A fluorescent dye based on benzothiadiazole as a parent and application thereof

Abstract

The application discloses a fluorescent dye based on a benzothiadiazole matrix and application thereof, and introduces a benzotriazole ring into a benzothiadiazole structure matrix to obtain a fluorescent probe in the NIR-II region, so that the fluorescent probe has a longer fluorescent emission wavelength; and the introduction of an alkyl side chain and a (trimethyl) propyl ammonium group into the benzotriazole ring can improve the stability of the three-ring structure of the benzothiadiazole-triazole and the water solubility of the benzothiadiazole-triazole, and the spacer thiophene between the electron donor and the electron acceptor can make the fluorescent dye have a higher quantum yield, so that the fluorescent dye can better recognize and detect ferric ions in an environment and a biological sample, and provides a new thought for the detection of Fe 3+ .

Description

Technical Field

[0001] This invention relates to the fields of organic synthesis, analytical chemistry and biological imaging, and in particular to a fluorescent dye based on benzothiadiazole and its applications. Background Technology

[0002] Iron, the most abundant transition metal in the human body, plays a vital role in many biological processes, such as oxygen transport, energy production, DNA synthesis, and cell growth and replication via iron-dependent proteins. Besides acting as a static cofactor embedded within protein active sites, iron ions also exist in loose and unstable pools of ions that participate in key biological processes, such as signaling pathways. Iron homeostasis is strictly regulated by cells and tissues; disruption of homeostasis can lead to disease, and vice versa. Therefore, to better understand the mechanisms of intracellular iron transport and the biological functions of ionic iron, it is essential to develop metal ion detection methods that can provide real-time monitoring and reveal metal ion dynamics.

[0003] Traditional methods for detecting iron ions include titration, electrochemical analysis, indicator methods, and photochemical methods. However, these methods are time-consuming, complex to operate, costly, have low sensitivity, and poor selectivity. For example, in electrochemical methods, the current itself has a significant impact on the homeostasis of the organism; in indicator methods, the indicators are generally highly toxic to organisms; and photochemical methods require expensive and inconvenient large instruments such as spectrophotometers. This limits the use of these methods in living organisms. Compared to these traditional analytical methods, fluorescence spectroscopy can overcome these problems well, offering advantages such as high sensitivity, a relatively wide linear range, superior selectivity, non-invasive detection, low operational requirements, and rapid and accurate detection. As an extremely important spectrochemical analysis method, fluorescence spectroscopy has attracted increasing attention in recent years. This method utilizes the interaction between the recognition group in the fluorescent probe molecule and the analyte, causing a significant change in the probe's fluorescence signal. This signal change is used to perform qualitative and quantitative analysis of the sample. In recent years, the combination of fluorescent probes and microscopic imaging technology has become a powerful tool for real-time imaging of organisms due to their high specificity, non-invasiveness, and high spatiotemporal resolution, which can display biomolecules in situ in real time.

[0004] However, although fluorescent probes are widely used for in vitro metal ion detection, their in vivo application is hindered by limitations in the penetration depth of excitation light. Therefore, developing probes with redshift absorption is a future direction for fluorescent probe design. Most fluorescent probes based on photoelectron transfer (PET) mechanisms have short emission shifts and are susceptible to autofluorescence background. Near-infrared (NIR-I) fluorescent probes also exhibit strong light attenuation and scattering, and their short fluorescence wavelengths have adverse effects on the organism itself. Therefore, developing NIR-II probes with longer fluorescence absorption and emission wavelengths is urgently needed. Furthermore, although small organic molecule fluorescent probes have significant advantages over traditional instrumental testing methods, most organic dyes are affected by photobleaching. In addition, lesion sites may have adverse factors such as low pH, high reactive oxygen species, or enzyme concentrations, which can affect the stability of organic dyes and reduce probe activity and detection efficiency. Therefore, improving the stability of organic dyes is another problem that needs to be addressed. Moreover, current DAD (Donor-Acceptor-Donor) type NIR-II fluorophores have limited application value due to their inherent defects. For example, most DAD-type NIR-II fluorophores suffer from problems such as low quantum yield (QYs), low molar extinction coefficient, and poor water solubility. Therefore, the development of NIR-II fluorophores that meet the requirements of brightness and biocompatibility is urgently needed for precise biological and cellular imaging. Summary of the Invention

[0005] Therefore, in view of the above background, the present invention provides a fluorescent dye based on benzothiadiazole and its application. It obtains a fluorescent probe in the NIR-II region by introducing a benzotriazole ring into the benzothiadiazole structural parent to achieve a longer fluorescence emission wavelength. Furthermore, the stability and water solubility of the tricyclic structure of benzothiadiazole-triazole can be improved by introducing alkyl side chains and (trimethyl)propylammonium groups into the benzotriazole ring. In addition, the spacer thiophene group located between the electron donor and electron acceptor results in a higher quantum yield, which enables better identification and detection of ferric ions in environmental and biological samples.

[0006] The technical solution of the present invention is as follows:

[0007] A fluorescent dye based on benzothiadiazole has the following structural formula:

[0008] .

[0009] Furthermore, it includes the following steps:

[0010] S1: Synthesis of intermediate B

[0011] S1-1: Under ice bath conditions, concentrated nitric acid is added dropwise to fuming trifluoromethanesulfonic acid;

[0012] S1-2: Add 4,7-dibromo-2,1,3-benzothiadiazole to the mixed acid of S1-1 in portions;

[0013] S1-3: React at 50℃ with stirring until the reaction is complete;

[0014] S1-4: Slowly pour the reaction mixture of S1-3 into ice water, then add sodium hydroxide solution to neutralize excess acid, filter after precipitation, and rinse with water;

[0015] S1-4: Finally, recrystallization and purification from ethanol yields pale yellow flaky crystals, which is intermediate B;

[0016] S2: Synthesis of intermediate C

[0017] S2-1: Dissolve intermediate B, 2-thienyltributyltinane and tetra(triphenylphosphine)palladium obtained in S1 in tetrahydrofuran to remove oxygen;

[0018] S2-2: The mixture of S2-1 was heated under reflux for 36 hours. After the reaction was completed and cooled, saturated potassium fluoride was added. The mixture was stirred at room temperature until a large amount of solid precipitated. After filtration, the solid phase was extracted with dichloromethane and washed with saturated brine. The solid phase was then dried with anhydrous sodium sulfate, and the solvent was concentrated under reduced pressure. Finally, the solid phase was purified by recrystallization with ethyl acetate to obtain an orange flocculent solid, i.e., intermediate C.

[0019] S3: Synthesis of intermediate D

[0020] S3-1: Dissolve intermediate C obtained from S2 in a mixed solvent of N,N-dimethylformamide and acetonitrile, wrap the reaction apparatus with tin foil, heat the solution to 65°C, add N-bromosuccinimide, and then stir the reaction in the dark, adding HBr during the reaction.

[0021] S3-2: After the reaction has been going on for 3 hours, add N-bromosuccinimide and continue the reaction until it is complete;

[0022] S3-3: After the reaction is complete, cool the reaction mixture, add hydrochloric acid, stir at room temperature for a certain period of time, filter and collect the precipitate; wash the precipitate with water and methanol to obtain an orange-red powder, which is intermediate D;

[0023] S4: Synthesis of intermediate E

[0024] S4-1: Add compound D synthesized in S3, reduced iron powder, and acetic acid to the mixture and heat to 100℃;

[0025] S4-2: After reacting for one end of time, cool to room temperature, add distilled water, and a large amount of yellow solid precipitates out. After filtering, the solid is washed with the organic extract obtained by dichloromethane extraction with brine and dried with anhydrous sodium sulfate. Then remove the solvent, and the resulting orange solid is intermediate E.

[0026] S5: Synthesis of intermediate F

[0027] S5-1: Tetrahydrofuran and acetic acid are mixed to prepare a mixed solvent. The mixed solvent is then degassed by two cycles of thawing and circulation using a refrigeration pump. After heating to room temperature under nitrogen conditions, compound E prepared in S4 is quickly added. After it is completely dissolved, sodium nitrite aqueous solution is added dropwise over two hours under ice bath conditions.

[0028] S5-2: The reaction is carried out at room temperature. After the reaction is complete, the reaction solution is poured into water and filtered to obtain the precipitate.

[0029] S5-3: Dissolve the precipitate in a mixture of dichloromethane and triethylamine, add silica gel for adsorption, and then pack it into a chromatography column;

[0030] First, elute with a 1:1 mixture of petroleum ether / dichloromethane, then with ethyl acetate, and finally with a 20:1 mixture of dichloromethane / acetic acid.

[0031] Finally, the purple fraction was collected and the solvent was removed under reduced pressure to obtain a dark purple solid, which is intermediate F.

[0032] S6: Synthesis of fluorescent dye G based on benzothiadiazole

[0033] S6-1: Dissolve compound F obtained in S5 and 3-bromopropyltrimethylammonium bromide in dimethyl sulfoxide;

[0034] S6-2: Under nitrogen protection, add potassium tert-butoxide, heat to 60℃ and stir for 24 hours;

[0035] S6-3: After the reaction is complete, cool the reaction solution to room temperature and add excess distilled water. A lot of solid will precipitate out. After filtration, dichloromethane slurrying, and washing, a dark blue solid can be obtained, which is the fluorescent dye G based on benzothiadiazole.

[0036] This invention also provides an application of a benzothiadiazole-based fluorescent dye as a fluorescent material in bioimaging.

[0037] This invention also provides a fluorescent dye based on benzothiadiazole as a parent compound for Fe 3+ Application of fluorescent molecular probes in metal ion recognition.

[0038] The present invention also provides an organic fluorescent molecular probe, the structure of which is as follows:

[0039] .

[0040] The present invention also provides a kit for detecting ferric ions in a sample, comprising a compound having the following structure:

[0041] .

[0042] The beneficial effects achieved by using this invention are as follows:

[0043] (1) This invention introduces a benzotriazole ring into the benzothiadiazole structure parent, and combines benzothiadiazole and benzotriazole, both of which have excellent electron-withdrawing ability, to further enhance the electron-withdrawing ability of the electron acceptor unit and reduce the band gap of the fluorophore, thereby giving the fluorescent probe a longer fluorescence emission wavelength to obtain a fluorescent probe in the NIR-II region.

[0044] (2) The molecular structure of the present invention has high stability:

[0045] ①This invention increases the stability of the molecular structure by introducing alkyl side chains onto the benzotriazole ring, thereby overcoming the problem of the instability of the tricyclic structure of simple benzothiadiazole-triazole;

[0046] ② Furthermore, this invention increases the water solubility of the entire molecular structure by introducing a (trimethyl)propanenium group onto the benzotriazole ring, that is, by substituting water-soluble groups and target groups onto the benzotriazole ring.

[0047] (3) Due to the electropositive nature of quaternary ammonium salt, the target probe has the ability to locate mitochondria. Therefore, the molecular structure of the present invention has a certain antibacterial ability and can reduce the toxicity of the probe to normal cells.

[0048] (4) The present invention uses thiophene with a high electron cloud density as a spacer between electron donor and electron acceptor to improve quantum yield and further increase fluorescence emission wavelength.

[0049] (5) This invention can quench fluorescence by complexing with iron ions, thereby affecting Fe... 3+ It has excellent recognition capabilities;

[0050] (6) This invention is applicable to environmental or biological samples for Fe 3+ The test was performed to determine the Fe content. 3+ This provides new insights into the detection process. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention.

[0052] Appendix Figure 1 The UV absorption spectra of the organic fluorescent molecular probe of the present invention for different concentrations of ferric chloride are shown.

[0053] Appendix Figure 2 The fluorescence response of the organic fluorescent molecular probe of the present invention to different concentrations of ferric chloride is shown. Detailed Implementation

[0054] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0055] Therefore, this invention is intended to cover such modifications and variations falling within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the invention are disclosed in or will be apparent from the following detailed description. Those skilled in the art will understand that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the invention. The invention is further described below with reference to embodiments.

[0056] The technical solution of this invention is as follows:

[0057] An organic fluorescent molecular probe, the structure of which is as follows:

[0058]

[0059] The method for preparing organic fluorescent molecular probes includes the following steps:

[0060] S1: Synthesis of intermediate B

[0061] S1-1: Under ice bath conditions, concentrated nitric acid is added dropwise to fuming trifluoromethanesulfonic acid;

[0062] S1-2: Add 4,7-dibromo-2,1,3-benzothiadiazole to the mixed acid of S1-1 in portions;

[0063] S1-3: React at 50℃ with stirring until the reaction is complete;

[0064] S1-4: Slowly pour the reaction mixture of S1-3 into ice water, then add sodium hydroxide solution to neutralize excess acid, filter after precipitation, and rinse with water;

[0065] S1-4: Finally, recrystallization and purification from ethanol yields pale yellow flaky crystals, which is intermediate B;

[0066] S2: Synthesis of intermediate C

[0067] S2-1: Dissolve intermediate B, 2-thienyltributyltinane and tetra(triphenylphosphine)palladium obtained in S1 in tetrahydrofuran to remove oxygen;

[0068] S2-2: The mixture of S2-1 was heated under reflux for 36 hours. After the reaction was completed and cooled, saturated potassium fluoride was added. The mixture was stirred at room temperature until a large amount of solid precipitated. After filtration, the solid phase was extracted with dichloromethane and washed with saturated brine. The solid phase was then dried with anhydrous sodium sulfate, and the solvent was concentrated under reduced pressure. Finally, the solid phase was purified by recrystallization with ethyl acetate to obtain an orange flocculent solid, i.e., intermediate C.

[0069] S3: Synthesis of intermediate D

[0070] S3-1: Dissolve intermediate C obtained from S2 in a mixed solvent of N,N-dimethylformamide and acetonitrile, wrap the reaction apparatus with tin foil, heat the solution to 65°C, add N-bromosuccinimide, and then stir the reaction in the dark, adding HBr during the reaction.

[0071] S3-2: After the reaction has been going on for 3 hours, add N-bromosuccinimide and continue the reaction until it is complete;

[0072] S3-3: After the reaction is complete, cool the reaction mixture, add hydrochloric acid, stir at room temperature for a certain period of time, filter and collect the precipitate; wash the precipitate with water and methanol to obtain an orange-red powder, which is intermediate D;

[0073] S4: Synthesis of intermediate E

[0074] S4-1: Add compound D synthesized in S3, reduced iron powder, and acetic acid to the mixture and heat to 100℃;

[0075] S4-2: After reacting for one end of time, cool to room temperature, add distilled water, and a large amount of yellow solid precipitates out. After filtering, the solid is washed with the organic extract obtained by dichloromethane extraction with brine and dried with anhydrous sodium sulfate. Then remove the solvent, and the resulting orange solid is intermediate E.

[0076] S5: Synthesis of intermediate F

[0077] S5-1: Tetrahydrofuran and acetic acid are mixed to prepare a mixed solvent. The mixed solvent is then degassed by two cycles of thawing and circulation using a refrigeration pump. After heating to room temperature under nitrogen conditions, compound E prepared in S4 is quickly added. After it is completely dissolved, sodium nitrite aqueous solution is added dropwise over two hours under ice bath conditions.

[0078] S5-2: The reaction is carried out at room temperature. After the reaction is complete, the reaction solution is poured into water and filtered to obtain the precipitate.

[0079] S5-3: Dissolve the precipitate in a mixture of dichloromethane and triethylamine, add silica gel for adsorption, and then pack it into a chromatography column;

[0080] First, elute with a 1:1 mixture of petroleum ether / dichloromethane, then with ethyl acetate, and finally with a 20:1 mixture of dichloromethane / acetic acid.

[0081] Finally, the purple fraction was collected and the solvent was removed under reduced pressure to obtain a dark purple solid, which is intermediate F.

[0082] S6: Synthesis of fluorescent dye G based on benzothiadiazole

[0083] S6-1: Dissolve compound F obtained in S5 and 3-bromopropyltrimethylammonium bromide in dimethyl sulfoxide;

[0084] S6-2: Under nitrogen protection, add potassium tert-butoxide, heat to 60℃ and stir for 24 hours;

[0085] S6-3: After the reaction is complete, cool the reaction solution to room temperature and add excess distilled water. A lot of solid will precipitate out. After filtration, dichloromethane slurrying and washing, a dark blue solid can be obtained, which is the fluorescent dye G (organic fluorescent molecular probe G) based on benzothiadiazole.

[0086] The specific reaction diagram for the preparation of the organic fluorescent molecular probe is shown below:

[0087]

[0088] Where A is 4,7-dibromo-2,1,3-benzothiadiazole;

[0089] Intermediate B is 5,6-dinitro-4,7-dibromo-[2,1,3]-benzothiadiazole;

[0090] Intermediate C is 4,7-bis(2-thienyl)-5,6-dinitro-2,1,3-benzothiadiazole;

[0091] Intermediate D is 4,7-bis(5-bromothiophene-2-)-5,6-dinitro-2,1,3-benzothiadiazole;

[0092] Intermediate E is 4,7-bis(5-bromothiophene-2-)-5,6-diamino-2,1,3-benzothiadiazole;

[0093] Intermediate F is 4,8-bis(5-bromothiophene-2-)-6H-[1,2,3]triazole[4,5-f]-2,1,3-benzothiadiazole.

[0094] The following specific examples further illustrate the present invention.

[0095] Example 1: A method for preparing an organic fluorescent molecular probe, comprising the following steps:

[0096] S1: Synthesis of intermediate B

[0097] Under ice bath conditions, concentrated nitric acid (3 g, 47.6 mmol, 2.8 eq) was added dropwise to fuming trifluoromethanesulfonic acid (30 g, 200 mmol, 12 eq) in a three-necked flask.

[0098] 4,7-Dibromo-2,1,3-benzothiadiazole (5 g, 17 mmol, 1 eq) was added in portions to a mixed acid. After stirring overnight at 50 °C, TLC monitoring showed almost a single product spot. The mixture was slowly poured into ice water, followed by the addition of sodium hydroxide solution to neutralize excess acid. The precipitate was filtered and washed with water. Recrystallization from ethanol yielded pale yellow flaky crystals (6.03 g, 93% yield), which was intermediate B.

[0099] S2: Synthesis of intermediate C

[0100] Intermediate B (1.92 g, 5 mmol, 1 eq), 2-thienyltributyltinane (4.66 g, 12.5 mmol, 2.2 eq), and tetra(triphenylphosphine)palladium (289 mg, 0.25 mmol, 0.05 eq) were dissolved in tetrahydrofuran (50 mL). Oxygen was removed using the Schlenk operation, and the mixture was then heated under reflux for 36 hours. After the reaction was complete and cooled, saturated potassium fluoride (100 mL) was added, and the mixture was stirred at room temperature for 1 hour, resulting in the precipitation of a large amount of solid. The solid phase was extracted with dichloromethane after filtration, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was concentrated under reduced pressure. Finally, the solid was purified by recrystallization from ethyl acetate to give an orange flocculent solid (1.75 g, 90% yield), which was intermediate C.

[0101] S3: Synthesis of intermediate D

[0102] Intermediate C (780 mg, 2 mmol, 1 eq) was dissolved in a mixed solvent of DMF (10 mL) and CH3CN (5 mL). The reaction apparatus was wrapped with aluminum foil, and the solution was heated to 65 °C. One part of N-bromosuccinimide (783 mg, 4.4 mmol, 2.2 eq) was added, and the reaction was stirred in the dark. Four drops of HBr (48%) were added to the reaction mixture. After 3 hours, another part of N-bromosuccinimide (480 mg, 2.7 mmol, 1.4 eq) was added, and the reaction was allowed to continue for 2 hours. When TLC analysis showed that compound C was completely converted, the reaction mixture was cooled, and hydrochloric acid (180 mL, 2 M) was added, and the mixture was stirred at room temperature for 2 hours. The precipitate was collected by filtration, washed with water and methanol, and an orange-red powder (1 g, 92% yield) was given, which was intermediate D.

[0103] S4: Synthesis of intermediate E

[0104] Intermediate D (600 mg, 1.1 mmol, 1 eq), reduced iron powder (736 mg, 13 mmol, 12 eq), and 50 mL of acetic acid were added to a three-necked flask and heated to 100 °C. After 3 h, the mixture was cooled to room temperature, and 100 mL of distilled water was added. A large amount of yellow solid precipitated out. The solid was filtered and extracted with dichloromethane. The organic extract was washed with brine, dried over anhydrous sodium sulfate, and then the solvent was removed on a rotary evaporator. An orange solid (480 mg, 90% yield) was obtained, which was intermediate E.

[0105] S5: Synthesis of intermediate F

[0106] In a 500 mL round-bottom flask, a mixed solvent of tetrahydrofuran (125 mL) and acetic acid (125 mL) was added. The solution was degassed by two refrigeration pump cycles, then heated to room temperature under nitrogen, followed by the rapid addition of intermediate E (2.000 g, 4.10 mmol, 1 eq). After complete dissolution, sodium nitrite (0.339 g, 4.92 mmol, 1.2 eq) dissolved in water was added dropwise over two hours in an ice bath (10 mL). The reaction was heated to room temperature overnight. After the reaction was completed by TLC monitoring, the reaction solution was poured into water (500 mL), and filtered to obtain a deep purple precipitate. The precipitate was then dissolved in dichloromethane (100 mL) and triethylamine (10 mL), adsorbed onto silica gel, and packed into a chromatography column. Elution was performed first with a 1:1 petroleum ether / dichloromethane solution, then with ethyl acetate, and finally with a 20:1 dichloromethane / acetic acid solution. The purple fraction eluted at the end was collected, and the solvent was removed under reduced pressure to obtain a dark purple solid (0.947 g, yield 46%), which is intermediate F.

[0107] S6: Synthesis of fluorescent dye G based on benzothiadiazole

[0108] Intermediate F (150 mg, 0.3 mmol, 1 eq) and 3-bromopropyltrimethylammonium bromide (94 mg, 0.36 mmol, 1.2 eq) were dissolved in dimethyl sulfoxide (2 mL). Under nitrogen protection, potassium tert-butoxide (40 mg, 0.3 mmol, 1 eq) was added, the mixture was heated to 60 °C and stirred for 24 h. After the reaction was completed by TLC monitoring, the reaction solution was cooled to room temperature, excess distilled water was added, and a large amount of solid precipitated. The solid was filtered, slurried with dichloromethane, and washed to obtain a dark blue solid (98 mg, yield 47%), which is the fluorescent dye G (organic fluorescent molecular probe G) based on benzothiadiazole.

[0109] The organic fluorescent molecular probe G prepared in this embodiment was used to detect iron ions as follows:

[0110] After the fluorescent dye G is pre-prepared into a probe solution, it is added to the solution containing iron ions to be tested. Under ultraviolet light irradiation, the red fluorescence can be immediately observed to be quenched.

[0111] The principle is that the probe molecule complexes with the ferric ion, thereby quenching the fluorescence.

[0112]

[0113] The specific steps are as follows:

[0114] The fluorescent dye was dissolved and diluted in methanol to prepare a target probe solution with a concentration of 40 μmol / L. Then, 1.5 mL of the target probe solution was mixed with 1.5 mL of ferric chloride solution of different concentrations.

[0115] Figure 1 This example illustrates the UV response of the organic molecular probe solution prepared in this embodiment to solutions (aqueous solutions) of iron ions at different concentrations, and the relationship between its absorbance and the concentration of ferric chloride in the solution; wherein... Figure 1 As shown in (A), the UV absorbance at 365 nm gradually increases as the ferric chloride concentration increases from 0 eq to 9 eq (the concentration of ferric chloride increases sequentially from top to bottom according to the peak values ​​of different curves). This indicates that the organic fluorescent molecular probe G is highly sensitive to changes in ferric chloride concentration. Figure 1 (B) As the concentration of ferric chloride increases, the UV absorbance (A365) of the probe also increases in a certain curve, indicating a linear correlation (R0) between the probe absorbance and the concentration of ferric chloride. 2 =0.91).

[0116] Figure 2 The fluorescence response of the organic molecular probe solution prepared in this embodiment to iron ion solutions (aqueous solutions) of different concentrations and the relationship between the fluorescence intensity and the concentration of ferric chloride in the solution are shown.

[0117] like Figure 2 (A) The results showed that the fluorescence emission spectrum of the probe exhibited similar enhancement characteristics. At an excitation wavelength of 365 nm, the probe IAY produced an emission peak at a wavelength of 732 nm. As the ferric chloride concentration increased from 0 eq to 9 eq (the concentration of ferric chloride increased sequentially from top to bottom according to the peak values ​​of different curves), the fluorescence intensity at this wavelength gradually increased.

[0118] Importantly, from Figure 2 (B), R 2 =0.90, indicating a good linear correlation between fluorescence intensity and iron ion concentration. Within a certain range of ferric chloride concentration, the fluorescence emission intensity (I732) of the probe exhibits a certain linear relationship. This suggests that G can serve as a sensitive sensor for detecting ferric iron in environmental and biological samples.

[0119] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are 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.

Claims

1. A fluorescent dye based on benzothiadiazole, characterized in that, Its structural formula is as follows: 。 2. The method for preparing a fluorescent dye based on benzothiadiazole as a parent compound as described in claim 1, characterized in that, It includes the following steps: S1: Synthesis of intermediate B S1-1: Under ice bath conditions, concentrated nitric acid is added dropwise to fuming trifluoromethanesulfonic acid; S1-2: Add 4,7-dibromo-2,1,3-benzothiadiazole to the mixed acid of S1-1 in portions; S1-3: React at 50℃ with stirring until the reaction is complete; S1-4: Slowly pour the reaction mixture of S1-3 into ice water, then add sodium hydroxide solution to neutralize excess acid, filter after precipitation, and rinse with water; S1-4: Finally, recrystallization and purification from ethanol yields pale yellow flaky crystals, which is intermediate B; The structural formula of intermediate B is as follows: ； S2: Synthesis of intermediate C S2-1: Dissolve intermediate B, 2-thienyltributyltinane and tetra(triphenylphosphine)palladium obtained in S1 in tetrahydrofuran to remove oxygen; S2-2: The mixture of S2-1 was heated under reflux for 36 hours. After the reaction was completed and cooled, saturated potassium fluoride was added. The mixture was stirred at room temperature until a large amount of solid precipitated. After filtration, the solid phase was extracted with dichloromethane and washed with saturated brine. The solid phase was then dried with anhydrous sodium sulfate, and the solvent was concentrated under reduced pressure. Finally, the solid phase was purified by recrystallization with ethyl acetate to obtain an orange flocculent solid, i.e., intermediate C. The structural formula of intermediate C is as follows: ； S3: Synthesis of intermediate D S3-1: Dissolve intermediate C obtained from S2 in a mixed solvent of N,N-dimethylformamide and acetonitrile, wrap the reaction apparatus with tin foil, heat the solution to 65°C, add N-bromosuccinimide, and then stir the reaction in the dark, adding HBr during the reaction. S3-2: After the reaction has been going on for 3 hours, add N-bromosuccinimide and continue the reaction until it is complete; S3-3: After the reaction is complete, cool the reaction mixture, add hydrochloric acid, stir at room temperature for a certain period of time, filter and collect the precipitate; wash the precipitate with water and methanol to obtain an orange-red powder, which is intermediate D; The structural formula of intermediate D is as follows: ； S4: Synthesis of intermediate E S4-1: Add compound D synthesized in S3, reduced iron powder, and acetic acid to the mixture and heat to 100℃; S4-2: After reacting for a period of time, the mixture was cooled to room temperature. After adding distilled water, a large amount of yellow solid precipitated out. After filtering the solid, it was washed with the organic extract obtained by dichloromethane extraction with brine and dried with anhydrous sodium sulfate. Then the solvent was removed, and the resulting orange solid was intermediate E. The structural formula of intermediate E is shown below: ； S5: Synthesis of intermediate F S5-1: Tetrahydrofuran and acetic acid are mixed to prepare a mixed solvent. The mixed solvent is then degassed by two cycles of thawing and circulation using a refrigeration pump. After heating to room temperature under nitrogen conditions, compound E prepared in S4 is quickly added. After it is completely dissolved, sodium nitrite aqueous solution is added dropwise over two hours under ice bath conditions. S5-2: The reaction is carried out at room temperature. After the reaction is complete, the reaction solution is poured into water and filtered to obtain the precipitate. S5-3: Dissolve the precipitate in a mixture of dichloromethane and triethylamine, add silica gel for adsorption, and then pack it into a chromatography column; First, elute with a 1:1 mixture of petroleum ether / dichloromethane, then with ethyl acetate, and finally with a 20:1 mixture of dichloromethane / acetic acid. Finally, the purple fraction was collected and the solvent was removed under reduced pressure to obtain a dark purple solid, which is intermediate F. The structural formula of intermediate F is shown below: ； S6: Synthesis of fluorescent dye G based on benzothiadiazole S6-1: Dissolve compound F obtained in S5 and 3-bromopropyltrimethylammonium bromide in dimethyl sulfoxide; S6-2: Under nitrogen protection, add potassium tert-butoxide, heat to 60℃ and stir for 24 hours; S6-3: After the reaction is complete, cool the reaction solution to room temperature and add excess distilled water. A lot of solid will precipitate out. After filtration, dichloromethane slurrying, and washing, a dark blue solid can be obtained, which is the fluorescent dye G based on benzothiadiazole.

3. The application of the fluorescent dye based on benzothiadiazole as described in claim 1 in the preparation of fluorescent materials for bioimaging.

4. The fluorescent dye based on benzothiadiazole as described in claim 1, used in the preparation of Fe metal ion recognition... 3 + Applications of fluorescent molecular probes.

5. An organic fluorescent molecular probe, characterized in that, The structure of the organic fluorescent molecular probe is as follows: 。 6. A reagent kit for detecting ferric ions in a sample, characterized in that, It includes compounds having the following structures: 。

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