JPH0427996B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel peptides. More specifically, the formula H-Lys ⁵³ -Lys-Glu ⁵⁵ -Asp-Asn-Vsl-Leu
−Val ⁶⁰ −Glu−Ser−His−Glu−Lys ⁶⁵ −Ser−
Leu−Gly−Glu−Ala ⁷⁰ −Asp−Lys−Ala−Asp
−Val ⁷⁵ −Asn−Val−Leu−Thr−Lys ⁸⁰ −Ala−
It relates to a peptide represented by Lys-Ser-Gln ⁸⁴ -OH [1] or a salt thereof. The novel peptide of the present invention represented by the formula [] (hereinafter simply referred to as peptide) or its salt is constructed by condensing individual amino acids or lower peptides in the amino acid order represented by the formula [], and the final step of the condensation reaction is It can be obtained by removing the protective group of the functional group on the side chain. The condensation reaction itself is carried out by repeating the attachment and detachment of a protecting group and the condensation reaction in accordance with conventional methods for peptide synthesis. That is,
The various protecting groups used in the production of the raw materials for the target compound and all intermediates are those known in peptide synthesis, such as hydrolysis, acidolysis, reduction,
Protecting groups are used that are easily removable by known means such as aminolysis or hydrazinolysis. 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 and t-amyloxycarbonyl group, 2
-phenyl-isopropoxycarbonyl group, 2-
Tolyl-isopropoxycarbonyl group, 2-p-
Examples include aralkyloxycarbonyl groups such as diphenyl-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
- Phenols such as trichlorophenol, 2,4,5-trichlorophenol, 3,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, thiophenol, p-nitrothiophenol, etc. is substituted by thiophenol, etc. The hydroxyl groups of the serine and threonine can be protected, for example, by esterification or etherification. Groups suitable for this esterification include, for example, acetyl group, benzoyl group, benzyloxycarbonyl group, and ethyloxycarbonyl group. Groups suitable for etherification include, for example, benzyl group, tetrahydropyranyl group, t
-butyl group. To protect these hydroxyl groups,
Also suitable are the 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl group and the 2,2,2-trifluoro-1-benzyloxycarbonylamino group. However, it is not necessary to protect these hydroxyl groups. 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. In the synthesis of the peptides of the present invention represented by the formula [], the condensation of individual amino acids or lower peptides is carried out, for example, with amino acids or peptides having a protected α-amino group and an activated terminal carboxyl group, and a free α-amino group. and an amino acid or peptide with a protected terminal carboxyl group, or 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 α-amino group. This can be carried out by reacting amino acids or peptides with groups. 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-hydroxyphthalic imide ester, 8-hydroxyquinoline ester or N-hydroxypiperidine ester, or carbodiimide, N,N'-carbonyl-diimidazole or isoxolium salt, For example, it can be activated 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 Wunsch method [Z.Naturforsch., 21b , 426
(1966)] or the Geiger method [Chem.Ber., 103 788
(1970)] in particular as a condensing agent.
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 peptide (65-84) has a C-terminal fragment 69-84 and an N-terminal fragment 65-84.
68 is preferably condensed using a modified Geiger method using WSCI. N-terminal fragment 65-68 is preferably obtained by condensing fragment 66-68 with the 65th amino acid by the active ester method or the modified Geiger method using WSCI. In addition, the C-terminal fragment 69-84 is obtained by adding the 71st amino acid, the 70th amino acid, and the 69th amino acid to fragment 72-84 using the active ester method.
Condensation is preferably carried out by a modified Geiger method using WSCI. Fragment 72−84 is fragment 77−84
76th amino acid, 75th amino acid, 74th amino acid,
The amino acid No. 1 and fragments 72-73 are preferably condensed by the active ester method or a modified Geiger method using WSCI. Fragment 77-84 is WSCI fragment 82-84 and fragment 77-81
The condensation is preferably carried out by the modified Geiger method using . The protected peptide (62-84) consists of the previously described C-terminal fragment 69-84 and the N-terminal fragment 62-
68 is preferably condensed by a modified Geiger method using WSCI. Fragment 62−68 is fragment 65
-68 is preferably condensed with the 64th amino acid, fragment 62-63, by the modified Geiger method using an active ester or WSCI and the azide method. The protected peptide (53-84) also consists of a C-terminal fragment 55-84 and an N-terminal fragment 53-54.
It is preferable to condense them by a modified Geiger method using WSCI. C-terminal fragment 55-84 is fragment 61-84 obtained by condensing the 61st amino acid to the above-mentioned protected peptide (62-84), and fragment 59-60 with the 58th amino acid and the 57th amino acid. Fragment 57 obtained by condensing amino acids
It is preferable to condense 60 by a modified Geiger method using WSCI, and sequentially condense the 56th amino acid and the 55th amino acid by an active ester method. 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 solution and converted to a hydrazide, and a benzyl ester group can be cleaved with anhydrous hydrogen fluoride or hydrogenolysis.
The α-amino groups of these peptides are protected by conventional protecting groups such as benzyloxycarbonyl, t-butoxycarbonyl, and t-aminooxycarbonyl groups, but the benzyloxycarbonyl group is susceptible to hydrogenolysis. and was separated,
The t-butoxycarbonyl group and t-amyloxycarbonyl group are removed with trifluoroacetic acid. The hydroxyl groups of serine and threonine are suitably protected with a benzyl group, and the ε-amino group of lysine is suitably protected with an o-chlorobenzyloxycarbonyl group. These protecting groups are removed with anhydrous hydrogen fluoride. The protected peptide (53-84) is thus obtained. These protecting groups are preferably protected against acid decomposition,
For example, it is eliminated in one step by treatment with anhydrous hydrogen fluoride to obtain the target compound of formula []. The above target compound [ ] can be purified by known means for purifying peptides. For example, Cephadex LH-20, Cephadex G-
It can be carried out by column chromatography using a carrier such as 50, Dowexl, or carboxymethylcellulose. 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. Enzyme immunoassay (EIA) can be performed by antibody obtained using the peptide of the present invention [] as an antigen or by an immune reaction using the present peptide [] as an antigen.
Radioimmunoassay (RIA) can be used to measure the concentration of human parathyroid hormone (h-PTH). First, the usefulness of the peptide represented by the formula [] of the present invention in immune reactions will be described. (1) Preparation of peptides and antibodies for antibody production [Preparation method of BSA-[Cys ⁴⁵ ]-peptide (45-84) conjugate] 71.5 mg of BSA (bovine serum albumin) was added to 1mMEDTA.
Dissolved in 25 ml of 100 mM phosphate buffer (PH 8.0) containing 436 μg of 5,5′-dithiobis(2-nitrobenzoic acid) and reacted at 37°C for 15 minutes.
Free -SH groups were protected. 3 ml of a dimethylformamide solution containing 7.59 mg of 3-(2'-benzothiazolyl-dithio)propionic acid succinimide ester was added to the reaction solution, and the mixture was reacted at 5°C for 1 hour.
After adjusting the pH to 5.0 with acetic acid, the reaction solution was added to a column of Sephadex G-25 (45 x 70 cm), and gel filtration was performed with 50 mM acetate buffer (PH 5.0). The flow-through fraction was collected, and 3-(2'-
A derivative having a benzothiazolyl-dithio/propionyl group introduced therein (5.67 mol bond relative to BSAI mol) was obtained. Next, after adjusting the pH of this derivative-containing liquid to 7.0,
10.85 mg of [Cys ⁴⁵ ]-peptide (45-84) was added, and the reaction was stirred at room temperature for 24 hours. The reaction solution was dialyzed against distilled water, the dialyzed solution was freeze-dried, and BSA-
A [Cys ⁴⁵ ]-peptide (45-84) conjugate (binding molar ratio 1:5.17) was obtained. [Method for producing antibodies] 500 μg of each antigen of peptide (46-84), peptide (51-84), and peptide (53-84) was added to BSA-
[Cys ⁴⁵ ]-peptide (45-84) is 1 mg ([Cys ⁴⁵ ]-
Approximately 250 μg of peptide (45-84) was added to 10 mM phosphate buffer (PH7.4) containing 0.15M NaCl.
Dissolve in 0.5ml and add Freunds Complete Adjuvant to this.
0.5 ml was added and thoroughly mixed to obtain an emulsion. One ml of the obtained emulsion was injected into each guinea pig into the skin of the limbs and several subcutaneous locations on the back. The same amount of emulsion was subcutaneously injected 5 times every 10 days, and whole blood was collected by cardiac puncture on the 10th day after the final immunization, left at room temperature for 1 hour to solidify, and then centrifuged at 3000 rpm for 5 minutes. Peptide (4-84), Peptide (51-84),
Antisera against peptide (53-84) and BSA-[ ^Cys45 ]-peptide-(45-84) were obtained. (2) Peptide for labeling [Preparation method of β-galatosidase-[Cys ⁴⁵ ]-peptide (45-84)] 1 mg of [Cys ⁴⁵ ]-peptide (45-84)
lmMEDTA containing 50mM phosphate buffer (PH7.0) 1
ml of dimethylformamide solution containing 165.3 μg of N-[2-(2'-pyridyl-dithio)ethyl]-3-(2'-benzothiazolyl-dithio)propionamide. The reaction was allowed to proceed at ℃ for 2 hours. After adjusting the reaction solution to pH 5.0 with acetic acid, it was added to a column of Sephadex G-15 (15 x 40 cm), and gel filtration was performed with 50 mM acetate buffer (PH 5.0). Collect the fraction that passes through,
[Cys(2'-pyridyl-S-S-( _CH2 ) _2- NH-CO
-( _CH2 ) ₂ -S) ⁴⁵ ] peptide (45-84) derivative was obtained. Next, 2 mg of β-galactosidase was added to 2 ml of 100 mM phosphate buffer (PH80.) containing 38.9 μg of this derivative, and the mixture was allowed to react at room temperature for 3 hours. The reaction solution was added to a Sephadex G-150 (1.5 x 90 cm) column, gel filtrated with 10 mM phosphate buffer (PH7.4) containing 0.15 M NaCl, and the flow-through fraction was collected, and β-galactosidase [Cys ⁴⁵ ]-peptide (45-84) conjugate (purity 94%) was obtained. [ ¹²⁵ I-labeled product preparation method] In a reaction test tube, add ¹²⁵ I with 2 mCi of radioactivity.
-500mM phosphate buffer containing NaI (PH7.5)
50μ, [Tyr ⁴⁵ ]-peptide (45-84), [Tyr ⁵⁰ ]
-Peptide (50-84), [Tyr ⁵² ]-Peptide (52
−84) and [Tyr ⁶⁴ ]-peptide (64−84), respectively.
10μ of 50mM phosphate buffer (PH7.5) containing 2μg
, and 50mM containing 20μg of chloramine T
20μ of phosphate buffer (PH7.5) was added, and the reaction was stirred for 30 seconds. Next, 10μ of 1% potassium iodide aqueous solution and 5% human serum albumin were added.
After adding 0.5ml of 0.1N acetic acid,
Add the reaction solution to a 25 (1.0 x 50 cm) column,
10mM phosphate buffer (PH
Perform gel filtration in step 7.4) to remove the final reaction ^125I -NaI to obtain ^125I- [ ^Tyr45 ]-peptide (45-84), ^125I
-[ ^Tyr50 ]-peptide (50-84), ^125I- [ ^Tyr52 ]-
Labeled compounds of peptide (52-84) and ¹²⁵ I-[Tyr ⁶⁴ ]-peptide (64-84) were obtained. The obtained ^125I- [ ^Tyr45 ]-peptide (45-84)
The specific activity of the labeled compound was 540 μCi/μg, the reaction yield was 76.5%, and ¹²⁵ I-[Tyr ⁵⁰ ]-peptide (50
-84) The specific activity of the labeled compound is 535 μCi/μg, reaction yield 75.5%, and the specific activity of the ¹²⁵ I-[Tyr ⁵² ]-peptide (52-84) labeled compound is 560 μCi/μg,
The reaction yield was 74.8%, and the specific activity of the ¹²⁵ I-[Tyr ⁶⁴ ]-peptide (64-84) labeled compound was 555 μCi/μg.
The reaction yield was 77.3%. Similarly, h-
PTH (1-34) or bovine PTH (1-84) (extract)
The specific activity is 2-3 compared to the result of labeling using
The reaction yield was 3 to 4 times better. (3) Immune reaction [Immunoreactivity of antiserum] Measurement of antibody titer by EIA Each of the antisera mentioned above was added to the immune reaction medium (0.25%
Using 10mM phosphate buffer (PH7.4) containing BSA, 3mMEDTA, 0.1% _NaN3 , 0.15M NaCl,
200, 400, 800, 1600, 3200, 6400, 12800,
Add 100μ of a 25,600-fold diluted solution, 100μ of β-galactosidase [Cys ⁴⁵ ]-peptide (45-84) solution (containing about 20 pg as [Cys ⁴⁵ ]-peptide (45-84)), and 100μ of the immunoreaction medium to a small plate. Add to the test tube and react at 5°C for 24 hours, then add 100 μl of normal guinea pig serum diluted and 100 μl of 100 μl anti-guinea pig γ-globulin serum (rabbit) diluted 10 times, and react at 5°C for another 24 hours. I let it happen. After adding 3ml of 0.5M NaCl to the reaction solution,
The precipitate was collected by centrifugation at 3000 rpm for 15 minutes. Next, this precipitate was added with a β-galactosidase activity measurement solution [o-nitrophenyl-β-D-
0.1% BSA with galactopyranoside 5 mg/ml,
85 parts of 10mM phosphate buffer (PH6.7) containing _1mMgCI2 , 0.1% _NaN3 , 0.15M NaCl, and
Methanol solution containing 200mM mercaptoethanol
Solution consisting of 15 parts] Add 200μ and incubate at 37℃ for 60 minutes.
Allowed to react for minutes. After the reaction is complete, add the reaction stop solution [100mM glycine-NaOH buffer (PH11.0)].
Add 2.3ml to stop the reaction, and measure the reaction solution at 420nm.
The absorbance at each dilution of the antiserum was measured, and the binding rate due to the immunoreaction with β-galactosidase [Cys ⁴⁵ ]-peptide (45-84) at each dilution concentration of the antiserum was determined.
The results for the 3200-fold dilution are shown in Table 1, but similar trends were observed for other dilutions.

[Table] As a result, all antigens were found to be useful immunizing antigens capable of producing specific antibodies. Furthermore, judging from the immunoreactivity with various h-PTH-related substances described below, it is clear that this is an antigen capable of producing antibodies specific to the C-terminus of h-PTH. Immunoreactivity with ¹²⁵ I-labeled substances Three antisera showed high titers when measured using the EIA system [anti-peptide (46-84); No. 1-(A), anti-peptide (53-84); ); No. 3-(B), and anti-BSA-[ ^Cys45 ]-
Peptide (45-84); No. 1-(C)], each ¹²⁵ I
-Immunoreactivity with the labeled substance was investigated. The above three types of serum were mixed with immunoreaction medium [50mM veronal buffer containing 0.5% BSA (PH8.0)] at 200%
100μ of solutions diluted 400, 800, 1600, 3200, 6400, 12800, 25600 times: each ¹²⁵ I-labeled substance (0.01μci
Add 100μ of each labeled substance with radioactivity) and 100μ of immunoreaction medium to a small test tube,
React for 45 hours at 5°C, then add 100μ of a 100-fold dilution of normal guinea pig serum and 100μ of a 10-fold dilution of rabbit anti-guinea pig γ-globulin serum.
In addition, the reaction was further carried out at 5°C for 24 hours. The reaction solution was centrifuged at 3000 rpm for 15 minutes to collect the precipitate, and the radioactivity of the precipitate was measured using a γ-counter, and the immunoreaction of each label with the antiserum at each diluted concentration was determined. The binding rate was determined.
The results for the 6400-fold dilution are shown in Table 2, but similar trends were observed for other dilutions.

[Table] As a result, both labeled antigens showed that the C of h-PTH
It shows good reactivity with end-specific antibodies,
It was found to be useful as a labeled antigen for RIA. (4) Immunoreactivity with various h-PTH-related substances [Extraction and purification of h-PTH (1-84)] h-PTH (1-84) was extracted from patients with hyperparathyroidism. The parathyroid gland was removed and extracted and purified according to the method of Keutmann et al. (ACS, 17, 26 , 1978). [Preparation method of natural h-PTH (53-84)] The purified h-PTH (1-84) described above was digested with trypsin, and the method of Keutmann et al. (ACS, 17, 26 ,
(1978). [Synthesis [Asp ⁷⁶ ]-h-PTH (53-84)] Synthesis [Asp ⁷⁶ ]-h-PTH (53-84)
It was synthesized according to the specification of No. 53-187686. [Measurement of immunological cross-reactivity] Various h-PTH related substances (natural h-PTH (1-
84), natural h-PTH (53-84), synthetic [ ^Asp76 ]-h
-PTH (53-84), peptide of the present invention (53-84)
each containing 0.2 to 2000 fmoles) solution 100 μ, β
-galactosidase- [Cys ⁴⁵ ] peptide (45-
84) (approximately 10 pg as [Cys ⁴⁵ ] peptide (45−84)
containing) solution 100μ, and antiserum diluted solution (A is
Add 100μ of a 15,000-fold dilution, B: a 5,000-fold dilution, and C: a 9,000-fold dilution) to a reaction test tube and allow to react at 5°C for 24 hours, then add 100μ of a 100-fold dilution of guinea pig normal serum, anti-guinea pig gamma
- 100 μl of a 10-fold dilution of globulin serum (rabbit)
was added, and the reaction was further carried out at 5°C for 24 hours. After adding 3 mml of 0.5M NaCl to the reaction solution, it was heated at 3000 rpm for 15 minutes.
The precipitate was collected by centrifugation for a minute. Next, 200μ of the β-galactosidase activity measurement solution (described above) was added to the precipitate, and the mixture was reacted at 37°C for 120 minutes.
After the reaction is complete, add 2.3 ml of the reaction stop solution (above),
The reaction solution was stopped, and the absorbance of the reaction solution at 420 nm was measured. The results are as shown in Figure 1 (antiserum-A), Figure 2 (antiserum-B), and Figure 3 (antiserum C).
In each figure, 〇-〇 indicates the case of the peptide of the present invention (53-84), ●-● indicates the case of natural h-PTH (53-84), and △-△ indicates the case of the synthetic [Asp ⁷⁶ ]- h-PTH
(53-84) is shown, □-□ is natural h-
The case of PTH (1-84) is shown. From the results of the above immune reactions, etc., various peptides represented by the formula [] of the present invention are peptide hormones consisting of 84 amino acids, h-
It was a useful reagent for diagnosing PTH-related diseases in measuring the blood concentration of PTH on the C-terminal side. Therefore, these various peptides are also known
It may be appropriately used in various quantitative methods in EIA and RIA, such as competitive methods, Sand-Deutsch methods, and combinations of these with solid-phase methods and antibody methods. Furthermore, in EIA, enzyme-labeled peptides may be obtained by appropriately selecting and using various known enzymes, such as peroxidase and alkaline phosphatase, as labels in addition to β-galactosidase, which is the labeling compound described above. Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto. In addition, the abbreviations described in this specification have the following meanings. 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-CI; 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 BuCH; Butanol ether; Diethyl ether WSCI; N-ethyl, N'-3-dimethylaminopropyl-carbodiimide HOBT; 1-hydroxybenzotriazole Next, examples of the production of the present invention will be described with reference to Examples and Reference Examples. I will explain in detail. The carrier and developing solvent for thin layer chromatography (TLC) used in the examples are as follows. Support: Silica Gel G manufactured by Merck & Co., Ltd. Developing solvent: 1; CHCI 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: Cellulose manufactured by Merck & Co., Ltd. Developing solvent: 9; BuOH-pyridine-acetic acid-water
(2:2:2:3) 10;BuOH-pyridine-acetic acid-water
(1:1:1:2) 11; BuOH-pyridine-acetic acid-water
(15:10:3:12) The conditions for amino acid analysis are as follows. The specimen was hydrolyzed with 6N hydrochloric acid (10% anisole was added if necessary in the case of a protected peptide) at 110°C for 24 to 45 hours in a sealed tube, dried under reduced pressure, and subjected to amino acid analysis. Reference example 1 [Tyr ⁶⁴ ]-peptide (65-84); H-Tyr-
Lys−Ser−Leu−Gly−Glu−Ala−Asp−Lys
−Ala−Asp−Val−Asn−Val−Leu−Thr−
Lys−Ala−Lys−Ser−Gln−OH (1) P(83−84); BOC−Ser(Bzl)−Gln−OBzl
[] BOC−Gln−OBzl81.4g (0.242M)
Dissolved in 270ml of TFA and stirred at room temperature for 45 minutes.
TFA was distilled off under reduced pressure. Add ether to the residue,
The resulting precipitate was collected. Dissolve this in 270ml of DMF, add 32.67g of HOBT (0.242M) and BOC-Ser.
(Bzl)-OH67.35g (0.242M) and WSCI44.29
ml (0.242M) and stirred overnight. After the reaction,
DMF was distilled off under reduced pressure. Add the residue to 440ml of ethyl acetate.
The solution was washed with 1N hydrochloric acid, 5% aqueous sodium bicarbonate, and water in this order. It was dried with anhydrous sodium sulfate and evaporated to dryness. Recrystallization from ethyl acetate-hexane gave 101.43 g (yield: 81.6%). Melting point 121-123℃ TLC; Rf ₇ = 0.75 [α] ²⁷ D-14.12 (C = 1.0, 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, HOBT28.6
g, DMF solution of BOC-Lys(Z-Cl)-OH (BOC-Lys(Z-Cl)-OH・TBA103.33g(1.1
M) 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 110ml DMF) and 83.72ml WSCI
(1.1 times the mole) was added, and the mixture was stirred at room temperature for 2 days. After the reaction, DMF was distilled off under reduced pressure, ice water was added to the residue,
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°C [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 successively with 1N hydrochloric acid and water.
After drying with anhydrous sodium sulfate and drying under reduced pressure, 400ml of DMF
It was dissolved in To this, H-Ala-OMe・HCl67.0
g, 64.8 g of HOBT, and 87.84 ml of WSCI were added, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in ethyl acetate 2, and 5% aqueous sodium bicarbonate, 1N
Washed with hydrochloric acid and water in that order. After drying with anhydrous sodium sulfate and drying under reduced pressure, it was dissolved in 400 ml of DMF. H to this
-Ala-OMe・HCl67.0g, HOBT64.8g,
87.84 ml of WSCI was added and stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was diluted with ethyl acetate.
The solution 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. Recrystallized from ethyl acetate-hexane to give 231.8 g of [3] (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 Calculated value 55.25 6.85 8.40 (4) P(79−81); BOC−Thr(Bzl)−Lys(Z−
Cl)-Ala-OMe [4] [3] Add 174.99g (0.35M) to 500ml of TFA,
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, followed by washing with 5% aqueous sodium bicarbonate and water in this order. After drying with anhydrous sodium sulfate, it was dried under reduced pressure. Dissolve the residue in 400ml of DMF and add
HOBT49.95g (1.05x M), BOC−Thr(Bzl)−
OH114.33g (1.05x M) and WSCI67.7ml
(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, and the residue was dissolved in chloroform and washed successively with 5% sodium bicarbonate solution (4 times), 1N hydrochloric acid (2 times), and water (2 times). After drying with anhydrous sodium sulfate, it was dried under reduced pressure. The residue was subjected to silica gel column chromatography [eluent: chloroform-ethanol-
Acetic acid (5:1:5)] [4] 35.09
I got g. The fraction containing impurities was used for column chromatography purification in the next additional synthesis. Next, [3] 22.5g (45mM) of TFA70
ml and stirred at room temperature for 45 minutes. After the reaction,
After TFA was distilled off under reduced pressure and the residue was dissolved in 50 ml of DMF, the pH was adjusted to 7 with NMM. Then,
HOBT6.68g (1.1x M), BOC−Thr(Bzl)−
OH15.31g (1.1xM), WSCI9.06ml (1.1xM)
was added and stirred overnight. After the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in 200 ml of chloroform.
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 purified by silica gel column chromatography together with the previous impure fraction. 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,
Dissolve WSCI86.01ml (0.47M) in THF400ml,
Stir overnight. After the reaction, THF was distilled off under reduced pressure, the residue was dissolved in 400 ml of ethyl acetate, and then 5% aqueous sodium bicarbonate solution,
Washed with 1N hydrochloric acid and water in that 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] 34.43g (0.375M) in 400ml of ethanol
Dissolve in 1N-NaOH412.5ml (1.1x M) under ice-cooling.
was added and 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. Wash with ether and aqueous layer
400 ml of 1N hydrochloric acid was added, 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 (yield) of [6].
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 added to 350 ml of DMF.
It was dissolved in Adjust this to PH6.5 with NMM under cooling, HOBT42.61g (1.2 times M), [6] 104.28g
(1.2x M) and WSCI57.8ml (1.2x M) were added.
After stirring for 1.5 hours at room temperature, the mixture solidified and could not be stirred even after adding 200 ml of DMF, so it was left at room temperature overnight and then at 30°C for 4 hours. Ice water was added to the reaction mixture and the precipitate was collected. Add this to chloroform 2.5
The solution was washed with 5% sodium bicarbonate solution, 1N hydrochloric acid, and water in this order. Chloroform was distilled off under reduced pressure, and the residue was reconsolidated from chloroform-etherhexane [7]
224.68g (yield 94.9%) was obtained. Melting point: 219-221℃ 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 720ml of chloroform
800 ml of a 90% ethanol solution of 1N-KCH was added under cooling, and the mixture was stirred at 0 to 5°C for 40 minutes. Next, add 800ml of 1N hydrochloric acid under cooling, and add chloroform.
Added 500 ml and extracted. The chloroform layer was washed with water, dried over anhydrous sodium sulfate, and then dried under reduced pressure. The residue was subjected to silica gel column chromatography [eluent: chloroform-ethanol-ethyl acetate (5:
1:5)]. The corresponding fractions were dried under reduced pressure to form chloroform-methanol-ether.
[8] was obtained by recrystallization three times from hexane. The above operation was repeated three times, and a total of 216.9 g of [7] was hydrolyzed to obtain 156.07 g of [8] (yield 73.1%). 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), Alal, Val0.93(1), Leu0.94(1),
Lys1.01(1) (9) (77−84);BOC−Val−Leu−Thr(Bzl)−
Lys(Z-Cl)-Ala-Lys(Z-Cl)-Ser(Bzl)
-Gln-OBzl [9] [2] Add 104.5g (129mM) to 400ml of TFA,
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. This includes HOBT20.9
g (1.2 times M), [8] 137.7 g (1.2 times M),
28.3 ml of WSCI (1.2 times M) was added, the pH was adjusted with NMM, and the mixture was stirred overnight. Since the reaction solution became gel-like, 150 ml of DMF and 12.8 ml of WSCI were further added and stirred overnight. Ice water was added to the reaction solution, and the precipitate was collected and washed five times with hot methanol to obtain 185.36 g (yield: 90.68%) of [9]. 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 60.75 6.82 9.74 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−Asn−Val−Leu−Thr
(Bzl)−Lys(Z−C1)−Ala−Lys(Z−Cl)−
Ser(Bzl)-Gln-OBzl [10] [9] Add 174.0g (110mM) to 550ml of TFA,
After stirring at room temperature for 60 minutes, TFA was distilled off under reduced pressure.
Add ether to the residue, collect the resulting precipitate,
Dissolved in 950ml of DMF. In addition, BOC−Asn−
77.7 g (2.0 times M) of ONP was added, and the mixture was stirred at room temperature for 48 hours while adjusting the pH to 7.5 with NMM. After reaction
DMF was distilled off under reduced pressure, and ice water was added to the residue. The resulting precipitate was collected and washed with hot methanol.
After cooling, insoluble matter was collected. The same treatment was performed two more times to obtain 179.6 g (yield: 96.3%) of [10]. Melting point; 248-251℃ TLC; Rf ₂ = 0.65 Rf ₃ = 0.40 Elemental analysis [as C ₈₄ H ₁₁₃ O ₂₀ N ₁₃ Cl ₂ ] C% H% N% Measured value 59.23 6.89 10.88 Calculated value 59.50 6.72 10.74 (11) P(75−84); BOC−Val−Asn−Val−Leu
−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z−
Cl)-Ser(Bzl)-Gln-OBzl [11] [10] Add 169.58g (0.1M) to 500ml of TFA,
After stirring at room temperature for 60 minutes, TFA is removed under reduced pressure.
Add ether to the residue, collect the resulting precipitate,
Dissolved in DMF1.3. While stirring this
HOBT17.6g (1.3x M), BOC-Val-OH28.2
g (1.3xM) 2.4-dinitrophenol 23.9g
(1.3 times M) and 23.8 ml of WSCI (1.3 times M) were added, the pH was adjusted to 7 with NMM, and the mixture was stirred at room temperature. After 1 hour, the reaction solution became gel-like, so it was left overnight. Next, NMP400ml, WSCI23.8ml
(1.3xM) 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 washed four times with hot methanol [11]
171.06g (yield 95.3%) was obtained. Melting point: 256-269℃ TLC; Rf ₃ = 0.36 Elemental analysis [as C ₈₉ H ₁₂₂ O ₂₁ N ₁₄ Cl ₂・H ₂ O] C% H% N% Measured value 59.14 6.71 10.86 Calculated value 58.96 6.89 10.82 Amino acid analysis [ Sample 3.1mg/1ml 6N hydrochloric acid + 0.1ml
Anisole, 48 hours, 110℃ hydrolysis]; Asp1.01(1), Thr0.82(1), Ser0.76(1), Glu1.05(1),
Ala1.03(1), Leu1, Val1.85(2), Lys2.15(2) (12) P(74−84); BOC−Asp(OBzl)−Val−Asn
−Val−Leu−Thr(Bzl)−Lys(Z−Cl)−Ala
-Lys(Z-Cl)-Ser(Bzl)-Gln-OBzl [12] [11] 143.60g (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 500ml of DMF and NMP1,
BOC-Asp(OBzl)-OSU43.7g (1.3x M),
1.14 g of HOBT (0.13 times M) and 11.4 ml of NMM (1.3 times M) were added, and the mixture was 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 treatment was repeated twice to obtain 149.5 g (yield 93.4%) of [12]. TLC; Rf ₃ = 0.35 Elemental analysis [as C ₁₀₀ H ₁₃₃ O ₂₄ N ₁₅ Cl ₂ ] C% H% N% Measured value 60.01 6.79 10.91 Calculated value 60.05 6.70 10.50 Amino acid analysis [6N hydrochloric acid/anilyl, 110℃,
48 hours]; Asp1.90(2), Thr0.88(1), Ser0.75(1),
Glu1.03(1), Ala1.01(1), Val1.95(2), Leu1,
Lys2.09(2) (13) P(72−73); BOC−Lys(Z−Cl)−Ala−
OH [13] [3] Dissolve 48.0g (96mM) in 100ml of ethanol, and add 115.2ml of 1N-NaOH to this while cooling to 0℃.
(1.2xM) and stirred for 50 minutes. After the reaction,
After adjusting the pH to 6 by adding 19 ml of 1N hydrochloric acid, 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. The ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, and then dried under reduced pressure.
The residue was reconsolidated from ethyl acetate-hexane [13]
45.90g (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−Asn−Val−Leu−Thr
(Bzl)−Lys(Z−Cl)−Ala−Lys(Z−Cl)−
Ser(Bzl)−Gln−OBzl [14] [12] 120.01 (60mM) with 60ml of methylene chloride,
After adding 360ml of TFA and stirring at room temperature for 55 minutes,
TFA was distilled off under reduced pressure. Add ether to the residue,
The resulting precipitate was collected. Add 600ml of DMF to this
600ml of NMP was added and dissolved. Add this to ice +5%
Added to baking soda solution. The resulting precipitate was collected by filtration, washed with water three times, then washed with methanol and ether, and then dried. Add 1200ml of NMP and 600ml of DMF to this and dissolve, HOBT10.54g (1.3x M), [13] 37.92g
(1.3 times M) and 14.28 ml of WSCI (1.3 times M) were added, and the mixture was stirred at room temperature for 3 days. The reaction solution was added to ice water, and the resulting precipitate was collected, washed three times with water, and then twice with hot methanol. Then after washing with ether,
After drying, 135.83 g (yield 95.6%) of [14] was obtained. Elemental analysis [as C ₁₁₇ H ₁₅₅ O ₂₈ N ₁₈ Cl ₃ ] C% H% N% Measured value 59.12 6.73 10.87 Calculated value 59.35 6.60 10.65 Amino acid analysis [6N hydrochloric acid/anisole, 110℃,
48 hours]; Asp1.09(2), Thr0.85(1), Ser0.72(1),
Glu1.02(1), Ala1.95(2), Val1.94(2), Leu1,
Lys2.99(3) (15) P(71−84); BOC−Asp(OBzl)−Lys(Z
−Cl)Ala−Asp(OBzl)−Val−Asn−Val−
Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z
-Cl)-Ser(Bzl)-Gln-OBzl [15] [14] Add 118.40g (50mM) to 500ml of TFA,
After stirring at room temperature for 55 minutes, TFA was distilled off under reduced pressure.
Add ether to the residue, collect the resulting precipitate,
700 ml of NMP and 700 ml of DMF were added and dissolved. This was added to ice + 5% sodium bicarbonate solution, the resulting precipitate was collected by filtration, and 3 times with water and 1 time each with methanol and ether.
Washed twice. Add 800ml of NMP and 800ml of DMF to this sediment.
11.05 g of 2,4-dinitrophenol (1.2 times M), 0.81 g of HOBT (0.12 times M), 25.22 g of BOC-Asp(OBzl)-OSU (1.2 times M), and 6.60 ml of NMM. (1.4x M) and
The mixture was stirred for several days. 120ml of NMP and 60ml of DMF in the reaction solution
Add and dissolve BOC−Asp(OBzl)−
OSU4.20g (0.2xM), NMM1.09ml (0.2xM)
was 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.
Next, it was suspended in hot methanol, cooled, and collected by filtration. Repeat this operation three times to obtain [15] 117.47
g (yield 91.3%) was obtained. Elemental analysis [as C ₁₂₈ H ₁₆₆ O ₃₁ N ₁₉ Cl ₃ ] C% H% N% Measured value 59.52 6.32 10.60 Calculated value 59.75 6.50 10.34 Amino acid analysis [6N hydrochloric acid/anisole, 110℃,
48 hours]; Asp2.71(3), Thr0.85(1), Ser0.70(1),
Glu1.01(1), Ala1.95(2), Val1.92(2), Leu1,
Lys2.92(3) (16) P(70−84); BOC−Ala−Asp(OBzl)−
Lys(Z−C1)−Ala−Asp(OBzl)−Val−Asn
−Val−Leu−Thr(Bzl)−Lys(Z−Cl)−Ala
-Lys(Z-Cl)-Ser(Bzl)-Gln-OBzl [16] [15] Add 102.9g (40mM) to 400ml of TFA,
After stirring at room temperature for 70 minutes, TFA was distilled off under reduced pressure.
Add ether to the residue, collect the resulting precipitate,
After adding and dissolving 600ml of NMP and 600ml of DMF,
% sodium bicarbonate solution. Collect the resulting precipitate and dilute with water
Washed once with methanol. Next, 720ml of NMP and 720ml of DMF were added to this precipitate and dissolved.
Add to this 11.0 g (1.5 g) of 2,4-dinitrophenol.
xM), HOBT0.54g (0.1xM), BOC-Ala-
OSU17.18g (1.5x M), NMM6.60ml (1.5x M)
was added and stirred at room temperature overnight. Then, BOC−
Ala-OSU2.29g (0.2xM), NMM0.88ml (0.2
M) was added and stirred at room temperature for 5 days. The reaction solution was added to ice water, the resulting precipitate was collected and diluted with water three times.
Washed twice with methanol [16] 98.90g (yield
93.5%). Melting point: 280℃ or higher (decomposition) Amino acid analysis [6N hydrochloric acid/anisole, 110℃,
48 hours]; Asp2.75(3), Thr0.84(1), Ser0.72(1),
Glu1.01(1), Ala2.80(3), Val1.92(2), Leu1,
Lys2.92(3) (17) P(69−84); BOC−Glu(OBzl)−Ala−
Asp(OBzl)−Lys(Z−Cl)−Ala−Asp(OBzl)
−Val−Asn−Val−Leu−Thr(Bzl)−Lys(Z
−Cl) −Ala−Lys(Z−Cl)−Ser(Bzl)−Gln−
Add 92.55g (35mM) of OBzl [17] [16] to 350ml of TFA,
After stirring at room temperature for 60 minutes, TFA was distilled off under reduced pressure.
Add ether to the residue, collect the resulting precipitate,
600 ml of DMF and 600 ml of NMP were added and dissolved, and this was added to 5% sodium bicarbonate water + ice. Collect the resulting precipitate,
Washed three times with water and once with methanol. to this
Add 600ml and 800ml of DMF and dissolve, add 2,
4-dinitrophenol 7.73g (1.2x M),
HOBT0.59g (0.14x M), BOC-Glu(OBzl)-
OSU21.29g (1.4xM), NMM5.4ml (1.4xM)
was added and stirred at room temperature for 3 days. Then BOC−
Glu (OBzl) - OSU3.04g (0.2x M) and DMF60ml
＋NMP60ml added and dissolved solution and
Add 0.77ml of NMM (0.2x M) and
The mixture was stirred for several days. Next, add the same amount of BOC-Glu as before.
(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, cooled, and collected by filtration. This operation was repeated three times [17]92.71g (yield
92.5%). Amino acid analysis [6N hydrochloric acid/anisole, 110℃,
24 hours]; Asp2.72(3), Thr0.85(1), Ser0.76(1),
Glu1.85(2), Ala2.80(3), Val1.93(2), Leu1,
Lys2.95(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
Dissolve in 200ml of THR and cool with 33.69ml of WSCI.
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. Add this to ethyl acetate 600
ml, 5% sodium bicarbonate solution, 1N hydrochloric acid, and water.
Wash twice. The organic layer was dried over anhydrous sodium sulfate and dried under reduced pressure to obtain 69.0 g (yield: 99%) of an oily substance. Dissolve this in 10 ml of methylene chloride, add 250 ml of TFA while cooling to 5°C, and then heat at room temperature while stirring.
The reaction was allowed to proceed for 20 minutes. 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. This includes HOBT20.7g (0.19M),
BOC-Ser(Bzl)-OH56g (0.19M) and
After adding 34.8 ml of WSCI (0.19 M), the mixture was 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 by filtration. After recrystallizing from ethyl acetate-hexane,
Recrystallization from ethyl acetate-ether-hexane gave 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) and 20ml of methylene chloride
After cooling to 5° C., 250 ml of TFA was added, and the mixture was 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 100 ml of DMF was added. This is cooled to 5℃.
Neutralized by adding NMM. On the other hand, BOC-Lys(Z-Cl)-OH・TBA70g
(0.143M), add 200ml of ethyl acetate, add 1N hydrochloric acid,
Washed twice with water. The ethyl acetate layer was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. Add 100 ml of DMF to the obtained oil, and add BOC-Lys(Z-Cl)-
It was made into a DMF solution of OH. 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 diluted with 5% aqueous sodium bicarbonate (1N) three times.
Washed twice with 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, and the resulting precipitate was collected and recrystallized from ethyl acetate-methanol-hexane to obtain 104 g (yield 94.6%) of [19]. 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/anisole,
105℃, 48 hours]; Ser0.92(1), Gly0.98(1), Leu1,
Lys0.99(1) (20) P(65−68); BOC−Lys(Z−C1)−Ser
(Bzl)-Leu-Gly-OH [20] [19] Add 600 ml of methanol to 85.3 g and heat at 5℃.
While stirring under cooling, 120 ml of 1N-NaOH was added, and the mixture was stirred at room temperature for 3 hours. After the reaction, 20 ml of 1N hydrochloric acid was added while cooling to 5°C, and methanol was distilled off under reduced pressure. 100 ml of 1N hydrochloric acid was added to the aqueous solution of the residue while cooling at 5°C, and the mixture was extracted with 500 ml of chloroform.
The chloroform layer was washed with water, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. Hexane was added to the residue, the resulting precipitate was collected, and recrystallized from ethyl acetate [20]
66.21 g (yield 87%) was obtained. 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 [0.5 μM/6N hydrochloric acid / Anisole,
105℃, 48 hours]; Ser0.92(1), Gly0.97(1), Leu1,
Lys0.99(1) (21) P(65−84); BOC−Lys(Z−Cl)−Ser
(Bzl)−Leu−Gly−Glu(OBzl)−Ala−Asp
(OBzl)−Lys(Z−Cl)−Ala−Asp(OBzl)−
Val−Asn−Val−Leu−Thr(Bzl)−Lys(Z−
Cl) −Ala−Lys(Z−Cl)−Ser(Bzl)Gln−
Add 100ml of TFA to OBzl [21] [17] 14.32g (5mM),
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 100 ml of NMP was added to dissolve it. Ice this
It was added to 5% sodium bicarbonate solution, and the resulting precipitate was collected by filtration and washed with water and methanol in that order. to this precipitate
Add and dissolve 200ml of NMP, add 1.10g of 2,4-dinitrophenol, and 0.81g of HOBT [20]
After 4.66 g was added and dissolved, 1.10 ml of WSCI was added at -10°C, and the mixture was stirred at room temperature for 3 days. After the reaction, the reaction solution was poured into ice-5% sodium bicarbonate solution, the resulting precipitate was collected by filtration, and water,
Wash with methanol (4 times), [21] 15.00g
(yield 85.5%). Amino acid analysis; Asp2.72(3), Thr0.93(1), Ser1.65(2), Glu1.82
(2), Gly0.99(1), Ala2.82(3), Val1.98(2), Leu2,
Lys3.92(4), (22) [Tyr ⁶⁴ ]P(64−84); BOC−Tyr(Bzl−
Cl ₂ )−Lys(Z−Cl)−Ser(Bnl)−Leu−Gly−
Glu(OBzl)−*;Asp2.74(3), Thr0.91(1),
Ser1.67(2), Glu1.86(2), Gly0.98(1), Ala2.86
(3), Val1.99(2), Leu2, Lys3.95(4), Tyr0.95(1) (2) [ ^Tyr64 ]-peptide (64-84); [22] 3.06g (0.8mM) 5 ml of anisole was added to the mixture, and 50 ml of anhydrous HF was introduced thereinto, followed by stirring at 0° C. for 1 hour. Next, HF was distilled off under reduced pressure, ether was added to the residue, the precipitate was collected, and 0.1N acetic acid was added.
The solution was dissolved in 50 ml and passed through a Dowex 1×2 column (3.0×40 cm, acetic acid type), and the effluent was freeze-dried to obtain 2.41 g. Dissolve this in 70ml of 8M urea aqueous solution, adjust the pH to 9.5 with aqueous ammonia, and then
It was left at ℃ for 1 hour. This solution was then charged to a CM-cellulose column (3 x 30 cm),
0.01M ammonium acetate buffer (PH4.5) 800ml~
Elution was performed using a value linear concentration gradient of 800 ml of 0.2 M ammonium acetate buffer (PH4.5), and the eluate was 10 ml.
The 79th to 99th fractions were collected, and after freeze-drying, they were separated using a Sephadex LH-20 column (3.0 x 120 cm, 0.1N
acetic acid), fractionated into 9.8 ml portions, collected the 42nd to 49th portions, and freeze-dried to obtain 483 mg of [Tyr ⁶⁴ ]-peptide (64-84). TLC; Rf ₁₁ = 0.15 Amino acid analysis (6N-hydrochloric acid, 105°C, 24 hours);
Asp2.96(3), Thr0.95(1), Ser1.79(2), Glu2.03(2),
Gly0.97(1), Ala2.94(3), Val1.97(2), Leu2,
Lys4.06(4), Tyr0.96(1) Example 1 Peptide (53-84); H-Lys-Lys-Glu-
Asp－Asn－Val－Leu－Val－Glu－Ser－His
−Glu−Lys−Ser−Leu−Cly−Glu−Ala−
Asp−Lys−Ala−Asp−Val−Asn−Val−Leu
−Thr−Lys−Ala−Lys−Ser−Gln−OH (1) P(64−68); BOC−Glu(OBzl)−Lys(Z−
Cl)-Ser(Bzl)-Leu-Gly-OH [23] Add 30 ml of methylene chloride to 66.2 g (87 mM) of [20] described in Reference Example 1, and add 250 ml of TFA while cooling to 5°C.
was added, and the mixture was 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 was neutralized with NMM while cooling to 5°C. 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 obtained precipitate was washed with water and dried. Recrystallization was performed twice from acetone-methanol to obtain 63.85 g (yield: 73.5%) of [23]. 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/anisole, 105℃ 48 hours]; Ser0.92(1), Glu0.99(1),
Gly0.97(1), Leu1, Lys0.99(1) (2) P(62−63); BOC−Ser(Bzl)−His−
NHNH ₂ [24] 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. Further, 4.4 g of HOBT and 6 ml of WSCI were added, and the mixture was 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 the crude product.
BOC−Ser(Bzl)−His−OMe(TlC; Rf ₃ =0.56)
46.1g was obtained. 44.6 g (0.1 M) of the above product was dissolved in 300 ml of DMF, 100 ml of hydrazine hydrate (100%) was added thereto, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure. The residue was extracted eight times with ethyl acetate, 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 subjected to silica gel column chromatography [elution solvent: chloroform-ethyl acetate (1:1) to chloroform-methanol-ethyl acetate (5:1:
5)]. The corresponding sections were dried under reduced pressure, and the residue was dissolved in benzene-hexane (very small amount).
It was crystallized from After being placed in an ice chamber, the precipitated crystals were recrystallized twice from ethyl acetate-methanol-hexane to obtain [24]. 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 (3) P (62−68);BOC−Ser(Bzl)−His−Glu
(OBzl)−Lys−(Z−C1)−Ser(Bzl)−Leu−
Dissolve 33.5g of Gly-OH [25] [24] in 200ml of DMRF, and add 4.32N hydrogen chloride in dioxane solution while cooling to -50℃.
After adding 52 ml and 20 ml of isoamyl nitrite, −20
Stirred at °C for 10 minutes. Then cooled to -50°C,
Added 31.6 ml of _Et3N . On the other hand, [23] 63.85g (64mM) was added to methylene chloride.
After adding 20 ml of TFA to this while cooling to 5° C., 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 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 the reaction, DMF was distilled off under reduced pressure, and the residue was dissolved in methanol. This was poured into water, and the resulting precipitate was collected by filtration, washed with water, and dried. Reconcrete with DMF-ether and methanol
After washing twice, 59.16 g (yield 60.1%) of [25] 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/anisole, 105℃, 45 hours]; Ser1.82(2), Glu1.01(1),
Gly0.98(1), Leu1, Lys1.01(1), His0.94(1) (4) 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−Asn−Val−
Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z
-Cl)-Ser(Bzl)-Gln-OBzl [26] [17] 250ml of TFA was added to 71.59g (25.0mM) 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 and dissolved by adding 500 ml of NMP and 500 ml of DMF. This was added to ice-5% sodium bicarbonate solution, and the resulting precipitate was collected by filtration and washed successively with water and methanol.
Add NMPl and DMF1 to this precipitate, dissolve it, and add 5.52 g of 2,4-dinitrophenol.
(1.2xM), HOBT4.05g (12xM), [25] 38.84
g (1.2 times M) and 5.5 ml of WSCI (1.2 times M) were added and stirred at room temperature for 4 days. After the reaction, the reaction solution was
% sodium bicarbonate water-ice, and the resulting precipitate was collected by filtration.
Washing with water, hot methanol, and methanol (twice) in this order gave 89.21 g (yield: 88.3%) of [26]. Amino acid analysis [0.5μM/6N hydrochloric acid-anisole
110℃ 48 hours]; Asp2.90(3), Thr0.91(1), Ser1.92
(3), Glu2.89(3), Gly0.89(1), Ala2.95(3),
Val2.20, Leu2, Lys4.02(4), His0.88(1) (5) 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−
Asn−Val−Leu−Thr(Bzl)Lys(Z−Cl)−
Ala−Lys(Z−Cl)−Ser(Bzl)−Gln−OBzl
[27] [26] Add 250ml of TFA to 48.49g (12mM),
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 350 ml of DMF and 550 ml of NMP were added to dissolve it.
This was poured into 5% sodium bicarbonate water-ice, and the resulting precipitate was collected by filtration, washed with water, and then with methanol. Add and dissolve 800 ml of DMF and 700 ml of NMP to this precipitate, add 2.7 g of 2,4-dinitrophenol (1.2 times M), 7.30 g of BOC-Glu (OBzl)-OSU (1.4 times M),
HOBT0.23g (0.14xM) and NMM1.85ml
(1.4xM) was added and stirred at room temperature overnight. Next, BOC-Glu(OBzl)-OSU1.04 was added to this reaction solution.
g (0.2 times M) and 0.26 ml of NMM (0.2 times M) were added, and the mixture was further stirred overnight. Add reaction solution to ice-5%
Pour into baking soda, filter the resulting precipitate, wash thoroughly with water, and then wash with hot methanol three times [27] 45.3
g (yield: 88.6%). Amino acid analysis [/6N hydrochloric acid, anisole, 110
°C, 48 hours]; Asp2.98(3), Thr0.93(1), Ser1.83
(1), Glu3.62(4), Gly0.89(1), Ala2.97(3),
Val2.23, Leu2, Lys4.06(4), His0.85(1) (6) P(59−60); BOC−Leu−Val−OBzl [28] H−Val−OBzl・TOSOH17.8g (47mM ) was added with 100 ml of ethyl acetate, washed successively with 5% sodium bicarbonate solution and water, and the ethyl acetate layer was dried over anhydrous sodium sulfate, and then ethyl acetate was distilled off under reduced pressure. Dissolve the residue in 100ml of DMF and add 11.7g of BOC-Leu-OH・H ₂ O to this.
(56.4mM), HOBT9.3g (1.2x M) and
10.3 ml of WSCI (1.3 times M) was added and stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in 300 ml of ethyl acetate, and 5% aqueous sodium bicarbonate, 1N hydrochloric acid,
Washed with water. After drying with anhydrous sodium sulfate, it was dried under reduced pressure. The residue was crystallized from ethyl acetate-hexane to obtain 17.55 g (yield: 88.8%) of [28]. TLC; Rf ₆ = 0.85 (7) P (58-60); BOC-Val-Leu-Val-OBzl
[29] [28] 17.2g (41mM) of TFA (50ml) was added, and after stirring at room temperature for 30 minutes, TFA was distilled off under reduced pressure.
Ether was added to the residue, and the resulting product was collected by filtration and then dissolved in 60 ml of DMF. 7.7g of HOBT for this
(1.2xM), BOC-Val-OH10.7g (1.2xM) and WSCI9.0ml (1.2xM), and PH with NMM.
After adjusting the temperature to 7, 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, IN 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 [29]. Melting point; 149-152℃ TLC; Rf ₅ = 0.82 [α] D ²² ; -32.36 (C = 1.0DMF) 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 (8) P(57−60); BOC−Asn−Val−Leu−Val
−OBzl [30] [29] Add 50ml of TFA 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. HOBT0.62g for this
(0.14xM) BOC-Asn-ONP16.32g (1.4xM)
was added, the pH was adjusted to 7 with NMM, and 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 methanol-ether [30]
13.82g (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 (9) P (57−60); BOC−Asn−Val −Leu−Val
−OH [31] [30] 13.2g (25mM) with 50ml of ethanol
It was dissolved in a mixture of 60 ml of DMF, 5% PD/clg was added thereto, and hydrogenated for 3 hours. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure. The residue was recrystallized twice from ethanol-ether to obtain 9.70 g (yield 71.4%) of [31]. TLC; Rf ₂ = 0.56 Amino acid analysis [0.5 μM/6N hydrochloric acid-anisole,
110℃ 48 hours]; 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 (10) P(57−84); BOC−Asn−Val−Leu−Val
−Glu(0Bzl)−Ser(Bzl)−His−Glu(OBzl)−
Lys(Z−Cl)−Ser(Bzl)−Leu−Gly−Glu
(OBzl)-Ala-Asp(OBzl)-Lys(Z-Cl)-
Ala−Asp(OBzl)−Val−Asn−Val−Leu−
Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z−Cl)
-Ser(Bzl)-Gln-OBzl [32] [27] Add 220ml of TFA to 42.60g (10mM),
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, and the resulting precipitate was filtered and washed three times with water and once with methanol. Add and dissolve NMP1.2 and DMF1.2 to this precipitate, add 1.76 g of HOBT (0.3 times M), 2,
4-dinitrophenol 2.39g (1.3x M), [31]
7.05 (1.3 times M) and 4.76 ml of WSCI (2.6 times M) were added and stirred at room temperature for 2 days. Furthermore [31] 1.09
A solution of g (0.2 times M) dissolved in 100 ml of DMF was added, and the mixture was stirred at room temperature overnight. The reaction solution was mixed with 5% sodium bicarbonate water.
The resulting precipitate was collected on ice, suspended in 100 ml of DMF, and heated. After cooling, insoluble matter was filtered off. Furthermore, heat-treated methanol and ethanol in the same manner as above [32] 43.30g
(yield 92.4%). Amino acid analysis [0.5μM/6N hydrochloric acid-anisole,
110℃ 48 hours]; Asp3.81(4), Thr0.90(1), Ser1.82
(3), Glu3.62(4), Gly0.88(1), Ala3, Val3.65(4),
Leu2.58(3), Lys4.21(4), His0.86(1) (11) P(56−84); BOC−Asp(OBzl)−Asn−Val
−Leu−ValGlu(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−Asn−Val−
Leu−Thr(Bzl)−Lys(Z−Cl)Ala−Lys(Z
-Cl)-Ser(Bzl)-Gln-OBzl [33] [32] 220 ml of TFA was added to 42.2 g (9 mM), and 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 residue, and the resulting precipitate was collected and dried overnight in a NaOH desiccator.
Add and dissolve 400 ml of DMF and 600 ml of NMP to this precipitate, add 0.24 g of HOBT (0.2 times M), BOC-
Add Asp(OBzl)-OSU8.00g (2xM),
After adjusting the pH to 7.0 with NMM, the mixture was stirred at room temperature overnight. In addition, BOC-Asp(OBzl)-OSU4.00g (1
M) was added and stirred overnight. The reaction solution was poured into ice water, and the resulting precipitate was collected, washed with water, added to methanol, and heated. After cooling, insoluble matter was filtered off. The above heat treatment was performed three times [33] 39.97g
(Yield 90.8% was obtained. Amino acid analysis [0.5 μM/6N hydrochloric acid-anisole,
110℃ 48 hours]; Asp4.82(5), Thr0.95(1), Ser1.90
(3), Glu3.72(4), Gly0.92(1), Ala3, Val3.67(4),
Leu2.73(3), Lys4.10(4), His0.78(1) (12) 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−Asn−Val−Leu−Thr(Bzl)−Lys(Z
−Cl) −Ala−Lys(Z−Cl)−Ser(Bzl)Gln−
Add 220ml of TFA to OBzl [34] [33] 39.13g (8.0mM),
Stirred at room temperature for 60 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 in a NaOH desiccator for 2 days. Add 500 ml of DMF and 600 ml of NMP to this precipitate, dissolve it, and add 2,4-dinitrophenol.
1.47g (1xM), 0.11g HOBT (0.10xM) and 4.52g (1.3xM) of BOC-Glu(OBzl)-OSU were added and stirred overnight at room temperature. Furthermore, BOC−Glu
(OBzl)-OSU 4.52 g (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, heat treated, and after cooling, insoluble matter was filtered off. The above heat treatment was performed three times to obtain 37.25 g (yield: 91.1%) of [34]. Amino acid analysis [0.5μM/6M hydrochloric acid-anisole,
110℃ 48 hours]; Asp4.78(5), Thr0.96(1), Ser1.95
(3), Glu4.79(5), Gly0.89(1), Ala3, Val3.70(4),
Leu2.71(3), Lys4.09(4), His0.77(1) (13) P(53−54); BOC−Lys(Z−Cl)−Lys(Z
−Cl) PAC [35] BOC−Lys(Z−Cl)−OH・TBA36.6g
(75mM) in 300ml of ethyl acetate, ice + 1N
After washing with hydrochloric acid, it was washed with water. The ethyl acetate layer was dried over anhydrous sodium sulfate, dried under reduced pressure, and the residue was dissolved in 50 ml of DMF.
It was dissolved in 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. Add 3.68 g of sodium acetate (0.5 times M) to the reaction solution and 500 ml of ethyl acetate.
After adding a solution dissolved in water, the solution was washed three times each with 5% sodium bicarbonate solution, 1N hydrochloric acid, and water in that order. The ethyl acetate layer was dried over anhydrous sodium sulfate and then dried under reduced pressure. The residue was purified by silica gel column chromatography [eluent benzene-ethyl acetate (2:1)],
The fractions near Rf ₁ =0.77 on TLC were collected and dried under reduced pressure. The residue was recrystallized from ether-hexane.
BOC-Lys(Z-Cl)-PAC23.46g (yield 60.5%)
I got it. Melting point: 52-54°C TLC: Rf ₁ =0.86 60 ml of TFA was added to 20.68 g (40 mM) of the above product, and the mixture was stirred at room temperature for 20 minutes. After the reaction, 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 to obtain DMF solution A. On the other hand, BOC−Lys(Z−Cl)−OH・TBA23.42
g (1.2 times M) in 200 ml of ethyl acetate, and
Washed with 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. Solution B adjusted to the DMF solution A,
HOBT6.48g (1.2xM), WSCI8.78ml (1.2xM)
was added, the pH was adjusted to 7 with NMM, and the mixture was stirred at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in 500 ml of ether, and then diluted with 5% sodium bicarbonate solution for 30 minutes.
Washed twice. 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 give [35] 18.73 g (yield
57.5%). 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 (14) P (53-54); BOC-Lys (Z−Cl)−Lys(Z
-Cl)-OH [36] [33] Dissolve 4.07g (5mM) in 30ml of acetic acid,
This was added to 20 g of zinc powder/30 ml of acetic acid and stirred at room temperature for 1.5 hours. After the reaction, the zinc dust was filtered off, and acetic acid was distilled off from the filtrate 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 give [36] 3.08 g (yield 85.8%)
I got it. 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 (15) 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−Asn−Val−
Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z
-Cl)-Ser(Bzl)-Gln-OBzl [37] [34] 80 ml of TFA was added to 10.22 g (2 mM), and the mixture was 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 200 ml of DMF and 200 ml of NMP were added and dissolved. Add to this HOBT0.41g (1.5x M) under ice cooling, [36]
2.13g (1.5xM) and WSCI0.55ml (1.5xM)
was added and stirred at 5 to 10°C for 3 days. The reaction solution was poured into ice water, and the resulting precipitate was collected, washed with water, and then heat-treated with methanol. After cooling, insoluble matter was filtered off. This heat treatment was performed three times [37] 10.41g
(yield 91.25%). Amino acid analysis [0.5μM/6N hydrochloric acid-anisole,
110℃ 48 hours]; Asp4.82(5), Thr0.92(1), Ser1.89
(3), Glu4.77(5), Gly0.91(1), Ala3.00, Val3.79
(4), Leu2.62(3), Lys5.83(6), His0.75(1) (16) Peptide (53−84) [37] Add 5ml of anisole to 3.4g (0.6mM), and add Introducing 50ml of anhydrous hydrogen fluoride (HF),
Stirred at 0°C for 60 minutes. After the reaction, HF is distilled off under reduced pressure, and ether is added to the residue. Collect the precipitate, dissolve it in 50ml of 0.1N acetic acid, and add Dowex 1
Pass through 2 x columns (2.7 x 36 cm acetic acid type). The effluent was freeze-dried to obtain 2.1 g of crude product. this
Dissolve in 50ml of 8M urea solution and adjust pH with ammonia water.
After adjusting the temperature to 9.5, it was left at 0°C for 1 hour. This solution was then applied to a CM-cellulose column (4.4×
After washing with 200ml of 0.01M ammonium acetate buffer (PH4.5), 700ml of 0.01M ammonium acetate buffer (PH4.5) to 700ml of 0.1M ammonium acetate buffer (PH4.5) linear type. Elution is performed with a concentration gradient and then with 300 ml of 0.2M ammonium acetate buffer (PH4.5). Eluate is 13.5
The mixture was fractionated in ml portions, each fraction was measured by the Folin-Lowry method (750 nm), and the 76th to 110th fractions were collected and freeze-dried to obtain 600 mg. Column of Sephadex LH-20 (3.4 x 120 cm, 0.1N acetic acid)
to desalinate. The eluate was fractionated into 8.5 ml portions, and the 47th to 53rd eluates were collected and freeze-dried to obtain 415 mg.
Furthermore, it was charged to a CM-cellulose column (4.4 x 15 cm) and 0.01M ammonium acetate buffer (PH
4.5) 700ml ~ 0.1M ammonium acetate buffer (PH
4.5) Perform elution with a 700 ml linear concentration gradient,
Fractionate into 13.5ml portions, collect 80th to 96th tubes, freeze-dry, and apply to Sephadex LH-20 column (3.4 x 12cm).
The mixture was charged to 100 ml, eluted with 0.1N acetic acid, fractionated into 5 ml portions, and the 76th to 85th fractions were collected and freeze-dried to obtain 220 mg of peptide (53-84). TLC; Rf ₉ = 0.76 Amino acid analysis; Asp4.80(5), Thr0.95(1),
Ser2.34(3), Glu4.99(5), Gly0.95(1), Ala3,
Val3.95(4), Leu2.96(3), Lys6.28(6), His0.97(1) Reference example 2 [Tyr ⁵² ]-peptide (52-84); H-Tyr-
Lys−Lys−Glu−Asp−Asn−Val−Leu−Val
−Glu−Ser−His−Glu−Lys−Ser−Leu−
Gly−Glu−Ala−Asp−Lys−Ala−Asp−Val
−Asn−Val−Leu−Thr−Lys−Ala−Lys−
Ser−Gln−OH (1) [Tyr ⁵² ]P(52−84); BOC−Tyr(Bzl−
Cl ₂ )−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−Asn−Val−Leu−
Thr(Bzl)−Lys(Z−Cl)−Ala−Lys(Z−Cl)
−Ser(Bzl)−Gln−OBzl [38]. [37] 5.82g (1.0mM) described in Example 1
50 ml of TFA was added and stirred at room temperature for 50 minutes. TFA was distilled off under reduced pressure, ether was added to the residue,
After collecting the resulting precipitate, add 150ml of DMF and 150ml of MNP.
was added and dissolved. HOBT0.27g (2xM) and
After adding and dissolving 0.88 g of BOC-Tyr (Bzl-Cl ₂ )-OH (2x M), cool it at -20°C, and add 0.37 g of WSCI.
(2xM) was added and stirred at room temperature for 2 days.
The reaction solution was poured into ice water, the resulting precipitate was collected, washed with water, suspended in methanol, heated, cooled, and the insoluble matter was filtered off. This precipitate was further heated and suspended in methanol, then cooled and collected to give 5.15 g of [38]
(yield 88.4%). TLC; R9=0.75 Amino acid analysis Asp4.43(5), Thr0.88(1), Ser1.86(3), Glu4.63
(5), Gly0.92(1), Ala3(3), Val3.59(4), Leu2.62
(3), Tyr0.88(1), Lys5.58(6), His0.76(1), (2) [ ^Tyr52 ]-peptide (52-84) in 50ml of anhydrous hydrogen fluoride (HF) at 0°C. under cooling (1)
Add 3.68g (0.6mM) and 10ml of anisole and cool at 0°C.
and stirred for 60 minutes. After the reaction, HF was distilled off under reduced pressure,
Add ether to the residue, collect the resulting precipitate,
Dissolve in 50 ml of 0.1N vinegar and pass through a Dowex 1 column (acetate type, 3 x 48 cm). Elute with 0.1N acetic acid and lyophilize the effluent to obtain the crude product.
2.43g was obtained. Dissolve this in 50ml of 8M urea aqueous solution, adjust the pH to 9.5 with aqueous ammonia, and then store at 0℃.
It was left for 60 minutes. Furthermore, this solution was added to a CM-cellulose column (3 x 30
cm) and drained with about 100ml of 0.01M ammonium acetate solution (PH4.5), then linear concentration of 700ml of 0.01M ammonium acetate solution (PH4.5) to 700ml of 0.1M ammonium acetate solution (PH4.5). Gradient elution was performed, followed by elution with 300 ml of 0.2M aqueous ammonium acetate. Eluate: 13.5ml
Each fraction was measured at UV280nm, and 75~
The 121st eluate was collected and lyophilized. This lyophilized product was dissolved in 5 ml of 0.1N acetic acid, passed through a column of Sephadex LH-20 (3.4 x 120 cm), the effluent was fractionated into 8.5 ml portions, the 46th to 53rd eluates were collected, and lyophilized. and obtained 532 mg. Dissolve this in 40 ml of 0.1N acetic acid, and add a CM-cellulose column (4.5
x 11 cm) and elute with a linear concentration gradient of 500 ml of 0.01 M ammonium acetate aqueous solution (PH4.5) - 500 ml of 0.1 M ammonium acetate aqueous solution (PH4.5). The eluate was fractionated into 6.0 ml portions, each fraction was measured at UV 280 nm, and the 121st to 133rd eluates were collected and freeze-dried. This freeze-dried product
Dissolve in 5 ml of 0.1N acetic acid and add Sephadex LH-
The mixture was passed through 20 columns (3.4 x 120 cm), fractionated into 8.4 ml portions, and the 47th to 52nd fractions were collected and lyophilized to obtain 129 mg of [Tyr ⁵² ]-peptide (52-84). TLC, RF ₉ =0.75 1 spot Amino acid analysis Asp4.78(5), Thr0.95(1), Ser2.48(3), Glu5.01
(5), Gly0.96(1), Ala3, Val3.97(4), Leu2.95(3),
Tyr0.91(1), Lys6.03(6), His0.98(1), Reference example 3 Peptide (51-84); H-Pro-Arg-Lys-
Lys−Glu−AspAsn−Val−Leu−Val−Glu−
Ser−His−Glu−Lys−Ser−Leu−Gly−Glu
−Ala−Asp−Lys−Ala−Asp−Val−Asn−
Val−Leu−Thr−Lys−Ala−Lys−Ser−Gln
−OH (1) P(52−54); AOC−Arg(Tos)−Lys(Z−
Cl)-Lys(Z-Cl)-PAC [39] 14.65g (18mM) of [35] described in Example 1
50 ml of TFA was added and 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 (1.2x M)-HOBT2.92g (1.2x M) and WSCI3.95
ml (1.2xM) 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. Recrystallized from ethyl acetate-ether [39] 14.46g (yield 69.6%)
I got it. Melting point: 79-82℃ TLC; Rf ₇ = 0.76 Elemental analysis [as C ₅₅ H ₇₀ O ₁₃ N ₈ SCI] C% H% N% Measured value 57.06 6.26 9.82 Calculated value 57.23 6.11 9.71 (2) P (51-54 )；BOC−Pro−Arg(Tos)−Lys
(Z-Cl)-Lys(Z-Cl)-PAC [40] [39] Add 40ml of TFA to 14.43g (12.5mM),
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. to this
HOBT2.03g (1.2x M), BOC-Pro-OH3.23
g (1.2 times M) and 2.75 ml of WSCI (1.2 times M) 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 and 1N hydrochloric acid solution in this order. The ethyl acetate layer was dried over anhydrous sodium sulfate and then dried under reduced pressure. The residue was recrystallized twice from ethyl acetate-ether to obtain 14.71 g (yield 95.1%) of [40]. Melting point: 90-93℃ TLC; Rf ₇ = 0.64 Elemental analysis [as C ₅₉ H ₇₅ O ₁₄ N ₉ SCI ₂ ] C% H% N% Measured value 57.33 6.20 9.79 Calculated value 57.27 6.11 10.19 (3) P(51− 54);BOC−Pro−Arg(Tos)−Lys
(Z-Cl)-Lys(Z-Cl)-OH [41] 6.19 g (5mM) in 20 g of zinc powder/30 ml of acetic acid [40]
A solution of 40 ml of acetic acid was added thereto, and the mixture was stirred at room temperature for 2 hours. After the reaction, the zinc dust was filtered off, and the filtrate was concentrated under reduced pressure. The residue was extracted with 5% aqueous sodium bicarbonate and ether, and the separated aqueous layer was adjusted to _H2 with 1N hydrochloric acid, and then extracted 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 to obtain 5.40 g (yield 96.5%) of [41]. Melting point: 110-113℃ TLC; Rf ₄ = 0.43 Elemental analysis [as C ₅₁ H ₆₉ O ₁₃ N ₉ SCI ₂ ] C% H% N% Measured value 54.56 6.46 11.22 Calculated value 54.73 6.21 11.27 (4) 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−Asn−Val−Leu−Thr(Bzl)−Lys(Z
−Cl) −Ala−Lys(Z−Cl)−Ser(Bzl)−Gln−
OBzl [42] [34]10.22 (2.0mM) described in Example 1
80 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. Collect the resulting precipitate and add with 180ml of DMF.
180ml of NMP' was added and dissolved. to this
HOBT0.41g (1.5xM), [41]3.35g (1.5xM)
and 0.55 ml of WSCI (1.5x M) and
The mixture was stirred for several 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, wash with ether [42] 10.65g
(yield 87.1%). Amino acid analysis (0.5μM/6N hydrochloric acid-anisole,
110℃ 48 hours); Asp4.62(5), Thr0.98(1), Ser2.32
(3), Glu4.52(5), Pro0.83(1), Gly0.94(1), Ala3,
Val3.82(4), Leu2.68(3), Lys5.81(6), His0.78(1),
Arg0.86(1) (5) Peptide (51-84) [42] Add 5 ml of anisole to 3.7 g (0.6 mM), introduce 60 ml of anhydrous hydrogen fluoride (HF),
The mixture was stirred at 0°C for 60 minutes. After the reaction, HF was distilled off under reduced pressure, ether was added to the residue, the precipitate was collected, dissolved in 50 ml of 0.1N acetic acid, and washed with Dowex 1x.
2 column (2.7×40 cm acetic acid type) and the effluent was lyophilized to obtain 2.2 g of crude product. this
Dissolve in 50ml of 8M urea solution and add with ammonia water.
After adjusting the pH to 9.5, it was left at 0°C for 1 hour. This solution was then applied to a CM-cellulose column (4.2
x 13cm), washed with 200ml of 0.01M ammonium acetate buffer (PH4.5), and then a straight line between 700ml of 0.01M ammonium acetate buffer (PH4.5) and 700ml of 0.1M ammonium acetate buffer (PH4.5). Elution was performed using a type concentration gradient, followed by elution with 300 ml of 0.2M ammonium acetate buffer (PH4.5). The eluate is
Fractionate into 8.5 ml portions, collect the 120th to 160th tubes, freeze-dry, pass through a Sephadex LH-20 column (3.4 x 110 cm, 0.1N acetic acid), fractionate into 5.2 ml portions, and collect the 51st to 62nd tubes. was collected and freeze-dried to obtain 392 mg. Furthermore, charge a CM-cellulose column (2.0 x 36 cm) in the same manner, and elute with a linear concentration gradient of 500 ml of 0.01 M ammonium acetate buffer (PH4.5) to 500 ml of 0.2 M ammonium acetate buffer (PH4.5). conduct. The eluate was fractionated into 7.5 ml portions and 92
~Collect the 110th section, freeze-dry it, and apply it to a Sephadex LH-20 column (3.0 x 123cm 0.1N acetic acid).
The eluate was fractionated into 6 ml portions, the 37th to 47th tubes were collected, and lyophilized to yield 213 mg of peptide (51-84).
I got it. TLC; R ₁₀ = 0.89 Amino acid analysis; Asp4.98(5), Thr0.92(1),
Ser2.45(3), Glu5.13(5), Gly1.00(1), Ala3,
Val3.98(4), Leu2.99(3), Lys6.08(6), His0.97(1),
Arg0.99(1), Pro1.02(1) Reference example 4 [Tyr ⁵⁰ ] peptide (50-84); H-Tyr-Pro
−Arg−Lys−Glu−Asp−Asn−Val−Leu−
Val−Glu−Ser−His−Glu−Lys−Ser−Leu
−Gly−Glu−Ala−Asp−Lys−Ala−Asp−
Val−Asn−Val−Leu−Thr−Lys−Ala−Lys
−Ser−Gln−OH (1) [Tyr ⁵⁰ ]P(50−84); BOC−Tyr(Bzl−
Cl ₂ )−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−Asn−
Val−Leu−Thr(Bzl)−Lys(Z−Cl)−Ala−
Lys(Z-Cl)-Ser(Bzl)-Gln-OBzl [43] 6.31g (1.0mM) of [42] described in Reference Example 3
50 ml of TFA was added and stirred at room temperature for 55 minutes. TFA was distilled off under reduced pressure, ether was added to the residue,
After collecting the resulting precipitate, add 160ml of DMF and 160ml of NMP.
was added and dissolved. HOBT0.27g (2xM) and
After adding and dissolving 0.88 g of BOC-Tyr (Bzl-Cl ₂ )-OH (2x M), it was cooled to -20°C, and 0.37 g of WSCI was added.
(2xM) was added and stirred at room temperature for 3 days. The reaction solution was poured into ice, and the resulting precipitate was collected, washed with water, suspended in methanol, heated, cooled, and the insoluble matter was filtered off. This precipitate was further heated and suspended in methanol, and then cooled to collect the precipitate. After performing this operation once more, wash with ether [43]
5.97g (yield 90%) was obtained. Amino acid analysis Asp4.45(5), Thr0.94(1), Ser2.25(3), Glu4.67
(5), Pro0.89(1), Gly0.92(1), Ala3, Val3.66(4),
Leu2.81(3), Tyr0.81(1), Lys5.53(6), His0.77(1),
Arg0.79(1) (2) [Tyr ⁵⁰ ] h-PTH (50-84) 3.98 g in 60 ml of anhydrous HF at 0°C cooling [43]
(0.6mM) and 10ml of anisole, and
Stir for a minute. After the reaction, HF was distilled off under reduced pressure, ether was added to the residue, and the resulting precipitate was collected. This precipitate was dissolved in 50 ml of 0.1N acetic acid and passed through a 1×2 Dowex column (acetate type, 3×45 cm). The effluent was freeze-dried to obtain 2.41 g of crude product. This was dissolved in 60 ml of 8M urea aqueous solution, adjusted to pH 9.5 with aqueous ammonia, and then left at 0° C. for 60 minutes. Next, this solution was charged to a CM-cellulose column (3 x 30 cm) packed with 8M urea aqueous solution, and 0.01M ammonium acetate aqueous solution (PH4.5) was charged.
After draining the urea, elution was performed using a linear concentration gradient of 700 ml of 0.01M ammonium acetate solution (PH4.5) to 700 ml of 0.1M ammonium acetate solution (PH4.5), and then 0.2M ammonium acetate solution (PH4.5). ) eluted at 250 ml. The eluate was fractionated into 8.5ml portions, and each fraction was measured at UV280nm to yield ~142 fractions.
The 171st effluent was collected and freeze-dried. This lyophilized product was dissolved in 6 ml of 0.1M acetic acid, passed through a Cephadex LH-20 column (3.4 x 120 cm), fractionated into 8.5 ml portions, and the 46th to 52nd effluent was collected and lyophilized to obtain 410 mg. Obtained. Dissolve this in 30 ml of 0.1N acetic acid, and
x 35cm) and elute with a linear concentration gradient of 500ml of 0.01M aqueous ammonium acetate (PH4.5) to 500ml of 0.2M aqueous ammonium acetate (PH4.5). The eluate is fractionated into 7.5 ml portions, 112 or more.
The 136th effluent was collected and lyophilized, then 0.1N
The effluent was dissolved in 6 ml of acetic acid and passed through a Sephadex LH-20 column (3.0 x 120 cm) into 6 ml fractions. The 35th to 43rd fractions were collected and lyophilized to obtain [Tyr ⁵⁰ ] peptide ( 50−84) 134 mg was obtained. TLC; Rf ₁₀ = 0.88 1 spot Amino acid analysis Asp4.98(5), Thr0.94(1), Ser2.43(3), Glu5.11
(5), Pro0.97(1), Gly0.97(1), Ala3, Val3.98(4),
Leu2.96(3), Tyr0.91(1), LyS6.05(6), His0.93(1),
Arg0.98(1) Reference example 5 Peptide (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−Asn−Val−Leu−
Thr−Lys−Ala−Lys−Ser−Gln−OH (1) P(49−50); BOC−Gln−Arg(Tos)−OMe
[44] H-Arg(Tos)-OMe・HCl11.37g (30mM)
and BOC-Gln-ONP13.21g (1.2x M)
Dissolve in 200ml of DMF and cool to 0℃ with NMM to pH7
After adjusting the temperature, the mixture was stirred overnight. After the reaction, DMF was distilled off under reduced pressure, the residue was dissolved in chloroform, and then diluted with 5% sodium bicarbonate solution three times, 1M hydrochloric acid twice, and water three times.
Washed twice. The chloroform layer was dried with anhydrous sodium sulfate, and chromatography was performed on a silica gel column filled with chloroform. Chloroform-
The mixture was flushed with methanol-methyl 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. Dissolve the residue in ethyl acetate and
Crystallize by adding hexane under cooling at °C [44]
get. Yield: 11.86g Melting point: 103-107℃ (2) P(48-50); BOC-Ser(Bzl)-Gln-Arg
After adding 50 ml of TFA to 9.39 g (16.5 mM) of (Tos)-OMe [45] [44] and stirring at room temperature for 20 minutes, TFA was distilled off under reduced pressure.
Add ether to the residue, filter the resulting precipitate,
Dissolved in 50ml of DMF. 3.24g of HOBT in this solution
(1.45 times M), BOC-Ser(Bzl)-OH7.07g (1.45
Add 4.39 ml of WSCI (1.45 times M) and
Stir 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.
% 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,
[45] 10.0 g (yield 81.0%) was obtained. 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 (3) P (46−47 ); BOC−Ala−Gly−OH [46] BOC−Ala−OH7.57g, HOBT5.4g
Dissolved in THF60ml, H-Gly-OET・HCl5.58g
After that, 8.05 ml of WSCI was added dropwise over 5 minutes while cooling at -20°C, and the mixture was stirred for 1 hour and then at room temperature for 24 hours. After distilling the reaction solution under reduced pressure, ethyl acetate
After washing with 5% sodium bicarbonate solution (3 times), water, N-hydrochloric acid solution (3 times), saturated saline solution (3 times), and water, the ethyl acetate layer was separated and anhydrous Na ₂ SO ₄ was added. It was dried. The desiccant was removed and the mixture was concentrated to dryness to obtain pale yellow oily BOC-Ala-Gly-OET (Rf ₁ =0.77).
Dissolve this as it is in 20 ml of ethanol, add 44 ml of N-NaOH over 10 minutes at 0℃, and leave it at room temperature for 1 hour.
Stirred for 30 minutes. Add 4 ml of N-hydrochloric acid at 0°C,
After distilling off ethanol under reduced pressure, add N-hydrochloric acid at 0°C.
42 ml was added, and the oil was extracted with 200 ml of ethyl acetate. After washing the ethyl acetate layer, it was concentrated, and the precipitated crystals were
Recrystallization was performed with ETOH-ether-n-hexane to obtain 6.12 g (yield 5.08%) of colorless crystals [46]. TLC; Rf ₁ = 0.14 Melting point; 85-89°C (decomposition) [α] ²⁶ D-9.28 (C = 1.0, DMF) Elemental analysis [as C ₁₀ H ₁₈ O ₅ N ₂・H ₂ O] C% H% N% Measured value 45.49 7.71 10.78 Calculated value 45.45 7.63 10.60 (4) P(46−50);BOC−Ala−Gly−Ser(Bzl)−
50 ml of TFA was added to 9.72 g (13 mM) of Gln-Arg(Tos)-OMe [47] [45], 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 by filtration and dissolved in 100 ml of DMF. In this solution
BOC-Ala-Gly-OH [46] 3.84g (1.2x M),
HOBT2.11g (1.2xM) and WSCI2.85ml (1.2
M) was added, and the mixture was stirred at room temperature for 2 days. After the reaction,
DMF was distilled off under reduced pressure, the residue was dissolved in 200 ml of ethyl acetate, and then washed with water. After drying over anhydrous sodium sulfate and concentration under reduced pressure, the residue was crystallized twice from ethanol-ether to obtain 10.46 g (yield 91.9%) of [47]. 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 (5) P (46-50); BOC-Ala- Gly−Ser(Bzl)−
9.64 g (11 mM) of Gln-Arg(Tos)-NH.NH ₂ [48] [47] was dissolved in 50 ml of ethanol, and 6.4 ml of 50% NH ₂ MH ₂ was added thereto and stirred overnight at room temperature. Add 100ml of ethanol to the reaction solution,
Insoluble matter was filtered off. This was suspended in 100 ml of ethanol and heated. After cooling, filter [48]9.02g
(yield 93.6%). 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 (6) P (46-50); BOC-Ala- Gly−Ser(Bzl)−
Gln−Arg(Tos)−Pro−Arg(Tos)−Lys(Z−
Cl)-Lys(Z-Cl)-PAC [49] 8.04g (6.6mM) of [40] described in Reference Example 3
After adding 40 ml of TFA and stirring at room temperature for 20 minutes,
TFA was distilled off under reduced pressure. Add ether to the residue,
The resulting precipitate was collected by filtration to obtain crude H-Pro-Arg.
(Tos)−Lys(Z−Cl)−Lys(Z−Cl)−PAC・
Got TFA. On the other hand, 6.83g (7.8mM) of [49] 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. , 20 at −20℃
Stir for a minute. Then the above H-Pro-Arg(Tos)
-Lys(Z-Cl)-PAC/TFA was added and heated to -35℃.
After adding 5.46ml (39mM) of Et ₃ N, the
The mixture was stirred for several days. After the reaction, DMF was distilled off under reduced pressure.
The residue was dissolved in 300 ml of chloroform, and washed successively with 5% sodium bicarbonate, 1N hydrochloric acid, and water. 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 [50]. TLC; Rf=0.64 [chloroform-methanol-
Acetic 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 Calculated value 54.72 6.29 12.49 Amino acid analysis (0.5 μM /Hydrochloric acid, anisole,
110℃ 48 hours); Ser0.65(1), Glu1.10(1), Pro1(1),
Gly1.02(1), Ala1.00(1), Lys1.89(2), Arg2.02(2) (7) P(46−54); BOC−Ala−Gly−Ser(Bzl)−
Gln−Arg(Tos)−Pro−Arg(Tos)−Lys(Z−
Cl)-Lys(Z-Cl)-OH [50] 10.62 g of acetic acid in 30 g of zinc powder/60 ml of acetic acid [49]
40 ml of solution was added and stirred at room temperature for 2 hours. After the reaction, the zinc dust was filtered off, and the filtrate was concentrated under reduced pressure. Ether was added to the residue, and the resulting precipitate was diluted with ethanol.
Purification was performed once with ether and twice with ethanol-ethyl acetate to obtain 9.60 g (91.4% yield) of [50]. TLC; Rf ₄ = 0.22 Elemental analysis [as C ₈₄ H ₁₁₄ O ₂₂ N ₁₈ S ₂ 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 (0.5μM/6N -Hydrochloric acid-anisole, 110℃ 48 hours); Ser0.88(1), Glu1.11(1),
Pro1.02(1), Gly1.02(1), Ala1(1), Lys2.00(2),
Arg2.09(2) (8) 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−Ash−Val−Leu−Thr(Bzl)−Lys(Z−
Cl) −Ala−Lys(Z−Cl)−Ser(Bzl)−Gln−
OBzl [51] [34] 15.33g (3mM) described in Example 1
After adding 130 ml of TFA and stirring at room temperature for 60 minutes,
TFA was distilled off under reduced pressure. Add ether to the residue,
The resulting precipitate was dissolved in a mixture of 200 ml of DMF and 200 ml of NMP. This was cooled to 0°C [50] 6.70 (1.2 times M), HOBT 0.49 g (1.2 times M) and WSCI 0.66.
ml (1.2 times M) and 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 by filtration. Add 300ml of methanol to this, heat it, cool it, and then filter out the insoluble matter twice. [51] 18.30g
(yield 89.0%). (7) Peptide (46-84) [51] Add 3 ml of anisole to 4.11 g (0.6 mM), introduce 60 ml of anhydrous hydrogen fluoride (HF),
The mixture was stirred at 0°C for 60 minutes. After the reaction, HF was distilled off under reduced pressure, ether was added to the residue, and the resulting precipitate was collected.
Dissolve in 50 ml of 1M acetic acid and pass through a Dowex 1 x 2 column (3.0 x 45 cm acetic acid type). The effluent was freeze-dried to obtain 2.8 g of crude product. Then add this to 8M
Dissolve in 50ml of urea aqueous solution and pH with ammonia water.
After adjusting the temperature to 9.5, it was left at 0°C for 60 minutes. This solution was then applied to a CM-cellulose column (2.0 x 36
cm) and washed with 200ml of 0.01M ammonium acetate buffer (PH4.5), then 500ml of the same buffer.
~0.3M ammonium acetate buffer (PH4.5) 500ml
Perform elution using a linear concentration gradient. The eluate is
Fractionate into 7.5 ml portions, collect 56th to 84th tubes, freeze-dry, and transfer to Sephadex LH-20 column (3.0 x 120
cm), eluted with 0.1N acetic acid, fractionated into 6 ml portions, collected 41st to 52nd tubes, and freeze-dried to obtain 620 ml. Further, this was charged to a CM-cellulose recolumn (2.0 x 38 cm), and a linear concentration gradient of 500 ml of 0.01N ammonium acetate buffer (PH4.5) to 500 ml of 0.3M ammonium acetate buffer (PH4.5) was applied in the same manner. Elution was performed using Fractionate 7.5ml each, 59
~Collect the 80th bottle, freeze-dry it, and then ceph-dex.
Pass through LH-20 column (3.0 x 120cm 0.1N acetic acid),
The mixture was fractionated into 8 ml portions, 31st to 36th portions were collected, and lyophilized to obtain 235 mg of peptide (46-84). TLC; Rf=0.77 Amino acid analysis; Asp4.98(5), Thr0.95(1),
Ser3.62(4), Glu6.14(6), Pro0.99(1), Gly1.96(2),
Ala4, Val4.01(4), Leu2.98(3), Lys6.12(6),
His0.93(1), Arg1.92(2) Reference example 6 [Tyr ⁴⁵ ]-peptide (45-84); H-Tyr-
Ala−Gly−SerGln−Arg−Pro−Arg−Lys−
Lys−Glu−Asp−Asn−Val−Leu−Val−Glu
−Ser−His−Glu−LysSer−Leu−Gly−Glu
−Ala−Asp−Lys−Ala−Asp−Val−Asn−
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−Asn−Val−Leu−
Thr(Bzl)−Lys(Z−C1)−Ala−Lys(Z−Cl)
-Ser(Bzl)-Gln-OBzl [52] 13.71g (2.0mM) of [51] described in Reference Example 5
180 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. Filter the resulting precipitate and remove BOC 13.80
I got g. 6.90g (1mM) of the above BOC removed product in 135ml DMF
Dissolved in a mixture of 135 ml of NMP and 0.53 g of BOC-Tyr(Bzl-Cl ₂ )-OH (1.2 times M), 0.16 g of HOBT (1.2 times M) and WSCI0.22 while cooling to 0°C.
ml (1.2 times M) and stirred overnight at room temperature.
After the reaction, DMF was distilled off under reduced pressure, and ice water was added to the residue. The resulting precipitate was collected by filtration and washed with DMF-methanol-ether to give 6.12 g (yield 85.3) [52].
%) was obtained. (2) 1.5 ml of anisole was added to 4.31 g (0.60 mM) of [Tyr ⁴⁵ ]-peptide (45-84) [52], anhydrous HF was introduced, and the mixture was stirred at 0°C for 1 hour. Next, HF was distilled off under reduced pressure, ether was added to the residue, the precipitate was collected, dissolved in 25 ml of 0.1N acetic acid, and loaded onto a Dowex 1x2 column (acetic acid type,
2.5 x 30cm). The effluent was freeze-dried to obtain 3.21 g of crude product. Add to this 8M urea aqueous solution (PH
9.5) Dissolve in 50ml and leave at 0℃ for 1 hour. This solution was then applied to a CM cellulose column (4.3 x 16
cm) and elute with a linear concentration gradient of 800 ml of 0.01 M ammonium acetate buffer (PH4.5) to 800 ml of 0.3 M ammonium acetate buffer (PH4.5). The eluate was fractionated into 6.5ml portions, 118th to 151st tubes were collected, lyophilized, and charged to a Sephadex LH-20 column (3.0 x 120cm, 0.1N acetic acid).
Fractionate the effluent into 7.5ml portions, collect the 26th to 37th bottles,
Lyophilization yielded 521 mg. CM this further
- Charge a cellulose column (2.1 x 48 cm) and add 0.01N ammonium acetate buffer (PH4.5) at 500%
ml~0.3M ammonium acetate buffer (PH4.5) 500
Perform elution using a linear concentration gradient of 800 ml, collect the 59th to 70th aliquots, freeze-dry, charge to a Sephadex LH-20 column (3.0 x 92 cm, 0.1N acetic acid), and collect the effluent. Fractionate 8.0ml each,
The 21st to 30th strands were collected and freeze-dried to obtain 281 mg of [Tyr ⁴⁵ ]-peptide (45-84). TLC; Rf ₉ = 0.75 Amino acid analysis; Asp4.97(5), Thr0.97(1),
Ser3.62(4), Glu6.11(6), Pro0.96(1), Gly1.93(2),
Ala4, Val3.98(4), Leu2.95(3), Thr0.91(1),
Lys6.04(6), His0.92(1), Arg1.91(2), Reference example 7 [ ^Cys45 ]-peptide (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−Asn−Val−Leu−Thr−Lys−Ala−
Lys−Ser−Glu−OH (1) P(45−84); BOC−Cys(Acm)−Ala−Gly
−Ser(Bzl)−Gln−Arg(Tos)−Pro−Arg
(Tos)−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−Asn−Val−Leu−Thr(Bzl)−Lys(Z
−Cl) −Ala−Lys(Z−Cl)−Ser(Bzl)−Gln−
OBzl [53] 6.90 g of the remaining BOC removed product obtained in Reference Example 6
(1.0mM) was dissolved in a mixture of 130ml of DMF and 130ml of NMP, and in this was added 0.16g of HOBT (1.2xM) and 0.35g of BOC-Cys(Acm)-OH (1.2xM) under cooling to 0℃.
After adding 0.22 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, and the resulting precipitate was collected. This was suspended in ethanol, heated, cooled, and then the insoluble matter was filtered off. This operation was repeated twice to obtain 5.93 g (yield: 84.4%) of [53]. (2) 15 ml of anisole was added to 4.22 g (0.6 mM) of [Cys(Acm) ⁴⁵ ]-peptide (45-84) [53], 80 ml of anhydrous HF was introduced therein, and the mixture was stirred at 0° C. for 1 hour. Next, HF was distilled off under reduced pressure, ether was added to the residue, the resulting precipitate was collected, dissolved in 50 ml of 20% acetic acid, and transferred to a Dowex 1 x 2 column (2.8 x 45
cm, acetic acid form). Freeze-dry the effluent, dissolve it in 60 ml of 8M urea aqueous solution, adjust the pH to 9.5 with aqueous ammonia, and add CM-cellulose (3 x 45 cm).
and elute with a linear concentration gradient of 700 ml of 0.01 M ammonium acetate buffer (PH4.5) to 700 ml of 0.3 M ammonium acetate buffer (PH4.5), and fractionate the eluate into 8.0 ml portions. The ~55th tube was collected, lyophilized, passed through a Sephadex LH-20 column (3.0 x 120 cm, 0.1N acetic acid), and the 35th to 41st tubes were collected and freeze-dried to obtain 580 mg. This was further coated with a CM-cellulose column (5x
13 cm), and elution was performed using a linear concentration gradient of 400 ml of 0.01 M ammonium acetate buffer (PH4.5) to 400 ml of 0.3 M ammonium acetate buffer (PH4.5). Fractionate the eluate into 6.0 ml portions, 120 to 131
After collecting the ingredients and freeze-drying, Sephadex LH-
20 columns (4.0 x 120 cm, 0.1N acetic acid),
Fractionate the effluent into 8.0ml portions, collect 38th to 53rd bottles,
Freeze-dry to obtain [Cys(Acm) ⁴⁵ ]-peptide(46-
84) Obtained 233 mg. TLC; Rf ₉ = 0.74 Amino acid analysis; Asp4.97(5), Thr1.00(1),
Ser3.67(4), Glu6.07, Pro1.01(1), Gly1.96(2),
Ala4, Val3.99(4), Cys0.43(0.5), Leu2.94(3),
Lys6.03(6), His0.91(1), Arg1.92(2), (3) [CysAcm) ⁴⁵ ]-Peptide (45−84) mg
(0.02mM) dissolved in 58mg of mercuric acetate
2 ml of 50% acetic acid was added and dissolved, and after stirring at room temperature for 90 minutes, 4 ml of β-mercaptoethanol was added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was centrifuged, and the supernatant was transferred to Sephadex G-25 (3.0 x 90 cm, 0.1N
acetic acid), fractionate the eluate into 10.5 ml portions, collect the 24th to 29th fractions, freeze-dry them,
64.1 mg of [Cys ⁴⁵ ]-peptide (45-84) was obtained. TLC; Rf ₉ = 0.73

[Brief explanation of drawings]

Figures 1 to 3 show cross-reactivity measurement curves with various h-PTH-related substances.

Claims (1)

[Claims] 1 Formula H-Lys-Lys-Glu-Asp-Asn-Val-Leu-
Val−Glu−Ser−His−Glu−Lys−Ser−Leu−
Gly−Glu−Ala−Asp−Lys−Ala−Asp−Val−
Asn−Val−Leu−Thr−Lys−Ala−Lys−Ser−
Peptide represented by Gln-OH or its salt.