A tetrazole heterocyclic complex containing an N-phenylcarbazole fluorophore and Tb(III) complex, its synthesis method and application

Abstract

This invention provides a tetrazole heterocycle containing an N-phenylcarbazole fluorophore and a Tb(III) complex, along with its synthesis method and applications, relating to the technical field of ATP fluorescent probes. The invention uses N-phenylcarbazole as an energy donor, linking it to a tetrazole heterocycle with three amide arms via an amide bond to obtain the ligand CBP-Cy, which is then combined with Tb... 3+ The reaction yielded a tetrazole heterocyclic Tb(III) complex containing an N-phenylcarbazole fluorophore, namely CBP-Cy-Tb. This complex exhibits enhanced fluorescence response to recognize ATP under physiological conditions, with a detection limit as low as 3.02 nM. CBP-Cy-Tb can be used to prepare ratiometric fluorescent probes for the quantitative detection of ATP in cells. This invention offers advantages such as inexpensive raw materials, simple synthesis process, easy separation procedure, high yield, and stable and easy storage. The complex fluorescent probe of this invention specifically activates a fluorescence response with ATP, enabling the quantitative detection of ATP in cells and its application in disease diagnosis, demonstrating excellent prospects for biological applications.

Description

Technical Field

[0001] This invention relates to the technical field of ATP fluorescent probes, and more particularly to a tetrazole heterocyclic complex containing an N-phenylcarbazole fluorophore and Tb(III) complex, its synthesis method, and its application. Background Technology

[0002] Adenosine triphosphate (ATP) exists within cells as a coenzyme and is the smallest molecular unit for energy transfer processes within cells. ATP plays a vital physiological role in cells, providing the power for most cellular activities, such as the synthesis and transport of biomolecules, cell signaling, maintaining cell structure, DNA or RNA biosynthesis, and cell contraction. Normally, a healthy adult body contains an average of 250g of ATP. Abnormal ATP levels are often associated with various diseases, such as cardiovascular disease, ischemia, tissue hypoxia, hypoglycemia, Parkinson's disease, cancer, and inflammation.

[0003] Currently, commonly used methods for ATP detection include bioluminescence, chromatography, fluorescence, and electrochemical methods. Among these methods, fluorescent probes are considered powerful tools for identifying ATP due to their advantages such as simple operation, high sensitivity, low detection limit, and high temporal resolution. Since ATP detection and monitoring are crucial for understanding many biological processes, fluorescent probes need to possess high selectivity and sensitivity for rapid ATP detection.

[0004] Lanthanide ions possess large Stokes shifts, narrow ffr transition spectral bands, and long fluorescence lifetimes, effectively avoiding interference from background fluorescence and improving signal-to-noise ratio and detection sensitivity. Therefore, their unique photophysical properties have led to their extensive research in fluorescence detection and imaging. However, ATP fluorescent probes carrying lanthanide ions are prone to aggregation in water due to their rigid, flat structure, leading to quenching and fluorescence loss. Therefore, a solution is urgently needed to address these issues. Summary of the Invention

[0005] The purpose of this invention is to provide a tetrazole heterocyclic complex containing an N-phenylcarbazole fluorophore and Tb(III) complex, its synthesis method, and its application.

[0006] In a first aspect, the present invention provides a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III), the structural formula of which is shown in Formula I:

[0007] .

[0008] The beneficial effect of the tetrazole heterocyclic N-phenylcarbazole complex with Tb(III) provided by this invention is that N-phenylcarbazole belongs to aggregation-induced emission dyes and can be used as photosensitizers, through energy transfer to Tb. 3+ Fluorescence occurs on the ions, and the complex exhibits fluorescence based on Tb due to the presence of coordinated water molecules. 3+ The fluorescence intensity at 547 nm is very weak, but in the presence of ATP, it displaces water molecules, thus making the Tb-based fluorescence... 3+ The fluorescence intensity is enhanced at 547 nm, thereby achieving the purpose of ATP recognition.

[0009] At the same time, due to Tb 3+ One of the coordination sites is occupied by a water molecule that has fluorescence quenching properties. When ATP is recognized, the ATP replaces the coordination site where the water molecule is located, which can also prevent the complex from exhibiting fluorescence quenching.

[0010] Secondly, the present invention provides a method for synthesizing a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III), comprising the following steps: in the presence of a solvent and under alkaline conditions, 4-aminoN-phenylcarbazole is amidated and nucleophilically substituted, followed by a complex with Tb(III). 3+ Coordination was performed to obtain the complex shown in Formula I.

[0011] The advantages of the method for synthesizing a tetrazole heterocycle containing N-phenylcarbazole and a Tb(III) complex provided by the present invention are that the raw materials are inexpensive and the synthesis process is simple.

[0012] Optionally, the process includes the following steps: In a first solvent and alkaline environment, 4-amino-N-phenylcarbazole is amidated with bromoacetyl bromide to obtain compound one; in a second solvent and alkaline environment, compound one is nucleophilically substituted with cyclopentadiene to obtain compound two, and compound two is amidated to obtain the precursor; in a third solvent and alkaline environment, the precursor is reacted with Tb... 3+ The complex shown in Formula I was obtained after coordination. The advantages are: the separation procedure is simple, the yield is high, and the obtained intermediate and product complexes are stable and easy to store.

[0013] Optionally, the process of preparing compound one by amidation of 4-aminoN-phenylcarbazole and bromoacetyl bromide includes: dissolving 4-aminoN-phenylcarbazole and a first basic promoter in a first solvent to prepare a first mixture; adding bromoacetyl bromide solution dropwise to the first mixture at 0±5℃ to prepare a first reaction solution; heating the first reaction solution to 25±5℃ and stirring for 6-10 hours, then separating and purifying to obtain compound one.

[0014] Optionally, after adding a bromoacetyl bromide solution to the first mixture to prepare the first reaction solution, the molar ratio of 4-aminoN-phenylcarbazole to bromoacetyl bromide in the first reaction solution is 1:(1-1.5).

[0015] Optionally, the process of nucleophilic substitution of compound 1 with cyclotinocyanine to obtain compound 2 includes: dissolving compound 1, cyclotinocyanine and a second basic promoter in a second solvent to obtain a second mixture; and then separating and purifying the second mixture after stirring and reflux for 46-50 hours to obtain compound 2.

[0016] Optionally, the first catalyst is added to the second mixture before the second mixture is stirred and refluxed.

[0017] Optionally, after dissolving compound one, cyclohexane and the second basic promoter in the second solvent to prepare the second mixture, the molar ratio of compound one to cyclohexane in the second mixture is 1:(4-4.5).

[0018] Optionally, the process of preparing the precursor by amidation of compound 2 includes: dissolving compound 2, bromoacetamide and a third basic promoter in a second solvent to prepare a third mixture; and then separating and purifying the third mixture after stirring and reflux for 46-50 hours to obtain the precursor.

[0019] Optionally, a second catalyst may be added to the third mixture before the third mixture is stirred and refluxed.

[0020] Optionally, after dissolving compound 2, bromoacetamide and the third alkaline promoter in the second solvent to prepare the third mixture, the molar ratio of compound 2 to bromoacetamide in the third mixture is 1:(3-3.2).

[0021] Optionally, the precursor and Tb 3+ The process of preparing the complex shown in Formula I after coordination includes: dissolving the precursor in a third solvent to prepare a fourth mixture; adding TbCl3 solution dropwise to the fourth mixture to prepare a second reaction solution; heating the second reaction solution under reflux for 12-24 hours and then separating and purifying it to obtain the complex shown in Formula I.

[0022] Optionally, after adding TbCl3 solution dropwise to the fourth mixture to prepare the second reaction solution, the precursor in the second reaction solution reacts with TbCl3. 3+ The molar ratio is 1:(1-1.2).

[0023] Alternatively, the reaction formula is as follows:

[0024] .

[0025] Thirdly, this invention also provides an application of a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III) in ATP recognition. The beneficial effects are: it can exhibit a specific fluorescent response to ATP and can perform quantitative and qualitative detection of ATP, showing excellent prospects for biological applications. Attached Figure Description

[0026] Figure 1 This is a flowchart of a method for synthesizing a tetrazole heterocyclic N-phenylcarbazole complex with Tb(III) provided in an embodiment of the present invention;

[0027] Figure 2 The image shows the hydrogen nuclear magnetic spectrum (CDCl3) of compound 1 synthesized in Example 1 of this invention.

[0028] Figure 3 The 1H NMR spectrum of compound 2 synthesized in Example 1 of this invention (Acetonitrile-d3).

[0029] Figure 4 This is the carbon NMR spectrum (Acetonitrile-d3) of compound 2 synthesized in Example 1 of the present invention.

[0030] Figure 5 The 1H NMR spectrum (Acetonitrile-d3) of the precursor (CBP-Cy) synthesized in Example 1 of this invention.

[0031] Figure 6 The carbon NMR spectrum (Acetonitrile-d3) of the precursor (CBP-Cy) synthesized in Example 1 of this invention.

[0032] Figure 7 The fluorescence spectra of the precursor (CBP-Cy) and complex (CBP-Cy-Tb) synthesized in Example 1 of this invention are shown.

[0033] Figure 8 The fluorescence intensity change curves of the complex (CBP-Cy-Tb) synthesized in Example 1 of the present invention during fluorescence titration with different anions;

[0034] Figure 9 The fluorescence intensity curve of the complex (CBP-Cy-Tb) synthesized in Example 1 of the present invention as a function of ATP concentration during fluorescence titration with ATP concentration.

[0035] Figure 10 The curve showing the change of the fluorescence emission peak at 547 nm of the complex (CBP-Cy-Tb) synthesized in Example 1 of this invention as a function of ATP titration.

[0036] Figure 11The curve shows the relationship between the fluorescence intensity of the complex (CBP-Cy-Tb) synthesized in Example 1 of this invention and the ATP concentration (4.5 μmol / L-8.25 μmol / L). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art. The terms "comprising" and similar expressions used herein mean that the element or object preceding the word covers the element or object listed after the word and its equivalents, but does not exclude other elements or objects.

[0038] This invention provides a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III) complex, the structural formula of which is shown in Formula I:

[0039] .

[0040] N-phenylcarbazole belongs to the aggregation-induced emission dye category. It can be used as a photosensitizer and also as an energy donor to transfer energy to Tb. 3+ Fluorescence occurs on the ions, and the complex exhibits fluorescence based on Tb due to the presence of coordinated water molecules. 3+ The fluorescence intensity at 547 nm is very weak, but in the presence of ATP, it displaces water molecules, thus making the Tb-based fluorescence... 3+ The fluorescence intensity is enhanced at 547 nm, thereby achieving the purpose of ATP recognition, and due to Tb 3+ One of the coordination sites is occupied by a water molecule that has fluorescence quenching properties. When ATP is recognized, the ATP replaces the coordination site where the water molecule is located, which can also prevent the complex from exhibiting fluorescence quenching.

[0041] This invention also provides a method for synthesizing a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III), comprising the following steps: in the presence of a solvent and under alkaline conditions, 4-aminoN-phenylcarbazole is amidated and nucleophilically substituted, followed by reaction with Tb. 3+ Coordination was performed to obtain the complex shown in Formula I above.

[0042] In some embodiments, see Figure 1 The synthesis of a tetrazide heterocycle containing N-phenylcarbazole and a Tb(III) complex includes the following steps:

[0043] S1. Preparation of Compound 1: Compound 1 was prepared by amidation of 4-aminoN-phenylcarbazole and bromoacetyl bromide in a first solvent and alkaline environment.

[0044] S2. Preparation of precursor: Compound 1 was nucleophilically substituted with cyclotinocyanine in a second solvent and alkaline environment to obtain compound 2; compound 2 was amidated to obtain the precursor.

[0045] S3. Preparation of the complex: Under a third solvent and alkaline environment, the precursor reacts with Tb 3+ After coordination, the complex shown in Formula I was obtained.

[0046] In fact, after obtaining compound one, compound two and the precursor, compound one, compound two and the precursor can be stored in the dark to allow for stepwise preparation of the complex.

[0047] In some embodiments, step S1 includes the following sub-steps:

[0048] S11. Dissolve 4-aminoN-phenylcarbazole and the first alkaline accelerator in the first solvent to prepare the first mixture;

[0049] S12. Add bromoacetyl bromide solution dropwise to the first mixture at 0±5℃ to obtain the first reaction solution;

[0050] S13. The first reaction solution is heated to 25±5℃ and stirred for 6-10 hours. After separation and purification, compound one is obtained.

[0051] In some embodiments, when performing sub-step S11, the first alkaline promoter can be one or a mixture of any of KOH, NaOH, K2CO3, Cs2CO3, and Na2CO3. In practice, the first alkaline promoter can be an inorganic alkaline promoter conventionally used in the art, as long as it is necessary to provide an alkaline environment for the reaction system.

[0052] In some embodiments, the first solvent may be chloroform, acetonitrile, or methanol when performing sub-step S11. In practice, the first solvent may be an organic solvent conventionally used in the art, provided that it does not react with the reactants and products and can dissolve the reactants.

[0053] Specifically, in sub-step S11, 4-aminoN-phenylcarbazole is dissolved in the first solvent, then the first alkaline accelerator is added, and the mixed solution is vigorously stirred to ensure that 4-aminoN-phenylcarbazole and the first alkaline accelerator are completely dissolved and fully mixed, thereby obtaining the first mixed solution.

[0054] In some embodiments, the solvent in the bromoacetyl bromide solution is the first solvent when performing sub-step S12.

[0055] Specifically, in performing sub-step S12, the amount of bromoacetyl bromide solution added is necessary to ensure that the molar ratio of 4-aminoN-phenylcarbazole to bromoacetyl bromide in the first reaction solution is 1:(1-1.5).

[0056] Specifically, in sub-step S12, after cooling the first mixed solution to 0±5℃, bromoacetyl bromide solution is added dropwise to the first mixed solution, and the dropwise addition time of bromoacetyl bromide solution is controlled to be 30 min, thereby obtaining the first reaction solution.

[0057] In fact, during the dropwise addition of the bromoacetyl bromide solution in sub-step S12, the first mixture is continuously stirred during the dropwise addition process so that the bromoacetyl bromide can be evenly distributed.

[0058] In some embodiments, the separation and purification techniques used in performing sub-step S13 include washing, extraction, filtration, drying, recrystallization, and column chromatography. In practice, conventional separation and purification techniques used in the art, including but not limited to those described above, can be employed when separating and purifying compound one.

[0059] In some embodiments, during step S13, the first reaction solution is heated to 25±5°C by stirring in a water bath, then kept at this temperature and stirred for 6-10 hours. After the reaction is complete, the solvent is removed by rotary evaporation and passed through V 石油醚 V 二氯甲烷 Compound 1 was obtained by separation and purification using silica gel chromatography with a ratio of 5:1.

[0060] Specifically, after performing step S1 to obtain compound one, compound one is a pale yellow solid compound.

[0061] Specifically, the following reaction occurs during step S1:

[0062] .

[0063] In some embodiments, the process of obtaining compound two by performing step S2 includes the following sub-steps:

[0064] S21. After dissolving compound one, cyclohexanetin and the second basic accelerator in the second solvent, a second mixture is prepared.

[0065] S22. The second mixture was stirred and refluxed for 46-50 hours, and then separated and purified to obtain compound two.

[0066] In some embodiments, when performing sub-step S21, the second alkaline promoter may be the same substance as the first alkaline promoter, or it may be an inorganic alkaline promoter different from the first alkaline promoter, in order to achieve the necessary provision of an alkaline environment for the reaction system.

[0067] In some embodiments, when performing sub-step S21, the second solvent may be the same substance as the first solvent, or it may be an organic solvent different from the first solvent, as long as it does not react with the reactants and products and can dissolve the reactants.

[0068] Specifically, in sub-step S21, compound one and cyclohexanetin are added to the second solvent and stirred to dissolve. Then, the second alkaline promoter is added to the second solvent and stirred to make the three substances uniformly dispersed in the second solvent, thereby obtaining the second mixture.

[0069] In some embodiments, after performing sub-step S21, the molar ratio of compound one to cyclohexane in the second mixture is 1:(4-4.5).

[0070] In some embodiments, a first catalyst is added to the second mixture before performing step S22, thereby increasing the reaction rate.

[0071] Specifically, KI is selected as the first catalyst.

[0072] Specifically, the amount of the first catalyst added to the second mixture is necessary to make the molar ratio of the first catalyst to compound one 1:(0.9-1.1).

[0073] In some embodiments, during the separation and purification of compound two in step S22, the reaction solution after the reflux reaction is completed is filtered and the solvent is rotary evaporated, and then passed through V 二氯甲烷 V 甲醇 Compound II was obtained by separation and purification using silica gel chromatography with a ratio of 20:1.

[0074] Specifically, after performing step S22 to obtain compound two, compound two is a white solid compound.

[0075] In some embodiments, the process of amidating compound two in step S2 to obtain the precursor includes the following sub-steps:

[0076] S23. After dissolving compound II, bromoacetamide and the third alkaline accelerator in the second solvent, a third mixture is prepared.

[0077] S24. After stirring and refluxing the third mixture for 46-50 hours, the precursor is obtained by separation and purification.

[0078] In some embodiments, when performing step S23, the third alkaline promoter can be the same substance as the first alkaline promoter and the second alkaline promoter, or it can be an inorganic alkaline promoter that is different from the first alkaline promoter and the second alkaline promoter, so as to achieve the necessary provision of an alkaline environment for the reaction system.

[0079] Specifically, in step S23, compound two and bromoacetamide are dissolved in the second solvent, and then the third alkaline promoter is dissolved in the second solvent. The mixture is stirred continuously to ensure that the three substances are evenly dispersed in the second solvent, thereby obtaining the third mixture.

[0080] In some embodiments, after performing step S23, the molar ratio of compound 2 to bromoacetamide in the third mixture is 1:(3-3.2).

[0081] In some embodiments, a second catalyst is added to the third mixture before performing step S24. Specifically, the second catalyst may be the same substance as the first catalyst.

[0082] Specifically, the amount of the second catalyst added to the third mixture is necessary to make the molar ratio of the second catalyst to compound two 1:(0.9-1.1).

[0083] In some embodiments, during the separation and purification of the precursor in step S24, the reaction solution after the reflux reaction is completed is filtered and the solvent is rotary evaporated, and then passed through V 二氯甲烷 V 甲醇 The precursor was obtained by separation and purification using silica gel chromatography at a ratio of 20:1.

[0084] Specifically, after performing step S24 to obtain the precursor, the precursor is a pale yellow solid compound.

[0085] Specifically, during step S2, the following reaction occurs:

[0086] .

[0087] In some embodiments, step S3 includes the following sub-steps:

[0088] S31. Dissolve the precursor in a third solvent to prepare a fourth mixture;

[0089] S32. Add TbCl3 solution dropwise to the fourth mixture to prepare the second reaction solution;

[0090] S33. After heating the second reaction solution under reflux for 12-24 hours, separate and purify it to obtain the complex shown in Formula I.

[0091] In some embodiments, when performing sub-step S31, the third solvent is the same substance as the first solvent and the second solvent, or an organic solvent different from the first solvent and the second solvent may be used, as long as it does not react with the reactants and products and can dissolve the reactants.

[0092] In some embodiments, after performing sub-step S32, the precursor in the second reaction solution reacts with Tb. 3+ The molar ratio is 1:(1-1.2).

[0093] In some embodiments, during the separation and purification process in step S33, the refluxed solvent is evaporated to 1 / 5 of its original volume, and then diethyl ether is added dropwise until the solution begins to become turbid. Afterward, the solution is transferred to an ether atmosphere and allowed to stand for 10-12 hours. The precipitate is then collected by filtration and washed with diethyl ether to obtain the complex (CBP-Cy-Tb) shown in Formula I. Specifically, the refluxed solvent is evaporated to 1 mL.

[0094] Specifically, the following reaction occurs during step S3:

[0095] .

[0096] The present invention also provides the application of the tetrazole heterocyclic Tb(III) complex (CBP-Cy-Tb) containing an N-phenylcarbazole fluorophore prepared by any of the above synthetic methods.

[0097] Specifically, CBP-Cy-Tb can be used to prepare ratiometric fluorescent probes for qualitative and quantitative detection of ATP.

[0098] Specifically, the quantitative detection of ATP using CBP-Cy-Tb includes the following steps: dissolving CBP-Cy-Tb in DMSO to prepare a 2×10⁻⁶ solution. -3 The mother liquor was prepared in mol / L, and HEPES buffer at pH 7.4 was used to prepare 20 μmol / L CBP-Cy-Tb fluorescent probe solutions. ATP-containing solutions were added to the fluorescent probe solutions, and their fluorescence intensity was tested. The ATP content was calculated based on the linear relationship.

[0099] Example 1:

[0100] This embodiment 1 provides a method for synthesizing CBP-Cy-Tb, including the following steps:

[0101] S1. Dissolve 0.7 g (2.71 mmol) of 4-aminoN-phenylcarbazole in 30 mL of chloroform, and add 0.29 g (2.71 mmol) of sodium carbonate. After vigorous stirring and mixing, cool to 0 °C, and add 10 mL of chloroform solution containing 0.35 mL (4.07 mmol) of bromoacetyl bromide dropwise. Heat the mixture to room temperature and stir for 8 h. Filter and rotary evaporate. After purification by silica gel chromatography (petroleum ether to dichloromethane volume ratio 5:1), weigh to obtain 0.61 g of compound one as a pale yellow solid. The yield is calculated to be 87%.

[0102] S2. 0.6 g (1.58 mmol) of compound one and 1.09 g (6.33 mmol) of cyclohexane were dissolved in acetonitrile. 0.22 g (1.58 mmol) of potassium carbonate and 0.26 g (1.58 mmol) of potassium iodide were added. The mixture was stirred and refluxed for 48 h, then filtered and evaporated. After purification by silica gel chromatography (dichloromethane to methanol volume ratio 20:1), 0.37 g of compound two as a white solid was weighed, and the yield was calculated to be 62%.

[0103] S3. 0.3 g (0.64 mmol) of compound II and 0.28 g (2.04 mmol) of bromoacetamide were dissolved in acetonitrile, and 0.31 g (2.23 mmol) of potassium carbonate and 0.11 g (0.64 mmol) of potassium iodide were added. After stirring and refluxing for 48 h, the mixture was filtered and evaporated. After purification by silica gel chromatography (dichloromethane to methanol volume ratio 20:1), 0.19 g of the precursor (CBP-Cy) as a pale yellow solid was weighed, and the yield was calculated to be 63%.

[0104] S4. Dissolve 50 mg (0.078 mmol) of the precursor (CBP-Cy) in 3 mL of methanol, and add dropwise 3 mL of a methanol solution containing 29 mg (0.078 mmol) TbCl3·6H2O. Reflux overnight and evaporate the solvent to 1 ± 0.1 mL. Add diethyl ether dropwise until the solution becomes turbid. Place the solution in an ether atmosphere overnight, filter to collect the precipitate, wash the precipitate with diethyl ether, and weigh to obtain a white solid complex (CBP-Cy-Tb). The yield is calculated to be 62%.

[0105] The reaction formula for synthesizing CBP-Cy-Tb in Example 1 is shown below:

[0106] .

[0107] See Figure 2 Compound 2 was characterized using a Varian instrument (400 MHz), and the data are as follows:

[0108] 1 H NMR (400 MHz, Chloroform-d) δ (ppm): 8.32 (s, 1H), 8.14 (d, J =7.7 Hz, 2H), 7.78 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.3 Hz, 2H), 7.38 (q, J =7.9, 7.5 Hz, 4H), 7.30 (d, J = 7.4 Hz, 2H), 4.08 (s, 2H).

[0109] See Figure 3 and Figure 4 Compound 3 was characterized using a Varian instrument (400 MHz), and the data are as follows:

[0110] 1 H NMR (400 MHz, Acetonitrile-d3) δ (ppm): 8.20 (d, J = 7.7 Hz, 2H), 8.02 (d, J = 8.5 Hz, 2H), 7.58 – 7.54 (m, 2H), 7.46 – 7.38 (m, 4H), 7.30 (td,J = 7.4, 6.8, 1.4 Hz, 2H), 3.48 (s, 2H), 2.90 – 2.82 (m, 16H). 13 C NMR (101MHz, Acetonitrile-d3) δ (ppm): 169.80, 140.90, 136.74, 134.91, 127.46,126.12, 120.87, 120.30, 119.93, 117.33, 109.69, 60.42, 52.61, 47.01, 46.13,45.07.

[0111] See Figure 5 and Figure 6 CBP-Cy was measured using a nuclear magnetic resonance instrument (Varian instrument 400 MHz), and the characterization data are as follows:

[0112] 1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.39 (d, J = 108.4 Hz, 1H), 8.20 (d, J = 7.8 Hz, 2H), 7.99 – 7.82 (m, 4H), 7.52 (t, J = 8.1 Hz, 4H), 7.40 (t,J = 7.7 Hz, 2H), 7.24 (ddd, J = 39.2, 27.1, 10.5 Hz, 6H), 4.15 – 3.74 (m,6H), 3.33 – 2.61 (m, 16H). 13C NMR (101 MHz, DMSO-d6) δ (ppm): 175.02,173.00, 168.04, 140.71, 138.39, 132.22, 127.58, 126.68, 122.98, 121.22, 120.97, 120.39, 109.99, 62.28, 61.65, 59.91, 54.88, 50.99, 53.70, 53.68, 53.60, 53.52, 49.19.

[0113] Effect verification:

[0114] The precursor (CBP-Cy) and complex (CBP-Cy-Tb) prepared in Example 1 were dissolved in dimethyl sulfoxide (DMSO) to prepare a concentration of 2 × 10⁻⁶. -3 The mother liquor was prepared at mol / L, and HEPES buffer at pH 7.4 was used to prepare 20 μmol / L CBP-Cy and 20 μmol / L CBP-Cy-Tb test solutions, respectively. The fluorescence intensity of the two test solutions was then measured, and the results are as follows: Figure 7 As shown.

[0115] The complex (CBP-Cy-Tb) prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a concentration of 2 × 10⁻⁶. -3 The stock solution was prepared at mol / L, and HEPES buffer at pH 7.4 was used to prepare 20 μmol / L CBP-Cy-Tb test solutions. 20 μM 0.01M CTP, ATP, UTP, PPi, ADP, Pi, AMP, and Cl were added sequentially to the test solutions. - ,Br - SO4 2- HCO3 - and NO2 - Anions are used in a fluorescence titration experiment to measure the change in fluorescence intensity during the titration process, such as... Figure 8 As shown.

[0116] The complex (CBP-Cy-Tb) prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a concentration of 2 × 10⁻⁶. -3 The stock solution was prepared at mol / L, and HEPES buffer at pH 7.4 was used to prepare 20 μmol / L CBP-Cy-Tb test solutions. ATP was added to the test solutions for fluorescence titration experiments, and the changes in fluorescence emission spectra during the titration process were recorded as follows: Figure 9 As shown, the changes in the fluorescence emission peak at 527 nm during the titration process are recorded as follows: Figure 10 As shown, a curve was plotted showing the relationship between the fluorescence intensity of the test solution and the ATP concentration (4.5 μmol / L - 8.25 μmol / L). Figure 11 As shown.

[0117] Attached Figure Analysis:

[0118] See Figure 7 The fluorescence intensity of the CBP-Cy test solution without Tb coordination was 7957 a.u., while the fluorescence intensity of the coordinated CBP-Cy-Tb decreased to 1225 a.u., and Tb... 3+ The presence of a characteristic fluorescence emission peak indicates that CBP can transfer some energy to Tb. 3+ This makes Tb 3+ It has a characteristic fluorescence emission peak, and the energy transfer efficiency is calculated using the formula ϕ. Et =1-I DA / I D The energy transfer efficiency of CBP-Cy-Tb was calculated to be 84.6%.

[0119] See Figure 8 Tb in CBP-Cy-Tb can be found 3+ The characteristic fluorescence emission intensity of CBP-Cy-Tb was significantly enhanced after the addition of ATP, indicating that CBP-Cy-Tb has high selectivity for ATP. Therefore, CBP-Cy-Tb can be used as a probe for the qualitative detection of ATP.

[0120] See Figure 9 It can be seen that the fluorescence emission spectrum of CBP-Cy-Tb increases with the increase of ATP, further verifying... Figure 8 The conclusion, and see also Figure 10 It can be seen that the fluorescence emission peak of CBP-Cy-Tb at 547 nm increases with the increase of ATP concentration and reaches saturation when the ATP concentration reaches 12 μmol / L, and its fluorescence intensity is increased by 2.5 times.

[0121] See Figure 11It can be seen that the fluorescence intensity of CBP-Cy-Tb has a good linear relationship with the concentration of ATP in the range of 4.5 μmol / L to 8.25 μmol / L, and the linear equation is Y = 0.44677X + 1.70865, R 2 =0.99514, and based on the 3σ / K rule, the detection limit of CBP-Cy-Tb for ATP is calculated to be 3.02 nmol / L. Therefore, CBP-Cy-Tb can be used as a probe for the quantitative detection of ATP.

[0122] While 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 can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III), characterized in that, The structural formula of the complex is shown in Formula I: 。 2. A method for synthesizing the tetrazole heterocycle containing N-phenylcarbazole and Tb(III) complex as described in claim 1, characterized in that, Includes the following steps: Compound 1 was prepared by amidation of 4-amino-N-phenylcarbazole and bromoacetyl bromide in a first solvent and alkaline environment; In a second solvent and alkaline environment, compound 1 was nucleophilically substituted with cyclopentadiene to obtain compound 2, and then compound 2 was amidated to obtain the precursor. In a third solvent and alkaline environment, the precursor reacts with Tb. 3+ After coordination, the complex shown in Formula I was obtained; 。 3. The method for synthesizing a tetrazolium heterocyclic complex containing N-phenylcarbazole and Tb(III) according to claim 2, characterized in that, The process of preparing compound one by amidation of 4-amino-N-phenylcarbazole and bromoacetyl bromide includes: A first mixture was prepared by dissolving 4-amino-N-phenylcarbazole and a first basic accelerator in a first solvent. The first reaction solution was prepared by adding bromoacetyl bromide solution dropwise to the first mixture at 0±5℃. The first reaction solution was heated to 25±5℃ and stirred for 6-10 hours, then separated and purified to obtain compound one.

4. The method for synthesizing a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III) according to claim 3, characterized in that, After adding bromoacetyl bromide solution dropwise to the first mixture to prepare the first reaction solution, the molar ratio of 4-amino-N-phenylcarbazole to bromoacetyl bromide in the first reaction solution is 1:(1-1.5).

5. The method for synthesizing a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III) according to claim 2, characterized in that, The process of nucleophilic substitution of compound 1 with cyclotinocyanine to obtain compound 2 includes: The second mixture was prepared by dissolving compound 1, cyclohexanetin and the second basic accelerator in the second solvent; Compound II was obtained by stirring and refluxing the second mixture for 46-50 hours, followed by separation and purification.

6. The method for synthesizing a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III) according to claim 5, characterized in that, Before stirring and refluxing the second mixture, the first catalyst is added to the second mixture.

7. The method for synthesizing a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III) according to claim 5, characterized in that, After dissolving compound 1, cyclohexanetin and the second basic accelerator in the second solvent to prepare the second mixture, the molar ratio of compound 1 to cyclohexanetin in the second mixture is 1:(4-4.5).

8. The method for synthesizing a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III) according to claim 2, characterized in that, The process of preparing the precursor by amidation of compound 2 includes: The third mixture was prepared by dissolving compound 2, bromoacetamide and the third basic accelerator in the second solvent; The precursor was obtained by stirring and refluxing the third mixture for 46-50 hours, followed by separation and purification.

9. The method for synthesizing a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III) according to claim 8, characterized in that, Before stirring and refluxing the third mixture, a second catalyst is added to the third mixture.

10. The method for synthesizing a tetrazolium heterocyclic complex containing N-phenylcarbazole and Tb(III) according to claim 8, characterized in that, After dissolving compound 2, bromoacetamide and a third basic accelerator in a second solvent to prepare a third mixture, the molar ratio of compound 2 to bromoacetamide in the third mixture is 1:(3-3.2).

11. The method for synthesizing a tetrazole heterocyclic complex containing N-phenylcarbazole and Tb(III) according to claim 2, characterized in that, Precursor and Tb 3+ The process of preparing the complex shown in Formula I after coordination includes: The precursor is dissolved in a third solvent to prepare a fourth mixture; The second reaction solution was prepared by adding TbCl3 solution dropwise to the fourth mixture; The second reaction solution was heated under reflux for 12-24 hours and then separated and purified to obtain the complex shown in Formula I.

12. The method for synthesizing a tetrazolium heterocycle containing N-phenylcarbazole and a Tb(III) complex according to claim 11, characterized in that, After adding TbCl3 solution dropwise to the fourth mixture to prepare the second reaction solution, the precursor in the second reaction solution reacts with TbCl3. 3+ The molar ratio is 1:(1-1.2).

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