JPH04169598A - New peptide, its salt and production thereof - Google Patents
New peptide, its salt and production thereofInfo
- Publication number
- JPH04169598A JPH04169598A JP2294536A JP29453690A JPH04169598A JP H04169598 A JPH04169598 A JP H04169598A JP 2294536 A JP2294536 A JP 2294536A JP 29453690 A JP29453690 A JP 29453690A JP H04169598 A JPH04169598 A JP H04169598A
- Authority
- JP
- Japan
- Prior art keywords
- peptide
- casoxin
- followed
- tyr
- salt
- 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.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 49
- 150000003839 salts Chemical class 0.000 title claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 title claims description 4
- 101800002044 Casoxin-C Proteins 0.000 claims abstract description 22
- 108010006303 Carboxypeptidases Proteins 0.000 claims abstract description 7
- 102000005367 Carboxypeptidases Human genes 0.000 claims abstract description 7
- 239000004475 Arginine Substances 0.000 claims abstract 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract 2
- 210000004899 c-terminal region Anatomy 0.000 claims abstract 2
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 abstract description 33
- 230000000694 effects Effects 0.000 abstract description 10
- 108010076119 Caseins Proteins 0.000 abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 102000004142 Trypsin Human genes 0.000 abstract description 4
- 108090000631 Trypsin Proteins 0.000 abstract description 4
- 235000013336 milk Nutrition 0.000 abstract description 4
- 239000008267 milk Substances 0.000 abstract description 4
- 210000004080 milk Anatomy 0.000 abstract description 4
- 239000012588 trypsin Substances 0.000 abstract description 4
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 238000010438 heat treatment Methods 0.000 abstract description 3
- HPGXYQZKIMWRAM-UHFFFAOYSA-N 4-ethylmorpholin-4-ium;acetate Chemical compound CC(O)=O.CCN1CCOCC1 HPGXYQZKIMWRAM-UHFFFAOYSA-N 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 102000011632 Caseins Human genes 0.000 abstract 1
- 241000282898 Sus scrofa Species 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 230000009849 deactivation Effects 0.000 abstract 1
- 238000011534 incubation Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 210000000496 pancreas Anatomy 0.000 abstract 1
- 235000021246 κ-casein Nutrition 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 230000002401 inhibitory effect Effects 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 10
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 description 10
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 10
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 6
- 239000005018 casein Substances 0.000 description 6
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 6
- 235000021240 caseins Nutrition 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 229910000040 hydrogen fluoride Inorganic materials 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 108090000087 Carboxypeptidase B Proteins 0.000 description 3
- 102000003670 Carboxypeptidase B Human genes 0.000 description 3
- 239000002220 antihypertensive agent Substances 0.000 description 3
- 230000004531 blood pressure lowering effect Effects 0.000 description 3
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 102000015427 Angiotensins Human genes 0.000 description 2
- 108010064733 Angiotensins Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 229940030600 antihypertensive agent Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- -1 octadecylsilyl Chemical group 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108010085169 Lysine carboxypeptidase Proteins 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101150021948 SAM2 gene Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 125000005042 acyloxymethyl group Chemical group 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 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
- 238000000605 extraction Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、新規なノナペプチド、その塩及びその製法に
関する０本発明のペプチド及びその塩は、アンジオテン
シン転換酵素阻害活性を有し、血圧降下剤等の医薬とし
て有用である。Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a novel nonapeptide, a salt thereof, and a method for producing the same. It is useful as a medicine.
食品中に含まれる蛋白質は栄養効果のみならず種々の生
理活性を有することが知られている。It is known that proteins contained in foods have not only nutritional effects but also various physiological activities.
例えば牛乳蛋白質であるカゼインを酵素分解に付するこ
とにより、血圧降下作用を有するペプチドが得られるこ
とが特公昭６０−２３０８５号公報、特公昭６０−２３
０８６号公報、特公昭６０−２３０８７号公報等に開示
されている。For example, by subjecting casein, which is a milk protein, to enzymatic decomposition, a peptide having a blood pressure lowering effect can be obtained, as disclosed in Japanese Patent Publication No. 60-23085 and Japanese Patent Publication No. 60-23.
It is disclosed in Japanese Patent Publication No. 086, Japanese Patent Publication No. 60-23087, etc.
最近種々の蛋白質を酵素分解して得られるペプチドが種
々の生理活性を有することが確認されており、食品由来
の蛋白質のアミノ酸配列には、潜在的に生体機能をもつ
ことが推定されている。It has recently been confirmed that peptides obtained by enzymatically decomposing various proteins have various physiological activities, and the amino acid sequences of food-derived proteins are presumed to have potential biological functions.
本発明者らは、牛乳にカゼインのトリプシン分解物中よ
り強いオピオイド活性を有するペプチドを見出している
（吉用他、Ｊｏｕｒｎａｌ ｏｆ ＤａｉｒｙＲｅｓｅ
ｒｃｈ ５６巻、３６３〜３６６頁、 １９８９年）。The present inventors have discovered a peptide in milk that has stronger opioid activity than in the tryptic digest of casein (Yoshiyuki et al., Journal of DairyRese
rch vol. 56, pp. 363-366, 1989).
このペプチドのうちカソキシンＣ（Ｃａｓｏｘｉｎ Ｃ
）と命名されたデカペプチドは、次の一次式（ＩＩ）で
示されるペプチドで、オピオイド活性とともに強いアン
ジオテンシン変換酵素阻害活性を有しておリ、血圧降下
作用を示すので、血圧降下剤として有用であることが確
認された。本発明者らはこのカソキシンＣを存効成分と
する血圧降下剤を特許出願している（特願平２−１２８
８８）。Among these peptides, casoxin C (Casoxin C
) is a peptide represented by the following primary formula (II), which has both opioid activity and strong angiotensin-converting enzyme inhibitory activity, and exhibits a blood pressure lowering effect, making it useful as a blood pressure lowering agent. It was confirmed that The present inventors have filed a patent application for a hypotensive agent containing casoxin C as an active ingredient (Patent Application No. 128/1999).
88).
Ｈ−Ｔｙｒ−Ｉ ｉ、 ｅ−Ｐｒｏ−Ｉｌｅ−Ｇ Ｉ！
、ｎ−Ｔｙｒ−Ｖａ ｌ −Ｌｅｕ−Ｓｅｒ−Ａｒｇ−
ＯＨ（ＩＩ ）本発明者らはこのカソキシンＣの活性と
構造との関係についてさらに研究を進めた結果、より強
力なアンジオテンシン転換酵素阻害活性を有する新規ペ
プチドを見出し、本発明を完成するに至った。H-Tyr-I i, e-Pro-Ile-G I!
, n-Tyr-Val-Leu-Ser-Arg-
OH(II) As a result of further research into the relationship between the activity and structure of casoxin C, the present inventors discovered a new peptide with more potent angiotensin converting enzyme inhibitory activity and completed the present invention. .
カソキシンＣは、上述した式（ｎ）の構造を有するデカ
ペプチドである０本発明者らは、このＣ末端に存在する
アルギニン残基を酵素処理により除去したノナペプチド
〔デスアルギニル カソキシンＣ（Ｄｅｓ−Ａｒｇ−Ｃ
ｏｓｏｘｉｎ Ｃ） ）がカソキシンＣに比較し約２倍
のアンジオテンシン変換酵素阻害活性を有することを見
出した。カソキシンＣの末端を酵素処理をすることによ
り、アンジオテンシン阻害活性が上昇することは、これ
まで知られていないまったく新しい知見である。Casoxin C is a decapeptide having the structure of formula (n) described above. The present inventors have developed a nonapeptide [desarginyl casoxin C (Des-Arg- C
It has been found that osoxin C) has approximately twice the angiotensin converting enzyme inhibitory activity compared to casoxin C. It is a completely new finding that the angiotensin inhibitory activity is increased by enzymatically treating the terminal end of casoxin C.
本発明は、カソキシンＣのＣ末端に存在するＡｒｇを欠
失する新規で有用なノナペプチド及びその塩を提供する
ことを課題とする。An object of the present invention is to provide a novel and useful nonapeptide lacking Arg present at the C-terminus of casoxin C and a salt thereof.
本ペプチドは、食品由来のペプチドの中でも、最も強い
アンジオテンシン転換酵素阻害活性を有するペプチドの
一つで、血圧降下剤として特に有用である。This peptide is one of the peptides having the strongest angiotensin converting enzyme inhibitory activity among food-derived peptides, and is particularly useful as an antihypertensive agent.
（課題を解決するための手段）
本発明の新規ペプチドは牛にカゼインの配列に存在し、
次の式（１）で表されるペプチドまたは、その塩に関す
る。(Means for Solving the Problems) The novel peptide of the present invention exists in the sequence of casein in cattle, and
The present invention relates to a peptide represented by the following formula (1) or a salt thereof.
Ｈ−Ｔｙｒ−Ｉ １． ｅ−Ｐｒｏ−Ｉ Ｉ！ ｅ−Ｇ
ｌ ｎ−Ｔｙｒ−Ｖａ ｊ！ −Ｌｅｕ−Ｓｅｔ−Ｏ
Ｈ（’Ｉ ）
本発明のペプチドは、−Ｃに知られているペプチドの化
学合成によって得ることができる。また牛乳に一カゼイ
ンを酵素分解に付し、さらに高速液体クロマトグラフィ
ー処理を行って得られたカソキシンＣをカルボンキシペ
プチダーゼにより処理することによりカソキシンＣ末端
のＡｒｇを除去し、目的とするデスアルギニルカソキシ
ンＣを得ることができる。H-Tyr-I 1. e-Pro-I I! e-G
l n-Tyr-Va j! -Leu-Set-O
H('I) The peptides of the invention can be obtained by chemical synthesis of peptides known to -C. In addition, by enzymatically decomposing monocasein in milk and further performing high-performance liquid chromatography treatment, the casoxin C obtained is treated with carboxypeptidase to remove Arg at the casoxin C-terminus and obtain the desired desarginyl. Casoxin C can be obtained.
さらに化学合成により作成したカソキシンＣを同じくカ
ルボキシペプチダーゼを作用させることによっても得る
こともできる。カポキシペプチダーゼとしてはカルボキ
シペプチドダーゼＰのように、特定の末端配列を認識す
る酵素以外であればどのようなものでも使用できるが、
特に末端のＡｒｇに特異的に作用するカルボキシペプチ
ダーゼＢ、カルボキシペプチダーゼＮが好適である。Furthermore, casoxin C created by chemical synthesis can also be obtained by the action of carboxypeptidase. Any capoxypeptidase can be used as long as it is not an enzyme that recognizes a specific terminal sequence, such as carboxypeptidedase P.
In particular, carboxypeptidase B and carboxypeptidase N, which act specifically on terminal Arg, are preferred.
化学合成を行う場合には以下の方法で合成できる。When performing chemical synthesis, it can be synthesized by the following method.
合成装置として、バイオサーチ社製のＳＡＭ２ペプチド
合成装置を用い、ペプチドの担体としての樹脂からの脱
離と保護基の除去は、１０％アニソールを含む無水フッ
化水素中で、０°Ｃの温度条件下に１時間撹拌すること
により行う。A SAM2 peptide synthesizer manufactured by BioSearch was used as a synthesis apparatus, and the desorption of the peptide from the resin as a carrier and the removal of the protecting group were performed at a temperature of 0°C in anhydrous hydrogen fluoride containing 10% anisole. This is done by stirring for 1 hour under these conditions.
上記フッ化水素を留去した後、樹脂をエーテルで洗浄し
、３０％酢酸によりペプチドを抽出する。After distilling off the hydrogen fluoride, the resin is washed with ether and the peptides are extracted with 30% acetic acid.
抽出により得られたペプチドは、オクタデシルシリル（
ＯＤＳ）カラム（Ｃｏｓ＋＋＋ｏｓｉｌ ５ＣＩｌ＋＋
２ Ｘ ２５ ＣＩ）による逆相高速液体クロマトグ
ラフィにより精製する。The peptides obtained by extraction are octadecylsilyl (
ODS) column (Cos+++osil 5CIl++
Purify by reverse phase high performance liquid chromatography on 2×25 CI).
目的とする本ペプチドは、０．１％トリフルオロ酢酸を
含むアセトニトリルの直線的濃度勾配による展開の際に
、３７％アセトニトリルの濃度でカラムから溶出するこ
とによって得ることができる。The desired peptide can be obtained by elution from the column at a concentration of 37% acetonitrile during development with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid.
ペプチドをカゼインから得る場合には上述したジャーナ
ル オブ デイリー リサーチ誌に記載される方法に従
って酵素加水分解し、ＨＰＬＣ処理を行って精製して得
ることができる。即ち、に−カゼインをトリプシンによ
り３７°Ｃで酵素分解し、次いで酵素を１００°Ｃで１
０分間加熱処理し、失活させた後、遠心を行い上清を得
る。この上清をオクタデシルシリル（ＯＤＳ）カラム（
Ｃｏｓ＋＋＋ｏｓｉｌ ５Ｃ＋ｓ）などの逆相ＨＰＬＣ
カラムにより、アセトニトリル０〜６０％濃度勾配で溶
出し、アセトニトリル濃度２３％でカソキシンＣを得る
ことができる。このようにして得られたカソキシンＣを
カルボキシペプチダーゼを加え、ｐＨ８，５前後のアル
カリ性下で３７°Ｃで１〜５時間反応させ、次いでＯＤ
Ｓカラムを装着した高速液体クロマトグラフにて０．１
％トリフルオロ酢酸を含むアセトニトリルの直線的濃度
勾配にて展開し、３７％アセトニトリルで溶出する両分
を回収することで目的とするペプチドを得ることができ
る。When peptides are obtained from casein, they can be obtained by enzymatic hydrolysis and purification by HPLC treatment according to the method described in the above-mentioned Journal of Daily Research. Namely, casein was enzymatically digested with trypsin at 37°C, and then the enzyme was digested at 100°C for 1 time.
After inactivation by heat treatment for 0 minutes, centrifugation is performed to obtain a supernatant. This supernatant was applied to an octadecylsilyl (ODS) column (
Reverse phase HPLC such as Cos+++osil 5C+s)
The column is eluted with an acetonitrile concentration gradient of 0 to 60%, and casoxin C can be obtained at an acetonitrile concentration of 23%. Carboxypeptidase was added to the casoxin C obtained in this way, and the reaction was carried out at 37°C for 1 to 5 hours under alkaline conditions around pH 8.5, and then the OD
0.1 using a high performance liquid chromatograph equipped with an S column.
The target peptide can be obtained by developing with a linear concentration gradient of acetonitrile containing 37% trifluoroacetic acid and collecting both fractions eluted with 37% acetonitrile.
又トリプシン分解後の分解液をさらにカルボキシペプチ
ダーゼ、３７°Ｃ１〜５時間処理し、Ｉ（ＰＬＣを行う
ことによっても目的とするペプチドを得ることができる
。The target peptide can also be obtained by further treating the decomposition solution after trypsin digestion with carboxypeptidase at 37°C for 1 to 5 hours and performing I (PLC).
このようにして得られたペプチドをアミノ酸シーケンサ
−による配列分析およびアミノ酸組成により特定するこ
とができる。The peptide thus obtained can be identified by sequence analysis using an amino acid sequencer and amino acid composition.
また、本発明のペプチドは、酢酸、塩酸等と塩を形成す
る。本発明では、これらの塩をも包含する。Furthermore, the peptide of the present invention forms salts with acetic acid, hydrochloric acid, and the like. The present invention also includes these salts.
このようにして得られた新規ペプチドは血圧降下作用を
有する。これらの生理活性は以下のように確認された。The novel peptide thus obtained has a blood pressure lowering effect. These physiological activities were confirmed as follows.
■アンジオテンシン転換酵素阻害作用
ウサギ肺アセトンパウダーから抽出したアンジオテンシ
ン転換酵素を用い、ＣｕｓｈｍａｎとＣｈｅｕｎｇの方
法（Ｂｉｏｃｈｅｍ、、 Ｐｈａｒｍ、 、２０．１６
３７〜１６４８ （１９７１） ）に従って測定した。■ Angiotensin converting enzyme inhibitory effect Using angiotensin converting enzyme extracted from rabbit lung acetone powder, the method of Cushman and Cheung (Biochem, Pharm, 20.16)
37-1648 (1971)).
本発明のペプチドのＩＣ，。は２．０μｈであった。IC of the peptide of the invention. was 2.0 μh.
同時に測定したカソキシンＣのＩＣ５゜は３．８μＨで
あった。従って、本発明のペプチドはカソキシンＣにく
らべて約２倍のアンジオテンシン変換酵素阻害作用を有
する。本発明のペプチドのこのように高いアンジオテン
シン変換酵素阻害活性は食品由来の同種のペプチド中で
も特に強い活性を有するものの一つである。The IC5° of casoxin C measured at the same time was 3.8 μH. Therefore, the peptide of the present invention has an angiotensin converting enzyme inhibitory effect approximately twice that of casoxin C. The peptide of the present invention has such high angiotensin-converting enzyme inhibitory activity, making it one of the most potent peptides among similar peptides derived from food.
以下に実施例を示し、本発明の詳細な説明する。EXAMPLES The present invention will be explained in detail by way of examples below.
実施例１
（カルボキシペプチダーゼによるＨ−Ｔｙｒ−Ｉｌｅ−
Ｐｒ。Example 1 (H-Tyr-Ile- by carboxypeptidase)
Pr.
−Ｉ Ｉ！ ｅ−Ｇ １． ｎ−Ｔｙｒ−Ｖａ ４２−
Ｌｅｕ−３ｅｒ−ＯＨの調製）牛乳にカゼイン１０ｇ／
ｍに１／１００量のブタ膵臓トリプシン（シグマ社製タ
イプ■）を添加し、ｐＨ７で３７°Ｃ５時間反応させ、
次いで１００°Ｃ１０分間の加熱により酵素を失活させ
て得た加水分解物を吉川らの方法（Ｊｏｕｒｎａｌ ｏ
ｆ Ｄａｉｎｙ Ｒｅ５ｅａｒｃｈ５６巻３６３〜３６
６．１９８９年）に従って行い、ＨＰＬＣで精製してカ
ソキシンＣを得た。 このカソキシンＣを５１ｇ／Ｉ１
１となるように２００＋＊Ｍ Ｎ−エチルモルホリン
酢酸緩衝液（ｐＨ８，５）に溶解し、次いでカソキシン
Ｃの１／２０量のブタ腎臓カルボキシペプチダーゼＢ（
シグマ社製タイプｎ［−ＣＰ）を添加し、３７°Ｃで５
時間インキュベートした。次いでＯＤＳカラム（Ｃｏｓ
ｍｏｓｉｆ ５Ｃ＋ｓ 、４．６ Ｘ １５０１１１．
ナカライテスク製）を装置した高速液体クロマトグラフ
（ＨＰＬＣ）にて、０．１％トリフルオロ酢酸を含むア
セトニトリルの直線的濃度勾配（０〜４０％／４０ｍ１
ｎ 、 １ ｄ／ｓｉｎ ）にて展開した。３７％ア
セトニトリルで溶出する画分（Ａ）（第１図参照）を回
収し、目的とする本発明のペプチドを得た。-I I! e-G 1. n-Tyr-Va 42-
Preparation of Leu-3er-OH) 10g casein/milk
Add 1/100 amount of porcine pancreatic trypsin (type ■ manufactured by Sigma) to m and react at 37°C for 5 hours at pH 7.
Next, the enzyme was inactivated by heating at 100°C for 10 minutes, and the resulting hydrolyzate was subjected to the method of Yoshikawa et al.
f Dainy Research Volume 56 363-36
6.1989) and purified by HPLC to obtain casoxin C. This casoxin C is 51g/I1
1 in 200+*M N-ethylmorpholine acetate buffer (pH 8.5), and then 1/20 amount of porcine kidney carboxypeptidase B (
Sigma type n [-CP) was added and
Incubated for hours. Next, an ODS column (Cos
mosif 5C+s, 4.6 X 150111.
A linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid (0 to 40%/40ml
n, 1 d/sin). Fraction (A) (see Figure 1) eluted with 37% acetonitrile was collected to obtain the desired peptide of the present invention.
この画分は強いアンジオテンシン転換酵素の阻害活性を
有していたが、動脈収縮活性はなく、この点においてカ
ソキシンＣと活性が相違していた。Although this fraction had strong angiotensin converting enzyme inhibitory activity, it did not have arterial constriction activity, and in this respect it differed in activity from casoxin C.
得られたペプチドをペプチドシーケンサ（アブライドバ
イオシステムズ社４７７ Ａ型）によす分析したところ
Ｔｙｒ−Ｉｌｅ−Ｐｒｏ−Ｉｌｅ−Ｇ １． ｎ−Ｔｙ
ｒ−Ｖａ ｉ！、 −Ｌｅｕ−３ｅｒの配列を有してい
た。又ペプチドのアミノ酸組成比をアミノ酸分析計によ
り分析したところＴｙｒ： Ｉ ｌ ｅ：Ｐｒｏ：Ｇ
ｌ ｎ：Ｖａ １ ：Ｌｅｕ：Ｓｅｒの各アミノ酸比が
２：２：１：１：１：１：１となり理論値と一致した。When the obtained peptide was analyzed using a peptide sequencer (Abride Biosystems, Type 477 A), Tyr-Ile-Pro-Ile-G 1. n-Ty
r-Va i! , -Leu-3er sequence. In addition, when the amino acid composition ratio of the peptide was analyzed using an amino acid analyzer, it was found that Tyr:Ile:Pro:G
The amino acid ratio of ln:Va1:Leu:Ser was 2:2:1:1:1:1:1, which coincided with the theoretical value.
又本ペプチドをＫｉｓｅ１２ｇｅｌｚｓａ （メルク
製）を用いた１属クロマトグラフィーを行った。展開溶
媒としてｎブタノール：酢酸：ピリジン：水＝１５：３
：１０：１２を使用し、展開させたところＲｆ値は０．
６９を示した。This peptide was also subjected to genus 1 chromatography using Kise12gelzsa (manufactured by Merck). n-butanol:acetic acid:pyridine:water=15:3 as developing solvent
:10:12 and when expanded, the Rf value was 0.
It showed 69.
実施例２
（合成による製造）
Ｓａｗ ｔｗｏペプチド合成装置（Ｂｉｏｓｅａｒｃｈ
社製）により、同装置の標準プロトコールに従って合成
した。Example 2 (Production by synthesis) Saw two peptide synthesizer (Biosearch
(manufactured by S.A., Inc.) according to the standard protocol of the same device.
即ち１ｇ当り０．４ｍｍｏ１のｔ−Ｂｏｃ−Ｓｅｒ （
Ｂｚ ｌ ）を結合したアシルオキシメチル樹脂２ｇを
ペプチド合成装置の反応容器にセットし、４５％（ｖ／
ｖ） ）リフルオロ酢酸、２．５％（ｖ／ｖ）アニソ
ール、５２．５％（ｖ／ｖ）ジクロロメタンを含むデブ
ロツタ液と２０分間接触させｔ−Ｂｏｃ基を除いた。ジ
クロロメタンによる洗浄の後、１０％（ｖ／ｖ）ジイソ
プロピルエチルアミンを含むジクロロメタンにて樹脂中
和し、ジクロロメタンにより洗浄した。その後、５．３
ｍ閣ｏｌのｔ−Ｂｏｃ−Ｐｒｏおよび５．３＋ｍｏＩ！
、のジイソプロピルカルボジイミド（それぞれ理論当量
の６．７倍）を含む２７Ｍ１のジクロロメタン、ジメチ
ルフォルムアミド混合液中で２時間室温にて反応せしめ
た。That is, 0.4 mmol of t-Boc-Ser per 1 g (
2 g of acyloxymethyl resin bound with Bz l ) was set in the reaction vessel of the peptide synthesizer, and the
v)) The t-Boc group was removed by contacting with a debrotta solution containing lifluoroacetic acid, 2.5% (v/v) anisole, and 52.5% (v/v) dichloromethane for 20 minutes. After washing with dichloromethane, the resin was neutralized with dichloromethane containing 10% (v/v) diisopropylethylamine, and washed with dichloromethane. After that, 5.3
m cabinet ol's t-Boc-Pro and 5.3+ moI!
, diisopropylcarbodiimide (each 6.7 times the theoretical equivalent) was reacted for 2 hours at room temperature in a 27M1 mixture of dichloromethane and dimethylformamide.
ジメチルフォルムアミドおよびジクロロメタンにて順次
洗浄した後、上記と同様にデブロッキングを行い、以下
同様にＣ末端側からｔ−Ｂｏｃ−Ｌｅｕ−、ｔ−Ｂｏｃ
−Ｖａ ｆ 、 ｔ−Ｂｏｃ−Ｔｙｒ（Ｃｌ ｚ−Ｂｚ
１ ）＋ ｔ−Ｂｏｃ−Ｇ ｌ ｎ、 ｔ−Ｂｏｃ−
Ｉｊ２ｅ、Ｂｏｃ−Ｐｒｏ、Ｂｏｃ−ＩＩ！、ｅ、Ｂｏ
ｃ−Ｔｙｒ（ＣＪＩ！ｚ−Ｂｚｆ）を順次結合せしめ、
ｔ−Ｂｏｃ−Ｔｙｒ （Ｃ１ｚ Ｂｚｌ）−Ｉｌｅ−Ｐ
ｒｏ−Ｉｌｅ−Ｇ ｉ！ ｎ−Ｔｙｒ （Ｃｌ ｚ−Ｂ
ｚ １ ）−Ｖａ ｌ −Ｌｅｕ−５ｅｒ（Ｂｚ １．
）樹脂を得た。After sequentially washing with dimethylformamide and dichloromethane, deblocking was performed in the same manner as above, and t-Boc-Leu-, t-Boc
-Va f , t-Boc-Tyr(Cl z-Bz
1) + t-Boc-G ln, t-Boc-
Ij2e, Boc-Pro, Boc-II! ,e,Bo
sequentially combine c-Tyr (CJI!z-Bzf),
t-Boc-Tyr (C1z Bzl)-Ile-P
ro-Ile-G i! n-Tyr (Cl z-B
z1)-Val-Leu-5er(Bz1.
) Resin was obtained.
この樹脂を１０％アニソールを含む無水フッ化水素中で
２時間０°Ｃにて反応させた後、フッ化水素の留去およ
びエーテルによる洗浄を行った。得られたペプチドおよ
び樹脂の混合物から３０％酢酸にてペプチドを抽出し凍
結乾燥によって約７００■の粗ペプチドを得た。This resin was reacted in anhydrous hydrogen fluoride containing 10% anisole at 0°C for 2 hours, and then the hydrogen fluoride was distilled off and washed with ether. The peptide was extracted from the resulting mixture of peptide and resin with 30% acetic acid and freeze-dried to obtain about 700 μg of crude peptide.
粗ペプチドを０．１％トリフルオロ酢酸に溶解した後、
オクタデシルシラン（ＯＤＳ）カラム（Ｃｏｓｗｏｓｉ
１５Ｃ＋ｓ、２５０Ｘ２０ｓｍナカライテスク社製）を
接続した高速液体クロマトグラフ（Ｍ２Ｏ３型、日本ウ
ォータース社製）により、０．１％のトリフルオロ酢酸
を含むアセトニトリルの直線的濃度勾配（０〜５０％１
５０分、１０ｄ／分ニテ展開した。目的とするペプチド
は第１図と同様にアセトニトリル濃度的３７％にて溶出
された。このようにして得られた物質がＴｙｒ−１Ｉｌ
ｅ−Ｐｒｏ４１ｅ−Ｇｌｎ−Ｔｙｒ−Ｖａ １−Ｌｅｕ
−３ｅｒであることはアミノ酸分析およびプロティンシ
ーケンサ（アプライドバイオシステムズ製４７７Ａ）に
よりｉ認された。After dissolving the crude peptide in 0.1% trifluoroacetic acid,
Octadecylsilane (ODS) column (Coswosi
A linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid (0 to 50% 1
It was developed for 50 minutes at 10 d/min. The target peptide was eluted at an acetonitrile concentration of 37% as in FIG. The substance thus obtained is Tyr-1Il
e-Pro41e-Gln-Tyr-Va 1-Leu
-3er was confirmed by amino acid analysis and protein sequencer (Applied Biosystems 477A).
（発明の効果）
本発明の実施により強いアンジオテンシン変換酵素阻害
活性を有する新規なペプチドが提供される。このペプチ
ドは強いアンジオテンシン阻害作用を有し、血圧降下剤
等の医薬などに有用に利用される。(Effects of the Invention) By carrying out the present invention, a novel peptide having strong angiotensin-converting enzyme inhibitory activity is provided. This peptide has a strong angiotensin inhibitory effect and is useful in medicines such as antihypertensive agents.
第１図は、カソキシンＣをカルボキシペプチダーゼＢで
酵素分解した液の１（ＰＬＣパターンを示す。
図中Ａのピークが本発明ペプチドに相当する。FIG. 1 shows a PLC pattern of a solution obtained by enzymatically decomposing casoxin C with carboxypeptidase B. The peak A in the figure corresponds to the peptide of the present invention.
Claims (2)
その塩 Ｈ−Ｔｙｒ−Ｉｌｅ−Ｐｒｏ−Ｉｌｅ−Ｇｌｎ−Ｔｙｒ
−Ｖａｌ−Ｌｅｕ−Ｓｅｒ−ＯＨ（ I ）(1) A peptide represented by the following general formula (I) or its salt H-Tyr-Ile-Pro-Ile-Gln-Tyr
-Val-Leu-Ser-OH (I)
ペプチダーゼにより処理し、Ｃ末端のアルギニンを除去
することを特徴とする一般式（ I ）で表されるペプチ
ドの製造方法(2) A method for producing a peptide represented by general formula (I), which comprises treating Casoxin C with carboxypeptidase to remove C-terminal arginine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2294536A JPH04169598A (en) | 1990-10-31 | 1990-10-31 | New peptide, its salt and production thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2294536A JPH04169598A (en) | 1990-10-31 | 1990-10-31 | New peptide, its salt and production thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04169598A true JPH04169598A (en) | 1992-06-17 |
Family
ID=17809056
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2294536A Pending JPH04169598A (en) | 1990-10-31 | 1990-10-31 | New peptide, its salt and production thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04169598A (en) |
-
1990
- 1990-10-31 JP JP2294536A patent/JPH04169598A/en active Pending
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