3-bromo-5-methylene pyrrolidone heterotype bifunctional reagent and preparation and application thereof in biological coupling

Abstract

The invention discloses a 3-bromine-5-methylene pyrrolidone heterotype bifunctional reagent and preparation and application thereof in biological coupling, and 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 steps of reactions, and a compound containing a specific functional group is used as a raw material to prepare the compound through a series of reactions. The reagent has excellent performance in biological coupling, and can be used for biological coupling of oligonucleotides, cell penetrating peptides and proteins and polypeptide cyclization. In the aspect of biological coupling of oligonucleotide cell penetrating peptide and protein, efficient modification and coupling can be realized, and a conjugate can keep a relatively good biological function; in polypeptide cyclization, efficient cyclization of linear peptides can be promoted, and cell targeting can be maintained. A new way is provided for a biological coupling technology, and the method has important application prospects in the fields of biomedical research, drug research and development and the like. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of bioconjugation, and particularly relates to a 3-bromo-5-methylenepyrrolidone-based heterobifunctional reagent and its preparation and application in bioconjugation, especially in oligonucleotide bioconjugation and polypeptide cyclization. Background Art

[0002] Bioconjugation technology is crucial in the fields of biomedicine, drug development, disease diagnosis, etc. It can achieve the connection between biomolecules, endow biomolecules with new functions or optimize their properties. As important biomolecules, oligonucleotides and polypeptides have great potential in the therapeutic field but face many challenges.

[0003] Oligonucleotides can specifically bind to target RNA or DNA molecules and participate in gene regulation and transcriptional expression, showing great potential in treating diseases related to single-gene mutations or abnormal gene expression. However, their charged nature and nuclease sensitivity make it difficult for them to cross the cell membrane. Naked nucleic acids have poor stability in vivo, are easily enzymatically degraded or cleared by the kidneys, resulting in difficulty in effectively reaching target cells to exert their effects. Although delivery technologies such as lipid nanoparticles and GalNAc have been developed, there are still problems such as difficulty in specifically delivering to extrahepatic cells / tissues and insufficient effective uptake of oligonucleotides.

[0004] Polypeptides have the advantages of high selectivity, biological target specificity, low side effects, and low accumulation in tissues and organs. However, linear peptides have poor stability, are easily hydrolyzed by proteases, and have relatively large polarity and hydrophilicity, resulting in low cell membrane permeability and bioavailability. Although cyclic peptides have advantages such as high activity, anti-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. Heterobifunctional crosslinkers are widely used in bioconjugation and can reduce unnecessary polymerization or self-coupling. However, existing crosslinkers such as SMCC (Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) have problems with poor stability. Its maleimide moiety is easily hydrolyzed in alkaline solutions, and the formed thioether bond is also unstable and prone to thiol exchange reactions, limiting the scope of application. Therefore, it is of great significance to develop bioconjugation reagents with high stability, good reaction activity, and a wide range of applications. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a 3-bromo-5-methylenepyrrolidone (3-Br-5MPs) heterobifunctional reagent and its application in bioconjugation in view of the problems faced by oligonucleotides and polypeptides in biotherapeutic applications in the above-mentioned prior art, as well as the problems such as poor stability of traditional bioconjugation reagents. The reagent uses 3-Br-5MPs as the core to connect NHS (N-hydroxysuccinimide) ester. By reasonably designing the linker structure, the overall stability and reactivity of the reagent are improved. This method utilizes the high thiol selectivity and dual-site reactivity of 3Br-5MP, as well as the excellent amino reactivity and specificity of NHS ester to achieve precise conjugation of biomolecules.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] In the first aspect of the present invention, there is provided a 3-bromo-5-methylenepyrrolidone heterobifunctional reagent, which comprises a compound represented by the general formula (I), or its optical isomers, racemates, single enantiomers, possible diastereoisomers, or its pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, solvates.

[0009]

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

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

[0012] In the second aspect of the present invention, there is provided a method for preparing the above-mentioned 3-bromo-5-methylenepyrrolidone heterobifunctional reagent, which comprises the following steps:

[0013] (1) React 4-bromofuran-2-carbaldehyde with sodium borohydride to obtain intermediate 2;

[0014] (2) React intermediate 2 with acetic anhydride to obtain compound 3;

[0015] (3) React compound 3 with N-bromosuccinimide and the like, and add different aminobenzoic acid derivatives a-c for reaction to obtain compounds 4a-4c;

[0016] (4) React compounds 4a-4c with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) respectively, and after treatment, obtain compounds 5a-5c, namely the compounds represented by the general formula (I).

[0017]

[0018] More specifically, the compounds represented by the general formula (I) of the present invention can be prepared by the methods described above. However, the conditions of this method, such as reactants, solvents, amounts of compounds used, reaction temperature, reaction time required, etc., are not limited to the above explanations. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art. Such combinations can be easily carried out by those skilled in the art to which the present invention pertains.

[0019] In the third aspect of the present invention, there is provided the use of the above-mentioned 3-bromo-5-methylene pyrrolidone-based heterobifunctional reagents in bioconjugation.

[0020] Preferably, it is specifically the use in oligonucleotide bioconjugation and polypeptide cyclization. In the bioconjugation of oligonucleotide cell-penetrating peptides and proteins, efficient modification and conjugation can be achieved, and the conjugates can maintain good biological functions; in polypeptide cyclization, it can promote the efficient cyclization of linear peptides and maintain cell targeting. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] In the 3-bromo-5-methylene pyrrolidone (3-Br-5MPs)-based heterobifunctional reagents designed by the present invention, the 3-Br-5MPs parent nucleus has higher chemical stability, and has high selectivity for sulfhydryl (-SH) and dual-site reaction activity, while the NHS ester part has high reactivity with amino (-NH2), thereby achieving precise conjugation, reducing non-specific side reactions, and improving the conjugation efficiency and stability. By reasonably designing the linker structure, unnecessary polymerization or self-conjugation phenomena are reduced, the generation of by-products is decreased, and the success rate and reliability of bioconjugation are improved.

[0022] Due to its heterobifunctional characteristics, the reagent provided by the present invention can be widely used for the conjugation of biological macromolecules such as proteins, antibodies, drug molecules, etc., providing a new approach for bioconjugation technology, and is applicable to a variety of biomedical and drug R & D applications, such as targeted drug delivery, development of diagnostic probes, etc. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 5 It is the result diagram of the optimal conditions for the reaction of compound 5b with oligonucleotide Os.

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

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

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

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

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

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

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

[0032] Figure 13 Fluorescence imaging of cell uptake of CyPB-FPH. Detailed implementation methods

[0033] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the described embodiments.

[0034] Example 1:

[0035] The preparation of compound 5 includes the following steps:

[0036]

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

[0038] 2) Synthesis of Intermediate 3: In an ice-water bath at 0 °C, acetic anhydride (2.64 mL, 25.8 mmol, 4.0 eq.) was added dropwise to a dry pyridine (30 mL) solution of Compound 2 (1.13 g, 6.46 mmol, 1.0 eq.). After stirring at room temperature for 5 hours, the reaction was quenched with water and concentrated under reduced pressure. The resulting mixture was diluted with ethyl acetate and washed and extracted with saturated sodium bicarbonate, dried, and the organic phase was concentrated. The crude product was separated by silica gel column chromatography (petroleum ether:ethyl acetate = 95:5, v:v) to obtain Compound 3 as a yellow liquid (1.19 g, yield 84.1%). 1 H NMR (400 MHz, DMSO-d6) δ 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) δ 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 of a mixture of tetrahydrofuran:0.25 M sodium phosphate buffer (pH 7.5) = 1:1. The mixed solution was cooled to 0 °C. N-Bromosuccinimide (206.5 mg, 1.16 mmol, 1.2 eq.) was added, and the mixture was stirred at 0 °C until TLC monitoring showed that all the starting materials had been converted to the intermediate. m-Aminomethylbenzoic acid (217.6 mg, 1.16 mmol, 1.2 eq.) was added, and the pH was adjusted to 7. The reaction mixture was stirred at room temperature for 10 hours. After the reaction was completed, the pH was adjusted to less than 7 with dilute hydrochloric acid, and then the reaction mixture was concentrated under reduced pressure, extracted 3 times with ethyl acetate / water and 1 time with saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate:acetic acid = 70:30:0.1, v:v:v) to obtain Compound 4a as a pale yellow solid (135.5 mg, yield 45.7%). 1 H NMR (500 MHz, DMSO-d6) δ 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, and purified by silica gel column chromatography (petroleum ether: ethyl acetate: acetic acid = 70:30:0.1, v:v:v) to obtain compound 4b as a white solid (123.3 mg, yield 41%). 1 HNMR(500MHz,Chloroform-d)δ7.10(s,1H),4.90(dd,J=15.8,2.1Hz,2H),3.50(d,J=7.2Hz,2H),2.25(tt,J=12.2,3.6Hz,1H),2.06–1.99(m,2H),1.76(d,J=3.5Hz,2H),1.68(dtt,J=11.1,7.3,3.6Hz,1H),1.37(dd,J=12.6,3.4Hz,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, and purified by silica gel column chromatography (petroleum ether: ethyl acetate: acetic acid = 70:30:0.1, v:v:v) to obtain compound 4c as a pale yellow solid (179.9 mg, yield 70%). 1 HNMR(500MHz,DMSO-d6)δ7.90(d,J=8.3Hz,2H),7.72(s,1H),7.31(d,J=8.4Hz,2H),5.18(d,J=1.9Hz,1H),5.10(d,J=2.0Hz,1H),4.94(s,2H).

[0042] 6) Synthesis of Compound 5a: Dissolve Intermediate 4a (130 mg, 0.42 mmol, 1 eq.) in 10 ml of anhydrous tetrahydrofuran solution, and successively add N-hydroxysuccinimide (57.54 mg, 0.50 mmol, 1.2 eq) and 1-ethyl-carbonyldiimide hydrochloride (95.85 mg, 0.5 mmol, 1.2 eq). The reaction mixture is reacted overnight at room temperature. After the reaction is completed, the reaction solution is concentrated under reduced pressure, extracted 3 times with ethyl acetate / water, extracted 1 time with saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic phase is concentrated under reduced pressure to obtain the crude product. Purified by silica gel column chromatography (petroleum ether: ethyl acetate: acetic acid = 75:25, v:v:v) to obtain compound 5a as a pale yellow solid (57.3 mg, 33.8% yield). 1HNMR(400MHz, Chloroform-d) δ 8.05 (d, J = 7.4Hz, 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(101MHz, 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 from intermediate 4b according to the synthesis method of Compound 5a, and purified by silica gel column chromatography (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(500MHz, Chloroform-d) δ 7.11 (s, 1H), 4.90 (d, J = 9.7Hz, 2H), 3.52 (d, J = 7.2Hz, 2H), 2.83–2.79 (m, 4H), 2.63–2.53 (m, 1H), 2.16 (dd, J = 13.8, 3.6Hz, 2H), 1.80 (dd, J = 13.5, 3.6Hz, 2H), 1.73 (tt, J = 7.8, 3.7Hz, 1H), 1.52 (qd, J = 13.1, 3.5Hz, 2H), 1.10 (qd, J = 13.2, 3.6Hz, 2H).

[0045]

[0046] 8) Synthesis of Compound 5c: The synthesis of Compound 5c can be prepared from intermediate 4c according to the synthesis method of Compound 5a, and purified by silica gel column chromatography (petroleum ether: ethyl acetate: acetic acid = 70:30:0.1, v:v:v) to obtain Compound 5c as a pale yellow solid (108 mg, 44.5% yield). 1HNMR(500MHz,Chloroform-d)δ8.11–8.02(m,2H),7.33(d,J=8.2Hz,2H),7.17(d,J=6.7Hz,1H),4.94(d,J=12.6Hz,2H),4.86(d,J=2.4Hz,1H),4.77(d,J=2.4Hz,1H),2.90(s,4H).

[0047]

[0048] Example 2:

[0049] Stability test experiments of compounds 5a, 5b, and 5c include the following steps:

[0050] 1 Experimental reagents

[0051] Dissolve the control compound 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid N-succinimidyl ester (SMCC), 5a - 5c in DMSO to prepare a stock solution of 100 mM, and then dilute them to a final concentration of 5 mM with buffers of different pH values (pH 6.0 10 mM sodium phosphate buffer, pH 7.5 10 mM sodium phosphate buffer, pH 9 10 mM sodium phosphate buffer).

[0052] 2 Experimental methods

[0053] Take a certain amount of the mixture at different time intervals for LCMS analysis.

[0054] 3 Experimental results and discussion

[0055] Test 5a, 5b, 5c and the control compound SMCC according to the above experimental method. The results are as Figures 1-4As shown. By testing the stability of compounds SMCC, 5a - 5c at pH 6 - 9, we found that the degradation rates of the compounds were: SMCC > 5c > 5a > 5b. In neutral buffer solution, more than half of SMCC was hydrolyzed within 24 h. LC - MS analysis showed that at pH 6, SMCC mainly underwent hydrolysis of the NHS ester moiety, while at pH > 7, hydrolysis of the NHS ester moiety occurred first, followed by hydrolysis of the maleimide moiety. Compounds 5a - 5c mainly underwent hydrolysis of the NHS ester moiety at pH 6 - 9, and the degree of hydrolysis increased with the increase of pH value. The parent nucleus part was basically not decomposed, showing superior stability compared with SMCC. Among them, compounds 5c and 5a with an aminomethylbenzoic acid spacer had relatively low stability. The reason was that the electron - withdrawing conjugation of the benzene ring increased the deficiency of the carbonyl carbon, making the NHS ester more prone to hydrolysis. Compound 5b with a cyclohexane spacer arm was the most stable, which was consistent with the theoretical results. The reason was that the electron - withdrawing conjugation of the benzene ring increased the deficiency of the carbonyl carbon, making the NHS ester more prone to hydrolysis. According to the stability study results, compound 5b was selected for subsequent bioconjugation research.

[0056] Example 3: Study on the bioconjugation of oligonucleotides mediated by compound 5b:

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

[0058]

[0059] The NHS ester moiety of 5b can specifically react with amino - modified oligonucleotides to form oligonucleotide - modified products. This product has reactivity and can react with two kinds of thiol - containing biomolecules successively. Through NaBH4 reduction, stable thioether bonds are formed to achieve the conjugation of oligonucleotides with proteins or polypeptides, and finally a conjugate product for fluorescence imaging or characterization is obtained.

[0060] Screening of reaction conditions for the bioconjugation of oligonucleotides mediated by 5b:

[0061] 1 Experimental reagents

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

[0063] The oligonucleotide sequences are as follows:

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

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

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

[0067] 2 Experimental methods

[0068] A 0.2 mM Os solution was mixed with compound 5b solutions at different concentrations in different types and concentrations of buffer solutions and reacted for a certain time to explore the effects of reaction time, 5b concentration, and the type and concentration of buffer solution on the reaction. The formation of the product OsB was detected by LCMS to screen the optimal reaction system.

[0069] 3 Experimental results

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

[0071] Coupling, characterization, cellular uptake and imaging of oligonucleotide-polypeptide mediated by 5b:

[0072] 1 Experimental reagents

[0073] Hela cell line, PBS, DMEM medium, Trypsin, 1640 medium, 4% paraformaldehyde tissue cell fixative, 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 methods

[0079] (1) Os was mixed with a 10-fold equivalent of 5b in 100 mM PBS buffer and incubated for 2 h. According to the reported reactivity of 3-bromo-5-methylenepyrrolidone, the optimal reaction equivalent screened out - an 8-fold equivalent of TAT solution - was added, and the reaction was carried out at 37 °C for 30 min. The formation of the product OsB-TAT was detected by LCMS.

[0080] (2) Since 3-bromo-5-methylenepyrrolidone is known to have the property of dithiol reaction, after monitoring the complete conversion of OsB-TAT according to the above steps, 10-fold equivalent of mercaptofluorescein was added to undergo a thiol addition reaction with another reaction site of 3-bromo-5-methylenepyrrolidone. The reactants were vortexed and mixed evenly, and then continuously incubated in a 37 °C constant temperature mixer for 1 h. 10-fold equivalent of sodium borohydride was added for reduction for 15 min. LCMS was used to analyze the formation of the product, and the obtained fluorescence-modified conjugate was named OsB-TAT-FPH.

[0081] (3) Preparation of the positive control cell-penetrating peptide TAT-RP: An aqueous solution of TAT was mixed with a DMSO solution of 2-fold equivalent of 5-carboxytetramethylrhodamine succinimidyl ester and reacted at 37 °C for 1 h. Desalting and purification were carried out to obtain the product TAT-RP.

[0082] (4) Cell uptake experiment:

[0083] (i) Take out the well-conditioned Hela cells from the incubator and inoculate them into a 12-well cell culture plate containing coverslips. Control the number of cells to be tested to be 10 5 cells / dish, and place it in a 37 °C, 5% CO2 constant temperature incubator for culture. It is better when the cell density reaches about 60%-70%.

[0084] (ii) Take out the 12-well cell culture plate, aspirate and discard the old medium, and wash the coverslips with PBS two to three times. Set up a negative control group, a positive control group, and an experimental group for cell drug administration as follows.

[0085] a. Negative control group: Add 1 ml of DMEM complete medium containing 0.5 mM of OsFN to the culture dish, mix well and place it in a 37 °C, 5% CO2 constant temperature incubator for incubation for 2 h;

[0086] b. Positive control group: Add 1 ml of DMEM complete medium containing 0.5 mM of TAT-RP to the culture dish, mix well and place it in a 37 °C, 5% CO2 constant temperature incubator for incubation for 2 h;

[0087] c. Experimental group: Add 1 ml of DMEM complete medium containing 0.5 mM of OsB-TAT-FPH to the culture dish, mix well and place it in a 37 °C, 5% CO2 constant temperature incubator for incubation for 2 h;

[0088] (5) Cell imaging: Fixation: Aspirate the culture medium, wash the cell slides with 1 ml of PBS three times, 2 min each time. Add 500 μL of pre-cooled 4% paraformaldehyde fixative, fix in the dark for 15 min. Aspirate the fixative and wash the cell slides with 1 ml of PBS 3 times. Mounting: Use pointed forceps to take out the cell slides, place them face down carefully on a glass slide with a small amount of anti-fluorescence quencher dropped on it, and prevent the formation of air bubbles. Seal the slide with nail polish. After solidification, image with a laser confocal microscope.

[0089] 3 Experimental Results and Discussion

[0090] (i) Conjugation and Characterization of Oligonucleotide-Polypeptide Mediated by 5b

[0091] As Figure 6 shown, through LC-MS characterization, it was found that the peak with a retention time of 7.1 min was the conjugate OsB-TAT of oligonucleotide-penetrating peptide. The obtained molecular weight was 5202.7, which was basically consistent with the theoretical molecular weight of 5202.4. Using the peak area of the HPLC product, the yield was calculated by the area normalization method, and the final yield was >92%.

[0092] Furthermore, for the fluorescence-modified product OsB-TAT-FPH, the secondary thiol addition reaction was very slow. After reacting for 2 - 3 h, the reactants were monitored by LCMS. It was found that the peak with a retention time of 7.9 min was the fluorescence-modified product OsB-TAT-FPH. The obtained molecular weight was 5784.28, which was basically consistent with the theoretical molecular weight of 5783.8. Using the peak area of the HPLC product, the yield was calculated by the area normalization method, and the final yield was >90%.

[0093] (ii) Cellular Uptake and Imaging of Oligonucleotide-Penetrating Peptide Conjugates

[0094] To study whether the conjugation of oligonucleotides mediated by 5b with cell-penetrating peptides would affect the biological activity of the polypeptides, a cellular imaging experiment of the conjugates was carried out in living cells. As Figure 7 shown, the fluorescence-modified oligonucleotide OsFN without a linked penetrating peptide could not penetrate the cell membrane and enter the cells, while green fluorescence could be observed inside the cells for OsB-TAT-FPH, indicating that the oligonucleotide could be carried into the cells after conjugation with the penetrating peptide through 5b, and the penetrating peptide still maintained good physiological activity.

[0095] Conjugation, Characterization, Cellular Uptake and Imaging of Oligonucleotide-Protein Mediated by 5b:

[0096] 1 Experimental Materials

[0097] Same as before.

[0098] Histone H3 sequence:

[0099] Histone3V35C / C110A

[0100] ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGCKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIMPKDIQLARRIRGERA

[0101] 2 Experimental methods

[0102] (1) Preparation of positive control fluorescent H3: An aqueous solution of H3 was mixed with a DMSO solution of 10-fold equivalent 5-carboxytetramethylrhodamine succinimidyl ester and reacted at 37 °C for 1 h. Desalting and purification were carried out to obtain the product Hs-RP.

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

[0104] (3) After monitoring the complete conversion of OsB-Hs according to the above steps, 10-fold equivalent mercaptofluorescein was added, the reactants were vortexed and mixed evenly, and then incubated in a 37 °C constant temperature mixer for 1 h. 10-fold equivalent sodium borohydride was added for reduction for 15 min. SDS-PAGE was used to analyze the formation of the product, and the obtained fluorescently modified conjugate was named OsB-Hs-FPH.

[0105] (4) The experimental procedures for cell uptake and cell imaging were the same as above.

[0106] 3 Results and discussion

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

[0108] First, SDS-PAGE gel electrophoresis was used to characterize the conjugation of protein-oligonucleotide. As Figure 8 shown, the oligonucleotide could be conjugated with H3 through 5b to form a band of the OsB-Hs conjugate. Due to the increase in the overall volume and mass of the conjugate and the decrease in the charge-mass ratio, the migration speed slowed down. Compared with H3, the migration of the lane band was significantly delayed, thus proving the binding of the oligonucleotide to histone 3.

[0109] In addition, the conjugation of OsB-Hs with H3 showed a concentration dependence. After optimizing the conjugation conditions, it was finally determined that H3 could basically react completely with 4eq of OsB.

[0110] The product of OsB-Hs modified by fluorescence, OsB-Hs-FPH, was also characterized by SDS-PAGE( Figure 9 ). To further confirm the conjugate, the conjugate was desalted by a desalting column (7 kDa) to remove salts and small molecules, and characterized by high-resolution liquid chromatography-mass spectrometry. The mass spectrometry results showed that the molecular weight of OsB-Hs was 18945.44, which was basically consistent with the theoretical molecular weight of 18945.5. The molecular weight of OsB-Hs-FPH was 19528.15, which was basically consistent with the theoretical molecular weight of 19528.12. This indicated that the oligonucleotide and H3 could be conjugated through 5b and modified by the fluorescent probe.

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

[0112] Since H3 has the property of a penetratin peptide, as Figure 10 shown, in the positive control group, red fluorescence could be observed in the nuclear part of the cells treated with Hs-RP. In the negative control group, no fluorescence was observed after the cells were treated with OsFN, indicating that the oligonucleotide itself did not have the ability to enter the cells. In the experimental group, green fluorescence could be observed in the nuclear part of the cells treated with OsB-Hs-FPH, indicating that the oligonucleotide could penetrate the cell membrane after being conjugated with histone.

[0113] Example 4: Study on the cyclization of polypeptide mediated by compound 5b:

[0114] The overall experimental design idea is as follows:

[0115]

[0116] The NHS ester part of the starting material 5b specifically binds to the amino group-containing polypeptide, and at the same time its 3Br-5MP part reacts with the thiol group on the polypeptide to jointly form a cyclic intermediate product. This intermediate product has reactivity and can react with another molecule of thiol group-containing fluorescent molecule, and is reduced by NaBH4 to form a stable thioether bond, realizing the modification of the polypeptide and obtaining a labeled final product, which can be used for subsequent fluorescence imaging or related characterization.

[0117] Screening of the conditions for polypeptide cyclization mediated by 5b:

[0118] 1 Experimental materials

[0119] Polypeptides:

[0120] (i) Series 1:

[0121] P1: NH2-AAACF-CONH2

[0122] P2: NH2-AAAACF-CONH2

[0123] P3: NH2-AAAAACF-CONH2

[0124] (ii) Series two:

[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 of the model peptides were dissolved in buffers of different pH values (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, and then stirred at room temperature for 15 - 20 min and monitored by LC-MS until the reaction was completed.

[0131] 3 Experimental results and discussion

[0132] Through LC-MS characterization of the crude reaction mixture, it was found that for the 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 rapid. The cyclization of all polypeptides proceeded stoichiometrically, with complete conversion within 15 min and no by-products. However, when the pH was greater than 9, the hydrolysis of the NHS ester increased, resulting in a decrease in the reaction conversion rate. For the intra-chain amino peptides (P4 - P7), as shown in (b) of Figure 8 , an increase in the distance between the two reactive groups would reduce the conversion rate and affect the reaction extent. By increasing the pH to 7 - 8, a conversion rate > 98% could basically be achieved. The results showed that for the terminal amino peptides, the reactivity between the amino group and the thiol group was not greatly affected by the distance, and the conversion rates were all > 90%; the reaction between the ε-amino group and the thiol group was affected by the distance, and the greater the distance, the smaller the conversion rate. A greater pH was required to achieve a conversion rate greater than 98%.

[0133] Cyclization, cellular uptake and imaging of bioactive polypeptides:

[0134] 1 Experimental reagents

[0135] Linear bioactive polypeptide CyP:

[0136] AcNH-CPIEDRPMK-CONH2

[0137] 2 Experimental methods

[0138] (1) Preparation of Fluorescently Modified Cyclic CyP: 0.5 mM polypeptide CyP and 1 mM 5b were stirred at room temperature for 15 min. Then, 4-fold equivalent of mercaptofluorescein was added, and the reaction was carried out in a constant temperature mixer at 37 °C for 1 h. 10-fold equivalent of sodium borohydride was added for reduction. The reaction was monitored by analytical LCMS until completion, and the resulting conjugate was named CyPB-FP.

[0139] (2) Cell Uptake Experiment: It is known that the cyclic peptide CyP can specifically bind to the cell membrane of human colon cancer differentiated cells (Caco2 cells). Therefore, 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 well-conditioned Caco2 cells, skbr3 cells, and Hela cells and inoculate them into a 12-well cell culture plate with coverslips placed inside. Control the number of test cells to be 10 5 cells / dish, and place them in a constant temperature incubator at 37 °C and 5% CO2 for culture until the cell density reaches about 60%-70%. Take out the 12-well cell culture plate, aspirate the old medium, and wash the coverslips with PBS two to three times. Add 1 ml of complete medium containing 1 mM CyPB-FP to each cell culture dish and incubate in a constant temperature incubator at 37 °C and 5% CO2 for 2 h.

[0140] (3) Cell Imaging: Staining: Take out the 12-well plate, aspirate the medium, and wash the coverslips with PBS three times, 2 min each time. Then add 1 ml of nuclear dye Hoechst 33342 (final concentration 2.5 μg / mL) and cell membrane dye Dill (final concentration 5 mM) diluted with PBS, and place it in a constant temperature incubator at 37 °C and 5% CO2 for light-avoiding staining for 10 min. Fixation: Aspirate the staining solution, wash the cell coverslips with 1 ml of PBS three times, 2 min each time. Add 500 μL of pre-cooled 4% paraformaldehyde fixative and fix in the dark for 15 min. Aspirate the fixative and wash the cell coverslips with 1 ml of PBS 3 times. Sealing: Use pointed forceps to take out the cell coverslips, place them face down carefully on a glass slide with a small amount of anti-fluorescence quencher dropped on it to prevent the generation of bubbles. Seal with nail polish. After solidification, image with a laser confocal microscope.

[0141] 3 Experimental Results and Discussion

[0142] (1) 5b-mediated Cyclization and Fluorescent Modification of CyP

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

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

[0145] The binding of the cyclically modified polypeptide CyPB-FH to Caco2 cells was detected using a laser confocal fluorescence microscope. As Figure 13 shown, the image of the fluorescein-conjugated cyclic peptide CyPB-FH overlapped well with the image of the cell membrane dye Dil. Compared with skbr3 cells and Hela cells, the fluorescein-modified cyclic peptide showed specific binding to Caco2 cells, indicating that the cyclization of the active polypeptide mediated by 5b could effectively mimic disulfide bonds and maintain cell targeting ability. These studies demonstrated that the cyclization of polypeptides mediated by 5b could be further modified with functional groups such as fluorescein. By changing the functional groups and recognition groups of the cyclic peptides, there was potential for synthesizing functional cyclic peptide biomolecules.

[0146] In summary, the present invention designed and synthesized a heterobifunctional reagent (3Br-5MP-NHS ester) based on 3-bromo-5-methylenepyrrolidone (3Br-5MP). By optimizing the linker structure, the cyclohexane linker 5b with the best stability was screened out, and its application in bioconjugation was systematically studied. The results showed that the NHS ester part of 5b could efficiently modify aminoated oligonucleotides, and the formed active intermediate OsB could further couple with thiol polypeptides / proteins. The conjugate successfully penetrated the Hela cell membrane and achieved fluorescence localization. At the same time, the polypeptide cyclization reaction mediated by 5b under pH 7-8 conditions was efficient with few by-products, and the cyclized product maintained the targeting ability to Caco2 cells, providing a new tool for oligonucleotide delivery and cyclic peptide synthesis.

[0147] The above are only embodiments of the present invention and should not be construed as limiting the scope of the present invention. Those skilled in the art can modify, innovate, or apply the claims and the content of the drawings to other technical fields. However, it should be noted that all modifications to the present invention should be included within the scope of the patent protection of the present invention.

Claims

1. A 3-bromo-5-methylenepyrrolidine heterobifunctional reagent, characterized in that: The compound comprises a compound represented by the general formula (I), or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof: Among them, R 2 Selected from the following structural fragments: Represents R 2 The site of attachment to the 3Br-5MP moiety, Represents R 2 Site of attachment to the NHS ester moiety.

2. The method for preparing the 3-bromo-5-methylenepyrrolidine heterobifunctional reagent according to claim 1, characterized in that: The preparation method comprises the following steps: Step (1), mixing 4-bromofuran-2-carboxaldehyde with sodium borohydride for reduction reaction to obtain intermediate 2; Step (2), acetylation reaction of intermediate 2 with acetic anhydride to obtain compound 3; Step (3), mixing compound 3 with N-bromosuccinimide, adding an aminobenzoic acid derivative to react, and obtaining compound 4; Where R 1 Selected from the following structural fragments: Step (4), compound 4 is mixed with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for condensation reaction to obtain a compound represented by general formula (I).

3. The preparation method according to claim 2, characterized in that: The solvent for 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: Step (2) was reacted at room temperature for 2 h.

5. The preparation method according to claim 2, characterized in that: In step (3), compound 3 and N-bromosuccinimide are dissolved in a mixed solution of tetrahydrofuran and PBS, reacted at 0°C, and then an aminobenzoic acid derivative is added and reacted at room temperature for 10 hours.

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

7. Use of the 3-bromo-5-methylenepyrrolidine heterobifunctional reagent as claimed in claim 1 in biocoupling.

8. The use according to claim 7, characterized in that: Specifically, it is used in oligonucleotide bioconjugation or polypeptide cyclization.

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