A thiosericin-based active molecular probe based on AfBPP and its preparation and application

Abstract

This invention discloses an AfBPP-based active molecular probe for thiostreptin, its preparation, and its application, belonging to the field of chemical biology. This invention introduces a bifunctional tag integrating a photocrosslinking group (bisacrididine) and a bioorthogonal reactive group (alkynyl group) into the structure of thiostreptin. While retaining the original biological activity of the parent compound, it endows the probe with highly efficient probe function, solving the technical problem of target loss during washing and purification of traditional non-covalent probes. After target labeling, the probe can specifically connect to reporter groups such as fluorescein or biotin through click-chemical reactions, achieving efficient enrichment of drug targets. Combined with mass spectrometry analysis, it enables global identification of potential targets in cells or complex biological samples at the omics level, such as chemical proteomics, providing a powerful molecular tool for in-depth revelation of the potential targets and pharmacological mechanisms of thiostreptin.

Description

Technical Field

[0001] This invention belongs to the field of chemical biology, specifically relating to an AfBPP molecular probe based on the active molecule thiostreptin and its preparation and application. Background Technology

[0002] Thiostrapton (TS) is a natural cyclic oligopeptide antibiotic produced and isolated from Streptomyces, belonging to the thiopeptide family. Its traditional antibacterial mechanism is believed to be through altering the structure of the rRNA-L11 protein complex, preventing the binding of GTPase elongation factors to the 70S ribosome, thereby blocking protein translation elongation. Through this mechanism, thiostrepton exhibits significant antibacterial effects against various Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). However, with further research, thiostrepton has demonstrated biological activities beyond its traditional antibiotic definition. Recent studies have found that although thiostrepton does not exhibit direct killing or bacteriostatic effects against certain bacteria (such as Gram-negative bacteria), it can significantly inhibit the expression of virulence factors in these bacteria, weakening their pathogenicity. Furthermore, studies have reported potential applications of thiostrepton in antitumor and immunomodulatory fields. These newly discovered bioactivities suggest that thiosericin may have other unknown targets or multi-target regulatory mechanisms within cells besides ribosomes. Due to the lack of effective molecular tools, the precise molecular targets of thiosericin in complex biological systems and the molecular mechanisms by which it exerts its multiple biological functions remain unclear, which greatly limits the expansion of the clinical application of this natural product and the design and development of novel drugs.

[0003] Affinity-based protein profiling (AfBPP) is a cutting-edge proteomics technique that integrates chemistry, biology, and mass spectrometry. It involves chemically modifying active molecules to create functionalized probes that capture targets by leveraging the affinity between the probes and target proteins, while preserving the molecules' biological activity. A typical AfBPP probe contains at least an affinity group and a reporter group. The affinity group is usually part of the active molecule's parent structure and is used to recognize and bind to the target protein; the reporter group is used for subsequent tracing and enrichment. In recent years, probe design schemes based on bioorthogonal reactions (such as click chemistry) have gained significant popularity. This approach introduces functionalized groups (such as alkynyl or azide groups) onto active molecules, performs target labeling in live cells or cell lysates, and then connects fluorescein or biotin via click chemistry. This design significantly reduces the steric hindrance of the probe, enabling a more accurate reflection of the active molecule's state under physiological conditions. Therefore, designing and synthesizing a thiostreptin affinity molecular probe that can maintain biological activity and efficiently capture targets is of great scientific significance and application value for elucidating the multiple mechanisms of action of this compound in different biological systems, discovering new targets, and promoting its pharmacological research. Summary of the Invention

[0004] The purpose of this invention is to provide a thiosericin active molecular probe, which, by introducing bioorthogonal reactive groups, is intended to serve as a universal molecular tool for exploring the molecular interaction network of thiosericin in various biological systems (including but not limited to Gram-positive bacteria, Gram-negative bacteria, and eukaryotic cells).

[0005] The technical solution adopted in this invention is: On the one hand, the AfBPP-based thiosericin-active molecular probe has the following structure:

[0006] Part 1 is the affinity group: This part retains the biologically active parent structure of thiostrepton. The thiostrepton backbone contains electron-rich or electron-deficient reaction sites (such as dehydroalanine Dha residues). Probes mainly attach to subsequent groups through addition reactions (such as aza-Michael conjugation) or cycloaddition reactions at these sites. This part retains the core pharmacophore for thiostrepton to exert its biological activity, responsible for specifically recognizing and binding to target proteins in complex biological samples.

[0007] Part 2 is the linker (L): This part is the molecular structure that connects part 1 (affinity group) and part 3 (functional group). The linker acts as a spatial separator and bridge, effectively increasing the spatial distance between the affinity group and the functional group; at the same time, by adjusting the chain length, rigidity, and / or hydrophilicity / hydrophobicity of the linker, the overall cell membrane permeability and physicochemical properties of the probe can be optimized. In a broader sense, any structural unit that can effectively connect part 1 and part 3 is within the scope of protection of this invention. The linker arm L is selected from: substituted or unsubstituted C1-C20 alkylene, C2-C20 alkenylene, C2-C20 alkyneene, polyethylene glycol (PEG) chain, cycloalkylene, arylene, heteroarylene, polypeptide chain, aminobenzamide structure or any combination thereof, and the substituent is selected from halogen atom, C1-C6 alkyl, amino, alkoxy, cycloalkyl, aryl, heteroaryl; and the main chain of the linker arm optionally contains one or more amide bonds, ester bonds, ether bonds, thioether bonds, disulfide bonds, amino groups or heterocycles formed by cycloaddition reactions (such as 1,2,3-triazole rings formed by click chemistry).

[0008] Part 3 comprises functional groups: This part integrates a photocrosslinking group and a bioorthogonal reporter group. Specifically, the three-membered ring structure containing a nitrogen-nitrogen double bond (N=N) in the middle of the carbon chain is a diazirine photocrosslinking group, which can release nitrogen gas under ultraviolet light of a specific wavelength (e.g., 365 nm) to generate a highly active carbene intermediate, thereby undergoing rapid and irreversible covalent crosslinking with spatially adjacent target proteins, avoiding target loss during elution due to non-covalent binding; the rightmost alkyne group serves as a reporter group, which can specifically attach a fluorescein or biotin tag with an azide group through a copper-catalyzed azido-alkynyl click chemistry reaction (CuAAC) after the probe is covalently crosslinked with the target protein, achieving fluorescence imaging tracking and affinity enrichment of the target protein. n1 and n2 are independently 0 or positive integers, preferably integers from 1 to 3.

[0009] On the other hand, the present invention also provides a method for preparing the AfBPP-based thiosericin active molecular probe. The preparation method uses aralkylamide units as the linker backbone and completes the probe assembly through an aza-Michael conjugated addition reaction, specifically including the following steps: 1) Construction of intermediate L2, an aryl linker containing a free amino group: An aminobenzoic acid derivative with an amino protecting group and a substituent R on the benzene ring was dissolved in an organic solvent. Then, a compound L1 containing diacylpropidine and an alkynyl group was added, and an amidation reaction was carried out under the combined action of a condensing agent and a basic reagent. After the reaction, post-treatment was performed to obtain intermediate S1. Intermediate S1 was dissolved in an organic solvent, and then a deprotecting agent (e.g., trifluoroacetic acid) was added to carry out the reaction. After complete removal of the protecting group, post-treatment was performed to obtain intermediate L2, an aryl linker containing a free amino group.

[0010]

[0011] 2) Synthesis of the target molecular probe: Thioxetin (TS), the aryl linker intermediate L2 containing a free amino group obtained in step 1), and a basic catalyst (such as triethylamine) were added to an organic solvent and stirred. The free amino group of L2 underwent an aza-Michael conjugated addition reaction with the dehydroalanine (Dha) residue on the thioxetin backbone. After the reaction was complete, the target molecular probe was obtained through post-processing steps including water washing and quenching, extraction, concentration, and purification.

[0012]

[0013] n1 and n2 are each independently 0 or a positive integer, preferably n1 and n2 are integers from 1 to 3. R is independently selected from: hydrogen atom, halogen, cyano, nitro, hydroxyl, amino, carboxyl, C1-C6 alkoxy, C1-C6 acyl, C1-C6 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaryl; wherein the alkoxy, acyl, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaryl may optionally be independently substituted by one or more substituents selected from: halogen, amino, hydroxyl, cyano, nitro, carboxyl, C1-C6 alkyl or aryl.

[0014] Further, the organic solvent is selected from one or more mixed solvents selected from alcohol solvents, halogenated hydrocarbon solvents, ketone solvents, ether solvents, ester solvents, nitrile solvents, and polar aprotic solvents. Specifically, the organic solvent is selected from one or more mixed solvents selected from methanol, ethanol, isopropanol, dichloromethane, chloroform, 1,2-dichloroethane, acetone, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), 1,4-dioxane, acetonitrile, and ethyl acetate.

[0015] Further, the post-processing steps specifically include: after the reaction is complete, adding water (or ice water) to the reaction system to quench the reaction, and extracting with an organic solvent (preferably dichloromethane, chloroform, or ethyl acetate); combining the organic phases, washing sequentially with saturated sodium bicarbonate aqueous solution or water as needed, followed by washing with saturated brine, and drying with a drying agent (preferably anhydrous sodium sulfate or anhydrous magnesium sulfate); removing the organic solvent by vacuum concentration using a rotary evaporator to obtain the crude product; purifying the crude product by silica gel column chromatography and / or a high-pressure preparative purification system (preparative HPLC) to finally obtain a high-purity target molecular probe.

[0016] Furthermore, this invention also provides the application of an AfBPP-based thiostreptin active molecular probe in the preparation of antibacterial drugs or antibacterial virulence drugs, as well as its application in the preparation of thiostreptin target identification probe samples and in the study of the pharmacological mechanism of thiostreptin.

[0017] Furthermore, the target labeling and detection mechanism of the probe in biological samples (such as live cells, tissues, or protein lysates) are as follows: The portion of the probe structure that has the same structure as the parent compound thiotetracycline serves as an affinity recognition group, capable of selectively binding to specific target proteins (including but not limited to a class of enzymes) in the proteome of cells or cell lysates based on affinity non-covalent binding. Subsequently, under ultraviolet light irradiation at a specific wavelength (preferably 365 nm), the photocrosslinking group (bisacrididine) in the probe structure is activated and generates a highly active carbene intermediate, thereby rapidly and irreversibly binding directly to the spatially adjacent target protein to form a stable affinity-based covalent label. Afterward, in the cell lysate, through a bioorthogonal reaction of click chemistry, a fluorescent group or a biotin group with an azide group is attached to the alkyne site of the probe.

[0018] Furthermore, methods for detecting and identifying target proteins include: When a fluorescent group is attached, the protein labeled with the probe molecule can be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and specific fluorescent signal bands can be displayed by an imaging system, enabling visualization, localization, and molecular weight determination of the target protein. When a biotin group is attached, the protein covalently labeled with the probe molecule can be specifically captured, enriched, and eluted by streptavidin, and then analyzed in high-throughput by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to accurately determine the type of labeled protein.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention marks the first successful integration of a bifunctional tag, combining a photocrosslinking group (biacrylidine) and a bioorthogonal reactive group (alkynyl), into the structure of the natural macrocyclic oligopeptide thiosericin, synthesizing a novel AfBPP-based thiosericin active molecular probe. This design ingeniously overcomes the challenge of easily losing efficacy when modifying complex natural product structures, preserving the original biological activity of the parent thiosericin while endowing it with highly efficient probe functionality. Under UV light induction, the probe undergoes rapid and irreversible covalent crosslinking with spatially adjacent targets via biacrylidine, effectively solving the technical problem of target loss during washing and purification of traditional non-covalent probes. After target labeling, the probe can specifically connect to reporter groups such as fluorescein or biotin via click-through chemical reactions, achieving highly efficient enrichment of intracellular drug targets. Furthermore, combined with mass spectrometry analysis, this probe can achieve global identification of potential targets in cells or complex biological samples at the omics level, such as chemical proteomics, providing a powerful molecular tool for further revealing the potential targets and pharmacological mechanisms of thiotetracycline. Attached Figure Description

[0020] Figure 1 The reaction formula of probe TSP2 and the possible reaction sites of TS; Figure 2 For probe TSP2 1 H- 1 Enlarged view of a portion of the H TOCSY NMR spectrum; Figure 3 A comparison of the proton NMR spectra of thiotetracycline TS (top) and probe TSP2 (bottom); Figure 4 The structure of the probe TSP2 was identified; (a) HRMS UV spectrum (254 nm) of pure TSP2; (b) HRMS of purified TSP2. Figure 5 The inhibition curves and IC50 values ​​of thioseridin (TS) and probe TSP2 against fluorescent heparin are shown. 50 Value; repeat the experiment 3 times; Figure 6 Fluorescent labeling of different concentrations of probe TSP2 in Pseudomonas aeruginosa and staining of total protein in gels were shown. (a) Fluorescent labeling of different concentrations of probe TSP2 in Pseudomonas aeruginosa; (b) Coomassie Brilliant Blue staining of the gel in (a). Note: Flu represents the fluorescent marker for protein; Fluorescence indicates fluorescent SDS-PAGE gel; Coomassie indicates SDS-PAGE gel stained with Coomassie Brilliant Blue; 0 indicates an equal amount of DMSO added to the sample instead of the compound. Detailed Implementation

[0021] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0022] Unless otherwise specified, the experimental methods in the following examples are performed according to conventional methods and conditions or according to the product instructions; the materials and reagents used are all commercially available products unless otherwise specified.

[0023] Example 1

[0024] Synthesis of AfBPP-based thiosericin-active molecular probes: Step 1: Synthesis of compound L2-1; 200 mg of N-Boc-4-aminobenzoic acid was dissolved in 3 mL of DMF, followed by the addition of 127.1 mg of 3-aminoethyl-3-(but-3-ynyl)bisacrylidine (2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethan-1-amine, CAS No.: 1450752-97-2), 170.8 mg of 1-hydroxybenzotriazole (HOBt), 242.3 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), and 85.3 mg of triethylamine (TEA). The reaction was carried out at room temperature for 5 hours. After the reaction was completed, 12 mL of water was added to quench the reaction mixture, and the mixture was extracted with ethyl acetate (6 mL × 3). The organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The organic solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified using a flash purification system to obtain 230.1 mg of a white solid, S1-1. Subsequently, 100 mg of S1-1 was dissolved in 3 mL of dichloromethane, followed by the addition of 1 mL of trifluoroacetic acid (TFA). The reaction was carried out at room temperature for 1 hour. After the reaction was complete, the solvent was removed using a rotary evaporator to obtain the crude product. The crude product was then purified using a flash purification system to obtain 64.4 mg of a white solid, L2-1.

[0025]

[0026] Step 2: Synthesis of probe TSP2; 300.0 mg of thiostreptoxin (TS), 55.3 mg of L2-1, and 273.2 mg of triethylamine were added sequentially to 5 mL of chloroform. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by pre-HPLC (prep-HPLC, mobile phase: acetonitrile-water system, gradient elution program: 5% acetonitrile (v / v) linearly up to 95% acetonitrile, elution time 30 min) to obtain 105.8 mg of white solid.

[0027]

[0028] The specific reaction formula and possible reaction sites of probe TSP2 are as follows: Figure 1 As shown, there are a total of 4 potential reaction sites. The product of this invention is generated by the reaction of the Dha17 site of thiostreptin TS. Figure 2 For probe TSP2 1 H- 1 Enlarged view of a portion of the H TOCSY nuclear magnetic resonance spectrum. Figure 3 The image shows a comparison of the proton NMR spectra of thiotetracycline TS (top) and probe TSP2 (bottom). Analysis of the proton NMR spectra of thiotetracycline and probe TSP2 revealed that the signal peaks for Dha3 and Dhb8 were present in both the TS and TSP2 spectra, ruling out the possibility of modification at these sites. From TSP2... 1 H- 1 In the 1H TOCSY spectrum, a correlation was observed between the characteristic peak of Dha16-NH (9.75 ppm) and the Dha16 methylene signal (5.43 ppm and 6.62 ppm), indicating that the structure of Dha16 is well preserved. Figure 4 (a) is the HRMS UV spectrum (254 nm) of pure TSP2. Figure 4 (b) HRMS of purified TSP2.

[0029] Example 2

[0030] The probe TSP2 inhibits the detection of fluorescent heptaphors: 5 µL of Pseudomonas aeruginosa glycerol culture solution was inoculated into 5 mL of LB medium and incubated overnight at 37°C and 200 rpm. The OD value of bacterial growth was measured using a multi-functional microplate reader. 600 Value. Centrifuge at 5000 g for 1 min to remove LB, resuspend the bacterial pellet in CAA low-iron culture medium and dilute to OD. 600 = 0.01. 100 µL of bacterial culture was aliquoted into 96-well plates, and different concentrations of the compound (0 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, and 5 μM of thiotetracycline or probe TSP2) were added. The 96-well plates were incubated at 37°C for 18 hours. After incubation, the fluorescence value (excitation wavelength 398 nm, emission wavelength 465 nm) and the OD of bacterial growth were measured using a multi-functional microplate reader. 600 The values ​​were calculated, and the inhibition rate of the compound against the fluorescent heptaphora was determined. The data were then imported into GraphPad Prism 9.5 software to plot the IC values. 50 Curve and calculate IC 50 value.

[0031] like Figure 5 As shown, the in vitro antibacterial activity assay results indicate that TSP2 has an IC50 value of [missing information]. 50 =0.56 μM, with the parent compound thiosericin (IC50). 50 The activity of the probe TSP2 (0.40 μM) was comparable, indicating that the prepared probe TSP2 retained the antibacterial activity of the parent compound thiostreptin, significantly inhibited fluorescent iron, and showed a concentration-dependent effect.

[0032] In addition, TSP2 has a bipropidium photosensitizing group that the parent compound thiostreptin does not possess, which can be covalently linked to the target protein after light exposure. It also has an alkynyl group, which can be linked to a reporter group through a click chemical reaction, thereby enabling the intracellular localization of thiostreptin and the analysis and identification of the target protein.

[0033] Example 3

[0034] Gel-based fluorescent labeling of AfBPP protein: Bacterial culture: 5 µL of Pseudomonas aeruginosa was inoculated into 5 mL of LB medium and incubated overnight at 37°C and 200 rpm. OD was then measured. 600 After centrifugation (6000×g, 4℃, 8 min), the supernatant was discarded, and the bacterial pellet was washed once with CAA medium and resuspended in CAA medium. The bacterial culture was then inoculated into a shake flask containing 200 mL of CAA medium. The shake flask was placed in a constant temperature shaking incubator and cultured at 37℃ and 200 rpm. OD was then measured every hour. 600 Value. When bacteria grow to OD... 600 When the value remains unchanged, a plateau is reached. Record the OD (Original Demand) at this point. 600 Collect all bacteria (6000×g, 4℃, 8 min), remove the culture medium, and wash the bacterial pellet twice with PBS. Store the bacterial pellet at -80℃ for later use.

[0035] Probe incubation: The bacterial pellet is resuspended in PBS to allow its OD to rise. 600 The theoretical value is 8.

[0036] Dispense 1 mL of OD 600 =8% bacterial culture was placed in 6-well plates, and different concentrations of thiotetracycline probe TSP2 or DMSO were added. The 6-well plates were then incubated at 37°C for 1 hour. The experiment was divided into six groups, and the dosage of the drug added to each group was as follows:

[0037] Photocrosslinking: After incubation, irradiate the sample with 365 nm light for 20 min in a UV crosslinker. Place ice packs under the sample to prevent overheating. Collect the bacterial culture in a 1.5 mL sterile centrifuge tube, centrifuge (6000×g, 4℃, 8 min), and discard the supernatant. Wash the bacterial pellet once with PBS.

[0038] Bacterial lysis: The bacterial pellet was resuspended in 200 µL of PBS and lysed using an ultrasonic homogenizer. The homogenization was repeated six times, with each homogenization followed by 2 min cooling on ice (hybridization parameters: 25 kHz, 90% maximum power, 2 s operation time, 4 s interval, total 30 s). After homogenization, the sample was centrifuged (13000 g, 4℃, 30 min). 100 µL of the supernatant was transferred to a new 1.5 mL centrifuge tube (the remaining supernatant was discarded). The pellet was dissolved in 100 µL of PBS containing 0.8% SDS.

[0039] For each sample (100 µL), a click chemistry reaction was performed: 2 µL of RhN3 / TAMRA-azide (5 mM DMSO), 6 µL of 1xTBTA ligand (1.667 mM TBTA, t-BuOH / DMSO = 4:1) and 2 µL of TCEP (52 mM inddH2O, freshly prepared) were premixed and added to the sample, and finally 2 µL of CuSO4 was added and mixed well; the sample was incubated at room temperature for 1 hour.

[0040] Sample loading, gel electrophoresis, and analysis: Add 5×SDS loading buffer (sample to buffer volume ratio 4:1) to the sample and mix well to prepare the gel electrophoresis sample. Take 10 μL of the sample for SDS-PAGE electrophoresis analysis. The fluorescence signal bands of the gel were measured in a chemiluminescence imaging system with an EPI excitation wavelength of 546 nm.

[0041] like Figure 6 As shown, the gel-based AfBPP protein fluorescence labeling results indicate that the TSP2-labeled protein can be detected by fluorescence scanning. Furthermore, the fluorescence signal intensifies with increasing TSP2 concentration, indicating that TSP2 binds to the target protein of Pseudomonas aeruginosa in a concentration-dependent manner.

[0042] Target identification: Because the probe retains the active structural portion of thiostreptin, it can specifically bind to its potential protein targets through covalent binding of the photosensitive group on the probe to the target protein. Subsequently, using a terminal alkyne tag (such as a fluorescent tag or biotin tag) on ​​the probe, the specific protein targets interacting with thiostreptin can be identified using appropriate detection techniques (fluorescence imaging, avidin-biotin system enrichment and binding mass spectrometry, etc.), thereby further clarifying the molecular mechanism by which thiostreptin exerts its pharmacological effects.

[0043] Drug action mechanism study: By observing the enrichment of probe-labeled proteins, the potential targets for the pharmacological effects of thiostreptin can be inferred.

[0044] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A thiosericin-based active molecular probe based on AfBPP, characterized in that, The structure is as follows: Linker refers to a connecting arm selected from: substituted or unsubstituted C1-C20 alkylene, C2-C20 alkenylene, C2-C20 alkyneene, polyethylene glycol chain, cycloalkylene, arylene, heteroarylene, polypeptide chain, aminobenzamide structure, or any combination thereof; the substituent is selected from halogen atom, C1-C6 alkyl, amino, alkoxy, cycloalkyl, aryl, heteroaryl; and the main chain of the connecting arm optionally contains one or more amide bonds, ester bonds, ether bonds, thioether bonds, disulfide bonds, amino groups, or heterocycles formed by cycloaddition reactions; n1 and n2 are independently 0 or positive integers.

2. The AfBPP-based thiosericin-active molecular probe according to claim 1, characterized in that, The structure is as follows: n1 and n2 are each independently 0 or a positive integer; R is independently selected from: hydrogen atom, halogen, cyano, nitro, hydroxyl, amino, carboxyl, C1-C6 alkoxy, C1-C6 acyl, C1-C6 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaryl; wherein the alkoxy, acyl, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaryl may optionally be independently substituted by one or more substituents selected from: halogen, amino, hydroxyl, cyano, nitro, carboxyl, C1-C6 alkyl or aryl.

3. The AfBPP-based thiosericin-active molecular probe according to claim 2, characterized in that, n1 and n2 are integers from 1 to 3.

4. A method for preparing the AfBPP-based thiosericin-active molecular probe as described in claim 2 or 3, characterized in that, Includes the following steps: 1) Construction of intermediate L2 containing a free amino group: An aminobenzoic acid derivative with an amino protecting group and a substituent R on the benzene ring and compound L1 were dissolved in an organic solvent and subjected to an amidation reaction. After the reaction was completed, the intermediate compound S1 was obtained by post-treatment. The intermediate compound S1 was dissolved in an organic solvent and subjected to a deprotection reaction. After post-treatment, intermediate L2 containing a free amino group was obtained. 2) Synthesis of molecular probe: Thioxetin (TS) and intermediate L2 containing an aryl linker arm with a free amino group were added to an organic solvent and stirred to undergo an aza-Michael conjugate addition reaction. After the reaction was completed, the molecular probe was obtained after post-processing. n1, n2 and R are as described in claim 2.

5. The method for preparing the AfBPP-based thiosericin-active molecular probe according to claim 4, characterized in that, The organic solvent is selected from one or more of the following solvents: methanol, ethanol, isopropanol, dichloromethane, chloroform, 1,2-dichloroethane, acetone, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, acetonitrile, and ethyl acetate.

6. The method for preparing the AfBPP-based thiosericin-active molecular probe according to claim 4, characterized in that, The post-processing steps include: after the reaction is complete, adding water or ice water to the reaction system to quench the reaction, and extracting with an organic solvent; combining the organic phases, washing sequentially with saturated sodium bicarbonate aqueous solution or water as needed, followed by washing with saturated brine, and drying with a drying agent; removing the organic solvent by rotary evaporation to obtain the crude product; purifying the crude product by silica gel column chromatography and / or high-pressure preparative purification system to obtain a high-purity molecular probe.

7. The use of the AfBPP-based thiostreptin active molecular probe according to any one of claims 1-3 in the preparation of antibacterial drugs or antibacterial virulence drugs.

8. The application of the AfBPP-based thiostreptin active molecular probe according to any one of claims 1-3 in the preparation of samples for identification of thiostreptin target sites.

9. The application of the AfBPP-based active molecular probe of thiostreptin as described in any one of claims 1-3 in the study of the pharmacological mechanism of thiostreptin.

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