A maleimide functionalized polyethyleneimine and its preparation and application

Abstract

The present invention belongs to the technical field of biomedical functional materials, and more specifically, relates to a maleimide-functionalized polyethyleneimine and its preparation and application. The present invention utilizes an amidation reaction of maleimidoacetic acid activated by an organic base with polyethyleneimine adjusted to a weak acid or neutral pH to prepare maleimide-functionalized polyethyleneimine. The preparation method is simple to operate, and the reaction conditions are mild. The prepared PEI-Mal has good water solubility, stable chemical properties, and good compatibility with biological tissues, and has important application value in multiple fields such as biomedical nanoparticle coating modification, polypeptide fixation, and drug loading.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical functional materials, and more specifically, relates to maleimide-functionalized polyethyleneimine and the preparation and application thereof. Background Art

[0002] Polyethyleneimine (PEI) is a typical water-soluble cationic polymer obtained by ring-opening polymerization of ethyleneimine (EI) monomer in the presence of an acidic catalyst and polymer additives. It is the cationic polymer with the highest charge density known so far. PEI exists in the form of polycations in water, can neutralize and adsorb a large number of anionic substances, and can also chelate heavy metal ions, so it has outstanding cationic properties. The amine groups in the PEI molecule can form hydrogen bonds with the hydroxyl groups in the system and react with the carboxyl groups to form covalent bonds, and there are rich functionalization pathways. At the same time, due to the presence of both hydrophilic polar groups (amine groups) and hydrophobic groups (vinyl groups), PEI has extremely high adhesion and adsorption properties; most importantly, there are a large number of theoretically protonable amine groups in the PEI structure, which makes PEI have a unique "proton sea effect". Based on the above properties, PEI can be used as an efficient gene transfection carrier and is widely used in cutting-edge fields such as bioengineering, immobilized enzymes and gene therapy.

[0003] However, since the functional groups of PEI are too single, many important compounds (such as thiol-containing peptides) cannot be loaded onto PEI to achieve biomedical functions. Previous studies have attempted to cross-link PEI with other functionalized polymers to achieve the purpose of introducing multiple functional groups, but other polymers are mixed in the product, weakening the inherent advantages of PEI itself and causing unnecessary trouble. Some studies have also attempted to directly connect small molecule compounds containing functional groups to PEI through substitution reactions, but there are a large number of positive charges on the surface of PEI, and the PEI solution is highly alkaline, which aggravates the reverse reaction and makes it difficult to effectively carry out functionalized PEI.

[0004] Click chemistry has the advantages of mild reaction conditions, high reaction yield, and easy removal of by-products, and is widely used in the biomedical field, especially in the loading of thiol-containing peptides. Among the many click chemistry reactions, the (α, β)-unsaturated double bonds in maleic anhydride react with thiol groups in a particularly efficient manner, and have great application potential. Therefore, the development of a method for preparing maleimide-functionalized polyethyleneimine with simple operation and mild conditions is of great value in promoting the application of PEI in the biomedical field. Summary of the invention

[0005] In view of the defects of the prior art, the purpose of the present invention is to provide a maleimide-functionalized polyethyleneimine and its preparation and application, so as to solve the technical problems of the prior art that biomedical functionalized polyethyleneimine needs to be mixed with other polymers, weakens the inherent advantages of PEI, and is difficult to effectively carry out functionalized PEI.

[0006] To achieve the above object, the present invention provides a maleimide-functionalized polyethyleneimine, which is a maleimide-functionalized polyethyleneimine obtained by amidation reaction of an amine group of polyethyleneimine with a carboxyl group of maleimidoacetic acid, wherein the mass fraction of the maleimide group in the maleimide-functionalized polyethyleneimine molecule is 1% to 30%.

[0007] According to another aspect of the present invention, there is provided a method for preparing the maleimide functionalized polyethyleneimine, comprising the following steps:

[0008] (1) mixing maleimidoacetic acid, a fifth organic solvent and an organic base and stirring for activation for several hours to obtain activated maleimidoacetic acid;

[0009] (2) dissolving polyethyleneimine in a sixth organic solvent and adjusting the pH thereof to weak acidity or neutrality with a dilute acid to obtain a polyethyleneimine solution with adjusted pH;

[0010] (3) mixing the activated maleimidoacetic acid obtained in step (1) with the pH-adjusted polyethyleneimine solution obtained in step (2), and stirring the mixture for several hours, so that the amine group of the pH-adjusted polyethyleneimine undergoes an amidation reaction with the carboxyl group of the activated maleimidoacetic acid to obtain maleimide-functionalized polyethyleneimine.

[0011] Preferably, the method for preparing maleimidoacetic acid comprises the following steps:

[0012] S1: maleic anhydride and compound A are reacted in an organic solvent under reflux for several hours under the protection of an inert gas, and 4-[(carboxymethyl)amino]-4-oxo-2-butenoic acid is obtained by separation and purification; the chemical formula of the compound A is NH2-(CH2) n -COOH, wherein n=1 to 10;

[0013] S2: dissolving the 4-[(carboxymethyl)amino]-4-oxo-2-butenoic acid obtained in step S1 in a third organic solvent, adding an organic base under stirring, reacting under reflux for several hours under heating, then extracting with a fourth organic solvent, and then removing the fourth organic solvent by rotary evaporation to obtain maleimidoacetic acid.

[0014] More preferably, the reflux reaction time in step S1 is 2 to 5 hours.

[0015] Further preferably, the heating temperature in step S2 is 100-150° C., and the reflux reaction time is 1-3 hours.

[0016] Preferably, the activation time in step (1) is 0.5 to 2 hours; and the molar ratio of the maleimidoacetic acid to the organic base is 1:1 to 1.5.

[0017] Preferably, in step (2), the pH is adjusted to 5-7, and the concentration of the polyethyleneimine solution for adjusting the pH is 0.01-0.02 mol / L.

[0018] Preferably, the reaction time of step (3) is 1.5 to 3 hours; and the molar ratio of the activated maleimidoacetic acid to the weakly acidic polyethyleneimine is 10:1 to 3:1.

[0019] Preferably, the organic base in step S1 and step (1) is independently at least one of triethylamine, N-ethyltetrahydropyrrolylmethylamine, N-ethyldiisopropylamine, pyridine, 1,8-diazabicycloundec-7-ene, 2,6-lutidine, imidazole and N-methylimidazole.

[0020] According to another aspect of the present invention, there is provided an application of the maleimide-functionalized polyethyleneimine for carrying out a thiol-ene click reaction with a biological functional substance.

[0021] In general, the above technical solution conceived by the present invention has the following advantages compared with the prior art:

[0022] Beneficial effects:

[0023] (1) The maleimide-functionalized polyethyleneimine provided by the present invention can undergo a click reaction with a thiol compound, and has good water solubility and compatibility with biological tissues. It can be coated with hydrophobic inorganic nanoparticles or carry hydrophobic drugs or polypeptides to achieve biomedical applications of aqueous systems.

[0024] (2) The present invention utilizes the amino group of the amphiphilic substance glycine to undergo a nucleophilic substitution reaction with the ester group of maleic anhydride, so that the maleic anhydride is ring-opened to obtain a dicarboxyl compound, and then the dicarboxyl compound is dehydrated and ring-closed under the action of an alkaline catalyst to obtain maleimidoacetic acid, and finally maleimide-functionalized polyethyleneimine is obtained by an amidation reaction with polyethyleneimine. The preparation method of maleimide-functionalized polyethyleneimine provided by the present invention is simple to operate and has mild reaction conditions. The prepared PEI-Mal has good water solubility and stable chemical properties, and can be used for various biomedical applications such as hydrophobic inorganic nanoparticle coating modification, polypeptide fixation, and drug loading. Under physiological conditions, PEI-Mal exhibits positive charge and can be combined with negatively charged functional substances to exert corresponding biological functions without toxic effects on organisms.

[0025] (3) The present invention synthesizes a maleimide-functionalized polyethyleneimine (PEI-Mal) by optimizing and screening suitable synthesis routes and reaction conditions, thereby solving the problem that PEI is difficult to functionalize due to its high positive charge density and strong alkalinity, and providing controllable reaction sites for the subsequent thiol-ene click reaction with biological functional substances such as polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic structural formula of the functionalized polyethyleneimine of the present invention.

[0027] Figure 2 This is the synthetic route of PEI-Mal.

[0028] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of GMI prepared in Example 1.

[0029] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of PEI-Mal prepared in Example 1. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] The present invention provides a maleimide-functionalized polyethyleneimine, which is obtained by amidation reaction between the amine group of polyethyleneimine and the carboxyl group of maleimidoacetic acid, wherein the mass fraction of the maleimide group in the maleimide-functionalized polyethyleneimine molecule is 1% to 30%. The maleimide-functionalized polyethyleneimine of the present invention has the formula (I) and Figure 1 The schematic structural formula shown is: The molecular weight of the preferred polyethyleneimine is 600-10000.

[0032]

[0033] In some embodiments, the method for preparing maleimide functionalized polyethyleneimine of the present invention comprises the following steps:

[0034] (1) Synthesis of dicarboxyl compound GMA: maleic anhydride and compound A are reacted in an organic solvent under inert gas protection for several hours under reflux, and 4-[(carboxymethyl)amino]-4-oxo-2-butenoic acid (GMA) is obtained by separation and purification; the chemical formula of compound A is NH2-(CH2) n -COOH, wherein n=1 to 10;

[0035] (2) Synthesis of maleimidoacetic acid (GMI): dissolving the 4-[(carboxymethyl)amino]-4-oxo-2-butenoic acid obtained in step (1) in a third organic solvent, adding an organic base under stirring conditions, reflux the reaction under heating conditions for several hours, then extracting with a fourth organic solvent, and then removing the fourth organic solvent by rotary evaporation to obtain maleimidoacetic acid (GMI);

[0036] (3) mixing the maleimidoacetic acid obtained in step (2), the fifth organic solvent and the organic base and stirring them for activation for several hours to obtain activated maleimidoacetic acid;

[0037] (4) dissolving polyethyleneimine in a sixth organic solvent and adjusting the pH thereof to weak acidity or neutrality with a dilute acid to obtain a polyethyleneimine solution with adjusted pH;

[0038] (5) mixing the activated maleimidoacetic acid obtained in step (3) with the pH-adjusted polyethyleneimine solution obtained in step (4), and stirring the mixture for several hours, so that the amine group of the pH-adjusted polyethyleneimine undergoes an amidation reaction with the carboxyl group of the activated maleimidoacetic acid to obtain maleimide-functionalized polyethyleneimine (PEI-Mal).

[0039] In some embodiments, step (1) is specifically as follows: maleic anhydride is dissolved in a first organic solvent, compound A is added under stirring, and then refluxed for several hours under the protection of an inert gas; after the reaction is completed, the first organic solvent is removed by rotary evaporation, and then the concentrated product is redissolved in a second organic solvent, solid-liquid separation is performed, the liquid is collected, and finally the second organic solvent is removed by rotary evaporation, and dried to obtain 4-[(carboxymethyl)amino]-4-oxo-2-butenoic acid. Compound A includes but is not limited to aminoformic acid, aminoacetic acid (glycine), aminopropionic acid, aminobutyric acid, etc.

[0040] In some embodiments, in step (1), the first organic solvent is selected from acetone, ethyl acetate and dimethyl sulfoxide; the second organic solvent is selected from methanol, ethanol and isopropanol. The inert atmosphere is nitrogen, argon, etc.; the drying is vacuum drying, and the drying time is 12 to 48 hours. The reflux reaction time in step (1) is 2 to 5 hours.

[0041] In some embodiments, the heating temperature in step (2) is 100-150° C., and the reflux reaction time is 1-3 hours. The third organic solvent in step (2) is selected from toluene, carbon tetrachloride and benzene, and the fourth organic solvent is selected from ethyl acetate, acetone and dimethyl sulfoxide. The molar ratio of the 4-[(carboxymethyl)amino]-4-oxo-2-butenoic acid to the organic base is 1:1-1.5.

[0042] In some embodiments, the activation time in step (3) is 0.5 to 2 hours; the molar ratio of maleimidoacetic acid to the organic base is 1:1 to 1.5. The fifth organic solvent is selected from dichloromethane, dinaphthol and dimethyl sulfoxide.

[0043] In some embodiments, step (4) adjusts the pH to 5-7, and the concentration of the polyethyleneimine solution for adjusting the pH is 0.01-0.02 mol / L. The sixth organic solvent is selected from methanol, ethanol and isopropanol. The dilute acid is dilute hydrochloric acid, dilute sulfuric acid or dilute nitric acid, and the concentration of the dilute acid is 0.5-1.5 mol / L.

[0044] In some embodiments, the reaction time of step (5) is 1.5 to 3 hours; the molar ratio of the activated maleimidoacetic acid to the polyethyleneimine for adjusting the pH is 3:1 to 10:1, preferably 4:1 to 8:1.

[0045] In some embodiments, the organic base in step (2) and step (3) is used as a catalyst, and the organic base in step (2) and step (3) is independently at least one of triethylamine, N-ethyltetrahydropyrrolmethylamine, N-ethyldiisopropylamine, pyridine, 1,8-diazabicycloundec-7-ene, 2,6-lutidine, imidazole and N-methylimidazole.

[0046] The purpose of the present invention is to construct a water-soluble polymer platform suitable for thiol-ene click reaction, so as to facilitate the loading of thiol-containing polypeptides and biomedical applications. The maleimide-functionalized polyethyleneimine provided by the present invention can be used for thiol-ene click reaction with biological functional substances such as polypeptides, providing controllable reaction sites for the reaction.

[0047] The synthetic route of maleamide functionalized polyethyleneimine in some embodiments of the present invention is as follows Figure 2As shown, in the embodiment of the present invention, the amino group of the amphiphilic substance glycine and the ester group of maleic anhydride are used to undergo a nucleophilic substitution reaction, so that the maleic anhydride is ring-opened to obtain a dicarboxyl compound, and then the dicarboxyl compound is dehydrated and ring-closed under the action of an alkaline catalyst to obtain maleimidoacetic acid (GMI), and finally maleimide-functionalized polyethyleneimine (PEI-Mal) is obtained by an amidation reaction with polyethyleneimine. The preparation method is simple to operate, the reaction conditions are mild, the prepared PEI-Mal has good water solubility, stable chemical properties, good compatibility with biological tissues, and has important application value in multiple fields such as biomedical nanoparticle coating modification, polypeptide fixation, and drug loading.

[0048] On the one hand, there are a large number of positive charges on the surface of polyethyleneimine itself, and the PEI solution is highly alkaline, and the strong alkaline conditions will aggravate the reverse reaction of the amidation reaction, making it difficult for the functionalized PEI to be effectively carried out; on the other hand, if the PEI is adjusted to acidity to promote the amidation reaction, it is found that the inherent advantages brought by the alkaline functional groups on the surface of PEI are greatly reduced. How to overcome this contradiction, whether it is possible to overcome this contradiction, and prepare maleimide functionalized polyethyleneimine is the key technical problem to be solved by the present invention. The present invention designs the synthesis process route, and screens and optimizes the process conditions of each operation step. It is found that the selection of catalyst types, purification conditions, especially the range of polyethyleneimine to adjust the pH, and the feed ratio with the activated maleimide acetic acid small molecule, etc., have a greater impact on the yield of the functionalized polyethyleneimine target product obtained in the end. For example, experiments have found that when the feed ratio of activated maleimide acetic acid small molecules to weakly acidic polyethyleneimine is 1:1, not only can polyethyleneimine not be functionalized to obtain the target product PEI-Mal, but the amino group on PEI will also cause GMI to open the ring; only when the feed ratio of activated maleimide acetic acid small molecules to polyethyleneimine adjusted to weakly acidic or neutral pH is greater than or equal to 3:1, can the functionalized polyethyleneimine target product PEI-Mal be prepared. The possible reason is that there is a competitive reaction when the two react. Only when the activated maleimide acetic acid small molecules are grafted enough, the adjacent amine groups cannot contact the N on the ring due to steric hindrance, so it is not easy to open the maleimide group.

[0049] The following are examples:

[0050] Comparative Example 1

[0051] In this comparative example, the preparation method of GMA is as follows: 9.8 g / 0.1 mol of maleic anhydride is dissolved in 100 mL of acetone, 7.5 g / 0.1 mol of glycine is added under vigorous stirring, and reflux reaction is carried out under nitrogen protection for 3 hours. After removing the acetone, the concentrated product is redissolved in methanol, filtered, the liquid is collected and the methanol is removed, and vacuum dried for 24 hours to obtain GMA as a light yellow solid.

[0052] In this comparative example, the preparation method of GMI is as follows: 3.0 g / 0.017 mol of GMA is dissolved in 70 mL of toluene, 3.9 g / 0.03 mol of N-ethyldiisopropylamine is added under vigorous stirring, the reaction is refluxed at 130° C. for 2 hours, and the brown oily substance of GMI is obtained after extraction with ethyl acetate and removal of ethyl acetate by rotary evaporation.

[0053] In this comparative example, the preparation method of PEI-Mal is as follows:

[0054] 0.0310 g / 0.0002 mol of GMI was added to 10 mL of dichloromethane, and 0.208 g / 0.0016 mol of N-ethyldiisopropylamine (DIPEA) was added under vigorous stirring, and the mixture was stirred and activated at room temperature for 0.5 hours to obtain activated GMI;

[0055] Take 2.000g / 0.0002mol of PEI and dissolve it in 10mL of methanol, add 1.0mol / L of dilute hydrochloric acid to adjust the pH to about 6 to obtain weakly acidic PEI;

[0056] The two reaction solutions were mixed and stirred at room temperature for 3 hours to stop the reaction. The reactant was oily and no solid was generated.

[0057] In this comparative example, PEI-Mal cannot be produced because the molar ratio of GMI to PEI of 1:1 does not meet the requirement.

[0058] Comparative Example 2

[0059] The preparation method of GMA is as follows: 19.6 g / 0.2 mol of maleic anhydride is dissolved in 150 mL of acetone, 15 g / 0.2 mol of glycine is added under vigorous stirring, and the mixture is refluxed for 3 hours under nitrogen protection, acetone is removed by rotary evaporation of the reaction mixture, and the residue is redissolved in methanol, filtered, the liquid is collected and methanol is removed by rotary evaporation, and vacuum dried for 24 hours to obtain GMA as a light yellow solid.

[0060] The preparation method of GMI is as follows: 3.0 g / 0.017 mol of GMA is dissolved in 70 mL of toluene, 0.03 mol of triethylamine (TEA) is added under vigorous stirring, the mixture is refluxed at 130° C. for 2 hours, extracted with ethyl acetate, and the ethyl acetate is removed by rotary evaporation to obtain a brown oily substance of GMI.

[0061] The preparation method of PEI-Mal is as follows:

[0062] 0.2172 g / 0.0014 mol of GMI was added to 10 mL of dichloromethane, and 0.208 g / 0.0016 mol of DIPEA was added under vigorous stirring, and the mixture was stirred and activated at room temperature for 0.5 hours to obtain activated GMI;

[0063] 2.000 g / 0.0002 mol of PEI was dissolved in 10 mL of methanol, and 1.0 mol / L of dilute hydrochloric acid was added to adjust the pH to about 10 to obtain pH-adjusted PEI;

[0064] The two reaction solutions were mixed and stirred at room temperature for 3 hours. The reaction was stopped and the liquid was removed by rotary evaporation to obtain no product.

[0065] In this comparative example, since the pH of the solution was too high, the prepared PEI-Mal was rapidly decomposed and could not exist stably.

[0066] Example 1

[0067] In this embodiment, the preparation method of GMA is as follows: 9.8 g / 0.1 mol of maleic anhydride is dissolved in 100 mL of acetone, 7.5 g / 0.1 mol of glycine is added under vigorous stirring, and the mixture is refluxed for 3 hours under nitrogen protection, the acetone is removed by rotary evaporation of the reaction mixture, and the residue is redissolved in methanol, filtered, the liquid is collected and the methanol is removed by rotary evaporation, and vacuum dried for 24 hours to obtain GMA as a light yellow solid.

[0068] In this embodiment, the preparation method of GMI is as follows: 3.0 g / 0.017 mol of GMA is dissolved in 70 mL of toluene, 3.9 g / 0.03 mol of DIPEA is added under vigorous stirring, and the reaction is refluxed at 130° C. for 2 hours. The reaction mixture is extracted with ethyl acetate, and the ethyl acetate is removed by rotary evaporation to obtain a brown oily substance of GMI.

[0069] In this embodiment, the preparation method of PEI-Mal is as follows:

[0070] 0.2172 g / 0.0014 mol of GMI was added to 10 mL of DCM, and 0.208 g / 0.0016 mol of DIPEA was added under vigorous stirring, and the mixture was stirred at room temperature for 0.5 hours to activate the mixture; activated GMI was obtained;

[0071] Take 2.000g / 0.0002mol of PEI and dissolve it in 10mL of methanol, add 1.0mol / L of dilute hydrochloric acid to adjust the pH to about 6 to obtain weakly acidic PEI;

[0072] The two reaction solutions were mixed, stirred at room temperature for 3 hours, the reaction was stopped, the liquid was removed by rotary evaporation, the residue was washed with methyl nitrile, the rinsed liquid was removed by pouring, and vacuum drying was performed to obtain a light yellow solid of PEI-Mal. The mass fraction of maleimide groups in PEI-Mal was 20%.

[0073] Example 2

[0074] In this embodiment, the preparation method of GMA is as follows: 19.6 g / 0.2 mol of maleic anhydride is dissolved in 150 mL of acetone, 15 g / 0.2 mol of glycine is added under vigorous stirring, and the mixture is refluxed for 3 hours under nitrogen protection, the acetone is removed by rotary evaporation of the reaction mixture, and the residue is redissolved in methanol, filtered, the liquid is collected and the methanol is removed by rotary evaporation, and vacuum dried for 24 hours to obtain GMA as a light yellow solid.

[0075] In this embodiment, the preparation method of GMI is as follows: 3.0 g / 0.017 mol of GMA is dissolved in 70 mL of toluene, 0.03 mol of TEA is added under vigorous stirring, and the reaction is refluxed at 130° C. for 2 hours. The reaction mixture is extracted with ethyl acetate, and the ethyl acetate is removed by rotary evaporation to obtain a brown oily substance of GMI.

[0076] In this embodiment, the preparation method of PEI-Mal is as follows:

[0077] 0.2172 g / 0.0014 mol of GMI was added to 10 mL of DCM, and 0.208 g / 0.0016 mol of DIPEA was added under vigorous stirring. The mixture was stirred and activated at room temperature for 0.5 hours to obtain activated GMI.

[0078] 2.000 g / 0.0002 mol of PEI was dissolved in 10 mL of methanol, and 1.0 mol / L of dilute hydrochloric acid was added to adjust the pH to about 7 to obtain pH-adjusted PEI;

[0079] The two reaction solutions were mixed, stirred at room temperature for 3 hours, the reaction was stopped, the liquid was removed by rotary evaporation, the residue was washed with methyl nitrile, the rinsed liquid was removed by pouring, and vacuum drying was performed to obtain a light yellow solid of PEI-Mal. The mass fraction of maleimide groups in PEI-Mal was 18%.

[0080] Example 3

[0081] In this embodiment, the preparation method of GMA is as follows: 9.8 g / 0.1 mol of maleic anhydride is dissolved in 100 mL of acetone, 7.5 g / 0.1 mol of glycine is added under vigorous stirring, and the reaction mixture is refluxed for 3 hours under nitrogen protection, and the acetone is removed by rotary evaporation of the reaction mixture. The residue is redissolved in methanol, filtered, the liquid is collected and the methanol is removed by rotary evaporation, and vacuum dried for 24 hours to obtain GMA as a light yellow solid.

[0082] In this embodiment, the preparation method of GMI is as follows: 1.5 g / 0.0085 mol of GMA is dissolved in 50 mL of toluene, 0.02 mol of TEA is added under vigorous stirring, and the reaction is refluxed at 130° C. for 2 hours. The reaction mixture is extracted with ethyl acetate, and the ethyl acetate is removed by rotary evaporation to obtain a brown oily substance of GMI.

[0083] In this embodiment, the preparation method of PEI-Mal is as follows:

[0084] 0.1551 g / 0.0010 mol of GMI was added to 10 mL of DCM, and 0.208 g / 0.0016 mol of DIPEA was added under vigorous stirring, and the mixture was stirred and activated at room temperature for 0.5 h to obtain activated GMI;

[0085] 2.000 g / 0.0002 mol of PEI was dissolved in 10 mL of methanol, and 1.0 mol / L of dilute hydrochloric acid was added to adjust the pH to about 7 to obtain pH-adjusted PEI;

[0086] The two reaction solutions were mixed, stirred at room temperature for 2.5 hours, the reaction was stopped, the liquid was removed by rotary evaporation, the residue was washed with methyl nitrile, the rinsed liquid was removed by pouring, and vacuum drying was performed to obtain a light yellow solid of PEI-Mal. The mass fraction of maleimide groups in PEI-Mal was 12%.

[0087] Example 4

[0088] In this embodiment, the preparation method of GMA is as follows: 9.8 g / 0.1 mol of maleic anhydride is dissolved in 100 mL of acetone, 7.5 g / 0.1 mol of glycine is added under vigorous stirring, and the reaction mixture is refluxed for 3 hours under nitrogen protection, and the acetone is removed by rotary evaporation of the reaction mixture. The residue is redissolved in methanol, filtered, the liquid is collected and the methanol is removed by rotary evaporation, and vacuum dried for 24 hours to obtain GMA as a light yellow solid.

[0089] In this embodiment, the preparation method of GMI is as follows: 3.0 g / 0.017 mol of GMA is dissolved in 70 mL of toluene, 3.9 g / 0.03 mol of MNI is added under vigorous stirring, and the reaction is refluxed at 130° C. for 2 hours. The reaction mixture is extracted with ethyl acetate, and the ethyl acetate is removed by rotary evaporation to obtain a brown oily substance of GMI.

[0090] In this embodiment, the preparation method of PEI-Mal is as follows:

[0091] 0.2172 g / 0.0014 mol of GMI was added to 10 mL of DCM, and 0.208 g / 0.0016 mol of TEA was added under vigorous stirring, and the mixture was stirred and activated at room temperature for 0.5 h to obtain activated GMI;

[0092] 2.000 g / 0.0002 mol of PEI was dissolved in 10 mL of methanol, and 1.0 mol / L of dilute hydrochloric acid was added to adjust the pH to about 5 to obtain pH-adjusted PEI;

[0093] The two reaction solutions were mixed, stirred at room temperature for 3 hours, the reaction was stopped, the liquid was removed by rotary evaporation, the residue was washed with methyl nitrile, the rinsed liquid was removed by pouring, and vacuum drying was performed to obtain a light yellow solid of PEI-Mal. The mass fraction of maleimide groups in PEI-Mal was 16%.

[0094] Comparative Example 3

[0095] In this comparative example, the preparation method of GMA is as follows: 9.8 g / 0.1 mol of maleic anhydride is dissolved in 100 mL of acetone, 7.5 g / 0.1 mol of glycine is added under vigorous stirring, and the reaction mixture is refluxed for 3 hours under nitrogen protection, and the acetone is removed by rotary evaporation of the reaction mixture. The residue is redissolved in methanol, filtered, the liquid is collected and the methanol is removed by rotary evaporation, and vacuum dried for 24 hours to obtain GMA as a light yellow solid.

[0096] In this comparative example, the preparation method of GMI is as follows: 3.0 g / 0.017 mol of GMA is dissolved in 70 mL of toluene, 3.9 g / 0.03 mol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) is added under vigorous stirring, and the reaction is refluxed at 130° C. for 2 hours. The reaction mixture is extracted with ethyl acetate, and the ethyl acetate is removed by rotary evaporation to obtain a brown oily substance of GMI.

[0097] In this comparative example, the preparation method of PEI-Mal is as follows:

[0098] 0.2172 g / 0.0014 mol of GMI was added to 10 mL of DCM, and 0.208 g / 0.0016 mol of EDC was added under vigorous stirring. The mixture was stirred at room temperature for 0.5 hours to obtain activated GMI.

[0099] 2.000 g / 0.0002 mol of PEI was dissolved in 10 mL of methanol, and 1.0 mol / L of dilute hydrochloric acid was added to adjust the pH to about 5 to obtain pH-adjusted PEI;

[0100] The two reaction solutions were mixed and stirred at room temperature for several hours (3 hours), and the reaction was stopped. The reactant was oily and no solid was generated.

[0101] The other conditions of this comparative example are the same as those of Example 1, except that the organic amine catalyst is replaced by EDC from DIPEA in Example 1. Experiments show that PEI-Mal cannot be produced due to improper catalyst selection that does not meet the requirements.

[0102] The technical effects of the present invention will be described below based on experimental evidence.

[0103] Figure 3 Shown is the hydrogen nuclear magnetic resonance spectrum of GMI prepared in Example 1, 1 H NMR (600 MHz) δ 6.90 (s, 2H), 4.31 (s, 2H), the chemical shift 6.90 is the characteristic peak of H on the carbon-carbon double bond, and 4,31 are the characteristic peaks of H on the methylene group, which proves the successful preparation of GMI.

[0104] Figure 4 Shown is the hydrogen nuclear magnetic resonance spectrum of PEI-Mal prepared in Example 1, 1 H NMR (600 MHz) δ 6.87 (s, 2H), 3.82 (s, 1H), 3.67 (m, 4H), 3.29 (s, 4H), 3.22 (s, 5H), 3.16 (q, 5H), 3.02 (d, J = 18.4 Hz, 1H). The chemical shift of 6.87 is the characteristic peak of H on the carbon-carbon double bond, and that of 3.82-3.02 is the characteristic peak of H on the polymer, which proves the successful preparation of PEI-Mal.

[0105] By adjusting the feed ratio of the reaction raw materials, the mass fraction of the maleimide groups in PEI-Mal is 1% to 30%.

[0106] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A maleimide-functionalized polyethyleneimine, characterized in that The maleimide-functionalized polyethyleneimine is obtained by amidation reaction between the amine group of polyethyleneimine and the carboxyl group of maleimidoacetic acid, wherein the mass fraction of the maleimide group in the maleimide-functionalized polyethyleneimine molecule is 1% to 30%; The method for preparing maleimide functionalized polyethyleneimine comprises the following steps: (1) mixing maleimidoacetic acid, a fifth organic solvent and an organic base and stirring for activation for several hours to obtain activated maleimidoacetic acid; (2) dissolving polyethyleneimine in a sixth organic solvent and adjusting the pH thereof to weak acidity or neutrality with a dilute acid to obtain a polyethyleneimine solution with adjusted pH; (3) mixing the activated maleimidoacetic acid obtained in step (1) with the pH-adjusted polyethyleneimine solution obtained in step (2), and stirring the mixture for several hours, so that the amine group of the pH-adjusted polyethyleneimine undergoes an amidation reaction with the carboxyl group of the activated maleimidoacetic acid to obtain maleimide-functionalized polyethyleneimine; The organic bases in step (1) are each independently at least one of triethylamine, N-ethyltetrahydropyrrolmethylamine, N-ethyldiisopropylamine, pyridine, 1,8-diazabicycloundec-7-ene, 2,6-lutidine, imidazole and N-methylimidazole; Step (2) adjusting the pH to 5-7, wherein the concentration of the polyethyleneimine solution for adjusting the pH is 0.01-0.02 mol / L; The reaction time of step (3) is 1.5 to 3 hours; the molar ratio of the activated maleimidoacetic acid to the weakly acidic polyethyleneimine is 10:1 to 3:

1.

2. The method for preparing maleimide functionalized polyethyleneimine according to claim 1, characterized in that: The steps include: (1) mixing maleimidoacetic acid, a fifth organic solvent and an organic base and stirring for activation for several hours to obtain activated maleimidoacetic acid; (2) dissolving polyethyleneimine in a sixth organic solvent and adjusting the pH thereof to weak acidity or neutrality with a dilute acid to obtain a polyethyleneimine solution with adjusted pH; (3) mixing the activated maleimidoacetic acid obtained in step (1) with the pH-adjusted polyethyleneimine solution obtained in step (2), and stirring the mixture for several hours, so that the amine group of the pH-adjusted polyethyleneimine undergoes an amidation reaction with the carboxyl group of the activated maleimidoacetic acid to obtain maleimide-functionalized polyethyleneimine; The organic bases in step (1) are each independently at least one of triethylamine, N-ethyltetrahydropyrrolmethylamine, N-ethyldiisopropylamine, pyridine, 1,8-diazabicycloundec-7-ene, 2,6-lutidine, imidazole and N-methylimidazole; Step (2) adjusting the pH to 5-7, wherein the concentration of the polyethyleneimine solution for adjusting the pH is 0.01-0.02 mol / L; The reaction time of step (3) is 1.5 to 3 hours; the molar ratio of the activated maleimidoacetic acid to the weakly acidic polyethyleneimine is 10:1 to 3:

1.

3. The preparation method according to claim 2, characterized in that: The preparation method of maleimidoacetic acid comprises the following steps: S1: reacting maleic anhydride and compound A in an organic solvent under the protection of an inert gas for several hours under reflux, separating and purifying to obtain 4-[(carboxymethyl)amino]-4-oxo-2-butenoic acid; the chemical formula of compound A is NH2-CH2-COOH; S2: dissolving the 4-[(carboxymethyl)amino]-4-oxo-2-butenoic acid obtained in step S1 in a third organic solvent, adding an organic base under stirring, reacting under reflux for several hours under heating, then extracting with a fourth organic solvent, and then removing the fourth organic solvent by rotary evaporation to obtain maleimidoacetic acid.

4. The preparation method according to claim 3, characterized in that: The reflux reaction time in step S1 is 2 to 5 hours.

5. The preparation method according to claim 3, characterized in that: The heating temperature in step S2 is 100-150° C., and the reflux reaction time is 1-3 hours.

6. The preparation method according to claim 2, characterized in that: The activation time in step (1) is 0.5 to 2 hours; the molar ratio of the maleimidoacetic acid to the organic base is 1:1 to 1.

5.

7. The preparation method according to claim 3, characterized in that: The organic base in step S2 is at least one of triethylamine, N-ethyltetrahydropyrrolmethylamine, N-ethyldiisopropylamine, pyridine, 1,8-diazabicycloundec-7-ene, 2,6-lutidine, imidazole and N-methylimidazole.

8. The use of maleimide functionalized polyethyleneimine as claimed in claim 1, characterized in that: Used for thiol-ene click reactions with biological functional substances.

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