JPS5855137B2 - Method for producing polypeptides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a protected peptide useful as an intermediate for producing a novel nonapeptide having a strong ovulation control effect.

More specifically, the present invention is based on the general formula [wherein, if an amino acid may have an optical isomer, unless otherwise specified, it represents one isomer.

X is D-Leu, D-NVa, D-Abu, D
-Phgla-A ibu or D-8er, R represents a lower alkyl group.

] Among the amino acid residues, the imino group of His is benzyl, tosyl, 2,4-dinitrophenyl, t-7'' thyloxycarbonyl or carbobenzoxy, the hydroxyl group of Ser and Tyr is benzyl or t-butyl, and the guanidino group of Arg is In the method for producing a protected peptide in which at least one group is protected with nitro, tosyl, carbobenzoxy, inbonyloxycarbonyl, adamantyloxycarbonyl or hydrohalide, the group is a partial amino acid or peptide that can constitute the above polypeptide. This is a method for producing the above-mentioned protected polypeptide (I), which comprises condensing both fragments in which at least one amino acid residue is protected in the remaining portions.

In this specification, amino acids, peptides, protecting groups, etc.
IUPAC-IUB Comm1ssion onB
They are indicated by iological nomenclature abbreviations and common names in the field.

Examples of these are listed below.

NVa: norvaline Abu: 2-aminobutyric acid Phg: phenylglycine α-Aibu; α-aminoisobutyric acid Z: carbobenzoxy HONBI: N-hydroxy-5-norbornene 2
・3 dicarboxinimide DCC: N-N'-synchrohexylcarbodiimide OMe: Methyl ester 0Bzl: Benzyl ester In the above formula (I), the lower alkyl group represented by R includes, for example, methyl, ethyl, propyl, isopropyl group. etc.

These may be attached to a hydroxyl group.

In general, in peptide synthesis, many methods have been established for forming the desired amino acid sequence by protecting amino groups or carboxyl groups that do not participate in the condensation reaction of raw amino acids or peptides, and then carrying out the condensation reaction. Any of these may be used in carrying out the method of the present invention.

Advantageous specific examples of the method of the present invention include the following means.

(1) (a) The carboxyl group at the C-terminus is free and the N-terminus is pyroglutamic acid or pyroglutamic acid, which is protected or unprotected, and the following formula (I)
a partial peptide constituting the amino acid sequence of (b) N-
The terminal amino group is free, and the remaining portion of the amino acid sequence of the target polypeptide (e.g., partial peptide substituted amide, proline substituted amide, or substituted amine constituting the remaining portion), and at least one amino acid residue of both fragments is A means of condensing protected groups in the presence of a condensing agent.

(2) (a) The carboxyl group at the C-terminus is activated, and the above formula (
A partial peptide constituting the amino acid sequence of I), and (b)
The N-terminus is a free amino group, and the remaining portion of the amino acid sequence of the target polypeptide (I) (e.g., partial peptide substituted amide, proline substituted amide, or substituted amine constituting the remaining portion), and both fragments thereof means for condensing protected at least one amino acid residue of .

(3) (a) A partial peptide having a free carboxyl group at the C-terminus and having protected or unprotected pyroglutamic acid or pyroglutamic acid at the N-terminus and constituting the amino acid sequence of the following formula (I); b) The N-terminal amine group is activated, and the remaining portion of the amino acid sequence of the target polypeptide (I) (e.g., partial peptide-substituted amide, proline-substituted amide, or substituted amine constituting the remaining portion) and a means of condensing both fragments in which at least one amino acid residue is protected.

The pyroglutamyl group of pyroglutamic acid or the pyroglutamyl group in the partial peptide having pyroglutamic acid at the N-terminus and constituting the amino acid sequence of formula (I) above is γ-
Carboxylic acids are easily converted into pyroglutamyl groups such as esterified or amidated glutamyl groups5
This includes cases where the

In addition to the intramolecular acylamino group of pyroglutamic acid and the side chain amino group of arginine, the polypeptide amide represented by general formula (I) includes histidine, tributophane, serine,
The molecule of pyroglutamic acid has a side chain reactive group such as tyrosine. The protecting group for the inner acylamino group and the protecting group for the amino group of the α-amino group of the intermediate peptide include benzyloxycarbonyl group 1. Protecting groups for the imino group of histidine include t-butoxycarbonyl group, inbornyloxycarbonyl group, etc., for example, benzyl, tosyl, 2
・4-dinitrophenyl, t-butyloxycarbonyl, carbobenzoquine, etc. are used as protective groups for the hydroxyl group of serine, such as benzyl, ethers such as t-butyl, and as protective groups for the hydroxyl group of tyrosine, such as benzyl. Examples of protective groups for the guanidino group of arginine include nitro group, tosyl group, carbobenzoxy, inbonyloxycarbonyl, adamantyloxycarbonyl, and the like.

However, protection of the intramolecular acylamino group of pyroglutamic acid has positive significance in terms of producing pyroglutamic acid itself and influencing the solubility of intermediates.

As described above, the protected peptide (I) of the present invention can be produced in which at least one side chain functional group of an amino acid residue of the peptide (I) is protected.

The protecting group of the protected peptide (I) of the present invention can be removed by any suitable means known per se that does not impair the amino acid sequence of the resulting polypeptide, and the free polypeptide shown in formula (I) can be removed by any suitable means known per se. Give peptides.

Means for this purpose include, for example, catalytic reduction using catalysts such as palladium black, palladium on carbon, platinum, etc., acid decomposition using, for example, hydrogen fluoride, trifluoroacetic acid, etc., reduction using, for example, metallic sodium in liquid ammonia, and formic acid decomposition. Examples include reduction with stannous chloride.

In addition, the above C-terminal carboxyl group is activated and the N-terminal imino group is protected or unprotected pyroglutamic acid or pyroglutamic acid is used as the N-terminus to constitute the amino acid sequence of the above formula (I). Partial peptides can also be produced by known methods or methods known per se.

Activated forms of the C-terminal carboxyl group of such compounds include, in particular, the corresponding acid anhydrides, e.g. mixed acid anhydrides with mono-lower alkyl esters of carbonic acid, azides, activated esters (penta Chlorphenol, 2, 4, 5-)'J dichlorenol, 2, 4-
Dinitrophenol, cyanomethyl alcohol, paranitrophenol, N-hydroxyphthalimide, N
-hydroxyphthalimide, esters with alcohols such as N-hydroxybenztriazole), and the like.

According to the knowledge of the present inventor, as an active ester,
N-hydroxy-5-norbornene-2,3-dicarboximide ester and 1-7-hydroxybenztriazole are particularly useful for this purpose.

The use of such esters allows the reaction to be carried out conveniently without racemization phenomena or side reactions in the case of N--idroquine succinimide ester.

In addition, when using N-hydroxy-5-norbornene-2,3-dicarboximide ester or 1-hydroxybenzolyazole, (a) the C-terminal carboxyl group is free and is protected or unprotected. (b) a partial peptide having an N-terminus of glutamic acid or pyroglutamic acid and constituting the amino acid sequence of the above formula (I); (b) an amino group at the N-terminus is free; , a partial peptide (substituted amide, proline, substituted amide or substituted amine) constituting the remaining portion, in which at least one amino acid residue of both fragments is protected, (C) a dehydrating agent, and (d) an N-hydrocarbon. Kin-5 norbornene-
It is also particularly recommended that 2,3-dicarboximide and 1-hydroxybenztrianol be reacted in one phase to produce the imide ester in the reaction solution and allow the condensation reaction to proceed in one operation.

The amino acid or partial peptide having an activated amino group may be of a known type, and specific examples thereof include the corresponding ring phosphoric acid amide.

Any dehydrating agent that can be used for peptide synthesis can be used, but so-called carbodiimide reagents such as dicyclohexylcarbodiimide (DCC) are particularly suitable.

Although there are many possible steps for producing peptides, the most preferred step often depends on the crystallinity of the intermediate.

This is because purification of intermediates particularly affects the purity of the final product.

In this regard, a preferable example is illustrated below.

In the above, R1 is a conventional C-terminal carboxyl group protecting group such as methyl, ethyl, t-butyl, benzyl, etc., and X and R have the same meanings as above.

The condensation reaction is generally carried out in the presence of an appropriate solvent, such as anhydrous or hydrous dimethylformamide, dimethyl sulfoxide,
Examples include pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, and mixtures thereof.

The reaction temperature is usually about -20°C to 30°C, but the reaction may be carried out with cooling or heating as appropriate.

The two raw materials to form an amide bond are usually used in corresponding molar amounts, but this may be changed as desired.

Usually, for every 1 mole of one raw material, about 1 to 2 moles of the other raw material is used.
molar equivalent, more preferably about 1 to 1.4 molar equivalent, and the amount of dehydrating agent and racemization inhibitor used is usually about 1 to 2 molar equivalents.
It is a molar equivalent.

A reaction time of about 6 to 10 hours is often sufficient, but α
-Aminoim It is preferred that sterically hindered amino acids such as butyric acid be used to prolong the reaction time.

After the reaction is complete, the reaction product can be collected by adding a solvent in which the reaction product is difficult to dissolve, such as ether or ethyl acetate, to precipitate it and filtering it.

If this reaction product has a protecting group, it is removed by means known per se as described above to obtain the desired formula (
The polypeptide of I) can be produced.

This can be collected by means known per se, such as the above-mentioned precipitation method.

The target products obtained in this way are, for example, carboxymethylcellulose, carboxymethylcephatex,
It may be purified by an appropriate means such as column chromatography using Amberlite XAD 2 (trade name).

Depending on the reaction or purification conditions used, this novel protected compound (I) may be obtained in the form of a base or a salt. From this salt, a base can be obtained in accordance with conventional methods, and this base can be used for therapeutic purposes. It can be converted into a salt by reaction with a suitable acid to form a salt that can be used for.

These acids include, for example, inorganic acids such as hydrohalic acid (e.g. hydrochloric acid or hydrobromic acid), perchloric acid, nitric acid or thiocyanic acid, sulfuric acid, phosphoric acid or organic acids such as formic acid, acetic acid, phlopionic acid, Examples include gelcholic acid, lactic acid, acid-free grape acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, and sulfanilic acid.

Polypeptide (I) and its salts produced in this manner have extremely low toxicity and can be safely administered.

For example, by intravenously, intramuscularly, or subcutaneously injecting a minute amount of approximately 1 to 20 ng/100f into rats, LH in the blood can be increased.
Also, the effect of strongly increasing FSH concentration is observed.

For example, R is an ethyl group and X is D Leu, D-NV
al, D-Abu or D-Ser polypeptide (I is about 2 to 3.5 ng per liter using rats with diestrus, which is the period when ovulation is most difficult).
When injected subcutaneously at a rate of 50% of test animals, an ovulatory effect is observed.

On the other hand, the amount of natural peptide (I[) required to exhibit the same effect is about 200 ng.

Therefore, it can be seen that ice crystals exhibit about 80 to 100 times more physiological activity than natural peptide ([[) in terms of ovulatory effects.

Polypeptide (I) in which R is in70 pyl or propyl is useful because it has an ovulation-inducing effect about 50 times that of natural peptide (II) and has a sustained effect.

Peptide (I) in which R is a methyl group is a natural peptide (I[
) has an ovulation-inducing effect about 10-30 times more effective.

Peptide (I) in which X is α-aminoisobutyric acid and R is an ethyl group was found to have a 50% ovulatory effect at 15 ng in the above test method, and in the case of XIJ''=D-phenylglycine, the ED5o was approximately 7ng.

Therefore, the peptide (I) of the present invention can be used, for example, in diagnosing pituitary function in humans or animals (e.g., sheep, pigs, cows, quail, knit IJ, etc.), treating hypofunction of the pituitary gland, and as an ovulation promoter. can be used.

The dosage varies depending on the target animal, the symptoms to which it is applied, etc., but it is generally about 1 ng to 1 µm of compound (I). /kg
be selected as appropriate from the range.

The administration is mainly carried out parenterally, such as by injection, and in some cases, rectally.

An injection solution can be prepared by dissolving compound (I) in physiological saline by a method known per se.

Compound (I) can also be used as a frozen ampoule using, for example, mannitol as a bulking agent.

Example 1 (Pyr) Glu-His-Trp-5er-
Tyr(D)-Leu-Leu-Arg-Pr
Production of o -NHCH2CH3 (a) Z-(D)-jeu-Leu-Arg(NO2)
Preparation of Pro-NH-CH2-CH3 Z -(D)-Leu-OH (oil) 1.51
' and 1.2 g of HONBI were dissolved in 50 ml of dichloromethane, and then H-Leu -Arg (NO2) -
Pr.

Add 2.8 g of NH-CH2-CH3 and cool to 0°C.
Add DCC1,4P to this and stir at room temperature for 8 hours.
The resulting dicyclohexylurea is filtered off and dried under reduced pressure, and the residue is dissolved in 150 ml of ethyl acetate, washed twice with 4% aqueous sodium bicarbonate and twice with water, dried over sodium sulfate, and dried under reduced pressure.

3. The residue was reprecipitated with ethyl acetate-petroleum benzyl.
6 P (85,5%) of the target product is obtained.

Melting point 134℃ (decomposition) [α] -47.3 (C=O15
4% in methanol) Rf' (silica gel thin layer chromatography, chloroform:methanol:acetic acid, 9:1
:0.1) -0,57 (Hereinafter, Rf' indicates the case of the same chromatography operation.

) Elemental analysis Calculated value as C33H5308N9 C156,31; Hl 7.59; N, 17
．． 91 Actual measurement value C, 56,08; H, 7,82; N, 17.6
9 (b) (Pyr)Glu-Hls-Trp-8
er -Tyr-(D) -Leu -Leu -A
rg(NO2)-Pr.

-NH-CH-CH3 Z -(D) -Leu -Leu-Arg (NO
2) Pro -NH-CH2-CH33,6f? 2
5% hydrogen bromide-dissolved in 30 rnl of glacial acetic acid and diluted with 50% hydrogen bromide at room temperature.
Shake for a minute to mix.

Add 300 ml of ether to this, collect the resulting precipitate by filtration, dry it, then dissolve it in 5 ml of water.
erlite) CG-410 (free base) column (2.5 x 20 crfL) and wash thoroughly with 30% methanol water. Combine the distillate and washing liquid, concentrate under reduced pressure to remove methanol, and freeze-dry to obtain H-(D). -Leu
Leu -Arg (NO2) -Pro -NH-C
The free base of H2CH3 is obtained 3.11.

Rf2 (silica gel thin layer chromatography, ethyl acetate; pyridine:acetic acid:water, 60:20:6:10) = 0
．． 24 Rf3 (silica gel thin layer chromatography n-butanol:ethyl acetate:water, 1:1:l:1)=
0.68 (In the following description, Rf2 and Rf3 indicate the same chromatography operation.

) crystal 228m9 and (Pyr) Glu-Hls
-Trp -8er -TyrOH290m9 was dissolved in 4 ml of dimethylformamide, and HONBI 144
Add m9 and cool to -10°C.

Add 90m9 of DCC to this and keep at -10℃ for 2 hours at 0℃.
Stir at C for 4 hours and at room temperature for 4 hours.

The reaction solution is filtered to remove dicyclohexylurea, ethyl acetate is added, and the resulting precipitate is collected by filtration.

This precipitate was dried, dissolved in 20% ethanol water, and poured into an Amberlite XAD-2 column (200 mesh, φ3 x 20 cm).
20% ethanol and 100% ethanol (300m/3
Perform gradient elution.

The target is 360m1 rather than 260m1! This fraction was concentrated under reduced pressure and further lyophilized to obtain 346m9 of protected peptide (Pyr) G.
lu -H1sTrp -8er -Tyr -(D
) -Leu -LeuArg(NO2) -Pro-
NH-CH2-CH3 is obtained.

[α] -38,28° (C=0.105%, in methanol) Rf2=0.235 Rf3=0.765
) (Pyr)Glu-His-Trp-8er
-Tyr-(D) -Leu -Leu -Arg-P
ro -NHCH2-CH3 production (b) 300 m9 of NO2 was added to 20 m1 of 6
Dissolve in 0% formic acid water, add 5nC12.600m9 of H2O, and heat at 80-85°C for 2 hours.

After drying under reduced pressure and extracting the residue with 50 mJ of warm water (80'C) and filtering, it was poured into a column (φ3 x 20 CIrL) of Amberlite XAD-2 (200 mesh), washed with water, and washed with water. Gradient elution is performed with % ethanol (400 mJ each).

The target substance elutes between 150ml and 260mJ, so this fraction is collected and concentrated under reduced pressure to remove ethanol, and then added to a carboxymethylcellulose column (φ2X33CrIl).
) and perform gradient elution with 0.005M to 0.2M ammonia acetate (pH 6,8).

The target peptide is eluted at 520 mA from 330 rILl, so when this fraction is lyophilized, a white feather-like substance 28
Get 2 ■.

[α]”3-32.66° (C=0.545.5% acetic acid in water) Rf2=0.10 Rf3=0.75 Amino acid analysis (hydrochloric acid hydrolysis in the presence of thioglycolic acid)
, Hisl, 00: Argo, 96: TrpO,
96:5er1.00; Glul, 00: Prol
, 04; Leu2.00: Tyrl, 00 Three Examples 2 (Pyr) Glu-His-Trp-8e
r-Tyr-a-Aibu-Leu-Arg
-Production of -Pro -NH-CH2CH3) Z -a -A ibu -Leu -Arg
Production of (NO2)Pro-NH-CH2-CH3 Z -a -A ibu -OH0,6? and H-Leu
Arg (NO2) -Pro -NH-CH2-C
7ml of H30,92 in dimethylformamide! The water-soluble carbodiimide [1-ethyl 3
-(3-dimethylaminopropyl)-carbodiimide)
Add 0.7 IP and stir for 12 hours.

The solvent was distilled off, and the residue was dissolved in 1 oomg of chloroform, washed with 4% aqueous sodium bicarbonate and N-hydrochloric acid, washed with water, and dried over magnesium sulfate.

The solvent was distilled off under reduced pressure, and the residue was filtered as a powder with ether.

When this was reprecipitated from ethyl acetate, the target product 47
Get 3■ (35%).

Melting point: 104-107°C [α] -45.8° (C=0
．． 55 in DMF) Rf”=0.50 Elemental analysis C3, H5oO8N9・0.5H20) BOC-Tyr (BzL) -a-Aibu
-LeuArg (NO2)-Pro-Nu-CH2-
Production of CH3 Za -Aibu -Leu -Arg (NO2)
Dissolve PrO-NH-CH2-CH3423m9 in 25% hydrogen bromide and 3 mJ of glacial acetic acid, and shake and mix at room temperature for 40 minutes.

Add 50 rnl of dry ether to this and collect the resulting precipitate by filtration.1. Dry under reduced pressure in a sodium hydroxide desiccator.

This powder was dissolved in DMF 3d, neutralized with 0.24 ml of N-ethylmorpholine, cooled to 0°C, and BOC-T
yr (Bzl) -OH557■ and HONB I
Add 298 mg, add 280 m9 of DCC and stir at room temperature for 6 days.

After removing dicyclohexylurea by filtration, DMF was distilled off under reduced pressure, and the residue was dissolved in 100 mJ of chloroform, washed with N-hydrochloric acid water and 4% sodium bicarbonate water, washed with water, dried over Mac)ium sulfate, and dried under reduced pressure. It solidifies and the residue is crystallized from ethyl acetate.

Yield 275 ■ (49.5%), melting point 127-129 °C [α) 2 g 3-27.6 ° (C = 0.54, in DMF)
Rf1=0.60 Elemental analysis C44H6701ON10 ” N20 (c) BOC-8er -Tyr (Bzl
) -a-AibuLeu -A rg (NO2)
-Pro -NH-CH2CH3 production BOC-Tyr (Bzl) -Aibu -Leu
Arg (NO2) -Pro -NH-CH2-CH
Dissolve 32507'llp in trifluoroacetic acid containing 1 ml of anisole and shake at room temperature for 30 minutes.

30rrLt of dry ether is added to this, and the resulting precipitate is filtered and dried.

Dissolve this powder in 5 ml of DMF and add 0.08 ml of N-ethylmorpholine. Neutralize with BOC-S e
Add trichlorophenyl ester 15 of r (Up) and let it swell for 2 days.

DMF was distilled off, the residue was filtered as a powder with ether, and reprecipitation was repeated twice with ethyl acetate.

Yield 100m9 (38%) Melting point 140-142℃ (decomposed), [α] -34.8℃ (C=0.44 in DMF) Rf1=0.74 Elemental analysis C4□H7□0□2
Calculated value as N1, 3H20 C, 54, 43, H,
7,58, N114.86% Actual value C, 54,66; H, 7,45; N, 14.9
1% (d) (Pyr)Glu-Hls-Trp-8
er -Tyrα-A ibu -Leu -Arg
-Production of -NHCH2-CH3 BOC-8er -Tyr (Bzl) -a-Ai
bu -Leu-Arg(NO2)-Pro-NH-C
Dissolve 377 m9 of H2CH in 3 ml/l of trifluoroacetic acid containing 0.1 ml of anisole and shake at room temperature for 30 minutes.

Add 3 ml of dry ether to this, filter the resulting precipitate, and dry.

This powder was dissolved in 3 ml of DMF, neutralized with 0.21 mJ of 10% N-ethylmorpholine-DMF solution, and (Pyr) G lu -Hls -T rp -O
Add 37cm of H crystals, cool to -10°C, and add HONBI
21 mL? Add 30 m9 of DCC and stir at 12 hours.

Dicyclohexyl urea was removed by filtration, and the filtrate was dried under reduced pressure.
Dissolve the residue in 20% ethanol and add Amberlite C.
, Amberlite) XAD2 column (φl
Xl0cIrL) and 1 from 20% ethanol.
Perform gradient elution to 0.00% ethanol (in 2 oomz increments).

Collect target classifications with ethanol concentration between 40% and 70%,
After vacuum concentration and lyophilization, the protected nonapeptide amide (Pyr)Glu-H1sTrp-Se
r -Tyr (Bzl) -a -A ibuLe
u-Arg(NO2)-Pro-NH-CH2CH33
Obtain 0m9 (Rf2=0.31) of the h-protected nonapeptide amide in 0.1 ml of anisole.

Add 0.1 ml of mercaptoethanol, stir with 3 ml of anhydrous hydrogen fluoride at 0°C for 60 minutes, distill off the hydrogen fluoride under reduced pressure, and then dissolve in 1 omz of water.

This was extracted three times with 2 ml of ether, and the aqueous layer was passed through a column of Amberlite CG-410 (acetic acid type) (φ2×4 (X)) and passed through water.

The effluent and washing solution were combined and applied to a carboxymethylcellulose column and an Amberlite XAI-2 column respectively in the same manner as in Example 1, and lyophilized to obtain white feather-like 14m9.

[α] Wart-54,10 (C=0.31.5% in acetic acid)
Rf2=0.099 Rf3=0.715 Amino acid analysis: Hiso, 94; Argo, 9
7 rTrpo, 81: 5erl, 06: Glu
o, 97; Pr.

0, 97: Leul, 03: Tyrl, 03
;Abul, 00 Example 3 (Pyr) Glu-Hls-Trp-8er
-Phe(D) -Leu -Leu -Arg
-Pro -NH-CH2-CH3 production a) (Pyr)Glu -Hls -Trp -8
Production of er -Phe-OH Z -S er -Phe -OMe 4. Dissolve Off in 100ml of methanol and add 500ml of palladium black.
was used as a catalyst for reduction at normal pressure for 1.5 hours.

The catalyst was removed by filtration, methanol was quickly distilled off under reduced pressure, and the residue was dissolved in DMF 25r/'Ll.
) Add crystal 4.11 of Glu-His -Trp -OH, cool to -5°C, and add HONBI 3.8P and DCC.
, l' and stirred at -5°C for 2 hours, at 0°C for 2 hours, and then at room temperature for 8 hours.

The resulting dicyclohexyl urea was filtered off, and the filtrate was dried under reduced pressure and filtered as a powder with ethyl acetate (6.71°) This powder was dissolved in 30 rnl of 15% methanol-chloroform, and the solution was also prepared with 15% methanol-chloroform. Pour into a silica gel column (φ6 x 12 cyrt),
Elute with 600 ml of 15% methanol-chloroform, 25% methanol-chloroform IJ, and 30% methanol-chloroform IJ, respectively.

400ml of 25% methanol-chloroform
Collect up to 7 ooml of 0% methanol-chloroform, dry it under reduced pressure, add ethyl acetate to it, and filter it as a powder to obtain the desired product in a yield of 4.21.

Rf2=0.29 3.52 of this methyl ester was suspended in 20 ml of methanol, cooled to 0°C, 8 ml of N-caustic soda water was added, stirred for 1 hour, and 8 ml of N-hydrochloric acid water was added.
Add to neutralize.

Methanol was distilled off under reduced pressure, and 20 ml of cold water was added to cool the precipitate. The precipitate was collected by filtration, washed with cold water, then washed with ethanol-ether (4:6), and dried to obtain the desired product, 2.32P ( 66%).

[α] Dorsal, 5-3.3° (C=0.55, in glacial acetic acid)
Rf=0.61 :b) (Pyr) Glu-Hls-Trp
-8er -(D)-Leu-Lau-Arg
(NO2) -Pro -NHCH2-CH3 production (Pyr) Glu -Hls -T rp -S
er -Phe-OH290m9 and H-(D)-L
eu -LeuArg(NO2) Pro NHC
Dissolve H2CH3228■ in DMF 4rul,
Add ONBll, 407Q and cool to -10°C.

Add DCC90 to this and stir at -10°C for 2 hours, at 0°C for 3 hours, and then at room temperature for 5 hours.

This reaction solution was developed on Amberlite XAD-20 column chromatography in exactly the same manner as in Example (1), and the protected peptide (Pyr)G1u-His-Trp-Se
r-Phe-(D)-Leu-I, eu-A
rg (NO2) Pr.

-NH-CH2-CH3342m9 was obtained.

[α]2D5-41.74° (C = 0.115%, in methanol), Rf2 = 0.265, Rf” = 0.78 Dissolve this product 300In9 in 20ml of 60% formic acid water and
After adding 600 cm of nC12.H2O and leaving it at 80-85°C for 2 hours, it was dried under reduced pressure and purified using an Amberlite XAD-20 column and a carboxymethylcellulose column in the same manner as in Example (1). .

A white feathery substance 211■ is obtained.

[α] -38,49° (C=0.53.5% in acetic acid)
Rf2=0.12 Rf3=0.74 Amino acid analysis (hydrochloric acid melt in the presence of thioglycolic acid), Hisl
,OO:Argl,04;TrpO,98:5er0
．． 92: Glul, 00: Pro 1.00; Le
u'2.04: Phe 1.00 downstream 4 (Pyr) Glu-Hls-Trp-8er
-Tyr(D) -Abu -Leu -Arg-P
ro -NH-CH-CH3L)Z-(D)-Abu-Leu-Arg
Production of (NO2) -Pro-NH-CH2-CH3 Z -(D) -Abu-OHO,3? and H-Leu
-Arg(NO2)-Pro-NH-CH2-CH30
, 45S' was dissolved in 5 ml of dimethylformamide, and 0
Cool to ℃ and add HONB I O, 25g and DCCo, 2
Add 91 and stir at 0°C for 1 hour at room temperature for 5 hours.

The urea compound is filtered off, and the filtrate is dried under reduced pressure, dissolved in 80 ml of chloroform, washed with N-hydrochloric acid and 4% sodium bicarbonate, washed with water, dried over magnesium sulfate, and dried under reduced pressure.

The powder was filtered with ether and reprecipitated with ethyl acetate-petroleum benzine.

Yield: 489 ml, melting point: 111-114°C.

[α]￥-51,2° (C=0.6 in methanol), Rf1=0.66 b) (Pyr) Glu-Hls-Trp
-Ser -Tyr(D) -Abu -Leu
-Arg -Pro -NH-CH2-CH5 Z (D) -Abu -Leu -Arg (N
O2) -Pro -NH-CH2-CH3 at 25%
Treated with hydrogen bromide and glacial acetic acid, precipitated with ether and collected by filtration.
This is Amberlite CG
H-(D)-Abu-Leu-Arg (N
O2) Pro-NH-CH2-CHs free base 220
■ and (Pyr) Glu-His -Trp-8er
-Tyr-OH2901nJ? and DMF 3ml
Add HONBI 1501n9 to -10℃
Cool to , add 85m9 of DCC, and heat at -10°C for 2 hours.
Stir at 0°C for 4 hours and at room temperature for 7 hours.

Ethyl acetate was added to the reaction solution, the resulting precipitate was collected by filtration, and 2
When heated and dissolved in 0% ethanol water and cooled, a precipitate is deposited.

This is collected by filtration and dried to obtain the above-mentioned target product (Pyr)G.
lu -Hls -Trp -8er -Tyr(D
) -Abu -Leu -Arg (NO2) -P
r.

-NH-CH2-CH3 is obtained.

Yield 284■. Rf2=0.240. Rf3=0.7
72 Dissolve 200 μm of ice crystals (containing about 10% of small Rf impurities in both solvent systems) in 15 md of 60% formic acid water, add 400 mg of SnCl2-H20, and heat at 80° to 80°C.
Warm at 5°C for 2 hours.

It was dried under reduced pressure, the residue was extracted by adding 50rrLl of warm water, filtered to remove insoluble matter, and Amberlite (Am
berlite) XAD-2, carboxymethyl cellulose, and Sephadex LH-20 columns, and finally freeze-dried to obtain 104 cm of white feather-like material.

[α] -43.6° (C = 0.50, 5% acetic acid in water) Rf2 = 0.08, Rf3 = 0.52 Amino acid analysis (in the presence of thioglycolic acid), Hiso, 98:
Argo, 96; Trpo, 94; 5erO, 9
2: Glul, OO: Prol, OO: LeuO, 9
8: Abul, 00: Tyrl, 00: Ethylamine 1.02 Example 5 (Pyr) Glu-Hls-Trp-Se
r -Tyr -(D) -NVa -Leu -A
Production of rg -Pro -NHCH2-CH3 a) Z (D) Production of NVa-OH H-(D)-
NVa-OH is reacted with carbobenzoxy chloride in 2N caustic soda water in a conventional manner, and the product is recrystallized from ethyl acetate-petroleum benzine to obtain needle crystals.

Melting point 83-84℃ [α]+12.1° (C=1.0, in methanol) (b) Z-(D)-NVa-Leu-Arg
(NO2)Pro-NH-CH-CH3 Z-(D)-NVa-OH376m9, H-L
eu -Arg (NO2) Pro -NH-CH2
-CH3685■, HONBI 295m9 DMF
Dissolve in 5 mA and add DCC340 at 0°C.
Stir for 2 hours at room temperature and 8 hours at room temperature.

The resulting urea compound was filtered off, 100 m4 of ethyl acetate was added, and the mixture was washed with 0.5N hydrochloric acid and 4% sodium bicarbonate, and then dried with sulfuric acid.

This is dried to dryness under reduced pressure, filtered with ether as a powder, and purified by reprecipitation with ethyl acetate-ether.

Yield 900■ Melting point 105-107℃ (decomposition), [α]
Eagle-51,2° (C-1, in methanol) Rf-0,
60 (c) (Pyr) Glu -Hls -Tr
p-Ser-Tyr-(D)-NVa-Le
Z obtained in u -Arg -Pro -NHCH2-CH3 (b) (D) -NVa-Leu -
Arg(No2)-Pro-NH-CH2-CH3 and (
Pyr ) G 1u-His -Trp -8er
-Tyr-OH, the reaction was carried out in exactly the same manner as described in Example 4-(b), and the purified peptide (Pyr) Glu-Hls-Trp-
8erTyr (D) NVa -Leu -Arg
(NO2)Pro-NH-CH2-CH3 is obtained.

This is further processed in the same manner as in 4-(b) to obtain the desired product.

[α] -39.8° (C = 0.51.5% acetic acid in water) Rf2 = 0.094, Rf3 = 0.74 Amino acid analysis (in the presence of thioglycolic acid); Hisl, 00:
ArgO, 98: TrpO, 93; 5erO, 94:
Glul, 08: Prol, 02: NVaO, 98
;Leul, OO: Tyro, 98;Ethylamine 1.04 Example 6 (Pyr) Glu-Hls-Trp-Se
r-Phe-(D)-NVa-Leu-A
Production of rg -Pro -NHCH2-CH2-CH3 (a) Z -(D) -NVa -Leu -A
rg (N02)Pro-NH-CH2-CH2-CH
Production of 3 Z -Leu-A rg (NO2) -P
ro -NH-CH2-CH2-CH3720m2
Dissolve in 2 ml of 5% hydrogen bromide-glacial acetic acid and shake for 40 minutes.

To this was added 20 ml of dry ether, and the resulting precipitate was collected by filtration, thoroughly washed with dry ether, and dried under reduced pressure in a sodium hydroxide desiccator.

After drying, dissolve in 5 ml of DMF, and add Z-(D
)-NVa 250m9 and triethylamine 0
．． Add 15 mJ and 200 mg of HONBI, cool to 0°C, add 230 m9 of DCC, and stir at room temperature for 8 hours.

After adding 80 ml of ethyl acetate and filtering out insoluble matter, the mixture was washed with N-hydrochloric acid water and 4% sodium bicarbonate water, and after washing with water, it was dried over sodium bicarbonate.

This is dried under reduced pressure, and the residue is purified by powdering it with petroleum ether and reprecipitating it with ethyl acetate-petroleum ether.

Yield 718 m9 [α1 section -51,00 (C=1.0, in methanol), Rf1=0.64 Elemental analysis Calculated value as C33H5308N9 C156,23;) (17,58; N117.
89% Actual value C156,14; H, 7,70; N, 17.5
8% (b) (Pyr) Glu-Hls-Trp
-8er-Phe(D)-NVa-Leu-Arg-P
Production of ro-NHCH2-CH2-CH3 Z -(D)-NVa-Leu-Arg(N
O2) Pro -NH-CH2-CH2-CH350
Dissolve 0Tug in 3 ml of 25% hydrogen bromide-glacial acetic acid,
Mix for 0 minutes.

Add OQml of dry ether to this, collect the precipitate by filtration, dissolve it in 40 mJ of water, and make Amberlite.
e) CG-410 (free base) column (φ1×
141Z771) and a5 with 30% methanol water.
゜The effluent and washing liquid are combined, methanol is distilled off under reduced pressure, and lyophilized to yield H-(D)-NVa-Leu-A.
rg (NO2)Pro NHCH2CH2CH3 free base 3207119 is obtained.

This product 225■ and (Pyr)G 1u-His -Tr
DM p -S er -Phe -OR280m9
Dissolve in 2ml of F, add HONBI 1307Q and cool to -10°C.

Add 87m9 of DCC to this and keep at -10℃ for 3 hours at 0℃.
Stir at room temperature for 2 hours, then at room temperature for 6 hours.

Add 40 ml of ethyl acetate to the reaction solution, collect the resulting precipitate by filtration, dissolve it in 30% ethanol water by heating, and filter off the insoluble matter.
Upon cooling, a precipitate separates out.

The protected peptide (
Pyr) GluHis-Trp-8er-Ph
e −(D) −NVa−Leu −Arg (NO
2) -Pro -NH-CH2CH2-CH3340
Get m9.

Dissolve 300 m9 of this product in 20 m1 (7) of 60% formic acid water, add 580 r/l of C1□H2O, and heat at 85°C for 2 hours.

The reaction solution was dried under reduced pressure and extracted with warm water.
(Amberlite)
Obtain 70m.

[α]￥'-37.7° (C=0.505.5% acetic acid in water), Rf2=0.13, Rf3=0.76 Amino acid analysis (in the presence of thioglycolic acid): Hisl, 02:
ArgO,98:Trpo,97:5erO,9
8; GILll, 02: Prol, OO: Leul
,0ONVa1.02; PheO,98 Example 7 By solid phase method (Pyr) GIu-Hls-
TrpSer -Tyr-(D) -Leu-Leu
-ArgPro -NH-CH2-CH3- production (a
) Production of BOC-Pro resin Chloromethyl resin 15P (CI content 9.45rrLmol)
50rn1. and BOC-Pr.

Add 6.1 g of OH and 3.33 fd of triethylamine and shake for 5 days.

The resin is collected by filtration, washed thoroughly with DMF, washed with ethanol, water, ethanol, and ether, and dried.

Yield: 17.55P, this resin has a yield of 0.3 from amino acid analysis.
Contains 1477 Lmol/P of BOC-Pro.

(b) (Pyr)Glu-His(Tos)-T
rp -8er (Bzl) -Tyr (Bzl
) -D -Leu -Leu -Arg (Tos
)-Pro-Resin production BOC-Pr obtained in (a)
The o-resin 3.145S' was placed in a reactor of an automatic peptide synthesizer (Shimadzu Corporation, APS = 800), swollen with dichloromethane for 12 hours, and each amino acid was introduced in the following cycle.

Dichloromethane (3 minutes x 3 times) 150% trifluoroacetic acid/dichloromethane (10 minutes and 30 minutes) → dichloromethane (3 minutes x 3 times) → ethanol (3 minutes x
3 times) → dichloromethane (3 minutes x 3 times) 110% triethylamine/chloroform (10 minutes) → chloroform (3 minutes x 3 times) → dichloromethane (3 minutes x 2 times)
→ BOC-amino acid-anhydride synthesized from BOC-amino acid and DCC) (30 and 60 minutes) → Acetylation (using dichloromethane, triethylamine, and acetic anhydride) (1 hour) → Dichloromethane (3 minutes x 3 times) ) [(Pyr)Glu-OH only is directly DCC in DMF.
Condensation reaction was carried out] Finally, the resin was washed with ethanol, chloroform, and dimethylformamide, and then with ether and dried.

Yield 3.81'. The resin 3.52P obtained in (b) was suspended in 20 ml of methanol and cooled to 20°C, and the ethylamine produced by heating 80% ethylamine water in a hot water bath was blown through sodium hydroxide until it was sufficiently saturated.
Stir for 40 minutes at room temperature.

The resin is collected by filtration, the filtrate is dried under reduced pressure and powdered with ether to obtain a crude product of 835 mp.

This crude product contained a large amount of impurities thought to be methyl esters, so this 503■ was added to methanol-D.
Ethylamine was introduced into MF (8 mJ-8 mJ) in the same manner as described above.

After stirring for 34 hours, the methyl ester will disappear.
The protected peptide (Pyr) Glu-Hls (To
s)-Trp-8er (Bzl)-Tyr
(Bzl) -DLeu -Leu -Arg (T
os ) -Pro -NHCH2-CH3 is obtained.

4657%d) (Pyr) Glu-His −
Trp-8er-Tyr-D-Leu-Leu-A
Preparation of rg-Pro -NH-CH2CH3 The protected peptide 327.8m9 obtained in (e) was mixed with 0.
Dissolve in 8 ml of anhydrous hydrogen fluoride in the presence of 4 ml of anisole and 0.4 rnl of mercaptoethanol and stir at 0° C. for 1 hour.

It was dried under reduced pressure, dissolved in 20ml of water, and filtered to remove insoluble matter.Amberlite IRA-
410 (acetic acid type) column (φ1.5X20crrL)
and then carboxymethylcellulose and Amberlite XAD.
-2 and further Sephadex LH-20
Gel filtration was performed using a 0 column to obtain 98 m2 of the target product.

[α] Back-32,00 (C = 0.52.5% in acetic acid water)
Rf”=0.101. Rf3=0.75 Amino acid analysis (in the presence of thioglycolic acid): Hiso, 98:A
rgl, 00: Trpo, 87: 5erO, 9
0: Glul, 02: Prol O2: Leu2.
01: TyrO, 98; Ethylamine 1.10° Example 8 (Pyr) Glu-His-Trp-8er
-Tyr (D) -8er (OBu") -Leu
-Arg(Tos)-Production of -NHCH2CH3 (a) Z-(D)-8et (0But)
-LeuArg (Tos) -Pro -NH
-CH2CH3 production Z (D) -8er (0B
ut) -OH1,48? and HONBI 1.08′
? Dissolve in 30ml of DMF, and further add H-Leu
-Arg (Tos) -Pro -NHCH2CH
32.83g was added and cooled to 0°C, and DCCl,
Add 24P and stir at room temperature for 8 hours.

The resulting dicyclohexylurea is filtered off and dried under reduced pressure, and the residue is dissolved in 1 oomz of ethyl acetate, washed three times with 4% aqueous sodium bicarbonate and three times with saturated brine, and dried over sodium sulfate.

This was again dried to dryness under reduced pressure, and the residue was reprecipitated with ethyl acetate-petroleum benzene to obtain 3.5 CI (83.1%) of the desired product.

Rf'=0.83 Elemental analysis Calculated value as C4, H6209N8S C,
58,41;H,7,41,N, 13.29;S
, 3.80 actual value C158,66; N1751; N112.91; S, 3.60 (b) (Pyr) Glu-His-Trp-
8er -Tyr(D) -Ser(0But)
-Leu -Arg(Tos)-Pro -NHC
Production of H2CH3 Z-(D)-8et (0But)-LeuAr
g (Tos) -Pro -NHCH2CH344
3m9 was dissolved in 20rrLl of methanol, and catalytic reduction was carried out at room temperature and pressure for 5 hours using palladium black as a catalyst.

The catalyst was removed by filtration and the filtrate was dried under reduced pressure to form a residue (
Pyr) Glu-Hls-Trp-8er-
Dissolved in Tyr -OH351mti and DMF 5rlLl, N-hydroxybenztriazole 135
Add ① and cool to -5℃.

DCC206■ was added to the mixture and stirred at -5°C for 4 hours and at room temperature for 8 hours, and the precipitated dicyclohexylurea was filtered off and dried under reduced pressure.

Dissolve this in 5% ethanol and
berlite) XAD2 column (1,5x20
CrIL) and gradient elution with 5% ethanol (300ml) and 80% ethanol (300ml).

The main fractions are collected, ethanol is distilled off and lyophilized.

Yield 103■.

Add 0 more crystals. Dissolve in IN acetic acid water and gel filtrate with Sephadec LH-20 column (IX55crrL).

Lyophilize to obtain the pure target product of 781119.

[α] -27.9° (C = 0.5.5% acetic acid water) R
f-0,15, Rf3=0.85 Amino acid analysis (in the presence of thioglycolic acid), Hisll
, 00: Arg, 0.92: Trp, 0.
21: Set, 1.82: Glull, 00:
Pro, 1.04; Leu, 1.00; Ty
r, 0.98 Example 9 (Pyr) Glu-Hls-Trp-8er
-Tyr -(D) -Phg -Leu -Ar
Production of g-Pro -NH-CH2CH3 (a) Z-(D) -Phg -Leu-Arg(
No2) -Z-(D)-Phg-OH580■ and H-Leu-Arg (NO2) Pro NT(
CH2CH31,14r with dimethylformamide 10r
rLl and dichloromethane (20rlll) mixture,
Add 400mg of HONBI and cool on ice to DCC500.
Add 1n9 and stir at room temperature for 4 hours.

Dicyclohexylurea produced is filtered off, dried under reduced pressure, and dissolved in 100 ml of ethyl acetate.

This is washed with 4% sodium bicarbonate and water, dried over sodium sulfate, and the ethyl acetate is distilled off.

When the residue was reprecipitated with ethyl acetate petroleum ether, powder 1 was obtained.
．． Get 41.

Rf=0.65, Rf2=0.83 [α)2D5-80.2 (C=0.5 in methanol) b) (Pyr)Glu -Hls -Trp-
8er -(D)-Phg -Leu -Arg -P
ro -NH-CH2-CH3 Z-(D) Phg -Leu -Arg (No2
) -Pro -NH-CH2-Cf (3150 m9 was dissolved in 20 ml of acetic acid, 100 ml of palladium black was added and reduced by passing hydrogen under normal pressure.

After 4 hours, the catalyst was removed by filtration, 0.4 ml of N-hydrochloric acid was added, and the mixture was dried under reduced pressure, and then dissolved in 20 liters of water and freeze-dried.

The obtained powder was dissolved in 8 ml of dimethylformamide,
0.25 ml of 10% N-ditylmorpholine-dimethylformamide solution was added and further (Pyr)Gl
u -Hls -Trp -8er -0H141■ was added thereto, and 1-hydroxybenztriazole 5-OIn9 was added thereto at -5°C, followed by DCC40In9.
Add and stir at -5°C for 2 hours, at 0°C for 2 hours, and at room temperature for 8 hours.

Ether was added to the reaction solution, the precipitate was collected by filtration, and the precipitate was poured into 2 oz of water.
Dissolve in rnl.

Insoluble materials were filtered off and Amberlite
e) Thoroughly wash the XAD-20 column with water.

When this was eluted with 80% ethanol and the solvent was distilled off (
Pyr) Glu-HlsTrp-8er-Ty
r -(D) -Phg -Leu -A rg -
Pro -NHCH2CH3 hydrochloride 1317+19 is obtained.

Rf2=0.10 Amino acid analysis (in the presence of thioglycolic acid) Hiss, 96: ArgO, 96: Tr
pO192: 5erO,90:Glul,02;P
rol, 04, Leul, OO; PhgO, 92:T
yrO,93 Dissolve this hydrochloride in water and add Amberlite.
lite) Pass through a column of IRA-410 (acetic acid type) and wash with water.

The pass through solution and washing solution were combined and adsorbed on a carboxymethyl cellulose column (1.5 x 25 crIL), and 0.0
05M ammonia acetate and 0.175M ammonia acetate (
Perform gradient elution of 300 ml each at pH 6, 8).

The target substance is eluted in the 240 rnl section from 170 rrLl, so this is collected and applied to an Amberlite XAD-2 column (2
Pour the adsorbed target substance into water (20cfrL).
When gradient elution is performed using 50 ml) and ethanol (280 ml), the pure product is eluted in the 240 ml section from 190/nl.

This is concentrated under reduced pressure and freeze-dried to obtain the target product 1101v.

Rf2 - 0.098, Rf3 = 0.73 [α] -57.0 (C = 0.56 5% acetic acid core amino acid analysis (in the presence of thioglycolic acid), HisO, 96
:Argl,04 :TrpO,92:5erO,9
3; Glul, OO; Prol, OO: Lenl, 01
:PhgO, 98:Tyrl, 01 Example 10 (Pyr) Glu-Hls-Trp-8er
-Tyr -(D) -8er-Leu -Arg
-Pro -NH-CH2-CH3 production (a) Z-(D) -8er -Leu-Arg(
Production of NO2) -Pro -NH-CH2-CH3 Z -(D) -8er -OH9551n9, H-
Len-Arg (NO2) -Pro -NH-C
H2-CH31, 82? Oyohi HONBI 788■Woshi-A-xane 10ml and dimethylformamide 10r
DCC907 was dissolved in a mixture of rLl and cooled to 0°C.
Add (2) and stir at 0°C for 2 hours and at room temperature for an additional 5 hours.

The resulting dicyclohexylurea is filtered off, dried under reduced pressure, and the residue is dissolved in 100 m7 of chloroform.

The product is washed with 0.5N hydrochloric acid and 4% sodium bicarbonate in that order, dried over magnesium sulfate, dried under reduced pressure, filtered with petroleum ether as a powder, and reprecipitated from ethanol and ether.

Yield 2.4'i? Melting point 123-126 (decomposition) [α:l'i 49.8 (C=0.51.Iri/-
/L/Medium] Elemental analysis C30H4709N9 ” N2
Calculated value as 0: C, 51,79; H, 7,09; N
, 18.12% Actual value: C152.01; N16.96; N118.0
4% Rf1=0.40 (b) (Pyr)Glu-Hls-Trp-8
er -Tyr-(D) -3er -Leu -A
Production of rg-Pro-NH-CH2-CH3 Z-(D)-8er-Leu-Arg (No
□) -Pro -MH-CH2-CH3334m& was dissolved in a mixture of 20 ml of methanol and 1 ml of glacial acetic acid,
Catalytic reduction is carried out at room temperature and pressure for 4 hours using palladium black as a catalyst.

The catalyst was removed by filtration, and the filtrate was dried under reduced pressure to a 0. IN hydrochloric acid water 9.6
Dissolve the residue in ml and lyophilize.

This dry powder and (Pyr)Glu-Hls-
Trp-8er-Tyr-OH280m9 in DMF
Dissolve the solution in 3 ml, add 100~ of N-hydroxybenztriazole and 0.615 mA of 10% N-ethylmorpholine-DMF solution, and cool to -5°C.

DCC165■ was added to the mixture and stirred at -5°C for 4 hours and at room temperature for 8 hours, and the precipitated urea compound was filtered off and dried under reduced pressure.

The residue was dissolved in 20 ml of water, insoluble matter was removed by filtration, and the mixture was poured onto an Amberlite XAD-20 column and thoroughly washed with water.

This was eluted with 60% ethanol, the main fraction was collected, and when the solvent was distilled off, (Pyr) Glu-Hls-Trp-8er-
Tyr(D)-8er-Leu-Arg-Pro
-NHCH2CH3hydrochloride 178% is obtained.

Rf2-O,06 Amino acid analysis (in the presence of thioglycolic acid), HisO,
94:Argl, 08;Trpo, 94:5er
1.81: Glul, 01: Prol, 02: L
Dissolve the hydrochloride of eu 1.00Tyr0.9 in water and prepare Amberlite IRA-410 (acetic acid type)
column (10 ml), and the effluent and washing liquid were combined and adsorbed onto a carboxymethyl cellulose column (1,5X 27 crIL).

This was mixed with 0.005M ammonium acetate buffer (3
00mJ) and 0.15M ammonia acetate (300mJ)
Gradient elution with 280ml to 322ml
Since the desired product was found in the elution fraction up to, this was collected and freeze-dried to obtain 135 ml of crude product.

Dissolve it in a small amount of water and add Amber light.
lite) XAD-2 column (1,5X17c
rrL) and gradient elution with 5% ethanol water (300 ml) and 0% ethanol water (33 omi).

A 231 ml portion from 196 mJ is collected, ethanol is distilled off, and lyophilized.

Yield: 621n9° crystal. Dissolve in IN-acetic acid water and gel filtrate with Sephadex LH-20 column (1 x 54 crrL).

Lyophilize to obtain 40 m9 of pure target material.

[α] Trade-46.4 (C = 0.5.5% acetic acid water) Rf”
=0.055, Rf3=0.70 Amino acid analysis (in the presence of thioglycolic acid), Hisl,
OO; Argl, 04: Trp 1.00:
5er1.81 :Glul, OO:Prol, OO:
Leul, 04Tyre, 96: Ethylamine 1.
04 Example 11 (Pyr) Glu-Hls-Trp-8er
-Tyr -(D) -Abu -Leu -Arg
-Production of -Pro -NHCH(CH3)2 (a) Z-(D) -Abu -Aeu -Ar
g (NO2)Pro Production of NHCH(CH3)2 Z -Leu -Arg (NO2) -Pro -
2700 m9 of NHCH (CH3) is dissolved in 3 ml of 25% hydrogen bromide-glacial acetic acid, shaken for 60 minutes, 50 ml of dry ether is added thereto, and the resulting precipitate is filtered and dried.

After drying, dissolve in 6 ml of DMF and further add Z-(D)
-Abu -Add OH240rtbp and Torie fAy
7 Neutralize with 70.15 ml of HONBI200m
Add 9 and cool to 0°C, add 230m9 of DCC and stir at room temperature for 6 hours.

Add 100ml of ethyl acetate to this, filter out insoluble matter,
Wash with 0.5 N hydrochloric acid and 4% sodium bicarbonate, and dry over sodium sulfate.

After drying under reduced pressure and powdering with ether, reprecipitating with ethyl acetate-ether yielded 725 m9 [α] -52.0° (C = 0.5, in methanol), Rf' = 0.62 Elemental analysis C3□ 2σ calculated value as H5□08N9 C, 55, 71; H, 7, 45; N, 18.2
7 Actual measurement value C155,42;H,7,59;・N,18.
01 (b) (Pyr)Glu-Hls-Trp-8
er −Tyr(D) −Abu −Leu −Ar
Z-(D)-Abu-Leu obtained in production (a) of g-Pro-NH-CH(CH3)2
-Arg(NO2) Pro NHCH(CH3
)2 and (Pyr)Glu-Hls-Trp-8
er -Tyr -OH and (Pyr) Glu -Hls in exactly the same manner as shown in Example 5.
-Trp-8erTyr -(D) -Abu -L
eu -A rg -P r.

The hydrochloride of NHCH(CH3)2 is obtained.

Rf2=0.10 Amino acid analysis (in the presence of thioglycolic acid), Hisl,
OO; Argo, 92; TrpO, 88; 5erO
, 90; Glul, 02: Prol, 00; Abu
1,00Leu1.00: Tyr 0.88 This hydrochloride is operated in exactly the same manner as shown in Example 5 to obtain the desired product.

(α)2,9 41.00 (C=0.62.5% in acetic acid) Rf2=O8 ■1, Rf3=0.74 Amino acid analysis (
(in the presence of thioglycolic acid), Hisl, OO:Arg
O, 90; TrpO, 87: 5erO, 97: Glu
l, 00: Proo, 94: Abul, OO: L
eul, 02: Tyr O, 96°

Claims (1)

[Scope of Claims] 1 General formula [In the formula, if the amino acid may have optical isomers, unless otherwise specified, it represents one isomer. X is D-Leu, D-NVa, D-Abu, D-
Phg, aAibu or D-Set, R represents a lower alkyl group. ] Among the amino acid residues, the imino group of His is benzyl, tosyl, 2,4 dinitrophenyl, t-7” tyloxycarbonyl or carbobenzoxy, Ser and T
The hydroxyl group of yr is benzyl or t-butyl, and the guanidino group of Arg is nitro, tosyl, carbobenzoxy, inbonyloxycarbonyl, adamantyloxycarbonyl, or at least one protected with hydrologenide. A method for producing a peptide, which comprises condensing a partial amino acid or a peptide that can constitute the polypeptide with the remaining portion, both of which have at least one amino acid residue protected. Method for producing polypeptides.