JP3746181B2 - Phenyldiazirine compounds and photoaffinity labeling reagents - Google Patents

Description

【００１０】
一般式（I）中、Ｒ¹は−ＮＨＰ¹又は−ＣＯＸ¹を表し、Ｒ²は、アルデヒド基、ヒドロキシ低級アルキル基、ハロゲン化低級アルキル基、フタルイミドオキシ低級アルキル基、又は−(ＣＨ₂)_x−Ｏ−ＮＨＰ¹を表し、Ｐ¹はアミノ保護基を表し、Ｘ¹はアルコキシ基，ヒドロキシ基，又は−ＮＨＳＯ₂−Ｘ²−−ＣＯ−Ｘ³を表し、Ｘ²はアリーレン基又はアルキレン基を表し、Ｘ³はアルコキシ基又はヒドロキシ基を表し、ｍは１〜４、好ましくは１〜２の整数を表し、ｎは１〜６、好ましくは１〜４の整数を表し、ｘは１〜４、好ましくは１〜２の整数を表す。
【００１１】
本発明において、低級アルキル基とは、メチル、エチル、プロピル、ブチル等の炭素数１〜４のアルキル基を表し、直鎖状が好ましい。また、アルコキシ基としては、炭素数１〜６のアルコキシ基であり、例えばメトキシ基、エトキシ基、プロポキシ基、ブトキシ基、ペントキシ基等が挙げられる。アリーレン基としては、メチル基、エチル基等の置換基を有していてもよいフェニレン基、ナフチレン基等の炭素数５〜１４のアリーレン基が挙げられるが、フェニレン基が好ましい。アルキレン基としては、メチレン基、エチレン基、プロピレン基、ブチレン基等の炭素数１〜６のアルキレン基が挙げられ、直鎖状の基が好ましい。
アミノ保護基としては、ｔ−ブトキシカルボニル基、トリフルオロアセチル基、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、ｐ−トルエンスルホニル基、９−フルオレニルメチルオキシカルボニル基、トリチル基、フタロイル基、ｏ−ニトロフェニルスルフェニル基、３−ニトロ−２−ピリジンスルフェニル基等が挙げられる。これらの内ｔ−ブトキシカルボニル基が簡便である。
【００１２】
本発明のビオチン化フェニルジアジリン化合物は、下記一般式（II）で表される化合物であり、上記一般式（I）の化合物をビオチン化し、所望によりＲ²をアミノオキシ低級アルキル基に変換することにより得られる。
【００１３】
【化９】

【００１４】
一般式（II）中、Ｒ³は、アミノ基、−ＮＨＰ¹又はアミノ基の塩を表し、Ｐ¹はアミノ保護基を表し、ｍは１〜４、好ましくは１〜２の整数を表し、ｎは１〜６、好ましくは１〜４の整数を表し、ｘは１〜４、好ましくは１〜２の整数を表す。又、アミノ保護基は、上記一般式（I）におけるものと同義を表す。アミノ基の塩としては、塩酸、硫酸、硝酸、リン酸等の無機酸の塩、或いは酢酸、トリフルオロ酢酸、プロピオン酸、メタンスルホン酸等の有機酸の塩が挙げられる。これらの内、トリフルオロ酢酸は、一般式（V）の化合物を生成するのに有利である。
【００１５】
Ｙ¹は−ＮＨ−又は−ＣＯＮＨＳＯ₂−Ｘ²−ＣＯ−を表し、Ｘ²はアリーレン基又はアルキレン基を表し、Ｙ²はビオチン又はビオチン誘導体残基を表す。又、アリーレン基及びアルキレン基は上記一般式(I)におけるものと同義を表す。本発明でビオチン誘導体とは、ビオチンの末端基（−ＯＨ）がアミノ基又はカルボキシル基と反応し得る基に置き換わったビオチンを意味し、例えば、カルボキシル基と反応し得る基を有するものとして、(ビオチン)−ＮＨ−Ｚｑ−ＮＨ₂で表される末端にアミノ基を有する化合物、好ましくはビオチンヒドラジドが挙げられる。
本発明の一般式(II)で示される化合物のより具体的な化合物は、下記一般式(III)及び(IV)で示される化合物である。
【００１６】
【化１０】

一般式(III)中、Ｒ³、Ｐ¹、ｍ、ｎ及びｘは、一般式(II)におけるものと同義を表す。
【００１７】
【化１１】

一般式(IV)中、Ｒ³、Ｐ¹、ｍ、ｎ及びｘは、上記一般式(II)におけるものと同義を表し、Ｘ²はアリーレン基又はアルキレン基を表し、上記一般式(I)におけるものと同義を表す。Ｚは、酸素、硫黄、窒素等のヘテロ原子で中断されていてもよい炭素数１〜２０のアルキレン基を表し、例えば、メチレン基、エチレン基、プロピレン基、（ポリ）オキシエチレン基等が挙げられる。ｑは０又は１を表す。
【００１８】
本発明の下記一般式(V)〜(VII)で表される、糖結合ビオチン化フェニルジアジリン化合物は、一般式（II）〜(IV)において、末端アミノ基或いはアミノ基の塩を有するビオチン化フェニルジアジリン化合物に、還元末端を有する糖化合物の還元末端のアルデヒド基を反応させることにより得られる化合物である。
【００１９】
【化１２】

【００２０】
一般式(V)中、Ｒ⁴は、還元末端をもつ糖化合物の残基を表し、ｍは１〜４、好ましくは１〜２の整数を表し、ｎは１〜６、好ましくは１〜４の整数を表し、ｘは１〜４、好ましくは１〜２の整数を表す。Ｙ¹は−ＮＨ−又は−ＣＯＮＨＳＯ₂−Ｘ²−ＣＯ−を表し、Ｘ²はアリーレン基又はアルキレン基を表し、Ｙ²はビオチン又はビオチン誘導体残基を表し、上記一般式(II)におけるものと同義を表す。
本発明の下記一般式(VI)及び(VII)で示される化合物は、一般式(V)で示される化合物のより具体的な化合物である。
【００２１】
【化１３】

一般式(VI)中、Ｒ⁴、ｍ、ｎ、ｘは上記一般式(V)におけるものと同義を表す。
【００２２】
【化１４】

一般式（VII）中、Ｒ⁴、ｍ、ｎ、ｘ及びＸ²は、上記一般式(V)におけるものと同義を表し、Ｚ及びｑは、上記一般式(IV)におけるものと同義を表す。
一般式(VII)で示される化合物は、切断性スペーサーを有する糖結合ビオチン化フェニルジアジリン化合物であり、特にＸ²がフェニレン基、ｑ＝０の化合物は、ラベル蛋白質の分離・回収に有用な光親和性標識試薬を構成する化合物として好ましい。
【００２３】
一般式（II）〜(IV)で示される化合物と反応させる糖化合物としては、還元末端を持つ多糖類、オリゴ糖類、単糖類、例えば、ムチン型糖類、Asn型糖類、シアリル糖類、グリコサミノグリカン、ラクトサミン、N-アセチルラクトサミン、ラクトサミンオリゴ糖、シアリルラクトサミン、グルカン、マンナン、フルクタン、ガラクタン、ポリウロン酸、オリゴアミノ酸、ポリアミノ糖、ガラクトオリゴ糖などが挙げられる。Ｒ⁴のより具体的な例としては、N-アセチルラクトシル基、シアリル−α2-3-ラクトシル基、シアリル−α2-3-N-アセチルラクトサミニル基、ルイスX型三糖残基、シアリルルイスX型四糖残基、又はキトビオース残基が挙げられるが、下記模式図に示す結合が可能な糖化合物であればこれらに限定されない。即ち、本発明の一般式（II）で示される化合物において、Ｒ³がアミノ基又はアミノ基の塩である化合物と還元末端を有する糖化合物との結合は選択的で、その結合は以下のように示される。
【００２４】
【化１５】

【００２５】
本発明の一般式(II)〜(IV)で示される化合物において、Ｒ³がアミノ基又はアミノ基の塩である化合物と、還元末端を有する糖化合物との反応はｐＨ依存性であり、該反応は弱酸性下有利に進行する。通常該反応はｐＨ１〜７で進行するが、好ましくは緩衝液を用いｐＨ４〜５に調整されて行われる。
本発明の一般式（II)〜(IV)で示される光反応性ビチオン化フェニルジアジリン化合物の一種は、そのフェニル骨格にスペーサーを介してオキシアミノ基が導入されている特徴を有し、このオキシアミノ基は、還元末端を有する糖化合物の還元末端のアルデヒド基と、糖鎖水酸基の保護などを特に必要とせず、また従来必要とされているＤＣＣ等の縮合剤も存在せしめることなく、単に糖化合物と混合するだけでシッフ塩基形成反応により結合し、一般式(V)〜(VII)で示される糖結合ビオチン化フェニルジアジリン化合物を生成する。そして、この化合物によって本発明の光親和性標識試薬を構成することができる。該試薬は、この化合物の機能を害さない限り、水、緩衝剤、安定化剤，塩等の添加物を含んでいてもよい。また、一般式(II)〜(IV)の化合物のオキシアミノ基は、アルデヒド基以外にケトン基、カルボキシル基とも結合し得るので、糖化合物がこれらの反応基を有する場合はその官能基とオキシアミノ基を反応させてもよい。
なお、光親和性標識試薬とは、広義には、光反応性基を有する化合物にリガンドを結合した標識試薬を指称するが、本明細書において「光親和性標識試薬」とは、光反応性糖受容体親和性標識試薬を意味するものとし、糖受容体とは糖と親和性を有する、すなわち糖との特異的な相互作用により糖に結合性を示す蛋白質を意味し、具体的には糖に特異的なレクチン、レセプター、酵素、抗体などを包含する。
【００２６】
本発明の光親和性標識試薬と種々の蛋白質を含む混合系において、該標識試薬の糖化合物と糖受容体との相互作用をおこなわせると同時又はその後に、光を照射すると、光親和性標識試薬の糖化合物に特異的に相互作用する蛋白質（糖受容体）の、該糖化合物との相互作用部位（アミノ酸）に該標識試薬のジアジリン基が光反応によりクロスリンクする。更に、このビオチンを含む該光親和性標識試薬でラベル化された蛋白質（ラベル化蛋白質）は、ビオチンを検出する自体公知の検出系によって高感度での検出が可能であり、アビジン、ストレプトアビジン等を担体に結合したアビジンカラムを利用することにより、アフィニティークロマトグラフィーによって目的蛋白質を容易に精製することが可能になる。更に、本発明においては、光親和性標識試薬を構成する糖化合物としてビオチンが切断性スペーサーを介して結合された化合物を適用することにより、アビジンカラムによるアフィニティークロマトグラフィーを行う際、緩和な条件でビオチン部分を容易に切り離すことができ、目的蛋白質を高い回収率で取得することが可能である。それ故、糖化合物を結合した本発明の光親和性標識試薬は、糖鎖関連蛋白質の構造及び機能解析に極めて有用なプローブ又は精製手段となることが期待されるのである。
【００２７】
本発明の一般式（II）で示され、オキシアミノ基をフェニル骨格に有する光反応性ビチオン化フェニルジアジリン化合物に、そのオキシアミノ基と選択的に反応する基、例えばアルデヒド基、ケトン基、カルボキシル基などを有するか、或いは必要に応じこれらの基が導入された薬物或いは糖化合物以外の生体由来成分をリガンドとして結合させることにより、広義な意味での光親和性標識試薬に誘導することもできる。そして、この試薬は、薬物あるいは糖化合物以外の生体由来物質に親和性を持つ蛋白質の構造及び機能解析のプローブとなり又単離精製も可能とする。この様にして、多種多様なリガンドを連接する手法で得ることができプローブライブラリーと、質量分析装置による超微量解析とを組み合わせる応用は、蛋白質機能の高速度・高精度解析への展望を開くものとして期待される。従って、一般式（II）で示される本発明化合物は極めて広範な有用性を有する化合物である。
【００２８】
本発明化合物の合成の一例を図１〜６に示す反応スキームに基づいて以下に説明する。
まず、図１に示す反応工程に従い、一般式（I）で示される本発明化合物、即ち、図中化合物(4)〜(7)の製造工程を説明する。
出発原料である化合物(1)、［2-メトキシ-4-(1-アジ-2,2,2-トリフルオロエチル）ベンズアルデヒド］は、橋本らの方法（M.Hashimoto,Y.Kanaoka,Y.Hatanaka,Heterocycles.46,119-122(1997))により調製することができる。
化合物(1)は、例えばジクロロメタンなどの溶媒中、不活性ガス雰囲気下、−２０℃付近でBBr₃を作用させることにより化合物(2)を生成する。
生成した化合物(2)に、化合物(3)を無水炭酸カリウムなどの塩基及びBu₄NIなどの相関移動触媒の存在下反応させることにより、化合物(4)を生成することができる。この反応は、ジメチルホルムアミド(DMF)中、不活性ガス雰囲気下、６０℃付近で行うことができる。なお、化合物(3)は、式Ｐ¹ＮＨ(ＣＨ₂)_m（ＯＣ₂Ｈ₄）_n−Ｂｒにおいて、m=n=2、Ｐ¹がt-ブトキシカルボニル基である化合物であり、畑中らの方法（Y.Hatanaka,M.Hashimoto,Y.Kanaoka,Bioorg.Med.Chem.2,1367-1373(1994)）により調製することができる。
【００２９】
化合物(4)は、例えばエタノールなどの溶媒中、NaBH₄により室温付近で還元して化合物(5)とすることができる。還元反応生成物は、精製処理することなく、次の反応工程に供することができる。
生成した化合物(5)は、四臭化炭素とトリフェニルホスフィンにより、例えばジクロルメタンなどの溶媒中でブロム化することにより、化合物(6)へと導く。この反応は、０℃付近に冷却して行うことが好ましい。
化合物(6)は、N-ヒドロキシフタルイミドと無水炭酸カリウムなどの塩基の存在下反応させることにより、化合物(7)を得ることができる。この反応は、ジメチルスルホキシド（DMSO）などの溶媒中、室温付近で行うことができる。
【００３０】
次いで、図２に則り、化合物(7)をビオチン化し、一般式（II）中、特に一般式(III)で示される本発明化合物、即ち、図中化合物(8)〜(10)の製造工程を説明する。
化合物(7)に、トリフルオロ酢酸を例えばジクロロメタンなどの有機溶媒中、０℃付近で作用させた後、トリエチルアミン（Et₃N)などの有機塩基の存在下、例えばDMFなどの溶媒中、ビオチンのN-ヒドロキシスクシンイミドエステル（biotin-OSu）を室温付近で反応させることによりビオチン化した化合物(8)を得ることができる。
【００３１】
化合物(8)に、例えばメタノール溶媒中、無水ヒドラジンを室温付近で作用させることにより化合物(9)を生成することができるが、この化合物(9)はカラムクロマトグラフィーによる精製中に分解し易いので、精製処理することなく次の反応に用いることが好ましい。
化合物(8)とヒドラジンとの反応生成物である化合物(9)をそのまま、例えばクロロホルムとアセトニトリルとの混合溶媒中で、トリエチルアミンなどの有機塩基の存在下、ジ−ｔ−ブチルジカルボナート｛[(CH₃)₃ COCO]₂O｝と室温付近で反応させることにより化合物(10)を得ることができる。
【００３２】
更に、図３及び４に示すように、上記工程で得たビオチン化フェニルジアジリン化合物に還元末端を有する糖化合物を導入し、一般式(V)中、特に一般式（VI）で示される本発明化合物、即ち化合物(12)〜(16)を生成することができる。
化合物(10)に、トリフルオロ酢酸を、例えばジクロロメタンなどの有機溶媒中、０℃付近で作用させることにより化合物(11)を得ることができる。生成した化合物(11)は、直ちに還元末端を有する各種の糖化合物と反応させて、それぞれの糖化合物の結合したビオチン化フェニルジアジリン化合物を得ることができる。例えば、化合物(11)を含水アセトニトリル中でN-アセチルラクトサミン（Galβ1-4ＧlcNAc）と３７℃付近で反応させることにより化合物(12)を取得することができる。同様に、シアリル-α2-3-ラクトース（NeuNAcα2-3Galβ1-4Glc）、シアリル-α2-3-N-アセチルラクトサミン（NeuNAcα2-3Galβ1-4GlcNAc）、ルイスX型三糖（Galβ1-4（Fucα1-3）GlcNAc）、またはシアリルルイスX型四糖（NeuNAcα2-3Galβ1-4（Fucα1-3）GlcNAc）と各々反応させることにより化合物(13)〜(16)をそれぞれ得ることができる。又、化合物(10)に作用させるトリフルオロ酢酸に代えて、塩酸／酢酸、塩酸／ジオキサン混合物等を使用し、無機酸の塩を生成することもできる。
【００３３】
次に、図５及び図６に示す反応工程に従い、一般式(I)で示される化合物、即ち、図中化合物(18)〜(20),(22)、(23)、(25)、(26)、一般式（IV）及び(VII)で示される切断性スペーサーを有する化合物、即ち、図中化合物(28)及び(30)、加えて一般式(VI)の化合物(29)の製造工程を説明する。
化合物(2)は、図１に示す工程により化合物(1)から合成する。化合物(2)に化合物(17)を無水炭酸カリウムなどの塩基及びBu₄NIなどの相関移動触媒の存在下反応させることにより、化合物(18)を生成することができ、この反応は、ジメチルスルホキシド(DMSO)中、不活性ガス雰囲気下、６０℃付近で行うことができる。なお、化合物(18)は、一般式(I)において、Ｒ²がアルデヒド基、Ｒ¹がＣＯＸ¹で、Ｘ¹がエトキシ基、m=1、n=３である化合物である。
【００３４】
一方、化合物(17)は以下のようにして合成出来る。即ち、2-[2-(2-クロロエトキシ)エトキシ]エタノールを水素化ナトリウムを含むトルエン溶媒中、−７０℃でブロム酢酸エチルと反応させ、得られた反応混合物から溶媒を留去して得た残渣をカラムクロマトグラフィーにより精製する。精製した残渣をＮ−メチルピロリドン等の溶媒に溶解した後、臭化エチル及び臭化ナトリウムを加えて65℃で反応させ、反応物から溶媒を留去後、更にカラムクロマトグラフィーにより精製する。
【００３５】
化合物(18)は、例えばエタノールなどの溶媒中、NaBH₄により室温付近で還元して化合物(19)とすることができる。生成した化合物(19)は、四臭化炭素とトリフェニルホスフィンにより、例えばジクロルメタンなどの溶媒中でブロム化することにより、化合物(20)へと導かれる。この反応は、０℃付近に冷却して行うことが好ましい。
化合物(20)は、N-ヒドロキシカルバミン酸 t-ブチルエステル等のN-ヒドロキシカルバミン酸アルキルエステル（化合物21）と水素化ナトリウムなどの塩基の存在下反応させることにより、化合物(22)を得ることができる。この反応は、テトラヒドロフラン（THF）などの溶媒中、０℃付近で行うことができる。
【００３６】
化合物(22)は、例えばエタノールとアルカリ水溶液との混合溶媒中で加水分解することにより化合物(23)を生成する。化合物(23)とカルボニルジイミダゾールをTHF等の溶媒に溶解し、これにスペーサーに切断性を付与するための化合物であるスルファモイル安息香酸エステルなどの化合物(24)をジイソプロピルエチルアミンなどの有機塩基の存在下反応させ、化合物(25)に導く。
切断性を付与するために使用される化合物(24)は、本発明の標識試薬に結合している糖化合物の種類、並びに該糖化合物に特異的に相互作用する糖の受容体である蛋白質の種類等を考慮して適宜選択されるが、スルファモイル安息香酸エステルは、緩和な条件で切断され、且つ安定性も良いので好ましい。
【００３７】
化合物(25)は、例えばエタノールとアルカリ水溶液との混合溶媒中で加水分解することにより化合物(26)を生成し、化合物(26)をＮ−ヒドロキシスクシンイミドとカルボジイミド類（ＤＣＣ等）を反応させ活性エステルとした後、ビオチンヒドラジドである化合物(27)などビオチン誘導体を反応させてビオチン化し、切断性スペーサーを有するビオチン化フェニルジアジリン化合物(28)とする。
この化合物(28)に対し、前記図３及び図４に示す工程と同様にして糖化合物（キトビオース：GlcNAcβ1-4GlcNAc）を導入して切断性スペーサーを有する糖結合ビオチン化フェニルジアジリン化合物(30)を形成し、他方、前記化合物(11)に還元末端を有する糖化合物としてキトビオースを導入して、切断性スペーサーを有しない化合物(29)を生成する。
【００３８】
【実施例】
以下、本発明を実施例によりさらに詳細に説明するが、本発明はその要旨を超えない限り、これらの実施例に限定されるものではない。なお、実施例中、各化合物番号は、図１〜図６に記載の化合物番号に対応する。
【００３９】
実施例１ （化合物(1) → (2)）
化合物(1)3.66ｇ(0.015mol)を30mlのジクロロメタンに溶解し、アルゴンガス雰囲気下、-20℃でこの溶液にBBr₃3.76ｇ(0.015mol)をゆっくり滴下した。滴下後、溶液を０℃で１時間かくはんした後、水を０℃で加えた。反応混合物をジクロロメタンで抽出し、ジクロロメタン層を無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；ジクロロメタン：ヘキサン＝１：２）で精製して3.18ｇの白色固体として化合物(2)｛2-ヒドロキシ-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンズアルデヒド｝を得た（収率：92%）。
IR(nujol):v=3170,1665cm^-1;
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=11.05(s,1H),9.92(s,1H),7.60(d,J=8.0Hz,1H),6.78(d,J=8.0Hz,1H),6.77(br s,1H);
ＭＳ（ＥＩ⁺）：ｍ／ｚ（％）:230(38)[Ｍ⁺];
ＨＲＭＳ：Ｃ₉Ｈ₅Ｆ₃Ｎ₂Ｏ₂ ［Ｍ⁺］＝230.0303(計算値),230.0291(実測値)
【００４０】
実施例２ （化合物(2)+(3) → (4)）
化合物(２）２．７６ｇ（０．０１２ｍｏｌ）、化合物（３)｛2-[2-(2-tert-ブトキシカルボニルアミノエトキシ)エトキシ]エチル ブロミド｝４．５０ｇ（０．０１４４ｍｏｌ）、無水炭酸カリウム１．６６ｇ（０．０１２ｍｏｌ）、Ｂｕ₄ＮＩ５５４ｍｇ（１．５ｍｍｏｌ）、及びＤＭＦ１５ｍｌの混合物をアルゴンガス雰囲気下、６０℃に１４時間かくはんした。その後、反応混合物の溶媒を減圧留去し、得られた残渣に水を加え、酢酸エチルで抽出した。酢酸エチル層を水、０．１Ｎ ＨＣ１、及び水で順次洗浄して無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；酢酸エチル：ヘキサン＝１：１）で精製して５．３３ｇの淡黄色油状物質として化合物（４)｛2-[2-[2-(2-tert-ブトキシカルボニルアミノエトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンズアルデヒド｝を得た（収率：９６％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=10.5(s,1H),7.85(d,J=8.1Hz,1H),6.86(d,J=8.1Hz,1H),6.75(br s,1H),5.00(br,1H),4.27(t,J=4.8Hz,2H),3.92(t,J=4.8Hz,2H),3.7(m,2H),3.6(m,2H),3.54(t,J=5.1Hz,2H),3.3(m,2H),1.43(s,9H);
ＭＳ（ＦＡＢ⁺）：ｍ／ｚ（％）：484（84）［Ｍ＋Ｎａ］⁺，462（15）［Ｍ＋Ｈ］⁺；
ＨＲＭＳ：Ｃ₂₀Ｈ₂₇Ｆ₃Ｎ₃Ｏ₆ ［Ｍ＋Ｈ］⁺＝462.1852(計算値),462.1874(実測値)
【００４１】
実施例３（化合物（４）→（５））
化合物（４）３．６９ｇ（８ｍｍｏ１）をエタノール４０ｍ１に溶解し、この溶液にＮａＢＨ₄３０３ｍｇ（８ｍｍｏ１）をゆっくり加えた．反応混合物を室温で３０分かくはんした後、溶媒を減圧留去して得られた残渣に水を加え、０℃で１Ｎ ＨＣｌを加えることによりｐＨ３付近に調節した後、酢酸エチルて抽出した。酢酸エチル層を水、飽和重曹水、及び飽和食塩水で順次洗浄して無水硫酸マグネシウムて乾燥後、溶媒を減圧留去し、淡黄色油状物質として粗化合物(５)｛2-[2-[2-(2-tert-ブトキシカルボニルアミノエトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンジルアルコール｝を得た。粗化合物（５）は更に精製することなく直ちに次の反応に用いた。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=7.26(d,J=8.0Hz,1H),6.79(d,J=8.0Hz,1H),6.66(br s,1H),6.01(br,1H),5.02(br,1H),4.66(s,2H),4.22(t,J=4.8Hz,2H),3.84(t,J=4.8Hz,2H),3.7(m,2H),3.6(m,2H),3.51(t,J=5.1Hz,2H),3.3(m,2H),1.45(s,9H);
ＭＳ（ＦＡＢ⁺）:ｍ／ｚ（％）:486（100）［Ｍ＋Ｎａ］⁺，464（16）［Ｍ＋Ｈ］⁺;
ＨＲＭＳ：Ｃ₂₀Ｈ₂₉Ｆ₃Ｎ₃Ｏ₆ ［Ｍ＋Ｈ］⁺＝464.2008(計算値),464.1997(実測値)
【００４２】
実施例４（化合物（５）→（６））
上記の方法で化合物（４）３．６９ｇ（８ｍｍｏ１）から得られた粗化合物（５）と四臭化炭素３．３２ｇ（０．０１ｍｏｌ）とを１６ｍｌのジクロロメタンに溶解し、０℃に冷却しながらトリフェニルホスフィン３．１５ｇ（０．０１２ｍｏｌ）をゆっくりと加えた。１０分後、無水炭酸カリウム１．６６ｇ（０．０１２ｍｏｌ）を反応混合物に加え、０℃でさらに３０分かくはんした。その後、反応混合物に水を加え、ジクロロメタンで抽出し、ジクロロメタン層を無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；酢酸エチル：ヘキサン＝１：１）で精製して３．６２ｇの淡黄色油状物質として化合物(６)｛2-[2-[2-(2-tert-ブトキシカルボニルアミノエトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンジルブロミド｝を得た（収率：８６％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=7.35(d,J=8.1Hz,1H),6.77(d,J=8.1Hz,1H),6.65(br s,1H),5.00(br,1H),4.51(s,2H),4.21(t,J=4.8Hz,2H),3.91(t,J=4.8Hz,2H),3.75(m,2H),3.65(m,2H),3.55(t,J=5.1Hz,2H),3.3(m,2H),1.43(s,9H);
ＭＳ(ＦＡＢ⁺）:ｍ／ｚ(％):550（41）［Ｍ＋Ｎａ］⁺,548(40),528(12)［Ｍ＋Ｈ］⁺,526(11);
ＨＲＭＳ：(⁷⁹Ｂｒ)Ｃ₂₀Ｈ₂₈ＢｒＦ₃Ｎ₃Ｏ₅ ［Ｍ＋Ｈ］⁺＝526.1164(計算値),526.1193(実測値)
【００４３】
実施例５（化合物（６）→（７））
化合物（６）２．６３ｇ（５ｍｍｏｌ）、N-ヒドロキシフタルイミド９７９ｍｇ（６ｍｍｏｌ）、無水炭酸カリウム６９１ｍｇ（５ｍｍｏｌ）、及びDMSO１０ｍｌの混合物を室温で１２時間かくはんした。その後、反応混合物の溶媒をオイルポンプを用いて減圧留去し、得られた残渣に水を加え、酢酸エチルで抽出した。酢酸エチル層を水、ｌＮ ＮａＯＨ及び飽和食塩水で順次洗浄して無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；ヘキサン：アセトン＝３：１）で精製して２．５９ｇの淡黄色油状物質として化合物(７)｛2-[2-[2-(2-tert-ブトキシカルボニルアミノエトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンジルオキシフタルイミド｝を得た（収率：８５％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=7.82-7.73(AB,4H),7.54(d,J=7.9Hz,1H),6.81(d,J=7.9Hz,1H),6.66(br s,1H),5.27(s,2H),5.05(br,1H),4.15(t,J=4.8Hz,2H),3.86(t,J=4.8Hz,2H),3.7(m,2H),3.6(m,2H),3.54(t,J=5.2Hz,2H),3.3(m,2H),1.42(s,9H);
ＭＳ（ＦＡＢ⁺）:ｍ／ｚ（％）:631（100）［Ｍ＋Ｎａ］⁺,609(9)［Ｍ＋Ｈ］⁺;ＨＲＭＳ：Ｃ₂₈Ｈ₃₂Ｆ₃Ｎ₄Ｏ₈ ［Ｍ＋Ｈ］⁺＝609.2172(計算値),609.2164(実測値)
【００４４】
実施例６（化合物（７）→（８））
化合物（７）３０４ｍｇ（０．５ｍｍｏｌ）をジクロロメタン０．５ｍｌに溶解し、０℃に冷却しながらトリフルオロ酢酸０．５ｍｌをゆっくりと加え、反応混合物を０℃で１時間かくはんした。溶媒を減圧留去して得られた残渣にトルエン１ｍｌを加え、減圧留去した。この操作を計３回繰り返し、残存する過剰のトリフルオロ酢酸を除いて得られる黄色油状残渣をＤＭＦ０．５ｍｌに溶解し、ビオチンＮ−ヒドロキシスクシンイミドエステル１７１ｍｇ（０．５ｍｍｏｌ）を２ｍｌのＤＭＦに溶解した溶液を加え、触媒量のトリエチルアミンの存在下反応混合物を室温で１４時間かくはんした。その後、溶媒を減庄留去し、得られた残渣をジクロロメタンとメタノール１：１の混合溶媒に溶解し、１Ｎ ＮａＯＨ、飽和食塩水、０．１Ｎ ＨＣｌ、及び飽和食塩水で順次洗浄して無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤：クロロホルム：エタノール＝１０：１）で精製して３２０ｍｇの無色固体として化合物(８)｛2-[2-[2-(2-ビオチニルアミノエトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンジルオキシフタルイミド｝を得た（収率：８７％）。
¹HNMR(400MHz,CDCl₃,25℃):δ=7.83-7.73(AB,4H),7.53(d,J=7.9Hz,1H),6.84(s,1H),6.81(d,J=7.9Hz,1H),6.64(s,1H),6.61(s,1H),5.64(s,1H),5.26(s,2H),4.5(m,1H),4.3(m,1H),4.15(t,J=4.8Hz,2H),3.90(t,J=4.8Hz,2H),3.7(m,2H),3.65(m,2H),3.55(m,2H),3.4(br m,2H),3.1(m,1H),2.9(m,1H),2.72(d,J=12.5Hz,1H),2.19(t,J=7.5Hz,2H),1.8-1.6(m,4H),1.4(m,2H);
MS(FAB⁺):m/z(%):757(16),[M+Na]⁺,735(62)[M+H]⁺;
HRMS:C₃₃H₃₈F₃N₆O₈S [M+H]⁺=735.2424(計算値),735.2458(実測値)
【００４５】
実施例７（化合物（８）→（９））
化合物（８）７３ｍｇ（０．１ｍｍｏｌ）を無水ヒドラジンの１Ｍメタノール溶液１ｍｌに溶解し、室温で３０分間反応させた。その後、溶媒を減圧留去して得られた残渣にトルエン１ｍｌを加え、減圧留去した。この操作を計３回繰り返し、残存する過剰のヒドラジンを除いて無色固体として粗化合物（９）を得た。粗化合物（９）は精製することなく直ちに次の反応に用いた。
【００４６】
実施例８（化合物：（９）→（１０））
上記の方法で化合物（８）７３ｍｇ（０．１ｍｍｏｌ）から得られた粗化合物（９）をクロロホルムとアセトニトリル１：１の混合溶媒１ｍｌに溶解し、［（ＣＨ₃）₃ＣＯＣＯ］₂Ｏ １０９ｍｇ（０．５ｍｍｏｌ）とトリエチルアミン５１ｍｇ（０．５ｍｍｏｌ）を加えて室温で１４時間反応させた。その後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；クロロホルム：エタノール＝１０：１）で精製して６４ｍｇの無色固体として化含物(１０)｛2-[2-[2-(2-ビオチニルアミノエトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンジルオキシカルバミン酸 tert-ブチルエステル｝を得た（収率：９５％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=8.31(s,1H),7.39(d,J=7.9Hz,1H),6.97(br,1H),6.81(d,J=7.9Hz,1H),6.64(s,1H),6.58(s,1H),5.66(s,1H),4.91(s,2H),4.5(m,1H),4.3(m,1H),4.16(t,J=4.5Hz,2H),3.87(t,J=4.5Hz,2H),3.7(m,2H),3.65(m,2H),3.55(m,2H),3.4(br m,2H),3.1(m,1H),2.9(m,1H),2.70(d,J=12.8Hz,1H),2.17(t,J=7.3Hz,2H),1.8-1.6(m,4H),1.45(s,9H),1.4(m,2H);
ＵＶ／ＶＩＳ（ＭｅＯＨ）:λ_max（ε）＝360(400),283(2600);
ＭＳ（ＦＡＢ⁺）:ｍ／ｚ（％）:727(100),［Ｍ＋Ｎａ］⁺,705(12)［Ｍ＋Ｈ］⁺;ＨＲＭＳ：Ｃ₃₀Ｈ₄₄Ｆ₃Ｎ₆Ｏ₈Ｓ ［Ｍ＋Ｈ］⁺＝705.2893(計算値),705.2853(実測値)
【００４７】
実施例９（化合物（１０）→（１１））
化合物（１０）７ｍｇ（０．０１ｍｍｏｌ）をジクロロメタン０．１ｍｌに溶解し、０℃に冷却しながらトリフルオロ酢酸０．１ｍｌをゆっくりと加え、０℃で１時間反応させた。その後、溶媒を減圧留去して得られた残渣にトルエン０．１ｍｌを加え、減圧留去した。この操作を計３回繰り返し、残存する過剰のトリフルオロ酢酸を除き、淡黄色固体として化合物（１１）を得た。化合物（１１）は精製することなく直ちに糖化合物類との縮合反応に用いた。
【００４８】
実施例１０（化合物（１１）→（１２））
上記の方法で化合物（１０）７ｍｇ（０．０１ｍｍｏｌ）から調製した粗化合物（１１）をアセトニトリル０．１ｍｌに溶解し、Ｎ−アセチルラクトサミン１．９ｍｇ（０．００５ｍｍｏｌ）の水溶液を加え、３７℃で４０時間反応させた。反応混合物をシリカゲルカラム（センシュー科学AQUASILＳＳ−１２５１−１２０、４．６ｆ ｘ ２５０ｍｍ）を用いてＨＰＬＣにより精製した。収量は、メタノール溶液のＵＶスペクトルを測定し、化合物（１１）のＵＶスペクトル（３６１ｎｍ（ｅ＝３８６））を基準として求め０．００３１ｍｍｏｌの化合物（１２）を得た。〈収率６３％）。
ＨＰＬＣ条件:90％aq.ＣＨ₃ＣＮ→75％aq.ＣＨ₃ＣＮ、２０min、１ｍｌ／min
ＭＳ（ＦＡＢ^-）:m/z(%):968(64)［Ｍ−Ｈ］^-;
ＨＲＭＳ;Ｃ₃₉Ｈ₅₇Ｆ₃Ｎ₇０₁₆Ｓ（[Ｍ−Ｈ]^-）:968.3535(計算値)、968.3586(実測値)
【００４９】
実施例１１（化合物（１１）→（１３））
実施例９の方法で化合物（１０）３．５ｍｇ（０．００５ｍｍｏｌ）から調製した化合物（１１）とシアリル−α２−３−ラクトース１．６ｍｇ（０．００２５ｍｍｏｌ）を実施例１０と同様の条件で反応させた。同様にＨＰＬＣ精製後０．００１２ｍｍｏｌの化合物（１３）を得た。（収率４８％）。
ＨＰＬＣ条件:95％aq.ＣＨ₃ＣＮ→50％aq.ＣＨ₃ＣＮ、１０min、１ｍｌ／min
Negative ion FAB-MS（３-nitrobenzylalcohol）ｍ／ｚ：１２１８[Ｍ−Ｈ]^-ＨＲ-ＦＡＢ-ＭＳ （３-nitrobenzylalcohol）
Ｃ₄₈Ｈ₇₁Ｆ₃Ｎ₇Ｏ₂₄Ｓ（[Ｍ‐Ｈ]^-）：1218.4223(計算値)、1218.4257(実測値)
【００５０】
実施例１２（化合物（１１）→（１４））
実施例９の方法で化合物（１０）４．３ｍｇ（０．００６ｍｍｏｌ）から調製した化合物（１１）とシアリル−α２−３−Ｎ−アセチルラクトサミン（純度９５％）１．０ｍｇ（０．００１４ｍｍｏｌ）を実施例１０と同様の条件で反応させた。同様にＨＰＬＣ精製後０．０００８５ｍｍｏｌの化合物（１４）を得た。（収率６１％）。
ＨＰＬＣ条件:85%aq.ＣＨ₃ＣＮ→70%aq.ＣＨ₃ＣＮ、15ｍｉｎ、1ｍｌ/ｍｉｎ
Negative ion FAB-MS（３-nitrobenzylalcohol）ｍ／ｚ：１２５９[Ｍ−Ｈ]^-ＨＲ-ＦＡＢ-ＭＳ （３-nitrobenzylalcohol）
Ｃ₅₀Ｈ₇₄Ｆ₃Ｎ₈Ｏ₂₄Ｓ（[Ｍ−Ｈ]^-）：1259.4489(計算値)、1259.4507(実測値)
【００５１】
実施例１３（化合物（１１）→（１５））
実施例９の方法で化合物（１０）ｌ．４ｍｇ（０．００２ｍｍｏｌ）から調製した化合物（ｌｌ）とルイスＸ型三糖０．５ｍｇ（０．０００９４ｍｍｏｌ）を実施例１０と同様の条件で反応させた。同様にＨＰＬＣ精製後 ０．０００７３ｍｍｏｌの化合物（１５）を得た。（収率７８％）。
HPLC条件:９０％ａｑ．CH₃CN→７０％ａｑ．CH₃CN、１５min、１ｍｌ／min
ＭＳ（ＦＡＢ^-）:m/z(%):1114(47)［Ｍ−Ｈ］^-;
ＨＲＭＳ; Ｃ₄₅Ｈ₆₆Ｆ₃Ｎ₇Ｏ₂₀Ｓ（[Ｍ−Ｈ]^-）:1114.4114(計算値)、1114.4163(実測値)
【００５２】
実施例１４（化合物（１１）→（１６））
実施例９の方法で化合物（１０）１．７ｍｇ（０．００２４ｍｍｏｌ）から調製した化合物（１１）とシアリルルイスＸ型四糖（純度９５％）１ｍｇ（０．００１２ｍｍｏｌ）を実施例１０と同様の条件で反応させた。同様にＨＰＬＣ精製後０．０００８５ｍｍｏｌの化含物（１６）を得た。（収率７１％）。
ＨＰＬＣ条件:85%aq.ＣＨ₃ＣＮ→65%aq.ＣＨ₃ＣＮ、１５min、１ｍｌ／min
ＭＳ（ＦＡＢ^-）:m/z(%):1405(29)［Ｍ−Ｈ］^-;
ＨＲＭＳ; Ｃ₅₆Ｈ₈₄Ｆ₃Ｎ₈Ｏ₂₈Ｓ（[Ｍ−Ｈ]^-）：1405.5068(計算値)、1405.5143(実測値)
【００５３】
参考例１ （レクチンの光親和性標識実験）
化合物（１２）〜化合物（１６）による光親和性標識実験は畑中ら（Y.Hatanaka、M.Hashimoto、H.Nishihara、H.Narimatsu、Y.Kanaoka、Carbohydr.Res.,294、95-108(1996)）の方法に準じて以下の通り行った。
０．１Ｍリン酸緩衝液、ｐＨ７.６中に、化合物（１２）〜化合物（１６）のいずれかの光親和性標識試薬（０．１ｍＭ）及びレクチン（サブユニット濃度０．１ｍＭ）、を含む光親和性標識試科を各０．０５ｍｌ調製した。別に対照試料として、化合物（１２）〜化合物（１６）のいずれかの光親和性標識試薬（０．１ｍＭ）、レクチン（サブユニットあたり０．１ｍＭ）、及び阻害剤０．１Ｍを含む０．１Ｍリン酸緩衝液、ｐＨ７．６を各0.05ml調製した。光親和性標識、レクチン、及び対照実験における阻害剤の組み合わせは、この順に以下の通りである。
【００５４】
化合物(12) ヒママメレクチン(RCA) N-アセチルラクトサミン
化合物(13) 小麦胚芽レクチン(WGA) シアリル-α2-3-ラクトース
化合物(14) イヌエンジュレクチン(MAL) シアリル-α2-3-ラクトース
化合物(15) ロータスレクチン(Lotus) メチル-α-フコピラノシド
化合物(16) イヌエンジュレクチン(MAL) シアリル-α2-3-ラクトース
以上のようにして調製した、光親和性標識試薬試料及び対照試料を遮光下、２５℃で３０分間インキュベートした後、３０Ｗ長波長ＵＶランプ（フナコシ、XX-15）を用いて、上方５ｃｍの距離から、氷上０℃で１時間照射した。照射後の各試料は、常法に従い12% SDS-ポリアクリルアミド電気泳動で分離後、PVDF膜に転写して化学発光解析により光標識バンドを検出した。対照実験により、光標識が阻害されることから、標識は各レクチンに特異的であることを確認した。結果を表−１に示す。
【００５５】
【表１】

【００５６】
実施例１５（化合物（１７））
水素化ナトリウム８.０ｇ(６０%油状分散液 ０.２ｍｏｌ)をトルエン２００ｍｌに懸濁し、-７０℃で2-[2-(2-クロロエトキシ)エトキシ]エタノール３３.７ｇ(０.２ｍｏｌ)をゆっくり加えた。この混合物にブロム酢酸エチル３３.４ｇ(０.２ｍｏｌ)を-７０℃でゆっくり滴下し、３０分後反応混合物を室温に放置した。２時間後、反応混合物に０℃で酢酸を加えて中和後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；酢酸エチル：ヘキサン＝３：２）で精製して３８.８ｇの無色油状物質を得た。これをＮ−メチル−２−ピロリドン２００ｍｌに溶解し、臭化エチル１６３ｇ(１.５ｍｏｌ)と臭化ナトリウム３.１ｇ(０.０３ｍｏｌ)を加えて６５℃に４８時間加熱した。溶媒を減圧留去して残渣をシリカゲルカラム（溶離剤；酢酸エチル：ヘキサン＝３：２）で精製して３２.３ｇの無色油状物質として化合物（１７)｛2-[2-(2-ブロムエトキシ)エトキシ]エトキシ酢酸エチル｝を得た（収率：７６％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=1.28(3H,t,J=7.3Hz,-CH₂-CH ₃ ),3.47(2H,t,J=6.3Hz,BrCH₂),3.68-3.75(8H,m,O-CH₂-),3.81(2H,t,J=6.3Hz,BrCH₂ CH ₂ O),4.15(2H,s,O-CH₂-CO-),4.23(2H,q,J=7.3Hz,-CH ₂ -CH₃)
【００５７】
実施例１６（化合物(２)+(１７)→(１８)）
化合物(２)４.６２ｇ(２０ｍｍｏｌ)、化合物(１７)７.１８ｇ(２４ｍｍｏｌ)、無水炭酸カリウム２.７６ｇ(２０ｍｍｏｌ)、Ｂｕ₄ＮＩ ７.３９ｇ(２０ｍｍｏｌ)、及びＤＭＳＯ ２０ｍｌの混合物をアルゴンガス雰囲気下、６０℃に１４時間撹拌した。その後、反応液に水を加え、酢酸エチルで抽出した。酢酸エチル層を水、０．１Ｎ ＨＣｌ、及び水で順次洗浄して無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；ジエチルエーテル：ヘキサン＝３：１）で精製して６.５８ｇの淡黄色油状物質として化合物（１８）｛2-[2-[2-(2-(エトキシカルボニルメトキシ)エトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンズアルデヒド｝を得た（収率：７６％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=1.28(3H,t,J=7.3Hz,-CH₂-CH ₃ ),3.67-3.93(12H,m,O-CH₂-),4.14(2H,s,O-CH₂-CO-),4.20(2H,q,J=7.3Hz,-CH ₂ -CH₃),4.52(2H,s,Ph-CH₂-),6.74(1H,s,Ph-H),6.85(1H,d,J=8.1Hz,Ph-H),7.84(1H,d,J=8.1Hz,Ph-H);
ＵＶ(ＥｔＯＨ)ｎｍ(ε):210(16,368),289.5(1,272)
【００５８】
実施例１７（化合物(１８)→(１９)）
化合物(１８)２.１７ｇ（５ｍｍｏｌ）を、エタノール１０ｍｌに溶解し、この溶液にＮａＢＨ₄ １８９ｍｇ（５ｍｍｏｌ）をゆっくり加えた。反応混合物を室温で３０分撹拌した後、反応液に０℃で酢酸を加えることによりｐＨ３付近に調節した後溶媒を減圧留去し、残渣を酢酸エチルで抽出した。酢酸エチル層を０.１Ｎ ＨＣｌ、水、及び飽和食塩水で順次洗浄して無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；酢酸エチル：ヘキサン＝１：１）で精製して１.７２ｇの淡黄色油状物質として化合物（１９)｛2-[2-[2-(2-(エトキシカルボニルメトキシ)エトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンジルアルコール｝を得た（収率：７６％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=1.28(3H,t,J=7.3Hz,-CH₂-CH ₃ ),3.43-3.87(12H,m,O-CH₂-),4.12(2H,s,O-CH₂-CO-),4.20(2H,q,J=7.3Hz,-CH ₂ -CH₃),4.52(2H,s,Ph-CH₂-),6.62(1H,s,Ph-H),6.79(1H,d,J=7.8Hz,Ph-H),7.30(1H,d,J=7.8Hz,Ph-H);
ＵＶ(ＥｔＯＨ)ｎｍ(ε):206.5(24,660),227(10,244),280(2,311)
【００５９】
実施例１８（化合物(１９)→(２０)）
化合物(１９)３.６０ｇ（８ｍｍｏｌ）と四臭化炭素３.３２ｇ（１０ｍｍｏｌ）とを、１６ｍｌのジクロロメタンに溶解し、０℃に冷却しながらトリフェニルホスフィン３.１５ｇ（１２ ｍｍｏｌ）をゆっくりと加えた。３０分後、反応混合物に水を加え、ジクロロメタンで抽出し、ジクロロメタン層を無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；酢酸エチル：ヘキサン＝１：１）で精製して３.３７ｇの淡黄色油状物質として化合物（２０）｛2-[2-[2-(2-(エトキシカルボニルメトキシ)エトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンジルブロミド｝を得た（収率：82％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=1.28(3H,t,J=7.3Hz,-CH₂-CH ₃ ),3.67-3.91(12H,m,O-CH₂-),4.14(2H,s,O-CH₂-CO-),4.20(2H,q,J=7.3Hz,-CH ₂ -CH₃),4.52(2H,s,Ph-CH₂-),6.65(1H,s,Ph-H),6.77(1H,d,J=8.0Hz,Ph-H),7.35(1H,d,J=8.0Hz,Ph-H);
Positive ion FABMS (3-nitrobenzylalcohol)ｍ／ｚ：５３５（Ｍ＋Ｈ）⁺
ＵＶ(ＥｔＯＨ)ｎｍ(ε):209(23,783),238.5(8,873),290(2,927)
【００６０】
実施例１９（化合物（２０）＋（２１）→（２２））
水素化ナトリウム２１６ｍｇ（６０%油状分散液 ５.４ｍｍｏｌ）をＴＨＦ１０ｍｌに懸濁し、-２０℃に冷却しながらＮ-ヒドロキシカルバミン酸 tert-ブチル(２１)７１８ｍｇ（６ｍｍｏｌ）を加えた。１５分後、化合物(２０)２.３１ｇ（４．５ｍｍｏｌ）を−１５℃に冷却しながら加え、反応混合物を０℃で撹拌した。２時間後、反応液に０℃で１Ｎ ＨＣｌを加えることによりｐＨ３付近に調節した後、酢酸エチルで抽出した。酢酸エチル層を無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；ヘキサン：ジエチルエーテル＝１：１０）で精製して２.１１ｇの淡黄色油状物質として化合物（２２）｛2-[2-[2-[2-(エトキシカルボニルメトキシ)エトキシ]エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンジルオキシカルバミン酸 tert-ブチルエステル｝を得た（収率：83％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=1.28(3H,t,J=7.1Hz,-CH₂-CH ₃ ),1.46(9H,s,C(CH₃)₃),3.67-3.88(12H,m,O-CH₂-),4.12(2H,s,O-CH₂-CO-),4.20(2H,q,J=7.1Hz,-CH ₂ -CH₃),4.91(2H,s,Ph-CH₂-),6.65(1H,s,Ph-H),6.79(1H,d,J=8.0Hz,Ph-H),7.38(1H,d,J=8.0Hz,Ph-H),7.67(1H,s,NH);
Positive ion FABMS (3-nitrobenzylalcohol)ｍ／ｚ：５８８（Ｍ＋Ｈ）⁺
ＵＶ:２０７ｎｍ(ε３４０２９),２２８.５ｎｍ(ε１３２２８),２８０.５ｎｍ(ε２９５９)
【００６１】
実施例２０ （化合物（２２）→（２３））
化合物（２２）２．２６ｇ（４ｍｍｏｌ）を１Ｎ ＮａＯＨ ２０ｍｌとエタノール１８ｍｌの混合溶媒に溶かし、０℃で３０分間撹拌した。反応液に０℃で１Ｎ ＨＣｌを加えることによりｐＨ３付近に調節した後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；クロロホルム：メタノール＝５：１）で精製して２．１ｇの淡黄色油状物質として化合物（２３）｛2-[2-[2-[2-(カルボキシメトキシ)エトキシ]エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-ジアジリン-3-イル]ベンジルオキシカルバミン酸 tert-ブチルエステル｝を得た（収率：98％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=1.46(9H,s,C(CH₃)₃),3.69-3.90(12H,m,O-CH₂-),4.13(2H,bs,O-CH₂-CO-),4.91(2H,s,Ph-CH₂-),6.65(1H,s,Ph-H),6.81(1H,d,J=7.8Hz,Ph-H),7.37(1H,d,J=7.8Hz,Ph-H),8.06(1H,bs,NH)
【００６２】
実施例２１ （化合物（２３）＋ （２４） →（２５））
化合物（２３）５３７ｍｇ（１ｍｍｏｌ）とカルボニルジイミダゾール １９４ｍｇ（１．２ｍｍｏｌ）を無水ＴＨＦ １ｍｌに溶解し、室温で撹拌しながらメチル スルファモイルベンゾエート（２４） ２５８ｍｇ（１．２ｍｍｏｌ）とジイソプロピルエチルアミン １５８ｍｇ（１．２ｍｍｏｌ）を加え、室温で１８時間反応させた。その後、溶媒を減圧留去して得られた残渣に水を加え、酢酸エチルで抽出した。酢酸エチル層を０．５Ｎ ＨＣｌ、飽和重曹水、及び飽和食塩水で順次洗浄して無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；酢酸エチル：エタノール＝１０：１）で精製して７３４ｍｇの淡黄色油状物質として化合物（２５）を得た（収率：74％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=1.40(9H,s,-C(CH₃)₃),3.69-3.90(12H,m,O-CH₂-),3.94(3H,s,CH₃),4.2(2H,bs,O-CH₂-CO-),5.02(2H,s,Ph-CH₂-),6.67(1H,s,Ph-H),6.80(1H,d,J=7.8Hz,Ph-H),7.34(1H,d,J=7.8Hz,Ph-H),7.69(1H,s,NH),8.14(4H,s,Ph-H)
【００６３】
実施例２２ （化合物（２５）→（２６））
化合物（２５）１２０ｍｇ（０．１５ｍｍｏｌ）を１Ｎ ＮａＯＨ ０．７５ｍｌとエタノール３．５ｍｌの混合溶媒に溶かし、０℃で３０分間撹拌した。０℃に冷却しながら５Ｎ ＨＣｌ ０．１５ｍｌを加えてｐＨ３付近に調節した後、酢酸エチルで抽出した。酢酸エチル層を飽和食塩水で洗浄して無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；クロロホルム：メタノール＝５：１）で精製して９９ｍｇの淡黄色油状物質として化合物（２６）を得た（収率：８９％）。
¹ＨＮＭＲ(400MHz,CDCl₃,25℃):δ=1.43(9H,s,-C(CH₃)₃),3.68-4.10(12H,m,O-CH₂-),4.23(2H,s,O-CH₂-CO-),4.91(2H,s,Ph-CH₂-),6.65(1H,s,Ph-H),6.77(1H,d,J=8.1Hz,Ph-H),7.35(1H,d,J=8.1Hz,Ph-H),8.17-8.23(4H,m,Ph-H),8.03(1H,bs,-NH)
【００６４】
実施例２３ （化合物（２６）＋（２７）→（２８））
化合物（２６）７２ｍｇ（０．１ｍｍｏｌ）をジクロロロメタン１ｍｌに溶解し、Ｎ−ヒドロキシスクシンイミド１３ｍｇ（０．１１ｍｍｏｌ）とＤＣＣ ２３ｍｇ （０．１１ｍｍｏｌ）を加え、室温で１時間撹拌した。溶媒を減圧留去し、残渣をＤＭＳＯ １ｍｌに溶解し、ビオチンヒドラジド（２７）２６ｍｇ（０．１ｍｍｏｌ）を加えて室温で一夜攪拌した。反応液を直接シリカゲルカラム（溶離剤；クロロホルム：メタノール＝５：１）で精製して１０ｍｇの淡黄色固体として化合物（２８）を得た（収率：１０％）
Positive ion FABMS (3-nitrobenzylalcohol) ｍ／ｚ：961（Ｍ＋Ｈ）⁺
【００６５】
実施例２４ （化合物（１１）→（２９））
実施例９の方法で化合物（１０）３．５ｍｇ（５μｍｏｌ）から調製した化合物（１１）とキトビオース １．１ｍｇ（２．５μｍｏｌ）を実施例１０と同様な条件で反応させた。同様にＨＰＬＣ精製後、１．２μｍｏｌの化合物（２９)を得た（収率：４８％）。
ＨＰＬＣ条件：８５％ aq.ＣＨ₃ＣＮ、１ml/min
Positive ion FABMS (3-nitrobenzylalcohol) ｍ／ｚ：1011（Ｍ＋Ｈ）⁺，1033（Ｍ＋Ｎａ）⁺
【００６６】
実施例２５ （化合物（２８）→（３０））
実施例９の方法で化合物（２８）９．６ｍｇ（１０μｍｏｌ）を脱保護し、キトビオース ２．１ｍｇ（５μｍｏｌ）を実施例１０と同様な条件で反応させた。同様にＨＰＬＣ精製後、１．０μｍｏｌの化合物（３０)を得た（収率：２％）。
ＨＰＬＣ条件：90％ aq.ＣＨ₃ＣＮ→80％ ａｑ.ＣＨ₃ＣＮ 20min,１ml/min
Positive ion FABMS (3-nitrobenzylalcohol)ｍ/ｚ:1266(Ｍ+Ｈ)⁺,1288(Ｍ+Ｎａ)⁺
【００６７】
参考例２ （ＷＧＡの光親和性標識実験）
化合物(２９)、化合物(３０)の光親和性標識実験を参考例１と同様な方法で行った。 ０.１Ｍリン酸緩衝液、ｐＨ７.６中に、化合物(２９)及び化合物(３０)のいずれかの光親和性標識試薬（０.１ｍＭ）及びＷＧＡ（小麦胚芽レクチン(wheat germ agglutinin);サブユニット濃度０.１ｍＭ）、を含む光親和性標識試料を各０.０５ｍｌ調製した。別に対照試料として、化合物(２９)及び化合物(３０)のいずれかの光親和性標識試薬（０.１ｍＭ）、ＷＧＡ（サブユニット濃度０.１ｍＭ）、及びキトビオース０.１Ｍを含む０.１Ｍリン酸緩衝液、ｐＨ７．６を各０.０５ｍｌ調製した。
以上のようにして調製した、光親和性標識試薬試料及び対照試料を遮光下、２５℃で３０分間インキュベートした後、３０Ｗ 長波長ＵＶランプ（フナコシ、XX-15）を用いて、上方５ｃｍの距離から、氷上０℃で１時間照射した。照射後の各試料は、常法に従い12% SDS-ポリアクリルアミド電気泳動で分離後、PVDF膜に転写して化学発光解析により光標識バンドを検出した。対照実験により、分子量２２ＫのＷＧＡバンドの光標識が阻害されることから、標識はＷＧＡに特異的であることを確認した。
【００６８】
スペーサーの切断は、照射後の各試料 １０μｌをとり、７Ｍグアニジン,ＥＤＴＡ ２Ｎａ、１０ｍＭを含む０.５Ｍトリス塩酸緩衝液、ｐＨ８.５、１０μｌに溶かし、０.７Ｍ ＤＴＴ １０μｌを加えて室温２時間反応させた。さらに、１Ｍ ヨード酢酸ナトリウム １０μｌを加えて室温４時間、さらに４Ｍ メルカプトエタノールを加えて５時間反応させた後、０.１Ｍリン酸緩衝液、ｐＨ７.６に対して透析した後、同様に電気泳動により解析した。切断反応後は、化合物(３０)によるＷＧＡラベルバンドの分子量２２Ｋが消失することにより、切断を確認した。
【００６９】
【発明の効果】
本発明の一般式(II)で示される化合物、特にオキシアミノ基およびその塩を有する上記化合物(11)は、還元末端を有する糖化合物に、特別の保護処理を施すことなく、一段階で反応して光反応基（ジアジリン）とビオチンを有する一般式(V)の糖結合フェニルジアジリン化合物を導くことができ、この一般式(V)の化合物は、光親和性標識試薬として、糖と相互作用する蛋白質（糖受容体）のビオチン標識に極めて有用である。しかも、該一般式(V)の化合物において、ビオチンが切断性スペーサーを介してフェニルジアリジン骨格に結合している化合物は、固定化アビジンからのラベル化した蛋白質の遊離・回収が容易である点で優れている。従って、一般式(II)の化合物は、光親和性標識試薬を簡便に製造するための安定した有用な化合物であり、この一般式(II)の化合物の合成中間体である、一般式(I)の化合物も有用性の高い物質である。
【図面の簡単な説明】
【図１】 本発明の一般式(I)化合物を合成する一例を示す反応スキームの概略図である。
【図２】 本発明の一般式(II)化合物を合成する一例を示す反応スキームの概略図である。
【図３】 本発明の一般式(Ｖ)化合物を合成する一例を示す反応スキームの概略図である。
【図４】 本発明の一般式(Ｖ)化合物を合成する一例を示す反応スキームの概略図である。
【図５】 本発明の一般式(I)化合物及び(IV)化合物を合成する一例を示す反応スキームの概略図である。
【図６】 本発明の一般式(Ｖ)化合物を合成する一例を示す反応スキームの概略図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel photoreactive phenyldiazirine compound, a photoreactive biotinylated phenyldiazirine compound, and a photoreactive sugar-linked biotinylated phenyldiazirine compound. Related to the photoreactive labeling reagent composed of the photoreactive biotinylated phenyldiazirine compound and the photoaffinity labeling reagent composed of the photoreactive sugar-linked biotinylated phenyldiazirine compound, which are expected to be used for analysis probes It is.
[0002]
[Prior art]
A photoaffinity labeling reagent is known as a useful probe capable of analyzing an interaction site between a drug or a ligand and a protein. An azido group has long been known as a photoreactive group for labeling proteins by photoreaction, but a diazirine group has attracted attention as a more excellent photoreactive group. Introduction of a diazirine group into a ligand derived from a drug or a biological substance is generally performed by an amide bond or an ester bond, and is further converted to a radioactive derivative for detection in a trace amount.
[0003]
Very recently, phenyldiazirine derivatives having biotin residues in the molecule via a spacer compound have been developed as a means to simultaneously detect trace amounts of proteins and affinity purification. For example, as a ligand derived from biological substances, It has been reported that a non-radioactive photoaffinity labeling reagent can be synthesized by linking a sugar compound to the carboxyl group of the diazirine derivative (Journal of Organic Synthetic Chemistry, Vol. 56, No. 7, 581). ˜590 (1998); Pharmacia, Vol. 34, No. 8, pp 772-776 (1998); Biochem. J. (1998) 330, 1209-1215). However, in this method, when a saccharide compound is bonded to the diazirine derivative, an amino group is introduced into the saccharide compound in advance, in the presence of a carboxyl group possessed by the diazirine derivative and a condensing agent such as dicyclohexylcarbodiimide (DCC). The reaction was necessary and the operation was complicated.
On the other hand, the use of an oxyamino group has attracted attention as a method for specifically modifying a biological substance, but attempts to introduce the oxyamino group into a photoreactive compound and bond it to a sugar compound have not yet been made. It has not been.
[0004]
[Problems to be solved by the invention]
The present invention relates to a photoreactive labeling reagent containing a novel photoreactive biotinylated phenyldiazirine compound that can lead to a photoreactive probe that does not need to be radioactively labeled, and a protein that can interact with a sugar ( To provide a photoaffinity labeling reagent containing a non-radioactive photoreactive sugar-linked biotinylated phenyldiazirine compound that is expected to be useful for studying the functions and structures of biopolymers such as sugar receptors) With the goal.
A further object of the present invention is to provide novel biotinylated phenyldiazirine compounds and sugar-linked biotinylated phenyldiazirine compounds useful for constructing these labeling reagents, and optionally via a spacer where the biotin residue is cleavable. The compound linked to the phenyldiazirine compound, and in addition to providing a novel phenyldiazirine compound that is a synthetic intermediate for obtaining these compounds, as well as this novel sugar-linked biotinylated phenyldiazirine compound And a method for labeling a sugar receptor with the compound.
[0005]
[Means for Solving the Problems]
The gist of the present invention is a biotinylated phenyldiazirine compound represented by at least one of the above general formulas (II) to (IV), and a phenyldia represented by the above general formula (I), which is a synthetic intermediate thereof. A glycine-linked biotin derived from at least one of the dilin compound and the general formulas (II) to (IV) and represented by at least one of the general formulas (V) to (VII) corresponding to each formula The present invention relates to a photoreactive labeling reagent containing a compound represented by at least one of the general formulas (II) to (IV), and the general formulas (V) to (V) VII) is a photoaffinity labeling reagent comprising a sugar-linked biotinylated phenyldiazirine compound represented by at least one.
A further subject matter of the present invention is a reaction comprising reacting a biotinylated phenyldiazirine compound represented by at least one of general formulas (II) to (IV) with a sugar compound having a reducing end. A method for producing a sugar-linked biotinylated phenyldiazirine compound represented by at least one of (VII) and a method for labeling a sugar receptor using the sugar-linked biotinylated phenyldiazirine compound.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The intermediate compound for producing the compound constituting the photoaffinity labeling reagent in the present invention is essentially trifluoromethylphenyl having a diazirine group as a photoreactive group for labeling a sugar receptor protein by a photoreaction. Any phenyldiaziline derivative may be used as long as it has diazirine as a basic skeleton and a group having an amino group to which a ligand such as sugar can be bonded to a benzene nucleus and a group having a labeling compound. Specifically, in the phenyldiazirine derivative, a group selected from an amino group, an amino group salt, a protected amino group and the like is bonded to a benzene nucleus via a spacer such as an alkylene group and affects the ability to bind to a ligand. In order to reduce the influence of the photoreactive group of the type, it is bonded to the P-position with respect to the bonding position of the photoreactive group.
[0007]
In addition, the group having a labeling compound has a labeling compound such as biotin bonded to the benzene nucleus through a hydrophilic spacer containing a polyoxyethylene group or the like. In that case, the labeling compound, ie biotin, can be bound to the benzene nucleus via the terminal amino group of the spacer. On the other hand, biotinylated sugar receptor (labeled protein) is affinity-purified using avidin-immobilized carrier (immobilized avidin), and immobilization usually used for separation and purification when re-release / recovery is desired. Since the binding force between avidin and biotin is strong and the recovery rate of the labeled protein may decrease, the labeled protein and biotin may be bound via a cleavable spacer so that they can be separated under mild conditions. For example, biotin can be linked to the benzene nucleus through a spacer introduced at the polyoxyethylene group end with a cleavable spacer such as acylsulfonamide.
[0008]
The photoreactive biotinylated phenyldiazirine compound represented by the general formula (II) of the present invention represents this derivative.
The compound represented by the following general formula (I) of the present invention is an intermediate for synthesizing the biotinylated phenyldiazirine compound represented by the general formula (II), and is a novel compound.
[0009]
[Chemical 8]

[0010]
In general formula (I), R¹Is -NHP¹Or -COX¹Represents R²Is an aldehyde group, a hydroxy lower alkyl group, a halogenated lower alkyl group, a phthalimidooxy lower alkyl group, or-(CH₂)_x-O-NHP¹Represents P¹Represents an amino protecting group and X¹Is an alkoxy group, a hydroxy group, or -NHSO₂-X²--CO-X^ThreeX²Represents an arylene group or an alkylene group, and X^ThreeRepresents an alkoxy group or a hydroxy group, m represents an integer of 1 to 4, preferably 1 to 2, n represents an integer of 1 to 6, preferably 1 to 4, and x represents 1 to 4, preferably 1 Represents an integer of ~ 2.
[0011]
In the present invention, the lower alkyl group represents an alkyl group having 1 to 4 carbon atoms such as methyl, ethyl, propyl and butyl, and is preferably linear. Moreover, as an alkoxy group, it is a C1-C6 alkoxy group, for example, a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a pentoxy group etc. are mentioned. Examples of the arylene group include arylene groups having 5 to 14 carbon atoms such as a phenylene group and a naphthylene group which may have a substituent such as a methyl group and an ethyl group, and a phenylene group is preferable. As an alkylene group, C1-C6 alkylene groups, such as a methylene group, ethylene group, a propylene group, a butylene group, are mentioned, A linear group is preferable.
As amino protecting group, t-butoxycarbonyl group, trifluoroacetyl group, benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl, p-toluenesulfonyl group, 9-fluorenylmethyloxycarbonyl group, trityl group, phthaloyl group , O-nitrophenylsulfenyl group, 3-nitro-2-pyridinesulfenyl group and the like. Of these, the t-butoxycarbonyl group is convenient.
[0012]
The biotinylated phenyldiazirine compound of the present invention is a compound represented by the following general formula (II), and the compound of the above general formula (I) is biotinylated and optionally R²Is converted to an aminooxy lower alkyl group.
[0013]
[Chemical 9]

[0014]
      In general formula (II), R^ThreeIsAMino group, -NHP¹Or a salt of an amino group, P¹Represents an amino protecting group, m represents an integer of 1 to 4, preferably 1 to 2, n represents an integer of 1 to 6, preferably 1 to 4, and x represents 1 to 4, preferably 1 to 2. Represents an integer. The amino protecting group has the same meaning as in the above general formula (I). Examples of the amino group salt include salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid, and salts of organic acids such as acetic acid, trifluoroacetic acid, propionic acid, and methanesulfonic acid. Of these, trifluoroacetic acid is advantageous for producing compounds of general formula (V).
[0015]
Y¹Is —NH— or —CONHSO₂-X²-CO- represents X²Represents an arylene group or an alkylene group, Y²Represents biotin or a biotin derivative residue. The arylene group and the alkylene group have the same meaning as in the general formula (I). In the present invention, the biotin derivative means biotin in which a terminal group (-OH) of biotin is replaced with a group capable of reacting with an amino group or a carboxyl group. For example, the biotin derivative has a group capable of reacting with a carboxyl group, Biotin) -NH-Zq-NH₂And a compound having an amino group at the terminal, preferably biotin hydrazide.
More specific compounds of the compound represented by the general formula (II) of the present invention are compounds represented by the following general formulas (III) and (IV).
[0016]
Embedded image

In general formula (III), R^Three, P¹, M, n and x have the same meaning as in general formula (II).
[0017]
Embedded image

In general formula (IV), R^Three, P¹, M, n and x have the same meanings as in the general formula (II), and X²Represents an arylene group or an alkylene group, and has the same meaning as in general formula (I) above. Z represents an alkylene group having 1 to 20 carbon atoms which may be interrupted by a heteroatom such as oxygen, sulfur or nitrogen, and examples thereof include a methylene group, an ethylene group, a propylene group and a (poly) oxyethylene group. It is done. q represents 0 or 1;
[0018]
The sugar-linked biotinylated phenyldiazirine compound represented by the following general formulas (V) to (VII) of the present invention is a biotin having a terminal amino group or a salt of an amino group in the general formulas (II) to (IV). It is a compound obtained by reacting a reduced phenyldiazirine compound with an aldehyde group at the reducing end of a sugar compound having a reducing end.
[0019]
Embedded image

[0020]
In general formula (V), R^FourRepresents a residue of a sugar compound having a reducing end, m represents an integer of 1 to 4, preferably 1 to 2, n represents an integer of 1 to 6, preferably 1 to 4, and x represents 1 to 4, preferably an integer of 1 to 2. Y¹Is —NH— or —CONHSO₂-X²-CO- represents X²Represents an arylene group or an alkylene group, Y²Represents biotin or a biotin derivative residue and has the same meaning as in general formula (II) above.
The compounds represented by the following general formulas (VI) and (VII) of the present invention are more specific compounds of the compounds represented by the general formula (V).
[0021]
Embedded image

In general formula (VI), R^Four, M, n, and x have the same meaning as in the general formula (V).
[0022]
Embedded image

In general formula (VII), R^Four, M, n, x and X²Represents the same meaning as in the general formula (V), and Z and q represent the same meaning as in the general formula (IV).
The compound represented by the general formula (VII) is a sugar-linked biotinylated phenyldiazirine compound having a cleavable spacer, particularly X²A compound having a phenylene group and q = 0 is preferable as a compound constituting a photoaffinity labeling reagent useful for separation and recovery of a labeled protein.
[0023]
Examples of sugar compounds to be reacted with the compounds represented by the general formulas (II) to (IV) include polysaccharides having a reducing end, oligosaccharides, monosaccharides such as mucin-type saccharides, Asn-type saccharides, sialyl saccharides, and glycosaminos. Examples include glycans, lactosamines, N-acetyllactosamines, lactosamine oligosaccharides, sialyllactosamines, glucans, mannans, fructans, galactans, polyuronic acids, oligoamino acids, polyaminosaccharides, galactooligosaccharides, and the like. R^FourMore specific examples of N-acetyl lactosyl group, sialyl-α2-3-lactosyl group, sialyl-α2-3-N-acetyllactosaminyl group, Lewis X type trisaccharide residue, sialyl Lewis X type Examples thereof include tetrasaccharide residues and chitobiose residues, but are not limited to these as long as they are sugar compounds capable of binding shown in the following schematic diagram. That is, in the compound represented by the general formula (II) of the present invention, R^ThreeThe bond between a compound in which is an amino group or a salt of an amino group and a sugar compound having a reducing end is selective, and the bond is shown as follows.
[0024]
Embedded image

[0025]
In the compounds represented by the general formulas (II) to (IV) of the present invention, R^ThreeThe reaction between a compound in which is an amino group or a salt of an amino group and a sugar compound having a reducing end is pH-dependent, and the reaction proceeds advantageously under weak acidity. Usually, the reaction proceeds at pH 1 to 7, but is preferably performed by adjusting the pH to 4 to 5 using a buffer solution.
One of the photoreactive bithionized phenyldiazirine compounds represented by the general formulas (II) to (IV) of the present invention is characterized in that an oxyamino group is introduced into the phenyl skeleton via a spacer. The oxyamino group simply does not require the protection of the aldehyde group at the reducing end of the sugar compound having a reducing end and the hydroxyl group of the sugar chain, or the presence of a condensing agent such as DCC, which is conventionally required. By simply mixing with a sugar compound, it is bound by a Schiff base forming reaction to produce a sugar-linked biotinylated phenyldiazirine compound represented by the general formulas (V) to (VII). And the photoaffinity labeling reagent of this invention can be comprised with this compound. The reagent may contain additives such as water, a buffer, a stabilizer, and a salt as long as the function of the compound is not impaired. In addition, since the oxyamino group of the compounds of the general formulas (II) to (IV) can be bonded to a ketone group or a carboxyl group in addition to the aldehyde group, when the sugar compound has these reactive groups, the functional group and the oxy An amino group may be reacted.
The photoaffinity labeling reagent broadly refers to a labeling reagent in which a ligand is bound to a compound having a photoreactive group. In this specification, the term “photoaffinity labeling reagent” refers to photoreactivity. It means a sugar receptor affinity labeling reagent, and a sugar receptor means a protein that has affinity for sugar, that is, a protein that binds to sugar by specific interaction with sugar. Includes sugar-specific lectins, receptors, enzymes, antibodies, and the like.
[0026]
In a mixed system containing the photoaffinity labeling reagent of the present invention and various proteins, photoaffinity labeling is carried out by irradiating light at the same time or after the interaction between the sugar compound of the labeling reagent and the sugar receptor. The diazirine group of the labeling reagent is cross-linked by a photoreaction at the site of interaction (amino acid) with the sugar compound of a protein (sugar receptor) that specifically interacts with the sugar compound of the reagent. Furthermore, the protein (labeled protein) labeled with the photoaffinity labeling reagent containing biotin can be detected with high sensitivity by a detection system known per se for detecting biotin, such as avidin, streptavidin, etc. By using an avidin column in which is bound to a carrier, the target protein can be easily purified by affinity chromatography. Furthermore, in the present invention, by applying a compound in which biotin is bound via a cleavable spacer as a sugar compound constituting the photoaffinity labeling reagent, when performing affinity chromatography with an avidin column, under mild conditions. The biotin moiety can be easily separated, and the target protein can be obtained with a high recovery rate. Therefore, the photoaffinity labeling reagent of the present invention bound with a sugar compound is expected to be a very useful probe or purification means for analyzing the structure and function of a sugar chain-related protein.
[0027]
A group which is represented by the general formula (II) of the present invention and has a oxyamino group in the phenyl skeleton and selectively reacts with the oxyamino group, such as an aldehyde group, a ketone group, It may be induced to a photoaffinity labeling reagent in a broad sense by binding a biological component other than a drug or a sugar compound having a carboxyl group or the like, if necessary, as a ligand. it can. This reagent becomes a probe for analyzing the structure and function of a protein having affinity for a biological substance other than a drug or a sugar compound, and also enables isolation and purification. In this way, the application that combines a probe library and ultra-trace analysis using a mass spectrometer that can be obtained by concatenating a wide variety of ligands opens up the prospect of high-speed and high-precision analysis of protein functions. Expected as a thing. Therefore, the compound of the present invention represented by the general formula (II) is a compound having extremely wide utility.
[0028]
An example of the synthesis of the compound of the present invention will be described below based on the reaction scheme shown in FIGS.
First, according to the reaction process shown in FIG. 1, the manufacturing process of this invention compound shown by general formula (I), ie, compound (4)-(7) in a figure, is demonstrated.
The starting material compound (1), [2-methoxy-4- (1-azi-2,2,2-trifluoroethyl) benzaldehyde], was prepared by the method of Hashimoto et al. (M. Hashimoto, Y. Kanaoka, Y. Hatanaka, Heterocycles. 46, 119-122 (1997)).
Compound (1) is a compound of BBr at around −20 ° C. in an inert gas atmosphere in a solvent such as dichloromethane._ThreeTo produce compound (2).
To the compound (2) produced, the compound (3) was added to a base such as anhydrous potassium carbonate and Bu._FourCompound (4) can be produced by reacting in the presence of a phase transfer catalyst such as NI. This reaction can be performed in dimethylformamide (DMF) at around 60 ° C. under an inert gas atmosphere. Compound (3) is represented by the formula P¹NH (CH₂)_m(OC₂H_Four)_n-Br, m = n = 2, P¹Is a compound having a t-butoxycarbonyl group and can be prepared by the method of Hatanaka et al. (Y. Hatanaka, M. Hashimoto, Y. Kanaoka, Bioorg. Med. Chem. 2, 1367-1373 (1994)). .
[0029]
Compound (4) is a NaBH in a solvent such as ethanol._FourThe compound (5) can be obtained by reduction at around room temperature. The reduction reaction product can be subjected to the next reaction step without purification.
The resulting compound (5) is led to compound (6) by bromination with carbon tetrabromide and triphenylphosphine in a solvent such as dichloromethane. This reaction is preferably performed by cooling to around 0 ° C.
Compound (6) can be obtained by reacting compound (6) with N-hydroxyphthalimide in the presence of a base such as anhydrous potassium carbonate. This reaction can be carried out in a solvent such as dimethyl sulfoxide (DMSO) at around room temperature.
[0030]
Next, according to FIG. 2, the compound (7) is biotinylated, and the compound of the present invention represented by the general formula (II), particularly the general formula (III), that is, the production steps of the compounds (8) to (10) in the figure Will be explained.
Compound (7) is allowed to react with trifluoroacetic acid in an organic solvent such as dichloromethane at around 0 ° C., and then triethylamine (Et_ThreeIn the presence of an organic base such as N), biotinylated compound (8) can be obtained by reacting N-hydroxysuccinimide ester of biotin (biotin-OSu) in a solvent such as DMF at around room temperature.
[0031]
Compound (9) can be produced by reacting compound (8) with, for example, anhydrous hydrazine in a methanol solvent at around room temperature, but this compound (9) is easily decomposed during purification by column chromatography. It is preferably used for the next reaction without purification treatment.
Compound (9), which is a reaction product of compound (8) and hydrazine, is used as it is, for example, in a mixed solvent of chloroform and acetonitrile in the presence of an organic base such as triethylamine, di-t-butyl dicarbonate {[ (CH_Three)_ThreeCOCO]₂Compound (10) can be obtained by reacting with O} at around room temperature.
[0032]
Further, as shown in FIGS. 3 and 4, a sugar compound having a reducing end is introduced into the biotinylated phenyldiazirine compound obtained in the above step, and the general formula (V), especially the general formula (VI) Inventive compounds, ie compounds (12) to (16), can be produced.
Compound (11) can be obtained by allowing compound (10) to react with trifluoroacetic acid in an organic solvent such as dichloromethane at around 0 ° C. The produced compound (11) can be immediately reacted with various sugar compounds having a reducing end to obtain biotinylated phenyldiazirine compounds to which the respective sugar compounds are bound. For example, compound (12) can be obtained by reacting compound (11) with N-acetyllactosamine (Galβ1-4GlcNAc) at about 37 ° C. in water-containing acetonitrile. Similarly, sialyl-α2-3-lactose (NeuNAcα2-3Galβ1-4Glc), sialyl-α2-3-N-acetyllactosamine (NeuNAcα2-3Galβ1-4GlcNAc), Lewis X-type trisaccharide (Galβ1-4 (Fucα1-3 ) GlcNAc) or sialyl Lewis X-type tetrasaccharide (NeuNAcα2-3Galβ1-4 (Fucα1-3) GlcNAc), respectively, to thereby obtain compounds (13) to (16). Further, instead of trifluoroacetic acid acting on the compound (10), a salt of an inorganic acid can be produced by using hydrochloric acid / acetic acid, a hydrochloric acid / dioxane mixture or the like.
[0033]
Next, according to the reaction steps shown in FIGS. 5 and 6, the compound represented by the general formula (I), that is, the compounds (18) to (20), (22), (23), (25), ( 26), production process of compounds having a cleavable spacer represented by general formulas (IV) and (VII), that is, compounds (28) and (30) in the figure, in addition to compound (29) of general formula (VI) Will be explained.
Compound (2) is synthesized from compound (1) by the process shown in FIG. Compound (2) is converted to compound (17) with a base such as anhydrous potassium carbonate and Bu._FourBy reacting in the presence of a phase transfer catalyst such as NI, compound (18) can be produced, and this reaction can be performed in dimethyl sulfoxide (DMSO) under an inert gas atmosphere at around 60 ° C. . In addition, compound (18) is R in general formula (I).²Is an aldehyde group, R¹Is COX¹And X¹Is an ethoxy group, m = 1, n = 3.
[0034]
On the other hand, the compound (17) can be synthesized as follows. That is, 2- [2- (2-chloroethoxy) ethoxy] ethanol was reacted with ethyl bromoacetate in a toluene solvent containing sodium hydride at −70 ° C., and the solvent was distilled off from the obtained reaction mixture. The residue is purified by column chromatography. After the purified residue is dissolved in a solvent such as N-methylpyrrolidone, ethyl bromide and sodium bromide are added and reacted at 65 ° C. After the solvent is distilled off from the reaction product, the residue is further purified by column chromatography.
[0035]
Compound (18) is, for example, NaBH in a solvent such as ethanol._FourThe compound (19) can be reduced by reduction at around room temperature. The produced compound (19) is led to the compound (20) by bromination with carbon tetrabromide and triphenylphosphine in a solvent such as dichloromethane. This reaction is preferably performed by cooling to around 0 ° C.
Compound (20) is obtained by reacting N-hydroxycarbamic acid alkyl ester (Compound 21) such as N-hydroxycarbamic acid t-butyl ester in the presence of a base such as sodium hydride. Can do. This reaction can be performed in a solvent such as tetrahydrofuran (THF) at around 0 ° C.
[0036]
Compound (22) is hydrolyzed in, for example, a mixed solvent of ethanol and an aqueous alkali solution to produce compound (23). Compound (23) and carbonyldiimidazole are dissolved in a solvent such as THF, and compound (24) such as sulfamoylbenzoate, which is a compound for imparting cleavability to the spacer, is present in the presence of an organic base such as diisopropylethylamine. The reaction is allowed to proceed to the compound (25).
The compound (24) used for imparting the cleavage property includes the kind of the sugar compound bound to the labeling reagent of the present invention, and the protein that is a receptor for the sugar that specifically interacts with the sugar compound. The sulfamoyl benzoate is preferably selected because it is cleaved under mild conditions and has good stability.
[0037]
Compound (25) is produced by, for example, hydrolyzing in a mixed solvent of ethanol and an aqueous alkali solution to produce compound (26), and reacting compound (26) with N-hydroxysuccinimide and carbodiimides (DCC, etc.) After making into an ester, a biotin derivative such as biotin hydrazide compound (27) is reacted to biotinylate to obtain a biotinylated phenyldiazirine compound (28) having a cleavable spacer.
A sugar-linked biotinylated phenyldiazirine compound (30) having a cleavable spacer by introducing a sugar compound (chitobiose: GlcNAcβ1-4GlcNAc) into this compound (28) in the same manner as in the steps shown in FIGS. On the other hand, chitobiose is introduced as a sugar compound having a reducing end into the compound (11) to produce a compound (29) having no cleavable spacer.
[0038]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples, unless the summary is exceeded. In the examples, each compound number corresponds to the compound number shown in FIGS.
[0039]
Example 1 (Compound (1) → (2))
3.66 g (0.015 mol) of compound (1) is dissolved in 30 ml of dichloromethane, and BBr is added to this solution at −20 ° C. under an argon gas atmosphere._Three3.76 g (0.015 mol) was slowly added dropwise. After the dropwise addition, the solution was stirred at 0 ° C. for 1 hour, and water was added at 0 ° C. The reaction mixture was extracted with dichloromethane, the dichloromethane layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent; dichloromethane: hexane = 1: 2) to obtain 3.18 g of a white solid. As compound (2) {2-hydroxy-4- [3- (trifluoromethyl) -3H-diazilin-3-yl] benzaldehyde} was obtained (yield: 92%).
IR (nujol): v = 3170,1665cm^-1;
¹HNMR (400MHz, CDCl_Three, 25 ° C): δ = 11.05 (s, 1H), 9.92 (s, 1H), 7.60 (d, J = 8.0Hz, 1H), 6.78 (d, J = 8.0Hz, 1H), 6.77 (br s, 1H);
MS (EI⁺): M / z (%): 230 (38) [M⁺];
HRMS: C₉H_FiveF_ThreeN₂O₂ [M⁺] = 230.0303 (calculated value), 230.0291 (actually measured value)
[0040]
Example 2 (Compound (2) + (3) → (4))
Compound (2) 2.76 g (0.012 mol), Compound (3) {2- [2- (2-tert-butoxycarbonylaminoethoxy) ethoxy] ethyl bromide} 4.50 g (0.0144 mol), anhydrous potassium carbonate 1.66 g (0.012 mol), Bu_FourA mixture of NI554 mg (1.5 mmol) and DMF 15 ml was stirred at 60 ° C. for 14 hours under an argon gas atmosphere. Thereafter, the solvent of the reaction mixture was distilled off under reduced pressure, water was added to the resulting residue, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with water, 0.1N HCl, and water, dried over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified with a silica gel column (eluent; ethyl acetate: hexane = 1: 1). Purify compound (4) as a pale yellow oily substance (5.3) {2- [2- [2- (2-tert-butoxycarbonylaminoethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-diazilin-3-yl] benzaldehyde} (yield: 96%).
¹HNMR (400MHz, CDCl_Three, 25 ° C): δ = 10.5 (s, 1H), 7.85 (d, J = 8.1Hz, 1H), 6.86 (d, J = 8.1Hz, 1H), 6.75 (br s, 1H), 5.00 (br, 1H), 4.27 (t, J = 4.8Hz, 2H), 3.92 (t, J = 4.8Hz, 2H), 3.7 (m, 2H), 3.6 (m, 2H), 3.54 (t, J = 5.1Hz, 2H), 3.3 (m, 2H), 1.43 (s, 9H);
MS (FAB⁺): M / z (%): 484 (84) [M + Na]⁺, 462 (15) [M + H]⁺;
HRMS: C₂₀H₂₇F_ThreeN_ThreeO₆ [M + H]⁺= 462.1852 (calculated value), 462.1874 (actually measured value)
[0041]
Example 3 (compound (4) → (5))
3.69 g (8 mmol) of compound (4) was dissolved in 40 ml of ethanol, and NaBH was added to this solution._Four303 mg (8 mmol) was added slowly. After stirring the reaction mixture at room temperature for 30 minutes, the solvent was distilled off under reduced pressure, water was added to the residue, and the pH was adjusted to around 3 by adding 1N HCl at 0 ° C., followed by extraction with ethyl acetate. The ethyl acetate layer was washed successively with water, saturated aqueous sodium hydrogen carbonate and saturated brine and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure to give crude compound (5) {2- [2- [ 2- (2-tert-butoxycarbonylaminoethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-diazilin-3-yl] benzyl alcohol} was obtained. The crude compound (5) was immediately used in the next reaction without further purification.
¹HNMR (400MHz, CDCl_Three, 25 ° C): δ = 7.26 (d, J = 8.0Hz, 1H), 6.79 (d, J = 8.0Hz, 1H), 6.66 (br s, 1H), 6.01 (br, 1H), 5.02 (br, 1H), 4.66 (s, 2H), 4.22 (t, J = 4.8Hz, 2H), 3.84 (t, J = 4.8Hz, 2H), 3.7 (m, 2H), 3.6 (m, 2H), 3.51 ( t, J = 5.1Hz, 2H), 3.3 (m, 2H), 1.45 (s, 9H);
MS (FAB⁺): M / z (%): 486 (100) [M + Na]⁺, 464 (16) [M + H]⁺;
HRMS: C₂₀H₂₉F_ThreeN_ThreeO₆ [M + H]⁺= 464.2008 (calculated value), 464.1997 (actually measured value)
[0042]
Example 4 (compound (5) → (6))
The crude compound (5) obtained from 3.69 g (8 mmol) of compound (4) and 3.32 g (0.01 mol) of carbon tetrabromide were dissolved in 16 ml of dichloromethane by the above method and cooled to 0 ° C. Then, 3.15 g (0.012 mol) of triphenylphosphine was slowly added. After 10 minutes, 1.66 g (0.012 mol) of anhydrous potassium carbonate was added to the reaction mixture and stirred at 0 ° C. for an additional 30 minutes. Then, water was added to the reaction mixture, extracted with dichloromethane, the dichloromethane layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent; ethyl acetate: hexane = 1: 1). 3.62 g of light yellow oily compound (6) {2- [2- [2- (2-tert-butoxycarbonylaminoethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl)- 3H-diazilin-3-yl] benzyl bromide} was obtained (yield: 86%).
¹HNMR (400MHz, CDCl_Three, 25 ° C): δ = 7.35 (d, J = 8.1Hz, 1H), 6.77 (d, J = 8.1Hz, 1H), 6.65 (br s, 1H), 5.00 (br, 1H), 4.51 (s, 2H), 4.21 (t, J = 4.8Hz, 2H), 3.91 (t, J = 4.8Hz, 2H), 3.75 (m, 2H), 3.65 (m, 2H), 3.55 (t, J = 5.1Hz, 2H), 3.3 (m, 2H), 1.43 (s, 9H);
MS (FAB⁺): M / z (%): 550 (41) [M + Na]⁺, 548 (40), 528 (12) [M + H]⁺, 526 (11);
HRMS :(⁷⁹Br) C₂₀H₂₈BrF_ThreeN_ThreeO_Five [M + H]⁺= 526.1164 (calculated value), 526.1193 (actual value)
[0043]
Example 5 (compound (6) → (7))
A mixture of 2.63 g (5 mmol) of Compound (6), 979 mg (6 mmol) of N-hydroxyphthalimide, 691 mg (5 mmol) of anhydrous potassium carbonate, and 10 ml of DMSO was stirred at room temperature for 12 hours. Thereafter, the solvent of the reaction mixture was distilled off under reduced pressure using an oil pump, water was added to the resulting residue, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with water, 1N NaOH and saturated brine, dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent; hexane: acetone = 3: 1). 2.59 g of compound (7) {2- [2- [2- (2-tert-butoxycarbonylaminoethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H as a pale yellow oily substance -Diazilin-3-yl] benzyloxyphthalimide} was obtained (yield: 85%).
¹HNMR (400MHz, CDCl_Three, 25 ° C): δ = 7.82-7.73 (AB, 4H), 7.54 (d, J = 7.9Hz, 1H), 6.81 (d, J = 7.9Hz, 1H), 6.66 (br s, 1H), 5.27 ( s, 2H), 5.05 (br, 1H), 4.15 (t, J = 4.8Hz, 2H), 3.86 (t, J = 4.8Hz, 2H), 3.7 (m, 2H), 3.6 (m, 2H), 3.54 (t, J = 5.2Hz, 2H), 3.3 (m, 2H), 1.42 (s, 9H);
MS (FAB⁺): M / z (%): 631 (100) [M + Na]⁺, 609 (9) [M + H]⁺; HRMS: C₂₈H₃₂F_ThreeN_FourO₈ [M + H]⁺= 609.2172 (calculated value), 609.2164 (actually measured value)
[0044]
Example 6 (Compound (7) → (8))
Compound (7) 304 mg (0.5 mmol) was dissolved in 0.5 ml of dichloromethane, 0.5 ml of trifluoroacetic acid was slowly added while cooling to 0 ° C., and the reaction mixture was stirred at 0 ° C. for 1 hour. To the residue obtained by distilling off the solvent under reduced pressure, 1 ml of toluene was added and distilled off under reduced pressure. This operation was repeated 3 times in total, and the yellow oily residue obtained by removing the remaining excess trifluoroacetic acid was dissolved in 0.5 ml of DMF, and 171 mg (0.5 mmol) of biotin N-hydroxysuccinimide ester was dissolved in 2 ml of DMF. The solution was added and the reaction mixture was stirred for 14 hours at room temperature in the presence of a catalytic amount of triethylamine. Thereafter, the solvent was distilled off, and the resulting residue was dissolved in a mixed solvent of dichloromethane and methanol 1: 1, washed successively with 1N NaOH, saturated brine, 0.1N HCl, and saturated brine, and anhydrous. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent: chloroform: ethanol = 10: 1) to give 320 mg of colorless solid as compound (8) {2- [2- [2 -(2-Biotinylaminoethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-diazilin-3-yl] benzyloxyphthalimide} was obtained (yield: 87%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 7.83-7.73 (AB, 4H), 7.53 (d, J = 7.9Hz, 1H), 6.84 (s, 1H), 6.81 (d, J = 7.9Hz, 1H), 6.64 (s , 1H), 6.61 (s, 1H), 5.64 (s, 1H), 5.26 (s, 2H), 4.5 (m, 1H), 4.3 (m, 1H), 4.15 (t, J = 4.8Hz, 2H) , 3.90 (t, J = 4.8Hz, 2H), 3.7 (m, 2H), 3.65 (m, 2H), 3.55 (m, 2H), 3.4 (br m, 2H), 3.1 (m, 1H), 2.9 (m, 1H), 2.72 (d, J = 12.5Hz, 1H), 2.19 (t, J = 7.5Hz, 2H), 1.8-1.6 (m, 4H), 1.4 (m, 2H);
MS (FAB⁺): m / z (%): 757 (16), [M + Na]⁺, 735 (62) [M + H]⁺;
HRMS: C₃₃H₃₈F_ThreeN₆O₈S [M + H]⁺= 735.2424 (calculated value), 735.2458 (actually measured value)
[0045]
Example 7 (compound (8) → (9))
73 mg (0.1 mmol) of compound (8) was dissolved in 1 ml of 1M methanol solution of anhydrous hydrazine and reacted at room temperature for 30 minutes. Thereafter, 1 ml of toluene was added to the residue obtained by distilling off the solvent under reduced pressure, followed by distilling off under reduced pressure. This operation was repeated 3 times in total, and the excess hydrazine remaining was removed to obtain a crude compound (9) as a colorless solid. The crude compound (9) was immediately used for the next reaction without purification.
[0046]
Example 8 (compound: (9) → (10))
The crude compound (9) obtained from 73 mg (0.1 mmol) of the compound (8) by the above method was dissolved in 1 ml of a mixed solvent of chloroform and acetonitrile 1: 1, and [(CH_Three)_ThreeCOCO]₂109 mg (0.5 mmol) of O and 51 mg (0.5 mmol) of triethylamine were added and reacted at room temperature for 14 hours. Thereafter, the solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent; chloroform: ethanol = 10: 1) to obtain a compound (10) {2- [2- [2- ( 2-Biotinylaminoethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-diazilin-3-yl] benzyloxycarbamic acid tert-butyl ester} was obtained (yield: 95%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 8.31 (s, 1H), 7.39 (d, J = 7.9Hz, 1H), 6.97 (br, 1H), 6.81 (d, J = 7.9Hz, 1H), 6.64 (s, 1H ), 6.58 (s, 1H), 5.66 (s, 1H), 4.91 (s, 2H), 4.5 (m, 1H), 4.3 (m, 1H), 4.16 (t, J = 4.5Hz, 2H), 3.87 (t, J = 4.5Hz, 2H), 3.7 (m, 2H), 3.65 (m, 2H), 3.55 (m, 2H), 3.4 (br m, 2H), 3.1 (m, 1H), 2.9 (m , 1H), 2.70 (d, J = 12.8Hz, 1H), 2.17 (t, J = 7.3Hz, 2H), 1.8-1.6 (m, 4H), 1.45 (s, 9H), 1.4 (m, 2H) ;
UV / VIS (MeOH): λ_max(Ε) = 360 (400), 283 (2600);
MS (FAB⁺): M / z (%): 727 (100), [M + Na]⁺, 705 (12) [M + H]⁺; HRMS: C₃₀H₄₄F_ThreeN₆O₈S [M + H]⁺= 705.2893 (calculated value), 705.2852 (actual value)
[0047]
Example 9 (compound (10) → (11))
7 mg (0.01 mmol) of the compound (10) was dissolved in 0.1 ml of dichloromethane, 0.1 ml of trifluoroacetic acid was slowly added while cooling to 0 ° C., and the mixture was reacted at 0 ° C. for 1 hour. Thereafter, 0.1 ml of toluene was added to the residue obtained by distilling off the solvent under reduced pressure, followed by distilling off under reduced pressure. This operation was repeated 3 times in total, and the remaining excess trifluoroacetic acid was removed to obtain Compound (11) as a pale yellow solid. Compound (11) was immediately used for the condensation reaction with sugar compounds without purification.
[0048]
Example 10 (compound (11) → (12))
The crude compound (11) prepared from 7 mg (0.01 mmol) of the compound (10) by the above method was dissolved in 0.1 ml of acetonitrile, an aqueous solution of 1.9 mg (0.005 mmol) of N-acetyllactosamine was added, and 37 The reaction was carried out at 40 ° C. for 40 hours. The reaction mixture was purified by HPLC using a silica gel column (Senshu Scientific AQUASILSS-1251-120, 4.6 fx 250 mm). The yield was determined by measuring the UV spectrum of the methanol solution and using the UV spectrum of the compound (11) (361 nm (e = 386)) as a reference to obtain 0.0031 mmol of the compound (12). <Yield 63%).
HPLC conditions: 90% aq. CH_ThreeCN → 75% aq.CH_ThreeCN, 20min, 1ml / min
MS (FAB^-): M / z (%): 968 (64) [MH]^-;
HRMS; C₃₉H₅₇F_ThreeN₇0₁₆S ([MH]^-): 968.3535 (calculated value), 968.3586 (actual value)
[0049]
Example 11 (compound (11) → (13))
Compound (11) prepared from 3.5 mg (0.005 mmol) of Compound (10) by the method of Example 9 and 1.6 mg (0.0025 mmol) of sialyl-α2-3-lactose were used under the same conditions as in Example 10. Reacted. Similarly, 0.0012 mmol of compound (13) was obtained after HPLC purification. (Yield 48%).
HPLC conditions: 95% aq. CH_ThreeCN → 50% aq.CH_ThreeCN, 10min, 1ml / min
Negative ion FAB-MS (3-nitrobenzylalcohol) m / z: 1218 [M−H]^-HR-FAB-MS (3-nitrobenzylalcohol)
C₄₈H₇₁F_ThreeN₇O_{twenty four}S ([MH]^-): 1218.4223 (calculated value), 1218.4257 (actually measured value)
[0050]
Example 12 (compound (11) → (14))
Compound (11) prepared from 4.3 mg (0.006 mmol) of Compound (10) by the method of Example 9 and 1.0 mg (0.0014 mmol) of sialyl-α2-3-N-acetyllactosamine (purity 95%) Was reacted under the same conditions as in Example 10. Similarly, 0.00085 mmol of compound (14) was obtained after HPLC purification. (Yield 61%).
HPLC conditions: 85% aq. CH_ThreeCN → 70% aq.CH_ThreeCN, 15min, 1ml / min
Negative ion FAB-MS (3-nitrobenzylalcohol) m / z: 1259 [M−H]^-HR-FAB-MS (3-nitrobenzylalcohol)
C₅₀H₇₄F_ThreeN₈O_{twenty four}S ([MH]^-): 1259.4489 (calculated value), 1259.4507 (actual value)
[0051]
Example 13 (compound (11) → (15))
According to the method of Example 9, compound (10) l. Compound (ll) prepared from 4 mg (0.002 mmol) and Lewis X-type trisaccharide 0.5 mg (0.00094 mmol) were reacted under the same conditions as in Example 10. Similarly, 0.00073 mmol of compound (15) was obtained after HPLC purification. (Yield 78%).
HPLC conditions: 90% aq. CH_ThreeCN → 70% aq. CH_ThreeCN, 15min, 1ml / min
MS (FAB^-): M / z (%): 1114 (47) [MH]^-;
HRMS; C₄₅H₆₆F_ThreeN₇O₂₀S ([MH]^-): 1114.4114 (calculated value), 1114.4163 (actually measured value)
[0052]
Example 14 (compound (11) → (16))
Compound (11) prepared from 1.7 mg (0.0024 mmol) of compound (10) by the method of Example 9 and 1 mg (0.0012 mmol) of sialyl Lewis X-type tetrasaccharide (purity 95%) were subjected to the same conditions as in Example 10. It was made to react with. Similarly, 0.00085 mmol of compound (16) was obtained after HPLC purification. (Yield 71%).
HPLC conditions: 85% aq. CH_ThreeCN → 65% aq.CH_ThreeCN, 15min, 1ml / min
MS (FAB^-): M / z (%): 1405 (29) [MH]^-;
HRMS; C₅₆H₈₄F_ThreeN₈O₂₈S ([MH]^-): 1405.5068 (calculated value), 1405.5143 (actually measured value)
[0053]
Reference example 1 (photoaffinity labeling experiment of lectin)
The photoaffinity labeling experiment with the compound (12) to the compound (16) was conducted by Hatanaka et al. (Y. 1996)) was performed as follows.
0.1M phosphate buffer, pH 7.6, containing a photoaffinity labeling reagent (0.1 mM) and a lectin (subunit concentration of 0.1 mM) of any one of compounds (12) to (16) 0.05 ml of each photoaffinity labeling trial was prepared. Separately, as a control sample, a photoaffinity labeling reagent (0.1 mM) of any of compounds (12) to (16), lectin (0.1 mM per subunit), and 0.1 M containing 0.1 M inhibitor A 0.05 ml phosphate buffer solution, pH 7.6, was prepared. The combinations of photoaffinity label, lectin, and inhibitor in the control experiment are as follows in this order.
[0054]
Compound (12) Castor lectin (RCA) N-acetyllactosamine
Compound (13) Wheat Germ Lectin (WGA) Sialyl-α2-3-Lactose
Compound (14) Dog Endurectin (MAL) Sialyl-α2-3-Lactose
Compound (15) Lotus lectin (Lotus) Methyl-α-fucopyranoside
Compound (16) Dog Endurectin (MAL) Sialyl-α2-3-Lactose
After the photoaffinity labeling reagent sample and the control sample prepared as described above were incubated at 25 ° C. for 30 minutes in the dark, using a 30 W long wavelength UV lamp (Funakoshi, XX-15), a distance of 5 cm above And then irradiated on ice at 0 ° C. for 1 hour. Each sample after irradiation was separated by 12% SDS-polyacrylamide electrophoresis according to a conventional method, transferred to a PVDF membrane, and a photolabeled band was detected by chemiluminescence analysis. Control experiments confirmed that the label was specific for each lectin since photolabeling was inhibited. The results are shown in Table-1.
[0055]
[Table 1]

[0056]
Example 15 (compound (17))
8.0 g of sodium hydride (0.2 mol of 60% oil dispersion) was suspended in 200 ml of toluene, and 33.7 g (0.2 mol) of 2- [2- (2-chloroethoxy) ethoxy] ethanol was suspended at −70 ° C. Slowly added. To this mixture, 33.4 g (0.2 mol) of ethyl bromoacetate was slowly added dropwise at −70 ° C., and after 30 minutes, the reaction mixture was allowed to stand at room temperature. After 2 hours, the reaction mixture was neutralized by adding acetic acid at 0 ° C., the solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent; ethyl acetate: hexane = 3: 2) to obtain 38.8 g of A colorless oil was obtained. This was dissolved in 200 ml of N-methyl-2-pyrrolidone, 163 g (1.5 mol) of ethyl bromide and 3.1 g (0.03 mol) of sodium bromide were added, and the mixture was heated to 65 ° C. for 48 hours. The solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent; ethyl acetate: hexane = 3: 2) to give compound (17) {2- [2- (2-bromo Ethoxy) ethoxy] ethoxyethyl acetate} was obtained (yield: 76%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 1.28 (3H, t, J = 7.3Hz, -CH₂-CH _Three ), 3.47 (2H, t, J = 6.3Hz, BrCH₂), 3.68-3.75 (8H, m, O-CH₂-), 3.81 (2H, t, J = 6.3Hz, BrCH₂ CH ₂ O), 4.15 (2H, s, O-CH₂-CO-), 4.23 (2H, q, J = 7.3Hz,-CH ₂ -CH_Three)
[0057]
Example 16 (compound (2) + (17) → (18))
Compound (2) 4.62 g (20 mmol), Compound (17) 7.18 g (24 mmol), anhydrous potassium carbonate 2.76 g (20 mmol), Bu_FourA mixture of 7.39 g (20 mmol) of NI and 20 ml of DMSO was stirred at 60 ° C. for 14 hours under an argon gas atmosphere. Thereafter, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with water, 0.1N HCl, and water and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified with a silica gel column (eluent; diethyl ether: hexane = 3: 1). Compound (18) {2- [2- [2- (2- (ethoxycarbonylmethoxy) ethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) as a pale yellow oil after purification -3H-diazilin-3-yl] benzaldehyde} (yield: 76%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 1.28 (3H, t, J = 7.3Hz, -CH₂-CH _Three ), 3.67-3.93 (12H, m, O-CH₂-), 4.14 (2H, s, O-CH₂-CO-), 4.20 (2H, q, J = 7.3Hz,-CH ₂ -CH_Three), 4.52 (2H, s, Ph-CH₂-), 6.74 (1H, s, Ph-H), 6.85 (1H, d, J = 8.1Hz, Ph-H), 7.84 (1H, d, J = 8.1Hz, Ph-H);
UV (EtOH) nm (ε): 210 (16,368), 289.5 (1,272)
[0058]
Example 17 (compound (18) → (19))
2.17 g (5 mmol) of the compound (18) was dissolved in 10 ml of ethanol, and NaBH was added to this solution._Four 189 mg (5 mmol) was added slowly. The reaction mixture was stirred at room temperature for 30 minutes, adjusted to pH 3 by adding acetic acid to the reaction solution at 0 ° C., then the solvent was distilled off under reduced pressure, and the residue was extracted with ethyl acetate. The ethyl acetate layer was washed successively with 0.1N HCl, water and saturated brine, dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by a silica gel column (eluent; ethyl acetate: hexane = 1: 1). ) To give 1.72 g of a pale yellow oil (19) {2- [2- [2- (2- (ethoxycarbonylmethoxy) ethoxy) ethoxy] ethoxy] -4- [3- (trifluoro Methyl) -3H-diazilin-3-yl] benzyl alcohol} was obtained (yield: 76%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 1.28 (3H, t, J = 7.3Hz, -CH₂-CH _Three ), 3.43-3.87 (12H, m, O-CH₂-), 4.12 (2H, s, O-CH₂-CO-), 4.20 (2H, q, J = 7.3Hz,-CH ₂ -CH_Three), 4.52 (2H, s, Ph-CH₂-), 6.62 (1H, s, Ph-H), 6.79 (1H, d, J = 7.8Hz, Ph-H), 7.30 (1H, d, J = 7.8Hz, Ph-H);
UV (EtOH) nm (ε): 206.5 (24,660), 227 (10,244), 280 (2,311)
[0059]
Example 18 (compound (19) → (20))
3.60 g (8 mmol) of compound (19) and 3.32 g (10 mmol) of carbon tetrabromide were dissolved in 16 ml of dichloromethane, and 3.15 g (12 mmol) of triphenylphosphine was slowly added while cooling to 0 ° C. added. After 30 minutes, water was added to the reaction mixture, extracted with dichloromethane, the dichloromethane layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was silica gel column (eluent; ethyl acetate: hexane = 1: 1). (20) {2- [2- [2- (2- (ethoxycarbonylmethoxy) ethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) as 3.37 g of a pale yellow oil. ) -3H-diazilin-3-yl] benzyl bromide} (yield: 82%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 1.28 (3H, t, J = 7.3Hz, -CH₂-CH _Three ), 3.67-3.91 (12H, m, O-CH₂-), 4.14 (2H, s, O-CH₂-CO-), 4.20 (2H, q, J = 7.3Hz,-CH ₂ -CH_Three), 4.52 (2H, s, Ph-CH₂-), 6.65 (1H, s, Ph-H), 6.77 (1H, d, J = 8.0Hz, Ph-H), 7.35 (1H, d, J = 8.0Hz, Ph-H);
Positive ion FABMS (3-nitrobenzylalcohol) m / z: 535 (M + H)⁺
UV (EtOH) nm (ε): 209 (23,783), 238.5 (8,873), 290 (2,927)
[0060]
Example 19 (compound (20) + (21) → (22))
216 mg of sodium hydride (5.4 mmol of 60% oil dispersion) was suspended in 10 ml of THF, and 718 mg (6 mmol) of tert-butyl N-hydroxycarbamate (21) was added while cooling to −20 ° C. After 15 minutes, 2.31 g (4.5 mmol) of compound (20) was added while cooling to −15 ° C., and the reaction mixture was stirred at 0 ° C. After 2 hours, the reaction mixture was adjusted to pH 3 by adding 1N HCl at 0 ° C. and extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent; hexane: diethyl ether = 1: 10) to give 2.11 g of a compound (light yellow oily substance) 22) {2- [2- [2- [2- (Ethoxycarbonylmethoxy) ethoxy] ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-diazilin-3-yl] benzyloxycarbamic acid tert -Butyl ester} was obtained (yield: 83%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 1.28 (3H, t, J = 7.1Hz, -CH₂-CH _Three ), 1.46 (9H, s, C (CH_Three)_Three), 3.67-3.88 (12H, m, O-CH₂-), 4.12 (2H, s, O-CH₂-CO-), 4.20 (2H, q, J = 7.1Hz,-CH ₂ -CH_Three), 4.91 (2H, s, Ph-CH₂-), 6.65 (1H, s, Ph-H), 6.79 (1H, d, J = 8.0Hz, Ph-H), 7.38 (1H, d, J = 8.0Hz, Ph-H), 7.67 (1H, s, NH);
Positive ion FABMS (3-nitrobenzylalcohol) m / z: 588 (M + H)⁺
UV: 207 nm (ε34029), 228.5 nm (ε13228), 280.5 nm (ε2959)
[0061]
Example 20 (Compound (22) → (23))
2.26 g (4 mmol) of the compound (22) was dissolved in a mixed solvent of 20 ml of 1N NaOH and 18 ml of ethanol and stirred at 0 ° C. for 30 minutes. After adjusting the pH of the reaction solution to about 3 by adding 1N HCl at 0 ° C., the solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent: chloroform: methanol = 5: 1) to obtain 2.1 g of Compound (23) as a pale yellow oily substance {2- [2- [2- [2- (carboxymethoxy) ethoxy] ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-diazilin-3-yl ] Benzyloxycarbamic acid tert-butyl ester} was obtained (yield: 98%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 1.46 (9H, s, C (CH_Three)_Three), 3.69-3.90 (12H, m, O-CH₂-), 4.13 (2H, bs, O-CH₂-CO-), 4.91 (2H, s, Ph-CH₂-), 6.65 (1H, s, Ph-H), 6.81 (1H, d, J = 7.8Hz, Ph-H), 7.37 (1H, d, J = 7.8Hz, Ph-H), 8.06 (1H, bs, NH)
[0062]
Example 21 (Compound (23) + (24) → (25))
Compound (23) (537 mg, 1 mmol) and carbonyldiimidazole (194 mg, 1.2 mmol) were dissolved in anhydrous THF (1 ml), and stirred at room temperature, methyl sulfamoylbenzoate (24) (258 mg, 1.2 mmol) and diisopropylethylamine (158 mg) 1.2 mmol) was added and allowed to react at room temperature for 18 hours. Thereafter, the solvent was distilled off under reduced pressure, water was added to the resulting residue, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with 0.5N HCl, saturated aqueous sodium hydrogen carbonate, and saturated brine, dried over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified with a silica gel column (eluent; ethyl acetate: ethanol = 10 The compound (25) was obtained as a pale yellow oily substance (734 mg) (yield: 74%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 1.40 (9H, s, -C (CH_Three)_Three), 3.69-3.90 (12H, m, O-CH₂-), 3.94 (3H, s, CH_Three), 4.2 (2H, bs, O-CH₂-CO-), 5.02 (2H, s, Ph-CH₂-), 6.67 (1H, s, Ph-H), 6.80 (1H, d, J = 7.8Hz, Ph-H), 7.34 (1H, d, J = 7.8Hz, Ph-H), 7.69 (1H, s, NH), 8.14 (4H, s, Ph-H)
[0063]
Example 22 (Compound (25) → (26))
120 mg (0.15 mmol) of the compound (25) was dissolved in a mixed solvent of 0.75 ml of 1N NaOH and 3.5 ml of ethanol, and stirred at 0 ° C. for 30 minutes. While cooling to 0 ° C., 0.15 ml of 5N HCl was added to adjust the pH to around 3, and then extracted with ethyl acetate. The ethyl acetate layer was washed with saturated brine and dried over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified with a silica gel column (eluent; chloroform: methanol = 5: 1) to give 99 mg of a pale yellow oil. Compound (26) was obtained as a substance (yield: 89%).
¹HNMR (400MHz, CDCl_Three, 25 ℃): δ = 1.43 (9H, s, -C (CH_Three)_Three), 3.68-4.10 (12H, m, O-CH₂-), 4.23 (2H, s, O-CH₂-CO-), 4.91 (2H, s, Ph-CH₂-), 6.65 (1H, s, Ph-H), 6.77 (1H, d, J = 8.1Hz, Ph-H), 7.35 (1H, d, J = 8.1Hz, Ph-H), 8.17-8.23 ( 4H, m, Ph-H), 8.03 (1H, bs, -NH)
[0064]
Example 23 (Compound (26) + (27) → (28))
72 mg (0.1 mmol) of the compound (26) was dissolved in 1 ml of dichloromethane, 13 mg (0.11 mmol) of N-hydroxysuccinimide and 23 mg (0.11 mmol) of DCC were added, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure, the residue was dissolved in 1 ml of DMSO, 26 mg (0.1 mmol) of biotin hydrazide (27) was added, and the mixture was stirred overnight at room temperature. The reaction solution was directly purified with a silica gel column (eluent; chloroform: methanol = 5: 1) to obtain 10 mg of a compound (28) as a pale yellow solid (yield: 10%).
Positive ion FABMS (3-nitrobenzylalcohol) m / z: 961 (M + H)⁺
[0065]
Example 24 (Compound (11) → (29))
Compound (11) prepared from 3.5 mg (5 μmol) of compound (10) by the method of Example 9 and 1.1 mg (2.5 μmol) of chitobiose were reacted under the same conditions as in Example 10. Similarly, 1.2 μmol of compound (29) was obtained after HPLC purification (yield: 48%).
HPLC conditions: 85% aq. CH_ThreeCN, 1ml / min
Positive ion FABMS (3-nitrobenzylalcohol) m / z: 1011 (M + H)⁺, 1033 (M + Na)⁺
[0066]
Example 25 (Compound (28) → (30))
9.6 mg (10 μmol) of the compound (28) was deprotected by the method of Example 9, and 2.1 mg (5 μmol) of chitobiose was reacted under the same conditions as in Example 10. Similarly, 1.0 μmol of compound (30) was obtained after HPLC purification (yield: 2%).
HPLC conditions: 90% aq.CH_ThreeCN → 80% aq.CH_ThreeCN 20min, 1ml / min
Positive ion FABMS (3-nitrobenzylalcohol) m / z: 1266 (M + H)⁺, 1288 (M + Na)⁺
[0067]
Reference Example 2 (WGA photoaffinity labeling experiment)
The photoaffinity labeling experiment of compound (29) and compound (30) was carried out in the same manner as in Reference Example 1. Photoaffinity labeling reagent (0.1 mM) of either compound (29) or compound (30) and WGA (wheat germ agglutinin) in 0.1 M phosphate buffer, pH 7.6; 0.05 ml of each photoaffinity-labeled sample containing 0.1 mM) was prepared. Separately, as a control sample, a photoaffinity labeling reagent (0.1 mM) of either compound (29) or compound (30), WGA (subunit concentration: 0.1 mM), and 0.1 M phosphorus containing chitobiose 0.1 M 0.05 ml each of acid buffer solution, pH 7.6 was prepared.
After the photoaffinity labeling reagent sample and the control sample prepared as described above were incubated at 25 ° C. for 30 minutes in the dark, using a 30 W long wavelength UV lamp (Funakoshi, XX-15), a distance of 5 cm above And then irradiated on ice at 0 ° C. for 1 hour. Each sample after irradiation was separated by 12% SDS-polyacrylamide electrophoresis according to a conventional method, transferred to a PVDF membrane, and a photolabeled band was detected by chemiluminescence analysis. A control experiment inhibited the photolabeling of the WGA band with a molecular weight of 22K, confirming that the label was specific for WGA.
[0068]
The spacer is cut by taking 10 μl of each sample after irradiation, dissolving in 7 M guanidine, EDTA 2Na, 10 mM in 0.5 M Tris-HCl buffer, pH 8.5, 10 μl, adding 0.7 M DTT 10 μl, and room temperature for 2 hours. Reacted. Further, 10 μl of 1M sodium iodoacetate was added, and the mixture was reacted for 4 hours at room temperature and further for 5 hours by adding 4M mercaptoethanol, dialyzed against 0.1M phosphate buffer, pH 7.6, and then electrophoresed in the same manner. Was analyzed. After the cleavage reaction, cleavage was confirmed by the disappearance of the molecular weight 22K of the WGA label band due to the compound (30).
[0069]
【The invention's effect】
The compound represented by the general formula (II) of the present invention, particularly the compound (11) having an oxyamino group and a salt thereof, reacts in one step without subjecting the sugar compound having a reducing end to a special protection treatment. Thus, a sugar-linked phenyldiazirine compound of the general formula (V) having a photoreactive group (diazirine) and biotin can be derived, and this compound of the general formula (V) can interact with sugar as a photoaffinity labeling reagent. It is extremely useful for biotin labeling of the acting protein (sugar receptor). In addition, in the compound of the general formula (V), the compound in which biotin is bonded to the phenyldilysine skeleton via a cleavable spacer facilitates the release and recovery of the labeled protein from the immobilized avidin. Is excellent. Therefore, the compound of the general formula (II) is a stable and useful compound for easily producing a photoaffinity labeling reagent, and is a synthetic intermediate of the compound of the general formula (II), the general formula (I ) Is also a highly useful substance.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of general formula (I) of the present invention.
FIG. 2 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of the general formula (II) of the present invention.
FIG. 3 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of the general formula (V) of the present invention.
FIG. 4 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of the general formula (V) of the present invention.
FIG. 5 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of general formula (I) and compound (IV) of the present invention.
FIG. 6 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of the general formula (V) of the present invention.

Claims (19)

The phenyl diazirine compound represented by the following general formula (I).

(In the formula, R ¹ represents —NHP ¹ or —COX ¹ , and R ² represents an aldehyde group, a hydroxy lower alkyl group, a halogenated lower alkyl group, a phthalimidooxy lower alkyl group, or — (CH ₂ ) _x —O. —NHP ¹ , P ¹ represents an amino protecting group, X ¹ represents an alkoxy group, a hydroxy group, or —NHSO ₂ —X ² —CO—X ³ , X ² represents an arylene group or an alkylene group, X ³ represents an alkoxy group or a hydroxy group, m represents an integer of 1 to 4, n represents an integer of 1 to 6, and x represents an integer of 1 to 4.) A biotinylated phenyldiazirine compound represented by the following general formula (II).

(Wherein, R ³ represents amino group, a salt of -NHP ¹ or amino group, P ¹ represents an amino protecting group, Y ¹ is -NH- or -CONHSO ₂ -X ² -CO- a represents , X ² represents an arylene group or an alkylene group, Y ² represents biotin or a biotin derivative residue, m represents an integer of 1 to 4, n represents an integer of 1 to 6, and x represents 1 to 4 Represents an integer.) The biotinylated phenyldiazirine compound according to claim 2 represented by the following general formula (III).

(Wherein, R ³ is amino group, a salt of -NHP ¹ or amino group, P ¹ represents an amino protecting group, m represents an integer of 1 to 4, n is an integer of 1 to 6 And x represents an integer of 1 to 4.) The biotinylated phenyldiazirine compound according to claim 2 represented by the following general formula (IV).

(Wherein, R ³ is amino group, a salt of -NHP ¹ or amino group, P ¹ represents an amino protecting group, m represents an integer of 1 to 4, n is an integer of 1 to 6 X ² represents an arylene group or an alkylene group, Z represents an alkylene group having 1 to 20 carbon atoms which may be interrupted by a hetero atom, x represents an integer of 1 to 4, and q represents 0 or 1 Represents.) A sugar-linked biotinylated phenyldiazirine compound represented by the following general formula (V).

(Wherein R ⁴ represents a residue of a sugar compound having a reducing end, Y ¹ represents —NH— or —CONHSO ₂ —X ² —CO—, X ² represents an arylene group or an alkylene group, Y ² represents biotin or a biotin derivative residue, m represents an integer of 1 to 4, n represents an integer of 1 to 6, and x represents an integer of 1 to 4.) A sugar-linked biotinylated phenyldiazirine compound represented by the following general formula (VI).

(In the formula, R ⁴ represents a residue of a sugar compound having a reducing end, m represents an integer of 1 to 4, n represents an integer of 1 to 6, and x represents an integer of 1 to 4. ) A sugar-linked biotinylated phenyldiazirine compound represented by the following general formula (VII).

(In the formula, R ⁴ represents a residue of a sugar compound having a reducing end, X ² represents an arylene group or an alkylene group, and Z represents an alkylene group having 1 to 20 carbon atoms which may be interrupted by a hetero atom. M represents an integer of 1 to 4, n represents an integer of 1 to 6, x represents an integer of 1 to 4, and q represents 0 or 1.) A photoreactive labeling reagent comprising a biotinylated phenyldiazirine compound represented by the general formula (II) according to claim 2. However, in the general formula (II), R ³ represents an amino group or a salt of an amino group. A photoreactive labeling reagent comprising a biotinylated phenyldiazirine compound represented by the general formula (III) according to claim 3. However, in the general formula (III), R ³ is representative of a salt of an amino group, or an amino group. A photoreactive labeling reagent comprising a biotinylated phenyldiazirine compound represented by the general formula (IV) according to claim 4. However, in the general formula (IV), R ³ represents a salt of an amino group, or an amino group. A photoaffinity labeling reagent comprising a sugar-linked biotinylated phenyldiazirine compound represented by the general formula (V) according to claim 5. A photoaffinity labeling reagent comprising a sugar-linked biotinylated phenyldiazirine compound represented by the general formula (VI) according to claim 6. A photoaffinity labeling reagent comprising a sugar-linked biotinylated phenyldiazirine compound represented by the general formula (VII) according to claim 7. The biotinylated phenyldiazirine compound represented by the general formula (II) is reacted with a sugar compound having a reducing end, and the aldehyde group of the reducing end of the sugar compound having the reducing end and the amino acid of the biotinylated phenyldiazirine compound A method for producing a sugar-linked biotinylated phenyldiazirine compound represented by the above general formula (V), comprising forming a Schiff base with a group.
However, in the general formula (II), R ³ represents a salt of an amino group, or an amino group. The method according to claim 14, wherein the biotinylated phenyldiazirine compound is represented by the general formula (III), and the sugar-linked biotinylated phenyldiazirine compound is represented by the general formula (VI). The production method according to claim 14, wherein the biotinylated phenyldiazirine compound is represented by the general formula (IV), and the sugar-linked biotinylated phenyldiazirine compound is represented by the general formula (VII). The sugar-linked biotinylated phenyldiazirine compound represented by the above general formula (V) and a sugar receptor are mixed and bound by specific interaction between the sugar compound residue of the phenyldiazirine compound and the sugar receptor. At the same time, the mixture is irradiated with light, and the compound of the general formula (V) is bound by a photoreaction to the site of the sugar receptor that interacts with the sugar compound bound to the compound of the general formula (V). And labeling the sugar receptor. 18. The method for labeling a sugar receptor according to claim 17, wherein the sugar-linked biotinylated phenyldiazirine compound is represented by the general formula (VI). 18. The method for labeling a sugar receptor according to claim 17, wherein the sugar-linked biotinylated phenyldiazirine compound is represented by the general formula (VII).

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