JPS648640B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to human chorionic gonadotropin (hCG) C-
Regarding terminal fragments.

More specifically, the present invention provides hCG(112-145),
That is, the expression It is a peptide represented by or a salt thereof.

hCG is a glycoprotein and is a sex hormone secreted from the placenta during pregnancy and plays an important role in maintaining pregnancy. By qualitatively or quantitatively measuring this hCG, it becomes possible to detect and diagnose the presence or absence of pregnancy, ectopic pregnancy, hair cancer, etc. at an early stage. However, gonadotropins include luteinizing hormone (LH), follicle stimulating hormone (FSH), and CG, which have similar molecular structures and are glycoproteins. Furthermore, it consists of two subunits, an α chain and a β chain, and the α chain in particular is so similar in molecular structure that there is no change in activity even if it is exchanged. The β chain is more specific for each gonadotropin, but is similar to the β chain of LH.
It is similar to the β chain of CG. However, the amino acid composition near the C-terminus of the CGβ chain is significantly different from that of the LH β-chain, and therefore, if a measurement system that detects the C-terminus of the CGβ chain were developed, it would be possible to measure the amino acid composition near the C-terminus of the CGβ chain. In particular, a highly reliable CG measurement system that does not intersect with LH at all becomes possible.

The present invention was completed focusing on the above, and an antibody obtained using a novel peptide represented by the formula [] as an antigen has immunological cross-activity with hCG.
Therefore, this peptide is useful as an antibody preparation reagent for hCG measurement systems.

hCG (112-145) of the present invention is constructed by condensing individual amino acids or lower peptides in the amino acid sequence shown by the formula [], and removing the protective group of the functional group of the side chain in the final step of the condensation reaction. It can be obtained by 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 preparation of the raw materials for the compound of interest as well as all intermediates are those known in peptide synthesis and therefore can be carried out by known means such as hydrolysis, acidolysis, reduction, aminolysis or hydrazinolysis. Therefore, a protecting group that can be easily removed is used.

For example, as a protecting group for an amino group,
formyl group, trifluoroacetyl group, phthaloyl group, benzenesulfonyl group, p-toluenesulfonyl group, o-nitrophenylsulfenyl group,
2,4-dinitrophenylsulfenyl group, 2,
Acyl groups such as 4-dinitrophenylsulfenyl groups, benzyl groups, diphenylmethyl groups, triphenylmethyl groups (these groups may be o
- aralkyl groups such as (substituted with lower alkoxy groups such as methoxy group and p-methoxy group), benzyloxycarbonyl group, o-bromobenzyloxycarbonyl group, p-bromobenzyloxycarbonyl group, o-chloro benzyloxycarbonyl group, p-chlorobenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group,
benzyloxycarbonyl groups such as p-phenylazo-benzyloxycarbonyl group, p-(p′-methoxyphenylazo)-benzyloxycarbonyl group, cyclopentyloxycarbonyl group,
Aliphatic oxycarbonyl groups such as trichloroethyloxycarbonyl group, t-amyloxycarbonyl group, t-butoxycarbonyl group, diisopropylmethoxycarbonyl group, 2-phenyl-isopropoxycarbonyl group, 2-tolyl-isopropoxycarbonyl group, 2 Examples include aralkyloxycarbonyl groups such as -p-diphenyl-isopropoxycarbonyl group. These amino groups can also be protected by the formation of enamines obtained by reacting with 1,3-diketones such as benzoylacetone, acetylacetone, and dimedone.

Carboxyl groups are protected by amide formation, hydratide formation or esterification. That is, the amide group is substituted with a 3,4-dimethoxybenzyl group, a bis-(p-methoxyphenyl)methyl group, or the like. Hydratide group is benzyloxycarbonyl group, trichloroethyloxycarbonyl group, trifluoroacetyl group, t-butoxycarbonyl group, trityl group, 2-p-diphenyl group.
Substituted with isopropoxycarbonyl group, etc. Ester groups include methanol, ethanol, t
-Alkanols such as butanol and cyanomethyl alcohol, benzyl alcohol, p-promobenzyl alcohol, p-chlorobenzyl alcohol, 2,6-dichlorobenzyl alcohol, p-
Aralkanols such as methoxybenzyl alcohol, p-nitrobenzyl alcohol, 2,4,6-trimethylbenzyl alcohol, benzhydryl alcohol, benzoylmethyl alcohol, p-bromobenzoylmethyl alcohol, p-chlorobenzoylmethyl alcohol, 2,4 , 6-trichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, p-
It is substituted with phenols such as cyanophenol and p-methanesulfonylphenol, and thiophenols such as thiophenol, thiocresol and p-nitrothiophenol.

The hydroxyl groups of the serine and threonine can be protected, for example, by esterification or etherification. Examples of groups suitable for this esterification include lower alkanoyl groups such as acetyl groups,
These are groups derived from carbonic acid such as aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethyloxycarbonyl groups. Examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, and t-butyl group. 2,2,2-trifluoro-
Also suitable are the 1-tert-butyloxycarbonylaminoethyl group and the 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group.

However, it is not necessary to protect these hydroxyl groups.

Examples of the group used to protect the amino group in the guanidino group of arginine include a nitro group, tosyl group, and benzyloxycarbonyl group, but it is not necessary to protect this guanidino group.

In the synthesis of the present target compound [1], condensation of individual amino acids or lower peptides is carried out, for example, with an amino acid or peptide having a protected α-amino group and an activated terminal carboxyl group, and a free α-amino group and a protected terminal carboxyl group. or react an amino acid or peptide with an activated α-amino group and a protected terminal carboxyl group with an amino acid or peptide with a free terminal carboxyl group and a protected α-amino group. This can be carried out by reacting amino acids or peptides.

The carboxyl group in this case is an azide, an acid anhydride, an acid imidazolide or an active ester derived from a hydrazide or an N'-protected hydrazide, such as t-butyloxycarbonylhydrazide, benzyloxycarbonylhydrazide, etc. with nitrous acid, such as an acid anhydride, an acid imidazolide, such as cyanomethyl ester. , p-nitrothiophenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 2,
4,5-trichlorophenyl ester, 2,4,
6-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, etc., or N,N'-dicyclohexyl-carbodiimide,
Carbodiimides such as N-ethyl-N'-3-dimethylaminopropyl-carbodiimide, N,
Activation can be achieved by reaction using isoxazolium salts such as N'-carbonyl-diimidazole or Woodward reagents, diphenylphosphorazidate, and the like.

Preferred condensation methods in the present invention are the carbodiimide method, the azide method and the active ester 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 or the active ester method.

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.

Preferably, the protected hCG(112-145) is condensed by the azide method with the C-terminal fragment 116-145, sequentially with fragments 114-115 and 112-113.

C-terminal fragments 116-145 are sequentially divided into C-terminal fragments 130-145 and fragments 127-
129, fragment 123-126, fragment 120-
Preferably, 122 and fragments 116-119 are condensed by the azide method.

C-terminal fragment 130-145 is the 139th amino acid of C-terminal fragment 140-145,
Amino acid 138, fragment 136-137, fragment 133-135 and fragment 130-132
It is preferable to condense them by the azide 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 or converted to a hydratide or N'-protected hydratide, and a benzyl ester can be cleaved with anhydrous hydrogen fluoride or hydrogenolysis.

The α-amino group of these peptides is suitably protected with a benzyloxycarbonyl group. These are eliminated by hydrogenolysis.

The hydroxyl groups of serine and threonine are not necessarily protected, but if they are,
Protection with tert-butyl or benzyl groups is suitable. The side chain carboxyl group of aspartic acid is
Although protection is not always required, in the case of protection, it is suitable to protect with a benzyl ester group. The ε-amino group of lysine is a t-butyloxycarbonyl group, and the amino group in the guanidino group of arginine is suitably protected with a nitro group.

said C-terminal fragment 116-145, i.e. protected hCG(116-145) and said C-terminal fragment 130-145, i.e. protected hCG(130-145).
and for their intermediate fragments,
Chem., Pharm. Bull., 28 (9), 2707-2713 (1980),
28 (9), 2714-2719 (1980).

Protected hCG (112-145) is thus obtained, and these protecting groups are preferably removed in one step by acid decomposition, e.g. trifluoroacetic acid, hydrogen fluoride anhydride, etc., to give hCG (112-145) of formula []. 112-145) are obtained.

The above hCG (112-145) can be separated and purified by known peptide purification methods. For example, Sefadex G-25, Sefadex G-50, Dowex X1, Sefadex LH
-20, can be performed by column chromatography using a carrier such as carboxymethylcellulose.

The target compound [ ] can be obtained in the form of a free base or a salt depending on the conditions of the method. Usually, it can be preserved in the form of a salt with an inorganic acid such as hydrochloric acid or a salt with an organic acid such as acetic acid.

Enzyme immunoassay, radioimmunoassay, four-ball agglutination inhibition reaction, four-ball agglutination reaction, hCG can be measured using latex agglutination reactions, etc. For example, this hCG (112-145) is used as an antigen for antibodies used in pregnancy diagnosis. For example, when hCG (112-145) is conjugated to bovine serum albumin (BSA) using carbodiimide and brutalaldehyde and administered to New Zealand white rabbits, antibodies are detected approximately 8 weeks after the initial immunization, and ¹²⁵ I- It showed approximately 30% binding with hCG at 1000-fold dilution. As a result of examining this antibody using the hCG-RIA (radioimmunoassay) and hCG-β-RIA systems, the dose-response curves due to hCG showed a parallel relationship, and the results were positive at 500 mIU of hCG. Furthermore, when comparing this hCG (112-145) and hCG, they are inhibited at approximately the same molar ratio. This hCG (112-145)
The antibody is specific for hCG and has no cross-reactivity with LH.

Note that the abbreviations described in this specification have the following meanings.

Asp; L-aspartic acid Pro; L-proline Arg; L-arginine Phe; L-phenylalanine Gln; L-glutamine Ser; L-serine Lys; L-lysine Ala; L-alanine Leu; L-leucine Gly; Glycine Thr; L-threonine Ile; L-isoleucine Boc; t-butyloxycarbonyl Z; benzyloxycarbonyl OBu ^t ; t-butyl ester OBzl; benzyl ester ONP; p-nitrophenyl ester Bu ^t ; t-butyl MeOH; Methanol DMF; dimethylformamide CHCl ₃ ; chloroform ether; diethyl ether TFA; trifluoroacetic acid Et ₃ N; triethylamine DCC; N,N'-dicyclohexyl-carbodiimide HOBt; 1-hydroxybenzotriazole A manufacturing example will be specifically explained. The thin layer chromatography (TLC) carrier and developing solvent used in the examples were as follows:
Unless otherwise specified, they are as follows.

Support: Silica gel 60GArt7731 manufactured by Merck & Co., Ltd. Developing solvent: A: CMCl ₃ -MeOH-acetic acid (90:8:2) B: Lower layer of CHCl ₃ -MeOH-water (8:3:1) C: Butanol-pyridine-acetic acid- Water (4:1:
1:2) D: Butanol-pyridine-acetic acid-water (30:20:
6:24) Example 1 Production of hCG (112-145) (1) Fragment 114-115; Z-Arg(NO ₂ )-
Phe―NHNHBoc〔1〕 Z―Phe―NHNHBoc〔E.Wunsch and G.
Wendlberger, Chem. Ber., 97 , 2504 (1964)]
H-Phe-NHNHBoc and Z-Arg (NO ₂ ) obtained from 2.0g by hydrogenolysis in the presence of Pd catalyst
-Dissolve 1.7g of OH in 20ml of DMF and add to this under water cooling.
Add 1.2 g of DCC and 0.9 g of HOBt and stir overnight. After the reaction, the precipitated urea derivative is removed and the liquid is concentrated under reduced pressure. The residue was extracted with ethyl acetate,
Wash with 10% sodium bicarbonate solution, 10% citric acid solution, and water in this order.
After drying with anhydrous sodium sulfate, concentrate under reduced pressure. Add ether to the residue and collect the resulting precipitate. This is dissolved in a small amount of chloroform, charged to a silica gel column, and chromatographed using chloroform containing 3% methanol. Rf _A = TLC
Fractions around 0.41 were collected and dried under reduced pressure to obtain 1.6 g (yield 44.5%) of [1].

Melting point: 120-125℃ [α] ²⁷ _D -28.92 (c = 1.0, MeOH) TCL; Rf _A = 0.41, Rf _B = 0.79 Elemental analysis [as C ₂₈ H ₃₈ N ₈ O ₈ ] C% H% N% Calculated value 54.7 6.23 18.8 Measured value 54.5 6.23 18.5 (2) Fragment 114―145; Z―Arg(NO ₂ )―
Phe―Gln―Asp―Ser―Ser―Ser―Ser―Lys
(Boc) ―Ala―Pro―Pro―Pro―Ser―Leu―
Pro―Ser―Pro―Ser―Arg―Leu―Pro―Gly
―Pro―Ser―Asp―Thr (Bu ^t )―Pro―Ile―
Leu－Pro－Gln－OBu ^t [2] Z－Gln－Asp(OBzl)－Ser－Ser－Ser－Ser
―Lys (Boc)―Ala―Pro―Pro―Pro―Ser―
Leu―Pro―Ser―Pro―Ser―Arg―Leu―Pro―
Gly－Pro－Ser－Asp－Thr（Bu ^t ）－Pro－Ile－
Leu-Pro-Gln-OBu ^t [3] is dissolved by adding 10 ml of ethanol and 10 ml of 5% acetic acid, and hydrogenolysis is performed in the presence of a Pd catalyst. The catalyst was removed and the liquid was concentrated under reduced pressure. Dissolve the residue in 5 ml of DMF,
Adjust pH to 8 with Et ₃ N.

On the other hand, [1] 316 mg and 0.05 ml of anisole and
Add 1 ml of TFA, leave at room temperature for 40 minutes, then add ether. The resulting white precipitate is collected by centrifugation, washed with ether, and then dried under reduced pressure over KOH. Dissolve this hydrazide in 4ml of DMF,
To this was added 0.16 ml of a 6.4 hydrogen chloride/dioxane solution while cooling to -20°C, and then 0.072 ml of isopentyl nitrite. After 5 minutes, check the pH of the reaction solution.
Adjust the pH to 8 with 0.15ml of Et ₃ N. This azide solution was added to the above pH-adjusted DMF solution and heated to 48°C at 4°C.
Stir for an hour. The reaction mixture was passed through a Sephadex LH-20 column (1.5 x 180 cm) packed with DMF and eluted with DMF. The effluent is fractionated into 3g portions, followed by TLC, and the 25th to 30th fractions are collected and concentrated under reduced pressure. Add ether to the residue,
Collect the white precipitate that forms. This precipitate was gel-filtered in the same manner as above to obtain 310 mg of white solid (yield:
94.7%). This was dissolved in as little as possible of the upper layer of butanol-acetic acid-water (4:1:5), and silica gel (1.3
x 35cm) column and elute with the same solvent as above. The eluate is fractionated into 3 g portions, followed by TLC, and the 14th to 33rd fractions are collected and concentrated under reduced pressure. Ether was added to the residue, and the resulting white precipitate was collected by centrifugation [2] 287 mg (yield
87.7%).

Melting point: wet at 160℃, decomposed at 187℃ [α] ²⁷ _D -63.0 (c=0.2, DMF) TLC; Rf _A =0.20 Elemental analysis [as C ₁₇₅ H ₂₇₀ N ₄₂ O ₅₄・HCl・2H ₂ O] C% H% N% Calculated value 53.4 7.15 15.0 Measured value 53.52 7.72 15.36 Amino acid analysis; Asp2.03(2), Thr0.95(1),
Ser7.77(8), Glu2.17(2), Pro8.96(9), Gly1.00(1),
Ala1.01(1), Ile0.93(1), Leu2.96(3), Phe1.30(1),
Lys1.05(1), Arg1.84(2) The physical properties of the above peptide fragment [3] are:
Chem.Pharm.Bull., 28 (9), 2714-2719 (1980), and methods for producing the fragment itself and its intermediate fragments are described in that document and in the same journal, 28
(9), 2707-2713 (1980).

(3) Fragment 112-113; Z-Asp(OBzl)-
Pro-NHNHBoc [4] Z-Asp (OBzl)-ONP4.8g and H-Pro-
Dioxane containing NHNHoc2.3g _Et3N1.4ml
Dissolve in 22 ml and stir overnight at room temperature. After the reaction, dioxane is distilled off under reduced pressure, the residue is dissolved in ethyl acetate, and then washed with 10% citric acid water, 5% sodium bicarbonate water, and water in this order. After drying with anhydrous sodium sulfate, it was concentrated under reduced pressure, and petroleum ether was added to the residue. The resulting precipitate was collected to obtain 4.45 g (yield 78.2%) of amorphous powder [4].

[α] ²⁷ _D -78.9゜ (C = 1.0, MeOH) TLC; Rf _A = 0.49, Rf _B = 0.79 Elemental analysis [as C ₂₉ H ₃₆ N ₄ O ₈ ] C% H% N% Calculated value 61.3 6.38 9.9 Measured value 61.2 6.60 9.6 (4) Fragment 112―145; Z―Asp(OBzl)―
Pro―Arg―Phe―Gln―Asp―Ser―Ser―Ser
―Ser―Lys (Boc)―Ala―Pro―Pro―Pro―
Ser―Leu―Pro―Ser―Pro―Ser―Arg―Leu
―Pro―Gly―Pro―Ser―Asp―Thr (Bu ^t )―
Add 10 ml of ethanol and 10 ml of 5% acetic acid water to 250 mg of Pro-Ile-Leu-Pro-Gln-OBu ^t [5] [2] to dissolve, and perform hydrogenolysis in the presence of a Pd catalyst. The catalyst was removed and the liquid was concentrated under reduced pressure. Add water containing 0.19 ml of 0.1N hydrochloric acid to the residue and freeze-dry. The obtained peptide was dissolved in 3 ml of DMF and the pH was adjusted to 8 with Et ₃ N.

On the other hand, 183 mg of [4] was dissolved in 0.21 ml of 6.4N hydrogen chloride/dioxane solution, and after 5 minutes, 0.2 ml of dioxane was added thereto, and the mixture was left at room temperature for 30 minutes. Next, add 5 ml of DMF, and add 0.045 ml of isopentyl nitrite while cooling to -20°C. After 5 minutes, after adjusting the pH of the solution to 8 with Et ₃ N, the azide solution was added to the above solution.
Add to pH-adjusted DMF solution and stir at 4°C for 48 hours. The reaction mixture was passed through a Sephadex G-25 column (1.8 x 190 cm) packed with DMF.
Outflow with DMF. The effluent was fractionated into 3g portions,
Collect fractions 23 to 29 and concentrate under reduced pressure. Ether is added to the residue, and the resulting precipitate is collected by centrifugation to obtain 226 mg (yield: 81.7%) of [5].

Melting point: 191℃ (decomposition), with semi-melting at 178℃.

[α] ²⁷ _D -69.5゜ (c=0.2, DMF) TLC; Rf _B =0.19, Rfc=0.51 Elemental analysis [as C ₁₉₁ H ₂₈₉ N ₄₃ O ₅₆・2HCl・11H ₂ O] C% H% N% Calculated value 52.7 7.25 13.8 Measured value 52.4 7.20 13.8 Amino acid analysis; Asp3.20(3), Thr1.02(1),
Ser7.41(8), Glu2.12(2), Pro10.38(10), Gly1.00(1),
Ala1.03(1), Ile0.93(1), Leu2.89(3), Phe1.16(1),
Lys1.08(1), Arg1.95(2), NH ₃ 2.94 (5) hCG (112-145) [5] 150mg plus TFA3ml and anisole 0.5ml
Add and leave at room temperature for 3 hours. Ether is added to this reaction solution, and the resulting white precipitate is collected by centrifugation, washed with ether, and then dried over KOH under reduced pressure. Add 7 ml of ethanol and 5 ml of water to the dried product obtained.
was added and dissolved, and hydrogenolysis was performed for 8 hours in the presence of a Pd catalyst. The catalyst is removed, the liquid is concentrated under reduced pressure, water is added to the residue, and the mixture is freeze-dried. This dried product was dissolved in 2 ml of 5% acetic acid, and this was added to a Sephadex G-25 column (2.5 x 135
cm) and eluted with 5% acetic acid. 3g of effluent
The 67th to 75th fractions are concentrated under reduced pressure and then freeze-dried. The obtained dried product was subjected to column chromatography again in the same manner as above, and 95 mg of fluffy powder hCG (112-145) was obtained.
(yield 68%). This is dissolved in a small amount of water containing 1.06 ml of 0.1N hydrochloric acid and lyophilized to convert it to the tetrahydrochloride.

[α] ²⁷ _D -153.8゜ (c=0.2, water) TLC; Rf _D =0.27 Elemental analysis [as C ₁₅₆ H ₂₄₇ N ₄₃ O ₅₂・4HCl・20H ₂ O] C% H% N% Calculated value 46.1 7.22 14.8 Measured value 46.0 6.67 14.4 Amino acid analysis [acid hydrolysis]; Asp2.83(3),
Thr0.95(1), Ser0.46(8), Glu2.03(2). Pro10.14(10),
Gly1.00(1), Ala1.00(1), Ile0.92(1), Leu2.80(1),
Phe1.03(1), Lys1.06(1), Arg1.93(2) Amino acid analysis [Pierce Chemical, AP-
M, hydrolyzed with LotO8307.33]; Asp2.67(3), Thr
+Gln2.20 (1+1), Ser6.67(8), Glu0.96(1),
Pro8.85(10), Gly1.00(1), Ala0.86(1), Ile0.97(1),
Leu2.80(3), Phe1.01(1), Lys0.85(1), Arg1.93(2),
Arg was not quantified. Since Gln appears at the same position as Thr, it was calculated as Thr.

Claims (1)

[Claims] 1 formula H-Asp-Pro-Arg-Phe-Gln-Asp ―Ser―Ser―Ser―Ser―Lys―Ala ―Pro―Pro―Pro―Ser―Leu―Pro ―Ser―Pro―Ser―Arg―Leu―Pro ―Gly―Pro―Ser―Asp―Thr―Pro ―Ile―Leu―Pro―Gln―OH A peptide represented by or a salt thereof.