Click chemistry reaction-activated aggregation-induced fluorescent probe and preparation method thereof, Treponema pallidum labeling method and application thereof

Abstract

The present invention discloses a click chemistry-activated aggregation-induced fluorescent probe, a preparation method thereof, and a Treponema pallidum labeling method and application. The preparation steps include: reacting 4-bromotriphenylamine and 5-formyl-2-thiopheneboronic acid to obtain TPA-A, reacting TPA-A with ethynylmagnesium bromide to obtain an oily product TPA-TH with blue fluorescence, and then treating it with manganese dioxide to obtain 5-(4-(diphenylamino)phenyl)thiophene-2-alkynyl ketone (TPA-TA). Advantages of the present invention include: through the rational design and synthesis of a click chemistry-activated aggregation-induced fluorescent probe TPA-TA, it can then undergo a click chemistry reaction with amino proteins on the outer membrane of Treponema pallidum, thereby achieving stable labeling of T. pallidum.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen marking and imaging, and in particular to a one-step marking technology for Treponema pallidum. Background Art

[0002] Click chemistry, a bioorthogonal chemical bonding method pioneered by scientists such as Bertozzi and Sharpless, is widely used for introducing functional group modifications or biomolecule coupling. Notably, this reaction does not affect related physiological processes within cells, and has therefore been used by numerous researchers for labeling, tracing, imaging, and targeted therapy of tumor cells and pathogens. It effectively avoids the shortcomings of traditional biocoupling, such as heavy metal catalysis and reactivity, and has greatly promoted the cross-development between biofunctional materials and biology. Currently, the in vitro culture technology for Treponema pallidum is still immature and cannot be effectively expanded, which affects the labeling efficiency when the state is poor. Therefore, it is of great significance to develop a labeling and tracing method that is not affected by the activity state of Treponema pallidum.

[0003] In recent years, a metal-free, catalytic click bioconjugation strategy based on activated alkynes has been used in research in chemistry, biology, and other fields, enriching the modification methods for natural products and bioactive substances. This method is simple to operate, has minimal impact on the activity of biomolecules, and the groups can react directly with the activated alkynes without any modification. This strategy can achieve efficient modification and potential functionalization in natural polysaccharides, biocompatible polyethylene glycol (PEG), synthetic polymers, cell-penetrating peptides, proteins, rapid whole-cell mapping, and even rapid differentiation and specific staining of Gram-positive bacteria. Therefore, the metal-free click bioconjugation strategy based on activated alkynes currently has broad application prospects in the development of rapid fluorescent labeling and functional modification of human.

[0004] The molecular structure of Treponema pallidum is currently recognized to consist of an outer membrane, peptidoglycan, periplasmic space, and inner membrane. The outer membrane, which contains various structural lipoproteins (such as TP0453) and pore proteins (such as Tprs), plays a crucial role in Treponema pallidum's self-protection and immune escape, and is also a potential antigen for the development of a syphilis vaccine. Summary of the Invention

[0005] The primary purpose of the present invention is to provide a click chemistry reaction-activated aggregation-induced fluorescent probe and its preparation method, as well as a Treponema pallidum labeling method and application, to solve the problem that the existing probe technology is inconvenient to label Treponema pallidum.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A click chemistry reaction-activated aggregation-induced fluorescent probe, the structural formula of which is shown below:

[0008]

[0009] The present invention also provides a method for preparing a click chemistry reaction-activated aggregation-induced fluorescent probe, which comprises the following steps: reacting 4-bromotriphenylamine with 5-formyl-2-thiopheneboronic acid to obtain 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde, reacting 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde with ethynylmagnesium bromide to obtain an oily product TPA-TH with blue fluorescence, and then treating the product with manganese dioxide to obtain 5-(4-(diphenylamino)phenyl)thiophene-2-ynyl ketone (TPA-TA).

[0010] Furthermore, the steps include: first weighing 4-bromotriphenylamine, 5-formyl-2-thiopheneboronic acid, and potassium carbonate, dissolving them in a mixed solution of tetrahydrofuran and water, adding a catalyst tetrakistriphenylphosphine palladium Pd(PPh3)4, and then stirring and reacting at 80°C for 24 hours to obtain compound 15-(4-(diphenylamino)phenyl)thiophene-2-aldehyde (TPA-A);

[0011] The compound 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde was weighed and dissolved in THF, and then ethynylmagnesium bromide solution was added dropwise in an ice bath. The reaction was stirred at room temperature for 12 hours to obtain an oily product TPA-TH with blue fluorescence. TPA-TH was dissolved in DCM, manganese dioxide was added, and the reaction was stirred at room temperature to obtain 5-(4-(diphenylamino)phenyl)thiophene-2-ynyl ketone TPA-TA.

[0012] Furthermore, the detailed steps include: first weighing 1.29 g of 4-bromotriphenylamine, 936 mg of 5-formyl-2-thiopheneboronic acid, and 1.19 g of potassium carbonate and dissolving them in 60 mL of a mixed solution of tetrahydrofuran and water with a volume ratio of V / V=3 / 1, then adding 4462 mg of tetrakistriphenylphosphine palladium Pd(PPh3) as a catalyst, stirring the above mixture at 80°C for 24 hours, cooling to room temperature, extracting with dichloromethane, and purifying on a silica gel column using petroleum ether and dichloromethane as eluents to obtain compound 15-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde TPA-A.

[0013] 335.4 mg of the compound 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde TPA-A was weighed and dissolved in 10 mL of THF. Then, 3 mL of a 0.5 M ethynylmagnesium bromide solution in THF was added dropwise in an ice bath. The solution was transferred to room temperature and stirred for 12 h. The reaction was then quenched with saturated ammonium chloride and extracted with dichloromethane to obtain a crude product. The product was purified on a silica gel column using a mixture of petroleum ether and ethyl acetate in a volume ratio of V1 / V2 = 10 / 1 as the eluent to obtain an oily product TPA-TH with blue fluorescence. TPA-TH was dissolved in 25 mL of DCM, 1.304 g of manganese dioxide was added, and the mixture was stirred at room temperature. After the reaction, manganese dioxide was removed with diatomaceous earth. The crude product was purified on a silica gel column using n-hexane and dichloromethane in a volume ratio of 3 / 1 as the eluent to obtain the final product 5-(4-(diphenylamino)phenyl)thiophene-2-ynyl ketone TPA-TA.

[0014] The present invention also provides a one-step fluorescent labeling and tracing imaging strategy for Treponema pallidum using the click chemistry reaction-activated aggregation-induced fluorescent probe.

[0015] The steps include: directly labeling Treponema pallidum and TPA-TA through a one-step incubation method, causing a click chemistry reaction, and realizing tracer imaging of Treponema pallidum.

[0016] More preferably, the detailed steps include: adding 1 mL 10 7 The Treponema pallidum suspension of 1000 tb / mL was incubated with TPA-TA at a final concentration of 5 μM in a 34°C tri-gas incubator for 30 min; the Treponema pallidum was then centrifuged to precipitate the Treponema pallidum, unused TPA-TA was removed, the Treponema pallidum was washed and resuspended, the Treponema pallidum was centrifuged to precipitate the Treponema pallidum, and the Treponema pallidum was resuspended to achieve visual labeling of the Treponema pallidum.

[0017] More preferably, the detailed steps include: mixing 1 mL of 10 7 The suspension of Treponema pallidum was incubated with TPA-TA at a final concentration of 5 μM in a 34°C tri-gas incubator for 30 min; then, the suspension was centrifuged at 14,000 g for 5 min to precipitate the Treponema pallidum, and the unused TPA-TA was removed. The suspension was then washed with 1 mL of PBS and resuspended in PBS. The suspension was centrifuged at 14,000 g for 5 min to precipitate T. pallidum, and resuspended in 500 μL of culture medium to achieve visual labeling of the Treponema pallidum.

[0018] More preferably, the culture medium components for culturing / suspending Treponema pallidum include: sterile water for culture, CMRL1066 without glutathione, sodium pyruvate, resazurin, morpholinopropanesulfonic acid, sodium bicarbonate, glutamine, glucose, histidine, dithiothreitol, and inactivated fetal bovine serum.

[0019] More preferably, the culture medium for culturing / suspending Treponema pallidum includes: 10 g / L CMRL1066 without glutathione, 0.7 mM sodium pyruvate, 0.1% resazurin by mass, 1 M morpholinopropanesulfonic acid, 7.5% sodium bicarbonate by mass, 200 mM glutamine, 17.6 mM glucose, 0.52 mM histidine, 0.52 mM dithiothreitol, 20% volume fraction of inactivated fetal bovine serum, and sterile water for culture, the volume being made up to 500 mL.

[0020] The present invention provides an application of a click chemistry reaction-activated aggregation-induced fluorescent probe in the preparation of a reagent or a kit.

[0021] Advantages of the present invention include:

[0022] By utilizing the structural characteristics of Treponema pallidum and through click chemistry reaction, a one-step fluorescent labeling and tracer imaging strategy for T. pallidum is achieved; the probe can effectively label Treponema pallidum for a long time without being affected by its activity, and can provide a powerful support and imaging monitoring tool for basic research on the pathogenic mechanism of syphilis, immune escape, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of this application, and do not constitute an improper limitation of the present invention. In the drawings:

[0024] Figure 1 Schematic diagram of the strategy for labeling T. pallidum using an aggregation-inducing fluorescent probe activated by click chemistry reaction.

[0025] Figure 2 This is the H NMR spectrum of TPA-A.

[0026] Figure 3 This is the carbon spectrum of TPA-A.

[0027] Figure 4 This is the mass spectrum of TPA-A.

[0028] Figure 5 This is the mass spectrum of TPA-TA.

[0029] Figure 6 This is the H NMR spectrum of TPA-TA.

[0030] Figure 7 This is the NMR carbon spectrum of TPA-TA.

[0031] Figure 8These are the results of the investigation into the spectral properties of the probe; Figures A and B are the ultraviolet fluorescence spectra of TPA-A and TPA-TA; Figure C is the fluorescence spectrum of TPA-A in water / methanol systems with different volume ratios and its statistics, and the inset figure is the actual fluorescence graph; Figure D is the fluorescence spectrum of TPA-TA in water / methanol systems with different volume ratios and its statistics, and the inset figure is the actual fluorescence graph.

[0032] Figure 9 The following are graphs evaluating the biocompatibility of the probes TPA-A and TPA-TA. Figures AB show the CCK8 results of the fluorescent probes TPA-A and TPA-TA on cell activity; Figure C is a confocal image of the effect of TPA-TA on Treponema pallidum activity.

[0033] Figure 10 A is the laser confocal image of T. pallidum labeled with fluorescent probe TPA-TA and TPA-A. Figure 10 B is the super-resolution imaging of TPA-TA labeled T. pallidum.

[0034] Figure 11 This is a co-localization image of T. pallidum labeled with TPA-TA and anti-T. pallidum antibody.

[0035] Figure 12 This is a graph showing the labeling efficiency of T. pallidum using the fluorescent probe TPA-TA as detected by flow cytometry.

[0036] Figure 13 This is the SDS-PAGE result and fluorescence imaging diagram after TPA-TA binds to the outer membrane protein of Treponema pallidum.

[0037] Figure 14 These are the fluorescence confocal results of T. pallidum labeled with different concentrations of TPA-TA.

[0038] Figure 15 This figure shows the flow cytometric evaluation of the labeling efficiency of T. pallidum using different concentrations of TPA-TA.

[0039] Figure 16 These are confocal fluorescence images of T. pallidum labeled with the fluorescent probe TPA-TA before and after inactivation. DETAILED DESCRIPTION

[0040] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0041] The experimental methods in the following examples where specific experimental conditions are not specified are generally performed according to conventional experimental conditions or the experimental conditions recommended by the manufacturer.

[0042] Example 1

[0043] Prepare the bioorthogonal fluorescent labeling probe TPA-A, whose structure is as follows:

[0044]

[0045] The synthesis steps for the bioorthogonal fluorescent probe TPA-A provided in this example are as follows: 4-bromotriphenylamine (1.29 g, 4 mmol), 5-formyl-2-thiopheneboronic acid (936 mg, 6 mmol), and potassium carbonate (1.19 g, 8 mmol) were dissolved in a mixture of tetrahydrofuran (THF) and water (60 mL, V / V = 3:1). The catalyst, tetrakistriphenylphosphine palladium (Pd(PPh3)4) (462 mg, 0.4 mmol), was then added. The mixture was stirred at 80°C for 24 hours. After cooling to room temperature, it was extracted with dichloromethane (DCM) and purified on a silica gel column using petroleum ether and dichloromethane as eluents. The resulting product, an orange solid, is the compound 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde (TPA-A).

[0046] like Figure 2 As shown, the nuclear magnetic hydrogen spectrum characterization data of compound TPA-A: 1 HNMR (400MHz, CDCl3) δ: 9.85 (s, 1H), 7.70 (d, J = 4.0Hz, 1H), 7.52 (d, J = 8.7Hz, 2H), 7.29 (t, J = 7.9Hz, 5H), 7.17–7.02 (m, 9H).

[0047] like Figure 3 As shown, the NMR carbon spectrum characterization data of compound TPA-A: 13 C NMR (101MHz, CDCl3) δ: 182.61, 154.61, 149.18, 147.00, 141.35, 137.72, 129.51, 127.27, 126.17, 125.20, 123.90, 122.88, 122.39.

[0048] like Figure 4 As shown, the mass spectrum characterization data of compound TPA-A is: MH=355.10MH=355.10+1.0078=358.1078.

[0049] The compound 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde (TPA-A, 1 mmol, 335.4 mg) was weighed and dissolved in 10 mL of THF. Then, 3 mL of ethynylmagnesium bromide (0.5 M THF) solution was added dropwise under ice bath conditions. The solution was transferred to room temperature and stirred for 12 hours, followed by quenching the reaction with saturated ammonium chloride. The crude product was extracted with dichloromethane and purified on a silica gel column using petroleum ether and ethyl acetate (V / V=10 / 1) as eluents to obtain an oily product TPA-TH with blue fluorescence, which was used directly in the next reaction without further purification. The synthesized TPA-TH was dissolved in 25 mL of DCM, manganese dioxide (1.304 g, 15 mmol) was added, stirred at room temperature, and the reaction progress was monitored by thin layer chromatography. After the reaction, manganese dioxide was removed with diatomaceous earth, and the crude product was purified by silica gel column using n-hexane / dichloromethane (volume ratio = 3 / 1) as eluent to obtain an orange solid, which was the final product, 5-(4-(diphenylamino)phenyl)thiophene-2-ynyl ketone TPA-TA.

[0050] The reaction process is as follows:

[0051]

[0052] like Figure 5 As shown, the mass spectrometry characterization data of compound TPA-TA: MH+Na + =379.1031+23.9976=403.1007MH=379.1031+1.0078=380.1109HRMS(TOF-MS):m / z calcd.for C 25 H7NOS.[MH + ]:380.1109;found:380.1109.

[0053] like Figure 6 As shown, TPA-TA H NMR spectrum characterization data: 1 H NMR (400MHz, CDCl3) δ7.90(d,J=3.5Hz,1H),7.51(d,J=8.2Hz,2H),7.29(t,J=7.6Hz,4H),7.20–6.97(m,8H),3.33(s,1H).

[0054] like Figure 7 As shown, TPA-TA NMR carbon spectrum characterization data: 13C NMR (100MHz, CDCl3) δ168.49,155.50,149.31,146.95,141.58,137.50,129.52,127.26,125.99,125.26,123.97,122.99,122.28,79.99,78.95.

[0055] From the corresponding NMR and mass spectra, it can be seen that the actual mass spectrum of the obtained TPA-TA is 380.1109, which is completely consistent with the theoretical calculated value. In addition, the NMR hydrogen spectrum and NMR carbon spectrum are consistent with expectations. Based on this, it can be judged that the click chemistry reaction-activated aggregation-induced fluorescent probe TPA-TA has been successfully synthesized.

[0056] Example 2

[0057] The ultraviolet absorption of the fluorescent labeled probes TPA-A and TPA-TA obtained in Example 1 was measured, and their aggregation-induced emission characteristics in a mixed medium of water and methanol were analyzed.

[0058] From the UV-visible absorption results Figure 8 As can be seen from AB, the characteristic absorption peaks of probe TPA-A range from approximately 400-500nm, and those of probe TPA-TA range from approximately 400-600nm, showing a certain red shift compared to the precursor TPA-A. This is likely due to the addition of a carbon-carbon triple bond to the TPA-TA molecular structure, which increases the conjugated system and thus results in a red shift in absorption. In particular, the emission wavelength of probe molecule TPA-A ranges from 500-600nm, with a maximum emission peak at 530nm, while the emission wavelength of probe molecule TPA-TA ranges from 500-700nm, with a maximum emission peak at 598nm, also red-shifted compared to the emission peak of TPA-A, as expected.

[0059] Since TPA-A and TPA-TA are typical AIE molecules with triphenylamine as their molecular framework, fluorescence spectroscopy was then used to investigate their fluorescence behavior in water / methanol mixtures with different volume ratios to explore their aggregation-induced emission properties. Figure 8 As shown in C, the results of TPA-TA are Figure 8 As shown in Figure D, when the water content gradually increases from 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% to 95%, the fluorescence intensity of the two probes also gradually increases. In summary, the synthesized fluorescent probes TPA-A and TPA-TA have aggregation-induced emission properties.

[0060] Example 3:

[0061] In this example, the biocompatibility of the fluorescent probe was evaluated. Different concentrations of the probes TPA-A and TPA-TA were incubated with different cell lines. A concentration gradient of 0, 0.3, 0.6, 1.2, 2.5, and 5.0 μM TPA-A and TPA-TA was set up and incubated with Sf1Ep, HUVEC, and AD293 cells, respectively. The cell activity was detected by CCK-8 assay. The results are shown in Figure 2. Figure 9 As shown in AB, the cell viability after treatment with fluorescent probes TPA-A and TPA-TA remained greater than 85%.

[0062] Treponema pallidum was treated with TPA-TA probe at concentration gradients of 0, 0.6, 1.2, 2.5, and 5.0 μM, and the activity of T. pallidum was detected using the LIVE / DEAD bacterial activity detection kit. The results are shown in Figure 9 As shown in C, it was found that the activity of T. pallidum could be maintained above 80% under treatment with different concentrations of fluorescent probes, indicating that the fluorescent labeled probe is relatively safe for T. pallidum.

[0063] Combined with the above experiments, it can be shown that these two substances have no obvious effect on cell viability and Treponema pallidum itself within a certain range, that is, they have good cell compatibility and no obvious cytotoxicity.

[0064] Example 4

[0065] In order to evaluate whether the probe TPA-TA can label T.pallidum through click chemistry reaction, the reference probe TPA-A without activated alkyne was selected for labeling exploration. TPA-TA and TPA-A were incubated with the same amount of T.pallidum in a tri-gas incubator for 30 minutes to react and cross-link with the outer membrane protein of T.pallidum, and the fluorescent probe was labeled on the surface of T.pallidum. Figure 10 As shown in A, the probe TPA-TA is incubated with T. pallidum for 30 minutes to achieve labeling of T. pallidum, and the labeled T. pallidum is imaged using a super-resolution microscope. Figure 10 B shows the helical structure of T. pallidum and its binary fission state. The two experimental groups with only the probe but no T. pallidum showed no fluorescence. While the group co-incubated with TPA-A and T. pallidum exhibited faint fluorescence, the labeling efficiency was very low and almost negligible. These results demonstrate that the fluorescent probe TPA-TA effectively labels T. pallidum.

[0066] Example 5

[0067] The effectiveness of the fluorescently labeled probe TPA-TA obtained in Example 1 in labeling T. pallidum was evaluated. The experimental groups were as follows:

[0068] (1) No treatment, blank control group;

[0069] (2) staining with anti- T. pallidum antibody only;

[0070] (3) incubation with probe TPA-A;

[0071] (4) incubation with the probe TPA-TA;

[0072] (5) Incubate with probe TPA-A, then stain with anti-T. pallidum antibody for 1 h and wash;

[0073] (6) Based on the successful live staining of T. pallidum with the probe TPA-TA, T. pallidum was first fixed, then stained with anti-T. pallidum antibody for 1 h, and washed;

[0074] Confocal imaging was performed on glass slides for each experimental group. Figure 11 As shown, the green fluorescence displayed by TPA-TA labeling and the Cy5 fluorescence labeled with the anti-T. pallidum antibody can achieve good co-localization. From the details, it can be seen that immunofluorescence staining can only stain the relevant antibodies of Treponema pallidum itself, while the activated alkyne probe TPA-TA can stain and label the entire Treponema pallidum. In addition, as a control group, the probe TPA-A cannot stain Treponema pallidum because it does not have an activated alkyne structure and cannot label Treponema pallidum through a click chemistry reaction. In summary, the probe TPA-TA can effectively label live T. pallidum.

[0075] Example 6

[0076] On this basis, the efficiency of the fluorescent labeled probe TPA-TA obtained in Example 1 in labeling T. pallidum was evaluated. The experiment was carried out according to the grouping in Example 5, and then each experimental group was incubated and analyzed by flow cytometry fluorescence. The results are shown in Figure 2. Figure 12 As shown, it can be found that the efficiency of TPA-TA probe labeling T. pallidum is slightly higher than that of antibody staining. This may be because immunofluorescence staining depends largely on the amount of antigens present in the pathogen T. pallidum itself.

[0077] Example 7

[0078] Subsequently, the feasibility of labeling outer membrane proteins was preliminarily verified by using Coomassie Brilliant Blue staining and fluorescence imaging methods for the fluorescent labeled probe TPA-TA obtained in Example 1. Figure 13 As shown, there is no obvious difference between the outer membrane protein extracted from Treponema pallidum labeled with TPA-TA and the outer membrane protein extracted from the unlabeled pathogen. In particular, under the irradiation of excitation light with a wavelength of 488nm, only the Treponema pallidum protein labeled with the fluorescent probe has fluorescence, indicating that TPA-TA can achieve the labeling of Treponema pallidum by modifying the outer membrane protein through bioorthogonal reaction.

[0079] Example 8

[0080] The same conditions as above were used, and TPA-TA was incubated with T. pallidum at different concentrations (0, 0.6, 1.3, 2.5, 5.0, 10.0 μM) for 30 min, followed by confocal imaging and flow cytometry. Figure 14 Fluorescence confocal results and Figure 15 It can be seen from the flow cytometry fluorescence intensity results that as the probe concentration increases, the fluorescence intensity also increases until it maintains a relatively stable value at 5-10μM. Later experiments also selected 5μM as the experimental concentration.

[0081] Example 9

[0082] The present invention points out that the important starting point of the one-step labeling of Treponema pallidum is that it is not affected by the state of Treponema pallidum. Then, it is verified and evaluated by labeling live T. pallidum and heat-inactivated T. pallidum. T. pallidum inactivated at 56°C is incubated with the probe TPA-TA and the dye PI, followed by buffer washing and confocal observation. Figure 16 As shown, heat-inactivated T. pallidum can be stained with PI, while live T. pallidum cannot, demonstrating that heat inactivation effectively kills T. pallidum. Furthermore, both dead and live T. pallidum can be stably labeled with TPA-TA, strongly demonstrating that this one-step labeling strategy is independent of T. pallidum viability. While in vitro culture of Treponema pallidum is still immature, this method could provide a new tool and research strategy for basic syphilis research.

[0083] The components of the culture medium used for suspending / culturing Treponema pallidum in the above experiment were: 10 g / L CMRL1066 without glutathione, 0.7 mM sodium pyruvate, 0.1% resazurin by mass, 1 M morpholinopropanesulfonic acid, 7.5% sodium bicarbonate by mass, 200 mM glutamine, 17.6 mM glucose, 0.52 mM histidine, 0.52 mM dithiothreitol, 20% by volume inactivated fetal bovine serum, and sterile water for culture to 500 mL.

[0084] Advantages of the present invention include: utilizing a metal-free click bioconjugation strategy with activated alkynes to develop and design a click-chemistry-activated aggregation-induced fluorescent probe, TPA-TA, for labeling and imaging Treponema pallidum, enabling structural analysis and imaging studies of syphilis intercellular interactions. Incubating syphilis with TPA-TA allows amino groups on its outer membrane protein to undergo a click chemistry reaction with the activated alkyne on TPA-TA, achieving stable labeling of the syphilis. Notably, the viability of T. pallidum remained comparable before and after labeling, and fluorescence confocal microscopy was used to monitor the interaction between T. pallidum and cells from various organisms. This strongly demonstrates that the probe provides a novel labeling and imaging monitoring strategy for fundamental research on the pathogenic mechanisms and immune escape of syphilis.

[0085] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A method for preparing a click chemistry-activated aggregation-induced fluorescent probe, characterized in that: The steps include: reacting 4-bromotriphenylamine with 5-formyl-2-thiopheneboronic acid to obtain 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde; reacting 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde with ethynylmagnesium bromide to obtain an oily product TPA-TH with blue fluorescence; and then treating with manganese dioxide to obtain 5-(4-(diphenylamino)phenyl)thiophene-2-ynyl ketone (TPA-TA), the structural formula of which is shown below: 。 2. The method for preparing a click chemistry reaction-activated aggregation-induced fluorescent probe according to claim 1, wherein: The steps include: first, weighing 4-bromotriphenylamine, 5-formyl-2-thiopheneboronic acid, and potassium carbonate, dissolving them in a mixture of tetrahydrofuran and water, adding a catalyst, tetrakistriphenylphosphine palladium (Pd(PPh3)4), and then stirring and reacting at 80°C for 24 hours to obtain 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde (TPA-A); The compound 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde was weighed and dissolved in THF. Then, ethynylmagnesium bromide solution was added dropwise in an ice bath and stirred at room temperature for 12 h to obtain an oily product TPA-TH with blue fluorescence. TPA-TH was dissolved in DCM, manganese dioxide was added, and stirred at room temperature to obtain 5-(4-(diphenylamino)phenyl)thiophene-2-ynyl ketone TPA-TA.

3. The method for preparing a click chemistry reaction-activated aggregation-induced fluorescent probe according to claim 2, wherein: The detailed steps include: first weighing 1.29 g of 4-bromotriphenylamine, 936 mg of 5-formyl-2-thiopheneboronic acid, and 1.19 g of potassium carbonate and dissolving them in 60 mL of a volume ratio of V 四氢呋喃 / V 水 = 3 / 1 tetrahydrofuran and water mixed solution, and then added tetrakistriphenylphosphine palladium Pd(PPh3)4 462 mg catalyst, the above mixture was stirred at 80 ° C for 24 hours, cooled to room temperature, extracted with dichloromethane, and purified on a silica gel column using petroleum ether and dichloromethane as eluents to obtain 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde TPA-A, 335.4 mg of the compound 5-(4-(diphenylamino)phenyl)thiophene-2-aldehyde TPA-A was weighed and dissolved in 10 mL of THF. Then, 3 mL of a 0.5 M ethynylmagnesium bromide THF solution was added dropwise in an ice bath. The solution was transferred to room temperature and stirred for 12 h. The reaction was then quenched with saturated ammonium chloride and extracted with dichloromethane to obtain a crude product. The volume ratio was V 石油醚 / V 乙酸乙酯 The product was purified on a silica gel column using a mixture of petroleum ether and ethyl acetate in a ratio of 10 / 1 as the eluent to obtain an oily product TPA-TH with blue fluorescence. TPA-TH was dissolved in 25 mL of DCM, 1.304 g of manganese dioxide was added, and the mixture was stirred at room temperature. After the reaction, manganese dioxide was removed with diatomaceous earth, and the crude product was purified on a silica gel column using n-hexane and dichloromethane in a volume ratio of 3 / 1 as the eluent to obtain the final product, 5-(4-(diphenylamino)phenyl)thiophene-2-ynyl ketone TPA-TA.

4. A one-step fluorescent labeling and tracer imaging method for Treponema pallidum using the click chemistry reaction-activated aggregation-induced fluorescent probe according to claim 1, characterized in that: The steps include: directly labeling Treponema pallidum and TPA-TA through a one-step incubation method, causing a click chemistry reaction, and realizing tracer imaging of Treponema pallidum.

5. A one-step fluorescent labeling and tracer imaging method for Treponema pallidum using a click chemistry reaction-activated aggregation-induced fluorescent probe according to claim 4, characterized in that: The detailed steps include: 1 mL 10 7 A suspension of Treponema pallidum (1000 pieces / mL) was incubated with TPA-TA at a final concentration of 5 µM in a 34°C tri-gas incubator for 30 min. The Treponema pallidum was then centrifuged to precipitate the treponema pallidum, unused TPA-TA was removed, the treponema pallidum was washed and resuspended, the treponema pallidum was centrifuged and resuspended to achieve visual labeling of the treponema pallidum.

6. A one-step fluorescent labeling and tracer imaging method for Treponema pallidum using a click chemistry reaction-activated aggregation-induced fluorescent probe according to claim 5, characterized in that: The detailed steps include: 1 mL of 10 7 A suspension of Treponema pallidum at 500 nm / mL was incubated with TPA-TA at a final concentration of 5 μM in a 34°C tri-gas incubator for 30 min. The Treponema pallidum was then precipitated by centrifugation at 14,000 g for 5 min, and unused TPA-TA was removed. The suspension was then washed with 1 mL of PBS and resuspended in PBS. The suspension was then centrifuged at 14,000 g for 5 min and resuspended in 500 μL of culture medium to achieve visual labeling of the Treponema pallidum.

7. A one-step fluorescent labeling and tracing imaging method for Treponema pallidum using a click chemistry reaction-activated aggregation-induced fluorescent probe according to claim 4, characterized in that: The components of the culture medium for culturing / suspending Treponema pallidum include: sterile water for culture, CMRL1066 without glutathione, sodium pyruvate, resazurin, morpholinopropanesulfonic acid, sodium bicarbonate, glutamine, glucose, histidine, dithiothreitol, and inactivated fetal bovine serum.

8. A one-step fluorescent labeling and tracer imaging method for Treponema pallidum using a click chemistry reaction-activated aggregation-induced fluorescent probe according to claim 7, characterized in that: The culture medium for culturing / suspending Treponema pallidum includes the following components: 10 g / L CMRL1066 without glutathione, 0.7 mM sodium pyruvate, 0.1% resazurin, 1 M morpholinopropanesulfonic acid, 7.5% sodium bicarbonate, 200 mM glutamine, 17.6 mM glucose, 0.52 mM histidine, 0.52 mM dithiothreitol, 20% inactivated fetal bovine serum, and sterile water for culture to 500 mL.

9. Use of the click chemistry reaction-activated aggregation-induced fluorescent probe TPA-TA prepared by the method of claim 1 in preparing a reagent or kit for fluorescent labeling and tracing imaging of Treponema pallidum.

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