JPS6345680B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a peptide for measuring human parathyroid hormone. More specifically, the present invention provides the formula (In the formula, R is H or H-R ₁ group, R ₁ is Cys or
Tyr group) or a salt thereof. Human parathyroid hormone (h-PTH) is a peptide hormone consisting of 84 amino acids, and it has been found that its biological activity lies in the N-terminal 34 residues. GWTregear et al; Hoppe−Seyler's
N.Physiol.Chem., 355 , 415 (1974)]. For this reason
For the diagnosis of PTH-related diseases, it has been thought that it is important to measure the blood concentration of biologically active N-terminal peptides [Kiichiro Ota et al.; History of Medicine, 109 (7), 375 ( 1979)]. However, in 1978
FPDiBella etc. are PTH-C with no biological activity.
We discovered that measuring the terminal blood concentration is important for diagnosing PTH-related diseases [F.
P. DiBela et al; J. Clin. Endocrinol. Metab.; 46
(4), 604 (1978)]. Therefore, the present inventor investigated the C-terminal side 39 of h-PTH.
C by synthesizing residue h-PTH (46-84) and sensitizing rabbits with these peptides as antigens.
We succeeded in obtaining an antibody specific to the terminal. On the other hand h
-PTH(46-84) does not contain tyrosine, an amino acid that is easily labeled with ^I125 , but
[Try ⁴⁵ ]-h-PTH (46-84) was found to provide a sensitive tracer. Also, h-PTH (46
-84) does not contain cysteine, an amino acid that is labeled with an enzyme that binds to an SH group, but
[Cys ⁴⁵ ]-h-PTH (46-84) was found to provide a sensitive tracer. As mentioned above, this peptide [ ] is useful as a peptide for preparing h-PTH antibodies and as a labeling reagent for quantifying h-PTH. The object compound [] of the present invention is constructed by condensing individual amino acids or lower peptides in the amino acid order represented by the formula [], and removing the protective group of the functional group of the side chain in the final step of the condensation reaction. It is obtained by The condensation reaction itself is carried out by repeating the attachment/detachment of a protecting group and the condensation reaction in accordance with a known conventional method for peptide synthesis. That is, the various protecting groups used in the preparation of the raw materials for the compound of interest as well as all intermediates are those known in peptide synthesis and therefore can be carried out by known means such as hydrolysis, acid hydrolysis, reduction, aminolysis or hydrazinolysis. Therefore, a protecting group that can be easily removed is used. Such protecting groups are described in the literature and reference books in the field of peptide synthetic chemistry. For example, protective groups used for amino groups include acyl groups such as formyl group, trifluoroacetyl group, phthaloyl group, P-toluenesulfonyl group, O-nitrophenylsulfenyl group, benzyloxycarbonyl group, o (or p )-bromobenzyloxycarbonyl group, o (or p)-chlorobenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, benzyloxycarbonyl group such as p-methoxybenzyloxycarbonyl group, trichloroethyloxycarbonyl group, t-
Aliphatic oxycarbonyl groups such as butyloxycarbonyl group, t-amyloxycarbonyl group, diisopropylmethyloxycarbonyl group, 2-phenyl-isoproboxycarbonyl group, 2-tolyl-isopropoxycarbonyl group, 2-p-diphenyl- Examples include aralkyloxycarbonyl groups such as isopropoxycarbonyl groups. These amino groups can also be protected by forming enamines obtained by reacting with 1,3-diketones such as benzoylacetone and acetylacetone. Carboxyl groups are protected by amide formation, hydratide formation or esterification. That is, the amide group is a 3,4-dimethoxybenzyl group, a bis-
Substituted with (p-methoxyphenyl)methyl group, etc. The hydratide group is substituted with a benzyloxycarbonyl group, a trichloroethyloxycarbonyl group, a trifluoroacetyl group, a t-butyloxycarbonyl group, a trityl group, a 2-p-diphenyl-isopropoxycarbonyl group, and the like. Ester groups include methanol, ethanol,
Alkanols such as t-butanol and cyanomethyl alcohol, benzyl alcohol, p-bromobenzyl alcohol, p-chlorobenzyl alcohol, 2,6-dichlorobenzyl alcohol, p
- aralkanols such as methoxybenzyl alcohol, p-nitrobenzyl alcohol, benzhydryl alcohol, benzoylmethyl alcohol, p-bromobenzoylmethyl alcohol, p-chlorobenzoylmethyl alcohol, 2,4,6
- substituted with phenols such as trichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, thiophenols such as thiophenol, p-nitrothiophenol, etc. Ru. The hydroxyl groups of the serine threonine and tyrosine can be protected, for example, by esterification or etherification. Groups suitable for this esterification include, for example, acetyl group, benzoyl group,
These include benzyloxycarbonyl group and ethyloxycarbonyl group. Further, examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, and t-butyl group. To protect these hydroxyl groups, 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl group, 2,2,2
-Trifluoro-1-benzyloxycarbonylamino groups are also suitable. However, it is not necessary or necessary to protect these hydroxyl groups. Groups used to protect the amino group in the guanidino group of arginine include, for example, a nitro group, a tosyl group, a benzyloxycarbonyl group, a mesitylene-2-sulfonyl group, but the guanidino group is not necessarily protected. There's no need. Examples of the group used to protect the imino group of histidine include benzyl group, trityl group, benzyloxycarbonyl group, tosyl group,
2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl group, 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group, etc., but it is not necessary to protect this imino group. There isn't. Groups used to protect cysteine mercapto groups include benzyl, p-methoxybenzyl, p-nitrobenzyl, trityl, benzylthiomethyl, ethylcarbamoyl, acetamidomethyl, and the like. In the synthesis of the present target compound [ ], condensation of individual amino acids or lower peptides is carried out, for example, with an amino acid or peptide having a protected α-amino group and an activated terminal carboxyl group, and a free α
- reacting an amino acid or peptide with an amino group and a protected terminal carboxyl group, or reacting an amino acid or peptide with an activated α-amino group and a protected terminal carboxyl group with a free terminal carboxyl group and a protected terminal carboxyl group; This can be carried out by reacting an amino acid or peptide having an α-amino group. In this case, the carboxyl group is an azide, an acid anhydride, an acid imidazolide or an active ester, such as cyanomethyl ester, thiophenyl ester,
p-nitrothiophenyl ester, p-methanesulfonylphenyl ester, thiodyl ester,
p-nitrophenyl ester, 2,4-dinitrophenyl ester, 2,4,5-trichlorophenyl ester, 2,4,6-trichlorophenyl ester, pentachlorophenyl ester, N-
or by converting into hydroxysuccinimide ester, N-hydroxaphthalic imide ester, 8-hydroxyquinoline ester or N-hydroxypiperidine ester, or carbodiimide, N,N'-carbonyl-diimidazole or isoxolium salt. , for example, by reacting with a Woodward reactant or the like. Preferred condensation methods in the present invention are the carbodiimide method, azide method, active ester method and anhydride method. In each step of the condensation, it is desirable to use a method that does not cause racemization or a method that minimizes racemization, preferably the azide method, active ester method, or Wu¨nseh method [Z.Naturforsch., 21b , 426
(1966)] or the Geige method [Chem.Ber., 103 , 788
(1970)] N-ethyl-
A modified method using N'-3-dimethylaminopropyl-carbodiimide (WSCI) is used. The condensation order can be synthesized in any order as long as it is the amino acid order shown by the formula [], but C-
It is advantageous to synthesize from the terminal side. For example, the protected h-PTH(46-84) is
The -terminal fragment 55-84 and the N-terminal fragment 46-54 are preferably condensed by a modified Geiger method using WSCI. The N-terminal fragment 46-54 is
Fragments 51-54 and 46-50 are preferably condensed by the azide method. C-terminal fragment 55-84 is similar to fragment 61-84 and fragment
It is preferable to condense 57-60 by a modified Geiger method using WSCI, and then condense the 56th amino acid and the 55th amino acid sequentially by an active ester method. fragment 61−84 is fragment 61−84
may be obtained by condensing the 71st amino acid, the 70th amino acid, the 69th amino acid, the fragments 62-68, and the 61st amino acid to fragment 72-84 by the active ester method or the modified Geiger method using WSCI. Fragments 62-68 are preferably obtained by condensing fragments 64-68 and 62-63 by the azide method. Fragment 72−84
The active ester method or
Condensation is preferably carried out by a modified Geiger method using WSCI. Fragment 77-84 is preferably obtained by condensing fragment 82-84 and fragment 77-81 by a modified Geiger method using WSCI. Protected [Tyr ⁴⁵ ]-h-PTH (46-84) and protected [Cys ⁴⁵ ]-h-PTH (46-84) are
Corresponds to the previously mentioned protected h-PTH (46−84)
The 45th amino acid is preferably condensed by a modified Geiger method using WSCI. During the synthesis of the above peptides, the terminal carboxyl group does not necessarily have to be protected. For example, when condensation is carried out by an azide method or an active ester method, protection is not required. However, by esterification of these groups as described above, e.g. methyl ester,
It can also be protected with ethyl ester, benzyl ester, etc. In addition, these ester groups are
For example, a methyl ester group can be cleaved with dilute aqueous sodium hydroxide or converted to a hydratide, and a benzyl ester group can be cleaved with anhydrous hydrogen fluoride or hydrogenolysis.
The α-amino groups of these peptides can be protected by conventional protecting groups such as benzyloxycarbonyl groups.
It is protected with t-butoxycarbonyl group and t-amyloxycarbonyl group, but benzyloxycarbonyl group is removed by hydrogenolysis and t-
The butoxycarbonyl group and t-amyloxycarbonyl group are removed with trifluoroacetic acid. The hydroxyl group of serine and threoline is a benzyl group, the hydroxyl group of tyrosine is a 2,6-dichlorobenzyl group, the ε-amino group of lysine is an o-chlorobenzyloxycarbonyl group, and the amino group in the guanidino group of arginine is a tosyl group. The mercapto group of cysteine is preferably protected with a p-methoxybenzyl group. These protecting groups are removed with anhydrous hydrogen fluoride. An acetamidomethyl group can be used as a protecting group for the mercapto group of cysteine. This group is stable to anhydrous hydrogen fluoride, so once all other protecting groups have been removed, PH4
It is desorbed with mercury acetate. Protected h-PTH (46-84), protected [Tyr ⁴⁵ ]-h-PTH (46-84) and protected [Cys ⁴⁵ ]-h-PTH (46-84) are thus obtained.
These protecting groups are preferably removed in one step by acid decomposition, for example by treatment with anhydrous hydrogen fluoride,
The target compound of formula [] is obtained. When an acetamidomethyl group is used as a protecting group for the mercapto group of cysteine 45, it can be removed by mercuric acetate at pH 4 after all other protecting groups are removed by anhydrous hydrogen fluoride treatment. . The above target compound [] can be purified by known means for purifying peptides. For example, Cephadex LH-20 (trade name, manufactured by Pharmacia, gelling agent, hydroxypropylated dextran gel), Cephadex G-50 (trade name).
This can be carried out by column chromatography using a carrier such as Pharmacia (gelling agent, dextran gel), Dowex1 (trade name, Dow Chemical Company, strong basic anion exchange resin), carboxymethyl cellulose, or the like. The peptide [ ] of the present invention can be obtained in the form of a base or a salt thereof depending on the conditions of the method. Usually, it can be preserved in the form of a salt with an organic acid such as acetic acid. h-PTH can be measured using an enzyme immunoassay or a radioimmunoassay using an antibody obtained using the peptide of the present invention [ ] as an antigen or any immune reaction using the present peptide [ ] as an antigen. Next, h-PTH (53-84), h-PTH (51-84)
and h-PTH (46-84) antibody formation. <Preparation of antibodies> Add 300 μg each of h-PTH (53-84), h-PTH (51-84), and h-PTH (46-84) to 0.1N acetic acid.
Dissolve in 250μ and equal volume of Freunds Complate
Prepare a mixed emulsion with Adjuvant. Five male guinea pigs each weighing 280 to 340 g are immunized with this. The first time is on the legs of the legs and buttocks, and the
From the second administration, subcutaneous injections are repeated in the flank every three weeks, and blood is collected by cardiac puncture one week after the fifth administration. <Labeled antigen> Take I ¹²⁵ /mCi in a reaction tube, add 0.5 ml of 0.5 M phosphate buffer (PH7.4), and add [Tyr ⁴⁵ ]-h-PTH.
(46−84) with the addition of 5 μg and 50 μg of chloramine T.
After stirring for 30 seconds, add 120 μg of sodium pyrosulfite. This includes a small amount of potassium iodide and 5mg of BSA.
After adding , column chromatography is performed using a Sephadex G-50 column (1.0 x 20 cm). Collect 7-10 ml fractions. Veronal buffer (PH8.6, 0.05M, containing 0.5% BSA) is used as a diluent, and approximately 0.01 μCi is used per analysis. <Analysis method> Add 100μ of the antibody to 100μ of the sample solution, add 100μ of the labeled peptide, and react at 4°C for 3 days. Next, add 100μ of normal guinea pig serum and 100μ of anti-guinea pig rabbit serum and incubate at 25°C.
After reacting for 30 minutes, centrifuge at 3000 rpm for 30 minutes, remove the supernatant, and count the precipitate. Dilute the antibody 500 to 8000 times and perform the above analysis.
Measure the dilution ratio at which the binding rate is 30-40%. <Measurement results> h-PTH (53-84) 3000 times h-PTH (51-84) 500 times h-PTH (46-84) 8000 times The abbreviations described in this specification have the following meanings. have Gln; L-glutamine Ser; L-serine Lys; L-lysine Ala; L-alanine Thr; L-threonine Leu; L-leucine Val; L-valine Asp; L-aspartic acid Glu; L-glutamic acid Gly; glycine His ; L-histidine Asn; L-asparagine Arg; L-arginine Pro; L-proline Tyr; L-tyrosine Cys; L-cysteine BOC; t-butyloxycarbonyl AOC; t-amyloxycarbonyl Z-Cl; o-chloro Benzyloxycarbonyl Bzl; benzyl Tos; tosyl OMe; methyl ester OEt; ethyl ester OBzl; benzyl ester OSU; N-hydroxysuccinimide ester ONP; p-nitrophenyl ester PAC; phenacyl ester Acm; acetamidomethyl TosoH; p -Toluenesulfonic acid TFA; trifluoroacetic acid _Et3N ; triethylamine TBA; tribenzylamine NMM; N-methylmorpholine HOBT; 1-hydroxybenzotriazole DMF; dimethylformamide THF; tetrahydrofuran NMP; N-methyl-2-pyrrolidone MeOH; Methanol EtOH; Ethanol BuOH; Butanol ether; Diethyl ether WSCI; N-ethyl, N'-3-dimethylaminopropyl-carbodiimide HOBT; 1-hydroxybenzotriazole Next, examples will be given to specifically explain the production examples of the present invention. explain. The carrier and developing solvent for thin layer chromatography (TLC) used in the examples and the hydrolysis conditions for amino acid analysis were as follows, unless otherwise specified.
It is as follows. Support: Silica Gel G manufactured by Merck & Co., Ltd. Developing solvent: 1; CHCl ₃ -MeOH-acetic acid (95:5:3) 2; (85:15:
5) 3; 〃 (85:10:
5) 4; 〃 (80:25:
2) 5; Benzene-ethyl acetate (1:1) 6; 〃 (2:1) 7; CHCl ₃ -EtOH-ethyl acetate (5; 2:5) 8; 〃 (10:
1:5) Support: Merck cellulose developing solvent; 9; BuOH-pyridine-acetic acid-water (2:2:2:
3) 10; BuOH-pyridine-acetic acid-water (1:1:1:
2) Hydrolysis conditions for amino acid analysis 0.5 μM sample was mixed with 1 ml of 6N hydrochloric acid and 0.1 ml of anisole.
Hydrolysis was carried out at 110°C for 48 hours. Example 1 h-PTH (46-84); H-Ala-Gly-Ser-Gln
−Arg−Pro−Arg−Lys−Lys−Glu−Asp−
Asn−Val−Leu−Val−Glu−Ser−His−Glu
−Lys−Ser−Leu−Gly−Glu−Ala−Asp−
Lys−Ala−Asp−Val−Asp−Val−Leu−Thr
−Lys−Ala−Lys−Ser−Gln−OH (1) P(83−84); BOC−Ser(Bzl)−Gln−OBzl
[1] BOC-Gln-OBzl81.4g (0.242M)
After dissolving in 270 ml of TFA and stirring at room temperature for 45 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue and the resulting precipitate was collected. Add this to 270ml of DMF
Dissolve in, add 32.67g (0.242M) of HOBT to this,
BOC-Ser(Bzl)-OH67.35g (0.242M) and WSCI44.29ml (0.242M) were added and stirred overnight. After the reaction, DMF was distilled off under reduced pressure. The residue was dissolved in 440 ml of ethyl acetate, washed successively with 1N hydrochloric acid, 5% aqueous sodium bicarbonate, and water, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Recrystallization from ethyl acetate-hexane gave 101.43 g (yield: 81.6%) of [1]. Melting point 121-123℃ TLC; Rf ₇ = 0.75 [α] ²⁷ _D -14.12 (C = 0.1, DMF) Elemental analysis [as C ₂₇ H ₃₅ O ₇ N ₃ ] C% H% N% Measured value 62.98 7.03 8.01 Calculation Value 63.14 6.87 8.18 (2) P(82−84);BOC−Lys(Z−Cl)−Ser
(Bzl)-Gln-OBzl [2] [1] 98.87g (192.5mM) was added to 440ml of TFA, and after stirring at room temperature for 30 minutes, TFA was distilled off under reduced pressure. Dissolve the residue in 330ml of DMF,
HOBT28.6g, BOC-Lys(Z-Cl)-OH
DMF solution (BOC-Lys(Z-Cl)-OH.
103.33 g (1.1 times M) of TBA was treated with ethyl acetate-1N hydrochloric acid, and the ethyl acetate layer was dried over anhydrous sodium sulfate and then dried under reduced pressure. Dissolve the residue in 110 ml of DMF) and add 38.72 ml of WSCI (1.1 times molar),
Stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, ice water was added to the residue, and the resulting precipitate was collected. Reconsolidation was performed three times with ethanol-hexane to obtain 111.06 g (yield 71.2%) of [2]. TLC; Rf ₁ = 0.32, Rf ₇ = 0.76 Melting point; 145-147℃ [α] ²⁷ _D −13.1 (C = 1.0, DMF) Elemental analysis [as C ₄₁ H ₅₂ O ₁₀ N ₅ Cl] C% H% N % Measured value 61.02 6.65 8.74 Calculated value 60.77 6.47 8.65 (3) P(80−81);BOC−Lys(Z−Cl)−Ala−
OMe[3] 234.24 g of BOC-Lys(Z-Cl)-OH·TBA was suspended in ethyl acetate and washed with 1N hydrochloric acid and water in that order. After drying with anhydrous sodium sulfate and drying under reduced pressure,
Dissolved in 400ml of DMF. To this H-Ala-
OMe・HCl67.0g, HOBT64.8g, WSCI87.84
ml and stirred at room temperature overnight. After the reaction,
DMF was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate 2 and washed sequentially with 5% aqueous sodium bicarbonate, 1N hydrochloric acid, and water. After drying with anhydrous sodium sulfate, it was dried under reduced pressure. Recrystallized from ethyl acetate-hexane [3] 231.8g
(yield 96.6%). Melting point: 58-60℃ TLC; Rf ₁ = 0.77 [α] ²⁷ _D -17.16 (C = 1.0, DMF) Elemental analysis [as C ₂₃ H ₃₄ O ₇ N ₃ Cl] C% H% N% Measured value 54.96 6.78 8.56 Calculation statement 55.25 6.85 8.40 (4) P(79−81); BOC−Thr(Bzl)−Lys(Z−
174.99 g (0.35 M) of Cl)-Ala-OMe [4] [3] was added to 500 ml of TFA and stirred at room temperature for 50 minutes. After the reaction, TFA was distilled off under reduced pressure and the residue was dissolved in ethyl acetate.
It was washed with 5% sodium bicarbonate solution and then water. After drying with anhydrous sodium sulfate, it was dried under reduced pressure. Dissolve the residue in 400 ml of DMF and add 49.95 g of HOBT (1.05 times M) and BOC-
Thr(Bzl)-OH114.33g (1.05x M) and
67.7 ml of WSCI (1.05 times M) was added and stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and ice water was added to the residue. The resulting precipitate was collected and reprecipitated four times with hot ethanol to obtain 99.31 g of [4]. The mother liquor was concentrated under reduced pressure, the residue was dissolved in chloroform, 5% sodium bicarbonate solution (4 times), 1N hydrochloric acid (2 times),
Washed with water (twice). After drying with anhydrous sodium sulfate, it was dried under reduced pressure. The residue was purified by silica gel column chromatography [eluent: chloroform-ethanol-acetic acid (5:1:5)] to obtain 35.09 g of [4]. Classifications containing impurities are
It is used in conjunction with column chromatography purification during the next additional synthesis. Next, 22.5 g (45 mM) of [3] was added to 70 ml of TFA, and the mixture was stirred at room temperature for 45 minutes. After reaction, TFA
After distilling off under reduced pressure and dissolving the residue in 50 ml of DMF,
The pH was adjusted to 7 with NMM. Then HOBT6.68
g (1.1x M), BOC-Thr(Bzl)-OH15.31g
(1.1 times M) and 9.06 ml of WSCI (1.1 times M) were added and stirred overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 200 ml of chloroform, and then washed with 5% aqueous sodium bicarbonate, 1N hydrochloric acid, and water in this order. After drying with anhydrous sodium sulfate, it was dried under reduced pressure. The residue was combined with the previous impure fraction and purified by silica gel column chromatography. The corresponding fraction was dried under reduced pressure and reprecipitated twice with chloroform-hexane to obtain 48.80 g of [4]. Total amount 183.2g Melting point: 132-134℃ TLC; Rf ₇ = 0.85 Elemental analysis [as C ₃₄ H ₄₇ O ₉ N ₄ Cl] C% H% N% Measured value 59.06 7.05 7.41 Calculated value 59.08 6.85 8.11 (5) P( 77-78); BOC-Val-Leu-OEt [5] BOC-Val-OH101.99g (0.47M), H-
Leu−OEt・HCl91.98g (0.47M),
HOBT63.45g, WSCI86.01ml (0.47M)
It was dissolved in 400 ml of THF and stirred overnight. After the reaction, THF was distilled off under reduced pressure, and the residue was dissolved in 400 ml of ethyl acetate, and then washed with 5% aqueous sodium bicarbonate, 1N hydrochloric acid, and water in this order. After drying with anhydrous sodium sulfate, it was dried under reduced pressure. Recrystallization from ethyl acetate-hexane gave 153.7 g (yield 91.2%) of [5]. Melting point: 108-110℃ TLC; Rf ₁ = 0.63 [α] ²⁷ _D -25.78 (C = 1.0, DMF) Elemental analysis [as C ₁₈ H ₃₄ O ₅ N ₂ ] C% H% N% Measured value 60.35 9.42 8.39 Calculated value 60.31 9.56 7.82 (6) P (77-78); BOC-Val-Leu-OH [6] [5] 134.43g (0.375M) in ethanol 400
ml, 12.5 ml of 1N-NaOH4 (1.1 times M) was added under ice-cooling, and the mixture was stirred. After an hour and a half, 1N−
NaOH37.5 (0.1xM) was added and stirred for 1 hour. 75 ml of 1N hydrochloric acid was added to the reaction solution, and the pH was adjusted to 5 with a small amount of hydrochloric acid. After washing with ether, 400 ml of 1N hydrochloric acid was added to the aqueous layer, and the mixture was extracted with ethyl acetate. After drying the ethyl acetate layer, it was dried under reduced pressure and reconsolidated from ethyl acetate-hexane to give 119.66 g of [6].
(yield 96.6%). TLC; Rf ₁ = 0.35, Rf ₇ = 0.58 Elemental analysis [as C ₁₆ H ₃₀ O ₅ N ₂ ] C% H% N% Measured value 57.83 9.40 8.78 Calculated value 58.16 9.15 8.48 (7) P (77−81); BOC−Val−Leu−Thr(Bzl)
-Lys(Z-Cl)-Ala-OMe [7] [4] 182 g (0.263 M) was added to 500 ml of TFA and stirred at room temperature for 50 minutes. After the reaction, TFA was distilled off under reduced pressure, and hexane was added to the residue. The resulting oil was separated from the solvent by decantation and dissolved in 350 ml of DMF. cool this down
Adjust to PH6.5 with NMM, HOBT42.61g (1.2x M), [6] 104.28g (1.2x M) and
After adding 57.8 ml of WSCI (1.2 times M) and stirring at room temperature for 1.5 hours, it solidified, and even after adding 200 ml of DMF, it could not be stirred, so it was left at room temperature overnight, and then stirred at 30°C.
I left it for 4 hours. Ice water was added to the reaction mixture and the precipitate was collected. This was dissolved in 2.5 ml of chloroform and washed successively with 5% sodium bicarbonate solution, 1N hydrochloric acid, and water.
Chloroform was distilled off under reduced pressure, and the residue was reconstituted from chloroform-ether-hexane [7]
224.68g (yield 94.9%) was obtained. Melting point: 119-221℃ TLC: Rf ₁ = 0.15, Rf ₈ = 0.68 [α] ²⁷ _D -11.72 (C = 1.0, DMF) Elemental analysis [as C ₄₅ H ₆₇ O ₁₁ N ₆ Cl] C% H% N % Measured value 59.80 7.56 9.83 Calculated value 59.82 7.48 9.30 (8) P(77−81); BOC−Val−Leu−Thr(Bzl)
-Lys(Z-Cl)-Ala-OH [8] [7] 72.3g (80mM) in chloroform 720
ml, 800 ml of a 90% ethanol solution of 1N KOH was added under ice-cooling, and the mixture was stirred at 0 to 5°C for 40 minutes. Next, 800 ml of 1N hydrochloric acid was added under ice cooling, and 500 ml of chloroform was added for extraction. The chloroform layer was washed with water, dried over anhydrous sodium sulfate, and then dried under reduced pressure. The residue was purified by silica gel column chromatography [eluent: chloroform-ethanol-ethyl acetate (5:1:5)]. The corresponding fraction was dried to dryness under reduced pressure and recrystallized three times from chloroform-methanol-ether-hexane to obtain [8]. Repeat the above operation 3 times, [7] Total 216.9g
Hydrolyze [8] 156.07g (yield 73.1%)
I got it. Melting point: 180-183℃ TLC; Rf ₃ = 0.69 [α] ²⁷ _D −7.54 (C = 1.0, DMF) Elemental analysis [as C ₄₄ H ₆₅ O ₁₁ N ₆ Cl・1/2H ₂ O] C% H% N% Measured value 58.94 7.67 9.64 Calculated value 58.82 7.40 9.35 Amino acid analysis [Sample 3.1 mg/1 ml 6N hydrochloric acid + 0.1
ml anisole, 45 hours, 110℃ hydrolysis);
Thr0.96(1), Ala1, Val0.93(1), Leu0.94(1),
Lys1.01(1) (9) P(77−84); BOC−Val−Leu−Thr(Bzl)
-Lys(Z-Cl)-Ala-Lys(Z-Cl)-Ser
(Bzl)−Gln−OBzl

[9] [2] 104.5 g (129 mM) was added to 400 ml of TFA and stirred at room temperature for 40 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected and dissolved in 500 ml of DMF. to this
Add HOBT20.9g (1.2xM), [8]137.7g (1.2xM), WSCI28.3ml (1.2xM), NMM
The pH was adjusted to 7 and stirred overnight. Since the reaction solution became gel-like, add 150 ml of DMF.
12.8 ml of WSCI was added and stirred overnight. Add ice water to the reaction solution, collect the precipitate, and wash with hot methanol 5 times.

[9] 185.36g (yield 90.68%) was obtained. TLC; Rf ₂ = 0.85 [α] ²⁷ _D −12.84 (C = 1.0, DMF) Elemental analysis [as C ₈₀ H ₁₀₇ O ₁₈ N ₁₁ Cl ₂ ] C% H% N% Measured value 60.61 7.04 10.38 Calculated value 60.74 6.82 99.74 Amino acid analysis [sample 3.1 mg/1 ml 6N hydrochloric acid + 0.1
ml anisole, 110℃, 45 hours hydrolysis);
Thr0.93(1), Ser0.91(1), Glu1.01(1), Alal,
Val0.74(1), Leu0.75(1), Lys2.01(2) (10) P(76−84); BOC−Asp(OBzl)−Val−Leu
−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z−
Cl)−Ser(Bzl)−Gln−OBzl[10]

[9] 183.5 g (116 mM) was added to 500 ml of TFA, and after stirring at room temperature for 65 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected and dissolved in 900 ml of DMF. to this
HOBT18.8g (1.2x M), BOC-Asp (OBzl)
-OH45.0g (1.2x M), BOC-Asp(OBzl)-
OH45.0g (1.2xM) and WSCI25.5ml (1.2
After adding M) and adjusting the pH to 7 with NMM, the mixture was stirred at room temperature overnight. The reaction solution and DMF were distilled off under reduced pressure, and ice water was added to the residue. The resulting precipitate was collected and washed twice with hot methanol. The insoluble matter was dissolved in hot DMF and ethanol was added. After collecting the resulting precipitate and suspending it in methanol,
205.5 g (yield 99.14%) of [10] was obtained. Melting point: 259-262℃ TLC; Rf ₂ = 0.81 [α] ²⁷ _D -13.7 (C = 1.0, DMF) Elemental analysis [as C ₉₁ H ₁₁₈ O ₂₁ N ₁₂ Cl ₂ ] C% H% N% Measured value 61.01 6.84 9.54 Calculated value 61.16 6.66 9.41 (11) P(75−84); BOC−Val−Asp(OBzl)−Val
−Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys
(Z-Cl)-Ser(Bzl)-Gln-OBzl [11] [10] 196.55 g (0.11 M) was added to 500 ml of TFA, and after stirring at room temperature for 60 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected and dissolved in DMF1.3. Add 19.3 g of HORT (1.3 times M) and BOC-Val- while stirring.
OH31.1g (1.3xM) and WSCl26.2ml (1.3
After adding M) and adjusting the pH to 7 with NMM, the mixture was stirred at room temperature. After 1 hour, the reaction solution became gel-like, so it was left overnight. Next, 400ml of NMP,
2,4-dinitrophenol 20.2g,
26.2 ml of WSCI (1.3x M) was added. It became gel-like again in about 10 minutes, and was left overnight at room temperature. After the reaction, ice and 5% sodium bicarbonate solution were added, and the resulting precipitate was collected, washed three times with water, and then four times with hot methanol to obtain 203.74 g (yield 98.2%) of [11]. . Melting point: 267-270℃ TLC; Rf ₃ = 0.56 Elemental analysis [as C ₉₆ H ₁₂₇ O ₂₂ N ₁₃ Cl ₂ H ₂ O] C% H% N% Measured value 60.25 6.78 9.82 Calculated value 60.55 6.83 9.56 Amino acid analysis [ Sample 3.1mg/1ml 6N hydrochloric acid + 0.1
ml anisole, 48 hours, 110℃ hydrolysis];
Asp1.04(1), Thr0.83(1), Ser0.78(1), Glu1.03
(1), Ala1.08(1), Leu1, Val1.87(2), Lys2.19(2) (12) P(74−84); BOC−Asp(OBzl)−Val−Asp
(OBzl)−Val−Leu−Thr(Bzl)−Lys(Z−Cl)
−Ala−Lys(Z−Cl)−Ser(Bzl)−Gln−OBzl
[12] [11] 151.2g (0.08M) of methylene chloride 100
After adding 450 ml of TFA and stirring at room temperature for 40 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and 500 ml of DMF and NMP1 were added to the resulting precipitate to dissolve it. Ice and 5% sodium bicarbonate solution were added to this, and the resulting precipitate was collected, thoroughly washed with water, and then dried. Dissolve this in a mixture of 500 ml of DMF and NMP1, and dissolve 44.0 g of BOC-Asp (OBzl)-OSU (1.3
times mol), HOBT1.4g (0.13 times M), NMM11.4
ml (1.3 times M) and stirred at room temperature overnight.
The reaction solution was added to ice water, the resulting precipitate was collected, methanol was added, and the mixture was heated. This process 2
This was repeated twice to obtain 157.6 g (yield 94.2%) of [12]. TLC; Rf ₃ = 0.56 Elemental analysis [as C ₁₀₇ H ₁₃₅ O ₂₅ N ₁₄ Cl ₂ ] C% H% N% Measured value 61.35 6.66 9.90 Calculated value 61.45 6.65 9.38 Amino acid analysis [6N hydrochloric acid/anisole, 110
°C, 48 hours]; Asp1.86(2), Thr0.87(1), Ser0.71
(1), Glu1.01(1), Ala1.00(1), Val1.91(2),
Leu1, Lys2.01(2) (13) P(72−73); BOC−Lys(Z−Cl)−Ala−
OH [13] [3] 48.0g (96mM) in 100ml of ethanol
and cooled to 0°C with 1N−
115.2 ml of NaOH (1.2 times M) was added and stirred for 50 minutes. After the reaction, 19 ml of 1N hydrochloric acid was added to adjust the pH to 6, and then ethanol was distilled off under reduced pressure at 35°C. Ethyl acetate, ice water, and 96 ml of 1N hydrochloric acid were added to the residue for extraction. Ethyl acetate carbonate was washed with water, dried over anhydrous sodium sulfate, and then dried under reduced pressure. The residue was recrystallized from ethyl acetate-hexane to give [13] 45.90 g (yield 98.4
%) was obtained. Melting point: 116-119℃ TLC; Rf ₇ = 0.44 Elemental analysis [as C ₂₂ H ₃₂ O ₇ N ₃ Cl] C% H% N% Measured value 54.57 6.91 8.95 Calculated value 54.37 6.64 8.65 (14) P (72-84 )；BOC−Lys(Z−Cl)−Ala−
Asp(OBzl)−Val−Asp(OBzl)−Val−Leu−
Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z−Cl)
-Ser(Bzl)-Gln-OBzl [14] [12] 125.46 (60mM) with methylene chloride 60
ml and 360 ml of TFA were added, and after stirring at room temperature for 55 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue and the resulting precipitate was collected. DMF600 for this
ml and 600 ml of NMP were added and dissolved. This was added to ice + 5% sodium bicarbonate solution. The resulting precipitate was collected, washed with water three times, then washed with methanol and ether, and then dried. Add 1200ml of NMP to this
Add 600ml of DMF and dissolve, 10.56g of HOBT
(1.3 times M), [13] 37.92g (1.3 times M),
14.28 ml of WSCI (1.3 times M) was added and stirred at room temperature for 3 days. The reaction solution was added to ice water, the resulting precipitate was collected, washed three times with water, and then diluted with hot methanol for two
Washed twice. After washing with ether, the product was dried to obtain 142.74 g (yield: 96.7%) of [14]. Elemental analysis [as C ₁₂₄ H ₁₆₁ D ₂₉ N ₂₇ Cl ₃ ] C% H% N% Measured value 60.30 6.79 9.70 Calculated value 60.54 6.60 9.68 Amino acid analysis [6N hydrochloric acid/anisole, 110
°C, 48 hours]; Asp1.86(2), Thr0.85(1), Ser0.67
(1), Glu1.02(1), Ala1.90(2), Val1.93(2),
Leu1, Lys2.93(3) (15) P(71−84); BOC−Asp(OBzl)−Lys(Z
−Cl) −Ala−Asp(OBzl) −Val−Asp(OBzl)
−Val−Leu−Thr(Bzl)−Lys(Z−Cl)−Ala
-Lys(Z-Cl)-Ser(Bzl)-Gln-OBzl [15] [14] 132.84g (54mM) was added to 420ml of TFA, and after stirring at room temperature for 55 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, the resulting precipitate was collected, and 720 ml of NMP and 720 ml of DMF were added and dissolved. This was added to ice + 5% sodium bicarbonate solution, and the resulting precipitate was collected and washed three times with water and once each with methanol and ether. to this precipitate
Add and dissolve 840 ml of NMP and 840 ml of DMF, and add 11.93 g of 2,4-dihydrophenol (1.2 times M), 0.732 g of HOBT (0.14 times M), and BOC-Asp.
(OZzl) - OSU3.18g (1.2x M), NMM5.94ml
(1.4 times M) and stirred at room temperature for 2 days. 120 ml of NMP and 60 ml of DMF were added to the reaction solution to dissolve it, 4.56 g of BOC-Asp(OBzl)-OSU (0.2 times M) and 1.19 ml of NMM (0.2 times M) were added, and the mixture was further stirred at room temperature for 2 days. The reaction solution was added to ice water, and the resulting precipitate was collected and washed with water. Then,
It was suspended in hot methanol and collected after cooling. Repeat this operation 3 times [15] 136.72g
(yield 95.0%). Elemental analysis [as C ₁₃₅ H ₁₇₂ O ₃₂ N ₁₈ Cl ₃ ] C% H% N% Measured value 60.33 6.55 9.76 Calculated value 60.83 6.51 9.46 Amino content analysis [6N hydrochloric acid/anisole, 110
°C, 48 hours] Asp2.60(3), Thr0.83(1), Ser0.65
(1), Glu1.00(1), Ala1.81(2), Val1.92(2),
Leu1, Lys2.85(3) (16) P(70−84); BOC−Ala−Asp(OBzl)−
Lys(Z-Cl)-Ala-Asp(OBzl)-Val-Asp
(OBzl)−Val−Leu−Thr(Bzl)−Lys(Z−Cl)
−Ala−Lys(Z−Cl)−Ser(Bzl)−Gln−OBzl
[16] [15] 108.74 g (40.8 mM) was added to 325 ml of TFA, and after stirring at room temperature for 70 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, the resulting precipitate was collected, dissolved by adding 600 ml of NMP and 600 ml of DMF, and then added to 5% aqueous sodium bicarbonate. The resulting precipitate was collected and washed three times with water and once with methanol. Next, add 720ml of NMP to this precipitate.
Add and dissolve 720ml of DMF, add 9.0g of 2,4-dinitrophenol, 0.55g of HOBT (0.1x M), 17.52g of BOC-Ala-OSU (1.5x M),
6.72 ml of NMM (1.5 times M) was added and stirred at room temperature overnight. Next, 2.34 g of BOC-Ala-OSU,
0.9 ml of NMM was added and stirred at room temperature for 5 days.
Add the reaction solution to ice water, collect the resulting precipitate, and wash it three times with water and twice with methanol [16]
109.78g (yield 98.3%) was obtained. Melting point: 280℃ or higher (decomposition) Amino acid analysis [6N hydrochloric acid/anisole, 110℃, 48 hours]; Asp2.57(3), Thr0.84(1), Ser0.67
(1), Glu1.00(1), Ala2.53(3), Val1.98(2),
Leu1, Lys2.79(3) (17) P(69−84); BOC−Glu(OBzl)−Ala−
Asp(OBzl)−Lys(Z−Cl)−Ala−Asp(OBzl)
−Val−Asp(OBzl)−Val−Leu−Thr(Bzl)−
Lys(Z-Cl)-Ala-Lys(Z-Cl)-Ser(Bzl)
-Gln-OBzl [17] [16] 85.38g (31.2mM) was added to 280ml of TFA, and after stirring at room temperature for 60 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, the resulting precipitate was collected, dissolved by adding 540 ml of DMF and 540 ml of NMP, and added to 5% sodium bicarbonate water + ice. The resulting precipitate was collected and washed three times with water and once with methanol. Add 600ml of DMF and 780ml of NMP to this, dissolve, and add 2,4-dinitrophenol.
6.89g (1.2xM), HOBT0.59g (0.14xM),
BOC-Glu(OBzl)-OSU18.97g (1.4x M),
4.8 ml of NMM (1.4 times M) was added and stirred at room temperature for 3 days. Then BOC−Glu(OBzl)−
OSU2.71g (0.2x M) DMF60ml + NMP50
ml solution and NMM0.64ml
(1.4 times M) was added, and the mixture was further stirred at room temperature for 3 days. Next, the same amount of BOC−Glu(OBzl)−
OSU and NMM were added and stirred at room temperature overnight.
After the reaction, the reaction solution was added to ice water, and the resulting precipitate was collected and washed three times with water. This precipitate was suspended in hot methanol and collected after cooling. Repeat this operation three times [17] 88.97g (yield
96.5%). Amino acid analysis [6N hydrochloric acid/anisole, 110℃, 24 hours]; Asp2.66(3), Thr0.91(1), Ser0.78
(1), Glu1.76(2), Ala2.65(3), Val1.97(2),
Leu1, Lys2.88(3) (18) P(66−68); BOC−Ser(Bzl)−Leu−Gly
−OBzl [18] BOC−Leu−OH・H ₂ O45.64g, H−Gly−
OBzl・TosOH61.87g and HOBT24.87g
Dissolved in THF200ml and cooled in this WSCI33.69
ml was added dropwise, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to obtain an oily substance. This was dissolved in 600 ml of ethyl acetate and washed three times each with 5% sodium bicarbonate solution, 1N hydrochloric acid, and water. The organic layer was dried over anhydrous sodium sulfate and dried under reduced pressure to give 69.0 g of oil (yield:
99%). This was dissolved in 10 ml of methylene chloride, and 250 ml of TFA was added while cooling to 5°C, followed by reaction at room temperature for 20 minutes with stirring. TFA was distilled off under reduced pressure, and 200 ml of DMF was added to the residue, which was neutralized by adding NMM while cooling to 0°C. to this
HOBT20.7g (0.19M), BOC−Ser(Bzl)−
OH56g (0.19M) and WSCI34.8ml
(0.19M) and stirred at room temperature overnight.
DMF was distilled off under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed successively with 5% aqueous sodium bicarbonate, 1N hydrochloric acid, and water.
After drying over anhydrous magnesium sulfate, it was concentrated under reduced pressure. Hexane was added to the residue, and the resulting precipitate was collected. It was recrystallized from ethyl acetate-hexane and then from ethyl acetate-ether-hexane to obtain 72.47 g (yield 72.0%) of [18]. Melting point: 112-113℃ TLC; Rf ₅ = 0.55 Elemental analysis [as C ₃₀ H ₄₁ O ₇ N ₃ ] C% H% N% Measured value 65.06 7.75 7.36 Calculated value 64.84 7.44 7.56 (19) P (65-68) ;BOC−Lys(Z−Cl)−Ser
(Bzl)-Leu-Gly-OBzl [19] [18] 72.47g (0.13M) of methylene chloride 20
ml, added 250 ml of TFA while cooling to 5°C, and stirred at room temperature for 20 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. Collect the resulting precipitate and add 100 ml of DMF. 5 for this
The mixture was neutralized by adding NMM while cooling at ℃. On the other hand, BOC-Lys(Z-Cl)-OH・TBA70g
(0.143M) was added with 200ml of ethyl acetate, and washed twice with 1N hydrochloric acid and water. The ethyl acetate layer was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. Add 100ml of DMF to the obtained oil, and add BOC-Lys.
A DMF solution of (Z-Cl)-OH was prepared. To the above neutralized DMF solution were added 19.3 g of HOBT, the DMF solution of BOC-Lys(Z-Cl)-OH obtained above, and 26.2 ml (0.143 M) of WSCI, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure.
400 ml of ethyl acetate was added to the residue, and the mixture was washed three times with 5% aqueous sodium bicarbonate, twice with 1N hydrochloric acid, and twice with water. The organic layer was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. Ether and hexane were added to the residue, the resulting precipitate was collected, and recrystallized from ethyl acetate-methanol-hexane to give 104 g of [19].
(yield 94.6%). Melting point: 128-130℃ TLC: Rf ₁ = 0.51, Rf ₂ = 0.88 Elemental analysis [as C ₄₄ H ₅₈ O ₁₀ N ₅ Cl] C% H% N% Measured value 61.96 7.02 7.91 Calculated value 62.00 6.86 8.22 Amino acid analysis [ 1μM/6N hydrochloric acid 0.3ml, 105℃, 20 hours]; Ser0.92(1), Gly0.98(1), Leu1,
Lys0.99(1) (20) P(65−68); BOC−Lys(Z−Cl)−Ser
(Bzl)-Leu-Gly-OH [20] [19] Add 600 ml of methanol to 85.3 g,
While stirring under cooling at °C, 120 ml of 1N-NaOH was added, followed by stirring at room temperature for 3 hours. After reaction, 5
After adding 20 ml of 1N hydrochloric acid while cooling at ℃, methanol was distilled off under reduced pressure. Add 100 ml of 1N hydrochloric acid to the aqueous solution of the residue while cooling at 5°C, and add 500 ml of chloroform.
Extracted with. The chloroform layer was washed with water, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. Hexane was added to the residue, and the resulting precipitate was collected and recrystallized from ethyl acetate to obtain 66.21 g (87% yield) of [20]. Melting point: 156-158℃ TLC; Rf ₂ = 0.63 Elemental analysis [as C ₃₇ H ₅₂ O ₁₀ N ₅ Cl] C% H% N% Measured value 58.49 6.95 9.09 Calculated value 58.30 6.88 9.19 Amino acid analysis [1 μM/0.3 ml 6N hydrochloric acid , 105 ℃, 20 hours]; Ser0.92(1), Gly0.97(1), Leu1,
Lys0.99(1) (21) P(64−68); BOC−Glu(OBzl)−Lys(Z−
Cl)-Leu-Gly-OH [21] [20] 66.2g (87mM) and 30ml of methylene chloride
After adding 250ml of TFA while cooling to 5℃,
Stirred at room temperature for 45 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected and dissolved in 100 ml of DMF. This is 5
Neutralized with NMM while cooling to ℃. Since a precipitate had separated out, an additional 500 ml of DMF was added. BOC-Glu(OBzl)-OSU 50g (113mM) was added to this solution.
After adding 1.53 g (11.3 mM) of HOBT and neutralizing with NMM, the mixture was stirred at room temperature overnight. After the reaction,
DMF was distilled off under reduced pressure, and the residue was poured into water. The resulting precipitate is washed with water and dried. Acetone-
Recrystallize twice with methanol [21] 63.85g
(yield 73.5%). Melting point: 165-167℃ (decomposition) TLC; Rf ₄ = 0.72 Elemental analysis [as C ₄₉ H ₆₅ O ₁₄ N ₆ Cl] C% H% N% Measured value 59.61 6.76 8.31 Calculated value 59.00 6.57 8.42 Amino acid decomposition [0.927 mg /0.3ml 6N hydrochloric acid, 105℃ 24 hours]; Ser0.92(1), Glu0.99(1), Gly0.97
(1), Leu1, Lys0.99(1) (22) P(62−63); BOC−Ser(Bzl)−His−
NHNH ₂ [22] BOC−Ser(Bzl)−OH37g (0.125M)
THF150ml, H-His-OMe・2HCl31.5g
(0.13M) and HOBT17.6g (0.13M), and WSCI23.8ml (0.13M) under cooling to 5℃.
After adding 150 ml of DMF, the mixture was stirred at room temperature overnight. Additionally HOBT4.4g and WSCI6ml
was added and stirred at room temperature overnight. After the reaction, the solvent was distilled off under reduced pressure, and the residual oil was dissolved in ethyl acetate and washed three times with 5% aqueous sodium bicarbonate and three times with water. It was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Hexane was added to the residue, the resulting precipitate was collected, and recrystallized from ethyl acetate-ether-hexane to obtain crude BOC-Ser(Bzl)-His-
46.1 g of OMe (TLC; Rf ₃ =0.56) was obtained. Dissolve 44.6 g (0.1 M) of the above product in 300 ml of DMF and add 100 g of hydrazine hydrate (100%) to this.
ml and stirred overnight at room temperature. After the reaction,
DMF was distilled off under reduced pressure. The residue was extracted eight times with ethyl acetate, and the extract was washed with a small amount of water and then dried over anhydrous magnesium sulfate. It was concentrated under reduced pressure, hexane was added to the residue, and the resulting precipitate was collected. This was purified by silica gel column chromatography [elution solvent: chloroform-methanol-ethyl acetate (5:0 to 1:5)]. The corresponding sections were dried under reduced pressure and the residue was crystallized from benzene-hexane (very small amount). After being placed in an ice chamber, the precipitated crystals were recrystallized twice from ethyl acetate-methanol-hexane to obtain [22]. Melting point: 125-129℃ TLC: Rf ₄ = 0.70, Rf ₇ = 0.14 Elemental analysis [as C ₂₁ H ₃₀ O ₅ N ₆ ] C% H% N% Measured value 56.30 6.98 18.54 Calculated value 56.49 6.77 18.82 (23) P (62−68);BOC−Ser(Bzl)−His−Glu
(OBzl)−Lys(Z−Cl)−Ser(Bzl)−Leu−Gly
Dissolve 33.5 g of -OH [23] [22] in 200 ml of DMF, and add 52 ml of a 4.32N dioxane solution of hydrogen chloride and 20 ml of isoamyl nitrile while cooling to -50°C.
Stirred at -20°C for 10 minutes. It was then cooled to −50° C. and 31.6 ml of Et ₃ N was added. On the other hand, add 20 ml of methylene chloride to 63.85 g (64 mM) of [21], and add 200 ml of TFA while cooling to 5°C.
After addition, the mixture was stirred at room temperature for 50 minutes. After the reaction,
TFA was distilled off under reduced pressure, ether was added to the residual oil, and the resulting precipitate was collected. this
It was dissolved in 200 ml of DMF and neutralized with NMM while cooling to 5°C. The neutralized DMF solution was added to the triethylamine-treated solution cooled to 0° C. and stirred overnight in an ice chamber and overnight at room temperature. After reaction, DMF
was distilled off under reduced pressure, and the residue was dissolved in methanol. Pour this into water, remove the resulting precipitate,
Washed with water and dried. Recombine with DMF-ether,
After washing twice with methanol, 59.16 g (yield 60.1%) of [23] was obtained. Melting point: 209-213℃ (decomposition) TLC: Rf ₄ = 0.66 Elemental analysis [as C ₆₅ H ₈₃ O ₁₇ N ₁₀ Cl] C% H% N% Measured value 59.43 6.58 10.67 Calculated value 59.51 6.38 10.68 Amino acid analysis [1.549 mg /0.5ml 6N hydrochloric acid, 105℃, 21 hours]; Ser1.82(2), Glu1.01(1), Gly0.98
(1), Leu1, Lys1.01(1), His0.94(1) (24) P(62−84); BOC−Ser(Bzl)−His−Glu
(OBzl)−Lys(Z−Cl)−Ser(Bzl)−Leu−Gly
−Glu(OBzl)−Ala−Asp(OBzl)−Lys(Z−
Cl) −Ala−Asp(OBzl)−Val−Asp(OBzl)−
Val−Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−
250 ml of TFA was added to 75.35 g (25.5 mM) of Lys(N-Cl)-Ser(Bzl)-Gln-OBzl [24] [17], and the mixture was stirred at room temperature for 60 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected and dissolved by adding 500 ml of NMP and 500 ml of DMF. Add this to ice-5% sodium bicarbonate solution,
The resulting precipitate was collected and washed with water and methanol in that order. NMP1 and DMF1 were added to this precipitate and dissolved, and to this was added 5.61 g of 2,4-dinitrophenol (1.2 times M), 4.08 g of HOBT (1.2 times M), [23] 40.16 g (1.2 times M) and WSCI5. 6
ml (1.2 times M) and stirred at room temperature for 4 days.
After the reaction, the reaction solution was poured into 5° C. sodium bicarbonate water-ice, and the resulting precipitate was collected. Washed with water, hot methanol, and methanol (twice) in order [24] 93.53g
(yield 88.4%). Amino acid analysis; Asp2.67(3), Thr0.81(1),
Ser1.37(3), Glu2.59(3), Gly0.76(1), Ala2.68
(3), Val2, Leu1.74(2), Lys3.66(4), His0.66
(1) (25) P(61−84); BOC−Glu(OBzl)−Ser(Bzl)
-His-Glu(OBzl)-Lys(Z-Cl)-Ser(Bzl)
−Leu−Gly−Glu(OBzl)−Ala−Asp(OBzl)
−Lys(Z−Cl)−Ala−Asp(OBzl)−Val−
Asp(OBzl)−Val−Leu−Thr(Bzl)−Lys(Z
−Cl) −Ala−Lys(Z−Cl)−Ser(Bzl)−Gln−
150 ml of TFA was added to 41.5 g (10 mM) of OBzl [25] [24], and after stirring at room temperature for 60 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, the resulting precipitate was collected, and 300 ml of DMF and 500 ml of NMP were added to dissolve it. This was poured into 5% sodium bicarbonate water and ice, and the resulting precipitate was collected, washed with water, and then washed with methanol. Add and dissolve 700 ml of DMF and 600 ml of NMP to this precipitate, add 22 g of 2,4-dinitrophenol (1.2 times M), BOC-Glu(OBzl)-
6.08 g of OSU (1.4 times M), 0.23 g of HOBT (0.14 times M) and 1.52 ml of NMM (1.4 times M) were added and stirred at room temperature overnight. Next, add this reaction solution to
0.87 g (0.2 times M) of BOC-Glu(OBzl)-OSU and 0.22 ml (0.2 times M) of NMM were added, and the mixture was further stirred overnight. Pour the reaction solution into ice-5% sodium bicarbonate,
The resulting precipitate was collected, thoroughly washed with water, and then washed with hot methanol three times to obtain 40.7 g (yield) of [25].
93.2%). Amino acid analysis [/6N hydrochloric acid, anisole, 110 °C, 48 hours]; Asp2.66(3), Thr0.85(1), Ser1.56
(3), Glu3.14(4), Gly0.71(1), Ala2.65(3),
Val2, Leu1.73(2), Lys3.67(4), His0.63(1) (26) P(59−60); BOC−Leu−Val−OBzl [26] H−Val−OBzl・TOSOH17. 8g (47mM)
100 ml of ethyl acetate was added to the solution, and the mixture was washed successively with 5% sodium bicarbonate water and water. The ethyl acetate layer was dried over anhydrous sodium sulfate, and then ethyl acetate was distilled off under reduced pressure. residue
Dissolve in 100ml of DMF and add BOC-Leu-
OH・H ₂ O11.7g (56.4mM), HOBT9.3g
(1.2xM) and 10.3ml of WSCI (1.3xM) were added and stirred overnight at room temperature. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 300 ml of ethyl acetate.
It was washed with 5% sodium bicarbonate solution, 1N hydrochloric acid, and water in this order. After drying with anhydrous sodium sulfate, it was dried under reduced pressure. The residue was crystallized from ethyl acetate-hexane to give 17.55 g of [26]
(yield 88.8%). TLC; Rf ₆ = 0.85 (27) P (58−60); BOC−Val−Leu−Val−
Add 50ml of TFA to OBzl [27] [26] 17.2g (41mM),
After stirring at room temperature for 30 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and the resulting crystals were collected and dissolved in 60 ml of DMF. to this
HOBT7.7g (1.2x M), BOC-Val-OH10.7
After adding 9.0 ml of WSCI (1.2 times M) and 9.0 ml of WSCI (1.2 times M) and adjusting the pH to 7 with NMM, the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 300 ml of ethyl acetate, and then washed successively with 5% aqueous sodium bicarbonate, 1N hydrochloric acid, and water. It was dried with anhydrous sodium sulfate and evaporated to dryness. The residue was recrystallized twice from ethyl acetate-hexane to obtain 17.38 g (yield: 81.6%) of [27]. Melting point: 149-152℃ TLC; Rf ₅ = 0.82 [α] ²² _D -32.36 (C = 1.0, DMF) Elemental analysis [as C ₂₈ H ₄₅ O ₆ N ₃ ] C% H% N% Measured value 64.80 8.81 8.20 Calculated value 64.71 8.73 8.09 (28) P(57−60); BOC−Asn−Val−Leu−Val
−OBzl [28] [27] Add 50ml of TA to 17.15g (33mM),
After stirring at room temperature for 25 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected and dissolved in 100 ml of DMF. to this
HOBT0.62g (0.14x M), BOC-Asn-
Add 16.32g of ONP (1.4x M) and PH7 with NMM
After adjusting the temperature, the mixture was stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, and the residue was poured into 5% sodium bicarbonate water-ice. The resulting precipitate was dissolved in chloroform and washed successively with 5% sodium bicarbonate water and water. It was dried with anhydrous sodium sulfate and evaporated to dryness. The residue was recrystallized twice from ethanol-ether [28] 13.82
g (yield 79.5%) was obtained. TLC; Rf ₂ = 0.70 Elemental analysis [as C ₃₂ H ₅₁ O ₈ N ₅ ] C% H% N% Measured value 60.83 8.19 11.09 Calculated value 60.64 8.11 11.05 (29) P (57-60); BOC-Asn-Val −Leu−Val
-OH [29] [28] 13.2g (25mM) with 50ml of ethanol
Dissolve in a mixture of 60 ml of DMF and add 5% Pd/C1
g and hydrogenated for 3 hours. The catalyst was separated and the liquid was concentrated under reduced pressure. The residue is ethanol-
It was recrystallized twice from ether to obtain 9.70 g (yield 71.4%) of [29]. TLC; Rf ₂ = 0.56 Amino acid analysis; Asp1.02(1), Val1.95(2), Leu1
(1) Elemental analysis [as C ₂₅ H ₄₅ O ₈ N ₅ ] C% H% N% Measured value 55.02 8.13 13.06 Calculated value 55.23 8.34 12.88 (30) P(57−84); BOC−Asn−Val−Leu− Val
−Glu(OBzl)−Ser(Bzl)−His−Glu(OBzl)−
Lys(Z−Cl)−Ser(Bzl)−Leu−Gly−Glu
(OBzl)-Ala-Asp(OBzl)-Lys(Z-Cl)-
Ala−Asp(OBzl)−Val−Asp(OBzl)−Val−
Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys
150 ml of TFA was added to 39.75 g (9.1 mM) of (ZCl)-Ser(Bzl)-Gln-OBzl [30] [25], and the mixture was stirred at room temperature for 60 minutes. After the reaction, TFA was distilled off under reduced pressure, ether was added to the residue, the resulting precipitate was collected, and 600 ml of DMF and 600 ml of NMP were added and dissolved. This was added to ice-5% sodium bicarbonate solution, the resulting precipitate was collected, and washed three times with water and once with methanol. This precipitate contains NMP1.2 and
Add DMF1.2 and melt, add HOBT1.60 to this
g (1.3x M), 2,4-dinitrophenol
Add 2.18 g (1.3 times M), [29] 6.43 g (1.3 times M) and 4.32 ml of WSCI (2.6 times M), and incubate at room temperature for 2 hours.
The mixture was stirred for several days. Furthermore [29] 0.99g (0.2x M)
A solution prepared by dissolving the above in 100 ml of DMF was added, and the mixture was stirred at room temperature overnight. Add the reaction solution to 5% sodium bicarbonate water-ice,
The resulting precipitate was collected, suspended in 100 ml of DMF, and heated. After cooling, the insoluble matter was removed. Furthermore, methanol and ethanol were each heat-treated in the same manner as above [30] 42.25 g (yield:
97.2%). Amino acid analysis; Asp3.34(4), Thr0.93(1),
Ser1.60(3), Glu3.24(4), Gly0.85(1), Ala3,
Val3.39(4), Leu2.58(3), Lys4.24(4), His0.79
(1) (31) P(56−84); BOC−Asp(OBzl)−Asn−
Val−Leu−Val−Glu(OBzl)−Ser(Bzl)−His
−Glu(OBzl)−Lys(Z−Cl)−Ser(Bzl)−Leu
−Gly−Glu(OBzl)−Ala−Asp(OBzl)−Lys
(Z−Cl)−Ala−Asp(OBzl)−Val−Asp
(OBzl)−Val−Leu−Thr(Bzl)−Lys(Z−Cl)
−Ala−Lys(Z−Cl)−Ser(Bzl)Gln−OBzl
[31] [30] 150 ml of TFA was added to 28.7 g (6 mM) and stirred at room temperature for 50 minutes. After the reaction, TFA was distilled off under reduced pressure, ether was added to the residue, and the resulting precipitate was collected and dried overnight in a NaOH desiccator. Add 300ml of DMF to this sediment.
Add 500ml of NMP and dissolve it.
HOBT0.16g (0.2x M), BOC-Asp (OBzl)
- Add OSu5.04g (2x M), PH7.0 with NMM
After adjusting the temperature, the mixture was stirred at room temperature overnight. moreover
BOC-Asp(OBzl)-OSU (2.50 g (1xM)) was added and stirred overnight. Pour the reaction solution into ice water,
The resulting precipitate was collected, washed with water, added to methanol, and heated. After cooling, the insoluble matter was removed. Perform the above heat treatment 3 times [31] 27.75g
(yield 92.5%). Amino acid analysis; Asp4.00(5), Thr0.92(1),
Ser1.69(3), Glu3.57(4), Gly0.81(1), Ala3,
Val3.22(4), Leu2.62(3), Lys4.12(4), His0.62
(1) (32) P(55−84); BOC−Glu(OBzl)−Asp
(OBzl) −Asn−Val−Leu−Val−Glu(OBzl)
−Ser(Bzl)−His−Glu(OBzl)−Lys(Z−Cl)
−Ser(Bzl)−Leu−Gly−Glu(OBzl)−Ala−
Asp(OBzl)−Lys(Z−Cl)−Ala−Asp(OBzl)
−Val−Asp(OBzl)−Val−Leu−Thr(Bzl)−
Lys(Z-Cl)-Ala-Lys(Z-Cl)-Ser(Bzl)
-Gln-OBzl [32] [31] 150 ml of TFA was added to 27.50 g (5.5 mM) and stirred at room temperature for 55 minutes. After the reaction, TFA was distilled off under reduced pressure, ether was added to the residue, the resulting precipitate was collected, and then washed in a NaOH desiccator for 2 hours.
Dry for days. Add 300ml of DMF to this sediment.
Add 500ml of NMP to dissolve, and add 2,4-
Dinitrophenol 1.01g (1xM),
HOBT0.11g (0.15xM) and BOC-Glu
(OBzl)-OSU 3.10 g (1.3 times M) was added and stirred overnight at room temperature. Furthermore, BOC−Glu(OBzl)−
3.10 g of OSU (1.3 times M) was added and stirred for 2 days. The reaction solution was poured into ice + 5% aqueous sodium bicarbonate, and the resulting precipitate was washed twice with 5% aqueous sodium bicarbonate and three times with water. Next, methanol was added and heat treated, and after cooling, insoluble matter was removed. The above heat treatment was carried out three times [32] to obtain 26.77 g (yield 93.3%). Amino acid analysis; Asp4.11(5), Thr0.92(1),
Ser1.59(3), Glu3.99(5), Gly0.83(1), Ala3,
Val3.28(4), Leu2.49(3), Lys4.08(4), His0.71
(1) (33) P(53−54); BOC−Lys(N−Cl)−Lys(Z
−Cl)−PAC [33] BOC−Lys(Z−Cl)−OH・TBA36.6g (75
mM) was suspended in 300 ml of ethyl acetate, washed with ice and 1N hydrochloric acid, and then with water. The ethyl acetate layer was dried over anhydrous sodium sulfate, dried under reduced pressure, and the residue was dissolved in DMF50.
Dissolved in ml. To this were added 19.40 g of phenacyl bromide and 13.60 ml of Et ₃ N (1.3 times M) while cooling at 0° C., and the mixture was stirred at room temperature for 4 hours. After adding a solution of 3.68 g (0.5 times M) of sodium acetate dissolved in 500 ml of ethyl acetate to the reaction solution, 5%
It was washed three times each in the order of sodium bicarbonate solution, 1N hydrochloric acid, and water. The ethyl acetate layer was dried over anhydrous sodium sulfate and then dried under reduced pressure. The residue was subjected to silica gel column chromatography [eluent: benzene-ethyl acetate (2:
1)], and the fractions near Rf ₁ =0.77 on TLC were collected and dried under reduced pressure. The residue was recrystallized from ether-hexane to give BOC-Lys(Z-Cl)-
23.46 g (yield 60.5%) of PAC was obtained. Melting point: 52-54℃ TLC; Rf ₁ = 0.86 20.68g (40mM) of the above product and 60ml of TFA
was added and stirred at room temperature for 20 minutes. After the reaction,
TFA was distilled off under reduced pressure, ether was added to the residue,
The resulting precipitate was collected and dissolved in 50 ml of DMF to obtain DMF solution A. On the other hand, BOC−Lys(Z−Cl)−OH・
23.42 g (1.2 times M) of TBA was suspended in 200 ml of ethyl acetate and washed with ice + 200 ml of 1N hydrochloric acid and 100 ml of water. The ethyl acetate layer was dried over anhydrous sodium sulfate and then dried under reduced pressure. The obtained oil was dissolved in 50 ml of DMF to obtain DMF solution B. DMF solution B prepared in the DMF solution A,
After adding 6.48 g of HOBT (1.2 times M) and 8.78 ml of WSCI (1.2 times M) and adjusting the pH to 7 with NMM, the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 500 ml of ether.
Washed three times with sodium bicarbonate solution. 300 ml of ethyl acetate was added to the ether layer, and the mixture was washed twice with 1N hydrochloric acid and three times with water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was recrystallized three times from ether to obtain 18.73 g (yield 57.5%) of [33]. Melting point: 72-75℃ Elemental analysis [as C ₄₁ H ₅₀ O ₁₀ N ₄ Cl ₂ ] C% H% N% Measured value 59.22 5.98 6.81 Calculated value 59.35 6.07 6.75 (34) P (53-54); BOC-Lys (Z−Cl)−Lys(Z
-Cl)-OH[34] 4.07g (5mM) of [34] was dissolved in 30ml of acetic acid, this was added to 20g of zinc powder/30ml of acetic acid, and the mixture was stirred at room temperature for 1.5 hours. After the reaction, the zinc powder was separated, and acetic acid was distilled off from the solution under reduced pressure. The residue was dissolved in 100 ml of ether and extracted three times with 70 ml of 5% aqueous sodium bicarbonate. The aqueous layer was adjusted to pH 2 with 1N hydrochloric acid under ice cooling, and extracted with ethyl acetate. The ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, and then dried under reduced pressure.
The residue was recrystallized from ether-hexane to obtain 3.08 g (yield: 85.8%) of [34]. Melting point: 59-62℃ TLC; Rf ₂ = 0.45 Elemental analysis [as C ₃₃ H ₄₄ O ₉ N ₄ Cl ₂ ] C% H% N% Measured value 55.58 6.40 7.58 Calculated value 55.69 6.23 7.87 (35) P (53− 84);BOC−Lys(Z−Cl)−Lys(Z
−Cl)−Glu(OBzl)−Asp(OBzl)−Asn−Val
−Leu−Val−Glu(OBzl)−Ser(Bzl)−His−
Glu(OBzl)−Lys(Z−Cl)−Ser(Bzl)−Leu−
Gly−Glu(OBzl)−Ala−Asp(OBzl)−Lys(Z
−Cl) −Ala−Asp(OBzl) −Val−Asp(OBzl)
−Val−Leu−Thr(Bzl)−Lys(Z−Cl)−Ala
-Lys(Z-Cl)-Ser(Bzl)-Gln-OBzl [35] [32] Add 50ml of TFA to 7.83g (1.5mM),
Stirred at room temperature for 55 minutes. After the reaction, TFA was distilled off under reduced pressure, ether was added to the residue, the resulting precipitate was collected, and 150 ml of DMF and 150 ml of NMP were added and dissolved. Add to this HOBT0.41g (2.0g) under ice cooling.
times M), [34] 2.13g (2.0 times M) and
Add 0.55 ml of WSCI (2.0x M) and incubate at 5-10℃.
The mixture was stirred for several days. The reaction solution was poured into ice water, and the resulting precipitate was collected, washed with water, and then heated with methanol. After cooling, the insoluble matter was removed. This heat treatment was performed three times [35] to obtain 8.31 g (yield 95.3%). Amino acid analysis; Asp4.22(5), Thr0.92(1),
Ser1.60(3), Glu4.06(5), Gly0.85(1), Ala3.00
(3), Val3.33(4), Leu2.48(3), Lys5.41(6),
His0.70(1) (36) P(52−54); AOC−Arg(Tos)−Lys(Z−
50 ml of TFA was added to 14.65 g (18 mM) of Cl)-Lys(Z-Cl)-PAC [36] [33], and the mixture was stirred at room temperature for 30 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected and dissolved in 10 ml of DMF. To this, AOC-Arg(Tos)-OH9.19g (1.2 times M)-
HOBT2.92g (1.2xM) and WSCI3.95ml
(1.2xM) was added and stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure and the residue was dissolved in ethyl acetate.
After dissolving in 300 ml, the solution was washed three times each in the order of 5% sodium bicarbonate solution, 1N hydrochloric acid, and water. The ethyl acetate layer was dried over anhydrous sodium sulfate and then dried under reduced pressure. Recrystallization from ethyl acetate-ether gave 14.46 g (yield 69.6%) of [36]. Melting point: 79-82℃ TLC; Rf ₇ = 0.76 Elemental analysis [as C ₅₅ H ₇₀ O ₁₃ N ₈ SCl] C% H% N% Measured value 57.06 6.26 9.82 Calculated value 57.23 6.11 9.71 (37) P (51-54 )；BOC−Pro−Arg(Tos)−Lys
40 ml of TFA was added to 14.43 g (12.5 mM) of (Z-Cl)-Lys(Z-Cl)-PAC [37] [36] and stirred at room temperature for 20 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected and dissolved in 80 ml of DMF. Add to this HOBT2.03g (1.2x M), BOC-Pro-
OH3.23g (1.2xM) and WSCI2.75ml (1.2
M) was added, and the mixture was stirred at room temperature overnight. reaction solution,
DMF was distilled off under reduced pressure and the residue was dissolved in 300ml of ethyl acetate.
After dissolving in water, the solution was washed with 5% sodium bicarbonate solution and 1N hydrochloric acid solution in this order. After drying the ethyl acetate layer with anhydrous sodium sulfate,
It was dried under reduced pressure. The residue was recrystallized twice from ethyl acetate-ether [37] 14.71 g (yield 95.1%)
I got it. Melting point: 90-93℃ TLC; Rf ₇ = 0.64 Elemental analysis [as C ₅₉ H ₇₅ O ₁₄ N ₉ SCl ₂ ] C% H% N% Measured value 57.33 6.20 9.79 Calculated value 57.27 6.11 10.19 (38) P (51- 54);BOC−Pro−Arg(Tos)−Lys
(Z-Cl)-Lys(Z-Cl)-OH [38] 20 g of zinc powder/30 ml of acetic acid [37] 6.19 g (5 m
A solution of M) dissolved in 40 ml of acetic acid was added, and the mixture was stirred at room temperature for 2 hours. After the reaction, separate the zinc powder,
The liquid was concentrated under reduced pressure. The residue was extracted by adding 5% sodium bicarbonate solution and ether, and the separated aqueous layer was extracted with 1N hydrochloric acid.
After adjusting the pH to 2, extraction was performed with ethyl acetate. The ethyl acetate layer was washed three times with water, dried over anhydrous sodium sulfate, and then dried under reduced pressure. The residue was recrystallized from ethyl acetate-ether [38] 5.40g (yield 96.5%)
I got it. Melting point: 110-113℃ TLC; Rf ₄ = 0.43 Elemental analysis [as C ₅₁ H ₆₉ O ₁₃ N ₉ SCl ₂ ] C% H% N% Measured value 54.56 6.46 11.22 Calculated value 54.73 6.21 11.27 (39) P (51- 84);BOC−Pro−Arg(Tos)−Lys
(Z-Cl)-Lys(Z-Cl)-Glu(OBzl)-Asp
(OBzl) −Asn−Val−Leu−Val−Glu(OBzl)
−Ser(Bzl)−His−Glu(OBzl)−Lys(Z−Cl)
−Ser(Bzl)−Leu−Gly−Glu(OBzl)−Ala−
Asp(OBzl)−Lys(Z−Cl)−Ala−Asp(OBzl)
−Val−Asp(OBzl)−Val−Leu−Thr(Bzl)−
Lys(Z-Cl)-Ala-Lys(Z-Cl)-Ser(Bzl)
-Gln-OBzl [39] [32] Add 50ml of TFA to 7.83g (1.5mM),
Stirred at room temperature for 60 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected and dissolved by adding 120 ml of DMF and 120 ml of NMP. Add to this HOBT0.30g (1.5x M),
[38] 2.52g (1.5x M) and WSCI0.41ml
(1.5xM) was added and stirred at room temperature for 2 days. The reaction solution was added to ice water, and the resulting precipitate was washed with water, then methanol was added and heat treated. After cooling, insoluble matter was collected. After repeating the above heat treatment twice, washing with ether [39] 8.20 g (yield
87.9%). Amino acid analysis; Asp4.09(5), Thr1.05(1),
Ser2.30(3), Glu4.08(5), Pro0.52(1), Gly0.83
(1), Ala3, Val3.42(4), Leu2.53(3), Lys5.11
(6), His0.69(1), Arg0.51(1) (40) P(49−50); BOC−Gln−Arg(Tos)−
OMe [40] H-Arg(Tos)-OMe・HCl11.37g (30m
M) and BOC-Gln-ONP13.21g (1.2 times M)
Dissolve in 200ml of DMF and PH with NMM while cooling to 0℃.
After adjusting the temperature to 7, the mixture was stirred overnight. After the reaction,
After evaporating DMF under reduced pressure and dissolving the residue in chloroform, it was diluted with 5% sodium bicarbonate solution three times and 1N hydrochloric acid twice.
Washed twice with water and three times with water. The chloroform layer was dried with anhydrous sodium sulfate, and chromatography was performed on a silica gel column packed with chloroform.
The mixture was flushed with chloroform-ethanol-ethyl acetate, and when the target product began to elute, it was eluted with chloroform-ethanol-ethyl acetate (1:1:1). Corresponding fractions were collected and concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and while cooling at 0°C, hexane was added to crystallize it to obtain [40]. yield
11.86g Melting point; 103-107℃ (41) P(48-50); BOC-Ser(Bzl)-Gln-Arg
After adding 50 ml of TFA to 9.39 g (16.5 mM) of (Tos)-OMe [41] [40] and stirring at room temperature for 20 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and the resulting precipitate was collected and dissolved in 50 ml of DMF. In this solution
HOBT3.24g (1.45x M), BOC−Ser(Bzl)−
OH7.07g (1.45x M) and WSCI4.39ml
(1.45 times M) was added and stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 300 ml of ethyl acetate, and then washed with 5% sodium bicarbonate, 1N hydrochloric acid, and water in this order. The ethyl acetate layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was crystallized twice from ethyl acetate-ether to obtain 10.0 g (yield: 81.0%) of [41]. Melting point: 97-102℃ Elemental _analysis [as _C34H49O10N7S・1 _{/ 2H2O} ] _C % H% N% Measured value 54.07 6.90 13.29 Calculated value 53.95 6.66 13.00 (42) P (46−50 )；BOC−Ala−Gly−Ser(Bzl)
-Gln-Arg(Tos)-OMe [42] [41] Add 50ml of TFA to 9.72g (13mM),
Stirred at room temperature for 30 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected and dissolved in 100 ml of DMF. This solution contains 3.84 g of BOC-Ala-Gly-OH (1.2 times M), 2.11 g of HOBT (1.2 times M) and
2.85 ml of WSCI (1.2 times M) was added and stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in 200 ml of ethyl acetate, and then washed with water.
After drying with anhydrous sodium sulfate and concentrating under reduced pressure, the residue was crystallized twice from ethanol-ether [42]
10.46g (yield 91.9%) was obtained. Melting point: 154-157℃ Elemental analysis [as C ₃₉ H ₅₇ O ₁₂ N ₉ S] C% H% N% Measured value 53.21 6.90 14.38 Calculated value 53.47 6.56 14.39 (43) P (46−50); BOC−Ala− Gly-Ser (Bzl)
-Gln-Arg(Tos)-NHNH ₂ [43] [42] 9.64 g (11 mM) was dissolved in 50 ml of ethanol, and 6.4 ml of 50% NH ₂ NH ₂ was added thereto and stirred overnight at room temperature. 100 ml of ethanol was added to the reaction solution to remove insoluble matter. Add this to ethanol
It was suspended in 100 ml and heated. After cooling and filtering, 9.02 g (yield 93.6%) of [43] was obtained. Melting point: 178-180℃ Elemental analysis [as C ₃₈ H ₅₇ O ₁₁ N ₁₁ S] C% H% N% Measured value 52.13 6.86 16.65 Calculated value 52.10 6.56 17.59 (44) P (46−54); BOC−Ala− Gly-Ser (Bzl)
−Gln−Arg(Tos)−Pro−Arg(Tos)−Lys(Z
-Cl)-Lys(Z-Cl)-PAC [44] [37] Add 40ml of TFA to 8.04g (6.6mM),
After stirring at room temperature for 20 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, the resulting precipitate was collected, and crude H-Pro-Arg(Tos)-Lys(Z
-Cl)-PAC・TFA was obtained. On the other hand, 6.83g (7.8mM) of [43] was dissolved in 30ml of DMF, and 5.42ml (23.4mM) of a 4.32N dioxane solution of hydrogen chloride and 1.10ml (8.09mM) of isoamylnitrile were added to this while cooling at -50°C. ,
Stirred at -20°C for 20 minutes. Then the above H-Pro
-Arg(Tos)-Lys(Z-Cl)-PAC·TFA was added, and 5.46 ml (39 mM) of Et ₃ N was added at -35°C, followed by stirring at 0 to 5°C for 2 days. After the reaction,
DMF was distilled off under reduced pressure, and the residue was dissolved in chloroform at 300 ml.
ml, and washed with 5% sodium bicarbonate solution, 1N hydrochloric acid, and water in this order. The chloroform layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification with ethanol-ether and chloroform-ether gave 13.42 g of [44]. TLC; Rf=0.64 [chloroform-methanolacetic acid (83:18:3.5)] Elemental analysis [as C ₉₂ H ₁₂₀ O ₂₂ N ₁₈ Cl ₂ S・3H ₂ O] C% H% N% Measured value 54.68 6.21 12.72 Calculation Value 54.72 6.29 12.49 Amino acid analysis; Ser0.65(1), Glu1.10(1), Pro1
(1), Gly1.02(1), Ala1.00(1), Lys1.89(2),
Arg2.02(2) (45) P(46−54); BOC−Ala−Gly−Ser(Bzl)
−Gln−Arg(Tos)−Pro−Arg(Tos)−Lys(Z
-Cl)-Lys(Z-Cl)-OH [45] 5.31 g of acetic acid in 15 g of zinc powder/30 ml of acetic acid [44]
40 ml of solution was added and stirred at room temperature for 2 hours. After the reaction, the zinc powder was separated and the liquid was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was purified once with ethanol-ether and twice with ethanol-ethyl acetate to obtain 4.41 g (87.7% yield) of [45]. TLC; Rf ₄ = 0.22 Elemental analysis [as C ₈₄ H ₁₁₄ O ₂₂ N ₁₈ S ₃ Cl ₂・2H ₂ O] C% H% N% Measured value 52.89 6.16 13.22 Calculated value 53.12 6.26 13.28 Amino acid analysis; Ser0.88( 1), Glu1.11(1),
Pro1.02(1), Gly1.02(1), Ala1(1), Lys2.00(2),
Arg2.09(2) (46) P(46−84); BOC−Ala−Gly−Ser(Bzl)
−Gln−Arg(Tos)−Pro−Arg(Tos)−Lys(Z
−Cl)−Lys(Z−Cl)−Glu(OBzl)−Asp
(OBzl) −Asn−Val−Leu−Val−Glu(OBzl)
−Ser(Bzl)−His−Glu(OBzl)−Lys(Z−Cl)
−Ser(Bzl)−Leu−Gly−Glu(OBzl)−Ala−
Asp(OBzl)−Lys(Z−Cl)−Ala−Asp(OBzl)
−Val−Asp(OBzl)−Val−Leu−Thr(Bzl)−
Lys(Z-Cl)-Ala-Lys(Z-Cl)-Ser(Bzl)
80 ml of TFA was added to 10.44 g (2 mM) of -Gln-OBzl [46] [32], and after stirring at room temperature for 60 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and the resulting precipitate was dissolved in a mixture of 160 ml of DMF and 160 ml of NMP. This was cooled to 0°C [45] 4.28 g (1.15 times M), HOBT 0.32 g (1.2 times M) and
After adding 0.44 ml of WSCI (1.2x M),
The mixture was stirred for several days. After the reaction, DMF was distilled off under reduced pressure.
After adding ice water to the residue, the resulting precipitate was collected. Add 200ml of methanol to this and heat.
The operation of removing insoluble matter after cooling was repeated twice to obtain 12.17 g (yield: 87.4%) of [46]. (47) h-PTH (46-84) Anhydrous HF 60ml Cooled to 120℃ [46] 4.18g
(0.6mM) and 10ml of anisole were added and stirred for 60 minutes. After the reaction, HF was distilled off under reduced pressure, and ether was added to the residue. Collect the resulting precipitate and add 10%
It was dissolved in 50 ml of acetic acid and passed through a Dowex x 1 column (acetate type, 2.5 x 24 cm). The effluent was freeze-dried to obtain 2.82 g of crude product. This was dissolved in 50 ml of 8M urea aqueous solution (PH9.0) and left at room temperature for 60 minutes. Then this solution
Charge a CM-cellulose column (2.0 x 33 cm) packed with 8M urea aqueous solution, and add 500ml to 0.3M of 0.01M ammonium acetate aqueous solution (PH4.5).
Elution was performed using a linear concentration gradient of 500 ml of ammonium acetate aqueous solution (PH4.5). Eluate is 7.5
Fractionate by ml, each fraction is measured by Folin-Lowry method (500nm) and divided into 1st to 22nd sections.
Eluates of _C1 , 23rd to 45th _C2 , 46th to 80th _C3 , and 81st to _{120th C4} eluates were obtained. Each fraction was desalted by passing through a Sephadex LH-20 column. Section C ₁ was passed through a 3.0 x 120 cm column, and the effluent was fractionated into 7.5 ml portions to obtain the 28th to 42nd sections C ₁ L. Category C ₂ is suitable for 3.4×120cm,
Fractionate the effluent into 7.6ml portions and divide into 33rd to 40th sections.
C ₂ L ₁ and 41st to 62nd sections C ₂ L ₂ were obtained. Section C ₃ passes the effluent through a 3.0 x 120 cm column.
It was fractionated into 6.0 ml portions to obtain the 31st to 40th sections C ₃ L ₁ and the 41st to 51st sections C ₃ L ₂ . Category C ₄ is
The effluent was passed through a 3.4 x 120 cm column and fractionated into 7.5 ml portions to obtain ₄ L of the 35th to 48th fractions. Freeze-dry each section to obtain 312 mg for C ₁ L section and ₁ C ₂ L section.
142.3mg, _{C2L 2} divisions 1380mg, _{C3L 1} division 104mg,
510 mg of C ₃ L ₂ fraction and 130 mg of C ₄ L fraction were obtained. Dissolve 1380 mg of the above C ₂ L ₂ sections in 13 ml of 0.1N acetic acid and apply it to a CM-cellulose column (4.3 x 6.0 cm).
Elution was performed using a linear concentration gradient from 500 ml of 0.01 M ammonium acetate aqueous solution (PH 4.5) to 500 ml of 0.3 M ammonium acetate aqueous solution (PH 4.5). The eluate was fractionated into 7.6 ml portions, and the 40th to 50th sections C ₂ L ₂ -C ₁ and the 53rd to 77th sections
_{C2L2 - C2} was obtained. Safe index LH for each category
-20 column for desalting. Category C ₂ L ₂ −
C ₁ was passed through a 2.9 x 120 cm column and the effluent was 8.0
Fractionate by ml, 26th to 30th division C ₂ L ₂ −C ₁ L ₁
And _the 31st to 39th sections _{C2L2 - C1L2} were obtained.
Section C ₂ L ₂ −C ₂ is passed through a 3.4 x 120 cm column, and the effluent is fractionated into 8.0 ml portions.
C ₂ L ₂ − C ₂ L ₁ and 45th to 53rd divisions C ₂ L ₂ −
C ₂ L ₂ was obtained. Freeze-dry each section to produce C ₂ L ₂ −
C ₁ L ₁ category 79.0 mg, C ₂ L ₂ −C ₁ L ₂ category 455 mg, C ₂ L ₂
−C ₂ L ₁ category 157.3 mg and C ₂ L ₂ −C ₂ L ₂ category 551.7
I got mg. Amino acid analysis of C ₂ L ₂ −C ₂ L ₂ division; Asp4.65(5),
Thr0.97(1), Ser3.47(4), Glu5.99(6), Pro0.93
(1), Gly1.96(2), Ala4(4), Val3.97(4),
Leu3.01(3), Lys6.13(6), His0.93(1), Arg1.96
(2) Dissolve the above C ₂ L ₂ - C ₂ L ₂ sections in 5 ml of 0.1N acetic acid and apply it to a CM-cellulose column (4.2 x 7.0 cm).
Elution was performed using a linear concentration gradient from 300 ml of 0.01 M ammonium acetate aqueous solution (PH4.5) to 0.3 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 8.0 ml portions to obtain the 39th to 45th fractions C ₂ L ₂ -C ₂ L ₂ -C. This fraction is desalted by passing it through a Sephadex LH-20 column (2.9 x 120 cm). The effluent was fractionated into 8.0 ml portions, and the 24th to 30th divisions C ₂ L ₂ −C ₂ L ₂ −CL ₁ ,
31st to 35th division C ₂ L ₂ −C ₂ L ₂ −CL ₂ and 36~
The 39th segment C ₂ L ₂ −C ₂ L ₂ −CL ₃ was obtained. Freeze-dry each portion to obtain 200 mg of C ₂ L ₂ −C ₂ L ₂ −CL ₂ portions and ₁ portion of C ₂ L ₂ −C ₂ L ₂ −CL [h-PTH (46−
84)] 94.9 mg was obtained. TLC; Rf ₉ = 0.76 Amino acid analysis; Asp4.86(5), Thr0.99(1),
Ser3.58(4), Glu6.17(6), Pro0.96(1), Gly1.97
(2), Ala4(4), Val4.02(4), Leu2.93(3),
Lys6.05(6), His0.90(1), Arg1.87(2) Example 2 [ ^Tyr45 ]h-PTH(46-84); H-Tyr-Ala-
Gly−Ser−Gln−Arg−Pro−Arg−Lys−Lys
−Glu−Asp−Asn−Val−Leu−Val−Glu−
Ser−His−Glu−Lys−Ser−Leu−Gly−Glu
−Ala−Asp−Lys−Ala−Asp−Val−Asp−
Val−Leu−Thr−Lys−Ala−Lys−Ser−Gln
−OH (1) P(45−84); BOC−Tyr(Bzl−Cl ₂ )−Ala−
Gly−Ser(Bzl)−Gln−Arg(Tos)−Pro−Arg
(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−Glu
(OBzl)−Asp(OBzl)−Asn−Val−Leu−Val
−Glu(OBzl)−Ser(Bzl)−His−Glu(OBzl)−
Lys(Z−Cl)−Ser(Bzl)−Leu−Gly−Glu
(OBzl)-Ala-Asp(OBzl)-Lys(Z-Cl)-
Ala−Asp(OBzl)−Val−Asp(OBzl)−Val−
Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z
-Cl)-Ser(Bzl)-Gln-OBzl [47] 6.27 g (0.9 mM) of [46] described in Example 1
60 ml of TFA was added and stirred at room temperature for 60 minutes. After the reaction, TFA was distilled off under reduced pressure, and ether was added to the residue. The resulting precipitate was collected to obtain 6.30 g of a BOC-free product. 2.10g (0.3mM) of the above BOC removed product was added to DMF35
ml and NMP (35 ml), and add 0.16 _g (1.2
times M), HOBT0.05g (1.2 times M) and
After adding 0.07 ml of WSCI (1.2 times M), the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure.
Ice water was added to the residue. The resulting precipitate was collected to obtain 2.00 g [47]. (2) [Try ⁴⁵ ]-h-PTH (46-84) 2.00 g of [47] cooled to 0℃ in 20 ml of anhydrous HF
(0.27mM) and 1.0ml of anisole, 60
Stir for a minute. After the reaction, HF was distilled off under reduced pressure,
Ether was added to the residue. Collect the resulting precipitate, dissolve it in 20 ml of 0.1N acetic acid, and add Dowex
1 column (acetate type, 2.5 x 15 cm). The effluent was freeze-dried to obtain 1.37 g of crude product. Dissolve this in 50ml of 8M urea aqueous solution (PH9.5) and leave at room temperature for 60 minutes. Then this solution
Charge a CM-cellulose column (4.3 x 8.0 cm) packed with 8M urea aqueous solution, and add 400ml to 0.3M 0.01M ammonium acetate aqueous solution (PH4.5).
Elution was performed using a linear concentration gradient of 400 ml of ammonium acetate aqueous solution (PH4.5). Eluent is 6.5
Fractionate by ml, each fraction is measured by Folin-Lowry method (500nm), and the 30th to 43rd division
C ₁ , 51st to 68th division C ₂ , 69th to 83rd division
C ₃ and 84th to 100th segment C ₄ were obtained. Each fraction was desalted by passing it through a Sephadex LH-20 column. Pass the fraction C ₁ through a 3.4 x 120 cm column, fractionate the effluent into 7.5 ml portions, and divide the 25th to 41st fraction C ₁ L.
I got it. Section C ₂ is passed through a 3.0 x 120 cm column,
Fractionate the effluent into 7.5ml portions and divide into 25th to 36th sections.
Obtained _C2L . Section C ₃ was passed through a 2.9 x 120 cm column, and the effluent was fractionated into 8.0 ml portions to obtain the 24th to 27th sections C ₃ L ₁ and the 28th to 38th sections C ₃ L ₂ .
Section C ₄ passes the effluent through a 2.9 x 95 cm column.
It was fractionated into 7.6 ml portions to obtain the 20th to 25th sections C ₄ L ₁ and the 26th to 30th sections C ₄ L ₂ . Freeze-dry each section to obtain 521.6 mg for C ₁ L section, 230.2 mg for C ₂ L section,
C ₃ L ₁ category 41.8 mg, C ₃ L ₂ category 222.4 mg, C ₄ L ₁ category
74.3 mg and 48.9 mg of C ₄ L ₂ fractions were obtained. Dissolve the above C ₂ L fraction in 3 ml of 0.1N acetic acid,
Charge a CM-cellulose column (2.1 x 25 cm) and add 0.01M ammonium acetate aqueous solution (PH
4.5) Elution was performed using a linear concentration gradient from 300 ml to 300 ml of a 0.3 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 8.0 ml portions to obtain the 30th to 36th fractions C ₂ L-C. This fraction was desalted by passing it through a Sephadex LH-20 column (2.9 x 90 cm). Fractionate the effluent into 8.0ml portions,
Freeze-dry the 20th to 29th sections [Tyr ⁴⁵ ]-h
-163.3 mg of PTH (46-84) was obtained. TLC; Rf ₉ = 0.75 Amino acid analysis; Asp4.86(5), Thr1.02(1),
Ser3.51(4), Glu6.05(6), Pro0.93(1), Gly1.90
(2), Ala4(4), Val4.00(4), Leu2.93(3),
Tyr0.88(1), Lys6.02(6), His0.86(1), Arg1.81
(2) Example 3 [Cys(Acm) ⁴⁵ ]-h-PTH(46-84)];H-
Cys(Acm)−Ala−Gly−Ser−Gln−Arg−Pro
−Arg−Lys−Lys−Glu−Asp−Asn−Val−
Leu−Val−Gln−Ser−His−Glu−Lys−Ser
−Leu−Gly−Glu−Ala−Asp−Lys−Ala−
Asp−Val−Asp−Val−Leu−Thr−Lys−Ala
−Lys−Ser−Gln−OH (1) P(45−84); BOC−Cys(Acm)−Ala−Gly
−Ser(Bzl)−Glu−Arg(Tos)−Pro−Arg
(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−Glu
(OBzl)−Asp(OBzl)−Asn−Val−Leu−Val
−Glu(OBzl)−Ser(Bzl)−His−Glu(OBzl)−
Lys(Z−Cl)−Ser(Bzl)−Leu−Gly−Glu
(OBzl)-Ala-Asp(OBzl)-Lys(Z-Cl)-
AlaAsp(OBzl)−Val−Asp(OBzl)−Val−
Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z
-Cl)-Ser(Bzl)-Gln-OBzl [48] 4.20 mg of the remaining BOC-removed product obtained in Example 2
(0.6mM) was dissolved in a mixture of 70ml of DMF and 70ml of NMP, and 0.10g of HOBT (1.2
After adding 0.20 g (1.2 times M) of BOC-Cys(Acm)-OH (1.2 times M) and 0.13 ml (1.2 times M) of WSCI, the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, ice water was added to the residue, and the resulting precipitate was collected. This was suspended in ethanol, heated, cooled, and then the insoluble matter was removed. Repeat this operation twice [48] 4.07g (yield 95.0%)
I got it. (2) [Cys (Acm) ⁴⁵ ]-h-PTH (46-84) 4.00 g of [48] in 60 ml of anhydrous HF while cooling to 0℃
(0.57mM) and 10ml of anisole, 60
Stir for a minute. After the reaction, HF was distilled off under reduced pressure,
Ether was added to the residue. Collect the resulting precipitate, dissolve it in 40ml of 20% acetic acid, and add Dowex x 1.
column (acetate type, 2.8 x 35 cm).
The effluent was lyophilized. Add this to 8M urea aqueous solution.
After dissolving in 50 ml and adjusting the pH to 9.0 with aqueous ammonia, it was left for 30 minutes. This solution was then diluted to 8M
CM-cellulose filled with urea aqueous solution (3.4×
35 cm), and elution was performed using a concentration gradient of 700 ml of 0.01 M ammonium acetate aqueous solution (PH4.5) to 700 ml of 0.3 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 8.0ml portions, and each fraction was measured using the Folin-Lowry method (500nm).
A 25th to 35th segment _C1 , a 36th to 45th segment _C2 , and a 46th to 84th segment _C3 were obtained. Each fraction was desalted by passing it through a Sephadex LH-20 column. Section C ₂ is passed through a 3.0 x 120 cm column, the effluent is fractionated into 8.0 ml portions, and the 27th to 33rd sections C ₂ L ₁
and the 34th to 40th _{divisions C2L2} were obtained. Category C ₃
is passed through a 3.4 x 120 cm column and the effluent is 8.0 ml.
The 35th to 47th fractions C ₃ L ₁ and 48
~53rd segment _C3L2 was obtained _. Freeze-dry each section to obtain 148 mg of C ₂ L ₁ section, 620 mg of C ₂ L ₂ sections, and 620 mg of C ₃ L _1.
212 mg of fraction and 605 mg of C ₃ L ₂ fraction were obtained. Dissolve the ₂ portions of C ₂ L in 6 ml of 0.1N acetic acid,
This is a CM-molulose column (5.0 x 12 cm)
Elution was performed using a linear concentration gradient from 400 ml of 0.01 M ammonium acetate aqueous solution (PH 4.5) to 400 ml of 0.3 M ammonium acetate aqueous solution (PH 4.5). The eluate was fractionated into 6.0 ml portions, 110 to 126
The main classification C ₂ L ₂ -C was obtained. This was desalted by passing it through a Sephadex LH-20 column (4.0 x 120 cm). The effluent was fractionated into 8.0 ml portions, and
The 54th segment C ₂ L ₂ −CL was obtained. This fraction was freeze-dried to obtain [Cys(Acm) ⁴⁵ ]-h-PTH(46-
84) Obtained 246.8 mg. TCL; Rf ₉ = 0.74 Amino acid analysis; Asp4.91(5), Thr0.98(1),
Ser3.50(1), Glu6.11(6), Pro0.98(1), Gly1.98
(2), Ala4(4), Val4.04(4), Cys0.42(0.5),
Leu2.90(3), Lys5.99(6), His0.87(1), Arg1.87
(2) Example 4 [Cys ⁴⁵ ]-h-PTH (46-84); H-Cys-Ala-
Gly−Ser−Gln−Arg−Pro−Arg−Lys−Lys
−Glu−Asp−Asn−Val−Leu−Val−Glu−
Ser−His−Glu−Lys−Ser−Leu−Gly−Glu
−Ala−Asp−Lys−Ala−Asp−Val−Asp−
Val−Leu−Thr−Lys−Ala−Lys−Ser−Gln
-OH [Cys(Acm) ⁴⁵ ]-h-PTH obtained in Example 3
(46-84) 88mg (0.02mM) was dissolved in 2ml of 50% acetic acid, and 57.24mg (0.18mM) of mercuric acetate was added to this.
was added, and the mixture was stirred at room temperature for 70 minutes. Then β
− Add 3.4 ml of mercaptoethanol and
Stir for hours. The reaction solution was centrifuged, and the supernatant liquid was charged to a Sephadex LH-20 column (3.2 x 42 cm) and eluted with 0.1M acetic acid. Eluate is 5ml
The 9th to 14th fractions that were positive for ninhydrin reaction were collected. Freeze-dry this [Cys ⁴⁵ ]−
76.1 mg of h-PTH (46-84) was obtained. TLC; Rf ₉ = 0.73 Reference example 1 h-PTH (53-84); H-Lys-Lys-Glu-
Asp－Asn－Val－Leu－Val－Glu－Ser－His
−Glu−Lys−Ser−Leu−Gly−Glu−Ala−
Asp−Lys−Ala−Asp−Val−Asp−Val−Leu
-Thr-Lys-Ala-Lys-Ser-Gln-OH (a) Add 3.49 g (0.6 mM) of [35] described in Example 1 and 3 ml of anisole to 25 ml of anhydrous hydrogen fluoride (HF) while cooling to 0°C. and stirred for 75 minutes. After the reaction, HF was distilled off under reduced pressure, and ether was added to the residue. Collect the resulting precipitate and add 50ml of 0.1N acetic acid.
2.7× column (2.7×
35cm). The effluent was freeze-dried to obtain 2.18 g of crude product. After dissolving this in 50ml of 8M urea aqueous solution and adjusting the pH to 9.5 with aqueous ammonia,
It was left for 50 minutes. This solution was then transferred to a CM-cellulose column (4.4
x 12cm) and drained with approximately 100ml of 0.01M ammonium acetate aqueous solution (PH4.5),
0.01M ammonium acetate aqueous solution (PH4.5) 700ml
~0.1M ammonium acetate aqueous solution (PH4.5) 700
Perform elution with a linear concentration gradient of ml, then
0.2M ammonium acetate aqueous solution (PH4.5) 300ml
It was eluted. The eluate was fractionated into 13.5 ml portions, and each fraction was measured using the Folin-Lowry method (500 nm) to divide the 30th to 50th sections C ₁ and the 56th to 119th sections.
_C2 and 120th to 150th segment _C3 eluates were obtained. Each fraction was desalted by passing it through a Sephadex LH-20 column. The effluent was fractionated into 8.5ml portions.
Each fraction was measured in the same manner as above. Category C ₁
was passed through a 3.4×113 cm column to obtain the 31st to 40th sections _L1 , the 41st to 44th sections _L2 , and the 45th to 54th sections _L3 . Segment _C2 was passed through a 3.4 x 120 cm column to obtain the 35th to 45th segment _L1 , the 46th to 52nd segment _L2 , and the 53rd to 60th segment _L3 . Segment C ₃ was passed through a 3.4 x 120 cm column to obtain 31st to 44th sections L ₁ and 45th to 52nd sections L ₂ .
Each section was freeze-dried to obtain the following components.

[Table] (b) Purification of C ₂ L ₂ Dissolve 565 mg of the above C ₂ L ₂ in 5 ml of 0.1N acetic acid,
Charge a CM-cellulose column (4.4 x 70 cm) and add 0.01M ammonium acetate aqueous solution (PH
4.5) Elution was performed using a linear concentration gradient from 500 ml to 500 ml of a 0.1 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 6.0ml portions, and each fraction was
Measured by Folin-Lowry method: 113th to 136th segment C ₂ L ₂ − C ₁ , 137th to 1551st segment C ₂ L ₂ −
_C2 and the 152nd to 190th sections _{C2L2 - C3} were obtained. Each fraction was desalted by passing it through a Sephadex LH-20 column. The effluent was fractionated into 5.2 ml portions.
Each fraction was measured in the same manner as above. classification
C ₂ L ₂ −C ₁ is passed through a 3.4 x 120 cm column and 55 to 72
The main division _{C2L2 - C1L1} and _the 73rd to 80th divisions _{C2L2 - C1L2 were} obtained. Division C ₂ L ₂ −C ₂ is 3.4×
Pass through a 110cm column and divide the 50th to 59th sections C ₂ L ₂
−C ₂ L ₁ and the 60th to 67th sections C ₂ L ₂ −C ₂ L ₂ were obtained. Segments C ₂ L ₂ - C ₃ are passed through a 3.4 x 110 cm column, and C ₂ L ₂ - C ₃ L ₁ and 61 of the 45th to 60th sections are passed through a 3.4 x 110 cm column.
~72nd segment C ₂ L ₂ −C ₃ L ₂ was obtained. Each section was freeze-dried to obtain the following components. C ₂ L ₂ −C ₁ L ₁ 108.7mg C ₂ L ₂ −C ₁ L ₂ 95.9mg C ₂ L ₂ −C ₂ L ₁ 53.6mg C ₂ L ₂ −C ₂ L ₂ 44.1mg C ₂ L ₂ −C ₃ L ₁ 97.0mg C ₂ L ₂ −C ₃ L ₂ 95.1mg (c) Purification of C ₂ L ₂ −C ₁ L ₁ Dissolve the above C ₂ L ₂ −C ₁ L ₁ in 1 ml of 0.1N acetic acid,
Charge a CM-cellulose column (2.0 x 15 cm) and add 0.01M ammonium acetate aqueous solution (PH
4.5) Elution was performed using a linear concentration gradient from 300 ml to 300 ml of a 0.1 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 7.4ml portions, and each fraction was
46 measured by Folin-Lowry method (500nm)
~56th segment _{C2L2 - C1L1 -} C was obtained. This was concentrated under reduced pressure, charged to a Sephadex LH-20 column (3.0 x 90 cm), and eluted with 0.1N acetic acid. The eluate was fractionated into 6.0 ml portions and measured using the _same method as above _{.
C1L1 -} CL was obtained. This is freeze-dried and h-
90.0 mg of PTH (53-84) was obtained. TLC; R ₉ = 0.76 1 spot Amino acid analysis; Asp4.45(5), Thr0.92(1),
Ser2.16(3), Glu4.94(5), Gly0.96(1), Ala3,
Val3.96(4), Leu2.92(3), Lys6.22(6), His1.01
(1) Reference example 2 h-PTH (51-84); H-Pro-Arg-Lys-
Lys−Glu−Asp−Asn−Val−Leu−Val−Glu
−Ser−His−Glu−Lys−Ser−Leu−Gly−
Glu−Ala−Asp−Lys−Ala−Asp−Val−Asp
−Val−Leu−Thr−Lys−Ala−Lys−Ser−
Gln-OH 3.73 g (0.6 mM) of [39] described in Example 1 and 4 ml of anisole were added to 40 ml of anhydrous HF while cooling to 0°C, and the mixture was stirred for 60 minutes. After the reaction, HF was distilled off under reduced pressure, and ether was added to the residue. Collect the resulting precipitate, dissolve it in 50 ml of 0.1N acetic acid, and add Dowex
1 column (acetate type - 2.7 x 33 cm). The effluent was freeze-dried to obtain 2.41 g of crude product. This was dissolved in 50 ml of 8M urea aqueous solution, adjusted to pH 10.0 with aqueous ammonia, and then left for 30 minutes. This solution was then poured into a CM− filled with 8M urea aqueous solution.
Charge a cellulose column (4.2 x 11.5 cm) and drain the urea with 0.01M ammonium acetate aqueous solution (PH4.5), then add 700ml of 0.01M ammonium acetate aqueous solution (PH4.5) to 0.1M ammonium acetate aqueous solution (PH4. 5) Perform elution with a 700 ml linear concentration gradient, then elute with 0.2 M ammonium acetate aqueous solution (PH
4.5) Eluted at 250ml. The eluate was fractionated into 8.5 ml portions, and each fraction was measured using the Folin-Lowry method (500 nm) to indicate the 30th to 63rd division _C1 , the 105th to 150th division _C2 , and the 151st to 195th division C. ₃ solutions were obtained.
Sections _C2 and _C3 were desalted by passing through a Sephadex LH-20 column. The effluent is fractionated into 5.2 ml portions, and division C ₂ is suitable for a 3.4 x 110 cm column, with 51 to
63rd division C ₂ L ₁ and 64th to 80th division C ₂ L ₂
I got it. Segment C ₃ was suitable for 3.4 x 120 cm, and 50th to 69th sections C ₃ L ₁ and 70th to 78th sections C ₃ L ₂ were obtained.
Each section was freeze-dried to obtain the following components.

[Table] Dissolve 375 mg of the above C ₂ L in ₄ ml of 0.1 N acetic acid, charge it to a CM-cellulose column (2.0 x 31 cm), and add 0.01 M ammonium acetate aqueous solution (PH
4.5) 500ml ~ 0.2M ammonium acetate aqueous solution (PH
4.5) Elution was performed using a 500 ml linear concentration gradient. The eluate was fractionated into 7.5 ml portions to obtain the 85th to 103rd fractions C ₂ L ₂ -C. Secure this classification
Desalting was carried out through an LH-20 column (3.0 x 123 cm). The effluent is fractionated into 6 ml portions and divided into 34th to 42nd sections.
_{C2L2 - CL1} and 43rd to 50th sections _{C2L2 - CL2} were obtained. Each section was freeze-dried to obtain the following components. C ₂ L ₂ −CL ₁ 80.0mg Amino acid analysis; Asp4.95(5), Thr0.98(1), Ser2.47
(3), Glu5.14(5), Gly1.01(1), Ala3(3), Val4.05
(4), Leu3.02(3), Lys6.16(6), His0.96(1),
Arg0.97(1), Pro1.06(1) C ₂ L ₂ −CL ₂ 197.6mg Amino acid analysis; Asp5.00(5), Thr0.93(1), Ser2.30
(3), Glu5.17(5), Gly1.01(1), Ala3(3), Val4.03
(4), Leu3.00(3), Lys6.15(6), His0.95(1),
Arg1.01(1), Pro1.04(1) Dissolve 195 mg of the above C ₂ L ₂ -CL ₂ sections in 2 ml of 0.1N acetic acid, and add it to a CM-cellulose column (2.0 x 15 cm).
Elution was performed using a linear concentration gradient of 300 ml of 0.01 M ammonium acetate aqueous solution (PH 4.5) to 300 ml of 0.2 M ammonium acetate aqueous solution (PH 4.5). The eluate was fractionated into 6.4 ml portions, and the 55th to 64th sections C ₂ L ₂ −CL ₂ −C ₁ and the 56th to 72nd sections
_{C2L2 - CL2} - _C2 was obtained. Securely index each category
Desalting was carried out through an LH-20 column. Category C ₂ L ₂ −
CL ₂ −C ₁ was passed through a 3.0 x 123 cm column, and the effluent was
Fractionate into 7.4ml portions and divide into 31st to 38th sections C ₂ L ₂ −CL ₂
−C ₁ L ₁ and the 39th to 43rd divisions C ₂ L ₂ −CL ₂ −C ₁ L ₂ were obtained. Freeze-dry the division C ₂ L ₂ −CL ₂ −C ₁ L ₁ and h−
77.2 mg of PTH (51-84) was obtained. TLC; R ₁₀ = 0.89 1 spot

Claims (1)

[Claims] 1 Formula R-Ala-Gly-Ser-Gln-Arg-Pro-Arg-Lys-Lys-G
lu−Asp−Asn−Val−Leu −Val−Glu−Ser−His−Glu−Lys−Ser−Leu−Gly−G
lu−Ala −Asp−Lys−Ala−Asp−Val−Asp−Val−Leu−Thr−L
ys-Ala-Lys-Ser-Gln-OH (wherein, R is H or H-R ₁ group, R ₁ is Cys or
Tyr group) or its salt.