A dual-emission fluorescent probe and a synthesis method and application thereof

Abstract

The application provides a dual-emission fluorescent probe and a synthesis method and application thereof. A structural formula of the dual-emission fluorescent probe is shown in formula (1): wherein R is methyl or methoxy. The dual-emission fluorescent probe has adjustable fluorescence emission behavior, can realize ratio sensing for different solvents, and can realize visual detection of a solvent (for example, methane chloride).

Description

Technical Field

[0001] This invention belongs to the field of lyochromic organic small molecule fluorescent probe technology, specifically relating to a dual-emission fluorescent probe, its synthesis method, and its application. Background Technology

[0002] Chlorinated methane (such as dichloromethane, trichloromethane, and carbon tetrachloride) are commonly used organic solvents in laboratories and are widely used in biopharmaceuticals, agricultural and veterinary drug production, organic cleaning agents, and extractants. Chlorinated methane is highly toxic and volatile, and poses a potential carcinogenic risk, causing multiple hazards to human health, the environment, and safety. For example, chlorinated methane can decompose to produce highly toxic products (such as phosgene), and its photochemical reactions in the atmosphere have a significant destructive effect on the ozone layer. Furthermore, the physicochemical properties of chlorinated methane (such as boiling point, solubility, reactivity, and decomposition products) vary depending on the number of chlorine atoms, leading to differences in storage and transportation conditions and applicable ranges. Therefore, the detection and identification of chlorinated methane during production, storage, transportation, and use are extremely important. However, the lack of photoelectric activity in chlorinated methane makes highly sensitive detection and visual differentiation a significant challenge for the scientific community.

[0003] For the detection and differentiation of methane chloride, commonly used techniques include gas chromatography, liquid chromatography, nuclear magnetic resonance, and surface acoustic wave sensing. While these techniques offer advantages such as high selectivity and reliability, they often suffer from drawbacks such as cumbersome operation, high cost, insufficient sensitivity, lack of visualization, and slow speed, hindering their widespread adoption. Therefore, developing a highly sensitive, discriminative, fast-responding, non-corrosive, and physically unrestricted visual detection method is of great significance.

[0004] Fluorescence sensing technology boasts advantages such as simple operation, low cost, high sensitivity, sensitivity to microenvironment, non-erosion of samples, and fast response speed, making it a widely adopted detection technique. However, according to literature review, there are few reports on fluorescence sensing techniques for methane chloride. Furthermore, traditional fluorescent probe molecules emit a single emission signal, exhibit no excitation wavelength dependence, and lack sufficient visual discrimination and recognition capabilities. Therefore, the development of high-performance fluorescent probes is extremely important.

[0005] Excited-state intramolecular proton transfer compounds have attracted widespread attention in the field of fluorescence sensing due to their variety, ease of structural modification, and ability to emit dual fluorescence. However, the proton transfer products of existing excited-state intramolecular proton transfer compounds are prone to double bond rotation in solution, resulting in fluorescence quenching. Furthermore, their fluorescence emission is poorly tunable, and they have poor ability to distinguish and recognize similar compounds within a certain polarity range. These shortcomings limit their practical applications. Summary of the Invention

[0006] To overcome the shortcomings of existing methane chloride detection technologies, such as insufficient sensitivity and lack of visualization, as well as the problems of fluorescence quenching, poor controllability, and poor ability to distinguish and identify similar compounds in existing fluorescent probes, this invention provides a dual-emission fluorescent probe, its synthesis method, and its application.

[0007] This invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a dual-emission fluorescent probe, the structure of which is shown in formula (1):

[0009]

[0010] Where R is methyl or methoxy.

[0011] Secondly, the present invention provides a method for preparing the aforementioned dual-emission fluorescent probe, comprising:

[0012] Step 1: 2-Aminothiophenol is reacted with 2-amino-5-methylbenzoic acid or 2-amino-5-methoxybenzoic acid in the presence of triphenyl phosphite and tetrabutylammonium bromide to obtain the intermediate shown in formula (2).

[0013]

[0014] Step 2: The intermediate shown in formula (2) is reacted with cholesterol chloroformate in the presence of triethylamine to obtain a dual-emission fluorescent probe.

[0015] Preferably, in step 1, the molar ratio of 2-aminothiophenol to 2-amino5-methylbenzoic acid or 2-amino-5-methoxybenzoic acid is 1:(1~1.1), and the molar ratio of 2-aminothiophenol to triphenyl phosphite and tetrabutylammonium bromide is 1:(1~1.2):(1~1.2).

[0016] Preferably, in step 1, the reaction temperature is 120~130℃ and the reaction time is 8~10 hours.

[0017] Preferably, in step 2, the molar ratio of the intermediate shown in formula (2) to cholesterol chloroformate and triethylamine is 1:(20~30):(20~30).

[0018] Preferably, in step 2, the reaction temperature is 60~70℃ and the reaction time is 4~6 hours.

[0019] Thirdly, the present invention provides a method for regulating the luminescence behavior of the dual-emission fluorescent probe, which regulates the luminescence behavior of the dual-emission fluorescent probe by changing the type of solvent used to dissolve the dual-emission fluorescent probe, or by changing the excitation wavelength.

[0020] Fourthly, the present invention provides the application of the dual-emission fluorescent probe in distinguishing types of methane chloride.

[0021] Preferably, the application of the dual-emission fluorescent probe in distinguishing types of methane chloride further includes: adding the dual-emission fluorescent probe to methane chloride to obtain a dual-emission fluorescent probe solution; irradiating the dual-emission fluorescent probe solution with a laser, and identifying the type of methane chloride based on the fluorescence color emitted by the dual-emission fluorescent probe solution.

[0022] Preferably, the application of the dual-emission fluorescent probe in distinguishing types of methane chloride further includes: adding the dual-emission fluorescent probe to methane chloride to obtain a dual-emission fluorescent probe solution; performing fluorescence emission spectroscopy on the dual-emission fluorescent probe solution to obtain a first fluorescence emission intensity corresponding to a first maximum emission wavelength and a second fluorescence emission intensity corresponding to a second maximum emission wavelength; and identifying the type of methane chloride based on the ratio of the first fluorescence emission intensity to the second fluorescence emission intensity.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention discloses a dual-emission fluorescent probe that uses a moderately acidic imine (NH) as a proton donor and a moderately basic benzothiazole (N) atom as a proton acceptor, and incorporates a cholesterol structural fragment with strong van der Waals interactions. First, the appropriately acidic and basic proton donor and acceptor induce an excited-state intramolecular proton transfer reaction in the fluorescent probe molecule, producing two proton transfer isomers that maintain mutual equilibrium, thereby emitting dual fluorescence. The ratio of the fluorescence emission intensities at the two different wavelengths can be modulated by changing the solvent or excitation wavelength, allowing for the detection and identification of different solvents based on this ratio, thus achieving ratio sensing. Furthermore, the dual-emission fluorescent probe emits different fluorescence colors in different solvents, exhibiting solvent dependence and belonging to the lyochromic fluorescent molecule category, thus enabling the visual detection of solvents (e.g., methane chloride). Second, the introduction of the cholesterol structure effectively suppresses the double bond rotation of the proton transfer products, preventing fluorescence quenching and enhancing fluorescence emission, thereby improving the sensitivity of solvent detection and the ability to distinguish similar compounds.

[0025] The method for synthesizing the dual-emission fluorescent probe described in this invention is simple to operate, uses readily available raw materials, has low equipment requirements, and is suitable for large-scale production.

[0026] This invention provides a dual-emission fluorescent probe for distinguishing different types of methane chlorides. By adding the dual-emission fluorescent probe as a lyochromic fluorescent molecule to methane chlorides and using light of a suitable wavelength as excitation, solvent-dependent dual fluorescence emission can be achieved. It exhibits ratiometric response capability, particularly for structurally similar methane chlorides such as dichloromethane, trichloromethane, and carbon tetrachloride, enabling visual detection of methane chlorides and effectively solving the problem of visual detection of methane chlorides that lack photoelectric activity and have similar structures. Attached Figure Description

[0027] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The hydrogen NMR spectrum of compound 1 obtained in this invention;

[0029] Figure 2 This is a high-resolution mass spectrum of compound 1 obtained in this invention;

[0030] Figure 3 The excitation-emission spectrum of compound 1 obtained in this invention in solution;

[0031] Figure 4 This is a potential energy profile of the ground state and the first excited state of compound 1 obtained in this invention along the proton transfer coordinates;

[0032] Figure 5 This is a potential energy profile of the ground state and the first excited state of the proton transfer product of compound 1 obtained in this invention, showing the change in coordinates along the rotational motion of the C=C double bond.

[0033] Figure 6 This is a potential energy profile of the ground state, the first excited state, and the second excited state of compound 1 obtained in this invention along the proton transfer coordinates.

[0034] Figure 7 The fluorescence emission spectra of compound 1 obtained in this invention in different solvents are shown.

[0035] Figure 8 The fluorescence emission spectra of compound 1 obtained in this invention in dichloromethane, trichloromethane and carbon tetrachloride are shown.

[0036] Figure 9 The images show the fluorescence of compound 1 obtained in this invention under 365 nm ultraviolet light irradiation in different methane chloride solvents. Detailed Implementation

[0037] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0038] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0039] It should be noted that the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products, or apparatuses. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not intended to limit the order of the method steps or define the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0040] The dual-emission fluorescent probe described in this invention is a fluorescent probe based on the intramolecular proton transfer reaction of the anti-Kasha excited state, and it is a dual-emission fluorescent probe with tunable luminescence behavior. Its structural formula is shown in formula (1):

[0041]

[0042] Where R is methyl or methoxy.

[0043] Specifically, the dual-emission fluorescent probe of this invention is compound 1 or compound 2 as shown below:

[0044] .

[0045] The method for synthesizing the dual-emission fluorescent probe of the present invention includes the following steps:

[0046] Step 1: 2-Aminothiophenol is reacted with 2-amino-5-methylbenzoic acid or 2-amino-5-methoxybenzoic acid in the presence of triphenyl phosphite and tetrabutylammonium bromide to obtain the intermediate shown in formula (2).

[0047]

[0048] Step 2: The intermediate shown in formula (2) is reacted with cholesterol chloroformate in the presence of triethylamine to obtain the dual-emission fluorescent probe.

[0049] In some preferred embodiments of the present invention, in step 1, the molar ratio of 2-aminothiophenol to 2-amino-5-methylbenzoic acid or 2-amino-5-methoxybenzoic acid is (1~1.1).

[0050] In some preferred embodiments of the present invention, in step 1, the molar ratio of 2-aminothiophenol to triphenyl phosphite and tetrabutylammonium bromide is 1:(1~1.2):(1~1.2). Triphenyl phosphite and tetrabutylammonium bromide act as catalysts to catalyze the reaction of 2-aminothiophenol with 2-amino-5-methylbenzoic acid or 2-amino-5-methoxybenzoic acid.

[0051] In some preferred embodiments of the present invention, in step 1, the reaction temperature is 120~130℃ and the reaction time is 8~10 hours.

[0052] Specifically, in some embodiments of the present invention, step 1 is as follows: 2-aminothiophenol, 2-amino-5-methylbenzoic acid or 2-amino-5-methoxybenzoic acid, triphenyl phosphite and tetrabutylammonium bromide are added to toluene and reacted under an inert atmosphere. After the reaction is completed, the reaction solution is cooled to room temperature, filtered, and the filtrate is concentrated under reduced pressure. The concentrate is separated by column chromatography to obtain the intermediate.

[0053] In this invention, the 2-aminothiophenol reacts with 2-amino-5-methylbenzoic acid or 2-amino-5-methoxybenzoic acid under an inert atmosphere to avoid side reactions caused by the presence of oxygen in the air.

[0054] In the preparation of the intermediates in this invention, toluene is used as a solvent. Its high boiling point ensures that the intermediate preparation can be carried out under heating conditions. The molar ratio of 2-aminothiophenol to toluene is 1:(100~120).

[0055] In step 1 of this invention, column chromatography separation uses a hexane-dichloromethane system as the eluent, wherein the volume ratio of hexane to dichloromethane is preferably 2:1.

[0056] In some embodiments of the present invention, in step 2, the molar ratio of the intermediate shown in formula (2) to cholesterol chloroformate is 1:(20~30).

[0057] In some embodiments of the present invention, in step 2, the molar ratio of the intermediate shown in formula (2) to the acid scavenger triethylamine is 1:(20~30).

[0058] Specifically, in some embodiments of the present invention, step 2 is as follows: under an inert atmosphere, the intermediate shown in formula (2), anhydrous triethylamine and ultra-dry dichloromethane are mixed, refluxed and stirred, and then a cholesterol chloroformate solution dissolved in ultra-dry dichloromethane is added dropwise to carry out the reaction. After the reaction is completed, the reaction solution is cooled to room temperature, concentrated under reduced pressure, and the concentrated solution is separated by column chromatography to obtain the dual-emission fluorescent probe.

[0059] In the process of mixing the intermediate shown in formula (2), anhydrous triethylamine and ultra-dry dichloromethane, the molar ratio of the intermediate shown in formula (2) to ultra-dry dichloromethane is 1:(1000~1200); wherein, the ultra-dry dichloromethane mentioned in this invention refers to dichloromethane with a water content ≤20ppm.

[0060] In step 2 of this invention, column chromatography separation uses a petroleum ether-ethyl acetate system as the eluent, wherein the volume ratio of petroleum ether to ethyl acetate is preferably 10:1.

[0061] In some embodiments of the present invention, in step 2, the reaction temperature is 60~70℃ and the reaction time is 4~6 hours.

[0062] The compound represented by formula (1) of this invention uses a moderately acidic imine NH as a proton donor and a moderately basic benzothiazole N atom as a proton acceptor, and introduces a cholesterol structural fragment with strong van der Waals interactions. The appropriately acidic and basic proton donor and proton acceptor cause the fluorescent probe molecule to undergo an excited-state intramolecular proton transfer reaction, and the resulting proton transfer products are two proton transfer isomers that can maintain mutual equilibrium, thereby emitting dual fluorescence, that is, there are two maximum fluorescence emission wavelengths at the maximum excitation wavelength, which are 400 nm and 580 nm, respectively. Therefore, the compound represented by formula (1) of this invention is a dual-emission fluorescent probe. The fluorescence emission intensity of the dual-emission fluorescent probe in different solvents and the relative fluorescence emission intensity at the two maximum fluorescence emission wavelengths are different. Therefore, different solvents can be detected and identified based on the ratio of the fluorescence emission intensities at the two different wavelengths, realizing ratio sensing; at the same time, the fluorescence color emitted by the dual-emission fluorescent probe in different solvents is different, which is solvent-dependent and can be used for visual detection. Meanwhile, the introduction of the cholesterol structure effectively inhibits the double bond rotation of the proton transfer product, avoids fluorescence quenching, and enhances fluorescence emission, thereby improving the sensitivity and distinguishability of solvent detection.

[0063] The dual-emission fluorescent probe has two maximum excitation wavelengths (Ex), which are 290 nm and 340 nm, respectively. After being excited to the second excited state, the energy gap between the first and second excited states is large, which allows the proton transfer process in the second excited state to compete with the internal conversion process. This can lead to an intramolecular proton transfer reaction in the excited state that follows the anti-Kasha rule, providing a reaction channel for the proton transfer product in the first excited state. Thus, the luminescence behavior of the dual-emission fluorescent probe in solution can be tuned by changing the excitation wavelength.

[0064] Specifically, when the dual-emission fluorescent probe is excited with 290 nm light, the contribution of fluorescence emission originating from 580 nm to the fluorescence spectrum is greater than that of fluorescence emission at 580 nm when the dual-emission fluorescent probe is excited with 340 nm light. Therefore, the luminescence behavior of the dual-emission fluorescent probe can be modulated by switching the excitation wavelength. Simultaneously, the fluorescence color of the dual-emission fluorescent probe in different solvents and the relative fluorescence emission intensity at the two maximum fluorescence emission wavelengths are also different. Therefore, the luminescence behavior of the dual-emission fluorescent probe can also be modulated by changing the solvent.

[0065] In other words, the luminescence behavior of the dual-emission fluorescent probe of the present invention can be regulated. Specifically, the luminescence behavior of the dual-emission fluorescent probe can be regulated by changing the type of solvent used to dissolve it, or by changing the excitation wavelength.

[0066] Based on the tunable luminescence behavior of the dual-emission fluorescent probe described in this invention, the luminescence behavior of the dual-emission fluorescent probe differs among different types of methane chlorides. Specifically, the fluorescence color emitted by the dual-emission fluorescent probe differs among different types of methane chlorides, and the relative fluorescence emission intensity at the two maximum fluorescence emission wavelengths differs. Based on this, the dual-emission fluorescent probe described in this invention can be used to visually detect methane chlorides and distinguish different types of methane chlorides, such as dichloromethane, trichloromethane, and carbon tetrachloride.

[0067] Specifically, the application of the dual-emission fluorescent probe of the present invention in the detection of methane chloride includes: adding the dual-emission fluorescent probe to methane chloride to obtain a dual-emission fluorescent probe solution; irradiating the dual-emission fluorescent probe solution with a laser, and identifying the type of methane chloride based on the fluorescence color emitted by the dual-emission fluorescent probe solution.

[0068] The concentration of the dual-emission fluorescent probe solution is 0.01-0.1 mmol / L.

[0069] To further improve the accuracy of the detection results, further detection can be performed: the dual-emission fluorescent probe is added to methane chloride to obtain a dual-emission fluorescent probe solution; the dual-emission fluorescent probe solution is subjected to fluorescence emission spectroscopy to obtain the first fluorescence emission intensity corresponding to the first maximum emission wavelength (400 nm) and the second fluorescence emission intensity corresponding to the second maximum emission wavelength (580 nm); the type of methane chloride is identified based on the ratio of the first fluorescence emission intensity to the second fluorescence emission intensity.

[0070] Of course, the type of methane chloride can also be identified directly based on the ratio of the first fluorescence emission intensity to the second fluorescence emission intensity.

[0071] Example 1

[0072] Preparation of dual-emission fluorescent probe compound 1:

[0073] 1) Synthetic intermediate 1

[0074] 0.30 g (0.002 mol) of 2-amino-5-methylbenzoic acid, 0.62 g (0.002 mol) of triphenyl phosphite (TPP), 0.78 g (0.0024 mol) of tetrabutylammonium bromide (TBAB), and 0.25 g (0.002 mol) of 2-aminothiophenol were placed in a 100 mL double-necked flask. Toluene (30 mL, 0.282 mol) was added to the flask. Under argon protection, the mixture was stirred and heated to 120 °C and refluxed for 8 hours. The resulting reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the crude product was separated by column chromatography using dichloromethane-n-hexane (volume ratio of dichloromethane to n-hexane 1:2) as the eluent. The collected fraction was evaporated to dryness to obtain a pale yellow powder intermediate 1.

[0075] The reaction equation is as follows:

[0076]

[0077] 2) Preparation of compound 1

[0078] Under argon (Ar) protection, intermediate 1 (0.096 g, 0.0004 mol), anhydrous triethylamine (NEt3) (1.12 mL, 0.008 mol), and ultra-dry dichloromethane (20 mL, 0.313 mol) were added to a flask. After stirring under reflux at 60 °C for 30 minutes, 10 mL of cholesterol chloroformate solution dissolved in ultra-dry dichloromethane (5.41 g, 0.012 mol) was added dropwise. The mixture was stirred under reflux for 4 hours. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated under reduced pressure. Column chromatography was performed using petroleum ether-ethyl acetate (volume ratio of petroleum ether to ethyl acetate was 10:1) as the eluent. The collected fraction was evaporated to dryness to obtain dual-emission fluorescent probe compound 1.

[0079] The reaction equation is as follows:

[0080]

[0081] The 1H NMR spectrum and high-resolution mass spectrum of compound 1 prepared in this embodiment are shown below. Figure 1 and Figure 2 As shown.

[0082] Example 2

[0083] Preparation of dual-emission fluorescent probe compound 2

[0084] 1) Synthetic intermediate 2

[0085] 2-Amino-5-methoxybenzoic acid (0.34 g, 0.0022 mol), triphenyl phosphite (0.62 g, 0.002 mol), tetrabutylammonium bromide (0.78 g, 0.0024 mol), and 2-aminothiophenol (0.25 g, 0.002 mol) were placed in a 100 mL double-necked flask. Toluene (30 mL, 0.282 mol) was added to the flask. Under argon protection, the mixture was stirred and heated to 130 °C, refluxed for 10 hours, and then the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the crude product was separated by column chromatography using dichloromethane-n-hexane (dichloromethane to n-hexane volume ratio 1:2) as the eluent. The collected fraction was evaporated to dryness to obtain a white solid intermediate 2.

[0086] The reaction equation is as follows:

[0087]

[0088] 2) Preparation of compound 2

[0089] Under argon protection, intermediate 2 (0.10 g, 0.0004 mol), anhydrous triethylamine (1.12 mL, 0.008 mol), and ultra-dry dichloromethane (20 mL, 0.313 mol) were added to a flask. After stirring under reflux for 30 minutes, 10 mL of cholesterol chloroformate solution dissolved in ultra-dry dichloromethane (5.41 g, 0.012 mol) was added dropwise. The mixture was stirred under reflux at 70 °C for 6 hours. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated under reduced pressure. Column chromatography was performed using petroleum ether-ethyl acetate (volume ratio of petroleum ether to ethyl acetate was 10:1) as the eluent. The collected fraction was evaporated to dryness to obtain dual-emission fluorescent probe compound 2.

[0090] The reaction equation is as follows:

[0091]

[0092] The dual-emission fluorescent probe solution was prepared using compound 1 obtained in Example 1 above, and the following steps were included:

[0093] 1) Prepare a 0.1 mmol / L solution of compound 1 with a solvent, seal it, and let it stand for 10 minutes for later use;

[0094] 2) Filter the above solution using an organic microporous membrane with a pore size of 0.22 μm. After freezing the filtrate with liquid nitrogen, immerse it in acetone at room temperature for 10 minutes. Once the solution temperature returns to room temperature, repeat this hot and cold cycle three times to remove the gas from the solution. Seal and store for later use.

[0095] To verify the effectiveness of the present invention, a large number of experimental studies were conducted on the dual-emission fluorescent probe solution prepared using compound 1, and the experimental results are as follows.

[0096] 1. Characterization of basic fluorescence behavior

[0097] The dual-emission fluorescent probe solution prepared above was characterized by excitation and emission spectra using an Edinburgh Instruments FS 5 single-photon counting fluorescence spectrometer. The results are shown in the figure. Figure 3 .Depend on Figure 3 It can be seen that compound 1 has two maximum excitation wavelengths (Ex), namely 290 nm and 340 nm; under each maximum excitation wavelength, compound 1 has two maximum fluorescence emission wavelengths, namely 400 nm and 580 nm, that is, compound 1 is a dual-emission fluorescent probe. Figure 3 The results provide light source information and detection wavelength information for the identification of methane chloride in this invention. According to... Figure 3 As a result, this invention uses a 340 nm light source as the excitation source and detects the fluorescence emission intensity at 400 nm and 580 nm to conduct subsequent methane chloride detection experiments.

[0098] 2. Calculation of excited-state processes of compound 1

[0099] Fluorescence emission is a fundamental factor determining the properties and practical applications of fluorescent probes. Therefore, using time-dependent density functional theory (TD-)PBE0 / 6-31G(d,p) (time-dependent density functional theory calculations using the PBE0 functional and the 6-31G(d,p) basis set), the potential energy profiles of compound 1 along the proton transfer coordinate system and its proton transfer product along the C=C double bond rotation coordinate system were simulated. The calculation results are as follows: Figures 4-6 As shown.

[0100] Figure 4 This is a potential energy profile of compound 1 obtained in this invention along the proton transfer coordinates in its ground state (S0) and first excited state (S1). Figure 4 It is known that when compound 1 is vertically excited to the first excited state (FC region), it relaxes to the local minimum (where it emits 400 nm fluorescence). Then, after overcoming the 4.7 kcal / mol barrier in the first excited state (S1), a proton transfer product is generated (where it emits 580 nm fluorescence). There are two proton transfer isomers in the first excited state. The two proton transfer isomers can exist stably and emit 400 nm and 580 nm fluorescence, respectively. This lays a solid foundation for the detection and identification of ratiometric dual-emission fluorescent probes in different solvents.

[0101] Figure 5 This is a potential energy profile of the ground state and the first excited state (S1) of the proton transfer product of compound 1, showing the coordinate changes along the C=C double bond rotation. Figure 5 It is known that the proton transfer product generated by compound 1 is not as prone to losing fluorescence activity as common excited-state proton transfer fluorescent molecules due to the double bond rotation without a barrier. The C=C double bond rotation of the proton transfer product generated by compound 1 needs to overcome a barrier of 1.5 kcal / mol, making it relatively stable and possessing better fluorescence activity. This lays a solid foundation for compound 1 to emit dual fluorescence signals.

[0102] Figure 6 This is a potential energy profile of compound 1 along the proton transfer coordinate system, showing its ground state (S0), first excited state (S1), and second excited state (S2). Figure 6It is known that when compound 1 is excited to the second excited state, the energy gap between the first and second excited states is relatively large (0.63 eV), which allows the proton transfer process in the second excited state to compete with the internal conversion process. This allows for the occurrence of an intramolecular proton transfer reaction in the excited state that violates the Kassa rule, providing a reaction channel for the proton transfer products in the first excited state. This lays a solid foundation for adjusting the luminescence behavior of compound 1 in solution by changing the excitation wavelength.

[0103] 3. Light emission behavior regulation experiment

[0104] The prepared dual-emission fluorescent probe solution was subjected to excitation wavelength-dependent and solvent-dependent fluorescence emission spectroscopy experiments using an Edinburgh Instruments FS 5 single-photon counting fluorescence spectrometer. The results are as follows: Figure 3 and Figure 7 As shown. By Figure 3 It is known that when the sample (dual-emission fluorescent probe solution) is excited by 290 nm light, the contribution of fluorescence emission from 580 nm to the fluorescence spectrum is greater than that of fluorescence emission from 580 nm when the sample is excited by 340 nm light. Therefore, the luminescence behavior of compound 1 can be controlled by switching the excitation wavelength. This method is not limited by physical space, does not corrode the sample, and is easy to operate, providing a new idea for the regulation of the luminescence behavior of dual-emission fluorescent probe molecules.

[0105] Figure 7 The fluorescence emission spectra of compound 1 in different solvents using 340 nm wavelength light as excitation are shown. The solvents used were water (H2O), N,N-dimethylformamide (DMF), ethanol (EtOH), acetone, tetrahydrofuran (THF), chloroform (CHCl3), dichloromethane (CH2Cl2), carbon tetrachloride (CCl4), toluene, and n-hexane. Figure 7 It is known that the fluorescence emission intensity of compound 1 and the relative intensity of the two sets of fluorescence emission are solvent-dependent. Therefore, the luminescence behavior of the dual-emission fluorescent probe can be controlled by changing the type of solvent. This method is simple to operate, not limited by time and space, and the sample can be recovered.

[0106] 4. Detection test for methane chloride

[0107] Following the above method for preparing dual-emission fluorescent probe solutions, dual-emission fluorescent probe solutions with dichloromethane, carbon trichloride, and carbon tetrachloride as solvents were prepared using different types of methane chlorides (dichloromethane, carbon trichloride, and carbon tetrachloride). Fluorescence emission spectroscopy experiments were then conducted using an Edinburgh Instruments FS 5 single-photon counting fluorescence spectrometer. The results are as follows: Figure 8 As shown. By Figure 8It can be seen that in different methane chloride solvents, as the number of chlorine atoms in the solvent increases, when using light with a wavelength of 340 nm as the excitation light, the contribution of fluorescence emission at 400 nm to the fluorescence spectrum gradually decreases. Dividing the fluorescence emission intensity at 400 nm by the fluorescence emission intensity at 580 nm yields I. 440 / I 580 The values ​​are shown in Table 1. Table 1 shows that for different methane chloride solvents, the obtained I... 440 / I 580 The values ​​differ, according to I 440 / I 580 The difference in values ​​can distinguish between dichloromethane, carbon trichloride, and carbon tetrachloride.

[0108] Table 1

[0109]

[0110] Figure 9 These are fluorescence images of compound 1 of the present invention under 365 nm ultraviolet light irradiation in different methane chloride solvents. Figure 9 As shown, three dual-emission fluorescent probe solutions with dichloromethane, carbon trichloride, and carbon tetrachloride as solvents exhibit different fluorescence colors, enabling visual detection of dichloromethane, carbon trichloride, and carbon tetrachloride. This method is simple, inexpensive, rapid, and allows for immediate detection; it is a novel ratiometric visual detection method based on dual fluorescence emission.

[0111] In summary, this invention selects imine NH as the proton donor and benzothiazole N atom as the proton acceptor, and incorporates the cholesterol structure into the design of a dual-emission fluorescent probe molecule. Utilizing the intramolecular proton transfer reaction of the anti-Kasha excited state and the strong van der Waals interaction of cholesterol, a novel dual-emission fluorescent probe based on the intramolecular proton transfer reaction of the anti-Kasha excited state is developed. A method for controlling the luminescence behavior by changing the excitation wavelength and solvent type is proposed. This method is simple to operate, does not corrode or contact the sample, and is not limited by physical space. Simultaneously, the visual detection of dichloromethane, carbon trichloride, and carbon tetrachloride is achieved, developing a new visual detection method for methane chlorides.

[0112] The preparation method of the dual-emission fluorescent probe of this invention is simple to operate and has mild reaction conditions. The prepared compound has a stable dual-fluorescence emission signal, is sensitive to the solvent environment and is wavelength-dependent. The proposed visual detection method for methane chloride is simple to operate, has low requirements for equipment and testing environment, and has a fast response speed. With further development, it can be developed into a portable methane chloride detection device.

[0113] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A dual-emission fluorescent probe, characterized in that, The structure of the dual-emission fluorescent probe is shown in formula (1): Where R is methyl or methoxy.

2. The method for preparing the dual-emission fluorescent probe according to claim 1, characterized in that, include: Step 1: 2-Aminothiophenol is reacted with 2-amino-5-methylbenzoic acid or 2-amino-5-methoxybenzoic acid in the presence of triphenyl phosphite and tetrabutylammonium bromide to obtain the intermediate shown in formula (2). Step 2: The intermediate shown in formula (2) is reacted with cholesterol chloroformate in the presence of triethylamine to obtain a dual-emission fluorescent probe.

3. The method for preparing a dual-emission fluorescent probe according to claim 2, characterized in that, In step 1, the molar ratio of 2-aminothiophenol to 2-amino5-methylbenzoic acid or 2-amino-5-methoxybenzoic acid is 1:(1~1.1), and the molar ratio of 2-aminothiophenol to triphenyl phosphite and tetrabutylammonium bromide is 1:(1~1.2):(1~1.2).

4. The method for preparing a dual-emission fluorescent probe according to claim 2, characterized in that, In step 1, the reaction temperature is 120~130℃ and the reaction time is 8~10 hours.

5. The method for preparing a dual-emission fluorescent probe according to claim 2, characterized in that, In step 2, the molar ratio of the intermediate shown in formula (2) to cholesterol chloroformate and triethylamine is 1:(20~30):(20~30).

6. The method for preparing a dual-emission fluorescent probe according to claim 2, characterized in that, In step 2, the reaction temperature is 60~70℃ and the reaction time is 4~6 hours.

7. The method for regulating the luminescence behavior of the dual-emission fluorescent probe according to claim 1, characterized in that, The luminescence behavior of the dual-emission fluorescent probe can be modulated by changing the type of solvent used to dissolve it, or by changing the excitation wavelength.

8. The application of the dual-emission fluorescent probe of claim 1 in distinguishing types of methane chloride.

9. The application according to claim 8, characterized in that, The dual-emission fluorescent probe is added to methane chloride to obtain a dual-emission fluorescent probe solution; the dual-emission fluorescent probe solution is irradiated with a laser, and the type of methane chloride is identified based on the fluorescence color emitted by the dual-emission fluorescent probe solution.

10. The application according to claim 8, characterized in that, The dual-emission fluorescent probe is added to methane chloride to obtain a dual-emission fluorescent probe solution; the dual-emission fluorescent probe solution is subjected to fluorescence emission spectroscopy to obtain the first fluorescence emission intensity corresponding to the first maximum emission wavelength and the second fluorescence emission intensity corresponding to the second maximum emission wavelength; the type of methane chloride is identified based on the ratio of the first fluorescence emission intensity and the second fluorescence emission intensity.

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