Enzymatic self-anchored pre-targeting molecule P-TCO as well as preparation method and application thereof

Abstract

The invention discloses an enzymatic self-anchored pre-targeting molecule P-TCO as well as a preparation method and application thereof.The enzymatic self-anchored pre-targeting molecule P-TCO provided by the invention is subjected to a dephosphorylation reaction under the action of high-expression alkaline phosphatase of a tumor cell membrane, and an active intermediate with a QM group is generated. The intermediate can be used for covalently labeling ALP and membrane proteins around the ALP so as to ensure that a TCO group is stably anchored on a cell membrane. Subsequently, the TCO group covalently labeled on the membrane protein can be subjected to an efficient biological orthogonal reaction with the tetrazine probe, so that a targeted delivery platform of the multi-modal imaging probe is constructed, high-sensitivity visualization of living tumors is realized, and huge application potential in the field of precise diagnosis of clinical tumors is shown.

Description

Technical Field

[0001] The present invention belongs to the field of biological probes, and in particular relates to an enzymatic self-anchoring pre-targeting molecule P-TCO and a preparation method and application thereof. Background Art

[0002] By leveraging the metabolic properties of tumor cells, the introduction of chemical tags, such as fluorescent or isotope-labeled molecules, onto the cell membrane allows for precise localization and quantitative analysis of tumor tissue. This technology has achieved significant progress in multimodal cell membrane imaging probes. However, practical in vivo applications still face challenges, including specific recognition of target proteins, labeling time limitations, the influence of labeling concentration, differential expression of membrane protein receptors, and interference from physiological changes such as protein translation. Compared to metabolic labeling, proximity labeling techniques offer not only higher specificity but also greater translational potential and flexibility in cell membrane editing. However, the range of action of exogenous enzymes limits the application of conventional contact enzymatic proximity labeling techniques for in situ labeling of tissues in vivo. Quinonemethide, a chemical functional group generated by the "caging" of endogenous enzymes, is an emerging proximity labeling chemical tool that can undergo nucleophilic attack reactions with neighboring molecules in situ, achieving covalent attachment of target proteins. This technology has been successfully applied in a variety of fields, including the development of cell membrane imaging molecular probes, protein interaction studies, and the study of specific protein localization, providing new perspectives and tools for cell membrane editing and protein research. Summary of the Invention

[0003] The purpose of the present invention is to develop a tumor-specific enzyme-responsive anchored pre-targeting molecule P-TCO for constructing a multimodal imaging platform such as near-infrared fluorescence, bioluminescence, positron emission tomography, and magnetic resonance imaging in vivo to achieve accurate visualization of living tumors.

[0004] The technical problem to be solved by the present invention is to provide an enzymatic anchored pre-targeting molecule P-TCO and a preparation method thereof in view of the deficiencies in the prior art.

[0005] The technical problem that the present invention also aims to solve is to provide an application of an enzymatic anchored pre-targeting molecule P-TCO in protein labeling.

[0006] A further technical problem to be solved by the present invention is to provide an application of an enzymatically anchored pre-targeting molecule P-TCO in cell imaging.

[0007] The final technical problem to be solved by the present invention is to provide a multimodal imaging application of the above-mentioned enzymatic anchored pre-targeting molecule P-TCO in tumor-bearing mice.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: the present invention combines the self-anchoring strategy of specific enzyme response with the pre-targeting strategy of biocompatibility, and designs and synthesizes a small molecule P-TCO containing a targeted ALP recognition group, a self-anchoring scaffold and a bioorthogonal TCO group.

[0009] In order to solve the above-mentioned first technical problem, the present invention discloses a structure as shown in the formula P-TCO:

[0010]

[0011] Wherein, the R group is Any one of: R2 is

[0012] The enzymatic anchored pre-targeting molecule P-TCO comprises the following parts:

[0013] (1) a phosphate (-PO3H2) group for specific enzyme response, wherein the target recognition group is capable of being recognized by a biomacromolecule and interacting with the target recognition group to generate a phenolic hydroxyl group;

[0014] (2) o-(difluoromethyl)-p-hydroxybenzylethylcarbamate for protein anchoring, which is sensitive to light, heat or chemical induction and can cause the p-hydroxybenzylethylcarbamate to break to produce a phenolate anion intermediate. The o-(difluoromethyl)-p-hydroxybenzylethylcarbamate is capable of being hydrolyzed after activation by a trigger, undergoing a 1,4-elimination reaction or a 1,6-elimination reaction and releasing ortho-Quinone methide (o-QM) and para-quinonemethide (p-QM) intermediates;

[0015] (3) Bioorthogonal groups for bioorthogonal reactions, including trans-cyclooctene groups (TCO, ), azide compounds (-N3), alkynyl compounds Cyclooctyne derivatives Tetrazine compounds trans-cyclooctene derivatives wait.

[0016] The present invention further discloses a method for preparing the above-mentioned molecular P-TCO, comprising the following steps:

[0017] Step a: Compound 1 reacts with sodium azide to obtain compound 2;

[0018] Step b: Compound 2 undergoes substitution reaction to obtain compound 3;

[0019] Step c: Compound 3 is subjected to oxidation reaction to obtain compound 4;

[0020] Step d: Compound 4 undergoes addition and substitution reaction to obtain compound 5;

[0021] Step e: Compound 5 is subjected to reduction reaction to obtain compound 6;

[0022] Step f: Compound 6 undergoes substitution reaction to obtain compound 7;

[0023] Step g: Compound 7 undergoes substitution reaction to obtain compound 8;

[0024] Step h: Compound 8 undergoes addition reaction to obtain compound 9;

[0025] Step i: Compound 9 is subjected to a condensation reaction to obtain an enzymatic self-anchored pre-targeting molecule P-TCO;

[0026]

[0027] Specifically, in step a, the reaction is that compound 2-bromo-1-[4-hydroxy-3-(hydroxymethyl)phenyl]ethan-1-one (Compound 1) and sodium azide (NaN3) are dissolved in a solvent and reacted to obtain a reaction solution containing Compound 2.

[0028] Wherein, the molar ratio of the compound 1 to NaN3 is 1:1-1:1.6, preferably 1:1.4.

[0029] Wherein, the concentration of the compound 1 is 0.01-0.08 mmol / mL, preferably 0.04 mmol / mL.

[0030] The solvent includes but is not limited to N,N-dimethylformamide, preferably N,N-dimethylformamide.

[0031] Wherein, the reaction temperature is 20-30°C, preferably room temperature.

[0032] Wherein, the reaction is carried out under stirring.

[0033] The reaction time is more than 12 hours, preferably 12 to 16 hours, and more preferably 16 hours.

[0034] After the reaction is completed, the reaction solution containing compound 2 is extracted, separated and purified by column chromatography, and recrystallized to obtain compound 2.

[0035] Wherein, the extraction solvent includes but is not limited to ethyl acetate (EA), preferably EA.

[0036] Wherein, the column chromatography separation developing solvent is a mixture of petroleum ether (PE) and EA, preferably a mixture of PE and EA in a volume ratio of 1:1.

[0037] In step b, the reaction is to dissolve compound 2, N,N-diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP) in a first solvent and stir at -20°C; then diethyl phosphite and carbon tetrachloride (CCl4) are added dropwise to the above reaction solution, react for a certain time, and transfer the reaction solution to room temperature for reaction to obtain a reaction solution containing compound 3.

[0038] Wherein, the molar ratio of the compound 2, DIPEA and DMAP is 5:10:1-5:12:1, preferably 5:12:1.

[0039] Wherein, the concentration of compound 2 is 0.024-0.241 mmol / mL, preferably 0.121 mmol / mL.

[0040] Wherein, the solvent includes but is not limited to anhydrous acetonitrile, preferably anhydrous acetonitrile.

[0041] Wherein, the reaction temperature is -20 to -10°C, preferably -20°C.

[0042] Wherein, the reaction is carried out under stirring.

[0043] The stirring time in the first step is 5-15 minutes, preferably 10 minutes.

[0044] Wherein, the dropping time is 10-30 minutes, preferably 30 minutes.

[0045] The time for transferring to room temperature is when the dropwise addition of diethyl phosphite and carbon tetrachloride (CCl4) is completed.

[0046] The reaction time to room temperature is 1-4 hours, preferably 2 hours.

[0047] After the reaction is completed, the reaction solution containing compound 3 is extracted, separated and purified by column chromatography, and recrystallized to obtain compound 3.

[0048] Wherein, the extraction solvent includes but is not limited to dichloromethane (DCM), preferably DCM.

[0049] The column chromatography separation developing solvent is a mixture of petroleum ether (PE) and EA, preferably a mixture of PE and EA in a volume ratio of 1:3.

[0050] In step c, the reaction is to dissolve compound 3 and Dess-Martin periodinane (DMP) in a solvent to react to obtain a reaction solution containing compound 4.

[0051] Wherein, the molar ratio of the compound 3 to DMP is 1:1.2-1:1.5, and is preferably 1:1.5.

[0052] Wherein, the concentration of the compound 3 is 0.10-0.30 mmol / mL, preferably 0.23 mmol / mL.

[0053] The solvent includes but is not limited to anhydrous DCM, preferably anhydrous DCM.

[0054] Wherein, the reaction temperature is 20-30°C, preferably 30°C.

[0055] Wherein, the reaction is carried out under stirring.

[0056] Wherein, the reaction time is 1-4 hours, preferably 2 hours.

[0057] After the reaction is completed, the reaction solution containing compound 4 is extracted and distilled under reduced pressure to obtain compound 4.

[0058] Wherein, the extraction solvent includes but is not limited to dichloromethane (DCM), preferably DCM.

[0059] In step d, the reaction is performed by dissolving compound 4 and diethylaminosulfur trifluoride (DAST) in a solvent to obtain a reaction solution containing compound 5.

[0060] Wherein, the molar ratio of compound 4 to DAST is 1:2-1:3, and 1:3 is selected.

[0061] Wherein, the concentration of compound 4 is 0.15-0.30 mmol / mL, preferably 0.25 mmol / mL.

[0062] Wherein, the solvent is redistilled dichloromethane.

[0063] The reaction temperature is below 0°C, preferably -20°C.

[0064] Wherein, the reaction is carried out under stirring.

[0065] Wherein, the reaction time is 2-4 hours, preferably 4 hours.

[0066] Wherein, deionized water needs to be added to quench the reaction at the end of the reaction.

[0067] After the reaction is completed, the reaction solution containing compound 5 is extracted, distilled under reduced pressure, and separated and purified by column chromatography to obtain compound 5.

[0068] Wherein, the extraction solvent includes but is not limited to dichloromethane (DCM), preferably DCM.

[0069] Wherein, the column chromatography separation developing solvent is a mixture of petroleum ether (PE) and EA, preferably a mixture of PE and EA in a volume ratio of 2:5.

[0070] In step e, the reaction is to dissolve compound 5 and sodium borohydride (NaBH4) in a solvent to react to obtain a reaction solution containing compound 6.

[0071] Among them, the molar ratio of compound 5 and NaBH4 is 1:1.5-1:1.8, and 1:1.8 is selected.

[0072] Wherein, the concentration of compound 5 is 0.20-0.27 mmol / mL, preferably 0.27 mmol / mL.

[0073] Wherein, the solvent is methanol.

[0074] The reaction temperature is below 0°C, preferably 0°C.

[0075] Wherein, the reaction is carried out under stirring.

[0076] Wherein, the reaction time is 0.5-1 hour, preferably 1 hour.

[0077] Wherein, at the end of the reaction, a saturated ammonium chloride solution needs to be added to quench the reaction.

[0078] The volume of saturated ammonium chloride solution added after the reaction is completed is 2-10 mL, preferably 5 mL.

[0079] After the reaction is completed, the reaction liquid containing compound 6 is extracted and distilled under reduced pressure to obtain compound 6.

[0080] Wherein, the extraction solvent includes but is not limited to dichloromethane (DCM), preferably DCM.

[0081] In step f, the reaction is performed by dissolving compound 6, ethyl isocyanate, and triethylamine in a solvent to react to obtain a reaction solution containing compound 7.

[0082] The molar ratio of compound 6, ethyl isocyanate and triethylamine is 1:7:10-1:15:20, and the preferred molar ratio is 1:15:15.

[0083] The concentration of compound 6 is 0.13-0.20 mmol / mL, preferably 0.20 mmol / mL.

[0084] Wherein, the solvent is anhydrous dichloromethane.

[0085] Wherein, the reaction is carried out under stirring.

[0086] Wherein, the reaction temperature is 40°C reflux.

[0087] Wherein, the reaction time is 10-30 hours, preferably 24 hours.

[0088] Wherein, at the end of the reaction, a saturated sodium chloride solution needs to be added to quench the reaction.

[0089] Wherein, the volume of saturated sodium chloride solution added after the reaction was completed was 5 mL.

[0090] After the reaction is completed, the reaction solution containing compound 7 is extracted, distilled under reduced pressure, and separated and purified by column chromatography to obtain compound 7.

[0091] Wherein, the extraction solvent includes but is not limited to dichloromethane (DCM), preferably DCM.

[0092] Wherein, the column chromatography separation developing solvent is a mixture of petroleum ether (PE) and EA, preferably a mixture of PE and EA in a volume ratio of 1:20.

[0093] In step g, the reaction is to dissolve compound 7 and trimethylsilyl bromide in a solvent to react to obtain a reaction solution containing compound 8.

[0094] Wherein, the molar ratio of compound 7 to trimethylsilyl bromide is 1:20-1:30, preferably 1:20.

[0095] The concentration of compound 7 is 0.10–0.27 mmol / mL, preferably 0.20 mmol / mL.

[0096] Wherein, the solvent is anhydrous acetonitrile.

[0097] Wherein, the reaction is carried out under stirring.

[0098] The reaction temperature is below 0°C, preferably -10°C.

[0099] Wherein, the reaction time is 10-16 hours, preferably 16 hours.

[0100] After the reaction is completed, the reaction solution containing compound 8 is separated and purified by high performance liquid chromatography to obtain compound 8.

[0101] The liquid elution solvent is a mixture of deionized water (H2O) and acetonitrile (MeCN) containing 1‰ trifluoroacetic acid (TFA), preferably a mixture of H2O and MeCN in a volume ratio of 3:7.

[0102] In step h, the reaction is performed by dissolving compound 8, propargylamine, tris(3-hydroxypropyltriazolemethyl)amine, copper sulfate, and sodium ascorbate in a solvent to obtain a reaction solution containing compound 9.

[0103] The molar ratio of compound 8, propargylamine, tris(3-hydroxypropyltriazolemethyl)amine, copper sulfate and sodium ascorbate is 1:1.2:0.2:0.2:0.2.

[0104] Wherein, the concentration of compound 8 is 0.05-0.12 mmol / mL, preferably 0.05 mmol / mL.

[0105] Wherein, the solvent is a mixture of water and DMF.

[0106] Wherein, the volume ratio of water and DMF in the solution is 10:1.

[0107] Wherein, the reaction is carried out under stirring.

[0108] Wherein, the reaction temperature is room temperature.

[0109] Wherein, the reaction time is 0.5-1 hour.

[0110] After the reaction is completed, the reaction solution containing compound 9 is separated and purified by high performance liquid chromatography and freeze-dried to obtain compound 9.

[0111] The liquid elution solvent is a mixture of deionized water (H2O) and acetonitrile (MeCN) containing 1‰ trifluoroacetic acid (TFA), preferably a mixture of H2O and MeCN in a volume ratio of 1:3.

[0112] In step i, the reaction is to dissolve compound 9, the activated ester-modified bioorthogonal compound, and DIPEA in a solvent to react to obtain a reaction solution containing compound P-TCO.

[0113] Wherein, the compound 9 is an activated ester-modified bioorthogonal compound, and the molar ratio of DIPEA is 1:1.2:5.

[0114] The concentration of compound 9 is 0.05-0.10 mmol / mL, preferably 0.05 mmol / mL.

[0115] Wherein, the activated ester-modified bioorthogonal compound is preferably (4E)-trans-cyclooctene active ester.

[0116] Wherein, the solvent is a mixed solution of anhydrous DMF.

[0117] Wherein, the reaction is carried out under stirring.

[0118] Wherein, the reaction temperature is room temperature.

[0119] Wherein, the reaction time is 0.5 hours.

[0120] After the reaction is completed, the reaction solution containing the compound P-TCO is separated and purified by high performance liquid chromatography and freeze-dried to obtain the compound P-TCO.

[0121] The liquid elution solvent is a mixture of deionized water (H2O) and acetonitrile (MeCN) containing 1‰ trifluoroacetic acid (TFA), preferably a mixture of H2O and MeCN in a volume ratio of 2:3.

[0122] In order to solve the second technical problem mentioned above, the present invention discloses the application of the compound P-TCO in protein labeling, especially cell membrane protein labeling.

[0123] Specifically, the enzymatic anchoring pre-targeting molecule P-TCO realizes covalent labeling of proteins under the action of alkaline phosphatase (ALP).

[0124] Furthermore, under the action of ALP, P-TCO realizes the covalent labeling application of albumin (BSA).

[0125] In order to solve the third technical problem mentioned above, the present invention discloses the application of an enzymatically anchored pre-targeting molecule P-TCO in cell imaging.

[0126] The enzymatically anchored pre-targeting molecule P-TCO is combined with a tetrazine probe and used in cell imaging of multimodal imaging at the cellular level.

[0127] Furthermore, the present application discloses the application of the enzymatic self-anchoring pre-targeting molecule P-TCO in multimodal imaging in vivo, wherein the P-TCO is combined with a tetrazine probe to achieve multimodal imaging in vivo.

[0128] The tetrazine probe comprises a tetrazine bioorthogonal group and an imaging reporter molecule, wherein the tetrazine bioorthogonal group is The imaging reporter molecules include small molecules, proteins or nanoparticles used for positron emission tomography, fluorescence imaging, bioluminescence or magnetic resonance imaging.

[0129] In some specific embodiments, the tetrazine probe is a fluorescent small molecule probe FITC-Tz, a fluorescent small molecule probe IR780-ZW-Tz, a positron emission tomography small molecule probe [ 68 Ga]-Tz, bioluminescent protein probe GFP-Tz, fluorescent protein probe BSA-Cy5-Tz, bioluminescent enzyme protein probe RLuc-Tz and fluorescence / magnetic resonance nanoprobe GdNPs-Tz, wherein the tetrazine probe has the following structural formula:

[0130]

[0131] Among them, Rluc represents Rluc8.6-535.

[0132] When used for multimodal imaging at the cellular level, the tetrazine probe is any one of FITC-Tz, GFP-Tz, BSA-Cy5-Tz, and GdNPs-Tz.

[0133] The enzymatic anchored pre-targeting molecule P-TCO forms a covalent bond with the protein under the action of a specific enzyme, and is then combined with a tetrazine probe for application in cell imaging.

[0134] Wherein, the enzyme is alkaline phosphatase (ALP), preferably endogenous ALP on the surface of human cervical cancer HeLa cell membrane.

[0135] The molar concentration of the enzymatic self-anchoring pre-targeting molecule P-TCO in cell imaging is 0.5-10 μmol / L, and the experimental time is 5-360 minutes. Preferably, the most suitable molar concentration of the enzymatic self-anchoring pre-targeting molecule P-TCO in cell imaging is 10 μmol / L, and the experimental time is 30 minutes.

[0136] The molar concentration ratio of the enzymatic self-anchoring pre-targeting molecule P-TCO to the tetrazine probe, which is any one of FITC-Tz, GFP-Tz, BSA-Cy5-Tz, and GdNPs-Tz, is 10:1-1:1, preferably 1:1.

[0137] In some specific embodiments, the present application discloses the multimodal imaging application of the enzymatically anchored pre-targeting molecule P-TCO in tumor-bearing mice.

[0138] The enzymatic anchored pre-targeting molecule P-TCO is combined with a tetrazine probe for multimodal imaging applications in tumor-bearing mice. Specifically, P-TCO is injected into the tail vein in advance, and then the tetrazine probes FITC-Tz, IR780-ZW-Tz, [ 68Any one of [Ga]-Tz, GFP-Tz, BSA-Cy5-Tz, RLuc-Tz, and GdNPs-Tz was used for in vivo imaging.

[0139] Wherein, the enzyme is alkaline phosphatase (ALP), preferably endogenous ALP enzyme highly expressed in HeLa tumor-bearing mice.

[0140] The concentration of the enzymatic self-anchoring pre-targeting molecule P-TCO in in vivo imaging of tumor-bearing mice ranged from 200 to 600 μmol / L, and the experimental duration was 30 to 240 minutes. The optimal molar concentration of the enzymatic self-anchoring pre-targeting molecule P-TCO for in vivo imaging of tumor-bearing mice was 400 μmol / L, with a tail vein injection volume of 200 μL, resulting in an optimal molar amount of 80 nmol, and the experimental duration was 120 minutes.

[0141] Wherein, the enzymatic self-anchored pre-targeting molecule P-TCO and the tetrazine probe are IR780-ZW-Tz, BSA-Cy5-Tz, [ 68 The molar concentration ratio of any one of Ga]-Tz and GdNPs-Tz is 40:1-10:1, and the preferred molar concentration ratio is 20:1.

[0142] Wherein, the enzymatic self-anchored pre-targeting molecule P-TCO and the tetrazine probe are IR780-ZW-Tz, BSA-Cy5-Tz, [ 68 The intravenous injection volume of either Ga]-Tz or GdNPs-Tz was 200 μL.

[0143] Wherein, when the tetrazine probe is a compound [ 68 When PET probe [Ga]-Tz and IR780-ZW-Tz are used together, 68 When the radioactive dose of Ga]-Tz is 7.4 MBq, i.e., a molar amount of 2 nmol, and the molar concentration of the fluorescent probe IR780-ZW-Tz is 20 μmol / L, i.e., a molar amount of 4 nmol, the molar ratio of the two is 1:2. Preferably, the molar ratio of P-TCO to the mixture of the two is 80:3, which can be used for in vivo PET and fluorescence synergistic imaging of tumors.

[0144] Beneficial effects: Compared with the existing technology, the present invention provides an enzymatic self-anchoring pre-targeting molecule P-TCO, which can quickly respond to the catalytic action of ALP on the tumor cell membrane and efficiently form a covalent bond with the tumor cell membrane protein. At the same time, P-TCO can efficiently combine with tetrazine-based imaging reporter molecules of different sizes and imaging reporter groups through bioorthogonal reactions, achieving precise enrichment of reporter molecules of different sizes and imaging modes on the tumor cell membrane, with a high labeling efficiency of 4.06×10 per cell.7 TCOs, thus providing a powerful multimodal imaging tool for in vivo tumor diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 Schematic diagram of the enzymatically self-anchored pretargeting molecule P-TCO for tumor pretargeting multimodal imaging, including: (a) the chemical structure of P-TCO and its application in E-SIM for labeling TCO groups on tumor cell membranes; (b) a flow chart of the bioorthogonal (IEDDA) reaction between TCO and Tz groups, the covalent labeling of membrane proteins with the enzymatically self-anchored pretargeting molecule P-TCO, and the subsequent IEDDA reaction to achieve two-step tumor pretargeting for fluorescence (FL), magnetic resonance imaging (MRI), positron emission tomography (PET), and bioluminescence (BL) imaging;

[0146] Figure 2 Specific response and enzymatic cleavage rate of P-TCO to alkaline phosphatase (ALP), including: (a) High-performance liquid chromatography (HPLC) analysis (UV = 214 nm) of P-TCO, P-TCO incubated with ALP, and P-TCO incubated with ALP and its inhibitor Na3VO4; (b) High-resolution mass spectrometry (HRMS) analysis of peak A in (a) (positive and negative ion modes); (c) H-ionization of different concentrations of P-TCO (1, 2, 5, 10, 20, and 40 μmol / L) in Tris buffer. PLC graph (UV = 214 nm), where the injection volume for each concentration was maintained at 1 mL; (d) Relationship between HPLC peak area and P-TCO concentration; (e) High-performance liquid chromatography (HPLC) graph (UV = 214 nm) after incubation of different concentrations of P-TCO (5, 7.5, 10, 20, and 30 μmol / L) with alkaline phosphatase (ALP, 1 U / L ≈ 0.0001 mg / mL) in Tris buffer for 1 minute; (f) Enzyme kinetics of P-TCO against ALP;

[0147] Figure 3. Schematic diagram of two-step protein labeling using P-TCO combined with tetrazine probe, including: (a) Schematic diagram of two-step labeling of ALP and bovine serum albumin (BSA) using P-TCO and FITC-Tz; (bc) Coomassie brilliant blue (CB) staining and in-gel fluorescence (FL) analysis of ALP or BSA labeled with P-TCO and FITC-Tz; (d) labeling efficiency of BSA with P-TCO; (e) quadrupole time-of-flight (QTOF) mass spectra of BSA and TCO-labeled BSA (BSA-TCO), respectively. The molecular weights of single TCO labeling (peak 2) and double TCO labeling (peak 3) are 66,825 Da and 67,221 Da respectively; (f) Coomassie brilliant blue (CB) staining and in-gel fluorescence (FL) analysis of different concentrations of BSA labeled with P-TCO and FITC-Tz; (gh) analysis of the enzymatic catalytic activity of ALP enzyme after P-TCO labeling; (i) protein BSA-GFP coupling experiment of P-TCO combined with GFP-Tz; (j) dynamic light scattering (DLS) analysis of GdNPs-Tz and BSA-GdNPs conjugates. BSA (1.0 mg / mL) and P-TCO (10 μmol / L) were incubated for 30 min and then incubated with GdNPs-Tz (10 μmol / L) for 60 min to form BSA-GdNPs with an average size of approximately 69.39 nm for GdNPs-Tz and 238.07 nm for BSA-GdNPs. (k) Fluorescence size exclusion chromatography (SEC) analysis of BSA-GFP (left) and BSA-GdNPs (right), both generated under the conditions described in detail in g and h.

[0148] Figure 4P-TCO combined with tetrazine probes for multimodal imaging of tumor cell membranes, including: (a) Western blot analysis of ALP expression in different tumor cells; (b) cytotoxicity analysis of HeLa cells with P-TCO; (c) Schematic diagram and fluorescence imaging of HeLa, NIH3T3, and U87MG cells labeled with P-TCO, followed by IEDDA reaction with FITC-Tz; (d) Schematic diagram and fluorescence imaging of HeLa cells labeled with P-TCO, followed by IEDDA reaction with FITC-Tz, with or without incubation with P-TCO (10 μmol / L, 30 minutes) and then with FITC-Tz (10 μmol / L, 30 minutes). Incubation, the inhibitor group was pretreated with Na3VO4 (1 mM, 30 minutes); (ef) Flow cytometry (FACS) analysis and quantification of the fluorescence intensity of HeLa and NIH3T3 cells incubated with or without P-TCO (10 μmol / L, 30 minutes) and then incubated with FITC-Tz (10 μmol / L, 30 minutes). Data are expressed as mean ± SD (n = 3 independent cell samples, two-tailed t-test); (g) Fluorescence imaging of co-cultured HeLa and NIH3T3 cells. HeLa cells (pre-treated with Hoechst 33342 staining) and NIH3T3 cells were incubated with P-TCO (10 μmol / L, 30 minutes) and then incubated with FITC-Tz (10 μmol / L, 30 minutes); (h) Coomassie brilliant blue and fluorescent in-gel analysis of membrane proteins and cytoplasmic proteins extracted from HeLa cells fluorescently labeled with (P-TCO+FITC-Tz); (i) HeLa cells were incubated with P-TCO (10 μM, 30 minutes) and then incubated with G Fluorescence imaging of HeLa cells incubated with FP-Tz (1 mg / mL) or BSA-Cy5-Tz (1 mg / mL) for 30 minutes; (j) Dual-modal fluorescence (FL) and T1-weighted magnetic resonance (MR) imaging and quantitative analysis of HeLa cells incubated with P-TCO (10 μM, 30 minutes) and GdNPs-Tz (10 μM, 30 minutes); (k) Orthogonal fluorescence imaging of HeLa cells labeled with E-SIM and metabolic sugars;

[0149] Figure 5.P-TCO combined with IR780-ZW-Tz is used for in vivo pre-targeted tumor fluorescence imaging, wherein, (a) P-TCO and IR780-ZW-Tz are used for pre-targeted near-infrared fluorescence (NIR-FL) imaging of HeLa tumors, and mice bearing HeLa tumors are intravenously injected with P-TCO (preferably 400 μmol / L) or not, and preferably injected with IR780-ZW-Tz (20 μmol / L) 2 hours later. Fluorescence images were acquired before (Pre) and at 0.5, 2, 4, 8, and 24 h after IR780-ZW-Tz injection; (b) normalized fluorescence intensity of tumor (T) and background (B), and (c) tumor-to-background ratio (TBR) over time, comparing mice treated with P-TCO + IR780-ZW-Tz (P-TCO+) versus IR780-ZW-Tz alone (P-TCO-), data are presented as mean ± SD (n = 5 P-TCO+ animals, n = 3 P-TCO- animals; one-way analysis of variance (ANOVA) and post hoc Tukey test for normalized fluorescence intensity at 0.5 and 24 h; two-tailed t-test for TBR); (de) fluorescence imaging of ex vivo tissues of tumors and major organs; (f) Coomassie brilliant blue (CB) and fluorescent in-gel analysis of labeled tumors. Mice were intravenously injected with P-TCO (400 μmol / L) or not, and 2 hours later were injected with IR780-ZW-Tz (20 μmol / L). 4 hours later, the tumors were excised and lysed; (g) shows the normalized fluorescence intensity analysis of the labeled tumors.

[0150] Figure 6 P-TCO combined with BSA-Cy5-Tz for in vivo pretargeted tumor fluorescence imaging. (a) Pretargeted near-infrared fluorescence imaging of HeLa tumors using P-TCO and BSA-Cy5-Tz. (bc) Normalized fluorescence intensity and TBR quantitative analysis of tumors in groups I-III. Data are expressed as mean ± SD (n = 3 animals; two-tailed t-test for normalized fluorescence intensity; one-way analysis of variance (ANOVA) with post hoc Tukey test for TBR).

[0151] Figure 7 P-TCO combined with GdNPs-Tz for in vivo pretargeted tumor fluorescence / magnetic resonance dual-modality imaging, wherein: (a) Pretargeted dual-modality fluorescence and T1-weighted magnetic resonance imaging of HeLa tumors using P-TCO and GdNPs-Tz. The white dots in the magnetic resonance images are the internal standard: Dotarem (1 mM); (bc) Normalized fluorescence intensity and percentage signal enhancement (%SE) of HeLa tumors. Data are expressed as mean ± standard deviation (n = 3 animals, two-tailed t-test);

[0152] Figure 8 .P-TCO combined with RLuc-Tz for in vivo pre-targeted tumor bioluminescence imaging, including: (a) Schematic diagram of covalent labeling of P-TCO and RLuc-Tz for bioluminescence imaging; (b) Comparative analysis of the bioluminescence efficiency of RLuc and Tz-modified RLuc-Tz; (c) Bioluminescence imaging of HeLa and NIH3T3 cells after treatment with different dosing groups, including blank, RLuc-Tz, P-TCO, P-TCO+RLuc-Tz, RLuc, and P-TCO+Rluc; (d) Quantitative analysis of the bioluminescence intensity of HeLa and NIH3T3 cells after the specified treatments in Figure c; (e) Pre-targeted bioluminescence imaging of HeLa tumors; (f) Quantitative analysis of the bioluminescence intensity of normalized tumor (T) and background (B) at different time points; (g) Quantitative analysis of tumor-to-background ratio (TBR).

[0153] Figure 9 .P-TCO combination[ 68 Ga]-Tz / IR780-ZW-Tz was used for in vivo pre-targeted tumor PET / fluorescence dual-modality imaging, where (a) [ 68 Ga]-Tz radiosynthesis; (b) [ 68 Analysis of radioactive stability of Ga]-Tz in PBS buffer; (cd) PET images and quantitative analysis of HeLa cells after treatment with different drugs, data are expressed as mean ± SD (n = 3 independent cell samples, two-tailed t test); (e) Using P-TCO, [ 68 Experimental steps for pretargeted PET and FL imaging of HeLa tumors using P-TCO and [ 68 Representative coronal and axial PET images of HeLa tumors treated with [Ga]-Tz (attenuation-corrected); boxes indicate magnified areas, circles indicate tumors; (gh) Quantified mean radioactivity uptake (ID% / g) in HeLa tumors based on attenuation correction and quantitative analysis of tumor-to-background ratio (TBR) in PET imaging; (i) Graph showing near-infrared fluorescence imaging of mice using IR780-ZW-Tz 24 hours after PET imaging; mice from groups A and B that underwent PET imaging were injected with IR780-ZW-Tz (20 μmol / L) via the tail vein 24 hours after injection; fluorescence images before (Pre) and 2 hours after injection; (jk) Normalized fluorescence intensity of tumors and quantitative analysis of TBR. Data are expressed as mean ± SD (n = 3 animals, two-tailed t-test). DETAILED DESCRIPTION

[0154] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0155] Unless otherwise specified, the "%" in the following examples refers to molar percentage.

[0156] Reagents and Instruments: All chemical reagents and solvents were purchased from Shanghai Bidex Pharmaceuticals, McLean, and Admas Ltd. Cy5-Tz was purchased from Xi'an Kangfuno Biotechnology Co., Ltd. Analytical solvents and reagents were chromatographically pure, and conventional reagents were all analytically pure and were not further purified. Alkaline phosphatase (ALP, ~10 DEA units / mg solid) was obtained from bovine intestinal mucosa. D Fluorescein potassium salt was purchased from Sigma-Aldrich. Biotechnology-grade bovine serum albumin (BSA, LOT#3015C493) was purchased from Amresco-Inc. Coelenterazine (CTZ) was purchased from Yuanye Biotechnology Co., Ltd. High-glucose Dulbecco's modified Eagle's medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit, BCA protein quantification kit, Hoechst 33342 stain, and phosphate-buffered saline (PBS) were all purchased from Nanjing Keygen Biotechnology Co., Ltd. InstaBlue protein staining solution (Cat. No. B8226) for Coomassie brilliant blue staining was purchased from APExBIO. The molecular weights of the compounds were determined using an electrospray quadrupole-orbitrap high-resolution mass spectrometer (Q Exactive) equipped with a Vanquish Flex binary ultra-high performance liquid chromatograph (Thermo Fisher, Germany). Protein molecular weight was determined using an ultrafleXtreme with a 1kHz smartbeam-II laser. TM Matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF / TOF, Bruker Daltonics) and ACQUITY ProteinBEH C4 was analyzed by ultra-high performance liquid chromatography-high resolution quadrupole time-of-flight mass spectrometry (Waters Xevo G2-QTOF, USA). 1 H NMR and 13C NMR (NMR) was analyzed using a Bruker 400 MHz or Avance 600 NMR spectrometer (Bruker, Germany). Column chromatography was performed using 40-63 μm silica gel. Analytical high-performance liquid chromatography (HPLC) and semi-preparative HPLC were performed using a Thermo Scientific Dionex Ultimate 3000 chromatograph, using HPLC-grade CH3CN / H2O (containing 1‰ CF3COOH) as the eluent. Radio-HPLC detection was performed using a pump (Waters, USA) equipped with a dual-wavelength UV absorption detector and a radioactivity detector. All UV-visible spectra (UV-Vis) were measured using an Ocean Optics Maya 2000 Pro spectrometer. Fluorescence spectra were measured using a HORIBA Jobin Yvon Fluoromax-4 fluorescence spectrophotometer in 1 cm quartz cuvettes. Dynamic light scattering (DLS) analysis was performed using a Brook Haven 90 Plus / BI-MAS instrument (USA). Transmission electron microscopy (TEM) images were taken using a high-throughput transmission electron microscope (JEM-2800, JEOL, Japan). The MTT assay was performed on a microplate reader (Tcan) with an optical density (OD) of 490 nm. SDS-PAGE analysis was performed according to standard gel electrophoresis procedures, and gel images were obtained using a fluorescent ChemiDoc. TM MP imager (Bio-Rad) analysis was performed. Magnetic resonance (MR) relaxivities were measured on an MR scanner (0.5T, NMI20-015 VI, NIUMAG). All fluorescence and brightfield (BF) images of cells and tissue sections were acquired using a Leica SP8 STED 3X confocal laser scanning microscope or an Olympus IX73 fluorescence inverted microscope. Cell fluorescence intensity was measured using a FACS Calibur (Beckman Coulter) flow cytometer and quantitatively analyzed using CytExpert software. In vitro bioluminescence counts were performed using a microplate reader (Tcan) with BL scanning capability. In vivo MR imaging was performed using a small animal MR scanner (1.0T, Bruker ICON, Germany). Dynamic and static PET images were acquired using a dedicated Inveon small animal whole-body microPET scanner (Siemens, Germany). Radioactive cellular uptake was detected using a 2470 Wizard gamma radioimmunoassay counter (γ counter, Perkin Elmer, USA). All in vitro or in vivo fluorescence and bioluminescence images were recorded by the IVIS Lumina XR III system, and region of interest (ROI) measurements of fluorescence intensity or bioluminescence radiation intensity were processed by Living Image software (PerkinElmer).

[0157] This application proposes an enzyme-triggered membrane protein self-anchoring compound (P-TCO), which can rapidly and selectively modify tumor cell membranes in vivo, giving them bioorthogonal functions. The P-TCO compound structure includes an alkaline phosphatase (ALP) response group and a trans-cyclooctene (TCO) functional group. It reacts with ALP highly expressed on the surface of tumor cell membranes to dephosphorylate ALP, covalently labeling ALP and nearby membrane proteins, allowing the TCO group to be efficiently anchored on the cell membrane surface. At the same time, a class of tetrazine (Tz)-modified multimodal probe molecules was developed, including small molecule probes FITC-Tz, [ 68 Ga]-Tz, IR780-ZW-Tz; protein probes GFP-Tz, BSA-Cy5-Tz, RLuc-Tz; and nanoprobes GdNPs-Tz. P-TCO molecules have high specificity and good biocompatibility, do not affect the physiological properties of cell membranes, and can achieve high labeling efficiency. By utilizing TCO groups anchored to tumor cell membranes, a platform for efficient targeted delivery of multimodal imaging probes is established, simplifying the design and synthesis steps of probes, generating enhanced multimodal imaging signals, and improving the accuracy of tumor diagnosis.

[0158] Under the action of ALP, P-TCO is converted into active o-QM and p-QM intermediates. These intermediates provide multiple nucleophilic attack sites on the tumor cell membrane through Michael addition, thereby improving labeling efficiency. Ultimately, the tumor cell membrane is rapidly modified with a high density of TCO groups, enabling efficient IEDDA reactions with various Tz-containing imaging probes (R-Tz). This two-step process significantly immobilizes and enriches reporter molecules on tumor cells, enabling in vivo multimodal imaging of FL, MRI, BL, and PET.

[0159] like Figure 1 The enzymatic self-anchoring pre-targeting strategy described herein utilizes an enzymatic self-anchoring pre-targeting molecule P-TCO. Figure 1 Under the action of ALP enzyme of a, an intermediate containing phenolic acid anion can be formed, which can form active ortho-QM and para-QM electrophiles through 1,4- and 1,6-elimination. These can provide additional reaction sites for subsequent Michael addition reactions, thereby improving the labeling efficiency of protein residues (such as thiols and amines) on the cell membrane. This process is called E-SIM (Enzyme-triggered Self-Immobilization). E-SIM utilizes an alkaline phosphatase (ALP)-responsive quinone methyl (QM) precursor P-TCO, which carries a TCO group and quickly connects high-density TCO groups to the tumor cell membrane through ortho-labeling. These TCO groups are then rapidly bio-orthogonal reacted ( Figure 1b), enabling it to undergo efficient IEDDA reactions with various Tz-containing imaging probes (R-Tz). This two-step process significantly fixed and enriched the reporter molecules on tumor cells, thereby achieving in vivo FL, MRI, BL and PET multimodal imaging. This application further verified the effectiveness of E-SIM labeling and bioorthogonal reactions in tumor pre-targeting multimodal imaging. In particular, selective and efficient covalent attachment of Tz-modified Renilla luciferase to the cell membrane was successfully achieved in vivo, providing a highly sensitive bioluminescent signal for detecting and guiding the surgical resection of small HepG2 human liver cancer peritoneal metastases. The enzymatic self-anchoring pre-targeting molecule P-TCO is a powerful tool ( Figure 1 b) It can achieve precise labeling of tumor cells in complex in vivo environments and can be used for pre-targeted enrichment of multiple imaging reporter molecules in tumors, facilitating multimodal imaging applications.

[0160] The present invention is described in detail below through specific embodiments.

[0161] Example 1: Design, synthesis and characterization of compounds.

[0162] Path S1 shows the structural design and synthesis method of P-TCO.

[0163]

[0164] Reaction conditions: 2-Bromo-1-[4-hydroxy-3-(hydroxymethyl)phenyl]ethanone (Compound 1) was purchased from Shanghai Bid Pharmaceutical Co., Ltd. Compounds 2-8 were synthesized according to previous research methods (Chem. Eur. J. 2019, 25, 13994).

[0165] Synthesis of Compound 9: Compound 8 (24.0 mg, 0.063 mmol), 3-aminopropyne (4.8 μL, 0.075 mmol), CuSO (2.1 mg, 0.012 mmol), and sodium L-(+)-ascorbate (2.4 mg, 0.012 mmol) were mixed in DMSO (600 μL) and stirred at room temperature for 0.5 hour. The mixture was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column to yield the title compound as a white powder (31.0 mg, 90%). 1H NMR (600MHz, DMSO-d6) δ8.40(s,3H),8.05(s,1H),7.59–7.50(m,2H),7.39(d,J=8.7Hz,1H),7.32(d,J=8.7Hz,1H),7.2 3–7.03(m,1H),5.99(d,J=4.5Hz,1H),4.85–4.71(m,2H),4.08–4.01(m,2H),2.97–2.90(m,2H),0.95(t,J=7.2Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ155.03,151.21,139.42,133.61,130.88,125.48,123.97,121.6 1,114.65,112.17,110.62,72.78,54.11,35.48,34.29,15.22.HRMS(ESI)m / zcalcd.for C 15 H 21 F2N5O6P[M+H] + :436.1192,found HRMS:m / z 436.1192.

[0166] Synthesis of compound P-TCO: Compound 9 (20.0 mg, 0.036 mmol), (4E)-TCO-NHS ester (11.7 mg, 0.044 mmol), and DIPEA (25 μL, 0.180 mmol) were mixed in DMF (800 μL) and stirred at room temperature for 0.5 h. The product was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column to yield P-TCO as a white powder (18.5 mg, 87%). 1 H NMR (400MHz, DMSO-d6) δ7.92(d,J=2.3Hz,1H),7.59(s,1H),7.51(d,J=8.8Hz,1 H),7.46–7.38(m,2H),7.24–6.92(m,2H),6.05–5.97(m,1H),5.64–5.55(m,1H) ,5.46–5.40(m,1H),4.81–4.66(m,2H),4.24–4.14(m,2H),2.95–2.89(m,2H),2 .35–2.19(m,4H),1.94–1.85(m,4H),1.59–1.55(m,3H),0.95(t,J=7.2Hz,3H). 13C NMR(151MHz,DMSO-d6)δ156.22,154.99,145.82,135.34,135.34,134.59,132.93,132.93,130.51,124.4 3,123.57,121.25,111.91,79.75,72.84,53.86,41.08,38.56,36.23,35.46,34.15,32.58,31.01,15.23. 19 F NMR(376MHz,DMSO)δ-113.30,-113.46.HRMS(ESI)m / z calcd.for C 24 H 31 F2N5O8P - [MH] - :586.1884; found HRMS:m / z 586.1896.

[0167] Example 2: Design, synthesis and characterization of tetrazine imaging molecules.

[0168] Paths S2-S9 show FITC-Tz, IR780-ZW-Tz, [ 68 Structural design and synthesis methods of [Ga]-Tz, GFP-Tz, BSA-Cy5-Tz, RLuc-Tz and GdNPs-Tz.

[0169] (1) Synthesis of FITC-Tz:

[0170]

[0171] Reaction conditions: (a) Mix fluorescein 5-isothiocyanate (19 mg, 0.05 mmol), 3-(4-benzylamino)-1,2,4,5-tetrazine (10 mg, 0.05 mmol), and DIPEA (28 μL, 30 mmol), dissolve in DMF (3 mL), and stir at room temperature for 4 hours. The reaction mixture was purified by semi-preparative HPLC and lyophilized to obtain FITC-Tz as an orange solid (27.4 mg, yield: 79.3%). HRMS (ESI) m / z calcd. for C 30 H 20 N6O5S[M+H] + :577.1216; found HRMS:m / z577.1263.

[0172] (2) Synthesis of IR780-ZW-Tz:

[0173]

[0174] Reaction conditions: (a) CuSO4, sodium L-(+)-ascorbate, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), reaction at room temperature for 1 hour. (b) NaN3, stirring at room temperature for 10 minutes; propargyl acetic acid, CuSO4, sodium L-(+)-ascorbate, reaction at room temperature for 30 minutes. (c) EDC, NHS, DIPEA, 3-(4-benzylamino)-1,2,4,5-tetrazine, reaction at room temperature for 4 hours. After lyophilization, IR780-ZW-Tz was obtained as a green solid (15.0 mg, yield: 47.6%). HRMS (ESI) m / z calcd. for C 68 H 89 N 18 O7S2 + [M] + :1333.6598,found HRMS:m / z1333.7467.

[0175] (3)[ 68 Synthesis of Ga]-Tz labeling precursor NODA-Tz:

[0176]

[0177] Reaction conditions: (a) HBTU, DIPEA, DCM, reaction time 6 h. (b) 95% TFA, 1% TIPS, 4% anhydrous DCM, reaction time 4 h. (c) DMF, DIPEA, reaction time 3 h.

[0178] Synthesis of compound NODA-Tz: 2-(2-(2-aminoethoxy)ethoxy)acetic acid (Compound 10) was purchased from Xi'an Kangfuno Biotechnology Co., Ltd. Compound 11 was synthesized according to the literature method (Chem. Sci., 2015, 6, 6256-6261). Compound 10 (130.0 mg, 0.45 mmol), 11 (214 mg, 0.40 mmol) and HBTU (160 mg, 0.42 mmol) were dissolved in DCM (5 mL), and DIPEA (200 μL) was added to adjust the pH to 8-9. After stirring at room temperature for 2-3 hours, the mixture was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using a C18 column to obtain compound 12 as a colorless oil (73 mg, 37%). Subsequently, compound 12 (38 mg, 0.04 mmol) was reacted in 95% TFA and 1% triisopropylsilane (TIPS) in 4% DCM for 4 hours to remove the tert-butyl group. After removing the solvent, the crude product 13 was precipitated by adding n-hexane. The precipitate was dried under vacuum and dissolved in anhydrous DMSO (0.5 mL). DIPEA (5 μL) was added, followed by methyl tetrazine-NHS ester (14 mg, 0.04 mmol) and DIPEA (20 μL) in anhydrous DMF (2 mL). After stirring at room temperature for 3 hours, the product was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column to obtain NODA-Tz (or Tz-NODA) as a pink oil (10 mg, 60%). 1 H NMR (400MHz, DMSO-d6) δ8.40(d,J=8.4Hz,2H),8.27–8.21(m,1H),7.90–7.84(m,1H),7.54(d,J=8.4Hz,2H),3.90(d,J=6.2Hz,2H),3.76–3.6 9(m,4H),3.57(s,3H),3.51(d,J=1.2Hz,9H),3.45–3.37(m,6H),3.23(d,J=5.7Hz,2H),2.99(s,12H),2.23–2.17(m,2H),1.98–1.75(m,2H). 13CNMR(101MHz,DMSO-d6)δ173.45,171.91,171.25,169.89,169.69,167.05 ,163.27,141.26,130.06,130.02,127.30,79.28,78.96,78.63,69.76,69. 74,69.61,69.57,69.12,69.06,62.94,54.52,54.22,50.70,49.97,49.38 ,48.03,46.65,42.20,38.53,31.93,25.15,24.46,20.81.HRMS:calcd.for C 34 H 51 N9O 11 [M+H] + :762.3708; found HRMS:m / z 762.3771.

[0179] (4)[ 68 Radiosynthesis of [Ga]-Tz:

[0180]

[0181]

[0182] Reaction conditions: Radioisotope [ 68 Ga] 3+ By using HCl (0.05 mol / L, 5 mL) from 68 Ge / 68 Elute from the Ga generator. 68 The volume of GaCl3 was 0.5, 0.5, 0.5, 1.4, 0.7 and 1.4 mL of each fraction. The intermediate fraction (1.4 mL) contained more than 90% of the radioactivity and was transferred to a hot chamber in a lead tank after radioactivity dose detection. After adjusting the pH of the reaction system to 4.0 with sodium acetate buffer (0.25 mol / L, 340 μL), NODA-Tz (20 μg) was added and the reaction was incubated at 37°C for 15 minutes, shaking once every 5 minutes. A small amount of the final solution was taken for HPLC analysis and then used directly after further purification by a C18 light Sep-Pak column (Waters, USA).

[0183] (5) Synthesis of GFP-Tz:

[0184]

[0185] Reaction conditions: GFP (~27 kDa, 1 mg / mL) and tetrazine activated ester (Tz-NHS, 10 equivalents) were mixed in Tris buffer (1 mL) and shaken at 4°C for 8 hours, followed by ultrafiltration (10 kDa molecular weight cutoff, MWCO) to remove unreacted Tz-NHS. The protein concentration of GFP-Tz was determined using the BCA protein assay. The molecular weight was characterized by QTOF-MS analysis. Subsequently, the presence of tetrazine in GFP-Tz was further verified using gel fluorescence analysis by IEDDA reaction with Cy5-TCO. The gel was analyzed using a fluorescence imager (excitation wavelength: 480 nm, emission wavelength: 530 ± 15 nm) and then stained with InstaBlue protein dye solution.

[0186] (6) Synthesis of BSA-Cy5-Tz:

[0187]

[0188] Reaction conditions: BSA (~66.5 kDa, 1 mg / mL) and tetrazine activated ester (Tz-NHS, 10 equivalents) were mixed in Tris buffer (1 mL) and incubated on a shaker at 4°C overnight. Unreacted Tz-NHS was subsequently removed by ultrafiltration (10 kDa molecular weight cutoff, MWCO). The concentration of BSA-Tz was determined using the BCA protein assay, and its molecular weight was characterized by QTOF-MS analysis. Next, BSA-Tz (1 mmol / L, 1 mL) and Cy5-TCO (2.5 mM, 2.5 equivalents) were added to PBS buffer (pH 7.4, 1×) and incubated at 4°C with shaking for 60 minutes. Unreacted Cy5-TCO was then removed by ultrafiltration (10 kDa molecular weight cutoff, MWCO). The residue was washed three times with PBS, concentrated, and the protein concentration was determined by the BCA protein assay. BSA-Cy5-Tz was characterized by MALDI-TOF-MS and gel fluorescence analysis.

[0189] (7) Synthesis of RLuc-Tz:

[0190]

[0191] Reaction Conditions: RLuc8.6-535 was prepared as described in the literature (Nat Methods 2007, 4, 641-643). RLuc8.6-535 was cloned into the pBAD vector with a His-tag added to the C-terminus, and the manufacturer's protocol was followed. The recombinant pBAD-RLuc8.6-535 plasmid was transformed into Escherichia coli LMG194 and cultured in Luria-Bertani (LB) medium containing 100 μg / mL ampicillin at 37°C. Cells were then induced with 0.2% L-(+)-arabinose overnight at 32°C. Cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, pH 8.0, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF)). The cell suspension was lysed using an ultrasonic cell disruptor, and cell debris was removed by centrifugation at 4°C. The resulting supernatant was incubated with Ni-NTA agarose beads and subsequently washed sequentially with wash buffer containing 5 mM, 20 mM, and 250 mM imidazole. The imidazole was removed by passing through a PD-10 column. The protein concentration was determined to be 10.4 mg / mL using the BCA protein assay. RLuc8.6-535 (abbreviated as RLuc, concentration 0.5 mg / mL) was added to PBS buffer (1×, pH = 7.4, 1 mL) containing a tetrazine activated ester (Tz-NHS, approximately 10 equivalents). The mixture was stirred at 4°C for 8 hours, after which unreacted Tz-NHS was removed by ultrafiltration. The concentration of RLuc or RLuc-Tz was quantified using the BCA protein assay. The RLuc-Tz protein was characterized by QTOF-MS and verified by in-gel fluorescence analysis by reaction with Cy5-TCO.

[0192] (8) Synthesis of GdNPs-Tz:

[0193] Reaction conditions: First, the synthesis method of FL / MRI dual-modality nanoprobe GdNPs was based on previous research (Angew. Chem. Int. Ed. 2022, 61, e202111759). 2000 -OMe (6 mg), DSPE-PEG 2000-NH2 (1 mg), MEH-PPV (0.25 mg), NIR775 (0.02 mg), and DTPA-BSA (Gd) (3 mg) were dissolved in tetrahydrofuran (THF, 1 mL), and deionized water (9 mL) was added under vigorous ultrasonic conditions. After ultrasonic treatment, the solvent THF was dried with a vacuum pump. The resulting aqueous solution was washed three times with deionized water and centrifuged at 4000 rpm for 15 minutes through a centrifugal filter (molecular weight cutoff value was 10 kDa). The content of MEH-PPV and NIR775 was determined by indirect measurement of the UV-vis-NIR absorption spectrum of the filtrate, and the results showed that almost all of NIR775 was encapsulated in the nanoparticles. Gd was determined by inductively coupled plasma mass spectrometry (ICP-MS) analysis. 3+ Finally, GdNPs were stored in PBS buffer (1×, pH 7.4), in which MEH-PPV, NIR775 and Gd 3+ The concentrations of Tz in GdNPs were approximately 280, 22, and 350 μg / mL, respectively. For subsequent Tz surface modification of the nanoparticles, NHS-PEG4-MAL (2 mg) was added to the prepared GdNP solution (PBS buffer, 1×, pH 7.4, 1 mL). The aqueous solution was stirred at room temperature for 4 hours to promote the coupling of free NH2 groups. Unreacted NHS-PEG4-MAL was removed by ultrafiltration to obtain GdNPs-MAL. To covalently attach Tz groups to the surface of GdNPs, tris(2-carboxyethyl)phosphonate chloride (TCEP, 0.205 mg) and sodium bicarbonate (NaHCO3, 0.241 mg) were first reacted at room temperature for 30 minutes to reduce Tz-PEG6-SS (Angew. Chem. Int. Ed. 2023, 63, e202314039), generating free sulfhydryl groups. The reaction solution was then added directly to the GdNPs-MAL solution (1×, pH = 7.4, 0.5 mL) and stirred at room temperature in the dark for 4 hours. Unreacted Tz-PEG6-SS was removed by centrifugal filter (10 kDa MWCO). The GdNPs-Tz was obtained and resuspended in deionized water and stored at 4°C. The final stock solution concentrations of MEH-PPV, NIR775, and Gd 3+ The concentrations of GdNPs and GdNPs-Tz were approximately 280, 22, and 350 μg / mL, respectively. The particle sizes of GdNPs and GdNPs-Tz were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). UV-Vis and fluorescence spectra of GdNPs and GdNPs-Tz were obtained by spectrophotometry. The click reaction of GdNPs-Tz with Cy5-TCO directly reflected the Tz groups modified on the nanoprobes using UV-Vis spectroscopy.

[0194] Example 3: Performance test of the enzymatically anchored pre-targeting molecule P-TCO.

[0195] 1. Study on the specific response of P-TCO to ALP

[0196] (1) First, the response of P-TCO to ALP in Tris buffer was studied. P-TCO (10 μmol / L) was incubated with ALP (20 U / L≈0.002 mg / mL) in Tris buffer (pH=8.0) for 30 minutes. Figure 2 As shown in a, P-TCO, P-TCO and ALP, P-TCO and ALP and its inhibitor Na3VO4 were analyzed by high performance liquid chromatography (HPLC) at a wavelength of 214 nm to observe the changes before and after the enzyme response, and the enzymatic addition products were verified by mass spectrometry.

[0197] Figure 2 aThe results showed that P-TCO could be cleaved under the action of ALP. HPLC showed that P-TCO (t R =14.7min) into enzyme cleavage addition products (t R =16.8min). Figure 2 HRMS analysis of peak A in (a) (positive and negative ion modes): ESI+ detected a molecular weight of 415.1958, and ESI- detected a molecular weight of 413.1801. Possible chemical transformations after dephosphorylation of P-TCO by low-concentration ALP: In aqueous solution, P-TCO rapidly converts to p-QM and o-QM intermediates upon dephosphorylation. When the ALP concentration is low and cannot completely capture all p-QM and o-QM intermediates, these intermediates react with water molecules in the buffer (approximately 55 M), forming water adducts through the proposed steps.

[0198] (2) Kinetic study of P-TCO on ALP: Different concentrations of P-TCO (5, 7.5, 10, 20, and 30 μmol / L) were incubated with ALP (1 U / L ≈ 0.0001 mg / mL) in enzyme digestion buffer (10 mmol / L Tris containing 1% DMSO, pH 8.0) for 1 minute. Subsequently, the incubation solution was detected by high performance liquid chromatography (HPLC) at a wavelength of 214 nm. The injection volume was 1 mL. Figure 2 The concentration of residual P-TCO was obtained from the HPLC spectra using a standard curve, and the amount of dephosphorylated P-TCO was then calculated. Kinetic parameters (including Km and Vmax) were determined by Lineweaver-Burk plotting.

[0199] Figure 2The cf results show that the Michaelis constant (K m ) is about 17.22 μmol / L, and the maximum reaction rate (V max ) is approximately 2.91 μmol L -1 min-1. According to calculation, the catalytic efficiency of P-TCO to ALP (k cat / K m ) is approximately 2.25×10 6 M -1 s -1 .

[0200] 2. Study on covalent labeling of ALP and BSA proteins with P-TCO

[0201] (1) Covalent labeling of ALP with P-TCO: In-gel fluorescence analysis of ALP labeled with P-TCO and FITC-Tz. ALP (104 U / L≈1.0 mg / mL) was mixed with P-TCO (10 μmol / L) and incubated at 37°C for 30 minutes. Denatured ALP was obtained by heating at 95°C for 5 minutes. FITC-Tz (10 μmol / L) was then added, and the mixture was incubated for another 30 minutes. The labeled protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For each sample, reducing loading buffer (5×) was added, and after heating at 95°C for 5 minutes, a total of 5 μg of protein sample was loaded onto a 12% Tris-Gly gel. The protein separation process was: run at 90 V for 30 minutes, followed by running at 120 V for 90 minutes. The gel was analyzed by fluorescence imaging (excitation wavelength: 475 / 15 nm, emission wavelength: 530 / 15 nm) and stained with InstaBlue protein staining solution (ApexBio) without destaining.

[0202] (2) Determination of covalent labeling and labeling efficiency of BSA with P-TCO: BSA of different concentrations (0.01-20.0 mg / mL) was mixed with ALP (20 U / L), and P-TCO (10 μmol / L) was added and incubated for 30 minutes. FITC-Tz (10 μmol / L) was then added and incubated for another 30 minutes. Each sample was tested by Unbound FITC-Tz was removed by ultrafiltration using an Ultra (10 kDa NMWCO). The supernatant after ultrafiltration was separated by SDS-PAGE and subjected to fluorescence imaging analysis and Coomassie Brilliant Blue staining, ensuring consistent loading volumes for all samples. Unbound FITC-Tz in the filtrate was assessed by measuring the fluorescence intensity of FITC. The unreacted FITC-Tz concentration was estimated based on the fluorescence intensity, and the relative BSA labeling efficiency, defined as the percentage of reacted FITC-Tz relative to the total FITC-Tz, was calculated.

[0203] (3) Study on the effect of P-TCO on enzyme activity after protein labeling: Fluorescence analysis of the reaction of P-CyFF-NODA with ALP and ALP-TCO. A previously reported ALP-activated near-infrared fluorescent probe, P-CyFF-NODA (Nano Lett. 21, 24, 10377–10385 (2021); excitation / emission wavelength: 680 / 720 nm), was incubated with ALP or TCO-labeled ALP modified by P-TCO (ALP-TCO). The experimental groups were as follows: Group I: P-CyFF-NODA (10 μmol / L) in Tris buffer (pH 8.0). Group II: P-CyFF-NODA (10 μmol / L) was incubated with ALP (100 U / L). Group III: P-TCO (10 μmol / L) was preincubated with ALP (100 U / L) and then incubated with P-CyFF-NODA (10 μmol / L). Fluorescence intensity was monitored using a T-CAN microplate reader with an excitation wavelength of 680 nm and an emission wavelength of 720 ± 20 nm.

[0204] like Figure 3 The results of ab are shown in Figures bc. The symbols (★, ●, ▲ and ◆) represent addition / containment, the symbols (☆, ○, Δ and ◇) represent non-addition / lack, and the symbols It represents ALP that loses its enzymatic activity at 95°C. Only when ALP is incubated with P-TCO and FITC-Tz in sequence, the ALP band shows strong fluorescence, confirming that the labeling is effective. P-TCO or FITC-Tz alone does not produce fluorescence. No fluorescence is observed after denatured ALP, indicating that the enzymatic activity of ALP is crucial for labeling. 4 U / L) and P-TCO (10 μmol / L) were co-incubated, and the BSA (~67 kDa) and ALP (~56 kDa) bands showed fluorescence in SDS-PAGE analysis ( Figure 3c). The labeling effect increases with the increase of ALP concentration and incubation time. The labeling effect is optimal when the ALP concentration reaches 20 U / L within 30 minutes. As the BSA concentration increases, more than 99% of TCO is covalently labeled to BSA ( Figure 3 d), each BSA molecule is labeled with an average of approximately 0.7 TCOs, which was verified by mass spectrometry analysis ( Figure 3 e). SDS-PAGE gel fluorescence analysis showed that when the BSA concentration increased from 0.01 to 1.0 mg / mL, the fluorescence intensity of the BSA band gradually increased ( Figure 3 f). In contrast, the labeling efficiency using the one-step method (P-DHP-FITC, 10 μmol / L) was only ~79%. Further studies showed that the E-SIM labeling of BSA by P-TCO was highly specific for ALP. Importantly, after labeling, the enzymatic activity of ALP remained largely unchanged, thus supporting further E-SIM labeling cycles (Supplementary Figure 2). Figure 3 TCO-labeled BSA (BSA-TCO) was reacted with green fluorescent protein (GFP-Tz, 1.0 mg / mL) and Gd nanoparticles (GdNPs-Tz) with Tz groups, respectively. Figure 3 i SDS-PAGE showed that GFP-Tz was converted into BSA-GFP conjugate (~100kDa). Similarly, BSA-TCO reacted with GdNPs-Tz to generate BSA-GdNPs, whose particle size increased from ~69.4nm to ~238.1nm ( Figure 3 j). Fluorescence size exclusion chromatography (SEC) analysis further confirmed the formation of BSA-GFP and BSA-GdNPs conjugates ( Figure 3 k) P-TCO can achieve covalent modification of proteins with various imaging labels through ALP-triggered E-SIM labeling and subsequent IEDDA reaction.

[0205] 3. Study on the specific labeling of ALP-overexpressing cancer cell membrane proteins by P-TCO

[0206] (1) Cell Culture: The cells involved in the present invention include human cervical cancer HeLa cells, human glioblastoma U87MG cells, human liver cancer HepG2 cells, HepG2 cells transfected with firefly luciferase (HepG2-fLuc), and mouse embryonic fibroblast NIH3T3 cells. All cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS), 100 units / mL penicillin, and 100 units / mL streptomycin. The cells were cultured at 37°C in a humidified atmosphere with 5% CO2.

[0207] (2) Screening of ALP expression in different cancer cells: Cells were lysed with RIPA lysis buffer (Biyuntian, P0013C) to prepare protein samples, and the protein concentration was determined using a BCA protein concentration assay kit (Keygen, KGP902). Gel electrophoresis was performed in a Bio-Rad Criterion vertical electrophoresis tank and a Bio-Rad PowerPac Basic power supply using a 12% Tris-glycine polyacrylamide gel (SDS / Tris-Gly). The initial voltage was set to 90 V for 30 minutes and then adjusted to approximately 120 V until the target protein was fully separated. After electrophoresis, the gel was removed from the prefabricated box and transferred to a PVDF transfer membrane (Immobilon, ISEQ00010) at a constant current of 300 A for 90 minutes. After transfer, the PVDF membrane was blocked with 5% skim milk powder and then washed with TBST (1×). ALP primary antibody (Hua'an Bio, ET#:1601-21) was added and incubated overnight at 4°C with gentle shaking. The membranes were then washed three times with TBST (1×) and incubated with an HRP-conjugated secondary antibody. ALP protein expression was detected using an ultrasensitive chemiluminescence (ECL) kit (Thermo Scientific Pierce™) on an imaging analysis system (Tanon).

[0208] (3) Cell biocompatibility (MTT) study: HeLa, U87MG, and NIH3T3 cells were seeded in 96-well plates at 5,000 cells per well and incubated at 37°C overnight. DMEM medium (100 μL) containing different concentrations of P-TCO (0, 1, 2, 5, 10, 20, 50, 100, 150, and 200 μmol / L) was then added to each well. After 24 hours of incubation, MTT solution (1 mg / mL, PBS solution, 50 μL) was added to each well and incubated at 37°C for another 4 hours. Subsequently, the liquid in each well was carefully removed, and DMSO (150 μL) was added to dissolve the formed purple formazan crystals. The absorbance (OD) was measured at 490 nm using a microplate reader (Tcan). The absorbance of blank cells (control group) was used as a reference, and the percentage of cell viability was calculated by the ratio of the OD value of the experimental group to the OD value of the control group.

[0209] (4) Fluorescence imaging study of tumor cells with high ALP expression selectively labeled by P-TCO: First, HeLa cells with high ALP expression were selected for fluorescence imaging study. 5HeLa cells were seeded in 4-well glass-bottomed culture dishes (Cellvis, thickness 0.13-0.16 mm). After the cells attached, they were incubated with different concentrations of P-TCO (0.5, 1, 2, 5, 10, and 20 μmol / L) for 30 minutes, followed by the addition of FITC-Tz (10 μmol / L) for another 30 minutes. Furthermore, the incubation time for P-TCO (10 μmol / L) was varied (5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours), followed by the addition of FITC-Tz (10 μmol / L) for 30 minutes. Cell nuclei were stained with Hoechst 33342. After incubation, the cells were washed three times with PBS and fresh DMEM medium was added for microscopic imaging. Fluorescence images were collected using a Leica SP8 STED 3X confocal laser scanning microscope, including the FITC channel (excitation wavelength: 488 nm, emission wavelength: 530 / 15 nm) and the Hoechst 33342 channel (excitation wavelength: 405 nm, emission wavelength: 460 / 15 nm). HeLa cells were then incubated in P-TCO and FITC-Tz, washed three times with PBS, and collected as a cell suspension into a FACS tube. A FACS Calibur instrument was used for detection, and 1×10 4 Cells were fluorescently labeled using the mean fluorescence intensity (MFI) of FITC (excitation wavelength: 488 nm, emission wavelength: 518 nm). Normalized fluorescence intensity (FL intensity) data of HeLa cells were processed and analyzed using CytExpert 2.4 software.

[0210] (5) Determination of the number of TCO groups modified by P-TCO on the HeLa cell membrane: HeLa cells were seeded in a 96-well plate at a density of 1×104 cells per well. After incubation, P-TCO (10 μmol / L, 30 minutes) and Cy5-Tz (10 μmol / L, 30 minutes) were added in sequence. After incubation, the fluorescence intensity of P-TCO and Cy5-Tz labeled cells was detected using a Tcan microplate reader (excitation wavelength: 630 nm, emission wavelength: 680 nm). At the same time, the fluorescence intensity of different concentrations of Cy5-Tz in the culture medium was detected to draw a standard curve. The concentration of Cy5-Tz bound to the cell membrane was detected and calculated. Since the reaction between TCO and Tz is a stoichiometric ratio of 1:1, it is equal to the concentration of TCO labeled on the cell membrane.

[0211] The results of cytotoxicity experiments showed that ( Figure 4ab), P-TCO has almost no toxicity to human cervical adenocarcinoma HeLa cells with high ALP expression, human glioma U87MG with low ALP expression, and mouse embryonic fibroblast NIH3T3 cells. High concentrations have almost no effect on cell growth, indicating that P-TCO has good biocompatibility. Next, the preferred concentration of P-TCO in cell imaging experiments is 10 μmol / L, and the preferred incubation time is 30 minutes. Under these conditions, P-TCO can specifically label the HeLa cell membrane and react with FITC-Tz on the cell membrane, so that the HeLa cell membrane shows bright green fluorescence. In contrast, in cells with low ALP expression ( Figure 4 c), or use ALP enzyme inhibitors (Na3VO4, Figure 4 df) After pretreatment, the cell membrane had almost no green fluorescence, which indicated that P-TCO specifically labeled HeLa cells with high ALP expression. In the co-culture system of HeLa and NIH3T3, P-TCO could also selectively label HeLa cell membranes ( Figure 4 g). Subsequent gel fluorescence analysis showed that labeling was limited to membrane proteins and did not involve intracellular proteins ( Figure 4 h). After a single HeLa cell was treated with P-TCO (10 μmol / L, 30 minutes), 4.06±0.12×107 TCO groups were detected. This indicates that P-TCO has a high labeling efficiency for cell membrane proteins and can stably anchor TCO groups to the cell membrane, enabling multimodal imaging of cell membrane proteins.

[0212] 4. Research on the multimodal imaging application of P-TCO in tumor-bearing mice.

[0213] (1) Animal husbandry and animal model establishment: The nude mouse model involved in the patent was purchased from the Model Animal Research Center (MARC) of Nanjing University, which were 4-5 weeks old female BALB / c nude mice with an average weight of 20 grams. The mice were kept in an environment with suitable conditions and provided with sufficient food and water. All animal experimental operations were approved by the Animal Experiment Ethics Committee (IACUC) of Nanjing University (approval number: IACUC-2204019). The small animal PET imaging experiment was conducted in accordance with the ethical principles established by the Jiangsu Institute of Nuclear Medicine. The subcutaneous tumor model involved in the patent was a HeLa cell (approximately 2×10 6 ) or U87MG cells (approximately 4×10 6 ) were suspended in a mixture of matrigel and DMEM (50 μL, 50%, volume ratio) and injected subcutaneously into the right upper leg of nude mice. The tumor diameter was monitored every other day. When the tumor volume reached 100-120 mm3 (volume formula: length × width), the tumor was resected. 2 / 2) in vivo experiments were conducted. The patent involves a tumor peritoneal metastasis model in which HepG2 or HepG2-fLuc cells (approximately 1×10 6 ) were suspended in serum-free medium (100 μL) and injected directly into the mouse peritoneal cavity. Bioluminescence imaging was performed via intraperitoneal injection of D-Luciferin to monitor HepG2-fLuc tumor growth and metastasis. Two weeks later, imaging confirmed the successful establishment of a peritoneal liver metastasis model.

[0214] (2) P-TCO combined with IR780-ZW-Tz to achieve near-infrared fluorescence imaging of living tumors: First, the preferred concentration and dosing interval of P-TCO used in living tumor imaging were optimized. The experimental setting was that mice were injected with different concentrations of P-TCO (200, 400 or 600 μmol / L, 200 μL) through the tail vein. 2 hours after injection, IR780-ZW-Tz (20 μmol / L, 200 μL) was injected through the tail vein. Secondly, mice were injected with P-TCO (400 μmol / L, 200 μL) through the tail vein. 0.5, 2 or 4 hours after injection, IR780-ZW-Tz (20 μmol / L, 200 μL) was injected through the tail vein again. Whole-body fluorescence images were acquired using the IVIS Lumina XR III imaging system with an excitation filter of 790 nm and an emission filter of 845 / 20 nm. Each experiment was performed in three mice, and the fluorescence intensity was quantified using the ROI measurement function of Living Image Software (4.5.2, PerkinElmer, MA, USA).

[0215] The results of the fluorescence experiment on tumor-bearing mice showed that the optimal concentration of P-TCO injected into the tail vein was 400 μmol / L and the injection volume was 200 μL. Therefore, the optimal amount of P-TCO injected into the tail vein was 80 nmol. Figure 5 a, Mice bearing subcutaneous (sc) HeLa tumors were injected with P-TCO (400 μmol / L) followed by IR780-ZW-Tz (20 μmol / L) 2 hours later. In the P-TCO-injected experimental group, IR780-ZW-Tz injected tumors showed strong near-infrared (NIR) fluorescence 0.5 hours after injection, which then slowly weakened but persisted for more than 24 hours. In contrast, tumors injected with IR780-ZW-Tz alone showed only weak fluorescence. At 24 hours, the average tumor fluorescence intensity of the (P-TCO+IR780-ZW-Tz) group was approximately 3.6 times that of the IR780-ZW-Tz group, and its tumor-to-background ratio (TBR) was 4.7±0.9, which is approximately 4.2 times the TBR of the IR780-ZW-Tz group (1.1±0.2). Figure 5c). Ex vivo fluorescence imaging of tumors and major organs ( Figure 5 de), normalized fluorescence intensity of tumors and major organs removed 8 hours after administration (H: heart, Lu: lung, Li: liver, Sp: spleen, K: kidney, In: intestine, T: tumor, M: muscle). Data are expressed as mean ± standard deviation (n = 3 animals, two-tailed t-test); the results confirmed that the tumor fluorescence intensity in the (P-TCO + IR780-ZW-Tz) group was the highest, and fluorescence imaging of tissue sections further supported this result. Protein gel analysis showed that there was a strong fluorescent band in the tumor lysate treated with (P-TCO + IR780-ZW-Tz), indicating that P-TCO achieved covalent labeling of tumor proteins. The use of ALP inhibitors (such as L When P-TCO was pretreated with either IR780-ZW-Tz or U87MG tumors, both resulted in a significant decrease in NIR fluorescence and TBR. When P-TCO was used with IR780-ZW-Tz in mice bearing both subcutaneous HeLa and U87MG tumors, strong fluorescence was observed only in HeLa tumors, confirming that P-TCO labeling is dependent on ALP activity. Figure 5 (f) Coomassie Brilliant Blue (CB) and fluorescent in-gel analysis of labeled tumors. Mice were intravenously injected with or without P-TCO (400 μmol / L) and then injected with IR780-ZW-Tz (20 μmol / L) 2 hours later. Tumors were excised and lysed 4 hours later.

[0216] (3) Fluorescence imaging of live tumors using P-TCO combined with BSA-Cy5-Tz: In HeLa subcutaneous tumor-bearing mice, P-TCO (400 μmol / L, 200 μL) was injected through the tail vein. Two hours after injection, BSA-Cy5-Tz (25 μmol / L, 200 μL) was injected again through the tail vein. For comparison, mice were injected with either BSA-Cy5-Tz (25 μmol / L, 200 μL) or the single-step self-immobilized probe P-DHP-BSA-Cy5 (25 μmol / L, 200 μL) through the tail vein. Fluorescence images were acquired using an IVIS Lumina XR III imaging system before or 4 hours after BSA-Cy5-Tz injection, with an excitation filter of 620 nm and an emission filter of 670 nm.

[0217] Specifically, the experimental group was divided into three groups: Group I: P-TCO (400 μmol / L) was injected with BSA-Cy5-Tz (25 μmol / L) 2 hours after injection; Group II: BSA-Cy5-Tz (25 μmol / L) alone was injected; and Group III: P-DHP-BSA-Cy5 (25 μmol / L) alone was injected with the control fluorescent probe. Fluorescence images were acquired before (Pre) and 4 hours after injection.

[0218] The results showed that P-TCO can be used to pre-targeted fluorescence imaging of subcutaneous HeLa tumors using protein imaging reporter molecules (such as BSA-Cy5-Tz). Figure 6 ac). After intravenous injection of P-TCO + BSA-Cy5-Tz in tumor-bearing mice, HeLa tumors exhibited bright Cy5 fluorescence, with a TBR reaching 4.1 after 4 hours. In contrast, mice injected with BSA-Cy5-Tz or P-DHP-BSA-Cy5 alone displayed weaker tumor fluorescence and lower TBR. These results indicate that P-TCO can effectively capture BSA-Cy5-Tz in vivo and maintain a strong fluorescence signal at the tumor site. However, P-DHP-BSA-Cy5 alone was unable to effectively label HeLa tumor membranes. This may be due to the large size of P-DHP-BSA-Cy5, which provides high steric hindrance during protein labeling.

[0219] (4) P-TCO combined with GdNPs-Tz to achieve near-infrared / magnetic resonance dual-modality imaging of living tumors: Mice bearing subcutaneous HeLa tumors were injected with P-TCO (400 μmol / L, 200 μL saline) through the tail vein. Two hours later, FL / MR dual-modality nanoparticles GdNPs-Tz (MEHPPV / NIR775 / Gd: 0.28 / 0.022 / 0.35 mg / mL, 0.2 mL) were injected. Fluorescence images were acquired using an IVIS Lumina XR III imaging system with an excitation filter of 740 nm and an emission filter of 790 / 20 nm. Fluorescence intensity was quantified by ROI measurement. T1-weighted magnetic resonance imaging (MRI) was performed before and 24 hours after GdNPs-Tz injection. All MRI experiments were performed on a Bruker I-CON 1.0T small animal MRI scanner, and mice were scanned in the tail-first prone position. Images were acquired using a T1-RARE imaging sequence. Each experimental group included three mice. MRI data were exported to DICOM format and quantitative image analysis was performed using the RadiAnt DICOM Viewer. The percentage signal enhancement (%SE) at each time point was calculated based on the signal intensity (SI) of the tumor region: %SE(t) = (SI(t) - SI(t = 0)) / SI(t = 0). This formula was applied to each imaging result for each mouse.

[0220] Figure 7ac results showed that P-TCO can be effectively combined with Tz-modified nanoparticles (such as GdNPs-Tz) for pre-targeted MR and NIR fluorescence dual-modality imaging of HeLa tumors in vivo. The maximum signal enhancement (%SE) and NIR fluorescence intensity of HeLa tumors treated with (P-TCO+GdNPs-Tz) were significantly higher than those treated with GdNPs-Tz alone or P-TCO combined with GdNPs. In addition, dynamic 3D images of ex vivo tumors showed that the MR signal was higher in tumors pretreated with P-TCO, which also confirmed that P-TCO can efficiently label living tumors and efficiently bind to Tz-containing nano-imaging reporter groups.

[0221] (5) P-TCO combined with RLuc-Tz to achieve bioluminescence imaging of living tumors: First, in the pre-targeted bioluminescence imaging experiment of HeLa tumors, mice were injected with P-TCO (400 μmol / L, 200 μL) through the tail vein. Two hours later, RLuc-Tz (0.2 mg / mL, 200 μL) was injected through the tail vein. Before the injection of RLuc-Tz and 4 hours, 8 hours, 24 hours, 72 hours and 120 hours after the injection, CTZ (20 μg) was injected intraperitoneally to collect bioluminescence (BL) images. Fluorescence images were acquired at the above time points using the IVIS Lumina XR III imaging system using the open filter mode. Secondly, in the dual-mode (fLuc and RLuc) pre-targeted BL imaging experiment of the HepG2-fLuc tumor peritoneal metastasis model, mice were injected with P-TCO (400 μmol / L) through the tail vein and RLuc-Tz (40 μg) was injected 2 hours later. Subsequently, potassium salt of luciferin was injected intraperitoneally. D Following intraperitoneal injection of CTZ (1 mg / kg), fLuc BL images were acquired at 8, 24, and 120 hours (day 5) after RLuc-Tz injection. BL intensity in peritoneal HepG2-Luc tumors was quantified using the ROI measurement function of Living Image Software.

[0222] The experimental results showed that, given the high density of TCO groups labeled on the cell membrane (4.06±0.12×10 7 ), and used these groups to covalently capture Tz-modified Renilla luciferase (RLuc-Tz) to achieve sensitive bioluminescence (BL) imaging of ALP-positive tumors ( Figure 8 a). First, it was confirmed that when reacted with CTZ, RLuc-Tz exhibited similar luminescence efficiency and BL spectrum to native RLuc ( Figure 8b). After incubation with P-TCO (10 μmol / L), RLuc-Tz (approximately 0.05 mg / mL) was added to HeLa cells. After the addition of CTZ (0.025 mg / mL), strong BL was observed in HeLa cells treated with both P-TCO and RLuc-Tz, while no BL was observed in NIH3T3 cells treated with RLuc-Tz alone or ALP-deficient cells. Figure 8 cd). This demonstrates that RLuc can be efficiently chemically bound to HeLa cells by E-SIM labeling combined with IEDDA reaction, with a limit of detection (LOD) of approximately 9 cells in vitro and approximately 45 cells in vivo.

[0223] Subsequently, nude mice bearing HeLa tumors were injected with P-TCO (400 μmol / L, 200 μL) and then RLuc-Tz (2.0 mg / kg, intraperitoneal injection) 2 hours later. The HeLa tumors treated with (P-TCO+RLuc-Tz) emitted a strong signal, reaching a peak at 4 hours and remaining detectable for more than 5 days. The target-to-target ratio (TBR) on day 5 was 29.4±7.1 ( Figure 8 e.g.) where Figure 8 e) Pretargeted bioluminescence imaging of HeLa tumors. Group I was first injected with P-TCO, followed by RLuc-Tz 2 hours later; Group II was injected with RLuc-Tz alone. After CTZ injection, bioluminescence images were captured before, and 4, 8, 24, 72, and 120 hours after RLuc-Tz injection. In contrast, tumors treated with RLuc-Tz alone exhibited weaker BL and a lower TBR (1.5 ± 0.5). BL intensity in tumors treated with (P-TCO + RLuc-Tz) was over 1000-fold higher than in tumors treated with RLuc-Tz alone. These results demonstrate that RLuc-Tz is captured by tumors and remains stable over time.

[0224] (6) P-TCO combination 68 Ga]-Tz and IR780-ZW-Tz to achieve radioactive PET / near-infrared fluorescence synergistic imaging of living tumors: HeLa tumor-bearing mice were first divided into two groups: P-TCO group and control group (n=3). Mice were injected with P-TCO (400μmol / L, 200μL) through the tail vein. Two hours later, the radiotracer [ 68[Ga]-Tz (~7.4 MBq, 200 μL). During the experiment, mice were maintained under anesthesia under 2% isoflurane mixed with oxygen (flow rate of 1.5 L / min). Imaging was performed using the Siemens Inveon Dedicated microPET animal scanner, and real-time and static PET images were processed in the professional image processing software ASIPro. Static images were acquired at 0.5, 1, 2, and 3 hours. ROI measurements of the tumor site and other organs were analyzed, and the quantitative absorption values were calculated using the attenuation correction factor in units of %ID / g (percentage of injected dose per gram of tissue). After radiation decay and clearance were completed (24 hours), the NIR fluorescent probe IR780-ZW-Tz (20 μmol / L, 200 μL) was injected into the tail vein. Whole-body fluorescence images were acquired using the IVIS Lumina XR III imaging system with an excitation filter of 790 nm and an emission filter of 845 nm. Fluorescence intensity was measured and quantified using Living Image Software (4.5.2, PerkinElmer). Three mice were included in each experiment to ensure reproducibility.

[0225] For positron emission tomography (PET) imaging, the radionuclide 68Ga 3+ Chelated NODA-Tz was used to synthesize Tz ([ 68 Ga]-Tz)( Figure 9 a), the compound is stable and has high radiochemical purity ( Figure 9 b). Specifically, HeLa cells were incubated with or without P-TCO (10 μmol / L, 30 min) and then with [ 68 Isotope abundance analysis of co-incubation with Ga]-Tz (~0.19 MBq, 30 min). Group I: [ 68 Ga]-Tz; Group II: P-TCO+[ 68 Ga]-Tz; Group III: Na3VO4 pretreatment (1mmol / L, 30 minutes) inhibitor group, followed by addition of P-TCO and [ 68 Ga]-Tz. HeLa cells pretreated with P-TCO (10 μmol / L) for 30 minutes showed a significant difference compared with those treated with [ 68 Compared with cells treated with Ga]-Tz, cells showed stronger PET signals (0.19 MBq≈0.05 nmol, 30 min) ( Figure 9 c). About 12.71% of [68Ga]-Tz was captured by cells pretreated with P-TCO, which was significantly higher than that of cells pretreated with [ 68 Ga]-Tz treated cells (about 0.51%, Figure 9cd), which was only about 25 times higher. However, pretreatment of HeLa cells with Na3VO4 to inhibit alkaline phosphatase (ALP) activity significantly reduced the PET signal and decreased the cellular uptake to about 4.22%. When used for in vivo HeLa tumor pretargeted PET imaging ( Figure 9 e, f), specifically, Figure 9 f is the use of P-TCO and [ 68 Representative coronal and axial PET images of HeLa tumors (attenuation corrected) with [Ga]-Tz, Group A: only [ 68 Ga]-Tz (7.4 MBq); Group B: P-TCO (400 μmol / L), injected 2 hours later [ 68 Ga]-Tz (7.4 MBq), 9f showed that after injection of [ 68 PET images of mice at 0.5, 1, 2, and 3 hours after treatment with [Ga]-Tz. 68 Compared with the tumors treated with P-TCO pretreatment and injection of [ 68 Tumors treated with [Ga]-Tz (~7.4 MBq, i.e., 2.1 nmol) showed higher radioactivity and TBR (6.18 ± 0.82% ID / g and ~5.4, respectively), while tumors treated with [ 68 The radioactivity and TBR of the Ga]-Tz treated tumors were 2.38±0.53% ID / g and ∼2.6 ( Figure 9 gh). The radioactivity of the tumors pretreated with P-TCO decreased more slowly, remaining at 4.26±0.31% ID / g after 3 hours, while the radioactivity of the tumors in the control group decreased rapidly (1.08±0.46% ID / g after 3 hours). 68 Ga]-Tz was rapidly cleared from the muscles, and the TBR of P-TCO pretreated mice increased to over 7.0 after 1 hour ( Figure 9 h). These results indicate that the enzymatic self-anchoring and pre-targeting strategy can make more [ 68 Ga]-Tz can be captured by P-TCO-pretreated HeLa tumors, thereby prolonging the retention time of the radionuclide in the tumor and improving the TBR of PET imaging.

[0226] because[ 68 The molar amount of Ga]-Tz (~2.1 nmol) is much lower than the molar amount of injected P-TCO (~80 nmol). 68 The remaining TCO handles in the Ga]-Tz-treated tumors could further capture additional injected IR780-ZW-Tz for dual-modality PET and near-infrared fluorescence imaging of HeLa tumors ( Figure 9e, i). Consistent with the PET imaging results, P-TCO pretreated tumors showed significantly stronger near-infrared fluorescence (4.5 times) and higher TBR (2.6 times) compared with untreated tumors, indicating that [ 68 Ga]-Tz and IR780-ZW-Tz for dual-modality PET and fluorescence imaging ( Figure 9 j, k). This method can be used for preoperative PET imaging and intraoperative fluorescence-guided surgery.

[0227] The present invention provides an enzymatic self-anchoring pre-targeting molecule, P-TCO, and its preparation and application. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Components not specified in this embodiment may be implemented using existing technologies.

Claims

1. An enzymatic self-anchoring pre-targeting molecule P-TCO, characterized in that: Its structural formula is shown in the formula P-TCO: Wherein, the R group is Any one of: R2 is 2. The method for preparing the enzymatic self-anchoring pre-targeting molecule according to claim 1, characterized in that: The steps include: Step a: Compound 1 reacts with sodium azide to obtain compound 2; Step b: Compound 2 undergoes substitution reaction to obtain compound 3; Step c: Compound 3 is subjected to oxidation reaction to obtain compound 4; Step d: Compound 4 undergoes addition and substitution reaction to obtain compound 5; Step e: Compound 5 is subjected to reduction reaction to obtain compound 6; Step f: Compound 6 undergoes substitution reaction to obtain compound 7; Step g: Compound 7 undergoes substitution reaction to obtain compound 8; Step h: Compound 8 undergoes addition reaction to obtain compound 9; Step i: Compound 9 is subjected to a condensation reaction to obtain an enzymatic self-anchored pre-targeting molecule P-TCO; 3. Use of the enzymatic self-anchoring pre-targeting molecule P-TCO according to claim 1 in cell membrane protein labeling and multimodal imaging of cell membrane proteins.

4. Use of the enzymatic self-anchoring pre-targeting molecule P-TCO according to claim 1 in multimodal imaging at the cellular level, wherein: The P-TCO is combined with a tetrazine probe to achieve multimodal imaging at the cellular level.

5. Use of the enzymatic self-anchoring pre-targeting molecule P-TCO according to claim 1 in multimodal imaging in vivo, wherein: The P-TCO is combined with a tetrazine probe to achieve multimodal imaging in vivo.

6. The use according to claim 4 or 5, characterized in that The tetrazine probe comprises a tetrazine bioorthogonal group and an imaging reporter molecule, wherein the tetrazine bioorthogonal group is The imaging reporter molecules include small molecules, proteins or nanoparticles used for positron emission tomography, fluorescence imaging, bioluminescence or magnetic resonance imaging.

7. The use according to claim 6, characterized in that The tetrazine probes are FITC-Tz, IR780-ZW-Tz, [ 68 Any one of [Ga]-Tz, GFP-Tz, BSA-Cy5-Tz, RLuc-Tz and GdNPs-Tz, with the following structural formula:

8. The use according to claim 3 or 4, characterized in that The molar concentration of the enzymatic self-anchoring pre-targeting molecule P-TCO in cell imaging is 0.5-10 μmol / L, and the experimental time is 5-360 minutes.

9. The use according to claim 5, characterized in that The molar concentration of the enzymatic self-anchoring pre-targeting molecule P-TCO in in vivo imaging of tumor-bearing mice was 200-600 μmol / L, and the experimental time was 30-240 minutes.

10. The use according to claim 4 or 5, characterized in that: The molar concentration ratio of the P-TCO to the tetrazine probe is 40:1 to 1:1.

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