Star-shaped bionic peptoid cryoprotectant as well as preparation method and application thereof

Abstract

The invention belongs to the field of biopolymer synthesis, and discloses a star-shaped bionic peptoid cryoprotectant as well as a preparation method and application thereof. According to the bionic peptoid cryoprotectant, a star-shaped structure with an isopropyl alcohol group as an amphiphilic side chain, an alkyl chain as a hydrophobic side chain and tris (2-aminoethyl) amine as a central pivot is designed, and the star-shaped bionic peptoid cryoprotectant containing different hydrophilic and hydrophobic sequences is synthesized through a solid-phase sub-monomer synthesis method; the star-shaped bionic peptoid cryoprotectant can form hydrogen bond interaction with the surface of an ice crystal, and hydrophobic alkyl contained in the star-shaped bionic peptoid cryoprotectant generates hydrophobic interaction to prevent movement of water molecules and inhibit aggregation of the water molecules, so that growth of the ice crystal is effectively controlled.

Description

Technical Field

[0001] The invention belongs to the field of biopolymer synthesis, and in particular relates to a star-shaped bionic peptide cryoprotectant and a preparation method and application thereof. Background Art

[0002] The metabolism of organisms slows down as the temperature decreases. People have long tried to use low temperatures to preserve living organisms for a long time. However, the growth of ice crystals at low temperatures will cause cell dehydration or intracellular ice necrosis, and this damage is irreversible. At present, we still cannot ensure that cells, tissues, and organs can maintain their original vitality and function after long-term freezing and thawing. The reason is that they will be damaged by ice crystals during the freezing and thawing process. Effectively reducing ice crystal damage is crucial for deep-cryopreservation of living cells, tissues, and organs, and it is also the key to effectively improving the efficiency and quality of cryopreservation. With the continuous development of cryobiology, researchers have discovered natural antifreeze proteins (AFPs) and various types of synthetic cryoprotectants, all of which have the effects of lowering the freezing point, slowing down the growth of ice crystals, and preventing ice recrystallization.

[0003] Natural antifreeze proteins (AFPs) found in some low-temperature-resistant organisms have excellent antifreeze properties, but they are prone to induce rejection reactions and cause biological hazards, and the extraction cost is huge. Therefore, artificial synthetic cryoprotectants have received widespread attention from researchers. Currently, commonly used artificial synthetic cryoprotectants include dimethyl sulfoxide, glycerol, polyethylene glycol, polyvinyl alcohol, etc., but they have the disadvantages of poor antifreeze effects and certain toxicity. They cannot be widely used in fields that require high safety, such as biomedicine and food processing. In order to achieve application and development in biological-related fields, especially in the field of low-temperature cryopreservation, we still need to continue to meet the challenge of finding efficient and safe cryoprotectants.

[0004] Peptoids, also known as N-substituted polyglycine, are a type of peptoid biomaterial. What distinguishes peptoids from polypeptides is that the substituents on the peptoid main chain structure are transferred from the α-carbon of the polypeptide to the N atom. Therefore, there are no chiral centers and intermolecular hydrogen bonds in the peptoid main chain. The disappearance of the chiral center makes it difficult for the peptoid to form the same secondary structure as the polypeptide through hydrogen bond interactions, making the peptoid have better solubility and thermal processability. Changing the peptoid side chain structure can adjust the steric hindrance and electronic effects, affecting the spatial conformation of the peptoid. At the same time, different side chain groups enable the peptoid to obtain different properties, such as solubility, crystallinity, degradability, etc., and the peptoid has the advantages of good biocompatibility, resistance to enzymatic hydrolysis, low cytotoxicity, etc., making the peptoid material applicable to many fields such as biomedicine, biosensors, chemical catalysis and nanomaterials.

[0005] Star polymers are widely studied in the biomedical field due to their unique topological structures and excellent physical and chemical properties, such as enhanced stimuli responsiveness, biocompatibility and biodegradability. Currently, star polymers are used as drug delivery, gene delivery, tissue engineering, diagnosis, medical devices and antibacterial / antifouling biomaterials. Designing star polymers with appropriate functional groups is particularly important for biomedical applications. Summary of the invention

[0006] The present invention uses peptoids as raw materials, simulates the side chain groups of natural antifreeze proteins and designs a star-shaped topological structure, and uses a solid-phase sub-monomer synthesis method to prepare amphiphilic star-shaped peptoid oligomers. Compared with the most commonly used antifreeze agent DMSO, the prepared peptoids not only have excellent antifreeze properties, but are also non-toxic to cells and have higher biocompatibility, so they can be widely used in food preservation, medical engineering, agriculture and other fields.

[0007] The invention provides a star-shaped bionic peptide cryoprotectant and a preparation method and application thereof.

[0008] The bionic peptoid cryoprotectant of the present invention is designed with a star-shaped structure having an isopropanol group as an amphiphilic side chain, an alkyl chain as a hydrophobic side chain, and tris(2-aminoethyl)amine as a central pivot. The star-shaped bionic peptoid cryoprotectant containing different hydrophilic and hydrophobic sequences is synthesized by a solid-phase sub-monomer synthesis method. The star-shaped bionic peptoid cryoprotectant can form hydrogen bond interactions with the surface of ice crystals, and the hydrophobic alkyl groups contained therein produce hydrophobic interactions, thereby preventing the movement of water molecules and inhibiting the aggregation of water molecules, thereby effectively controlling the growth of ice crystals.

[0009] The present invention first provides a star-shaped bionic peptoid cryoprotectant, the structural formula of which is shown below:

[0010]

[0011] Among them, u is 0 to 12, v is 0 to 12, w is 0 to 4, and u and v cannot be 0 at the same time;

[0012] m is 0 to 12, n is 0 to 12, p is 0 to 4, m and n cannot be 0 at the same time;

[0013] x is 1 to 12, and y is 1 to 12.

[0014] The present invention also provides a method for preparing the above-mentioned star-shaped bionic peptide cryoprotectant, the steps of which are as follows:

[0015] (1) Preparation of primary amine monomers by hydroxyl protection of isopropanolamine:

[0016] Anhydrous dichloromethane, isopropanolamine and triethylamine were added to a round-bottom flask, a magnetic rod was placed, the bottle mouth was sealed with a rubber stopper, and the first stirring reaction was carried out in an ice bath environment; then trimethylchlorosilane was slowly added with a syringe, the needle was left on the rubber stopper for degassing, and the second stirring reaction was continued; after removing the ice bath, the mixture was stirred at room temperature and the third stirring reaction was carried out; the reaction solution was transferred to a separatory funnel, distilled water was added and shaken to degas, the lower layer solution was transferred to a conical flask, and an appropriate amount of anhydrous magnesium sulfate was added for drying; the liquid part was filtered into a new round-bottom flask, and the solvent was removed by rotary evaporation to obtain a white transparent liquid product, which was isopropanolamine monomer.

[0017] (2) Resin swelling: Add amide resin into a synthesis tube, add DMF, blow nitrogen gas and stir to make the resin fully swell, then filter;

[0018] (3) Deprotection: Add a DMF solution of 4-methylpiperidine to the product obtained in step (2), blow nitrogen gas through it, and then filter it; add a DMF solution of 4-methylpiperidine again, blow nitrogen gas through it, filter it, and then add DMF for washing.

[0019] (4) Synthesis of the first arm:

[0020] Acylation: Add a DMF solution of bromoacetic acid and N,N'-diisopropylcarbodiimide to the deprotected resin obtained in step (3), blow nitrogen until the acylation reaction is complete, filter and wash to obtain product A;

[0021] Primary amine substitution: add a DMF solution of primary amine to product A, blow nitrogen until the substitution reaction is complete, filter and wash to obtain product B;

[0022] Repeat the acylation and primary amine substitution operations (u+v)×x-1 times according to the designed structure of product B to obtain product C;

[0023] (5) Substitution of the central pivot tris(2-aminoethyl)amine:

[0024] Acylation: subjecting product C to the acylation reaction in step (4) to obtain product D;

[0025] Tris(2-aminoethyl)amine substitution: Add a DMF solution of tris(2-aminoethyl)amine to product D, blow nitrogen gas until the substitution reaction is complete, filter and wash to obtain product E;

[0026] (6) Synthesis of the last three arms:

[0027] Acylation: Add bromoacetic acid in DMF and N,N'-diisopropylcarbodiimide to product E, blow nitrogen until the reaction is complete, filter and wash to obtain product F;

[0028] Primary amine substitution: Add a DMF solution of a primary amine to the product F to carry out a substitution reaction, blow nitrogen gas until the reaction is complete, filter and wash to obtain the product G;

[0029] Repeat the acylation and primary amine substitution operations (m+n)×y-1 times for product G according to the designed structure, wash with DMF and DCM to obtain product H; transfer to a sample bottle after drying, and seal and freeze for storage;

[0030] (7) Cracking: adding the cracking solution to the product H, stirring and filtering, and blowing nitrogen gas over the filtrate to dryness to obtain the crude product of the star-shaped biomimetic peptoid cryoprotectant.

[0031] In step (1), the volume ratio of anhydrous dichloromethane, isopropanolamine and triethylamine is 300:7.75:21.06; the volume ratio of trimethylchlorosilane to isopropanolamine is 15.5:7.75;

[0032] The first stirring reaction time was 10 min, the second stirring time was 20 min, and the third stirring reaction time at room temperature was 16 h.

[0033] In step (2), the usage ratio of the amide resin to N,N'-dimethylformamide is 100 mg:2 mL; and the swelling time is 10 min.

[0034] In step (3), in the DMF solution of 4-methylpiperidine, the volume ratio of 4-methylpiperidine to DMF is 1:4;

[0035] The volume ratio of the DMF solution added with 4-methylpiperidine for the first time to the DMF solution added with 4-methylpiperidine for the second time is 1:1;

[0036] When the DMF solution of 4-methylpiperidine was added for the first time, bubbling was performed for 2 minutes; when the DMF solution of 4-methylpiperidine was added for the second time, bubbling was performed for 12 minutes.

[0037] In step (4), the primary amine is isopropanolamine monomer or NH2-(CH2) w -CH3, where w is 0 to 4;

[0038] The amount ratio of the DMF solution of bromoacetic acid, the N,N'-diisopropylcarbodiimide solution, the DMF solution of the primary amine and the amide resin of step (2) is 1 mL:86 μL:1 mL:100 mg; wherein the concentration of the DMF solution of bromoacetic acid is 0.6 M; the concentration of the DMF solution of the primary amine is 1 M;

[0039] The acylation reaction time is 10 min;

[0040] The time of the substitution reaction is 10 min;

[0041] The w is 0-4, u is 0-12, v is 0-12, and x is 1-12.

[0042] In step (5), the volume ratio of the DMF solution of tris(2-aminoethyl)amine to the DMF solution of the primary amine in step (4) is 1:1; wherein the concentration of the DMF solution of tris(2-aminoethyl)amine is 0.6 M;

[0043] The time of the substitution reaction is 30 min.

[0044] In step (6), the primary amine is isopropanolamine monomer or NH2-(CH2) p -CH3, where p is 0 to 4;

[0045] The amount ratio of the DMF solution of bromoacetic acid, the N,N'-diisopropylcarbodiimide solution, the DMF solution of the primary amine and the amide resin of step (2) is 1 mL: 258 μL: 3 mL: 100 mg; wherein the concentration of the DMF solution of bromoacetic acid is 1.8 M; the concentration of the DMF solution of the primary amine is 1 M;

[0046] The acylation reaction time is 20 min; the substitution reaction time is 30 min;

[0047] The w is 0-4, m is 0-12, n is 0-12, and y is 1-12.

[0048] In step (7), the lysis solution is a mixed solution comprising triisopropylsilane, pure water and trifluoroacetic acid, wherein the volume ratio of triisopropylsilane, pure water and trifluoroacetic acid is 2.5:2.5:95;

[0049] The ratio of the lysate to the amide resin in step (2) is 4 mL:100 mg;

[0050] The stirring time is 10 min.

[0051] The star-shaped bionic peptide prepared by the invention is used as a cryoprotectant for biological samples.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The present invention designs and prepares an amphiphilic star-shaped peptoid oligomer with controllable molecular weight and unique topological structure; the star-shaped biomimetic peptoid oligomer uses an isopropanol group as an amphiphilic side chain, an alkyl chain as a hydrophobic side chain, and tris(2-aminoethyl)amine as a central pivot, and is synthesized by a solid-phase sub-monomer synthesis method according to the designed structure to obtain a star-shaped biomimetic peptoid oligomer containing different hydrophilic and hydrophobic sequences; the star-shaped biomimetic peptoid oligomer has excellent ice recrystallization inhibitory activity, has the effect of changing the morphology of ice crystals and reducing the growth rate of ice crystals, and can be used as a biomimetic peptoid cryoprotectant.

[0054] (2) The star-shaped biomimetic peptide cryoprotectant of the present invention uses a mixed solution of triisopropylsilane, pure water and trifluoroacetic acid as a lysing solution during the preparation process, which can smoothly remove the carrier without affecting the product molecules;

[0055] (3) The preparation process of the present invention has the characteristics of easy operation, high repeatability and high accuracy;

[0056] (4) The star-shaped biomimetic peptide cryoprotectant of the present invention has broad application prospects in biomedicine, low-temperature preservation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the solid phase submonomer synthesis method.

[0058] Figure 2 : This is a comparison chart of ice crystal sizes of PBS solutions containing the star-shaped peptoid described in Example 1.

[0059] Figure 3 This is a comparison chart of ice crystal sizes of PBS solutions containing the star-shaped peptoid described in Example 2.

[0060] Figure 4 : is a comparison chart of ice crystal sizes of PBS solutions containing the star-shaped peptoid described in Example 3.

[0061] Figure 5 This is a morphology diagram of the ice crystal growth process of the aqueous solution containing the star-shaped peptide described in Example 1.

[0062] Figure 6 This is a morphology diagram of the ice crystal growth process of the aqueous solution containing the star-shaped peptide described in Example 2.

[0063] Figure 7 This is a morphology diagram of the ice crystal growth process of the aqueous solution containing the star-shaped peptide described in Example 3. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0065] Embodiment 1:

[0066] In this embodiment, the Figure 1 The solid phase sub-monomer synthesis method shown in the figure prepared a star-shaped biomimetic peptide cryoprotectant with the structural formula shown below:

[0067]

[0068] The specific preparation method is as follows:

[0069] 1. Synthesis of crude product:

[0070] (1) Cleaning: Add the silanization solution (C2H6Cl2Si / DCM, v / v=1:19) prepared in advance into the synthesis tube, let it stand for 30 min, then remove it, add DCM and CH3OH in turn, filter and wash, and dry it for later use.

[0071] (2) Swelling: 100 mg of amide resin was weighed using an electronic balance and added to a synthesis tube. 2 mL of DMF was added and nitrogen was blown through for 10 min to allow the resin to fully swell. The mixture was then filtered using a vacuum circulation pump.

[0072] (3) Deprotection: In order to remove the protecting groups on the resin, add 1 mL of 4-methylpiperidine / DMF (v / v=1:4) solution to the synthesis tube, blow nitrogen for 2 min, and then filter; add 1 mL of 4-methylpiperidine / DMF solution again, blow nitrogen for 12 min, and then filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to remove residues.

[0073] (4) Synthesis of the first arm:

[0074] Acylation: Add 1 mL of 0.6 M bromoacetic acid / DMF solution and 86 μL of N,N'-diisopropylcarbodiimide to the synthesis tube, blow nitrogen for 10 min, filter, add 2 mL of DMF for washing, repeat the washing 5 times and filter to obtain product A;

[0075] Isopropanolamine monomer substitution: add 1 mL of 1 M isopropanolamine monomer / DMF solution into the synthesis tube, blow nitrogen for 10 min, filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to obtain product B;

[0076] The product B was subjected to the acylation and isopropanolamine monomer substitution operations for two times according to the designed structure to obtain the product C;

[0077] (5) Substitution of the central pivot tris(2-aminoethyl)amine:

[0078] Acylation: subjecting product C to the acylation reaction in step (4) to obtain product D;

[0079] Tris(2-aminoethyl)amine substitution: add 1 mL of 0.6 M tris(2-aminoethyl)amine / DMF solution to the synthesis tube, blow nitrogen for 30 min, filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to obtain product E;

[0080] (6) Synthesis of the last three arms:

[0081] Acylation: Add 1 mL of 1.8 M bromoacetic acid / DMF solution and 258 μL of N,N'-diisopropylcarbodiimide to the synthesis tube, blow nitrogen for 20 min, filter, then add 2 mL of DMF for washing, repeat the washing 5 times and filter to obtain product F;

[0082] Isopropanolamine monomer substitution: add 3 mL of 1 M isopropanolamine monomer / DMF solution into the synthesis tube, blow nitrogen for 30 min, filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to obtain product G;

[0083] The product G was acylated and replaced with isopropanolamine monomers twice according to the designed structure, rinsed with DMF 5 times and filtered, then rinsed with DCM 3 times, dried at room temperature and transferred to a sample bottle to obtain product H, which was sealed and stored at -18°C.

[0084] (7) Cracking: Add 4 mL of lysate to the product H, shake at room temperature for 10 min, transfer the resulting solution to a new sample bottle after filtration, and blow nitrogen to completely evaporate the lysate to obtain a relatively viscous yellow transparent colloid, i.e., the crude product of the star-shaped biomimetic peptoid cryoprotectant. The lysate is a mixed solution comprising triisopropylsilane, pure water and trifluoroacetic acid, wherein the volume ratio of triisopropylsilane, pure water and trifluoroacetic acid is 2.5:2.5:95.

[0085] 2. Purification of crude product: The crude product was purified by preparative high performance liquid chromatography.

[0086] Embodiment 2:

[0087] In this example, a star-shaped biomimetic peptide cryoprotectant with the structural formula shown below was prepared:

[0088]

[0089] The specific preparation method is as follows:

[0090] 1. Synthesis of crude product:

[0091] (1) Cleaning: Add the silanization solution (C2H6Cl2Si / DCM, v / v=1:19) prepared in advance into the synthesis tube, let it stand for 30 min, then remove it, add DCM and CH3OH in turn, filter and wash, and dry it for later use.

[0092] (2) Swelling: 100 mg of amide resin was weighed using an electronic balance and added to a synthesis tube. 2 mL of DMF was added and nitrogen was blown through for 10 min to allow the resin to fully swell. The mixture was then filtered using a vacuum circulation pump.

[0093] (3) Deprotection: In order to remove the protecting groups on the resin, add 1 mL of 4-methylpiperidine / DMF (v / v=1:4) solution to the synthesis tube, blow nitrogen for 2 min, and then filter; add 1 mL of 4-methylpiperidine / DMF solution again, blow nitrogen for 12 min, and then filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to remove residues.

[0094] (4) Synthesis of the first arm:

[0095] Acylation: Add 1 mL of 0.6 M bromoacetic acid / DMF solution and 86 μL of N,N'-diisopropylcarbodiimide to the synthesis tube, blow nitrogen for 10 min, filter, add 2 mL of DMF for washing, repeat the washing 5 times and filter to obtain product A;

[0096] Primary amine substitution: add 1 mL of 1 M primary amine (methylamine / isopropanolamine monomer) / DMF solution into a synthesis tube, blow nitrogen for 10 min, filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to obtain product B;

[0097] Repeat the acylation and primary amine (methylamine / isopropanolamine monomer) substitution operations of product B 5 times according to the designed structure to obtain product C;

[0098] (5) Substitution of the central pivot tris(2-aminoethyl)amine:

[0099] Acylation: subjecting product C to the acylation reaction in step (4) to obtain product D;

[0100] Tris(2-aminoethyl)amine substitution: add 1 mL of 0.6 M tris(2-aminoethyl)amine / DMF solution to the synthesis tube, blow nitrogen for 30 min, filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to obtain product E;

[0101] (6) Synthesis of the last three arms:

[0102] Acylation: Add 1 mL of 1.8 M bromoacetic acid / DMF solution and 258 μL of N,N'-diisopropylcarbodiimide to the synthesis tube, blow nitrogen for 20 min, filter, then add 2 mL of DMF for washing, repeat the washing 5 times and filter to obtain product F;

[0103] Primary amine substitution: add 3 mL of 1 M primary amine (methylamine / isopropanolamine monomer) / DMF solution into the synthesis tube, blow nitrogen for 30 min, filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to obtain product G;

[0104] The product G was acylated and replaced with primary amine (methylamine / isopropanolamine monomer) 5 times according to the designed structure, rinsed with DMF 5 times and filtered, then rinsed with DCM 3 times, dried at room temperature and transferred to a sample bottle to obtain product H, which was sealed and stored at -18°C.

[0105] (7) Cracking: Add 4 mL of lysate to the product H, shake at room temperature for 10 min, transfer the resulting solution to a new sample bottle after filtration, and blow nitrogen to completely evaporate the lysate to obtain a relatively viscous yellow transparent colloid, i.e., the crude product of the star-shaped biomimetic peptoid cryoprotectant. The lysate is a mixed solution comprising triisopropylsilane, pure water and trifluoroacetic acid, wherein the volume ratio of triisopropylsilane, pure water and trifluoroacetic acid is 2.5:2.5:95.

[0106] 2. Purification of crude product: The crude product was purified by preparative high performance liquid chromatography.

[0107] Embodiment 3:

[0108] In this example, a star-shaped biomimetic peptide cryoprotectant with the structural formula shown below was prepared:

[0109]

[0110] The specific preparation method is as follows:

[0111] 1. Synthesis of crude product:

[0112] (1) Cleaning: Add the silanization solution (C2H6Cl2Si / DCM, v / v=1:19) prepared in advance into the synthesis tube, let it stand for 30 min, then remove it, add DCM and CH3OH in turn, filter and wash, and dry it for later use.

[0113] (2) Swelling: 100 mg of amide resin was weighed using an electronic balance and added to a synthesis tube. 2 mL of DMF was added and nitrogen was blown through for 10 min to allow the resin to fully swell. The mixture was then filtered using a vacuum circulation pump.

[0114] (3) Deprotection: In order to remove the protecting groups on the resin, add 1 mL of 4-methylpiperidine / DMF (v / v=1:4) solution to the synthesis tube, blow nitrogen for 2 min, and then filter; add 1 mL of 4-methylpiperidine / DMF solution again, blow nitrogen for 12 min, and then filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to remove residues.

[0115] (4) Synthesis of the first arm:

[0116] Acylation: Add 1 mL of 0.6 M bromoacetic acid / DMF solution and 86 μL of N,N'-diisopropylcarbodiimide to the synthesis tube, blow nitrogen for 10 min, filter, add 2 mL of DMF for washing, repeat the washing 5 times and filter to obtain product A;

[0117] Primary amine substitution: add 1 mL of 1 M primary amine (methylamine / isopropanolamine monomer) / DMF solution into a synthesis tube, blow nitrogen for 10 min, filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to obtain product B;

[0118] Repeat the acylation and primary amine (methylamine / isopropanolamine monomer) substitution operations of product B 5 times according to the designed structure to obtain product C;

[0119] (5) Substitution of the central pivot tris(2-aminoethyl)amine:

[0120] Acylation: subjecting product C to the acylation reaction in step (4) to obtain product D;

[0121] Tris(2-aminoethyl)amine substitution: add 1 mL of 0.6 M tris(2-aminoethyl)amine / DMF solution to the synthesis tube, blow nitrogen for 30 min, filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to obtain product E;

[0122] (6) Synthesis of the last three arms:

[0123] Acylation: Add 1 mL of 1.8 M bromoacetic acid / DMF solution and 258 μL of N,N'-diisopropylcarbodiimide to the synthesis tube, blow nitrogen for 20 min, filter, then add 2 mL of DMF for washing, repeat the washing 5 times and filter to obtain product F;

[0124] Primary amine substitution: add 3 mL of 1 M primary amine (ethylamine / isopropanolamine monomer) / DMF solution into the synthesis tube, blow nitrogen for 30 min, filter, then add 2 mL of DMF for rinsing, repeat rinsing 5 times and filter to obtain product G;

[0125] The product G was acylated and replaced with primary amine (ethylamine / isopropanolamine monomer) three times according to the designed structure, rinsed with DMF five times and filtered, then rinsed with DCM three times, dried at room temperature and transferred to a sample bottle to obtain product H, which was sealed and stored at -18°C.

[0126] (7) Cracking: Add 4 mL of lysate to the product H, shake at room temperature for 10 min, transfer the resulting solution to a new sample bottle after filtration, and blow nitrogen to completely evaporate the lysate to obtain a relatively viscous yellow transparent colloid, i.e., the crude product of the star-shaped biomimetic peptoid cryoprotectant. The lysate is a mixed solution comprising triisopropylsilane, pure water and trifluoroacetic acid, wherein the volume ratio of triisopropylsilane, pure water and trifluoroacetic acid is 2.5:2.5:95.

[0127] 2. Purification of crude product: The crude product was purified by preparative high performance liquid chromatography.

[0128] Embodiment 4:

[0129] In this example, the ice recrystallization inhibition (IRI) activity of the star-shaped biomimetic peptide cryoprotectant was investigated by ice recrystallization experiment. The specific investigation steps are as follows:

[0130] The ice recrystallization experiment used the Splat cooling assay (SCA) method to detect IRI activity. A 10 μL drop of PBS solution of star-shaped biomimetic peptoid cryoprotectant was quickly dropped from a fixed height (h = 1.5m) to the center of a glass slide (precooled to -60°C) to form an ultra-thin solid ice layer, which was then heated to -6°C and maintained for 30 minutes to allow the ice layer to recrystallize and grow. The size of the ice crystals during the recrystallization process was observed "in situ and in real time" under a microscope. In the next 20 minutes, a high-speed camera was used to record micrographs of the sample ice crystals to obtain the particle size defined by the two largest orthogonal dimensions of the ice particle surface. For three concentrations of the sample (1 mg·mL -1 , 5mg·mL -1 , 10mg·mL -1 ) were performed in parallel for at least three times.

[0131] Depend on Figure 2 , Figure 3 , Figure 4 It can be seen that the samples of Example 1, Example 2 and Example 3 all have ice recrystallization inhibition activity, and as the sample concentration increases, the IRI activity increases.

[0132] Embodiment 5:

[0133] In this example, the ability of the star-shaped biomimetic peptide cryoprotectant to change the morphology and growth rate of ice crystals was investigated by a nanoliter osmotic pressure experiment. The specific investigation steps are as follows:

[0134] Prepare 10 mg mL -1 A pure aqueous solution of the star-shaped biomimetic peptide cryoprotectant was injected into a six-well plate filled with immersion oil using a microsyringe, and the temperature was adjusted to rapidly cool to about -20°C until it froze. The temperature was slowly raised to the melting temperature (T m ), melt until only stable single ice crystals remain, then adjust the temperature to supercool. When the ice crystals just begin to grow, use a high-speed camera to record the growth process of the ice crystals, and take photos of the ice crystals at multiple different time periods. The ice crystal growth rate is the growth amount of a single ice crystal divided by the time spent in the growth process.

[0135] Depend on Figure 5 , Figure 6 , Figure 7 It can be seen that the samples of Example 1, Example 2, and Example 3 can interact with ice crystals, change the morphology of pure water ice crystals, make the ice crystals grow from round to hexagonal, and have the function of reducing the growth rate of ice crystals.

[0136] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A star-shaped biomimetic peptide cryoprotectant, characterized in that: Its structural formula is as follows: in, u is 0 to 12, v is 0 to 12, w is 0 to 4, u and v cannot be 0 at the same time; m is 0 to 12, n is 0 to 12, p is 0 to 4, m and n cannot be 0 at the same time; x is 1 to 12, and y is 1 to 12.

2. The method for preparing the star-shaped biomimetic peptoid cryoprotectant according to claim 1, characterized in that: Here are the steps: (1) Preparation of primary amine monomers by hydroxyl protection of isopropanolamine: Anhydrous dichloromethane, isopropanolamine and triethylamine are added to a round-bottom flask, a magnetic rod is placed, the bottle mouth is sealed with a rubber stopper, and the first stirring reaction is carried out in an ice bath environment; then trimethylsilyl chloride is slowly added with a syringe, the needle is left on the rubber stopper for degassing, and the second stirring reaction is continued; after removing the ice bath, the mixture is stirred at room temperature and the third stirring reaction is carried out; the reaction solution is transferred to a separatory funnel, distilled water is added and shaken to degas, the lower layer solution is transferred to a conical flask, and an appropriate amount of anhydrous magnesium sulfate is added for drying; the liquid part is filtered into a new round-bottom flask, and the solvent is removed by rotary evaporation to obtain a white transparent liquid product which is isopropanolamine monomer; (2) Resin swelling: Add amide resin into a synthesis tube, add DMF, blow nitrogen gas and stir to make the resin fully swell, then filter; (3) Deprotection: adding a DMF solution of 4-methylpiperidine to the product obtained in step (2), blowing nitrogen gas through it, and then filtering it with suction; adding a DMF solution of 4-methylpiperidine again, blowing nitrogen gas through it, filtering it with suction, and then adding DMF for washing; (4) Synthesis of the first arm: Acylation: Add a DMF solution of bromoacetic acid and N,N'-diisopropylcarbodiimide to the deprotected resin obtained in step (3), blow nitrogen until the acylation reaction is complete, filter and wash to obtain product A; Primary amine substitution: add a DMF solution of primary amine to product A, blow nitrogen until the substitution reaction is complete, filter and wash to obtain product B; Repeat the acylation and primary amine substitution operations (u+v)×x-1 times according to the designed structure of product B to obtain product C; (5) Substitution of the central pivot tris(2-aminoethyl)amine: Acylation: subjecting product C to the acylation reaction in step (4) to obtain product D; Tris(2-aminoethyl)amine substitution: Add a DMF solution of tris(2-aminoethyl)amine to product D, blow nitrogen gas until the substitution reaction is complete, filter and wash to obtain product E; (6) Synthesis of the last three arms: Acylation: Add bromoacetic acid in DMF and N,N'-diisopropylcarbodiimide to product E, blow nitrogen until the reaction is complete, filter and wash to obtain product F; Primary amine substitution: Add a DMF solution of a primary amine to the product F to carry out a substitution reaction, blow nitrogen gas until the reaction is complete, filter and wash to obtain the product G; Repeat the acylation and primary amine substitution operations (m+n)×y-1 times for product G according to the designed structure, wash with DMF and DCM to obtain product H; transfer to a sample bottle after drying, and seal and freeze for storage; (7) Cracking: adding the cracking solution to the product H, stirring and filtering, and blowing nitrogen gas over the filtrate to dryness to obtain the crude product of the star-shaped biomimetic peptoid cryoprotectant.

3. The preparation method according to claim 2, characterized in that: In step (1), the volume ratio of anhydrous dichloromethane, isopropanolamine and triethylamine is 300:7.75:21.06; the volume ratio of trimethylchlorosilane to isopropanolamine is 15.5:7.75; The first stirring reaction time was 10 min, the second stirring time was 20 min, and the third stirring reaction time at room temperature was 16 h.

4. The preparation method according to claim 2, characterized in that: In step (2), the usage ratio of the amide resin to N,N'-dimethylformamide is 100 mg:2 mL; and the swelling time is 10 min.

5. The preparation method according to claim 2, characterized in that: In step (3), in the DMF solution of 4-methylpiperidine, the volume ratio of 4-methylpiperidine to DMF is 1:4; The volume ratio of the DMF solution added with 4-methylpiperidine for the first time to the DMF solution added with 4-methylpiperidine for the second time is 1:1; When the DMF solution of 4-methylpiperidine was added for the first time, bubbling was performed for 2 minutes; when the DMF solution of 4-methylpiperidine was added for the second time, bubbling was performed for 12 minutes.

6. The preparation method according to claim 2, characterized in that: In step (4), the primary amine is isopropanolamine monomer or NH2-(CH2) w -CH3, where w is 0 to 4; The amount ratio of the DMF solution of bromoacetic acid, the N,N'-diisopropylcarbodiimide solution, the DMF solution of the primary amine and the amide resin of step (2) is 1 mL:86 μL:1 mL:100 mg; wherein the concentration of the DMF solution of bromoacetic acid is 0.6 M; the concentration of the DMF solution of the primary amine is 1 M; The acylation reaction time is 10 min; The time of the substitution reaction is 10 min; The w is 0-4, u is 0-12, v is 0-12, and x is 1-12.

7. The preparation method according to claim 2, characterized in that: In step (5), the volume ratio of the DMF solution of tris(2-aminoethyl)amine to the DMF solution of the primary amine in step (4) is 1:1; wherein the concentration of the DMF solution of tris(2-aminoethyl)amine is 0.6 M; The substitution reaction time is 30 min.

8. The preparation method according to claim 2, characterized in that: In step (6), the primary amine is isopropanolamine monomer or NH2-(CH2) p -CH3, where p is 0 to 4; The amount ratio of the DMF solution of bromoacetic acid, the N,N'-diisopropylcarbodiimide solution, the DMF solution of the primary amine and the amide resin of step (2) is 1 mL: 258 μL: 3 mL: 100 mg; wherein the concentration of the DMF solution of bromoacetic acid is 1.8 M; the concentration of the DMF solution of the primary amine is 1 M; The acylation reaction time is 20 min; the substitution reaction time is 30 min; The w is 0-4, m is 0-12, n is 0-12, and y is 1-12.

9. The preparation method according to claim 2, characterized in that: In step (7), the lysis solution is a mixed solution comprising triisopropylsilane, pure water and trifluoroacetic acid, wherein the volume ratio of triisopropylsilane, pure water and trifluoroacetic acid is 2.5:2.5:95; The ratio of the lysate to the amide resin in step (2) is 4 mL:100 mg; The stirring time is 10 min.

10. Use of the star-shaped biomimetic peptoid as claimed in claim 1 as a cryoprotectant for biological samples.

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