JP3821494B2 - Fluorescent labeling method for sugar and method for producing artificial glycoconjugate - Google Patents
Fluorescent labeling method for sugar and method for producing artificial glycoconjugate Download PDFInfo
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- JP3821494B2 JP3821494B2 JP04154594A JP4154594A JP3821494B2 JP 3821494 B2 JP3821494 B2 JP 3821494B2 JP 04154594 A JP04154594 A JP 04154594A JP 4154594 A JP4154594 A JP 4154594A JP 3821494 B2 JP3821494 B2 JP 3821494B2
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- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical group O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 238000003239 susceptibility assay Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Saccharide Compounds (AREA)
Description
【０００１】
【産業上の利用分野】
本発明は、糖の蛍光標識方法および人工複合糖質の製造法に関し、特に、２−アミノピリジン誘導体を用いた該方法に関するものである。
【０００２】
【従来の技術】
近年、糖蛋白質等の複合糖質の構造とその機能の関係を研究するために、より高感度でしかも微量の試料で定量検定が可能な方法が種々提案されている。
例えば、糖鎖の微量分析の方法としては、還元末端を糖アルコールに変換する際にトリチウムを導入し、その放射活性を利用することが挙げられる。この検出感度は、ｐｍｏｌレベルであり、良好であるが、放射性物質であるために種々の制約がある。
【０００３】
この制約がない高感度検定法として、２−アミノピリジン等の有機化合物を蛍光標識剤として使用する方法が公知である（例えば、Ｓ．ハセ等、ジャーナル・オブ・バイオケミストリー、第９５巻、第１９７〜２０３頁（１９８４））。
これは、糖又は糖鎖の還元末端に該２−アミノ基を反応させてシッフ塩基とし、次いで還元して、蛍光により検出するものであり、感度は上述と同様にｐｍｏｌレベルであることが開示されている。
【０００４】
しかしながら、該蛍光標識剤は、糖鎖の構造解析には有効であるが、一官能性であるため、他の生体物質、例えば、蛋白質、ペプチド、アミノ酸、脂質、核酸、合成高分子等と糖との複合体等を形成させることはできず、従ってそれらを総合的に解析するには、十分であるとは言えなかった。
そこで、本出願人は特開平５−２５５２５３号公報に６位に前記生体物質と反応し得る官能基を導入した２−アミノピリジン誘導体を開示し、種々の人工複合糖質が合成できることを示した。
【０００５】
しかし、２−アミノピリジン誘導体にシアノ基を導入したものを使用して標識した糖鎖を分離精製後、シアノ基の還元で生成するアミノ基を介して人工複合糖質を合成する方法は、該シアノ基の還元においては、濃アンモニア水中、Ｐｄを用いて長時間の加圧接触還元しなければならないという問題が生じた。
【０００６】
【発明が解決しようとする課題】
本発明の第一の目的は、温和な条件で糖に２−アミノピリジン誘導体を導入する蛍光標識方法を提供すると共に人工複合糖質の合成に使用し得る反応剤としての２−アミノピリジン誘導体と糖との結合物を容易に製造する方法を提供することであり、第２に該反応剤を用いた人工複合糖質の製造法を提供することにある。
【０００７】
【課題を解決するための手段】
本発明は、
１） アミノ保護基を有するアミノアルキル基を６位に有する２−アミノピリジン誘導体と末端が還元糖からなる単糖、オリゴ糖、多糖またはグリコサミノグリカンとを還元アミノ化反応により結合し、次いでアミノ保護基を脱離することを特徴とする糖の蛍光標識方法、
２） アミノ保護基は、ウレタン型アミノ保護基またはトリハロアセチル基であることを特徴とする前記１）記載の糖の蛍光標識方法、
３） アミノ保護基は、ｔ−ブトキシカルボニル基、ベンジルオキシカルボニル基またはトリフルオロアセチル基であることを特徴とする前記１）記載の糖の蛍光標識方法、
４） アミノ保護基の脱離反応は、酸性条件もしくは塩基性条件による処理、または還元反応により行われる前記１）〜３）のいずれかに記載の糖の蛍光標識方法、
５） 酸性条件による処理は、トリフルオロ酢酸を用いて行われる前記４）記載の糖の蛍光標識方法、
６） 塩基性条件による処理は、ピペリジン水溶液を用いて行われる前記４）記載の糖の蛍光標識方法、
７） 還元反応は、接触還元により行われる前記４）記載の糖の蛍光標識方法、
８） アミノ保護基を有するアミノアルキル基を６位に有する２−アミノピリジン誘導体と末端が還元糖からなる単糖、オリゴ糖、多糖またはグリコサミノグリカンとを還元アミノ化反応により結合し、次いでアミノ保護基を脱離して６位にアミノアルキル基を有する２−アミノピリジン誘導体と単糖、オリゴ糖、多糖またはグリコサミノグリカンとの結合物を得、次いで該結合物のピリジンの６位のアミノアルキル基の該アミノ基を、該アミノ基と結合し得る官能基を有する有機化合物と直接反応させるか、またはアミノ基と結合し得る官能基を有するスペーサーを介して有機化合物と反応させて、アミド結合反応、シッフ塩基形成反応、ウレタン結合反応、アルキル化反応、ジスルフィド結合反応、ジスルフィド交換反応、アミジン結合反応または炭素−炭素結合反応により結合させることを特徴とする人工複合糖質の製造法、
９） 有機化合物は、糖、蛋白質、ペプチド、アミノ酸、脂質、核酸、ヌクレオチド、ヌクレオシド、ビオチン、または合成高分子化合物である前記８）記載の人工複合糖質の製造法、
１０）有機化合物またはスペーサーがカルボキシル基を有する化合物であり、該カルボキシル基を活性化してピリジンの６位のアミノアルキル基の該アミノ基と反応させる前記８）または９）記載の人工複合糖質の製造法である。
【０００８】
本発明の糖の蛍光標識方法は、アミノ保護基を有するアミノアルキル基を６位に有する２−アミノピリジン誘導体（以下、保護基含有２−アミノピリジン誘導体と略すこともある。）と末端が還元糖からなる単糖、オリゴ糖、多糖またはグリコサミノグリカン（以下、糖化合物ともいう）とを還元アミノ化反応により結合し、次いでアミノ保護基を脱離するものである。本発明は、この脱離により結果的に６位にアミノアルキル基を有した２−アミノピリジン誘導体と糖化合物との結合物が得られ、この結合物は、他の任意の有機化合物を６位のアミノ基に結合させて人工複合糖質を得るための蛍光標識反応剤としての機能を有するものであるから、本発明の方法は、蛍光標識反応剤の製造法を提供するものでもある。
【０００９】
本発明に使用される保護基含有２−アミノピリジン誘導体としては、下記一般式（１）で表される化合物（以下、化合物１ともいう）を挙げることができる。
【００１０】
【化３】
式中、Ｒは、有機基である保護基を示し、ｎは、２〜２０、好ましくは２〜８の整数である。該保護基としては、ウレタン型アミノ保護基またはトリハロアセチル基が例示され、ウレタン型アミノ保護基としてｔ−ブトキシカルボニル基（以下、Ｂｏｃと記す）またはベンジルオキシカルボニル基（以下、Ｚと記す）が特に好ましく、トリハロアセチル基としてはトリフルオロアセチル基（以下、Ｔｆａと記す）が特に好ましいが、これに制限されるものではない。
【００１２】
また、式中、３〜５位の水素は、任意の有機基、好ましくは炭化水素基、特に好ましくは炭素数１〜４の低級アルキル基により置換されていてもよい。化合物１と糖化合物との還元アミノ化反応は、化合物１の２位のアミノ基と糖化合物とを縮合させて、シッフ塩基を形成し、次いでこれを還元することにより−ＣＨ２ＮＨ−結合を形成させることである。得られる化合物１と糖化合物との反応生成物（結合物）を糖残基含有化合物１とも言う。
【００１３】
得られた糖残基含有化合物１は、６位のアミノ保護基を脱離する反応に供される。この脱離反応は、特に制限はないが、好ましくは以下の方法が挙げられる。
▲１▼ トリフルオロ酢酸、無水塩化水素、蟻酸等を用いて酸性条件で糖残基含有化合物１を処理する方法。特に、保護基がＢｏｃの場合に好適である。
▲２▼ 水素を常圧または加圧下、Ｐｄ（パラジウム）等の触媒存在下、糖残基含有化合物１を接触還元する方法、あるいはＰｄ存在下、有機水素供与体を用いた接触水素移動還元する方法。特に、保護基がＺの場合に好適である。
▲３▼ 塩基、例えば、ピペリジン、アンモニア、水酸化ナトリウム等の水溶液により塩基性条件で糖残基含有化合物１を処理する方法が挙げられる。特に、保護基がＴｆａの場合に好適である。
【００１４】
▲１▼でトリフルオロ酢酸を使用して保護基を脱離する場合の条件は、糖残基含有化合物１のトリフルオロ酢酸溶液を室温、具体的には１０〜２５℃で、１０〜３０分間攪拌し、その後、窒素ガスを吹きつけながらトリフルオロ酢酸を除くことが挙げられる。
▲２▼でＰｄ触媒を用いて保護基を脱離する場合の条件は、糖残基含有化合物１の水溶液にＰｄを加え、１〜１０気圧の水素雰囲気下、室温で３０〜９０分間攪拌することが挙げられる。
【００１５】
▲３▼でピペリジンを用いて保護基を脱離する場合の条件は、糖残基含有化合物１の０．２〜２Ｍピペリジン水溶液を０〜１０℃で０．５〜２時間攪拌することが挙げられる。
上記▲１▼〜▲３▼の反応で得られた６位がアミノアルキル基である糖残基含有２−アミノピリジン誘導体（以下、「糖残基含有化合物１ｒ」とも記す）は、任意の方法で精製され得るが、好ましくは水溶液としてＨＰＬＣ（高速液体クロマトグラフィー）により高収率（約９５％以上）で高度精製、単離することができる。上記本発明の方法は、極めて温和な方法であるため上記高収率を達成したものである。
【００１６】
ＨＰＬＣを用いた精製は、例えば、Ｃｏｓｍｏｓｉｌ（登録商標）５Ｃ １８ −ＡＲを用いた逆相ＨＰＬＣあるいはＴＳＫ ｇｅｌ（登録商標） Ａｍｉｄｅ−８０を用いたイオン交換ＨＰＬＣを用いて行うことができる。
この糖残基含有化合物１ｒは、６位に反応性のアミノ基を有しているので、他の任意の有機化合物を標識する蛍光標識剤の機能を有していると共に人工複合糖質を作成するための反応剤の機能を有している。
【００１７】
この人工複合糖質の製造条件としては、特に、制限はないが、具体的には以下の方法が例示できる。
ａ．所望の糖残基構造を有する糖残基含有化合物１ｒのアミノ基と反応する官能基（例えば、カルボキシル基、ホルミル基等）を有する所望の有機化合物とを結合させることにより所望の人工複合糖質を得る方法。
ｂ．ａ．の有機化合物として糖残基含有化合物１ｒのアミノ基と反応する官能基以外に他の有機化合物と反応する官能基を有した化合物（例、スペーサー）を選択し、得られた糖残基含有化合物１ｒ誘導体（例、糖残基含有化合物１ｒ−スペーサー結合物）と所望の有機化合物とを反応させて人工複合糖質を得る方法。糖残基含有化合物１ｒ誘導体は官能基を複数有することができ、これに反応する有機化合物も複数であってもかまわない。
【００１８】
複数の官能基を有するスペーサーとしては、グリオキサール、ジスクシンイミジルスベラート、スクシンイミジル ３−（２−ピリジルジチオ）プロピオネート、スクシンイミジル ６−マレイミドヘキサノエート等に由来するものが例示される。
ｃ．ｂで得られた人工複合糖質に導入もしくは予め存在する官能基に所望の有機化合物を結合させる方法。
【００１９】
ａにおける糖残基含有化合物１ｒと有機化合物との反応としては、アミド結合反応、シッフ塩基形成反応、ウレタン結合反応、アルキル化反応等を挙げることができる。
ｂにおける糖残基含有化合物１ｒ誘導体は、一種の人工複合糖質あるいは他の人工複合糖質を作成するための反応剤として機能する。この反応剤を得るための有機化合物は単に他の有機化合物を結合するためのスペーサーとしてのみの機能を有したものであっても他の有用な作用を有する化合物であってもよい。また、この糖残基含有化合物１ｒ誘導体において、導入される有機化合物を選定することにより、それ自体で有用な試薬となり得るものを合成することができる。例えば、有機化合物として、ビオチン誘導体を選択すれば、標識化アビジン等を用いて行うレクチン等の解析（ＥＬＩＳＡ等）に有用な試薬が得られる。
【００２０】
ここで、糖残基含有化合物１ｒと有機化合物との反応により糖残基含有化合物１ｒ誘導体に導入される官能基としては、有機化合物と前記ａの結合反応が生じるような官能基の他、ジスルフィド結合反応、ジスルフィド交換反応、アミジン結合反応、炭素−炭素結合反応などが生じる官能基、例えば、カルボキシル基、アミノ基、水酸基、ジスルフィド基、アクリロイル基等を挙げることができる。
【００２１】
なお、上記ａ〜ｃの反応において、官能基を予め活性化して反応に供することができる。例えば、官能基がカルボキシル基である場合、スクシンイミド基によって活性化することができ、メルカプト基の場合、３−ニトロ−２−ピリジルスルフェニル基によって活性化することができる。
前記、ａ〜ｃにおける有機化合物としては、任意であるが、特に、糖、蛋白質、ペプチド、アミノ酸、脂質、核酸、ヌクレオチド、ヌクレオシド、ビオチン、または合成高分子（そのモノマーを含む）が好ましく、種々、有用な人工複合糖質を容易に得ることができる。
【００２２】
本発明における還元アミノ化反応による結合とは、糖化合物の還元末端に化合物１の２位のアミノ基を反応させてシッフ塩基を形成させ、次いで還元することによって−ＣＨ２ＮＨ−結合を形成させるものであり、前期反応により得られた糖残基含有化合物１を、必要に応じて高速液体クロマトグラフィー（ＨＰＬＣ）等の分別手段によって分別し、紫外線もしくは蛍光スペクトルによって検出することにより、目的とする糖残基含有化合物１を検出すると共に分離精製することができる。
【００２３】
なお、上記シッフ塩基形成反応及びそれに続く還元反応は、公知の方法（例えば、特開昭６４−１０１７７号、特開平１−１４１３５６号、Ｊ．Ｂｉｏｃｈｅｍ．，第９５巻，第１９７〜２０３頁（１９８４）、「蛋白質 核酸 酵素」第３６巻、第１号、第６３〜６８頁（１９９１年）参照）に従って行うことができる。すなわち、塩酸、フッ化水素酸等の無機酸もしくは酢酸、トリフルオロ酢酸等の有機酸及びピリジン等の有機溶媒中、常温〜１００℃、数分〜数時間（好ましくは、約９０℃、１〜３時間、ｐＨ３〜６．４）の反応条件下、糖類に対して２０〜１００当量程度の化合物１を使用して反応させることによってシッフ塩基を生成させることができる。シッフ塩基の還元には、通常シッフ塩基の還元に使用されている還元剤を使用することができ、とりわけ揮発性のボランコンプレックス（例えば、ボランジメチルアミンコンプレックス（ＢＨ3 ・Ｍｅ2 ＮＨ）、ボラントリエチルアミンコンプレックス、ボランピリジンコンプレックス等）、ナトリウムボロハイドライド、シアノ水素化ホウ素ナトリウム（ＮａＢＨ3 ＣＮ）等が好ましい。還元反応は、常温〜１００℃、１時間〜１０時間（好ましくは約８０〜９０℃、１時間程度）で完了する。
【００２４】
【実施例】
以下、本発明の実施例を具体的に説明するが、本発明はこれにより限定されるものではない。
合成例１ 化合物１−１（ｎ＝３、Ｒ＝Ｂｏｃ）の合成
２−トリチルアミノ−６−（２−シアノエチル）ピリジンをＬｉＡｌＨ4 により還元して得られる２−トリチルアミノ−６−（３−アミノプロピル）ピリジンを（Ｂｏｃ）2 Ｏ（ジ−第３ブチル ジカーボネート）と反応して得られる２−トリチルアミノ−６−（３−ｔ−ブトキシカルボニルアミノプロピル）ピリジン（２．００ｇ、４．０５ｍｍｏｌ）の酢酸−蒸留ＭｅＯＨ〔１：１（ｖ／ｖ）〕、１４ｍｌ〕の溶液を５０℃で５時間半攪拌した。減圧濃縮し、残渣を中圧シリカゲルクロマトグラフィー〔シリカゲル５０ｇ、クロロホルム−メタノール（１５：１）〕で精製した。一部が酢酸塩となっていたため、脱塩のために酢酸エチルを加えて溶かし、飽和炭酸水素ナトリウム水溶液と飽和食塩水で順次洗浄した後、硫酸マグネシウムで乾燥した。乾燥剤を濾去後、減圧濃縮して淡黄色オイルを得た。そのオイルを２週間冷蔵庫に放置して針状晶の標記化合物１−１を得た。収量９３１ｍｇ（２−トリチルアミノ−６−（３−ｔ−ブトキシカルボニルアミノプロピル）ピリジンよりの収率９１．６％）、ｍｐ ７２−７４℃。1Ｈ−ＮＭＲ（２７０ＭＨｚ、ＣＤＣｌ3 ）で化合物１−１の構造を確認した。また、Ｃ13Ｈ21Ｏ2 Ｎ3 の計算値、Ｃ ６２．１３；Ｈ ８．４２；Ｎ １６．７２において、実測値、Ｃ ６１．９０；Ｈ ８．４８；Ｎ １６．５９であった。
【００２５】
合成例２ 化合物１−２（ｎ＝３、Ｒ＝Ｚ）の合成
２−トリチルアミノ−６−（２−シアノエチル）ピリジンをＬｉＡｌＨ4 により還元して得られる２−トリチルアミノ−６−（３−アミノプロピル）ピリジンをＺＯＳｕ（ベンジル Ｎ−スクシンイミジル カーボネート）と反応して得られる２−トリチルアミノ−６−（３−ベンジルオキシカルボニルアミノプロピル）ピリジン（６９７ｍｇ、１．３２ｍｍｏｌ）の酢酸−ＭｅＯＨ〔１：１（ｖ／ｖ）、１０ｍｌ〕の溶液を５０℃で１８時間半攪拌した。減圧濃縮し、残渣を中圧シリカゲルクロマトグラフィー〔シリカゲル１０ｇ、クロロホルム−メタノール（１５：１）〕で精製した。一部が酢酸塩となっていたため、合成例１と同様にして淡黄色オイルの標記化合物１−２を得た。収量３６１ｍｇ（２−トリチルアミノ−６−（３−ベンジルオキシカルボニルアミノプロピル）ピリジンよりの収率９６．２％）。1Ｈ−ＮＭＲ（２７０ＭＨｚ、ＣＤＣｌ3 ）で化合物１−２の構造を確認した。また、Ｃ16Ｈ19Ｏ2 Ｎ3 の計算値、Ｃ ６５．８３；Ｈ ６．８１；Ｎ １４．３８において、実測値、Ｃ ６５．８３；Ｈ ６．７２；Ｎ１４．３８であった。
【００２６】
合成例３ 化合物１−３（ｎ＝３、Ｒ＝Ｔｆａ）の合成
２−トリチルアミノ−６−（２−シアノエチル）ピリジンをＬｉＡｌＨ4 により還元して得られる２−トリチルアミノ−６−（３−アミノプロピル）ピリジンを（Ｔｆａ）2 Ｏ（トリフルオロ酢酸無水物）と反応して得られる２−トリチルアミノ−６−（３−トリフルオロアセチルアミノプロピル）ピリジン（１４０ｍｇ、２８５μｍｏｌ）の酢酸−ＭｅＯＨ〔１：１（ｖ／ｖ）〕、４ｍｌ〕の溶液を５０℃で２２時間攪拌した。減圧濃縮し、残渣を中圧シリカゲルクロマトグラフィー〔シリカゲル８ｇ、ジクロロメタン−メタノール（２０：１）〕で精製した。一部が酢酸塩となっていたため、合成例１と同様にして淡黄色針状晶の標記化合物１−３を得た。収量６１．４ｍｇ（２−トリチルアミノ−６−（３−トリフルオロアセチルアミノプロピル）ピリジンよりの収率９６．２％）。1Ｈ−ＮＭＲ（２７０ＭＨｚ、ＣＤＣｌ3 ）で化合物１−３の構造を確認した。また、Ｃ10Ｈ12ＯＮ3 Ｆ3 の計算値、Ｃ ４８．５８；Ｈ ４．８９；Ｎ １７．００において、実測値、Ｃ ４８．６２；Ｈ ４．８１；Ｎ １６．８７であった。
【００２７】
実施例１−１〜３
乾燥した糖化合物（表１に記載のもの）（５．５５μｍｏｌ）に化合物１−１〜３（２７．８μｍｏｌ、表１記載）と酢酸−ピリジン〔１：１７（ｖ／ｖ）、４８．０μＬ〕を加え、封管して９０℃で、３時間加熱し、放冷した後、還元反応溶液〔ＢＨ３・Ｍｅ２ＮＨ（６．５５ｍｇ、１１１μｍｏｌ）の酢酸（３３．５μＬ）溶液〕（Ｍｅ：メチル基）を加え、封管して８０℃で１時間加熱した。放冷後、水を加えて、ＨＰＬＣ〔カラム：コスモシール（登録商標）５Ｃ １８ −ＡＲ ２０ｍｍＩＤ×２５０ｍｍＬ；溶出溶媒：アセトニトリル−０．１Ｍ蟻酸アンモニウム（ｐＨ４．５）；濃度勾配：〔０−６０％アセトニトリル（２％／分）〕；流速：８ｍＬ／分；検出：ＵＶ ３００ｎｍ〕によって各糖残基含有化合物１を精製後、凍結乾燥を行った。各収率を表１に示した。
【００２８】
【表１】
次に実施例１−３で得られた次に示す３種の糖残基含有化合物１（ａ〜ｃ）、即ち
【００３０】
【化４】
の保護基を次のように脱離した。
（１）化合物ａのＢｏｃ除去
化合物ａ（１．１８ｍｇ、１．６０μｍｏｌ）のトリフルオロ酢酸（２００μＬ）溶液を室温で３０分間攪拌した。その後、窒素ガスを吹きつけてトリフルオロ酢酸を除き、残渣に水を加えて溶かし、ＨＰＬＣで分離精製後、凍結乾燥を行いＢｏｃがＨで置換された次式で示される化合物ａｒを得た。収率はＨＰＬＣ分析により、定量的で略１００％であった。
【００３２】
【化５】
（２）化合物ｂのＺの除去
化合物ｂ（１．３１ｍｇ、１．６９μｍｏｌ）に水（５００μＬ）を加えて溶かし、パラジウム黒（１１．２ｍｇ）を加え、４気圧の水素雰囲気下室温で９０分間攪拌した。その後、ＨＰＬＣで分離精製後、凍結乾燥を行いＺがＨで置換された化合物ａｒを得た。収率はＨＰＬＣ分析により、定量的で略１００％であった。
（３）化合物ｃのＴｆａ除去
化合物ｃ（０．１５ｍｇ、０．２μｍｏｌ）の１Ｍピペリジン水溶液（２５０μＬ）溶液を０℃で１時間攪拌した。その後、ＨＰＬＣで分離精製後、凍結乾燥を行いＴｆａがＨで置換された化合物ａｒを得た。収率はＨＰＬＣ分析により、定量的で略１００％であった。
【００３４】
比較例
化合物ａｒにおいて、ピリジン環の６位がシアノエチル基である化合物３２μｍｏｌを２５％アンモニア水溶液（１４ｍＬ）中パラジウム黒（５０ｍｇ）の存在下、水素を９ｋｇ／ｃｍ２の条件で室温下６６時間水素化し、化合物ａｒを合成した。この触媒を濾別し、濾過物を濃縮した。その濃縮残渣を水（１０ｍＬ）に溶解し、ＨＰＬＣ〔カラム：コスモシール（登録商標）５Ｃ１８ −ＡＲ、２０ｍｍ×２５０ｍｍ；溶媒：ＭｅＣＮ−０．１Ｍ ＣＨ３ＣＯ２ＮＨ４（ｐＨ６．９）；勾配：５％ＭｅＣＮ−５０％ＭｅＣＮ（１．５％／ｍｉｎ）；流速：８ｍＬ／ｍｉｎ；検知：ＵＶ ３０７ｎｍ〕で精製した。化合物ａｒを含む分画を凍結乾燥し、その得られた粉末を水（１０ｍＬ）に溶解した。この溶液に混在するアンモニウム塩を除くためにＨＰＬＣ〔カラム：コスモシール（登録商標）５Ｃ１８ −ＡＲ、２０ｍｍ×２５０ｍｍ；溶媒：１５％ＭｅＣＮ−０．０１％ＴＦＡ；流速：８ｍＬ／ｍｉｎ；検知：ＵＶ３０７ｎｍ〕で再精製した。収率は、７７％であった。
【００３５】
実施例２
実施例２−１ ウシ血清アルブミン（ＢＳＡ）と化合物ａｒとからなる人工複合糖質の合成
下記化４に示す合成ルートにより標記人工複合糖質を合成した。
【００３６】
【化６】
化合物ａｒとスクシンイミジル ３−（３−ニトロ−２−ピリジルジチオ）プロピオネート（１）をテトラヒドロフラン（ＴＨＦ）−水（２：１）のＴＥＡ（トリエチルアミン）溶液（ｐＨ９）中で結合させ、逆相ＨＰＬＣ（カラム：コスモシール（登録商標）５Ｃ １８ −ＡＲ、溶媒：ＭｅＣＮ−０．１％ＴＦＡ水溶液）により化合物（２）を得た。化合物（２）は２トリフルオロ酢酸塩として得られた。
【００３８】
次に６個のシステイン残基を有するＢＳＡ（３）および化合物（２）を種々のモル比〔化合物（２）：ＢＳＡ＝１〜６：１〕で０．５Ｍトリス−０．００５ＭＥＤＴＡナトリウム塩緩衝液（ｐＨ７．５）に共に溶解して４℃でＢＳＡの−ＳＨ基と化合物（２）のジスルフィド基の交換反応に供し、人工複合糖質（４）を得た。得られる人工複合糖質（４）は、ゲル濾過ＨＰＬＣ〔カラム：アサヒパックＧＳ−５１０、溶媒：３０％ＭｅＣＭ−０．２Ｍリン酸ナトリウム緩衝液（ｐＨ７．０）〕により化合物（２）より分離した。
【００３９】
実施例２−２ ビオチンと化合物ａｒとからなる人工複合糖質の合成
下記化５に示す合成ルートにより標記人工複合糖質を合成した。
【００４０】
【化７】
化合物ａｒ（１５．０ｍｇ、１６．６μｍｏｌ）の０．５％ＮａＨＣＯ３水溶液−ＤＭＦ〔１：１（ｖ／ｖ），５００μＬ〕水溶液にＮ−ヒドロキシスクシンイミドビオチン（５）（４０．０ｍｇ、１１７μｍｏｌ）を加え、この混合物を室温で３．５時間攪拌した。その後、化合物（５）（２４．０ｍｇ、７０．４ｍｍｏｌ）を加え、その混合物を濾過し、かつその濾過物をＨＰＬＣ〔カラム：コスモシール（登録商標）５Ｃ１８ −ＡＲ、２０ｍｍ×２５０ｍｍ；溶媒：ＭｅＣＮ−０．１Ｍ ＨＣＯ２ＮＨ４（ｐＨ６．３）；勾配：５％ＭｅＣＮ−５０％ＭｅＣＮ（１．５％／ｍｉｎ）；流速：８ｍＬ／ｍｉｎ；検知：ＵＶ ３００ｎｍ〕にかけた。２０．９分後に溶出した分画を集め、凍結乾燥し、無色の粉末である化合物（６）を得た（収量９．３ｍｇ（収率６５％））。化合物（６）は、１Ｈ−ＮＭＲ（Ｄ２Ｏ）で確認され、かつポジティブＦＡＢ−ＭＳでｍ／ｚ８６６．７〔（Ｍ＋Ｈ）＋〕に擬分子イオンピークを示した。
【００４２】
実施例２−３ アクリル酸と化合物ａｒとからなる人工複合糖質の合成
下記化６に示す合成ルートにより標記人工複合糖質を合成した。
【００４３】
【化８】
化合物ａｒ（１５ｍｇ、１６．６μｍｏｌ）および４−アクリロイルオキシフェニルジメチルスルホニウムメチルスルフェイト（７）（２０．０ｍｇ、６２．５μｍｏｌ）を飽和ＮａＨＣＯ３溶液（３００μＬ）に加え、この溶液を室温で５．５時間攪拌し、該化合物（７）１５．０ｍｇの水（１００μＬ）水溶液１６．６μＬを追加した。この混合物を更に一晩攪拌し、次いで濾過した。この濾過物をＨＰＬＣ〔カラム：コスモシール（登録商標）５Ｃ１８ −ＡＲ、２０ｍｍ×２５０ｍｍ；溶媒：ＭｅＣＮ−０．１Ｍ ＨＣＯ２ＮＨ４（ｐＨ６．３）；勾配：５％ＭｅＣＮ−５０％ＭｅＣＮ（１．５％／ｍｉｎ）；流速：８ｍＬ／ｍｉｎ；検知：ＵＶ ３００ｎｍ〕にかけた。１８．７分後に溶出した分画を集め、凍結乾燥し、無色の粉末である化合物（８）を得た（収量９．２ｍｇ（収率８０％））。化合物（８）の構造は、１Ｈ−ＮＭＲ（Ｄ２Ｏ）で確認され、かつポジティブＦＡＢ−ＭＳでｍ／ｚ６９４．４〔（Ｍ＋Ｈ）＋〕に擬分子イオンピークを示した。化合物（８）は、アクリルアミドとともに容易にコポリマー化して高分子化合物とすることができる。
【００４５】
【発明の効果】
本発明の糖の蛍光標識方法は、極めて温和な条件で２位が還元糖残基に結合した６位にアルキルアミノ基を有する糖のピリジン誘導体、即ち、蛍光標識剤を収率よく製造でき、かつ６位のアミノ基と所望の蛋白質、脂質、糖、核酸等の生体物質を結合させることにより、容易に蛍光標識された人工複合糖質が生成できるので、各種物質の同定、定量、調製、精製等に極めて有用な手段を提供することができる。[0001]
[Industrial application fields]
The present invention relates to a method for fluorescently labeling sugars and a method for producing artificial glycoconjugates, and more particularly to the method using a 2-aminopyridine derivative.
[0002]
[Prior art]
In recent years, in order to study the relationship between the structure of glycoconjugates such as glycoproteins and their functions, various methods have been proposed that can be quantitatively assayed with a higher sensitivity and with a small amount of sample.
For example, a method for microanalysis of a sugar chain includes introducing tritium when converting the reducing end to a sugar alcohol and utilizing its radioactivity. This detection sensitivity is at the pmol level and is good, but there are various limitations due to being a radioactive substance.
[0003]
As a high-sensitivity assay without this restriction, a method using an organic compound such as 2-aminopyridine as a fluorescent labeling agent is known (for example, S. Hase et al., Journal of Biochemistry, Vol. 95, Vol. 197-203 (1984)).
It is disclosed that the 2-amino group is reacted with the reducing end of a sugar or sugar chain to form a Schiff base, then reduced and detected by fluorescence, and the sensitivity is at the pmol level as described above. Has been.
[0004]
However, although the fluorescent labeling agent is effective for structural analysis of sugar chains, it is monofunctional, so it can be used with other biological substances such as proteins, peptides, amino acids, lipids, nucleic acids, synthetic polymers and sugars. Therefore, it was not sufficient to analyze them comprehensively.
Therefore, the present applicant disclosed in JP-A-5-255253 a 2-aminopyridine derivative introduced with a functional group capable of reacting with the biological substance at the 6-position, and showed that various artificial glycoconjugates can be synthesized. .
[0005]
However, a method for synthesizing an artificial complex carbohydrate via an amino group produced by reduction of a cyano group after separating and purifying a labeled sugar chain using a cyano group introduced into a 2-aminopyridine derivative, In the reduction of the cyano group, there has been a problem that the pressure contact reduction must be performed for a long time using Pd in concentrated aqueous ammonia.
[0006]
[Problems to be solved by the invention]
The first object of the present invention is to provide a fluorescent labeling method for introducing a 2-aminopyridine derivative into a sugar under mild conditions, and a 2-aminopyridine derivative as a reactive agent that can be used for the synthesis of artificial glycoconjugates. It is to provide a method for easily producing a conjugate with sugar, and secondly, to provide a method for producing an artificial complex carbohydrate using the reactant.
[0007]
[Means for Solving the Problems]
The present invention
1) A 2-aminopyridine derivative having an aminoalkyl group having an amino protecting group at the 6-position and a terminalMonosaccharide, oligosaccharide, polysaccharide or glycosaminoglycan consisting of reducing sugarA method for fluorescent labeling of sugars, wherein the amino protecting group is removed by reductive amination reaction;
2) The amino protecting group is a urethane type amino protecting group or a trihaloacetyl group.1)Fluorescent labeling method for sugar according to the description,
3) The amino protecting group is a t-butoxycarbonyl group, a benzyloxycarbonyl group or a trifluoroacetyl group,1)Fluorescent labeling method for sugar according to the description,
4) The elimination reaction of the amino protecting group is carried out by treatment under acidic conditions or basic conditions, or by reduction reaction.1)~3)Or a method for fluorescently labeling a sugar according to any one of
5) The treatment under acidic conditions is performed using trifluoroacetic acid.4)Fluorescent labeling method for sugar according to the description,
6) The treatment under basic conditions is performed using an aqueous piperidine solution.4)Fluorescent labeling method for sugar according to the description,
7) The reduction reaction is performed by catalytic reduction.4)Fluorescent labeling method for sugar according to the description,
8) A 2-aminopyridine derivative having an aminoalkyl group having an amino protecting group at the 6-position and a terminalMonosaccharide, oligosaccharide, polysaccharide or glycosaminoglycan consisting of reducing sugarAnd a 2-aminopyridine derivative having an aminoalkyl group at the 6-position by removing the amino protecting group and reductive amination reactionMonosaccharides, oligosaccharides, polysaccharides or glycosaminoglycansAnd then directly reacting the amino group of the aminoalkyl group at the 6-position of the pyridine of the conjugate with an organic compound having a functional group capable of binding to the amino groupMakeOr reacted with an organic compound through a spacer having a functional group capable of binding to an amino groupAmide bond reaction, Schiff base formation reaction, urethane bond reaction, alkylation reaction, disulfide bond reaction, disulfide exchange reaction, amidine bond reaction or carbon-carbon bond reactionA method for producing an artificial glycoconjugate characterized by binding,
9) The organic compound is a sugar, protein, peptide, amino acid, lipid, nucleic acid, nucleotide, nucleoside, biotin, or synthetic polymer compound8)A method for producing the described artificial glycoconjugate,
10)The organic compound or the spacer is a compound having a carboxyl group, and the carboxyl group is activated to react with the amino group of the aminoalkyl group at the 6-position of pyridine8)Or9)It is a manufacturing method of the described artificial glycoconjugate.
[0008]
The method for fluorescently labeling a saccharide of the present invention comprises a 2-aminopyridine derivative having an aminoalkyl group having an amino protecting group at the 6-position (hereinafter sometimes abbreviated as a protecting group-containing 2-aminopyridine derivative) and a terminal.ReturnedMonosaccharides, oligosaccharides, polysaccharides or glycosaminoglycans composed of primary sugars (hereinafter also referred to as sugar compounds))WhenAre coupled by reductive amination reaction, and then the amino protecting group is removed. In the present invention, as a result of this elimination, a conjugate of a 2-aminopyridine derivative having an aminoalkyl group at the 6-position and a sugar compound is obtained, and this conjugate is a 6-position of any other organic compound. Therefore, the method of the present invention also provides a method for producing a fluorescent labeling reagent.
[0009]
Examples of the protecting group-containing 2-aminopyridine derivative used in the present invention include a compound represented by the following general formula (1) (hereinafter also referred to as compound 1).
[0010]
[Chemical Formula 3]
In the formula, R represents a protecting group which is an organic group, and n is an integer of 2 to 20, preferably 2 to 8. Examples of the protecting group include a urethane-type amino protecting group or a trihaloacetyl group, and the urethane-type amino protecting group includes a t-butoxycarbonyl group (hereinafter referred to as Boc) or a benzyloxycarbonyl group (hereinafter referred to as Z). The trihaloacetyl group is particularly preferably a trifluoroacetyl group (hereinafter referred to as Tfa), but is not limited thereto.
[0012]
In the formula, hydrogen at the 3-5 position may be substituted with any organic group, preferably a hydrocarbon group, particularly preferably a lower alkyl group having 1 to 4 carbon atoms.ConversionCompound 1WhenIn the reductive amination reaction with a sugar compound, the amino group at the 2-position of compound 1 and the sugar compound are condensed to form a Schiff base, which is then reduced to —CH2Forming an NH-bond. The resulting reaction product (conjugate) of compound 1 and a sugar compound is also referred to as sugar residue-containing compound 1.
[0013]
The resulting sugar residue-containing compound 1 is subjected to a reaction for removing the amino protecting group at the 6-position. This elimination reaction is not particularly limited, but the following method is preferable.
(1) A method of treating a sugar residue-containing compound 1 under acidic conditions using trifluoroacetic acid, anhydrous hydrogen chloride, formic acid or the like. It is particularly suitable when the protecting group is Boc.
(2) A method of catalytically reducing hydrogen residue-containing compound 1 in the presence of a catalyst such as Pd (palladium) under normal pressure or pressure, or catalytic hydrogen transfer reduction using an organic hydrogen donor in the presence of Pd Method. It is particularly suitable when the protecting group is Z.
(3) A method of treating the sugar residue-containing compound 1 with a base, for example, an aqueous solution of piperidine, ammonia, sodium hydroxide or the like under basic conditions. It is particularly suitable when the protecting group is Tfa.
[0014]
The condition for removing the protecting group using trifluoroacetic acid in (1) is that the trifluoroacetic acid solution of the sugar residue-containing compound 1 is used at room temperature, specifically at 10 to 25 ° C. for 10 to 30 minutes. Stirring and then removing trifluoroacetic acid while blowing nitrogen gas.
The condition for removing the protecting group using Pd catalyst in (2) is that Pd is added to the aqueous solution of the sugar residue-containing compound 1 and stirred at room temperature for 30 to 90 minutes in a hydrogen atmosphere of 1 to 10 atm. Can be mentioned.
[0015]
The condition for removing the protecting group using piperidine in (3) is that the 0.2 to 2M piperidine aqueous solution of the sugar residue-containing compound 1 is stirred at 0 to 10 ° C. for 0.5 to 2 hours. It is done.
The sugar residue-containing 2-aminopyridine derivative (hereinafter also referred to as “sugar residue-containing compound 1r”) in which the 6-position obtained by the reactions (1) to (3) is an aminoalkyl group can be obtained by any method. However, it can be highly purified and isolated in high yield (about 95% or more) preferably by HPLC (high performance liquid chromatography) as an aqueous solution. Since the method of the present invention is a very mild method, the high yield is achieved.
[0016]
Purification using HPLC is, for example, Cosmosil.(Registered trademark)5C 18 -Reversed phase HPLC or TSK gel using AR(Registered trademark) It can be performed using ion exchange HPLC with Amide-80.
Since this sugar residue-containing compound 1r has a reactive amino group at the 6-position, it has the function of a fluorescent labeling agent for labeling any other organic compound and creates an artificial complex carbohydrate It has the function of a reactive agent.
[0017]
There are no particular restrictions on the conditions for producing this artificial glycoconjugate, but specific examples include the following methods.
a. Desired artificial glycoconjugate by binding desired organic compound having functional group (for example, carboxyl group, formyl group, etc.) that reacts with amino group of sugar residue-containing compound 1r having desired sugar residue structure How to get.
b. a. As the organic compound, a compound having a functional group that reacts with another organic compound in addition to the functional group that reacts with the amino group of the sugar residue-containing compound 1r (eg, a spacer) is selected, and the resulting sugar residue-containing compound A method of obtaining an artificial complex carbohydrate by reacting a 1r derivative (eg, sugar residue-containing compound 1r-spacer conjugate) with a desired organic compound. The sugar residue-containing compound 1r derivative may have a plurality of functional groups, and there may be a plurality of organic compounds that react therewith.
[0018]
Examples of the spacer having a plurality of functional groups include those derived from glyoxal, disuccinimidyl suberate, succinimidyl 3- (2-pyridyldithio) propionate, succinimidyl 6-maleimidohexanoate, and the like.
c. A method in which a desired organic compound is bonded to a functional group introduced or previously present in the artificial glycoconjugate obtained in b.
[0019]
Examples of the reaction between the sugar residue-containing compound 1r and the organic compound in a include an amide bond reaction, a Schiff base formation reaction, a urethane bond reaction, and an alkylation reaction.
The sugar residue-containing compound 1r derivative in b functions as a reactive agent for preparing a kind of artificial glycoconjugate or other artificial glycoconjugate. The organic compound for obtaining this reactant may be a compound having a function only as a spacer for binding another organic compound or a compound having other useful actions. Further, in this sugar residue-containing compound 1r derivative, by selecting an organic compound to be introduced, it is possible to synthesize what can be a useful reagent by itself. For example, if a biotin derivative is selected as the organic compound, a reagent useful for analysis of lectins or the like (ELISA or the like) performed using labeled avidin or the like can be obtained.
[0020]
Here, as a functional group introduced into the sugar residue-containing compound 1r derivative by the reaction of the sugar residue-containing compound 1r with the organic compound, in addition to a functional group that causes a bonding reaction between the organic compound and the a, a disulfide Examples include functional groups that cause a bonding reaction, a disulfide exchange reaction, an amidine bonding reaction, a carbon-carbon bonding reaction, such as a carboxyl group, an amino group, a hydroxyl group, a disulfide group, and an acryloyl group.
[0021]
In the above reactions a to c, the functional group can be activated in advance and used for the reaction. For example, when the functional group is a carboxyl group, it can be activated by a succinimide group, and when it is a mercapto group, it can be activated by a 3-nitro-2-pyridylsulfenyl group.
The organic compounds in a to c are arbitrary, but sugars, proteins, peptides, amino acids, lipids, nucleic acids, nucleotides, nucleosides, biotin, or synthetic polymers (including monomers thereof) are particularly preferable. A useful artificial glycoconjugate can be easily obtained.
[0022]
BookBinding by reductive amination reaction in the inventionWhenIs compound 1 at the reducing end of the sugar compound.Amino group at position 2To form a Schiff base and then reduced to —CH2Form an NH-bondObtained from the previous reactionSugar residue-containing compound 1,If necessary, fractionate by high-performance liquid chromatography (HPLC), etc.ThedetectionByThe target sugar residue-containing compound 1 can be detected and separated and purified.
[0023]
The Schiff base formation reaction and the subsequent reduction reaction are carried out by a known method (for example, JP-A 64-10177, JP-A 1-1141356, J. Biochem., 95, 197-203 ( 1984), “Protein Nucleic Acid Enzyme”, Vol. 36, No. 1, pp. 63-68 (1991)). That is, in an inorganic acid such as hydrochloric acid and hydrofluoric acid, or an organic acid such as acetic acid and trifluoroacetic acid, and an organic solvent such as pyridine, normal temperature to 100 ° C., several minutes to several hours (preferably about 90 ° C., 1 to A Schiff base can be produced by reacting the saccharide with about 20 to 100 equivalents of Compound 1 under the reaction conditions of pH 3 to 6.4) for 3 hours. For the reduction of the Schiff base, a reducing agent usually used for the reduction of the Schiff base can be used, and in particular, a volatile borane complex (for example, borane dimethylamine complex (BH)).Three・ Me2NH), borane triethylamine complex, borane pyridine complex, etc.), sodium borohydride, sodium cyanoborohydride (NaBH)ThreeCN) and the like are preferable. The reduction reaction is completed at room temperature to 100 ° C. for 1 hour to 10 hours (preferably about 80 to 90 ° C. for about 1 hour).
[0024]
【Example】
Examples of the present invention will be specifically described below, but the present invention is not limited thereby.
Synthesis Example 1 Synthesis of Compound 1-1 (n = 3, R = Boc)
2-Tritylamino-6- (2-cyanoethyl) pyridine is LiAlHFour2-tritylamino-6- (3-aminopropyl) pyridine obtained by reduction with (Boc)2O(Di-tert-butyl dicarbonate)2-tritylamino-6- (3-t-butoxycarbonylaminopropyl) pyridine (2.00 g, 4.05 mmol) obtained by reaction with acetic acid-distilled MeOH [1: 1 (v / v)], 14 ml The solution was stirred at 50 ° C. for 5 and a half hours. The residue was purified by medium pressure silica gel chromatography [silica gel 50 g, chloroform-methanol (15: 1)]. Since a part of the solution was acetate, ethyl acetate was added for dissolution for desalting, and the mixture was washed successively with saturated aqueous sodium hydrogencarbonate and saturated brine, and then dried over magnesium sulfate. The desiccant was filtered off and concentrated under reduced pressure to give a pale yellow oil. The oil was left in the refrigerator for 2 weeks to obtain needle-shaped title compound 1-1. Yield 931 mg (91.6% yield from 2-tritylamino-6- (3-t-butoxycarbonylaminopropyl) pyridine), mp 72-74 ° C.1H-NMR (270 MHz, CDClThree) Confirmed the structure of compound 1-1. C13Htwenty oneO2NThreeCalculated value of C 62.13; H 8.42; N 16.72, found value C 61.90; H 8.48; N 16.59.
[0025]
Synthesis Example 2 Synthesis of Compound 1-2 (n = 3, R = Z)
2-Tritylamino-6- (2-cyanoethyl) pyridine is LiAlHFour2-Tritylamino-6- (3-aminopropyl) pyridine obtained by reduction with ZOSu(Benzyl N-succinimidyl carbonate)A solution of 2-tritylamino-6- (3-benzyloxycarbonylaminopropyl) pyridine (697 mg, 1.32 mmol) in acetic acid-MeOH [1: 1 (v / v), 10 ml] obtained by reaction with Stir at 18 ° C for 18 and a half hours. The residue was purified by medium pressure silica gel chromatography [silica gel 10 g, chloroform-methanol (15: 1)]. Since a part of it was an acetate salt, the title compound 1-2 as a pale yellow oil was obtained in the same manner as in Synthesis Example 1. Yield 361 mg (96.2% yield from 2-tritylamino-6- (3-benzyloxycarbonylaminopropyl) pyridine).1H-NMR (270 MHz, CDClThree) Confirmed the structure of compound 1-2. C16H19O2NThreeCalculated value of C, C 65.83; H 6.81; N 14.38, found value C 65.83; H 6.72; N 14.38.
[0026]
Synthesis Example 3 Synthesis of Compound 1-3 (n = 3, R = Tfa)
2-Tritylamino-6- (2-cyanoethyl) pyridine is LiAlHFour2-Tritylamino-6- (3-aminopropyl) pyridine obtained by reduction with (Tfa)2O(Trifluoroacetic anhydride)A solution of 2-tritylamino-6- (3-trifluoroacetylaminopropyl) pyridine (140 mg, 285 μmol) in acetic acid-MeOH [1: 1 (v / v)], 4 ml] obtained by reacting with 50 ° C. For 22 hours. The residue was purified by medium pressure silica gel chromatography [silica gel 8 g, dichloromethane-methanol (20: 1)]. Since a part of it was an acetate salt, the title compound 1-3 having pale yellow needles was obtained in the same manner as in Synthesis Example 1. Yield 61.4 mg (96.2% yield from 2-tritylamino-6- (3-trifluoroacetylaminopropyl) pyridine).1H-NMR (270 MHz, CDClThree) Confirmed the structure of compound 1-3. CTenH12ONThreeFThreeCalculated value of C 48.58; H 4.89; N 17.00, found value C 48.62; H 4.81; N 16.87.
[0027]
Examples 1-1 to 1-3
Compound 1-1 to 1-3 (27.8 μmol, described in Table 1) and acetic acid-pyridine [1:17 (v / v), 48.0 μL) to the dried sugar compound (as described in Table 1) (5.55 μmol) ], Sealed and heated at 90 ° C. for 3 hours, allowed to cool, and then reduced reaction solution [BH3・ Me2NH (6.55 mg, 111 μmol) in acetic acid (33.5 μL) solution] (Me: methyl group) was added, sealed, and heated at 80 ° C. for 1 hour. After standing to cool, water was added and HPLC [Column: Cosmo Seal(Registered trademark)5C 18 −AR 20 mm ID × 250 mm L; elution solvent: acetonitrile-0.1 M ammonium formate (pH 4.5); concentration gradient: [0-60% acetonitrile (2% / min)]; flow rate: 8 mL / min; detection: UV 300 nm] Each sugar residue-containing compound 1 was purified and then lyophilized. Each yield is shown in Table 1.
[0028]
[Table 1]
Next, the following three sugar residue-containing compounds 1 (ac) obtained in Example 1-3, that is,
[0030]
[Formula 4]
The protecting group was removed as follows.
(1) Boc removal of compound a
A solution of compound a (1.18 mg, 1.60 μmol) in trifluoroacetic acid (200 μL) was stirred at room temperature for 30 minutes. Thereafter, nitrogen gas was blown to remove trifluoroacetic acid, and water was added to the residue to dissolve it. After separation and purification by HPLC, lyophilization was performed to obtain a compound ar represented by the following formula in which Boc was replaced with H. The yield was quantitative and approximately 100% by HPLC analysis.
[0032]
[Chemical formula 5]
(2) Removal of Z from compound b
Compound (b) (1.31 mg, 1.69 μmol) was dissolved in water (500 μL), palladium black (11.2 mg) was added, and the mixture was stirred at room temperature for 90 minutes under a hydrogen atmosphere of 4 atm. Then, after separation and purification by HPLC, lyophilization was performed to obtain a compound ar in which Z was replaced with H. The yield was quantitative and approximately 100% by HPLC analysis.
(3) Tfa removal of compound c
A solution of compound c (0.15 mg, 0.2 μmol) in 1M piperidine aqueous solution (250 μL) was stirred at 0 ° C. for 1 hour. Then, after separation and purification by HPLC, lyophilization was performed to obtain compound ar in which Tfa was replaced with H. The yield was quantitative and approximately 100% by HPLC analysis.
[0034]
Comparative example
In the compound ar, 32 μmol of the compound in which the 6-position of the pyridine ring is a cyanoethyl group was added with 9 kg / cm of hydrogen in the presence of palladium black (50 mg) in a 25% aqueous ammonia solution (14 mL).2The compound ar was synthesized by hydrogenation at room temperature for 66 hours under the following conditions. The catalyst was filtered off and the filtrate was concentrated. The concentrated residue was dissolved in water (10 mL) and HPLC [column: Cosmosi-Le(Registered trademark)5C18 −AR, 20 mm × 250 mm; Solvent: MeCN-0.1M CH3CO2NH4(PH 6.9); gradient: 5% MeCN-50% MeCN (1.5% / min); flow rate: 8 mL / min; detection: UV 307 nm]. The fraction containing compound ar was lyophilized and the resulting powder was dissolved in water (10 mL). In order to remove ammonium salts mixed in this solution, HPLC [column: Cosmosi-Le(Registered trademark)5C18 −AR, 20 mm × 250 mm; solvent: 15% MeCN-0.01% TFA; flow rate: 8 mL / min; detection: UV307 nm]. The yield was 77%.
[0035]
Example 2
Example 2-1 Synthesis of artificial glycoconjugate consisting of bovine serum albumin (BSA) and compound ar
The title artificial glycoconjugate was synthesized by the synthetic route shown in the following chemical formula 4.
[0036]
[Chemical 6]
Compound ar and succinimidyl 3- (3-nitro-2-pyridyldithio) propionate (1) were combined in a tetrahydrofuran (THF) -water (2: 1) TEA (triethylamine) solution (pH 9), and reverse phase HPLC ( Column: Cosmosi-Le(Registered trademark)5C 18 −AR, solvent: MeCN-0.1% aqueous TFA solution) gave compound (2). Compound (2) was obtained as 2 trifluoroacetate salt.
[0038]
Next, BSA (3) having 6 cysteine residues and compound (2) are mixed at various molar ratios [compound (2): BSA = 1 to 6: 1] with 0.5 M Tris-0.005 MEDTA sodium salt buffer. The resultant was dissolved together in a liquid (pH 7.5) and subjected to an exchange reaction between the -SH group of BSA and the disulfide group of compound (2) at 4 ° C. to obtain an artificial complex carbohydrate (4). The resulting artificial glycoconjugate (4) is separated from compound (2) by gel filtration HPLC [column: Asahi Pack GS-510, solvent: 30% MeCM-0.2M sodium phosphate buffer (pH 7.0)]. did.
[0039]
Example 2-2 Synthesis of artificial glycoconjugate consisting of biotin and compound ar
The title artificial glycoconjugate was synthesized by the synthetic route shown in Chemical Formula 5 below.
[0040]
[Chemical 7]
Compound ar (15.0 mg, 16.6 μmol) 0.5% NaHCO 33N-hydroxysuccinimide biotin (5) (40.0 mg, 117 μmol) was added to an aqueous solution-DMF [1: 1 (v / v), 500 μL] aqueous solution, and the mixture was stirred at room temperature for 3.5 hours. Compound (5) (24.0 mg, 70.4 mmol) is then added, the mixture is filtered, and the filtrate is purified by HPLC [column: Cosmosi.-Le(Registered trademark)5C18 −AR, 20 mm × 250 mm; Solvent: MeCN-0.1M HCO2NH4(PH 6.3); gradient: 5% MeCN-50% MeCN (1.5% / min); flow rate: 8 mL / min; detection: UV 300 nm]. Fractions eluted after 20.9 minutes were collected and lyophilized to obtain Compound (6) as a colorless powder (yield 9.3 mg (yield 65%)). Compound (6) is1H-NMR (D2O) and m / z 866.7 [(M + H) by positive FAB-MS+] Shows a quasi-molecular ion peak.
[0042]
Example 2-3 Synthesis of Artificial Complex Carbohydrate Composed of Acrylic Acid and Compound ar
The title artificial glycoconjugate was synthesized by the synthetic route shown in Chemical Formula 6 below.
[0043]
[Chemical 8]
Compound ar (15 mg, 16.6 μmol) and 4-acryloyloxyphenyldimethylsulfonium methylsulfate (7) (20.0 mg, 62.5 μmol) were saturated with NaHCO 3.3In addition to the solution (300 μL), the solution was stirred at room temperature for 5.5 hours, and 16.6 μL of an aqueous solution of 15.0 mg of the compound (7) in water (100 μL) was added. The mixture was further stirred overnight and then filtered. This filtrate was subjected to HPLC [column: Cosmosi.-Le(Registered trademark)5C18 −AR, 20 mm × 250 mm; Solvent: MeCN-0.1M HCO2NH4(PH 6.3); gradient: 5% MeCN-50% MeCN (1.5% / min); flow rate: 8 mL / min; detection: UV 300 nm]. Fractions eluted after 18.7 minutes were collected and lyophilized to obtain Compound (8) as a colorless powder (yield 9.2 mg (yield 80%)). The structure of compound (8) is1H-NMR (D2O) and m / z 694.4 [(M + H) by positive FAB-MS+] Shows a quasi-molecular ion peak. Compound (8) can be easily copolymerized with acrylamide to give a polymer compound.
[0045]
【The invention's effect】
The sugar fluorescent labeling method of the present invention can produce a pyridine derivative of a sugar having an alkylamino group at the 6-position bonded to a reducing sugar residue at the 2-position under extremely mild conditions, that is, a fluorescent labeling agent in a high yield. In addition, it is possible to easily produce fluorescently labeled artificial complex carbohydrates by combining 6-position amino group and biological substances such as desired proteins, lipids, sugars, nucleic acids, etc. An extremely useful means for purification and the like can be provided.
Claims (10)
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| JP2005241389A (en) * | 2004-02-25 | 2005-09-08 | Ochiyanomizu Jiyoshi Univ | Specific immobilization reagent for fluorescence-labeled sugar chain and immobilization method |
| US9239329B2 (en) | 2006-12-18 | 2016-01-19 | Japan Science And Technology Agency | Method of measuring interaction between biomaterial and sugar chain, method of evaluating biomaterial in sugar chain selectivity, method of screening biomaterial, method of patterning biomaterials, and kits for performing these methods |
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