KR100643192B1 - Novel dinuclear metal complex and pyrophosphate assay using the same - Google Patents

Abstract

New coordination complexes with dinuclear metal complexes are presented. The coordination complex is a heteronuclear metal complex of a compound, wherein the compound is a conjugation ring system, which is substituted by the following substituents: a) an electron donating group selected from -OH, -SH and -NH ₂ ; b) an indicator selected from chromophores, fluorescent and electrochemical groups; And c) two binding aids, each of which is coordinated with the metal in combination with the electron donor to provide an anion binding site. As the complex binds with the anion, the coordination between the electron donor and the metal is weakened, the electromagnetic excitation to the conjugation ring system by the electron donor is enhanced, and the enhanced electromagnetic excitation by the electron donor is the conjugation. The indicator is delivered to the indicator via a ring system, which generates an indication signal detectable by a change in electron density. The coordination complex is superior in sensitivity and selectivity to pyrophosphate over other ions over a wide pH range in aqueous solution. Thus, the coordination complex is useful for the analysis of pyrophosphate as a pyrophosphate sensor.

Description

New Binary Metal Complexes and Methods of Analysis of Pyrophosphate Using the Same {NOVEL DINUCLEAR METAL COMPLEX AND PYROPHOSPHATE ASSAY USING THE SAME}

1 is a UV-vis spectrum showing the change in absorbance according to the type of metal, (a) is a sensor 1 / 2Cu , (b) is a sensor 1 / 2Mg , (c) is a sensor 1 / 2Pb , (d) is a sensor 1, 2Zn and (e) are sensors 1 and 2Co (II) and (f) and 1 and 2Cd are sensors. The spectra were respectively measured at 25 ° C. 10 mM HEPES buffered aqueous solution (pH 7.4).

FIG. 2 (a) is UV-vis spectrum showing absorbance change of sensor 1,2Zn (30 μM) upon addition of PPi (sodium salt), [PPi] = 0, 2, 4, 6, 8, 11, 14 , 17, 20, 23, 26, 29, 32 μM. The absorbance spectrum was measured in a 10 mM HEPES buffered pure aqueous solution (pH 7.4) at 25 ℃.

FIG. 2 (b) is the UV-vis spectrum of the sensor 1,2Zn (30 μM) in the presence of various anions (30 μM) in a 10 mM HEPES buffered pure aqueous solution (pH 7.4) at 25 ° C. FIG.

FIG. 3 is a UV-vis spectrum showing the change in absorbance of sensor 1,2Zn (30 μM) in the presence of various anions (60 μM) in 100 mM HEPES buffered pure aqueous solution (pH 7.4) at 25 ° C. FIG.

FIG. 4 is a UV-vis spectrum showing the absorbance change of sensor 1,2Zn (30 μM) in the presence of various anions (60 μM) in a buffered pure aqueous solution (pH 7.4) at 25 ° C. (a) pH 6.5 (MES 10 mM) (b) pH 7.0 (HEPES 10 mM) (c) pH 7.4 (HEPES 10 mM) (d) pH 8.3 (Tris.HCl 10 mM).

FIG. 5 is a UV-vis spectrum showing absorbance change of sensor 1,2Zn (30 μM) upon addition of PPi (sodium salt) in the presence of excess HPO ₄ ^2- (300 μM), [PPi] = 0, 4 , 8, 12, 16, 20, 23, 28, 32, 40, 65 μM. Here, the absorbance spectrum was measured in a 10 mM HEPES buffered pure aqueous solution (pH 7.4) at 25 ℃.

Figure 6 is a diagram showing the color change of the sensor 1,2Zn in 10 mM HEPES buffered aqueous solution (pH 7.4) at 25 ℃ ([Sensor 1Z2Zn ] = 60 μM, [Anion] = 60 μM); From left to right, anion free, PPi, citrate, HPO ₄ ^2- , H ₂ PO ₄ ⁻ , acetate, F ⁻ .

FIG. 7 is a UV-vis spectrum showing the change in absorbance of sensor 2 · Zn (30 μM) in the presence of various anions (900 μM) in 10 mM HEPES buffered pure aqueous solution (pH 7.4) at 25 ° C. FIG.

8 (a) and 8 (b) are X-ray diffraction diagrams showing the crystal structure and bonding method of the binary nucleated zinc metal complex of Compound 1 and PPi complex confirmed by X-ray analysis. Hydrogen atoms have been omitted for brevity.

Figure 9 is a fluorescence spectrum showing the change in fluorescence intensity, where (a) shows PPi (sodium salt) in 10 mM HEPES buffered aqueous solution (pH 7.4) at 25 ° C [PPi] = 0, 0.6, 1.2, 1.8, 2.4, shows the fluorescence intensity change of 3.0, 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, the sensor 7 of the addition to 7.8 μM and 2Zn (6 μ M), wherein the insertion degree of the July 2Zn and (◆ ) PPi, (▲) and the job plot between ATP anion, (b) is different anions (8 μ sensor at 25 ℃ 10 mM HEPES buffer solution (pH 7.4) in the presence of M) 7 and 2Zn (6 μ M Change in fluorescence intensity.

Figure 10 is a PPi (sodium salt) in the presence of ATP (300 μ M) [PPi ] = 0, 1.2, 2.4, 3.6, 4.8, 6.0, 8.0, 11, 17, 24, 34, 45, 65, 85 μM the fluorescence spectrum shows the fluorescent intensity change in the July 2Zn (6 μ M) of the addition to the. The spectra were measured in 25 ° C. 10 mM HEPES buffered aqueous solution (pH 7.4).

Figure 11 is a fluorescence spectrum of different anions (12 μ M) at 25 ℃ pure buffer sensors 7 and 2Zn (6 μ M) in the presence of, (a) pH 6.5 (MES 10 mM) (b) pH 7.4 (HEPES 10 mM) (c) pH 8.3 (Tris.HCl 10 mM) (d) pH 10.1 (CHES 10 mM).

12 is a cyclic voltammogram of the sensor 12.2Zn in the presence of various anions.

The present invention relates to binuclear metal complexes and, more particularly, to binuclear metal complexes useful for pyrophosphate analysis. The present invention also relates to a method for analyzing pyrophosphate using the dinuclear metal complex.

Development of sensors or receptors for biologically important anions has emerged as an important area ¹ . In particular, pyrophosphate ("PPi") anions participate in various bioenergy and metabolic processes ^{2, 3} . Therefore, detection of PPi has been studied in several research groups. In PPi analysis, methods such as ion chromatography are important, but there are many demands for new analytical methods using selective chemosensors ^{1b, 4} . In particular, systems that recognize PPi in aqueous solutions and convert the results into various signals (eg, optical signals, fluorescent signals, and electrical signals) will be very useful. PPi sensor which so far works in the aqueous solution is a situation mere several ^5,6. And even the PPi sensors disclosed therein did not provide satisfactory results.

Suitable PPi sensors must meet the following conditions:

(1) high selectivity to pyrophosphate anions;

(2) high sensitivity to pyrophosphate anions;

(3) good adaptability to aqueous solutions systems; And

(4) insensitivity to pH change or applicability in a wide range of pH.

An object of the present invention is to provide a new pyrophosphate sensor that satisfies the above conditions. Pyrophosphate sensors according to the invention are coordination complexes produced by binuclear metal complexation. More specifically, the coordination complex is a heteronuclear metal complex of the compound, wherein the compound is a conjugation ring system, which is substituted by the following substituents: a) an electron selected from -OH, -SH and -NH ₂ Excitation (electron donating group); b) an indicating group selected from chromophores, fluorescent and electrochemical groups; And c) two binding auxiliary groups, each of which is coordinated with the electron in combination with the electron donor to provide an anion binding site. As the complex binds to the anion, the coordination between the electron donor and the metal is weakened, and the enhanced electromagnetic excitation by the electron donor is transferred to the indicator through the conjugation ring system, and the indicator changes in electron density. Generates an indication signal detectable by This was supported by X-ray analysis. As the PPi anion binds to the anion binding site of the binuclear metal complex, the coordination between the electron donor and the metal is weakened. This induces a negative charge characteristic on the electron donor, and the electromagnetic contribution by the electron donor is increased. The increased electromagnetic excitation by the electron donor is transferred to the indicator through the conjugation ring system, which increases the electron density (or placement) of the indicator. The altered electron density (or placement) of the indicator produces detectable indicator signals such as color changes, fluorescence signals, and electrical signals. According to a preferred embodiment of the invention, said conjugation ring system is an aromatic ring system. More preferably, the conjugation ring system is a benzene ring system, and based on the electromagnetic excitation, the two bonding aids are each substituted at the ortho position, and the indicator is substituted at the para position.

According to another preferred embodiment of the present invention, there is provided a heteronuclear metal complex of a compound having formula I:

는 컨쥬게이션 링 시스템이다.Wherein X is selected from —OH, —SH and —NH ₂ as the electron donating group; Y is an indicator selected from chromophores, fluorescent groups, and electrochemical groups; Z ₁ and Z ₂ are bonding aids and are hydrocarbons containing at least one element selected from the group consisting of N, O, S and P;

Is a conjugation ring system.

According to another preferred embodiment of the present invention there is provided a heteronuclear metal complex of a compound having the formula II:

In formula II, X is selected from -OH, -SH and -NH ₂ as the electron donating group; Y is an indicator selected from chromophores, fluorescent groups, and electrochemical groups; Z ₃ , Z ₄ , Z ₅ and Z ₆ are independently hydrocarbons containing at least one element selected from the group consisting of N, O, S and P; R ₁ and R ₂ independently of one another are hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy, thioalkyl, alkylamino, imine, amide, phosphate, phosphine, carbonyl, carboxyl, silyl, ether, thioether, Selected from the group consisting of sulfonyl, selenoether, ketone; a, b, c, d, e and f are each independently an integer of 1 to 3, preferably 1 or 2.

The present invention also provides a method for analyzing pyrophosphate anions. The analysis method includes adding a pyrophosphate sensor to the sample solution to generate an indication signal, and detecting the indication signal to perform quantification of the pyrophosphate, wherein the pyrophosphate sensor described above. It is a binuclear metal complex.

I. Definition

Unless specifically defined otherwise, the terms used in the description, examples, and claims are defined as follows.

As used herein, the term "conjugation ring system" refers to a system in which conjugation occurs within a ring to counteract the local electron abundance or deficiency when a local electron abundance or deficiency occurs at a particular location within the ring. For example, when local electron abundance occurs, conjugation stabilizes the ring by dispersing it to other locations in the ring.

As used herein, "aromatic ring system" means a ring system having aromaticity. These are systems satisfying the so-called "Shunkel 4n + 2 rule" (where n is a natural number), including hydrocarbon aromatic ring systems and heteroaromatic ring systems containing at least one to four non-carbon atoms selected from oxygen, sulfur and nitrogen. Include. Examples of hydrocarbon aromatic ring systems include aryl ring systems such as benzene, indene, naphthalene, anthracene, phenanthrene and the like. Examples of heteroaromatic ring systems include thiophene, purine, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, triazole, thiadiazole, oxadiazole, tetrazole, tithia And heteroaryl ring systems such as sol, oxatriazole, pyridine, pyrimidine, pyrazine, pyridazine, triazine, tetraazine, quinoline, isoquinoline, 1,2-dihydroquinoline, purine.

As used herein, "electron donating group" means a functional group that provides electrons beyond that of hydrogen atoms providing electrons at the same location. Such examples include -OH, -SH, -NH ₂ and the like.

As used herein, "indicator" refers to a functional group that can indicate the presence and / or amount of anion to be analyzed. In other words, the "indicator" refers to a functional group capable of producing a detectable signal depending on the presence of the anion to be analyzed. A "chromic group" refers to a functional group that generates an optical signal, such as a color change, in response to a change in electron density (or placement), which is easily detected by vision or in the absorption spectrum. "Fluorescent group" refers to a functional group that results in a change in fluorescence quantum yield corresponding to a change in electron density (or placement), and "electrochemical group" to produce an electrical signal corresponding to a change in electron density (or placement) Say functional group.

As used herein, "alkyl" includes substituted or unsubstituted alkyl, wherein substituted alkyl means an alkyl moiety having a substituent that substitutes for hydrogen at one or more carbons of the hydrocarbon backbone. Such substituents include, for example, halogen, hydroxyl, alkenyl, alkynyl, carbonyl, alkoxy, ester, phosphoryl, amine, amide, imine, thiol, thioether, thioester, sulfonyl, amino, nitro, Or organometallic moieties. Those skilled in the art will appreciate that substituted residues on the hydrocarbon chain may be substituted as appropriate. For example, substituents of substituted alkyl include substituted, unsubstituted forms of amines, imines, amides, silyl groups, as well as ethers, thioethers, selenoethers, carbonyls (ketones, aldehydes, carboxylates, and esters). Including), -CF ₃ and -CN and the like. Preferred alkyls include straight or branched chain saturated carbon chains having 1 to 6 carbon atoms. Such examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, sec-pentyl, isopentyl, and n-hexyl. "Alkylene" means alkyl having a two point contact. Examples include methylene, ethylene and propylene. "Alkenyl" means a straight or branched carbon chain having at least one carbon-carbon double bond and having 2-6 carbon atoms. Such examples include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, and hexenyl. The term "alkynyl" means a straight or branched carbon chain having at least one carbon-carbon triple bond and having 2-6 carbon atoms. Such examples include ethynyl, propynyl, butynyl and hexynyl.

As used herein, "amino" refers to -NH ₂ ; "Nitro" means -NO ₂ and "halogen" means F, Cl, Br or I; "Hydroxy" means -OH, the term "thiol" means -SH, "alkoxy" means -OR (R: alkyl), "thioalkyl" means -SR (R: alkyl), "Carboxyl" means -COOH and "alkylamine" means an amine substituted with an alkyl group.

As used herein, the term "substituted" includes substituents of all possible organic compounds. In broad terms, possible substituents include acyclic and cycle, straight and branched, carbocycle and heterocycle, aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, the substituents described above. Possible substituents may be substituted, one or more times, identically or differently in an appropriate organic compound. In the present invention, heteroatoms such as nitrogen may have hydrogen substituents and / or possible substituents of the organic compounds described herein that meet the valency of the heteroatoms.

As used herein, "heteroaryl" or "heteroaromatic ring" refers to an aromatic ring containing at least one to three elements selected from the group consisting of oxygen, nitrogen, and sulfur, such as thienyl, furyl, blood Rollyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, tithiazolyl, oxatriazolyl, pyridyl, pyrimidyl , Pyrazinyl, pyridazinyl, triazinyl, tetrazinyl and similar rings.

The terms cited herein, including the terms defined above, should be construed in the organic chemical field as widely accepted meanings.

II. sensor

According to a first aspect of the present invention, the present invention provides a novel coordination complex produced by the formation of a binuclear metal complex. The coordination complex is a heteronuclear metal complex of the compound, wherein the compound is a conjugation ring system, which is substituted by the following substituents: a) an electron donating group selected from -OH, -SH and -NH _2; ); b) an indicating group selected from chromophores, fluorescent and electrochemical groups; And c) two binding auxiliary groups, each of which is coordinated with the electron in combination with the electron donor to provide an anion binding site. Preferably the anion is pyrophosphate.

Examples of such conjugation ring systems include aromatic ring systems. The aromatic ring system includes a hydrocarbon aromatic ring system consisting only of carbon and hydrogen and a heteroaromatic ring system containing at least one to four non-carbon atoms selected from oxygen, sulfur and nitrogen. Examples of hydrocarbon aromatic ring systems include aryl ring systems such as benzene, naphthalene, indene, anthracene, phenanthrene and the like. Examples of heteroaromatic ring systems include thiophene, purine, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, triazole, thiadiazole, oxadiazole, tetrazole, tithia And hetero ring systems such as sol, oxatriazole, pyridine, pyrimidine, pyrazine, pyridazine, triazine, tetrazine, quinoline, isoquinoline, 1,2-dihydroquinoline, purine.

Examples of the color generator are not particularly limited, and a color generator widely used in the field of analysis and screening may be used. Representative examples of the color group include azo compounds. In the examples of the present invention, a p-nitrophenylazo group was used as a chromophore, and the system provided visually detectable color change in aqueous solution and accurate quantification of pyrophosphate anions by absorbance test. Specific examples of the fluorescent group is a naphthyl group, an anthryl group, (4-dicyanomethylene-2-methyl -6- [p- dimethylamino styryl] -4 H-pyran), 2'-bis (amino-methyl DCM derivatives such as) biphene, pyrenyl group and porphyrin group. Specific examples of the electrochemical group include a ferrocenoylethynylene group, a furyl group, a thienyl group, and derivatives thereof.

The metal which can be used for forming the complex is not particularly limited. It may be appropriately selected in consideration of the kind of substituents present in the conjugation ring system. According to an embodiment of the present invention, Zn ²⁺ , Fe ²⁺ , Fe ³⁺ , Mn ²⁺ , Mn ³⁺ , Cu ²⁺ , Co ²⁺ , Co ³⁺ , Hg ²⁺ , Pb ²⁺ , Ce ^{2 +} Cd ²⁺ and Mg ²⁺ were used to measure selectivity and sensitivity to pyrophosphate. Dinuclear metal complexes of Zn ²⁺ , Co ³⁺ , Cd ²⁺ , Fe ³⁺ and Cu ²⁺ were particularly useful for pyrophosphate analysis. Most preferably Zn ²⁺ .

The bonding auxiliary group contains at least an element having an unshared electron pair, and examples of such elements include nitrogen, oxygen, sulfur, and the like. They are coordinated with the metal along with any of the oxygen, sulfur and nitrogen atoms of the electron donating group. For example, when the conjugation ring system is a benzene ring system and the electron donating group is hydroxy, a phenoxo-bridged dinuclear metal complex is formed. Even when the electron donating group is a thiol or amino group, a heteronuclear metal complex will be formed in a similar manner. When the complex formed is added to the sample in which the anion to be analyzed is present, the complex binds to the anion. At this time, the binding assistant is coordinated with the metal ion to provide an anion binding site. According to X-ray analysis, as the anion binds to the anion binding site of the complex, the bond between the electron donor and the metal is weakened. This induces a negatively charged character to the electron donor. For example, in phenox-bridged dinuclear metal complexes, the complex induces a negatively charged nature to phenolate oxygen as it binds to the anion. The induced negative charge nature increases the electromagnetic contribution to the conjugation ring system by the electron donor, and the increased electromagnetic excitation is transmitted through the conjugation ring system to an indicator substituted in the system. In particular, when the indicator is substituted, for example, in the para position of the benzene ring, the effect of the electromagnetic donation by the electron donor group is maximized. As a result, the electron density of the indicator is greatly increased. This increase in electron density produces detectable indication signals such as changes in color, changes in fluorescence quantum yield and emission of electrons.

According to a preferred embodiment of the invention, the dinuclear deposits of compounds having the formula I are markedly suitable for pyrophosphate analysis:

는 컨쥬게이션 링 시스템이다. 상기 컨쥬게이션 링 시스템은 방향족 링 시스템인 것이 바람직하고, 상기 두 개의 결합보조기 Z₁ 및 Z₂가 상기 X에 대해 각각 오르쏘 위치에 치환되고, 파라 위치에 상기 지시기가 치환되는 것이다. 상기 두 개의 결합보조기는 산소, 질소, 황과 같은 비공유전자쌍을 갖는 원소를 적어도 하나 이상 갖고 있으며, 금속과 컴플렉세이션하여 음이온 결합부위를 제공한다.Wherein X is selected from —OH, —SH and —NH ₂ as the electron donating group; Y is an indicator selected from chromophores, fluorescent groups, and electrochemical groups; Z ₁ and Z ₂ are bonding aids and are hydrocarbons containing at least one element selected from the group consisting of N, O, S and P;

Is a conjugation ring system. The conjugation ring system is preferably an aromatic ring system, wherein the two bonding aids Z ₁ and Z ₂ are each substituted at the ortho position relative to X and the indicator is substituted at the para position. The two bond aids have at least one element having a non-covalent electron pair such as oxygen, nitrogen, and sulfur, and are complexed with a metal to provide an anion bond site.

More preferably, the pyrophosphate sensor is a dinuclear metal complex of a compound having formula II below.

In Formula II, X is selected from -OH, -SH and -NH ₂ as an electron donating group, Y is an indicator selected from a chromophore, a fluorescent group and an electrochemical group, Z ₃ , Z ₄ , Z ₅ and Z ₆ Is independently a hydrocarbon containing at least one element selected from the group consisting of N, O, S and P, and R ₁ and R ₂ are independently of each other hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy , Thioalkyl, alkylamino, imine, amide, phosphate, phosphine, carbonyl, carboxyl, silyl, ether, thioether, sulfonyl, selenoether, ketone, and a, b, c , d, e, f are each independently an integer of 1 to 3, preferably 1 or 2. Specific examples of Z ₃ , Z ₄ , Z ₅ and Z ₆ include -NR ₃ R ₄ , -OR ₅ , -SR ₆ , -PR ₇ R ₈ and heteroaliphatic rings and heteroaromatic rings, where R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are alkyl or substituted alkyl. Preferably Z ₃ , Z ₄ , Z ₅ and Z ₆ are heteroaromatic rings having the formula:

Wherein at least one of A, B, D, E and G is nitrogen, the remainder is oxygen and carbon, at least one of I, J, M and N is oxygen and the remainder is nitrogen and carbon. R ₃ and R ₄ independently of one another are hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy, thioalkyl, alkylamino, imine, amide, phosphate, phosphine, carbonyl, carboxyl, silyl, ether, thioether, It is selected from the group consisting of sulfonyl, selenoether and ketone. Specific examples of the heteroaromatic ring include thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxdiazolyl, tetrazolyl, Thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl. These may be substituted by substituents, and substituents that provide a bonding site capable of forming a self-assembled monolayer are preferred. Such examples include carboxyl groups. Immobilization to a solid phase can be carried out by methods well known in the art, which are well known. In this regard, John J. Lavigne and Eric V. Anslyn, Angew. Chem. Int. Ed. 2001, 40, 3118-3130; Abraham Ulman, Chem. Rev. 1996, 96, 1533-1554; Mercedes Crego-Calama and David N. Reinhoudt, Adv. Mater. 2001, 13, No. 15, 1171-1®; Victor Chechik, Richard M. Crooks, and Charles JM Stirling, Adv. Mater. 2000, 12, No. 16, 1161-1171; Simon Flink, Frank CJM van Veggel, and David N. Reinhoudt, Adv. Mater. 2000, 12, No. 18, 1315-1328.

III. Example

A. Synthesis of p- (p-nitrophenylazo) -bis [(bis (2-pyridylmethyl) amino) methyl] phenol and binuclear Zn metal complexes using the same

Synthesis of p- (p-nitrophenylazo) -bis [(bis (2-pyridylmethyl) amino) methyl] phenol (hereinafter Compound 1 ) proceeded as follows.

Illustration 1

(a) bis- (2-pyridylmethyl) amine, K ₂ CO ₃ , KI, CH ₃ CN (b) aq. NaOH, MeOH, THF (c) i. aq. NaOH, MeOH, rt, ii. p- nitroaniline, NaNO ₂ , aq. HCl, Acetone, 0 ° C. (d) Zn (NO ₃ ) ₂ , H ₂ O, MeOH.

A-1: Compound 4 Synthesis of

To a solution of compound 3 (360 mg, 1.12 mmol) in acetonitrile 4 equivalents of KI (742 mg, 4.4 mmol), 4 equivalents of K ₂ CO ₃ (610 mg, 4.4 mmol) and 2.2 equivalents of bis- (2-pyridylmethyl) amine (510 mg, 2.24 mmol) were added. After the reaction mixture was adequately heated and refluxed for 12 hours, all volatile components were evaporated and the residue was partitioned between ethyl acetate and water. The organic phase was washed three times with water and dried over Na ₂ SO ₄ . Purification by flash chromatography (CH ₂ Cl ₂ : methanol = 20: 1 (v / v)) afforded Compound 4 (497 mg, 80% yield).

¹ H NMR (300 MHz, CDCl ₃ ): δ 2.16 (3H, s), 3.57 (4H, s), 3.77 (8H, s), 7.09-7.13 (4H, m), 7.24 (1H, t, J = 7.5 Hz), 7.52 (4H, br d, J = 7.5 Hz), 7.57-7.65 (6H, m), 8.49 (4H, br d, J = 4.2 Hz). ¹³ C NMR (75 MHz, CDCl ₃ ): δ 20.9, 53.2, 60.6, 122.4, 123.2, 126.5, 129.4, 131.7, 136.8, 148.5, 149.3, 159.7, 169.4. HRMS (FAB): calcd 558.2743, found 558.2750.

A-2: Compound One Synthesis of

P -nitroaniline (83 mg, 0.61 mmol) was dissolved in a 5 mL vial containing a mixture of 0.13 mL concentrated hydrochloric acid and 0.13 mL water. The vial was placed in an ice saline bath and cooled to 0 ° C. with vigorous stirring. A cold solution of sodium nitrite salt (56 mg, 0.81 mmol) was added slowly to 0.08 mL of water with stirring. Compound 5 (113 mg, 0.20 mmol) was dissolved in 0.25 mL of an aqueous NaOH (70 mg, 1.75 mmol) solution and cooled in an ice bath. To the obtained Compound 5 solution, the diazotized solution was slowly added while stirring. Thereafter, concentrated hydrochloric acid was slowly added to the cold mixture with vigorous stirring. The color of the solution changed from purple to dark reddish brown. After stirring for 2 minutes, the solution was partitioned between ethyl acetate and water. The organic extract was dried over Na ₂ SO ₄ , concentrated and subjected to chromatography (eluent concentration change: 20: 1 to 10: 1 CH ₂ Cl ₂ / methanol) to obtain the title compound 1 (40 mg, 40%).

¹ H NMR (300 MHz, CDCl ₃ ): δ 3.94 (12H, s), 7.23 (4H, dd, J = 5.1, 6.6 Hz), 7.56 (4H, br d, J = 7.8 Hz), 7.68-7.75 ( 4H, m), 8.01 (2H, s), 8.03 (2H, d, J = 12 Hz), 8.41 (2H, d, J = 12 Hz), 8.53 (4H, d, J = 4.5 Hz).

¹³ C NMR (75 MHz, CDCl ₃ ): δ 54.5, 59.7, 122.5, 123.2, 123.3, 125.1, 125.6, 125.9, 126.2, 136.9, 145.6, 148.5, 149.2, 156.5, 159.4, 161.4, MS (ESI): calculated 666.3, Found 666.9. Elemental Analysis of C ₃₈ H ₃₅ N ₉ O ₃ : calcd C, 68.56; H, 5. 30; N, 18.94. Found C, 67.16; H, 5. 31; N, 17.85.

A-3: Compound One Dinuclear zinc metal complexes of "1 · 2Zn" Manufacturing

To 20 mL of a methanol solution of Compound 1 (100 mg, 0.15 mmol) was added dropwise an aqueous solution of Zn (NO ₃ ) ₂ .6H ₂ O (0.5 M; 0.65 mL, 0.32 mmol) and the mixture was stirred at room temperature for 30 minutes. . After concentration in vacuo, the aqueous solution was lyophilized. The obtained solid was recrystallized from methanol-water (1: 1) to give a heteronuclear zinc metal complex of Compound 1 (simply " 1.2Zn "). (70 mg, 45%) was obtained.

¹ H NMR (300 MHz, MeOH-d ₄ + D ₂ O): δ 3.78 (4H, s), 4.18 (8H, dd, J = 15.6, 45 Hz), 7.23 (4H, br s), 7.39 (4H , br s), 7.45 (4H, br s), 7.70 (4H, br s), 7.87 (4H, d, J = 9 Hz), 8.36 (4H, d, J = 9 Hz), 8.50 (4H, br s). MALDI-TOF-MS: calcd 916.11, found 916.84. Elemental analysis calcd for C ₃₈ H ₃₄ N ₁₁ 0 ₉ 2Zn ₃ NO ₃ 2H ₂ O: C, 44.85; H, 3.76; N, 16.52. Found: C, 43.16; H, 3.55; N, 16.60

For the purpose of comparison, a 1: 1 metal complex of p- (p-nitrophenylazo)-[(bis (2-pyridylmethyl) amino) methyl] phenol (hereafter compound 2 ) and compound 2 and zinc was synthesized. .

Figure 2

(a) aq. NaOH, MeOH, THF, (b) i. aq. NaOH, MeOH, rt, ii. p-nitroaniline, NaN, aq. HCl, Acetone, (c) Zn (NO ₃ ) ₂ , MeOH.

A'-1: Compound 6 Synthesis of

In the same manner as described in the synthesis of compound 4 , compound 6 (232 mg, 80%) was obtained: 1 H NMR (300 MHz, acetone-d ₆ ): δ 2.24 (3H, s), 3.67 (2H, s ), 3.76 (4H, s), 7.05 (1H, dd, J = 3.2, 6.6 Hz), 7.21-7.28 (4H, m), 7.62 (2H, d, J = 7.8 Hz), 7.72-7.77 (3H, m), 8.51 (2H, d, J = 4.2 Hz). HRMS (FAB): calcd 348.1634, found 348.1785.

A'-2: Compound 2 Synthesis of

Compound 2 (68 mg, 52%) was obtained by the same method as A-2, except that compound 6 (100 mg, 0.29 mmol) was used. ¹ H NMR (300 MHz, Acetone-d ₆ ): δ 3.98 (6H, s), 7.05 (1H, d, J = 9.3 Hz), 7.30-7.33 (2H, m), 7.45 (2H, d, J = 7.8 Hz), 7.77 (2H, dd, J = 1.8, 7.8 Hz), 7.91-7.94 (2H, m), 8.07 (2H, d, J = 9.0 Hz), 8.44 (2H, d, J = 9.0 Hz) , 8.59 (2H, doublet, J = 4.8 Hz). ¹³ C NMR (75 MHz, CDCl ₃ ): δ 56.1, 52.9, 54.9, 113.9, 119.1, 119.4, 119.7, 120.1, 121.1, 122.2, 122.7, 133.9, 142.1, 144.3, 144.7, 152.6, 154.1, 158.6. HRMS (FAB): found 455.1832, found 455.1850. Elemental Analysis Calcd for C ₂₅ H ₂₂ N ₆ O ₃ : C, 66.07; H, 4.88; N, 18.49. Found: C, 65.20; H, 4.96; N, 17.63.

A'-3: Compound 2 Preparation of 1: 1 Metal Complexes with Zn

Using compound 2 (50 mg, 0.11 mmol) in the same manner as described for A-3, A 1: 1 metal complex of Compound 2 with Zn (simply " 2.Zn ") (39 mg, 55%) was obtained.

¹ H NMR (300 MHz, Acetone-d ₆ ): δ 3.96 (2H, s), 4.39 (4H, d, J = 3.6 Hz), 6.60 (1H, br s), 7.66-7.73 (5H, m), 7.79 (1H, s), 7.96 (2H, d, J = 8.7 Hz), 8.14 (2H, t, J = 7.8 Hz), 8.38 (2H, d, J = 8.7 Hz), 8.78 (2H, d, J = 5.4 Hz). MS (ESI): calcd 517.1, found 517.6. Elemental analysis calculated for C ₂₅ H ₂₁ N ₆ O ₃ ZnNO ₃ H ₂ O: C, 50.14; H, 3.87; N, 16.37. Found: 49.94; H, 3.77; N, 16.42

B. Synthesis of p-naphthyl-bis [(bis (2-pyridylmethyl) amino) methyl] phenol and binuclear Zn metal complexes using the same

Synthesis of p-naphthyl-bis [(bis (2-pyridylmethyl) amino) methyl] phenol (hereinafter Compound 7 ) proceeded as follows.

Illustration 3

B-1: Compound 10 Manufacture

To 9 mL of a benzene solution of compound 11 (500 mg, 1.83 mmol) and (tetrakistriphenylphosphine) palladium (0) (113 mg, 0.09 mmol), added 4.0 mL of an aqueous 2N sodium carbonate solution in an argon atmosphere, followed by 2- 4 mL of naphthalene boronic acid (630 mg, 3.66 mmol) ethanol solution was added. The mixture was refluxed for 16 hours, diluted with 50 mL of water and extracted with 3 mL of 50 mL of ethyl acetate. The combined organic extracts were washed with 50 mL of brine, dried and evaporated. The residue was subjected to silica gel chromatography using hexane / ethyl acetate 3/1 to give compound 10 (563 mg, 96% yield).

¹ H NMR (300 MHz, Acetone- d ₆ ): δ 1.56 (6H, s), 4.76-4.78 (2H, d, J = 6.0 Hz), 4.96 (2H, s), 7.40 (1H, s), 7.47 -7.52 (2H, m), 7.81-7.95 (5H, m), 8.12 (1H, s). ¹³ C NMR (75 MHz, CDCl ₃ ): δ 24.7, 59.9, 61.0, 100.1, 119.9, 122.3, 125.0, 125.1, 125.6, 126.1, 126.7, 128.0, 128.5, 128.8, 130.9, 132.8, 132.9, 134.4, 138.7, 148.5.

B-2: Compound 9 Manufacture

To 10 mL of a methanol solution of compound 10 (320 mg, 1.0 mmol), 5 mL of 10% HCl was added. After the reaction mixture was stirred at room temperature for 12 hours, all volatile components were evaporated and the residue was partitioned between ethyl acetate and water. The organic phase was washed three times with water and dried over Na ₂ SO ₄ . After the obtained solution was concentrated under reduced pressure, the obtained residue was triturated with hexane. When a white solid precipitated, the precipitate was filtered, washed with hexane and dried under vacuum to give compound 9 (252 mg, 90% yield).

¹ H NMR (300 MHz, Acetone-d ₆ ): δ 4.91 (4H, s), 7.44-7.53 (2H, m), 7.64 (2H, s), 7.81 (1H, d, J = 9.0 Hz), 7.88 -7.95 (3H, m), 8.11 (1H, s). ¹³ C NMR (75 MHz, CDCl ₃ ): δ 61.8, 124.8, 125.3, 125.6, 125.9, 126.6, 127.9, 128.0, 128.4, 128.7, 132.0, 132.8, 134.4, 138.8, 154.0.

B-3: Compound 8 Manufacture

To 10 mL of THF stirred solution of compound 9 (252 mg, 0.9 mmol), 10 mL of 0.5 N NaOH aqueous solution was added at 0 ° C. To the obtained solution was added 10 mL of THF in which para-toluenesulfonyl chloride (532 mg, 2.79 mmol) was dissolved. After the reaction mixture was stirred at 0 ° C. for 4 hours, all volatile components were evaporated and the residue was partitioned between ethyl acetate and water. The organic phase was washed three times with water and dried over Na ₂ SO ₄ . Purification by flash chromatography (hexane: ethyl acetate = 3: 1 (v / v)) gave Compound 8 (334 mg, 50% yield).

¹ H NMR (300 MHz, Acetone-d ₆ ): δ 2.07 (6H, s), 2.39 (3H, s), 5.13 (4H, s), 7.42 (4H, d, J = 9.0 Hz), 7.57-7.60 (4H, m), 7.69 (1H, d, J = 9.0 Hz), 7.75-7.83 (8H, m), 7.97 (1H, d, J = 9.0 Hz), 8.05 (2H, d, J = 8.7 Hz) , 8.10 (1 H, s).

B-4: Compound 7 Manufacture

In an acetonitrile solution of compound 8 (334 mg, 0.45 mmol), 4 equivalents of KI (298 mg, 1.8 mmol), 4 equivalents of K ₂ CO ₃ (248 mg, 1.8 mmol) and 2.2 equivalents of DPA (197 mg, 0.99 mmol) was added. The reaction mixture was moderately heated and refluxed for 12 h. Insoluble inorganic salts were filtered off and all volatile components were evaporated. The obtained residue was dissolved in 10 mL of methanol. For hydrolysis, 5 mL of 2N NaOH aqueous solution was added to the solution with stirring. The reaction mixture was stirred at room temperature for 2 hours. The mixture was neutralized with 1N HCl and partitioned between ethyl acetate and water. The organic phase was washed three times with water and dried over Na ₂ SO ₄ . Purification by flash chromatography (CH ₂ Cl ₂ : methanol = 20: 1 (v / v)) afforded compound 7 (231 mg, 80% yield).

¹ H NMR (300 MHz, Acetone-d ₆ ): δ 3.94 (12H, s), 7.21 (4H, t, J = 5.7 Hz), 7.48 (2H, m), 7.61 (4H, d, J = 7.8 Hz ), 7.71 (4H, t, J = 7.2 Hz), 7.78 (2H, s), 7.85-7.96 (4H, m), 8.54 (1H, s), 8.54 (4H, d, J = 3.9 Hz). ¹³ C NMR (75 MHz, CDCl ₃ ): δ 54.8, 59.8, 122.5, 123.2, 124.7, 125.1, 125.7, 125.8, 126.3, 126.6, 127.9, 128.4, 128.7, 130.9, 132.7, 134.4, 136.9, 138.9, 149.3, 156.6, 159.8. HRMS (FAB): calcd 643.3107, found 643.3185.

B-5: compound 7 Dinuclear zinc metal complexes of "7 · 2Zn" Manufacturing

To 20 mL of a methanol solution of compound 7 (64 mg, 0.10 mmol) was added dropwise an aqueous solution of Zn (NO ₃ ) ₂ .6H ₂ O (0.5M; 0.42 mL, 0.21 mmol) and the mixture was stirred at room temperature for 30 minutes. All. After concentration in vacuo, the aqueous solution was lyophilized. The obtained solid was recrystallized in methanol-water (1: 1) to give 7.2Zn (48 mg, 48%).

¹ H NMR (300 MHz, Acetone-d ₆ ): δ 4.16 (4H, s), 4.45 (8H, dd, J = 18, 54 Hz), 7.41 (2H, br s), 7.47-7.54 (9H, m ), 7.62 (2H, d, J = 12 Hz), 7.89-7.93 (4H, m), 7.98 (4H, br s), 8.62 (4H, br)

C. Synthesis of ferrocenoylethynylene-bis [(bis (2-pyridylmethyl) amino) methyl] phenol and the heteronuclear Zn metal complex using the same

The synthesis of ferrocenoylethynylene-bis [(bis (2-pyridylmethyl) amino) methyl] phenol (hereinafter Compound 12 ) proceeded as follows.

Figure 4

C-1: Compound 14 Manufacture

Compound 13 (400 mg, 0.98 mmol) with (dichloridebistriphenylphosphine) palladium (0) (72 mg, 0.1 mmol), capperiodide (38 mg, 0.2 mmol), triphenylphosphine (75 mg , 0.29 mmol), and 5.0 mL of triethylamine solution in argon atmosphere were added to 10 mL of methylene chloride solution of ferrocenoylethynylene (256 mg, 1.2 mmol). The mixture was refluxed for 1 hour, diluted with 50 mL of water and extracted three times with 50 mL of ethyl acetate. The combined organic extracts were washed with 50 mL of brine, dried and evaporated. The residue was subjected to silica gel chromatography using hexane / ethyl acetate 4/1 to give compound 14 (455 mg, 95% yield).

¹ H NMR (300 MHz, Acetone-d ₆ ): δ 2.07 (6H, s), 2.37 (3H, s), 4.27 (5H, s), 4.33-4.34 (2H, m), 4.55-4.56 (2H, m), 5.06 (4H, s), 7.59 (2H, s).

C-2: Compound 15 Manufacture

Compound 14 (455 mg, 0.94 mmol) was dissolved in 20 mL of THF solution, and after cooling to 0 ° C., lithium aluminum hydride (71 mg, 1.88 mmol) was added. After the reaction mixture was stirred at 0 ° C. for 10 minutes, water and 15% NaOH solution were added to complete the reaction, followed by stirring at room temperature for 10 minutes. 10 mL of water and NaOH (376 mg, 9.4 mmol) were added to the mixed solution, followed by further stirring at 0 ° C. for 30 minutes. To the obtained solution was added 10 mL of THF in which para-toluenesulfonyl chloride (1064 mg, 5.58 mmol) was dissolved. After the reaction mixture was stirred at 0 ° C. for 4 hours, all volatile components were evaporated and the residue was partitioned between ethyl acetate and water. The organic phase was washed three times with water and dried over Na ₂ SO ₄ . Purification by flash chromatography (hexane: ethyl acetate = 3: 1 (v / v)) gave compound 15 (327 mg, 40% yield).

¹ H NMR (300 MHz, Acetone-d ₆ ): δ 2.46 (s, 6H), 2.53 (s, 3H), 4.28 (s, 5H), 4.35 (d, J = 3Hz, 2H), 4.58 (d, J = 3 Hz, 2H), 4.99 (s, 4H), 7.45-7.48 (m, 6H), 7.55 (d, J = 6 Hz, 2H), 7.56-7.80 (m, 6H). ¹³ C NMR (75 MHz, acetone-d ₆ ): δ 14.00, (21.16), 21.26, (21.41), 22.86, 31.85, (64.40), 66.34, 69.76, 70.25, 70.39, 71.92, 83.59, 91.59, 124.21, 128.26, 128.69, 130.12, 130.54, 131.06, 131.77, 133.34, 133.84, 143.50, 145.80, 147.62.

C-3: compound 12 Manufacture

In an acetonitrile solution of compound 15 (327 mg, 0.40 mmol) 4 equivalents of KI (264 mg, 1.6 mmol), 4 equivalents of K ₂ CO ₃ (220 mg, 1.6 mmol) and 2.2 equivalents of DPA (160 mg, 0.80) mmol) was added. The reaction mixture was moderately heated and refluxed for 12 h. Insoluble inorganic salts were filtered off and all volatile components were evaporated. The obtained residue was dissolved in 10 mL of methanol. For hydrolysis, 5 mL of 2N NaOH aqueous solution was added to the solution with stirring. The reaction mixture was stirred at room temperature for 2 hours. The mixture was neutralized with 1N HCl and partitioned between ethyl acetate and water. The organic phase was washed three times with water and dried over Na ₂ SO ₄ . Purification by flash chromatography (CH ₂ Cl ₂ : methanol = 20: 1 (v / v)) gave compound 12 (230 mg, 80% yield).

¹ H NMR (300 MHz, Acetone-d ₆ ): δ 3.83 (s, 4H), 3.89 (s, 8H), 4.23 (s, 5H), 4.24 (s, 2H), 4.47 (s, 2H), 7.24 (d, J = 6 Hz), 7.44 (s, 2H), 7.57 (d, J = 9 Hz, 4H), 7.72 (t, J = 9 Hz, 4H), 8.53 (d, J = 6 Hz, 4H). ¹³ C NMR (75 MHz, Acetone-d ₆ ): δ 13.97, 22.85, 31.85, 54.39, 59.63, (66.49), 68.99, 70.19, 71.46, 85.95, 86.58, 113.77, 122.53, 123.18, 125.19, 132.90, 136.99, 149.23, 156.96, 159.60

C-4: compound 12 Dinuclear zinc metal complexes of "12 2Zn" Manufacturing

To 20 mL of a methanol solution of compound 12 (74 mg, 0.10 mmol) was added dropwise an aqueous solution of Zn (NO ₃ ) ₂ .6H ₂ O (0.5M; 0.42 mL, 0.21 mmol) and the mixture was stirred at room temperature for 30 minutes. . After concentration in vacuo, the aqueous solution was lyophilized. The resulting solid was recrystallized in methanol-water (1: 1) to give 12.2Zn (43 mg, 50% yield).

¹ H NMR (300 MHz, MeOH-d ₄ + D ₂ O): δ 3.88 (4H, s), 4.23 (s, 5H), 4.32 (8H, dd, J = 13.6, 40 Hz), 4.42 (s, 2H), 4.65 (s, 2H), 7.10 (2H, br s), 7.47 (4H, br s), 7.70 (4H, br s), 7.87 (4H, br s), 8.50 (4H, br s).

D. Absorbance Test of Binuclear Metal Complex of Compound 1

D-1) Absorbance Changes According to Metals

The effect on the anion was investigated by changing various metals in compound 1 . Each dinuclear metal complex was easily obtained by adding 2 equivalents of each metal dissolved in water to Compound 1 dissolved in methanol in the same manner as the zinc complex. The effect of the change of anion on each metal coordination complex was investigated in pH 7.4 10 mM HEPES buffer at 25 ° C, each using an Na salt. As metal ions Zn ²⁺ , Fe ²⁺ , Fe ³⁺ , Mn ²⁺ , Mn ³⁺ , Cu ²⁺ , Co ²⁺ , Co ³⁺ , Hg ²⁺ , Pb ²⁺ , Ce ²⁺ , Cd ²⁺ And Mg ²⁺ , the selectivity and sensitivity to pyrophosphate were measured. UV-vis absorption spectra for Cu ²⁺ , Mg ²⁺ , Pb ²⁺ , Zn ²⁺ , Co ²⁺ , Cd ²⁺ are shown in FIG. 1. 1 shows that Zn ²⁺ , Cd ²⁺ and Cu ²⁺ as metal ions are more useful for pyrophosphate analysis. Co ³⁺ and Fe ³⁺ gave similar results as Cd ²⁺ . The remaining metal ions gave similar results as Mg ²⁺ , Pb ²⁺ , Co ²⁺ . The effect on a wider variety of metal ions will be further revealed, and the results show that dinuclear metal complexes of Zn ²⁺ , Co ³⁺ , Cd ²⁺ , Fe ³⁺ and Cu ²⁺ are particularly useful for pyrophosphate analysis. . Most preferably Zn ²⁺ .

D-2) Absorbance Test of Binary Zinc Metal Complex (1 · 2Zn) of Compound 1

Based on the above results, the absorbance change of the sensor 1,2Zn was examined more closely. The effect of the anion (sodium salt) on the absorption spectrum of the sensor 1,2Zn was pH 7.4 10 mM HEPES (HEPES = 2- [4- (2-hydroxyl-ethyl) -1-piperazinyl] ethane-sulfur at 25 ° C. Phonic acid) buffer solution (Fig. 2). Before adding an anion, the absorbance spectrum of the sensor 1,2Zn shows a strong band at 417 nm. Sensor 1 · 2Zn is ^{_{H 2 PO 4 -, CH 3}} CO 2 -, F -, HCO 3 -, Cl - and a monovalent anion or H ₂ PO ₄ same ^- even if the addition of more than 100 equivalent change in the spectrum was almost invisible . In addition, there is little change in the spectrum even if divalent HPO ₄ ^2- or trivalent citrate is added. However, when PPi was added, the red shift was shown from 417 nm to 465 nm. Absorption spectra showed that no more noticeable changes occur when more than 1 equivalent of PPi is added. As shown in the UV-vis absorption data of FIG. 6, when PPi is added to the sensor 1 · 2Zn , a color change occurs from yellow to red. Job's Plot on the coupling of sensor 1,2Zn and PPi shows that these two materials are stoichiometrically 1: 1 (inset of Fig. 2 (a)). Sensor 1,2Zn shows similar PPi detection capability even when 10 equivalents of HPO ₄ ^2- are present. The binding constant (Ka) for the PPi-sensor 1,2Zn was determined by standard computational methods for competition experiments in the presence of excess HPO ₄ ^2- in pH 7.4 10 mM HEPES buffer solution at 25 ° C. and the value was (6.6 ±. 1.2) × 10 ⁸ M ⁻¹ . These results show that sensor 1,2Zn has significantly higher selectivity for PPi than other anions.

Similar results were obtained with 25 ° C. pH 7.4 100 mM HEPES buffered pure aqueous solution (FIG. 3). The addition of PPi changes the color from yellow (λmax = 417 nm) to red (λmax = 463 nm). In 100 mM buffer solution, the binding capacity to PPi was somewhat reduced compared to that of 10 mM buffer solution ( K a = (8.3 ± 1.8) × 10 ⁷ M ⁻¹ ).

The pH dependence of the sensing capacity of the PPi of the sensor 1,2Zn was examined, and the results are shown in FIG. 4. Similar trends were seen at pH 6.5-8.3 as shown in Figure 2 (b). This shows that sensor 1 · 2Zn can still detect PPi even when pH changes.

FIG. 5 is a UV-vis spectrum showing the change in absorbance of sensor 1,2Zn upon addition of PPi (sodium salt) in the presence of excess HPO ₄ ²⁻ , with results similar to those shown in FIG. 2 (b). there was. These results show that PPi sensing by sensor 1Zn is possible even in the presence of excess HPO ₄ ^2- .

E. Crystal Structure Using X-ray Diffraction of Binary Zinc Metal Complex of Compound 1 and PPi

E-1) Crystal Formation and Identification of Crystal Structure

Crystals suitable for X-ray diffraction studies were grown by slowly dispersing a pyrophosphate potassium salt and an aqueous methanol solution of 1,2 Zn complex in diethyl ether at room temperature. X-ray data for single crystals were Enraf-Nonius Kappa CCD single crystals at room temperature using graphite-monochromated Moka radiation (λ = 0.71073). Collected using an X-ray diffractometer). X-ray crystal data were collected at -100 ° C using Oxford low temperature flow low temperature equipment, which did not help to improve R value. Specific details of the decision data, data collection and structure modification are shown in Table 1. The structure was solved by the direct method (SHELXS-97) and structure modifications were made to all F ² data (SHELXS-97).

E-2) Analysis of Crystal Structure

FIG. 8 (a) shows the crystal structure of the complexes of the sensors 1 · 2Zn and PPi confirmed by the X-ray analysis, and FIG. 8 (b) shows the new coupling scheme for the PPi and the sensors 1 · 2Zn . The crystal structure shown in Figure 8 (a) is two oxygen anions to two Zn ^2+, respectively coupled to the sensor 1, two Zn ²⁺ coordination number of six and two 2Zn connected to each of the (P) of PPi To form. The combination of HPO ₄ ^2- and sensor 1,2Zn is likely to be the same as HPO ₄ ^2- -H-bpp. However, HPO ₄ ^2- does not change the absorbance spectra even when combined with sensor 1,2Zn . Instead, only PPi combines with sensor 1,2Zn to show selective red shift. This is because the bond between p-nitrophenylazo phenolate oxygen and Zn ²⁺ is weakened to increase the negative charge property of phenolate oxygen. According to previous literature, HPO ₄ ^2- is not able to quadruple like PPi. This is why HPO ₄ ^2- does not change the absorbance spectrum of the sensor 1,2Zn . Stronger bonding to PPi's sensor 1,2Zn is the reason for color change to sensor 1,2Zn and selectivity for HPO ₄ ^2- (FIG. 6). Hex coordination of Zn ²⁺ causes the PPi-sensor 1,2Zn to have a very strong binding constant in water. PPi binds 1000 times more strongly to sensor 1,2Zn than HPO ₄ ^2- .

The comparative group sensor 2 · Zn having one Zn ²⁺ shows no change in color or wavelength even when PPi is added (FIG. 7). This result shows that the selective detection of PPi is possible only when two Zn ²⁺ and the co-assisted bis (2-pyridylmethyl) amine (simply “Dpa”) work together.

F. Fluorescence Test

First, the effect of the anion (sodium salt) on the fluorescence spectrum of the fluorescent sensor 7 · 2Zn was investigated in pH 7.4 10 mM HEPES buffer solution at 25 ° C., and the results are shown in FIG. 9. The concentration of the fluorescence sensor was adjusted to 6 μΜ. Before adding an anion, the fluorescence spectrum of the sensor 7Zn exhibits a strong band at 436 nm.

It was a fluorescent sensor 7 and 2Zn did almost no change in the spectrum to the addition of ^{_{AMP, HPO 4 2-, CH 3}} CO 2 -, F -, HCO 3 -, Cl - and 1 is added to the anion equivalent of more than 100, such as Did not change. When PPi was added to the 7 · 2Zn solution, the fluorescence emission spectrum shifted to long wavelengths in a dose dependent manner. As shown in FIG. 9, λ _max shifted from 436 nm to 456 nm. Increasing the PPi concentration to 1 equivalent resulted in a 9.5-fold increase in fluorescence. However, even when ATP was added in an amount of 4 equivalents or more, only a 2-fold increase was accompanied by a red shift of 12 nm. In the case of ADP, even when ADP was added in 50 times the excess, the sensor 7Zn showed only a slight emission change (1.5-fold increase). The addition of AMP, HPO ₄ ^2- did not cause an increase in emissions even after adding 100 equivalents of each ion. Job's Plot for the coupling of the sensors 7 · 2Zn and PPi shows that these two materials combine stoichiometrically in 1: 1 (inset of Fig. 9 (a)). The binding constant (Ka) for PPi-7.2Zn was determined by standard computational methods for competition experiments in the presence of excess HPO ₄ ^2- in pH 7.4 10 mM HEPES buffer solution at 25 ° C. and the value was (2.9 ± 0.7 ) X10 ⁸ M ^-1 . In the same way, the binding constant Ka for ATP- 7 · 2Zn was found to be (7.2 ± 1.0) × 10 ⁶ M ⁻¹ , which is 40 times lower than PPi. This indicates that PPi can be detected even if 7 · 2Zn is present in the nanomolar concentration in water.

FIG. 10 shows that even if ATP is present 50 times, 250 times, or excessively, based on the PPi to be detected, 7 · 2Zn can detect 1 equivalent or less of PPi. In other words, 7 · 2Zn can selectively detect PPi with selectivity significantly superior to ATP in aqueous solution within the detection limit of micromolar concentration. There are various biochemical reactions that release PPi in the presence of ATP. Therefore, to develop a PPi sensor for bioanalytical applications, a sensor must be developed that can detect a small amount of PPi even in the presence of ATP in excess. The results show that our sensors can be used for bioanalytical applications.

It is believed that the coupling mode of PPi- 7 · 2Zn is almost the same as that of PPi-1 · 2Zn . The complex is formed by two oxygen anions connected to each phosphorus (P) of PPi to two Zn ²⁺ , respectively , to form two Zn ²⁺ and two sixth coordination of the sensor 1,2Zn . As the bond between phenolate oxygen and Zn ²⁺ is weakened and Zn ²⁺ strengthens the negatively charged nature of phenolate oxygen, PPi causes significant red shift of λ _max of 7 · 2Zn . At the same time, the increased negatively charged nature of phenolate oxygen is increased through the benzene ring to the naphthyl group, causing a fluorescence increase of the naphthyl group.

PPi selectivity superior to ATP can be understood based on the guest's structure. In the case of ATP, neutral oxygen of one of the PO bonds participates in the bond with 7 · 2Zn . Therefore, the binding ability of ATP is markedly lowered, and the degree of fluorescence change is stately smaller than that of PPi binding.

The control group with one Zn ²⁺ showed no red shift or increase in fluorescence intensity even when PPi was added. The results show that selective detection of PPi is possible only when the two Zn ²⁺ and Dpa cooperatively act.

The pH dependence in PPi sensing of 7.2Zn was investigated. Fluorescence intensity changes were observed at pH 6.5-10.1 in a form similar to that shown in FIG. 9. This shows that when this sensor is used, sensor 7 · 2Zn can still detect PPi even in the case of changing pH (Fig. 11).

G. Cyclic Voltammetry Test

The electrochemical properties of the anion (sodium salt) of the electrochemical sensor 12.2Zn were determined by cyclic voltammetry. Irradiated in pH 7.4 10 mM HEPES buffer solution at 25 ° C., a three-electrode system (working electrode: glassy carbon working electrode, reference electrode: Ag / Ag ⁺ , counter electrode: platinum wire) was employed. The potential was increased at a rate of 50 mV / s in the 0-0.8 V range. The concentration of the electrochemical sensor was set at 0.3 mM. The results are shown in FIG. As shown in FIG. 12, the cyclic voltammetry of the electrochemical sensor 12.2Zn before adding the anion showed a reversible redox pair of ferrocene / ferricinium. Electrochemical sensor 12 and 2Zn is _{^{H 2 PO 4 - CH 3 CO}} 2 - when the first, such as to be added to the anion did not bring a little change in the cyclic voltammetry Fig. In addition, almost no change was observed even when the divalent anion HPO ₄ ^2- was added. However, when PPi is added, oxidation and reduction waves are shown at lower potentials. The cyclic voltammetry shows a remarkable phenomenon which hardly changes when more than 1.2 equivalents of PPi is added. Since the sensors 1, 2Zn , 7 , 2Zn and 12.2Zn all have the same anion binding site, the coupling structure and stoichiometry of the sensor 12.2Zn to PPi and the coupling constant ( K a) to PPi are described above. One sensor will be equal to 1 · 2Zn and 7 · 2Zn . Sensor 12.2Zn is stoichiometrically coupled to PPi 1: 1, and the coupling structure will tend to be the same as that of FIG. The coupling constant ( K a) for the PPi-sensor 12 · 2Zn was too large to be measured by cyclic voltammetry, but from the discussion above, the value in pH 7.4 10 mM HEPES buffer solution at 25 ° C. It will have a size of 1.0 × 10 ⁸ M ^-1 . These results show that sensor 12 · 2Zn has higher selectivity for PPi than other anions, and that 12 · 2Zn is a sensor that converts the presence of PPi into an electrochemical signal.

In summary, we have synthesized a novel heteronuclear metal complex useful for pyrophosphate analysis, and as the complex binds to the pyrophosphate, the coordination between the electron donor and the metal of the heteronuclear metal complex is weakened, and the electron donation by the electron donor Is delivered to the indicator through the conjugation ring system of the binuclear metal complex and generates an indication signal by a change in electron density (or placement). The heteronuclear metal complex has better sensitivity and selectivity to pyrophosphate than other ions in a wide pH range in aqueous solution. The sensor can detect PPi even in the presence of strong competitor HPO ₄ ^2- or ATP. Therefore, it can be widely used as a pyrophosphate sensor in the bioenergy and metabolism process in which pyrophosphate is involved. Specifically, the sensor is an enzyme analysis including the reaction ATP → AMP + PPi, DNA sequencing accompanied with the release of PPi during the pyrosequencing process, and polymerase chain reaction (PCR) It can be applied to the monitoring of PCR accompanied with the release of PPi. In addition, the sensor can be applied to the development of biochips for them.

Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. Therefore, the above-described embodiments are to be understood as illustrative in all respects and not as restrictive. The scope of the present invention is represented by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and equivalent concepts thereof are included in the scope of the present invention.

References

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Claims (17)

As a dinuclear metal complex of a compound, the compound is a conjugation ring system, substituted by the following substituents: a) an electron donating group selected from -OH, -SH, and -NH ₂ ; b) a group consisting of a chromophore indicating a change in color in response to a change in electron density, a fluorine indicating a change in fluorescence quantum yield in response to a change in electron density, and an electrochemical group generating an electrical signal in response to a change in electron density. An indicator selected from the groups for generating a detectable indicator signal depending on the presence of the anion to be analyzed; And c) two binding aids, each of which is coordinated with the electron donor to coordinate with the metal to provide an anion binding site, and as the complex binds to the anion, the coordination between the electron donor and the metal Weakens, the electromagnetic excitation to the conjugation ring system by the electron donor is enhanced, and the enhanced electromagnetic excitation by the electron donor is transmitted to the indicator through the conjugation ring system, and the indicator is adapted to the change in electron density. Generating an indication signal that is detectable by means of a dinuclear metal complex. The complex of claim 1 wherein the compound has the formula I:

Wherein X is selected from —OH, —SH and —NH ₂ as the electron donating group; Y is a group consisting of a chromophore indicating a color change in response to a change in electron density, a fluorophore indicating a fluorescence quantum yield change in response to a change in electron density, and an electrochemical group generating an electrical signal in response to a change in electron density. Is an indicator that selects from and produces a detectable signal depending on the presence of the anion to be analyzed; Z ₁ and Z ₂ are bonding aids and are hydrocarbons containing at least one element selected from the group consisting of N, O, S and P;

Is a conjugation ring system. The complex of claim 2 wherein said conjugation ring system is an aromatic ring system. 3. The conjugation ring system of claim 2, wherein the conjugation ring system is a benzene ring system, wherein, based on the electron donor, the two bonding aids are each substituted at an ortho position relative to the electron donor, and the indicator is substituted at a para position. Complexes. The complex of claim 1 wherein said compound has formula II:

In formula II, X is selected from -OH, -SH and -NH ₂ as the electron donating group; Y is a group consisting of a chromophore indicating a color change in response to a change in electron density, a fluorophore indicating a fluorescence quantum yield change in response to a change in electron density, and an electrochemical group generating an electrical signal in response to a change in electron density. Is an indicator that selects from and produces a detectable signal depending on the presence of the anion to be analyzed; Z ₃ , Z ₄ , Z ₅ and Z ₆ are independently hydrocarbons containing at least one element selected from the group consisting of N, O, S and P; R ₁ and R ₂ independently of one another are hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy, thioalkyl, alkylamino, imine, amide, phosphate, phosphine, carbonyl, carboxyl, silyl, ether, thioether, Selected from the group consisting of sulfonyl, selenoether and ketone; a, b, c, d, e and f are each independently an integer from 1 to 3. The method according to claim 5, wherein Z ₃ , Z ₄ , Z ₅ and Z ₆ are independently of each other -NR ₃ R ₄ , -OR ₅ , -SR ₆ , -PR ₇ R ₈ , wherein R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are alkyl or substituted alkyl), a heteroaliphatic ring and a heteroaromatic ring. The complex of claim 6 wherein Z ₃ , Z ₄ , Z ₅ and Z ₆ are independently of each other a heteroaromatic ring having the formula:

Wherein at least one of A, B, D, E and G is nitrogen, the remainder is oxygen or carbon, at least one of I, J, M and N is oxygen, the remainder is nitrogen or carbon, R ₃ and R ₄ independently of one another is hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy, thioalkyl, alkylamino, imine, amide, phosphate, phosphine, carbonyl, carboxyl, silyl, ether, thioether, sulfonyl, Selected from the group consisting of selenoether and ketone. The complex of claim 1 wherein said anion is pyrophosphate. The complex of claim 1 wherein the metal is Zn, Fe, Mn, Cu, Co, Hg, Pb, Ce, Cd or Mg. 10. The complex of claim 9 wherein said metal is Zn, Co, Fe, Cd or Cu. 10. The complex of claim 9 wherein said metal is coordination. The complex of claim 1 wherein said electron donating group is -OH. A pyrophosphate analysis method comprising adding a pyrophosphate sensor to a sample solution to generate an indication signal, and detecting the indication signal to perform pyrophosphate analysis. The method according to any one of claims 12 to 12, characterized in that the binuclear metal complex. The method of claim 13, wherein said pyrophosphate analysis is performed in an aqueous solution system. The method of claim 13, wherein the pyrophosphate assay is used for bioanalytical applications involving the release of pyrophosphate. The method of claim 15, wherein said bioanalytical application is enzymatic analysis, DNA sequencing and monitoring of polymerase chain reaction. The method of claim 13, wherein pyrophosphate analysis is performed while the pyrophosphate sensor is fixed to the biochip.

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