Use of divinyltetrazine in linear polypeptide cyclization

Abstract

The application discloses application of a divinyl tetrazine in cyclization of a linear polypeptide, and a structure formula of the divinyl tetrazine is; the linear polypeptide has at least two free mercapto groups, and the vinyl groups of the divinyl tetrazine are subjected to Michael addition reaction with corresponding mercapto groups to form a ring. The application provides a new use of the divinyl tetrazine, the divinyl tetrazine can be subjected to high-efficiency Michael addition reaction with two natural cysteine mercapto groups on the linear polypeptide through the vinyl groups, so that double-mercapto peg cyclization of the linear polypeptide is realized, and the purpose of limiting the conformation of the linear polypeptide and improving the stability of the linear polypeptide is achieved. Moreover, the divinyl tetrazine can be subjected to a reverse electron demand Diels-Alder reaction with trans-cyclooctene (TCO) after the two vinyl groups are subjected to reaction with mercapto groups, so that functional polypeptides can be modularly constructed by coupling with functional molecules modified with TCO, and related applications are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of divinyltetrazine in the cyclization of linear polypeptides. Background Technology

[0002] While peptide drugs have become important candidate drugs for diseases such as cancer due to their high targeting and low toxicity, their linear structure has long faced two major bottlenecks in clinical translation: First, their conformational flexibility is high, and linear peptides are difficult to maintain a stable secondary structure in the physiological environment, making them easy to be rapidly degraded and inactivated by entering the active pocket of proteolytic enzymes; second, their poor stability results in a short half-life and low bioavailability in vivo. Both of these factors limit their application in refractory diseases (such as cancer).

[0003] Cyclization modification (or pinning) is an important strategy for improving the stability of linear peptides. For example, patent application CN120665137A discloses a method for arylizing and cyclizing peptides, which uses a dichloro-containing nitrogen-containing heteroaryl ring reagent to selectively arylate cysteine ​​residues of the peptide. Besides this, there are other cyclization strategies such as all-carbon pinning and dichlorotetraazine pinning, each with its own advantages. Developing new cyclization strategies can provide more pathways for improving the stability of linear peptides. Summary of the Invention

[0004] The purpose of this invention is to provide the application of divinyltetrazine in the cyclization of linear peptides, thus providing a new strategy for improving the performance of linear peptides.

[0005] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0006] This invention first provides the application of divinyltetrazine in the cyclization of linear peptides, wherein the structural formula of the divinyltetrazine is as follows: ;

[0007] The linear polypeptide has at least two free thiol groups, and the vinyl group of the bisvinyltetraazine undergoes a Michael addition reaction with the corresponding thiol group to form a ring.

[0008] This invention provides a novel use for divinyltetrazine, which can undergo an efficient Michael addition reaction with two natural cysteine ​​thiol groups on a linear polypeptide via its vinyl group, thereby achieving dithiol pinning cyclization of the linear polypeptide and thus restricting the conformation of the linear polypeptide and improving its stability.

[0009] This invention does not have special requirements for the structure of the linear polypeptide, and the structural formula of the linear polypeptide can be represented as follows:

[0010] ;

[0011] Where X1, X2, and X3 represent any amino acid, C represents D-type or L-type cysteine, a and c represent any natural number, and b is a natural number ≥0 and ≤11.

[0012] That is, cysteine ​​in a linear polypeptide can be either a D-type amino acid or an L-type amino acid; cysteine ​​can be located at the terminal or central position of the polypeptide; the number of amino acids between two adjacent cysteine ​​residues can be between 0 and 11 (inclusive); the N-terminus or C-terminus of the polypeptide can be protected or unprotected.

[0013] Preferably, the application includes: mixing a linear peptide solution and a divinyltetrazine solution in a linear peptide to divinyltetrazine equivalent ratio of 1:(1.1-3) in a PB, HEPES, or PBS buffer containing 0-30% acetonitrile, N,N-dimethylformamide, or dimethyl sulfoxide and with a pH of 6-8.5, and reacting at room temperature for 0.5-6 hours. The pinning cyclization reaction conditions of this invention are mild, the reaction efficiency is high, and the substrate versatility is strong.

[0014] Preferably, the linear polypeptide solution is obtained by dissolving the linear polypeptide in PBS buffer;

[0015] The divinyltetraazine solution is obtained by dissolving divinyltetraazine in dimethyl sulfoxide.

[0016] The present invention also provides a stapled peptide, which is obtained by a two-site Michael addition reaction between the above-mentioned divinyltetraazine and two thiol groups on a linear polypeptide.

[0017] The amino acid sequence of the linear polypeptide is selected from any one of the following three:

[0018] NH2-ETFSDLWCLLPCN-COOH;

[0019] NH2-ETFCDLWCLLPEN-COOH;

[0020] NH2-ETFCDLWRLLCEN-COOH.

[0021] These three linear peptides are p53-derived peptides targeting mouse dual microsome 2 homolog (MDM2). This invention found that when pinning and cyclizing with divinyltetrazine, the resulting pinned peptides not only have significantly improved stability, but also effectively enhance their interaction with the MDM2 target and significantly increase their cytotoxicity to target cells.

[0022] This invention also provides the application of the above-mentioned divinyltetrazine in conjugated antibodies and functionalized modules, the application including:

[0023] S1. Reduce the interchain disulfide bonds of the antibody to obtain the reduced antibody.

[0024] S2. A two-site Michael addition reaction is performed between divinyltetrazine and the thiol group on the reduced antibody to obtain a heavily bridged antibody.

[0025] S3. This enables the re-bridging antibody to be coupled with the functionalized module.

[0026] The functionalized module includes a fluorescently labeled molecule or therapeutic drug molecule having a trans-cyclooctene group, which undergoes a cycloaddition reaction with a tetrazine on the heavy-bridged antibody to couple the heavy-bridged antibody to the functionalized module.

[0027] When the interchain disulfide bonds are reduced, the reduced antibody has at least four pairs of free thiol groups. At this point, the interchain linkage can be reconstructed by reacting divinyltetraazine with the thiol groups. Even in stabilized peptides and heavy-bridged antibodies, the tetraazine retains highly efficient bioorthogonal reactivity (the second-order reaction kinetic constant K2 can reach 10). 3 M -1 S -1 It can undergo a Diels-Alder reaction with trans-cyclooctene (TCO) with an electron-demanding reaction, thereby enabling the modular construction of functional peptides (including linear peptides and antibodies) by coupling with functional molecules modified with TCO, and realizing related applications; for example, coupling with fluorescent labeling molecules can realize live-cell fluorescence imaging of target cells, and coupling with therapeutic drug molecules can realize multiple therapies for target cells.

[0028] Based on this, the present invention also provides a polypeptide derivative, which is formed by coupling a polypeptide and a functional module, wherein the polypeptide is: a bridging peptide obtained by a two-site Michael addition reaction between the above-mentioned divinyltetrazine and two thiol groups on a linear polypeptide; or a heavily bridged antibody obtained by a two-site Michael addition reaction between the above-mentioned divinyltetrazine and thiol groups on a reduced antibody.

[0029] The functionalized module includes a fluorescently labeled molecule or a therapeutic drug molecule with a trans-cyclooctene group, which undergoes a cycloaddition reaction with a tetrazine on the polypeptide to couple the polypeptide to the functionalized module.

[0030] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0031] 1. This invention provides a novel application for divinyltetrazine, which can undergo a highly efficient Michael addition reaction with two natural cysteine ​​thiol groups on a linear polypeptide via its vinyl group, thereby achieving dithiol-pinned cyclization of the linear polypeptide and restricting its conformation, thus improving its stability. When using divinyltetrazine to pinned cyclize linear polypeptides, the reaction conditions are mild, the reaction efficiency is high, and the substrate versatility is strong; thus providing a new route for the cyclization of linear polypeptides.

[0032] 2. After using the divinyltetraazine of the present invention to perform pinning cyclization on p53-derived peptides, the obtained pinned peptides not only have significantly improved stability, but also effectively enhance their interaction with the MDM2 target site, and significantly enhance their cytotoxicity to target cells.

[0033] 3. This invention discovers that the divinyltetrazine, after both vinyl groups react with thiol groups, can still undergo a Diels-Alder reaction with anti-electron demanding trans-cyclooctene (TCO). This allows for the modular construction of functional peptides (including linear peptides and antibodies) through coupling with functional molecules modified with TCO, enabling related applications. For example, coupling with fluorescent labeling molecules can achieve live-cell fluorescence imaging of target cells, and coupling with therapeutic drug molecules can achieve multiple therapies for target cells. Attached Figure Description

[0034] Figure 1 This is a synthetic route diagram for divinyltetraazine;

[0035] Figure 2 The NMR spectrum of bisvinyltetraazine;

[0036] Figure 3 This is a schematic diagram illustrating the cyclization of a linear polypeptide using the bisvinyltetraazine of the present invention;

[0037] Figure 4 The results of the 1H NMR spectrum of the divinyltetrazine of the present invention before and after cyclization with a linear polypeptide are shown.

[0038] Figure 5 This is a schematic diagram of the structure of the p53-derived polypeptide targeting the mouse two-microsome 2 homologous protein and the corresponding pinning peptide.

[0039] Figure 6 The results of circular dichroism analysis of p53 wild-type peptide, various p53-derived peptides and corresponding ligated peptides are shown.

[0040] Figure 7 The results show the cytotoxicity of p53 wild-type peptide and Tz-EN-4-L stapled peptide against target cells.

[0041] Figure 8 The second-order reaction kinetics curves of the ligand peptide and trans-cyclooctene are shown.

[0042] Figure 9 Live-cell fluorescence imaging results of the fluorescent peptides SIR-EN-4-L and SIR-EN-4-R targeting MDM2 protein;

[0043] Figure 10 A flowchart illustrating the preparation of fluorescently labeled functionalized antibodies;

[0044] Figure 11 Mass spectrometry analysis results of the heavily bridged antibody prepared using divinyltetraazine;

[0045] Figure 12 The results of in vitro ELISA analysis of the heavily bridged antibody prepared using divinyltetraazine are shown.

[0046] Figure 13 The results show the stability analysis of the heavily bridged antibody prepared using divinyltetraazine.

[0047] Figure 14 The results of in vivo imaging of mice with fluorescently labeled functionalized antibodies. Detailed Implementation

[0048] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0049] Example 1 Synthesis of divinyltetraazine

[0050] This embodiment describes a bisvinyltetraazine, the synthetic route of which is as follows: Figure 1 As shown, it includes the following steps:

[0051] (1) Using 3-hydroxypropionitrile as a raw material, a cyclization reaction was first carried out under the action of hydrazine hydrate and mercaptopropionic acid to construct a 1,2,4,5-tetraazine core; and then a diazotization reaction was carried out to obtain 3,6-bis(2-hydroxyethyl)-1,2,4,5-tetraazine;

[0052] Specifically, 205.5 μL (3 mmol) of 3-hydroxypropionitrile, 87 μL (1.5 mmol) of 3-mercaptopropionic acid, 0.3 mL of ethanol, and 0.75 mL (15 mmol) of N2H4·H2O were added to a 10 mL reaction flask containing a magnetic stirrer. The flask was placed in an oil bath at 40 °C and stirred overnight under argon protection. The next day, the reaction was transferred to an ice-water bath, and 30 mL of ice water containing dissolved sodium nitrite (1.05 g, 15 mmol) was added. Then, 1 M HCl was slowly added dropwise while stirring to adjust the pH to 2.0–3.0. During the process, gas was released and the solution gradually turned red. After the gas release stopped, stirring was continued for 10 minutes. The mixture was extracted with ethyl acetate (50 mL), dried, concentrated, and purified to obtain 178 mg of a pink solid product 1a, which is 3,6-bis(2-hydroxyethyl)-1,2,4,5- Tetraazine, yield 70%.

[0053] (2) The hydroxyl group on 3,6-bis(2-hydroxyethyl)-1,2,4,5-tetraazine is converted to a methanesulfonate group to obtain a methanesulfonate intermediate; then, an elimination reaction is carried out under the induction of an organic base to obtain a divinyltetraazine with the following structural formula:

[0054] ;

[0055] Specifically, under argon protection at 0°C, MsCl (184 µL, 2.4 mmol) was slowly added dropwise to a dichloromethane solution (4 mL) of 1a (136 mg, 0.8 mmol) and diisopropylethylamine (417 µL, 2.4 mmol). After the addition was complete, the mixture was transferred to room temperature and stirred, and the reaction was monitored by TLC. When 1a was completely converted to the methanesulfonate intermediate, diisopropylethylamine (DIPEA, 417 µL, 2.4 mmol) was added to the reaction flask. After the reaction was completed, water (20 mL) was added, and the mixture was extracted with dichloromethane (DCM, 20 mL). The combined organic phases were washed with brine, dried, concentrated, and purified to obtain 63 mg of red oily product 1, which is the divinyltetraazine of this example, with a yield of 59%.

[0056] The NMR spectrum of the divinyltetraazine is as follows: Figure 2 As shown.

[0057] Example 2: Preparation and performance characterization of stabilizing peptides

[0058] 1. Divinyltetraazine for the pinning cyclization of linear peptides

[0059] In this embodiment, the divinyltetraazine prepared in Example 1 is used to staple cyclize a linear polypeptide to prepare a stapled peptide (such as...). Figure 3 (As shown).

[0060] This embodiment selected multiple linear polypeptides covering all natural amino acids, containing D-cysteine, with N-terminal / C-terminal protection or unprotection, cysteine ​​located at the terminal position, and 0–11 amino acids separated by two cysteine ​​residues. The specific information of each linear polypeptide is shown in Table 1.

[0061] Table 1. Information on the tested linear peptides

[0062] polypeptide sequence length Stapling interval GL-1 NH2-GCCGAVL-COOH (SEQ ID No. 1) 7 1 YA-3 NH2-YCNPCA-NH2 (SEQ ID No. 2) 6 3 VA-4 NH2-VCGPGCA-NH2 (SEQ ID No.3) 7 4 IC-4 Ac-IPPKYCELLC-NH2 (SEQ ID No.4) 10 4 EN-4-L Ac-ETFCDLWCLLPEN-NH2 (SEQ ID No.5) 13 4 EN-4-R Ac-ETFSDLWCLLPCN-NH2 (SEQ ID No.6) 13 4 fT-5 NH2-fCYwKTCT-COOH (SEQ ID No. 7) 8 5 AK-7 NH2-ACPSMAALCK-COOH (SEQ ID No.8) 10 7 LS-7 NH2-LTFcHYWAQLcS-COOH (SEQ ID No. 9) 12 7 SY-7 NH2-SKICDFVLKNCY-COOH (SEQ ID No.10) 12 7 EN-7 Ac-ETFCDLWRLLCEN-NH2 (SEQ ID No. 11) 13 7 GC-11 NH2-GACNWSFFKTWTSC-COOH (SEQ ID No.12) 14 11

[0063] In the “Sequence” column of the table, except for “Ac” which represents acetyl, lowercase letters represent D-type amino acids and uppercase letters represent L-type amino acids.

[0064] The specific steps of stapled ring formation are as follows:

[0065] First, weigh an appropriate amount of linear peptide powder, dissolve it in PBS, and prepare a 20 mM peptide stock solution. Simultaneously, weigh an appropriate amount of divinyltetraazine, dissolve it in DMSO, and prepare a 50 mM tetraazine stock solution. Accurately pipette an appropriate amount of the peptide stock solution and add it to PB buffer (30% acetonitrile, 20 mM, pH 8.0) to achieve a final peptide concentration of 50 μM. Then, add 1.1 equivalents of the divinyltetraazine stock solution, mix well, and allow the reaction to proceed at room temperature. Considering that linear peptides containing two Cys groups may form a small amount of disulfide bond products during storage, the peptide stock solution was analyzed by HPLC-MS before the reaction. The peptide purity (a) was obtained by integrating the HPLC chromatogram at 214 nm. The reaction solution was then analyzed by HPLC-MS, and the HPLC yield (b) was obtained by integrating the HPLC chromatogram at 214 nm. The corrected actual labeled yield was b / a.

[0066] HPLC conditions: Porosell 120, EC-C18 column (3.0 mm × 150 mm, 2.7 μm); column temperature 35℃; elution gradient 0 min, 5% B, 0–8 min, 5–100% B; flow rate 0.5 mL / min.

[0067] from Figure 4 The 1H NMR spectrum results shown indicate that after the reaction, the vinyl double bond hydrogen signal (6.0–7.5 ppm) of the divinyltetraazine completely disappeared, confirming that both double bonds were successfully added by thiol groups, thus achieving the cyclization of the linear polypeptide.

[0068] The cyclization results of each linear polypeptide are shown in Table 2.

[0069] Table 2. Cycling results of each linear polypeptide

[0070] polypeptide sequence Yield % GL-1 NH2-GCCGAVL-COOH 94 YA-3 NH2-YCNPCA-NH2 90 VA-4 NH2-VCGPGCA-NH2 95 IC-4 Ac-IPPKYCELLC-NH2 94 EN-4-L Ac-ETFCDLWCLLPEN-NH2 97 EN-4-R Ac-ETFSDLWCLLPCN-NH2 90 fT-5 NH2-fCYwKTCT-COOH 90 AK-7 NH2-ACPSMAALCK-COOH 95 LS-7 NH2-LTFcHYWAQLcS-COOH 97 SY-7 NH2-SKICDFVLKNCY-COOH 92 EN-7 Ac-ETFCDLWRLLCEN-NH2 98 GC-11 NH2-GACNWSFFKTWTSC-COOH 93

[0071] As can be seen from Table 2, the pinning cyclization in this embodiment is not only mild but also highly efficient and has strong substrate universality.

[0072] 2. Secondary configuration analysis of the stabilizing peptide

[0073] This embodiment selected p53-derived peptides targeting mouse dual microsome 2 homolog (MDM2): EN-7, EN-4-L (i.e., EN-L in Tables 1 and 2), and EN-4-R (i.e., EN-R in Tables 1 and 2). The corresponding pinning peptides Tz-EN-4-L, Tz-EN-4-R, and Tz-EN-7 (structures shown in Tables 1 and 2) were also analyzed. Figure 5 Perform circular dichroism analysis (as shown); Figure 6 The analysis results show that Tz-EN-7 and Tz-EN-4-L exhibit a significant increase in the characteristic peaks of the α-helical structure (negative peaks at 208 nm and 222 nm) compared to EN-7 and EN-4-L.

[0074] 3. Stability analysis of the stabilizing peptide

[0075] p53, Tz-EN-4-L, Tz-EN-4-R, and Tz-EN-7 were added to pure fetal bovine serum to achieve a final peptide concentration of 100 μM. The mixture was then incubated at 37°C for 72 hours, and the stability of p53 and each peptide was monitored during the incubation process. The results are shown in Table 3.

[0076] In addition, p53, Tz-EN-4-L, Tz-EN-4-R and Tz-EN-7 were added to 20 μg / mL trypsin to make the final peptide concentration 100 μM; then incubated for 90 minutes, and the stability of p53 and each peptide was monitored during the incubation process. The results are shown in Table 4.

[0077] Table 3. Stability of p53 wild-type peptide and various stapled peptides in pure fetal bovine serum.

[0078] time Tz-EN-7 Tz-EN-4-L Tz-EN-4-R p53 （h） （%） （%） （%） （%） 06 10096 ± 3 10093 ± 1 10088 ± 2 10081 ± 4 10 94 ± 2 88 ± 4 81 ± 3 73 ± 2 24 86 ± 3 82 ± 1 58 ± 3 60 ± 6 36 81 ± 2 74 ± 1 53 ± 5 52 ± 2 48 78 ± 5 68 ± 4 43 ± 5 32 ± 4 60 71 ± 1 65 ± 5 33 ± 5 8 ± 1 72 66 ± 7 62 ± 5 27 ± 3 -

[0079] Table 4. Stability of p53 wild-type peptide and various stapled peptides in pancreatic enzymes.

[0080] time Tz-EN-7 Tz-EN-4-L Tz-EN-4-R p53 (min) （%） （%） （%） （%） 05 10095 ± 1 10087 ± 4 10083 ± 3 10044 ± 3 10 86 ± 2 78 ± 2 58 ± 2 14 ± 2 20 82 ± 3 74 ± 2 38 ± 3 4 ± 1 40 77 ± 3 43 ± 2 20 ± 2 - 90 42 ± 2 31 ± 2 7 ± 2 -

[0081] As shown in Table 3, after 72 hours of incubation in pure fetal bovine serum, the p53 wild-type peptide was completely degraded, while the residual rates of Tz-EN-7 and Tz-EN-4-L both exceeded 60%, and the residual rate of Tz-EN-4-R was 26.8%. Table 4 shows that after 90 minutes of incubation in 20 μg / mL trypsin, the p53 wild-type peptide was completely degraded, while the stapled peptides were not completely degraded, with the residual rate of Tz-EN-7 exceeding 40%. These results indicate that the formation of the α-helix structure in stapled peptides is closely related to peptide stability, and the divinyltetraazine stapled strategy can significantly enhance the peptide's resistance to enzymatic degradation.

[0082] 4. Analysis of the interaction between the stabilizing peptide and the target site

[0083] The in vitro binding affinity of p53, Tz-EN-4-L, Tz-EN-4-R, and Tz-EN-7 to the MDM2 target was analyzed, and the results are shown in Table 5.

[0084] Table 5. Results of in vitro binding affinity analysis of p53 wild-type peptide and various stapled peptides to the target site.

[0085] polypeptide <![CDATA[K d (nM)]]> P53 69.7 Tz-EN-4-R 16.4 Tz-EN-4-L 0.764 Tz-EN-7 6.79

[0086] As shown in Table 5, compared with the p53 wild-type peptide, the in vitro binding ability of each ligation peptide to the MDM2 target was significantly improved. Among them, Tz-EN-4-L showed the best binding ability to the K+ target of MDM2. d The value is as low as 0.764 nM.

[0087] 5. Cytotoxicity analysis of stabilizing peptides

[0088] The cytotoxicity of p53 and Tz-EN-4-L against SJSA-1 human osteosarcoma cells that highly express the MDM2 target was analyzed using the CCK-8 cytotoxicity assay. The results are as follows: Figure 7 As shown.

[0089] from Figure 7 It can be seen that as the concentration of p53 wild peptide increases, the survival rate of SJSA-1 cells does not decrease significantly; however, as the concentration of Tz-EN-4-L pinning peptide increases, the survival rate of SJSA-1 cells decreases significantly; indicating that Tz-EN-4-L pinning peptide has greater cytotoxicity to target cells and can effectively kill target cells.

[0090] 6. Bioorthogonal functionalization modification of pinned peptides

[0091] Weigh appropriate amounts of different TCO-SIR fluorescent probes (1:1 molar ratio) and dissolve them in acetonitrile to achieve a final reaction concentration of 2.5 mM. After mixing, add 3 equivalents of N,N-diisopropylethylamine, mix well, and react at room temperature. After complete conversion as detected by HPLC-MS, add 2 equivalents of the stabbing peptides Tz-EN-4-L and Tz-EN-4-R to the reaction solution, mix well, and react at room temperature. After the reaction is complete, separate and purify the stabbing peptides SIR-EN-4-L and SIR-EN-4-R using semi-preparative HPLC.

[0092] like Figure 8 As shown, the second-order reaction kinetic constant K2 of the stabilizing peptide and trans-cyclooctene (TCO) in pure PBS at 37°C can reach 10. 3 M -1 S -1 This indicates that it retains highly efficient bioorthogonal reaction activity.

[0093] TCO-SiR, SIR-EN-4-L, and SIR-EN-4-R were co-incubated with SJSA-1, MCF-7, and HCT116 cells expressing MDM2 protein, respectively. Intracellular fluorescence signals were detected, and the results are as follows: Figure 9 As shown. From Figure 9 As can be seen, in various tumor cell lines expressing MDM2 protein (SJSA-1, MCF-7, HCT116), incubation with either the fluorescent pinning peptide SIR-EN-4-L or SIR-EN-4-R specifically illuminated the target cells; while no fluorescence signal was observed in the over-excess wild-type p53 competition group and the TCO-SiR-only incubation group, confirming the targeting specificity of the fluorescent pinning peptide. This indicates that the fluorescent pinning peptide derived from p53 can achieve live-cell fluorescence imaging targeting the MDM2 protein.

[0094] Example 4: Preparation and performance characterization of fluorescently labeled functionalized antibodies

[0095] 1. Preparation of fluorescently labeled functionalized antibodies

[0096] The preparation route of fluorescently labeled functionalized antibodies is as follows: Figure 10 As shown, it includes the following steps:

[0097] S1. Reduce the interchain disulfide bonds of the antibody to obtain the reduced antibody.

[0098] Specifically, a suitable amount of trastuzumab stock solution (144 μM, dissolved in PBS) was pipetted into PB buffer (100 mM, pH = 8.0) to bring the final reaction concentration to 50 μM. After mixing by pipetting, 10 equivalents of tris(2-carboxyethyl)phosphine stock solution (100 mM, dissolved in dimethyl sulfoxide) was pipetted in, mixed, and then placed in a 37°C water bath for 2 hours. After the reaction, the reaction solution was ultrafiltered three times using an ultrafiltration tube with a molecular weight cutoff of 10K. The protein solution in the inner tube after the last ultrafiltration was collected, and the concentration of reduced trastuzumab was determined by UV-Vis.

[0099] S2. A two-site Michael addition reaction is performed between divinyltetrazine and the thiol group on the reduced antibody to obtain a heavily bridged antibody.

[0100] Specifically, the reduced trastuzumab of the above-determined concentration was diluted to a final reaction concentration of 20 μM with PB buffer (100 mM, pH = 8.0) containing 10% dimethyl sulfoxide. 20 equivalents of divinyltetraazine stock solution (50 mM, dissolved in dimethyl sulfoxide) were added, mixed well, and reacted in a room temperature water bath for 2 hours. After the reaction, the reaction solution was ultrafiltered 3 times using an ultrafiltration tube with a molecular weight cutoff of 10K. The protein solution in the inner tube after the last ultrafiltration was collected, and the concentration of the heavily bridged antibody was determined by UV-Vis.

[0101] S3. This enables the re-bridging antibody to be coupled with the functionalized module.

[0102] Specifically, by reacting the heavy bridging antibody with 10 equivalents of the TCO-Cy5 fluorescent probe in PBS for 4 hours and then removing the TCO-Cy5 by ultrafiltration, the fluorescently labeled functionalized antibody (hereinafter referred to as fluorescently labeled antibody) can be obtained.

[0103] Mass spectrometry analysis of trastuzumab and heavy-bridged antibody revealed that each heavy-bridged antibody was labeled with 4 tetrazine molecules (DAR=4) (see...). Figure 11 Furthermore, in vitro ELISA analysis revealed that the heavy-bridged antibody maintained its binding ability to the target HER2 protein, and there was no significant difference compared to the unmodified trastuzumab (see [link to ELISA]). Figure 12 Stability analysis revealed that after incubating the heavy-bridged antibody in fetal bovine serum for 9 days (final concentration 10 μM), the stability remained above 50% (see [link to relevant documentation]). Figure 13 ).

[0104] In the SKOV3 tumor-bearing mouse model, after administration of fluorescently labeled antibodies via the tail vein, tumor enrichment was observed within 3 hours, with the signal persisting for more than 12 days. The tumor-to-muscle signal ratio reached its optimal level on day 8, providing an experimental basis for determining the interval for in vivo pre-targeted imaging (see [link to article]). Figure 14 ）。

Claims

1. The application of divinyltetraazine in the cyclization of linear polypeptides, characterized in that, The structural formula of the aforementioned divinyltetraazine is: ； The linear polypeptide has at least two free thiol groups, and the vinyl group of the bisvinyltetraazine undergoes a Michael addition reaction with the corresponding thiol group to form a ring.

2. The application as described in claim 1, characterized in that, The structural formula of the linear polypeptide is as follows: ； Where X1, X2, and X3 represent any amino acid, C represents D-type or L-type cysteine, a and c represent any natural number, and b is a natural number ≥0 and ≤11.

3. The application as described in claim 1, characterized in that, include: The linear peptide solution and the divinyltetrazine solution were mixed in a ratio of linear peptide to divinyltetrazine of 1:(1.1-3) into a PB, HEPES or PBS buffer containing 0-30% acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide and with a pH of 6-8.5, and reacted at room temperature for 0.5-6 hours.

4. A styling peptide, characterized in that, It is obtained by a two-site Michael addition reaction between divinyltetraazine and two thiol groups on a linear polypeptide; The structural formula of the aforementioned divinyltetraazine is: .

5. The stabilizing peptide as described in claim 4, characterized in that, The amino acid sequence of the linear polypeptide is selected from any one of the following three: NH2-ETFSDLWCLLPCN-COOH; NH2-ETFCDLWCLLPEN-COOH; NH2-ETFCDLWRLLCEN-COOH.

6. The application of bisvinyltetraazine in conjugated antibodies and functionalized modules, characterized in that, include: S1. Reduce the interchain disulfide bonds of the antibody to obtain the reduced antibody. S2. A two-site Michael addition reaction is performed between divinyltetrazine and the thiol group on the reduced antibody to obtain a heavily bridged antibody. The structural formula of the aforementioned divinyltetraazine is: ; S3. This enables the re-bridging antibody to be coupled with the functionalized module. The functionalized module includes a fluorescently labeled molecule or therapeutic drug molecule having a trans-cyclooctene group, which undergoes a cycloaddition reaction with a tetrazine on the heavy-bridged antibody to couple the heavy-bridged antibody to the functionalized module.

7. A polypeptide derivative, characterized in that, The product is composed of a polypeptide and a functionalized module, wherein the polypeptide is: a bridging peptide obtained by a two-site Michael addition reaction between divinyltetrazine and two thiol groups on a linear polypeptide; or a heavily bridged antibody obtained by a two-site Michael addition reaction between divinyltetrazine and thiol groups on a reduced antibody. The structural formula of the aforementioned divinyltetraazine is: ; The functionalized module includes a fluorescently labeled molecule or therapeutic drug molecule having a trans-cyclooctene group, which undergoes a cycloaddition reaction with a tetrazine on the polypeptide to couple the polypeptide to the functionalized module.

Images