Cyclo-metallated platinum complexes and their use

Abstract

The application discloses a kind of cyclometallated platinum complexes, the cyclometallated platinum complex of the application can be used as fluorescent probe in different detection three biological mercaptans, realize "one probe, multi-color" differentiation distinction;The cyclometallated platinum complex can also be used as fluorescent probe in detecting anion and DNA molecule, can with green, brown, orange red fluorescence enhancement type detection DNA in aqueous solution, with red fluorescence enhancement type detection RNA in aqueous solution, the detection limit of DNA and RNA is 32.69 µM and 62.5 µM respectively, and concentration and fluorescence intensity present excellent linear relationship;The cyclometallated platinum complex of the application is simple to prepare, and has high sensitivity, low detection cost, easy operation, rapid determination and real-time detection and the like.

Description

Technical Field

[0001] This invention relates to a class of cyclic metallized platinum complexes and their applications in the detection of biothiols, anions, nucleic acid molecules, and fluorescence imaging, belonging to the field of biomedical detection and imaging. Background Technology

[0002] Cyclic platinum(II) complexes possess excellent photophysical and photochemical properties. Their molecular configurations are easily modified, exhibiting large Stokes shifts, long phosphorescence lifetimes, and high photostability. They also demonstrate specific organelle targeting capabilities, high cellular uptake levels, excellent anticancer activity, and environmentally sensitive emission characteristics, attracting widespread attention in recent years for chemisensing, bioimaging, and antitumor drug development. In particular, their ability to form supramolecular assemblies through intermolecular Pt-Pt and π-π stacking interactions, followed by depolymerization under external stimuli, resulting in significant changes in luminescence color or intensity, makes them a highly promising class of smart responsive fluorescent probes.

[0003] Biothiols mainly refer to three biomolecules: cysteine ​​(CYS), homocysteine ​​(HCY), and glutathione (GSH). They are the most abundant low-weight molecules in cells and play a crucial role in human physiology. Existing complexes can only recognize GSH individually and cannot achieve differentiated "one probe, multiple colors" detection. Various trace anions in the human body play an important role in regulating physiological and biochemical functions. While existing complexes can recognize multiple anions (such as ClO4), they are not suitable for all anions. - NO3 - ,Br - While some reagents (such as ethidium bromide EB and DAPI) may exhibit a response, this is often a non-specific enhancement or alteration of fluorescence. Nucleic acids are the primary materials for storing, replicating, and transmitting genetic information. Gene sequencing plays a crucial role in DNA damage research, gene therapy, mutation analysis, bacterial infection, drug development, and clinical diagnosis. Currently, fluorescence detection of nucleic acids heavily relies on commercially available intercalation dyes (such as ethidium bromide EB and DAPI) or complex polymer nanomaterials. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a class of cyclic platinum metallization complexes that can specifically detect three biogenic thiocysteines (CYS), homocysteine ​​(HCY), and glutathione (GSH) in aqueous solutions, achieving differential differentiation with a single probe and multiple colors. Furthermore, these cyclic platinum metallization complexes exhibit aggregation-induced emission (AIE), enabling the specific detection of anions; they can also detect DNA in aqueous solutions with green, brown, and orange-red fluorescence enhancement, and RNA with red fluorescence enhancement. The detection limits for DNA and RNA are 32.69 µM and 62.5 µM, respectively, and the concentration exhibits an excellent linear relationship with fluorescence intensity.

[0005] The structural formula of the cyclic metallized platinum complex of the present invention is shown in Formula I: Formula I ; in Selected from ; Selected from .

[0006] The preparation method of the cyclic metallized platinum complex of the present invention is as follows: (1) Under an inert atmosphere, potassium tetrachloroplatinate and aromatic ring compounds are dissolved in solvent A to obtain mixed solution A. Mixed solution A is refluxed and stirred at 80-85℃ for 48-54h to precipitate. The precipitate is washed and dried to obtain platinum bridging precursor. The aromatic ring compounds are selected from 2-[(4-trifluoromethyl)phenyl]pyridine, 2-[2,4-bis(trifluoromethyl)phenyl]pyridine, 4-chloro-2,2'-bipyridine, and 4-fluoro-2,2'-bipyridine, i.e. ; The inert atmosphere is selected from N2, Ar, and He; the molar ratio of potassium tetrachloroplatinate to aromatic ring compounds is 1:2-2.5; solvent A is a mixed solvent of ethylene glycol ethyl ether and ultrapure water, and the volume ratio of ethylene glycol ethyl ether to ultrapure water is 2:1. (2) Under an inert atmosphere, the platinum precursor obtained in step (1) and the silver salt are dissolved in solvent B to obtain mixed solution B. Mixed solution B is stirred and dechlorinated at room temperature for 20-24 hours. Solid-liquid separation is performed, and the liquid phase is the intermediate product. The molar ratio of the platinum-bridged precursor to the silver salt is 1:2; solvent B is acetonitrile; (3) Under an inert atmosphere, the intermediate product obtained in step (2) is mixed with a bipyridine compound and refluxed at 60-70°C for 24-36 h to precipitate. The precipitate is washed and dried to obtain a cyclic metallized platinum complex.

[0007] The bipyridine compounds are selected from 2,2'-bipyridine and 1,10-phenanthroline, i.e. The molar ratio of the platinum-bridged precursor to the bipyridine compound is 1:2-2.5.

[0008] The cyclic metallized platinum complexes of this invention are selected from one of the following: .

[0009] Another objective of this invention is to apply the above-mentioned cyclic platinum metallization complex as a fluorescent probe for the specific detection of three biothiols, wherein the cyclic platinum metallization complex is... It uses red fluorescence to detect cysteine, yellow-brown fluorescence to detect homocysteine, and yellow-green fluorescence to detect glutathione.

[0010] Another objective of this invention is to apply the above-mentioned cyclic metallized platinum complex as a fluorescent probe for the detection of anions. Specifically, the complex Pt1 uses an orange fluorescence-enhanced method to detect ClO4. - SO4 2- ,Br - NO3 - HCO3 - Green fluorescence enhanced detection of SO3 2- The complex Pt2 was used for the detection of orange-red fluorescence enhanced ClO4. - SO4 2- HSO3 - ,Br - NO3 - CO3 2- Green fluorescence enhanced detection of SO3 2- The Pt3 complex was used to detect NO2 using a red fluorescence-enhanced assay. - I was detected using light blue fluorescence enhancement. - CO3 2- SO3 was detected using green fluorescence enhancement. 2- The Pt4 complex was used to detect SO4 using an orange fluorescence-enhanced assay. 2- NO2 - Cl - ClO4 - HCO3 - NO3 - Orange-red fluorescence-enhanced detection of Br was employed. - I - Green fluorescence enhanced detection of SO3 2- The complex Pt5 was detected using a red fluorescence-enhanced assay for ClO. - Yellow fluorescence-enhanced detection of CO3 2- HCO3 -The complex Pt6 was detected using an orange-red fluorescence-enhanced Br-type assay. - SO4 2- ClO4 - NO3 - quenching detection of SO3 2- .

[0011] Another objective of this invention is to apply the aforementioned cyclic metallized platinum complexes as fluorescent probes for the detection of nucleic acid molecules. Complex Pt1 uses green fluorescence enhancement to detect DNA molecules; complex Pt2 uses orange-red fluorescence enhancement to detect DNA molecules and red fluorescence enhancement to detect RNA molecules; complex Pt3 uses brown fluorescence enhancement to detect DNA molecules; complex Pt4 uses orange fluorescence enhancement to detect DNA or RNA molecules; complex Pt5 uses orange-red fluorescence enhancement to detect DNA molecules and red fluorescence enhancement to detect RNA molecules; and complex Pt6 uses orange-red fluorescence enhancement to detect DNA molecules and quenching to detect RNA molecules.

[0012] Another objective of this invention is to use the above-mentioned cyclic metallized platinum complex Pt4 as a fluorescent probe in the detection of nucleic acid concentrations, with detection limits of 32.69 µM for DNA and 62.5 µM for RNA.

[0013] Advantages and technical effects of the present invention: The cyclic platinum metallized complex provided by this invention can be used as a fluorescent probe to differentiate three biothiols, achieving "one probe, multiple colors" for differential differentiation. The cyclic platinum metallized complex can also be used as a fluorescent probe to detect anions and DNA molecules. It can detect DNA in aqueous solution with green, brown, and orange-red fluorescence enhancement, and RNA in aqueous solution with red fluorescence enhancement. The detection limits for DNA and RNA are 32.69 µM and 62.5 µM, respectively, and the concentration and fluorescence intensity exhibit an excellent linear relationship. The cyclic platinum metallized complex of this invention is simple to prepare and features high sensitivity, low detection cost, convenient operation, rapid measurement, and real-time detection. Attached Figure Description

[0014] Figure 1 This is a high-resolution mass spectrum of Pt1; Figure 2 This is a high-resolution mass spectrum of Pt3; Figure 3 These are the fluorescence emission spectra of Pt1 in aqueous solutions of different anions, with the left figure being the fluorescence emission spectrum and the right figure being the corresponding fluorescence intensity bar chart; Figure 4 These are the fluorescence emission spectra of Pt2 in aqueous solutions of different anions, with the left figure being the fluorescence emission spectrum and the right figure being the corresponding fluorescence intensity bar chart; Figure 5 These are the fluorescence emission spectra of Pt3 in aqueous solutions of different anions, with the left figure being the fluorescence emission spectrum and the right figure being the corresponding fluorescence intensity bar chart; Figure 6 These are the fluorescence emission spectra of Pt4 in aqueous solutions with different anions, where the left figure is the fluorescence emission spectrum and the right figure is the corresponding fluorescence intensity bar chart; Figure 7 These are the fluorescence emission spectra of Pt5 in aqueous solutions with different anions, where the left figure is the fluorescence emission spectrum and the right figure is the corresponding fluorescence intensity bar chart; Figure 8 These are the fluorescence emission spectra of Pt6 in aqueous solutions with different anions, where the left figure is the fluorescence emission spectrum and the right figure is the corresponding fluorescence intensity bar chart; Figure 9 This is the time-response fluorescence emission spectrum of Pt3 to three biothiols; Figure 10 These are the fluorescence emission spectra of Pt1 with DNA and RNA; Figure 11 These are the fluorescence emission spectra of Pt2 with DNA and RNA; Figure 12 These are the fluorescence emission spectra of Pt3 with DNA and RNA; Figure 13 These are the fluorescence emission spectra of P4 with DNA and RNA; Figure 14 These are the fluorescence emission spectra of Pt5 with DNA and RNA; Figure 15 These are the fluorescence emission spectra of Pt6 with DNA and RNA; Figure 16 This is a schematic diagram simulating the molecular docking of Pt5 with deoxyribonucleic acid (DNA); Figure 17 This is a bar chart showing the fluorescence intensity of the Pt4 complex reacting with different concentrations of DNA or RNA. Figure 18 This is the detection limit result for the Pt4 complex in detecting DNA or RNA; Figure 19 These are the fluorescence emission spectra of Pt2 in mixed solutions of water and dimethyl sulfoxide in different proportions; Figure 20 These are fluorescence emission spectra of Pt6 in mixed solutions of water and dimethyl sulfoxide in different proportions; Figure 21 The results show the dual-channel uptake of complex Pt3 after co-incubation with HeLa cells and HaCaT cells for 6 hours. Figure 22 This is a dual-channel localization confocal imaging image after co-incubation of the complex Pt3 with HeLa cells. Detailed Implementation

[0015] The present invention will be further described in detail below through examples, but the scope of protection of the present invention is not limited to the content described herein. The structures of the compounds prepared in the examples of the present invention were determined by proton nuclear magnetic resonance spectroscopy and mass spectrometry. Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be purchased, and methods used are conventional methods unless otherwise specified.

[0016] Example 1: Synthesis of complex Pt1 1. Under a nitrogen atmosphere, potassium tetrachloroplatinate and 2-[(4-trifluoromethyl)phenyl]pyridine were dissolved in a mixed solvent of ethylene glycol ethyl ether and ultrapure water (volume ratio 2:1) at a molar ratio of 1:2 to obtain solution A. Solution A was refluxed and stirred at 80°C for 48 h for coordination reaction. After the reaction was completed, a large amount of ultrapure water was added and the mixture was placed in an ice bath until a light green flocculent precipitate appeared. The precipitate was filtered to obtain the precipitate. The precipitate was washed three times with ultrapure water and ether respectively and dried at 50°C to obtain light green platinum bridging precursor 1. 2. Under a nitrogen atmosphere, platinum bridging precursor 1 (60 mg, 0.067 mmol) and AgCF3SO3 (34.11 mg, 0.134 mmol) were dissolved in 15-20 mL of acetonitrile to obtain solution B. Solution B was reacted at room temperature for 24 h. The solid and liquid phases were separated, and the AgCl precipitate was removed. The liquid phase was the intermediate product. 3. Under a nitrogen atmosphere, 2,2'-bipyridine (bpy) (20.73 mg, 0.134 mmol) was added to the intermediate product, and the mixture was refluxed at 60 °C for 24 h. After the reaction was complete, the solution was concentrated to 1-2 mL by rotary evaporation, and diethyl ether was added to precipitate the mixture. The precipitate was filtered, washed successively with water and diethyl ether, and dried at 50 °C to obtain an orange-yellow solid complex Pt1; the yield was 41.7%. The high-resolution mass spectrum of complex Pt1 is shown in [image missing]. Figure 1 .

[0017] The structural formula of the complex Pt1 is as follows: ; Its 1H NMR and mass spectrometry data are as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 11.52 (d, J = 5.9Hz, 1H), 11.30 (d, J = 5.5 Hz, 1H), 11.22 (d, J = 5.8 Hz, 1H), 10.94 (dt, J=24.9, 12.4 Hz, 2H), 10.79 – 10.65 (m, 2H), 10.62-10.42 (m, 2H), 10.26 (d, J =8.0 Hz, 1H), 10.19 (t, J = 6.7 Hz, 1H), 10.14 (t, J = 6.7 Hz, 1H), 9.88-9.76(m, 2H), 9.77-9.66 (m, 1H). ESI-MS m / z: [M]±573.09. Example 2: Synthesis of complex Pt2 The preparation method is the same as in Example 1, except that in step (3), 2,2'-bipyridine is replaced with 1,10-phenanthroline; a red solid complex Pt2 is obtained; the yield is 28.3%; The structural formula of the complex Pt2 is as follows:

[0018] Its 1H NMR and mass spectrometry data are as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 9.54 (d, J = 5.3Hz, 1H), 9.38 (d, J = 5.0 Hz, 1H), 8.92 (dd, J =24.5, 7.3 Hz, 3H), 8.36-8.10(m, 4H), 8.07-7.97 (m, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.42 (dd, J = 12.4, 4.9Hz, 3H). ESI-MS m / z: [M]±597.09. Example 3: Synthesis of complex Pt3 The preparation method is the same as in Example 1, except that in step (1), 2-[(4-trifluoromethyl)phenyl]pyridine is replaced with 2-[2,4-bis(trifluoromethyl)phenyl]pyridine; a yellow solid complex Pt3 is obtained; the yield is 51.0%. The high-resolution mass spectrum of complex Pt3 is shown in [reference needed]. Figure 2 .

[0019] The structural formula of the complex Pt3 is as follows:

[0020] Its 1H NMR and mass spectrometry data are as follows: 1 H NMR (600 MHz, DMSO-d 6) δ 9.31 (s, 1H), 9.17 (d, J = 25.0 Hz, 2H), 8.85-8.76 (m, 2H), 8.55 (d, J =8.8Hz, 2H), 8.45(s, 1H), 8.24 (d, J = 8.3Hz, 1H), 8.05 -7.96 (m, 3H), 7.79 (s, 1H). ESI-MS m / z: [M]±641.07. Example 4: Synthesis of complex Pt4 The preparation method is the same as in Example 1, except that in step (1), 2-[(4-trifluoromethyl)phenyl]pyridine is replaced with 4-chloro-2,2'-bipyridine to obtain an orange solid complex Pt4; the yield is 45.0%.

[0021] The structural formula of the complex Pt4 is as follows:

[0022] Its 1H NMR and mass spectrometry data are as follows: 1 H NMR (500 MHz, DMSO- d 6) δ 9.28 (d, J = 5.9Hz, 1H), 9.02 (d, J = 5.5 Hz, 1H), 8.89 (d, J = 5.9 Hz, 1H), 8.69 (dd, J =19.4, 8.2 Hz, 2H), 8.47 (dt, J = 16.6, 7.7 Hz, 2H), 8.27-8.14 (m, 2H), 7.92(dt, J = 24.6, 6.8 Hz, 2H), 7.82 (d, J = 8.3 Hz, 1H), 7.51 (ddd, J = 7.3,5.7, 1.6 Hz, 1H), 7.30 (dd, J = 8.2, 1.9 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H).ESI-MS m / z: [M]±540.06. Example 5: Synthesis of complex Pt5 The preparation method is the same as in Example 1, except that in step (1), 2-[(4-trifluoromethyl)phenyl]pyridine is replaced with 4-chloro-2,2'-bipyridine, and in step (3), 2,2'-bipyridine is replaced with 1,10-phenanthroline to obtain the orange solid complex Pt5; the yield is 45.0%.

[0023] The structural formula of the complex Pt5 is as follows:

[0024] Its 1H NMR and mass spectrometry data are as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 9.48 (d, J = 5.3Hz, 1H), 9.26 (d, J = 5.0 Hz, 1H), 8.84 (dd, J = 17.6, 8.1 Hz, 2H), 8.75-8.68(m, 1H), 8.19 (dd, J = 19.8, 7.8 Hz, 2H), 8.12-8.08 (m, 1H), 8.00-7.89 (m,1H), 7.69 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.3 Hz, 1H), 7.23 (t, J = 6.5 Hz, 1H), 7.08 (s, 1H), 7.04-6.98 (m, 1H). ESI-MS m / z: [M]±564.06. Example 6: Synthesis of complex Pt6 The preparation method is the same as in Example 1, except that in step (1), 2-[(4-trifluoromethyl)phenyl]pyridine is replaced with 4-fluoro-2,2'-bipyridine, and in step (3), 2,2'-bipyridine is replaced with 1,10-phenanthroline to obtain the red solid complex Pt6; the yield is 43.0%.

[0025] The structural formula of the complex Pt6 is as follows:

[0026] Its 1H NMR and mass spectrometry data are as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 9.57 (s, 1H), 9.31 (s, 1H), 8.93-8.87 (m, 2H), 8.77 (d, J = 6.1 Hz, 1H), 8.24 (q,J = 8.9,8.3 Hz, 2H), 8.14-8.10 (m, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 8.6Hz, 1H), 7.51 (dd, J = 10.0, 4.7 Hz, 1H), 7.24-7.20 (m, 1H), 6.97 (d, J = 9.6Hz, 1H), 6.93-6.80 (m, 1H). ESI-MS m / z: [M]±547.08. Example 7: Anion Detection Experiment of Cyclic Metallized Platinum Complexes Weigh 3 mg of the complex obtained in Example 1 and add it to dimethyl sulfoxide to prepare a stock solution of 80 mmol / L. Weigh 1.5 mg of the complexes obtained in Examples 2, 4, and 5 and add them to dimethyl sulfoxide to prepare a stock solution of 20 mmol / L. Weigh 5 mg of the complex obtained in Example 3 and add it to dimethyl sulfoxide to prepare a stock solution of 200 mmol / L. Weigh 1 mg of the complex obtained in Example 6 and add it to dimethyl sulfoxide to prepare a stock solution of 5 mmol / L.

[0027] Take 3 μL of the complex stock solution into a 5 mL centrifuge tube, add 27 μL of dimethyl sulfoxide, and then dilute the complex of Example 1 with 3 mL of water to a complex solution of 80 μmol / L; dilute the complexes of Examples 2, 4, and 5 to a complex solution of 20 μmol / L; dilute the complex of Example 3 to a complex solution of 200 μmol / L; and dilute the complex of Example 6 to a complex solution of 5 μmol / L.

[0028] Then add CO3 separately. 2- H2PO4 - HCO3 - NO3 - SO4 2- I - CH3COO - Cl - NO2 - ,Br - F - SO3 2- ,ClO - ClO4 - HSO3 -The plasma was used, with a final ion concentration of 5 mmol / L. The fluorescence spectrum of the above solution was measured in the range of 400-800 nm. At the same time, a control (free) was set up with no added anions in the complex. The cuvette was photographed under UV light in a dark environment with a black baffle as the background.

[0029] See results Figure 3-8 As can be seen from the figure, the complex Pt1 can specifically recognize ClO4. - SO3 2- SO4 2- ,Br - NO3 - HCO3 - The control without anion showed weak orange fluorescence, with the fluorescence emission peak having the maximum fluorescence intensity at 450±20 nm. ClO4 was added to the Pt1 solution of the complex. - SO4 2- ,Br - NO3 - HCO3 - Afterwards, the solution emitted strong orange fluorescence, with the strongest fluorescence intensity at 570±20 nm. Compared to the control without anion, the fluorescence intensity increased by 56 times, 35 times, 28 times, 18 times, and 10 times, and the fluorescence shifted by 120 nm. SO3 anion was added to the Pt1 complex solution. 2- Afterwards, the solution exhibited green fluorescence, with the strongest fluorescence intensity at the green fluorescence emission peak at 500±20nm, which was 134 times stronger and red-shifted by 50nm compared to the control without anions.

[0030] The complex Pt2 can specifically recognize ClO4 - SO4 2- HSO3 - ,Br - NO3 - CO3 2- SO3 2- The control without anion showed weak red fluorescence, with the fluorescence emission peak exhibiting maximum intensity at 450±20 nm; ClO4 was added to the Pt2 complex solution. - SO4 2- HSO3 - ,Br - NO3 - CO3 2-Subsequently, the solution turned orange-red fluorescence, with the strongest fluorescence intensity at the orange-red fluorescence emission peak at 570±20 nm. Compared to the control without anion, the fluorescence intensity increased by 157, 139, 85, 80, 60, 58, and 52 times, respectively, and the fluorescence shifted by 120 nm. Adding SO3 anion to the Pt2 complex solution... 2- Afterwards, the solution turned green fluorescence, with the strongest fluorescence intensity at the green fluorescence emission peak at 500±20nm, which was 5 times stronger than the control without anion and red-shifted by 50nm.

[0031] The complex Pt3 can specifically recognize NO2. - I - CO3 2- SO3 2- The control without anion showed no fluorescence emission; NO2 was added to the Pt3 complex solution. - The solution emitted strong red fluorescence, with the strongest fluorescence intensity at 600±20 nm, representing an 18-fold increase compared to the control without anion. Adding I to the Pt3 complex solution... - CO3 2- The solution emitted blue fluorescence, with the strongest fluorescence intensity at 450±20 nm, which was 15 times and 8 times stronger than the control without anion. SO3 was added to the Pt3 complex solution. 2- The green fluorescence of the solution was enhanced, with the strongest green fluorescence emission peak at 520±20nm, which was 9 times stronger than the control without anions.

[0032] The complex Pt4 can specifically recognize NO2. - I - CO3 2- SO3 2- Without the addition of anions, it exhibits weak blue fluorescence, with the fluorescence emission peak showing maximum intensity at 450±20 nm. Adding SO4 to the Pt4 complex solution... 2- NO2 - Cl - ClO4 - HCO3 - NO3 - Subsequently, the solution changed from blue to orange fluorescence, with the strongest fluorescence intensity at 570±20 nm, representing an increase of 87, 35, 30, 26, 22, and 22 times compared to the control without anion, and a red shift of 120 nm. The addition of SO3 anion to the Pt4 complex solution further enhanced the fluorescence. 2-Subsequently, the solution changed from blue to green fluorescence, with the strongest green fluorescence emission peak at 500±20 nm, representing a 37-fold increase and a 50 nm red shift compared to the control without anion. Br was then added to the Pt4 complex solution. - I - Afterwards, the solution changed from blue to orange-red fluorescence. The orange-red fluorescence emission peak had the strongest fluorescence intensity at 570±20nm, which was 28 times and 26 times stronger than the control without anions, and red-shifted by 120nm.

[0033] The complex Pt5 can specifically recognize ClO - CO3 2- HCO3 - The control without anion showed weak red fluorescence, with the fluorescence emission peak having the maximum fluorescence intensity at 580±20 nm. Adding ClO to the Pt5 complex solution... - Afterwards, it emitted a strong red fluorescence, which was 6 times stronger than the control without anion; CO3 was added to the Pt5 complex solution. 2- HCO3 - Afterwards, the solution changed from red fluorescence to yellow fluorescence. The fluorescence intensity of the yellow fluorescence emission peak was strongest at 560±20nm, which was 3 times and 2 times stronger than the control without anion, respectively, and blue shifted by 20nm.

[0034] The complex Pt6 can specifically recognize Br - SO4 2- ClO4 - NO3 - SO3 2- The control without anion emitted red fluorescence, with the fluorescence emission peak exhibiting maximum intensity at 600±20 nm. Adding Br to the Pt6 complex solution... - SO4 2- ClO4 - NO3 - Subsequently, the solution changed from red fluorescence to orange-red fluorescence, with the strongest fluorescence intensity at the orange-red fluorescence emission peak at 600±20 nm, representing increases of 8, 6, 5, and 5 times compared to the control without anion addition, respectively. SO3 was added to the Pt6 complex solution. 2- Afterwards, the red fluorescence of the solution was quenched and turned into blue fluorescence. The blue fluorescence emission peak was strongest at 475±20nm, which was twice as strong as the control without anion and shifted by 125nm.

[0035] Example 8: Experiment on the detection of three biogenic thiols using the cyclic metallized platinum complex Pt3. Weigh 2 mg of complex Pt3 from Example 3 and add it to dimethyl sulfoxide to prepare a 150 mmol / L stock solution. Take 3 μL of the stock solution and transfer it to a 5 mL centrifuge tube. Add 27 μL of dimethyl sulfoxide, and then dilute the complex Pt3 with 3 mL of water to a 150 μmol / L solution. Add aqueous solutions of GSH, HCY, and CYS to the complex solution, respectively, to achieve a final thiol concentration of 5 mmol / L. Then, measure the fluorescence at 0 min, 1 min, 2 min, 3 min, 4 min, 5 min, 10 min, 15 min, 20 min, and 30 min in the 400-800 nm range to obtain fluorescence spectra. A control (thiol-free) was set up, along with controls containing 5 mmol / L of GSH, HCY, or CYS aqueous solutions. Photographs were taken under UV light in a dark environment with a black background.

[0036] The results are as follows Figure 9-10 As shown in the figure, the Pt3 complex solution showed no fluorescence emission before the addition of thiol. After adding GSH, the solution immediately emitted yellow-green fluorescence. After 20 minutes, the fluorescence intensity of the yellow-green fluorescence emission peak at 530±20 nm was the strongest, and the fluorescence intensity at 530±20 nm was 123 times stronger than before the addition of GSH. After adding HCY, the solution emitted yellow-brown fluorescence after about 25 minutes, with the strongest fluorescence intensity at 550±20 nm, and the fluorescence intensity at 550±20 nm was 10 times stronger than before the addition of HCY. After adding CYS, the solution emitted red fluorescence after about 25 minutes, with the strongest red fluorescence emission peak at 550±20 nm, and the fluorescence intensity at 550±20 nm was 15 times stronger than before the addition of CYS.

[0037] Example 9: Experiments using cyclic metallized platinum complexes Pt1-Pt6 as fluorescent probes for nucleic acid molecule detection. 1. Weigh 0.5 mg of the complexes from Examples 1, 2, 4, and 5 respectively, and add them to dimethyl sulfoxide to prepare a stock solution of 20 mmol / L. Weigh 2 mg of the complex from Example 3 and add it to dimethyl sulfoxide to prepare a stock solution of 150 mmol / L. Weigh 0.5 mg of the complex from Example 6 and add it to dimethyl sulfoxide to prepare a stock solution of 5 mmol / L.

[0038] Take 3 μL of the complex stock solution into a 5 mL centrifuge tube, add 27 μL of dimethyl sulfoxide, and then dilute the complexes of Examples 1, 2, 4, and 5 with 3 mL of water to a complex solution of 20 μmol / L; dilute the complex of Example 3 to a complex solution of 150 μmol / L; and dilute the complex of Example 6 to a complex solution of 5 μmol / L. Then, add aqueous solutions of DNA and RNA to the complex solutions, respectively, to a final concentration of 100 μmol / L. Measure the fluorescence in the 400-800 nm range to obtain fluorescence spectra. Set up a control (complexes without added nucleic acid) and a control of 100 μmol / L DNA or RNA aqueous solution. Take comparative photos of the cuvettes in a dark environment under UV light with a black background.

[0039] See results Figure 10-15 As shown in the figure, the Pt1 complex solution was colorless before the addition of DNA, and the fluorescence emission peak had the maximum fluorescence intensity at 450±20 nm. After the addition of DNA to the Pt1 complex solution, the solution changed from colorless to green fluorescence, and the green fluorescence emission peak had the strongest fluorescence intensity at 520±20 nm, which was 14 times stronger than before the addition of DNA. There was no fluorescence response after the addition of RNA to the Pt1 complex solution.

[0040] Before the addition of nucleic acid, the Pt2 complex solution exhibited weak red fluorescence, with the fluorescence emission peak showing the maximum fluorescence intensity at 580±20 nm. After the addition of DNA to the Pt2 complex solution, the solution changed from red to orange-red fluorescence, with the orange-red fluorescence emission peak showing the strongest fluorescence intensity at 600±20 nm, representing a 30-fold increase compared to before the addition of DNA. After the addition of RNA to the Pt2 complex solution, the red fluorescence of the solution increased, representing a 15-fold increase compared to before the addition of RNA.

[0041] The Pt3 complex solution showed no fluorescence emission before the addition of nucleic acid. After the addition of DNA to the Pt3 complex solution, the solution emitted light brown fluorescence, with the strongest fluorescence intensity at 500±20 nm, which was 11 times stronger than before the addition of DNA. The Pt3 complex solution showed no response upon the addition of RNA.

[0042] Before the addition of nucleic acids, the Pt4 complex solution exhibited weak blue fluorescence, with the fluorescence emission peak having the maximum fluorescence intensity at 450±20 nm. After the addition of DNA and RNA to the Pt4 complex solution, the solution changed from blue to orange fluorescence, with the orange fluorescence emission peak having the strongest fluorescence intensity at 570±20 nm. Compared with before the addition of DNA and RNA, the fluorescence intensity was enhanced by 9 times and 20 times, respectively, and the fluorescence intensity was red-shifted by 120 nm.

[0043] Before the addition of nucleic acid, the Pt5 complex solution exhibited weak red fluorescence, with the fluorescence emission peak reaching its maximum intensity at 580±20 nm. After the addition of DNA to the Pt5 complex solution, the solution changed from red fluorescence to orange-red fluorescence, with the orange-red fluorescence emission peak reaching its strongest intensity at 600±20 nm, representing an 8-fold increase compared to before the addition of DNA. After the addition of RNA to the Pt5 complex solution, it exhibited strong red fluorescence, representing a 3-fold increase compared to before the addition of RNA.

[0044] Before the addition of nucleic acid, the Pt6 complex solution exhibited weak red fluorescence, with the fluorescence emission peak having the maximum fluorescence intensity at 600±20 nm. After the addition of DNA to the Pt6 complex solution, the solution changed from red fluorescence to orange-red fluorescence, with the orange-red fluorescence emission peak having the strongest fluorescence intensity at 600±20 nm, which was 52 times stronger than before the addition of DNA. After the addition of RNA to the Pt6 complex solution, the red fluorescence of the solution was quenched.

[0045] Molecular docking was performed using complex Pt5 with deoxyribonucleic acid (DNA) (PBD: 126D). The structure of complex Pt5 was optimized using Chem3D 21.0 to minimize energy; results are shown below. Figure 16 The complex Pt5 can form strong hydrogen bonds with nucleotide residues in DNA, such as DT-4 and DC-3, with a binding energy of -8.11 kcal / mol.

[0046] 2. Response titration experiment of complex Pt4 with DNA / RNA solutions of different concentrations Weigh 2 mg of complex Pt4 and add it to dimethyl sulfoxide to prepare a 20 mmol / L stock solution. Then, take 3 μL of the complex Pt4 stock solution and add it to DNA or RNA aqueous solutions of different concentrations (concentration gradients of 0 μmol / L, 20 μmol / L, 40 μmol / L, 80 μmol / L, 100 μmol / L, 120 μmol / L, 140 μmol / L, 160 μmol / L, 180 μmol / L, and 200 μmol / L). Measure the fluorescence of the above solutions in the range of 400-800 nm to obtain fluorescence spectra.

[0047] See results Figure 17 As the concentration of DNA or RNA gradually increased from 0 μmol / L to 200 μmol / L, the absorption peak of Pt4 at 570 nm ± 20 nm showed a significant red shift of 120 nm, and the absorbance showed a linear increasing trend with the increase of DNA or RNA concentration. Figure 18The detection limit is based on the change in fluorescence intensity to determine the interaction between Pt4 and DNA and RNA. The lowest detection limit concentration of Pt4 for DNA is 32.69 μM, and the lowest detection limit concentration of Pt4 for RNA is 62.5 μM.

[0048] Example 10: Aggregation-induced emission (AIE) determination of cyclic metallized platinum complexes Pt1-Pt6 0.5 mg of the synthesized complexes from Examples 1, 4, 5, and 6 were weighed and added to dimethyl sulfoxide to prepare a 20 mmol / L stock solution. 1 mg of the synthesized complex from Example 2 was weighed and added to dimethyl sulfoxide to prepare an 80 mmol / L stock solution. 1.5 mg of the synthesized complex from Example 3 was weighed and added to dimethyl sulfoxide to prepare a 100 mmol / L stock solution. Then, 3 µL of each solution was added to a 5 mL centrifuge tube, and 27 µL of dimethyl sulfoxide was added. 3 mL of a mixture of water and dimethyl sulfoxide was added according to the following water contents: 0%, 10%, 30%, 50%, 70%, 90%, and 100%. Fluorescence was detected in the 400-800 nm range to obtain fluorescence emission spectra. The contrast cuvettes were photographed under UV light in a dark environment with a black background.

[0049] In Pt1 solution, when the water content is 50%, strong red fluorescence is emitted at 612 nm, exhibiting strong aggregation-induced emission (AIE). Subsequently, the red fluorescence drops sharply with increasing water content, and at 100% water content, the red fluorescence is quenched. Aggregation-induced emission (AIE) and aggregation fluorescence quenching (ACQ) coexist. In Pt2 solution, when the water content is 100%, bright red fluorescence is observed at around 600 nm, significantly stronger than in other water content ratios, without ACQ interference, exhibiting strong AIE activity. Figure 19 In Pt3 solution, blue fluorescence continuously decreased with increasing water content, and almost no fluorescence was observed at 100% water content. This indicates that 2-[2,4-bis(trifluoromethyl)phenyl]pyridine ligand failed to suppress the ACQ effect compared to 2-[(4-trifluoromethyl)phenyl]pyridine ligand. In Pt4 solution, a weak blue-green fluorescence was emitted at 445 nm at 30% water content, followed by a sharp drop in fluorescence until quenching with increasing water content. In Pt5 solution, a bright red fluorescence was observed at around 590 nm at 70% water content, exhibiting strong AIE activity, which subsequently decreased with increasing water content. In Pt6 solution, a bright red fluorescence was observed at around 620 nm at 100% water content, significantly stronger than other ratios, with no ACQ interference, and exhibiting strong AIE activity. Figure 20 ); Example 11: Determination of the antitumor activity of cyclic platinum metallide complexes in four cell lines (HeLa, HepG2, HCT116, HaCaT) Complexes Pt1, Pt2, Pt3, Pt4, and Pt5 were used as experimental groups, with cisplatin (cis) as the control group. Their cytotoxicity against HeLa (human cervical cancer cells), HepG2 (human liver cancer cells), HCT116 (human colorectal cancer cells), and HaCaT (human immortalized keratinocytes) was determined. The specific assay methods are as follows: The MTT assay was used to determine the concentration of tumor cells. The tested tumor cells were digested with trypsin into single-cell suspensions, and cell counts were performed using a hemocytometer. The cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 4 The drug concentration was 160 μL / well in 96-well plates and cultured for 24 h. Then, different concentrations of the drug were added, and the plates were incubated in a 5% CO2 incubator at 37°C for 48 h. Four h before the end of incubation, 20 μL of MTT was added per well. After 4 h, the supernatant was discarded, and 150 μL of DMSO was added per well. After shaking for 5 min, the OD value was measured using a microplate reader at a wavelength of 492 nm. The survival rate of the tested tumor cells was calculated, and the IC50 was plotted and determined. 50 Values ​​were used to evaluate the antitumor activity of the complexes.

[0050] The results are shown in Table 1. The table shows that Pt5 exhibited the best cytotoxicity in HeLa cells, followed by Pt2, IC50, and Pt2. 50 The values ​​were 7.58±1.94 and 10.01±2.92, respectively, indicating that the cytotoxicity was increased by 2.8 times and 2.1 times compared with the control group cisplatin. In HepG2 cells, the cytotoxicity of Pt2 and Pt5 was similar, with IC50 values ​​of 7.58±1.94 and 10.01±2.92, respectively. 50 The values ​​were 10.24±2.39 and 10.86±2.97, respectively. Compared with the control group cisplatin, the cytotoxicity was increased by 1.9 times and 1.8 times, respectively. The IC50 of Pt1 was... 50 The value was 18.35±1.75, and its cytotoxicity was comparable to that of the control group cisplatin, exhibiting similar antitumor activity. In HCT116 cells, Pt5 showed the best cytotoxicity, followed by Pt2, with an IC50 value of 18.35±1.75. 50 The values ​​were 16.84±3.03 and 18.24±2.4, respectively, which showed 1.2-fold and 1.1-fold increased cytotoxicity compared with the control group cisplatin. In HaCaT cells, Pt2 showed the best cytotoxicity, followed by Pt5, with an IC50 of 16.84±3.03 and 18.24±2.4, respectively. 50 The values ​​were 5.05±2.34 and 7.61±1.61, respectively. Compared with the control group cisplatin, the cytotoxicity was increased by 4.1 times and 2.7 times, respectively. The toxicity IC50 value of Pt1 was 19.21±1.99, which was comparable to that of the control group cisplatin, and it had similar antitumor activity as cisplatin.

[0051] Table 1 .

[0052] Example 12: Dual-channel fluorescence monitoring of the uptake and localization of ring nanoparticles in tumor cells HeLa and HaCaT cells in good growth condition were digested with trypsin and seeded into confocal culture dishes. The cells were cultured at 37°C in an incubator containing 5% CO2. When the HeLa and HaCaT cell density reached 70%, the complex Pt3 was added, resulting in a final drug concentration of 20 μmol / L. After culturing for another 6 hours, the cells were washed twice with PBS and observed using a laser confocal microscope. (Green channel: λex = 468±30nm; λem = 488±30nm; Orange channel: λex = 540±30nm; λem = 561±30nm).

[0053] The results are as follows Figure 21 The results showed that after co-incubating the complex Pt3 with HeLa cells for 6 hours, strong yellow fluorescence was observed in the cytoplasm; after co-incubating the complex Pt3 with HaCaT cells for 6 hours, orange fluorescence was observed in both the cell nucleus and cytoplasm.

[0054] HeLa cells in good growth condition were digested with trypsin and seeded into confocal culture dishes. They were cultured at 37°C in an incubator containing 5% CO2. When the HeLa cell density reached 70%, the complex Pt3 was added. After 6 hours of culture, the cells were gently washed twice with PBS. Then, they were incubated for 15-20 minutes in a constant temperature incubator using nuclear blue probes, endoplasmic reticulum blue probes, lysosomal blue probes, and mitochondrial red probes, respectively. After incubation, the cells were gently washed twice with PBS and observed under a confocal microscope. The results are as follows: Figure 22 The figure shows that after co-incubating the complex Pt3 with HeLa cells for 6 hours, Pt3 was mainly located in the mitochondria.

Claims

1. A class of cyclic metallized platinum complexes, whose chemical structural formula is shown in Formula I: Formula I ; in Selected from ; Selected from .

2. The application of the cyclic metallized platinum complex of claim 1 as a fluorescent probe in the specific detection of three biothiols, characterized in that: Cyclic metallized platinum complexes are It uses red fluorescence to detect cysteine, yellow-brown fluorescence to detect homocysteine, and yellow-green fluorescence to detect glutathione.

3. The application of the cyclic metallized platinum complex of claim 1 as a fluorescent probe in the detection of anions, characterized in that: Orange fluorescence-enhanced detection of ClO4 - SO4 2- ,Br - NO3 - HCO3 - Green fluorescence enhanced detection of SO3 2- ; Used for orange-red fluorescence enhancement of detected ClO4 - SO4 2- HSO3 - ,Br - NO3 - CO3 2- Green fluorescence enhanced detection of SO3 2- ; NO2 detection using red fluorescence enhancement - I was detected using a light blue fluorescence-enhanced detector. - CO3 2- A green fluorescence-enhanced detection method was used for SO3. 2- ; SO4 detection using orange fluorescence enhancement 2- NO2 - Cl - ClO4 - HCO3 - NO3 - Orange-red fluorescence-enhanced detection of Br was employed. - I - Green fluorescence enhanced detection of SO3 2- ; Detection of ClO using red fluorescence enhancement - Yellow fluorescence-enhanced detection of CO3 2- HCO3 - ; Orange-red fluorescence-enhanced detection type Br - SO4 2- ClO4 - NO3 - quenching detection of SO3 2- .

4. The application of the cyclic metallized platinum complex of claim 1 as a fluorescent probe in the detection of nucleic acid molecules, characterized in that: DNA molecules are detected using a green fluorescence-enhanced method. Orange-red fluorescence enhancement was used to detect DNA molecules, and red fluorescence enhancement was used to detect RNA molecules. DNA molecules were detected using brown fluorescence enhancement. Orange fluorescence enhancement is used to detect DNA or RNA molecules; Orange-red fluorescence enhancement was used to detect DNA molecules, and red fluorescence enhancement was used to detect RNA molecules. Orange-red fluorescence enhancement was used to detect DNA molecules, while quenching was used to detect RNA molecules.

5. The application of the cyclic metallized platinum complex of claim 1 as a fluorescent probe in the detection of nucleic acid concentration.

6. The use of the cyclic metallized platinum complex according to claim 1 in the preparation of cell diagnostic and imaging reagents.

7. The use of the cyclic metallized platinum complex according to claim 1 in antitumor drugs.

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