CN103865012A - Preparation of polymer-polypeptide bioconjugate with comb-shaped structure - Google Patents

Abstract

The invention provides a preparation method of a comb structure polymer-polypeptide bioconjugate. The process is to use reversible addition-cracking chain transfer radical polymerization technology to synthesize diblock copolymers containing polyhydroxyethyl methacrylate segments. After removing dithiobenzoate end groups, block copolymers and Acryloyl chloride was reacted to synthesize a block copolymer modified with acrylate groups in the side chain. Then, at room temperature, the polymer is reacted with reduced glutathione in N,N-dimethylformamide/water solution to obtain a comb-like structure polymer-polypeptide bioconjugate. The comb-like structure polymer-polypeptide bioconjugate prepared by the present invention can self-assemble into nanomicelles in aqueous solution, has pH responsiveness, biorecognition and degradability, and is used in drug controlled release, biomedicine and biological detection, etc. It has broad application prospects.

Description

Preparation of comb-like polymer-polypeptide bioconjugates

technical field

The invention relates to a preparation method of a novel comb structure polymer-polypeptide bioconjugate.

Background technique

In recent years, bioconjugates (Bioconjugates) formed by combining polymers with proteins and polypeptides can not only become new therapeutic agents and biological probes, but also serve as new functional materials in biotechnology, biomedicine, and functional nanomaterials. It has broad and far-reaching application prospects. Living radical polymerization technology provides a convenient and effective way to synthesize various polymers with controllable molecular weight and structure, and promotes the development of polymer-protein/polypeptide bioconjugates with precise structures. There are many methods for synthesizing polymer-protein/polypeptide bioconjugates reported in the literature, which are mainly divided into "grafting from" and "grafting to". Using protein or polypeptide molecularly modified initiators or chain transfer agents, polymer bioconjugates can be directly obtained through the "grafting from" approach (Boyer, C., Bulmus, V., Liu, J.Q., Davis, T.P., Stenzel, M.H., Barner-Kowollik, C., J.Am.Chem.Soc.2007, 129:7145.), but considering the biological activity of proteins and polypeptides, this method is more suitable for polymerization in water systems at lower temperatures, This limits the range of applicable monomers. Combining living radical polymerization technology with various coupling chemistries, especially the simple and efficient "click" chemistry (Sumerlin, B.S., Vogt, A.P., Macromolecules 2010, 43: 1.), synthetic polymerization through the "grafting to" approach Bioconjugates (Li, M., De, P., Gondi, S.R., Sumerlin, B.S., Macromol. Rapid Commun. 2008, 29: 1172.), mild reaction conditions, good selectivity, high yield, abundant With the structural and functional design of bioconjugates, biomaterials with various topological structures and advanced functionalities can be obtained.

Contents of the invention

The present invention provides a new method for preparing comb-like structure polymer-polypeptide bioconjugates, which uses reversible addition-fragmentation chain transfer (RAFT) free radical polymerization technology to synthesize block copolymers containing hydroxyl groups in side chains, After removal of the terminal dithiobenzyl ester group, an acrylate-modified block polymer was obtained by reacting with acryloyl chloride, and reduced glutathione was bonded to the side chain of the polymer by mercapto-alkene click chemistry to prepare Comb-like polymer-polypeptide bioconjugates. This comb-like bioconjugate self-assembles in aqueous solution to form spherical micelles with uniform size and pH responsiveness. It can release glutathione under acidic conditions and can be used as a drug carrier for drug controlled release. The adopted conjugation method has the advantages of mild reaction conditions, high yield, suitable for conjugating other biomolecules containing free sulfhydryl groups, and the like.

Technical solutions

Use RAFT polymerization technology to polymerize methacrylate monomers to synthesize macromolecular RAFT reagents with narrow molecular weight distribution, and then carry out block copolymerization with hydroxyethyl methacrylate (HEMA) as the second monomer to obtain two block segment copolymers. At 80℃, the block copolymer was reacted with excess azobisisobutylcyanide (AIBN) to remove the terminal dithiobenzyl group. In anhydrous THF, the determinated polymer was reacted with acryloyl chloride to obtain an acrylate-modified block copolymer. The acrylate-modified polymer was dissolved in N, N-dimethylformamide (DMF), and reduced glutathione (GSH) was dissolved in secondary water. Under a nitrogen atmosphere, the GSH aqueous solution was added dropwise to the polymer solution, The reaction was carried out under stirring at room temperature for 3 days, followed by secondary water dialysis (MWCO 3500) for 2 days, and freeze-drying to obtain the polymer-polypeptide bioconjugate.

Specific reaction process:

The molecular weight Mn of the two-block copolymer synthesized in the invention ranges from 5,000 to 10,000 g/mol, the molecular weight polydispersity index PDI<1.3, and the polymerization degree of polyhydroxyethyl methacrylate is 7 to 10. The monomers are polyethylene glycol methacrylate (PEGMA), 3-methacryloyl-1,2:5,6-bis-O-isopropyl-D-glucofuranose (MAIpGlc), 6 -O-methacryloyl-1,2:3,4-bis-O-isopropylidene-D-galactopyranose (MAIpGal).

The preparation method of the comb structure polymer-polypeptide bioconjugate of the present invention comprises the following steps:

1) Synthesize a macromolecular chain transfer agent, and use CPADB as a chain transfer agent to carry out RAFT polymerization of methacrylate monomers. The method is to add monomer, free radical initiator and chain transfer agent into the reaction bottle, add organic solvent to dissolve, freeze with liquid nitrogen - vacuumize - fill with nitrogen, cycle three times, then heat to 70 ° C in a water bath, polymerize for 24 hours, and terminate Reaction, the polymer is precipitated, centrifuged and purified to remove unreacted monomers, dried in a vacuum oven to constant weight, and a macromolecular chain transfer agent is obtained;

2) Dissolve the macromolecular chain transfer agent synthesized in the first step in an organic solvent, add hydroxyethyl methacrylate, initiator, liquid nitrogen freezing-vacuumizing-nitrogen filling, cycle three times, and then heat to 70 ° C in a water bath, After 24 hours of polymerization, the reaction was terminated, the polymer was precipitated, centrifuged and purified to remove unreacted monomers, dried in a vacuum oven to constant weight, and a diblock copolymer was obtained;

3) Dissolve the block copolymer synthesized in the second step in DMF, add excess azobisisobutyronitrile (AIBN), react at 80°C for 3 hours, precipitate, centrifuge, and dry in a vacuum oven to constant weight to obtain Diblock copolymers with thiobenzyl end groups;

4) Dissolve the block copolymer obtained in the third step in tetrahydrofuran (THF), add pyridine, slowly add acryloyl chloride/THF (0.5mL) solution dropwise at 0°C, react for 2h at 0°C, and then react for 24h at room temperature. After the reaction, filter, spin dry THF solution, dissolve with CHCl ₃ , then extract three times with saturated NaHCO ₃ solution, spin CHCl ₃ dry, dissolve with THF solution, put the sample solution in a dialysis bag (MWCO 3500), and dialyze After 2 days, it was freeze-dried to obtain an acrylate-modified diblock copolymer.

5) Dissolving the acrylate-modified diblock copolymer obtained in the fourth step in DMF, adding pyridine and reduced glutathione (GSH) aqueous solution. After flowing nitrogen for 20 min, the reaction was carried out at room temperature for 3 days. Unreacted GSH was removed by dialysis, and the sample was freeze-dried to obtain a comb-shaped polymer-polypeptide bioconjugate. For the block copolymer obtained by polymerization of protective sugar-containing monomers, after acrylate modification, the protective group is removed in 80% formic acid solution, and then reacted with the polypeptide.

The organic solvent is dioxane, N,N-dimethylformamide.

The free radical initiator is 4,4'-azobis-(4-cyanovaleric acid) (ACPA), azobisisobutyronitrile (AIBN).

The consumption ratio of described methacrylate monomer and chain transfer agent, free radical initiator:

Monomer concentration: 10-30wt%, mass %: total monomer mass/(total monomer mass+organic solvent mass);

Monomer/chain transfer agent: 20-100mol/mol, the number of moles of monomer/the number of moles of chain transfer agent;

Free radical initiator/chain transfer agent: 0.1-0.25mol/mol, number of moles of initiator/mole number of chain transfer agent.

In the present invention, diblock copolymers with different properties, molecular weights and compositions can be synthesized by changing the type of the first monomer, the monomer/chain transfer agent molar ratio, and the amount of hydroxyethyl methacrylate.

The invention uses RAFT polymerization combined with mercapto-alkene click chemistry to synthesize a comb-shaped polymer-polypeptide bioconjugate. The present invention uses polyethylene glycol methacrylate (PEGMA), 3-methacryloyl-1,2:5,6-bis-O-isopropyl-D-glucofuranose (MAIpGlc) or 6-O- Methacryloyl-1,2:3,4-bis-O-isopropylidene-D-galactopyranose as the first monomer (MAIpGal), hydroxyethyl methacrylate (HEMA) as the second monomer Synthesize a two-block copolymer, modify the acrylate group on the side chain of the copolymer by reacting with acryloyl chloride, and then perform a click reaction with a polypeptide containing a sulfhydryl group under mild conditions to prepare a comb-shaped structure polypeptide bioconjugate . The click reaction has the advantages of simple conditions, high reaction efficiency, and pure product. The obtained sugar-containing copolymer-polypeptide bioconjugate has pH responsiveness, biorecognition, and biodegradability, and can be applied to the fields of targeted drug controlled release, bionano materials, bioimitation materials, and the like.

Description of drawings

Figure 1: Schematic diagram of the synthetic route of the polymer-peptide bioconjugate.

Figure 2: Transmission electron micrographs of micelles formed by comb-like poly(polyethylene glycol methacrylate) copolymer-glutathione bioconjugate in aqueous solution.

Figure 3: Transmission electron micrographs of micelles formed by the comb-like glycopolymer-glutathione bioconjugate in aqueous solution.

Figure 4: Transmission electron micrographs of micelles formed by the comb-like glycoside copolymer-glutathione bioconjugate in pH 5.0 sodium acetate buffer solution.

Detailed ways

Raw materials used in the present invention: monomers, initiators, solvents and other reagents are all analytically pure.

Example 1: In a 25mL reaction bottle, polyethylene glycol methacrylate (PEGMA) (Mw 475g/mol) 2.5g, chain transfer agent CPADB 37mg and initiator ACPA 6.2mg were dissolved in 7mL N, N-dimethyl In methyl formamide (DMF), freeze-vacuum-inflate nitrogen cycle three times, react at 70°C for 24h, cool with ice water and ventilate to the atmosphere to terminate the reaction. Then the polymer was sunken in ether, the precipitate was centrifuged, washed three times, and dried in a vacuum oven at room temperature to constant weight to obtain the pink polymer PPEGMA.

Example 2: In a 25mL reaction bottle, 1.7g of MAIpGlc monomer, 31mg of chain transfer agent 4-cyanopentanoic acid dithiobenzoate (CPADB) and initiator ACPA 3.2mg were dissolved in 13.5mL of 1,4- In dioxane. After three cycles of freezing-vacuumizing-nitrogen filling, react at 70°C for 7 hours, cool with ice water and ventilate the air to terminate the polymerization reaction, then sink the polymer in petroleum ether, centrifuge the precipitate, wash three times, and dry at room temperature in a vacuum oven to constant weight , to obtain the pink polymer PMAIpGlc.

Example 3: In a 10mL reaction flask, polymer PMAIpGlc 0.15g, hydroxyethyl methacrylate (HEMA) 16mg and initiator ACPA 0.6mg were dissolved in 1.5mL DMF. After three cycles of freezing-vacuumizing-nitrogen filling, react at 70°C for 24h. After the polymerization reaction was terminated, the polymer was sunk in petroleum ether, precipitated, centrifuged, washed three times, and dried in a vacuum oven at room temperature to constant weight to obtain the block copolymer PMAIpGlc-b-PHEMA.

Example 4: In a 10mL reaction bottle, 0.1g of poly(ethylene glycol methacrylate) (P(PEGMA)), 36mg of hydroxyethyl methacrylate (HEMA), and 0.6mg of initiator ACPA were dissolved in 1mL of DMF . Freezing-vacuumizing-nitrogen gas cycled three times, reacted at 70°C for 24 hours, cooled with ice water and vented to the atmosphere to terminate the reaction. The polymer was sunk in n-hexane, precipitated and centrifuged, washed three times, and dried in a vacuum oven at room temperature to constant weight to obtain the block polymer PPEGMA-b-PHEMA.

Example 5: Block copolymer PMAIpGlc-b-PHEMA 0.14g and AIBN 36mg were dissolved in 1.5mL of DMF, after three cycles of freezing-vacuumizing-nitrogen filling, placed in an oil bath at 80°C for 2.5h. After the reaction, the polymer was sunk in a large amount of petroleum ether, the precipitate was centrifuged, washed three times, and dried in a vacuum oven at room temperature to constant weight to obtain a white powder of the block copolymer from which the end groups were removed.

Example 6: 0.18 mg of the block copolymer PMAIpGlc-b-PHEMA after removing the end group was dissolved in 4 mL of THF. Add 26 μL of pyridine, then cool to 0°C, slowly add 2mL of acryloyl chloride (26 μL)/THF solution dropwise, after the dropwise addition, react at 0°C for 3 hours and at room temperature for 24 hours. The sample solution was dialyzed in a dialysis bag (Mw 3500) for 2 days, the water was changed every 4 hours, and freeze-dried to obtain the acrylate-modified block copolymer PMAIpGlc-b-P (HEMA-acrylate).

Example 7: Dissolve 86mg of block copolymer PMAIpGlc-b-P(HEMA-acrylate) in 3mL of 80% formic acid/water solution, react at 25°C for 24h, then add 1mL of secondary water and stir at room temperature for 3h. After the reaction is stopped, the solvent is spin-dried, and a small amount of DMF is added to dissolve the polymer. The sample solution is dialyzed in a dialysis bag (Mw 3500) for 2 days, and the water is changed every 4 hours, and freeze-dried to obtain the sugar-containing block copolymer PMAGlc-b-P (HEMA-acrylate).

Example 8: Add 50 mg of PMAGlc-b-P (HEMA-acrylate) to a 10 mL reaction bottle, dissolve it in 1 mL of DMF, add 2 μL of pyridine and 0.18 mL of reduced glutathione (GSH) aqueous solution (54 mg/mL), and blow nitrogen After 20 min, react at room temperature for 3 days, the sample solution was dialyzed in a dialysis bag (Mw 3500) for 2 days, the water was changed every 4 hours, and freeze-dried to obtain a comb-shaped structure sugar-containing block copolymer-glutathione bioconjugate.

Example 9: In a 10mL reaction bottle, 93mg of the block copolymer PPEGMA-b-PHEMA and 42mg of AIBN were dissolved in 2mL of DMF. Freezing-vacuumizing-nitrogen gas cycled three times, and reacted at 80°C for 3h. After the reaction, the polymer was sunk in a large amount of petroleum ether, precipitated, centrifuged, washed three times, and dried in a vacuum oven at room temperature to constant weight to obtain a block copolymer PPEGMA-b-PHEMA without end groups.

Example 10: In a 50mL reaction flask, 93mg of the block copolymer P(PEGMA)-b-PHEMA with the terminal group removed was dissolved in 15mL of toluene, distilled at atmospheric pressure and spin-dried to dry the solvent. Under the protection of nitrogen, 5 mL of tetrahydrofuran and 0.18 mL of pyridine were added, and 1 mL of acryloyl chloride (0.19 mL)/THF solution was slowly added dropwise at 0°C. After the addition was completed, the mixture was reacted at 0°C for 3 h and at room temperature for 24 h. The sample solution was dialyzed in a dialysis bag (MWCO 3500) for 2 days, the water was changed every 4 hours, and freeze-dried to obtain the acrylate-modified block copolymer P(PEGMA)-b-P(HEMA-acrylate).

Example 11: Add 60 mg of P(PEGMA)-b-P(HEMA-acrylate) to a 10 mL reaction bottle, dissolve it in 15 mL of DMF, add 25 μL of pyridine and 2 mL of reduced glutathione (GSH) aqueous solution (50 mg/mL), Nitrogen gas was applied for 20 minutes, and the reaction was carried out at room temperature for 3 days. The sample solution was dialyzed in a dialysis bag (Mw 3500) for 2 days, and the water was changed every 4 hours. The polymer-glutathione bioconjugate with a comb structure was obtained by freeze-drying.

Claims (7)

1. A polymer-polypeptide bioconjugate with a comb structure, characterized in that it has pH responsiveness, biorecognition, and forms spherical micelles in aqueous solution, and can release conjugated polypeptide molecules under acidic conditions. The polymer is a block copolymer synthesized by reversible addition-cracking chain transfer radical polymerization of methyl methacrylate monomers, with a number-average molecular weight of 5,000-10,000 g/mol and a molecular weight polydispersity index PDI<1.3. 2. The polymer-polypeptide bioconjugate according to claim 1, characterized in that the methyl methacrylate monomer is polyethylene glycol methacrylate (PEGMA), 3-methylpropene Acyl-1,2:5,6-bis-O-isopropyl-D-glucofuranose (MAIpGlc), 6-O-methacryloyl-1,2:3,4-bis-O-isopropylidene Base-D-galactopyranose (MAIpGal), hydroxyethyl methacrylate (HEMA). 3. The synthetic method of polymer-polypeptide bioconjugate according to claim 1, is characterized in that comprising the following steps: 1) Synthesis of macromolecular chain transfer agent, the method is to dissolve methyl methacrylate monomer, initiator, and chain transfer agent in an organic solvent, freeze in liquid nitrogen--vacuumize-inflate nitrogen for three times, and then polymerize at 70°C After 24 hours, the reaction was terminated, the polymer was precipitated, centrifuged and purified to remove unreacted monomers, dried in a vacuum oven to constant weight, and a macromolecular chain transfer agent was obtained; 2) Dissolve the macromolecular chain transfer agent synthesized in the first step, hydroxyethyl methacrylate, and the initiator in an organic solvent, and after three cycles of liquid nitrogen freezing-vacuumizing-nitrogen filling, polymerize at 70°C for 24 hours to terminate the reaction , the polymer is precipitated, centrifuged and purified to remove unreacted monomers, dried in a vacuum oven to constant weight, and a diblock copolymer is obtained; 3) Heat the block copolymer synthesized in the second step to 80°C together with excess azobisisobutyronitrile and solvent. After reacting for 3 hours, the polymer is purified by precipitation and centrifugation to obtain dithiobenzyl ester-free Terminated block copolymers; 4) react the block copolymer prepared in the third step with acryloyl chloride at room temperature for 24 hours, dialyze the product for 2 days for purification, and freeze-dry to obtain an acrylate-modified block copolymer; 5) If the block copolymer prepared in the third step contains a sugar-containing polymer with a protective group, the protective group is removed in 80% formic acid solution, the hydrolyzate is purified by dialysis for 2 days, and freeze-dried to obtain a sugar-containing block polymer thing; 6) dissolving the polymer prepared in the fourth step or the sugar-containing block polymer obtained in the fifth step and reduced glutathione in N,N-dimethylformamide/water solution, and reacting with weak alkalinity for 24 hours, The product was purified by dialysis and freeze-dried to obtain a comb-shaped structure polymer-polypeptide bioconjugate. 4. the synthetic method of comb structure polymer-polypeptide bioconjugate according to claim 3 is characterized in that described methyl methacrylate monomer and organic solvent, chain transfer agent, free radical initiator Dosage ratio: Monomer concentration: 10-30wt%, mass %: total monomer mass/(total monomer mass+organic solvent mass); Monomer/chain transfer agent: 30-60mol/mol, molar ratio: monomer moles/chain transfer agent moles; Chain transfer agent/free radical initiator: 6-10mol/mol, molar ratio: chain transfer agent moles/free radical moles. 5. the synthetic method of comb structure polymer-polypeptide bioconjugate according to claim 3 is characterized in that in the described block copolymer, the degree of polymerization of the first block polymer is 10-40, and the second block polymer is 10-40. Segment polymerization degree 7-10. 6. The method for synthesizing the comb-shaped polymer-polypeptide bioconjugate according to claim 3, characterized in that the side chain of the sugar-containing segment polymer is glucose or galactosyl. 7. the synthetic method of comb structure polymer-polypeptide bioconjugate according to claim 3 is characterized in that described chain transfer agent is 4-cyanovaleric acid dithiobenzoate, free radical The initiator is azobisisobutyronitrile.

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