JPS6110598A - Peptide compound - Google Patents

Abstract

NEW MATERIAL:A compound (salt) expressed by formula I (R<4> is H or a protecting group of the mercapto group) and formula II. USE:An antigen composition for analyzing hepatitis B viruses and hepatitis B vaccines. PREPARATION:The protecting group of N-tert-butoxycarbonyl-O-benzylserine resin as a starting material is eliminated therefrom, and amino acids having amino group protected with tert-butoxycarbonyl group are condensed therewith using dicyclohexylcarbodiimide, etc. as a condensing agent according to the amino acid sequence. The above-mentioned operation is repeated to extend the peptide chain. In the process, functional groups in the side chain are protected as follows; Benzyl group for Ser and Thr, Acetamidomethyl group for Cys, cyclohexyl ester for Asp and 2-chlorobenzyloxycarbonyl group for Lys to carry out the condensation reaction. After completing the peptide chain synthesis, the protecting groups are eliminated from the resin to afford the aimed compound expressed by formulas I and II.

Description

[Detailed Description of the Invention] This invention relates to a novel peptide compound,
In particular, the present invention relates to a novel peptide useful for analysis of hepatitis B virus antigens and also useful as antigen compositions for Bg hepatitis vaccines, and pharmaceutically acceptable salts thereof.

The novel peptides of this invention have the following formulas [I] to EI
I].

[I] H-5et-Cys(R4)-Cys(R4)-Cy,
s(R4)-Thr-Lys-Pro-Ser-Asp
-Gly-Asn-Cyg(R4)-Thr-Cys(
R4)-Ile-Pro-118-Pro-Ser-3
er-0)! [II] H-Ser-Cys(R4)-Cys(R4)-Cys
(R4)-Thr-Lys-Pro-3et-Asp-
Gly-Asn-Cys(R4)-Thr-C:ys(
R4)-Ile-Pro-Ile-Pro-9et-S
er-Tyr-Leu-Leu-Tyr-Cys (R4
)-DH (However, in each of the above formulas, R4 means hydrogen or a mercapto protecting group) In this specification, amino acids, protecting groups, active groups, solvents, etc.
onan Biological Nomencl
It may be indicated by an abbreviation based on ature or a common abbreviation in the field, examples of which are as follows. Cys: cysteine, Tyr: tyrosine, P
ro nibroline, Ice: isoleucine, Asp: aspartic acid, Asn: asparagine, Thr: threonine, Leu: leucine, Gly nigericin, Lys:
Lysine, Ser: serine, Boc: t-butoxycarbonyl, CI-Z: 2-chlorobenzyloxycarbonyl, Bzl: benzyl, CI2 B21 dichlorobenzyl, Acm: acetamidomethyl, 0-cHex dichlorohexyl ester. The compound of this invention [II
, [TI] and pharmaceutically acceptable salts thereof can be produced by various methods, which are described below.

(1) Method-1 (Peptide synthesis by solid phase method): 1) Method 1-1 R1-Ser(R3)-Resin [Engineering a]→R'-Ser
(R8)-Cys(Ra4)-Cys(Ra4)-Cy
s(Ra4)-Thr(R3)-Lys(R1)-Pr
o-Ser(R”)-Asp-(OR2)-G17-A
sn-Cys(Ra)-Thr(R)-Gys(Ra
)-Ile-Pro-Ile-Pro-Ser (R8
)-Ser-(R8)-resin [Ib] 2) Method 1-2 R1-Cys(Ra4)-resin [IIa]+R1-5e
t(R3)-Cys(Ra4)-Cys(Ra4)-C
:ys(Rp4)-Thr(R3)-Lys(R4)-
Pro-Ser(R”)-Asp(OR”)-Gly-
Asn-Cys(Ra4)-Thr(R3)-Cys(
Ra4)-I 1e-Pro-Ile-Pro-9et
(R”)-Ser(R”)-Tyr(R8)-Leu-
Leu-Tyr(R”)-Cys(Ra)-resin [I
IbCo(2) Method 2 (removal method of retaining group): 1) Method 2-1 R'-9et(R'')-Cys(Ra4)-Cys(R
a4)-Cys(Ra')-Thr(R3)-Lys
(R')-Pro-9et(R8)-Asp(OR"
)-Gly-Asn-Cys(Ra4)-Thr(R”
)-C:ys(Ra4)-Ile-Pro-Ile-P
ro-3er(R3)-3er(R3)-resin [I b
]H-3et-Cys(Ra)-Cys(Ra)-
C: ys(Ra)-Thr-Lys-Pro-3er
-Asp-Gly-Asn-Cys(Ra4)-Thr
-Cys(Ra4)-Ile-Pro-Ile-Pro
-3er-3er-OH[Ic] 2) Method 2-2 R'-5et (R'')-Cys(Ra')-C:y
s(Ra4)-Crys(Ra4)-Thr(R”)-
L7s(R”)-Pro-9et(R3)-Asp(O
R2) -Gly-Asn-Gys(Ra4)-Thr
(R8)-Cys(Ra4)-Ile-Pro-Ile
-Pro-3er(R8)-Ser(R”)-Tyr(
R3)-Leu-Leu-Tyr(R3)-Gys(R
a4)-Resin [ITb]+H-3er-Cys(Ra4
)-Gys(Ra4)-C7s(Ra4)-Thr-L
ys-Pro-9et-Asp-Gly-Asm-Cy
s(Ra4)-Thr-Cys(Ra)-Ile-P
ro-Ile-Pro-Ser-Ser-Tyr-Le
u-Leu-Tyr-C:ys(Ra4)-0H[II
c] (3) Method 3 (elimination reaction of mercapto protecting group)
:l) Method 3-1 H-3er-Cys(Ra4)-Cys(Ra4)-C
ys(Ra4)-Thr-Lys-Pro-Ser-A
sp-Gly-Asn-Cys(Ra4)-Thr-C
ys(Ra 4)-Ile-Pro-Ile-Pro-
Ser-Ser-OH[Ic] ↓ H-9e r-Cys-Cys-Cys-Thr-Ly
s-Pro-9et-Asp-Gly-Asn-fll
:ys-Thr-Cys-Ile-Pro-Ile-P
ro-9et-Ser-OH[Id] 2) Method 3-2 H-3er(:ys(Ra)-Cys(Ra)-C
ys(Ra4)-Thr-Lys-Pro-Ser-A
sp-Gly-Asn-Cys(Ra)-Thr-
Cys(Ra4)-Ile-Pro-Ile-Pro-
Ser-Ser-Tyr-Leu-Leu-T7r-C
ys(Ra4)-0H[II cl'↓ H-3er-Cys-Cys-C7s-Thr-Lys
-Pro-Ser-Asp-Gly-Asn-C7s-
Thr-(4g-Ile-Pro-Ile-Pro-9
et-Ser-Tyr-Leu-Leu-Tyr-Cy
s-OH [II d ] In the above formula, R1 is an amino protecting group, R2 is a carboxy protecting group, R3 is a hydroxy protecting group,
Ra' each represents a mercapto protecting group. These definitions are explained below.

(1) Regarding the amino protecting group in R: Amine protecting groups include common amine protecting groups commonly used in the fields of amino acid and peptide chemistry, and preferred examples of such amine 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, unsubstituted allenesulfonyl (eg, benzenesulfonyl, P-toluenesulfonyl, etc.), and the like.

(2) Carboxy protecting group in R: Carboxy protecting groups include common carboxy protecting groups commonly used in the fields of amino acid and peptide chemistry, and preferable examples of such protecting groups include, for example, lower alkyl (e.g. alkyl such as methyl, ethyl, etc.), cycloalkyl (e.g. cyclopentyl, cyclohexyl etc.), aralkyl such as 7- or di-phenyl lower alkyl (e.g. benzyl, diphenylmethyl etc.), aroyl alkyl (
Examples include phenacyl, toluoyl, and the like.

(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 7ralkyl (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, amino 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-butyl mercapto, etc.), etc. The removal conditions for each protecting group are appropriately selected. It can be used by

(5) Regarding pharmaceutically acceptable salts: Compound [II
The pharmaceutically acceptable salts in [11] include, for example, alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g., calcium salts, etc.), ammonium salts, organic amine salts (e.g., ethanolamine salts, etc.). salts with inorganic or organic bases such as triethylamine salts, dicyclohexylamine salts, etc.) and acetic acid,
Included are 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) About method 1: In this method, compounds [Ia] and [IIa] are coupled with each protected constituent amino acid in sequence according to the conventional method of peptide synthesis by solid phase method, and compound [Ib
This is a method to obtain l and [■b].

The compounds [Ia] and [IIa] used here as starting materials include known substances (for example, Biopolymer Vol. 12, p. 2513, 1973) and new substances, and the new substances are described in the literature. It can be manufactured in the same manner as described. Furthermore, as the resin used, any resin used in peptide synthesis by the solid phase method can be used, and examples of such resins include, for example, chloromethylated styrene-divinylbenzene copolymer. , polystyrene-type resins such as hydroxymethylated styrene-divinylbenzene copolymer, amine-methylated styrene-divinylbenzene copolymer, and polyamide-type resins such as polydimethylacrylamide resin.

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 the starting material, the protected amino acid-resin, and to swell the resin. Suitable examples of solvents used for this purpose include methylene chloride, Examples include chloroform, dimethylformamide, or a mixed solvent thereof.

2) Step 2: This step is a step of removing the amino 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 acid oil 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, formic acid, p-toluenesulfonic acid, hydrochloric acid, or hydrobromic acid, preferably ethanedithiol or This is done by adding anisole.

Among the acids listed above, trifluoroacetic acid and formic 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. It is preferable to treat with

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 Step 1 above.

6) Step 6: This step is a step carried out for desalting when the amine 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.

9) 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 1: This step is carried out for washing and swelling the resin, and is carried out by substantially the same method as Step 1 above.

Each of the above steps is generally performed at about room temperature, and each step (excluding step 8) is preferably repeated about 2 to 3 times.

(2) Regarding method 2: This method involves subjecting compounds [Ib] and [lIb1 to elimination reactions of amine-protecting groups, carboxy-protecting groups, and hydroxy-protecting groups, respectively ((), and converting compounds [IC]t-
1 and [II c].

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 (for example, formic acid, acetic acid, trifluoroacetic acid, propionic acid, benzenesulfonic acid, P-toluenesulfonic 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 with solvents that do not adversely affect the reaction [e.g., water, wood-philic solvents such as alcohols (e.g., methanol, ethanol, propatool, etc.), 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
-) combinations with luenesulfonic 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.), haladium catalysts (palladium sponge, palladium black, palladium oxide, palladium on carbon). , palladium colloid, palladium-barium sulfate, palladium-barium carbonate, etc.), nickel catalyst (reduced nickel, nickel oxide, Raney nickel, etc.), cobalt catalyst (e.g. reduced co/<rut, Raney cobalt, etc.)
, iron catalysts (for example, reduced iron, Raney iron, etc.), copper catalysts (for example, reduced copper, Raney copper, Ullmann copper, etc.), and the like. Reduction is usually carried out in a solvent. Preferred solvents include, for example, water, alcohols (eg 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,
A method of treating with alcohol such as trifluoromethanesulfonic acid is mentioned, 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 anisole or the like. Further, the above acid can also be used as a solvent. This reaction is usually preferably carried out at around 0.degree. C. by cooling.

(3) Regarding method 3: In this method, compounds [C], t-1 and [IIc are subjected to a mercapto-protecting group elimination reaction, and the corresponding mercapto-protecting group is removed from the compound [I d]. and [IId]
This is the way to obtain.

In this method, the compound [Ic] or [IIc] is mixed with a metal salt such as mercury acetate, mercury trifluoroacetate, or silver acetate, an organic phosphorus compound such as triphenylphosphine or tributylphosphine, mercaptoethanol, mercaptoacetic acid, ethanedithiol, This is a method of obtaining the corresponding compound [Id] or compound [IId] or compound, respectively, by treating with a reagent such as an alkyl or arylthiol such as thiophenol, dithiothreitol, or dithioerythritol.

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.

The compound of this invention [! ] , [nl includes one or more isomers due to asymmetric carbon atoms in the molecule, and all such isomers are included within the scope of the present invention.

The peptide of this invention, that is, the compound [■].

[II] and pharmaceutically acceptable salts thereof are useful for the analysis of hepatitis B virus antigens as described above,
It is also useful as an antigen composition for hepatitis B vaccines.

The pharmaceutical compositions of the invention can be used, for example, in the formulation layers of solid, semi-solid or liquid forms, which layers contain the active ingredients of the invention in organic or inorganic formulations suitable for topical, oral or parenteral administration. It is contained in a mixed state with a carrier or excipient. The active ingredient can be, for example, tablets, pellets,
They can be formulated with conventional non-toxic pharmaceutically acceptable carriers for capsules, half-opens, solutions, emulsions, suspensions, and any other formulation 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 being present in an amount sufficient to produce the desired therapeutic effect on the treatment or medical condition. present in the pharmaceutical composition.

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+ ag/kg of animal body weight per day. Administer the ingredients for therapeutic purposes. Also, the average dose per dose is approximately 50m
g, 100 mg, 250 mg and 500+*g 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-0-benzylserine resin (2.15 g) [Serine content: 0.82 mmol/g, using styrene-1% divinylbenzene copolymer as a starting material, the first The reaction order of the amino acids subjected to the peptide bond-forming reaction is Ser, Pro, and Ile according to the solid-phase method schedule shown in the table.

Pro, Ie, Cys, Thr, Cys
, Asn , G ly , Asp , Ser , Pr
o, Lys.

A peptide having 16 amino acid residues was synthesized by repeating the schedule shown in Table 1 for 15 cycles. The α-amino group of these amino acids is a t-butoxycarbonyl group, and regarding the other side chain functional groups, Ser and Thr are benodyl groups, Cys is an acetamidomethyl group, Asp is a cyclohexyl ester, and Lys is a benodyl group. Each was protected with a 2-chlorobenzyloxycarbonyl group.

Starting material (or intermediate material obtained by elongating the peptide chain by coupling an amino acid to the N-terminal amino group of the starting material) according to the procedure shown in Table 1, that is, Steps 1-11 of Table 1. Then, the peptide chain was extended by sequentially applying the solvents and reagents shown in the same table.

The coupling reaction, which is the eighth step in the same procedure, was generally carried out in a methylene chloride solution, but only when reacting Cys protected with an S-acetamidomethyl group, a mixture of methylene chloride and dimethylformamide was used. (2: l
) mixture was used. In addition, in the coupling reaction of Asn, N-t-butoxycarbonylasparaquine is
-Hydroxybenzotriazole (1:1 equivalent) and dicyclohexylcarbodiimide (1 equivalent) in dimethylformamide-methylene chloride (1:1) mixture,
It was activated by stirring for 1e minutes at 'C and used in the 8th step. When introducing the seventh and subsequent amino acids (after completion of the first reaction using cys), the second step shown in the table is to introduce 5% by volume of the amino acid into a methylene chloride-trifluoroacetic acid mixture. This was done by adding ethanedithiol. Furthermore, when introducing the 12th and subsequent amino acids after coupling Asp, the neutralization in the 6th step was carried out using a water-cooled solution of triethylamine, and the treatment time was reduced to 1.5 minutes. Shortened.

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 Table 1 were repeated in the order listed. In the experiment, the above-mentioned repeating process was performed for each of the coupling reactions of Cys (6th), Gln, Asp, and Ser. 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) was obtained.

Bo c-Th r (Bz l )-(CI-Z)-
Pro-Ser(Bzl)-Asp(OcHe
x) -G ly-Asr+-C:ys (Acm
)-Thr(Bz l)-C7s(Acm)-I 1
e-Pro-Ile-Pro-9et (Bzl)-3e
1.38 g of the r(Bzl)-resin was taken out and placed in a solid-phase reactor, and treated in the same manner to further produce (4s, Cys).

Cys and Ser were coupled to obtain 1.677 g of a peptide resin represented by the following formula.

Boc-3er(Bzl)-Cys(Acm)-f:y
s(Acm)-Cys(Acm)-Thr(Bz
1)-Lys (CI-Z)-Pro-5e t
(Bz 1)-Asp (OcHex)-Gly-A
sn-C7s (Acm)-Thr (Bz 1)-C
ys (Ac11) -Ile-Pro-Ile-Pr
o-Ser(Bzl)-3er(Bzl)-Resin Example 1-2 The peptide resin (1-1) (1,62g) obtained above was treated with hydrogen fluoride (40ml) in the presence of anisole (3,0ml).
1), the mixture was stirred at -5 to -0°C for 1 hour. Thereafter, hydrogen fluoride was distilled off under reduced pressure at the same temperature for 2 hours and at room temperature for 1 hour. 0.7N acetic acid was added to the residue, and the mixture was stirred for 1 hour while cooling with water. After removing the resin by filtration and washing the filtrate with ether, it was passed through a column of DOWEX 1×2 (acetic acid form, 150 μl) and lyophilized to obtain crude H-Ser-Cys.
(Acm)-C nears(Acm)-Cys(Acs+)
-Thr-Lys-Pro-Ser-Asp-Gly-
Asn-Cys(Acm)-Thr-Cys(Acm)
-Ile-Pro-Ile-Pro-Ser-Ser-
OH (700 mg) was obtained [% yield based on starting material CI)].

This crude product (125+ag) was subjected to semi-preparative separation using a microcosm tube (7.8mm x 300m).
m) From the column (manufactured by Waters), elute with 0.1% aqueous trifluoroacetic acid-acetonitrile (75:25) at a flow rate of 2.5 ml and 17 minutes, perform fractionation in 8 times, and retain. Fractions with a time period of 5 to 8 minutes were collected and lyophilized to obtain a purified product (109 mg) of the above peptide.

High performance liquid chromatography column: Nucleozil 1OcIB (250X 4a
m) Holding IJ7: 21.4 minutes Eluent: 0.1% trifluoroacetic acid acetonitrile linearity gradient position % → 50% (30 minutes) Flow rate: 1.0 +al/min Detection: UV 210 n+* Example 2 H-Ser-Cys(Acm)-C obtained in Example 1-2
ys(Acm)-Cys(Ac++)-Thr-Lys
-Pro-3er-Asp-Gly-Asn-Cys(
Acm)-Thr-Cys(Ac+1)-Ile-Pr
o-Ile-Pro-9ar-Ser-OR (100m
g) in 0.1% acetic acid (301) followed by a solution of mercuric acetate (10 mg) in water (51) under nitrogen atmosphere and the reaction mixture was stirred at room temperature for 3 hours. Then, mercaptoethanol (El, 58 g) was added, and the reaction mixture was stirred gently overnight, and the reaction solution was concentrated to 10 ml, and the precipitate was filtered off. Column of Sephadex G25 (3,2c
m× 115 cm) and eluted with 0.1% acetic acid. The main fraction (355 ml-420 ml) was collected and lyophilized to yield H-Ser-Cy s-C:ys-
fll Near 5-Thr-Lys-Pro-Ser-
Asp-G l 1-Asn-Gys-Thr-Cy
s-Ile-Pro-Ile-Pro-Ser-Ser
-OH (E14 mg) was obtained. Ellman's reagent (5
, 5-dithiobis-2-nitrobenzoic acid), it was confirmed that five thiol groups were present in one molecule.

(1) Acid hydrolyzate (6N hydrochloric acid at 110°C, 24°C)
Amino acid analysis of time treated product: Asp: 1.895
Thr: 1.3tt? Ser:3.027
Gly: 1.000Lys: 0.925
Pro: 2.728 Ile: 1.775 (2) Thin layer chromatography Rf = 0.44 (tailing) [Cellulose Merck No. 5552, n-butanol-acetic acid-water-pyridine (15:3:12:10) Detected by ninhydrin] (3) High performance liquid polymerization retention time: 12.0 minutes Column: Nucleosil 1OC18 (Machey10Cl8 (: 250 x 4+am)
Eluent: 0.1 in water and acetonitrile (80:20)
% of trifluoroacetic acid Flow rate: 1.0 + nl/min Detection: UV 210 nm Example 3-1 N-t-butoxy to styrene-divinylbenzene copolymer with 1% crosslinking N-t-butoxycarbonyl-5-7cetamidomethylcysteine resin into which carbonyl-5-acetamidomethylcysteine has been introduced via an ester bond (
4.0 g) (1.92 mmol) [Cystine content: Q, 48 mmol/gl was placed in a solid phase reactor,
The peptide chains were sequentially extended according to the solid phase synthesis schedule shown in Table 2. The reaction order of the amino acids used in the reaction was Tys, Leu.

Leu, Tyr, Ser, Ser, Pro, I
Ie, Pro, Iie, CyS, Thr, Cy
s.

Asn, Gly, Asp, Ser, Pro, Lys, T
A peptide having 21 amino acid residues was synthesized by repeating the schedule shown in Table 2 for 20 cycles. The α-amino group of these amino acids is a 1-butoxycarbonyl group, and regarding the other side chain functional groups, Ser and Thr are benzyl groups, Cys is an acetamidomethyl group, Asp is a cyclohexyl ester, and Lys is a 2-butoxycarbonyl group. -chlorobenzyloxycarbonyl group, Tyr is dichlorobenzyl group, and the protected amino acids in Table 2 are Boc-Tyr (CI2 Bz
l)-0H.

Boc-Leu-OH, Boc-3e r(Bz I
)-OH, Boc-Crys (Acm)-OH.

Boc-Thr(Bzl)-0H, Boc-Asp(O
cHex)-0H, Boc-Lys(CI-Z)-0H
was used. The condensation reaction in Step 8 was carried out in methylene chloride using 3 equivalents each of these protected amino acids and dicyclohexylcarbodiimide. However, 4 equivalents were used for each of asparagine and later. Also, Boc-C:
For the condensation reaction of ys (Acm, ) JH, a mixed solution of dimethylformamide and methylene chloride (1:2) was used. B
The condensation reaction of oC-Asn-CI)l was carried out in a mixed solution of dimethylformamide-methylene chloride (1:1) in the presence of l-hydroxybenzotriazole (equimolar). After completion of the condensation reaction of all amino acids, the resulting resin was washed with methylene chloride to obtain 11.20 g of a protected peptide resin represented by the following formula (theoretical weight gain: 117χ).

Boc-Thr(Bzl)-Lyg(CI-Z)-Pr
o-3er(Bzl)-Asp(OcHex)-G17
-Asn-Cys(Acm)-Thr(Bzl)-Cy
s(Acm)-Ile-Pro-I 1e-Pro-S
er(Bzl)-5et(Bzl)-Tyr(C12B
zl)-Leu-Leu-Tyr(C12Bzl)-C
ys(Ac+s)-resin propiono-resistant-salt alcohol (1:I) 110°C 24hr,
Amino acid analysis of hydrolyzate in the presence of 2% phenol [
(Theoretical values are in parentheses).

Asp: 1.998 (2) Thr: 1.081
(2) Ser: 1.510 (3) Gly: 0.1118
5 (1) Ile: 1.818 (2) Leu: 1
．． 574 (2) Tyr: 1.519 (2) Ly
s:1.0GPro:3.087(3) (Ly
s 0.1513 IL mol/mg) Example 3-2 Protected peptide resin CI obtained in Example 3-1) 5.52 g
was placed in a solid-phase reactor, and the peptide chain was further extended in the same manner as in Example 3-1. However, the protected amino acid in each condensation reaction is Boc-Cys(Acm)-0H
is 3 degrees, Boc-Ser (Bzl)-0H is 1 degree,
4 times the molar amount of each was used. Boc-Cys(Act)-O
When condensing H, a methylene chloride-dimethylformamide ('2:l) + 7) mixed solvent was used to condense BoC-Ser (
During the condensation of Bzl)-0)1, the reaction was carried out using only methylene chloride.

After the Cps(Acm)6 condensation reaction, a ninhydrin test was performed according to Kaiser's method and the result was positive, so the condensation reaction was repeated. After completion of the final condensation reaction, the resin was washed with methylene chloride and dried under reduced pressure.8.09
Boc-3er(Bzl)-Cys(Acm)-C
ys(Act)-Ill:ys(Acji)-Thr(
BzI)-Lys(CI-Z)-Pro-3et(Bz
I)-Asp(OcHey)-Gly-Asn-C
ys (ACW)-Thr (Bzl)-Cys
(Acm) -Ile-Pro-Ile-Pro-5e
t(BzI)-5et(BzI)-Tyr
(C12Bzl)-Leu-Leu-Tyr(C12B
zl)-Cys(Acm)-resin was obtained.

Hydrolyzate (conc, HCI: propionic acid = 1:
1.2% phenol, 110°C 24 hours) Amino acid analysis values [Theoretical values are in parentheses].

^gp:2.0f3B(2) Thr:1.13
0(2)Ser:1.81B(4)Gly:1
．． 0151:1) Ile:1.64B(2) L
eu: 1.588 (2) Tyr: 1.540 (2)
, Lrs: 1.00 (J) Pro: 3.488 (3
) [Lys 0.181 pL mol/mg (theoretical value 1o3
] Example 3-3 7.0 liters of anisole and 50 liters of anhydrous hydrogen fluoride were added to 4.18 g of the protected peptide resin obtained in Example 3-2,
Treat with water salt under cooling (below 0°C) for 1 hour.

After removing the hydrogen fluoride under reduced pressure at 0°C (requires approximately 2 hours), 100 ml of IN-acetic acid and 100 ml of ether are added.
+*l and stir under water cooling to extract. After filtering off the resin using a glass filter, acetic acid 301 and ether 1001 were added to the emulsion-state filtrate to separate the liquid.
The lower layer is passed through a DOWEX IX2 (oAc-type) column (300I*1), and the ninhydrin-positive portion of the eluate is collected and freeze-dried. The obtained crude product was subjected to Sephadex LH-20 (eluted with IN-acetic acid), and 1.0 g of peptide was obtained from the ninhydrin-positive main fraction. Acm)-Cys(A
c)-Cys(Acm)-Thr-Lys-Pro-
Ser-Asp-Gly-Asn-Cys(Acm)-
Thr-Cys(Acm)-Ile-Pro-Ile-
Pro-Se r-5e r-Tyr-Leu-Le
u-Tyr-C2s (Act) -OH (yield: 50.0% calculated from Baa-(4s(Act)-resin).
7%), 1.0 g of the above peptide purified by Sephadex LH-20 was added to Whatman's carboxymethyl cellulose CM-52 (3.2 x 60 cm, 0.1
M-pH 5,4 pyridine-acetate buffer) and the same buffer 0.1M to 0.4M (each 10010
Elute with a 0O linear concentration gradient and fractionate into 10g portions. 8QQ mg from 30th to 40th fraction
A purified peptide was obtained.

Hydrolyzate (IN-HCI, 2% phenol 11
Amino acid analysis of 0”c 24hr) [Theoretical values are in parentheses].

Asp: 1.855 (2) Thr: 1.33e
(2) Ser: 3.032 (4) Gly: 1.
00B(1)Ile:1.8213(2)Le
u: 1.897 (2) Tyr: 1.8E11 (2)
Lys: 1.000 (1) Pro: 2.803 (3
) [Lys O, 339 mol/g (theoretical value 78.8
%)] Example 3-4 Purified peptide obtained in Example 3-3 2901g72.81
The aqueous solution was divided into 150 to 180 JLM portions and applied to Waters Associate's semi-preparative column: Micron Bondapak c1g (7,8X2O3 intestinal layer), and L1% trifluoroacetic acid, H2O-acetonitrile (83:3)
7) Fractionate under the conditions of 2.2 ml/sin, concentrate and dry the eluate containing the target product to obtain 228 mg (78.8%
A purified peptide (recovered) was obtained.

Hydrolyzate (EIN-HC: l 110℃ 24hr
) Amino acid analysis value of (2-88 mg) [Theoretical values are in parentheses].

Asp: 2.2B (2) Thr: 2-01 (2
) Ser:4.52(4) Pro=3-27(
3) Gly: 1.20 (1) Ile: 2.03
(2) Leu: 2.00 (2) Lys: 1.0
9 (1) Tyr: 0.48 (2) NH3: 5
．． 34 (1) [Lys 1.38 JL mol/g (72.8% of theoretical value)] Example 4 20.3 mg (8
, 68 pmol) in 0.1% acetic acid 51, and
A solution of 38.3 g of mercury (0.12 #L mol, 18 yields) dissolved in 2 liters of 0.1% acetic acid is added at room temperature in a nitrogen stream and allowed to react for 3 hours. Add 1.4 liters of 2-mercaptoethanol and leave it under nitrogen atmosphere overnight.
The solution was concentrated to 3 liters. The precipitate is filtered through a Bast Wool Het (Corning Inc.) filled with a cotton plug.

The filtrate was applied to Sephadex G-25 (2.2×44c set), eluted with 0.1% acetic acid, and fractionated into 3.5 g portions.

Check each fraction in 28 On intestine, 24-3
Collect the fourth fraction. A portion (0.8 x 1) was taken and the thiol group was determined using Ehrman's method, yielding 84.4% of the theoretical value.
The chemical formula of the product obtained is H-Ser (84.9%).
-Cys-(ys-[ys-Th r-Lys-Pro
-3e t-Asp-G l 1-Asn-C1s-T
br-C1s-Ile-Pro-Ile-Pro
-Ser-Ser-τyr-Leu-Leu-Tyr-
It is C7g-OH.

Each peptide obtained in the above example was combined and subjected to the following quantitative analysis.

1) Hydrolysis after performic acid oxidation - amino acid analysis 3.1fg
After mixing 0.5 l of 30% hydrogen peroxide and 10 ml of formic acid, add Solution 21 which has been used for 2.5 hours and react at -5°C for 2 hours. After adding 5 drops of 48% hydrobromic acid, it was concentrated and dried using an evaporator, and the residue was dissolved in 8N-HCl2.
Dissolve in porcelain and hydrolyze at 110℃ for 24 hours [()
The values inside are theoretical values].

Asp: 1.88 (2) Thr: 1.9B (2
) Ser: 3.38 (4) Pro: 2.72 (
3) Gly: 1.00 (1) Ile: 1.6i
11(2)Leu:1.64('2)Lys:
0.138(1)Cys03H:e, 09(8)T
yr: O(2) (disappeared due to Br conversion) [Gly
t, g8#L mol/mg (89,2% recovery)] 2) Take 1.04o+g of normal hydrolysis and add 1.2+wl of 0N-HCI
, Hydrolysis was carried out in 2% phenol at 110°C for 24 hours [theoretical values in parentheses].

Claims (1)

[Claims] H-Ser-Cys(R^4)-Cys(R^4)-C
ys(R^4)-Thr-Lys-Pro-Ser-A
sp-Gly-Asn-Cys(R^4)-Thr-C
ys(R^4)-Ile-Pro-Ile-Pro-S
er-Ser-OH and H-Ser-Cys(R^4)-Cys(R^4)-C
ys(R^4)-Thr-Lys-Pro-Ser-A
sp-Gly-Asn-Cys(R^4)-Thr-C
ys(R^4)-Ile-Pro-Ile-Pro-S
er-Ser-Tyr-Leu-Leu-Tyr-Cy
A peptide compound selected from s(R^4)-OH (wherein R^4 in each of the above formulas means hydrogen or a mercapto protecting group) or a pharmaceutically acceptable salt thereof.