JPH0134239B2 - - Google Patents

Description

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

Rhodosporidium toruloides and Tremella mesenterica, which belong to heterobasidiomycete yeasts
However, it is well known that yeast secretes a hormone-like substance during the transition from haploid to diploid yeast in its life history, and induces cell fusion by elongating the mating tube. It is a physiological phenomenon.

Already among these hormone-like substances, the former A-factor is rhodotorcin A.
(rhodotorucine A) and the latter as tremerogen A-10 (tremerogen A-10), and their structures have been determined [Agric.Biol.
Chem., 43 , 363 (1979); ibid., 43 , 2643
(1979)].

The present inventors isolated and determined the structure of the above-mentioned a-factor secreted from the isomerocytes of Tremela mesenterica, and herein named it Tremelogen a-13.

Structure of tremelogen a-13 Furthermore, the present inventors succeeded in chemically synthesizing tremelogen a-13, and at the same time synthesized various derivatives, and revealed that these also have the same strong activity. These substances can not only be used effectively for breeding and improving seeds of normal yeast and yeast prepared by genetic engineering, but also can serve as important reagents for biochemistry and molecular biochemistry research. .

That is, the present invention provides a novel general formula that has a-factor activity in the mating tube forming action of yeast. [In the formula, R is hydrogen, alkyl having 1 to 10 carbon atoms with or without a sulfur atom, or the general formula H-[ _CH2C ( _-CH3 )= _CHCH2- ] _o (in the formula,
n is an integer of 1 to 5); It provides a manufacturing method.

As used herein, amino acids or residues thereof,
IUPAC-IUB for peptides, protecting groups, reagents, etc.
Abbreviations adopted by the Nomenclature Committee or abbreviations commonly used in the field may be used; examples of such abbreviations include the following.

Arg: Arginine Asn: Asparagine Asp: Aspartic acid Cys: Cysteine Glu: Glutamic acid Gly: Glycine Pro: Proline Ser: Serine Val: Valine Boc: t-Butoxycarbonyl Aoc: Amyloxycarbonyl But: ^t -Butyl Z: Benzyloxycarbonyl Me: Methyl Bzl: Benzyl 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 abbreviations for each amino acid above are also used as they are to indicate the corresponding amino acid residue. When the above abbreviations are used, the symbols other than glycine mean the L-form.

In the formula, examples of the alkyl having 1 to 10 carbon atoms represented by R include methyl, propyl, i-propyl, 2-hexenyl, octyl, and decyl. R is the general formula H-[CH ₂ C (-CH ₃ )=CHCH ₂
-] _o (wherein n is an integer of 1 to 5), polyprenyl is preferably bonded directly to the sulfur atom of the polypeptide without intervening a sulfur atom.

The esterification of carboxyl represented by
butyl, i-butyl, t-butyl), and amidation refers to amidation with an unsubstituted amino group.

The compound of the present invention may exist as a salt with an acid (e.g., acetate, etc.) or a salt with a base (e.g., sodium salt, etc.) depending on extraction and purification conditions, and these salts are also included in the present invention. It is something.

The compound () of the present invention can be chemically produced by condensing a partial amino acid that may constitute the polypeptide of the target compound () or the peptide with a compound that may constitute the remainder thereof using peptide synthesis means. The peptide synthesis means may be according to any known method, such as Bodansky and Ondechi, Peptide Synthesis, Interscience, 1966; Huynh and Hofhon, The Proteins, Volume 2, Academic Press, 1976; "Peptide Synthesis" by Nobuo Izumiya et al., Maruzen Co., Ltd., 1975, methods described in Japanese Patent Application Laid-open No. 55-28948 or Japanese Patent Application No. 113663-1983, such as the azide method and 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/
Examples include the HONB method. In some cases,
The NCA method (N-carboxyanhydride; a method using an intramolecular cyclic carbonyl compound corresponding to an amino acid without using a protecting group) may be applied.

Before carrying out the main condensation reaction, carboxyl groups that do not participate in the reaction of the raw materials are removed by means known per se.
The amino group may be protected, or the carboxyl group or amino group involved in the reaction may be activated.

Examples of the protecting group for the raw material include the above-mentioned protecting groups. The carboxyl group of the raw material is, for example, a metal salt (e.g., sodium, potassium salt, etc.), a t-alkylamine salt (e.g., triethylamine, N-methylmorpholine, etc.), or an ester (e.g., methyl, ethyl, benzyl, p-nitrobenzyl). ,t
-butyl, t-amyl, etc.). As the protecting group for the amino group of the raw material, for example, benzyloxycarbonyl group, t
-butoxycarbonyl group, amyloxycarbonyl group, isobornyloxycarbonyl group, etc., but examples of protecting groups for the guanidino group of arginine include nitro, tosyl, carbobenzoxyl, isobornyloxycarbonyl, adamantyloxycarbonyl, p -Methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, while benzyl, p-methoxybenzyl, p-methylbenzyl and Examples include thioethers having a protecting group such as t-butyl.

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 general formula R ¹ ―Glu(―R ² )―Gly―Gly―Gly―Asn― Arg(―R ³ )―Gly―Asp(―R ² )―Pro ―Ser―Gly―Val― obtained by these methods (L) NHCH(-X′)CH ₂ SR ⁴
() [In the formula, R ¹ , R ² and R ³ represent a protecting group, R ⁴ represents an alkyl or a protecting group, and X' represents a carboxyl protected by R ² or amidated or esterified] The protecting group of the cysteine-containing polypeptide represented by is removed to form the compound ()' [In the formula, R' is hydrogen or alkyl, and X has the same meaning as above] can be produced.

In the above general formula (), the protecting group represented by R ¹ is a protecting group for an amino group used in a peptide condensation reaction, such as t-butoxycarbonyl, benzyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl. , adamantyloxycarbonyl, O-nitrophenylthio, diphenylphosphinothioyl,
Examples include dimethylphosphinothioyl, chloro- or nitro-substituted benzyloxycarbonyl group, and the like. The protecting group represented by R ² includes carboxyl group protecting groups used in peptide condensation reactions, such as methyl, ethyl, t-butyl,
benzyl, chloro- or nitro-substituted benzyl,
sec-butyl, cyclopentyl, cyclohexyl,
Examples include diphenylmethyl (as an ester).

Examples of the protecting group represented by R ³ include nitro, tosyl, carbobenzoxyl, isobornyloxycarbonyl, adamantyloxycarbonyl, methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl, 4-
Examples include methoxy-2,3,6-trimethylbenzenesulfonyl group. Further, examples of the protecting group represented by R ⁴ include benzyl, p-methoxybenzyl, p-methylbenzyl, and t-butyl.

For removal of the 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 from -20°C to +40°C.
It is carried out at a temperature of about ℃. After the reaction, the cysteine-containing peptide produced in this manner can be purified by known peptide separation means such as partitioning, extraction, reprecipitation, column chromatography, etc.

Compound () [where R is a chain hydrocarbon group with or without a sulfur atom]
The cysteine-containing peptide ()' obtained here is reacted with a compound represented by the general formula R-Z () [wherein R has the same meaning as above and Z represents hydrogen or halogen]. It can be obtained by Examples of the halogen represented by Z here include bases, bromine, and iodine.

In the case of a chain hydrocarbon group via a sulfur atom, the method for introducing a substituent represented by R into the -SH group of the cysteine-containing peptide of the present invention is to convert the cysteine-containing peptide into, for example, a lower alkanol (methanol, ethanol, butanol, etc.). compound ()
and reacted with air oxidation or in the presence of an oxidizing agent (eg, iodine, 1,2-diiodide ethane).

In the case of a chain hydrocarbon without a sulfur atom, the cysteine-containing peptide is dissolved in, for example, water or water-containing dimethylformamide, and if necessary, the reaction is carried out with the compound (2) using magnesium oxide or dilute hydrochloric acid as a catalyst. Hydrous dimethylformamide solutions generally have a water content of 10~
The amount is 70% by volume, preferably 30 to 60% by volume, and when magnesium oxide is added, it is used in a molar ratio of 1 to 10 equivalents, preferably 2 to 4 equivalents, relative to the peptide. Use dilute hydrochloric acid of about 0.05N to 0.5N.

Compound () has a molar ratio of 1 to peptide
10 equivalents, generally 2 to 4 equivalents are preferred. The reaction is generally carried out at -10°C to 60°C, preferably 0°C to 30°C, and the reaction time is usually preferably about 3 hours to 20 hours.

The polypeptides thus produced can be isolated from the reaction solution by means of separation and purification known per se (eg, partitioning, extraction, reprecipitation, column chromatography).

The polypeptide () produced by the method of the present invention is Tremella mesenterica (IFO-9310).
On the other hand, it shows a promoting effect on junctional tube formation at a low concentration of 0.02 to 2 ng/ml, has a strong yeast sex hormone effect, and is safe for the human body and easy to handle.
Therefore, based on this, it is useful for yeast breeding, seed improvement, etc., and can also be used as a reagent for biochemistry and molecular biochemistry 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 a file.

Next, reference examples and examples will be shown, in which thin layer chromatography was used under the following conditions.

Rf ¹ = chloroform: methanol: acetic acid (9:
1:0.5) ^*1 Rf ² = n-butanol:acetic acid:water (12:3:
5) ^*1 Rf ³ = n-butanol:pyridine:acetic acid:water (30:20:6:24) ^*2 *1 Silica gel 60F ₂₅₄ (manufactured by Merck) *2 Avicel (manufactured by Funakoshi Pharmaceutical) Reference example 1 BOC- Production of Val-Cys(MBzl)-OBzl: Dissolve 4.3g of BOC-Cys(MBzl)-OBzl in 30ml of TFA and stir at room temperature for 5 minutes. under reduced pressure
Distill off TFA, add a small amount of ether and petroleum ether to the residue to form an oily precipitate, and remove the solvent.
Dissolve in 20 ml of DMF and neutralize with 1.4 ml of triethylamine. Add 2.2g of BOC-Val-OH to this solution.
Add BOC-Val-ONB obtained by reacting 2.2 g of HONB and 2.3 g of DCC in acetonitrile and stir for 18 hours. The reaction solvent was distilled off, and the residue was dissolved in ethyl acetate.
Dissolve in 200ml. Add this solution to 0.2N hydrochloric acid, 4%
-Wash with sodium bicarbonate and water, dry over anhydrous sodium sulfate, and then distill off the solvent to obtain crystals. It is further recrystallized from ethyl acetate and ether.

Yield 3.2g, melting point 111-112℃, [α] ²⁴ _D −
64.5° (c 0.59, methanol).

Elemental analysis C ₂₈ H ₃₈ O ₆ N ₂ S: Calculated value C63.37;
H7.22; N5.28; S6.04. Analysis value C63.53; H7.24;
N5.41; S6.04.

Reference example 2 Production of BOC-Gly-Val-Cys (MBzl)-OBzl: 3g of BOC-Val-Cys (MBzl)-OBzl was added to TFA20
ml and stir for 5 minutes at room temperature. Distill TFA under reduced pressure, add a small amount of ether and petroleum ether to the residue to form an oily precipitate, and remove the solvent.
Dissolve in 20ml DMF. After neutralizing by adding 4 ml of triethylamine, add 2.3 g of BOC-Gly-ONB and stir for 16 hours. The solvent was distilled off, and the residue was dissolved in 200 ml of ethyl acetate, washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate and water, and dried over anhydrous sodium sulfate. The solvent is distilled off to obtain crystals, which are then recrystallized from ethyl acetate and ether.

Yield 2.8g, melting point 123-126℃, [α] ²⁴ _D −
62.2° (c 0.55, methanol).

Elemental analysis C ₃₀ H ₄₁ O ₇ N ₃ S: Calculated value C61.30;
H7.03; N7.15; S5.46. Analysis value C61.56;
H7.02; N7.08; S5.53.

Reference example 3 BOC―Ser―Gly―Val―Cys (MBzl)―OBzl
Production of: BOC-Gly-Val-Cys (MBzl)-OBzl2g
Dissolve in 10 ml of TFA, stir for 5 minutes at room temperature, then distill off the TFA, add ether to the residue, and collect by filtration. After dissolving this in 15 ml of DMF and neutralizing it with 0.5 ml of triethylamine, BOC-Ser-OH838
BOC-Ser-ONB synthesized from mg and HONB832mg
Add and stir for 16 hours. The reaction solution was distilled off, ethyl acetate was added to the residue, and the precipitate was collected by filtration. After drying, it is recrystallized from acetonitrile.

Yield 1.12g, melting point 131-132℃, [α] ²⁴ _D -
55.7° (c0.67, methanol) Elemental analysis
C ₃₃ H ₄₆ O ₉ N ₄ S: Calculated value C58.73; H6.87;
N8.30; S4.75. Analysis value C59.09; H7.08;
N8.38; S4.52.

Reference example 4 BOC―Asp(OBzl)―Pro―Ser―Gly―Val―
Production of Cys(MBzl)-OBzl: BOC-Asp(OBzl)-Pro-OH・DCHA910mg
Suspend in 50 ml of ethyl acetate, add 13 ml of 0.2N sulfuric acid, and mix well.The ethyl acetate layer is washed thoroughly with water, dried over anhydrous sodium sulfate, and the solvent is distilled off. On the other hand, BOC―Ser―Gly―Val―Cys
Dissolve 1.02 g of (MBzl)-OBzl in 7 ml of TFA, evaporate the solvent after 5 minutes, add equimolar amount of hydrochloric acid and dioxane, and stir. Further, ether is added to obtain a precipitate, which is filtered and dried. Dissolve this in 15ml of DMF,
After neutralizing with 0.21 ml of triethylamine, add the above residue and 816 mg of HONB, and cool on ice. next
Add 468mg of DCC and cool on ice for 4 hours, then at room temperature.
Stir for 12 hours. After filtering off the precipitate and distilling off the solvent, 100 ml of ethyl acetate was added to dissolve.
The ethyl acetate layer was washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water, and dried over anhydrous sodium sulfate. The solvent was distilled off, and the residue was dissolved in a solvent system of ethyl acetate-pyridine-acetic acid-water (1422:10:3:5), applied to a silica gel column (4 x 6.5 cm) packed with the same solvent, and developed. do. 300-750
Collect ml fractions, distill off the solvent, add ether to the residue, and filter it as a powder.

Yield 500mg, melting point 111℃, [α] ^23D _-42.0゜(c
0.26, DMF).

Elemental analysis C ₄₉ H ₆₄ O ₁₃ N ₆ S: Calculated value C60.23;
H6.60; N8.60; S3.28. Analysis value C60.23;
H6.82; N8.58; S3.30.

Reference example 5 Production of Aoc-Arg(Tos)-Gly-OH: Aoc-Arg(Tos)-OH17.7g and HONB8.64g
Dissolve in 300 ml of DMF, add 9.1 g of DCC under ice cooling, stir for 4 hours, and continue to cool to room temperature.
Stir for 12 hours. The precipitate was filtered off, and the filtrate was diluted with H-
Mix with 200 ml of an aqueous solution containing 4.5 g of Gly-OH and 4.2 g of sodium bicarbonate, and stir vigorously. 3
After an hour, the solvent was evaporated and the residue was extracted into ethyl acetate and water (200ml-500ml). Cool the aqueous layer on ice, N
- After adding hydrochloric acid to adjust the pH to 2-3, extract again with 500 ml of ethyl acetate, wash thoroughly with water, dry over anhydrous sodium sulfate, and distill off the solvent. The residue was dissolved in ethyl acetate-pyridine-acetic acid-water (146:
The desired product was dissolved in a solvent system of 10:3:5) and applied to a silica gel column (6 x 22 cm) packed with the same solvent, a fraction of the target product was collected, and the solvent was distilled off to obtain an oil.

Yield 10g, Rf ¹ 0.20.

Reference example 6 Aoc―Arg(Tos)―Gly―Asp(OBzl)―Pro―
Production of Ser―Gly―Val―Cys (MBzl)―OBzl: BOC―Asp (OBzl)―Pro―Ser―Gly―Val―
Cys (MBzl) - Dissolve 400mg of OBzl in 3ml of TFA,
After 10 minutes, the solvent was distilled off and ether was added to the residue.
Filter it as a powder. Dissolve this in 15ml of DMF,
After neutralizing by adding 0.6 ml of triethylamine,
Aoc―Arg(Tos)―Gly―OH246mg and HONB177
Aoc－Arg(Tos)－Gly－ synthesized from mg and
Add 15 ml of ONB in DMF and stir at room temperature for 16 hours. After filtering off the insoluble matter and distilling off the solvent,
The residue is filtered as a powder with ethyl acetate and ether, washed with hot ethyl acetate, and filtered as a powder with ether.

Yield 400mg, melting point 115-120℃ (decomposition), [α]
²³ _D -33.2° (c 0.18, DMF).

Elemental analysis C ₆₅ H ₈₇ O ₁₇ N ₁₁ S ₂・2H ₂ O: Calculated value
C55.98; H6.58; N11.05; S4.60. Analysis value
C56.06; H6.83; N11.02; S3.85.

Reference example 7 BOC―Asn―Arg(Tos)―Gly―Asp(OBzl)
-Pro-Ser-Gly-Val-Cys (MBzl)-OBzl
Production of: Aoc―Arg(Tos)―Gly―Asp(OBzl)―Pro―
Ser―Gly―Val―Cys (MBzl)―OBzl350mg
Dissolve in 2 ml of TFA and evaporate the solvent after 10 minutes. Add ether to the residue and filter it. After drying
Dissolve in 0.5 ml of DMF, add 0.1 ml of triethylamine, stir well, add 50 ml of ether, filter as a powder, and dry. This is BOC―Asn―
Dissolve in 20 ml of DMF with 90 mg of OH and 140 mg of HONB, add 80 mg of DCC under ice cooling, and stir for 4 hours.
Stir for another 16 hours at room temperature. Insoluble matter is filtered off, the solvent is distilled off, and the precipitated residue is filtered off with acetonitrile. The solvent of the filtrate was distilled off, and the residue was diluted with ethyl acetate-pyridine-acetic acid-water (146:10:
The precipitate is collected by filtration using a mixed solvent of 3:5).

Yield 220mg, melting point 154-160℃ (decomposition), [α]
²³ _D -38.9° (c 0.45, DMF).

Elemental analysis C ₆₈ H ₉₁ O ₁₉ N ₁₃ S ₂・H ₂ O: Calculated value
C55.31; H6.35; N12.33; S4.34. Analysis value
C55.25; H6.45; N12.36; S4.34.

Reference example 8 Production of BOC-Glu(OBzl)-Gly-OH: 4.5g of H-Gly-OH and 4.2g of sodium hydrogen carbonate
Dissolve in 100ml of water, add BOC-Glu(OBzl)
-BOC synthesized from 13.5g of OH and 8.6g of HONB
- Add 200ml of DMF solution of Glu(OBzl)-ONB,
Stir vigorously. After 6 hours, the solvent was distilled off,
Dissolve the residue by adding 250ml of water and 100ml of ether.
Extract with Cool the aqueous layer on ice, add N-hydrochloric acid to pH 2
-3 and then extracted with 300 ml of ethyl acetate. Wash the ethyl acetate layer thoroughly with water and dry with anhydrous sodium sulfate. The solvent was distilled off, petroleum benzine was added to the residue, and the crystals were collected by filtration.

Yield: 12.1 g, melting point: 94-96°C, [α] ²³ _D -8.4° (c 0.55, methanol).

Elemental analysis C ₁₉ H ₂₆ O ₇ N ₂ : Calculated value C57.86;
H6.64; N7.10. Analysis value C57.94; H6.58; N7.19.

Reference example 9 Production of BOC-Glu(OBzl)-Gly-Gly-Gly-OH: 1.32g of H-Gly-Gly-OH and triethylamine
Dissolve 1.4ml in a mixed solvent of water-DMF (20ml-30ml), and dissolve 3.9g of BOC-Glu(OBzl)-Gly-OH.
BOC-Glu (OBzl) synthesized from 2.2g of HONB
-Gly- Mix with 20ml of DMF solution of ONB and stir vigorously. After 16 hours, the solvent was distilled off, and the residue was dissolved in cold 0.2N hydrochloric acid to adjust the pH to 2-3. This is extracted with 300 ml of ethyl acetate, and the ethyl acetate layer is washed with water, dried over anhydrous sodium sulfate, and the solvent is distilled off. The residue is filtered with ether and recrystallized from methanol and ether.

Yield: 2.8 g, melting point: 138-139°C, [α] ²³ _D -1.6° (c 0.74, methanol).

Elemental analysis C ₂₃ H ₃₂ O ₉ N ₄ : Calculated value C54.32;
H6.34; N11.02. Analysis value C54.56; H6.42;
N11.30.

Reference example 10 BOC―Glu(OBzl)―Gly―Gly―Gly―Asn―
Arg(Tos)―Gly―Asp(OBzl)―Pro―Ser―
Production of Gly-Val-Cys (MBzl)-OBzl: BOC-Asn-Arg (Tos)-Gly-Asp (OBzl)
―Pro―Ser―Gly―Val―Cys (MBzl)―
Dissolve 180 mg of OBzl in 1 ml of TFA, and evaporate the solvent after 10 minutes. Ether was added to the residue, filtered, dried, dissolved in DMF 0.4ml, and triethylamine 0.1ml.
Add and stir well, then add 50 ml of ether and filter it as a powder. Dissolve this in 10ml of DMF and add BOC-Glu(OBzl)-Gly-Gly-Gly-
Add 94mg of OH and 133mg of HONB and cool on ice. next
Add 76 mg of DCC, cool on ice for 4 hours, and then keep at room temperature for 16 hours.
Stir for a while, filter off insoluble matter, and distill off the solvent. The residue is filtered as a powder with ether, washed with a mixed solvent of acetonitrile and ethyl acetate (1:1), and filtered.

Yield 180mg, melting point 165-168℃ (decomposition), [α]
²³ _D -30.5° (c 0.26, DMF).

Elemental analysis C ₈₆ H ₁₁₃ O ₂₅ N ₁₇ S ₂ H ₂ O: Calculated value
C55.32; H6.21; N12.75; S3.43. Analysis value
C55.30; H6.24; N12.73; S3.32.

Reference example 11 BOC―Glu(OBzl)―Gly―Gly―Gly―Asn―
Arg(Tos)―Gly―Asp(OBzl)―Pro―Ser―
Production of Gly-Val-Cys(MBzl) _-NH2 : The same operations as in Reference Examples 2 to 10 are repeated using BOC-Cys(MBzl) _-NH2 as a starting material to obtain the target compound.

Yield 499mg, melting point 190-193℃ (decomposition), [α]
²⁶ _D -36.7° (c 0.22, DMF).

Elemental analysis C ₇₉ H ₁₀₈ O ₂₄ N ₁₈ S ₂・2H ₂ O: Calculated value
C52.89; H6.29; N14.06; S3.57. Analysis value
C53.10; H6.29; N13.85; S3.62.

Reference example 12 Production of S-decyl-cysteine: 12.1g of cysteine was added to 100% of 2N-sodium hydroxide.
ml and add 330ml of ethanol. Next, 22.1 g of decyl bromide was dissolved in 30 ml of ether, added to the above solution under a nitrogen stream, and stirred at room temperature for 8 hours. Neutralize by adding 25 ml of 4N hydrochloric acid, filter out the precipitated crystals, wash thoroughly with water, and dry.

Yield 23.4g, melting point 217-218℃ (decomposed),
[α] ²³ _D -64.5° (c 0.12, acetic acid).

Elemental analysis C ₁₃ H ₂₇ O ₂ NS: Calculated value C59.72;
H10.41; N5.36; S12.27. Analysis value C59.83;
H10.41; N5.45; S12.21.

Reference example 13 Production of BOC-Cys(S-decyl)-OH: Dissolve 2.6 g of S-decylcysteine in 400 ml of 50% aqueous DMF with 2 ml of triethylamine,
Add 8 g of -t-butoxycarbonyloxyimino-2-phenylacetonitrile and stir at room temperature for 24 hours. Insoluble materials are filtered off, the solvent is distilled off, and the residue is extracted into a mixed solution of 100 ml of 4% sodium bicarbonate water and 100 ml of ethyl acetate. cool the water layer,
Add N-hydrochloric acid to adjust the pH to 2, extract with 100 ml of ethyl acetate, wash the ethyl acetate layer with water, and then dry over anhydrous sodium sulfate. The solvent was distilled off to obtain an oil.

Yield 1.0g, Rf ¹ 0.71 Reference example 14 BOC―Glu(OBzl)―Gly―Gly―Gly―Asn―
Arg(Tos)―Gly―Asp(OBzl)―Pro―Ser―
Production of Gly-Val-Gys (MBzl)-OMe: Using BOC-Cys (MBzl)-OMe as the starting material,
The same operations as in Reference Examples 2 to 10 are repeated to obtain the target compound.

Yield 140mg, melting point 120-123℃ (decomposition), [α]
²² _D -22.3° (c 0.27, DMF).

Elemental analysis C ₈₀ H ₁₀₉ O ₂₅ N ₁₇ S ₂・H ₂ O: Calculated value
C53.65; H6.25; N13.30; S3.58. Analysis value
C53.41; H6.45; N13.00; S3.33.

Example 1 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Production of Asp-Pro-Ser-Gly-Val-Cys-OH: BOC-Glu(OBzl)-Gly-Gly-Gly-Asn-
Arg(Tos)―Gly―Asp(OBzl)―Pro―Ser―
Treat 15 mg of Gly-Val-Cys (MBzl)-OBzl with 1 ml of anisole in 10 ml of HF at 0°C for 60 minutes. The HF is distilled off, the residue is dissolved in 10 ml of water and extracted with 3 ml of ethyl acetate. amber light water layer
IRA-410 (acetic acid type) resin column (1 x 5 cm)
Wash the column thoroughly with water. All flowthrough is collected and lyophilized.

Yield 95 mg, [α] ²³ _D -55.2° (c 0.12, N-acetic acid).

Amino acid analysis values: Arg1.08, Asp1.91, Ser0.87,
Glu1.05, Pro0.88, Gly5.0, Cys0.81, Val0.91 Average recovery rate 78.3%, Rf ³ 0.24 Example 2 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp-Pro-Ser-Gly-Val-Cys (s-farne
-syl)-OH (tremelogen a-13) production: H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Dissolve 36 mg of Asp-Pro-Ser-Gly-Val-Cys-OH in 2 ml of degassed water and 2 ml of DMF, add 6 mg of MgO, and then dropwise add 1 ml (0.1 mnol) of isopropyl ether solution of farnesyl bromide (trans form). , stir for 16 hours. Distill the solvent and add 1ml of DMF.
Sephadex, which was added and dissolved and filled with DMF.
Apply to LH-20 column (1.5 x 88 cm) and develop. 43 to 48 ml portions are collected, the solvent is distilled off, water is added, and the mixture is freeze-dried. This was dissolved in 1 ml of the upper layer solution and applied to a Sephadex G-25 column filled with the lower layer solution of n-butanol-acetic acid-water (4:1:5), and developed with the upper layer solution. Collect 34-45 ml portions, distill off the solvent, add water and freeze-dry.

Yield 22 mg, [α] ²² _D -42.0° (c 0.10, 5N-acetic acid), Rf ² 0.19, Rf ³ 0.48.

Amino acid analysis: Arg1.02, Asp1.96, Ser0.87,
Glu1.04, Pro0.95, Gly5.0, Cys0.21, Val0.92 average recovery rate (74.0%).

Example 3 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Production of Asp―Pro―Ser―Gly―Val―Cys―NH ₂ : Boc―Glu (OBzl)―Gly―Gly―Gly―Asn―
Arg(Tos)―Gly―Asp(OBzl)―Pro―Ser―
176 mg of Gly-Val-Cys (MBzl)-NH ₂ is treated with 0.6 ml of anisole and 10 ml of HF at 0°C for 60 minutes, and the HF is distilled off. Dissolve the residue in 10 ml of water and add 5 ml of ethyl acetate.
After extracting with ml, Amberlight IRA-410
(acetic acid form) through a resin column (1 x 5 cm) and wash the column thoroughly with water. All flowthrough is collected and lyophilized. This was dissolved in 1 ml of N-acetic acid, and a Sephadex LH-20 column (3×
131 cm), and collect 295-347 ml sections and freeze-dry them.

Yield 72 mg, [α] ²² _D -78.0° (c 0.21, N-acetic acid).

Amino acid analysis value Arg1.01, Asp1.89, Ser0.89,
Glu0.95, Pro1.0, Gly4.76, Cys0.73, Val0.89.
Average recovery rate (75.0%), Rf ³ 0.28.

Example 4 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp―Pro―Ser―Gly―Val―Cys (S―
farnesyl)-NH ₂ production: H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp―Pro―Ser―Gly―Val―Cys―NH ₂ 34mg
Dissolve in 2 ml of 50% aqueous DMF and add 10 mg of MgO.
Next, 0.5 ml (0.3 mnol) of isopropyl ether of farnesyl bromide (trans form) was added dropwise and stirred for 18 hours. The solvent was distilled off, the residue was dissolved in 90% methanol and water, and applied to a Sephadex LH-20 column (1.5 x 88 cm) packed with the same solvent.The desired fraction was collected and rechromatographed on the same column to obtain the desired product. obtained as a lyophilized product.

Yield 18 mg, [α] ²³ _D -43.8° (c 0.13, 5N-acetic acid).

Amino acid analysis values: Arg1.0, Asp1.99, Ser0.97,
Glu0.78, Pro1.05, Gly4.93, Cys0.11, Val0.93.
Average recovery rate (70.5%). Rf ² 0.28, Rf ³ 0.50 Example 5 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp―Pro―Ser―Gly―Val―Cys (S―
geranylfarnesyl)-NH ₂ production: H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp―Pro―Ser―Gly―Val―Cys―NH ₂ 22mg
Dissolve in 3 ml of 50% aqueous DMF and add 5 mg of MgO.
Next, 0.5 ml (0.18 mnol) of an isopropyl ether solution of geranyl falnesyl bromide (trans isomer) was added dropwise, and the mixture was stirred for 18 hours. The solvent was distilled off, the residue was dissolved in 0.5 ml of DMF, and applied to a Sephadex LH-20 column (1.4 x 83 cm) packed with the same solvent. Obtain the product as a lyophilized product.

Yield 10 mg, [α] ²⁶ _D -59.3° (c 0.15, 5N-acetic acid), Rf ³ 0.53 Example 6 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp―Pro―Ser―Gly―Val―Cys (S―
Production of methyl)-OH: H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Dissolve 10 mg of Asp-Pro-Ser-Gly-Val-Cys-OH in 1 ml of water, add 0.1 ml of 0.1N hydrochloric acid, and adjust the pH to approximately 4. Add 0.2 ml of methyl iodide to this solution and stir at room temperature for 18 hours. Add 1M sodium carbonate water and adjust the pH to 9-10, then add 0.3ml of mercaptoethanol and stir for 12 hours. The reaction solvent was distilled off, the residue was dissolved in 0.5 ml of N-acetic acid,
The target fraction was collected by applying it to a Sephadex G-25 column (1.4 x 80 cm) packed with the same solvent and freeze-dried.

Yield 6 mg, Rf ³ 0.27.

Example 7 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp-Pro-Ser-Gly-Val-Cys (S-S-
Production of decyl)-OH: H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp－Pro－Ser－Gly－Val－Cys－OH11mg n
-Dissolve in a mixed solvent of n-butanol, methanol, and 0.1N ammonium acetate aqueous solution (PH6.9) with 0.03ml of decylmercaptan. After adding 3 mg of ethylene iodide to this solution and stirring at room temperature for 4 hours, the solvent was distilled off and the residue was dissolved in N-acetic acid. Sephadex filled with the same solvent
Apply to LH-20 column (1 x 87 cm) and collect the desired fraction and lyophilize.

Yield 6 mg, Rf ³ 0.47.

Example 8 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp-Pro-Ser-Gly-Val-Cys (S-decyl)
-OH production: Using BOC-Cys (S-decyl)-OH as the starting material,
Repeat the operations of Reference Examples 2 to 10 and Example 1,
LH-20 column packed with 3N-acetic acid (1 x 87
cm) and eluted with the same solvent, collect and freeze-dry.

Yield 20mg, Rf ³ 0.48.

Example 9 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Production of Asp-Pro-Ser-Gly-Val-Cys-OMe: BOC-Glu(OBzl)-Gly-Gly-Gly-Asn-
Arg(Tos)―Gly―Asp―Pro―Ser―Gly―Val
-Cys(MBzl)-OMe120mg with anisole 0.8ml
Treat with 10 ml of HF at 0°C for 60 minutes, and HF is distilled off.
After dissolving the residue in 10 ml of water and extracting with 5 ml of ethyl acetate, Amberlite IRA-410 (acetic acid type)
The solution was passed through a resin column (1 x 5 cm), and the flow-through was collected and freeze-dried. Dissolve this in 1 ml of 1N acetic acid,
The mixture was applied to a Sephadex LH-20 column (2 x 94 cm) packed with the same solvent, and aliquots of 92 to 105 ml were collected and freeze-dried.

Yield 33 mg, [α] ²² _D -52.5° (c 0.12, N-acetic acid), Rf ³ 0.33.

Example 10 H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Asp―Pro―Ser―Gly―Val―Cys (S―
production of OMe: H-Glu-Gly-Gly-Gly-Asn-Arg-Gly-
Dissolve 24mg of Asp-Pro-Ser-Gly-Val-Cys-OMe in 2ml of degassed water and 2ml of DMF, and 10mg of MgO.
Then, 1 ml (0.2 mnol) of an isopropyl ether solution of farnesyl bromide (trans isomer) was added dropwise, and the mixture was stirred for 16 hours. Distill the solvent and add 1ml of DMF.
Sephadex, which was added and dissolved and filled with DMF.
Attach to LH-20 (1.5 x 83cm) and unfold. 37.5～
After collecting 50 ml portions and distilling off the solvent, water is added and freeze-dried. This was dissolved in 1 ml of the upper layer solution and applied to a Sephadex G-25 column (1.8 x 47 cm) filled with the lower layer solution of n-butanol-acetic acid-water (4:1:5), and developed with the upper layer solution. Collect 34-49 ml portions, distill off the solvent, add water and freeze-dry.

Yield 10 mg, [α] ²² _D -49.8° (c 0.10, 5N-acetic acid), Rf ² 0.29, Rf ³ 0.58.

Claims (1)

[Claims] 1 [In the formula, R is hydrogen, alkyl having 1 to 10 carbon atoms with or without a sulfur atom, or the general formula H-[ _CH2C ( _-CH3 )= _CHCH2- ] _o (in the formula,
n is an integer of 1 to 5);