JP2014012654A - Fluorescent compound consisting of tetraphenyl ethene derivative - Google Patents
Fluorescent compound consisting of tetraphenyl ethene derivative Download PDFInfo
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本発明は、新規な発蛍光性化合物に関し、特に、各種の生体物質または生体関連物質の存在下に蛍光を発し、それらの物質の検出や測定に用いることのできる発蛍光性化合物（蛍光プローブ）に関する。 The present invention relates to a novel fluorescent compound, and in particular, emits fluorescence in the presence of various biological substances or biological substances, and can be used for detection and measurement of these substances (fluorescent probe). About.
臨床検査や新薬の開発には各種の生体物質（生体関連物質）の分析が必須であり、高感度に微量分析が可能な手段として蛍光分析が用いられており、また、そのための発蛍光性化合物も案出されている。 Analysis of various biological substances (biologically related substances) is indispensable for the development of clinical tests and new drugs. Fluorescence analysis is used as a means capable of high-sensitivity trace analysis. Has also been devised.
例えば、シグナル伝達系における情報伝達の制御など生体内で重要な役割を果たすリン酸イオン（リン酸基）を分析するための発蛍光性化合物としては、ルテニウム−ビピリジルポリアザ化合物（P.D. Beer他、Angew. Chem.
Int. Ed., 40, 486 (2001)：非特許文献１）、亜鉛−ジピコリルアミン二核錯体（特許第４１３８３３１号公報：特許文献１）などが提案されている。しかし、リン酸（リン酸イオン）を選択的に認識する発蛍光性化合物は少なく、特に、特定のリン酸構造、例えば、重要な生体物質でＡＴＰに見られる三リン酸構造を認識して蛍光を発し得るような発蛍光性化合物の例は見られない。
For example, as a fluorescent compound for analyzing phosphate ions (phosphate groups) that play an important role in vivo such as control of signal transmission in signal transduction system, ruthenium-bipyridyl polyaza compound (PD Beer et al., Angew. Chem.
Int. Ed., 40 , 486 (2001): Non-patent document 1), zinc-dipicolylamine binuclear complex (Japanese Patent No. 4138331: Patent document 1) and the like have been proposed. However, there are few fluorescent compounds that selectively recognize phosphate (phosphate ion), and in particular, it recognizes a specific phosphate structure, for example, a triphosphate structure found in ATP in an important biological substance, and thus fluoresces. There is no example of a fluorescent compound capable of emitting.
また、糖尿病の診断マーカーとして知られているグルコースをはじめとする糖を検出・測定するための発蛍光性化合物としては、フェニルボロン酸構造を含有する各種の化合物が本発明者らによって案出されている（例えば、特許第２７９９８３７号公報：特許文献２、特許第２８８３８２４号公報：特許文献３、特許第２８８９４７６号公報：特許文献４など）。 In addition, various compounds containing a phenylboronic acid structure have been devised by the present inventors as fluorescent compounds for detecting and measuring sugars such as glucose, which are known as diagnostic markers for diabetes. (For example, Japanese Patent No. 2799837: Patent Document 2, Japanese Patent No. 2883824: Patent Document 3, Japanese Patent No. 2889476: Patent Document 4).
その他の重要な生体物質の例として、生体内の多くの酸化還元反応において補酵素として機能するＮＡＤ＋／ＮＡＤＨおよびＮＡＤＰ＋／ＮＡＤＰＨがある。これらの補酵素系は、酸化還元反応の生成物や酵素活性を調べるなどの目的に利用され、例えば、ＮＡＤＨの蛍光が測定されることがある（例えば、特開平２−２６５５００号公報：特許文献５）が、それらの補酵素のうちの特定のものを認識して発光する発蛍光性化合物は知られていない。 Examples of other important biological materials are NAD + / NADH and NADP + / NADPH that function as coenzymes in many redox reactions in vivo. These coenzyme systems are used for the purpose of examining oxidation-reduction reaction products and enzyme activities. For example, NADH fluorescence may be measured (for example, JP-A-2-265500: Patent Documents). However, there is no known fluorescent compound that recognizes a specific one of these coenzymes and emits light.
上に例示したような生体物質を検出、測定する従来の発蛍光性化合物の多くは、溶液中の被検出物質の濃度に応じて、その蛍光強度が漸次変化することに基くものであり、低濃度域では蛍光が無く、一方、高濃度になると凝集体を形成して発光しなくなる（消光する）ことがあることが知られている。 Many of the conventional fluorescent compounds that detect and measure biological substances as exemplified above are based on the fact that the fluorescence intensity gradually changes according to the concentration of the substance to be detected in the solution. It is known that there is no fluorescence in the concentration range, and on the other hand, when the concentration is high, aggregates are formed and light emission is stopped (quenching).
これに対して、最近、凝集にともなって蛍光が増大する凝集誘起発光（Aggregation-induced emission：AIE）という現象が見出されている（Y.
Hong. J. W. Y. Lam およびB.Z. Tang,
Chem. Soc. Rev., 2011, 40, 5361-5388：非特許文献２）（J. Wu, W. Liu, J. Ge, H. ZhangおよびP. Wang,
Chem. Soc. Rev., 2011, 40, 3483-3495：非特許文献３）。このＡＩＥ利用すれば、これまでにない新しい方式の蛍光分析ができるものと期待されているが、ＡＩＥを示す発蛍光性化合物の例はきわめて少ない。
In contrast, recently, a phenomenon called aggregation-induced emission (AIE) has been found in which fluorescence increases with aggregation (Y.
Hong. JWY Lam and BZ Tang,
Chem. Soc. Rev., 2011, 40, 5361-5388: Non-Patent Document 2) (J. Wu, W. Liu, J. Ge, H. Zhang and P. Wang,
Chem. Soc. Rev., 2011, 40, 3483-3495: Non-Patent Document 3). If this AIE is used, it is expected that a new type of fluorescence analysis will be possible, but there are very few examples of fluorescent compounds showing AIE.
本発明の目的は、各種の生体物質の検出、測定に適用されるように設計することができる基本構造を有しＡＩＥを発現する新規な発蛍光性化合物を提供することにある。 An object of the present invention is to provide a novel fluorescent compound having a basic structure that can be designed to be applied to detection and measurement of various biological substances and expressing AIE.
本発明者らは、鋭意検討を重ねた結果、アリールエテン誘導体のうち上記目的を達成し得るものがあることを見出し本発明を導き出した。
かくして、本発明は、下記の一般式（Ｉ）で表されることを特徴とする発蛍光性化合物を提供するものである。
As a result of intensive studies, the present inventors have found that some of the arylethene derivatives can achieve the above object, and have derived the present invention.
Thus, the present invention provides a fluorescent compound characterized by being represented by the following general formula (I).
式（Ｉ）中、ＸおよびＹは、同一または異なっており、それぞれ独立して、下記の式（Ａ）、（Ｂ）、（Ｃ）または（Ｄ）で示される原子団を表す。ｎおよびｍは１から６の整数を表し、(−ＣＨ２−)ｎおよび(−ＣＨ２−)ｍ中の１つまたは２つの−ＣＨ２−は、それぞれ独立して、−Ｏ−、−Ｓ−、−ＣＯ−、−ＣＯＯ−、−ＯＣＯ−または−ＣＨ＝ＣＨ−で置き換えられてもよい。 In the formula (I), X and Y are the same or different and each independently represent an atomic group represented by the following formula (A), (B), (C) or (D). n and m represents an integer from 1 to 6, (- CH 2 -) n and (-CH 2 -) one in m or two -CH 2 - are each independently, -O -, - It may be replaced by S-, -CO-, -COO-, -OCO- or -CH = CH-.
上記の式（Ｉ）で表されるように、本発明の発蛍光性化合物は、アリールエテン（テトラフェニルエテン）構造から成る発光部位、ＸおよびＹで表される認識部位（目的の生体物質を認識しそれらと結合し得る部位）、および発光部位と認識部位との間のスペーサー部位から構成されている。 As represented by the above formula (I), the fluorescent compound of the present invention comprises a light emitting site comprising an arylethene (tetraphenylethene) structure, a recognition site represented by X and Y (a target biological substance A site that can recognize and bind to them), and a spacer site between the light emitting site and the recognition site.
認識部位のうち、上記（Ａ）で表される原子団は、グアニジウム基であり、この原子団を含む本発明の発蛍光性化合物は、生体液または生理的塩類中でリン酸イオンまたはリン酸基を有する生体物質を認識して蛍光を発する。ＸまたはＹとして、この（Ａ）で表される原子団を有する発蛍光性化合物は、特に、三リン酸（トリフォスフェート）構造を認識してＡＴＰを高感度に検出、測定することができるという特性を有する。さらに、（Ａ）で表されるグアニジウム基を有する本発明の発蛍光性化合物は、補酵素系ＮＡＤ＋／ＮＡＤＨおよびＮＡＤＰ＋／ＮＡＤＰＨのうち、特にＮＡＤＰＨに対して特異的にＡＩＥを示す特性を有し、その検出、測定に用いることができる。そして、本発明の発蛍光性化合物を用いれば、測定系に適当な付加化合物（adduct）を発生させることにより、その特異的反応性を変化させ、その付加化合物の存在しない場合に検出できないような物質の検出も可能にする。 Among the recognition sites, the atomic group represented by (A) above is a guanidinium group, and the fluorescent compound of the present invention containing this atomic group is a phosphate ion or phosphate in a biological fluid or physiological salt. It recognizes a biological substance having a group and emits fluorescence. The fluorescent compound having the atomic group represented by (A) as X or Y can particularly detect and measure ATP with high sensitivity by recognizing the triphosphate structure. It has the characteristic. Further, the fluorescent compound of the present invention having a guanidinium group represented by (A) has a characteristic of showing AIE specifically for NADPH among the coenzyme systems NAD + / NADH and NADP + / NADPH. It can be used for detection and measurement. And, when the fluorescent compound of the present invention is used, the specific reactivity is changed by generating an appropriate adduct in the measurement system, which cannot be detected when the adduct is not present. It also enables detection of substances.
また、認識部位として上記（Ｂ）で表される原子団は、ビピリジン類縁基であり、この原子団を含む本発明の発蛍光性化合物も、リン酸イオンまたはリン酸基を有する生体物質を認識して蛍光を発することができる。 The atomic group represented by (B) as a recognition site is a bipyridine analog, and the fluorescent compound of the present invention containing this atomic group also recognizes a biological substance having a phosphate ion or a phosphate group. And can emit fluorescence.
認識部位として上記（Ｃ）で表される原子団は、ジピコリルアミン基である。この原子団を含む本発明の原子団は、その原子団を介して、適当な金属、特に亜鉛または銅との錯体の形態で用いられることにより、リン酸イオンまたはリン酸基を有する生体物質を認識して蛍光を発することができる。 The atomic group represented by the above (C) as a recognition site is a dipicolylamine group. The atomic group of the present invention containing this atomic group is used in the form of a complex with an appropriate metal, particularly zinc or copper, through the atomic group, thereby allowing a biological substance having a phosphate ion or a phosphate group. It can recognize and emit fluorescence.
認識部位として、上記（Ｄ）で表される原子団は、フェニルボロン酸含有原子団であり、生体液または生理的塩類中で、グルコースに代表される糖類を認識し糖類と結合するので、この原子団を有する本発明の発蛍光性化合物は生体液または生理的塩類中の糖類の検出、測定に用いることができる。 As a recognition site, the atomic group represented by (D) above is a phenylboronic acid-containing atomic group, and recognizes and binds to a saccharide represented by glucose in a biological fluid or physiological salt. The fluorescent compound of the present invention having an atomic group can be used for detection and measurement of saccharides in biological fluids or physiological salts.
上記の式（Ｉ）で表される本発明の発蛍光性化合物のうち、合成の容易さなどから、好ましいものとして下記の式（II）で表される化合物を挙げることができる。式（II）中、Ｘは、前記の式（Ａ）、（Ｂ）、（Ｃ）または（Ｄ）で示される原子団の一つを表す。 Among the fluorescent compounds of the present invention represented by the above formula (I), preferred are compounds represented by the following formula (II) because of ease of synthesis and the like. In formula (II), X represents one of the atomic groups represented by the formula (A), (B), (C) or (D).
上記の式（Ｉ）で表される本発明の発蛍光性化合物の別の好ましい例として、下記の式（III）で表される化合物を挙げることができる。式（III）中、Ｘは前記の式（Ａ）、（Ｂ）または（Ｃ）で示される原子団の１つを表し、Ｙは前記の式（Ａ）、（Ｂ）または（Ｃ）で示される原子団の１つであって、Ｘで表される原子団とは異なる原子団を表す。 Another preferred example of the fluorescent compound of the present invention represented by the above formula (I) is a compound represented by the following formula (III). In the formula (III), X represents one of the atomic groups represented by the formula (A), (B) or (C), and Y represents the formula (A), (B) or (C). One of the atomic groups shown, and represents an atomic group different from the atomic group represented by X.
上述した本発明の発蛍光性化合物を用いる生体物質の検出、測定においては、被検物質が一定以上の濃度になると凝集（Aggregation）に伴う急激な蛍光の発生が認められる。すなわち、蛍光発生に閾値が存在して、被検物質のOn／Off検出が可能となる。 In the detection and measurement of a biological substance using the above-described fluorescent compound of the present invention, when a test substance has a concentration of a certain level or more, rapid fluorescence accompanying aggregation is recognized. That is, there is a threshold for fluorescence generation, and it is possible to detect on / off of the test substance.
本発明の発蛍光性化合物は、既知の反応を工夫することにより合成することができる。
例えば、図１には、上記の式（II）に属する本発明の化合物の例として、テトラフェニルエテン構造の末端（Ｘ）にスペーサー部位を介して、前記（Ａ）で表されるグアニジウム基が結合した発蛍光性化合物（ＴＰＥ−Ｇ）の合成スキームが示されている。
The fluorescent compound of the present invention can be synthesized by devising a known reaction.
For example, in FIG. 1, as an example of the compound of the present invention belonging to the above formula (II), the guanidinium group represented by the above (A) is present at the terminal (X) of the tetraphenylethene structure via a spacer site. A synthetic scheme for the bound fluorescent compound (TPE-G) is shown.
図２には、式（II）に属する本発明の化合物の別の例として、テトラフェニル構造の末端（Ｘ）にスペーサー部位を介して、前記（Ｂ）で表されるビピリジン類縁基が結合した発蛍光性化合物（ＴＰＥ−bipy）の合成スキームが示されている。 In FIG. 2, as another example of the compound of the present invention belonging to the formula (II), the bipyridine analog represented by (B) is bonded to the terminal (X) of the tetraphenyl structure via a spacer site. A synthetic scheme for a fluorescent compound (TPE-bipy) is shown.
図３には、式（II）に属する本発明の化合物の別の例として、テトラフェニル構造の末端（Ｘ）にスペーサー部位を介して、前記（Ｃ）で表されるジピコリルアミン基が結合し、この基を介してＺｎ錯体を形成している発蛍光性化合物（ＴＰＥ−Ｚｎ）の合成スキームが示されている。 In FIG. 3, as another example of the compound of the present invention belonging to the formula (II), the dipicolylamine group represented by the above (C) is bonded to the terminal (X) of the tetraphenyl structure via a spacer site. A synthesis scheme of a fluorescent compound (TPE-Zn) forming a Zn complex via this group is shown.
図４には、式（II）に属する本発明の化合物の更に別の例として、テトラフェニルエテン構造の末端（Ｘ）にスペーサー部位を介して、前記（Ｄ）で表されるフェニルボロン酸含有原子団が結合した発蛍光性化合物（ＴＰＥ−Ｂ）の合成スキームが示されている。 FIG. 4 shows, as still another example of the compound of the present invention belonging to the formula (II), containing phenylboronic acid represented by the above (D) via a spacer site at the terminal (X) of the tetraphenylethene structure. A synthesis scheme of a fluorescent compound (TPE-B) to which atomic groups are bonded is shown.
さらに、図５には、上記の式（III）に属する本発明の化合物の例として、テトラフェニルエテン構造の末端（Ｘ）および（Ｙ）に、スペーサー部位を介して、それぞれ、前記（Ａ）で表されるグアニジウム基および（Ｃ）で表されるジピコリルアミン基が結合した発蛍光性化合物（hetero−ＴＰＥ）の合成スキームが示されている。
以下、本発明をさらに具体的に説明するために実施例を記すが、本発明はこれらの実施例によって限定されるものではない。
Further, FIG. 5 shows, as an example of the compound of the present invention belonging to the above formula (III), the above (A), respectively, via a spacer site at the terminals (X) and (Y) of the tetraphenylethene structure. A synthesis scheme of a fluorescent compound (hetero-TPE) in which a guanidinium group represented by (C) and a dipicolylamine group represented by (C) are bonded is shown.
EXAMPLES Hereinafter, examples will be described in order to more specifically describe the present invention, but the present invention is not limited to these examples.
ＴＰＥ−Ｇの合成
図１に示す合成スキームに従って、本発明の発蛍光性化合物ＴＰＥ−Ｇを合成した。
＜化合物２）の合成＞
４，４’−dimethoxybenzophenone１（５．００ｇ，２０．６ｍｍｏｌ）とそれに亜鉛粉末（６．６６ｇ，１０．７ｍｍｏｌ）を１００ｍｌのＴＨＦに加え、ＴｉＣｌ４（７．５０ｍＬ，６８．４ｍｍｏｌ）をゆっくりと滴下した。その後、１５時間加熱還流した。反応溶液が室温まで冷却した後、１００ｍｌの水を加えた。溶液をクロロホルム１００ｍｌで三回抽出し、それぞれの抽出液を纏めて無水硫酸マグネシウムで乾燥させ、ろ液を減圧濃縮した。粗製物はシリカゲルカラムクロマトグラフで分離精製し、分離物をクロロホルム：ヘキサン＝２：１の溶媒から再結晶した。白色結晶として化合物２を３．４３グラム得た。収率７４％。
1H NMR（300MHz, DMSO-d6）δ6.85（d, J=8.7Hz, 8H）,6.69（d, J=8.7Hz, 8H）,3.68（s, 12H）
MS（ESI）m/z 452.2(M+).
Synthesis of TPE-G The fluorescent compound TPE-G of the present invention was synthesized according to the synthesis scheme shown in FIG.
<Synthesis of Compound 2)
4,4′-dimethoxybenzophenone 1 (5.00 g, 20.6 mmol) and zinc powder (6.66 g, 10.7 mmol) were added to 100 ml of THF, and TiCl 4 (7.50 mL, 68.4 mmol) was slowly added dropwise. did. Thereafter, the mixture was heated to reflux for 15 hours. After the reaction solution cooled to room temperature, 100 ml of water was added. The solution was extracted three times with 100 ml of chloroform, and the extracts were combined and dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography, and the separated product was recrystallized from a solvent of chloroform: hexane = 2: 1. 3.43 g of compound 2 was obtained as white crystals. Yield 74%.
1 H NMR (300 MHz, DMSO-d 6 ) δ 6.85 (d, J = 8.7 Hz, 8H), 6.69 (d, J = 8.7 Hz, 8H), 3.68 (s, 12H)
MS (ESI) m / z 452.2 (M + ).
＜化合物３）の合成＞
化合物２（６．０１ｇ，１３．３ｍｍｏｌ）を１００ｍｌのクロロホルムに溶解し、寒剤を入れた氷浴で冷却しながら、２ＭＢＢｒ３の塩化メチレン溶液（３７．２ｍＬ，７４．４ｍｍｏｌ）をゆっくりと滴下した。氷浴を外し、室温で１２時間撹拌した後、水５０ｍｌを加えた。生じた沈殿物をろ取し、アセトン：水＝１：１で再結晶した。白色結晶として化合物３を５．２６グラム得た。収率９２％。
1H NMR（300MHz, DMSO-d6）δ9.24（s, 4H）,6.70（d, J=8.6Hz, 8H）,6.48（d, J=8.6Hz, 8H）
<Synthesis of Compound 3)
Compound 2 (6.01 g, 13.3 mmol) was dissolved in 100 ml of chloroform, and 2 MBBr 3 in methylene chloride (37.2 mL, 74.4 mmol) was slowly added dropwise while cooling in an ice bath containing a cryogen. . After removing the ice bath and stirring at room temperature for 12 hours, 50 ml of water was added. The resulting precipitate was collected by filtration and recrystallized with acetone: water = 1: 1. 5.26 grams of compound 3 was obtained as white crystals. Yield 92%.
1 H NMR (300 MHz, DMSO-d 6 ) δ 9.24 (s, 4H), 6.70 (d, J = 8.6Hz, 8H), 6.48 (d, J = 8.6Hz, 8H)
＜化合物５）の合成＞
３−bromopropylamine hydrobromide４（５．００ｇ，２２．８ｍｍｏｌ）を乾燥した１００ｍｌのジクロロメタンに溶解し、それにＢｏｃ２Ｏ（５．４８ｇ，２５．１ｍｍｏｌ）とtriethylamine（３．５ｍＬ，２５．１ｍｍｏｌ）を加えた。反応混合物は室温で１４時間撹拌した。その後、１００ｍｌのジクロロメタンを加え、１Ｍ塩酸水溶液、水、食塩水の順に洗浄した。有機相は無水硫酸マグネシウムで乾燥し、ろ液を減圧濃縮した。粗製物はシリカゲルカラムクロマトグラフで精製し、無色オイル状物として化合物５を５．１７グラム得た。収率９５％。
1H NMR（CDCl3, 300MHz）δ4.65（br s, 1H）,3.45（t, J=6.5Hz, 2H）,3.28（q, J=6.5Hz, 2H）,2.05（quint, J=6.5Hz, 2H）,1.45（s, 9H）.
<Synthesis of Compound 5)
3-bromopropylamine hydrobromide 4 (5.00 g, 22.8 mmol) was dissolved in 100 ml of dry dichloromethane, and Boc 2 O (5.48 g, 25.1 mmol) and triethylamine (3.5 mL, 25.1 mmol) were added thereto. . The reaction mixture was stirred at room temperature for 14 hours. Thereafter, 100 ml of dichloromethane was added, and the mixture was washed with 1M hydrochloric acid aqueous solution, water and brine in this order. The organic phase was dried over anhydrous magnesium sulfate and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 5.17 grams of compound 5 as a colorless oil. Yield 95%.
1 H NMR (CDCl 3 , 300 MHz) δ 4.65 (br s, 1H), 3.45 (t, J = 6.5Hz, 2H), 3.28 (q, J = 6.5Hz, 2H), 2.05 (quint, J = 6.5 Hz, 2H), 1.45 (s, 9H).
＜化合物６）の合成＞
化合物３（１．００ｇ，２．３３ｍｍｏｌ）、炭酸カリウム（２．６０ｇ，１８．８ｍｍｏｌ）および化合物５（２．５ｇ，１０．５ｍｍｏｌ）を２０ｍｌの乾燥ＤＭＦに加え、７０℃で１０時間加熱した。室温まで反応液の温度を戻した後、水５０ｍｌを加えた。生じた沈殿物をろ取し水で洗浄した。得られた固体を１００ｍｌのジクロロメタンに溶解し、無水硫酸マグネシウムで乾燥させ、ろ液を減圧濃縮した。粗製物はシリカゲルカラムクロマトグラフで精製し、白色固体として化合物６を１．３４グラム得た。収率５６％。
1H NMR（300MHz, CDCl3）δ6.91（d, J=8.8Hz, 8H）,6.62（d, J=8.8Hz, 8H）,4.78（br s, 4H）,3.95（t, J=6.1Hz, 8H）,3.30（q, J=6.1Hz, 8H）,1.94（quint, J=6.1Hz,
8H）,1.44（s, 36H）;
<Synthesis of Compound 6)
Compound 3 (1.00 g, 2.33 mmol), potassium carbonate (2.60 g, 18.8 mmol) and compound 5 (2.5 g, 10.5 mmol) were added to 20 ml of dry DMF and heated at 70 ° C. for 10 hours. . After returning the temperature of the reaction solution to room temperature, 50 ml of water was added. The resulting precipitate was collected by filtration and washed with water. The obtained solid was dissolved in 100 ml of dichloromethane, dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1.34 g of compound 6 as a white solid. Yield 56%.
1 H NMR (300 MHz, CDCl 3 ) δ6.91 (d, J = 8.8 Hz, 8H), 6.62 (d, J = 8.8 Hz, 8H), 4.78 (br s, 4H), 3.95 (t, J = 6.1 Hz, 8H), 3.30 (q, J = 6.1Hz, 8H), 1.94 (quint, J = 6.1Hz,
8H), 1.44 (s, 36H);
＜化合物７）の合成＞
化合物６（８２５ｍｇ，０．８０５ｍｍｏｌ）を５ｍｌのジクロロメタンに溶解し、ＴＦＡ（５ｍＬ，６５．３ｍｍｏｌ）を加え、室温で１４時間撹拌した。その後、反応液をエーテルに空け、生じた白色沈殿をろ取し、エーテルで洗浄後乾燥した。白色固体として化合物７を６６０ｍｇ得た。収率７９％。
1H NMR（300MHz, DMSO-d6）δ7.98（s, 12H）,6.85（d, J=8.8 Hz, 8H）,6.70（d, J=8.8Hz, 8H）,3.97（t, J=5.8Hz, 8H）,2.95（t, J=7.4Hz, 8H）,1.97（quint, J=6.6Hz,
8H）
<Synthesis of Compound 7)
Compound 6 (825 mg, 0.805 mmol) was dissolved in 5 ml of dichloromethane, TFA (5 mL, 65.3 mmol) was added, and the mixture was stirred at room temperature for 14 hours. Thereafter, the reaction solution was poured into ether, and the resulting white precipitate was collected by filtration, washed with ether and dried. 660 mg of compound 7 was obtained as a white solid. Yield 79%.
1 H NMR (300 MHz, DMSO-d 6 ) δ 7.98 (s, 12H), 6.85 (d, J = 8.8 Hz, 8H), 6.70 (d, J = 8.8 Hz, 8H), 3.97 (t, J = 5.8Hz, 8H), 2.95 (t, J = 7.4Hz, 8H), 1.97 (quint, J = 6.6Hz,
8H)
＜化合物８）の合成＞
化合物７（６００ｍｇ，０．５６ｍｍｏｌ）を１５ｍｌのジクロロメタンに懸濁し、Ｅｔ３Ｎ（０．６ｍＬ，４．３３ｍｍｏｌ）と１−Ｈ−pyrazole−１−（Ｎ，Ｎ’−bis（tert−butyloxycarbonyl））carboxamidine（１．２１ｇ，３．８９ｍｍｏｌ）を加えた。反応混合溶液を室温で７２時間撹拌し、溶媒を減圧溜去した。粗製物をシリカゲルカラムクロマトグラフで精製し、白色固体として化合物８を５２３ｍｇ得た。収率５９％。
1H NMR（300MHz, CDCl3）δ11.5（s, 4H）,8.66（t, J=4.7Hz, 4H）,6.91（d, J=8.6Hz, 8H）,6.69（d, J=8.6Hz, 8H）,3.98（t, J=5.3Hz, 8H）,3.62（q, J=5.8Hz, 8H）,2.03（quint, J=5.8Hz, 8H）,1.50（s, 36H）,1.49（s, 36H）;
<Synthesis of Compound 8)
Compound 7 (600 mg, 0.56 mmol) was suspended in 15 ml of dichloromethane, Et 3 N (0.6 mL, 4.33 mmol) and 1-H-pyrazole-1- (N, N′-bis (tert-butyloxycarbonyl) ) Carboxamidine (1.21 g, 3.89 mmol) was added. The reaction mixture solution was stirred at room temperature for 72 hours, and the solvent was distilled off under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 523 mg of Compound 8 as a white solid. Yield 59%.
1 H NMR (300 MHz, CDCl 3 ) δ 11.5 (s, 4H), 8.66 (t, J = 4.7 Hz, 4H), 6.91 (d, J = 8.6 Hz, 8H), 6.69 (d, J = 8.6 Hz , 8H), 3.98 (t, J = 5.3Hz, 8H), 3.62 (q, J = 5.8Hz, 8H), 2.03 (quint, J = 5.8Hz, 8H), 1.50 (s, 36H), 1.49 (s , 36H);
＜ＴＰＥ−Ｇの合成＞
化合物８（４９２ｍｇ，０．３１ｍｍｏｌ）を５ｍｌのジクロロメタンに溶解し、ＴＦＡ（５ｍＬ，６５．３ｍｍｏｌ）を加え、室温で１３時間撹拌した。反応混合溶液をエーテルに空け、生じた沈殿物をろ取し、エーテル洗浄した後、乾燥した。粗製物はＴＦＡのジクロロメタン溶液に溶解し、エーテルに空け、生じた沈殿物をろ取し乾燥した。白色固体としてＴＰＥを１５３ｍｇ得た。収率４０％。
1H NMR（300MHz, CDCl3）δ7.87（t, J=5.4 Hz, 4H）,7.29（br-s, 16H）,6.85（d, J=8.7Hz, 8H）,6.69（d, J=8.7Hz, 8H）,3.93（t, J=5.9Hz, 8H）,3.24（q, J=6.4Hz, 8H）,1.89（quint, J=6.4Hz, 8H）;
MS（ESI）m/z 1135.2（[M-CF3COO-]+）
Calc. for C50H60F12N12O12（TPE）:C, 48.08; H, 4.84; N, 13.46. Found: C, 47.78; H, 4.86; N, 13.22.
<Synthesis of TPE-G>
Compound 8 (492 mg, 0.31 mmol) was dissolved in 5 ml of dichloromethane, TFA (5 mL, 65.3 mmol) was added, and the mixture was stirred at room temperature for 13 hours. The reaction mixture was poured into ether, and the resulting precipitate was collected by filtration, washed with ether, and dried. The crude product was dissolved in a solution of TFA in dichloromethane, poured into ether, and the resulting precipitate was collected by filtration and dried. 153 mg of TPE was obtained as a white solid. Yield 40%.
1 H NMR (300 MHz, CDCl 3 ) δ 7.87 (t, J = 5.4 Hz, 4H), 7.29 (br-s, 16H), 6.85 (d, J = 8.7 Hz, 8H), 6.69 (d, J = 8.7Hz, 8H), 3.93 (t, J = 5.9Hz, 8H), 3.24 (q, J = 6.4Hz, 8H), 1.89 (quint, J = 6.4Hz, 8H);
MS (ESI) m / z 1135.2 ([M-CF 3 COO -] +)
Calc. For C 50 H 60 F 12 N 12 O 12 (TPE): C, 48.08; H, 4.84; N, 13.46. Found: C, 47.78; H, 4.86; N, 13.22.
ＴＰＥ−bipyの合成
図２に示す合成スキームに従って、本発明の発蛍光性化合物ＴＰＥ−bipyを合成した。
＜化合物１１）の合成＞
２−クロロ−５−メトキシカルボニルピリジン（１．３ｇ、７．６ｍｍｏｌ）をｍ−キシレン２０ｍｌに溶解し、ＰｄＣｌ２（ＰＰｈ３）２（０．２７ｇ、０．０４ｍｍｏｌ）と２−トリメチルスズピリジン（２．１ｇ、８．６ｍｍｏｌ）を加え、２０時間加熱還流した。放冷後エーテルで希釈し、中性アルミナを通してろ過した。ろ液を数回水洗し、無水硫酸ナトリウムで乾燥した。溶媒を留去後シリカゲルカラムクロマトグラフで精製し、白色固体として１．１３ｇの化合物１０を得た。収率７０％。
次いで、得られた化合物１０（１．１３ｇ、５．３ｍｍｏｌ）をエタノール／水１：１ ２０ｍｌに懸濁し、ＫＯＨ（０．３５ｇ、６．３ｍｍｏｌ）を水２ｍｌに溶解したものを添加し、室温で３時間撹拌した。その後、濃縮してエタノールを留去し、１ｍｏｌ／Ｌ塩酸を中性になるまで添加した。得られた懸濁液をクロロホルムで三回抽出し、抽出液を水で洗浄後、無水硫酸ナトリウムで乾燥、溶媒を留去した。粗製物をシリカゲルカラムクロマトグラフで精製し、淡黄色固体として化合物１１を０．８ｇ得た。収率７５％。
Synthesis of TPE-bipy The fluorescent compound TPE-bipy of the present invention was synthesized according to the synthesis scheme shown in FIG.
<Synthesis of Compound 11)
2-Chloro-5-methoxycarbonylpyridine (1.3 g, 7.6 mmol) was dissolved in 20 ml of m-xylene, and PdCl2 (PPh3) 2 (0.27 g, 0.04 mmol) and 2-trimethyltinpyridine (2. 1 g, 8.6 mmol) was added and the mixture was heated to reflux for 20 hours. After standing to cool, it was diluted with ether and filtered through neutral alumina. The filtrate was washed with water several times and dried over anhydrous sodium sulfate. After the solvent was distilled off, the residue was purified by silica gel column chromatography to obtain 1.13 g of compound 10 as a white solid. Yield 70%.
Subsequently, the obtained compound 10 (1.13 g, 5.3 mmol) was suspended in 20 ml of ethanol / water 1: 1, and KOH (0.35 g, 6.3 mmol) dissolved in 2 ml of water was added. For 3 hours. Then, it concentrated, ethanol was distilled off, and 1 mol / L hydrochloric acid was added until it became neutral. The obtained suspension was extracted three times with chloroform, and the extract was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off. The crude product was purified by silica gel column chromatography to obtain 0.8 g of Compound 11 as a pale yellow solid. Yield 75%.
＜ＴＰＥ−Bipyの合成＞
化合物７（６００ｍｇ、０．５６ｍｍｏｌ）を塩化メチレン（１５ｍｌ）に懸濁し、化合物１１（０．５ｇ、２．５ｍｍｏｌ）、トリエチルアミン（０．６ｍｌ、４．３３ｍｍｏｌ）、ＤＭＡＰ（０．１ｍｌ）を加えた。溶液を氷冷下、ＷＳＣ（１．７ｇ、８．９ｍｍｏｌ）を加え２４時間撹拌した。溶媒を濃縮乾固し、粗製物をシリカゲルカラムクロマトグラフで精製し、ＴＰＥ−Bipy０．５ｇ（収率３６％）を得た。
1H NMR（300MHz, CDCl3）δ9.22（s, 4H）,8.54-8.88（m, 8H）,8.15-8.40（m, 8H）,7.67-7.84（m, 4H）,7.15-7.33（m, 4H）,6.90（d, J=8.7Hz, 8H）,6.72（d, J=8.7Hz, 8H）,3.65（t, J=6.4Hz, 8H）,3.06（q, J=6.4Hz, 8H）,1.77（quint, J=6.4Hz, 8H）;
MS（ESI）m/z 1353.4.(M+1)
Calc. for C82H72N12O4（TPE-bipy）:C, 72.76; H, 5.36; N, 12.41. Found: C, 72.55; H, 5.48; N, 12.20.
<Synthesis of TPE-Bipy>
Compound 7 (600 mg, 0.56 mmol) was suspended in methylene chloride (15 ml), and compound 11 (0.5 g, 2.5 mmol), triethylamine (0.6 ml, 4.33 mmol) and DMAP (0.1 ml) were added. It was. Under ice cooling, WSC (1.7 g, 8.9 mmol) was added to the solution and stirred for 24 hours. The solvent was concentrated to dryness, and the crude product was purified by silica gel column chromatography to obtain 0.5 g of TPE-Bipy (yield 36%).
1 H NMR (300 MHz, CDCl 3 ) δ 9.22 (s, 4H), 8.54-8.88 (m, 8H), 8.15-8.40 (m, 8H), 7.67-7.84 (m, 4H), 7.15-7.33 (m , 4H), 6.90 (d, J = 8.7Hz, 8H), 6.72 (d, J = 8.7Hz, 8H), 3.65 (t, J = 6.4Hz, 8H), 3.06 (q, J = 6.4Hz, 8H ), 1.77 (quint, J = 6.4Hz, 8H);
MS (ESI) m / z 1353.4. (M + 1)
Calc. For C 82 H 72 N 12 O 4 (TPE-bipy): C, 72.76; H, 5.36; N, 12.41. Found: C, 72.55; H, 5.48; N, 12.20.
ＴＰＥ−Ｚｎの合成
図３に示す合成スキームに従って、本発明の発蛍光性化合物ＴＰＥ−Ｚｎを合成した。
＜化合物１２）の合成＞
化合物７（１．０５ｇ、１．６８ｍｍｏｌ）をアセトニトリル（１００ｍｌ）に懸濁し、２−クロロメチルピリジン塩酸塩（２．２１ｇ、１３．５ｍｍｏｌ）を加えた。炭酸カリウム（４．６５ｇ、３３．６ｍｍｏｌ）とヨウ化カリウム（２．２３ｇ、１３．４ｍｍｏｌ）を加え、２２時間加熱還流した。放冷し沈殿物をろ別後、溶液を濃縮乾固し、粗製物をシリカゲルカラムクロマトグラフで精製し、淡黄色固体として化合物１２を１．３３ｇを得た。収率５８％。
1H NMR
(300 MHz, DMSO-d6) δ8.43(m, 8H), 7.59(m, 8H), 7.43(m, 8H), 7.16(m, 8H), 6.86(d, J=8.8
Hz, 8H), 6.59(d, J=8.8 Hz, 8H), 3.87(t, J=6.2 Hz, 8H), 3.72(s, 16H), 2.55(t,
J=6.2 Hz, 8H), 1.85(quint, J=6.2 Hz, 8H);
13C
NMR(75 MHz, DMSO-d6) δ159.5, 156.9, 148.9, 138.3, 136.6, 136.4, 132.1, 122.7, 122.2,
113.8, 65.1, 59.9, 50.0, 26.7.
Synthesis of TPE-Zn The fluorescent compound TPE-Zn of the present invention was synthesized according to the synthesis scheme shown in FIG.
<Synthesis of Compound 12)
Compound 7 (1.05 g, 1.68 mmol) was suspended in acetonitrile (100 ml) and 2-chloromethylpyridine hydrochloride (2.21 g, 13.5 mmol) was added. Potassium carbonate (4.65 g, 33.6 mmol) and potassium iodide (2.23 g, 13.4 mmol) were added, and the mixture was heated to reflux for 22 hours. The mixture was allowed to cool and the precipitate was filtered off. The solution was concentrated to dryness, and the crude product was purified by silica gel column chromatography to obtain 1.33 g of Compound 12 as a pale yellow solid. Yield 58%.
1 H NMR
(300 MHz, DMSO-d 6 ) δ8.43 (m, 8H), 7.59 (m, 8H), 7.43 (m, 8H), 7.16 (m, 8H), 6.86 (d, J = 8.8
Hz, 8H), 6.59 (d, J = 8.8 Hz, 8H), 3.87 (t, J = 6.2 Hz, 8H), 3.72 (s, 16H), 2.55 (t,
J = 6.2 Hz, 8H), 1.85 (quint, J = 6.2 Hz, 8H);
13 C
NMR (75 MHz, DMSO-d 6 ) δ159.5, 156.9, 148.9, 138.3, 136.6, 136.4, 132.1, 122.7, 122.2,
113.8, 65.1, 59.9, 50.0, 26.7.
＜ＴＰＥ−Ｚｎの合成＞
化合物１２（５０ｍｇ、０．０３７０ｍｍｏｌ）をメタノール５ｍｌに溶解し、硝酸亜鉛Ｚｎ(ＮＯ３)２−６Ｈ２Ｏ（４４ｍｇ、０．１４８ｍｍｏｌ）を水（１ｍｌ）に溶解した溶液を添加した。得られた溶液を室温で３０分撹拌後、溶媒を蒸発させて乾燥し、次いで水（３ｍｌ）を添加した。得られた水溶液をろ過し、凍結乾燥して、淡黄色固体としてＴＰＥ−Ｚｎを得た（収率：定量）。
元素分析C86H88N20O28Zn4（TPE-Zn）：計算値：C, 48.92; H, 4.20; N, 13.27。実測値：C, 49.03; H, 4.11; N, 13.00。
<Synthesis of TPE-Zn>
Compound 12 (50 mg, 0.0370 mmol) was dissolved in 5 ml of methanol, and a solution of zinc nitrate Zn (NO 3 ) 2 -6H 2 O (44 mg, 0.148 mmol) in water (1 ml) was added. The resulting solution was stirred at room temperature for 30 minutes, then the solvent was evaporated to dryness and then water (3 ml) was added. The obtained aqueous solution was filtered and freeze-dried to obtain TPE-Zn as a pale yellow solid (yield: quantitative).
Elemental analysis C 86 H 88 N 20 O 28 Zn 4 (TPE-Zn): Calculated: C, 48.92; H, 4.20 ; N, 13.27. Found: C, 49.03; H, 4.11; N, 13.00.
ＴＰＥ−Ｂの合成
図４に示す合成スキームに従って、本発明の発蛍光性化合物ＴＰＥ−Ｂを合成した。
＜化合物１３）の合成＞
化合物７（６００ｍｇ、０．５６ｍｍｏｌ）を塩化メチレン（１５ｍｌ）に懸濁し、トリエチルアミン（０．６ｍｌ、４．３３ｍｍｏｌ）、ＤＭＡＰ（０．１ｍｌ）を加えた。塩化メチレン（３ｍｌ）に溶解した３−クロロカルボニルピリジン塩酸塩（０．５ｇ、２．８ｍｍｏｌ）を５℃以下で滴下した。その後室温で３時間撹拌した。溶媒を濃縮乾固し、得られた粗製物をシリカゲルカラムクロマトグラフで精製し、白色の化合物１３を０．３７ｇ得た。収率６３％。
1H NMR（300MHz, CDCl3）δ9.05（s, 4H）,8.10-8.85（m, 12H）,6.86（d, J=8.7Hz, 8H）,6.73（d, J=8.7Hz, 8H）,3.70（t, J=5.9Hz, 8H）,3.11（q, J=6.4Hz, 8H）,1.82（quint, J=6.4Hz, 8H）;
MS (ESI) m/z 1045.2(M+1)
Calc. for C82H72N12O4
(13): C, 71.25; H, 5.79; N, 10.72. Found: C, 71.04; H, 5.99; N, 10.54.
Synthesis of TPE-B The fluorescent compound TPE-B of the present invention was synthesized according to the synthesis scheme shown in FIG.
<Synthesis of Compound 13)
Compound 7 (600 mg, 0.56 mmol) was suspended in methylene chloride (15 ml), and triethylamine (0.6 ml, 4.33 mmol) and DMAP (0.1 ml) were added. 3-Chlorocarbonylpyridine hydrochloride (0.5 g, 2.8 mmol) dissolved in methylene chloride (3 ml) was added dropwise at 5 ° C. or lower. Thereafter, the mixture was stirred at room temperature for 3 hours. The solvent was concentrated to dryness, and the resulting crude product was purified by silica gel column chromatography to obtain 0.37 g of white compound 13. Yield 63%.
1 H NMR (300 MHz, CDCl 3 ) δ 9.05 (s, 4H), 8.10-8.85 (m, 12H), 6.86 (d, J = 8.7Hz, 8H), 6.73 (d, J = 8.7Hz, 8H) , 3.70 (t, J = 5.9Hz, 8H), 3.11 (q, J = 6.4Hz, 8H), 1.82 (quint, J = 6.4Hz, 8H);
MS (ESI) m / z 1045.2 (M + 1)
Calc. For C 82 H 72 N 12 O 4
(13): C, 71.25; H, 5.79; N, 10.72. Found: C, 71.04; H, 5.99; N, 10.54.
化合物１３（０．３７ｇ、０．３５ｍｍｏｌ）をアセトニトリル（２０ｍｌ）に懸濁し、２−ブロモメチルフェニルボロン酸プロピレンエステル（０．４５ｇ、１．８ｍｍｏｌ）を加え、５時間加熱還流する。その後溶媒を濃縮乾固し、得られた粗製物シリカゲルカラムクロマトグラフで精製し、ＴＰＥ−Ｂ０．３２ｇ（収率４４％）を得た。
1H NMR（300MHz, CD3OD）δ9.44（s, 4H）,9.05-9.22（m, 4H）,8.35-8.90（m, 8H）,7.37-7.86（m, 20H）,6.80（d, J=8.7Hz, 8H）,6.65（d, J=8.7Hz, 8H）,4.65（d, J=6.8Hz, 16H）,3.66（t, J=5.9Hz, 8H）,3.04（q, J=6.4Hz, 8H）,2.35（d, J=6.8Hz, 8H）,1.80（quint, J=6.4Hz, 8H）;
Calc. for C82H72N12O4
(TPE-B): C, 59.33; H, 10.42; N, 10.72. Found: C, 59.08; H, 10.66; N, 10.36.
Compound 13 (0.37 g, 0.35 mmol) is suspended in acetonitrile (20 ml), 2-bromomethylphenylboronic acid propylene ester (0.45 g, 1.8 mmol) is added, and the mixture is heated to reflux for 5 hours. The solvent was then concentrated to dryness, and the resulting crude product was purified by silica gel column chromatography to obtain 0.32 g (yield 44%) of TPE-B.
1 H NMR (300 MHz, CD 3 OD) δ 9.44 (s, 4H), 9.05-9.22 (m, 4H), 8.35-8.90 (m, 8H), 7.37-7.86 (m, 20H), 6.80 (d, J = 8.7Hz, 8H), 6.65 (d, J = 8.7Hz, 8H), 4.65 (d, J = 6.8Hz, 16H), 3.66 (t, J = 5.9Hz, 8H), 3.04 (q, J = 6.4Hz, 8H), 2.35 (d, J = 6.8Hz, 8H), 1.80 (quint, J = 6.4Hz, 8H);
Calc. For C 82 H 72 N 12 O 4
(TPE-B): C, 59.33; H, 10.42; N, 10.72.Found: C, 59.08; H, 10.66; N, 10.36.
hetero−ＴＰＥの合成
図５に示す合成スキームに従って、本発明の発蛍光性化合物hetero−ＴＰＥを合成した。
＜化合物１５）の合成＞
ビス（４−ヒドロキシフェニル）ケトン（５ｇ、２３．４ｍｍｏｌ）をＤＭＦ（１００ｍｌ）に溶解し、炭酸カリウム（１０ｇ、７２．５ｍｍｏｌ）を加えた。Ｎ−（３−ブロモプロピル）−Ｎ−ブトキシカルボニルアミド（７．８ｇ、３２．８ｍｍｏｌ）を添加し、室温で２４時間撹拌した。沈殿物をろ別し、濾液を濃縮乾固したのち、粗製物をシリカゲルカラムクロマトグラフで精製した。白色固体として化合物１５を８．９ｇ得た。収率７２％。
1H NMR（300MHz, CDCl3）δ8.20（s, 2H）,7.13（d, J=8.6Hz, 4H）,6.92（d, J=8.6Hz, 4H）,4.08（t, J=6.2Hz, 4H）,3.44（q, J=6.2Hz, 4H）,2.04（quint, J=6.2Hz,
4H）,1.42（s, 18H）
Synthesis of hetero-TPE The fluorescent compound hetero-TPE of the present invention was synthesized according to the synthesis scheme shown in FIG.
<Synthesis of Compound 15)
Bis (4-hydroxyphenyl) ketone (5 g, 23.4 mmol) was dissolved in DMF (100 ml) and potassium carbonate (10 g, 72.5 mmol) was added. N- (3-bromopropyl) -N-butoxycarbonylamide (7.8 g, 32.8 mmol) was added and stirred at room temperature for 24 hours. The precipitate was filtered off, the filtrate was concentrated to dryness, and the crude product was purified by silica gel column chromatography. 8.9 g of compound 15 was obtained as a white solid. Yield 72%.
1 H NMR (300 MHz, CDCl 3 ) δ8.20 (s, 2H), 7.13 (d, J = 8.6Hz, 4H), 6.92 (d, J = 8.6Hz, 4H), 4.08 (t, J = 6.2Hz , 4H), 3.44 (q, J = 6.2Hz, 4H), 2.04 (quint, J = 6.2Hz,
4H), 1.42 (s, 18H)
＜化合物１６）の合成＞
化合物１５（８．９ｇ、１６．９ｍｍｏｌ）を塩化メチレン（１００ｍｌ）に溶解し、氷冷下トリフルオロ酢酸（７．７ｇ、６７．５ｍｍｏｌ）を滴下した。その後室温で３時間撹拌したのち、溶媒を濃縮乾固した。得られた粗製物をシリカゲルカラムクロマトグラフで精製し、白色固体として化合物１６を７．０ｇ得た。収率７４％。
<Synthesis of Compound 16)
Compound 15 (8.9 g, 16.9 mmol) was dissolved in methylene chloride (100 ml), and trifluoroacetic acid (7.7 g, 67.5 mmol) was added dropwise under ice cooling. Then, after stirring at room temperature for 3 hours, the solvent was concentrated to dryness. The obtained crude product was purified by silica gel column chromatography to obtain 7.0 g of compound 16 as a white solid. Yield 74%.
＜化合物１７）の合成＞
化合物１６（７ｇ、１２．６ｍｍｏｌ）を塩化メチレン（１５０ｍｌ）に懸濁し、トリエチルアミン（４．９ｇ、４９ｍｍｏｌ）と１−Ｈ−ピラゾール−１−（Ｎ，Ｎ’−ビス（tert−ブトキシカルボニル））カルボキサミジン（１１．６ｇ、３７．５ｍｍｏｌ）を加えた。室温で７２時間撹拌したのち、溶媒を濃縮乾固した。得られた粗製をシリカゲルカラムクロマトグラフで精製し、白色固体として化合物１７を５．３ｇ得た。収率５２％。
1H NMR（300MHz, CDCl3）δ10.2（s, 2H）,9.04（t, J=4.7Hz, 2H）,7.22（d, J=8.6Hz, 4H）,7.05（d, J=8.6Hz, 4H）,4.03（t, J=5.3Hz, 4H）,3.89（q, J=5.8Hz, 4H）,2.03（quint, J=5.8Hz, 4H）,1.49（s, 36H）
<Synthesis of Compound 17)
Compound 16 (7 g, 12.6 mmol) was suspended in methylene chloride (150 ml), triethylamine (4.9 g, 49 mmol) and 1-H-pyrazole-1- (N, N′-bis (tert-butoxycarbonyl)) Carboxamidine (11.6 g, 37.5 mmol) was added. After stirring at room temperature for 72 hours, the solvent was concentrated to dryness. The obtained crude product was purified by silica gel column chromatography to obtain 5.3 g of Compound 17 as a white solid. Yield 52%.
1 H NMR (300 MHz, CDCl 3 ) δ 10.2 (s, 2H), 9.04 (t, J = 4.7 Hz, 2H), 7.22 (d, J = 8.6 Hz, 4H), 7.05 (d, J = 8.6 Hz , 4H), 4.03 (t, J = 5.3Hz, 4H), 3.89 (q, J = 5.8Hz, 4H), 2.03 (quint, J = 5.8Hz, 4H), 1.49 (s, 36H)
＜化合物１８）の合成＞
ビス（４−ヒドロキシフェニル）ケトン（５ｇ、２３．４ｍｍｏｌ）をＤＭＦ（１００ｍｌ）に溶解し、炭酸カリウム（１０ｇ、７２．５ｍｍｏｌ）を加えた。Ｎ−（３−ブロモプロピル）フタルイミド（１５．０ｇ、５６．２ｍｍｏｌ）を添加し、７０℃で２４時間撹拌した。沈殿物をろ別後、ろ液を濃縮乾固した。得られた粗製物をシリカゲルカラムクロマトグラフで精製し、淡黄色固体として化合物１８を７．３ｇ得た。収率５３％。
1H NMR（300MHz, CDCl3）δ8.13（d, J=7.8, 4H）,7.71(d, J=7.7,
4H),7.27（d, J=8.6Hz, 4H）,7.06（d, J=8.6Hz, 4H）,4.11（t, J=6.2Hz, 4H）,3.85（q, J=6.2Hz, 4H）,2.13（quint, J=6.2Hz, 4H）
<Synthesis of Compound 18)
Bis (4-hydroxyphenyl) ketone (5 g, 23.4 mmol) was dissolved in DMF (100 ml) and potassium carbonate (10 g, 72.5 mmol) was added. N- (3-bromopropyl) phthalimide (15.0 g, 56.2 mmol) was added and stirred at 70 ° C. for 24 hours. After the precipitate was filtered off, the filtrate was concentrated to dryness. The obtained crude product was purified by silica gel column chromatography to obtain 7.3 g of compound 18 as a pale yellow solid. Yield 53%.
1 H NMR (300 MHz, CDCl 3 ) δ 8.13 (d, J = 7.8, 4H), 7.71 (d, J = 7.7,
4H), 7.27 (d, J = 8.6Hz, 4H), 7.06 (d, J = 8.6Hz, 4H), 4.11 (t, J = 6.2Hz, 4H), 3.85 (q, J = 6.2Hz, 4H) , 2.13 (quint, J = 6.2Hz, 4H)
＜化合物１９）の合成＞
化合物１７（５．３ｇ、６．５ｍｍｏｌ）と化合物１８（３．８３ｇ、６．５ｍｍｏｌ）をＴＨＦ（１００ｍｌ）に溶解し、亜鉛粉末（０．４２ｇ、６．５ｍｍｏｌ）を加え、４塩化チタン（４．７ｍｌ、４２．９ｍｍｏｌ）を滴下したのち２４時間加熱還流した。放冷後、１００ｍｌの水を徐々に加え過剰の４塩化チタンを加水分解し、水層をクロロホルムで３回抽出した。有機層を無水硫酸ナトリウムで乾燥し、溶液を濃縮乾固した。粗製物をシリカゲルカラムクロマトグラフで精製し、白色固体として化合物１９を１．１ｇ得た。収率１２．２％。
1H NMR（300MHz, CDCl3）δ9.74（s, 2H）,9.09（t, J=4.7Hz, 2H）,8.08（d, J=7.8, 4H）,7.88（d, J=7.7, 4H）,7.28-7.35（m, 4H）,7.02-7.10（m, 4H）,4.02-4.10（m, 8H）,3.63-3.90（m, 8H）,2.00-2.13（m, 8H）,1.51（s, 36H）
<Synthesis of Compound 19)
Compound 17 (5.3 g, 6.5 mmol) and compound 18 (3.83 g, 6.5 mmol) are dissolved in THF (100 ml), zinc powder (0.42 g, 6.5 mmol) is added, and titanium tetrachloride ( (4.7 ml, 42.9 mmol) was added dropwise, followed by heating under reflux for 24 hours. After allowing to cool, 100 ml of water was gradually added to hydrolyze excess titanium tetrachloride, and the aqueous layer was extracted three times with chloroform. The organic layer was dried over anhydrous sodium sulfate and the solution was concentrated to dryness. The crude product was purified by silica gel column chromatography to obtain 1.1 g of compound 19 as a white solid. Yield 12.2%.
1 H NMR (300 MHz, CDCl 3 ) δ 9.74 (s, 2H), 9.09 (t, J = 4.7 Hz, 2H), 8.08 (d, J = 7.8, 4H), 7.88 (d, J = 7.7, 4H ), 7.28-7.35 (m, 4H), 7.02-7.10 (m, 4H), 4.02-4.10 (m, 8H), 3.63-3.90 (m, 8H), 2.00-2.13 (m, 8H), 1.51 (s , 36H)
＜化合物２０の合成＞
化合物１（２ｇ、１．４６ｍｍｏｌ）をエタノール（５０ｍｌ）に溶解し、ヒドラジン１水和物（０．３６ｇ、７．２ｍｍｏｌ）を入れ、５時間加熱還流した。放冷後、析出したフタルヒドラジドをろ別し、ろ液を濃縮乾固した。得られた粗製をシリカゲルカラムクロマトグラフで精製し、白色固体として化合物２０を１．０ｇ得た。収率６１．７％。
1H NMR（300MHz, CD3OD）δ8.43（s-br, 2H）,7.07（d, J=8.6Hz, 4H）,6.89（d, J=8.6Hz, 4H）,3.87-4.11（m, 8H）,3.63-3.90（m, 8H）,2.00-2.13（m, 8H）,1.49（s, 36H）
<Synthesis of Compound 20>
Compound 1 (2 g, 1.46 mmol) was dissolved in ethanol (50 ml), hydrazine monohydrate (0.36 g, 7.2 mmol) was added, and the mixture was heated to reflux for 5 hours. After allowing to cool, the precipitated phthalhydrazide was filtered off, and the filtrate was concentrated to dryness. The obtained crude product was purified by silica gel column chromatography to obtain 1.0 g of compound 20 as a white solid. Yield 61.7%.
1 H NMR (300 MHz, CD 3 OD) δ 8.43 (s-br, 2H), 7.07 (d, J = 8.6Hz, 4H), 6.89 (d, J = 8.6Hz, 4H), 3.87-4.11 (m , 8H), 3.63-3.90 (m, 8H), 2.00-2.13 (m, 8H), 1.49 (s, 36H)
＜化合物２１の合成＞
化合物２０（１ｇ、０．９ｍｍｏｌ）をアセトニトリル（２０ｍｌ）に溶解し、炭酸カリウム（３ｇ、２１．８ｍｍｏｌ）とヨウ化カリウム（２ｇ、１２．０ｍｍｏｌ）及び２−クロロメチルピリジン塩酸塩（０．７ｇ、４．２ｍｍｏｌ）を加え、２４時間加熱還流した。放冷後沈殿物をろ別し、ろ液を濃縮乾固した。得られた粗製をシリカゲルカラムクロマトグラフで精製し、淡黄色固体として化合物２１を０．８５ｇ得た。収率６４％。
1H NMR（300MHz, CDCl3）δ11.5（s, 4H）,8.50-8.70（m, 8H）,7.06-7.88（m, 8H）,6.75-6.93（m, 8H）,6.56-6.71（m, 8H）,3.70-3.99（m, 8H）,3.24-3.60（m, 8H）,1.88-2.06（m, 8H）,1.48（s, 36H）
<Synthesis of Compound 21>
Compound 20 (1 g, 0.9 mmol) was dissolved in acetonitrile (20 ml), potassium carbonate (3 g, 21.8 mmol), potassium iodide (2 g, 12.0 mmol) and 2-chloromethylpyridine hydrochloride (0.7 g). 4.2 mmol) was added and heated to reflux for 24 hours. After allowing to cool, the precipitate was filtered off, and the filtrate was concentrated to dryness. The resulting crude product was purified by silica gel column chromatography to obtain 0.85 g of compound 21 as a pale yellow solid. Yield 64%.
1 H NMR (300 MHz, CDCl 3 ) δ 11.5 (s, 4H), 8.50-8.70 (m, 8H), 7.06-7.88 (m, 8H), 6.75-6.93 (m, 8H), 6.56-6.71 (m , 8H), 3.70-3.99 (m, 8H), 3.24-3.60 (m, 8H), 1.88-2.06 (m, 8H), 1.48 (s, 36H)
＜hetero−ＴＰＥの合成＞
化合物２１（０．８５ｇ、０．５８ｍｍｏｌ）を塩化メチレン（３０ｍｌ）に溶解し、トリフルオロ酢酸（５ｍｌ、６５．３ｍｍｏｌ）を加え、室温で１８時間撹拌した。反応後、エーテルに溶液を添加し、生じた沈殿をろ別したのち少量のエーテルでろ洗後減圧乾燥した。粗製物を少量の塩化メチレンに溶解し、その溶液をエーテルに添加し再沈してろ取、エーテルで洗浄、乾燥し、白色固体として化合物２２を２４９ｍｇ得た。収率４０％。
1H NMR（300 MHz, CDCl3）δ11.5（s, 4H）,8.50-8.70（m, 8H）,7.06-7.88（m, 12H）,6.75-6.93（m, 8H）,6.56-6.71（m, 8H）,3.70-3.99（m, 8H）,3.24-3.60（m, 8H）,1.88-2.06（m, 8H）,1.48（s, 36H）
MS (ESI) m/z 1077.8([M-CF3COO-]+)
Calc. for C64H76N12O4
(13): C, 51.32; H, 4.55; N, 10.25. Found: C, 51.15; H, 4.82; N, 10.03.
<Synthesis of hetero-TPE>
Compound 21 (0.85 g, 0.58 mmol) was dissolved in methylene chloride (30 ml), trifluoroacetic acid (5 ml, 65.3 mmol) was added, and the mixture was stirred at room temperature for 18 hours. After the reaction, a solution was added to ether, and the resulting precipitate was filtered off, washed with a small amount of ether, and dried under reduced pressure. The crude product was dissolved in a small amount of methylene chloride, the solution was added to ether, reprecipitated, collected by filtration, washed with ether, and dried to obtain 249 mg of Compound 22 as a white solid. Yield 40%.
1 H NMR (300 MHz, CDCl 3 ) δ 11.5 (s, 4H), 8.50-8.70 (m, 8H), 7.06-7.88 (m, 12H), 6.75-6.93 (m, 8H), 6.56-6.71 ( m, 8H), 3.70-3.99 (m, 8H), 3.24-3.60 (m, 8H), 1.88-2.06 (m, 8H), 1.48 (s, 36H)
MS (ESI) m / z 1077.8 ([M-CF 3 COO-] + )
Calc. For C 64 H 76 N 12 O 4
(13): C, 51.32; H, 4.55; N, 10.25. Found: C, 51.15; H, 4.82; N, 10.03.
ＴＰＥ−Ｇの特性試験（１）
実施例１で合成した本発明の発蛍光性化合物ＴＰＥ−Ｇについて、その特性を調べた。
＜ヌクレオチドに対する蛍光特性＞
ＴＰＥ−Ｇに、ヌクレオチドとしてＡＭＰ、ＡＤＰまたはＡＴＰを加えて蛍光を測定した。測定条件は以下のとおりである。
ＴＰＥ−Ｇの濃度：６μＭ、ＨＥＰＥＳバッファー（５ｍＭ、ｐＨ７．４）中。
ＡＴＰ，ＡＤＴおよびＡＴＰの濃度：それぞれ、２０μＭ、１２μＭおよび８μＭ。
励起波長λｅｘ：３３５ｎｍ
測定装置：Perkin−ElmerＬＳ５５
測定温度：２５℃
測定セル：１ｃｍ石英セル
測定結果を図６に示す。図６（ａ）に示されるように本発明の発蛍光性化合物ＴＰＥ−ＧはＡＴＰを選択的に認識して蛍光を発することが理解される。このことは、紫外線照射下（λｅｘ：３３５ｎｍ）で肉眼でも観察された（図６（ｂ）参照）
TPE-G characteristics test (1)
The characteristics of the fluorescent compound TPE-G of the present invention synthesized in Example 1 were examined.
<Fluorescence properties for nucleotides>
Fluorescence was measured by adding AMP, ADP or ATP as nucleotides to TPE-G. The measurement conditions are as follows.
TPE-G concentration: 6 μM in HEPES buffer (5 mM, pH 7.4).
ATP, ADT and ATP concentrations: 20 μM, 12 μM and 8 μM, respectively.
Excitation wavelength λ ex : 335 nm
Measuring device: Perkin-Elmer LS55
Measurement temperature: 25 ° C
Measurement cell: 1 cm quartz cell The measurement results are shown in FIG. As shown in FIG. 6 (a), it is understood that the fluorescent compound TPE-G of the present invention selectively recognizes ATP and emits fluorescence. This was also observed with the naked eye under ultraviolet irradiation (λ ex : 335 nm) (see FIG. 6B).
＜凝集体の確認＞
ＴＰＥ−ＧにＡＴＰを添加して紫外可視スペクトルを観察したところ、吸収最大ピークが拡がり且つ３１０ｎｍから３３５ｎｍにシフトした。このことは、凝集体（aggregates）が形成していることを示唆していると考えられたので、ＤＬＳ（dynamic light scattering：ダイナミック光散乱）測定を行ったところ（測定装置：Malvern Zeta sizer Nano-ZS）、水性分散液中に平均直径６７７±２５２ｎｍの凝集体の形成が認められた（図７（ａ）参照）。また、ＳＥＭ（走査電子顕微鏡：HitachiＳ−５０００）観察によっても、〜１μｍ程度の凝集体の形成が認められた（図７（ｂ）参照）。
<Confirmation of aggregate>
When ATP was added to TPE-G and an ultraviolet-visible spectrum was observed, the maximum absorption peak broadened and shifted from 310 nm to 335 nm. Since this was thought to suggest that aggregates were formed, DLS (dynamic light scattering) measurement was performed (measurement apparatus: Malvern Zeta sizer Nano- ZS), formation of aggregates having an average diameter of 677 ± 252 nm was observed in the aqueous dispersion (see FIG. 7A). Moreover, formation of an aggregate of about ˜1 μm was also observed by SEM (scanning electron microscope: Hitachi S-5000) observation (see FIG. 7B).
＜蛍光滴定試験＞
ＴＰＥ−Ｇ（濃度：６μＭ）に、ＡＭＰ、ＡＤＰまたはＡＴＰを逐次添加して蛍光滴定試験を行った。バッファー、励起波長、測定装置、測定温度、測定セルなどの測定条件は、前述の「ヌクレオチドに対する蛍光特性」の項で述べたのと同じである。
その結果を図８に示す。図中、Ｆ０はヌクレオチドが存在しない場合の蛍光強度を表し、Ｆはヌクレオチドが存在する場合の蛍光強度を表し、△Ｆ＝Ｆ−Ｆ０である。したがって、△Ｆ／Ｆ０は蛍光強度の変化の度合いを意味することになる。
<Fluorescence titration test>
A fluorescent titration test was performed by sequentially adding AMP, ADP or ATP to TPE-G (concentration: 6 μM). The measurement conditions such as the buffer, excitation wavelength, measurement device, measurement temperature, and measurement cell are the same as those described in the above section “Fluorescence characteristics for nucleotides”.
The result is shown in FIG. In the figure, F 0 represents the fluorescence intensity in the absence of nucleotides, F represents the fluorescence intensity in the presence of nucleotides, and ΔF = F−F 0 . Therefore, ΔF / F 0 means the degree of change in fluorescence intensity.
図８（ｂ）に示されるように、ＡＭＰおよびＡＤＰを添加しても実質的な蛍光強度の変化は見られない。これに対して、ＡＴＰの添加に対しては、濃度の増加に対して急激に蛍光が発生し、その強度が大きくなり飽和している。図８の（ｂ）はＡＴＰの場合の低濃度域を拡大して示されているものであるが、この図から、ＴＰＥ−Ｇの蛍光強度はＡＴＰの濃度に対してシグモイド状に変化している。すなわち、従来の均一溶液系において蛍光が濃度に応じて漸増する場合に比べて、本発明の発蛍光性化合物を用いると、一定のＡＴＰ濃度においてターンオンまたはスイッチオン式にＡＴＰを検出し得ることが理解される。 As shown in FIG. 8B, even if AMP and ADP are added, no substantial change in fluorescence intensity is observed. On the other hand, when ATP is added, fluorescence is rapidly generated as the concentration increases, and the intensity increases and is saturated. FIG. 8B is an enlarged view of the low concentration region in the case of ATP. From this figure, the fluorescence intensity of TPE-G changes in a sigmoid manner with respect to the concentration of ATP. Yes. In other words, when the fluorescent compound of the present invention is used, the ATP can be detected in a turn-on or switch-on manner at a constant ATP concentration as compared with the case where the fluorescence gradually increases with the concentration in the conventional homogeneous solution system. Understood.
ＴＰＥ−Ｇの特性試験（２）
＜補酵素に対する蛍光特性＞
実施例１で合成したＴＰＥ−Ｇに補酵素として、ＮＡＤ＋、ＮＡＤＨ、ＮＡＤＰ＋、ＮＡＤＰＨを加えて蛍光を測定した。補酵素の濃度：１５μＭ、その他の測定条件は、実施例１の場合と同じである。
測定結果を図９に示す。図９（ａ）に示されるように本発明の発蛍光化合物ＴＰＥ−ＧはＮＡＤＰＨを選択的に認識して蛍光を発することが理解される。このことは、紫外線照射下（λｅｘ：３３５ｎｍ）で肉眼でも観察された（図９（ｂ）参照）。
TPE-G characteristic test (2)
<Fluorescence properties for coenzymes>
Fluorescence was measured by adding NAD + , NADH, NADP + , and NADPH as coenzymes to TPE-G synthesized in Example 1. The concentration of the coenzyme: 15 μM, and other measurement conditions are the same as in Example 1.
The measurement results are shown in FIG. As shown in FIG. 9A, it is understood that the fluorescent compound TPE-G of the present invention selectively recognizes NADPH and emits fluorescence. This was also observed with the naked eye under ultraviolet irradiation (λex: 335 nm) (see FIG. 9B).
＜蛍光滴定試験＞
ＴＰＥ−Ｇ（濃度：６μＭ）に、ＮＡＤ＋、ＮＡＤＨ、ＮＡＤＰ＋、ＮＡＤＰＨを逐次添加して蛍光滴定試験を行った。測定条件は、実施例１の場合と同じである。結果を図１０に示す。
図１０に示されるように、ＮＡＤ＋、ＮＡＤＰ＋を添加しても実質的な蛍光強度の変化は見られない。これに対して、ＮＡＤＨ、ＮＡＤＰＨの添加に対しては、蛍光強度の増加が見られた。ＴＰＥ−Ｇの蛍光強度は、ＮＡＤＨに対しては漸増するのに対して、ＮＡＤＰＨに対しては濃度の増加に伴って急激に蛍光が発生し、シグモイド状の応答曲線を示している。
このように、補酵素に対して本発明の発蛍光性化合物を用いると、一定のＮＡＤＰＨ濃度においてターンオンまたはスイッチオン式にＮＡＤＰＨを検出し得ることが理解される。
<Fluorescence titration test>
NAD + , NADH, NADP + , and NADPH were sequentially added to TPE-G (concentration: 6 μM) to perform a fluorescence titration test. The measurement conditions are the same as in Example 1. The results are shown in FIG.
As shown in FIG. 10, no substantial change in fluorescence intensity is observed even when NAD + or NADP + is added. On the other hand, an increase in fluorescence intensity was observed with the addition of NADH and NADPH. The fluorescence intensity of TPE-G gradually increases with respect to NADH, whereas with NADPH, fluorescence is rapidly generated as the concentration increases, indicating a sigmoid response curve.
Thus, it is understood that NADPH can be detected in a turn-on or switch-on manner at a constant NADPH concentration when the fluorescent compound of the present invention is used for a coenzyme.
＜凝集体形成挙動の追跡＞
ＴＰＥ−Ｇ（濃度：６μＭ）に、ＮＡＤ＋、ＮＡＤＨ、ＮＡＤＰ＋、ＮＡＤＰＨを逐次添加し、ＤＬＳ測定によって凝集体の形成挙動を追跡した。図１１に示されるように、ＮＡＤ＋、ＮＡＤＨを添加しても実質的な光散乱強度の変化は見られないことから、ＴＰＥ−Ｇと凝集体を形成しないことが分かる。これに対して、ＮＡＤＰ＋、ＮＡＤＰＨの添加に対しては、光散乱強度の増加が見られたことから、ＴＰＥ−Ｇとの凝集体形成が認められた。
光散乱強度は、ＮＡＤＰ＋に対しては４０μＭから漸増するのに対して、ＮＡＤＰＨに対しては発蛍光応答濃度と連動した急激な増加が認められた。このことは、ＮＡＤＰＨに対しては、蛍光応答閾値はＡＩＥ（凝集誘起発光）に因る臨界会合濃度を反映することを意味している。
<Tracking of aggregate formation behavior>
NAD + , NADH, NADP + , and NADPH were sequentially added to TPE-G (concentration: 6 μM), and the aggregate formation behavior was followed by DLS measurement. As shown in FIG. 11, even when NAD + or NADH is added, no substantial change in light scattering intensity is observed, indicating that no aggregate is formed with TPE-G. On the other hand, when NADP + and NADPH were added, an increase in light scattering intensity was observed, and thus an aggregate formation with TPE-G was observed.
The light scattering intensity gradually increased from 40 μM for NADP +, whereas a sharp increase associated with the fluorescence response concentration was observed for NADPH. This means that for NADPH, the fluorescence response threshold reflects the critical association concentration due to AIE (aggregation induced luminescence).
＜ＮＡＤ＋、ＮＡＤＰ＋の蛍光検出＞
上記のように、ＴＰＥ−Ｇ（濃度：６μＭ）に、ＮＡＤ＋、ＮＡＤＰ＋を添加しても実質的な蛍光強度の変化は見られない。しかし、亜硫酸ナトリウムを添加（最終濃度：５ｍＭ）することで、発蛍光強度の差異が認められた。これは、亜硫酸ナトリウムとの付加反応により得られる、ＮＡＤ−ＳＯ３、ＮＡＤＰ−ＳＯ３の負電荷数の差異を識別し、結果としてＮＡＤＰ＋を認識したものと理解される（図１２参照）。このように、本発明の発蛍光性化合物を用いれば、付加化合物（adduct）を発生させることにより、被測定物質に対する特異的反応性を変えることができる。
測定条件：ＴＰＥ−Ｇ濃度：６μＭ、ＮＡＤ＋、ＮＡＤＰ＋濃度：８０μＭ、亜硫酸ナトリウム濃度：５ｍＭ、ＨＥＰＥＳバッファー（５ｍＭ，ｐＨ７．４）中。
<Fluorescence detection of NAD + and NADP + >
As described above, even if NAD + or NADP + is added to TPE-G (concentration: 6 μM), no substantial change in fluorescence intensity is observed. However, a difference in fluorescence intensity was observed by adding sodium sulfite (final concentration: 5 mM). This is understood to be that the difference in the number of negative charges of NAD-SO 3 and NADP-SO 3 obtained by the addition reaction with sodium sulfite was identified, and as a result, NADP + was recognized (see FIG. 12). As described above, when the fluorescent compound of the present invention is used, the specific reactivity to the substance to be measured can be changed by generating an adduct.
Measurement conditions: TPE-G concentration: 6 μM, NAD + , NADP + concentration: 80 μM, sodium sulfite concentration: 5 mM, in HEPES buffer (5 mM, pH 7.4).
ＴＰＥ−Ｚｎの特性試験
＜ヌクレオチドに対する蛍光特性＞
実施例３で合成したＴＰＥ−Ｚｎに、イオン強度１００ｍＭの条件下で、ヌクレオチドとして、ＡＭＰ、ＡＤＰ、ＡＴＰを加えて蛍光を測定した。ヌクレオチドの濃度：１５μＭ、ＮａＣｌ濃度：１００ｍＭ、その他の測定条件は、実施例１の場合と同じである。
測定結果を図１３に示す。図１３（ａ）に示されるように本発明の発蛍光化合物ＴＰＥ−Ｚｎは、特に高塩濃度条件下で、ＡＤＰ、ＡＴＰを選択的に認識して蛍光を発することが理解される。このことは、紫外線照射下（λｅｘ：３３５ｎｍ）で肉眼でも観察された（図１３（ｂ）参照）。
Characterization of TPE-Zn <Fluorescence properties for nucleotides>
Fluorescence was measured by adding AMP, ADP, and ATP as nucleotides to TPE-Zn synthesized in Example 3 under conditions of an ionic strength of 100 mM. Nucleotide concentration: 15 μM, NaCl concentration: 100 mM, and other measurement conditions are the same as in Example 1.
The measurement results are shown in FIG. As shown in FIG. 13 (a), it is understood that the fluorescent compound TPE-Zn of the present invention selectively recognizes ADP and ATP and emits fluorescence particularly under high salt concentration conditions. This was also observed with the naked eye under ultraviolet irradiation (λex: 335 nm) (see FIG. 13B).
Claims (4)
X or Y represents an atomic group represented by the formula (C), and forms a complex with Cu or Zn via the atomic group. The fluorescent compound described in 1.
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