JPS6354396A - S-polyprenyl peptide - Google Patents

Abstract

NEW MATERIAL:A compound of formula (X is peptide chain of H-Tyr-Pro-Glu- Ile-Ser-Trp-Thr-Arg-Asn-Gly; Y is carboxyl group which may be amidated with an unsubstituted amino group or esterified with a lower alkyl group; n is 3, 4 or 5). USE:An enzyme breeding improving agent having an action of accelerating the formation of the conjugation tube of the enzyme. PREPARATION:For example, a carboxyl-protected amino acid is made to react with an amino-protected amino acid in a solvent in the presence of a condensation agent, the amino protecting group is eliminated from the reaction product and the above procedures are repeated in accordance with the sequence of the amino acids in the peptide chain to be used as a raw material to obtain a cysteine-containing peptide. The obtained peptide is condensed with pentaprenyl bromide (trans-isomer) in the presence of MgO to provide the compound of formula.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel S-
Concerning polyprenyl peptides.

Rhodosporidiu belonging to the basidiomycete yeast
mtoruloides and Tremella mes
It is well known that during the life history of S. enterica, a hormone-like substance is secreted during the transition from haploid yeast to diploid yeast, and that this substance induces elongation of the mating tube and cell fusion. This is a phenomenon.

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-11e-3er-Trp-
Thr -Arg-Asn-Gly-Cys-
OH or H-Glu-His-Asp-Pro-9et-
Ala-Pro-G 1y-Asn-G ly-
Veptides, such as Tyr-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 yeasts. If substances that act on these yeasts can be obtained, they can not only be used effectively for yeast breeding and N improvement, but also serve as important reagents for bimolecular biochemical research. It is.

The present inventors assumed that designated residues were bound to these active substances, and as a result of introducing various finger-soluble residues into 9HW of Sunde and Inn, we found that S- The present invention was completed by discovering that polyprenyl peptides have a stronger effect on promoting junctional tube formation than natural products.

That is, the present invention provides a general formula in which X is H-Tyr-Pro-Glu -11e-
It represents a peptide chain represented by Ser-Trp-Thr-Arg-Asn-Gly-, and Y represents a carboxyl group which may be amidated with an unsubstituted amino group or esterified with a lower alkyl group. n is an integer of 3.1 or 5].

Regarding general formula (1), Y is optionally amidated (
It represents a carboxyl group which may be esterified with an unsubstituted amide) or a lower alkyl group such as methyl, edyl or propyl. In this specification, amino acids, their residues, bevsendoer protecting groups, used reagents, etc. are referred to as [UPAC-
The abbreviations (adopted by the NIB Nomenclature and Attitude Committee) or the abbreviations commonly used in the relevant IF may be used, such as the following abbreviations.

Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Cys: Cystine Glu: Glutamic acid Gly: Glycine His: Histidine 11e: Isoleucine Pro: Proline Ser: Serine Thr: Threonine Trp:) Liptophan Tyr: Tyrosine Boa: t-butoxy Carbonyl Aoc: amyloxycarbonyl But
: t-Butyl Z : Benzyloxycarbonyl Me ・Methyl Bzl : Benzyl Bzl (C1) : p-Chlorobenzyl MBzl
+p~methoxybennol Tos: Tonle DCC・N,N'-dicyclohexylcarbodiimide DCHA: Nocyclohexylamine 110N
T3: N-hydroxy-5-norbornene-2
,3-dicarboximide ONB: N~hydroxy-5-norbornene-
2,3-Dicarboximide ester TFA: trifluoroacetic acid DMF: trimethylformamide 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. , except for glycine, which has the color of I7.

Cysteine-containing peptides, which are raw materials for producing S-polyprenyl peptides 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. These known methods are described in “The Peptides,” Part 1.
Volume (1966), 5 Chroder and Lubke, Academic Press, New
York, U.

SA1, “Peptide synthesis” Susumu Azumaya, Maruzen Co., Ltd. (
1975), or edited by the Japanese Biochemical Society, Biochemistry Experimental Course■, ``Protein Chemistry■, Chemical Modification and Peptide Synthesis''
It is explained in detail in literature such as Tokyo Kagaku Dojin (1977).

The following is a brief description of the method for producing raw peptides.
First, a partial amino acid that may constitute the target polypeptide or the peptide and a compound that may constitute the remainder thereof are condensed using known peptide synthesis methods. Examples of the condensation method include the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, method using Woodward reagent K, carboimidazole method,
Redox method.

Examples include DCC-additive method 0 (ON B, 1-hydroxybenzotriazole, N-hydroxyscunnimide, etc. are used as an additive). Before carrying out the main condensation reaction, it is preferable to protect the carboxyl groups and amino groups that are not involved in the reaction of the raw materials by means known per se, or to protect the hydroxyl groups and thiol groups. Carboxyl groups can be substituted with tertiary alkylamine salts (e.g. triethylamine, N
-methylmorpholine, etc.) or metal salts (e.g., sodium, potassium, lithium salts, etc.), or esters (e.g., methyl, ethyl, benzyl, p-chlorobenzyl, t-butyl, t-amyl, etc.). may be protected as an ester). Protective groups for amino groups include benzyloxycarbonyl, t-butoxycarbonyl, isobornyloxycarbonyl groups, etc. Protective groups for imidazole group of histidine include benzyl, tonor, etc.
Examples include 2,4-dinitroph5nyl, t-butyloxycarbonyl, and carbobenzoxyl groups. A protecting group for the guanidino group of arginine and 1. So, for example, nitro, tosyl.

carbobenzoxyl, isobornyloxycarbonyl,
Examples include an adamantyloxycarbonyl group. Also, benzyl and 1-butyl are used to protect the hydroxyl groups of tyrosine and serine. Examples of thiol protecting means include ethers having a protecting group such as 1-amyl, and thioethers having benzyl, p-methquinbenzyl, p-methylbenzyl, t-butyl, etc. as a protecting group.

The peptide condensation reaction can be appropriately carried out in a commonly used solvent, such as anhydrous or water-containing dimethylformamide, dimethylsulfoquine, pyridine, chloroform, and dioxane. dichloromethane,
Tetrahydrofuran, ethyl acetate or a suitable mixture thereof is used. The reaction generally takes place between 0°C and +60°C.
It is carried out at temperatures in the range of about °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 the reaction as a thiol peptide by removing the protecting group. 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 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 distribution, extraction, reprecipitation membrane, column chromatography, etc.

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

Water-containing Tsumedelporumamide solution generally has a water-containing M10 to 7
0% by volume, preferably 30 to 60% by volume, and the amount of magnesium oxide is 1 to 10 equivalents in molar ratio to the peptide.
Preferably, 2 to 11 injections are used. Furthermore, the molar ratio of halogenated polyprenyl to the peptide is 1 to 10 tons,
Generally, 2 to 4 equivalents are preferred. The reaction generally takes place at -lO℃
~60℃.

Preferably, the reaction is carried out at 0°C to 30°C, and the reaction time is usually about 3 hours to 12 hours.

The S-polyprenyl peptides thus produced can be isolated from the reaction solution by separation and purification means known per se (eg, partition, extraction, reprecipitation membrane, column chromatography).

The S-polyprenyl peptide (1) produced by the method of the present invention is a Rhodosporidium peptide.
It exhibits a zygotic tube formation-promoting effect on P. uloides at a low concentration of 0.1 to IOng/geese, has a strong yeast sex hormone effect, and is safe and easy to handle for the human body. Therefore, these S~voribrenyl peptides exhibit stronger effects than natural hormones with the same effect, and based on this, fj is used for the improvement of two yeast breeding species.
It is also used for biochemistry.

Reagents for molecular biochemistry research (7) can also be used.

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 can be labeled with radioactive iodine or a fluorescent reagent. 17 can be used.

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

rir, -chloroporum:methanol:acetic acid (9:I:
0.5) [,-acetic acid nibble:pyridine/acetic acid:water (60:20
:6・10) Rf3-ethyl acetate: n-butanol, acetic acid: water (1:
l:l:I) Tlr4=n-butanol:pyridine:acetic acid:water (30
:20・6:24) Unless otherwise specified, Rr is based on Merck silica gel plate 60 F ts-.

Reference example 1 1) Synthesis of raw materials 20.5 g of OH and 9 triethylamine were DM F2
00 rut and cooled in water. To this, benzlua bromide, 2
Add dropwise a solution of DMF35 dissolved in DMF, and stir while gradually returning the temperature 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. The residue was dissolved in 400% ethyl acetate, washed with 4% aqueous sodium carbonate and water, and dried over 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°C, [α] 21-45
．． 8° (c O, 53, methanol) elemental analysis Ct
aHtso sNS: Calculated value C64,01; H6,7
7, N 3.25. S 7.43. Analysis value C64,
10: H 6,70; N 3.20; S 7
．． 61°Bz 1 b) Production of Boc-Gly-Cys-OBzl: B
Stir for 5 minutes at oc-warm temperature. 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, the precipitate is dissolved in 50% ethyl acetate, and the precipitate is neutralized with triethylamine 2.3-. B in this solution
Add 6 g of oc-Gly-ONB and stir for 12 hours. The reaction solution was washed with 0.2N hydrochloric acid, 1% 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, 0.7°! ! IBZ 1 c) Boc -Asn-G Iy -Cys-OB
Production of zl: 6.7 g of Bz 1 Boc-Gly-Cys-OBZI was added to TF
Dissolve in A32.5- and stir at room temperature for 10 minutes.

2.5 d of 6.4N hydrochloric acid/dioxane solution is added, 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% DMF. After cooling with water, 2.75 g of triethylamine was added, and the precipitated triethylamine hydrochloride was filtered off. The filtrate was converted to Boc-Asn-0)-+
After adding 3.18 g and 13.7 g of HONl and cooling to -5°C, 3.11 g of DCC was added and stirred. After stirring for about 16 hours, insoluble precipitates were filtered off, and the filtrate was dried under reduced pressure.

The residue was dissolved in 300ml of ethyl acetate, and dissolved in 0.2N hydrochloric acid.
Wash with 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 5.7g, melting point 121-123°C, [αj23-3
9.8° (cO, 54, methanol).

Elemental analysis CzeIl, eOeN, S: Calculated value C57,
79; H6, 36; N 9JO; S 5.32.
Calculated value C58, 11°Ll 6.49; N 9.1
2; Production of S5.15Bz1: Boc-Asn-Gly-Cys-O
Dissolve 5.42 g of Bzl in 30 cm of TFA, stir at room temperature for 20 minutes, distill off TFA under reduced pressure, and add 6.
After adding 1.5 d of 4N hydrochloric acid/dioxane and stirring, ether was added and the resulting precipitate was filtered. DM this
0.94 d of triethylamine dissolved in F50- is added under cooling to neutralize the solution, and the precipitated triethylamine hydrochloride is filtered off. Add Aoc to the filtrate and cool to -5℃, DCC1.6
Add 6g. The mixture was stirred at room temperature for 16 hours, the precipitate was filtered off, and the solvent was distilled off. The residue was extracted with 300 ml of ethyl acetate, 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 filtered as a powder with ether. This is reprecipitated with acetonitrile and ether.

Yield 5.52g, melting point 70°C (decomposed), Cαgo23-3
0.36 (cO,535, methanol).

Yef, 0.41 OBzl 5. Dissolve Ig in 35 mffi of TFA, stir at room temperature for 15 minutes, and then dry under reduced pressure.1. ,6,4
1.6 cycles of N-hydrochloric acid/dioxane are added, and ether is further added to form a precipitate which is collected by filtration. DMF40+J
Neutralize by adding 0.78% triethylamine dissolved in
The precipitated triethylamine hydrochloride is filtered off. B to the filtrate
oc-Thr-OHI, 27g and HON B1.25
Add the dioxane solution of Boc-Thr-ON B synthesized in step g and stir for 16 hours. After the reaction was completed, the solvent was distilled off, and the residue was dissolved in 500 Jl of ethyl acetate.
Wash with 2N hydrochloric acid, 4% aqueous 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 80-83°C (decomposed).

, 23[α''D-33,0'(cO,52, methanol).

Rr, 0.20 os f) Boc-Trp-Thr-Arg-Asn-G
ly -Bz1 -(, Iy-Cys-OBzl 4.07g was added to TFA
25. After the solution was dissolved in d and left at room temperature for 15 minutes, TFA was distilled off under reduced pressure, ether was added to the residue, and the mixture was filtered.

Dissolve this in 3 volumes of acetonitrile, neutralize by adding 0.8-triethylamine, and add ether to precipitate. After removing the supernatant ether, the precipitate was dissolved in DMF and cooled with water. To this, 1.28 g of Boc-Trp-OH and HON
l3oc-'rrp~0 synthesized from 907mg of B
Add a dioxane solution of Nr3 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, washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water, and dried over 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: 13.55g, melting point: 120°C (decomposition), [α]2
4-25.8° (cO, 585, methanol).

Rf, 0.18 os g) Boc-Ser-Trp-Thr-Arg-A
Preparation of sn =Bzl-Gly-Cys-OBzl: Roe-T
rp-Thr20%-1,2-ethanedithiol was dissolved in dioxane 6.5% containing 6.4N hydrochloric acid/dioxane 13% and left 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 D~IF 10d 12, add 0.4d triethylamine and neutralize 1. The precipitated triethylamine hydrochloride is filtered off. Boc-9er-OH to the filtrate
Add 621a+g and I (ONB 990B, add DCC749ig and stir while cooling with water. After stirring at room temperature for 16 hours, insoluble matter is filtered off. The solvent is distilled off under reduced pressure, and ether is added to the residue. This was mixed with ethyl acetate/bilinone/acetic acid:water (120:10:
A silica gel column (5,5x
l 2.5 cm) and elutes with the same solvent, 1
The eluted portion of 490-2785- is collected and concentrated, ether is added and the resulting powder is filtered.

Yield 1.9 g, melting point 150-153°C (decomposition).

[α]25-28.2° (c O, 34, methanol) Elemental analysis C8° HtsOIIIN lts t: Calculated value C55,97, H6,11, N 13.06. S
4.9g, analysis value C55,57,H6,27: N
12.67: S 4.77゜h) Boc
--Tyr -Pro --Production notice of OH: H-P
4 g of ro-OH was dissolved in 80 layers of water-containing dioxane, 4.75 g of triethylamine was added, and the mixture was cooled with water. Add 11.1 g of '3oe-Tyr-ONB to this solution and stir vigorously. The solvent was distilled off, dissolved in 80 turns of 4% sulfur hydrogen carbonate/lium water, and washed with ether 50 times.
After cooling with water, adjust the pH to 2 and 1 with 0.2N hydrochloric acid. Treat the precipitated oil with ethyl acetate l 00! Extract to. The ethyl acetate layer is washed with water, dried over anhydrous sodium sulfate, and the solvent is distilled off.

Precipitate: Add ether to the crystals and collect by filtration.

Yield 4.3 g, melting point 123-126°C (decomposed).

22.5 [α] -23,6° (cO, 525, methanol).

Elemental analysis C+eHtoo aN t: Calculated value C60
, 30:1 (6,93; N 7.40. Analysis value C6
0,15,H6,80;N 7.30°Buti) Production of Z-Glu-11e-OBzl: H-1
1e-OBzl p-toluenesulfonate 14.2
g in ethyl acetate 409d, 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 of Z-But Glu-0)-(-DCHA was dissolved in 3001 n1 of ethyl acetate!! DCHA was extracted and removed with 0.2N sulfuric acid after turbidity. After further washing with water and drying with anhydrous sodium sulfate, the solvent was removed. Distill under reduced pressure. Dissolve the rain sound in 140 d of a mixed solvent of tetrahydrofuran and ethyl acetate (121), add 8.1 g of HONB, cool on ice, and dissolve in DCC7.
Add 44g and stir. The mixture was stirred at room temperature for 12 hours, the precipitate was filtered off, the solvent was distilled off, and the residue was dissolved in ethyl acetate.
Dissolves in 00 ruts.

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°C, [α] 23-24.5
° (c O, 60, methanol), [.

0.92 7ut j) l3oc-Tyr-Pro-Glu-11e =
OH But Manufacture: Z-Glu-1ie-OBzl 5.4g
is dissolved in methanol together with N-hydrochloric acid lO-, 1 g of palladium black is added, and hydrogen is passed through the solution to reduce the solution. Palladium black was filtered off, methanol was distilled off under reduced pressure, and the residue was dissolved in DMF1.
oog and add triethylamine 2.8- while cooling with water. Separately Boc-Tyr-Pr.

-0H3,788 and 2.16 g of HONB are dissolved in 100 d of a mixed solvent of ethyl acetate and dioxane (1:1), 2.27 g of DCC is added, and the mixture is stirred at room temperature for 6 hours. The precipitate was filtered off, this filtrate was added to the above DMF solution, and 16
Stir the time. After the reaction is completed, the solvent is distilled off under reduced pressure, extracted into 200ml of water containing 2.5g of sodium hydrogen carbonate, and washed with ether. The aqueous layer was cooled with water, acidified with 35 ml of N-hydrochloric acid, and the precipitated oil was extracted twice with 200 Ml of ethyl acetate. The ethyl acetate layers were 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, and water (107:4:2), eluted with the same solvent system, and the eluted portion of 68 (1-980d) was collected. The solvent was distilled off, and the residue was filtered as a powder with ether.

Yield 3.45 g, melting point 115-119°C (decomposed).

[α]”-40,2° (cO,465, methanol).

Elemental analysis C34Hszo ION 4+calculated value C60
,34;1-(7,74,N 8.28.Analysis value C6
0,22,H8,02°N 7.82. 0? Ut k) Boc-Tyr-Pro-Glu-11e-8
Production of et -os: Boc-9er-Trp-Thr-Arg-A
snBz 1 -Gly-Cys-OBzl 644mg in DMFo
, 5- and dioxane 2d, add 1,2-ethanediol 0.3- and 6.4N hydrochloric acid/dioxane 6, 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. DMF this
5d and neutralize by adding 0.3i of triethylamine. The precipitated triethylamine hydrochloride was filtered off, and 406 mg of Boc-Pro-But Glu-11e-OH and HONB 36 were added to the filtrate.
Add 0 mg and cool to -10 to -5°C. DCC206
mg 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: 650 mg, melting point: 100-105°C (decomposed).

[α Koku 5-26.6° (co, 365.methanol)
.

Elemental analysis CasH+toOtyN +sS t”
2 H20+calculated value C56,79,H6,64,N
11.91. S 3.41゜Analysis value C56,23,
H6,67, N 11.67, S 3.90° Amino acid division value close to 1 minute (theoretical value): Arg 0.99 (1).

Asp 1.03 (1), Thr O, 9g (1),
Set 0.92(1).

Glu O, 87(1), Pro 1.09(1),
Gly 1,0 (1).

Cys O, 37 (1), I le O, 85 (1)
), Tyr O, 6 g (1), average recovery rate 72% ii) H-Tyr-Pro-Glu -r le-
Ser-Trp-Thr-Arg-Asn-Gly-C
Production of ys-OH: But Boc-Tyr-Pro-Glu-I 1e-Ser-
554 mg of Trp was dissolved in 20 - of hydrogen fluoride in the presence of 1,6 - of anisole and stirred at 0°C for 60 minutes. Hydrogen fluoride was distilled off under reduced pressure, and the residue was extracted with 10 d of water and 5M ether.
1. 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 permeate is combined and lyophilized to give 405 mg. This was applied to a column of Sephadex LH-20 (2.4 x 107 cm) and eluted with 0° IN-acetic acid.
The eluate was collected and lyophilized to obtain 106 mg of SH peptide.

[α]26-63.5°(cO,60,IN-acetic acid), IRr4 (cellulose) 0,64
Amino acid analysis 1 analysis value (theoretical value): Arg 1.14
(1).

Trp O,59(1), Asp 1.00(1),
Thr 1.00 (1).

Set 0.95(1), Glu O,95(1),
Pro 1.02(1).

G ly 1.00 (1), Cys O, 86 (1)
, I le O, 95 (+).

Tyr O, 98 (1), average recovery rate 79.1% Reference example 2 H-Glu-His-Asp-Pro-9et-Ala
-Pro-Gly-Asn-Gly-Tyr -Cy
Production of S-Nl (t and S H-peptide whose C-terminus is a methyl ester: i) Synthesis of raw materials Bz1 a) Production of Boc-Asp-Pro-OH/DCHA: 6.9 g of H-Pro-OH Dissolve triethylamine in 20% water-containing DMF7 (Jml), B
z I Under cooling, [3oc-Asp -OH13g and HON B
Boc, Asp ONB synthesized from Bz l J 864g
Add dioxane solution and stir for 12 hours. The reaction solution was distilled off under reduced pressure, and the residue was dissolved in saturated aqueous sodium bicarbonate, washed with ether, cooled to 0°C, and dissolved in N
-Add hydrochloric acid to r102~3 and add ethyl acetate to 200ml.
Extract times. The ethyl acetate layer is washed with water and dried over anhydrous sodium sulfate. The solvent was distilled off and the residue was dissolved in ether 300 ml while stirring.
Add rl. The precipitated crystals are collected by filtration and washed with ether.

Yield 21g, melting point 1!8-+2 pC, [α]21-30
．． 3' (c O, 595, methanol) elemental analysis C
5ffH, +07 N3: Calculated value C65,8
6; H8,54; N 6.96 Analysis value C65,
89; H8゜59; N 6.84゜Bz 1 Boc-Asp-Pro-01-I ・DC) iA
4 g was suspended in 150 g of ethyl acetate, washed with 0.2 N sulfuric acid and water, and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure (13), and the remaining oil was dissolved in rFA (15). After 15 minutes, the solvent was distilled off, ether was added, and the gel was collected by filtration. Dissolve this in 50 addition ρ of DMF', cool it, and add triethyl, ? 'ζn 14862.86g
) synthesized from 1.51 g of ioNLl t-62 Boc
Add a dioxane solution of os-H45-ONR and incubate at room temperature for 16
Stir the time. After the reaction is complete, the solvent is distilled off, and the residue is dissolved in saturated aqueous sodium bicarbonate, washed with ether, cooled with water, and converted to pi-I2-3 with N-hydrochloric acid. The precipitated oil was extracted twice with ethyl acetate+00d. 1-1 The ethyl acetate layers were combined, washed with water, and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure, and the residue was filtered as a powder with ether.

Since it is a mixture of , it is used as it is in the next reaction without further purification.

OH OH Hydroxyhentriazole 684 Ing was dissolved in tetrahydrofuran and stirred for 30 minutes, then the tetrahydrofuran was distilled off and the residue was dissolved in T F
Dissolve in A 7 hri. After 20 minutes, T r'' A is distilled off, ether is added and the powder is collected by filtration.
~ Triethylamine 106 dissolved in IF20d and cooled with water
d and further F3oc-4;lu-OH 896
A dioxane solution of Boc-Glu-ONB synthesized from 573 mg of IONB and 573 mg of IONB is added and stirred for 16 hours. The solvent is distilled off, the extract is extracted with saturated sodium bicarbonate water, washed with needles and ethyl acetate, and N-hydrochloric acid is added while cooling with water to give a pI of 12 to 3. Extract twice with ethyl acetate food and combine the extracts.
Wash with water and dry with anhydrous sodium sulfate. The solvent was distilled off, the residue was filtered as a powder with ether, and reprecipitated from ethyl acetate and ether.

Yield: 1.42g. Melting point 118-121°C (decomposed).

[α] 19-36.8° (c O.565.methanol).

[, 0.42. Rf. 0.70.

d) Production of Eoc-Ala-Pro-OH: Boc
-Ala-OH9,26g and HON B9,0
Dissolve g in 50° of acetonitrile and dilute with DCCl1 under water cooling.
Add g and stir for 4 hours. The resulting urea body is filtered off, and the filtrate is dried under reduced pressure. Dissolve the residue in I) MP20-.

On the other hand, 6.9 g of H-Pro-OH was dissolved in 20 ml of DMF and 10 d of water, 7 ml of triethylamine was added, and the mixture was cooled with water. This solution contains the active ester 'Fi'.
Add DMF solution and stir for 6 hours. Under reduced pressure D
MF was distilled off and 200% ethyl acetate was added. After adding and dissolving nIl and washing with a saturated aqueous citric acid solution and water, the ethyl acetate layer was dried over sodium sulfate, and the ethyl acetate was distilled off under reduced pressure. The residue was crystallized from ethyl acetate and petroleum ether and filtered to give 12 g of needle-like crystals. Melting point 150.5-15
1.5℃ elemental analysis CI3Htto 5N = Calculated as follows: C54,53; H7,75; N 9.7
8 analysis value: C54,75; H7,93; N9
．． 63e) Boc-Ala-Pro-Gly-OB
Production of zl (CI): Dissolve 5.73 g of Boa-Ala-Pro-OH in 40 bottles of acetonitrile, cool to 0°C, and dissolve 4 g of HON B.
Add 4.6 g of DCC and stir for 6 hours. .

The resulting urea body was filtered and the filtrate was added H-I-Gly-OBzl.
(C1) Add 7 g of TFA and 3 g of triethylamine and stir for 6 hours. Distill the solvent under reduced pressure 17, ethyl acetate 2
After washing with 4% hydrogen carbonate→-thorium and saturated aqueous citric acid solution, drying over sulfuric acid and sulfuric acid, the solvent was distilled off under reduced pressure. The residue is crystallized from ethyl acetate and petroleum ether to give 9.1 g of needles i-). Melting point 108-, -110°C, ref, = 0.77° Elemental analysis Calculated as Ct2H3oOeN3CI: C5B, 46; H6, 46; N8
．． 98; CI 7.58 Analysis value 2 G 58.57: H6,63: N 8
．． 95; CI 746 D Boc-Set-Ala-Pro-Gly-OB
Production of ZI (CI): Boc-Ala-Pro-G
7.02 g of ly-OBzl (CI) was dissolved in TFA 30 years old, 6N-hydrochloric acid-dioxane dioxane was added thereto, and the mixture was stirred for 20 minutes at lO<0>C. Ether 1OO1n1.
The precipitate formed is filtered, washed with ether and dried.

The dry powder is dissolved in 20m DMF, cooled and triethylamine 2.4- is added.

Meanwhile, Boc-ser-ot (3.08 g was dissolved in 30% acetonitrile, and under water cooling, 3 g of HON8 and DCC3 were dissolved.
, 3 g was added, stirred for 4 hours, and the resulting urea body was filtered off. Add the filtrate to the above DMF solution and stir at room temperature for 10 hours. The solvent was distilled off under reduced pressure, and the residue was diluted with ethyl acetate.
oy and washed with 4% sodium bicarbonate water and saturated citric acid water.

The product was dried under reduced pressure and dissolved in a mixture of 8 parts of Rf and ethyl acetate. Add this to a silica gel column (
6X 15cm, solvent system shown in Rft and 1 of ethyl acetate
: Filled with 12 mixed liquid), expand to 468 919
A 54-inch eluate section was collected, washed with water, and dried under reduced pressure. The residue is washed with a mixture of ether and petroleum ether II and filtered. 6,8 g.

Melting point: Does not have a clear melting point and decomposes. [,=0.56 Elemental analysis Ct s H* s Oe N Calculated value as 4CI: C54,10,H6,3B, N 9.
51°CI 6.40 Analysis value: C54,29, H6,52, N 9.81
゜CI 6.16 g) T3oc-Set-Ala-Pro-Gly-
Production of OH: Boc-8er-Ala-Pro-Gly-Or3zl
(C1) 1.4g was dissolved in 50% t-butanol and hydrogenated for 4 hours using 500mg of palladium black as a catalyst.
(The catalyst is filtered off and dried under reduced pressure.

Yield: Quantitative, Rf, = 0.08.11f, = 0.28
゜Rz 1 h) Boc-Tyr-Cys-Nl- (Production of 2:
Boc-officially distilled off under reduced pressure, ether and petroleum ether were added to the residue, and the crystals were collected by filtration. This crystal is DNIF5
Add 16.7 g of triethylamine, 1.7 g of Tyr-ONB, and stir at room temperature for 16 hours. The solvent was distilled off under reduced pressure, and the residue was dissolved in 250+n1 ethyl acetate, washed with 02N hydrochloric acid, 4% aqueous sodium bicarbonate and water, and dried over anhydrous sodium sulfate. The solvent was distilled off, and the residue was filtered as gel crystals from ethyl acetate and ether. Further, suspend in ethyl acetate and heat to boiling point.1. Cool and then remove.

Yield 8.2g, melting point 141147°C, [α]21-24
．． 3' (cO,61, methanol), Rf.

0.54 Elemental analysis CzsH3sOsNsS: Calculated value: C59,
62: H6,61; N 8.34; S 6.37
．． Analysis value: C59,47: ) (6,67; N 7.
98: S 6.13°! IIBZ 1 i) Production of Boc-Gly-Tyr-Cys-NHz: 7.55 g of NHz I Boc-Tyr-Cys-NHz was dissolved in TFA45d containing anisole 5d, left at room temperature for 15 minutes, and then distilled off under reduced pressure. The residue is filtered as a powder with ether. This was dissolved in 50 DMF, neutralized by adding 2.4 d of triethylamine, and then Roc-Gly
- Add 5.2 g of ONB and stir for 12 hours. The solvent was distilled off and the residue was dissolved in ethyl acetate 200 nJ l, -.
, extracted, washed with 0.2N hydrochloric acid, 4% aqueous sodium bicarbonate, and water, and dried over anhydrous sodium sulfate.

Ethyl acetate is distilled off under reduced pressure, and the residue is filtered as crystals from ethyl acetate and ether, and further recrystallized from the same solvent system.

Yield 6.4g, melting point 119-120°C, [α]19-3
2.4° (c O, 515, methanol), Rr.

0.56゜Elemental analysis Cz7I-13607N,"3 Calculated value:
C57,84, H6,47, N 9.99. S5
．． 72. Analysis value: C57,99: H6,62; N
10.08: S 6.00゜NHz1 j) Boc-Asn-Gly-Tyr-Cys-N
Production of i12 Bz 1 Production: Boc-Giy-Tyr-Cys-NHz 6
g was dissolved in TFA30 containing 5-anisole, and 10
After distillation under partial pressure, 2 volumes of 6.4N hydrochloric acid/dioxane were added to the residue, stirred thoroughly, and then ether was added and the mixture was filtered as a powder. This is dissolved in 30 mN of DMF, neutralized by adding 1.5 ml of triethylamine, and the precipitated triethylamine hydrochloride is filtered off. Boc-Asn in the filtrate
Add 61 g of -OH2 and 2.9 g of HON8 and cool to -5°C. While cooling, add 2.43 g of DCC and stir for 16 hours. Insoluble materials were filtered off, the solvent was distilled off, and the residue was dissolved in 200 ml of ethyl acetate. Add this to 0.2N-hydrochloric acid,
Wash with 4% aqueous sodium bicarbonate and water, and dry over anhydrous sodium sulfate. The solvent is distilled off and the precipitated gel is collected by filtration, suspended in ethyl acetate, heated, cooled and filtered, then treated in the same manner with acetonitrile and filtered.

Yield 3.6 g, melting point 139-141°C (decomposed).

[α12′ −49,3°(c O,45, methanol).

[, 0,33° Elemental analysis Cs 1H-t O, N s S: Calculated value: C55,18: H6,27; N 12.46;
s4.75. Analysis value.

C54,89; H6,48; N 12.33
; S 4.63゜k) Boc-8er-Al
Production of a-Pro-Gly-Asn-NHzIaly-Cys-Nttt: Boc-
69 g of Asn-G lyBz 1 TYr Cys NHz 1 was dissolved in 5 d of TFA 7 containing 0.8 ml of anisole and allowed to stand for 10 minutes. Remove TFA by distillation under reduced pressure,
The residue is filtered as a powder with ether. I)
Dissolve in M P2 solution, neutralize by adding 0.7 d of triethylamine, add ether to this solution, wash the resulting precipitate thoroughly with ether, and then add DMFIO! dissolve in Add Boc -S cr-A 1a-Pro -Gl to this solution.
After adding and dissolving 1.08 g of y-OH and 560 mg of HONB, the mixture is cooled with water. Add 620 mg of DCC under water cooling and stir at room temperature for 16 hours. Insoluble matter is filtered off, the solvent is distilled off, ethyl acetate is added to the residue, the powder is collected by filtration, and further washed with acetonitrile to obtain 2.35 g of powder. This was mixed with ethyl acetate, pyridine/acetic acid:water (4:2/
Silica gel column (5x
350-67 and eluted with the same solvent system.
The eluted portion of 2n4 is collected, the solvent is distilled off under reduced pressure, and the residue is filtered as a powder with ether.

Yield 1.15 g, melting point 87-90°C (decomposition).

[α] 21-60.2° (e O, 54, methanol).

Rft 0.42 Bz 1 Ala Pro cly Asn Gly
Production of T'lr Cys-N I (, B oc -
S er -A la -Pro -G ly -B
z 1 嘱Asn-Gly -Tyr-Cys-Nl2 4
9 Dissolve 4mg of TFA2di: 6.4N-hydrochloric acid/
Add dioxane, 1Tn1, and wait for 15 minutes.The resulting precipitate is collected by filtration. Dissolve this in DMF3-, neutralize with 0.16 d of triethylamine, and filter off the precipitated triethylamine hydrochloride, filtrate OBz l
Boc-G111-Iris-Asp-Pro-OH in OBz 1
488,111 g and 180 mg of IONB were added, dissolved, and cooled. Add 206mg of DCC to 16
Stir the time. Insoluble matter was filtered off and the solvent was distilled off under reduced pressure)7
Then, add ethyl acetate to the residue and make a powder. Take a telegram (
830 mg) D This was developed on a silica gel column (3 x 29 cm) chromatograph using a mixed solvent of ethyl acetate: pyridine: acetic acid: water (60:20:6:10).
The eluted portion of 0 to 543 d is collected, the solvent is distilled off, ether is added, and the powder is collected by filtration.

Yield 400 mg, melting point 160-164°C (decomposed).

[α]” −56,8° (c O,50, methanol).

[t O, 23 Amino acid analysis 1 analysis value (theoretical value): ll is 1.0
6(1).

Asp 2.00 (2), Set 0.93 (1),
Glu 1.03(1).

Pro 1.9 to 2), Gly 1.98(2),
Ala 1.10(1).

Cys O, 41(1), Tyr O, 45(1),
Average recovery rate 84.4% Bz 1 A la-Pro-G ly-Asn-G ly-Ty
Production of r-Cys-QMe: Cool 50 ml of water to -10°C and stir. To this, 13thl of thionyl chloride was added dropwise, stirred for 10 minutes, then 2g of H-Cys-OHl was added, and the mixture was stirred for 10 minutes at room temperature.
Stir for 6 hours. The precipitated crystals were collected by filtration and thoroughly washed with ether to obtain the desired product. Yield: 12 g, melting point: 151-153°C, [α]

-25,1'(c O,912, methanol), rt
fto, 69 Elemental analysis C1tH+, 0sNs' HCI・Total X [
Value C49,39: H6,22; N 4.80;
S 10.99; CI 12.15 Analysis value
C49,55: H6,20; N 4,7? , S
11.02. CI 12.23゜Bz I Ala-Pro-G ly-Asn-G ly-Ty
Production of r-Cys-Bz IOMe: Repeat the same operation as h-1) using H-Cys-OMe HCI as a starting material (7, to obtain the target compound.

Yield 238 mg, melting point +20-124°C (decomposition).

[α]2' -47,9° (cO,29, methanol).

[,0,31゜ii) H-Glu-His-ASpPro 5e
r-AlaPro Giy Asn Gly
T'lr CYS NI(tBz 1 薯-Ala -Pro -G ly -Asn -G 1
Dissolve 345 mg of y-Tyr -Cys -NHy in hydrogen fluoride 13-methyl together with anisole Id and stir at 0°C for 60 minutes. Distill the fluoride under reduced pressure, add 15 ml of water to the residue, dissolve it, and dissolve in ether. Wash twice with 5 turns. The aqueous layer was passed through a column (1 x 5 cm) of Amberlite IRA-410 (acetic acid type), the resin was thoroughly washed with water, and the passed liquid was combined. and lyophilize (250 mg). Add this to O, IN-acetic acid or j2 Sephadex L H-2
Developed on an O column (2,4X I I Ocm) and
Elute with IN-acetic acid. The 196th to 221st batches were collected and freeze-dried.

Revenue! it126mg, [α]2'-90.6°(c
O,2゜O,IN-acetic acid), RL (cell o-se) 0.
48, Amino acid minute hand 1 minute near value (theoretical value): His O,
9Kl).

Asp 1.95 (2), Set 0.96 (1),
Glu O, 99(1).

Pro 2.51 (2), Gly 2.0 (2),
Ala 1.09(1).

Cys O, 29(1), Tyr O, 94(1),
Average recovery rate 61.0%2 iii) H-Gtu-His-Asp-Pro-9
er-Ala-Pro-G ly-Asn-G
t of Iy −Tyr −Cys −OMe,! Structure: Pro-5et -Ala -Pro-Gly-Asn
The desired S H-peptide is obtained from -Gly-Bz 1 Tyr -CYs -OMe.

[α] 21-82.9° (e O, 205, O, lN
Monoacetic acid), Rfa (cellulose) 0.44 Amino acid analysis 5 analysis value (theoretical value): His O, 85 (1), As
p 2.02(2), Ser O, 97(1), G
lu 1,11(1), Pro 2.17(2), G
ly 2.0 (2), Ala 0.95 (1).

Cys O, 95(1), Tyr O, 99(1),
Average recovery rate (91.7%) Reference example 3 H-Glu-=His-Asp -Pro -5er-
Ala-Pro-Gly-Asn-Gly-Tyr-
Production of Cys(S-tetraprenyl)-N1-1: H-Glu-His-Asp-Pro-8er
-Ala-Pro-Gly-Asn -Gl)' T
~1 g of Yr Cys-NHt 125m.

201 and 50% water-containing D M P 4 J, an isopropyl ether solution I J (0.15 mmol) of tetraprenyl bromide (trans isomer) was added dropwise thereto, and the mixture was stirred for 16 hours. The solvent was concentrated under reduced pressure, dissolved in 0.5 d of 60% methanol aqueous solution, and developed on a Sephadex L H=20 column (1.4 x 83.5 cm).

The eluted portion of 4O-60d was collected and lyophilized to obtain 66111
get g. Dissolve this in water and use Amberlight XAD-2.
column (IX 3 cm) and elute with a gradient of water and ethanol, and the fraction with an ethanol concentration of 50 to 85% is collected and freeze-dried to obtain 30 mg of the target product.

Rr, (cell o-s) 0.73 Reference example 4! 1-Glu-His-Asp-Pro-5et-A
ha-Pro-Gly-Asn-Gly-Tyr-C
'A structure of ys(S-pentaprenyl)-NH,: I
(-Glu-His-Asp-Pro-Set-Al
a-Pro-Gly-Asn-Gly-Tyr-Cy
s-NH, 62mg with 0mg of MgOl in hydrated DM
Dissolved in F'2, pentaprenyl bromide (trans form)
Add 0.5 J (0,075 mmol) dropwise and stir for 16 hours. The solvent was distilled off under reduced pressure, and a 60% aqueous methanol solution was added.
．． Dissolve in 5Kl, Sephadex I, H-20 (1,
4x 83.5cm). The eluted portion from 60 to 72- was collected and lyophilized to obtain 5 mg of the target product.

Rr, (cell a-s) 0.74 Reference example 5 II-Glu-His-Asp-Pro-Ser-Al
a -Pro -G Iy-Asn -G ly -
Production of Tyr-Cys(S-pentaprenyl)-OMe: H-Glu-11is-Asp-Pro-S
et-Ala-Pro-Gly-Asn-Gly-T
yr-Cys-QC)-[360mg ~1g01(1
Pentaprenyl bromide (trans isomer) 0.5+J (0.07
Add 5 mmol) dropwise and stir for 16 hours. The solvent was distilled off under reduced pressure, dissolved in 0.5 volume of 60% methanol aqueous solution, and Sephadex Ll-[,20 (1,4X 83.5 molar volume)
). 62-757.1. The eluted portion is collected and freeze-dried to obtain 4 mg of the target product.

Rf4 (cell o-s) 0.82 Reference example 6 l-1-Glu-His-Asp-Pro-3et-A
la = Pro - G ly - Asn - (';
ly -Tyr-Cys(S-hexaprenyl') -
Production of NI-12: 1l-Glu-1(is-A
sp-Pro-9et-Ala-Pro-Gly-As
Dissolve 31 mg of n-Gly-Tyr-Cys-Nlt in 70% water-containing DMF3+J with 5 mg of Mg0, and dissolve hexaprenyl bromide (trans form) in isopropyl ether a0, 5 hlI2<0, 0411Im
ol) was added dropwise and stirred for 16 hours. The solvent was distilled off under reduced pressure, dissolved in 85% n-butanol aqueous solution 0.5 ha, and washed with Sephadex L L-(-20 column (1.4X
30 cm), collect 20 to 30 fractions, concentrate, and then develop again in the same column, collect the target fraction, and distill off the solvent. Collect the residue and obtain the objective 5B.

Rr4 (cell a-s) 0.76 Example 1 H-Tyr-Pro -Glu -11e -5er-
Trp -T hr -A rg -A sn -G
Preparation of 1y-Cys(S-pentaprenyl)-oit: H-TYr-Pro-Glu-11e-
S er −T rp −T hr −−A rg −
, 85n-Gly-Cys-o it, i o m
g with 6 mg of MgO in 70% hydrated D M P 3
Dissolved in 0.5 mg of isopropyl ether solution of pentaprenyl bromide (trans form) (0.05 mmol)
Dropwise and stir for 16 hours.

Distill the solvent under reduced pressure 1.85% n-sitanol aqueous solution 0.
57n0. Dissolve in Sephadex 1. , l(-2
0 column (2.3 x 28.5 cm). 61
The fractions from 0 to 56 rotations are collected and concentrated, developed on the same column as rtG, and the target fractions are collected and the solvent is distilled off to obtain 10 mg of the target product.

[, 0,70 Procedural amendments to be amended September 10, 1988 1.11 indication Showa h] 1962 Patent Application No. 128684 2 Name of the invention S-polyprenyl peptides 3, Consideration case for amendment Relationship with Patent Applicant Address 2-27 Doshomachi, Higashi-ku, Osaka Name Name
(293) Takeda Pharmaceutical Co., Ltd. Representative Umemoto
Genuine/1 Agent Address: 2-17-85-5 Jusohonmachi, Yodoyo-ku, Osaka City
, VJ appendix 6 of the hli regular instruction, title of the invention of the specification to be amended, scope of claims, and detailed description of the invention φ column 7, content of the amendment

Claims (1)

[Claims] General formula ▲ Numerical formula, chemical formula, table, etc. ▼ [In the formula, X is H-Tyr-Pro-Glu-Ile-S
A peptide chain represented by er-Trp-Thr-Arg-Asn-Gly- is shown, and Y represents a carboxyl group which may be amidated with an unsubstituted amino group or esterified with a lower alkyl group. n represents an integer of 3, 4 or 5].