A platinum complex fluorescent probe and its application in hypochlorite detection and trivalent metal ion detection

Abstract

The application relates to a platinum complex fluorescent probe and application thereof in hypochlorite detection and trivalent metal ion detection. The structure formula of the platinum complex fluorescent probe is shown in the following formula. The platinum complex fluorescent probe described in the application has good selectivity and high sensitivity to hypochlorite and trivalent metal ions, fills the blank that no compound capable of detecting both hypochlorite and trivalent metal ions is found in the prior art, and meanwhile, the preparation method of the platinum complex fluorescent probe has mild reaction conditions.

Description

Technical Field

[0001] This application relates to the field of fluorescent probe technology, specifically to a platinum complex fluorescent probe and its application in the detection of hypochlorite and trivalent metal ions. Background Technology

[0002] Intracellular bioactive components / signaling molecules are a hot topic in life science research. For example, reactive oxygen species (ROS) refer to all highly reactive oxygen free radicals, peroxides, and singlet oxygen species derived from oxygen molecule metabolites. This includes superoxide radicals (O2). – ), hydrogen peroxide (H2O2), singlet oxygen ( 1 O2), hydroxyl radicals (·OH), hypochlorous acid (HClO), and hypochlorite ions (ClO) – Among these, hypochlorite is a relatively common reactive oxygen species, mainly produced by white blood cells in the human immune system, such as neutrophils, for phagocytosis and killing of pathogens. Abnormal hypochlorite levels are closely related to damage, aging, inflammation, and cancer. On the other hand, the rapid development and expansion of modern industry has led to a large amount of Fe... 3+ Al 3+ Gr 3+ and Ce 3+ When trivalent metal ions enter the environment, they can cause significant health problems if absorbed by the human body through the food chain. For example, excessive Fe... 3+ It may accumulate in organs such as the liver and heart, leading to liver cell damage, liver fibrosis, cirrhosis, and myocardial cell necrosis, increasing the risk of heart attack. 3+ In addition to causing indigestion, anemia, osteomalacia, osteoporosis, and cognitive impairment, poisoning can also lead to Alzheimer's disease and chronic kidney failure. Excessive Cr... 3+ Ingestion can cause serious harm to the skin and respiratory system, such as skin erythema, edema, ulceration, and nasal mucosal swelling, cough, and headache. Long-term exposure to Ce... 3+ This can also damage nerve cells, leading to symptoms such as memory loss, insomnia, and excessive dreaming. In severe cases, it can even cause nervous system dysfunction, while also damaging the kidneys and liver, and causing lung inflammation and pulmonary fibrosis. Therefore, efficient detection of hypochlorite ions in cells and trivalent metal ions in the environment is of great significance.

[0003] Most reported methods for detecting hypochlorite and trivalent metal ions utilize small-molecule fluorescent probes, which offer advantages such as high efficiency, low cost, and ease of use. However, in biological systems, organic fluorescent probes also face challenges such as significant background interference, insufficient stability, and relatively low accuracy. Transition metal complex probes, on the other hand, exhibit higher quantum efficiency due to the spin-orbit coupling effect caused by heavy atoms. Especially those with d... 6 d 8 and d 10 Electronically configured Ru(II)-, Os(II)-, Re(I)-, Ir(III)-, Cu(I)-, Pt(II)-, and Au(I)- complexes not only possess real-time detection, trace detection, and even micro-level detection capabilities comparable to fluorescent probes, but also offer advantages such as high stability, high luminescence efficiency, long luminescence lifetime, large Stokes shift, and easily tunable excitation and emission spectra, making them promising candidates for biosensing and cell imaging. Literature review results show that current fluorescent probe research mainly focuses on Ru(II) or Ir(III) complexes, while research on platinum complex fluorescent probes is still limited and in its early stages. To date, no papers have been published on the simultaneous detection of hypochlorite and trivalent metals using platinum complex molecular probes.

[0004] In summary, no compound has been found in the existing technology that can detect both hypochlorite and trivalent metal ions. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the purpose of this application is to provide a platinum complex fluorescent probe and its application in the detection of hypochlorite and trivalent metal ions, so as to fill the gap in the prior art that no compound has been found that can detect both hypochlorite and trivalent metal ions.

[0006] The platinum complex fluorescent probe described in this application has the following structural formula:

[0007]

[0008] R is one or more of H, -CH3, -C2H5, -C(CH3)3, -OH and -CHO.

[0009] Preferably, R is C(CH3)3.

[0010] This application also provides a method for preparing the platinum complex fluorescent probe as described above, comprising the following steps:

[0011] Step 1, take Dissolve in anhydrous THF and cool to 0–5°C, then add phenyllithium solution dropwise under an inert atmosphere; The molar ratio of the phenyllithium to the organic phase is 1:1 to 1.5. After adding the phenyllithium solution dropwise, the mixture is brought to room temperature and stirred for 5 to 24 hours to obtain solution A. A saturated ammonium chloride solution is added to solution A, the organic phase is collected, manganese dioxide is added to the organic phase, and the mixture is stirred for 16 hours to obtain solution B. Solution B is filtered to remove the solvent from the filtered solution. The solvent-free solution is then subjected to column chromatography to obtain compound a, the structural formula of which is shown below:

[0012]

[0013] Step 2: Dissolve compound a and K2PtCl4 in a CH3CN / H2O mixed solution at a molar ratio of 1:1 to 1.5 to obtain solution C, with a concentration of 0.1 mmol / mL; the volume ratio of CH3CN to H2O in the CH3CN / H2O mixed solution is 1:1 to 2; reflux under an inert atmosphere for 14 to 36 hours to obtain solution D. Remove CH3CN from solution D and filter, collect the solid, and perform column chromatography to obtain compound b. The structural formula of compound b is shown below:

[0014]

[0015] Step 3: Dissolve 4-ethynylbenzaldehyde and 2-aminoanthracene in anhydrous DMF at a molar ratio of 1:1 to 1.2 to obtain solution E, with a concentration of 1 mmol / mL. Add activated 4A molecular sieve and react at an inert atmosphere and 100–140 °C for 4–16 h. After reacting for 4–16 h, cool to room temperature to obtain solution F. Add methanol to solution F, filter, collect the solid, and wash the solid with methanol to obtain compound c. The structural formula of compound c is shown below:

[0016]

[0017] Step 4: Dissolve compound b and compound c in DCM at a molar ratio of 1:1 to 1.2 to obtain solution G. The concentration of solution G is determined by adding CuI and TEA. The molar ratio of compound b to CuI is 1:0.1 to 0.5, and the molar ratio of compound b to TEA is 1:60 to 100. React for 15 to 24 hours under light-protected, inert atmosphere and room temperature to obtain solution H. Remove the solvent from solution H and perform column chromatography to obtain the platinum complex fluorescent probe.

[0018] Preferably, in step 1, the The molar ratio of the phenyllithium to the phenyllithium is 1:1.1, the solvent of the phenyllithium solution is diethyl ether, the concentration of the phenyllithium solution is 1 mol / L, after the phenyllithium solution is added dropwise, the mixture is brought to room temperature and stirred for 6 hours.

[0019] Preferably, in step 2, the molar ratio of compound a to K2PtCl4 is 1:1; the volume of the CH3CN / H2O mixed solution is [volume value missing], and the volume ratio of CH3CN to H2O is 1:1.

[0020] Preferably, in step 3, the molar ratio of 4-ethynylbenzaldehyde and 2-aminoanthracene is 1:1; the reaction is carried out under an inert atmosphere at 100°C for 5 hours.

[0021] Preferably, in step 4, the molar ratio of compound b to compound c is 1:1; the molar ratio of compound b to CuI is 1:0.1; the molar ratio of compound b to TEA is 1:60; and the reaction is carried out for 16 hours under light-protected, inert atmosphere and room temperature conditions.

[0022] Preferably, the inert atmosphere is nitrogen.

[0023] This application also provides an application of the platinum complex fluorescent probe as described above or the platinum complex fluorescent probe prepared by the method described above, wherein the platinum complex fluorescent probe is used to detect hypochlorite in living cells.

[0024] This application also provides an application of the platinum complex fluorescent probe as described above or the platinum complex fluorescent probe prepared by the method described above, wherein the platinum complex fluorescent probe is used to detect Fe in the environment. 3+ Al 3+ Gr 3+ and Ce 3+ .

[0025] The advantages of the platinum complex fluorescent probe described in this application and its application in the detection of hypochlorite and trivalent metal ions are as follows:

[0026] Since the platinum complex fluorescent probe described in this invention is inherently yellow fluorescent, the addition of hypochlorite ions, due to the strong oxidizing effect of hypochlorous acid, oxidizes the four-coordinate platinum to a six-coordinate platinum, causing a change in the structure of the platinum complex fluorescent probe and altering its ultraviolet absorption, resulting in the disappearance of the yellow fluorescence; while the addition of trivalent metal ions Fe 3+ Al 3 +, Gr 3+ and Ce 3+Subsequently, the trivalent metal ions cause the imine group in the probe molecule to break, generating anthracene that emits green fluorescence. This alters the ultraviolet absorption, causing the yellow fluorescence to change to green fluorescence. Therefore, the platinum complex fluorescent probe described in this application can detect both hypochlorite and trivalent metal ions. It exhibits good selectivity and high sensitivity for both hypochlorite and trivalent metal ions, filling the gap in the existing technology for compounds that can detect both hypochlorite and trivalent metal ions. Furthermore, the preparation method of this platinum complex fluorescent probe uses mild reaction conditions. Attached Figure Description

[0027] Figure 1 This is the UV absorption spectrum of the platinum complex fluorescent probe in Experiment Example 1 of this invention after adding different equivalents of hypochlorite ions;

[0028] Figure 2 The platinum complex fluorescent probe in Experimental Example 2 of this invention, after the addition of Fe... 3+ Al 3+ Gr 3+ and Ce 3+ UV absorption spectra before and after;

[0029] Figure 3 This is a graph showing the ion competition experimental data of the platinum complex fluorescent probe against various reactive oxygen species in Experiment Example 3 of this invention;

[0030] Figure 4 This is a graph showing the experimental data of the platinum complex fluorescent probe competing with anions and metal cations in Experiment Example 4 of this invention;

[0031] Figure 5 These are images of the platinum complex fluorescent probe used to test trivalent metal ions in Experimental Example 5 of this invention;

[0032] Figure 6 This is a cell imaging image of hypochlorite ions detected by the platinum complex fluorescent probe in Experiment Example 6 of this invention. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments.

[0034] The raw materials involved in this application can be found in Table 1.

[0035] Table 1

[0036]

[0037]

[0038] The instruments mentioned in this application can be found in Table 2.

[0039] Table 2

[0040]

[0041] The description of "room temperature" in this application refers to a temperature range of 20 to 25°C; the "column chromatography" operation mentioned uses 200 to 300 mesh silica gel as the packing material for the chromatography column.

[0042] Example 1

[0043] The preparation method of the platinum complex fluorescent probe in this embodiment includes the following steps:

[0044] (1) Take 2.68g (10mmol, 1.0eq) Dissolved in 50 mL of anhydrous THF (tetrahydrofuran) and cooled to 0–5 °C, 11 mL of phenyllithium solution (1 M / ethyl ether, 11 mmol, 1.1 eq) was slowly added dropwise under nitrogen protection. The mixture was then brought to room temperature and stirred for 6 h to obtain solution A. Saturated ammonium chloride solution was added to solution A to quench excess phenyllithium. The organic phase was collected, and manganese dioxide was added to the organic phase. The mixture was stirred for 16 h to obtain solution B. Solution B was filtered to remove the solvent from the filtered solution. The solvent-free solution was subjected to column chromatography to obtain compound a, a white solid. The mass of compound a obtained was 2.86 g, with a yield of 83%. The structural formula of compound a is shown below:

[0045]

[0046] (2) 344.5 mg (1.0 mmol, 1.0 eq) of compound a and 415.1 mg (1.0 mmol, 1.0 eq) of K2PtCl4 were dissolved in a CH3CN / H2O (20 mL, volume ratio 1:1) mixed solution to obtain solution C. Under nitrogen protection, solution C was refluxed for 16 h. After the reaction was completed, solution D was obtained. CH3CN in solution D was removed and filtered. The solid was collected and subjected to column chromatography to obtain compound b. Compound b was a yellow solid. The mass of compound b obtained was 447 mg, and the yield was 78%. The structural formula of compound b is shown below:

[0047]

[0048] (3) 130.2 mg of 4-ethynylbenzaldehyde (1.0 mmol, 1.0 eq) and 193.3 mg of 2-aminoanthracene (1.0 mmol, 1.0 eq) were dissolved in 2 mL of anhydrous DMF (N,N-dimethylformamide), and several activated 4A molecular sieves were added. The mixture was reacted at 100 °C for 5 h under nitrogen protection, then cooled to room temperature, and methanol was added to precipitate the solid. The solid was filtered, washed with methanol, and 278 mg of brownish-yellow solid, namely compound c, was obtained, with a yield of 91%. The structural formula of compound c is shown below:

[0049]

[0050] (4) 57.4 mg (0.1 mmol, 1.0 eq) of compound b and 30.5 mg (0.1 mmol, 1.0 eq) of compound c were dissolved in 10 mL of DCM. 1.9 mg (0.01 mmol, 0.1 eq) of CuI and 607 mg (6 mmol, 60 eq) of TEA were added. The mixture was reacted at room temperature for 16 h under nitrogen protection in the dark. After the reaction was completed, the solvent was removed, and column chromatography yielded 67 mg of a yellow solid, which was the platinum complex fluorescent probe, with a yield of 79%. The structural formula of the platinum complex fluorescent probe is shown below:

[0051]

[0052] Experimental Example 1: Ultraviolet Absorption Spectroscopy Measurement of Hypochlorous Acid Response Based on Platinum Complex Fluorescent Probe

[0053] The platinum complex fluorescent probe prepared in Example 1 was used. The test concentration of the platinum complex fluorescent probe was 50 μM, and the test solvent was a mixed solution of THF / PB = 1 / 1. Figure 1 As shown, with increasing hypochlorite concentration, the absorption peak of the platinum complex fluorescent probe at 209 nm significantly increases, absorption is enhanced in the 200–250 nm range, while absorption decreases in the 280–500 nm range. This is because the strong oxidizing effect of hypochlorous acid oxidizes the tetracoordinate platinum to the hexacoordinate platinum, resulting in a change in the structure of the platinum complex fluorescent probe and thus altering its ultraviolet absorption.

[0054] Experimental Example 2: Fe based on platinum complex fluorescent probe 3+ Al 3+ Gr 3+ and Ce 3+ Response UV absorption spectroscopy test

[0055] The platinum complex fluorescent probe prepared in Example 1 was used. The test concentration of the platinum complex fluorescent probe was 20 μM, and the test solvent was a mixed solution of THF / H2O = 1 / 1. Figure 2 It is a platinum complex fluorescent probe with the addition of 40 μM Fe 3+ Al 3+ Gr 3+ and Ce 3+ The UV absorption before and after. For example... Figure 2 As shown, the UV absorption of the probe solution was significantly enhanced after the addition of trivalent metal ions. This is because the trivalent metal ions cause the imine groups in the probe molecules to break, generating anthracene that emits green fluorescence, thus altering the UV absorption.

[0056] Experiment Example 3: Competition Experiment of Platinum Complex Fluorescent Probes for Various Reactive Oxygen Ions

[0057] The platinum complex fluorescent probe prepared in Example 1 was used. The concentration of the platinum complex fluorescent probe was 20 μM. The test solvent was a 1 / 1 THF / PB mixed solution. The analytes were H2O2, ·OH, and ClO. - , 1 O2, ·OtBu, and TBHP were tested at 560 nm to measure the fluorescence intensity of the platinum complex fluorescent probe on the analyte. The test results are as follows: Figure 3 As shown, Figure 3 The results show that hypochlorite alters the structure of the platinum complex fluorescent probe, leading to a decrease in fluorescence intensity, while the absorbance of other reactive oxygen species remains around 1750 a.u. after the addition of the platinum complex fluorescent probe. In other words, apart from hypochlorite, other reactive oxygen species do not cause significant fluorescence changes in the platinum complex probe, demonstrating its high selectivity for hypochlorite.

[0058] Experiment Example 4: Competition Experiment of Platinum Complex Fluorescent Probes for Anions and Metal Cations

[0059] The platinum complex fluorescent probe prepared in Example 1 was used. Figure 4 In the diagram, origin 0 represents a platinum complex fluorescent probe without any added ions, and analytes 1-24 represent Na... + 2 is K + 3 is Mg 2+ 4 is Cu 2+ 5 is Cr 3+ 6 is Ce 3+ 7 is Fe 3+ 8 is Al 3+ 9 is Zn 2+ 10 is Ni 2+ 11 is Mn 2+ 12 is Ca 2+ 13 is CH3COO - 14 is NO3 - 15 is NO 2- 16 is Cl - 17 is Br - 18 is HCO3 - 19 is CO3 2- 20 is SiO3 2- 21 is SO4 2- 22 is SO3 2- 23 is PO4 3- 24 is HPO4 2-The platinum complex probe concentration was 50 μM, the test solvent was a 1 / 1 THF / H₂O mixture, and the added ion concentration was 100 μM. The results are as follows: Figure 4 As shown, apart from trivalent metal ions, other ions do not cause significant fluorescence changes in the fluorescent probe, proving that the platinum complex probe has high selectivity for trivalent metal ions.

[0060] Experiment 5: Ion Recognition Performance of Platinum Complex Fluorescent Probe

[0061] The platinum complex fluorescent probe prepared in Example 1 was used to test the luminescence of the probe under 365 nm UV light irradiation with the addition of various ions. Specifically, the analyte solution was placed in a cuvette, the platinum complex fluorescent probe was added, and the color change of the solution in the cuvette was observed under 365 nm UV light irradiation. Analytes 1-24 are Na... + 2 is K + 3 is Mg 2+ 4 is Cu 2+ 5 is Cr 3+ 6 is Ce 3+ 7 is Fe 3+ 8 is Al 3+ 9 is Zn 2+ 10 is Ni 2+ 11 is Mn 2+ 12 is Ca 2+ 13 is CH3COO - 14 is NO3 - 15 is NO 2- 16 is Cl - 17 is Br - 18 is HCO3 - 19 is CO3 2- 20 is SiO3 2- 21 is SO4 2- 22 is SO3 2- 23 is PO4 3- 24 is HPO4 2- The platinum complex probe concentration was 50 μM, the test solvent was a 1 / 1 THF / H₂O mixture, and the added ion concentration was 100 μM. Figure 5 As shown, Al was added to the probe. 3+ Cr 3+ Fe 3+ and Ce 3+ Subsequently, under 365nm ultraviolet light irradiation, the test system changed from yellow fluorescence to yellow-green fluorescence, while the addition of other ions showed no significant change, indicating that the platinum complex fluorescent probe described in this application is effective against Al. 3+ Cr 3+Fe 3+ and Ce 3+ It has a certain degree of "naked eye" recognition effect.

[0062] Experiment Example 6: Detection of hypochlorite ions in cells using platinum complex fluorescent probes.

[0063] The platinum complex fluorescent probe prepared in Example 1 was used to investigate its application in cell imaging using laser confocal microscopy. After resuscitation, 1×10⁶ cells were collected. 5 HeLa cells were seeded into confocal culture dishes and incubated at 37°C for 24 h. After changing the culture medium, 20 μM of fluorescent probe was added and incubated for 24 h. The culture medium was removed and the cells were washed three times with PBS. Then, 50 μM of hypochlorite was added (no such addition was added to the control group), and the cells were incubated for 2 h. The cells were washed three times with PBS, and the cells were observed and imaged using a confocal microscope. Figure 6 Cell imaging results of the platinum complex probe confirm that the probe can effectively enter cells and detect hypochlorite ions in the cells.

[0064] In the description of this application, it should be understood that the orientation or positional relationship indicated by directional terms such as "front, back, up, down, left, right", "horizontal, vertical, horizontal" and "top, bottom" is usually based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing this application and simplifying the description. Unless otherwise stated, these directional terms do not indicate or imply that the device or element referred to must have a specific orientation or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the scope of protection of this application.

[0065] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this application.

Claims

1. A platinum complex fluorescent probe, characterized in that, The structural formula of the platinum complex fluorescent probe is shown below: ； R is one or more of H, -CH3, -C2H5, and -C(CH3)3.

2. The platinum complex fluorescent probe according to claim 1, characterized in that, The R is C(CH3)3.

3. A method for preparing a platinum complex fluorescent probe as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1, take Dissolve in anhydrous THF and cool to 0–5°C, then add phenyllithium solution dropwise under an inert atmosphere; The molar ratio of the phenyllithium to the phenyllithium is 1:1 to 1.5; after adding the phenyllithium solution, the temperature is raised to 20 to 25°C and stirred for 5 to 24 hours to obtain solution A; saturated ammonium chloride solution is added to solution A, the organic phase is collected, manganese dioxide is added to the organic phase, and the mixture is stirred for 16 hours to obtain solution B; The solution B was filtered to remove the solvent from the filtered solution. The solvent-free solution was then subjected to column chromatography to obtain compound a, the structural formula of which is shown below: ； Step 2: Dissolve compound a and K2PtCl4 in a CH3CN / H2O mixed solution at a molar ratio of 1:1 to 1.5 to obtain solution C, with a concentration of 0.1 mmol / mL; the volume ratio of CH3CN to H2O in the CH3CN / H2O mixed solution is 1:1 to 2; reflux under an inert atmosphere for 14 to 36 hours to obtain solution D. Remove CH3CN from solution D and filter, collect the solid, and perform column chromatography to obtain compound b. The structural formula of compound b is shown below: ； Step 3: Dissolve 4-ethynylbenzaldehyde and 2-aminoanthracene in anhydrous DMF at a molar ratio of 1:1 to 1.2 to obtain solution E, with a concentration of 1 mmol / mL. Add activated 4A molecular sieve and react at an inert atmosphere and 100–140 °C for 4–16 h. After reacting for 4–16 h, cool to room temperature to obtain solution F. Add methanol to solution F, filter, collect the solid, and wash the solid with methanol to obtain compound c. The structural formula of compound c is shown below: ； Step 4: Dissolve compound b and compound c in DCM at a molar ratio of 1:1 to 1.2 to obtain solution G. Add CuI and TEA to solution G; the molar ratio of compound b to CuI is 1:0.1 to 0.5; the molar ratio of compound b to TEA is 1:60 to 100. React for 15 to 24 hours under light-protected, inert atmosphere and room temperature conditions to obtain solution H. Remove the solvent from solution H and perform column chromatography to obtain the platinum complex fluorescent probe.

4. The preparation method according to claim 3, characterized in that, In step 1, the The molar ratio of the phenyllithium to the phenyllithium is 1:1.1, the solvent of the phenyllithium solution is diethyl ether, the concentration of the phenyllithium solution is 1 mol / L, after the phenyllithium solution is added dropwise, the mixture is brought to room temperature and stirred for 6 hours.

5. The preparation method according to claim 3, characterized in that, In step 2, the molar ratio of compound a to K2PtCl4 is 1:1; the volume of the CH3CN / H2O mixed solution is [volume value missing], and the volume ratio of CH3CN to H2O is 1:

1.

6. The preparation method according to claim 3, characterized in that, In step 3, the molar ratio of 4-ethynylbenzaldehyde to 2-aminoanthracene is 1:1; the reaction is carried out under an inert atmosphere at 100°C for 5 hours.

7. The preparation method according to claim 3, characterized in that, In step 4, the molar ratio of compound b to compound c is 1:1; the molar ratio of compound b to CuI is 1:0.1; the molar ratio of compound b to TEA is 1:60; and the reaction is carried out for 16 hours under light-protected, inert atmosphere and room temperature conditions.

8. The application of the platinum complex fluorescent probe according to any one of claims 1-2 or the platinum complex fluorescent probe prepared by any one of claims 3-7, characterized in that, The platinum complex fluorescent probe was used to prepare a reagent for detecting hypochlorite in living cells.

9. The application of the platinum complex fluorescent probe according to any one of claims 1-2 or the platinum complex fluorescent probe prepared by any one of claims 3-7, characterized in that, The platinum complex fluorescent probe was applied to the preparation and detection environment for Fe. 3+ Al 3+ Gr 3+ and Ce 3+ The reagent.

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