JPH036160B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing peptides by protecting guanidino groups.

In order to produce a peptide using a raw material compound (such as arginine) containing [Formula], it is necessary to protect the guanidino group. Protection of the guanidino group has conventionally been carried out by introducing a nitro group or a tosyl group. In these conventional methods, the yield when removing the protecting group is low, and in order to remove the tosyl group, it must be carried out under strong conditions using liquid ammonia-metallic sodium or anhydrous hydrogen fluoride. As a result, other parts of the peptide are decomposed and by-products are produced, resulting in a reduction in the yield of the desired peptide. The present inventors have proposed a method to overcome these drawbacks by first using p-methoxybenzenesulfonyl, meditylenesulfonyl, etc., which can be easily removed with methanesulfonic acid, as a protecting group for the guanidino group. (Japanese Unexamined Patent Publication No. 1973-100030) was introduced and put into practical use. After that, the present inventors continued their research on the protection of guanidino groups, and found that 4-methoxy-2,6-
It was discovered that when a dimethylbenzenesulfonyl group is used, the protecting group can be removed even by mild acid treatment after peptide condensation, and as a result of further research, the present invention was completed. That is, in producing a peptide having a guanidino group, the present invention protects the guanidino group of a guanidino group-containing raw material compound (e.g., arginine) with a 4-methoxy-2,6-dimethylbenzenesulfonyl group to synthesize the peptide. This is a method for producing a peptide, which is characterized by removing the post-protecting group with an acid. In the present invention, the following general formula () Arginine derivatives represented by the formula (wherein R represents H or a protecting group for an -amino group) and salts thereof are advantageously used. In the present invention, when introducing a 4-methoxy-2,6-dimethylbenzenesulfonyl group into the guanidino group of a guanidino group-containing compound, the α-amino group of the guanidino group-containing compound is subjected to a protected reaction. This α-amino group protecting group can be a conventionally known protecting group, such as the general formula ()
As the protecting group represented by R in, carbobenzoxy group, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group,
t-butoxycarbonyl group, t-amyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group, isonicotinyloxycarbonyl group, o-nitrophenylsulfenyl group, 2-(p-biphenyl)-isopropyloxycarbonyl group Examples include those in which arginine is introduced by a conventional method, and those in which arginine is protected with a carbobenzoxy group or a t-butoxycarbonyl group are particularly advantageously used. Next, 4-methoxy-2,
6-dimethylbenzenesulfonyl group is reacted. This reaction involves adding 4-methoxy-2,6-dimethylbenzenesulfonic acid groups to a guanidino group-containing compound at approximately 1 to
The reaction is preferably carried out in an amount of 5 equivalents, more preferably about 1 to 2 equivalents. The 4-methoxy-2,6-dimethylbenzenesulfonic acid group is usually subjected to the reaction in its halide form. As the halogenide, any of chloride, fluoride, bromide, and iodide can be used. Usually, it is preferable to carry out the reaction in the presence of a base. Examples of the base include sodium hydroxide, potassium hydroxide, lithium hydroxide, etc.
It is used in an amount of about 1 to 10 equivalents, more preferably about 1 to 5 equivalents, per equivalent of the guanidino group-containing compound.
The reaction is usually carried out in a suitable solvent such as water, acetone, dioxane, dimethylformamide, tetrahydrofuran, or a mixed solvent thereof. The reaction is carried out at -10°C to 25°C, preferably -5°C to 10°C. 4-methoxy-2,6 thus obtained
A compound having a guanidino group protected by a -dimethylbenzenesulfonyl group is subjected to peptide condensation in its free state or as a salt of cyclohexylamine, dicyclohexylamine, sodium, etc. according to a conventional method. In the present invention, the compound having a guanidino group protected with a 4-methoxy-2,6-dimethylbenzenesulfonyl group is an arginine derivative represented by the above general formula () and a salt thereof. 4-methoxy-2,6 thus obtained
- A compound having a guanidino group protected with a dimethylbenzenesulfonyl group is subjected to a peptide condensation reaction by a conventional method. This common method is
For example, M. Bodansky and MA Ondetti, Peptide Synthesis, Inter
science, New York, 1966; FMFinn and K.
The Proteins by Hofmann,
Volume 2, edited by H. Nenrath and R. Hill, Academic
Press Inc, New York, 1976; methods described in "Peptide Synthesis" by Nobuo Izumiya et al., Maruzen Co., Ltd., 1975, such as the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, Active ester method,
Examples include a method using Woodward's reagent K, a carbodiimidazole method, a redox method, and a DCC/HONB method. Next, after peptide condensation, the protecting group of the present invention is removed with an acid. As this desorption method, known acid treatment methods such as anhydrous hydrogen fluoride method, methanesulfonic acid method, and trifluoromethanesulfonic acid method can be applied. Furthermore, in the case of the method of the present invention, trifluoroacetic acid can be advantageously used as a new acid treatment method, and in particular, when trifluoroacetic acid is used in the presence of thioanisole or anisole, the elimination reaction proceeds very favorably. The above-mentioned trifluoroacetic acid, thioanisole, and anisole may be used in amounts sufficient to serve as a solvent and remove the protecting group. For example, 1 to 1 equivalent of a compound having a protected guanidino group
10 ⁵ equivalents, more preferably 1 to 10 ³ equivalents are used, and the elimination reaction may be carried out in a solvent such as acetic acid, chloroform, methylene chloride, etc., and the temperature is about -10°C to 300°C, more preferably It is carried out at a temperature of about 10° to 100°C. The method of the present invention can be applied to the production of any peptide having a guanidino group. As a specific example, as shown in the example below, Dea-
Gly ¹⁰ −[D-Leu ⁶ ]−LH−RH−ethylamide (see Japanese Patent Publication No. 53-14072), Des−Gly ¹⁰ −LH−RH−
ethylamide (see Special Publication No. 53-24423), Tuftsin
[See Nature, 228, 672 (1970)], Substance
Physiologically active peptides such as P and Kyotorphin can be advantageously produced. Other peptides include MSH,
ACTH, Glucagon-Secretin Bradykinin,
Dynorphin, a-Neoendorphin and their active fragments can also be advantageously produced. 4-methoxy-2, used in the method of the present invention
The 6-dimethylsulfonyl group can be easily removed not only by the conventional method of removing the guanidino protecting group by acid treatment but also under milder conditions. For example, mild acid treatment such as trifluoroacetic acid cannot be applied to remove conventionally known guanidino protecting groups, but when protected with 4-methoxy-2,6-dimethylsulfonyl group, Desorption progresses well and can be applied satisfactorily. By the way, when the guanidino group is protected with a p-methoxybenzenesulfonyl group or meditylenesulfonyl group and the protecting group is removed using methanesulfonic acid after peptide condensation by the conventional method, Furthermore, when asparagine or aspartic acid residues are contained, succinimide-type side reactions may occur, and when serine or threonine residues are present, N→O acyl rearrangement occurs. In the method of the present invention, even peptides containing these amino acid residues can be removed by using a mild acid such as trifluoroacetic acid without causing side reactions as described above. Next, the present invention will be explained in more detail with reference to Examples and Test Examples. The synthesis steps for each example are summarized in the table at the end. In this specification, 4-methoxy-2,6-dimethylbenzenesulfonyl group may be abbreviated as MDS, and regarding amino acids, peptides, protecting groups, active groups, etc., the IUPAC-IUB commission on
It may be indicated by an abbreviation based on Biological or Nomenclature or by an abbreviation commonly used in the field. Illustrate them. pGlu: pyroglutamic acid; His: histidine;
Trp: tryptophan; Ser: serine; Tyr: tyrosine; Leu: leucine; Gly: glycine;
Arg: arginine; Pro: proline; Lys: lysine; Gln: glutamine; Phe: phenylalanine; Met: methionine; Thr: threonine (unless otherwise specified, the amino acids refer to the L-form, and the D-form (excluding Gly); Z: carbobenzoxy; Boc: t-butoxycarbonyl; Et: ethyl; HONB and
ONB: N-hydroxy-5-norbornene-
2,3-dicarboximide and its ester; HOBt: N-hydroxybenztriazole; DCC: N,N-dicyclohexylcarbodiimide; H ₂ /Pd: catalytic reduction; TFA: trifluoroacetic acid; CHA: cyclohexylamine;
OTCP: 2,4,5-trichlorophenyl ester; OSu: N-hydroxysuccinimide ester Reference example 1 (1) Synthesis of Z-Arg(MDS)-OH-CHA salt Z-Arg-OH46.2g (0.15M) 4N
Dissolve in a mixture of 150ml of NaOH and 400ml of acetone at room temperature and cool on ice. To this, 4-methoxy-2,6
-Dimethylbenzenesulfonyl chloride 70.7g
(0.30M) in 150ml of acetone was added dropwise over 1 hour. After stirring at room temperature for 2 hours, crystalline citric acid was added to the reaction mixture to make it acidic. Acetone is distilled off and the resulting oil is extracted with ethyl acetate. The ethyl acetate layer is washed twice with water. Extract the target product from the ethyl acetate layer with sodium bicarbonate solution. After acidifying the aqueous layer with citric acid, the resulting oil is extracted with ethyl acetate. The ethyl acetate layer was thoroughly washed with water, dried, and the ethyl acetate was distilled off under reduced pressure.
Obtain 63g of oil. Dissolve this in 300 ml of ethyl acetate, and when cold, cyclohexylamine (CHA)
Add 14.3 ml, leave at room temperature overnight, collect the resulting crystals, and recrystallize from acetotrile. Yield 48.0g (52.8%”, melting point 140-141°C Optical rotation: [α] ²³ _D +5.7° (c = 0.5, in methanol) Thin layer chromatography (TLC): Rf1 (CHCl ₃ : methanol : acetic acid) =9:1:0.5)=
0.25 Elemental analysis value: C ₂₃ H ₃₀ O ₇ N ₄ S・C ₆ H ₁₃ N Calculated value: C 57.50; H 7.15; N 11.56; S 5.29 Experimental value: C 57.23; H 6.96; N 11.66 S 5.32 (2 ) Synthesis of H-Arg(MDS)-OH) Z-Afg(MDS)-OH-CHA salt4.23g
(0.007M) was dissolved in 150ml of methanol, and catalytic reduction was carried out using palladium black as a catalyst according to a conventional method. The catalyst is separated and the liquid is concentrated under reduced pressure. Add water to the residue and remove the resulting crystals. Recrystallize from water. Yield 2.15g (80.5%), melting point 120-122℃ (decomposition) Optical rotation: [α] ²³ _D -7.8° (c = 0.7, methanol) TLC: Rf ₂ (ethyl acetate: pyridine {acetic acid:
water = 30:20:6:11) = 0.16, Rf ₄ (n-butanol: ethyl acetate: acetic acid: water =
1:1:1:1)=0.52 Elemental analysis value: C ₁₅ H ₂₄ O ₅ N ₄ S. Calculated value as 1/2H ₂ O: C 47.23; H 6.61; N 14.69; S 8.41 Experimental value: C 47.68; H 6.58; N 14.69; S 8.47 (3) Synthesis of Boc-Arg(MDS)-OH H-Afg (MDS-OH1.12g (0.003M) in 1.65ml
Dissolved in water, cold, triethylamine 0.63ml
Add (0.0045M). To this, t-butyl S-
793 mg (0.0033 M) of 4,6-dimethylpyridin-2-ylthiocarbonate was added to 1.65 mg of dioxane.
ml of solution while stirring vigorously. After stirring at room temperature for 12 hours, dioxane is distilled off, the residue is diluted with water, and the aqueous layer is washed with ethyl acetate. The aqueous layer is acidified with 6M hydrochloric acid while cold, and the resulting oil is extracted with ethyl acetate.
The ethyl acetate layer was washed twice with water and then dried.
Ethyl acetate is distilled off under reduced pressure. Add petroleum benzine to the resulting crystals and collect. Recrystallize from ethyl acetate. Yield 1.35g (95.7%), melting point 175-176℃ (decomposition) Optical rotation: [α] ²⁶ _D + 3.5° (c = 0.5, in methanol) TLC: Rf ₁ = 0.34 Elemental analysis value: C ₂₀ H ₃₂ As O ₇ N ₄ S Calculated value: C 50.83; H 6.83; N 11.86; S 6.79 Experimental value: C 50.96; H 7.07; N 11.56; S 6.63 Test example (2) Trifluoroacetic acid (2) in a mixture of acetic acid (2 ml) and thioanisole (0.1 ml) at 50°C for 1 hour.
ml) and thioanisole (0.1 ml) at room temperature (21°C) for 5 hours; (3) in a mixed solution of trifluoroacetic acid (2 ml) and anisole (0.1 ml) at 50°C for 1 hour;
(4) Treated in trifluoroacetic acid (2 ml) at 50°C for 1 hour, and after distilling off the trifluoroacetic acid under reduced pressure, the residue was dissolved in water and washed once with ether. The whole was weighed to 100 ml, subjected to amino acid analysis, and the amount of arginine produced was measured. The results are shown in Table 1.

[Table] Example 1 Z-Arg (MDS). Synthesis of Pro-NHEt Dissolve 2.76 g (0.01M) of Z-Pro-NHEt in 70 ml of methanol and add p-toluenesulfonic acid.
Add 1.90g (0.01M) and perform catalytic reduction using palladium black as a catalyst according to a conventional method. Separate the catalyst and concentrate the liquid. Dissolve the residue in 50 ml of dimethylformamide and add triethylamine under ice cooling.
Add 1.40〓(0.01M), then Z-Arg
(MDS)-OH [Z-Arg(MDS)-OH-CHA salt
Prepared from 6.06g (0.01M)] Add and dissolve 1.54g (0.01M) of toto hydroxybenztriazole.
Dicyclohexylcarbodiimide 2.06g (0.01M)
Add and stir for 24 hours. The resulting urea is separated off and the liquid is concentrated in vacuo. Add ethyl acetate to the residue, and wash the ethyl acetate layer with aqueous sodium bicarbonate and 0.2N hydrochloric acid. The ethyl acetate is distilled off under reduced pressure, and the residue is pulverized with petroleum benzine. This is purified by silica gel chromatography. (Developing solvent: chloroform) Yield 3.5g (56.6%), melting point 65-67°C Optical rotation: [α] ²³ _D -13.8° (c = 0.5, dimethylformamide) TLC: Rf ₁ = 0.47 Elemental analysis value: C ₂₉ As H ₄₂ O ₇ N ₆ S Calculated value: C 56.29; H 6.84; N 13.58; S 5.18 Experimental value: C 56.56; H 6.81; N 13.40; S 4.93 Example 2 Z-Leu-Arg(MDS)-Pro- Synthesis of NHEt Z-Arg(MDS)-Pro-NHEt3.16g (0.0051M)
is dissolved in 70 ml of methanol, 969 mg (0.0051 M) of p-toluenesulfonic acid is added, and catalytic reduction is carried out using palladium black as a catalyst according to a conventional method.
The decomposition medium is separated and the liquid is concentrated. The residue was dissolved in 30 ml of dimethylformamide, and while cold, 0.71 ml of triethylamine (0.0051 M) was added and then Z-
Add Leu-ONB2.40g (0.0051M x 1.1) and stir as is for 12 hours. The solvent is removed in vacuo and the residue is dissolved in ethyl acetate. The ethyl acetate layer is washed with aqueous sodium bicarbonate and 0.2N hydrochloric acid, dried, and then evaporated under reduced pressure.
Add ether to the residue and remove the resulting precipitate. Reprecipitate from ethyl acetate and ether. Yield 2.5g (67.0%), melting point 95-100℃ Optical rotation: [α] ²³ _D -22.0゜ (c = 0.6, dimethylformamide) TLC: Rf ₁ = 0.50 Elemental analysis value: C ₃₅ H ₅₃ O ₈ N ₇ As S Calculated value: C 57.44; H 7.31; N 13.40; S 4.38 Experimental value: C 57.60; H 7.29; N 13.15; S 4.28 Example 3 pGlu-His-Trp-Ser-Tyr-(D)Leu-
Synthesis of Leu-Arg(MDS)-Pro-NHEt Z-Leu-Arg(MDS)-Pro-NHEt732mg
(0.001M) was dissolved in 50ml of methanol, and catalytic reduction was performed using palladium black as a catalyst according to a conventional method. Separate the catalyst and concentrate the liquid. residue
Dissolve in 10 ml of dimethylformamide and add to this
pGlu-His-Trp-Ser-Tyr-(D)-Leu-
OH816mg (0.001M) and HONB717mg (0.004M)
Add and melt, cool to -10℃ with ice-salt, and dicyclohexylcarbodiimide 412mg (0.002M)
and incubate at -10℃ for 3 hours, at 0℃ for 10 hours, and at room temperature.
Stir for 24 hours. The resulting urea is separated off and the liquid is concentrated in vacuo. Add ether to the residue and remove the resulting precipitate. Reprecipitate twice from acetonitrile. Yield 1.15g (81.6%), melting point 105-110℃ (decomposition) Optical rotation: [α] ²³ _D -28.2゜ (c = 0.6, dimethylformamide), TLC: Rf ₂ = 0.20 Elemental analysis value: C ₆₈ H ₉₃ As O ₁₅ N ₁₆ SH ₂ O Calculated value: C 57.33; H 6.72; N 15.73; S 2.25 Experimental value: C 57.29; H 7.18; N 15.32; S 2.02 Example 4 pGLu−His−Trp−Ser−Tyr−( D) Leu-
Synthesis of Leu-Arg-Pro-NHEt pGlu-His-Trp-Ser-Tyr-(D)-Leu-
300 mg of Leu-Afg(ADS)-Pro-NHEt is dissolved in a mixture of 0.5 ml of thioanisole and 10 ml of trifluoroacetic acid and left at 50 to 55°C for 1 hour. Trifluoroacetic acid is distilled off under reduced pressure, ether is added to the residue, and the resulting precipitate is collected. Dissolve this in a small amount of water and pass through an Amberlite RA-410 (acetic acid type) column (1.5 x 10 cm). Combine the effluent and washing liquid and pour them directly into a carboxymethyl cellulose column (1.5 x 12 cm). After washing the column with 50 ml of water, elute with a linear gradient between water (500 ml) and 0.15 M ammonium acetate (500 ml, pH 6.9). The main elution fractions (240-390 ml) are collected and lyophilized. Dissolve this in a small amount of N acetic acid,
Sephadex LH-20 column (2.5 x 120cm)
and develop with the same solvent system. The main elution fraction (290-350 ml) is collected and lyophilized. Yield 120mg Optical rotation: [α] ²³ _D -34.1° (c = 0.4, 5% acetic acid) TLC: Rf ₃ (n-butanol:pyridine:acetic acid:water = 30:20:6:24) = 0.56 Amino acid analysis ( Acid decomposition): His 1.01(1); Arg+ethylamine 1.75(1+1); Trp0.90
(1); Ser0.86(1); Glu1.05(1); Pro1.04(1);
Leu2.03; Tyr1.11(1), average recovery rate 85.0% Example 5 pGlu−His−Trp−Ser−Tyr−Gly−Leu−
Synthesis of Arg(MDS)-Pro-NHEt Z-Leu-Arg(MDS)-Pro-NHEt476mg
(0.00065M) and pGlu−His−Trp−Ser−Tyr−
Example 3 using 494mg (0.00065M) of Gly-OH
Prepared in a similar manner. Yield: 710 mg (80.9%), melting point: 135-140°C (decomposed) Optical rotation: [α] ²³ _D -26.7° (c = 0.5, dimethylformamide), Rf ₂ = 0.16 Elemental analysis: C ₆₄ H ₈₅ O ₁₅ N ₁₆ As S・H ₂ O Calculated value: C 56.16; H 6.41; N 16.38; S 2.34 Experimental value: C 56.15; H 6.71; N 16.11; S 2.18 Example 6 pGlu−His−Trp−Ser−Tyr−Gly− Leu×
Synthesis of Arg−Pro−NHEt pGlu−His−Trp−Ser−Tyr−Gly−Leu−
Using 300 mg of Arg(MDS)-Pro-NHEt. It was synthesized in the same manner as in Example 4. Yield 122 mg Optical rotation: [α] ²³ _D -55.2° (c = 0.5, 5% acetic acid water) TLC: Rf ₄ = 0.48 (Avicel) Amino acid analysis (acid decomposition): His 1.02 (1); Arg + ethylamine, 1.83 (1+1);Trp1.01(1);Ser0.81
(1); Glu1.04(1); Pro1.00(1); Leu1.03(1); Tyr1.03
(1); Gly1.06(1), average recovery rate 90.0% Example 7 Boc−Arg(MDS)−Pro−Lys(Boc)−Pro−
Synthesis of OH H-Pro-OMe, Z-Lys(Boc)-ONB and Z-
590 mg (0.001 M) of oily Z-Pro-ONB synthesized by successive condensation of Pro-ONB was dissolved in 50 ml of methanol, and palladium black was used as a catalyst.
Catalytic reduction is carried out according to conventional methods. Separate the catalyst
Concentrate the liquid under reduced pressure. Dissolve the residue in 10 ml of dimethylformamide and add Boc-Arg to this.
Add 473 mg (0.001 M) of (MDS)-OH and 153 mg (0.001 M) of hydroxybenztriazole, and react at room temperature for 12 hours. The resulting urea is separated off and the liquid is concentrated in vacuo. Dissolve the residue in ethyl acetate,
The ethyl acetate layer is washed with aqueous sodium bicarbonate and 0.2N hydrochloric acid.
After drying, ethyl acetate is distilled off under reduced pressure. Residual oil [Boc-Arg(MDS)-Pro-Lys(Boc)-
Pro-OMe] was thoroughly washed with petroleum benzine and 10ml
Dissolve in methanol. When cool, add 2 ml of N-caustic soda and saponify at room temperature for 2 hours. 2 ml of N-hydrochloric acid was added dropwise while cold, the reaction mixture was diluted with water, and the resulting oil was extracted with ethyl acetate. The ethyl acetate layer is washed with water and dried. The ethyl acetate is distilled off under reduced pressure and the residue is triturated with petroleum benzine. Reprecipitate from ethyl acetate and ether. Yield: 530 mg (58.3%), melting point: 85-90°C Optical rotation: [α] ²⁶ _D -35.6° (c = 0.5, dimethylformamide), TLC: Rf ₁ = 0.33 Elemental analysis: C ₄₂ H ₆₈ O ₁₂ N ₈ As S Calculated value: C 54.94; H 7.58; N 12.21; S 3.49 Experimental value: C 55.02; H 7.65; N 12.08 S 3.63 Example 8 Boc-Arg(MDS)-Pro-Lys(Boc)-Pro-
Gln−Gln−Phe−Phe−Gly−Leu−Met−
Synthesis of NH ₂ Boc−Gln−Gln−Phe−Phe−Gly−Leu−Met
−NH ₂ [J.Bergmann, M., Bienert, H.
Niedvich, B. Mehlis and P. Oehme,
Experientia, 30 , 401 (1974)] 485mg (0.0005M)
Dissolve in a mixture of trifluoroacetic acid (4.5 ml) and water (0.5 ml) and shake at 10°C for 20 minutes. Add 0.5 ml of N-hydrochloric acid and evaporate under reduced pressure. Add ether to the residue, remove the resulting precipitate, and dry over caustic soda. Dissolve the dry powder in 15 ml of dimethylformamide and, when cold, add 0.07 ml of triethylamine.
Add (0.0005M). In this, Boc-Arg (MDS)
−Pro−Lys(Boc)−Pro−OH448mg (0.0005M)
Add and dissolve 179mg (0.001M) of HONB. Dicyclohexylcarbosiimide 155mg (0.0075M)
Add and stir for 24 hours. Separate the urea produced, concentrate the liquid, add water to the residue,
Collect the resulting precipitate. Precipitate twice from ethanol. Yield 500mg (56.8%), melting point 245-247℃) (decomposition) Optical rotation: [α] ²⁶ _D -34.0° (c = 0.5 dimethylformamide), TLC: Rf ₂ = 0.83 Elemental analysis value: C ₈₃ CH ₁₂₆ O ₂₀ N ₁₈ S ₂ Calculated value: C 56.64; H 4.22; N 14.32; S 3.63 Experimental value: C 56.50; H 7.15; N 14.28 S 3.73 Example 9 H-Arg-Pro-Lys-Pro-Gln-Gln- Phe−
Phe−Gly−Leu−Met−NH ₂ (SubStancep)
Synthesis of Boc−Arg(MDS)−Pro−Lys(Boc)−Pro−
Gln−Gln−Phe−Phe−Gly−Leu−Met−
Dissolve 100 mg of NH ₂ in a mixture of 1 ml of thioanisole and 10 ml of trifluoroacetic acid, and shake at 50°C for 2 hours. Trifluoroacetic acid is distilled off under reduced pressure, ether is added to the residue, and the resulting precipitate is collected. After drying in vacuum over caustic soda, dissolve in a small amount of water, add 3 ml of Amberlite IRA-410 (acetic acid type), and shake for a while. The resin is separated and the liquid is freeze-dried. Dissolve the lyophilized powder in a small amount of 30% acetic acid water and apply it to a Sephadex G-25 column (2.5
x 120cm). Develop with the same solvent system, collect the main elution fraction (240-280 ml) and lyophilize. Yield 67 mg Optical rotation: [α] ²⁴ _D -80.7° (c = 0.5, 5% acetic acid) TLC: Rf ₃ = 0.53 (Avicel) Amino acid analysis (acid decomposition): Lys0.99(1); Arg0.99(1) )；Gln2.03
(2);Pro2.10(2);Gly0.99(1);Met0.99(1);
Leu0.98(1); Phe1.96(2) Normal recovery rate 90.4% Example 10 Synthesis of Z-Pro-Arg(MDS)-OH 3.72g (0.01M) of H-Arg(MDS)-OH was dissolved in dimethylformamide. Dissolve in 50ml, add 1.40ml (0.01M) of triethylamine when cold, then add Z-Pro
-Add 4.5g (0.011M) of ONB and stir at room temperature for 12 hours. 5 ml of acetic acid are added to the reaction mixture and the solvent is distilled off in vacuo. The residue is dissolved in ethyl acetate and the ethyl acetate layer is washed twice with water. Ethyl acetate was distilled off under reduced pressure, and petroleum benzine was added to the residue to obtain a powder. Reprecipitate from ethyl acetate and petroleum benzine. Yield 5.6g (93.3%), melting point 75-80℃ Optical rotation: [α] ²³ _D -16.0゜ (c = 0.6, dimethylformamide), TLC: Rf ₁ = 0.22 Elemental analysis value: C ₂₈ H ₃₇ O ₈ N ₅ As S Calculated value: C 55.71; H 6.18; N 11.60; S 5.31 Experimental value: C 56.18; H 6.58; N 11.26 S 4.73 Example 11 Synthesis of H-Pro-Arg (MDS)-OH Z-Pro-Arg (MDS)−OH5.4g (0.009M)
Dissolve in 100ml of methanol and perform catalytic reduction using palladium black as a catalyst according to a conventional method. Separate the catalyst and concentrate the liquid. Dissolve the residue in water,
Leave it in a cool place. Collect the resulting crystals. Yield 2.80g (66.0%) Melting point 173-174℃ (decomposition) Optical rotation: [α] ²³ _D -15.8゜ (c = 0.6, dimethylformamide), TLC: Rf ₂ = 0.03, Rf ₄ = 0.46 Elemental analysis values: As C ₂₀ H ₃₂ O ₆ N ₅ S Calculated value: C 51.05; H 6.86; N 14.88; S 6.81 Experimental value: C 51.22; H 6.95; N 14.99 S 6.86 Example 12 Z-Lys(Boc)-Pro-Arg Synthesis of (MDS)-OH H-Pro-Arg(MDS)-OH2.35g (0.005M)
Dissolve in 30 ml of dimethylformamide, cool,
Add 0.7 ml (0.005 M) of triethylamine, then 2.80 g (0.005 M) of Z-Lys(Boc)-OTCP, and stir at room temperature for 12 hours. 3 ml of acetic acid are added to the reaction mixture and the dimethylformamide is distilled off in vacuo. The residue is dissolved in ethyl acetate and the ethyl acetate layer is washed twice with water. The solvent was distilled off under reduced pressure, ether was added to the residue, and the resulting precipitate was collected. Reprecipitate from ethyl acetate and ether. Yield 3.30g (79.3%), melting point 85-90℃ Optical rotation: [α] ²³ _D -19.3゜ (c = 0.6, dimethylformamide) TLC: Rf1 = 0.25 Elemental analysis value: C ₃₉ H ₅₇ O ₁₁ N ₇ S・As 1/2H ₂ O Calculated value: C 55.70; H 6.95; N 11.66; S 3.81 Experimental value: C 55.77; H 7.03; N 11.77 S 3.65 Example 13 Boc-Thr-Lys(Boc)-Pro-Arg( MDS)−
Synthesis of OH Z-Lys(Boc)-Pro-Arg(MDS)-OH3.0g
(0.0036M) was dissolved in 100ml of methanol, and catalytic reduction was performed using palladium black as a catalyst according to a conventional method. The catalyst is separated and the liquid is concentrated under reduced pressure. Dissolve the residue in 50 ml of dimethylformamide and add 0.50 ml (0.0036 M) of triethylamine when cold. To this, Boc−Thr−ONB [Boc;Thr−
Prepared from 877mg (0.004M) of OH] and stirred at room temperature.
Stir for 12 hours. Add 3 ml of acetic acid to the reaction solution,
The solvent is removed in vacuo. The residue is dissolved in ethyl acetate and the ethyl acetate layer is washed twice with water. Ethyl acetate was distilled off under reduced pressure and the residue was triturated with ether. Reprecipitate from ethyl acetate and ether. Yield 2.3g (71.0%), melting point 85-90℃ Optical rotation: [α] ²³ _D -20.2゜ (c = 0.6, dimethylformamide), TLC: Rf ₁ = 0.14 Elemental analysis value C ₄₀ H ₆₆ O ₁₃ N ₈ As S Calculated value: C 53.44; H 7.40; N 12.46; S 3.57 Experimental value: C 53.30; H 7.65; N 12.47; S 3.36 Example 14 Synthesis of H-Thr-Lys-Pro-Arg-OH (Tuftsin) BoC −Thr−Lys(Boc)−Pro−Arg(MDS)−
Dissolve 300 mg of OH in a mixture of 0.5 ml of thioanisole and 10 ml of trifluoroacetic acid and leave at 50°C for 1 hour. Trifluoroacetic acid is distilled off under reduced pressure, ether is added to the residue, and the resulting precipitate is collected. After drying, dissolve in a small amount of water and use Amberlite IRA-
Mix with 10ml of 410 (acetic acid type) for 30 minutes. The resin is separated and the liquid is freeze-dried. The dry powder is dissolved in a small amount of water and poured onto a carboxymethyl cellulose column (1.5 x 10 cm), eluted with a linear gradient between water (300 ml) and 0.2 M ammonium acetate (300 ml, PH 6.9). Main elution part (290-350
ml) and freeze-dried. The powder was dissolved in N acetic acid, poured into a Sephadex LH-20 (2.5 x 120 cm) column, and developed with the same solvent system. The main elution fraction (270-310 ml) is collected and lyophilized. Yield 180mg Optical rotation: [a] ²³ _D -63.1° (c = 0.6, 5% acetic acid) TLC: Rf ₃ = 0.22 (Azecel) Amino acid analysis (acid analysis): Lys1.00(1); Arg1.02
(1); Thr1.01(1); Pro0.67(1), average recovery rate 92.0% Example 15 Synthesis of Boc-Tyr-Arg(MDS)-OH H-Arg(MDS)-OH1.91g (0.005 Dissolve M) in 30 ml of dimethylformamide, add 0.7 ml (0.005 M) of triethylamine when cold, and add Boc-Tyr-
Add 1.89g (0.005M) of OSu and stir at room temperature for 12 hours. 5 ml of acetic acid are added and the dimethylformamide is distilled off in vacuo. Dissolve the residue in ethyl acetate,
The ethyl acetate layer is washed twice with water. Ethyl acetate was distilled off under reduced pressure, ether was added to the residue, and the resulting precipitate was collected. Reprecipitate from ethyl acetate and ether. Yield 2.70g (84.9%), melting point 85-90℃ Optical rotation: [α] ²³ _D +0.6° (c = 0.5, dimethylformamide), TLC: Rf ₁ = 0.14 Elemental analysis value: C ₂₉ H ₄₁ O ₉ As N ₅ S Calculated value: C 54.79; H 6.50; N 11.02; S 5.04 Experimental value: C 54.94; H 7.01; N 10.66 S 4.54 Example 16 Synthesis of H-Tyr-Arg-OH (Kyotorphin) Boc-Tyr- Dissolve 300 mg of Arg(MDS)-OH in a mixture of 0.5 ml of thioanisole and 10 ml of trifluoroacetic acid and leave at 50-55°C for 1 hour. Trifluoroacetic acid was distilled off under reduced pressure, ether was added to the residue,
Collect the resulting precipitate. Dissolve this in a small amount of water, add 10ml of Amberlite IRA-410 (acetic acid type) and 30ml
Stir for a minute. The resin is separated and the liquid is freeze-dried. Dissolve the dry powder in a small amount of water and pour into a carboxymethyl cellulose column (1.5 x 10 cm). Elution is performed using a linear gradient between water (300 ml) and 0.1 M ammonium acetate (300 ml, pH 6.9). Collect the main elution fraction (100-150 ml) and
Freeze dry. The dry powder is dissolved in a small amount of N acetic acid and poured into a column of Sephadex LH-20 (2.5 x 120 cm). Develop with the same solvent system, collect the main eluted fractions, and lyophilize. Yield 150mg Optical rotation: [α] ²¹ _D −17.6° (c = 0.4, H ₂ O) TLC: Rf ₃ = 0.45 (Azecel) Amino acid analysis (acid decomposition): Arg1.06(1); Tyr0.64
(1), average recovery rate 90.6%

[Table] ↓

pGlu−His−Trp−Ser−Tyr
−X−Leu−Arg−Pro−NHEt

[Table] ↓

H−Arg−Pro−Lys−Pro−Gl _N −Gl _N
−Phe−Phe−Gly−Leu−Met−NH ₂

[Table] ↓
H−Thr−Lys−Pro−Agr−OH

[Table] ↓
H−Tyr−Arg−OH

Claims (1)

[Claims] 1. In producing a peptide having a guanidino group, the guanidino group of a guanidino group-containing raw material compound is protected with a 4-methoxy-2,6-dimethylbenzenesulfonyl group, and after peptide condensation, the protecting group is Method 2 for producing a peptide, characterized in that it is removed with an acid.Production method 3 according to claim 1, using trifluoroacetic acid as the acid.Removal of the protecting group in the presence of thioanisole or anisole. A manufacturing method according to claim 1 or 2, characterized in that: