Thiophene alanine aggregation-induced emission fluorescent probe as well as synthesis method and application thereof

Abstract

The invention discloses a thiophene alanine aggregation-induced emission fluorescent probe as well as a synthesis method and application thereof, and belongs to the technical field of fluorescent probes, the fluorescent probe is obtained by directly coupling alanine and fluorescent molecules and introducing thiophene in the middle to be in conjugate connection with alanine, and the fluorescent probe can mark a target object through alanine. The novel functional probe is synthesized for the first time, compared with a probe without thiophene, the probe with thiophene functional groups added in the middle has red shift in emission wavelength, and the probe with longer emission wavelength has the advantages of deeper penetration, higher imaging signal-to-noise ratio and the like. Experiments prove that the probe can successfully mark bacteria, especially pneumonia bacteria.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and relates to a fluorescent probe, its synthesis method and application, specifically to a thiophene alanine aggregation-induced emission fluorescent probe, its synthesis method and application. Background Technology

[0002] Bioorthogonal chemical reactions are a class of chemical reactions that can occur under physiological conditions. They are simple, efficient, and highly specific, and are widely used in biomedical research. Based on metabolic engineering of natural biological processes, biomolecules can be modified non-destructively and efficiently, making it an ideal biomodification technique. After modifying a target analyte (such as bacteria) with bioorthogonal functional groups and incubating it, the active chemical groups are expressed. Subsequently, fluorophores modified with the corresponding bioorthogonal groups are introduced, achieving effective fluorescent labeling of the target analyte through covalent bonds. However, this labeling method generally requires two steps: the labeling process necessitates pretreatment of the target analyte and may even require complex catalytic reactions.

[0003] Chinese patent application CN116143749A discloses an alanine aggregation-induced emission fluorescent probe that directly couples alanine to fluorescent molecules. Based on current labeling technology advancements, this direct coupling one-step labeling method offers advantages such as simple operation and high efficiency. However, its luminescence properties are relatively weak, making it unsuitable for effectively labeling certain specific targets, such as bacteria. Summary of the Invention

[0004] The purpose of this invention is to provide a thiophene alanine aggregation-induced emission fluorescent probe, its synthesis method, and its application. This fluorescent probe is a novel functional probe synthesized for the first time, with a novel structure, stable luminescent properties, and the ability to effectively label bacteria.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention discloses a thiophene alanine aggregation-induced emission fluorescent probe, which is obtained by directly coupling alanine and a fluorescent molecule, with thiophene conjugated to alanine in between. This fluorescent probe can label targets via alanine and is named TPAPy-S-Ala, with the following structural formula:

[0007]

[0008] The present invention also discloses the application of the above-mentioned thiophene alanine aggregation-induced emission fluorescent probe in the preparation of luminescent materials.

[0009] This invention also discloses the application of the above-mentioned thiophene alanine aggregation-induced emission fluorescent probe as a fluorescent label for bioorthogonal chemical reactions.

[0010] The present invention also discloses the application of the above-mentioned thiophene alanine aggregation-induced emission fluorescent probe in the preparation of bacterial markers.

[0011] Preferably, the bacterial markers achieve real-time location and tracking of bacteria through fluorescent labeling with dyes.

[0012] More preferably, the bacteria are pneumonia bacteria.

[0013] More preferably, the pneumonia bacteria is Klebsiella pneumoniae.

[0014] Therefore, the thiophene alanine aggregation-induced luminescence probe is a probe capable of labeling Klebsiella pneumoniae.

[0015] This invention also discloses a method for synthesizing the above-mentioned thiophene alanine aggregation-induced luminescent fluorescent probe, comprising the following steps:

[0016] 1) TPA-S was prepared by a Suzuki coupling reaction of 4-halotriphenylamine and 5-aldehyde-2-thiophene borate pinacol ester in the presence of an alkaline substance.

[0017] The structural formula of TPA-S is as follows:

[0018]

[0019] 2) Using TPA-S and 4-methylpyridine as raw materials, TPAPy-S was prepared by reaction in the presence of p-toluenesulfonic acid;

[0020] The structural formula of TPAPy-S is as follows:

[0021]

[0022] 3) TPAPy-S was reacted with 4-bromo-1-butyne to prepare TPAPy-S-Butyne;

[0023] The structural formula of TPAPy-S-Butyne is as follows:

[0024]

[0025] 4) TPAPy-S-Butyne and 6-azido-alanine were reacted in the presence of cuprous bromide and trimethylamine to prepare a thiophene alanine aggregation-induced emission fluorescent probe.

[0026] In some preferred embodiments, in step 1), 4-halotriphenylamine and 5-aldehyde-2-thiophene borate pinacol ester are dissolved in an organic solvent, an alkaline substance is added, nitrogen or an inert gas is introduced, and a catalyst is added to carry out the reaction. The reaction solution is purified to obtain TPA-S.

[0027] Optionally, the 4-halotriphenylamine is 4-bromotriphenylamine or 4-iodotriphenylamine;

[0028] Optionally, the alkaline substance is potassium carbonate or sodium carbonate;

[0029] Optionally, the catalyst is a palladium-based catalyst;

[0030] Optionally, the inert gas is argon;

[0031] Optionally, the organic solvent is DMF;

[0032] Optionally, the reaction time is 8-16 hours;

[0033] Optionally, the purification process includes extraction, washing, drying, and elution.

[0034] Alternatively, dichloromethane can be used as the extractant during extraction, and ethyl acetate and n-hexane in a volume ratio of 1:10-20 can be used as the eluent during elution.

[0035] Further optionally, the mass ratio of 4-halotriphenylamine to 5-aldehyde-2-thiophene borate pinacol ester is 2-3:1.

[0036] In some preferred embodiments, in step 2), 4-methylpyridine and p-toluenesulfonic acid are mixed in an organic solvent, and TPA-S prepared in step 1) is added to react. After the reaction is completed, the reaction solution is extracted, and the organic layer is collected for purification.

[0037] Optionally, the organic solvent is DMF;

[0038] Optionally, the reaction solution can be extracted using DCM and saturated saline solution;

[0039] Optionally, the reaction is a reflux reaction, and the reaction time is 8-24 hours;

[0040] Optionally, after merging the organic phases, purification can be carried out using DCM and methanol at a volume ratio of 99:1 as eluents;

[0041] Further optionally, the molar ratio of 4-methylpyridine, p-toluenesulfonic acid and TPA-S is (4.5-7):(4.5-7):(0.8-1.5).

[0042] In some preferred embodiments, in step 3), TPAPy-S and 4-bromo-1-butyne are added to an organic solvent, heated to react, and purified to obtain TPAPy-S-Butyne;

[0043] Optionally, the organic solvent is DMF;

[0044] Optionally, the heating reaction temperature is 70-90℃, and the heating reaction time is 8-24h;

[0045] Optionally, the purification process includes extraction, drying, and elution. Dichloromethane is used as the extractant during extraction, and dichloromethane and methanol in a volume ratio of 10:0.5-2 are used as the eluent during elution.

[0046] Optionally, in step 3), the molar ratio of TPAPy-S to 4-bromo-1-butyne is 1:4-6.

[0047] In some preferred embodiments, in step 4), TPAPy-S-Butyne and 6-azido-alanine are dissolved in an organic solvent to obtain a solution, and cuprous bromide is dispersed in triethylamine and added to the above solution for reaction. The reaction solution is purified to obtain a fluorescent probe with aggregation-induced emission function, namely TPAPy-S-Ala.

[0048] Optionally, the organic solvent is DMSO;

[0049] Optionally, the purification process includes filtration, collection of the organic layer, drying, and recrystallization, wherein the recrystallization reagent is methanol;

[0050] Further optionally, the molar ratio of TPAPy-S-Butyne, 6-azido-alanine, and cuprous bromide is 1:(0.8-1.2):(0.8-1.2).

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] This invention proposes a strategy for direct labeling of target analytes using the fluorophore-thiophene-alanine. Alanine and a fluorescent molecule are directly coupled, with thiophene introduced as a conjugated linker to further improve the luminescence properties. Based on this strategy, we designed and synthesized a thiophene-alanine aggregation-induced emission fluorescent probe (TPAPy-S-Ala). This fluorescent probe is expected to achieve target labeling by involving alanine in the metabolism of the target analyte.

[0053] This invention presents a novel functional probe synthesized for the first time. The probe with the addition of a thiophene functional group exhibits a redshift in emission wavelength compared to probes without thiophene. This longer emission wavelength provides advantages such as deeper penetration and a higher signal-to-noise ratio in imaging. Experimental verification has shown that this probe can successfully label bacteria, particularly *Pneumocystis pneumoniae*.

[0054] The synthesis method of the thiophene alanine aggregation-induced emission fluorescent probe provided by this invention has a simple and efficient process, high yield, and is easy to purify, resulting in a high-purity target product. It directly couples alanine and fluorescent molecules, and through the participation of alanine in the metabolism of the target, it achieves one-step direct labeling of the target. Compared with biological orthogonal labeling, the direct labeling of this invention is more convenient because it bypasses two steps and the complex catalysts that may be required for the reaction. Attached Figure Description

[0055] Figure 1 This is the 1H NMR spectrum of TPA-S obtained in Example 1 of this invention;

[0056] Figure 2 This is the 1H NMR spectrum of TPAPy-S obtained in Example 1 of this invention;

[0057] Figure 3 This is the 1H NMR spectrum of TPAPy-S-Butyne obtained in Example 1 of this invention;

[0058] Figure 4 The above are the 1H NMR spectra of TPAPy-S-Ala prepared in Example 1 of this invention; wherein, (a) and (b) are the 1H NMR spectra of the two isomers, respectively.

[0059] Figure 5 This is a high-resolution mass spectrum of TPAPy-S-Ala obtained in Example 1 of the present invention; wherein, (a) and (b) are high-resolution mass spectra of the two isomers, respectively.

[0060] Figure 6 This is the ultraviolet absorption spectrum of TPAPy-S-Ala prepared in Example 1 of the present invention; the horizontal and vertical axes are wavelength and absorption.

[0061] Figure 7 This is the fluorescence emission spectrum of TPAPy-S-Ala prepared in Example 1 of this invention; x and y axes: wavelength, y axis: FL intensity.

[0062] Figure 8This is the fluorescence emission spectrum of TPAPy-S-Ala in different proportions of DMSO / tetrahydrofuran (THF) mixtures in Experiment Example 2 of the present invention; x and y axes: wavelength, y axis: FL intensity.

[0063] Figure 9 This is a graph showing the relationship between the volume percentage of tetrahydrofuran in the mixture and the strongest fluorescence intensity in Experiment Example 2 of the present invention.

[0064] Figure 10 This is a photograph of bacteria imaged using a Leica super-resolution structured light microscope in Experiment Example 3 of the present invention. Detailed Implementation

[0065] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. 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 should fall within the scope of protection of the present invention.

[0066] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises 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 such processes, methods, products, or apparatus.

[0067] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0068] The present invention will now be described in further detail with reference to the accompanying drawings:

[0069] Example 1

[0070] A method for synthesizing a thiophene alanine aggregation-induced luminescent fluorescent probe, the specific reaction equation and steps are as follows:

[0071] 1) TPA-S was prepared by a Suzuki coupling reaction of 4-halotriphenylamine and 5-aldehyde-2-thiophene borate pinacol ester in the presence of an alkaline substance.

[0072]

[0073] Specifically, 2.58 g of 4-bromotriphenylamine (TPA-Br) and 1.05 g of 5-aldehyde-2-thiophene borate pinacol ester were dissolved in DMF. 2.2 g of K₂CO₃ was added, and the mixture was deoxygenated three times by purging with argon gas. 0.09 g of Pd(PPh₃)₄ was then added. The reaction mixture was refluxed at 150 °C for 12 h, then extracted with dichloromethane and washed three times with saturated brine. The organic phase was collected, dried over anhydrous Na₂SO₄, and the solvent was evaporated. The product, denoted as TPA-S, was purified by silica gel column chromatography using ethyl acetate / n-hexane (1:20–1:10, v / v) as the eluent, with a yield of 90%.

[0074] The 1H NMR spectrum data of TPA-S are as follows, the 1H NMR spectrum is shown below. Figure 1 As shown.

[0075] 1 H NMR (400MHz, CDCl3) δ (ppm) 9.88 (s, 1H), 7.73 (d, J = 4.0Hz, 1H), 7.58-7.50 (m, 2H), 7.35-7.30 (m, 5H), 7.20-7.05 (m, 8H).

[0076] 2) Using TPA-S and 4-methylpyridine as raw materials, TPAPy-S was prepared by reaction in the presence of p-toluenesulfonic acid;

[0077]

[0078] Specifically, compound TPA-S (1 mmol) and p-toluenesulfonic acid (5 mmol) were added to dry DMF (10 mL), followed by the addition of 4-methylpyridine (5 mmol). The mixture was refluxed for 24 h, cooled to room temperature, and the reaction solution was extracted with DCM and saturated brine. The organic phases were then combined. The crude product was purified by silica gel column chromatography using DCM / methanol (99:1 v / v) as the eluent. A bright yellow TPAPy-S powder was obtained in 85% yield and designated as TPAPy-S.

[0079] The 1H NMR data of TPAPy-S are shown below. Figure 2 As shown.

[0080] 1H NMR (400MHz, CDCl3) δ (ppm) 8.61-8.55 (m, 2H), 7.50 (d, J = 8.7Hz, 2H), 7.41 (d, J=16.0Hz,1H),7.35-7.29(m,5H),7.20-7.06(m,11H),6.80(d,J=16.0Hz,1H).

[0081] 3) TPAPy-S was reacted with 4-bromo-1-butyne to prepare TPAPy-S-Butyne;

[0082]

[0083] Specifically, compound TPAPy-S (0.1 mmol) and 4-bromo-1-butyne (66 mg, 0.5 mmol) were dissolved in DMF (5 mL), heated at 80 °C for 14 h, cooled to room temperature, and the compound was extracted with DCM. The collected organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The residue was subjected to column chromatography with dichloromethane / methanol = 10 / 1 (v / v) to give an orange solid, designated TPAPy-S-Butyne, in 80% yield.

[0084] The 1H NMR data of TPAPy-S-Butyne are shown below. Figure 3 As shown.

[0085] 1 H NMR (400MHz, DMSO) δ (ppm) 8.98 (dt, J = 7.2, 3.4Hz, 2H), 8.36–8.22 (m, 3H), 7.63 (d, J = 8.5Hz, 2H), 7.52 (s, 2H), 7.35 (t, J = 7.8Hz, 4H), 7.10(dd,J=16.9,6.6Hz,7H), 6.98(d,J=8.5Hz,2H), 4.66(dt,J=8.4,4.2Hz,2H), 3.10(d,J=2.6Hz,1H), 2.96(td,J=6.6,2.6Hz,2H).

[0086] 4) TPAPy-S-Butyne and 6-azido-alanine (purchased from Jena Bioscience) were reacted in the presence of cuprous bromide and trimethylamine to prepare the thiophene alanine aggregation-induced emission fluorescent probe, namely TPAPy-S-Ala.

[0087]

[0088] Specifically, compound TPAPy-S-Butyne (0.1 mmol) and 6-azido-alanine (38 mg, 0.1 mmol) were dissolved in DMSO (2 mL) and placed in a 5 mL round-bottom flask. Cuprous bromide (14 mg, 0.1 mmol) was dispersed in 0.5 mL of trimethylamine and injected into the solution. After stirring at room temperature for 12 h, the mixture was filtered through a 0.45 μm polyethersulfone membrane filter. The collected organic layer was evaporated and dried. The product, denoted as TPAPy-S-Ala, was obtained by recrystallization from methanol with a yield of 92%.

[0089] The TPAPy-S-Ala was systematically characterized, including 1H NMR spectroscopy and high-resolution mass spectrometry, and the results are as follows: Figure 4 and Figure 5 As shown, this proves that the above synthetic method is feasible and the target compound is correct. The 1H NMR and high-resolution mass spectrometry data are as follows:

[0090] 1 H NMR(400MHz,DMSO-d6 and 1 / 5CF3CO2D)δ(ppm)8.77(s,2H),8.22–8.03(m,3H),7.93(s,1H),7.57(d,J=7.8Hz ,2H),7.53–7.19(m,8H),7.20–6.75(m,12H),4.76(d,J=28.9Hz,4H),4.55(s,1H).

[0091] 8.77 (d, J = 5.9 Hz, 2H), 8.19–8.07 (m, 3H), 7.92 (s, 1H), 7.57 (d, J = 7.9 Hz, 2H), 7.45 (s, 2H), 7.30 (t, J = 7.4 Hz, 6H), 7.04 (d, J = 7.4 Hz, 9H), 6.94 (d, J = 8.1 Hz, 3H), 4.84–4.70 (m, 4H), 4.55 (d, J = 5.1 Hz, 1H). Mass spectrometry data are from HRMS (ESI) calcd. for C 36 H 33 N6O2S + [M] + 613.23802,found:613.23792or613.23825.

[0092] This invention takes the thiophene alanine aggregation-induced emission fluorescent probe TPAPy-S-Ala prepared in Example 1 above as an example to test its related luminescence properties.

[0093] Effect Experiment Example 1

[0094] The absorption and photoemission (PL) spectra of the fluorescent probe TPAPy-S-Ala in DMSO were obtained. The specific method involved preparing a 10 μM TPAPy-S-Ala solution in DMSO. The absorption and emission spectra were detected using a UV spectrophotometer and a fluorescence spectrophotometer. The results are shown below. Figure 6 and Figure 7 As shown, from Figure 6 As can be seen, the absorption wavelength (left peak) of TPAPy-S-Ala is at 485nm; from Figure 7 As can be seen, the photoemission wavelength (right peak) is 680nm.

[0095] Effect Experiment Example 2

[0096] The aggregation-induced emission characteristics of TPAPy-S-Ala in DMSO / tetrahydrofuran mixtures with different volume ratios were investigated. A series of 10 μM TPAPy-S-Ala solutions were prepared by adding TPAPy-S-Ala to DMSO / tetrahydrofuran mixtures with different volume ratios, and their emission spectra were detected using a fluorescence spectrophotometer. The results are shown in [link to study]. Figure 8 As can be seen, the fluorescence emission intensity of TPAPy-D-Ala increases with the gradual increase of tetrahydrofuran concentration in the DMSO / tetrahydrofuran mixture from 0% to 80%. A graph was plotted with the volume percentage of tetrahydrofuran in the DMSO / tetrahydrofuran mixture as the x and y axes, and the highest fluorescence intensity of each group of TPAPy-S-Ala solutions at 675 nm as the y-axis. (See figure). Figure 9 As shown, it can be observed that the strongest fluorescence intensity of TPAPy-D-Ala is significantly increased when the proportion of tetrahydrofuran is 80% compared to 0%.

[0097] Effect Experiment Example 3

[0098] Klebsiella pneumoniae was transferred to 5 mL of liquid culture medium and incubated at 37 °C for 12 h. The bacterial concentration was determined at 600 nm (OD 600). 7 CFU of bacteria were transferred to 1.5 mL centrifuge tubes. The bacteria were collected by centrifugation at 4000 rpm for 3 min. 200 μL of the bacterial culture was transferred to a 35 mm confocal dish, and TPAPy-S-Ala solution (containing 1% DMSO aqueous solution as solvent to prepare a stock solution) was added to control the staining concentration at 2.5 μM. The bacteria were imaged using a Leica structured light illuminated visible microimaging (SIM) microscope with excitation at 561 nm and emission range of 570-750 nm. Results are as follows: Figure 10 As shown, from Figure 10As can be seen from the confocal image, the outline of the pneumonia bacteria is clearly visible and emits bright red light, indicating that the thiophene alanine aggregation-induced emission fluorescent probe prepared in this invention can participate in the metabolism of bacteria and thus successfully label the pneumonia bacteria. Therefore, it is expected that the real-time location and tracking of the pneumonia bacteria can be achieved through the fluorescent labeling experiment of the dye.

[0099] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A thiophene alanine aggregation-induced luminescent fluorescent probe, characterized in that, This fluorescent probe is obtained by directly coupling alanine and a fluorescent molecule, with thiophene conjugated to alanine in between. This fluorescent probe can label targets via alanine and is named TPAPy-S-Ala, with the following structural formula:

2. The application of the thiophene alanine aggregation-induced emission fluorescent probe according to claim 1 in the preparation of luminescent materials.

3. The application of the thiophene alanine aggregation-induced emission fluorescent probe according to claim 1 as a fluorescent label for bioorthogonal chemical reactions.

4. The application of the thiophene alanine aggregation-induced emission fluorescent probe according to claim 1 in the preparation of bacterial markers.

5. The application as described in claim 4, characterized in that, The bacterial markers described herein enable real-time location and tracking of bacteria through fluorescent labeling with dyes.

6. The method for synthesizing the thiophene alanine aggregation-induced emission fluorescent probe according to claim 1, characterized in that, Includes the following steps: 1) TPA-S was prepared by a Suzuki coupling reaction of 4-halotriphenylamine and 5-aldehyde-2-thiophene borate pinacol ester in the presence of an alkaline substance. The structural formula of TPA-S is as follows: 2) Using TPA-S and 4-methylpyridine as raw materials, TPAPy-S was prepared by reaction in the presence of p-toluenesulfonic acid; The structural formula of TPAPy-S is as follows: 3) TPAPy-S was reacted with 4-bromo-1-butyne to prepare TPAPy-S-Butyne; The structural formula of TPAPy-S-Butyne is as follows: 4) TPAPy-S-Butyne and 6-azido-alanine were reacted in the presence of cuprous bromide and trimethylamine to prepare a thiophene alanine aggregation-induced emission fluorescent probe.

7. The method for synthesizing the thiophene alanine aggregation-induced emission fluorescent probe according to claim 6, characterized in that, In step 1), 4-halotriphenylamine and 5-aldehyde-2-thiophene borate pinacol ester are dissolved in an organic solvent, an alkaline substance is added, nitrogen or an inert gas is introduced, and a catalyst is added to carry out the reaction. The reaction solution is purified to obtain TPA-S. Optionally, the 4-halotriphenylamine is 4-bromotriphenylamine or 4-iodotriphenylamine; Optionally, the alkaline substance is potassium carbonate or sodium carbonate; Optionally, the catalyst is a palladium-based catalyst; Optionally, the inert gas is argon; Optionally, the organic solvent is DMF; Optionally, the reaction time is 8-16 hours; Optionally, the purification process includes extraction, washing, drying, and elution. Alternatively, dichloromethane can be used as the extractant during extraction, and ethyl acetate and n-hexane in a volume ratio of 1:10-20 can be used as the eluent during elution. Further optionally, the mass ratio of 4-halotriphenylamine to 5-aldehyde-2-thiophene borate pinacol ester is 2-3:

1.

8. The method for synthesizing the thiophene alanine aggregation-induced emission fluorescent probe according to claim 6, characterized in that, In step 2), 4-methylpyridine and p-toluenesulfonic acid are added to an organic solvent and mixed. The TPA-S prepared in step 1) is then added to react. After the reaction is complete, the reaction solution is extracted and the organic layer is collected for purification. Optionally, the organic solvent is DMF; Optionally, the reaction solution can be extracted using DCM and saturated saline solution; Optionally, the reaction is a reflux reaction, and the reaction time is 8-24 hours; Optionally, after merging the organic phases, purification can be carried out using DCM and methanol at a volume ratio of 99:1 as eluents; Further optionally, the molar ratio of 4-methylpyridine, p-toluenesulfonic acid and TPA-S is (4.5-7):(4.5-7):(0.8-1.5).

9. The method for synthesizing the thiophene alanine aggregation-induced emission fluorescent probe according to claim 6, characterized in that, In step 3), TPAPy-S and 4-bromo-1-butyne are added to an organic solvent, heated and reacted, and then purified to obtain TPAPy-S-Butyne. Optionally, the organic solvent is DMF; Optionally, the heating reaction temperature is 70-90℃, and the heating reaction time is 8-24h; Optionally, the purification process includes extraction, drying, and elution. Dichloromethane is used as the extractant during extraction, and dichloromethane and methanol in a volume ratio of 10:0.5-2 are used as the eluent during elution. Optionally, in step 3), the molar ratio of TPAPy-S to 4-bromo-1-butyne is 1:4-6.

10. The method for synthesizing the thiophene alanine aggregation-induced emission fluorescent probe according to claim 6, characterized in that, In step 4), TPAPy-S-Butyne and 6-azido-alanine are dissolved in an organic solvent to obtain a solution. Cuprous bromide is dispersed in triethylamine and added to the above solution for reaction. The reaction solution is purified to obtain a fluorescent probe with aggregation-induced emission function, namely TPAPy-S-Ala. Optionally, the organic solvent is DMSO; Optionally, the purification process includes filtration, collection of the organic layer, drying, and recrystallization, wherein the recrystallization reagent is methanol; Further optionally, the molar ratio of TPAPy-S-Butyne, 6-azido-alanine, and cuprous bromide is 1:(0.8-1.2):(0.8-1.2).

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