JP2005201773A - Secondary copper ion measuring probe and measuring method of secondary copper ion using the same - Google Patents
Secondary copper ion measuring probe and measuring method of secondary copper ion using the same Download PDFInfo
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本発明は、第二銅イオン測定用プローブ及びそれを用いた第二銅イオンの測定方法に関する。 The present invention relates to a cupric ion measurement probe and a cupric ion measurement method using the same.
銅イオンは、人体における必須重金属イオンの中で三番目に多く、ヘモグロビン合成などの酵素活性に関与しており、銅の欠乏によって貧血、過剰によってヘモグロビン尿症などの症状が起こる。また、環境や人体への影響から水田中の銅濃度に関して基準が定められている。また、ブドウ酒中の銅の定量、工業材料における銅の定量などが行われている。 Copper ions are the third most important heavy metal ion in the human body and are involved in enzyme activities such as hemoglobin synthesis. Symptoms such as anemia due to copper deficiency and hemoglobinuria due to excess are caused. In addition, a standard has been established regarding the copper concentration in paddy fields because of its impact on the environment and human body. In addition, determination of copper in wine, determination of copper in industrial materials, and the like are performed.
吸光、蛍光といった分光学的手法では、高速、簡便、高感度に測定対象を定量可能である。銅の定量においては、従来、比色抽出試薬として、Bathocuproine (2,9-ジメチル -4,7-ジフェニル-1,10-フェナントロリン）などが使われてきた。BathocuproineはCu+に選択的である。また、Cu2+に対してのクロモイオノフォアおよびフルオロイオノフォアの報告例もいくつか存在する。 Spectroscopic methods such as absorption and fluorescence can quantitate the measurement target at high speed, simply and with high sensitivity. In the determination of copper, hitherto, Bathocuproine (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) has been used as a colorimetric extraction reagent. Bathocuproine is selective to Cu + . There are also several reports of chromoionophores and fluoroionophores for Cu 2+ .
しかしながら、公知のCu2+選択性クロモイオノフォアおよびフルオロイオノフォアでは選択性、感度が低く、他のイオンの共存による妨害や、着色物質の妨害を受けやすかった。 However, known Cu 2+ selective chromoionophores and fluoroionophores have low selectivity and sensitivity, and are susceptible to interference due to the coexistence of other ions and coloring substances.
従って、本発明の目的は、第二銅イオンに対する選択性が高く、第二銅イオンを高感度に測定することができる第二銅イオン測定用プローブ及びそれを用いた第二銅イオンの測定方法を提供することである。 Accordingly, an object of the present invention is to provide a cupric ion measurement probe having high selectivity to cupric ions and capable of measuring cupric ions with high sensitivity, and a method for measuring cupric ions using the same. Is to provide.
本願発明者らは、鋭意研究の結果、環構造上のラクトンを構成する酸素原子と、該ラクトンに隣接する炭素原子に、１個の炭素原子を介して結合するイオウ原子とを含む構造が、第二銅イオンと高度に選択的に結合することを見出し、さらに、この構造を吸光性又は蛍光性の色素原子団に結合すれば、前記構造が第二銅イオンと結合した際の吸光度又は蛍光の変化を測定することにより試料中の第二銅イオンを選択的かつ高感度に測定できることを見出し、本発明を完成した。 As a result of earnest research, the inventors of the present application have a structure including an oxygen atom constituting a lactone on a ring structure and a sulfur atom bonded to a carbon atom adjacent to the lactone via one carbon atom. We find that it binds to cupric ions with high selectivity, and if this structure is bound to a light-absorbing or fluorescent dye atomic group, the absorbance or fluorescence when the structure is bound to cupric ions. By measuring this change, it was found that cupric ions in the sample can be measured selectively and with high sensitivity, and the present invention has been completed.
すなわち、本発明は、下記一般式[I] That is, the present invention provides the following general formula [I]
（ただし、式中、R1は-CH2-を表し、R2は水素原子若しくは一般式[I]で表される化合物と第二銅イオンとの結合を妨害しない任意の基を表し、又はR1とR2は協働して環を形成し、Ａは式中の炭素原子１及び２と共に環式構造を形成する原子団、Ｘは色素原子団であって、Ａを含む環と縮合環を形成していてもよい）
で表される構造を有する第二銅イオン測定用プローブを提供する。また、本発明は、上記本発明の第二銅イオン測定用プローブを、第二銅イオンを含む検体と接触させ、検体中の第二銅イオンと結合した該プローブの吸光度又は蛍光を測定することを含む、検体中の第二銅イオンの測定方法を提供する。
(Wherein, R 1 represents —CH 2 —, R 2 represents a hydrogen atom or any group that does not interfere with the binding between the compound represented by the general formula [I] and the cupric ion, or R 1 and R 2 cooperate to form a ring, A is an atomic group that forms a cyclic structure with carbon atoms 1 and 2 in the formula, X is a dye atomic group, and is condensed with the ring containing A A ring may be formed)
A probe for measuring cupric ion having a structure represented by the formula: Further, the present invention comprises contacting the probe for measuring cupric ion of the present invention with a specimen containing cupric ion, and measuring the absorbance or fluorescence of the probe bound to cupric ion in the specimen. A method for measuring cupric ions in a specimen is provided.
本発明によれば、第二銅イオンに対する選択性が高く、第二銅イオンを高感度に測定することができる新規な第二銅イオン測定用プローブ及びそれを用いた第二銅イオンの測定方法が提供された。下記実施例において具体的に記載されるように、本発明のプローブは、第二銅イオンと接触すると、他の金属陽イオンと接触した場合には見られない吸光ピークが特定の波長において現れ、また、他の金属陽イオンと接触した場合には見られない蛍光強度の変化が見られる。従って、本発明のプローブにより、第二銅イオンを選択的に高感度に測定することができる。 ADVANTAGE OF THE INVENTION According to this invention, the selectivity for a cupric ion is high, the novel cupric ion measuring probe which can measure a cupric ion with high sensitivity, and the measuring method of the cupric ion using the same Was provided. As specifically described in the examples below, when the probe of the present invention is contacted with cupric ions, an absorption peak that does not appear when contacted with other metal cations appears at a specific wavelength, Moreover, the change of the fluorescence intensity which cannot be seen when it contacts with another metal cation is seen. Therefore, cupric ions can be selectively measured with high sensitivity by the probe of the present invention.
上記の通り、本発明の第二銅イオン測定用プローブは、上記一般式[I]で表される構造を有する。一般式[I]中に示されるラクトンを構成する酸素原子と、炭素原子２に結合している基中のイオウ原子とが第二銅イオンとの結合に重要であり、これらの原子の間に第二銅イオンが挟まれて錯結合されるものと考えられる。従って、R2は水素原子又は一般式[I]で表される化合物と第二銅イオンとの結合を妨害しない基であれば任意の基であってよい。このような基の例として、ハロゲン、低級（炭素数１〜６、以下同じ）アルキル基、低級アルコキシル基、アリール基（フェニル基、ナフチル基等）を挙げることができる。もっとも、イオウ原子が堅固に保持されることが好ましいので、R1とR2は協働して環を形成することが好ましい。環は５員環又は６員環が好ましく、特に、プローブが下記一般式[II]で表される構造を有するものであることが好ましい。 As described above, the cupric ion measurement probe of the present invention has a structure represented by the above general formula [I]. The oxygen atom constituting the lactone represented by the general formula [I] and the sulfur atom in the group bonded to the carbon atom 2 are important for binding to the cupric ion, and between these atoms It is thought that cupric ions are sandwiched and complex bonded. Therefore, R 2 may be any group as long as it does not interfere with the bond between the hydrogen atom or the compound represented by the general formula [I] and the cupric ion. Examples of such groups include halogen, lower (C1-C6, the same applies hereinafter) alkyl group, lower alkoxyl group, and aryl group (phenyl group, naphthyl group, etc.). However, since it is preferable that the sulfur atom is held firmly, it is preferable that R 1 and R 2 cooperate to form a ring. The ring is preferably a 5-membered ring or a 6-membered ring, and in particular, the probe preferably has a structure represented by the following general formula [II].
式中、Ａ及びＸは一般式[I]と同義、Y1は-O-、-S-、-CR3R4-（ただし、R3及びR4は互いに独立に水素原子又は一般式[II]で表される化合物と第二銅イオンとの結合を妨害しない任意の基）を表す。ここで、「任意の基」の好ましい例としては、ハロゲン、アルキル基（好ましくは低級（炭素数１〜６、以下同じ）アルキル基）、置換アルキル基（好ましくは置換低級アルキル基（置換基の例としては１又は複数のニトロ基、ハロゲノ基、スルホン酸基等）、アルコキシル基（好ましくは低級アルコキシル基）、アリール基（フェニル基、ナフチル基等）、置換アリール基（置換基の例としてはニトロ基、アルキル基（好ましくは低級アルキル基）、ハロゲノ基、スルホン酸基）アミノ基、ニトロ基等を挙げることができる。ｎは１又は２の整数を表し、すなわち、Ｓを含む環が５員環又は６員環である。また、Ｓを含む環を構成する任意の１又は２以上の炭素原子に結合している各水素原子は、一般式[II]で表される化合物と第二銅イオンとの結合を妨害しない任意の基で置換されていてもよい。ここで、「任意の基」の好ましい例としては、上記したR3及びR4の「任意の基」の好ましい例を挙げることができる。なお、Ｓを含む環を構成する炭素原子（Y1が-CR3R4-である場合には、-CR3R4-中の炭素原子も包含する）には、上記の通り置換基が結合していてもよいが、これらの置換基は不要であるので、合成の簡便さやコストを考慮すると、これらの置換基は存在しないことが好ましい。 In the formula, A and X have the same meanings as those in the general formula [I], Y 1 is —O—, —S—, —CR 3 R 4 — (wherein R 3 and R 4 are each independently a hydrogen atom or a general formula [ II] represents an arbitrary group that does not interfere with the binding between the compound represented by II and the cupric ion. Here, preferable examples of the “arbitrary group” include a halogen, an alkyl group (preferably a lower (C1-C6, the same shall apply hereinafter) alkyl group), a substituted alkyl group (preferably a substituted lower alkyl group (of the substituent). Examples include one or more nitro groups, halogeno groups, sulfonic acid groups, etc., alkoxyl groups (preferably lower alkoxyl groups), aryl groups (phenyl groups, naphthyl groups, etc.), substituted aryl groups (examples of substituents) Examples thereof include a nitro group, an alkyl group (preferably a lower alkyl group), a halogeno group, a sulfonic acid group, an amino group, a nitro group, etc. n represents an integer of 1 or 2, that is, a ring containing S is 5 Each of the hydrogen atoms bonded to any one or more carbon atoms constituting the ring containing S is a compound represented by the general formula [II] and a second ring. Copper ion The preferred examples of “optional group” include the preferred examples of “optional group” of R 3 and R 4 described above. The carbon atom constituting the ring containing S (when Y 1 is —CR 3 R 4 —, also includes the carbon atom in —CR 3 R 4 —) is as described above. Although substituents may be bonded, these substituents are unnecessary. Therefore, in view of the convenience of synthesis and cost, it is preferable that these substituents do not exist.
上記一般式[II]中のＳを含む環は、Y1が-S-でｎが１であり、Ｓを含む環構造を構成する炭素原子に置換基が結合していないものが特に好ましい。 The ring containing S in the general formula [II] is particularly preferably one in which Y 1 is —S— and n is 1, and no substituent is bonded to the carbon atom constituting the ring structure containing S.
一般式[I]中、Ａを含む環構造は、ラクトンの酸素原子が結合している炭素原子１及びイオウを含む基が結合している炭素原子２を堅固に保持することに役立っており、これに色素原子団Ｘが結合している。色素原子団Ｘは、Ａを含む環と縮合環を形成していてもよく、この場合には色素原子団ＸとＡを含む環との縮合環が色素を構成する。従って、「色素原子団」は、それ自体で色素を構成するものであってもよいし、Ａを含む環と協働して色素を形成するものであってもよい。ここで、「色素」は、吸光性又は蛍光性を示すものである。一般に、吸光性又は蛍光性を示す化合物は、金属イオン等と結合するとその吸光特性（吸光度及び／若しくは最大吸収波長）又は蛍光特性（蛍光強度及び／若しくは蛍光波長）が変化するので、その変化を利用して金属イオン等を測定することが可能である。従って、Ａを含む環及び色素原子団Ｘの構造は、吸光性又は蛍光性を有するものであれば特に限定されるものではない。プローブの好ましい例として、下記一般式[III]又は[IV]で表されるものを挙げることができる。 In the general formula [I], the ring structure containing A serves to firmly hold the carbon atom 1 to which the oxygen atom of the lactone is bonded and the carbon atom 2 to which the group containing sulfur is bonded, The dye atomic group X is bonded to this. The dye atom group X may form a condensed ring with the ring containing A. In this case, the condensed ring of the dye atom group X and the ring containing A constitutes the dye. Therefore, the “dye atom group” may constitute a dye by itself, or may form a dye in cooperation with a ring containing A. Here, the “dye” indicates absorbance or fluorescence. In general, a compound exhibiting absorbance or fluorescence changes its absorbance characteristics (absorbance and / or maximum absorption wavelength) or fluorescence characteristics (fluorescence intensity and / or fluorescence wavelength) when bound to a metal ion or the like. It is possible to measure metal ions and the like. Therefore, the structure of the ring containing A and the dye atomic group X is not particularly limited as long as it has light absorption or fluorescence. Preferred examples of the probe include those represented by the following general formula [III] or [IV].
式中、Y1及びｎは一般式[II]と同義、Y2は-O-、-S-、-CR3R4-（ただし、R3及びR4は互いに独立に水素原子又は一般式[II]で表される化合物と第二銅イオンとの結合を妨害しない任意の基）を示す。ここで、「任意の基」の好ましい例としては、上記した、一般式[II]におけるＳを含む環上の置換基の好ましい例を挙げることができるが、水素原子が好ましい。式中の縮合環が色素であるので、X'は存在していても存在していなくてもよく、存在する場合には一般式[III]中の縮合環構造と協働して色素を形成する原子団であってベンゼン環と縮合していてもよい。一般式[III] 中の環構造を構成する任意の１又は２以上の炭素原子に結合している各水素原子は、一般式[III]で表される化合物と第二銅イオンとの結合を妨害しない任意の基で置換されていてもよい。ここで、「任意の基」の好ましい例としては、上記した、一般式[II]における環上の置換基の好ましい例を挙げることができるが、上記と同様、このような置換基は不要であるので存在しない方が合成の簡便さやコストの観点から好ましい。 In the formula, Y 1 and n are as defined in the general formula [II], Y 2 is —O—, —S—, —CR 3 R 4 — (wherein R 3 and R 4 are each independently a hydrogen atom or a general formula An arbitrary group that does not interfere with the binding between the compound represented by [II] and the cupric ion). Here, preferred examples of the “arbitrary group” include the above-mentioned preferred examples of the substituent on the ring containing S in the general formula [II], and a hydrogen atom is preferred. Since the condensed ring in the formula is a dye, X ′ may or may not be present, and if present, forms a dye in cooperation with the condensed ring structure in the general formula [III]. Which may be condensed with a benzene ring. Each hydrogen atom bonded to any one or more carbon atoms constituting the ring structure in the general formula [III] forms a bond between the compound represented by the general formula [III] and the cupric ion. It may be substituted with any group that does not interfere. Here, preferred examples of the “arbitrary group” include the above-mentioned preferred examples of the substituent on the ring in the general formula [II], but as described above, such a substituent is unnecessary. Since it exists, it is preferable that it does not exist from the viewpoint of the ease of synthesis and cost.
式中、Y1及びｎは一般式[II]と同義、Y3は-O-、-S-、-CR3R4-（ただし、R3及びR4は互いに独立に水素原子又は一般式[IV]で表される化合物と第二銅イオンとの結合を妨害しない任意の基）を示す。ここで、「任意の基」の好ましい例としては、上記した一般式[II]におけるＳを含む環上の置換基の好ましい例を挙げることができるが、水素原子が好ましい。Ｚは-CH=又は-N=を表わす。一般式[IV] 中の環構造を構成する任意の１又は２以上の炭素原子に結合している各水素原子は、一般式[IV]で表される化合物と第二銅イオンとの結合を妨害しない任意の基で置換されていてもよい。ここで、「任意の基」の好ましい例としては、上記した、一般式[II]におけるＳを含む環上の置換基の好ましい例を挙げることができるが、上記と同様、このような置換基は不要であるので存在しない方が合成の簡便さやコストの観点から好ましい。 In the formula, Y 1 and n are as defined in the general formula [II], Y 3 is —O—, —S—, —CR 3 R 4 — (wherein R 3 and R 4 are each independently a hydrogen atom or a general formula Any group that does not interfere with the binding between the compound represented by [IV] and the cupric ion). Here, preferred examples of the “arbitrary group” include preferred examples of the substituent on the ring containing S in the above general formula [II], and a hydrogen atom is preferred. Z represents -CH = or -N =. Each hydrogen atom bonded to any one or more carbon atoms constituting the ring structure in the general formula [IV] forms a bond between the compound represented by the general formula [IV] and the cupric ion. It may be substituted with any group that does not interfere. Here, preferred examples of the “arbitrary group” include the above-mentioned preferred examples of the substituent on the ring containing S in the general formula [II]. Is not necessary, and is preferably not present from the viewpoint of the ease of synthesis and cost.
一般式[III]及び[IV]において、aで示す炭素原子に置換基が結合している場合、該置換基は、上記した「任意の基」であってよいが、特にNR5R6（ただし、R5及びR6は互いに独立に水素原子又は一般式[III]又は[IV]で表される化合物と第二銅イオンとの結合を妨害しない任意の基（ここで「任意の基」の好ましい例としては、上記した、一般式[II]におけるＳを含む環上の置換基の好ましい例）であり、水素原子が好ましい）、水酸基、アリール基（フェニル基、ナフチル基等）、置換アリール基（ここで、置換基の好ましい例としては１又は複数のニトロ基、アルキル基、ハロゲノ基、スルホン酸基等）、アミノ基、ニトロ基、アルキル基（好ましくは低級アルキル基）、置換アルキル基（好ましくは低級置換アルキル基、置換基の好ましい例としては１又は複数のニトロ基、ハロゲノ基、スルホン酸基等）を好ましい例として挙げることができる。 In the general formulas [III] and [IV], when a substituent is bonded to the carbon atom represented by a, the substituent may be the above-mentioned “arbitrary group”, but particularly NR 5 R 6 ( However, R 5 and R 6 are each independently a hydrogen atom or any group that does not interfere with the binding between the compound represented by the general formula [III] or [IV] and the cupric ion (here, “arbitrary group” Preferred examples of the above are preferred examples of the substituent on the ring containing S in the general formula [II] described above, preferably a hydrogen atom), hydroxyl group, aryl group (phenyl group, naphthyl group, etc.), substitution An aryl group (wherein, as preferred examples of the substituent, one or more nitro groups, alkyl groups, halogeno groups, sulfonic acid groups, etc.), amino groups, nitro groups, alkyl groups (preferably lower alkyl groups), substituted alkyls Groups (preferably lower substituted alkyl groups, preferred examples of substituents and Can be exemplified one or more nitro group, a halogeno group, a sulfonic acid group, etc.) Preferred examples Te.
上記一般式[III]で表される化合物の好ましい例として、下記一般式[V]で表されるものを挙げることができる。 Preferable examples of the compound represented by the general formula [III] include those represented by the following general formula [V].
式中、Y1、ｎ及びY2は一般式[III]と同義である。一般式[V] 中の環構造を構成する任意の１又は２以上の炭素原子に結合している各水素原子は、一般式[V]で表される化合物と第二銅イオンとの結合を妨害しない任意の基で置換されていてもよい。ここで、「任意の基」の好ましい例としては、上記した、一般式[II]における環上の置換基の好ましい例を挙げることができるが、上記と同様、このような置換基は不要であるので存在しない方が合成の簡便さやコストの観点から好ましい。一般式[V]で表される化合物のうち、特に好ましい例として、下記式[VI]で表されるもの(「KCU-1」と命名）を挙げることができる。 In the formula, Y 1 , n and Y 2 have the same meaning as in the general formula [III]. Each hydrogen atom bonded to any one or more carbon atoms constituting the ring structure in the general formula [V] forms a bond between the compound represented by the general formula [V] and the cupric ion. It may be substituted with any group that does not interfere. Here, preferred examples of the “arbitrary group” include the above-mentioned preferred examples of the substituent on the ring in the general formula [II], but as described above, such a substituent is unnecessary. Since it exists, it is preferable that it does not exist from the viewpoint of the ease of synthesis and cost. Among the compounds represented by the general formula [V], particularly preferred examples include those represented by the following formula [VI] (named “KCU-1”).
KCU-1は、可視領域に吸収を持ち明るい蛍光を発するクマリン骨格を有し、ラクトンのα位にジチオアセタール構造を有するものである。下記実施例において具体的に示されるように、KCU-1は、第二銅イオンの存在下において、波長533nm及び578nmに特異的な（すなわち、他の金属イオンの存在下においては見られない）吸収極大を示す。従って、これらの特性を利用して第二銅イオンを選択的に測定することが可能である。また、第二銅イオンの存在下において、波長480nmの蛍光強度が特異的に減少するので、検体中に第二銅イオン以外の金属イオンが存在しないことがわかっている場合には蛍光強度の減少を利用して検体中の第二銅イオンを測定することができる。 KCU-1 has a coumarin skeleton that absorbs in the visible region and emits bright fluorescence, and has a dithioacetal structure at the α-position of the lactone. As specifically shown in the examples below, KCU-1 is specific for wavelengths 533 nm and 578 nm in the presence of cupric ions (ie, not seen in the presence of other metal ions). The absorption maximum is shown. Therefore, cupric ions can be selectively measured using these characteristics. In addition, since the fluorescence intensity at a wavelength of 480 nm specifically decreases in the presence of cupric ions, if it is known that metal ions other than cupric ions are not present in the sample, the fluorescence intensity decreases. Can be used to measure cupric ions in the specimen.
本発明のプローブは、炭素原子２にシアノ基等の官能基が結合した環式色素を出発物質とし、これにアルキルチオアルコールを反応させる公知の手法に基づいて合成することができ、その具体例は下記実施例に詳述されている。 The probe of the present invention can be synthesized based on a known method in which a starting material is a cyclic dye having a functional group such as a cyano group bonded to carbon atom 2 and an alkylthioalcohol is reacted therewith. Detailed in the examples below.
本発明のプローブは、検体にプローブを作用させ、吸光度を測定するか又は励起光を当てて蛍光を測定するという、従来の吸光プローブ又は蛍光プローブと全く同様な方法によって使用することができる。例えば、アセトニトリルのような、極性有機溶媒に溶解したものを検体に加え（又は検体にこれを加え）て、吸光を測定するか又は励起光を当てて蛍光を測定することができる。極性有機溶媒中のプローブ濃度は、特に限定されないが、通常、1 nMないし1 mM程度、好ましくは、1μMないし１０μM程度である。反応温度は、特に限定されず、各検体に適した温度が適宜選択できるが、通常、０℃〜４０℃程度であり、室温が簡便で好ましい。室温で混合するだけで速やかに錯形成が起きる。なお、本発明における「測定」には、定量と検出の両者が包含される。 The probe of the present invention can be used in the same manner as a conventional light-absorbing probe or fluorescent probe, in which a probe is allowed to act on a specimen and the absorbance is measured or fluorescence is measured by applying excitation light. For example, a substance dissolved in a polar organic solvent such as acetonitrile can be added to the specimen (or added to the specimen), and the absorbance can be measured or the fluorescence can be measured by applying excitation light. The probe concentration in the polar organic solvent is not particularly limited, but is usually about 1 nM to 1 mM, preferably about 1 μM to 10 μM. The reaction temperature is not particularly limited, and a temperature suitable for each specimen can be selected as appropriate, but is usually about 0 ° C. to 40 ° C., and room temperature is convenient and preferable. Complexation occurs quickly just by mixing at room temperature. The “measurement” in the present invention includes both quantification and detection.
検体は、第二銅イオンを測定することが望まれるいずれのものであってもよく、例えば水田の水のような種々の環境由来の検体、中間体を包含する各種工業材料、ワイン等の飲食品、体液等の生体由来試料等を挙げることができる。 The specimen may be any specimen for which it is desired to measure cupric ions. For example, specimens derived from various environments such as paddy water, various industrial materials including intermediates, food and drink such as wine And biological samples such as products and body fluids.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
KCU-1の合成
(1)10-オキソ-2,3,5,6-テトラヒドロ-1H,4H,10H-11-オキサ-3a-アザ-ベンゾ [デ]アントラセン-9-カルボアルデヒドの合成
Synthesis of KCU-1
(1) Synthesis of 10-oxo-2,3,5,6-tetrahydro-1H, 4H, 10H-11-oxa-3a-aza-benzo [de] anthracene-9-carbaldehyde
アルゴン置換した50 ml二口ナスフラスコに，Coumarin337 (1) (市販品、500 mg, 1.88 mmol, 1 eq.)を入れ、ギ酸 (4.5 ml) に溶解した。この溶液にRaney Ni (0.625 ml, 4.51 mmol)を加え、130℃で1.5時間還流した。反応系をろ過し、水を加えてクロロホルムで2回抽出し、有機層をNaHCO3aq、NaClaqで2回洗浄後、芒硝乾燥した。溶媒を減圧留去し、黒褐色の化合物4 (441 mg, 収率；87.1 ％)を得た。 Coumarin337 (1) (commercial product, 500 mg, 1.88 mmol, 1 eq.) Was placed in a 50 ml two-necked eggplant flask purged with argon, and dissolved in formic acid (4.5 ml). Raney Ni (0.625 ml, 4.51 mmol) was added to this solution and refluxed at 130 ° C. for 1.5 hours. The reaction system was filtered, water was added, and the mixture was extracted twice with chloroform. The organic layer was washed twice with NaHCO 3 aq and NaClaq, and dried with sodium sulfate. The solvent was distilled off under reduced pressure to obtain black-brown compound 4 (441 mg, yield; 87.1%).
薄層クロマトグラフィー(TLC); Rf=0.5(溶離液;クロロホルム/酢酸エチル= 10/1, v/v=1/1)
1H-NMR (300 MHz, CDCl3, TMS, r. t.)
δ(ppm) 1.98 (s, 4H, -CH2-), 2.76 (t, J = 6.2Hz, 2H, -CH2-), 2.89 (t, J = 6.3 Hz, 2H, -CH2-), 3.24 (q, J = 5.5 Hz, 4H, -CH2-), 3.37 (q, J = 4.2 Hz, 2H, -CH2-), 6.98 (s, 1H, ArH), 8.14 (s, 1H, ArH)
Thin layer chromatography (TLC); Rf = 0.5 (eluent: chloroform / ethyl acetate = 10/1, v / v = 1/1)
1 H-NMR (300 MHz, CDCl 3 , TMS, rt)
δ (ppm) 1.98 (s, 4H, -CH 2- ), 2.76 (t, J = 6.2Hz, 2H, -CH 2- ), 2.89 (t, J = 6.3 Hz, 2H, -CH 2- ), 3.24 (q, J = 5.5 Hz, 4H, -CH 2- ), 3.37 (q, J = 4.2 Hz, 2H, -CH 2- ), 6.98 (s, 1H, ArH), 8.14 (s, 1H, ArH )
(2) 9-[1,3]ジチオラン-2-イル-2,3,5,6-テトラヒドロ-1H,4H-11-オキサ-3a-アザ-ベンゾ[デ]アントラセン-10-オン (KCU-1)の合成 (2) 9- [1,3] Dithiolan-2-yl-2,3,5,6-tetrahydro-1H, 4H-11-oxa-3a-aza-benzo [de] anthracen-10-one (KCU- Synthesis of 1)
Ar置換した100 ml三口ナスフラスコに，化合物4 (200 mg, 0.743 mmol, 1 eq.)を加え、塩化メチレン (2.78 ml) に溶かした。さらに1,2-エタンジチオール (5) (0.111 g, 1.18 mmol, 1.5 eq.)を加えた。この溶液に三フッ化ホウ素エーテレート (18.6 mg, 0.131 mmol, 0.176 eq.) を加え、65℃で加熱した。24 時間後に三フッ化ホウ素エーテレート (33.4 mg, 0.235mol, 0.317 eq.)加え、さらに15時間後に1,2-ジクロロエタン (10 ml)，および塩化メチレン (2.78 ml) を加えた。その後105 時間還流した。反応系に飽和NaHCO3水溶液を加え、クロロホルムで2回抽出し、NaClaqで2回洗浄後、芒硝乾燥した。溶媒を減圧留去し、粗生成物6 を得た。カラムクロマトグラフィ(SiO2, クロロホルム：ヘキサン = 4 : 1 v/v)で精製し、褐色固体 6 (136.2 mg, 収率 : 53.1 %) を得た。
Compound 4 (200 mg, 0.743 mmol, 1 eq.) Was added to an Ar-substituted 100 ml three-necked eggplant flask and dissolved in methylene chloride (2.78 ml). Further 1,2-ethanedithiol (5) (0.111 g, 1.18 mmol, 1.5 eq.) Was added. Boron trifluoride etherate (18.6 mg, 0.131 mmol, 0.176 eq.) Was added to this solution and heated at 65 ° C. After 24 hours, boron trifluoride etherate (33.4 mg, 0.235 mol, 0.317 eq.) Was added, and after 15 hours, 1,2-dichloroethane (10 ml) and methylene chloride (2.78 ml) were added. Thereafter, the mixture was refluxed for 105 hours. A saturated aqueous NaHCO 3 solution was added to the reaction system, extracted twice with chloroform, washed twice with NaClaq, and dried with sodium sulfate. The solvent was distilled off under reduced pressure to obtain a
TLC ; Rf = 0.2 (溶離液; クロロホルム/酢酸エチル= 4/1, v/v=1/1)
1H-NMR (300 MHz, CDCl3, TMS, r. t.)
δ (ppm) 1.96 (m, J = 6.0 Hz, 4H, -CH2-), 2.75 (t, J = 6.2 Hz, 2H, -CH2-), 2.87 (t, J = 6.3 Hz, 2H, -CH2-), 3.25 (q, J = 5.5 Hz, 4H, -CH2-), 3.31 (s, 4H, -CH2-), 5.76 (s, 1H, -CH-), 6.87 (s, 1H, ArH), 7.84 (s, 1H, ArH)
TLC; R f = 0.2 (eluent: chloroform / ethyl acetate = 4/1, v / v = 1/1)
1 H-NMR (300 MHz, CDCl 3 , TMS, rt)
δ (ppm) 1.96 (m, J = 6.0 Hz, 4H, -CH 2- ), 2.75 (t, J = 6.2 Hz, 2H, -CH 2- ), 2.87 (t, J = 6.3 Hz, 2H,- CH 2- ), 3.25 (q, J = 5.5 Hz, 4H, -CH 2- ), 3.31 (s, 4H, -CH 2- ), 5.76 (s, 1H, -CH-), 6.87 (s, 1H , ArH), 7.84 (s, 1H, ArH)
KCU- 1のさまざまなイオンによる吸収・蛍光スペクトル
実施例１で合成したKCU-1を、種々の金属イオンと接触させた際の色素の吸収と蛍光スペクトルを測定した。測定条件、装置条件などの詳細は以下のとおりである。
Absorption and fluorescence spectra of KCU-1 by various ions The absorption and fluorescence spectra of the dye when KCU-1 synthesized in Example 1 was brought into contact with various metal ions were measured. Details such as measurement conditions and apparatus conditions are as follows.
測定条件
KCU-1終濃度： 1.0 x 10-5 M
金属塩終濃度： 1 x 10-5 M
測定温度： 室温
溶媒： アセトニトリル
金属塩： Al(ClO4)3・9H2O, MgCl2, CaCl2, CuCl2, FeCl3, CoCl2, ZnCl2, AgCl,
CrCl3,CdCl2, PbCl2
Measurement condition
KCU-1 final concentration: 1.0 x 10 -5 M
Metal salt final concentration: 1 x 10 -5 M
Measurement temperature: Room temperature Solvent: Acetonitrile metal salt: Al (ClO 4 ) 3 · 9H 2 O, MgCl 2 , CaCl 2 , CuCl 2 , FeCl 3 , CoCl 2 , ZnCl 2 , AgCl,
CrCl 3 , CdCl 2 , PbCl 2
装置設定
U-2001(吸光光度計)
スキャン速度： 200 [nm/min]
Device setting
U-2001 (Absorptiometer)
Scan speed: 200 [nm / min]
F-4500(蛍光光度計)
スキャン速度： 240 [nm/min]
応答： 自動
バンドパス： 蛍光: 2.5 [nm]
光学倍増管： 700 [V]
F-4500 (Fluorometer)
Scan speed: 240 [nm / min]
Response: Automatic bandpass: Fluorescence: 2.5 [nm]
Optical multiplier: 700 [V]
結果を図１及び図２に示す。図１に示されるように、KCU-1は、第二銅イオンと接触すると、波長510nm以上でも吸光性を有し、533nm及び578nmに吸光度のピークが現れる。これに対し、他の金属イオンと接触した場合には、波長510nm以上では吸光度はほとんど０になる。従って、510nm以上の波長、好ましくは吸収極大のある波長533nm又は578nmの吸光度を測定することにより、検体中の第二銅イオンを選択的に測定することができる。また、波長400nmの励起光で励起した場合、約480nmにある蛍光強度の極大値が、第二銅イオンと接触した場合にはブランク（金属塩を含まない）に比べて約２５％減少するが、他の金属イオンと接触した場合には、これほどの減少は見られない。従って、蛍光特性も第二銅イオンの存在下では特異的に変化することがわかる。 The results are shown in FIGS. As shown in FIG. 1, when contacted with cupric ions, KCU-1 has absorbance even at wavelengths of 510 nm or more, and absorbance peaks appear at 533 nm and 578 nm. On the other hand, when contacting with other metal ions, the absorbance is almost zero at a wavelength of 510 nm or more. Therefore, by measuring the absorbance at a wavelength of 510 nm or longer, preferably a wavelength of 533 nm or 578 nm having an absorption maximum, cupric ions in the specimen can be selectively measured. In addition, when excited with excitation light having a wavelength of 400 nm, the maximum value of fluorescence intensity at about 480 nm is reduced by about 25% when contacted with cupric ions compared to a blank (not including a metal salt). When contacted with other metal ions, such a decrease is not observed. Therefore, it can be seen that the fluorescence characteristics also change specifically in the presence of cupric ions.
種々の濃度の塩化第二銅アセトニトリル溶液を検体とし、実施例２と同様にして励起波長400 nmにおける蛍光スペクトルを測定した。 Fluorescence spectra at an excitation wavelength of 400 nm were measured in the same manner as in Example 2 using cupric chloride acetonitrile solutions having various concentrations.
結果を図３に示す。また、図３に示すデータの塩化第二銅濃度の対数を横軸に、ピーク波長における蛍光強度を縦軸にとったグラフを図４に示す。図４に示されるように、塩化第二銅濃度が5x10-6Mから5x10-3Mの間では、蛍光強度がほぼ直線的に減少しているので、この範囲であれば第二銅イオンを定量できることがわかる。 The results are shown in FIG. FIG. 4 is a graph in which the logarithm of the cupric chloride concentration in the data shown in FIG. 3 is plotted on the horizontal axis and the fluorescence intensity at the peak wavelength is plotted on the vertical axis. As shown in FIG. 4, when the cupric chloride concentration is between 5 × 10 −6 M and 5 × 10 −3 M, the fluorescence intensity decreases almost linearly. It turns out that it can be quantified.
Claims (8)
で表される構造を有する第二銅イオン測定用プローブ。 The following general formula [I]
The probe for a cupric ion measurement which has a structure represented by these.
で表される構造を有する請求項２記載のプローブ。 The following general formula [II]
The probe according to claim 2, which has a structure represented by:
又は下記一般式[IV]
で表わされる構造を有する請求項３記載のプローブ。 The following general formula [III]
Or the following general formula [IV]
The probe of Claim 3 which has a structure represented by these.
で表わされる構造を有する請求項５記載のプローブ。 The following general formula [V]
The probe of Claim 5 which has a structure represented by these.
The probe for measuring cupric ion according to any one of claims 1 to 7 is brought into contact with a specimen containing cupric ions, and the absorbance or fluorescence of the probe bound to cupric ions in the specimen is measured. A method for measuring cupric ions in a specimen, comprising measuring.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101022518B1 (en) | 2008-09-05 | 2011-03-16 | 고려대학교 산학협력단 | Anthracene derivatives having CuII ion selectivity, method for preparing therefor and detecting method using the same |
| CN104745194A (en) * | 2015-03-24 | 2015-07-01 | 南昌大学 | Preparation method of quantum dot@Cu nano-cluster ratiometric fluorescent sensor and application thereof in Cu<2+> detection |
| CN109632746A (en) * | 2018-12-29 | 2019-04-16 | 吉林大学 | A kind of method that fluorescence probe detects sarcosine, sarcosine oxidase and its concentration |
| CN112010865A (en) * | 2020-09-23 | 2020-12-01 | 周学军 | Micromolecular fluorescent probe for detecting hypochlorous acid and preparation method and application thereof |
| CN115417881A (en) * | 2022-09-23 | 2022-12-02 | 郑州大学 | Fluorescent probe for rapidly detecting mercury ions and application thereof |
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- 2004-01-15 JP JP2004008475A patent/JP2005201773A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101022518B1 (en) | 2008-09-05 | 2011-03-16 | 고려대학교 산학협력단 | Anthracene derivatives having CuII ion selectivity, method for preparing therefor and detecting method using the same |
| CN104745194A (en) * | 2015-03-24 | 2015-07-01 | 南昌大学 | Preparation method of quantum dot@Cu nano-cluster ratiometric fluorescent sensor and application thereof in Cu<2+> detection |
| CN109632746A (en) * | 2018-12-29 | 2019-04-16 | 吉林大学 | A kind of method that fluorescence probe detects sarcosine, sarcosine oxidase and its concentration |
| CN112010865A (en) * | 2020-09-23 | 2020-12-01 | 周学军 | Micromolecular fluorescent probe for detecting hypochlorous acid and preparation method and application thereof |
| CN115417881A (en) * | 2022-09-23 | 2022-12-02 | 郑州大学 | Fluorescent probe for rapidly detecting mercury ions and application thereof |
| CN115417881B (en) * | 2022-09-23 | 2023-07-25 | 郑州大学 | Fluorescent probe for rapidly detecting mercury ions and application thereof |
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