JP4098839B2 - Indocyanine compounds - Google Patents

Description

（式中、A¹及びA²はそれぞれ独立にベンゼン環又はナフタレン環を示し、R¹及びR²はそれぞれ独立に水素原子、アルキル基、アリール基、遊離若しくは解離したスルホン酸基、又はアルコキシ基を示し、R³はアルキル基、遊離若しくは解離したスルホン酸アルキル基、又はアミノアルキル基若しくはアンモニオアルキル基を示し、n は 1〜3 の整数を示し、X ^- は必要に応じてアニオン種を示し、Z は以下の式：
【化４】

からなる群から選ばれる基であり、M ⁺ はアルカリ金属イオンであり、W 及びY はそれぞれ独立に炭素数が1 〜10のアルキレン基を示すか、あるいはW 及びY は酸素原子、窒素原子、及び硫黄原子からなる群から選ばれる１種以上の原子を含む炭素数が1 〜10のアルキレン基を示す）で示される化合物を提供するものである。
【００１１】
本発明の好ましい態様によれば、A¹及びA²がともにナフタレン環であり、R¹及びR²がともに水素原子であり、R³が解離した炭素数 2〜5 の直鎖スルホン酸アルキル基であり、n が3 であり、Y が炭素数 2〜5 の直鎖アルキレン基であり、Z がスクシンイミドオキシ基又はアルカリ金属スルホスクシンイミドオキシ基である上記化合物；及び、X ^- がヨウ素イオンであり、R³がナトリウムイオンと塩を形成した炭素数 2〜5 の直鎖スルホン酸アルキル基である上記化合物が提供される。
【００１２】
また、本発明の別の態様によれば、上記化合物からなる蛍光標識用試薬；アミノ基、水酸基、カルボニル基、及びスルフヒドリル基からなる群から選ばれる１個以上の官能基を含む化合物の蛍光標識に用いる上記試薬；官能基を含む該化合物が生体高分子である上記試薬；並びに、生体高分子がタンパク、糖タンパク、脂質、リン脂質、多糖類、及び核酸からなる群から選ばれる上記試薬が提供される。
【００１３】
【発明の実施の形態】
上記一般式中、A¹及びA²はそれぞれ独立にベンゼン環又はナフタレン環を示し、好ましくはナフタレン環を用いることができる。A¹及び／又はA²がナフタレン環を示す場合、A¹及び／又はA²が縮合するピロール環に対する該ナフタレン環の縮合位置は特に限定されることはない。また、A¹及び／又はA²がナフタレン環を示す場合、R¹及びR²はナフタレン環を構成する２個のフェニル環のうちのいずれかの環の任意の位置に置換することができる。A¹及び／又はA²がフェニル環を示す場合、R¹及びR²はフェニル環の任意の位置に置換していてもよい。
【００１４】
R¹及びR²は、それぞれ独立に水素原子、アルキル基、アリール基、遊離若しくは解離したスルホン酸基、又はアルコキシ基を示す。アルキル基としては、炭素数 1〜6 程度、好ましくは炭素数1 〜4 の直鎖又は分枝鎖の低級アルキル基を用いることができる。例えば、メチル基、エチル基、イソプロピル基、n-ブチル基、sec-ブチル基、tert- ブチル基などを好適に使用できる。アリール基としては、例えば、フェニル基、ナフチル基、ピリジル基、イミダゾリル基、ピロリル基などを挙げることができる。解離したスルホン酸基には対イオンとしてアルカリ金属イオンなど、例えば、ナトリウムイオンあるいはカリウムイオンなどが存在していてもよい。アルコキシ基としては、炭素数 1〜6 程度、好ましくは炭素数 1〜4 の直鎖又は分枝鎖の低級アルコキシ基を用いることができ、より具体的には、メトキシ基、エトキシ基、プロポキシ基、n-ブトキシ基、sec-ブトキシ基、tert- ブトキシ基などを用いることが可能である。
【００１５】
R³はアルキル基、遊離若しくは解離したスルホン酸アルキル基、又はアミノ若しくはアンモニオアルキル基を示す。アルキル基としては炭素数 1〜6 程度、好ましくは炭素数1 〜4 の直鎖又は分枝鎖の低級アルキル基を用いることができる。スルホン酸アルキル基のスルホン酸、又はアミノ若しくはアンモニオアルキル基のアミノ基若しくはアンモニオ基はアルキル基の任意の位置に置換することができるが、アルキル基の末端に置換したものを用いるのが好適である。アミノ基若しくはアンモニオ基は、１個又は２個（アンモニオ基の場合には１ないし３個）の同一若しくは異なる低級アルキル基（好ましくは炭素数 1〜6 程度のアルキル基）を有していてもよい。
【００１６】
例えば、R³として-(CH₂)_k -SO₃- （k は3 〜5 の整数を示す）で示される基などが好適である。R³が解離したスルホン酸アルキル基である場合には、スルホン酸イオンの対イオンとしてアルカリ金属イオン、例えば、ナトリウムイオンあるいはカリウムイオンなどが存在していてもよい。R³がアンモニオアルキル基である場合、対イオンとしてハロゲンイオン、過塩素酸イオンなどの陰イオン、好ましくはハロゲンイオンが存在していてもよい。
【００１７】
X ^- は必要に応じて存在していてもよいアニオン種、例えばハロゲンイオン、酢酸イオン、過塩素酸イオン、炭酸イオン、またはチオシアン酸イオンを示す。X ^- で示されるアニオン種は、母核内の窒素原子上の陽電荷を打ち消して化合物を全体として中性に維持するために作用する。例えば、R¹, R², 及びR³のうちのいずれか一つに解離したスルホン酸部分が存在すると、その解離したスルホン酸の陰電荷と母核内の窒素原子上の陽電荷が打ち消しあうので、X ^- は存在しなくてもよい。一方、例えば、R¹及びR²のうちのいずれか一方が解離したスルホン酸基であり、R³がアンモニオアルキル基である場合には、これらの基の電荷が釣り合うので、母核内の窒素原子上の陽電荷を打ち消すためにX ^- が必要になることがある。X ^- としては、好ましくは、塩素イオン、臭素イオン、ヨウ素イオンなどを用いることができる。これらのうち、ヨウ素イオンが特に望ましい。n は1 〜3 の整数を示すが、n が3 であることが好ましい。
【００１８】
Y は、炭素数１〜10の直鎖若しくは分枝鎖のアルキレン基、好ましくは炭素数 2〜5 の直鎖又は分枝鎖のアルキレン基を好適に用いることができる。特に好ましくは、トリメチレン基、テトラメチレン基、ペンタメチレン基を用いることができる。また、Y は、酸素原子、窒素原子、及び硫黄原子からなる群から選ばれる１以上の原子を含む炭素数 1〜10の直鎖若しくは分枝鎖のアルキレン基を示す。
【００１９】
具体的には、-Y-CO-で示される基として、例えば、-CH₂-CO-; -(CH₂)₂-CO-; -(CH₂)₃-CO-; -(CH₂)₄-CO-; -(CH₂)₅-CO-; -CH₂-CO-NH-(CH₂)₅-CO-; -(CH₂)₂-CO-NH-(CH₂)₅-CO-; -(CH₂)₃-CO-NH-(CH₂)₅-CO-; -(CH₂)₄-CO-NH-(CH₂)₅-CO-; -CH₂-CO-NH-(CH₂)₅-CO-NH-(CH₂)₂-CO-; -(CH₂)₄-CO-(N,N'-piperadinyl)-(CH₂)₂-CO (N,N'-piperadinylは、ピペラジンの1-位に-(CH₂)₄-CO- 基が置換されており、4-位に-(CH₂)₂-Z 基が置換されていることを示す。以下、同様である。）; または-CH₂-CO-NH-(CH₂)₅-CO-(N,N'-piperadinyl)-(CH₂)₂-CO-などを利用することができる。
【００２０】
Z で示される基において、W としては上記 Y-CO の例示中のY に相当する基を好適に用いることができる。また、N-スルホスクシンイミジルオキシ基のスルホン酸基の対イオンの M⁺ と X^- の対イオンの M⁺ とは、同一でも異なっていてもよいが、両者がナトリウムイオンであることが好ましい。さらに、R¹及び／又はR²が解離したスルホン酸基を示し、及び／又はR³が解離したスルホン酸アルキル基を示す場合には、該スルホン酸の対イオンは X^- の対イオンの M⁺ 及び／又はN-スルホサクシイミジルオキシ基のスルホン酸基の対イオンの M⁺ と同一でも異なっていてもよいが、いずれもナトリウムイオンであることが好ましい。
【００２１】
いかなる特定の理論に拘泥するわけではないが、例えばR³が解離したスルホン酸アルキル基の場合には、そのスルホン酸基の陰電荷と窒素原子上の陽電荷（式中 N⁺ で表す）とが打ち消し合い、いわゆる分子内塩（ツビッター・イオン型化合物）が形成される。このような分子内塩型の化合物では、分子全体のイオン性が低下して化合物の水溶性が損なわれることがある。一方、このような化合物において、スルホン酸基の陰電荷に対してアルカリ金属イオンが対イオンとして存在し、かつ、窒素原子上の陽電荷に対して対イオンとしてX ^- で表されるアニオン種が存在すると、スルホン酸基の陰電荷と窒素原子上の陽電荷とによる分子内塩の生成が阻害されるので水溶性が顕著に増大することがある。従って、X ^- がヨウ素イオンなどのアニオンであり、かつ、R³がナトリウムイオンと塩を形成したスルホン酸アルキル基である化合物は、本発明の好ましい態様である。
【００２２】
なお、上記の一般式、並びに下記の好ましい態様の化合物及び実施例中のスキームの化合物では、便宜上、母核内の一の窒素原子上に陽電荷を固定して記載したが、共役二重結合を介して陽電荷が母核内の他の窒素原子に移動可能であることは当業者に容易に理解されよう。従って、このような共役に基づく異性体はすべて本発明の範囲に包含されることはいうまでもない。
【００２３】
本発明の化合物は、2,3,3-トリメチルインドレニン、2,3,3-トリメチルベンゾインドレニン又はそれらの誘導体を原料として合成することができる。これらの原料はフィッシャーの合成法（E. Fischer and O. Hess: Berichte, 17, 559, 1883 など）に従って合成するか、又は市販品を入手することができる。例えば、原料化合物の窒素原子上にアルキル基を導入した後、オルトぎ酸トリエチルなどを反応させてシアニン色素骨格を形成し、その後にZ で示される標識基を導入することにより製造可能である。
【００２４】
X ^- がヨウ素イオンなどのアニオンであり、かつ、R³がナトリウムイオンと塩を形成したスルホン酸アルキル基である化合物などの分子内塩を形成しない化合物を製造するためには、まず、解離したスルホン酸アルキル基の陰電荷と母核内の窒素原子上の陽電荷とが打ち消しあって分子内塩を形成した化合物（X ^- の存在しない化合物）を製造した後、この化合物を例えばジメチルホルムアミドやジメチルスルホキシドなどの有機溶媒に高濃度で溶解し、その溶液にヨウ化ナトリウムなどの塩を添加すればよい。もっとも、本発明の化合物の製造方法はこれらの方法に限定されることはない。
【００２５】
本発明の化合物の代表例として化合物(A) 〜(P) を以下に示す。
【化５】

【００２６】
【化６】

【００２７】
【化７】

【００２８】
【化８】

【００２９】
【化９】

【００３０】
本発明の化合物は、例えば、反応性のアミノ基、水酸基、チオール基、カルボニル基、スルフヒドリル基などを有する動植物由来の生体高分子の標識に用いることができる。生体高分子としては、例えば、タンパク、糖タンパクなどの複合タンパク、脂質、リン脂質などの複合脂質、多糖類、核酸などを挙げることができる。例えば、ガンなどに特異性の高い抗体や酵素などは本発明の化合物の好適な標識対象である。また、本発明の化合物は、反応性のアミノ基、水酸基、チオール基、カルボニル基、スルフヒドリル基などを有する低分子化合物と容易に反応するので、例えば、ビオチン−アビジンなどの増幅系に用いられる増幅系物質を標識することも可能である。
【００３１】
例えば、Z としてN-サクシイミジルオキシ基およびN-スルホサクシイミジルオキシ基を用いる場合、これらの基はZ が結合するカルボニル基とともに反応性の活性エステルを形成しており、例えば、被標識化合物に含まれるアミノ基と反応してアミド結合を形成する。また、Z としてN-マレイミドアルキル基を用いる場合には、例えば、被標識化合物に含まれるチオール基(HS-R)が反応してチオエーテルが形成される。さらに、Z が-NH-NH₂ である場合には、被標識化合物に含まれる還元糖末端のアルデヒド基(OHC-R) が反応して-Y-CO-NH-NH-CO-Rが形成される。従って、本発明の化合物を適宜選択して用いることにより、官能基特異的に被標識化合物を標識することが可能である。もっとも、本発明の化合物の用途は上記に例示したものに限定されることはない。
【００３２】
本発明の化合物は、モル吸光係数及び蛍光量子収率の高いインドシアニン化合物に活性部位を導入した化合物であり、被標識化合物にインドシアニンの分光学的性質を付加させることができる。本発明の化合物により標識された被標識化合物は、励起波長500 〜800nm 、蛍光波長550 〜850nm の蛍光特性を備えているので、高出力の半導体レーザーやHe・Neレーザーを励起光源として用いることにより極めて高感度な検出が可能になる。
【００３３】
【実施例】
以下、実施例により本発明をさらに具体的に説明するが、本発明の範囲は以下の実施例に限定されることはない。また、この実施例を参照した当業者には、出発原料、試薬、反応条件などに適宜の修飾・改変を加えることにより、本発明の範囲に包含される所望の化合物が同様に容易に製造することが容易に理解されよう。なお、実施例中の化合物番号はスキーム中の化合物番号に対応しており、化合物(A) などは前記の好ましい化合物として示された化合物の番号に対応している。
【００３４】
例１：化合物(A) 及び(B) の製造
【化１０】

【００３５】
化合物１ (2,3,3-トリメチルインドレニン) 5.0 g (31.4 ミリモル) をアセトニトリル100 mlに溶解し、ヨードエタン 5.9 g (37.8ミリモル) を加えて一夜加熱還流した。反応液を減圧下濃縮し、残渣にエーテルを加えて化合物２の灰赤色結晶を濾取した(9.1 g, 92%)。別途、化合物１ (5.0 g, 31.4 ミリモル) をジメチルホルムアミド(DMF)100mlに溶かし、6-ブロモヘキサン酸エチル 20.0 g (89.6 ミリモル) を加えて窒素雰囲気下に80℃で一夜加熱した。反応液を減圧下濃縮し、得られたオイルをエーテル 50 mlで洗浄した。次いで、得られたオイルをメタノール100 mlに溶解し、水酸化ナトリウム 11.5 g (287.5ミリモル) を水に溶解して加え、室温で５時間攪拌して加水分解した。メタノールを減圧下に留去した後、 1N 塩酸で中和した。クロロホルムで洗浄して非水溶性のオイルを除いた後、減圧下で濃縮乾固した。エタノールに溶解して濾過することにより無機塩を除き、得られた濾液を減圧下に濃縮乾固し、さらに減圧下で乾燥して化合物３の淡赤色粉末を得た(3.2 g, 37.3%)。
【００３６】
化合物２ (3.2g, 10.2ミリモル) と化合物３ (2.8 g, 10.2 ミリモル) をピリジン 60 mlに溶解して110 ℃に加熱した後、オルトギ酸トリエチル 2.5 ml (15.0 ミリモル) を徐々に滴下して１時間加熱した。反応液を減圧下濃縮し、クロロホルムに溶解して水洗した。クロロホルム溶液を減圧下で濃縮し、得られた残渣をシリカゲルカラムクロマトグラフィー（溶媒：メタノール／クロロホルム) に付して分離精製した。溶離液を減圧下で濃縮乾固し、得られた固体を減圧乾燥して化合物４の緑赤色金属光沢結晶を得た(1.6 g, 33%)。
【００３７】
化合物４ (20 mg, 0.042ミリモル) とN-ヒドロキシこはく酸イミド(NHS) 6.4 mg (0.056 ミリモル) を無水DMF 100 μl に溶解して 0℃に保ち、ジシクロヘキシルカルボジイミド(DCC) 180 mg (0.874 ミリモル) を無水DMF 50μl に溶解して加え、0 ℃で一夜反応させた。反応液に塩化水素ガスを吹込み、エーテルを加えて、目的物である化合物(A) を得た(18.0 mg, 70.1%)。化合物(A) をpH 7.4のPBS (NaCl: 8.0g, KCl: 0.2g, Na₂HPO₄: 11.5g, KH₂PO₄: 0.2g／水 1000 ml) に溶かした溶液の吸収スペクトルを図１に示す。
【００３８】
化合物４ (300 mg, 0.64ミリモル) をDMF 20 ml に溶かし、ピペラジノエチルマレイミド二塩酸塩一水和物(PEM) 242 mg (0.81ミリモル) 及びトリエチルアミン 120μl (0.88 ミリモル) を加えて 0℃に冷却した。この溶液に DCC 680 mg (3.30 ミリモル) を加えて 0℃で一夜反応させた。反応液を濾過し、濾液を減圧下に濃縮し、得られた残渣をシリカゲルカラムクロマトグラフィー（溶媒：メタノール／クロロホルム) に付して分離精製し、化合物(B) の緑赤色金属光沢結晶を得た (350 mg, 78.6%)。
【００３９】
例２：化合物(C) の製造
【化１１】

【００４０】
化合物１ (2.0 g, 6.3ミリモル) をDMF 100 mlに溶かし、6-ブロモヘキサン酸エチル 8.4 g (37.6ミリモル) を加えて窒素雰囲気下に80℃で一夜加熱した。反応液を減圧下濃縮し、残渣をシリカゲルクロマトグラフィー（溶媒：メタノール／クロロホルム) に付して分離精製し、化合物５の赤褐色オイルを得た (3.4g, 40%)。化合物２ (2.0 g, 6.3ミリモル) と化合物５ (2.4 g, 6.3ミリモル) をピリジン 10 mlに溶解して110 ℃に加熱した後、オルトぎ酸トリエチル 1.6 ml (10.0 ミリモル) を徐々に滴下し、1 時間加熱した。反応液を減圧下濃縮し、残渣をクロロホルムに溶解して水洗した。クロロホルム溶液を減圧下で濃縮し、得られた残渣をシリカゲルカラムクロマトグラフィー（溶媒：メタノール／クロロホルム) に付して分離精製した。溶離液を減圧下で濃縮乾固し、得られた固体を減圧乾燥して化合物６の緑赤色結晶を得た (960 mg, 26.1%)。
【００４１】
化合物６ (200 mg, 0.35ミリモル) をアセトニトリル 10 mlに溶解し、ヒドラジン一水和物 10 ml (205.7 ミリモル) を加えて室温で一夜攪拌した。1N塩酸で反応液を弱酸性とし、減圧下で濃縮した後、残渣をセファデックスLH20／メタノールで精製して化合物(C) を得た (130 mg, 72.3%)。
【００４２】
例３：化合物(H) の製造
【化１２】

【００４３】
化合物２ (3.2 g, 10.2 ミリモル) と化合物３ (2.8 g, 10.2 ミリモル) をピリジン 60 mlに溶解し、110 ℃に加熱した後、1,3,3-トリメトキシプロペン 2.0g (15.1 ミリモル) を加え、1 時間加熱した。反応液を減圧下で濃縮し、得られた残渣をクロロホルムに溶解して水洗した。クロロホルム溶液を減圧下で濃縮し、得られた残渣をシリカゲルカラムクロマトグラフィー（溶媒：メタノール／クロロホルム) に付して分離精製した。溶離液を減圧下で濃縮乾固し、得られた残渣を減圧乾燥して化合物７の緑色金属光沢結晶を得た(1.2 g, 23.8%)。例１と同様にして化合物７ (200 mg, 0.40ミリモル) とNHS 60 mg(0.52ミリモル) とを反応させ、反応物を単離精製して化合物(H) を得た(1.2 g, 23.8%)。
【００４４】
例４：化合物(J) の製造
【化１３】

【００４５】
化合物８ (4.0 g, 19.1 ミリモル) をDMF 60 ml に溶解し、6-ブロモヘキサン酸エチル 13.0 g (58.3 ミリモル) を加え、窒素雰囲気下に80℃で一夜加熱した。反応液を減圧下で濃縮し、残渣のオイルをエーテル 50 mlで洗浄した。次いで、得られたオイルをメタノール 100 ml に溶解し、水酸化ナトリウム 7.0 g (175 ミリモル) を水に溶解して加え、室温で5 時間攪拌して加水分解した。メタノールを減圧下留去した後 1N 塩酸で中和した。クロロホルムで洗浄して非水溶性のオイルを除いた後、減圧下濃縮乾固した。残渣をエタノールに溶解して濾過することにより無機塩を除き、その濾液を減圧下濃縮乾固して減圧乾燥し化合物９の淡青色粉末を得た(4.2 g, 67.9%)。
【００４６】
化合物８ (7.0 g, 19.1 ミリモル) をアセトニトリル 100 ml に溶解し、ヨードエタン 4.5 g (28.9ミリモル) を加え、一夜加熱還流した。反応液を減圧下に濃縮し、残渣にエーテルを加えて化合物10の灰青色結晶を得た(7.4 g, 84.8%)。グルタコンアルデヒドジアニル塩酸塩 6.0 g (24.2ミリモル) を無水酢酸 100 ml に溶解し、撹袢下に化合物10 (7.4 g, 20.3 ミリモル) を加えた。混合液を100 ℃に加熱して１時間反応させた後、反応液を減圧下に濃縮し、残渣にエーテルを加えて緑色結晶を得た。
【００４７】
上記の緑色結晶をピリジン 60 mlに溶解し、化合物９ (5.6 g, 17.3 ミリモル) を加えて、60℃で5 時間反応させた。反応液を減圧下に濃縮し、残渣をシリカゲルカラムクロマトグラフィー（溶媒：メタノール／クロロホルム) に付して分離精製した。溶離液を減圧下で濃縮乾固し、得られた固体を減圧乾燥して化合物12の緑色結晶を得た(3.8 g, 35.2%)。化合物12 (20 mg, 0.032ミリモル) とNHS 4.9 mg (0.043 ミリモル) を無水DMF 100 mlに溶解して 0℃に保ち、DCC 140 mg (0.68ミリモル) を無水DMF 50 ml に溶解して加え、一夜反応させた。反応液に塩化水素ガスを吹込み、エーテルを加えて目的とする化合物(J) を得た(10.5 mg, 43.2%)。
【００４８】
例５：化合物(K) 及び(L) の製造
【化１４】

【００４９】
化合物８ (5 g, 23.9 mmol) ／DMF 100 mlに 6- ブロモヘキサン酸エチルエステル (6.32 ml, 35.5 mmol) を加えて 80 ℃で 16 時間加熱した。反応液を減圧下に濃縮し、残渣にエーテル 200 ml を加えて結晶化した後、得られた結晶を濾過してエーテルで洗浄し、減圧下で乾燥して化合物８´を得た。化合物８´に1 M 水酸化ナトリウム水溶液／メタノール＝１／１混合溶液 30 mlを加えて室温で２時間攪拌した。メタノールを減圧留去し、水溶液を 4 M塩酸水溶液で中和したのち、クロロホルムで３回洗浄した。水溶液を減圧下に濃縮し、化合物９の赤色固体を得た。収量 5.75 g ( 収率 74.4 %)。
【００５０】
化合物13 (5.0 g, 14.5 mmol) とグルタコンアルデヒドジアニル塩酸塩 (4.23 g, 14.5 mmol)を無水酢酸 20 mlと酢酸 300 ml の混合溶液に懸濁し、還流温度で５時間加熱した。赤色溶液を減圧濃縮し、得られた残渣に酢酸エチルと水の混合溶液 200 ml を加えて懸濁させ、黒赤色固体を濾取した。水で洗浄した後、減圧下に乾燥し、化合物14の黒赤色粉末を得た。収量 5.20 g ( 収率 66.1 %)。
【００５１】
化合物14 (300 mg, 0.553 mmol) と化合物９ (179 mg, 0.553 mmol) をピリジン 5 ml に溶解し、50℃で１時間攪拌した。反応混合物を減圧下に濃縮し、得られた残渣を水 10 mlに溶解して溶液の pH を３に合わせた後、セファデックス LH20 カラム( 溶媒：メタノール) で精製して、化合物15の黒緑色固体を得た。収量 105 mg ( 収率 25.9 %)。
【００５２】
化合物15 (106 mg, 0.145 mmol)/50 v/v % THF水溶液 3 ml に N- ヒドロキシ無水こはく酸スルホン酸ナトリウム (65.2 mg, 0.287 mmol)と N,N'-ジシクロヘキシルカルボジイミド(129 mg, 0.596 mmol)を加えて４℃で一晩反応させた後に濾過し、濾液を 20 ℃以下で減圧下濃縮した。残渣に酢酸エチル 10 mlを加え、生じた結晶を更にエーテルで３回洗浄し、化合物(K) の黒緑色固体を得た(110 mg, 80.7%) 。MS (FAB) m/e =906 (M^- ) ；蛍光スペクトル :λ_ex=768 nm,λ_em=807 nm (H₂O)
【００５３】
上記の化合物(K) 100 mg (0.11 mmol)をメタノール 2 ml に溶かし、ヨウ化ナトリウム 1 g (6.7 mmol) のメタノール 5 ml 溶液を加えた。メタノールを1/2 に減圧濃縮し 4℃で一夜冷却させた後、析出結晶を濾取した。得られた緑色結晶を減圧デシケーターで乾燥して化合物(L) 85 mg を得た( 収率 72%) 。ヨウ化ナトリウムの塩形成前（分子内対イオン型) の化合物(K) 及び塩形成後の化合物(L) の水溶性および赤外線吸収スペクトルデータ(IR)を測定した。また、ヨウ素含量をシェーニガー法より求めた。
【００５４】
ヨウ化ナトリウムを塩として取り込んだ化合物(L) と原料として用いた対イオン型の化合物(K) とでは赤外吸収スペクトルが変化しており、分子内対イオン型化合物において観測された 2360 及び 1740 cm^-1の吸収が化合物(L) では消失していた（図５参照： a) 化合物(L):塩形成後、 b) は化合物(K):分子内対イオン型、すなわち塩形成前のものを示す) 。これらの結果から、分子内対イオンを形成している場合とヨウ化ナトリウムの塩を形成した場合とでは、結晶構造が変化していることが明らかである。
【００５５】
例６：化合物(Q) 及び(R) の合成
例５の方法に準じてインドシアニングリーン-N- ブタン酸スルホコハク酸イミドエステルを製造した。この化合物 150 mg(0.17 mmol)をメタノール 3 ml に溶かし、ヨウ化ナトリウム 1.5 g (10 mmol)のメタノール 8 ml 溶液を加えた。メタノールを1/2 に減圧濃縮し 4℃で一夜冷却させた後、析出結晶を濾取した。得られた緑色結晶を減圧デシケーターで乾燥して化合物(Q) 117 mgを得た(66%) 。ヨウ化ナトリウムの塩形成前後での水溶性データおよび赤外線吸収スペクトルデータ(IR)を測定した。また、ヨウ素含量をシェーニガー法より求めた。
【００５６】
同様に、インドシアニングリーン-N- ヘキサン酸スルホコハク酸イミドエステル 100 mg(0.11 mmol)をメタノール 2 ml に溶かし、過塩素酸ナトリウム 0.3 g (2.5 mmol) のメタノール 20 ml 溶液を加えた。メタノールを1/4 に減圧濃縮し 4℃で一夜冷却させた後、析出結晶を濾取した。得られた緑色結晶を減圧デシケーターで乾燥して化合物(R) 23 mg を得た( 収率 21%) 。過塩素酸ナトリウムの塩形成前後での水溶性データおよび IR を測定した。
【００５７】
【表１】

【００５８】
例７：化合物(I) の合成
【化１５】

【００５９】
化合物16 (5.0 g, 15.1 mmol) とグルタコンアルデヒドジアニル塩酸塩 (4.40 g, 15.1 mmol)を無水酢酸 20 mlと酢酸 300 ml の混合溶液に懸濁し、還流温度で５時間加熱した。赤色溶液を減圧濃縮し、得られた残渣に酢酸エチルと水の混合溶液 200 ml を加えて懸濁させ、黒赤色固体を濾取した。水で洗浄した後、減圧下に乾燥し、化合物17の黒赤色粉末を得た。収量 5.33 g ( 収率 65.2 %)。
【００６０】
化合物17 (167 mg, 0.308 mmol) と上記例６で得た化合物９ (100 mg, 0.309 mmol) をピリジン 3 ml に溶解し、50℃で１時間攪拌した。反応混合物を減圧下に濃縮し、得られた残渣を水 10 mlに溶解して溶液の pH を３に合わせた後、セファデックス LH20 カラム( 溶媒：メタノール) で精製して、化合物18の黒緑色固体を得た。収量 51 mg( 収率 23.1 %)。
【００６１】
化合物18(30 mg, 41.8μmol)／ 50 v/v % THF 水溶液 1 ml に N- ヒドロキシ無水こはく酸スルホン酸ナトリウム (16.8 mg, 77.4 μmol)と N,N'-ジシクロヘキシルカルボジイミド(25.8 mg, 0.125 mmol) を加えて４℃で一晩反応させた後に濾過し、濾液を 20 ℃以下で減圧下濃縮した。残渣に酢酸エチル 10 mlを加え、生じた結晶を更にエーテルで３回洗浄し、化合物19の黒緑色固体を得た。収量 34 mg( 収率 88.8 %)。MS (FAB) m/e = 892(M^- ) ；蛍光スペクトル :λ_ex=771 nm,λ_em=822 nm ( 水)
【００６２】
上記の化合物19を、例６の方法に従ってヨウ化ナトリウムのメタノール溶液で処理して化合物(I) を得た。化合物19の赤外線吸収スペクトルと化合物(I) の赤外線吸収スペクトルを比較したところ、化合物19において観測された 2360 及び 1740 cm^-1の吸収が化合物(I) においては観測されなかった。
【００６３】
例８：化合物(A) を用いたタンパクの標識及び蛍光検出
牛血清アルブミン(BSA) 3.0 mgをpH 8.5の 0.1 M炭酸緩衝液 5.0 ml に溶かし、50ミリモルの化合物(A) の DMSO 溶液 5 ml を加え、37℃で１時間放置した。HPLC（ゲルカラム、蛍光検出：励起波長 550nm、検出波長 569nm）を用いてこの溶液を測定したところ、化合物(A) のピーク以外に標識BSA のピークが認められた。図２は化合物(A) をpH 7.4のPBS に溶かした溶液の蛍光スペクトルを示し、図３は化合物(A) を用いて標識されたBSA をpH 7.4の PBSに溶かした溶液の蛍光スペクトルを示す。さらに、BSA の量を変えて標識して同様の測定を行ったところ、図４に示すように蛍光強度とBSA 量との間に良好な比例関係が認められ、BSA にして約 0.1フェムトモルの検出限界であることが分かった。このときのシグナル／ノイズ比は3 であった。
【００６４】
例９：本発明の化合物による抗体の標識
化合物(L) 1 mgを水 0.94 mlに溶解して 1 mM 溶液とした。ヒト IgG 1 mg を pH 8.5 の50 mM 炭酸ナトリウムバッファー 1 ml に溶解し、この溶液に化合物(L) を含む上記の溶液 0.2 ml を加えて 30 ℃で１時間反応させた。反応液をセファデックス G-25 カラムに付して標識化されたヒトIgG を分離した。分離用のバッファーとしては 50 mMリン酸バッファー(pH 7.4)を用いた。未反応の化合物(L) はセファデックス・ゲルの上端に残り、標識化IgG との分離は良好であった。得られた標識化IgG を凍結乾燥して-20 ℃で冷凍保存した。この標識化IgG 1 mgをとり100 mlの50 mM リン酸バッファー(pH 7.4)に溶解して蛍光スペクトルを測定した結果を図６に示す。
【００６５】
【発明の効果】
本発明の化合物は、高い蛍光量子収率を有するので高蛍光性であり、水溶性が高いという特徴を有している。また、本発明の化合物は官能基選択的に被標識化合物を標識できるという特徴がある。さらに、分子内に標識基を１個のみ有するので、被標識化合物を架橋せず、標識後にも被標識化合物の特性をそのまま維持できるという特徴がある。従って、本発明の化合物を用いると、タンパクなどの生体高分子の立体構造や生理活性を保持したまま蛍光標識することができ、標的分子にインドシアニンの分光学的性質を付加させることができる。
【図面の簡単な説明】
【図１】 本発明の化合物(A) をpH 7.4のPBS (NaCl: 8.0g, KCl: 0.2g, Na₂HPO₄: 11.5g, KH₂PO₄: 0.2g／水 1000 ml) に溶かした溶液の吸収スペクトルを示す図である。
【図２】 本発明の化合物(A) をpH 7.4のPBS に溶かした溶液の蛍光スペクトルを示す図である。
【図３】 本発明の化合物(A) を用いて標識されたBSA をpH 7.4の PBSに溶かした溶液の蛍光スペクトルを示す図である。
【図４】 本発明の化合物(A) を用いて標識した標識化BSA の蛍光強度と反応に供したBSA 量との間の相関関係を示す図である。
【図５】 ヨウ化ナトリウムを塩として取り込んだ化合物(L) と分子内対イオン型の化合物(K) の赤外線吸収スペクトルを示す図である。図中、 a) は化合物(L) の赤外線吸収スペクトルを示し、b)は化合物(K) の赤外線吸収スペクトルを示す。
【図６】 本発明の化合物(L) を用いて標識されたヒト IgGの蛍光スペクトルを示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to indocyanine derivatives. The compound of the present invention is useful as a fluorescent labeling reagent for detecting a compound having an amino group, a sulfhydryl group, and / or a carbonyl group with high sensitivity.
[0002]
[Prior art]
A labeling reagent is a reagent that gives a specific property to a target compound so that it can be detected. As a typical labeling method using a labeling reagent, for example, a fluorescent labeling method, a method of introducing a radioisotope, and a method of using an enzyme can be mentioned. Although the method using a radioisotope enables highly sensitive measurement, there is a risk of exposure to workers due to the use of an unsealed radioisotope. In addition, facilities that use radioisotopes are limited, and there are problems in the treatment of radioactive waste and the management of radioactive materials, as well as significant investment in measuring equipment and facility management. As a method using an enzyme, an ELISA method is known. However, since sufficient sensitivity cannot be obtained by a conventional colorimetric method, higher-sensitivity measurement using a fluorescent labeling reagent has been attempted.
[0003]
For the reasons described above, labeling methods using fluorescent labeling reagents have become the mainstream of labeling methods for microanalysis. The fluorescent labeling reagent is a reagent for detecting a labeling compound by using a fluorescence property added to a fluorescent substance bound to a specific functional group in the labeling compound. Conventionally, various fluorescent labeling reagents have been proposed, and in detection using high-performance liquid chromatography (HPLC), the detection sensitivity is increased to about several femtomole (sample injection amount). However, the sensitivity is still not sufficient for the detection of trace amounts of biopolymers such as proteins and nucleic acids, and single-molecule measurement is possible by image processing using a fluorescence microscope or the like. Therefore, there is a demand for a fluorescent labeling reagent with higher sensitivity.
[0004]
In addition to high fluorescence intensity, fluorescent labeling reagents used for labeling biopolymers such as proteins do not change the properties of labeled biopolymers after binding, by selectively binding to specific functional groups Various properties such as that are required. In addition, since a labeling reagent having a plurality of binding sites in the molecule may change the structure or properties of the compound to be labeled due to cross-linking, the labeling reagent has only one labeling group in the molecule. There must be. For example, JP-A-2-91674 discloses an indocyanine-type highly fluorescent compound, but the compound has a plurality of reactive groups, and there is a possibility that a crosslinking reaction proceeds upon labeling. is there. Similarly, the fluorescent labeling dye disclosed in JP-A-5-40097 may cause a crosslinking reaction. Further, since the compound itself cannot react with various functional groups, it is necessary to activate the reagent in advance, so that there is a problem that the operation is complicated.
[0005]
In addition to the above requirements, the labeling reagent is also required to have high water solubility. Indocyanine green, which is a kind of indocyanine compound, is a substance that exhibits specific absorption by infrared irradiation and emits fluorescence, and is used in liver function tests (indocyanine green load test) and the like. Attempts have been made to fluorescently label proteins using the fluorescence of indocyanine green, and various derivatives have been proposed in which the mother nucleus of indocyanine green is modified or reactive groups are introduced. However, a reactive indocyanine green derivative having a reactive group introduced therein is extremely hardly soluble in water, and when used as a labeling reagent, it is necessary to dissolve the compound in an organic solvent such as dimethyl sulfoxide. For this reason, when protein aqueous solution was added to the reaction system, the problem that the reagent precipitated or the labeled protein precipitated had arisen.
[0006]
For example, the compound disclosed in Japanese Patent Application No. 6-222059 is inferior in water solubility and needs to be used by dissolving the compound in an organic solvent. The compound disclosed in Japanese Patent Application Laid-Open No. 2-91674 is a compound in which water solubility is improved by directly introducing a sulfonic acid group into an aromatic ring. Even though this compound is used, protein labeling is performed in a pure aqueous system. Does not have sufficient water solubility.
[0007]
On the other hand, when labeling a protein using an indocyanine green derivative, it is necessary to label so as not to inhibit the physiological activity of the protein itself. In the reported indocyanine green derivatives, the distance between the labeled protein and the indocyanine green label is short, and part of the physiological activity of the labeled protein may be impaired. is there. For example, an indocyanine green derivative developed by Patoney et al. Has an isothiocyano group directly introduced into the benzene ring and has a very short distance between the label and the protein.
[0008]
[Problems to be solved by the invention]
An object of the present invention is to provide a fluorescent labeling reagent in which the above problems are reduced or eliminated. More specifically, a fluorescent labeling reagent having a high fluorescence quantum effect can be introduced into a labeled compound in a simple and functional group-specific manner, and a fluorescent labeling reagent that does not change the properties of the labeled compound. It is an object of the present invention to provide. Another object of the present invention is to provide a fluorescent labeling reagent having the above characteristics and excellent in water solubility.
[0009]
[Means for Solving the Problems]
The inventors of the present invention have made utmost efforts to solve the above-mentioned problems, and synthesized various derivatives of indocyanine green to emit fluorescence by near-infrared to far-infrared excitation light and have high water solubility. Succeeded in manufacturing. In addition, the present inventors have proved that the above indocyanine green derivative is useful for labeling biopolymers such as antibodies. Furthermore, when sodium iodide or the like is allowed to act on the above compound capable of forming an intramolecular counter ion (zitter ion), a salt anion is added to the cation portion of the molecule, and a salt cation is added to the anion portion. It has been found that as a result of the inhibition of the formation of intramolecular counter ions, a compound in which the ionicity of the whole molecule is maintained and the water solubility is remarkably increased can be obtained. The present invention has been completed based on these findings.
[0010]
That is, the present invention provides the following formula:
[Chemical 3]

(Where A ¹ And A ² Each independently represents a benzene ring or a naphthalene ring, R ¹ And R ² Each independently represents a hydrogen atom, an alkyl group, an aryl group, a free or dissociated sulfonic acid group, or an alkoxy group, and R ^Three Represents an alkyl group, a free or dissociated alkyl sulfonate group, an aminoalkyl group or an ammonioalkyl group, n represents an integer of 1 to 3, ^- Indicates an anionic species as required and Z is the following formula:
[Formula 4]

A group selected from the group consisting of ⁺ Is an alkali metal ion, and W 1 and Y 2 each independently represent an alkylene group having 1 to 10 carbon atoms, or W 1 and Y 2 are one or more selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom And an alkylene group having 1 to 10 carbon atoms containing the above-mentioned atoms).
[0011]
According to a preferred embodiment of the present invention, A ¹ And A ² Are both naphthalene rings and R ¹ And R ² Are both hydrogen atoms and R ^Three Is a dissociated linear sulfonic acid alkyl group having 2 to 5 carbon atoms, n is 3, Y is a linear alkylene group having 2 to 5 carbon atoms, and Z is a succinimideoxy group or an alkali metal sulfosuccinimideoxy group. The above compound as a group; and X ^- Is iodine ion and R ^Three Is a linear alkyl sulfonate group having 2 to 5 carbon atoms which forms a salt with sodium ion.
[0012]
According to another aspect of the present invention, a fluorescent labeling reagent comprising the above compound; a fluorescent labeling of a compound comprising one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carbonyl group, and a sulfhydryl group The reagent used in the above; the reagent containing the functional group as a biopolymer; and the reagent selected from the group consisting of a protein, glycoprotein, lipid, phospholipid, polysaccharide, and nucleic acid. Provided.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
In the above general formula, A ¹ And A ² Each independently represents a benzene ring or a naphthalene ring, preferably a naphthalene ring. A ¹ And / or A ² A represents a naphthalene ring, A ¹ And / or A ² The condensation position of the naphthalene ring with respect to the pyrrole ring to which is condensed is not particularly limited. A ¹ And / or A ² R represents a naphthalene ring, R ¹ And R ² Can be substituted at any position of any one of the two phenyl rings constituting the naphthalene ring. A ¹ And / or A ² R represents a phenyl ring, R ¹ And R ² May be substituted at any position on the phenyl ring.
[0014]
R ¹ And R ² Each independently represents a hydrogen atom, an alkyl group, an aryl group, a free or dissociated sulfonic acid group, or an alkoxy group. As the alkyl group, a linear or branched lower alkyl group having about 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, can be used. For example, a methyl group, an ethyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group and the like can be suitably used. Examples of the aryl group include a phenyl group, a naphthyl group, a pyridyl group, an imidazolyl group, and a pyrrolyl group. In the dissociated sulfonic acid group, an alkali metal ion such as sodium ion or potassium ion may exist as a counter ion. As the alkoxy group, a linear or branched lower alkoxy group having about 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms can be used, and more specifically, a methoxy group, an ethoxy group, or a propoxy group. N-butoxy group, sec-butoxy group, tert-butoxy group, and the like can be used.
[0015]
R ^Three Represents an alkyl group, a free or dissociated alkyl sulfonate group, or an amino or ammonioalkyl group. As the alkyl group, a linear or branched lower alkyl group having about 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, can be used. The sulfonic acid of the alkyl sulfonate group, or the amino group or ammonio group of the amino or ammonioalkyl group can be substituted at any position of the alkyl group, but it is preferable to use those substituted at the terminal of the alkyl group. is there. The amino group or ammonio group may have one or two (1 to 3 in the case of an ammonio group) the same or different lower alkyl group (preferably an alkyl group having about 1 to 6 carbon atoms). Good.
[0016]
For example, R ^Three As- (CH ₂ ) _k -SO _Three A group represented by-(k represents an integer of 3 to 5) is preferred. R ^Three When is a dissociated sulfonate alkyl group, an alkali metal ion such as sodium ion or potassium ion may be present as a counter ion of the sulfonate ion. R ^Three When is an ammonioalkyl group, a counter ion such as a halogen ion or an anion such as a perchlorate ion, preferably a halogen ion may be present.
[0017]
X ^- Denotes an anionic species which may be present as required, for example, a halogen ion, acetate ion, perchlorate ion, carbonate ion or thiocyanate ion. X ^- The anionic species represented by the formula acts to counteract the positive charge on the nitrogen atom in the mother nucleus and keep the compound neutral as a whole. For example, R ¹ , R ² , And R ^Three If there is a dissociated sulfonic acid moiety in any one of these, the negative charge of the dissociated sulfonic acid and the positive charge on the nitrogen atom in the mother nucleus cancel each other, so X ^- May not exist. On the other hand, for example, R ¹ And R ² Any one of is a dissociated sulfonic acid group, R ^Three When is an ammonioalkyl group, the charge of these groups is balanced, so X can be used to counter the positive charge on the nitrogen atom in the nucleus. ^- May be required. X ^- Preferably, chlorine ion, bromine ion, iodine ion, or the like can be used. Of these, iodine ions are particularly desirable. n represents an integer of 1 to 3, and n is preferably 3.
[0018]
Y 1 can suitably be a linear or branched alkylene group having 1 to 10 carbon atoms, preferably a linear or branched alkylene group having 2 to 5 carbon atoms. Particularly preferably, a trimethylene group, a tetramethylene group, or a pentamethylene group can be used. Y 1 represents a C 1-10 linear or branched alkylene group containing one or more atoms selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom.
[0019]
Specifically, as the group represented by -Y-CO-, for example, -CH ₂ -CO-;-(CH ₂ ) ₂ -CO-;-(CH ₂ ) _Three -CO-;-(CH ₂ ) _Four -CO-;-(CH ₂ ) _Five -CO-; -CH ₂ -CO-NH- (CH ₂ ) _Five -CO-;-(CH ₂ ) ₂ -CO-NH- (CH ₂ ) _Five -CO-;-(CH ₂ ) _Three -CO-NH- (CH ₂ ) _Five -CO-;-(CH ₂ ) _Four -CO-NH- (CH ₂ ) _Five -CO-; -CH ₂ -CO-NH- (CH ₂ ) _Five -CO-NH- (CH ₂ ) ₂ -CO-;-(CH ₂ ) _Four -CO- (N, N'-piperadinyl)-(CH ₂ ) ₂ -CO (N, N'-piperadinyl is the 1-position of piperazine-(CH ₂ ) _Four The -CO- group is substituted and the 4-(- ₂ ) ₂ -Z Indicates that the group is substituted. The same applies hereinafter. ); Or -CH ₂ -CO-NH- (CH ₂ ) _Five -CO- (N, N'-piperadinyl)-(CH ₂ ) ₂ -CO- can be used.
[0020]
In the group represented by Z 1, a group corresponding to Y 1 in the above examples of Y—CO 2 can be suitably used as W 1. In addition, the M of the counter ion of the sulfonic acid group of the N-sulfosuccinimidyloxy group ⁺ And X ^- Counter ion M ⁺ And may be the same or different, but it is preferable that both are sodium ions. In addition, R ¹ And / or R ² Represents a dissociated sulfonic acid group and / or R ^Three Represents a dissociated alkyl sulfonate group, the counter ion of the sulfonic acid is X ^- Counter ion M ⁺ And / or M of the counter ion of the sulfonic acid group of the N-sulfosuccinimidyloxy group ⁺ May be the same as or different from each other, but both are preferably sodium ions.
[0021]
Not bound by any particular theory, for example R ^Three Is a dissociated alkyl sulfonate group, the negative charge of the sulfonate group and the positive charge on the nitrogen atom (where N ⁺ And the so-called inner salt (zitter-ion type compound) is formed. In such an intramolecular salt type compound, the ionicity of the whole molecule is lowered, and the water solubility of the compound may be impaired. On the other hand, in such a compound, an alkali metal ion exists as a counter ion with respect to the negative charge of the sulfonic acid group, and X as a counter ion with respect to the positive charge on the nitrogen atom. ^- In the presence of the anionic species represented by the formula, the formation of an inner salt due to the negative charge of the sulfonic acid group and the positive charge on the nitrogen atom is inhibited, so the water solubility may be significantly increased. Therefore, X ^- Is an anion such as iodine ion, and R ^Three A compound in which is a sulfonic acid alkyl group that forms a salt with sodium ion is a preferred embodiment of the present invention.
[0022]
In addition, in the compound of the above general formula and the following preferred embodiment and the scheme in the examples, a positive charge is fixed on one nitrogen atom in the mother nucleus for the sake of convenience, but a conjugated double bond It will be readily appreciated by those skilled in the art that a positive charge can be transferred to other nitrogen atoms in the mother nucleus via. Therefore, it goes without saying that all isomers based on such conjugation are included in the scope of the present invention.
[0023]
The compound of the present invention can be synthesized using 2,3,3-trimethylindolenine, 2,3,3-trimethylbenzoindolenine or a derivative thereof as a raw material. These raw materials can be synthesized according to Fischer's synthesis method (E. Fischer and O. Hess: Berichte, 17, 559, 1883, etc.), or can be obtained commercially. For example, it can be produced by introducing an alkyl group onto a nitrogen atom of a raw material compound, reacting with triethyl orthoformate to form a cyanine dye skeleton, and then introducing a labeling group represented by Z.
[0024]
X ^- Is an anion such as iodine ion, and R ^Three In order to produce a compound that does not form an intramolecular salt, such as a compound that is a sulfonate alkyl group that forms a salt with sodium ions, first, the negative charge of the dissociated sulfonate alkyl group and the nitrogen atom in the mother nucleus The compound (X ^- After the preparation of the compound, the compound is dissolved in an organic solvent such as dimethylformamide or dimethyl sulfoxide at a high concentration, and a salt such as sodium iodide may be added to the solution. But the manufacturing method of the compound of this invention is not limited to these methods.
[0025]
As typical examples of the compound of the present invention, compounds (A) to (P) are shown below.
[Chemical formula 5]

[0026]
[Chemical 6]

[0027]
[Chemical 7]

[0028]
[Chemical 8]

[0029]
[Chemical 9]

[0030]
The compound of the present invention can be used, for example, for labeling biological macromolecules derived from animals and plants having a reactive amino group, hydroxyl group, thiol group, carbonyl group, sulfhydryl group and the like. Examples of biopolymers include complex proteins such as proteins and glycoproteins, complex lipids such as lipids and phospholipids, polysaccharides, and nucleic acids. For example, antibodies and enzymes with high specificity for cancer and the like are suitable labels for the compounds of the present invention. In addition, since the compound of the present invention easily reacts with a low molecular weight compound having a reactive amino group, hydroxyl group, thiol group, carbonyl group, sulfhydryl group, etc., for example, amplification used in amplification systems such as biotin-avidin It is also possible to label system substances.
[0031]
For example, when N-succinimidyloxy group and N-sulfosuccinimidyloxy group are used as Z, these groups form a reactive active ester together with a carbonyl group to which Z is bonded. It reacts with an amino group contained in the compound to form an amide bond. When an N-maleimidoalkyl group is used as Z 1, for example, a thiol group (HS-R) contained in the labeled compound reacts to form a thioether. And Z is -NH-NH ₂ In this case, the reducing sugar terminal aldehyde group (OHC-R) contained in the compound to be labeled reacts to form -Y-CO-NH-NH-CO-R. Therefore, by appropriately selecting and using the compound of the present invention, it is possible to label the compound to be labeled specifically with a functional group. But the use of the compound of this invention is not limited to what was illustrated above.
[0032]
The compound of the present invention is a compound in which an active site is introduced into an indocyanine compound having a high molar extinction coefficient and a high fluorescence quantum yield, and can add the spectroscopic properties of indocyanine to a labeled compound. The labeled compound labeled with the compound of the present invention has fluorescence characteristics with an excitation wavelength of 500 to 800 nm and a fluorescence wavelength of 550 to 850 nm. By using a high-power semiconductor laser or He / Ne laser as an excitation light source, Extremely sensitive detection is possible.
[0033]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to a following example. Moreover, those skilled in the art who refer to this example can easily produce a desired compound included in the scope of the present invention by appropriately modifying and altering starting materials, reagents, reaction conditions and the like. It will be easily understood. The compound numbers in the examples correspond to the compound numbers in the scheme, and the compound (A) and the like correspond to the compound numbers shown as the preferred compounds.
[0034]
Example 1: Preparation of compounds (A) and (B)
[Chemical Formula 10]

[0035]
Compound 1 (2,3,3-trimethylindolenine) (5.0 g, 31.4 mmol) was dissolved in acetonitrile (100 ml), iodoethane (5.9 g, 37.8 mmol) was added, and the mixture was heated to reflux overnight. The reaction mixture was concentrated under reduced pressure, ether was added to the residue, and gray red crystals of compound 2 were collected by filtration (9.1 g, 92%). Separately, Compound 1 (5.0 g, 31.4 mmol) was dissolved in 100 ml of dimethylformamide (DMF), 20.0 g (89.6 mmol) of ethyl 6-bromohexanoate was added, and the mixture was heated at 80 ° C. overnight under a nitrogen atmosphere. The reaction solution was concentrated under reduced pressure, and the resulting oil was washed with 50 ml of ether. Next, the obtained oil was dissolved in 100 ml of methanol, 11.5 g (287.5 mmol) of sodium hydroxide was dissolved in water and added, and the mixture was stirred at room temperature for 5 hours for hydrolysis. Methanol was distilled off under reduced pressure, and then neutralized with 1N hydrochloric acid. After washing with chloroform to remove the water-insoluble oil, it was concentrated to dryness under reduced pressure. Inorganic salts were removed by dissolving in ethanol and filtering, and the resulting filtrate was concentrated to dryness under reduced pressure, and further dried under reduced pressure to obtain a pale red powder of Compound 3 (3.2 g, 37.3%). .
[0036]
Compound 2 (3.2 g, 10.2 mmol) and Compound 3 (2.8 g, 10.2 mmol) were dissolved in 60 ml of pyridine and heated to 110 ° C., and then 2.5 ml (15.0 mmol) of triethyl orthoformate was slowly added dropwise. Heated for hours. The reaction solution was concentrated under reduced pressure, dissolved in chloroform and washed with water. The chloroform solution was concentrated under reduced pressure, and the resulting residue was subjected to silica gel column chromatography (solvent: methanol / chloroform) for separation and purification. The eluent was concentrated to dryness under reduced pressure, and the resulting solid was dried under reduced pressure to obtain compound 4 greenish red metallic luster crystals (1.6 g, 33%).
[0037]
Compound 4 (20 mg, 0.042 mmol) and N-hydroxysuccinimide (NHS) 6.4 mg (0.056 mmol) were dissolved in 100 μl of anhydrous DMF and kept at 0 ° C., and dicyclohexylcarbodiimide (DCC) 180 mg (0.874 mmol) Was dissolved in 50 μl of anhydrous DMF, and the mixture was reacted at 0 ° C. overnight. Hydrogen chloride gas was blown into the reaction solution, and ether was added to obtain the target compound (A) (18.0 mg, 70.1%). Compound (A) was added to PBS pH 7.4 (NaCl: 8.0g, KCl: 0.2g, Na ₂ HPO _Four : 11.5g, KH ₂ PO _Four : The absorption spectrum of the solution dissolved in 0.2 g / 1000 ml of water) is shown in FIG.
[0038]
Compound 4 (300 mg, 0.64 mmol) was dissolved in 20 ml of DMF, and 242 mg (0.81 mmol) of piperazinoethylmaleimide dihydrochloride monohydrate (PEM) and 120 μl (0.88 mmol) of triethylamine were added to 0 ° C. Cooled down. To this solution, 680 mg (3.30 mmol) of DCC was added and reacted at 0 ° C. overnight. The reaction solution is filtered, the filtrate is concentrated under reduced pressure, and the resulting residue is separated and purified by silica gel column chromatography (solvent: methanol / chloroform) to obtain a green-red metallic glossy crystal of compound (B). (350 mg, 78.6%).
[0039]
Example 2: Preparation of compound (C)
Embedded image

[0040]
Compound 1 (2.0 g, 6.3 mmol) was dissolved in 100 ml of DMF, 8.4 g (37.6 mmol) of ethyl 6-bromohexanoate was added, and the mixture was heated at 80 ° C. overnight under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure, and the residue was separated and purified by silica gel chromatography (solvent: methanol / chloroform) to obtain a reddish brown oil of compound 5 (3.4 g, 40%). Compound 2 (2.0 g, 6.3 mmol) and Compound 5 (2.4 g, 6.3 mmol) were dissolved in 10 ml of pyridine and heated to 110 ° C., and then 1.6 ml (10.0 mmol) of triethyl orthoformate was gradually added dropwise. Heated for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in chloroform and washed with water. The chloroform solution was concentrated under reduced pressure, and the resulting residue was subjected to silica gel column chromatography (solvent: methanol / chloroform) for separation and purification. The eluent was concentrated to dryness under reduced pressure, and the obtained solid was dried under reduced pressure to obtain greenish red crystals of Compound 6 (960 mg, 26.1%).
[0041]
Compound 6 (200 mg, 0.35 mmol) was dissolved in 10 ml of acetonitrile, 10 ml (205.7 mmol) of hydrazine monohydrate was added, and the mixture was stirred overnight at room temperature. The reaction mixture was made weakly acidic with 1N hydrochloric acid, concentrated under reduced pressure, and the residue was purified with Sephadex LH20 / methanol to give compound (C) (130 mg, 72.3%).
[0042]
Example 3: Preparation of compound (H)
Embedded image

[0043]
Compound 2 (3.2 g, 10.2 mmol) and Compound 3 (2.8 g, 10.2 mmol) are dissolved in 60 ml of pyridine, heated to 110 ° C., and 2.0 g (15.1 mmol) of 1,3,3-trimethoxypropene is added. In addition, it was heated for 1 hour. The reaction solution was concentrated under reduced pressure, and the resulting residue was dissolved in chloroform and washed with water. The chloroform solution was concentrated under reduced pressure, and the resulting residue was subjected to silica gel column chromatography (solvent: methanol / chloroform) for separation and purification. The eluent was concentrated to dryness under reduced pressure, and the resulting residue was dried under reduced pressure to obtain green metallic luster crystals of Compound 7 (1.2 g, 23.8%). In the same manner as in Example 1, compound 7 (200 mg, 0.40 mmol) was reacted with NHS 60 mg (0.52 mmol), and the reaction product was isolated and purified to obtain compound (H) (1.2 g, 23.8%). .
[0044]
Example 4: Production of compound (J)
Embedded image

[0045]
Compound 8 (4.0 g, 19.1 mmol) was dissolved in 60 ml of DMF, 13.0 g (58.3 mmol) of ethyl 6-bromohexanoate was added, and the mixture was heated at 80 ° C. overnight under a nitrogen atmosphere. The reaction solution was concentrated under reduced pressure, and the residual oil was washed with 50 ml of ether. Subsequently, the obtained oil was dissolved in 100 ml of methanol, 7.0 g (175 mmol) of sodium hydroxide was dissolved in water and added, and the mixture was hydrolyzed by stirring at room temperature for 5 hours. Methanol was distilled off under reduced pressure and then neutralized with 1N hydrochloric acid. After washing with chloroform to remove water-insoluble oil, the solution was concentrated to dryness under reduced pressure. The residue was dissolved in ethanol and filtered to remove inorganic salts, and the filtrate was concentrated to dryness under reduced pressure and dried under reduced pressure to obtain a pale blue powder of Compound 9 (4.2 g, 67.9%).
[0046]
Compound 8 (7.0 g, 19.1 mmol) was dissolved in 100 ml of acetonitrile, 4.5 g (28.9 mmol) of iodoethane was added, and the mixture was heated to reflux overnight. The reaction mixture was concentrated under reduced pressure, and ether was added to the residue to obtain grayish blue crystals of compound 10 (7.4 g, 84.8%). Glutaconaldehyde dianyl hydrochloride 6.0 g (24.2 mmol) was dissolved in acetic anhydride 100 ml, and compound 10 (7.4 g, 20.3 mmol) was added with stirring. After the mixture was heated to 100 ° C. and reacted for 1 hour, the reaction was concentrated under reduced pressure, and ether was added to the residue to obtain green crystals.
[0047]
The above green crystals were dissolved in 60 ml of pyridine, compound 9 (5.6 g, 17.3 mmol) was added, and the mixture was reacted at 60 ° C. for 5 hours. The reaction solution was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (solvent: methanol / chloroform) for separation and purification. The eluent was concentrated to dryness under reduced pressure, and the obtained solid was dried under reduced pressure to obtain green crystals of compound 12 (3.8 g, 35.2%). Compound 12 (20 mg, 0.032 mmol) and NHS 4.9 mg (0.043 mmol) were dissolved in 100 ml of anhydrous DMF and kept at 0 ° C., and DCC 140 mg (0.68 mmol) was dissolved in 50 ml of anhydrous DMF and added overnight. Reacted. Hydrogen chloride gas was blown into the reaction solution, and ether was added to obtain the target compound (J) (10.5 mg, 43.2%).
[0048]
Example 5: Preparation of compounds (K) and (L)
Embedded image

[0049]
6-Bromohexanoic acid ethyl ester (6.32 ml, 35.5 mmol) was added to 100 ml of compound 8 (5 g, 23.9 mmol) / DMF and heated at 80 ° C. for 16 hours. The reaction solution was concentrated under reduced pressure, and 200 ml of ether was added to the residue for crystallization. The resulting crystals were filtered, washed with ether, and dried under reduced pressure to obtain Compound 8 ′. To the compound 8 ′, 30 ml of 1 M sodium hydroxide aqueous solution / methanol = 1/1 mixed solution was added and stirred at room temperature for 2 hours. Methanol was distilled off under reduced pressure, the aqueous solution was neutralized with 4 M aqueous hydrochloric acid solution, and then washed with chloroform three times. The aqueous solution was concentrated under reduced pressure to obtain a red solid of compound 9. Yield 5.75 g (Yield 74.4%).
[0050]
Compound 13 (5.0 g, 14.5 mmol) and glutaconaldehyde dianil hydrochloride (4.23 g, 14.5 mmol) were suspended in a mixed solution of 20 ml of acetic anhydride and 300 ml of acetic acid and heated at reflux temperature for 5 hours. The red solution was concentrated under reduced pressure, 200 ml of a mixed solution of ethyl acetate and water was added to the resulting residue to suspend it, and a black red solid was collected by filtration. After washing with water, it was dried under reduced pressure to obtain a black-red powder of compound 14. Yield 5.20 g (66.1% yield).
[0051]
Compound 14 (300 mg, 0.553 mmol) and compound 9 (179 mg, 0.553 mmol) were dissolved in 5 ml of pyridine and stirred at 50 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in 10 ml of water to adjust the pH of the solution to 3. Then, the solution was purified with a Sephadex LH20 column (solvent: methanol) to obtain the blackish green color of Compound 15. A solid was obtained. Yield 105 mg (25.9% yield).
[0052]
Compound 15 (106 mg, 0.145 mmol) / 50 v / v% 3 ml of aqueous THF solution in 3 ml of sodium N-hydroxysuccinate sulfonate (65.2 mg, 0.287 mmol) and N, N'-dicyclohexylcarbodiimide (129 mg, 0.596 mmol) ) And reacted at 4 ° C. overnight, followed by filtration. The filtrate was concentrated at 20 ° C. or lower under reduced pressure. To the residue was added 10 ml of ethyl acetate, and the resulting crystals were further washed three times with ether to obtain a black-green solid of compound (K) (110 mg, 80.7%). MS (FAB) m / e = 906 (M ^- ; Fluorescence spectrum: λ _ex = 768 nm, λ _em = 807 nm (H ₂ O)
[0053]
100 mg (0.11 mmol) of the above compound (K) was dissolved in 2 ml of methanol, and a solution of sodium iodide 1 g (6.7 mmol) in 5 ml of methanol was added. Methanol was concentrated under reduced pressure to 1/2 and cooled at 4 ° C. overnight, and then the precipitated crystals were collected by filtration. The obtained green crystals were dried with a vacuum desiccator to obtain 85 mg of Compound (L) (yield 72%). The water solubility and infrared absorption spectrum data (IR) of the compound (K) before the salt formation of sodium iodide (intramolecular counterion type) and the compound (L) after the salt formation were measured. The iodine content was determined by the Schöniger method.
[0054]
Infrared absorption spectra change between the compound (L) incorporating sodium iodide as a salt and the counterion type compound (K) used as a raw material, and 2360 and 1740 observed in the intramolecular counterion type compound. cm ^-1 (See Fig. 5: a) Compound (L): After salt formation, b) shows compound (K): intramolecular counter ion type, that is, before salt formation ) From these results, it is clear that the crystal structure changes between the case where an intramolecular counter ion is formed and the case where a sodium iodide salt is formed.
[0055]
Example 6: Synthesis of compounds (Q) and (R)
Indocyanine green-N-butanoic acid sulfosuccinimide ester was prepared according to the method of Example 5. 150 mg (0.17 mmol) of this compound was dissolved in 3 ml of methanol, and a solution of 1.5 g (10 mmol) of sodium iodide in 8 ml of methanol was added. Methanol was concentrated under reduced pressure to 1/2 and cooled at 4 ° C. overnight, and then the precipitated crystals were collected by filtration. The obtained green crystals were dried with a vacuum desiccator to obtain 117 mg of Compound (Q) (66%). Water solubility data and infrared absorption spectrum data (IR) before and after salt formation of sodium iodide were measured. The iodine content was determined by the Schöniger method.
[0056]
Similarly, 100 mg (0.11 mmol) of indocyanine green-N-hexanoic acid sulfosuccinimide ester was dissolved in 2 ml of methanol, and a solution of sodium perchlorate 0.3 g (2.5 mmol) in 20 ml of methanol was added. Methanol was concentrated under reduced pressure to 1/4 and cooled at 4 ° C. overnight, and then the precipitated crystals were collected by filtration. The obtained green crystals were dried using a vacuum desiccator to obtain 23 mg of Compound (R) (yield 21%). Water solubility data and IR before and after salt formation of sodium perchlorate were measured.
[0057]
[Table 1]

[0058]
Example 7: Synthesis of compound (I)
Embedded image

[0059]
Compound 16 (5.0 g, 15.1 mmol) and glutaconaldehyde dianil hydrochloride (4.40 g, 15.1 mmol) were suspended in a mixed solution of acetic anhydride 20 ml and acetic acid 300 ml, and heated at reflux temperature for 5 hours. The red solution was concentrated under reduced pressure, 200 ml of a mixed solution of ethyl acetate and water was added to the resulting residue to suspend it, and a black red solid was collected by filtration. After washing with water, it was dried under reduced pressure to obtain a black-red powder of Compound 17. Yield 5.33 g (65.2% yield).
[0060]
Compound 17 (167 mg, 0.308 mmol) and compound 9 (100 mg, 0.309 mmol) obtained in Example 6 were dissolved in 3 ml of pyridine and stirred at 50 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in 10 ml of water to adjust the pH of the solution to 3. Then, the solution was purified with a Sephadex LH20 column (solvent: methanol) to obtain the blackish green color of Compound 18. A solid was obtained. Yield 51 mg (23.1% yield).
[0061]
Compound 18 (30 mg, 41.8 μmol) / 50 v / v% THF aqueous solution in 1 ml N-hydroxysuccinic anhydride sodium sulfonate (16.8 mg, 77.4 μmol) and N, N'-dicyclohexylcarbodiimide (25.8 mg, 0.125 mmol ) And reacted at 4 ° C. overnight, followed by filtration. The filtrate was concentrated under reduced pressure at 20 ° C. To the residue was added 10 ml of ethyl acetate, and the resulting crystals were further washed with ether three times to obtain Compound 19 as a black-green solid. Yield 34 mg (Yield 88.8%). MS (FAB) m / e = 892 (M ^- ; Fluorescence spectrum: λ _ex = 771 nm, λ _em = 822 nm (water)
[0062]
Compound 19 was treated with a solution of sodium iodide in methanol according to the method of Example 6 to give compound (I). Comparing the infrared absorption spectrum of Compound 19 with the infrared absorption spectrum of Compound (I), the observed 2360 and 1740 cm ^-1 Absorption was not observed in compound (I).
[0063]
Example 8: Protein labeling and fluorescence detection using compound (A)
Bovine serum albumin (BSA) (3.0 mg) was dissolved in pH 8.5, 0.1 M carbonate buffer (5.0 ml), 50 mmol of compound (A) in DMSO (5 ml) was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. When this solution was measured using HPLC (gel column, fluorescence detection: excitation wavelength 550 nm, detection wavelength 569 nm), a peak of labeled BSA was observed in addition to the peak of compound (A). Fig. 2 shows the fluorescence spectrum of a solution prepared by dissolving Compound (A) in PBS at pH 7.4, and Fig. 3 shows the fluorescence spectrum of a solution prepared by dissolving BSA labeled with Compound (A) in PBS at pH 7.4. . Furthermore, when the same measurement was performed by changing the amount of BSA and labeling, a good proportional relationship was observed between the fluorescence intensity and the amount of BSA as shown in FIG. 4, and about 0.1 femtomole was detected as BSA. It turned out to be a limit. The signal / noise ratio at this time was 3.
[0064]
Example 9: Labeling of an antibody with a compound of the invention
1 mg of compound (L) was dissolved in 0.94 ml of water to give a 1 mM solution. 1 mg of human IgG was dissolved in 1 ml of 50 mM sodium carbonate buffer at pH 8.5, 0.2 ml of the above solution containing the compound (L) was added to this solution, and reacted at 30 ° C. for 1 hour. The reaction solution was applied to a Sephadex G-25 column to separate labeled human IgG. As a separation buffer, 50 mM phosphate buffer (pH 7.4) was used. The unreacted compound (L) remained at the top of the Sephadex gel, and the separation from labeled IgG was good. The resulting labeled IgG was lyophilized and stored frozen at -20 ° C. 6 mg of this labeled IgG was dissolved in 100 ml of 50 mM phosphate buffer (pH 7.4), and the result of measuring the fluorescence spectrum is shown in FIG.
[0065]
【The invention's effect】
Since the compound of the present invention has a high fluorescence quantum yield, it is characterized by high fluorescence and high water solubility. In addition, the compound of the present invention is characterized in that the compound to be labeled can be labeled selectively with a functional group. Furthermore, since only one labeling group is present in the molecule, the property of the labeled compound can be maintained as it is even after labeling without crosslinking the labeled compound. Therefore, when the compound of the present invention is used, it can be fluorescently labeled while retaining the three-dimensional structure and physiological activity of biopolymers such as proteins, and the spectroscopic properties of indocyanine can be added to the target molecule.
[Brief description of the drawings]
FIG. 1 A compound of the present invention (A) was added to PBS of pH 7.4 (NaCl: 8.0 g, KCl: 0.2 g, Na ₂ HPO _Four : 11.5g, KH ₂ PO _Four : 0.2g / water 1000ml) is a diagram showing an absorption spectrum of a solution.
FIG. 2 is a graph showing a fluorescence spectrum of a solution obtained by dissolving the compound (A) of the present invention in PBS at pH 7.4.
FIG. 3 is a graph showing a fluorescence spectrum of a solution obtained by dissolving BSA labeled with the compound (A) of the present invention in PBS at pH 7.4.
FIG. 4 is a graph showing the correlation between the fluorescence intensity of labeled BSA labeled with the compound (A) of the present invention and the amount of BSA subjected to the reaction.
FIG. 5 is an infrared absorption spectrum of a compound (L) in which sodium iodide is incorporated as a salt and an intramolecular counter ion type compound (K). In the figure, a) shows the infrared absorption spectrum of compound (L), and b) shows the infrared absorption spectrum of compound (K).
FIG. 6 shows a fluorescence spectrum of human IgG labeled with the compound (L) of the present invention.

Claims (5)

The following formula:

(In the formula, A ¹ and A ² both represent a benzene ring, or both represent a naphthalene ring, R ¹ and R ² both represent a hydrogen atom, R ³ represents an alkyl group (provided that both A ¹ and A ² are Except for a naphthalene ring), or a free or dissociated alkyl sulfonate group, n represents an integer of 1 to 3, X ⁻ represents an anionic species, and Z represents the following formula:

A group selected from the group consisting of: M ⁺ is an alkali metal ion, and Y is an alkylene group having 2 to 5 carbon atoms). 2. The compound according to claim 1, wherein X ⁻ is a halogen ion, and R ³ is a linear sulfonic acid alkyl group having 2 to 5 carbon atoms that forms a salt with a sodium ion.  A fluorescent labeling reagent comprising the compound according to claim 1 or 2.  The reagent according to claim 3, which is used for fluorescent labeling of biopolymers.  The reagent according to claim 4, wherein the biopolymer is selected from the group consisting of proteins, glycoproteins, lipids, phospholipids, polysaccharides, and nucleic acids.

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