Organic room-temperature phosphorescent molecular compound as well as preparation method and application thereof

Abstract

The invention provides an organic room-temperature phosphorescent molecular compound as well as a preparation method and application thereof. The organic room-temperature phosphorescent molecular compound provided by the invention takes a hexa-substituted aromatic hydrocarbon phosphorescent core as a core molecule, a group capable of inducing an intermolecular hydrogen bond is modified on the core molecule, and different aggregation states are formed through a pi-pi stacking effect and the hydrogen bond. The preparation method of the compound comprises the following steps: based on hexa-substituted benzene, further synthesizing to obtain a hexa-substituted aromatic hydrocarbon structure as a compound skeleton, and then introducing groups including carboxyl, carboxyl derivatives, hydroxyl or amide to the skeleton. The organic room-temperature phosphorescent molecular compound can be used for cell life imaging and can be used as a pH sensing probe for detecting intracellular pH change. The service life of the compound provided by the invention is as long as 30 microseconds when the water content is 99%, the compound can respond to pH, and accurate and reliable life bioimaging is provided in clinical biomedical application; and when being used as a pH sensing probe, the probe can accurately reflect the change of pH in cells.

Description

Technical Field

[0001] The invention belongs to the technical field of luminescent materials, and in particular relates to an organic room temperature phosphorescent molecular compound and a preparation method and application thereof. Background Art

[0002] Cellular pH is a key parameter for the normal operation of various biological processes. The stability of pH is crucial in these physiological and pathological processes, including cell proliferation, apoptosis and endocytosis. Therefore, monitoring pH changes is very important in the fields of chemistry, biology and medicine. Among them, changes in intracellular pH are closely related to cancer occurrence and cell aging. Studies have found that cancer cells show a pH gradient opposite to that of normal somatic cells, that is, intracellular pH (pH i ) is higher than the extracellular pH (pH e ), which can promote cancer cell proliferation, escape apoptosis, and accelerate cancer development (Nature Reviews Cancer 11,671–677(2011). In the study of neurodegenerative diseases caused by cell aging, it was found that the intracellular pH acidified (pH i <pH e ), changes in intracellular pH can be used as a marker for diagnosing early cell aging and cancer. (Cell.Mol.Life Sci.79,380(2022). Therefore, intracellular pH sensing probes came into being.

[0003] With the support of fluorescence imaging technology, many fluorescent probes have been developed to sense changes in intracellular pH. So far, the literature has reported the detection of intracellular pH changes by changes in fluorescence intensity (Dyes and Pigments 205 (2022) 110545.), but the results are biased due to the fluctuation of fluorescence intensity, excitation intensity, and sample concentration (ACS sensors, 2019, 4 (4): 883-891). In contrast, fluorescence lifetime imaging microscopy (FLIM) shows obvious superiority because it is not affected by changes in excitation luminescence intensity and fluorophore concentration. Therefore, there is an urgent need to invent a long-life adjustable FLIM fluorescent probe to sense changes in intracellular pH and reflect the cell state in real time.

[0004] Organic room temperature phosphorescent materials have attracted increasing attention due to their unique optical properties and good applications in bioimaging, chemical sensing, information encryption, etc., but the lack of molecular systems that can integrate them into solution aggregates has greatly limited their related biological applications (ACS Applied Materials & Interfaces, 2023, 15(18): 22415-22425.).

[0005] Based on the above research background, a long-lived adjustable molecular probe that senses intracellular pH has been developed. It is of great significance to use a detection probe whose emission wavelength and lifetime change with pH in the molecular system. This can reflect the intracellular pH and then the cell state according to the changes in the emission wavelength and lifetime of the molecular system, and is suitable as a simple means of clinical diagnosis.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide an organic room temperature phosphorescent molecular compound and a preparation method and application thereof to solve the above problems.

[0008] In order to achieve the above objectives, the present invention particularly adopts the following technical solutions:

[0009] An organic room temperature phosphorescent molecular compound, wherein the compound takes a hexasubstituted aromatic hydrocarbon phosphorescent core as a core molecule, a group capable of inducing intermolecular hydrogen bonds is modified on the core molecule, and different aggregation states are formed through π-π stacking and hydrogen bonding.

[0010] The hexasubstituted aromatic skeleton is used as the core structure, and then the groups that can induce hydrogen bonds between molecules are modified on the periphery. Different aggregation states are further formed through π-π stacking and hydrogen bonds, and the luminescence and lifetime properties of the molecule are regulated through the diverse self-assembly induced by hydrogen bonds. The emission wavelength and lifetime of the molecule will be sensitive to pH, and can be used for lifetime imaging to sense pH changes in cells.

[0011] Optionally, the general structural formula of the compound includes Formula I, wherein Formula I is:

[0012]

[0013] Wherein, R includes any one or more of carboxyl, carboxyl derivatives, hydroxyl, and amide.

[0014] Preferably, the organic room temperature phosphorescent molecular compound includes any one of hexacarboxylic acid ethyl hexathiobenzene, hexacarboxyl hexathiobenzene, hexahydroxy hexathiobenzene, and hexaamide hexathiobenzene. The structural formulas thereof are:

[0015]

[0016] The general structural formula of the compound also includes Formula II, which is:

[0017]

[0018] Wherein, R is a carboxyl group.

[0019] Preferably, the organic room temperature phosphorescent molecular compound includes hexacarboxyhexaselenobenzene, and its structural formula is

[0020] The present application provides a method for preparing the organic room temperature phosphorescent molecular compound, which comprises: taking hexasubstituted benzene as the basis, further synthesizing a hexasubstituted aromatic hydrocarbon structure as the compound skeleton, and then introducing groups including carboxyl, carboxyl derivatives, hydroxyl or amide on the skeleton to obtain the compound.

[0021] Optionally, the preparation method further comprises: adding ethyl p-mercaptobenzoate, hexachlorobenzene and anhydrous potassium carbonate into a reaction vessel, injecting N,N-dimethylformamide under nitrogen protection, wherein the molar mass ratio of ethyl p-mercaptobenzoate, hexachlorobenzene and anhydrous potassium carbonate is 16-20:0.5-2.5:16-20, and reacting at 60-70°C for 40-45h; further optionally, after the reaction is completed and cooled, dripping into ice water to generate a yellow precipitate, filtering, repeatedly washing the solid with a large amount of water, ethanol and acetone, and drying to obtain the hexacarboxylic acid ethyl hexathiobenzene; further optionally, after the reaction is completed and cooled, dripping into ice water to generate a yellow precipitate, filtering, repeatedly washing the solid with a large amount of water, ethanol and acetone, drying, dissolving in THF, and dripping sodium hydroxide aqueous solution, stirring at room temperature for 18h, adding dilute hydrochloric acid solution to adjust the solution to acidity and precipitate, filtering the obtained solid, washing with water, ethanol and acetone, and drying to obtain the hexacarboxylic hexathiobenzene;

[0022] Alternatively, in a reaction container, hexa(3-methoxy-1-phenylthio)benzene is added under nitrogen protection and dissolved in dry dichloromethane, and BBr3 is quickly added to the solution at -10°C, wherein the molar mass ratio of hexa(3-methoxy-1-phenylthio)benzene to BBr3 is 1-1.5:20-25, and then stirred. After stirring for 15 hours, the reaction mixture is poured into cold water and vigorously stirred until no precipitate is formed, and the hexahydroxyhexathiobenzene is obtained after filtering and drying;

[0023] Alternatively, hexafluorobenzene and p-mercaptobenzamide are added to a reaction container under nitrogen protection and dissolved in 1,3-dimethyl-2-imidazolidinone, and sodium hydride powder is slowly added at 0°C, wherein the molar mass ratio of hexafluorobenzene to p-mercaptobenzamide is 1-3:20-25, and the reaction is carried out at 0°C for 2 hours and then at room temperature for 24 hours. Ice water and a saturated aqueous NaOH solution (100 mL) are added to the reaction solution, and the solid is collected by filtration, and washed with a large amount of distilled water, ethanol, ethyl acetate, and acetone in sequence to obtain the hexafluoroamide hexathiobenzene.

[0024] Optionally, the preparation method further comprises: adding ethyl 4-aminobenzoate, water and concentrated hydrochloric acid in a reaction container under ice bath conditions, dropping a NaNO2 aqueous solution, and stirring at 0°C for 10 minutes;

[0025] Then, sodium acetate, KSeCN and NaHCO3 aqueous solution were added and stirred for 30 minutes, and the solution was heated to 55°C and stirred for 30 minutes. A mixture of zinc powder, Et2O and H2SO4 was added and stirred at 40°C for 1.5 hours. The obtained product and NaH were added to HMPA under nitrogen protection, stirred at 0°C for 3 hours, and hexabromobenzene was added and stirred at room temperature for 24 hours. Water was added for filtration, dissolved in tetrahydrofuran, and then NaOH aqueous solution was added and stirred at 70°C for 18 hours; the mixture was neutralized with dilute hydrochloric acid, and then the obtained solid was washed with deionized water and ethanol to obtain the hexacarboxyhexaselenobenzene;

[0026] Among them, the molar mass ratio of ethyl 4-aminobenzoate, NaNO2, sodium acetate, KSeCN, NaHCO3, zinc powder, NaH and hexabromobenzene is 3-5:3-5:16-17:3-5:1-2:10-11:23-25:0.3-0.5.

[0027] The present application also provides an application of the organic room temperature phosphorescent molecular compound, which includes: being used for cell life imaging and being used as a pH sensing probe for detecting intracellular pH changes.

[0028] Preferably, the application method of the pH sensing probe for detecting intracellular pH changes comprises: dissolving the organic room temperature phosphorescent molecular compound in a mixed solution of water and dimethyl sulfoxide, and detecting pH changes by measuring changes in the emission wavelength and lifetime of the system when the volume fraction of water is 99%.

[0029] Beneficial effects of the present invention:

[0030] The present application provides a series of compounds based on hexasubstituted aromatics, wherein the compound is a hexasubstituted aromatic phosphorescent core as the core structure, and then a group that can induce hydrogen bonds between molecules is modified on its periphery, and the luminescence and life properties of the molecule are regulated by self-assembly induced by hydrogen bonds. The compound has the characteristics of aggregation-induced luminescence, is sensitive to the presence of water in organic solvents, and the molecular emission wavelength and life change are affected by pH. Specifically, within a certain range, as the water fraction increases, its emission wavelength first blue-shifts and then red-shifts, and the life gradually increases. When the water content reaches 99%, the life is as long as 30μs. In the process of pH changing from 6.07 to 8.06, the emission wavelength blue-shifts, and the life decays to the ns level. In the process of pH changing from 7.78 to 5.78, the molecular emission wavelength red-shifts and the life can be restored. Therefore, the compound can be used as a molecular probe for real-time detection of intracellular pH changes by emission wavelength and life change.

[0031] Compared with the existing pH sensing probes, the series of compounds provided by the present invention have the characteristics of pH sensitivity, can sense the changes in intracellular pH according to the changes in emission wavelength and lifetime, and have the advantages of cheap and readily available raw materials, simple synthesis, convenient detection, etc. It solves the technical problems of high detection cost and complex synthesis route in the prior art, and effectively improves the detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 The H NMR spectrum of the compound provided in Example 2;

[0034] Figure 2 The fluorescence emission diagram of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with different water volume contents;

[0035] Figure 3 A lifetime decay diagram of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with a water volume fraction of 40%;

[0036] Figure 4 A lifetime decay diagram of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with a water volume fraction of 70%;

[0037] Figure 5 A lifetime decay graph of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with a water volume fraction of 99%;

[0038] Figure 6 A hydrogen nuclear magnetic spectrum of the compound provided in Example 2 in a water volume content of 0% to 99%;

[0039] Figure 7 The fluorescence emission graphs of the compound provided in Example 2 when NaOH solutions of different concentrations are added to a mixed solution of dimethyl sulfoxide and water having a water volume content of 99%;

[0040] Figure 8 The life decay graph of the compound provided in Example 2 when NaOH solutions of different concentrations are added to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99%;

[0041] Fig. 9The life decay graph of the compound provided in Example 2 when NaOH solutions of different concentrations are added to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99%;

[0042] Fig.10 Fluorescence emission graphs of the compound provided in Example 2 after adding 160 μmol / L NaOH solution to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99% and then adding CF3COOH solutions of different concentrations;

[0043] Fig.11 The life decay graph of the compound provided in Example 2 after adding 160 μmol / L NaOH solution to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99% and then adding CF3COOH solutions of different concentrations;

[0044] Fig.12 The life decay graph of the compound provided in Example 2 after adding 160 μmol / L NaOH solution to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99% and then adding CF3COOH solutions of different concentrations;

[0045] Fig.13 This is a graph showing the change in afterglow color of a film made using the compound provided in Example 2 after CF3COOH acid evaporation;

[0046] Fig.14 A test diagram showing the effect of different excitation lights on the material provided in Comparative Example 1;

[0047] Fig.15 This is a test diagram of the pH sensing range of the material provided in Comparative Example 1;

[0048] Fig.16 This is a working curve of the compound provided in Example 2 sensing pH changes in a mixed solution of dimethyl sulfoxide and water with a water volume content of 99%. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0050] The room temperature described in the following examples is: 25-28°C; the raw materials and reagents used are all commercially available.

[0051] Example 1

[0052] This embodiment provides an organic room temperature phosphorescent molecular compound and a preparation method thereof, specifically hexacarboxylic acid ethyl ester hexathiobenzene, and the preparation steps are as follows:

[0053] S1: Add p-mercaptobenzoic acid (1.52 g, 10 mmol), 150 mL of ethanol, and 0.2 mL of concentrated sulfuric acid as catalyst into a 250 mL round-bottom flask, and reflux under condensation for 20 h;

[0054] S2: The S1 system was naturally cooled to room temperature, added dropwise into water, extracted with ethyl acetate three times, and concentrated in vacuo to obtain oily ethyl p-mercaptobenzoate;

[0055] S3: Add ethyl p-mercaptobenzoate (1.68 g, 18 mmol, 9 eq.) obtained in S2, hexachlorobenzene (0.568 g, 2 mmol, 1 eq.) and anhydrous potassium carbonate (2.48 g, 18 mmol, 9 eq.) into a three-necked flask, protect with nitrogen, inject 15 mL of dry DMF, react at 60 ° C for 40 h, cool and drop into 150 mL of ice water to form a yellow precipitate, filter, wash the solid repeatedly with large amounts of water, ethanol and acetone, and dry to obtain ethyl hexacarboxylate hexathiobenzene.

[0056] Calculated yield: 55%. Product characterization: 1H NMR (400 MHz, C2D6OS) δ (ppm): 7.85 (d, J = 8.1 Hz, 12H), 7.20 (d, J = 8.1 Hz, 12H), 4.34 (q, J = 7.1 Hz, 12H), 1.37 (t, J = 7.1 Hz, 18H).

[0057] Example 2

[0058] This embodiment provides an organic room temperature phosphorescent molecular compound and a preparation method thereof, specifically hexacarboxyhexathiobenzene, and the preparation steps are as follows:

[0059] S1-S3 are the same as in Example 1;

[0060] S4: The product of S3, hexacarboxylic acid ethyl ester hexathiobenzene (1.15 g, 1 mmol), was dissolved in 20 mL THF, and sodium hydroxide aqueous solution (2.0 mol / L, 10 mL) was added dropwise to the solution, stirred at room temperature for 18 h, and diluted hydrochloric acid solution (1.0 mol / L, 250 mL) was added to acidify the solution and precipitate, filtered, and the solid was washed with water, ethanol and acetone in turn to obtain a yellow powder, which was hexacarboxyhexathiobenzene, marked as S6, with the structural formula

[0061] The calculated yield is 60%, and its H NMR spectrum is as follows Figure 1 shown. 1H NMR (400MHz, C2D6OS) δ (ppm): 12.90 (s, 6H), 7.82-7.75 (m, 12H), 7.17-7.10 (m, 12H). 13C NMR (101MHz, C2D6OS) δ (ppm): 167.09, 147.66, 143.04, 130.64, 128.75, 126.86.

[0062] The hexacarboxyl hexasulfobenzene provided in this example was prepared into a mixed solution of dimethyl sulfoxide and water, the proportion of water was 0%, 40%, 70%, 90%, 99% in sequence, and the concentration was 10 μmol / L. 2 mL of the solution was added to a 1 cm×1 cm×4 cm stoppered cuvette for performance testing:

[0063] 1. Test the fluorescence emission spectrum, λex = 365nm, the results are as follows Figure 2 As shown, Figure 2 This is a fluorescence emission diagram of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with different water volume contents, with the horizontal axis being the emission wavelength and the vertical axis being the fluorescence intensity value. Figure 2 It can be seen that within a certain range, as the water content in the solution increases, its fluorescence intensity gradually increases, and the emission wavelength first blue-shifts and then red-shifts.

[0064] 2. Test the lifetime decay spectrum, λex = 365nm, and observe the relationship between lifetime and water volume fraction. The results are as follows: Figure 3-5 As shown, Figure 3 A lifetime decay diagram of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with a water volume fraction of 40%; Figure 4 A lifetime decay diagram of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with a water volume fraction of 70%; Figure 5 This is a lifetime decay diagram of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with a water volume fraction of 99%, with the horizontal axis representing time and the vertical axis representing intensity value; Figure 3-5 It can be seen that within a certain range, the life span increases with the increase of water content.

[0065] 3. The mixed solution with different water volume fractions was subjected to NMR titration test, and its hydrogen NMR spectrum was as follows: Figure 6 As shown, NMR titration shows that adding D2O (concentration of 10 -4 M), within a certain range, with the increase of water content, hydrogen bonds are formed between water molecules and hexacarboxyhexasulfide molecules, resulting in an integral shielding effect, which causes the aromatic protons on the hexacarboxyhexasulfide molecules to move to the high field. At the same time, with the formation of hydrogen bonds, the proton resonance of the hexacarboxyhexasulfide molecules becomes broadened, which indicates that there may be an aggregation effect.

[0066] The hexacarboxyl hexasulfobenzene provided in this embodiment was prepared into a mixed solution of dimethyl sulfoxide and water, the volume fraction of water being 99%, and NaOH solutions of different concentrations were added to the solution. 2 mL of the solution was added to a 1 cm×1 cm×4 cm stoppered cuvette to test its performance:

[0067] 4. Test the fluorescence emission spectrum, λex = 365nm, and observe the relationship between the emission wavelength and the concentration of NaOH added. The results are as follows: Figure 7 As shown, Figure 7 The fluorescence emission diagram of the compound provided in Example 2 when NaOH solutions of different concentrations are added to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99%; Figure 7 It can be seen that within a certain range, as the concentration of the added NaOH solution increases, the emission wavelength blue shifts;

[0068] 5. Test the lifetime decay spectrum, λex = 365nm, and observe the relationship between the lifetime and the added NaOH concentration. The results are as follows: Figure 8-9 As shown, Figure 8-9 The life decay diagram of the compound provided in Example 2 when NaOH solutions of different concentrations are added to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99%; Figure 8-9 It can be seen that within a certain range, the lifetime decays from microseconds to nanoseconds as the NaOH concentration increases.

[0069] The hexacarboxyl hexasulfobenzene obtained in this example was prepared into a mixed solution of dimethyl sulfoxide and water, with a water volume fraction of 99%. A NaOH solution with a concentration of 160 μmol / L was added to the solution, followed by CF3COOH solutions of different concentrations. 2 mL of the solution was added to a 1 cm×1 cm×4 cm stoppered cuvette, and its performance was tested:

[0070] 6. Test the fluorescence emission spectrum, λex = 365nm, and observe the relationship between the emission wavelength and the added CF3COOH solution. The results are as follows: Fig.10 As shown, Fig.10 The fluorescence emission diagram of the compound provided in Example 2 after adding 160 μmol / L NaOH solution to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99% and then adding CF3COOH solutions of different concentrations; Fig.10 It can be seen that within a certain range, as the concentration of CF3COOH solution increases, the emission wavelength red-shifts.

[0071] 7. Test the lifetime decay spectrum, λex = 365nm, and observe the relationship between the lifetime and the added CF3COOH solution. The results are as follows: Figure 11-12 As shown, Figure 11-12The lifetime decay diagram of the compound provided in Example 2 after adding 160 μmol / L NaOH solution to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99% and then adding CF3COOH solutions of different concentrations; Figure 11-12 It can be seen that within a certain range, as the concentration of CF3COOH solution increases, the lifetime gradually increases from nanoseconds to microseconds.

[0072] The hexacarboxyl hexasulfobenzene provided in this embodiment is prepared into a dimethyl sulfoxide solution, which is mixed with an aqueous solution of PVA and stirred evenly to form a film, such as Fig.13 As shown, Fig.13 The graph is a graph showing the change in afterglow color of a film made using the compound provided in Example 2 after CF3COOH acid evaporation; Fig.13 It can be seen that the original film afterglow is yellow. After CF3COOH acid evaporation, the film afterglow changes from yellow to green. The film with green afterglow is then treated with C6H 15 After alkali N is evaporated, the afterglow returns to yellow.

[0073] Example 3

[0074] This embodiment provides an organic room temperature phosphorescent molecular compound and a preparation method thereof, specifically hexahydroxyhexathiobenzene, and the preparation steps are as follows:

[0075] In a 250mL round-bottom flask, hexylbenzene (3-methoxy-1-phenylbenzene) (1.0 g, 1.1 mmol) and 70 mL of dry dichloromethane were added to dissolve and stirred under nitrogen atmosphere, and then BBr3 (2.0 mL, 22.0 mmol) was quickly added to the -10°C solution. After stirring the solution for 15 hours, the reaction mixture was poured into cold water and stirred vigorously until no precipitation was formed. After filtering and drying, a yellow solid was obtained, which was the product hexahydroxyhexathiobenzene, with a yield of 90%. The product hydrogen spectrum is 1 H NMR (600 MHz, DMSO-d6) δ (ppm) 6.27 (m, 6H), 6.42 (s, 6H), 6.54 (m, 6H), 7.03 (t, 6H), 9.55 (s, 6H). 13C nuclear magnetic resonance (600 MHz, DMSO-d6) δ (ppm) 113.27, 113.65, 117.36, 130.18, 138.56, 147.23, 157.84. MS (TOF-MS, m / z): MS (ESI+, m / z): 823.0445 [M+H]+.

[0076] Example 4

[0077] This embodiment provides an organic room temperature phosphorescent molecular compound and a preparation method thereof, specifically hexaamide hexathiobenzene, and the preparation steps are as follows:

[0078] In a 250ml three-necked flask under nitrogen protection, add hexafluorobenzene (0.372g, 2.0mmol) and p-mercaptobenzamide (4.08g, 24mmol) dissolved in DMI (20mL), cool to 0℃, slowly add sodium hydride powder (purity 60%, 0.96g, 24mmol), react the mixture at this temperature for 2h, then react at room temperature for 24h, add ice water and saturated NaOH aqueous solution (100mL) to the reaction solution, filter and collect the solid, wash the product with a large amount of distilled water, ethanol, ethyl acetate, and acetone in turn, and obtain a white powder, which is the product hexafluorobenzene hexathioamide, with a mass of 1.00g, and a calculated yield of 46%. Product hydrogen spectrum 1 H NMR (400MHz, DMSO-d6): δ = 2.01 (s, 18H), 7.14 (d, J = 8.0Hz, 12H), 7.47 (d, J = 8.0Hz, 12H), 9.93 (s, 6H). 13C NMR (100MHz, DMSO-d6): δ = 168.80, 138.95, 129.99, 127.76, 120.29, 110.00, 24.46.MS: MALDI-TOF MS, m / z: [M+H]+calcd for C54H48N6O6S6,1068.20; foundm / z,1068.48.

[0079] Example 5

[0080] A mixture of ethyl 4-aminobenzoate (0.73 g, 4.8 mmol), 14 mL of water and concentrated hydrochloric acid (12 M, 0.86 mL) was added to a 250 ml three-necked flask and stirred in an ice bath. Then, an aqueous solution (2 mL) of sodium nitrite (0.33 g, 4.8 mmol) was added dropwise, and the mixture was stirred at 0°C for 10 minutes; a solution of sodium acetate (1.33 g, 16.2 mmol), KSeCN (0.58 g, 4.00 mmol) and sodium bicarbonate (0.10 g, 1.19 mmol) in water (2 mL) was added at 0°C, stirred at 0°C for 30 minutes, and then heated to 55°C and stirred for 30 minutes; after cooling, the mixture was extracted with dichloromethane, the combined extracts were washed with brine, dried over anhydrous magnesium sulfate, the solvent was removed, and the crude product was further purified by silica gel column chromatography with gasoline ether / ethyl acetate (v / v, 5:1) as the eluent, and then recrystallized from hexane to obtain white crystals, whose hydrogen spectrum is 1H NMR (400MHz, CDCl3) δ = 8.04 (d, J = 8.3, 2H), 7.66 (d, J = 8.3, 2H), 3.93 (s, 3H).13C NMR(100MHz, CDCl3)δ=165.89,132.51,131.26,131.24,131.11,130.45,128.08,100.44,52.54.MALDI-TOF MS,m / z:calcd.for C9H7NO2Se[MH]+241.9642,found 242.3337.

[0081] A mixture of the white crystals (682 mg, 2.84 mmol) obtained in the above step, zinc powder (689 mg, 10.5 mmol), Et2O (14 mL) and sulfuric acid (20%, 6 mL) was stirred at 40°C for 1.5 hours. After filtration, the residue was extracted with dichloromethane, the extract was washed, dried over anhydrous magnesium sulfate and evaporated, and the crude product was purified by column chromatography using gasoline ether / ethyl acetate (v / v, 5:1) as eluent to obtain 530 mg of yellow crystals with a yield of 87%. The hydrogen spectrum 1 H NMR (400MHz, CDCl3) δ = 7.94 (d, J = 7.3, 4H), 7.67 (d, J = 7.3, 4H), 3.92 (s, 6H). 13C NMR (100MHz, CDCl3) δ166.48, 136.48, 130.28, 130.10, 129.34, 52.24. MALDI-TOF MS, m / z: calcd. for C16H14NO4Se2[M]+429.9223, found 429.9868.

[0082] A mixture of the yellow crystals obtained above (514 mg, 1.2 mmol) and NaH (60%, 960 mg, 24 mmol) was added to HMPA (6 mL) and stirred for 3 hours under a nitrogen atmosphere. A solution of hexabromobenzene (165 mg, 0.4 mmol) was added to HMPA (6 mL), followed by stirring at room temperature for 24 h. The crude product was precipitated after adding water (250 mL), filtered, and purified by silica gel column chromatography with dichloromethane / ethyl acetate (v / v, 50:1) as the eluent to obtain red crystals (173 mg, 32%). 1H NMR (400MHz, CDCl3) δ7.83 (d, J = 8.4Hz, 12H), 7.14 (d, J = 8.4Hz, 12H), 3.95 (s, 18H). 13C NMR (100MHz, CDCl3)δ166.31,148.36,141.10,130.50,129.80,128.76,52.22.MALDI-TOF MS,m / z:calcd.for C54H42O12Se6[MH]+1357.7488,found 1357.6754.

[0083] To a tetrahydrofuran (10 mL) solution of the above-obtained red crystal (173 mg, 0.13 mmol), an aqueous solution of sodium hydroxide (2 M, 10 mL) was added, and after stirring at 70°C for 18 h, the mixture was neutralized with dilute hydrochloric acid, and the obtained solid was washed with deionized water and ethanol, and then a red solid was obtained, namely hexacarboxyhexaselenobenzene, with a mass of 133 mg and a yield of 60%. Hydrogen spectrum 1 H NMR (400MHz, DMSO) δ12.94 (s, 6H), 7.79 (d, J = 8.5 Hz, 12H), 7.26 (d, J = 8.4 Hz, 12H). 13C NMR (100MHz, DMSO) δ167.18,148.34,141.77,130.80,129.44,129.35.MS: MALDI-TOF MS, m / z: calcd.for C48H30O12Se6[MH]+1273.5868, found 1273.4639.

[0084] Comparative Example 1

[0085] The prior art reports a new type of fluorescent silicon nanoparticles (SiNPs) as a pH-sensitive probe. The fluorescence intensity is positively correlated with the pH value of the solution, making SiNPs a feasible pH-sensitive sensing probe. The probe's response to pH was found in the pH range of 5.0-10.0. This method can sense changes in the pH of living cells and provide an effective means for visual diagnosis of intracellular pH-related diseases ( . Analytical Chemistry, 2021,93(12):5185-5193.).

[0086] This comparison reflects pH changes by changes in fluorescence intensity and its ratio, and the pH range is wide. However, in actual detection, factors such as fluorophore concentration and excitation light will affect the fluorescence intensity, such as Fig.14 , which is the effect of different excitation lights on the fluorescence intensity of the material provided by the comparative example, and the comparative example is not accurate enough in detecting small pH changes, such as Fig.15, indicating that the minimum change gradient of this material in response to pH is 0.5.

[0087] The present invention can accurately reflect small changes in pH according to changes in life span, such as Fig.16 As shown, Fig.16 The working curve of the compound provided in Example 2 inducing pH changes in a mixed solution of dimethyl sulfoxide and water with a water volume content of 99%, wherein the horizontal axis is the response to pH, and the vertical axis is the ratio of the lifespan at different pH values ​​to the lifespan when the water volume fraction is 99%, wherein the minimum pH change gradient can reach 0.05.

[0088] Please note that the technical features of the above embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above embodiments only express several implementation methods of the present application, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all belong to the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be based on the attached claims.

Claims

1. An organic room temperature phosphorescent molecular compound, characterized in that: The compound uses a hexasubstituted aromatic phosphorescent core as a core molecule, and a group capable of inducing intermolecular hydrogen bonds is modified on the core molecule to form different aggregation states through π-π stacking and hydrogen bonds.

2. The organic room temperature phosphorescent molecular compound according to claim 1, characterized in that: The general structural formula of the compound includes Formula I, which is: Wherein, R includes any one or more of carboxyl, carboxyl derivatives, hydroxyl, and amide.

3. The organic room temperature phosphorescent molecular compound according to claim 2, characterized in that: The organic room temperature phosphorescent molecular compound includes any one of hexacarboxylic acid ethyl hexathiobenzene, hexacarboxyl hexathiobenzene, hexahydroxy hexathiobenzene, and hexaamide hexathiobenzene.

4. The organic room temperature phosphorescent molecular compound according to claim 1, characterized in that: The general structural formula of the compound also includes Formula II, wherein Formula II is: Wherein, R is a carboxyl group.

5. The organic room temperature phosphorescent molecular compound according to claim 4, characterized in that: The organic room temperature phosphorescent molecular compound includes hexacarboxyhexaselenobenzene.

6. A method for preparing an organic room temperature phosphorescent molecular compound as claimed in any one of claims 1 to 5, characterized in that: The preparation method comprises: taking hexasubstituted benzene as the basis, further synthesizing a hexasubstituted aromatic hydrocarbon structure as the compound skeleton, and then introducing groups including carboxyl, carboxyl derivatives, hydroxyl or amide on the skeleton to obtain the compound.

7. The preparation method according to claim 6, characterized in that: The preparation method further comprises: adding ethyl p-mercaptobenzoate, hexachlorobenzene and anhydrous potassium carbonate into a reaction container, injecting N,N-dimethylformamide under nitrogen protection, wherein the molar mass ratio of ethyl p-mercaptobenzoate, hexachlorobenzene and anhydrous potassium carbonate is 16-20:0.5-2.5:16-20, and reacting at 60-70°C for 40-45h; Alternatively, in a reaction vessel, hexa(3-methoxy-1-phenylthio)benzene is added under nitrogen protection and dissolved in dry dichloromethane, and BBr3 is quickly added to the solution at -10°C, wherein the molar mass ratio of hexa(3-methoxy-1-phenylthio)benzene to BBr3 is 1-1.5:20-25, and then stirred; Alternatively, hexafluorobenzene and p-mercaptobenzamide are added to 1,3-dimethyl-2-imidazolidinone in a reaction container under nitrogen protection, and sodium hydride powder is slowly added at 0°C, wherein the molar mass ratio of hexafluorobenzene to p-mercaptobenzamide is 1-3:20-25, and the reaction is carried out at 0°C for 2 hours and then at room temperature for 24 hours.

8. The preparation method according to claim 6 or 7, characterized in that: The preparation method further comprises: adding ethyl 4-aminobenzoate, water and concentrated hydrochloric acid to a reaction container under ice bath conditions, dropping a NaNO2 aqueous solution, and stirring at 0°C for 10 minutes; Then add sodium acetate, KSeCN and NaHCO3 aqueous solution and stir for 30 minutes, heat the solution to 55℃ and stir for 30 minutes, add zinc powder, Et2O and H2SO4 mixture, stir at 40℃ for 1.5 hours, add HMPA to the obtained product and NaH under nitrogen protection, stir at 0℃ for 3 hours, add hexabromobenzene, stir at room temperature for 24 hours. Add water and filter, dissolve in tetrahydrofuran, add NaOH aqueous solution, stir at 70℃ for 18 hours; Among them, the molar mass ratio of ethyl 4-aminobenzoate, NaNO2, sodium acetate, KSeCN, NaHCO3, zinc powder, NaH and hexabromobenzene is 3-5:3-5:16-17:3-5:1-2:10-11:23-25:0.3-0.

5.

9. Use of the organic room temperature phosphorescent molecular compound according to any one of claims 1 to 5, characterized in that: The applications include: use in cell lifespan imaging and as a pH sensing probe for detecting intracellular pH changes.

10. The use according to claim 9, characterized in that: The application method of the pH sensing probe for detecting intracellular pH changes includes: dissolving the organic room temperature phosphorescent molecular compound in a mixed solution of water and dimethyl sulfoxide, and detecting pH changes by measuring changes in the system emission wavelength and lifetime when the volume fraction of water is 99%.

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