An organic room-temperature phosphorescent molecular compound, and a preparation method and application thereof

Abstract

The application provides an organic room-temperature phosphorescent molecular compound and a preparation method and application thereof. The organic room-temperature phosphorescent molecular compound provided in the application takes a six-substituted arene phosphorescent core as a core molecule, and a group capable of inducing intermolecular hydrogen bonding is modified on the core molecule to form different aggregation states through pi-pi stacking and hydrogen bonding. The preparation method of the compound comprises the following steps: taking a six-substituted benzene as a basis, further synthesizing a six-substituted arene structure as a compound skeleton, and then introducing groups including a carboxyl group, a carboxyl group derivative, a hydroxyl group or an amide on the skeleton. The organic room-temperature phosphorescent molecular compound can be used for cell life imaging and as a pH sensing probe for detecting intracellular pH changes. The compound provided in the application has a life of up to 30 mu s when the water content is 99%, can respond to pH, and provides accurate and reliable life biological imaging in clinical biomedical applications; and when used as a pH sensing probe, the compound can accurately respond to intracellular pH changes.

Description

Technical Field

[0001] This invention belongs to the field of luminescent materials technology, and particularly relates to an organic room temperature phosphorescent molecular compound, its preparation method and application. Background Technology

[0002] Cellular pH is a critical parameter for the proper functioning of various biological processes, and its stability is essential for these physiological and pathological processes, including cell proliferation, apoptosis, and endocytosis. Therefore, monitoring pH changes is crucial in chemistry, biology, and medicine. Intracellular pH changes are closely related to cancer development and cellular senescence; studies have found that cancer cells exhibit a pH gradient opposite to that of normal somatic cells, i.e., an intracellular pH (pH value is not specified in the original text). i The pH should be higher than the extracellular pH (pH). e This gradient can promote cancer cell proliferation and evade apoptosis, thus accelerating cancer development (Nature Reviews Cancer 11, 671–677 (2011)). Studies on neurodegenerative diseases related to cellular senescence have revealed intracellular pH acidification (pH...). i <pH e Changes in intracellular pH can serve as a marker for diagnosing early cellular senescence and cancer. (Cell. Mol. Life Sci. 79, 380 (2022). Therefore, intracellular pH-sensing probes have emerged.

[0003] Supported by fluorescence imaging technology, many fluorescent probes have been developed to sense changes in intracellular pH. To date, the literature has reported detecting intracellular pH changes through changes in fluorescence intensity (Dyes and Pigments 205(2022)110545.), but the results can be biased due to fluctuations in fluorescence intensity, excitation intensity, and sample concentration (ACS Sensors, 2019, 4(4):883-891). In contrast, fluorescence lifetime imaging microscopy (FLIM) shows significant advantages because it is unaffected by changes in excitation intensity and fluorophore concentration. Therefore, there is an urgent need to invent a long-lifetime tunable FLIM fluorescent probe to sense changes in intracellular pH and thus reflect the cell state in real time.

[0004] Organic room temperature phosphorescent materials have attracted increasing attention due to their unique optical properties and their potential applications in bioimaging, chemical sensing, and information encryption. However, 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, tunable molecular probe that senses intracellular pH has been developed. The detection probe that utilizes the changes in the emission wavelength and lifetime of the molecular system with pH is of great significance. This probe can reflect the intracellular pH and thus the cell state based on the changes in the emission wavelength and lifetime of the molecular system, making it suitable as a simple means of clinical diagnosis.

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

[0007] The purpose of this invention is to provide an organic room-temperature phosphorescent molecular compound, its preparation method, and its application, in order to solve the above-mentioned problems.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] An organic room-temperature phosphorescent molecular compound, wherein the compound has a hexasubstituted aromatic phosphorescent core as the core molecule, and the core molecule is modified with groups that can induce intermolecular hydrogen bonds, forming different aggregation states through π-π stacking and hydrogen bonding.

[0010] Using a hexasubstituted aromatic skeleton as the core structure, and then modifying its periphery with groups that can induce intermolecular hydrogen bonds, different aggregation states are further formed through π-π stacking and hydrogen bonding. The luminescence and lifetime properties of this molecule are modulated through diverse self-assemblies induced by hydrogen bonds. The emission wavelength and lifetime of the molecule are pH-sensitive, and can therefore be used for lifetime imaging to sense changes in intracellular pH.

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

[0012]

[0013] R includes one or more of carboxyl groups, carboxyl derivatives, hydroxyl groups, and amides.

[0014] Preferably, the organic room-temperature phosphorescent molecule compound includes any one of: ethyl hexacarboxylate hexathiobenzene, hexacarboxylated hexathiobenzene, hexahydroxyhexathiobenzene, and hexaamide hexathiobenzene. Their structural formulas are as follows:

[0015]

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

[0017]

[0018] Where R is a carboxyl group.

[0019] Preferably, the organic room-temperature phosphorescent molecular compound comprises hexacarboxyhexaselenobenzene, with the structural formula as follows:

[0020] This application provides a method for preparing the aforementioned organic room temperature phosphorescent molecular compound. The method includes: using a hexasubstituted benzene as a base, further synthesizing a hexasubstituted aromatic hydrocarbon structure as the compound skeleton, and then introducing groups including carboxyl groups, carboxyl derivatives, hydroxyl groups, or amides onto the skeleton to obtain the compound.

[0021] Optionally, the preparation method further includes: adding ethyl p-mercaptobenzoate, hexachlorobenzene, and anhydrous potassium carbonate to 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℃ for 40-45 h; further optionally, after the reaction is completed and cooled, a yellow precipitate is generated by dripping into ice water, filtered, and the solid is repeatedly washed with a large amount of water, ethanol, and acetone, and dried to obtain the hexacarboxylic acid ethyl ester hexathiobenzene; even further optionally, after the reaction is completed and cooled, a yellow precipitate is generated by dripping into ice water, filtered, and the solid is repeatedly washed with a large amount of water, ethanol, and acetone, dried, dissolved in THF, and sodium hydroxide aqueous solution is added dropwise. After stirring at room temperature for 18 h, dilute hydrochloric acid solution is added to adjust the solution to acidity and precipitate, the obtained solid is filtered, washed with water, ethanol, and acetone, and dried to obtain the hexacarboxylic acid hexathiobenzene;

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

[0023] Alternatively, under nitrogen protection in a reaction vessel, hexafluorobenzene and p-mercaptobenzamide dissolved in 1,3-dimethyl-2-imidazolinone are added, and sodium hydride powder is slowly added at 0°C. The molar mass ratio of hexafluorobenzene to p-mercaptobenzamide is 1-3:20-25. After reacting at 0°C for 2 hours, the reaction is continued at room temperature for 24 hours. Ice water and saturated NaOH aqueous solution (100 mL) are added to the reaction solution. The solid is collected by filtration and washed successively with large amounts of distilled water, ethanol, ethyl acetate, and acetone to obtain the hexaamide hexathiobenzene.

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

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

[0026] The molar 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] This application also provides an application of the aforementioned organic room-temperature phosphorescent molecular compound, the application including: for cell lifetime imaging and as a pH-sensing probe for detecting intracellular pH changes.

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

[0029] The beneficial effects of this invention are:

[0030] This application provides a series of compounds based on hexasubstituted aromatic hydrocarbons. These compounds have a hexasubstituted aromatic hydrocarbon phosphorescent core as their core structure, with surrounding structures modified with groups that induce intermolecular hydrogen bonds. The luminescence and lifetime properties of the molecule are regulated through hydrogen bond-induced self-assembly. These compounds exhibit aggregation-induced luminescence (AIE) characteristics, are sensitive to the presence of water in organic solvents, and their emission wavelength and lifetime are affected by pH. Specifically, within a certain range, with increasing water content, the emission wavelength first exhibits a blue shift followed by a red shift, while the lifetime gradually increases, reaching 30 μs at 99% water content. During a pH change from 6.07 to 8.06, the emission wavelength exhibits a blue shift, and the lifetime decays to the nanosecond level. During a pH change from 7.78 to 5.78, the emission wavelength exhibits a red shift, and the lifetime recovers. Therefore, these compounds can serve as molecular probes for real-time detection of intracellular pH changes based on variations in emission wavelength and lifetime.

[0031] Compared to existing pH-sensing probes, the series of compounds provided by this invention are pH-sensitive, capable of sensing intracellular pH changes based on variations in emission wavelength and lifetime. Furthermore, they offer advantages such as readily available and inexpensive raw materials, simple synthesis, and convenient detection. This solves the technical problems of high detection costs and complex synthetic routes in existing technologies, and effectively improves detection accuracy. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

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

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

[0035] Figure 3 The 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 The 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 The 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%;

[0038] Figure 6 The hydrogen nuclear magnetic resonance spectrum of the compound provided in Example 2 in water volume contents of 0%-99%;

[0039] Figure 7 Fluorescence emission patterns of the compound provided in Example 2 with different concentrations of NaOH solution added to a mixed solution of dimethyl sulfoxide and water with a water volume content of 99%;

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

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

[0042] Figure 10 Fluorescence emission patterns 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 solution of different concentrations;

[0043] Figure 11 The lifetime 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 solution of different concentrations;

[0044] Figure 12 The lifetime 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 solution of different concentrations;

[0045] Figure 13 The graph shows the change in afterglow color of the film prepared using the compound provided in Example 2 after acid evaporation with CF3COOH.

[0046] Figure 14 Test diagrams showing the effect of different excitation light on the materials provided in Comparative Example 1;

[0047] Figure 15 A test diagram showing the pH sensitivity range of the material provided for Comparative Example 1;

[0048] Figure 16 The working curve of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with a water volume content of 99% is shown. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0051] Example 1

[0052] This embodiment provides an organic room-temperature phosphorescent molecular compound and its preparation method, specifically ethyl hexacarboxylate 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 to a 250 mL round-bottom flask, and reflux for 20 h.

[0054] S2: Cool the S1 system to room temperature naturally, add it dropwise to water, extract it three times with ethyl acetate, and concentrate it under vacuum to obtain oily ethyl p-mercaptobenzoate.

[0055] S3: 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.) were added to a three-necked flask under nitrogen protection. 15 mL of dry DMF was injected, and the mixture was reacted at 60 °C for 40 h. After cooling, a yellow precipitate was formed by adding it dropwise to 150 mL of ice water. The precipitate was filtered, and the solid was washed repeatedly with a large amount of water, ethanol and acetone. After drying, hexachlorobenzene hexacarboxylic acid ethyl ester was obtained.

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

[0057] Example 2

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

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

[0060] S4: Dissolve ethyl hexacarboxylate hexathiobenzene (1.15 g, 1 mmol) obtained in S3 in 20 mL of THF, and add sodium hydroxide aqueous solution (2.0 mol / L, 10 mL) dropwise to the solution. Stir at room temperature for 18 h, then add dilute hydrochloric acid solution (1.0 mol / L, 250 mL) to adjust the solution to acidity and precipitate. Filter, and wash the solid successively with water, ethanol, and acetone to obtain a yellow powder, which is hexacarboxylated hexathiobenzene, labeled as S6, with the structural formula [notation missing].

[0061] The calculated yield is 60%, and its proton NMR spectrum is as follows: Figure 1 As 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 hexacarboxyhexathiobenzene provided in this embodiment was prepared into a mixed solution of dimethyl sulfoxide and water, with water proportions of 0%, 40%, 70%, 90%, and 99%, respectively, and a concentration of 10 μmol / L for each solution. 2 mL of each solution was added to a 1 cm × 1 cm × 4 cm stoppered cuvette for performance testing.

[0063] 1. The fluorescence emission spectrum was tested at λex = 365 nm, and the results are as follows: Figure 2 As shown, Figure 2 The image shows the fluorescence emission of the compound provided in Example 2 in mixed solutions of dimethyl sulfoxide and water with different water volume contents. The horizontal axis represents the emission wavelength, and the vertical axis represents the fluorescence intensity value. Figure 2 It can be seen that, within a certain range, as the water content in the solution increases, the fluorescence intensity gradually increases, and the emission wavelength first undergoes a blue shift and then a red shift.

[0064] 2. Test the lifetime decay spectrum, λex = 365 nm, and observe the relationship between its lifetime and water volume fraction. The results are as follows: Figure 3-5 As shown, Figure 3 The 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 The 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 graph of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with a water volume fraction of 99%. The horizontal axis represents time, and the vertical axis represents the intensity value. Figure 3-5 It can be seen that, within a certain range, the lifespan increases with increasing water content.

[0065] 3. The mixed solutions with different water volume fractions were subjected to nuclear magnetic resonance titration tests, and their proton NMR spectra are shown below. Figure 6 As shown, NMR titration indicates that the addition of D2O (concentration of 10) to a DMSO-d6 solution of hexacarboxyhexathiobenzene... -4 Within a certain range, as the water content increases, hydrogen bonds are formed between water molecules and hexacarboxyhexathiobenzene molecules, resulting in an integral shielding effect. This causes the aromatic protons on the hexacarboxyhexathiobenzene molecules to move to a higher field. At the same time, with the formation of hydrogen bonds, the proton resonance of the hexacarboxyhexathiobenzene molecules widens, which suggests that there may be an aggregation effect.

[0066] The hexacarboxyhexathiobenzene provided in this embodiment was prepared as a mixed solution of dimethyl sulfoxide and water, with a water volume fraction of 99%. Different concentrations of NaOH solution were added to the solution, and 2 mL of each solution was added to a 1cm×1cm×4cm stoppered cuvette to test its performance.

[0067] 4. Measure the fluorescence emission spectrum, λex = 365 nm, and observe the relationship between the emission wavelength and the concentration of added NaOH. The results are as follows: Figure 7 As shown, Figure 7 Fluorescence emission patterns of the compound provided in Example 2 were obtained by adding different concentrations of NaOH solution 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, the emission wavelength undergoes a blue shift as the concentration of NaOH solution increases;

[0068] 5. Test the lifetime decay spectrum, λex = 365 nm, and observe the relationship between its lifetime and the concentration of added NaOH. The results are as follows: Figure 8-9 As shown, Figure 8-9 The lifetime decay graph of the compound provided in Example 2 is obtained by adding different concentrations of NaOH solution 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 decreases from the microsecond level to the nanosecond level as the NaOH concentration increases.

[0069] The hexacarboxyhexathiobenzene obtained in this example was prepared as a mixed solution of dimethyl sulfoxide and water, with a water volume fraction of 99%. A 160 μmol / L NaOH solution was added to the solution, followed by CF3COOH solutions of different concentrations. 2 mL of each 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 = 365 nm, and observe the relationship between the emission wavelength and the added CF3COOH solution. The results are as follows: Figure 10 As shown, Figure 10 Fluorescence emission patterns 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 10 It can be seen that, within a certain range, the emission wavelength redshifts as the concentration of the added CF3COOH solution increases.

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

[0072] The hexacarboxyhexathiophene provided in this example was configured into a dimethyl sulfoxide solution, which was mixed and stirred evenly with an aqueous solution of PVA to form a film, as Figure 13 shown, Figure 13 The figure shows the change in the afterglow color of the film prepared using the compound provided in Example 2 after being steamed with CF3COOH acid; from Figure 13 It can be seen that the original film has a yellow afterglow. After being steamed with CF3COOH acid, the afterglow of the film changes from yellow to green. The film with the green afterglow is then steamed with C6H 15 N base, and the afterglow returns to yellow.

[0073] Example 3 [[ID=ID=18]]

[0074] This example provides an organic room temperature phosphorescent molecular compound and its preparation method, specifically hexahydroxyhexathiophene. The preparation steps are as follows:

[0075] Add hexylbenzene (3-methoxy-1-phenylbenzene) (1.0 g, 1.1 mmol) to a 250 mL round-bottom flask under a nitrogen atmosphere, dissolve and stir with 70 mL of dry dichloromethane. Then quickly add BBr3 (2.0 mL, 22.0 mmol) to the solution at -10 °C. After stirring the solution for 15 h, pour the reaction mixture into cold water and stir vigorously until no more precipitate forms. After filtration and drying, a yellow solid is obtained as the product hexahydroxyhexathiophene, with a yield of 90%. The 1H NMR spectrum of the product is 1 1H 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 example provides an organic room temperature phosphorescent molecular compound and its preparation method, specifically hexamidohexathiophene. The preparation steps are as follows:

[0078] In a nitrogen-protected 250 mL three-necked flask, hexafluorobenzene (0.372 g, 2.0 mmol) and p-mercaptobenzamide (4.08 g, 24 mmol) dissolved in DMI (20 mL) were added. The mixture was cooled to 0 °C, and sodium hydride powder (60% purity, 0.96 g, 24 mmol) was slowly added. The mixture was allowed to react at this temperature for 2 h, followed by 24 h at room temperature. Ice water and saturated NaOH aqueous solution (100 mL) were added to the reaction solution. The solid was collected by filtration and washed successively with large amounts of distilled water, ethanol, ethyl acetate, and acetone to obtain a white powder, which was the product hexafluorobenzene hexathioamide, with a mass of 1.00 g. The calculated yield was 46%. (The product's proton NMR spectrum is shown.) 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 of sodium nitrite (0.33 g, 4.8 mmol) (2 mL) was added dropwise, and the mixture was stirred at 0 °C for 10 min. 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, and the mixture was stirred at 0 °C for 30 min. After stirring at 55 °C for 30 min, the mixture was heated and stirred for another 30 min. After cooling, the mixture was extracted with dichloromethane. The combined extract was washed with brine, dried on anhydrous magnesium sulfate, and the solvent was removed. The crude product was further purified by silica gel column chromatography using gasoline ether / ethyl acetate (v / v, 5:1) as the eluent. The crude product was then recrystallized from hexane to obtain white crystals with the following 1H NMR spectrum: 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] The mixture of white crystals (682 mg, 2.84 mmol), zinc powder (689 mg, 10.5 mmol), Et₂O (14 mL), and sulfuric acid (20%, 6 mL) obtained in the above steps was stirred at 40 °C for 1.5 hours. After filtration, the residue was extracted with dichloromethane, the extract was washed, dried on anhydrous magnesium sulfate, and evaporated. The crude product was purified by column chromatography using gasoline ether / ethyl acetate (v / v, 5:1) as the eluent to give 530 mg of yellow crystals, with a yield of 87%. (1H NMR 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] The mixture of the above-obtained yellow crystals (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. Hexabromobenzene (165 mg, 0.4 mmol) solution was added to HMPA (6 mL), and the mixture was stirred at room temperature for 24 hours. The crude product was precipitated after the addition of 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] In a tetrahydrofuran (10 mL) solution of the obtained red crystals (173 mg, 0.13 mmol), an aqueous solution of sodium hydroxide (2 M, 10 mL) was added. After stirring at 70 °C for 18 h, the mixture was neutralized with dilute hydrochloric acid. The resulting solid was washed with deionized water and ethanol, yielding a red solid, which was hexacarboxyhexaselenobenzene, with a mass of 133 mg and a yield of 60%. (Hydrogen spectroscopy) 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] Existing technology reports a novel fluorescent silicon nanoparticle (SiNP) 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. A pH response to the probe was observed in the pH range of 5.0-10.0. This method can sense changes in the pH of living cells, providing an effective means for the visual diagnosis of intracellular pH-related diseases. . Analytical Chemistry, 2021,93(12):5185-5193.).

[0086] This comparative example reflects pH changes through changes in fluorescence intensity and its ratio, and covers a wide pH range. However, in actual detection, factors such as fluorophore concentration and excitation light can affect fluorescence intensity. Figure 14 This illustrates the effect of different excitation lights on the fluorescence intensity of the material in this comparative example, and this comparative example is not accurate enough for detecting small pH changes, such as... Figure 15This indicates that the minimum pH response gradient of this material is 0.5.

[0087] This invention can accurately reflect minute changes in pH based on changes in lifetime, such as... Figure 16 As shown, Figure 16 The working curves of the compound provided in Example 2 in a mixed solution of dimethyl sulfoxide and water with a water volume content of 99% are shown. The horizontal axis represents the response to pH, and the vertical axis represents the ratio of lifetime at different pH values ​​to lifetime at a water volume fraction of 99%. The minimum pH change gradient can reach 0.05.

[0088] Please note that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The above embodiments only illustrate several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. The application of an organic room-temperature phosphorescent molecular compound in the preparation of a pH-sensing probe, characterized in that, The applications include: using it as a pH-sensing probe to detect intracellular pH changes; the general structural formula of the organic room-temperature phosphorescent molecular compound is Formula I, wherein Formula I is: ； Where R is a carboxyl group.

2. The application according to claim 1, characterized in that, The organic room-temperature phosphorescent molecular compound is hexacarboxyhexathiobenzene.

3. The application according to claim 1, characterized in that, The method for preparing the organic room temperature phosphorescent molecular compound includes: using a hexasubstituted benzene as a base, further synthesizing a hexasubstituted aromatic hydrocarbon structure as the compound skeleton, and then introducing a carboxyl group onto the skeleton to obtain the compound.

4. The application according to claim 2 or 3, characterized in that, The method for preparing the organic room-temperature phosphorescent molecular compound includes: adding ethyl p-mercaptobenzoate, hexachlorobenzene, and anhydrous potassium carbonate to 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 40-45 h; After the reaction was completed and cooled, a yellow precipitate was formed by dripping into ice water. The precipitate was filtered, and the solid was repeatedly washed with a large amount of water, ethanol and acetone. After drying, it was dissolved in THF, and sodium hydroxide aqueous solution was added dropwise. After stirring at room temperature for 18 hours, dilute hydrochloric acid solution was added to adjust the solution to acidity and precipitate was formed. The obtained solid was filtered, washed with water, ethanol and acetone, and dried to obtain hexacarboxyhexathiobenzene.

5. The application according to claim 1, characterized in that, The method of using the organic room-temperature phosphorescent molecular compound as a pH sensing probe to detect 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 the changes in the emission wavelength and lifetime of the system when the water volume fraction is 99%.

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