3-bromo-5-methylene pyrrolone hetero-bifunctional reagent, its preparation and use in bioconjugation

Abstract

The application discloses a 3-bromo-5-methylene pyrrolone hetero-difunctional reagent, a preparation method thereof and application thereof in biological coupling. The reagent comprises a specific structure as shown in a general formula (I) and covers various variant forms. The preparation method comprises a plurality of reaction steps, and the reagent is prepared from a compound containing a specific functional group through a series of reactions. The reagent performs well in biological coupling and can be used in biological coupling of oligonucleotides and cell-penetrating peptides and proteins and polypeptide cyclization. In the biological coupling of oligonucleotides and cell-penetrating peptides and proteins, efficient modification and coupling can be realized, and the conjugate can maintain good biological functions. In polypeptide cyclization, linear peptides can be efficiently cyclized and cell targeting can be maintained. The application provides a new way for biological coupling technology and has important application prospects in the fields of biomedical research and drug research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioconjugation, and particularly relates to a 3-bromo-5-methylene pyrrolone hetero-bifunctional reagent, and preparation and application thereof in bioconjugation, especially oligonucleotide bioconjugation and polypeptide cyclization. BACKGROUND

[0002] Bioconjugation technology is crucial in the fields of biomedical science, drug development, disease diagnosis, etc., and it can realize the connection between biomolecules, endow biomolecules with new functions or optimize their performance. Oligonucleotides and polypeptides, as important biomolecules, have great potential in the field of treatment, but face many challenges.

[0003] Oligonucleotides can specifically bind to target RNA or DNA molecules, participate in gene regulation and transcription expression, and have great potential in treating diseases related to single gene mutation or abnormal gene expression. However, their charge and nuclease sensitivity make it difficult for them to cross the cell membrane, and naked nucleic acids have poor stability in vivo, are easily enzymatically degraded or cleared by the kidneys, making it difficult to effectively reach target cells and play a role. Although delivery technologies such as lipid nanoparticles and GalNAc have been developed, there are still problems such as difficulty in specifically delivering to cells / tissues outside the liver and insufficient uptake of oligonucleotides.

[0004] Polypeptides have the advantages of high selectivity, biological target specificity, low side effects, and difficulty in accumulating in tissues and organs. However, linear peptides have poor stability, are easily hydrolyzed by proteases, and have high polarity and hydrophilicity, resulting in low cell membrane permeability and bioavailability. Although cyclic peptides have the advantages of high activity, resistance to enzymatic hydrolysis, and strong membrane permeability, traditional cyclic peptide synthesis methods have problems such as harsh reaction conditions and many side reactions.

[0005] Bioconjugation reagents are the key to bioconjugation, and hetero-bifunctional cross-linking agents are widely used in bioconjugation, which can reduce unnecessary polymerization or self-coupling. However, existing cross-linking agents such as SMCC (Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) have poor stability, and the maleimide moiety is easily hydrolyzed in alkaline solution, forming a thioether bond that is not stable and is prone to thiol exchange reactions, limiting its application range. Therefore, it is of great significance to develop bioconjugation reagents with high stability, good reactivity, and wide application range. SUMMARY

[0006] The technical problem solved by the present application is to provide a 3-bromo-5-methylene pyrrolone (3-Br-5MPs) hetero-bifunctional reagent and its application in biological coupling, aiming at the problems of poor stability of traditional biological coupling reagents and the problems of oligonucleotides and polypeptides in biological treatment applications.

[0007] In order to solve the above technical problems, the technical scheme adopted by the present application is:

[0008] In the first aspect of the present application, a 3-bromo-5-methylene pyrrolone hetero-bifunctional reagent is provided, comprising a compound as shown in general formula (I), or an optical isomer, a racemate, a single enantiomer, a possible diastereoisomer thereof, or a pharmaceutically acceptable salt, a prodrug, a deuterium derivative, a hydrate, a solvate thereof.

[0009]

[0010] R 2 is selected from the following structural fragments:

[0011] R represents R 2 is the site connected with the 3Br-5MP part, represents R 2 is the site connected with the NHS ester part.

[0012] In the second aspect of the present application, a preparation method of the above-mentioned 3-bromo-5-methylene pyrrolone hetero-bifunctional reagent is provided, comprising the following steps:

[0013] (1) obtaining an intermediate 2 by reacting 4-bromo furan-2-carboxaldehyde with sodium borohydride;

[0014] (2) obtaining compound 3 by reacting the intermediate 2 with acetic anhydride;

[0015] (3) obtaining compounds 4a-4c by reacting compound 3 with different amino benzoic acid derivatives a-c under the action of N-bromosuccinimide, etc.;

[0016] (4) obtaining compounds 5a-5c, i.e. the compound as shown in general formula (I), by reacting compounds 4a-4c with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) respectively and then treating.

[0017]

[0018] More specifically, the compounds of the present application of general formula (I) can be prepared by the above process, however, the conditions of the process, such as the reactants, solvents, amounts of compounds used, reaction temperature, time required for the reaction, etc. are not limited to the above explanations. The compounds of the present application can also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, such combination can be easily made by a person skilled in the art to which the present application pertains.

[0019] In a third aspect, the present application provides the use of the above-mentioned 3-bromo-5-methylene pyrrolone hetero-bifunctional reagent in biological coupling.

[0020] As preferred, the application is particularly used in the biological coupling of oligonucleotides and the cyclization of polypeptides. In the biological coupling of oligonucleotide cell-penetrating peptides and proteins, efficient modification and coupling can be achieved, and the conjugate can maintain good biological function; in the cyclization of polypeptides, linear peptides can be efficiently cyclized and cell targeting can be maintained. Compared with the prior art, the present application has the following beneficial effects:

[0021] In the 3-bromo-5-methylene pyrrolone (3-Br-5MPs) hetero-bifunctional reagent designed by the present application, the 3-Br-5MPs parent nucleus has higher chemical stability, and has high selectivity and double-site reactivity to sulfhydryl (-SH), and the NHS ester part has high reactivity to amino (-NH2), thereby realizing precise coupling, reducing non-specific side reactions, improving coupling efficiency and stability. By reasonably designing the structure of the connecting arm, unnecessary polymerization or self-coupling phenomenon is reduced, the generation of by-products is reduced, and the success rate and reliability of biological coupling are improved.

[0022] Due to its hetero-bifunctional characteristics, the reagent provided by the present application can be widely used for the coupling of proteins, antibodies, drug molecules and other biological macromolecules. The biological coupling technology provides a new way and is suitable for various biomedical and drug research and development applications, such as targeted drug delivery and development of diagnostic probes. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figures 1-4 are the degradation rate diagrams of compounds 5a, 5b, 5c and SMCC at different pH values, wherein (a) is pH = 6, (b) is pH = 7.5, and (c) is pH = 9.

[0024] Figure 5 is a diagram of the results of the optimal reaction conditions of compound 5b and oligonucleotide Os.

[0025] Figure 6HPLC characterization of OsB-TAT-FPH.

[0026] Figure 7 Fluorescence imaging of Hela cell uptake of OsB-TAT-FPH.

[0027] Figure 8 SDS-PAGE characterization of OsB-Hs-FPH.

[0028] Figure 9 SDS-PAGE characterization of OsB-Hs-FPH.

[0029] Figure 10 Fluorescence imaging of Hela cell uptake of Os-Hs-FPH.

[0030] Figure 11 Conversion of compound 5b mediated cyclization of a series of polypeptides, where (a) is polypeptide P1-P3 and (b) is polypeptide P4-P7.

[0031] Figure 12 HPLC characterization of compound 5b mediated cyclization of CyP and fluorescence modification.

[0032] Figure 13 Fluorescence imaging of cell uptake of CyPB-FPH. DETAILED DESCRIPTION

[0033] The application is further described below in connection with the drawings and examples, but the application is not limited to the described examples.

[0034] Example 1:

[0035] Preparation of compound 5, comprising the following steps:

[0036]

[0037] 1) Synthesis of intermediate 2: To a solution of 4-bromofuran-2-carboxaldehyde (1.0 g, 5.71 mmol, 1.0 eq.) in anhydrous tetrahydrofuran (30 ml) was added sodium borohydride (226.6 mg, 5.99 mmol, 1.05 eq.) at 0 °C in an ice water bath, then the reaction mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC, after completion of the reaction, the reaction was quenched with saturated aqueous ammonium chloride solution, the tetrahydrofuran was removed by rotary evaporation, the organic layer was extracted with ethyl acetate in a separatory funnel for 3 times. The combined organic phase was further extracted with saturated sodium chloride solution for once, dried over anhydrous sodium carbonate, after the drying agent was removed by filtration, the filtrate was concentrated under vacuum to give the crude product 1.13 g. The crude product was used directly for the next step without purification.

[0038] 2) Synthesis of intermediate 3: Acetic anhydride (2.64 mL, 25.8 mmol, 4.0 eq.) was added dropwise to a solution of compound 2 (1.13 g, 6.46 mmol, 1.0 eq) in dry pyridine (30 ml) at ice water bath of 0 °C. After stirring at room temperature for 5 h, the reaction was quenched with water and concentrated under reduced pressure. The resulting mixture was diluted with ethyl acetate and washed with saturated sodium bicarbonate, the organic phase was dried and concentrated, the crude was purified by silica gel column (petroleum ether: ethyl acetate = 95:5, v:v) to give compound 3 as a yellow liquid (1.19 g, yield 84.1%). 1 H NMR (400 MHz, DMSO-d6) d 7.15 (d, J = 0.6 Hz, 1H), 5.94 (s, 1H), 4.23 (s, 2H), 1.25 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) d 175.12, 156.07, 147.33, 118.78, 104.69, 62.62, 25.72.

[0039] 3) Synthesis of intermediate 4a: Intermediate 3 (300 mg, 0.97 mmol, 1.0 eq) was dissolved in 6 ml tetrahydrofuran: 0.25 M sodium phosphate buffer (pH 7.5) = 1 : 1, the mixture solution was cooled to 0 °C. N-bromosuccinimide (206.5 mg, 1.16 mmol, 1.2 eq.) was added, stirred at 0 °C until TLC monitoring of the starting material was completely converted to intermediate. M- aminomethylbenzoic acid (217.6 mg, 1.16 mmol, 1.2 eq.) was added, the pH was adjusted to 7, the reaction mixture was stirred at room temperature for 10 h. After the reaction was completed, the pH was adjusted to less than 7 with dilute hydrochloric acid, then the reaction mixture was concentrated under reduced pressure, extracted with ethyl acetate / water 3 times, saturated sodium chloride 1 time, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to give the crude product. Purified by silica gel column purification (petroleum ether: ethyl acetate: acetic acid = 70:30:0.1, v:v:v) to give compound 4a as a light yellow solid (135.5 mg, yield 45.7%). 1 H NMR (500 MHz, DMSO-d6) d 7.87 - 7.81 (m, 1H), 7.78 (d, J = 1.7 Hz, 1H), 7.72 (s, 1H), 7.51 - 7.43 (m, 2H), 5.24 (d, J = 2.0 Hz, 1H), 5.11 (d, J = 2.0 Hz, 1H), 4.94 (s, 2H).

[0040] 4) Synthesis of intermediate 4b: The synthesis of compound 4b can be prepared according to the synthesis method of compound 4a, purified by silica gel column separation (petroleum ether: ethyl acetate: acetic acid = 70:30:0.1, v:v:v) to give compound 4b as a white solid (123.3 mg, yield 41%). 1 HNMR (500 MHz, Chloroform-d) δ 7.10 (s, 1H), 4.90 (dd, J = 15.8, 2.1 Hz, 2H), 3.50 (d, J = 7.2 Hz, 2H), 2.25 (tt, J = 12.2, 3.6 Hz, 1H), 2.06 - 1.99 (m, 2H), 1.76 (d, J = 3.5 Hz, 2H), 1.68 (dtt, J = 11.1, 7.3, 3.6 Hz, 1H), 1.37 (dd, J = 12.6, 3.4 Hz, 2H), 1.11 - 0.99 (m, 2H).

[0041] 5) Synthesis of intermediate 4c: The synthesis of compound 4c can be prepared according to the synthesis method of compound 4a, purified by silica gel column separation (petroleum ether: ethyl acetate: acetic acid = 70:30:0.1, v:v:v) to give compound 4c as a light yellow solid (179.9 mg, yield 70%). 1 HNMR (500 MHz, DMSO-d6) δ 7.90 (d, J = 8.3 Hz, 2H), 7.72 (s, 1H), 7.31 (d, J = 8.4 Hz, 2H), 5.18 (d, J = 1.9 Hz, 1H), 5.10 (d, J = 2.0 Hz, 1H), 4.94 (s, 2H).

[0042] 6) Synthesis of compound 5a: Intermediate 4a (130 mg, 0.42 mmol, 1 eq.) was dissolved in 10 ml of anhydrous tetrahydrofuran solution, and N-hydroxysuccinimide (57.54 mg, 0.50 mmol, 1.2 eq), 1-ethyl- carbodiimide hydrochloride (95.85 mg, 0.5 mmol, 1.2 eq) were added in turn, and the reaction mixture was reacted at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, extracted with ethyl acetate / water 3 times, saturated sodium chloride 1 time, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to give the crude product. Purified by silica gel column separation (petroleum ether: ethyl acetate: acetic acid = 75:25, v:v:v) to give compound 5a as a light yellow solid (57.3 mg, 33.8% yield). 1HNMR (400 MHz, Chloroform-d) δ 8.05 (d, J = 7.4 Hz, 1H), 7.97 (s, 1H), 7.55 - 7.44 (m, 2H), 7.17 (s, 1H), 4.93 (s, 2H), 4.90 - 4.81 (m, 2H), 2.93 - 2.89 (m, 4H). 13 CNMR (101 MHz, Chloroform-d) δ 169.20, 165.24, 161.56, 143.32, 137.68, 135.70, 133.67, 129.93, 129.55, 129.01, 125.65, 118.64, 98.52, 43.29, 25.70.

[0043]

[0044] 7) Synthesis of compound 5b: The synthesis of compound 5b can be prepared according to the synthesis method of compound 5a, using intermediate 4b as raw material, purified by silica gel column separation (petroleum ether: ethyl acetate: acetic acid = 70:30:0.1, v:v:v) to obtain compound 5b as a white solid (57.5 mg, yield 35%). 1 HNMR (500 MHz, Chloroform-d) δ 7.11 (s, 1H), 4.90 (d, J = 9.7 Hz, 2H), 3.52 (d, J = 7.2 Hz, 2H), 2.83 - 2.79 (m, 4H), 2.63 - 2.53 (m, 1H), 2.16 (dd, J = 13.8, 3.6 Hz, 2H), 1.80 (dd, J = 13.5, 3.6 Hz, 2H), 1.73 (tt, J = 7.8, 3.7 Hz, 1H), 1.52 (qd, J = 13.1, 3.5 Hz, 2H), 1.10 (qd, J = 13.2, 3.6 Hz, 2H).

[0045]

[0046] 8) Synthesis of compound 5c: The synthesis of compound 5c can be prepared according to the synthesis method of compound 5a, using intermediate 4c as raw material, purified by silica gel column separation (petroleum ether: ethyl acetate: acetic acid = 70:30:0.1, v:v:v) to obtain compound 5c as a light yellow solid (108 mg, 44.5% yield). 1HNMR (500 MHz, Chloroform-d) δ 8.11 - 8.02 (m, 2H), 7.33 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 6.7 Hz, 1H), 4.94 (d, J = 12.6 Hz, 2H), 4.86 (d, J = 2.4 Hz, 1H), 4.77 (d, J = 2.4 Hz, 1H), 2.90 (s, 4H).

[0047]

[0048] Example 2:

[0049] Stability test experiment of compounds 5a, 5b, 5c, including the following steps:

[0050] 1 Experimental reagents

[0051] The control compound 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), 5a-5c were dissolved with DMSO, prepared 100 mM stock solution, and then diluted with different pH buffers (pH 6.0 10 mM sodium phosphate buffer, pH 7.5 10 mM sodium phosphate buffer, pH 9 10 mM sodium phosphate buffer) to a final concentration of 5 mM.

[0052] 2 Experimental method

[0053] A certain amount of mixture was taken at different time intervals for LCMS analysis.

[0054] 3 Experimental results and discussion

[0055] 5a, 5b, 5c and the control compound SMCC were tested according to the above experimental method. The results are as follows Figures 1-4The stability of the tested compounds SMCC, 5a-5c at pH 6-9 was tested. We found that the degradation rate of the compounds was: SMCC > 5c > 5a > 5b. In neutral buffer solution, SMCC hydrolyzed more than half within 24 h. LC-MS analysis showed that at pH 6, SMCC mainly hydrolyzed the NHS ester part, while at pH > 7, the NHS ester part hydrolyzed first, followed by the maleimide part. Compounds 5a-5c mainly hydrolyzed the NHS ester part at pH 6-9, and the degree of hydrolysis increased with increasing pH, and the parent nucleus part was basically not decomposed, showing superior stability compared to SMCC. Compounds 5c and 5a with an aminomethylbenzoic acid spacer were both less stable, and the analysis showed that the electron-withdrawing conjugation of the benzene ring increased the electron deficiency of the carbonyl carbon, making the NHS ester more prone to hydrolysis. Compound 5b with a cyclohexane spacer was the most stable, consistent with the theoretical results. The analysis showed that the electron-withdrawing conjugation of the benzene ring increased the electron deficiency of the carbonyl carbon, making the NHS ester more prone to hydrolysis. Based on the stability study results, compound 5b was selected for subsequent biological coupling studies.

[0056] Example 3: Study on compound 5b mediated oligonucleotide biological coupling:

[0057] The overall experimental design idea is as follows:

[0058]

[0059] The NHS ester part of 5b can specifically react with amino-modified oligonucleotides to generate oligonucleotide modification products. The product has reactivity and can react with two thiol-containing biomolecules in turn, and through NaBH4 reduction to form stable thioether bonds, realizing the coupling of oligonucleotides with proteins or polypeptides, and ultimately obtaining a coupling product that can be used for fluorescence imaging or characterization.

[0060] Screening of 5b mediated oligonucleotide biological coupling reaction conditions:

[0061] 1 Experimental reagents

[0062] 10 mM, 100 mM, 500 mM, 1 M buffer solution at different pH (pH 6.0 MES buffer, pH 7.5 HEPES buffer, pH 9.0 TE buffer, pH 11 sodium phosphate buffer).

[0063] The oligonucleotide sequence is as follows:

[0064] Os: 5'-NH2-ss11-mer (5'-NH2-C6-TTATACATCTA-3')

[0065] Dissolve Os in PBS buffer to prepare a 1 mM stock solution;

[0066] Compound 5b was dissolved in DMSO to prepare a 10 mM stock solution.

[0067] 2 Experimental method

[0068] A 0.2 mM Os solution was mixed with different concentrations of compound 5b solution in different types and concentrations of buffer for a certain period of time to investigate the effects of reaction time, 5b concentration, and buffer type and concentration on the reaction. LCMS was used to detect the generation of product OsB to screen the best reaction system.

[0069] 3 Experimental results

[0070] As shown in Figure 5 , by optimizing the reaction time, 5b concentration, and buffer type and concentration, the best reaction conditions were determined as follows: when 5b was reacted at 10 times the equivalent in 100 mM PBS for 90 min, the relatively pure oligonucleotide modification product, named OsB, was obtained with a conversion rate of 90%.

[0071] 5b-mediated coupling of oligonucleotide-polypeptide, characterization, and cell uptake and imaging:

[0072] 1 Experimental reagents

[0073] Hela cell strain, PBS, DMEM medium, Trypsin, 1640 medium, 4% paraformaldehyde tissue cell fixation solution, DAPI staining solution, Dil staining agent, StainsAll staining agent, chromatographic grade acetonitrile, chromatographic grade methanol, trifluoroacetic acid, mercaptofluorescein, etc.

[0074] Cell penetrating peptide TAT sequence:

[0075] GRKKRRQRRRC

[0076] Negative control oligonucleotide OsFN sequence:

[0077] 5'-NH2-FITC-ss11-mer(5'-NH2-C6-TTA TAC ATC TA-3')

[0078] 2 Experimental method

[0079] (1) Os was mixed with 10 times the equivalent of 5b in 100 mM PBS buffer and incubated for 2 h. According to the reported reactivity of 3-bromo-5-methylene pyrrolone, the best reaction equivalent, 8 times the equivalent of TAT solution, was added, and the reaction was carried out at 37°C for 30 min. LCMS was used to detect the generation of product OsB-TAT.

[0080] (2) Since 3-bromo-5-methylene pyrrolinone is known to have a dual thiol reactivity, after the conversion of OsB-TAT is monitored to be complete, 10 times equivalent of thiol fluorescein is added to react with the other reactive site of 3-bromo-5-methylene pyrrolinone, the reactants are vortexed and mixed, and then incubated in a constant temperature mixer at 37°C for 1 h, and 10 times equivalent of sodium borohydride is added for reduction for 15 min. The production of the product is analyzed by LCMS, and the obtained fluorescein-modified conjugate is named as OsB-TAT-FPH.

[0081] (3) Preparation of positive control cell penetrating peptide TAT-RP: TAT aqueous solution is mixed with 2 times equivalent of 5-carboxy tetramethyl rhodamine succinimidyl ester in DMSO, and reacted at 37°C for 1 h. Desalination and purification are performed to obtain the product TAT-RP.

[0082] (4) Cell uptake experiment:

[0083] (i) The well-conditioned Hela cells are taken out from the incubator, inoculated in a 12-well cell culture plate in which the coverslips have been placed, and the number of cells to be tested is controlled to be 10 5 cells / well, and then placed in a constant temperature incubator at 37°C and 5% CO2 for culture, and when the cell density reaches about 60%-70%, it is optimal.

[0084] (ii) The 12-well cell culture plate is taken out, the old culture medium is aspirated, and the coverslips are washed with PBS for two to three times. The negative control group, the positive control group and the experimental group are set as follows for cell administration.

[0085] a. The negative control group: 1 ml of DMEM complete culture medium containing 0.5 mM OsFN is added to the culture dish, mixed, and then placed in a constant temperature incubator at 37°C and 5% CO2 for incubation for 2 h;

[0086] b. The positive control group: 1 ml of DMEM complete culture medium containing 0.5 mM TAT-RP is added to the culture dish, mixed, and then placed in a constant temperature incubator at 37°C and 5% CO2 for incubation for 2 h;

[0087] c. The experimental group: 1 ml of DMEM complete culture medium containing 0.5 mM OsB-TAT-FPH is added to the culture dish, mixed, and then placed in a constant temperature incubator at 37°C and 5% CO2 for incubation for 2 h;

[0088] (5) Cell Imaging: Fixation: Discard the culture medium and wash the cell slide three times with 1 ml PBS, 2 min each time. Add 500 mL of pre-cooled 4% paraformaldehyde fixative and fix for 15 min in the dark. Discard the fixative and wash the cell slide three times with 1 ml PBS. Mounting: Use pointed forceps to remove the cell slide, cell side down, and carefully place it on a glass slide with a small amount of anti-fluorescence quencher to prevent air bubbles. Mount with nail polish and, after solidification, image using a laser confocal microscope.

[0089] 3. Experimental Results and Discussion

[0090] (i) 5b-mediated oligonucleotide-peptide coupling and characterization

[0091] like Figure 6 As shown, LC-MS characterization revealed that the peak with a retention time of 7.1 min was the oligonucleotide-penetrating peptide conjugate OsB-TAT, with a molecular weight of 5202.7, which is basically consistent with the theoretical molecular weight of 5202.4. The yield was calculated using the area normalization method based on the peak area of ​​the HPLC product, and the final yield was >92%.

[0092] Further monitoring of the fluorescently modified product OsB-TAT-FPH revealed that the secondary thiol addition reaction was very slow. After 2-3 hours of reaction, LCMS monitoring of the reactants showed that the peak with a retention time of 7.9 min was the fluorescently modified product OsB-TAT-FPH. The obtained molecular weight was 5784.28, which was basically consistent with the theoretical molecular weight of 5783.8. The yield was calculated using the area normalization method based on the peak area of ​​the product in HPLC, and the final yield was >90%.

[0093] (ii) Cellular uptake and imaging of oligonucleotide-penetrating peptide conjugates

[0094] To investigate whether 5b-mediated coupling of oligonucleotides to cell-penetrating peptides affects the bioactivity of the peptides, cell imaging experiments of the conjugates were performed in living cells, such as... Figure 7 As shown, the fluorescently modified oligonucleotide OsFN without the connection to the penetrating peptide cannot pass through the cell membrane into the cell, while OsB-TAT-FPH can be observed to have green fluorescence inside the cell. This indicates that the oligonucleotide can be carried into the cell after being coupled to the penetrating peptide via 5b, and the penetrating peptide still maintains good physiological activity.

[0095] 5b mediates oligonucleotide-protein coupling, characterization, cellular uptake, and imaging:

[0096] 1. Experimental Materials

[0097] Same as before.

[0098] Histone H3 sequence:

[0099] Histone3 V35C / C110A

[0100] ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGCKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIMPKDIQLARRIRGERA

[0101] 2Experimental Methods

[0102] (1) Preparation of positive control fluorescent H3: H3 aqueous solution was mixed with 10 times equivalent 5-carboxy tetramethyl rhodamine succinimidyl ester in DMSO solution, and reacted at 37°C for 1 h. Desalination and purification were performed to obtain the product Hs-RP.

[0103] (2) Os was mixed with 10 times equivalent 5b in 100 mM PBS buffer for 2 h to obtain OsB. 0.4 mM H3 solution was mixed with different equivalents of OsB solution, and reacted at 37°C for 30 min. SDS-PAGE was used to detect the generation of product OsB-Hs.

[0104] (3) After the complete conversion of OsB-Hs was monitored according to the above steps, 10 times equivalent mercaptofluorescein was added, the reactants were vortexed, and the incubation was continued at 37°C for 1 h. 10 times equivalent sodium borohydride was added for reduction for 15 min. SDS-PAGE was used to analyze the generation of the product, and the obtained fluorescently modified conjugate was named OsB-Hs-FPH.

[0105] (4) The steps of cell uptake and cell imaging experiment were the same as above.

[0106] 3Results and Discussion

[0107] (1) SDS-PAGE characterization of oligonucleotide-histone H3 conjugate

[0108] Firstly, the coupling of protein-oligonucleotide was characterized by SDS-PAGE gel electrophoresis, as shown in Figure 8 , the oligonucleotide can be coupled with H3 through 5b to form OsB-Hs conjugate band. Due to the increase in overall volume and mass of the conjugate, the charge-to-mass ratio decreases, and the migration speed slows down, so the lane band migrates significantly slower compared to H3, thereby proving the binding of oligonucleotide to histone 3.

[0109] In addition, the coupling of OsB-Hs and H3 showed concentration dependence, and after optimization of the coupling conditions, it was finally determined that H3 could basically react completely with 4 eq of OsB.

[0110] The fluorescently modified product OsB-Hs, OsB-Hs-FPH, was also characterized by SDS-PAGE. Figure 9 To further confirm the conjugates, salts and small molecules were removed by passing them through a desalting column (7 kDa), followed by high-resolution liquid chromatography-mass spectrometry (LC-MS / MS) characterization. Mass spectrometry results showed that the molecular weight of OsB-Hs was 18945.44, which is consistent with the theoretical molecular weight of 18945.5. The molecular weight of OsB-Hs-FPH was 19528.15, which is consistent with the theoretical molecular weight of 19528.12. This indicates that the oligonucleotide and H3 can be conjugated via 5β and modified by a fluorescent probe.

[0111] (2) Cellular uptake and imaging of oligonucleotide-histone H3 conjugates

[0112] H3 has the properties of a penetrating peptide, therefore, as Figure 10 As shown, in the positive control group, Hs-RP treated cells exhibited red fluorescence in the nucleus. In the negative control group, OsFN treated cells showed no fluorescence, indicating that oligonucleotides themselves do not have the ability to enter cells. In the experimental group, OsB-Hs-FPH treated cells exhibited green fluorescence in the nucleus, indicating that oligonucleotides can cross the cell membrane after being coupled with histones.

[0113] Example 4: Study on peptide cyclization mediated by compound 5b:

[0114] The overall experimental design is as follows:

[0115]

[0116] The NHS ester moiety of starter 5b specifically binds to an amino-containing peptide, while its 3Br-5MP moiety reacts with the thiol group on the peptide to form a cyclic intermediate. This intermediate is reactive and can react with another thiol-containing fluorescent molecule, which is reduced by NaBH4 to form a stable thioether bond, thereby modifying the peptide and obtaining a labeled final product that can be used for subsequent fluorescence imaging or related characterization.

[0117] Screening of 5b-mediated peptide cyclization conditions:

[0118] 1. Experimental Materials

[0119] polypeptide:

[0120] (i) Series 1:

[0121] P1: NH2-AAACF-CONH2

[0122] P2: NH2-AAAACF-CONH2

[0123] P3: NH2-AAAAACF-CONH2

[0124] (ii) Series II:

[0125] P4: AcNH-KAAACF-CONH2

[0126] P5: AcNH-KAAAACF-CONH2

[0127] P6: AcNH-KAAAAACF-CONH2

[0128] P7: AcNH-KAAAAAACF-CONH2

[0129] 2. Experimental Methods

[0130] In the cyclization of all polypeptides, 0.1 mM of P1-P7 was dissolved in different pH buffer (10 mM sodium phosphate buffer at pH 8, PBS buffer at pH 7, sodium phosphate buffer at pH 6). An equal amount of 5b dissolved in DMSO was added to the reaction mixture, which was then stirred at room temperature for 15-20 min and monitored by LC-MS until the reaction was complete.

[0131] 3. Experimental Results and Discussion

[0132] By LC-MS characterization of the crude reaction mixture, it was found that for terminal amino peptides (P1-P3), as shown in (a) of Figure 11 , under the conditions of pH 6-8, the cyclization reaction was very simple and fast, and the cyclization of all polypeptides was stoichiometric, completely converted within 15 min and without any by-products, but pH greater than 9 would increase the hydrolysis of NHS ester, reducing the reaction conversion. For intra-chain amino peptides (P4-P7), as shown in (b) of Figure 8 , increasing the distance between the two reactive groups will reduce the conversion rate and affect the reaction degree, and by increasing the pH to 7-8, the conversion rate can be basically > 98%. The results show that for terminal amino peptides, the reactivity of amino and thiol is not much affected by the distance, and the conversion rate is > 90%; the reaction of ε-amino and thiol is affected by the distance, the greater the distance, the smaller the conversion rate, and a larger pH is required for a conversion rate greater than 98%.

[0133] Cyclization of active polypeptides and cell uptake and imaging:

[0134] 1. Experimental Reagents

[0135] Linear active polypeptide CyP:

[0136] AcNH-CPIEDRPMK-CONH2

[0137] 2. Experimental Methods

[0138] (1) Preparation of fluorescent modified cyclic CyP: 0.5 mM polypeptide CyP was stirred with 1 mM 5b at room temperature for 15 min. Then 4 equivalents of mercaptophthalene were added, and the reaction was carried out in a constant temperature mixer at 37°C for 1 h. Ten equivalents of sodium borohydride were added for reduction, and the reaction was monitored by analytical LCMS until completion. The obtained conjugate was named CyPB-FP.

[0139] (2) Cell uptake experiment: The cyclic peptide CyP is known to specifically bind to the cell membrane of human colon cancer differentiated cells (Caco2 cells), so Caco2 cells were selected for routine culture, and human breast cancer cells (skbr3 cells) and human cervical cancer cells (Hela cells) were selected as negative controls. Take the Caco2 cells, skbr3 cells, and Hela cells in good condition and inoculate them in 12-well cell culture plates with pre-placed cell slides. Control the number of cells to be tested to be 10 5 cells / well, and place them in a constant temperature incubator at 37°C, 5% CO2. When the cell density reaches about 60%-70%, remove the 12-well cell culture plate, discard the old culture medium, and wash the cell slides with PBS for two to three times. Add 1 ml of complete culture medium containing 1 mM CyPB-FP to each cell culture dish, and incubate in a constant temperature incubator at 37°C, 5% CO2 for 2 h.

[0140] (3) Cell imaging: Take out the 12-well plate, discard the culture medium, and wash the cell slides with PBS for three times, each for 2 min. Then add 1 ml of cell nucleus dye Hoechst 33342 (final concentration 2.5 μg / mL) and cell membrane dye Dil (final concentration 5 mM) diluted with PBS, and incubate in a constant temperature incubator at 37°C, 5% CO2 for 10 min in the dark. Fixation: Discard the staining solution, and wash the cell slides with 1 ml of PBS for three times, each for 2 min. Add 500 mL of pre-cooled 4% paraformaldehyde fixing solution, and fix in the dark for 15 min. Discard the fixing solution, and wash the cell slides with 1 ml of PBS for three times. Mounting: Take out the cell slides with pointed tweezers, with the cell side facing down, and carefully place them on a glass slide with a small amount of anti-fluorescence quencher dropped on it to prevent air bubbles. Seal the glass slide with nail polish, and image with a laser confocal microscope after solidification.

[0141] 3 Experimental results and discussion

[0142] (1) 5b mediated CyP cyclization and fluorescent modification

[0143] As Figure 12As shown, LCMS results indicate that CyP and 5b can cyclize within 15 min, with a relatively single product peak and a retention time of 2.9 min. The obtained molecular weight is 1362.6548, which is basically consistent with the theoretical molecular weight. The yield was calculated using the area normalization method based on the peak area of ​​the HPLC product, and the final yield was >90%. LCMS results for the fluorescently modified product CyPB-F show a retention time of 3.9 min and a molecular weight of 899.3705, which is basically consistent with the theoretical molecular weight of 899.67, and the final yield was >95%. After reduction, a more stable cyclized product CyPB-FH was obtained, with a retention time of 3.7 min and a molecular weight of 900.8799, which is basically consistent with the theoretical molecular weight of 900.60, and the final yield was >90%.

[0144] (2) Cellular uptake and cell imaging of CyPB-FH

[0145] The binding of the cyclized peptide CyPB-FH to Caco2 cells was detected using laser confocal fluorescence microscopy. Figure 13 As shown, the images of the fluorescein-bound cyclic peptide CyPB-FH overlap well with those of the cell membrane dye Dil. Compared with skbr3 and HeLa cells, the fluorescein-modified cyclic peptide exhibits specific binding to Caco2 cells, indicating that 5b-mediated cyclization of active peptides can effectively mimic disulfide bonds and maintain cell targeting ability. These studies demonstrate that 5b-mediated peptide cyclization can be further modified with functional groups such as fluorescein. By altering the functional and recognition groups of cyclic peptides, there is potential for synthesizing functional cyclic peptide biomolecules.

[0146] In summary, this invention designed and synthesized a heterobifunctional reagent (3Br-5MP-NHS ester) based on 3-bromo-5-methylenepyrrolidone (3Br-5MP). By optimizing the linker arm structure, the cyclohexane linker 5b with the best stability was screened, and its application in bioconjugation was systematically studied. The results show that the NHS ester moiety of 5b can efficiently modify amino oligonucleotides, and the resulting active intermediate OsB can be further conjugated with thiol-based peptides / proteins. The conjugate successfully penetrated the HeLa cell membrane and achieved fluorescent localization. Simultaneously, the peptide cyclization reaction mediated by 5b under pH 7-8 conditions was highly efficient with few byproducts, and the cyclized product maintained its targeting ability to Caco2 cells, providing a novel tool for oligonucleotide delivery and cyclic peptide synthesis.

[0147] The above description is merely an embodiment of the present invention and should not be construed as limiting the scope of the present invention. For those skilled in the art, modifications and innovations can be made to the claims and drawings, or the invention can be applied to other technical fields. However, it should be noted that all modifications to the present invention should be included within the scope of patent protection of the present invention.

Claims

1. A 3-bromo-5-methylene pyrrolone heterobifunctional reagent characterized by, ###0001### 1 A compound comprising a compound as shown in general formula (I), or a pharmaceutically acceptable salt thereof: wherein R 2 is selected from the following structural fragments: represents R 2 the site of attachment to the 3Br-5MP moiety, represents R 2 the site of attachment to the NHS ester moiety.

2. The process for the preparation of 3-bromo-5-methylene pyrrolone heterobifunctional reagent as claimed in claim 1, wherein, The preparation method comprises the following steps: Step (1), a reduction reaction is carried out by mixing 4-bromofuran-2-carboxaldehyde and sodium borohydride to obtain an intermediate 2; Step (2), an acetylation reaction is carried out by mixing the intermediate 2 and acetic anhydride to obtain a compound 3; Step (3), the compound 3 is mixed with N-bromosuccinimide, and a reaction is carried out by adding m-aminomethylbenzoic acid, p-aminomethylbenzoic acid or 4-(aminomethyl)cyclohexane carboxylic acid to obtain a compound 4; wherein R 1 is selected from the following structural fragments: Step (4), a condensation reaction is carried out by mixing the compound 4, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to obtain a compound as shown in general formula (I).

3. The preparation method according to claim 2, characterized in that, The solvent of the reduction reaction in step (1) is tetrahydrofuran, and the reaction is carried out at room temperature for 2 h.

4. The preparation method according to claim 2, characterized in that, The reaction in step (2) is carried out at room temperature for 2 h.

5. The preparation method according to claim 2, characterized in that, In step (3), the compound 3 and N-bromosuccinimide are dissolved in a mixed solution of tetrahydrofuran and PBS, and after the reaction is carried out at 0℃, m-aminomethylbenzoic acid, p-aminomethylbenzoic acid or 4-(aminomethyl) cyclohexane carboxylic acid is added, and the reaction is carried out at room temperature for 10 h.

6. The preparation method according to claim 2, characterized in that, The condensation reaction in step (4) is carried out in tetrahydrofuran, and the reaction is carried out at room temperature for 8 h.

7. Use of the 3-bromo-5-methylene pyrrolone hetero-bifunctional reagent in biological coupling according to claim 1.

8. Use according to claim 7, characterized in that, Specifically, use in oligonucleotide biological coupling or polypeptide cyclization.

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