JPS5855134B2 - Peptide noseizoho - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing peptides by protecting the guanide group.

In order to produce a peptide using a compound (for example, arginine), it is necessary to protect the guanide group.

Protection of guanido groups is conventionally carried out by nitro or tosyl groups (
(hereinafter sometimes abbreviated as TO8).

In these conventional methods, the yield when removing the protecting group is low, and in order to remove the tosyl group, it must be carried out under strong conditions using liquid ammonia-metallic sodium or anhydrous hydrogen fluoride. As a result, other parts of the peptide are decomposed and by-products are generated, resulting in a decrease in the yield of the desired peptide.

In view of the above-mentioned drawbacks, the present inventor conducted intensive research with the aim of finding a method with a high yield during elimination, and as a result, the present inventor used a lower alkoxy- or tri-lower alkylbenzenesulfonyl group as a protecting group for the guanide group. As a result of further research, we discovered that this is a distant relative of the initial objective and that halogeno or lower alkyl sulfonic acids are advantageous for removing the protecting group after peptide condensation.
The invention has been completed.

That is, in the production of a peptide having a guanido group, the present invention protects the guanido group of a guanido group-containing raw material compound with a lower alkoxy or tri-lower alkylbenzenesulfonyl group, performs peptide condensation, and then converts the protecting group into a halogeno or lower alkyl sulfonic acid. This is a method for producing a peptide, which is characterized in that the peptide is eliminated by

Among the protecting groups used in the present invention, the lower alkoxy of the lower alkoxybenzenesulfonyl group has 1 to 3 carbon atoms.
Preferred examples include methoxy, ethoxy, propoxy, impropoxy, and the like.

The lower alkoxy may be present at any position on the benzene ring, but the 4-position of the sulfonyl is particularly preferred.

Specific examples include p-methoxybenzenesulfonyl group (hereinafter sometimes abbreviated as MBS)
, p-ethoxybenzenesulfonyl group, p-propoxybenzenesulfonyl group, p-isopropoxybenzenesulfonyl group, and the like.

The lower alkyl of the tri-lower alkylbenzenesulfonyl group preferably has the same or different carbon atoms of 1 to 5, such as methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, n-amyl, t-amyl, etc. Can be mentioned.

The tri-lower alkyl may be located at any position on the benzene ring, but the 2-, 4- and 6-positions of the sulfonyl are particularly preferred.

Specific examples include 2,4,6-toIJ methylbenzenesulfonyl group, 2,4,6-dryethylbenzenesulfonyl group, 2,4,6-to'Jpropylbenzenesulfonyl group, 2, 4,6-dolyisopropylbenzenesulfonyl group, 2,4,6-41J -t
-phthylbenzenesulfonyl group and the like.

Lower alkoxy-
Alternatively, in order to introduce a tri-lower alkylbenzenesulfonyl group, a lower alkoxy or tri-lower alkylbenzenesulfonic acid group is chopped into 1 equivalent of the guanido group-containing compound to give about 1 to 5 equivalents, more preferably about 1 to 5 equivalents. It is preferable to act so that the amount is 2 equivalents.

The lower alkoxy- or tri-lower alkylbenzenesulfonic acid group is usually subjected to the reaction in its halide form.

Halogenides are chloride, fluoride, bromide, and yosito.

Preferably, the reaction is carried out in the presence of a base.

Examples of the base include sodium hydroxide, potassium hydroxide, lithium hydroxide, etc., and are used in an amount of about 1 to 10 equivalents, more preferably about 1 to 5 equivalents, per equivalent of the guanide group-containing compound.

The reaction is usually carried out in a suitable solvent such as acetone, dioxane, dimethylformamide, tetrahydrofuran and the like.

The reaction is carried out at -10'C to 25°C, preferably -5°C.
The temperature is between 10°C and 10°C.

After the peptide condensation reaction using the thus obtained compound having a guanide group protected with a lower alkoxy- or tri-lower alkylbenzenesulfonyl group, the protecting group is removed from the product.

In the elimination reaction, lower alkylsulfonic acids (eg, methanesulfonic acid, ethanesulfonic acid, etc.) or halogenosulfonic acids (eg, fluorosulfonic acid) are advantageously used.

The protecting group removal reaction can also be performed using Lewis acids such as poron tris trifluoroacetate CB (CF 3COO).
3) (hereinafter sometimes abbreviated as BTFA) etc. can also be advantageously used.

The above acid may be used in an amount sufficient to serve as a solvent and remove the protecting group.

For example, 1 to 105 equivalents, more preferably 1 to 1000 equivalents per equivalent of a compound having a protected guanido group.
Equivalent amounts are used.

The elimination reaction involves trifluoroacetic acid, acetic acid, chloroform,
It may also be carried out in a solvent such as methylene chloride.

The temperature is -100 to 100°C, more preferably -15°C.
The temperature is between 50°C and 50°C.

As shown in the following experimental examples, the method of the present invention allows easier removal of the protecting group than the conventional methods of protecting with tosyl or nitro groups.

Therefore, the method of the present invention is an advantageous method for producing peptides having a guanido group.

Test Example 1 0.1 mmol of tosyl arginine and p-methoxybenzenesulfonyl arginine were each dissolved in 0.15 ml of trifluoroacetic acid and cooled to 0°C, followed by 6 equivalents, 8 equivalents, and 10 equivalents of BTFA in trifluoroacetic acid. Each solution was added and stirred at 0° C. for 1 hour, then 10 times the volume of water was added and freeze-dried. The residue was analyzed for amino acids and the arginine content was calculated. Table 1 shows the results.

While paramethoxybenzenesulfonyl is mostly removed with 8 equivalents of reagent, only 3.2% of the tosyl group is removed.

In the case of the nitro group, similar results were obtained as with the tosyl group.

Test Example 2 (1) H-Arg (MBS)-OH and H-Arg (
Tos )-OH 0.5 to about 100 μmole each
ml of methanesulfonic acid (CH3SO3H) was added and treated at room temperature (22°C) for 40 minutes.

Ether was added to the reaction solution, and the resulting oil was thoroughly washed with ether, followed by amino acid analysis (see Table 2).

For H-Arg(MBS)-OH, only the peak of H-Arg-OH was observed, and no peak of the raw material H-Arg(MBS)OH was observed, indicating that the elimination of the MBS group was complete. Ta.

On the other hand, in HArg(Tos)-OH, a fairly large peak of H>Arg(Tos)-OH, the raw material, was observed, indicating that the elimination of the tosyl group was incomplete.

(2) H-Arg (MBS)-OH and H-Arg
(Tos)OH 100μmole each 0.25ml
Dissolved in trifluoroacetic acid (CF3COOH) 0
．． Add 25 ml of methanesulfonic acid and bring to room temperature (22°C).
) for 35 minutes.

After adding ether and washing the resulting oil thoroughly with ether, amino acid analysis was performed (see Table 2).

H-Arg (MBS)OH is 1-2% raw material H-A
rg(MBS)OH peak was observed, but H-A rg
Most of the -OH peak was present, indicating that the MBS group was almost completely eliminated.

On the other hand, HArg (Tos) -OH is a large raw material H
Arg(Tos)-OH peak and a small amount of H-A
An rgOH peak was observed, indicating that the elimination of the tosyl group was very incomplete.

Example l Synthesis of bradykinin is carried out as follows.

The respective synthesis steps are summarized in Table 3 at the end.

Furthermore, the meanings of the abbreviations of the raw materials and products used below are summarized after Table 3.

MBS (1) Synthesis of Z-Arg-OH-DCHA-CH3CN Z-Arg-OH55,5f? (0,18M) to 4
180 ml of N-NaOH and 7 setsone 1.3. Dissolve in the mixture of e and cool with water.

744 g (0.36 M) of p-methoxybenzenesulfonyl chloride was dissolved in 7300 ml of acetate and added dropwise over 30 minutes.

After stirring for 2 hours, citric acid was added to make it acidic, and acetone was distilled off under reduced pressure.

The remaining oil is dissolved in ethyl acetate, and the ethyl acetate layer is washed twice with water and then extracted with 5% aqueous NaHCO3.

The extracted layer is acidified with citric acid, and the precipitated oil is extracted with ethyl acetate.

After washing the ethyl acetate layer twice with water, the solvent was distilled off under reduced pressure.

Remaining oily substance 86.0'? (lo-0%)' was dissolved in 600 ml of ethyl acetate, dicyclohexylamine (D
CHA) 36.0ml (0.18M) is added and left in the refrigerator to crystallize.

This was collected and recrystallized twice using CH3CN.

Yield near 7, Og (61,1%), melting point 1100-11
2.0'C (decomposition) Optical rotation: [α)23+5.1°cc=i, 3s, Meta elemental analysis value: C33H4907N5S ・As CH3CN; Take the oily Z-Arg-OH2g and dissolve it in methanol. Catalytic reduction was carried out in the presence of palladium black.

Methanol was distilled off, and the remaining oil was recrystallized from hot water.

Reaction amount: 900m9 (63.0%), melting point 144.0~
1460℃ (decomposed) Optical rotation: [α] 2 - 61° (C = 0.71, in methanol) Elemental analysis value ' C13H2005N4S '' + H20: Treated with 002NH2SO4, Z-Arg-O
Let it be H.

The ethyl acetate was distilled off and the remaining oil was dissolved in 30 ml of dimethylformamide.

1.68 ml (0,012 M) of triethylamine and 2.59 g (0,012 M) of p-nitrobenzyl bromide were cooled with water.
012M).

The reaction solution was stirred at 80°C for 2 hours and then left at room temperature overnight.

The reaction solution was poured into water, extracted with ethyl acetate, and washed with 1N-HCl 5% NaHCO3 according to a conventional method.

The ethyl acetate layer was dried over anhydrous Na2SO4 and then distilled off.

Add petroleum benzene to the remaining oil and rub it with a glass rod to precipitate a powder.

Yield: 6.2 g (100%), melting point 90.0-110.
0°C (decomposition) Optical rotation: [α)21:s4° (C=0.59, in dimethylformamide) Elemental analysis value: As C28Ha, 09N5S; Calculated value C54.80, N5.09, N11.41, S5.23; Experimental value C55,49, N5.15, N11.15, S 436 Rfl: 0.91 (chloroform: methanol: acetic acid - 9:1:0.5) [The following Rfl refers to the above solvent. This is the Rf value when used.

)Z -Arg -0NBzl 6.1? (0,0
1M) with 35 ml of 25% HBr/acetic acid at room temperature and, after 40 minutes, powdered with ether.

After drying overnight in a desigguter containing NaOH,
Dissolve in 30'ml of dimethylformamide, add 2.8ml (0.02M) of triethylamine while cooling with water, and remove the resulting salt.

Z -Phe -0NB 4.6 in P solution? (0, O
LM) and stir overnight at room temperature.

The reaction solution was diluted with water and extracted with ethyl acetate.

The ethyl acetate layer was diluted with 1N-HCl 5% NaHC according to the usual method.
After washing with O3 and drying with anhydrous Na2SO4, it is distilled off.

Petroleum benzine is added to the remaining oil to cause precipitation and crystallization.

Recrystallize from ethanol. Yield: 4.5ft (59,
2%), melting point 130.0-1320°C Optical rotation: [α]21 6.8° (C=0.66, in dimethylformamide) Elemental analysis value: C3□H4oO11N6S; calculated value C58,41,N5 .30, N11.05, S4.22; Experimental value C58,52, N5.19, N 10.98, S4゜36 Rfl = 0.82 Z-Phe-Arg-ONBZ13.8t (0,005
M) with 20 ml of 25% HBr/acetic acid for 40 minutes at room temperature.

After adding ether to form a powder, it was dried overnight in a desiccator containing NaOH.

Dissolve this in 20 ml of dimethylformamide, add 1.4 ml (0.01 M) of triethylamine while cooling with water, and separate the resulting salt.

BOC-8er-Pro-OH [melting point 147.0
~148.0°C (decomposition)] 1.51 (0,005M)
, HONB9001n9 (0,005M) and dissolve.

N-N'-dicyclohexylcarbodiimide 1.031
(0,005M) and stirred at 4°C for 2 days.

After further stirring at room temperature for one day, dimethylformamide is distilled off, the residue is dissolved in ethyl acetate, and washed with 0.2 N-HC15% Na HCOs according to a conventional method.

After drying over anhydrous Na2SO4, ethyl acetate is distilled off.

When petroleum benzene is added to the remaining oily substance, it forms crystals.

Recrystallize from ethyl acetate/petroleum benzene.

Yield: 31' (83.4%), melting point 155.0-160
．． 0°C (decomposition) Optical rotation: [α) 21-26.0° (C = 0.52, dimethylformamide) Elemental analysis value: as C42H54C13NaS-+H20; (6) BOC ・Phe-8er-Pro-Phe-ArgONBZl
Synthesis of BOC-8er-Pro-Phe-ArgONBZl3
．． 64 ft (0,004M) to 30m1 of CF3CO
Treat with OH for 30 minutes at room temperature.

After CF3COOH is distilled off under reduced pressure, ether is added to the residue to form a powder.

After drying overnight in a desiccator containing NaOH,
Dissolve in 2 oml of dimethylformamide.

Cool on ice and add 0.56 ml of triethylamine (0,004
Add M).

BOC-Phe-0NB1.72 ft (0,004
Add M) and stir at room temperature for 6 hours.

Dimethylformamide was distilled off in vacuo, the residue was dissolved in ethyl acetate, and 0.2N-HCI 5% was added according to a conventional method.
Wash with NaHCO3.

After drying over anhydrous Na2SO4, ethyl acetate was distilled off, and the remaining crystalline residue was taken up with ether.

Recrystallize from ethyl acetate.

Yield: 4. Og (946%), melting point 165.0-170
．． 0'C Optical rotation: [α]''-22.2° (C=0.59, in dimethylformamide) Elemental analysis value: as C51H53014N9S-H2O; (7) z-Arg-Pro-Pro-Gly-OtB
70ml of synthetic oily Z-Pro-Pro-Gly-OtBu4.9g (0,0106M) (Rfl=0.80)
of methanol and perform catalytic reduction in the presence of palladium black for 3 days.

The catalyst is distributed from door to door, and the p liquid is concentrated.

The remaining oil was dioxane:tetrahydrofuran (9:
Dissolve the mixture of 1) in 100 ml and add MBS MBS 1 Z-Arg・OH [Z-Arg-OH-DCHA-CH
3CN7.42'? (0,0106M) by a conventional method.

] and HONH2.0g (0.0111M) and dissolve.

Water-cooled N-N'-dicyclohexylcarbodiimide 2
．． 41 (0,0117M) and stirred at 4°C for 2 days.

The produced urea is distributed door to door, and the r-liquid is concentrated.

The oily residue was dissolved in ethyl acetate and diluted with 0.2N by a conventional method.
- Wash with HCI 5% NaHCO3.

After drying over anhydrous Na2SO4, ethyl acetate is distilled off under reduced pressure.

The remaining oil is mixed with ether and rubbed with a glass rod to precipitate a powder.

Reprecipitate from ethyl acetate/ether.

Yield: 5.5 g (66.2%), melting point 120.0-13
0.0°C (decomposed) Optical rotation: [α) 21-47.7° (C=0.53, dimethylformamide) Elemental analysis value: C37H5101ON7S −+H20; Calculated value C5 1 Experimental value C5 I Rfl=0. 70 5.90. H6,5 2,33, S 4.0 5.89, H6,6 2,31, S 4.0 4.72P (0,006M) in 30m1 of CF3CO
Treat with OH for 30 minutes at room temperature.

The CF3COOH is distilled off under reduced pressure and the remaining oily residue is dissolved in ethyl acetate and washed twice with water.

After drying over anhydrous Na2SO4, ethyl acetate is distilled off.

The residue is collected as a powder by adding ether.

Purify by reprecipitation from ethyl acetate.

Yield: 3.5f (85.4%), melting point 80.0-9
0.0°C (decomposed) Optical rotation: [α) 21-46.4° (C = 0.56, in dimethylformamide) Elemental analysis value: as C33H4301oN7S-H20; calculated value C53,00, H6,06, N13.11, S4.29; Experimental value C53.13, H6.03, N 12.60. S 4.26 Rfl =0.44 Takashi BOC-Phe-8er-Pro-Phe-Arg-O
NBzll, 06f (1mM) in cold CF a C0OH
Dissolve in 12ml and leave at room temperature for 30 minutes, then add 5.85NH
Add 70.17ml of CI/dioxa, CF3COOH
to remove.

Add ether to the remaining oil to make a powder.

After drying overnight in a NaOH desicguter, 20 ml of dimethylformamide! Stir and add 1.28 ml of 10% N-ethylmorpholine dimethylformamide solution under cooling.
So middle BS sum.

Z-Arg-Pro-Pro-Gly-OH73017
V (1mM) and HONB2707Q (1.5mM) were added and dissolved, and N-N'-dichlorohexylcarbodiimide 247■ (1.2mM) was added at 0°C, stirred at 4°C for 3 days, and further incubated at room temperature for one day. Stir.

The produced dicyclohexylurea (DCurea) is separated from each other, dimethylformamide is distilled off, and the residue is washed with ether using a decanting method.

Recrystallize three times from hot ethanol. Yield: 1.4P (8
4.0%), melting point 120.0-125.0°C (decomposition) Optical rotation: [α) 22 37.1° (C = 0.56, in dimethylformamide) Rf2 = 0.06 (ethyl acetate) Pyridine: Acetic acid:
Water-60:20:6:11) Elemental analysis value: C79H9602□Nl6S2・2H20
as; Calculated value C55,62, H5,91, N 1314, 376; Experimental value 5.62, 5.87, 12.97, 3.87 Amino acid analysis: Arg 1.94 (21: Ser
O,96(1): *Pro3.08(3): Gly
0.96(1): Phe 1.98(2) Synthesis 4 with average recovery rate of 98.0%, Off (2.4mM) in 80% acetic acid aqueous solution 10
Dissolve to 0 ml and reduce in the presence of palladium black for 8 hours.

Separate the palladium black and concentrate the liquid f under reduced pressure.

Add acetone/ether (1:1) to the remaining oil and rub with a glass rod to precipitate a powder.

Wash thoroughly with hot acetone.

Yield: 34g (100%), melting point 170.0-180.
0°C (decomposed) Optical rotation: [α)”-51.3° (C = 0.50, in acetic acid) *Elemental analysis value: C64H8501, N15S2/H20; Calculated value C54,19, H618, N 14.81.S 4.52; Experimental value C54,16, H6,35, N 14.76, S 458 Amino acid analysis: Arg 2.07(2):Ser
O,93(1): Pro 2.90(3): Gl
y 1, OO(1): Phe 1.95(2): Average recovery rate 100% 280m9 (0.2mM) was added to anisole (0.12m1
) and methanesulfonic acid (3.0 m/liter) and stir at room temperature for 40 minutes.

After adding ether, remove the oily residue with a slant, wash thoroughly with ether, and dissolve in water to obtain Amberlite (
Amberlite) IRA-410 (acetic acid type)
ion exchange through a column (2.5 x 10 crI'L).

Collect the effluent and lyophilize.

(Yield: 233 In9) This powder was dissolved in water, and carboxymethyl cellulose (CMC) Kara A (2,5
After washing with 0.0 IN ammonium acetate (100 ml), add 0.0 IN ammonium acetate (500 ml) and 0.3 N ammonium acetate A (500 ml) to create a linear concentration gradient. Elute.

The main elution fractions are collected and lyophilized (yield 178 µ).

Further purification is performed under the same conditions. Yield: 170m9 Optical rotation: [α]24-808° (C=0.49, in water) Amino acid ratio of acid decomposition product: Arg 2.03 :Set
0.96 (1): Pro 3.03 (3): Gl
y 1.00: Phe 2.02 (21: Average recovery rate 943%: l-5 paper electrophoresis: pH 1.9 (formic acid: acetate buffer) 4.2 cm Thin layer chromatography Rf = 0.11 (developing solvent; Ethyl acetate: n-phthanol: acetic acid - 1:1:1:1) Rf = 0.72 (developing solvent: n-butanol: pyridine: acetic acid = 30:20:6:24) However, in the table, Arg: arginine: Pronibroline; G
ly nigericinr Phe: Phenylalanine: S
er: serine; Z: carbobenzoxy group, MBS: p-
, l xybenzenesulfonyl group, MBS-C1: p
-Methoxybenzenesulfonyl chloride, 0NBzl
: p-nitrobenzyl ester, 0Bzl: benzyl ester, ONB: N-hydroxy-5-norbornene-2,3-dicarboximide ester, BOC:
tertiary-butyloxycarbonyl group, 0tBu: tertiary-butyl ester, HONB: N-hydroxy-5-norbornene-2,3-dicarboximide,
DCC: N・N'-dicyclohexylcarbodiimide,
HBr/AcOH: acetic acid raw hydrobromic acid, H2/pd: catalytic reduction, CF3COOH nitrifluoroacetic acid.

In addition, the numbers in parentheses in the table correspond to the numbers of each reaction step.

The bradykinin synthesized above showed chemically and *biologically the same behavior as the standard product.

Bradykinin is a peptide that exhibits effects such as lowering blood pressure ("Amino Acid Series, Separate Volume, Active Peptides, Volume 2", published by World Health News, 1966).
6), pp. 72-82) and are useful as antihypertensive agents.

When used as a blood pressure lowering agent, the usual adult dose is 1 to 1 per day.
000γ is administered in a dosage form such as an injection.

Example 2 Angiotensin converting enzyme inhibitor
nzyme 1 nhibitor) or as follows.

Each synthesis step is summarized in Table 4 at the end.

In addition, the meanings of the abbreviations of raw materials and products used below are summarized after Table 3 of Example 10 and after Table 4 of this example.

In addition, the target product of this example has a blood pressure lowering enhancing effect,
Useful as a blood pressure lowering agent.

Regarding this object, see Biochemistry, Vol. 10, 403.
3 (1971).

When used as a blood pressure lowering agent, the usual adult dose is 1 to 2 doses per day.
00■ is administered in the form of an injection or the like.

(1) Synthesis of Z-Ileu-Pro-Pro-OtBu z-Pro-Pro-OtBu (11.6 g, 0.03
H-Pro-Pro-OtBu and z・■1eu-ONB obtained by catalytically reducing M) in methanol according to a conventional method and concentrating the r liquid.
CZ ・l1euOH (7,96P, o, o3M), H
condensate with ONB (5,4Of, 0.03M)] and DCC (6,19g, 0.03M) with dioxa7C8o
180° C. for 7 hours.

Dioxane is distilled off, dissolved in ethyl acetate, and the residue is washed with acid and alkali in the usual manner.

Ethyl acetate was distilled off, and the resulting oil was subjected to silica gel column chromatography (solvent: CHCl3).
Purify according to conventional methods.

Yield 12.0 g (oil) (77.4%) (2) Z-G
Synthesis of lu-Ileu-Pro-Pro-OtBu
■1eu-Pro-Pro-OtBu 12,0g (0
, 0233M) in methanol in a conventional manner.

The obtained H.■1eu-Pro.Pro.0tBu.

Z-Glu-OH (6,5 g, 0.0233 M) and HONB (4,17 f, 0.0233 M) were dissolved in dimethylformamide (50 ml), cooled to 0 °C, and DC
Add C (4.81g, 0.0233M) and stir for 12 hours.

The resulting urea is separated from each other, and the P solution is diluted with water and extracted with ethyl acetate.

The ethyl acetate layer was washed with 0.2N HCl and NaHCO3, dried, and then ethyl acetate was distilled off.

When petroleum benzene is added to an oily substance, it forms crystals.

Collect a large amount, dissolve it in ethyl acetate, add NN-dimethylaminopropylamine 2.5-, leave it for 3 hours, and wash the ethyl acetate layer with 0ZNHCI.

Ethyl acetate is distilled off, petroleum benzine is added, and a powder is collected.

Recrystallize from ethanol. Yield 3.89 (25,3
%), melting point 98-100'C optical rotation: [α) 21-82
．． 2° (C=0.69, in dimethylformamide) Elemental analysis: C33H4908N5・+H20 Calculated value: C
60,72; H7,72; N10.73 Experimental value: C60,38; H7,57; N10.69 3) Synthesis of Z-Arg(MBS)・Pro・0tBu Z
-Pro -01Bu (10,0P, 0.03M)
is subjected to catalytic reduction according to a conventional method to obtain H-Pro-0tBu.

H-Pro-0tBu. Z-Arg(MBS)・OH [Z-Arg(MBS)・0H-DCHA-CH3CN
(21,0?, 0.03M)] and HONB
(5.4g, 0.03M) was dissolved in dioxane (50m7) and DCC (6.2g, 0.03M) was dissolved in dioxane (50m7) in a cold room (4°C).
Add M) and stir for 48 hours.

The urea is separated off, the dioxane is distilled off, and the residue is dissolved in ethyl acetate and washed with acid and Alzuli in the usual manner.

After drying, ethyl acetate is distilled off, and petroleum benzine is added to the residue for crystallization.

Recrystallize twice from 50% ethanol.

Yield 13.1' (69,0%), melting point 137139°C Optical rotation: [α]''-28,00 (C = 0.53, in dimethylformamide) Elemental analysis: C30H4108H5S Calculated value: C57,04; H6 ,54; N 11.09; S 5.07 Experimental value: C56,70; H6,67; N 11.20; S 5.10 (4) Z-Pro-Arg (MBS) Pro-0tB
Synthesis of u z-Arg(MBS)・Pro-0tBu(1
2,6 (0.02M) in methanol according to a conventional method.
Restored for 6 hours.

The obtained H-Arg(MBS)・Pro-0tBu and Z
-Pro-0NB (8,2?, 0.02M) is dissolved in dioxane and stirred at room temperature for 3 hours.

Dioxane is distilled off, the residue is dissolved in ethyl acetate, and washed with acid and alkali in the usual manner.

After drying, ethyl cyanide is distilled off, and ether is added to the residue to give a final solution.

Reprecipitate from ethyl acetate-ether.

Yield 12.3r (4,2?), melting point 90-95°C
(Decomposition) Optical rotation: [α] 21-55.2° (C = 0.54, dimethylformamide) Elemental analysis' C35H490111H6S calculated value: C5
7,60; H6,77; N 11.52; S 4.39 Experimental value: C57,40; H6,77; N 11.30; S 4.27 (5) Z-Trp-Pro-Arg (MBS)・Pro
-0tBu synthesis Z-Pro-Arg (MB mark Pro-0tBu (11
, 7g, 0.016M) in methanol and reduced according to a conventional method.

The obtained H-Pro-Arg(MBS)・Pro-0t
Bu and Z = Trp-ONBCZ-Trp -OH(
5, 4? , 0.016M), HONB (2,9@, 0
．． 016M), prepared from DCC (3.3 g, 0.016M)] was dissolved in dioxane (150 mO, 12
It takes time.

Dioxane is distilled off under reduced pressure, the residue is dissolved in ethyl acetate, and the ethyl acetate layer is washed with acid and alkali in the usual manner.

After drying, ethyl acetate is distilled off, and ether is added to the residue to form a powder.

This powder was dissolved in ethyl acetate, N-N dimethylaminopropylamine (1 m0) was added, and after standing for 1 hour, the ethyl acetate layer was washed with 0.2N HCl, the ethyl acetate was distilled off under reduced pressure, and the residue was diluted with ether. Collect as a powder.

Yield 11.51 (79.0%), melting point 120125℃ (
Decomposition) Optical rotation: [α] 2153.5° (C = 0.54, dimethylformamide) Elemental analysis: C46H580 to Na S ” +H2
0 Calculated value: C59,79; H6,43; N 12.13; S 3.47 Experimental value: C59,73; H6,37; N 11.90; S 3.43 BS ■ (6) p-Glu- Trp-Pro-Arg-Pro-
Synthesis of OHz-Trp-Pro-Arg(MBS)・P
ro·0tBu (9.15 g, 0.01M) is reduced in methanol according to conventional methods.

The obtained H-Trp-Pro-Arg (MBS).Pr
o-0tBu and p-Glu ・OH (1,29ft,
0.01 M), HONB (1,79'f!, 0
．． 01M) in dimethylformamide (30 mO), add DCC (2,061, Q, 01M) at 0°C, and stir at room temperature for 12 hours.

The resulting urea is separated and the dimethylformamide is distilled off in vacuo.

Add water and remove the resulting oil by decanting.

Dissolve this in ethanol, add water and decant the resulting oil again.

Further, dissolve in ethanol and add ethyl acetate to make a powder.

This was dissolved in trifluoroacetic acid (80ml (containing 1ml of thioglycolic acid and 4ml of anisole)), allowed to stand at room temperature for 40 minutes, then most of the trifluoroacetic acid was distilled off under reduced pressure, and ether was added to make a powder. do.

Yield 7.8P (83.3%), melting point 130-135℃ (
Decomposition) Optical rotation: [α] -29.2° (C = 0.60. in dimethylformamide) Elemental analysis: C39H4901oN9S Calculated value: C53,72; H N 14.46; S Experimental value: C53,68; Synthesis of Z-Glu・■1eu-Pro-Pro°0tBu(1
, 29g, 0.002M) in methanol in the presence of an equivalent amount of hydrochloric acid according to a conventional method.

The obtained H-Gln・■1eu-Pro-Pro・0t
Bu-HCl and p-Glu-Tr-p-Pro-Arg
(MBS)・Pro-0H (1,67P, 0.00
2M), HONB (537In9.0.003M), N
-Ethylmorpholine (026ml, Q, 002M)
Dissolved in dimethylformamide (15m0), added DCC (495rru?, 0.0024M) at 0°C, stirred in a cold room (4°C) for 122 hours, then at room temperature for 3 days, and then distributed the urea from door to door. Concentrate the p solution.

The residue was made into a powder by adding chill and powdered using a silica gel column chromaphy (3,0 cm x 2
2.0C771).

Yield 2.2 P (83,0%), melting point 175-1 °C (
Decomposition) Optical rotation: [α] 24-68.10 (C=0.56 in methyl formaldehyde) (8) Synthesis (example of removing the protecting group with methanesulfonic acid) 266m9 was converted into aniso-# (0, 121rL1.),
Dissolve in a mixture of thiocricholic acid (0.03ml) and methanesulfonic acid (3.0ml) and stir at room temperature for 30 minutes.

Increase the ether to 11 degrees and remove the resulting oily substance with a slant.
Wash thoroughly with ether.

Dissolve this in water and add Amberlite.
e) IRA-410 (acetic acid type) column (25 x 12
．． 0crrL) for ion exchange.

IN ammonium acetate (20 ml) was added to the effluent, and Amberlite XAD-20
Load into a column (2.5 x 10.0 cIrL).

After washing the column with 0.01N ammonium acetate (100mJ), add 0.01N ammonium acetate (5oomz
) and 50% ethanol (containing 0.5% acetic acid) (F,
nnm1) between F≦W name self-intoxication 1 parable [Otko West P to t
x Elutes into the filter soil.

(Yield: 1301719) This was dissolved in 0.01N ammonium acetate and passed through a carboxymethyl cellulose (CMC) column (2.5 x 10 cIrL).

Collect the effluent and lyophilize.

Yield: 125mσ Optical rotation: [α]2'-160,4° (C=0.48,
(in water) Amino acid ratio of acid decomposition product: Arg 1.02 (1);
Trp 0.90 (1): Glu 2.10 (21
Pro3.96 (4CI leu 1.02(1), average recovery 910°C thin layer chromatography, Rf = 0
．． Synthesis of 67 (developing solvent; n-butanol: pyridine: acetic acid: water = 30:20: 6: 24) (example of removing the protecting group with fluorosulfonic acid) 266m9, anisole (0.12ml), thioglycol Dissolve in a mixture of acid (0,03rrLl) and fluorosulfonic acid (3,QmO) and shake for 30 minutes at room temperature.

Ether is added and the resulting oil is decanted and washed thoroughly with ether.

Dissolve this in water and add Amberlite.
e) IRA-410 (acetic acid type) column (2,5Xi
2. ocrrL) for ion exchange.

To the effluent, add IN ammonium acetate (20 m0) and add Amberlite X A D
-2 column (2,5X i O, 0crIl).

After washing the column with 0.01N ammonium acetate (100ml), add 0.01N ammonium acetate (500ml).
) and 50% ethanol (containing 0.5% acetic acid) (50%
Elute into water to form a linear concentration gradient between 0m00 and 0m00.

(Yield: 130In9) Dissolve this in 0.01N ammonium acetate to obtain carboxymethylcellulose (CMC)
column (2.5X 10crrL).

Collect the effluent and lyophilize.

Yield 125m9 Optical rotation: [α] -159.3° (C = 0.601 in water) Amino acid ratio of acid decomposition product: Arg 1.01 (1):
Trp O,89(1):Glu 1.98(2):P
ro 4.00 (4); I leu 1. O0(1)
, average recovery rate 90.2% thin layer chromatography, Rf
=0.67 CJ: open solvent; n-butanol: pyridine: acetic acid: water 30: 20: 6: 24) , 2mM) in 30ml of trifluoroacetic acid and treated with 75 equivalents of BTFA at 0°C for 1 hour.

Trifluoroacetic acid was distilled off under reduced pressure, and the residue was dissolved in 100ml of water.
Wash away insoluble matter.

The p solution is freeze-dried.

Dissolve the freeze-dried powder in a small amount of water and add Amberlite C.
AmberliteIRA-410 (acetic acid type)
) (2,OX 10.0crIL) for ion exchange.

Add 1.5 liters of ammonium acetate to 200ml of the effluent to prepare Amberlite
- Pour into a 20 column (2,5x14, Ocm).

After purging the column with 100 ml of 0.01M ammonium acetate (5% acetic acid included), elution is carried out by column gradient elution between 1.01MIO and 5% acetic acid.

The main elution fractions are collected, ethanol is distilled off, and then lyophilized.

The lyophilized powder is dissolved in water and passed through a column of carboxymethylcellulose (2.0X 7.5 cm).

The main fractions are collected and lyophilized. Yield 130 ■ Optical rotation: [α] 2 degrees Celsius 1528°C = 0.19, in water) Amino acid ratio of acid decomposition product: Arg 1.02 (1):
Trp 0.94 (1); Gln 2.04 (2);
Pro 3.98 (4); I leu 1. O0(1
), average recovery rate 92.0% Example 3 (1) Z-Arg (MSS)/OHO combined synthesis Arg
-OH3,08f (0,OLM) is dissolved in a mixture of 10 ml of 4N sodium hydroxide and 80 ml of acetone and cooled on ice.

Mesitylenesulfonyl chloride 4.38 P(0,02
M) dissolved in 10 ml of acetone was added dropwise over 10 minutes.

After stirring for 2 hours, citric acid was added to make the mixture acidic, and acetone was distilled off under reduced pressure.

The remaining oil is dissolved in ethyl acetate, and the ethyl acetate layer is washed twice with water and then extracted with 5% aqueous sodium bicarbonate.

The sodium bicarbonate extract is made acidic with citric acid, and the precipitated oil is extracted with ethyl acetate. The ethyl acetate is washed twice with water and then dried under reduced pressure.

The remaining oily substance is powdered by adding petroleum benzene.

Yield 4.01 (81.8%), melting point 110-112 °C
(Decomposition) Optical rotation: [α] -〇, 41° (C = 0.97, in methanol) Elemental analysis: C23H280a N4 S calculated value: C5
6,54; H N 11.47; S Experimental value: C56,28; H 5,78; 6.56 6.10; (ilEIE) MSS: mesitylenesulfonyl group (2)
Synthesis of H-Arg(MSS)・OH Z-Arg(MSS)
S) ・OH2,0? is dissolved in methanol and catalytically reduced in the presence of palladium black.

The palladium black is separated from each other, and the liquid f is concentrated. Ethyl acetate is added to the residue to collect it as a powder.

Yield 1.3P (89.2%), melting point 144-146℃ (
Decomposition) Optical rotation: [α)''; -3,37° (C=0.89, in methanol) Elemental analysis value: C15H2404N4 S calculated value: C5
0,54; H6,79; N 15.27; S 9.00 Experimental value: C50,39; H6,78; N 14.89; S 8.79 (3) Desorption of MSS (a) [Methanesulfone Desorption by acid] H-Arg (MSS) -OH35,6 (100 μm
ole) was added with 0.5 ml of methanesulfonic acid containing 5% anisole and treated at room temperature (22°C) for 40 minutes.

Add ether to the reaction solution and wash the resulting oil well with ether.

The oil was dissolved in 10 ml of water, a portion was analyzed for amino acids, and the arginine content was calculated, and the yield was 78.0%.
It is.

(b) Desorption by CBTFA] (Reference example) 35.6 mI each for three colbens? (100 μmole) of H-Arg (MS S )-OH was added and the
1 of trifluoroacetic acid and cooled to 0°C.
To this, 6 equivalents, 8 equivalents, and 10 equivalents of trifluoroacetic acid solutions of BTFA are added, and the mixture is stirred at 0° C. for 1 hour, and then 10 times the amount of water is added and freeze-dried.

The residue was dissolved in 10 ml of water and a portion was analyzed for amino acids as shown in the table on the next page.

It was produced in the same manner as in Example 3-(1) using 2,4,6-triisopropylbenzenesulfonyl chloride.

Yield 78.0%, melting point 130-135°C Optical rotation: [α:]”-0,54 (C = 1.11, in methanol) Elemental analysis value C29H4206N4 S calculated value: C6
0,60; H N9.72; S Experimental value: C61,20; H N: 8,68; S (Note) TPS: 2, 4, 6 Rubenzenesulfonyl group 7.37; 5.58 7.41; 5.70 Triisopropyl 2) Synthesis of H-Arg(TPS)-OH Produced in the same manner as Example 3-(2+).

Yield 82.0%, melting point 225-226℃ (decomposition) Optical rotation: [α] -3.8° (C = 1.02, in methanol) Elemental analysis value: C21H3604N4S ” +H20
Calculated value: C56,10; H8,07; N 12.46; S 7.13 Experimental value: C56,12; H8,24; N 12.04; S 7.21. 31 Desorption of TPS (a) [Desorption with methanesulfonic acid] H-Arg (TPS) -OH44,Omg (1
00 μmole) containing 5% anisole
1 of methanesulfonic acid was added and the mixture was heated at room temperature (22°C) for 40
Process for minutes.

Add ether to the reaction solution and wash the resulting oil well with ether.

The oil was dissolved in 1QmA water, a portion was analyzed for amino acids, and the arginine content was calculated, and the yield was 89.6%.
It is.

(b) [Desorption using BTFA] (Reference example) 44.0 In9 (100 μmole) of H-Arg(TPS)-OWx was placed in each of three colbens, dissolved in 0 and 5 ml of trifluoroacetic acid, and cooled to 0°C. Then, 6 equivalents, 8 equivalents, and 10 equivalents of BTFA in trifluoroacetic acid solutions were added to this, and after stirring at 0°C for 1 hour,
Add 0 times the volume of water and freeze-dry.

The residue was dissolved in 10 ml of water, a portion was analyzed for amino acids, and the arginine content was calculated as shown in the following table.

structure, it stimulates the anterior pituitary gland to produce luteinizing hormone (
lutenizing hormone (LH) and follicle stimulating hormone (folliclestimula)
ting hormone-FSH).

Therefore, Gn-RH is useful as a secretory function test agent for luteinizing hormone and follicle** stimulating hormone,
Usually, for adults, 0.01 m9 to 1.0 m9 is administered in the form of an injection or the like.

Gn-RH was synthesized as shown below.

(1) Z −Arg (MBS) ・Pro −
Synthesis of OH (a) Z-Arg (MBS) =
OH[Z-Arg(MBS) 0H-DCHA-
CH3CN (14.0g, 0.03M)] was dissolved in dioxane (100mJ) and HONB (4.0P) was dissolved in dioxane (100mJ).
, 0.022M), DCC (4.33g, 0.-021M) was added under cooling, and the mixture was stirred overnight at 4°C.
) from door to door and the P solution is H-Pro・OMe [H-Pro-
[Prepared from OMe-HCl (4981, 0.03M) and triethylamine (Et3N) (4.2ml, 0.03M)] in DMF (10Qm7) and stirred at room temperature for 2 days.

After distilling off the dioxane, rinse the remaining DMF with water.
The resulting oil is extracted with ethyl acetate.

The ethyl acetate layer was washed in a conventional manner, and N-N-dimethylaminopropylamine (2.5 ml) was added thereto. After standing for 1 hour, the ethyl acetate layer was washed with NHCt.

After drying with anhydrous sodium sulfate and distilling off the ethyl acetate, an oily substance (1
0f) is obtained.

Dissolve this in methanol (100 ml), add 50 ml of IN NaOH, and leave at room temperature for 4 hours.

Add an appropriate amount of crystalline citric acid to make it acidic, and distill off methanol.

The remaining oil is extracted with ethyl acetate.

The ethyl acetate layer was washed with water, dried, and evaporated under reduced pressure to obtain an oil.

This is purified by silica gel column chromatography.

(Solvent: Chloroform, Column: 6.OX 15.0
cIrL).

The main eluted fractions are collected and concentrated, and the desired product is powdered using ether.

Yield 3.5P (30.5%), melting point 90.0-93.0
'C (decomposition) Optical rotation: [α)''-27.5° (C = 0.59, methanol) Elemental analysis: C26H3308N5S-H20 words +19f
Straight: C52,60; H5,94; N 11.80;
S 5.40 Experimental value: C52,64;) (5,56; N 11.21; S 5.43 (b) Z-Arg(MBS)-Pro-0+Bu(4
00m9) Trifluoroacetic acid (dissolve to 2m0 and leave at room temperature for 45 minutes.

Most of the trifluoroacetic acid was distilled off under reduced pressure, the remaining oil extract was dissolved in ethyl acetate, and the ethyl acetate layer &'! After washing and drying, ethyl acetate was distilled off under reduced pressure.

The remaining oil was powdered with ether. Yield: 310cm, melting point: 93.0-95.0℃ Degree of rotation: [α ￥-29.2°
(C=0.55, methanol) Elemental analysis: C26H3308N5S-H20 calculated value: C
52,60; H5,94; N 11.80; S 5.40 4 Experimental value: C52,64; H5,56; N 11.69; S 5.17 (2) Z-Arg(MBS) ・Pro − Gly
Synthesis of -NH2 Z-Arg (MBS) -Pro-O
H (3,45?, 6mM) and HONB (1,29?, 7
．． 2mM) in dioxane and diluted with DCC (1mM) under cooling.
, 36P, 6.6mM) and stir for 5 hours.

The generated DGUrea was taken from house to house and added to the liquid H-Gty-NH.
2 (740cm, lOpanM)' and stir overnight.

The dioxane is distilled off and the remaining oil is dissolved in ethyl acetate and washed in the usual manner.

After drying, ethyl acetate is distilled off, and petroleum benzine is added to the remaining oil to powder.

Reprecipitate from ethyl acetate and petroleum benzine.

Yield 2.8? (74,0%) Melting point 90-95.0℃
(Decomposition) Optical rotation: [α] -19.2° (C = 0.53, methanol) Elemental analysis: C28H3□08N7S Calculated value: C53,24; H5,90; N 15.52; S 5.08 Experimental value: C53,95; H6,32; N 14.97; S 4.40 (3) Z-Leu-Arg(MBS) -Pro-
Synthesis of Glv-NH2 Z-Arg(MBS)-Pro-Gly-NH2(2
, 71,0,0042M) was dissolved in a volume of 5 ml and subjected to catalytic reduction according to a conventional method.

The methanol was distilled off, and the remaining oil was dissolved in dioxane (50%
ml) and Z-Leu-ONB (Z-Leu-OH(1,11f
i, 0.0042M), HONBC752mg, 0.0
042M), DCC (8661n9, o, 0042M)
Add the garnish and stir overnight.

The dioxane is distilled off under reduced pressure and the remaining oil is extracted with a 1:1 mixture of ethyl acetate and n-butanol.

The organic layer is washed in the usual manner.

After drying, the solvent is distilled off and the remaining oil is triturated with ether.

Reprecipitate twice from ethyl acetate.

Yield 2.2y (7o3%), melting point 125.0-130.
0°C Optical rotation: [α co'3-35.6° (C = 0.57, methanol) Elemental analysis: C34H4909Ns S - + H20 calculated value: C54,10; H6,68; Amino acid ratio of acid decomposition product: Arg , 1.03 (1) : Pro, * 1,00 (1) ; Gly, 0.97 (1) : L
Synthesis of Z-Lsu-Arg(MBS)・Pro-Gly-NH with eu, 0.93(1) average recovery rate of 98.0%
2 (1,34P, 1.8mM) in methanol (70ml
Nitokashi 1. Perform catalytic reduction as usual.

After removing the catalyst, the liquid is concentrated under reduced pressure.

The remaining oily residue is dried in a vacuum desiccator overnight.

Dissolve this in DMF (20 ml) and prepare PGlu-His-Trp-8er-Tyr-Gly-
OH (1,37g, tsmM) and HONB (645■,
3.6mM; and cooled to -10°C.

Add DCC (743■, 3.6mM) and stir overnight.

The resulting DCUrea is separated, and the p liquid is distilled off in vacuo.

Add acetonitrile to the residue and collect the resulting powder.

Reprecipitate twice from ethanol to 1.

Yield 1.78@(73.0%), melting point 180-185℃
(Resolution) Optical rotation: [α” -27.4° (C = 1.07, DMF
) Elemental analysis: C62Hat Ota Nl 7 S
- H20 calculated value: C54,33; H6,10; N 17.38; S 2.34 Experimental value: C54,47; H6,23; N 16.64; ) to methanesulfonic acid (7
mA'), 7=sol (0,5mt: ), thioglycolic acid (o, osmo), and stir at room temperature for 1 hour.

After adding ether and washing off the resulting oil by decanting,
Water (sharpened into lomg, Amberlite IRA-4
10 (acetic acid form) column (2,5X 10. Ocm)
pass.

The eluate (200 mJ) was poured into a carboxymethyl cellulose column (2.5 x 14.Ocm), the column was washed with 0.005M ammonium acetate (soomg), and then 0.005M ammonium acetate (500rrL0
Elution was performed by a linear gradient method between and 6.2 M ammonium acetate (500 mA').

The main elution fraction was collected and poured onto an Amberlite XAD-20 column (2,5X 12.QcrrL).

Wash the column with 0.005M ammonium acetate (200ml).
Elution was performed by a linear gradient method between m0 and 60% aqueous ethanol (500 rI10).

The main eluted fractions were collected, ethanol was distilled off, and then lyophilized. The dried powder was dissolved in 0.1M acetic acid and passed through a Sephadenx LH-200 column.

Fractions containing the target product were collected and lyophilized.

Yield: 375 m9 Optical rotation [α -50.5° (C=0.
49.5% acetic acid water) Amino acid ratio of acid decomposition product: His, 0.96 (1)
;Ar:g, 0.96 (1), Trp, 0
．． 83(1): Ser.

0.89(1): Glu, 1.06(1);
Proll, 02(1); Gly, 2.06(2)
; Leull, 00(1) : Tyr, 1.0
0(1) Average recovery rate 89.0% This product showed the same chemical and biological behavior as the standard product.

Example 6 Synthesis of Tuftsin Tuftsin is H-Thr-Lys-Pro-Ar
It has an o-OH structure and has biological activity that promotes phagocytosis* in phagocytes such as white blood cells.

Therefore, tuftsin is useful as a leukocyte phagocytosis promoter, and is usually administered to adults at a daily dose of 0.1 m9 to 100 m2 in the form of an injection or the like.

Tuftsin was synthesized as shown below.

(1) Z Pro-Arg (D, 1Bs) O
Synthesis of H H-Arg(MBS) -OH(2,76?,
8mM) was dissolved in DMF (15ml), triethylamine (1,12mt:, smM) was added under water cooling, and then Z-Pro-0NB (3,28f?, 8mM) was dissolved in DMF (15ml).
m M ) and stir at room temperature for 6 hours.

Acetic acid (1.5 mO) was added to the reaction solution, and DMF was distilled off in vacuo.

The remaining oil is dissolved in ethyl acetate and the ethyl acetate layer is washed three times with water.

Ethyl acetate was distilled off under reduced pressure and the remaining oil was triturated with ether.

Reprecipitate from chloroform ether.

Yield 3.9P (35.0%), melting point 80.0-85.
0°C (decomposition) Optical rotation: [α cos -16.3° (C = 0.58, DM
F) Elemental analysis: C26H3308N5S 1yH20 calculated value: C53,41; H5,86; N 12.00; S 5.49 Experimental value: C53,37; H5,78; N 11.66; S 5.20 (2) BOC-Lys(Z)-Pro-Arg(MBS
) Synthesis of -OH Z-Pro -Arg (MBS) -OH(: 3.
46? , 6mM) in methanol, water and acetic acid (6:3:
The mixed solution of 1) is dissolved to 100 mJ, and catalytic reduction is performed according to a conventional method.

The catalyst is separated from each other and the liquid is concentrated under reduced pressure.

Water is added to the residue and the water is then distilled off under reduced pressure.

After repeating this operation three times to remove acetic acid, the residue was
Triethylamine (0.84 ml, 6 mM) and BOC-Lys (
Z ) -〇TCP (3,36f/, 6mM) was added and stirred at room temperature for 12 hours.

Add acetic acid (2 m0) to the reaction solution and evaporate DMF in vacuo.

The residue was dissolved in ethyl acetate, and the ethyl acetate layer was diluted with water.
After washing twice and drying, ethyl acetate is distilled off under reduced pressure.

The residue is purified by chromatography on silica gel.

[Column: 6, OX 6. Ocm, solvent: chloroform:methanol (97:3)] yield 3.41 (70,
9%), melting point 103.0-109.0 (decomposition) Optical rotation: [α]'4-19.4° (C = 0.70, DM
F) Elemental analysis: C3□H53011N7S -+H20 Calculated value: C54,67; H6,69; N 12.06: S 394 Experimental value: C54,72; H6,70; N 11.78; S 3.80 (3 ) Z-Thr-Lys(Z)-Pro-Arg
(MBS) -OH synthesis OC-Lys (Z) -Pro-Arg (MB
S)-0H (1,85S', 2.3'mM) is dissolved in trifluoroacetic acid (10m/?) and stirred at 10°C for 20 minutes.

Most of the trifluoroacetic acid was distilled off under reduced pressure, ether was added, and one portion of the resulting precipitate was taken and dried.

This was dissolved in DMF (1 (Jrrre)), triethylamine (0.64ml, 1.46mM) was added under water cooling, and then Z-Thr-0NB (952A, 2.3mM) was added.
Add and stir overnight at room temperature.

Add acetic acid (2 m0) and remove DMF in vacuo.

Add ether to the remaining oil and make a powder.

Reprecipitate from ethyl acetate and ether. Yield 2. IP(
95.5%), melting point 90.0-95.0°C (decomposition) Optical rotation: [α] rock -15.3° (C = 0.62, DMF
) Elemental analysis: C44H58013"l S Calculated value: C56,28; H6,23; N 11.93; S 3.42 Experimental value: C55,99; H6,22; N 11.56; S 3.36 (4 ) H-Thr-Lys-Pro-Arg
Synthesis of -OH (Tuftsin) Z-Thr-Lys (Z) -Pro -Arg
(MB S ) −()H(1,88s', 2771
Treat M) with a mixture of anine-/L/ (1.0 ml) and methanesulfonic acid (17 m, g) at room temperature for 45 minutes, add ether to the reaction solution, and wash the resulting oil thoroughly with ether using a decanting method. .

This oil was dissolved in water (10 mJ) and added to a column of Amberlite IRA-410 (acetic acid form) (2.5 x 10
．． Pour into Ocm).

Collect the eluate (200 mO) and pour into a column of carboxymethylcellulose (2,5X 15.0 crrL).

After washing the column with water (500ml), wash the column with water (500w).
Elution is performed using a linear gradient method with 0.2M ammonium acetate (500 wL0).

The main elution fractions are collected and lyophilized.

Dissolve this in 0.1M acetic acid and use Sephadex LH-2.
,0 column (3,0×130.0 Crn).

The main elution fractions are collected and lyophilized.

Yield: 840 m9 Optical rotation: [α -60.2° (C7 -0.55,5%
Acetic acid] Amino acid ratio of acid decomposition product: Lys, 10I (1); Arg
, 1. O0(1); T)tr, 0.99(1)
: Pro, 1.06 (1) Average recovery rate 100% This product showed strong chemotaxis activity.

Example 7 Synthesis of tripeptide H-Thr -Lys-Arg ・OH Tripeptide H-Thr -Lys ・Arg ・
OH is luteinizing hormone
rmone = LH) has a secretion-stimulating effect.

Therefore, it is useful as a drug for testing the secretion function of luteinizing hormone, and is usually administered in the form of an injection or the like in an amount of 0.1 m9 to 700 rrL9 per dose for adults.

This tripeptide was synthesized as shown below.

(1) BOC-Lys (Z) -Arg (M
Synthesis of B S )-()H H-Arg(MBS )
-OH (2,76?, 8mM) in DMF (15mJ
) and add triethylamine (1.12 m
1.8 mM) then BOC-Lys (Z)
-0TCP (4,48?, 8mM) is added and stirred at room temperature for 48 hours.

Acetic acid (3 m0) was added to the reaction solution, and DMF was distilled off in vacuo.

Dissolve the remaining oil in ethyl acetate and wash the ethyl acetate layer with water.

Ethyl acetate was distilled off under reduced pressure and the residue was triturated with ether.

Reprecipitate from acetonitrile ether.

Yield 4.8 f (84,4%), melting point 95.0-10
0.0°C (decomposition)) Z -Thr-Lys(Z)-Arg(MBS)
-()H synthesis OC-Lys (Z) -Arg(
Dissolve MBS)-0H (4, 24, 6mM) in 30ml of trifluoroacetic acid and stir at 10°C for 20 minutes.

Most of the trifluoroacetic acid is distilled off under reduced pressure, ether is added, and the resulting precipitate is collected and dried.

This was dissolved in DMF (20m) and triethylamine (1.65mg, 12mM) was added under water cooling, followed by Z-Thr-ONB (2.49?, 6mM).
Stir at room temperature for 12 hours.

Acetic acid (3 ml) was added to the reaction solution, and DMF was distilled off in vacuo.

The remaining oil was dissolved in ethyl acetate, the ethyl acetate layer was washed with water, and the ethyl acetate was distilled off under reduced pressure.

Triturate the remaining oil with ether.

Re-precipitate from acetic acid and ether.

Yield 4.7 P (93.1%), melting point 77-82.0°
C (decomposition) Optical rotation IJj: Ca]”d-0.9° (C=0.5
3. Synthesis of H-Thr-Lys -Arg -OH Z-
Thr −Lys (Z) −Arg (MB S
) -0H (1,68S', 2mM) in aniso-/L/
(1,0ml) and methanesulfonic acid (17,0ml)
Treat in the mixture of step 1) at room temperature for 45 minutes, add ether, and thoroughly wash the resulting oil by decanting.

Water (dissolved to 10m0, Amberlite IRA-41
0 (acetic acid type) column (2,5X 10.0CrrL)
Pour into.

The eluate (200ml) was collected and poured onto a carboxymethylcellulose column (2,5X 15.0cx).
After washing the column with water (200mA), rinse it with water (500mA).
Elute with a linear gradient between J) and 0.2M ammonium acetate.

The main elution fractions are collected and lyophilized.

Dissolve this in 0.1M acetic acid and use Sephadex LH-
Pass through 20 columns (3,0 x 130.0 cfrL).

The main elution fractions are collected and lyophilized.

Distance: 773m9 Optical rotation: [α]'￥3-8.8° (C=0.55.5%
acetic acid) Amino acid ratio of acid decomposition product: Lys, 1. OO(1)
;Arg.

1.03(1); Thr, 1.00(1) Average recovery rate 100%

Claims (1)

[Claims] 1. In the production of peptides having guanido groups, the guanido groups of the guanido group-containing raw material compounds are converted to lower alkoxy-
Alternatively, a method for producing a peptide, which comprises protecting the peptide with a tri-lower alkylbenzenesulfonyl group, condensing the peptide, and then removing the protecting group with a halogeno or lower alkylsulfonic acid.