CN120208848A - A 3-bromo-5-methylenepyrrolone heterobifunctional reagent and its preparation and application in bioconjugation - Google Patents

Abstract

The present invention discloses a 3-bromo-5-methylene pyrrolidine heterobifunctional reagent and its preparation and application in bioconjugation, wherein the reagent comprises a specific structure as shown in the general formula (I), covering a variety of variant forms. Its preparation method comprises a multi-step reaction, and a compound containing a specific functional group is prepared by a series of reactions as a raw material. The reagent performs well in bioconjugation and can be used for the bioconjugation and polypeptide cyclization of oligonucleotides, cell-penetrating peptides and proteins. In terms of the bioconjugation of oligonucleotide cell-penetrating peptides and proteins, efficient modification and coupling can be achieved, and the conjugate can maintain good biological functions; in polypeptide cyclization, efficient cyclization of linear peptides can be promoted and cell targeting can be maintained. This provides a new approach for bioconjugation technology, and has important application prospects in the fields of biomedical research, drug development, etc.

Description

3-Bromo-5-methylenepyrrolidone heterobifunctional reagent, preparation method thereof and application thereof in bioconjugate

Technical Field

The invention belongs to the technical field of biological coupling, and particularly relates to a 3-bromo-5-methylenepyrrolidone heterobifunctional reagent, a preparation method thereof and application thereof in biological coupling, in particular to oligonucleotide biological coupling and polypeptide cyclization.

Background

The biological coupling technology is important in the fields of biomedicine, drug research and development, disease diagnosis and the like, and can realize the connection between biomolecules, endow the biomolecules with new functions or optimize the performances thereof. Oligonucleotides and polypeptides have great potential as important biomolecules in the therapeutic field, but face a number of challenges.

The oligonucleotide can be specifically combined with target RNA or DNA molecules, is involved in gene regulation and transcription expression, and has great potential in treating diseases related to single gene mutation or abnormal gene expression. However, its chargeability and nuclease sensitivity make it difficult to cross cell membranes, and bare nucleic acids are poorly stable in vivo, are easily enzymatically or renally cleared, and result in difficulty in reaching target cells effectively for function. Although lipid nanoparticle, galNAc and other delivery technologies have been developed, there are problems such as difficulty in specific delivery to extrahepatic cells/tissues and insufficient efficient uptake of oligonucleotides.

The polypeptide has the advantages of high selectivity, biological target specificity, low side effect and difficult accumulation in tissues and organs. However, linear peptides are poorly stable, are readily hydrolyzed by proteases, and are more polar and hydrophilic, resulting in lower cell membrane permeability and bioavailability. The cyclic peptide has the advantages of high activity, enzymolysis resistance, strong membrane permeability and the like, but the traditional cyclic peptide synthesis method has the problems of severe reaction conditions, multiple side reactions and the like.

The biological coupling reagent is the key of biological coupling, and the heterobifunctional crosslinking reagent is widely applied in biological coupling, so that unnecessary polymerization or self-coupling can be reduced. However, the existing crosslinking agents such as SMCC (Succinimidyl- (N-maleimidomethyl) cyclohexane-1-carboxylate) have the problem of poor stability, the maleimide part of the crosslinking agents is easy to hydrolyze in alkaline solution, the formed thioether bond is also unstable, thiol exchange reaction is easy to occur, and the application range is limited. Therefore, the development of the biological coupling reagent with high stability, good reaction activity and wide application range is significant.

Disclosure of Invention

The invention aims to solve the technical problems of the prior art that the oligonucleotide and the polypeptide are difficult to apply in biological treatment and the stability of the traditional biological coupling reagent is poor, and provides a 3-bromo-5-methylenepyrrolidone (3-Br-5 MPs) heterobifunctional reagent and application thereof in biological coupling. The reagent takes 3-Br-5MPs as a mother nucleus to be connected with NHS (N-hydroxysuccinimide) ester, and the whole stability and the reaction activity of the reagent are improved by reasonably designing a connecting arm structure. The method utilizes the high sulfydryl selectivity and double-site reactivity of 3Br-5MP and the excellent amino reaction activity and specificity of NHS ester to realize the precise coupling of biomolecules.

The invention aims to solve the technical problems, and adopts the following technical scheme:

In a first aspect of the present invention, there is provided a 3-bromo-5-methylenepyrrolidone type heterobifunctional reagent comprising a compound of formula (I), or an optical isomer, racemate, single enantiomer, possible diastereomer thereof, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof.

Wherein R ² is selected from the following structural fragments:

Wherein the method comprises the steps of Represents the site where R ² is linked to the 3Br-5MP moiety,Represents the site where R ² is linked to the NHS ester moiety.

In a second aspect of the present invention, a method for preparing the 3-bromo-5-methylenepyrrolone heterobifunctional reagent is provided, comprising the steps of:

(1) 4-bromofuran-2-formaldehyde is reacted with sodium borohydride to obtain intermediate 2;

(2) Reacting the intermediate 2 with acetic anhydride to obtain a compound 3;

(3) Under the action of N-bromosuccinimide and the like, adding different aminobenzoic acid derivatives a-c into the compound 3 for reaction to obtain compounds 4a-4c;

(4) And respectively reacting the compounds 4a-4c with N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI), and treating to obtain the compounds 5a-5c shown as the general formula (I).

More specifically, the compound represented by the general formula (I) of the present invention can be produced by the above-mentioned method, however, the conditions of the method, such as reactants, solvents, amounts of the compounds used, reaction temperature, time required for the reaction, etc., are not limited to the above-mentioned explanation. The compounds of the present invention may also be conveniently prepared by optionally combining the various synthetic methods described in this specification or known in the art, such combinations being readily apparent to those skilled in the art to which the present invention pertains.

In a third aspect, the invention provides the use of the 3-bromo-5-methylenepyrrolone heterobifunctional reagent described above in bioconjugates.

Preferably, the use of oligonucleotide bioconjugates and polypeptide circularization is specified. In the aspect of biological coupling of oligonucleotide cell penetrating peptide and protein, the conjugate can realize efficient modification and coupling, can maintain better biological function, and can promote efficient cyclization of linear peptide and maintain cell targeting in polypeptide cyclization. Compared with the prior art, the invention has the beneficial effects that:

In the 3-bromo-5-methylenepyrrolidone (3-Br-5 MPs) heteromorphic bifunctional reagent designed by the invention, the 3-Br-5MPs parent nucleus has higher chemical stability, high selectivity and double-site reaction activity on sulfhydryl (-SH), and high reaction activity on amino (-NH ₂) of NHS ester part, thereby realizing accurate coupling, reducing non-specific side reaction and improving coupling efficiency and stability. Through reasonable design of the connecting arm structure, unnecessary polymerization or self-coupling phenomena are reduced, the generation of byproducts is reduced, and the success rate and reliability of biological coupling are improved.

Because of the heterobifunctional characteristics, the reagent provided by the invention can be widely used for coupling biological macromolecules such as proteins, antibodies, drug molecules and the like, and the biological coupling technology provides a new way, so that the reagent is suitable for various biomedical and drug research and development applications, such as targeted drug delivery, development of diagnostic probes and the like.

Drawings

Figures 1-4 are graphs of the degradation rates of compounds 5a, 5b, 5c and SMCC at different pH, respectively, where (a) is ph=6, (b) is ph=7.5, and (c) is ph=9.

FIG. 5 is a graph showing the results of optimal conditions for the reaction of Compound 5b with oligonucleotide Os.

FIG. 6 is a HPLC characterization of OsB-TAT and OsB-TAT-FPH.

FIG. 7 is a graph of uptake fluorescence imaging of Hela cells of OsB-TAT-FPH.

FIG. 8 is a SDS-PAGE characterization of OsB-Hs.

FIG. 9 is a SDS-PAGE characterization of OsB-Hs-FPH.

FIG. 10 is a graph showing uptake fluorescence imaging of Hela cells of Os-Hs-FPH.

FIG. 11 shows the conversion of compound 5b mediated cyclization of a series of polypeptides, wherein (a) is polypeptides P1-P3 and (b) is polypeptides P4-P7.

FIG. 12 is a HPLC characterization of compound 5b mediated cyclization of CyP and fluorescent modification.

FIG. 13 is a cell uptake fluorescence imaging of CyPB-FPH.

Detailed Description

The invention is further illustrated in the following figures and examples, which are not intended to limit the invention to the examples.

Example 1:

The preparation of compound 5 comprises the following steps:

1) Synthesis of intermediate 2 sodium borohydride (226.6 mg,5.99mmol,1.05 eq) was added to a solution of 4-bromofuran-2-carbaldehyde (1.0 g,5.71mmol,1.0 eq.) in anhydrous tetrahydrofuran (30 ml) in a 0℃ice-water bath and the reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction by TLC, the reaction was quenched with saturated aqueous ammonium chloride, tetrahydrofuran was removed by rotary evaporation, ethyl acetate was added and extracted 3 times in a separating funnel. The combined organic phases were extracted 1 further with saturated sodium chloride solution, dried over anhydrous sodium carbonate, filtered to remove the drying agent and the filtrate was concentrated under vacuum to give crude 1.13g. The next step of synthesis was carried out directly without purification.

2) Synthesis of intermediate 3 acetic anhydride (2.64 mL,25.8mmol,4.0 eq.) was added dropwise to a solution of compound 2 (1.13 g,6.46mmol,1.0 eq.) in dry pyridine (30 ml) in a 0℃ice-water bath. 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 extracted with saturated sodium bicarbonate, the organic phase was dried and concentrated, and the crude product was separated by column on silica gel (petroleum ether: ethyl acetate=95:5, v:v) to give compound 3 as a yellow liquid (1.19 g, yield 84.1％).¹H NMR(400MHz,DMSO-d₆)δ7.15(d,J＝0.6Hz,1H),5.94(s,1H),4.23(s,2H),1.25(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ175.12,156.07,147.33,118.78,104.69,62.62,25.72.

3) Intermediate 4a was synthesized by dissolving intermediate 3 (300 mg,0.97mmol,1.0 eq) in 6ml tetrahydrofuran in 0.25M sodium phosphate buffer (ph 7.5) =1:1 and cooling the mixed solution to 0 ℃. N-bromosuccinimide (206.5 mg,1.16mmol,1.2 eq.) was added and stirred at 0deg.C until TLC monitored complete conversion of starting material to intermediate. M-aminomethylbenzoic acid (217.6 mg,1.16mmol,1.2 eq.) was added, the pH was adjusted to 7 and 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, then the reaction mixture was 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 was concentrated under reduced pressure to give a crude product. Purification by column chromatography on silica gel (petroleum ether: ethyl acetate: acetic acid=70:30:0.1, v: v) afforded compound 4a as a pale yellow solid (135.5 mg, yield 45.7％).¹H NMR(500MHz,DMSO-d₆)δ7.87–7.81(m,1H),7.78(d,J＝1.7Hz,1H),7.72(s,1H),7.51–7.43(m,2H),5.24(d,J＝2.0Hz,1H),5.11(d,J＝2.0Hz,1H),4.94(s,2H).

4) Synthesis of intermediate 4b Synthesis of Compound 4b may be prepared according to the synthetic method of Compound 4a, and purified by silica gel column separation (Petroleum ether: ethyl acetate: acetic acid=70:30:0.1, v: v) to give Compound 4b as a white solid (123.3 mg, yield 41％).¹HNMR(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).

5) Synthesis of intermediate 4c Synthesis of Compound 4c may be prepared according to the synthetic method of Compound 4a, and purified by silica gel column separation (Petroleum ether: ethyl acetate: acetic acid=70:30:0.1, v: v) to give Compound 4c as a pale yellow solid (179.9 mg, yield 70％).¹HNMR(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).

6) Synthesis of Compound 5a intermediate 4a (130 mg,0.42mmol,1 eq.) was dissolved in 10ml of anhydrous tetrahydrofuran, N-hydroxysuccinimide (57.54 mg,0.50mmol,1.2 eq.) was added sequentially, 1-ethyl-carbodiimide hydrochloride (95.85 mg,0.5mmol,1.2 eq.) and the reaction mixture was reacted at room temperature overnight. After the reaction was completed, the reaction mixture was 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 was concentrated under reduced pressure to obtain a crude product. Purification by column chromatography on silica gel (petroleum ether: ethyl acetate: acetic acid=75:25, v: v) afforded compound 5a as a pale yellow solid (57.3 mg,33.8% yield ).¹HNMR(400MHz,Chloroform-d)δ8.05(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).¹³CNMR(101MHz,Chloroform-d)δ169.20,165.24,161.56,143.32,137.68,135.70,133.67,129.93,129.55,129.01,125.65,118.64,98.52,43.29,25.70.

7) Synthesis of Compound 5b may be carried out according to the method for synthesizing Compound 5a starting from intermediate 4b, and purification by silica gel column separation (Petroleum ether: ethyl acetate: acetic acid=70:30:0.1, v: v) gives Compound 5b as a white solid (57.5 mg, yield 35％).¹HNMR(500MHz,Chloroform-d)δ7.11(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).

8) Synthesis of Compound 5c may be carried out according to the method for synthesizing Compound 5a starting from intermediate 4c, and purification by silica gel column separation (Petroleum ether: ethyl acetate: acetic acid=70:30:0.1, v: v) gives 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).

Example 2:

stability test experiments for compounds 5a, 5b, 5c, comprising the steps of:

1 Experimental reagent

Control compounds 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), 5a-5c were dissolved in DMSO to prepare 100mM stock solutions, which were diluted to a final concentration of 5mM with buffers of different pH (pH 6.010mM sodium phosphate buffer, pH7.510mM sodium phosphate buffer, pH910mM sodium phosphate buffer), respectively.

2 Experimental methods

At various time intervals, a certain amount of the mixture was taken for LCMS analysis.

3 Experimental results and discussion

The control compounds SMCC were tested as described above for 5a, 5b, 5 c. The results are shown in FIGS. 1-4. By testing the stability of the compounds SMCC, 5a-5c at pH6-9, we found that the degradation rate of the compounds was SMCC >5c >5a >5b. In neutral buffer solution, SMCC hydrolyzes more than half within 24 h. Analysis by LC-MS showed that hydrolysis of the NHS ester moiety occurred predominantly to SMCC at pH6, whereas hydrolysis of the NHS ester moiety occurred first at pH >7, followed by hydrolysis of the maleimide moiety. The compounds 5a-5c mainly hydrolyze NHS ester part at pH6-9, the degree of hydrolysis increases with the increase of pH value, the parent nucleus part is not decomposed basically, and the compound has superior stability compared with SMCC. The stability of the compounds 5c and 5a with the aminomethylbenzoic acid as the intermediate group is lower, and the analysis reason is that the electron-withdrawing conjugation of the benzene ring increases the lack of electricity of carbonyl carbon, so that the NHS ester is easier to hydrolyze. Compound 5b, in which the spacer is cyclohexane, is the most stable, consistent with theoretical results. The analytical reasons are that electron-withdrawing conjugation of the benzene ring increases the electron deficiency of carbonyl carbon, so that the NHS ester is easier to hydrolyze. According to the stability study result, compound 5b was selected for subsequent bioconjugation studies.

Example 3 study of Compound 5b mediated oligonucleotide bioconjugation:

The whole experimental design thought is as follows:

The NHS ester moiety of 5b can react specifically with amino modified oligonucleotides to form oligonucleotide modified products. The product has reactivity, can sequentially act with two biomolecules containing sulfhydryl groups, and is reduced to form stable thioether bond through NaBH ₄, so that the coupling of the oligonucleotide and the protein or polypeptide is realized, and finally, the coupling product which can be used for fluorescence imaging or characterization is obtained.

5B mediated oligonucleotide bioconjugate reaction condition screening:

1 Experimental reagent

10MM,100mM,500mM,1M buffer (pH 6.0MES buffer, pH7.5HEPES buffer, pH9.0TE buffer, pH11 sodium phosphate buffer) at different pH.

The oligonucleotide sequences were as follows:

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

Dissolving Os in PBS buffer solution to prepare 1mM mother solution;

compound 5b was dissolved in DMSO and a 10mM stock solution was prepared.

2 Experimental methods

Mixing 0.2mM of Os solution with different concentrations of compound 5b solution in different types and concentrations of buffer solutions for reacting for a certain time, exploring the influence of the reaction time, 5b concentration and the types and concentrations of the buffer solutions on the reaction, detecting the generation condition of the product OsB by LCMS, and screening the optimal reaction system.

3 Results of experiments

As shown in FIG. 5, by optimizing the reaction time, 5b concentration and the kind and concentration of the buffer, it was confirmed that the optimal condition of the reaction was that in 100mMPBS, when 5b was reacted at 10-fold equivalent for 90min, a relatively pure oligonucleotide-modified product, designated OsB, was obtained at 90% conversion.

5B mediate oligonucleotide-polypeptide coupling, characterization, and cellular uptake and imaging:

1 Experimental reagent

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 and the like.

Cell penetrating peptide TAT sequence:

GRKKRRQRRRC

negative control oligonucleotide OsFN sequence:

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

2 Experimental methods

(1) Os were incubated with 10-fold equivalents of 5b in 100mM PBS buffer for 2h. According to the reported reactivity of 3-bromo-5-methylenepyrrolidone, the screened optimal reaction equivalent, 8 times equivalent of TAT solution, was added for reaction at 37 ℃ for 30min. LCMS detects the formation of OsB-TAT product.

(2) Since 3-bromo-5-methylenepyrrolidone is known to have dimercapto reaction property, after the completion of OsB-TAT conversion is monitored according to the steps, 10 times equivalent of mercaptofluorescein is added to carry out mercapto addition reaction with the other reaction site of 3-bromo-5-methylenepyrrolidone, the reactants are uniformly mixed by vortex, incubation is continued for 1h at a constant temperature of 37 ℃, and 10 times equivalent of sodium borohydride is added to reduce for 15min. LCMS analyzed the formation of the product and the resulting fluorescence-modified conjugate was designated OsB-TAT-FPH.

(3) Preparation of the Positive control cell penetrating peptide TAT-RP an aqueous solution of TAT was mixed with 2 equivalents of 5-carboxytetramethyl rhodamine succinimidyl ester in DMSO and reacted at 37℃for 1h. Desalting and purifying to obtain the product TAT-RP.

(4) Cell uptake assay:

(i) Taking out the Hela cells with good state from the incubator, inoculating the Hela cells into a 12-hole cell culture plate on which the climbing plates are placed, controlling the number of cells to be detected to be 10 ⁵ cells/dish, placing the cells to be detected into a constant-temperature incubator with the temperature of 37 ℃ and the concentration of 5% CO ₂ for culture, and preferably, keeping the cell density to be about 60% -70%.

(Ii) The 12-well cell culture plate was removed, old medium was aspirated, and the slide was washed two to three times with PBS. A negative control group was set, and the positive control group and the experimental group were subjected to cell administration treatment as follows.

A. A negative control group, namely adding 1ml of DMEM complete medium containing 0.5mM OsFN into a culture dish, uniformly mixing, and then placing the culture dish into a constant-temperature incubator with 37 ℃ and 5% CO ₂ for incubation for 2 hours;

b. positive control group, namely adding 1ml of DMEM complete medium containing 0.5mM TAT-RP into a culture dish, uniformly mixing, and then placing the culture dish into a constant temperature incubator with 37 ℃ and 5% CO ₂ for incubation for 2 hours;

c. the experimental group is that 1ml of DMEM complete medium containing 0.5mM of OsB-TAT-FPH is added into a culture dish, and after being mixed evenly, the culture dish is put into a constant temperature incubator with 37 ℃ and 5% CO ₂ for incubation for 2 hours;

(5) Cell imaging, fixing, namely sucking and removing the culture medium, and washing the cell climbing plate three times for 2min each time by using 1ml PBS. 500mL of pre-chilled 4% paraformaldehyde fixing solution was added and fixed in the dark for 15min. The fixative was aspirated off and the cell slide was washed 3 times with 1ml PBS. The sealing piece is that the cell climbing piece is taken out by using a pointed tweezers, the cells face downwards, and the cell climbing piece is carefully placed on a glass slide with a small amount of anti-fluorescence quenching agent to prevent bubbles. And sealing the sheet with a nail oil, and imaging by using a laser confocal microscope after solidification.

3 Experimental results and discussion

(I) 5 b-mediated oligonucleotide-polypeptide coupling and characterization

As shown in FIG. 6, the conjugate OsB-TAT, which was found to have a retention time of 7.1min as an oligonucleotide-penetrating peptide conjugate, was characterized by LC-MS, and had a molecular weight of 5202.7, which substantially matches the theoretical molecular weight 5202.4, and the yield was calculated by area normalization using the HPLC product peak area, with a final yield of >92%.

Further monitoring the fluorescent modification product OsB-TAT-FPH, wherein the secondary mercaptan addition reaction is very slow, monitoring the reactant by LCMS after 2-3 hours of reaction, finding that the peak with the retention time of 7.9min is the fluorescent modification product OsB-TAT-FPH, the obtained molecular weight is 5784.28, basically conforming to the theoretical molecular weight 5783.8, calculating the yield by using an HPLC product peak area and adopting an area normalization method, and the final yield is more than 90%.

(Ii) Cellular uptake and imaging of oligonucleotide-penetrating peptide conjugates

In order to investigate whether 5b mediated coupling of an oligonucleotide to a cell penetrating peptide would affect the biological activity of a polypeptide, cell imaging experiments of the conjugate were performed in living cells, as shown in fig. 7, fluorescence modified oligonucleotide OsFN, to which the penetrating peptide was not attached, was unable to penetrate the cell membrane into the cell, while OsB-TAT-FPH could observe green fluorescence in the cell, indicating that the oligonucleotide could be carried into the cell after coupling of 5b to the penetrating peptide, which still maintained good physiological activity.

5B mediate oligonucleotide-protein coupling, characterization, and cellular uptake and imaging:

1 Experimental materials

As before.

Histone H3 sequence:

Histone3V35C/C110A

ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGCKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVALFEDTNLAAIHAKRVTIMPKDIQLARRIRGERA

2 Experimental methods

(1) Preparation of positive control fluorescence H3 aqueous solution was mixed with 10-fold equivalent of DMSO solution of 5-carboxytetramethyl rhodamine succinimidyl ester and reacted at 37℃for 1H. Desalting and purifying to obtain the product Hs-RP.

(2) Os was incubated with 10 equivalents of 5b in 100mM PBS buffer for 2h to give OsB.0.4mM H3 solution was mixed with different equivalents of OsB solution and reacted at 37℃for 30min. SDS-PAGE detects the formation of OsB-Hs.

(3) After the completion of the conversion of OsB-Hs is monitored according to the steps, 10 times equivalent of mercaptofluorescein is added, the reactants are mixed by vortex, the incubation is continued for 1h at a constant temperature of 37 ℃ by a mixer, and 10 times equivalent of sodium borohydride is added for 15min. SDS-PAGE analyzes the formation of the product, and the resulting fluorescence-modified conjugate was designated as OsB-Hs-FPH.

(4) Cell uptake and cell imaging experimental procedures were as above.

3 Results and discussion

(1) SDS-PAGE characterization of oligonucleotide-histone H3 conjugates

The protein-oligonucleotide coupling was first characterized by SDS-PAGE gel electrophoresis, as shown in FIG. 8, and the oligonucleotide was capable of coupling to H3 via 5b to form OsB-Hs conjugate bands. The binding of the oligonucleotide to histone 3 is demonstrated by a significantly delayed lane band migration compared to H3 due to the increased overall volume and mass of the conjugate, reduced charge-to-mass ratio and slower migration.

In addition, the coupling of the OsB-Hs and H3 shows concentration dependence, and the OsB of H3 and 4eq can be basically completely reacted through the optimization of coupling conditions.

The products of OsB-Hs modified by fluorescence OsB-Hs-FPH were also characterized by SDS-PAGE (FIG. 9). To further confirm the conjugate, the conjugate was passed through a desalting column (7 kda) to remove salts and small molecules, characterized by high resolution liquid chromatography-mass spectrometry. The mass spectrum results show that the molecular weight of the OsB-Hs is 18945.44, which is basically consistent with the theoretical molecular weight 18945.5. The molecular weight of OsB-Hs-FPH is 19528.15, which is basically consistent with the theoretical molecular weight 19528.12. It is illustrated that the oligonucleotide and H3 can be coupled by 5b and modified with a fluorescent probe.

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

H3 has the property of penetrating peptide, so in the positive control group, hs-RP treated cells can observe red fluorescence in the nucleus portion as shown in FIG. 10. In the negative control group, osFN treated cells were not fluorescent, indicating that the oligonucleotide itself was not able to enter the cells. In the experimental group, the OsB-Hs-FPH treated cells can observe green fluorescence at the cell nucleus, which indicates that the oligonucleotide can penetrate the cell membrane after being coupled with histone.

Example 4 study of Compound 5b mediated cyclization of a polypeptide:

The whole experimental design thought is as follows:

The NHS ester moiety of initiator 5b specifically binds to the amino-containing polypeptide while its 3Br-5MP moiety reacts with the thiol group on the polypeptide to collectively form a cyclic intermediate. The intermediate product has reactivity, can react with another molecule of thiol-containing fluorescent molecule, and is reduced by NaBH ₄ to form stable thioether bond, so that the modification of polypeptide is realized, and the final product with a label is obtained and can be used for subsequent fluorescence imaging or related characterization.

5 B-mediated screening of polypeptide cyclization conditions:

1 Experimental materials

Polypeptide:

(i) Series one:

P1:NH₂-AAACF-CONH₂

P2:NH₂-AAAACF-CONH₂

P3:NH₂-AAAAACF-CONH₂

(ii) Series two:

P4:AcNH-KAAACF-CONH₂

P5:AcNH-KAAAACF-CONH₂

P6:AcNH-KAAAAACF-CONH₂

P7:AcNH-KAAAAAACF-CONH₂

2 Experimental methods

In cyclization of all polypeptides, model peptides 0.1mM P1-P7 were dissolved in buffers of different pH (10 mM pH8 sodium phosphate buffer, pH7PBS buffer, pH6 sodium phosphate buffer). 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.

3 Experimental results and discussion

By LC-MS characterization of the crude reaction mixture, it was found that for the terminal amino peptides (P1-P3), as shown in fig. 11 (a), the cyclization reaction was very simple and rapid at pH6-8, with all polypeptide cyclizations proceeding stoichiometrically, complete conversion within 15min and no byproducts, but pH greater than 9 increased hydrolysis of NHS ester, resulting in a decrease in reaction conversion. For the intra-chain aminopeptides (P4-P7), as shown in FIG. 8 (b), an increase in the distance between both reactive groups decreases the conversion, affecting the extent of the reaction, and a conversion of >98% can be achieved essentially by increasing the pH to 7-8. The results show that for the terminal amino peptide, the reactivity of amino and sulfhydryl groups is not greatly influenced by distance, the conversion rate is more than 90%, the reaction of epsilon-amino and sulfhydryl groups is influenced by distance, the conversion rate is smaller as the distance is larger, and the pH is required to be larger as the conversion rate is larger than 98%.

Cyclization and cellular uptake and imaging of the active polypeptide:

1 Experimental reagent

Linear active polypeptide CyP:

AcNH-CPIEDRPMK-CONH₂

2 Experimental methods

(1) Preparation of fluorescence-modified Cyclic CyP 0.5mM polypeptide CyP and 1mM 5b were stirred at room temperature for 15min. Then, 4-fold equivalents of mercaptofluorescein were added, reacted in a constant temperature mixer at 37℃for 1 hour, 10-fold equivalents of sodium borohydride were added for reduction, monitored by analytical LCMS until the reaction was completed, and the resulting conjugate was designated CyPB-FP.

(2) Cell uptake experiments it is known that cyclopeptide CyP can specifically bind to the cell membrane of human colon cancer differentiated cells (Caco 2 cells), so that Caco2 cells are selected for routine culture, and human breast cancer cells (skbr cells) and human cervical cancer cells (HeLa cells) are selected as negative controls. Taking Caco2 cells, skbr cells and Hela cells with good states, inoculating the cells into a 12-hole cell culture plate on which a climbing plate is placed, controlling the number of cells to be detected to be 10 ⁵ cells/dish, placing the cells into a constant-temperature incubator with the temperature of 37 ℃ and the temperature of 5% CO ₂ for culture, and keeping the cell density to be about 60% -70%. The 12-well cell culture plate was removed, old medium was aspirated, and the slide was washed two to three times with PBS. To each cell culture dish was added 1ml of complete medium containing 1mMCyPB-FP and incubated in an incubator at 37℃with 5% CO ₂ for 2h.

(3) Cell imaging, namely, staining, namely, taking out a 12-hole plate, sucking and removing the culture medium, and washing the climbing plate three times with PBS (phosphate buffered saline) for 2min each time. Then 1mL of the nuclear dye Hoechst 33342 (final concentration 2.5. Mu.g/mL) diluted with PBS and the cell membrane dye Dill (final concentration 5 mM) were added, and the mixture was stained in a constant temperature incubator at 37℃with 5% CO ₂ in the absence of light for 10 minutes. Fixing, namely absorbing and discarding the staining solution, and washing the cell climbing sheet with 1ml PBS for three times for 2min each time. 500mL of pre-chilled 4% paraformaldehyde fixing solution was added and fixed in the dark for 15min. The fixative was aspirated off and the cell slide was washed 3 times with 1ml PBS. The sealing piece is that the cell climbing piece is taken out by using a pointed tweezers, the cells face downwards, and the cell climbing piece is carefully placed on a glass slide with a small amount of anti-fluorescence quenching agent to prevent bubbles. And sealing the sheet with a nail oil, and imaging by using a laser confocal microscope after solidification.

3 Experimental results and discussion

(1) 5B mediated CyP cyclization and fluorescent modification

As shown in fig. 12, LCMS results showed that CyP and 5b can cyclize within 15min, the product peak is single, the retention time is 2.9min, the molecular weight is 1362.6548, the molecular weight substantially matches the theoretical molecular weight, the yield is calculated by using HPLC product peak area, and the final yield is >90% by area normalization. LCMS results for fluorescence-modified product CyPB-F showed a retention time of 3.9min, resulting molecular weight 899.3705 substantially consistent with theoretical molecular weight 899.67, final yield >95%. After reduction, a more stable cyclized product CyPB-FH was obtained, with a retention time of 3.7min, a molecular weight of 900.8799, substantially corresponding to the theoretical molecular weight 900.60, and a final yield of >90%.

(2) CyPB-FH cellular uptake and cellular imaging

And detecting the combination of the cyclized modified polypeptide CyPB-FH and Caco2 cells by using a laser confocal fluorescence microscope. As shown in fig. 13, the image of fluorescein-binding cyclopeptide CyPB-FH overlapped well with the image of cell membrane dye Dil, and the fluorescein-modified cyclopeptide showed specific binding to Caco2 cells compared to skbr cells and Hela cells, indicating that cyclization of 5 b-mediated active polypeptide can effectively mimic disulfide bonds, preserving cell targeting ability. These studies demonstrate that 5 b-mediated cyclization of polypeptides can be further modified with functional groups such as fluorescein. The potential for synthesizing functional cyclopeptide biomolecules can be used by altering the functional and recognition groups of the cyclopeptide.

In summary, the invention designs and synthesizes a heterobifunctional reagent (3 Br-5MP-NHS ester) based on 3-bromo-5-methylenepyrrolidone (3 Br-5 MP), and selects cyclohexane linker 5b with optimal stability by optimizing a connecting arm structure, and systematically researches the application of the heterobifunctional reagent in bioconjugation. The result shows that the NHS ester part of 5b can efficiently modify the amination oligonucleotide, the formed active intermediate OsB can be further coupled with sulfhydryl polypeptide/protein, the conjugate successfully penetrates Hela cell membranes and realizes fluorescent localization, meanwhile, the polypeptide cyclization reaction mediated by 5b under the condition of pH 7-8 is efficient and has few byproducts, and the cyclization product maintains the targeting capability on Caco2 cells, thereby providing a novel tool for oligonucleotide delivery and cyclopeptide synthesis.

The foregoing is merely exemplary embodiments of the present invention and should not be construed as limiting the scope of the present invention, and it is obvious to those skilled in the art that modifications, innovations, or other technical applications may be made in the claims and drawings, however, it should be noted that all modifications are included in the scope of the present invention.

Claims (8)

1. A 3-bromo-5-methylenepyrrolone heterobifunctional reagent comprising a compound of formula (I), or an optical isomer, racemate, single enantiomer, possible diastereomer thereof, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof:

wherein R ² is selected from the following structural fragments:

Represents the site where R ² is linked to the 3Br-5MP moiety, Represents the site where R ² is linked to the NHS ester moiety.

2. The method for preparing the 3-bromo-5-methylenepyrrolone heterobifunctional reagent of claim 1, comprising the steps of:

step (1), mixing 4-bromofuran-2-formaldehyde with sodium borohydride for reduction reaction to obtain an intermediate 2;

Step (2), performing an acetylation reaction on the intermediate 2 and acetic anhydride to obtain a compound 3;

step (3), mixing the compound 3 with N-bromosuccinimide, and adding an aminobenzoic acid derivative for reaction to obtain a compound 4;

Wherein R ¹ is selected from the following structural fragments:

And (4) mixing the compound 4 with N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide for condensation reaction to obtain the compound shown in the general formula (I).

3. The method according to claim 2, wherein the solvent for the reduction reaction in step (1) is tetrahydrofuran, and the reaction is carried out at room temperature for 2 hours.

4. The method of claim 2, wherein step (2) is carried out at room temperature for 2 hours.

5. The preparation method according to claim 2, wherein in the step (3), the compound 3 and the N-bromosuccinimide are dissolved together in a mixed solution of tetrahydrofuran and PBS, and the aminobenzoic acid derivative is added after the reaction at 0 ℃ and the reaction is carried out for 10 hours at room temperature.

6. The process according to claim 2, wherein the condensation reaction of step (4) is carried out in tetrahydrofuran at room temperature for 8 hours.

7. Use of a 3-bromo-5-methylenepyrrolidone heterobifunctional reagent of claim 1 in bioconjugate.

8. Use according to claim 7, in particular for oligonucleotide bioconjugate or polypeptide cyclisation.