto protecting group) or a pharmaceutically acceptable salt thereof.