A GUSB photocrosslinking probe, its preparation method and application

Abstract

This invention belongs to the field of photocrosslinking probe technology, specifically relating to a GUSB photocrosslinking probe, its preparation method, and its application. The GUSB photocrosslinking probe has the general chemical formula C0. 13 H 18 N2O6-R, wherein R is any one of the oxalic group elements or peptide bonds. The R group covalently connects the glucuronic acid recognition core and the photosensitive unit in the GUSB photocrosslinking probe, and the R group contains no more than 4 atoms, resulting in a small size and low steric hindrance. The GUSB photocrosslinking probe provided by this invention uses glucuronic acid as the recognition core, combined with a small-volume diazacyclopropane photosensitive unit and a terminal alkyne group, to achieve µM-level in-situ covalent capture of active GUSB in complex systems such as cells or tissues. The probe of this invention significantly improves positioning accuracy, sensitivity, data availability, quantification, and enrichment, while also possessing advantages such as modular synthesis, mild conditions, and strong system compatibility, effectively addressing the technical shortcomings of existing technologies that struggle to accurately obtain GUSB activity in situ under low-dose conditions.

Description

Technical Field

[0001] This invention belongs to the field of photocrosslinking probe technology, specifically relating to a GUSB photocrosslinking probe, its preparation method, and its application. Background Technology

[0002] β-Glucuronase is a key glycosidic hydrolase in lysosomes, specifically cleaving β-1,4-glucuronide glycosidic bonds and maintaining the stereochemistry of the reducing end after the reaction. β-Glucuronase is abbreviated as GUSB. Human GUSB belongs to the GH2 family and is a 332 kDa tetrameric glycoprotein. GUSB deficiency leads to the accumulation of glycosaminoglycans such as chondroitin sulfate, quercetin sulfate, and heparin sulfate in lysosomes, triggering severe pathological reactions such as mucopolysaccharidosis type VII. Besides genetic diseases, elevated GUSB expression or activity is commonly found in necrotic areas of various tumors and in body fluid samples, and abnormalities have also been reported in infection, metabolic, and inflammation-related diseases.

[0003] There are three main types of existing methods for detecting GUSB activity. The first is traditional biochemical methods such as gel zymography and colorimetry, which can determine the overall GUSB activity, but have limited sensitivity and are difficult to spatially locate in complex systems. The second is hydrolyzable fluorescent probes constructed using glucuronic acid as the smallest nucleus, utilizing the fluorescence difference before and after hydrolysis for indirect detection. The third is active probes combined with photoaffinity probes, which achieve direct imaging and enrichment identification through covalent labeling at the active site.

[0004] Among them, active probes and photoaffinity probes achieve direct imaging and enrichment identification through covalent labeling at active sites. However, some reported schemes use probe molecules with relatively large sizes, limiting the probability of entering the active site and requiring high working concentrations, resulting in low sensitivity. For example, Bi Wenjing, "Design and Synthesis of Photocrosslinked Molecular Probes Based on β-Glucuronase Activity [D]. Shenzhen University, 2023. DOI:10.27321 / d.cnki.gszdu.2023.003039," uses oxyglycosidic bonds and benzophenone photocrosslinking groups to synthesize photoaffinity probes. In this case, the photoaffinity probe is linked by oxyglycosidic bonds, which are easily hydrolyzed. Alternatively, existing technologies use diazacyclopropane linkages, which easily quench the photoaffinity probe in the aqueous phase, making the molecule unstable. On the other hand, the large size of photosensitive groups such as benzophenone leads to large steric hindrance in the photoaffinity probe, resulting in low sensitivity.

[0005] In summary, the current active probes and photoaffinity probes have unstable connections, and the photocatalytic groups are relatively large, resulting in low sensitivity. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a GUSB photocrosslinking probe, its preparation method, and its application.

[0007] To facilitate understanding of this invention, the materials used in this invention and their abbreviations are listed below: N,N-Dimethylformamide, abbreviated as DMF. N,N,N',N'-Tetramethylurea hexafluorophosphate, abbreviated as HATU. N,N-Diisopropylethylamine, abbreviated as DIPEA.

[0008] 2,3,4-Tri-O-acetyl-α-D-bromoglucose, abbreviated as Compound 1. (2S,3R,4S,5S,6S)-2-guanidinothio-6-methoxycarbonyl-tetrahydro-2H-pyran-3,4,5-triyltriacetate, abbreviated as Compound 2. (2S,3R,4S,5S,6S)-2-mercapto-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate, abbreviated as Compound 3. (2S,3R,4S,5S,6S)-2-((2-(3-(but-3-yn-1-yl)-3H-diazacyclopropen-3-yl)ethyl)thio)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate, abbreviated as Compound 4. (2R,3R,4S,5S,6S)-2-azido-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate, abbreviated as compound 5. (2R,3R,4S,5S,6S)-2-amino-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate, abbreviated as compound 6. (2R,3R,4S,5S,6S)-2-[3-(3-(but-3-yn-1-yl)-3H-diazapropylcyclo-3-yl)propamidopropaneamide]-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate, abbreviated as compound 7. (2R,3R,4S,5S,6S)-2-[2-(but-3-yn-1-yl)-3H-diazapropylcyclo-3-yl]ethoxy-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate, abbreviated as compound 8.

[0009] The purpose of this invention is to provide a GUSB photocrosslinking probe, wherein the general chemical formula of the GUSB photocrosslinking probe is C0. 13 H 18 N2O6-R, chemical structural formula is .

[0010] Wherein, R is any one of the oxalic group elements or peptide bonds.

[0011] The R group stably connects the glucuronic acid recognition core and the photosensitive unit in the GUSB photocrosslinking probe through covalent bonds, and the R group contains less than or equal to 4 atoms, which are small in size and have low steric hindrance.

[0012] Preferably, when R is S, the structural formula of the GUSB photocrosslinking probe is as follows: Its name is GUSB-S-Probe.

[0013] Preferably, when R is NH-CO, the structural formula of the GUSB photocrosslinking probe is as follows: Its name is GUSB-N-Probe.

[0014] Preferably, when R is O, the structural formula of the GUSB photocrosslinking probe is as follows: Its name is GUSB-O-Probe.

[0015] Preferably, the method for preparing the GUSB-S-Probe includes the following steps: 2,3,4-Tri-O-acetyl-α-D-bromoglucose was dissolved in acetone, and thiourea was added, resulting in a substitution reaction to give (2S,3R,4S,5S,6S)-2-guanidinothio-6-methoxycarbonyl-tetrahydro-2H-pyran-3,4,5-trimethyltriacetate. Compound 2 and sodium metabisulfite were reduced to give (2S,3R,4S,5S,6S)-2-mercapto-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate. Compound 3 was dissolved in acetonitrile, and 3-(but-3-yn-1-yl)-3-(2-iodoethyl)-3H-bisacrylidine was added for substitution to give (2S,3R,4S,5S,6S)-2-((2-(3-(but-3-yn-1-yl)-3H-diazacyclopropen-3-yl)ethyl)thio)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate). (2S,3R,4S,5S,6S)-2-((2-(3-(but-3-yn-1-yl)-3H-diazacyclopropen-3-yl)ethyl)thio)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate) was dissolved in methanol, and sodium methoxide was added. The mixture was then hydrolyzed to give GUSB-S-Probe.

[0016] Preferably, the method for preparing the GUSB-N-Probe includes the following steps: 2,3,4-Tris-O-acetyl-α-D-bromoglucose was dissolved in DMF, and NaN3 was added. A substitution reaction was then carried out to give (2R,3R,4S,5S,6S)-2-azido-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate. (2R,3R,4S,5S,6S)-2-azido-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate was dissolved in dichloromethane, and a catalyst was added. The reaction was carried out under a hydrogen atmosphere to give (2R,3R,4S,5S,6S)-2-amino-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate. 4,5-Triacetate: (2R,3R,4S,5S,6S)-2-amino-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate was dissolved in DMF, and 3-(3-(butyl-3-yn-1-yl)-3H-bisacrylidine-3-yl)propionic acid, HATU, and DIPEA were added. A condensation reaction was carried out to give (2R,3R,4S,5S,6S)-2-[3-(3-(but-3-yn-1-yl)-3H-diazapropylcyclo-3-yl)propamidopropaneamide]-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate. (2R,3R,4S,5S,6S)-2-[3-(3-(but-3-yn-1-yl)-3H-diazapropylcyclo-3-yl)propamidopropaneamide]-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate was dissolved in methanol, and sodium methoxide was added. The mixture was then hydrolyzed to give GUSB-N-Probe.

[0017] Preferably, the method for preparing the GUSB-O-Probe includes the following steps: 2,3,4-Tris-O-acetyl-α-D-bromoglucose was dissolved in DCM, and 2-(3-but-3-ynyl-3H-bis(acrylidine-3-yl)-ethanol and AgOTf were added. A substitution reaction was then carried out to give (2R,3R,4S,5S,6S)-2-[2-(but-3-ynyl-1-yl)-3H-diazapropylcyclo-3-yl]ethoxy-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate. (2R,3R,4S,5S,6S)-2-[2-(but-3-ynyl-1-yl)-3H-diazapropylcyclo-3-yl]ethoxy-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate was dissolved in methanol, and sodium methoxide was added. A hydrolysis reaction was then carried out to give GUSB-O-Probe.

[0018] Another object of the present invention is to provide an application of a GUSB photocrosslinking probe, comprising at least one of the following: (1) Use the GUSB photocrosslinking probe for GUSB in situ imaging or quantitative analysis.

[0019] (2) Prepare GUSB photocrosslinking probes into GUSB detection kits for in situ imaging or quantitative analysis of GUSB.

[0020] Preferably, the method for in-situ imaging of GUSB involves mixing the GUSB probe solution with the GUSB solution, performing a photocrosslinking reaction and a click chemistry reaction, adding acetone, centrifuging, collecting the precipitate, dissolving the precipitate, heating to denature the protein, electrophoresis, developing, and achieving in-situ imaging.

[0021] Preferably, the kit consists of a 100µM GUSB photocrosslinking probe solution, a buffer solution, and a reaction termination solution.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. The general chemical formula of the GUSB photocrosslinking probe of the present invention is C0 13 H 18 In the N2O6-R crosslinking probe, the R group covalently connects the glucuronic acid recognition core and the photosensitive unit in the GUSB photocrosslinking probe, and the R group contains no more than 4 atoms, resulting in small volume and low steric hindrance. The R group of this invention is stably linked in aqueous solution. The GUSB photocrosslinking probe provided by this invention exhibits stable properties and stable linkage in solution. The GUSB photocrosslinking probe provided by this invention has a small molecular weight, low steric hindrance, easily binds to substrates, and has high sensitivity; it can label and recognize GUSB at a concentration of 100 μM. Therefore, the GUSB photocrosslinking probe of this invention combines stability with high sensitivity.

[0023] The GUSB photocrosslinking probe provided by this invention uses glucuronic acid as the recognition core, combined with a small-volume diazacyclopropane photosensitive unit and a terminal alkyne group, to achieve μM-level in-situ covalent capture of active GUSB in complex systems such as cells or tissues, followed by CuAAC incorporation. Fluorescence or biotin is used for imaging and affinity enrichment, and mass spectrometry identification is performed. This invention designs hydrolysis-resistant R groups through C / N-glycosidic bonds, avoiding the problems of photosensitive fragment release and nonspecificity caused by the hydrolysis of the GUSB photocrosslinking probe. Simultaneously, it minimizes steric hindrance, improves entry into active grooves and short-range labeling efficiency, and obtains high selectivity and low background signal under short-term 350nm~380nm irradiation. Compared with fluorescent substrates that rely on hydrolysis differences and bulky benzophenone probes, the probe of this invention significantly improves localization accuracy, sensitivity, data availability, quantification, and enrichment, while also possessing advantages such as modular synthesis, mild conditions, and strong system compatibility. It effectively solves the technical shortcomings of existing technologies that struggle to accurately obtain GUSB activity in situ under low-dose conditions.

[0024] 2. The GUSB photocrosslinking probe of this invention can be used for in-situ imaging and quantification of active GUSB in cells and tissues, and achieves affinity enrichment—LC-MS / MS identification—through click chemo-linked biotin, for chemical proteomics and crosslinking site analysis. It is suitable for inhibitor competition validation and high-throughput screening, evaluating selectivity and efficacy. It can be used for biomarker monitoring and efficacy assessment in tumor necrosis areas and related body fluids. It can be used for enzyme replacement or gene therapy response assessment in mucopolysaccharidosis type VII models and clinical samples. It can be used to compare source differences in the host's gut microbiota system to study the reactivation and enterohepatic circulation of drug glucuronide conjugates. It supports activity tracking in models of inflammation or infection, low-abundance capture in liquid biopsies, and validation of prodrugs and pharmacokinetic mechanisms, serving multiple applications in precision medicine and drug discovery. Attached Figure Description

[0025] Figure 1 This is a diagram illustrating the synthesis steps of compound 3 of the present invention.

[0026] Figure 2 This is the mass spectrum of compound 3 of the present invention.

[0027] Figure 3 This is a diagram illustrating the synthesis steps of compound 4 of the present invention.

[0028] Figure 4 The mass spectrum of compound 4 of this invention.

[0029] Figure 5 Compound 4 of the present invention 1 H NMR spectrum.

[0030] Figure 6 Compound 4 of the present invention 13 C10 NMR spectrum.

[0031] Figure 7 This is a diagram illustrating the synthesis steps of the GUSB-S-Probe photocrosslinking probe of the present invention.

[0032] Figure 8 This is the mass spectrum of the GUSB-S-Probe of this invention.

[0033] Figure 9 For the GUSB-S-Probe of this invention 1 H NMR spectrum.

[0034] Figure 10 For the GUSB-S-Probe of this invention 13 C10 NMR spectrum.

[0035] Figure 11 This is a diagram illustrating the synthesis steps of compound 5 of the present invention.

[0036] Figure 12 This is the mass spectrum of compound 5 of the present invention.

[0037] Figure 13 Compound 5 of the present invention 1 H NMR spectrum.

[0038] Figure 14 This diagram illustrates the synthesis steps of compounds 6 and 7 of the present invention.

[0039] Figure 15 This is the mass spectrum of compound 6 of the present invention.

[0040] Figure 16 This is the mass spectrum of compound 7 of the present invention.

[0041] Figure 17 Compound 7 of the present invention 1 H NMR spectrum.

[0042] Figure 18 Compound 7 of the present invention 13 C10 NMR spectrum.

[0043] Figure 19 This diagram illustrates the synthesis steps of the GUSB-N-Probe photocrosslinking probe of the present invention.

[0044] Figure 20 The mass spectra and NMR spectra of the GUSB-N-Probe of this invention are shown.

[0045] Figure 21 For the GUSB-N-Probe of the present invention 1 H NMR spectrum.

[0046] Figure 22 For the GUSB-N-Probe of the present invention 13 C10 NMR spectrum.

[0047] Figure 23 This is a diagram illustrating the synthesis steps of compound 8 of the present invention.

[0048] Figure 24 This is the mass spectrum of compound 8 of the present invention.

[0049] Figure 25 Compound 8 of the present invention 1 H NMR spectrum.

[0050] Figure 26 Compound 8 of the present invention 13 C10 NMR spectrum.

[0051] Figure 27 This diagram illustrates the synthesis steps of the GUSB-O-Probe photocrosslinking probe of the present invention.

[0052] Figure 28 This is the mass spectrum of the GUSB-O-Probe of the present invention.

[0053] Figure 29 The GUSB-O-Probe of the present invention 1 H NMR spectrum.

[0054] Figure 30 The GUSB-O-Probe of the present invention 13 C10 NMR spectrum.

[0055] Figure 31 This is a diagram showing the docking of the GUSB-S-Probe with the GUSB molecule in this invention.

[0056] Figure 32 This is a diagram showing the docking of the GUSB-N-Probe with the GUSB molecule in this invention.

[0057] Figure 33 This is a diagram showing the docking of the GUSB-O-Probe with the GUSB molecule in this invention.

[0058] Figure 34 This is a comparison of the results of GUSB probes with different glycosidic bonds according to the present invention.

[0059] Figure 35 The figure shows the experimental results of the GUSB-S-Probe protein labeling of the present invention.

[0060] Figure 36 The figure shows the experimental results of GUSB-O-Probe protein labeling in this invention.

[0061] Figure 37 The figure shows the experimental results of GUSB-N-Probe protein labeling in this invention.

[0062] Figure 38 This is a flowchart of the competitive ABPP experiment of the present invention.

[0063] Figure 39 This is a structural diagram of the inhibitor ganoderic acid A of the present invention.

[0064] Figure 40 The figure shows the experimental results of the competitive ABPP test of the GUSB-S-Probe of this invention.

[0065] Figure 41 The figure shows the experimental results of the competitive ABPP test of GUSB-N-Probe in this invention.

[0066] Figure 42 The figure shows the experimental results of the competitive ABPP test of GUSB-O-Probe of the present invention.

[0067] Figure 43 The diagram shows the experimental results of the GUSB photocrosslinking probe IC50 of this invention. In the diagram, A is the GUSB-S-Probe, B is the GUSB-N-Probe, and C is the GUSB-O-Probe.

[0068] Figure 44 This is a diagram showing the results of the photocrosslinking labeling experiment of mouse intestinal flora according to the present invention. Detailed Implementation

[0069] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the following detailed description, in conjunction with preferred embodiments and accompanying drawings, provides a clear and complete account of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0070] It should be noted that all technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0071] The main materials used in this invention are ganoderic acid A and mice.

[0072] The purity of ganoderic acid A was %, CAS number 100665-40-5, and it was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Mice, model C57BL / 6J, were purchased from Guangdong Provincial Medical Laboratory Animal Center.

[0073] Example 1 A method for preparing a GUSB-S-Probe includes the following steps: 2.53 mmol of compound 1 was dissolved in 10 mL of dry acetone. Then, 3.80 mmol of thiourea was added. The mixture was stirred at 60 °C for 2.5 h, and then the solvent was removed by vacuum distillation to give compound 2.

[0074] The synthesis steps of compound 3 are as follows: Figure 1As shown. Compound 2 was added to 15 mL of a mixed solvent prepared from dichloromethane and water in a 1:1 volume ratio, followed by 3.04 mmol of sodium metabisulfite. The mixture was heated to 60 °C and refluxed for 2.5 h. After the reaction, the aqueous phase was extracted three times with 15 mL of dichloromethane. The organic extracts were combined, and 10 g of anhydrous Na₂SO₄ was added. The mixture was dried at room temperature for 3 min, and then the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using a mixture of petroleum ether and ethyl acetate in a 3:1 volume ratio as the eluent, yielding 0.55 g of white solid compound 3 with a purity of 62%. The mass spectrometry analysis of compound 3 was HRMS (ESI) [M+Na]. + C 13 H 18 O9SNa + , calculate: 373.0564, found: 373.0128 and Figure 2 Consistent.

[0075] The synthesis steps of compound 4 are as follows: Figure 3 As shown. 0.86 mmol of compound 3 was dissolved in 5 mL of dry acetonitrile. 1.03 mmol of 3-(but-3-yn-1-yl)-3-(2-iodoethyl)-3H-bisacrylidine was added. The mixture was allowed to react overnight at room temperature. After the reaction was complete, insoluble matter was removed by filtration, and the solvent was removed by vacuum distillation. Purification was then performed by column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 4–3:1 to give compound 4 as a white solid with a purity of 91%, weighing 368 mg. The mass spectrometry and NMR results of compound 4 are shown in the figure. HRMS (ESI) [M+Na] + C 20 H 26 N2O9SNa + , calculate: 493.1251, found: 493.1231. 1 H NMR (600MHz, CDCl3) δ5.27 (t, J=9.4Hz, 1H), 5.20 (t, J=9.7Hz, 1H), 5.04 (t, J=9.6Hz, 1H), 4.51 (d, J=10.0Hz, 1H), 4.04 (d, J=9.9Hz, 1H), 3.76 (s, 3H), 2. 56(ddd, J=13.0, 8.7, 6.6Hz, 1H), 2.48 (ddd, J=13.0, 8.6, 6.9Hz, 1H), 2.07 (s, 3H), 2.02 (d, J=7.8Hz, 9H), 1.81~1.72 (m, 2H), 1.67 (t, J=7.2Hz, 2H), such as Figure 5 As shown. 13C NMR (151MHz, CDCl3) δ 170.0, 169.4, 169.3, 166.8, 83.6, 82.7, 76.3, 72.9, 69.4, 69.3, 53.0, 33.4, 32.1, 27.6, 24.1, 20.7, 20.6, 20.5, 13.3, etc. Figure 6 As shown.

[0076] The synthesis steps of GUSB-S-Probe are as follows: Figure 7 As shown. 0.021 mmol of compound 4 was dissolved in 2 mL of methanol, and 0.0053 mmol of sodium methoxide was added. The reaction was allowed to proceed for 1 h. After the reaction, the solvent was removed under reduced pressure using an Amberlite IR20. 2 mL of 50% (v / v) aqueous methanol solution was added to dissolve the compound, followed by the addition of 1 M NaOH to bring the pH to 11. After reacting at room temperature for 1 h, the solvent was removed under reduced pressure, and the compound was purified by HPLC to obtain 7.1 mg of GUSB-S-Probe with a purity of 90%. The mass spectrometry and NMR analysis results of GUSB-S-Probe were HRMS (ESI) [M+Na]. + C 13 H 18 N2O6SNa + , calculate: 353.0778, found: 353.0782, and Figure 8 Consistent. 1 H NMR (600MHz, DMSO) δ5.25 (s, 1H), 4.38 (d, J=9.7Hz, 1H), 3.63 (d, J=9.8Hz, 1H), 3.32 (d, J=9.3Hz, 1H), 3.16 (t, J=8.8Hz, 1H), 3.01 (t , J=9.1Hz, 1H), 2.81 (t, J=2.6Hz, 1H), 2.46~2.33 (m, 2H), 2.00 (td, J=7.3, 2.6Hz, 2H), 1.70 (t, J=7.7Hz, 2H), 1.59 (t, J=7.3Hz, 2H), such as Figure 9 As shown. 13 C NMR (151MHz, DMSO) δ 170.6, 86.0, 83.7, 79.6, 77.8, 72.9, 72.2, 71.8, 33.4, 31.9, 28.6, 23.8, 13.1, etc. Figure 10 As shown.

[0077] Example 2 A method for preparing a GUSB-N-Probe includes the following steps: The synthesis steps of compound 5 are as follows: Figure 11 As shown. 0.76 mmol of compound 1 was dissolved in 3 mL of dry DMF, followed by the addition of 3.03 mmol of NaN3. The mixture was stirred for 36 h, diluted with 50 mL of water, and the aqueous phase was extracted three times with dichloromethane. The precipitate was washed with saturated NaCl solution. All organic phases were combined, dried over anhydrous Na2SO4, filtered, and the solvent was removed by vacuum distillation. Purification was then performed by column chromatography. Eluent was obtained using petroleum ether and ethyl acetate in a volume ratio of 4–3:1 to give 177 mg of compound 5 with a purity of 65%. The mass spectrometry and NMR analysis results of compound 5 were HRMS (ESI) [M+Na]. + C 13 H 17 N3O9Na + , calculate: 382.0857, found: 382.0860, and Figure 12 Consistent. 1 H NMR (600MHz, CDCl3) δ5.32~5.21 (m, 2H), 4.96 (t, J=8.9Hz, 1H), 4.72 (d, J=8.7Hz, 1H), 4.12 (d, J=9.5Hz, 1H), 3.78 (s, 3H), 2.08 (s, 3H), 2.03 (s, 6H), such as Figure 13 As shown.

[0078] The synthesis steps of compounds 6 and 7 are as follows: Figure 14 As shown. 0.28 mmol of compound 5 was dissolved in dichloromethane, and 10 wt% Pd / C was added. The reaction flask was sealed with a rubber stopper, a hydrogen balloon was inserted, and the reaction was carried out under a hydrogen atmosphere for 5 h. After the reaction was complete, diatomaceous earth was poured onto filter paper, and the reaction solution was filtered to remove insoluble matter. Then, the solvent was removed by vacuum distillation to obtain compound 6. The product can be proceeded to the next step without purification. The mass spectrometry analysis result of compound 6 was HRMS (ESI) [M+Na]. + C 11 H 17 NO8 + , calculate: 314.0846, found: 314.1173, and Figure 15 Consistent.

[0079] Compound 6 was dissolved in 1 mL of dry DMF, followed by the addition of 0.34 mmol of 3-(3-(butyl-3-yn-1-yl)-3H-bisacrylidine-3-yl)propionic acid, 0.36 mmol of HATU, and 1.12 mmol of DIPEA. After reacting overnight at room temperature, the mixture was diluted with water, and the aqueous phase was extracted three times with dichloromethane. The extracts were combined and washed with brine. The organic phase was dried over Na₂SO₄, filtered, and the solvent was removed under reduced pressure. Column chromatography was used, eluting with a 2:1 volume ratio of petroleum ether and ethyl acetate to give 108 mg of compound 7 with a purity of 81%. Mass spectrometry and NMR analysis of compound 7 were performed using HRMS (ESI) [M+Na]. + C 21 H 27 N3O 10 + , calculate: 482.1769, found: 482.1775, such as Figure 16 As shown. 1 HNMR (600MHz, CDCl3) δ6.52 (d, J=9.3Hz, 1H), 5.39 (t, J=9.6Hz, 1H), 5.31 (t, J=9.4Hz, 1H), 5.14 (t, J=9.7Hz, 1H), 4.96 (t, J=9.6Hz, 1H), 4.17 (d, J =10.0Hz, 1H), 3.73(s, 3H), 2.04(d, J=3.7Hz, 6H), 2.02~2.00(m, 2H), 1.8 7(ddt, J=14.4, 10.4, 7.0Hz, 2H), 1.82~1.74(m, 1H), 1.69~1.58(m, 2H), such as Figure 17 As shown. 13 C NMR (151MHz, CDCl3) δ 171.7, 171.0, 169.7, 169.6, 167.2, 82.6, 77.9, 73.8, 71.9, 70.2, 69.6, 69.4, 53.0, 32.4, 30.1, 27.7, 27.5, 20.7, 20.6, 20.5, 13.2, etc. Figure 18 As shown.

[0080] The synthesis steps of GUSB-N-Probe are as follows: Figure 19As shown. 0.021 mmol of compound 7 was dissolved in 2 mL of methanol, and 0.0053 mmol of sodium methoxide was added. The reaction was allowed to proceed for 1 h. After the reaction, the mixture was post-treated using an Amberlite IR20 to remove the solvent under reduced pressure. 2 mL of a 1:1 methanol-water solution was added to dissolve the compound, followed by the addition of 1 M NaOH to bring the pH to 11. The reaction was allowed to proceed for 1 h, and the solvent was removed. Purification was performed by HPLC to obtain 6.38 mg of GUSB-N-Probe with a purity of 90%. The mass spectrometry and NMR analysis results of GUSB-N-Probe were HRMS (ESI) [M+Na]. + C 14 H 19 N3O7Na + , calculate: 364.1115, found: 364.1116, such as Figure 20 As shown. 1 H NMR (600MHz, MeOD) δ4.92 (s, 1H), 3.85 (d, J=9.7Hz, 1H), 3.49 (t, J=9.4Hz, 1H), 3.42 (t, J=9.0Hz, 1H), 3.28 (d, J=9.0Hz, 1 H), 2.26(t, J=2.7Hz, 1H), 2.20~2.06(m, 2H), 2.02(td, J=7.4, 2.7Hz, 2H), 1.74(q, J=7.3Hz, 2H), 1.67~1.56(m, 2H), such as Figure 21 As shown. 13 C NMR (151MHz, MeOD) δ 174.1, 171.1, 82.2, 79.7, 77.1, 76.7, 72.1, 71.7, 68.9, 31.9, 29.6, 28.2, 27.4, 12.4, etc. Figure 22 As shown.

[0081] Example 3 A method for preparing a GUSB-O-Probe includes the following steps: The synthesis steps of compound 8 are as follows: Figure 23 As shown. 0.25 mmol of compound 1 was dissolved in 2 mL of dry DCM, and 1.25 mmol of 2-(3-but-3-ynyl-3H-bisacrylidine-3-yl)-ethanol was added, followed by 0.63 mmol of AgOTf. The reaction was allowed to proceed for 2.5 h. Diatomaceous earth was poured onto filter paper, and the reaction solution was filtered through the diatomaceous earth. The solvent was then removed by vacuum distillation. The solution was purified by column chromatography using petroleum ether and ethyl acetate in a 3:1 volume ratio to obtain 26 mg of compound 8 with a purity of 23%. The mass spectrometry and NMR analysis results of compound 8 are as follows: HRMS (ESI) [M+NH4]+ C 20 H 30 N3O 10 + , calculate: 472.1926, found: 472.2501, such as Figure 24 As shown. 1 H NMR (600MHz, CDCl3) δ5.30~5.17 (m, 2H), 5.03 (t, J=8.4Hz, 1H), 4.52 (d, J=7.7Hz, 1H) , 4.14~3.99(m, 1H), 3.84(dt, J=10.1, 5.2Hz, 1H), 3.77(d, J=4.4Hz, 3H), 3.23(td, J=9.3, 4.7Hz, 1H), 2.04 (d, J=4.0Hz, 6H), 2.02~1.95 (m, 2H), 1.84 (ddd, J=14.6, 8. 8, 5.2Hz, 2H), 1.70 (dt, J=14.6, 7.1Hz, 1H), 1.61 (ddt, J=22.1, 14.5, 6.0Hz, 2H), such as Figure 25 As shown. 13 C NMR (151MHz, CDCl3) δ 170.1, 169.4, 169.4, 167.2, 100.6, 82.7, 72.6, 72.0, 71.0, 69.4, 69.3, 64.5, 52.9, 32.6, 32.6, 26.6, 20.8, 20.6, 20.5, 13.3, etc. Figure 26 As shown.

[0082] The synthesis steps of GUSB-O-Probe are as follows: Figure 27 As shown. 0.022 mmol of compound 8 was dissolved in 2 mL of methanol, and 0.0055 mmol of sodium methoxide was added. The reaction was allowed to proceed for 1 h. After the reaction, the mixture was post-treated using an Amberlite IR20 to remove the solvent under reduced pressure. 2 mL of a 1:1 methanol-water solution was then added to dissolve the compound, followed by the addition of 1 M NaOH to bring the pH to 11. The reaction was allowed to proceed for 1 h, and the solvent was removed under reduced pressure. Purification was performed by HPLC to obtain 6.22 mg of GUSB-O-Probe with a purity of 90%. The mass spectrometry and NMR analysis results of GUSB-O-Probe were HRMS (ESI) [M+Na]. + C 13 H 18 N2O7Na + , calculate: 337.1006, found: 337.1008, such as Figure 28 As shown. 1H NMR (600MHz, MeOD) δ4.31(d, J=7.8Hz, 1H), 3.82–3.75(m, 2H), 3.53(t, J=9.4Hz, 1H), 3.45(dt, J=10.1, 6.7Hz, 1H), 3.38( t, J=9.2Hz, 1H), 3.23 (dd, J=9.2, 7.9Hz, 1H), 2.24 (t, J=2.7Hz, 1H), 2.04 (td, J=7.5, 2.7Hz, 2H), 1.75~1.60 (m, 4H), such as Figure 29 As shown. 13 CNMR (151MHz, MeOD) δ 171.1, 103.3, 82.4, 76.1, 75.2, 73.3, 71.7, 68.8, 64.4, 32.9, 32.2, 26.3, 12.5, etc. Figure 30 As shown.

[0083] Example 4 A GUSB photocrosslinking probe molecular docking method includes the following steps: 1. Structure Selection and Software Platform: The GUSB crystal structure selected in this invention is derived from RCSB's PDB 5G0Q, which is a complex of GUSB and an N-alkynyl cyclic polyol aziridine inhibitor. All computational and molecular simulation work was performed using computer-aided drug design platforms provided by Schrödinger, Inc. in New York, including Glide, LigPrep, Maestro, and PyMOL.

[0084] 2. Protein and Probe Pretreatment: The software's default parameter settings were used throughout the protein and probe pretreatment process. Preliminary treatment of GUSB and probes included molecular optimization and water molecule removal to ensure experimental accuracy.

[0085] 3. Molecular docking operation: Molecular docking was performed using the SP mode of Glide version 9.0. The docking grid was constructed using Glide 9.0, with N-alkynyl cyclopolyol aziridine as the docking center. The dimensions of the boundary box and the surrounding box were set to 10 Å × 10 Å × 10 Å and 55 Å × 55 Å × 55 Å, respectively, to ensure that the docking region covered the catalytic active center of GUSB.

[0086] 4. Ligand Preparation and Pretreatment: Ligand structures were built using Maestro 14.0, and pretreatment of all compounds was performed using LigPrep 3.5 with default parameters for optimization. Each ligand underwent independent docking runs.

[0087] 5. Docking Result Analysis: After docking, the docking conformation was analyzed using Maestro and PyMOL.

[0088] The docking results of the GUSB-S-Probe with GUSB molecules in this invention are as follows: Figure 31 As shown. The docking results of the GUSB-N-Probe with the GUSB molecules in this invention are as follows. Figure 32 As shown. The docking results of the GUSB-O-Probe and GUSB molecules in this invention are as follows. Figure 33 As shown in the figure, the results indicate that all three types of glycosidic probes are located near the catalytic active site of GUSB, consistent with the reported co-crystal structure of GUSB and the compound N-alkynylcyclopolyol aziridine, and are all located in the same catalytic active region. Further analysis revealed that among the three types of glycosidic probes, the thioglycosidic probes had the highest simulated binding fraction, exhibiting the strongest binding affinity. In contrast, the nitrogenous glycosidic probes had the lowest simulated binding fraction, indicating weaker binding affinity.

[0089] Table 1 GUSB probe docking results Example 5 A method for labeling GUSB probe proteins includes the following steps: 1. GUSB probe-labeled protein 1. Prepare GUSB solution: Prepare 10 μL of a solution containing 0.1 mg / mL GUSB. Mix the probe with the GUSB solution according to experimental requirements and incubate at 37°C for 30 min.

[0090] 2. Photocrosslinking treatment: After incubation, place the probe and protein mixture sample on ice and put it into the photocrosslinking instrument. Irradiate it at a wavelength of 365nm for a certain period of time according to the experimental requirements.

[0091] 3. Click reaction: After photocrosslinking, the sample was removed and a fresh mixed solution was added. The final concentrations were: 1 mM CuSO4, 1 mM NaVc, 100 μM THPTA, 100 μM TMARA-N3, and 0.4% SDS solution. The reaction was continued at 37 °C for 1 h.

[0092] 4. Precipitation and centrifugation: After the reaction was complete, pre-cooled acetone was added to each centrifuge tube, and the tubes were placed at -80°C for 20 min. Subsequently, the tubes were centrifuged at 21000g for 10 min at 4°C, and the supernatant was removed.

[0093] 5. Protein dissolution: Dissolve the precipitate in 1% SDS solution and add 5× loading buffer. Then, boil in a 100°C metal bath for 5 minutes until the protein is completely denatured.

[0094] 6. SDS-PAGE analysis: After gel separation, the gel was placed in an imaging system for development and fluorescence band observation was performed.

[0095] 2. Probe comparison experiment Three different glycosidic bond probes were added to 0.1 mg / mL GUSB solution in different centrifuge tubes, and a DMSO blank control group and a control group for the photocrosslinking group linkage site were set up. Subsequently, the experiment was carried out according to the standard experimental procedure described above.

[0096] The comparison results of different glycosidic bond GUSB probes of the present invention are as follows: Figure 34 As shown in the figure, the results indicate that thioglycosidic bonds exhibit significantly higher labeling efficiency than the other two types of glycosidic bond probes under the pre-defined experimental conditions. This suggests that thioglycosidic bonds possess stronger affinity and higher labeling capacity, enabling them to bind to GUSB and stably label the target protein. Their superior labeling effect may stem from the strong hydrolysis resistance of thioglycosidic bonds, which makes them more stable during enzyme labeling and effectively avoids the influence of other factors on labeling efficiency.

[0097] 3. Probe concentration gradient and photocrosslinking time gradient Each probe stock solution had a concentration of 20 mM. It was diluted to the target concentration, ensuring that the same volume of probe solution was added to each centrifuge tube. A DMSO blank control group and a control group for the photocrosslinking group linkage sites were set up. The UV irradiation time was consistent for each group, and the experiment was then conducted according to the above procedure.

[0098] The probe concentration was kept consistent in each centrifuge tube, but the exposure time in the photocrosslinker was different; the rest of the experimental steps were kept consistent.

[0099] The experimental results of the GUSB-S-Probe protein labeling of this invention are as follows: Figure 35 As shown. The experimental results of GUSB-O-Probe protein labeling of the present invention are as follows. Figure 36 As shown. The experimental results of GUSB-N-Probe protein labeling of the present invention are as follows. Figure 37 As shown in the figure, the binding of the three types of probes to GUSB exhibited both concentration-dependent and photocrosslinking time-dependent effects. This indicates that the binding ability of the probes to the target protein not only increases with increasing probe concentration, but also becomes more significant with prolonged photocrosslinking time.

[0100] 4. Competitive ABPP experiment The competitive ABPP experimental procedure of the present invention is as follows: Figure 38As shown. Competitive activity proteomics analysis, abbreviated as cABPP. The purpose of cABPP is to verify the specificity and accuracy of probe recognition by having the probe compete with known inhibitors for binding to the target protein. This experiment can clarify whether the binding site of the probe to the target protein is consistent with the action site of the known inhibitor, thereby confirming the competitive binding ability and effectiveness of the probe. In addition, cABPP can be used to screen and identify potential protein inhibitors, providing an effective tool and basis for drug development. Due to the limited variety of commercially available GUSB inhibitors, the selection space is small. Based on availability, we selected ganoderic acid A as a GUSB inhibitor for our study to evaluate its effect on enzyme activity and the competitive binding ability of the probe. The structure of ganoderic acid A is shown below. Figure 39 As shown.

[0101] First, 0µM, 10µM, 100µM, and 1000µM of ganoderic acid A were incubated at 37°C for 30 min. Then, probe solution of equal concentration was added, and incubation was continued for another 30 min. The experimental procedure described above was then followed.

[0102] The competitive ABPP experimental results of the GUSB-S-Probe of this invention are as follows: Figure 40 As shown. The competitive ABPP experimental results of the GUSB-N-Probe of the present invention are as follows. Figure 41 As shown. The competitive ABPP experimental results of the GUSB-O-Probe of the present invention are as follows. Figure 42 As shown in the figure, all three types of probes competitively bind to GUSB with commercially available GUSB inhibitors. Notably, the thioglycolic acid probe exhibited stronger competitive binding ability than the other two probes. This result validates that the thioglycolic acid probe can not only effectively label GUSB but also screen for potential inhibitors, indicating that this probe can serve as a powerful tool for drug screening and inhibitor development.

[0103] 5. IC 50 experiment 4-Nitrophenyl-β-D-glucuronic acid (PNPG) was selected as the hydrolysis substrate, and GUSB derived from *E. coli* was used as the hydrolase. First, 10 μL of a 1 mg / mL sodium acetate buffer solution of *E. coli* GUSB was added to a centrifuge tube. The sodium acetate buffer solution had a pH of 5.0, a concentration of 40 mM, and additional sodium acetate was added. Next, 10 μL of probe solutions at concentrations of 1 µM, 10 µM, and 100 µM were added to their respective centrifuge tubes, followed by 0.526 μL of a 2 mM PNPG solution. After incubation at 37 °C for 30 min, the reaction was terminated by adding sodium hydroxide solution; the reaction solution changed from colorless and transparent to clear yellow. Finally, the absorbance was measured at 405 nm, and the IC50 of the probe was plotted. 50 The curve graphs show that all experiments were repeated three times.

[0104] The present invention provides three types of GUSB photocrosslinking probe ICs 50 Experimental results are as follows Figure 43 As shown in the figure, the results indicate that, compared to nitrogen- and oxygen-glycosidic probes, thioglycosidic probes exhibited the strongest inhibitory ability in suppressing the hydrolytic activity of GUSB on its substrates. The inhibitory efficacy of the three types of probes showed a clear gradient relationship, in the order of S>O>N. This trend is consistent with the labeling intensity observed in previous GUSB probe labeling experiments, further validating that thioglycosidic probes possess higher affinity and stability in binding to GUSB. This result may be related to the unique structural properties of thioglycosidic bonds; their higher resistance to hydrolysis and spatial adaptation to enzyme active sites may enhance the binding stability with the GUSB active pocket. Therefore, thioglycosidic probes not only have advantages in labeling efficiency but also show promising application prospects in functional inhibition, demonstrating their greater potential in the study of GUSB-related mechanisms and the screening of inhibitors.

[0105] 6. Mouse gut microbiota protein labeling experiment Animal experiments were conducted in accordance with the guidelines approved by the Animal Protection Ethics Committee of Shenzhen University (ethics number: IACUC-202500048). The steps for extracting proteins from mouse intestinal flora were as follows: Collected fecal samples were thawed in 3 mL of pre-chilled PBS and thoroughly dispersed by vigorous vortexing. Subsequently, the samples were centrifuged at 4°C, 100 g for 2 min to remove insoluble impurities. The supernatant containing bacterial cells was collected and aliquoted into 1 mL portions. Each fraction was centrifuged at 4°C, 6500 g for 15 min to precipitate the cells. After washing twice with cold PBS, the cells were resuspended in 450 μL of lysis buffer, which consisted of a mixture of M-PER and Roche protease inhibitors. Next, the cells were sonicated at 4°C for 15 min using an ultrasonic cell disruptor. Insoluble fragments were then removed by centrifugation at 4°C, 10000 g for 5 min, and the concentration of soluble proteins in the supernatant was determined using a Pierce BCA protein quantification kit. Subsequently, 20g of total protein was added to each centrifuge tube, and the pH of the system was adjusted to 5.0. GUSB-S-Probe was then added, and the mixture was incubated for 30 minutes. The subsequent photocrosslinking experiments were performed as before. The results of the mouse gut microbiota photocrosslinking labeling experiment are shown below. Figure 44 As shown.

[0106] Based on the preliminary exploratory experiments of this invention, we used GUSB-S-Probe to perform target protein capture experiments in a complex gut microbiota protein environment to verify its labeling performance in a real system. The results showed that this photocrosslinked probe with a thioglycolic bond structure can effectively label GUSB target proteins derived from gut microbiota at a concentration of 100 µM, and exhibits a clear specific band around 70 kDa in SDS-PAGE, indicating that the probe has good targeting ability and practical application value.

[0107] However, the experiment also observed several non-specific bands generated by the probe labeling, indicating that the probe exhibits some non-specific binding in complex biological samples. Possible reasons for this phenomenon include: the probe binding non-specifically to other non-target proteins when it fails to completely selectively recognize the target enzyme; and during photocrosslinking, the activated photosensitive group reacts non-selectively with non-target residues in nearby proteins, resulting in decreased crosslinking efficiency and enhanced background signal.

[0108] In summary, although the probe has shown preliminary practicality in terms of labeling specificity, further optimization of its structure or experimental conditions is needed to improve selectivity and reduce background interference, thereby enhancing its practical application in complex biological systems.

[0109] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0110] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A GUSB photocrosslinking probe, characterized in that, The chemical formula of the GUSB photocrosslinking probe is C 13 H 18 N2O6-R, chemical structural formula is ; Wherein, R is any one of the oxalic group elements or peptide bonds; The R group stably connects the glucuronic acid recognition core and the photosensitive unit in the GUSB photocrosslinking probe through covalent bonds, and the R group contains less than or equal to 4 atoms, resulting in a small size and low steric hindrance.

2. The GUSB photocrosslinking probe according to claim 1, characterized in that, When R is S, the structural formula of the GUSB photocrosslinking probe is: Its name is GUSB-S-Probe.

3. The GUSB photocrosslinking probe according to claim 1, characterized in that, The structural formula of the GUSB photocrosslinking probe when R is O is: Its name is GUSB-O-Probe.

4. The GUSB photocrosslinking probe according to claim 2, characterized in that, The method for preparing the GUSB-S-Probe includes the following steps: 2,3,4-Tris-O-acetyl-α-D-bromoglucose was dissolved in acetone, and thiourea was added, resulting in a substitution reaction to give (2S,3R,4S,5S,6S)-2-guanidinothio-6-methoxycarbonyl-tetrahydro-2H-pyran-3,4,5-triyltriacetate; Compound 2 was reduced with sodium metabisulfite to give (2S,3R,4S,5S,6S)-2-mercapto-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate; Compound 3 was dissolved in acetonitrile, and 3-(but-3-yn-1-yl)-3-(2-iodoethyl)-3H-bisacrylidine was added, resulting in a substitution reaction. (2S,3R,4S,5S,6S)-2-((2-(3-(but-3-yn-1-yl)-3H-diazacyclopropen-3-yl)ethyl)thio)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate) was obtained. (2S,3R,4S,5S,6S)-2-((2-(3-(but-3-yn-1-yl)-3H-diazacyclopropen-3-yl)ethyl)thio)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate) was dissolved in methanol, and sodium methoxide was added. The mixture was then hydrolyzed to obtain GUSB-S-Probe.

5. The GUSB photocrosslinking probe according to claim 1, characterized in that, When R is a peptide bond, the preparation method of the GUSB photocrosslinking probe includes the following steps: 2,3,4-Tris-O-acetyl-α-D-bromoglucose was dissolved in DMF, and NaN3 was added to induce a substitution reaction, yielding (2R,3R,4S,5S,6S)-2-azido-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate. (2R,3R,4S,5S,6S)-2-azido-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate was dissolved in dichloromethane, and a catalyst was added. The reaction was carried out under a hydrogen atmosphere to yield (2R,3R,4S,5S,6S)-2-amino-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate. (2R,3R,4S,5S,6S)-2-amino-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate was dissolved in DMF, and NaN3 was added to induce a substitution reaction, yielding (2R,3R,4S,5S,6S)-2-amino-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate. 3-(3-(butyl-3-yn-1-yl)-3H-bis(acrylidine-3-yl)propionic acid, HATU, and DIPEA were condensed to give (2R,3R,4S,5S,6S)-2-[3-(3-(but-3-yn-1-yl)-3H-diazapropylcyclo-3-yl)propamidopropaneamide]-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate; (2R,3R,4S,5S,6S)-2-[3-(3-(but-3-yn-1-yl)-3H-diazapropylcyclo-3-yl)propamidopropaneamide]-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate]-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate was dissolved in methanol, sodium methoxide was added, and hydrolysis was carried out to give the GUSB photocrosslinking probe, denoted as GUSB-N-Probe.

6. The GUSB photocrosslinking probe according to claim 3, characterized in that, The method for preparing the GUSB-O-Probe includes the following steps: 2,3,4-Tris-O-acetyl-α-D-bromoglucose was dissolved in DCM, and 2-(3-but-3-ynyl-3H-bis(acrylidine-3-yl)-ethanol and AgOTf were added. A substitution reaction was then carried out to give (2R,3R,4S,5S,6S)-2-[2-(but-3-ynyl-1-yl)-3H-diazapropylcyclo-3-yl]ethoxy-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate. (2R,3R,4S,5S,6S)-2-[2-(but-3-ynyl-1-yl)-3H-diazapropylcyclo-3-yl]ethoxy-6-methoxycarbonyl-tetrahydropyran-3,4,5-triacetate was dissolved in methanol, and sodium methoxide was added. A hydrolysis reaction was then carried out to give GUSB-O-Probe.

7. The application of the GUSB photocrosslinking probe according to claim 1, characterized in that, Includes at least one of the following: (1) Use the GUSB photocrosslinking probe for GUSB in situ imaging or quantitative analysis; (2) Prepare GUSB photocrosslinking probes into GUSB detection kits for in situ imaging or quantitative analysis of GUSB.

8. The application of the GUSB photocrosslinking probe according to claim 7, characterized in that, The method for GUSB in situ imaging involves mixing the GUSB probe solution with the GUSB solution, performing photocrosslinking and click chemistry reactions, adding acetone, centrifuging, collecting the precipitate, dissolving the precipitate, heating to denature the protein, electrophoresis, and developing the image to achieve in situ imaging.

9. The GUSB photocrosslinking probe according to claim 7, characterized in that, The kit consists of a 100µM GUSB photocrosslinking probe solution, a buffer solution, and a reaction termination solution.

Images