Water-soluble polypeptide cross-linking agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a water-soluble polypeptide cross-linking agent and a preparation method and application thereof. The water-soluble polypeptide cross-linking agent provided by the invention is a random copolymer of oligomeric ethylene glycol monomethyl ether glutamate and 4-boric acid phenylalanine, and both ends of the random copolymer are double-bond end caps. The water-soluble polypeptide cross-linking agent can be subjected to pH-induced conformational transformation from alpha-helix to random curling, is of an alpha-helix structure under a neutral pH condition, and is transformed into a random curling structure under an alkaline pH condition. The intelligent polypeptide cross-linking agent has important application value in the field of biomedical materials, and is especially suitable for preparing biomedical materials which can be used under physiological conditions.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a water-soluble polypeptide crosslinking agent, its preparation method, and its application. Background Technology

[0002] Similar to the structural characteristics of proteins, peptides or polyamino acids also possess secondary structures such as α-helices, β-sheets, and random coils. Some peptides can also undergo a pH-induced helix-coil transition. For example, polyglutamic acid folds into an α-helix under acidic conditions but transforms into a random coil conformation under neutral and alkaline conditions. Conversely, polylysine folds into an α-helix under alkaline conditions but transforms into a random coil conformation under neutral and acidic conditions. Crosslinking agents based on these peptides can introduce peptide segments into polymers, utilizing the pH-induced helix-random coil conformational transition to achieve specific functions, resulting in novel polymer materials with unique functionalities.

[0003] When such materials are used in vivo as biomedical agents, it may be necessary for the peptide cross-linking agent to fold into an α-helix under neutral conditions, but transform into a random coil conformation under acidic or alkaline conditions. One study reported a tertiary amine-modified polyglutamate ester, with the tertiary amine and glutamate bridged by ethylene glycol. This polyamino acid underwent a pH-induced helical-random coil conformational transition, with the transition point within the physiological pH range (5.3-6.5). Other studies have reported that partially imidazole-modified polylysine can undergo a pH-induced helical-random coil conformational transition, with the transition point around neutral pH. However, peptides that are water-soluble, fold into an α-helix under neutral conditions, and form a random coil under acidic or alkaline conditions, are rarely reported. Summary of the Invention

[0004] In view of this, the present invention provides a water-soluble polypeptide crosslinking agent, its preparation method and application. The water-soluble polypeptide crosslinking agent has unique pH-responsive characteristics, and these pH-responsive characteristics are reversible, providing a new material for the development of intelligent biomedical products.

[0005] To solve the above technical problems, the first aspect of the present invention provides a water-soluble polypeptide crosslinking agent, wherein the water-soluble polypeptide crosslinking agent is a random copolymer of oligoethylene glycol monomethyl ether glutamate and 4-boronic acid phenylalanine, and both ends of the random copolymer are double-bonded.

[0006] The water-soluble polypeptide crosslinking agent provided by this invention is primarily a random copolymer formed by copolymerizing oligoethylene glycol monomethyl ether glutamate and 4-boronic acid phenylalanine as monomers, with polymerizable double bonds introduced at both ends of the random copolymer. This polypeptide crosslinking agent exhibits unique pH-responsive characteristics: it maintains a stable α-helical structure under physiological pH conditions, while transforming into a random coil structure in an alkaline environment, and this conformational change is reversible.

[0007] In conjunction with the first aspect, the oligoethylene glycol monomethyl ether glutamate and 4-boronic acid phenylalanine are both L-configuration or both D-configuration; wherein the degree of polymerization n of the oligoethylene glycol is: 2≤n≤100; and the degree of polymerization of the random copolymer is 10~150.

[0008] Preferably, the degree of polymerization n of the oligoethylene glycol is 2≤n≤10, and more preferably 3≤n≤8.

[0009] Preferably, the degree of polymerization of the random copolymer is 15 to 100, more preferably 20 to 80, and even more preferably 25 to 75.

[0010] For example, the oligoethylene glycol monomethyl ether glutamate can be triethylene glycol monomethyl ether glutamate, tetraethylene glycol monomethyl ether glutamate, pentaethylene glycol monomethyl ether glutamate, hexaethylene glycol monomethyl ether glutamate, heptaethylene glycol monomethyl ether glutamate, octaethylene glycol monomethyl ether glutamate, etc., with D or L configuration.

[0011] In conjunction with the first aspect, the water-soluble polypeptide crosslinking agent has an α-helix structure under neutral conditions.

[0012] In conjunction with the first aspect, the water-soluble polypeptide crosslinking agent transforms from an α-helix structure to a random coil structure under alkaline conditions.

[0013] In conjunction with the first aspect, the general structural formula of the water-soluble polypeptide crosslinking agent is as follows:

[0014]

[0015] Where x, y, and n are all integers greater than 0;

[0016] R1 and R2 are keys, or R1 and R2 are independently selected from the keys R. 11 The following groups, substituted or unsubstituted: C1-C 10 Alkyl, C2-C 10 alkenyl, C1-C 10 alkoxy, or R 12 Substituted or unsubstituted C3-C8 cycloalkyl groups, wherein R 11 It is a halogen, C3-C8 cycloalkyl or hydroxyl group, R 12It is a C1-C6 alkyl, halogen, or hydroxyl group;

[0017] R3 and R4 are each independently selected from hydrogen, or each independently selected from halogenated, nitro, amino, R 11 Substituted or unsubstituted C1-C6 alkyl or alkoxy groups.

[0018] Here, "alkyl" refers to straight-chain and branched saturated aliphatic hydrocarbon groups. For example, C1-C 10 Alkyl groups refer to saturated straight-chain or branched hydrocarbon groups with 1 to 10 carbon atoms. Preferably, they are C1-C6 alkyl groups, more preferably C1-C3 alkyl groups. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, and 2-ethylbutyl, etc.

[0019] "Alkenyl" refers to an unsaturated aliphatic hydrocarbon group containing at least one carbon-carbon double bond of a compound or its stereoisomers, which may be straight-chain or branched, or a combination thereof, or have a cycloalkyl structure. Non-limiting examples of alkenyl groups include vinyl, propenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 2-methyl-3-butenyl, 1-hexenyl, 2-hexenyl, and 2-hexenyl, etc.

[0020] "Alkoxy" refers to an alkyl group that is attached to the rest of the molecule by an oxygen atom, wherein the alkyl group has the meaning of "alkyl" as described above in this application. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, and isopropoxy, or butoxy (including n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy), etc.

[0021] "Cycloalkyl" refers to a saturated monocyclic, bicyclic, or polycyclic system that can be fused with aryl or heteroaryl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0022] A second aspect of the present invention provides a method for preparing the above-mentioned water-soluble polypeptide crosslinking agent, comprising the steps of:

[0023] S1. Oligoethylene glycol monomethyl ether glutamate and 4-boronic acid phenylalanine are reacted with triphosgene to generate the corresponding NCA monomers.

[0024] S2. The two NCA monomers mentioned above are subjected to ring-opening polymerization using an initiator containing double bonds to obtain a random copolymer with double bonds at one end of the molecular chain.

[0025] S3. The random copolymer is capped with a capping agent containing double bonds to obtain the water-soluble polypeptide crosslinking agent.

[0026] The method for preparing a water-soluble polypeptide crosslinking agent provided by the present invention uses oligoethylene glycol monomethyl ether glutamate and 4-boronic acid phenylalanine as polymerizing monomers. First, they are reacted with triphosgene to generate corresponding N-carboxylic anhydride (NCA) monomers. Then, the two NCA monomers are ring-opening polymerized using an initiator containing double bonds to obtain a random copolymer. Finally, the random copolymer is end-capped with a reagent containing double bonds to obtain a water-soluble polypeptide crosslinking agent with double bonds at both ends of the molecular chain.

[0027] For example, the steps of the above-mentioned method for preparing a water-soluble peptide crosslinking agent include:

[0028] S1. Oligopolyethylene glycol monomethyl ether glutamate and 4-boronic acid phenylalanine were dispersed in ultra-dry tetrahydrofuran, and appropriate amounts of triphosgene were added to each. The mixture was reacted at room temperature for 5-7 hours. The reaction solution was then concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain oligopolyethylene glycol monomethyl ether glutamate NCA monomer and 4-boronic acid phenylalanine NCA monomer, respectively.

[0029] S2. Dissolve the two NCA monomers in ultra-dry N,N-dimethylformamide (DMF) in proportion, add an appropriate amount of initiator containing double bonds, and initiate the ring-opening polymerization of the two NCA monomers at 48-52℃ for 70-74h. Then, precipitate, centrifuge, wash and dry the product in methyl tert-butyl ether solution to obtain a random copolymer with double bonds at one end of the molecular chain.

[0030] S3. Dissolve the random copolymer in ultra-dry dichloromethane (DCM), add an appropriate amount of end-capping agent containing double bonds, stir and react at room temperature for 5-7 hours, then continue to precipitate and centrifuge the product in methyl tert-butyl ether solution, remove impurities by dialysis, and finally freeze dry to obtain a water-soluble polypeptide crosslinking agent with double bonds at both ends.

[0031] In conjunction with the second aspect, in step S2, the molar percentage of the monomer corresponding to the 4-boronicophenylalanine is 5% to 70%, preferably 10% to 20%.

[0032] In conjunction with the second aspect, the double-bonded initiator is selected from at least one of allylamine, 3-buten-1-amine, 4-penten-1-amine, and 5-hexen-1-amine. Other types of double-bonded initiators may also be used, and the available double-bonded initiators are not limited to the aforementioned types.

[0033] Preferably, the initiator containing a double bond is 3-butene-1-amine.

[0034] In conjunction with the second aspect, the double-bonded end-capping agent is selected from at least one of acrylic acid, acryloyl chloride, methacrylic acid, and methacryl chloride. Other types of double-bonded end-capping agents may also be used, and the available double-bonded initiators are not limited to the aforementioned types.

[0035] Preferably, the end-capping agent containing double bonds is acryloyl chloride.

[0036] A third aspect of the present invention provides an intelligent biomedical material comprising the above-mentioned water-soluble polypeptide crosslinking agent or a water-soluble polypeptide crosslinking agent prepared according to the above-mentioned method for preparing the water-soluble polypeptide crosslinking agent.

[0037] For example, the smart biomedical material includes, but is not limited to, molecularly imprinted polymers.

[0038] The beneficial effects of this invention are as follows: The water-soluble polypeptide crosslinking agent provided by this invention not only possesses good water solubility and biocompatibility, but also exhibits an α-helix structure under physiological pH conditions and can transform into a random coil structure under alkaline conditions, and this pH-induced helix-random coil transformation is reversible. This intelligent water-soluble polypeptide crosslinking agent has significant application value in the field of biomedical materials, and is particularly suitable for preparing biomedical materials that can be used under physiological conditions. Attached Figure Description

[0039] Figure 1 The molecular structural formula of the water-soluble polypeptide crosslinking agent obtained in Example 1 of this invention is shown below.

[0040] Figure 2 The following are circular dichroism chromatograms of the water-soluble polypeptide crosslinking agent obtained in Example 1 of this invention at physiological pH and high pH.

[0041] Figure 3 The water-soluble polypeptide crosslinking agent obtained in Example 1 of this invention at different pH values ​​is θ. 222 value. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0043] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the meaning consistent with their meaning in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined.

[0044] The preparation method provided by the present invention will be described below through specific embodiments.

[0045] Unless otherwise specified, the raw materials, reagents and equipment used in this invention are all conventional commercially available reagents and equipment.

[0046] Example 1

[0047] This embodiment provides a water-soluble polypeptide crosslinking agent with the following molecular structure: Figure 1 As shown, it is prepared according to the following method:

[0048] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0049] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine (as an acid-binding and neutralizing agent) and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 10%.

[0050] The circular dichroism chromatogram and θ of the obtained water-soluble peptide crosslinking agent were tested. 222 The pH-value curves are shown below. Figures 2-3 As shown. By Figure 2 It can be seen that the obtained water-soluble polypeptide cross-linking agent has an α-helix structure under physiological pH conditions, and transforms into a random coil structure under alkaline conditions. Figure 3It can be seen that the pH of the resulting water-soluble polypeptide crosslinking agent is approximately 10.

[0051] Example 2

[0052] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0053] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 10.4 μL of allylamine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helical structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 10%.

[0054] Example 3

[0055] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0056] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 16.9 μL of 4-penten-1-amine was added, and ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 10%.

[0057] Example 4

[0058] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0059] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 19.6 μL of 5-hexene-1-amine was added, and ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helical structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 10%.

[0060] Example 5

[0061] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0062] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DMF solution. 50 μL of acrylic acid, 0.02 g of 1-hydroxybenzotriazole (as a catalyst), and 0.15 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (as a condensing agent) were added, and the mixture was stirred at room temperature for 24 h. The product was then precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and the product was finally freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25; 4-Borate phenylalanine content: 10%.

[0063] Example 6

[0064] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0065] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 44 μL of methacrylamide chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 10%.

[0066] Example 7

[0067] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0068] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DMF solution. 59 μL of methacrylic acid, 0.02 g of 1-hydroxybenzotriazole, and 0.15 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added, and the mixture was stirred at room temperature for 24 h. The product was then precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and the product was finally freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25; 4-Borate phenylalanine content: 10%.

[0069] Example 8

[0070] 13.7 g of tetraethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0071] 1.1 g of tetraethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 61 μL of triethylamine and 32 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 10%.

[0072] Example 9

[0073] 16.6 g of pentaethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0074] 1.3 g of pentaethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 54 μL of triethylamine and 28 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 10%.

[0075] Example 10

[0076] 19.5 g of hexaethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0077] 1.4 g of hexaethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-buten-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 41 μL of triethylamine and 23 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helical structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 10%.

[0078] Example 11

[0079] 25.3 g of octaethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0080] 1.6 g of octaethylene glycol monomethyl ether glutamate NCA and 0.081 g of 4-boronico-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-buten-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 35 μL of triethylamine and 19 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronico-phenylalanine content: 10%.

[0081] Example 12

[0082] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0083] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 7.0 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 50. 4-boronic acid-L-phenylalanine content: 10%.

[0084] Example 13

[0085] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0086] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 5.2 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 75. 4-boronic acid-L-phenylalanine content: 10%.

[0087] Example 14

[0088] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0089] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.081 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 3.6 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 100. 4-boronic acid-L-phenylalanine content: 10%.

[0090] Example 15

[0091] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0092] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.315 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 18.2 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 30%.

[0093] Example 16

[0094] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0095] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 0.736 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 25.5 μL of 3-buten-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 50%.

[0096] Example 17

[0097] 10.8 g of triethylene glycol monomethyl ether-L-glutamate and 1.25 g of 4-boronic acid-L-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0098] 1.0 g of triethylene glycol monomethyl ether-L-glutamate NCA and 1.715 g of 4-boronic acid-L-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 42.4 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-L-phenylalanine content: 70%.

[0099] Example 18

[0100] 10.8 g of triethylene glycol monomethyl ether-D-glutamate and 1.25 g of 4-boronic acid-D-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0101] 1.0 g of triethylene glycol monomethyl ether-D-glutamate NCA and 0.081 g of 4-boronic acid-D-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 38 μL of acryloyl chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-D-phenylalanine content: 10%.

[0102] Example 19

[0103] 10.8 g of triethylene glycol monomethyl ether-D-glutamate and 1.25 g of 4-boronic acid-D-phenylalanine were suspended in 300 mL of ultradry THF, respectively. 4.1 g and 0.89 g of triphosgene were added, respectively. The reaction mixture was reacted at room temperature for 6 h. The reaction solution was concentrated by rotary evaporation, precipitated in n-hexane solution, filtered, and dried to obtain the corresponding NCA monomers.

[0104] 1.0 g of triethylene glycol monomethyl ether-D-glutamate NCA and 0.081 g of 4-boronic acid-D-phenylalanine NCA were dissolved in 20 mL of ultra-dry DMF. 14.1 μL of 3-butene-1-amine was added. Ring-opening polymerization was carried out at 50 °C for 72 h. The product was precipitated in methyl tert-butyl ether solution, centrifuged, washed, and dried to obtain a random copolymer. 1.0 g of the random copolymer was dissolved in 40 mL of ultra-dry DCM, and 84 μL of triethylamine and 44 μL of methacrylamide chloride were added. The mixture was stirred at room temperature for 6 h. Then, the product was precipitated in methyl tert-butyl ether solution and centrifuged. Impurities were removed by dialysis, and finally, the product was freeze-dried to obtain a water-soluble polypeptide crosslinking agent that has an α-helix structure under neutral conditions and transforms into a random coil structure under alkaline conditions. Degree of polymerization: 25. 4-boronic acid-D-phenylalanine content: 10%.

[0105] The circular dichroism chromatograms of the water-soluble polypeptide crosslinking agents obtained in Examples 2 to 19 at physiological pH and high pH are similar to those in Example 1, θ 222 The pH-value graphs are similar to those in Example 1, and will not be listed one by one.

[0106] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A water-soluble polypeptide cross-linking agent, characterized in that, The water-soluble polypeptide crosslinking agent is a random copolymer of oligoethylene glycol monomethyl ether glutamate and 4-boronic acid phenylalanine, wherein both ends of the random copolymer are double-bonded.

2. The water-soluble polypeptide crosslinking agent as described in claim 1, characterized in that, The oligoethylene glycol monomethyl ether glutamate and 4-boronic acid phenylalanine are both L-configuration or both D-configuration; The degree of polymerization n of the oligoethylene glycol is 2 ≤ n ≤ 100; the degree of polymerization of the random copolymer is 10 to 150.

3. The water-soluble polypeptide crosslinking agent according to any one of claims 1 to 2, characterized in that, The water-soluble polypeptide crosslinking agent transforms from an α-helix structure to a random coil structure under alkaline conditions.

4. The water-soluble polypeptide crosslinking agent as described in claim 1, characterized in that, The general structural formula of the water-soluble polypeptide crosslinking agent is: Where x, y, and n are all integers greater than 0; R1 and R2 are keys, or R1 and R2 are independently selected from the keys R. 11 The following groups, substituted or unsubstituted: C1-C 10 Alkyl, C2-C 10 alkenyl, C1-C 10 alkoxy, or R 12 Substituted or unsubstituted C3-C8 cycloalkyl groups, wherein R 11 It is a halogen, C3-C8 cycloalkyl or hydroxyl group, R 12 It is a C1-C6 alkyl, halogen, or hydroxyl group; R3 and R4 are each independently selected from hydrogen, or each independently selected from halogenated, nitro, amino, R 11 Substituted or unsubstituted C1-C6 alkyl or alkoxy groups.

5. The method for preparing the water-soluble polypeptide crosslinking agent according to any one of claims 1 to 4, characterized in that, step include: S1. Oligoethylene glycol monomethyl ether glutamate and 4-boronic acid phenylalanine are reacted with triphosgene to generate the corresponding NCA monomers. S2. The two NCA monomers mentioned above are subjected to ring-opening polymerization using an initiator containing double bonds to obtain a random copolymer with double bonds at one end of the molecular chain. S3. The random copolymer is capped with a capping agent containing double bonds to obtain the water-soluble polypeptide crosslinking agent.

6. The method for preparing the water-soluble polypeptide crosslinking agent as described in claim 5, characterized in that, In step S2, the molar percentage of the monomer corresponding to the 4-boronicophenylalanine is 5% to 70%.

7. The method for preparing the water-soluble polypeptide crosslinking agent as described in claim 5, characterized in that, The initiator containing a double bond is selected from at least one of allylamine, 3-buten-1-amine, 4-penten-1-amine and 5-hexen-1-amine.

8. The method for preparing the water-soluble polypeptide crosslinking agent as described in claim 5, characterized in that, The double-bond-containing end-capping agent is selected from at least one of acrylic acid, acryloyl chloride, methacrylic acid, and methacryloyl chloride.

9. A smart biomedical material, characterized in that, The water-soluble polypeptide crosslinking agent includes the water-soluble polypeptide crosslinking agent according to any one of claims 1 to 4, or the water-soluble polypeptide crosslinking agent prepared according to the preparation method of the water-soluble polypeptide crosslinking agent according to any one of claims 5 to 8.

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