JPS6332145B2 - - Google Patents
Info
- Publication number
- JPS6332145B2 JPS6332145B2 JP6998481A JP6998481A JPS6332145B2 JP S6332145 B2 JPS6332145 B2 JP S6332145B2 JP 6998481 A JP6998481 A JP 6998481A JP 6998481 A JP6998481 A JP 6998481A JP S6332145 B2 JPS6332145 B2 JP S6332145B2
- Authority
- JP
- Japan
- Prior art keywords
- hcg
- group
- reduced pressure
- under reduced
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
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- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 12
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- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 10
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- RTEXIPZMMDUXMR-UHFFFAOYSA-N benzene;ethyl acetate Chemical compound CCOC(C)=O.C1=CC=CC=C1 RTEXIPZMMDUXMR-UHFFFAOYSA-N 0.000 description 9
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 8
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- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 8
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- NSADPSVGPKADJE-OALUTQOASA-N benzyl (2s)-1-[(2s)-4-methyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanoyl]pyrrolidine-2-carboxylate Chemical compound CC(C)(C)OC(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)OCC1=CC=CC=C1 NSADPSVGPKADJE-OALUTQOASA-N 0.000 description 7
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 7
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 7
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 6
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 6
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- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 6
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- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 6
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 5
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 5
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- 239000000969 carrier Substances 0.000 description 5
- ZKQFHRVKCYFVCN-UHFFFAOYSA-N ethoxyethane;hexane Chemical compound CCOCC.CCCCCC ZKQFHRVKCYFVCN-UHFFFAOYSA-N 0.000 description 5
- 125000000524 functional group Chemical group 0.000 description 5
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- QILSFLSDHQAZET-UHFFFAOYSA-N diphenylmethanol Chemical compound C=1C=CC=CC=1C(O)C1=CC=CC=C1 QILSFLSDHQAZET-UHFFFAOYSA-N 0.000 description 1
- 125000005982 diphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 1
- 229940043264 dodecyl sulfate Drugs 0.000 description 1
- 201000003511 ectopic pregnancy Diseases 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 150000002081 enamines Chemical class 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 description 1
- UREBWPXBXRYXRJ-UHFFFAOYSA-N ethyl acetate;methanol Chemical compound OC.CCOC(C)=O UREBWPXBXRYXRJ-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- 230000001456 gonadotroph Effects 0.000 description 1
- 229940094892 gonadotropins Drugs 0.000 description 1
- RRAMGCGOFNQTLD-UHFFFAOYSA-N hexamethylene diisocyanate Chemical compound O=C=NCCCCCCN=C=O RRAMGCGOFNQTLD-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000006698 hydrazinolysis reaction Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- JBFYUZGYRGXSFL-UHFFFAOYSA-N imidazolide Chemical compound C1=C[N-]C=N1 JBFYUZGYRGXSFL-UHFFFAOYSA-N 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 108010004902 lactose oxidase Proteins 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- 108010033881 maltose dehydrogenase Proteins 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- BRMYZIKAHFEUFJ-UHFFFAOYSA-L mercury diacetate Chemical compound CC(=O)O[Hg]OC(C)=O BRMYZIKAHFEUFJ-UHFFFAOYSA-L 0.000 description 1
- 125000004492 methyl ester group Chemical group 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- MXHTZQSKTCCMFG-UHFFFAOYSA-N n,n-dibenzyl-1-phenylmethanamine Chemical compound C=1C=CC=CC=1CN(CC=1C=CC=CC=1)CC1=CC=CC=C1 MXHTZQSKTCCMFG-UHFFFAOYSA-N 0.000 description 1
- CZFNISFYDPIDNM-UHFFFAOYSA-N n,n-dimethylformamide;oxolane Chemical compound CN(C)C=O.C1CCOC1 CZFNISFYDPIDNM-UHFFFAOYSA-N 0.000 description 1
- 125000002560 nitrile group Chemical group 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 125000005740 oxycarbonyl group Chemical group [*:1]OC([*:2])=O 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229960003540 oxyquinoline Drugs 0.000 description 1
- 125000006503 p-nitrobenzyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1[N+]([O-])=O)C([H])([H])* 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 150000002989 phenols Chemical group 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 125000003170 phenylsulfonyl group Chemical group C1(=CC=CC=C1)S(=O)(=O)* 0.000 description 1
- 125000000612 phthaloyl group Chemical group C(C=1C(C(=O)*)=CC=CC1)(=O)* 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 229960002429 proline Drugs 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000004756 silanes Chemical class 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- DVKJHBMWWAPEIU-UHFFFAOYSA-N toluene 2,4-diisocyanate Chemical compound CC1=CC=C(N=C=O)C=C1N=C=O DVKJHBMWWAPEIU-UHFFFAOYSA-N 0.000 description 1
- LDHQCZJRKDOVOX-UHFFFAOYSA-N trans-crotonic acid Natural products CC=CC(O)=O LDHQCZJRKDOVOX-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Reproductive Health (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Description
本発明は、ヒトじゆう毛性性腺刺激ホルモン
〔human chorionic gonadotropin（hCG）〕の新
規な測定法に関する。
hCGは糖蛋白であり、妊娠とともに胎盤より分
泌され、妊娠の維持に重要な役割を果している性
ホルモンである。このhCGを定性または定量する
ことにより妊娠の有無、異所性妊娠、じゆう毛性
性癌などの早期発見並びに診断が可能となる。し
かしながら、性腺刺激ホルモンには黄体形成ホル
モン（LH）、卵胞刺激ホルモン（FSH）やCGな
どがあり、それらの分子構造は類似しており、糖
蛋白であり、しかもα鎖、β鎖の２種のサブユニ
ツトからなり、特にそのα鎖においては各々のα
鎖を交換しても活性に変化がない程分子構造的に
類似しているものである。またβ鎖は各々の性腺
刺激ホルモンにより特異的であるが、LHのβ鎖
とCGのβ鎖とは類似している。
このような性腺刺激ホルモンにおいて、特に
hCGの測定に関して、ラジオイミユノアツセイ、
エンザイムイミユノアツセイ、血球凝集阻止反
応、血球凝集反応、ラテツクス凝集反応などを利
用して特にLHと交叉性の少ない信頼性の高い測
定系が種々検討されている。また特にエンザイム
イミユノアツセイにおけるhCGの測定系において
は、測定のための酵素標識体に用いられるhCGま
たはそのフラグメントによつて測定系の感度も不
満足なものであつた。
本発明者は、全く意外にも、このhCGのエンザ
イムイミユノアツセイにおいて、hCGの分子中に
結合しているシアリン酸（sialic acid）を脱離せ
しめてなる脱シアリン酸化hCG（ds―hCG）と酵
素との結合体をその酵素標識体として用いること
により、著しく感度よく測定し得ることを見い出
した。
本発明は上記の知見に基いが完成されたもの
で、hCGのエンザイムミミユノアツセイにおい
て、ds―hCG―酵素結合体を酵素標識体として用
いることを特徴とするhCGの測定法である。
まず本発明に用いられるエンザイムイミユノア
ツセイの手法としては、ds―hCG―酵素結合体と
被検液とをその抗体に対して競争反応せしめ、次
いでds―hCG―酵素結合体と抗体との免疫結合体
と、未反応のds―hCG―酵素結合体とを分離せし
め、その後そのいずれか一方または両方の酵素活
性を測定することにより被検液中のhCGの測定を
行なう競争反応に基く方法が簡便な手法として例
示される。さらにこの競争反応に基く方法は、好
ましくはds―hCG―酵素結合体と抗体との免疫結
合体と、未反応のds―hCG―酵素結合体との分離
の際に、その抗体に対する抗体であるいわゆる第
２抗体を用いる方法や、その抗体を不溶化せしめ
た不溶化抗体として用いる方法にて実施される。
まず本発明のds―hCG―酵素結合体を得るに用
いられるds―hCGとしては、hCGの分子内のシア
リン酸分子を弱酸などによる化学的加水分解や、
酵素による加水分解にてシアリン酸をhCG分子よ
り脱離せしめればよい。例えばhCGを含有する
0.05M酢酸緩衝液に、ニユラミニダーゼ
（neuraminidase；シグマ社製）を加えて37℃に
て１時間以上反応せしめて加水分解後、次いで一
夜透析後、クロマトグラフイーにて精製して目的
とするds―hCG分画を回収すればよい。さらにds
―hCG―酵素結合体を得るに用いられる酵素とし
ては、酸化還元酵素、加水分解酵素、転位酵素、
リアーゼ、イソメラーゼ、リガーゼが適宜使用さ
れるもので、例えばラクテートデヒドロゲナー
ゼ、マレイトデヒドロゲナーゼ、リンゴ酸デヒド
ロゲナーゼ、マルトースデヒドロゲナーゼ、ペル
オキシダーゼ、ラクトースオキシダーゼ、マレイ
トオキシダーゼ、グルコースオキシダーゼ、コリ
ンオキシダーゼ、キサンチンオキシダーゼ、アミ
ノ酸オキシダーゼ、ザルコシンオキシダーゼ、カ
タラーゼ、α―アミラーゼ、β―ガラクトシダー
ゼ、リゾチーム、リパーゼ、アルカルホスフアタ
ーゼ、アミノペプチダーゼ、トリプシン、パパイ
ン、α―キモトリプシン、アミダーゼ、ヘキソキ
ナーダ、グリセロキナーゼなどが挙られる。さら
にこれらの酵素やds―hCGは、あらかじめ任意の
スペーサー導入を行なつてもよく、例えばスクシ
ンアルデヒド、グルタルアルデヒド、アジポアル
デヒドなどのジアルデヒド、ω―アミノ酸の酸ク
ロライドやスクシンイミドエステルなどの反応性
誘導体、ジカルボン酸の反応性誘導体、ヘキサメ
チレンジアミン、デカメチレンジアミンなどのジ
アミン、Ｓ―アセチルメルカプトサクシニツク・
アンハイドライド、ジアルデヒドと２―アミノエ
タンチオールなどのスペーサー試薬を一種または
２種以上用いて新たにアルデヒド基、アミノ基、
チオール基、カルボキシル基をスペーサー導入せ
しめてもよい。次いでこのようなds―hCGと酵素
とを結合せしめるのであるが、ds―hCG、酵素の
有するアミノ基、水酸基、チオール基、カルボキ
シル基などや、さらに導入したアルデヒド基、ア
ミノ基、チオール基やカルボキシル基などに基い
て両者を結合せしめればよい。結合に当つては、
両者を直接または結合剤を用いて間接的に結合せ
しめてもよい。両者を直接結合せしめるに当つて
は、例えばds―hCGの分子内アミノ基と酵素の分
子内カルボキシル基とを水溶性カルボジイミドの
存在下反応せしめればよい。また結合剤を用いて
反応せしめるに当つては公知の種々の多官能性化
合物、例えばヘキサメチレンジイソシアナート、
２，４―トルエンジイソシアナートなどのジイソ
シアナート化合物、ヘキサメチレンジイソチオシ
アナートなどのジイソチオシアナート、ジアルデ
ヒド、Ｎ，N′―エチレンビスマレイミド、Ｎ，
N′―ｏ―フエニレンジマレイミド、メタマレイ
ミドベンゾイル・Ｎ―オキシスクシンイミドエス
テル、ビスジアゾベンジジン、Ｎ，N′―ポリメ
チレンビスヨードアセトアミド、ジエチルマロン
イミデート、ジメチルアジピンイミデート、３―
（2′―ベンゾチアゾリル―ジチオ）プロピオン酸、
３―（2′―ピリジル―Ｎ―オキシド―ジチオ）プ
ロピオン酸、６―Ｎ〔３―（2′―ベンゾチアゾリ
ル―ジチオ）プロピオニル〕カプロン酸などのス
ルフイドカルボン酸またはそのスクシンイミドエ
ステル、ｐ―ニトロフエニルエステル、酸クロラ
イドやイミデードなどの反応性誘導体、マレイミ
ド安息香酸、マレイミドフエニル酢酸、マレイミ
ドフエニルプロピオン酸などのマレイミドカルボ
ン酸またはその反応性誘導体などを用いればよ
い。またds―hCG―酵素結合体を得るに当つて
は、通常PH６〜８の緩衝液中メタノール、エタノ
ール、アセトン、ジオキサン、テトラビドロフラ
ン、ジメチルアセトアミド、ジメチルホルムアミ
ドなどの有機溶媒の存在下、または有機溶媒中、
０〜40℃にて、例えばds―hCGと多官能性化合物
を反応せしめ、次いで酵素を加えてさらに反応せ
しめる。反応後、必要に応じて精製すればよく、
好ましくはクロマトグラフイーやゲル過手段を
用いればよい。さらにds―hCG―酵素結合体を得
るに当つて、結合の際、介在物質を１種または２
種以上介して両者を結合せしめてもよい。即ちds
―hCG―〔介在物質〕―酵素結合体となして得て
もよいものである。この介在物質としては、ds―
hCGの分子量より大きく、また用いる酵素の分子
量より小さく、通常ds―hCGの分子量の５〜９倍
程度であればよく、またアフイニテイ―クロマト
グラフイーにて特異的吸着を示すものが好まし
い。この介在物質の使用目的としては、まずds―
hCGと介在物質とを、前記の如くの多官能性化合
物にて結合せしめてds―hCG―介在物質結合体と
なし、この際未反応のds―hCG、介在物質などを
その分子量の差異によりゲル過にて分子篩せし
め、さらに介在物質の特異的吸着によりアフイニ
テイ―クロマトグラフイーにて精製し、さらに必
要に応じて別の介在物質を結合せしめて同様に精
製し、次いで目的とする酵素を結合せしめて精製
してなるもので、介在物質の分子量および特異的
吸着性に基いて精製することにより、目的とする
ds―hCG―〔介在物質〕―酵素結合体の結合性の
良好なものが得られる。特にds―hCGに対して用
いる酵素の分子量が著しく大きい場合、例えば分
子量約50万のβ―ガラクトシダーゼの場合には、
ds―hCG―β―ガラクトシダーゼ結合体と未反応
のβ―ガラクトシダーゼとの分離は著しく困難で
あるに対し、介在物質として、例えばコンカナバ
リンＡに対して特異的吸着性を示すペルオキシダ
ーゼを用いれば、ds―hCG―ペルオキシダーゼ結
合体を得るに当つて、ゲル過およびコンカナバ
リンＡによるアフイニテイ―クロマトグラフイー
にて精製し得、さらにこれを用いて目的とするds
―hCG―〔ペルオキシダーゼ〕―酵素結合体を得
るに当つて同様に精製することにより良好に精製
し得るものである。
また本発明に用いられる抗体を得るに当つて
は、好ましくはhCGのβ鎖またはそのＣ末端フラ
グメント、例えば後述参考例に示すhCGのβ鎖の
127―145のＣ末端フラグメント（hCG〔127―145〕
と略す）、hCG〔118―145〕や種々のβ鎖のＣ末端
フラグメントが用いられる。
特に、hCG〔127―145〕、hCG〔118―145〕とし
ては、下記式〔〕
（ただし式中、ＲはＨ、または
基を示す）にて表わされるペプチドである。
まずこのβ鎖またはそのＣ末端フラグメント
は、高分子蛋白質、例えば牛血清アルブミン
（BSA）やラビツト血清アルブミン（RSA）、ま
たはそれらのアルカリ処理もしくはゾジウムラウ
リルサルフエートとメルカプトエタノール処理に
よる分子内ジスルフイド基を開裂せしめた処理物
に、高分子蛋白質１分子当り、前記の多官能性化
合物を用いて、５〜30分子程度結合せしめた結合
体となすか、または水溶性カルボジイミドやグル
タルアルデヒドなどのジアルデヒドを用いてβ鎖
またはそのＣ末端フラグメントの重合体となす。
次いでこれを種々の哺乳動物、例えばβ鎖または
そのＣ末端フラグメントと高分子蛋白質との結合
体、またはβ鎖またはそのＣ末端フラグメントの
重合体をフロイント・コンプリート・アジユバン
トに乳化せしめ、ラビツト、ラツト、モルモツト
やマウスなどの適宜選択した哺乳動物に皮下注射
せしめ、１〜２週間間隔にて数回投与するか、ま
たは１回多量投与して感作せしめればよく、感作
後適当な時期にてその哺乳動物より採血し、その
抗体力価を測定し、抗体産生が良好な時期にその
動物より採血し、これを常法により遠心処理など
を行なつてその抗血清を得ればよい。またこの抗
血清は十分高濃度の目的たる抗体を含有してなる
もので、適宜そのまま保存してもよく、また使用
時に必要に応じて希釈して用いればよい。さらに
この抗血清は、塩析、等電点沈澱、透析、クロマ
トグラフイー、ゲル過手段などの抗体の常法に
よる精製手段により、その抗体を得てもよい。
さらにこの精製された抗体は、不溶性担体と結
合せしめて不溶化抗体となすことにより、不溶化
抗体を用いる競争反応に基く方法に利用される。
まずこの不溶化抗体となすに用いられる不溶性担
体としては、例えばグルタルアルデヒドなどにて
処理してなるアルブミンやゼラチンなどの不溶性
蛋白質担体、アガロース、セルロースやデキスト
リンなどのエピクロルヒドリン処理または臭化シ
アン処理、さらにそれらのアミノ基導入のための
アミノ化試薬処理してなる不溶性半合成高分子担
体、アクリロニトリル、アクリル酸、アクリル酸
エステル、メタアクリル酸、メタアクリル酸エス
テル、ビニルアルコール、酢酸ビニル、スチレ
ン、アミノスチレン、ジビニルベンゼン、アクリ
ルアミド、エチレン、無水マレイン酸、クロトン
酸などのポリマーまたはコポリマー、さらにそれ
らのアミノ化試薬処理してなる不溶性合成高分子
担体、その他アミノ化試薬処理してなるシラン化
合物などの不溶性無機担体などが挙られる。また
これら不溶性担体にアミノ基を導入するためのア
ミノ化試薬処理としては、例えばニトリル基の還
元によるアミノ化のための試薬、水酸基をγ―ア
ミノプロピルトリエトキシシランの反応によるア
ミノアルキル基の導入のための試薬、ヒドロキシ
メチル基の過ヨウ素酸酸化にてアルデヒド基に変
換せしめ、次いでこのアルデヒド基にジアミンを
反応せしめてなるアミノ基導入のための試薬など
が適宜用いられ、さらに多糖類水酸基の臭化シア
ン反応によるイミドカルボナート基への変換、ア
ミド基のハロゲン化リン化合物によるイミノハロ
ゲニド基への変換、さらにこれらの官能基は公知
の種々の手段により新たな官能基を導入してもよ
い。またこのような不溶性担体と前記抗体とを結
合せしめて不溶化抗体を得るに当つては、通常水
性媒体、例えば緩衝液中にて、抗体の有する官能
基、不溶性担体の有する官能基に基いて、適宜選
択した前記多官能性化合物を用いて、０〜40℃に
て反応せしめればよい。
さらに第２抗体を用いる競争反応に基く方法を
実施するに当つては、前記抗体を得るに用いた哺
乳動物種の免疫グロブリンを、別種の哺乳動物に
投与して感作せしめればよい。この別種の哺乳動
物への投与、感作に当つては、前記の抗体を得る
方法と同様にして行なえばよく、さらに感作後、
採血し、抗血清を、さらに必要に応じて抗体を採
取すればよく、これを本発明の第２抗体として使
用するものである。
次いで本発明を実施するに当つて例示すれば、
例えば適宜希釈した抗体含有液、通常抗血清
200μ、被検液50μ、PH7.2〜7.4の緩衝液、通
常0.01Mリン酸緩衝液（PH7.2）100μ、および
ds―hCG―酵素結合体、例えばds―hCG―β―ガ
ラクトシダーゼ結合体含有液50μを４℃にて一
夜インキユベイトし、次いでこれに抗血清を得た
哺乳動物種の免疫グロブリン含有液100μおよ
び第２抗体含有液100μを加えて４℃、一夜イ
ンキユベイトした後これを遠心分離する。次いで
その上澄液と沈澱物とを分離し、これをその酵素
活性測定用媒体に加えて、その上澄液または沈澱
物の酵素活性を測定する。例えばds―hCG―酵素
結合体の酵素がβ―ガラクトシダーゼの場合に
は、ｏ―ニトロフエノール―β―Ｄ―ガラクトピ
ラノシドの0.1％程度の基質溶液を用いて、37℃
にて15分〜一夜反応せしめて後反応を停止し、次
いで反応によつて生じた呈色を420nmの波長にて
吸光度を測定すればよい。また不溶化抗体を用い
る場合には、例えば被検液50μ、緩衝液300μ
、不溶化抗体のビース状物１〜２粒、ds―hCG
―酵素結合体含有液50μを、同時または順次イ
ンキユベイトせしめ、その後そのビース状物と液
層とを分離し、以下そのいずれか一方または両方
の酵素活性を測定すればよい。またこの際に用い
れる不溶化抗体はビーズ状物に限られるものでな
く、免疫反応のために用いる試験管壁の面をもつ
て不溶化したものであつてもよく、適宜種々の形
状にて用いられるものである。さらに酵素活性を
測定するに当つては、用いる酵素に従つて公知の
酵素活性測定法により活性を測定すればよく、通
常紫外線吸収、螢光反応、呈色反応、発光反応、
酸素電極、過酸化水素電極などの電気的反応等に
基いて測定される。
次いで本発明の実施例および参考例を挙げて具
体的に述べるが、本発明はこれらによつて何んら
限定されるものではない。
実施例
(1) 抗体作成について
(イ) hCG（127―145）の３mg含有水溶液0.25mlに、
75mgの水溶性カルボジイミド（WSCD）水溶
液0.25mlを加えた。次いでそのPHを5.5に調整
した後、室温にて20時間反応せしめた。反応
後、これを、セフアデツクスＧ―150のカラム
にチヤージして、hCG（127―145）の重合体分
画を得、凍結乾燥した。
(ロ) 上記のhCG（127―145）の代りにhCG（118―
145）の３mgを用い、以下上記方法と同様にし
て、hCG（118―145）の重合体を得た。
(ハ) ２mgのBSA含有0.1Mリン酸緩衝液（PH7.0）
に、2.5％グルタルアルデヒド溶液0.2mlを加え
て室温にて18時間反応せしめた。反応後、セフ
アデツクスＧ―25のカラムにチヤージしてその
流出分画を得、これに、１mgのhCG（118―145）
含有生理食塩水１mlを加え、さらに1M炭酸ナ
トリウム水溶液（PH9.5）0.1mlを加えて、４℃
にて24時間反応せしめ、反応後セフアデツクス
Ｇ―100のカラムにチヤージして、BSA―hCG
（118―145）結合体の分画を得た。
(ニ) 0.7mgのhCG（118―145）含有250μ水溶液
に、2.5mgのRSA含有2.50μ水溶液を加え、こ
れに75mgの水溶性カルボジイミド（WSCD）
含有500μ水溶液を加えて、そのPHを５〜5.5
と調整した後、室温にて20時間反応せしめ、次
いでこれを、セフアデツクスＧ―100のカラム
にチヤージして、RSA―hCG（118―145）結合
体の分画を得た。
(ホ) 前記(ハ)のhCG（118―145）の代りにhCGのβ
鎖５mgを用い、以下同様にして、BSA―hCGβ
鎖結合体を得た。
(ヘ) 前記の如くして得られたhCG（127―145）の
重合体、hCG（118―145）の重合体、BSA―
hCG（118―145）結合体、RSA―hCG（118―
145）結合体、BSA―hCGβ鎖結合体を用いて、
これを等量のフロイント・コンプリート・アジ
ユバントと混和混合して乳化物となし、この
0.5mlづつをラビツト背部皮下に注射して感作
せしめ、その後採血し、遠心分離してその抗血
清を得た。
なお、hCG（127―145）重合体を用いてなる感
作に当つて、感作後４週間にて320U／mlを示し
た。またhCG（118―145）重合体を用いた場合に
は感作後５週間にて320U／mlを示した。さらに
BSA―hCG（118―145）結合体を用いた場合には
感作後５週間後40U／ml、７週間後80U／mlであ
つた。またBSA―hCGβ鎖結合体を用いた場合に
は感作後５週間で320U／ml、６週間で1280U／
mlであつた。
(2) ds―hCGの調整について
9.5mgの精製されたhCGを含有する0.05M酢酸
緩衝液（PH4.6；2mM CaCl2および0.2ｍ Ｍの
EDTA含有）0.75mlに６mgのニユラミニダーゼ
（シグマ社製）を加えて37℃にて２時間反応せし
めた。反応後上記と同一緩衝液に対して37℃にて
一夜透析せしめた。次いでこれをDEAE―セルロ
ースによるカラムクロマトグラフイーを行なつて
ds―hCG分画（収量4.7mg）を得た。
(3) ds―hCG―β―ガラクトシダーゼ結合体など
について
(イ) 0.05Mリン酸緩衝液（PH7.0）の0.25mlに0.5
mgのds―hCGを加え、これに無水テトラヒドロ
フランに溶解したメタマレイミド・ベンゾイ
ル・Ｎ―オキシスクシンイミドエステルのほぼ
等モル量を有する溶液20μを加えて30℃にて
30分間反応せしめ、次いでこれに1Mのクエン
酸緩衝液0.25mlを加え、セフアデツクスＧ―25
のカラムにチヤージして未反応試薬の除去をし
た。次いでその流出区分に、１mgのβ―ガラク
トシダーゼ含有1Mリン酸緩衝液（PH7.0）0.5
mlを加えて、室温にて２時間反応せしめ、その
後これに10-5Mメルカプトエタノールの50μ
溶液を加えて反応を停止せしめた。次いでこれ
を、コンカナバリンＡ―セフアロース4Bにて
アフイニテイ―クロマトグラフイーを行ない、
さらにウルトロゲルAcA44のカラムにてゲル
過して、ds―hCG―β―ガラクトシダーゼ結
合体分画を得た。
(ロ) 上記ds―hCGの代りに、hCGを用いて、以下
同様に行なつて、hCG―β―ガラクトシダーゼ
結合体を得た。
(ハ) 上記ds―hCGの代りに、hCGのβ鎖を用い
て、以下同様に行なつて、hCGβ鎖―β―ガラ
クトシダーゼ結合体を得た。
(ニ) 0.5mgのds―hCG含有0.1Mリン酸緩衝液（PH
8.0）0.5mlに、5mMのEDTAおよび３―（2′―
ベンゾチアゾリル―ジチオ）プロピオン酸スク
シンイミドエステルのほぼ等モル量を含有する
ジメチルアセトアミド溶液0.1mlを加えて、０
℃にて60分間反応せしめた。反応後、反応液
6μを分取し、0.15M NaCl含有0.01Mリン酸
緩衝液（PH7.2）500μおよびβ―ガラクトシ
ダーゼ1.3mgを加え、０℃、30分間反応せしめ
た。反応後、これをゲル過して、ds―hCG―
β―ガラクトシダーゼ結合体を得た。
(4) hCGの測定について
(イ) 前記のBSA―hCGβ鎖結合体を用いて得られ
た抗血清を用いて、hCGの各種濃度（100〜
16000mIU／ml）に対するエンザイムイミユノ
アツセイにおける各種酵素標識体による差異を
求めた。なお酵素標識体としては、ds―hCG―
β―ガラクトシダーゼ結合体〔３項(イ)に記載の
方法で得られたもの；第１図中、○―○にて示
される場合である〕、hCG―β―ガラクトシダ
ーゼ結合体〔３項(ロ)に記載の方法で得られたも
の；第１図中、△―△にて示される場合であ
る〕、hCGβ鎖―β―ガラクトシダーゼ結合体
〔３項(ハ)に記載の方法で得られたもの；第１図
中、×―×にて示される場合である〕の各々を
用い、その測定結果は第１図に示す通りであ
る。
(ロ) 前記のhCG（127―145）重合体を用いて得ら
れた抗血清を用いて、hCGの各種濃度（200〜
32000mIU／ml）に対するエンザイムイミユノ
アツセイにおける各種酵素標識体による差異を
求めた。なお酵素標識体としては、ds―hCG―
β―ガラクトシダーゼ結合体〔３項(イ)に記載の
方法で得られたもの；第２図中、○―○にて示
される場合である〕、hCG―β―ガラクトシダ
ーゼ結合体〔３項(ロ)に記載の方法にて得られた
もの；第２図中、△…△にて示される場合であ
る〕の各々を用い、その測定結果は第２図に示
す通りであり、その結果、hCG（127―145）重
合体の如くのhCGのＣ末端フラグメントを用い
る抗体または抗血清の場合には、特にds―hCG
の酵素標識体との結合性が良好であることが明
らかである。
なお、このエンザイムイミユノアツセイに用い
た測定法は、以下の通りである。
まず200μの抗血清、50μのhCGの各種濃度
の被検液、100μの0.01Mリン酸緩衝液（PH7.2）
および酵素標識体含有液50μを４℃にて一夜イ
ンキユベイトし、次いでこれにラビツトの正常血
清の希釈液100μおよび第２抗体含有液（ラビ
ツトの免疫グラブリンに対する抗体を含有する抗
血清）100μを加えて、４℃にて一夜インキユ
ベイトした。次いでこれを遠心分離し、その沈澱
物を回収し、これを、0.1％のｏ―ニトロフエノ
ール―β―Ｄ―ガラクトシドの0.1Mリン酸緩衝
液（PH7.2）の500μに加え、37℃にて一夜イン
キユベイトし、その後反応を停止せしめ、次いで
その呈色を420nmの波長にて吸光度測定した。
以上の通り、本発明のds―hCG―酵素結合体
は、hCGβ鎖を用いて得られる抗体に対してもそ
の結合性は良好であり、特にhCGβ鎖のＣ末端フ
ラグメントを用いて得られる抗体に対して極めて
良好な結合性を示すものであつた。
(5) 検量線について
前記のBSA―hCGβ鎖結合体を用いて得られた
抗血清（8000倍希釈）を用い、ds―hCG―β―ガ
ラクトシダーゼ結合体によるhCGの検量線を求め
た。その結果、第３図に示す通りであつた。
なお、本発明に用いられる式〔〕で表わされ
るペプチドを得るに当つては、その式〔〕で示
されるアミノ酸順序に個々のアミノ酸または低級
ペプチドを縮合して構成せしめ、縮合反応の最終
段階で側鎖の官能基の保護基を脱離することによ
り得られる。縮合反応自体はペプチド合成のため
の常法手段に従つて、保護基の着脱、縮合反応を
繰り返すことにより行なわれる。即ち、本目的化
合物の原料ならびにすべての中間体の製造におい
て使用される各種保護基はペプチド合成で既知な
もの、従つて加水分解、酸分解、還元、アミノリ
シスまたはヒドラジノリシスのような既知手段に
よつて容易に脱離することのできる保護基が用い
られる。例えば、アミノ基に使用する保護基とし
ては、ホルミル基、トリフルオロアセチル基、フ
タロイル基、ベンゼンスルホニル基、ｐ―トルエ
ンスルホニル基、ｏ―ニトロフエニルスルフエニ
ル基、２，４―ジニトロフエニルスルフエニル
基、２，４―ジニトロフエニルスルフエニル基な
どのアシル基、ベンジル基、ジフエニルメチル
基、トリフエニルメチル基（これらの基は場合に
よつてはｏ―メトキシ基、ｐ―メトキシ基などの
低級アルコキシ基によつて置換されている）など
のアラルキル基、ベンジルオキシカルボニル基、
ｏ―ブロモベンジルオキシカルボニル基、ｐ―ブ
ロモベンジルオキシカルボニル基、ｏ―クロロベ
ンジルオキシカルボニル基、ｐ―クロロベンジル
オキシカルボニル基、ｐ―ニトロベンジルオキシ
カルボニル基、ｐ―メトキシベンジルオキシカル
ボニル基、ｐ―フエニルアゾ―ベンジルオキシカ
ルボニル基、ｐ―（p′―メトキシフエニルアゾ）
―ベンジルオキシカルボニル基などのベンジルオ
キシカルボニル基、シクロペンチルオキシカルボ
ニル基、トリクロロエチルオキシカルボニル基、
ｔ―アミルオキシカルボニル基、ｔ―ブトキシカ
ルボニル基、ジイソプロピルメトキシカルボニル
基などの脂肪族オキシカルボニル基、２―フエニ
ル―イソプロポキシカルボニル基、２―トリル―
イソプロポキシカルボニル基、２―ｐ―ジフエニ
ル―イソプロポキシカルボニル基などのアラルキ
ルオキシカルボニル基などがある。またこれらア
ミノ基をベンゾイルアセトン、アセチルアセト
ン、ジメドンなどの１，３―ジケトンと反応させ
ることによつて得られるエナミンの形成により保
護することができる。
カルボキシル基は、アミド形成、ヒドラチド形
成またはエステル化によつて保護される。すなわ
ちアミド基は３，４―ジメトキシベンジル基、ビ
ス―（ｐ―メトキシフエニル）メチル基などによ
つて置換される。ヒドラチド基はベンジルオキシ
カルボニル基、トリクロロエチルオキシカルボニ
ル基、トリフルオロアセチル基、ｔ―ブトキシカ
ルボニル基、トリチル基、２―ｐ―ジフエニル―
イソプロポキシカルボニル基などによつて置換さ
れる。エステル基はメタノール、エタノール、ｔ
―ブタノール、シアノメチルアルコールなどのア
ルカノール、ベンジルアルコール、ｐ―プロモベ
ンジルアルコール、ｐ―クロロベンジルアルコー
ル、２，６―ジクロロベンジルアルコール、ｐ―
メトキシベンジルアルコール、ｐ―ニトロベンジ
ルアルコール、２，４，６―トリメチルベンジル
アルコール、ベンズヒドリルアルコール、ベンゾ
イルメチルアルコール、ｐ―ブロモベンゾイルメ
チルアルコール、ｐ―クロロベンゾイルメチルア
ルコールなどのアラルカノール、２，４，６―ト
リクロロフエノール、２，４，５―トリクロロフ
エノール、ペンタクロロフエノール、ｐ―ニトロ
フエノール、２，４―ジニトロフエノール、ｐ―
シアノフエノール、ｐ―メタンスルホニルフエノ
ールなどのフエノール、チオフエノール、チオク
レゾール、ｐ―ニトロチオフエノールなどのチオ
フエノールなどによつて置換される。
前記セリン、スレオニンおよびチロシンの水酸
基は、例えばエステル化またはエーテル化によつ
て保護することができる。このエステル化に適す
る基としては例えばアセチル基などの低級アルカ
ノイル基、ベンゾイル基などのアロイル基、ベン
ジルオキシカルボニル基、エチルオキシカルボニ
ル基などの炭酸から誘導される基である。またエ
ーテル化に適する基としては、例えばベンジル
基、テトラヒドロピラニル基、ｔ―ブチル基など
である。これら水酸基の保護には２，２，２―ト
リフルオロ―１―ｔ―ブチルオキシカルボニルア
ミノエチル基、２，２，２―トリフルオロ―１―
ベンジルオキシカルボニルアミノエチル基も適す
る。
しかしながら、これら水酸基を必ずしも保護す
る必要はない。
前記アルギニンのグアニジノ基中のアミノ基を
保護するのに使用する基としては例えばニトロ
基、トシル基、ベンジルオキシカルボニル基、な
どであるが、このグアニジノ基を必ずしも保護す
る必要はない。
前記ヒスチジンのイミノ基を保護するのに使用
する基としては、例えばベンジル基、トリチル
基、ベンジルオキシカルボニル基、トシル基、ア
ダマンチルオキシカルボニル基、２，２，２―ト
リフルオロ―１―ｔ―ブチルオキシカルボニルア
ミノエチル基、２，２，２―トリフルオロ―１―
ベンジルオキシカルボニルアミノエチル基などで
あるが、このイミノ基を必ずしも保護する必要は
ない。
システインのメルカプト基を保護するのに使用
する基としては、ベンジル基、ｐ―メトキシベン
ジル基、ｐ―ニトロベンジル基、トリチル基、ベ
ンジルチオメチル基、エチルカルバモイル基、ア
セトアミドメチル基などである。
本目的化合物〔〕の合成においては、個々の
アミノ酸もしくは低級ペプチドの縮合は、例え
ば、保護されたα―アミノ基および活性化末端カ
ルボキシル基をもつアミノ酸またはペプチドと遊
離α―アミノ基および保護された末端カルボキシ
ル基をもつアミノ酸またはペプチドとを反応させ
るか、あるいは活性化α―アミノ基および保護さ
れた末端カルボキシル基をもつアミノ酸またはペ
プチドと遊離の末端カルボキシル基および保護さ
れたα―アミノ基をもつアミノ酸またはペプチド
を反応させることにより実施することができる。
この場合カルボキシル基は、アジド、酸無水
物、酸イミダゾリドまたは活性エステル、例えば
シアノメチルエステル、チオフエニルエステル、
ｐ―ニトロチオフエニルエステル、ｐ―メタンス
ルホニルフエニルエステル、チオジルエステル、
ｐ―ニトロフエニルエステル、２，４―ジニトロ
フエニルエステル、２，４，５―トリクロロフエ
ニルエステル、２，４，６―トリクロロフエニル
エステル、ペンタクロロフエニルエステル、Ｎ―
ヒドロキシコハク酸イミドエステル、Ｎ―ヒドロ
キサフタル酸イミドエステル、８―ヒドロキシキ
ノリンエステルまたはＮ―ヒドロキシピペリジン
エステルなどに変換することによつて、あるいは
カルボジイミド、Ｎ，N′―カルボニル―ジイミ
ダゾールまたはイソオキゾリウム塩、例えばウツ
ドワード反応剤などを使用して反応させることに
よつて活性化することができる。
本発明において好ましい縮合方法は、カルボジ
イミド法、アジド法、活性エステル法および無水
物法である。縮合の各段階では、ラセミ化が起ら
ない方法またはラセミル化が最小になる方法を用
いるのが望ましく、好ましくはアジド法、活性エ
ステル法、Wu¨nsch法〔Z.Naturforsch.，21b，
426（1966）〕またはGeiger法〔Chem.Ber.，103，
788（1970）〕、とりわけ縮合剤としてＮ―エチル―
N′―３―ジメチルアミノプロピル―カルボジイ
ミド（WSCI）を用いる変法などを用いる。
縮合順序は式〔〕で示されるアミノ酸順序で
あれば、如何なる順序からも合成し得るがＣ―末
端側から合成するのが有利である。
保護されたhCG〔127―145〕は、Ｃ―末端フラ
グメント132―145とＮ―末端フラグメント127―
131をWSCIを用いるGeiger変法による方法で縮
合するのが好ましい。Ｃ―末端フラグメント132
―145はフラグメント132―137とフラグメント138
―144をWSCIを用いるGeiger変法による方法で
縮合するのが好ましい。Ｎ―末端フラグメント
127―131はフラグメント127―129とフラグメント
130―131をWSCIを用いるGeiger変法による方法
で縮合するのが好ましい。
保護されたhCG〔118―145〕は、Ｃ―末端フラ
グメント127―145、即ち保護されたhCG〔127―
145〕とＮ―末端フラグメント118―126をWSCI
を用いるGeiger変法による方法で縮合するのが
好ましい。Ｃ―末端フラグメント118―126はフラ
グメント118―121とフラグメント122―126をアジ
ド法により縮合するのが好ましい。
保護されたhCG〔105―145〕は、Ｃ―末端フラ
グメント112―145とＮ―末端フラグメント105―
111をWSCIを用いるGeiger変法による方法で縮
合するのが好ましい。Ｃ―末端フラグメント112
―145は保護されたhCG〔118―145〕にフラグメン
ト116―117、フラグメント113―115および第112
番目のアミノ酸を活性エステル法により順次縮合
して連絡するのが好ましい。Ｎ―末端フラグメン
ト105―111はフラグメント110―111とフラグメン
ト105―109をアジド法により縮合するのが好まし
い。
保護されたhCG〔100―145〕は、Ｃ―末端フラ
グメント112―145とＮ―末端フラグメント100―
111を活性エステル法により縮合するのが好まし
い。Ｎ―末端フラグメント100―111はフラグメン
ト100―104とフラグメント105―111をアジド法に
より縮合するのが好ましい。
保護された〔Tyr100〕−hCG〔100―145〕は、Ｃ
―末端フラグメント112―145とＮ―末端フラグメ
ント100―111を活性エステル法により縮合するの
が好ましい。Ｎ―末端フラグメント100―111はフ
ラグメント100―104とフラグメント105―111をア
ジド法により縮合するのが好ましい。
上記のペプチドの合成に際して、その末端カル
ボキシル基は、これを必らずしも保護しなければ
ならないわけではない。例えば、アジド法、活性
エステル法によつて縮合させる場合には、保護し
なくてもよい。
しかしながら、これらの基を前記で述べたよう
なエステル化によつて、例えばメチルエステル、
エチルエステル、ベンジルエステルなどで保護す
ることもできる。また、これらのエステル基は、
例えばメチルエステル基はこれを希薄な水酸化ナ
トリウム水溶液で***し、またはヒドラチドに変
え、またベンジルエステル基は無水沸化水素また
は水素添加分解によつて***することができる。
これらペプチドのα―アミノ基は、これらを通
常の保護基、例えばベンジルオキシカルボニル
基、ｔ―ブトキシカルボニル基、ｔ―アミルオキ
シカルボニル基で保護されるが、ベンジルオキシ
カルボニル基は水素添加分解によつて脱離され、
ｔ―ブトキシカルボニル基、ｔ―アミルオキシカ
ルボニル基はトリフルオロ酢酸で脱離される。
セリンおよびスレオニンの水酸基はベンジル基
で、チロシンの水酸基は２，６―ジクロロベンジ
ル基で、リジンのε―アミノ基はｏ―クロロベン
ジルオキシカルボニル基で、アルギニンのグアニ
ジノ基中のアミノ基はトシル基で、システインの
メルカプト基はｐ―メトキシベンジル基で保護す
るのが適する。これらの保護基は無水沸化水素で
脱離される。システインのメルカプト基の保護基
としてアセトアミドメチル基を使用することがで
きる。この基は無水沸化水素に対して安定である
ため、他の全ての保護基が脱離された時点でPH４
において酢酸水銀で脱離される。
こうして保護されたhCG〔127―145〕、保護され
たhCG〔118―145〕、保護されたhCG〔105―145〕、
保護されたhCG〔100―145〕、保護された
〔Tyr100〕―hCG〔100―145〕が得られる。これら
の保護基は、好ましくは、酸分解、例えば無水沸
化水素の処理による方法によつて一段階で脱離さ
れ、式〔〕の目的化合物が得られる。第110番
目および／または第100番のシステインのメルカ
プト基の保護基としてアセトアミドメチル基を使
用した場合、無水沸化水素処理により他の全ての
保護基が脱離した後に、PH４において酢酸第二水
銀により脱離してもよい。
上記の目的化合物〔〕は、公知のペプチドを
精製する手段により精製することができる。例え
ばセフアデツクスLH―20、セフアデツクスＧ―
50、Dowex1、カルボキシメチルセルロース等の
担体を用いるカラムクロマトグラフイーにより行
うことができる。
本発明のペプチド〔〕は、その方法の条件に
より塩基またはその塩の形で得られる。通常は酢
酸の如き有機酸との塩の形で保存され得る。
次いで、本発明に用いられるペプチド〔〕の
合成例について挙げるが、何んら本発明を限定す
るものではない。
なお、本明細書に記載の各記号は、次の意味を
有する。
Gln；Ｌ―グルタミン
Pro；Ｌ―プロリン
Leu；Ｌ―ロイシン
Ile；Ｌ―イソロイシン
Thr；Ｌ―スレオニン
Asp；Ｌ―アスパラギン酸
Ser；Ｌ―セリン
Gly；グリシン
Arg；Ｌ―アルギニン
Ala；Ｌ―アラニン
Lys；Ｌ―リジン
Phe；Ｌ―フエニルアラニン
Cys；Ｌ―システイン
His；Ｌ―ヒスチジン
Tyr；Ｌ―チロシン
BOC；ｔ―ブチルオキシカルボニル
AOC；ｔ―アミルオキシカルボニル
Ｚ；ベンジルオキシカルボニル
Ｚ―Cl；Ｏ―クロロベンジルオキシカルボニル
Bzl；ベンジル
MBzl；ｐ―メトキシベンジル
Bzl―Cl2；２，６―ジクロロベンジル
Acm；アセトアミドメチル
Tos；トシル
OMe；メチルエステル
OEt；エチルエステル
OBzl；ベンジルエステル
OSU；Ｎ―ヒドロキシコハク酸イミドエステ
ル
ONP；ｐ―ニトロフエニルエステル
TFA；トリフルオロ酢酸
TEA；トリエチルアミン
TBA；トリベンジルアミン
DCHA；ジシクロヘキシルアミン
NMM；N′―メチルモルホリン
THF；テトラヒドロフラン
DMF；ジメチルホルムアミド
WSCI；Ｎ―エチル，N′―３―ジメチルアミノ
プロピル―カルボジイミド
HOBT；１―ヒドロキシベンゾトリアゾール
また、本明細書中で使用した薄層クロマトグラ
フイー（TLC）の担体および溶媒系は、次の通
りである。
担体；シリカゲルＧ
溶媒系；
１ クロロホルム―メタノール―酢酸（95：５：
３）
２ クロロホルム―メタノール―酢酸（85：15：
５）
３ クロロホルム―メタノール―酢酸（85：10：
５）
４ クロロホルム―メタノール―酢酸（80：25：
２）
５ クロロホルム―メタノール―酢酸（24：６：
１）
６ クロロホルム―エタノール―酢酸エチル
（５：２：５）
７ 酢酸エチル
８ 酢酸エチル―メタノール（10：１）
９ 酢酸エチル―ベンゼン（１：１）
10 ブタノール―酢酸―水（３：１：１）
担体；メルク社製セルロース
溶媒系；
11 ブタノール―ピリジン―酢酸―水（15：10：
３：12）
参考例 １
hCG〔127―145〕；Ｈ―Ser―Leu―Pro―Ser―
Pro―Ser―Arg―Leu―Pro―Gly―Pro―Ser
―Asp―Thr―Pro―Ile―Leu―Pro―Gln―
OH
１ Ｐ（143―144）；BOC―Leu―Pro―OBzl〔１〕
BOC―Leu―OH.H2O149.59ｇ（0.6M）に
THF300mlを加え、これにHOBT81.06ｇ
（0.6M）、THF150ml、DMF100mlを加えて溶液
とする。次いでＨ―Pro―OBzl152.28ｇ
（0.63M）を加え、−10℃に冷却下WSCI120.8ml
（0.66M）を滴下した後、DMF100mlを追加し、
室温で一夜撹拌する。
反応後、減圧下で溶媒を留去し、残渣に酢酸エ
チル600mlを加え、５％重曹水（３回）、1N塩酸
（２回）、水（２回）の順に洗浄する。酢酸エチル
層を無水芒硝で乾燥後、減圧濃縮する。油状物を
ベンゼン１に溶かし、減圧濃縮して油状の
〔１〕を得る。
TLC；Rf1＝0.89
２ Ｐ（142―144）；BOC―Ile―Leu―Pro―
OBzl〔２〕
〔１〕192mMを塩化メチレン100mlに溶かし、
これにTFA300mlを加え、室温で30分間撹拌す
る。TFAを減圧下留去し、残渣にヘキサンを加
え、減圧留去する。残渣の油状物をTHF200mlに
溶かし、これに氷冷下NMM56.1mlでPH７に中和
した後、BOC―Ile―OH・H2O38.33ｇ
（159.5mM）およびHOBT21.55ｇ（159.5mM）
を加える。次いでDMF100mlを加えて溶かし、氷
冷下WSCI34.1mlを滴下する。
滴下後、室温で一夜撹拌する。
反応後、減圧下で溶媒を留去し、残渣に酢酸エ
チル500mlを加え、５％重曹水（３回）、飽和食塩
水（１回）、1N塩酸（２回）、飽和食塩水（２
回）、水（１回）の順に洗浄し、無水芒硝で乾燥
後、減圧濃縮する。残渣にヘキサンを加え、沈澱
物をデカンテーシヨンにより採取する。酢酸エチ
ル―ヘキサンより再結晶を行う。過中に融ける
ので一部過し、残りはベンゼンに溶解して凍結
乾燥して〔２〕76.98ｇ（収率90.9％）を得る。
融点；60〜64℃
〔α〕24 D；−66.34゜（Ｃ＝１，DMF）
TLC；Rf1＝0.63
３ Ｐ（142―144）；BOC―Ile―Leu―Pro―OH
〔３〕
〔２〕76.98ｇ（145mM）にｎ―ブタノール20
ml、エタノール300mlを加えて溶かし、これに５
％Pd／C15ｇを加え、３時間水素添加する。触媒
を去し、母液を減圧濃縮する。残渣にジエチル
エーテル（エーテル）300mlを加え、５％重曹水
500ml、200mlで各１回づつ抽出する。抽出液を氷
冷下1N塩酸でPH４に調節すると、生成物がダン
ゴ状となり析出する。酢酸エチルで抽出し、酢酸
エチル層を水で２回洗浄する。無水芒硝で乾燥
後、減圧濃縮する。残渣をエーテル―ヘキサンで
固化する。酢酸エチル―エーテル―ヘキサンから
再結晶して〔３〕60.32ｇ（収率94.2％）を得る。
融点；114〜119℃
〔α〕24 D；−61.98゜（Ｃ＝１，DMF）
TLC；Rf1＝0.53、Rf6＝0.47
元素分析〔C22H39O6N3として〕
Ｃ％ Ｈ％ Ｎ％
測定値 59.82 9.42 9.56
計算値 59.84 8.90 39.52
４ Ｐ（142―145）；BOC―Ile―Leu―Pro―Gln
―OBzl〔４〕
BOC―Gln―OBzl36.41ｇ（108mM）を塩化メ
チレン100mlに溶かし、これにTFA150mlを加え、
室温で30分間撹拌する。反応後、減圧下でTFA
を留去し、残渣をTHF100mlに溶かし、寒剤で冷
却下NMM40mlでPH７とした後、〔３〕47.69ｇ
（108mM）のTHF100ml溶液およびHOBT14.59
ｇ（108mM）のDMF溶液を加える。この溶液に
寒剤で冷却下WSCI19.76ml（108mM）を10分間
で滴下した後、一夜撹拌する。反応液に酢酸エチ
ル500mlを加え、５％重曹水で洗浄する。水層を
酢酸エチル300mlで抽出し、先の酢酸エチル層と
合わせて５％重曹水（１回）、飽和食塩水（１
回）、1N塩酸（２回）、飽和食塩水（２回）、水
（１回）の順に洗浄する。無水芒硝で乾燥し、減
圧濃縮する。残渣にエーテル―ヘキサンを加えて
固化し、集める。酢酸エチル400mlで再結晶して
〔４〕56.98ｇ（収率80.0％）を得る。
融点；141〜143℃
元素分析〔C34H53O8N5として〕
Ｃ％ Ｈ％ Ｎ％
測定値 61.76 8.41 10.84
計算値 61.89 8.10 10.61
５ Ｐ（140―141）；BOC―Thr（Bzl）―Pro―
OBzl〔５〕
BOC―Thr（Bzl）―OH32.88ｇ（0.15M）を
THF100mlに溶かし、これにHOBT20.94ｇ
（0.155M）およびＨ―Pro―OBzl・HCl37.47ｇ
（0.155M）を加え、DMF200mlを加えて溶液とす
る。次いで寒剤で冷却下WSCI28.37ml（0.155M）
を10分間で滴下した後、室温で一夜撹拌する。反
応後、減圧下で溶媒を留去し、残渣に酢酸エチル
500mlを加え、５％重曹水（２回）、飽和食塩水
（１回）、1N塩酸（２回）、飽和食塩水（２回）、
水（１回）の順に洗浄する。無水芒硝で乾燥した
後、減圧濃縮して油状の〔５〕57.41ｇ（収率
77.1）を得る。
TLC；Rf6＝0.85、Rf1＝0.63
６ Ｐ（140―141）；BOC―Thr（Bzl）―Pro―
OH〔６〕
〔５〕52.78ｇ（106.29mM）をメタノール300
mlに溶かし、氷冷下1N―NaOH150ml（1.4倍Ｍ）
を20分間で加えた後、室温で１時間半撹拌する。
反応後、氷冷下1N塩酸43.71ml（0.4倍Ｍ）を加え
てPH７に調節し、減圧下でメタノールを留去す
る。得られた水溶液をエーテル150mlで洗浄し、
水層を氷冷下1N塩酸110mlでPH３に調節する。酢
酸エチル300ml、150mlで２回抽出し、抽出液を食
塩水（２回）、水（１回）の順に洗浄する。酢酸
エチル層を無水芒硝で乾燥後、減圧濃縮する。残
渣にエーテルとヘキサンを加え、生じた沈澱物を
得る。この操作を２回行なつた後、酢酸エチルに
溶かし、減圧乾固して発泡固化した〔６〕32.27
ｇ（収率77.75％）を得る。
融点；50〜55℃
〔α〕24 D；−36.64゜（Ｃ＝１，DMF）
TLC；Rf6＝0.34
元素分析〔C21H30O5N2・H2Oとして〕
Ｃ％ Ｈ％ Ｎ％
測定値 61.12 7.62 6.67
計算値 61.75 7.90 6.86
７ Ｐ（140―145）；BOC―Thr（Bzl）―Pro―Ile
―Leu―Pro―Gln―OBzl〔７〕
〔６〕54.77ｇ（83mM）を塩化メチレン80ml
に加え、これに氷冷下TFA240mlを加え、室温で
25分間撹拌する。反応後、減圧下０℃でTFAを
留去し、残渣にエーテルを加える。生じた沈澱物
を取し、液を減圧濃縮する。濃縮物にエーテ
ルを加え、さらに生じた沈澱物を得、先の沈澱物
と合せてアルカリデシケータ中で一夜減圧乾燥す
る。この沈澱物をDMF200mlに溶かし、これに寒
剤で冷却下NMM（9.5ml）でPH７とした後、
HOBT11.21ｇ（83mM）およびBOC―Thr（Bzl）
―Pro―OH32.27ｇ（83mM）を加え、DMF40ml
を追加して溶液とする。これに寒剤で冷却下
WSCI15.19ml（83mM）を10分間で滴下し、２時
間後、室温で一夜撹拌する。反応後、減圧下で
DMFを留去し、残渣に５％重曹水300mlを加え、
酢酸エチル800mlおよび300mlで２回抽出する。酢
酸エチル層を５％重曹水（２回）、飽和食塩水
（１回）、1N塩酸（２回）、飽和食塩水（２回）、
水（１回）の順に洗浄する。無水芒硝で乾燥した
後、減圧濃縮する。放置により沈澱物が析出した
ら、エーテルを加えて沈澱物を分離する。酢酸エ
チルから再結晶化し、エーテルを加えて取して
〔７〕63.98ｇ（収率81.3％）を得る。
融点；101〜103℃
〔α〕24 D；−61.78゜（Ｃ＝１，DMF）
元素分析〔C50H72O11N7・H2Oとして〕
Ｃ％ Ｈ％ Ｎ％
測定値 62.21 7.89 10.03
計算値 62.16 7.82 10.15
アミノ酸分析〔1.330mg／塩酸0.3ml、酢酸0.3
ml、アニソール0.1ml、105℃、24時間〕；
Thr0.84(1)、Pro2、Ile0.97(1)、Leu1.00(1)、
Gln0.98(1)
８ Ｐ（139―145）；BOC―Asp（Bzl）―Thr
（Bzl）―Pro―Ile―Leu―Pro―Gln―OBzl
〔８〕
〔７〕63.98ｇ（67.48mM）に塩化メチレン150
mlを加え、これに氷冷下TFA300mlを加えた後、
室温で30分間撹拌する。反応後、減圧下０℃で
TFAを留去し、残渣にエーテルを加える。生じ
た沈澱物を取し、母液より生じた二次結晶と合
せてアルカリデシケーター中で一夜減圧乾燥す
る。収量67.19ｇ。この沈澱物をDMF350mlに溶
かし、これに寒剤で冷却下NMM7.42mlでPH７に
調節した後、BOC―Asp（OBzl）―OH29.77ｇ
（87.72mM）およびHOBT11.85ｇ（87.72mM）
を加える。次いで寒剤で冷却下WSCI16.05ml
（87.72mM）を滴下し、２時間後、室温に戻し一
夜撹拌する。反応後、減圧下で溶媒を留去し、残
渣に水を加え、生じた沈澱物を氷冷下デカンテー
シヨンにより水層と分離する。沈澱物を酢酸エチ
ル1.5に溶かし、５％重曹水（２回）、飽和食塩
水（１回）、1N塩酸（１回）、飽和食塩水（２
回）、水（１回）の順に洗浄する。無水芒硝で乾
燥後、減圧濃縮する。濃縮液を放置して、生じた
沈澱物を取する。酢酸エチル―エーテル２回再
結晶を行い、〔８〕73.78ｇ（収率94.8％）を得
る。
融点；99〜103℃
〔α〕24 D；−82.44゜（Ｃ＝１，DMF）
TLC；Rf3＝0.62、Rf2＝0.79
元素分析〔C6H84O14N8として〕
Ｃ％ Ｈ％ Ｎ％
測定値 63.38 7.56 9.80
計算値 63.52 7.34 9.72
９ Ｐ（138―145）；BOC―Ser（Bzl）―Asp
（OBzl）―Thr（Bzl）―Pro―Ile―Leu―Pro
―Gln―OBzl
The present invention relates to a novel method for measuring human chorionic gonadotropin (hCG). hCG is a glycoprotein and a sex hormone that is 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, gonadotropic hormones include luteinizing hormone (LH), follicle stimulating hormone (FSH), and CG, which have similar molecular structures and are glycoproteins, and there are two types, α chain and β chain. In particular, in the α chain, each α
They are so similar in molecular structure that there is no change in activity even if the chains are exchanged. Although the β chain is more specific to each gonadotropin, the LH β chain and the CG β chain are similar. Among these gonadotropins, especially
Regarding the measurement of hCG, radioimmunoassay,
Various highly reliable measurement systems with little cross-reactivity with LH have been studied using enzyme immunoassay, hemagglutination inhibition reaction, hemagglutination reaction, latex agglutination reaction, etc. In particular, in the measurement system for hCG in enzyme immunoassay, the sensitivity of the measurement system is also unsatisfactory depending on the hCG or its fragment used as the enzyme label for measurement. Surprisingly, the present inventor discovered that, in this hCG enzyme immunoassay, desialylated hCG (ds-hCG), which is formed by removing sialic acid bound to the hCG molecule, and enzyme It has been found that by using a conjugate with the enzyme as its enzyme label, measurement can be performed with extremely high sensitivity. The present invention has been completed based on the above findings, and is a method for measuring hCG, which is characterized in that a ds-hCG-enzyme conjugate is used as an enzyme label in an enzyme antibody assay for hCG. First, the enzyme immunoassay method used in the present invention involves competitively reacting the ds-hCG-enzyme conjugate with the test solution against the antibody, and then immunizing the ds-hCG-enzyme conjugate with the antibody. A competitive reaction-based method involves separating the conjugate from the unreacted ds-hCG-enzyme conjugate, and then measuring the enzymatic activity of either or both of them to measure hCG in the test solution. This is exemplified as a simple method. Furthermore, the method based on this competitive reaction preferably uses an antibody against the antibody during separation of the immunoconjugate of the ds-hCG-enzyme conjugate and the antibody from the unreacted ds-hCG-enzyme conjugate. This is carried out by a method using a so-called second antibody or a method using the antibody as an insolubilized antibody. First, the ds-hCG used to obtain the ds-hCG-enzyme conjugate of the present invention is obtained by chemically hydrolyzing the sialic acid molecule within the hCG molecule with a weak acid, etc.
Sialic acid may be released from hCG molecules by enzymatic hydrolysis. For example, containing hCG
Neuraminidase (manufactured by Sigma) was added to 0.05M acetate buffer and reacted at 37°C for more than 1 hour to hydrolyze it, followed by overnight dialysis and purification by chromatography to obtain the desired ds- What is necessary is to collect the hCG fraction. More ds
-Enzymes used to obtain the hCG-enzyme conjugate include oxidoreductases, hydrolases, transposases,
Lyase, isomerase, and ligase are used as appropriate, such as lactate dehydrogenase, maleate dehydrogenase, malate dehydrogenase, maltose dehydrogenase, peroxidase, lactose oxidase, maleate oxidase, glucose oxidase, choline oxidase, xanthine oxidase, amino acid oxidase, Examples include cosine oxidase, catalase, α-amylase, β-galactosidase, lysozyme, lipase, alkaline phosphatase, aminopeptidase, trypsin, papain, α-chymotrypsin, amidase, hexoquinada, and glycerokinase. Furthermore, these enzymes and ds-hCG may be preliminarily introduced with an arbitrary spacer, such as dialdehydes such as succinic aldehyde, glutaraldehyde, and adipaldehyde, and reactions such as acid chlorides and succinimide esters of ω-amino acids. reactive derivatives of dicarboxylic acids, diamines such as hexamethylene diamine and decamethylene diamine, S-acetyl mercaptosuccinic
New aldehyde groups, amino groups,
A thiol group or carboxyl group may be introduced as a spacer. Next, such ds-hCG and enzyme are bonded, and ds-hCG, the amino group, hydroxyl group, thiol group, carboxyl group, etc. of the enzyme, and the introduced aldehyde group, amino group, thiol group, carboxyl group, etc. The two may be combined based on a group or the like. Regarding the connection,
Both may be bonded directly or indirectly using a binder. To directly bond the two, for example, the intramolecular amino group of ds-hCG and the intramolecular carboxyl group of the enzyme may be reacted in the presence of water-soluble carbodiimide. In addition, when reacting using a binder, various known polyfunctional compounds such as hexamethylene diisocyanate,
Diisocyanate compounds such as 2,4-toluene diisocyanate, diisothiocyanates such as hexamethylene diisothiocyanate, dialdehyde, N,N'-ethylene bismaleimide, N,
N'-o-phenylene dimaleimide, metamaleimidobenzoyl N-oxysuccinimide ester, bisdiazobenzidine, N,N'-polymethylene biiodoacetamide, diethylmalonimidate, dimethyladipineimidate, 3-
(2′-benzothiazolyl-dithio)propionic acid,
Sulfide carboxylic acids such as 3-(2'-pyridyl-N-oxide-dithio)propionic acid, 6-N[3-(2'-benzothiazolyl-dithio)propionyl]caproic acid or its succinimide ester, p-nitro Reactive derivatives such as phenyl ester, acid chloride and imide, maleimidocarboxylic acids such as maleimidobenzoic acid, maleimidophenyl acetic acid, maleimidophenylpropionic acid or reactive derivatives thereof, etc. may be used. Furthermore, in order to obtain the ds-hCG-enzyme conjugate, it is usually carried out in a buffer solution with a pH of 6 to 8 in the presence of an organic solvent such as methanol, ethanol, acetone, dioxane, tetrahydrofuran, dimethylacetamide, dimethylformamide, or an organic solvent. in solvent,
For example, ds-hCG and a polyfunctional compound are reacted at 0 to 40°C, and then an enzyme is added for further reaction. After the reaction, it may be purified as necessary.
Preferably, chromatography or gel filtration means may be used. Furthermore, in order to obtain a ds-hCG-enzyme conjugate, one or two intervening substances may be used during binding.
The two may be combined via a species or more. i.e. ds
- hCG - [Intervening substance] - It may be obtained as an enzyme conjugate. This intervening substance is ds-
The molecular weight may be larger than that of hCG and smaller than the molecular weight of the enzyme used, usually about 5 to 9 times the molecular weight of ds-hCG, and preferably shows specific adsorption in affinity chromatography. The purpose of using this intervening substance is firstly ds-
hCG and the intervening substance are combined with the above-mentioned polyfunctional compound to form a ds-hCG-intervening substance conjugate. After passing through a molecular sieve, it is further purified by affinity chromatography by specific adsorption of an intervening substance, and if necessary, another intervening substance is bound and purified in the same manner, and then the target enzyme is bound. The target substance is purified based on the molecular weight and specific adsorption properties of intervening substances.
A ds-hCG-[intervening substance]-enzyme conjugate with good binding properties can be obtained. In particular, when the molecular weight of the enzyme used for ds-hCG is extremely large, for example, in the case of β-galactosidase with a molecular weight of approximately 500,000,
While it is extremely difficult to separate the ds-hCG-β-galactosidase conjugate from unreacted β-galactosidase, if peroxidase, which exhibits specific adsorption to concanavalin A, is used as an intervening substance, for example, ds- To obtain the hCG-peroxidase conjugate, it can be purified by gel filtration and affinity chromatography using concanavalin A, and further used to obtain the desired ds
-hCG-[peroxidase]-Enzyme conjugate can be purified in a similar manner to obtain the enzyme conjugate. In addition, in obtaining the antibody used in the present invention, preferably the β chain of hCG or its C-terminal fragment, for example, the β chain of hCG shown in the reference examples below.
C-terminal fragment of 127-145 (hCG[127-145]
), hCG [118-145], and various C-terminal fragments of β-chains are used. In particular, for hCG [127-145] and hCG [118-145], the following formula [] (However, in the formula, R is H, or This is a peptide represented by (indicating a group). First, this β chain or its C-terminal fragment is extracted from high molecular weight proteins such as bovine serum albumin (BSA) and rabbit serum albumin (RSA), or by treating them with alkali or with Zodium lauryl sulfate and mercaptoethanol to obtain an intramolecular disulfide group. The cleaved product can be made into a conjugate in which about 5 to 30 molecules are bound using the above-mentioned polyfunctional compound per molecule of high-molecular protein, or dialdehyde such as water-soluble carbodiimide or glutaraldehyde can be used. to form a polymer of the β chain or its C-terminal fragment.
This is then applied to various mammals, such as rabbits, rats, etc., by emulsifying the conjugate of the β chain or its C-terminal fragment with a high-molecular protein, or the polymer of the β chain or its C-terminal fragment in Freund's complete adjuvant. It is sufficient to sensitize a suitably selected mammal such as a guinea pig or a mouse by subcutaneously injecting it several times at intervals of 1 to 2 weeks, or by administering a large amount once, or at an appropriate time after sensitization. Blood may be collected from the mammal, its antibody titer may be measured, and the blood may be collected from the animal at a time when antibody production is good, and the antiserum may be obtained by centrifugation or the like in a conventional manner. Furthermore, this antiserum contains the target antibody at a sufficiently high concentration, and may be stored as appropriate, or may be diluted as necessary before use. Furthermore, this antiserum may be obtained by conventional antibody purification methods such as salting out, isoelectric precipitation, dialysis, chromatography, and gel filtration. Furthermore, this purified antibody is combined with an insoluble carrier to form an insolubilized antibody, thereby being utilized in a method based on a competitive reaction using an insolubilized antibody.
First, the insoluble carriers used for this insoluble antibody include, for example, insoluble protein carriers such as albumin or gelatin treated with glutaraldehyde, agarose, epichlorohydrin treatment or cyanogen bromide treatment such as cellulose or dextrin, and the like. An insoluble semi-synthetic polymer carrier treated with an amination reagent for the introduction of amino groups, acrylonitrile, acrylic acid, acrylic ester, methacrylic acid, methacrylic ester, vinyl alcohol, vinyl acetate, styrene, aminostyrene, Polymers or copolymers of divinylbenzene, acrylamide, ethylene, maleic anhydride, crotonic acid, etc., as well as insoluble synthetic polymer carriers prepared by treating these with an aminating reagent, and other insoluble inorganic carriers such as silane compounds treated with an aminating reagent. etc. In addition, treatments with amination reagents for introducing amino groups into these insoluble carriers include, for example, reagents for amination by reduction of nitrile groups, and introduction of aminoalkyl groups by reaction of hydroxyl groups with γ-aminopropyltriethoxysilane. A reagent for introducing an amino group, which is obtained by converting a hydroxymethyl group into an aldehyde group by periodic acid oxidation and then reacting this aldehyde group with a diamine, is used as appropriate. New functional groups may be introduced into these functional groups by conversion into an imidocarbonate group by a cyanide reaction, conversion of an amide group into an iminohalogenide group by a halogenated phosphorus compound, or by various known methods. In addition, in order to obtain an insolubilized antibody by binding such an insoluble carrier and the antibody, usually in an aqueous medium such as a buffer solution, based on the functional group of the antibody and the functional group of the insoluble carrier, The reaction may be carried out at 0 to 40°C using an appropriately selected polyfunctional compound. Furthermore, when implementing a method based on a competitive reaction using a second antibody, the immunoglobulin of the mammalian species used to obtain the antibody may be administered to a different species of mammal to sensitize it. Administration and sensitization to this different species of mammal may be carried out in the same manner as the method for obtaining antibodies described above, and further, after sensitization,
Blood may be collected, and antiserum and, if necessary, antibodies may be collected, which are used as the second antibody of the present invention. Next, to illustrate when carrying out the present invention,
For example, appropriately diluted antibody-containing solution, usually antiserum
200μ, test solution 50μ, PH7.2-7.4 buffer, usually 0.01M phosphate buffer (PH7.2) 100μ, and
50μ of a solution containing a ds-hCG-enzyme conjugate, such as a ds-hCG-β-galactosidase conjugate, was incubated overnight at 4°C, then 100μ of a solution containing an immunoglobulin of the mammalian species from which the antiserum was obtained and a second Add 100μ of antibody-containing solution, incubate overnight at 4°C, and then centrifuge. Next, the supernatant and the precipitate are separated and added to the medium for measuring the enzyme activity, and the enzyme activity of the supernatant or the precipitate is measured. For example, if the enzyme in the ds-hCG-enzyme conjugate is β-galactosidase, use a substrate solution of about 0.1% o-nitrophenol-β-D-galactopyranoside at 37°C.
The reaction may be stopped for 15 minutes to overnight, and then the absorbance of the color produced by the reaction may be measured at a wavelength of 420 nm. In addition, when using insolubilized antibodies, for example, 50μ of test solution, 30μ of buffer
, 1-2 beads of insolubilized antibody, ds-hCG
- Incubate 50μ of the enzyme conjugate-containing solution simultaneously or sequentially, then separate the beads and the liquid layer, and then measure the enzyme activity of either or both of them. Furthermore, the insolubilized antibodies used in this case are not limited to beads, but may be insolubilized with the surface of the test tube wall used for the immune reaction, and can be used in various shapes as appropriate. It is something. Furthermore, when measuring enzyme activity, it is sufficient to measure the activity by a known enzyme activity measuring method depending on the enzyme used, and usually involves ultraviolet absorption, fluorescence reaction, color reaction, luminescence reaction,
It is measured based on electrical reactions such as oxygen electrodes and hydrogen peroxide electrodes. Next, the present invention will be specifically described with reference to Examples and Reference Examples, but the present invention is not limited thereto. Example (1) Regarding antibody production (a) Add 0.25 ml of an aqueous solution containing 3 mg of hCG (127-145) to
0.25 ml of a 75 mg water-soluble carbodiimide (WSCD) aqueous solution was added. Then, after adjusting the pH to 5.5, the reaction was carried out at room temperature for 20 hours. After the reaction, this was charged to a Sephadex G-150 column to obtain a polymer fraction of hCG (127-145), which was freeze-dried. (b) Instead of hCG (127-145) above, hCG (118-
Using 3 mg of 145), a polymer of hCG (118-145) was obtained in the same manner as above. (c) 0.1M phosphate buffer containing 2mg of BSA (PH7.0)
0.2 ml of 2.5% glutaraldehyde solution was added to the mixture and reacted at room temperature for 18 hours. After the reaction, the effluent fraction was obtained by charging it to a Sephadex G-25 column, and 1 mg of hCG (118-145) was added to this.
Add 1 ml of physiological saline, add 0.1 ml of 1M sodium carbonate aqueous solution (PH9.5), and heat at 4°C.
After the reaction, charge it to a column of Sephadex G-100 to collect BSA-hCG.
A fraction of the (118−145) conjugate was obtained. (d) Add a 2.50μ aqueous solution containing 2.5mg of RSA to a 250μ aqueous solution containing 0.7mg of hCG (118-145), and add 75mg of water-soluble carbodiimide (WSCD) to this.
Add 500μ aqueous solution and adjust its pH to 5 to 5.5.
After adjusting the reaction mixture, the mixture was allowed to react at room temperature for 20 hours, and then charged to a Sephadex G-100 column to obtain a fraction of the RSA-hCG (118-145) conjugate. (e) β of hCG instead of hCG (118-145) in (c) above
Using 5 mg of the chain, BSA-hCGβ was prepared in the same manner.
A chain conjugate was obtained. (F) Polymer of hCG (127-145), polymer of hCG (118-145), BSA-
hCG(118-145) conjugate, RSA-hCG(118-
145) Using the conjugate, BSA-hCG β chain conjugate,
This is mixed with an equal amount of Freund's Complete Adjuvant to form an emulsion.
Rabbits were sensitized by injecting 0.5 ml subcutaneously into their backs, and then their blood was collected and centrifuged to obtain the antiserum. In addition, in sensitization using hCG (127-145) polymer, 320 U/ml was shown 4 weeks after sensitization. Furthermore, when hCG (118-145) polymer was used, it showed 320 U/ml 5 weeks after sensitization. moreover
When BSA-hCG (118-145) conjugate was used, the concentration was 40 U/ml 5 weeks after sensitization and 80 U/ml 7 weeks after sensitization. In addition, when using BSA-hCG β chain conjugate, 320U/ml at 5 weeks after sensitization and 1280U/ml at 6 weeks after sensitization.
It was hot in ml. (2) Preparation of ds-hCG 0.05M acetate buffer (PH4.6; 2mM CaCl 2 and 0.2mM
6 mg of Nyulaminidase (manufactured by Sigma) was added to 0.75 ml (containing EDTA) and reacted at 37°C for 2 hours. After the reaction, the mixture was dialyzed overnight at 37°C against the same buffer as above. This was then subjected to column chromatography using DEAE-cellulose.
A ds-hCG fraction (yield 4.7 mg) was obtained. (3) Regarding ds-hCG-β-galactosidase conjugate, etc. (a) 0.5% in 0.25ml of 0.05M phosphate buffer (PH7.0)
mg of ds-hCG was added, and to this was added 20μ of a solution containing approximately equimolar amounts of metamaleimide benzoyl N-oxysuccinimide ester dissolved in anhydrous tetrahydrofuran, and the mixture was heated at 30°C.
After reacting for 30 minutes, 0.25 ml of 1M citrate buffer was added to it, and Sephadex G-25 was added.
The unreacted reagent was removed by charging the column. Then, 1M phosphate buffer (PH7.0) containing 1mg of β-galactosidase (0.5%) was added to the outflow section.
ml and allowed to react at room temperature for 2 hours, followed by adding 50μ of 10 -5 M mercaptoethanol.
The reaction was stopped by adding the solution. This was then subjected to affinity chromatography using Concanavalin A-Sepharose 4B.
Further, gel filtration was performed on an Ultrogel AcA44 column to obtain a ds-hCG-β-galactosidase conjugate fraction. (b) Using hCG in place of the above ds-hCG, the same procedure was repeated to obtain an hCG-β-galactosidase conjugate. (c) Using hCG β chain instead of the above ds-hCG, the same procedure was repeated to obtain an hCG β chain-β-galactosidase conjugate. (d) 0.1M phosphate buffer (PH
8.0) 0.5ml of 5mM EDTA and 3-(2′-
Add 0.1 ml of a dimethylacetamide solution containing approximately equimolar amounts of benzothiazolyl-dithio)propionic acid succinimide ester to
The reaction was carried out at ℃ for 60 minutes. After reaction, reaction solution
6μ was taken out, 500μ of 0.01M phosphate buffer (PH7.2) containing 0.15M NaCl and 1.3mg of β-galactosidase were added, and the mixture was reacted at 0°C for 30 minutes. After the reaction, this is passed through a gel and ds-hCG-
A β-galactosidase conjugate was obtained. (4) Measurement of hCG (a) Using the antiserum obtained using the BSA-hCG β chain conjugate described above, various concentrations of hCG (100 to
Differences between various enzyme labels in the enzyme immunoassay for 16,000 mIU/ml) were determined. In addition, as an enzyme label, ds-hCG-
β-galactosidase conjugate [obtained by the method described in Section 3 (A); cases indicated by ○-○ in Figure 1], hCG-β-galactosidase conjugate [obtained by the method described in Section 3 (A); ); cases indicated by △-△ in Figure 1], hCG β chain-β-galactosidase conjugate [obtained by the method described in Section 3 (c); The measurement results are as shown in FIG. 1. (b) Using the antiserum obtained using the hCG (127-145) polymer described above, various concentrations of hCG (200 to
Differences between various enzyme labels in the enzyme immunoassay for 32,000 mIU/ml) were determined. In addition, as an enzyme label, ds-hCG-
β-galactosidase conjugate [obtained by the method described in Section 3 (A); cases indicated by ○-○ in Figure 2], hCG-β-galactosidase conjugate [obtained by the method described in Section 3 (A); ); the cases indicated by △...△ in Figure 2] were used, and the measurement results are as shown in Figure 2. As a result, hCG (127−145) In the case of antibodies or antisera using C-terminal fragments of hCG, such as polymers, ds-hCG
It is clear that the binding property with the enzyme label is good. The measurement method used for this enzyme immunoassay is as follows. First, 200μ of antiserum, 50μ of hCG test solution with various concentrations, and 100μ of 0.01M phosphate buffer (PH7.2)
50μ of the solution containing the enzyme label was incubated overnight at 4°C, and then 100μ of a diluted rabbit normal serum and 100μ of the second antibody-containing solution (antiserum containing an antibody against rabbit immunoglobulin) were added. , and incubated overnight at 4°C. This was then centrifuged to collect the precipitate, which was added to 500μ of 0.1% o-nitrophenol-β-D-galactoside in 0.1M phosphate buffer (PH7.2) and incubated at 37°C. After incubating overnight, the reaction was stopped, and the color development was measured by absorbance at a wavelength of 420 nm. As described above, the ds-hCG-enzyme conjugate of the present invention has good binding properties even to antibodies obtained using hCGβ chain, and particularly to antibodies obtained using C-terminal fragment of hCGβ chain. It showed extremely good bonding properties. (5) Regarding the calibration curve Using the antiserum (8000-fold diluted) obtained using the BSA-hCG β chain conjugate described above, a calibration curve of hCG using the ds-hCG-β-galactosidase conjugate was determined. The results were as shown in FIG. In order to obtain the peptide represented by the formula [] used in the present invention, individual amino acids or lower peptides are condensed in the amino acid order represented by the formula [], and in the final step of the condensation reaction, It is obtained by removing the protective group from the side chain functional group. 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, protective groups used for amino groups include formyl group, trifluoroacetyl group, phthaloyl group, benzenesulfonyl group, p-toluenesulfonyl group, o-nitrophenylsulfenyl group, 2,4-dinitrophenylsulfenyl group. acyl groups such as enyl group, 2,4-dinitrophenylsulfenyl group, benzyl group, diphenylmethyl group, and triphenylmethyl group (these groups may also include lower groups such as o-methoxy group and p-methoxy group). aralkyl groups such as (substituted by alkoxy groups), benzyloxycarbonyl groups,
o-bromobenzyloxycarbonyl group, p-bromobenzyloxycarbonyl group, o-chlorobenzyloxycarbonyl group, p-chlorobenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, p- Phenylazo-benzyloxycarbonyl group, p-(p'-methoxyphenylazo)
-benzyloxycarbonyl group such as benzyloxycarbonyl group, cyclopentyloxycarbonyl group, trichloroethyloxycarbonyl group,
Aliphatic oxycarbonyl groups such as t-amyloxycarbonyl group, t-butoxycarbonyl group, diisopropylmethoxycarbonyl group, 2-phenyl-isopropoxycarbonyl group, 2-tolyl-
Examples include aralkyloxycarbonyl groups such as isopropoxycarbonyl group and 2-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 serine, threonine and tyrosine 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. For protection of these hydroxyl groups, 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 groups 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. Examples of the group used to protect the imino group of histidine include benzyl group, trityl group, benzyloxycarbonyl group, tosyl group, adamantyloxycarbonyl group, and 2,2,2-trifluoro-1-t-butyl group. Oxycarbonylaminoethyl group, 2,2,2-trifluoro-1-
This imino group may be a benzyloxycarbonylaminoethyl group, but it is not necessary to protect this imino group. Groups used to protect the mercapto group of cysteine include benzyl group, p-methoxybenzyl group, p-nitrobenzyl group, trityl group, benzylthiomethyl group, ethylcarbamoyl group, acetamidomethyl group, and the like. In the synthesis of the present target compound [ ], condensation of individual amino acids or lower peptides is carried out, for example, with an amino acid or peptide having a protected α-amino group and an activated terminal carboxyl group, and a free α-amino group and a protected terminal carboxyl group. 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. In this case, the carboxyl group is an azide, an acid anhydride, an acid imidazolide or an active ester, such as cyanomethyl ester, thiophenyl ester,
p-nitrothiophenyl ester, p-methanesulfonylphenyl ester, thiodyl ester,
p-nitrophenyl ester, 2,4-dinitrophenyl ester, 2,4,5-trichlorophenyl ester, 2,4,6-trichlorophenyl ester, pentachlorophenyl ester, N-
or by converting into hydroxysuccinimide ester, N-hydroxaphthalic imide ester, 8-hydroxyquinoline ester or N-hydroxypiperidine ester, or carbodiimide, N,N'-carbonyl-diimidazole or isoxolium salt. , for example, by reacting with a Woodward reactant or the like. Preferred condensation methods in the present invention are the carbodiimide method, azide method, active ester method and anhydride method. In each step of the condensation, it is desirable to use a method that does not cause racemization or a method that minimizes racemization, preferably the azide method, active ester method, or Wu¨nsch method [Z.Naturforsch., 21b ,
426 (1966)] or the Geiger method [Chem.Ber., 103 ,
788 (1970)], especially N-ethyl as a condensing agent.
A modified method using N'-3-dimethylaminopropyl-carbodiimide (WSCI) is used. As long as the condensation order is the amino acid order shown by the formula [], the synthesis can be performed in any order, but it is advantageous to synthesize from the C-terminal side. The protected hCG [127-145] consists of a C-terminal fragment 132-145 and an N-terminal fragment 127-
Preferably, 131 is condensed by a modified Geiger method using WSCI. C-terminal fragment 132
-145 is fragment 132-137 and fragment 138
-144 is preferably condensed by a modified Geiger method using WSCI. N-terminal fragment
127-131 are fragments 127-129 and fragments
Preferably, 130-131 is condensed by a modified Geiger method using WSCI. Protected hCG[118-145] is the C-terminal fragment 127-145, i.e. protected hCG[127-
145] and N-terminal fragments 118-126 by WSCI
Preferably, the condensation is carried out by a modified Geiger method using . C-terminal fragments 118-126 are preferably obtained by condensing fragments 118-121 and 122-126 by the azide method. The protected hCG[105-145] consists of a C-terminal fragment 112-145 and an N-terminal fragment 105-
Preferably, 111 is condensed by a modified Geiger method using WSCI. C-terminal fragment 112
-145 is protected hCG [118-145] with fragments 116-117, fragments 113-115 and 112
It is preferable to sequentially condense and connect the th amino acid by an active ester method. N-terminal fragments 105-111 are preferably obtained by condensing fragments 110-111 and 105-109 by the azide method. The protected hCG [100-145] consists of a C-terminal fragment 112-145 and an N-terminal fragment 100-
Preferably, 111 is condensed by the active ester method. N-terminal fragments 100-111 are preferably obtained by condensing fragments 100-104 and 105-111 by the azide method. The protected [Tyr 100 ]-hCG[100-145] is C
-Terminal fragments 112-145 and N-terminal fragments 100-111 are preferably condensed by an active ester method. N-terminal fragments 100-111 are preferably obtained by condensing fragments 100-104 and 105-111 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 or converted to a hydratide, and a benzyl ester group can be cleaved with anhydrous hydrogen fluoride or hydrogenolysis. The α-amino groups of these peptides can be protected with conventional protecting groups such as benzyloxycarbonyl, t-butoxycarbonyl, and t-amyloxycarbonyl groups, but the benzyloxycarbonyl group can be protected by hydrogenolysis. and was separated,
The t-butoxycarbonyl group and t-amyloxycarbonyl group are removed with trifluoroacetic acid. The hydroxyl group of serine and threonine is a benzyl group, the hydroxyl group of tyrosine is a 2,6-dichlorobenzyl group, the ε-amino group of lysine is an o-chlorobenzyloxycarbonyl group, and the amino group in the guanidino group of arginine is a tosyl group. Therefore, it is suitable to protect the mercapto group of cysteine with a p-methoxybenzyl group. These protecting groups are removed with anhydrous hydrogen fluoride. An acetamidomethyl group can be used as a protecting group for the mercapto group of cysteine. This group is stable to anhydrous hydrogen fluoride, so once all other protecting groups have been removed, PH4
It is desorbed with mercury acetate. Thus protected hCG [127-145], protected hCG [118-145], protected hCG [105-145],
Protected hCG [100-145] and protected [Tyr 100 ]-hCG [100-145] are obtained. These protecting groups are preferably removed in one step by acid decomposition, for example by treatment with anhydrous hydrogen fluoride, to yield the target compound of formula []. When an acetamidomethyl group is used as a protecting group for the mercapto group of cysteine 110 and/or 100, mercuric acetate is added at PH4 after removal of all other protecting groups by anhydrous hydrogen fluoride treatment. It may be desorbed by 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, Dowex 1, or carboxymethyl cellulose. 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. Next, examples of synthesis of the peptide [] used in the present invention will be given, but this is not intended to limit the present invention in any way. In addition, each symbol described in this specification has the following meaning. Gln; L-glutamine Pro; L-proline Leu; L-leucine Ile; L-isoleucine Thr; L-threonine Asp; L-aspartic acid Ser; L-serine Gly; glycine Arg; L-arginine Ala; L-alanine Lys ; L-lysine Phe; L-phenylalanine Cys; L-cysteine His; L-histidine Tyr; L-tyrosine BOC; t-butyloxycarbonyl AOC; t-amyloxycarbonyl Z; benzyloxycarbonyl Z-Cl; O -chlorobenzyloxycarbonyl Bzl; benzyl MBzl; p-methoxybenzyl Bzl-Cl 2 ; 2,6-dichlorobenzyl Acm; acetamidomethyl Tos; tosyl OMe; methyl ester OEt; ethyl ester OBzl; benzyl ester OSU; N-hydroxysucci Acid imide ester ONP; p-nitrophenyl ester TFA; trifluoroacetic acid TEA; triethylamine TBA; tribenzylamine DCHA; dicyclohexylamine NMM; N'-methylmorpholine THF; tetrahydrofuran DMF; dimethylformamide WSCI; N-ethyl, N' -3-dimethylaminopropyl-carbodiimide HOBT; 1-hydroxybenzotriazole In addition, the carrier and solvent system for thin layer chromatography (TLC) used in this specification are as follows. Support: Silica gel G Solvent system: 1 Chloroform-methanol-acetic acid (95:5:
3) 2 Chloroform-methanol-acetic acid (85:15:
5) 3 Chloroform-methanol-acetic acid (85:10:
5) 4 Chloroform-methanol-acetic acid (80:25:
2) 5 Chloroform-methanol-acetic acid (24:6:
1) 6 Chloroform-ethanol-ethyl acetate (5:2:5) 7 Ethyl acetate 8 Ethyl acetate-methanol (10:1) 9 Ethyl acetate-benzene (1:1) 10 Butanol-acetic acid-water (3:1: 1) Carrier: Cellulose manufactured by Merck & Co., Ltd. Solvent system: 11 Butanol-pyridine-acetic acid-water (15:10:
3:12) Reference example 1 hCG [127-145]; H-Ser-Leu-Pro-Ser-
Pro―Ser―Arg―Leu―Pro―Gly―Pro―Ser
―Asp―Thr―Pro―Ile―Leu―Pro―Gln―
OH 1 P (143-144); BOC-Leu-Pro-OBzl [1] BOC-Leu-OH.H 2 O149.59g (0.6M)
Add 300ml of THF and add 81.06g of HOBT
(0.6M), THF150ml, and DMF100ml to make a solution. Next, H-Pro-OBzl152.28g
(0.63M) and cooled to -10℃ WSCI120.8ml
After dropping (0.66M), add 100ml of DMF,
Stir overnight at room temperature. After the reaction, the solvent is distilled off under reduced pressure, and 600 ml of ethyl acetate is added to the residue, which is washed successively with 5% sodium bicarbonate solution (3 times), 1N hydrochloric acid (2 times), and water (2 times). The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Dissolve the oil in benzene 1 and concentrate under reduced pressure to obtain oil [1]. TLC; Rf 1 = 0.89 2 P (142-144); BOC-Ile-Leu-Pro-
Dissolve 192mM of OBzl [2] [1] in 100ml of methylene chloride,
Add 300 ml of TFA to this and stir at room temperature for 30 minutes. TFA was distilled off under reduced pressure, hexane was added to the residue, and the mixture was distilled off under reduced pressure. Dissolve the residual oil in 200 ml of THF, neutralize it to pH 7 with 56.1 ml of NMM under ice cooling, and add 38.33 g of BOC-Ile-OH・H 2 O.
(159.5mM) and HOBT21.55g (159.5mM)
Add. Next, add 100 ml of DMF to dissolve, and add 34.1 ml of WSCI dropwise while cooling on ice. After addition, stir overnight at room temperature. After the reaction, the solvent was distilled off under reduced pressure, 500 ml of ethyl acetate was added to the residue, and 5% aqueous sodium bicarbonate (3 times), saturated brine (1 time), 1N hydrochloric acid (2 times), and saturated brine (2 times) were added.
Wash with water (once) and water (once), dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Hexane is added to the residue and the precipitate is collected by decantation. Recrystallize from ethyl acetate-hexane. Since it melts during the process, part of it is filtered, and the rest is dissolved in benzene and freeze-dried to obtain 76.98 g (yield: 90.9%) of [2]. Melting point; 60-64℃ [α] 24 D ; -66.34゜ (C = 1, DMF) TLC; Rf 1 = 0.63 3 P (142-144); BOC-Ile-Leu-Pro-OH
[3] [2] 76.98g (145mM) of n-butanol 20
ml, add 300ml of ethanol to dissolve, and add 5
Add 15g of %Pd/C and hydrogenate for 3 hours. The catalyst is removed and the mother liquor is concentrated under reduced pressure. Add 300ml of diethyl ether (ether) to the residue and add 5% sodium bicarbonate solution.
Extract once each with 500ml and 200ml. When the extract is cooled with ice and adjusted to pH 4 with 1N hydrochloric acid, the product becomes lumpy and precipitates. Extract with ethyl acetate and wash the ethyl acetate layer twice with water. After drying with anhydrous sodium sulfate, concentrate under reduced pressure. The residue is solidified with ether-hexane. Recrystallization from ethyl acetate-ether-hexane gives 60.32 g (yield 94.2%) of [3]. Melting point: 114-119℃ [α] 24 D : -61.98゜ (C=1, DMF) TLC: Rf 1 = 0.53, Rf 6 = 0.47 Elemental analysis [as C 22 H 39 O 6 N 3 ] C% H% N% Measured value 59.82 9.42 9.56 Calculated value 59.84 8.90 39.52 4 P (142-145); BOC-Ile-Leu-Pro-Gln
-OBzl [4] Dissolve 36.41g (108mM) of BOC-Gln-OBzl in 100ml of methylene chloride, add 150ml of TFA to this,
Stir for 30 minutes at room temperature. After reaction, TFA under reduced pressure
was distilled off, the residue was dissolved in 100 ml of THF, and the pH was adjusted to 7 with 40 ml of NMM while cooling with a cryogen. [3] 47.69 g
(108mM) in THF100ml solution and HOBT14.59
g (108 mM) in DMF solution. To this solution, 19.76 ml (108 mM) of WSCI was added dropwise over 10 minutes while cooling with a cryogen, and the mixture was stirred overnight. Add 500 ml of ethyl acetate to the reaction solution and wash with 5% sodium bicarbonate solution. The aqueous layer was extracted with 300 ml of ethyl acetate, and combined with the previous ethyl acetate layer, 5% sodium bicarbonate solution (1 time) and saturated brine (1 time) were extracted.
Wash with 1N hydrochloric acid (twice), saturated saline (twice), and water (once). Dry with anhydrous sodium sulfate and concentrate under reduced pressure. Add ether-hexane to the residue to solidify and collect. Recrystallize with 400 ml of ethyl acetate to obtain 56.98 g (yield: 80.0%) of [4]. Melting point: 141-143℃ Elemental analysis [as C 34 H 53 O 8 N 5 ] C% H% N% Measured value 61.76 8.41 10.84 Calculated value 61.89 8.10 10.61 5 P(140-141); BOC-Thr(Bzl)- Pro―
OBzl [5] BOC―Thr(Bzl)―OH32.88g (0.15M)
Dissolve in 100ml of THF and add 20.94g of HOBT
(0.155M) and H-Pro-OBzl・HCl37.47g
(0.155M) and 200ml of DMF to make a solution. Then cooled with cryogen WSCI28.37ml (0.155M)
was added dropwise over 10 minutes, and then stirred at room temperature overnight. After the reaction, the solvent was distilled off under reduced pressure and ethyl acetate was added to the residue.
Add 500ml, 5% sodium bicarbonate solution (twice), saturated saline (once), 1N hydrochloric acid (twice), saturated saline (twice),
Wash with water (once). After drying with anhydrous sodium sulfate, it was concentrated under reduced pressure to obtain 57.41 g of oily [5] (yield
77.1). TLC; Rf 6 = 0.85, Rf 1 = 0.63 6 P (140-141); BOC-Thr (Bzl)-Pro-
OH [6] [5] 52.78g (106.29mM) in methanol 300
ml, cooled on ice with 150ml of 1N-NaOH (1.4x M)
was added for 20 minutes, and then stirred at room temperature for 1.5 hours.
After the reaction, 43.71 ml of 1N hydrochloric acid (0.4 times M) was added under ice cooling to adjust the pH to 7, and methanol was distilled off under reduced pressure. The resulting aqueous solution was washed with 150 ml of ether,
The aqueous layer was adjusted to pH 3 with 110 ml of 1N hydrochloric acid under ice cooling. Extract twice with 300 ml and 150 ml of ethyl acetate, and wash the extract with brine (twice) and water (once) in that order. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Add ether and hexane to the residue to obtain the resulting precipitate. After performing this operation twice, it was dissolved in ethyl acetate and dried under reduced pressure to foam and solidify [6] 32.27
g (yield 77.75%). Melting point; 50-55℃ [α] 24 D ; -36.64゜ (C = 1, DMF) TLC; Rf 6 = 0.34 Elemental analysis [as C 21 H 30 O 5 N 2・H 2 O] C% H% N % Measured value 61.12 7.62 6.67 Calculated value 61.75 7.90 6.86 7 P(140-145); BOC-Thr(Bzl)-Pro-Ile
-Leu-Pro-Gln-OBzl [7] [6] 54.77g (83mM) in 80ml of methylene chloride
In addition to this, add 240ml of TFA under ice-cooling, and let it cool at room temperature.
Stir for 25 minutes. After the reaction, TFA is distilled off at 0°C under reduced pressure, and ether is added to the residue. Collect the resulting precipitate and concentrate the liquid under reduced pressure. Ether is added to the concentrate to obtain a precipitate, which is combined with the previous precipitate and dried under reduced pressure in an alkaline desiccator overnight. This precipitate was dissolved in 200 ml of DMF, cooled with a cryogen, and adjusted to pH 7 with NMM (9.5 ml).
HOBT11.21g (83mM) and BOC-Thr (Bzl)
-Add 32.27g (83mM) of Pro-OH and 40ml of DMF
Add to make a solution. This is then cooled with a cryogen.
Add 15.19 ml (83 mM) of WSCI dropwise over 10 minutes, and after 2 hours, stir overnight at room temperature. After reaction, under reduced pressure
DMF was distilled off, 300ml of 5% sodium bicarbonate solution was added to the residue,
Extract twice with 800 ml and 300 ml of ethyl acetate. The ethyl acetate layer was mixed with 5% sodium bicarbonate solution (twice), saturated saline (once), 1N hydrochloric acid (twice), saturated saline (twice),
Wash with water (once). After drying with anhydrous sodium sulfate, it is concentrated under reduced pressure. When a precipitate is deposited by standing, ether is added to separate the precipitate. Recrystallization from ethyl acetate and addition of ether gave 63.98 g (yield: 81.3%) of [7]. Melting point; 101-103 ℃ [α] 24 D ; -61.78゜ (C=1, DMF) Elemental analysis [as C50H72O11N7・H2O ] C % H% N % Measured value 62.21 7.89 10.03 Calculated value 62.16 7.82 10.15 Amino acid analysis [1.330mg/hydrochloric acid 0.3ml, acetic acid 0.3
ml, anisole 0.1ml, 105℃, 24 hours];
Thr0.84(1), Pro2, Ile0.97(1), Leu1.00(1),
Gln0.98(1) 8 P(139-145); BOC-Asp(Bzl)-Thr
(Bzl)-Pro-Ile-Leu-Pro-Gln-OBzl
[8] [7] 63.98g (67.48mM) of methylene chloride 150
After adding 300ml of TFA under ice-cooling,
Stir for 30 minutes at room temperature. After reaction, at 0℃ under reduced pressure.
TFA is distilled off and ether is added to the residue. The resulting precipitate is collected, combined with secondary crystals generated from the mother liquor, and dried under reduced pressure in an alkaline desiccator overnight. Yield: 67.19g. This precipitate was dissolved in 350 ml of DMF, and after cooling with a cryogen and adjusting the pH to 7 with 7.42 ml of NMM, 29.77 g of BOC-Asp(OBzl)-OH was added.
(87.72mM) and HOBT11.85g (87.72mM)
Add. Then WSCI16.05ml under cooling with cryogen.
(87.72mM) was added dropwise, and after 2 hours, the mixture was brought to room temperature and stirred overnight. After the reaction, the solvent is distilled off under reduced pressure, water is added to the residue, and the resulting precipitate is separated from the aqueous layer by decantation under ice cooling. Dissolve the precipitate in 1.5 ml of ethyl acetate, add 5% sodium bicarbonate (twice), saturated saline (once), 1N hydrochloric acid (once), and saturated saline (twice).
Wash with water (1 time) and water (1 time). After drying with anhydrous sodium sulfate, concentrate under reduced pressure. Leave the concentrated solution to stand and remove the resulting precipitate. Recrystallize twice with ethyl acetate-ether to obtain 73.78 g (yield 94.8%) of [8]. Melting point; 99-103℃ [α] 24 D ; -82.44゜ (C = 1, DMF) TLC; Rf 3 = 0.62, Rf 2 = 0.79 Elemental analysis [as C 6 H 84 O 14 N 8 ] C% H% N% Measured value 63.38 7.56 9.80 Calculated value 63.52 7.34 9.72 9 P(138-145); BOC―Ser(Bzl)―Asp
(OBzl) - Thr (Bzl) - Pro - Ile - Leu - Pro
―Gln―OBzl
〔９〕
〔８〕73.78ｇ（63.97mM）に塩化メチレン100
mlを加え、これに氷冷下TFA300mlを加えた後、
室温で30分間撹拌する。反応後、減圧下０℃で
TFAを留去し、残渣にエーテルを加え、生じた
沈澱物を取した後、カセイカリデシケーター中
で一夜減圧乾燥する。この沈澱物をDMF200mlに
溶かし、寒剤で冷却下NMM5mlでPH７に調節し
た後、BOC―Ser（Bzl）―OH24.56ｇ
（83.16mM）、HOBT11.23ｇ（83.16mM）および
DMF50mlを加える。さらに寒剤で冷却下
WSCI15.22mlを滴下した後、NMM2.03mlを追加
してPH６に調節する。２時間後、室温に戻し、一
夜撹拌する。反応後、減圧下でDMFを留去し、
残渣を冷水２中に注入する。生じた沈澱物を
取する。次いでこの沈澱物にCHCl35旺５％重
曹水２を加え抽出する。CHCl3層を５％重曹
水、飽和食塩水、1N塩酸、飽和食塩水、水の順
に洗浄した後、無水芒硝で乾燥する。減圧乾固
し、残渣に酢酸エチル―エーテル―ヘキサンを加
える。生じた沈澱物をクロロホルム300mlに溶か
し、酢酸エチル１を加えた後、減圧下クロロホ
ルムを留去する。得られた懸濁液にエーテル―ヘ
キサンを加え沈澱物を得る。同様の操作で再結晶
を行つて[9] [8] 73.78g (63.97mM) of methylene chloride 100
After adding 300ml of TFA under ice-cooling,
Stir for 30 minutes at room temperature. After the reaction, at 0°C under reduced pressure.
TFA is distilled off, ether is added to the residue, the resulting precipitate is collected, and then dried under reduced pressure in a caustic desiccator overnight. This precipitate was dissolved in 200 ml of DMF, cooled with a cryogen, and adjusted to pH 7 with 5 ml of NMM.
(83.16mM), HOBT11.23g (83.16mM) and
Add 50ml of DMF. Further cooling with cryogen
After dropping 15.22 ml of WSCI, add 2.03 ml of NMM to adjust the pH to 6. After 2 hours, return to room temperature and stir overnight. After the reaction, DMF was distilled off under reduced pressure.
Pour the residue into cold water 2. Collect the resulting precipitate. Next, 5% CHCl 3 and 22% aqueous sodium bicarbonate solution were added to the precipitate for extraction. The CHCl 3 layer is washed with 5% sodium bicarbonate solution, saturated brine, 1N hydrochloric acid, saturated brine, and water in this order, and then dried with anhydrous sodium sulfate. Dry under reduced pressure and add ethyl acetate-ether-hexane to the residue. The resulting precipitate was dissolved in 300 ml of chloroform, 1 portion of ethyl acetate was added, and the chloroform was distilled off under reduced pressure. Ether-hexane is added to the resulting suspension to obtain a precipitate. Perform recrystallization using the same procedure.
〔９〕80.95ｇ（収率95.1％）を得る。
融点；120〜122℃
TLC；Rf2＝0.70
元素分析〔C71H95O16N9として〕
Ｃ％ Ｈ％ Ｎ％
測定値 63.87 7.38 9.62
計算値 64.09 7.20 9.47
10 Ｐ（136―137）；BOC―Gly―Pro―OBzl〔10〕
BOC―Gly―OH26.28ｇ（0.15M）にTHF100
ml、DMF100mlおよびHOBT20.94ｇ0.115Mを加
え、これにＨ―Pro―OBzl・Ｈ Cl37.47ｇ
（0.155M）のDMF20mlの溶液を加え、−10℃に冷
却下、WSCI28.37ml（0.155M）を10分間で滴下
する。DMF30mlを加え、室温で一夜撹拌する。
反応後、減圧下で溶媒を留去し、残渣に酢酸エチ
ル500mlを加え、５％重曹水（２回）、飽和食塩水
（１回）、1N塩酸（２回）、飽和食塩水（２回）、
水（１回）の順に洗浄する。酢酸エチル層を無水
芒硝で乾燥し、減圧濃縮して油状物67ｇを得る。
これをベンゼン100mlに溶かし、ベンゼンで充填
したシリカゲル500ｇのカラム（７×33cm）にチ
ヤージし、ベンゼン300mlを流す。次いでベンゼ
ン―酢酸エチル（10：１→５：１→２：１→１：
１）〜酢酸エチルで溶出する。TLCでRf1＝0.86
付近のフラクシヨンを集め減圧乾固して油状の
[9] Obtain 80.95 g (yield 95.1%). Melting point: 120-122℃ TLC; Rf 2 = 0.70 Elemental analysis [as C 71 H 95 O 16 N 9 ] C% H% N% Measured value 63.87 7.38 9.62 Calculated value 64.09 7.20 9.47 10 P (136-137); BOC ―Gly―Pro―OBzl〔10〕 THF100 in BOC―Gly―OH26.28g (0.15M)
ml, DMF 100ml and HOBT20.94g0.115M, and to this H-Pro-OBzl・H Cl37.47g
(0.155M) in 20ml of DMF, and while cooling to -10°C, 28.37ml of WSCI (0.155M) was added dropwise over 10 minutes. Add 30 ml of DMF and stir overnight at room temperature.
After the reaction, the solvent was distilled off under reduced pressure, 500 ml of ethyl acetate was added to the residue, and 5% aqueous sodium bicarbonate (twice), saturated brine (once), 1N hydrochloric acid (twice), and saturated brine (twice) were added. ),
Wash with water (once). The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 67 g of an oil.
Dissolve this in 100 ml of benzene, charge it to a 500 g silica gel column (7 x 33 cm) packed with benzene, and flow 300 ml of benzene. Then benzene-ethyl acetate (10:1→5:1→2:1→1:
1) ~Elute with ethyl acetate. Rf 1 = 0.86 at TLC
The nearby fractions are collected and dried under reduced pressure to form an oily
〔９〕46.16ｇを得る。さらに残りのフラクシヨン
を減圧濃縮し、シリカゲル100ｇのカラム（4.5×
16cm）のカラムにチヤージし、前記と同様にカラ
ムクロマトグラフイーを行い、油状の〔10〕8.78
ｇを得る。
全量54.94ｇ
TLC；Rf1＝0.86
11 Ｐ（134―135）；BOC―Leu―Pro―OH〔11〕
BOC―Leu―Pro―OBzl82.76ｇ（0.252M）を
メタノール600mlに溶かし、これに氷冷下1N―
NaOH327.6ml（1.3倍Ｍ）を滴下した後、室温で
１時間撹拌する。反応液に氷冷下1N塩酸の６ml
を加えた後、減圧下メタノールを留去する。水層
をベンゼン、酢酸エチルの順に洗浄した後、氷冷
下1N塩酸260mlを加えると油状物が生じる。酢酸
エチルで抽出し、飽和食塩水、水の順に洗浄した
後、無水芒硝で乾燥する。減圧乾固し、残渣をベ
ンゼンに溶かし、凍結乾燥して〔11〕58.57ｇ
（収率70.8％）を得る。
融点；63〜67℃
〔α〕24 D；−66.42゜（Ｃ＝１，DMF）
元素分析〔C16H28O5N2として〕
Ｃ％ Ｈ％ Ｎ％
測定値 58.29 8.87 8.59
計算値 58.52 8.59 8.53
12 Ｐ（134―137）；BOC―Leu―Pro―Gly―Pro
―OBzl〔12〕
〔10〕54.36ｇ（0.15M）に塩化メチレン100
ml、TFA200mlを加え、室温で25分間撹拌する。
反応後、減圧下TFAを留去し、残渣をカセイカ
リデシケーター中で減圧乾燥する。次いで、
THF100mlに溶かし、−10℃に冷却下トリエチル
アミン48mlを加えてPH７とした後、〔11〕49.26ｇ
（0.15M）のTHF100ml溶液とHOBT20.27ｇ
（0.15M）を加える。さらにTHF50mlを追加して
溶液とし、これに−10℃に冷却下WSCI27.45mlを
10分間で滴下する。２時間後、室温に戻し、一夜
撹拌を続ける。反応後、減圧下溶媒を留去し、残
渣に酢酸エチル700mlを加え、５％重曹水500mlで
洗浄する。水層をさらに酢酸エチル300mlで抽出
し、抽出液を先の酢酸エチル層と合わせ、５％重
曹水（１回）、飽和食塩水（１回）、1N塩酸（２
回）、飽和食塩水（２回）、水１回の順で洗浄す
る。無水芒硝で乾燥後、減圧濃縮し、残渣にベン
ゼンを加えて減圧濃縮する。濃縮液をベンゼンで
充填したシリカゲル300ｇのカラム（6.5×20cm）
にチヤージし、ベンゼン→ベンゼン―酢酸エチル
（３：１→２：１）→酢酸エチルで溶出する。
TLCでRf1＝0.57付近のフラクシヨンを集め、減
圧乾固する。残渣を酢酸エチルに溶かし、減圧乾
固すると発泡して固化して〔12〕45.73ｇを得る。
残りのフラクシヨンを集め、減圧濃縮し、前記と
同様にカラムクロマトグラフイーを行い〔12〕18
ｇを得る。さらにその残りのフラクシヨンを減圧
濃縮し、シリカゲル100ｇのカラム（4.5×15cm）
にチヤージし、前記と同様に溶出して〔12〕7.11
ｇを得る。全量70.84ｇ（収率82.46％）
融点；65〜69℃
〔α〕24 D；−93.64゜（C21，DMF）
TLC；Rf1＝0.57、Rf6＝0.54
元素分析〔C30H44O7N4として〕
Ｃ％ Ｈ％ Ｎ％
測定値 62.98 7.94 10.07
計算値 62.92 7.74 9.78
13 Ｐ（133―137）；BOC―Arg（Tos）―Leu―
Pro―Gly―Pro―OBzl〔12〕
〔12〕68.72ｇ（120mM）を塩化メチレン100
mlに溶かし、これにTFA220mlを加え、室温で20
分間撹拌する。反応後、減圧下０℃でTFAを留
去し、残渣にヘキサンを加えて減圧乾固する。残
渣をカセイカリゲシケーター中で減圧乾燥した
後、DMF200mlに溶かし、−10℃で冷却下
NMM59.4mlを加えてPH６に調節する。これに
BOC―Arg（Tos）―OH53ｇ（123mM）の
DMF50ml溶液、HOBT16.89ｇ（125mM）を加
えて溶解した後、−10℃に冷却下WSCI22.86ml
（125mM）を10分間で滴下する。２時間後、室温
で一夜撹拌する。次いで、反応液に−10℃に冷却
下WSCI3.66ml（0.2mM）を追加し、室温で４時
間撹拌する。反応後、減圧下DMFを留去し、残
渣に５％重曹水600mlを加える。得られた沈澱物
を酢酸エチル500mlで２回抽出し、抽出液を５％
重曹水（１回）、飽和食塩水（１回）、1N塩酸
（２回）、飽和食塩水（３回）、水（１回）の順に
洗浄する。無水芒硝で乾燥し、減圧濃縮する。残
渣にエーテル―ヘキサンを加え、生じた沈澱物を
取する。酢酸エチル―エーテル―ヘキサンから
再結晶して〔13〕100.77ｇ（収率95.1％）を得
る。
融点；107〜112℃（分解）
〔α〕24 D；−64.84゜（Ｃ＝１，DMF）
TLC；Rf1＝0.19
アミノ酸分析〔0.709mg／塩酸0.3ml、酢酸0.3
ml、アニソール0.1ml、10.5℃、24時間）；
Pro(2)、Gly0.95(1)、Leu1.01(1)、Arg0.90
(1)
14 Ｐ（132―137）；BOC―Ser（Bzl）―Arg
（Tos）―Leu―Pro―Gly―Pro―OBzl〔14〕
〔13〕100.77ｇ（114mM）に塩化メチレン100
ml、TFA400mlを加え、室温で35分間撹拌する。
反応後、減圧下０℃でTFAを留去し、残渣にエ
ーテルを加え、生じた沈澱物を取する。母液を
減圧濃縮して二次沈澱物を得る。両方合わせ、カ
セイカリデシケーター中で一夜減圧乾燥する。こ
れをDMF200mlに溶かし、−10℃に冷却下
NMM10mlを加えてPH８とした後、HOBF16.9ｇ
（125.4mM）およびBOC―Ser（Bzl）―OH37.03
ｇ（125.4mM）を加える。次いで、氷冷下
WSCI22.95ml（125.4mM）を滴下し（PH５）、２
時間後、室温で一夜撹拌する。反応後、減圧下
DMFを留去し、残渣を氷水1.5に注ぎ入れ、沈
澱物を得る。これを酢酸エチル１、500mlで２
回抽出し、酢酸エチル層を1N塩酸（１回）、飽和
食塩水（１回）、1N―NaOH水溶液（２回）、飽
和食塩水（２回）、水１回の順に洗浄する。無水
芒硝で乾燥後、減圧濃縮する。残渣にエーテルを
加え、生じた沈澱物を取する。酢酸エチル―エ
ーテルから２回再結晶して〔14〕107.34ｇ（収率
87.0％）を得る。
融点；101〜105℃
TLC；Rf6＝0.71、Rf3＝0.49
アミノ酸分析〔1.818mg／塩酸0.3ml、酢酸0.3
ml、アニソール0.1ml、105℃、24時間〕；
Ser0.72(1)、Arg0.95(1)、Leu1.03(1)、Pro
(2)
15 Ｐ（132―137）；BOC―Ser（Bzl）―Arg
（Tos）―Leu―Pro―Gly―Pro―OH〔15〕
〔14〕105.44ｇ（97.43mM）をメタノール350
mlに溶かし、これに氷冷下、1N―NaOH水溶液
125.36ml（1.3倍Ｍ）を滴下した後、室温で２時
間半撹拌する。反応液に氷冷下さらに1N―
NaOH水溶液20mlを追加し、室温で１時間半撹
拌する。反応液に氷冷下1N塩酸49mlを加えPH６
とし、減圧下メタノールを留去する。水溶液に水
50mlを加え、エーテル100mlで洗浄する。次いで
水層に氷冷下1N塩酸50mlを滴下すると、生じた
粘性沈澱物のため撹拌不能となつたため、酢酸エ
チル300mlを加えた後、1N塩酸60mlを滴下して水
層のPHを２とする。酢酸エチル１、500mlで２
回抽出し、酢酸エチル層を飽和食塩水（２回）、
水の順に洗浄する。無水芒硝で乾燥後、減圧濃縮
し、得られた油状物にエーテルを加えて固化す
る。酢酸エチル―エーテルから再結晶して〔15〕
95.12ｇ（収率100％）を得る。
TLC；Rf2＝0.10、Rf4＝0.32
16 Ｐ（132―145）；BOC―Ser（Bzl）―Arg
（Tos）―Leu―Pro―Gly―Pro―Ser（Bzl）―
Asp（OBzl）―Thr（Bzl）―Pro―Ile―Leu―
Pro―Gln―OBzl〔16〕[9] Obtain 46.16g. Furthermore, the remaining fraction was concentrated under reduced pressure, and the column of 100 g of silica gel (4.5×
Charge a 16cm) column and perform column chromatography in the same manner as above to obtain an oily [10]8.78
get g. Total amount: 54.94g TLC; Rf 1 = 0.86 11 P (134-135); BOC-Leu-Pro-OH [11] Dissolve 82.76g (0.252M) of BOC-Leu-Pro-OBzl in 600ml of methanol and cool with ice. Lower 1N-
After dropping 327.6 ml of NaOH (1.3 times M), the mixture was stirred at room temperature for 1 hour. Add 6 ml of 1N hydrochloric acid to the reaction solution under ice cooling.
After adding , methanol is distilled off under reduced pressure. After washing the aqueous layer with benzene and ethyl acetate in that order, 260 ml of 1N hydrochloric acid was added under ice cooling to produce an oily substance. Extract with ethyl acetate, wash sequentially with saturated brine and water, and then dry with anhydrous sodium sulfate. Dry under reduced pressure, dissolve the residue in benzene, and freeze-dry to obtain 58.57 g of [11]
(yield 70.8%). Melting point; 63-67℃ [α] 24 D ; -66.42゜ (C=1, DMF) Elemental analysis [as C 16 H 28 O 5 N 2 ] C% H% N% Measured value 58.29 8.87 8.59 Calculated value 58.52 8.59 8.53 12 P (134-137); BOC-Leu-Pro-Gly-Pro
-OBzl [12] [10] 54.36g (0.15M) of methylene chloride 100
ml, add 200 ml of TFA, and stir at room temperature for 25 minutes.
After the reaction, TFA is distilled off under reduced pressure, and the residue is dried under reduced pressure in a caustic desiccator. Then,
Dissolved in 100 ml of THF, added 48 ml of triethylamine under cooling to -10°C to adjust the pH to 7, then [11] 49.26 g
(0.15M) THF100ml solution and HOBT20.27g
Add (0.15M). Add 50ml of THF to make a solution, and add 27.45ml of WSCI while cooling to -10℃.
Drop in 10 minutes. After 2 hours, return to room temperature and continue stirring overnight. After the reaction, the solvent is distilled off under reduced pressure, 700 ml of ethyl acetate is added to the residue, and the mixture is washed with 500 ml of 5% aqueous sodium bicarbonate. The aqueous layer was further extracted with 300 ml of ethyl acetate, and the extract was combined with the previous ethyl acetate layer.
(times), saturated saline (twice), and water once. After drying over anhydrous sodium sulfate, the mixture is concentrated under reduced pressure. Benzene is added to the residue, and the mixture is concentrated under reduced pressure. 300g silica gel column (6.5 x 20cm) packed with concentrated liquid with benzene
Charge and elute with benzene → benzene-ethyl acetate (3:1 → 2:1) → ethyl acetate.
Fractions around Rf 1 =0.57 were collected by TLC and dried under reduced pressure. The residue was dissolved in ethyl acetate and dried under reduced pressure, foaming and solidifying to obtain 45.73 g of [12].
The remaining fractions were collected, concentrated under reduced pressure, and subjected to column chromatography in the same manner as above [12] 18
get g. Furthermore, the remaining fraction was concentrated under reduced pressure, and a column of 100 g of silica gel (4.5 x 15 cm) was collected.
and elute in the same manner as above [12] 7.11
get g. Total amount 70.84g (yield 82.46%) Melting point: 65-69℃ [α] 24 D ; -93.64゜ (C 21 , DMF) TLC; Rf 1 = 0.57, Rf 6 = 0.54 Elemental analysis [C 30 H 44 O 7 As N 4 ] C% H% N% Measured value 62.98 7.94 10.07 Calculated value 62.92 7.74 9.78 13 P(133-137); BOC-Arg(Tos)-Leu-
Pro-Gly-Pro-OBzl [12] [12] 68.72g (120mM) in methylene chloride 100
ml, add 220ml of TFA to this, and stir at room temperature for 200ml.
Stir for a minute. After the reaction, TFA is distilled off at 0°C under reduced pressure, hexane is added to the residue, and the mixture is dried to dryness under reduced pressure. The residue was dried under reduced pressure in a caustic gesicator, then dissolved in 200 ml of DMF and cooled at -10°C.
Add 59.4ml of NMM to adjust the pH to 6. to this
BOC-Arg(Tos)-OH53g (123mM)
After adding and dissolving 50 ml of DMF solution and 16.89 g (125 mM) of HOBT, 22.86 ml of WSCI was cooled to -10℃.
(125mM) over 10 minutes. After 2 hours, stir overnight at room temperature. Next, 3.66 ml (0.2 mM) of WSCI was added to the reaction solution while cooling to -10°C, and the mixture was stirred at room temperature for 4 hours. After the reaction, DMF is distilled off under reduced pressure, and 600 ml of 5% sodium bicarbonate solution is added to the residue. The obtained precipitate was extracted twice with 500 ml of ethyl acetate, and the extract was diluted with 5%
Wash in the following order: sodium bicarbonate solution (once), saturated saline (once), 1N hydrochloric acid (2 times), saturated saline (3 times), and water (once). Dry with anhydrous sodium sulfate and concentrate under reduced pressure. Add ether-hexane to the residue and collect the resulting precipitate. Recrystallization from ethyl acetate-ether-hexane gives 100.77 g (yield 95.1%) of [13]. Melting point: 107-112℃ (decomposition) [α] 24 D ; -64.84゜ (C = 1, DMF) TLC; Rf 1 = 0.19 Amino acid analysis [0.709mg/hydrochloric acid 0.3ml, acetic acid 0.3
ml, anisole 0.1ml, 10.5℃, 24 hours);
Pro(2), Gly0.95(1), Leu1.01(1), Arg0.90
(1) 14 P(132-137); BOC-Ser(Bzl)-Arg
(Tos)-Leu-Pro-Gly-Pro-OBzl [14] [13] 100.77g (114mM) of methylene chloride 100
ml, add 400 ml of TFA, and stir at room temperature for 35 minutes.
After the reaction, TFA is distilled off at 0°C under reduced pressure, ether is added to the residue, and the resulting precipitate is collected. The mother liquor is concentrated under reduced pressure to obtain a secondary precipitate. Combine both and dry under reduced pressure in a caustic desiccator overnight. Dissolve this in 200ml of DMF and cool to -10℃.
After adding 10ml of NMM to adjust the pH to 8, HOBF16.9g
(125.4mM) and BOC-Ser(Bzl)-OH37.03
g (125.4mM). Then cooled on ice
Drop 22.95ml (125.4mM) of WSCI (PH5),
After an hour, stir at room temperature overnight. After reaction, under reduced pressure
DMF is distilled off and the residue is poured into 1.5 g of ice water to obtain a precipitate. Add 1 part of this to ethyl acetate, and 2 parts to 500 ml of this.
Extract twice, and wash the ethyl acetate layer in the following order: 1N hydrochloric acid (once), saturated brine (once), 1N-NaOH aqueous solution (twice), saturated brine (twice), and water once. After drying with anhydrous sodium sulfate, concentrate under reduced pressure. Add ether to the residue and collect the resulting precipitate. Recrystallized twice from ethyl acetate-ether [14] 107.34 g (yield
87.0%). Melting point: 101-105℃ TLC: Rf 6 = 0.71, Rf 3 = 0.49 Amino acid analysis [1.818 mg/hydrochloric acid 0.3 ml, acetic acid 0.3
ml, anisole 0.1ml, 105℃, 24 hours];
Ser0.72(1), Arg0.95(1), Leu1.03(1), Pro
(2) 15 P(132-137); BOC-Ser(Bzl)-Arg
(Tos)-Leu-Pro-Gly-Pro-OH [15] [14] 105.44g (97.43mM) in methanol 350
ml, add 1N-NaOH aqueous solution to this under ice-cooling.
After dropping 125.36 ml (1.3 times M), the mixture was stirred at room temperature for 2 and a half hours. Add another 1N to the reaction solution under ice cooling.
Add 20 ml of NaOH aqueous solution and stir at room temperature for 1.5 hours. Add 49 ml of 1N hydrochloric acid to the reaction solution under ice cooling to adjust the pH to 6.
and methanol was distilled off under reduced pressure. water in aqueous solution
Add 50 ml and wash with 100 ml of ether. Next, when 50 ml of 1N hydrochloric acid was added dropwise to the aqueous layer under ice cooling, stirring became impossible due to the viscous precipitate formed, so 300 ml of ethyl acetate was added, and then 60 ml of 1N hydrochloric acid was added dropwise to bring the pH of the aqueous layer to 2. . ethyl acetate 1, 2 in 500ml
Extract twice, and remove the ethyl acetate layer with saturated brine (twice).
Rinse with water. After drying with anhydrous sodium sulfate, it was concentrated under reduced pressure, and ether was added to the obtained oil to solidify it. Recrystallized from ethyl acetate-ether [15]
Obtain 95.12 g (100% yield). TLC; Rf 2 = 0.10, Rf 4 = 0.32 16 P (132-145); BOC-Ser (Bzl)-Arg
(Tos) -Leu-Pro-Gly-Pro-Ser (Bzl)-
Asp (OBzl) - Thr (Bzl) - Pro - Ile - Leu -
Pro―Gln―OBzl〔16〕
〔９〕49.66ｇ（37.32mM）を塩化メチレン130
mlに溶かし、これに氷冷下TFA220mlを加え、室
温で45分間撹拌する。反応後、減圧濃縮し、残渣
にエーテルを加える。生じた沈澱物を取し、ア
ルカリデシケーター中で一夜減圧乾燥する。これ
をDMF220mlに溶かし、−10℃に冷却下
NMM4.10mlでPH７に調節した後、〔15〕43.72ｇ
（44.79mM）、HOBT6.56ｇ（48.52mM）を加え
る。溶解後、−10℃に冷却下WSCI8.88ml
（48.52mM）を滴下し、室温で２日間撹拌する。
反応後、減圧下DMFを留去し、残渣を氷水２
に注ぎ入れる。得られた沈澱物を取し、水に懸
濁、洗浄して取する。この操作を５回行なつた
後（PH６）、デシケーター中で減圧乾燥して固形
物109ｇを得る。これをメタノールに溶解後、減
圧濃縮し、さらにベンゼンを加えて共沸により残
留する水分を除去する。この操作を３回行つた
後、酢酸エチル―エーテルから再結晶する。
90.09ｇこれをクロロホルム270mlに溶かし、クロ
ロホルムで充填したシリカゲル600ｇのカラムに
チヤージし、クロロホルム―メタノール（20：
１）で溶出する。TLCで１スポツトのフラクシ
ヨンのみを集めて減圧乾固して〔15〕を得る。残
りのフラクシヨンは減圧濃縮してシリカゲル220
ｇのカラムにチヤージし、前記と同様に溶出して
TLCで１スポツトのフラクシヨンのみを集めて
減圧乾固する。このような再クロマトグラフイー
操作を４回行い、〔16〕を得る。全量64.26ｇ（収
率78.9％）
TLC；Rf3＝0.40、Rf2＝0.60
融点；120〜125℃
元素分析〔C12H152O25N18S・2H2Oとして〕
Ｃ％ Ｈ％ Ｎ％
測定値 60.66 7.20 11.50
計算値 60.63 7.09 11.36
〔α〕22 D；−63.3゜（Ｃ＝１，DMF）
17 Ｐ（130―131）；BOC―Ser（Bzl）―Pro―
OBzl〔17〕
BOC―Ser（Bzl）―OH35.4ｇ（0.12M）に
THF100ml、Ｈ―Pro―OBzl・HCl31.5ｇ
（0.13M）、HOBT17.5ｇ（0.13M）を加え、さら
にDMF50mlを加えて溶液とする。これに０℃に
冷却下WSCI24.8mlを滴下し、０℃で１時間、室
温で一夜撹拌する。反応後、減圧下で溶媒を留去
し、残渣にベンゼン加え、５％重曹水（３回）、
1N塩酸（３回）、水（３回）の順に洗浄する。無
水芒硝で乾燥後、減圧乾固して油状の〔17〕58ｇ
（収率100％）を得る。
TLC；Rf1＝0.68
18 Ｐ（127―129）；BOC―Ser（Bzl）―Leu―
Pro―OBzl〔18〕
BOC―Leu―Pro―OBzl71.27ｇ（156mM）に
塩化メチレン30mlを加え、これに０℃に冷却下
TFA350mlを加えた後、室温で30分間撹拌する。
反応後、減圧下でTFAを留去し、得られた油状
物をカセイカリゲシケーター中で３時間減圧乾燥
する。この油状物をDMF170mlに溶かし、これに
０℃に冷却下NMM60mlを加えて中和した後、
BOC―Ser（Bzl）―OH46.0ｇ（156mM）、
HOBT21.1ｇ（156mM）を加えて溶液とする。
これに０℃に冷却下WSCI28.6ml（156mM）を滴
下し、０℃で１時間、室温で一夜撹拌する。反応
後、減圧下でDMFを留去し、残渣に酢酸エチル
500mlを加え、５％重曹水（３回）、1N塩酸（３
回）、水（３回）の順に洗浄する。酢酸エチル層
を無水芒硝で乾燥後、減圧濃縮する。油状物をで
きるだけ少量のベンゼンに溶かし、ベンゼンで充
填したシリカゲル500ｇのカラムにチヤージし、
ベンゼン１、ベンゼン―酢酸エチル（10：１）
２、ベンゼン―酢酸エチル（５：１）１、ベ
ンゼン―酢酸エチル（１：１）２の順で溶出す
る。TLCにより相当するフラクシヨンのみを減
圧濃縮し、残渣をベンゼンに溶かして、凍結乾燥
して〔18〕81ｇ（収率87.0％）を得る。
TLC；Rf1＝0.81
元素分析〔C33H45O7N3として〕
Ｃ％ Ｈ％ Ｎ％
測定値 66.34 7.81 6.80
計算値 66.53 7.61 7.05
19 Ｐ（127―129）；BOC―Ser（Bzl）―Leu―
Pro―OH〔19〕
〔18〕81ｇ（135.5mM）をエタノール300mlに
溶かし、これに０℃に冷却下1N―NaOH水溶液
176.15ml（1.3倍Ｍ）を30分間で滴下した後、室
温で３時間撹拌する。反応後、０℃に冷却下1N
塩酸で中和し、減圧下でエタノールを留去する。
水層に水300mlを加え、エーテルで２回洗浄した
後、０℃に冷却下1N塩酸150mlを加え、酢酸エチ
ル400mlで抽出する。酢酸エチル層を水洗し、無
水硫酸マグネシウムで乾燥後、減圧濃縮する。得
られた油状物をデシケーター中で減圧乾燥する。
得られた粉末をエーテルヘキサンで再沈澱して
〔19〕65.0ｇ（収率94.5％）を得る。
融点；56〜63℃
TLC；Rf1＝0.58
〔α〕22 D；−47.1゜（Ｃ＝１，DMF）
元素分析〔C26H39O7N3として〕
Ｃ％ Ｈ％ Ｎ％
測定値 61.52 7.71 8.50
計算値 61.76 7.78 8.31
20 Ｐ（127―131）；BOC―Ser（Bzl）―Leu―
Pro―Ser（Bzl）―Pro―OBzl〔20〕
〔17〕58ｇ（0.12M）に塩化メチレン30mlを加
え、０℃に冷却下TFA350mlを加えた後、室温で
30分間撹拌する。反応後、減圧下でTFAを留去
し、残渣をDMF150mlに溶かす。これを０℃に冷
却下NMM50mlで中和し、〔19〕61.1ｇ（0.12M）、
HOBT16.2ｇ（0.12M）を加え、次いで０℃に冷
却下WSCI22ml（0.12M）を滴下する。０℃で１
時間、室温で一夜撹拌する。反応後、減圧下で
DMFを留去し、残渣に酢酸エチル400mlを加え
る。５％重曹水（３回）、1N塩酸（３回）、水
（３回）の順に洗浄し、無水硫酸マグネシウムで
乾燥後、減圧濃縮する。得られた油状物をできる
だけ少量のベンゼンに溶かし、ベンゼンで充填し
たシリカゲル500ｇのカラムにチヤージする。ベ
ンゼン500ml、ベンゼン―酢酸エチル（10：１）
２、ベンゼン―酢酸エチル（10：１）２、ベ
ンゼン―酢酸エチル（３：１）２、ベンゼン―
酢酸エチル（１：１）４、酢酸エチル３の順
で溶出し、TLCで相当するフラクシヨンを集め
て減圧乾固して〔20〕84.07ｇ（収率80.8％）を
得る。
融点；58〜65℃
TLC；Rf1＝0.51
〔α〕D；−63.7゜（Ｃ＝１、DMF）
元素分析〔C48H63O10N5として〕
Ｃ％ Ｈ％ Ｎ％
測定値 66.31 7.25 8.09
計算値 66.26 7.30 8.05
アミノ酸分析；Ser1.42(2)、Pro(2)、Leu1.01(2)
21 Ｐ（127―131）；BOC―Ser（Bzl）―Leu―
Pro―Ser（Bzl）―Pro―OH〔21〕
〔20〕74.4ｇ（85.4mM）をメタノール30mlに
溶かし、これに０℃に冷却下1N―NaOH水溶液
110ml（1.3倍Ｍ）を滴下した後、室温で３時間撹
拌する。反応後、０℃に冷却下1N塩酸26mlを加
えて中和し、減圧下でメタノールを留去する。水
溶液をエーテルで２回洗浄し、０℃に冷却下1N
塩酸86mlを加え、酢酸エチルで抽出する。酢酸エ
チル層を２回水洗し、無水硫酸マグネシウムで乾
燥後、減圧濃縮する。残渣にヘキサンを加える。
生じた沈澱物を取し、できるだけ少量のクロロ
ホルムに溶かし、クロロホルムで充填したシリカ
ゲルのカラムにチヤージする。クロロホルム、ク
ロロホルム―酢酸エチル（１：１）、クロロホル
ム―エタノール―酢酸エチル（５：１：５）の順
で溶出する。TLCで相当するフラクシヨンを集
め減圧乾固して〔21〕53.75ｇ（収率81％）を得
る。
TLC；Rf1＝0.34
〔α〕D；−66.4゜（Ｃ＝１、DMF）
元素分析〔C41H57O10N10として〕
Ｃ％ Ｈ％ Ｎ％
測定値 63.11 7.66 8.85
計算値 63.14 7.37 8.98
アミノ酸分析〔0.779mg／6N塩酸0.3ml、105℃、
20時間〕；Ser1.84(2)、Pro2.06(2)、Leu(1)
22 Ｐ（127―145）；BOC―Ser（Bzl）―Leu―
Pro―Ser（Bzl）―Pro―Ser（Bzl）―Arg
（Tos）―Leu―Pro―Gly―Pro―Ser（Bzl）―
Asp（OBzl）―Thr（Bzl）―Pro―Ile―Leu―
Pro―Gln―OBzl〔22〕
〔16〕63.66ｇ（29.17mM）に塩化メチレンを
加え、これに０℃に冷却下TFA260mlを加えた
後、室温で40分間撹拌する。反応後、減圧下で
TFAを留去し、残渣にエーテルを加える。生じ
た沈澱物を取し、カセイカリデシケーター中で
一夜減圧乾燥する。67.6ｇ。これをDMF200mlに
溶かし（PH４）、−10℃に冷却下NMM4.21mlを加
えてPH７に調節する。次いでHOBT4.73ｇ
（35mM）、〔21〕27.30ｇ（35mM）および
DMF100mlを加え、これに−10℃に冷却下
WSCI6.41ml（35mM）を滴下した後、−10℃で２
時間、室温で一夜撹拌する。反応後、減圧下で溶
媒を留去し、残渣を氷冷水1.5に注ぐ。生じた
沈澱を水に懸濁し、取する。この懸濁、取の
操作を５回繰り返す（PH６）。沈澱物を２日間減
圧乾燥した後、メタノール500mlに溶かし、減圧
濃縮する。残渣にベンゼンを加え、減圧濃縮す
る。このベンゼン処理を２回行い、濃縮物にエー
テルを加え、生じた沈澱物を取する。前記のメ
タノール処理からエーテル処理までを８回繰り返
し、〔22〕79.85ｇ（収率96.2％）を得る。
融点；127〜130℃
TLC；Rf2＝0.61
〔α〕22 D；−67.58゜（Ｃ＝１、DMF）
元素分析〔C148H207O36N23Sとして〕
Ｃ％ Ｈ％ Ｎ％
測定値 61.05 6.96 11.03
計算値 60.95 7.15 11.05
アミノ酸分析〔1.893mg／6N塩酸0.5ml、105℃、
21時間〕；Asp0.99(1)、Thr0.96(1)、Ser3.74
(4)、Glu1.04(1)、Pro6.00(6)、Gly0.98(1)、
Ile0.95(1)、Leu(3)、Arg0.94(1)
23 hCG〔127―145〕
〔22〕１ｇ（0.34mM）にアニソール５mlを加
え、これに０℃に冷却下無水弗化水素（HF）30
mlを加え、１時間撹拌する。反応後、減圧下で
HFを速やかに留去し、得られた残渣にエーテル
100mlと0.1N酢酸水50mlを加えて振盪する。水層
をDowex １（酢酸型）のカラム（3.3×18cm）に
チヤージし、0.1N酢酸水で流出する。流出液と
洗液を合わせて凍結乾燥して粗生成物660mgを得
る。これを8M尿素水溶液30mlに溶かし、アンモ
ニア水でPH９に調節した後、セフアデツクスLH
―20のカラム（4.5×120cm）にチヤージし、0.1N
酢酸水で溶出する。溶出液を10mlづつ分画し、58
〜64本目の区分を集めて凍結乾燥して540mgを得
る。これを0.1N酢酸水30mlに溶かし、カルボキ
シメチルセルロースのカラム（５×13cm）にチヤ
ージし、0.01M酢酸アンモニウム緩衝液（PH4.5）
で洗浄後、酢酸アンモニウム緩衝液（PH4.5）
0.01Mと0.1Mの各400mlの間で直線濃度勾配をか
けて溶出する。溶出液を６mlづつ分画し、79〜86
本目の区分を集め、凍結乾燥して180mgを得る。
これを0.1N酢酸水に溶かし、セフアデツクスLH
―20のカラム（３×17cm）にチヤージし、0.1N
酢酸水で溶出する。溶出液は10.5mlづつ分画し、
25〜30本目の区分を集めて凍結乾燥してhCG〔127
―145〕136.4mgを得る。
融点；215〜225℃
〔α〕28 D；＋65゜ （Ｃ＝0.1，0.1N酢酸水）
TLC；Rf11＝0.71
アミノ酸分析；Asp1.05(1)、Thr0.97(1)、
Ser3.44（４）、Glu1.10(1)、Pro6.50(6)、
Gly1.04(1)、Ile0.89(1)、Leu3(3)、Arg1.02
(1)
参考例 ２
hCG〔118―145〕；Ｈ―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 Ｐ（118―145）；BOC―Ser（Bzl）―Ser（Bzl）
―Ser（Bzl）―Ser（Bzl）―Lys（Ｚ―Cl）―
Ala―Pro―Pro―Pro―Ser（Bzl）―Leu―Pro
―Ser（Bzl）―Pro―Ser（Bzl）―Arg（Tos）
―Leu―Pro―Gly―Pro―Ser（Bzl）―Asp
（OBzl）―Thr（Bzl）―Pro―Ile―Leu―Pro
―Gln―OBzl〔34〕
参考例１記載の〔22〕79ｇ（28mM）に塩化メ
チレン200mlを加え、これに０℃に冷却下
TFA400mlを加えた後、室温で50分間撹拌する。
反応後、減圧下でTFAを留去し、残渣にエーテ
ルを加え、生じた沈澱物を取した後、カセイカ
リデシケーター中で一夜減圧乾燥する。85.35ｇ。
この生成物をDMF270mlに溶かし（PH３）、−10℃
に冷却下NMM4.58mlでPH６に調節する。次いで
これに、Ｐ（118―126）〔BOC―Ser（Bzl）―Ser
（Bzl）―Ser（Bzl）―Ser（Bzl）―Lys（Ｚ―Cl）
―Ala―Pro―Pro―Pro―OH、融点；125〜135
℃、〔α〕D；−56.4゜（Ｃ＝１、DMF）、TLC；Rf6
＝原点付近、Rf5＝0.45、アミノ酸分析；Ser3.23
（４）、Pro3(3)、Ala0.98(1)、Lys0.97(1)〕49.93ｇ
（33.6mM）を加え、さらにDMF100mlを追加し
て溶液とする。これに−10℃で冷却下WSCI6.20
mlを滴下した（PH５）後、−10℃で２時間、室温
で２時間撹拌する。反応後、減圧下で溶媒を留去
し、残渣を冷水1.5に注ぎ入れる。生じた沈澱
物を得、水に懸濁して洗浄を繰り返した後、デシ
ケーター中で２日間減圧乾燥する。130ｇ。次い
でメタノールを加えて減圧濃縮し、残渣にベンゼ
ンを加えて減圧濃縮し、残渣にエーテルを加えて
再結晶化する操作を12回行なう。さらにメタノー
ルを加えて減圧濃縮し、残渣にエーテルを加えて
再結晶化して〔34〕108.43ｇ（収率91.9％）を得
る。
融点；129〜131℃
〔α〕24 D；−72.76゜（Ｃ＝１、DMF）
TLC；Rf2＝0.56
元素分析〔C220H294O51N33SClとして〕
Ｃ％ Ｈ％ Ｎ％
測定値 61.49 7.07 11.02
計算値 61.67 6.92 10.79
アミノ酸分析〔0.978mg／6N塩酸0.5ml、105℃、
21時間〕；Asp1.00(1)、Thr0.96(1)、Ser6.87
(8)、Glu1.05(1)、Pro9.63(9)、Gly1.03(1)、
Ala1.09(1)、Ile0.94(1)、Leu3(3)、Lys1.09
(1)、Arg0.97(1)
２ hCG〔118―145〕
〔34〕3.0ｇ（0.71mM）にアニソール10mlを加
え、これに無水弗化水素（HF）80mlを導入し、
０℃で１時間撹拌する。反応後、減圧下でHFを
留去する。残渣にエーテルを加え、生じた沈澱物
を取する。この粉末を0.1N酢酸水50mlに溶解
し、Dowex １（酢酸型）のカラム（3.5×35cm）
にチヤージし、0.1N酢酸水で洗う。流出液と洗
液を合わせ、凍結乾燥して1.46ｇを得る。これを
8M尿素水溶液30mlで溶解し、０℃でアンモニア
水にてPH９に調節し、セフアデツクスLH―20の
カラム（4.5×120cm）にチヤージし、0.1N酢酸水
で溶出する。溶出液を７mlづつ分画し、55〜83本
目の区分を集め、凍結乾燥して990mgを得る。こ
れを0.1N酢酸水30mlで溶解し、カルボキシメチ
ルセルロースのカラム（５×14cm）にチヤージす
る。0.01M酢酸アンモニウム緩衝液（PH4.5）200
mlで洗浄後、酢酸アンモニウム緩衝液（PH4.5）
0.01Mと0.1Mの各400mlの間で直線濃度勾配をか
けて溶出を行う。溶出液を６mlづつ分画し、85〜
115本目の区分を集め、凍結乾燥して850mgを得
る。これを0.1N酢酸水で溶解し、セフアデツク
スLH―20のカラム（４×120cm）にチヤージし、
0.1N酢酸水で溶出する。溶出液を10.5mlづつ分画
し、35〜43本目の区分を集め、凍結乾燥して650
mgを得る。これを0.1N酢酸水30mlで溶解し、再
度カルボキシメチルセルロースのカラム（５×15
cm）にチヤージする。0.01M酢酸アンモニウム緩
衝液（PH4.5）200mlで洗い、次いで酢酸アンモニ
ウム緩衝液（PH4.5）0.01Mと0.1Mの各400mlの間
で直線濃度勾配をかけて溶出を行う。溶出液を
6.5mlづつ分画し、100〜111本目の区分を集め、
凍結乾燥して480mgを得る。これを0.1N酢酸水で
溶解し、セフアデツクスLH―20のカラム（３×
117cm）にチヤージし、0.1N酢酸水で溶出する。
溶出液を10.5mlづつ分画し、24〜30本目の区分を
集め、凍結乾燥して、hCG〔118―145〕320mgを得
る。
融点；220℃以上で分解
TLC；Rf11＝0.57
アミノ酸分析；Asp1.07(1)、Thr0.97(1)、
Ser5.23（８）、Glu1.13(1)、Pro9.86(9)、
Gly1.07(1)、Ala1.06(1)、Ile0.89(1)、Leu3
(3)、Lys1.08(1)、Arg1.07(1)[9] 49.66g (37.32mM) of methylene chloride 130
ml, add 220 ml of TFA under ice-cooling, and stir at room temperature for 45 minutes. After the reaction, concentrate under reduced pressure and add ether to the residue. The resulting precipitate is collected and dried under reduced pressure in an alkaline desiccator overnight. Dissolve this in 220ml of DMF and cool to -10℃.
After adjusting the pH to 7 with NMM4.10ml, [15]43.72g
(44.79mM) and HOBT6.56g (48.52mM). After dissolving, cool to -10℃ and add 8.88ml of WSCI.
(48.52mM) dropwise and stirred at room temperature for 2 days.
After the reaction, DMF was distilled off under reduced pressure, and the residue was poured into ice water 2
Pour into. The obtained precipitate is taken, suspended in water, washed and collected. After performing this operation five times (PH6), the mixture was dried under reduced pressure in a desiccator to obtain 109 g of a solid. After dissolving this in methanol, it is concentrated under reduced pressure, and benzene is further added to remove residual water by azeotropy. After performing this operation three times, it is recrystallized from ethyl acetate-ether.
90.09g of this was dissolved in 270ml of chloroform, charged to a 600g column of silica gel packed with chloroform, and chloroform-methanol (20:
Elute with 1). Only one fraction of the fraction was collected by TLC and dried under reduced pressure to obtain [15]. The remaining fraction was concentrated under reduced pressure to silica gel 220.
Charge the column of g and elute in the same manner as above.
Only one fraction of the fraction was collected by TLC and dried under reduced pressure. This re-chromatography operation was performed four times to obtain [16]. Total amount 64.26g (yield 78.9%) TLC; Rf 3 = 0.40, Rf 2 = 0.60 Melting point; 120-125°C Elemental analysis [as C 12 H 152 O 25 N 18 S・2H 2 O] C% H% N% Measured value 60.66 7.20 11.50 Calculated value 60.63 7.09 11.36 [α] 22 D ; −63.3° (C=1, DMF) 17 P (130−131); BOC―Ser(Bzl)―Pro―
OBzl〔17〕 BOC―Ser(Bzl)―OH35.4g (0.12M)
THF100ml, H-Pro-OBzl・HCl31.5g
(0.13M), HOBT17.5g (0.13M), and further add DMF50ml to make a solution. 24.8 ml of WSCI was added dropwise to this while cooling to 0°C, and the mixture was stirred at 0°C for 1 hour and at room temperature overnight. After the reaction, the solvent was distilled off under reduced pressure, and benzene was added to the residue, followed by 5% aqueous sodium bicarbonate (3 times),
Wash with 1N hydrochloric acid (3 times) and water (3 times) in this order. Dry with anhydrous sodium sulfate and dry under reduced pressure to obtain 58g of oily [17]
(yield 100%). TLC; Rf 1 = 0.68 18 P (127-129); BOC―Ser(Bzl)―Leu―
Pro-OBzl[18] Add 30ml of methylene chloride to 71.27g (156mM) of BOC-Leu-Pro-OBzl and cool to 0℃.
After adding 350 ml of TFA, stir at room temperature for 30 minutes.
After the reaction, TFA is distilled off under reduced pressure, and the resulting oil is dried under reduced pressure in a caustic gesicator for 3 hours. This oil was dissolved in 170 ml of DMF and neutralized by adding 60 ml of NMM while cooling at 0°C.
BOC-Ser(Bzl)-OH46.0g (156mM),
Add 21.1g (156mM) of HOBT to make a solution.
28.6 ml (156 mM) of WSCI was added dropwise to this while cooling to 0°C, and the mixture was stirred at 0°C for 1 hour and at room temperature overnight. After the reaction, DMF was distilled off under reduced pressure and ethyl acetate was added to the residue.
Add 500ml, 5% sodium bicarbonate solution (3 times), 1N hydrochloric acid (3 times)
Wash with water (3 times) and then with water (3 times). The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Dissolve the oil in as little benzene as possible and charge it to a 500g column of silica gel packed with benzene.
Benzene 1, benzene-ethyl acetate (10:1)
2. Elute in the following order: benzene-ethyl acetate (5:1) 1, benzene-ethyl acetate (1:1) 2. Only the corresponding fraction was concentrated under reduced pressure by TLC, and the residue was dissolved in benzene and lyophilized to obtain 81 g (yield: 87.0%) of [18]. TLC; Rf 1 = 0.81 Elemental analysis [as C 33 H 45 O 7 N 3 ] C% H% N% Measured value 66.34 7.81 6.80 Calculated value 66.53 7.61 7.05 19 P (127-129); BOC-Ser (Bzl)- Leu――
Dissolve 81g (135.5mM) of Pro-OH [19] [18] in 300ml of ethanol, and add 1N-NaOH aqueous solution to this while cooling to 0℃.
After dropping 176.15 ml (1.3 times M) over 30 minutes, the mixture was stirred at room temperature for 3 hours. After the reaction, cool to 0℃ for 1N.
Neutralize with hydrochloric acid and distill off the ethanol under reduced pressure.
Add 300 ml of water to the aqueous layer, wash twice with ether, add 150 ml of 1N hydrochloric acid while cooling to 0°C, and extract with 400 ml of ethyl acetate. The ethyl acetate layer is washed with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained oil is dried under reduced pressure in a desiccator.
The obtained powder was reprecipitated with etherhexane to obtain 65.0 g (yield 94.5%) of [19]. Melting point; 56-63℃ TLC; Rf 1 = 0.58 [α] 22 D ; -47.1° (C = 1, DMF) Elemental analysis [as C 26 H 39 O 7 N 3 ] C% H% N% Measured value 61.52 7.71 8.50 Calculated value 61.76 7.78 8.31 20 P (127-131); BOC―Ser(Bzl)―Leu―
Pro-Ser (Bzl)-Pro-OBzl [20] [17] Add 30 ml of methylene chloride to 58 g (0.12 M), add 350 ml of TFA while cooling to 0°C, and then cool at room temperature.
Stir for 30 minutes. After the reaction, TFA is distilled off under reduced pressure and the residue is dissolved in 150 ml of DMF. This was cooled to 0°C and neutralized with 50ml of NMM, [19] 61.1g (0.12M),
16.2 g (0.12 M) of HOBT is added, and then 22 ml (0.12 M) of WSCI is added dropwise while cooling to 0°C. 1 at 0℃
Stir at room temperature overnight. After reaction, under reduced pressure
DMF is distilled off, and 400 ml of ethyl acetate is added to the residue. Wash with 5% sodium bicarbonate solution (3 times), 1N hydrochloric acid (3 times), and water (3 times) in this order, dry over anhydrous magnesium sulfate, and concentrate under reduced pressure. The resulting oil was dissolved in as little benzene as possible and charged to a 500 g column of silica gel packed with benzene. Benzene 500ml, benzene-ethyl acetate (10:1)
2. Benzene-ethyl acetate (10:1) 2. Benzene-ethyl acetate (3:1) 2. Benzene-
Elute with ethyl acetate (1:1) (4) and ethyl acetate (3) in this order, collect the corresponding fractions by TLC, and dry under reduced pressure to obtain 84.07 g (yield: 80.8%) of [20]. Melting point; 58-65℃ TLC; Rf 1 = 0.51 [α] D ; -63.7゜ (C = 1, DMF) Elemental analysis [as C 48 H 63 O 10 N 5 ] C% H% N% Measured value 66.31 7.25 8.09 Calculated value 66.26 7.30 8.05 Amino acid analysis; Ser1.42(2), Pro(2), Leu1.01(2) 21 P(127-131); BOC―Ser(Bzl)―Leu―
Dissolve 74.4g (85.4mM) of Pro-Ser (Bzl)-Pro-OH [21] [20] in 30ml of methanol, and add 1N-NaOH aqueous solution while cooling to 0℃.
After dropping 110 ml (1.3 times M), stir at room temperature for 3 hours. After the reaction, 26 ml of 1N hydrochloric acid is added to neutralize the mixture while cooling to 0°C, and methanol is distilled off under reduced pressure. The aqueous solution was washed twice with ether and cooled to 0°C for 1N.
Add 86 ml of hydrochloric acid and extract with ethyl acetate. The ethyl acetate layer is washed twice with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Add hexane to the residue.
Collect the resulting precipitate, dissolve it in as little chloroform as possible, and charge it to a silica gel column filled with chloroform. Elute in the following order: chloroform, chloroform-ethyl acetate (1:1), and chloroform-ethanol-ethyl acetate (5:1:5). The corresponding fractions were collected by TLC and dried under reduced pressure to obtain 53.75 g (yield: 81%) of [21]. TLC; Rf 1 = 0.34 [α] D ; -66.4゜ (C = 1, DMF) Elemental analysis [as C 41 H 57 O 10 N 10 ] C% H% N% Measured value 63.11 7.66 8.85 Calculated value 63.14 7.37 8.98 Amino acid analysis [0.779mg/6N hydrochloric acid 0.3ml, 105℃,
20 hours]; Ser1.84(2), Pro2.06(2), Leu(1) 22 P(127-145); BOC―Ser(Bzl)―Leu―
Pro―Ser(Bzl)―Pro―Ser(Bzl)―Arg
(Tos) -Leu-Pro-Gly-Pro-Ser (Bzl)-
Asp (OBzl) - Thr (Bzl) - Pro - Ile - Leu -
Add methylene chloride to 63.66 g (29.17 mM) of Pro-Gln-OBzl [22] [16], add 260 ml of TFA while cooling to 0°C, and stir at room temperature for 40 minutes. After reaction, under reduced pressure
TFA is distilled off and ether is added to the residue. The resulting precipitate is collected and dried under reduced pressure in a caustic desiccator overnight. 67.6g. Dissolve this in 200 ml of DMF (PH 4) and adjust the pH to 7 by adding 4.21 ml of NMM while cooling to -10°C. Next is HOBT4.73g
(35mM), [21] 27.30g (35mM) and
Add 100ml of DMF and cool to -10℃.
After dropping 6.41ml (35mM) of WSCI, incubate at -10℃ for 2 hours.
Stir at room temperature overnight. After the reaction, the solvent is distilled off under reduced pressure and the residue is poured into 1.5 g of ice-cold water. The resulting precipitate is suspended in water and collected. This suspension and removal operation is repeated 5 times (PH6). After drying the precipitate under reduced pressure for 2 days, it was dissolved in 500 ml of methanol and concentrated under reduced pressure. Add benzene to the residue and concentrate under reduced pressure. This benzene treatment is carried out twice, ether is added to the concentrate, and the resulting precipitate is collected. The above methanol treatment to ether treatment was repeated 8 times to obtain 79.85 g (yield 96.2%) of [22]. Melting point; 127-130℃ TLC; Rf 2 = 0.61 [α] 22 D ; -67.58゜ (C = 1, DMF) Elemental analysis [as C 148 H 207 O 36 N 23 S] C% H% N% Measured value 61.05 6.96 11.03 Calculated value 60.95 7.15 11.05 Amino acid analysis [1.893mg/6N hydrochloric acid 0.5ml, 105℃,
21 hours]; Asp0.99(1), Thr0.96(1), Ser3.74
(4), Glu1.04(1), Pro6.00(6), Gly0.98(1),
Ile0.95(1), Leu(3), Arg0.94(1) 23 hCG [127-145] [22] Add 5 ml of anisole to 1 g (0.34 mM), and add anhydrous hydrogen fluoride while cooling to 0°C. (HF)30
ml and stir for 1 hour. After reaction, under reduced pressure
HF is quickly distilled off and the resulting residue is mixed with ether.
Add 100ml and 50ml of 0.1N acetic acid and shake. Charge the aqueous layer to a column (3.3 x 18 cm) of Dowex 1 (acetic acid form) and eluted with 0.1N aqueous acetic acid. The effluent and washings are combined and lyophilized to obtain 660 mg of crude product. Dissolve this in 30ml of 8M urea aqueous solution, adjust the pH to 9 with aqueous ammonia, and then
-Charge 20 columns (4.5 x 120cm), 0.1N
Elute with acetic acid water. Fractionate the eluate into 10 ml portions and
The ~64th section was collected and lyophilized to yield 540 mg. Dissolve this in 30ml of 0.1N acetic acid water, charge it to a carboxymethylcellulose column (5 x 13cm), and add 0.01M ammonium acetate buffer (PH4.5).
After washing with ammonium acetate buffer (PH4.5)
Elute by applying a linear concentration gradient between 400 ml each of 0.01M and 0.1M. Fractionate the eluate into 6 ml portions, 79-86
The main section is collected and freeze-dried to obtain 180 mg.
Dissolve this in 0.1N acetic acid water and use Sephadex LH.
-Charge 20 columns (3 x 17cm) to 0.1N
Elute with acetic acid water. The eluate was fractionated into 10.5ml portions.
Collect the 25th to 30th sections and freeze-dry them to obtain hCG [127
-145] Obtain 136.4 mg. Melting point; 215-225℃ [α] 28 D ; +65゜ (C = 0.1, 0.1N acetic acid water) TLC; Rf 11 = 0.71 Amino acid analysis; Asp1.05(1), Thr0.97(1),
Ser3.44(4), Glu1.10(1), Pro6.50(6),
Gly1.04(1), Ile0.89(1), Leu3(3), Arg1.02
(1) Reference example 2 hCG [118-145]; H-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 P(118-145); BOC-Ser(Bzl)-Ser(Bzl)
-Ser(Bzl)-Ser(Bzl)-Lys(Z-Cl)-
Ala―Pro―Pro―Pro―Ser (Bzl)―Leu―Pro
―Ser(Bzl)―Pro―Ser(Bzl)―Arg(Tos)
―Leu―Pro―Gly―Pro―Ser(Bzl)―Asp
(OBzl)-Thr(Bzl)-Pro-Ile-Leu-Pro
-Gln-OBzl[34] Add 200ml of methylene chloride to 79g (28mM) of [22] described in Reference Example 1, and cool to 0°C.
After adding 400 ml of TFA, stir at room temperature for 50 minutes.
After the reaction, TFA is distilled off under reduced pressure, ether is added to the residue, the resulting precipitate is collected, and then dried under reduced pressure in a caustic desiccator overnight. 85.35g.
Dissolve this product in 270 ml of DMF (PH3) and -10℃
Adjust the pH to 6 with 4.58ml of NMM under cooling. This is then followed by P(118-126) [BOC-Ser(Bzl)-Ser
(Bzl)-Ser(Bzl)-Ser(Bzl)-Lys(Z-Cl)
-Ala-Pro-Pro-Pro-OH, melting point: 125-135
℃, [α] D ; -56.4゜ (C=1, DMF), TLC; Rf 6
= Near the origin, Rf 5 = 0.45, amino acid analysis; Ser3.23
(4), Pro3(3), Ala0.98(1), Lys0.97(1)〕49.93g
(33.6mM) and then add 100ml of DMF to make a solution. WSCI6.20 under cooling at −10℃
ml was added dropwise (PH5), and the mixture was stirred at -10°C for 2 hours and at room temperature for 2 hours. After the reaction, the solvent is distilled off under reduced pressure and the residue is poured into 1.5 liters of cold water. The resulting precipitate is obtained, suspended in water, washed repeatedly, and then dried under reduced pressure in a desiccator for 2 days. 130g. Next, methanol is added and concentrated under reduced pressure, benzene is added to the residue and concentrated under reduced pressure, and ether is added to the residue for recrystallization, which is repeated 12 times. Furthermore, methanol was added and concentrated under reduced pressure, and the residue was recrystallized by adding ether to obtain 108.43 g (yield: 91.9%) of [34]. Melting point: 129-131℃ [α] 24 D : -72.76゜ (C=1, DMF) TLC: Rf 2 = 0.56 Elemental analysis [as C 220 H 294 O 51 N 33 SCl] C% H% N% Measured value 61.49 7.07 11.02 Calculated value 61.67 6.92 10.79 Amino acid analysis [0.978mg/6N hydrochloric acid 0.5ml, 105℃,
21 hours]; Asp1.00(1), Thr0.96(1), Ser6.87
(8), Glu1.05(1), Pro9.63(9), Gly1.03(1),
Ala1.09(1), Ile0.94(1), Leu3(3), Lys1.09
(1), Arg0.97(1) 2 hCG [118-145] [34] Add 10 ml of anisole to 3.0 g (0.71 mM), introduce 80 ml of anhydrous hydrogen fluoride (HF),
Stir at 0°C for 1 hour. After the reaction, HF is distilled off under reduced pressure. Add ether to the residue and collect the resulting precipitate. Dissolve this powder in 50 ml of 0.1N acetic acid water, and apply it to a Dowex 1 (acetic acid type) column (3.5 x 35 cm).
Charge and wash with 0.1N acetic acid. Combine the effluent and washing solution and freeze-dry to obtain 1.46 g. this
Dissolve in 30 ml of 8M urea aqueous solution, adjust the pH to 9 with aqueous ammonia at 0°C, charge to a Sephadex LH-20 column (4.5 x 120 cm), and elute with 0.1N aqueous acetic acid. The eluate was fractionated into 7 ml portions, and the 55th to 83rd fractions were collected and lyophilized to obtain 990 mg. This was dissolved in 30 ml of 0.1N acetic acid water and charged to a carboxymethyl cellulose column (5 x 14 cm). 0.01M ammonium acetate buffer (PH4.5) 200
After washing with ml ammonium acetate buffer (PH4.5)
Elution is performed by applying a linear concentration gradient between 400 ml each of 0.01M and 0.1M. Fractionate the eluate into 6 ml portions, 85~
The 115th section was collected and lyophilized to obtain 850 mg. This was dissolved in 0.1N acetic acid water and charged to a Sephadex LH-20 column (4 x 120 cm).
Elute with 0.1N acetic acid water. Fractionate the eluate into 10.5 ml portions, collect the 35th to 43rd fractions, and freeze-dry to 650 ml.
Get mg. Dissolve this in 30 ml of 0.1N acetic acid water, and re-dissolve it in a carboxymethyl cellulose column (5 x 15
cm). Wash with 200 ml of 0.01M ammonium acetate buffer (PH4.5), and then elute by applying a linear concentration gradient between 400 ml each of 0.01M and 0.1M ammonium acetate buffer (PH4.5). eluate
Fractionate 6.5ml each, collect the 100th to 111th fractions,
Lyophilize to obtain 480 mg. This was dissolved in 0.1N acetic acid water, and a Sephadex LH-20 column (3x
117cm) and elute with 0.1N acetic acid water.
Fractionate the eluate into 10.5 ml portions, collect the 24th to 30th fractions, and freeze-dry to obtain 320 mg of hCG [118-145]. Melting point: Decomposes above 220℃ TLC: Rf 11 = 0.57 Amino acid analysis: Asp1.07(1), Thr0.97(1),
Ser5.23(8), Glu1.13(1), Pro9.86(9),
Gly1.07(1), Ala1.06(1), Ile0.89(1), Leu3
(3), Lys1.08(1), Arg1.07(1)
第１図は、BSA―hCGβ鎖を用いて得られた抗
血清を用いる測定結果を示し、第２図は、hCG
（127―145）重合体を用いて得られた抗血清を用
いる測定結果を示し、第３図は検量線を示す。
Figure 1 shows the measurement results using antiserum obtained using BSA-hCG β chain, and Figure 2 shows the results of measurements using the antiserum obtained using BSA-hCG β chain.
The results of measurements using antiserum obtained using the (127-145) polymer are shown, and FIG. 3 shows the calibration curve.
Claims (1)
エンザイムイミユノアツセイにおいて、脱シアリ
ン酸化hCG―酵素結合体を酵素標識体として用い
ることを特徴とするhCGの測定法。 ２ エンザイムイミユノアツセイが、競争反応に
基く方法である特許請求の範囲第１項記載のhCG
の測定法。 ３ 競争反応に基く方法が、第２抗体を用いる競
争反応に基く方法である特許請求の範囲第２項記
載のhCGの測定法。 ４ 競争反応に基く方法が、不溶化抗体を用いる
競争反応に基く方法である特許請求の範囲第２項
記載のhCGの測定法。 ５ 酵素が、β―ガラクトシダーゼである特許請
求の範囲第１項，第２項，第３項または第４項記
載のhCGの測定法。[Scope of Claims] 1. A method for measuring hCG, which comprises using a desialylated hCG-enzyme conjugate as an enzyme label in an enzyme immunoassay for human gonadotropin (hCG). 2. The hCG according to claim 1, wherein the enzyme immunoassay is a method based on a competitive reaction.
measurement method. 3. The method for measuring hCG according to claim 2, wherein the method based on a competitive reaction is a method based on a competitive reaction using a second antibody. 4. The method for measuring hCG according to claim 2, wherein the method based on a competitive reaction is a method based on a competitive reaction using an insolubilized antibody. 5. The method for measuring hCG according to claim 1, 2, 3, or 4, wherein the enzyme is β-galactosidase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6998481A JPS57184970A (en) | 1981-05-08 | 1981-05-08 | Measuring method for hcg |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6998481A JPS57184970A (en) | 1981-05-08 | 1981-05-08 | Measuring method for hcg |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS57184970A JPS57184970A (en) | 1982-11-13 |
| JPS6332145B2 true JPS6332145B2 (en) | 1988-06-28 |
Family
ID=13418433
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6998481A Granted JPS57184970A (en) | 1981-05-08 | 1981-05-08 | Measuring method for hcg |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS57184970A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5990054A (en) * | 1982-11-15 | 1984-05-24 | Takeda Chem Ind Ltd | Method and reagent for immunochemical mesurement of human chorionic gonadotropin |
| CA1340927C (en) * | 1987-10-09 | 2000-03-14 | Peter S. Linsley | Method for increasing the sensitivity of assays for target ligand |
-
1981
- 1981-05-08 JP JP6998481A patent/JPS57184970A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS57184970A (en) | 1982-11-13 |
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