Anti-ice nucleus peptide

Abstract

To provide a novel peptide with anti-ice nucleation activity; and also, to provide an anti-ice nucleation active agent, antifreeze solution, a coating composition, an anti-ice nucleation activity treated product, an article, and a method for preserving biological materials using the peptide.SOLUTION: Provided are any of the following peptides (a) or (b): (a) a peptide consisting of an amino acid sequence represented by Ser-His-Ile-Ala-Arg-Ser-Val or Arg-Pro-Ala-Val-Tyr-His-His, or (b) a peptide consisting of an amino acid sequence in which one amino acid is replaced with alanine in the above amino acid sequence; and an anti-ice nucleation agent, antifreeze solution, an anti-ice nucleation activity treated product, a coating composition, an article, and a method for preserving biological materials using the peptide.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a peptide. Further, the present invention relates to an anti-ice nucleation activator, a non-freezing solution, an anti-ice nucleation activated product, a coating composition, an article, and a method for preserving biological materials using the peptide.

Background Art

[0002] Since foreign substances are present in water, these foreign substances serve as nuclei for ice, and water freezes at 0°C. Such foreign substances are called ice nucleation active substances, and typical ice nucleation active substances include bacteria of the genus Pseudomonas and silver iodide. On the other hand, since pure water does not contain foreign substances, ice nuclei cannot be generated, and even when cooled to a temperature lower than the freezing point (0°C), for example, -39°C, it may not freeze (solidify). Such a phenomenon is generally referred to as the "supercooling phenomenon."

[0003] Several anti-ice nucleation activators (supercooling accelerators) that promote such a supercooling phenomenon have been reported so far. The anti-ice nucleation activator can create water that does not freeze even below the freezing point. As a result, expansion due to freezing does not occur, so plant or animal cells can be preserved without being destroyed. Even when once frozen, minute ice nuclei are formed and minute ice crystal formation occurs, so applications in fields such as foods and biological materials (organ preservation) are expected.

[0004] For example, it has been reported that low molecular weight compounds such as eugenol (see Non-Patent Document 1), which is a component of spices, and high molecular weight compounds such as polysaccharides derived from Bacillus thuringiensis (see Non-Patent Document 2) exhibit anti-ice nucleation activity.

[0005] However, although these anti-ice nucleation activators exhibit anti-ice nucleation activity against bacteria such as Pseudomonas fluorescens, which is an ice nucleation active substance, they have low anti-ice nucleation activity against silver iodide. Further, due to safety problems, it has been difficult to use these anti-ice nucleation activators in fields such as foods and biological materials.

[0006] In contrast, the present inventors have reported that tyrosine peptides and complexes composed of tyrosine peptides and polymers exhibit broad anti-ice nucleation activity against ice nucleation active substances, making them applicable to fields such as food, biomaterials, and the environment (see Patent Document 1). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2016 / 178426 [Non-patent literature]

[0008] [Non-Patent Document 1] H.Kawahara et al, J. Antibact. Antifung. Agents, 1996, Vol. 24, pp.95-100 [Non-Patent Document 2] Y. Yamashita et al, Biosci. Biotech. Biochem., 2002, Vol. 66, pp.948-954 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention aims to provide a novel peptide having anti-ice nucleation activity. Furthermore, the present invention aims to provide an anti-ice nucleation activator, an antifreeze solution, an anti-ice nucleation treated product, a coating composition, an article, and a method for preserving biological materials using the peptide. [Means for solving the problem]

[0010] As a result of diligent research to achieve the above objective, the inventors of this invention have discovered that two new peptide sequences possess anti-ice nucleus activity. Furthermore, when they synthesized peptides in which the amino acids in these peptide sequences were replaced with alanine and investigated their anti-ice nucleus activity, they found that the activity was enhanced.

[0011] Based on these findings and further investigations, the present invention provides the following peptides, anti-ice nucleation activators, antifreeze solutions, anti-ice nucleation activated products, coating compositions, articles, and methods for preserving biological materials.

[0012] Item 1. Either (a) or (b) below: (a) A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2 (b) A peptide consisting of an amino acid sequence in which one amino acid is replaced by alanine in the amino acid sequence represented by Sequence ID No. 1 or 2. Item 2. The peptide described in Item 1, which consists of an amino acid sequence represented by any of Sequence IDs 1 to 8. Item 3. A peptide according to Item 1, which consists of an amino acid sequence represented by any of Sequence IDs 1 to 4. Item 4. An anti-ice nucleating agent containing at least one of the peptides described in any one of items 1 to 3. Item 5. An antifreeze solution containing at least one of the peptides described in any one of items 1 to 3. Item 6. An anti-ice nucleation active product containing at least one of the peptides described in any one of items 1 to 3. Item 7. A coating composition containing at least one of the peptides described in any one of items 1 to 3. Article 8. An article having at least one of the peptides described in any one of the articles 1 to 3 on its surface. Item 9. A method for preserving a biological material, comprising the step of bringing at least one of the peptides described in any one of items 1 to 3 into contact with the biological material. [Effects of the Invention]

[0013] According to the present invention, a new peptide having anti-ice nucleation activity can be provided. Further, according to the present invention, an anti-ice nucleation active agent, a non-freezing solution, an anti-ice nucleation active treated product, a coating composition, an article, and a method for storing biological materials using the peptide can be provided. By using the peptide of the present invention, it is expected that long-term low-temperature storage of foods, biological materials, etc. will be possible.

Brief Description of Drawings

[0014] [Figure 1] It is a figure showing a peptide synthesis scheme by the Fmoc solid-phase synthesis method in an example. [Figure 2] It is a graph showing the anti-ice nucleation activity values (°C) of each peptide. The values in the graph are mean values ± SD. n = 3 [Figure 3] It is a graph showing the anti-ice nucleation activity values (°C) after alanine scanning. The values in the graph are mean values ± SD. n = 3 [Figure 4] It is a photograph showing the state of ice crystals in which the ice recrystallization inhibition (RI) activity after alanine scanning was measured. [Figure 5] It is a graph showing the results (RI values) of RI measurement after alanine scanning.

Modes for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described in detail.

[0016] In the present specification, "containing, comprising" includes the meaning of "essentially consisting of" and the meaning of "consisting of only".

[0017] In this specification, the abbreviations used to represent amino acids shall conform to the provisions of IUPAC-IUB (IUPAC-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)), the "Guidelines for the Preparation of Specifications Including Base Sequences or Amino Acid Sequences" (edited by the Japan Patent Office), and commonly used symbols in the relevant art. Furthermore, if optical isomers exist for amino acids, the L-isomer shall be indicated unless otherwise specified.

[0018] In this specification, the "amino acids" present in the various amino acid sequences listed herein are identified by known three-letter or one-letter abbreviations (see Table 1).

[0019] [Table 1]

[0020] 1. Peptides The peptide of the present invention is characterized by being either (a) or (b) below. (a) A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2 (b) A peptide consisting of an amino acid sequence in which one amino acid is replaced by alanine in the amino acid sequence represented by Sequence ID No. 1 or 2.

[0021] Hereinafter, when "the peptide of the present invention" is referred to in this specification, it means the peptide of (a) or (b) above.

[0022] The peptide of the present invention has high anti-ice nucleation activity. This is demonstrated in the examples described later by performing microdroplet freezing and ice recrystallization inhibitory activity measurements.

[0023] The peptides consisting of the amino acid sequences represented by SEQ ID NO: 1 or 2 are Ser-His-Ile-Ala-Arg-Ser-Val and Arg-Pro-Ala-Val-Tyr-His-His, respectively.

[0024] Peptides consisting of an amino acid sequence in which one amino acid in the amino acid sequence represented by Sequence ID No. 1 in (b) above is replaced with alanine include, specifically, Ala-His-Ile-Ala-Arg-Ser-Val (Sequence ID No. 3), Ser-Ala-Ile-Ala-Arg-Ser-Val (Sequence ID No. 4), Ser-His-Ala-Ala-Arg-Ser-Val (Sequence ID No. 5), Ser-His-Ile-Ala-Ala-Ser-Val (Sequence ID No. 6), Ser-His-Ile-Ala-Arg-Ala-Val (Sequence ID No. 7), and Ser-His-Ile-Ala-Arg-Ser-Ala (Sequence ID No. 8).

[0025] From the viewpoint of anti-ice nucleation activity, the peptide of the present invention is more preferably a peptide consisting of an amino acid sequence represented by any of SEQ ID NOs: 1 to 8, even more preferably a peptide consisting of an amino acid sequence represented by any of SEQ ID NOs: 1 to 4, and particularly preferably a peptide consisting of an amino acid sequence represented by SEQ ID NO: 3.

[0026] The peptides of the present invention also include salts thereof. Here, "salt" refers to any salt, such as sodium salt, potassium salt, calcium salt, hydrochloride salt, sulfate salt, nitrate salt, magnesium salt, ammonium salt, phosphate salt, hydrobromide salt, perchlorate salt, and organic acid salts (acetate salt, trifluoroacetate salt, citrate salt, maleate salt, oxalate salt, malate salt, lactate salt, succinate salt, propionate salt, tartrate salt, fumarate salt, formate salt, picrate salt, benzoate salt, p-toluenesulfonate salt, benzenesulfonate salt, methanesulfonate salt, etc.).

[0027] The peptide of the present invention may be a solvate. Examples of solvates include water (in the case of a hydrate), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, and dimethoxyethane.

[0028] Furthermore, the peptides of the present invention also include their derivatives. Here, "derivative" refers to a peptide of the present invention that has been modified by modifying, adding, changing, substituting, deleting, or otherwise altering its functional groups by known methods. For example, a derivative of the peptide of the present invention may have undergone chemical modification, biological (enzymatic) modification, or the like at its N-terminus, C-terminus, or amino acid side chain. Examples of modifications include, but are not limited to, alkylation, acylation (more specifically, acetylation, etc.), hydroxylation, esterification, halogenation, amination, amidation, and other functional group introductions (conversions).

[0029] The amino acids constituting the peptide of the present invention may be L-forms, D-forms, or DL-forms. Preferably, the peptide consists only of L-form amino acids or only of D-form amino acids, and particularly preferably, the peptide consists only of L-form amino acids.

[0030] The peptides of the present invention can be synthesized according to their amino acid sequence by general chemical synthesis methods such as condensation methods. These methods include conventional liquid-phase and solid-phase peptide synthesis methods. More specifically, such peptide synthesis methods include stepwise erogation, in which each amino acid is sequentially linked to extend the chain, and fragment condensation, in which fragments consisting of several amino acids are synthesized beforehand and then each fragment is subjected to a coupling reaction. The peptides of the present invention can be synthesized by either of these methods.

[0031] The condensation method used in the above peptide synthesis can also follow various known methods. Specific examples include the DMT-MM method, the azide method, the mixed acid anhydride method, the DCC method, the activated ester method, the redox method, the DPPA (diphenylphosphoryl azide) method, the DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-5-norbornene-2,3-dicarboximide, etc.), and the Woodward method. The solvents that can be used in each of these methods can be appropriately selected from common solvents that are well known to be used in this type of peptide condensation reaction. Examples include dimethylformamide (DMF), acetonitrile, dimethyl sulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate, and mixtures thereof.

[0032] In the peptide synthesis reaction described above, carboxyl groups in amino acids and peptides that do not participate in the reaction can generally be protected by esterification as lower alkyl esters such as methyl esters, ethyl esters, tertiary butyl esters, benzyl esters, p-methoxybenzyl esters, p-nitrobenzyl ester aralkyl esters, etc. In the solid-phase method, the C-terminal carboxyl group is bonded to a support such as chlorotrityl resin, chloromethyl resin, oxymethyl resin, or p-alkoxybenzyl alcohol resin. Examples of protecting groups for amino groups in amino acids and peptides that do not participate in the reaction include benzyloxycarbonyl (CBZ), t-butyloxycarbonyl (Boc), and 9-fluorenylmethyloxycarbonyl (Fmoc). Deprotection reactions of these protecting groups in amino acids, peptides, and the final peptide of the present invention can also be carried out according to conventional methods, such as catalytic reduction or methods using liquid ammonia / sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, trifluoromethanesulfonic acid, etc. In the solid-phase method, the bond between the C-terminus of the peptide and the resin is further cleaved.

[0033] The produced peptides can be purified using affinity chromatography, ion exchange chromatography, hydroxyapatite column chromatography, ammonium sulfate salting-out, silica gel chromatography, reversed-phase chromatography, HPLC, dialysis membrane chromatography, and other methods.

[0034] The peptides of the present invention can be used individually or in combination of two or more.

[0035] 2. Anti-ice nucleation activators The anti-ice nucleation agent of the present invention is characterized by containing at least one of the peptides described in (a) or (b) above.

[0036] The anti-ice nucleating agent of the present invention may contain other components in addition to the peptide described in either (a) or (b) above. The other components are not particularly limited and include, for example, known anti-ice nucleating agents (e.g., the anti-ice nucleating agent described in Japanese Patent Application Publication No. 2010-121052), bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.

[0037] When formulating the anti-ice nucleation agent of the present invention, there are no particular limitations on the dosage form, and examples include solutions, suspensions, emulsions, tablets, capsules, granules, powders, creams, ointments, and the like.

[0038] The content (on a solid content basis) of either peptide (a) or (b) of the present invention's anti-ice nucleation activator is not particularly limited and can be set according to the intended use, and can be adjusted as needed from low to high concentrations.

[0039] The applications of the anti-ice nucleation agent and peptide of the present invention are, for example, as follows.

[0040] The anti-ice nucleation activator and peptides of the present invention can be widely applied to the food industry, such as food preservatives (or liquids) for food and beverages (food preservatives (or liquids), beverage preservatives (or liquids)); the medical industry, such as cell preservatives (or liquids), blood preservatives (or liquids), and organ preservatives (or liquids); the cosmetics industry; and the environmental industry, such as frost damage prevention agents (or liquids) and paints. Furthermore, by using the anti-ice nucleation activator and peptides of the present invention, it is also possible to provide methods for improving the anti-ice nucleation activity of biological materials and food, and methods for preserving biological materials and food.

[0041] Examples of foods that can be preserved include fresh foods such as vegetables, fish, and meat (chicken, pork, beef, etc.); and processed foods such as juices, tofu, and freeze-dried tofu. By using the anti-ice nucleation activator or peptide of the present invention, preservation (preservation of freshness) is possible, and when importing or transporting these foods, it is possible to switch from frozen preservation to supercooled preservation, which can reduce electricity and other energy consumption.

[0042] The cells to be preserved are not particularly limited to plant or animal cells, but include, for example, human cells, sperm, and eggs. By using the anti-ice nucleation activator or peptide of the present invention, it is possible to preserve cells without damaging them.

[0043] The blood to be preserved is not limited to human or animal (excluding human) blood, and examples include whole blood, plasma, serum, etc. Furthermore, the anti-ice nucleation activator and peptide of the present invention can also be used for blood components such as white blood cells, red blood cells, plasma, platelets, etc.

[0044] The organs to be preserved are not particularly limited, as long as they are human or animal (excluding human) organs or parts thereof. The anti-ice nucleation activator and compounds of the present invention can be used in preservation solutions for organs removed during organ transplantation; preservation solutions for long-term storage of organs, etc.

[0045] Examples of products targeted for frost damage prevention include coolants for computers and car engines; defrosting agents for freezers, anti-fogging agents for car windows, and anti-condensation agents for tunnels.

[0046] In the following description, unless otherwise specified, the anti-ice nucleation agent of the present invention will be described as being in solid form, but the anti-ice nucleation agent of the present invention is not limited to solid form. That is, the anti-ice nucleation agent of the present invention may be in either liquid or solid form. For example, if the anti-ice nucleation agent of the present invention is solid, contacting the solid anti-ice nucleation agent of the present invention with biological materials (e.g., food (e.g., edible fish and shellfish; plants such as vegetables; edible meat such as beef, pork, and chicken; protein-denatured processed products such as tofu and yogurt; beverages), biomaterials (e.g., plant or animal cells (tissues); human or animal blood; human or animal organs or parts thereof)) or liquids (e.g., water, refrigerants, etc.) will cause the peptide to dissolve in the water or liquid of the biological material, thereby exhibiting anti-ice nucleation activity. Furthermore, if the anti-ice nucleation agent of the present invention is liquid, contacting the liquid anti-ice nucleation agent of the present invention with the above-mentioned biological materials or liquids (e.g., by spraying, dropping, or immersion) will allow it to be used as an anti-ice nucleation agent for said biological materials or liquids.

[0047] 3.Antifreeze solution The antifreeze solution of the present invention is characterized by containing at least one of the peptides described in (a) or (b) above. An antifreeze solution means a solution that does not freeze at the freezing point of ice (0°C).

[0048] The antifreeze solution of the present invention can be obtained by adding either peptide (a) or (b) above to a solvent or solution (for example, water or a refrigerant). The antifreeze solution of the present invention is less likely to freeze even at lower temperatures, and even if it does freeze, the size of the ice crystals at the moment of freezing can be reduced, and the formation of ice nuclei and ice crystals that can be induced by vibration is further suppressed.

[0049] The content (on a solid content basis) of either peptide (a) or (b) in the antifreeze solution of the present invention is not particularly limited, and for example, concentrations of 0.001 to 100 mg / mL can be given, preferably with a lower limit of 0.01 mg / mL and an upper limit of 50 mg / mL.

[0050] By immersing biological materials (for example, food (edible fish and shellfish; plants such as vegetables; edible meat such as beef, pork, and chicken; protein-denatured processed products such as tofu and yogurt; beverages), biomaterials (plant or animal cells (tissues); human or animal organs or parts thereof)) in the antifreeze solution of the present invention, or by spraying or dropping the antifreeze solution of the present invention onto the biological materials and then cooling, the biological materials will not freeze (be destroyed) at temperatures below 0°C, especially in the range of about 0°C to -15°C, or even if they do freeze, tiny ice nuclei will form and tiny ice crystals will be formed, thus enabling long-term low-temperature storage.

[0051] 4. Anti-ice nucleation activated material The anti-ice nucleation active product of the present invention is characterized by containing at least one of the peptides described in (a) or (b) above.

[0052] The anti-ice nucleation active treated material of the present invention can be obtained by treating a target material (for example, the above-mentioned biological material) with the peptide of the present invention. The anti-ice nucleation active treated material of the present invention is less likely to freeze even at lower temperatures, and even if it does freeze, the size of the ice crystals at the moment of freezing can be reduced, and the formation of ice nuclei and ice crystals that can be induced by vibration is further suppressed.

[0053] 5. Compositions for coating The coating composition of the present invention is characterized by containing at least one of the peptides described in (a) or (b) above. Furthermore, the article of the present invention is characterized by having at least one of the peptides described in (a) or (b) above on its surface.

[0054] The coating composition of the present invention can be obtained by preparing the coating composition using the peptide of the present invention. Surfaces treated with the coating composition of the present invention exhibit suppressed snow and ice accumulation.

[0055] The content (on a solid content basis) of either peptide (a) or (b) in the coating composition of the present invention is not particularly limited, and for example, concentrations of 0.001 to 100 mg / mL can be given, preferably with a lower limit of 0.01 mg / mL and an upper limit of 50 mg / mL.

[0056] The coating composition of the present invention preferably contains, in addition to the peptide described in either (a) or (b) above, other components necessary for coating. Examples of other components include binders and solvents.

[0057] The articles to which the coating composition of the present invention can be applied are not particularly limited. Preferably, such articles are those used in environments where ice may adhere. Specific examples of such articles include vehicles such as aircraft, ships, trains, and automobiles; roofs and exterior walls of buildings; antennas; power lines; winter clothing; traffic lights; heat exchangers; and parts thereof (especially parts that constitute surfaces that may come into contact with the outside air).

[0058] 6. Methods for preserving biological materials The present invention provides a method for preserving biological materials, characterized by including the step of bringing at least one of the peptides described in (a) or (b) above into contact with the biological material.

[0059] The biological material is the same as the biological material described above. To bring either the peptide described in (a) or (b) above into contact with the biological material, an anti-ice nucleating agent or antifreeze solution containing the peptide can be used.

[0060] Contact, in the case of the anti-ice nucleating agent of the present invention, involves bringing this solid into contact with a biological material (for example, by sprinkling it evenly), causing it to dissolve in the moisture of the biological material and exhibit anti-ice nucleating activity, thereby allowing the biological material to be stored at low temperatures without freezing (destruction). Alternatively, if the anti-ice nucleating agent of the present invention is a liquid or if the antifreeze solution of the present invention is used, contacting the above-mentioned biological material with the liquid (for example, by immersion, spraying, or dropping) will cause it to exhibit anti-ice nucleating activity, allowing the biological material to be stored at low temperatures without freezing (destruction). [Examples]

[0061] Examples are given below to further explain the present invention. However, the present invention is not limited in any way to these examples.

[0062] <Reagents> The reagents used are listed below.

[0063] [Synthesis of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM)] Methanol (MeOH) (Fujifilm Wako Pure Chemical Corporation) Sodium bicarbonate (AGC Inc.) • 2,4,6-Trichloro-1,3,5-Triazine (Fujifilm Wako Pure Chemical Corporation) • N-methylmorpholine (NMM) (Fujifilm Wako Pure Chemical Corporation) Acetone (Fujifilm Wako Pure Chemical Corporation) • α-Cyano-4-hydroxycinnamic acid (α-CHCA) (Sigma-Aldrich) • Angiotensin II (human) MALDI-TOF-MS calibrant (Fujifilm Wako Pure Chemical Corporation)

[0064] [Peptide synthesis using Fmoc solid-phase synthesis] The amino acid reagents used for peptide synthesis were manufactured by Watanabe Chemical Engineering Co., Ltd. The amino acids used are listed below. N-(9-Fluorenylmethoxycarbonyl)-Valine-Alcopolyethylene glycol resin (Fmoc-Val-Alko-PEG Resin) N-(9-Fluorenylmethoxycarbonyl)-trityl-histidine-trityl A-polyethylene glycol resin (Fmoc-His(Trt)-TrtA-PEG Resin) N-(9-Fluorenylmethoxycarbonyl)-alanine-alcopolyethylene glycol resin (Fmoc-Ala-Alko-PEG Resin) N-(9-Fluorenylmethoxycarbonyl)-t-butylserine (Fmoc-Ser(tBu)-OH) N-(9-Fluorenylmethoxycarbonyl)-L-alanine (Fmoc-Ala-OH) N-(9-Fluorenylmethoxycarbonyl)-t-butyl-L-threonine (Fmoc-Thr(tBu)-OH) N-(9-Fluorenylmethoxycarbonyl)-t-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH) N-(9-Fluorenylmethoxycarbonyl)-N-ω-2,2,4,6,7Pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Leu-OH) N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH) • N-(9-Fluorenylmethoxycarbonyl)-trityl-L-histidine (Fmoc-His(Trt)-OH) N-(9-Fluorenylmethoxycarbonyl)-L-isoleucine (Fmoc-Ile-OH) N-(9-Fluorenylmethoxycarbonyl)-L-valine (Fmoc-Val-OH) Except for the amino acid reagents, the following products manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. were used. • N-methylmorpholine (NMM) • N,N-dimethylformamide (DMF) • Dimethyl sulfoxide (DMSO) • Piperidine (PPD) · Methanol

[0065] [TNBS test] · 2,4,6-Trinitrobenzene-1-sulfonic acid (TNBS) (FUJIFILM Wako Pure Chemical Corporation) · Sodium hydrogen carbonate (AGC Corporation) · Phosphate buffer solution (PBS) (DS Pharma Biomedical Co., Ltd.)

[0066] [Chloranil test] · N,N-Dimethylformamide (DMF) (FUJIFILM Wako Pure Chemical Corporation) · Tetrachloro-p-benzoquinone (approx. 2% N,N-dimethylformamide solution) (Chloranil) (Tokyo Chemical Industry Co., Ltd.) · Acetaldehyde (FUJIFILM Wako Pure Chemical Corporation)

[0067] [Final deprotection] · Trifluoroacetic acid (TFA) (Watanabe Chemical Industries, Ltd.) · 1,2-Ethanedithiol (EDT) (Tokyo Chemical Industry Co., Ltd.) · Diethyl ether (FUJIFILM Wako Pure Chemical Corporation)

[0068] [Peptide identification and purification] · α-Cyano-4-hydroxycinnamic acid (α-CHCA) (Sigma-Aldrich) · Angiotensin II (human) MALDI-TOF-MS calibrant (FUJIFILM Wako Pure Chemical Corporation)

[0069] [Anti-ice nucleation activity measurement] · Dipotassium hydrogen phosphate (FUJIFILM Wako Pure Chemical Corporation) · Potassium dihydrogen phosphate (FUJIFILM Wako Pure Chemical Corporation) · Silver iodide (Sigma-Aldrich) · Aluminum plate 2.0×20×20 · Aluminum plate 2.0×20×40

[0070] [Synthesis of DMT-MM] 40 g of sodium hydrogen carbonate was added to a 500 mL Erlenmeyer flask and dissolved with 120 mL of methanol and 12 mL of ultrapure water. After stirring in an ice bath for 10 minutes, 29.6 g of 2,4,6-trichloro-1,3,5-triazine was added and stirred in the ice bath for 15 minutes. Then, it was stirred in a water bath at 30 - 35 °C for 7.5 hours. After stirring, pure water was added to precipitate 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), and suction filtration was carried out using an aspirator. Then, to wash the CDMT obtained by suction filtration, CDMT and 1000 mL of ultrapure water were added to a 1000 mL Erlenmeyer flask and stirred for 30 minutes. Suction filtration was carried out again, and CDMT was dried under reduced pressure. CDMT was dissolved in acetone, and 1.5 equivalents of NMM with respect to CDMT was slowly added dropwise while stirring. After stirring for 30 minutes, suction filtration was carried out to obtain DMT-MM. The obtained DMT-MM was identified by a matrix-assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF-MS) (Microflex manufactured by Bruker corporation) manufactured by Bruker corporation.

[0071] α-CHCA was dissolved in a mixed solution of acetonitrile:pure water = 1:1 to obtain a saturated solution of α-CHCA (matrix solution). The matrix solution and the DMT-MM / methanol solution were mixed at a ratio of 1:1, and 1 μL was dropped onto the sample stage. By drying under reduced pressure, a mixed crystal of the sample and the matrix was prepared. Measurement was carried out using this with MALDI-TOF-MS.

[0072] <Peptide synthesis by Fmoc solid-phase synthesis method> RPAVYHH (RP peptide) and SHIARSV (SH peptide) were synthesized using the Fmoc solid-phase method. The synthesis scheme is shown in Figure 1, and the charged amounts of amino acid reagents are shown in Tables 2 and 3.

[0073] Resin was placed in a column and washed three times each with methanol and DMF. The resin was then stirred with 25% DMSO / DMF for 30 minutes to swell. After swelling, it was washed six times with DMF. Then, 20% PPD / DMF was added and stirred for 30 minutes to deprotect the Fmoc group. After deprotection, the completion of Fmoc group deprotection was confirmed by the TNBS test. The column was washed six times with DMF to remove any remaining PPD. Three equivalents of amino acids, 80 μL of NMM, and 0.30 g of DMT-MM were added to the resin and condensed for 3 hours. After condensation, it was washed six times with DMF to remove any unreacted amino acids, DMT-MM, and NMM. The completion of condensation was confirmed by the TNBS test. Furthermore, after proline deprotection, the completion of the reaction was confirmed by the chloranil test. By repeating these operations and sequentially condensing amino acid derivatives, H-Arg(Pbf)-Pro-Ala-Val-Tyr(tBu)-His(Trt)-His(Trt)-TrtA-PEG Resin (RP peptide-PEG Resin) and H-Ser(tBu)-His(Trt)-Ile-Ala-Arg(Pbf)-Ser(tBu)Val-Alko-PEG Resin (SH peptide-PEG Resin) were synthesized. The resulting resins and side-chain protected peptides were prepared as Cleavage Mixture B and then deprotected.

[0074] [Table 2]

[0075] [Table 3]

[0076] TNBS exam TNBS solution (100 μL), PBS (200 μL), and NaHCO3 aqueous solution (200 μL) were taken using a micropipette and transferred to microtubes. A few granules of resin were taken with a spatula and dispersed in the solutions in the microtubes. The presence or absence of color change indicated the presence or absence of primary amino groups, thereby confirming the completion of condensation and deprotection.

[0077] Chloranil test A small amount of resin was added to a solution prepared by mixing 200 μL of 2% chloranil / DMF and 200 μL of 2% acetaldehyde / DMF, and the color change of the resin was confirmed. The completion of the deprotection of the Fmoc group was confirmed by the resin turning blue.

[0078] Final Deprotection 8.0 mL of TFA, 0.25 mL of ultrapure water, 0.25 mL of EDT, 0.50 mL of thioanisole, and 0.75 g of crystalline phenol were mixed and stirred in an ice bath for 10 minutes to obtain cleavage mixture B. The resin synthesized above was added thereto and stirred at room temperature for 3 hours. Then, to precipitate the peptide, 50 mL of diethyl ether was added and stirred at room temperature for 30 minutes. The resin and the peptide were recovered by suction filtration and washed with diethyl ether to remove EDT. Then, after dissolving the peptide in TFA, concentration under reduced pressure was performed, decantation was carried out with diethyl ether, and the peptide was recovered by suction filtration. The recovered peptide was dissolved in ultrapure water, dialyzed using a dialysis membrane with a molecular weight cut-off of 100 - 500 D (Biotech CE Tubing MWCO: 100 - 500 D, manufactured by Spectrum Laboratories, Inc.), the precipitate was removed by suction filtration, and RP peptide and SH peptide in the form of a white powder were obtained through lyophilization.

[0079] <Identification of the target product by MALDI-TOF-MS spectrum> The synthesized RP peptide and SH peptide were dissolved in methanol, and the mass was measured using a MALDI-TOF-MS spectrum to identify the target product.

[0080] <Measurement of anti-ice nucleation activity> In order to examine the ability of the synthesized peptide to inhibit ice recrystallization, anti-ice nucleation activity measurement was performed.

[0081] Preparation of potassium-based phosphate buffer (KPB) A potassium dihydrogen phosphate aqueous solution was prepared by dissolving 5.2 g of potassium dihydrogen phosphate in 200 mL of pure water. A potassium dihydrogen phosphate aqueous solution was prepared by dissolving 2.6 g of potassium dihydrogen phosphate in 130 mL of pure water. Potassium dihydrogen phosphate aqueous solution was added to 200 mL of the potassium dihydrogen phosphate aqueous solution to adjust the pH to 7.0. The mixture was passed through a 0.45 μL filter.

[0082] Preparation of AgI aqueous solution, which acts as a pseudo-ice nucleus. AgI was mixed with potassium phosphate buffer (KPB) to a concentration of 1.0 mg / mL to prepare an AgI aqueous solution. The AgI aqueous solution and ultrapure water were mixed in a volume ratio of 9:1 to obtain a blank sample. AgI was used as an ice nucleus mimic.

[0083] Anti-ice nucleation activity test The microdroplet freezing method developed by Vali et al. was used to measure anti-ice nucleation activity. An aluminum plate was placed inside a programmable ultra-low temperature aluminum block constant temperature bath cryoporter (manufactured by Cynics Co., Ltd.). Twelve drops of evaluation sample (10 μL each) and a blank sample were seeded onto the surface of the aluminum plate, and the temperature of the cold plate was lowered at a rate of 1.0 °C / min. The temperature at which 6 out of 12 drops froze (T 50 The freezing temperature of the sample was recorded. Each sample was measured three times, and the anti-ice nucleus activity (ANA) = sample (T 50 )-Control(T 50 It was calculated using the formula shown.

[0084] Each evaluation sample was prepared by mixing an aqueous AgI solution with an aqueous solution containing a peptide at a concentration of 8.0 mg / mL in a volume ratio of 9:1. The results are shown in Figure 2.

[0085] <Alanine Scan> Fabrication of AHIARSV, SAIARSV, SHAARSV, SHIAASV, SHIARAV, and SHIARSA To identify effective amino acids within the SH peptide sequence that showed the highest anti-ice nucleation activity in the anti-ice nucleation activity measurement described above, an alanine scan was performed. First, AHIARSV, SAIARSV, SHAARSV, SHIAASV, SHIARAV, and SHIARSA were prepared by substituting each residue with Ala. The preparation method was the same as described above, using the Fmoc solid-phase synthesis method. The TNBS test and chloranil test, which confirm condensation and deprotection, were performed using the same procedure as described above.

[0086] The quantities of amino acid reagents used are shown in Tables 4-9.

[0087] [Table 4]

[0088] [Table 5]

[0089] [Table 6]

[0090] [Table 7]

[0091] [Table 8]

[0092] [Table 9]

[0093] AHIARSV, SAIARSV, SHAARSV, SHIARAV, SHIARSA's final deprotection Cleverage mixture B was prepared using the same procedure as above, and final deprotection was performed to obtain white powders of AHIARSV, SAIARSV, SHAARSV, SHIARAV, and SHIARSA.

[0094] SHIAASV's final deprotection 9.5 mL of TFA, 0.25 mL of ultrapure water, and 0.25 mL of EDT were mixed and stirred in an ice bath for 10 minutes to obtain Cleavage Mixture C. Resin and side-chain protected SHIAASV were added to this mixture and stirred at room temperature for 3 hours. Then, 50 mL of diethyl ether was added to precipitate the peptide and stirred at room temperature for 30 minutes. The resin and peptide were recovered by suction filtration and washed with diethyl ether to remove the EDT. After that, the peptide was dissolved in TFA, concentrated under reduced pressure, decanted with diethyl ether, and the peptide was recovered by suction filtration. The recovered peptide was dissolved in ultrapure water and dialyzed using a dialysis membrane with a molecular weight cutoff of 100-500 D (Biotech CE Tubing MWCO: 100-500 D, manufactured by Spectrum Laboratories, Inc.). After removing the precipitate by suction filtration, SHIAASV was obtained as a white powder after freeze-drying.

[0095] Identification of target product by MALDI-TOF-MS spectroscopy The synthesized AHIARSV, SAIARSV, SHAARSV, SHIAASV, SHIARAV, and SHIARSA were dissolved in methanol, and the target compounds were identified by measuring their mass using MALDI-TOF-MS spectroscopy.

[0096] Measurement of anti-ice nucleus activity The anti-ice nucleus activity of synthesized SH peptides, AHIARSV, SAIARSV, SHAARSV, SHIAASV, SHIARAV, and SHIARSA, was measured using the same procedure as described above, and an attempt was made to identify effective amino acids. Furthermore, concentration dependence was confirmed by measuring the activity at concentrations of 1.0 mg / mL, 2.0 mg / mL, 4.0 mg / mL, and 8.0 mg / mL. The results are shown in Figure 3.

[0097] Measurement of ice recrystallization inhibition (RI) activity To observe the ice crystals formed when an aqueous solution containing peptides was frozen, the RI activity was measured using the sucrose sandwich method. A microscope equipped with a temperature control device (Linkam Corporation) was used. Synthesized RP peptide, SH peptide, AHIARSV, SAIARSV, SHAARSV, SHIAASV, SHIARAV, and SHIARSA were each dissolved in ultrapure water at a concentration of 1 mg / mL to prepare samples. Each sample was mixed with a 60% (w / v) sucrose aqueous solution in a 1:1 ratio, and 1 μL of the mixed solution was sandwiched between two coverslips. The coverslips were placed on a temperature control device and rapidly frozen to -40°C at 100°C / min. The temperature was immediately increased at the same rate to -6°C. Ice crystals were photographed with a camera 30 minutes after reaching -6°C. The captured images were cropped to a size of 500 × 500, and the number of ice crystals and the total area of ​​ice crystals within that area were determined, and the average size of each ice crystal (unit crystal area) was calculated. Based on the measured size per unit area, the RI activity was calculated using the following formula. A lower RI value indicates higher activity. Only ultrapure water was used as the blank solution. The results are shown in Figures 4 and 5. RI value = Unit crystal area of ​​sample solution / Unit crystal area of ​​blank solution

Claims

1. A peptide consisting of an amino acid sequence represented by one of SEQ ID NOs: 1 to 8.

2. The peptide according to claim 1, which is a peptide comprising an amino acid sequence represented by any of SEQ ID NOs: 1 to 4.

3. An anti-ice nucleating agent containing at least one of the peptides described in claim 1 or 2.

4. An antifreeze solution containing at least one of the peptides described in claim 1 or 2.

5. An anti-ice nucleation active product containing at least one of the peptides described in claim 1 or 2.

6. A coating composition containing at least one of the peptides described in claim 1 or 2.

7. An article having at least one of the peptides described in claim 1 or 2 on its surface.

8. A method for preserving a biological material, comprising the step of contacting the biological material with at least one of the peptides described in claim 1 or 2.

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