JPH0228599B2 - - Google Patents

Description

[Detailed description of the invention]

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

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 traditionally been
This has been done 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. However, other parts of the peptide that cannot be obtained are degraded to produce by-products, resulting in a decrease 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 Patent Application Laid-Open No. 1973-100030) was introduced and put into practical use. After that, the present inventors continued research on the protection of guanidino groups, and found that 4-methoxy-2,3,
The present invention was completed based on the discovery that the protecting group of the 6-trimethylbenzenesulfonyl group can be removed by further mild acid treatment. That is, the present invention protects the guanidino group of a guanidino group-containing raw material compound (e.g., arginine) with a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group to produce a peptide having a guanidino group. This is a method for producing a peptide, which is characterized by removing the protecting group with an acid after condensation. In the present invention, the general formula () Arginine derivatives and salts thereof represented by the formula (wherein R represents H or a protecting group for an α-amino group) and 4-methoxy-2,3,6-trimethylbenzenesulfonyl halide are used as starting compounds. . In the present invention, when introducing a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group into the guanidino group of a guanidino group-containing compound, the α-amino group of the guanidino group-containing compound is protected and subjected to the reaction. be given The α-amino group can be protected using conventionally known protective groups, such as carbobenzoxy group, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, t-butoxycarbonyl group, t-amyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group, isonicotinyloxycarbonyl group, O
Examples include those in which a -nitrophenylsulfenyl group or a 2-(p-biphenyl)-isopropyloxycarbonyl group is introduced by a conventional method, and those in which arginine is protected with a carbobenzoxy group or a t-butoxycarbonyl group are particularly advantageous. used for. Next, 4-methoxy-2,
3,6-trimethylbenzenesulfonyl group is reacted. In this reaction, a 4-methoxy-2,3,6-trimethylbenzenesulfonic acid group is added to a guanidino group-containing compound in an amount of about 1 to 5 equivalents, more preferably about 1 to 2 equivalents per equivalent of the guanidino group-containing compound.
It is best to react in equivalent amounts. The 4-methoxy-2,3,6-trimethylbenzenesulfonic 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.
4-Methoxy-2,3,6-trimethylbenzenesulfonyl chloride is obtained as a crystal by reacting 2,3,5-trimethylanisole with chlorsulfonic acid without producing isomers. be able to. 4-methoxy-2,
The introduction of the 3,6-trimethylbenzenesulfonyl group is preferably carried out 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 obtained in this way,
A compound having a guanidino group protected by a 3,6-trimethylbenzenesulfonyl group is subjected to peptide condensation either as a free form or as a salt of cyclohexylamine, dicyclohexylamine, sodium, etc. according to a conventional method. In the present invention, a compound having a guanidino group protected with a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group refers to an arginine derivative represented by the above general formula () and a salt thereof. be. 4-methoxy-2 obtained in this way,
A compound having a guanidino group protected with a 3,6-trimethylbenzenesulfonyl group is subjected to a peptide condensation reaction by a conventional method. As a common method, for example, M. Bodansky and MA Ondetti
Author, Peptide Synthesis
Synthesis), Inter science, New York, 1966
Year; The Proteins by FMFinn and K. Hofmann, Volume 2, H. Nenrath, RL
Edited by Hill, Academic Press Inc. New York,
1976: Methods described in "Peptide Synthesis" by Nobuo Izumiya et al., Maruzen Co., Ltd., 1975, such as azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, Woodward reagent Examples include a method using 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 10 3 equivalents.゜～
It is carried out at around 100℃. The method of the invention can be applied to the production of any peptide having a guanidino group. A specific example is Des-Gly ¹⁰ − [D-Leu ⁶ ]-LH-RH-
ethylamide (see Special Publication No. 53-14072), Des-Gly ¹⁰
-LH-RH-ethylamide (see Japanese Patent Publication No. 53-24423), Tuftsin [see Nature, 228, 672 (1970)],
Physiologically active peptides such as Substance P and Kyotorphin can be advantageously produced. Other peptides include MSH, ACTH, Glucagon-Secretin
Bradykinin, Dynorphin, α-Neoendorphin and their active fragments can also be advantageously produced. 4-methoxycy used in the method of the present invention
The 2,3,6-trimethylbenzenesulfonyl group can be easily removed not only by the conventional method of removing the guanidino protecting group by acid treatment but also by milder conditions. For example, mild acid treatment such as trifluoroacetic acid cannot be applied to the removal of conventionally known guanidino protecting groups, but When protected, 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 by giving reference examples, examples, and test examples. In this specification, 4-methoxy-2,3,6-trimethylbenzenesulfonyl group may be abbreviated as Mtr, 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 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; Gl _N : Glutamine; Phe: Phenylalanine; Met: Methionine; Thr: Threonine (Unless otherwise specified, 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 2,3,5-trimethylanisole 10 g of 2,3,5-trimethylphenol and 10.4 ml of methyl iodide were mixed with 100 g of dimethyl sulfoxide.
ml, cool on ice, add 5.6 g of 60% oily sodium hydride, and stir for 10 hours.
After adding water to this, it is extracted with ether, and the ether layer is washed with water and dried over anhydrous sodium sulfate. Evaporation of the solvent gives an oil. yield
12.9g (quantitative). (2) Synthesis of 4-methoxy-2,3,6-trimethylbenzenesulfonyl chloride 4.5 g of 2,3,5-trimethylanisole,
After dissolving in 500 ml of methylene chloride and cooling to -5° to -10°C, 400 ml of a methylene chloride solution containing 6.0 ml of chlorosulfonic acid was added dropwise. after that,
Bring to room temperature and place on ice containing 5% sodium bicarbonate water. The methylene chloride layer is washed with water and then dried over anhydrous magnesium sulfate. After distilling off the solvent, the crystals are collected by filtration from n-hexane. Yield 5.0g (67.0%) Melting point 56-58℃ Elemental analysis C ₁₀ H ₁₃ O ₃ Calculated value as SC1: C, 48.29; H, 5.27;
S, 12.89; C1, 14.26 Experimental value: C, 48.42; H, 5.21;
S, 12.61; C1, 14.25 (3) Synthesis of Z-Arg(Mtr)OH・CHA Dissolve 2.83 g of Z-Arg OH in a mixture of 10 ml of 4N-sodium hydroxide and 40 mL of acetone, and cool on ice. To this was added 10 ml of an acetone solution containing 4.0 g of 4-methoxy-2,3,6-trimethylbenzenesulfonyl chloride, and the mixture was stirred for 33 hours. As citric acid, acetone is distilled off,
Extract with ethyl acetate. When the solvent is distilled off, 4.8 g of an oil is obtained. This is dissolved in a small amount of ethyl acetate, 1.04 ml of cyclohexylamine is added, the crystals are collected by filtration, and recrystallized from MeOH-ethyl acetate. Yield: 4.10g (72.1%). Melting point 195
−197℃〔α〕 ²³ _D +6.5゜(C=1.18, MeOH) Elemental analysis As C ₃₀ H ₄₅ O ₇ N ₅ S Calculated value: C, 58.14; H, 7.32;
N, 11.30; S, 5.17 Experimental value: C, 58.08; H, 7.34;
N, 11.58; S, 5.32 (4) Synthesis of H-Arg(Mtr)-OH 1.5g of Z-Arg(Mtr)-OH・CHA was mixed with 30ml of ethyl acetate, and 0.2N-H ₂ SO ₄ 15
ml, shake well and wash with water. After distilling off the solvent, the residue was dissolved in memethanol and Pd
Catalytic reduction is carried out using black as a catalyst. When the catalyst is filtered off, the solvent is distilled off, and water is added to the residue, crystallization occurs, which is collected by filtration. Yield 0.77g
(81%). Melting point 100−103℃ [α] ²³ _D −4.8゜ (C=1.30, MeOH) Elemental analysis C ₁₆ H ₂₆ O ₅ N ₄ S・1/2H ₂ O Calculated value: C, 48.59; H, 6.88;
N, 14.18; S, 8.11 Experimental value: C, 48.78; H, 7.16;
N, 13.88; S, 8.29 Test Example Approximately 20 mg of H-Arg(Mtr)-OH was dissolved in 2 ml of trifluoroacetic acid-thioanisole (9:1) and left under the conditions shown in Table 1.
A 100 μ sample was taken, the whole was weighed to 10 ml, and amino acid analysis was performed to measure the amount of arginine produced. The results are shown in Table 1.

[Table] As a result, the Mtr group can be sufficiently cleaved in the TFA-thioanisole system even at 23°C for about 1 hour. Example 1 (1) Boc-Tyr-Arg(Mtr)-OH Dissolve 0.80 g of H-Arg(Mtr)-OH in 20 ml of tetrahydrofuran and add triethylamine when cold.
0.34ml, Boc-Tyr-ONB (Boc-Tyr-
(prepared from 0.57 g of OH, 0.40 g of HONB, and 0.50 g of DCC) and stir at room temperature for 15 hours. After evaporating the solvent, the mixture is acidified with citric acid and extracted with ethyl acetate. After washing the ethyl acetate layer with water, the solvent was distilled off, dissolved in chloroform, and applied to a silica gel column (4 x 6 cm). 5%
Elute with MeOH/CHCl ₃ , collect fractions of the desired product, concentrate, and filter off as a powder from ether.
Yield 0.67g (51.5%) Melting point 114-121℃ [α] ²³ _D +1.2゜ (C = 0.4, dimethylformamide) Elemental analysis C ₃₀ H ₄₃ O ₉ N ₅ S Calculated value: C, 55.45; H, 6.67;
N, 10.78; S, 4.94 Experimental value: C, 55.12; H, 6.83;
N, 10.53; S, 4.54 (2) Synthesis of H-Tyr-Arg-OH (Kyotorphin) Dissolve 400 mg of Boc-Tyr-Arg (Mtr) OH in 5 ml of trifluoroacetic acid-thioanisole (9:1), Leave it for a while. After the trifluoroacetic acid is distilled off under reduced pressure, ether is added to the residue and the resulting precipitate is filtered. This is dissolved in a small amount of water, passed through a column (1 x 10 cm) of Amberlite IRA-410 (acetic acid type), and then freeze-dried. This was dissolved in a small amount of water and applied to a carboxymethyl cellulose column (2.2 x 8 cm), followed by elution using a linear gradient between water (300 ml) and 0.1M ammonium acetate (300 ml). Fractions of 100-150 ml are collected and lyophilized. Yield 175 mg, [α] ²¹ _D −17.4° (C = 0.5,
_H2O ) Amino acid decomposition (acid decomposition): Arg1.00(1);
Tyr0.94(1) Average recovery rate 86.5%. Example 2 (1) Z-Arg(Mtr)-Pro-Lys(Boc)-Pro-
Synthesis of OH H-Pro-OMe, Z-Lys(Boc)-ONB and Z
-Oily Z-Pro-Lys(Boc)-Pro- synthesized by sequential condensation of -Pro-ONB
Dissolve 0.59 g of OMe in 30 methanol and perform catalytic reduction using palladium black as a catalyst. After filtering off the catalyst and concentrating the filtrate, the residue was dissolved in 10 ml of dimethylformamide, and Z-
Arg(Mtr)OH・[Z−Arg(Mtr)・OH・
Prepared from CHA0.56g], HOBt0.15g,
Add 0.23 g of DCC and stir at room temperature for 15 hours. After filtering off the resulting DCU and evaporating the solvent, the residue is dissolved in ethyl acetate and washed with aqueous sodium bicarbonate and 0.2N hydrochloric acid. After drying, the solvent was distilled off and the remaining oil [Z-Arg(Mtr)-Pro-Lys(Boc)-
Dissolve Pro OMe] in 10 ml of methanol.
When cold, add 2 ml of 1N sodium hydroxide solution,
Saponification is carried out for 2 hours at room temperature. 1N-hydrochloric acid 2ml
was added while cold to neutralize, methanol was distilled off, and the precipitated oil was extracted with ethyl acetate.
After distilling off the solvent, petroleum benzine was added and the powder was collected by filtration and reprecipitated from ethyl acetate-ether. Yield 610 mg (66.1%) Melting point 90-95°C [α] ²³ _D -32.7° (C = 0.6, dimethylformamide) Elemental analysis C ₄₃ H ₇₀ O ₁₂ N ₈ Calculated value as S: C, 55.94; H, 7.64;
N, 12.14; S, 3.47 Experimental value: C, 55.62; H, 7.56;
N, 11.98; S, 3.19 (2) H―Arg―Pro―Lys―Pro―Gl _N ―Gl _N ―Phe
―Phe―Gly―Leu―Met―NH ₂ (Substance
Synthesis of P) Boc―Gl _N ―Gl _N ―Phe―Phe―Gly―Leu―
Met-NH ₂ 0.49g in trifluoroacetic acid (4.5ml)
- Dissolve in a mixture of water (0.5ml) and stir at 10℃ for 20 minutes. Add 0.5 ml of 1N hydrochloric acid and evaporate, add ether to the residue, filter it as a powder, and dry. Add this to 15ml of dimethylformamide
Stir, add 0.1ml of triethylamine when cold,
Furthermore, Z-Arg (Mtr)-Pro-Lys (Boc)-
Add 0.45g of Pro-OH, 0.18g of HONB, and 0.20g of DCC and stir for 24 hours. generated
Filter off the DCU and concentrate the filtrate. Add water to the residue and filter the resulting precipitate. This 100mg
was dissolved in 1 ml of trifluoroacetic acid-thioanisole (9:1), shaken at 50°C for 1 hour, the trifluoroacetic acid was distilled off under reduced pressure, the residue was added to ether, and the resulting precipitate was collected by filtration. and dry. Dissolve this in a small amount of water and add it to an Amberlite-IRA-410 (acetic acid type) column (1
x 10cm) and then freeze-dried. This was applied to a Sephadex G-25 column (2.5 x 120 cm) and eluted with 30% aqueous acetic acid. The main elution fractions (230-260 ml) are collected and lyophilized. yield
58 mg, [α] ²³ _D -78.8° (C = 0.5, 5% acetic acid) Amino acid analysis (acid decomposition): Lys1.00(1);
Arg1.04(1); Glu2.05(2); Pro2.20(2); Gly0.91
(1); Met 0.89 (1); Leu 1.05 (1); Phe 1.89 (2), average recovery rate 82.3%.

Claims (1)

[Claims] 1. In the production of a peptide having a guanidino group, the guanidino group of a guanidino group-containing raw material compound is protected with a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, and then the peptide is condensed. A method for producing a peptide, characterized by removing a protecting group with an acid. 2. The manufacturing method according to claim 1, wherein trifluoroacetic acid is used as the acid. 3. The manufacturing method according to claim 1 or 2, characterized in that the protecting group is removed in the presence of thioanisole or anisole.