JPS6360040B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel S-polyprenyl peptides that have the effect of promoting mating tube formation in yeast.

Rhodosporidium, which belongs to the heterobasidiomycete yeast
It is well known that during the life history of toruloides and Tremella mesenterica, a hormone-like substance is secreted during the transition from haploid yeast to diploid yeast, and by the induction of this substance, they elongate the mating tube and cause cell fusion. This is a physiological phenomenon of yeast. It has become clear from the search for its active substance that this substance is a peptide, but simple peptides such as H
―Tyr―Pro―Glu―Ile―Ser―Trp―Thr―Arg
-Asn-Gly-Cys-OH and H-Glu-His-Asp-
Pro―Ser―Ala―Pro―Gly―Asn―Gly―Tyr―
Peptides such as Cys-OH, whose structure is thought to be related to hormone-like substances, have no physiological effects at all. This fact indicates that simple peptides have no effect on heterobasidiomycete yeast. If substances that act on these yeasts can be obtained, they can not only be used effectively for yeast breeding and seed improvement, but also serve as important reagents for biochemistry and molecular biochemistry research. be.

Based on the assumption that fat-soluble residues are bound to these active substances, the present inventors investigated by introducing various fat-soluble residues into the -SH group of cysteine. The present invention was completed by discovering that prenyl peptides have a stronger effect on promoting junctional tube formation than natural products.

That is, the present invention has the following formula: [In the formula, X is H-Tyr-Pro-Glu-Ile-Ser-
The present invention relates to an S-polyprenyl peptide represented by a peptide chain represented by Trp-Thr-Arg-Asn-Gly.

Regarding the above general formula (), Y represents a carboxyl group which may optionally be amidated (unsubstituted amide) or esterified with a lower alkyl group such as methyl, ethyl, propyl. In this specification, the abbreviations adopted by the IUPAC-IUB nomenclature committee or the abbreviations commonly used in the field may be used for amino acids or their residues, peptides, protecting groups, reagents used, etc. For example, the following abbreviations are given.

Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Cys: Cysteine Clu: Glutamic acid Gly: Glycine His: Histidine Ile: Isoleucine Pro: Proline Ser: Serine Thr: Threonine Trp: Tryptophan Tyr: Tyrosine Boc: t-Butoxycarbonyl Aoc :Amyloxycarbonyl Bu ^t :t-Butyl Z:Benzyloxycarbonyl Me: Methyl Bzl: Benzyl Bzl(Cl): p-chlorobenzyl MBzl: p-methoxybenzyl Tos: Tosyl DCC: N,N'-dicyclohexylcarbodiimide DCHA: Dicyclohexylamine HONB: N-hydroxy-5-norbornene-2,3-dicarboximide ONB: N-hydroxy-5-norbornene
2,3-dicarboximide ester TFA: trifluoroacetic acid DMF: dimethylformamide The above amino acid abbreviations are also used as they are to indicate the corresponding amino acid residues, and when an amino acid or its residue is indicated by the above abbreviations, Other than glycine shall mean L-form.

Cysteine-containing peptides, which are raw materials for producing the S-polyprenyl peptide of the present invention, are produced by removing the protecting group from the corresponding protected peptide, and the protected peptide is produced by a method known per se. For information on these known methods, see “The
Peptides” Volume 1 (1966), Schroder and Lubke
Author, Academic Press, New York, USA;
“Peptide Synthesis” by Izumiya et al., Maruzen Co., Ltd. (1975)
2007), or Biochemical Experiment Course 1, edited by the Japanese Biochemical Society,
“Protein chemistry, chemical modification and peptide synthesis”
It is explained in detail in literature such as Tokyo Kagaku Dojin (1977).

Briefly describing the method for producing the raw peptide below, first, a partial amino acid that can constitute the target polypeptide or the peptide and a compound that can constitute the remainder thereof are condensed by known peptide synthesis means. Condensation methods include, for example, the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, method using Woodward's reagent K, carbodiimidazole method, redox method, DCC-additive method ( HONB, 1
- Hydroxybenzotriazole, N-hydroxysuccinimide, etc. are used as additives). Before carrying out the main condensation reaction, it is preferable to protect carboxyl groups and amino groups that do not participate in the reaction of the raw materials by means known per se, or to protect hydroxyl groups and thiol groups. may be protected as tertiary alkylamine salts (e.g., triethylamine, N-methylmorpholine, etc.) or metal salts (e.g., sodium, potassium, lithium salts, etc.), or as esters (e.g., methyl,
It may be protected as esters such as ethyl, benzyl, p-chlorobenzyl, t-butyl, t-amyl, etc.). Protective groups for amino groups include benzyloxycarbonyl, t-butoxycarbonyl, isobornyloxycarbonyl groups, etc. Protective groups for imidazole groups of histidine include benzyl, tosyl, 2,4-dinitrophenyl, t-butyloxy, etc. Examples include carbonyl and carbobenzoxyl groups. Protecting groups for the guanidino group of arginine include, for example, nitro, tosyl, carbobenzoxyl, isobornyloxycarbonyl,
Examples include an adamantyloxycarbonyl group. In addition, as means for protecting the hydroxyl groups of tyrosine and serine, ethers with benzyl, t-butyl, t-amyl, etc. are used as protecting groups, and as means for protecting thiols, benzyl, p-methoxybenzyl, p-methylbenzyl, t- Examples include thioethers having butyl as a protecting group.

The peptide condensation reaction can be appropriately carried out in a commonly used solvent, such as anhydrous or hydrous dimethylformamide, dimethyl sulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, ethyl acetate, or an appropriate mixture thereof. is used. The reaction is generally carried out at a temperature in the range of about -20°C to +60°C. Further, the raw material compounds in the present invention can also be easily produced by a so-called solid phase synthesis method.

The protected cysteine-containing peptide thus obtained is subjected to a reaction as a thiol peptide by removing the protecting group. For removal of protecting group,
Catalytic reduction is not preferred due to the presence of sulfur, and a method using an acid decomposition reaction using, for example, hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, etc. is preferred. These reactions are generally carried out in the presence of anisole at temperatures of about -20°C to +40°C. After the reaction, the raw peptide produced in this manner can be purified by known peptide separation means such as partitioning, extraction, reprecipitation, column chromatography, and the like.

Now, the cysteine-containing peptide of the present invention...
A method for introducing a polyprenyl group into an SH group is carried out by dissolving the cysteine-containing peptide as a raw material in, for example, hydrous dimethylformamide and reacting it with a halogenated polyprenyl using magnesium oxide or the like as a catalyst.

Aqueous dimethylformamide solutions generally have a water content of
10-70% by volume, preferably 30-60% by volume,
The amount of magnesium oxide is 1 molar ratio to the peptide.
~10 equivalents are used, preferably 2 to 4 equivalents. Further, the molar ratio of the halogenated polyprenyl to the peptide is 1 to 10 equivalents, preferably 2 to 4 equivalents. The reaction is generally carried out at -10°C to 60°C, preferably 0°C.
It is carried out at ~30℃, and the reaction time is usually 3 hours to 12
About an hour is preferable.

The S-polyprenyl peptide thus produced can be separated and purified by known separation and purification means (e.g., distribution, extraction,
It can be isolated from the reaction solution by reprecipitation, column chromatography).

The S-polyprenyl peptide () produced by the method of the present invention is Rhodosporidium
It exhibits a zygotic tube formation promoting effect on toruloides at low concentrations of 0.1 to 10 ng/ml, has a strong yeast sex hormone effect, and is safe for the human body and easy to handle. Therefore, these S-polyprenyl peptides exhibit stronger effects than hormones with the same effect isolated from nature, and based on this, they are useful for yeast breeding, seed improvement, etc., and are also useful for biochemistry and molecular research. It can also be used as a reagent in biochemical research.
Generally, when using this compound, it is sufficient to add this compound to yeast culture solution at least at the active concentration described above, and as a biochemical reagent, this compound may be labeled with radioactive iodine or a fluorescent reagent. It can also be used as

Next, Reference Examples and Examples are shown, in which the following abbreviations are used for developing solvent systems for thin layer chromatography.

Rf ₁ = chloroform: methanol: acetic acid (9:
1:0.5) Rf ₂ = ethyl acetate: pyridine: acetic acid: water (60:
20:6:10) Rf ₃ = ethyl acetate: n-butanol: acetic acid: water (1:1:1:1) Rf ₄ = n-butanol: pyridine: acetic acid:
Water (30:20:6:24) Unless otherwise specified, Rf is based on Merck silica gel plate 60F ₂₅₄ .

Reference example 1 1) Synthesis of raw materials a) Dissolve 20.5 g and 9 ml of triethylamine in 200 ml of DMF and cool on ice. Add this to 7.2ml of benzyl bromide.
Add dropwise a solution of DMF (35 ml) and stir while gradually returning to room temperature. After 16 hours, the precipitated insoluble matter was filtered off, and the solvent of the filtrate was distilled off under reduced pressure. Dissolve the residue in 400 ml of ethyl acetate,
% - Wash with sodium carbonate and water and dry with anhydrous sodium sulfate. Ethyl acetate is distilled off and ether is added to the residue to form crystals. It is filtered and recrystallized from ether and petroleum ether.

Yield 16.1g, melting point 61-64℃, [α] ²¹ _D -45.8゜ (c 0.53, methanol) Elemental analysis C ₂₃ H ₂₉ O ₅ NS: Calculated value C
64.01; H 6.77; N 3.25; S 7.43.Analysis value
C 64.10; H 6.70; N 3.20; S 7.61 b) Dissolve 7 g in 35 ml of TFA and stir at room temperature for 5 minutes. TFA is distilled off under reduced pressure, and a small amount of ether and petroleum ether are added to the residue to obtain an oily precipitate.The solvent is removed, dissolved in 50 ml of ethyl acetate, and neutralized with 2.3 ml of triethylamine. Add 6 g of Boc-Gly-ONB to this solution and stir for 12 hours. The reaction solution was washed with 0.2N hydrochloric acid, 4% sodium bicarbonate and water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure, leaving an oily residue.

Yield 6.7g, Rf ₁ 0.7c) Dissolve 6.7g in 32.5ml of TFA and stir at room temperature for 10 minutes. 6.4N-hydrochloric acid/dioxane solution 2.5ml
The solvent is distilled off under reduced pressure, and the residue is washed with petroleum ether containing a small amount of ether and dissolved in 50 ml of DMF. Cool on ice, add 2.75 ml of triethylamine, and filter off the precipitated triethylamine hydrochloride. Add the filtrate to 3.18g of Boc-Asn-OH.
After adding 3.7g of HONB and cooling to -5℃
Add 3.11 g of DCC and stir. Stir for about 16 hours, filter off the insoluble precipitate, and dry the filtrate under reduced pressure. Dissolve the residue in 300ml of ethyl acetate and add 0.2N-
Wash with hydrochloric acid, 4% aqueous sodium bicarbonate, and water, and dry over anhydrous sodium sulfate. The solvent is distilled off under reduced pressure, and the precipitated solid is filtered with ether.

Yield 5.7 g, melting point 121-123°C, [α] ²³ _D -39.8° (c 0.54, methanol) Elemental analysis C ₂₉ H ₃₈ O ₈ N ₄ S: Calculated value C 57.79;
H 6.36; N 9.30; S 5.32, calculated value C
58.11; H 6.49; N 9.12; S 5.15 d) Dissolve 5.42 g in 30 ml of TFA and stir at room temperature for 20 minutes. TFA is distilled off under reduced pressure to give a residue of 6.4 N.
- Add 1.5 ml of hydrochloric acid/dioxane and stir, then add ether and filter the resulting precipitate.
Dissolve this in 50ml of DMF and use triethylamine.
Add 0.94 ml under cooling to neutralize and filter off the precipitated triethylamine hydrochloride. to the filtrate Add 2.96g and 2.41g of HONB and cool to -5℃.
Add 1.66g of DCC. Stir at room temperature for 16 hours, filter off the precipitate, and evaporate the solvent. The residue was extracted into 300 ml of ethyl acetate and diluted with 0.2N hydrochloric acid, 4%.
Wash with sodium bicarbonate and water and dry with anhydrous sodium sulfate. The solvent was distilled off, and the residue was filtered as a powder with ether. This is reprecipitated with acetonitrile and ether.

Yield 5.52g, melting point 70℃ (decomposition), [α] ²³ _D −
30.3゜(c0.535, methanol), Rf ₁ 0.41 e) Dissolve 5.1g in 35ml of TFA, stir at room temperature for 15 minutes, and dry under reduced pressure to form 6.4N-hydrochloric acid/dioxane.
Add 1.6 ml and further ether to form a precipitate, which is collected by filtration. Dissolve this in 40 ml of DMF, add 0.78 ml of triethylamine to neutralize it, and filter off the precipitated triethylamine hydrochloride. to the filtrate
Add a dioxane solution of Boc-Thr-ONB synthesized with 1.27 g of Boc-Thr-OH and 1.25 g of HONB and stir for 16 hours. After the reaction, the solvent was distilled off, the residue was dissolved in 500ml of ethyl acetate, and diluted with 0.2N
Wash with hydrochloric acid, 4% sodium bicarbonate and water, dry over anhydrous sodium sulfate and evaporate the solvent. Add a small amount of ethyl acetate to the residue and rub it with a glass rod to form gel-like crystals. Add ether and filter this.

Yield 5.42 g, melting point 80-80°C (decomposition), [α] ²³ _D -33.0° (c 0.52, methanol), Rf ₁ 0.20 f) Dissolve 4.07 g in 25 ml of TFA and leave at room temperature for 15 minutes. TFA is distilled off under reduced pressure, ether is added to the residue, and the solution is filtered. Dissolve this in 3 mm of acetonitrile, add 0.8 ml of triethylamine to neutralize it, and add ether to precipitate. Remove the supernatant ether, dissolve the precipitate in DMF, cool on ice, and add 1.28 g of Boc-Trp-OH to this.
Boc-Trp-ONB synthesized from 907mg of HONB
Add dioxane solution and stir at room temperature for 16 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was extracted with 500 ml of ethyl acetate.
- Wash with sodium bicarbonate and water and dry with anhydrous sodium sulfate. The solvent is distilled off and a small amount of ethyl acetate is added to the residue to form a gel. This is filtered using a mixed solvent of ethyl acetate and ether. The precipitate collected by filtration is suspended in ethyl acetate, heated to boiling point, cooled, and collected.

Yield 3.55g, melting point 120℃ (decomposition), [α] ²⁴ _D −
25.8° (c 0.585, methanol), Rf ₁ 0.18 g) Dissolve 3.3 g in 6.5 ml of dioxane containing 20%-1,2-ethanedithiol, add 13 ml of 6.4N-hydrochloric acid/dioxane, and leave at room temperature for 35 minutes. The solvent was distilled off, ether was added to the residue, and the powder was collected by filtration. Dissolve this in 10 ml of DMF, neutralize by adding 0.4 ml of triethylamine, and filter off the precipitated triethylamine hydrochloride.
Boc-Ser-OH 621mg and HONB 990mg in filtrate
Add 749mg of DCC and stir while cooling on ice. After stirring at room temperature for 16 hours, insoluble matter was filtered off. The solvent was distilled off under reduced pressure, and ether was added to the residue, which was filtered as a powder. Add this to ethyl acetate:pyridine:acetic acid:water (120:10:3:5)
Silica gel column (5.5×
12.5cm) and elutes with the same solvent, 1490
The ~2785 ml eluate is collected and concentrated, ether is added and the resulting powder is filtered off.

Yield 1.9g, melting point 150-153℃ (decomposed), [α] ²⁵ _D -28.2゜ (c 0.34, methanol) Elemental analysis C ₆₀ H ₇₈ O ₁₆ N ₁₂ S ₂ : Calculated value C 55.97; H 6.11; N 13.06 ;S 4.98,
Analysis value C 55.57; H 6.27; N 12.67; S
4.77 h) Production of Boc-Tyr-Pro-OH: H-Pro-
Dissolve 4.4 g of OH in 80 ml of aqueous dioxane, add 4.75 ml of triethylamine, and cool on ice. Add 11.1 g of Boc-Tyr-ONB to this solution and stir vigorously. The solvent was distilled off, the solution was dissolved in 80 ml of 4% sodium bicarbonate water, and washed with 50 ml of ether.
Cool on ice, adjust the pH to 2 with 0.2N hydrochloric acid, and extract the precipitated oil into 100 ml of ethyl acetate. The ethyl acetate layer is washed with water, dried over anhydrous sodium sulfate, and the solvent is distilled off. Add ether to the precipitated crystals and collect by filtration.

Yield 4.3g, melting point 123-126℃ (decomposition), [α] ^22.5 _D -23.6゜ (c 0.525, methanol), elemental analysis C ₁₉ H ₂₆ O ₆ N ₂ : Calculated value C
60.30; H 6.93; N 7.40. Analysis value C
60.15; H 6.80; N 7.30. i) Production: 14.2 g of H-Ile-OBzl p-toluenesulfonate was suspended in 400 ml of ethyl acetate, washed with saturated sodium carbonate water, further washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. next 15.6 g was suspended in 300 ml of ethyl acetate, and DCHA was extracted and removed with 0.2N sulfuric acid. After further washing with water and drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. Both were dissolved in 140 ml of a mixed solvent of tetrahydrofuran and ethyl acetate (1:1),
Add 8.1g of HONB, cool on ice, add 7.44g of DCC, and stir. Stir at room temperature for 12 hours, filter off the precipitate, distill off the solvent, and dissolve the residue in ethyl acetate.
Dissolve in 300ml. This is washed with IN-hydrochloric acid, 4% aqueous sodium bicarbonate, and water, and dried over anhydrous sodium sulfate. When the solvent is distilled off and the mixture is left at room temperature, it crystallizes, and petroleum benzine is added thereto and the mixture is filtered.

Yield 13g, melting point 65-68℃, [α] ^23D _-24.5゜(c
0.60, methanol), Rf ₁ 0.92 j) Dissolve 5.4 g in methanol with 10 ml of N-hydrochloric acid, add 1 g of palladium black, and reduce through hydrogen. Palladium black is filtered off, methanol is distilled off under reduced pressure, the residue is dissolved in 100 ml of DMF, and 2.8 ml of triethylamine is added under ice cooling. separately
Boc―Tyr―Pro―OH 3.78g and HONB 2.16g
was dissolved in 100 ml of a mixed solvent of ethyl acetate and dioxane (1:1), 2.27 g of DCC was added, and the solution was dissolved at room temperature.
Stir the time. Filter off the precipitate, add this filtrate to the above DMF solution, and stir for 16 hours. After the reaction is complete, the solvent is distilled off under reduced pressure, extracted into 200 ml of water containing 2.5 g of sodium hydrogen carbonate, and washed with ether. The aqueous layer is cooled with ice, acidified with 35 ml of N-hydrochloric acid, and the precipitated oil is extracted twice with 200 ml of ethyl acetate. The ethyl acetate layers are combined, washed with water, and then dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure, and the residue was filtered as a powder with ether. This was applied to a silica gel column (5.5 x 12.5 cm) using a mixed solvent of chloroform:methanol:water (107:4:2) and eluted with the same solvent system to obtain 680
-980 ml of the eluate is collected, the solvent is distilled off, and the residue is filtered off as a powder with ether.

Yield 3.45g, melting point 115-119℃ (decomposition), [α]
²³ _D −40.2゜(c 0.465, methanol) Elemental analysis C ₃₄ H ₅₂ O ₁₀ N ₄ : Calculated value C
60.34; H 7.74; N 8.28, analysis value C
60.22; H 8.02; N 7.82, k) Dissolve 644mg in DMF 0.5ml and dioxane 2ml,
1,2-ethanediol 0.3ml and 6.4N-hydrochloric acid/
Add 6 ml of dioxane and stir at room temperature for 30 minutes. The solvent was distilled off under reduced pressure, ether was added to the residue, and the powder was collected by filtration. Dissolve this in 5 ml of DMF and neutralize by adding 0.3 ml of triethylamine.
The precipitated triethylamine hydrochloride is filtered off and added to the filtrate. Add 406 mg and 360 mg of HONB and cool to -10 to -5°C. Add 206 mg of DCC and stir for 16 hours. The precipitated insoluble matter is removed by filtration, the solvent is distilled off, and the residue is filtered as a powder using acetonitrile and ethyl acetate.

Yield 650mg, melting point 100-105℃ (decomposition), [α] ²⁵ _D
−26.6° (c 0.365, methanol), elemental analysis C ₈₉ H ₁₂₀ O ₂₃ N ₁₆ S ₂・2H ₂ O: Calculated value C56.79; H6.64; N11.91; S3.41. Analysis value C56.23 ;H6.67;N11.67;S3.90. Amino acid analysis, analysis value (theoretical value): Arg 0.99
(1), Asp 1.03(1), Thr 0.98(1), Ser 0.92(1),
Glu 0.87(1), Pro 1.09(1), Glx 1.0(1), Cys
0.37(1), Ile 0.85(1), Tyr 0.68(1), Average recovery rate
72%) H-Tyr-Pro-Glu-Ile-SerTrp-Thr
-Production of Arg-Asn-Gly-Cys-OH: 554 mg in the presence of 1.6 ml of anisole and 20 ml of hydrogen fluoride
and stir at 0°C for 60 minutes. Hydrogen fluoride was distilled off under reduced pressure, and the residue was extracted with 10 ml of water and 5 ml of ether.
wash with The aqueous layer was passed through a resin column (1 x 5 cm) of Amberlite IRA-410 (acetic acid type), washed with water,
All the flow throughs are combined and lyophilized to give 405 mg. This was applied to a Sephadex LH-20 (2.4 x 107 cm) column, eluted with 0.1N acetic acid, and 237-281 ml of the eluate was collected and lyophilized to obtain 106 mg of SH peptide.

[α] ²⁶ _D −63.5° (c 0.60, 1N-acetic acid), Rf ₄
(Cellulose) 0.64 Amino acid analysis, analysis value (theoretical value): Arg1.14(1),
Trp0.59(1), Asp 1.00(1), Thr 1.00(1), Ser 0.95
(1), Glu 0.95(1), Pro 1.02(1), Gly 1.00(1), Cys
0.86(1), Ile 0.95(1), Tyr 0.98(1), Average recovery rate
79.1% Example 1 H-Tyr-Pro-Glu-Ile-Ser-Trp-Thr-
Arg-Asn-Gly-Cys (S-pentaprenyl)-
Production of OH: H-Tyr-Pro-Glu-Ile-Ser-
Trp―Thr―Arg―Asn―Gly―Cys―OH 40mg
Dissolve in 3 ml of 70% hydrated DMF with 6 mg of MgO,
Add dropwise 0.5 ml (0.05 mmol) of an isopropyl ether solution of pentaprenyl bromide (trans isomer) and stir for 16 hours. The solvent was distilled off under reduced pressure, dissolved in 0.5 ml of 85% n-butanol aqueous solution, and Sephadex LH
- Develop into 20 columns (2.3 x 28.5 cm). 40～
Collect a 56 ml fraction, concentrate it, apply it again to the same column, collect the target fraction, and distill off the solvent to obtain 10 mg of the target product.

_Rf3 0.70

Claims (1)

[Claims] 1 formula [In the formula, X is H-Tyr-Pro-Glu-Ile-Ser-
An S-polyprenyl peptide represented by a peptide chain represented by Trp-Thr-Arg-Asn-Gly.