A pyrophosphate indicator and its preparation method and application

Abstract

The present invention provides a pyrophosphate indicator, a preparation method, and applications thereof, relating to the field of measurement and detection technology. The pyrophosphate indicator of the present invention contains a Schiff base structure that can electrostatically interact with pyrophosphate, enhancing fluorescence at 370 nm and weakening fluorescence at 450 nm, thereby enabling qualitative and quantitative detection of pyrophosphate. The indicator exhibits high selectivity, high sensitivity, and a low detection limit for pyrophosphate, and also exhibits strong stability.

Description

Technical Field

[0001] The present invention relates to the technical field of determination and detection, and in particular to a pyrophosphate indicator and a preparation method and application thereof. Background Art

[0002] Inorganic pyrophosphate (PPi), also known as diphosphate or bisphosphate, participates in important physiological processes such as energy conversion and metabolism in organisms and is a product or essential substrate for many biochemical reactions. PPi is produced in enzymatic reactions such as ATP hydrolysis, DNA and RNA polymerization, cyclic AMP formation, and the enzymatic activation of fatty acids to form their CoA esters. It plays an irreplaceable role in life sciences, environmental sciences, pharmaceuticals, and chemical processes. The presence of PPi in joint fluid, plasma, and urine prevents calcification, and PPi in extracellular fluid can inhibit the formation of hydroxyapatite. Therefore, the PPi content in plasma, serum, and other biological fluids is a crucial indicator in the study of bone metabolism, kidney stone disease, and arthritis treatment.

[0003] Given the significant research value of PPi, its detection has become a research hotspot in recent years. Existing methods for PPi detection include heavy metal ion analysis, colorimetry, fluorescence, conductivity, and pH meter analysis. Pyrophosphate detection kits are commonly used commercially to test samples. However, current technology has a detection limit of 0.1 μmol / L for pyrophosphate, resulting in low sensitivity and requiring high precision from the fluorescence sensor. Summary of the Invention

[0004] The present invention aims to provide a pyrophosphate indicator and a preparation method and application thereof. The indicator can specifically respond to pyrophosphate, can perform qualitative and quantitative detection of pyrophosphate, and has a low detection limit, high sensitivity, and a certain stability.

[0005] In a first aspect, the present invention provides a pyrophosphate indicator having a structure as shown in formula (I):

[0006]

[0007] The pyrophosphate indicator provided by the present invention contains a Schiff base structure. When the sample to be tested contains pyrophosphate, the pyrophosphate will generate an electrostatic interaction with the -C=N- group in the indicator. During fluorescence measurement, an emission peak can be detected at 370nm. At the same time, the fluorescence at 370nm is enhanced and the fluorescence at 450nm is weakened, thereby achieving qualitative and quantitative detection of pyrophosphate.

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned pyrophosphate indicator, comprising the following steps:

[0009] In an alkaline solvent environment, in the presence of a catalyst, 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid undergo a coupling reaction, followed by extraction to obtain 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine;

[0010] In a polar organic solvent, pyrophosphate indicator was prepared by nucleophilic addition of 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine to pyridine-2,6-dicarbaldehyde.

[0011] The method for preparing a pyrophosphate indicator provided by the present invention is simple to operate and highly efficient.

[0012] Optionally, during the coupling reaction of 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid, the reaction atmosphere is a non-oxidizing atmosphere, thereby reducing the influence of oxygen in the air.

[0013] Optionally, when 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid are subjected to a coupling reaction, a weak base is present in the alkaline solvent environment, and the weak base includes at least one of potassium carbonate, sodium carbonate, and sodium bicarbonate. The use of a strong base will produce excessive by-products.

[0014] Optionally, when the coupling reaction is carried out between 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid, the basic solvent environment includes tetrahydrofuran.

[0015] Optionally, when 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid undergo coupling reaction, the catalyst includes triphenylphosphine palladium.

[0016] Optionally, when 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid are subjected to a coupling reaction, 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid are mixed in an alkaline solvent environment in advance, and then refluxed at 80-90° C. for 6-8 h.

[0017] Optionally, in the process of extracting 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine, ethyl acetate, dichloromethane or chloroform is used to extract and separate 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine.

[0018] Optionally, when 4(9H-carbazol-9-yl)phenyl)pyridin-2-amine reacts with pyridine-2,6-dicarbaldehyde, the polar organic solvent includes at least one of methanol and ethanol.

[0019] Optionally, when 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine and pyridine-2,6-dicarbaldehyde react, the molar ratio of the 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine is (2.1-2.3):1.

[0020] Optionally, the nucleophilic addition of 4-(4-4(9H-carbazole-9-yl)phenyl)pyridin-2-amine and pyridine-2,6-dicarbaldehyde comprises: mixing 4-(4-4(9H-carbazole-9-yl)phenyl)pyridin-2-amine and pyridine-2,6-dicarbaldehyde, and reacting at 65-75° C. for 12-14 hours.

[0021] In a third aspect, the present invention provides use of the pyrophosphate indicator prepared by any of the above optional preparation methods in pyrophosphate detection.

[0022] The pyrophosphate indicator provided by the present invention can specifically respond to pyrophosphate when used for the detection of pyrophosphate, can perform qualitative and quantitative detection of pyrophosphate, has a low detection limit, high sensitivity, and has certain stability.

[0023] Optionally, applications include qualitative and quantitative detection.

[0024] Optionally, the qualitative detection of pyrophosphate includes: mixing the sample to be tested with an indicator and then performing fluorescence measurement; when an emission peak is detected at 370 nm, the sample to be tested contains pyrophosphate.

[0025] Optionally, the quantitative detection of pyrophosphate includes: measuring the fluorescence intensity after mixing the sample to be tested with the indicator, calculating the fluorescence ratio of the fluorescence intensity at 370 nm to the fluorescence intensity at 450 nm, and calculating the concentration of pyrophosphate in the sample to be tested based on the fluorescence ratio and a titration curve; wherein the titration curve is a relationship curve between the molar ratio of the pre-determined pyrophosphate standard solution and the indicator and the fluorescence ratio.

[0026] Optionally, the molar ratio of pyrophosphate to indicator in the titration curve is 1:1.

[0027] Optionally, the linear indication range of pyrophosphate in the sample to be tested is 4 to 11 μmol / L, and when σ = 0.2, the detection limit of pyrophosphate is 4.989×10 -3 μ mol / L . BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly introduce the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0029] Figure 1 The structural formula of the pyrophosphate indicator provided by the present invention;

[0030] Figure 2 A schematic diagram of the process of detecting pyrophosphate using the pyrophosphate indicator provided by the present invention;

[0031] Figure 3 The synthetic route of the pyrophosphate indicator provided by the present invention;

[0032] Figure 4 This is the H NMR spectrum of the pyrophosphate indicator in Example 1 of the present invention;

[0033] Figure 5 This is a graph showing the fluorescence intensity changes of the pyrophosphate indicator prepared in Example 1 of the present invention in response to different concentrations of pyrophosphate;

[0034] Figure 6 Calculation of the detection limit for a solution of 10 μmol / L indicator and 0-15 μmol / L pyrophosphate in the performance test of the present invention;

[0035] Figure 7 is the test result of the selectivity of the indicator to pyrophosphate in the selectivity test of the present invention;

[0036] Figure 8 This is the test result of the time stability of the pyrophosphate indicator in the stability test of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described through the following examples in conjunction with the accompanying drawings. However, the following examples are illustrative and are only used to specifically describe the present invention, rather than to limit the present invention.

[0038] See also Figure 1 The present invention provides a pyrophosphate indicator having a structural formula as shown in formula (1):

[0039]

[0040] The present invention also provides a method for preparing a pyrophosphate indicator, comprising the following steps:

[0041] S1. In an alkaline solvent environment, in the presence of a catalyst, 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid undergo a coupling reaction, followed by extraction to obtain 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine;

[0042] S2. In a polar organic solvent, 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine is subjected to nucleophilic addition reaction with pyridine-2,6-dicarbaldehyde to obtain a pyrophosphate indicator.

[0043] In some embodiments, when performing the coupling reaction in step S1, the reaction atmosphere is a non-oxidizing atmosphere. Specifically, the non-oxidizing atmosphere can be an inert gas atmosphere, a nitrogen atmosphere, or a mixed atmosphere of nitrogen and inert gas.

[0044] In some embodiments, when performing step S1, the alkaline solvent environment is a weak base environment. Specifically, the weak base can be potassium carbonate, sodium carbonate, or sodium bicarbonate.

[0045] In some embodiments, when performing step S1, the alkaline solvent environment includes tetrahydrofuran.

[0046] In some embodiments, the catalyst used in step S1 includes triphenylphosphine palladium.

[0047] In fact, when executing step S1, the following sub-steps are included:

[0048] S1.1. Place 4-bromo-2-aminopyridine, 4-(9H-carbazol-9-yl)phenylboronic acid, a weak base, and a catalyst in a solvent environment, stir and mix, and reflux in a non-oxidizing atmosphere at 80-90°C for 6-8 hours;

[0049] S1.2. After the reaction is completed, extraction is performed and the mixture is dried by spin column to separate the obtained 4-(4-(9H-carbazol-9-yl)phenyl)pyridin-2-amine.

[0050] In some embodiments, when performing step S2, the polar organic solvent used includes at least one of methanol and ethanol.

[0051] In some embodiments, when performing the nucleophilic addition in step S2 , 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine and pyridine-2,6-dicarbaldehyde are mixed at a molar ratio of (2.1-2.3):1.

[0052] In fact, when executing step S2, the following sub-steps are included:

[0053] S2.1. Add 4-(4-(9H-carbazol-9-yl)phenyl)pyridin-2-amine and pyridine-2,6-dicarbaldehyde (CAS: 5431-44-7) to a polar organic solvent and reflux at 65-75°C for 12-14 hours.

[0054] S2.2. After the reaction is completed, cool and filter, wash the filter cake, and dry it to obtain a pyrophosphate indicator.

[0055] The present invention also provides the use of the pyrophosphate indicator in pyrophosphate detection, including qualitative detection and quantitative detection:

[0056] (1) Qualitative detection of pyrophosphate includes: mixing a sample to be tested with an indicator and then performing fluorescence measurement. When an emission peak is detected at 370 nm, the sample to be tested contains pyrophosphate.

[0057] (2) Quantitative detection of pyrophosphate includes: mixing the sample to be tested with the indicator and then measuring the fluorescence intensity, calculating the fluorescence ratio of the fluorescence intensity at 370 nm to the fluorescence intensity at 450 nm, and calculating the concentration of pyrophosphate in the sample to be tested based on the fluorescence ratio and a titration curve; wherein the titration curve is a curve showing the relationship between the molar ratio of the pyrophosphate standard solution and the indicator and the fluorescence ratio determined in advance.

[0058] In some embodiments, when performing application (2), the molar ratio of pyrophosphate to indicator in the titration curve is 1:1.

[0059] In some embodiments, when performing application (2), the linear indication range of pyrophosphate in the sample to be tested is 4 to 11 μmol / L, and when σ=0.2, the detection limit of pyrophosphate is 4.989×10 -3 μ mol / L .

[0060] The schematic diagram of the process of detecting pyrophosphate by the pyrophosphate indicator provided by the present invention is as follows Figure 2 shown.

[0061] Example 1

[0062] See also Figure 3 The method for preparing a pyrophosphate indicator provided in Example 1 of the present invention comprises the following steps:

[0063] S1.1. Add 0.3142 g (1.816 mmol) of 4-bromo-2-aminopyridine (CAS: 84249-14-9), 1.044 g (7.568 mmol) of potassium carbonate, 4.434 mg (w=1%) of triphenylphosphine palladium (CAS: 14221-01-3), and 0.4344 g (1.513 mmol) of 4-(9H-carbazol-9-yl)phenylboronic acid (CAS: 419536-33-7) to a 50 mL side-mouth flask, add 10 mL of tetrahydrofuran and 2 mL of deionized water, and reflux at 90°C under a nitrogen atmosphere for 6 h.

[0064] S1.2. After the reaction was completed, 50 mL of saturated brine was added to quench the reaction. The mixture was extracted three times with 25 mL of dichloromethane. The combined extracts were dried and passed through a column (SiO2, silicone rubber, eluent: a mixture of petroleum ether and ethyl acetate in a volume ratio of 10:1) to obtain 1 g (2.985 mmol) of 4-(4-(9H-carbazol-9-yl)phenyl)pyridin-2-amine. The calculated yield was 83%.

[0065] S2.1. Add 1 g (2.985 mmol) of 4-(4-(9H-carbazol-9-yl)phenyl)pyridin-2-amine and 0.1918 g (1.421 mmol) of pyridine-2,6-dicarbaldehyde (CAS: 5431-44-7) to a 50 mL side-necked flask, add 25 mL of ethanol, and reflux at 70°C for 12 h.

[0066] S2.2. After the reaction is completed, the mixture is cooled and filtered, the filter cake is washed with ethanol, and the filter cake is dried to obtain 0.9129 g of a light yellow pyrophosphate indicator (KZ-Py-Schiff). The calculated yield is 76.6%.

[0067] KZ-Py-Schiff was characterized by H NMR spectroscopy. Figure 4 As shown, its characterization data is: 1 H NMR (400MHz, DMSO-d6) 68.03 (s, 2H), δ 7.91 (s, 1H), 7.57-7.52 (m, 5H), 7.30 (s, 10H), 7.08-6.97 (m, 14H), 6.92-6.85 (m, 3H).

[0068] Performance testing

[0069] The response of the indicator KZ-Py-Schiff to pyrophosphate:

[0070] Prepare 2 mL of 10 μmol / L indicator solution with pure water, add pyrophosphate to the indicator solution, and perform fluorescence measurement after adding 1 μmol / L of pyrophosphate until saturation is reached. The fluorescence intensity changes as the pyrophosphate concentration increases. Figure 5 As shown, the results showed that the emission peak at 370 nm increased with the increase of pyrophosphate content in the solution, while the emission peak at 450 nm decreased with the increase of pyrophosphate content in the solution, and reached saturation when the pyrophosphate content reached 10 μmol / L.

[0071] According to the different pyrophosphate concentrations and the corresponding fluorescence intensity ratio I 370 / I 450 Draw the titration curve, the result is as follows Figure 6 As shown, from Figure 6It can be seen that in the range of 4 to 11 μmol / L, the ratio of pyrophosphate to fluorescence intensity I 370 / I 450 It has a good linear relationship and can be used for quantitative detection of pyrophosphate. It is calculated that when σ = 0.2, the detection limit of the pyrophosphate indicator is 4.989×10 -3 μmol / L.

[0072] Optional testing

[0073] Selective test of indicator KZ-Py-Schiff for pyrophosphate (PPi):

[0074] Use pure water to prepare a solution containing 10 μmol / L indicator KZ-Py-Schiff, and add 550 μmol / L of PPi, Pi, mPPi, F - 、Cl - Br - , I - 、CO3 2- 、SO4 2- 、CA、NO3 - Anions. Figure 7 As shown, the fluorescence spectra after adding the above anions to the indicator were measured. The results showed that after adding PPi to the indicator, the emission peak at 370nm increased, while the emission peak at 450nm decreased. After adding other anions, the emission peak at 450nm decreased, while the emission peak at 370nm did not change significantly, indicating that the indicator KZ-Py-Schiff has excellent selectivity for pyrophosphate.

[0075] Stability testing

[0076] Stability test of indicator KZ-Py-Schiff:

[0077] Use pure water to prepare a solution containing 10 μmol / L indicator KZ-Py-Schiff and measure the fluorescence intensity from 0 to 10 hours. Figure 8 As shown, the results showed that the fluorescence intensity of KZ-Py-Schiff had no obvious fluctuation from 0 to 10 h, indicating that the indicator KZ-Py-Schiff has excellent stability in aqueous medium.

[0078] While the embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations of these embodiments are possible. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the claims. Furthermore, the invention described herein is susceptible to other embodiments and may be practiced or implemented in a variety of ways.

Claims

1. A pyrophosphate indicator having a structure as shown in formula (I): 。 2. A method for preparing a pyrophosphate indicator as claimed in claim 1, characterized in that: The following steps are involved: In an alkaline solvent environment, in the presence of a catalyst, 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid undergo a coupling reaction, followed by extraction to obtain 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine; A pyrophosphate indicator was prepared by nucleophilic addition of 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine to pyridine-2,6-dicarbaldehyde in a polar organic solvent.

3. The method for preparing a pyrophosphate indicator according to claim 2, wherein When 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid undergo a coupling reaction: the reaction atmosphere is a non-oxidizing atmosphere; or, the alkaline solvent environment contains a base, and the base is at least one of potassium carbonate, sodium carbonate, and sodium bicarbonate; or, the alkaline solvent environment is tetrahydrofuran; or, the catalyst is triphenylphosphine palladium.

4. The method for preparing a pyrophosphate indicator according to claim 2, wherein In the coupling reaction of 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid, 4-bromo-2-aminopyridine and 4-(9H-carbazol-9-yl)phenylboronic acid are mixed in advance in an alkaline solvent environment and refluxed at 80-90°C for 6-8 hours.

5. The method for preparing a pyrophosphate indicator according to claim 2, wherein In the process of extracting 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine, ethyl acetate, dichloromethane or chloroform is used for extraction and separation.

6. The method for preparing a pyrophosphate indicator according to claim 2, wherein When (4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine reacts with pyridine-2,6-dicarbaldehyde: the polar organic solvent is at least one of methanol and ethanol; or, 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine and pyridine-2,6-dicarbaldehyde react at a molar ratio of (2.1-2.3):

1.

7. The method for preparing a pyrophosphate indicator according to claim 2, wherein The nucleophilic addition reaction of 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine and pyridine-2,6-dicarbaldehyde comprises: mixing 4-(4-4(9H-carbazol-9-yl)phenyl)pyridin-2-amine and pyridine-2,6-dicarbaldehyde and reacting the mixture at 65-75° C. for 12-14 hours.

8. Use of the pyrophosphate indicator according to claim 1 or the pyrophosphate indicator prepared by the preparation method according to any one of claims 2 to 7 in preparing a reagent for pyrophosphate detection, characterized in that: Includes qualitative testing and quantitative testing.

9. The use according to claim 8, characterized in that The qualitative detection of pyrophosphate includes: mixing the sample to be tested with an indicator and then performing fluorescence measurement. When an emission peak is detected at 370 nm, the sample to be tested contains pyrophosphate.

10. The use according to claim 8, characterized in that The quantitative detection of pyrophosphate includes: mixing a sample to be tested with an indicator, measuring the fluorescence intensity, calculating the fluorescence ratio of the fluorescence intensity at 370 nm to the fluorescence intensity at 450 nm, and calculating the concentration of pyrophosphate in the sample to be tested based on the fluorescence ratio and a titration curve; wherein the titration curve is a relationship curve between the molar ratio of a pre-determined pyrophosphate standard solution and the indicator and the fluorescence ratio; the molar ratio of pyrophosphate to the indicator in the titration curve is 1:1; the linear indication range of pyrophosphate in the sample to be tested is 4 to 11 μmol / L, and when σ=0.2, the detection limit of pyrophosphate is 4.989×10 -3 μmol / L.

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