KR101381201B1 - Supramolecular Complexes for Detecting Hydrogenpyrophosphate Anion and Detecting Methods Using the Same - Google Patents

Abstract

(상기 화학식 1에서, R은 탄소수 1 내지 6의 알킬기를 나타내고, X^-는 할로, PF₆ ^-, SbCl₆ ^- SCN^-, CH₃COO^-, HSO₄ ^-, BF₄ ^-, 및 ClO₄ ^- 중에서 선택된 음이온을 나타낸다)
본 발명의 초분자 착화합물은 하이드로젠파이로포스페이트 음이온이 포함되어 있을수록 그와 비례하여 형광이 강하게 살아나기 때문에 ppb 이하의 극히 낮은 농도의 하이드로젠파이로포스페이트 음이온을 고감도로 검출할 수 있으며, 또한 유기 용매 뿐 아니라 수용액에서도 정량이 가능하여 미량의 하이드로젠파이로포스페이트 음이온을 검출하는 데 특히 유용하다.The present invention relates to a supramolecular complex for detecting hydrogen pyrophosphate anion represented by Formula 1 and a method for selectively detecting and quantifying hydrogen pyrophosphate anion using the supramolecular complex.
[Chemical Formula 1]

(In Formula 1, R represents an alkyl group having 1 to 6, _{^{X-from-halo, PF 6 -, SbCl 6 -}} SCN -, CH 3 COO -, HSO 4 -, BF 4 -, and ClO ₄ Represents the selected anion)
Since the ultramolecular complex of the present invention contains a hydrogen pyrophosphate anion, the fluorescence is strongly increased in proportion to the hydrogen pyrophosphate anion, and thus, a very low concentration of hydrogen pyrophosphate anion of ppb or less can be detected with high sensitivity. It is particularly useful for detecting trace hydrogen pyrophosphate anions because it can be quantified in aqueous solutions as well as in solvents.

Description

Supramolecular Complexes for Detecting Hydrogenpyrophosphate Anion and Detecting Methods Using the Same}

The present invention relates to a supramolecular complex for detecting hydrogen pyrophosphate anion and a method for selectively detecting hydrogen pyrophosphate anion using the complex compound.

Research into the development of sensors or receptors capable of detecting biologically important ions has been very active. Recently, as the understanding and research on supramolecular chemistry is progressed, the possibility of developing a sensor for detecting an ion is increased, and various approaches are being studied. Among them, research on chemical sensors using the principle of emitting fluorescence using an affinity with an analyte using a supramolecular structure in which a supramolecular compound is combined with a fluorescent material is quenched. It is possible to develop a sensitive chemical sensor because of the turn-on 'phenomenon. Recently, researches on fluorescence chemical sensors capable of detecting cations, anions, nanomaterials or neutral organic molecules using these mechanisms have been published one after another.

Hydrogenpyrophosphate (hereinafter referred to as 'PPi') is a hydrolyzate of ATP in cells and is a biologically important target anion and participates in various bioenergy and metabolism processes. In various diseases, PPi is overexpressed. There are many cases. Thus, detection of PPi anions can be an important technique for diagnosing a variety of biological diseases. Recently, PPi ion measurement technology has been used for cancer research. (Xu S et al., Anal. Biochem. 2001, 299, 188)

However, compared to other ion detection sensors, much of the research on chemical sensors capable of detecting PPi anions is based on Zn-DPA complexes. When the PPi anion complexes with Zn-DPA, the fluorescence of the phosphor introduced into the probe decreases. However, little is known about the method of detecting a small amount of PPi anion using an increase in fluorescence using a supramolecular complex (on).

It is an object of the present invention to provide a novel supramolecular complex that detects hydrogen pyrophosphate (PPi) anions with high sensitivity even at concentrations up to ppb.

Another object of the present invention is to provide a method for detecting hydrogen pyrophosphate (PPi) anion using the supramolecular complex compound described above.

The present invention is characterized by a supramolecular complex compound for detecting hydrogen pyrophosphate (PPi) anion represented by the following formula (1).

In Formula 1, R represents an alkyl group having 1 to 6 carbon atoms; X ^- is selected from halo, hexafluorophosphate (PF ₆ ^-), hexachloro antimonate (SbCl ₆ ^-), thiocyanate (SCN ^-), acetate (CH ₃ COO ^-), sulfonate (HSO ₄ ^-), An anion selected from tetrafluoroborate (BF ₄ ⁻ ), and perchlorate (ClO ₄ ⁻ ).

In addition, the present invention is characterized in that the method of using the chromenolate complex represented by the following formula (4) in which the supramolecular complex represented by the formula (1) is complexed with chromenolate, for the detection of hydrogen pyrophosphate (PPi) anion. It is done. That is, the present invention comprises hydrogen pyrophosphate (PPi) anion comprising the step of measuring the luminescence intensity by adding a complex represented by the following formula (4) to a solution containing or not containing hydrogen pyrophosphate (PPi) anion The detection method is characterized by.

[Chemical Formula 4]

In Formula 4, R and X ⁻ are as defined in Formula 1, and R ₁ represents a trifluoromethyl group or a pentafluoroethyl group.

The supramolecular complex of the present invention can detect PPi anion with a concentration of ppb or less with high sensitivity because the fluorescence of the supramolecular complex compound in perfect quenching state is strongly in proportion to the content of PPi anion.

The supramolecular complex of the present invention exhibits no luminescence at all other ions other than the PPi anion, and is capable of quantifying the PPi anion even under solution conditions dissolved in water or an organic solvent, so that a small amount of PPi anion is present in the sample having mixed ions. It is particularly useful for selectively detecting.

1 is a ¹ H NMR spectrum of the supramolecular complex according to the present invention.
Figure 2 is the result of measuring the luminescence intensity according to the concentration of the PPi anion complexed with the supramolecular complex.
3 is a result of measuring the luminescence intensity according to the concentration of the PPi anion while varying the type of anion (X) of the supramolecular complex.
4 is a result of measuring the emission intensity according to the PPi anion concentration.
5 is a graph showing a correlation between PPi anion concentration and luminescence intensity.
6 is a result of measuring the absorbance according to the concentration of supramolecular complex compound in the UV-vis wavelength range.
7 is a result of measuring the absorbance according to the concentration of PPi in the UV-vis wavelength range.
8 is a graph showing the binding constants according to the types of anions in 100% acetonitrile solvent.
9 is a graph showing the binding constants according to the kinds of anions in the 70% acetonitrile aqueous solution.

The present invention relates to a supramolecular complex for detecting the PPi anion represented by Chemical Formula 1.

In the supramolecular complex represented by Formula 1, preferably,

5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole difluoro salt,

5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole dichloro salt,

5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole diiodine salt,

5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole dibromo salt,

5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole dihexafluorophosphate salt,

5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole diacetate salt,

5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole disulfonate salt.

The preparation method of the supramolecular complex compound represented by Chemical Formula 1 is briefly shown in Scheme 1 below.

[Reaction Scheme 1]

In Reaction Scheme 1, R, and X ⁻ are as defined in Formula 1, respectively.

The compound represented by Chemical Formula 2 used as a starting material in the preparation method according to Scheme 1 was prepared by heating and refluxing 4-acetylpyridine and pyrrole under a methanesulfonic acid catalyst.

In addition, the compound represented by Chemical Formula 2 is prepared by reacting the compound represented by Chemical Formula 2 with trifluoroborane (BF ₃ ) at room temperature of 20 ° C. to 30 ° C. and under a mixed solvent of acetone and ethanol.

In addition, the compound represented by Chemical Formula 3 may be reacted with an alkyl halide compound to prepare a compound represented by Chemical Formula 1 in which an alkyl group R is substituted at the N 1 position of pyridine (X = halo). And X is PF ₆ ^-, SbCl ₆ ^{-, _{SCN -, CH 3 COO -,}} HSO 4 -, BF 4 -, or ClO ₄ ^- in order to replaced by another anion such as, X = halo ion-exchange compound of the formula (1) as containing a suitable anion compound And various anions may be substituted. The anion exchange method is a method commonly used in the art, and the present invention places no particular limitation on the anion exchange method.

In addition, the present invention relates to a chromenolate complex represented by the following formula (4) prepared by reacting the supramolecular complex represented by the formula (1) with a chromenolate compound represented by the following formula (5).

[Reaction Scheme 2]

In Scheme 2, R, X ⁻ , and R ₁ are each as defined above.

The chromenolate complex represented by Formula 4 exists in a state where fluorescence is completely quenched, but when PPi anion is present, PPi anion having greater affinity emits chromenolate anion while generating a complex, thereby exhibiting fluorescence. In addition, the intensity of luminescence is determined by the amount of chromanolate anion released in proportion to the concentration of the PPi anion. Eventually, as the concentration of PPi anion increases, the degree of luminescence increases in proportion to the concentration of PPi anion, so that the PPi anion concentration can be quantified by measuring the intensity of luminescence of the chromenolate anion.

Accordingly, the present invention relates to a method for detecting PPi anions in a solution by measuring the luminescence intensity of fluorescence which occurs after the chromolate complex represented by the formula (4) is added to a solution containing or not containing a PPi anion.

That is, when the chromenolate complex represented by Chemical Formula 4 is injected into a solution containing or not containing PPi anions, light is emitted while forming a complex with PPi anions. In this case, when the emission intensity is measured using a fluorescence spectrometer, the PPi anion Can be easily detected. The solvent of the solution used to detect the PPi anion may be water or a conventional organic solvent, and the present invention does not limit the type of solvent.

When measuring the emission intensity, it is preferable to measure the intensity at 450 to 550 nm, which is the emission wavelength of the phosphor, because the concentration of the PPi anion can be measured with the highest sensitivity.

In addition, since the luminescence intensity is directly proportional to the PPi anion at low concentration, that is, in the range of 0 to 200 nM, the quantitative measurement is also possible using this correlation.

The present invention as described above will be described in more detail through the following Examples and Experimental Examples, the following Examples and Experimental Examples are only for illustrating the present invention, not to limit the present invention to understand Will only do.

[Example]

Example 1.Synthesis of 5- (4-pyridine) -5-methyl-dipyromethane

3.0 g (24.8 mmol) of 4-acetylpyridine was dissolved in 25 mL of ethanol, and then 17.2 mL (0.24 mol) of pyrrole was added under a stream of nitrogen. A catalytic amount of methanesulfonic acid 0.8 mL (12.4 mmol) was added and refluxed for 3 days to change the color of the reaction mixture to a dark red color. The reaction mixture was cooled to room temperature, 50 mL of 0.1 N aqueous sodium hydroxide solution was added to terminate the reaction, and then extracted with chloroform (50 mL × 2). The organic layer was dried over anhydrous sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluate CHCl ₃ / EtOAc = 5.5 / 4.5) to obtain 1.47 g (yield 25%) of the title compound.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ [ppm] 10.46 (br s, 2H, pyrrole-NH), 8.47-8.45 (m, 2H, pyridyl-H), 6.99-6.97 (m, 2H, pyridyl -H), 6.70-6.68 (m, 2H, pyrrole-H), 5.94-5.92 (m, 2H, pyrrole-H), 5.60-5.58 (m, 2H, pyrrole-H), 1.99 (s, 3H, CH ₃ ); ^{13 C NMR (100 MHz, DMSO-} d 6) δ [ppm] 157.6, 149.6, 136.6, 122.9, 117.9, 106.8, 106.6, 44.5, 27.4; MALDI-MS Calcd. for C ₁₅ H ₁₅ N ₃ 237.13, Found 238.17 (M + 1).

Example 2. Synthesis of cis-5,15- [bis- (4-pyridine)]-5,10,10,15,20,20-hexamethylcalix [4] pyrrole

Dissolve 5- (4-pyridine) -5-methyl-dipyromethane (1.0 g, 4.21 mmol) in 30 mL of a 1: 1 mixed solution of acetone and ethanol, and then 1.0 mL (8.43 mmol) of BF ₃ · OEt ₂ . Was added and stirred at room temperature for 24 hours. 2.53 mL (18.12 mmol) of triethylamine was added to the reaction mixture to terminate the reaction, and the solvent was distilled under reduced pressure. The residue was extracted with dichloromethane (100 mL × 2). The organic layer was dried over anhydrous sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography to separate the isomers. First, 0.07 g (3% yield) of a trans-isomer compound was isolated using chloroform / methanol (9.9 / 0.1) as the elution solution. And chloroform / methanol (9.8 / 0.2) was used to separate 0.19 g (8% yield) of cis-isomer compound as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) δ [ppm] 8.46-8.45 (m, 4H, pyridyl-H), 7.26 (br s, 4H, pyrrole-NH), 6.91-6.89 (m, 4H, pyridyl-H ), 5.95-5.94 (m, 4H, pyrrole-H), 5.63-5.61 (m, 4H, pyrrole-H), 1.88 (s, 6H, CH ₃ ), 1.63 (s, 6H, CH ₃ ), 1.55 ( s, 6H, CH ₃ ); ¹³ C NMR (100 MHz, CDCl ₃ ) δ [ppm] 156.7, 149.4, 138.8, 134.9, 122.5, 106.3, 103.5, 44.5, 35.2, 30.0, 27.8, 27.1; MALDI-MS Calcd. for C ₃₆ H ₃₈ F ₂ N ₆ 554.32, Found 555.29 (M + l).

Example 3. Synthesis of cis-5,15- [bis (4- N -methylpyridinium)]-5,10,10,15,20,20-hexamethylcalix [4] pyrrole diiodine salt

0.15 g (0.27 mmol) of cis-5,15- [bis- (4-pyridyl)]-5,10,10,15,20,20-hexamethylcalix [4] pyrrole in 10 mL of anhydrous dichloromethane. After dissolving, 1.68 mL (2.70 mmol) of iodomethane was added and stirred at room temperature for 24 hours. The yellow solid precipitate was isolated by filtration and washed with dichloromethane (10 mL × 3) to give 0.14 g (60% yield) of the title compound.

¹ H NMR (400 MHz, CD ₃ CN) δ [ppm] 9.61 (br s, 4H, pyrrole-NH), 8.40 (d, J = 6.8 Hz, 4H, pyridyl-H), 7.46 (d, J = 6.7 Hz, 4H, pyridyl-H), 5.99-5.98 (m, 4H, pyrrole-H), 5.81-5.80 (m, 4H, pyrrole-H), 4.15 (s, 3H, N ⁺ -CH ₃ ), 4.12 ( s, 3H, N ⁺ -CH ₃ ), 1.89 (s, 6H, CH ₃ ), 1.73 (s, 6H, CH ₃ ), 1.70 (s, 6H, CH ₃ ); ¹³ C NMR (100 MHz, CD ₃ CN) δ [ppm] 170.1, 145.0, 141.6, 134.3, 127.6, 105.8, 102.4, 47.7, 46.0, 35.1, 31.2, 29.0, 26.8; MALDI-MS Calcd. for C ₃₈ H ₄₄ I ₂ N ₆ 838.17, Found 711.26 (MI).

Example 4. Synthesis of 5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole dihexafluorophosphate salt

0.12 g (0.14 mmol) of 5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole diiodine salt in 2 mL of acetonitrile. It was heated and melted. Then an aqueous ammonium hexafluorophosphate (NH ₄ PF ₆ ) solution was added for ion exchange. The reaction mixture was stirred at room temperature for 1 hour, and the pale yellow solid precipitate was filtered and washed with 100 mL of water. The residue was recrystallized from water and acetonitrile to give 65 mg (yield 52%) of the title compound as pale yellow crystals.

¹ H NMR (400 MHz, CD ₃ CN) δ [ppm] 8.44 (d, J = 6.7 Hz, 4H, pyridyl-H), 7.96 (br s, 4H, pyrrole-NH), 7.48 (d, J = 6.7 Hz, 4H, pyridyl-H), 5.96-5.95 (m, 4H, pyrrole-H), 5.68-5.66 (m, 4H, pyrrole-H), 4.22 (s, 6H, N ⁺ -CH ₃ ), 1.95 ( s, 6H, CH ₃ ), 1.66 (s, 6H, CH ₃ ), 1.53 (s, 6H, CH ₃ ); ¹³ C NMR (100 MHz, CD ₃ CN) δ [ppm] 167.3, 144.3, 139.9, 132.7, 126.0, 106.2, 103.2, 47.2, 45.3, 34.7, 29.1, 26.3, 25.9; MALDI-MS Calcd. for C ₃₈ H ₄₄ I ₂ N ₆ 874.29, Found 729.31 (M-PF ₆ ).

Example 5 Synthesis of cis-5,15- [bis (4- N -methylpyridinium)]-5,10,10,15,20,20-hexamethylcalix [4] pyrrole chromenolate complex

Cis-5,15- [bis (4- N -methylpyridinium)]-5,10,10,15,20,20-hexamethylcalix [4] pyrrole hexafluorophosphate salt and tetrabutyl ammonium- (4 -Trifluoromethyl) -chromenolate was mixed in a 1: 1 molar ratio and dissolved in acetonitrile. It can be seen that this solution produced a 1/1 complex completely in the NMR spectrum. The supramolecular complexes thus produced were used for PPi quantification.

[Experimental Example]

Experimental Example 1 Hydrogen Pyrophosphate (PPi) Anion Detection Performance Verification

Chromenolate complex synthesized in Example 5 was adjusted to acetonitrile to a concentration of 2.1 μM and 4.8 μM, respectively. Tri (tetrabutylammonium) hydrogen pyrophosphate was added dropwise to 1 to 4.9 μM, and the emission intensity in the 410 nm wavelength region was measured and shown in FIG. 2. As shown in FIG. 2, it can be seen that the emission intensity increases as the concentration of the PPi anion increases.

Experimental Example 2 Comparison of the Detection Performance of the Supermolecular Complexes According to the Kinds of Anions

Chromenolate complexes having an anion (X) of F ⁻ , H ₂ PO ₄ ⁻ , CH ₃ COO ⁻ , Cl ⁻ , HSO ₄ ⁻ , or Br ^- were adjusted to acetonitrile to concentrations of 2.1 μM and 4.8 μM, respectively. . After adding dropwise tri (tetrabutylammonium) hydrogen pyrophosphate to 1 to 4.9 μM, the emission intensity in the 410 nm wavelength region was measured and shown in FIG. 3.

As shown in FIG. 3, although there are some differences in luminescence intensity depending on the type of anion (X), it can be seen that PPi anions can be detected with high sensitivity.

Experimental Example 3 Luminescence Intensity According to PPi Anion Concentration

Chromenolate complex synthesized in Example 5 was adjusted to acetonitrile to a concentration of 2.1 μM and 4.8 μM, respectively. The tri (tetrabutylammonium) hydrogen pyrophosphate was added dropwise while changing the concentration from 0 to 110 nM, and the emission intensity in the 410 nm wavelength region was measured and shown in FIG. 4. In addition, the correlation between the concentration of the hydrogen pyrophosphate anion and the luminescence intensity is shown graphically in FIG. 5. As shown in Figure 5 it can be seen that the concentration of PPi anion and the emission intensity in the range of 0 to 110 nM concentration is directly proportional.

Experimental Example 4 Stability Constant Using Changes of Absorbance in the UV-vis Wavelength Range

Chromenolate complex synthesized in Example 5 was adjusted to a concentration of 0 to 33.6 μM in acetonitrile. Tri (tetrabutylammonium) hydrogen pyrophosphate was added dropwise while changing to a concentration of 0 to 35.8 μM, and then absorbance in the UV-vis wavelength range was measured and shown in FIGS. 6 and 7.

6 and 7 show that the formation of complexes with hydrogen pyrophosphate is reversible and complexes are generated in proportion to concentration.

In addition, when the cromenoleate complex reacts with the anion shown in Table 1 in the UV-vis wavelength region, the cromenoleate anion bound to the complex is released and ion exchanged into another anion in place. Table 1 shows the binding strength of the anion (X) bound to the supramolecular complex of Formula 1 by measuring the stability constant (K _a ).

Anion (X) Coupling Constants (K _a , M ^-1 ) HP ₂ O ₇ ^3- (2.55 ± 0.12) × 10 ⁷ Chromenolate Anion (7.25 ± 0.12) × 10 ⁶ F ^- (6.94 ± 0.12) × 10 ⁶ H ₂ PO ₄ ^- (6.71 ± 0.11) × 10 ⁶ CH ₃ COO ^- (4.29 ± 0.09) × 10 ⁵ Cl ^- (4.02 ± 0.11) × 10 ⁵ HSO ₄ ^- (5.21 ± 0.46) × 10 ⁴ Br ^- (4.72 ± 0.44) × 10 ⁴ Acetonitrile solvent, 25 ° C. temperature, concentration of acceptor = 1.00 × 10 ⁻⁵ M.

Experimental Example 5: Selectivity to Hydrogenpyrophosphate Anion According to Solvent

Chromenolate complex synthesized in Example 5 was adjusted to a concentration of 10 μM in acetonitrile alone solvent or 70% acetonitrile aqueous solution. Tri (tetrabutylammonium) hydrogen pyrophosphate was added dropwise at a ratio of 0 to 3 equivalents, and then absorbance in the UV-vis wavelength range was measured to investigate the change in selectivity for anions. A croissant menol rate complexes in each solvent _{^{HP 2 O 7 3-, F -}} , and H ₂ PO ₄ ^- anion when the reaction selected from among, chroman menol anion is glass and the ion exchange with an anion of the formula (1) It will form a supramolecular complex. As a result of measuring the binding constant (K _a) of this case it is shown in FIG. 9 and FIG.

8 and 9, it can be seen that the selectivity for hydrogen pyrophosphate anion is greatly increased in the aqueous solution. In other words, this means that the present invention can quantify hydrogen pyrophosphate in an aqueous solution.

Claims (6)

A supramolecular complex for detecting hydrogen pyrophosphate (PPi) anion represented by Formula 1 below.
[Chemical Formula 1]

(In Formula 1, R represents an alkyl group having 1 to 6, X ^- is halo, phosphate (PF ₆ ^{hexafluoro-),} hexachloro antimonate (SbCl ₆ ^-), thiocyanate (SCN ^-) , acetate (CH ₃ COO ^-), sulfonate (HSO ₄ ^-) represents an anion selected from a), tetrafluoroborate (BF ₄ ^{- -)} to, and perchlorate (ClO ₄₎
The method according to claim 1,
5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole polyfluoro salt,
5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole dichloro salt,
5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole diiodine salt,
5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole dibromo salt,
5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole dihexafluorophosphate salt,
5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole diacetate salt,
5,15-bis (4- N -methylpyridinium) -5,10,10,15,20,20-hexamethylcalix [4] pyrrole disulfonate salt
A supramolecular complex for detecting hydrogen pyrophosphate (PPi) anion, characterized in that selected from among.
Chromenolate complex for the detection of hydrogen pyrophosphate (PPi) anion represented by the following formula (4).
[Chemical Formula 4]

(In Formula 4, R and X ⁻ are as defined in claim 1, and R ₁ represents a trifluoromethyl group or a pentafluoroethyl group.)
Hydrogenpyrophosphate (PPi) of the hydrogen pyrophosphate (PPi) anion comprising the step of measuring the luminescence intensity of the fluorescence by adding a chromolate complex represented by the following formula (4) to a solution containing or not Detection and Quantification Methods.
[Chemical Formula 4]

(In Formula 4, R and X ⁻ are as defined in claim 1, and R ₁ represents a trifluoromethyl group or a pentafluoroethyl group.)
5. The method of claim 4,
The emission intensity is a method for detecting and quantifying hydrogen pyrophosphate (PPi) anion, characterized in that for measuring the intensity of light of 450 ~ 550 nm wavelength.
Preparing a compound represented by the following Chemical Formula 3 by reacting the compound represented by the following Chemical Formula 2 with trifluoroborane (BF ₃ ) at room temperature and in a mixed solvent of acetone and ethanol;

Reacting a compound represented by Chemical Formula 3 with an alkyl halide compound to prepare a supramolecular complex represented by Chemical Formula 1 (X = halo) in which an alkyl group (R) is substituted at the N 1 position of pyridine; And

(In the above scheme, R represents an alkyl group having 1 to 6 carbon atoms, and X ⁻ represents a halo anion.)
Ion-exchange the supramolecular complex represented by Formula 1 wherein X ⁻ is a halo anion, and X ⁻ is PF ₆ ⁻ , SbCl ₆ ⁻ , _{^{SCN -, CH 3 COO -,}} HSO 4 -, BF 4 -, or ClO ₄ ^- process for preparing a supramolecular complex compound represented by the above formula (1);
Method of producing a supramolecular complex, characterized in that it comprises a.

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