Preparation method of polyethylene glycol monomethyl ether-polyproline block copolymer rich in PP II

Abstract

The invention belongs to the technical field of high polymer materials, and relates to a preparation method of a polyethylene glycol monomethyl ether-polyproline block copolymer rich in PP II. The method aims at solving the problems that an existing natural PPII helical material is complex in extraction process, high in cost, large in batch difference and low in yield and has a pathogen pollution risk, a PPII helical conformation with a definite and stable structure is difficult to construct by a synthetic material, the biological activity and biocompatibility of the material are insufficient, and the cost is low. The high-end biomedical application requirements of targeted drug delivery, in-vivo long-acting circulation, tissue engineering and the like are difficult to meet. Polyethylene glycol monomethyl ether is activated by methylsulfonyl chloride and then reacts with ammonia water to prepare amino-terminated polyethylene glycol monomethyl ether; the preparation method comprises the following steps: by taking N-t-butyloxycarbonyl-L-proline (BOC-L-Pro) as a raw material, reacting with triphosgene in the presence of epoxypropane to prepare L-proline-N-carboxylic acid anhydride; under the protection of inert gas, amino-terminated polyethylene glycol monomethyl ether is used as an initiator, and the amino-terminated polyethylene glycol monomethyl ether and L-proline-N-carboxylic acid anhydride are subjected to ring-opening polymerization in an organic solvent to prepare the target block copolymer.

Description

Technical Field

[0001] This invention relates to the field of polymer materials and provides a method for preparing a polyethylene glycol monomethyl ether-polyproline block copolymer rich in PPⅡ. Background Technology

[0002] In recent years, with the rapid development of biomedical engineering and nanomedicine, biomedical polymer materials with specific advanced structures have shown great application potential in disease diagnosis and treatment. Among them, polyproline type II (PPII) helices, as a left-handed helical protein secondary structure, are widely found in collagen, antimicrobial peptides, and certain natural proteins with cell-penetrating capabilities. Studies have shown that the PPII helical structure plays a crucial role in mediating physiological processes such as protein-protein interactions, cell surface receptor recognition, and signal transduction. In the field of disease treatment, materials with PPII helical conformations exhibit excellent resistance to protein nonspecific adsorption, extremely low immunogenicity, and efficient transmembrane transport potential, making them irreplaceable in targeted drug delivery, long-acting circulating carriers in vivo, and tissue engineering scaffolds.

[0003] However, extracting and separating materials containing stable PPII helical structures from natural biological tissues (such as animal cartilage and cortex) faces insurmountable technical bottlenecks in practical applications. First, natural extracts are complex in composition, and their separation and purification processes are cumbersome, costly, and have extremely low yields, making large-scale preparation difficult. Second, naturally derived biomolecules exhibit significant batch-to-batch structural variations and uncontrollable molecular weight distribution, and may carry harmful pathogens, triggering immune rejection responses in the human body. This makes it difficult to meet the stringent requirements of modern clinical medicine for pharmaceutical excipients and implantable materials that require clearly defined components, controllable structures, and high safety.

[0004] Therefore, it is of great significance to artificially prepare polymer materials with a well-defined and stable PPII helical structure through chemical synthesis. Summary of the Invention

[0005] The purpose of this invention is to address the problems in existing technologies, such as the complex and costly extraction process of naturally derived polyproline type II (PPII) helical materials, significant batch-to-batch variations, low yields, and the risk of pathogen contamination. Furthermore, traditional synthetic medical polymers struggle to construct well-defined and stable PPII helical conformations, resulting in materials lacking specific biological activity (such as resistance to non-specific protein adsorption, low immunogenicity, and efficient transmembrane transport) and insufficient biocompatibility. These issues make it difficult to meet the stringent requirements of high-end biomedical applications such as targeted drug delivery, long-term in vivo circulation, and tissue engineering, which demand well-defined material composition, controllable structure, precise function, and clinical safety.

[0006] To achieve the above objectives, the present invention employs the following technical means:

[0007] This invention provides a method for preparing a polyethylene glycol monomethyl ether-polyproline block copolymer rich in PPⅡ, comprising the following steps:

[0008] Step (1): Using polyethylene glycol monomethyl ether mPEG-OH as the starting material, after activation with methanesulfonyl chloride, it reacts with ammonia to obtain amino-terminated polyethylene glycol monomethyl ether mPEG. 22 -NH2;

[0009] Step (2): Using N-tert-butoxycarbonyl-L-proline (BOC-L-Pro) as a raw material, react with triphosgene in the presence of propylene oxide to prepare L-proline-N-carboxylic anhydride (L-Pro-NCA).

[0010] Step (3): Under the protection of an inert gas, the mPEG is... 22 Using -NH2 as an initiator, L-proline-N-carboxylic anhydride is subjected to ring-opening polymerization in an organic solvent to obtain a polyethylene glycol monomethyl ether-polyproline block copolymer with the molecular formula of Formula I. The copolymer has a polymer with a significant polyproline type II (PPII) helical conformation.

[0011] (Formula I)

[0012] In the above scheme, step (1) includes the following steps:

[0013] Step (1.1): Dissolve polyethylene glycol monomethyl ether mPEG-OH completely in 250 mL of double-distilled deionized water, extract with dichloromethane multiple times, precipitate with frozen diethyl ether, filter and dry to constant weight;

[0014] Step (1.2): Dissolve the product obtained in step (1.1) in 600 mL of freshly distilled dichloromethane and toluene in an ice bath, add 14.7 mL of triethylamine, stir until fully cooled, slowly add 3.7 mL of methanesulfonyl chloride under vigorous stirring, react for 12 hours under nitrogen protection, filter to remove the light yellow precipitate, remove dichloromethane by rotary evaporation, filter again to remove the precipitate, precipitate with frozen diethyl ether and dry to constant weight. The yellow precipitate is a thioolefin polymer.

[0015] Step (1.3): Add 800 mL of ammonia water to the product obtained in step (1.2), react under vigorous stirring for 72 hours, extract with dichloromethane multiple times, then precipitate with frozen diethyl ether and dry to constant weight to obtain a white solid powder of amino-terminated polyethylene glycol monomethyl ether (mPEG). 22 -NH2.

[0016] In the above scheme, step (2) includes the following steps:

[0017] Step (2.1): Take 5 g of BOC-L-proline into a 250 mL flask, then add acetonitrile and propylene oxide and stir, and finally add triphosgene. Stir the reaction under reflux for 4.5 h.

[0018] Step (2.2): After the reaction is complete, the solvent is removed by rotary evaporation of the reaction solution to obtain L-Pro-NCA with the molecular formula as shown in Formula II and then frozen overnight;

[0019] (Formula II)

[0020] Step (2.3): Take out the frozen yellow oily substance, add 25 mL of freshly distilled tetrahydrofuran, stir for 0.5 h, add 250 mL of frozen petroleum ether, freeze for several days, filter and dry to constant weight to obtain the yellow product.

[0021] In the above scheme, step (3) includes the following steps:

[0022] 1 mmol of mPEG 22 -NH2 was added to a 100 mL single-necked flask and purged with nitrogen. Then, 6 mL of freshly distilled trichloromethane and DMF mixture was added and stirred until dissolved under nitrogen protection. 15 mmol of L-proline-N-carboxylic anhydride (L-Pro-NCA) was added and the mixture was reacted at room temperature for 24 h under nitrogen protection. After the reaction was completed, the mixture was precipitated with frozen diethyl ether, filtered, and dried under vacuum to constant weight to obtain a polymer with a polyproline type II (PPII) helical conformation.

[0023] Because the present invention employs the above-mentioned technical means, it has the following beneficial effects:

[0024] This invention utilizes monoamino polyethylene glycol monomethyl ether (mPEG) 22 Using NH2 as a macromolecular initiator, L-proline-N-carboxylic anhydride (L-Pro-NCA) undergoes ring-opening polymerization. After precipitation purification and drying, a polymer rich in the PPⅡ conformation is obtained. This solves the technical problems in existing technologies, such as the lack of specific bioactive secondary structures in traditional medical polymer materials, and the cumbersome process, high cost, large batch-to-batch variability, and risk of immune rejection in extracting proteins containing this structure from natural tissues. This achieves the effect of artificially controllable and large-scale preparation of polymer materials with biomedical application value. Attached Figure Description

[0025] Figure 1 It is a polyethylene glycol monomethyl ether-polyproline block copolymer (mPEG) 22 -P15 The synthesis reaction of ).

[0026] Figure 2 Polyethylene glycol monomethyl ether (mPEG) with one-sided amino end capping by a macromolecular initiator 22 The synthesis reaction of -NH2).

[0027] Figure 3 This is the synthesis reaction of L-proline-N-carboxylic anhydride (L-Pro-NCA).

[0028] Figure 4 Polyethylene glycol monomethyl ether (mPEG) with one-sided amino end capping by a macromolecular initiator 22 NMR of -NH2) 1 H spectrum.

[0029] Figure 5 NMR of L-proline-N-carboxylic anhydride (L-Pro-NCA) 1 In the H spectrum, a represents the chemical shift of the proton H at the junction of the pyrrole ring and the anhydride ring of L-Pro-NCA; b1 and b2 represent the chemical shifts of the two proton Hs belonging to the adjacent N on the pyrrole ring; c2, c2, d1, and d2 represent the chemical shifts of -CH2- on the pyrrole ring.

[0030] Figure 6 It is a polyethylene glycol monomethyl ether-polyproline block copolymer (mPEG) 22 -P 15 ) NMR 1 H spectrum.

[0031] Figure 7 It is a polyethylene glycol monomethyl ether-polyproline block copolymer (mPEG) 22 -P 15 Circular dichroism (CD) spectrum of aqueous solution. Detailed Implementation

[0032] The embodiments of the present invention will be described in detail below. Although the present invention will be described and illustrated in conjunction with some specific embodiments, it should be noted that the present invention is not limited to these embodiments. On the contrary, any modifications or equivalent substitutions made to the present invention should be covered within the scope of the claims of the present invention.

[0033] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art will understand that the present invention can be practiced without these specific details.

[0034] Example 1

[0035] Step 1: Preparation of single-sided end-capped polyethylene glycol monomethyl ether: mPEG22 The preparation steps of -NH2 are as follows: 0.08 parts by weight of mPEG-OH are fully dissolved in one part of double-distilled deionized water, extracted multiple times with dichloromethane, precipitated with 1.43 parts of frozen diethyl ether, filtered, and dried to constant weight. The product is dissolved in a mixture of 2.13 parts of freshly distilled dichloromethane and 0.69 parts of toluene and placed in an ice bath. Then, 0.04 parts of triethylamine are added, stirred until fully cooled, and 0.02 parts of methanesulfonyl chloride are slowly added dropwise under vigorous stirring. After reacting for 12 hours under nitrogen protection, the pale yellow precipitate is removed by filtration, dichloromethane is removed by rotary evaporation, the precipitate is removed again by filtration, precipitated with 1.43 parts of frozen diethyl ether, and dried to constant weight. 2.91 parts of ammonia are added to the product, and after reacting for 72 hours under vigorous stirring, it is extracted multiple times with dichloromethane, precipitated with frozen diethyl ether, and dried to constant weight to obtain a white solid powder.

[0036] Step 2: The preparation steps of L-proline-N-carboxylic anhydride (L-Pro-NCA) are as follows: Take 1 part of BOC-L-Pro into a 250ml flask, then add 7.86 parts of acetonitrile and 3.32 parts of propylene oxide and stir. Finally, add 0.69 parts of triphosgene and stir under reflux for 4.5h. After the reaction is completed, remove the solvent by rotary evaporation to obtain a yellow oily substance and freeze it overnight. Take out the frozen yellow oily substance, add 4.45 parts of freshly distilled tetrahydrofuran, stir for 0.5h, add 33.5 parts of frozen petroleum ether, freeze for several days, filter and dry to constant weight to obtain the yellow product.

[0037] Step 3: Synthesis of polyethylene glycol monomethyl ether-polyproline

[0038] Add 1 part mPEG to a 100ml single-necked flask 22 -NH2 was replaced with nitrogen gas, followed by the addition of a mixed solution of 5.92-8.88 parts of freshly distilled chloroform and 1.89-3.78 parts of freshly distilled DMF. The mixture was stirred under nitrogen protection until dissolved, and then 0.99-2.12 parts of L-proline-NCA were added. The mixture was reacted at room temperature under nitrogen protection for 24 hours. The solid was then precipitated with 142.6 parts of frozen diethyl ether, filtered, and dried under vacuum to constant weight to obtain a yellow solid.

[0039] Example 2

[0040] Add 1 part mPEG to a 100ml single-necked flask 22-NH2 was purged with nitrogen, followed by the addition of a mixed solution of 5.92 parts freshly distilled chloroform and 1.89 parts freshly distilled DMF. The mixture was stirred until dissolved under nitrogen protection, then 0.99 parts L-proline-NCA were added, and the reaction was carried out at room temperature for 24 hours under nitrogen protection. The solution was then precipitated with 142.6 parts of frozen diethyl ether, filtered, and dried under vacuum to constant weight to obtain a yellow solid. Example 2

[0041] Same as in Example 1, except that the parameter 5.92 parts of freshly distilled chloroform was 7.40 parts of freshly distilled chloroform, the parameter 1.89 parts of freshly distilled DMF was 2.83 parts of freshly distilled DMF, and the parameter 0.99 parts of L-proline-NCA was 1.41 parts of L-proline-NCA;

[0042] Example 3

[0043] Same as in Example 1, except that the parameter 5.92 parts of freshly distilled chloroform was 8.88 parts of freshly distilled chloroform, the parameter 1.89 parts of freshly distilled DMF was 3.78 parts of freshly distilled DMF, and the parameter 0.99 parts of L-proline-NCA was 2.12 parts of L-proline-NCA;

[0044] Data and effect analysis, Figures 4-7 Corresponding Example 3

[0045] 1. For example Figure 4 The single-sided end-capped polyethylene glycol monomethyl ether (mPEG) shown 22 -NH2) nuclear magnetic resonance 1 H-chromatogram, using deuterated chloroform as solvent, mPEG at room temperature 22 -OH and mPEG 22 The chemical shift δ (ppm) of each proton hydrogen in -NH2 is as follows: Figure 4 As shown. The proton hydrogen chemical shift of methoxy CH3-O- is at 3.38 ppm; the peak at 3.65 ppm is attributed to the proton hydrogen chemical shift of the methylene-CH2-CH2- in the main chain. Compared with mPEG-OH, mPEG-NH2 shows a triplet at 2.89 ppm, which is attributed to the proton hydrogen peak of the methylene-CH2-NH2 directly linked to the terminal amino group -NH2.

[0046] 2. For example Figure 5 The nuclear magnetic resonance of L-Pro-NCA shown 1 H-map

[0047] Using deuterated DMSO as a solvent, the obtained 1 H-NMR spectra (such as H-NMR spectra) Figure 5This data can also be corroborated by the data obtained from Fourier transform infrared spectroscopy. The value at 4.33 ppm belongs to the proton H at the junction of the L-Pro-NCA pyrrole ring and the anhydride ring; 3.80 ppm and 3.34 ppm belong to the two proton H adjacent to the N on the pyrrole ring; and 2.33 ppm, 2.22 ppm, 2.13 ppm, and 1.96 ppm belong to the four proton H at positions c and d on the pyrrole ring, respectively. Therefore, 1 The results of H-NMR showed that L-Pro-NCA was successfully prepared.

[0048] 3. For example Figure 6 Nuclear magnetic resonance of polyethylene glycol monomethyl ether-polyproline 1 H-map

[0049] Polyethylene glycol monomethyl ether-polyproline (hereinafter referred to as mPEG) with different block lengths (7, 10, 15) 22 -P7, mPEG 22 -P 10 mPEG 22 -P 15 ) by mPEG 22 L-Pro-NCA was obtained by ring-opening polymerization initiated by -NH2. Polyethylene glycol monomethyl ether-polyproline compounds with different block lengths were obtained at room temperature using deuterated trifluoroacetic acid as a solvent. 1 H-NMR spectra (such as H-NMR spectra) Figure 6 (As shown). 3.88 ppm (peak a) belongs to the CH2 resonance peak (4H, CH2-CH2-) in the mPEG chain, 3.54 ppm (peak b) belongs to the methoxy proton resonance peak (3H, CH3-O-) in the mPEG chain, and 4.90 ppm (peak c) belongs to the proton hydrogen on the pyrrole ring of the proline fragment (1H, CH-).

[0050] 4. For example Figure 7 Circular dichroism of polyethylene glycol monomethyl ether-polyproline

[0051] like Figure 7 As shown, the spectrum contains a strong negative peak at 205 nm and a weak positive peak at 227 nm. This spectral feature is the PPⅡ conformation in the main secondary structure of polyproline. This conformation is mainly found in aqueous solutions and is the most common and important secondary structure in aqueous solutions of polyproline polymers.

Claims

1. A method for preparing a polyethylene glycol monomethyl ether-polyproline block copolymer rich in PPⅡ, characterized in that, Includes the following steps: Step (1): Using polyethylene glycol monomethyl ether mPEG-OH as the starting material, after activation with methanesulfonyl chloride, it reacts with ammonia to obtain amino-terminated polyethylene glycol monomethyl ether mPEG. 22 -NH2; Step (2): Using BOC-L-Pro as raw material, react with triphosgene in the presence of propylene oxide to prepare L-proline-N-carboxylic anhydride; Step (3): Under the protection of an inert gas, the mPEG is... 22 -NH2 is used as an initiator to carry out a ring-opening polymerization reaction with L-proline-N-carboxylic anhydride in an organic solvent to obtain a polyethylene glycol monomethyl ether-polyproline block copolymer with the molecular formula of Formula I. The copolymer has a polymer with a significant polyproline type II helical conformation. (Formula I).

2. The method according to claim 1, characterized in that: Step (1) includes the following steps: Step (1.1): Dissolve polyethylene glycol monomethyl ether mPEG-OH completely in 250 mL of double-distilled deionized water, extract with dichloromethane multiple times, precipitate with frozen diethyl ether, filter and dry to constant weight; Step (1.2): Dissolve the product obtained in step (1.1) in 600 mL of freshly distilled dichloromethane and toluene in an ice bath, add 14.7 mL of triethylamine, stir until fully cooled, slowly add 3.7 mL of methanesulfonyl chloride under vigorous stirring, react for 12 hours under nitrogen protection, filter to remove the light thioolefin polymer, remove dichloromethane by rotary evaporation, filter again to remove the precipitate, precipitate with frozen diethyl ether and dry to constant weight; Step (1.3): Add 800 mL of ammonia water to the product obtained in step (1.2), react under vigorous stirring for 72 hours, extract with dichloromethane multiple times, then precipitate with frozen diethyl ether and dry to constant weight to obtain a white solid powder of amino-terminated polyethylene glycol monomethyl ether (mPEG). 22 -NH2.

3. The method according to claim 1, characterized in that: Step (2) includes the following steps: Step (2.1): Take 5 g of BOC-L-Pro into a 250 mL flask, then add acetonitrile and propylene oxide and stir, and finally add triphosgene. Stir the reaction under reflux for 4.5 h. Step (2.2): After the reaction is complete, the solvent is removed by rotary evaporation of the reaction solution to obtain L-proline-N-carboxylic anhydride with the molecular formula as shown in Formula II and then frozen overnight; (Formula II). Step (2.3): Take out the frozen yellow oily substance, add 25 mL of freshly distilled tetrahydrofuran, stir for 0.5 h, add 250 mL of frozen petroleum ether, freeze for several days, filter and dry to constant weight to obtain the yellow product.

4. The method according to claim 1, characterized in that: Step (3) includes the following steps: 1 mmol of mPEG 22 -NH2 was added to a 100 mL single-necked flask and purged with nitrogen. Then, 6 mL of freshly distilled trichloromethane and DMF mixture was added and stirred until dissolved under nitrogen protection. 15 mmol of L-proline-N-carboxylic anhydride was added and the mixture was reacted at room temperature for 24 h under nitrogen protection. After the reaction was complete, the product was precipitated with frozen diethyl ether, filtered, and dried under vacuum to constant weight to obtain a polymer with a polyproline type II helical conformation.

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