JPH0232273B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to 4-methoxy-2,3,6-trimethylbenzenesulfonyl halide useful as a protecting group for 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 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 separated and by-products are generated, 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 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-2,3,
It was discovered that the protective group of 6-trimethylbenzenesulfonyl group can be removed by further mild acid treatment, and the arginine derivative of the present invention useful for peptide production was completed. That is, the present invention provides 4-methoxy-2,3,6
-trimethylbenzenesulfonyl halide. The compound of the present invention is a guanidino group-containing compound,
For example, by introducing the guanidino group of arginine, the following general formula () It is used in peptide synthesis as an arginine derivative represented by the formula (wherein R represents H or a protecting group for an α-amino group). 4-Methoxy-2,3,6-trimethylbenzenesulfonyl halide includes chloride,
Any of fluoride, bromide, and iodide may be used. For example, 4-methoxy-2,3,6-trimethylbenzenesulfonyl chloride can be converted into an isomer by reacting 2,3,5-trimethylanisole with chlorsulfonic acid, as shown in the example below. , it can be obtained as a crystal without forming. 4- of the guanidino group of a guanidino group-containing compound
When introducing a methoxy-2,3.6-trimethylbenzenesulfonyl group, the α-amino group of the guanidino group-containing compound is protected and subjected to the reaction. 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-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,
3,6-trimethylbenzenesulfonyl group is reacted. In this reaction, 4-methoxy-2,3,6-trimethylbenzenesulfonic acid group is added to the 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 introduction of the 4-methoxy-2,3,6-trimethylbenzenesulfonyl group is preferably carried out in the presence of a base. Examples of bases include sodium hydroxide,
Potassium hydroxide, lithium hydroxide, etc. are mentioned, and about 1 to 1 equivalent of guanidino group-containing compound is used.
10 equivalents are used, more preferably about 1 to 5 equivalents. 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,
It is preferably carried out at -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 used in a peptide condensation reaction by a conventional method. For example, M. Bodansky and M.A.
Ondetti, 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; “Peptide Synthesis” by Nobuo Izumiya et al. Maruzen Co., Ltd.
Methods described in 1975, such as the azide method, chloride method, acid anhydride method, mixed acid anhydride method,
DCC method, active ester method, Woodward reagent K
, the carbodiimidazole method, the redox method, and the 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 remove the protecting group as a solvent. 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℃
It is carried out at a temperature of about 100°C. The compounds of the present invention are applicable to the production of any peptide having a guanidino group. A specific example is Des−Gly ¹⁰ −[D−eu 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, 228672 (1970)],
Physiologically active peptides such as Substance P.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. The 4-methoxy-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 under milder conditions. For example, mild acid treatments such as trifluoroacetic acid cannot be applied to remove conventionally known guanidino protecting groups, but 4-methoxy-
When protected with a 2,3,6-trimethylbenzenesulfonyl group, elimination proceeds well and can be applied satisfactorily. By the way, by the conventional method, after protecting the guanidino group with a p-methoxybenzenesulfonyl group or meditylenesulfonyl group and condensing the peptide,
When removing a protecting group using methanesulfonic acid, succinimide-type side reactions may occur if the peptide contains asparagine or aspartic acid residues, and serine or threonine residues may occur. In the presence of , N→O acyl rearrangement occurs. In the method of the present invention, even peptides containing these amino group 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. 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 Biological
It may be indicated by an abbreviation based on 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; _H2 /Pd: catalytic reduction; TFA: trifluoroacetic acid; CHA: cyclohexylamine;
OTCP: 2,4,5-trichlorophenyl ester; OSu: N-hydroxysuccinimide ester Example 1 (1) Synthesis of 2,3,5-trimethylanisole 2,3,5-trimethylphenol 10g, methyl iodide 10.4 Dissolve ml in 100ml of dimethyl sulfoxide, cool on ice, add 5.6g of 60% oily sodium hydroxide, 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 Dissolve 4.5 g of 2,3,5-trimethylanisole in 500 ml of methylene chloride and cool to -5° to -10°C. , dropwise add 400 ml of methylene chloride solution containing 6.0 ml of chlorsulfonic acid. Thereafter, the mixture was brought to room temperature and placed 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 SCl: C, 48.29; H, 5.27; S, 12.89; Cl,
14.26 Experimental value: C, 48.42; H, 5.21; S, 12.61; Cl,
14.25 Reference Example 1 Synthesis of Z-Arg(Mtr)OH・CHA (1) 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 3 hours. As citric acid acidifies, 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 C ₃₀ H ₄₅ O ₇ N ₅ Calculated value as S: 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 (2) Synthesis of H-Arg(Mtr)-OH Add 1.5 g of Z-Arg(Mtr)-OH・CHA to ethyl acetate.
Dilute to 30ml, add 15ml of 0.2N-H ₂ SO ₄ , shake well, and wash with water. After distilling off the solvent, the residue was dissolved in methanol and subjected to catalytic reduction using Pd black as a catalyst. When the catalyst is filtered off, the solvent is distilled off, and water is added to the residue, it will crystallize.
Filter this out. 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. Reference example 2 (1) Boc-Tyr-Arg(Mtr)-OH H-Arg(Mtr)-OH 0.80 is dissolved in 20ml of tetrahydrofuran, and when cold, triethylamine 0.34
ml, Boc-TyrONB (Boc-Tyr-OH 0.57g,
(prepared from 0.40g of HONB and 0.50g of DCC) and stir at room temperature for 15 hours. After distilling off the solvent,
Acidify with citric acid and extract with ethyl acetate.
After washing the ethyl acetate layer with water, the solvent was distilled off, dissolved in chloroform, and transferred to a silica gel column (4 x 6
cm). Elute with 5% 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 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) 400 mg of Boc-Tyr-Arg(Mtr)OH is dissolved in 5 ml of trifluoroacetic acid-thioanisole (9:1) and left at room temperature for 2 hours. 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), and then water (300
ml) and 0.1M ammonium acetate (300 ml). Fractions of 100-150 ml are collected and lyophilized. Yield 175mg. [α] ²¹ _D −17.4° (C=0.5, H ₂ O) Amino acid decomposition (acid decomposition): Arg 1.00(1); Tyr 0.94 (1) Average recovery rate 86.5%. Reference example 3 (1) Z−Arg(Mtr)−Pro−Lys(Boc)−Pro−
Synthesis of OH H-Pro-OMe, Z-Lys(Boc)-ONB and Z-
0.59 g of oily Z-Pro-Lys(Boc)-Pro-OMe synthesized by successive condensation of Pro-ONB was dissolved in 30 ml of methanol, and catalytic reduction was performed using palladium black as a catalyst. After filtering off the catalyst and concentrating the filtrate, the residue was dissolved in dimethylformamide 10
ml and add Z−Arg(Mtr)OH・[Z−
Prepared from 0.56g of Arg (Mtr)・OH・CHA], HOBt
Add 0.15g and 0.23g of DCC and stir at room temperature for 15 hours. After filtering off the generated DCU and distilling off the solvent,
Dissolve the residue in ethyl acetate and wash 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)-Pro-
OMe] in 10 ml of menol. 2ml when cold
Add 1N aqueous sodium hydroxide and saponify at room temperature for 2 hours. After cold addition of 2 ml of 1N hydrochloric acid 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 610mg
(66.1%), melting point 90-95℃ [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
-Dissolve 0.49 g of NH ₂ in a mixture of trifluoroacetic acid (4.5 ml) and water (0.5 ml) and stir at 10°C 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. Dissolve this in 15 ml of dimethylformamide, add 0.1 ml of triethylamine when cold, and add Z-Arg.
(Mtr)-Pro-Lys(Boc)-Pro-OH 0.45g,
Add 0.18g of HONB and 0.20g of DCC and stir for 24 hours. The generated DCU is filtered off and the filtrate is concentrated. Add water to the residue and filter the resulting precipitate. Take 100mg of this and trifluoroacetic acid-
Dissolve in 1 ml of thioanisole (9:1) and shake 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 filtered and dried. This is dissolved in a small amount of water, passed through an Amberlite-IRA-410 (acetic acid type) column (1 x 10 cm), and then freeze-dried. Column of Sephadex G-25 (2.5 x 120cm)
and eluted with 30% acetic acid water. 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): Lys 1.00(1); Arg 1.04
(1); Glu 2.05(2); Pro 2.20(2); Gly 0.91(1);
Met 0.89(1); Leu 1.05(1); Phe 1.89(2), average recovery 82.3%.

Claims (1)

[Claims] 1 4-Methoxy-2,3,6-trimethylbenzenesulfonyl halide.