JP3569966B2

JP3569966B2 - New peptide derivatives

Info

Publication number: JP3569966B2
Application number: JP19204594A
Authority: JP
Inventors: 直之山本; 巧田中
Original assignee: Wako Pure Chemical Industries Ltd; Fujifilm Wako Pure Chemical Corp
Current assignee: Fujifilm Wako Pure Chemical Corp
Priority date: 1994-07-22
Filing date: 1994-07-22
Publication date: 2004-09-29
Anticipated expiration: 2019-09-29
Also published as: JPH0834796A

Description

【０００１】
【発明の利用分野】
本発明は、プロテアーゼ、アミダーゼ、凝固酵素等の活性測定用基質として、特にエンドトキシンとカブトガニ血球成分（ａｍｏｅｂｏｃｙｔｅｌｙｓａｔｅ）との反応により活性化される凝固酵素の活性測定用基質として有用な新規なペプチド誘導体に関する。
【０００２】
【発明の背景】
エンドトキシンは、グラム陰性菌の細胞壁外膜に存在するリポ多糖（Ｌｉｐｏｐｏｌｙ− ｓａｃｃｈａｒｉｄｅ，ＬＰＳ）であり、強い発熱性物質として知られている。このため、注射用医薬品等におけるエンドトキシンの検出は重要と考えられており、米国や日本の薬局方にもエンドトキシン試験法が収載されている。また、エンドトキシンはグラム陰性菌感染症におけるショックの主な原因と考えられており、臨床診断上では、血漿中エンドトキシンの測定がグラム陰性菌感染症の診断、グラム陰性菌感染症の治療効果及び予後の判定、エンドトキシンショックの早期診断等に用いられている。
【０００３】
カブトガニの血球成分は、エンドトキシンによって活性化されて、凝固酵素（プロテアーゼ等）等の活性化反応やゲル化反応を生じる性質を有しており、医学、薬学、微生物学の分野ではこれを利用した、簡便で、安価なエンドトキシン検出法が、広く用いられている。この代表的な方法としては、例えば米国薬局法［Ｕ．Ｓ．ＰｈａｒｍａｃｏｐｅｉａＸＸ．８８８（１９８０）］に採用されたゲルの固さを肉眼で判定する方法、ゲル化に基づく濁度を測定する方法、クロット蛋白定量法等があるが、いずれもゲル化現象に基づいて判定を行う方法であるため測定精度の面で若干問題が生じる場合がある。
【０００４】
一方、カブトガニ血球成分とエンドトキシンとの反応により活性化される凝固酵素（プロテアーゼ）の作用により、例えばニトロフェノール、ニトロアニリン、メチルクマリン誘導体、インドキシル誘導体等の発色性化合物又は蛍光性化合物を遊離する合成ペプチド誘導体をエンドトキシン測定用の基質として使用する測定方法も報告されている（特公昭５９−１９５３２号公報、特公昭６１−５４４００号公報、特開昭５７−５０２２６６号公報等）。
【０００５】
このような方法に使用される合成基質に於いては、該凝固酵素による発色性基或は蛍光性基とペプチド残基との結合を開裂させるためには一般に２つのアミノ酸配列、即ちグリシルアルギニン残基（−Ｇｌｙ−Ａｒｇ−）が必要であることが判明しており、その代表的なものとしては例えば以下のようなものが挙げられる。
（１）Ｂｏｃ−Ｌｅｕ−Ｇｌｙ−Ａｒｇ−ｐＮＡ
（２）Ｂｏｃ−Ｓｅｒ（ＯＢｎ）−Ｇｌｙ−Ａｒｇ−ｐＮＡ
（３）Ｂｏｃ−Ｓｅｒ−Ｇｌｙ−Ａｒｇ−ｐＮＡ
（上記式中、「Ｂｏｃ」はｔ−ブトキシカルボニル基を、「Ｌｅｕ」はロイシン残基を、「ｐＮＡ」はｐ−ニトロアニリノ基を、「Ｓｅｒ」はセリン残基を、また「Ｂｎ」はセリン残基側鎖の水酸基と結合したベンジル基を夫々示す。）
【０００６】
しかしながら、これらペプチド誘導体はエンドトキシン定量の際の基質として用いるには問題が多い。
即ち、例えば上記（１）及び（２）で示されるペプチド誘導体には、該凝固酵素との反応性は高いものの水溶性が悪いため、該凝固酵素活性を効率よく測定するために必要な濃度の水溶液を得ることが困難であるという問題点が、また、上記（３）で示されるペプチド誘導体には、水溶性はよいものの該凝固酵素との反応性が低いため、これを基質として用いた場合には微量のエンドトキシンの測定が困難になるという問題点がある。
【０００７】
【発明の目的】
本発明は、上記した如き状況に鑑みなされたもので、プロテアーゼ、アミダーゼ、凝固酵素等の活性測定用基質として、特にエンドトキシンとカブトガニ血球成分（ａｍｏｅｂｏｃｙｔｅｌｙｓａｔｅ）との反応により活性化される凝固酵素の活性を簡便に且つ高感度に測定するための基質として好適なペプチド誘導体を提供することを目的とする。
【０００８】
【発明の構成】
本発明は、一般式［Ｉ］
Ｘ−Ｔｈｒ−Ｇｌｙ−Ａｒｇ−Ｙ［Ｉ］
（式中、Ｘはアミノ酸のＮ−末端保護基を表し、Ｔｈｒはスレオニン残基を表し、Ｇｌｙはグリシン残基を表し、Ａｒｇはアルギニン残基を表し、Ｙはアルギニン残基のＣ−末端と共有結合を形成している発色性基又は蛍光性基を表す。）
で示されるペプチド誘導体又はその酸付加塩、の発明である。
【０００９】
また、本発明は、一般式［Ｉ］で示されるペプチド誘導体又はその酸付加塩を基質として使用することを特徴とする、エンドトキシンの定量方法、の発明である。
【００１０】
更に、本発明は、エンドトキシンを含む試料と、カブトガニ血球成分と、一般式［Ｉ］で示されるペプチド誘導体又はその酸付加物とを反応させ、その結果遊離する発色性化合物又は蛍光性化合物の増加量を測定し、その測定結果に基づいて試料中のエンドトキシン量を定量することを特徴とするエンドトキシンの測定方法、の発明である。
【００１１】
更にまた、本発明は、カブトガニ血球成分と、一般式［Ｉ］で示されるペプチド誘導体又はその酸付加物とを、構成成分として含んで成るエントドキシン定量用試薬キット、の発明である。
【００１２】
即ち、本発明者らは、エンドトキシンとカブトガニ血球成分との反応の結果活性化される、例えばクロッティングエンザイム等の凝固酵素（プロテアーゼ）の活性を簡便に且つ高感度に測定することができる基質を求めて鋭意研究を重ねた結果、グリシルアルギニン残基（−Ｇｌｙ−Ａｒｇ−）のＮ−末端に更にスレオニンが結合しているペプチドを基本骨格とする一般式［Ｉ］で示されるペプチド誘導体は、水溶性に優れているため、酵素反応に用いる水溶液又は緩衝液等に目的の濃度に容易に溶解させることができ、しかも目的の凝固酵素との反応性が高いのでこれを基質として用いれば該酵素を高感度に測定することが可能となることを見出し本発明を完成するに至った。
【００１３】
一般式［Ｉ］で示されるペプチド誘導体に於いて、Ｘで表されるアミノ酸のＮ−末端保護基としては、アミノ酸やペプチドのＮ−末端保護基として通常この分野で用いられる基であれば特に限定されることなく挙げられるが、例えばアセチル基、ベンゾイル基等のアシル基、ベンジルオキシカルボニル基、ｔ−ブトキシカルボニル基、トシル基、グルタリル基等が好ましく挙げられる。
【００１４】
また、一般式［Ｉ］で示されるペプチド誘導体に於いて、Ｙで示される発色性基又は蛍光性基としては、エンドトキシンとカブトガニ血球成分との反応の結果活性化される凝固酵素によりペプチド残基との結合が切断されて、発色性或は蛍光性を示す化合物を生じさせ得る基（或は適当な化合物とカップリング等させることにより発色性或は蛍光性を示す物質となる化合物を生じさせ得る基）であれば特に限定されることなく挙げられるが、例えばｐ−ニトロアニリノ基、３−ヒドロキシメチル−４−ニトロアニリノ基、４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリノ基、４−（Ｎ，Ｎ−ジエチル）アミノアニリノ基等の発色性基、例えば７−アミノ−４−メチルクマリノ基等の蛍光性基等が好ましく挙げられる。
【００１５】
更に、一般式［Ｉ］で示されるペプチド誘導体は、無機酸又は有機酸と酸付加塩を形成していても良く、そのような酸付加塩の具体例としては、例えば塩酸塩、硫酸塩、硝酸塩、リン酸塩等の無機酸塩、例えば酢酸塩、シュウ酸塩、酒石酸塩、コハク酸塩、クエン酸塩、ｐ−トルエンスルホン酸塩等の有機酸塩等が好ましく挙げられる。
【００１６】
本発明のペプチド誘導体は、ペプチド合成に慣用されている方法を用いれば容易に合成できるが、例えば以下のようにして合成すれば良い。
即ち、先ず、一般式［ＩＩ］
Ｈ−Ａｒｇ−Ｙ［ＩＩ］
（式中、Ｙは前記に同じ。）
で示されるペプチド誘導体又はその酸付加塩を、例えば以下のようにして合成する。
【００１７】
即ち、適当な保護基［例えばｔ−ブトキシカルボニル基（Ｂｏｃ基）、ベンジルオキシカルボニル基（Ｚ基）等］によりアミノ基が保護されたアルギニン、又は、適当な保護基（例えばＢｏｃ基、Ｚ基、ニトロ基等）によりアミノ基及びグアニジノ基が保護されたアルギニンと、アルギニンに対して１〜２倍モル量の例えばｐ−ニトロアニリン、３−ヒドロキシメチル−４−ニトロアニリン、４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリン、４−（Ｎ，Ｎ−ジエチル）アミノアニリン等の発色性化合物或は例えば７−アミノ−４−メチルクマリン等の蛍光性化合物とを、ペプチド誘導体の合成に於いて通常用いられる溶媒、例えばジメチルホルムアミド（ＤＭＦ）、ジメチルスルホキシド（ＤＭＳＯ）、テトラヒドロフラン（ＴＨＦ）、或はこれらの混合溶媒中、アルギニンに対して１〜２倍モル量の例えばジシクロヘキシルカルボジイミド（ＤＣＣ）等のペプチド合成に慣用される縮合剤の存在下、０〜２５℃で、１２〜２４時間反応させる。反応終了後、反応液を減圧濃縮し、次いでこれをペプチド誘導体の精製方法として通常用いられている方法、例えばシリカゲルカラムクロマトグラフィー、イオン交換カラムクロマトグラフィー等により精製した後、脱保護の処理を行うことにより、一般式［ＩＩ］で示されるアルギニン誘導体が容易に得られる。尚、一般式［ＩＩ］で示されるアルギニン誘導体は、上記した方法以外のペプチド化学の分野で汎用される方法、例えば活性エステル法、混合酸無水物法、アジド法、ホスファゾ法等により合成してもよいことは言うまでもない。
【００１８】
次いで、一般式［ＩＩ］で示されるアルギニン誘導体をペプチド合成に於いて通常用いられる溶媒、例えばＤＭＦ、ＤＭＳＯ、ＴＨＦ或はこれらの混合溶媒中、一般式［ＩＩ］で示されるアルギニン誘導体に対して１〜２倍モルのＮ−末端が適当な保護基［例えばＢｏｃ基、Ｚ基、ベンゾイル基（Ｂｚ基）、アセチル基（Ａｃ基）等］により保護されたスレオニルグリシンと、一般式［ＩＩ］で示されるアルギニン誘導体に対して１〜２倍モルの例えばＤＣＣ等のペプチド合成に慣用される縮合剤の存在下に、０〜２５℃で１２〜２４時間反応させるか、或は、一般式［ＩＩ］で示されるアルギニン誘導体をペプチド合成に於いて通常用いられる溶媒、例えばＤＭＦ、ＤＭＳＯ、ＴＨＦ、或はこれらの混合溶媒中、一般式［ＩＩ］で示されるアルギニン誘導体に対して１〜２倍モルのＮ−末端が適当な保護基（例えばＢｏｃ基、Ｚ基、Ｂｚ基、Ａｃ基等）により保護され且つＣ−末端に例えばＮ−ヒドロキシスクシンイミドエステル、Ｎ−ヒドロキシ−５−ノルボルネン−２，３−ジカルボキシイミドエステル等が結合したスレオニルグリシンの活性エステル体と０〜２５℃で１２〜２４時間反応させる。反応終了後、反応液を減圧濃縮し、次いでこれをペプチド誘導体の精製方法として通常用いられている方法、例えばシリカゲルカラムクロマトグラフィー、イオン交換カラムクロマトグラフィー等により精製し、必要に応じて脱保護処理を行えば、本発明のペプチド誘導体が容易に得られる。尚、一般式［ＩＩ］で示されるアルギニン誘導体とスレオニルグリシンとを結合させるのは、上記した方法以外のペプチド化学の分野で汎用される方法、例えば混合酸無水物法、アジド法、ホスファゾ法等で行ってもよいことは言うまでもない。
【００１９】
かくして得られた本発明のペプチド誘導体は、特定酵素、例えばカブトガニ血球成分とエンドトキシンとの反応の結果活性化される凝固酵素（プロテアーゼ、アミダーゼ様酵素等）の基質として有用なものである。
【００２０】
本発明のペプチド誘導体を基質として用いてエンドトキシンを定量するには、例えば、本発明のペプチド誘導体を基質として用いる以外は、通常この分野で本発明のペプチド誘導体の如き合成基質を使用してエンドトキシンの定量を行う、所謂合成基質法に準じて操作を行えばよい。また、エンドトキシンとカブトガニ血球成分と本発明のペプチド誘導体とを混合して反応を開始させ、その結果遊離する発色性或は蛍光性を示す化合物に基づく吸光度変化量（或は蛍光強度変化量）が一定の値に到達するまでに要する時間（到達時間）を求め、この値を、到達時間とエンドトキシン濃度との関係を表す検量線に当てはめることにより行ってもよい。
【００２１】
この際に用いられるカブトガニの血球成分を含む溶液としては、通常のエンドトキシンの測定に使用できるものであれば特に限定されることなく用いることができるが、例えば、ＡＣＣ社、ヘマケム社、ウィタカーバイオプロダクト社、帝国臓器（株）、エンドセイフ社等によって製造された市販のカブトガニ血球成分の凍結乾燥品から調製されたものを用いてもよいし、リムルス（Ｌｉｍｕｌｕｓ）属、タキプレウス（Ｔａｃｈｙｐｌｅｕｓ）属或はカルシノスコルピウス（Ｃａｒｃｉｎｏｓｃｏｒｐｉｕｓ）属に属するカブトガニの血球から得られたもので、エンドトキシンとの反応により酵素（プロテアーゼ等）の活性化が生じるものであれば、特に限定されることなく挙げられる。
【００２２】
また、上記の如き本発明のペプチド誘導体を使用するエンドトキシン定量用試薬中には、例えば緩衝剤その他の適当な試薬類が含まれていても良く、これら試薬類は所謂合成基質法等で使用されるものの中から適宜選択して用いれば足りるし、その使用濃度も所謂合成基質法用試薬等を調製する際に選択される濃度範囲から適宜選択すれば足りる。尚、上記のエンドトキシン定量法に於ける本発明のペプチド誘導体の使用濃度としては、エンドトキシンの測定範囲をどの程度にするかによっても異なるが、反応時の濃度として通常０．５〜５ｍＭ、好ましくは１〜３ｍＭの範囲から適宜選択される。
【００２３】
本発明のペプチド誘導体を基質とするエンドトキシンの定量方法をより具体的に述べれば例えば以下の如くである。
【００２４】
ａ）合成基質法
エンドトキシンを含む試料とカブトガニ血球成分を含む溶液とを混合した後、２５〜４０℃保温下に所定時間反応させた後、該溶液に本発明のペプチド誘導体を加え、更に２５〜４０℃保温下に所定時間反応させるか、或はまた、エンドトキシンを含む試料とカブトガニ血球成分を含む溶液と本発明のペプチド誘導体とを混合し、２５〜４０℃保温下に所定時間反応させる。その後、反応液に反応停止液（例えば塩酸溶液、酢酸溶液等）を添加して反応を停止させ、得られた最終溶液の所定の吸光度（或は蛍光強度）を適当な測定機器（例えば分光光度計、マイクロプレートリーダー、蛍光光度計等）を使用して測定する。
得られた測定値を、濃度既知のエンドトキシン溶液を使用して予め作成しておいたエンドトキシン濃度と該測定値との関係を表す検量線に当てはめ、試料中のエンドトキシン濃度を求める。
【００２５】
ｂ）到達時間分析法
エンドトキシンを含む試料と、カブトガニ血球成分を含む溶液と、本発明のペプチド誘導体とを混合した後、２５〜４０℃保温下で該混合液の所定の吸光度変化（或は蛍光強度変化）を適当な測定機器（例えば分光光度計、マイクロプレートリーダー、蛍光光度計等）を使用して測定し、混合直後からの吸光度変化量（或は蛍光強度変化量）が所定の値に到達する迄の時間（到達時間）を求める。
得られた到達時間を、濃度既知のエンドトキシン溶液を使用して予め作成しておいた到達時間とエンドトキシン濃度との関係を表す検量線に当てはめ、試料中のエンドトキシン濃度を求める。
【００２６】
これらの定量法に於いて、本発明のペプチド誘導体から遊離する発色性（或は蛍光性）の化合物を測定するに当たっては、該化合物自身が発色性或は蛍光性を有する場合はその性質に基づいて測定を行えば良い。該化合物自身の発色性或は蛍光性がそれほど強くない場合（或は殆どない場合）には、適当な化合物とカップリング等させることにより発色性或は蛍光性を示す化合物を生じさせ、それに基づく発色或は蛍光を測定すれば良い。後者の場合についてより具体的に述べれば、本発明のペプチド誘導体から遊離する化合物が例えば４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリン、４−（Ｎ，Ｎ−ジエチル）アミノアニリン等である場合には、これらを適当な酸化剤等の存在下、フェノール、ナフトール等と酸化縮合させ、生成する青色のインドフェノール型色素とした後に、生じた色素に基づく吸光度を測定すれば良い。また、本発明のペプチド誘導体から遊離する化合物が例えばｐ−ニトロアニリンである場合には、それ自身が発色性を有しているのでそのままで測定を行っても良いが、測定感度を上げるために適当な化合物とジアゾカップリング反応させ、その結果生じた化合物に基づく吸光度を測定しても良い。
【００２７】
本発明のエンドトキシン定量用試薬キットは、上記した如き方法によりエンドトキシンを定量する際に用いられるもので、カブトガニ血球成分と、一般式［Ｉ］で示されるペプチド誘導体又はその酸付加物とを、構成成分として含んで成るものであり、夫々の構成要素の好ましい態様、具体例については上で述べたとおりである。
以下に実施例、参考例等により本発明を更に詳細に説明するが、本発明はこれら実施例等により何等限定されるものではない。
【００２８】
【実施例】
実施例１．ｔ−ブトキシカルボニルスレオニルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩の合成
１）ベンジルオキシカルボニルアルギニン−ｐ−ニトロアニリド塩酸塩の合成
ｐ−ニトロアニリン（２６ｇ，１８８ｍｍｏｌ）をピリジン（４００ｍｌ）に溶解し、−２０〜−１０℃に冷却下、三塩化リン（８．２ｍｌ，９４ｍｍｏｌ）を含むピリジン溶液（１００ｍｌ）を３０分間を要して滴下した後、同温度で３０分間攪拌反応させた。反応液を室温に戻し、更に２時間攪拌反応させた後、ベンジルオキシカルボニルアルギニン（５８ｇ，１８８ｍｍｏｌ。和光純薬工業（株）製。）を加え、４０〜５０℃で３時間攪拌反応させた。反応終了後、反応液を減圧濃縮し、得られた残渣を酢酸エチル：ｎ−ブタノール＝７：３の混合溶媒で抽出し、抽出液を１規定塩酸、飽和食塩水で順次洗浄し、有機層を無水硫酸マグネシウムで乾燥後、溶媒を留去した。得られた残渣をアセトンで結晶化して、５６．５ｇのベンジルオキシカルボニルアルギニン−ｐ−ニトロアニリド塩酸塩（収率６４．６％）を得た。
２）ｔ−ブトキシカルボニルスレオニルグリシンエチルエステルの合成
グリシンエチルエステル塩酸塩（８．４ｇ，６０ｍｍｏｌ、和光純薬工業（株）製。）とｔ−ブトキシカルボニルスレオニン（１１．０ｇ，５０ｍｍｏｌ、和光純薬工業（株）製。）とを塩化メチレン（３００ｍｌ）に懸濁し、これに氷冷下トリエチルアミン（８．４ｍｌ，６０ｍｍｏｌ）とＤＣＣ（１２．４ｇ，６０ｍｍｏｌ）とを加えた後、室温で２０時間攪拌反応させた。反応終了後、不溶物を濾去し、濾液を減圧濃縮し、得られた残渣を酢酸エチルで抽出処理した。抽出液を１規定塩酸、飽和重曹水、飽和食塩水で順次洗浄し、無水硫酸マグネシウムで乾燥後、溶媒を留去した。得られた残渣は、特に精製を行わず、次反応に供した。
３）ｔ−ブトキシカルボニルスレオニルグリシンの合成
上記２）で得たｔ−ブトキシカルボニルスレオニルグリシンエチルエステル（１５．０ｇ，４９ｍｍｏｌ）をメタノール（６００ｍｌ）に溶解し、これに１規定水酸化ナトリウム水溶液（７５ｍｌ）を加え、室温で２時間攪拌反応させた。反応終了後、反応液を１規定塩酸（７５ｍｌ）で中和し、これを減圧濃縮した。得られた残渣を飽和重曹水に溶解し、酢酸エチルで洗浄した。飽和重曹水層を、１規定塩酸で酸性とした後、酢酸エチルで抽出した。酢酸エチル層を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した後、酢酸エチルを留去する事により、１０．０ｇのｔ−ブトキシカルボニルスレオニルグリシン（収率７２％）を油状物として得た。
４）ｔ−ブトキシカルボニルスレオニルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩の合成
上記１）で得たベンジルオキシカルボニルアルギニン−ｐ−ニトロアニリド塩酸塩（１６．３ｇ，３５ｍｍｏｌ）を２５％臭化水素−酢酸溶液（５０ｍｌ）に懸濁し、氷冷下で４時間攪拌反応させた。反応終了後、反応液にエーテルを注ぎ、析出した沈澱物を濾取し、更にエーテルで洗浄した。これを、減圧下で１０時間乾燥させた後、ＤＭＦ（５００ｍｌ）に溶解し、氷浴中トリエチルアミン（４．９ｍｌ，３５ｍｍｏｌ）、ＤＣＣ（７．２ｇ，３５ｍｍｏｌ）、１−オキシベンゾトリアゾール（５．４ｇ，３５ｍｍｏｌ）、及び上記３）で得たｔ−ブトキシカルボニルスレオニルグリシン（９．７ｇ，３５ｍｍｏｌ）を順次添加し、氷冷下で２時間、更に室温下で２０時間攪拌反応させた。反応終了後、不溶物を濾去し、濾液を減圧濃縮し、得られた残渣を酢酸エチル：ｎ−ブタノ−ル＝８：２の混合溶媒で抽出した。抽出液を１規定塩酸、飽和重曹水、飽和食塩水で順次洗浄し、無水硫酸マグネシウムで乾燥した後、溶媒を留去した。得られた残渣をシリカゲルカラムクロマトグラフィー（ＷａｋｏｇｅｌＣ−３００；和光純薬工業（株）商品名、７５０ｇ，溶出液；酢酸エチル：ピリジン：酢酸：水＝６０：２０：６：１１の混合溶媒）にて精製し、目的物の溶出画分を減圧濃縮して得られた残渣を酢酸エチル：ｎ−ブタノール＝８：２の混合溶媒で抽出した。抽出液を１規定塩酸、飽和重曹水、飽和食塩水で順次洗浄し、無水硫酸マグネシウムで乾燥した後、溶媒を留去した。得られた残渣をイオン交換カラムクロマトグラフィー（ＡｍｂｅｒｌｉｔｅＩＲＡ−４１０酢酸型；アンバーライト社製、５００ｃｍ^３，溶出液；水）にて酢酸塩型へと変換した。目的物の溶出画分を凍結乾燥して得られた粉末をゲル濾過カラムクロマトグラフィー（ＳｅｐｈａｄｅｘＬＨ−２０，ファルマシア社商品名。溶出液；メタノール）にて精製した。更に、目的物の溶出画分を減圧濃縮して得られた残渣を、注射用蒸留水（大塚製薬（株）製，７００ｍｌ）に溶解し、限外濾過（Ｓａｒｔｏｒｉｕｓ社製；ＵｌｔｒａｚａｒｔＤ−２０、及びそのユニット）にて発熱性物質を除去し、濾液を凍結乾燥することにより、１１．５ｇのｔ−ブトキシカルボニルスレオニルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩（収率５３．７％）を得た。
元素分析値（Ｃ_２５Ｈ_４０Ｎ_８Ｏ_１０として）
計算値（％）：Ｃ４９．０１，Ｈ６．５８，Ｎ１８．２９
測定値（％）：Ｃ４７．３１，Ｈ６．４３，Ｎ１７．６８
【００２９】
実施例２．ｔ−ブトキシカルボニルスレオニルグリシルアルギニン−３−ヒドロキシメチル−４−ニトロアニリド酢酸塩の合成
１）ベンジルオキシカルボニルアルギニン−３−アセトキシメチル−４−ニトロアニリド塩酸塩の合成
特公平６−２７１１１号公報に記載された合成方法に準じて３−アミノベンジルアルコールを原料としてアセチル化、ニトロ化等を行うことにより合成された３−アセトキシメチル−４−ニトロアニリン（２．１ｇ，１０ｍｍｏｌ）をピリジン（５０ｍｌ）に溶解したものを−２０〜−１０℃に冷却し、三塩化リン（０．４４ｍｌ，５ｍｍｏｌ）を含むピリジン溶液（５ｍｌ）を５分間で滴下し、−２０〜−１０℃で３０分間攪拌反応させた。反応後、室温に戻し、更に１時間攪拌反応させた後、ベンジルオキシカルボニルアルギニン（３．１ｇ，１０ｍｍｏｌ、和光純薬工業（株）製。）を加え、４０〜５０℃で３時間攪拌反応させた。反応終了後、反応液を減圧濃縮し、得られた残渣を酢酸エチルで抽出した。抽出液を１規定塩酸、飽和食塩水で順次洗浄し、無水硫酸マグネシウムで乾燥後、溶媒を留去した。得られた残渣を、シリカゲルカラムクロマトグラフィー（ＷａｋｏｇｅｌＣ−３００；和光純薬工業（株）商品名、２５０ｇ，溶出液；酢酸エチル：ピリジン：酢酸：水＝６０：２０：６：１１の混合溶媒）にて精製した。目的物の溶出画分を減圧濃縮し、得られた残渣を酢酸エチルで抽出した。抽出液を１規定塩酸、飽和重曹水、飽和食塩水で順次洗浄し、無水硫酸マグネシウムで乾燥した後、溶媒を留去して、３．８ｇのベンジルオキシカルボニルアルギニン−３−アセトキシメチル−４−ニトロアニリド塩酸塩（収率７６．１％）を油状物として得た。
２）ベンジルオキシカルボニルアルギニン−３−ヒドロキシメチル−４−ニトロアニリド塩酸塩の合成
上記１）で得たベンジルオキシカルボニルアルギニン−３−アセトキシメチル−４−ニトロアニリド塩酸塩（３．６ｇ，６．７ｍｍｏｌ）をメタノ−ル（１５０ｍｌ）に溶解し、氷冷下、これにナトリウムメトキシドの２８％メタノール溶液（０．１５ｍｌ）を添加して、氷冷下で３時間攪拌反応させた。反応終了後、反応液を１規定塩酸で中和し、これを減圧濃縮した。得られた残渣を酢酸エチル：ｎ−ブタノール＝６：４の混合溶媒で抽出し、抽出液を１規定塩酸、蒸留水で順次洗浄した後、溶媒を留去した。得られた残渣をエタノール／エーテルの混合溶媒で結晶化し、３．０ｇのベンジルオキシカルボニルアルギニン−３−ヒドロキシメチル−４−ニトロアニリド塩酸塩（収率９１．０％）を得た。３）ｔ−ブトキシカルボニルスレオニルグリシルアルギニン−３−ヒドロキシメチル−４−ニトロアニリド酢酸塩の合成
上記２）で得たベンジルオキシカルボニルアルギニン−３−ヒドロキシメチル−４−ニトロアニリド塩酸塩（２．５ｇ，５ｍｍｏｌ）を２５％臭化水素−酢酸溶液（５ｍｌ）に懸濁し、氷冷下で４時間攪拌反応させた。反応終了後、反応液にエーテルを注ぎ、析出した沈澱物を濾取し、エーテルで洗浄した。これを、減圧下で１０時間乾燥させた後、ＤＭＦ（１００ｍｌ）に溶解し、氷冷下トリエチルアミン（０．７ｍｌ，５ｍｍｏｌ）、ＤＣＣ（１．０ｇ，５ｍｍｏｌ）、１−オキシベンゾトリアゾール（０．８ｇ，５ｍｍｏｌ）、及び上記実施例１の３）で得たｔ−ブトキシカルボニルスレオニルグリシン（１．４ｇ，５ｍｍｏｌ）を順次添加し、氷冷下で２時間、更に室温に戻し２０時間攪拌反応させた。反応終了後、不溶物を濾去し、濾液を減圧濃縮し、得られた残渣を酢酸エチル：ｎ−ブタノール＝７：３の混合溶媒で抽出した。抽出液を１規定塩酸、飽和重曹水、飽和食塩水で順次洗浄し、無水硫酸マグネシウムで乾燥した後、溶媒を留去した。得られた残渣を、シリカゲルカラムクロマトグラフィー（ＷａｋｏｇｅｌＣ−３００；和光純薬工業（株）商品名、３００ｇ，溶出液；酢酸エチル：ピリジン：酢酸：水＝６０：２０：６：１１の混合溶媒）にて精製した。目的物の溶出画分を減圧濃縮し、得られた残渣を、酢酸エチル：ｎ−ブタノール＝７：３の混合溶媒で抽出した。抽出液を１規定塩酸、飽和重曹水、飽和食塩水で順次洗浄し、無水硫酸マグネシウムで乾燥した後、溶媒を留去した。得られた残渣を、イオン交換カラムクロマトグラフィー（ＡｍｂｅｒｌｉｔｅＩＲＡ−４１０酢酸型；アンバーライト社製、５０ｃｍ^３，溶出液；水）にて酢酸塩型へと変換した。目的物の溶出画分を凍結乾燥し、得られた粉末を、ゲル濾過カラムクロマトグラフィー（ＳｅｐｈａｄｅｘＬＨ−２０，ファルマシア社商品名。溶出液；メタノール）にて精製した。更に、目的物の溶出画分を減圧濃縮して得られた残渣を、注射用蒸留水（大塚製薬（株）製、５０ｍｌ）に溶解し、限外濾過（Ｓａｒｔｏｒｉｕｓ社製；ＵｌｔｒａｚａｒｔＤ−２０、及びそのユニット）にて発熱性物質を除去し、濾液を凍結乾燥することにより、２．１ｇのｔ−ブトキシカルボニルスレオニルグリシルアルギニン−３−ヒドロキシメチル−４−ニトロアニリド酢酸塩（収率６５．１％）を得た。
元素分析値（Ｃ_２６Ｈ_４２Ｎ_８Ｏ_１１として）
計算値（％）：Ｃ４８．５９，Ｈ６．５９，Ｎ１７．４４
測定値（％）：Ｃ４７．３１，Ｈ６．４３，Ｎ１７．６８
【００３０】
実施例３．ｔ−ブトキシカルボニルスレオニルグリシルアルギニン−４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリド酢酸塩の合成
１）ｔ−ブトキシカルボニル−Ｎ^Ｇ−ニトロアルギニン−４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリドの合成
ｔ−ブトキシカルボニル−Ｎ^Ｇ−ニトロアルギニン（２０．２ｇ，６３ｍｍｏｌ、和光純薬工業（株）製）をＤＭＦ（１３０ｍｌ）に溶解し、ピリジン（５．０ｍｌ，６３ｍｍｏｌ）とＮ−エチルピペリジン（８．７ｍｌ，６３ｍｍｏｌ）とを加え、−２０〜−１５℃に冷却した後、塩化ピバロイル（７．７ｍｌ，６３ｍｍｏｌ）を含むＤＭＦ溶液（３０ｍｌ）を１０分間で滴下し、０〜５℃で１０分間攪拌した。これを再び−２０〜−１５℃に冷却した後、４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリン（１１．４ｇ，６３ｍｍｏｌ、和光純薬工業（株）製。）を含むＤＭＦ溶液（３０ｍｌ）を添加し、−１０〜５℃で２時間、更に室温に戻し１２時間攪拌反応させた。反応終了後、反応液を減圧濃縮し、得られた残渣を１０％クエン酸水溶液に溶解し、酢酸エチルで洗浄した。水層を、飽和重曹水でアルカリ性とした後、酢酸エチルで抽出した。抽出液を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した後、溶媒を留去した。得られた残渣を、シリカゲルカラムクロマトグラフィー（ＷａｋｏｇｅｌＣ−３００；和光純薬工業（株）商品名、１０００ｇ，溶出液；クロロホルム：メタノール＝９：１の混合溶媒）にて精製した。目的物の溶出分画を減圧濃縮し、得られた残渣をヘキサンにて粉末化することにより、２３．８ｇのｔ−ブトキシカルボニル−Ｎ^Ｇ−ニトロアルギニン−４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリド（収率７８．２％）を得た。
２）ｔ−ブトキシカルボニルスレオニルグリシン−５−ノルボルネン−２，３−ジカルボキシイミドエステルの合成
上記実施例１の３）で得たｔ−ブトキシカルボニルスレオニルグリシン（３．３ｇ，１２ｍｍｏｌ）とＮ−ヒドロキシ−５−ノルボルネン−２，３−ジカルボキシイミド（２．２ｇ，１２ｍｍｏｌ）とをＤＭＦ（５０ｍｌ）に溶解し、氷冷下ＤＣＣ（２．５ｇ，１２ｍｍｏｌ）を加え、室温で２０時間攪拌反応させた。反応終了後、不溶物を濾去し、濾液を減圧濃縮して、ｔ−ブトキシカルボニルスレオニルグリシン−５−ノルボルネン−２，３−ジカルボキシイミドエステルを油状物として得た。得られた油状物は、特に精製を行わず、次反応に供した。
３）ｔ−ブトキシカルボニルスレオニルグリシル−Ｎ^Ｇ−ニトロアルギニン−４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリドの合成
上記１）で得たｔ−ブトキシカルボニル−Ｎ^Ｇ−ニトロアルギニン−４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリド（４．８ｇ，１０ｍｍｏｌ）をトリフルオロ酢酸（２５ｍｌ）に懸濁し、アニソール（１ｍｌ）を加えた後、室温で１時間攪拌反応させた。反応終了後、反応液を減圧濃縮し、得られた残渣にエーテルを注入した。析出した沈澱物を濾取し、エーテルで洗浄した。これを、減圧下で１０時間乾燥させた後、ＤＭＦ（２５ｍｌ）に溶解し、氷冷下トリエチルアミン（２．８ｍｌ，２０ｍｍｏｌ）、及び上記２）で得たｔ−ブトキシカルボニルスレオニルグリシン−５−ノルボルネン−２，３−ジカルボキシイミドエステル（１２ｍｍｏｌ）を含むＤＭＦ溶液（２０ｍｌ）を添加し、氷冷下で２時間、更に室温に戻し２０時間攪拌反応させた。反応終了後、反応液を減圧濃縮し、得られた残渣を酢酸エチル：ｎ−ブタノール＝８：２の混合溶媒で抽出した。抽出液を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した後、溶媒を留去した。得られた残渣を、シリカゲルカラムクロマトグラフィー（ＷａｋｏｇｅｌＣ−３００；和光純薬工業（株）商品名、６００ｇ，溶出液；クロロホルム：メタノール＝９：１の混合溶媒）にて精製した。目的物の溶出画分を減圧濃縮し、得られた残渣をエーテルにて結晶化し、４．４ｇのｔ−ブトキシカルボニルスレオニルグリシル−Ｎ^Ｇ−ニトロアルギニン−４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリド（収率６８．１％）を得た。
４）ｔ−ブトキシカルボニルスレオニルグリシルアルギニン−４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリド酢酸塩の合成
上記３）で得たｔ−ブトキシカルボニルスレオニルグリシル−Ｎ^Ｇ−ニトロアルギニン−４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリド（３．２ｇ，５ｍｍｏｌ）をメタノール（３５０ｍｌ）に溶解し、パラジウムカーボン触媒（５００ｍｇ）を加えて、室温で４昼夜水添反応を行った。反応終了後、不溶物を濾去し、濾液を減圧濃縮した。得られた残渣をイオン交換カラムクロマトグラフィー（ＡｍｂｅｒｌｉｔｅＩＲＡ−４１０酢酸型；アンバーライト社製、５０ｃｍ^３，溶出液；水）にて酢酸塩型へと変換した。目的物の溶出画分を、更にイオン交換カラムクロマトグラフィー（ＣＭ−ＴＯＹＯＰＥＡＲＬ６５０Ｍ；東ソー（株）製、４０ｃｍ^３，溶出液；蒸留水〜０．２Ｍ酢酸アンモニウム、リニアグラジエント）で精製を行った。目的物の溶出画分を凍結乾燥し、得られた粉末を、ゲル濾過カラムクロマトグラフィー（ＳｅｐｈａｄｅｘＬＨ−２０，ファルマシア社商品名、溶出液；メタノール）にて精製した。目的物の溶出画分を減圧濃縮し、得られた残渣を注射用蒸留水（大塚製薬（株）製、３０ｍｌ）に溶解し、限外濾過（Ｓａｒｔｏｒｉｕｓ社製；ＵｌｔｒａｚａｒｔＤ−２０、及びそのユニット）にて発熱性物質を除去し、濾液を凍結乾燥することにより、３．３ｇのｔ−ブトキシカルボニルスレオニルグリシルアルギニン−４−（Ｎ−エチル−Ｎ−β−ヒドロキシエチル）アミノアニリド酢酸塩（収率４８．８％）を得た。
元素分析値（Ｃ_２９Ｈ_５０Ｎ_８Ｏ_９として）
計算値（％）：Ｃ５３．２０，Ｈ７．７０，Ｎ１７．１１
測定値（％）：Ｃ５１．３７，Ｈ７．５６，Ｎ１６．４９
【００３１】
参考例１．ｔ−ブトキシカルボニル−Ｏ−ベンジルセリルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩の合成
１）ｔ−ブトキシカルボニル−Ｏ−ベンジルセリルグリシンエチルエステルの合成
グリシンエチルエステル塩酸塩（６．７ｇ，４８ｍｍｏｌ、和光純薬工業（株）製）とｔ−ブトキシカルボニル−Ｏ−ベンジルセリン（１１．８ｇ，４０ｍｍｏｌ、ペプチド研究所製）とをＴＨＦ（１５０ｍｌ）に懸濁し、氷冷下トリエチルアミン（６．７ｍｌ，４８ｍｍｏｌ）とＤＣＣ（９．９ｇ，４８ｍｍｏｌ）を加えた後、室温で２０時間攪拌反応させた。反応終了後、実施例１の２）に準じて後処理を行った。得られた残渣は、特に精製を行わず、次反応に供した。
２）ｔ−ブトキシカルボニル−Ｏ−ベンジルセリルグリシンの合成
上記１）で得たｔ−ブトキシカルボニル−Ｏ−ベンジルセリルグリシンエチルエステル（１５．２ｇ，４０ｍｍｏｌ）をメタノール（４００ｍｌ）に溶解し、これに１規定水酸化ナトリウム水溶液（６０ｍｌ）を加え、室温で２時間攪拌反応させた。反応終了後、実施例１の３）に準じて後処理を行い、５．７ｇのｔ−ブトキシカルボニル−Ｏ−ベンジルセリルグリシン（収率４０％）を得た。
３）ｔ−ブトキシカルボニル−Ｏ−ベンジルセリルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩の合成
実施例１の１）で得たベンジルオキシカルボニルアルギニン−ｐ−ニトロアニリド塩酸塩（４．６ｇ，１０ｍｍｏｌ）を２５％臭化水素−酢酸溶液（１５ｍｌ）に懸濁し、氷冷下で４時間攪拌反応させた。反応終了後、反応液にエーテルを注入し、析出した沈澱物を濾取し、エーテルで洗浄した。これを、減圧下で１０時間乾燥させた後、ＤＭＦ（５０ｍｌ）に溶解し、氷浴中トリエチルアミン（１．４ｍｌ，１０ｍｍｏｌ）、ＤＣＣ（２．１ｇ，１０ｍｍｏｌ）、１−オキシベンゾトリアゾール（１．５ｇ，１０ｍｍｏｌ）、及び上記２）で得たｔ−ブトキシカルボニル−Ｏ−ベンジルセリルグリシン（３．５ｇ，１０ｍｍｏｌ）を順次添加し、氷冷下で２時間、更に室温に戻し２０時間攪拌反応させた。反応終了後、実施例１の４）に準じて後処理、及び精製を行い、５．８ｇのｔ−ブトキシカルボニル−Ｏ−ベンジルセリルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩（収率８５％）を凍乾粉末として得た。
元素分析値（Ｃ_３１Ｈ_４４Ｎ_８Ｏ_１０として）
計算値（％）：Ｃ５４．０６，Ｈ６．４４，Ｎ１６．２７
測定値（％）：Ｃ５３．８０，Ｈ６．３３，Ｎ１６．１３
【００３２】
参考例２．ｔ−ブトキシカルボニルロイシルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩の合成
１）ｔ−ブトキシカルボニルロイシルグリシンエチルエステルの合成
グリシンエチルエステル塩酸塩（６．７ｇ，４８ｍｍｏｌ、和光純薬工業（株）製）とｔ−ブトキシカルボニルロイシン１水和物（１０．０ｇ，４０ｍｍｏｌ、和光純薬工業（株）製）とを塩化メチレン（１５０ｍｌ）に懸濁し、氷冷下トリエチルアミン（６．７ｍｌ，４８ｍｍｏｌ）とＤＣＣ（９．９ｇ，４８ｍｍｏｌ）を加えた後、室温で２０時間攪拌反応させた。反応終了後、実施例１の２）に準じて後処理を行った。得られた残渣は、特に精製を行わず、次反応に供した。
２）ｔ−ブトキシカルボニルロイシルグリシンの合成
上記１）で得たｔ−ブトキシカルボニルロイシルグリシンエチルエステル（１２．５ｇ，４０ｍｍｏｌ）をメタノール（４００ｍｌ）に溶解し、これに１規定水酸化ナトリウム水溶液（６０ｍｌ）を加え、室温で２時間攪拌反応させた。反応終了後、実施例１の３）に準じて後処理を行い、９．０ｇのｔ−ブトキシカルボニルロイシルグリシン（収率７８％）を得た。
３）ｔ−ブトキシカルボニルロイシルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩の合成
実施例１の１）で得たベンジルオキシカルボニルアルギニン−ｐ−ニトロアニリド塩酸塩（４．６ｇ，１０ｍｍｏｌ）を２５％臭化水素−酢酸溶液（１５ｍｌ）に懸濁し、氷冷下で４時間攪拌反応させた。反応終了後、反応液にエーテルを注入し、析出した沈澱物を濾取し、エーテルで洗浄した。これを、減圧下で１０時間乾燥させた後、ＤＭＦ（５０ｍｌ）に溶解し、氷浴中トリエチルアミン（１．４ｍｌ，１０ｍｍｏｌ）、ＤＣＣ（２．１ｇ，１０ｍｍｏｌ）、１−オキシベンゾトリアゾール（１．５ｇ，１０ｍｍｏｌ）、及び上記２）で得たｔ−ブトキシカルボニルロイシルグリシン（２．９ｇ，１０ｍｍｏｌ）を順次添加し、氷冷下で２時間、更に室温に戻し２０時間攪拌反応させた。反応終了後、実施例１の４）に準じて後処理、及び精製を行い、５．５ｇのｔ−ブトキシカルボニルロイシルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩（収率８８％）を凍乾粉末として得た。
元素分析値（Ｃ_２７Ｈ_４４Ｎ_８Ｏ_９として）
計算値（％）：Ｃ５１．９１，Ｈ７．１０，Ｎ１７．９４
測定値（％）：Ｃ５１．１８，Ｈ７．０２，Ｎ１７．１８
【００３３】
参考例３．ｔ−ブトキシカルボニルセリルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩の合成
１）ｔ−ブトキシカルボニルセリルグリシンエチルエステルの合成
グリシンエチルエステル塩酸塩（６．７ｇ，４８ｍｍｏｌ、和光純薬工業（株）製）とｔ−ブトキシカルボニルセリン（８．２ｇ，４０ｍｍｏｌ、ペプチド研究所製）とをＴＨＦ（１５０ｍｌ）に懸濁し、氷冷下トリエチルアミン（６．７ｍｌ，４８ｍｍｏｌ）とＤＣＣ（９．９ｇ，４８ｍｍｏｌ）を加えた後、室温で２０時間攪拌反応させた。反応終了後、実施例１の２）に準じて後処理を行った。得られた残渣は、特に精製を行わず、次反応に供した。
２）ｔ−ブトキシカルボニルセリルグリシンの合成
上記１）で得たｔ−ブトキシカルボニルセリルグリシンエチルエステル（１１．６ｇ，４０ｍｍｏｌ）をメタノール（４００ｍｌ）に溶解し、これに１規定水酸化ナトリウム水溶液（６０ｍｌ）を加え、室温で２時間攪拌反応させた。反応終了後、実施例１の３）に準じて後処理を行い、７．０ｇのｔ−ブトキシカルボニルセリルグリシン（収率６７％）を得た。
３）ｔ−ブトキシカルボニルセリルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩の合成
実施例１の１）で得たベンジルオキシカルボニルアルギニン−ｐ−ニトロアニリド塩酸塩（４．６ｇ，１０ｍｍｏｌ）を２５％臭化水素−酢酸溶液（１５ｍｌ）に懸濁し、氷冷下で４時間攪拌反応させた。反応終了後、反応液にエーテルを注入し、析出した沈澱物を濾取し、エーテルで洗浄した。これを、減圧下で１０時間乾燥させた後、ＤＭＦ（５０ｍｌ）に溶解し、氷冷下、トリエチルアミン（１．４ｍｌ，１０ｍｍｏｌ）、ＤＣＣ（２．１ｇ，１０ｍｍｏｌ）、１−オキシベンゾトリアゾール（１．５ｇ，１０ｍｍｏｌ）、及び上記２）で得たｔ−ブトキシカルボニルセリルグリシン（２．６ｇ，１０ｍｍｏｌ）を順次添加し、氷冷下で２時間、更に室温に戻し２０時間攪拌反応させた。反応終了後、実施例１の４）に準じて後処理及び精製を行い、４．３ｇのｔ−ブトキシカルボニルセリルグリシルアルギニン−ｐ−ニトロアニリド酢酸塩（収率７３％）を凍乾粉末として得た。
元素分析値（Ｃ_２４Ｈ_３８Ｎ_８Ｏ_１０として）
計算値（％）：Ｃ４８．１６，Ｈ６．４０，Ｎ１８．７２
測定値（％）：Ｃ４７．６１，Ｈ６．３１，Ｎ１８．７０
【００３４】
実施例４．基質の反応性の検討
エンドトキシンテストワコー（和光純薬工業（株）製）中のカブトガニ血球成分試薬（リムルス属由来、凍結乾燥品、２ｍｌ用。）を４．０ｍｌの０．２Ｍ−トリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液（ｐＨ８．０、エンドトキシンフリー）に溶解した（使用時迄氷冷下で保存。）。これに、１ＥＵ／ｍｌのエンドトキシン水溶液（４．０ｍｌ）を添加して、３７℃で１０分間加温下に反応させたものを酵素液とした。
所定の基質の所定濃度の水溶液（１００μｌ）と、酵素液（１００μｌ）とを混合し、３７℃で２分間反応させた後、３０％酢酸水溶液（８００μｌ）で反応を停止させた。この溶液の４０５ｎｍの吸光度を測定した結果に基づいて求めた各基質のＫｍ値、Ｖｍａｘ値、Ｖｍａｘ値／Ｋｍ値を表１に示す。尚、表１には各基質の水溶性（水にどの程度溶解するか）のデータも併せて示す。
【００３５】
【表１】

【００３６】
表１の結果から、参考例１及び２で得られた基質は水溶性が極めて低いことが、また、参考例３で得られた基質は水溶性は良好ではあるが、エンドトキシンとカブトガニ血球成分との反応の結果活性化される凝固酵素（プロテアーゼ）との反応性が他の基質に比較して悪い（Ｋｍ値が大きく、且つＶｍａｘ／Ｋｍ値が小さい。）ことが判る。
これに対し、本発明のペプチド誘導体は、これら参考例で得られた基質にはない性質、即ちエンドトキシンとカブトガニ血球成分との反応の結果活性化される凝固酵素（プロテアーゼ）との反応性が良好で且つ良好な水溶性を有しており、該凝固酵素の基質として最も適したものであることが判る。
【００３７】
実施例５．本発明のペプチド誘導体を基質として用いたエンドトキシンの定量
（１）カブトガニ血球成分溶液の調製
エンドトキシンテストワコー（和光純薬工業（株）製）中のカブトガニ血球成分試薬（リムルス属由来、凍結乾燥品、２ｍｌ用。）を１２．０ｍｌの０．１Ｍトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液（ｐＨ８．０、エンドトキシンフリー、また、基質として実施例１又は２で得られた本発明のペプチド誘導体を１ｍＭ含有。）に溶解したものをカブトガニ血球成分溶液とした（使用時迄氷冷下で保存。）。
（２）操作法
所定濃度のエンドトキシン（ＵＳＰＲｅｆｅｒｅｎｃｅＥｎｄｏｔｏｘｉｎＳｔａｎｄａｒｄＥＣ−５）を含む水溶液（１００μｌ）と、上記のカブトガニ血球成分溶液（１００μｌ）とを、９６穴マイクロプレートの所定の試料ウェルに分注、混合した後、マイクロプレートリーダーＴ−ＭＡＸ（モレキュラーデバイス社製）にセットし、３７℃加温下で４０５ｎｍの吸光度を１５秒毎に測定した。
測定結果に基づいて到達時間（エンドトキシン溶液と上記カブトガニ血球成分溶液とを混合後から４０５ｎｍの吸光度が０．１００に到達するまでに必要な時間）を求めた。
（３）結果
実施例１で得られた本発明のペプチド誘導体を基質として用いた場合の到達時間とエンドトキシン濃度との関係を示す検量線を図１に示す（縦軸、横軸共に対数目盛り）。
また、実施例２で得られた本発明のペプチド誘導体を基質として用いた場合の到達時間とエンドトキシン濃度との関係を示す検量線を図２に示す（縦軸、横軸共に対数目盛り）。
図１及び図２から明かな如く、本発明のペプチド誘導体を基質として用いることにより、到達時間とエンドトキシン濃度との良好な検量関係が得られること、言い換えれば、エンドトキシンを低濃度から高濃度まで精度よく測定し得ることが判る。
【００３８】
【発明の効果】
以上に述べた如く、本発明は、プロテアーゼ、アミダーゼ、凝固酵素等の活性測定用基質として、特にエンドトキシンとカブトガニ血球成分との反応により活性化される凝固酵素の活性測定用基質として有用な新規なペプチド誘導体を提供するものであり、本発明のペプチド誘導体は、エンドトキシンとカブトガニ血球成分との反応の結果活性化される凝固酵素との反応性が高いのでこれをエンドトキシン定量用の基質として用いれば低濃度のエンドトキシンを検出することが可能であり、しかも水溶性も高いので、エンドトキシン定量用試薬用の凍結乾燥品を調製するための原液を容易に調製することができるという、従来の基質では得られなかった効果を奏するので、斯業に貢献するところ大なる発明である。
【図面の簡単な説明】
【図１】実施例５で得られた、実施例１で得られた本発明のペプチド誘導体を基質として用いた場合の到達時間とエンドトキシン濃度との関係を示す検量線である（縦軸、横軸共に対数目盛り）。
【図２】実施例５で得られた、実施例２で得られた本発明のペプチド誘導体を基質として用いた場合の到達時間とエンドトキシン濃度との関係を示す検量線である（縦軸、横軸共に対数目盛り）。[0001]
Field of application of the invention
The present invention relates to a novel peptide derivative useful as a substrate for measuring the activity of protease, amidase, coagulation enzyme, etc., and particularly useful as a substrate for measuring the activity of coagulation enzyme activated by the reaction between endotoxin and amoebocyte lysate. About.
[0002]
BACKGROUND OF THE INVENTION
Endotoxin is a lipopolysaccharide (Lipopoly-saccharide, LPS) present in the cell wall outer membrane of Gram-negative bacteria, and is known as a strong pyrogen. For this reason, it is considered important to detect endotoxin in injectable drugs and the like, and endotoxin test methods are also listed in the United States and Japanese Pharmacopoeia. In addition, endotoxin is considered to be the main cause of shock in Gram-negative bacterial infections, and in clinical diagnosis, measurement of endotoxin in plasma is useful for diagnosis of Gram-negative bacterial infection, therapeutic effect of Gram-negative bacterial infection, and prognosis. And early diagnosis of endotoxin shock.
[0003]
The blood cell component of horseshoe crab is activated by endotoxin and has the property of activating clotting enzymes (such as proteases) and gelling reactions, and has been used in the fields of medicine, pharmacy, and microbiology. Simple, inexpensive endotoxin detection methods are widely used. Typical examples of this method include, for example, the United States Pharmacopeia Act [U. S. Pharmacopeia XX. 888 (1980)], a method for visually determining the hardness of a gel, a method for measuring turbidity based on gelation, a method for quantifying a clot protein, and the like, all of which are based on the gelation phenomenon. This method may cause some problems in terms of measurement accuracy.
[0004]
On the other hand, by the action of a coagulation enzyme (protease) activated by the reaction between the horseshoe crab blood cell component and endotoxin, a color-forming compound or a fluorescent compound such as nitrophenol, nitroaniline, a methylcoumarin derivative, or an indoxyl derivative is released. Measurement methods using synthetic peptide derivatives as substrates for endotoxin measurement have also been reported (JP-B-59-19532, JP-B-61-54400, JP-A-57-502266, etc.).
[0005]
In the synthetic substrate used in such a method, two amino acid sequences, ie, glycylarginine, are generally used to cleave the bond between the chromogenic group or the fluorescent group and the peptide residue by the coagulation enzyme. It has been found that a residue (-Gly-Arg-) is necessary, and typical examples thereof include the following.
(1) Boc-Leu-Gly-Arg-pNA
(2) Boc-Ser (OBn) -Gly-Arg-pNA
(3) Boc-Ser-Gly-Arg-pNA
(In the above formula, “Boc” represents a t-butoxycarbonyl group, “Leu” represents a leucine residue, “pNA” represents a p-nitroanilino group, “Ser” represents a serine residue, and “Bn” represents serine. A benzyl group bonded to a hydroxyl group of the residue side chain is shown.)
[0006]
However, there are many problems when these peptide derivatives are used as substrates for endotoxin quantification.
That is, for example, the peptide derivatives represented by the above (1) and (2) have high reactivity with the coagulation enzyme but poor water solubility, so that the concentration of the peptide derivative required for efficiently measuring the coagulation enzyme activity is low. It is difficult to obtain an aqueous solution, and the peptide derivative shown in the above (3) has good water solubility but low reactivity with the coagulation enzyme. Has a problem that it is difficult to measure a trace amount of endotoxin.
[0007]
[Object of the invention]
The present invention has been made in view of the above-mentioned circumstances, and has been proposed as a substrate for measuring an activity of a protease, an amidase, a coagulation enzyme, or the like, particularly a coagulation enzyme activated by a reaction between endotoxin and a horseshoe crab hemocyte component (amoebocyte lysate). An object of the present invention is to provide a peptide derivative suitable as a substrate for easily and highly measuring the activity.
[0008]
Configuration of the Invention
The present invention provides a compound represented by the general formula [I]:
X-Thr-Gly-Arg-Y [I]
(Wherein, X represents an N-terminal protecting group of an amino acid, Thr represents a threonine residue, Gly represents a glycine residue, Arg represents an arginine residue, and Y represents the C-terminal of the arginine residue. Represents a color-forming group or a fluorescent group forming a covalent bond.)
Or an acid addition salt thereof.
[0009]
Further, the present invention is an invention of a method for quantifying endotoxin, which comprises using a peptide derivative represented by the general formula [I] or an acid addition salt thereof as a substrate.
[0010]
Furthermore, the present invention comprises reacting a sample containing endotoxin, a horseshoe crab blood cell component with a peptide derivative represented by the general formula [I] or an acid adduct thereof, and increasing the amount of a chromogenic compound or a fluorescent compound released as a result. It is an invention of a method for measuring endotoxin, comprising measuring the amount and quantifying the amount of endotoxin in a sample based on the measurement result.
[0011]
Furthermore, the present invention is an invention of a reagent kit for quantifying an endotoxin, comprising, as constituents, a horseshoe crab blood cell component and a peptide derivative represented by the general formula [I] or an acid adduct thereof.
[0012]
That is, the present inventors have developed a substrate capable of simply and highly sensitively measuring the activity of a coagulation enzyme (protease) such as a clotting enzyme, which is activated as a result of the reaction between endotoxin and a horseshoe crab blood cell component. As a result of intensive studies, the peptide derivative represented by the general formula [I] having a basic skeleton of a peptide in which threonine is further bound to the N-terminal of a glycylarginine residue (-Gly-Arg-) is obtained. Since it has excellent water solubility, it can be easily dissolved in an aqueous solution or a buffer solution used for an enzyme reaction at a target concentration, and has high reactivity with a target clotting enzyme. The inventors have found that the enzyme can be measured with high sensitivity, and have completed the present invention.
[0013]
In the peptide derivative represented by the general formula [I], as the N-terminal protecting group of the amino acid represented by X, a group usually used in this field as an N-terminal protecting group of an amino acid or a peptide is used. Examples include, but are not limited to, acyl groups such as acetyl and benzoyl, benzyloxycarbonyl, t-butoxycarbonyl, tosyl, and glutaryl.
[0014]
In the peptide derivative represented by the general formula [I], the chromogenic group or the fluorescent group represented by Y includes a peptide residue by a coagulation enzyme activated as a result of a reaction between endotoxin and a horseshoe crab blood cell component. And a group capable of generating a compound exhibiting a color or fluorescence by being cleaved (or a compound capable of being a substance exhibiting a color or fluorescence by coupling with an appropriate compound or the like). Examples thereof include, but are not limited to, a p-nitroanilino group, a 3-hydroxymethyl-4-nitroanilino group, a 4- (N-ethyl-N-β-hydroxyethyl) aminoanilino group, Preferable examples include a coloring group such as-(N, N-diethyl) aminoanilino group and a fluorescent group such as 7-amino-4-methylcoumarino group.
[0015]
Further, the peptide derivative represented by the general formula [I] may form an acid addition salt with an inorganic acid or an organic acid. Specific examples of such an acid addition salt include, for example, hydrochloride, sulfate, Inorganic acid salts such as nitrates and phosphates, for example, organic acid salts such as acetate, oxalate, tartrate, succinate, citrate and p-toluenesulfonate are preferred.
[0016]
The peptide derivative of the present invention can be easily synthesized by using a method commonly used for peptide synthesis. For example, the peptide derivative may be synthesized as follows.
That is, first, the general formula [II]
H-Arg-Y [II]
(In the formula, Y is the same as described above.)
Is synthesized as follows, for example, as follows.
[0017]
That is, arginine in which an amino group is protected by a suitable protecting group [for example, t-butoxycarbonyl group (Boc group), benzyloxycarbonyl group (Z group), etc.], or a suitable protecting group (for example, Boc group, Z group) Arginine in which the amino group and guanidino group are protected by nitro group, etc .; and 1 to 2 times the molar amount of arginine, for example, p-nitroaniline, 3-hydroxymethyl-4-nitroaniline, 4- (N- A chromogenic compound such as ethyl-N-β-hydroxyethyl) aminoaniline or 4- (N, N-diethyl) aminoaniline or a fluorescent compound such as 7-amino-4-methylcoumarin is used as a derivative of a peptide derivative. Solvents commonly used in the synthesis, for example, dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran THF) or a mixed solvent thereof in the presence of a condensing agent commonly used for peptide synthesis such as dicyclohexylcarbodiimide (DCC) in a molar amount of 1 to 2 times the amount of arginine at 0 to 25 ° C and 12 to 12 times. Incubate for 24 hours. After completion of the reaction, the reaction solution is concentrated under reduced pressure, and then purified by a method generally used as a method for purifying a peptide derivative, for example, silica gel column chromatography, ion exchange column chromatography, etc., and then subjected to a deprotection treatment. Thereby, the arginine derivative represented by the general formula [II] can be easily obtained. The arginine derivative represented by the general formula [II] is synthesized by a method widely used in the field of peptide chemistry other than the above-mentioned methods, for example, an active ester method, a mixed acid anhydride method, an azide method, a phosphazo method and the like. Needless to say, it is good.
[0018]
Then, the arginine derivative represented by the general formula [II] is added to a arginine derivative represented by the general formula [II] in a solvent usually used in peptide synthesis, for example, DMF, DMSO, THF or a mixed solvent thereof. A threonylglycine in which the N-terminal is protected by a suitable protecting group [for example, a Boc group, a Z group, a benzoyl group (Bz group), an acetyl group (Ac group) or the like] in an amount of 1 to 2 moles, and a general formula [ II] in the presence of a condensing agent commonly used for peptide synthesis such as DCC, for example, in a concentration of 1 to 2 moles relative to the arginine derivative of the formula (II), at 0 to 25 ° C for 12 to 24 hours, or The arginine derivative represented by the formula [II] is converted to a compound represented by the general formula [II] in a solvent usually used in peptide synthesis, for example, DMF, DMSO, THF, or a mixed solvent thereof. The N-terminus of 1 to 2 moles relative to the luginine derivative is protected by a suitable protecting group (for example, Boc group, Z group, Bz group, Ac group, etc.) and at the C-terminus, for example, N-hydroxysuccinimide ester, Reaction with an active ester of threonylglycine to which -hydroxy-5-norbornene-2,3-dicarboximide ester or the like is bound is carried out at 0 to 25 ° C for 12 to 24 hours. After completion of the reaction, the reaction solution is concentrated under reduced pressure, and then purified by a method generally used for purifying a peptide derivative, for example, silica gel column chromatography, ion exchange column chromatography, etc., and if necessary, deprotection treatment. Is carried out, the peptide derivative of the present invention can be easily obtained. The arginine derivative represented by the general formula [II] may be bound to threonylglycine by a method other than the above-mentioned methods widely used in the field of peptide chemistry, for example, a mixed acid anhydride method, an azide method, a phosphazoline method. Needless to say, the method may be used.
[0019]
The peptide derivative of the present invention thus obtained is useful as a substrate for a specific enzyme, for example, a coagulation enzyme (protease, amidase-like enzyme, etc.) activated as a result of the reaction between the horseshoe crab blood cell component and endotoxin.
[0020]
In order to quantify endotoxin using the peptide derivative of the present invention as a substrate, for example, except that the peptide derivative of the present invention is used as a substrate, endotoxin is usually used in this field using a synthetic substrate such as the peptide derivative of the present invention. The operation may be performed according to a so-called synthetic substrate method for performing quantification. Further, the endotoxin, the horseshoe crab blood cell component and the peptide derivative of the present invention are mixed to start the reaction, and as a result, the amount of change in absorbance (or the amount of change in fluorescence intensity) based on the released chromogenic or fluorescent compound is reduced. The time required to reach a certain value (arrival time) may be determined, and this value may be applied to a calibration curve representing the relationship between the arrival time and the endotoxin concentration.
[0021]
The solution containing the hemocyte component of horseshoe crab used at this time can be used without any particular limitation as long as it can be used for usual measurement of endotoxin. For example, ACC, Hemachem, Whittaker Bioproducts A commercially available lyophilized product of the horseshoe crab blood cell component manufactured by the company, Imperial Organ Co., Ltd., Endosafe Co., etc. may be used, or Limulus, Tachypleus, or Calci may be used. There is no particular limitation as long as it is obtained from the blood cells of horseshoe crab belonging to the genus Carcinoscorpius and activates an enzyme (such as a protease) by a reaction with endotoxin.
[0022]
The reagent for quantifying endotoxin using the peptide derivative of the present invention as described above may contain, for example, a buffer or other appropriate reagents, and these reagents are used in a so-called synthetic substrate method or the like. It suffices if it is appropriately selected from those used and the concentration used is appropriately selected from the concentration range selected when preparing a so-called synthetic substrate method reagent or the like. The concentration of the peptide derivative of the present invention used in the above-mentioned method for quantifying endotoxin varies depending on the measurement range of endotoxin, but is usually 0.5 to 5 mM, preferably 0.5 to 5 mM, preferably at the time of reaction. It is appropriately selected from the range of 1 to 3 mM.
[0023]
The method for quantifying endotoxin using the peptide derivative of the present invention as a substrate is more specifically described, for example, as follows.
[0024]
a) Synthetic substrate method
After mixing a sample containing endotoxin and a solution containing a horseshoe crab blood cell component, the mixture is allowed to react for a predetermined time while keeping the temperature at 25 to 40 ° C, and then the peptide derivative of the present invention is added to the solution, and the solution is further kept at a temperature of 25 to 40 ° C. The sample is allowed to react for a predetermined time, or a sample containing endotoxin, a solution containing a horseshoe crab blood cell component, and a peptide derivative of the present invention are mixed, and reacted at 25 to 40 ° C. for a predetermined time. Thereafter, a reaction stop solution (for example, a hydrochloric acid solution, an acetic acid solution, or the like) is added to the reaction solution to stop the reaction, and a predetermined absorbance (or fluorescence intensity) of the obtained final solution is measured with an appropriate measuring device (for example, a spectrophotometer) Meter, microplate reader, fluorometer, etc.).
The obtained measured value is applied to a calibration curve previously prepared using an endotoxin solution having a known concentration and representing the relationship between the measured endotoxin concentration and the endotoxin concentration in the sample.
[0025]
b) Arrival time analysis method
After mixing a sample containing endotoxin, a solution containing a horseshoe crab blood cell component, and the peptide derivative of the present invention, the mixture is subjected to a predetermined change in absorbance (or change in fluorescence intensity) while keeping the temperature at 25 to 40 ° C. The measurement is performed using a measuring instrument (for example, a spectrophotometer, a microplate reader, a fluorometer, or the like), and the time until the change in absorbance (or the change in fluorescence intensity) immediately after mixing reaches a predetermined value ( Arrival time).
The obtained arrival time is applied to a calibration curve that represents the relationship between the arrival time and the endotoxin concentration prepared in advance using an endotoxin solution with a known concentration, and the endotoxin concentration in the sample is determined.
[0026]
In these quantification methods, when a color-forming (or fluorescent) compound released from the peptide derivative of the present invention is measured, if the compound itself has color-forming or fluorescent properties, it is determined on the basis of its properties. Measurement. If the compound itself is not so strong (or hardly any), a compound exhibiting color or fluorescence is produced by coupling with an appropriate compound and the like. Color development or fluorescence may be measured. More specifically, in the latter case, the compound released from the peptide derivative of the present invention is, for example, 4- (N-ethyl-N-β-hydroxyethyl) aminoaniline or 4- (N, N-diethyl) aminoaniline. In the case of these, in the presence of a suitable oxidizing agent or the like, these are oxidatively condensed with phenol, naphthol, or the like, and the resulting blue indophenol-type dye is formed.The absorbance based on the generated dye may be measured. . Further, when the compound released from the peptide derivative of the present invention is, for example, p-nitroaniline, it may be measured as it is because it itself has a coloring property. A diazo coupling reaction may be performed with an appropriate compound, and the absorbance based on the resulting compound may be measured.
[0027]
The endotoxin quantification reagent kit of the present invention is used for quantifying endotoxin by the method described above, and comprises a horseshoe crab blood cell component, a peptide derivative represented by the general formula [I], or an acid adduct thereof. It is included as a component, and preferred embodiments and specific examples of each component are as described above.
Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the present invention is not limited to these Examples and the like.
[0028]
【Example】
Embodiment 1 FIG. Synthesis of t-butoxycarbonylthreonylglycylarginine-p-nitroanilide acetate
1) Synthesis of benzyloxycarbonylarginine-p-nitroanilide hydrochloride
p-Nitroaniline (26 g, 188 mmol) was dissolved in pyridine (400 ml), and a pyridine solution (100 ml) containing phosphorus trichloride (8.2 ml, 94 mmol) was required for 30 minutes while cooling to -20 to -10 ° C. Then, the mixture was allowed to react by stirring at the same temperature for 30 minutes. The reaction solution was returned to room temperature, and after further stirring and reacting for 2 hours, benzyloxycarbonylarginine (58 g, 188 mmol; manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred and reacted at 40 to 50 ° C for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was extracted with a mixed solvent of ethyl acetate: n-butanol = 7: 3. The extract was washed successively with 1N hydrochloric acid and saturated saline, and the organic layer was washed. After drying over anhydrous magnesium sulfate, the solvent was distilled off. The obtained residue was crystallized from acetone to obtain 56.5 g of benzyloxycarbonylarginine-p-nitroanilide hydrochloride (yield 64.6%).
2) Synthesis of t-butoxycarbonylthreonylglycine ethyl ester
Glycine ethyl ester hydrochloride (8.4 g, 60 mmol, manufactured by Wako Pure Chemical Industries, Ltd.) and t-butoxycarbonylthreonine (11.0 g, 50 mmol, manufactured by Wako Pure Chemical Industries, Ltd.) are methylene chloride ( 300 ml), and triethylamine (8.4 ml, 60 mmol) and DCC (12.4 g, 60 mmol) were added thereto under ice-cooling, and the mixture was stirred and reacted at room temperature for 20 hours. After completion of the reaction, insolubles were removed by filtration, the filtrate was concentrated under reduced pressure, and the obtained residue was extracted with ethyl acetate. The extract was washed successively with 1N hydrochloric acid, saturated aqueous sodium hydrogen carbonate and saturated saline, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The obtained residue was subjected to the next reaction without purification.
3) Synthesis of t-butoxycarbonylthreonylglycine
The t-butoxycarbonylthreonylglycine ethyl ester (15.0 g, 49 mmol) obtained in 2) above was dissolved in methanol (600 ml), 1N aqueous sodium hydroxide solution (75 ml) was added thereto, and the mixture was stirred at room temperature for 2 hours. The mixture was stirred and reacted. After completion of the reaction, the reaction solution was neutralized with 1N hydrochloric acid (75 ml), and concentrated under reduced pressure. The obtained residue was dissolved in saturated aqueous sodium hydrogen carbonate and washed with ethyl acetate. The saturated aqueous sodium bicarbonate layer was acidified with 1N hydrochloric acid, and then extracted with ethyl acetate. The ethyl acetate layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and then ethyl acetate was distilled off to obtain 10.0 g of t-butoxycarbonylthreonylglycine (72% yield) as an oil. Obtained.
4) Synthesis of t-butoxycarbonylthreonylglycylarginine-p-nitroanilide acetate
The benzyloxycarbonylarginine-p-nitroanilide hydrochloride (16.3 g, 35 mmol) obtained in 1) above was suspended in a 25% hydrogen bromide-acetic acid solution (50 ml), and the mixture was stirred and reacted under ice cooling for 4 hours. . After completion of the reaction, ether was poured into the reaction solution, and the deposited precipitate was collected by filtration and further washed with ether. This was dried under reduced pressure for 10 hours, then dissolved in DMF (500 ml), and triethylamine (4.9 ml, 35 mmol), DCC (7.2 g, 35 mmol), 1-oxybenzotriazole (5. 4 g, 35 mmol) and t-butoxycarbonylthreonylglycine (9.7 g, 35 mmol) obtained in 3) above were sequentially added, and the mixture was stirred and reacted under ice-cooling for 2 hours and further at room temperature for 20 hours. After completion of the reaction, insolubles were removed by filtration, the filtrate was concentrated under reduced pressure, and the obtained residue was extracted with a mixed solvent of ethyl acetate: n-butanol = 8: 2. The extract was washed successively with 1N hydrochloric acid, saturated aqueous sodium hydrogen carbonate and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The obtained residue is subjected to silica gel column chromatography (Wakogel C-300; trade name of Wako Pure Chemical Industries, Ltd., 750 g, eluent; a mixed solvent of ethyl acetate: pyridine: acetic acid: water = 60: 20: 6: 11). The residue obtained by concentrating the eluted fraction of the desired product under reduced pressure was extracted with a mixed solvent of ethyl acetate: n-butanol = 8: 2. The extract was washed successively with 1N hydrochloric acid, saturated aqueous sodium hydrogen carbonate and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The obtained residue was subjected to ion exchange column chromatography (Amberlite IRA-410 acetic acid type; Amberlite, 500 cm). ³ , Eluate; water). The powder obtained by freeze-drying the eluted fraction of the desired product was purified by gel filtration column chromatography (Sephadex LH-20, trade name of Pharmacia; eluent; methanol). Further, the residue obtained by concentrating the eluted fraction of the target substance under reduced pressure is dissolved in distilled water for injection (700 ml, manufactured by Otsuka Pharmaceutical Co., Ltd.), and subjected to ultrafiltration (manufactured by Sartorius; Ultrazart D-20, And its unit) to remove the exothermic substance, and freeze-dry the filtrate to obtain 11.5 g of t-butoxycarbonylthreonylglycylarginine-p-nitroanilide acetate (yield 53.7%). Obtained.
Elemental analysis value (C ₂₅ H ₄₀ N ₈ O ₁₀ As)
Calculated value (%): C 49.01, H 6.58, N 18.29
Measured value (%): C 47.31, H 6.43, N 17.68
[0029]
Embodiment 2. FIG. Synthesis of t-butoxycarbonylthreonylglycylarginine-3-hydroxymethyl-4-nitroanilide acetate
1) Synthesis of benzyloxycarbonylarginine-3-acetoxymethyl-4-nitroanilide hydrochloride
3-acetoxymethyl-4-nitroaniline (2.1 g) synthesized by performing acetylation, nitration, etc. using 3-aminobenzyl alcohol as a raw material according to the synthesis method described in Japanese Patent Publication No. 6-27111. , 10 mmol) in pyridine (50 ml) was cooled to −20 to −10 ° C., and a pyridine solution (5 ml) containing phosphorus trichloride (0.44 ml, 5 mmol) was added dropwise over 5 minutes. The reaction was stirred at -10 ° C for 30 minutes. After the reaction, the temperature is returned to room temperature, and the mixture is further stirred and reacted for 1 hour. Then, benzyloxycarbonylarginine (3.1 g, 10 mmol, manufactured by Wako Pure Chemical Industries, Ltd.) is added, and the mixture is stirred and reacted at 40 to 50 ° C. for 3 hours. Was. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was extracted with ethyl acetate. The extract was washed sequentially with 1N hydrochloric acid and saturated saline, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The obtained residue is subjected to silica gel column chromatography (Wakogel C-300; trade name of Wako Pure Chemical Industries, Ltd., 250 g, eluent; ethyl acetate: pyridine: acetic acid: water = 60: 20: 6: 11 mixed solvent) ). The eluted fraction of the desired product was concentrated under reduced pressure, and the obtained residue was extracted with ethyl acetate. The extract was washed successively with 1N hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated saline, dried over anhydrous magnesium sulfate, and then the solvent was distilled off to obtain 3.8 g of benzyloxycarbonylarginine-3-acetoxymethyl-4-. Nitroanilide hydrochloride (76.1% yield) was obtained as an oil.
2) Synthesis of benzyloxycarbonylarginine-3-hydroxymethyl-4-nitroanilide hydrochloride
The benzyloxycarbonylarginine-3-acetoxymethyl-4-nitroanilide hydrochloride (3.6 g, 6.7 mmol) obtained in 1) above was dissolved in methanol (150 ml), and sodium methoxy was added thereto under ice-cooling. Then, a 28% methanol solution (0.15 ml) of the compound was added, and the mixture was stirred and reacted under ice cooling for 3 hours. After completion of the reaction, the reaction solution was neutralized with 1 N hydrochloric acid, and concentrated under reduced pressure. The obtained residue was extracted with a mixed solvent of ethyl acetate: n-butanol = 6: 4, and the extract was washed with 1N hydrochloric acid and distilled water sequentially, and then the solvent was distilled off. The obtained residue was crystallized with a mixed solvent of ethanol / ether to obtain 3.0 g of benzyloxycarbonylarginine-3-hydroxymethyl-4-nitroanilide hydrochloride (yield 91.0%). 3) Synthesis of t-butoxycarbonylthreonylglycylarginine-3-hydroxymethyl-4-nitroanilide acetate
The benzyloxycarbonylarginine-3-hydroxymethyl-4-nitroanilide hydrochloride (2.5 g, 5 mmol) obtained in 2) above was suspended in a 25% hydrogen bromide-acetic acid solution (5 ml), and the suspension was added under ice-cooling. The mixture was stirred for a period of time. After completion of the reaction, ether was poured into the reaction solution, and the deposited precipitate was collected by filtration and washed with ether. After drying under reduced pressure for 10 hours, this was dissolved in DMF (100 ml), and triethylamine (0.7 ml, 5 mmol), DCC (1.0 g, 5 mmol), 1-oxybenzotriazole (0. 8 g, 5 mmol) and t-butoxycarbonylthreonylglycine (1.4 g, 5 mmol) obtained in 3) of Example 1 described above were successively added, and the mixture was stirred for 2 hours under ice-cooling, further returned to room temperature, and stirred for 20 hours. Reacted. After completion of the reaction, insolubles were removed by filtration, the filtrate was concentrated under reduced pressure, and the obtained residue was extracted with a mixed solvent of ethyl acetate: n-butanol = 7: 3. The extract was washed successively with 1N hydrochloric acid, saturated aqueous sodium hydrogen carbonate and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The obtained residue was subjected to silica gel column chromatography (Wakogel C-300; trade name of Wako Pure Chemical Industries, Ltd., 300 g, eluent; a mixed solvent of ethyl acetate: pyridine: acetic acid: water = 60: 20: 6: 11). ). The eluted fraction of the target substance was concentrated under reduced pressure, and the obtained residue was extracted with a mixed solvent of ethyl acetate: n-butanol = 7: 3. The extract was washed successively with 1N hydrochloric acid, saturated aqueous sodium hydrogen carbonate and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The obtained residue was subjected to ion exchange column chromatography (Amberlite IRA-410 acetic acid type; Amberlite, 50 cm). ³ , Eluate; water). The eluted fraction of the desired product was freeze-dried, and the obtained powder was purified by gel filtration column chromatography (Sephadex LH-20, trade name of Pharmacia; eluent; methanol). Furthermore, the residue obtained by concentrating the eluted fraction of the target product under reduced pressure is dissolved in distilled water for injection (50 ml, manufactured by Otsuka Pharmaceutical Co., Ltd.) and subjected to ultrafiltration (manufactured by Sartorius; Ultrazart D-20, And its unit), and the filtrate was lyophilized to give 2.1 g of t-butoxycarbonylthreonylglycylarginine-3-hydroxymethyl-4-nitroanilide acetate (yield 65). .1%).
Elemental analysis value (C ₂₆ H ₄₂ N ₈ O ₁₁ As)
Calculated value (%): C 48.59, H 6.59, N 17.44
Measured value (%): C 47.31, H 6.43, N 17.68
[0030]
Embodiment 3 FIG. Synthesis of t-butoxycarbonylthreonylglycylarginine-4- (N-ethyl-N-β-hydroxyethyl) aminoanilide acetate
1) t-butoxycarbonyl-N ^G Synthesis of -nitroarginine-4- (N-ethyl-N-β-hydroxyethyl) aminoanilide
t-butoxycarbonyl-N ^G -Nitroarginine (20.2 g, 63 mmol, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in DMF (130 ml), and pyridine (5.0 ml, 63 mmol) and N-ethylpiperidine (8.7 ml, 63 mmol) were added. In addition, after cooling to −20 to −15 ° C., a DMF solution (30 ml) containing pivaloyl chloride (7.7 ml, 63 mmol) was added dropwise over 10 minutes, followed by stirring at 0 to 5 ° C. for 10 minutes. After cooling this to -20 to -15 ° C again, DMF containing 4- (N-ethyl-N-β-hydroxyethyl) aminoaniline (11.4 g, 63 mmol, manufactured by Wako Pure Chemical Industries, Ltd.). A solution (30 ml) was added, and the mixture was reacted at −10 to 5 ° C. for 2 hours and then returned to room temperature for 12 hours with stirring. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was dissolved in a 10% aqueous citric acid solution and washed with ethyl acetate. The aqueous layer was made alkaline with saturated aqueous sodium hydrogen carbonate and extracted with ethyl acetate. The extract was washed with brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The obtained residue was purified by silica gel column chromatography (Wakogel C-300; trade name of Wako Pure Chemical Industries, Ltd., 1000 g, eluent; chloroform: methanol = 9: 1 mixed solvent). The eluted fraction of the target substance was concentrated under reduced pressure, and the obtained residue was triturated with hexane to give 23.8 g of t-butoxycarbonyl-N. ^G -Nitroarginine-4- (N-ethyl-N-β-hydroxyethyl) aminoanilide (yield 78.2%) was obtained.
2) Synthesis of t-butoxycarbonylthreonylglycine-5-norbornene-2,3-dicarboximide ester
The t-butoxycarbonylthreonylglycine (3.3 g, 12 mmol) obtained in 3) of Example 1 and N-hydroxy-5-norbornene-2,3-dicarboximide (2.2 g, 12 mmol) were combined with each other. It was dissolved in DMF (50 ml), DCC (2.5 g, 12 mmol) was added under ice cooling, and the mixture was stirred and reacted at room temperature for 20 hours. After completion of the reaction, insolubles were removed by filtration, and the filtrate was concentrated under reduced pressure to obtain t-butoxycarbonylthreonylglycine-5-norbornene-2,3-dicarboximide ester as an oil. The obtained oil was subjected to the next reaction without purification.
3) t-butoxycarbonylthreonylglycyl-N ^G Synthesis of -nitroarginine-4- (N-ethyl-N-β-hydroxyethyl) aminoanilide
T-butoxycarbonyl-N obtained in 1) above ^G -Nitroarginine-4- (N-ethyl-N-β-hydroxyethyl) aminoanilide (4.8 g, 10 mmol) was suspended in trifluoroacetic acid (25 ml), and anisole (1 ml) was added. The mixture was stirred for an hour. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and ether was poured into the obtained residue. The deposited precipitate was collected by filtration and washed with ether. This was dried under reduced pressure for 10 hours, then dissolved in DMF (25 ml), and triethylamine (2.8 ml, 20 mmol) under ice cooling and t-butoxycarbonylthreonylglycine-5 obtained with 2) above. A DMF solution (20 ml) containing -norbornene-2,3-dicarboximide ester (12 mmol) was added, and the mixture was allowed to react under stirring for 2 hours under ice cooling and further to room temperature for 20 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was extracted with a mixed solvent of ethyl acetate: n-butanol = 8: 2. The extract was washed with brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The obtained residue was purified by silica gel column chromatography (Wakogel C-300; trade name of Wako Pure Chemical Industries, Ltd., 600 g, eluent; mixed solvent of chloroform: methanol = 9: 1). The eluted fraction of the desired product was concentrated under reduced pressure, and the obtained residue was crystallized from ether to obtain 4.4 g of t-butoxycarbonylthreonylglycyl-N ^G -Nitroarginine-4- (N-ethyl-N-β-hydroxyethyl) aminoanilide (yield 68.1%) was obtained.
4) Synthesis of t-butoxycarbonylthreonylglycylarginine-4- (N-ethyl-N-β-hydroxyethyl) aminoanilide acetate
T-butoxycarbonylthreonylglycyl-N obtained in 3) above ^G -Nitroarginine-4- (N-ethyl-N-β-hydroxyethyl) aminoanilide (3.2 g, 5 mmol) was dissolved in methanol (350 ml), and a palladium carbon catalyst (500 mg) was added. A hydrogenation reaction was performed. After completion of the reaction, insolubles were removed by filtration, and the filtrate was concentrated under reduced pressure. The obtained residue was subjected to ion exchange column chromatography (Amberlite IRA-410 acetic acid type; Amberlite, 50 cm). ³ , Eluate; water). The eluted fraction of the target substance is further subjected to ion exchange column chromatography (CM-TOYOPEARL 650M; manufactured by Tosoh Corporation, 40 cm ³ , Eluate; distilled water to 0.2 M ammonium acetate, linear gradient). The eluted fraction of the target substance was freeze-dried, and the obtained powder was purified by gel filtration column chromatography (Sephadex LH-20, trade name of Pharmacia, eluent; methanol). The eluted fraction of the target substance is concentrated under reduced pressure, the obtained residue is dissolved in distilled water for injection (30 ml, Otsuka Pharmaceutical Co., Ltd.), and ultrafiltration (Sartorius; Ultrazart D-20, and its unit) )), And the filtrate was freeze-dried to obtain 3.3 g of t-butoxycarbonylthreonylglycylarginine-4- (N-ethyl-N-β-hydroxyethyl) aminoanilide acetate. (48.8% yield).
Elemental analysis value (C ₂₉ H ₅₀ N ₈ O ₉ As)
Calculated value (%): C 53.20, H 7.70, N 17.11
Measured value (%): C 51.37, H 7.56, N 16.49
[0031]
Reference Example 1. Synthesis of t-butoxycarbonyl-O-benzylserylglycylarginine-p-nitroanilide acetate
1) Synthesis of t-butoxycarbonyl-O-benzylserylglycine ethyl ester
Glycine ethyl ester hydrochloride (6.7 g, 48 mmol, manufactured by Wako Pure Chemical Industries, Ltd.) and t-butoxycarbonyl-O-benzylserine (11.8 g, 40 mmol, manufactured by Peptide Research Laboratories) in THF (150 ml). After suspending and adding ice-cooled triethylamine (6.7 ml, 48 mmol) and DCC (9.9 g, 48 mmol), the mixture was stirred and reacted at room temperature for 20 hours. After completion of the reaction, a post-treatment was performed according to 2) of Example 1. The obtained residue was subjected to the next reaction without purification.
2) Synthesis of t-butoxycarbonyl-O-benzylserylglycine
The t-butoxycarbonyl-O-benzylserylglycine ethyl ester (15.2 g, 40 mmol) obtained in the above 1) was dissolved in methanol (400 ml), and a 1N aqueous sodium hydroxide solution (60 ml) was added thereto. The reaction was stirred for 2 hours. After completion of the reaction, post-treatment was carried out according to 3) of Example 1 to obtain 5.7 g of t-butoxycarbonyl-O-benzylserylglycine (yield: 40%).
3) Synthesis of t-butoxycarbonyl-O-benzylserylglycylarginine-p-nitroanilide acetate
The benzyloxycarbonylarginine-p-nitroanilide hydrochloride (4.6 g, 10 mmol) obtained in 1) of Example 1 was suspended in a 25% hydrogen bromide-acetic acid solution (15 ml), and the mixture was stirred under ice cooling for 4 hours. Reacted. After completion of the reaction, ether was poured into the reaction solution, and the deposited precipitate was collected by filtration and washed with ether. This was dried under reduced pressure for 10 hours, then dissolved in DMF (50 ml), and triethylamine (1.4 ml, 10 mmol), DCC (2.1 g, 10 mmol), 1-oxybenzotriazole (1. 5 g, 10 mmol) and the t-butoxycarbonyl-O-benzylserylglycine (3.5 g, 10 mmol) obtained in the above 2) are sequentially added, and the mixture is stirred and reacted for 2 hours under ice-cooling and further returned to room temperature for 20 hours. Was. After completion of the reaction, post-treatment and purification were carried out according to 4) of Example 1, and 5.8 g of t-butoxycarbonyl-O-benzylserylglycylarginine-p-nitroanilide acetate (yield 85%). Was obtained as a lyophilized powder.
Elemental analysis value (C ₃₁ H ₄₄ N ₈ O ₁₀ As)
Calculated value (%): C 54.06, H 6.44, N 16.27
Measured value (%): C 53.80, H 6.33, N 16.13
[0032]
Reference example 2. Synthesis of t-butoxycarbonylleucylglycylarginine-p-nitroanilide acetate
1) Synthesis of t-butoxycarbonylleucylglycine ethyl ester
Glycine ethyl ester hydrochloride (6.7 g, 48 mmol, manufactured by Wako Pure Chemical Industries, Ltd.) and t-butoxycarbonylleucine monohydrate (10.0 g, 40 mmol, manufactured by Wako Pure Chemical Industries, Ltd.) are salified. After suspending in methylene (150 ml) and adding triethylamine (6.7 ml, 48 mmol) and DCC (9.9 g, 48 mmol) under ice-cooling, the mixture was stirred and reacted at room temperature for 20 hours. After completion of the reaction, a post-treatment was performed according to 2) of Example 1. The obtained residue was subjected to the next reaction without purification.
2) Synthesis of t-butoxycarbonylleucylglycine
The t-butoxycarbonylleucylglycine ethyl ester (12.5 g, 40 mmol) obtained in 1) above was dissolved in methanol (400 ml), 1N aqueous sodium hydroxide solution (60 ml) was added thereto, and the mixture was stirred at room temperature for 2 hours. Reacted. After completion of the reaction, post-treatment was carried out according to 3) of Example 1 to obtain 9.0 g of t-butoxycarbonylleucylglycine (yield 78%).
3) Synthesis of t-butoxycarbonylleucylglycylarginine-p-nitroanilide acetate
The benzyloxycarbonylarginine-p-nitroanilide hydrochloride (4.6 g, 10 mmol) obtained in 1) of Example 1 was suspended in a 25% hydrogen bromide-acetic acid solution (15 ml), and the mixture was stirred under ice cooling for 4 hours. Reacted. After completion of the reaction, ether was poured into the reaction solution, and the deposited precipitate was collected by filtration and washed with ether. This was dried under reduced pressure for 10 hours, then dissolved in DMF (50 ml), and triethylamine (1.4 ml, 10 mmol), DCC (2.1 g, 10 mmol), 1-oxybenzotriazole (1. 5 g, 10 mmol) and t-butoxycarbonylleucylglycine (2.9 g, 10 mmol) obtained in the above 2) were sequentially added, and the mixture was stirred and reacted for 2 hours under ice-cooling and further returned to room temperature for 20 hours. After completion of the reaction, post-treatment and purification were carried out according to 4) of Example 1, and 5.5 g of t-butoxycarbonylleucylglycylarginine-p-nitroanilide acetate (88% yield) was lyophilized. Obtained as a powder.
Elemental analysis value (C ₂₇ H ₄₄ N ₈ O ₉ As)
Calculated value (%): C 51.91, H 7.10, N 17.94
Measured value (%): C 51.18, H 7.02, N 17.18
[0033]
Reference example 3. Synthesis of t-butoxycarbonylserylglycylarginine-p-nitroanilide acetate
1) Synthesis of t-butoxycarbonylserylglycine ethyl ester
Glycine ethyl ester hydrochloride (6.7 g, 48 mmol, manufactured by Wako Pure Chemical Industries, Ltd.) and t-butoxycarbonylserine (8.2 g, 40 mmol, manufactured by Peptide Research Laboratories) were suspended in THF (150 ml), and iced. After adding triethylamine (6.7 ml, 48 mmol) and DCC (9.9 g, 48 mmol) under cooling, the mixture was stirred and reacted at room temperature for 20 hours. After completion of the reaction, a post-treatment was performed according to 2) of Example 1. The obtained residue was subjected to the next reaction without purification.
2) Synthesis of t-butoxycarbonylserylglycine
The t-butoxycarbonylserylglycine ethyl ester (11.6 g, 40 mmol) obtained in 1) above was dissolved in methanol (400 ml), 1N aqueous sodium hydroxide solution (60 ml) was added thereto, and the mixture was stirred and reacted at room temperature for 2 hours. I let it. After completion of the reaction, post-treatment was carried out according to 3) of Example 1 to obtain 7.0 g of t-butoxycarbonylserylglycine (yield 67%).
3) Synthesis of t-butoxycarbonylserylglycylarginine-p-nitroanilide acetate
The benzyloxycarbonylarginine-p-nitroanilide hydrochloride (4.6 g, 10 mmol) obtained in 1) of Example 1 was suspended in a 25% hydrogen bromide-acetic acid solution (15 ml), and the mixture was stirred under ice cooling for 4 hours. Reacted. After completion of the reaction, ether was poured into the reaction solution, and the deposited precipitate was collected by filtration and washed with ether. This was dried under reduced pressure for 10 hours, then dissolved in DMF (50 ml), and triethylamine (1.4 ml, 10 mmol), DCC (2.1 g, 10 mmol), 1-oxybenzotriazole (1 0.5 g, 10 mmol) and t-butoxycarbonylserylglycine (2.6 g, 10 mmol) obtained in 2) above were sequentially added, and the mixture was stirred and reacted for 2 hours under ice-cooling, and further returned to room temperature for 20 hours. After completion of the reaction, post-treatment and purification were carried out according to 4) of Example 1, and 4.3 g of t-butoxycarbonylserylglycylarginine-p-nitroanilide acetate (yield 73%) was obtained as a lyophilized powder. Obtained.
Elemental analysis value (C ₂₄ H ₃₈ N ₈ O ₁₀ As)
Calculated value (%): C 48.16, H 6.40, N 18.72
Measured value (%): C 47.61, H 6.31, N 18.70
[0034]
Embodiment 4. FIG. Examination of substrate reactivity
A Limulus amoebocyte blood cell component reagent (derived from Limulus spp., Lyophilized product, for 2 ml) in Endotoxin Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used in 4.0 ml of 0.2 M-tris (hydroxymethyl) aminomethane-hydrochloric acid. It was dissolved in a buffer (pH 8.0, endotoxin-free) (stored under ice cooling until use). A 1 EU / ml endotoxin aqueous solution (4.0 ml) was added thereto, and the mixture was reacted at 37 ° C. for 10 minutes under heating to obtain an enzyme solution.
An aqueous solution (100 μl) having a predetermined concentration of a predetermined substrate and an enzyme solution (100 μl) were mixed, reacted at 37 ° C. for 2 minutes, and then stopped with a 30% acetic acid aqueous solution (800 μl). Table 1 shows the Km value, Vmax value, and Vmax value / Km value of each substrate obtained based on the results of measuring the absorbance at 405 nm of this solution. Table 1 also shows data on the water solubility of each substrate (how much it dissolves in water).
[0035]
[Table 1]

[0036]
From the results shown in Table 1, the substrates obtained in Reference Examples 1 and 2 have extremely low water solubility, and the substrate obtained in Reference Example 3 has good water solubility, but the endotoxin and horseshoe crab blood cell components It can be seen that the reactivity with the coagulation enzyme (protease) activated as a result of the reaction is worse than other substrates (the Km value is large and the Vmax / Km value is small).
On the other hand, the peptide derivative of the present invention has a property not found in the substrates obtained in these reference examples, that is, good reactivity with a coagulation enzyme (protease) activated as a result of the reaction between endotoxin and a horseshoe crab blood cell component. It has good water solubility and is found to be the most suitable as a substrate for the coagulation enzyme.
[0037]
Embodiment 5 FIG. Determination of endotoxin using the peptide derivative of the present invention as a substrate
(1) Preparation of horseshoe crab blood cell component solution
A horseshoe crab blood cell component reagent (derived from Limulus spp., Lyophilized product, for 2 ml) in Endotoxin Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was buffered with 12.0 ml of 0.1 M tris (hydroxymethyl) aminomethane-hydrochloride buffer. A solution of a horseshoe crab blood cell component solution dissolved in a solution (pH 8.0, endotoxin-free, and containing 1 mM of the peptide derivative of the present invention obtained in Example 1 or 2 as a substrate) was used (ice-cooled until use). Save below.)
(2) Operation method
After dispensing and mixing an aqueous solution (100 μl) containing a predetermined concentration of endotoxin (USP Reference Endotoxin Standard EC-5) and the aforementioned horseshoe crab blood cell component solution (100 μl) into a predetermined sample well of a 96-well microplate, It was set on a microplate reader T-MAX (manufactured by Molecular Devices), and the absorbance at 405 nm was measured every 15 seconds while heating at 37 ° C.
The arrival time (the time required after the endotoxin solution and the horseshoe crab blood cell component solution were mixed until the absorbance at 405 nm reached 0.100) was determined based on the measurement results.
(3) Result
FIG. 1 shows a calibration curve showing the relationship between the arrival time and the endotoxin concentration when the peptide derivative of the present invention obtained in Example 1 is used as a substrate (both vertical and horizontal axes are logarithmic scales).
FIG. 2 shows a calibration curve showing the relationship between the arrival time and endotoxin concentration when the peptide derivative of the present invention obtained in Example 2 was used as a substrate (both vertical and horizontal axes are logarithmic scales).
As is clear from FIGS. 1 and 2, by using the peptide derivative of the present invention as a substrate, a good calibration relationship between the arrival time and the endotoxin concentration can be obtained. In other words, the accuracy of endotoxin from low to high concentrations can be improved. It turns out that it can measure well.
[0038]
【The invention's effect】
INDUSTRIAL APPLICABILITY As described above, the present invention is a novel substrate useful for measuring the activity of protease, amidase, coagulation enzyme, etc., and particularly useful as a substrate for measuring the activity of coagulation enzyme activated by the reaction between endotoxin and a horseshoe crab blood cell component. The present invention provides a peptide derivative, and the peptide derivative of the present invention has high reactivity with a coagulation enzyme that is activated as a result of the reaction between endotoxin and a horseshoe crab blood cell component. Since it is possible to detect endotoxin at a high concentration and has high water solubility, it is possible to easily prepare a stock solution for preparing a lyophilized product for a reagent for quantifying endotoxin. It is a great invention that contributes to the industry because it has an effect that has not been achieved.
[Brief description of the drawings]
FIG. 1 is a calibration curve showing the relationship between the arrival time and endotoxin concentration obtained when the peptide derivative of the present invention obtained in Example 1 was used as a substrate, obtained in Example 5 (vertical axis, horizontal axis) Logarithmic scale for both axes).
FIG. 2 is a calibration curve showing the relationship between the arrival time and endotoxin concentration obtained when the peptide derivative of the present invention obtained in Example 2 was used as a substrate, obtained in Example 5 (vertical axis, horizontal axis). Logarithmic scale for both axes).

Claims

General formula [I]
X-Thr-Gly-Arg-Y [I]
(Wherein, X represents an N-terminal protecting group of an amino acid, Thr represents a threonine residue, Gly represents a glycine residue, Arg represents an arginine residue, and Y represents the C-terminal of the arginine residue. Represents a color-forming group or a fluorescent group forming a covalent bond.)
Or an acid addition salt thereof.

The peptide derivative according to claim 1, wherein the N-terminal protecting group of the amino acid is an acetyl group, a benzoyl group, a benzyloxycarbonyl group, a tosyl group or a t-butoxycarbonyl group, or an acid addition salt thereof.

The color-forming or fluorescent group forming a covalent bond with the C-terminal of the arginine residue is a p-nitroanilino group, a 3-hydroxymethyl-4-nitroanilino group, or a 4- (N-ethyl-N-β- The peptide derivative according to claim 1, which is a (hydroxyethyl) aminoanilino group, a 4- (N, N-diethyl) aminoanilino group or a 7-amino-4-methylcoumarino group, or an acid addition salt thereof.

A method for quantifying endotoxin, comprising using the peptide derivative according to claim 1 or an acid adduct thereof as a substrate.

A sample containing endotoxin, a horseshoe crab blood cell component, and a peptide derivative or an acid adduct thereof according to claim 1 are reacted with each other, and the amount of a released chromogenic compound or fluorescent compound is measured, and the measurement result is measured. A method for measuring endotoxin, comprising quantifying the amount of endotoxin in a sample based on the method.

A reagent kit for quantifying an endotoxin comprising a horseshoe crab blood cell component and the peptide derivative or the acid adduct thereof according to claim 1 as constituent components.