JPH06157504A - Fluorescent labelling agent for near-infrared excitation and its application - Google Patents
Fluorescent labelling agent for near-infrared excitation and its applicationInfo
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- JPH06157504A JPH06157504A JP5447892A JP5447892A JPH06157504A JP H06157504 A JPH06157504 A JP H06157504A JP 5447892 A JP5447892 A JP 5447892A JP 5447892 A JP5447892 A JP 5447892A JP H06157504 A JPH06157504 A JP H06157504A
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【発明の詳細な説明】
本発明は、微量成分の検出のための一般式（Ｉ）で表さ
れるシアノピロニン誘導体（以下「本発明化合物」と略
記す）および本発明化合物を近赤外光励起が可能な新規
蛍光ラベル化剤として用いる分析法に関するものであ
る。上記の本発明化合物は、いずれも文献未載の新規か
つ有用な物質で、長波長域での高感度分析が行える蛍光
ラベル化剤として有用である。現在までに蛍光性ラベル
化剤としては代表的なＦＩＴＣ（フルオレセインイソチ
オシアネート）やＴＲＩＴＣ（テトラメチルローダミン
イソチオシアネート）、クマリン誘導体をはじめとして
数多くの化合物が開発され、アミノ酸やカルボン酸分析
あるいはモノクローナル抗体などのタンパク標識に用い
られ、高感度分析用試薬として欠かせないものとなって
いる。また、従来は放射性同位元素標識が一般的であっ
たＤＮＡ（デオキシリボ核酸）の標識にも蛍光ラベル化
剤が用いられるようになり塩基配列決定の自動化も可能
になってきている。蛍光標識化合物をより高感度に検出
するためには、検出波長領域でのバックグラウンド蛍光
が少ないことが要求される。一般に生体試料は紫外部領
域で励起したときに蛍光を示す物質が多く、バックグラ
ウンド蛍光をおさえるためには蛍光波長をより長波長側
に持っていく必要がある。すなわち、従来の４００ない
し６００ｎｍ領域で発光極大を持つ蛍光ラベル化剤では
なく６００ｎｍ以上の領域で発光極大をもつようなラベ
ル化剤が望ましい。また装置の面から考えれば、検出感
度を向上させるためには、光源もより励起光エネルギー
強度の強いレーザーを用いるほうが望ましい。但し、ア
ルゴン−ネオンレーザーなどの一般的なレーザー装置は
価格も高く、維持管理が用意ではない等の問題点もあ
り、ＨＰＬＣなどの汎用機に用いることはできないのが
現状である。近年、半導体レーザーの開発によって６０
０−９００ｎｍの近赤外領域の高出力光源が安価に得ら
れるようになり、この領域での蛍光分析が注目されるよ
うになってきた。半導体レーザーとしては例えば、シャ
ープ製ＬＴ０２４ＭＤ（７８０ｎｍ，２０ｍＷ）、ＬＴ
０１５ＭＦＯ（８３２ｎｍ，３０ｍＷ）、浜松ホトニク
ス社製Ｌ２３７６（９０４ｎｍ，１０Ｗ，パルス光）あ
るいは、ＮＥＣ社製ＮＤＬ３２００（６７０ｎｍ，３ｍ
Ｗ）など多くの半導体レーザーがすでに市販されてい
る。半導体レーザーを用いるもう一つの利点は検出装置
がコンパクトにできることである。一方、この近赤外領
域に吸収波長を持つ蛍光性標識化合物は、水溶液の状態
で非常に不安定なポリメチン化合物以外には特に知られ
ておらず実用に至っていないのが現状である。従って、
この領域に極大吸収波長を持つ蛍光性標識化合物の開発
は高感度分析技術の面から重要である。長波長領域で分
析を行う利点は、こうした半導体レーザーが利用できる
以外に、前にも述べたように生体サンプル由来の蛍光性
化合物に妨害されにくいということがあげられる。以上
の状況に基づき、基本骨格をシアノピロニンとする近赤
外蛍光ラベル化剤の合成を検討した結果、本発明化合物
を開発した。これらの化合物はアミノ酸分析では１０
−１２Ｍレベルの検出が可能であることがわかった。
（実施例５参照）本発明化合物の一般的な合成方法につ
いて述べる。原料はｍアニシジン誘導体で、はじめにラ
ベルするために必要な側鎖官能基を導入する。側鎖官能
基を導入するための原料としてブロモカルボン酸エステ
ルを用い、このアルキル化剤とｍ−アニシジン誘導体を
反応させＮ−モノアルキル−ｍ−アニシジン誘導体を合
成する。次に、アルキルハライド、アルキルジハライド
を用い、アミノ基に導入し三級アミンとする。アルキル
ジハライドの場合は三級アミンになると同時にもう一方
のハロゲン化アルキル部分でアニシジンのフェニル基と
反応し環状構造をとる。アミノ基にアルキルカルボン酸
エステルが導入された化合物は次のステップでメトキシ
基のメチル基の脱保護とエステルの加水分解を行い、の
ちＮ−ジアルキル−ｍ−アミノフェノールとホルムアル
デヒドを用いカップリングさせメタンベース誘導体とす
る。この場合脱保護およびエステルの加水分解反応とカ
ップリング反応の順序を逆にしてもよい。次に、熱濃硫
酸中で脱水縮合を行いロイコピロニン誘導体に変換す
る。この際、酸化されピロニン誘導体も生成するが、精
製せず塩化第二鉄−塩酸で酸化しピロニン誘導体とす
る。このピロニン誘導体をシアン化カリウムでニトリル
化しロイコシアノピロニン化合物としたのち塩化第二鉄
−塩酸で酸化しシアノピロニン誘導体とする。この後、
カルボキシル基はスクシンイミドエステル、ヨードアル
キルアミド、マレイミドアルキルエステル、アミノアル
キルアミドとし、アミノ基、チオール基、カルボキシル
基と反応できる形にする。このようにして得られたシア
ノピロニン誘導体は置換基によって励起波長、蛍光発光
波長が異なる。一般にアミノ基に付加しているアルキル
鎖の長さが長くなると長波長にシフトしベンゼン環に付
加するアルキル基の数が多いほど長波長にシフトする傾
向にある。また、蛍光強度はアルキル鎖が環状構造をと
っているほうが強く、試薬としての安定性も向上する傾
向にある。本研究で開発された近赤外蛍光ラベル化剤の
代表的な化合物の構造式とその吸収スペクトルおよび蛍
光スペクトルデータを示す。これらの化合物はそれぞれ
黒青色固体として得られた。吸収スペクトルは試薬濃度
１ｘ１０−５Ｍ（ｐＨ７．０ ２０ｍＭＨＥＰＥＳ緩衝
液）、また、蛍光スペクトルは試薬濃度１ｘ１０−７Ｍ
（ｐＨ７．０ ２０ｍＭ ＨＥＰＥＳ緩衝液）で測定し
た。
実施例により本発明をさらに詳細に述べる。
実施例１．化合物９の合成
合成スキームを図１に示す。
１−１．化合物２の合成
ｍ−アニシジン６１．６ｇ（０．５００ｍｏｌ）をアセ
トニトリル６２０ｍｌに溶かし、６−ブロモヘキサン酸
エチル１６７ｇ（０．７４９ｍｏｌ）および炭酸水素ナ
トリウム８４．１ｇ（１．００ｍｏｌ）を加え、６時間
加熱還流した。冷却後、反応液を濾過し濾液を減圧濃縮
して得られたオイルをベンゼン８００ｍｌに溶かし、水
で３回洗浄した。硫酸マグネシウムで乾燥後濾過し減圧
濃縮し粗製物を得た。得られた粗製物はシリカゲルカラ
ム（ベンゼン：酢酸エチル＝４：１）で分離し、目的化
合物２を１０７ｇ得た。収率８１％ 淡褐色油状物 １
Ｈ−ＮＭＲ（ＣＤＣｌ３δｐｐｍ）１．２５（ｔＪ＝
７．２Ｈｚ３Ｈ）１．３０−１．５０（ｍ２Ｈ）１．５
５−１．７５（ｍ４Ｈ）２．３０（ｔＪ＝７．３Ｈｚ２
Ｈ）３．１０（ｔＪ＝６．９Ｈｚ２Ｈ）３．７７（ｓ３
Ｈ）４．１３（ｑＪ＝７．２Ｈｚ２Ｈ）６．１５−６．
３０（ｍ３Ｈ）７．０７（ｔＪ＝８．０Ｈｚ１Ｈ）ＭＳ
（ＦＡＢ ｐｏｓｉｔｉｖｅ）Ｍ＋１＝２６６
１−２．化合物３の合成
化合物２２０．０ｇ（７５．４ｍｍｏｌ）をアセトニト
リル３００ｍｌに溶かし、ヨードエタン１２．０ｍｌ
（０．１５０ｍｏｌ）および炭酸水素ナトリウム１２．
７ｇ（０．１５１ｍｏｌ）を加え一夜加熱還流した。反
応液を濾過し濾液を減圧濃縮し、得られたオイルをベン
ゼンに溶かし、水で３回洗浄した。硫酸マグネシウムで
乾燥後濾過し減圧濃縮し、粗製物を２１．８ｇ得た。収
率９９％淡褐色油状物 １Ｈ−ＮＭＲ（ＣＤＣｌ３δ
ｐｐｍ）１．１３（ｔＪ＝７．１Ｈｚ ３Ｈ）１．２５
（ｔＪ＝７．２Ｈｚ ３Ｈ）１．３０−１．４０（ｍ２
Ｈ）１．５０−１．７５（ｍ ４Ｈ）２．３０（ｔＪ＝
７．５Ｈｚ ２Ｈ）３．２３（ｔＪ＝７．６Ｈｚ ２
Ｈ）３．３１（ｑＪ＝７．１Ｈｚ２Ｈ）３．７８（ｓ３
Ｈ）４．１２（ｑＪ＝７．２Ｈｚ２Ｈ）６．２０−６．
３０（ｍ３Ｈ）７．１０（ｔＪ＝７．５Ｈｚ１Ｈ）ＭＳ
（ＦＡＢ ｐｏｓｉｔｉｖｅ）Ｍ＋１＝２９４
１−３．化合物４の合成
化合物３１６ｇ（５４．５ｍｍｏｌ）を１６０ｍｌの４
７％臭化水素酸水溶液に溶かし、１３０℃で２４時間加
熱した。反応液を約５００ｇの氷に注ぎ、水酸化ナトリ
ウムでｐＨ４付近に調節し減圧濃縮したのち、メタノー
ルに懸濁させ無機塩を除いた。減圧濃縮し得られたオイ
ル状物をさらにエタノールに懸濁させ、濾過で不溶物を
除き、再び減圧濃縮し、目的化合物４を１４．１ｇ得た
た。収率１０３％ 褐色油状物 １Ｈ−ＮＭＲ（ＣＤＣ
ｌ３＋ＭｅＯＨ δｐｐｍ）１．１９（ｔＪ＝７．１Ｈ
ｚ ３Ｈ）１．２５−１．４０（ｍ ２Ｈ）１．４０−
１．６５（ｍ ４Ｈ）２．２８（ｔＪ＝７．３Ｈｚ ２
Ｈ）３．２３（ｔＪ＝７．６Ｈｚ ２Ｈ）３．３０−
３．６０（ｍ ２Ｈ）６．７０−６．８０（ｍ ３Ｈ）
７．３０（ｔＪ＝８．３Ｈｚ １Ｈ）ＭＳ（ＦＡＢ ｎ
ｅｇａｔｉｖｅ）Ｍ−１＝２５０
１−４． 化合物５の合成
Ｎ，Ｎ−ジエチル−ｍ−アミノフェノール３．００ｇ
（１８．２ｍｍｏｌ）および化合物４４．６０ｇ（１
８．３ｍｍｏｌ）を１５ｍｌのテトラヒドロフラン（以
下ＴＨＦと略記す）：水＝７：３の混合溶媒に溶かし、
３７％ホルムアルデヒド水溶液１．５２ｍｌ（１８．７
ｍｍｏｌ）を加え、６時間加熱還流した。反応液を減圧
濃縮したあと３％メタノール／クロロホルム１００ｍｌ
に溶かし、水洗を３回行った。硫酸マグネシウムで乾燥
し濾過、減圧濃縮し得られた紫色オイル状物をシリカゲ
ルカラム（クロロホルム−５％メタノール／クロロホル
ム）で分離して、目的化合物５を２．１２ｇ得た。収率
２７％ 紫色粘性液体１Ｈ−ＮＭＲ（ＣＤＣｌ３＋Ｍ
ｅＯＨ δｐｐｍ）１．０９（ｔＪ＝７．６Ｈｚ ９
Ｈ）１．２５−１．４０（ｍ ２Ｈ）１．４０−１．６
５（ｍ ４Ｈ）２．２８（ｔＪ＝７．４Ｈｚ ２Ｈ）
３．１０−３．４０（ｍ ８Ｈ）４．８６（ｓ ２Ｈ）
６．２０−６．３０（ｍ ４Ｈ）６．９０−７．００
（ｍ２Ｈ）ＭＳ（ＦＡＢｎｅｇａｔｉｖｅ）Ｍ−１＝４
２７
１−５．化合物６の合成
化合物５２．００ｇ（４．６７ｍｍｏｌ）を濃硫酸２０
ｍｌに溶かし１００℃で３０分加熱した。硫酸溶液を氷
１００ｇに注ぎ、４Ｎの水酸化ナトリウム水溶液でｐＨ
４付近に調節し減圧濃縮した。メタノール１００ｍｌに
懸濁し、濾過してメタノールに可溶な成分だけ取り出し
た。粗製物をシリカゲルカラム（５％メタノール／クロ
ロホルム）で精製し目的化合物６を１．２６ｇ得た。収
率６６％赤紫色固体１Ｈ−ＮＭＲ（ＭｅＯＨ δｐｐ
ｍ）１．０２−１．４０（ｍ ９Ｈ）１．４５−１．７
０（ｍ ６Ｈ）２．２５（ｔＪ＝７．２Ｈｚ ２Ｈ）
３．４０−３．７０（ｍ ８Ｈ）４．８６（ｓ ２Ｈ）
６．７６−７．００（ｍ ４Ｈ）７．８０−７．９５
（ｍ２Ｈ）ＭＳ（ＦＡＢ ｎｅｇａｔｉｖｅ）Ｍ−１＝
４０９
１−６．化合物７の合成
化合物６１．００ｇ（２．４４ｍｍｏｌ）に、塩化第二
鉄２．００ｇ（１２．４ｍｍｏｌ）を２０ｍｌの１Ｍ塩
酸水溶液に溶かした溶液を加え、室温で一夜攪拌した。
生じた赤褐色の沈殿を濾取し少量の冷水で洗浄し乾燥
し、目的化合物７を１．１５ｇ得た。収率１０６％ 赤
褐色固体１Ｈ−ＮＭＲ（ＭｅＯＨ δｐｐｍ）１．００
−１．３０（ｍ ９Ｈ）１．３５−１．４０（ｍ ２
Ｈ）１．７０−１．９０（ｍ ４Ｈ）２．２４（ｔＪ＝
７．２Ｈｚ ２Ｈ）３．７５−４．００（ｍ ８Ｈ）
７．２５−７．４０（ｍ ４Ｈ）７．６０（ｓ１Ｈ）
８．１０−８．２５（ｍ２Ｈ）ＭＳ（ＦＡＢ ｐｏｓｉ
ｔｉｖｅ）Ｍ＋＝４０９
１−７．化合物８の合成
化合物７１．００ｇ（２．２５ｍｍｏｌ）をエタノール
１２ｍｌと水６ｍｌの混合溶媒に溶かし、酢酸１８０μ
ｌ（３．１５ｍｍｏｌ）を加えたのち、シアン化カリウ
ム３８０ｍｇ（５．８４ｍｍｏｌ）を加え室温で２時間
撹拌した。反応液を減圧濃縮し、それにクロロホルムを
加え濾過し濾液を再度減圧濃縮し、シリカゲルカラム
（１０％メタノール／クロロホルム）で精製し目的化合
物８を５６０ｍｇ得た。収率５７％ 青黒色固体１
Ｈ−ＮＭＲ（ＭｅＯＨ δｐｐｍ）１．１０−１．３
５（ｍ ９Ｈ）１．３５−１．５５（ｍ ２Ｈ）１．６
５−１．８０（ｍ ４Ｈ）２．３０（ｔＪ＝７．０Ｈｚ
２Ｈ）３．４５−３．７５（ｍ ８Ｈ）６．７５−
６．９５（ｍ ５Ｈ）７．６０−７．８０（ｍ２Ｈ）Ｍ
Ｓ（ＦＡＢ ｎｅｇａｔｉｖｅ）Ｍ−１＝４３４
１−８． 化合物９の合成
化合物８４８０ｍｇ（１．１０ｍｍｏｌ）に、塩化第二
鉄８８０ｍｇ（５．４３ｍｍｏｌ）を１Ｍ塩酸８．８ｍ
ｌに溶かした溶液を加え室温で一夜攪拌した。生じた黒
青色の沈殿を濾取し少量の冷水で洗浄したのち乾燥し、
目的化合物９を５２０ｍｇ得た。収率１０１％ 黒青
色固体１
Ｈ−ＮＭＲ（ＭｅＯＨ δｐｐｍ）１．００−１．３
５（ｍ ９Ｈ）１．４０−１．５５（ｍ ２Ｈ）１．６
０−１．８０（ｍ ４Ｈ）２．２０−２．４０（ｍ ２
Ｈ）３．７０−４．００（ｍ ８Ｈ）７．４０−７．６
５（ｍ ４Ｈ）８．１５−８．３０（ｍ ２Ｈ）ＭＳ
（ＦＡＢ ｐｏｓｉｔｉｖｅ）Ｍ＋１＝４３４
実施例２．化合物１０の合成
合成スキームを図２に示す。化合物９２５０ｍｇ（０．
５３２ｍｍｏｌ）をジメチルホルムアミド５ｍｌに溶か
し、Ｎ−ヒドロキシスクシンイミド１８５ｍｇ（１．６
１ｍｍｏｌ）およびジシクロヘキシルカルボジイミド
１．００ｇ（４．８５ｍｍｏｌ）を加え室温で５時間撹
拌した。反応物を濾過し濾液を４０℃以下で減圧濃縮
し、エーテルを加えデカンテーションした。さらに得ら
れた固体を水に溶かし濾過し４０℃以下で減圧濃縮し
た。濃縮物にエーテルを加え結晶化させ濾取し目的化合
物１０を２３５ｍｇ得た。収率７８％ 黒青色固体 １
Ｈ−ＮＭＲ（ＭｅＯＨ δｐｐｍ）１．０５−１．３５
（ｍ ９Ｈ）１．４０−１．５５（ｍ ２Ｈ）１．５５
−１．７０（ｍ ４Ｈ）２．２５−２．４０（ｍ ２
Ｈ）２．６０（ｓ ４Ｈ）３．６０−４．００（ｍ ８
Ｈ）７．３５−７．６０（ｍ ４Ｈ）８．１０−８．２
５（ｍ ２Ｈ） ＭＳ（ＦＡＢ ｐｏｓｉｔｉｖｅ）Ｍ
＋＝５３１ 化合物１０の吸収スペクトルを図９に、蛍
光スペクトルを図１０に示す。
実施例３．化合物１１の合成
合成スキームを図３に示す。化合物９２５０ｍｇ（０．
５３２ｍｍｏｌ）をジメチルホルムアミド５ｍｌに溶か
し、Ｎ−ヨードアセチルピペリジン４１３ｍｇ（１．６
１ｍｍｏｌ）およびジシクロヘキシルカルボジイミド
（以下ＤＣＣと略記す）１．０ｇ（４．８５ｍｍｏｌ）
を加え室温で一夜攪拌した。反応物を濾過し濾液を減圧
濃縮し、残渣に酢酸エチルを加え冷蔵庫に一夜放置し、
生じた白色沈殿を濾過で除き減圧濃縮した。濃縮残渣に
エーテルを加えデカンテーションし、さらに得られた固
体を水に溶かし濾過し減圧濃縮した。濃縮残渣をシリカ
ゲルカラムクロマトグラフ（３％メタノール／クロロホ
ルム）で分離し目的フラクションを集め減圧濃縮した。
濃縮物にエーテルを加え結晶化させ濾取し目的化合物１
１を２１０ｍｇ得た。収率５６％ 黒青色固体１Ｈ−Ｎ
ＭＲ（ＭｅＯＨ δｐｐｍ）１．１０−１．１５（ｍ
１１Ｈ）１．６０−１．７５（ｍ ４Ｈ）２．３２（ｔ
Ｊ＝７．２Ｈｚ２Ｈ）３．２０−３．９０（ｍ １６
Ｈ）４．２２（ｓ２Ｈ）７．４０−７．６５（ｍ ４
Ｈ）８．１５−８．３０（ｍ ２Ｈ）ＭＳ（ＦＡＢ ｐ
ｏｓｉｔｉｖｅ）Ｍ＋＝６７１
実施例４．化合物１７の合成
合成スキームを図４に示す。実施例１および２で示した
合成法と同様の操作で行った。原料に化合物４４．６０
ｇ（１８．３ｍｍｏｌ）および８−ヒドロキシジュロリ
ジン３．４６ｇ（１８．３ｍｍｏｌ）を用い、ホルムア
ルデヒドでカップリングさせ、化合物１２を収量１．９
０ｇ（収率２３％）で得た。この化合物を用いて硫酸に
よる脱水縮合を行い、化合物１３を収量１．１３ｇ（収
率６２％）で得た。次に塩化第二鉄／塩酸で酸化し、ピ
ロニン化合物１４を９８０ｍｇ（収率８０％）得た。更
にこの化合物をシアン化カリウムでシアノ化しシリカゲ
ルカラムで精製して目的化合物１５を５６０ｍｇ（収率
６０％）得、次に塩化第二鉄／塩酸で酸化し収量５８０
ｍｇ（収率９６％）で化合物１６を得た。この化合物２
５０ｍｇを用いてＮ−ヒドロキシスクシンイミドで活性
エステル化合物１７とした。収量２８０ｍｇ（収率８０
％）。黒青色固体 １Ｈ−ＮＭＲ（ＭｅＯＨ δｐｐ
ｍ）１．１０−１．５５（ｍ ５Ｈ）１．６０−１．７
５（ｍ ４Ｈ）２．０５−２．４０（ｍ ６Ｈ）２．６
６（ｓ ４Ｈ）２．７０−２．９０（ｍ ４Ｈ）３．１
０−３．４０（ｍ ６Ｈ）７．５０−７．７０（ｍ ３
Ｈ）８．０５−８．１０（ｍ １Ｈ）ＭＳ（ＦＡＢ ｐ
ｏｓｉｔｉｖｅ）Ｍ＋＝５５５ 化合物１７の吸収スペ
クトルを図１１に、蛍光スペクトルを図１２に示す。
実施例５．化合物１８の合成法
合成スキームを図５に示す実施例４で示した化合物１６
２５０ｍｇ（０．４６０ｍｍｏｌ）を乾燥した５ｍｌの
ＤＭＦに溶かし、０℃に冷却しながら１０４ｍｇ（０．
５０４ｍｍｏｌ）のＤＣＣを加えたのち２０分後、Ｎ−
ヒドロキシエチルマレイミド７１ｍｇ（０．５０３ｍｍ
ｏｌ）を０．５ｍｌのＤＭＦに溶かし加えた。そのまま
室温で一夜攪拌し、反応液を濾過し濾液を減圧濃縮した
のち、エーテルを加えデカンテーションした。さらに得
られた固体を水に溶かして濾過し濾液を減圧濃縮した。
濃縮物にエーテルを加え結晶化させ濾取し、目的化合物
１８を２１０ｍｇ得た。収率７４％ 黒青色固体 １Ｈ
−ＮＭＲ（ＭｅＯＨ δｐｐｍ）１．１０−１．５５
（ｍ ５Ｈ）１．６０−１．８０（ｍ ４Ｈ）２．１０
−２．４０（ｍ ６Ｈ）２．７５−２．９５（ｍ ４
Ｈ）３．０５−３．３０（ｍ ４Ｈ）３．３０−３．６
０（ｍ ４Ｈ）４．０７（ｔＪ＝６．８Ｈｚ ２Ｈ）
７．４６（ｓ ２Ｈ）７．５５−７．７０（ｍ ３Ｈ）
８．０５−８．１０（ｍ １Ｈ） ＭＳ（ＦＡＢ ｐｏ
ｓｉｔｉｖｅ）Ｍ＋＝５８１
実施例６．化合物２６の合成
合成スキームを図６に示す。
６−１．化合物１９ａ、化合物１９ｂの合成
化合物２１０．０ｇ（３７．７ｍｍｏｌ）をアセトニト
リル１００ｍｌに溶かし、炭酸水素ナトリウム１２．７
ｇ（０．１５１ｍｏｌ）および３−ブロモクロロプロパ
ン５０ｍｌを加え、１０日間加熱還流した。生じた白色
沈殿を濾過で除き、濾液を減圧濃縮したのちベンゼン２
００ｍｌに溶かし，水で３回洗浄した。硫酸マグネシウ
ムで乾燥後濾過し減圧濃縮し粗製物を得た。得られた粗
製物はシリカゲルカラム（ベンゼン：酢酸エチル＝５：
１）で分離し、二種類の目的化合物１９ａ，１９ｂをそ
れぞれ３．６５ｇ（収率３２％）および２．３５ｇ（収
率１６％）得た。 いずれも淡褐色油状物 化合物１９
ａ １Ｈ−ＮＭＲ（ＭｅＯＨ δｐｐｍ）１．２７（ｔ
Ｊ＝７．２Ｈｚ ３Ｈ）１．５０−１．８５（ｍ６Ｈ）
１．９０−２．１５（ｍ ４Ｈ）２．６０−２．９０
（ｍ ２Ｈ）３．００−３．３０（ｍ ４Ｈ）３．７８
（ｓ ３Ｈ）４．１３（ｑＪ＝７．２Ｈｚ２Ｈ）６．５
０−６．８０（ｍ ２Ｈ）７．００−７．１０（ｍ １
Ｈ） ＭＳ（ＦＡＢ ｐｏｓｉｔｉｖｅ）Ｍ＋＝３０６
化合物１９ｂ １Ｈ−ＮＭＲ（ＭｅＯＨ δｐｐｍ）
１．２５（ｔＪ＝７．２Ｈｚ ３Ｈ）１．５５−１．９
０（ｍ ６Ｈ）１．９５−２．２５（ｍ ４Ｈ）２．６
０−２．９０（ｍ ２Ｈ）３．０５−３．３５（ｍ ４
Ｈ）３．８９（ｓ ３Ｈ）４．１６（ｑＪ＝７．２Ｈｚ
２Ｈ）６．８０（ｄＪ＝７．５Ｈｚ １Ｈ）７．１０
（ｍ １Ｈ）７．３６（ｄＪ＝２．０Ｈｚ １Ｈ） Ｍ
Ｓ（ＦＡＢ ｐｏｓｉｔｉｖｅ）Ｍ＋＝３０６
６−２． 化合物２６の合成
実施例１と同様に、化合物１９ａ３．６０ｇ（１１．７
ｍｍｏｌ）を臭化水素酸で処理し化合物２０を３．２８
ｇ（収率１０６％）得た。次にこの化合物３．００ｇ
（１１．４ｍｍｏｌ）と８−ヒドロキシジュロリジン
２．１５ｇ（１１．４ｍｍｏｌ）とをホルムアルデヒド
でカップリングさせ、化合物２１を収量１．４３ｇ（収
率２７％）で得た。この化合物を用いて硫酸による脱水
縮合を行い、化合物２２を収量１．２０ｇ（収率８７
％）で得た。次に塩化第二鉄／塩酸で酸化し、ピロニン
化合物２３を１．０５ｇ（収率８１％）得た。さらにこ
の化合物をシアン化カリウムでシアノ化しシリカゲルカ
ラムで精製して目的化合物２４を６４０ｍｇ（収率６２
％）得、次に塩化第二鉄／塩酸で酸化し収量６００ｍｇ
（収率８８％）で化合物２５を得た。この化合物２５０
ｍｇを用いてＮ−ヒドロキシスクシンイミドで活性エス
テル化合物２６とした。収量２５５ｍｇ（収率８６
％）。黒青色固体 １Ｈ−ＮＭＲ（ＭｅＯＨ δｐｐ
ｍ）１．５０−１．８５（ｍ ６Ｈ）２．００−２．３
５（ｍ ８Ｈ）２．６７（ｓ ４Ｈ）２．７５−２．９
５（ｍ ６Ｈ）３．００−３．４５（ｍ ８Ｈ）７．５
１（ｄＪ＝６．２Ｈｚ １Ｈ）７．６８（ｄＪ＝６．２
Ｈｚ １Ｈ）８．１２（ｓ１Ｈ）ＭＳ（ＦＡＢ ｐｏｓ
ｉｔｉｖｅ）Ｍ＋＝５６７
実施例７．化合物２７の合成
合成スキームを図７に示す。包水ヒドラジン０．２６ｍ
ｌ（３．３３ｍｍｏｌ）を水２ｍｌと混合し、４Ｎ塩酸
水溶液でｐＨ＝６にした後、実施例６で合成した化合物
２６１００ｍｇ（０．１６５ｍｍｏｌ）をＤＭＦ３ｍｌ
に溶かした溶液を、室温で攪拌しながら少しづつ加え
た。一夜攪拌後、反応液を減圧濃縮し水５ｍｌを加えて
４Ｎの水酸化ナトリウム水溶液でｐＨ＝１０にし、クロ
ロホルムで５回抽出した。無水硫酸マグネシウムで乾燥
したのち減圧濃縮し、目的化合物２７を８６ｍｇ得た。
収率９５％ 黒青色固体 １Ｈ−ＮＭＲ（ＭｅＯＨ δ
ｐｐｍ）１．５５−１．９０（ｍ６Ｈ）１．９５−２．
２５（ｍ ８Ｈ）２．８０−３．０５（ｍ６Ｈ）３．１
５−３．８０（ｍ １０Ｈ）７．４８（ｄＪ＝６．２Ｈ
ｚ １Ｈ）７．５５（ｄＪ＝６．２Ｈｚ １Ｈ）８．０
４（ｓ １Ｈ） ＭＳ（ＦＡＢ ｐｏｓｉｔｉｖｅ）Ｍ
＋＝５１２
実施例８
化合物３７の合成
合成スキームを図８に示す。
８−１．化合物２９の合成
３−メトキシ−６−メチルアニリン２５．０ｇ（０．１
８２ｍｏｌ）をアセトニトリル２００ｍｌに溶かし、ブ
ロモ酢酸エチル４５．６ｇ（０．２７３ｍｏｌ）および
炭酸水素ナトリウム３０．６ｇ（０．３６４ｍｏｌ）を
加え、６時間加熱還流した。冷却後、反応液を濾過し濾
液を減圧濃縮して得られたオイルをベンゼン３００ｍｌ
に溶かし、水で３回洗浄した。硫酸マグネシウムで乾燥
後濾過し減圧濃縮し粗製物を得た。得られた粗製物はシ
リカゲルカラム（ベンゼン：酢酸エチル＝４：１）で分
離し、目的化合物２９を２０．１ｇ得た。収率６３％
淡褐色油状物１
Ｈ−ＮＭＲ（ＣＤＣｌ３ δｐｐｍ）１．２２（ｔＪ
＝７．６Ｈｚ ３Ｈ）２．１８（ｓ ３Ｈ）３．７８
（ｄＪ＝７．５Ｈｚ ２Ｈ）３．８２（ｓ ３Ｈ）４．
２２（ｑＪ＝７．５Ｈｚ ２Ｈ）６．００−６．２５
（ｍ ２Ｈ）７．０１（ｄＪ＝７．６Ｈｚ １Ｈ）ＭＳ
（ＦＡＢ ｐｏｓｉｔｉｖｅ）Ｍ＋１＝２２４
８−２． 化合物３０の合成
化合物２９１０．０ｇ（５７．１ｍｍｏｌ）をアセトニ
トリル１００ｍｌに溶かし、炭酸水素ナトリウム９．６
０ｇ（０．１１４ｍｏｌ）および３−ブロモクロロプロ
パン５０ｍｌを加え、７日間加熱還流した。生じた白色
沈殿を濾過で除き、濾液を減圧濃縮したのちベンゼン２
００ｍｌに溶かし，水で３回洗浄した。硫酸マグネシウ
ムで乾燥後濾過し減圧濃縮し粗製物を得た。得られた粗
製物はシリカゲルカラム（ベンゼン：酢酸エチル＝５：
１）で分離し、目的化合物３０を１２．９ｇ得た。収率
８６％ 淡褐色油状物 化合物３０ １Ｈ−ＮＭＲ（Ｃ
ＤＣｌ３ δｐｐｍ）１．２５（ｔＪ＝７．８Ｈｚ ３
Ｈ）１．９５−２．０５（ｍ ２Ｈ）２．１９（ｓ ３
Ｈ）２．７０−２．９０（ｍ ２Ｈ）３．３５−３．５
０（ｍ ２Ｈ）３．９６（ｓ ３Ｈ）４．０２（ｓ ２
Ｈ）４．２３（ｑＪ＝７．８Ｈｚ ２Ｈ）６．２３（ｄ
Ｊ＝７．６Ｈｚ １Ｈ）６．８３（ｄＪ＝７．６Ｈｚ
１Ｈ）ＭＳ（ＦＡＢ ｐｏｓｉｔｉｖｅ）Ｍ＋１＝２６
４
８−３． 化合物３７の合成
以下、実施例１および２と同様の枠作で行った。実施例
１と同様に、化合物３０１２．９ｇ（４９．０ｍｍｏ
ｌ）を臭化水素酸で処理し化合物３１を１０．６ｇ（収
率９８％）得た。次にこの化合物５．００ｇ（２２．６
ｍｍｏｌ）と８−ヒドロキシジュロリジン４．２８ｇ
（２２．６ｍｍｏｌ）とをホルムアルデヒドでカップリ
ングさせ、化合物３２を収量２．３９ｇ（収率２５％）
で得た。この化合物を用いて１００℃で３時間、５０％
硫酸中で脱水縮合を行い、化合物３３を収量１．７９ｇ
（収率７８％）で得た。次に塩化第二鉄／塩酸で酸化
し、ピロニン化合物３４を１．７３ｇ（収率８９％）得
た。さらにこの化合物をシアン化カリウムでシアノ化し
シリカゲルカラムで精製して目的化合物３５を１．０７
ｇ（収率６３％）得、次に塩化第二鉄／塩酸で酸化し収
量１．０６ｇ（収率９２％）で化合物３６を得た。実施
例２と同様にこの化合物２５０ｍｇを用いてＮ−ヒドロ
キシスクシンイミドで活性エステル化合物３７とした。
収量２４２ｍｇ（収率８０％）。黒青色固体 １Ｈ−Ｎ
ＭＲ（ＭｅＯＨ δｐｐｍ）１．８５−２．０５（ｍ６
Ｈ）２．６１（ｓ ３Ｈ）２．６５（ｓ ４Ｈ）２．８
０−３．００（ｍ ６Ｈ）３．７０−３４．０５（ｍ
６Ｈ）４．３２（ｓ ２Ｈ）７．５８（ｓ １Ｈ）７．
７６（ｓ１Ｈ）８．１２（ｓ １Ｈ） ＭＳ（ＦＡＢ
ｐｏｓｉｔｉｖｅ）Ｍ＋＝５２５
実施例９
Ｎ，Ｎ，Ｎ′−トリエチル−Ｎ−（５′−スクシンイミ
ドオキシカルボニルペンチル）−３，６−ジアミノ−９
−シアノキサンテンクロリド（化合物１０）を用いたア
ミノ酸の分析
４−１． アミノ酸のラベル化法
被分析アミノ酸としてグリシン、アラニン、グルタミン
酸を用いた。１ｐＭから１μＭの濃度既知のアミノ酸水
溶液を用い検量線を作成した。アミノ酸を含む水溶液を
１０μｌとりｐＨ７．０２０ｍＭＨＥＰＥＳ緩衝液２０
μｌを加え、１ｍＭのＮ，Ｎ，Ｎ′−トリエチル−Ｎ−
（５′−スクシンイミドオキシカルボニルペンチル）−
３，６−ジアミノ−９−シアノキサンテンクロリド水溶
液１０μｌを加え、３７℃で１時間インキュベートし
た。水５０μｌを加え氷水に入れクロロホルム１００μ
ｌで２回抽出した。水相は分析するまで−２０℃で凍結
保存した。
４−２． ラベル化アミノ酸の分析法
ラベル化アミノ酸は液体クロマトグラフ（ＨＰＬＣ）で
分析した。ＨＰＬＣ本体は島津社製ＬＣ４Ａを用い、カ
ラムに逆相カラム（ＧＬサイエンス社製Ｎｕｃｌｅｏｓ
ｉｌ ＯＤＳ：カラムサイズ４．６ｍｍｘ２５０ｍｍ粒
子径５μｍ）を使用し、流速１ｍｌ／ｍｉｎでアセトニ
トリル：１０ｍＭリン酸水溶液＝１：１混合溶媒を流し
た。蛍光測定には簡易型蛍光分析装置を用いた。光源に
はＮＥＣ社製半導体レーザーＮＤＬ３２００（６７０ｎ
ｍ ３ｍＷ）を使用した。レーザー光を顕微鏡用対物レ
ンズで集光し、フローセルに当て出てきた蛍光をモノク
ロメーター（日本分光ＣＴ−１０）で分光したのち、光
電子倍増管（浜松ホトニクスＲ９４３−０２）で検出し
た。ラベル化した検量線作成用サンプル溶液１０μｌを
ＨＰＬＣに注入し各ピークの面積とアミノ酸濃度との関
係をグラフにした。作成したグリシンの検量線を図１３
に、アラニンの検量線を図１４に、グルタミン酸の検量
線を図１５に示す。
４−３． 未知濃度アミノ酸混合溶液の分析
グリシン、アラニンおよびグルタミン酸の１μＭ大溶液
を作成し、３種を適当量混合し約１０００倍に希釈し
た。この溶液を１０μｌとりｐＨ７．０２０ｍＭＨＥＰ
ＥＳ緩衝液２０μｌ加え、１ｍＭのＮ，Ｎ，Ｎ′−トリ
エチル−Ｎ−（５′−スクシンイミドオキシカルボニル
ペンチル）−３，６ジアミノ−９−シアノキサンテンク
ロリド水溶液１０μｌを加え、３７℃で１時間インキュ
ベートした。水５０μｌを加え氷水に入れクロロホルム
１００μｌで２回抽出した。水相は分析するまで−２０
℃で凍結保存した。サンプルを１０μｌとり実施例４−
２と同様の条件でＨＰＬＣ分析し１０００倍希釈時のア
ミノ酸含量を検量線から求めた。その結果を図１６に示
す。
アミノ酸量 グリシン １８．３ｐＭ
アラニン ９．５ｐＭ
グルタミン酸 ５０．１ｐＭDetailed Description of the Invention
The present invention is represented by the general formula (I) for detecting trace components.
Cyanopyrronine derivative (hereinafter abbreviated as “the compound of the present invention”)
And a novel compound capable of near-infrared light excitation of the compound of the present invention
Relates to analytical methods used as fluorescent labeling agents
It Is the above-mentioned compound of the present invention novel in any unpublished literature?
Fluorescent light, which is one of the most useful substances and enables highly sensitive analysis in the long wavelength region.
It is useful as a labeling agent. Fluorescent label to date
FITC (fluorescein
Cyanate) and TRITC (tetramethylrhodamine)
Isothiocyanate) and coumarin derivatives
Numerous compounds have been developed and analyzed for amino acids and carboxylic acids
Or used to label proteins such as monoclonal antibodies
And become an indispensable reagent for high-sensitivity analysis.
There is. In addition, conventionally, radioisotope labeling is common.
Labeling of DNA (deoxyribonucleic acid)
Now that agents are used, it is possible to automate nucleotide sequencing
Is becoming. Highly sensitive detection of fluorescently labeled compounds
To detect background fluorescence in the detection wavelength range
Is required to be small. Generally, biological samples are in the ultraviolet region
Many substances exhibit fluorescence when excited in the region
The longer wavelength side to suppress the unbound fluorescence.
Need to bring it to. That is, there is no conventional 400
In the fluorescent labeling agent that has a maximum emission in the 600 nm region,
Without having a maximum emission in the region of 600 nm or more
Lubricant is desirable. From the perspective of the device, the sense of detection
In order to improve the
It is preferable to use a strong laser. However,
Common laser devices such as the Lugon-Neon laser
There are also problems such as high prices and lack of maintenance.
Therefore, it cannot be used for general-purpose machines such as HPLC.
The current situation. In recent years, with the development of semiconductor lasers, 60
A high-power light source in the near infrared region of 0-900 nm can be obtained at low cost.
And the fluorescence analysis in this area will attract attention.
It's growing. As a semiconductor laser, for example,
LT024MD (780nm, 20mW), LT
015MFO (832nm, 30mW), Hamamatsu Photonik
L2376 (904nm, 10W, pulsed light)
Ruiha, NEC NDL3200 (670nm, 3m
Many semiconductor lasers such as W) are already on the market
It Another advantage of using a semiconductor laser is a detection device
Can be made compact. On the other hand, this near infrared region
A fluorescent labeling compound with an absorption wavelength in the region is in the state of an aqueous solution.
Is especially known except for the highly unstable polymethine compound
The current situation is that it has not been put to practical use. Therefore,
Development of fluorescent labeling compound with maximum absorption wavelength in this region
Is important in terms of high sensitivity analysis technology. Min in the long wavelength region
The advantage of performing analysis is that these semiconductor lasers can be used
In addition, as mentioned earlier, fluorescence from biological samples
It is difficult to be interfered by compounds. that's all
Based on the situation of
As a result of studying the synthesis of the extrafluorescent labeling agent, the compound of the present invention
Was developed. These compounds are 10 in amino acid analysis.
-12It was found that M level detection was possible.
(See Example 5)
I will explain. The raw material is m-anisidine derivative.
Introduce the side-chain functional groups required for belling. Side chain functionality
Bromocarboxylic acid ester as a raw material for introducing a group
The alkylating agent and m-anisidine derivative
The reaction is carried out and the N-monoalkyl-m-anisidine derivative is combined.
To achieve. Next, alkyl halide and alkyl dihalide
Is introduced into the amino group to give a tertiary amine. Alkyl
In the case of dihalide, it becomes a tertiary amine and at the same time the other
With the phenyl group of anisidine in the halogenated alkyl part of
Reacts to form a ring structure. Alkylcarboxylic acid on the amino group
The compound with the ester introduced will be
Deprotection of the methyl group of the group and hydrolysis of the ester,
Chi N-dialkyl-m-aminophenol and formal
Coupling with dehydration to form a methane-based derivative
It In this case, deprotection and ester hydrolysis reaction
The order of the pulling reactions may be reversed. Next, hot sulfur
Conversion to leukopyronine derivative by dehydration condensation in acid
It At this time, it is oxidized and a pyronin derivative is also produced, but
Not prepared, oxidized with ferric chloride-hydrochloric acid to give a pyronin derivative
It This pyronin derivative was nitrile with potassium cyanide.
Ferric chloride after being made into a leucocyanopyrronine compound
-Oxidize with hydrochloric acid to give a cyanopyrronine derivative. After this,
Carboxyl group is succinimide ester, iodoal
Killamide, maleimide alkyl ester, aminoal
Kilamide, amino group, thiol group, carboxyl
Make it a form that can react with the group. Shea obtained in this way
Excitation wavelength and fluorescence emission of nopyrronine derivatives depending on the substituents
The wavelength is different. Alkyl generally attached to amino groups
As the chain length becomes longer, it shifts to longer wavelengths and attaches to the benzene ring.
Increasing the number of added alkyl groups causes a shift to longer wavelength
In the direction. In addition, the fluorescence intensity is such that the alkyl chain has a cyclic structure.
Is stronger and the stability as a reagent is improved.
In the direction. Of the near-infrared fluorescent labeling agent developed in this study
Structural formulas of typical compounds and their absorption spectra and firefly
The optical spectrum data is shown. Each of these compounds
Obtained as a black-blue solid. Absorption spectrum is reagent concentration
1x10-5M (pH 7.0 20 mM HEPES buffer
Liquid), and the fluorescence spectrum shows a reagent concentration of 1 × 10-7M
(PH 7.0 20 mM HEPES buffer)
It was
The invention will be described in more detail by way of examples.
Example 1. Compound9Synthesis of
The synthetic scheme is shown in FIG.
1-1. CompoundTwoSynthesis of
61.6 g (0.500 mol) of m-anisidine was
Dissolved in 620 ml of trinitrile, 6-bromohexanoic acid
Ethyl 167 g (0.749 mol) and hydrogen carbonate
Add 84.1 g (1.00 mol) of thorium for 6 hours
Heated to reflux. After cooling, the reaction solution is filtered and the filtrate is concentrated under reduced pressure.
Dissolve the oil obtained in 800 ml of benzene and add water.
It was washed 3 times. After drying over magnesium sulfate, filtering and reducing the pressure
Concentration gave a crude product. The resulting crude product is silica gel color.
Separation (benzene: ethyl acetate = 4: 1)
CompoundTwoWas obtained. Yield 81% Light brown oil1
H-NMR (CDClThreeδppm) 1.25 (tJ =
7.2Hz3H) 1.30-1.50 (m2H) 1.5
5-1.75 (m4H) 2.30 (tJ = 7.3Hz2
H) 3.10 (tJ = 6.9 Hz 2H) 3.77 (s3
H) 4.13 (qJ = 7.2Hz2H) 6.15-6.
30 (m3H) 7.07 (tJ = 8.0Hz1H) MS
(FAB positive) M + 1 = 266
1-2. CompoundThreeSynthesis of
CompoundTwo20.0 g (75.4 mmol) of acetonite
Dissolve in 300 ml of ril, 12.0 ml of iodoethane
(0.150 mol) and sodium hydrogen carbonate 12.
7 g (0.151 mol) was added, and the mixture was heated under reflux overnight. Anti
The reaction solution is filtered and the filtrate is concentrated under reduced pressure.
It was dissolved in Zen and washed 3 times with water. With magnesium sulfate
After drying, it was filtered and concentrated under reduced pressure to obtain 21.8 g of a crude product. Income
99% light brown oil1H-NMR (CDClThreeδ
ppm) 1.13 (tJ = 7.1Hz 3H) 1.25
(TJ = 7.2Hz 3H) 1.30-1.40 (m2
H) 1.50-1.75 (m 4 H) 2.30 (tJ =
7.5 Hz 2 H) 3.23 (tJ = 7.6 Hz 2
H) 3.31 (qJ = 7.1 Hz 2H) 3.78 (s3
H) 4.12 (qJ = 7.2 Hz 2H) 6.20-6.
30 (m3H) 7.10 (tJ = 7.5Hz1H) MS
(FAB positive) M + 1 = 294
1-3. CompoundFourSynthesis of
CompoundThree16 g (54.5 mmol) of 160 ml of 4
Dissolve in 7% hydrobromic acid aqueous solution and add at 130 ° C for 24 hours.
I got hot. Pour the reaction solution into approximately 500 g of ice and add sodium hydroxide.
After adjusting the pH to around 4 with sodium and concentrating under reduced pressure, methanol
And the inorganic salts were removed. Oy obtained by vacuum concentration
Insoluble matter is suspended in ethanol and filtered to remove insoluble matter.
Remove and concentrate again under reduced pressure to obtain the target compoundFour14.1g was obtained
It was Yield 103% brown oil1H-NMR (CDC
lThree+ MeOH δppm) 1.19 (tJ = 7.1H
z 3H) 1.25-1.40 (m 2H) 1.40-
1.65 (m 4H) 2.28 (tJ = 7.3Hz 2
H) 3.23 (tJ = 7.6Hz 2H) 3.30-
3.60 (m 2H) 6.70-6.80 (m 3H)
7.30 (tJ = 8.3 Hz 1H) MS (FAB n
negative) M-1 = 250
1-4. Compound5Synthesis of
3.00 g of N, N-diethyl-m-aminophenol
(18.2 mmol) and compoundsFour4.60 g (1
8.3 mmol) in 15 ml of tetrahydrofuran (below
(Hereinafter abbreviated as THF): dissolved in a mixed solvent of water = 7: 3,
1.52 ml of 37% aqueous formaldehyde solution (18.7
mmol) was added and the mixture was heated under reflux for 6 hours. Reduce the reaction pressure
After concentration, 3% methanol / chloroform 100 ml
And was washed 3 times with water. Dried over magnesium sulfate
Filtered, and concentrated under reduced pressure.
Column (chloroform-5% methanol / chloroform
Target compound52.12 g was obtained. yield
27% purple viscous liquid1H-NMR (CDClThree+ M
eOH δppm) 1.09 (tJ = 7.6Hz 9
H) 1.25-1.40 (m2H) 1.40-1.6
5 (m 4H) 2.28 (tJ = 7.4Hz 2H)
3.10-3.40 (m 8H) 4.86 (s 2H)
6.20-6.30 (m4H) 6.90-7.00
(M2H) MS (FAB negative) M-1 = 4
27
1-5. Compound6Synthesis of
Compound52.00 g (4.67 mmol) of concentrated sulfuric acid 20
It was dissolved in ml and heated at 100 ° C. for 30 minutes. Sulfuric acid solution on ice
Pour to 100 g and pH with 4N aqueous sodium hydroxide
It was adjusted to around 4 and concentrated under reduced pressure. To 100 ml of methanol
Suspend and filter to extract only the components soluble in methanol
It was The crude product was applied to a silica gel column (5% methanol / chroma).
Target compound6Was obtained 1.26 g. Income
66% red-purple solid1H-NMR (MeOH δpp
m) 1.02-1.40 (m 9H) 1.45-1.7
0 (m 6H) 2.25 (tJ = 7.2Hz 2H)
3.40-3.70 (m 8H) 4.86 (s 2H)
6.76-7.00 (m4H) 7.80-7.95
(M2H) MS (FAB negative) M-1 =
409
1-6. Compound7Synthesis of
Compound6To 1.00 g (2.44 mmol), the second chloride
Iron 2.00 g (12.4 mmol) 20 ml 1M salt
A solution dissolved in an aqueous acid solution was added, and the mixture was stirred at room temperature overnight.
The reddish brown precipitate that formed was collected by filtration, washed with a small amount of cold water and dried.
Target compound7Was obtained. Yield 106% Red
Brown solid1H-NMR (MeOH δppm) 1.00
-1.30 (m 9H) 1.35-1.40 (m 2
H) 1.70-1.90 (m 4 H) 2.24 (tJ =
7.2Hz 2H) 3.75-4.00 (m 8H)
7.25-7.40 (m4H) 7.60 (s1H)
8.10-8.25 (m2H) MS (FAB posi
Tive) M + = 409
1-7. Compound8Synthesis of
Compound71.00 g (2.25 mmol) of ethanol
Dissolve in a mixed solvent of 12 ml and 6 ml of water, and add 180μ of acetic acid.
l (3.15 mmol) was added, and then potassium cyanide was added.
380 mg (5.84 mmol) was added for 2 hours at room temperature.
It was stirred. The reaction solution was concentrated under reduced pressure, and chloroform was added to it.
The mixture was filtered, the filtrate was concentrated again under reduced pressure, and the silica gel column was added.
Purify with (10% methanol / chloroform) and target compound
object8560 mg was obtained. Yield 57% Blue-black solid1
H-NMR (MeOH δppm) 1.10-1.3
5 (m 9H) 1.35-1.55 (m 2H) 1.6
5-1.80 (m 4H) 2.30 (tJ = 7.0Hz
2H) 3.45-3.75 (m 8H) 6.75-
6.95 (m5H) 7.60-7.80 (m2H) M
S (FAB negative) M-1 = 434
1-8. Compound9Synthesis of
Compound8480 mg (1.10 mmol), the second chloride
Iron 880 mg (5.43 mmol) was added with 1 M hydrochloric acid 8.8 m.
The solution dissolved in 1 was added, and the mixture was stirred at room temperature overnight. Black produced
The blue precipitate is collected by filtration, washed with a small amount of cold water and then dried,
Target compound9520 mg was obtained. Yield 101% Black Blue
Color solid1
H-NMR (MeOH δppm) 1.00-1.3
5 (m 9H) 1.40-1.55 (m 2H) 1.6
0-1.80 (m4H) 2.20-2.40 (m2
H) 3.70-4.00 (m8H) 7.40-7.6.
5 (m 4H) 8.15-8.30 (m 2H) MS
(FAB positive) M + 1 = 434
Example 2. Compound10Synthesis of
The synthetic scheme is shown in FIG. Compound9250 mg (0.
532 mmol) dissolved in 5 ml of dimethylformamide
185 mg of N-hydroxysuccinimide (1.6
1 mmol) and dicyclohexylcarbodiimide
Add 1.00 g (4.85 mmol) and stir at room temperature for 5 hours.
I stirred. The reaction product is filtered and the filtrate is concentrated under reduced pressure at 40 ° C or lower.
Then, ether was added and decantation was performed. Further got
The solid obtained was dissolved in water, filtered, and concentrated under reduced pressure below 40 ° C.
It was Ether is added to the concentrate to crystallize it and collect by filtration to obtain the desired compound.
object10235 mg was obtained. Yield 78% black blue solid1
1 H-NMR (MeOH δppm) 1.05-1.35
(M 9H) 1.40-1.55 (m 2H) 1.55
-1.70 (m4H) 2.25-2.40 (m2
H) 2.60 (s 4H) 3.60-4.00 (m 8
H) 7.35-7.60 (m4H) 8.10-8.2.
5 (m 2H) MS (FAB positive) M
+ = 531 compounds10Fig. 9 shows the absorption spectrum of
The optical spectrum is shown in FIG.
Example 3. Compound11Synthesis of
The synthetic scheme is shown in FIG. Compound9250 mg (0.
532 mmol) dissolved in 5 ml of dimethylformamide
, N-iodoacetylpiperidine 413 mg (1.6
1 mmol) and dicyclohexylcarbodiimide
(Hereinafter abbreviated as DCC) 1.0 g (4.85 mmol)
Was added and the mixture was stirred overnight at room temperature. The reaction product is filtered and the filtrate is depressurized.
Concentrate, add ethyl acetate to the residue and leave in the refrigerator overnight,
The white precipitate formed was removed by filtration and concentrated under reduced pressure. To concentrate residue
Ether was added and decanted, and the solid
The body was dissolved in water, filtered, and concentrated under reduced pressure. Concentrated residue is silica
Gel column chromatograph (3% methanol / chlorophore
The desired fractions were collected and concentrated under reduced pressure.
Ether was added to the concentrate to crystallize and collect by filtration to obtain the target compound.1
1210 mg was obtained. Yield 56% Black blue solid1H-N
MR (MeOH δppm) 1.10-1.15 (m
11H) 1.60-1.75 (m 4H) 2.32 (t
J = 7.2 Hz 2H) 3.20-3.90 (m 16)
H) 4.22 (s2H) 7.40-7.65 (m4)
H) 8.15-8.30 (m 2 H) MS (FAB p
aggressive) M + = 671
Example 4. Compound17Synthesis of
The synthetic scheme is shown in FIG. Shown in Examples 1 and 2
The procedure was the same as the synthetic method. Compound as raw materialFour4.60
g (18.3 mmol) and 8-hydroxyjuloli
Using 3.46 g (18.3 mmol) of gin,
Coupling with aldehyde12Yield 1.9
Obtained in 0 g (23% yield). To sulfuric acid using this compound
Dehydration condensation byThirteenYield 1.13g
Rate 62%). Then oxidize with ferric chloride / hydrochloric acid and
Ronin compound14To obtain 980 mg (yield 80%). Change
This compound was cyanated with potassium cyanide to give silica gel.
Target compound15560 mg (yield
60%) and then oxidized with ferric chloride / hydrochloric acid to give a yield of 580
Compound in mg (96% yield)16Got This compound 2
Active with N-hydroxysuccinimide using 50 mg
Ester compound17And Yield 280 mg (Yield 80
%). Black blue solid1H-NMR (MeOH δpp
m) 1.10-1.55 (m 5H) 1.60-1.7
5 (m 4H) 2.05-2.40 (m 6H) 2.6
6 (s 4H) 2.70-2.90 (m 4H) 3.1
0-3.40 (m6H) 7.50-7.70 (m3
H) 8.0-8.10 (m 1H) MS (FAB p
aggressive) M + = 555 compound17Absorption capacity
The spectrum is shown in Fig. 11 and the fluorescence spectrum is shown in Fig. 12.
Example 5. Compound18Synthesis method of
The compound shown in Example 4 whose synthetic scheme is shown in FIG.16
250 mg (0.460 mmol) of dried 5 ml
Dissolve in DMF and cool to 0 ° C. to obtain 104 mg (0.
(504 mmol) DCC was added, and 20 minutes later, N-
71 mg of hydroxyethyl maleimide (0.503 mm
ol) was dissolved in 0.5 ml of DMF and added. As it is
The mixture was stirred overnight at room temperature, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure.
After that, ether was added and decantation was performed. Further gain
The obtained solid was dissolved in water, filtered, and the filtrate was concentrated under reduced pressure.
Ether was added to the concentrate to crystallize and collect by filtration.
18210 mg was obtained. Yield 74% Black blue solid1H
-NMR (MeOH delta ppm) 1.10-1.55
(M 5H) 1.60-1.80 (m 4H) 2.10
-2.40 (m6H) 2.75-2.95 (m4
H) 3.05-3.30 (m4H) 3.30-3.6.
0 (m 4H) 4.07 (tJ = 6.8Hz 2H)
7.46 (s 2H) 7.55-7.70 (m 3H)
8.0-5.10 (m 1H) MS (FAB po
positive) M + = 581
Example 6. Compound26Synthesis of
The synthetic scheme is shown in FIG.
6-1. Compound19a,Compound19bSynthesis of
CompoundTwo10.0 g (37.7 mmol) of acetonite
Dissolve in 100 ml of Lil, sodium bicarbonate 12.7
g (0.151 mol) and 3-bromochloropropa
Solution (50 ml) was added and the mixture was heated under reflux for 10 days. White color
The precipitate is removed by filtration, the filtrate is concentrated under reduced pressure, and then benzene 2 is added.
It was dissolved in 00 ml and washed 3 times with water. Magnesium sulfate
After drying with a vacuum filter and concentrating under reduced pressure, a crude product was obtained. Obtained coarse
The product is a silica gel column (benzene: ethyl acetate = 5:
Two types of target compounds separated in 1)19a，19bSo
3.65 g (yield 32%) and 2.35 g (yield respectively)
16%) was obtained. All are light brown oil compounds19
a 11 H-NMR (MeOH δppm) 1.27 (t
J = 7.2 Hz 3H) 1.50-1.85 (m6H)
1.90-2.15 (m4H) 2.60-2.90
(M 2H) 3.00-3.30 (m 4H) 3.78
(S 3H) 4.13 (qJ = 7.2Hz 2H) 6.5
0-6.80 (m2H) 7.00-7.10 (m1
H) MS (FAB positive) M + = 306
Compound19b 1H-NMR (MeOH δppm)
1.25 (tJ = 7.2Hz 3H) 1.55-1.9
0 (m 6H) 1.95-2.25 (m 4H) 2.6
0-2.90 (m2H) 3.05-3.35 (m4
H) 3.89 (s 3H) 4.16 (qJ = 7.2Hz
2H) 6.80 (dJ = 7.5Hz 1H) 7.10
(M 1H) 7.36 (dJ = 2.0Hz 1H) M
S (FAB positive) M + = 306
6-2. Compound26Synthesis of
Compound as in Example 119a3.60 g (11.7
(mmol) treated with hydrobromic acid20To 3.28
g (yield 106%) was obtained. Then 3.00 g of this compound
(11.4 mmol) and 8-hydroxyjulolidine
2.15 g (11.4 mmol) and formaldehyde
Compound with21Yield 1.43g
The rate was 27%). Dehydration with sulfuric acid using this compound
Compounds that undergo condensation22Yield 1.20 g (yield 87
%). Then oxidize with ferric chloride / hydrochloric acid to give pyronin
Compound23Was obtained (1.05 g, yield 81%). More
Cyanide the above compound with potassium cyanide.
Target compound after purification with rum24640 mg (yield 62
%), And then oxidized with ferric chloride / hydrochloric acid to give a yield of 600 mg.
Compound with (yield 88%)25Got This compound 250
mg of N-hydroxysuccinimide
Tell compounds26And Yield 255 mg (yield 86
%). Black blue solid1H-NMR (MeOH δpp
m) 1.50-1.85 (m 6H) 2.00-2.3
5 (m 8H) 2.67 (s 4H) 2.75-2.9
5 (m 6H) 3.00-3.45 (m 8H) 7.5
1 (dJ = 6.2 Hz 1H) 7.68 (dJ = 6.2
Hz 1H) 8.12 (s1H) MS (FAB pos
itive) M + = 567
Example 7. Compound27Synthesis of
The synthetic scheme is shown in FIG. 7. Water hydrazine 0.26m
1 (3.33 mmol) was mixed with 2 ml of water and 4N hydrochloric acid was added.
After adjusting the pH to 6 with an aqueous solution, the compound synthesized in Example 6 was used.
26100 mg (0.165 mmol) of DMF 3 ml
The solution dissolved in is added little by little while stirring at room temperature.
It was After stirring overnight, concentrate the reaction mixture under reduced pressure and add 5 ml of water.
Adjust the pH to 10 with 4N aqueous sodium hydroxide and
Extracted 5 times with Loform. Dried over anhydrous magnesium sulfate
After that, concentrate under reduced pressure to obtain the target compound.2786 mg was obtained.
Yield 95% black blue solid1H-NMR (MeOH δ
ppm) 1.55-1.90 (m6H) 1.95-2.
25 (m8H) 2.80-3.05 (m6H) 3.1
5-3.80 (m 10H) 7.48 (dJ = 6.2H
z 1H) 7.55 (dJ = 6.2Hz 1H) 8.0
4 (s 1H) MS (FAB positive) M
+ = 512
Example 8
Compound37Synthesis of
The synthetic scheme is shown in FIG.
8-1. Compound29Synthesis of
2-Methoxy-6-methylaniline 25.0 g (0.1
82 mol) in 200 ml of acetonitrile,
45.6 g (0.273 mol) of ethyl lomoacetate and
30.6 g (0.364 mol) of sodium hydrogen carbonate
In addition, the mixture was heated under reflux for 6 hours. After cooling, the reaction solution is filtered and filtered.
The oil obtained by concentrating the liquid under reduced pressure is 300 ml of benzene.
And was washed 3 times with water. Dried over magnesium sulfate
After filtration and concentration under reduced pressure, a crude product was obtained. The crude product obtained is
Minutes on a Rica gel column (benzene: ethyl acetate = 4: 1)
Release, target compound2920.1 g was obtained. 63% yield
Light brown oil1
H-NMR (CDClThree δppm) 1.22 (tJ
= 7.6 Hz 3H) 2.18 (s 3H) 3.78
(DJ = 7.5 Hz 2H) 3.82 (s 3H) 4.
22 (qJ = 7.5Hz 2H) 6.00-6.25
(M 2H) 7.01 (dJ = 7.6Hz 1H) MS
(FAB positive) M + 1 = 224
8-2. Synthesis of compound 30
Compound2910.0 g (57.1 mmol) of acetoni
Dissolve in 100 ml of tolyl, sodium bicarbonate 9.6
0 g (0.114 mol) and 3-bromochloropro
Bread (50 ml) was added, and the mixture was heated under reflux for 7 days. White color
The precipitate is removed by filtration, the filtrate is concentrated under reduced pressure, and then benzene 2 is added.
It was dissolved in 00 ml and washed 3 times with water. Magnesium sulfate
After drying with a vacuum filter and concentrating under reduced pressure, a crude product was obtained. Obtained coarse
The product is a silica gel column (benzene: ethyl acetate = 5:
Target compound separated in 1)Thirty12.9 g was obtained. yield
86% light brown oil compoundThirty 1H-NMR (C
DClThree δppm) 1.25 (tJ = 7.8Hz 3
H) 1.95-2.05 (m 2 H) 2.19 (s 3
H) 2.70-2.90 (m2H) 3.35-3.5.
0 (m 2H) 3.96 (s 3H) 4.02 (s 2
H) 4.23 (qJ = 7.8Hz 2H) 6.23 (d
J = 7.6Hz 1H) 6.83 (dJ = 7.6Hz
1H) MS (FAB positive) M + 1 = 26
Four
8-3. Compound37Synthesis of
Hereinafter, the same frame work as in Examples 1 and 2 was performed. Example
Similar to 1Thirty12.9 g (49.0 mmo
l) is treated with hydrobromic acid to give a compound3110.6 g
Rate 98%). Next, 5.00 g (22.6 g) of this compound
mmol) and 8-hydroxyjulolidine 4.28 g
(22.6 mmol) with formaldehyde.
Compound322.39 g (yield 25%)
Got with. Using this compound at 100 ° C for 3 hours, 50%
Compound is obtained by dehydration condensation in sulfuric acid.33Yield 1.79g
(Yield 78%). Then oxidize with ferric chloride / hydrochloric acid
And pyronine compound341.73 g (yield 89%) was obtained
It was Furthermore, this compound is cyanated with potassium cyanide
Target compound after purification with silica gel column35To 1.07
g (yield 63%), then oxidized with ferric chloride / hydrochloric acid to obtain
Amount of compound 1.06 g (yield 92%)36Got Implementation
Using 250 mg of this compound as in Example 2, N-hydro
Active ester compound with xysuccinimide37And
Yield 242 mg (80% yield). Black blue solid1H-N
MR (MeOH δppm) 1.85-2.05 (m6
H) 2.61 (s 3H) 2.65 (s 4H) 2.8
0-3.00 (m 6H) 3.70-34.05 (m
6H) 4.32 (s 2H) 7.58 (s 1H) 7.
76 (s1H) 8.12 (s1H) MS (FAB
positive) M + = 525
Example 9
N, N, N'-triethyl-N- (5'-succinimine
Dooxycarbonylpentyl) -3,6-diamino-9
-Cyanoxanthene chloride (compound10)
Analysis of mino acids
4-1. Amino acid labeling method
Glycine, alanine, glutamine as amino acids to be analyzed
Acid was used. Amino acid water with a known concentration of 1 pM to 1 μM
A calibration curve was prepared using the solution. Aqueous solution containing amino acid
10 μl aliquot pH 7.020 mM HEPES buffer 20
μl was added and 1 mM N, N, N′-triethyl-N-
(5'-succinimidooxycarbonylpentyl)-
3,6-diamino-9-cyanoxanthene chloride water solution
Add 10 μl of the solution and incubate at 37 ° C for 1 hour
It was Add 50 μl of water and put in ice water. Chloroform 100 μm.
Extracted twice with 1. Aqueous phase frozen at -20 ° C until analyzed
saved.
4-2. Analytical method for labeled amino acids
Labeled amino acids can be analyzed by liquid chromatography (HPLC)
analyzed. The main body of the HPLC is Shimadzu LC4A
Reverse phase column on ram (GL Science Nucleos
il ODS: Column size 4.6 mm x 250 mm particles
(Diameter 5 μm) and flow rate of 1 ml / min.
Trill: 10 mM phosphoric acid aqueous solution = 1: 1
It was A simple fluorescence analyzer was used for the fluorescence measurement. To the light source
Is an NEC semiconductor laser NDL3200 (670n
m 3 mW) was used. The laser beam is used to
The fluorescence collected from the flow cell is focused on
After the light is dispersed with a lometer (JASCO CT-10), the light
Detected with an electron multiplier (Hamamatsu Photonics R943-02)
It was 10 μl of labeled sample solution for calibration curve
The relationship between the area of each peak and the amino acid concentration was injected into the HPLC.
I made a graph of the clerk. Figure 13 shows the calibration curve of glycine prepared.
Fig. 14 shows the calibration curve for alanine, and the calibration curve for glutamic acid.
The line is shown in FIG.
4-3. Analysis of amino acid mixed solution of unknown concentration
Large 1 μM solution of glycine, alanine and glutamic acid
Is prepared, and the three kinds are mixed in an appropriate amount and diluted about 1000 times.
It was 10 μl of this solution is taken and pH is 7.020 mM HEP
Add 20 μl of ES buffer and add 1 mM N, N, N′-tri
Ethyl-N- (5'-succinimidooxycarbonyl
Pentyl) -3,6 diamino-9-cyanoxanthene
Add 10 μl of aqueous solution of chloride and incubate at 37 ° C for 1 hour.
I got it. Add 50 μl of water, add to ice water and add chloroform.
Extracted twice with 100 μl. The water phase is -20 until analysis
It was stored frozen at ℃. A sample (10 μl) was taken from Example 4-
HPLC analysis was performed under the same conditions as in 2, and the 1000-fold dilution was performed.
The mino acid content was determined from the calibration curve. The result is shown in Fig. 16.
You
Amino acid amount Glycine 18.3 pM
Alanine 9.5 pM
Glutamic acid 50.1 pM
【図面の簡単な説明】
図１は化合物９の合成スキームである。
図２は化合物１０の合成スキームである。
図３は化合物１１の合成スキームである。
図４は化合物１７の合成スキームである。
図５は化合物１８の合成スキームである。
図６は化合物２６の合成スキームである。
図７は化合物２７の合成スキームである。
図８は化合物３７の合成スキームである。
図９は実施例１に記載の化合物９の吸収スペクトルで、
横軸に波長、縦軸にモル吸光係数をとってある。
図１０は化合物９の蛍光スペクトルで、横軸に波長、縦
軸に蛍光強度をとってある。
図１１は実施例４に記載の化合物１７の吸収スペクトル
で、横軸に波長、縦軸にモル吸光係数をとってある。
図１２は実施例４に記載の化合物１７の蛍光スペクトル
で、横軸に波長、縦軸に蛍光強度をとってある。
図１３、１４、１５はそれぞれ化合物９を用いてラベル
したグリシン、アラニン、グルタミン酸の検量線で、横
軸にアミノ酸の濃度の対数、縦軸にＨＰＬＣのピーク面
積の対数をとってある。
図１６は未知濃度アミノ酸のＨＰＬＣ分析結果である。
これからピコモルレベルの検出が可能であることがわか
る。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a synthetic scheme of compound 9 . FIG. 2 is a synthetic scheme of Compound 10 . FIG. 3 is a synthetic scheme of compound 11 . FIG. 4 is a synthetic scheme of compound 17 . FIG. 5 is a synthetic scheme of Compound 18 . FIG. 6 is a synthetic scheme of compound 26 . FIG. 7 is a synthetic scheme of compound 27 . FIG. 8 is a synthetic scheme of compound 37 . FIG. 9 is an absorption spectrum of the compound 9 described in Example 1,
The horizontal axis represents wavelength and the vertical axis represents molar absorption coefficient. FIG. 10 is a fluorescence spectrum of Compound 9 , in which the horizontal axis represents wavelength and the vertical axis represents fluorescence intensity. FIG. 11 is an absorption spectrum of the compound 17 described in Example 4, in which the horizontal axis shows the wavelength and the vertical axis shows the molar absorption coefficient. FIG. 12 is a fluorescence spectrum of the compound 17 described in Example 4, with the horizontal axis representing wavelength and the vertical axis representing fluorescence intensity. 13, 14, and 15 are calibration curves of glycine, alanine, and glutamic acid labeled with compound 9 , respectively, with the horizontal axis representing the logarithm of the amino acid concentration and the vertical axis representing the logarithm of the HPLC peak area. FIG. 16 shows the results of HPLC analysis of amino acids of unknown concentration.
This shows that it is possible to detect picomolar levels.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｇ０１Ｎ 33/58 Ａ 7055−2Ｊ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification number Office reference number FI technical display location G01N 33/58 A 7055-2J
Claims (1)
ルキル基、あるいはＲ１はＲ８と、Ｒ２はＲ３と、Ｒ９
はＲ６またはＲ７とアルキル鎖で結合した環状構造をと
ってもよい。Ｒ３、Ｒ４、Ｒ６、Ｒ７、Ｒ８は水素また
はメチル、エチル等の低級アルキル基である。ｎは１か
ら１０までの整数で、Ｙはカルボキシル基、ハロゲン、
アミノ基、マレイミド基、または、スクシンイミドエス
テルである。Ｘはハロゲンイオンである）で示されるシ
アノピロニン誘導体。 ２） 一般式 （式中、Ｒ１、Ｒ２、Ｒ９はメチル、エチル等の低級ア
ルキル基、あるいはＲ１はＲ８と、Ｒ２はＲ３と、Ｒ９
はＲ６またはＲ７とアルキル鎖で結合した環状構造をと
ってもよい。Ｒ３、Ｒ４、Ｒ６、Ｒ７、Ｒ８は水素また
はメチル、エチル等の低級アルキル基である。ｎは１か
ら１０までの整数で、Ｙはカルボキシル基、ハロゲン、
アミノ基、マレイミド基、または、スクシンイミドエス
テルである。Ｘはハロゲンイオンである）で示されるシ
アノピロニン誘導体を近赤外光励起が可能な新規蛍光ラ
ベル化剤。[Claims] 1) General formula (In the formula, R 1 , R 2 , and R 9 are lower alkyl groups such as methyl and ethyl, or R 1 is R 8 , R 2 is R 3 , and R 9 is
May have a cyclic structure in which R 6 or R 7 is bonded to an alkyl chain. R 3 , R 4 , R 6 , R 7 , and R 8 are hydrogen or a lower alkyl group such as methyl or ethyl. n is an integer from 1 to 10, Y is a carboxyl group, halogen,
It is an amino group, a maleimide group, or a succinimide ester. X is a halogen ion). 2) General formula (In the formula, R 1 , R 2 , and R 9 are lower alkyl groups such as methyl and ethyl, or R 1 is R 8 , R 2 is R 3 , and R 9 is
May have a cyclic structure in which R 6 or R 7 is bonded to an alkyl chain. R 3 , R 4 , R 6 , R 7 , and R 8 are hydrogen or a lower alkyl group such as methyl or ethyl. n is an integer from 1 to 10, Y is a carboxyl group, halogen,
It is an amino group, a maleimide group, or a succinimide ester. X is a halogen ion), which is a novel fluorescent labeling agent capable of exciting near-infrared light with a cyanopyrronine derivative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4054478A JP2593121B2 (en) | 1992-01-28 | 1992-01-28 | Fluorescent labeling agent excited by near-infrared light and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4054478A JP2593121B2 (en) | 1992-01-28 | 1992-01-28 | Fluorescent labeling agent excited by near-infrared light and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH06157504A true JPH06157504A (en) | 1994-06-03 |
| JP2593121B2 JP2593121B2 (en) | 1997-03-26 |
Family
ID=12971778
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4054478A Expired - Lifetime JP2593121B2 (en) | 1992-01-28 | 1992-01-28 | Fluorescent labeling agent excited by near-infrared light and its application |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2593121B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005532434A (en) * | 2002-07-01 | 2005-10-27 | グアヴァ テクノロジーズ，インコーポレーテッド | Fluorescent dyes, energy transfer pairs and methods |
| JP2012167153A (en) * | 2011-02-10 | 2012-09-06 | Fujifilm Corp | Curable colored composition, color filter and method for producing the same, liquid crystal display, solid-state image sensor, and pigment compound |
| CN115636761A (en) * | 2021-07-20 | 2023-01-24 | 中国石油天然气股份有限公司 | Oil-soluble surfactant, oil displacement agent and application thereof |
-
1992
- 1992-01-28 JP JP4054478A patent/JP2593121B2/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005532434A (en) * | 2002-07-01 | 2005-10-27 | グアヴァ テクノロジーズ，インコーポレーテッド | Fluorescent dyes, energy transfer pairs and methods |
| JP2012167153A (en) * | 2011-02-10 | 2012-09-06 | Fujifilm Corp | Curable colored composition, color filter and method for producing the same, liquid crystal display, solid-state image sensor, and pigment compound |
| CN115636761A (en) * | 2021-07-20 | 2023-01-24 | 中国石油天然气股份有限公司 | Oil-soluble surfactant, oil displacement agent and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2593121B2 (en) | 1997-03-26 |
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