JP6958781B2 - Fluorescent compound or salt thereof, ionic compound detection agent and ionic compound detection method - Google Patents
Fluorescent compound or salt thereof, ionic compound detection agent and ionic compound detection method Download PDFInfo
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- JP6958781B2 JP6958781B2 JP2016024189A JP2016024189A JP6958781B2 JP 6958781 B2 JP6958781 B2 JP 6958781B2 JP 2016024189 A JP2016024189 A JP 2016024189A JP 2016024189 A JP2016024189 A JP 2016024189A JP 6958781 B2 JP6958781 B2 JP 6958781B2
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- 239000007850 fluorescent dye Substances 0.000 title claims description 61
- 150000003839 salts Chemical class 0.000 title claims description 37
- 150000008040 ionic compounds Chemical class 0.000 title claims description 31
- 239000003795 chemical substances by application Substances 0.000 title claims description 20
- 238000001514 detection method Methods 0.000 title claims description 19
- 238000012360 testing method Methods 0.000 claims description 28
- 125000002091 cationic group Chemical group 0.000 claims description 22
- 125000004432 carbon atom Chemical group C* 0.000 claims description 20
- 125000000217 alkyl group Chemical group 0.000 claims description 12
- 125000000129 anionic group Chemical group 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 11
- -1 2- but -O- Chemical class 0.000 claims description 10
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- 150000001449 anionic compounds Chemical class 0.000 claims description 9
- 125000005843 halogen group Chemical group 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 125000002947 alkylene group Chemical group 0.000 claims description 6
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 125000003545 alkoxy group Chemical group 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 3
- 125000002843 carboxylic acid group Chemical group 0.000 claims description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 2
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- 125000004429 atom Chemical group 0.000 claims 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims 1
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- 230000015572 biosynthetic process Effects 0.000 description 30
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- 239000007787 solid Substances 0.000 description 22
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 21
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 15
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- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 9
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- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 8
- 229920001287 Chondroitin sulfate Polymers 0.000 description 8
- 229940059329 chondroitin sulfate Drugs 0.000 description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 8
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 4
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- AOSZTAHDEDLTLQ-AZKQZHLXSA-N (1S,2S,4R,8S,9S,11S,12R,13S,19S)-6-[(3-chlorophenyl)methyl]-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-azapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one Chemical compound C([C@@H]1C[C@H]2[C@H]3[C@]([C@]4(C=CC(=O)C=C4[C@@H](F)C3)C)(F)[C@@H](O)C[C@@]2([C@@]1(C1)C(=O)CO)C)N1CC1=CC=CC(Cl)=C1 AOSZTAHDEDLTLQ-AZKQZHLXSA-N 0.000 description 3
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Description
本発明は、発蛍光性化合物又はその塩、イオン性化合物の検出剤及びイオン性化合物の検出方法に関する。 The present invention relates to a fluorescent compound or a salt thereof, a detection agent for an ionic compound, and a method for detecting an ionic compound.
例えば、臨床検査や新薬の開発等における各種生体分子の分析や、河川、水道水、工業用水等の環境水中におけるイオン性化合物の定量・半定量分析等、生体中や環境中において、特定化合物を高感度に微量分析する需要がある。 For example, analysis of various biomolecules in clinical tests and development of new drugs, quantitative / semi-quantitative analysis of ionic compounds in environmental water such as rivers, tap water, industrial water, etc. There is a demand for highly sensitive microanalysis.
特定化合物を高感度に微量分析する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。 Fluorescence analysis is used as a means for highly sensitive microanalysis of a specific compound, and a fluorescent compound for that purpose 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等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等が提案されている(例えば、特許文献1及び非特許文献1を参照。)。
For example, a ruthenium-bipyridyl polyaza compound or the like has been proposed as a fluorescent compound for analyzing a phosphate ion (phosphate group) that plays an important role in a living body such as control of information transmission in a signal transduction system. (See, for example,
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、フェニルボロン酸構造を含有する各種の化合物が案出されている(例えば、特許文献2〜4を参照。)。 In addition, various compounds containing a phenylboronic acid structure have been devised as fluorescent compounds for detecting and measuring glucose and other sugars, which are known as diagnostic markers for diabetes (for example, patents). See Documents 2-4).
また、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及びNADP+/NADPHは、酸化還元反応の生成物や酵素活性を調べる等の目的に利用され、例えば、NADHの蛍光が測定されることがある(例えば、特許文献5を参照。)。 In addition, NAD + / NADH and NADP + / NADPH, which function as coenzymes in many redox reactions in the living body, are used for the purpose of investigating the product and enzyme activity of the redox reaction, for example, fluorescence of NADH. May be measured (see, for example, Patent Document 5).
上に例示したような特定化合物を検出・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づくものであり、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。 Most of the conventional fluorescent compounds that detect and measure a specific compound 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, and the fluorescence intensity is low. It is known that there is no fluorescence in the concentration range, and on the other hand, when the concentration is high, aggregates may be formed and the light may not be emitted (quenched).
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(例えば、非特許文献2及び3を参照。)。このAIEを利用すれば、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。
On the other hand, recently, a phenomenon called agglutination-induced emission (AIE) in which fluorescence increases with aggregation has been found (see, for example,
そこで、本発明は、凝集誘起発光(AIE)を示す新たな発蛍光性化合物を提供することを目的とする。本発明はまた、イオン性化合物の検出剤を提供することを目的とする。 Therefore, an object of the present invention is to provide a new fluorescent compound exhibiting aggregation-induced luminescence (AIE). It is also an object of the present invention to provide a detection agent for an ionic compound.
本発明は以下の通りである。
[1]下記式(1)で表される発蛍光性化合物又はその塩。
[2]下記式(P1)で表される、[1]に記載の発蛍光性化合物又はその塩。
[3][1]又は[2]に記載の発蛍光性化合物又はその塩を有効成分とする、イオン性化合物の検出剤。
[4]前記式(1)又は前記式(P1)におけるXがカチオン性基であり、前記イオン性化合物がアニオン性化合物である、[3]に記載のイオン性化合物の検出剤。
[5]溶媒と、被検試料と、[1]又は[2]に記載の発蛍光性化合物又はその塩とを含む混合液を調製する工程と、前記混合液の蛍光を検出する工程と、を備える、被検試料中のイオン性化合物の検出方法。
The present invention is as follows.
[1] A fluorescent compound represented by the following formula (1) or a salt thereof.
[2] The fluorescent compound or salt thereof according to [1], which is represented by the following formula (P1).
[3] An ionic compound detection agent containing the fluorescent compound according to [1] or [2] or a salt thereof as an active ingredient.
[4] The agent for detecting an ionic compound according to [3], wherein X in the formula (1) or the formula (P1) is a cationic group, and the ionic compound is an anionic compound.
[5] A step of preparing a mixed solution containing a solvent, a test sample, and the fluorescent compound according to [1] or [2] or a salt thereof, a step of detecting fluorescence of the mixed solution, and a step of detecting fluorescence of the mixed solution. A method for detecting an ionic compound in a test sample.
本発明により、凝集誘起発光(AIE)を示す新たな発蛍光性化合物を提供することができる。また、イオン性化合物の検出剤を提供することができる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a novel fluorescent compound exhibiting aggregation-induced luminescence (AIE). In addition, an agent for detecting an ionic compound can be provided.
[発蛍光性化合物]
1実施形態において、本発明は、下記式(1)で表される発蛍光性化合物又はその塩を提供する。
In one embodiment, the present invention provides a fluorescent compound represented by the following formula (1) or a salt thereof.
式(1)中、R1はそれぞれ独立に、水素原子又はシアノ基、ハロゲン原子若しくはフェニル基を表し、R2はそれぞれ独立に、存在しないか又はハロゲン原子、炭素数1〜3のアルキル基、アミノ基若しくは炭素数1〜3のアルコキシ基を表し、R3はそれぞれ独立に、存在しないか又は−O−、−S−、−NH−、−CO−若しくは−CONH−で中断されていてもよい炭素数1〜20のアルキレン基を表し、Xはそれぞれ独立に、存在しないか又はカチオン性基若しくはアニオン性基を表し、R4はそれぞれ独立に、存在しないか又は−O−、−S−、−NH−、−CO−、−CONH−で中断されていてもよい炭素数1〜20のアルキル基若しくは炭素数1〜20のアルコール基若しくは−N(R5)2(ここで、R5はそれぞれ独立に、水素原子又は−O−、−S−、−NH−、−CO−、−CONH−で中断されていてもよい炭素数1〜20のアルキル基を表す。)を表す。pはそれぞれ独立に0〜4の整数を表し、qはそれぞれ独立に1〜5の整数を表し、少なくとも1つのXはカチオン性基又はアニオン性基である。 In formula (1), R 1 independently represents a hydrogen atom or a cyano group, a halogen atom or a phenyl group, and R 2 is an independent or non-existent or halogen atom, an alkyl group having 1 to 3 carbon atoms, respectively. an amino group or an alkoxy group having 1 to 3 carbon atoms, R 3 are each independently absent or -O -, - S -, - NH -, - CO- or -CONH- be interrupted by represents an alkylene group having a carbon number of 1 to 20, X is independently, represent an absent or a cationic group or an anionic group, R 4 are each independently absent or -O -, - S- , -NH-, -CO-, -CONH-, an alkyl group having 1 to 20 carbon atoms or an alcohol group having 1 to 20 carbon atoms or -N (R 5 ) 2 (here, R 5). Represent each independently as a hydrogen atom or an alkyl group having 1 to 20 carbon atoms which may be interrupted by a hydrogen atom or -O-, -S-, -NH-, -CO-, -CONH-). p independently represents an integer of 0 to 4, q independently represents an integer of 1 to 5, and at least one X is a cationic group or an anionic group.
式(1)中、R3で表されるアルキレン基は分岐鎖を有していてもよい。また、当該分岐鎖の炭素数は1〜10であってもよい。分岐鎖としては、アミノ酸の側鎖に由来する基が挙げられ、例えば、メチル基、ベンジル基等が挙げられる。 In the formula (1), the alkylene group represented by R 3 may have a branched chain. Further, the number of carbon atoms of the branched chain may be 1 to 10. Examples of the branched chain include a group derived from the side chain of an amino acid, and examples thereof include a methyl group and a benzyl group.
後述する実施例で示すように、本実施形態の発蛍光性化合物又はその塩は、イオン性化合物の存在下で凝集誘起発光(AIE)を示し、イオン性化合物の非存在下では蛍光を発しない。 As shown in Examples described later, the fluorescent compound of this embodiment or a salt thereof exhibits aggregation-induced emission (AIE) in the presence of the ionic compound and does not fluoresce in the absence of the ionic compound. ..
本実施形態の発蛍光性化合物又はその塩において、上記式(1)におけるXがカチオン性基である場合、アニオン性化合物の存在下でAIEを示し、蛍光を発する。また、上記式(1)におけるXがアニオン性基である場合、カチオン性化合物の存在下でAIEを示し、蛍光を発する。 In the fluorescent compound of the present embodiment or a salt thereof, when X in the above formula (1) is a cationic group, it exhibits AIE in the presence of an anionic compound and fluoresces. When X in the above formula (1) is an anionic group, it exhibits AIE in the presence of a cationic compound and emits fluorescence.
上記のカチオン性基としては、例えば下記式(2)〜(4)で表される基が挙げられる。
また、上記のアニオン性基としては、例えばリン酸基、カルボン酸基、硫酸基等が挙げられる。 Further, examples of the above-mentioned anionic group include a phosphoric acid group, a carboxylic acid group, a sulfuric acid group and the like.
本実施形態の発蛍光性化合物又はその塩において、上記式(1)で表されるR1はシアノ基であることが好ましい。また、上記式(1)で表されるR2は存在しないことが好ましい。また、上記式(1)で表されるXはカチオン性基であることが好ましい。また、上記式(1)で表されるqは1〜3であることが好ましく、1であることがより好ましい。 In the fluorescent compound of the present embodiment or a salt thereof, R 1 represented by the above formula (1) is preferably a cyano group. Further, it is preferable that R 2 represented by the above formula (1) does not exist. Further, X represented by the above formula (1) is preferably a cationic group. Further, q represented by the above formula (1) is preferably 1 to 3, and more preferably 1.
本実施形態の発蛍光性化合物の塩の種類は、本発明の効果が得られる限り特に限定されない。具体的には、上記式(1)におけるXがカチオン性基である場合には、例えば、硫酸、硝酸等の無機酸との塩、例えば塩素、臭素、ヨウ素等のハロゲン原子との塩、例えば酢酸、トリフルオロ酢酸、クエン酸、グルコン酸、プロピオン酸、パントテン酸等の有機酸との塩等が挙げられる。また、上記式(1)におけるXがアニオン性基である場合には、例えば、アルカリ金属、アルカリ土類金属等の金属との塩、アンモニアとの塩、テトラブチルアンモニウム等の有機アミン若しくは有機アンモニウムとの塩等が挙げられる。 The type of salt of the fluorescent compound of the present embodiment is not particularly limited as long as the effects of the present invention can be obtained. Specifically, when X in the above formula (1) is a cationic group, for example, a salt with an inorganic acid such as sulfuric acid or nitrate, for example, a salt with a halogen atom such as chlorine, bromine or iodine, for example. Examples thereof include salts with organic acids such as acetic acid, trifluoroacetic acid, citric acid, gluconic acid, propionic acid and pantothenic acid. When X in the above formula (1) is an anionic group, for example, a salt with a metal such as an alkali metal or an alkaline earth metal, a salt with ammonia, an organic amine such as tetrabutylammonium, or an organic ammonium. And salt and the like.
[イオン性化合物の検出剤]
1実施形態において、本発明は、上述した発蛍光性化合物又はその塩を有効成分とする、イオン性化合物の検出剤を提供する。
[Detective agent for ionic compounds]
In one embodiment, the present invention provides a detection agent for an ionic compound containing the above-mentioned fluorescent compound or a salt thereof as an active ingredient.
後述する実施例で示すように、上述した発蛍光性化合物又はその塩は、イオン性化合物の存在下で蛍光を発し、イオン性化合物の非存在下では蛍光を発しない。したがって、本実施形態の検出剤を用いることにより、生体由来試料や環境由来試料中におけるイオン性化合物を検出することができる。 As shown in Examples described later, the above-mentioned fluorescent compound or salt thereof fluoresces in the presence of the ionic compound and does not fluoresce in the absence of the ionic compound. Therefore, by using the detection agent of the present embodiment, it is possible to detect an ionic compound in a biological sample or an environment-derived sample.
本実施形態の検出剤が検出することができるイオン性化合物としては、アニオン性化合物又はカチオン性化合物が挙げられる。 Examples of the ionic compound that can be detected by the detection agent of the present embodiment include an anionic compound and a cationic compound.
アニオン性化合物としては、例えば、モノカルボン酸、ジカルボン酸、トリカルボン酸、ポリカルボン酸(フミン酸等)等のカルボン酸系化合物;リン酸、オリゴリン酸、ポリリン酸等のリン酸系化合物;オリゴヌクレオチド、ポリヌクレオチド、デオキシリボ核酸、リボ核酸、ニコチンアミドアデニンジヌクレオチドリン酸(酸化型(NADP+)又は還元型(NADPH))、ニコチンアミドアデニンジヌクレオチド(酸化型(NAD+)又は還元型(NADH))等のヌクレオチド類;ヘパリン、コンドロイチン硫酸、ヒアルロン酸等のアニオン性の多糖類;アニオン性ペプチド、プロテイン等が挙げられる。 Examples of the anionic compound include carboxylic acid compounds such as monocarboxylic acid, dicarboxylic acid, tricarboxylic acid and polycarboxylic acid (humic acid and the like); phosphoric acid compounds such as phosphoric acid, oligophosphate and polyphosphate; oligonucleotides. , Polynucleotide, deoxyribonucleic acid, ribonucleic acid, nicotinamide adenine dinucleotide phosphate (oxidized (NADP + ) or reduced (NADPH)), nicotinamide adenin dinucleotide (oxidized (NAD + ) or reduced (NADH)) ) And the like; anionic polysaccharides such as heparin, chondroitin sulfate, hyaluronic acid; anionic peptides, proteins and the like.
一方、カチオン性化合物としては、例えば、ナトリウムイオン、カルシウムイオン等の金属イオン;ポリアミン等のアミン系化合物;カチオン性ペプチド、プロテイン等が挙げられる。 On the other hand, examples of the cationic compound include metal ions such as sodium ion and calcium ion; amine compounds such as polyamines; cationic peptides and proteins.
実施例において後述するように、例えば、上記式(1)で表されるXがカチオン性基である発蛍光性化合物又はその塩を用いることにより、ジカルボン酸、ヌクレオチド、アニオン性多糖類等のアニオン性化合物の検出を行うことができる。 As will be described later in the examples, for example, by using a fluorescent compound or a salt thereof in which X represented by the above formula (1) is a cationic group, anions such as dicarboxylic acids, nucleotides, and anionic polysaccharides are used. It is possible to detect sex compounds.
例えば、ジカルボン酸は、クエン酸回路等の代謝経路において生成される中間体でもある。代謝の機能不全により増加した体内ジカルボン酸は、最終的に尿中に排泄される。したがって、尿中の高濃度ジカルボン酸群は、代謝疾患のマーカーとなり、この高濃度ジカルボン酸群をターンオン又はスイッチオン式に検出する発蛍光性化合物(蛍光プローブ、蛍光センサ)を代謝疾患のスクリーニング検査に利用することにより、検査を簡略化することができる。 For example, dicarboxylic acids are also intermediates produced in metabolic pathways such as the citric acid cycle. The dicarboxylic acid in the body, which is increased due to metabolic dysfunction, is finally excreted in the urine. Therefore, the high-concentration dicarboxylic acid group in urine serves as a marker for metabolic diseases, and a fluorescent compound (fluorescent probe, fluorescent sensor) that detects this high-concentration dicarboxylic acid group in a turn-on or switch-on manner is used as a screening test for metabolic diseases. The inspection can be simplified by using the above.
本明細書において、ターンオン式とは、発蛍光性化合物が、検出対象化合物の濃度依存的に、無蛍光又は無視できるほど微弱な蛍光を発する状態から、発蛍光状態に直線的に切り替わることを意味する。また、スイッチオン式とは、発蛍光性化合物が、検出対象化合物の濃度が所定の閾値以上になった場合に、無蛍光又は無視できるほど微弱な蛍光を発する状態から、発蛍光状態に切り替わることを意味する。 In the present specification, the turn-on type means that the fluorescent compound linearly switches from a non-fluorescent state or a state in which a negligibly weak fluorescence is emitted to a fluorescent state depending on the concentration of the compound to be detected. do. The switch-on type means that the fluorescent compound switches from a non-fluorescent state or a state in which a negligibly weak fluorescence is emitted to a fluorescent state when the concentration of the detection target compound exceeds a predetermined threshold value. Means.
[イオン性化合物の検出方法]
1実施形態において、本発明は、溶媒と、被検試料と、上述した発蛍光性化合物又はその塩とを含む混合液を調製する工程と、前記混合液の蛍光を検出する工程と、を備える、被検試料中のイオン性化合物の検出方法を提供する。
[Method for detecting ionic compounds]
In one embodiment, the present invention comprises a step of preparing a mixed solution containing a solvent, a test sample, and the above-mentioned fluorinated compound or a salt thereof, and a step of detecting the fluorescence of the mixed solution. , Provide a method for detecting an ionic compound in a test sample.
本実施形態の検出方法において、溶媒は、被検試料に由来する液体であってもよく、発蛍光性化合物又はその塩を溶解した液体であってもよく、被検試料、発蛍光性化合物又は蛍光性化合物の塩とは別に用意された液体であってもよい。 In the detection method of the present embodiment, the solvent may be a liquid derived from the test sample, a liquid in which a fluorescent compound or a salt thereof is dissolved, the test sample, the fluorescent compound, or the like. It may be a liquid prepared separately from the salt of the fluorescent compound.
また、上記の混合液は、pH緩衝作用を有していることが好ましい。溶媒のpHは目的に応じて適宜調整すればよく、例えば中性であってもよく、例えば酸性であってもよく、例えば塩基性であってもよい。 Moreover, it is preferable that the above-mentioned mixed solution has a pH buffering action. The pH of the solvent may be appropriately adjusted according to the intended purpose, and may be, for example, neutral, for example, acidic, or basic.
被検試料としては、特に制限されず、血液、血漿、血清、尿、生体由来組織、培養細胞、培養微生物、培養細胞又は培養微生物の培地等の生体試料;河川から採取された水、水道水、工業用水等の試料等が挙げられる。また、検出対象とするイオン性化合物としては上述したものが挙げられる。 The test sample is not particularly limited, and is a biological sample such as blood, plasma, serum, urine, biological tissue, cultured cells, cultured microorganisms, cultured cells or culture medium of cultured microorganisms; water collected from a river, tap water. , Samples of industrial water and the like. Further, examples of the ionic compound to be detected include those described above.
蛍光の検出は、具体的には、上記の混合液に励起光を照射し、発生する蛍光を検出することにより行うことができる。混合液の蛍光の検出は、例えば肉眼で行ってもよいし、例えば分光光度計を用いた測定により行ってもよい。蛍光を肉眼で検出する場合には、例えば、UVハンディーランプ等を光源として励起光の照射を行ってもよい。 Specifically, the fluorescence can be detected by irradiating the above-mentioned mixed solution with excitation light and detecting the generated fluorescence. The fluorescence of the mixed solution may be detected by, for example, the naked eye, or by measurement using a spectrophotometer, for example. When the fluorescence is detected with the naked eye, for example, the excitation light may be irradiated using a UV handy lamp or the like as a light source.
実施例において後述するように、例えば、被検試料の非存在下における、発蛍光性化合物又はその塩の蛍光強度と比較して、被検試料の存在下における、発蛍光性化合物又はその塩の蛍光強度が上昇した場合には、被検試料中にイオン性化合物が存在すると判断することができる。また、蛍光強度の上昇の程度により、イオン性化合物の濃度を定量することもできる。 As will be described later in the examples, for example, the fluorescence intensity of the fluorescent compound or its salt in the presence of the test sample is compared with the fluorescence intensity of the fluorescent compound or its salt in the absence of the test sample. When the fluorescence intensity increases, it can be determined that the ionic compound is present in the test sample. In addition, the concentration of the ionic compound can be quantified by the degree of increase in fluorescence intensity.
あるいは、実施例において後述するように、被検試料の存在下における、発蛍光性化合物又はその塩の分光学的特徴が、被検試料の非存在下における、発蛍光性化合物又はその塩の分光学的特徴から変化した場合に、被検試料中にイオン性化合物が存在すると判断することができる。分光学的特徴の変化としては、例えば、蛍光波長の変化、吸光特性の変化等が挙げられる。分光学的特徴の変化の検出は、例えば肉眼で行ってもよいし、例えば分光光度計を用いた測定により行ってもよい。 Alternatively, as will be described later in the examples, the spectroscopic characteristics of the fluorescent compound or its salt in the presence of the test sample are the components of the fluorescent compound or its salt in the absence of the test sample. It can be determined that the ionic compound is present in the test sample when it changes from the optical characteristics. Examples of changes in spectroscopic characteristics include changes in fluorescence wavelength and changes in absorption characteristics. The change in spectroscopic characteristics may be detected by, for example, the naked eye, or by measurement using a spectrophotometer, for example.
また、実施例において後述するように、分光学的特徴の変化に基づいて、被検試料中のイオン性化合物の種類を特定することもできる。 Further, as will be described later in the examples, the type of the ionic compound in the test sample can be specified based on the change in the spectroscopic characteristics.
次に実験例を示して本発明を更に詳細に説明するが、本発明は以下の実験例に限定されるものではない。 Next, the present invention will be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.
[実験例1]
下記合成スキーム1にしたがって、実施例1の発蛍光性化合物(以下、「OPV−G」という場合がある。)を合成した。
[Experimental Example 1]
The fluorescent compound of Example 1 (hereinafter, may be referred to as “OPV-G”) was synthesized according to the following
(化合物2の合成)
2−(2−aminoethoxy)ethanol1(16.0g,152mmol)を乾燥ジクロロメタン(120mL)に溶解し、それにBoc2O(33.2g,150mmol)を加えた。反応混合物を室温で3時間撹拌した後、溶媒を減圧留去した。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2を25.6g得た。収率82%。
(Synthesis of Compound 2)
2- (2-aminoethoxy) etherol1 (16.0 g, 152 mmol) was dissolved in dry dichloromethane (120 mL) and Boc 2 O (33.2 g, 150 mmol) was added thereto. The reaction mixture was stirred at room temperature for 3 hours, and then the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 25.6 g of
1H NMR(300MHz,CDCl3):δ4.92(br−s,1H),3.74(m,2H),3.56(m,2H),3.34(m,2H),2.14(s,1H),1.45(s,9H). 1 1 H NMR (300 MHz, CDCl 3 ): δ4.92 (br-s, 1H), 3.74 (m, 2H), 3.56 (m, 2H), 3.34 (m, 2H), 2. 14 (s, 1H), 1.45 (s, 9H).
(化合物3の合成)
化合物2(25.6g,125mmol)及び四臭化炭素(41.4g,125mmol)を乾燥ジクロロメタン(200mL)に溶解し、それにトリフェニルホスフィン(32.8g,125mmol)を加えた。反応混合物を室温で22時間撹拌した後、溶媒を減圧留去した。粗製物はシリカゲルカラムクロマトグラフで精製し、無色固体として化合物3を28.6g得た。収率85%。
(Synthesis of Compound 3)
Compound 2 (25.6 g, 125 mmol) and carbon tetrabromide (41.4 g, 125 mmol) were dissolved in dry dichloromethane (200 mL), to which triphenylphosphine (32.8 g, 125 mmol) was added. The reaction mixture was stirred at room temperature for 22 hours, and then the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 28.6 g of
1H NMR(300MHz,CDCl3):δ4.91(br−s,1H),3.76(m,2H),3.55(m,2H),3.47(m,2H),3.33(m,2H),1.45(s,9H). 1 1 H NMR (300 MHz, CDCl 3 ): δ4.91 (br-s, 1H), 3.76 (m, 2H), 3.55 (m, 2H), 3.47 (m, 2H), 3. 33 (m, 2H), 1.45 (s, 9H).
(化合物4の合成)
p−ヒドロキシベンズアルデヒド(1.82g,14.9mmol)、炭酸カリウム(4.12g,29.8mmol)及び化合物3(4.00g,14.9mmol)を乾燥DMF(20mL)に懸濁させ、65℃で6時間加熱した。室温まで放冷したのち、酢酸エチル(200mL)を加え、水、飽和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4を3.97g得た。収率86%。
(Synthesis of Compound 4)
P-Hydroxybenzaldehyde (1.82 g, 14.9 mmol), potassium carbonate (4.12 g, 29.8 mmol) and compound 3 (4.00 g, 14.9 mmol) were suspended in dry DMF (20 mL) at 65 ° C. Was heated for 6 hours. After allowing to cool to room temperature, ethyl acetate (200 mL) was added, and the mixture was washed in the order of water and saturated brine. 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 3.97 g of
1H NMR(300MHz,CDCl3):δ9.89(s,1H),7.84(d,J=8.9Hz,2H),7.04(d,J=8.9Hz,2H),4.96(br−s,1H),4.21(m,2H),3.86(m,2H),3.62(t,J=5.2Hz,2H),3.35(q,J=5.2Hz,2H),1.44(s,9H). 1 1 H NMR (300 MHz, CDCl 3 ): δ9.89 (s, 1H), 7.84 (d, J = 8.9 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 4 .96 (br-s, 1H), 4.21 (m, 2H), 3.86 (m, 2H), 3.62 (t, J = 5.2Hz, 2H), 3.35 (q, J) = 5.2Hz, 2H), 1.44 (s, 9H).
(化合物5の合成)
化合物4(3.97g,12.8mmol)及びxylylene dicyanide(1.00g,6.42mmol)を乾燥エタノール(50mL)に溶解した。そこにBu4NOH(40% in water)を5滴加え、3時間加熱還流した。得られた沈殿物を回収し、エタノールで洗浄し、黄色固体として化合物5を4.28g得た。収率90%。
(Synthesis of Compound 5)
Compound 4 (3.97 g, 12.8 mmol) and xylylene dicyanide (1.00 g, 6.42 mmol) were dissolved in dry ethanol (50 mL). Five drops of Bu 4 NOW (40% in water) were added thereto, and the mixture was heated under reflux for 3 hours. The obtained precipitate was collected and washed with ethanol to obtain 4.28 g of
1H NMR(300MHz,CDCl3):δ7.92(d,J=8.9Hz,4H),7.72(s,4H),7.52(s,2H),7.02(d,J=8.9Hz,4H),4.96(s,2H),4.20(m,4H),3.86(m,4H),3.63(t,J=5.1Hz,4H),3.36(q,J=5.1Hz,4H),1.45(s,18H). 1 1 H NMR (300 MHz, CDCl 3 ): δ7.92 (d, J = 8.9 Hz, 4H), 7.72 (s, 4H), 7.52 (s, 2H), 7.02 (d, J) = 8.9Hz, 4H), 4.96 (s, 2H), 4.20 (m, 4H), 3.86 (m, 4H), 3.63 (t, J = 5.1Hz, 4H), 3.36 (q, J = 5.1Hz, 4H), 1.45 (s, 18H).
(化合物6の合成)
化合物5(3.0g,4.06mmol)を乾燥ジクロロメタン(15mL)に懸濁させ、それにトリフルオロ酢酸(10mL,130mmol)を加えた。反応混合物を室温で21時間撹拌した後、乾燥ジエチルエーテルを加え、黄色固体として化合物6を2.99g得た。収率96%。
(Synthesis of Compound 6)
Compound 5 (3.0 g, 4.06 mmol) was suspended in dry dichloromethane (15 mL) and trifluoroacetic acid (10 mL, 130 mmol) was added thereto. After stirring the reaction mixture at room temperature for 21 hours, dry diethyl ether was added to obtain 2.99 g of
1H NMR(300MHz,DMSO−d6):δ8.09(s,2H),8.00(d,J=9.0Hz,4H),7.87(s,4H),7.84(s,6H),7.16(d,J=9.0Hz,4H),4.25(m,4H),3.84(m,4H),3.68(t,J=5.2Hz,4H),3.03(t,J=5.2Hz,4H). 1 1 H NMR (300 MHz, DMSO-d 6 ): δ8.09 (s, 2H), 8.00 (d, J = 9.0 Hz, 4H), 7.87 (s, 4H), 7.84 (s) , 6H), 7.16 (d, J = 9.0Hz, 4H), 4.25 (m, 4H), 3.84 (m, 4H), 3.68 (t, J = 5.2Hz, 4H) ), 3.03 (t, J = 5.2Hz, 4H).
(化合物7の合成)
化合物6(1.53g,2.00mmol)を乾燥ジクロロメタン(25mL)に溶解し、それにトリエチルアミン(0.61mL,4.40mmol)及び1−H−pyrazole−1−(N,N’−bis(tert−butyloxycarbonyl))carboxamidine(1.86g,6.00mmol)を加えた。反応混合物を室温で3日間撹拌した。その後、クロロホルム(100mL)を加え、水、飽和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、黄色固体として化合物7を1.40g得た。収率68%。
(Synthesis of Compound 7)
Compound 6 (1.53 g, 2.00 mmol) was dissolved in dry dichloromethane (25 mL) with triethylamine (0.61 mL, 4.40 mmol) and 1-H-pyrazole-1- (N, N'-bis (tert). -Butyloxycarbonyl)) carboxamide (1.86 g, 6.00 mmol) was added. The reaction mixture was stirred at room temperature for 3 days. Then, chloroform (100 mL) was added, and the mixture was washed with water and saturated 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 1.40 g of Compound 7 as a yellow solid. Yield 68%.
1H NMR(300MHz,CDCl3):δ11.49(s,2H),8.69(br−s,2H),7.91(d,J=8.9Hz,4H),7.72(s,4H),7.52(s,2H),7.03(d,J=8.9Hz,4H),4.22(m,4H),3.89(m,4H),3.70(m,8H),1.51(s,18H),1.49(s,18H). 1 1 H NMR (300 MHz, CDCl 3 ): δ11.49 (s, 2H), 8.69 (br-s, 2H), 7.91 (d, J = 8.9 Hz, 4H), 7.72 (s) , 4H), 7.52 (s, 2H), 7.03 (d, J = 8.9Hz, 4H), 4.22 (m, 4H), 3.89 (m, 4H), 3.70 ( m, 8H), 1.51 (s, 18H), 1.49 (s, 18H).
(OPV−G(実施例1)の合成)
化合物7(1.27g,1.24mmol)を乾燥ジクロロメタン(15mL)に懸濁させ、それにトリフルオロ酢酸(5.0mL,65.0mmol)を加えた。反応混合物を室温で17時間撹拌した後、乾燥ジエチルエーテルを加え、黄色固体としてOPV−Gを1.00g得た。収率95%。
(Synthesis of OPV-G (Example 1))
Compound 7 (1.27 g, 1.24 mmol) was suspended in dry dichloromethane (15 mL) and trifluoroacetic acid (5.0 mL, 65.0 mmol) was added thereto. After stirring the reaction mixture at room temperature for 17 hours, dry diethyl ether was added to obtain 1.00 g of OPV-G as a yellow solid. Yield 95%.
1H NMR(300MHz,DMSO−d6):δ8.09(s,2H),8.00(d,J=8.9Hz,4H),7.87(s,4H),7.60(s,2H),7.15(d,J=8.9Hz,4H),4.23(m,4H),3.82(m,4H),3.59(t,J=5.2Hz,4H),3.32(t,J=5.2Hz,4H).
MS(MALDI):m/z=623.37([M−H+−2CF3COO−]+).
calcd(%)for C38H40F6N8O8(OPV−G):C53.65,H4.74,N13.17;found:C53.51,H4.82,N13.11.
1 1 H NMR (300 MHz, DMSO-d 6 ): δ8.09 (s, 2H), 8.00 (d, J = 8.9 Hz, 4H), 7.87 (s, 4H), 7.60 (s) , 2H), 7.15 (d, J = 8.9Hz, 4H), 4.23 (m, 4H), 3.82 (m, 4H), 3.59 (t, J = 5.2Hz, 4H) ), 3.32 (t, J = 5.2Hz, 4H).
MS (MALDI): m / z = 623.37 ([M-H + -
calcd (%) for C 38 H 40 F 6 N 8 O 8 (OPV-G): C53.65, H4.74, N13.17; found: C53.51, H4.82, N13.11.
[実験例2]
下記合成スキーム2にしたがって、実施例2〜7の発蛍光性化合物を合成した。以下、実施例2〜7の化合物を、それぞれ「OPV−A」(実施例2)、「OPV−ImMe」(実施例3)、「OPV−ImC2OMe」(実施例4)、「OPV−ImC2OH」(実施例5)、「OPV−ImDEO」(実施例6)、「OPV−ImTEO」(実施例7)という場合がある。
[Experimental Example 2]
The fluorescent compounds of Examples 2 to 7 were synthesized according to the following
(化合物8の合成)
p−ヒドロキシベンズアルデヒド(4.69g,38.4mmol)及びxylylenedicyanide(3.00g,19.2mmol)を乾燥エタノール(50mL)に溶解した。そこに酢酸(3.0mL)及びピペリジン(5.0mL)を加え、20時間加熱還流した。得られた沈殿物を回収した後、エタノールで洗浄し、黄色固体として化合物8を5.84g得た。収率83%。
(Synthesis of Compound 8)
P-Hydroxybenzaldehyde (4.69 g, 38.4 mmol) and xyllenedicyanide (3.00 g, 19.2 mmol) were dissolved in dry ethanol (50 mL). Acetic acid (3.0 mL) and piperidine (5.0 mL) were added thereto, and the mixture was heated under reflux for 20 hours. After recovering the obtained precipitate, it was washed with ethanol to obtain 5.84 g of
(化合物9の合成)
化合物8(2.00g,5.49mmol)、炭酸カリウム(3.00g,21.7mmol)及び1,1’−Oxybis(2−bromoethane)(12.8g,55.2mmol)を乾燥アセトン(50mL)に懸濁させ、65℃で6時間加熱還流した。室温まで放冷した後、固形物をろ別し、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、黄色固体として化合物9を1.57g得た。収率43%。
(Synthesis of Compound 9)
Compound 8 (2.00 g, 5.49 mmol), potassium carbonate (3.00 g, 21.7 mmol) and 1,1'-Oxybis (2-bromoethane) (12.8 g, 55.2 mmol) in dry acetone (50 mL). It was suspended in 1 and refluxed by heating at 65 ° C. for 6 hours. After allowing to cool to room temperature, the solid matter was filtered off, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1.57 g of
1H NMR(400MHz,CDCl3):δ7.91(d,J=9.0Hz,4H),7.72(s,4H),7.52(s,2H),7.02(d,J=9.0Hz,4H),4.22(m,4H),3.93−3.89(m,8H),3.51(t,J=6.4Hz,4H). 1 1 H NMR (400 MHz, CDCl 3 ): δ7.91 (d, J = 9.0 Hz, 4H), 7.72 (s, 4H), 7.52 (s, 2H), 7.02 (d, J) = 9.0Hz, 4H), 4.22 (m, 4H), 3.93-3.89 (m, 8H), 3.51 (t, J = 6.4Hz, 4H).
(OPV−A、OPV−ImMe、OPV−ImC2OMe、OPV−ImC2OH、OPV−ImDEO、OPV−ImTEOの合成)
化合物9(350mg,0.53mmol)をアセトニトリル‐エタノール(2:1、v/v)の混合溶媒(15mL)に溶解し、それに10等量のアミン化合物あるいはイミダゾール化合物を加えた。反応混合物は24時間加熱還流した。室温まで放冷した後、析出した固体をろ取し、黄色固体を得た。収率44〜84%。
(Synthesis of OPV-A, OPV-ImMe, OPV-ImC2OMe, OPV-ImC2OH, OPV-ImDEO, OPV-ImTEO)
Compound 9 (350 mg, 0.53 mmol) was dissolved in a mixed solvent (15 mL) of acetonitrile-ethanol (2: 1, v / v), and 10 equal amounts of an amine compound or an imidazole compound was added thereto. The reaction mixture was heated to reflux for 24 hours. After allowing to cool to room temperature, the precipitated solid was collected by filtration to obtain a yellow solid. Yield 44-84%.
《OPV−A(実施例2)》
1H NMR(400MHz,DMSO−d6):δ8.09(s,2H),7.99(d,J=9.0Hz,4H),7.87(s,4H),7.15(d,J=9.0Hz,4H),5.27(t,J=5.0Hz,2H),4.25(m,4H),3.94(br−s,4H),3.84(br−t,8H),3.63(m,4H),3.48(m,4H),3.13(s,12H).
calcd(%)forC40H52Br2N4O6(OPV−A):C56.88,H6.21,N6.63;found:C56.62,H6.18,N6.62.
<< OPV-A (Example 2) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ8.09 (s, 2H), 7.99 (d, J = 9.0 Hz, 4H), 7.87 (s, 4H), 7.15 (d) , J = 9.0Hz, 4H), 5.27 (t, J = 5.0Hz, 2H), 4.25 (m, 4H), 3.94 (br-s, 4H), 3.84 (br) -T, 8H), 3.63 (m, 4H), 3.48 (m, 4H), 3.13 (s, 12H).
calcd (%) forC 40 H 52 Br 2 N 4 O 6 (OPV-A): C56.88, H6.21, N6.63; found: C56.62, H6.18, N6.62.
《OPV−ImMe(実施例3)》
1H NMR(400MHz,DMSO−d6):δ9.09(s,2H),8.09(s,2H),7.99(d,J=9.0Hz,4H),7.87(s,4H),7.75(t,J=1.8Hz,2H),7.70(t,J=1.8Hz,2H),7.12(d,J=9.0Hz,4H),4.39(t,J=5.0Hz,4H),4.20(m,4H),3.86(t,J=5.0Hz,4H),3.84(s,6H),3.81(m,4H).
calcd(%)forC40H42Br2N6O4(OPV−ImMe):C57.84,H5.10,N10.12;found:C57.94,H5.13,N10.04.
<< OPV-IMMe (Example 3) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ9.09 (s, 2H), 8.09 (s, 2H), 7.99 (d, J = 9.0 Hz, 4H), 7.87 (s) , 4H), 7.75 (t, J = 1.8Hz, 2H), 7.70 (t, J = 1.8Hz, 2H), 7.12 (d, J = 9.0Hz, 4H), 4 .39 (t, J = 5.0Hz, 4H), 4.20 (m, 4H), 3.86 (t, J = 5.0Hz, 4H), 3.84 (s, 6H), 3.81 (M, 4H).
calcd (%) forC 40 H 42 Br 2 N 6 O 4 (OPV-IMMe): C57.84, H5.10, N10.12; found: C57.94, H5.13, N10.04.
《OPV−ImC2OMe(実施例4)》
1H NMR(400MHz,DMSO−d6):δ9.14(s,2H),8.09(s,2H),7.99(d,J=9.2Hz,4H),7.87(s,4H),7.77(t,J=1.8Hz,2H),7.76(t,J=1.8Hz,2H),7.12(d,J=9.2Hz,4H),4.42(t,J=5.2Hz,4H),4.36(t,J=5.0Hz,4H),4.19(m,4H),3.87(t,J=5.2Hz,4H),3.81(m,4H),3.68(t,J=5.0Hz,4H),3.25(s,6H).
calcd(%)forC44H50Br2N6O6(OPV−ImC2OMe):C57.52,H5.49,N9.15;found:C57.32,H5.44,N9.12.
<< OPV-ImC2OMe (Example 4) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ9.14 (s, 2H), 8.09 (s, 2H), 7.99 (d, J = 9.2 Hz, 4H), 7.87 (s) , 4H), 7.77 (t, J = 1.8Hz, 2H), 7.76 (t, J = 1.8Hz, 2H), 7.12 (d, J = 9.2Hz, 4H), 4 .42 (t, J = 5.2Hz, 4H), 4.36 (t, J = 5.0Hz, 4H), 4.19 (m, 4H), 3.87 (t, J = 5.2Hz, 4H), 3.81 (m, 4H), 3.68 (t, J = 5.0Hz, 4H), 3.25 (s, 6H).
calcd (%) forC 44 H 50 Br 2 N 6 O 6 (OPV-ImC2OMe): C57.52, H5.49, N9.15; found: C57.32, H5.44, N9.12.
《OPV−ImC2OH(実施例5)》
1H NMR(400MHz,DMSO−d6):δ9.13(s,2H),8.09(s,2H),7.99(d,J=9.2Hz,4H),7.87(s,4H),7.76(t,J=1.8Hz,2H),7.75(t,J=1.8Hz,2H),7.12(d,J=9.2Hz,4H),5.18(t,J=5.3Hz,2H),4.42(t,J=5.2Hz,4H),4.23−4.19(m,8H),3.87(t,J=5.2Hz,4H),3.82(m,4H),3.72(q,J=5.3Hz,4H).
calcd(%)forC42H46Br2N6O6(OPV−ImC2OH):C56.64,H5.21,N9.44;found:C56.66,H5.24,N9.42.
<< OPV-ImC2OH (Example 5) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ9.13 (s, 2H), 8.09 (s, 2H), 7.99 (d, J = 9.2 Hz, 4H), 7.87 (s) , 4H), 7.76 (t, J = 1.8Hz, 2H), 7.75 (t, J = 1.8Hz, 2H), 7.12 (d, J = 9.2Hz, 4H), 5 .18 (t, J = 5.3Hz, 2H), 4.42 (t, J = 5.2Hz, 4H), 4.23-4.19 (m, 8H), 3.87 (t, J = 5.2Hz, 4H), 3.82 (m, 4H), 3.72 (q, J = 5.3Hz, 4H).
calcd (%) forC 42 H 46 Br 2 N 6 O 6 (OPV-ImC2OH): C56.64, H5.21, N9.44; found: C56.66, H5.24, N9.42.
《OPV−ImDEO(実施例6)》
1H NMR(400MHz,DMSO−d6):δ9.12(s,2H),8.08(s,2H),7.99(d,J=8.8Hz,4H),7.87(s,4H),7.77(t,J=1.8Hz,2H),7.75(t,J=1.8Hz,2H),7.12(d,J=8.8Hz,4H),4.42(t,J=5.0Hz,4H),4.35(t,J=5.0Hz,4H),4.19(m,4H),3.87(t,J=5.0Hz,4H),3.81(m,4H),3.76(t,J=5.0Hz,4H),3.53(m,4H),3.40(m,4H),3.21(s,6H).
calcd(%)forC48H58Br2N6O8(OPV−ImDEO):C57.26,H5.81,N8.35;found:C57.22,H5.87,N8.21.
<< OPV-ImDEO (Example 6) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ9.12 (s, 2H), 8.08 (s, 2H), 7.99 (d, J = 8.8 Hz, 4H), 7.87 (s) , 4H), 7.77 (t, J = 1.8Hz, 2H), 7.75 (t, J = 1.8Hz, 2H), 7.12 (d, J = 8.8Hz, 4H), 4 .42 (t, J = 5.0Hz, 4H), 4.35 (t, J = 5.0Hz, 4H), 4.19 (m, 4H), 3.87 (t, J = 5.0Hz, 4H), 3.81 (m, 4H), 3.76 (t, J = 5.0Hz, 4H), 3.53 (m, 4H), 3.40 (m, 4H), 3.21 (s) , 6H).
calcd (%) forC 48 H 58 Br 2 N 6 O 8 (OPV-ImDEO): C57.26, H5.81, N8.35; found: C57.22, H5.87, N8.21.
《OPV−ImTEO(実施例7)》
1H NMR(400MHz,DMSO−d6):δ9.13(s,2H),8.08(s,2H),7.99(d,J=9.0Hz,4H),7.87(s,4H),7.77(t,J=1.8Hz,2H),7.76(t,J=1.8Hz,2H),7.12(d,J=9.0Hz,4H),4.39(t,J=5.0Hz,4H),3.77(t,J=5.0Hz,4H),3.55−3.53(m,4H),3.49−3.45(m,8H),3.42−3.40(m,4H),3.23(s,6H).
<< OPV-ImTEO (Example 7) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ9.13 (s, 2H), 8.08 (s, 2H), 7.99 (d, J = 9.0 Hz, 4H), 7.87 (s) , 4H), 7.77 (t, J = 1.8Hz, 2H), 7.76 (t, J = 1.8Hz, 2H), 7.12 (d, J = 9.0Hz, 4H), 4 .39 (t, J = 5.0Hz, 4H), 3.77 (t, J = 5.0Hz, 4H), 3.55-3.53 (m, 4H), 3.49-3.45 ( m, 8H), 3.42-3.40 (m, 4H), 3.23 (s, 6H).
[実験例3]
下記合成スキーム3にしたがって、化合物10a、10b及び10cを出発材料として、それぞれ実施例8〜10の発蛍光性化合物を合成した。スキーム3中、「*」は結合手を表す。以下、実施例8〜10の化合物を、それぞれ「G2」(実施例8)、「G4」(実施例9)、「G6」(実施例10)という場合がある。各化合物の合成手順は、上述した合成スキーム1と同様であった。より具体的には、化合物10aから11a、12a、13aを経てG2(実施例8)を得た。また、化合物10bから11b、12b、13bを経てG4(実施例9)を得た。また、化合物10cから11c、12c、13cを経てG6(実施例8)を得た。
[Experimental Example 3]
According to the following
《G2(実施例8)》
1H NMR(400MHz,DMSO−d6):δ8.08(s,2H),7.99(d,J=9.2Hz,4H),7.86(s,4H),7.63(t,J=5.4Hz,2H),7.14(d,J=9.2Hz,4H),7.13(br−s,8H),4.13(t,J=6.2Hz,4H),3.30(q,J=6.2Hz,4H),1.97(quint,J=6.2Hz,4H).
MS(MALDI):m/z=563.1[M−H+−2CF3COO−]+.
calcd(%)for C36H36F6N8O6(G2):C54.68,H4.59,N14.17;found:C54.30,H4.55,N14.07.
<< G2 (Example 8) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ8.08 (s, 2H), 7.99 (d, J = 9.2 Hz, 4H), 7.86 (s, 4H), 7.63 (t) , J = 5.4Hz, 2H), 7.14 (d, J = 9.2Hz, 4H), 7.13 (br-s, 8H), 4.13 (t, J = 6.2Hz, 4H) , 3.30 (q, J = 6.2Hz, 4H), 1.97 (quint, J = 6.2Hz, 4H).
MS (MALDI): m / z = 563.1 [M-H + -
calcd (%) for C 36 H 36 F 6 N 8 O 6 (G2): C54.68, H4.59, N14.17; found: C54.30, H4.55, N14.07.
《G4(実施例9)》
1H NMR(400MHz,DMSO−d6):δ8.07(s,2H),7.86(s,4H),7.78(t,J=5.4Hz,2H),7.77(t,J=5.4Hz,2H),7.73(d,J=2.0Hz,2H),7.61(dd,J=8.8,2.0Hz,2H),7.21(br−s,16H),7.20(d,J=8.8Hz,2H),4.13(t,J=6.4Hz,4H),4.10(t,J=6.4Hz,4H),3.28(m,8H),2.00(quint,J=6.4Hz,4H),1.98(quint,J=6.4Hz,4H).
MS(MALDI):m/z=793.0[M−3H+−4CF3COO−]+.
HRMS(ESI):m/z calcd for[M−CF3COO−]+:1135.4155;found:1135.4157.
<< G4 (Example 9) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ8.07 (s, 2H), 7.86 (s, 4H), 7.78 (t, J = 5.4 Hz, 2H), 7.77 (t) , J = 5.4Hz, 2H), 7.73 (d, J = 2.0Hz, 2H), 7.61 (dd, J = 8.8, 2.0Hz, 2H), 7.21 (br- s, 16H), 7.20 (d, J = 8.8Hz, 2H), 4.13 (t, J = 6.4Hz, 4H), 4.10 (t, J = 6.4Hz, 4H), 3.28 (m, 8H), 2.00 (quint, J = 6.4Hz, 4H), 1.98 (quint, J = 6.4Hz, 4H).
MS (MALDI): m / z = 793.0 [M-3H + -
HRMS (ESI): m / z calcd for [M-
《G6(実施例10)》
1H NMR(400MHz,DMSO−d6):δ8.09(s,2H),7.88(s,4H),7.86(t,J=5.6Hz,4H),7.75(t,J=5.6Hz,2H),7.40(s,4H),7.25(br−s,24H),4.09(t,J=6.4Hz,8H),4.05(t,J=6.4Hz,4H),3.35(q,J=6.4Hz,4H),3.30(q,J=6.4Hz,8H),2.01(quint,J=6.4Hz,8H),1.88(quint,J=6.4Hz,4H).
MS(MALDI):m/z=1023.2[M−5H+−6CF3COO−]+.
HRMS(ESI):m/z calcd for[M−CF3COO−]+:1593.5503;found:1593.5510.
<< G6 (Example 10) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ8.09 (s, 2H), 7.88 (s, 4H), 7.86 (t, J = 5.6 Hz, 4H), 7.75 (t) , J = 5.6Hz, 2H), 7.40 (s, 4H), 7.25 (br-s, 24H), 4.09 (t, J = 6.4Hz, 8H), 4.05 (t) , J = 6.4Hz, 4H), 3.35 (q, J = 6.4Hz, 4H), 3.30 (q, J = 6.4Hz, 8H), 2.01 (quint, J = 6. 4Hz, 8H), 1.88 (quint, J = 6.4Hz, 4H).
MS (MALDI): m / z = 1023.2 [M-5H + -
HRMS (ESI): m / z calcd for [M-
[実験例4]
下記合成スキーム4にしたがって、実施例11の発蛍光性化合物を合成した。スキーム4中、「*」は結合手を表す。以下、実施例11の化合物を「DEO−OPV−CN−G」という場合がある。
[Experimental Example 4]
The fluorescent compound of Example 11 was synthesized according to the following
(化合物14の合成)
アニリン(1.61g,17.3mmol)、炭酸カリウム(9.56g,69.2mmol)、ヨウ化カリウム(1.43g,8.65mmol)及び1−bromo−2−(2−methoxyethoxy)ethane(9.50g,51.9mmol)を乾燥DMF(5.0mL)に懸濁させ、80℃で24時間加熱した。室温まで放冷したのち、酢酸エチル(200 mL)を加え、水、飽和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14を4.27g得た。収率83%。
(Synthesis of Compound 14)
Aniline (1.61 g, 17.3 mmol), potassium carbonate (9.56 g, 69.2 mmol), potassium iodide (1.43 g, 8.65 mmol) and 1-bromo-2- (2-methoxyethoxy) ethane (9). .50 g, 51.9 mmol) was suspended in dry DMF (5.0 mL) and heated at 80 ° C. for 24 hours. After allowing to cool to room temperature, ethyl acetate (200 mL) was added, and the mixture was washed in the order of water and saturated brine. 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 4.27 g of Compound 14 as a colorless oily product. Yield 83%.
1H NMR(400MHz,CDCl3):δ7.20(dd,J=8.4,7.6Hz,2H),6.71(d,J=8.4Hz,2H),6.66(t,J=7.6Hz,1H),3.63−3.519(m,16H),3.39(s,6H). 1 1 H NMR (400 MHz, CDCl 3 ): δ7.20 (dd, J = 8.4,7.6 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H), 6.66 (t, J = 7.6Hz, 1H), 3.63-3.519 (m, 16H), 3.39 (s, 6H).
(化合物15の合成)
化合物14(4.26g,14.3mmol)を乾燥DMF(5.0mL)に溶解し、それにオキシ三塩化リン(2.0mL,21.5mmol)を加えた。反応混合物を60℃で3時間加熱した。その後、水(100mL)、酢酸エチル(200mL)を加え、有機相を水、飽和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、黄色オイル状物として化合物15を3.48g得た。収率75%。
(Synthesis of Compound 15)
Compound 14 (4.26 g, 14.3 mmol) was dissolved in dry DMF (5.0 mL) and phosphorus oxytrichloride (2.0 mL, 21.5 mmol) was added thereto. The reaction mixture was heated at 60 ° C. for 3 hours. Then, water (100 mL) and ethyl acetate (200 mL) were added, and the organic phase was washed with water and saturated 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 3.48 g of
1H NMR(400MHz,CDCl3):δ9.73(s,1H),7.71(d,J=9.2Hz,2H),6.76(d,J=9.2Hz,2H),3.68(m,8H),3.61(m,4H),3.52(m,4H),3.38(s,6H). 1 1 H NMR (400 MHz, CDCl 3 ): δ9.73 (s, 1H), 7.71 (d, J = 9.2 Hz, 2H), 6.76 (d, J = 9.2 Hz, 2H), 3 .68 (m, 8H), 3.61 (m, 4H), 3.52 (m, 4H), 3.38 (s, 6H).
(化合物16の合成)
Diethyl(4−Cyanobenzyl)phosphonate(3.24g,12.8mmol)、水素化ナトリウム(60% in oil,642mg,16.1mmol)を乾燥THF(15mL)に懸濁させ、そこに化合物15(3.47g,10.7mmol)を加えた。反応混合物を室温で4時間撹拌した。溶媒を減圧留去した後、水を加え、反応生成物をクロロホルムで抽出した。有機相を水、飽和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、黄色固体として化合物16を3.64g得た。収率80%。
(Synthesis of Compound 16)
Diethyl (4-Cyanobenzyl) phosphonate (3.24 g, 12.8 mmol), sodium hydride (60% in oil, 642 mg, 16.1 mmol) was suspended in dry THF (15 mL), wherein compound 15 (3. 47 g, 10.7 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours. After distilling off the solvent under reduced pressure, water was added and the reaction product was extracted with chloroform. The organic phase was washed with water and saturated brine in that 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 3.64 g of compound 16 as a yellow solid.
1H NMR(400MHz,CDCl3):δ7.58(d,J=8.4Hz,2H),7.51(d,J=8.4Hz,2H),7.38(d,J=8.8Hz,2H),7.12(d,J=16Hz,1H),6.85(d,J=16Hz,1H),6.71(d,J=8.8Hz,2H),3.68−3.52(m,16H),3.39(s,6H).
13C NMR(100MHz,CDCl3):δ148.1,142.8,132.4,132.3,128.3,126.1,124.2,121.9,119.3,111.7,109.1,71.9,70.6,68.3,59.1,50.9.
1 1 H NMR (400 MHz, CDCl 3 ): δ7.58 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8. 8Hz, 2H), 7.12 (d, J = 16Hz, 1H), 6.85 (d, J = 16Hz, 1H), 6.71 (d, J = 8.8Hz, 2H), 3.68- 3.52 (m, 16H), 3.39 (s, 6H).
13 C NMR (100 MHz, CDCl 3 ): δ148.1, 142.8, 132.4, 132.3, 128.3, 126.1, 124.2, 121.9, 119.3, 111.7, 109.1, 71.9, 70.6, 68.3, 59.1, 50.9.
(化合物17の合成)
化合物16(3.64g,8.57mmol)を乾燥THF(10mL)に溶解し、それにDIBAL(1.0M THF solution,17.1mL)を加えた。反応混合物を室温で18時間撹拌した後、10%酢酸水溶液を加え、反応を停止させた。反応生成物をクロロホルムで抽出し、有機相を水、飽和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、橙色固体として化合物17を1.65g得た。収率45%。
(Synthesis of Compound 17)
Compound 16 (3.64 g, 8.57 mmol) was dissolved in dry THF (10 mL) and DIBAL (1.0 M THF solution, 17.1 mL) was added to it. The reaction mixture was stirred at room temperature for 18 hours, and then a 10% aqueous acetic acid solution was added to stop the reaction. The reaction product was extracted with chloroform, and the organic phase was washed with water and saturated brine in that 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 1.65 g of Compound 17 as an orange solid.
1H NMR(400MHz,CDCl3):δ9.96(s,1H),7.83(d,J=8.4Hz,2H),7.60(d,J=8.4Hz,2H),7.41(d,J=9.0Hz,2H),7.19(d,J=16Hz,1H),6.92(d,J=16Hz,1H),6.72(d,J=9.0Hz,2H),3.68−3.53(m,16H),3.39(s,6H).
13C NMR(100MHz,CDCl3):δ191.6,148.1,144.5,134.4,132.3,130.2,128.3,126.2,124.5,122.5,111.7,71.9,70.6,68.3,59.1,50.9.
1 1 H NMR (400 MHz, CDCl 3 ): δ9.96 (s, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7 .41 (d, J = 9.0Hz, 2H), 7.19 (d, J = 16Hz, 1H), 6.92 (d, J = 16Hz, 1H), 6.72 (d, J = 9. 0 Hz, 2H), 3.68-3.53 (m, 16H), 3.39 (s, 6H).
13 C NMR (100 MHz, CDCl 3 ): δ191.6, 148.1, 144.5, 134.4, 132.3, 130.2, 128.3, 126.2, 124.5, 122.5, 111.7, 71.9, 70.6, 68.3, 59.1, 50.9.
(化合物18の合成)
化合物17(800mg,1.87mmol)及び化合物21(600mg,1.87mmol)を乾燥エタノール(10mL)に溶解した。そこにBu4NOH(40% in water)を3滴加え、12時間加熱還流した。得られた沈殿物を回収し、エタノールで洗浄し、橙色固体として化合物18を1.11g得た。収率81%。
(Synthesis of Compound 18)
Compound 17 (800 mg, 1.87 mmol) and compound 21 (600 mg, 1.87 mmol) were dissolved in dry ethanol (10 mL). Three drops of Bu 4 NOW (40% in water) were added thereto, and the mixture was heated under reflux for 12 hours. The obtained precipitate was collected and washed with ethanol to obtain 1.11 g of compound 18 as an orange solid. Yield 81%.
1H NMR(400MHz,CDCl3):δ7.86(d,J=8.4Hz,2H),7.61(d,J=8.8Hz,2H),7.54(d,J=8.4Hz,2H),7.40(d,J=8.8Hz,2H),7.39(s,1H),7.12(d,J=16Hz,1H),6.99(d,J=8.8Hz,2H),6.91(d,J=16Hz,1H),6.72(d,J=8.8Hz,2H),4.97(br−s,1H),4.17(m,2H),3.85(m,2H),3.68−3.53(m,18H),3.39(s,6H),3.36(m,2H),1.45(s,9H).
13C NMR(100MHz,CDCl3):δ159.3,155.9,147.7,140.3,139.9,132.0,130.5,129.5,128.1,127.5,127.1,126.2,124.9,123.0,118.5,115.0,111.7,109.4,79.3,71.9,70.6,70.4,69.3,68.3,67.4,59.1,50.9,40.3,28.4.
MS(MALDI):m/z=752.71([M+Na]+).
1 1 H NMR (400 MHz, CDCl 3 ): δ7.86 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8. 4Hz, 2H), 7.40 (d, J = 8.8Hz, 2H), 7.39 (s, 1H), 7.12 (d, J = 16Hz, 1H), 6.99 (d, J = 8.8Hz, 2H), 6.91 (d, J = 16Hz, 1H), 6.72 (d, J = 8.8Hz, 2H), 4.97 (br-s, 1H), 4.17 ( m, 2H), 3.85 (m, 2H), 3.68-3.53 (m, 18H), 3.39 (s, 6H), 3.36 (m, 2H), 1.45 (s) , 9H).
13 C NMR (100 MHz, CDCl 3 ): δ159.3, 155.9, 147.7, 140.3, 139.9, 132.0, 130.5, 129.5, 128.1, 127.5. 127.1, 126.2, 124.9, 123.0, 118.5, 115.0, 111.7, 109.4, 79.3, 71.9, 70.6, 70.4, 69. 3,68,3,67,4,59.1,50.9,40.3,28.4.
MS (MALDI): m / z = 752.71 ([M + Na] + ).
(化合物19の合成)
化合物18(980mg,1.34mmol)を乾燥ジクロロメタン(3.0mL)に懸濁させ、それにトリフルオロ酢酸(2.0mL,26.1mmol)を加えた。反応混合物を室温で6時間撹拌した後、乾燥ジエチルエーテルを加え、橙色固体として化合物19を904mg得た。収率91%。
(Synthesis of Compound 19)
Compound 18 (980 mg, 1.34 mmol) was suspended in dry dichloromethane (3.0 mL) and trifluoroacetic acid (2.0 mL, 26.1 mmol) was added thereto. After stirring the reaction mixture at room temperature for 6 hours, dry diethyl ether was added to obtain 904 mg of compound 19 as an orange solid. Yield 91%.
1H NMR(400MHz,DMSO−d6):δ7.90(d,J=8.4Hz,2H),7.88(s,1H),7.73(br−s,3H),7.71(d,J=8.8Hz,2H),7.67(d,J=8.4Hz,2H),7.44(d,J=8.8Hz,2H),7.28(d,J=16Hz,1H),7.09(d,J=8.8Hz,2H),7.01(d,J=16Hz,1H),6.72(d,J=8.8Hz,2H),4.21(m,2H),3.83(m,2H),3.67(t,J=5.2Hz,2H),3.56−3.43(m,16H),3.25(s,6H),3.02(t,J=5.2Hz,2H).
13C NMR(100MHz,DMSO−d6):δ159.1,158.1(q,2J=31Hz,CF3COO),147.7,140.2,140.1,131.9,130.7,129.4,128.2,127.1,126.6,126.1,124.1,122.3,118.4,117.3(q,1J=298Hz,CF3COO),115.1,111.5,108.1,71.3,69.7,68.8,67.9,67.2,66.8,58.1,50.2,38.6.
MS (MALDI):m/z=630.63([M−CF3COO]+).
1 1 H NMR (400 MHz, DMSO-d 6 ): δ7.90 (d, J = 8.4 Hz, 2H), 7.88 (s, 1H), 7.73 (br-s, 3H), 7.71 (D, J = 8.8Hz, 2H), 7.67 (d, J = 8.4Hz, 2H), 7.44 (d, J = 8.8Hz, 2H), 7.28 (d, J = 16Hz, 1H), 7.09 (d, J = 8.8Hz, 2H), 7.01 (d, J = 16Hz, 1H), 6.72 (d, J = 8.8Hz, 2H), 4. 21 (m, 2H), 3.83 (m, 2H), 3.67 (t, J = 5.2Hz, 2H), 3.56-3.43 (m, 16H), 3.25 (s, 6H), 3.02 (t, J = 5.2Hz, 2H).
13 C NMR (100 MHz, DMSO-d 6 ): δ159.1, 158.1 (q, 2 J = 31 Hz, CF 3 COO), 147.7, 140.2, 140.1, 131.9, 130. 7, 129.4, 128.2, 127.1, 126.6, 126.1, 124.1, 122.3, 118.4, 117.3 (q, 1 J = 298 Hz, CF 3 COO), 115.1, 111.5, 108.1, 71.3, 69.7, 68.8, 67.9, 67.2, 66.8, 58.1, 50.2, 38.6.
MS (MALDI): m / z = 630.63 ([M-CF 3 COO] + ).
(化合物20の合成)
化合物19(800mg,1.08mmol)を乾燥ジクロロメタン(10mL)に溶解し、それにEt3N(0.23mL,1.62mmol)及び1−H−pyrazole−1−(N,N−bis(tert−butyloxy−carbonyl))carboxamidine(503mg,1.62mmol)を加えた。反応混合物を室温で20時間撹拌した。その後、クロロホルム(100mL)を加え、水、飽和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、橙色固体として化合物20を745mg得た。収率79%。
(Synthesis of Compound 20)
Compound 19 (800 mg, 1.08 mmol) was dissolved in dry dichloromethane (10 mL) with Et 3 N (0.23 mL, 1.62 mmol) and 1-H-pyrazole-1- (N, N-bis (tert-). Butyloxy-carbonyl)) carboxamide (503 mg, 1.62 mmol) was added. The reaction mixture was stirred at room temperature for 20 hours. Then, chloroform (100 mL) was added, and the mixture was washed with water and saturated 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 745 mg of
1H NMR(400MHz,CDCl3):δ11.5(s,1H),8.68(br−t,1H),7.85(d,J=8.6Hz,2H),7.59(d,J=8.8Hz,2H),7.53(d,J=8.6Hz,2H),7.40(d,J=8.8Hz,2H),7.39(s,1H),7.12(d,J=16Hz,1H),6.99(d,J=8.8Hz,2H),6.90(d,J=16Hz,1H),6.72(d,J=8.8Hz,2H),4.19(m,2H),3.87(m,2H),3.70−3.53(m,20H),3.39(s,6H),1.51(s,9H),1.48(s,9H).
13C NMR(100MHz,CDCl3):δ163.5,159.4,156.3,153.0,147.7,140.2,139.8,132.0,130.5,129.5,128.1,127.4,127.1,126.2,124.9,123.1,118.5,115.1,111.7,109.5,83.0,79.2,71.9,70.6,69.5,69.3,68.4,67.5,59.1,50.9,40.5,28.3,28.0.
MS(MALDI):m/z=694.65([(DEO−OPV−CN−G)−CF3COOH+Na]+).
1 1 H NMR (400 MHz, CDCl 3 ): δ11.5 (s, 1H), 8.68 (br-t, 1H), 7.85 (d, J = 8.6 Hz, 2H), 7.59 (d) , J = 8.8Hz, 2H), 7.53 (d, J = 8.6Hz, 2H), 7.40 (d, J = 8.8Hz, 2H), 7.39 (s, 1H), 7 .12 (d, J = 16Hz, 1H), 6.99 (d, J = 8.8Hz, 2H), 6.90 (d, J = 16Hz, 1H), 6.72 (d, J = 8. 8Hz, 2H), 4.19 (m, 2H), 3.87 (m, 2H), 3.70-3.53 (m, 20H), 3.39 (s, 6H), 1.51 (s) , 9H), 1.48 (s, 9H).
13 C NMR (100 MHz, CDCl 3 ): δ163.5, 159.4, 156.3, 153.0, 147.7, 140.2, 139.8, 132.0, 130.5, 129.5 128.1, 127.4, 127.1, 126.2, 124.9, 123.1, 118.5, 115.1, 111.7, 109.5, 83.0, 79.2, 71. 9,70.6,69.5,69,3,68,4,67,5,59.1,50.9,40.5,28.3,28.0.
MS (MALDI): m / z = 694.65 ([(DEO-OPV-CN-G) -CF 3 COOH + Na] + ).
(DEO−OPV−CN−G(実施例11)の合成)
化合物20(700mg,0.80mmol)を乾燥ジクロロメタン(3.0mL)に懸濁させ、それにトリフルオロ酢酸(2.0mL,26.1mmol)を加えた。反応混合物を室温で4時間撹拌した後、乾燥ジエチルエーテルを加え、赤色固体としてDEO−OPV−CN−Gを381mg得た。収率61%。
(Synthesis of DEO-OPV-CN-G (Example 11))
Compound 20 (700 mg, 0.80 mmol) was suspended in dry dichloromethane (3.0 mL) and trifluoroacetic acid (2.0 mL, 26.1 mmol) was added thereto. After stirring the reaction mixture at room temperature for 4 hours, dry diethyl ether was added to obtain 381 mg of DEO-OPV-CN-G as a red solid. Yield 61%.
1H NMR(400MHz,DMSO−d6)δ7.90(d,J=8.6Hz,2H),7.87(s,1H),7.70(d,J=9.2Hz,2H),7.66(d,J=8.6Hz,2H),7.51(t,J=5.4Hz,1H),7.44(d,J=9.2Hz,2H),7.28(d,J=16Hz,1H),7.10(br−s,4H),7.09(d,J=9.2Hz,2H),7.01(d,J=16Hz,1H),6.72(d,J=9.2Hz,2H),4.18(m,2H),3.81(m,2H),3.60−3.43(m,18H),3.33(t,J=5.4Hz,2H),3.25(s,6H).
13C NMR(100MHz,DMSO−d6)δ159.1,158.6(q,2J=32Hz,CF3COO),157.1,147.7,140.2,140.1,131.9,130.7,129.4,128.1,127.1,126.6,126.1,124.1,122.3,118.4,116.7(q,1J=296Hz,CF3COO),115.1,111.5,108.1,71.3,69.7,68.8,68.6,67.9,67.3,58.1,50.3,40.8.
MS(MALDI):m/z=672.66([M−CF3COO]+).
1 1 H NMR (400 MHz, DMSO-d 6 ) δ7.90 (d, J = 8.6 Hz, 2H), 7.87 (s, 1H), 7.70 (d, J = 9.2 Hz, 2H), 7.66 (d, J = 8.6Hz, 2H), 7.51 (t, J = 5.4Hz, 1H), 7.44 (d, J = 9.2Hz, 2H), 7.28 (d) , J = 16Hz, 1H), 7.10 (br-s, 4H), 7.09 (d, J = 9.2Hz, 2H), 7.01 (d, J = 16Hz, 1H), 6.72 (D, J = 9.2Hz, 2H), 4.18 (m, 2H), 3.81 (m, 2H), 3.60-3.43 (m, 18H), 3.33 (t, J) = 5.4Hz, 2H), 3.25 (s, 6H).
13 C NMR (100 MHz, DMSO-d 6 ) δ159.1, 158.6 (q, 2 J = 32 Hz, CF 3 COO), 157.1, 147.7, 140.2, 140.1, 131.9 , 130.7, 129.4, 128.1, 127.1, 126.6, 126.1, 124.1, 122.3, 118.4, 116.7 (q, 1 J = 296Hz, CF 3) COO), 115.1, 111.5, 108.1, 71.3, 69.7, 68.8, 68.6, 67.9, 67.3, 58.1, 50.3, 40.8 ..
MS (MALDI): m / z = 672.66 ([M-CF 3 COO] + ).
[実験例5]
上述した合成スキーム4と同様な合成手順にしたがって、下記式(5)で表される実施例12〜15の発蛍光性化合物を合成した。
[Experimental Example 5]
The fluorescent compounds of Examples 12 to 15 represented by the following formula (5) were synthesized according to the same synthesis procedure as the
式(5)中、R5が−CH3であるものが実施例12の化合物であり(以下、「Me−OPV−CN−G」という場合がある。)、R5が−n−C4H9であるものが実施例13の化合物であり(以下、「C4−OPV−CN−G」という場合がある。)、R5が下記式(6)で表される基であるものが実施例14の化合物であり(以下、「C4 *−OPV−CN−G」という場合がある。)、R5が−n−C12H25であるものが実施例15の化合物である(以下、「C12−OPV−CN−G」という場合がある。)。
In formula (5), the compound in which R 5 is -CH 3 is the compound of Example 12 (hereinafter, may be referred to as "Me-OPV-CN-G"), and R 5 is -n-C 4 what is H 9 are the compound of example 13 (hereinafter sometimes referred to as "C 4 -OPV-CN-G".), those wherein R 5 is a group represented by the following formula (6) The compound of Example 14 (hereinafter, may be referred to as “C 4 * -OPV-CN-G”), and the compound in which R 5 is −n—C 12 H 25 is the compound of Example 15 (hereinafter, it may be referred to as “
《Me−OPV−CN−G(実施例12)》
1H NMR(400MHz,DMSO−d6):δ7.91(d,J=8.6Hz,2H),7.87(s,1H),7.70(d,J=9.0Hz,2H),7.67(d,J=8.6Hz,2H),7.52(t,J=5.4Hz,1H),7.47(d,J=8.8Hz,2H),7.30(d,J=16Hz,1H),7.10(br−s,4H),7.09(d,J=9.0Hz,2H),7.03(d,J=16Hz,1H),6.74(d,J=8.8Hz,2H),4.19(m,2H),3.81(m,2H),3.59(m,2H),3.33(q,J=5.4Hz,2H),2.95(s,6H).
13C NMR(100MHz,DMSO−d6):δ159.1,158.7(q,2J=31Hz,CF3COO),157.1,150.2,140.2,140.1,131.9,130.8,129.4,127.9,127.1,126.5,126.1,124.5,122.5,118.4,117.1(q,1J=298Hz,CF3COO),115.1,112.2,108.2,68.8,68.6,67.3,40.8(One peak is overlapped into solvent peak at ca.40ppm).
MS(MALDI):m/z=496.48([M−CF3COO]+).
<< Me-OPV-CN-G (Example 12) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ7.91 (d, J = 8.6 Hz, 2H), 7.87 (s, 1H), 7.70 (d, J = 9.0 Hz, 2H) , 7.67 (d, J = 8.6Hz, 2H), 7.52 (t, J = 5.4Hz, 1H), 7.47 (d, J = 8.8Hz, 2H), 7.30 ( d, J = 16Hz, 1H), 7.10 (br-s, 4H), 7.09 (d, J = 9.0Hz, 2H), 7.03 (d, J = 16Hz, 1H), 6. 74 (d, J = 8.8Hz, 2H), 4.19 (m, 2H), 3.81 (m, 2H), 3.59 (m, 2H), 3.33 (q, J = 5. 4Hz, 2H), 2.95 (s, 6H).
13 C NMR (100 MHz, DMSO-d 6 ): δ159.1, 158.7 (q, 2 J = 31 Hz, CF 3 COO), 157.1, 150.2, 140.2, 140.1, 131. 9, 130.8, 129.4, 127.9, 127.1, 126.5, 126.1, 124.5, 122.5, 118.4, 117.1 (q, 1 J = 298 Hz, CF 3 COO), 115.1, 112.2, 108.2, 68.8, 68.6, 67.3, 40.8 (One peak is overlapped into solvent peak at ca. 40 ppm).
MS (MALDI): m / z = 496.48 ([M-CF 3 COO] + ).
《C4−OPV−CN−G(実施例13)》
1H NMR(400MHz,DMSO−d6):δ7.90(d,J=8.4Hz,2H),7.85(s,1H),7.70(d,J=9.0Hz,2H),7.65(d,J=8.4Hz,2H),7.49(t,J=5.6Hz,1H),7.42(d,J=8.6Hz,2H),7.25(d,J=16Hz,1H),7.10(br−s,4H),7.08(d,J=9.0Hz,2H),6.97(d,J=16Hz,1H),6.66(d,J=8.6Hz,2H),4.19(m,2H),3.81(m,2H),3.60(t,J=5.6Hz,2H),3.33(q,J=5.6Hz,2H),3.30(t,J=7.6Hz,4H),1.52(quint,J=7.6Hz,4H),1.33(sext,J=7.6Hz,4H),0.93(t,J=7.6Hz,6H).
13C NMR(100MHz,DMSO−d6):δ159.0,158.3(q,2J=32Hz,CF3COO),157.1,147.7,140.13,140.11,131.7,130.8,129.3,128.1,127.0,126.6,126.0,123.7,122.0,118.3,116.8(q,1J=297Hz,CF3COO),115.1,111.6,108.1,68.7,68.6,67.3,49.9,40.8,29.0,19.6,13.7.
MS(MALDI):m/z=580.57([M−CF3COO]+).
<< C 4- OPV-CN-G (Example 13) >>
1 H NMR (400 MHz, DMSO-d 6 ): δ7.90 (d, J = 8.4 Hz, 2H), 7.85 (s, 1H), 7.70 (d, J = 9.0 Hz, 2H) , 7.65 (d, J = 8.4Hz, 2H), 7.49 (t, J = 5.6Hz, 1H), 7.42 (d, J = 8.6Hz, 2H), 7.25 ( d, J = 16Hz, 1H), 7.10 (br-s, 4H), 7.08 (d, J = 9.0Hz, 2H), 6.97 (d, J = 16Hz, 1H), 6. 66 (d, J = 8.6Hz, 2H), 4.19 (m, 2H), 3.81 (m, 2H), 3.60 (t, J = 5.6Hz, 2H), 3.33 ( q, J = 5.6Hz, 2H), 3.30 (t, J = 7.6Hz, 4H), 1.52 (quint, J = 7.6Hz, 4H), 1.33 (sext, J = 7) .6Hz, 4H), 0.93 (t, J = 7.6Hz, 6H).
13 C NMR (100 MHz, DMSO-d 6 ): δ159.0, 158.3 (q, 2 J = 32 Hz, CF 3 COO), 157.1, 147.7, 140.13, 140.11, 131. 7, 130.8, 129.3, 128.1, 127.0, 126.6, 126.0, 123.7, 122.0, 118.3, 116.8 (q, 1 J = 297Hz, CF 3 COO), 115.1, 111.6, 108.1, 68.7, 68.6, 67.3, 49.9, 40.8, 29.0, 19.6, 13.7.
MS (MALDI): m / z = 580.57 ([M-CF 3 COO] + ).
《C4 *−OPV−CN−G(実施例14)》
1H NMR(400MHz,DMSO−d6):δ7.90(d,J=8.6Hz,2H),7.87(s,1H),7.70(d,J=8.6Hz,2H),7.65(d,J=8.6Hz,2H),7.55(br−s,1H),7.42(d,J=9.0Hz,2H),7.26(d,J=16Hz,1H),7.10(br−s,4H),7.09(d,J=8.6Hz,2H),6.98(d,J=16Hz,1H),6.68(d,J=9.0Hz,2H),4.19(m,2H),3.81(m,2H),3.59(m,2H),3.40−3.29(m,4H),3.07(dd,J=15Hz,8.0Hz,2H),1.80(m,2H),1.40(m,2H),1.10(m,2H),0.88(t,J=7.6Hz,6H),0.84(d,J=6.4Hz,6H).
13C NMR(100MHz,DMSO−d6):δ159.0,158.4(q,2J=31Hz,CF3COO),157.1,148.0,140.12140.11,131.7,130.7,129.3,127.9,127.0,126.5,125.9,123.5,122.0,118.3,117.1(q,1J=298Hz,CF3COO),115.1,112.2,108.1,68.7,68.6,67.3,57.6,40.8,32.4,26.4,16.7,11.2.
MS(MALDI):m/z=608.62([M−CF3COO]+).
<< C 4 * -OPV-CN-G (Example 14) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ7.90 (d, J = 8.6 Hz, 2H), 7.87 (s, 1H), 7.70 (d, J = 8.6 Hz, 2H) , 7.65 (d, J = 8.6Hz, 2H), 7.55 (br-s, 1H), 7.42 (d, J = 9.0Hz, 2H), 7.26 (d, J = 16Hz, 1H), 7.10 (br-s, 4H), 7.09 (d, J = 8.6Hz, 2H), 6.98 (d, J = 16Hz, 1H), 6.68 (d, J = 9.0Hz, 2H), 4.19 (m, 2H), 3.81 (m, 2H), 3.59 (m, 2H), 3.40-3.29 (m, 4H), 3 .07 (dd, J = 15Hz, 8.0Hz, 2H), 1.80 (m, 2H), 1.40 (m, 2H), 1.10 (m, 2H), 0.88 (t, J) = 7.6Hz, 6H), 0.84 (d, J = 6.4Hz, 6H).
13 C NMR (100 MHz, DMSO-d 6 ): δ159.0, 158.4 (q, 2 J = 31 Hz, CF 3 COO), 157.1, 148.0, 140.12140.11, 131.7, 130.7, 129.3, 127.9, 127.0, 126.5, 125.9, 123.5, 122.0, 118.3, 117.1 (q, 1 J = 298 Hz, CF 3 COO ), 115.1, 112.2, 108.1, 68.7, 68.6, 67.3, 57.6, 40.8, 32.4, 26.4, 16.7, 11.2.
MS (MALDI): m / z = 608.62 ([M-CF 3 COO] + ).
《C12−OPV−CN−G(実施例15)》
1H NMR(400MHz,DMSO−d6):δ7.90(d,J=8.8Hz,2H),7.87(s,1H),7.70(d,J=9.0Hz,2H),7.65(d,J=8.8Hz,2H),7.50(t,J=5.6Hz,1H),7.42(d,J=8.8Hz,2H),7.26(d,J=16Hz,1H),7.10(br−s,4H),7.09(d,J=9.0Hz,2H),6.97(d,J=16Hz,1H),6.64(d,J=8.8Hz,2H),4.19(m,2H),3.81(m,2H),3.59(t,J=5.6Hz,2H),3.33(q,J=5.6Hz,2H),3.29(t,J=6.8Hz,4H),1.51(br−quint,4H),1.30−1.24(m,36H),0.85(t,J=6.8Hz,6H).
13C NMR(100MHz,DMSO−d6):δ159.0,158.8(q,2J=32Hz,CF3COO),157.1,147.6,139.9,139.8,131.8,130.5,129.4,128.0,126.9,126.5,125.9,123.7,121.9,118.3,116.8(q,1J=296Hz,CF3COO),115.0,111.3,108.0,68.7,68.6,67.2,50.2,40.8,31.4,29.24,29.19,29.0,28.9,26.9,26.5,22.2,13.8.(Two carbon peaks of dodecyl group were overlapped.)
MS(MALDI):m/z=804.94([M−CF3COO]+).
<< C 12- OPV-CN-G (Example 15) >>
1 1 H NMR (400 MHz, DMSO-d 6 ): δ7.90 (d, J = 8.8 Hz, 2H), 7.87 (s, 1H), 7.70 (d, J = 9.0 Hz, 2H) , 7.65 (d, J = 8.8Hz, 2H), 7.50 (t, J = 5.6Hz, 1H), 7.42 (d, J = 8.8Hz, 2H), 7.26 ( d, J = 16Hz, 1H), 7.10 (br-s, 4H), 7.09 (d, J = 9.0Hz, 2H), 6.97 (d, J = 16Hz, 1H), 6. 64 (d, J = 8.8Hz, 2H), 4.19 (m, 2H), 3.81 (m, 2H), 3.59 (t, J = 5.6Hz, 2H), 3.33 ( q, J = 5.6Hz, 2H), 3.29 (t, J = 6.8Hz, 4H), 1.51 (br-quint, 4H), 1.30-1.24 (m, 36H), 0.85 (t, J = 6.8Hz, 6H).
13 C NMR (100 MHz, DMSO-d 6 ): δ159.0, 158.8 (q, 2 J = 32 Hz, CF 3 COO), 157.1, 147.6, 139.9, 139.8, 131. 8,130.5,129.4,128.0,126.9,126.5,125.9,123.7,121.9,118.3,116.8 (q, 1 J = 296Hz , CF 3 COO), 115.0, 111.3, 108.0, 68.7, 68.6, 67.2, 50.2, 40.8, 31.4, 29.24, 29.19, 29. 0,28.9,26.9,26.5,22.2,13.8. (Two carbon peaks of dodecyl group were overlapped.)
MS (MALDI): m / z = 804.94 ([M-CF 3 COO] + ).
[実験例6]
下記合成スキーム5にしたがって、実施例16の発蛍光性化合物を合成した。以下、実施例16の化合物を「OPV−CN−G」という場合がある。
[Experimental Example 6]
The fluorescent compound of Example 16 was synthesized according to the following
(化合物21の合成)
4−ヒドロキシベンジルシアニド(2.48g,18.6mmol)、炭酸カリウム(5.14g,37.2mmol)及び化合物3(5.00g,18.6mmol)を乾燥DMF(20mL)に懸濁させ、65℃で5時間加熱した。室温まで放冷したのち、酢酸エチル(200mL)を加え、水、飽和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、無色固体として化合物21を4.25g得た。収率71%。
(Synthesis of Compound 21)
4-Hydroxybenzyl cyanide (2.48 g, 18.6 mmol), potassium carbonate (5.14 g, 37.2 mmol) and compound 3 (5.00 g, 18.6 mmol) were suspended in dry DMF (20 mL). It was heated at 65 ° C. for 5 hours. After allowing to cool to room temperature, ethyl acetate (200 mL) was added, and the mixture was washed in the order of water and saturated brine. 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 4.25 g of
1H NMR(400MHz,CDCl3):δ7.23(d,J=8.8Hz,2H),6.93(d,J=8.8Hz,2H),4.95(br−s,1H),4.12(m,2H),3.82(m,2H),3.61(t,J=5.6Hz,2H),3.35(q,J=5.6Hz,2H),1.44(s,9H).
13C NMR(100MHz,CDCl3):δ158.4,155.9,129.0,122.1,118.1,115.2,79.2,70.4,69.3,67.4,40.2,28.4,22.8.
MS(MALDI):m/z=342.71([M+Na]+).
1 1 H NMR (400 MHz, CDCl 3 ): δ7.23 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4.95 (br-s, 1H) , 4.12 (m, 2H), 3.82 (m, 2H), 3.61 (t, J = 5.6Hz, 2H), 3.35 (q, J = 5.6Hz, 2H), 1 .44 (s, 9H).
13 C NMR (100 MHz, CDCl 3 ): δ158.4, 155.9, 129.0, 122.1, 118.1, 115.2, 79.2, 70.4, 69.3, 67.4 40.2, 28.4, 22.8.
MS (MALDI): m / z = 342.71 ([M + Na] + ).
(化合物22の合成)
化合物21(2.00g,6.24mmol)及びテレフタルアルデヒド(419mg,3.12mmol)を乾燥エタノール(20mL)に溶解した。そこにBu4NOH(40% in water)を3滴加え、3時間加熱還流した。得られた沈殿物を回収し、エタノールで洗浄し、黄色固体として化合物22を1.03g得た。収率45%。
(Synthesis of Compound 22)
Compound 21 (2.00 g, 6.24 mmol) and terephthalaldehyde (419 mg, 3.12 mmol) were dissolved in dry ethanol (20 mL). Three drops of Bu 4 NOW (40% in water) were added thereto, and the mixture was heated under reflux for 3 hours. The obtained precipitate was collected and washed with ethanol to obtain 1.03 g of Compound 22 as a yellow solid.
1H NMR(400MHz,CDCl3):δ7.96(s,4H),7.64(d,J=8.8Hz,4H),7.44(s,2H),7.01(d,J=8.8Hz,4H),4.95(br−s,2H),4.18(m,4H),3.85(m,4H),3.63(t,J=5.6Hz,4H),3.36(q,J=5.6Hz,4H),1.45(s,18H).
13C NMR(100MHz,CDCl3):δ159.8,155.9,138.6,135.3,129.5,127.4,126.9,117.9,115.1,112.2,79.3,70.5,69.3,67.5,40.3,28.4.
MS(MALDI):m/z=760.92([M+Na]+).
1 1 H NMR (400 MHz, CDCl 3 ): δ7.96 (s, 4H), 7.64 (d, J = 8.8 Hz, 4H), 7.44 (s, 2H), 7.01 (d, J) = 8.8Hz, 4H), 4.95 (br-s, 2H), 4.18 (m, 4H), 3.85 (m, 4H), 3.63 (t, J = 5.6Hz, 4H) ), 3.36 (q, J = 5.6Hz, 4H), 1.45 (s, 18H).
13 C NMR (100 MHz, CDCl 3 ): δ159.8, 155.9, 138.6, 135.3, 129.5, 127.4, 126.9, 117.9, 115.1, 112.2. 79.3, 70.5, 69.3, 67.5, 40.3, 28.4.
MS (MALDI): m / z = 760.92 ([M + Na] + ).
(化合物23の合成)
化合物22(900mg,1.22mmol)を乾燥ジクロロメタン(5.0mL)に懸濁させ、それにトリフルオロ酢酸(3.0mL,39.2mmol)を加えた。反応混合物を室温で7時間撹拌した後、乾燥ジエチルエーテルを加え、黄色固体として化合物23を876mg得た。収率94%。
1H NMR(400MHz,DMSO−d6):δ8.04(s,4H),7.99(s,2H),7.79(br−s,6H),7.75(d,J=8.8Hz,4H),7.12(d,J=8.8Hz,4H),4.22(m,4H),3.83(m,4H),3.68(t,J=5.6Hz,4H),3.02(t,J=5.6Hz,4H).
13C NMR(100MHz,DMSO−d6):δ159.5,158.1(q,2J=32Hz,CF3COO),139.4,135.4,129.3,127.4,126.1,117.9,117.3(q,1J=298Hz,CF3COO),115.2,110.9,68.8,67.3,66.9,38.6.
MS(MALDI):m/z=538.82([M−H−2CF3COO]+).
(Synthesis of Compound 23)
Compound 22 (900 mg, 1.22 mmol) was suspended in dry dichloromethane (5.0 mL) and trifluoroacetic acid (3.0 mL, 39.2 mmol) was added thereto. After stirring the reaction mixture at room temperature for 7 hours, dry diethyl ether was added to obtain 876 mg of compound 23 as a yellow solid. Yield 94%.
1 1 H NMR (400 MHz, DMSO-d 6 ): δ8.04 (s, 4H), 7.99 (s, 2H), 7.79 (br-s, 6H), 7.75 (d, J = 8) 8.8Hz, 4H), 7.12 (d, J = 8.8Hz, 4H), 4.22 (m, 4H), 3.83 (m, 4H), 3.68 (t, J = 5.6Hz) , 4H), 3.02 (t, J = 5.6Hz, 4H).
13 C NMR (100 MHz, DMSO-d 6 ): δ159.5, 158.1 (q, 2 J = 32 Hz, CF 3 COO), 139.4, 135.4, 129.3, 127.4, 126. 1,117.9,117.3 (q, 1 J = 298Hz ,
MS (MALDI): m / z = 538.82 ([MH-2CF 3 COO] + ).
(化合物24の合成)
化合物23(782mg,1.02mmol)を乾燥ジクロロメタン(10mL)に溶解し、それにEt3N(0.43mL,3.06mmol)及び1−H−pyrazole−1−(N,N’−bis(tert−butyloxy−carbonyl))carboxamidine(950mg,3.06mmol)を加えた。反応混合物を室温で20時間撹拌した。その後、クロロホルム(100mL)を加え、水、飽和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、黄色固体として化合物24を479mg得た。収率46%。
(Synthesis of Compound 24)
Compound 23 (782 mg, 1.02 mmol) was dissolved in dry dichloromethane (10 mL) with Et 3 N (0.43 mL, 3.06 mmol) and 1-H-pyrazole-1- (N, N'-bis (tert). -Butyloxy-carbonyl)) carboxamide (950 mg, 3.06 mmol) was added. The reaction mixture was stirred at room temperature for 20 hours. Then, chloroform (100 mL) was added, and the mixture was washed with water and saturated 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 479 mg of compound 24 as a yellow solid. Yield 46%.
1H NMR(400MHz,CDCl3):δ11.48(s,2H),8.67(br−s,2H),7.96(s,4H),7.63(d,J=8.8Hz,4H),7.43(s,2H),7.01(d,J=8.8Hz,4H),4.21(m,4H),3.88(m,4H),3.70(m,8H),1.51(s,18H),1.48(s,18H).
13C NMR(100MHz,CDCl3):δ163.5,159.9,156.3,153.0,138.5,135.4,129.5,127.4,126.9,117.9,115.2,112.2,83.0,79.3,69.6,69.2,67.6,40.5,28.3,28.0.
MS(MALDI):m/z=644.88([(OPV−CN−G)+Na−2CF3COOH]+).
1 1 H NMR (400 MHz, CDCl 3 ): δ11.48 (s, 2H), 8.67 (br-s, 2H), 7.96 (s, 4H), 7.63 (d, J = 8.8 Hz) , 4H), 7.43 (s, 2H), 7.01 (d, J = 8.8Hz, 4H), 4.21 (m, 4H), 3.88 (m, 4H), 3.70 ( m, 8H), 1.51 (s, 18H), 1.48 (s, 18H).
13 C NMR (100 MHz, CDCl 3 ): δ163.5, 159.9, 156.3, 153.0, 138.5, 135.4, 129.5, 127.4, 126.9, 117.9, 115.2, 112.2, 83.0, 79.3, 69.6, 69.2, 67.6, 40.5, 28.3, 28.0.
MS (MALDI): m / z = 644.88 ([(OPV-CN-G) + Na-2CF 3 COOH] + ).
(OPV−CN−G(実施例16)の合成)
化合物24(400mg,0.39mmol)を乾燥ジクロロメタン(5.0mL)に懸濁させ、それにトリフルオロ酢酸(3.0mL,39.2mmol)を加えた。反応混合物を室温で10時間撹拌した後、乾燥ジエチルエーテルを加え、黄色固体としてOPV−CN−Gを300mg得た。収率90%。
(Synthesis of OPV-CN-G (Example 16))
Compound 24 (400 mg, 0.39 mmol) was suspended in dry dichloromethane (5.0 mL) and trifluoroacetic acid (3.0 mL, 39.2 mmol) was added thereto. After stirring the reaction mixture at room temperature for 10 hours, dry diethyl ether was added to obtain 300 mg of OPV-CN-G as a yellow solid. Yield 90%.
1H NMR(400MHz,DMSO−d6):δ8.04(s,4H),7.99(s,2H),7.75(d,J=8.8Hz,4H),7.57(br−s,2H),7.13(br−s,8H),7.11(d,J=8.8Hz,4H),4.20(m,4H),3.81(m,4H),3.59(t,J=5.6Hz,4H),3.33(t,J=5.6Hz,4H).
13C NMR(100MHz,DMSO−d6):δ159.5,158.7(q,2J=31Hz,CF3COO),157.4,139.4,135.4,129.3,127.4,126.1,117.9,117.1(q,1J=297Hz,CF3COO),115.2,110.9,68.8,68.6,67.4,40.8.
MS(MALDI):m/z=622.94([M−H−2CF3COO]+).
1 1 H NMR (400 MHz, DMSO-d 6 ): δ8.04 (s, 4H), 7.99 (s, 2H), 7.75 (d, J = 8.8 Hz, 4H), 7.57 (br) -S, 2H), 7.13 (br-s, 8H), 7.11 (d, J = 8.8Hz, 4H), 4.20 (m, 4H), 3.81 (m, 4H), 3.59 (t, J = 5.6Hz, 4H), 3.33 (t, J = 5.6Hz, 4H).
13 C NMR (100 MHz, DMSO-d 6 ): δ159.5, 158.7 (q, 2 J = 31 Hz, CF 3 COO), 157.4, 139.4, 135.4, 129.3, 127. 4,126.1, 117.9, 117.1 (q, 1 J = 297 Hz, CF 3 COO), 115.2, 110.9, 68.8, 68.6, 67.4, 40.8.
MS (MALDI): m / z = 622.94 ([MH-2CF 3 COO] + ).
[実験例7]
実施例1の発蛍光性化合物(OPV−G)について、ジカルボン酸に対する蛍光特性を検討した。
[Experimental Example 7]
The fluorescence characteristics of the fluorescent compound (OPV-G) of Example 1 with respect to the dicarboxylic acid were examined.
OPV−Gに、ジカルボン酸として、L−酒石酸又はmeso−酒石酸を加えて蛍光を測定した。測定条件は以下のとおりであった。 L-tartaric acid or meso-tartaric acid was added to OPV-G as a dicarboxylic acid, and the fluorescence was measured. The measurement conditions were as follows.
(測定条件)
OPV−Gの濃度:10μM、HEPESバッファー(10mM、pH7.4)中。
L−酒石酸及びmeso−酒石酸の濃度:5.0mM。
励起波長λex:388nm
測定装置:Perkin−ElmerLS55
測定温度:25℃
測定セル:1mm石英セル
(Measurement condition)
Concentration of OPV-G: 10 μM, in HEPES buffer (10 mM, pH 7.4).
Concentrations of L-tartaric acid and meso-tartaric acid: 5.0 mM.
Excitation wavelength λ ex : 388 nm
Measuring device: Perkin-Elmer LS55
Measurement temperature: 25 ° C
Measuring cell: 1 mm quartz cell
測定結果を図1に示す。図1(a)に示されるように、実施例1の発蛍光性化合物は酒石酸に対して蛍光を発することが肉眼で観察された。また、図1(b)に示されるように、同じ測定条件にも関わらず、L−酒石酸添加時の蛍光強度は、meso−酒石酸添加時のそれよりも、2.5倍強い蛍光強度を与えることが認められた。 The measurement results are shown in FIG. As shown in FIG. 1 (a), it was observed with the naked eye that the fluorescent compound of Example 1 fluoresces against tartaric acid. Further, as shown in FIG. 1 (b), despite the same measurement conditions, the fluorescence intensity when L-tartaric acid is added gives 2.5 times stronger fluorescence intensity than that when meso-tartaric acid is added. Was recognized.
[実験例8]
実施例1の発蛍光性化合物(OPV−G)について、その特性を更に検討した。OPV−GにL−酒石酸を添加して紫外可視吸収スペクトルを測定したところ、370nmの吸収最大ピークが減少し且つ新たな吸収ピークが425nmに認められた(図2(a)を参照。)。このことは、J−会合体の形成を示唆している。その水性分散液の蛍光顕微鏡観察によって、ファイバー状の凝集体形成が認められた(図2(b)を参照。)。これに対して、meso−酒石酸の添加に対しては、370nmの吸収最大ピークが大きく減少し且つ342nmに短波長シフトした(図2(c)を参照。)。このことは、H−会合体が形成されていることを示唆している。その水性分散液の蛍光顕微鏡観察によって、粒子状の凝集体形成が認められた(図2(d)を参照。)。
[Experimental Example 8]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. When L-tartaric acid was added to OPV-G and the ultraviolet-visible absorption spectrum was measured, the maximum absorption peak at 370 nm decreased and a new absorption peak was observed at 425 nm (see FIG. 2 (a)). This suggests the formation of J-aggregates. By observing the aqueous dispersion with a fluorescence microscope, the formation of fibrous aggregates was observed (see FIG. 2 (b)). On the other hand, with respect to the addition of meso-tartaric acid, the maximum absorption peak at 370 nm was greatly reduced and the wavelength was shifted to 342 nm by a short wavelength (see FIG. 2 (c)). This suggests that an H-aggregate is formed. By observing the aqueous dispersion with a fluorescence microscope, the formation of particulate aggregates was observed (see FIG. 2 (d)).
ここで、J−会合体及びH−会合体について説明する。図16(a)は、J−会合体の一例を示す模式図である。図16(a)は、後述する、OPV−G(20)のカチオン性基21とヒアルロン酸10の負電荷11との相互作用により、OPV−G(20)がJ−会合体100を形成し発蛍光している様子を示す。また、図16(b)は、H−会合体の一例を示す模式図である。図16(b)は、後述する、OPV−G(20)のカチオン性基21とヘパリン30の負電荷31との相互作用により、OPV−G(20)がH−会合体200を形成し発蛍光している様子を示す。
Here, the J-aggregate and the H-aggregate will be described. FIG. 16A is a schematic diagram showing an example of a J-aggregate. In FIG. 16A, OPV-G (20) forms a J-
図16(a)及び(b)に示すように、J−会合体では、発蛍光性化合物が、分子の長軸方向に滑りながら積み重なっている(slip−stacked fashion)。これに対し、H−会合体では、発蛍光性化合物が、分子の長軸方向に対して垂直方向に密に積み重なっている。 As shown in FIGS. 16A and 16B, in the J-aggregate, the fluorescent compounds are piled up while sliding in the long axis direction of the molecule (slip-stacked fashion). On the other hand, in the H-aggregate, the fluorescent compounds are densely stacked in the direction perpendicular to the long axis direction of the molecule.
[実験例9]
実施例1の発蛍光性化合物(OPV−G)について、その特性を更に検討した。OPV−G(濃度:10μM)に、ジカルボン酸としてL−酒石酸又はmeso−酒石酸を逐次添加して蛍光滴定試験を行った。測定条件は、実験例7と同様であった。
[Experimental Example 9]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. A fluorescence titration test was carried out by sequentially adding L-tartaric acid or meso-tartaric acid as a dicarboxylic acid to OPV-G (concentration: 10 μM). The measurement conditions were the same as in Experimental Example 7.
結果を図3に示す。図中、F0はジカルボン酸が存在しない場合の蛍光強度を表し、Fはジカルボン酸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。 The results are shown in FIG. In the figure, F 0 represents the fluorescence intensity in the absence of the dicarboxylic acid, F represents the fluorescence intensity in the presence of the dicarboxylic acid, and ΔF = FF 0 . Therefore, ΔF / F 0 means the degree of change in fluorescence intensity.
図3(a)に示されるように、L−酒石酸の添加に対しては、濃度の増加に対して250μMから蛍光(λem:518nm)が観測され、最大で45倍の蛍光増大を示した。これに対して、meso−酒石酸の添加に対しては、50μMから蛍光(λem:518nm)が観測され、最大で18倍の蛍光増大が認められた。図3(b)は図3(a)の低濃度域を拡大したものである。この結果から、OPV−Gは酒石酸の立体構造(L−体、meso−体)に依存した蛍光応答を与えていることが認められた。 As shown in FIG. 3A, with respect to the addition of L-tartaric acid, fluorescence (λ em : 518 nm) was observed from 250 μM with respect to the increase in concentration, showing a maximum of 45-fold increase in fluorescence. .. On the other hand, with respect to the addition of meso-tartaric acid, fluorescence (λ em : 518 nm) was observed from 50 μM, and a maximum of 18-fold increase in fluorescence was observed. FIG. 3 (b) is an enlargement of the low concentration region of FIG. 3 (a). From this result, it was confirmed that OPV-G imparted a fluorescence response depending on the three-dimensional structure (L-form, meso-form) of tartaric acid.
[実験例10]
実施例1の発蛍光性化合物(OPV−G)について、その特性を更に検討した。OPV−G(濃度:10μM)に、ジカルボン酸としてフマル酸又はマレイン酸を逐次添加して蛍光滴定試験を行った。測定条件は、実験例7と同様であった。
[Experimental Example 10]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. A fluorescence titration test was carried out by sequentially adding fumaric acid or maleic acid as dicarboxylic acids to OPV-G (concentration: 10 μM). The measurement conditions were the same as in Experimental Example 7.
結果を図4に示す。図4(a)に示されるように、フマル酸の添加に対しては、濃度の増加に対して200μMから蛍光(λem:520nm)が観測され、最大で54倍の蛍光増大を示した。これに対して、マレイン酸の添加に対しては、100μMから蛍光(λem:514nm)が観測され、最大で20倍の蛍光増大が認められた。図4(b)は図4(a)の低濃度域を拡大たものである。この結果から、OPV−Gはフマル酸、マレイン酸の位置異性体構造(trans−体、cis−体)に依存した蛍光応答を与えていることが認められた。 The results are shown in FIG. As shown in FIG. 4 (a), with respect to the addition of fumaric acid, fluorescence (λ em : 520 nm) was observed from 200 μM with respect to the increase in concentration, showing a maximum of 54-fold increase in fluorescence. On the other hand, with respect to the addition of maleic acid, fluorescence (λ em : 514 nm) was observed from 100 μM, and a maximum of 20-fold increase in fluorescence was observed. FIG. 4B is an enlargement of the low concentration region of FIG. 4A. From this result, it was confirmed that OPV-G provided a fluorescence response depending on the positional isomer structure (trans-form, cis-form) of fumaric acid and maleic acid.
[実験例11]
実施例1の発蛍光性化合物(OPV−G)について、その特性を更に検討した。OPV−G(濃度:10μM)に、ジカルボン酸として、シュウ酸、マロン酸、コハク酸、グルタル酸又はアジピン酸を逐次添加して蛍光滴定試験を行った。測定条件は、実験例7と同様であった。
[Experimental Example 11]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. A fluorescence titration test was carried out by sequentially adding oxalic acid, malonic acid, succinic acid, glutaric acid or adipic acid as dicarboxylic acids to OPV-G (concentration: 10 μM). The measurement conditions were the same as in Experimental Example 7.
結果を図5及び図6に示す。図5に示されるように、モノカルボン酸として酢酸を逐次添加した場合、蛍光が観測されなかった。これに対して、上記ジカルボン酸の添加に対しては、濃度の増加に対して蛍光(λem:515nm)が観測された。図6に示されるように、蛍光強度が観測される濃度(濃度閾値)と蛍光強度は、ジカルボン酸構造に特有のものであることが認められる。 The results are shown in FIGS. 5 and 6. As shown in FIG. 5, when acetic acid was sequentially added as a monocarboxylic acid, no fluorescence was observed. On the other hand, with respect to the addition of the dicarboxylic acid, fluorescence (λ em : 515 nm) was observed with respect to the increase in concentration. As shown in FIG. 6, it is recognized that the concentration at which the fluorescence intensity is observed (concentration threshold value) and the fluorescence intensity are peculiar to the dicarboxylic acid structure.
実験例6〜8の結果に示されるように、実施例1の発蛍光性化合物を用いると、一定のジカルボン酸濃度においてターンオン又はスイッチオン式にジカルボン酸を検出し得ることが明らかとなった。さらに、ジカルボン酸の化学構造情報が蛍光応答の閾値、蛍光強度、及び蛍光波長に変換・増幅されることで、ジカルボン酸を識別し得ることが明らかとなった。 As shown in the results of Experimental Examples 6 to 8, it was revealed that when the fluorescent compound of Example 1 was used, the dicarboxylic acid could be detected in a turn-on or switch-on manner at a constant dicarboxylic acid concentration. Furthermore, it was clarified that the dicarboxylic acid can be identified by converting and amplifying the chemical structure information of the dicarboxylic acid into the threshold value of the fluorescence response, the fluorescence intensity, and the fluorescence wavelength.
[実験例12]
実施例1の発蛍光性化合物(OPV−G)について、その特性を更に検討した。OPV−G(濃度:10μM)に、ポリカルボン酸としてポリアクリル酸(平均分子量5000)を逐次添加して蛍光滴定試験を行った。測定条件は、実験例7と同様であった。
[Experimental Example 12]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. A fluorescence titration test was carried out by sequentially adding polyacrylic acid (average molecular weight 5000) as a polycarboxylic acid to OPV-G (concentration: 10 μM). The measurement conditions were the same as in Experimental Example 7.
結果を図7に示す。図7(a)に示されるように、ポリアクリル酸の添加に対しては、濃度の増加に対してターンオン又はスイッチオン式に蛍光(λem:520nm)が観測された。また、図7(b)に示されるように、蛍光強度は0.1ppmから観測され、1.3ppmで飽和に達した。 The results are shown in FIG. As shown in FIG. 7 (a), with respect to the addition of polyacrylic acid, fluorescence (λ em : 520 nm) was observed in a turn-on or switch-on manner with respect to the increase in concentration. Further, as shown in FIG. 7 (b), the fluorescence intensity was observed from 0.1 ppm and reached saturation at 1.3 ppm.
このように、実施例1の発蛍光性化合物を用いると、濃度範囲0.1から1.3ppmにおいて、ポリアクリル酸濃度を蛍光強度から測定し得ることが明らかとなった。 As described above, it was clarified that the polyacrylic acid concentration can be measured from the fluorescence intensity in the concentration range of 0.1 to 1.3 ppm by using the fluorescent compound of Example 1.
[実験例13]
実施例3の発蛍光性化合物(OPV−ImMe)について、その特性を検討した。OPV−ImMe(濃度:20μM)に、ポリカルボン酸としてポリアクリル酸(平均分子量5000)を逐次添加して蛍光滴定試験を行った。測定条件は、実験例7と同様であった。
[Experimental Example 13]
The characteristics of the fluorescent compound (OPV-IMMe) of Example 3 were examined. A fluorescence titration test was carried out by sequentially adding polyacrylic acid (average molecular weight 5000) as a polycarboxylic acid to OPV-IMMe (concentration: 20 μM). The measurement conditions were the same as in Experimental Example 7.
結果を図8に示す。図8(a)に示されるように、ポリアクリル酸の添加に対しては、濃度の増加に対してターンオン又はスイッチオン式に蛍光(λem:515nm)が観測された。また、図8(b)に示されるように、蛍光強度は0.1ppmから観測され、4.0ppmで飽和に達した。 The results are shown in FIG. As shown in FIG. 8 (a), with respect to the addition of polyacrylic acid, fluorescence (λ em : 515 nm) was observed in a turn-on or switch-on manner with respect to the increase in concentration. Further, as shown in FIG. 8 (b), the fluorescence intensity was observed from 0.1 ppm and reached saturation at 4.0 ppm.
このように、実施例3の発蛍光性化合物を用いると、濃度範囲0.1から4.0ppmにおいて、ポリアクリル酸濃度を蛍光強度から測定し得ることが明らかとなった。 As described above, it was clarified that the polyacrylic acid concentration can be measured from the fluorescence intensity in the concentration range of 0.1 to 4.0 ppm by using the fluorescent compound of Example 3.
[実験例14]
実施例6の発蛍光性化合物(OPV−ImDEO)について、その特性を検討した。OPV−ImDEO(濃度:60μM)に、ポリカルボン酸としてポリアクリル酸(平均分子量5000)を逐次添加して蛍光滴定試験を行った。測定条件は、実験例7と同様であった。
[Experimental Example 14]
The characteristics of the fluorescent compound (OPV-ImDEO) of Example 6 were examined. A fluorescence titration test was carried out by sequentially adding polyacrylic acid (average molecular weight 5000) as a polycarboxylic acid to OPV-ImDEO (concentration: 60 μM). The measurement conditions were the same as in Experimental Example 7.
結果を図9に示す。図9(a)に示されるように、ポリアクリル酸の添加に対しては、濃度の増加に対してターンオン又はスイッチオン式に蛍光(λem:513nm)が観測された。また、図9(b)に示されるように、蛍光強度は0.1ppmから観測され、15ppmで飽和に達した。 The results are shown in FIG. As shown in FIG. 9A, with respect to the addition of polyacrylic acid, fluorescence (λ em : 513 nm) was observed in a turn-on or switch-on manner with respect to the increase in concentration. Further, as shown in FIG. 9B, the fluorescence intensity was observed from 0.1 ppm and reached saturation at 15 ppm.
このように、実施例6の発蛍光性化合物を用いると、濃度範囲0.1から15ppmにおいて、ポリアクリル酸濃度を蛍光強度から測定し得ることが明らかとなった。 As described above, it was clarified that the polyacrylic acid concentration can be measured from the fluorescence intensity in the concentration range of 0.1 to 15 ppm by using the fluorescent compound of Example 6.
[実験例15]
実施例1の発蛍光性化合物(OPV−G)及び実施例8の発蛍光性化合物(G2)について、ヌクレオチドに対する蛍光特性検討した。具体的には、OPV−G及びG2に、ヌクレオチドとして、ATP、NADPHを添加して蛍光を測定した。以下に、ATP及びNADPHの化学式を示す。
[Experimental Example 15]
The fluorescence characteristics of the fluorescent compound (OPV-G) of Example 1 and the fluorescent compound (G2) of Example 8 were examined for nucleotides. Specifically, ATP and NADPH were added as nucleotides to OPV-G and G2, and the fluorescence was measured. The chemical formulas of ATP and NADPH are shown below.
測定条件は、実験例7と同様であった。ただし、溶媒には、EtOH/HEPESバッファー(10mM、pH7.4)(1:9 v/v)を用いた。測定結果を図10(a)及び(b)に示す。ATP又はNADPHを発蛍光性化合物に添加したところ、図10(a)に示すように、OPV−Gは、ATP及びNADPHの両者に対して同程度の蛍光応答を示した。一方、図10(b)に示すように、G2では、ATPとNADPHとで明瞭な蛍光強度の差異が認められた。 The measurement conditions were the same as in Experimental Example 7. However, EtOH / HEPES buffer (10 mM, pH 7.4) (1: 9 v / v) was used as the solvent. The measurement results are shown in FIGS. 10 (a) and 10 (b). When ATP or NADPH was added to the fluorescent compound, OPV-G showed a similar fluorescence response to both ATP and NADPH, as shown in FIG. 10 (a). On the other hand, as shown in FIG. 10B, in G2, a clear difference in fluorescence intensity was observed between ATP and NADPH.
上述した実験例7及び8の結果から推察されるように、G2は、ATPとJ−会合体を形成し強い発蛍光を示すのに対し、NADPHとはH−会合体を形成するため、より弱い蛍光強度を示すものと考えられた。 As can be inferred from the results of Experimental Examples 7 and 8 described above, G2 forms a J-aggregate with ATP and exhibits strong fluorescence, whereas NADPH forms an H-aggregate with NADPH. It was considered to show weak fluorescence intensity.
実験例15の結果から、OPV−Gは、ヌクレオチド類を非特異的に検出することができ、G2は、ヌクレオチド類の中からATPを選択的に検出できることが明らかとなった。 From the results of Experimental Example 15, it was clarified that OPV-G can detect nucleotides non-specifically, and G2 can selectively detect ATP from among nucleotides.
[実験例16]
実施例8(G2)と実施例10(G6)の発蛍光性化合物について、発蛍光に対する塩濃度の影響を検討した。より具体的には、G2及びG6に、塩として塩化ナトリウム(NaCl)を逐次添加して蛍光滴定試験を行った。測定条件は、実験例7と同様であった。
[Experimental Example 16]
The influence of the salt concentration on the fluorescence of the fluorescent compounds of Example 8 (G2) and Example 10 (G6) was examined. More specifically, sodium chloride (NaCl) was sequentially added as a salt to G2 and G6 to carry out a fluorescence titration test. The measurement conditions were the same as in Experimental Example 7.
測定結果を図11に示す。図11に示されるように、G2は、塩化ナトリウムの添加により蛍光を発することが認められた。これに対し、G6では、塩化ナトリウムの添加による発蛍光は認められなかった。 The measurement results are shown in FIG. As shown in FIG. 11, G2 was found to fluoresce with the addition of sodium chloride. On the other hand, in G6, fluorescence due to the addition of sodium chloride was not observed.
実験例16の結果に示されるように、G2は、塩濃度の存在下で自己会合して蛍光を発するのに対し、G6は、生理的塩濃度(約150mM)条件下でも蛍光を発しない、すなわち、自己会合しないことが明らかとなった。 As shown in the results of Experimental Example 16, G2 self-associates and fluoresces in the presence of salt concentration, whereas G6 does not fluoresce even under physiological salt concentration (about 150 mM). That is, it became clear that they did not meet themselves.
[実験例17]
実施例10の発蛍光性化合物(G6)について、その特性を更に検討した。具体的には、生理的塩濃度条件下(NaCl:125mM、KCl:5mM、CaCl2:1mM、MgCl2:0.5mM)において、G6に、ヌクレオチドとして、AMP、ADP又はATPを添加して蛍光滴定試験を行った。測定条件は、実験例7と同様であった。
[Experimental Example 17]
The characteristics of the fluorescent compound (G6) of Example 10 were further investigated. Specifically, under physiological salt concentration conditions (NaCl: 125 mM, KCl: 5 mM, CaCl 2 : 1 mM, MgCl 2 : 0.5 mM), AMP, ADP or ATP is added as a nucleotide to G6 for fluorescence. A titration test was performed. The measurement conditions were the same as in Experimental Example 7.
測定結果を図12(a)及び(b)に示す。図12(a)に示されるように、G6は、ATPに対して選択的に発蛍光を示すことが肉眼で観察された。また、図12(b)に示されるように、G6は、ヌクレオチドの中でもATPに選択的に蛍光応答を示すことが明らかとなった。 The measurement results are shown in FIGS. 12 (a) and 12 (b). As shown in FIG. 12 (a), it was observed with the naked eye that G6 selectively fluoresces with respect to ATP. Further, as shown in FIG. 12 (b), it was revealed that G6 selectively exhibits a fluorescence response to ATP among nucleotides.
実験例16〜17の結果に示されるように、G6を用いると、生理的塩濃度条件下において、ターンオン又はスイッチオン式にATPを選択的に検出し、且つATPの濃度を蛍光強度から測定できることが明らかとなった。したがって、G6はATP検出剤であるということができる。 As shown in the results of Experimental Examples 16 to 17, when G6 is used, ATP can be selectively detected in a turn-on or switch-on manner under physiological salt concentration conditions, and the concentration of ATP can be measured from the fluorescence intensity. Became clear. Therefore, it can be said that G6 is an ATP detection agent.
[実験例18]
実施例11の発蛍光性化合物(DEO−OPV−CN−G)について、アニオン性多糖類に対する蛍光特性を検討した。具体的には、DEO−OPV−CN−Gに、アニオン性多糖類として、ヘパリン、コンドロイチン硫酸又はヒアルロン酸を添加して蛍光滴定試験を行った。以下に、ヘパリン、コンドロイチン硫酸及びヒアルロン酸の化学式を示す。
[Experimental Example 18]
The fluorescence characteristics of the fluorescent compound (DEO-OPV-CN-G) of Example 11 with respect to anionic polysaccharides were examined. Specifically, a fluorescence titration test was carried out by adding heparin, chondroitin sulfate or hyaluronic acid as anionic polysaccharides to DEO-OPV-CN-G. The chemical formulas of heparin, chondroitin sulfate and hyaluronic acid are shown below.
測定条件は以下のとおりであった。
(測定条件)
DEO−OPV−CN−Gの濃度:30μM、HEPESバッファー(10mM、pH7.4)中。
励起波長λex:400nm
測定装置:Perkin−ElmerLS55
測定温度:25℃
測定セル:1mm石英セル
The measurement conditions were as follows.
(Measurement condition)
Concentration of DEO-OPV-CN-G: 30 μM in HEPES buffer (10 mM, pH 7.4).
Excitation wavelength λ ex : 400 nm
Measuring device: Perkin-Elmer LS55
Measurement temperature: 25 ° C
Measuring cell: 1 mm quartz cell
測定結果を図13(a)及び(b)に示す。図13(a)に示されるように、DEO−OPV−CN−Gにアニオン性多糖類を添加すると、赤色蛍光の強度増大が観察された。また、図13(b)に示されるように、DEO−OPV−CN−Gは、アニオン性多糖類の濃度増加に対して直線的な蛍光応答を与えることが明らかとなった。 The measurement results are shown in FIGS. 13 (a) and 13 (b). As shown in FIG. 13 (a), when an anionic polysaccharide was added to DEO-OPV-CN-G, an increase in the intensity of red fluorescence was observed. Further, as shown in FIG. 13 (b), it was revealed that DEO-OPV-CN-G gives a linear fluorescence response to an increase in the concentration of anionic polysaccharides.
実験例18の結果に示されるように、DEO−OPV−CN−Gを用いると、一定のアニオン性多糖類濃度においてターンオン又はスイッチオン式にアニオン性多糖類、特に、ヘパリンを選択的に検出することができ、且つアニオン性多糖類の濃度を蛍光強度から測定できることが明らかとなった。したがって、DEO−OPV−CN−Gは、アニオン性多糖類検出剤、又はヘパリン検出剤であるということができる。 As shown in the results of Experimental Example 18, DEO-OPV-CN-G is used to selectively detect anionic polysaccharides, especially heparin, in a turn-on or switch-on manner at a constant anionic polysaccharide concentration. It was clarified that the concentration of anionic polysaccharides can be measured from the fluorescence intensity. Therefore, it can be said that DEO-OPV-CN-G is an anionic polysaccharide detection agent or a heparin detection agent.
[実験例19]
実施例1の発蛍光性化合物(OPV−G)について、アニオン性多糖類に対する蛍光特性を検討した。具体的には、OPV−Gに、アニオン性多糖類として、ヘパリン、コンドロイチン硫酸又はヒアルロン酸を添加して蛍光滴定試験を行った。測定条件は、実験例7と同様であった。
[Experimental Example 19]
The fluorescence characteristics of the fluorescent compound (OPV-G) of Example 1 with respect to anionic polysaccharides were examined. Specifically, a fluorescence titration test was carried out by adding heparin, chondroitin sulfate or hyaluronic acid as anionic polysaccharides to OPV-G. The measurement conditions were the same as in Experimental Example 7.
測定結果を図14(a)及び(b)に示す。図14(a)に示されるように、OPV−Gにアニオン性多糖類を添加すると、ヒアルロン酸添加に対して最も強い蛍光強度を与えた。また、図14(b)に示されるように、OPV−Gは、アニオン性多糖類の濃度増加に対して直線的な蛍光応答を与えることが明らかとなった。 The measurement results are shown in FIGS. 14 (a) and 14 (b). As shown in FIG. 14 (a), the addition of anionic polysaccharides to OPV-G gave the strongest fluorescence intensity to the addition of hyaluronic acid. Further, as shown in FIG. 14 (b), it was revealed that OPV-G gives a linear fluorescence response to an increase in the concentration of anionic polysaccharides.
[実験例20]
実施例1の発蛍光性化合物(OPV−G)について、その特性を更に検討した。具体的には、OPV−Gにヒアルロン酸及びヘパリンを添加して紫外可視吸収スペクトルを測定した。
[Experimental Example 20]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. Specifically, hyaluronic acid and heparin were added to OPV-G, and the ultraviolet-visible absorption spectrum was measured.
その結果、図15(a)に示すように、ヒアルロン酸の添加により、370nmの吸収最大ピークが減少し且つ新たな吸収ピークが430nmに認められた。このことは、J−会合体が形成されたことを示唆している。図16(a)にJ−会合体の模式図を示す。図16(a)においては、OPV−G(20)のカチオン性基21とヒアルロン酸10の負電荷11との相互作用により、OPV−G(20)がJ−会合体100を形成し発蛍光している。
As a result, as shown in FIG. 15A, the addition of hyaluronic acid reduced the maximum absorption peak at 370 nm and a new absorption peak was observed at 430 nm. This suggests that a J-aggregate was formed. FIG. 16A shows a schematic diagram of the J-aggregate. In FIG. 16 (a), OPV-G (20) forms a J-
一方、図15(b)に示すように、ヘパリンの添加により、370nmの吸収最大ピークが大きく減少した。このことは、H−会合体が形成されたことを示唆している。図16(b)にH−会合体の模式図を示す。図16(b)においては、OPV−G(20)のカチオン性基21とヘパリン30の負電荷31との相互作用により、OPV−G(20)がH−会合体200を形成し発蛍光している。
On the other hand, as shown in FIG. 15B, the addition of heparin significantly reduced the maximum absorption peak at 370 nm. This suggests that an H-aggregate was formed. FIG. 16B shows a schematic diagram of the H-aggregate. In FIG. 16 (b), OPV-G (20) forms an H-
実験例19及び20の結果に示されるように、OPV−Gを用いると、一定のアニオン性多糖類濃度においてターンオン又はスイッチオン式にアニオン性多糖類を検出できることが明らかとなった。また、OPV−Gは、アニオン性多糖類中最も電荷数の小さいヒアルロン酸の存在下でJ−会合体を形成することで、ヒアルロン酸を選択的に検出することができ、且つヒアルロン酸の濃度を蛍光強度から測定できることが明らかとなった。したがって、OPV−Gは、アニオン性多糖類検出剤、又はヒアルロン酸検出剤であるということができる。 As shown in the results of Experimental Examples 19 and 20, it was revealed that OPV-G can detect anionic polysaccharides in a turn-on or switch-on manner at a constant anionic polysaccharide concentration. In addition, OPV-G can selectively detect hyaluronic acid by forming J-aggregates in the presence of hyaluronic acid, which has the lowest charge number among anionic polysaccharides, and the concentration of hyaluronic acid. It became clear that can be measured from the fluorescence intensity. Therefore, OPV-G can be said to be an anionic polysaccharide detection agent or a hyaluronic acid detection agent.
[実験例21]
実施例1の発蛍光性化合物(OPV−G)について、その特性を更に検討した。具体的には、終濃度10μMのOPV−Gに、アニオン性多糖類として、ヘパリン(終濃度2.5ppm)、コンドロイチン硫酸(終濃度5.5ppm)又はヒアルロン酸(終濃度8.5ppm)を添加して蛍光を測定した。測定条件は、溶液のpHを1.5とした点以外は実験例7と同様であった。
[Experimental Example 21]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. Specifically, heparin (final concentration 2.5 ppm), chondroitin sulfate (final concentration 5.5 ppm) or hyaluronic acid (final concentration 8.5 ppm) was added to OPV-G having a final concentration of 10 μM as an anionic polysaccharide. And the fluorescence was measured. The measurement conditions were the same as in Experimental Example 7 except that the pH of the solution was 1.5.
図17(a)は、実験結果を示す写真である。また、図17(b)は、図17(a)の各試料の蛍光強度を測定した結果を示すグラフである。その結果、図17(a)及び(b)に示されるように、pH1.5の条件下でOPV−Gにアニオン性多糖類を添加すると、コンドロイチン硫酸の添加により最も強い蛍光強度を与えた。 FIG. 17A is a photograph showing the experimental results. Further, FIG. 17B is a graph showing the results of measuring the fluorescence intensity of each sample of FIG. 17A. As a result, as shown in FIGS. 17 (a) and 17 (b), when an anionic polysaccharide was added to OPV-G under the condition of pH 1.5, the strongest fluorescence intensity was given by the addition of chondroitin sulfate.
実験例21の結果に示されるように、OPV−Gを用いると、酸性条件において、ターンオン又はスイッチオン式にアニオン性多糖類、特に、コンドロイチン硫酸を選択的に検出し得ることが明らかとなった。したがって、OPV−Gは、アニオン性多糖類検出剤、又はコンドロイチン硫酸検出剤であるということができる。 As shown in the results of Experimental Example 21, it was clarified that OPV-G can selectively detect anionic polysaccharides, particularly chondroitin sulfate, in an acidic condition in a turn-on or switch-on manner. .. Therefore, OPV-G can be said to be an anionic polysaccharide detection agent or a chondroitin sulfate detection agent.
実験例18〜21の結果から、発蛍光性化合物の化学構造や溶液条件を適切に調整することにより、アニオン性多糖類に対する選択性をコントロールできることが明らかとなった。 From the results of Experimental Examples 18 to 21, it was clarified that the selectivity for anionic polysaccharides can be controlled by appropriately adjusting the chemical structure and solution conditions of the fluorescent compound.
[実験例22]
実施例16の発蛍光性化合物(OPV−CN−G)について、ジカルボン酸に対する蛍光特性を検討した。具体的には、OPV−CN−Gに、ジカルボン酸として、L−酒石酸又はmeso−酒石酸を添加して蛍光を測定した。測定条件は以下のとおりであった。
[Experimental Example 22]
The fluorescence characteristics of the fluorescent compound (OPV-CN-G) of Example 16 with respect to a dicarboxylic acid were examined. Specifically, L-tartaric acid or meso-tartaric acid was added as a dicarboxylic acid to OPV-CN-G, and the fluorescence was measured. The measurement conditions were as follows.
(測定条件)
OPV−CN−Gの濃度:10μM、DMSO/HEPESバッファー(10mM、pH7.4)中(1:2 v/v)。
L−酒石酸及びmeso−酒石酸の濃度:3.0mM。
励起波長λex:365nm(UVハンディーランプ)
(Measurement condition)
Concentration of OPV-CN-G: 10 μM, in DMSO / HEPES buffer (10 mM, pH 7.4) (1: 2 v / v).
Concentrations of L-tartaric acid and meso-tartaric acid: 3.0 mM.
Excitation wavelength λ ex : 365 nm (UV handy lamp)
測定結果を図18(a)〜(c)に示す。図18(a)は、実験結果を示す写真である。また、図18(b)及び(c)は、各試料の分光学的特徴を測定した結果を示すグラフである。図18(a)に示されるように、OPV−CN−Gは酒石酸の化学構造により異なる蛍光色を発することが肉眼で観察された。具体的には、OPV−CN−G単独では青色であるのに対し、L−酒石酸の存在下では緑色の蛍光を示し、meso−酒石酸の存在下では黄色の蛍光を示した。また、図18(b)及び(c)に示されるように、OPV−CN−Gは、ジカルボン酸の化学構造に対応した分光学的特徴を有することが明らかとなった。 The measurement results are shown in FIGS. 18 (a) to 18 (c). FIG. 18A is a photograph showing the experimental results. Further, FIGS. 18 (b) and 18 (c) are graphs showing the results of measuring the spectroscopic characteristics of each sample. As shown in FIG. 18 (a), it was observed with the naked eye that OPV-CN-G emits a different fluorescent color depending on the chemical structure of tartaric acid. Specifically, OPV-CN-G alone was blue, whereas it showed green fluorescence in the presence of L-tartaric acid and yellow fluorescence in the presence of meso-tartaric acid. Further, as shown in FIGS. 18 (b) and 18 (c), it was revealed that OPV-CN-G has spectroscopic characteristics corresponding to the chemical structure of the dicarboxylic acid.
実験例22の結果に示されるように、OPV−CN−Gを用いると、ジカルボン酸の化学構造を、発光色の違いにより肉眼で、又は分光光度計を用いて分光学的に識別できることが明らかとなった。 As shown in the results of Experimental Example 22, it is clear that the chemical structure of the dicarboxylic acid can be spectroscopically identified by the difference in emission color with the naked eye or using a spectrophotometer by using OPV-CN-G. It became.
[実験例23]
上記式(1)において、R3で表されるアルキレン基がアミノ酸に由来するスペーサーを含む、実施例17の発蛍光性化合物(以下、「OPV−CN−G*」という場合がある。)を合成した。下記式(7)にOPV−CN−G*の化学式を示す。式(7)中、「‡」で示す基はアミノ酸(アラニン)に由来する基である。また、「†」は不斉炭素原子を表す。
[Experimental Example 23]
In the above formula (1), comprising a spacer which alkylene group represented by R 3 is derived from amino acids, fluorogenic compound of Example 17 (hereinafter referred to as "OPV-CN-G *".) The Synthesized. The chemical formula of OPV-CN-G * is shown in the following formula (7). In the formula (7), the group represented by "‡" is a group derived from an amino acid (alanine). In addition, "†" represents an asymmetric carbon atom.
OPV−CN−G*に紫外線を照射して蛍光を肉眼で観察した結果、OPV−CN−G*は、固体状態と溶媒(DMSO)に溶解した状態とで異なる波長の蛍光を発することが明らかとなった。この結果は、OPV−CN−G*が凝集形態の変化に応じて異なる波長の蛍光を発することを示す。 As a result of irradiating OPV-CN-G * with ultraviolet rays and observing the fluorescence with the naked eye, it is clear that OPV-CN-G * emits fluorescence of different wavelengths in the solid state and the state dissolved in a solvent (DMSO). It became. This result indicates that OPV-CN-G * fluoresces at different wavelengths depending on the change in agglutination morphology.
本発明により、凝集誘起発光(AIE)を示す新たな発蛍光性化合物を提供することができる。また、イオン性化合物の検出剤を提供することができる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a novel fluorescent compound exhibiting aggregation-induced luminescence (AIE). In addition, an agent for detecting an ionic compound can be provided.
10…ヒアルロン酸、11,31…負電荷、20…OPV−G、21…カチオン性基、30…ヘパリン、100…J−会合体、200…H−会合体。 10 ... hyaluronic acid, 11,31 ... negative charge, 20 ... OPV-G, 21 ... cationic group, 30 ... heparin, 100 ... J-aggregate, 200 ... H-aggregate.
Claims (5)
Xが上記式(2)で表されるカチオン性基、又は、リン酸基、カルボン酸基及び硫酸基から選択されるアニオン性基である場合、当該Xに隣接するR4は存在せず、Xが上記式(3)又は(4)で表されるカチオン性基である場合、当該Xに隣接するR4は、水素原子、又は、−CH2−が、−O−、−S−、−NH−、−CO−、−CONH−に置換されることにより中断されていてもよく、ここで、−O−、−S−、−NH−、−CO−若しくは−CONH−が隣接することはない炭素数1〜20のアルキル基若しくは−CH2CH2OH若しくは−N(R5)2(ここで、R5はそれぞれ独立に、水素原子又は−CH2−が、−O−、−S−、−NH−、−CO−、−CONH−に置換されることにより中断されていてもよく、ここで、−O−、−S−、−NH−、−CO−若しくは−CONH−が隣接することはない炭素数1〜20のアルキル基を表す。)を表す。pはそれぞれ独立に0〜4の整数を表し、qはそれぞれ独立に0〜5の整数を表し、少なくとも1つのqは0ではなく、少なくとも1つのXは前記カチオン性基又は前記アニオン性基である。] A fluorescent compound represented by the following formula (1) or a salt thereof.
When X is a cationic group represented by the above formula (2) or an anionic group selected from a phosphate group, a carboxylic acid group and a sulfate group, R 4 adjacent to the X does not exist. When X is a cationic group represented by the above formula (3) or (4), R 4 adjacent to X is a hydrogen atom, or -CH 2- is -O-, -S-, It may be interrupted by being replaced by −NH−, −CO−, −CONH−, where −O−, −S−, −NH−, −CO− or −CONH− are adjacent. There is no alkyl group with 1 to 20 carbon atoms or -CH 2 CH 2 OH or -N (R 5 ) 2 (Here, R 5 is an independent hydrogen atom or -CH 2- is -O-,- It may be interrupted by being replaced by S-, -NH-, -CO-, -CONH-, where -O-, -S-, -NH-, -CO- or -CONH- Represents an alkyl group having 1 to 20 carbon atoms that is not adjacent to each other). p independently represents an integer of 0 to 4, q independently represents an integer of 0 to 5, at least one q is not 0, and at least one X is the cationic group or the anionic group. be. ]
前記混合液の蛍光を検出する工程と、を備える、被検試料中のイオン性化合物の検出方法。 A step of preparing a mixed solution containing a solvent, a test sample, and the fluorescent compound according to claim 1 or 2 or a salt thereof.
A method for detecting an ionic compound in a test sample, comprising a step of detecting the fluorescence of the mixed solution.
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