CN111790327A

CN111790327A - Molecular design method of ice control material

Info

Publication number: CN111790327A
Application number: CN201910282422.5A
Authority: CN
Inventors: 王健君; 金晟琳; 严杰; 乔杰; 闫丽盈; 李蓉
Original assignee: Institute of Chemistry CAS; Peking University Third Hospital
Current assignee: Beijing Dai Na Mi Ke Biotechnology Co ltd
Priority date: 2019-04-09
Filing date: 2019-04-09
Publication date: 2020-10-20
Anticipated expiration: 2039-04-09
Also published as: CN111790327B

Abstract

The invention relates to a molecular design method of an ice control material, which comprises the following steps: constructing a compound molecule structure library, wherein the compound molecules contain hydrophilic groups and ice-philic groups; evaluating the spreading performance of each compound molecule on an ice-water two-phase interface by adopting molecular dynamics simulation (MD simulation); and screening compound molecules with the required ice affinity and water affinity. The invention firstly provides a mechanism of the ice affinity and the hydrophilicity of the ice control material, introduces MD simulation into the molecular structure design of the ice control material, evaluates the ice affinity and the water affinity of the designed ice control material through molecular dynamics simulation, predicts the ice control performance of the ice control material and can realize structure optimization. The invention well solves the problem that the existing material can be subjected to performance analysis and material screening only by an experiment trial and error method in the research and development process of the ice control material by combining an ice control mechanism and an MD simulation mode, and provides a new thought of molecular structure design.

Description

Molecular design method of ice control material

Technical Field

The invention belongs to the technical field of materials, and particularly relates to a molecular design method of an ice control material.

Background

Cryopreservation refers to keeping the biological material at ultralow temperature to slow down or stop cell metabolism and division, and to continue to develop once normal physiological temperature is restored. Since the advent, this technique has become one of indispensable research methods in the field of natural science, and has been widely adopted. In recent years, with the increasing pressure of life, human fertility tends to decrease year by year, and fertility preservation is receiving more and more attention, and cryopreservation of human germ cells (sperm and oocyte), gonadal tissue, and the like is an important means for fertility preservation. In addition, as the world population ages, the need for cryopreservation of donated human cells, tissues or organs available for regenerative medicine and organ transplantation is also increasing dramatically. Therefore, how to efficiently store precious cells, tissues and organ resources in a freezing way becomes a scientific and technical problem to be solved urgently.

At present, the technology of human assisted reproductive medical treatment in the biomedical field has entered the full-scale freezing era. Cryopreservation of germ cells and embryos is an important technical support. The most common cryopreservation method currently used is vitrification freezing. The vitrification freezing technology can make the liquid inside and outside the cell become glass state directly in the fast freezing process, so as to avoid the damage caused by the formation of ice crystal in the freezing process. However, in the rewarming process, the existing cryopreservation reagents cannot effectively control the growth of ice crystals, thereby causing cell damage. In addition, the organic solvents such as dimethyl sulfoxide (DMSO) or N, N-Dimethylformamide (DMF) with high concentration (not less than 15%) used in the current vitrification freezing method, which are toxic to cells, cause toxic and side effects, and seriously affect the safety and functional expression of stored objects (filial generation). In conclusion, the prior cryopreservation reagent has the problems of no capability of effectively controlling the growth of ice crystals in the rewarming process and high reagent toxicity.

However, the existing anti-freezing materials or ice control materials are developed by screening the ice control capability of the known materials through experiments and searching for substances with better ice control capability through a trial and error method. The research and development path has low efficiency and large workload.

Disclosure of Invention

In order to solve the problems, the inventor of the invention firstly discovers and provides a molecular action mechanism of the ice control material on an ice water interface through research, and provides a molecular design method of the ice control material and the ice control material designed according to the method according to the mechanism.

The invention provides the following technical scheme:

a molecular design method of ice control material comprises the following steps:

(1) constructing a compound molecule structure library, wherein the compound molecules contain hydrophilic groups and ice-philic groups;

(2) adopting Molecular dynamics simulation (MD) to simulate and evaluate the spreading performance of each compound molecule on an ice-water two-phase interface;

(3) and screening ice control molecules with the required ice affinity and water affinity.

According to the invention, the main chain of the ice control molecule is of a carbon chain or peptide chain structure.

According to the invention, the hydrophilic group is a functional group capable of forming a non-covalent interaction with a water molecule, such as a hydrogen bond with water, van der waals interaction, electrostatic interaction, hydrophobic interaction, or pi-pi interaction; illustratively, the hydrophilic group may be selected from hydroxyl (-OH), amino (-NH)₂) Carboxylic acid group (-COOH), amide group (-CONH)₂) Or at least one of the above, or a compound selected from hydrophilic amino acids such as proline (L-Pro), arginine (L-Arg), and lysine (L-Lys), Gluconolactone (GDL), saccharides, and molecular fragments thereof.

According to the invention, the ice-philic group is compatible with iceFunctional groups that form non-covalent interactions, such as hydrogen bonding with ice, van der waals interactions, electrostatic interactions, hydrophobic interactions, or pi-pi interactions, among others; illustratively, the oxophilic group may be selected from the group consisting of hydroxyl (-OH), amino (-NH)₂) Phenyl (-C)₆H₅) Pyrrolidinyl (-C)₄H₈N), or an ice-philic amino acid selected from, for example, glutamine (L-Gln), threonine (L-Thr), aspartic acid (L-Asn), a benzene ring (C)₆H₆) Pyrrolidine (C)₄H₉N) or a molecular fragment thereof.

According to the invention, the ice control material can be formed by bonding hydrophilic groups and ice-philic groups through covalent bonds.

According to the invention, the ice control material can be formed by the ionic bond action of hydrophilic groups and ice-philic groups.

According to the invention, the MD simulation of step (2) can be performed by GROMACS, AMBER, CHARMM, NAMD, or LAMMPS.

According to the present invention, in the step (2) of MD simulation, the moisture submodel may be selected from TIP3P, TIP4P, TIP4P/2005, SPC, TIP3P, TIP5P, or SPC/E, preferably TIP4P/2005 moisture submodel.

According to the invention, in the step (2) of the MD simulation, the force field parameter is provided by one of a GROMOS, an ESFF, an MM form force field, an AMBER, a CHARMM, a COMPASS, a UFF, a CVFF, and the like force field.

According to the invention, in the step (2) of MD simulation, the simulation calculates the interaction between ice control molecules, the interaction between the ice control molecules and water molecules, and the interaction between the ice control molecules and ice-water molecules. The effects include whether hydrogen bonds are formed, van der waals effects, electrostatic effects, hydrophobic effects, pi-pi effects, and the like.

According to the invention, in the step (2) of MD simulation, the simulation calculates that the temperature and the pressure are adjusted when molecules interact. In one embodiment of the invention, the temperature and pressure are regulated using a V-regulated Berendsen temperature controller and a pressure controller.

According to the invention, in the step (2) of MD simulation, the molecular configuration of the compound molecules is maintained by selecting potential energy parameters. Preferably, the potential energy parameter is selected such that the molecular configuration of the compound molecule is maintained at a higher temperature.

According to the invention, in the step (2), when an aqueous solution system is simulated, periodic boundary conditions are adopted in the x, y and z directions; when an ice-water mixed system is simulated, periodic boundary conditions are adopted in the x direction and the y direction.

According to the invention, in the step (2) of MD simulation, a cubic or octahedral water box is selected, preferably 3.9 x 3.6 x 1.0nm³The cubic water box of (1).

According to the invention, the method also comprises a step of synthesizing the compound molecule, for example, the compound molecule can be synthesized by a known chemical synthesis method, such as a polymerization reaction, a condensation reaction, or a biological fermentation method of a genetically engineered bacterium.

The invention also provides the ice control material obtained by the molecular design method.

The ice control molecule is polyvinyl alcohol (PVA), the syndiotacticity (di adsyndiotacticity) r of the PVA is 50-60%, and the molecular weight is 10-500 kDa; preferably, the PVA syndiotacticity r is 50-55%, and the molecular weight is 10-30 kDa.

Advantageous effects

1. The invention discovers for the first time that in the process of controlling the growth of ice crystals in an ice-water mixed phase, ice and water both need to have good affinity for the material. Molecules have affinity with ice to ensure that the molecules are well adsorbed on the ice surface; the affinity of the molecule for water ensures better spreading at the ice-water interface to cover the maximum ice surface area with the least amount of material possible. Based on the ice control mechanism, a design idea of designing ice control molecules with an ice-philic group and a hydrophilic group is provided, and a new method is provided for synthesis of ice control materials.

2. The invention introduces MD simulation into the molecular structure design of the ice control material for the first time, evaluates the ice affinity and the water affinity of the designed ice control molecule through molecular dynamics simulation, predicts the ice control performance of the ice control material and can realize structure optimization.

3. The invention well solves the problem that the existing material can be subjected to performance analysis and material screening only by an experimental trial and error method in the research and development process of the ice control material by combining an ice control mechanism and MD simulation, provides a new thought of molecular structure design, and has great promotion effect on the development and application of the ice control material.

Drawings

FIG. 1: the molecular structure of the ice control material is shown schematically;

FIG. 2: MD simulates the aggregation state of atactic polyvinyl alcohol (a-PVA) and isotactic polyvinyl alcohol (i-PVA) in an ice-water interface;

FIG. 3: a NMR spectrum of a-PVA synthesized in example 1;

FIG. 4: the NMR spectra of PBVE and i-PVA synthesized in example 1, wherein A is PBVE and B is i-PVA;

FIG. 5: GPC curves for PBVE synthesized in example 1;

FIG. 6: the dispersion sizes of a-PVA (A) and i-PVA (B) in water at different concentrations in the DLS experiment;

FIG. 7: two kinds of optical microscopic pictures of PVA in the DPBS solution for the growth of ice crystals, wherein A is a-PVA, B is i-PVA, and C is a relation curve between the concentration and the maximum ice crystal size in the DPBS solution of the two kinds of PVA;

FIG. 8: a-PVA (FIG. A, B) and i-PVA (FIG. C, D) modify the morphological effects of ice crystals in pure water;

FIG. 9: molecular structure models of two kinds of PVA simulated by MD;

FIG. 10: the contactable surface areas of two PVA molecules simulated by MD and water molecules at an ice water interface at 240K are provided, wherein the upper part of the picture is provided with 3 a-PVA molecular chains, and the lower part of the picture is provided with 3 i-PVA molecular chains;

FIG. 11: the aggregation probability of two kinds of PVA in the water solution is calculated by MD simulation;

FIG. 12: the MD simulation calculates the number of intermolecular hydrogen bonds formed by the two PVAs with water in an aqueous solution and the number of intermolecular hydrogen bonds formed with water molecules and ice-water molecules at an ice-water interface at 240K.

Detailed Description

The ice control material core molecule can be designed into various groups with affinity with water and groups with affinity with ice which are connected through covalent bonds or non-covalent bonds.

As a specific embodiment of the present invention, the ice control material may be a compound having a carbon chain structure as a main chain and substituted with an ice-philic group and a hydrophilic group; the ice control material can contain hydrophilic and ice-philic groups, such as hydroxyl groups and amino groups, and can also contain ice-philic groups and hydrophilic groups, respectively. For example, the molecular structure of the ice control material is designed to have a- [ CH ]₂-CHOH]-a repeating unit of (a).

As a specific embodiment of the present invention, the ice control material is a polypeptide consisting of hydrophilic amino acids and ice-philic amino acids, such as: L-Thr-L-Arg (TR) or L-Arg-L-Thr (RT), L-Thr-L-Pro (TP) or L-Pro-L-Thr (PT), L-Thr-L-Arg-L-Thr (TRT), L-Thr-L-Pro-L-Thr (TPT), L-Ala-L-Ala-L-Thr (AAT), L-Thr-L-Cys-L-Thr (TCT), etc.

As a specific embodiment of the present invention, the ice control material is a molecule composed of hydrophilic Gluconolactone (GDL) and an ice-philic amino acid through chemical bonding, such as: GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Thr, and the like.

As a specific embodiment of the present invention, the ice control material may be a polyamino acid composed of the same amino acid containing both an ice-philic group and a hydrophilic group, such as: poly-L-proline (L-Pro)_n) poly-L-arginine (L-Arg)_n) poly-L-histidine (L-His)_n) And the like.

As a specific embodiment of the present invention, the ice control material may comprise a compound of the following structure:

wherein R is selected from substituted or unsubstituted alkyl, and the substituent can be selected from-OH, -NH₂、-COOH、-CONH₂Etc., e.g., R is substituted or unsubstituted C_1-6Alkyl, preferably R is-CH₃、-CH₂CH₃、-CH₂CH₂COOH; n is an integer of 1 to 1000 inclusive, and may be, for example, an integer in the range of 1 to 100. In some embodiments of the invention, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10.

Specifically, the ice control material can be designed into a compound with any one of the following structures:

as a specific embodiment of the invention, the MD simulation adjusts temperature and pressure by a V-regulated Berendsen temperature controller and a pressure controller during molecular dynamics calculation.

In the MD simulation calculation, the main criterion for judging the existence of the hydrogen bond is an energy criterion or a geometric criterion, and the geometric criterion is optimized; when the oxygen atom spacing is less than 0.35nm and the angle ho.

The invention will be further illustrated with reference to the following specific examples.

Example 1

(1) Designing a molecular structure of the compound:

designed to have- [ CH ]₂-CHOH]-to obtain a library of molecular structures comprising atactic and isotactic molecular models of polyvinyl alcohol.

(2) MD simulation experiment

The difference in the affinity of atactic polyvinyl alcohol and isotactic polyvinyl alcohol for ice and water was predicted by MD simulation experiments.

MD simulation was performed by GROMACS 5.1, and the water model was TIP4P/2005 with a melting point of about 252.5K. The interaction parameters of the PVA molecules are provided by the GROMOS54A7 force field, and the leapfrog integration algorithm is adopted, and the integration step size is 2 fs. The electrostatic interaction was calculated by the PME method, and the cutoff radii for the coulombic and L-J action potentials were both 1.0 nm. The temperature and pressure are regulated by a V-regulated berendsen temperature controller and a pressure controller. The time constant was set to 0.1 ps. .

b. Molecular chains of compounds containing 7 repeating units were selected for the simulation. The topological file of the PVA molecules is generated through ATB, and in order to keep the tacticity of the two PVA molecules, the dihedral angle potential function of the molecular carbon chain needs to be correspondingly adjusted.

c. When a PVA aqueous solution system is simulated, periodic boundary conditions are adopted in the x direction, the y direction and the z direction; when an ice-water mixed system is simulated, periodic boundary conditions are adopted in the x direction and the y direction. All systems were simulated for 120ns, and data taken 60ns later were analyzed.

Firstly, a molecular water solution system is researched, wherein 1491 water molecules are shared in the system with only one PVA chain, the pressure is 1atm, and the temperature is 240K, 250K, 260K, 270K, 300K and 330K.

In a system studying the interaction of PVA molecules with ice, x 1.0nm at 3.9 x 3.6 x³The water box of (1) is placed with 6 PVA molecular chains, an ice block containing 1100 water molecules is balanced for 10ns under 240K, and the ice block is placed under the water box along the z-axis direction. The z-direction size of the mixed system was increased to 10nm, and the ice-water mixed system was placed in the center of the water box.

The topological file of the PVA molecules is generated through ATB, the topological file is directly used, and in order to keep the tacticity of the two PVA molecules, the potential energy parameter is selected to be 50kcal/mol, so that the molecular configuration of the two PVA molecules can be kept even at higher temperature.

Molecular structure models of two PVAs simulated in MD are shown in fig. 9.

(3) Evaluating simulation results

The a-PVA can be effectively adsorbed on the ice surface by hydrogen bonding with the ice surface because the distance of three times of adjacent OH is matched with the crystal lattice size of the ice. The i-PVA only changes the direction of the hydroxyl group without changing the distance between adjacent OH groups, so that the i-PVA and the a-PVA have similar capacity of generating adsorption with ice. Meanwhile, the MD simulation result shows that the number of intermolecular hydrogen bonds formed by the a-PVA and water molecules is more than that formed by the i-PVA and the water molecules, so that the affinity of the a-PVA and water is stronger than that of the i-PVA. In addition, the state of 6 PVA molecular chains at an ice-water interface is simulated by MD, and the a-PVA is more prone to spreading at the ice-water interface due to good affinity with both ice and water; on the other hand, i-PVA tends to aggregate at the ice-water interface because of its weak affinity for water (FIG. 2).

TABLE 1

Moreover, MD simulation shows that 240K, the contact areas of two kinds of PVA and water molecules at the ice-water interface are larger than that of i-PVA, and the spreading performance of the a-PVA at the ice-water interface is further verified to be better than that of the i-PVA (see FIG. 10). The aggregation probability of two PVAs in aqueous solution calculated in MD, i-PVA was significantly higher than a-PVA (FIG. 11); at 240K, the number of hydrogen bonds formed by the two kinds of PVA and ice-water molecules is equivalent to that of hydrogen bonds formed by the two kinds of PVA and the ice-water molecules at the ice-water interface, but the number of hydrogen bonds formed between the a-PVA and water at the ice-water interface and in the water solution is obviously more than that of the i-PVA; the a-PVA spreads better at the ice-water interface, while the i-PVA aggregates (FIG. 12).

Therefore, multiple results of the MD simulation show that the a-PVA has better spreading performance at an ice-water interface due to better affinity of the molecular structure and water molecules, and has better ice control effect compared with the i-PVA.

(4) Synthesis of designed PVA molecules

(4.1) preparation of atactic polyvinyl alcohol a-PVA: the molecular weight is about 13-23 kDa, and the syndiotacticity r (diadsyndiaticity) is about 55%

Vinyl acetate (vinyl acetate, VAc, Sigma-Aldrich) from which inhibitors have been removed is dissolved in 100mL of solvent (methanol) in a 250mL round bottom flask under an argon atmosphere to obtain a 25% to 45% solution of VAc. After cooling the above solution to-5 ℃, 80mM of 2,2 '-Azobis (2-methylpropionitrile (2, 2' -Azobis, Sigma-Aldrich) was carefully added dropwise to the reaction solution, after allowing the solution to stand at room temperature and continuing stirring for 15 hours, the reaction solution was dissolved with 1L of acetone and added dropwise to methanol to obtain a white precipitate, the precipitate was washed with methanol and filtered, and then dried in oven vacuum at 60 ℃ for 6.0 hours to obtain a white solid, the white solid was dissolved in methanol solution (10 wt.%), and argon was introduced to remove oxygen in the solution, and a methanol solution of 25% potassium hydroxide was added dropwise to the above solution, and after continuing stirring for 4 hours, the reaction solution was dissolved in 2M hydrochloric acid solution and precipitated in 2.0M ammonia methanol solution to obtain atactic polyvinyl alcohol (a-hydrogen nuclear magnetic spectrum) (fig. 3) showing that the obtained compound was completely hydrolyzed a -PVA.

(4.2) preparation of isotactic polyvinyl alcohol i-PVA: the molecular weight is about 14-26 kDa, and the isotacticity m (isotacticity) is about 84%

a. Preparation of poly-tert-butyl vinyl ether (PBVE). Tert-butyl vinyl ether (tert-butyl vinyl ether, t-BVE, Sigma-Aldrich) was dissolved in 100mL of dry toluene in a 250mL round bottom flask under an argon atmosphere to obtain a 2.5% t-BVE solution in toluene. After cooling the above solution to-78 deg.C, 0.2mM boron trifluoride diethyl etherate (BF) was added₃·OEt₂Sigma-Aldrich) were carefully added dropwise to the cooled solution and supplemented with 0.2mM BF after 2.0 hours₃·OEt₂. After the solution was further stirred at-78 ℃ for 3.0 hours, the reaction was terminated with a small amount of methanol. And the reaction solution was added dropwise to 2.0L of methanol with rapid stirring to obtain a pale yellow precipitate. The precipitate was washed with methanol and filtered, and then dried in an oven at 60 ℃ for 6.0 hours under vacuum to obtain a pale yellow solid powder, and the resulting compound was PBVE as shown by NMR (FIG. 4A). The molecular weight of the synthesized PBVE is controlled by regulating the concentrations of boron trifluoride ethyl ether and tert-butyl vinyl ether. Gel permeation chromatography (GPC using Tetrahydrofuran (THF) system, flow rate of 1mL min^-1) Successful synthesis was shown to yield PBVE with different molecular weights (fig. 5).

b. Preparation of dry hydrogen bromide gas (HBr); in a 100mL two-neck flask, 5.0-30 mL of phosphorus tribromide (PBr)₃Alatin) was added dropwise to 10mL of 48% aqueous hydrogen bromide (HBr, Alfa Aesar). The gas produced is passed sequentially through carbon tetrachloride (CCl)₄) Red phosphorus (P, Alfa Aesar) and calcium chloride (CaCl)₂) To obtain dry HBr gas.

c. Preparation of isotactic polyvinyl alcohol (isotactic-PVA, i-PVA). 0.5g of PBVE was dissolved in 15mL of dry toluene under an argon atmosphere and dry argon was continuously introduced to remove oxygen from the solution. The dry HBr gas produced in step b was passed into the above oxygen-free PBVE solution in toluene at 0 ℃. After about 5.0 minutes, a pale yellow precipitate formed, and the passage of dry HBr gas was continued until no precipitate continued to form. The reaction solution was poured into a 2.0M solution of 200mL ammonia in methanol. The resulting precipitate was washed with methanol, filtered, and dried in oven vacuum at 60 ℃ for 6.0 hours to obtain a pale yellow solid powder. The NMR spectrum (FIG. 4B) showed that the hydrolysis of PBVE was complete to give solid i-PVA.

(5) Verification of ice control effect of synthesized PVA

(5.1) dynamic light Scattering DLS experiment

The particle size distributions of the two PVAs in an aqueous solution at 25 ℃ were measured using a Dynamic Light Scattering (DLS) experiment with a Nano ZS (Malvern Instruments) with a thermostatted chamber and a 4mW He-Ne laser (λ 632.8nm) with a scattering angle of 173 °. First, 1.0mg mL of each of the solutions was prepared^-1、4.0mg mL^-1、10mg mL^-1、20mg mL^-1The aqueous solutions of a-PVA and i-PVA of (a-PVA); about 1.0mL of the PVA solution was loaded into a 12mm disposable polystyrene cuvette for measurement.

Dynamic Light Scattering (DLS) results show that the dispersion size of the same concentration of a-PVA in aqueous solution is much smaller than that of i-PVA (FIG. 6). That is, i-PVA is more prone to exist in an aggregated state in aqueous solution than a-PVA. This is consistent with the results that the number of intramolecular hydrogen bonds of a-PVA in MD simulation is smaller than that of intramolecular hydrogen bonds of i-PVA, and the number of intermolecular hydrogen bonds of a-PVA and water molecules is larger than that of i-PVA and water molecules.

(5.2) measurement of Ice Crystal recrystallization inhibition (IRI) Activity

Dissolving and dispersing a sample into a DPBS solution by adopting a sputtering freezing method for inhibiting the recrystallization of ice crystals (IRI), dripping 10-30 mu L of the solution onto the surface of a clean silicon wafer precooled at minus 60 ℃ at a height of more than 1.0m, and utilizing a cold-hot table at 10 ℃ for min^-1Slowly increasing the temperature to-6 ℃ and keeping the temperature at the temperatureAnnealing for 30min, observing and recording the size of ice crystal with a polarizing microscope and a high-speed camera, and sealing a cold and hot platform to ensure that the internal humidity is about 50%. Each sample was replicated at least three times, and the ice crystal size was counted using a Nano Measurer 1.2 with standard deviation as the error of the statistical result.

(5.3) Ice Crystal morphology (DIS) Observation and Thermal Hysteresis (TH) measurement

Ice crystal morphology (DIS) observation and Thermal Hysteresis (TH) measurement Using a nanoliter osmometer, a capillary was first melted with an alcohol burner external flame and simultaneously stretched to produce a capillary of very fine pore size, which was connected to a microsyringe. The immersion lens oil with higher viscosity is injected into the micron-aperture wafer, and the water solution dissolved with the material is injected into the micropores by using a microsyringe. The liquid drop is quickly frozen by controlling the temperature, and slowly heated to obtain the single crystal ice, the temperature is slowly reduced with the precision of 0.01 ℃, and the appearance of the ice crystal is observed by utilizing a microscope equipped with a high-speed camera and a TH test is carried out.

a-PVA(M_w13-23 kD) has far better ice crystal growth inhibiting capability than i-PVA (M) with corresponding molecular weight _w14 to 26kD) (FIG. 7). As can be seen in FIG. 7A, the ice crystal size of a-PVA is significantly smaller than that of i-PVA at the same concentration; as can be seen in FIG. 7B, the maximum ice crystal size (MLGS) of a-PVA relative to DPBS was 2.0mg mL^-1Then reaches a minimum of about 20% of the maximum ice crystal size of DPBS; the different molecular weights of i-PVA relative to DPBS MLGS increased with increasing concentration and were at 10mg mL^-1Reaches a minimum of only about 50% of the maximum ice crystal size of DPBS and continues to increase to 20mgmL as the concentration continues to increase^-The MLGS is no longer decreasing and increases slightly. i-PVA (M) with a degree of polymerization of more than 333_w14-26 kD) in a concentration of more than 30mgmL^-1The solubility is difficult, so due to the limitation of i-PVA solubility, the IRI activity of i-PVA is optimally 10mg mL^-1MLGS of DPBS at 50%; while IRI activity of a-PVA is optimally 2.0mg mL^-1MLGS of DPBS at 20%. This is consistent with the fact that in the MD simulation, a-PVA spreads more easily at the ice-water interface than i-PVA, and the more easily spreading property enables a-PVA to achieve better ice crystal growth inhibition at lower dosages than i-PVALong effect.

As can be seen from the results of the MD simulation and the actual verification experiment, the results of the MD simulation and the actual verification experiment are good in consistency, the ice control performance of the ice control material can be accurately predicted through the MD simulation, and the molecular design of the ice control material can be effectively realized.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A molecular design method of an ice control material is characterized by comprising the following steps:

(2) evaluating the spreading performance of each compound molecule on an ice-water two-phase interface by adopting molecular dynamics simulation;

(3) and screening compound molecules with the required ice affinity and water affinity.

2. The molecular design method of claim 1, wherein the MD simulation of step (2) is performed by GROMACS, AMBER, CHARMM, NAMD, or LAMMPS;

preferably, in the step (2) MD simulation, the water molecule model is selected from TIP3P, TIP4P, TIP4P/2005, SPC, TIP3P, TIP5P, or SPC/E, preferably TIP4P/2005 moisture molecular model;

preferably, in the MD simulation of step (2), the force field parameter is provided by one of GROMOS, ESFF, MM form force field, AMBER, CHARMM, COMPASS, UFF, CVFF, and the like.

3. The molecular design method according to any one of claims 1 or 2, wherein in the step (2) MD simulation, the simulation calculates the interaction between the compound molecules, the interaction of the compound molecules with water molecules, the interaction of the compound molecules with ice-water molecules; for example, the effect includes whether a hydrogen bond is formed, van der waals effect, electrostatic effect, hydrophobic effect, pi-pi effect, etc.

4. The molecular design method of any one of claims 1 to 3, wherein in the step (2) of MD simulation, the simulation calculates the adjustment of temperature and pressure when molecules interact; preferably, a V-throttle temperature controller and a pressure controller are adopted to regulate the temperature and the pressure;

preferably, in the step (2) MD simulation, the molecular configuration of the compound molecule is maintained by selecting a potential energy parameter;

preferably, in the step (2), when an aqueous solution system is simulated, periodic boundary conditions are adopted in all three directions of x, y and z; when an ice-water mixed system is simulated, periodic boundary conditions are adopted in the x direction and the y direction;

preferably, in the step (2) MD simulation, a cubic or octahedral water box is selected, preferably 3.9 x 3.6 x 1.0nm³The water cartridge of (1).

5. The method of any one of claims 1 to 4, wherein the backbone of the compound molecule has a carbon chain or peptide chain structure.

6. A molecular design method according to any one of claims 1 to 5, wherein the hydrophilic group is a functional group capable of forming a non-covalent interaction with a water molecule, such as hydrogen bonding, Van der Waals interaction, electrostatic interaction, hydrophobic interaction or pi-pi interaction with water; for example, the hydrophilic group may be selected from hydroxyl (-OH), amino (-NH)₂) Carboxylic acid group (-COOH), amide group (-CONH)₂) At least one of the above, or, for example, a hydrophilic amino acid selected from proline (L-Pro), arginine (L-Arg), lysine (L-Lys), a compound molecule such as Gluconolactone (GDL) or a saccharide, or a molecular fragment thereof;

the ice-philic group is a functional group that can form a non-covalent interaction with ice, such as a hydrogen bond with ice, van der waals interaction, electrostatic interaction, hydrophobic interaction, or pi-pi interaction;illustratively, the oxophilic group may be selected from the group consisting of hydroxyl (-OH), amino (-NH)₂) Phenyl (-C)₆H₅) Pyrrolidinyl (-C)₄H₈N), or, for example, an ice-philic amino acid selected from the group consisting of glutamine (L-Gln), threonine (L-Thr), aspartic acid (L-Asn), a benzene ring (C)₆H₆) Pyrrolidine (C)₄H₉N) or a molecular fragment thereof.

7. The molecular design method of any one of claims 1 to 6, wherein the ice control material is formed by covalently bonding a block containing a hydrophilic group and a block containing an ice-philic group, or is formed by ionically bonding a molecule containing a hydrophilic group and a molecule containing an ice-philic group.

8. The method of any one of claims 1 to 7, further comprising a step of synthesizing the compound molecule, such as polymerization, dehydration condensation, or biological fermentation with genetically engineered bacteria.

9. An ice control material obtained by the molecular design method according to any one of claims 1 to 8.

10. The ice control material of claim 9, wherein the ice control material is PVA with a syndiotacticity r of 50% to 60%, and a molecular weight of 10kDa to 500 kDa; preferably, the PVA syndiotacticity r is 50-55%, and the molecular weight is 10-30 kDa.