JP2009186350A - Phosphate ion detection method and detection kit - Google Patents

Abstract

The present invention provides a method for easily detecting fluorescence of physiologically and chemically important phosphate anions in an aqueous medium and a kit capable of carrying out this method.
A method for detecting phosphate ions, wherein an analyte is a fluorescent dye having a diol moiety and a phenylboronic acid having a 2,2'-dipicolylamine metal complex moiety (provided that the metal of the metal complex moiety is Said method comprising: mixing with zinc or copper) and detecting the generated fluorescence. A phosphate ion detection kit comprising the following reagents: (1) Fluorescent dye having a diol moiety and (2) Phenylboronic acid having a 2,2'-dipiconylamine metal complex moiety, where the metal of the metal complex moiety is zinc or copper)
[Selection figure] None

Description

  The present invention relates to a fluorescence detection method and a detection kit that can be used for measurement of phosphate ions.

  In biological systems, phosphate compounds are found as various phosphate esters including DNA and RNA. Therefore, its biochemical importance has been pointed out, and the signal transduction system is involved in various information transmission controls through phosphorylated proteins and the like. In addition, there are some enzymes involved in metabolic reactions, for example, alkaline phosphatase (ALP) hydrolyzes phosphate esters under alkaline conditions, but abnormal ALP values suggest liver damage, It is also an important test substance for clinical tests.

  The phosphorus component is a regulatory factor in the environment. For example, phytic acid is a 6-phosphate ester of myo-inositol, which, as a familiar example, is contained in livestock cereal feed, but is not digested and raises the concentration of natural phosphate (eutrophication) . Therefore, the phosphoric acid compound is important from the viewpoint of environmental chemistry.

  The chemical test method for phosphate ions includes a molybdenum blue method, a biosensor, or a method using a chemical sensor using a synthetic molecule.

In the molybdenum blue method, a to-be-inspected sample is reacted with morbiddenic acid in an acidic solution to form a morbidden complex. This is reduced to molybdenum blue and optically quantified.
_{^{3NH 4 + + 12MoO 4 2- +}} H 2 PO 4 - + 22H + ← → (NH 4) 3 PO 4 · 12MoO 3 + 12H 2 O
Since this method is an orthophosphoric acid detection method, it has a drawback that it cannot be directly applied to other phosphoric acid species.

  In the method using a biosensor (enzyme sensor for measuring phosphoric acid), an enzyme substrate and necessary reagents such as coenzymes must coexist. A device for converting the detection information into a signal is required.

  In the method using a chemical sensor based on a synthetic molecule, an artificial molecule capable of signal processing of molecular recognition ability and recognition information is effective in proposing a bioimaging reagent and a simple measurement system.

Japanese Patent Laid-Open No. 2003-254909 (Patent Document 1), for example, is for detecting anions such as pyrophosphate ions comprising an anion host composed of a metal complex of a cyclic polyamine represented by the following formula and a compound having a fluorescent dye. A fluorescent sensor is disclosed.

Japanese Unexamined Patent Publication No. 2003-246788 (Patent Document 2) discloses a fluorescent sensor for detecting a phosphate anion such as a phosphate ion, which is made of a fluorescent compound composed of a zinc complex represented by the following formula, for example.

S. Aoki, et al., J. Am. Chem. Soc., 2005, 127, 9129-9139. (Non-patent Document 1) is composed of, for example, a fluorescent compound composed of a zinc complex represented by the following formula: Disclosed is a fluorescent sensor for detecting phosphate anions such as phosphate ions.

Furthermore, the present inventors have proposed self-organizing anion fluorescence sensing using dipicolylamine zinc complex-conjugated phenylboronic acid represented by the following formula (The 86th Annual Meeting of the Chemical Society of Japan, 1K1-49). (2006); The 87th Annual Meeting of the Chemical Society of Japan, 4E4-35 (2007), Non-Patent Document 2).
Japanese Patent Laid-Open No. 2003-254909 Japanese Patent Laid-Open No. 2003-246788 S. Aoki, et al., J. Am. Chem. Soc., 2005, 127, 9129-9139. The 86th Annual Meeting of the Chemical Society of Japan, 1K1-49 (2006); The 87th Annual Meeting of the Chemical Society of Japan, 4E4-35 (2007)

  However, the method using a chemical sensor using synthetic molecules described in Patent Documents 1 and 2 and Non-Patent Document 1 is not easy to express a plurality of functions in the molecule, and phosphoric acid that can function in water. There are few examples of ion fluorescent probes. Dipicolylamine zinc complex conjugated phenylboronic acid used in the method described in Non-Patent Document 2 contains a by-product in which the boronic acid moiety is substituted with a hydroxyl group at the stage of ligand synthesis, and thus is extremely difficult to purify and is a chemical product. There is a problem that the provision is hindered.

  Accordingly, an object of the present invention is to provide a method capable of easily detecting fluorescence of physiologically and chemically important phosphate anions in an aqueous medium and a kit capable of carrying out this method.

The present invention is as follows.
[1] A method for detecting phosphate ions, in which an analyte is a fluorescent dye having a diol moiety and a phenylboronic acid having a 2,2'-dipicolylamine metal complex moiety (provided that the metal at the metal complex moiety is zinc Or copper) and detecting the generated fluorescence.
[2] The method according to [1], wherein the phenylboronic acid having a 2,2′-dipiconylamine zinc complex moiety is at least one of the compounds represented by the following formulae.
[3] The method according to [1], wherein the phenylboronic acid having a 2,2′-dipiconylamine copper complex moiety is a compound represented by the following formula:
[4] The method according to any one of [1] to [3], wherein the fluorescent dye having a diol moiety is Alizarin Red S.
[5] The method according to any one of [1] to [4], wherein the mixing and fluorescence detection are performed in a neutral aqueous medium.
[6] The phosphate ion is a pyrophosphate ion, adenosine triphosphate ion (ATP), adenosine diphosphate ion (ADP), adenosine monophosphate ion (AMP), phytate ion, or inositol phosphate ion [1] ] The method according to any one of [5].
[7] The method according to any one of [1] to [6], wherein the fluorescence detection is measured as a fluorescence intensity near a fluorescence maximum wavelength.
[8] A phosphate ion detection kit containing the following reagents.
(1) a fluorescent dye having a diol moiety and
(2) Phenylboronic acid having a 2,2'-dipiconylamine metal complex site, where the metal of the metal complex site is zinc or copper)

  A feature of the present invention is that a self-organizing method is used. In other words, the test substance recognition site and the optical response site required for chemical sensors are provided as molecular components, and when phosphate ions, which are test substances, are added, spontaneous organization along the purpose occurs. Is a way to work.

The method of the present invention has the following advantages.
A sensor function can be achieved by simply mixing a fluorescent dye with a diol moiety and a phenylboronic acid with a 2,2'-dipicolylamine zinc complex moiety into a neutral aqueous medium. In the system using bis (dicopicolylzinc complex) -type phenylboronic acid (2 · 2Zn), we found ratio detection for phytate ions. Since the quantification can be performed with the fluorescence intensity ratios of two different wavelengths, it is possible to reduce the influence of the intensity difference, fading, and autofluorescence in the initial state, which is an advantageous method for precise measurement.

  The present invention is a method for detecting phosphate ions. In this method, an analyte is mixed with a fluorescent dye having a diol moiety and phenylboronic acid having a 2,2′-dipiconylamine metal complex moiety (where the metal of the metal complex moiety is zinc or copper), Detecting the fluorescence generated.

  In phenylboronic acid having a 2,2′-dipiconylamine metal complex site, the metal of the metal complex site is zinc or copper. Examples of phenylboronic acid having a 2,2′-dipicolylamine zinc complex moiety in which the metal of the metal complex moiety is zinc include compounds represented by the following formulae. These compounds are all novel compounds and can be synthesized by the methods described in the examples.

Examples of the phenylboronic acid having a 2,2′-dipicolylamine copper complex moiety in which the metal of the metal complex moiety is copper include compounds represented by the following formulae. This compound is a novel compound and can be synthesized by the methods described in the Examples.

  Fluorescent dyes having a diol moiety include alizarin dyes, pyrocatechol violet, flavonols, and coumarin derivatives esculetin and 4-methylesculetin. More specifically, for example, alizarin red S and alizarin complex-on are more preferable.

  In the method of the present invention, an analyte is mixed with a fluorescent dye having a diol moiety and a phenylboronic acid having a 2,2′-dipicolylamine metal complex moiety, and then fluorescence detection is performed. It can be carried out in a neutral aqueous medium. The ratio of the fluorescent dye having a diol moiety and the phenylboronic acid having a 2,2′-dipicolylamine metal complex moiety at the time of mixing is preferably, for example, in a range not exceeding 1 equivalent to 5 equivalents. Conditions (including the measured temperature) can be set as appropriate in mind.

The concentration of the fluorescent dye having a diol moiety is on the order of 10 ⁻⁵ mol / L, and the concentration of a phenylboronic acid having a 2,2′-dipicolylamine metal complex moiety is from 10 ⁻⁵ to 10 ⁻⁴ mol. Can be in the range of / L order.

  The neutral aqueous medium is, for example, an aqueous solution having a pH of 6 to 8, and may contain a buffer for adjusting the pH.

  The phosphate ions to be detected are, for example, pyrophosphate ions, adenosine triphosphate ions (ATP), adenosine diphosphate ions (ADP), adenosine monophosphate ions (AMP), phytate ions, inositol phosphate ions, etc. Can be.

  Of special note is the kit that combines alizarin red S, a fluorescent dye with a diol moiety, and 2,2'-dipicolylamine zinc complex-type phenylboronic acid (1Zn) against condensed phosphate ions. A selective response was observed, the order of which was pyrophosphate ion> ATP> ADP> AMP.

  In the case of fluorescence detection, in the case of qualitative measurement, the presence or absence of fluorescence can be simply confirmed with the naked eye. In addition, when quantification or semi-quantification is performed, it can be measured as the fluorescence intensity near the fluorescence maximum wavelength. In addition, depending on the combination of the type of phosphate ion to be detected, the fluorescent dye having a diol moiety, and the type of phenylboronic acid having a 2,2'-dipicolylamine metal complex moiety, the fluorescence intensity ratio of two different wavelengths In some cases, quantification can be performed. In that case, the influence of the intensity difference, fading, and autofluorescence in the initial state can be reduced, which is an advantageous method for precise measurement.

The present invention includes a phosphate ion detection kit containing the following reagents.
(1) a fluorescent dye having a diol moiety and
(2) Phenylboronic acid having a 2,2'-dipiconylamine metal complex site (provided that the metal of the metal complex site is zinc or copper)
Each of the reagents (1) and (2) can be an appropriate amount packed in a container.

  Furthermore, the kit of the present invention can also contain an aqueous solution containing a buffering agent in addition to the above reagents.

Example 1 Synthesis
1.Synthesis of N-3-boronobenzyl-N ', N'-bis (2-pyridylmethyl) ethylenediamine (1)

In an Ar atmosphere, N, N-bis (2-pyridylmethyl) ethylenediamine ¹ (4.60 g, 19.0 mmol) and 3-formyl phenylboronic acid (2.85 g, 19.0 mmol) were dissolved in dry ethanol (380 mL) that had been degassed by freezing. Stir overnight in the presence of Molecular Sieves 3A (10 g) at room temperature. After confirming the reaction by TLC, a dry ethanol solution of NaBH ₄ (1.44 mg, 38.1 mmol) is added, and the mixture is further stirred for 1 hour. After completion of the reaction, the molecular sieves are removed and the solvent is distilled off. The residue is extracted into the aqueous layer with AcOEt-H ₂ O and further extracted into the organic layer with methylene chloride. The organic layer is stirred and dried with anhydrous sodium sulfate, and the solvent is distilled off after fold filtration. The obtained crude product is separated by 6 wt% silica gel column chromatography (gradient: CH ₂ Cl ₂ -MeOH), and further washed with ethyl acetate and diethyl ether to obtain a pale yellow solid. (843.9 mg, 12%)

¹ H NMR (400 MHz, 50 mM in CD ₃ OD): δ 8.25 (d, J = 4.5 Hz, Pyr-H ₁ , 2H), 7.64 (td, J = 7.7, J = 1.7 Hz, Pyr-H, 2H), 7.63 (t, J = 7.4 Hz, Ar-H ₁₁ , 1H), 7.58 (s, Ar-H ₁₂ , 1H), 7.28 (t, J = 7.4 Hz, Ar-H ₁₀ , 1H), 7.17 -7.23 (m, Pyr-H _2,4 , Ar-H ₉ , 5H), 4.11 (s, Ar-CH ₂ -NH, 2H), 3.87 (s, Pyr-CH ₂ -N, 4H), 3.25 ( t, J = 5.4 Hz, NH-CH ₂ -CH ₂ , 2H), δ 3.04 (t, J = 5.5 Hz, CH ₂ -CH ₂ -NH ₂ , 2H); ¹³ C NMR (100.7 MHz, 50 mM in CH ₃ OH-d ₄ ): δ159.9, 149.8, 138.6, 135.3, 135.1, 131.4, 128.3, 127.2, 124.9, 123.8, 60.6, 53.0, 52.7, 46.4; ESI MS: m / z 376 ([M + H ^{.] +); elemental analysis:} anal calcd for C 21 H 25 BN 4 O 2 · 0.3H 2 O: C 66.09; H 6.76; N 14.68%, found: C 65.96; H 6.64; N 14.28%.

Synthesis of N-3-boronobenzyl-N ', N'-bis (2-pyridylmethyl) ethylenediamine zinc (II) complex (1Zn)

Compound (1) (514.4 mg, 1.37 mmol) and Zn (NO ₃ ) ₂ · 6H ₂ O (407.6 mg, 1.37 mmol) are dissolved in MeOH (45 ml) and stirred at room temperature for 60 minutes. The completion of the reaction is confirmed by TLC, evaporated and then reprecipitated with THF to obtain a yellow solid. Since THF could not be removed even after vacuum drying, the operation of dissolving in MeOH and freeze-drying was repeated, and then the solid was washed with diethyl ether and dried while heating at 40 ° C. (687.9 mg, 89%)

¹ H NMR (400 MHz, 98 mM in CD ₃ OD): δ 8.74 (d, J = 4.6 Hz, Pyr-H ₁ , 2H), 8.13 (app.t, J = 7.7 Hz, Pyr-H ₃ , 2H ), 7.66 (t, J = 6.4 Hz, Pyr-H ₂ , 2H), 7.61 (d, J = 7.9 Hz, Pyr-H ₄ , 2H), 7.54 (s, Ar-H ₁₂ , 1H), 7.50 ( d, J = 6.7 Hz, Ar-H ₁₁ , 1H), 7.33 (d, J = 7.4 Hz, Ar-H ₉ , 1H), 7.29-7.22 (m, Ar-H ₁₀ , 1H), 4.38, 4.25 ( dd, J = 17.0 Hz, Pyr-CH ₂ -N, 4H), 4.11 (brs, H ₈ , 1H), 3.49 (brs, H ₈ , 1H), 2.95 (br, NH-CH ₂ -CH ₂ , 2H ), 2.63 (s, CH ₂ -CH ₂ -NH ₂ , 2H); ¹³ C NMR (100.7 MHz, 98 mM in CH ₃ OH-d ₄ ): d 157.1, 149.7, 142.6, 136.3, 136.03, 135.6, 134.9 , 134.4, 132.4, 131.8, 129.1 , 126.2, 125.7, 59.1, 54.2, 53.4, 44.6; ESI Mass: m / z 219 ([M - 2 (NO 3 -)] 2+; elemental analysis:. anal calcd for C ₂₁ H ₂₅ BN ₆ O ₈ Zn ・ 0.5 H ₂ O ・ 0.5 MeOH: C 42.70; H 4.78; N 14.23%, found: C 42.50; H 4.42; N 14.11%.

Example 2 Synthesis
3,5-bis ((bis (pyridin-2-ylmethyl) amino) methyl) benzylamine (7)

Under an Ar atmosphere, dry DMF (100 ml) was mixed with monophthalimide (3) ² (8.00 g, 18.91 mmol) and potassium carbonate (11.10 g, 80.31 mmol) and stirred at room temperature, and then dry DMF (20 ml) ) 2,2′-dipicolylamine (8.67 g, 43.51 mmol) dissolved slowly was added dropwise and stirred for 90 minutes. After completion of the reaction, insoluble matters were removed by filtration, and the solvent was distilled off under reduced pressure to obtain a crude product (4). Subsequently, the crude product (4) and hydrazine monohydrate (2.0 ml, 40.77 mmol) were dissolved in dry EtOH (200 ml) and refluxed overnight under an Ar atmosphere. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was extracted into methylene chloride and washed with ion-exchanged water. The organic phase was dried over sodium sulfate and the solvent was distilled off under reduced pressure. The obtained residue was purified by column chromatography (SiO ₂ , eluent; CH ₂ Cl ₂ / MeOH / aqueous ammonia = 100: 5: 2) to obtain 5 as a yellow tar. (Yield 5.17 g, Yield 52%)

¹ H NMR (200 MHz, CDCl ₃ ) δ 3.69 (s, 4H, Ph-CH ₂ -N), 3.81 (s, 8H, N-CH ₂ ), 3.84 (s, 2H, NH ₂ -CH ₂ -Ph ), 7.09-7.16 (m, 4H, pyridine-H), 7.21 (s, 2H, Ph-H), 7.39 (s, 1H, Ph-H), 7.59-7.66 (m, 8H, pyridine-H), 8.49-8.52 (m, 4H, pyridine-H); FAB + MS (m / z) 530 [M + H] ⁺ (matrix; 3-nitrobenzyl alcohol)

3-((3,5-bis ((bis (pyridin-2-ylmethyl) amino) methyl) benzylamino) methyl) phenylboronic acid (2)

Under Ar atmosphere, bisdipicolylamine (5) (5.17 g, 9.76 mmol) and 3-formylphenylboronic acid (1.47 g, 9.80 mmol) were dissolved in freeze-degassed dry EtOH (150 ml), and MS3A ( The mixture was stirred at room temperature for 8 hours in the presence of ca.10 g). Thereafter, sodium borohydride (748.2 mg, 19.78 mmol) was added, and the mixture was further stirred for 1 hour. After completion of the reaction, insoluble matters were removed by Kiriyama filtration, the filtrate was concentrated, extracted into ethyl acetate, washed with ion-exchanged water, and dried over sodium sulfate. The organic phase was concentrated and column chromatography (SiO ₂ , eluent; gradient MeOH (0-100% (v / v)) in CH ₂ Cl ₂ ) gave 2 as a pale yellow powder. (Yield 2.95 g, Yield 47%)

¹ H NMR (400 MHz, CD ₃ OD) δ 3.68 (s, 4H, Ph-CH ₂ -N), δ 3.76 (s, 8H, N-CH ₂ ), 3.99 (s, 2H, NH-CH ₂ or CH ₂ -NH), 4.04 (s, 2H, NH-CH ₂ or CH ₂ -NH), 7.15 (d, J = 6.40 Hz, 1H, Ph-H), 7.22-7.27 (m, 5H, Ph- H, pyridine-H), 7.33 (s, 2H, Ph-H), 7.47 (s, 1H, Ph-H), 7.51 (s, 1H, Ph-H), 7.56 (d, J = 7.2 Hz, 2H , Ph-H), 7.65 (d, 4H, J = 7.8 Hz, pyridine-H), 7.74 (td, 4H, J = 7.7, 1.7 Hz, pyridine-H), 8.40-8.41 (m, 4H, pyridine- H); ESI MS (m / z) 664 [M + H] ⁺ (solvent; CH ₃ OH); Anal.Calc. For C ₄₀ H ₄₂ BN ₇ O ₂ .0.1 MeOH: C, 72.05; H, 6.41; N, 14.70. Found C, 71.97; H, 6.19; N, 14.94%

3-((3,5-bis ((bis (pyridin-2-ylmethyl) amino) methyl) benzylamino) methyl) phenylboronic acid zinc (II) complex (2 ・ 2Zn)

  Ligand (2) (1.87 g, 2.81 mmol) and zinc nitrate hexahydrate (1.70 g, 5.71 mmol) were dissolved in acetone (50 ml) and stirred at room temperature for 30 minutes. After completion of the reaction, the precipitated white solid was collected by filtration and dissolved in water. Thereafter, insoluble matters were removed by filtration and freeze-dried to obtain 2-2Zn as a white powder. (Yield 2.83 g, 97% yield)

¹ H NMR (400 MHz, D ₂ O) δ 3.82 (s, 4H, Ph-CH ₂ -N), 3.98 (s, 2H, NH-CH ₂ or CH ₂ -NH), 4.09 (s, 2H, NH -CH ₂ or CH ₂ -NH), 4.00, 4.19 (dd, J = 16.3 Hz, 4H, N-CH ₂ ), 7.18-7.22 (m, 2H, Ph-H), 7.41-7.50 (m, 7H, Ph-H, pyridine-H), 7.59 (t, J = 6.0 Hz, 4H, pyridine-H), 7.64 (s, 1H, Ph-H), 7.73 (d, 1H, J = 6.5 Hz, Ph-H ), 8.03 (t, J = 7.2 Hz, 4H, pyridine-H), 8.58 (d, J = 4.1 Hz, 4H, pyridine-H); ESI MS (m / z) 978 [M + H−NO ₃ ] ⁺ (solvent; CH ₃ OH); Anal.Calc.for C ₄₀ H ₄₂ BN ₁₁ O ₁₄ Zn ₂・ 5H ₂ O: C, 42.42; H, 4.63; N, 13.60. Found C, 42.60; H, 4.64; N, 13.83%

Literature
1. K. Hanaoka, K. Kikuchi, Y. Urano and T. Nagano, Chem. Soc., Perkin Trans. 2, 2001, 1840-1843.
2. WTS Huck, LJ Prins, RH Fokkens, NMM Nibbering, FCJM van Veggel and DN Reinhoudt, J. Am. Chem. Soc., 1998, 120, 6240-6246.

Example 3
Pyrophosphate ion detection system, an important biological species

Alizarin Red S (ARS) (50 μM) and 1 · Zn (250 μM) synthesized in Example 1 were mixed with MeOH-10 mM HEPES buffer (1: 1 v / v) containing NaCl (10 mM), pH 7.4. Fluorescence spectrum (λ _ex = 480 nm) with PPi addition (0-500 μM) was measured at 25 ° C. An increase in fluorescence intensity was observed (Figure 1).

  Figure 2 shows changes in fluorescence at 586 nm (excitation wavelength: 480) when various anions (PPi (pyrophosphate ion), Pi (phosphate ion), ATP, ADP, AMP, citric acid) are added and the concentration is changed. nm). Alizarin Red S (ARS) (50 μM) and 1 · Zn (250 μM) synthesized in Example 1 were mixed with MeOH-10 mM HEPES buffer (1: 1 v / v) containing NaCl (10 mM), pH 7.4. The system dissolved in was used. The selectivity to PPi was suggested, and the significance of the greater responsiveness than other interphosphate complexes was particularly significant.

Example 4
Related systems targeting inositol phosphates

Dissolve ARS (10 μM) and 2.2Zn (10 μM) in 10 mM HEPES buffer (pH 7.4), and obtain the fluorescence spectrum (λ _ex = 470 nm) with phytate ion addition (0-200 μM) at 25 ° C. (Figure 3). In the solution without phytate ion added, fluorescence showing an emission maximum near 610 nm is observed, but with the addition of phytate ion, the fluorescence band decreases and a new emission maximum near 560 nm is observed. A fluorescence spectrum was observed. Therefore, ratio detection (excitation wavelength 470 nm) was attempted (excitation wavelength 470 nm) by taking the ratio of I ₆₅₀ (fluorescence intensity at 650 nm) to I ₅₅₀ (fluorescence intensity at 550 nm) (I ₅₆₀ / I ₆₅₀ ). (Figure 4). It showed phytate ion selectivity compared to other inositol phosphate ions (cis, cis-1,3,5-cyclohexanetriol triphosphate).

Example 5
Synthesis of N-3-boronobenzyl-N ', N'-bis (2-pyridylmethyl) ethylenediamine copper (II) complex (1 ・ Cu)
Ligand (1) (30.0 mg, 0.080 mmol) and Cu (NO ₃ ) ₂ · 3H ₂ O (19.4 mg, 0.080 mmol) were dissolved in MeOH (3 ml) and stirred at room temperature for 60 minutes. After confirming the completion of the reaction by TLC, a blue solid was obtained by evaporating the solvent and freeze-drying. (45.2 mg, 100%)
ESI Mass: m / z 218.7354 ( [M - 2 (NO 3 -)] 2+.

Example 6
ARS (50 μM) and 1-Cu (250 μM) are dissolved in MeOH-10 mM HEPES buffer (1: 1, v / v) containing 10 mM NaCl (pH 7.4), and various anions (PPi (pyrophosphate ion) , Pi (phosphate ion), citric acid) was added (25 ° C., λex = 480 nm, λem = 590 nm, FIG. 5). The selectivity for PPi and ATP was suggested, and it was also found that the responsiveness to citrate ion was large.

  The present invention is useful in the field of biochemical tests such as clinical tests and the field of environmental chemistry tests.

The result of the increase in the fluorescence intensity obtained in Example 3 is shown. Changes in fluorescence when various anions (PPi (Pyrophosphate ion), Pi (Phosphate ion), ATP, ADP, AMP, Citric acid) are added. The result of the change of the fluorescence spectrum accompanying the phytate ion addition obtained in Example 4 is shown. Various anions (phytic acid, CTP3, Pi (phosphate ion), F ^-, acetate) shows the fluorescence change when added. The result of having measured the fluorescence change at the time of adding various anions (PPi (pyrophosphate ion), Pi (phosphate ion), citric acid) is shown.

Claims (8)

A method for detecting phosphate ions, wherein an analyte is a fluorochrome having a diol moiety and a phenylboronic acid having a 2,2'-dipicolylamine metal complex moiety (provided that the metal of the metal complex moiety is zinc or copper). And) detecting the fluorescence generated. The method according to claim 1, wherein the phenylboronic acid having a 2,2'-dipicolylamine zinc complex moiety is at least one of the compounds represented by the following formulae.
The method according to claim 1, wherein the phenylboronic acid having a 2,2'-dipiconylamine copper complex moiety is a compound represented by the following formula.
The method according to any one of claims 1 to 3, wherein the fluorescent dye having a diol moiety is alizarin red S. The method according to claim 1, wherein the mixing and fluorescence detection are performed in a neutral aqueous medium. The phosphate ion is pyrophosphate ion, adenosine triphosphate ion (ATP), adenosine diphosphate ion (ADP), adenosine monophosphate ion (AMP), phytate ion, or inositol phosphate ion. The method in any one of. The method according to claim 1, wherein the fluorescence detection is measured as a fluorescence intensity in the vicinity of a fluorescence maximum wavelength. A phosphate ion detection kit comprising the following reagents:
(1) a fluorescent dye having a diol moiety and
(2) Phenylboronic acid having a 2,2'-dipiconylamine metal complex site, where the metal of the metal complex site is zinc or copper)