N-carboxyanhydrides of phenylboronic acid-containing amino acids and polyamino acids, and methods of making and using the same

Abstract

The application belongs to the technical field of synthesis and application of responsive polymers, and particularly relates to N-carboxylic anhydride monomer containing phenylboronic acid and polyamino acid, and a preparation method and application thereof, and particularly relates to synthesis of cysteine N-carboxylic anhydride monomer functionalized by phenylboronic acid, and application of a series of polymers prepared by ring-opening polymerization of the monomer in drug delivery. The cysteine-N-carboxylic anhydride monomer functionalized by phenylboronic acid has a structure shown in formula I, and the polyamino acid prepared by ring-opening polymerization of the monomer has excellent biocompatibility. The application has the beneficial effect that the interaction between boronic acid and catechol, cis 1,2 or 1,3-diol small molecule drugs, polypeptide, protein drugs and nucleic acid drugs is utilized to realize efficient loading and delivery of drugs; and due to the active oxygen response characteristic of the boronic acid structure, the loaded drugs can be responsively released in an active oxygen environment.

Description

Technical Field

[0001] This invention belongs to the field of synthesis and application technology of responsive polymers, and particularly relates to an N-carboxylated intracyclic anhydride monomer containing phenylboronic acid and a polyamino acid, as well as their preparation methods and applications. Background Technology

[0002] Amino acid materials have been widely used in biomedicine due to their excellent biocompatibility, biodegradability, and ease of functionalization. Polyamino acid-based drug delivery systems have shown significant advantages in improving drug solubility and pharmacokinetics, reducing toxicity and side effects, and increasing efficacy; however, challenges remain, such as low drug loading capacity and uncontrollable drug release.

[0003] Chemical modification of amino acids to enable responsive drug release in response to various stimuli (such as pH, enzymes, and redox reactions) is of great significance in disease treatment. In particular, the reversible formation of boron ester bonds between phenylboronic acid and cis-1,2- or 1,3-diol structures enables the efficient loading and delivery of small molecule drugs containing cis-1,2- or 1,3-diol, catechol derivatives, peptides, proteins, and nucleic acid drugs. The constructed drug carriers exhibit glucose and reactive oxygen species (ROS) responsiveness, allowing for controlled drug release. Specific interactions exist between phenylboronic acid and sialic acid residues, which are highly expressed in tumor cells. The synthesized polyamino acids containing phenylboronic acid can be used for active targeted drug delivery to tumors, enhancing drug efficacy and reducing toxic side effects. Dynamic boron ester bonds between phenylboronic acids, and between phenylboronic acid and catechol or cis-diol functional groups, are frequently used to construct multifunctional hydrogel systems. Deng Chao et al. disclosed "an L-4-dihydroxyboronic acid-N-carboxylic acid anhydride monomer and polyamino acid and their preparation method and application", which synthesized a boron phenylalanine-N-carboxylic acid anhydride monomer and polyamino acid using 4-boronic acid-phenylalanine as raw material, and used it for the loading of dual antitumor drugs curcumin and sorafenib, confirming the unique advantages of phenylboronic acid functional groups in drug delivery. However, the prepared polymer lacks the characteristics of tumor microenvironment response.

[0004] Chinese patent CN105273205BA discloses "Block Polymers with Phenylboronic Acid Ester as Linking Units, Their Synthesis Methods and Applications," which uses 3,4-dihydroxyphenylacetic acid containing catechol groups to modify polyethylene glycol, and polyglutamic acid with phenylboronic acid at the end to be linked by phenylboronic acid catechol esters to synthesize a polyethylene glycol-polyglutamic acid diblock polymer. The prepared polymer is mainly used for loading lipid-soluble drugs. Chinese patent CN114409607A discloses "An N-Carboxycyclic Anhydride Containing a Thioether Group, Its Preparation Method and Applications," which uses an N-carboxycyclic anhydride containing a thioether group to prepare polyamino acid materials through ring-opening polymerization. The polyamino acids can self-assemble into nanoparticles in water, and the nanoparticles exhibit oxidation responsiveness, releasing the loaded drug in the highly reactive oxygen species environment of tumors. These two inventions utilize phenylboronic acid esters and thioether bonds as stimulus-responsive chemical bonds to prepare stimulus-responsive polymers, achieving responsive drug release. Its drawback is that it is mainly used for loading lipid-soluble drugs, and it has certain difficulties in efficiently loading water-soluble small molecule drugs, protein drugs, etc.

[0005] Therefore, there is a need for a material that is biocompatible, suitable for the efficient loading of hydrophilic / hydrophobic chemical drugs, drugs containing cis-1,2 or 1,3-diol, peptide drugs, protein drugs, and nucleic acid drugs, and that can be released in a controlled manner at the lesion site. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention provides an N-carboxylated intracyclic anhydride monomer containing phenylboronic acid and a polyamino acid, along with their preparation methods and applications. A phenylboronic acid-functionalized cysteine-N-carboxylated intracyclic anhydride monomer was designed and synthesized. A series of block copolymers were prepared by ring-opening polymerization of the phenylboronic acid-functionalized cysteine-N-carboxylated intracyclic anhydride monomer or / and other types of N-carboxylated intracyclic anhydride monomers initiated by polyethylene glycol. These block copolymers can be used to construct drug-loaded micelles and injectable polyamino acid hydrogels, achieving efficient drug loading and delivery and responsive release at lesion sites.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] An N-carboxylate intracyclic anhydride monomer containing phenylboronic acid, wherein the N-carboxylate intracyclic anhydride monomer containing phenylboronic acid is a phenylboronic acid-functionalized cysteine-N-carboxylate intracyclic anhydride monomer, having the structure shown in Formula I:

[0009]

[0010] This invention also provides a method for preparing an N-carboxyl ring anhydride monomer containing phenylboronic acid, wherein phenylboronic acid-functionalized cysteine ​​undergoes a cyclization reaction in the presence of triphosgene and α-pinene to obtain the phenylboronic acid-functionalized cysteine-N-carboxyl ring anhydride, wherein the phenylboronic acid-functionalized cysteine ​​has the structural formula shown in Formula II:

[0011]

[0012] Preferably, the cyclization reaction is carried out in an inert gas environment, the reaction temperature is 10-70℃, and the reaction time is 1-9h; the molar ratio of cysteine ​​functionalized with phenylboronic acid to triphosgene is 1:0.4-1; and the molar ratio of α-pinene to triphosgene is 2-5:1.

[0013] The cyclization reaction is carried out in a second solvent, which is at least one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide.

[0014] More preferably, the preparation process of the above-mentioned phenylboronic acid-functionalized cysteine-N-carboxylated intracyclic anhydride monomer is as follows: Phenyboronic acid-functionalized cysteine ​​and α-pinene are added to anhydrous tetrahydrofuran solution under an inert gas protection environment, followed by the dropwise addition of a tetrahydrofuran solution containing triphosgene. The reaction is carried out at 10–70°C for 1–9 hours. After the reaction, unreacted reactants are filtered off, and the reaction solution is concentrated by rotary evaporation and precipitated in icy hexane or petroleum ether to obtain a crude product. The crude product is redissolved in tetrahydrofuran and precipitated again. This process is repeated 2–3 times until a white powder solid is finally obtained, which is the phenylboronic acid-functionalized cysteine-N-carboxylated intracyclic anhydride. The synthetic route of the phenylboronic acid-functionalized N-carboxylated intracyclic anhydride of the present invention is shown below:

[0015]

[0016] Preferably, the compound of formula II is synthesized by the following steps:

[0017] In the presence of a first solvent and a base catalyst, L-cysteine ​​and 4-bromomethylphenylboronic acid or 4-bromomethylphenylboronic acid pinacol ester react at room temperature to give a compound having the formula shown in II.

[0018] More preferably, the specific preparation process of the compound of formula II is as follows: L-cysteine ​​is dispersed in a first solvent, an alkaline catalyst is added until the solution becomes clear, 4-bromomethylphenylboronic acid pinacol ester or 4-bromomethylphenylboronic acid is added, and the reaction is stirred at room temperature for 12-24 hours. After the reaction is completed, the mixture is filtered and dried to obtain phenylboronic acid-functionalized cysteine.

[0019] More preferably, the first solvent is at least one of tetrahydrofuran, ethanol, methanol, N,N-dimethylformamide, and water;

[0020] The alkaline catalyst is at least one selected from triethylamine, diethylamine, tetramethylguanidine, and sodium hydroxide;

[0021] The molar ratio of 4-bromomethylphenylboronic acid pinacol ester or 4-bromomethylphenylboronic acid to L-cysteine ​​is 1 to 2:1; the molar ratio of the base catalyst to L-cysteine ​​is 1 to 2:1.

[0022] The present invention also provides a linear polyamino acid block copolymer, wherein the monomers for synthesizing the polymer include the phenylboronic acid-functionalized cysteine-N-carboxycyclic anhydride as described in claim 1, and the polyamino acid block copolymer has the structure shown in Formula III:

[0023]

[0024] Wherein, R1 comes from the terminal functional group of the initiator polyethylene glycol; R2 comes from other types of N-carboxylic acid anhydrides; 10≤m≤400; 1≤x≤30, 0≤y≤30.

[0025] The initiator is a polyethylene glycol initiator, preferably one of polyethylene glycol amine, α-methoxy-ω-amino-polyethylene glycol, or α-maleimide-ω-amino-polyethylene glycol; the other types of N-carboxycyclic anhydrides are N-carboxycyclic anhydrides other than cysteine-N-carboxycyclic anhydrides, and the amino acid in the other types of N-carboxycyclic anhydrides is one of glutamic acid, tyrosine, N-ε-Boc-L-lysine, or β-benzyl-L-aspartic acid.

[0026] The present invention also provides a method for preparing the above-mentioned linear polyamino acid block copolymer, comprising the steps of: using polyethylene glycol as an initiator, using phenylboronic acid-functionalized cysteine-N-carboxycyclic anhydride as a monomer, or using phenylboronic acid-functionalized cysteine-N-carboxycyclic anhydride and other types of N-carboxycyclic anhydrides as comonomers, to carry out ring-opening polymerization.

[0027] Preferably, the ring-opening polymerization is carried out in an environment of organic solvent and inert gas; the organic solvent is at least one selected from dichloromethane, trichloromethane, N-methylpyrrolidone, N,N-dimethylformamide, and tetrahydrofuran, with N,N-dimethylformamide being the preferred solvent in this invention; the ring-opening polymerization temperature is 30–90°C, with 60°C being the preferred polymerization temperature in this invention; the polymerization reaction time is 1–5 days, with 3 days being the preferred reaction time in this invention.

[0028] As one embodiment of the linear polyamino acid block copolymer of the present invention, the block copolymer polyethylene glycol-b-polyamino acid is prepared by ring-opening polymerization of the phenylboronic acid-functionalized cysteine-N-carboxylic anhydride monomer initiated by polyethylene glycol. The chemical structural formula of the obtained block copolymer is shown below:

[0029]

[0030] Where m is 10–400, x is 1–30; R1 is the terminal functional group of the polyethylene glycol initiator, preferably a polyethylene glycol amino initiator, with the following molecular formula:

[0031]

[0032] As another way to construct the linear polyamino acid block copolymer of the present invention, the block copolymer is prepared by random copolymerization of polyethylene glycol as the initiator of the above-mentioned phenylboronic acid functionalization of cysteine-N-carboxylic acid anhydride monomer and other types of N-carboxylic acid anhydride monomers. The chemical structural formula of the block copolymer obtained by random copolymerization is shown below:

[0033]

[0034] Wherein, m is 10–400, x is 1–30, and y is 0–15; R1 is the terminal functional group of the polyethylene glycol initiator, preferably α-methoxy-ω-amino-polyethylene glycol and α-maleimide-ω-amino-polyethylene glycol as initiators; R2 comes from different types of amino acids, preferably glutamic acid, tyrosine, N-ε-Boc-L-lysine, and β-benzyl-L-aspartic acid.

[0035] The present invention also provides the application of the above-mentioned block copolymers self-assembling into nanomicelles in drug delivery.

[0036] Preferably, the linear polyamino acid block copolymer is self-assembled into nanomicelles for drug delivery, or used as a temperature-sensitive reactive oxygen species responsive hydrogel for drug delivery.

[0037] Preferably, the block copolymer is an amphiphilic polymer, polyethylene glycol-b-polyamino acid; the drug is a hydrophilic or hydrophobic chemical drug, a drug containing cis-1,2 or 1,3 diols, a polypeptide drug, a protein drug, or a nucleic acid drug.

[0038] For polymer nanomicelles, since the polymer of this invention has a suitable hydrophilic-hydrophobic ratio, a series of polymer micelles with controllable size can be prepared by directly dispersing the polymer solution in water or phosphate-buffered saline (PBS); or by solvent displacement method. The interaction between boric acid and catechol, cis-1,2 or 1,3-diol small molecule drugs, peptides, protein drugs and nucleic acid drugs can be used to achieve efficient drug loading and delivery.

[0039] For polymer hydrogels, the above-mentioned block copolymer is mixed with water under ice bath conditions, and then the temperature is increased to obtain a polypeptide hydrogel. The solvent is preferably water, physiological saline, a buffer solution, or tissue culture medium, more preferably a buffer solution or physiological saline, and most preferably a buffer solution. At lower temperatures, the block copolymer solution is a low-viscosity aqueous solution; when the temperature rises to near human body temperature, the aqueous solution transforms into a gel.

[0040] The cysteine ​​hydrophobic side chain functionalized with phenylboronic acid endows the block polymer with reactive oxygen species responsiveness (boronic acid structure has reactive oxygen species responsive properties), enabling the above-mentioned drugs to be released in response to reactive oxygen species environment; the injectability of thermosensitive peptide hydrogel makes it possible to form an in-situ gel for local drug delivery as a drug carrier.

[0041] Compared with the prior art, the present invention has the following advantages:

[0042] (1) This invention uses cysteine ​​as a raw material. First, cysteine ​​functionalized with phenylboronic acid or phenylboronic acid pinacol ester is obtained through a one-step reaction. Then, the corresponding N-carboxylic acid anhydride monomer is prepared through a cyclization reaction. The raw materials are widely available and the preparation method is simple. The thioether bond in the obtained monomer has an antioxidant effect, while the phenylboronic acid group is beneficial for efficient drug loading and controlled release. Therefore, it has potential application value in the field of drug carriers.

[0043] (2) The phenylboronic acid-functionalized cysteine-N-carboxy ring anhydride prepared in this invention is a novel anhydride monomer. It can be used to obtain polyamino acids with controllable performance through ring-opening polymerization. Compared with other polyamino acids, this type of polyamino acid has better bioresponsiveness and wider applicability, which greatly expands the types of amphiphilic polyamino acids.

[0044] (3) This invention uses polyethylene glycol amine as an initiator to obtain polymers with controllable molecular weight and narrow molecular weight distribution through ring-opening polymerization, which greatly expands the types of amphiphilic polyamino acids.

[0045] (4) The polymer disclosed in this invention has excellent biocompatibility and drug loading capacity, and can be used for efficient loading and delivery of hydrophilic / hydrophobic chemical drugs, drugs containing cis-1,2 or 1,3-diol, peptide drugs, protein drugs and nucleic acid drugs, thus having good application prospects. Attached Figure Description

[0046] Figure 1 The 1H NMR spectrum of phenylboronic acid-functionalized cysteine ​​synthesized in Example 1;

[0047] Figure 2The 1H NMR spectrum of the phenylboronic acid-functionalized cysteine-N-carboxy ring anhydride synthesized in Example 1;

[0048] Figure 3 The 1H NMR spectrum of the block copolymer PEG-b-(PPBA-co-PMLG) synthesized in Example 5;

[0049] Figure 4 The infrared spectrum of the block copolymer PEG-b-(PPBA-co-PMLG) synthesized in Example 5;

[0050] Figure 5 The 1H NMR spectrum of the block copolymer PEG-b-PPBA synthesized in Example 6;

[0051] Figure 6 Scanning electron microscope (SEM) images of drug-loaded micelle P-DOX NPs prepared in Example 7;

[0052] Figure 7 Particle size distribution of drug-loaded micelles P-VB2 NPs prepared in Example 8;

[0053] Figure 8 Sol-gel phase transition diagram (a) and photograph (b) of the drug-loaded gel for the preparation of polymer PEG-b-(PPBA-co-PMLG) solution in Example 9;

[0054] Figure 9 The release curve of Dox in the polymer PEG-b-(PPBA-co-PMLG) gel of Example 9 is shown. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0056] It should be noted that the raw materials used in this invention are all commercially available products, and the specific preparation and testing methods are all conventional techniques. The invention will be further described below.

[0057] Example 1

[0058] (1) Preparation of phenylboronic acid-functionalized cysteine ​​using L-cysteine ​​and 4-bromomethylphenylboronic acid as raw materials

[0059] L-cysteine ​​(3.6 g, 30 mmol) was dispersed in a mixed solvent of ethanol / water (V... 乙醇 V水 In a solution of sodium hydroxide (1.2 g, 30 mmol) in an ethanol / THF mixture (V = 3:2, 120 mL), add 10 mL of sodium hydroxide (1.2 g, 30 mmol) aqueous solution dropwise until the solution becomes clear. Add 10 mL of sodium hydroxide (1.2 g, 30 mmol) aqueous solution in an ethanol / THF mixture (V = 3:2, 120 mL) dropwise until the solution becomes clear. 乙醇 V THF A 3:2 (50 mL) solution was added dropwise to the above solution, and a white precipitate gradually formed. The reaction was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was filtered and dried to obtain phenylboronic acid-functionalized cysteine ​​in a yield of 40%.

[0060] (2) Preparation of phenylboronic acid-functionalized cysteine-N-carboxylic acid anhydride monomers

[0061] Under nitrogen atmosphere, phenylboronic acid-modified cysteine ​​(500 mg, 1.96 mmol) and α-pinene (0.5 mL, 3.15 mmol) were added to a dried three-necked round-bottom flask, followed by anhydrous tetrahydrofuran (150 mL). Then, a THF solution of triphosgene (237 mg, 0.80 mmol) was added dropwise to the suspension. The reaction mixture was stirred thoroughly in an oil bath at 55 °C for 5 hours. After 5 hours, the reaction solution was allowed to cool naturally to room temperature. The solution was then concentrated to 10 mL by rotary evaporation, followed by precipitation in pre-cooled n-hexane to obtain the crude product. The crude product was then redissolved in tetrahydrofuran, and precipitation was repeated three times to obtain a white powder solid, which was the phenylboronic acid-functionalized cysteine-N-carboxylated anhydride monomer, with a yield of 70%. The synthetic route of the phenylboronic acid-functionalized cysteine-N-carboxylated anhydride monomer is shown below:

[0062]

[0063] The products obtained from the above reaction were dissolved in DMSO-d6 and subjected to 1H NMR spectroscopy (Bruker, AVANCE 400). The obtained 1H NMR spectra are shown below. Figure 1 and Figure 2 .

[0064] Figure 1 Cysteine ​​functionalized with phenylboronic acid in DMSO-d6 1 The 1H-NMR spectrum shows the following peaks: δ2.91 ppm (proton hydrogen peak on the β-carbon of the carboxyl group adjacent to S), δ3.80 ppm (proton hydrogen peak of the methylene group between the benzene ring and S), and δ4.15 ppm (proton hydrogen peak of the methine group adjacent to the amino group). The peaks at δ7.30 ppm and δ7.75 ppm are proton hydrogen peaks on the benzene ring. The ratio of all peak areas matches the ratio of the various proton hydrogen numbers in phenylboronic acid-functionalized cysteine, confirming the successful synthesis of the phenylboronic acid-functionalized cysteine ​​monomer.

[0065] Figure 2 Cysteine-N-carboxylic anhydride functionalized with phenylboronic acid in DMSO-d6 1 The 1H-NMR spectrum shows the following peaks: δ2.80 ppm is the proton hydrogen peak on the methylene group between the sulfur (S) and the five-membered ring; δ3.80 ppm is the proton hydrogen peak on the methylene group between the benzene ring and the sulfur (S); δ4.78 ppm is the proton hydrogen peak on the carbon atom of the five-membered ring; and δ7.25 ppm and δ7.75 ppm are the proton hydrogen peaks on the benzene ring. The ratio of the peak areas in the spectrum matches the ratio of the number of proton hydrogens on the cysteine-N-carboxylic anhydride functionalized with phenylboronic acid, confirming the successful synthesis of the monomer.

[0066] Example 2

[0067] The difference between this embodiment and Example 1 is that the molar ratio of L-cysteine ​​and 4-bromomethylphenylboronic acid is 1:2.

[0068] (1) Preparation of phenylboronic acid-functionalized cysteine ​​using L-cysteine ​​and 4-bromomethylphenylboronic acid as raw materials

[0069] L-cysteine ​​(3.6 g, 30 mmol) was dispersed in a mixed solvent of ethanol / water (V... 乙醇 V 水 In a 3:2 (120 mL) solution, gradually add 10 mL of ethanol solution of tetramethylguanidine (3.75 mL, 30 mmol) until the solution becomes clear. Add 120 mL of ethanol / THF mixed solution of 4-bromomethylphenylboronic acid (12.9 g, 60 mmol) to the solution. 乙醇 V THF A 3:2 (50 mL) solution was added dropwise to the above solution, and a white precipitate gradually formed. The reaction was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was filtered and dried to obtain phenylboronic acid-functionalized cysteine ​​in 50% yield.

[0070] (2) Preparation of phenylboronic acid-functionalized cysteine-N-carboxylic acid anhydride monomers

[0071] Under nitrogen atmosphere, phenylboronic acid-modified cysteine ​​(500 mg, 1.96 mmol) and α-pinene (0.5 mL, 3.15 mmol) were added to a dried three-necked round-bottom flask, followed by anhydrous tetrahydrofuran (150 mL). Then, a THF solution of triphosgene (237 mg, 0.80 mmol) was added dropwise to the suspension. The reaction system was stirred evenly in an oil bath at 55 °C. After reacting for 5 hours, the reaction solution was naturally cooled to room temperature. The reaction solution was concentrated to 10 mL by rotary evaporation, and then precipitated in pre-cooled n-hexane to obtain the crude product. The crude product was then redissolved in tetrahydrofuran and precipitated again. This process was repeated 3 times to obtain a white powder solid, which is the phenylboronic acid-functionalized cysteine-N-carboxylic anhydride monomer, with a yield of 65%.

[0072] Example 3

[0073] The difference between this embodiment and Example 1 is that the molar ratio of L-cysteine ​​to 4-bromomethylphenylboronic acid is 1:1.5, and the molar ratio of phenylboronic acid-modified cysteine ​​to triphosgene is 1:1.

[0074] (1) Preparation of phenylboronic acid-functionalized cysteine ​​using L-cysteine ​​and 4-bromomethylphenylboronic acid as raw materials

[0075] L-cysteine ​​(3.6 g, 30 mmol) was dispersed in a mixed solvent of ethanol / water (V... 乙醇 V 水 Add 10 mL of ethanol solution of tetramethylguanidine (3.75 mL, 30 mmol) dropwise to a 3:2 (120 mL) solution until the solution becomes clear. Add 10 mL of ethanol / THF mixture of 4-bromomethylphenylboronic acid (9.7 g, 45 mmol) to the solution. 乙醇 V THF A 3:2 (50 mL) solution was added dropwise to the above solution, and a white precipitate gradually formed. The reaction was stirred at room temperature for 12 h. After the reaction was complete, the mixture was filtered and dried to obtain phenylboronic acid-functionalized cysteine ​​in 51% yield.

[0076] (2) Preparation of phenylboronic acid-functionalized cysteine-N-carboxylic acid anhydride monomers

[0077] Under nitrogen atmosphere, phenylboronic acid-modified cysteine ​​(500 mg, 1.96 mmol) and α-pinene (0.5 mL, 3.15 mmol) were added to a dried three-necked round-bottom flask, followed by anhydrous tetrahydrofuran (150 mL). Then, a THF solution of triphosgene (582 mg, 1.96 mmol) was added dropwise to the suspension. The reaction system was stirred evenly in an oil bath at 55 °C. After reacting for 5 hours, the reaction solution was naturally cooled to room temperature. The reaction solution was concentrated to 10 mL by rotary evaporation, and then precipitated in pre-cooled n-hexane to obtain the crude product. The crude product was then redissolved in tetrahydrofuran and precipitated again. This process was repeated 3 times to obtain a white powder solid, which is the phenylboronic acid-functionalized cysteine-N-carboxylic anhydride monomer, with a yield of 63%.

[0078] Example 4

[0079] The difference between this embodiment and Example 1 is that phenylboronic acid-functionalized cysteine ​​is prepared using L-cysteine ​​and 4-bromomethylphenylboronic acid pinacol ester as raw materials (molar ratio of 1:1), and the molar ratio of phenylboronic acid-functionalized cysteine ​​and triphosgene is changed (the molar ratio of the two is 1:0.7).

[0080] (1) Prepare phenylboronic acid-functionalized cysteine ​​using L-cysteine ​​and 4-bromomethylphenylboronic acid pinacol ester as raw materials.

[0081] L-cysteine ​​(3.6 g, 30 mmol) was dispersed in a mixed solvent of ethanol / water (V... 乙醇 V 水 In a 3:2 (120 mL) solution, triethylamine (4.15 mL, 30 mmol) was added dropwise until the solution became clear. A solution of 4-bromomethylphenylboronic acid pinacol ester (8.9 g, 30 mmol) in ethanol / THF was added (V... 乙醇 V THF A 3:2 (50 mL) solution was added dropwise to the above solution, and a white precipitate gradually formed. The reaction was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was filtered and dried to obtain phenylboronic acid-functionalized cysteine ​​in a yield of 45%.

[0082] (2) Preparation of phenylboronic acid-functionalized cysteine-N-carboxylic acid anhydride monomers

[0083] Under nitrogen atmosphere, phenylboronic acid-modified cysteine ​​(500 mg, 1.96 mmol) and α-pinene (0.7 mL, 4.40 mmol) were added to a dried three-necked round-bottom flask, followed by anhydrous tetrahydrofuran (150 mL). Then, a THF solution of triphosgene (445 mg, 1.50 mmol) was added dropwise to the suspension. The reaction system was stirred evenly in an oil bath at 55 °C. After reacting for 5 hours, the reaction solution was naturally cooled to room temperature. The reaction solution was concentrated to 10 mL by rotary evaporation, and then precipitated in pre-cooled n-hexane to obtain the crude product. The crude product was then redissolved in tetrahydrofuran and precipitated again. This process was repeated 3 times to obtain a white powder solid, which is the phenylboronic acid-functionalized cysteine-N-carboxylic anhydride monomer, with a yield of 60%.

[0084] Example 5: Preparation of block copolymer PEG-b-(PPBA-co-PMLG)

[0085] Using amino-terminated polyethylene glycol as an initiator, ring-opening polymerization of phenylboronic acid-functionalized N-carboxylic anhydride (PBA-NCA) with γ-methyl-L-glutamic acid-N-carboxylic anhydride (MLG-NCA), γ-ethyl-L-glutamic acid-N-carboxylic anhydride (ELG-NCA), or γ-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA) was initiated. By adjusting the molar ratio of polyethylene glycol, phenylboronic acid-functionalized N-carboxylic anhydride, and MLG-NCA, ELG-NCA, or BLG-NCA, polymers with different chain lengths, such as PEG-b-(PPBA-co-PMLG), PEG-b-(PPBA-co-PELG), and PEG-b-(PPBA-co-PBLG), can be prepared. Taking the synthesis of PEG-b-(PPBA-co-PMLG) as an example, its synthetic route is shown in the figure below:

[0086]

[0087] In this embodiment, the specific preparation process of PEG-b-(PPBA-co-PMLG) is as follows: Under a nitrogen atmosphere, PEG... 45 A DMF solution of -NH2 (0.25 g, 0.125 mmol) was added to a closed reactor. Then, under stirring, DMF solutions of phenylboronic acid-functionalized N-carboxylic anhydride (PBA-NCA) (0.21 g, 0.75 mmol) and MLG-NCA (0.24 g, 1.25 mmol) were added. The reaction was carried out in a constant temperature oil bath at 60 °C for 3 days (the corresponding carbonyl stretching peak of the PBA-NCA monomer in the infrared spectrum is 1770 cm⁻¹). -1 and 1845cm -1(Complete disappearance indicates complete polymerization). After the reaction, the polymer solution was precipitated with ice-cold anhydrous diethyl ether, centrifuged, and then reconstituted with methanol. The precipitation was repeated three times. The solid precipitate was collected and vacuum dried for 48 hours to obtain a white solid product with a yield of 75%.

[0088] The resulting white solid product was dissolved in DMSO-d6 / TFA (V DMSO-d6 V TFA The mixture was characterized by nuclear magnetic resonance (NMR) in a 5:2 (Bruker, Avance) solution, and the results were obtained. 1 The H-NMR spectrum is shown in [reference]. Figure 3 In the spectrum, the peak at chemical shifts δ 1.65 ppm - 2.0 ppm represents the proton hydrogen peak on the β carbon of the carboxyl group in the glutamic acid side chain; the peak at δ 2.25 ppm - 2.37 ppm represents the proton hydrogen peak on the α carbon of the carboxyl group in the glutamic acid side chain; the peak at δ 2.55 ppm - 2.90 ppm represents the proton hydrogen peak of the methylene group between the α carbon and S in the amino acid; and the peak at δ 3.40 ppm - 3.52 ppm is a characteristic peak of the polyethylene glycol segment. The peak at 0.65 ppm is the proton hydrogen peak of the methoxy group on the glutamic acid side chain; the peak at 3.66 ppm-3.81 ppm is the proton hydrogen peak of the methylene group between the benzene ring and S in the cysteine ​​side chain; the peak at 4.13 ppm-4.40 ppm is the proton hydrogen peak of the α-carbon of glutamic acid; the peak at 4.40 ppm-4.60 ppm is the proton hydrogen peak of the α-carbon of cysteine; and the peaks at 7.25 ppm and 7.70 ppm are the proton hydrogen peaks of the benzene ring. All representative peaks of the polymer were assigned, confirming the successful preparation of the copolymer. By comparing the peak integrals, the values ​​of m, x, and y of the polymer were determined to be 45, 6, and 10, respectively.

[0089] The PEG-b-(PPBA-co-PMLG) block copolymer, PBA-NCA, and MLG-NCA were characterized by infrared spectroscopy (Bruker, Vetor-22). The resulting infrared spectra are shown below. Figure 4 By comparing the infrared spectra of PBA-NCA and MLG-NCA monomers and polymers, it can be seen that the corresponding curves for MLG-NCA and PBA-NCA are in the range of 1726-1830 cm⁻¹. -1 Two strong peaks appeared between the two, corresponding to the anhydride groups on the NCA ring, while the copolymer curve showed peaks between 1625-1668 cm⁻¹. -1 The peak between 3278 cm⁻¹ represents the stretching vibration of the -CO- group in the amide bond, proving the presence of amide bonds in the polymer derived from the ring-opening polymerization of NCA monomers. -1 The sharp peak at that position represents the stretching vibration of -NH-, which is a result of both the amide bond and the free amino group. The peak positions above further confirm the structure of the substance.

[0090] Example 6: Preparation of the block copolymer PEG-b-PPBA

[0091] This embodiment describes a method for preparing the amphiphilic polymer polyethylene glycol-b-polyamino acid PEG-b-PPBA, specifically as follows:

[0092]

[0093] Under a nitrogen atmosphere, a 10 mL solution of DMF containing 1.5 g (0.75 mmol) of PEG-NH2 was added to a closed reactor. A 50 mL solution of DMF containing 4.22 g (15 mmol) of PBA-NCA was then added under stirring. The reaction was carried out for 3 days in a constant temperature oil bath at 30°C. After the reaction, the polymer solution was precipitated with ice-cold anhydrous diethyl ether, centrifuged, and then reconstituted with DMF. The ether precipitation was repeated 2-3 times. The solid precipitate was collected and vacuum dried for 48 hours to obtain a white solid product with a yield of 52%.

[0094] The obtained white solid product was dissolved in DMSO-d6 and characterized by nuclear magnetic resonance (Bruker, AVANCE 400). 1 The H-NMR spectrum is shown in [reference]. Figure 5 The peaks at δ 2.56 ppm–2.87 ppm in the spectrum represent the proton hydrogen peaks of the methylene group between the α carbon and S of the amino acid; the peak at δ 3.38 ppm–3.56 ppm is a characteristic peak of the polyethylene glycol segment; the peak at δ 3.63 ppm–3.75 ppm represents the proton hydrogen peaks of the methylene group of the cysteine ​​side chain between the benzene ring and S; the peak at δ 4.49 ppm–4.74 ppm represents the proton hydrogen peaks on the α carbon of cysteine; and the peaks at δ 7.13 ppm–7.3 ppm and δ 7.64 ppm–7.74 ppm represent the proton hydrogen peaks on the benzene ring. All representative peaks were assigned, demonstrating the successful preparation of the polymer. By comparing the peak integrals, the values ​​of m and x for the polymer were determined to be 45 and 17, respectively.

[0095] Example 7: Preparation of drug-loaded micelles of polymer PEG-b-(PPBA-co-PMLG)

[0096] Doxorubicin (Dox), an antitumor drug that interacts with boric acid on PEG-b-(PPBA-co-PMLG), was selected as a model drug, and drug-loaded micelles were prepared using a nanoprecipitation method. The steps for preparing drug-loaded micelles using the nanoprecipitation method were as follows: 100 mg of a block copolymer and 10 mg of Dox were dissolved in dimethyl sulfoxide (2 mL). The resulting solution (2 mL) was slowly added dropwise to deionized water (10 mL), and stirred in the dark for 0.5 h. The organic solvent and free Dox were removed by dialysis using a dialysis bag with a molecular weight cutoff of 3500. The mixture was then freeze-dried to obtain doxorubicin-loaded polymer micelles P-Dox NPs powder, which can be used for drug delivery. The Dox loading was found to be 8.5% using a UV spectrophotometer (TU-1900), with a loading efficiency of 93%. The morphology of the drug-loaded micelles P-Dox NPs was characterized using a field emission scanning electron microscope (FEI, Nano SEM450). Figure 6 As shown.

[0097] Example 8: Preparation of drug-loaded micelles of polymer PEG-b-PPBA

[0098] Vitamin B2 (Riboflavin or vitamin B2, VB2), containing cis-vicinal diols that interact with boric acid on PEG-b-PPBA, was selected as a model drug. Drug-loaded micelles were prepared using a nanoprecipitation method. The steps for preparing drug-loaded micelles by nanoprecipitation were as follows: 80 mg of the block copolymer PEG-b-PPBA was dissolved in dimethyl sulfoxide (or N,N-dimethylformamide) to obtain a polymer solution (5 mL). The polymer solution was then added dropwise to 10 mL of a 0.1 M sodium hydroxide aqueous solution containing 20 mg of vitamin B2. The mixture was stirred at 40 °C for 3 h. Finally, the organic solvent and free small molecules were removed by dialysis using a dialysis bag with a molecular weight cutoff (MWCO) of 5000 to obtain drug-loaded micelles P-VB2 NPs. The prepared drug-loaded micelles contained 15% BV2, and the loading efficiency was 70.6%. The particle size distribution of P-VB2 NPs (0.5 mg / mL) was characterized using dynamic light scattering (Zetasizer S90). Figure 7 As shown, the average particle size of the prepared drug-loaded micelles is 151 nm.

[0099] Example 9: Preparation of Thermosensitive Polymer Gel and Drug-Loaded Gel

[0100] The block copolymer of Example 5 was dissolved in one of water, physiological saline, PBS buffer solution, or tissue culture medium to obtain a block copolymer solution. When the temperature is raised to near human body temperature (37°C), the polymer solution transforms into a gel. PBS buffer solution is most preferred, and the optimal concentration of the polymer solution is 4 wt% to 15 wt%. The sol-gel phase transition diagram of the polymer solution is attached. Figure 8 (a) The gelation temperature range is 6-30℃ when the polymer solution concentration is 4wt% to 7wt%, and the copolymers with different degrees of polymerization, PEG-b-(PPBA3-co-PMLG), are shown to be effective. 15 ) and PEG-b-(PPBA6-co-PMLG 15 The copolymers exhibit the same sol-gel transition curves, which is due to the fact that gel formation is the result of a combination of factors, including intermolecular interactions, hydrophilicity-hydrophobicity balance, and the secondary structure of amino acid copolymers. The low gelation concentration (4wt%–7wt%) of the prepared copolymer at in vivo at the given temperature gives it significant practical application value.

[0101] In this embodiment, Dox was selected as the drug model and loaded into the polymer gel prepared above. First, a 5 mg / mL Dox aqueous solution was prepared, and triethylamine, in an equimolar amount of Dox, was added. This solution was then added to the block copolymer solution (polymer concentration 10 wt%) and mixed thoroughly, wherein the Dox content was 20% of the polymer content. The Dox-containing solution was placed in a 37°C water bath, and a drug-loaded gel was formed, which can be used for drug delivery. The drug-loaded gel is shown in the attached figure. Figure 8 (b)

[0102] Drug-loaded gels exhibit reactive oxygen species and pH responsiveness, enabling rapid drug release under acidic or H2O2 conditions. Taking a polymer gel loaded with the antitumor drug Dox as an example, its release curve is shown below. Figure 9 As shown, under PBS buffer solution (pH 7.4), drug release was slow, with only 10% of Dox released cumulatively after 60 hours. Under PBS buffer solution (pH 5.5), the interaction between Dox and the polymer was disrupted, and the cumulative release of Dox reached 50% after 60 hours. Adding 3 mmol of H2O2 to the PBS buffer solution (pH 7.4) also promoted drug release, with the cumulative release of Dox reaching 60% after 60 hours. These results demonstrate that the drug-loaded gel exhibits both reactive oxygen species and pH responsiveness.

[0103] The foregoing detailed examples of the present invention are merely preferred embodiments and should not be construed as limiting the scope of the invention. All equivalent variations and modifications made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. An N-carboxyl ring anhydride monomer containing phenylboronic acid, characterized in that: The N-carboxyl ring anhydride monomer containing phenylboronic acid is a phenylboronic acid-functionalized cysteine-N-carboxyl ring anhydride monomer, having the structure shown in Formula I: Formula I.

2. A method for preparing an N-carboxyl ring anhydride monomer containing phenylboronic acid as described in claim 1, characterized in that... Includes the following steps: Cysteine ​​functionalized with phenylboronic acid undergoes a cyclization reaction in the presence of triphosgene and α-pinene to obtain the cysteine-N-carboxyl anhydride containing phenylboronic acid functionalized with phenylboronic acid, wherein the phenylboronic acid functionalized cysteine ​​has the structural formula shown in Formula II: Formula II.

3. The preparation method according to claim 2, characterized in that: The cyclization reaction is carried out in an inert gas environment at a temperature of 10-70°C. o C, the reaction time is 1-9 h; the molar ratio of phenylboronic acid-functionalized cysteine ​​to triphosgene is 1:0.4-1; the molar ratio of α-pinene to triphosgene is 2-5:1; The cyclization reaction is carried out in a second solvent, which is at least one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide.

4. The preparation method according to claim 2, characterized in that: The method for synthesizing the compound of formula II includes the following steps: under the action of a first solvent and an alkaline catalyst, L-cysteine ​​and 4-bromomethylphenylboronic acid or 4-bromomethylphenylboronic acid pinacol ester are reacted at room temperature to obtain the compound having the formula II.

5. The preparation method according to claim 4, characterized in that: The first solvent is at least one of tetrahydrofuran, ethanol, methanol, N,N-dimethylformamide, and water; The alkaline catalyst is at least one selected from triethylamine, diethylamine, tetramethylguanidine, and sodium hydroxide; The molar ratio of 4-bromomethylphenylboronic acid pinacol ester or 4-bromomethylphenylboronic acid to L-cysteine ​​is 1~2:1; the molar ratio of the base catalyst to L-cysteine ​​is 1~2:

1.

6. A linear polyamino acid block copolymer, characterized in that: The monomers for synthesizing the polymer include the phenylboronic acid-functionalized cysteine-N-carboxycyclic anhydride as described in claim 1, and the linear polyamino acid block copolymer has the structure shown in Formula III: Formula III; Wherein, R1 comes from the terminal functional group of the initiator polyethylene glycol; R2 comes from other types of N-carboxylic acid anhydrides; 10≤m≤400; 1≤x≤30, 0≤y≤30.

7. A method for preparing the linear polyamino acid block copolymer according to claim 6, characterized in that... Includes the following steps: The product is obtained by ring-opening polymerization using polyethylene glycol as an initiator and phenylboronic acid-functionalized cysteine-N-carboxycyclic anhydride as a monomer, or phenylboronic acid-functionalized cysteine-N-carboxycyclic anhydride and other types of N-carboxycyclic anhydrides as comonomers.

8. The preparation method according to claim 7, characterized in that: The ring-opening polymerization is carried out in an environment of organic solvent and inert gas; the temperature of the ring-opening polymerization is 30-90℃; and the polymerization reaction time is 1-5 days. The organic solvent is at least one of dichloromethane, trichloromethane, N-methylpyrrolidone, N,N-dimethylformamide, and tetrahydrofuran; The initiator is one of polyethylene glycol amine, α-methoxy-ω-amino-polyethylene glycol, and α-maleimide-ω-amino-polyethylene glycol; the other type of N-carboxycyclic anhydride is an N-carboxycyclic anhydride other than cysteine-N-carboxycyclic anhydride, and the amino acid of the other type of N-carboxycyclic anhydride is one of glutamic acid, tyrosine, N-ε-Boc-L-lysine, and β-benzyl-L-aspartic acid.

9. The use of the linear polyamino acid block copolymer of claim 6 in the preparation of a drug delivery system.

10. The application according to claim 9, characterized in that: The linear polyamino acid block copolymer is self-assembled to form nanomicelles for drug delivery, or used as a temperature-sensitive reactive oxygen species responsive hydrogel for drug delivery. The drug is a hydrophilic or hydrophobic chemical drug, a drug containing cis-1,2 or 1,3 diol, a polypeptide drug, a protein drug, or a nucleic acid drug.

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