JPS6257200B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a peptide for measuring human gonadotropin (hCG) activity. More specifically, the present invention provides the formula (In the formula, X is a single bond or

[Formula] represents a group) 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.
have a similar molecular structure 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. Although the three chains are more specific for each gonadotropin, the β-chains of LH and CG are similar. However, the amino acid composition near the C-terminus of the CGβ chain is significantly different from that of the LH β-chain, so if a measurement system that recognizes the C-terminus of the CGβ chain were developed, it would be possible to detect other gonadotropins, especially Highly reliable, does not cross at all with LH
CG measurement system becomes possible. The present invention was completed focusing on the above, and the present peptide represented by the formula [] is useful as a labeling reagent for radioimmunoassay for an hCG measurement system. The peptide [] of the present invention is constructed by condensing individual amino acids or lower peptides in the amino acid order represented by the formula [], and removing the protective group of the functional group of the side chain in the final step of the condensation reaction. can get. 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 (substituted with lower alkoxy groups such as methoxy group, p-methoxy group), benzyloxycarbonyl group, o-bromobenzyloxycarbonyl group, p-bromobenzyloxycarbonyl group, o-chlorobenzyl group oxycarbonyl group, p-chlorobenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group,
Benzyloxycarbonyl groups such as p-phenylazobenzyloxycarbonyl group, p-(p'-methoxyphenylazo)-benzyloxycarbonyl group, cyclopentyloxycarbonyl group, trichloroethyloxycarbonyl group, t-amyloxycarbonyl group groups, aliphatic oxycarbonyl groups such as t-butoxycarbonyl group, diisopropylmethoxycarbonyl group, 2-phenyl-isopropoxycarbonyl group, 2-tolyl-isopropoxycarbonyl group, 2-p-diphenyl-isopropoxycarbonyl group, etc. Examples include aralkyloxycarbonyl groups. These amino groups can also be protected by the formation of enamines obtained by reacting with 1,3-diketones such as benzoylacetone, acetylacetone, dimedone and the like. 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-
Substituted with isopropoxycarbonyl group, etc. Ester groups include methanol, ethanol, t
- Alkanols such as butanol, 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, thiophenols such as thiophenol, thiocresol, and p-nitrothiophenol. The hydroxyl groups of the tyrosine, 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, aroyl groups such as benzoyl groups, and groups derived from carbonic acid such as benzyloxycarbonyl groups and ethyloxycarbonyl groups. Examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, and t-butyl group. Protection of these hydroxyl groups includes 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl group, 2,2,2-trifluoro-1-
Benzyloxycarbonylaminoethyl groups are also suitable. 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 [ ], 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 Reacting an amino acid or peptide with a terminal carboxyl group, or reacting an amino acid or peptide with an activated α-amino group and a protected terminal carboxyl group with an amino acid with a free terminal carboxyl group and a protected α-amino group. Alternatively, it can be carried out by reacting a peptide. The carboxyl group in this case can be an azide, an acid anhydride, an acid imidazolide or an active ester derived from a hydrazide or an N'-protected hydrazide, such as t-butyloxycarbonyl hydrazide, benzyloxycarbonyl hydrazide, etc. with nitrous acid, such as an acid anhydride, an acid imidazolide or an activated ester, 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, N-ethyl-N'-3-dimethylaminopropylene carbodiimides such as rucarbodiimide,
isoxazolium salts such as N,N'-carbonyl-diimidazole or Woodward reagents;
It can be activated by reacting with diphenylphosphoradidate or the like. Preferred condensation methods in the present invention are the azide method, active ester method and carbodiimide 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,
Carbodiimide method with addition of racemization prevention reagent,
For example, the Wu¨nsch method using DCC-HOSu [Z.
Naturforsch., 21b , 426 (1966)], DCC-HOBt
Geiger method [Chem.Ber., 103 , 788
(1970)], Izumiya method using WSCI-HOSu [Arch
Biochem. Biophys., 144 , 237 (1971)], WSCI-
It is suitable to use the method using HOBt. 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 preferable to connect amino acids or peptides sequentially from the terminal side. The protected [Tyr ¹¹⁵ ]-hCG [115-145] is
Preferably, the C-terminal fragment 116-145 and the tyrosine fragment 115 are condensed by the azide method. C-terminal fragments 116-145 are sequentially followed by C-terminal fragments 130-145 and fragments 127-145.
129, fragment 123−126, fragment 120−
Preferably, 122 and fragments 116-119 are condensed by the azide method. C-terminus 130-145 is C
-terminal fragment 140-145, sequentially amino acid 139th, amino acid 138th, fragment 136-
137, fragments 133−135 and fragments
Preferably, 130-132 is condensed by the azide method. The protected [Tyr ¹¹¹ ]-hCG [111-145] is
Preferably, the C-terminal fragment 112-145 and the tyrosine fragment 111 are condensed by the azide method. The C-terminal fragment 112-145 is the C-terminal fragment 112-145.
Sequential fragments from terminal fragments 116−145
Preferably, 114-115 and fragments 112-113 are condensed 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 is also possible to protect ethyl esters, t-butyl esters, benzyl esters, and the like. In addition, these ester groups, for example, methyl ester groups, can be split with a dilute aqueous sodium hydroxide solution,
Alternatively, the t-butyl ester group can be converted to a hydratide or N'-protected hydratide by cleavage with trifluoroacetic acid, and the benzyl ester can be cleaved by 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 tyrosine, serine, and threonine do not necessarily have to be protected, but if they are protected, it is suitable to protect them with a t-butyl group or a benzyl group. The side chain carboxyl group of aspartic acid does not necessarily need to be protected, but if it is protected, it is suitable to protect it 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). Thus protected [Tyr ¹¹⁵ ]−hCG[115−
145] or protected [Tyr ¹¹¹ ]−hCG[111−
145], these protecting groups are preferably removed in one step by acid decomposition, e.g. trifluoroacetic acid, anhydrous hydrogen fluoride, etc., to yield a peptide of formula [], i.e. [Tyr ¹¹⁵ ]-hCG [ 115-145] or [Tyr ¹¹¹ ]-hCG[111-145]. The above-mentioned peptides can be separated and purified by known peptide purification methods. For example, Cephadex G-25, Cephadex G-50,
This can be carried out by column chromatography using a carrier such as Dowex X1, Sephadex LH-20, or carboxymethyl cellulose. 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. In addition, the abbreviations described in this specification have the following meanings. Tyr; L-tyrosine 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; -Leucine Giy; glycine Thy; 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 _CHCl3 ; chloroform ether; diethyl ether TFA; trifluoroacetic acid _Et3N ; triethylamine DCC; N,N'-dicyclohexyl-carbodiimide HOBt; 1-hydroxybenzotriazole Examples are given below. Now, production examples of the present invention 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) E; upper layer of butanol-acetic acid-water (4:15) F; butanol-ethyl acetate-acetic acid-water (1:
1:1:1) Example 1 [Tyr ¹¹⁵ ]-hCG[115-145]; H-Tyr-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 (1) Fragment 115−145; Z−Tyr−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 [1] 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−
50 mg of Pro-Gln-OBu ^t [2] is dissolved in 5 ml of 40% aqueous ethanol containing 0.1 ml of acetic acid, and hydrogenated in the presence of a palladium catalyst. The catalyst is removed and the mother liquor is concentrated under reduced pressure.
The residue is dissolved in water containing 0.03 ml of 1N hydrochloric acid and lyophilized. On the other hand, 23 mg of Z-Tyr-NHNH ₂ was mixed with 1N hydrogen chloride/
Dissolve in 1 ml of DMF containing 0.14 ml of dioxane solution,
To this was added 0.01 ml of isoamyl nitrite under cooling at -30°C, and after stirring for 5 minutes, 0.02 ml of Et ₃ N was added; an azide solution was obtained. The above lyophilized product was added to DMF1 containing 0.006ml of _Et3N .
ml, add the azide solution obtained above under ice cooling, and stir for 2 days. Transfer the reaction solution to a column (1.2 x 180
cm) and elute with DMF. The eluate is fractionated into 3 g portions, followed by TLC, and the 43rd to 49th fractions are collected and concentrated under reduced pressure. Ether was added to the residue, and the resulting white powder was collected by centrifugation and washed with ether to obtain [1] 28 mg (yield 54.4
%). Melting point, 270℃ (decomposition), half-melted at 210℃ TLC; Rf _E = 0.75 Elemental analysis [as C ₁₆₉ H ₂₅₉ N ₃₇ O ₅₂ HCl・15H ₂ O] C% H% N% Calculated value 51.4 7.40 13.1 Measured value 51.3 7.06 13.2 Amino acid analysis [acid hydrolysis]; Asp2.11(2),
Thr0.97(1), Ser7.30(8), Glu2.28(2), Pro8.70(9),
Gly1.00(1), Ala1.06(1), Ile1.03(1), Leu2.76(3),
Tyr0.80(1), Lys1.08(1), Arg0.97(1) The physical properties and production method of the above peptide fragment [2] are described in Chem.Pharm.Bull., 28 (9), 2714~
2719 (1980). (2) Add 0.6 ml of TFA and 0.1 ml of anisole to 20 mg of [Tyr ¹¹⁵ ]-hCG [115-145] [2] and leave at room temperature for 3 hours. Ether is added to the reaction solution, and the precipitate is collected by centrifugation.
After washing with ether and drying under reduced pressure over KOH, 4 ml of etaler and 4 ml of 5% acetic acid are added and hydrogenated in the presence of a palladium catalyst. The catalyst is removed and the mother liquor is concentrated under reduced pressure. The residue is dissolved in water and lyophilized to obtain a fluffy powder. This powder was dissolved in 5% acetic acid, and this was added to a column (2.2
x 130 cm) and elute with 5% acetic acid. The eluate is fractionated into 4 g portions, followed by absorbance measurement at 275 nm, fractions 58 to 70 are collected, and the solvent is distilled off under reduced pressure. The residue was dissolved in water and lyophilized to obtain [Tyr ¹¹⁵ ]-[115-145] 17 mg (yield 85.2%).
get. [α ²⁷ _D −164.7° (C = 0.2, water) TLC; Rf _D = 0.13, Rf _F = 0.10 Amino acid analysis [acid hydrolysis]; Asp2.01(2),
Thr0.99(1), Ser7.00(8), Glu2.11(2), Pro9.82(9),
Gly1.00(1), Ala1.05(1), Ile0.99(1), Ile0.99(1),
Leu3.10(3), Tyr0.80(1), Lys1.02(1), Arg1.00(1) Example 2 [Tyr ¹¹¹ ]-hCG[111-145]; H-Tyr-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 (1) Fragment 114−115; Z−Arg(NO ₂ )−
Phe−NHNHBoc [3] Z−Phe−NHNHBoc [E.Wunsch and G.
Wendlberger, Chem. Ber., 97 , 2504 (1964)]
H-Phe-NHNHBoc and Z-Arg(NO ₂ )- obtained from 2.0 g by hydrogenolysis in the presence of Pd catalyst.
Dissolve 1.7g of OH in 20ml of DMF and add to this under ice 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
The fraction around 0.41 was collected and dried under reduced pressure to obtain 1.6 g (4.45% yield) of [3]. Melting point: 120-125℃ [α] ²⁷ _D -28.92 (c = 1.0, MeOH) TLC; 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 [4] 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 [5] 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 and PH with Et ₃ N.
Adjust to 8. On the other hand, [3] 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 6.4N 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 is added to the above pH-adjusted DMF solution and stirred at 4°C for 48 hours. The reaction mixture is passed through a Sephadex LH-20 column (1.5 x 180 cm) packed with DMF, and the effluent is fractionated into 3 g portions using DMF, followed by TLC, and the 25th to 30th fractions are collected and concentrated under reduced pressure. . Add ether to the residue and collect the resulting white precipitate. This precipitate was gel-filtered in the same manner as above to obtain 310 mg (yield 94.7%) of a white solid. Mix this with as little butanol-acetic acid as possible.
Dissolve in the upper layer of water (4:1:5), pass through a column of silica gel (1.3 x 35 cm) packed with the same solvent as above, 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 to obtain 287 mg (yield: 87.7%) of [4]. Melting point: wet at 160°C, decomposed at 187°C [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 [5] are:
Chem.Pharm.Bull., 28 (9), 2714-2719 (1980)
The method for producing the fragment itself and its intermediate fragments is described in the same document and in the same journal, 28.
(9), 2707-2713 (1980). (3) Fragment 112−113; Z−Asp(OBzl)−
Pro-NHNHBoc [6] Z-Asp (OBzl)-ONP4.8g and H-Pro-
Dioxane 22 containing NHNHoc2.3g _Et3N1.4ml
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 solution, 5% sodium bicarbonate solution, 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 [6]. [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 )−Pro
-Ile-Leu-Pro-Gln-OBu ^t [7] [4] Add 10 ml of ethanol and 10 ml of 5% acetic acid water to dissolve 250 mg, 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 [6] 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. Fractionate the effluent into 3g portions, 23
Collect fractions ~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 [7]. 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) fragment (111−145); Z−Tyr−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−Thy
(Bu ^t )−Pro−Ile−Leu−Pro−Gln−OBu ^t
[8] [7] 2 ml of ethanol and 2 ml of 5% acetic acid to 51 mg
ml to dissolve and perform hydrogenolysis in the presence of palladium catalyst for 11 hours. The catalyst is removed, the mother liquor is concentrated under reduced pressure, and the residue is dissolved in water containing 0.012 ml of 1N hydrochloric acid and lyophilized to obtain a fluffy powder. Dissolve this powder in 5ml of DMF and adjust the pH to 8 with 0.0033ml of Et ₃ N.
Adjust to On the other hand, dissolve 77.1 mg of Z-Tyr-NHNH ₂ in 3 ml of DMF containing 0.067 ml of 1N hydrogen chloride/dioxane solution, and add isoamyl nitrite to this solution while cooling to -30°C.
After adding 0.033ml and stirring for 10 minutes, 0.066ml of Et ₃ N
After adjusting the pH to 8, the azide solution was adjusted to the above pH.
Add to the adjusted DMF solution and stir under ice cooling for 2 days. The reaction mixture was passed through a Sephadex LH-20 column (1.6 x 170 cm) packed with DMF.
Elute with DMF. Fractionate the eluate into 3g portions,
Collect fractions ~35 and concentrate under reduced pressure.
Ether is added to the residue, and the resulting white precipitate is collected by centrifugation to obtain 36 mg (68% yield) of [8]. Melting point: 198-220℃ (decomposition) [α] ²⁷ _D -75.5゜ (c = 0.2, DMF) TLC; Rf _D = 0.76 Amino acid decomposition [acid hydrolysis]; Asp3.25(3),
Thr0.97(1), Ser6.71(8), Glu2.35(2), Pro9.77(10),
Gly0.96(1), Ala1.00(1), Ile0.98(1), Leu2.93(3),
Tyr1.03(1), Phe0.93(1), Lys0.91(1), Arg2.06(2) (6) [Tyr ¹¹¹ ]-hCG[111-145] [8] 20mg plus TFA0.6ml and Anisole 0.1ml
Add and leave at room temperature for 3 days. Ether is added to the reaction solution, and the precipitated white precipitate is collected by centrifugation, washed with ether, and then dried over KOH under reduced pressure. This powder is dissolved by adding 2 ml of ethanol and 2 ml of 5% acetic acid, and hydrogenolysis is performed for 6 hours in the presence of a palladium catalyst. The catalyst is removed, the mother liquor is concentrated under reduced pressure, and the residue is dissolved in water and freeze-dried. The obtained powder was dissolved in 1 ml of 5% acetic acid, passed through a Sephadex G-25 column (2.2 x 135 cm) packed with 5% acetic acid, and eluted with 5% acetic acid. The eluate is fractionated into 6 g portions, followed by absorbance measurement at 275 nm, and the 30th to 35th fractions are collected and concentrated under reduced pressure. The residue is dissolved in water and freeze-dried to form a fluffy powder [Tyr ¹¹⁵ ]-hCG [115-
145] 11 mg (yield 62%) is obtained. [α] ²⁶ _D −144.3° (c=0.2, water) TLC; Rf _D =0.25 Amino acid analysis [acid hydrolysis]; Asp3.31(3),
Thr0.95(1), Ser7.27(8), Glu2.37(2), Pro10.86
(10), Gly0.98(1), Ala1.00(1), Ile1.01(1), Leu3.01
(3), Tyr0.98(1), Phe0.95(1), Lys1.05(1),
Arg2.24(2).

Claims (1)

[Claims] 1 Formula H-Tyr-X-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 (wherein, X is a single bond or -Asp-Pro-Arg
-Phe- group) or a salt thereof.