A tetra-chain star-shaped peptide with anti-freezing activity, and a preparation method and application thereof

Abstract

The application belongs to the field of biological polymer synthesis, and discloses a four-chain star-shaped polymeric peptide with anti-freezing activity and a preparation method and application thereof. The four-chain star-shaped initiator is used to combine with R-NCA ring-opening polymerization to introduce different groups, such as methyl, ethyl, propyl, isopropyl and allyl, into the star-shaped polymeric peptide. The polymerization degree is controlled by regulating the monomer and initiator ratio. The synthesized polymeric peptide has the anti-freezing property and the excellent characteristics of biocompatibility and easy processability of the original peptide. The application mainly solves the problem of poor biocompatibility of the traditional cell freezing protectant.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological polymer synthesis, and particularly relates to a four-chain star-shaped polymeric peptide with anti-freezing activity and a preparation method and application thereof. BACKGROUND

[0002] Ice crystals can affect all aspects of people's lives. In the field of biological medicine, the metabolic activity of cells is inhibited at low temperatures, and ice crystals produced below the freezing point can cause irreversible damage to cells, making the cells lose activity. In order to achieve the cryopreservation of cells, people choose to add cell anti-freezing protective agents. Traditional protective agents include dimethyl sulfoxide, glycerol, polyvinyl pyrrolidone, etc., but such cell cryoprotective agents have certain toxicity and need to be removed before use. Taking the red blood cell cryoprotective agent glycerol as an example, according to statistics, the glycerol removal process of a unit (475 mL) of blood needs 30-60 minutes, which makes the blood cryopreserved by adding glycerol unable to be used in the case of a large number of urgent blood transfusions. Therefore, it is an urgent problem to find a new type of cell anti-freezing protective agent with good cryoprotective effect, non-toxicity, low cost and convenient and fast use.

[0003] Polymeric peptide material is a new type of biological polymer material with good anti-biochemical, degradability and biocompatibility. Compared with polypeptides, the substituents of polymeric peptides are located on the nitrogen atoms of the main chain, so that the main chain has no chiral center and hydrogen bond donor. This unique structure improves the resistance of polymeric peptides to protein enzyme hydrolysis, and at the same time allows high chemical diversity of sequence information to be encoded into the chain, and the structure is controllable. Ring-opening polymerization is a common method for preparing polymeric peptides, which usually involves synthesizing NCA / NTA monomers of target substituents, and then controlling the ring-opening polymerization of monomers by primary amine initiators. Different initiators will affect the configuration, molecular weight and molecular weight distribution of polymeric peptides, and the degree of polymerization can be controlled by controlling the feeding ratio of monomers and initiators.

[0004] The present application relates to a preparation method of a four-chain star-shaped anti-freezing polymeric peptide with anti-freezing activity, which is characterized by using a new four-chain star-shaped initiator, combining R-NCA ring-opening polymerization, and grafting different groups such as methyl, ethyl, propyl, isopropyl and allyl in the star-shaped polymeric peptide. At the same time, the degree of polymerization is controlled by adjusting the ratio of monomers and initiators. The synthesized polymeric peptide not only has anti-freezing performance, but also maintains the excellent properties of polymeric peptides such as biocompatibility and easy processability, and is expected to solve the problem of toxicity of traditional cell cryoprotective agents. SUMMARY

[0005] The present application aims to eliminate the shortcomings of toxic and cumbersome operation of existing cell cryoprotective agents, and aims to synthesize a four-chain star-shaped anti-freezing polymeric peptide.

[0006] A four-chain star-like peptide with anti-freezing activity, having a structural formula of:

[0007]

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

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

[0010] (A1) In a solvent, BOC-glycine, iodomethane and sodium hydride are sequentially added, stirred overnight, iodomethane and sodium hydride are supplemented, and stirred overnight; after the reaction is completed, ethyl acetate and distilled water are added to dissolve the reaction product, rotary evaporation, extraction, drying, rotary evaporation to remove the solvent, and then a white oily product 2-(N,N-tert-butyl carbonyl-methyl amino) acetic acid is obtained;

[0011] (A2) The product 2-(N,N-tert-butyl carbonyl-methyl amino) acetic acid in step (A1) is reacted with phosphorus trichloride in an atmosphere of N2 to complete ring formation, and the product Me-NCA monomer is obtained by multiple recrystallization in a glove box;

[0012] (A3) In a glove box, Me-NCA monomer and an initiator are added to perform a polymerization reaction, after the reaction is completed, a poor solvent is added for precipitation, and after separation and drying, anti-freezing four-chain star-like methyl peptides with different polymerization degrees are obtained.

[0013] In step (A1), the solvent is tetrahydrofuran, and the mass ratio of BOC-glycine, iodomethane and sodium hydride is 1:4:5, and after overnight, 2 equivalents of iodomethane and 1.5 equivalents of sodium hydride are supplemented.

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

[0015] In step (A2), the mass ratio of 2-(N,N-tert-butyl carbonyl-methyl amino) acetic acid and phosphorus trichloride is 1:1.2, the solvent is dichloromethane, and recrystallization is performed with dichloromethane and n-hexane 2 times, and tetrahydrofuran and n-hexane 1 time.

[0016] In step (A3), the Me-NCA monomer is reacted in acetonitrile, the reaction temperature is 50℃, the initiator is selected from PAMAM dendrimer, the mass ratio of monomer and 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 tetra-chain star-shaped peptide with antifreeze activity are as follows:

[0018] (B1) Add glyoxylic acid monohydrate, dichloromethane, and amine organic compounds in proportion, stir and react for 24 hours, and remove the solvent by rotary evaporation to obtain a yellowish-brown oily substance.

[0019] Among them, the amine organic compounds are one of ethylamine, propylamine, isopropylamine or allylamine;

[0020] (B2) Add hydrochloric acid to the product of step (B1), stir and reflux overnight, remove the solvent by rotary evaporation, recrystallize with methanol and diethyl ether, and filter to obtain product 1;

[0021] (B3) Triethylamine and ditert-butyl dicarbonate were added to the product of step (B2), and after multiple extractions, the product was dried with anhydrous magnesium sulfate and the solvent was evaporated by rotary evaporation to obtain product 2.

[0022] (B4) In step (B3), the product reacts with phosphorus trichloride to form a cyclization reaction in an N2 atmosphere. Multiple recrystallizations in a glove box yielded Et-NCA monomer, Pr-NCA monomer, and [other products]. i Pr-NCA monomer, Al-NCA monomer.

[0023] (B5) The polymerization reaction is carried out in an N2 atmosphere. Any monomer and initiator obtained in step (B4) are added. After the reaction is completed, a poor solvent is added for precipitation. After separation and drying, antifreeze polymerized peptides with different degrees of polymerization and different substituents are obtained.

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

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

[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. It is then dried over anhydrous magnesium sulfate.

[0027] In step (B4), the reaction is carried out in an N2 atmosphere, with the molar ratio of product 2 to phosphorus trichloride being 1:1.2, and dichloromethane as the solvent. Recrystallization is performed twice with dichloromethane and n-hexane, and once with tetrahydrofuran and n-hexane.

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

[0029] The structural formula of the initiator is:

[0030]

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

[0032] The beneficial effects of this invention are as follows:

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

[0034] (2) The preparation process of this invention has the characteristics of readily available raw materials, good repeatability, and high stability;

[0035] (3) The antifreeze prepared by the present invention has the characteristics of good antifreeze effect and non-toxicity. Attached Figure Description

[0036] Figure 1 The image shows the Me-NCA 1H NMR spectrum.

[0037] Figure 2 The image shows the Et-NCA 1H NMR spectrum.

[0038] Figure 3 The image shows the 1H NMR spectrum of Pr-NCA.

[0039] Figure 4 Infrared comparison images of monomers and polymers.

[0040] Figure 5 f-PNMG 10 The 1H NMR spectrum.

[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 Cytotoxicity. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. NCA monomers were determined by 1H NMR spectroscopy (Bruker 500MHz, CDCl3 as solvent). The number-average molecular weight of the polymer was determined by gel permeation chromatography (SSIpump 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 in the figure below:

[0046]

[0047] The reaction equation is:

[0048]

[0049] Example 1: Preparation of Me-NCA monomers and their polymeric peptides:

[0050] (1) A 500 mL round-bottom flask was placed in an ice-water bath at 0 °C. 300 mL of tetrahydrofuran was added to the flask, followed by 11.0 g BOC-glycine (62.9 mmol), 15.9 mL of iodomethane, and 7.8 g of sodium hydride. The mixture was stirred overnight. 7.9 mL of iodomethane and 2.6 g of sodium hydride were added, and the mixture was stirred overnight again. 150 mL of ethyl acetate was added to the flask, and 22.5 mL of pure water was added dropwise to break down the excess sodium hydride. The product was then rotary evaporated to obtain an oily substance. 150 mL of pure water and 90 mL of diethyl ether were added to the crude product, and the mixture was extracted layer by layer to obtain the lower aqueous phase a. The organic layer in diethyl ether was extracted with 75 mL of saturated sodium bicarbonate aqueous solution, and the lower aqueous phase b was collected. Aqueous phases a and b were combined, acidified to pH 2 with 1 mol / L hydrochloric acid, and extracted three times with 150 mL of ethyl acetate. The upper layer was collected. The product was extracted with 150 mL of water, and the upper organic phase was collected. The product was extracted twice with 150 mL of 5 wt% sodium thiosulfate aqueous solution and once with 150 mL of saturated brine. The supernatant was collected from both extractions, dried over anhydrous magnesium sulfate, filtered, and rotary evaporated to obtain 10.0 g of white oily product 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid.

[0051] (2) Connect a 500 mL two-necked flask to a Schlenk double-row tube, repeat the purging and charging process three times. In a 0°C ice-water bath under N2 atmosphere, add 265 mL of dichloromethane and 10.0 g of 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid (52.9 mmol). Slowly add 5.7 mL of phosphorus trichloride and stir for 2 hours. After the reaction is complete, turn on the oil pump, install a hydrazine coolant, remove the solvent by vacuum, transfer to a glove box, add sufficient good solvent dichloromethane to the flask, filter to remove insoluble impurities, evaporate under reduced pressure to saturation, add twice the volume of poor solvent n-hexane, place in a -23°C refrigerator overnight, filter, dry the product, transfer to a sample vial, and weigh to obtain 5.6 g of white powdery Me-NCA monomer. Figure 1 The structure was confirmed by NMR spectroscopy.

[0052] (3) Aggregation:

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

[0054] Add 250 mg of Me-NCA monomer (2.2 mmol) to a 40 mL reaction flask. Based on a monomer:initiator ratio of 40:1, maintain a monomer concentration of 0.8 mmol / mL and an initiator concentration of 0.2 mmol / L. Add 2536 μL of acetonitrile solution until the monomer is completely dissolved, then add 181 μL of initiator solution. Heat to 70 °C and stir for 24–72 hours. Detect the reaction at 1760 and 1860 cm⁻¹ using infrared spectroscopy. -1 To determine the extent of the reaction, observe whether the characteristic peak of the monomer at the specified location disappears. (See the appendix in the instruction manual.) Figure 4 After the reaction was complete, sufficient diethyl ether was added for precipitation. After separation and drying, a tetra-chain star-shaped methyl antifreeze peptide with a degree of polymerization of 10 per chain and an overall degree of polymerization of 40 was obtained. The structure and degree of polymerization were determined using NMR spectroscopy (see attached instructions). Figure 5 .

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

[0056] Example 2: Preparation of Et-NCA monomers and their peptides:

[0057] (1) A 1000 mL round-bottom flask was used. 18.41 g (200 mmol) of glyoxylic acid monohydrate and 500 mL of dichloromethane were added. Then, 5.63 mL (100 mmol) of ethylamine was added dropwise to the flask. The mixture was stirred on a magnetic stirrer at room temperature for 24 hours. After overnight, the dichloromethane was removed by rotary evaporation. Then, 500 mL of 1 mol / L hydrochloric acid was added, and the mixture was refluxed at 120 °C and stirred for 24 hours. After removing the solvent by rotary evaporation, sufficient methanol was added to dissolve the solvent completely. The mixture was then rotary evaporated to saturation. Subsequently, twice the volume of diethyl ether was added, and the mixture was placed in a refrigerator at -23 °C overnight. After filtration and drying, 18.6 g of pale yellow 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 containing 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 complete, extract twice with 516 mL of n-hexane and collect the supernatant. Acidify the supernatant with 4 mol / L hydrochloric acid aqueous solution to pH 2. Extract three times with 129 mL of ethyl acetate and once with 129 mL of saturated brine, collecting the supernatant from each extraction. Dry with anhydrous magnesium sulfate, filter, and rotary evaporate to obtain 17.0 g of the pale yellow oily product 2-(N,N-tert-butylcarbonyl-ethylamino)acetic acid.

[0059] (3) Connect a 1000 mL two-necked flask to a Schlenk double-row tube, repeatedly purging and charging it three times. In a 0°C ice-water bath under N2 atmosphere, add 418 mL of dichloromethane and 17.0 g of 2-(N,N-tert-butylcarbonyl-ethylamino)acetic acid (83.6 mmol), and slowly add 8.8 mL of phosphorus trichloride. Stir the reaction for 2 hours. After the reaction is complete, turn on the oil pump, install a cold hydrazine, remove the solvent by vacuum, transfer to a glove box, add sufficient good solvent dichloromethane to the flask, filter to remove insoluble impurities, evaporate under reduced pressure to saturation, add twice the volume of poor solvent n-hexane, place in a -23°C refrigerator overnight, remove the supernatant, remove the volatile solvent under vacuum, and weigh to obtain 4.3 g of yellow viscous Et-NCA monomer. Figure 2 The structure was confirmed by NMR spectroscopy.

[0060] (4) Aggregation:

[0061] Add 1040 mg of PAMAM dendrimer (2.0 mmol) to a 10 mL volumetric flask, and add tetrahydrofuran 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 flask. Based on a monomer:initiator ratio of 40:1, maintain a monomer concentration of 0.8 mmol / mL and an initiator concentration of 0.2 mmol / L. Add 2536 μL of tetrahydrofuran solution until the monomer is completely dissolved, then add 181 μL of initiator solution. Heat to 70 °C and stir for 24–72 hours. Detect the reaction at 1760 and 1860 cm⁻¹ using infrared spectroscopy. -1 The extent of the reaction was determined by whether the characteristic peaks of the monomers disappeared. After the reaction was complete, sufficient n-hexane was added for precipitation, and after separation and drying, tetra-chain star-shaped ethyl antifreeze peptides with a degree of polymerization of 10 per chain and a total degree of polymerization of 40 were obtained. NMR 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, tetra-chain star-shaped ethyl antifreeze polymeric peptides with a degree of polymerization of 20 per chain and a total degree of polymerization of 80, and a degree of polymerization of 40 per chain and a total degree of polymerization of 160 were prepared, respectively.

[0064] Example 3: Preparation of Pr-NCA monomers and their peptides

[0065] The synthesis process of Pr-NCA monomer is the same as in Example 2, except that ethylamine is replaced with propylamine, while the amounts of other substances remain unchanged. See the NMR spectrum. Figure 3 .

[0066] Example 4 i Preparation of Pr-NCA monomers and their polymeric peptides

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

[0068] Example 5: Preparation of Al-NCA monomers and their polymeric peptides

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

[0070] Example 6: Ice recrystallization inhibition (IRI) activity experiment

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

[0072] (2) Use a pipette to draw 15 μL of solution and drop it from a height of 1.5 meters onto a low-temperature stage pre-cooled to -60°C to form a thin solid ice film. Then raise the temperature to -6°C at a rate of 10°C / min and keep the frozen sample at -6°C for 30 minutes. Then take pictures of ice crystals at 10 different locations using a digital camera (Nikon Y-TV55, Japan). Each concentration is measured three times.

[0073] (3) The ice crystal photographs were measured using Nano Measurer software. Ten of the largest ice crystals from each photograph were selected for measurement, and the average maximum ice crystal size (MLGS) of each photograph was calculated. Then, the average maximum ice crystal size (MLGS) of the ten photographs was calculated sequentially. The average maximum ice crystal size (MLGS) of the three experiments was then calculated to evaluate the ice recrystallization inhibitory activity of the sample at that concentration. Ice crystal images are attached to the instruction manual. Figure 6 .

[0074] Example 7 Biocompatibility test of tetra-chain star-shaped antifreeze polymeric peptides

[0075] L929 cells were seeded into 96-well plates, 100 μL of culture medium was added, and the plates were incubated at 37°C and 5% CO2 for 24 h.

[0076] Take three 5mL sample vials and weigh out 1mg, 5mg, and 10mg of tetra-chain star-shaped methyl antifreeze peptides with a degree of polymerization of 10 per chain and a total degree of polymerization of 40, respectively. Add 1mL of culture medium to each vial, dissolve completely, and pass through a membrane to prepare sample solutions with concentrations of 1mg / mL, 5mg / mL, and 10mg / mL.

[0077] Remove the old culture medium, add 100 μL of sample solution, and incubate at 37°C and 5% CO2 for 24 h.

[0078] Add 10 μL of CCK8 solution, incubate for 2 hours, and measure the absorbance at 450 nm using a microplate reader to calculate the viability. See the instruction manual for cytotoxicity data. Figure 7 .

Claims

1. A tetra-stranded 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 takes a value between 10 and 100.

2. The method for preparing the four-chain star-shaped aggregated peptide with antifreeze activity as described in claim 1, characterized in that, When R is methyl, the preparation steps are as follows: (A1) In a solvent, BOC-glycine, iodomethane, and sodium hydride were added in sequence and stirred overnight. Iodomethane and sodium hydride were added again and stirred overnight. After the reaction was completed, ethyl acetate and distilled water were added to dissolve the reaction product. The product was extracted by rotary evaporation, dried, and the solvent was removed by rotary evaporation to obtain a white oily product 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid. (A2) In step (A1), the product 2-(N,N-tert-butylcarbonyl-methylamino)acetic acid and phosphorus trichloride complete cyclization in an N2 atmosphere, and recrystallize multiple times in a glove box to obtain the product Me-NCA monomer; (A3) The polymerization reaction was carried out in a glove box, with the addition of Me-NCA monomer and initiator. After the reaction was completed, a poor solvent was added for precipitation. After separation and drying, antifreeze tetra-chain star-shaped methyl peptides with different degrees of polymerization were obtained.

3. The method for preparing the four-chain star-shaped aggregated peptide with antifreeze activity as described in claim 2, characterized in that, In step (A1), the solvent is tetrahydrofuran, and the molar ratio of BOC-glycine, iodomethane, and sodium hydride is 1:4:

5. After overnight incubation, 2 equivalents of iodomethane and 1.5 equivalents of sodium hydride are added. The specific extraction steps are as follows: First, add diethyl ether and pure water for layer extraction and take aqueous phase a; then add saturated sodium bicarbonate aqueous solution to the diethyl 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 1 time, 5 wt% sodium thiosulfate 2 times, and saturated brine 1 time; dry with anhydrous magnesium sulfate.

4. The method for preparing the four-chain star-shaped aggregated peptide with antifreeze activity as described in claim 2, 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 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 aggregated peptide with antifreeze activity as described in claim 2, characterized in that, In step (A3), the Me-NCA monomer is reacted in anhydrous acetonitrile at a reaction temperature of 50 °C. The initiator is PAMAM dendritic polymer, and the molar ratio of monomer to initiator is 4n:

1. The unsuitable solvent is tetrahydrofuran or diethyl ether.

6. The method for preparing the four-chain star-shaped aggregated peptide with antifreeze activity as described in claim 1, characterized in that, When R is one of ethyl, propyl, isopropyl, or allyl, the preparation steps are as follows: (B1) Add glyoxylic acid monohydrate, dichloromethane, and amine organic compounds in proportion, stir and react for 24 hours, and remove the solvent by rotary evaporation to obtain a yellowish-brown oily substance. Among them, the amine organic compounds are one of ethylamine, propylamine, isopropylamine or allylamine; (B2) Add hydrochloric acid to the product of step (B1), stir and reflux overnight, remove the solvent by rotary evaporation, recrystallize with methanol and diethyl ether, and filter to obtain product 1; (B3) Triethylamine and ditert-butyl dicarbonate were added to the product of step (B2), and after multiple extractions, the product was dried with anhydrous magnesium sulfate and the solvent was evaporated by rotary evaporation to obtain product 2. (B4) In step (B3), the product reacts with phosphorus trichloride to form a cyclization reaction in an N2 atmosphere. Multiple recrystallizations yielded Et-NCA monomer, Pr-NCA monomer, and [other products]. i Pr-NCA monomers, Al-NCA monomers; (B5) The polymerization reaction is carried out in an N2 atmosphere. Any monomer and initiator obtained in step (B4) are added. After the reaction is completed, a poor solvent is added for precipitation. After separation and drying, antifreeze polymerized peptides with different degrees of polymerization and different substituents are obtained.

7. The method for preparing the four-chain star-shaped aggregated peptide with antifreeze activity as described in claim 6, characterized in that, In step (B1), the molar ratio of amine organic compounds to glyoxylic acid monohydrate is 1:2; In step (B2), the concentration of hydrochloric acid is 1 mol / L, and the molar ratio of hydrochloric acid to amine organic compounds is 5:1; the reflux temperature is 120°C.

8. The method for preparing the four-chain star-shaped aggregated peptide with antifreeze activity as described in claim 6, 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; the product is extracted once with n-hexane, once with 4 mol / L hydrochloric acid, three times with ethyl acetate, and once with saturated brine; and dried with anhydrous magnesium sulfate. In step (B4), the reaction is carried out in an N2 atmosphere, the molar ratio of product 2 to phosphorus trichloride is 1:1.2, and the solvent is dichloromethane; recrystallization is performed twice with dichloromethane and n-hexane, and once with tetrahydrofuran and n-hexane.

9. The method for preparing the four-chain star-shaped aggregated peptide with antifreeze activity as described in claim 6, characterized in that, In step (B5), the monomer reacts in anhydrous tetrahydrofuran at a reaction temperature of 50 °C. The initiator is PAMAM dendritic polymer, and the molar ratio of monomer to initiator is 4n:

1. The unsuitable solvent is n-hexane. The structural formula of the initiator is: 。 10. Using the four-chain star-shaped aggregate peptide with antifreeze activity as described in claim 1 as an antifreeze agent for cell cryopreservation.

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