JPS5927858A - Preparation of polypeptide - Google Patents

Abstract

PURPOSE:To prepare a polypeptide having the activity to suppress the secretion of gastric juice, in high purity and yield, by protecting a peptide having a definite amino acid sequence, subjecting successively to condensation reaction, removing the protecting groups of the resultant protected tripentacontapeptide, and oxidizing the product. CONSTITUTION:The objective polypeptide of formula II can be prepared by (1) protecting the peptide composed of 1-5 amino acids and having a definite amino acid sequence with protecting groups, (2) repeating the condensation reaction (e.g. azide method) successively from the C-terminal to obtain the tripentancontapeptide of formula I having protecting group, (3) removing the protecting group therefrom and (4) oxidizing six cysteine residues in the peptide chain. The oxidization reaction is carried out by using air, iodine or potassium ferricyanide, and S-S bond is formed by the reaction. The removal of the protecting group can be performed by using the conventional method used in the synthesis of peptide (e.g. methanesulfonic acid process).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a polypeptide [■] having the following amino acid sequence.
Concerning the manufacturing method.

H-Asn -Ser -Tyr -Pr.

＼ This peptide was discovered in the submandibular line of male mice by S. Cohen in the United States.
hFactor (EGF).

BGF is composed of 53+1 lil amino acid residues and exhibits a stable three-dimensional structure with three SS bonds in one molecule.

When this S-S bond is reduced, biological activity is lost, but when it is oxidized again, the activity is regained.

EGF has various physiological effects, such as suppressing gastric acid secretion and promoting growth of epithelial cells, and it has also been shown that these effects are not species-specific, making it extremely useful as a pharmaceutical agent.

However, EGF extracted from nature has many drawbacks, such as being difficult to collect due to extremely small amounts, and the purification process is complicated and the purity obtained is rarely satisfactory. In terms of this, it has been desired to provide a new EGF by synthesizing it.

In view of the above circumstances, 1. While researching a method for synthesizing EGF with high purity and high yield, the present inventor succeeded in developing an efficient production method as detailed below, although it was a bit of a hassle. I was able to complete it.

According to the present invention, a tripentacontapeptide (1) having a protecting group is first produced, and after removing the protecting group of the tripentacontapeptide, 06 cysteine residues in the peptide chain are oxidized to shorten the tripentacontapeptide (1). High purity EGF (11) can be produced in high yield and in a short period of time.

Generally, peptide synthesis methods are classified into two types: liquid phase method and solid phase method. In the case of liquid phase method, it is possible to purify synthetic intermediates, so it is easier to obtain products with higher purity than solid phase method. Can be synthesized. There are three important elements in peptide synthesis using the liquid phase method: (1) selection of a protecting group, (2) selection of a peptide bond forming reaction, and (3) selection of a method for removing the protecting group. , it is necessary to carry out synthesis while fully considering these factors.

According to the present invention, the protection of amino acids includes a benzyloxycarbonyl group and a methoxybenzyloxycarbonyl group.

To protect carboxyl groups such as tert-butyloxycarbonyl group, formyl group, and trifluoroacedel group, monoalkyl groups (ethyl, methyl, tert-butyl, etc.) and aralky/L/M (hensyl, p-methoxybenzyl) are used to protect carboxyl groups. , p-nitrobenzyl, etc.), which are commonly used in the field of peptide synthesis, can be used.

Further, an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tert-butyl group, etc. are used to protect side hydroxides such as threonine, serine, and tyrosine, but there are cases where no protection is used. The SH group of cysteine is a benzyl group.

Protected with p-methoxybenzyl group, trityl group, acetamidomethyl group, etc., and the guanide group of arginine is protected with
A nitro group, tosyl group, benzyloxycarbonyl group, adamantyloxycarbonyl group, isobornyloxycarbonyl group, mesitylenesulfonyl group, etc. are used.

Peptide synthesis methods include azide method and acid anhydride method.

Active ester method, diphenylphosphoboryl azide method, D
CC ten additive (1-hydroxybenztriazole, N
-Hydroxysuccinimide, etc.) method can be used, but the azide method is most suitable from the viewpoint of suppressing racemization.

In addition, the protective groups can be removed using methods commonly used in the field of Futatsufu peptide synthesis, such as methanesulfonic acid, trifluoromethanesulfonic acid, hydrogen fluoride, liquid ammonia/sodium, trifluoroacetic acid, hydrogen bromide, etc. can be used.

Furthermore, S-3 bond formation by oxidizing cysteine is performed by air oxidation, oxidation with iodine or potassium ferricyanide, and metal compounds such as iron chloride and copper sulfate are added as catalysts for air oxidation. There is.

Therefore, by appropriately combining the above-mentioned protecting groups, condensation methods, methods for removing protecting groups, and methods for oxidizing cysteine, it is possible to produce the desired EGF (U). For example, it can be manufactured as follows.

A tripeptide or pentapeptide fragment corresponding to the amino acid sequence of EGF (11) is synthesized, this is protected with the above-mentioned protecting group, and the fragment is isolated from the C-terminus using a method that does not cause racemization (mainly the azide method). By sequential condensation, a tripentaconc peptide with a protecting group is synthesized. The target EGF (n) can be obtained by removing the protective group of this tripentacon peptide by an appropriate method, purifying it by a method such as chromatography, and then oxidizing cysteine to form a disulfide bond.

According to experiments using rats, the compound of the present invention produced in this manner has been found to exhibit a remarkable inhibitory effect on W acid secretion. (11) It has also been confirmed that one compound of the present invention exhibits significant cross-reactivity with natural mouse EGF antibodies.

Examples of the present invention are shown below, but these methods are only preferred examples of the method of the present invention.

9 In this specification, primary abbreviations are used.

Rf, : Chloroborum methanol-water (8+3:
Silica gel thin layer chromatography value Rfz according to the system of 1)
: Silica gel thin layer chromatography value using the system of chloroporm-methanol (10:0:5) Rf3: Thin layer chromatography of silica gel using the system of chloroporm-methanol-acetic acid (9:1:0.5) [Comatogel rough value B, 1 Set :Pendylserine.

ell C÷5ap-methoxybenzylcysteine.

herg: N'-mesitylenesulfonylarginine TFA
: l-lifluoroacetic acid, IEt3N: ) ethylamine IAN: isoamyl nitrite.

DMF Nidimethylpolamide.

DNSO: dimethyl sulfoxide.

Boc: Butoxybenzyloxycarbonyl.

OBzl Knee 0-C I-12egH. -1 group P8:
Hexamethylbosbolic triamide example? H-Ser-Ser-Tyr-8sp
- Gly - Tyr - cps -
LeuHβi sn -Cys -Val - 11e
-Gly -Tyr -Ser -Gly-A
'r'g -Trp -Trp -G'fS''-Leu
-A7t, -Ollzl (protection ~102°c +
Rf2 = 0.48 + (α catty - 32.3”,
C=0.7 MeOIl) (10.0g)
was mixed with anisole (3.5 ml) and ethanedithiol (0
．． 7 ml) for 80 minutes under water cooling.
1 ml), powdered with ether, filtered and dried.

Next, melt this powder in DMF (50ml) and
Neutralize with t3N (1.1 mlopξl). On the other hand, sp to Boc-Leu-
/”r'g - NIINII, (6, 30g)
(Melting point 98-100°C Rf, = 0.69, (α imaginary -
14.1.

C=0.6 MeOII) in DMF solution (40ml)
4.18N hydrochloric acid=DMF solution (3.68ml)
and IAN (LL7 ml) to form azide, and neutralize with EJN (2.46 ml).

Add the above DMF solution to this solution. The liquid was made weakly alkaline (pH 7-8) and stirred at 4°C for 48 hours. After the reaction, the liquid is made neutral and the f6 medium is distilled off. When 5% citric acid water is added to the residue under water cooling, a powder is obtained. The obtained powder is washed in a mortar with 5% citric acid water, 5% NallCO3 water, and water in that order, and then recrystallized with vinegar varnish and isopropyl ether. Furthermore, silica gel chromatography (
7,2 x 17 cm). The eluate contains C
A mixed solvent of IICI and Meoll (40:1) is used. After elution, both fractions containing the target product were collected and the solvent was distilled off. Do not dissolve the residue in CIICI3.

The cut3i solution was washed with water and then distilled off. Crystals are obtained by adding vinegar varnish and ether to the residue.

- Trp-Trp-G name +-L・. -Yi-OB・1(
Synthesis of B...-(4253)-08zl): The Okuku peptide (3.79g > obtained in (1) was mixed with anisole (1.72ml) and ethanedithiol (0.33ml).
l) In the presence.

The mixture was treated with TFA (10.2 ml) for 60 minutes under water cooling, triturated with ether, filtered and dried. Next, this powder was dissolved in DMF (20 ml) and Et,xN (0.
Neutralize with 28 ml). On the other hand, BocN13. I
Mis-Cys-Gln-Th
r-Aug-N11NII2 (2,75g)
(Melting point 179-181°CRf, = 0.32.(α
)"I) 7.0°, C=0.6 DMSO]
4.78N hydrochloric acid-DMF to MFi'8 solution (15ml)
By adding liquid (1,37 ml) and I A N (0,44 ml), azide was obtained and Et3N (0,92
Neutralize with m1). The above-mentioned DMFi solution was added to this solution to make the solution slightly alkaline, and the mixture was stirred at 4°C for 48 hours. After the reaction, the solvent is distilled off, and 5% citric acid water is added to the residue to obtain a powder. 1M powder in 5% citric acid water, 5% Na1
After washing with lCO3 water and water in that order, DMF/MeOI
Recrystallize with I. Yield 3.02g (56.4%) R
f, =0.69-J'p'-Leu-Ar'g
-Trp -Trp-static' -Leu -Af'H-0
Bzl (Boc-(39'53) 0Bzl)
Synthesis: Dodecapepdide (5.0 g) obtained in (2) was mixed with anisole (2.4 ml) and ethanedithiol (0.47 g).
The mixture was treated with TFA (14 ml) for 60 minutes under water cooling in the presence of m1), powdered with ether, filtered and dried. Next, melt this powder in DMF (40ml) and Ht3N.
Neutralize with (0,26 ml). On the other hand, Boc-c+y-
Hekai Ikkou'g -NIINI+Yu (1,31g)
(Melting point 130-133°c Rf3 = 0.22.(
3.81 N hydrochloric acid-DMF solution (2 ml) was added to a DMF solution (2 ml) of
118 m1) and J A N ('0.30 m1) to form azide.

Neutralize with Et3N (0.63'ml). Add the above DMF solution to this solution and make the liquid slightly alkaline at 4°C.
Stir for 8 hours. After the reaction, the solvent is distilled off and 5% citric acid water is added to the residue to obtain a powder. After washing the obtained powder in the order of 5% citric acid water, 5% NatlCO3 water, and water, D and M
Recrystallize with F/MeOII.

-Leu -A'r”g-8Bzl (Boc -(3
7-53)-0Bz□).

Synthesis: Penkudetyl bebutide (2.48 g) obtained in (3) was mixed with anisole (1.0 ml) and ethanedithiol (0.19 g).
Treat with TFA (5 ml) for 60 minutes under water cooling in the presence of m1), powder with ether, filter and dry. Next, this powder was dissolved in DMF (20 ml) and EL3N (0
, 11m1). On the other hand, Boc-Tyr -5e
r-NllNIIz (0,36 g) (melting point 16
8-171°C Rf, = 0.36. (α)y+5.11°
, C=0.6 DMF) (7) D'MF solution (3 m
The azide was obtained by adding 3.81 N hydrochloric acid-DMF solution (0.52 ml) and IAN (0.14 ml) to 1).

Neutralize with E't3N (0,29ml). Add the above DMF solution to this solution, make the liquid slightly alkaline, and heat at 4°C.
Stir for 48 hours. After the reaction, the solvent is distilled off and 5% citric acid water is added to the residue to obtain a powder. The obtained powder is washed in a conventional manner and then crystallized with DMF/MeOIlTeN. Yield 2.28g
(85,0%), Rf, = 0.4734-53)
Synthesis of -08z I): Heptadecapepdide (2.27 g) obtained in (4) was treated with TEA (6.6 ml) in the presence of anisole (1.1 ml) and ethanedithiol (0.22 ml) for 60 minutes under water cooling. ), triturate with ether, filter and dry. Next, this powder was mixed with DMF (25ml
) and neutralized with Et3N (0,092 ml).

On the other hand, Boc-Val -11e -Gly -N
IINIIz (0,32g) (melting point 183-18
6°C, Rf, = 0.50. (α) dimension -5,5°
3.C=0.5DMSO) in a DMF solution (3 ml).
94N hydrochloric acid-DMF solution (0.44ml) and IAN
By adding (0.12ml), azide is obtained,
Neutralize with Et3N (0,082 ml). Add the above DMF solution to this solution to make the liquid slightly alkaline and
Stir for 48 hours. After the reaction, the solvent was distilled off and the residue contained 5%
Add citric acid water to obtain powder. The obtained powder is washed and recrystallized with 1&DMSO/Me011 in a conventional manner. Furthermore.

Purify by Sephadex 2+1-60 column chromatography (3, I x 142 cm). DMF is used as the eluent. Yield 1.6g (65.6%
) Rf+=0.43(6) Boc
-Thr -cps -8sn -clys
-Val -lie -Leu-8)g -0Bz
Synthesis of (Boc-(3053)0Bzl): Icozabetide (0.65g) obtained in (5) was mixed with anisole (0.30ml) and ethanedithiol (0.06ml).
m1) in the presence of

The mixture was treated with TFA (1.8 ml) for 60 minutes under water cooling, powdered with ether, filtered and dried. Next, add 10% of this powder
%D)ISOo, 17g) (melting point 204-206
°C, Rf+=0.57. (α)%? 39.5°C=
0.5 DMSO) in DMF solution (2 ml)
1N hydrochloric acid-DMF solution (0.12ml) and IAN (
0,031 ml) to make azide,
Neutralize with tJ (0,064 ml).

The above 10% DMSO-containing DMF solution was added to this solution to make the solution slightly alkaline, and the mixture was stirred at 4°C for 48 hours. After reaction.

The solvent is distilled off and 5% citric acid water is added to the residue to obtain a powder. After washing the obtained powder in a conventional manner, DMF + DM
SO/, M, p2' -Val -11e -G
ly -Tyr -5er -Gay -Ae
-Trp -Trp -Gl'u -Leu
-8rg -0Bzl'(Boc -(26
53) Synthesis of -0Bzl): The tetracosapeptide (305n+g) obtained in (6) was synthesized with anisole (0,18+nl) and ethanedithiol (0
, 035 ml) under water cooling in an argon gas stream.
Treated with TFA (0.3 ml) for 0 min and powdered with ether.
After that, filter and dry. Next, this powder is
Ut, N (0,0
Neutralize with >10ml. On the other hand, Boc-Leu-8'I
E-5Xr-Tyr-NIINH) (0,070
g) (Melting point 168-170''C, Rf, .=0
．． 35. Add 4.40N hydrochloric acid-DMF to a DMF solution (1.5ml) of (α)"8-30°C=0.8.0MF)
liquid (0,049ml) and IAN (0,014ml)
By adding IEJN (0,
Neutralize with 030 ml). Add the above II M to this solution.
After adding a DMF solution containing PA and reacting, the solvent is distilled off and water is added to the residue to obtain a powder. DM the obtained powder
Recrystallize with F/Meoll. Yield 304mg
(87,8%) -Thr -C+ys -8sn
-Cps -Val -11e -Gly
-Tyr♀H Cow 5 M19JI
MtS-Ser -Gly -8sp -8r
g -Cys -Gin -Thr -＾rg
-AT et al. 1-5e. -Ba'g-Trp-□1. - from
. . -Existence;-0Bzl (Boc -(23,53)
Synthesis of octacosapeptide (271 mg) obtained in (7) was treated with TFA (1 m) in the presence of anisole (0.14 ml) and ethanedithiol (0,027 ml) in an argon gas stream for 60 minutes under water cooling. The mixture is powdered with ether, filtered and dried.

Next, this powder was mixed with II MPA-containing DMF (2 m1
) and neutralized with Et3N (0,008 ml).
On the other hand + Boc -11e -Gl'u -3%r
NIINIIλ (0,058g) (melting point 156~
159°C, Rf2=0.30. (α)F'-13,9°
4 in a DMF solution (1.0 ml) of C=0.8DMF)
．． 40N hydrochloric acid-DMF solution (0,041ml) and IAN
The azide was obtained by adding (0,012 ml).

Neutralize with nt3N (0,025 ml). The above II MPA-containing DMF solution is added to this solution and reacted in a conventional manner. After the reaction, the solvent is distilled off, water is added to the residue, and the powder is recrystallized from DMF/Meoll containing DMSO. Yield if 270mg (89.3%) Rf+=
ts -Arg -0Bzl (Boc -(22-53)
-0Bzl) synthesis: Hentriacontapeptide (264+ng) obtained in (8) was added to anisole (0.12ml
) and ethanedithiol (0,024 ml) in an argon gas stream for 60 minutes under water cooling with TFA (0.5 ml).
), powdered with ether, filtered and dried.

Next, this powder was dissolved in IIMP^-containing DMF (1 ml) and neutralized with Et3N (0,007 ml). on the other hand,
Boc-11is NIINHx (0,019g)
Et3N (0,02
Neutralize with 0ml). Add the above II M P to this solution.
Add A-containing DMF solution and react in the usual manner. After the reaction, the solvent was distilled off, water was added to the residue, and the resulting powder was dissolved in DMSO.
Recrystallize with D' containing F/Merit. Collection M
242mg (90.5%) Rh=0.60(
10) Boc-Gly-Va□-Cps'-Ne
t −11is −1□. , name al-5er -Leu
-Asp -3er -Tyr -Thr -Cy'
s'-Asn? 1 Mp in H
9H-Cys-Val-lie-Gly
-Tyr -5er -Gly - sp phylum and s I [? ,1
Hmus 0Bal-8r
g -Cys -Gln Thr -^toc -8sp -Leu -Afg-Trp -T
rp -G7u' -Leu-Furuichi. 8゜I(Boc-
Synthesis of (18-53)-0Bzl): Todria conch peptide obtained in (9) (223 mg)
anisole (0.12 m1) and ethanedithiol (0
, 023 ml) under water cooling in an argon gas stream.
Treat with TFA (1 ml) for 0 minutes, powder with ether, filter and dry. Next, this powder was mixed with IIMPA-containing DM.
Dissolve in F (1 ml) and Et3N (0,006 ml).
) to neutralize it. On the other hand, B'oc-GlyHPi1 -Val -Cys -Met -NIINIlz(0
,038g) (melting point 205-208℃, Rt,
= 0.68. (α]g-18, IC=0.5
0M5O) DMF solution (1 ml), 4.40 N hydrochloric acid-DMF solution (0,030 ml) and ■ΔN (0,009
ml) to obtain the azide.

Neutralize with Et3N (0,018 ml). The above-mentioned DMF solution containing 11 M P is added to this solution and reacted in a conventional manner, and the obtained powder is recrystallized. Furthermore, Sephadex L11-60 column chromatography (3, I
142 cm). 30% D in the eluate
Use MSO-containing DMF. Yield 203n+g (8
3.5%) M, Ba1 (11) Boc-Cys-Leu-Asn
-Gly -Gly -Vat -Drop IH~1
Two Set -Tyr Thr - to ys -
8sn -Cys -Val -lie -□
hr -Mountain-A"f"p'-Leu -Ar'g -T
rp -Trp -G7u' -Leu-8f-g -
0Bzl (Boa” (14;53) 0Bz
Synthesis of l): Hexatriacontapeptide (203m) obtained in (10)
g) was treated with TFA (1 ml) in the presence of anisole (0.114 ml) and ethanedithiol (0.022 ml) in an argon gas stream under water cooling for 60 minutes, powdered with ether, filtered and dried. Next, add this powder to II
Melt in DMF (4゜0ml) containing MPA,
Neutralize with EtJ (0,005 ml). on the other hand.

196-198°C, Rf, = 0.47. (α book-19,07, C=0.7 DMSO) in DMF solution (
4.40N hydrochloric acid-DMF solution (0,033ml)
1) and IAN (0,010 ml) to form the azide, and neutralize with HJN (0,030 ml). The DMF solution containing the above-mentioned group was added to this solution and reacted in the usual manner.

The obtained powder is recrystallized. Furthermore, Sephadex Ll+
-60 column chromatography (3, I X 142
cm). DMF containing 30% DMSO is used as the eluent. Collection-Gly-Gly-Va
□ −,14,B2 −Met −11is −1
1e-G? "S"-Cd's-Val-1
1e -Gly -Tyr -3er -Gl
y - to χ2' nee Trp -Trp'-c't
u” -Leu -帛-8Bzl (Boa -(10
53) Synthesis of -0Bzl): Tetracontapeptide (195mg) obtained in (11)
Anisole (0,102 ml) and ethanedithiol (
The mixture was treated with TFA (1 ml) in the presence of 0,020 ml) in an argon gas stream for 60 minutes under water cooling, powdered with ether, filtered and dried. Next, this powder was mixed into HMPΔ-containing DM.
Dissolve in F (5 ml), EtJ (0,007 ml)
Neutralize with. On the other hand, Boc-TyrH-Asp-Gly-Tyr-NIINIlz(5
6mg) (Melting point 170-173℃, Rf, = 0
．． 013. (α)%F 146.96 G=0.5
DMF )’s DMF? S solution (1 ml), 4.40N hydrochloric acid-DMF solution (0,044 ml) and IAN (0,
013 ml) to make the azide, and
Neutralize with N (0,041 ml). The above II MPA-containing DMF solution was added to this solution and reacted in a conventional manner, resulting in 911 Mulberry Bat,
l”1BilSer -Tyr Thr
Cys -8sn -Cys -Val
-11e -Gly -Tyr -5er -
Gly -Jump -A Station -Cys' -Gln
-Thr-Δ';'g one point wise-Leu -A stomach-Trp
-Trp -San1-Leu -8)g 0
Bzl (Boc (653) -0B
Synthesis of tetratetracontapeptide (162m) obtained in (12)
g) was treated with TFA (1 ml) in the presence of anisole (0,088 ml) and ethanedithiol (0,017 ml) in an argon stream under water cooling for 90 minutes, powdered with ether, filtered and dried. Next, add this powder to Il? II'
A-containing DMF (3 m 198-100°C, Rfl = 0
．． 24. (Cl)ffi-3,97G=0.5
4.40N hydrochloric acid-DMF solution (0,057ml) and IAN (0,0
EbN
Neutralize with (0,052 ml). Add the above II to this solution.
A DMF solution containing MPA was added and reacted in the usual manner.

The obtained powder is recrystallized. Furthermore, Sephadex Ll+
-60 column chromatography (3,1X 142
cm). DMF containing 3o% DMSO is used as the eluent.

Yield 173 mg (97.6%). . -Asn -Gly -Gly -Val -C
';'s'-Met -11is -?l-1 and S
N, BJI Gly-Asp-Arg-Cys-G□. -T
hr -Ar'g -Alpken Boc -(1-53)
-0821) Synthesis of (having a protecting group [■]): (13) is an octatetracontapeptide (173
mg) was treated with TFA (1+nl) in the presence of anisole (0,096 ml) and ethanedithiol (0,019 ml) in an argon stream under water cooling for 90 minutes, powdered with ether, filtered and dried. Next, this powder is IIMPA
Containing DMF (5 ml) Melting point 130-133'C, Rf
, = 0.42. (α)D 10.61 G=
4.4 in a DMF solution (1.0 ml) of 0.90 MF
0N hydrochloric acid-DMF solution (0,066ml) and IAN
(0,019 ml) to obtain azide,
Neutralize with Et3'N ('0.061 ml). The aforementioned II MPA-containing DMF solution is added to this solution and reacted in a conventional manner, and the resulting powder is recrystallized. Furthermore, Sephadex L11-60 column chromatography (3, I
x 142 cm). Yield 90mg
(48,3%) (B) (Boc -(1-53)
-0Bzl) Deprotection and reduction of ([I] with protecting group): (A) - (1) with protecting group obtained in (14)
(36 mg) in the presence of metacresol (0,045 ml) and ethanedithiol (0,018 ml) at -5°C.
Treat with hydrogen fluoride for 60 minutes at ~0°C. After the reaction, hydrogen fluoride is distilled off and ether is added to obtain a powder. Filter the powder,
After drying 0.1M) Lisbuafy - (pH 8,0)
(1.5 ml), dithiothreitol (13
After adding 2 mg) and purging with argon, the mixture was stirred overnight.

Next, Sephadex G-25 column chromatography (1,8
x70cm). A 0.1N acetic acid solution is used as the eluate, and each fraction is 3rnl. Both amounts corresponding to 27 to 40 were collected and subjected to the first air oxidation reaction.

[C) Both portions corresponding to floors 27 to 40 obtained in S-S bond formation reaction CB) of (I) were heated at 4° under water cooling.
Dilute in C water to 200+++1. Further 0.01
After adding M ammonia acetate solution to bring the total ff to 400 mg, the pi was set to 7.0 with 15% ammonia water and left at room temperature for 4 days. Next, the pH was adjusted to 4 with normal hydrochloric acid under water cooling.
．． Lyophilize as 0.

The obtained powder was transferred to Sephadex G-25 column Chroma 1.
~Purified by graphie (1,8X 70 cm).

A 0.1N acetic acid solution is used as the eluate, and the -fraction is made 3 ml. Both fractions corresponding to Arashi 25-32 are collected and lyophilized to yield 6 mg (20%) of crude (II). Next, coarse (I
I) (6 mg) is purified by diethylaminoethylcellulose column chromatography (1,5X 6 cm). For the elution method, a linear gradient method from 0.02M ammonium acetate to 0.2M is used, and each fraction is 3 ml. Fractions corresponding to cases 28 to 35 are collected and lyophilized to obtain the desired product (n).

Yield: 3 mg' (deprotection yield: 10%, purification yield: 50%) According to the above example, each of the fragments (2) to (14) subjected to t4F and the final substance ° [■] was subjected to amino acid analysis. However, the amino acid analysis results shown in Table 1 were obtained.

Claims (1)

[Claims] A peptide consisting of 1 to 5 amino acids having a certain sequence is protected with a protecting group, and then subjected to a condensation reaction, and this condensation product is further added with 1 to 5 amino acids having a certain sequence. By protecting a peptide consisting of ~5 with a protecting group and post-condensation, and repeating these operations, a tripentaconc peptide (1) with a protecting group 11-^sn -5et -Tyr -Pro -Gl
y-Cys-Pr. -Ser -Ser -Tyr-Asp -Gly -
Tyr -Cys -Leu-8sn -Gly
-Gly -Val -Cys -Met -
Old s -11e-Glu -Ser -Leu-As
p Ser -Tyr -Thr -Cys-8sn
-Cys -Val -lie -Gly
-Tyr -5er -Gly-Asp -8rg -Cys -Gin -Thr -8r
g -Asp -Leu-Arg -Trp
-Trp -Glu -Leu -8rg -
A method for producing a polypeptide (II) having a ninth amino acid sequence, which comprises producing 011, deprotecting it, and then subjecting it to an oxidation reaction. 11-8 sn -Ser -Tyr -Pr. \