JP3600753B2 - Novel linker and solid-phase synthesis of glycopeptides using it - Google Patents

Description

【００１０】
（式中Ｒ^１ ，Ｒ^２ ，Ｒ^３ およびＲ^４ は、同一または相異なり、それぞれ置換基を有していてもよいアルキル基、アルケニル基、シクロアルキル基、シクロアルケニル基またはアリール基を示し、Ｘはハロゲン原子を示す）
で表わされるｐ−ニトロフェニルカルビニルシリルハライド化合物を提供する。
また、この出願の発明は、上記化合物について、第２には、Ｒ^１ およびＲ^２ の少くとも一方がメチル基である化合物を、第３には、Ｒ^３ およびＲ^４ の少くとも一方がフェニル基である化合物を、第４には、Ｘが塩素原子である化合物を、第５には、Ｒ^１ ，Ｒ^２ ，Ｒ^３ およびＲ^４ がメチル基を、Ｘが塩素原子である化合物を提供する。
【００１１】
この出願の発明は、第６には、一般式（ＩＩ）
【００１２】
【化６】

【００１３】
（式中Ｒ^１ ，Ｒ^２ ，Ｒ^３ およびＲ^４ は、同一または相異なり、それぞれ置換基を有していてもよいアルキル基、アルケニル基、シクロアルキル基、シクロアルケニル基またはアリール基を示し、Ｘはハロゲ原子を示す）
で表わされるフェニルカルビニルシリルハライド化合物を加水分解後、ニトロ化し、次いでハロゲン化することを特徴とする前記第１の発明のｐ−ニトロフェニルカルビニルシリルハライド化合物の製造方法を提供する。
【００１４】
そして、この出願の発明は、第７には、固相合成担体にシリルリンカーを介して糖鎖またはアミノ酸を固定させ、糖ペプチドまたはペプチド合成した後に、ケイ素−酸素結合を切断して糖またはアミノ酸を取り出す方法において、糖またはアミノ酸のアルコール性水酸基を前記第１の発明のｐ−ニトロフェニルカルビニルシリルハライド化合物で反応保護し、ニトロ基を還元した後にジカルボン酸無水物と縮合させてジカルボン酸モノアミド体としたものをシリルリンカーとして使用することを特徴とする糖ペプチドまたはオリゴペプチドの合成方法を提供する。
【００１５】
さらにまた、この出願の発明は、上記合成方法について、第８には、ジカルボン酸無水物として無水コハク酸または無水グルタル酸を用いる方法を、第９には、ケイ素−酸素結合をフッ素アニオンによって切断する方法を、第１０には、フッ化セシウムとともに、１８−クラウン−６または酢酸とを用いてケイ素−酸素結合を切断する方法をも提供する。
【００１６】
すなわち、以上のとおりのこの出願の発明は、発明者らによる糖鎖を介して固相合成担体に固定できるような合成素子についての検討から導かれたものであって、シリルエーテルを含む糖ペプチドユニットを合成し、そのシリル化合物の置換基を介して固相と連結させるべくリンカーとなる構造部分の検討と、糖ペプチド等の固相合成方法の検討の結果、立体障害の大きい、かさ高い置換基を有する新規なｐ−ニトロフェニルカルビニルシリルハライド化合物が、上記問題点を解決し、糖ペプチドあるいはペプチドの固相合成のリンカーとして有用であることを見いだしたことに基づいて完成されたものである。
【００１７】
【発明の実施の形態】
この出願の発明は以上のとおりの特徴を有するものであるが、以下に、その実施の形態について説明する。
まず、この発明において提供される前記の一般式（Ｉ）で表わされるｐ−ニトロフェニルカルビルシリルハライド化合物については、Ｒ^１ ，Ｒ^２ ，Ｒ^３ およびＲ^４ は、炭化水素基としてのアルキル基、アルケニル基、シクロアルキル基、シクロアルケニル基、またはアリール基、もしくは置換されたこれらの炭化水素の各種のものであってよいが、一般的には、炭素数が１〜８程度のものが適当であり、なかでも、メチル基、エチル基、プロピル基、イソプロピル基、ｎ−ブチル基、イソブチル基、ペンチル基等の低級アルキル基、またはフェニル基、トリル基等のアリール基から選択されたものが適当なものとして例示される。
【００１８】
またＸはハロゲン原子であるが、反応性、合成および取扱い性等の面からは塩素原子とすることが適当である。
たとえば、具体的に、一般式（Ｉ）の化合物を例示すると、（ｐ−ニトロフェニルジメチルカルビニル）ジメチルシリルクロリド、（ｐ−ニトロフェニルジメチルカルビニル）メチルエチルシリルクロリド、（ｐ−ニトロフェニルジメチルカルビニル）ジビニルシリルクロリド、（ｐ−ニトロフェニルジメチルカルビニル）ジフェニルシリルクロリド、（ｐ−ニトロフェニルジメチルカルビニル）メチルフェニルシリルクロリド、（ｐ−ニトロフェニルジメチルカルビニル）メチルトリルシリルクロリド、（ｐ−ニトロフェニルジエチルカルビニル）ジメチルシリルクロリド、（ｐ−ニトロフェニルフェニルメチルカルビニル）ジメチルシリルクロリド、（ｐ−ニトロフェニルメチルプロピルカルビニル）ジメチルシリルクロリド等が挙げられる。
【００１９】
なかでも、（ｐ−ニトロフェニルジメチルカルビニル）ジメチルシリルクロリド、（ｐ−ニトロフェニルジメチルカルビニル）メチルエチルシリルクロリド、（ｐ−ニトロフェニルジメチルカルビニル）ジエチルシリルクロリドが適当なものとして例示される。
たとえば以上のとおりのこの発明の前記一般式（Ｉ）で表わされるｐ−ニトロフェニルカルビルシリルハライド化合物は、シリルエーテル型リンカーを形成するものとして有用な新規な化合物である。
【００２０】
このｐ−ニトロフェニルカルビルシリルハライド化合物は、この発明においては、前記の一般式（ＩＩ）のフェニルカルビルシリルハライド化合物を原料とし、このものを加水分解し、ニトロ化およびハロゲン化反応させることによって製造する。この方法を反応式として示すと次の一般式（ＩＩＩ） のとおりとなる。
【００２１】
【化７】

【００２２】
原料となるフェニルカルビニルシリルハライド化合物としては、（フェニルジメチルカルビニル）ジメチルシリルクロリド、（フェニルジメチルカルビニル）メチルエチルシリルクロリド、（フェニルジメチルカルビニル）ジビニルシリルクロリド、（フェニルジメチルカルビニル）ジフェニルシリルクロリド、（フェニルジメチルカルビニル）メチルフェニルシリルクロリド、（フェニルジメチルカルビニル）メチルトリルシリルクロリド、（フェニルジエチルカルビニル）ジメチルシリルクロリド、（ジフェニルメチルカルビニル）ジメチルシリルクロリド、（フェニルメチルプロピルカルビニル）ジメチルシリルクロリドが例示される。好ましくは（フェニルジメチルカルビニル）ジメチルシリルクロリド、（フェニルジメチルカルビニル）メチルエチルシリルクロリド、（フェニルジメチルカルビニル）ジエチルシリルクロリドが使用される。
【００２３】
原料としてのフェニルカルビニルシリルハライド化合物の加水分解、ニトロ化、ハロゲン化には化学反応の常法が適宜に採用されるが、たとえば加水分解ではアルカリ水溶液による加水分解方法が、ニトロ化では硝酸アンモニウムとトリフルオロ酢酸無水物を用いたニトロ化方法が、ハロゲン化ではハロゲン化オキザリルを用いたハロゲン化方法が例示される。
【００２４】
また、この発明においては、前記一般式（Ｉ）で表わされるｐ−ニトロフェニルカルビニルシリルハライド化合物によって糖ペプチドあるいはオリゴペプチドの固相合成のためのシリルリンカーが提供されるが、このシリルリンカーは、たとえば次式
【００２５】
【化８】

【００２６】
（Ｒ^１ ，Ｒ^２ ，Ｒ^３ およびＲ^４ は、前記と同一のものを示し、Ａは、糖またはアミノ酸のアルコール性水酸基部位の残基を、Ｂはジカルボン酸の残基を示す）
で表わすことができる。
たとえば上記のとおりのこの発明のシリルリンカーは、前記の一般式（Ｉ）で表わされるｐ−ニトロフェニルカルビルシリルハライド化合物を糖もしくはアミノ酸のアルコール性水酸基と反応させてシリル−エーテル結合による保護構造を形成し、次いでニトロ基を還元してアミノ基とし、ジカルボン酸無水物と縮合反応させてジカルボン酸モノアミド体とすることにより形成される。
【００２７】
より具体的には、たとえば、固相合成に用いる出発物質ないし中間体として、有機化合水酸基を有する糖鎖またはアミノ酸を出発物質または中間体として用い、前記一般式（Ｉ）のｐ−ニトロフェニルカルビニルハライドを溶液中で脱酸剤の存在下に反応させると対応するシリルエーテルを容易に得ることができる。この反応は通常のシリル化反応の条件で実施できるが、ヨウ化ナトリウム等の反応促進剤の存在下で反応を実施することが望ましい。得られたシリルエーテルは化学的に安定で、クロマトグラフィー等の方法で容易に精製が可能であり、ＮＭＲ等で生成物の構造確認も容易にできる。
【００２８】
次いで得られたシリルエーテルのニトロ基は極めて穏和な条件、たとえば、亜鉛末の存在下で還元でき、対応するアニリン誘導体を得ることができる。この還元反応は基質の構造に影響されずに進行し、目的とするアニリン誘導体を得ることができる。
そして、得られたアニリン誘導体を環状の酸無水物、たとえば、コハク酸無水物やグルタル酸無水物と反応させると容易にアミド結合を有するモノカルボン酸（ジカルボン酸モノアミド）を得ることができる。もちろん、酸無水物は各種のものであってよい。このようにして前記のシリルエーテルは製造することができる。
【００２９】
このシリルエーテルは、適当な固相合成担体に固定されたアミノ酸のアミノ基と縮合剤を用いて反応させることによってアミド結合を形成し結合できる。
この発明で得られるシリルエーテル型縮合をもつリンカーは、従来のペプチド固相合成では行われなかったセリンやスレオニンの水酸基を利用して固相に結合し、縮合を繰り返してペプチド鎖の伸長を行った後に、Ｎ末端アミノ基、Ｃ末端カルボキシル基および側鎖の官能基に配した保護基を損なうことなく特異的に合成ペプチドを取りだすことを可能とする。さらにセリンやスレオニンばかりでなく糖鎖に多数存在する水酸基の一個所を固相への結合点とすることができる。この発明のシリルリンカーを用いる固相合成法によれば、ペプチド主鎖の両端がそれぞれの保護基を選択的に除去することによってペプチド合成の反応点として使用可能となることから、従来ほとんど試みられなかった固相上でのペプチドＣ末端側伸長も行うことができる。
【００３０】
この発明によるシリルリンカーはその調製が容易であること、切断時には全て樹脂上に残るという特性を持ち、目的生成物との分離は濾過操作のみという利点がある。また、力価が既知の市販のアミノ型樹脂（またはアミノ酸結合型樹脂）と結合することにより、ニンヒドリン法やＦｍｏｃ定量法などによってその結合効率の数値を容易に知ることができる。
【００３１】
この発明において、固定化反応の定量については特に特別な方法は必要なく、例えば、ニンヒドリンを用いた残存アミノ基の検出試験によって極めて容易に感度よく検出できる。
そして、この発明においては、特別な反応条件は必要なく固相合成反応を行うことができ、前記のとおり、Ｎ−末端、Ｃ−側酸末端の一方または双方にペプチド鎖を伸長させることができる。
【００３２】
固相合成を完成した後で特異的にケイ素−酸素結合を切断することで保護された糖ペプチドやオリゴペプチドが取り出せるが、通常のシリル系保護基の脱保護条件で固相上から遊離させることができる。フッ素イオンを発生する化合物、たとえば、フッ化セシウムにより、たとえば酢酸やクラウンエーテルの存在下または非存在下に反応を実施すると、極めて容易に定量的に切断することができる。
【００３３】
【実施例】
次にこの発明を実施例によりさらに具体的に説明するが、この発明はこれら実施例に限定されるものではない。
（実施例１）
＜（ｐ−ニトロフェニルジメチルカルビニル）ジメチルシリルクロリドの製造＞
（フェニルジメチルカルビニル）ジメチルシリルクロリド２６．５７ｇをジエチルエーテル７５ｍｌに溶解し、水酸化カリウム８．４ｇを溶解した水−メタノールの１：４（ｖ／ｖ）混合溶媒１００ｍｌを徐々に添加、攪拌した。混合溶液を室温で２０時間攪拌し、分液したのちジエチルエーテルで２度抽出し、エーテル溶液を洗浄、乾燥後、溶媒を減圧留去し無色油状物質として、２２．８９ｇの（フェニルジメチルカルビニル）ジメチルシラノールを得た。
【００３４】
このシラノール５．００ｇをアセトニトリル３５ｍｌに溶解し、硝酸アンモニウム２．６０ｇを加え、懸濁液を調整し、攪拌しながらそれにトリフルオロ酢酸無水物５．８ｍｌを少量ずつ加えた。添加終了後、さらに５０分後と６０分後にトリフルオロ酢酸無水物各２．０ｍｌずつ添加し、室温で一晩攪拌した。
得られた反応液に水を加え酢酸エチルで２回抽出した後、有機相を洗浄、乾燥し、溶媒を減圧で留去し、シリカゲルクロマトグラフィーで精製して無色結晶性化合物として３．９９ｇの（ｐ−ニトロフェニルジメチルカルビニル）ジメチルシラノールを得た。
【００３５】
次いで、この（ｐ−ニトロフェニルジメチルカルビニル）ジメチルシラノール３．５８ｇを塩化メチレン５０ｍｌに溶解し、塩化オキサリル１．５０ｍｌを加えた後、ジメチルフォルムアミド１滴加え、室温で１日攪拌した。反応液を減圧濃縮し、得られた結晶性残渣をヘキサンより再結晶し、無色結晶として、３．５６ｇの（ｐ−ニトロフェニルジメチルカルビニル）ジメチルシリルクロリド（ｍｐ．１１４．５−１１７℃（ヘキサン））を得た。
（実施例２）
実施例１の（フェニルジメチルカルビニル）ジメチルシリルクロリドを（フェニルジメチルカルビニル）メチルエチルシリルクロリドに変えて同様の反応を行うことにより、同等の収率で（ｐ−ニトロフェニルジメチルカルビニル）メチルエチルシリルクロリドが得られた。
【００３６】
また、同様の方法を実施することにより、以下の化合物が得られた。
（ｐ−ニトロフェニルジメチルカルビニル）ジビニルシリルクロリド、
（ｐ−ニトロフェニルジメチルカルビニル）ジフェニルシリルクロリド、
（ｐ−ニトロフェニルジメチルカルビニル）メチルフェニルシリルクロリド、
（ｐ−ニトロフェニルジメチルカルビニル）メチルトリルシリルクロリド、
（ｐ−ニトロフェニルジエチルカルビニル）ジメチルシリルクロリド、
（ｐ−ニトロフェニル−フェニルメチルカルビニル）ジメチルシリルクロリド、
（ｐ−ニトロフェニルメチルプロピルカルビニル）ジメチルシリルクロリド
（実施例３）
＜シリルリンカーの合成＞
次の反応式に従ってシリルリンカーの合成を行った。
【００３７】
【化９】

【００３８】
第１工程：シリルエーテルの製造
＜Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（４−ニトロフェニルジメチルカルビニル）ジメチルシリル〕−Ｌ−セリン アリルエステル＞
Ｎ−（９−フルオレニルメトキシカルボニル）−Ｌ−セリン アリルエステル（５００ｍｇ，１．３６ｍｍｏｌ）と（ｐ−ニトロフェニルジメチルカルビニル）ジメチルシリルクロリド（４２０ｍｇ，１．６３ｍｍｏｌ）およびヨウ化ナトリウム（６１０ｍｇ，４．０６ｍｍｏｌ）にジメチルホルムアミド（２０ｍｌ）を加え、室温で１５分反応させた。さらにＮ−メチルモルホリン（１７９μｌ，１．６２ｍｍｏｌ）を加えた後、２０分間反応させた。反応液をエーテル−飽和重曹水で分配し、有機層を飽和食塩水で洗浄した後、無水硫酸ナトリウムで乾燥した。減圧濃縮して得られた残渣をシリカゲルカラムクロマトグラフィー（ＳｉＯ_２ ６０ｇ，ｈｅｘａｎｅ−ＥｔＯＡｃ２：１）で精製し標記化合物を無色油状物として７７５ｍｇ（９７％）得た。
【００３９】
第２工程：アニリン誘導体の製造
＜Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（４−アミノフェニルジメチルカルビニル）ジメチルシリル〕−Ｌ−セリン アリルエステル＞
Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（４−ニトロフェニルジメチルカルビニル）ジメチルシリル〕−Ｌ−セリン アリルエステル（７８０ｍｇ，１．３２ｍｍｏｌ）をテトラヒドロフラン（２０ｍｌ）に溶解し、亜鉛末（５．００ｇ，ｅｘｃｅｓｓ）および酢酸（１ｍｌ）を加えて４５分間反応させた。反応液を酢酸エチルで希釈し、セライトの層を通して濾過した後、飽和重曹水、飽和食塩水で順次洗浄し、無水硫酸ナトリウムで乾燥した。減圧濃縮して得られた残渣をシリカゲルカラムクロマトグラフィー（ＳｉＯ_２ ６０ｇ，ｈｅｘａｎｅ−ＥｔＯＡｃ１：１）で精製し標記化合物を無色油状物として６５０ｍｇ（８８％）得た。
【００４０】
第３工程：アミドカルボン酸の製造
＜Ｎ−（９−フルオレニルメトキシカルボニル）｛Ｏ−〔（４−スクシン酸モノアミドフェニル）ジメチルカルビニル〕ジメチルシリル｝−Ｌ−セリン アリルエステル＞
９−フルオレニルメトキシカルボニル〔Ｏ−（４−アミノフェニルジメチルカルビニル）ジメチルシリル〕−Ｌ−セリン アリルエステル（６５０ｍｇ，１．１６ｍｍｏｌ）にコハク酸無水物（１２０ｍｇ，１．２０ｍｍｏｌ）とＮ−メチルモルホリン（１１１μｌ，０．８９ｍｍｏｌ）を加え、４時間反応させた。反応液を濃縮しＢｉｏｂｅａｄｓ ＳＸ−３（１００ｇ）のカラムに展着し、トルエン−酢酸エチル（１：１）の混合溶媒で溶出して精製し、標記化合物を７６０ｍｇ（９９％）得た。
（実施例４）
＜樹脂への導入＞
実施例３により得られたシリルリンカーを樹脂に導入して次式のとおり固定化した。
【００４１】
【化１０】

【００４２】
すなわち、Ｎ−（９−フルオレニルメトキシカルボニル）｛Ｏ−〔（４−スクシン酸モノアミドフェニル）ジメチルカルビニル〕ジメチルシリル｝−Ｌ−セリン アリルエステル（２５０ｍｇ，０．３８ｍｍｏｌ）のＮ−メチルモルホリン（３．０ｍｌ）溶液に、Ｏ−ベンゾトリアゾ−１−リル−１−Ｎ，Ｎ，Ｎ′，Ｎ′−テトラメチルウロニウムヘキサフルオロホスフェート（ＨＢＴＵ：２８８ｍｇ，０．７６ｍｍｏｌ）と０．５Ｍ １−ヒドロキシベンゾトリアゾール（ＨＯＢｔ：ジメチルホルムアミド溶液，０．７６ｍｌ，０．３８ｍｍｏｌ）および２Ｍジイソプロピルエチルアミン（ジメチルホルムアミド溶液，０．１９ｍｌ，０．３８ｍｍｏｌ）を加え、室温で１時間２０分反応させた。ついでグリシンＨＭＰ樹脂（ＡＢＩ製，０．９５ｍｍｏｌ／ｇ，２７１ｍｇ，０．２５３ｍｍｏｌ）を加え、一晩攪拌混和した。得られた樹脂を濾別し、Ｎ−メチルモルホリン，２−プロパノール、塩化メチレン、ジエチルエーテルで順次洗浄し、４３９ｍｇ（１０２％：重量よりの計算）の樹脂を得た。
（実施例５）
＜ペプチド固相合成＞
次の反応式に従って、実施例４に続いてペプチドの固相合成を行った。
【００４３】
【化１１】

【００４４】
９−フルオレニルメトキシカルボニル基（Ｆｍｏｃ）の除去（１）
Ｎ−（９−フルオレニルメトキシカルボニル）セリン（Ｏ−リンカー−グリシンＨＭＰ樹脂）アリルエステル（０．５９ｍｍｏｌ／ｇ，１５１ｍｇ，０．０８９ｍｍｏｌ）をＮ−メチルモルホリン−ピペリジン（１：１ｖ／ｖ，３．０ｍｌ）に懸濁し、１時間４０分攪拌混和した。得られた樹脂を濾別し、Ｎ−メチルモルホリン，２−プロパノール、塩化メチレン、ジエチルエーテルで順次洗浄し、１３４ｍｇの樹脂を得た。
【００４５】
Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕トレオニンの導入（２）
Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕−Ｌ−トレオニン（３３ｍｇ，０．０４９２ｍｍｏｌ）のＮ−メチルモルホリン（１．０ｍｌ）溶液に、Ｏ−ベンゾトリアゾ−１−リル−１−Ｎ，Ｎ，Ｎ′，Ｎ′−テトラメチルウロニウムヘキサフルオロホスフェート（３７．３ｍｇ，０．０９８ｍｍｏｌ），１−ヒドロキシベンゾトリアゾール（６６ｍｇ，０．４９ｍｍｏｌ）および２Ｍジイソプロピルエチルアミン（ジメチルホルムアミド溶液，０．４９μｌ，０．０９８ｍｍｏｌ）を加え３５分間反応させた。ついでセリン（Ｏ−リンカー−グリシンＨＭＰ樹脂）アリルエステル（０．６８ｍｍｏｌ／ｇ，６６ｍｇ，０．０４４７ｍｍｏｌ）を加え一晩攪拌混和した。得られた樹脂を濾別し、Ｎ−メチルモルホリン，２−プロパノール、塩化メチレン、ジエチルエーテルで順次洗浄し、８８．５ｍｇ（９６％：重量よりの計算）の樹脂を得た。
【００４６】
９−フルオレニルメトキシカルボニル基（Ｆｍｏｃ）の除去（３）
Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕−Ｌ−トレオニル−Ｌ−セリン（Ｏ−リンカー−グリシンＨＭＰ樹脂）アリルエステル（０．４７ｍｍｏｌ／ｇ，８４．３ｍｇ，０．０４２５ｍｍｏｌ）をＮ−メチルモルホリン−ピペリジン（１：１ｖ／ｖ，２．０ｍｌ）に懸濁、１時間２０分攪拌混和した。得られた樹脂を濾別し、Ｎ−メチルモルホリン，２−プロパノール、塩化メチレン、ジエチルエーテルで順次洗浄し、８０．４ｍｇの樹脂を得た。
【００４７】
Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕トレオニンの導入（４）
Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕−Ｌ−トレオニン（３３ｍｇ，０．０４９２ｍｍｏｌ）のＮ−メチルモルホリン（１．０ｍｌ）溶液に、Ｏ−ベンゾトリアゾ−１−リル−１−Ｎ，Ｎ，Ｎ′，Ｎ′−テトラメチルウロニウムヘキサフルオロホスフェート（３７．３ｍｇ，０．０９８ｍｍｏｌ），１−ヒドロキシベンゾトリアゾール（６６ｍｇ，０．４９ｍｍｏｌ）および２Ｍジイソプロピルエチルアミン（ジメチルホルムアミド溶液，０．４９μｌ，０．０９８ｍｍｏｌ）を加え３５分間反応させた。ついで〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕−Ｌ−トレオニル−Ｌ−セリン（Ｏ−リンカー−グリシンＨＭＰ樹脂）アリルエステル（０．５３ｍｍｏｌ／ｇ，８０．４ｍｇ，０．０４２５ｍｍｏｌ）を加え一晩攪拌した。上記（２）と同様の処理により１１２ｍｇ（１０２％：重量よりの計算）の樹脂を得た。
【００４８】
糖ペプチドの樹脂からの切り出し（５）
Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕−Ｌ−トレオニル−Ｌ−Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕−Ｌ−トレオニル−Ｌ−セリン（Ｏ−リンカー−グリシンＨＭＰ樹脂）アリルエステル（０．３９ｍｍｏｌ／ｇ，７７．４ｍｇ，０．０３０２ｍｍｏｌ）を０．２ｍｏｌ／ｌフッ化セシウム−１．０ｍｏｌ／ｌ酢酸ＤＭＦ溶液に懸濁し、一晩攪拌混和した。得られた樹脂を濾別し、Ｎ−メチルモルホリンで洗浄した後、２−プロパノール、塩化メチレン、ジエチルエーテルで順次洗浄した。２−プロパノール、塩化メチレン、ジエチルエーテルの洗液を濃縮しＮ−メチルモルホリンの洗液とあわせて酢酸エチルで希釈し、水と分配した。水層をさらに酢酸エチルで３回抽出したのち、有機層をあわせて飽和食塩水で洗浄、ついで無水硫酸ナトリウムで乾燥してから濃縮した。残渣を分取薄層クロマトグラフィー（１ｍｍ ｌａｙｅｒ，ＣＨＣｌ_３ ／ＭｅＯＨ９：１）で精製し、Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕−Ｌ−トレオニル−Ｎ−（９−フルオレニルメトキシカルボニル）〔Ｏ−（２−アセタミド−３，４，６−トリ−Ｏ−アセチル−２−デオキシ−α−Ｄ−ガラクトピラノシル〕−Ｌ−トレオニル−Ｌ−セリン アリルエステルを２７．２ｍｇ（７３％）得た。
【００４９】
【発明の効果】
この発明の（ｐ−ニトロフェニルカルビニル）シリルハライド化合物は固相合成のリンカーの形成に有用な化合物であり、糖またはアミノ酸のアルコール性水溶液と定量的に結合してシリルエーテル結合を形成し、さらに数段階を経て固相上に必要な化合物を固定することができる。このリンカーを使用した固相有機合成は機械的に進められ、コンビナトリアルケミストリーとして反応を展開後に穏和な条件で固相から離脱させることによって、酵素阻害剤となるオリゴペプチドやリセプターとなるペプチドホルモン等の生理活性糖ペプチドあるいはペプチドを得ることができる。[0001]
TECHNICAL FIELD OF THE INVENTION
The invention of this application relates to a novel linker and a solid phase synthesis method of a glycopeptide using the same.
More specifically, the invention of this application relates to a p-nitrophenylcarbinylsilyl halide compound which forms a novel linker having a large steric hindrance and a bulky substituent, a method for producing the same, and a method for preparing the same with a glycopeptide or a solid peptide. The present invention relates to a method used as a linker for phase synthesis.
[0002]
[Prior art and its problems]
In the currently common peptide solid-phase synthesis method, the carboxyl group of the amino acid serving as the C-terminal of the peptide is connected to a linker previously fixed to the solid phase by an ester bond, and then the required number of amino acid units are sequentially bonded in the N-terminal direction. In this method, a peptide chain is extended, and finally, an oligopeptide is cut out from a solid phase together with removal of a protecting group of an amino acid side chain. This cutting operation is generally performed under strong acidic conditions such as trifluoroacetic acid and hydrogen fluoride.
[0003]
However, since the acidic cutting-out operation in the conventional general method uses a strong acid, attention must be paid to its handling, which is not practical. Accordingly, linkers that can be cleaved under weaker acidic conditions have also been developed. In this case, it becomes possible to take out the oligopeptide while keeping the side chain protecting group. However, it is important to carefully consider the setting of this weak acid condition. Resins in which various amino acids are bound to a solid phase via a residue linker for these techniques have already been marketed, but the problem that the operation is not always easy has not been solved.
[0004]
In such a situation, linkers that do not require acid conditions when cleaving the solid phase and the synthetic peptide have also been developed.One of them is to use the affinity between the fluorine anion and silicon for the cleavage reaction. Several types of derived silicon-containing linkers are known (DG Mullen, G. Barany, J. Org. Chem. 1988, 53, 5240). However, those developed for peptide synthesis only use a silicon leaving group as a part of the substituent in the linker connecting the carboxyl group of the amino acid in an ester form.
[0005]
Furthermore, in recent years, attempts have been made to increase the efficiency of the synthesis of sugar chains using solid-phase synthesis, or to develop combinatorial chemistry. An example is known in which a sugar residue is immobilized by a silicon-oxygen bond between a hydroxyl group of the residue and cleavage with a fluorine anion after sugar chain elongation (JT Randolph, KF McClure). , SJ Danishefsky, J. Am. Chem. Soc, 1995, 117, 5712). However, in this case, it is difficult to directly detect and estimate the number of moles of the sugar residue immobilized on the resin. Glycoprotein research is prosperous and elucidation of the structure and function of glycoproteins composed of proteins and sugar chains and application of glycoprotein molecules to pharmaceuticals and agricultural chemicals are expected. There is also a need to establish a technology for precise synthesis. For this purpose, glycopeptides, which are small fragments to be used as components of glycoprotein molecule construction, are efficiently synthesized by protecting the functional groups on their side chains such as the sugar chain hydroxyl group, and then efficiently synthesized by block condensation or the like. Techniques for assembling into large molecules by technology are considered practical.
[0006]
However, although the silyl linkers developed for the conventional peptide synthesis as described above have the advantage that they can be cleaved out without depending on acidic conditions, their preparation is complicated, and some of the linkers are cleaved at the time of cleaving. Some of them have a disadvantage that they are mixed into the target product as by-products. In addition, in order to use these linkers, it is usually necessary to bind the required intermediate to the solid-phase synthesis support beforehand, and then immobilize it. However, there has been no definitive method for verifying this immobilization.
[0007]
Therefore, an object of the invention of the present application is to provide an excellent linker capable of overcoming these conventional problems in a solid phase synthesis reaction, and to use it as a linker for solid phase synthesis of a glycopeptide or a peptide.
[0008]
[Means for Solving the Problems]
The invention of this application solves the above-mentioned problems, and firstly, the general formula (I)
[0009]
Embedded image

[0010]
(Where R ¹ , R ² , R ³ And R ⁴ Are the same or different and each represents an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group which may have a substituent, and X represents a halogen atom)
A p-nitrophenylcarbinylsilyl halide compound represented by the formula:
Further, the invention of this application relates to the above compound, ¹ And R ² And at least one of them is a methyl group. ³ And R ⁴ A compound in which at least one is a phenyl group, a fourth compound in which X is a chlorine atom, and a fifth compound in which R is ¹ , R ² , R ³ And R ⁴ Is a methyl group and X is a chlorine atom.
[0011]
Sixth, the invention of the present application provides a compound represented by the general formula (II):
[0012]
Embedded image

[0013]
(Where R ¹ , R ² , R ³ And R ⁴ Are the same or different and each represents an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group or an aryl group which may have a substituent, and X represents a halogen atom)
Wherein the phenylcarbylsilyl halide compound represented by the formula is hydrolyzed, nitrated, and then halogenated to provide a method for producing a p-nitrophenylcarbylsilyl halide compound according to the first invention.
[0014]
Seventh, the invention of this application relates to a method of immobilizing a sugar chain or amino acid on a solid-phase synthesis support via a silyl linker, synthesizing a glycopeptide or peptide, and then cutting a silicon-oxygen bond to produce a sugar or amino acid. Wherein the alcoholic hydroxyl group of the sugar or amino acid is protected by a reaction with the p-nitrophenylcarbylsilyl halide compound of the first invention, and the nitro group is reduced and then condensed with a dicarboxylic anhydride to form a dicarboxylic monoamide. Provided is a method for synthesizing a glycopeptide or an oligopeptide, which comprises using the compound as a silyl linker.
[0015]
Furthermore, the invention of this application relates to the above synthesis method, in the eighth aspect, a method using succinic anhydride or glutaric anhydride as a dicarboxylic anhydride, and in the ninth, a silicon-oxygen bond is cleaved by a fluorine anion. And tenthly, a method for cleaving silicon-oxygen bonds using 18-crown-6 or acetic acid together with cesium fluoride.
[0016]
That is, the invention of this application as described above is derived from the inventors' study on a synthetic element that can be immobilized on a solid-phase synthesis support via a sugar chain, and is a glycopeptide containing a silyl ether. As a result of studying the structural part that becomes a linker to synthesize the unit and link it to the solid phase via the substituent of the silyl compound, and studying the solid-phase synthesis method for glycopeptides, etc., a bulky substitution with large steric hindrance was found. A novel p-nitrophenylcarbinylsilyl halide compound having a group has been completed based on the finding that it has solved the above problems and has been found to be useful as a linker for solid-phase synthesis of glycopeptides or peptides. is there.
[0017]
BEST MODE FOR CARRYING OUT THE INVENTION
The invention of this application has the features as described above, and embodiments thereof will be described below.
First, the p-nitrophenylcarbylsilyl halide compound represented by the above general formula (I) provided in the present invention is represented by R ¹ , R ² , R ³ And R ⁴ May be an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an aryl group as a hydrocarbon group, or various kinds of these substituted hydrocarbons. Are suitably about 1 to 8, and among them, a lower alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a pentyl group, or a phenyl group, a tolyl group, etc. Those selected from the aryl groups of are exemplified as suitable ones.
[0018]
X is a halogen atom, but is preferably a chlorine atom from the viewpoint of reactivity, synthesis and handling.
For example, specific examples of the compound of general formula (I) include (p-nitrophenyldimethylcarbyl) dimethylsilyl chloride, (p-nitrophenyldimethylcarbyl) methylethylsilyl chloride, and (p-nitrophenyldimethyl). (Carbinyl) divinylsilyl chloride, (p-nitrophenyldimethylcarbinyl) diphenylsilyl chloride, (p-nitrophenyldimethylcarbinyl) methylphenylsilyl chloride, (p-nitrophenyldimethylcarbinyl) methyltolylsilyl chloride, (p -Nitrophenyldiethylcarbinyl) dimethylsilyl chloride, (p-nitrophenylphenylmethylcarbinyl) dimethylsilyl chloride, (p-nitrophenylmethylpropylcarbinyl) dimethylsilyl chloride and the like. That.
[0019]
Among them, (p-nitrophenyldimethylcarbyl) dimethylsilyl chloride, (p-nitrophenyldimethylcarbyl) methylethylsilyl chloride and (p-nitrophenyldimethylcarbyl) diethylsilyl chloride are exemplified as suitable ones. .
For example, the p-nitrophenylcarbylsilyl halide compound represented by the general formula (I) of the present invention as described above is a novel compound useful as a compound forming a silyl ether type linker.
[0020]
In the present invention, the p-nitrophenylcarbylsilyl halide compound is obtained by using the above-mentioned phenylcarbylsilyl halide compound of the general formula (II) as a raw material, hydrolyzing the compound, and subjecting it to a nitration and halogenation reaction. Manufactured by This method is represented by the following general formula (III).
[0021]
Embedded image

[0022]
Examples of the phenylcarbylsilyl halide compound serving as a raw material include (phenyldimethylcarbyl) dimethylsilyl chloride, (phenyldimethylcarbyl) methylethylsilyl chloride, (phenyldimethylcarbyl) divinylsilyl chloride, and (phenyldimethylcarbyl) diphenyl. Silyl chloride, (phenyldimethylcarbyl) methylphenylsilyl chloride, (phenyldimethylcarbyl) methyltolylsilyl chloride, (phenyldiethylcarbinyl) dimethylsilyl chloride, (diphenylmethylcarbyl) dimethylsilyl chloride, (phenylmethylpropylcarbyl) (Vinyl) dimethylsilyl chloride is exemplified. Preferably, (phenyldimethylcarbyl) dimethylsilyl chloride, (phenyldimethylcarbyl) methylethylsilyl chloride and (phenyldimethylcarbyl) diethylsilyl chloride are used.
[0023]
For the hydrolysis, nitration, and halogenation of the phenylcarbylsilyl halide compound as a raw material, a conventional chemical reaction method is appropriately adopted.For example, a hydrolysis method using an aqueous alkali solution is used for hydrolysis, and ammonium nitrate is used for nitration. The nitration method using trifluoroacetic anhydride is exemplified, and the halogenation method is exemplified by the halogenation method using oxalyl halide.
[0024]
In the present invention, a silyl linker for solid-phase synthesis of glycopeptides or oligopeptides is provided by the p-nitrophenylcarbylsilyl halide compound represented by the general formula (I). , For example
[0025]
Embedded image

[0026]
(R ¹ , R ² , R ³ And R ⁴ Represents the same as above, A represents the residue at the alcoholic hydroxyl group site of the sugar or amino acid, and B represents the residue of the dicarboxylic acid)
Can be represented by
For example, as described above, the silyl linker of the present invention is obtained by reacting a p-nitrophenylcarbylsilyl halide compound represented by the above general formula (I) with an alcoholic hydroxyl group of a sugar or an amino acid to form a protected structure by a silyl-ether bond. And then reducing the nitro group to an amino group and subjecting it to a condensation reaction with a dicarboxylic anhydride to form a dicarboxylic acid monoamide.
[0027]
More specifically, for example, a sugar chain or an amino acid having an organic compound hydroxyl group is used as a starting material or an intermediate as a starting material or an intermediate used in solid phase synthesis, and the p-nitrophenyl carboxy group represented by the general formula (I) is used. When the vinyl halide is reacted in a solution in the presence of a deoxidizing agent, the corresponding silyl ether can be easily obtained. This reaction can be carried out under the conditions of ordinary silylation reaction, but it is desirable to carry out the reaction in the presence of a reaction accelerator such as sodium iodide. The obtained silyl ether is chemically stable, can be easily purified by a method such as chromatography, and the structure of the product can be easily confirmed by NMR or the like.
[0028]
The nitro group of the resulting silyl ether can then be reduced under very mild conditions, for example in the presence of zinc dust, to give the corresponding aniline derivative. This reduction reaction proceeds without being affected by the structure of the substrate, and the desired aniline derivative can be obtained.
Then, when the obtained aniline derivative is reacted with a cyclic acid anhydride, for example, succinic anhydride or glutaric anhydride, a monocarboxylic acid having an amide bond (dicarboxylic monoamide) can be easily obtained. Of course, the acid anhydride may be of various types. In this way, the above silyl ether can be produced.
[0029]
The silyl ether can form an amide bond by reacting with an amino group of an amino acid immobilized on a suitable solid phase synthesis carrier using a condensing agent.
The linker having silyl ether type condensation obtained in the present invention binds to the solid phase using the hydroxyl group of serine or threonine, which has not been performed in the conventional peptide solid phase synthesis, and repeats the condensation to extend the peptide chain. After that, it becomes possible to specifically take out the synthetic peptide without damaging the protecting groups disposed on the N-terminal amino group, the C-terminal carboxyl group and the functional group of the side chain. Further, not only serine and threonine but also one of a large number of hydroxyl groups present in the sugar chain can be used as a binding point to the solid phase. According to the solid-phase synthesis method using the silyl linker of the present invention, both ends of the peptide main chain can be used as reaction points for peptide synthesis by selectively removing the respective protecting groups, so that most attempts have been made in the past. The peptide C-terminal extension on a solid phase, which has not been performed, can also be performed.
[0030]
The silyl linker according to the present invention has the advantages that it can be easily prepared, and that it remains on the resin when cleaved, and that the separation from the target product is only a filtration operation. In addition, by binding to a commercially available amino-type resin (or amino acid-binding type resin) whose titer is known, the value of the binding efficiency can be easily known by the ninhydrin method, Fmoc quantification method, or the like.
[0031]
In the present invention, no particular method is required for the quantification of the immobilization reaction. For example, the immobilization reaction can be extremely easily and highly sensitively detected by a test for detecting residual amino groups using ninhydrin.
And in this invention, a solid-phase synthesis reaction can be performed without special reaction conditions, and as described above, a peptide chain can be extended to one or both of the N-terminal and the C-terminal acid terminal. .
[0032]
After the solid phase synthesis is completed, the protected glycopeptide or oligopeptide can be taken out by specifically cleaving the silicon-oxygen bond, but it must be released from the solid phase under the usual conditions for deprotection of the silyl protecting group. Can be. When the reaction is carried out with a compound generating fluorine ions, for example, cesium fluoride, for example, in the presence or absence of acetic acid or crown ether, the cleavage can be extremely easily performed quantitatively.
[0033]
【Example】
Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
(Example 1)
<Production of (p-nitrophenyldimethylcarbinyl) dimethylsilyl chloride>
26.57 g of (phenyldimethylcarbyl) dimethylsilyl chloride was dissolved in 75 ml of diethyl ether, and 100 ml of a 1: 4 (v / v) mixed solvent of water and methanol in which 8.4 g of potassium hydroxide was dissolved was gradually added, followed by stirring. did. The mixed solution was stirred at room temperature for 20 hours, separated, extracted twice with diethyl ether, washed and dried with an ether solution, and the solvent was distilled off under reduced pressure to obtain 22.89 g of (phenyldimethylcarbylvinyl) as a colorless oily substance. ) Dimethylsilanol was obtained.
[0034]
5.00 g of this silanol was dissolved in 35 ml of acetonitrile, 2.60 g of ammonium nitrate was added, the suspension was adjusted, and 5.8 ml of trifluoroacetic anhydride was added little by little with stirring. After the addition was completed, 50 minutes and 60 minutes later, 2.0 ml each of trifluoroacetic anhydride was added, and the mixture was stirred at room temperature overnight.
After adding water to the obtained reaction solution and extracting twice with ethyl acetate, the organic phase was washed and dried, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography to obtain 3.99 g of a colorless crystalline compound. (P-Nitrophenyldimethylcarbyl) dimethylsilanol was obtained.
[0035]
Next, 3.58 g of this (p-nitrophenyldimethylcarbyl) dimethylsilanol was dissolved in 50 ml of methylene chloride, and 1.50 ml of oxalyl chloride was added. Then, 1 drop of dimethylformamide was added, and the mixture was stirred at room temperature for 1 day. The reaction solution was concentrated under reduced pressure, and the obtained crystalline residue was recrystallized from hexane to give 3.56 g of (p-nitrophenyldimethylcarbyl) dimethylsilyl chloride (mp. 114.5 to 117 ° C as colorless crystals). Hexane)).
(Example 2)
The same reaction was carried out except that (phenyldimethylcarbyl) dimethylsilyl chloride of Example 1 was replaced with (phenyldimethylcarbyl) methylethylsilyl chloride, thereby obtaining (p-nitrophenyldimethylcarbyl) methyl in the same yield. Ethylsilyl chloride was obtained.
[0036]
The following compounds were obtained by performing the same method.
(P-nitrophenyldimethylcarbyl) divinylsilyl chloride,
(P-nitrophenyldimethylcarbyl) diphenylsilyl chloride,
(P-nitrophenyldimethylcarbyl) methylphenylsilyl chloride,
(P-nitrophenyldimethylcarbyl) methyltolylsilyl chloride,
(P-nitrophenyldiethylcarbinyl) dimethylsilyl chloride,
(P-nitrophenyl-phenylmethylcarbinyl) dimethylsilyl chloride,
(P-Nitrophenylmethylpropylcarbinyl) dimethylsilyl chloride
(Example 3)
<Synthesis of silyl linker>
A silyl linker was synthesized according to the following reaction formula.
[0037]
Embedded image

[0038]
First step: production of silyl ether
<N- (9-fluorenylmethoxycarbonyl) [O- (4-nitrophenyldimethylcarbyl) dimethylsilyl] -L-serine allyl ester>
N- (9-Fluorenylmethoxycarbonyl) -L-serine allyl ester (500 mg, 1.36 mmol), (p-nitrophenyldimethylcarbyl) dimethylsilyl chloride (420 mg, 1.63 mmol) and sodium iodide (610 mg) , 4.06 mmol) was added with dimethylformamide (20 ml), and the mixture was reacted at room temperature for 15 minutes. After further adding N-methylmorpholine (179 μl, 1.62 mmol), the reaction was carried out for 20 minutes. The reaction solution was partitioned with ether-saturated aqueous sodium bicarbonate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. The residue obtained by concentration under reduced pressure was subjected to silica gel column chromatography (SiO 2). ₂ Purification with 60 g, hexane-EtOAc 2: 1) gave 775 mg (97%) of the title compound as a colorless oil.
[0039]
Second step: production of aniline derivative
<N- (9-fluorenylmethoxycarbonyl) [O- (4-aminophenyldimethylcarbyl) dimethylsilyl] -L-serine allyl ester>
N- (9-Fluorenylmethoxycarbonyl) [O- (4-nitrophenyldimethylcarbyl) dimethylsilyl] -L-serine allyl ester (780 mg, 1.32 mmol) was dissolved in tetrahydrofuran (20 ml), and zinc dust was dissolved. (5.00 g, excess) and acetic acid (1 ml) were added and reacted for 45 minutes. The reaction solution was diluted with ethyl acetate, filtered through a celite layer, washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous sodium sulfate. The residue obtained by concentration under reduced pressure was subjected to silica gel column chromatography (SiO 2). ₂ The residue was purified by 60 g, hexane-EtOAc 1: 1) to obtain 650 mg (88%) of the title compound as a colorless oil.
[0040]
Third step: production of amidocarboxylic acid
<N- (9-fluorenylmethoxycarbonyl) {O-[(4-amidophenylphenylsuccinate) dimethylcarbyl] dimethylsilyl} -L-serine allyl ester>
9-Fluorenylmethoxycarbonyl [O- (4-aminophenyldimethylcarbyl) dimethylsilyl] -L-serine allyl ester (650 mg, 1.16 mmol) was added to succinic anhydride (120 mg, 1.20 mmol) and N- Methylmorpholine (111 μl, 0.89 mmol) was added and reacted for 4 hours. The reaction solution was concentrated, spread on a column of Biobeads SX-3 (100 g), and purified by elution with a mixed solvent of toluene-ethyl acetate (1: 1) to obtain 760 mg (99%) of the title compound.
(Example 4)
<Introduction to resin>
The silyl linker obtained in Example 3 was introduced into a resin and immobilized as in the following formula.
[0041]
Embedded image

[0042]
That is, N- (9-fluorenylmethoxycarbonyl) {O-[(4-amidophenylphenyl succinate) dimethylcarbyl] dimethylsilyl} -L-serine allyl ester (250 mg, 0.38 mmol) was N-methylmorpholine (3.0 ml) solution, O-benzotriazo-1-yl-1-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU: 288 mg, 0.76 mmol) and 0.5 M 1- Hydroxybenzotriazole (HOBt: dimethylformamide solution, 0.76 ml, 0.38 mmol) and 2M diisopropylethylamine (dimethylformamide solution, 0.19 ml, 0.38 mmol) were added, and the mixture was reacted at room temperature for 1 hour and 20 minutes. Then, a glycine HMP resin (ABI, 0.95 mmol / g, 271 mg, 0.253 mmol) was added, and the mixture was stirred and mixed overnight. The obtained resin was separated by filtration and washed sequentially with N-methylmorpholine, 2-propanol, methylene chloride and diethyl ether to obtain 439 mg (102%: calculated from weight) of the resin.
(Example 5)
<Peptide solid phase synthesis>
The solid phase synthesis of the peptide was performed following Example 4 according to the following reaction formula.
[0043]
Embedded image

[0044]
Removal of 9-fluorenylmethoxycarbonyl group (Fmoc) (1)
N- (9-Fluorenylmethoxycarbonyl) serine (O-linker-glycine HMP resin) allyl ester (0.59 mmol / g, 151 mg, 0.089 mmol) was converted to N-methylmorpholine-piperidine (1: 1 v / v, 3.0 ml) and stirred and mixed for 1 hour and 40 minutes. The obtained resin was separated by filtration and washed sequentially with N-methylmorpholine, 2-propanol, methylene chloride and diethyl ether to obtain 134 mg of a resin.
[0045]
Introduction of N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] threonine (2)
N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] -L-threonine (33 mg , 0.0492 mmol) in a solution of N-methylmorpholine (1.0 ml) in O-benzotriazo-1-yl-1-N, N, N ', N'-tetramethyluronium hexafluorophosphate (37.3 mg, 0.098 mmol), 1-hydroxybenzotriazole (66 mg, 0.49 mmol) and 2M diisopropylethylamine (dimethylformamide solution, 0.49 μl, 0.098 mmol) were added and reacted for 35 minutes, followed by serine (O-linker-glycine). HMP resin) allyl ester (0.68 mmol / g, 66 mg, 0.0447) The resulting resin was filtered off, washed successively with N-methylmorpholine, 2-propanol, methylene chloride and diethyl ether, and 88.5 mg (96%: calculated from weight). Resin was obtained.
[0046]
Removal of 9-fluorenylmethoxycarbonyl group (Fmoc) (3)
N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] -L-threonyl-L -Serine (O-linker-glycine HMP resin) allyl ester (0.47 mmol / g, 84.3 mg, 0.0425 mmol) suspended in N-methylmorpholine-piperidine (1: 1 v / v, 2.0 ml), The resulting resin was separated by filtration and washed sequentially with N-methylmorpholine, 2-propanol, methylene chloride and diethyl ether to obtain 80.4 mg of the resin.
[0047]
Introduction of N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] threonine (4)
N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] -L-threonine (33 mg , 0.0492 mmol) in a solution of N-methylmorpholine (1.0 ml) in O-benzotriazo-1-yl-1-N, N, N ', N'-tetramethyluronium hexafluorophosphate (37.3 mg, 0.098 mmol), 1-hydroxybenzotriazole (66 mg, 0.49 mmol) and 2M diisopropylethylamine (dimethylformamide solution, 0.49 μl, 0.098 mmol) were added and reacted for 35 minutes, followed by [O- (2-acetamide). -3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] -L Allyl ester of threonyl-L-serine (O-linker-glycine HMP resin) (0.53 mmol / g, 80.4 mg, 0.0425 mmol) was added and stirred overnight. %: Calculated from weight).
[0048]
Cleavage of glycopeptide from resin (5)
N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] -L-threonyl-L -N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl] -L-threonyl- L-serine (O-linker-glycine HMP resin) allyl ester (0.39 mmol / g, 77.4 mg, 0.0302 mmol) is suspended in 0.2 mol / l cesium fluoride-1.0 mol / l acetic acid DMF solution. The resulting resin was separated by filtration, washed with N-methylmorpholine, and then sequentially with 2-propanol, methylene chloride and diethyl ether. , Methylene chloride and diethyl ether were concentrated, combined with the N-methylmorpholine wash and diluted with ethyl acetate and partitioned with water.The aqueous layer was further extracted three times with ethyl acetate, and the organic layers were combined. The residue was washed with saturated saline, dried over anhydrous sodium sulfate and concentrated, and the residue was purified by preparative thin-layer chromatography (1 mm layer, CHCl 3). ₃ / MeOH 9: 1) and purified by N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyra). Nosyl] -L-threonyl-N- (9-fluorenylmethoxycarbonyl) [O- (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyrano 27.2 mg (73%) of [sil] -L-threonyl-L-serine allyl ester were obtained.
[0049]
【The invention's effect】
The (p-nitrophenylcarbyl) silyl halide compound of the present invention is a compound useful for forming a linker for solid phase synthesis, and quantitatively binds to an alcoholic aqueous solution of a sugar or amino acid to form a silyl ether bond, The necessary compound can be immobilized on the solid phase through several steps. Solid phase organic synthesis using this linker is mechanically advanced, and the reaction is developed and released from the solid phase under mild conditions as a combinatorial chemistry, so that oligopeptides serving as enzyme inhibitors and peptide hormones serving as receptors can be used. Bioactive glycopeptides or peptides can be obtained.

Claims (10)

General formula (I)

(Wherein R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an aryl group which may have a substituent. , X represents a halogen atom)
A p-nitrophenylcarbinylsilyl halide compound represented by the formula: The p-nitrophenylcarbinylsilyl halide compound according to claim 1, wherein at least ^one of R ¹ and R ² is a methyl group. At least one of R ³ and R ⁴ according to claim 1 or 2 p- nitrophenyl carbinyl silyl halide compound is a phenyl group. The p-nitrophenylcarbinylsilyl halide compound according to any one of claims 1 to 3, wherein X is a chlorine atom. The p-nitrophenylcarbinylsilyl halide compound according to claim ¹ , wherein R ¹ , R ² , R ³ and R ⁴ are a methyl group and X is a chlorine atom. General formula (II)

(Wherein R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an aryl group which may have a substituent. , X represents a halogen atom)
Wherein the phenylcarbylsilyl halide compound represented by the formula is hydrolyzed, nitrated, and then halogenated.

(Wherein R ¹ , R ² , R ³ , R ⁴ and X are the same as described above)
A method for producing a p-nitrophenylcarbinylsilyl halide compound represented by the formula: In a method in which a sugar chain or an amino acid is immobilized via a silyl linker to a solid-phase synthesis support, and a glycopeptide or peptide is synthesized, a silicon-oxygen bond is cleaved to remove the sugar or amino acid. General formula (I)

(Wherein R ¹ , R ² , R ³ and R ⁴ are the same or different and each represent a lower alkyl group, a lower alkenyl group or an aryl group which may have a substituent, and X represents a halogen atom. Show)
A sugar protected by reacting with a p-nitrophenylcarbinylsilyl halide compound represented by the formula (1), reducing the nitro group and then condensing with a dicarboxylic anhydride to form a dicarboxylic acid monoamide form as a silyl linker. A method for synthesizing a peptide or oligopeptide. 8. The method according to claim 7, wherein succinic anhydride or glutaric anhydride is used as the dicarboxylic anhydride. The method for synthesizing a glycopeptide or oligopeptide according to claim 7 or 8, wherein the silicon-oxygen bond is cleaved by a fluorine anion. The method for synthesizing a glycopeptide or oligopeptide according to claim 9, wherein the silicon-oxygen bond is cleaved using 18-crown-6 or acetic acid together with cesium fluoride.