JPH0592995A - 125 i-labeled radioactive substance of (nle8,nle18,tyr34)-h-pth(1-34)nh2 - Google Patents

Abstract

PURPOSE:To obtain the subject radioactive labeled substance composed of a new human parathyroid hormone derivative and useful for the parathyroid function assay, etc., by labeling tyrosine residue at a specific position of a peptide having a specific amino acid sequence with plural 125I atoms. CONSTITUTION:A protected peptide is synthesized by converting the carboxyl group of a C-terminal tyrosine group to amide group and successively condensing amino acids according to the amino acid sequence of the peptide by liquid-phase synthesis after protecting the alpha-amino group and side-chain functional group. The protected peptide is treated with anhydrous hydrogen fluoride, etc. to eliminate the protecting groups and purified by gel-filtration, ion-exchange column chromatography and highperformance liquid chromatography to obtain the human parathyroid hormone (h-PTH) expressed by formula. The objective radioactive substance containing the peptide of formula wheremn the 34th Tyr is labeled with one or two 125I atoms is produced by incorporating and reacting the h-PTH with a phosphate buffer solution containing 125-Nal and chloramine T.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel human parathyroid hormone (h-PTH) derivative. More specifically, the present invention is useful as a labeled compound for testing parathyroid function [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ] -hP.
TH (1-34) NH ₂ , that is, the formula H-SEQ ID NO: 1-NH ₂ [I] (wherein Ser is L-serine, Val is L-valine, G
lu is L-glutamic acid, Ile is L-isoleucine,
Gln is L-glutamine, Leu is L-leucine, NI
e is L-norleucine, His is L-histidine, As
n is L-asparagine, Gly is glycine, Lys is L
-Lysine, Arg is L-arginine, Trp is L-tryptophan, Asp is L-aspartic acid, Tyr is L
-Indicating tyrosine], the 34th T of the peptide represented by
It is a radioactive substance labeled with ¹²⁵ I having 1-2 yrs (hereinafter, may be simply referred to as ¹²⁵ I-labeled radioactive substance).

[0002]

2. Description of the Prior Art h-PTH is a peptide hormone consisting of 84 amino acids and its biological activity is h-PTH (1-
34) [Proc. Nat. A
cad. Sci. U. S. A. , 68 , 63-67)
(1971)]. However, h-PTH is unstable due to the presence of L-methionine (Met), and ¹²⁵ I
When labeled with, the hormone activity is lost [Recent
Prog. Hornone Res. , 18 , 269 ~
295 (1962)].

[0003]

In the conventional method for quantifying h-PTH, a specific antibody using h-PTH (1-34) as an antigen was prepared in order to quantify only a portion having h-PTH activity. It became so. However, h-PTH
(1-34) is unstable due to the presence of L-methionine (Met), and when labeled with ¹²⁵ I, Met at the 8th and 18th positions is oxidized and hormone activity is inactivated. There was a flaw.

Therefore, an h-PTH derivative having a PTH activity and an immunological activity against an antibody of PTH, and having a stable hormone activity even when labeled with ¹²⁵ I and having a stable radioactivity. As a result, Met at the 8th and 18th positions was changed to L-norleucine, and L-phenylalanine at the 34th position was changed to L-tyrosine [Nle ⁸ , Nle.
¹⁸ , Tyr ³⁴ ] -h-PTH (1-34) was found [JP-A-55-113753].

However, this [N1e ⁸ , Nl
e ¹⁸ , Tyr ³⁴ ] -h-PTH (1-34) does not inactivate even when labeled with ¹²⁵ I because Met does not exist in the molecule, but its hormone activity is at most natural h-PTH.
It was only as active as (1-34).

[0006]

The object compound [I] is P
Known h-PTH (1-34) for TH receptor
And [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ] -h-PTH
It has a stronger affinity than (1-34), has not only about 1.5 to 2 times the activity of h-PTH, but also has an immunological activity against the antibody of PTH and is labeled with ¹²⁵ I. However, the hormonal activity did not decrease, and [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ]-
It has about twice as much radioactivity as h-PTH (1-34), and its biological activity does not decrease even after long-term storage. Therefore, known h-PTH (1-34), [Nle ⁸ , Nl
e ¹⁸ , Tyr ³⁴ ] -h-PTH (1-34) exhibits a significantly superior effect. Therefore, the target compound [I]
Is a peptide that is extremely useful as a labeling compound for parathyroid function tests.

The peptide [I] of the present invention converts the carboxyl group of the C-terminal tyrosyl group into an amide group to give individual protected amino acids and / or protected lower amino acids in the amino acid sequence represented by the formula [I]. It can be obtained by condensing a peptide by a liquid phase synthesis method and removing the protecting group of the amino group at the N-terminal and the protecting group of the functional group of the side chain by acidolysis at the final stage of the condensation reaction. The condensation reaction itself is carried out by repeating the attachment / detachment of a protecting group and the condensation reaction according to a conventional method for peptide synthesis. That is, the various protective groups used in the production of the starting material of the peptide [I] and all the intermediates are known in peptide synthesis, and therefore can be easily prepared by known means such as hydrolysis, acid hydrolysis, reduction, aminolysis or hydrazinolysis. A protecting group that can be eliminated is used. Such protecting groups are described in literatures and reference books in the field of synthetic peptide chemistry.

For example, as a protecting group used for an amino group, an acyl group such as a formyl group, a trifluoroacetyl group, a phthaloyl group, a p-toluenesulfonyl group, an o-nitrophenylsulfenyl group, a benzyloxycarbonyl group, benzyloxycarbonyl group such as o (or p) -bromobenzyloxycarbonyl group, o (or p) -chlorobenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, trichloroethyloxycarbonyl group Group, t-butyloxycarbonyl group, t-amyloxycarbonyl group, aliphatic oxycarbonyl group such as diisopropylmethyloxycarbonyl group, 2-phenyl-isopropoxycarbonyl group, 2-tolyl-isopropoxycarbonyl group, 2 p- diphenyl - and the like aralkyloxycarbonyl groups such as isopropoxycarbonyl group. In addition, these amino groups are benzoylacetone,
It can be protected by the formation of the enamine obtained by reacting with a 1,3-diketone such as acetylacetone.

Carboxyl groups are protected by amide formation, hydratide formation or esterification. That is, the amide group is 3,4-dimethoxybenzyl group, bis- (p-
Methoxyphenyl) methyl group and the like.
The hydratide group is benzyloxycarbonyl group, trichloroethyloxycarbonyl group, trifluoroacetyl group, t-butyloxycarbonyl group, trityl group, 2-
It is substituted with a p-diphenyl-isopropoxycarbonyl group and the like. Ester groups include alkanols such as methanol, ethanol, t-butanol and cyanomethyl alcohol, benzyl alcohol, p-bromobenzyl alcohol, p-chlorobenzyl alcohol, 2,6-
Aralkanols such as dichlorobenzyl alcohol, p-methoxybenzyl alcohol, p-nitrobenzyl alcohol, benzhydryl alcohol, benzoylmethyl alcohol, p-bromobenzoylmethyl alcohol and p-chlorobenzoylmethyl alcohol, 2,4,6
-Substituted by trichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol, phenol such as 2,4-dinitrophenol, thiofenool, thiofenool such as p-nitrothiofenool, and the like.

The hydroxyl groups of serine and tyrosine can be protected by, for example, esterification or etherification. Suitable groups for this esterification include, for example, acetyl group, benzoyl group, benzyloxycarbonyl group, ethyloxycarbonyl group and the like. Examples of the group suitable for etherification include benzyl group and 2,6-
A dichlorobenzyl group, a tetrahydropyranyl group and a t-butyl group. To protect these hydroxyl groups 2,2,2-
A trifluoro-1-t-butyloxycarbonylaminoethyl group and a 2,2,2-trifluoro-1-benzyloxycarbonylamino group are also suitable. However, it is not always necessary to protect these hydroxyl groups.

Examples of the group used to protect the amino group in the guanidine group of arginine include a nitro group, a tosyl group, a benzyloxycarbonyl group and a mesitylene-2-sulfonyl group. It does not necessarily have to be protected. Examples of the group used for protecting the imino group of histidine include a benzyl group, a trityl group, a benzyloxycarbonyl group, a tosyl group, and a 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl group. , 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group and the like, but it is not always necessary to protect this imino group.

In the present invention, a t-butyloxycarbonyl group and a t-amyloxycarbonyl group are used to protect the α-amino group, and an o-chloro group is used to protect the side chain amino group, that is, the ε-amino group of lysine. A benzyloxycarbonyl group is used, a benzyl ester group, an ethyl ester group, and a phenacyl ester group are used to protect the α-carboxyl group.
Side chain carboxyl group, that is, glutamic acid, a benzyl ester group is used to protect the side chain carboxysyl group of aspartic acid, and a benzyl group is used to protect the hydroxyl group of serine.
It is preferable to use a 2,6-dichlorobenzyl group for protecting the hydroxyl group of tyrosine and a tosyl group or a mesitylene-2-sulfonyl group for protecting the amino group in the guanidine group of arginine.

In the synthesis of the object compound [I], the condensation of individual amino acids and / or lower peptides is
For example, reacting an amino acid or peptide having a protected α-amino group and an activated terminal carboxyl group with an amino acid or peptide having a free α-amino group and a protected terminal carboxyl group, or activating an activated α-amino group. It can be carried out by reacting an amino acid or peptide having a group and a protected terminal carboxyl group with an amino acid or peptide having a free terminal carboxyl group and a protected α-amino group.

In this case, the carboxyl group is, for example, an acid azide, an acid anhydride, an acid imidazolide or an active ester,
For example, cyanomethyl ester, thiophenyl ester,
p-nitrothiophenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 2,
Activation by converting into 4,5-trichlorophenyl ester, 2,4,6-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, N-hydroxyphthalic acid imide ester, etc. You can Also carbodiimide,
For example, N, N'-dicyclohexyl-carbodiimide,
N-Ethyl-N'-3-dimethylaminopropyl-carbodiimide, N, N'-carbonyl-diimidazole or isooxolium salts, eg activated by reacting with a condensing agent such as Woodward Reactant. it can.

Preferred condensation methods in the present invention are azide method, active ester method and carbodiimide method.
At each stage of the condensation, it is desirable to use a method in which racemization does not occur or a method in which racemization is minimized, and the azide method, active ester method, Bünsch method [Z. Na
Tursch. , 21b, 426 (1966)].
Alternatively, the Geiger method [Chem Ber. , 103, 78
8 (1970)] Especially as a condensing agent, N-ethyl-
It is suitable to use a modified method or the like using N′-3-dimethylaminopropyl-carbodiimide (WSC).

The condensation sequence may be any sequence as long as it is the amino acid sequence represented by the formula [I], but it is preferable to connect amino acids and / or peptides sequentially from the C-terminal side.

For example, a C-terminal fragment consisting of the 29th to 34th amino acid sequence and a peptide fragment consisting of the 23rd to 28th amino acid may be condensed.
This C-terminal fragment and hexapeptide 23-28
In order to condense, it is suitable to carry out by the Geiger modified method using WSC. The resulting C-terminal fragment 23-
A peptide fragment consisting of the amino acid sequence of the 18th to 22nd amino acids is ligated before 34, and it is suitable to use the Geiger modified method using WSC. The obtained C
A peptide fragment consisting of the sequence of amino acids 13 to 17 before the terminal fragment 18-34, 8
It is preferable to connect a peptide fragment consisting of the amino acid sequence of -12th and a peptide fragment consisting of the amino acid sequence of 1-7.

The protecting group for the α-amino group in the above condensation reaction, such as t-butyloxycarbonyl group and t-amyloxycarbonyl group, is eliminated with trifluoroacetic acid. The protecting group for the α-carboxyl group, such as ethyl ester, is decomposed with dilute sodium hydroxide solution or converted into a protective hydratide such as hydratide or trichloroethoxycarbonylhydratide, and the phenacyl ester group is Zn powder in acetic acid. When decomposed, the benzyl ester group can be decomposed by anhydrous hydrogen fluoride decomposition, hydrogenolysis, or converted to hydratide.

The N-terminal α-amino group thus protected,
A tetratriacontapeptide having an ε-amino group, a side chain carboxyl group, a guanidino group and / or a hydroxyl group can be obtained. These protecting groups are preferably acid-decomposed,
The target compound of the formula [I] is obtained by elimination in one step by a method using anhydrous hydrogen fluoride or trifluoromethanesulfonic acid.

The peptide [I] thus obtained
Can be separated and purified by known means for purifying peptides or proteins. For example, Sephadex G
-25, Sephadex G-50, Sephadex LH
It can be carried out by gel filtration using a gel filtration agent such as -20, column chromatography using carboxymethyl cellulose, ion exchange resin or the like, high performance liquid chromatography and the like.

The peptide [I] of the present invention can be obtained in the form of a base or a salt thereof depending on the conditions of the method. The salt is an inorganic acid salt, a salt with an organic acid such as formic acid, acetic acid, propionic acid, glycolic acid, succinic acid, malic acid, tartaric acid, and citric acid.

Further, the ¹²⁵ I-labeled radioactive substance of the peptide [I] of the present invention has, for example, a certain amount of radioactivity.
^The above peptide [I] and chloramine T were added to ¹²⁵ I-containing phosphate buffer (pH 7.1) and stirred, and then sodium bisulfite was added, and then a small amount of potassium iodide and serum albumin were added to perform chromatography. performed by collecting fractions labeled with ¹²⁵ I, ¹²⁵
A radioactive substance labeled with I is obtained.

The ¹²⁵ I-labeled radioactive substance can be purified by a purification means such as high performance liquid chromatography (HPLC). The obtained purified product is a labeled radioactive product in which hydrogen in the benzene ring of the side chain of the 34th Tyr is mono- or di-substituted with ¹²⁵ I. When the ^125- labeled radioactive substance of the present invention is used as a labeled compound for a parathyroid function test, the ¹²⁵ I mono-substituted product and the di-substituted product described above may be used separately. You may use it without separating.

Next, the parathyroid hormone (PTH) activity of the ¹²⁵ I-labeled radioactive substance of the peptide [I] of the present invention will be described. <Preparation of ¹²⁵ I-labeled product> Radioactivity of 2 mCi
0.5 M phosphate buffer containing ¹²⁵ I-NaI (pH
7.1) 50 μl of h-PTH (1-34), h-
PTH (1-34) NH ₂ , [Nle ⁸ , Nle ¹⁸ , T
yr ^34] -h-PTH (1-34) and [Nle ^8,
Nle ¹⁸ , Tyr ³⁴ ] -h-PTH (1-34) NH ₂
10 μl of a solution containing 2 μg of chloramine T and chloramine T (3.5 mg
/ Ml) containing solution (20 μl) and stirring for 30 seconds, and then adding sodium bisulfite (4.5 mg / ml) containing solution (50 μl)
The reaction was stopped by adding l. After adding 0.5 ml of a 0.1 N acetic acid solution containing 5% human serum albumin to this, the column was charged on a Sephadex G-10 column (1 x 50 cm), eluted with the acetic acid solution and labeled with ¹²⁵ I. The contained fraction of the test product was obtained.

<Method for measuring PTH activity> Preparation of PTH receptor Male SD rats (body weight: 200 to 250 g) were decapitated and exsanguinated, and after laparotomy, the kidney was removed and the surface film was removed.
Cut off the renal cortex and cool with ice. The following operations are performed at the lowest temperature (0 to 4 ° C). The above renal cortex part was added with 0.25 M sucrose and 1 mM EDTA.
The kidney cortex was dipped in mM Tris-hydrochloride buffer (pH 7.5) (hereinafter referred to as "A solution") with a glass mantle tube using Teflon pestle, and the amount of A was 3 times its wet weight (g) (ml). Add liquid and homogenize. 150X this homogenate
g, centrifuge for 10 minutes, and then add the supernatant to 2200X
g, centrifuge for 15 minutes. The supernatant is discarded, and the turbid color portion of the upper layer of the precipitate is suspended in solution A.
It is washed by centrifugation at Xg for 15 minutes, suspended again, dispensed in a container, frozen at -70 ° C and stored at -20 ° C.

Reaction of PTH and PTH receptor ATPMg 2mM, MgCl ₂ 10mM, KCl 60m was added to the test sample so that the concentrations were 2 μg / ml and 10 μg / ml.
M, GTP 20 μM, isobutylmethylxanthine 1 m
M, creatine phosphate 8 mM and bovine serum albumin (BSA) 0.2% in 100 mM Tris-hydrochloride buffer (pH 7.5) (hereinafter referred to as solution B), which was dissolved in standard bovine PTH (1-84) Do about.

50 μl of each of these 4 solutions is dispensed into a glass test tube, and 8 tubes each are prepared. Keep the sample in ice water to prevent decomposition of other substances such as ATP. Thaw the PTH receptor preparation stored at -20 ° C at room temperature, add creatine kinase previously dissolved in solution A, and further add creatine kinase 0.1 mg / ml in solution A and PTH receptor preparation protein amount 1.4 mg. / Ml, so that
Keep on ice. After the dispensed sample solution was placed in a constant temperature bath at 37 ° C. for several minutes, 50 μl of the PTH receptor-creatine kinase solution was added at 10 ° C. to 10 ° C.
Incubate for minutes. Then, 100 μl of 0.1 M acetate buffer (pH 4.0) is added and immediately put into ice water, and then the test tube is immediately heated with boiling water for 1 minute to stop the reaction.

Measurement of C-AMP Produced The above reaction-stopped sample was diluted 10 to 30 times with distilled water,
Deproteinization is performed by centrifugation at 2000XG for 15 minutes. The amount of c-AMP in the supernatant is measured with a RIA kit (manufactured by Yamasa Shoyu Co., Ltd.).

Measurement of PTH titer The measured value of C-AMP is PM / mg PTH receptor protein /
It is converted into the unit of minutes, and this is used as the value of the reaction, and the value obtained by the standard product is tested by the parallel line test 2 × 2 point method.

The results of PTH activity (U / mg) are shown in Table 1.

[Table 1]

The abbreviations used in this specification have the following meanings. Ser; L-serine Val; L-valine Glu; L-glutamic acid Ile; L-isoleucine Gln; L-glutamine Leu; L-leucine Nle; L-norleucine His; L-histidine Asn; L-asparagine Gly; glycine Lys; L-lysine Arg; L-arginine Trp; L-tryptophan Asp; L-aspartic acid Tyr; L-tyrosine Boc; t-butyloxycarbonyl Aoc; t-amyloxycarbonyl Z-Cl; o-chlorobenzyloxycarbonyl Bzl; benzyl Bzl-Cl _2; 2,6- dichlorobenzyl Tos; tosyl OEt; ethyl ester OBzl; benzyl ester ONP; p-nitrophenyl ester OPAC; Hue None ester TFA; trifluoroacetic acid TosO ; P-toluenesulfonic acid Et ₃ N; Triethylamine NMM; N-methylmorpholine TBA; t-butylamine DCHA; dicyclohexylamine NaOH; sodium hydroxide THF; tetrahydrofuran DMF; dimethylformamide DMSO; dimethyl sulfoxide ether diethyl ether DCC; N, N′-dicyclohexylcarbodiimide WSC; N-ethyl, N′-3-dimethylaminopropyl-carbodiimide HOBt; 1-hydroxybenzotriazole PF (); PF means a protected amino acid or peptide fragment, and the number in parentheses Represents the order of amino acids of formula [I].

[0032]

Reference Example Next, a production example of the present invention will be specifically described with reference to Reference Examples and Examples. The conditions for the carrier and developing solvent system of thin layer chromatography (TLC) used in the examples and the analysis of amino acids are as follows. <TLC>carrier; silica gel G developing solvent system; Chloroform-methanol-acetic acid (95: 5:
3), 2. Chloroform-methanol-acetic acid (85:15:
5), 3. Chloroform-methanol-acetic acid (80:25:
2), 4. Chloroform-ethanol-ethyl acetate (5: 2:
5), 5. Hexane-ethyl acetate (1: 1) Carrier: Cellulose (manufactured by Merck, DC-Alufoli
en) Developing solvent system; Butanol-pyridine-acetic acid-water (5: 3: 0.
1:11) Upper layer <Amino acid analysis> Unless otherwise specified, the sample was 1N with 6N hydrochloric acid.
Hydrolysis was performed in a sealed tube at 10 ° C. for 24 to 48 hours.

Reference Example 1 [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ] -h-PTH (1-
34) Preparation of _{NH 2 1) PF (34)} ; Boc-Tyr (Bzl-Cl 2)
-NH ₂ (1) _{Boc-Tyr (Bzl-Cl 2} ) -OH52.84g
(0.12M) and 16.69 g of p-nitrophenol
(0.12M) was dissolved in dry THF, and 24.76g (0.12M) dry THF of DCC was added thereto while cooling at -5 ° C.
After the solution was added dropwise, the mixture was stirred overnight. After the reaction, the precipitate was filtered off, the filtrate was saturated with NH ₃ gas, and the mixture was stirred for 5 hours. Although a precipitate was formed, DMF was added and dissolved, and then concentrated under reduced pressure. The residue was crystallized from ether, collected by filtration and dried to obtain the target product [1]. Yield: 44.77 g (yield 84.9%), melting point: 214
˜216 ° C. TLC; Rf ₁ = 0.62 [Α] ²⁵ D-5.54 ° (C = 1, DMF)

2) PF (33-34); Boc-Asn
Dissolved -Tyr the (Bzl-Cl ₂₎ -NH ₂ (2) Compound (1) 26.36g (60mM) in methylene chloride, after adding ice-cooling TFA100ml thereto, followed by stirring at room temperature for 30 minutes. After the reaction, methylene chloride and TFA
Was distilled off under reduced pressure, and the residue was crystallized with ether, collected by filtration and dried. Obtained crystals, Boc-Asn-OH1
3.93 g (60 mM) and HOBt 8.1 g (60
(mM) in DMF and WS at -15 ° C under cooling
After adding 10.98 ml (60 mM) of C, the mixture was stirred overnight. After the reaction, the precipitate was collected by filtration, washed with 5% aqueous sodium hydrogen carbonate (once), water (twice), and methanol (once) in that order, and dried to obtain crystals of the target product [2]. DMF was distilled off from the mother liquor under reduced pressure, and the obtained crystals were washed with water and methanol in this order and dried to obtain a compound [2], which was combined with the target compound [2]. Yield: 28.64 g (86.25% yield), melting point: 24
0-242 ° C [α] ²⁵ D-24.06 ° (C = 1, DMF)

3) PF (32-34); Boc-His
(Tos) -Asn-Tyr (Bzl -Cl 2) -NH
₂ [3] 22.14 g (40 mM) of the compound [2] was dissolved in a small amount of methylene chloride, and 100 ml of TFA was added thereto under ice cooling,
Stir for 30 minutes at room temperature. TFA was distilled off under reduced pressure to remove B.
oc compound was obtained. On the other hand, Boc-His (Tos) -O
28.36 g (48 mM) of H-DCHA was added to ethyl acetate 5
It was suspended in 00 ml, washed twice with 1N sulfuric acid and twice with water, dried over anhydrous sodium sulfate, and ethyl acetate was distilled off under reduced pressure. Dissolve the residue in dry DMF and add to it the de-Boc
DMF solution of compound and HOBt 6.48 g (48
mM), then WSC 8.78 under cooling at -15 ° C.
After adding ml (48 mM), the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was washed once with 5% aqueous sodium hydrogen carbonate,
It was washed with water twice in this order and then dried to obtain a crude product.
This was crystallized from methanol-ether to obtain the desired product [3]. The crystal mother liquor was concentrated under reduced pressure, and the residue was crystallized from methanol-hexane to obtain the compound [3], which was combined with the target compound [3]. Yield: 28.81 g (yield 85.1%), melting point: 170
˜175 ° C. TLC; Rf ₃ = 0.68, 0.42 Some Tos desorbed was obtained.

4) PF (31-34); Boc-SEQ ID NO: 2-NH ₂ [4] 28.81 g (34.06 mM) of compound [3] was dissolved in a small amount of methylene chloride, and 120 ml of TFA was added thereto while cooling with ice. Was added and the mixture was stirred at room temperature for 30 minutes, and then TFA was distilled off under reduced pressure. Ether was added to the residue, and the precipitated crystals were collected by filtration, dried and then dissolved in 140 ml of DMF. The solution was neutralized with NMM, and Boc-Val-OH (8.14 g, 3
7.47 mM) and HOBt 5.06 g (37.47)
(mM) was added to a solution of 60 ml of dry DMF, and then 6.86 ml (37.47 m) of WSC under cooling at -15 ° C.
M) was added and the mixture was stirred overnight at room temperature. After the reaction, DM under reduced pressure
F was distilled off, and the residue was washed once with a 5% aqueous sodium hydrogen carbonate solution and three times with water, and dried to obtain the target product [4]. Yield: 27.76 g (103.2% yield), melting point: 16
4 to 166 ° C. TLC; Rf ₃ = 0.65 [α] ²⁵ D−28.38 ° (C = 1, DMF)

5) PF (30-34); Boc-SEQ ID NO: 3-NH ₂ [5] Compound [4] 27.76 g (35.15 mM) was suspended in a small amount of methylene chloride, and this was cooled on ice. After adding 110 ml of TFA and stirring at room temperature for 30 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and the precipitated crystals were collected by filtration, dried and then dissolved in 120 ml of DMF. NMM1 in this solution
0 ml was added to neutralize, and Boc-Asp (OBz
1) -OH 12.5 g (38.67 mM) and HOB
t5.22 g (38.67 mM) was added to dry DMF 80 ml.
Was added to the solution, then cooled at -15 ° C under WSC.
After adding 7.08 ml (38.67 mM), the mixture was stirred overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure, and the residue was 5%.
After washing once with aqueous sodium hydrogen carbonate and twice with water, the product was suspended in methanol and ether was added for recrystallization to obtain the desired product [5]. Yield: 31.42 g (yield 89.8%), melting point: 214
˜215 ° C. TLC; Rf ₃ = 0.6 [α] ²⁵ D-23.28 ° (C = 1, DMF)

6) PF (29-34); Boc-SEQ ID NO: 4-NH ₂ [6] 31.11 g (31.27 mM) of compound [5] compound [5] was suspended in methylene chloride, and 120 ml of TFA was added thereto while cooling with ice. In addition,
After stirring at room temperature for 30 minutes, TFA and methylene chloride were distilled off under reduced pressure. Ether was added to the residue, and the precipitated crystals were collected by filtration, dried and then dissolved in 100 ml of dry DMF. This solution was neutralized with 8 ml of NMM, and Boc-Gln-ON was added to it.
P12.64 g (34.4 mM) and HOBt0.4
A solution prepared by dissolving 2 g (3.13 mM) in 100 ml of dry DMF was added, and then 3.78 ml of NMM was added under ice cooling, followed by stirring overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was washed once with a 5% aqueous sodium hydrogen carbonate solution and twice with water, then suspended in methanol and recrystallized with ether to obtain the target compound [6]. Yield: 33.19 g (yield 94.5%), melting point: 81-
83 ° C. TLC; Rf ₃ = 0.47 [α] ²⁴ D-23.98 ° (C = 1, DMF) Amino acid analysis; Asp2.19 (2), Glu1.05
(1), Val1 (1), Tyr0.73 (1), Hi
s0.85 (1)

7) PF (27-28); Boc-Lys
(Z-Cl) -Leu-OEt [7] Boc-Lys (Z-Cl) -OH.TBA 97.6 g
(0.2 M) was suspended in 500 ml of ethyl acetate, and this was added to 1
The mixture was washed with N hydrochloric acid and water in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give an oil. This was dissolved in 500 ml of dry THF, and 39.14 g of H-Leu-OEt.HCl was added thereto.
(0.2 M) and 27.0 g (0.2 M) HOBt were added, then 36.6 ml (0.
2M) was added, and the mixture was stirred overnight at room temperature. After the reaction, THF was distilled off under reduced pressure. The residue was dissolved in 600 ml of ethyl acetate, washed with 5% aqueous sodium hydrogen carbonate, water, 1N hydrochloric acid and water in this order, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was left to crystallize in the cold. Hexane was added and the product was collected by filtration to obtain the desired product [7]. Yield: 110.62 g (yield 99.5%), melting point: 77
~80 ℃ TLC; Rf ₃ = 0.48 [α] ^{24 D-19.08 ° (C =} 1, DMF)

8) PF (26-28); Boc-Lys
(Z-Cl) -Lys (Z-Cl) -Leu-OEt
[8] The compound [7] 110.62 g (0.199 M) was added to 50 ml of methylene chloride, 250 ml of TFA was added thereto under ice cooling, and the mixture was stirred at room temperature for 1 hour. After the reaction, TF under reduced pressure
A and methylene chloride were distilled off to obtain an oily de-Boc product.

On the other hand, Boc-Lys (Z-Cl) -OH
-TBA 97.1 g (0.199 M) was added to ethyl acetate 50
The product was suspended in 0 ml, washed with 300 ml of 1N hydrochloric acid and water in this order, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give an oil. This was dissolved in 150 ml of dry THF, and 26.9 g (0.199M) of the above-mentioned de-Boc product and HOBt was dried in TH.
After adding a solution dissolved in 250 ml of F, 36.4 ml (0.199M) of WSC was added dropwise while cooling to -15 ° C.
Stir overnight at room temperature. After the reaction, THF was distilled off under reduced pressure to precipitate agar crystals. Dissolve this in ethyl acetate, 5
% Sodium bicarbonate water, water, 1N hydrochloric acid, water in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting precipitate was treated with hexane and collected by filtration. This was recrystallized from ethyl acetate, ether and hexane to obtain the desired product [8]. Yield: 156.52 g (yield 92.2%), melting point: 11
4 to 116 ° C. TLC; Rf ₂ = 0.78 [α] ²⁹ D-20.72 ° (C = 1, DMF)

9) PF (25-28); Aoc-SEQ ID NO: 5-OEt

[9] 156.5 g (184 mM) of the compound [8] was added to 50 ml of methylene chloride, 250 ml of TFA was added thereto under ice cooling, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, the residue was dissolved in 300 ml of dry DMF and then neutralized with NMM, and 86.0 g of Aoc-Arg (Tos) -OH was added thereto.
A solution prepared by dissolving (202 mM) in 100 ml of dry DMF and 27.3 g (202 mM) of HOBt were added, and then 37.0 ml (202 mM) of WSC was added dropwise under cooling to -15 ° C, followed by stirring at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 1 l of ethyl acetate. This solution was washed twice with a 5% aqueous sodium hydrogen carbonate solution, twice with a saturated saline solution and twice with a 1N hydrochloric acid solution, and then saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Ether is added to the residue and filtered to obtain the desired product.

[9] was obtained. Yield: 217.91 g (100.6% yield), melting point; 7
5 to 78 ° C. TLC; Rf ₁ = 0.09, Rf ₂ = 0.67 [α] ²⁸ D-14.02 ° (C = 1, DMF)

10) PF (24-28); Boc-SEQ ID NO: 6-OEt [10] compound

[9] To 217.9 g (0.185 M), add 100 ml of methylene chloride and 250 ml of TFA, and add 80 at room temperature.
After stirring for 1 minute, methylene chloride and TFA were distilled off under reduced pressure. The obtained oily substance was dissolved in 300 ml of dry DMF, and NMM was added for neutralization. Boc-Le in this solution
u-OH.H ₂ O 50.9 g (0.204 M) and H
27.6 g (0.204 M) of OBt was added to dry DMF100.
Add the solution dissolved in ml, then cool at -15 ° C WS
C37.3 ml (0.204M) was added dropwise, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was added to water, and the precipitated product was collected by filtration. Recrystallization from methanol-ether-hexane twice gave the target product [10]. Yield: 213.63 g (90.5% yield), melting point: 15
7 to 160 ° C. TLC; Rf ₁ = 0.28, Rf ₂ = 0.77 [α] ²⁷ D-18.68 ° (C = 1, DMF)

11) PF (23-28); Boc-SEQ ID NO: 7-OEt [11] To 153.17 g (0.12 M) of compound [10], 100 ml of methylene chloride and 250 ml of TFA were added, and the mixture was allowed to stand at room temperature for 8 hours.
After stirring for 0 minutes, methylene chloride and TFA were distilled off under reduced pressure. The residue is dissolved in 250 ml of dry DMF and NMM is added.
Neutralized to pH. HOBt 17.84g in this solution
(0.132M) and Boc-Trp-OH 40.17g
(0.132M), then cooled to -15 ° C under W
After adding 24.2 ml (0.132M) of SC dropwise, the mixture was stirred overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure, the residue was poured into 5 L of 5% aqueous sodium hydrogen carbonate, and the precipitated product was collected by filtration. This was suspended in water, collected by filtration, and recrystallized twice from methanol-ether to obtain the desired product [11]. Yield: 142.57 g (81.2% yield), melting point: 16
8 to 170 ° C. TLC; Rf ₁ = 0.31, Rf ₂ = 0.82 [α] ²⁸ D-18.64 ° (C = 1, DMF)

12) PF (23-28); Boc-SEQ ID NO: 7-OH [12] Compound [11] 140.64 g (96.16 mM) was dissolved in 1200 ml of hot ethanol, and after cooling, a small amount of precipitate Was filtered off, 288 ml of 1N-NaOH aqueous solution (3 times M) was added, and the mixture was stirred at room temperature for 1 hour. 1N-T in the reaction solution
After adding 192 ml of an osOH aqueous solution (2 times M), the mixture was filtered and ethanol was distilled off under reduced pressure. 1N-Tos for concentrated liquid
After adding 96 ml of OH (equal to M) and then 2 l of water,
The resulting precipitate was collected by filtration. After washing twice with water, it was dried to obtain the desired product [12]. Yield: 142.98 g (yield 101.1%) Melting point: 125 to 130 ° C. TLC; Rf ₂ = 0.71 [α] ²⁷ D-37.24 ° (C = 1, DMF) Amino acid analysis; Leu2 (2), Lys2.08
(2), Arg1.10 (1), Trp0.83 (1)

13) PF (23-34); Boc-SEQ ID NO: 8-NH ₂ [13] 1.68 g (1.5 mM) of the compound [6] was suspended in a small amount of methylene chloride, and then 7 ml of TFA under ice cooling. After adding
Stir for 30 minutes at room temperature. After the reaction, TFA was distilled off under reduced pressure, ether was added to the residue, and the precipitated crystals were collected by filtration and dried. The crystals were dissolved in 30 ml of dry DMF and a small amount of N was added.
Neutralized with MM. 2.43 g of compound [12] in this solution
(1.65 mM), HOBt 0.22 g (1.65 m)
M) and 20 ml of dry DMF were added, and then 0.3 ml of WSC (1.1 times M) was added under cooling at -15 ° C, followed by stirring at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was washed once with a 5% aqueous sodium hydrogen carbonate solution and twice with water, suspended in methanol, added with ether, filtered and dried to obtain the desired product [1
3] was obtained. Yield: 3.62 g (99.1% yield), melting point: 260-
270 ° C. [α] ²⁵ D-4.66 ° (C = 0.3, DMF) amino acid analysis; Asp1.94 (2), Glu0.96
(1), Val0.71 (1), Leu2.00
(2), Tyr0.98 (1), Lys2.09
(2), His0.58 (1), Arg0.91
(1), Trp0.78 (1)

14) PF (22); Boc-Glu (O
Bzl) -OPAC [14] Boc-Glu (OBzl) -OH 128.2 g (0.
38M) was dissolved in 600 ml of DMF, 113.5 g (0.57M) of phenacyl bromide was added thereto under ice cooling, and then 79.3 ml (0.57M) of Et ₃ N was added dropwise. After dropping, the mixture was stirred at 30 ° C. for 4 hours, and then potassium acetate 30
After adding g, and stirring for 45 minutes, DMF was distilled off under reduced pressure. Add 600 ml of ethyl acetate to the residue and add 2 with 5% aqueous sodium hydrogen carbonate.
After washing twice with water and twice with water, the ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to precipitate crystals. Hexane was added to this and the product was collected by filtration to obtain the desired product [14]. Yield: 156.19 g (yield 90.2%) TLC; Rf ₅ = 0.73

15) PF (21-22); Boc-Va
l-Glu (OBzl) -OPAC [15] To 147.88 g (0.325M) of the compound [14], 50 ml of methylene chloride was added, 300 ml of TFA was added thereto under ice cooling, and the mixture was stirred at room temperature for 1 hour and then under reduced pressure. Methylene chloride and TFA were distilled off. Ether was added to the residue, and the precipitated crystals were collected by filtration and dried. This crystal was dried with DMF30.
It was dissolved in 0 ml and neutralized to pH 7 with NMM. 35.14 g (0.26 M) of HOBt and Boc-V were added to this solution.
56.49 g (0.26M) of al-OH was added, and -15
After adding 47.6 ml (0.26M) of WSC under cooling at ℃, the mixture was stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in 500 ml of chloroform, and washed with 5% aqueous sodium hydrogen carbonate, water, 1N hydrochloric acid and water in this order. The chloroform layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, hexane was added to the obtained crystals, and the crystals were collected by filtration and then recrystallized from ethyl acetate-ether to obtain the desired product [15]. Yield: 106.97 g (74.2% yield), melting point: 13
9 to 141 ° C. TLC; Rf ₃ = 0.63 [α] ²⁹ D-18.92 ° (C = 1, DMF)

16) PF (20-22); Aoc-Ar
g (Tos) -Val-Glu (OBzl) -OPAC
[16] To 99.83 g (0.18 M) of the compound [15], 50 ml of methylene chloride was added, 200 ml of TFA was added thereto under ice cooling, and the mixture was stirred at room temperature for 1 hour, and then methylene chloride and TFA were distilled off under reduced pressure. . Hexane was added to the residue for treatment, the hexane was removed by a gradient method, ether was added for treatment, and then ether was distilled off under reduced pressure. The oil obtained was dissolved in 200 ml of dry DMF and neutralized with NMM. 24.33 g (0.18 g) of HOBt was added to this solution.
M), Aoc-Arg (Tos) -OH 76.60g
(0.18M) and 200 ml of dry DMF were added, and 32.94 ml (0.18M) of WSC under cooling at -15 ° C.
Was added dropwise, and the mixture was stirred overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 1 l of ethyl acetate. This solution was washed with 5% aqueous sodium hydrogen carbonate, water, 1N hydrochloric acid, and water in this order,
After drying over anhydrous sodium sulfate, ethyl acetate was distilled off under reduced pressure. The obtained oily substance was crystallized from ethyl acetate-ether, and the obtained crystals were suspended in ether and collected by filtration three times to obtain the desired product [16]. Yield: 149.75 g (94.6% yield), melting point: 11
0 to 114 ° C. TLC; Rf ₁ = 0.74, Rf ₄ = 0.81 [α] ²⁹ D-11.5 ° (C = 1, DMF)

17) PF (19-22); Boc-SEQ ID NO: 9-OPAC [17] Compound [16] 149.40 g (0.17 M) was added with 50 ml of methylene chloride, and 300 ml of TFA was added thereto under ice cooling. After stirring at room temperature for 1 hour, methylene chloride and TFA were distilled off under reduced pressure. Ether was added to the residue for treatment, the ether was distilled off under reduced pressure, and the obtained oily substance was dissolved in 200 ml of dry DMF. To this, HOBt25.
27g (0.187M) and Boc-Glu (OBz
l) -OH 63.09 g (0.187 M) was added, dry DMF 100 ml was added, and WSC3 under cooling at -15 ° C.
4.22 ml (0.187M) was added, and the mixture was stirred at room temperature overnight. After the reaction, the solvent was distilled off, the residue was poured into 6 l of water,
The precipitated crystals were collected by filtration. Methanol-ether was added to the crystals to suspend, and the crystals were collected by filtration, dissolved in hot methanol, precipitated when cold, collected by filtration, further suspended in methanol, and collected by filtration. Got
The solvent was distilled off from the crystal mother liquor, and the product was crystallized from methanol-ether to obtain 25.02 g of the desired product. Yield: 141.44 g (76.7% yield), melting point: 11
9 to 121 ° C. TLC; Rf ₁ = 0.56, Rf ₄ = 0.82 [α] ²⁹ D-12.9 ° (C = 1, DMF)

18) PF (18-22); Boc-SEQ ID NO: 10-OPAC [18] To 6.51 g (6 mM) of compound [17], methylene chloride and 24 ml of TFA were added under ice cooling, and the mixture was stirred at room temperature for 40 minutes. After that, methylene chloride and TFA were distilled off under reduced pressure. Ether was added to the residue to crystallize and dry. The crystals were dissolved in dry DMF and neutralized to pH 7 with NMM under ice cooling. 1.67 g of Boc-Nle-OH (7.
2mM) and 0.97g (7.2mM) of HOBt were dissolved in 40ml of dry DMF, 1.3ml (7.2mM) of WSC was added under cooling to -15 ° C, and then stirred overnight. After the reaction, DMF was distilled off under reduced pressure, water was added to the residue, the resulting precipitate was collected by filtration, and 5% aqueous sodium hydrogen carbonate solution, water (3 times),
The mixture was washed with 1N hydrochloric acid water (three times) and methanol in that order. Then, reprecipitation was carried out from methanol-ether to obtain the target product [18]. Yield: 5.61 g (78% yield), TLC; Rf ₁ =
0.56

19) PF (18-22); Boc-SEQ ID NO: 10-OH [19] Compound [18] 5.03 g (4.2 mM) was added to acetic acid 30 ml.
8 g of zinc dust was added thereto, and the mixture was stirred at room temperature for 5.5 hours. After the reaction, zinc dust was filtered off, and acetic acid was distilled off under reduced pressure. Ether was added to the precipitated crystals and the crystals were collected by filtration to obtain the target product [19]. Yield; 4.42 g, melting point; 210 ° C. (decomposition) TLC; Rf ₁ = 0.18, Rf ₂ = 0.67 [α] ²⁷ D-4.66 ° (C = 0.3, DMF) amino acid analysis; Nle1.01 (1), Glu2.05
(2), Arg0.98 (1), Val1 (1)

20) PF (18-34); Boc-SEQ ID NO: 11-NH ₂ [20] Compound [13] 8.9 g (3.5 mM), skatole 0.5 g (3.5 mM), dimethyl sulfide. Add 25 ml, 2.5 ml ethanedithiol and 25 ml TFA,
After stirring at C for 10 minutes and at room temperature for 45 minutes, the reaction solution was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected by filtration, dried, and dissolved in 100 ml of dry DMF to obtain NMM.
It was neutralized to pH 7 with. 0.54 g of HOBt in this solution
(4 mM) and compound [19] (4.3 g, 4 mM) were added, and WSC (0.73 ml) was added to the mixture at -15 ° C under cooling.
The mixture was stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, 5% aqueous sodium hydrogen carbonate was added to the residue, and the resulting precipitate was collected by filtration and washed thoroughly with water. The step of dissolving this product in ethanol and adding ether to precipitate the product was performed twice to obtain the target product [20]. Yield: 11.12 g (94% yield), melting point: 250 ° C.
(Decomposition) TLC; Rf ₃ = 0.72 [α] ²⁸ D-4.73 ° (C = 0.53, DMF) Amino acid analysis; Asp1.98 (2), Glu3.04
(3), Val 1.69 (2), Leu2 (2), Ty
r1.07 (1), Lys1.93 (2), His0.
59 (1), Arg1.97 (2), Trp0.35
(1), Nle 1.07 (1)

21) PF (17); Boc-Ser (B
zl) -OPAC [21] Boc-Ser (Bzl) -OH 88.6 g (0.3
M) was dissolved in 400 ml of DMF, 89.6 g (0.45M) of phenacyl bromide was added thereto, and 62.6 ml (0.45M) of Et ₃ N was added dropwise thereto under ice cooling.
The mixture was stirred at ° C for 3.5 hours. Next, 22.1 g (0.225M) of potassium acetate was added to this reaction solution, and the mixture was stirred at room temperature for 1 hour. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in 500 ml of ethyl acetate, and washed with 5% weight water and water in this order. The ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was left in the refrigerator to crystallize, hexane was added, and the product was collected by filtration to obtain the desired product [21]. Yield: 122.97 g (99.1% yield), melting point: 45
˜47 ° C. TLC; Rf ₅ = 0.82 [α] ^29.5 D-11.88 ° (C = 1.0, DMF)

22) PF (16-17); Boc-As
n-Ser (Bzl) -OPAC [22] 50 ml of methylene chloride was added to 119.9 g (0.29M) of compound [21], 250 ml of TFA was added thereto under ice cooling, and the mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was concentrated under reduced pressure, ether was added to the residue, and the precipitated crystals were collected by filtration and dried.
Dissolve the crystals in 400 ml of dry DMF and adjust the pH with NMM.
Neutralized to 7. HOBt 31.35 g (0.
232M) and Boc-Asn-OH 53.88g.
(0.232M) was added, and the mixture was cooled to -15 ° C under WS.
After C42.46 ml (0.232M) was added dropwise, the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in 500 ml of ethyl acetate, and washed with 5% aqueous sodium hydrogen carbonate.
Crystals were precipitated during the separation, and the crystals were collected by filtration, washed with water, and then with ether to obtain 41.79 g of the target product [22] as crystals I. The ethyl acetate layer of the filtrate was concentrated under reduced pressure, and the residual oily substance was crystallized from ethyl acetate-ether to obtain 6.22 g of the target compound [22] as crystals II. Yield: 48.01 g (yield 39.2%), melting point: 174
˜176 ° C. TLC; Rf ₂ = 0.61, Rf ₄ = 0.62 [α] ^29.5 D-5.54 ° (C = 1.0, DMF) Amino acid analysis; Asp1.22 (1), Ser1.00
(1)

23) PF (15-17); Boc-Le
u-Asn-Ser (Bzl) -OPAC [23] To 80.91 g (0.153M) of compound [22], 50 ml of methylene chloride was added, and then 150 ml of TFA was added under ice cooling, followed by stirring at room temperature for 1 hour. The reaction solution is concentrated under reduced pressure,
Ether was added to the residue, and the resulting oily substance was separated by decantation, dissolved in 150 ml of dry DMF, and adjusted to pH with NMM.
Neutralized to 7. HOBt of 22.7 g (0.1
68M), Boc-Leu-OH · H 2 O41.9g
(0.168M) and DMF (100 ml) were added, and -15
30.7 ml (0.168 M) of WSC was added dropwise to the mixture while cooling to ℃, and the reaction solution stirred at room temperature gelled. After allowing it to stand in an ice room for 3 days, water was added and the resulting precipitate was collected by filtration. ,
The target product [23] was obtained by washing with 5% aqueous sodium hydrogen carbonate and water in this order and drying. Yield: 88.52 g (yield 90.3%), melting point: 192
_{~193 ℃ TLC; Rf 2 = 0.80} , Rf 3 = 0.88

24) PF (14-17); Boc-SEQ ID NO: 12-OPAC [24] To 87.55 g (0.137 M) of compound [23], 150 ml of methylene chloride was added, and then 200 ml of TFA under ice cooling. Then, the mixture was stirred at room temperature for 70 minutes. The reaction mixture was concentrated under reduced pressure, ether was added to the residue, the precipitate formed was collected by filtration, dried and then dissolved in 200 ml of dry DMF, and the pH was adjusted to 7 with NMM.
To neutralize to obtain a de-Boc solution.

On the other hand, Boc-His (Tos) -OH.
89.2 g of DCHA (0.151M) was added to 1 l of ethyl acetate.
The solid was suspended in water and washed with 1N sulfuric acid (500 ml), and the precipitated crystals were separated by filtration. The ethyl acetate layer was washed with water and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance is dried DM
A solution dissolved in 150 ml of F2 and 20.4 g of HOBt (0.
151M) was added to the de-Boc solution described above and -15
27.6 ml (0.151M) of WSC was added dropwise to the mixture while cooling to 0 ° C., and the mixture was stirred at room temperature for 3 days. After the reaction, the solvent was distilled off under reduced pressure, the residue was added to water, the resulting precipitate was collected by filtration, and then 5
% Sodium bicarbonate water and water in that order, and dried to obtain the desired product [24]. Yield; 108.63 g (yield 85.1%), melting point; 15
4 to 156 ° C. TLC; Rf ₂ = 0.20, 0.79, Rf ₃ = 0.5
5, 0.87 A part of Tos desorbed was obtained. [Α] ^29.5 D-18.58 ° (C = 1.0, DMF)

25) PF (13-17); Boc-SEQ ID NO: 13-OPAC [25] Compound [24] 107.96 g (0.116 M) was added with methylene chloride 100 ml, and then ice-cooled TFA 200 ml.
After adding, the mixture was stirred at room temperature for 70 minutes. The reaction mixture was concentrated under reduced pressure, ether was added to the residue, the precipitate formed was collected by filtration, dried and dissolved in 200 ml of dry DMF, and the pH was adjusted with NMM.
Neutralization to 7 gave a de-Boc solution.

On the other hand, Boc-Lys (Z-Cl) -OH
・ 62.46 g (0.128 M) of TBA was added to ethyl acetate 6
The mixture was suspended in 00 ml, washed with 1N hydrochloric acid and water in this order, the ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. A solution prepared by dissolving 17.30 g (0.128 M) of the obtained oil and HOBt in 100 ml of dry DMF was added to the de-Boc solution described above, and 24.42 ml of WSC under cooling to -15 ° C.
(0.128M) was added dropwise, and the mixture was stirred at room temperature overnight.
After the reaction, the solvent was distilled off under reduced pressure, the residue was added to 5 l of 3% sodium hydrogen carbonate solution, and the precipitated crystals were thoroughly washed with water and dried.
The crystals were dissolved in methanol, and ether was added for precipitation. The obtained precipitate was suspended in ethyl acetate and collected by filtration three times to obtain the desired product [25]. Yield: 114.42 g (91.8% yield), melting point: 20
0 to 202 ° C. TLC; Rf ₂ = 0.34, Rf ₃ = 0.68 [α] ²⁸ D-26.94 ° (C = 1.0, DMF)

26) PF (13-17); Boc-SEQ ID NO: 13-OH [25] Compound [25] 86.0 g (80 mM) was added to 500 ml of acetic acid.
150 g of zinc dust was added thereto, the mixture was stirred at room temperature for 5 hours, and the reaction liquid was filtered to remove zinc dust. The reaction filtrate was concentrated under reduced pressure, ether was added to the residue, and the precipitated crystals were collected by filtration to obtain the desired product [26]. Yield: 84.70 g (95.2% yield), melting point: 240
˜250 ° C. TLC; Rf ₂ = 0.47 [α] ³⁰ D-19.16 ° (C = 1.0, DMF) Elemental analysis [C ₄₅ H ₅₂ O ₁₂ N ₉ Cl.2CH ₃ COOH.
2H ₂ O] Amino acid analysis; Asp1.01 (1), Ser0.83
(1), Leu1 (1), Lys0.93 (1), Hi
s0.97 (1)

27) PF (13-34); Boc-SEQ ID NO: 14-NH ₂ [27] Compound [20] 10.77 g (3.2 mM) and 0.46 g (3.2 mM) skatole and dimethyl sulfone. Id 25
ml, ethanedithiol 2.5 ml and TFA 25 ml were added, and the mixture was stirred at 0 ° C. for 10 minutes and at room temperature for 60 minutes, and then the reaction solution was concentrated under reduced pressure. Ether was added to the residue, the resulting precipitate was collected by filtration, dried and then dissolved in 200 ml of dry DMF.
Neutralized to pH 7 with NMM. Then add HOBt
0.51 g (3.8 mM) and compound [26] 4.2
Add 3 g (3.8 mM) and cool to -15 ° C under WSC
After adding 0.70 ml, the mixture was stirred overnight at room temperature. After the reaction
DMF was distilled off under reduced pressure, water was added to the residue, and the resulting precipitate was collected by filtration, washed with water and dried to obtain the desired product [27]. Yield: 13.60 g (yield 100%), melting point: 138-
160.5 ° C. [α] ²⁸ D-1.96 ° (C = 0.56, DMF) Amino acid analysis; Asp2.96 (3), Ser0.62
(1), Glu3.02 (3), Val1.72
(2), Leu3 (3), Tyr1.06 (1), Ly
s3.01 (3), His1.43 (2), Arg1.
98 (2), Trp0.60 (1), NIe1.06
(1)

28) PF (11-12); Boc-Le
u-Gly-OBzl [28] Boc-Leu-OH.H ₂ O 4.99 g (20 mM)
Was dissolved in 30 ml of dry THF, and 50 ml of dry benzene was added to this.
ml was added and the solvent was distilled off azeotropically. The oily substance obtained was dissolved in 70 ml of dry THF, and added with H-Gly-OB.
zl TosOH (20 mM) and HOBt 2.7 g
(20 mM) and then WSC5 under cooling to -15 ° C.
After adding ml, the mixture was stirred overnight at room temperature. After the reaction, the solvent was distilled off under reduced pressure and the residue was dissolved in 100 ml of ethyl acetate.
It was washed twice with 1N hydrochloric acid, twice with 5% aqueous sodium hydrogen carbonate and twice with water. The ethyl acetate layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain an oily target product [28].

29) PF (10-12); Boc-As
n-Leu-Gly-OBzl [29] The oily substance [28] obtained above was cooled to -15 ° C under 4.39.
After adding 40 ml of N hydrogen chloride / dioxane solution and stirring for 90 minutes, the solution was concentrated under reduced pressure. Ether was added to the residue, the resulting precipitate was collected by filtration, dried and then dissolved in 30 ml of dry DMF. Added under cooling Et ₃ N to the -5 ° C. and adjusted to pH 7, then HOBt0.3g (2.2mM) and Boc-Asn-ONP7.77g (2.2mM) and Boc-Asn-ONP7.77g ( (22 mM) was added, and the mixture was stirred at room temperature for 3 days. Water was added to the reaction, and the deposited precipitate was extracted with 200 ml of chloroform. The chloroform layer was washed with 1N hydrochloric acid, 5% aqueous sodium hydrogen carbonate and water in that order, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was crystallized from ethyl acetate-hexane to obtain the desired product [29]. Yield: 8.0 g (yield 73.8%), melting point: 152-1
56 ° C [α] ²⁴ D-36.1 ° (C = 1.0, DMF)

30) PF (9-12); Boc-SEQ ID NO: 15-OBzl [30] Compound [29] 7.36 g (15.5 mM) was added with methylene chloride 5 ml and then ice-cooled with TFA 32 ml. After stirring for 60 minutes at room temperature, the reaction mixture was concentrated under reduced pressure, ether was added to the residue, and the deposited precipitate was collected by filtration, dried, dissolved in 40 ml of dry DMF, and adjusted to pH 7 with NMM to remove the Boc solution. Got

On the other hand, Boc-His (Tos) -OH.
DCHA 10.99 g (18.6 mM) and ethyl acetate 1
50 ml and 90 ml of 0.5N sulfuric acid were added, and the mixture was shaken. The ethyl acetate layer was washed 3 times with water and dried over anhydrous sodium sulfate, and ethyl acetate was evaporated under reduced pressure to give an oily substance. This oil and H
2.5 g of OBt (18.6 mM) was dissolved in 60 ml of dry DMF, the solution was added to the de-Boc solution described above, and then 3.4 ml of WSC (18.6 mM) was added under cooling to -15 ° C, followed by stirring at room temperature overnight. . After the reaction, the solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, and the mixture was washed with 5% aqueous sodium hydrogen carbonate three times,
After washing twice with water and drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. Ether was added to the residue, and the precipitated crystals were collected by filtration. Toss of His is partially desorbed in this crystal, but in order to completely desorb it, 100 ml of DMF was added to this crystal.
And HOBt 7.05 g was added to this, and the mixture was stirred at room temperature for 3 hours.
It was stirred for a day. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed twice with 5% aqueous sodium hydrogen carbonate and in this order with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Ether was added to the precipitated crystals and the crystals were collected by filtration to obtain the desired product [30]. Yield: 7.32 g (74.8% yield), TLC: Rf ₂
= 0.1

31) PF (8-12); Boc-SEQ ID NO: 16-OBzl [31] To 7.32 g (11.6 mM) of compound [30], 5 ml of methylene chloride was added, and then 30 ml of TFA under ice cooling. After that, the reaction mixture was concentrated under reduced pressure, ether was added to the residue, the deposited precipitate was collected by filtration, dried, dissolved in 40 ml of dry DMF, and adjusted to pH 7 with NMM. 9 g (13.92 mM) and Boc-Nle-OH 3.23 g (13.92 mM)
Was added to dry DMF, 2.5 ml (13.92 mM) of WSC was added under cooling to -15 ° C, and the mixture was stirred at room temperature overnight. After the reaction, the solvent was distilled off under reduced pressure, water was added to the residue, and the deposited precipitate was collected by filtration and washed twice with 5% aqueous sodium hydrogen carbonate,
The product was washed twice with 1N hydrochloric acid and then three times with water, and dried to obtain the desired product [31]. Yield: 3.70 g (yield 42.9%), TLC; Rf ₂
= 0.20

32) PF (8-12); Boc-SEQ ID NO: 16-OH [32] Compound [31] 2.8 g (3.8 mM) was added to ethanol 1
It was dissolved in 00 ml, 10% Pd / C (300 mg) was added thereto, and hydrogen gas was bubbled through at room temperature for 3 hours. Impurities were precipitated in the reaction solution, so they were filtered, washed with DMF, and the filtrate was concentrated under reduced pressure. Ethanol-ether was added to the residue and the precipitate was collected by filtration and dried to obtain the desired product [32]. Yield: 1.76 g (71.1% yield), melting point: 112.
5 ° C. TLC; Rf ₂ = 0.05 amino acid analysis; Asp 0.96 (1), Gly 0.98
(1), Leu1 (1), His0.95 (1), Nl
e 0.94 (1)

33) PF (8-34); Boc-SEQ ID NO: 17-NH ₂ [33] Compound [27] 10.60 g (2.5 mM) to skatole 0.33 g (2.5 mM), dimethylsulf Id 25
ml, ethanedithiol 2.5 ml and TFA 25 ml were added, and the mixture was stirred at 0 ° C. for 10 minutes and at room temperature for 50 minutes, and then the reaction solution was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected by filtration, dried, dissolved in 100 ml of dry DMF, and NMM was added thereto under ice cooling to adjust the pH to 7. To this solution was added HOBt 0.36 g (2.7 mM) and compound [32] 1.76 g (2.7 mM), and-
After adding 0.5 ml of WSC to 15 ° C. under cooling, the mixture was stirred overnight at room temperature. The deposited precipitate was collected by filtration, washed with water and dried, and then reprecipitated from ethanol-ether to obtain the desired product [3
3] was obtained. Yield: 10.94 g (91.7% yield), melting point: 14
0.5 to 162 ° C. [α] ²⁸ D-1.94 ° (C = 0.52, DMF) Amino acid analysis; Asp3.87 (4), Ser0.76
(1), Glu3.34 (3), Gly0.77
(1), Val1.84 (2), Leu4 (4), Ty
r1.04 (1), Lys3.28 (3), His2.
37 (3), Arg2.14 (2), Trp0.73
(1), Nle2.07 (2)

34) PF (7); Boc-Leu-OP
AC [34] _{Boc-Leu-OH · H 2} O15.0g (60mM)
DM with 17.9 g (90 mM) of phenacyl bromide
Dissolve in 100 ml of F and add 12.5 ml of Et ₃ N under ice cooling.
(90 mM) was added dropwise, and the mixture was stirred at 30 ° C for 2 hours.
Next, 4.42 g (45 mM) of potassium acetate was added, the mixture was stirred at room temperature for 45 minutes, and then DMF was distilled off under reduced pressure. The residue was dissolved in ethyl acetate and washed twice with 5% aqueous sodium hydrogen carbonate and twice with water. The ethyl acetate layer was dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure. The residue was left in an ice room, and the precipitated crystals were dried to obtain the desired product [34]. Yield; 21.23 g (yield 100%), TLC; Rf ₁
= 0.89

35) PF (6-7); Boc-Glu-
Leu-OPAC [35] 20 ml of methylene chloride was added to 20.96 g (60 mM) of compound [34], 80 ml of TFA was added thereto under ice cooling, and the mixture was stirred at room temperature for 40 minutes, and the reaction mixture was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected by filtration and dried,
Dissolve in 70 ml of dry DMF, add NMM under ice cooling and add pH.
Adjusted to 7. HOBt 8.1g (60m
M) and Boc-Gln-OH 14.78 g (60 m)
A solution of M) in 90 ml of dry DMF was added, and -15
WSC (10.9 ml, 60 mM) was added dropwise to the mixture while cooling to 0 ° C., and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, and the mixture was washed with 5% aqueous sodium hydrogencarbonate solution 2
It was washed twice with 1N hydrochloric acid, and then with water 3 times. After drying the ethyl acetate layer with anhydrous sodium sulfate, after distilling off the solvent under reduced pressure,
Hexane was added to the precipitated crystals, and the crystals were collected by filtration and dried to obtain the target product [35]. Yield; 17.25 g (60.2% yield), TLC; Rf
₁ = 0.38

36) PF (5-7); Boc-Ile-
Gln-Leu-OPAC [36] To 17.19 g (36 mM) of compound [35], 10 ml of methylene chloride was added, and then 70 ml of TFA was added under ice cooling, and the mixture was stirred at room temperature for 60 minutes, and the reaction mixture was concentrated under reduced pressure. The residue was dried under reduced pressure, dissolved in 130 ml of dry DMF, and cooled under ice cooling N
The pH was adjusted to 7 with MM. HOBt 5.3g in this solution
(39.6 mM) and Boc-Ile-OH.1 / 2
A solution prepared by dissolving 9.5 g of H ₂ O (39.6 mM) in 70 ml of dry DMF was added, and WSC (7.2 ml) was cooled to -15 ° C.
After (39.6 mM) was added dropwise, the mixture was stirred overnight at room temperature.
After the reaction, DMF was distilled off under reduced pressure, 5% aqueous sodium hydrogen carbonate was added to the residue, and the resulting precipitate was collected by filtration and washed with 5% aqueous sodium hydrogen carbonate twice, 1N hydrochloric acid twice and water three times in this order. Dried. This precipitate was reprecipitated from ethanol-ether to give the desired product [36].
Got Yield: 16.35 g (yield: 76.9%) TLC; Rf ₁ = 0.41, Rf ₂ = 0.68

37) PF (4-7); Boc-SEQ ID NO: 18-OPAC [37] To 16.24 g (27.5 mM) of compound [37], 10 ml of methylene chloride was added, and then 70 ml of TFA was added under ice cooling. After stirring at room temperature for 60 minutes, the reaction solution was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected by filtration, dried, dissolved in 100 ml of dry DMF, and then N-cooled under ice cooling.
The pH was adjusted to 7 by adding MM. HOBt in this solution
4.09 g (30.25 mM) and Boc-Glu
A solution of 10.2 g (30.25 mM) of (OBzl) -OH dissolved in 50 ml of dry DMF was added, and 5.5 ml of WSC was added dropwise to the mixture at -15 ° C under cooling, followed by stirring at room temperature overnight.
After the reaction, DMF was distilled off under reduced pressure, 5% aqueous sodium hydrogen carbonate was added to the residue, and the resulting precipitate was collected by filtration and washed with 5% aqueous sodium hydrogen carbonate twice, 1N hydrochloric acid twice and water four times in this order. Dried. Reprecipitation from ethanol-ether gave the desired product [37]. Yield: 21.68 g (yield 97.1%) TLC; Rf ₁ = 0.52

38) PF (3-7); Boc-SEQ ID NO: 19-OPAC [38] To 21.46 g (26.5 mM) of compound [37], 10 ml of methylene chloride was added, and then 90 ml of TFA was added under ice cooling. After stirring at room temperature for 1 hour, the reaction solution was concentrated under reduced pressure.
Ether was added to the residue, and the resulting precipitate was collected by filtration, dried, dissolved in 150 ml of dry DMF, and then NMM under ice cooling.
Was added to adjust the pH to 7. HOBt 3.9 was added to this solution.
g (29.15 mM) and Boc-Ser (Bzl)
-OH 8.6g (29.15mM) dry DMF 50ml
Was added to the solution and cooled to −15 ° C. under WSC5.3.
After adding ml (29.15 mM), the mixture was stirred overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure, 5% aqueous sodium hydrogen carbonate was added to the residue, and the deposited precipitate was collected by filtration. 5% sodium bicarbonate water,
After washing with 1N hydrochloric acid twice and water four times in this order, the product was suspended in ether and collected by filtration to obtain the desired product [38]. Yield: 24.8 g (yield 94.7%), TLC; Rf ₁
= 0.53

39) PF (2-7); Boc-SEQ ID NO: 20-OPAC [39] To 24.68 g (25 mM) of compound [38], 20 ml of methylene chloride was added, and then 100 ml of TFA was added under ice cooling. After stirring at room temperature for 50 minutes, the reaction solution was concentrated under reduced pressure,
Ether was added to the residue, and the resulting precipitate was collected by filtration, dried and dissolved in 120 ml of dry DMF, and then NMM under ice cooling.
Was added to adjust the pH to 7. Add HOBt 4.0 to this solution.
5 g (30 mM) and Boc-Val-OH 6.5 g
A solution prepared by dissolving (30 mM) in 80 ml of dry DMF was added, and 5.5 ml (30 mM) of WSC was added dropwise to the mixture at -15 ° C under cooling, followed by stirring at room temperature overnight. Since a precipitate had precipitated in the reaction solution, water was added to it, and the precipitate was collected by filtration and washed twice with 5% aqueous sodium hydrogen carbonate and 1N.
After washing twice with hydrochloric acid and then four times with water, the product was suspended in ether and collected by filtration to obtain the desired product [39]. Yield: 26.32 g (yield 96.8%), TLC; Rf
₁ = 0.49

40) PF (1-7); Boc-SEQ ID NO: 21-OPAC [40] To 26.07 g (24 mM) of compound [39], 20 ml of methylene chloride was added, and then 100 ml of TFA was added under ice cooling. After stirring at room temperature for 40 minutes, the reaction solution was concentrated under reduced pressure,
Ether was added to the residue, and the resulting precipitate was collected by filtration, dried, dissolved in 100 ml of dry DMF, and then NMM under ice cooling.
Was added to adjust the pH to 7. HOBt 3.9 was added to this solution.
g (28.8 mM) and Boc-Ser (Bzl)-
A solution prepared by dissolving 8.5 g (28.8 mM) of OH in 50 ml of dry DMF was added, and 5.3 ml of WSC under cooling to -15 ° C.
After adding (28.8 mM), the mixture was stirred at room temperature overnight.
Since a precipitate had precipitated in the reaction solution, water was added and the mixture was collected by filtration.
% Sodium bicarbonate water, 1N hydrochloric acid, and water in this order, and then the steps of suspending in ether and collecting by filtration were performed twice to obtain the target product [40]. Yield: 28.0 g (yield 92.3%), TLC; Rf ₁
= 0.53

41) PF (1-7); Boc-SEQ ID NO: 21-OH [41] Compound [40] 12.6 g (10 mM) was added to acetic acid 300 ml.
15 g of zinc dust was added thereto, the mixture was stirred at 50 ° C. for 4 hours, and the zinc dust was filtered off. Acetic acid was distilled off under reduced pressure, ether was added to the residue, and the precipitated crystals were collected by filtration and washed to obtain the desired product [41]. Yield: 11.15 g (97.4% yield), melting point: 260
C (decomposition) TLC; Rf ₁ = 0.14, Rf ₂ = 0.64 amino acid analysis; Ser1.81 (2), Glu2.02
(2), Val0.95 (1), Leu1 (1), Il
e 0.92 (1)

42) Protection- [Nle ⁸ , Nle ¹⁸ , Tr
y ³⁴ ] -h-PTH (1-34) NH ₂ ; Boc-SEQ ID NO: 22-NH ₂ [42] Compound [33] 10.86 g (2.28 mM) in ice-cooled skatole 0.30 g (2. 28 mM), dimethyl sulfide 25 ml, ethanedithiol 2.5 ml and TFA
After adding 25 ml and stirring at room temperature for 60 minutes, the mixture was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected by filtration, dried and dissolved in a mixed solution of 100 ml of dry DMF + 10 ml of DMSO, and then NMM was added under ice cooling to adjust the pH to 7. To this solution, 0.37 g (2.74 mM) of HOBt and 3.14 g (2.74 mM) of compound [41] were added, and then 0.50 ml (2.7%) of WSC under cooling to -15 ° C.
(4 mM) was added, and the mixture was stirred overnight at room temperature. Water was added to the reaction solution, the resulting precipitate was collected by filtration, washed thoroughly with water, and then washed with ethanol-ether to obtain the desired product [42]. Yield: 12.87 g (yield 97.3%), melting point: 13
9.5 to 175 ° C. [α] ²⁸ D-1.97 ° (C = 0.51, DMF) Amino acid analysis; Asp 3.72 (4), Ser 2.76
(3), Glu5.58 (5), Gly0.69
(1), Val2.86 (3), Ile1.11
(1), Leu5 (5), Tyr0.99 (1), Ly
s2.87 (3), His2.19 (3), Arg2.
06 (2), Trp0.65 (1), Nle1.96
(2)

43) [Nle ⁸ , Nle ¹⁸ , Try ³⁴ ]
-H-PTH (1-34) NH ₂ compound [42] 2.9 g (0.5 mM) under cooling to 0 ° C. anisole 3.5 ml, ethanedithiol 0.35 ml, dimethyl sulfide 3.5 ml and anhydrous HF 35 ml. Was added and stirred for 60 minutes. After the reaction, HF was distilled off under reduced pressure,
Ether was added to the residue, the resulting precipitate was collected, and
This solution dissolved in 1N acetic acid was passed through a Dowex × 1 (acetate type) column (3.5 × 12 cm), and only ninhydrin-positive fractions were collected and freeze-dried to obtain 1.87 g of a crude product. This was dissolved in 50 ml of 0.1N acetic acid and charged in a CM-cellulose column (2 × 33 cm), and 0.05M ammonium acetate (pH 5.1) was added.
Elution was performed with a linear concentration gradient of 1 liter of 1 liter to 0.4 M ammonium acetate (pH 6.0). Each fraction was fractionated into 9.0 ml, and the 74th to 84th fractions having spots near Rf ₆ = 0.30 were collected by TLC and dried. This was dissolved in as little 0.1N hydrochloric acid as possible, this solution was charged into a Sephadex G-25 column (3 × 115 cm), and eluted with 0.1N acetic acid. The absorbance of each fraction was measured at UV280 nm, and only the fractions having one large peak were collected and freeze-dried [Nle ⁸ , Nle ¹⁸ , Ty.
r ^34] was obtained _{-h-PTH (1-34) NH 2} . Yield: 140 mg, TLC; Rf ₆ = 0.30 amino acid analysis (hydrolyzed with 6N hydrochloric acid containing 3% thioglycolic acid); Asp3.98 (4), Ser2.10.
(3), Glu4.93 (5), Gly0.97
(1), Val2.66 (3), Ile 0.87
(1), Leu5.00 (5), Tyr1.11
(1), Lys3.26 (3), His2.30.
(3), Arg2.03 (2), Trp0.62
(1), Nle 2.22 (2) High Performance Liquid Chromatography; Column; Nucleosil ₅ C
₁₈ (4 mm ID × 150 mm) buffer solution; 0.1% acetic acid-acetonitrile containing 0.1 M phosphoric acid (ratio of acetonitrile is 20% for the first 5 minutes, 20 for the subsequent 20 minutes)
Flow rate: 1 ml / min Detection: 225 nm Measurement result: Peak detection only at 19.07 min.

Example 1 [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ] -h-PTH (1-
34) Preparation of ¹²⁵ I-labeled substance of NH _{2 In} 50 μl of 0.5 M phosphate buffer (pH 7.1) containing ¹²⁵ I-NaI having a radioactivity of ₂ mCi, [Nl
e ^8, Nle ^18, Tyr ^34] -h-PTH (1-34)
10 μl of a solution containing ₂ μg of NH ₂ and chloramine T
After adding 20 μl of a solution containing (3.5 mg / ml) and stirring for 30 seconds, 50 μl of a solution containing sodium bisulfite (4.5 mg / ml) was added to stop the reaction. After adding 0.5 ml of a 0.1 N acetic acid solution containing 5% human serum albumin to this, the column was charged on a Sephadex G-10 column (1 × 50 cm), eluted with the above acetic acid solution and labeled with ¹²⁵ I. Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ] -h-PTH (1-
34) NH ₂ was obtained.

<Peptide labeling with non-radioactive NaI>
Regarding the physical properties of the peptide labeled with ¹²⁵ I, the non-radioactive NaI was used for the I conversion as in the above method,
It was decided to clarify the physical properties of the product and to compare the peptide with ¹²⁵ I-labeled peptide. That is, [N
le ⁸ , Nle ¹⁸ , Tyr ³⁴ ] -h-PTH (1-3
4) Dissolving 5 mg of NH ₂ in 2.5 ml of water, 250 μl of 0.1M phosphate buffer (pH 7.1), NaI (2.2 mg /
ml) containing solution 200 μl and chloramine T (125 mg /
ml) containing solution (100 μl) and stirred for 30 seconds, and then sodium bisulfite (100 mg / ml) containing solution (65 μl)
The reaction was stopped by adding l. This is Sephadex G-
10 columns (1 × 50 cm) were charged and eluted with 0.1N acetic acid solution, and fractions having absorption at 280 nm were collected and lyophilized to give [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ] -h-
An I-form of PTH (1-34) NH ₂ was obtained.

The product obtained was subjected to HPL under the conditions shown below.
When C analysis was performed, two peaks were shown at an elution time of about 25.8 minutes (peak-1) and about 27.2 minutes (peak-2). ([Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ] of raw material-
h-PTH (1-34) NH ₂ elutes at about 24.2 minutes. ) Analytical conditions Instrument; Shimadzu LC-6A high pressure gradient system column; YMC-PACK R-ODS-5 (4.6 mm)
I. D. × 250 mm) Eluent: 0.1% TFA / CH ₃ CN 75/25 → 6
0/40 (v / v, 30 minutes) Flow rate: 1 ml / min Detection: 210 nm, 280 nm

Therefore, these two peaks were analyzed by HPLC.
And purified by the method described above, and enzymatically decomposing each of peak-1 and peak-2 with trypsin, and then HP
Analyzed by LC. Then, each of the separated trypsin decomposition peaks was subjected to amino acid analysis after hydrolysis for 24 hours at 6N-HCl and 110 ° C. As a result, in the amino acid analysis, the Tyr-free trypsin decomposition peak showed the amino acid composition, the elution time by HPLC was peak-1,
Peak-2 was equivalent. Since the trypsin decomposition peaks containing Tyr had different elution times on HPLC,
Further analysis by FAB-MASS was performed. FAB-
The trypsin-decomposed peak containing Tyr obtained from peak-1 by MASS had an MH ⁺ of 1013 and H-Leu-Gln-Asp-Val- of amino acid sequence 28 to 34.
It is converted to His-Asn-Tyr-NH ₂ once. Further, MH ⁺ obtained from peak-2 is 1139 converted into 2 isomers. Peak -1 by the above [NIe ^8, Nle ^18, Tyr ^34] -h
-PTH (1-34) NH ₂ Tyr is monoiodinated, and Peak-2 is diiodinated.

<[NIe ⁸ , Nle ¹⁸ , Tyr ³⁴ ] -h
-Analysis of ¹²⁵ I-labeled PTH (1-34) NH ₂ > Analysis was performed by HPLC. Analytical conditions Instrument: Shimadzu LC-6A high pressure gradient system column: YMC AM-302 ODS (4.6 mm I.D.
D. × 150mm) Mini Guard Column; Nucleosi (Nucleosi)
l) 100-5 C18 (4mmI.D. × 10mm ) _{eluent; 0.1% TFA / CH 3 CN} 69.3 / 3
0.7 (v / v) Flow rate; 1 ml / min Detector; Shimadzu SPD-6A (210 nm), RI detector RLC-701 (Aloka)

Under the above analysis conditions, [Nle ⁸ , Nle
The monoiodinated and diiodinated forms of ¹⁸ , Tyr ³⁴ ] -h-PTH (1-34) NH ₂ are about 13.5 each.
Minute, about 23.3 minutes. Therefore, the prepared [Nle ⁸ , Nle ¹⁸ , Tyr ³⁴ ] -h-PTH (1-
34) When ¹²⁵ I-labeled NH ₂ was analyzed under the same conditions, monoiodinated [ ¹²⁵ I] and diiodinated [ ¹²⁵ I ₂ ] were obtained at about 4: 1 (specific radioactivity). ¹²⁵ I-Na
This ratio varies depending on I, chloramine T, and reaction time, but a single monoiodine ( ¹²⁵ I) form or diiodine ( ¹²⁵ I ₂ ) form was obtained by purification by HPLC.

[Sequence list]

SEQ ID NO: 1 Sequence length: 34 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics: Other information: Carboxyl group of C-terminal Tyr is amidated. Sequence Ser Val Ser Glu Ile Gln Leu Nle His Asn Leu Gly Lys His Leu Asn Ser Nle 1 5 10 15 Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Tyr 20 25 30

SEQ ID NO: 2 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence characteristics: Other information: Carboxyl group of C-terminal Tyr is amide Has been converted. The N-terminal and C-terminal amino acids are protected by a protecting group. Sequence Val His Asn Tyr (Bzl-Cl ₂ ).

SEQ ID NO: 3 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence characteristics: Other information: Carboxyl group of C-terminal Tyr is amide Has been converted. The N-terminal and C-terminal amino acids are protected by a protecting group. Sequence Asp (OBzl) Val His Asn Tyr (Bzl-Cl ₂ ) 30

SEQ ID NO: 4 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence characteristics: Other information: Carboxyl group of C-terminal Tyr is amide Has been converted. The amino acid at the second position from the N-terminal and the C-terminal is protected by a protecting group.

SEQ ID NO: 5 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: Other information: Each amino acid is protected by a protecting group ing. Sequence Arg (Tos) Lys (Z-Cl) Lys (Z-Cl) Leu 25

SEQ ID NO: 6 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: Other information: Each amino acid is protected by a protecting group ing.

SEQ ID NO: 7 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: Other information: each except the second from the N-terminal The amino acid is protected by a protecting group.

SEQ ID NO: 8 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence characteristics: Other information: Carboxyl group amidation of C-terminal Tyr Has been done. Each amino acid may be protected by a protecting group. Sequence Trp Leu Arg (Tos) Lys (Z-Cl) Lys (Z-Cl) Leu Gln Asp (OBzl) Val His Asn 25 30 Tyr (Bzl-Cl ₂ ).

SEQ ID NO: 9 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: Other information: Each amino acid is protected by a protecting group There is a case.

SEQ ID NO: 10 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: Other information: Each amino acid is protected by a protecting group There is a case.

SEQ ID NO: 11 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence characteristics: Other information: Carboxyl group of C-terminal Tyr is amide It may have been Sequence Nle Glu (OBzl) Arg (Tos) Val Glu (OBzl) Trp Leu Arg (Tos) Lys (Z-Cl) Lys 20 25 (Z-Cl) Leu Glu Asp (OBzl) Val His Asn Tyr (Bzl-CL ₂ ) 30

SEQ ID NO: 12 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: Other information: Amino acids protected by protecting groups There are cases.

SEQ ID NO: 13 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence features: Other information: Amino acids protected by protecting groups There are cases.

SEQ ID NO: 14 Sequence length: 22 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence characteristics: Other information: Carboxyl group of C-terminal Tyr is amide Has been converted. The amino acid may be protected by a protecting group. Sequence Lys (Z-Cl) His Leu Asn Ser (Bzl) Nle Glu (OBzl) Arg (Tos) Val Glu (OBzl) Trp 15 20 Leu Arg (Tos) Lys (Z-Cl) Lys (Z-Cl) Leu Gln Asp (OBzl) Val His Asn Tyr (Bzl-C 25 30 -Cl ₂ ).

SEQ ID NO: 15 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence features: Other information: N-terminal and C-terminal amino acids It is protected by a protecting group.

SEQ ID NO: 16 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence features: Other information: N-terminal and C-terminal amino acids It may be protected by a protecting group.

SEQ ID NO: 17 Sequence length: 27 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence characteristics: Other information: Carboxyl group of C-terminal Tyr is amide Has been converted. The amino acid may be protected by a protecting group. Sequence Nle His Asn Leu Gly Lys (Z-Cl) His Leu Asn Ser (Bzl) Nle Glu (OBzl) Arg (To 10 15 20 s) Val Glu (OBzl) Trp Leu Arg (Tos) Lys (Z-Cl) Lys (Z-Cl) Leu Gln Asp (OB 25 30 zl) Val His Asn Tyr (Bzl-Cl ₂ )

SEQ ID NO: 18 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: Other information: N-terminal and C-terminal amino acids It is protected by a protecting group.

SEQ ID NO: 19 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence features: Other information: Amino acids protected by protecting groups There is a case.

SEQ ID NO: 20 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence features: Other information: Amino acids protected by protecting groups There is a case.

SEQ ID NO: 21 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence characteristics: Other information: Amino acid protected by protecting group There is a case.

SEQ ID NO: 22 Sequence length: 34 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Final fragment Sequence characteristics: Other information: Amino acids are protected by protecting groups There are cases. Sequence Ser (Bzl) Val Ser (Bzl) Glu (OBzl) Ile Gln Leu Nle His Asn Leu Gly Lys (ZC 1 5 10 1) His Leu Asn Ser (Bzl) Nle Glu (OBzl) Arg (Tos) Val Glu (OBzl ) Trp Leu Arg 15 20 25 (Tos) Lys (Z-Cl) Lys (Z-Cl) Leu Gln Asp (OBzl) Val His Asn Tyr (Bzl-Cl ₂ ) 30

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 33/53 B 8310-2J 33/534 8310-2J C07K 99:00

Claims (1)

[Claims] 1. A peptide represented by the formula H-SEQ ID NO: 1-NH ₂ has a Tyr at position 34 of 1-2.
Radioactive substance labeled with ¹²⁵ I.