Four-chain star-shaped clustered peptide with anti-freezing activity as well as preparation method and application of four-chain star-shaped clustered peptide

Abstract

The invention belongs to the field of biopolymer synthesis, and discloses a four-chain star-shaped cluster peptide with anti-freezing activity as well as a preparation method and application of the four-chain star-shaped cluster peptide. The method is characterized in that a novel four-chain star-shaped initiator is utilized, and different groups such as methyl, ethyl, propyl, isopropyl, allyl and the like are inoculated into a star-shaped clustered peptide in combination with an R-NCA ring opening polymerization method. Meanwhile, the polymerization degree is regulated and controlled by regulating and controlling the ratio of the monomer to the initiator. The synthesized clustered peptoid not only has anti-freezing performance, but also can maintain the excellent characteristics of biocompatibility, processability and the like of the peptoid. The invention mainly solves the problem of poor biocompatibility of the traditional cell cryoprotectant.

Description

Technical Field

[0001] The invention belongs to the field of biopolymer synthesis, and in particular relates to a four-chain star-shaped polypeptide with antifreeze activity and a preparation method and application thereof. Background Art

[0002] Ice crystals can have an impact on all aspects of people's lives. In the biomedical field, the metabolic activity of cells is inhibited at low temperatures, and ice crystals produced below the freezing point will cause irreversible damage to cells, making the cells inactive. In order to achieve the cryopreservation of cells, people choose to add cell antifreeze protectants. Traditional protectants include dimethyl sulfoxide, glycerol, polyvinyl pyrrole, etc., but these cell cryoprotectants have certain toxicity and need to be cleared before use. Taking erythrocyte cryoprotectant glycerol as an example, according to statistics, the cleaning and deglycerolization process of one unit (475mL) of blood takes 30-60 minutes, which causes the blood frozen and preserved with glycerol to be unusable in the case of a large number of blood transfusions in urgent need. Therefore, finding a new type of cell antifreeze protectant with good cryoprotective effect, non-toxic, low cost, and convenient and quick use has become a problem that needs to be solved urgently.

[0003] Polypeptide materials are a new type of biopolymer material with good biochemical resistance, degradability and biocompatibility. Compared with polypeptides, the substituents of polypeptides are located on the nitrogen atoms of the main chain, making the main chain free of chiral centers and hydrogen bond donors. This unique structure improves the resistance of polypeptides to protein enzymatic hydrolysis, while allowing highly chemically diverse sequence information to be encoded into the chain, and the structure is controllable. Ring-opening polymerization is a common method for preparing polypeptides. Usually, the NCA / NTA monomer of the target substituent is first synthesized, and then the monomer ring-opening polymerization is controlled by a primary amine initiator. Different initiators will affect the configuration, molecular weight and molecular weight distribution of polypeptides. The degree of polymerization is controlled by controlling the feed ratio of monomer and initiator.

[0004] The present invention relates to a method for preparing an antifreeze clustered peptide with a four-chain star-shaped structure having antifreeze activity, which is characterized in that a new type of four-chain star-shaped initiator is used, combined with an R-NCA ring-opening polymerization method, to graft different groups, such as methyl, ethyl, propyl, isopropyl, allyl, etc., into the star-shaped clustered peptide. At the same time, the degree of polymerization is regulated by regulating the ratio of monomers and initiators. The synthesized clustered peptide has both antifreeze performance and can maintain the excellent properties of the peptide itself, such as biocompatibility and easy processing, and is expected to solve the problem that traditional cell cryoprotectants have certain toxicity. Summary of the invention

[0005] The present invention aims to eliminate the disadvantages of existing cell cryoprotectants, such as toxicity and troublesome operation, and aims to synthesize an antifreeze polymer peptide with a four-chain star-shaped structure.

[0006] A four-chain star-shaped peptide with antifreeze activity, the structural formula is:

[0007]

[0008] Wherein, R is one of methyl, ethyl, propyl, isopropyl or allyl, and n is between 10-100.

[0009] (1) When R is methyl, the preparation steps of the four-chain star-shaped peptide with antifreeze activity are as follows:

[0010] (A1) adding BOC-glycine, methyl iodide and sodium hydride in a solvent in sequence, stirring overnight, adding more methyl iodide and sodium hydride, and continuing to stir overnight; after the reaction is completed, adding ethyl acetate and distilled water to dissolve the reaction product, rotary evaporating, extracting, drying, and rotary evaporating to remove the solvent to obtain a white oily product 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid;

[0011] (A2) the product 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid in step (A1) is reacted with phosphorus trichloride in an N2 atmosphere to complete cyclization, and recrystallized multiple times in a glove box to obtain the product Me-NCA monomer;

[0012] (A3) A polymerization reaction is carried out in a glove box, and Me-NCA monomer and an initiator are added. After the reaction is completed, a poor solvent is added for precipitation. After separation and drying, antifreeze four-chain star-shaped methyl-polypeptides with different polymerization degrees are obtained.

[0013] In step (A1), the solvent is tetrahydrofuran, and the molar ratio of BOC-glycine, methyl iodide and sodium hydride is 1:4:5. After overnight, 2 equivalents of methyl iodide and 1.5 equivalents of sodium hydride are added.

[0014] In step (A1), the specific steps of extraction are: first add ether and pure water to extract in layers to obtain aqueous phase a; then add saturated sodium bicarbonate aqueous solution to the ether layer for extraction to obtain aqueous phase b; acidify aqueous phases a and b with 1 mol / L hydrochloric acid to pH = 2; then extract with ethyl acetate 3 times, pure water once, 5wt% sodium thiosulfate twice, and saturated brine once; dry using anhydrous magnesium sulfate.

[0015] In step (A2), the molar ratio of 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid to phosphorus trichloride is 1:1.2, the solvent is dichloromethane, and the recrystallization is performed twice with dichloromethane and n-hexane, and once with tetrahydrofuran and n-hexane.

[0016] In step (A3), Me-NCA monomer is reacted in acetonitrile at a reaction temperature of 50° C., the initiator is PAMAM dendrimer, the molar ratio of the monomer to the initiator is 4n:1, and the poor solvent is tetrahydrofuran or diethyl ether.

[0017] (2) When R is one of ethyl, propyl, isopropyl or allyl, the preparation steps of the four-chain star-shaped peptide with antifreeze activity are:

[0018] (B1) adding glyoxylic acid monohydrate, dichloromethane and an amine organic compound in proportion, stirring and reacting for 24 hours, and removing the solvent by rotary evaporation to obtain a yellow-brown oil;

[0019] Wherein, the amine organic compound is one of ethylamine, propylamine, isopropylamine or allylamine;

[0020] (B2) adding hydrochloric acid to the product of step (B1), stirring and reflux overnight, removing the solvent by rotary evaporation, recrystallizing with methanol and diethyl ether, and filtering to obtain product 1;

[0021] (B3) adding triethylamine and di-tert-butyl dicarbonate to the product of step (B2), extracting for several times, drying over anhydrous magnesium sulfate, and rotary evaporating the solvent to obtain product 2.

[0022] (B4) The product in step (B3) is reacted with phosphorus trichloride in a N2 atmosphere to complete cyclization, and the products are recrystallized multiple times in a glove box to obtain Et-NCA monomer, Pr-NCA monomer, i Pr-NCA monomer, Al-NCA monomer.

[0023] (B5) performing a polymerization reaction in a N2 atmosphere, adding any one of the monomers and initiator obtained in step (B4), adding a poor solvent for precipitation after the reaction is completed, separating and drying, and obtaining antifreeze peptides with four-chain star structures of different polymerization degrees and different substituents.

[0024] In step (B1), the ratio of the amount of the amine organic compound to the amount of the glyoxylic acid monohydrate substance is 1:2.

[0025] In step (B2), the concentration of hydrochloric acid is 1 mol / L, wherein the molar ratio of hydrochloric acid to amine organic compound is 5:1; and the heating reflux temperature is 120°C.

[0026] In step (B3), the molar ratio of product 1, di-tert-butyl dicarbonate and triethylamine is 1:1.5:5, and the reaction solvent is pure water. The mixture is extracted once with n-hexane, once with 4 mol / L hydrochloric acid, three times with ethyl acetate, and once with saturated brine. The mixture is dried over anhydrous magnesium sulfate.

[0027] In step (B4), the reaction is carried out in a N2 atmosphere, the molar ratio of the product 2 to phosphorus trichloride is 1:1.2, and the solvent is dichloromethane. The recrystallization is carried out using dichloromethane and n-hexane twice, and tetrahydrofuran and n-hexane once.

[0028] In step (B5), the monomer is reacted in tetrahydrofuran at a reaction temperature of 50° C., the initiator is PAMAM dendritic polymer, the molar ratio of the monomer to the initiator is 4n:1, and the poor solvent is n-hexane.

[0029] The structural formula of the initiator is:

[0030]

[0031] The four-chain star-shaped antifreeze polymer peptide prepared by the invention is used as an antifreeze agent for cryopreservation of cells.

[0032] The beneficial effects of the present invention are:

[0033] (1) The present invention synthesizes a four-chain star-shaped antifreeze polymer peptide material with controllable structure and chain length;

[0034] (2) The preparation process of the present invention has the characteristics of easy availability of raw materials, good reproducibility and high stability;

[0035] (3) The antifreeze agent prepared by the present invention has the characteristics of good antifreeze effect and non-toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the 1H NMR spectrum of Me-NCA.

[0037] Figure 2 This is the 1H NMR spectrum of Et-NCA.

[0038] Figure 3 This is the 1H NMR spectrum of Pr-NCA.

[0039] Figure 4 Infrared comparison of monomer and polymer.

[0040] Figure 5 f-PNMG 10 1HNMR spectrum of.

[0041] Figure 6 Three concentrations of f-PNMG 10 Photograph of ice crystals after growth in PBS solution at -60°C for 30 minutes.

[0042] Figure 7 Three concentrations of f-PNMG 10 of cytotoxicity. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0044] The experimental methods used in the following examples are all conventional methods unless otherwise specified. NCA monomers were determined by H NMR spectroscopy, Bruker 500 MHz, CDCl3 as solvent, and polymer number average molecular weight was determined by gel permeation chromatography, SSI pump connected to Wyatt Optilab DSP, DMF as solvent, flow rate 1 mL / min, test temperature 50°C.

[0045] The structural formulas of several monomers are shown below:

[0046]

[0047] The reaction equation is:

[0048]

[0049] Example 1 Preparation of Me-NCA monomer and its polymerized peptides:

[0050] (1) Select a 500 mL round-bottom flask and place it in an ice-water bath at 0°C. Add 300 mL of tetrahydrofuran to the flask, add 11.0 g of BOC-glycine (62.9 mmol), 15.9 mL of iodomethane, and 7.8 g of sodium hydride to the solvent in sequence, and stir overnight. Add 7.9 mL of iodomethane and 2.6 g of sodium hydride and continue stirring overnight. Add 150 mL of ethyl acetate to the flask, add 22.5 mL of pure water dropwise to destroy the excess sodium hydride, and evaporate to obtain an oily substance. Add 150 mL of pure water and 90 mL of ether to the crude product, extract by stratification, and obtain the lower aqueous phase a. The ether organic layer is extracted with 75 mL of saturated sodium bicarbonate aqueous solution, and the lower aqueous phase b is taken. Combine the aqueous phases a and b, acidify to pH = 2 with 1 mol / L hydrochloric acid, and extract three times with 150 mL of ethyl acetate, and take the upper layer. Extract the product with 150 mL of water, and take the upper organic phase. The mixture was extracted twice with 150 mL of a 5 wt% aqueous sodium thiosulfate solution and once with 150 mL of saturated brine. The upper layers were taken and dried over anhydrous magnesium sulfate. The mixture was filtered and rotary evaporated to obtain 10.0 g of a white oily product, 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid.

[0051] (2) A 500 mL two-necked flask was connected to a Schlenk double-row tube, and the flask was repeatedly pumped and filled three times. In a 0°C ice-water bath and N2 atmosphere, 265 mL of dichloromethane and 10.0 g of 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid (52.9 mmol) were added to the flask, and 5.7 mL of phosphorus trichloride was slowly added dropwise. The reaction was stirred for 2 hours. After the reaction was completed, the oil pump was turned on, cold hydrazine was installed, the solvent was drained, and the flask was transferred to a glove box. A sufficient amount of good solvent dichloromethane was added to the flask, and insoluble impurities were removed by suction filtration. The flask was evaporated to saturation under reduced pressure, and two volumes of poor solvent n-hexane were added. The flask was placed in a -23°C refrigerator overnight. After filtering, the product was dried and transferred to a sample bottle. 5.6 g of white powdery Me-NCA monomer was weighed and used. Figure 1 The structure was confirmed by NMR.

[0052] (3) Aggregation:

[0053] In a 10 mL volumetric flask, 1040 mg of PAMAM dendrimer (2.0 mmol) was added, and acetonitrile was added to the mark to prepare an initiator solution with a concentration of 0.2 mmol / mL.

[0054] Add 250 mg Me-NCA monomer (2.2 mmol) to a 40 mL reaction bottle. According to the monomer: initiator feed ratio of 40:1, the monomer concentration is controlled at 0.8 mmol / mL and the initiator concentration is 0.2 mmol / L. Add 2536 μL of acetonitrile solution until the monomer is completely dissolved, add 181 μL of initiator solution, heat to 70 °C and stir for 24-72 hours. Use infrared detection at 1760 and 1860 cm -1 The degree of reaction can be judged by whether the monomer characteristic peak at Figure 4 After the reaction is completed, add enough ether to precipitate, separate and dry to obtain a four-chain star-shaped methyl antifreeze peptide with a polymerization degree of 10 for each chain and a total polymerization degree of 40. Use nuclear magnetic resonance to determine the structure and calculate the degree of polymerization, see the attached manual Figure 5 .

[0055] By changing the molar ratio of monomer to initiator to 80:1 and 160:1, four-chain star-shaped methyl antifreeze peptides with a polymerization degree of 20 for each chain and a total polymerization degree of 80 and a polymerization degree of 40 for each chain and a total polymerization degree of 160 were prepared respectively.

[0056] Example 2 Preparation of Et-NCA monomer and its polymerized peptides:

[0057] (1) A 1000 mL round-bottom flask was selected, 18.41 g (200 mmol) of glyoxylic acid monohydrate and 500 mL of dichloromethane were added, and then 5.63 mL (100 mmol) of ethylamine was added dropwise into the flask, and the mixture was stirred on a magnetic stirrer at room temperature for 24 hours. After overnight, dichloromethane was removed by rotary evaporation, and then 500 mL of 1 mol / L hydrochloric acid was added, and the mixture was refluxed and stirred at 120°C for 24 hours. After the solvent was removed by rotary evaporation, sufficient methanol was added to fully dissolve the mixture, and the mixture was rotary evaporated to saturation, followed by adding twice the volume of ether, and the mixture was placed in a refrigerator at -23°C overnight. After suction filtration and drying, 18.6 g of beige 2-(N-ethylamino)acetic acid hydrochloride was obtained.

[0058] (2) Dissolve 18.5 g (132.5 mmol) of 2-(N-ethylamino)acetic acid hydrochloride in a flask filled with 516 mL of pure water, add 43.4 g (198.8 mmol) of di-tert-butyl dicarbonate and 92 mL of triethylamine, and stir at room temperature for 24 hours. After the reaction is completed, extract twice with 516 mL of n-hexane and take the upper layer. Add 4 mol / L hydrochloric acid aqueous solution to the upper layer to acidify to pH = 2. Extract three times with 129 mL of ethyl acetate and once with 129 mL of saturated brine, and take the upper layer. Dry with anhydrous magnesium sulfate, filter and rotary evaporate to obtain 17.0 g of beige oily product 2-(N,N-tert-butylcarbonyl-ethylamino)acetic acid.

[0059] (3) A 1000 mL two-necked flask was connected to a Schlenk double-row tube, and the mixture was repeatedly pumped and filled for 3 times. In a 0°C ice-water bath and N2 atmosphere, 418 mL of dichloromethane and 17.0 g of 2-(N,N-tert-butylcarbonyl-ethylamino)acetic acid (83.6 mmol) were added to the flask, and 8.8 mL of phosphorus trichloride was slowly added dropwise. The mixture was stirred for 2 hours. After the reaction was completed, the oil pump was turned on, cold hydrazine was installed, the solvent was drained, and the mixture was transferred to a glove box. A sufficient amount of good solvent dichloromethane was added to the flask, and insoluble impurities were removed by suction filtration. The mixture was evaporated to saturation under reduced pressure, and two volumes of poor solvent n-hexane were added. The mixture was placed in a -23°C refrigerator overnight, the supernatant was removed, and the volatile solvent was removed by vacuum. After weighing, 4.3 g of yellow viscous Et-NCA monomer was obtained. Figure 2 The structure was confirmed by NMR.

[0060] (4) Aggregation:

[0061] In a 10 mL volumetric flask, 1040 mg of PAMAM dendrimer (2.0 mmol) was added, and tetrahydrofuran was added to the mark to prepare an initiator solution with a concentration of 0.2 mmol / mL.

[0062] Add 2.2 mmol of Et-NCA monomer to a 40 mL reaction bottle. According to the monomer: initiator feed ratio of 40:1, the monomer concentration is controlled at 0.8 mmol / mL and the initiator concentration is 0.2 mmol / L. Add 2536 μL of tetrahydrofuran solution until the monomer is completely dissolved, add 181 μL of initiator solution, heat to 70 °C and stir to react for 24-72 hours. Use infrared detection at 1760 and 1860 cm -1 The degree of reaction was determined by the disappearance of the monomer characteristic peak at the position. After the reaction was completed, sufficient n-hexane was added for precipitation. After separation and drying, a four-chain star-shaped ethyl antifreeze peptide with a polymerization degree of 10 for each chain and a total polymerization degree of 40 was obtained. Nuclear magnetic resonance was used to determine the structure and calculate the degree of polymerization.

[0063] By changing the molar ratio of monomer to initiator to 80:1 and 160:1, four-chain star-shaped ethyl antifreeze peptides with a polymerization degree of 20 for each chain and a total polymerization degree of 80 and a polymerization degree of 40 for each chain and a total polymerization degree of 160 were prepared respectively.

[0064] Example 3 Preparation of Pr-NCA monomer and its polypeptide

[0065] The synthesis process of Pr-NCA monomer is the same as that of Example 2, except that ethylamine is replaced by propylamine, and the amounts of other substances remain unchanged. Figure 3 .

[0066] Example 4 i Preparation of Pr-NCA Monomer and Its Polymerized Peptides

[0067] i The synthesis process of Pr-NCA monomer is the same as that of Example 2, except that ethylamine is replaced with isopropylamine, and the amounts of other substances remain unchanged.

[0068] Example 5 Preparation of Al-NCA monomer and its polypeptide

[0069] The synthesis process of Al-NCA monomer is the same as that of Example 2, except that ethylamine is replaced by allylamine, and the amounts of other substances remain unchanged.

[0070] Example 6 Ice Recrystallization Inhibition (IRI) Activity Experiment

[0071] (1) Take three 5 mL sample bottles, weigh 1 mg, 5 mg, and 10 mg of a four-chain star-shaped methyl antifreeze peptide with a degree of polymerization of 10 for each chain and a total degree of polymerization of 40, respectively, and add 1 mL of PBS solution to prepare sample solutions with concentrations of 1 mg / mL, 5 mg / mL, and 10 mg / mL.

[0072] (2) 15 μL of the solution was drawn up with a pipette and dropped from a height of 1.5 m onto a cryogenic cold stage precooled to −60 °C to form a thin layer of solid ice film. The temperature was then raised to −6 °C at a rate of 10 °C / min, and the frozen sample was kept at −6 °C for 30 min. A digital camera (Nikon Y-TV55, Japan) was then used to take photos of ice crystals at 10 different positions. Each concentration was measured three times.

[0073] (3) Use Nano Measurer software to measure the ice crystal photos taken. Select the ten largest ice crystals in each photo for measurement, calculate the average maximum ice crystal particle size (MLGS) of each photo, and then calculate the average maximum ice crystal particle size (MLGS) of the ten photos in turn. Calculate the average of the average maximum ice crystal particle size (MLGS) obtained in three experiments to evaluate the ice recrystallization inhibition activity of the sample at this concentration. See the attached instructions for ice crystal pictures Figure 6 .

[0074] Example 7 Biocompatibility test of four-chain star-shaped antifreeze peptide

[0075] L929 cells were seeded onto a 96-well plate, 100 μL of culture medium was added, and the cells were incubated at 37° C. and 5% CO 2 for 24 h.

[0076] Take three 5 mL sample bottles, weigh 1 mg, 5 mg, and 10 mg of a four-chain star-shaped methyl antifreeze peptide with a polymerization degree of 10 for each chain and a total polymerization degree of 40, respectively, add 1 mL of culture medium, and filter after complete dissolution to prepare sample solutions with concentrations of 1 mg / mL, 5 mg / mL, and 10 mg / mL.

[0077] The old culture medium was removed, 100 μL of the sample solution was added, and the cells were incubated at 37°C and 5% CO2 for 24 h.

[0078] Add 10 μL of CCK8 solution, incubate for 2 h, measure the absorbance at 450 nm with an enzyme reader, and calculate the survival rate. Figure 7 .

Claims

1. A four-chain star-shaped peptide with antifreeze activity, characterized in that: The structural formula is: Wherein, R is one of methyl, ethyl, propyl, isopropyl or allyl, and n is between 10-100.

2. The method for preparing the four-chain star-shaped peptide with antifreeze activity according to claim 1, characterized in that: When R is methyl, the preparation steps are: (A1) adding BOC-glycine, methyl iodide and sodium hydride in a solvent in sequence, stirring overnight, adding more methyl iodide and sodium hydride, and continuing to stir overnight; after the reaction is completed, adding ethyl acetate and distilled water to dissolve the reaction product, rotary evaporating, extracting, drying, and rotary evaporating to remove the solvent to obtain a white oily product 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid; (A2) the product 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid in step (A1) is reacted with phosphorus trichloride in an N2 atmosphere to complete cyclization, and recrystallized multiple times in a glove box to obtain the product Me-NCA monomer; (A3) A polymerization reaction is carried out in a glove box, and Me-NCA monomer and an initiator are added. After the reaction is completed, a poor solvent is added for precipitation. After separation and drying, antifreeze four-chain star-shaped methyl-polypeptides with different polymerization degrees are obtained.

3. The method for preparing the four-chain star-shaped peptide with antifreeze activity according to claim 1, characterized in that: In step (A1), the solvent is tetrahydrofuran, and the molar ratio of BOC-glycine, methyl iodide and sodium hydride is 1:4:

5. After overnight, 2 equivalents of methyl iodide and 1.5 equivalents of sodium hydride are added; The specific steps of extraction are: first add ether and pure water to extract in layers, and take water phase a; then add saturated sodium bicarbonate aqueous solution to the ether layer for extraction to obtain water phase b; acidify the water phases a and b to pH=2 with 1 mol / L hydrochloric acid; then extract with ethyl acetate 3 times, pure water once, 5wt% sodium thiosulfate twice, and saturated salt water once; and dry with anhydrous magnesium sulfate.

4. The method for preparing the four-chain star-shaped peptide with antifreeze activity according to claim 1, characterized in that: In step (A2), the molar ratio of 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid to phosphorus trichloride is 1:1.2, the solvent is dichloromethane, and the recrystallization is performed twice with dichloromethane and n-hexane, and once with tetrahydrofuran and n-hexane.

5. The method for preparing the four-chain star-shaped peptide with antifreeze activity according to claim 1, characterized in that: In step (A3), Me-NCA monomer is reacted in anhydrous acetonitrile at a reaction temperature of 50° C., the initiator is PAMAM dendrimer, the molar ratio of the monomer to the initiator is 4n:1, and the poor solvent is tetrahydrofuran or diethyl ether.

6. The method for preparing the four-chain star-shaped peptide with antifreeze activity according to claim 1, characterized in that: When R is one of ethyl, propyl, isopropyl or allyl, the preparation steps are: (B1) adding glyoxylic acid monohydrate, dichloromethane and an amine organic compound in proportion, stirring and reacting for 24 hours, and removing the solvent by rotary evaporation to obtain a yellow-brown oil; Wherein, the amine organic compound is one of ethylamine, propylamine, isopropylamine or allylamine; (B2) adding hydrochloric acid to the product of step (B1), stirring and reflux overnight, removing the solvent by rotary evaporation, recrystallizing with methanol and diethyl ether, and filtering to obtain product 1; (B3) adding triethylamine and di-tert-butyl dicarbonate to the product of step (B2), extracting for multiple times, drying over anhydrous magnesium sulfate, and rotary evaporating the solvent to obtain product 2; (B4) The product in step (B3) is reacted with phosphorus trichloride in a N2 atmosphere to complete cyclization, and the products obtained by multiple recrystallizations are Et-NCA monomer, Pr-NCA monomer, i Pr-NCA monomer, Al-NCA monomer; (B5) performing a polymerization reaction in a N2 atmosphere, adding any one of the monomers and initiator obtained in step (B4), adding a poor solvent for precipitation after the reaction is completed, separating and drying, and obtaining antifreeze peptides with four-chain star structures of different polymerization degrees and different substituents.

7. The method for preparing the four-chain star-shaped peptide with antifreeze activity according to claim 1, characterized in that: In step (B1), the ratio of the amount of the amine organic compound to the amount of the glyoxylic acid monohydrate substance is 1:2; In step (B2), the concentration of hydrochloric acid is 1 mol / L, wherein the molar ratio of hydrochloric acid to amine organic compound is 5:1; and the heating reflux temperature is 120°C.

8. The method for preparing the four-chain star-shaped peptide with antifreeze activity according to claim 1, characterized in that: In step (B3), the molar ratio of product 1, di-tert-butyl dicarbonate and triethylamine is 1:1.5:5, and the reaction solvent is pure water; extraction is performed with n-hexane once, 4 mol / L hydrochloric acid once, ethyl acetate three times, and saturated brine once; and drying is performed with anhydrous magnesium sulfate; In step (B4), the reaction is carried out in a N2 atmosphere, the molar ratio of the product 2 and phosphorus trichloride is 1:1.2, and the solvent is dichloromethane; the recrystallization is carried out twice with dichloromethane and n-hexane, and once with tetrahydrofuran and n-hexane.

9. The method for preparing the four-chain star-shaped peptide with antifreeze activity according to claim 1, characterized in that: In step (B5), the monomer is reacted in anhydrous tetrahydrofuran at a reaction temperature of 50° C., the initiator is a PAMAM dendritic polymer, the molar ratio of the monomer to the initiator is 4n:1, and the poor solvent is n-hexane; The structural formula of the initiator is:

10. Using the four-chain star-shaped peptide with antifreeze activity according to claim 1 as an antifreeze agent for cryopreservation of cells.

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