Drug in-situ synthesis method and application thereof

Abstract

The invention provides a drug in-situ synthesis method, and particularly relates to a kit and application thereof, the kit comprises: 1) phenylalanine boron trifluoride (Ph-BF3); 2) a compound A containing a silicon-based phenol ether group; and 3) a compound B derived from 2-formylphenylboronic acid (2-fPBA). The invention also relates to application of phenylalanine boron trifluoride (Ph-BF3), a compound B derived from 2-formyl phenylboronic acid (2-fPBA) and a compound A containing a silicon-based phenolic ether group in preparation of medicines.

Description

Technical Field

[0001] The present invention belongs to the field of in-situ drug synthesis, and relates to a desilylation and reconnection method and its application, specifically to a desilylation and reconnection method and its application in the presence of phenylalanine boron trifluoride (Phe-BF 3 ). Background Art

[0002] Inducing proximity interactions between proteins and cells is emerging as a novel approach in cancer therapy. Through specific binding, chemical-induced proximity can alter the fate of target proteins / cells, inducing protein degradation or cell death. However, most target antigens are usually co-expressed in non-malignant tumor tissues. This off-target non-oncotoxicity poses a major obstacle to the clinical progress of proximity-mediated therapies such as proteolysis-targeting chimeras (PROTACs) or T cell-mediated immunotherapy.

[0003] For example, toxicity is one of the bottlenecks in the clinical development of PROTACs because it may cause unexpected consequences by degrading entire proteins rather than simply inhibiting them. PROTACs are an effective way to selectively degrade pathogenic proteins in a catalytic manner. However, the powerful catalytic properties of PROTACs may narrow their therapeutic window, and their low selectivity for tumors will lead to severe systemic toxicity due to so-called "on-target toxicity". For example, BRD4 is an epigenetic regulator overexpressed in various types of tumors and is considered a potential therapeutic target for cancer treatment. However, the complete degradation of BRD4 may cause serious side effects, especially in small intestinal tissues. Some PROTAC drugs were originally prepared to enter clinical trials but were ultimately terminated.

[0004] As many as one-third of patients receiving immunotherapy suffer from the well-known cytokine release syndrome, which is caused by its powerful immune effector response. Human epidermal growth factor receptor 2 (HER2), a recognized therapeutic target in cancer treatment, is also expressed in healthy tissues, which has led to fatal cardiopulmonary toxicity in colorectal cancer patients who have previously received HER2-based chimeric antigen receptor (CAR) T cell therapy. Bispecific T cell engager (BiTE) is an important approach for T cell immunotherapy, which can connect T cells in vivo to target cells containing selected markers and induce T cell-mediated tumor cell killing. However, due to the lack of specific distribution of tumor-associated antigens, BiTE tumor targeting is insufficient, and T cell redirection therapy has off-tumor toxicity (OTOT), which limits its clinical application. Therefore, how to enhance the killing effect of immune cells on cancer cells while reducing toxic side effects is a key and difficult problem that needs to be solved in the field of immunotherapy.

[0005] Therefore, new strategies are urgently needed to mitigate the toxicities associated with PROTACs and T cell-mediated therapies in order to advance preclinical and clinical studies of related drug development. In theory, the function of PROTAC or T cell-mediated therapies depends on proximity-driven protein-protein interactions or cell-cell interactions, which highlights the need for precise regulation of the timing and location of these interactions. Therefore, if a technology platform that can locally induce proximity in a tumor-selective manner is successfully developed, it will provide theoretical and technical guidance for addressing the above challenges.

[0006] A large amount of literature has proved that bioorthogonal reactions can achieve selective chemical regulation of cells or target organs without interfering with normal life activities. Bioorthogonal chemistry is a method that can be used in the fields of biomolecule labeling and imaging, drug delivery, cell engineering, etc. Studies have shown that bioorthogonal chemistry has the ability to accurately connect two compounds in vitro. However, there is still a gap between the process of bioorthogonal connection and tumor selective intervention. The time and spatial position of exogenous light irradiation can be finely adjusted, giving light regulation ultra-high spatiotemporal resolution. Scientists have achieved controllable activation of BiTE regulated by light ( Figure 1 A in the figure). Although light-regulated immunotherapy has made some progress in the treatment of cells and superficial skin tumors, phototoxicity and penetration limit its further application in vivo. In contrast, chemical small molecules have the characteristics of structural adjustability and easy penetration into tissues, making them a more flexible means of regulation. LITE (Ligand-induced transient engagement) therapy ( Figure 1In B), the activity of T cells is regulated by selectively activating and controlling the formation of bispecific complexes through the injection of small molecule activators. However, small molecules are metabolized and cleared quickly, while antibodies have a long retention time, resulting in poor matching between the two. Therefore, long-term small molecule intervention and regulation are required, and the hook effect may occur after supersaturation, affecting the maximum drug efficacy. The pharmacokinetic properties of chemical small molecules and their ability to drive orthogonal reactions are key factors for achieving "enhanced efficacy and reduced toxicity" in immunotherapy. Therefore, how to achieve tumor-selective T cell targeting and regulation is a key scientific issue for realizing "enhanced efficacy and reduced toxicity" in immunotherapy. SUMMARY OF THE INVENTION

[0007] The inventors have found through research that by using exogenous small molecules with tumor targeting properties to construct a controllable ligation chemistry driven by them, the above-mentioned goal of "enhanced efficacy and reduced toxicity" can be achieved. To this end, the present disclosure has established a bioorthogonal ligation chemistry triggered by a tumor-selective probe to achieve in-situ drug synthesis for tumor localization ( Figure 2 ). The tumor-selective probe mentioned here is phenylalanine trifluoroborate (Phe-BF 3 , also known as boronic acid), which can mediate the removal of phenol silyl ether to release an effector molecule, and this effector molecule can further covalently bind to a pairing molecule to generate a target molecule ( Figure 3 ).

[0008] In one aspect of the present disclosure, a method for desilylation and re-ligation is provided, wherein the method comprises: reacting a compound A containing a silyl phenolic ether group with a compound B derived from 2-formylphenylboronic acid (2-fPBA) in the presence of phenylalanine trifluoroborate (Phe-BF 3 ).

[0009] In some specific embodiments, the compound A has the following structure:

[0010] The R 1 is selected from H, a protecting group, a drug active group, an antibody group, a fluorescent group, a staining group, or a gold nanoparticle.

[0011] In some preferred embodiments, the compound A is selected from:

[0012]

[0013] wherein Ab is an antibody group, Flu is a fluorescent group, Dye is a staining group, and AuNPs is a gold nanoparticle.

[0014] In some specific embodiments, the compound B has the following structure:

[0015] The R 2 is selected from H, a linking group, a fluorescent group or a dyeing group; the R 3 is selected from H, an alkyl group, an aryl group or a heteroaryl group, which may be optionally substituted.

[0016] In some preferred embodiments, the compound B is selected from:

[0017] wherein, Flu is a fluorescent group, Dye is a dyeing group, and the R 3 is selected from H, an alkyl group, an aryl group or a heteroaryl group, which may be optionally substituted.

[0018] In some specific embodiments, the desilylation and reconnection method has the following reaction formula:

[0019]

[0020] In some preferred embodiments, the reaction formula may be:

[0021]

[0022] In some preferred embodiments, the reaction formula may be:

[0023]

[0024] On the other hand, the present disclosure provides the use of any one of the above desilylation and reconnection methods in the fluorescence controllable labeling of antibodies.

[0025] That is, the present disclosure provides a method for fluorescence controllable labeling of antibodies, including performing any one of the above desilylation and reconnection methods.

[0026] On the other hand, the present disclosure provides the use of any one of the above desilylation and reconnection methods in the positive cell screening of antibodies.

[0027] That is, the present disclosure provides a method for positive cell screening of antibodies, including performing any one of the above desilylation and reconnection methods.

[0028] On the other hand, the present disclosure provides the use of any one of the above desilylation and reconnection methods in the controllable degradation of target proteins by antibodies.

[0029] That is, the present disclosure provides a method for controllable degradation of target proteins by antibodies, including performing any one of the above desilylation and reconnection methods.

[0030] On the other hand, the present disclosure provides the use of any one of the above desilylation and reconnection methods in bioorthogonal ligation chemistry.

[0031] That is, the present disclosure provides a method of bioorthogonal ligation chemistry, including performing any one of the above-mentioned desilylation and religation methods.

[0032] In some specific embodiments, the bioorthogonal ligation chemistry is selected from proteolysis-targeting chimera therapy or T cell-mediated immunotherapy. In some preferred embodiments, the proteolysis-targeting chimera therapy includes an in-situ synthesized proteolysis-targeting chimera drug. In some preferred embodiments, the T cell-mediated immunotherapy includes an in-situ synthesized nanodrug.

[0033] Another aspect of the present disclosure provides the use of any one of the above-mentioned desilylation and religation methods in precision anti-tumor therapy.

[0034] That is, the present disclosure provides a method of precision anti-tumor therapy, including performing any one of the above-mentioned desilylation and religation methods.

[0035] In some specific embodiments, the above method of the present disclosure can be carried out in vitro or in vivo; in a preferred embodiment, the method is carried out in vivo.

[0036] Yet another aspect of the present disclosure provides a method for treating cancer, which includes administering phenylalanine boron trifluoride (Phe-BF 3 ), compound A containing a silyl phenolic ether group, and compound B derived from 2-formylphenylboronic acid (2-fPBA) to a subject. In some embodiments, the above three substances are administered in sequence; in other embodiments, compound A containing a silyl phenolic ether group and compound B derived from 2-formylphenylboronic acid (2-fPBA) are first administered to the subject, and then phenylalanine boron trifluoride (Phe-BF 3 ) is administered to the subject; in other embodiments, Phe-BF 3 , compound A, and compound B are administered to the same site in the subject. The structures of compound A and compound B are as defined herein. The reactions of phenylalanine boron trifluoride (Phe-BF 3 ), compound A containing a silyl phenolic ether group, and compound B derived from 2-formylphenylboronic acid (2-fPBA) are as defined herein.

[0037] It should be understood that in other methods of the present disclosure, phenylalanine boron trifluoride (Phe-BF 3 ), compound A containing a silyl phenolic ether group, and compound B derived from 2-formylphenylboronic acid (2-fPBA) should also be provided to the subject or test sample in sequence.

[0038] The present disclosure also provides a kit, which includes: 1) phenylalanine boron trifluoride (Phe-BF3 ); 2) compound A containing a silicon-based phenol ether group; and 3) compound B derived from 2-formylphenylboronic acid (2-fPBA).

[0039] In some specific embodiments, compound A has the following structure:

[0040] The R 1 is selected from H, a protecting group, a pharmaceutically active group, an antibody group, a fluorescent group, a staining group, or a gold nanoparticle.

[0041] In some preferred embodiments, compound A is selected from:

[0042]

[0043] wherein Ab is an antibody group, Flu is a fluorescent group, Dye is a staining group, and AuNPs is a gold nanoparticle.

[0044] In some specific embodiments, compound B has the following structure:

[0045] The R 2 is selected from H, a linking group, a fluorescent group, or a staining group; the R 3 is selected from H, an alkyl group, an aryl group, or a heteroaryl group, which may be optionally substituted.

[0046] In some preferred embodiments, compound B is selected from:

[0047] wherein Flu is a fluorescent group, Dye is a staining group, and the R 3 is selected from H, an alkyl group, an aryl group, or a heteroaryl group, which may be optionally substituted.

[0048] In another aspect of the present disclosure, there is provided the use of any one of the above-mentioned kits in the fluorescence-controlled labeling of antibodies.

[0049] In another aspect of the present disclosure, there is provided the use of any one of the above-mentioned kits in the screening of positive cells for antibodies.

[0050] In another aspect of the present disclosure, there is provided the use of any one of the above-mentioned kits in the controllable degradation of target proteins by antibodies.

[0051] In another aspect of the present disclosure, there is provided the use of any one of the above-mentioned kits in bioorthogonal ligation chemistry.

[0052] In some specific embodiments, the bioorthogonal ligation chemistry is selected from proteolysis-targeting chimera therapy or T cell-mediated immunotherapy. In some preferred embodiments, the proteolysis-targeting chimera therapy comprises an in-situ synthesized proteolysis-targeting chimera drug. In some preferred embodiments, the T cell-mediated immunotherapy comprises an in-situ synthesized nanodrug.

[0053] In another aspect of the present disclosure, there is provided the use of any one of the above-mentioned kits in precision anti-tumor therapy.

[0054] In another aspect of the present disclosure, there is provided the use of phenylalanine boron trifluoride (Phe-BF 3 ), and / or compound A containing a silicon-based phenol ether group, and / or compound B derived from 2-formylphenylboronic acid (2-fPBA), or the aforementioned kit in the preparation of a drug.

[0055] In some specific embodiments, the drug is used for fluorescence controllable labeling of antibodies, positive cell screening of antibodies, controllable degradation of target proteins by antibodies, bioorthogonal ligation chemistry, and / or precision anti-tumor therapy.

[0056] In some specific embodiments, the bioorthogonal ligation chemistry is selected from proteolysis-targeting chimera therapy or T cell-mediated immunotherapy.

[0057] In some specific embodiments, the drug synthesizes a proteolysis-targeting chimera drug in-situ after administration.

[0058] In some specific embodiments, the drug synthesizes a nanodrug in-situ after administration.

[0059] In some specific embodiments, compound A has the following structure:

[0060] The R 1 is selected from a protecting group, a drug active group, an antibody group, a fluorescent group, a staining group, or a gold nanoparticle.

[0061] In some specific embodiments, compound A is selected from:

[0062]

[0063] wherein Ab is an antibody group, Flu is a fluorescent group, Dye is a staining group, and AuNPs is a gold nanoparticle.

[0064] In some specific embodiments, compound B has the following structure:

[0065] The R 2Selected from H, a linking group, a fluorescent group or a dye group; said R 3 Selected from H, an alkyl group, an aryl group or a heteroaryl group, which may be optionally substituted.

[0066] In some preferred embodiments, compound B is selected from:

[0067] Wherein, Flu is a fluorescent group and Dye is a dye group, and said R 3 Selected from H, an alkyl group, an aryl group or a heteroaryl group, which may be optionally substituted.

[0068] Advantages

[0069] In the present disclosure, the inventors utilized the desilylation-to-ligation method (abbreviated as D2L strategy) to achieve controllable ligation of tumor-selective diazaborolane (DAB (1-hydroxy-2,3,1-benzodiazaborine)). When phenylhydrazine is linked to a silyl phenolic ether system, it can be activated by phenylalanine trifluoroborate (Phe-BF 3 , an amino acid derivative with tumor targeting property), and then react with 2-formylphenylboronic acid (2-fPBA) to generate DAB. The inventors applied the D2L strategy to achieve fluorescent labeling of Cetuximab in HeLa cells and mixed it with HT-1080 wild-type cells to achieve selective imaging of HT-1080 FAP-positive cells. In addition, the inventors successfully degraded the target protein (BRD4) in tumors by in vivo synthesizing JQ1(+)-based PROTAC molecules, thereby reducing toxicity in the small intestine. At the same time, gold nanoparticles (AuNPs) are currently used in clinical research for hyperthermia and can be modified with cell-targeting peptides. The inventors achieved recruitment of T cells to attack cancer cells through D2L assembly. Brief Description of the Drawings

[0070] With reference to the following drawings, the present disclosure can be more fully understood.

[0071] Figure 1 Shows the existing chemically regulated BiTE strategy.

[0072] Figure 2 Shows an overview of the solution proposed in the present disclosure and some applicable scenarios.

[0073] Figure 3 Shows the reaction schematic diagram of the desilylation-to-ligation (D2L) strategy proposed in the present invention.

[0074] Figure 4Shows the targeted probe-mediated desilylation coupling reaction: (a) Schematic structural diagram of the desilylation reconnection reaction; (b) UPLC kinetic diagram of the reaction of TBS-DSA with 2-fPBA triggered by the targeted probe Phe-BF 3 ; (c) Substrate decline curve of TBS-DSA and product formation curve of DAB-DSA; (d) UPLC diagram of the stability of TBS-DSA with equivalent 2-fPBA in the PBS / DMSO system; (e) Table of single desilylation rate constant and comprehensive apparent rate constant of the desilylation reconnection reaction.

[0075] Figure 5 Shows the stability tests of unprotected phenylhydrazine and TBS-protected phenylhydrazine: (A) Schematic molecular structural diagram; (B) Kinetic diagram of the stability of unprotected phenylhydrazine and TBS-protected phenylhydrazine in PBS; (C) Stability comparison diagram after 12 hours.

[0076] Figure 6 Shows Phe-BF on the antibody 3 Triggered controllable ligation of fluorescent molecules: (a) Schematic process diagram; (b) Confocal imaging diagrams of different groups of cells; (c) Quantitative analysis bar chart of the comparison of the green fluorescence intensity of cells; where in group i, cells were co-incubated with TBS-Cetuximab and 2-fPBA-FITC for 12 hours, in group ii, TBS-Cetuximab was pretreated with NaF for 12 hours and then co-incubated with 2-fPBA-FITC for 12 hours, in group iii, cells were co-incubated with TBS-Cetuximab, 2-fPBA-FITC and Phe-BF 3 for 12 hours, and in group iv, cells were incubated with TBS-Cetuximab and 2-fPBA-FITC after pretreatment with NaF for 12 hours.

[0077] Figure 7 Shows selective imaging of HT-1080 FAP-positive cells: (a) Flow chart; (b) Fluorescent imaging diagrams of different channels. The control group was incubated with TBS-Sibrotuzumab and 2-fPBA-FITC, and the experimental group was co-treated with TBS-Sibrotuzumab, 2-fPBA-FITC and Phe-BF 3 on HT-1080 mixed cells. The RB channels of both groups represent cells incubated with Sibrotuzumab pre-labeled with rhodamine B (RB); (c) Quantitative analysis of the fluorescence intensity of the FITC channel in (b) (n = 20, mean ± standard error), where each group in (c) is: I. HT-1080 wild-type cells in the control group, II. HT-1080 FAP-positive cells in the control group, III. HT-1080 wild-type cells in the Phe-BF 3 group (i.e., the experimental group), IV. HT-1080 wild-type cells in the Phe-BF3 HT1080 FAP-positive cells in the group.

[0078] Figure 8 Shows Phe-BF 3 Mediates in-situ PROTAC synthesis: (A) Phe-BF 3 Schematic diagram of triggering bimolecular splicing to further induce biprotein ligation; (B) Schematic diagram of the specific reaction structural formula; where the JQ1(+) fragment non-covalently targets the BRD4 target protein, and the thalidomide Thd fragment non-covalently targets the E3 ligase CRBN.

[0079] Figure 9 Shows the evaluation diagram of the interaction between JQ1(+) and its derivatives and BRD4 protein: (A)-(C) are the two-dimensional interaction diagrams of JQ1(+), JQ1(+)-DAB-Thd, and TBS-NHNH-JQ1(+) with the BRD4 protein in the core pocket region of MOE molecular docking respectively; (D) is the fluorescence polarization diagram of JQ1(+) and its derivatives competing with JQ1(+)-FAM fluorescently labeled molecules to inhibit BRD4; (E) is the statistical table of the interaction forces after docking of JQ1(+) and its derivatives with the BRD4 target protein in the core region; (F) is the statistical table of the IC50 of JQ1(+) and its derivatives competing to inhibit the half concentration of the BRD4 target protein; Note a: N20 and NAP40 are the names of the same backbone nitrogen atom for JQ1(+)-DAB-Thd and JQ1(+) respectively.

[0080] Figure 10 Shows the immunoblot imaging and quantitative analysis diagram of BRD4: (A) is the immunoblot imaging diagram of BRD4 protein in HeLa and MV4-11 cells after treatment with different concentrations of JQ1(+)-DAB-Thd, where β-tubulin is the internal reference; (B)-(C) are the quantitative analysis curve diagrams of the BRD4 content in HeLa and MV4-11 cells respectively; (D) and (F) are the immunoblot imaging and quantitative analysis diagrams of BRD4 protein in HeLa and MV4-11 cells after treatment with different binary components respectively, where group a co-incubates TBS-NHNH-JQ1(+) and Phe-BF 3 , group b co-incubates 2-fPBA-Thd and Phe-BF 3 , group c co-incubates 2-fPBA-Thd and TBS-NHNH-JQ1(+); (E) and (G) are the immunoblot imaging and quantitative analysis diagrams of BRD4 protein in the three-component experimental group and the control group without Phe-BF 3 treatment.

[0081] Figure 11 Shows Phe-BF 3T cells can be recruited to attack tumors by assembling gold nanoparticles (AuNPs) decorated with cell-targeting peptides: (a) Phe-BF 3 Schematic diagram of inducing T cells to attack tumor cells; (b) Transmission electron microscopy (TEM) image of the experimental group; (c) Particle size distribution diagram.

[0082] Figure 12 Shows the TEM image (a) and the statistical chart of particle size distribution (b) of the mixed AuNPs at the cellular level, and the optical microscopy image (c) after different drug treatments after co-incubation of B16F10 cells and T cells (5 equivalents); among them, red circles: T cells, blue circles: B6F10 cells.

[0083] Figure 13 Shows the representative fluorescence images of Calcein-AM and propidium iodide (PI) staining of cells (a) after treatment with mixed AuNPs (and Phe-BF 3 ) for 48 hours. Green: live cells, red: dead cells. (b) The survival rate of B16F10 after treatment with T cells, mixed AuNPs and Phe-BF 3 respectively within 48 hours.

[0084] Figure 14 Shows the biodistribution of AuNPs and Phe-BF 3 demonstrating a dual-targeting regulation strategy: (a) At the specified time points, 89 89 Zr]TBS&RGD@AuNPs, 18 3 Zr]2-fPBA&anti-CD3@AuNPs or 89 89 Zr]F]Phe-BF 89 in B16F10 tumor-bearing mice; and (b)-(d) The average standardized uptake values (SUVs) of the main tissues; (e) 89

[0085] Figure 15 Shows the representative TEM images of the liver (a) and tumor (b) tissues of B16F10 tumor-bearing mice. Red arrows indicate AuNPs, and the AuNPs in regions 5 and 6 are significantly larger than those in the control group; (c) Figure 15 ​​​​Analysis of the diameters of AuNPs in (a) and (b) therein.

[0086] Figure 16 It shows that the D2L strategy successfully amplifies and activates the proportion of T cells in the cold tumor B16F10 and produces an anti-tumor effect: (a) experimental flow chart, (b) flow cytometry quantitative analysis of the proportion of double-positive CD3+CD8+ in the tumors of each experimental group, (c) tumor growth curve of mice. Specific implementation manners

[0087] The following description of the present disclosure is only intended to illustrate various different embodiments of the present disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various different equivalent, change and modification schemes can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments will be included herein. All references, including publications, patents and patent applications, cited herein are incorporated herein by reference in their entirety.

[0088] Definition

[0089] Unless otherwise specified, when disclosing or claiming any type of range, it is intended to separately disclose or claim each possible value that the range may reasonably cover, including any sub-ranges subsumed therein. For example, when the number of groups is from 1 to 6, it indicates integers within this range, where 1-6 should be understood to include 1, 2, 3, 4, 5, 6, and also sub-ranges of 1-5, 1-4, and 1-3.

[0090] The description of the present disclosure should be interpreted in accordance with the laws and principles of chemical bonds. In some cases, hydrogen atoms may be removed in order to accommodate substituents at a given position.

[0091] The similar words such as "comprising", "containing" or "including" used in the present disclosure mean that the elements appearing before the word cover the elements listed after the word and their equivalents, without excluding the unrecorded elements. The terms "containing" or "including (comprising)" used herein can be open-ended, semi-closed and closed-ended. In other words, the said terms also include "consisting essentially of...", or "consisting of...".

[0092] When "about" is followed by a value or ratio, it means such value or ratio ±10%, preferably ±5%, more preferably ±1%.

[0093] The term "group" refers to the part formed after a molecule removes one, two or more hydrogen radicals.

[0094] Phenylalanine trifluoroborate (Phe-BF 3)” refers to an amino acid derivative that has tumor targeting properties and has the chemical formula

[0095] The term "desilylation and reconnection" refers to the desilylation of a compound containing a silyl phenol ether group in the presence of Phe - BF 3 followed by connection with a compound derived from 2 - formylphenylboronic acid (2 - fPBA) to form an adduct containing a DAB structure through the methods or products of the present disclosure.

[0096] The term "pharmaceutically active group" refers to a unit derived from a "pharmaceutically active compound", and a "pharmaceutically active compound" refers to any compound that, when administered to a subject in a therapeutically effective amount, has a positive or beneficial effect on the disorder or disease condition of the subject. Preferably, the pharmaceutically active compound has a curative or palliative property and can be administered to improve, alleviate, mitigate, reverse one or more symptoms of a disease or disorder, delay its onset, or reduce its severity. The pharmaceutically active compound can have a prophylactic property and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. Pharmaceutically active compounds include pharmaceutically active peptides or proteins, pharmaceutically active nucleic acids (such as DNA or RNA), and other pharmaceutically active organic or inorganic molecules, such as small molecule compounds (i.e., bioactive organic compounds with a molecular weight less than 900 daltons).

[0097] The term "antibody group" means a unit derived from an "antibody", and an "antibody" includes intact antibodies and any antigen - binding fragment or single - chain thereof, its variants or derivatives. Thus, the term "antibody" includes any protein or peptide containing a molecule that comprises at least a portion of an immunoglobulin molecule. Examples thereof include, but are not limited to, the complementarity - determining regions (CDRs) of the heavy or light chains or their ligand - binding portions, the variable regions of the heavy or light chains, the constant regions of the heavy or light chains, the framework (FR) regions, or any portion thereof, or at least a portion of a binding protein, any of which can be incorporated into the antibodies of the present disclosure. The term "antibody" also aims to cover its digestion fragments, specific portions, derivatives, and variants, including antibody mimetics or antibody portions that contain the structure and / or function of a mimicked antibody or its specific fragment or portion (including single - chain antibodies and their fragments).

[0098] The term "fluorophore" refers to a compound structure that can absorb photons of a certain energy (wavelength) and emit fluorescence. It usually contains an aromatic group or a planar heterocyclic molecule with π bonds. In the present disclosure, it is sometimes also abbreviated as "Flu" for substitution. A fluorophore can be an independent organic small molecule or a fluorescent component of a certain functional system. For example, a specialized structure formed by chemical modification of three amino acids in a fluorescent protein is called a chromophore. The "fluorophore" used in the present disclosure can be any group known to those skilled in the art. For example, it can be a fluorescent protein (such as blue fluorescent protein mTagBFP2, cyan fluorescent protein mTurquoise2, green fluorescent protein EGFP, red fluorescent protein mCherry / tdTomato, far-red fluorescent protein mKate2, etc.), FITC (fluorescein isothiocyanate, such as fluorescein isothiocyanate), DAPI (4',6-diamidino-2-phenylindole), Texas Red, Cyanine5 (Cy5), FAM / SYBR GREEN (fluorescein / SYBR Green I, such as 5-carboxyfluorescein molecule (5-Carboxyfluorescein)), VIC / HEX / TET (viability dye 5, viability dye 6, tetramethylrhodamine), TET, HEX, JOE, ROX, etc., but not limited thereto.

[0099] The term "dye group" can be interchangeably understood in the broadest sense as any part of the above formula that provides visible staining. In the present disclosure, it is sometimes also abbreviated as "Dye" for substitution. The "dye group" used in the present disclosure can be, for example, rhodamine B (RB), congo red, hematoxylin-eosin (H&E), etc., but not limited thereto.

[0100] The term "gold nanoparticles ("nano-gold")" refers to tiny particles of gold with a diameter in the range of 1 to 100 nm, having high electron density, dielectric properties, and catalytic effects, capable of binding to a variety of biological macromolecules without affecting their biological activities. In the present disclosure, it is sometimes also abbreviated as "AuNPs" for substitution.

[0101] The term "linking group" in this context refers to a structural element that connects one part of a compound to another part of the same compound. As understood by those skilled in the art, the nature of the linking group depends on the type of organic reaction used to obtain the connection between the parts of the compound.

[0102] The term "click chemistry" is also known as "link chemistry", "combinatorial chemistry of rapid coupling", and is used to rapidly and reliably complete the chemical synthesis of various molecules through the splicing of small units.

[0103] The term "controllable degradation" in the present disclosure means that a degradation reaction occurs at a desired position by the methods or products of the present disclosure.

[0104] The term "bioorthogonal ligation chemistry" is also referred to as "bioorthogonal chemistry", which refers to a set of reactions that occur in a biological environment and have minimal impact on biomolecules or biochemical processes. It can carry out specific chemical reactions without interfering with the original biochemical processes in the living body.

[0105] The term "proteolysis-targeting chimera therapy" is also referred to as "targeted protein degradation chimera", "PROTAC", which is a heterobifunctional molecule composed of two ligands linked by a linker. One ligand can bind to the target protein, and the other ligand can target the E3 ligase. It is an event-driven mode that can bind to any position of the target protein and may induce the degradation of the target protein without requiring high affinity. PROTAC can induce the formation of a ternary complex of the target protein - PROTAC - E3 ligase.

[0106] The term "T cell-mediated immunotherapy" includes "CAR-T therapy", "chimeric antigen receptor T cell immunotherapy", which is a novel precision targeted therapy for treating tumors. By activating T cells and equipping them with a positioning and navigation device CAR (chimeric antigen receptor on tumors), ordinary "soldiers" T cells are transformed into "super soldiers", that is, CAR-T cells. Using its "positioning and navigation device" CAR, it specifically recognizes tumor cells in the body and releases a large number of various effector factors through immune action, which can efficiently kill tumor cells, thus achieving the purpose of treating malignant tumors.

[0107] The term "T cell-mediated immunotherapy" in this article also refers to BiTE (Bispecific T-cell Engager) therapy, which attacks cancer cells by activating the patient's own immune system (especially T cells). Its core is to use an artificially designed "bispecific antibody" that can simultaneously bind to two different targets: the CD3 molecule on the surface of T cells and the specific antigen of tumor cells, thus "bringing closer" T cells and cancer cells and directly activating T cells to kill tumor cells.

[0108] The term "in-situ synthesis" in the present disclosure means that a synthesis reaction occurs in-situ at a desired position by the methods or products of the present disclosure.

[0109] In this text, the term "drug" means any substance that can affect the physiological functions of an organism, and also includes substances that can measure physiological function indicators of an organism, and these substances are used for preventing diseases, treating diseases, diagnosing diseases, identifying targets, screening targets, and / or regulating the physiological functions of humans.

[0110] The present disclosure provides a method for desilylation and reconnection, wherein the method comprises: in the presence of phenylalanine boron trifluoride (Phe-BF 3 ), reacting compound A containing a silyl phenol ether group with compound B derived from 2-formylphenylboronic acid (2-fPBA).

[0111] In an embodiment of the present disclosure, compound A has the following structure:

[0112] The R 1 is selected from H, a protecting group, a drug active group, an antibody group, a fluorescent group, a staining group, or a gold nanoparticle. The antibody group can be, for example, but not limited to, derived from trastuzumab, Sibrotuzumab, anti-BRD4 primary antibody, anti-BRD4 secondary antibody, anti-tubulin primary antibody, anti-rabbit IgG secondary antibody, anti-mouse IgG secondary antibody, anti-lysozyme antibody; the fluorescent group can be, for example, but not limited to, FITC fluorescein, 5-carboxyfluorescein molecule (5-Carboxyfluorescein, FAM); the staining group can be, for example, but not limited to, rhodamine B, hematoxylin-eosin (H&E).

[0113] In a preferred embodiment of the present disclosure, compound A is selected from:

[0114]

[0115] wherein Ab is an antibody group, Flu is a fluorescent group, Dye is a staining group, and AuNPs is a gold nanoparticle. The antibody group can be, for example, but not limited to, derived from trastuzumab, Sibrotuzumab, anti-BRD4 primary antibody, anti-BRD4 secondary antibody, anti-tubulin primary antibody, anti-rabbit IgG secondary antibody, anti-mouse IgG secondary antibody, anti-lysozyme antibody; the fluorescent group can be, for example, but not limited to, FITC fluorescein, 5-carboxyfluorescein molecule (5-Carboxyfluorescein, FAM); the staining group can be, for example, but not limited to, rhodamine B, hematoxylin-eosin (H&E).

[0116] In an embodiment of the present disclosure, compound B has the following structure:

[0117] The R 2 is selected from H, a linking group, a fluorescent group or a staining group; the R 3 is selected from H, an alkyl group, an aryl group or a heteroaryl group, which may be optionally substituted. The fluorescent group may be, for example, but not limited to, FITC fluorescein, 5-carboxyfluorescein molecule (5-Carboxyfluorescein, FAM); the staining group may be, for example, but not limited to, rhodamine B, hematoxylin-eosin (H&E).

[0118] In a preferred embodiment of the present disclosure, the compound B is selected from:

[0119] wherein, Flu is a fluorescent group, Dye is a staining group, and the R 3 is selected from H, an alkyl group, an aryl group or a heteroaryl group, which may be optionally substituted. The fluorescent group may be, for example, but not limited to, FITC fluorescein, 5-carboxyfluorescein molecule (5-Carboxyfluorescein, FAM); the staining group may be, for example, but not limited to, rhodamine B, hematoxylin-eosin (H&E).

[0120] In an embodiment of the present disclosure, the method of desilylation and reconnection has the following reaction formula:

[0121]

[0122] In a preferred embodiment of the present disclosure, the reaction formula may be:

[0123]

[0124] In a preferred embodiment of the present disclosure, the reaction formula may be:

[0125]

[0126] The present disclosure also provides the use of any one of the above methods of desilylation and reconnection in the fluorescence controllable labeling of antibodies.

[0127] That is, the present disclosure provides a method for fluorescence controllable labeling of antibodies, including performing any one of the above methods of desilylation and reconnection.

[0128] The present disclosure also provides the use of any one of the above methods of desilylation and reconnection in the positive cell screening of antibodies.

[0129] That is, the present disclosure provides a method for positive cell screening of antibodies, including performing any one of the above methods of desilylation and reconnection.

[0130] The present disclosure also provides the use of any of the above-mentioned desilylation and reconnection methods in the controllable degradation of target proteins by antibodies.

[0131] That is, the present disclosure provides a method for controllably degrading target proteins by antibodies, including performing any of the above-mentioned desilylation and reconnection methods.

[0132] The present disclosure also provides the use of any of the above-mentioned desilylation and reconnection methods in bioorthogonal ligation chemistry.

[0133] That is, the present disclosure provides a method for bioorthogonal ligation chemistry, including performing any of the above-mentioned desilylation and reconnection methods.

[0134] In an embodiment of the present disclosure, the bioorthogonal ligation chemistry is selected from proteolysis-targeting chimera therapy or T cell-mediated immunotherapy. In a preferred embodiment of the present disclosure, the proteolysis-targeting chimera therapy includes an in-situ synthesized proteolysis-targeting chimera drug. In a preferred embodiment of the present disclosure, the T cell-mediated immunotherapy includes an in-situ synthesized nanodrug.

[0135] The present disclosure also provides the use of any of the above-mentioned desilylation and reconnection methods in precision anti-tumor therapy.

[0136] That is, the present disclosure provides a method for precision anti-tumor therapy, including performing any of the above-mentioned desilylation and reconnection methods.

[0137] In some specific embodiments, the above method of the present disclosure can be carried out in vitro or in vivo; in a preferred embodiment, the method is carried out in vivo.

[0138] In another aspect of the present disclosure, there is provided a method for treating cancer, which includes administering phenylalanine boron trifluoride (Phe-BF 3 ), compound A containing a silyl phenol ether group, and compound B derived from 2-formylphenylboronic acid (2-fPBA) to a subject. In some embodiments, the above three substances are administered in sequence; in other embodiments, compound A containing a silyl phenol ether group and compound B derived from 2-formylphenylboronic acid (2-fPBA) are first administered to the subject, and then phenylalanine boron trifluoride (Phe-BF 3 ) is administered to the subject; in other embodiments, Phe-BF 3 , compound A, and compound B are administered to the same site in the subject. The structures of compound A and compound B are as defined herein. The reactions of phenylalanine boron trifluoride (Phe-BF 3 ), compound A containing a silyl phenol ether group, and compound B derived from 2-formylphenylboronic acid (2-fPBA) are as defined herein.

[0139] It should be understood that in other methods of the present disclosure, phenylalanine boron trifluoride (Phe - BF 3 ), compound A containing a silicyl phenol ether group, and compound B derived from 2 - formylphenylboronic acid (2 - fPBA) should also be provided to the subject or test sample in sequence.

[0140] In another aspect of the present disclosure, a kit is provided, which comprises: 1) phenylalanine boron trifluoride (Phe - BF 3 ); 2) compound A containing a silicyl phenol ether group; and 3) compound B derived from 2 - formylphenylboronic acid (2 - fPBA).

[0141] In an embodiment of the present disclosure, compound A has the following structure:

[0142] The R 1 is selected from H, a protecting group, a pharmaceutically active group, an antibody group, a fluorescent group, a staining group, or gold nanoparticles. The antibody group can be, for example, but not limited to, derived from trastuzumab, Sibrotuzumab, anti - BRD4 primary antibody, anti - BRD4 secondary antibody, anti - tubulin primary antibody, anti - rabbit IgG secondary antibody, anti - mouse IgG secondary antibody, anti - lysozyme antibody; the fluorescent group can be, for example, but not limited to, FITC fluorescein, 5 - carboxyfluorescein molecule (5 - Carboxyfluorescein, FAM); the staining group can be, for example, but not limited to, rhodamine B, hematoxylin - eosin (H&E).

[0143] In a preferred embodiment of the present disclosure, compound A is selected from:

[0144]

[0145] wherein, Ab is an antibody group, Flu is a fluorescent group, Dye is a staining group, and AuNPs are gold nanoparticles. The antibody group can be, for example, but not limited to, derived from trastuzumab, Sibrotuzumab, anti - BRD4 primary antibody, anti - BRD4 secondary antibody, anti - tubulin primary antibody, anti - rabbit IgG secondary antibody, anti - mouse IgG secondary antibody, anti - lysozyme antibody; the fluorescent group can be, for example, but not limited to, FITC fluorescein, 5 - carboxyfluorescein molecule (5 - Carboxyfluorescein, FAM); the staining group can be, for example, but not limited to, rhodamine B, hematoxylin - eosin (H&E).

[0146] In an embodiment of the present disclosure, the compound B has the following structure:

[0147] The R 2 is selected from H, a linking group, a fluorescent group or a staining group; the R 3 is selected from H, an alkyl group, an aryl group or a heteroaryl group, which may be optionally substituted. The fluorescent group may be, for example, but not limited to, FITC fluorescein, 5-carboxyfluorescein molecule (5-Carboxyfluorescein, FAM); the staining group may be, for example, but not limited to, rhodamine B, hematoxylin-eosin (H&E).

[0148] In a preferred embodiment of the present disclosure, the compound B is selected from:

[0149] wherein, Flu is a fluorescent group, Dye is a staining group, and the R 3 is selected from H, an alkyl group, an aryl group or a heteroaryl group, which may be optionally substituted. The fluorescent group may be, for example, but not limited to, FITC fluorescein, 5-carboxyfluorescein molecule (5-Carboxyfluorescein, FAM); the staining group may be, for example, but not limited to, rhodamine B, hematoxylin-eosin (H&E).

[0150] The present disclosure also provides the use of any one of the above-mentioned kits in the fluorescence controllable labeling of antibodies.

[0151] The present disclosure also provides the use of any one of the above-mentioned kits in the screening of positive cells of antibodies.

[0152] The present disclosure also provides the use of any one of the above-mentioned kits in the controllable degradation of target proteins by antibodies.

[0153] The present disclosure also provides the use of any one of the above-mentioned kits in bioorthogonal ligation chemistry.

[0154] In an embodiment of the present disclosure, the bioorthogonal ligation chemistry is selected from proteolysis-targeting chimera therapy or T cell-mediated immunotherapy. In a preferred embodiment of the present disclosure, the proteolysis-targeting chimera therapy comprises an in-situ synthesized proteolysis-targeting chimera drug. In a preferred embodiment of the present disclosure, the T cell-mediated immunotherapy comprises an in-situ synthesized nanodrug.

[0155] The present disclosure also provides the use of any one of the above-mentioned kits in precision anti-tumor therapy.

[0156] The present disclosure also provides phenylalanine boron trifluoride (Phe-BF 3) and / or compound A containing a silicon-based phenol ether group, and / or compound B derived from 2-formylphenylboronic acid (2-fPBA), or the use of the aforementioned kit in the preparation of a drug.

[0157] In an embodiment of the present disclosure, the drug is used for fluorescence controllable labeling of antibodies, positive cell screening of antibodies, controllable degradation of target proteins by antibodies, bioorthogonal ligation chemistry, and / or precise anti-tumor therapy.

[0158] In an embodiment of the present disclosure, the bioorthogonal ligation chemistry is selected from proteolysis-targeting chimera therapy or T cell-mediated immunotherapy.

[0159] In an embodiment of the present disclosure, the drug in situ synthesizes a proteolysis-targeting chimera drug after administration.

[0160] In an embodiment of the present disclosure, the drug in situ synthesizes a nanodrug after administration.

[0161] In an embodiment of the present disclosure, compound A has the following structure:

[0162] The R 1 is selected from a protecting group, a drug active group, an antibody group, a fluorescent group, a staining group, or a gold nanoparticle.

[0163] In a preferred embodiment of the present disclosure, compound A is selected from:

[0164]

[0165] wherein Ab is an antibody group, Flu is a fluorescent group, Dye is a staining group, and AuNPs is a gold nanoparticle.

[0166] In an embodiment of the present disclosure, compound B has the following structure:

[0167] The R 2 is selected from H, a linking group, a fluorescent group, or a staining group; the R 3 is selected from H, an alkyl group, an aryl group, or a heteroaryl group, which may be optionally substituted.

[0168] In a preferred embodiment of the present disclosure, compound B is selected from:

[0169] wherein Flu is a fluorescent group, Dye is a staining group, and the R 3 is selected from H, an alkyl group, an aryl group, or a heteroaryl group, which may be optionally substituted.

[0170] Example

[0171] To enable those skilled in the art to better understand the present disclosure solution, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all of the embodiments.

[0172] I. Instrument information.

[0173] Table 1. Main instrument statistical table

[0174] Instrument Name Model or Manufacturer Microplate Reader Bio-Rad Analytical Balance A&D, GH-202 Rotary Evaporator IKA Cell Incubator Eppendorf, Galaxy 170S Ultra-pure Water System Millipak Express 40 Thermostatic Mixer TMS-200 Freeze Dryer LGJ-10E, Beijing Sihuan Ultra Performance Liquid Chromatography Waters FTN-H Ultra-micro UV Spectrophotometer Thermo NANO DROP ONE Dual-beam UV-Vis Spectrophotometer UV180, Muxi Instruments Fluorescence Spectrophotometer Hitachi F-7000 Heated Magnetic Stirrer IKA, RCT basic Ultra Performance Liquid Chromatography Waters, e2695, equipped with 2996PDA and QDA detectors Positron Emission Tomography Scanner Mediso nanoScan Laser Scanning Confocal Microscope Nikon, A1R-si

[0175] II. Reagent information.

[0176] Table 2. Main reagent statistical table

[0177]

[0178]

[0179] III. Experimental method

[0180] 1. Exemplary chemical synthesis method.

[0181] (1) Synthesis route of TBS-protected phenylhydrazine

[0182]

[0183] Dissolve 40 mg (1 eq) of p-nitrophenyl carbonate in anhydrous THF. Under liquid nitrogen freezing, add 2.0 eq of phenylhydrazine and triethylamine, immediately plug with a rubber stopper, evacuate the air and replace it with nitrogen. After the THF melts, freeze-thaw and replace the gas three times repeatedly. Finally, react at room temperature for 2 days under a nitrogen atmosphere. The whole process needs to ensure complete anaerobic and anhydrous conditions. After the reaction is completed, dilute the system with EA, wash with water to remove salts, dry with anhydrous sodium sulfate, concentrate and column, and purify with a petroleum ether-ethyl acetate system to obtain 28 mg of white solid with a yield of 76%. 1 H-NMR (400 MHz, CDCl 3 ) δ 7.32–7.18 (m, 4H), 6.89 (t, J = 7.4 Hz, 1H), 6.80 (d, J = 7.9 Hz, 4H), 6.57 (s, 1H), 5.75 (s, 1H), 5.08 (s, 2H), 0.98 (s, 9H), 0.20 (s, 6H). 13 C-NMR (101 MHz, CDCl 3)δ169.75,161.69,156.68,156.23,153.70,132.77,130.31,128.33,120.34,116.39,112.10,67.98,25.80,25.79,18.34,-4.28.HR-MS(ESI):m / z based on C 20 H 29 N 2 O 3 Calculated value of [M + H]+ for Si+ 373.19420, measured value 373.19389.

[0184] (2) Synthetic route of TBS-protected phenylhydrazide activated ester

[0185]

[0186] The synthesis of intermediate TBS-NHNH-Ph-COOH refers to the steps of Formula 1, and a white solid product is also obtained. 1 H-NMR(400MHz, DMSO-d 6 )δ12.21(s, 1H), 9.28(s, 1H), 8.35(s, 1H), 7.75(d, J = 8.6Hz, 2H), 7.08(dd, J = 173.1, 8.0Hz, 4H), 6.67(d, J = 8.6Hz, 2H), 5.02(s, 2H), 0.95(s, 9H), 0.19(s, 6H). 13 C-NMR(101MHz, DMSO)δ167.75, 157.16, 155.49, 153.60, 131.42, 130.36, 129.99, 120.46, 120.23, 111.00, 66.24, 26.03, 18.43, -4.07.HR-MS(ESI):m / z based on C 21 H 32 N 3 O 5 Calculated value of [M + NH 4 + + 434.21057, measured value 434.21033.

[0187] Dissolve the intermediate in anhydrous dichloromethane, add 1.5eq of N-hydroxysuccinimide and 2.0eq of EDC, and 3.0eq of triethylamine, and react at room temperature for 2 hours. After detecting the end of the reaction by TLC, dilute the system with dichloromethane, wash the system with water and brine, dry and concentrate, and then purify by column chromatography to obtain a white solid product, denoted as TBS-NHNH-NHS ester. 1 H-NMR(400MHz, CDCl 3)δ8.00–7.94(m,2H),7.23(s,2H),6.81(dd,J=11.3,8.1Hz,4H),6.71(s,1H),6.26(s,1H),5.09(s,2H),2.86(s,4H),0.98(s,9H),0.20(s,6H). 13 C-NMR(101MHz,CDCl 3 )δ169.75,161.69,156.68,156.23,153.70,132.77,130.31,128.33,120.34,116.39,112.10,67.98,25.80,25.79,18.34,-4.28.HR-MS(ESI):m / z based on C 25 H 35 N 4 O 7 Si+ calculated value of [M + NH 4 + + 531.22695, measured value 531.22700.

[0188] (3) Synthesis route of TBS-NHNH-Dansyl

[0189]

[0190] Dissolve the activated ester purified from Formula 2 in anhydrous THF, add a 1.2 eq THF solution of Dansyl-C4-NH 2 under an ice bath, stir for a few minutes, add a few drops of TEA, then displace with a water pump to a nitrogen atmosphere, react at room temperature for 2 hours, directly spin-dry the system, and purify by column chromatography with a dichloromethane-methanol system to obtain the light yellow solid product TBS-NHNH-Dansyl. 1 H-NMR(400MHz,CDCl 3)δ8.52(d,J=8.5Hz,2H),8.30(d,J=8.6Hz,1H),8.21(d,J=7.3Hz,1H),7.58(d,J=8.4Hz,2H),7.50(td,J=8.6,7.4Hz,2H),7.23(s,1H),7.15(d,J=7.5Hz,1H),6.89–6.78(m,3H),6.71(d,J=8.3Hz,2H),6.25(t,J=5.9Hz,1H),6.10(s,1H),5.33(s,1H),5.08(s,2H),3.26(q,J=6.3Hz,2H),2.90(d,J=6.5Hz,1H),2.86(s,6H),1.45(dq,J=11.7,6.1,5.2Hz,4H),1.25(s,1H),0.98(s,9H),0.19(s,6H). 13 C-NMR(101MHz,CDCl 3 )δ167.73,157.28,155.90,151.73,150.79,134.99,130.20,130.08,130.06,129.80,129.63,129.33,128.56,128.54,128.27,126.05,123.26,120.19,119.08,115.22,111.96,67.49,45.92,45.42,42.91,39.32,29.72,26.83,26.58,25.69,18.21,14.16,8.57,0.03,-4.39.HR-MS(ESI):m / z based on C 37 H 50 N 5 O 6 SSi+ calculated value of [M + H]+ 720.32456, measured value 720.32414.

[0191] (4) Synthesis route of 2-fPBA-Thd

[0192]

[0193] Dissolve 4-hydroxy thalidomide (2.7 g, 10 mmol) and 1.5 eq of tert-butyl 6-bromohexanoate in 50 mL of DMF, add 6.9 g of anhydrous potassium carbonate (5 eq), stop heating after reacting at 80 °C for 2 hours, dilute the system with a large excess of dichloromethane, wash with water to remove DMF, dry and concentrate by rotary evaporation, and purify by column chromatography to obtain 4 g of Thd-C6-OtBu. 1 H-NMR(400MHz,CDCl 3)δ8.35(s,1H),7.72–7.59(m,1H),7.42(d,J=7.2Hz,1H),7.19(d,J=8.5Hz,1H),4.94(dd,J=12.3,5.3Hz,1H),4.16(t,J=6.5Hz,2H),2.95–2.77(m,2H),2.77–2.66(m,1H),2.24(t,J=7.4Hz,2H),2.15–2.07(m,1H),1.89(p,J=6.8Hz,2H),1.66(p,J=7.3Hz,2H),1.55(q,J=4.2,3.3Hz,1H),1.42(s,9H). 13 C-NMR(101MHz,CDCl 3 )δ173.01,171.12,168.19,167.09,165.66,156.63,136.49,133.81,118.93,117.12,115.75,80.12,69.14,49.08,35.37,31.39,28.65,28.12,25.32,24.68,22.62.HR-MS(ESI):m / z based on C 23 H 29 N 2 O 7 + calculated value of [M + H]+ 445.19693, measured value 445.19694.

[0194] The tert-butyl protecting group of the above-obtained product was directly removed with a 50% TFA dichloromethane solution. After the disappearance of the raw material was monitored by TLC, the system was directly rotary evaporated to obtain an oily liquid. The oily substance was dissolved in DMF, and then triethylamine and HATU were added. After all the carboxylic acid derivatives of thalidomide were converted into the corresponding activated esters, the trifluoroacetate salt of the synthetic intermediate 2-fPBA was added and reacted at room temperature. After the reaction was completed, the reaction solution was directly separated and purified by preparative HPLC to obtain the product 2-fPBA-Thd. 1H-NMR (400 MHz, MeOD) δ 7.77 (ddd, J = 8.9, 7.3, 1.8 Hz, 1H), 7.53–7.36 (m, 2H), 7.21 (d, J = 8.1 Hz, 1H), 7.07–6.86 (m, 2H), 5.12 (dt, J = 12.3, 6.2 Hz, 1H), 4.14 (dp, J = 37.5, 5.8, 5.2 Hz, 4H), 3.60 (td, J = 5.5, 1.8 Hz, 2H), 3.36–3.34 (m, 2H), 2.98–2.64 (m, 4H), 2.30 (t, J = 7.3 Hz, 2H), 2.13 (dtd, J = 11.0, 5.2, 2.7 Hz, 1H), 1.89 (p, J = 6.8 Hz, 2H), 1.75 (p, J = 7.4 Hz, 2H), 1.66–1.52 (m, 2H), 1.43–1.31 (m, 2H). 13 C-NMR (101 MHz, MeOD) δ 175.09, 173.19, 170.05, 167.27, 165.98, 159.06, 156.59, 142.76, 136.56, 133.68, 131.59, 128.91, 119.15, 116.72, 114.93, 113.86, 111.63, 102.43, 68.95, 66.07, 38.71, 35.51, 30.78, 28.21, 25.20, 25.09, 22.27。

[0195] (5) Synthetic route of JQ1(+)-DAB-Thd

[0196]

[0197] Dissolve JQ1(+) in a 50% TFA dichloromethane solution. After the tert-butyl group is removed, the system is directly evaporated to dryness. Add triethylamine and HATU. After reacting for 30 minutes, add N-Boc-1,4-butanediamine and react at room temperature for 2 hours. Wash with saturated brine 2 - 3 times and then purify the product BocNH-JQ1(+) by column chromatography. 1 H-NMR (400 MHz, CDCl 3) δ 7.40 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 5.9 Hz, 1H), 4.77 (s, 1H), 4.64 (t, J = 7.0 Hz, 1H), 3.56 (dd, J = 14.3, 7.8 Hz, 1H), 3.32 (dq, J = 21.0, 6.4 Hz, 3H), 3.12 (d, J = 6.3 Hz, 2H), 2.68 (s, 3H), 2.40 (s, 3H), 1.67 (s, 3H), 1.61–1.48 (m, 4H), 1.44 (s, 9H). 13 C-NMR(101MHz, CDCl 3 ) δ 170.49, 163.91, 156.05, 155.70, 149.91, 136.81, 136.61, 132.13, 130.92, 130.85, 130.49, 129.83, 128.72, 54.54, 40.22, 39.41, 39.21, 28.46, 28.40, 27.37, 26.77, 14.38, 13.09, 11.82. HR-MS(ESI): m / z based on C 28 H 36 ClN 6 O 3 S+ calculated value [M + H]+ 571.22526, measured value 571.22425.

[0198] After removing the tert-butoxycarbonyl group from the product BocNH-JQ1(+) in the previous step with a 10% TFA dichloromethane solution, it was concentrated to obtain an oily liquid. After dissolving it in DMF, it was added to 4-(2-N-tert-butoxycarbonylhydrazino)benzoic acid that had been pretreated with triethylamine and HATU. After judging the completion of the reaction by TLC analysis, the same washing operation as before was carried out, and then the product BocNHNH-JQ1(+) was purified by column chromatography. 1 H-NMR(400MHz, CDCl 3 ) δ 7.67 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.17 (s, 1H), 6.93 (d, J = 20.7 Hz, 2H), 6.71 (d, J = 8.2 Hz, 3H), 4.65 (dd, J = 8.4, 5.7 Hz, 1H), 3.56 (dd, J = 14.5, 8.5 Hz, 1H), 3.29 (dd, J = 13.8, 5.4 Hz, 3H), 3.18 (s, 2H), 2.61 (s, 3H), 2.38 (s, 3H), 1.65 (s, 3H), 1.44 (s, 13H). 13C-NMR(101MHz,CDCl 3 ) δ 170.58, 167.53, 164.05, 156.30, 155.63, 151.28, 150.00, 136.80, 136.59, 132.06, 131.02, 130.86, 130.43, 129.87, 128.73, 128.63, 111.88, 81.30, 54.51, 39.53, 39.13, 29.70, 28.26, 26.67, 14.37, 13.09, 11.75. HR-MS(ESI): m / z calculated for C 35 H 42 ClN 8 O 4 S+ [M + H]+ 705.27328, found 705.27301.

[0199] Finally, TFA-treated BocNHNH-JQ1(+) was reacted with 2-fPBA-Thd, and the final product JQ1(+)-DAB-Thd was isolated and purified by thin-layer chromatography on silica gel plates. 1 H-NMR(400MHz,CD 3 SOCD 3 ) δ 11.09 (s, 1H), 9.02 (s, 1H), 8.43 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 8.3 Hz, 1H), 8.22 (t, J = 5.7 Hz, 1H), 8.13 (d, J = 11.2 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H), 7.82–7.72 (m, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.47 (t, J = 9.1 Hz, 3H), 7.42 (d, J = 1.9 Hz, 3H), 7.34 (d, J = 2.4 Hz, 1H), 7.25 (dd, J = 8.4, 2.4 Hz, 1H), 5.07 (dd, J = 12.8, 5.4 Hz, 1H), 4.51 (dd, J = 8.3, 5.8 Hz, 1H), 4.13 (t, J = 6.1 Hz, 4H), 3.48 (q, J = 5.7 Hz, 3H), 2.87 (ddd, J = 17.2, 14.0, 5.4 Hz, 1H), 2.59 (s, 4H), 2.39 (s, 3H), 2.14 (t, J = 7.3 Hz, 2H), 2.07–1.93 (m, 2H), 1.74 (p, J = 6.8 Hz, 2H), 1.61 (s, 9H), 1.43 (t, J = 7.4 Hz, 2H), 1.33 (s, 1H), 1.23 (s, 4H). 13 C-NMR(151MHz,CD 3SOCD 3 )δ173.27, 172.93, 170.45, 169.87, 167.32, 166.30, 165.79, 163.50, 161.50, 156.44, 155.61, 150.31, 149.15, 139.96, 137.46, 137.23, 135.71, 134.19, 133.70, 132.74, 131.17, 131.06, 130.59, 130.32, 130.06, 128.96, 127.72, 123.84, 120.15, 118.68, 116.65, 115.59, 110.27, 69.13, 67.01, 55.40, 54.37, 49.20, 39.39, 38.81, 38.59, 38.13, 35.69, 34.80, 31.43, 28.67, 27.28, 27.16, 25.42, 25.37, 22.48, 14.53, 13.15, 11.78. HR-MS(ESI): m / z based on C 58 H 60 BClN 11 O 10 Calculated value of [M + H]+ for S+ 1148.40214, measured value 1148.40214.

[0200] (6) Synthesis route of TBS-NHNH-JQ1(+)

[0201]

[0202] Similar to the previous operation, react the TFA-treated BocNH-JQ1(+) with the product TBS-NHNH-NHS ester in Formula 2 to obtain the target compound TBS-NHNH-JQ1(+). 1 H-NMR(400MHz, CDCl 3) δ 7.66 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.25–6.99 (m, 5H), 6.91 (t, J = 5.4 Hz, 1H), 6.81 (d, J = 7.8 Hz, 2H), 6.71 (d, J = 8.2 Hz, 2H), 5.07 (s, 2H), 4.71–4.59 (m, 1H), 3.56 (dd, J = 14.5, 8.6 Hz, 1H), 3.39–3.27 (m, 3H), 3.16 (d, J = 19.7 Hz, 2H), 2.60 (s, 3H), 2.38 (s, 3H), 1.65 (s, 3H), 1.35–1.19 (m, 4H), 0.97 (s, 9H), 0.19 (s, 6H). 13 C-NMR (101 MHz, CDCl 3 ) δ 170.68, 164.23, 157.28, 156.04, 155.74, 150.90, 150.13, 136.96, 136.68, 132.17, 131.16, 131.01, 130.56, 130.16, 129.98, 128.87, 128.75, 128.58, 120.28, 112.12, 67.64, 54.63, 47.19, 39.67, 39.30, 29.83, 26.86, 26.79, 25.79, 18.33, 14.49, 13.21, 11.87, 8.80, -4.28. HR-MS (ESI): m / z calculated for C 44 H 54 ClN 8 O 5 SSi + [M + H]+ 869.33902, found 869.33951.

[0203] (7) Synthetic route of DAB-DSA

[0204]

[0205] Dissolve the substrate in PBS / DMSO (1:1 ratio), add 3 equivalents of 2-fPBA, and then add Phe-BF 3 (final concentration of) 1 mM to obtain the target product DAB-DSA.

[0206] 2. Synthetic methods of exemplary materials: TBS&RGD@AuNP complex and 2-fPBA&anti-CD3@AuNP complex

[0207] Dissolve 6 mg of chloroauric acid in 100 mL of water, reflux for 1 hour. After cooling slightly, add 3 mL of 1% sodium citrate solution by mass concentration, and continue refluxing for 30 minutes. The solution turns purple-red, and after cooling, Citrate@AuNP is obtained.

[0208] In the above 100 mL solution, add 1 mL of 0.1 M NaHCO 3 aqueous solution, and add mPEG 5k -SH and NH 2 -PEG 5k -SH and DBCO-PEG 5k -SH. After reacting overnight, ultrafiltration and centrifugation are used to replace the aqueous solution to remove free PEG, and NH 2 @AuNP is obtained.

[0209] After the above solution is concentrated ten times and replaced with DMF, add triethylamine and the activated ester of the corresponding modified molecule. After reacting at room temperature for 2 hours, then centrifuge to remove the supernatant. Wash with DMF to remove free small molecules and then replace with an aqueous solution to obtain the modified gold nanoparticles, namely phenylhydrazine modified gold nanoparticles protected by TBS (TBS@AuNP) and 2-fPBA modified gold nanoparticles (2-fPBA@AuNP).

[0210] According to the principle of polypeptide synthesis, use a polypeptide synthesizer to synthesize the required polypeptides N 3 -RGD and N 3 -CD3 in sequence, and then react with the above-mentioned modified gold nanoparticles respectively to obtain the required TBS&RGD@AuNP complex and 2-fPBA&anti-CD3@AuNP complex.

[0211] 3. Ultra-high performance liquid chromatography test method

[0212] Add the DMSO solution of the test molecule (1 mM, 25 μL), the DMSO solution of o-formylphenylboronic acid (10 mM, 5 μL), and the DMSO solution of Phe-BF 3 (100 mM, 5 μL) into a microcentrifuge tube, add 215 μL of DMSO and 250 μL of PBS solution, and then add the same solution to two microcentrifuge tubes respectively. These three microcentrifuge tubes are used as the experimental group. Add the DMSO solution of the test molecule (1 mM, 25 μL) and the DMSO solution of o-formylphenylboronic acid (10 mM, 5 μL) into a microcentrifuge tube, add 220 μL of DMSO and 250 μL of PBS solution as the control group. After mixing the solution in each centrifuge tube, place it on a thermostatic mixer, and start the reaction at 37°C. Sample the above reaction system at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 18 h, and characterize the samples by UPLC.

[0213] 4. Chemical Post-modification Method of Antibodies

[0214] The common tumor cell-targeting Cetuximab was selected for experiments. First, PBS with pH = 7.4 and sodium carbonate / sodium bicarbonate buffer solution with pH = 9 were mixed at a volume ratio of 9:1 to prepare the solution required for the reaction. Subsequently, the DMSO solution of trastuzumab (100 μL, 5 mg / mL) was diluted in 895 μL of the above reaction solution in a microcentrifuge tube. Then, the PBS solution of tert-butyldimethylsilyl-protected phenylhydrazine-N-hydroxysuccinimide ester (TBS-NHNH-NHSester) (100 mM, 5 μL) was slowly added dropwise to the above buffer solution, and the mixture was incubated at 37 °C for 2 hours on a thermostatic mixer.

[0215] After the reaction, the solution was purified with a PD-10 desalting column and eluted with a phosphate-buffered saline solution with pH = 7.4. The eluate was aliquoted into different microcentrifuge tubes in chronological order. Subsequently, the protein concentration in the solution of each microcentrifuge tube was measured with a NanoDrop ONE, and the solution containing the protein was collected.

[0216] 5. Fluorescent Labeling Test Method for Model Antibodies

[0217] The modified antibody above was diluted in a PBS solution of 2 mM CTAC, with a final concentration of approximately 0.3 mg / mL. Subsequently, 1455 μL of the above antibody solution was taken into two microcentrifuge tubes respectively. The DMSO solution of 2-formylphenylboronic acid linked to fluorescein isothiocyanate (2-fPBA-FITC) (2.5 mM, 15 μL) and the DMSO solution of Phe-BF 3 (100 mM, 30 μL) were added to one of the microcentrifuge tubes as the experimental group, while the one without adding Phe-BF 3 was used as the control group.

[0218] After the solutions in the above two centrifuge tubes were shaken evenly, they were placed on a thermostatic mixer, and the reaction started at 37 °C. Samples were taken from the solutions in the two centrifuge tubes every 30 min, with 300 μL taken each time. The sample solution was purified with a PD-10 desalting column and eluted with a phosphate-buffered saline solution with pH = 7.4. The eluate was aliquoted into different microcentrifuge tubes in chronological order. Subsequently, the protein concentration in the solution of each microcentrifuge tube was measured with a NanoDropONE, and the solution containing the protein was collected.

[0219] The protein solutions collected at different times and different groups were diluted to 0.025 mg / mL with a PBS solution, and then added to a cuvette. The fluorescence intensity was measured with a fluorescence spectrophotometer and recorded.

[0220] 6. Immunofluorescence Assay Method

[0221] Incubate the cells in groups using an eight-well plate according to the experimental requirements. After they adhere to the wall, apply the drug in a timely manner. At the specified time point, remove the drug and culture medium incubated in the eight-well plate. Add 200 μL of PBS solution to each well, let it stand for washing, and then replace it with 200 μL of 4% paraformaldehyde solution. Fix at room temperature for 15 minutes and then remove. Wash each well three times with 200 μL of PBS solution, 3 minutes each time. Subsequently, add 200 μL of Triton X solution to each well and perform immunopermeabilization treatment at room temperature for 20 minutes, then remove and wash three times with PBS. Then add 200 μL of 10% goat serum to each well to block for 30 minutes. After removing the original solution, add 200 μL of anti-BRD4 primary antibody solution (product number: ab128874; dilution factor 1:500) to each well and incubate overnight at 4°C in the dark. After removing the original solution, wash each well three times with PBS solution. Then add 200 μL of anti-rabbit-FITC secondary antibody solution (product number: Beyotime A0562; dilution factor 1:360) to each well and incubate for 1 hour at room temperature in the dark. Subsequently, wash with PBS. Add 200 μL of DAPI solution (dilution factor 1:500) to each well and incubate for 15 minutes at room temperature in the dark. After removing the original solution, add 200 μL of PBS solution to each well for one wash. Image the prepared samples under a fluorescence confocal microscope.

[0222] 7. Immunoblotting experiment

[0223] Incubate the cells in groups using a six-well plate according to the experimental requirements. After they adhere to the wall, apply the drug in a timely manner. At the specified time point, remove the drug and culture medium incubated in the six-well plate. Add 1 mL of PBS solution to each well, pipette several times and then discard. Add 500 μL of 0.25% trypsin solution to each well and incubate at 37°C for 2 minutes. Add 500 μL of complete DMEM medium to each well, pipette and mix well to completely detach the cells. Collect the cell solution using a 1.5 mL EP tube and centrifuge for separation. Wash each well twice with 1 mL of PBS solution, and collect the washing solution into the EP tube each time and centrifuge for separation. Then aspirate the excess PBS solution in the EP tube until 50 μL remains, add 10 μL of 6×loading buffer, shake and mix well, and then activate at 95°C for 15 minutes. Store the samples at -20°C. For suspension cells, there is no need for trypsin digestion. Directly collect the culture medium and centrifuge for separation.

[0224] Use a commercial 10-well protein electrophoresis precast gel (4–15%) and precast gel electrophoresis solution. Sequentially add 5 μL of protein gradient Marker or 10–15 μL of protein sample solution into the electrophoresis wells. Set the electrophoresis instrument mode to constant voltage mode, with a voltage of 150 V and an electrophoresis time of 37 minutes. After electrophoresis is completed, take out the gel plate, separate it with a blade, and cut the gel at the 80 kDa band and 2 cm above and below it. Adopt the semi-dry transfer membrane method, and sequentially place from the positive electrode to the negative electrode: large filter paper (pre-saturated with transfer buffer), PVDF membrane (pre-activated with methanol), gel, large filter paper (the same as above). Set the transfer membrane instrument to constant current mode, with the current increasing by 0.1 A for every additional 16 square centimeters of PVDF membrane, and set the transfer membrane time to 40 minutes.

[0225] Place the PVDF membrane after transfer according to the protein bands in two culture dishes respectively, and add skim milk dissolved in 5% TBST to block for 1 hour. Pour it out, add anti-BRD4 or anti-tubulin primary antibody (dilution factor 1:1000) respectively, incubate overnight on a shaker, and supplement with an ice pack to keep it at a low temperature. Pour it out, rinse 3 times with 10% TBST solution, 15 minutes each time, then add anti-rabbit IgG or anti-mouse IgG secondary antibody solution (dilution factor 1:5000), incubate for 1 hour, and keep warm with an ice pack. Pour it out, rinse 3 times with 10% TBST solution, 15 minutes each time. Mix ECL chemiluminescent substrate solutions A and B in equal proportions, and in the dark, immerse the rinsed PVDF membrane in the ECL luminescent substrate for 5 minutes, then put it into an imager for exposure imaging, and the exposure time can vary from 10–90 seconds according to the protein content.

[0226] 8. Construction of tumor-bearing mice and related experimental methods

[0227] All animal experiments were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. All mice used were purchased from Vital River. The parts of this article involving mouse experiments can be operated with reference to this experimental method.

[0228] To construct a tumor model, implant HeLa cells (1×10 6 ) or B16F10 cells (1×10 6 ) into the right abdomen of Nu / Nu nude mice (for HeLa cells) or C57BL / 6N female mice (6–8 weeks old) in the form of 100 μL of PBS. For HeLa tumor mice, when the tumor grows to 200 mm 3 , the mice receive PBS (group a), TBS-JQ1(+)+2-fPBA-Thd (group b), TBS-JQ1(+)+2-fPBA-Thd+Phe-BF3 (Group c) or separate JQ1(+)-DAB-Thd (Group d) treatment. For Groups b and c, TBS-JQ1(+) (injection dose of 10 mg / kg) and 2-fPBA-Thd (dose of 20 mg / kg) were encapsulated in PLGA5k-PEG2k (1 mg / mL) and intravenously injected into mice. For Group c, Phe-BF 3 (dose of 30 mg / kg) was intravenously injected 24 hours and 48 hours after PLGA injection. For Group d, JQ1(+)-DAB-Thd (dose of 5 mg / kg) was encapsulated in PLGA5k-PEG2k (1 mg / mL) and intravenously injected into mice at the specified time. After three rounds of treatment, the mice were sacrificed, and tumor and small intestine sections were stained with anti-BRD4, hematoxylin-eosin (H&E), or anti-lysozyme.

[0229] To evaluate the concentration of JQ1(+)-DAB-Thd synthesized in HeLa tumors, for Groups b and c, tumor-bearing mice were treated according to the treatment protocol at one of the above doses. On the third day, the mice were sacrificed, the tumors were collected, quickly frozen in liquid nitrogen, ground into powder, and then extracted with methanol. After removing methanol, the precipitate was redissolved in acetonitrile, and the PROTAC concentration was quantified by standard methods.

[0230] For B16F10 tumor-bearing mice, when the tumor grew to 50 mm 3 , the mice received PBS (Group a), Phe-BF 3 (Group b), TBS&RGD@AuNPs (Group c), 2-fPBA&anti-CD3@AuNPs (Group d), mixed gold nanoparticles (AuNPs) (Group e), or mixed AuNPs + Phe-BF 3 (Group f) for administration. The doses of AuNPs and Phe-BF 3 were 5 mg / kg and 30 mg / kg, respectively (Phe-BF 3 was intravenously injected 24 hours and 36 hours after AuNPs injection).

[0231] For combination immunotherapy, mice with a tumor volume of 50 mm 3 received PBS (Group i), anti-PD-1 (Group ii), mixed AuNPs + Phe-BF 3 (Group iii), or mixed AuNPs + Phe-BF 3 + anti-PD-1 (Group iv) treatment. For Groups iii and iv, the doses of AuNPs and Phe-BF 3 were 5 mg / kg and 30 mg / kg, respectively. For Groups ii and iv, anti-PD-1 was intraperitoneally injected at a dose of 100 μg per mouse.

[0232] 9. Radioactive labeling experimental method

[0233] A DMSO solution of 3-amino-1-propanamine (10 mM, 7.5 μL) and a DMSO solution of p-NCS-Bz-DFO (10 mM, 5 μL) are first mixed together, and then diluted in a Na 2 CO 3 / NaHCO 3 buffer solution (pH = 9.0, 200 μL) and reacted at 37 °C for 1 hour. Without purification, the above solution is diluted with HEPES buffer (pH = 7.4, 500 μL) and mixed with 2 mCi of 89 Zr] oxalic acid solution and reacted at 37 °C for 30 to 60 minutes. Free 89 Zr] is removed by a short C18 column, and then 89 Zr-labeled N3-DFO is collected with 0.5 mL of ethanol. The above ethanol solution is mixed with TBS@AuNPs or 2-fPBA@AuNPs at 37 °C for 1 hour, and then an excess of N3-RGD or N3-CD3 is added to the reaction system respectively. Free peptides and N3-DFO are removed by centrifugation at 12000 g for 20 minutes three times. Finally, 89 Zr-labeled TBS&RGD@AuNPs and 2-fPBA&anti-CD3@AuNPs are obtained by resuspending the solid AuNPs.

[0234] 18 3 F]Phe-BF 18 F- 19 F isotope exchange (IEX) reaction for radiosynthesis.

[0235] PET / CT imaging studies are performed using a NanoPET / CT scanner . Subsequently, 100 - 200 μCi of 89 Zr-labeled AuNPs or 18 F-labeled Phe-BF 3 are injected into mice through the tail vein to observe their metabolism. Standard data acquisition and image reconstruction are performed in the PET data.

[0236] 10. Flow cytometry experimental method

[0237] ​​Tumor tissues were obtained from B16F10 tumor model mice and treated with an enzyme mixture (collagenase type I and type IV, at a concentration of 1 mg / mL). After various treatments, the cells were filtered through a 70-μm nylon mesh. The resulting single-cell suspension was then centrifuged and washed with PBS containing 1% BSA. The collected cells were stained with the following fluorescent dye-labeled antibodies: anti-mouse CD3 antibody labeled with PE (BioLegend, clone 17A2, catalog no. 100205) and anti-mouse CD8a antibody labeled with APC (BioLegend, clone 53-6.7, catalog no. 100712). During FACS analysis, cell viability was detected using the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (L10119, Invitrogen). Flow cytometry was performed using an LSR Fortessa cell analyzer (BD Biosciences), and data analysis was completed using FlowJo software.

[0238] IV. Examples

[0239] Example 1: Application of the desilylation-reconnection method in the targeted probe-mediated desilylation coupling reaction

[0240] Referring to (7) in the exemplary chemical synthesis method in the experimental method of Part III above, the reaction process was studied. The results are as Figure 4 shown, where (a) is a schematic structural diagram of the desilylation-reconnection reaction, (b) is the UPLC kinetic diagram of the reaction of TBS-DSA with 2-fPBA triggered by the targeting probe Phe-BF 3 , (c) is the decline curve of the TBS-DSA substrate and the production curve of the DAB-DSA product, (d) is the stability UPLC diagram of TBS-DSA and equivalent 2-fPBA in the PBS / DMSO system, and (e) is the table of the single desilylation rate constant and the comprehensive apparent rate constant of the desilylation-reconnection reaction.

[0241] It can be seen from Figure 4 that under the trigger of Phe-BF 3 , TBS-DSA gradually disappeared and the product DAB-DSA gradually formed. During this process, no derivatives of phenylhydrazine were observed, indicating that the rate-determining step of this stepwise reaction is the desilylation step, and the desilylated product immediately reacts with 2-fPBA to irreversibly form the cyclized product DAB-DSA.

[0242] Furthermore, the present disclosure analyzed the kinetic decline curve of the TBS-DSA substrate and the kinetic generation curve of the DAB-DSA product. The rate constant of the substrate decline, which is also the rate constant of the desilylation reaction, was calculated to be 0.044 ± 0.0015 M -1 s -1 by fitting with GraphPad Prism software. Additionally, the rate constant of the DAB-DSA product generation, which is also the comprehensive apparent rate constant of the desilylation and reconnection reaction, was 0.039 ± 0.0014 M -1 s -1 . The two are almost the same, which more fully indicates that the first desilylation process is the rate-determining step. Considering that the reaction rate constant of the Phe-BF 3 desilylation reaction has an order-of-magnitude difference compared with the reaction of phenylhydrazine and 2-fPBA, this result also conforms to the experimental expectation.

[0243] Example 2: Stability experiments of unprotected phenylhydrazine and TBS-protected phenylhydrazine

[0244] Stability experiments were respectively conducted on unprotected phenylhydrazine and TBS-protected phenylhydrazine, and the test methods are as follows:

[0245] Unprotected phenylhydrazine and TBS-protected phenylhydrazine (100 μM) were respectively dissolved in PBS. Samples were taken at the test time points and dissolved in an aqueous solution of 1 mM 2-fPBA. After shaking well for 20 - 30 seconds, UPLC characterization was performed. Quantitative analysis of the generated product or raw material peaks was carried out to obtain the stability data.

[0246] The results of the stability experiment are as Figure 5 shown. The results indicate that the silyl protection strategy can significantly improve the stability of phenylhydrazine (one of the synthons for the coupling reaction). The introduction of TBS silyl ether-protected phenol can achieve controllable "caging" of phenylhydrazine through a carbamate linker. It was experimentally found that unprotected phenylhydrazine decomposed rapidly in PBS, and nearly 54% of unprotected phenylhydrazine decomposed after 12 hours, while the stability of TBS-protected phenylhydrazine remained above 95%. Calculations showed that the half-life of unprotected phenylhydrazine was 8.2 ± 0.93 hours, while the half-life could be extended to more than 100 hours after TBS protection. The improved stability will greatly enrich the application scenarios of phenylhydrazine and lay a foundation for subsequent experiments.

[0247] Example 3: Application of the desilylation and reconnection method in the fluorescence controllable labeling and cell imaging of antibodies

[0248] In this example, HeLa cells were used as an example for research. The experiment was divided into four groups. Group i was used as a control group without treatment. Group ii was pretreated with NaF and could be regarded as monoclonal antibody Cetuximab modified with unprotected phenylhydrazine. The purpose of setting this group was to study whether unprotected phenylhydrazine could achieve cell labeling. Group iii, that is, the experimental group, was used to verify the feasibility of controllable labeling imaging. Group iv labeled FITC fluorescein on monoclonal antibody Cetuximab in a test tube as the positive control group. The specific operation methods are as follows:

[0249] Group i: (1) Cell preparation: Inoculate cells in the logarithmic growth phase at a density of 1×10 5 cells / well in a 6-well plate and culture at 37°C / 5% CO 2 until 80% confluence; (2) Reagent preparation: Dissolve TBS-Cetuximab (final concentration 50 nM) and 2-fPBA-FITC (final concentration 10 μM) in pre-warmed medium; (3) Pretreatment: Remove the original medium, add 2 mL of medium containing the mixed reagents, and incubate in the dark at 37°C / 5% CO 2 for 12 h. (4) Termination treatment: Wash 3 times with PBS, replace with complete medium for fluorescence imaging detection, and operate in the dark throughout the process to prevent fluorescence quenching.

[0250] Group ii: Premix TBS-Cetuximab (10 μM) and NaF (5 mM) in serum-free medium for 12 hours, and then perform co-incubation and subsequent treatments according to the steps of Group i.

[0251] Group iii: Dissolve TBS-Cetuximab (final concentration 50 nM), 2-fPBA-FITC (final concentration 10 μM), and Phe-BF 3 (final concentration 1 mM) in pre-warmed medium, and then perform co-incubation and subsequent treatments according to the steps of Group i.

[0252] Group iv: Premix TBS-Cetuximab (10 μM), 2-fPBA-FITC (20 μM), and NaF (5 mM) in serum-free medium for 12 hours, and then incubate the cells and perform subsequent treatments according to the steps of Group i.

[0253] In addition, the chemical post-modification method of the antibody, the fluorescence labeling test method of the antibody, and the immunofluorescence experiment method refer to Parts 4-6 of the experimental method in the aforementioned Section III.

[0254] The experimental results are as Figure 6 shown. The results show that the desilylation and reconnection method can act on the fluorescence controllable labeling of antibodies and finally be applied to cell imaging.

[0255] Example 4: Application of the desilylation and reconnection method in cell screening

[0256] To verify that the desilication reconnection method can be used in cell screening, the inventors designed an experiment for selective imaging of HT-1080 FAP-positive cells. The immunofluorescence experimental method refers to Example 3, and different-channel fluorescence imaging results are added compared with Example 3.

[0257] In the control group, cells were incubated with TBS-Sibrotuzumab and 2-fPBA-FITC; in the experimental group, cells were treated with TBS-Sibrotuzumab, 2-fPBA-FITC and Phe-BF 3 to co-treat HT-1080 mixed cells. The RB channel in both groups represents cells incubated with rhodamine B (RB)-prelabeled Sibrotuzumab. The role of rhodamine B (RB)-prelabeled Sibrotuzumab is to pre-screen FAP-positive cells for positive reference.

[0258] Control group: (1) Cell preparation: Logarithmic growth phase cells including HT-1080 FAP-positive cells and HT-1080 wild-type cells, i.e., FAP-negative cells, were mixed and seeded in a 6-well plate at a density of 1×10 5 cells / well, and cultured at 37°C / 5% CO 2 until 80% confluence; (2) Reagent preparation: Rhodamine B (RB)-prelabeled Sibrotuzumab (final concentration 50 nM), TBS-Sibrotuzumab (final concentration 50 nM) and 2-fPBA-FITC (final concentration 10 μM) were dissolved in pre-warmed medium; (3) Pretreatment: The original medium was removed, and 2 mL of medium containing the mixed reagents was added, and incubated at 37°C / 5% CO 2 in the dark for 12 h. (4) Termination treatment: Washed 3 times with PBS, replaced with complete medium for fluorescence imaging detection, and the operation was carried out in the dark throughout to prevent fluorescence quenching.

[0259] Experimental group: On the basis of the control group, Phe-BF with a final concentration of 1 mM was co-incubated 3 , and then the cells were incubated and subsequent treatments were carried out according to the steps of the control group. The experimental results are as Figure 7 shown, where (a) is the flow chart of this example; (b) is the fluorescence imaging map of different channels; (c) is the quantitative analysis of the fluorescence intensity of the FITC channel in (b) (n = 20, mean ± standard error); where, I: HT-1080 wild-type cells in the control group, II: HT-1080 FAP-positive cells in the control group, III: HT-1080 wild-type cells in the Phe-BF 3 group (i.e., the experimental group), IV: HT1080 FAP-positive cells in the Phe-BF 3 group.

[0260] Figure 7 The results indicate that the desilylation-reconnection method can effectively distinguish HT-1080 FAP-positive cells from negative cells.

[0261] Example 5: Application of the desilylation-reconnection method in in situ generation of PROTAC

[0262] (1) Mode of action

[0263] The inventors achieved in situ synthesis of PROTAC using the desilylation-reconnection method, and the reaction route is as Figure 8 shown. It should be noted that the reaction product of in situ generation of PROTAC in this example is the same as the product in (5) of the first part of the exemplary chemical synthesis method in the above Part III, except that in this example, Phe-BF 3 was used to trigger the reaction of TBS-protected JQ1(+) with 2-fPBA, and the reaction can be completed in one step under physiological conditions.

[0264] (2) Interaction forces between JQ1(+) and its derivatives and BRD4 target protein

[0265] The inventors performed molecular docking simulations on the interaction forces between JQ1(+) and its derivatives and BRD4 protein. Among them, the immunofluorescence experimental method refers to the sixth part of the experimental methods in the above Part III. The evaluation diagrams are as Figure 9 shown, where (A)–(C) are two-dimensional schematic diagrams of the interaction forces of JQ1(+), JQ1(+)-DAB-Thd, and TBS-NHNH-JQ1(+) with the core pocket region of BRD4 protein in the MOE molecular docking, (D) is the fluorescence polarization diagram of JQ1(+) and its derivatives competing with JQ1(+)-FAM fluorescently labeled molecule to inhibit BRD4, (E) is the statistical table of the interaction forces after molecular docking of JQ1(+) and its derivatives with BRD4 target protein in the core region, and (F) is the statistical table of the IC50 values of JQ1(+) and its derivatives competing to inhibit the half concentration of BRD4 target protein.

[0266] *N20 and NAP40 are the names of the same backbone nitrogen atoms of JQ1(+)-DAB-Thd and JQ1(+), respectively.

[0267] The experiment found that all three compounds have binding forces with BRD4, but there are differences, as Figure 9As shown, there are four simulated interaction sites between JQ1(+) and BRD4 protein. Among them, three coincide with those between JQ1(+)-DAB-Thd and BRD4 protein, and the interaction positions are concentrated in the aromatic ring region of the JQ1(+) backbone and the three sites of the protein receptor Asn140, Pro82, and Ile146 (light orange region). Due to the large steric hindrance and strong hydrophobicity of the TBS protecting group, only three interactions are fitted for TBS-NHNH-JQ1(+), and there is no overlap with the fitting of JQ1(+) (light blue region).

[0268] Steps of the simulated docking experiment:

[0269] The CRBN protein (PDB code: 4CI1) and BRD4 protein (PDB code: 3MXF) are obtained from the PDB database (https: / / www.rcsb.org / ). The conformations of JQ1-DAB-Thd or other molecules are randomly searched by LowModeMD to generate the csearch.mdb file. In the PROTAC plugin of the MOE 2020 software, the Method 4B method is used to generate the csearch.mdb file. Select the Method 4B method in the PROTAC-related plugin of the MOE 2020 software. First, perform protein-protein docking on the CRBN protein and BRD4 protein, and then combine the aforementioned conformation search file as a parameter file for three-body interaction docking.

[0270] To further evaluate the binding affinity of JQ1(+) and its three derivative compounds to the BRD4 protein, in this example, JQ1(+) modified with 5-carboxyfluorescein molecule (5-Carboxyfluorescein, FAM), denoted as JQ1(+)-FAM, is selected to test the binding behavior. The modification method can refer to, for example: Divakaran, A et al., Molecular Basis for the N-Terminal Bromodomain-and-Extra-Terminal-Family Selectivity of a Dual Kinase–Bromodomain Inhibitor. J. Med. Chem. 2018, 61(20), 9316-9334.

[0271] When this molecule binds to BRD4, the molecular vibration is affected, so there will be a higher fluorescence polarization value. When it is competitively excluded by other molecules, it will return to the free state, and at this time the fluorescence polarization value is weak. Based on this principle, in this example, the fluorescence polarization curves of different concentrations of JQ1(+) and its derivatives competing with the JQ1(+)-FAM fluorescently labeled molecule to inhibit BRD4 are studied ( Figure 9in D), from Figure 9 It can be analyzed that the blue JQ1(+) basically coincides with the red JQ1(+)-DAB-Thd, and the green TBS-NHNH-JQ1(+) is generally larger.

[0272] Fluorescence polarization method:

[0273] FAM-modified JQ1(+) and BRD4 protein were co-incubated with different concentrations of JQ1(+), TBS-JQ1(+), JQ1(+)-DAB-Thd or JQ1(-) for 1 hour. Then, they were transferred to a 384-well plate, and the fluorescence polarization value was measured by a multimode microplate reader. The excitation wavelength was selected as 485 nm, and the emission wavelength was selected as 535 nm. All molecules were dissolved in a buffer containing 50 mM HEPES, 100 mM NaCl and 4 mM CHAPS (pH 7.4). The final concentrations of BRD4 and FAM-JQ1(+) were 60 nM and 50 nM, respectively.

[0274] Half maximal inhibitory concentration (IC 50 ) results showed that JQ1(+) was 89 ± 21 nM, JQ1(+)-DAB-Thd was 93 ± 22 nM, and there was almost no difference between them. While TBS-NHNH-JQ1(+) was 206 ± 59 nM, which was 2.3 times that of JQ1(+). This indicates that although carrying a complete target molecule, introducing TBS silane can still widen the gap, demonstrating the universality of the silane protection strategy.

[0275] (3) Phe-BF 3 Role and efficiency in controllable degradation of target proteins

[0276] The inventors further evaluated the role and efficiency of Phe-BF in controllable degradation of target proteins by Western blotting technology. 3 The Western blotting experimental method can refer to the 7th part of the Western blotting experiment in the above-mentioned Part III.

[0277] First, in this example, the content of BRD4 protein in HeLa and MV4-11 cells treated with different concentrations of JQ1(+)-DAB-Thd was tested. The test method can refer to the 7th part of the Western blotting experiment in the above-mentioned Part III. As Figure 10As shown, the study found that JQ1(+)-DAB-Thd degraded 69% of the target protein at 100 nM in human cervical cancer HeLa cells and 58% of the target protein at only 1 nM in human acute monocytic cancer MV4-11 cells, showing a significant difference between the two. DC50 (the concentration causing 50% degradation of the target protein) analysis showed that JQ1(+)-DAB-Thd was 48 ± 17 nM and 0.5 ± 0.2 nM in HeLa and MV4-11, respectively.

[0278] This example further found that the ability of JQ1(+)-DAB-Thd to degrade BRD4 in HeLa and MV4-11 was increased by 1.9 times and 186 times, respectively, compared to its binding ability in vitro, which additionally demonstrated that PROTAC catalyzes protein degradation intracellularly ( Figure 10 ), and the test method can refer to the immunoblot experiment in Part 7 of Section III above.

[0279] The content of BRD4 in HeLa and MV4-11 cells treated with different binary components was further tested. The data showed that different mixed groups composed of TBS-NHNH-JQ1(+), 2-fPBA-Thd, and Phe-BF 3 could not degrade the target protein. Only JQ1(+)-DAB-Thd generated by the reaction of Phe-BF 3 mediating the desilylation of TBS-NHNH-JQ1(+) and reacting with 2-fPBA-Thd could effectively degrade the target protein ( Figure 10 ).

[0280] Example 6: Application of the desilylation and reconnection method in the in-situ synthesis of cell-targeted nanogels

[0281] (1) Phe-BF 3 can recruit T cells to attack tumors by assembling gold nanoparticles (AuNPs) decorated with cell-targeting peptides

[0282] A kind of gold nanoparticle (AuNPs) constructed in this example was modified with DBCO-PEG5k-SH, NH2-PEGk-SH, and mPEG5k-SH on its surface. The modification method refers to Part 2 of the experimental methods in Section III above. The synthesis method refers to the synthesis method of the exemplary gold nanoparticle material in Part 2 of Section III above.

[0283] Figure 11 It was demonstrated that the desilylation and reconnection method can be used for the in-situ synthesis of cell-targeted nanogels. It can bind two different types of cells (such as cancer cells and T cells) and act like a bispecific T cell engager (BiTE), that is, by redirecting the patient's immune T cells to attack cancer cells.

[0284] "2-fPBA" or "TBS" is covalently linked to AuNPs through amidation, while the azide-modified polypeptides targeting tumor antigen RGD and T cell receptor CD3 can be linked to the surface of AuNPs through "click chemistry", endowing it with the ability to target cells. Transmission electron microscopy data shows that after being triggered by Phe-BF 3 the hybrid AuNPs aggregated, and the corresponding size increased from 80 nm to the final 350 nm.

[0285] (2) In-situ synthesis of nanogels can occur at the cellular level

[0286] The experimental method of this example is as follows:

[0287] After euthanizing the mice, the spleen was removed under sterile conditions, placed in a Petri dish containing PBS, ground with a syringe piston, passed through a 70 μm cell sieve, and a single-cell suspension was collected. Subsequently, red blood cell lysis was performed: ACK buffer (5 mL / spleen) was added, lysed at room temperature for 5 minutes, centrifuged (300×g, 5 minutes), and the supernatant was discarded. Subsequently, CD3+ T cells were purified using a magnetic bead sorting kit (Pan T Cell Isolation Kit).

[0288] In this example, the T lymphocytes isolated from the mouse spleen were co-cultured with tumor cells. After 48 hours of grouping and drug administration, the cells were treated with a cell scraper, fixed with a cell fixative, and then subjected to electron microscopy analysis and particle size analysis.

[0289] Figure 12 The example shows that in-situ synthesis of nanogels can occur at the cellular level. The average diameter of AuNPs in the experimental group was 457.7 nm, which was 4.82 times that of the treatment with hybrid AuNPs alone. In addition, in this example, recruited T cells were detected by TEM, and it was observed that after treatment with hybrid AuNPs and Phe-BF 3 for 24 hours, the number of T cells around the tumor cells increased significantly under an optical microscope.

[0290] (3) The recruited T cells can be activated and have the ability to attack cancer cells

[0291] This example was carried out separately:

[0292] (a) Calcein-AM and propidium iodide (PI) staining experiments on cells treated with hybrid AuNPs and Phe-BF 3 respectively for 48 hours: Prepare the Calcein-AM and propidium iodide stock solutions in advance, and mix them in proportion to form a double-staining working solution. Set two control groups, namely T lymphocytes incubated with hybrid AuNPs (density 1×10 6(cells / well) and tumor cells (density 1×10 5 cells / well) co-culture group and mixed AuNPs+Phe-BF 3 double-cell co-culture group. After 48 hours, wash the supernatant medium, then incubate the cells with the double-staining working solution for about 30 min. Subsequently, aspirate the staining solution and gently wash the cells with PBS twice, and then fluorescence microscopy imaging can be used for recording, and the images are analyzed using Image J.

[0293] (b) Survival rate experiment of B16F10 after being treated with T cells, mixed AuNPs and Phe-BF 3 respectively within 48 hours: The protocol is the same as above. After 48-hour incubation, wash the supernatant medium, add 10% concentration of CCK8 working solution, and measure the absorbance of different experimental groups to calculate the corresponding survival rate.

[0294] The results are as Figure 13 shown, demonstrating that the recruited T cells can be activated and have the ability to attack cancer cells.

[0295] (4) Tumor targeting and pharmacokinetic properties of gold nanoparticles and small molecule Phe-BF 3

[0296] The experimental operation methods can refer to Parts 8 and 9 in the aforementioned Part III.

[0297] Among them, the synthesis methods of TBS&RGD@AuNP complex and 2-fPBA&CD3@AuNP complex refer to the synthesis method of exemplary materials in Part 2 of Part III, the mouse experiments refer to Part 8 in the aforementioned Part III, and the radioactive labeling experimental method refers to Part 9 in the aforementioned Part III.

[0298] Figure 14 The results show that there are significant differences in their metabolic behaviors. The nanomaterials are mainly metabolized by the liver, and the small molecule activator is mainly metabolized by the kidney. The two have the largest overlap at the tumor site, ensuring controllable activation of tissue selectivity.

[0299] (5) In-situ synthesis of nanogel only occurs in tumor tissues

[0300] For the related mouse experiments, refer to Part 8 in the aforementioned Part III. Among them, after one round of drug administration, the mice in group e (the control group in this example) and group f (the experimental group in this example) related to B16F10 tumor-bearing mice are sacrificed. The liver and tumor tissues of the two groups of mice are fixed with tissue fixative (fixed at 4°C for 2 - 4 hours), then processed by embedding and sectioning, and loaded on copper grids, and further stained for cells / structures to enhance the contrast. Finally, data are collected by photographing with a transmission electron microscope. The results are as​Figure 15 As shown, it indicates that the in-situ synthesis of nanoadhesive only occurs in tumor tissues, demonstrating the excellent selectivity of this technology.

[0301] Example 7: Role of the desilication and reconnection method in precise anti-tumor

[0302] In this example, the combined immunotherapy process plan was designed and optimized, and the flow cytometry experimental method referred to Part 10 of the above-mentioned Part III. Specifically, the combined treatment plan is as Figure 16 shown in a. Seven days before administration, 2×10 5 B16F10 cells were subcutaneously injected into the right shoulder of C57 mice. Seven days later, when the tumor was formed (about 50 mm 3 ), four groups were divided according to Part 10 of the above-mentioned Part III: PBS (group i), anti-PD-1 (group ii), mixed AuNPs + Phe-BF 3 (group iii) or mixed AuNPs + Phe-BF 3 + anti-PD-1 (group iv). The administration cycle was as follows: on day 0, AuNPs were injected (dose: 5 mg / kg), and on the next day, Phe-BF 3 was injected (dose: 30 mg / kg, 2 times, at an interval of 12 h). The first-round plan was repeated on days 2&3 and days 4&5. On days 5, 6, and 7, anti-PD-1 was intraperitoneally injected at a dose of 100 μg per mouse. After seven days, the drug administration was stopped, and parameters such as the tumor volume of the mice were continuously observed.

[0303] The results are as Figure 16 shown.

[0304] The proportions of CD3+CD8+ double-positive cytotoxic T cells in the combined treatment group and the single-drug treatment group were 5.6% and 1.4% respectively, while those in other groups were less than 0.5%. In addition, the B16F10 tumor is considered a "cold" tumor, which also means that it is usually resistant to immune checkpoint inhibitors such as anti-PD1. The D2L strategy demonstrated in this example can activate the immune response of cold tumors to immune checkpoint inhibitors. It was experimentally found that when T lymphocytes were activated by AuNP assembly, the combined treatment with anti-PD-1 antibody led to a significant reduction in tumor volume.

[0305] Incorporation by reference

[0306] The entire contents of each patent and scientific literature mentioned herein are incorporated herein by reference for all purposes.

[0307] Equivalence

[0308] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the above-described embodiments are to be considered in all respects as illustrative and not restrictive of the invention described herein. Thus, the scope of the present disclosure is indicated by the appended claims rather than by the foregoing description, and is intended to embrace all changes within the meaning and scope of equivalency of the claims.

Claims

1. A kit comprising: 1) Phenylalanine boron trifluoride (Phe-BF3); 2) Compound A containing a silylphenol ether group; and 3) Compound B derived from 2-formylphenylboronic acid (2-fPBA).

2. The kit according to claim 1, wherein The compound A has the following structure: The R1 is selected from a protecting group, a drug active group, an antibody group, a fluorescent group, a dyeing group or a gold nanoparticle.

3. The kit according to claim 2, wherein The compound A is selected from: Among them, Ab is an antibody group, Flu is a fluorescent group, Dye is a dye group, and AuNPs are gold nanoparticles.

4. The kit according to any one of claims 1 to 3, wherein The compound B is 2-fPBA or has the following structure: The R2 is selected from H, a linking group, a fluorescent group or a dyeing group; Said R3 is selected from H, alkyl, aryl or heteroaryl, which may be optionally substituted.

5. The kit according to claim 4, wherein The compound B is selected from: Among them, Flu is a fluorescent group, Dye is a dye group, Said R3 is selected from H, alkyl, aryl or heteroaryl, which may be optionally substituted.

6. Use of the kit according to any one of claims 1 to 5 in fluorescent controllable labeling of antibodies.

7. Use of the kit according to any one of claims 1 to 5 in antibody positive cell screening.

8. Use of the kit according to any one of claims 1 to 5 in the controllable degradation of target proteins of antibodies.

9. Use of the kit according to any one of claims 1 to 5 in bioorthogonal ligation chemistry.

10. The use according to claim 9, wherein The bioorthogonal ligation chemistry encompasses protein degradation targeted chimeric therapies or T cell-mediated immunotherapy.

11. The use according to claim 10, wherein The protein degradation targeted chimera therapy comprises a protein degradation targeted chimera drug synthesized in situ.

12. The use according to claim 10, wherein The T cell-mediated immunotherapy comprises nanomedicine synthesized in situ.

13. Use of the kit according to any one of claims 1 to 5 in precision anti-tumor therapy.

14. Use of phenylalanine boron trifluoride (Phe-BF3), compound B derived from 2-formylphenylboronic acid (2-fPBA) and compound A containing a silylphenol ether group, or the kit according to any one of claims 1 to 5 in the preparation of a drug.

15. The use according to claim 14, wherein The drug is used for controllable fluorescence labeling of antibodies, positive cell screening of antibodies, controllable degradation of target proteins of antibodies, bioorthogonal connection chemistry and / or precise anti-tumor therapy.

16. The use according to claim 15, wherein The bioorthogonal ligation chemistry is selected from protein degradation targeted chimera therapy or T cell mediated immunotherapy.

17. The use according to claim 14, wherein The drug synthesizes a protein degradation targeted chimeric drug in situ after administration.

18. The use according to claim 14, wherein The drug synthesizes nanomedicine in situ after administration.

19. The use according to any one of claims 14 to 18, wherein The compound A has the following structure: The R1 is selected from a protecting group, a drug active group, an antibody group, a fluorescent group, a dyeing group or a gold nanoparticle.

20. The use according to claim 19, wherein The compound A is selected from: Among them, Ab is an antibody group, Flu is a fluorescent group, Dye is a dye group, and AuNPs are gold nanoparticles.

21. The use according to any one of claims 14 to 20, wherein The compound B is 2-fPBA or has the following structure: The R2 is selected from H, a linking group, a fluorescent group or a dyeing group; Said R3 is selected from H, alkyl, aryl or heteroaryl, which may be optionally substituted.

22. The use according to claim 21, wherein The compound B is selected from: Among them, Flu is a fluorescent group, Dye is a dye group, Said R3 is selected from H, alkyl, aryl or heteroaryl, which may be optionally substituted.

Images