A biomimetic peptide anti-icing and deicing agent, and a preparation method and application thereof
The biomimetic peptide antifreeze agent prepared by solid-phase submonomer synthesis solves the problems of high cost and toxicity of existing antifreeze agents, and achieves efficient and safe antifreeze effect, which is suitable for fields such as biomedicine and food processing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-04
- Publication Date
- 2026-04-14
AI Technical Summary
Existing antifreeze agents are expensive, unstable, and have a certain degree of toxicity, which limits their widespread application in fields such as biomedicine and food processing.
Using biomimetic peptides as raw materials, amphiphilic polymeric peptides with hydroxyl side chains as hydrophilic side chains and alkyl chains as hydrophobic side chains were prepared by solid-phase submonomer synthesis. Hydrogen bonding and hydrophobic interactions were used to inhibit ice crystal growth.
The prepared biomimetic peptide antifreeze agent has excellent antifreeze properties, is non-toxic to cells, has good biocompatibility, and has better antifreeze performance and biosafety, making it suitable for fields such as biomedicine, life and health, and environmental protection.
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Figure CN116284240B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biopolymer synthesis, specifically relating to a biomimetic peptide antifreeze agent, its preparation method, and its application. Background Technology
[0002] Water freezing into ice at low temperatures can cause significant disruption to people's lives and industrial production. For example, ice crystals formed during cryogenic storage can severely damage cells and organs, leading to loss of biological function. Therefore, preventing the formation and growth of ice crystals is crucial. Antifreeze agents function by lowering freezing temperatures, reducing ice crystal size, and preventing ice recrystallization.
[0003] Natural antifreeze proteins (AFPs) found in organisms in extremely cold regions possess excellent antifreeze effects. These are biological antifreeze materials that regulate the growth of ice nuclei and ice crystals, allowing fish, microorganisms, and other organisms to survive in environments below 0°C without freezing damage. AFPs prevent the formation of large ice particles in cells and body fluids, thus maintaining the fluid state. However, extracting AFPs from organisms is extremely difficult and expensive, and AFPs can easily reduce cell viability, trigger rejection reactions, and cause biohazards, limiting their application in production and daily life. Therefore, researchers have shifted their focus to lower-cost synthetic antifreeze agents. Existing commonly used antifreeze agents, such as polyvinyl alcohol and dimethyl sulfoxide, while possessing some antifreeze effects, are inefficient and have a certain degree of toxicity, making them unsuitable for large-scale, widespread use, especially in biomedicine and food processing. Therefore, there is an urgent need to develop ideal, highly efficient, and low-toxicity antifreeze materials that can be effectively applied in biological fields, particularly in cryopreservation.
[0004] Peptides are N-substituted polyglycines with a similar backbone structure to peptides. They are polymers that mimic peptides, but differ in that the substituents are transferred from the α-carbon of the peptide backbone to the N atom. This difference eliminates inter- and intra-chain hydrogen bonds and the chirality of the backbone, allowing the spatial conformation of the peptide to be controlled by the electronic interactions and steric hindrance of the side chains. This makes it easier to form random coil structures, resulting in good solubility in most solvents. Furthermore, peptides exhibit good thermal processability and excellent biocompatibility, making them widely applicable in biomedicine, chemical catalysis, and nanomaterials.
[0005] This invention utilizes peptide-like materials to mimic the structure of natural antifreeze proteins, employing a solid-phase submonomer synthesis method to produce amphiphilic clustered peptides possessing both hydrophilic and hydrophobic side chains. The prepared clustered peptides not only exhibit excellent antifreeze properties but also demonstrate non-toxicity to cells and good biocompatibility. Furthermore, compared to the most commonly used cryoprotectant DMSO, this peptide-based cryoprotectant offers superior antifreeze performance and higher biocompatibility, thus enabling its widespread application in fields such as biomedicine, life sciences, and environmental protection. Summary of the Invention
[0006] To address the shortcomings of existing anti-icing and antifreeze agents, such as high cost, instability, and toxicity, this invention provides a biomimetic peptide anti-icing and antifreeze agent, its preparation method, and its application. The biomimetic peptide anti-icing and antifreeze agent of this invention uses hydroxyl-containing side chains as hydrophilic side chains and alkyl chains as hydrophobic side chains. It is synthesized according to a designed structure using a solid-phase submonomer synthesis method to obtain biomimetic peptide anti-icing and antifreeze agents containing different hydrophilic and hydrophobic sequences. The biomimetic peptide anti-icing and antifreeze agent can form hydrogen bonds with the ice crystal surface to inhibit water molecule movement, and the hydrophobic alkyl groups can influence and prevent water molecule aggregation through hydrophobic interactions, thereby effectively regulating ice crystal growth.
[0007] This invention first provides a biomimetic peptide anti-icing and antifreeze agent, the structural formula of which is shown below:
[0008] Where n is 0 to 4, m is 0 to 4, v is 1 to 6, w is 1 to 6, and x is 1 to 6.
[0009] This invention also provides a method for preparing the above-mentioned biomimetic peptide antifreeze agent, comprising the following steps:
[0010] (1) Swelling: N,N'-dimethylformamide (DMF) is added to the amide resin for swelling. After stirring with nitrogen bubbling, the mixture is filtered.
[0011] (2) Deprotection: Add 4-methylpiperidine / DMF solution to the product obtained in step (1) for the first time, bubble with nitrogen and filter, and wash; then add 4-methylpiperidine / DMF solution to it for the second time, bubble with nitrogen and filter, and wash to obtain deprotected resin;
[0012] (3)NH2-(CH2) n -CH3 substitution:
[0013] Acylation: Add bromoacetic acid / DMF solution and N,N'-diisopropylcarbodiimide solution to the deprotected resin in step (2) to carry out acylation reaction. Nitrogen gas is bubbled through the reaction. After the reaction is completed, filter and wash to obtain product A.
[0014] NH2-(CH2) n -CH3 substitution: Add NH2-(CH2) to product A. n -CH3 undergoes a substitution reaction, during which nitrogen gas is bubbled through the gas. After the reaction is complete, the mixture is filtered and washed to obtain product B.
[0015] Product B was repeatedly acylated and subjected to NH2-(CH2) according to the designed structure. n The -CH3 substitution operation is performed v-1 times to obtain product C;
[0016] (4)NH2-(CH2) m Replacement of -CH2O-THP:
[0017] Acylation: Product C is subjected to the acylation reaction in step (3) to obtain product D;
[0018] NH2-(CH2) m -CH2O-THP substitution: NH2-(CH2) is added to product D. m The substitution reaction was carried out in CH2O-THP solution, with nitrogen gas bubbled through the reaction. After the reaction was completed, the solution was filtered and washed to obtain product E.
[0019] Product E was repeatedly acylated and subjected to NH2-(CH2) according to the designed structure. m The -CH2O-THP substitution operation was performed w-1 times to obtain product F;
[0020] (5) Repeat the acylation and NH2-(CH2) treatment of product F according to the designed structure. m The -CH2O-THP substitution process was repeated x-1 times, followed by washing with DMF and dichloromethane, drying, sealing and freezing to obtain product G;
[0021] (6) Cleavage: Add lysis buffer to product G, stir, filter, and then blow dry with nitrogen to obtain the crude product of the biomimetic peptide antifreeze agent.
[0022] Preferably, in step (1), the ratio of the amide resin to N,N'-dimethylformamide is 100 mg: 2 mL;
[0023] The swelling time is 10 minutes.
[0024] Preferably, in step (2), the volume ratio of 4-methylpiperidine to DMF in the 4-methylpiperidine / DMF solution is 1:4;
[0025] The volume ratio of the first addition of 4-methylpiperidine / DMF solution to the second addition of 4-methylpiperidine / DMF solution is 1:1;
[0026] When adding the 4-methylpiperidine / DMF solution for the first time, bubble for 2 minutes, filter, and wash with DNF; when adding the 4-methylpiperidine / DMF solution for the second time, bubble for 12 minutes, filter, and wash with DNF.
[0027] Preferably, in step (3), the NH2-(CH2) n In -CH3, n ranges from 0 to 4;
[0028] The bromoacetic acid / DMF solution, N,N'-diisopropylcarbodiimide solution, and NH2-(CH2) are mentioned. n The ratio of CH3 solution to amide resin in step (1) is 1 mL: 86 μL: 1 mL: 100 mg;
[0029] The concentration of the bromoacetic acid / DMF solution is 0.6M; the NH2-(CH2) solution... n The concentration of -CH3 is 1M;
[0030] The acylation reaction takes 5-30 minutes;
[0031] The substitution reaction takes 5-60 minutes;
[0032] The value of v is 1 to 6.
[0033] Preferably, in step (4), the NH2-(CH2) m In -CH2O-THP, m ranges from 0 to 4.
[0034] The NH2-(CH2) m -CH2O-THP solution and NH2-(CH2) in step (3) n The volume ratio of the CH3 solution is 1:1;
[0035] The NH2-(CH2) m The concentration of -CH2O-THP is 1M; the substitution reaction takes 5-60 min.
[0036] The value of w is 1 to 6.
[0037] Preferably, in step (5), x is 1 to 6.
[0038] Preferably, the lysis solution comprises a mixed solution of triisopropylsilane, pure water and trifluoroacetic acid, wherein the volume ratio of triisopropylsilane, pure water and trifluoroacetic acid in the mixed solution is 2.5:2.5:95; and the stirring time is 10 min to 3 h.
[0039] The present invention also provides the application of the above-mentioned biomimetic peptide antifreeze agent as an antifreeze agent in the cryopreservation of cells, tissues, organs or frozen foods.
[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0041] (1) The present invention designs and prepares amphiphilic peptide oligomers with controllable molecular weight and highly designable structure; the amphiphilic peptide oligomers use hydroxyl-containing side chains as hydrophilic side chains and alkyl chains as hydrophobic side chains, and are synthesized according to the designed structure by solid-phase submonomer synthesis method to obtain different hydrophilic and hydrophobic sequences; the amphiphilic peptide oligomers have the effect of changing ice crystal morphology and reducing ice crystal growth rate, and have better ice recrystallization inhibition activity than DMSO, and can be used as biomimetic peptide anti-icing and antifreeze agents.
[0042] (2) The preparation process of the present invention has the characteristics of convenient operation, good cycleability and high accuracy;
[0043] (3) The biomimetic peptide antifreeze agent of the present invention has broad application prospects in biomedicine, cryopreservation and other fields.
[0044] (4) The biomimetic peptide antifreeze agent of the present invention uses a mixed solution of triisopropylsilane, pure water and trifluoroacetic acid as a lysis buffer during the preparation process. It does not affect the integrity of the product molecules and can successfully remove the carrier. Attached Figure Description
[0045] Figure 1 This is a schematic diagram of the solid-phase submonomer synthesis method.
[0046] Figure 2 The image shows the mass spectrum (MS) of the purified P-(Net)3-(Nhe)3 prepared in Example 1.
[0047] Figure 3 The image shows the high-performance liquid chromatography (HPLC) chromatogram of the purified P-(Net)3-(Nhe)3 prepared in Example 1.
[0048] Figure 4 The image shows the mass spectrum (MS) of the purified P-(Nbu-Nhe)3 prepared in Example 2.
[0049] Figure 5 The image shows the high-performance liquid chromatography (HPLC) chromatogram of the purified P-(Nbu-Nhe)3 prepared in Example 2.
[0050] Figure 6 The figure shows a comparison of the effects of the peptides and DMSO described in Example 3 on ice crystal morphology (a) and ice crystal growth rate (b).
[0051] Figure 7 The figure shows the comparison results of the effects of the peptides and DMSO described in Example 4 on ice crystal size (a) and the inhibition of ice crystal growth rate (b).
[0052] Figure 8The graph shows the detection results of the cytotoxicity of the peptides and DMSO determined by the CCK-8 assay as described in Example 5. Detailed Implementation
[0053] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.
[0054] Example 1:
[0055] This embodiment uses, as follows: Figure 1 The solid-phase submonomer synthesis method shown below yielded a biomimetic peptide antifreeze agent with the following structural formula:
[0056] The specific preparation method is as follows:
[0057] 1. Synthesis of the crude product:
[0058] (1) Cleaning: Pour the dichlorodimethylsilane / dichloromethane (v / v = 1:19) solution into the synthesis tube, wait for 30 min, filter with a vacuum pump, and then wash the synthesis tube with dichloromethane and methanol in sequence, and dry it for later use.
[0059] (2) Swelling: Weigh 100 mg of amide resin and place it in a synthesis tube. Add 2 mL of N,N'-dimethylformamide (DMF) to swell the resin. Stir with nitrogen for 10 min and then filter.
[0060] (3) Deprotection: In order to remove the protective Fmoc group, 1 mL of 4-methylpiperidine solution / DMF (v / v = 1:4) solution was added to the product obtained in step (2), the mixture was bubbled with nitrogen for 2 min and then filtered, and then 2 mL of DMF solution was added to wash 5 times and filtered again; then 1 mL of 4-methylpiperidine solution / DMF (v / v = 1:4) solution was added, the mixture was bubbled with nitrogen for 12 min and then filtered, and then 2 mL of DMF solution was added to wash 5 times and filtered again to remove impurities.
[0061] (4) Substitution of NH2-CH2-CH3:
[0062] Acylation: Add 1 mL of 0.6 M bromoacetic acid / DMF solution and 86 μL of N,N'-diisopropylcarbodiimide solution to the synthesis tube and carry out the acylation reaction with the product obtained in step (3). Purge with nitrogen gas for 20 min, filter, and wash 5 times with 2 mL of DMF solution to obtain product A.
[0063] NH2-CH2-CH3 substitution: Add 1 mL of 1 M NH2-CH2-CH3 solution to the synthesis tube and react it with the product obtained in step (4). Purge with nitrogen gas for 30 min, filter, and wash 5 times with 2 mL of DMF solution to obtain product B.
[0064] The acylation and NH2-CH2-CH3 substitution operations were repeated twice, resulting in the addition of a total of 3 -CH2-CH3 side chain units. Then, DMF was added and the mixture was washed 5 times to obtain product C.
[0065] (5) Substitution of NH2-CH2-CH2O-THP:
[0066] Acylation: Product C is subjected to the acylation reaction in step (4) to obtain product D;
[0067] NH2-(CH2) m -CH2O-THP substitution: Add 1 mL of 1 M NH2-(CH2) solution to product D. m The substitution reaction was carried out in CH2O-THP solution, and nitrogen gas was bubbled through the solution for 30 min during the reaction. Then the solution was filtered and washed 5 times with 2 mL LDM solution to obtain product E.
[0068] Product E was repeatedly acylated and subjected to NH2-(CH2) according to the designed structure. m The -CH2O-THP substitution operation was performed twice, resulting in the addition of a total of 3 -CH2-CH2O-THP side chain units, yielding product F. Product F was then washed with DMF 5 times and dichloromethane 3 times, dried at room temperature, and sealed to obtain product G, which was stored at -18℃.
[0069] (6) Pyrolysis:
[0070] A magnetic stir bar was added to product G, along with 4 mL of mixed lysis buffer. The mixture was magnetically stirred for 2 hours, filtered, and the resulting solution was transferred to a new sample vial. Excess lysis buffer was dried by purging with nitrogen gas, yielding a relatively viscous, transparent gel-like substance, which is the crude product of the biomimetic peptide antifreeze agent, denoted as P-(Net)3-(Nhe)3. The mixed lysis buffer was prepared from triisopropylsilane, purified water, and trifluoroacetic acid, with a volume ratio of 2.5:5.5:95.
[0071] 2. Purification of P-(Net)3-(Nhe)3: The synthesized sample was purified using preparative high performance liquid chromatography.
[0072] Figure 1 This is a schematic diagram of the solid-phase submonomer synthesis method.
[0073] like Figure 2This is the MS characterization of purified P-(Net)3-(Nhe)3. The theoretical molecular weight of P-(Net)3-(Nhe)3 is 575.66. From the MS spectrum, the actual measured molecular weight of purified P-(Net-Nhe)3 is [m+H]. + :576.25, [m+ 23 Na]:598.25, thus confirming that the purified component is indeed the target compound to be synthesized.
[0074] like Figure 3 The figure shows the HPLC characterization of purified P-(Net)3-(Nhe)3. As can be seen from the figure, the final sample contains very few impurities, indicating that the target compound with the required purity was successfully separated and purified. The purity meets the test requirements, and the purity of P-(Net)3-(Nhe)3 reaches 93%.
[0075] Example 2:
[0076] In this embodiment, a biomimetic peptide antifreeze agent with the following structural formula was prepared:
[0077] The specific preparation method is as follows:
[0078] Synthesis of crude product:
[0079] (1) Cleaning: Pour the dichlorodimethylsilane / dichloromethane (v / v = 1:19) solution into the synthesis tube, wait for 30 min, filter with a vacuum pump, and then wash the synthesis tube with dichloromethane and methanol in sequence, and dry it for later use.
[0080] (2) Swelling: Weigh 100 mg of amide resin and place it in a synthesis tube. Add 2 mL of N,N'-dimethylformamide (DMF) to swell the resin. Stir with nitrogen for 10 min and then filter.
[0081] (3) Deprotection: In order to remove the protective Fmoc group, 1 mL of 4-methylpiperidine solution / DMF (v / v = 1:4) solution was added to the product obtained in step (2), the mixture was bubbled with nitrogen for 2 min and then filtered, and then 2 mL of DMF solution was added to wash 5 times and filtered again; then 1 mL of 4-methylpiperidine solution / DMF (v / v = 1:4) solution was added, the mixture was bubbled with nitrogen for 12 min and then filtered, and then 2 mL of DMF solution was added to wash 5 times and filtered again to remove impurities.
[0082] (4) Substitution of NH2-CH2-CH3:
[0083] Acylation: Add 1 mL of 0.6 M bromoacetic acid / DMF solution and 86 μL of N,N'-diisopropylcarbodiimide solution to the synthesis tube and carry out the acylation reaction with the product obtained in step (3). Purge with nitrogen gas for 20 min, filter, and wash 5 times with 2 mL of DMF solution to obtain product A.
[0084] NH2-CH2-CH3 substitution: Add 1 mL of 1 M NH2-CH2-CH3 solution to the synthesis tube and react it with the product obtained in step (4). Purge with nitrogen gas for 30 min, filter, and wash 5 times with 2 mL of DMF solution to obtain product B.
[0085] (5) Substitution of NH2-CH2-CH2O-THP:
[0086] Acylation: Product C is subjected to the acylation reaction in step (4) to obtain product D;
[0087] NH2-(CH2) m -CH2O-THP substitution: Add 1 mL of 1 M NH2-(CH2) solution to product D. m The substitution reaction was carried out in CH2O-THP solution, and nitrogen gas was bubbled through the solution for 30 min during the reaction. Then the mixture was filtered and washed 5 times with 2 mL of DMF solution to obtain product E.
[0088] Product E was repeatedly acylated and subjected to NH2-(CH2) according to the designed structure. m The -CH2O-THP substitution operation was performed twice, resulting in the addition of a total of 3 -CH2-CH2O-THP side chain units, yielding product F. Product F was then washed with DMF 5 times and dichloromethane 3 times, dried at room temperature, and sealed to obtain product G, which was stored at -18℃.
[0089] (6) Pyrolysis:
[0090] A magnetic stir bar was added to product G, along with 4 mL of mixed lysis buffer. The mixture was magnetically stirred for 2 hours, filtered, and the resulting solution was transferred to a new sample vial. Excess lysis buffer was dried by purging with nitrogen gas, yielding a relatively viscous, transparent gel-like substance, which is the crude product of the biomimetic peptide antifreeze agent, denoted as P-(Nbu-Nhe)3. The mixed lysis buffer was prepared from triisopropylsilane, purified water, and trifluoroacetic acid, with a volume ratio of 2.5:5.5:95.
[0091] The crude product of the 0.5 wt% biomimetic peptide antifreeze agent was subjected to high performance liquid chromatography-mass spectrometry to determine the purity and quality of the synthesized crude product of the biomimetic peptide antifreeze agent.
[0092] 2. Purification of P-(Nbu-Nhe)3: The synthesized sample was purified by preparative high performance liquid chromatography.
[0093] Figure 4 This is the MS characterization of purified P-(Nbu-Nhe)3. The theoretical molecular weight of P-(Nbu-Nhe)3 is 659.83. From the MS spectrum, the actual measured molecular weight of purified P-(Nbu-Nhe)3 is [m+H]. + :660.30, [m+ 23 Na]: 682.25, thus confirming that the purified component is indeed the target compound to be synthesized.
[0094] Figure 5 The figure shows the HPLC characterization of purified P-(Nbu-Nhe)3. As can be seen from the figure, the final sample contains very few impurities, indicating that the target compound with the required purity was successfully separated and purified. The purity meets the test requirements, and the purity of P-(Net)3-(Nhe)3 reaches 90%.
[0095] Example 3:
[0096] In this embodiment, the ability of the biomimetic peptide anti-icing and antifreeze agent to alter ice crystal morphology and affect ice crystal growth rate was investigated using nanoliter osmotic pressure. The specific investigation steps are as follows:
[0097] A biomimetic peptide antifreeze solution was injected into a six-well plate filled with immersion oil. The droplets were rapidly cooled to approximately -20°C and frozen. The temperature was then slowly raised to the melting temperature (T). m Then slowly decrease to the freezing temperature (T). f When ice crystals first begin to grow, their growth is recorded using a digital camera. At least five snapshots are taken during the growth process to obtain the growth rate, which is the growth rate of a single ice crystal divided by the time taken for the crystal to grow.
[0098] Three concentrations of the sample (1 mg·mL) -1 5 mg·mL -1 10 mg·mL -1 Perform at least three corresponding experiments and calculate the final average value under different subcooling degrees ΔT. The test results are as shown in the attached instruction manual. Figure 6 As shown.
[0099] Figure 6 This is a comparison of the effects of peptides and DMSO on ice crystal morphology and ice crystal growth rate described in Example 3. Figure 6 (a) It can be seen that the ice crystal morphology in PBS solution is typically disc-shaped, while the ice crystal morphology in PBS solution containing peptides changes to hexagonal; from Figure 6 (b) It is known that the concentration is 10 mg·mL.-1 At different supercooling degrees ΔT, the ice crystal growth rate is: R PBS >R P-(Nbu-Nhe)3 ≈R DMSO >R P-(Net)3-(Nhe)3 It is evident that biomimetic peptide antifreeze agents have the effect of altering ice crystal morphology and reducing ice crystal growth rate.
[0100] Example 4:
[0101] In this embodiment, the inhibitory activity of the biomimetic peptide antifreeze agent against ice recrystallization was investigated through an ice recrystallization experiment. The specific investigation steps are as follows:
[0102] A 10 μL droplet of biomimetic peptide antifreeze solution was dropped from a fixed height (h = 1.5 m) onto a glass slide (pre-cooled to -60 °C) to obtain a thin solid ice film. The sample was then heated to -6 °C and held at -6 °C for at least 30 min to allow recrystallization and assess its activity. The size and morphology of the ice crystals during recrystallization were observed in situ and in real-time under a microscope. Over the next 20 min, microscopic images of the sample were recorded using a digital camera to obtain the particle size defined by the two largest orthogonal dimensions on the ice particle surface.
[0103] To quantitatively evaluate ice recrystallization activity, the mean maximum size (MLGS) statistical method was used to calculate the ice particle size. This involved selecting the 10 largest ice crystals and taking their average value. For each sample, the procedure was repeated at least three times. PBS buffer was typically used as a reference sample. The percentage of MLGS in the recrystallized sample relative to the standard PBS buffer was used to characterize the recrystallization inhibition effect. The test results are shown below. Figure 7 As shown.
[0104] Figure 7 This is a comparison of the effects of the peptides described in Example 4 and DMSO on inhibiting ice recrystallization. Figure 7 (a) It can be seen that the average maximum size of ice crystals (MLGS) is: MLGS PBS >MLGS DMSO >MLGS P-(Net)3-(Nhe)3 >MLGS P-(Nbu-Nhe)3 ;Depend on Figure 7 (b) It can be seen that the MLGS percentage of ice crystals is: P DMSO >P P-(Net)3-(Nhe)3 >P P-(Nbu-Nhe)3 It is evident that biomimetic peptide antifreeze agents exhibit better ice recrystallization inhibition activity compared to DMSO.
[0105] Example 5:
[0106] In this embodiment, the cytotoxicity of the biomimetic peptide antifreeze agent to cells was investigated by a cytotoxicity test of the solution. The specific investigation steps are as follows:
[0107] Mouse fibroblasts (L929, commercially purchased and cultured) were cultured at a rate of 1–2 × 10⁻⁶. -4 The density of cells / well was seeded in a 96-well plate.
[0108] Then, the cells were incubated for 24 hours in 100 μL of standard culture medium (RPMI-1640, containing 10 μL of fetal bovine serum). The biomimetic peptide antifreeze agent was dissolved in the standard culture medium to obtain different concentrations: 0 (negative control), 1.0 mg / mL... -1 5.0 mg·mL -1 and 10.0 mg·mL -1 The solution was then used. Mouse fibroblast cells were then incubated in 100 μL of culture medium containing a biomimetic peptide antifreeze agent for 24 h.
[0109] After culture, cells were washed with PBS, and 100 μL of standard culture medium and 10 μL of CCK-8 were added to each well. Cells were incubated for 1–4 hours. The absorbance of cells at 450 nm was then measured using a microplate spectrophotometer, and cell viability was calculated. The results are shown below. Figure 8 As shown.
[0110] Figure 8 The graph shows the detection results of the cytotoxicity of the peptide-like substance with DMSO determined by the CCK-8 assay as described in Example 5. As can be seen from the graph, at a concentration of 10 mg / mL... -1 Relative Cell Viability (RCV) P-(Nbu-Nhe)3 >RCV P-(Net)3-(Nhe)3 >RCV DMSO It is evident that peptide-based antifreeze materials exhibit virtually no cytotoxicity compared to DMSO.
[0111] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.
Claims
1. A biomimetic peptide antifreeze agent, characterized in that, The structural formula of the biomimetic peptide antifreeze agent is shown below: Where n is 0 to 4, m is 0 to 4, v is 1 to 6, w is 1 to 6, and x is 1 to 6.
2. The preparation method of the biomimetic peptide antifreeze agent according to claim 1, characterized in that, include: (1) Swelling: N,N'-dimethylformamide (DMF) is added to the amide resin for swelling. After stirring with nitrogen bubbling, the mixture is filtered. (2) Deprotection: Add 4-methylpiperidine / DMF solution to the product obtained in step (1) for the first time, filter after bubbling with nitrogen, and wash; then add 4-methylpiperidine / DMF solution to it for the second time, filter after bubbling with nitrogen, and wash to obtain deprotected resin; (3)NH2-(CH2) n -CH3 substitution: Acylation: Add bromoacetic acid / DMF solution and N,N'-diisopropylcarbodiimide solution to the deprotected resin in step (2) to carry out acylation reaction. Nitrogen gas is bubbled through the reaction. After the reaction is completed, filter and wash to obtain product A. NH2-(CH2) n -CH3 substitution: Add NH2-(CH2) to product A. n -CH3 undergoes a substitution reaction, during which nitrogen gas is bubbled through the gas. After the reaction is complete, the mixture is filtered and washed to obtain product B. Product B was repeatedly acylated and subjected to NH2-(CH2) according to the designed structure. n The -CH3 substitution operation is performed v-1 times to obtain product C; (4)NH2-(CH2) m Replacement of -CH2O-THP: Acylation: Product C is subjected to the acylation reaction in step (3) to obtain product D; NH2-(CH2) m -CH2O-THP substitution: NH2-(CH2) is added to product D. m The substitution reaction was carried out in CH2O-THP solution, with nitrogen gas bubbled through the reaction. After the reaction was completed, the solution was filtered and washed to obtain product E. Product E was repeatedly acylated and subjected to NH2-(CH2) according to the designed structure. m The -CH2O-THP substitution operation was performed w-1 times to obtain product F; (5) Perform the steps (3) and (4) on product F according to the designed structure x-1 times, then wash with DMF and dichloromethane, dry, seal and store in a freezer to obtain product G; (6) Cleavage: Add lysis buffer to product G, stir, filter, and then blow dry with nitrogen to obtain the crude product of the biomimetic peptide antifreeze agent.
3. The preparation method of the biomimetic peptide antifreeze agent according to claim 2, characterized in that, In step (1), the ratio of the amide resin to N,N'-dimethylformamide is 100 mg: 2 mL; The swelling time is 10 minutes.
4. The preparation method of the biomimetic peptide antifreeze agent according to claim 2, characterized in that, In step (2), the volume ratio of 4-methylpiperidine to DMF in the 4-methylpiperidine / DMF solution is 1:4; The volume ratio of the first addition of 4-methylpiperidine / DMF solution to the second addition of 4-methylpiperidine / DMF solution is 1:1; When adding the 4-methylpiperidine / DMF solution for the first time, bubble for 2 minutes, filter, and wash with DNF; when adding the 4-methylpiperidine / DMF solution for the second time, bubble for 12 minutes, filter, and wash with DNF.
5. The preparation method of the biomimetic peptide antifreeze agent according to claim 2, characterized in that, In step (3), the NH2-(CH2) n In -CH3, n ranges from 0 to 4; The bromoacetic acid / DMF solution, N,N'-diisopropylcarbodiimide solution, and NH2-(CH2) are mentioned. n The ratio of CH3 solution to amide resin in step (1) is 1 mL: 86 μL: 1 mL: 100 mg; The concentration of the bromoacetic acid / DMF solution is 0.6M; the NH2-(CH2) solution... n The concentration of -CH3 is 1M; The acylation reaction takes 5-30 minutes; The substitution reaction takes 5-60 minutes; The value of v is 1 to 6.
6. The preparation method of the biomimetic peptide antifreeze agent according to claim 2, characterized in that, In step (4), the NH2-(CH2) m In -CH2O-THP, m ranges from 0 to 4. The NH2-(CH2) m -CH2O-THP solution and NH2-(CH2) in step (3) n The volume ratio of the CH3 solution is 1:1; The NH2-(CH2) m The concentration of -CH2O-THP is 1M; the substitution reaction time is 5-60 min; The value of w is 1 to 6.
7. The preparation method of the biomimetic peptide antifreeze agent according to claim 2, characterized in that, In step (5), x is 1 to 6.
8. The preparation method of the biomimetic peptide antifreeze agent according to claim 2, characterized in that, In step (6), the lysis solution comprises a mixed solution of triisopropylsilane, pure water and trifluoroacetic acid; The stirring time is 10 min to 3 h.
9. The preparation method of the biomimetic peptide antifreeze agent according to claim 8, characterized in that, The volume ratio of triisopropylsilane, pure water, and trifluoroacetic acid in the mixed solution is 2.5:2.5:
95.
10. The biomimetic peptide antifreeze agent according to claim 1 is used as an antifreeze agent in the cryopreservation of cells, tissues, organs or frozen foods.