JPS6346080B2

JPS6346080B2 -

Info

Publication number: JPS6346080B2
Application number: JP59268537A
Authority: JP
Inventors: Kyoichi Sakakibara; Masaaki Gondo; Haruo Yamashita; Yoshikazu Isowa
Original assignee: Sagami Chemical Research Institute; Teikoku Hormone Manufacturing Co Ltd
Current assignee: Aska Pharmaceutical Co Ltd; Sagami Chemical Research Institute
Priority date: 1984-12-21
Filing date: 1984-12-21
Publication date: 1988-09-13
Also published as: JPS60248700A

Description

[Detailed description of the invention]

本発明は新規なポリペプチドに関し、さらに詳
しくは下記式式中、Ｒは水素原子又は低級アルキル基を表わ
し、X₁及びX₂はそれぞれチオールの保護基を表
わすか、或いはX₁とX₂は一緒になつて単結合を
表わし、Y₁、Y₄及びY₅はそれぞれ水素原子又は
保護基を表わす、で示されるペプチドに関する。本発明により提供される上記式（）のペプチ
ドは、下記式及びで示されるポリペプチド、又は１位−７位間のジ
スルフイド結合が開環している該ポリペプチドの
誘導体、或いはこれらポリペプチド化合物の官能
基保護誘導体の製造のための中間体として有用で
ある。上記式（−ａ）及び（−ｂ）で示されるポ
リペプチドは、強力な血清カルシウム及びリン低
下活性を有し、骨形成促進作用及び骨吸収抑制作
用、尿中リン排泄促進作用等の優れた薬理作用が
あり、例えば内因性甲状線カルシトニン欠乏によ
る過カルシウム血症、骨折、骨軟化症、くる病等
の予防又は治療剤として既に公知の化合物であ
る。しかして、上記ポリペプチドの製造方法として
は従来から多くの方法が提案されており、例え
ば、離脱可能なアミノ基を含有する樹脂支持体を
用いる固相合成法（例えば、特開昭49−80087号
公報参照）や、上記ポリペプチドに対応する部分
配列をもつペプチドフラグメントを液相中で形成
せしめ、各フラグメントを液相中でさらにカツプ
リングさせる液相合成法（例えば特公昭54−
29513号公報参照）等が知られている。しかしな
がら、これら従来知られている方法は、例えば、
固相合成法では各段階の反応が完全に100％進行
しない限り、特に長鎖ペプチドでは最終目的物の
純度が低下しその精製に多大の困難を伴うこと、
使用するアミノ酸誘導体は側鎖官能基をすべて完
全に保護しなければならず、最終段階でまたその
すべてを完全に除去しなくてはらならないが、副
反応を伴う危険性が常に存すること、また溶媒、
試薬を大過剰に用いるので不経済なこと、スケー
ルアツプし難いことなどの欠点がある。一方液相
合成法ではアミノ酸の数が増すに従つてその溶解
度が微妙に変化し適当な溶媒を見出すのが次第に
困難になりそれにつれて未反応物や副生成物との
分離の困難さも増大してくること、分子量が増え
るに従つて溶液中のモル濃度が低下しその結果ペ
プチド鎖延長反応がむずかしくなつてゆくこと、
原料の回収が殆ど不可能なことなどの欠点があ
り、工業的に充分に満足できる方法とはいえな
い。本発明者らは上記の如き欠点の少ない上記式
（−ａ）又は（−ｂ）のポリペプチド又はそ
の誘導体の製造方法を提供することを目的として
鋭意研究を行なつた結果、以下に述べる如き方法
により、その目的を達成しうることを見出した。
また、上記式（）のペプチドは従来の文献に未
載の新規な化合物であり、これらの方法を行なう
のに際し極めて有用な中間体となることを見出し
た。すなわち、本発明により提供される式式中、Ｒ、X₁、X₂、Y₁、Y₄及びY₅は前記の意
味を有する、で示されるペプチドを式Ｈ−Thr−Ａ−Gly−Thr−Pro−NH₂ （）式中、ＡはAsn−Thr−Gly−Ser又はAsp−
Val−Gly−Alaを表わすで示されるペプチド又はその官能基保護誘導体
と、PH４〜10の緩衝溶液を含む媒質中で、トリプ
シン又はトリプシン様蛋白分解酵素の存在下に反
応せしめることにより式式中、Ａ、X₁、X₂、Y₁、Y₄及びY₅は前記の意
味を有する、で示されるペプチドを有利に製造することができ
る。なお、本明細書においてアミノ酸単位を表わす
ために使用する各略号は下記の意味を有する。 Ala−＝アラニル基、 Arg−＝アラギニル基、 Asn−＝アスパラギニル基、 Asp−＝アスパルチル基、 Cys−＝システイニル基、 Gln−＝グルタミニル基、 Glu−＝グルタミル基、 Gly−＝グリシル基、 His−＝ヒスチジル基、 Leu−＝ロイシル基、 Lys−＝リジル基、 Pro−＝プロリル基、 Ser−＝セリル基、 Thr−＝スレオニル基、 Tyr−＝チロシル基、 Val−＝バリル基。また、略号 The present invention relates to a novel polypeptide, and more specifically, to a novel polypeptide having the following formula: In the formula, R represents a hydrogen atom or a lower alkyl group, X ₁ and X ₂ each represent a thiol protecting group, or X ₁ and X ₂ together represent a single bond, and Y ₁ , Y ₄ and Y ₅ each represent a hydrogen atom or a protecting group. The peptide of the above formula () provided by the present invention has the following formula: as well as It is useful as an intermediate for producing the polypeptide represented by the above formula, a derivative of the polypeptide in which the disulfide bond between the 1st and 7th positions is ring-opened, or a functional group-protected derivative of these polypeptide compounds. The polypeptides represented by the above formulas (-a) and (-b) have strong serum calcium and phosphorus lowering activities, and have excellent effects such as promoting bone formation, inhibiting bone resorption, and promoting urinary phosphorus excretion. It is a compound that has pharmacological effects and is already known as a preventive or therapeutic agent for hypercalcemia, bone fractures, osteomalacia, rickets, etc. caused by endogenous thyroid calcitonin deficiency. Therefore, many methods have been proposed for producing the above-mentioned polypeptides. For example, solid-phase synthesis using a resin support containing a removable amino group (for example, (Refer to Japanese Patent Publication No. 1999-11-1) or a liquid-phase synthesis method in which a peptide fragment having a partial sequence corresponding to the above-mentioned polypeptide is formed in a liquid phase, and each fragment is further coupled in the liquid phase (for example, Japanese Patent Publication No.
29513) etc. are known. However, these conventionally known methods, for example,
In solid-phase synthesis, unless the reaction at each step progresses to 100%, the purity of the final target product will decrease, especially for long-chain peptides, and its purification will be extremely difficult.
The amino acid derivatives used must have all side chain functional groups completely protected, and all of them must be completely removed in the final step, but there is always a risk of side reactions and solvent ,
This method has drawbacks such as being uneconomical because it uses a large excess of reagents and difficult to scale up. On the other hand, in liquid phase synthesis, as the number of amino acids increases, their solubility changes slightly, making it increasingly difficult to find a suitable solvent, and the difficulty of separating them from unreacted substances and by-products increases accordingly. As the molecular weight increases, the molar concentration in the solution decreases, and as a result, the peptide chain extension reaction becomes difficult.
This method has drawbacks such as the fact that it is almost impossible to recover raw materials, and cannot be said to be a fully satisfactory method industrially. The present inventors have conducted intensive research with the aim of providing a method for producing the polypeptide of the formula (-a) or (-b) or its derivatives, which has fewer drawbacks as described above, and as a result, the following results have been obtained. We have found that this objective can be achieved by a method.
Furthermore, it has been found that the peptide of the above formula () is a novel compound that has not been described in conventional literature, and can be an extremely useful intermediate in carrying out these methods. That is, the formula provided by the present invention In the formula, R, X ₁ , X ₂ , Y ₁ , Y ₄ and Y ₅ have the above meanings, and a peptide represented by the formula H-Thr-A-Gly-Thr-Pro-NH ₂ () in the formula , A is Asn-Thr-Gly-Ser or Asp-
By reacting a peptide represented by Val-Gly-Ala or its functional group-protected derivative in the presence of trypsin or a trypsin-like protease in a medium containing a buffer solution of pH 4 to 10, the formula In the formula, A, X ₁ , X ₂ , Y ₁ , Y ₄ and Y ₅ have the above-mentioned meanings, and a peptide represented by the following can be advantageously produced. In addition, each abbreviation used to represent an amino acid unit in this specification has the following meaning. Ala-=alanyl group, Arg-=araginyl group, Asn-=asparaginyl group, Asp-=aspartyl group, Cys-=cysteinyl group, Gln-=glutaminyl group, Glu-=glutamyl group, Gly-=glycyl group, His- = histidyl group, Leu- = leucyl group, Lys- = lysyl group, Pro- = prolyl group, Ser- = seryl group, Thr- = threonyl group, Tyr- = tyrosyl group, Val- = valyl group. Also, abbreviations

【式】とは、式[Formula] means the expression

【式】を表わすために用いる。本方法が特徴とする所は、前記式（−ａ）及
び（−ｂ）で示される、ペプチド類を合成する
に際して、１位から24位までのアミノ酸配列をも
つペプチドフラグメントと、残りの25位から32位
までのアミノ酸配列をもつペプチドフラグメント
とをそれぞれ予め合成しておき、24位のアルギニ
ンの遊離カルボキシル基又はエステル化されたカ
ルボキシル基と25位のスレオニンのアミノ基と
を、トリプシン又はトリプシン様蛋白分解酵素を
用いてカツプリング（アミド化）させる点にあ
る。従来、トリプシンの如き蛋白分解酵素は主とし
てペプチド結合の開裂に使用されてきたが、本方
法において、前記式（）のペプチドフラグメン
ト（式）のペプチドフラグメントのカツプリン
グ反応の縮合剤として極めて優れていることが意
外にも見い出された。本方法において使用されるトリプシンは蛋白分
解酵素として、国際生化学連合（I.U.B.）酵素委
員会に酵素番号EC3.4.21.4として登録された周知
の酵素であり、例えば、牛由来、豚由来等のトリ
プシン、またはトシル−Ｌ−フエニルアラニルク
ロロメチルケトン（TPCK）等で処理されたトリ
プシンとして市販されており、本方法ではこれら
はいずれも使用可能である。本方法においてはトリプシンのみならず、トリ
プシン様蛋白分解酵素もまた使用することができ
る。使用しうるトリプシン様蛋白分解酵素の例と
しては、例えばストレプトミセスグリセウス
（Streptomyces griseus）、ストレプトミセスフ
ラジエ（Streptomyces fradiae）等の産生する
プロテアーゼが知られており［「ザ・エンザイム
ズ」第巻（“THE ENZYMES”、Vol.）、
746頁、1971年アカデミツク・プレス
（ACADEMIC PRESS）発行参照］、商品名「プ
ロナーゼ」（科研化学社製）としてストレプトミ
セス・グリセウスＫ−１の産生するトリプシン様
蛋白分解酵素を含む酵素製品が市販されている。かかる特定の蛋白分解酵素の存在下における前
記式（）で示されるペプチド〔以下これを「Ｃ
成分」と呼ぶことがある〕と前記式（）で示さ
れるペプチド又はその官能基保護誘導体〔以下こ
れを「Ｎ成分」と呼ぶことがある〕とのカツプリ
ング反応は、PH４〜10、好ましくはPH５〜８の緩
衝液を含む媒質中で行なわれる。用いる緩衝液はPH値が上記範囲内のものであれ
ばその種類は特に制限されるものではなく、各種
のものを使用することができ、例えば、トリス塩
酸緩衝液、マツクイルベイン氏緩衝液、リン酸緩
衝液、酢酸アンモニウム緩衝液、アトキンス＆パ
ンチン氏緩衝液、ベロナール緩衝液等が挙げられ
る。中でもPH６〜８のトリス塩酸緩衝液が好適で
ある。これら緩衝液を反応媒質として使用する場合、
該緩衝液は通常、水混和性有機溶媒、殊にＣ成分
及びＮ成分の少なくとも一方を少なくとも部分的
に溶解する水混和性有機溶媒と混合して使用され
る。反応媒質の一部として用いうる該水混和性有
機溶媒としては、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）、ジ
メチルイミダゾリジノン（DMI）、ヘキサメチル
ホスホリルトリアミド（HMPA）等が包含され、
これらのうちでは就中ジメチルホルムアミドが好
適である。これら有機溶媒はそれぞれ単独で又は
２種もしくはそれ以上組合わせて使用してもよ
い。上記緩衝液と該水混和性有機溶媒の混合割合
は、緩衝液対有機溶媒の容積比で一般に２：８乃
至８：２好ましくは５：７乃至７：５の範囲内と
するのが有利である。かかる反応媒体中でのＣ成分とＮ成分との反応
は、前記蛋白分解酵素が作用する温度範囲、一般
には、約20〜約50℃、好ましくは約25〜約40℃の
範囲内の温度において行なうことができる。一方、Ｃ成分とＮ成分との使用割合は厳密に制
限されるものではなく、用いる他の反応条件等に
応じて変えることができるが、一般にはＣ成分／
Ｎ成分のモル比で１／１〜１／100、好適には
１／10〜１／20とするのが有利である。また、前記蛋白分解酵素の使用量も臨界的では
なく、反応条件に応じて変えることができるが、
Ｃ成分１ｇ当り一般に0.003〜0.2ｇ、好ましくは
0.05〜0.1ｇの量で使用するのが適当である。さらに、本方法を実施するに際して、トリプシ
ン等の蛋白分解酵素の活性安定化を図るため、反
応系に微量のカルシウムイオン等を存在させても
よい。かかる反応条件下に、カツプリング反応は通常
約１〜約20時間で終らせることができる。上記反応において、Ｃ成分として用いられる前
記式（）のペプチドは、そのＮ−末端及び側鎖
に存在する官能基（−OH、−COOH、−NH₂）は
無保護の状態、すなわち遊離型のものであつても
よく、或いは存在する官能基の一部又は全部がペ
プチド化学の分野で公知の保護基により保護され
ている。官能基保護誘導体の状態のものであつて
もよい。同様にＮ成分として用いられる前記式
（）のペプチドも遊離型又は官能基保護誘導体
型のいずれのものであつてもよい。式（）及び／又は（）のペプチド中に存在
する官能基を保護するために使用しうる保護基
は、本方法の反応条件下に安定であり且つ反応後
生成物から容易に離脱せしめることができ、しか
もその離脱に際して副反応を伴わないことが望ま
しく、例えば次のような保護基を例示することが
できる。 (a) アミノ基の保護基としては、例えば、ｔ−ブ
チルオキシカルボニル基、ベンジルオキシカル
ボニル基、ｐ−メトキシベンジルオキシカルボ
ニル基、トリチル基等。 (b) カルボキシル基の保護基としては、例えば、
ｔ−ブチル基、ベンジル基等。 (c) 水酸基の保護基としては、例えば、ベンジル
基、ｔ−ブチル基等。 (d) チオールの保護基としては、例えば、ベンジ
ル基、アセトアミドメチル基、トリチル基等。また、本方法において出発物質として使用され
る式（）のペプチド及び／又は式（）のペプ
チドは塩の形で使用することもでき、従つて、本
明細書において用いる「ペプチド」及び「ポリペ
プチド」なる語には、存在する酸性及び塩基性官
能基のすべてが遊離の状態にある（ポリ）ペプチ
ドのみならず、これら酸性及び塩基性官能基の少
なくとも一部が塩の形態にある（ポリ）ペプチド
をも包含する意味で使用する。かかる塩の例とし
ては、塩酸塩、臭化水素酸塩、トリフルオロ酢酸
塩、ｐ−トルエンスルホン酸塩、酢酸塩、トリエ
チルアンモニウム塩等を挙げることができる。以上述べた本方法によつて、前記式（）で示
されるポリペプチドを副反応なく高純度且つ高収
率で合成することができる。前記式（）において、X₁及びX₂が共にチオ
ールの保護基である場合の化合物は、該チオール
の保護基を離脱せしめた後に、酸化することによ
り、X₁及びX₂が一緒になつて単結合を表わす場
合の対応する式（）の化合物を生成せしめるこ
とができる。該酸化は、ポリペプチド中の２個のシステイン
単位間にジスルフイド結合を形成せしめるに際
し、ペプチド化学の分野で通常用いられている酸
化剤及び手段を用いて行なうことができ、その詳
細な方法については、例えばヘルベチカ・キミ
カ・アクタ（Helv.Chim.Acta）51巻、2061〜
2064頁（1968年）、ジヤーナル・オブ・ザ・アメ
リカン・ケミカル・ソサエテイー（J.Amer.
Chem.Soc.）、94巻、5456〜5461頁（1972年）等
の文献を参照すべきである。また、前記式（）の化合物が保護基を有する
場合には、ペプチド化学の分野で公知の方法、例
えば、イー・シユレーダー（E.Schro¨der）及び
ケイ・リユプケ（K.Lu¨bke）著「ザ・ペプタイ
ズ」（“The Peptides”）第１巻、３〜75頁、1965
年アカデミツク・プレス（Academic Press）発
行、に記載の方法により、該保護基を離脱せしめ
ることができる。該式（）の化合物を上記酸化
工程に付する場合、保護基の離脱は該酸化の前又
は後のいずれの時点に行なつてもよい。これにより前記式（−ａ）又は（−ｂ）で
示されるペプチド類を、従来法におけるよりもは
るかに有利に製造することができる。本方法において出発物質として用いらるＣ成
分、すなわち本発明の前記式（）のペプチド
は、前述したように従来の文献に未載の新規な物
質であり、例えばペプチド化学の分野で周知の液
相合成法（上記「ザ・ペプタイズ」（“The
Peptides”）の76〜136頁参照）によつて製造する
ことができる。例えば、周知の液相合成法によ
り、下記式式中、X₁及びX₂は前記の意味を有する、で示されるペプチド又はその官能基保護誘導体と
下記式Ｈ−Lys−Leu−Ser−Gln−Glu−Leu−His−Lys−Le
u−Gln−Thr−Tyr−Pro−Arg−OH で示されるペプチド又はその官能基保護誘導体を
合成し、しかる後これら両者を縮合させ、さらに
必要に応じて酸化することによつて１位−７位間
にジスルフイド結合を形成せしめることにより製
造することができる。前記式（）における低級アルキル基（Ｒ）と
しては炭素原子数６個まで、好ましくは１〜４個
のもの例えばメチル、エチル、ｎ−プロピル、ｎ
−ブチル基等が挙げられ、中でもメチル及びエチ
ル基が好適である。しかして、本発明の前記式（）のペプチドの
うち特に好適な群の化合物は、前記式（）にお
いて、X₁及びX₂がそれぞれトリチル基又はアセ
トアミドメチル基を表わすか、或いはX₁とX₂が
一緒になつて単結合を表わし、Y₁が水素原子、
ｔ−ブチルオキシカルボニル基、ｐ−メトキシベ
ンジルオキシカルボニル基又はトリチル基を表わ
し、Y₄が水素原子、ｔ−ブチルオキシカルボニ
ル基、ｐ−メトキシベンジルオキシカルボニル基
又はトリチル基を表わし、Y₅が水素原子又はｔ
−ブチル基を表わす場合の式（）の化合物であ
る。本方法によれば、同じポリペプチドを合成する
ための従来既知の方法に比べて以下に述べるよう
な利点があり、工業的に極めて有用である。 (イ) 酵素反応の性質上副反応を全く伴わずに反応
せしめることができ、しかもラセミ体を伴わ
ず、生成物を収率よく合成することができかつ
精製も容易である。 (ロ) Ｎ−成分、Ｃ−成分共に未反応分は完全に回
収再利用できるので経済的に有利である。 (ハ) 側鎖官能基の保護が不要又は必要最小限でよ
い。従つて原料の合成および最終の脱保護基操
作が不要であるか又は極めて容易である。 (ニ) 反応が短時間ですみ、反応装置も簡単なもの
でよい。以下実施例を掲げて本発明をさらに説明する。尚、以下の参考例および実施例中に記載の略号
は前述のアミノ酸の略号を除いて次の通りであ
る。 Boc−：ｔ−ブチルオキシカルボニルＺ：ベンジルオキシカルボニル Bzl：ベンジル Acm：アセトアミドメチル Trt：トリチル OMe：メチルエステル OEt：エチルエステル OTB：ｔ−ブチルエステル OSu：Ｎ−ヒドロキシスクシンイミドエステル ONp：パラニトロフエニルエステル OBzl：ベンジルエステル DMF：ジメチルホルムアミド DMSO：ジメチルスルホキシド DCC：ジシクロヘキシルカルボジイミド MeOH：メタノール EtOH：エタノール TsOH：トルエンスルホン酸 AcOH：酢酸 DCHA：ジシクロヘキシルアミンまた、以下の参考例および実施例中、「セフア
デツクスLH−20」はフアルマシア社の商品名
で、大部分の水酸基がアルキル化された架橋デキ
ストラン系ゲル濾過用分子篩剤であり、「セフア
デツクスＧ−25」はフアルマシア社の商品名で、
多数の水酸基を有する架橋デキストラン系ゲル濾
過用分子篩剤であり、「アンバーライトIRA−
410」はローム・アンド・ハース社製の商品名で、
Ｎ−（２−ヒドロキシエチル）−Ｎ，Ｎ−ジメチル
アミノ基を有するポリスチレン系イオン交換樹脂
である。参考例 (1) 22−23〔Ａ〕Boc−Tyr−Pro−OMe Boc−Tyr−OH・DCHA46.2ｇとＨ−Pro−
OMe・HCl16.5ｇとをクロロホルム200mlに溶
解し、氷冷撹拌しつつDCC20.6ｇをクロロホル
ム50mlに溶解して、滴下する。滴下後、約５時
間氷冷撹拌したのち、析出したジシクロヘキシ
ル尿素を別し、液を減圧濃縮する。得られ
た油状残渣を酢酸エチルに溶解し、クエン酸水
溶液および重曹水で洗つたのち、無水硫酸ナト
リウムで乾燥する。溶媒を留去して得た油状物
をエーテル／ヘキサンから再結晶してBoc−
Tyr−Pro−OMe35.5ｇ（90％）を得る。融点
122−４℃、〔α〕²⁶ _D−29.1゜（ｃ＝２、DMF）。参考例 (2) 22−23〔Ｂ〕Boc−Tyr−Pro−OH Boc−Tyr−Pro−OMe34.0ｇをメタノー
ル／ジオキサン（１：１）160mlに溶解し、2N
苛性ソーダ85mlを加えて室温に３時間放置す
る。氷冷して5N塩酸で中和したのち、約150ml
に減圧濃縮し、冷10％クエン酸溶液を加え、PH
を２〜３とする。析出した結晶を取、水洗、
乾燥するとBoc−Tyr−Pro−OH31.5ｇ（96
％）が得られる。融点114−５℃、〔α〕²⁶ _D−
22.9゜（ｃ＝２、DMF）。参考例 (3) 22−24〔Ａ〕Boc−Tyr−Pro−Arg（NO₂）−
OEt Boc−Tyr−Pro−OH30.3ｇとＮ−ヒドロキ
シスクシンイミド9.8ｇとをDMF150mlに溶か
し、氷冷撹拌しつつDCC16.5ｇをDMF50mlに
溶解して滴下する。更に５時間氷冷撹拌をつづ
けたのち、ジシクロヘキシル尿素を別し、
液を減圧濃縮し、残渣の油状物を酢酸エチルに
溶解し、重曹水で洗浄し、無水流酸ナトリウム
で乾燥する。溶媒を減圧で留去し、残渣を酢酸
エチル／エーテル／ヘキサンで処理して粉末状
のBoc−Tyr−Pro−OSu37.0ｇ（98％）を得
る。この活性エステルをＨ−Arg（NO₂）−OEt・
TsOH33.6ｇおよびトリエチルアミン11.2mlの
DMF溶液150mlに加え、室温で一夜放置する。
DMFを減圧で留去し、油状残渣を酢酸エチル
に溶解し、クエン酸水溶液および重曹水で洗浄
し、無水硫酸ナトリウムで乾燥する。酢酸エチ
ルを減圧留去し、残渣油状物を酢酸エチル／エ
ーテルから沈殿させ、Boc−Tyr−Pro−Arg
（NO₂）−OEt43.1ｇ（88％）を無水定形粉末と
して得る。〔α〕²⁶ _D−24.1゜（ｃ＝２、DMF）。参考例 (4) 22−24〔Ｂ〕Ｈ−Tyr−Pro−Arg−（NO₂）−
OEt・HCl Boc−Tyr−Pro−Arg（NO₂）−OEt36.4ｇに
約2.7Nの塩化水素／酢酸エチル250mlを加え、
室温で３時間放置したのち減圧濃縮する。残渣
に酢酸エチルを加え、減圧濃縮をくり返し、残
渣をエタノール／酢酸エチル／エーテルで沈殿
させ、Ｈ−Tyr−Pro−Arg（NO₂）−OEt・
HClの無定形粉末31.3ｇ（96％）を得る。参考例 (5) 19−21〔Ａ〕Boc−Leu−Gln−Thr−OMe Ｚ−Gln−Thr−OMe70ｇをｔ−ブタノー
ル／水（９：１）600mlに溶解し、５％パラジ
ウム炭素４ｇを添加して接触還元後、触媒を
別し、液を減圧濃縮する。残渣をDMF300ml
に溶解し、Boc−Leu−OSu53ｇを加え、室温
で一夜放置する。溶媒を減圧で留去し、残渣に
酢酸エチルを加えて加熱溶解し、放冷後エーテ
ルを加えて析出した沈殿を取し、エタノール
を含む水から再結晶して、目的のBoc−Leu−
Gln−Thr−OMe53.6ｇ（70％）を得る。融点
186−７℃、〔α〕²⁶ _D−25.0゜（ｃ＝１、DMF）。参考例 (6) 19−21〔Ｂ〕Boc−Leu−Gln−Thr−
NHNH₂ Boc−Leu−Gln−Thr−OMe47.4ｇをメタ
ノール300mlに溶解し、85％ヒドラジンヒドラ
ート40mlを加え室温で撹拌する。結晶析出後、
一晩放置し、エーテルを加え、結晶を取す
る。この粗結晶をDMF／水から再結晶して、
標記のヒドラジド37.6ｇ（79％）を得る。融点
203−４℃、〔α〕²² _D−44.6゜（ｃ＝２、80％
AcOH）。参考例 (7) 19−24〔Ａ〕Boc−Leu−Gln−Thr−Tyr−
Pro−Arg（NO₂）−OEt Boc−Leu−Gln−Thr−NHNH₂16.7ｇを
DMSO50mlとDMF150mlとに溶解した溶液を
−40℃に冷却し、2.72N塩化水素／酢酸エチル
35mlと、亜硝酸イソアミル5.7mlを加える。−20
℃で15分間撹拌したのち、反応液を−40℃に冷
却しトリエチルアミン13.3mlを加えて中和し、
さらに参考例(4)で得たＨ−Tyr−Pro−Arg
（NO₂）−OEt・HCl20ｇとトリエチルアミン5.4
mlをDMF50mlに溶解した溶液を加え、氷冷下
一夜撹拌する。トリエチルアミン塩酸塩を別
し、液を減圧濃縮し、得られた油状物をシリ
カゲルカラムにかけ、クロロホルム／メタノー
ル（10：１）で溶出する。溶媒を減圧で留去
し、残渣をメタノール／酢酸エチルで沈殿さ
せ、目的のBoc−Leu−Gln−Thr−Tyr−Pro
−Arg（NO₂）−OEt25.5ｇ（76％）を無定形粉
末として得る。〔α〕²¹ _D−36.4゜（ｃ＝１、DMF）。参考例 (8) 19−24〔Ｂ〕Ｈ−Leu−Gln−Thr−Tyr−
Pro−Arg（NO₂）−OEt・HCl Boc−Leu−Gln−Thr−Tyr−Pro−Arg
（NO₂）−OEt25.0ｇに約2.7N塩化水素／酢酸エ
チル150mlを加え、室温で３時間放置する。溶
媒を減圧で留去し、残渣に酢酸エチルを加え、
減圧濃縮を数回くり返す。残渣をエタノール／
酢酸エチル／エーテルで沈殿させ、無定形粉末
として目的物23.4ｇ（≒100％）を得る。参考例 (9) 17−24〔Ａ〕Ｚ−His−Lys（Boc）−Leu−
Gln−Thr−Tyr−Pro−Arg（NO₂）−OEt Ｚ−His−Lys（Boc）−NHNH₂10.2ｇを
DMF80mlに溶解した溶液を−20℃に冷却し、
2.78N塩化水素／酢酸エチル20mlと亜硝酸イソ
アミル3.15mlを加える。−20℃で15分間撹拌し
たのち、反応液を−40℃に冷却し、トリエチル
アミン7.78mlを加えて中和し、Ｈ−Leu−Gln
−Thr−Tyr−Pro−Arg（NO₂）−OEt・
HCl17.7ｇとトリエチルアミン3.36mlをDMF80
mlに溶解して加える。氷冷で一夜撹拌をつづけ
たのちトリエチルアミン塩酸塩を別し、液
を減圧濃縮し、油状残渣をシリカゲルカラムに
かけ、クロロホルム／メタノール（５：１）で
溶出する。目的の画分を減圧濃縮し、メタノー
ル／酢酸エチルで沈殿させると、目的のＺ−
His−Lys（Boc）−Leu−Gln−Thr−Tyr−Pro
−Arg（NO₂）−OEt19.2ｇ（74％）を無定形粉
末として得る。〔α〕^21.5 _D−23.5゜（ｃ＝２、DMF）。参考例 (10) 17−24〔Ｂ〕Ｈ−His−Lys（Boc）−Leu−
Gln−Thr−Tyr−Pro−Arg−OEt・3AcOH Ｚ−His−Lys（Boc）−Leu−Gln−Thr−
Tyr−Pro−Arg（NO₂）−OEt11.0ｇを80％酢酸
80mlに溶解し、５％パラジウム炭素1.0ｇを加
えて接触還元を行なつたのち、触媒を別す
る。液を減圧濃縮し、さらに数回、水を加え
て減圧濃縮したのちメタノールを加え減圧濃縮
する。得られた油状残渣をメタノール／エーテ
ルで沈殿させると、Ｈ−His−Lys（Boc）−Leu
−Gln−Thr−Tyr−Pro−Arg−OEt・
3AcOHの無定形粉末10.3ｇ（94％）を得る。アミノ酸分析 Lys1.10(1)、His1.02(1)、 Arg0.76(1)、Thr0.95(1)、 Gln1.03(1)、Pyr1.15(1)、 Leu1.00(1)、Tyr0.94(1)。参考例 (11) ４−５（12−13）Ｚ−Leu−Ser−NHNH₂ Ｚ−Leu−Ser−OMe51.5ｇをメタノール400
mlに溶解し、85％ヒドラジンヒドラート50mlを
加え、室温で撹拌し、結晶析出後、一夜放置す
る。エーテルを加え結晶を取し、メタノール
から再結晶してＺ−Leu−Ser−NHNH₂45.2ｇ
（87％）を得る。融点176−８℃、〔α〕²⁶ _D−8.6゜
（ｃ＝１、DMF）。参考例 (12) 12−16〔Ａ〕Ｚ−Leu−Ser−Gln−Glu
（OTB）−Leu−OMe Ｚ−Leu−Ser−NHNH₂13.9ｇをDMF100ml
に溶解した溶液を−40℃に冷却し、2.68N塩化
水素／酢酸エチル30mlと亜硝酸イソアミル６ml
を加える。−20℃で15分間撹拌したのち、−40℃
に冷却し、トリエチルアミン11.26mlを加えて
中和する。反応液にＨ−Gln−Glu（OTB）−
Leu−OMe17.4ｇを含むDMF溶液50mlを加え、
氷冷で一夜撹拌する。反応混合物を減圧濃縮
し、残渣に水を加えて沈殿物を取し、エタノ
ールから再結晶すると、目的のＺ−Leu−Ser
−Gln−Glu（OTB）−Leu−OMe28.0ｇ（93％）
が得られる。融点194−196℃、〔α〕¹⁸ _D−21.5゜
（ｃ＝２、DMF）。参考例 (13) 11−16〔Ａ〕Ｚ−Lys（Boc）−Leu−Ser−
Gln−Glu（OTB）−Leu−OMe Ｚ−Leu−Ser−Gln−Glu（OTB）−Leu−
OMe23.8ｇをｔ−ブタノール／水（９：１）
250mlに懸濁させ、５％パラジウム炭素１ｇを
加え接触還元後、触媒を別し、液を減圧濃
縮する。残渣をDMF300mlに溶解し、Ｚ−Lys
（Boc）−ONp15ｇを加え、室温で一夜放置し
たのち、減圧濃縮する。残渣に水を加えて生じ
た沈殿を取し、エタノール／水から再結晶し
て、目的のＺ−Lys（Boc）−Leu−Ser−Gln−
Glu（OTB）−Leu−OMe26.3ｇ（85％）を得
る。融点215−６℃、〔α〕^21.5 _D−25.0゜（ｃ＝２、
DMF）。参考例 (14) 11−16〔Ｂ〕Ｚ−Lys−（Boc）−Leu−Ser−
Gln−Glu（OTB）−Leu−NHNH₂ Ｚ−Lys（Boc）−Leu−Ser−Gln−Glu
（OTB）−Leu−OMe18.2ｇをDMF100mlに溶
解し、85％ヒドラジンヒドラート15mlを加え２
日間室温に放置後、反応液を減圧濃縮する。残
渣をエタノールで洗浄して目的のヘキサペプチ
ドヒドラジド16.7ｇ（92％）を得る。融点226
−７℃（分解）、〔α〕²¹ _D−45.0゜（ｃ＝２、80％
AcOH）。参考例 (15) 11−24〔Ａ〕Ｚ−Lys（Boc）−Leu−Ser−
Gln−Glu（OTB）−Leu−His−Lys（Boc）−
Leu−Gln−Thr−Tyr−Pro−Arg−OEt Ｚ−Lys（Boc）−Leu−Ser−Gln−Glu
（OTB）−Leu−NHNH₂5.2ｇをDMF50mlに溶
解し、溶液を−40℃に冷却したのち、2.7N塩
化水素／酢酸エチル５mlと亜硝酸イソアミル
0.8mlを加える。−20℃で15分間撹拌したのち、
再び−40℃に冷却し、トリエチルアミン1.9ml
を加えて中和する。この溶液に参考例(10)で得た
Ｈ−His−Lys（Boc）−Leu−Gln−Thr−Tyr
−Pro−Arg−OEt・3AcOH5.4ｇ、トリエチ
ルアミン1.4mlを含むDMF溶液30mlを加え、氷
冷で一夜撹拌する。反応液を減圧濃縮し、残渣
をシリカゲルカラムにかけクロロホルム／メタ
ノール／水（70：30：５）で溶出する。目的と
する標記のトリデカペプチドはメタノール／酢
酸エチル／エーテルで沈殿させると、5.4ｇ
（60％）の無定形粉末として得られる。アミノ酸分析： Lys2.24(2)、His1.08(1)、 Arg0.99(1)、Thr0.96(1)、 Ser0.89(1)、Glu3.17(3)、 Pro1.06(1)、Leu3.00(3)、 Tyr0.94(1)。参考例 (16) ４−６〔Ａ〕Ｚ−Leu−Ser−Thr−OMe 参考例(11)で得たＺ−Leu−Ser−NHNH₂25.6
ｇをDMF2.00mlに溶かし、−40℃に冷却したの
ち、2.35N塩化水素／酢酸エチル60ml、亜硝酸
イソアミル11.6mlを加え、−20℃で15分間撹拌
したのち再び−40℃に冷却してトリエチルアミ
ン19.8mlを加えて中和する。この溶液にＨ−Thr−OMe（Ｚ−Thr−
OMe18.7ｇを接触還元して得たもの）を
DMF50mlに溶かした溶液を加え氷冷下に一夜
撹拌をつづける。溶媒を減圧濃縮し油状の残渣
に水を加えてかきまぜ、取する。これをメタ
ノール／酢酸エチルから再結晶して目的のＺ−
Leu−Ser−Thr−OMe26.6ｇ（81％）を得る。
融点185−６℃、〔α〕²⁶ _D−9.8゜（ｃ＝１、DMF）。参考例 (17) ３−６〔Ａ〕Ｚ−Asn−Leu−Ser−Thr−
OMe Ｚ−Leu−Ser−Thr−OMe13.5ｇをｔ−ブ
タノール／水（９：１）400mlに懸濁させ、５
％パラジウム炭素２ｇを加え接触還元を行なつ
たのち、触媒を別し、液を減圧濃縮する。
残渣をDMF80mlに溶解し、Ｚ−Asn−
ONp11.2ｇを加え、室温で二夜放置する。析出
した結晶を加温溶解し、減圧濃縮し、残渣を
DMF／メタノールから再結晶すると、目的の
Ｚ−Asn−Leu−Ser−Thr−OMe13.7ｇ（81
％）を得る。融点218゜〜222℃、〔α〕²² _D−26.0゜
（ｃ＝２、DMF）。参考例 (18) ３−６〔Ｂ〕Ｈ−Asn−Leu−Ser−Thr−
OMe Ｚ−Asn−Leu−Ser−Thr−OMe5.8ｇをｔ
−ブタノール／水（９：１）150mlに懸濁し５
％−パラジウム炭素0.6ｇを加え接触還元した
のち触媒を別し、液を減圧乾固する。結晶
状の生成物は直ちに次の反応に使用する。参考例 (19) １−２〔Ａ〕Boc−Cys（Acm）−Ser−OMe Boc−Cys（Acm）−OH・DCHA8.8ｇ（0.03
モル）とＨ−Ser−OMe・HCl4.7ｇ（0.03モ
ル）とをクロロホルム100mlに溶解し、氷冷撹
拌しつつDCC6.18ｇ（0.03モル）を加える。氷
冷下に３時間、室温で２時間反応させたのち析
出したジシクロヘキシル尿素を別しクロロホ
ルムで洗う。、洗液をクエン酸水溶液および
重曹水で洗つたのち無水硫酸ナトリウムで乾燥
する。溶媒を留去して、油状の目的物9.6ｇ
（82％）を得る。参考例 (20) １−２〔Ｂ〕Boc−Cys（Acm）−Ser−
NHNH₂ Boc−Cys（Acm）−Ser−OMe9.6ｇを
DMF50mlに溶解し、容器を窒素ガスで置換し
たのち85％ヒドラジンヒドラート15mlを加え冷
蔵庫で一夜反応させる。反応液を減圧乾固し、
残る結晶をメタノールから再結晶して7.3ｇ
（75％）の目的物を得る。融点199−200℃、
〔α〕¹⁸ _D−24.0°（ｃ＝２、80％AcOH）。参考例 (21) １−６〔Ａ〕−Boc−Cys（Acm）−Ser−Asn
−Leu−Ser−Thr−OMe Boc−Cys（Acm）−Ser−NHNH₂3.94ｇを
DMF：DMSO（２：１）の混合溶媒60mlに加
温溶解後、−20℃に冷却し、撹拌下に2.72N塩
化水素／酢酸エチル20mlと亜硝酸イソアミル
2.37mlを加え15分間撹拌する。反応液を−40℃
に冷却しトリエチルアミン7.62mlを加えて中和
する。この溶液に参考例（18）で得たＨ−Asn
−Leu−Ser−Thr−OMe4.47ｇをDMF30mlに
溶解した溶液を加え、氷冷下に20時間反応させ
る。トリエチルアミン塩酸塩を別し、溶媒を
減圧乾固し、残渣をメタノールから再結晶し、
目的の標記化合物6.1ｇ（75％）を得る。融点
208−211℃、〔α〕²² _D−22.0゜（ｃ＝２、DMF）。参考例 (22) １−６〔Ｂ〕Boc−Cys（Acm）−Ser−Asn
−Leu−Ser−Thr−NHNH₂ Boc−Cys（Acm）−Ser−Asn−Leu−Ser−
Thr−OMe5ｇをDMF50mlに溶解し、85％ヒ
ドラジンヒドラート４mlを加え、窒素ふん囲気
下冷蔵庫中に一夜放置したのち減圧濃縮し、残
渣をメタノールで洗浄して目的物4.7ｇ（94％）
を得る。融点187−188℃、〔α〕²⁰ _D−34.5゜（ｃ＝
２、80％AcOH）。参考例 (23) ７−10〔Ａ〕Boc−Cys（Acm）−Val−Leu
−Gly−OEt Boc−Cys（Acm）−OH・DCHA29.34ｇとＨ
−Val−Leu−Gly−OEt・HCl21.8ｇとをクロ
ロホルム360mlに溶解し、氷冷撹拌しつつ
DCC12.77ｇをクロロホルム30mlに溶解した溶
液を滴下する。氷冷下３時間、室温で一夜撹拌
後、析出したジシクロヘキシル尿素を別し、
クロロホルムで洗う。、洗液をクエン酸水溶
液および重曹水で洗つたのち無水硫酸ナトリウ
ムで乾燥する。クロロホルムを留去し、残渣を酢酸エチルか
ら再結晶して目的の保護テトラペプチド29.3ｇ
（80％）を得る。融点140−141℃、〔α〕²² _D−
18.5゜（ｃ＝２、DMF）。参考例 (24) ７−10〔Ｂ〕Ｈ−Cys（Acm）−Val−Leu−
Gly−OEt・HCOOH Boc−Cys（Acm）−Val−Leu−Gly−OEt5.9
ｇを98％ギ酸100mlに溶かし、室温に一夜放置
する。減圧乾固し残渣をエーテルで充分洗浄、
乾燥して目的物5.3ｇ（100％）を粉末として得
る。参考例 (25) １−10〔Ａ〕Boc−Cys（Acm）−Ser−Asn
−Leu−Ser−Thr−Cys（Acm）−Val−Leu−
Gly−OEt 参考例（22）で得たBoc−Cys（Acm）−Ser
−Asn−Leu−Ser−Thr−NHNH₂4.05ｇを
DMF30mlに加温溶解する。０℃以下に冷却し
撹拌下に2.7N塩化水素／酢酸エチル10mlを加
え、ついで−20℃に冷却し亜硝酸イソアミル
1.1mlを加えて−15℃〜−10℃で10分間撹拌す
る。 −40℃に冷却し、トリエチルアミン3.81mlを
加えて中和し、これにＨ−Cys（Acm）−Val−
Leu−Gly−OEt・HCOOH2.7ｇをDMF30mlに
溶かし、トリエチルアミン0.7mlで中和した溶
液を加えて０℃で20時間反応させる。トリエチルアミン塩酸塩を別し、溶媒を減
圧濃縮し、残渣をDMF／メタノールで結晶化
し、さらにDMF／水から再結晶して目的物2.7
ｇ（40％）を得る。融点242−244℃（分解）、〔α〕²² _D−28.5゜（ｃ＝
１、DMF）。参考例 (26) １−10〔Ｂ〕Boc−Cys（Acm）−Ser−Asn
−Leu−Ser−Thr−Cys（Acm）−Val−Leu−
Gly−NHNH₂ Boc−Cys（Acm）−Ser−Asn−Leu−Ser−
Thr−Cys（Acm）−Val−Leu−Gly−OEt2.52
ｇをDMF30mlに溶かし、85％ヒドラジンヒド
ラート1.5mlを加え、冷蔵庫中で一夜反応させ
る。ヒドラジンヒドラートおよびDMFを減圧
濃縮し、残渣をメタノールで洗浄し、目的物
2.25ｇ（90％）を得る。融点236−237℃（分解）、〔α〕²² _D−43.3゜（ｃ＝
0.3、80％AcOH）。参考例 (27) 22−23Boc−Tyr（Bzl）−Pro−OH Boc−Tyr（Bzl）−OSu85ｇをDMF600mlに
溶かした液とプロリン23ｇを水100mlに溶かし
た液とを混合し、氷冷下撹拌しつつトリエチル
アミン28mlを滴下する。滴下後了後室温で３時
間撹拌し、ついで減圧濃縮し、得られたシロツ
プ状残渣を水300mlに溶かしクエン酸酸性にす
る。析出した生成物を酢酸エチルで抽出し、酢
酸エチル層を水洗、乾燥後、約1/3に濃縮し、
ヘキサンを加えると結晶が析出する。72ｇ（85
％）。融点141−２℃、〔α〕²⁰ _D−21.3゜（ｃ＝１、
EtOH）。参考例 (28) 22−24Boc−Tyr（Bzl）−Pro−Arg（NO₂）
−OEt 参考例（27）で得たBoc−Tyr（Bzl）Pro−
OH33ｇ、Ｈ−Arg（NO₂）−OEt・TsOH29.2
ｇ、Ｎ−ヒドロキシスクシンイミド9.0ｇ及び
トリエチルアミン10.9mlをDMF350mlに溶解
し、氷冷撹拌しつつDCC16.2ｇをDMF50mlに
溶かした液を滴下する。滴下終了後室温で12時
間撹拌をつづけたのち析出した結晶を別し、
液を減圧濃縮してシロツプ状残渣を得る。こ
れを酢酸エチルに溶かして４％クエン酸、４％
重曹水、水の順に洗浄し乾燥する。溶媒を留去
し、得られた残渣をシリカゲルカラムクロマト
（溶出液：クロロホルム／メタノール10：１）
に付し、酢酸エチル／ヘキサンから再結晶し
て、目的物43ｇ（79％）を得る。融点89−90
℃、〔α〕²⁰ _d−17.2゜（ｃ＝１、MeOH）。参考例 (29) 19−21〔Ａ〕Ｚ−Leu−Gln−Thr−OMe Ｚ−Gln−Thr−OMe39.5ｇをｔ−ブタノー
ル／水（９：１）１に溶解し、５％パラジウ
ム炭素２ｇを加えて接触還元後触媒を別し、
液を減圧濃縮する。残渣をDMF300mlに溶解
し、Ｚ−Leu−ONp38.3ｇを加え室温に一夜放
置する。溶媒を減圧で留去し、残る結晶を酢酸
エチルとよくすりつぶし取する。DMF−酢
酸エチルから再結晶して、目的のＺ−Leu−
Gln−Thr−OMe42ｇ（83％）を得る。融点
203−４℃、〔α〕²⁰ _D−18.3゜（ｃ＝１、DMF）。参考例 (30) 19−21〔Ｂ〕Ｚ−Leu−Gln−Thr−
NHNH₂ Ｚ−Leu−Gln−Thr−OMe25.4ｇを
DMF300mlに溶解し、０℃で85％ヒドラジンヒ
ドラート30mlを加え、室温に一夜放置したのち
減圧濃縮し、残渣を冷水でよくすりつぶし取
する。DMF／水から再結晶しての標記のヒド
ラジド23ｇ（90％）を得る。融点219−220℃、
〔α〕²⁰ _D−17.1゜（ｃ＝１、DMF）。参考例 (31) 19−24Z−Leu−Gln−Thr−Tyr（Bzl）−
Pro−Arg（NO₂）−OEt 参考例（28）で得たトリペプチドBoc−Tyr
（Bzl）−Pro−Arg（NO₂）−OEt7ｇを98％ギ酸
40mlに溶解し、室温に一夜放置したのち減圧乾
固する。残渣を冷水に溶かし、トリエチルアミ
ンを少量ずつ加えてPH９とし、析出した生成物
を酢酸エチルで抽出し、有機層を乾燥後溶媒を
留去してＨ−Tyr（Bzl）−Pro−Arg（NO₂）−
OEtを得る。一方参考例（30）で得たヒドラジ
ドＺ−Leu−Gln−Thr−NHNH₂5.1ｇを
DMF100mlにけん濁し、０℃で2.3N塩化水
素／酢酸エチル30mlを加えて溶解し、更に−15
℃に冷却して亜硝酸イソアミル1.2ｇを加える。
15分後−50℃に冷却しトリエチルアミン9.7ml
を加えたのち、上記のＨ−Tyr（Bzl）−Pro−
Arg（NO₂）−OEtをDMF30mlに溶解した液を
加え、ついで０℃にて16時間撹拌をつづける。
沈殿を別し液を減圧乾固し残渣をシリカゲ
ルのカラムクロマトで精製（溶出液クロロホル
ム／メタノール10：１）し、目的の画分をエー
テルで結晶化させて標記のヘキサペプチドＺ−
Leu−Gln−Thr（Bzl）−Pro−Arg（NO₂）−
OEt7.5ｇ（70％）を得る。融点120−２℃、
〔α〕²⁰ _D−37.2゜（ｃ＝１、EtOH）。参考例 (32) 17−24Z−His−Lys（Boc）−Leu−Gln−
Thr−Tyr−Pro−Arg−OEt 参考例（31）で得たＺ−Leu−Gln−Thr−
Tyr（Bzl）−Pro−Arg（NO₂）−OEt20ｇを80％
酢酸500mlに溶かし５％パラジウム炭素２ｇを
加えて接触還元する。終了後触媒を別し、
液を減圧乾固し残渣をエーテルとよくすりつぶ
して取し、水酸化ナトリウム上減圧乾燥す
る。この生成物のうち9.25ｇをDMF50mlに溶
解しトリエチルアミンで中和しておく。一方、
Ｚ−His−Lys（Boc）−NHNH₂6.36ｇを
DMF100mlに溶解し−20℃で2.3N塩化水素／
酢酸エチル20ml、ついで亜硝酸イソアミル1.2
ｇを加え、10分後−50℃に冷却してトリエチル
アミン6.4mlを加えたのち上記ヘキサペプチド
のDMF溶液を加え、０℃にて16時間撹拌をつ
づける。不溶物を別し液を減圧乾固し、残
渣をシリカゲルカラムにかけクロロホルム／メ
タノール／水（70：30：２）で溶出する。目的
の画分を濃縮しエーテルで沈殿させて標記のＺ
−His−Lys（Boc）−Leu−Gln−Thr−Tyr−
Pro−Arg−OEt10.2ｇ（78％）を得る。〔α〕²⁰ _D−25.7゜（ｃ＝１、DMF）。参考例 (33) 10−11〔Ａ〕Ｚ−Gly−Lys−（Boc）−OMe Ｈ−Lys（Boc）−OMe26ｇを酢酸エチル300
mlに溶かし、Ｚ−Gly−ONp33ｇを加えて室温
に16時間放置したのち溶液を0.5N苛性ソーダ、
水の順で洗浄し、乾燥、溶液をもとの1/5に濃
縮し、ヘキサンを加えると結晶が析出する。結
晶を取し、酢酸エチル／ヘキサンから再結晶
して、標記のジペプチド40.6ｇ（90％）を得
る。融点74−５℃、〔α〕²⁰ _D−8.8゜（ｃ＝１、
MeOH）。参考例 (34) 10−11〔Ｂ〕Ｚ−Gly−Lys（Boc）−
NHNH₂ 参考例（33）で得たＺ−Gly−Lys（Boc）−
ONe36ｇをDMF300ml中ヒドラジンヒドラー
ト30mlと一夜反応させたのち減圧濃縮し、残渣
をメタノール／水から再結晶して、目的のヒド
ラジドＺ−Gly−Lys（Boc）−NHNH₂32ｇ
（89％）を得る。融点139−140℃、〔α〕²⁰ _D−13.5゜（ｃ＝１、
MeOH）。参考例 (35) 10−16〔Ａ〕Ｚ−Gly−Lys（Boc）−Leu−
Ser−Gln−Glu（OTB）−Leu−OMe 参考例（34）で得たＺ−Gly−Lys（Boc）−
NHNH₂27.5ｇを300mlのDMFに溶かし、−15
℃に冷却して撹拌しつつ2.3N塩化水素／酢酸
エチル30ml、ついで亜硝酸イソアミル6.6ｇを
徐々に加える。10分後−50℃に冷却しトリエチ
ルアミン9.7mlを滴下する。一方、参考例(12)で
得たペンタペプチドＺ−Leu−Ser−Gln−Glu
（OTB）−Leu−OMe39.6ｇを５％パラジウム
炭素３ｇ存在下ｔ−ブタノール／水（９：１）
350ml中接触還元して得た生成物を、DMF100
mlに溶かして上記アジド溶液に加え、０℃にて
一夜撹拌をつづける。反応混合物を減圧乾固
し、残渣を充分すりつぶし沈殿を取、水洗、
乾燥後DMF／エタノールから再結晶して、標
記のＺ−Gly−Lys（Boc）−Leu−Ser−Gln−
Glu（OTB）−Leu−OMe45.3ｇ（84％）を得
る。融点224−５℃（分解）、〔α〕²⁰ _D−21.5゜（ｃ
＝１、DMF）。参考例 (36) 10−16〔Ｂ〕Ｚ−Gly−Lys（Boc）−Leu−
Ser−Gln−Glu（OTB）−Leu−NHNH₂ 参考例（35）で得たヘプタペプチド43ｇを
DMF500ml中ヒドラジンヒドラート30mlと24時
間室温で反応せしめたのち減圧濃縮し、残渣を
DMF／エタノールから再結晶して、目的のＺ
−Gly−Lys（Boc）−Leu−Ser−Gln−Glu
（OTB）−Leu−NHNH₂38ｇ（88％）、を得る。
融点225−６℃（分解）、〔α〕²⁰ _D−17.8゜（ｃ＝１、
DMF）。参考例 (37) 10−24Z−Gly−Lys（Boc）−Leu−Ser−
Gln−Glu（OTB）−Leu−His−Lys（Boc）−
Leu−Gln−Thr−Tyr−Pro−Arg−OEt 参考例（32）で得たオクタペプチドＺ−His
−Lys（Boc）−Leu−Gln−Thr−Tyr−Pro−
Arg−OEt20ｇを80％酢酸400mlに溶解し、５
％パラジウム炭素２ｇを加えて接触還元する。
反応終了後触媒を別し液を減圧乾固しし、
残渣をエーテルとよくすりつぶして取し水酸
化ナトリウム上減圧乾燥する。この生成物のう
ち6.8ｇをDMF50mlに溶解しトリエチルアミン
で中和しておく。一方、参考例（36）で得たヘプタペプチドヒ
ドラジドＺ−Gly−Lys（Boc）−Leu−Ser−
Gln−Glu（OTB）−Leu−NHNH₂6.5ｇを100
mlのDMFに溶解し、−15℃にて撹拌しつつ
2.3N塩化水素／酢酸エチル15ml、ついで亜硝
酸イソアミル0.77ｇを加える。10分後−50℃に
冷却し、トリエチルアミン4.8mlで中和し、上
記オクタペプチドのDMF溶液を加え、０℃に
て16時間撹拌をつづける。不溶物を別し、
液を減圧乾固し、残渣をシリカゲルのカムにか
け（溶出液：クロロホルム／メタノール／水
70：30：３）目的の画分を濃縮してエーテルと
すりつぶすと、標記のＺ−Gly−Lys（Boc）−
Leu−Ser−Gln−Glu（OTB）−Leu−His−Lys
（Boc）−Leu−Gln−Thr−Tyr−Pro−Arg−
OEt8.3ｇ（75％）が無定形の粉末として得ら
れる。〔α〕²⁰ _D−14.8゜（ｃ＝１、DMF）。参考例 (38) ７−9Trt−Cys（Trt）−Val−Leu−OMe Ｚ−Val−Leu−OMe37.8ｇをメタノール300
mlと5N塩化水素／メタノール50mlとに溶解し、
５％パラジウム炭素４ｇを加えて接触還元す
る。反応後触媒を別し、液を減圧乾固し残
渣を300mlのDMFに溶解し、Trt−Cys（Trt）−
OSu・C₆H₆78ｇを加えたのち０℃に冷却しト
リエチルアミン14mlを加える。室温に一夜放置
したのち減圧乾固し、残渣をクロロホルムに溶
解し、有機層を４％クエン酸、４％重曹、水の
順に洗浄し乾燥する。溶媒を留去し、残渣をメ
タノールから再結晶して、標記のTrt−Cys
（Trt）−Val−Leu−OMe71ｇ（85％）を得る。
融点108−９℃、〔α〕²⁰ _D＋22.3゜（ｃ＝２、クロロ
ホルム）。参考例 (39) １−２〔Ａ〕Boc−Cys（Trt）−Ser−OMe Boc−Cys（Trt）−OH・DCHA65ｇとＨ−
Ser−OMe・HCl15.6ｇとを500mlのクロロホル
ムに溶解し、冷却撹拌しつつDCC20.6ｇをクロ
ロホルム50mlに溶かした液を滴下する。滴下後
更に15時間室温で撹拌したのち不溶物を別
し、液を４％クエン酸、４酸％重曹、水の順
に洗浄し乾燥する。溶媒を留去し残渣を酢酸エ
チル／ヘキサンから再結晶して、標記のBoc−
Cys（Trt）−Ser−OMe39.5ｇ（70％）を得る。
融点181−２℃、〔α〕²⁰ _D＋23.7゜（ｃ＝１、
MeOH）。参考例 (40) １−２〔Ｂ〕Boc−Cys（Trt）−Ser−
NHNH₂ 参考例（39）で得たBoc−Cys（Trt）−Ser−
OMe56.4ｇをメタノール500mlに溶解し０℃で
ヒドラジンヒドラート25mlを加え、０℃に16時
間放置し析出した結晶を取し、水洗、乾燥し
て、目的のヒドラジドBoc−Cys（Trt）−Ser−
NHNH₂51ｇ（90％）を得る。融点104−８℃、
〔α〕²⁰ _D＋0.76゜（ｃ＝１、MeOH）。参考例 (41) １−６〔Ａ〕Boc−Cys（Trt）−Ser−Asn−
Leu−Ser−Thr−OMe 参考例（40）で得たヒドラジドBoc−Cys
（Trt）−Ser−NHNH₂32ｇをDMF250mlに溶
解し、−30℃で撹拌しつつ2.3N塩化水素／酢酸
エチル50ml、ついで亜硝酸イソアミル８ｇを
徐々に加える。10分後−50℃に冷却しトリエチ
ルアミン16.1mlを加えて中和する。一方、参考例（17）で得たテトラペプチドＺ
−Asn−Leu−Ser−Thr−OMe33.1ｇをｔ−ブ
タノール／水（９：１）800ml中５％パラジウ
ム炭素1.5ｇを用いて接触還元する。反応終了
後触媒を別、液を減圧乾固し残渣を
DMF100mlに溶解して上記アジド溶液に加え、
０℃にて20時間撹拌をつづける。反応混合物を
減圧乾固し、残渣に４％クエン酸400mlを加え
て充分すりつぶし沈殿を取、水洗、乾燥す
る。DMF／水から再結晶して、目的のBoc−
Cys（Trt）−Ser−Asn−Leu−Ser−Thr−
OMe42.8ｇ（76.7％）を得る。融点212−４℃、
〔α〕²⁰ _D＋0.44゜（ｃ＝１、DMF）。参考例 (42) １−６〔Ｂ〕Boc−Cys（Trt）−Ser−Asn−
Leu−Ser−Thr−NHNH₂ 参考例（41）で得たヘキサペプチド64.7ｇを
550mlのDMF中ヒドラジンヒドラート60mlと０
℃で一夜反応せしめたのち氷水３に注入す
る。沈殿を取し、メタノールついでエーテル
で洗つて、標記のヒドラジドBoc−Cys（Trt）
−Ser−Asn−Leu−Ser−Thr−NHNH₂56ｇ
（86％）を得る。融点205−６℃、〔α〕²⁰ _D＋6.2゜（ｃ＝１、DMF）。参考例 (43) １−９〔Ａ〕Boc−Cys（Trt）−Ser−Asn−
Leu−Ser−Thr−Cys（Trt）−Val−Leu−
OMe 参考例（42）で得たヒドラジドBoc−Cys
（Trt）−Ser−Asn−Len−Ser−Thr−
NHNH₂29.4ｇをDMF350mlに溶解し、−30℃
で撹拌しつつ2.3N塩化水素／酢酸エチル30ml、
ついで亜硝酸イソアミル4.2ｇを徐々に加える。
10分後−50℃に冷却し、トリエチルアミン9.7
mlを加えて中和する。一方、参考例（38）で得たトリペプチドTrt
−Cys（Trt）−Val−Leu−OMe30ｇを80％酢
酸に溶解し、室温に３時間放置したのち水160
mlを加える。生じた沈殿を別し液を減圧乾
固、残渣に飽和重曹水200mlを加え析出した油
状物を酢酸エチルで抽出する。有機層を乾燥し
溶媒を留去して得た油状物をDMF100mlに溶解
して上記アジド溶液に加える。反応液を０℃に
て２日間撹拌したのち減圧乾固し、残渣に５％
クエン酸を加えて生じた沈殿を取、水洗、乾
燥後クロロホルム／メタノールから再結晶し
て、目的のBoc−Cys（Trt）−Ser−Asn−Leu
−Ser−Thr−Cys（Trt）−Val−Leu−
OMe26.4ｇ（57％）を得る。融点227−230℃
（分解）、〔α〕²⁰ _D−7.7゜（ｃ＝１、DMF）。参考例 (44) １−９〔Ｂ〕Boc−Cys（Trt）−Ser−Asn−
Leu−Ser−Thr−Cys（Trt）−Val−Leu−
NHNH₂ 参考例（43）で得たノナペプチド26.1ｇを
DMF340mlとDMSO170mlとに溶解し、ヒドラ
ジンヒドラート63mlを加えて０℃に２日間放置
する。反応液を氷冷した飽和食塩水２に注入
し、沈殿を取、水洗、乾燥して、目的のヒド
ラジドBoc−Cys（Trt）−Ser−Asn−Leu−Ser
−Thr−Cys（Trt）−Val−Leu−NHNH₂2.6ｇ
（100％）を得る。融点242−５℃（分解）、〔α〕
²⁰ _D−6.5゜（ｃ＝１、DMF）。実施例１ Boc−Cys（Acm）−Ser−Asn−Leu−Ser−
Thr−Cys（Acm）−Val−Leu−Gly−Lys
（Boc）−Leu−Ser−Gln−Glu（OTB）−Leu−
His−Lys（Boc）−Leu−Gln−Thr−Tyr−Pro
−Arg−OEt 参考例（15）で得た保護テトラデカペプチド
2.5ｇを80％酢酸40mlに溶かし、５％パラジウム
炭素を加えて水素添加する。反応完了後触媒を
別し、減圧乾固し、残渣をエーテルで充分洗浄し
て粉末とする。一方参考例（26）で得たデカペプ
チドヒドラジド2.25ｇをDMF−DMSO（１：１）
40mlに溶かし−20℃に冷却して2.72N塩化水素／
酢酸エチル５mlと亜硝酸イソアミル0.23mlを加
え、15分撹拌したのち−40℃に冷却して、トリエ
チルアミン1.9mlを加えて中和する。これに上記
の還元生成物をDMF30mlに溶かし、トリエチル
アミン0.37mlを加えた溶液を加え、氷冷下一夜撹
拌をつづける。析出したトリエチルアミン塩酸塩
を別し、減圧濃縮し、残渣をDMF−0.5モル酢
酸（１：１）に溶かしてセフアデツクスLH−20
のカラムでゲル過を行う。同溶媒で溶出し、目
的の画分を減圧濃縮し、DMF／酢酸エチルから
再沈殿をくりかえし、標記化合物2.35ｇを得る。〔α〕²¹ _D−13.0゜（ｃ＝２、DMF）。実施例２実施例１で得た保護テトラコサペプチド300ml
をDMF3mlに溶かしメタノール30mlでうすめる。
この溶液をヨード400mgをメタノール40mlに溶か
した溶液中に室温で烈しく撹拌しながら滴下す
る。滴下終了後さらに１時間撹拌をつづけたのち
氷冷し1N−チオ硫酸ナトリウムを溶液が脱色す
るまで滴下する。メタノールを減圧濃縮し氷水を
加えて生じた沈殿を取、乾燥しメタノール／エ
ーテルから再沈殿をくり返す。これをDMF−0.5
モル酢酸（１：１）の系でセフアデツクスLH−
20のゲル過を行ない、メタノール／エーテルか
ら再沈殿して目的物280mgを得る。〔α〕²⁴ _D＋20.0゜（ｃ＝２、DMF）。アミノ酸分析： Lys1.98(2)、His0.91(1)、 Arg0.99(1)、Asp0.89(1)、 Thr1.82(2)、Ser2.51(3)、 Glu2.93(3)、Pro0.98(1)、 Gly1.01(1)、Cys0.89(1)、 Val1.00(1)、Leu5.07(5)、 Tyr1.01(1)。実施例３Ｈ−Cys（Acm）−Ser−Asn−Leu−Ser−Thr
−Cys（Acm）−Val−Leu−Gly−Lys−Len−
Ser−Gln−Glu−Leu−His−Lys−Leu−Gln
−Thr−Tyr−Pro−Arg−OH 実施例１で得た保護テトラコサペプチド50mgを
DMF0.5ml及び0.5モルトリス塩酸緩衝液（PH6.0）
0.3mlに溶解し、トリプシン２mgを加えて30℃で
１時間反応する。反応液を減圧乾固し、残渣をア
ニソール0.5mlを含むトリフルオロ酢酸３mlに溶
解し、室温で40分間反応したのち減圧乾固する。
残渣をエーテルで洗つたのち0.2N酢酸に溶かし、
セフアデツクスＧ−25（2.5×100cm）でゲル過
し、目的の画分を集めて凍結乾燥し、標記の化合
物39mgを得る。〔α〕²² _D−73.7゜（ｃ＝0.76、H₂O）。実施例４ Boc−Cys（Trt）−Ser−Asn−Leu−Ser−Thr
−Cys（Trt）−Val−Leu−Gly−Lys（Boc）−
Leu−Ser−Gln−Glu（OTB）−Leu−His−Lys
（Boc）−Leu−Gln−Thr−Tyr−Pro−Arg−
OEt 参考例（37）で得たペンタデカペプチドＺ−
Gly−Lys（Boc）−Leu−Ser−Gln−Glu（OTB）
−Leu−His−Lys（Boc）−Leu−Gln−Thr−Tyr
−Pro−Arg−OEt6.6ｇを80％酢酸150mlに溶か
し、５％パラジウム炭素を加えて接触還元する。
反応終了後触媒を別し、液を減圧乾固して残
渣をエーテルで充分洗浄して粉末とする。一方、参考例（44）で得たノナペプチドヒドラ
ジドBoc−Cys（Trt）−Ser−Asn−Leu−Ser−
Thr−Cys（Trt）−Val−Leu−NHNH₂4.6ｇを
DMF100ml及びDMSO100mlに溶かし、−10℃で
撹拌しつつ2.3N塩化水素／酢酸エチル10mlと亜
硝酸イソアミル0.36ｇとを徐々に加える。15分後
−50℃に冷却し、トリエチルアミン3.2mlで中和
する。これに上記の還元生成物を50mlのDMFに
溶解しトリエチルアミン0.42mlを加えた溶液を加
え、０℃で16時間撹拌をつづける。不溶物を別
し、液を減圧乾固し、残渣をシリカゲルのカラ
ムにかけ（溶出液：クロロホルム／メタノール／
水70：30：３）、目的の画分を濃縮しエーテルで
粉末として、標記のテトラコペサペプチド6.9ｇ
（64％）を得る。〔α〕²⁰ _D−8.0゜（ｃ＝１、DMF）。実施例５実施例４で得たテトラコサペプチド740mgをメ
タノール50mlに溶解し、この溶液をヨード510mg
をメタノール50mlに溶かした溶液中に20℃で撹拌
しつつ滴下する。５分後氷冷し1N−チオ硫酸ナ
トリウムを滴下して脱色し、氷水１中に注入す
る。生じた沈殿を遠心分離して集め、エタノール
に溶解して濃縮を三回くり返す。最後にエタノー
ルに溶かしエーテルを加えて沈殿を取乾燥し、
標記の目的化合物520mg（83％）を得る〔α〕²⁰ _D＋
19.8゜（ｃ＝１、DMF）。この化合物は、高速液体
クロマトグラフイー及び薄層クロマトグラフイー
による分析から実施例２の生成物と同一であるこ
とが認められた。実施例６実施例５で得たテトラコサペプチド100mgを
DMF1mlと、0.5モルトリス塩酸緩衝液（PH6.0）
0.6mlとに溶解し、トリプシン４mgを加えて室温
で１時間反応する。反応液をそのままセフアデツ
クスLH−20（2.5×100cm）のカラムに負荷し、
DMF／0.5モル酢酸（１：１）で溶出する。目的
の画分を集めて減圧濃縮しエーテルで洗浄して、
目的の標記化合物90mgを白色粉末として得る。〔α〕²⁰ _D＋6.2゜（ｃ＝１、DMF）。実施例７実施例５で得たテトラコサペプチド１ｇをアニ
ソール３mlを含むトリフルオロ酢酸30mlに溶解し
て、室温に30分間放置したのち減圧濃縮する。残
渣を0.2N酢酸30mlに溶解し、エーテル50mlを加
えてふりまぜる。水層と分取し約半量に減圧濃縮
したのち、セフアデツクスＧ−25（2.5×100cm）
でゲル過し、目的の画分を集めて減圧濃縮し、
エタノール／エーテルで粉末として、標記の無保
護テトラコサペプチドエステル730mgを得る。〔α〕²⁰ _D−13.5゜（ｃ＝0.25、50％AcOH）。実施例８実施例６で得た保護テトラコサペプチド90mgを
アニソール１mlを含むトリフルオロ酢酸５mlに溶
かし、室温で40分間反応したのち減圧濃縮し、残
渣をエーテルで洗浄する。これを0.2N酢酸に溶
かしてセフアデツクスＧ−25（2.5×100cm）でゲ
ル過し、目的の画分を集めて凍結乾燥し、標記
の完全無保護のテトラコサペプチド78mgを得る。〔α〕²⁰ _D−13.2゜（ｃ＝0.25、50％AcOH）。参考例 (45) 25−32〔Ａ〕Ｚ−Thr−Asn−Thr−Gly−
Ser−Gly−Thr−Pro−NH₂ Ｚ−Thr−Asn−Thr−Gly−NHNH₂5.4ｇ
をDMF50mlに溶かし、−20℃に冷却して2.70N
塩化水素／酢酸エチル20mlと亜硝酸イソアミル
1.55mlとを加え、−20℃に保つて10分間撹拌し
たのち−40℃に冷却しトリエチルアミン7.56ml
を加えて中和する。ついでＨ−Ser−Gly−
Thr−Pro−NH₂3.77ｇをDMF10mlに溶かした
溶液を加え、氷冷下に一夜撹拌をつづける。析
出したトリエチルアミン塩酸塩を別し、減圧
濃縮し残渣をメタノールから再沈殿をくりかえ
し目的物3.5ｇを得る。〔α〕²² _D−32.0（ｃ＝１、
DMF）。参考例 (46) 25−32〔Ｂ〕Ｈ−Thr−Asn−Thr−Gly−
Ser−Gly−Thr−Pro−NH₂・AcOH 参考例（45）で得た保護オクタペプチドアミ
ド２ｇを80％酢酸40ml中５％パラジウム−炭素
0.4ｇ存在下接触還元する。反応完了後触媒を
別し減圧乾固し、残渣をエーテルで洗浄して
粉末状の目的物1.7ｇを得る。〔α〕²² _D−45.3゜（ｃ
＝２、80％AcOH）。参考例 (47) 実施例２で得た保護テトラコサペプチド160
mgと参考例（46）で得たオクタペプチドアミド
315mgとをDMF2.5mlに溶かし、トリプシン10
mgとTPCK0.1mgとを含んだ0.3モルトリス塩酸
緩衝液（PH6.0）1.6mlを加えて1.5時間室温で撹
拌する。氷酢酸２mlを加えて反応を停止し
DMF2mlを追加したのちセフアデツクスLH−
20（2.5×100cm）のカラムに負荷し、DMF／0.5
モル酢酸（１：１）で溶出する。目的の画分を
集めて減圧濃縮し、残渣をエーテルで洗浄して
目的の標記の保護ドトリアコンタペプチドアミ
ドの白色粉末72mgを得る。〔α〕²⁸ _D−31.6゜（ｃ＝
２、DMF／H₂O（７：３））。参考例 (48) 参考例（47）で得た保護ドトリアコンタペプ
チドアミド100mgをアニソールを少量含んだト
リフルオロ酢酸3.5mlに溶かし室温で45分間放
置後エーテルを加えて約100mgの沈殿を得る。
これを1N酢酸に溶かしてアンバーライトIRA
−410（酢酸型）の小カラムに通し、流出液を凍
結乾燥する。これを0.2N酢酸に溶かしてセフ
アデツクスＧ−25（3.8×50cm）でゲル過し、
目的の画分を凍結乾燥し目的の標記ドトリアコ
ンタペプチドアミド55mgを得る。〔α〕²⁶ _D−41.0゜（ｃ＝１、50％AcOH）。アミノ酸分析： Lys1.99(2)、His1.01(1)、 Arg1.01(1)、Asp2.07(2)、 Thr4.67(5)、Ser3.61(4)、 Glu3.03(3)、Pro2.15(2)、 Gly2.92(3)、Cys0.91(1)、 Val1.02(1)、Leu5.00(5)、 Tyr1.00(1)。Used to represent [Formula]. The feature of this method is that when synthesizing peptides represented by the above formulas (-a) and (-b), a peptide fragment having the amino acid sequence from position 1 to position 24 and the remaining position 25 are synthesized. Peptide fragments having amino acid sequences from to position 32 are synthesized in advance, and the free carboxyl group or esterified carboxyl group of arginine at position 24 and the amino group of threonine at position 25 are combined with trypsin or trypsin-like The key point is that coupling (amidation) is performed using a proteolytic enzyme. Conventionally, proteolytic enzymes such as trypsin have been mainly used to cleave peptide bonds, but in this method, they are extremely excellent as condensing agents for the coupling reaction of the peptide fragment of the formula (). was surprisingly discovered. Trypsin used in this method is a well-known proteolytic enzyme registered with the International Union of Biochemistry (IUB) Enzyme Committee with enzyme number EC3.4.21.4. It is commercially available as trypsin or trypsin treated with tosyl-L-phenylalanyl chloromethyl ketone (TPCK), and any of these can be used in this method. In this method, not only trypsin but also trypsin-like proteolytic enzymes can be used. As examples of trypsin-like proteases that can be used, there are known proteases produced by Streptomyces griseus, Streptomyces fradiae, etc. ["The Enzymes" Vol. (“THE ENZYMES”, Vol.),
746, published by ACADEMIC PRESS in 1971], an enzyme product containing trypsin-like protease produced by Streptomyces griseus K-1 is commercially available under the trade name ``Pronase'' (manufactured by Kaken Chemical Co., Ltd.). ing. In the presence of such a specific proteolytic enzyme, the peptide represented by the above formula () [hereinafter referred to as "C
The coupling reaction between the peptide represented by the above formula ( It is carried out in a medium containing ~8 buffers. The type of buffer to be used is not particularly limited as long as the pH value is within the above range, and various types can be used.For example, Tris-HCl buffer, Matsukuilbain's buffer, phosphoric acid buffer, etc. Examples include buffer solutions, ammonium acetate buffer, Atkins & Punchin buffer, veronal buffer, and the like. Among these, a Tris-HCl buffer with a pH of 6 to 8 is preferred. When using these buffers as reaction media,
The buffer is usually used in admixture with a water-miscible organic solvent, in particular a water-miscible organic solvent which at least partially dissolves at least one of the C component and the N component. The water-miscible organic solvent that can be used as part of the reaction medium includes, for example, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylimidazolidinone (DMI), hexamethylphosphoryltriamide (HMPA), and the like. is,
Among these, dimethylformamide is particularly preferred. These organic solvents may be used alone or in combination of two or more. Advantageously, the mixing ratio of the buffer solution and the water-miscible organic solvent is generally in the range of 2:8 to 8:2, preferably 5:7 to 7:5 in volume ratio of buffer solution to organic solvent. be. The reaction between component C and component N in such a reaction medium is carried out at a temperature range in which the proteolytic enzyme acts, generally from about 20 to about 50°C, preferably from about 25 to about 40°C. can be done. On the other hand, the ratio of the C component to the N component is not strictly limited and can be changed depending on the other reaction conditions used, but in general, the ratio of the C component to the N component is
It is advantageous that the molar ratio of the N component is 1/1 to 1/100, preferably 1/10 to 1/20. Furthermore, the amount of the protease used is not critical and can be changed depending on the reaction conditions;
Generally 0.003 to 0.2 g per gram of component C, preferably
Suitably, it is used in an amount of 0.05 to 0.1 g. Furthermore, when carrying out this method, a trace amount of calcium ions or the like may be present in the reaction system in order to stabilize the activity of proteolytic enzymes such as trypsin. Under such reaction conditions, the coupling reaction can usually be completed in about 1 to about 20 hours. In the above reaction, the peptide of the formula () used as the C component has functional groups (-OH, -COOH, _-NH2 ) present at its N-terminus and side chain in an unprotected state, that is, in a free form. Alternatively, some or all of the functional groups present may be protected by protecting groups known in the field of peptide chemistry. It may be in the form of a functional group-protected derivative. Similarly, the peptide of formula () used as the N component may be either a free type or a functionally protected derivative type. The protecting groups that can be used to protect the functional groups present in the peptides of formula () and/or () are stable under the reaction conditions of the method and can be easily removed from the product after the reaction. It is desirable that the protective group can be removed without causing any side reactions upon removal, and the following protecting groups can be exemplified. (a) Examples of protecting groups for amino groups include t-butyloxycarbonyl group, benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, and trityl group. (b) As the carboxyl group protecting group, for example,
t-butyl group, benzyl group, etc. (c) Examples of the protecting group for hydroxyl group include benzyl group and t-butyl group. (d) Examples of thiol-protecting groups include benzyl group, acetamidomethyl group, and trityl group. Furthermore, the peptide of formula ( ) and/or the peptide of formula ( The term ``'' refers not only to (poly)peptides in which all of the acidic and basic functional groups present are in the free state, but also to (poly)peptides in which at least some of these acidic and basic functional groups are in the form of salts. The term is used to include peptides. Examples of such salts include hydrochloride, hydrobromide, trifluoroacetate, p-toluenesulfonate, acetate, triethylammonium salt, and the like. By the present method described above, the polypeptide represented by the above formula () can be synthesized with high purity and high yield without side reactions. In the above formula (), when X ₁ and X ₂ are both thiol-protecting groups, the compound is oxidized after removing the thiol-protecting group, so that X ₁ and X ₂ are combined. Compounds of the corresponding formula () when representing a single bond can be produced. The oxidation can be carried out using an oxidizing agent and means commonly used in the field of peptide chemistry to form a disulfide bond between two cysteine units in a polypeptide. , for example, Helvetica Chimica Acta (Helv.Chim.Acta) Volume 51, 2061~
2064 pages (1968), Journal of the American Chemical Society (J.Amer.
Chem.Soc.), Vol. 94, pp. 5456-5461 (1972). In addition, when the compound of formula () has a protecting group, methods known in the field of peptide chemistry, such as those written by E. Schroder and K. Lu¨bke, may be used. “The Peptides” Volume 1, pp. 3-75, 1965
The protecting group can be removed by the method described in 2010, published by Academic Press. When the compound of formula () is subjected to the above oxidation step, the protective group may be removed at any time before or after the oxidation. Thereby, the peptides represented by formula (-a) or (-b) can be produced much more advantageously than in conventional methods. As mentioned above, the C component used as a starting material in this method, that is, the peptide of the formula () of the present invention, is a novel substance that has not been described in the conventional literature. Phase synthesis method (“The Peptize” above)
For example, by the well-known liquid phase synthesis method, the following formula In the formula, X ₁ and X ₂ have the above-mentioned meanings, and a peptide represented by or a functional group protected derivative thereof and the following formula H-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Le
A peptide represented by u-Gln-Thr-Tyr-Pro-Arg-OH or its functional group-protected derivative is synthesized, and then the two are condensed, and if necessary, oxidized to form a peptide at position 1-7. It can be produced by forming disulfide bonds between positions. The lower alkyl group (R) in the above formula () has up to 6 carbon atoms, preferably 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n
-butyl group, among others, methyl and ethyl groups are preferred. Accordingly, a particularly preferred group of compounds among the peptides of the formula () of the present invention include those in which, in the formula (), X ₁ and X ₂ each represent _a trityl group or an acetamidomethyl group, or ₂ together represent a single bond, Y ₁ is a hydrogen atom,
t-butyloxycarbonyl group, p-methoxybenzyloxycarbonyl group or trityl group, Y ₄ represents a hydrogen atom, t-butyloxycarbonyl group, p-methoxybenzyloxycarbonyl group or trityl group, Y ₅ represents hydrogen atom or t
- This is a compound of formula () when representing a butyl group. This method has the following advantages over conventionally known methods for synthesizing the same polypeptide, and is extremely useful industrially. (a) Due to the nature of the enzymatic reaction, the reaction can be carried out without any side reactions, and moreover, the product can be synthesized in good yield without producing racemates, and the product can be easily purified. (b) Unreacted components of both the N-component and the C-component can be completely recovered and reused, which is economically advantageous. (c) Protection of side chain functional groups is unnecessary or may be minimal. Therefore, the synthesis of starting materials and the final deprotection operation are unnecessary or extremely easy. (d) The reaction takes a short time, and a simple reaction apparatus is sufficient. The present invention will be further explained below with reference to Examples. In addition, the abbreviations described in the following Reference Examples and Examples are as follows, excluding the abbreviations of the above-mentioned amino acids. Boc-: t-butyloxycarbonyl Z: benzyloxycarbonyl Bzl: benzyl Acm: acetamidomethyl Trt: trityl OMe: methyl ester OEt: ethyl ester OTB: t-butyl ester OSu: N-hydroxysuccinimide ester ONp: paranitrophenyl Ester OBzl: Benzyl ester DMF: Dimethylformamide DMSO: Dimethyl sulfoxide DCC: Dicyclohexylcarbodiimide MeOH: Methanol EtOH: Ethanol TsOH: Toluenesulfonic acid AcOH: Acetic acid DCHA: Dicyclohexylamine In addition, in the following reference examples and examples, "Sephadex LH- 20" is a product name of Pharmacia, which is a cross-linked dextran-based molecular sieve for gel filtration in which most of the hydroxyl groups are alkylated, and "Sephadex G-25" is a product name of Pharmacia,
A cross-linked dextran-based molecular sieve for gel filtration that has a large number of hydroxyl groups.
410" is a product name manufactured by Rohm and Haas.
It is a polystyrene-based ion exchange resin having an N-(2-hydroxyethyl)-N,N-dimethylamino group. Reference example (1) 22-23 [A] Boc-Tyr-Pro-OMe Boc-Tyr-OH・DCHA46.2g and H-Pro-
Dissolve 16.5 g of OMe.HCl in 200 ml of chloroform, and while stirring under ice cooling, dissolve 20.6 g of DCC in 50 ml of chloroform and add dropwise. After the dropwise addition, the mixture was stirred under ice cooling for about 5 hours, and the precipitated dicyclohexyl urea was separated, and the liquid was concentrated under reduced pressure. The obtained oily residue is dissolved in ethyl acetate, washed with an aqueous citric acid solution and an aqueous sodium bicarbonate solution, and then dried over anhydrous sodium sulfate. The oil obtained by distilling off the solvent was recrystallized from ether/hexane to obtain Boc-
35.5 g (90%) of Tyr-Pro-OMe are obtained. melting point
122-4°C, [α] ²⁶ _D -29.1° (c=2, DMF). Reference example (2) 22-23 [B] Boc-Tyr-Pro-OH Dissolve 34.0 g of Boc-Tyr-Pro-OMe in 160 ml of methanol/dioxane (1:1) and add 2N
Add 85 ml of caustic soda and leave at room temperature for 3 hours. After cooling on ice and neutralizing with 5N hydrochloric acid, approximately 150ml
Concentrate under reduced pressure, add cold 10% citric acid solution, and adjust the pH to
is set to 2 to 3. Take the precipitated crystals, wash them with water,
When dried, Boc-Tyr-Pro-OH31.5g (96
%) is obtained. Melting point 114-5℃, [α] ²⁶ _D −
22.9° (c=2, DMF). Reference example (3) 22-24 [A] Boc-Tyr-Pro-Arg( _NO2 )-
30.3 g of OEt Boc-Tyr-Pro-OH and 9.8 g of N-hydroxysuccinimide are dissolved in 150 ml of DMF, and 16.5 g of DCC is dissolved in 50 ml of DMF and added dropwise while stirring on ice. After continuing stirring under ice cooling for another 5 hours, dicyclohexyl urea was separated.
The liquid is concentrated under reduced pressure, and the residual oil is dissolved in ethyl acetate, washed with aqueous sodium bicarbonate, and dried over anhydrous sodium sulfate. The solvent is distilled off under reduced pressure and the residue is treated with ethyl acetate/ether/hexane to obtain 37.0 g (98%) of Boc-Tyr-Pro-OSu in powder form. This active ester is converted into H-Arg(NO ₂ )-OEt.
33.6g of TsOH and 11.2ml of triethylamine
Add to 150 ml of DMF solution and leave overnight at room temperature.
DMF is distilled off under reduced pressure, and the oily residue is dissolved in ethyl acetate, washed with aqueous citric acid and aqueous sodium bicarbonate, and dried over anhydrous sodium sulfate. The ethyl acetate was removed in vacuo and the residual oil was precipitated from ethyl acetate/ether to give Boc-Tyr-Pro-Arg.
43.1 g (88%) of (NO ₂ )-OEt are obtained as an anhydrous shaped powder. [α] ²⁶ _D −24.1° (c=2, DMF). Reference example (4) 22-24 [B] H-Tyr-Pro-Arg-( _NO2 )-
Add approximately 2.7N hydrogen chloride/ethyl acetate 250ml to OEt・HCl Boc−Tyr−Pro−Arg(NO ₂ )−OEt36.4g,
After standing at room temperature for 3 hours, it was concentrated under reduced pressure. Ethyl acetate was added to the residue, concentration under reduced pressure was repeated, and the residue was precipitated with ethanol/ethyl acetate/ether to form H-Tyr-Pro-Arg( _NO2 )-OEt.
31.3 g (96%) of amorphous powder of HCl are obtained. Reference example (5) 19-21 [A] Boc-Leu-Gln-Thr-OMe Dissolve 70 g of Z-Gln-Thr-OMe in 600 ml of t-butanol/water (9:1) and add 4 g of 5% palladium on carbon. After catalytic reduction, the catalyst is separated and the liquid is concentrated under reduced pressure. DMF300ml of the residue
Add 53 g of Boc-Leu-OSu and leave at room temperature overnight. The solvent was distilled off under reduced pressure, ethyl acetate was added to the residue, dissolved by heating, and after cooling, ether was added to collect the precipitate, which was recrystallized from water containing ethanol to obtain the desired Boc-Leu-
53.6 g (70%) of Gln-Thr-OMe are obtained. melting point
186-7°C, [α] ²⁶ _D -25.0° (c=1, DMF). Reference example (6) 19−21 [B] Boc−Leu−Gln−Thr−
47.4 g of NHNH ₂ Boc-Leu-Gln-Thr-OMe was dissolved in 300 ml of methanol, 40 ml of 85% hydrazine hydrate was added, and the mixture was stirred at room temperature. After crystal precipitation,
Leave it overnight, add ether, and collect the crystals. This crude crystal was recrystallized from DMF/water,
37.6 g (79%) of the title hydrazide are obtained. melting point
203-4℃, [α] ²² _D -44.6゜(c=2, 80%
AcOH). Reference example (7) 19-24 [A] Boc-Leu-Gln-Thr-Tyr-
Pro−Arg(NO ₂ )−OEt Boc−Leu−Gln−Thr−NHNH ₂ 16.7 g
A solution dissolved in 50 ml of DMSO and 150 ml of DMF was cooled to -40°C and diluted with 2.72N hydrogen chloride/ethyl acetate.
Add 35 ml and 5.7 ml of isoamyl nitrite. −20
After stirring at °C for 15 minutes, the reaction solution was cooled to -40 °C and neutralized by adding 13.3 ml of triethylamine.
Furthermore, H-Tyr-Pro-Arg obtained in Reference Example (4)
(NO ₂ )-OEt・HCl20g and triethylamine 5.4
Add a solution of 50 ml of DMF and stir overnight under ice cooling. The triethylamine hydrochloride was separated off, the liquid was concentrated under reduced pressure, and the resulting oil was applied to a silica gel column and eluted with chloroform/methanol (10:1). The solvent was distilled off under reduced pressure, and the residue was precipitated with methanol/ethyl acetate to obtain the desired Boc-Leu-Gln-Thr-Tyr-Pro.
-Arg( _NO2 )-OEt 25.5 g (76%) are obtained as an amorphous powder. [α] ²¹ _D −36.4° (c=1, DMF). Reference example (8) 19-24 [B] H-Leu-Gln-Thr-Tyr-
Pro−Arg(NO ₂ )−OEt・HCl Boc−Leu−Gln−Thr−Tyr−Pro−Arg
Approximately 2.7N hydrogen chloride/ethyl acetate (150 ml) was added to 25.0 g of (NO ₂ )-OEt and left at room temperature for 3 hours. The solvent was distilled off under reduced pressure, and ethyl acetate was added to the residue.
Repeat vacuum concentration several times. The residue is ethanol/
Precipitation with ethyl acetate/ether yields 23.4 g (≈100%) of the desired product as an amorphous powder. Reference example (9) 17-24 [A] Z-His-Lys(Boc)-Leu-
Gln−Thr−Tyr−Pro−Arg(NO ₂ )−OEt Z−His−Lys(Boc)−NHNH ₂ 10.2 g
The solution dissolved in 80ml of DMF was cooled to -20℃,
Add 20 ml of 2.78N hydrogen chloride/ethyl acetate and 3.15 ml of isoamyl nitrite. After stirring at -20℃ for 15 minutes, the reaction solution was cooled to -40℃, neutralized by adding 7.78ml of triethylamine, and H-Leu-Gln
−Thr−Tyr−Pro−Arg(NO ₂ )−OEt・
17.7g of HCl and 3.36ml of triethylamine in 80 DMF
Dissolve in ml and add. After stirring overnight under ice-cooling, the triethylamine hydrochloride was removed, the liquid was concentrated under reduced pressure, and the oily residue was applied to a silica gel column and eluted with chloroform/methanol (5:1). The desired fraction is concentrated under reduced pressure and precipitated with methanol/ethyl acetate to obtain the desired Z-
His−Lys(Boc)−Leu−Gln−Thr−Tyr−Pro
19.2 g (74%) of -Arg( _NO2 )-OEt are obtained as an amorphous powder. [α] ^21.5 _D −23.5° (c=2, DMF). Reference example (10) 17-24 [B] H-His-Lys(Boc)-Leu-
Gln−Thr−Tyr−Pro−Arg−OEt・3AcOH Z−His−Lys(Boc)−Leu−Gln−Thr−
Tyr−Pro−Arg(NO ₂ )−OEt11.0g in 80% acetic acid
After dissolving in 80 ml and adding 1.0 g of 5% palladium on carbon for catalytic reduction, the catalyst was separated. Concentrate the liquid under reduced pressure, add water several times and concentrate under reduced pressure, then add methanol and concentrate under reduced pressure. The resulting oily residue was precipitated with methanol/ether to yield H-His-Lys(Boc)-Leu.
−Gln−Thr−Tyr−Pro−Arg−OEt・
10.3 g (94%) of amorphous powder of 3AcOH are obtained. Amino acid analysis Lys1.10(1), His1.02(1), Arg0.76(1), Thr0.95(1), Gln1.03(1), Pyr1.15(1), Leu1.00(1) , Tyr0.94(1). Reference example (11) 4-5 (12-13) Z-Leu-Ser-NHNH ₂ Z-Leu-Ser-OMe 51.5 g in methanol 400
ml, add 50 ml of 85% hydrazine hydrate, stir at room temperature, and after crystal precipitation, leave overnight. Add ether to collect crystals and recrystallize from methanol to obtain Z-Leu-Ser-NHNH ₂ 45.2g
(87%). Melting point 176-8°C, [α] ²⁶ _D -8.6° (c=1, DMF). Reference example (12) 12-16 [A] Z-Leu-Ser-Gln-Glu
(OTB)-Leu-OMe Z-Leu-Ser-NHNH ₂ 13.9g in DMF100ml
Cool the solution dissolved in water to -40℃, add 30 ml of 2.68N hydrogen chloride/ethyl acetate and 6 ml of isoamyl nitrite.
Add. After stirring at -20℃ for 15 minutes, -40℃
Cool and neutralize by adding 11.26 ml of triethylamine. H-Gln-Glu(OTB)- in the reaction solution
Add 50 ml of DMF solution containing 17.4 g of Leu-OMe,
Stir overnight on ice. The reaction mixture was concentrated under reduced pressure, water was added to the residue to remove the precipitate, and recrystallization from ethanol yielded the desired Z-Leu-Ser.
-Gln-Glu(OTB)-Leu-OMe28.0g (93%)
is obtained. Melting point 194-196°C, [α] ¹⁸ _D -21.5° (c=2, DMF). Reference example (13) 11-16 [A] Z-Lys (Boc)-Leu-Ser-
Gln−Glu(OTB)−Leu−OMe Z−Leu−Ser−Gln−Glu(OTB)−Leu−
23.8g of OMe in t-butanol/water (9:1)
Suspend in 250 ml, add 1 g of 5% palladium on carbon, and after catalytic reduction, remove the catalyst and concentrate the liquid under reduced pressure. Dissolve the residue in 300ml of DMF and add Z-Lys
Add 15 g of (Boc)-ONp, leave it at room temperature overnight, and then concentrate under reduced pressure. Add water to the residue, collect the resulting precipitate, and recrystallize from ethanol/water to obtain the desired Z-Lys(Boc)-Leu-Ser-Gln-
26.3 g (85%) of Glu(OTB)-Leu-OMe are obtained. Melting point 215-6℃, [α] ^21.5D _- 25.0゜(c=2,
DMF). Reference example (14) 11−16 [B] Z−Lys−(Boc)−Leu−Ser−
Gln−Glu(OTB)−Leu−NHNH ₂ Z−Lys(Boc)−Leu−Ser−Gln−Glu
Dissolve 18.2 g of (OTB)-Leu-OMe in 100 ml of DMF and add 15 ml of 85% hydrazine hydrate.
After standing at room temperature for several days, the reaction solution was concentrated under reduced pressure. The residue is washed with ethanol to obtain 16.7 g (92%) of the desired hexapeptide hydrazide. melting point 226
-7°C (decomposition), [α] ²¹ _D -45.0° (c=2, 80%
AcOH). Reference example (15) 11-24 [A] Z-Lys (Boc)-Leu-Ser-
Gln−Glu(OTB)−Leu−His−Lys(Boc)−
Leu−Gln−Thr−Tyr−Pro−Arg−OEt Z−Lys(Boc)−Leu−Ser−Gln−Glu
Dissolve 5.2 g of (OTB)-Leu-NHNH ₂ in 50 ml of DMF, cool the solution to -40°C, and then add 5 ml of 2.7N hydrogen chloride/ethyl acetate and isoamyl nitrite.
Add 0.8ml. After stirring at -20℃ for 15 minutes,
Cool again to −40°C and add 1.9 ml of triethylamine.
Add to neutralize. Add to this solution H-His-Lys(Boc)-Leu-Gln-Thr-Tyr obtained in Reference Example (10).
Add 30 ml of a DMF solution containing 5.4 g of -Pro-Arg-OEt.3AcOH and 1.4 ml of triethylamine, and stir overnight on ice. The reaction solution was concentrated under reduced pressure, and the residue was applied to a silica gel column and eluted with chloroform/methanol/water (70:30:5). The desired title tridecapeptide was precipitated with methanol/ethyl acetate/ether, yielding 5.4 g.
(60%) obtained as an amorphous powder. Amino acid analysis: Lys2.24(2), His1.08(1), Arg0.99(1), Thr0.96(1), Ser0.89(1), Glu3.17(3), Pro1.06(1) ), Leu3.00(3), Tyr0.94(1). Reference Example (16) 4-6 [A] Z-Leu-Ser-Thr-OMe Z-Leu-Ser-NHNH ₂ obtained in Reference Example (11) 25.6
Dissolve g in 2.00 ml of DMF, cool to -40°C, add 2.35N hydrogen chloride/ethyl acetate 60 ml and 11.6 ml of isoamyl nitrite, stir at -20°C for 15 minutes, then cool to -40°C again. Neutralize by adding 19.8 ml of triethylamine. Add H-Thr-OMe (Z-Thr-
(obtained by catalytic reduction of 18.7g OMe)
Add the solution dissolved in 50 ml of DMF and continue stirring overnight under ice cooling. Concentrate the solvent under reduced pressure, add water to the oily residue, stir, and collect. This was recrystallized from methanol/ethyl acetate to obtain the desired Z-
26.6 g (81%) of Leu-Ser-Thr-OMe are obtained.
Melting point 185-6°C, [α] ²⁶ _D -9.8° (c=1, DMF). Reference example (17) 3-6 [A] Z-Asn-Leu-Ser-Thr-
Suspend 13.5 g of OMe Z-Leu-Ser-Thr-OMe in 400 ml of t-butanol/water (9:1),
After catalytic reduction by adding 2 g of % palladium on carbon, the catalyst was separated and the liquid was concentrated under reduced pressure.
Dissolve the residue in 80 ml of DMF and add Z-Asn-
Add 11.2 g of ONp and leave at room temperature for two nights. The precipitated crystals were dissolved by heating, concentrated under reduced pressure, and the residue was
Recrystallization from DMF/methanol yields the desired Z-Asn-Leu-Ser-Thr-OMe13.7g (81
%). Melting point: 218° to 222°C, [α] ²² _D -26.0° (c=2, DMF). Reference example (18) 3-6 [B] H-Asn-Leu-Ser-Thr-
OMe Z−Asn−Leu−Ser−Thr−OMe5.8g
- Suspended in 150 ml of butanol/water (9:1) 5
After catalytic reduction by adding 0.6 g of %-palladium on carbon, the catalyst was separated and the liquid was dried under reduced pressure. The crystalline product is used immediately in the next reaction. Reference example (19) 1-2 [A] Boc-Cys (Acm)-Ser-OMe Boc-Cys (Acm)-OH・DCHA8.8g (0.03
mol) and 4.7 g (0.03 mol) of H-Ser-OMe.HCl are dissolved in 100 ml of chloroform, and 6.18 g (0.03 mol) of DCC is added while stirring under ice cooling. After reacting for 3 hours under ice-cooling and 2 hours at room temperature, the precipitated dicyclohexyl urea was separated and washed with chloroform. After washing the washing liquid with an aqueous citric acid solution and an aqueous sodium bicarbonate solution, it is dried over anhydrous sodium sulfate. After distilling off the solvent, 9.6g of oily target product was obtained.
(82%). Reference example (20) 1-2 [B] Boc-Cys(Acm)-Ser-
NHNH ₂ Boc−Cys(Acm)−Ser−OMe9.6g
Dissolve in 50 ml of DMF, replace the container with nitrogen gas, add 15 ml of 85% hydrazine hydrate, and react overnight in the refrigerator. The reaction solution was dried under reduced pressure,
Recrystallize the remaining crystals from methanol to give 7.3g.
(75%) get the objective. Melting point 199-200℃,
[α] ¹⁸ _D −24.0° (c=2, 80% AcOH). Reference example (21) 1-6 [A]-Boc-Cys (Acm)-Ser-Asn
−Leu−Ser−Thr−OMe Boc−Cys(Acm)−Ser−NHNH ₂ 3.94 g
After heating and dissolving in 60 ml of a mixed solvent of DMF: DMSO (2:1), cool to -20℃ and mix with 2.72N hydrogen chloride/ethyl acetate 20 ml and isoamyl nitrite.
Add 2.37ml and stir for 15 minutes. The reaction solution was heated to -40℃
Cool and neutralize by adding 7.62 ml of triethylamine. Add H-Asn obtained in Reference Example (18) to this solution.
A solution of 4.47 g of -Leu-Ser-Thr-OMe dissolved in 30 ml of DMF is added, and the mixture is allowed to react for 20 hours under ice cooling. Triethylamine hydrochloride was separated, the solvent was dried under reduced pressure, and the residue was recrystallized from methanol.
6.1 g (75%) of the desired title compound are obtained. melting point
208-211°C, [α] ²² _D -22.0° (c=2, DMF). Reference example (22) 1-6 [B] Boc-Cys (Acm)-Ser-Asn
−Leu−Ser−Thr−NHNH ₂ Boc−Cys(Acm)−Ser−Asn−Leu−Ser−
Dissolve 5 g of Thr-OMe in 50 ml of DMF, add 4 ml of 85% hydrazine hydrate, leave it overnight in a refrigerator under a nitrogen atmosphere, concentrate under reduced pressure, wash the residue with methanol, and obtain 4.7 g (94%) of the desired product.
get. Melting point 187-188℃, [α] ²⁰ _D -34.5゜(c=
2, 80% AcOH). Reference example (23) 7-10 [A] Boc-Cys (Acm)-Val-Leu
−Gly−OEt Boc−Cys(Acm)−OH・DCHA29.34g and H
-Val-Leu-Gly-OEt・HCl21.8g was dissolved in 360ml of chloroform and stirred under ice cooling.
A solution of 12.77 g of DCC dissolved in 30 ml of chloroform is added dropwise. After stirring for 3 hours under ice cooling and overnight at room temperature, the precipitated dicyclohexyl urea was separated,
Wash with chloroform. After washing the washing liquid with an aqueous citric acid solution and an aqueous sodium bicarbonate solution, it is dried over anhydrous sodium sulfate. Chloroform was distilled off, and the residue was recrystallized from ethyl acetate to obtain 29.3 g of the desired protected tetrapeptide.
(80%). Melting point 140-141℃, [α] ²² _D −
18.5° (c=2, DMF). Reference example (24) 7-10 [B] H-Cys(Acm)-Val-Leu-
Gly−OEt・HCOOH Boc−Cys(Acm)−Val−Leu−Gly−OEt5.9
Dissolve g in 100 ml of 98% formic acid and leave at room temperature overnight. Dry under reduced pressure and wash the residue thoroughly with ether.
Dry to obtain 5.3 g (100%) of the target product as a powder. Reference example (25) 1-10 [A] Boc-Cys (Acm)-Ser-Asn
−Leu−Ser−Thr−Cys(Acm)−Val−Leu−
Gly−OEt Boc−Cys(Acm)−Ser obtained in reference example (22)
−Asn−Leu−Ser−Thr−NHNH ₂ 4.05g
Dissolve in 30ml of DMF by heating. Cool to below 0℃, add 10ml of 2.7N hydrogen chloride/ethyl acetate while stirring, then cool to -20℃ and add isoamyl nitrite.
Add 1.1 ml and stir at -15°C to -10°C for 10 minutes. Cool to -40°C, add 3.81 ml of triethylamine to neutralize, and add H-Cys(Acm)-Val-
Dissolve 2.7 g of Leu-Gly-OEt.HCOOH in 30 ml of DMF, add a solution neutralized with 0.7 ml of triethylamine, and react at 0°C for 20 hours. Triethylamine hydrochloride was separated, the solvent was concentrated under reduced pressure, and the residue was crystallized from DMF/methanol and then recrystallized from DMF/water to obtain the desired product 2.7.
g (40%). Melting point 242-244°C (decomposition), [α] ²² _D -28.5° (c=
1.DMF). Reference example (26) 1-10 [B] Boc-Cys (Acm)-Ser-Asn
−Leu−Ser−Thr−Cys(Acm)−Val−Leu−
Gly−NHNH ₂ Boc−Cys(Acm)−Ser−Asn−Leu−Ser−
Thr−Cys(Acm)−Val−Leu−Gly−OEt2.52
Dissolve g in 30 ml of DMF, add 1.5 ml of 85% hydrazine hydrate, and react overnight in the refrigerator. Concentrate hydrazine hydrate and DMF under reduced pressure, wash the residue with methanol, and obtain the desired product.
Obtain 2.25g (90%). Melting point 236-237°C (decomposition), [α] ²² _D -43.3° (c=
0.3, 80% AcOH). Reference example (27) 22−23Boc−Tyr(Bzl)−Pro−OH Boc−Tyr(Bzl)−OSu 85g dissolved in DMF 600ml and proline 23g dissolved in water 100ml were mixed and stirred under ice cooling. Add 28 ml of triethylamine dropwise while stirring. After the addition was completed, the mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure, and the resulting syrupy residue was dissolved in 300 ml of water and acidified with citric acid. The precipitated product was extracted with ethyl acetate, and the ethyl acetate layer was washed with water, dried, and concentrated to about 1/3.
Crystals precipitate when hexane is added. 72g (85
%). Melting point 141-2°C, [α] ²⁰ _D -21.3° (c=1,
EtOH). Reference example (28) 22−24Boc−Tyr(Bzl)−Pro−Arg(NO ₂ )
−OEt Boc−Tyr(Bzl)Pro− obtained in reference example (27)
OH33g, H-Arg( _NO2 )-OEt・TsOH29.2
9.0 g of N-hydroxysuccinimide and 10.9 ml of triethylamine are dissolved in 350 ml of DMF, and a solution prepared by dissolving 16.2 g of DCC in 50 ml of DMF is added dropwise while stirring under ice cooling. After the dropwise addition was completed, stirring was continued at room temperature for 12 hours, and the precipitated crystals were separated.
The liquid was concentrated under reduced pressure to obtain a syrupy residue. Dissolve this in ethyl acetate to make 4% citric acid, 4%
Wash with baking soda and water in that order and dry. The solvent was distilled off, and the resulting residue was subjected to silica gel column chromatography (eluent: chloroform/methanol 10:1).
and recrystallization from ethyl acetate/hexane to obtain 43 g (79%) of the desired product. Melting point 89−90
°C, [α] ²⁰ _d −17.2° (c=1, MeOH). Reference example (29) 19-21 [A] Z-Leu-Gln-Thr-OMe Dissolve 39.5 g of Z-Gln-Thr-OMe in t-butanol/water (9:1) 1, and add 2 g of 5% palladium on carbon. After catalytic reduction by adding
Concentrate the liquid under reduced pressure. The residue was dissolved in 300 ml of DMF, 38.3 g of Z-Leu-ONp was added, and the mixture was left at room temperature overnight. The solvent is distilled off under reduced pressure, and the remaining crystals are thoroughly triturated with ethyl acetate. Recrystallize from DMF-ethyl acetate to obtain the desired Z-Leu-
42 g (83%) of Gln-Thr-OMe are obtained. melting point
203-4°C, [α] ²⁰ _D -18.3° (c=1, DMF). Reference example (30) 19-21 [B] Z-Leu-Gln-Thr-
NHNH ₂ Z−Leu−Gln−Thr−OMe25.4g
Dissolve in 300 ml of DMF, add 30 ml of 85% hydrazine hydrate at 0°C, leave at room temperature overnight, concentrate under reduced pressure, and thoroughly triturate the residue with cold water. Recrystallization from DMF/water gives 23 g (90%) of the title hydrazide. Melting point 219-220℃,
[α] ²⁰ _D −17.1° (c=1, DMF). Reference example (31) 19−24Z−Leu−Gln−Thr−Tyr(Bzl)−
Pro−Arg(NO ₂ )−OEt Tripeptide Boc−Tyr obtained in Reference Example (28)
(Bzl)-Pro-Arg( _NO2 )-OEt7g in 98% formic acid
Dissolve in 40 ml, leave at room temperature overnight, and dry under reduced pressure. The residue was dissolved in cold water, triethylamine was added little by little to adjust the pH to 9, the precipitated product was extracted with ethyl acetate, the organic layer was dried, and the solvent was distilled off to give H-Tyr(Bzl)-Pro-Arg(NO ₂ )−
Get OEt. On the other hand, 5.1 g of hydrazide Z-Leu-Gln-Thr-NHNH ₂ obtained in Reference Example (30) was
Suspend in 100ml of DMF, add 30ml of 2.3N hydrogen chloride/ethyl acetate at 0℃ to dissolve, and then -15
Cool to ℃ and add 1.2 g of isoamyl nitrite.
After 15 minutes, cool to -50℃ and add 9.7ml of triethylamine.
After adding , the above H-Tyr(Bzl)-Pro-
A solution of Arg(NO ₂ )-OEt in 30 ml of DMF is added, and stirring is continued at 0° C. for 16 hours.
The precipitate was separated and the liquid was dried under reduced pressure. The residue was purified by silica gel column chromatography (eluent: chloroform/methanol 10:1), and the desired fraction was crystallized with ether to obtain the title hexapeptide Z-.
Leu−Gln−Thr(Bzl)−Pro−Arg(NO ₂ )−
Obtain OEt7.5g (70%). Melting point 120-2℃,
[α] ²⁰ _D −37.2° (c=1, EtOH). Reference example (32) 17−24Z−His−Lys(Boc)−Leu−Gln−
Thr−Tyr−Pro−Arg−OEt Z−Leu−Gln−Thr− obtained in Reference Example (31)
Tyr(Bzl)-Pro-Arg( _NO2 )-OEt20g at 80%
Dissolve in 500 ml of acetic acid and add 2 g of 5% palladium on carbon for catalytic reduction. After finishing, separate the catalyst,
The liquid was dried under reduced pressure, and the residue was thoroughly triturated with ether and dried over sodium hydroxide under reduced pressure. 9.25 g of this product was dissolved in 50 ml of DMF and neutralized with triethylamine. on the other hand,
Z-His-Lys(Boc) _-NHNH2 6.36g
2.3N hydrogen chloride/dissolved in 100ml of DMF at -20℃
20ml of ethyl acetate, then 1.2ml of isoamyl nitrite
After 10 minutes, the mixture was cooled to -50°C, and 6.4 ml of triethylamine was added thereto, followed by the DMF solution of the above hexapeptide, and stirring was continued at 0°C for 16 hours. Insoluble materials were separated, the liquid was dried under reduced pressure, and the residue was applied to a silica gel column and eluted with chloroform/methanol/water (70:30:2). The desired fraction was concentrated and precipitated with ether to obtain the indicated Z
−His−Lys(Boc)−Leu−Gln−Thr−Tyr−
10.2 g (78%) of Pro-Arg-OEt is obtained. [α] ²⁰ _D −25.7° (c=1, DMF). Reference example (33) 10-11 [A] Z-Gly-Lys-(Boc)-OMe H-Lys(Boc)-OMe 26g to ethyl acetate 300
ml, add 33g of Z-Gly-ONp and leave it at room temperature for 16 hours, then dilute the solution with 0.5N caustic soda,
Wash with water, dry, concentrate the solution to 1/5 of its original volume, and add hexane to precipitate crystals. The crystals are collected and recrystallized from ethyl acetate/hexane to yield 40.6 g (90%) of the title dipeptide. Melting point 74-5°C, [α] ²⁰ _D -8.8° (c=1,
MeOH). Reference example (34) 10−11 [B] Z−Gly−Lys(Boc)−
Z−Gly−Lys(Boc)− obtained in NHNH ₂ reference example (33)
36 g of ONe was reacted with 30 ml of hydrazine hydrate in 300 ml of DMF overnight, concentrated under reduced pressure, and the residue was recrystallized from methanol/water to obtain 32 g of the desired hydrazide Z-Gly-Lys (Boc)-NHNH ₂
(89%). Melting point 139-140°C, [α] ²⁰ _D -13.5° (c=1,
MeOH). Reference example (35) 10−16 [A] Z−Gly−Lys(Boc)−Leu−
Ser−Gln−Glu(OTB)−Leu−OMe Z−Gly−Lys(Boc)− obtained in Reference Example (34)
Dissolve 27.5 g of NHNH ₂ in 300 ml of DMF, -15
While cooling to 0.degree. C. and stirring, 30 ml of 2.3N hydrogen chloride/ethyl acetate and then 6.6 g of isoamyl nitrite are gradually added. After 10 minutes, the mixture was cooled to -50°C and 9.7ml of triethylamine was added dropwise. On the other hand, the pentapeptide Z-Leu-Ser-Gln-Glu obtained in Reference Example (12)
(OTB)-Leu-OMe 39.6g in the presence of 5% palladium carbon 3g t-butanol/water (9:1)
The product obtained by catalytic reduction in 350 ml was added to DMF100
ml and added to the above azide solution, and stirring was continued at 0°C overnight. The reaction mixture was dried under reduced pressure, the residue was sufficiently ground to remove the precipitate, and the precipitate was washed with water.
After drying, it was recrystallized from DMF/ethanol to obtain the title Z-Gly-Lys(Boc)-Leu-Ser-Gln-
45.3 g (84%) of Glu(OTB)-Leu-OMe are obtained. Melting point: 224-5°C (decomposition), [α] ²⁰ _D -21.5° (c
= 1, DMF). Reference example (36) 10−16 [B] Z−Gly−Lys(Boc)−Leu−
Ser−Gln−Glu(OTB)−Leu−NHNH ₂ 43g of the heptapeptide obtained in Reference Example (35) was
After reacting with 30 ml of hydrazine hydrate in 500 ml of DMF at room temperature for 24 hours, the residue was concentrated under reduced pressure.
Recrystallize from DMF/ethanol to obtain the desired Z
−Gly−Lys(Boc)−Leu−Ser−Gln−Glu
(OTB)-Leu- _NHNH2 38g (88%) is obtained.
Melting point 225-6°C (decomposition), [α] ²⁰ _D -17.8° (c=1,
DMF). Reference example (37) 10−24Z−Gly−Lys(Boc)−Leu−Ser−
Gln−Glu(OTB)−Leu−His−Lys(Boc)−
Leu−Gln−Thr−Tyr−Pro−Arg−OEt Octapeptide Z−His obtained in reference example (32)
−Lys(Boc)−Leu−Gln−Thr−Tyr−Pro−
Dissolve 20g of Arg-OEt in 400ml of 80% acetic acid,
% palladium on carbon is added for catalytic reduction.
After the reaction was completed, the catalyst was separated and the liquid was dried under reduced pressure.
The residue was triturated with ether and dried under reduced pressure over sodium hydroxide. 6.8 g of this product was dissolved in 50 ml of DMF and neutralized with triethylamine. On the other hand, the heptapeptide hydrazide Z-Gly-Lys(Boc)-Leu-Ser- obtained in Reference Example (36)
Gln−Glu(OTB)−Leu− _NHNH2 6.5g to 100
ml of DMF and stirred at -15°C.
Add 15 ml of 2.3N hydrogen chloride/ethyl acetate and then 0.77 g of isoamyl nitrite. After 10 minutes, the mixture was cooled to -50°C, neutralized with 4.8 ml of triethylamine, the DMF solution of the above octapeptide was added, and stirring was continued at 0°C for 16 hours. Separate the insoluble matter,
The liquid was dried under reduced pressure, and the residue was passed through a silica gel cam (eluent: chloroform/methanol/water).
70:30:3) Concentrate the desired fraction and triturate with ether to obtain the title Z-Gly-Lys(Boc)-
Leu−Ser−Gln−Glu(OTB)−Leu−His−Lys
(Boc)−Leu−Gln−Thr−Tyr−Pro−Arg−
8.3 g (75%) of OEt are obtained as an amorphous powder. [α] ²⁰ _D −14.8° (c=1, DMF). Reference example (38) 7-9Trt-Cys(Trt)-Val-Leu-OMe Z-Val-Leu-OMe 37.8g in methanol 300
ml and 50 ml of 5N hydrogen chloride/methanol,
Add 4 g of 5% palladium on carbon for catalytic reduction. After the reaction, the catalyst was separated, the liquid was dried under reduced pressure, the residue was dissolved in 300 ml of DMF, and Trt-Cys(Trt)-
After adding 78 g of OSu.C ₆ H ₆ , the mixture was cooled to 0°C and 14 ml of triethylamine was added. After standing at room temperature overnight, the mixture is dried under reduced pressure, the residue is dissolved in chloroform, and the organic layer is washed with 4% citric acid, 4% sodium bicarbonate, and water in this order, and dried. The solvent was distilled off, and the residue was recrystallized from methanol to obtain the title Trt-Cys.
(Trt)-Val-Leu-OMe 71 g (85%) is obtained.
Melting point 108-9°C, [α] ²⁰ _D +22.3° (c=2, chloroform). Reference example (39) 1-2 [A] Boc-Cys(Trt)-Ser-OMe Boc-Cys(Trt)-OH・DCHA65g and H-
15.6 g of Ser-OMe.HCl is dissolved in 500 ml of chloroform, and while cooling and stirring, a solution of 20.6 g of DCC in 50 ml of chloroform is added dropwise. After the dropwise addition, the mixture was further stirred at room temperature for 15 hours, and then the insoluble matter was separated, and the liquid was washed with 4% citric acid, 4% sodium bicarbonate, and water in this order, and dried. The solvent was distilled off and the residue was recrystallized from ethyl acetate/hexane to give the title Boc-
39.5 g (70%) of Cys(Trt)-Ser-OMe are obtained.
Melting point 181-2°C, [α] ²⁰ _D +23.7° (c=1,
MeOH). Reference example (40) 1-2 [B] Boc-Cys(Trt)-Ser-
Boc−Cys(Trt)−Ser− obtained in _NHNH2 reference example (39)
Dissolve 56.4 g of OMe in 500 ml of methanol, add 25 ml of hydrazine hydrate at 0°C, leave it at 0°C for 16 hours, collect the precipitated crystals, wash with water, dry, and prepare the desired hydrazide Boc-Cys(Trt)-Ser. −
51 g (90%) of _NHNH2 are obtained. Melting point 104-8℃,
[α] ²⁰ _D +0.76° (c=1, MeOH). Reference example (41) 1-6 [A] Boc-Cys (Trt)-Ser-Asn-
Leu−Ser−Thr−OMe Hydrazide Boc−Cys obtained in Reference Example (40)
32 g of (Trt)-Ser-NHNH ₂ is dissolved in 250 ml of DMF, and while stirring at -30°C, 50 ml of 2.3N hydrogen chloride/ethyl acetate and then 8 g of isoamyl nitrite are gradually added. After 10 minutes, cool to -50°C and neutralize by adding 16.1 ml of triethylamine. On the other hand, tetrapeptide Z obtained in Reference Example (17)
33.1 g of -Asn-Leu-Ser-Thr-OMe are catalytically reduced using 1.5 g of 5% palladium on carbon in 800 ml of tert-butanol/water (9:1). After the reaction is complete, separate the catalyst and dry the liquid under reduced pressure to remove the residue.
Dissolve in 100ml of DMF and add to the above azide solution,
Continue stirring at 0°C for 20 hours. The reaction mixture was dried under reduced pressure, and 400 ml of 4% citric acid was added to the residue, thoroughly ground to remove the precipitate, washed with water, and dried. Recrystallize from DMF/water to obtain the desired Boc-
Cys(Trt)−Ser−Asn−Leu−Ser−Thr−
Obtain 42.8 g (76.7%) of OMe. Melting point 212-4℃,
[α] ²⁰ _D +0.44° (c=1, DMF). Reference example (42) 1-6 [B] Boc-Cys (Trt)-Ser-Asn-
Leu-Ser-Thr-NHNH ₂ 64.7g of the hexapeptide obtained in Reference Example (41) was
60ml of hydrazine hydrate in 550ml of DMF and 0
After reacting overnight at °C, the mixture was poured into ice water 3. The precipitate was collected, washed with methanol and then ether, and the title hydrazide Boc-Cys (Trt) was obtained.
−Ser−Asn−Leu−Ser−Thr−NHNH ₂ 56g
(86%). Melting point 205-6°C, [α] ²⁰ _D +6.2° (c=1, DMF). Reference example (43) 1-9 [A] Boc-Cys (Trt)-Ser-Asn-
Leu−Ser−Thr−Cys(Trt)−Val−Leu−
OMe Hydrazide Boc−Cys obtained in reference example (42)
(Trt) −Ser−Asn−Len−Ser−Thr−
Dissolve 29.4 g of NHNH ₂ in 350 ml of DMF and cool at -30°C.
30 ml of 2.3N hydrogen chloride/ethyl acetate while stirring with
Then 4.2 g of isoamyl nitrite is gradually added.
After 10 minutes, cool to -50℃ and add triethylamine 9.7
Neutralize by adding ml. On the other hand, the tripeptide Trt obtained in reference example (38)
-Cys(Trt)-Val-Leu-OMe 30g was dissolved in 80% acetic acid, left at room temperature for 3 hours, and then dissolved in 160 g of water.
Add ml. Separate the resulting precipitate, dry the liquid under reduced pressure, add 200 ml of saturated sodium bicarbonate solution to the residue, and extract the precipitated oil with ethyl acetate. The organic layer was dried and the solvent was distilled off, and the obtained oil was dissolved in 100 ml of DMF and added to the above azide solution. After stirring the reaction solution at 0°C for 2 days, it was dried under reduced pressure, and the residue contained 5%
The precipitate formed by adding citric acid was washed with water, dried, and then recrystallized from chloroform/methanol to obtain the desired Boc-Cys(Trt)-Ser-Asn-Leu.
−Ser−Thr−Cys(Trt)−Val−Leu−
Obtain 26.4 g (57%) of OMe. Melting point 227-230℃
(decomposition), [α] ²⁰ _D −7.7° (c=1, DMF). Reference example (44) 1-9 [B] Boc-Cys (Trt)-Ser-Asn-
Leu−Ser−Thr−Cys(Trt)−Val−Leu−
26.1g of nonapeptide obtained from NHNH ₂ reference example (43)
Dissolve in 340 ml of DMF and 170 ml of DMSO, add 63 ml of hydrazine hydrate, and leave at 0°C for 2 days. The reaction solution was poured into ice-cooled saturated saline solution 2, and the precipitate was collected, washed with water, and dried to obtain the desired hydrazide Boc-Cys(Trt)-Ser-Asn-Leu-Ser.
−Thr−Cys(Trt)−Val−Leu−NHNH ₂ 2.6g
(100%). Melting point 242-5℃ (decomposition), [α]
²⁰ _D −6.5° (c=1, DMF). Example 1 Boc-Cys(Acm)-Ser-Asn-Leu-Ser-
Thr−Cys(Acm)−Val−Leu−Gly−Lys
(Boc) −Leu−Ser−Gln−Glu(OTB)−Leu−
His−Lys(Boc)−Leu−Gln−Thr−Tyr−Pro
-Arg-OEt Protected tetradecapeptide obtained in reference example (15)
Dissolve 2.5 g in 40 ml of 80% acetic acid, add 5% palladium on carbon, and hydrogenate. After the reaction is completed, the catalyst is separated and dried under reduced pressure, and the residue is thoroughly washed with ether to form a powder. On the other hand, 2.25 g of decapeptide hydrazide obtained in Reference Example (26) was mixed with DMF-DMSO (1:1).
Dissolve in 40ml and cool to -20℃ and add 2.72N hydrogen chloride/
Add 5 ml of ethyl acetate and 0.23 ml of isoamyl nitrite, stir for 15 minutes, cool to -40°C, and neutralize by adding 1.9 ml of triethylamine. A solution of the above reduction product dissolved in 30 ml of DMF and 0.37 ml of triethylamine is added to this, and stirring is continued overnight under ice cooling. The precipitated triethylamine hydrochloride was separated and concentrated under reduced pressure, and the residue was dissolved in DMF-0.5 mol acetic acid (1:1) and added to Sephadex LH-20.
Perform gel filtration using a column. Elute with the same solvent, concentrate the desired fraction under reduced pressure, and repeat the reprecipitation from DMF/ethyl acetate to obtain 2.35 g of the title compound. [α] ²¹ _D −13.0° (c=2, DMF). Example 2 300ml of protected tetracosapeptide obtained in Example 1
Dissolve in 3ml of DMF and dilute with 30ml of methanol.
This solution is added dropwise to a solution of 400 mg of iodine dissolved in 40 ml of methanol at room temperature with vigorous stirring. After the addition was completed, the mixture was stirred for another hour, cooled on ice, and 1N sodium thiosulfate was added dropwise until the solution was decolored. Concentrate the methanol under reduced pressure, add ice water to collect the resulting precipitate, dry it, and repeat the reprecipitation from methanol/ether. This is DMF−0.5
Sephadex LH- in a system of molar acetic acid (1:1)
Perform gel filtration for 20 minutes and reprecipitate from methanol/ether to obtain 280 mg of the desired product. [α] ²⁴ _D +20.0° (c=2, DMF). Amino acid analysis: Lys1.98(2), His0.91(1), Arg0.99(1), Asp0.89(1), Thr1.82(2), Ser2.51(3), Glu2.93(3 ), Pro0.98(1), Gly1.01(1), Cys0.89(1), Val1.00(1), Leu5.07(5), Tyr1.01(1). Example 3 H-Cys(Acm)-Ser-Asn-Leu-Ser-Thr
−Cys(Acm)−Val−Leu−Gly−Lys−Len−
Ser−Gln−Glu−Leu−His−Lys−Leu−Gln
-Thr-Tyr-Pro-Arg-OH 50 mg of the protected tetracosapeptide obtained in Example 1 was added to
DMF 0.5ml and 0.5M Tris-HCl buffer (PH6.0)
Dissolve in 0.3 ml, add 2 mg of trypsin, and react at 30°C for 1 hour. The reaction solution was dried under reduced pressure, the residue was dissolved in 3 ml of trifluoroacetic acid containing 0.5 ml of anisole, reacted at room temperature for 40 minutes, and then dried under reduced pressure.
After washing the residue with ether, it was dissolved in 0.2N acetic acid.
Gel filtrate through Sephadex G-25 (2.5 x 100 cm), collect the desired fractions, and freeze-dry to obtain 39 mg of the title compound. [α] ²² _D −73.7° (c=0.76, H ₂ O). Example 4 Boc-Cys(Trt)-Ser-Asn-Leu-Ser-Thr
−Cys(Trt)−Val−Leu−Gly−Lys(Boc)−
Leu−Ser−Gln−Glu(OTB)−Leu−His−Lys
(Boc)−Leu−Gln−Thr−Tyr−Pro−Arg−
OEt Pentadecapeptide Z- obtained in reference example (37)
Gly−Lys(Boc)−Leu−Ser−Gln−Glu(OTB)
−Leu−His−Lys(Boc)−Leu−Gln−Thr−Tyr
Dissolve 6.6 g of -Pro-Arg-OEt in 150 ml of 80% acetic acid and add 5% palladium on carbon for catalytic reduction.
After the reaction is completed, the catalyst is separated, the liquid is dried under reduced pressure, and the residue is thoroughly washed with ether to form a powder. On the other hand, the nonapeptide hydrazide Boc-Cys(Trt)-Ser-Asn-Leu-Ser- obtained in Reference Example (44)
Thr−Cys(Trt)−Val−Leu−NHNH ₂ 4.6g
Dissolve in 100 ml of DMF and 100 ml of DMSO, and gradually add 10 ml of 2.3N hydrogen chloride/ethyl acetate and 0.36 g of isoamyl nitrite while stirring at -10°C. After 15 minutes, cool to -50°C and neutralize with 3.2 ml of triethylamine. A solution prepared by dissolving the above reduction product in 50 ml of DMF and adding 0.42 ml of triethylamine was added thereto, and stirring was continued at 0° C. for 16 hours. Insoluble matter was separated, the liquid was dried under reduced pressure, and the residue was applied to a silica gel column (eluent: chloroform/methanol/
Water (70:30:3), concentrate the desired fraction and powder with ether, 6.9 g of the title tetracopesapeptide.
(64%). [α] ²⁰ _D −8.0° (c=1, DMF). Example 5 Dissolve 740 mg of the tetracosapeptide obtained in Example 4 in 50 ml of methanol, and add 510 mg of iodine to this solution.
is added dropwise to a solution of 50 ml of methanol at 20°C while stirring. After 5 minutes, cool on ice, add 1N sodium thiosulfate dropwise to decolorize, and pour into ice water 1. The resulting precipitate is collected by centrifugation, dissolved in ethanol, and concentrated three times. Finally, dissolve it in ethanol and add ether to remove the precipitate and dry it.
Obtain 520 mg (83%) of the title target compound [α] ²⁰ _D +
19.8° (c=1, DMF). This compound was found to be identical to the product of Example 2 from analysis by high performance liquid chromatography and thin layer chromatography. Example 6 100 mg of the tetracosapeptide obtained in Example 5
1ml of DMF and 0.5M Tris-HCl buffer (PH6.0)
Add 4 mg of trypsin and react for 1 hour at room temperature. Load the reaction solution as it is on a Sephadex LH-20 (2.5 x 100 cm) column,
Elute with DMF/0.5M acetic acid (1:1). Collect the desired fractions, concentrate under reduced pressure, wash with ether,
90 mg of the desired title compound are obtained as a white powder. [α] ²⁰ _D +6.2° (c=1, DMF). Example 7 1 g of the tetracosapeptide obtained in Example 5 is dissolved in 30 ml of trifluoroacetic acid containing 3 ml of anisole, left at room temperature for 30 minutes, and then concentrated under reduced pressure. Dissolve the residue in 30 ml of 0.2N acetic acid, add 50 ml of ether, and mix. After separating the aqueous layer and concentrating it under reduced pressure to about half the volume, Sephadex G-25 (2.5 x 100 cm)
Collect the desired fractions and concentrate under reduced pressure.
Powder with ethanol/ether gives 730 mg of the title unprotected tetracosapeptide ester. [α] ²⁰ _D −13.5° (c=0.25, 50% AcOH). Example 8 90 mg of the protected tetracosapeptide obtained in Example 6 was dissolved in 5 ml of trifluoroacetic acid containing 1 ml of anisole, reacted for 40 minutes at room temperature, concentrated under reduced pressure, and the residue was washed with ether. This is dissolved in 0.2N acetic acid and gel-filtered through Sephadex G-25 (2.5 x 100 cm), and the desired fractions are collected and lyophilized to obtain 78 mg of the title completely unprotected tetracosapeptide. [α] ²⁰ _D −13.2° (c=0.25, 50% AcOH). Reference example (45) 25-32 [A] Z-Thr-Asn-Thr-Gly-
Ser−Gly−Thr−Pro−NH ₂ Z−Thr−Asn−Thr−Gly−NHNH ₂ 5.4g
Dissolved in 50ml of DMF, cooled to -20℃ and made 2.70N.
Hydrogen chloride/ethyl acetate 20ml and isoamyl nitrite
Add 1.55 ml of triethylamine, stir at -20°C for 10 minutes, cool to -40°C, and add 7.56 ml of triethylamine.
Add to neutralize. Then H-Ser-Gly-
A solution of 3.77 g of Thr-Pro-NH ₂ dissolved in 10 ml of DMF is added, and stirring is continued overnight under ice cooling. The precipitated triethylamine hydrochloride was separated and concentrated under reduced pressure, and the residue was repeatedly reprecipitated from methanol to obtain 3.5 g of the desired product. [α] ²² _D −32.0 (c=1,
DMF). Reference example (46) 25-32 [B] H-Thr-Asn-Thr-Gly-
Ser-Gly-Thr-Pro-NH ₂ AcOH 2 g of the protected octapeptide amide obtained in Reference Example (45) was added to 5% palladium-carbon in 40 ml of 80% acetic acid.
Catalytic reduction is carried out in the presence of 0.4g. After the reaction was completed, the catalyst was separated and dried under reduced pressure, and the residue was washed with ether to obtain 1.7 g of the desired product in powder form. [α] ²² _D −45.3゜(c
= 2, 80% AcOH). Reference example (47) Protected tetracosapeptide 160 obtained in Example 2
mg and octapeptide amide obtained in reference example (46)
Dissolve 315mg of trypsin in 2.5ml of DMF,
Add 1.6 ml of 0.3 M Tris-HCl buffer (PH6.0) containing 0.1 mg of TPCK and 0.1 mg of TPCK, and stir at room temperature for 1.5 hours. Stop the reaction by adding 2 ml of glacial acetic acid.
After adding 2 ml of DMF, Cephadex LH-
20 (2.5 x 100 cm) column and DMF/0.5
Elute with molar acetic acid (1:1). The desired fractions are collected and concentrated under reduced pressure, and the residue is washed with ether to obtain 72 mg of the desired protected dotriacontapeptide amide as a white powder. [α] ²⁸ _D −31.6゜(c=
2, DMF/ _H2O (7:3)). Reference example (48) Dissolve 100 mg of the protected dotriacontapeptide amide obtained in Reference Example (47) in 3.5 ml of trifluoroacetic acid containing a small amount of anisole and leave to stand at room temperature for 45 minutes, then add ether to obtain about 100 mg of precipitate.
Dissolve this in 1N acetic acid and use Amberlite IRA.
Pass through a small column of -410 (acetic acid form) and lyophilize the effluent. This was dissolved in 0.2N acetic acid and gel-filtered through Sephadex G-25 (3.8 x 50cm).
The desired fraction is lyophilized to obtain 55 mg of the desired dotriacontapeptide amide. [α] ²⁶ _D −41.0° (c=1, 50% AcOH). Amino acid analysis: Lys1.99(2), His1.01(1), Arg1.01(1), Asp2.07(2), Thr4.67(5), Ser3.61(4), Glu3.03(3 ), Pro2.15(2), Gly2.92(3), Cys0.91(1), Val1.02(1), Leu5.00(5), Tyr1.00(1).

Claims

[Claims] 1 formula In the formula, R represents a hydrogen atom or a lower alkyl group, X ₁ and X ₂ each represent a thiol protecting group, or X ₁ and X ₂ together represent a single bond, and Y ₁ , Y ₄ and Y ₅ each represent a hydrogen atom or a protecting group. 2 X ₁ and X ₂ each represent a trityl group or an acetamidomethyl group, or X ₁ and X ₂ together represent a single bond, Y ₁ is a hydrogen atom, t
- represents a butyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, or trityl group, Y ₄ represents a hydrogen atom, t-butyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, or trityl group, Y ₅ represents a hydrogen atom or t
The peptide according to claim 1, which represents a -butyl group.