CN116284240A - Bionic peptide anti-icing antifreeze agent and preparation method and application thereof - Google Patents
Bionic peptide anti-icing antifreeze agent and preparation method and application thereof Download PDFInfo
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- CN116284240A CN116284240A CN202310351641.0A CN202310351641A CN116284240A CN 116284240 A CN116284240 A CN 116284240A CN 202310351641 A CN202310351641 A CN 202310351641A CN 116284240 A CN116284240 A CN 116284240A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 52
- 239000011664 nicotinic acid Substances 0.000 title claims abstract description 41
- 239000007798 antifreeze agent Substances 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 55
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 120
- 239000000243 solution Substances 0.000 claims description 72
- 239000000047 product Substances 0.000 claims description 60
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 52
- 238000006467 substitution reaction Methods 0.000 claims description 31
- 230000005587 bubbling Effects 0.000 claims description 27
- 238000000967 suction filtration Methods 0.000 claims description 26
- 238000005406 washing Methods 0.000 claims description 25
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 24
- UZOFELREXGAFOI-UHFFFAOYSA-N 4-methylpiperidine Chemical compound CC1CCNCC1 UZOFELREXGAFOI-UHFFFAOYSA-N 0.000 claims description 20
- 238000005917 acylation reaction Methods 0.000 claims description 19
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 18
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- 150000001408 amides Chemical class 0.000 claims description 8
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- 210000000056 organ Anatomy 0.000 claims description 3
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- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 2
- 238000005138 cryopreservation Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 44
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- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 2
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/36—Freezing; Subsequent thawing; Cooling
- A23L3/37—Freezing; Subsequent thawing; Cooling with addition of or treatment with chemicals
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a bionic peptide anti-icing antifreeze agent, a preparation method and application thereof, belonging to the field of biopolymer synthesis; the bionic peptide anti-icing antifreeze agent provided by the invention takes a hydroxyl-containing side chain as a hydrophilic side chain and an alkyl chain as a hydrophobic side chain, and is synthesized according to a designed structure by a solid-phase subunit synthesis method to obtain the bionic peptide anti-icing antifreeze agent containing different hydrophilic and hydrophobic sequences; the bionic peptide anti-icing antifreeze agent can form hydrogen bond interaction with the surface of ice crystals to inhibit movement of water molecules, and hydrophobic alkyl can influence and prevent aggregation of the water molecules through hydrophobic interaction, so that growth of the ice crystals can be effectively regulated and controlled.
Description
Technical Field
The invention belongs to the field of biopolymer synthesis, and in particular relates to a bionic peptide anti-icing antifreeze agent, a preparation method and application thereof.
Background
Freezing water into ice at low temperatures can cause significant disruption to people's life and industrial production, for example, ice crystals formed during ultra-low temperature storage can greatly damage cells and organs, resulting in loss of biological function. Therefore, prevention of ice crystal formation and growth is very important. The antifreezing agent has the functions of reducing freezing temperature, reducing ice crystal size, preventing ice recrystallization, etc.
The natural antifreeze protein (AFP) in organisms in extremely cold regions has good antifreeze effect, is a biological antifreeze material capable of regulating the growth of ice cores and ice crystals, and can prevent fishes, microorganisms and the like from being damaged by freezing and survive in an environment below 0 ℃. AFP can prevent the formation of large ice particles in cells and body fluids, thereby maintaining the body fluids in a liquid state. However, extraction of AFP from organisms is very difficult and expensive, and AFP tends to reduce cell viability, induce rejection reactions and cause biohazards, which limit its use in production and life. Therefore, scientific researchers shift the center of gravity to lower cost synthetic antifreeze agents. The existing common antifreeze agents such as polyvinyl alcohol, dimethyl sulfoxide and the like have certain antifreeze effect, but have low efficiency and certain toxicity, and are not suitable for a large number of wide applications, in particular to the fields of biological medicine, food processing and the like. Therefore, there is a need to develop ideal high-efficiency low-toxicity antifreeze materials which can be effectively applied to the field of biological correlation, in particular to the field of low-temperature freezing preservation.
The peptoid is N-substituted polyglycine with a similar main chain structure with the polypeptide, is a polymer simulating the peptide, and is different in that substituents on the main chain structure of the peptoid are transferred from alpha-carbon of the polypeptide to N atoms, and the difference eliminates inter-chain and intra-chain hydrogen bonds on a framework and chirality of the main chain, so that the space conformation of the peptoid is controlled by electronic action and steric effect of a side chain, thereby forming a random coil structure more easily, and having good solubility in most solvents. In addition, the clustered peptide also has good hot workability and excellent biocompatibility, and can be widely applied to the fields of biomedicine, chemical catalysis, nano materials and the like.
The invention uses peptoid as raw material to simulate natural antifreeze protein structure, and uses solid phase subunit synthesis method to prepare amphiphilic cluster peptide with hydrophilic side chain and hydrophobic side chain. The prepared clustered peptide not only has excellent anti-icing and anti-freezing properties, but also is non-toxic to cells and has better biocompatibility. In addition, compared with the most commonly used antifreezing agent DMSO, the peptide antifreezing agent has better antifreezing performance and higher biosafety, so that the peptide antifreezing agent can be widely applied to the fields of biological medicine, life health, environmental protection and the like.
Disclosure of Invention
Aiming at the defects of high cost, instability, toxicity and the like of the anti-icing and anti-freezing agent in the prior art, the invention provides a bionic peptide anti-icing and anti-freezing agent, and a preparation method and application thereof; the bionic peptide anti-icing antifreeze agent provided by the invention takes a hydroxyl-containing side chain as a hydrophilic side chain and an alkyl chain as a hydrophobic side chain, and is synthesized according to a designed structure by a solid-phase subunit synthesis method to obtain the bionic peptide anti-icing antifreeze agent containing different hydrophilic and hydrophobic sequences; the bionic peptide anti-icing antifreeze agent can form hydrogen bond interaction with the surface of ice crystals to inhibit movement of water molecules, and hydrophobic alkyl can influence and prevent aggregation of the water molecules through hydrophobic interaction, so that growth of the ice crystals can be effectively regulated and controlled.
The invention firstly provides a bionic peptide anti-icing antifreeze agent, which has the following structural formula:
wherein n is 0 to 4, m is 0 to 4, v is 1 to 6,w, x is 1 to 6.
The invention also provides a preparation method of the bionic peptide anti-icing antifreeze agent, which comprises the following steps:
(1) Swelling: adding N, N' -Dimethylformamide (DMF) into the amide resin for swelling, bubbling nitrogen, stirring and then carrying out suction filtration;
(2) Deprotection: adding 4-methylpiperidine/DMF solution into the product obtained in the step (1) for the first time, bubbling nitrogen, and then filtering and washing; then adding 4-methylpiperidine/DMF solution for the second time, bubbling nitrogen, filtering, and washing to obtain deprotected resin;
(3)NH 2 -(CH 2 ) n -CH 3 is substituted by:
acylation: adding bromoacetic acid/DMF solution and N, N' -diisopropylcarbodiimide solution into the deprotected resin in the step (2) for acylation reaction, introducing nitrogen in the reaction process for bubbling, and filtering and washing after the reaction is finished to obtain a product A;
NH 2 -(CH 2 ) n -CH 3 substitution: addition of NH to product A 2 -(CH 2 ) n -CH 3 Performing substitution reaction, introducing nitrogen for bubbling in the reaction process, and performing suction filtration and washing after the reaction is finished to obtain a product B;
the product B is sequentially and repeatedly acylated and NH according to the designed structure 2 -(CH 2 ) n -CH 3 The substituted operation is carried out for v-1 times, thus obtaining a product C;
(4)NH 2 -(CH 2 ) m -CH 2 substitution of O-THP:
acylation: carrying out an acylation reaction in the step (3) on the product C to obtain a product D;
NH 2 -(CH 2 ) m -CH 2 O-THP substitution: addition of NH to product D 2 -(CH 2 ) m -CH 2 Performing substitution reaction on the O-THP solution, introducing nitrogen for bubbling in the reaction process, and performing suction filtration and washing after the reaction is finished to obtain a product E;
the product E is sequentially and repeatedly acylated and NH according to the designed structure 2 -(CH 2 ) m -CH 2 O-THP substitution is carried out for w-1 times to obtain a product F;
(5) The product F is sequentially and repeatedly acylated and NH according to the designed structure 2 -(CH 2 ) m -CH 2 The O-THP substitution is operated for x-1 times, then DMF and dichloromethane are used for washing, airing,sealing and placing in a freezing storage way to obtain a product G;
(6) Cracking: and adding the pyrolysis liquid into the product G, stirring, filtering, introducing nitrogen and drying to obtain a crude product of the bionic peptide anti-icing antifreeze agent.
Preferably, in step (1), the amide resin and N, N' -dimethylformamide are used in a ratio of 100mg to 2mL;
the swelling time was 10min.
Preferably, in the 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 4-methylpiperidine/DMF solution to the second 4-methylpiperidine/DMF solution is 1:1;
bubbling for 2min when adding 4-methylpiperidine/DMF solution for the first time, suction filtering, and washing with DNF; the second time when 4-methylpiperidine/DMF solution was added, bubbling for 12min, suction filtration and DMF washing.
Preferably, in step (3), the NH 2 -(CH 2 ) n -CH 3 Wherein n is 0 to 4;
the bromoacetic acid/DMF solution, N' -diisopropylcarbodiimide solution and NH 2 -(CH 2 ) n -CH 3 The dosage ratio of the solution to the amide resin of step (1) was 1mL:86 μl:1mL:100mg;
the concentration of the bromoacetic acid/DMF solution is 0.6M; the NH is 2 -(CH 2 ) n -CH 3 Is 1M;
the time of the acylation reaction is 5-30min;
the time of the substitution reaction is 5-60min;
and v is 1-6.
Preferably, in step (4), the NH 2 -(CH 2 ) m -CH 2 M in O-THP is 0-4;
the NH is 2 -(CH 2 ) m -CH 2 O-THP solution and NH in step (3) 2 -(CH 2 ) n -CH 3 The volume ratio of the solution is 1:1;
the NH is 2 -(CH 2 ) m -CH 2 The concentration of O-THP was 1M; the time of the substitution reaction is 5-60min.
And w is 1-6.
Preferably, in step (5), x is 1 to 6.
Preferably, the cracking solution comprises a mixed solution of triisopropylsilane, pure water and trifluoroacetic acid, wherein the volume ratio of the triisopropylsilane to the pure water to the trifluoroacetic acid in the mixed solution is 2.5:2.5:95; the stirring time is 10min-3h.
The invention also provides application of the bionic peptide anti-icing antifreeze agent serving as an antifreeze agent in low-temperature cryopreservation of cells, tissues, organs or frozen foods.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention designs and prepares the amphiphilic peptide oligomer with controllable molecular weight and highly-designed structure; the amphiphilic peptide oligomer takes a hydroxyl-containing side chain as a hydrophilic side chain and an alkyl chain as a hydrophobic side chain, and is synthesized to obtain a polymer containing different hydrophilic and hydrophobic sequences according to a designed structure by a solid-phase subunit synthesis method; the amphiphilic peptide oligomer has the effects of changing the morphology of ice crystals and reducing the growth rate of the ice crystals, has better ice recrystallization inhibition activity compared with DMSO, and can be used as a bionic peptide anti-icing antifreeze agent.
(2) The preparation process has the characteristics of convenient operation, good circularity, high accuracy and the like;
(3) The bionic peptide anti-icing antifreeze agent has wide application prospect in the aspects of biological medicine, low-temperature preservation and the like;
(4) The bionic peptide anti-icing antifreeze agent disclosed by the invention uses a mixed solution comprising triisopropylsilane, pure water and trifluoroacetic acid as a cracking solution in the preparation process, has no influence on the integrity of product molecules, and can smoothly cut off a carrier.
Drawings
FIG. 1 is a schematic representation of a solid phase sub-monomer synthesis.
FIG. 2 is a P- (Net) prepared in example 1 3 -(Nhe) 3 Mass Spectrum (MS) plot after purification.
FIG. 3 shows the P- (Net) prepared in example 1 3 -(Nhe) 3 High Performance Liquid Chromatography (HPLC) profile after purification.
FIG. 4 shows the P- (Nbu-Nhe) prepared in example 2 3 Mass Spectrum (MS) plot after purification.
FIG. 5 shows the P- (Nbu-Nhe) prepared in example 2 3 High Performance Liquid Chromatography (HPLC) profile after purification.
FIG. 6 is a graph showing the results of comparing the effect of peptoids described in example 3 with DMSO on ice crystal morphology (a) and ice crystal growth rate (b).
FIG. 7 is a graph showing the comparison of the effect (b) of peptoid described in example 4 with DMSO on ice crystal particle size (a) and ice crystal growth rate inhibition.
FIG. 8 is a graph showing the results of the cytotoxicity assay of the peptoid and DMSO using the CCK-8 method described in example 5.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto.
Example 1:
in the embodiment, a solid-phase subunit synthesis method shown in figure 1 is adopted to prepare the bionic peptide anti-icing antifreeze agent with the structural formula shown as follows:
1. synthesis of crude product:
(1) Cleaning: the dichlorodimethylsilane/dichloromethane (v/v=1:19) solution was poured into the synthesis tube, filtered with a vacuum pump after waiting for 30min, and the synthesis tube was then washed sequentially with dichloromethane and methanol and dried for use.
(2) Swelling: 100mg of the amide resin was weighed into a synthesis tube, 2mL of N, N' -Dimethylformamide (DMF) was added to swell the resin, and after stirring for 10min with nitrogen bubbling, suction filtration was performed.
(3) Deprotection: to remove the protective Fmoc groups, 1mL of 4-methylpiperidine solution/DMF (v/v=1:4) solution was added to the product obtained in step (2), and after bubbling with nitrogen for 2min, suction filtration was performed, followed by washing 5 times with 2mL of DMF solution and suction filtration; then 1mL of 4-methylpiperidine solution/DMF (v/v=1:4) solution was added, bubbling with nitrogen gas for 12min followed by suction filtration, and then 2mL of DMF solution was added to wash 5 times and suction filtration to remove impurities.
(4)NH 2 -CH 2 -CH 3 Is substituted by:
acylation: 1mL of bromoacetic acid/DMF solution with the concentration of 0.6M and 86 mu L N of N' -diisopropylcarbodiimide solution are added into a synthesis tube, the acylation reaction is carried out on the product obtained in the step (3), nitrogen is introduced into the synthesis tube for bubbling for 20min, and the synthesis tube is subjected to suction filtration and then washed with 2mL of DMF solution for 5 times, so as to obtain a product A.
NH 2 -CH 2 -CH 3 Substitution: 1mL of NH at a concentration of 1M was added to the synthesis tube 2 -CH 2 -CH 3 And (3) carrying out substitution reaction on the solution and the product obtained in the step (4), bubbling nitrogen for 30min, and washing with 2mL of DMF solution for 5 times after suction filtration to obtain a product B.
Sequentially repeating the acylation and NH 2 -CH 2 -CH 3 The substitution operation is repeated twice, and 3-CH are connected in total 2 -CH 3 The side chain unit was then washed 5 times with DMF to give product C.
(5)NH 2 -CH 2 -CH 2 Substitution of O-THP:
acylation: carrying out an acylation reaction in the step (4) on the product C to obtain a product D;
NH 2 -(CH 2 ) m -CH 2 O-THP substitution: to product D was added 1mL of NH at a concentration of 1M 2 -(CH2) m -CH 2 Performing substitution reaction on the O-THP solution, introducing nitrogen to bubble for 30min in the reaction process, performing suction filtration, and then washing with the 2mLDMF solution for 5 times to obtain a product E;
the product E is sequentially and repeatedly acylated and NH according to the designed structure 2 -(CH 2 ) m -CH 2 O-THP substitution is carried out 2 times, and 3-CH are connected in total 2 -CH 2 O-THP side chain unit to obtain product F; DMF is added to the product F in sequenceWashing for 5 times, washing with dichloromethane for 3 times, air drying at room temperature, sealing to obtain product G, and storing at-18deg.C.
(6) Cracking:
adding magneton into the product G, adding 4mL of mixed lysate, magnetically stirring for 2h, transferring the solution obtained after suction filtration into a new sample bottle, introducing nitrogen gas to blow dry the redundant lysate, and obtaining a relatively viscous transparent jelly, namely a crude product of the bionic peptide anti-icing antifreeze, namely P- (Net) 3 -(Nhe) 3 . The mixed pyrolysis liquid is prepared from triisopropylsilane, pure water and trifluoroacetic acid, and the volume ratio of the triisopropylsilane to the pure water to the trifluoroacetic acid is 2.5:5.5:95.
2.P-(Net) 3 -(Nhe) 3 Is purified by (2): purifying the synthesized sample by using a preparative high performance liquid chromatography.
FIG. 1 is a schematic representation of a solid phase sub-monomer synthesis.
FIG. 2 shows the purified P- (Net) 3 -(Nhe) 3 MS characterization of P- (Net) 3 -(Nhe) 3 Is 575.66, can be obtained from MS spectra, and is purified to obtain P- (Net-Nhe) 3 The actual molecular weight was [ m+H ]] + :576.25，[m+ 23 Na]598.25, whereby the purified fraction is indeed the desired synthesized target compound.
FIG. 3 shows the purified P- (Net) 3 -(Nhe) 3 From the figure, the final sample contains few impurities, which indicates that the target compound with the required purity is obtained by successful separation and purification, and the purity meets the test requirement, and P- (Net) 3 -(Nhe) 3 The purity of (3) was 93%.
Example 2:
in the embodiment, the bionic peptide anti-icing antifreeze agent with the structural formula shown as follows is prepared:
synthesis of crude product:
(1) Cleaning: the dichlorodimethylsilane/dichloromethane (v/v=1:19) solution was poured into the synthesis tube, filtered with a vacuum pump after waiting for 30min, and the synthesis tube was then washed sequentially with dichloromethane and methanol and dried for use.
(2) Swelling: 100mg of the amide resin was weighed into a synthesis tube, 2mL of N, N' -Dimethylformamide (DMF) was added to swell the resin, and after stirring for 10min with nitrogen bubbling, suction filtration was performed.
(3) Deprotection: to remove the protective Fmoc groups, 1mL of 4-methylpiperidine solution/DMF (v/v=1:4) solution was added to the product obtained in step (2), and after bubbling with nitrogen for 2min, suction filtration was performed, followed by washing 5 times with 2mL of DMF solution and suction filtration; then 1mL of 4-methylpiperidine solution/DMF (v/v=1:4) solution was added, bubbling with nitrogen gas for 12min followed by suction filtration, and then 2mL of DMF solution was added to wash 5 times and suction filtration to remove impurities.
(4)NH 2 -CH 2 -CH 3 Is substituted by:
acylation: 1mL of bromoacetic acid/DMF solution with the concentration of 0.6M and 86 mu L N of N' -diisopropylcarbodiimide solution are added into a synthesis tube, the acylation reaction is carried out on the product obtained in the step (3), nitrogen is introduced into the synthesis tube for bubbling for 20min, and the synthesis tube is subjected to suction filtration and then washed with 2mL of DMF solution for 5 times, so as to obtain a product A.
NH 2 -CH 2 -CH 3 Substitution: 1mL of NH at a concentration of 1M was added to the synthesis tube 2 -CH 2 -CH 3 And (3) carrying out substitution reaction on the solution and the product obtained in the step (4), bubbling nitrogen for 30min, and washing with 2mL of DMF solution for 5 times after suction filtration to obtain a product B.
(5)NH 2 -CH 2 -CH 2 Substitution of O-THP:
acylation: carrying out an acylation reaction in the step (4) on the product C to obtain a product D;
NH 2 -(CH 2 ) m -CH 2 O-THP substitution: to product D was added 1mL of NH at a concentration of 1M 2 -(CH2) m -CH 2 Performing substitution reaction on the O-THP solution, bubbling nitrogen for 30min in the reaction process, performing suction filtration, and then washing with 2mL of DMF solution for 5 times to obtain a product E;
repeating the product E according to the designed structureAcylation and NH 2 -(CH 2 ) m -CH 2 O-THP substitution is carried out 2 times, and 3-CH are connected in total 2 -CH 2 O-THP side chain unit to obtain product F; and (3) sequentially adding DMF (dimethyl formamide) into the product F, washing for 5 times, washing with dichloromethane for 3 times, airing at room temperature, sealing to obtain a product G, and storing at-18 ℃.
(6) Cracking:
adding magneton into the product G, adding 4mL of mixed lysate, magnetically stirring for 2h, transferring the solution obtained after suction filtration into a new sample bottle, introducing nitrogen gas to blow dry the redundant lysate, and obtaining a relatively viscous transparent jelly, namely a crude product of the bionic peptide anti-icing antifreeze, namely P- (Nbu-Nhe) 3 . The mixed pyrolysis liquid is prepared from triisopropylsilane, pure water and trifluoroacetic acid, and the volume ratio of the triisopropylsilane to the pure water to the trifluoroacetic acid is 2.5:5.5:95.
And carrying out high performance liquid chromatography mass spectrometry on the crude product of the bionic peptide anti-icing and anti-freezing agent with the weight percentage of 0.5 percent to determine the purity and the quality of the crude product of the synthesized bionic peptide anti-icing and anti-freezing agent.
2.P-(Nbu-Nhe) 3 Is purified by (2): purifying the synthesized sample by using a preparative high performance liquid chromatography.
FIG. 4 is a purified P- (Nbu-Nhe) 3 MS characterization of P- (Nbu-Nhe) 3 Is 659.83, can be obtained from MS spectra, and is purified to obtain P- (Nbu-Nhe) 3 The actual molecular weight was [ m+H ]] + :660.30，[m+ 23 Na]682.25, whereby the purified fraction is indeed the desired synthesized target compound.
FIG. 5 is a purified P- (Nbu-Nhe) 3 From the figure, the final sample contains few impurities, which indicates that the target compound with the required purity is obtained by successful separation and purification, and the purity meets the test requirement, and P- (Net) 3 -(Nhe) 3 The purity of (2) reaches 90%.
Example 3:
in this embodiment, the ability of the bionic peptide anti-icing antifreeze agent to change ice crystal morphology and influence ice crystal growth rate is examined by nanoliter osmotic pressure, and specific examination steps are as follows:
injecting bionic peptide anti-icing antifreeze solution into six-hole plate filled with immersion lens oil, rapidly cooling liquid drop to about-20deg.C, freezing, and slowly increasing temperature to melting temperature (T) m ) Then slowly decrease to freezing temperature (T f ). When ice crystals just began to grow, the growth of ice was recorded with a digital camera, and at least five snapshots were taken during growth to obtain the growth rate, which was the amount of growth of an individual ice crystal divided by the time spent during growth.
Three concentrations of the sample (1 mg. ML) -1 、5mg·mL -1 、10mg·mL -1 ) At least three corresponding experiments are carried out, the final average value under different supercooling degrees delta T is calculated, and the test result is shown in figure 6 of the specification.
FIG. 6 is a graph showing the comparison of the effect of the peptoid described in example 3 and DMSO on ice crystal morphology and ice crystal growth rate, wherein the ice crystal morphology in PBS is typically disc-shaped, and the ice crystal morphology in PBS in the peptoid is transformed into hexagonal shape as shown in FIG. 6 (a); as shown in FIG. 6 (b), the concentration was 10 mg/mL -1 At different supercooling degree DeltaT, ice crystal growth rate R PBS ＞R P-(Nbu-Nhe)3 ≈R DMSO ＞R P-(Net)3-(Nhe)3 . Therefore, the bionic peptide anti-icing antifreeze agent has the effects of changing the ice crystal morphology and reducing the ice crystal growth rate.
Example 4:
in the embodiment, the inhibitory activity of the bionic peptide anti-icing antifreeze on ice recrystallization is examined through an ice recrystallization experiment, and the specific examination steps are as follows:
10. Mu.L of a solution drop of a bionic peptide anti-icing antifreeze agent was dropped onto a glass plate (pre-chilled to-60 ℃) from a fixed height (h=1.5 m) to give a thin solid ice film, which was then warmed to-6℃and the frozen sample was kept at-6℃for more than 30min to allow recrystallization and its activity to be assessed. The size and morphology of ice crystals during recrystallization were observed under a microscope "in situ real-time". And a micrograph of the sample was recorded with a digital camera over the next 20 minutes to obtain a particle size of the ice particle surface defined by the two largest orthogonal dimensions.
In order to quantitatively evaluate the ice recrystallization activity, calculating the particle size of ice particles by adopting an average maximum particle size (MLGS) statistical method, namely selecting 10 ice crystals with the largest particle size and taking the average value; for each sample, the corresponding procedure was repeated at least three times. The recrystallization inhibition effect was generally characterized by the percentage of MLGS of the recrystallized sample in the standard PBS buffer using PBS buffer as reference sample, and the detection results are shown in fig. 7.
FIG. 7 is a graph showing the comparison of the effect of the peptoid described in example 4 with DMSO in inhibiting ice recrystallization, and as can be seen from FIG. 7 (a), ice crystal average maximum particle size (MLGS): MLGS PBS ＞MLGS DMSO ＞MLGS P-(Net)3-(Nhe)3 ＞MLGS P-(Nbu-Nhe)3 The method comprises the steps of carrying out a first treatment on the surface of the From fig. 7 (b), the MLGS percentage of ice crystals: p (P) DMSO ＞P P-(Net)3-(Nhe)3 ＞P P-(Nbu-Nhe)3 . Therefore, compared with DMSO, the bionic peptide anti-icing antifreeze agent has better ice recrystallization inhibition activity.
Example 5:
in the embodiment, cytotoxicity test of the bionic peptide anti-icing antifreeze solution is used for investigating toxicity of the bionic peptide anti-icing antifreeze solution to cells, and specific investigation steps are as follows:
mouse fibroblasts (L929, obtained by culturing after commercial purchase) were cultured at 1 to 2X 10 -4 Density of individual/wells was seeded in 96-well plates,
then incubated in 100. Mu.L of conventional medium (RPMI-1640, containing 10. Mu.L of fetal bovine serum) for 24 hours. Dissolving bionic peptide anti-icing antifreeze agent in conventional culture solution to obtain 0 (negative control) and 1.0 mg.mL of different concentrations -1 、5.0mg·mL -1 And 10.0 mg.mL -1 Is a solution of (a) and (b). The mouse fibroblast cells were then incubated in 100 μl of culture medium containing the biomimetic peptoid anti-icing antifreeze for 24h.
After the cultivation is finished, the cells are washed by PBS solution, 100 mu L of conventional culture solution and 10 mu L of CCK-8 are added into each well, and the cells are incubated for 1 to 4 hours. Then, absorbance of the cells at 450nm was measured by a microplate spectrophotometer, and cell viability was calculated, and the detection results are shown in FIG. 8.
FIG. 8 is an embodiment5, wherein the cytotoxicity of the peptoid and DMSO is measured by the CCK-8 method, and the concentration of the peptoid is 10 mg/mL -1 Cell viability (Relative Cell Viability): RCV (RCV) P-(Nbu-Nhe)3 ＞RCV P-(Net)3-(Nhe)3 ＞RCV DMSO . It can be seen that the peptoid anti-icing antifreeze material is almost non-cytotoxic compared to DMSO.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (10)
1. The bionic peptide anti-icing antifreeze agent is characterized by having the following structural formula:
wherein n is 0 to 4, m is 0 to 4, v is 1 to 6,w, x is 1 to 6.
2. The method for preparing the bionic peptide anti-icing antifreeze agent according to claim 1, which is characterized by comprising the following steps:
(1) Swelling: adding N, N' -Dimethylformamide (DMF) into the amide resin for swelling, bubbling nitrogen, stirring and then carrying out suction filtration;
(2) Deprotection: adding 4-methylpiperidine/DMF solution into the product obtained in the step (1) for the first time, bubbling nitrogen, and then filtering and washing; then adding 4-methylpiperidine/DMF solution for the second time, bubbling nitrogen, filtering, and washing to obtain deprotected resin;
(3)NH 2 -(CH 2 ) n -CH 3 is substituted by:
acylation: adding bromoacetic acid/DMF solution and N, N' -diisopropylcarbodiimide solution into the deprotected resin in the step (2) for acylation reaction, introducing nitrogen in the reaction process for bubbling, and filtering and washing after the reaction is finished to obtain a product A;
NH 2 -(CH 2 ) n -CH 3 substitution: addition of NH to product A 2 -(CH 2 ) n -CH 3 Performing substitution reaction, introducing nitrogen for bubbling in the reaction process, and performing suction filtration and washing after the reaction is finished to obtain a product B;
the product B is sequentially and repeatedly acylated and NH according to the designed structure 2 -(CH 2 ) n -CH 3 The substituted operation is carried out for v-1 times, thus obtaining a product C;
(4)NH 2 -(CH 2 ) m -CH 2 substitution of O-THP:
acylation: carrying out an acylation reaction in the step (3) on the product C to obtain a product D;
NH 2 -(CH 2 ) m -CH 2 O-THP substitution: addition of NH to product D 2 -(CH 2 ) m -CH 2 Performing substitution reaction on the O-THP solution, introducing nitrogen for bubbling in the reaction process, and performing suction filtration and washing after the reaction is finished to obtain a product E;
the product E is sequentially and repeatedly acylated and NH according to the designed structure 2 -(CH 2 ) m -CH 2 O-THP substitution is carried out for w-1 times to obtain a product F;
(5) Sequentially carrying out the operations of the steps (3) and (4) for x-1 times according to the designed structure of the product F, washing with DMF and dichloromethane, airing, sealing and placing in a freezing storage way to obtain a product G;
(6) Cracking: and adding the pyrolysis liquid into the product G, stirring, filtering, introducing nitrogen and drying to obtain a crude product of the bionic peptide anti-icing antifreeze agent.
3. The method for preparing a bionic peptide anti-icing and anti-freeze agent according to claim 2, wherein in the step (1), the dosage ratio of the amide resin to the N, N' -dimethylformamide is 100 mg/2 mL;
the swelling time was 10min.
4. The method for preparing a bionic peptide anti-icing and anti-freeze agent according to claim 2, wherein in the 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 4-methylpiperidine/DMF solution to the second 4-methylpiperidine/DMF solution is 1:1;
bubbling for 2min when adding 4-methylpiperidine/DMF solution for the first time, suction filtering, and washing with DNF; the second time when 4-methylpiperidine/DMF solution was added, bubbling for 12min, suction filtration and DMF washing.
5. The method for preparing a biomimetic peptoid anti-icing anti-freeze agent according to claim 2, wherein in step (3), the NH 2 -(CH 2 ) n -CH 3 Wherein n is 0 to 4;
the bromoacetic acid/DMF solution, N' -diisopropylcarbodiimide solution and NH 2 -(CH 2 ) n -CH 3 The dosage ratio of the solution to the amide resin of step (1) was 1mL:86 μl:1mL:100mg;
the concentration of the bromoacetic acid/DMF solution is 0.6M; the NH is 2 -(CH 2 ) n -CH 3 Is 1M;
the time of the acylation reaction is 5-30min;
the time of the substitution reaction is 5-60min;
and v is 1-6.
6. The method for preparing a biomimetic peptoid anti-icing anti-freeze agent according to claim 2, wherein in step (4), the NH 2 -(CH 2 ) m -CH 2 M in O-THP is 0-4;
the NH is 2 -(CH 2 ) m -CH 2 O-THP solution and NH in step (3) 2 -(CH 2 ) n -CH 3 The volume ratio of the solution is 1:1;
the NH is 2 -(CH 2 ) m -CH 2 The concentration of O-THP was 1M; the time of the substitution reaction is 5-60min;
and w is 1-6.
7. The method for preparing a bionic peptide anti-icing anti-freeze agent according to claim 2, wherein in the step (5), x is 1-6.
8. The method for preparing a bionic peptide anti-icing antifreeze according to claim 2, wherein in the step (6), the lysate comprises a mixed solution of triisopropylsilane, pure water and trifluoroacetic acid;
the stirring time is 10min-3h.
9. The method for preparing the bionic peptide anti-icing antifreeze according to claim 8, wherein the volume ratio of triisopropylsilane, pure water and trifluoroacetic acid in the mixed solution is 2.5:2.5:95.
10. The use of the biomimetic peptoid anti-icing antifreeze agent of claim 1 as an antifreeze agent for the cryopreservation of cells, tissues, organs or frozen foods.
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