CN100509862C - Synthesis process of beta-cyclodextrin-poly-L-glutamic acid-benzyl ester grafted copolymer - Google Patents

Abstract

The invention relates to a method for synthesizing β-cyclodextrin-poly L-glutamic acid-benzyl ester graft copolymer. A certain amount of amino β-cyclodextrin and γ-benzyl ester L-glutamic acid cyclic anhydride are mixed in N,N-dimethylformamide and reacted at a reaction temperature of 25°C under nitrogen protection for 60-80 hours , the reaction mixture was poured into deionized water to precipitate, washed with distilled water, and dried in vacuum at 100°C to obtain the target product. The invention improves the water solubility of poly-L-glutamic acid, and at the same time, the hydrophobic poly-L-glutamic acid-benzyl ester segment can regulate the biodegradation speed and cycle of hydrophilic β-cyclodextrin, by changing each segment The content of the copolymer can control the solubility and degradability. The copolymer of the present invention can assemble hydrophobic drug molecules into its hydrophobic core, has the effect of improving the circulation time of drugs in the blood, and has good applications in biomedical fields such as controlled release of drugs and targeted drug delivery. prospect.

Description

Synthetic method of β-cyclodextrin-poly L-glutamic acid-benzyl ester graft copolymer

Technical field: The present invention relates to a method for synthesizing β-cyclodextrin-polyamino acid graft copolymer, in particular to a method for synthesizing β-cyclodextrin-poly-L- Method of glutamic acid-benzyl ester graft copolymer.

Background technology: Polyamino acids are a class of biodegradable polymers with low toxicity, good biocompatibility, and easy absorption and metabolism by the body. Therefore, they are widely used in medical fields such as controlled drug release and tissue engineering. However, the water solubility of its homopolymer is poor, and its application has certain limitations.

β-cyclodextrin (β-Cyclodextrin, referred to as β-CD) is produced by fermentation of starch and consists of seven D-(+)-glucopyranose, each of which takes a chair conformation, through α- The 1,4-glycosidic linkages connect end to end to form a ring-shaped molecule with a slightly truncated cone-shaped cylindrical structure.

The outer side of the β-CD molecule is hydrophilic because it is covered with hydroxyl groups. There are two rings of hydrogen atoms on carbon atoms inside the conical cylinder, and a ring of acetal oxygen atoms (ether rings) in the middle of the carbon-hydrogen bond. It has strong hydrophobicity, can contain various guest molecules, and has the reputation of molecular capsule. β-CD has good biocompatibility and has been widely used in many fields such as medicine and food. For example, hydrophobic drugs can be included to improve drug stability and solubility, prolong drug efficacy, and improve drug utilization. To sum up, the copolymerization of β-cyclodextrin and hydrophobic polyamino acid can improve the water solubility of polyamino acid, and obtain a new type of amphiphilic polymer with special properties, and the related content has not been reported in the literature.

Summary of the invention: The purpose of the present invention is to overcome the disadvantage of poor solubility of polyamino acid in water, and make it into a copolymer with β-cyclodextrin with good water solubility, so as to obtain energy by changing the content of each chain segment. Solubility controlled copolymer.

Structural formula of β-cyclodextrin as backbone:

Structural formula of polyamino acids as grafted chains:

Among them, R is:

In order to achieve the above object, the present invention provides a synthetic method of β-cyclodextrin-poly L-glutamic acid-benzyl ester graft copolymer, which is characterized in that a certain amount of amino β-cyclodextrin, γ-benzyl ester After mixing L-glutamic acid anhydride (NCA) in N,N-dimethylformamide (DMF), stir the reaction at a reaction temperature of 25°C under nitrogen protection for 60-80 hours, and pour the reaction mixture into deionized water Precipitate, filter with suction, wash the precipitate with distilled water, and dry under vacuum at 100°C to obtain the target product. in:

The molar ratio of β-cyclodextrin to amino acid anhydride (NCA) is 1:10~70;

The N,N-dimethylformamide used per gram of γ-benzyl ester-L-glutamic acid anhydride is 70-80mL;

The volume ratio of N,N-dimethylformamide to deionized water is 1:3~5

The reaction mechanism of the present invention is as follows: first, tosyl β-cyclodextrin is obtained through esterification reaction of β-cyclodextrin and p-toluenesulfonyl chloride; Dextrin; finally, ammonized β-cyclodextrin is obtained by aminolysis reaction; L-glutamic acid-benzyl ester is obtained by esterification of L-glutamic acid and benzyl alcohol, and then cyclized with solid phosgene Obtain γ-benzyl ester-L-glutamic acid anhydride (NCA); finally, the NH _group in the amino β-cyclodextrin molecule attacks the 5-carbonyl carbon atom of the NCA monomer to form intermediate A, which The intermediate quickly eliminates CO ₂ to generate intermediate B, and then B continues to react with NCA monomer to form a graft copolymer.

The inventor synthesized the β-cyclodextrin-polyamino acid graft copolymer through laboratory experiments, and confirmed the product through infrared spectroscopy and nuclear magnetic resonance.

Experimental example 1

Material: L-glutamic acid (AR, Sinopharm Chemical Reagent Co., Ltd.)

Benzyl alcohol (AR, Sinopharm Chemical Reagent Co., Ltd.)

Solid phosgene (AR, Haining Zhonglian Chemical Co., Ltd.)

β-cyclodextrin (AR, Sinopharm Chemical Reagent Co., Ltd.)

p-Toluenesulfonyl chloride (AR, Sinopharm Chemical Reagent Co., Ltd.)

Sodium azide (AR, Sinopharm Chemical Reagent Co., Ltd.)

N, N-Dimethylformamide (DMF) (AR, Sinopharm Chemical Reagent Co., Ltd.)

Triphenylphosphine (AR, Sinopharm Chemical Reagent Co., Ltd.)

Pyridine (AR, Sinopharm Chemical Reagent Co., Ltd.)

Potassium iodide (AR, Sinopharm Chemical Reagent Co., Ltd.)

Instrument: PE Spectrum one Fourier transform infrared spectrometer

Varian XL-300 NMR spectrometer

MalvernNano ZS Nanoparticle Size and Zeta Potential Analyzer

(1) Synthesis of L-glutamic acid-benzyl ester:

Weigh 11.1g of L-glutamic acid into a 250mL three-neck flask, add 53mL of benzyl alcohol and 18mL of hydrobromic acid. Then heat slowly under magnetic stirring, and the reaction temperature is controlled at about 70°C. After the L-glutamic acid is completely dissolved (about 1h), the reaction mixture is cooled to 30°C, and then poured into a mixture of 22mL pyridine and 147mL 95% In the mixed solution prepared by ethanol, place it at 3°C for 12 hours to make it fully precipitate. After suction filtration, the precipitate was washed with 30 mL of ethanol and 30 mL of ether, respectively, and the obtained white solid was the crude product. The crude product was recrystallized with 5% ethanol and dried under vacuum at 50°C to obtain 5.658g of product with a yield of 31.6%. The melting point of the product was 172-174°C.

(2) Synthesis of γ-benzyl ester-L-glutamic acid anhydride in the ring:

Weigh 5.658g of freshly prepared L-glutamic acid-benzyl ester into a 250mL three-necked flask equipped with a reflux condenser, a thermometer and an alkali absorption device, then add 75mL of anhydrous tetrahydrofuran, raise the temperature to 50°C, and add 8.5 g solid phosgene. After the reaction suspension became clear (about 40 min), nitrogen gas was passed for 30 min to remove the hydrogen chloride and remaining phosgene generated by the reaction. Part of the tetrahydrofuran was removed by rotary evaporation of the reaction mixture, cooled to room temperature, poured into 150 mL of petroleum ether, placed at -20°C for 12 h, and white needle-like crystals were obtained by suction filtration as the crude product. The crude product was dissolved in 30 mL of tetrahydrofuran, filtered, and the filtrate was poured into 70 mL of petroleum ether to obtain a precipitate. The precipitate was filtered, and after natural drying, 3.332 g of white needle-like crystals were obtained, which was the product. The yield is 53.1%, and the melting point is 95-96°C. The reaction formula is as follows:

(3) Synthesis of amino β-cyclodextrin:

Add 10g of β-cyclodextrin, 2.5g of p-toluenesulfonyl chloride and 140mL of pyridine into a 250mL three-necked flask, and under nitrogen protection, control the reaction temperature at 40°C and stir for 4h. Concentrate the reaction mixture with a rotary evaporator, then pour it into 100 mL of acetone for precipitation, filter it with suction after the precipitation is complete, wash the precipitate with acetone, and dry it in vacuum at 50°C to obtain 5.454 g of white powder, which is tosyl β-cyclodextrin. rate of 48.6%.

Add 5.454g of tosyl β-cyclodextrin, 2.00g of sodium azide, 0.20g of potassium iodide and 40mL of DMF into a 250mL three-neck flask, under nitrogen protection, control the reaction temperature at 80°C and stir for 24h. The DMF in the reaction mixture was evaporated to dryness, 100 mL of deionized water was added to dissolve the crude product, and then it was poured into 300 mL of ethanol to precipitate, filtered with suction, and dried in vacuum at 50°C to obtain 4.060 g of azide β-cyclodextrin, the yield 77.6%.

Add 4.060g of azidolated β-cyclodextrin, 1.80g of triphenylphosphine, 80mL of DMF, and 15mL of ammonia water into the reactor, and stir and react at 40°C for 24h under the protection of nitrogen. The reaction mixture was poured into 200 mL of acetone for precipitation, suction filtered, and vacuum-dried at 50° C. to obtain 3.568 g of aminated β-cyclodextrin, with a yield of 87.9%. The product was confirmed by infrared spectroscopy and nuclear magnetic resonance. The reaction formula is as follows:

(4) Synthesis of β-cyclodextrin-poly L-glutamic acid-benzyl ester graft copolymer (β-CDPBLG1):

Put 0.431g (0.38mmol) of amino β-cyclodextrin and 1.0g (3.8mmol) of γ-benzyl L-glutamic acid cyclic anhydride into the reaction flask, add 75mL of anhydrous DMF, at 25°C, nitrogen protection The reaction was stirred for 72h. The reaction mixture was poured into 300 mL deionized water to obtain a precipitate. Suction filtration, washing the precipitate with distilled water, and vacuum drying at 100°C gave the target product with a yield of 43.6%. The reaction formula is as follows:

The product was confirmed by infrared spectroscopy, nuclear magnetic resonance and the like. In the IR spectrum, the two characteristic absorptions at 600cm ^-1 ~ 800cm ^-1 are the characteristic absorptions of the benzene ring in the poly L-glutamic acid-benzyl ester segment; the two absorptions at 1600cm ^-1 and 1500cm ^-1 The peaks are two characteristic absorptions of the amide bond in the poly-L-glutamic acid-benzyl ester segment; commonly referred to as the amide I band and the amide II band; the absorption peak at ¹⁷⁰⁰ cm is characteristic of the carbonyl group on the ester bond in the benzyl ester Absorption: The absorption peak around 3200cm ^-1 is the characteristic absorption of the hydroxyl group in β-cyclodextrin and the amino group in poly-L-glutamic acid-benzyl ester. In the spectrogram, the characteristic absorption of the two segments of β-cyclodextrin and poly-benzyl glutamate can be observed, while the absorption peaks of the two carbonyl groups in the NCA molecule at 1855cm ^-1 and 1785cm ^-1 disappear, Preliminary indications were that a copolymer was formed.

In ^{the 1} H NMR spectrum, about δ=7.2ppm is the chemical shift of H on the benzene ring on the poly-L-glutamic acid-benzyl ester segment; about δ=5.0ppm is the methylene group (CH ₂ ) in the benzyl group The chemical shift of H on the top; the absorption peak at δ=2.4ppm is the chemical shift of H on the -CH ₂ CH ₂ -group in poly-L-glutamic acid benzyl ester; δ=3.9 is poly-L-glutamic acid benzyl The chemical shift of methine (CH)H in the ester segment; δ=8.0ppm is the chemical shift of H on the NH in the poly-L-benzyl glutamate segment; the absorption peak at δ=3.3～3.6ppm is the chemical shift of H at position 2, 3, 4, 5, and 6 in β-cyclodextrin molecule; the absorption peak at δ=4.8ppm is the chemical shift of H at position 1 in β-cyclodextrin molecule. It can be seen from the NMR spectrum of the polymer that the characteristic peaks of the corresponding groups in the β-cyclodextrin chain segment and the poly-L-benzyl glutamate chain segment all exist. Based on IR and ¹ H NMR analysis, it can be confirmed that the product is β-cyclodextrin-poly L-glutamic acid-benzyl ester graft copolymer.

The beneficial effects of the present invention are: through the present invention, the copolymerization of β-cyclodextrin and hydrophobic polyamino acid (poly-L-glutamic acid-benzyl ester) is realized, and a novel β-cyclodextrin with amphiphilic structure with special properties is obtained. Dextrin-poly-L-glutamic acid-benzyl ester graft copolymer. This copolymer can not only improve the water solubility of poly-L-glutamic acid-benzyl ester, but also the hydrophobic poly-L-glutamic acid-benzyl ester segment can also regulate the biodegradation rate and Period, so by changing the content of each segment to obtain a copolymer that can control solubility and degradability. The β-cyclodextrin-poly L-glutamic acid-benzyl ester graft copolymer prepared by the invention is an amphiphilic copolymer, which can self-assemble in aqueous solution to form micelles with a hydrophobic inner core and a hydrophilic outer shell. This copolymer micelle can assemble hydrophobic drug molecules into its hydrophobic core, which has the effect of improving the circulation time of drugs in the blood, and has good applications in biomedical fields such as controlled release of drugs and targeted drug delivery. Application prospects.

Description of drawings:

Fig. 1 is the micelle particle size distribution figure that the synthetic β-CDPBLG1 in Experimental Example 1 forms;

Fig. 2 is the micelle size distribution figure that the synthetic β-CDPBLG2 in embodiment 4 forms;

FIG. 3 is a particle size distribution diagram of micelles formed by β-CDPBLG3 synthesized in Example 5. FIG.

Detailed ways:

The synthesis of embodiment 1L-glutamic acid-benzyl ester:

Weigh 11.1g of L-glutamic acid into a 250mL three-neck flask, add 53mL of benzyl alcohol and 18mL of hydrobromic acid. Then heat slowly under magnetic stirring, and the reaction temperature is controlled at about 70°C. After the L-glutamic acid is completely dissolved (about 1h), the reaction mixture is cooled to 30°C, and then poured into a mixture of 22mL pyridine and 147mL95% ethanol In the prepared mixed solution, place it at 3°C for 12 hours to allow it to fully precipitate. The precipitate was suction-filtered, washed with 30 mL ethanol and 30 mL ether, respectively, and the obtained white solid was the crude product. The crude product was recrystallized with 5% ethanol and dried under vacuum at 50°C to obtain 5.658g of product with a yield of 31.6%. The melting point of the product was 172-174°C.

Synthesis of anhydride in the ring of embodiment 2 gamma-benzyl ester L-glutamic acid:

Weigh 5.658g of freshly prepared L-glutamic acid-benzyl ester into a 250mL three-necked flask, which is equipped with a reflux condenser, a thermometer, and an alkali absorption device. Add 75mL of anhydrous tetrahydrofuran, raise the temperature to 50°C, add 8.5g of solid phosgene under magnetic stirring, and after the reaction suspension becomes clear (about 40min), pass nitrogen gas for 30min to remove the hydrogen chloride and remaining phosgene generated by the reaction. Part of the tetrahydrofuran was removed by rotary evaporation of the reaction mixture, cooled to room temperature, poured into 150 mL of petroleum ether, placed at -20°C for 12 h, and white needle-like crystals were obtained by suction filtration as the crude product. The crude product was dissolved in 30 mL of tetrahydrofuran, filtered, and the filtrate was poured into 70 mL of petroleum ether to obtain a precipitate. The precipitate was filtered, and after natural drying, 3.332 g of white needle-like crystals were obtained, which was the product. The yield is 53.1%, and the melting point is 95-96°C. The product is confirmed by infrared spectrum and nuclear magnetic resonance.

Embodiment 3 The synthesis of amino β-cyclodextrin:

Add 10g of β-cyclodextrin, 2.5g of p-toluenesulfonyl chloride and 140mL of pyridine into a 250mL three-necked flask, and stir at 40°C for 4h under nitrogen protection. Concentrate the reaction mixture with a rotary evaporator, then pour it into 100mL of acetone to precipitate, filter it with suction after the precipitation is complete, wash the precipitate with acetone, and dry it in vacuum at 50°C to obtain a white powder, which is aminoβ-cyclodextrin, with a product weight of 5.454g. Yield 48.6%.

Add 5.454g of tosyl β-cyclodextrin, 2.00g of sodium azide, 0.20g of potassium iodide and 40mL of DMF into a 250mL three-necked flask, and stir and react at 80°C for 24h under nitrogen protection. The DMF in the reaction mixture was evaporated to dryness, 100 mL of deionized water was added to dissolve the crude product, precipitated with 300 mL of ethanol, filtered with suction, and dried under vacuum at 50°C to obtain 4.060 g of azide β-cyclodextrin with a yield of 77.6%.

Add 4.060g of β-cyclodextrin azide, 1.80g of triphenylphosphine, 80mL of DMF and 15mL of ammonia water into the reaction flask, and stir and react at 40°C for 24h under the protection of nitrogen. The reaction mixture was poured into 200 mL of acetone for precipitation, suction filtered, and vacuum-dried at 50° C. to obtain 3.568 g of aminated β-cyclodextrin, with a yield of 87.9%. The product was confirmed by infrared spectroscopy and nuclear magnetic resonance.

The synthesis of embodiment 4β-cyclodextrin-poly-L-benzyl glutamate graft copolymer (β-CDPBLG2):

Put 0.144g (0.127mmol) of amino β-cyclodextrin and 1.0g (3.8mmol) of γ-benzyl L-glutamic acid cyclic anhydride into the reaction flask, add 75mL of anhydrous DMF, at 25 ℃, nitrogen protection The reaction was stirred for 72h. The reaction mixture was poured into 300 mL deionized water to obtain a precipitate. Suction filtration, washing the precipitate with distilled water, and vacuum drying at 100°C gave the target product with a yield of 40.34%. The product was confirmed by infrared spectroscopy and nuclear magnetic resonance.

Example 5 Synthesis of β-cyclodextrin-poly-L-glutamic acid-benzyl ester graft copolymer (β-CDPBLG3):

Put 0.086g (0.0758mmol) of amino β-cyclodextrin and 1.0g (3.8mmol) of γ-benzyl ester L-glutamic acid cyclic anhydride into the reaction flask, add 80mL of anhydrous DMF, at 25 ℃, nitrogen protection The reaction was stirred for 60h. The reaction mixture was poured into 400 mL deionized water to obtain a precipitate. Suction filtration, washing the precipitate with distilled water, and vacuum drying at 100°C gave the target product with a yield of 34.2%. The product was confirmed by infrared spectroscopy and nuclear magnetic resonance.

Embodiment 6 The synthesis of β-cyclodextrin-poly L-glutamic acid-benzyl ester graft copolymer (β-CDPBLG4):

Put 0.062g (0.0547mol) of amino β-cyclodextrin and 1.0g (3.8mmol) of γ-benzyl L-glutamic acid cyclic anhydride into the reaction flask, add 70mL of anhydrous DMF, at 25 ℃, nitrogen protection The reaction was stirred for 80h. The reaction mixture was poured into 210 mL of deionized water to obtain a precipitate. Suction filtration, washing the precipitate with distilled water, and vacuum drying at 100°C gave the target product with a yield of 18.31%. The product was confirmed by infrared spectroscopy and nuclear magnetic resonance.

The preparation of embodiment 7 micelles:

Weigh 0.05g of β-CDPBLG1, β-CDPBLG2, and β-CDPBLG3 graft copolymers and dissolve them in 5mL of tetrahydrofuran respectively. After they are completely dissolved, slowly add 40mL of double-distilled water to the solution under stirring, and then remove them by rotary evaporation. Tetrahydrofuran (need about two hours), remaining solution is placed in 50mL volumetric flask, obtains the micelle solution of certain concentration with twice distilled water constant volume, utilizes the particle diameter of micelle to measure micelle.

As can be seen from Examples 4 to 6, as the molar ratio of amino β-cyclodextrin to amino acid anhydride (NCA) increases, β-cyclodextrin-poly L-glutamic acid-benzyl ester The yield of graft copolymer decreases, so the most suitable ratio is 1:10~50. And within the range of this ratio, in aqueous solution, micelles with a particle diameter of about 100nm can be formed. After testing, when the ratio is 1:10, micelles with an average particle diameter of 114nm can be formed (accompanying drawing 1). When the ratio is 1:30, micelles with an average particle size of 124nm can be formed (see Figure 2), and when the ratio is 1:50, micelles with an average particle size of 96nm can be formed (Figure 3). This kind of micelles formed by amphiphilic copolymers can assemble hydrophobic drug molecules into its hydrophobic core, which can improve the circulation time of drugs in the blood, and has great potential in the fields of controlled release of drugs and targeted drug delivery. Very good application prospects.

Claims (2)

1, a kind of synthetic method of β-cyclodextrin-poly L-glutamic acid-benzyl ester graft copolymer, it is characterized in that a certain amount of amino β-cyclodextrin, γ-benzyl ester-L-glutamic acid After mixing the acid anhydride in the ring in N,N-dimethylformamide, stir and react at a reaction temperature of 25°C under nitrogen protection for 60-80 hours; pour the reaction mixture into deionized water to precipitate; filter with suction, wash the precipitate with distilled water, and The target product was obtained by vacuum drying at 100°C; wherein: The molar ratio of amino β-cyclodextrin to γ-benzyl ester-L-glutamic acid anhydride is 1:10~50; the N,N- Dimethylformamide is 70-80mL; The volume ratio of N,N-dimethylformamide to deionized water is 1:3~5; The structure of amino β-cyclodextrin is: β-CD-NH ₂ , β-CD is β-cyclodextrin. 2. The synthesis method of β-cyclodextrin-poly-L-glutamic acid-benzyl ester graft copolymer according to claim 1, characterized in that the reaction time is 72 hours.

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