Sustainable antifreeze coatings by peptide self-assembly

Abstract

The technology disclosed in the application concerns antifreeze and anti-ice peptidic films.

Description

[0001] SUSTAINABLE ANTIFREEZE COATINGS BY PEPTIDE SELF-ASSEMBLY

[0002] TECHNOLOGICAL FIELD

[0003] The invention generally relates to anti-ice and anti-freeze films and uses thereof.

[0004] BACKGROUND

[0005] Global warming has intensified the frequency and severity of extreme weather events, with a notable increase in massive snowstorms and record-low temperatures, presenting challenges beyond daily life and impacting both natural and technological infrastructure. Ice and frost formation on surfaces, such as heat exchangers, insulators, and aircraft wings, poses significant threats to efficiency, safety, and overall performance. The consequences of these cold conditions extend to agriculture, with frost accumulation on plant surfaces causing crop injuries and agricultural disasters.

[0006] Numerous efforts have been invested to advance the development of coatings with anti-ice and anti-freeze properties. For instance, using superhydrophobic coatings reduces surface energy, consequently mitigating the adherence of water droplets to the surface. However, these coatings typically exhibit instability where icing, deicing, and high humidity occur. Another approach to anti-ice coatings involves the application of aqueous lubricating coatings, employing polymers with hydrophilic groups. This strategy effectively reduces ice adhesion by creating a smoother surface and exploits the relatively low freezing point of water within the lubricating layer compared to bulk water. Moreover, various materials, such as polydimethylsiloxane, fluorosilane-modified epoxy, and fluorine-silicone resin are employed for surface modification to confer anti-ice properties. However, many of these materials involve prolonged processes of application, are not environmentally friendly, and have high production costs.

[0007] Another promising material to mitigate the accumulation of ice and frost on surfaces involves utilizing anti-freeze proteins (AFP). These proteins are naturally produced by various organisms, such as fish, insects, and fungi, enabling the organisms to endure sub-zero conditions. AFPs exhibit diverse secondary structures, such as a- helices, P-sheets, and P-helices, individually or in combination, with sizes ranging from approximately 3 to 34 kDa. Furthermore, these proteins typically incorporate diverse amino acid sequences that actively contribute to their functionality. AFPs effectively control the growth and recrystallization of ice crystals by adsorbing to the front of the ice crystal planes, thereby impeding the crystal's growth, and decreasing the freezing process. Importantly, these proteins can depress the freezing point of water below the equilibrium melting point, thereby restricting ice growth for an extended duration.

[0008] Acquiring sufficient amounts of AFP for this purpose is a challenge, achievable through either extraction from living organisms or by employing recombinant expression in bacteria, a process that is relatively time-consuming and involves multiple purification steps. Additionally, attaching AFPs to the desired surface requires various strategies and involves multiple steps. Previous studies have shown diverse methods for surface attachment of AFPs, including using polymers and modified AFPs. For instance, adding a ketone group to AFPs facilitates their attachment to polymer chains on a glass surface. Alternatively, a modification enabling ionic attachment to aluminum surfaces treated with plasma has been explored. Another study utilized a peptide as a linker between AFP and the aluminum surface. Overall, the production and attachment of AFP to surfaces present complicated challenges, suggesting the need for innovative methods to simplify these processes.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors of the technology disclosed herein have developed a novel class of antifreeze materials, structured as short peptides with key structural elements that permit spontaneous self-assembly into anti-freeze coatings. The antifreeze peptides comprise between 4 and 50 amino acids and are configured for surface attachment, anchoring or adherence as films preventing ice formations on the surface. To achieve such capabilities, the peptides comprise at least one surface-binding amino acid, at least one amino acid promoting self-assembly and an anti-ice amino acid motif.

[0011] In a first of its aspects, the invention concerns a film or a coating on a surface, the film or coating being surface-bound and having a surface-binding side (or face) and an exposed side (or face), said exposed side being an anti-ice or anti-freeze surface, wherein binding of the film or coat to the surface comprises chemical interaction between a plurality of surface-binding moieties and surface localities, and wherein the film comprises a plurality of substantially perpendicularly aligned peptides having between 4 and 50 amino acids.

[0012] Also provided is a peptide composition comprising a plurality of peptides, each peptide comprises between 4 and 50 amino acids, of which at least one amino acid is a surface-binding group selected to bind to a target surface, at least one amino acid is or comprises an antifreeze motif that is selected to disrupt ice nucleation or that is selected to bind to ice crystal faces, and at least one amino acid is a self-assembly amino acid that is selected to promote formation of a dense and a continuous film on the surface, wherein the plurality of peptides being capable of forming, upon application to the surface from a formulation, an adherent antifreeze film that reduces ice nucleation and / or ice adhesion on the surface.

[0013] Surface-bound peptides of the invention, acting as antifreeze peptides and icebinding peptides offer an elegant route to both preventing ice formation and reducing ice adhesion on surfaces. Unlike bulk antifreeze agents that lower freezing point by colligative effects, peptides of the invention work at the interface: they adsorb to nascent ice nuclei or crystal faces and locally modify crystal growth, producing a combination of thermal hysteresis (a depressed freezing point relative to melting) and powerful inhibition of ice recrystallization. When these peptides are tethered to the surface, they present icerecognition motifs outward to influence heterogeneous nucleation directly at the substrate- water interface, where most problematic icing begins.

[0014] Mechanistically, the surface-bound peptides reduce the number and activity of effective nucleation sites by blocking water molecules from arranging into ice-like lattices at the interface and by stabilizing curved ice fronts that are thermodynamically disfavored. The peptides also disrupt growth by preferentially binding specific crystallographic faces, producing a blunt or irregular ice front that is mechanically weaker and less prone to strong adhesion. Due to the relatively thin peptide layer, having a maximum of 50 amino acids, the bound peptides further lower ice adhesion by reducing interfacial contact and mechanical interlocking.

[0015] Peptides of the invention solve several engineering challenges. They are immobilized with controlled orientation and sufficient surface density to retain icebinding activity. Their chemistries permit binding to a vast variety of surfaces; and films formed therefrom demonstrate durability under abrasion, UV exposure and fouling, which extend their functional life.

[0016] As used herein, films of the invention may be considered anti-ice or antifreeze films or coatings achieving the aforementioned properties by spontaneous self-assembly.. While the terms “anti-ice” and “antifreeze” may have different common definitions, wherein the term “anti-ice” means prevention of ice formations on the surface and / or prevention of ice adhesion to the surface; and the term “anti-freeze” refers to the capability of the film to reduce a solidification temperature of water or a water-based medium coming into contact with the film, such that the water or water medium does not substantially freeze on the film; each of the terms is used herein interchangeably to the other and encompasses one or more of the following:

[0017] -prevention of ice formations on the surface;

[0018] -prevention of ice adhesion to the surface;

[0019] -reduction in a solidification temperature of water or a water-based medium coming into contact with the film, such that the water or water medium does not substantially freeze on the film;

[0020] -blocking water molecules from arranging into ice-like lattices at the interface;

[0021] -disrupt growth of ice crystals by binding specific crystallographic faces thereof; and / or

[0022] -preventing or reducing interfacial contact of the surface with water, thereby preventing or reducing mechanical interlocking.

[0023] The ability to spontaneously self-assemble on a surface to form an ordered continuous film is derived from presence of one or more surface-binding amino acids, which nature and composition may depend on the surface composition and optionally on surface features or surface localities to which the surface-binding amino acids may associate. Generally speaking, the surface-binding amino acid is one having at least one atom or group of atoms capable of association, typically in a robust and an irreversible way to surface localities (atoms or functionalities) present on the surface or making up the surface composition. The association may be or may involve Van-der-Walls, coordinative, covalent, ionic, electrostatic, dipole-dipole, or hydrogen bonding or association or interaction.

[0024] The surface-binding amino acid may be replaced or may be substituted, or may have a functionality that can act as a surface anchoring group. Such a group may be a siloxane, a heteroatom such as sulfur (or a disulfur), a carboxylate (or a carboxylic acid) or any other atom or group known to surface associate in a manner disclosed herein.

[0025] In some embodiments, the surface-binding group is a silylated amino acid, having at least one siloxane group. In some embodiments, the silylated amino acid is of the form RSi-O-SiR or of the form -[I Si-O]-, in which R may be the amino acid or any atom or functionality thereof. Non-limiting examples of such silylated groups include Polydimethylsiloxane (PDMS), Vinyl-terminated PDMS, polymethylphenylsiloxane (PMS), aminopropyl -terminated PDMS and others. In some embodiments, the amino acid is an aminopropyl-terminated siloxane, such as aminopropyl -terminated PDMS.

[0026] In some embodiments, the surface-binding group is a siloxane moiety or a silylated amino acid. Non-limiting examples of silylated amino acids include O- trimethyl silyl serine, O-tert-butyldimethylsilyl serine, O-tert-butyldiphenylsilyl tyrosine, S-tert-butyldimethylsilyl cysteine, N-trimethylsilyl glycine, N,O-bis(trimethylsilyl) amino acids (such as N,O-bis(trimethylsilyl)-L-alanine and N,O-bis(trimethylsilyl)- serine methyl ester), Ne-(3 -tri ethoxy silylpropyl)-ly sine, (3 -tri ethoxy silyl)propyl-gly cine and others.

[0027] In some embodiments, the surface-binding group is a chemically modified amino acid. In some embodiments, the surface-binding amino acid is unmodified.

[0028] The surface-binding amino acid may for example be cysteine (capable of forming strong bonds to gold and silver surfaces and potentially react or interact with surface functionalities such as maleimides, iodoacetamides, disulfides and others), lysine (capable of providing covalent coupling to carboxylated functionalities, activated glass, and functionalized resins), tyrosine / tryptophan (capable of providing covalent coupling to carb on / graphi tic surfaces or to arylated substrates), serine / threonine, histidine (capable of coordinating to divalent metal ions or metal-chelate surfaces), aspartate / glutamate (capable of coordinating to metal oxide surfaces, such as TiCh, AI2O3, FeOx) and to metal ions / oxides and oxide ceramics), DOPA (capable of associating to a variety of surfaces such as glass, polymers, metals and others), and others.

[0029] In some embodiments, the surface-binding amino acid may be one or more of cysteine, lysine, tyrosine, tryptophan, serine, threonine, histidine, aspartate, glutamate, DOPA, and others.

[0030] Thus, the surface on which the antifreeze / anti-ice film may be formed does not need to be limited by composition in any way. The surface composition may be a metal, such as gold, silver, aluminum, stainless steel, and others; metal oxides, such as glass, silica, TiO2, AI2O3, and others; an activated glass or silanized surface, a polymeric film or plastics, such as polystyrene, PMMA, PDMS, PTFE and others; a carbonaceous material such as graphene, carbon nanotubes, graphite, and others. In some embodiments, the amino acid used for surface binding comprises one or more hydroxyl groups, such as hydroxyaryl groups having one, two or three or four or five hydroxyl functionalities.

[0031] In some embodiments, the hydroxyaryl is a catechol -based group, i.e., a phenyl having two ortho hydroxyl groups. In some embodiments, the surface-binding amino acid is a catechol-containing amino acid, such as 3,4-dihydroxy-L-phenylalanin (DOPA), and a DOPA containing short peptide. In some embodiments, the short peptide of the invention comprises one or more DOPA amino acids. Where a peptide comprises more than one DOPA amino acids, the DOPA amino acids may or may not be bonded to each other. In some cases, more than one DOPA amino acid may be spaced apart from each other along the peptide backbone.

[0032] To promote, enhance, or improve attachment of the peptide to the surface, the surface-binding amino acid may be in the form of an amino acid sequence comprising DOPA and at least one additional amino acid. The additional amino acid may comprise a surface-binding atom or group, optionally being or comprising a heteroatom such as S, N and O. Examples of such amino acids include lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, and serine. In some embodiments, the additional amino acid is lysine, and the amino acid sequence permitting surface binding is DOPA-Lys- or Lys-DOPA-, wherein one or both of the amino acids directly associate or interact with the surface.

[0033] In some embodiments, the amino acid sequence selected for surface binding is an amino acid sequence comprising two or more amino acids selected from cysteine, lysine, tyrosine, tryptophan, serine, threonine, histidine, aspartate, glutamate, and DOPA. In some embodiments, the amino acid sequence comprises DOPA and one or more amino acids, as selected. In some embodiments, the amino acids sequence comprises DOPA and an amino acid selected from lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, and serine.

[0034] In some embodiments, the surface binding amino acid sequence comprises DOPA and lysine. Without wishing to be bound by theory, it is believed that lysine can promote the adhesion to the surface due to the positive charge. However, without limitation, other natural and non-natural groups / amino acids can achieve such association as well. As such, the association to the surface may utilize other functionalities as known in the art. In some cases, the surface-binding amino acid(s) are provided at an end of the peptide, thereby permitting specific orientation of the peptides on the surface. However, the surface-binding amino acid(s) may also be provided along the peptide chain. The positioning of the surface-binding amino acid at an end of the peptide, and the anti-ice or antifreeze motif (or amino acid) at another end of the peptide, whereby both functionalities are associated via an amino acid or an amino acid sequence that promotes self-assembly, ensures formation of dense films whereby inner-film interactions between the substantially perpendicularly oriented peptide blocks water molecules from contacting the surface, achieving the aforementioned properties.

[0035] As used herein, the term substantially perpendicular or any lingual variation thereof, refers to the orientation of the film of peptides relative to the surface. As the actual orientation of the peptides relative to the surface may vary based on their length, chemical composition and possible interactions between atoms or groups along the peptide chain, the film is nevertheless oriented approximately normal to the substrate surface, within an angular tolerance that is 90°±10° of the substrate plane, measured as the average tilt relative to the surface normal. Such an orientation ensures continued and effective exposure of the anti-ice or antifreeze motif.

[0036] At least one amino acid that promotes or is capable of orienting the surface-bound peptides in a predetermined fashion are selected to have functional groups that form or increase inter- and intramolecular interactions or local interactions among the peptides, without external direction, to achieve an ordered structure that is dense and continuous and which is formed by noncovalent interactions. Amino acid(s) promoting self-assembly may be selected amongst such amino acids that comprise a functionality that interacts inter- or intramolecularly with other functionalities present in its vicinity. The interaction may be in a form of H-bond formations, ti~ ti stacking, hydrophobic interactions and others, that advance formation of a stable and ordered film. Typically, the amino acids are hydrophobic amino acids, amino acids having aromatic groups, or amino acids capable of generating intramolecular hydrogen bonding. The formation of intramolecular forces may alternatively or additionally by achieved by substituting an amino acid with such functionalities that are capable of inducing intramolecular interactions.

[0037] In some embodiments, the amino acid promoting self-assembly may be selected amongst aromatic amino acids or substituted forms thereof. These include tyrosine, phenylalanine and tryptophan. In some embodiments, the amino acid promoting selfassembly is phenylalanine, or a phenylalanine derivative.

[0038] In some embodiments, and without limitation, the surface-binding amino acid may also act to promote self-assembly. In such cases, the surface-binding amino acid and the at least one amino acid promoting self-assembly may be the same amino acid.

[0039] Thus, in some embodiments, the peptides used for assembling an anti-ice or antifreeze film on a surface are short peptides of between 4 and 50 amino acids, comprising at least one surface-binding amino acid selected from DOPA, Lys-DOPA- and DOPA- Lys, at least one amino acid promoting self-assembly selected from phenylalanine and phenylalanine derivatives and an anti-ice or antifreeze amino acid or motif.

[0040] The number of amino acids in the peptides is generally between 4 and 50, inclusive. In some cases, the number of amino acids may be between 4 and 10, or 4 and 15, or 4 and 20, or 4 and 25, or 4 and 30, or 4 and 35, or 4 and 40, or 4 and 45, or 10 and 50, or 10 and 40, or 10 and 30, or 10 and 40 amino acids. In some embodiments, the peptides have between 5 and 25 amino acids, or between 10 and 30 amino acids. In some cases, the number of amino acids is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids.

[0041] In some embodiments, the peptides may be of formula A-B-C, wherein A is a surface binding amino acid, e.g., DOPA or a sequence comprising DOPA and lysine; B is an amino acid or an amino acid sequence promoting self-assembly, e.g., phenylalanine or a derivative thereof, or an amino acid sequence comprising same; C is an anti -ice or an antifreeze amino acid; and each designates a peptide bond. In some cases, the surface binding peptide is of formula A-C-B, wherein each of A, B and C is as defined herein. The peptide may also be of a formula such as A-B-C-B, A-C-B-C and others.

[0042] In some embodiments, the peptide is of a structure A-[]-C, wherein [] designates an amino acid sequence comprising one or more of A, B and C. Some examples include A-A-B-C, A-B-A-C-C, A-B-B-C-A-C, and others.

[0043] In some embodiments, A is DOPA, or DOPA-Lys-, or Lys-DOPA- (Lys being the amino acid lysine).

[0044] In some embodiments, B comprises one or more Phe amino acid (Phe being phenylalanine).

[0045] In some embodiments, the peptide segment A-B- is selected from: DOPA-C

[0046] Lys-DOPA-Phe-C

[0047] Lys-DOPA-Phe-Phe-C

[0048] Lys-DOPA-Phe-C-Phe

[0049] Lys-DOPA-C-Phe-C

[0050] DOPA-Phe-C

[0051] DOPA-Phe-Phe-C wherein C is as defined herein.

[0052] The anti-ice or antifreeze amino acid C is an amino acid sequence of the form Thr- x, x-Thr or Thr-x-Thr, wherein Thr is a threonine (T) and x is a single amino acid or a sequence of two or more amino acids, such that the total number of amino acids in the peptide is maintained at 50 amino acids or less.

[0053] In some embodiments, the surface-binding peptide is of a form such as

[0054] DOPA-x-Thr-x

[0055] Lys-DOPA-Phe-x-Thr-x

[0056] Lys-DOPA-Phe-Phe-x-Thr-x

[0057] Lys-DOPA-Phe-x-Thr-x-Phe

[0058] Lys-DOPA-x-Thr-x-Phe-x-Thr-x

[0059] DOPA-Phe-x-Thr-x

[0060] DOPA-Phe-Phe-x-Thr-x

[0061] Lys-DOPA-Phe-Thr-x

[0062] Lys-DOPA-Phe-Phe-Thr-x

[0063] Lys-DOPA-Phe-Thr-x-Phe

[0064] Lys-DOPA-C-Phe-Thr-x

[0065] Lys-DOPA-Phe-Thr-x

[0066] DOPA-Phe-Thr-x

[0067] DOPA-Phe-Phe-Thr-x

[0068] Lys-DOPA-Phe-Thr-x-Thr

[0069] Lys-DOPA-Phe-Phe-Thr-x-Thr

[0070] Lys-DOPA-Phe-Thr-x-Thr-Phe

[0071] Lys-DOPA-C-Phe-Thr-x-Thr

[0072] Lys-DOPA-Phe-Thr-x-Thr

[0073] DOPA-Phe-Thr-x-Thr DOPA-Phe-Phe-Thr-x-Thr.

[0074] In some embodiments, the amino acid sequence x may comprise between 5 and 20 amino acids. The amino acid may be any natural or unnatural amino acid, an amino acid analog, a- or P-forms, or may be in either L- or D configurations. Amino acid analogs which may be used in a compound of the invention may be chemically modified at either or both C-terminal and / or N-terminal; or chemically modified at a side-chain functional group (e.g., positioned at the a-position or any other pendant group).

[0075] The amino acid may be selected amongst alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine valine, pyrrolysine and selenocysteine; and amino acid analogs such as homo-amino acids, N- alkyl amino acids, dehydroamino acids, aromatic amino acids and a, a-di substituted amino acids, e.g., cystine, 5 -hydroxylysine, 4-hydroxyproline, a-aminoadipic acid, a- amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, a-methyl serine, ornithine, pipecolic acid, ortho, meta or para-aminobenzoic acid, citrulline, canavanine, norleucine, d-glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3- benzothienylalanine and thyroxine.

[0076] In some embodiments, C is an amino acid sequence comprising the threonine- based motif, and any sequence of amino acids. Non-limiting examples include:

[0077] -Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-Pro

[0078] Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-Pro-

[0079] Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro-

[0080] -Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro-

[0081] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-

[0082] - Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala- Al a-Glu-Leu Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-

[0083] - Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala-Glu-Leu -Asp-Thr- Ala- Asp-Ala-Lys-Ala-Ala-Glu-Leu Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-

[0084] - Asp-Thr- Al a- S er- Al a-Ly s- Al a- Ala-Glu-Leu

[0085] -Asp-Thr- Al a- Asp- Al a-Ly s- Al a- Ala- Al a-Leu and others.

[0086] In some embodiments, the peptide is selected from Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 1), Lys-DOPA-Phe-Phe-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2),

[0087] Lys-DOPA-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3),

[0088] Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 4), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 5), Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 6),

[0089] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 7),

[0090] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 8),

[0091] Lys-DOPA-Phe-Phe-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 9),

[0092] Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 10),

[0093] Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO H),

[0094] Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO

[0095] 12),

[0096] Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO

[0097] 13),

[0098] Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro-Pro-DOPA-Lys (SEQ ID NO 14),

[0099] Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Pro-DOPA-Lys (SEQ ID NO 15),

[0100] Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Pro-DOPA-Lys (SEQ ID NO 16),

[0101] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 17),

[0102] Lys-DOPA-Pro-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 18),

[0103] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO Lys-DOPA-Pro-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 20),

[0104] Lys-DOPA-Pro-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 21), Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 22), Lys-DOPA-Pro-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 23), and

[0105] Lys-DOPA-Pro-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 24).

[0106] In some embodiments, the peptide is

[0107] Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro,

[0108] Lys-DOPA-Phe-Phe-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2), or

[0109] Lys-DOPA-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3).

[0110] The invention further provides a peptide selected from Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro, Lys-DOPA-Phe-Phe-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2),

[0111] Lys-DOPA-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3),

[0112] Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 4), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 5), Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 6),

[0113] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 7),

[0114] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 8),

[0115] Lys-DOPA-Phe-Phe-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 9),

[0116] Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 10),

[0117] Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO H),

[0118] Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 13),

[0119] Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro-Pro-DOPA-Lys (SEQ ID NO 14), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Pro-DOPA-Lys (SEQ ID NO 15), Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Pro-DOPA-Lys (SEQ ID NO 16),

[0120] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 17),

[0121] Lys-DOPA-Pro-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 18),

[0122] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO

[0123] 19),

[0124] Lys-DOPA-Pro-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO

[0125] 20),

[0126] Lys-DOPA-Pro-Asp-Thr- Ala- Asp- Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 21), Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 22), Lys-DOPA-Pro-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 23), and

[0127] Lys-DOPA-Pro-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 24).

[0128] In some embodiments of peptides of the invention, the peptide is

[0129] Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 1), Lys-DOPA-Phe-Phe-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2), or

[0130] Lys-DOPA-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3).

[0131] Each of the three-letter amino acid designations are as known in the art. For example, Ala is alanine, Arg is arginine, Asn is asparagine, Asp is aspartic acid (or aspartate), Cys is cysteine, Gin is glutamine, Glu is glutamic acid (or glutamate), Gly is glycine, His is histidine, He is isoleucine, Leu is leucine, Lys is lysine, Met is methionine, Phe is phenylalanine, Pro is proline, Ser is serine, Thr is threonine, Trp is tryptophan, Tyr is tyrosine, and Vai is valine.

[0132] In some embodiments, in each of the peptides disclosed herein, the DOPA group may be replaced with a siloxane moiety or with a silylated amino acid. For example, the invention provides a peptide of the form Lys-Z-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile- Thr-Gly-Pro, being equivalent to the sequence of SEQ ID NO 1, in which Z is a silylated amino acid, as defined. This similarly applies to all peptides disclosed herein.

[0133] Anti-freeze activity was assessed by recording droplet freezing using a Grant- Asymptote instrument (Grant Technologies, EF600M 106 cold stage, UK). A droplet freezing technique for the quantification of ice nucleation was used. Peptides of the invention demonstrated spontaneous self-assembly in water into fibrous or spherical shapes on surfaces. Without wishing to be bound by theory, the presence of the amino acids DOPA, Lysine, and diphenylamine, contributed to the formation of sustainable coating. The peptides delayed the drop-freezing process. The antifreeze activity may be attributed to the threonine-based motif, which mimics the arrangement of oxygen atoms in ice, allowing the peptide to align perfectly with ice planes and inhibit ice crystal growth, thereby lowering the freezing point. The designed peptides hold great potential in various fields such as cryopreservation, anti-icing coatings, and the development of frost-resistant materials for industrial and biomedical applications.

[0134] In another aspect, the invention provides a formulation comprising one or more peptides according to the invention. The formulation, solid or liquid, may be in a form of a solution or provided in a medium, comprising a carrier and surface-binding peptides for assembling an anti-ice or anti-freeze film on a surface. Each of the peptides comprises between 4 and 50 amino acids of which at least one amino acid is a surface-binding amino acid, at least one amino acid promotes self-assembly of said peptides and at least one amino acid comprises an anti-ice amino acid or motif.

[0135] Formulations comprising a peptide according to the invention may be used for forming a film or a coating on a surface region of a substrate. In some cases, the peptides are carried in an aqueous medium or in organic medium, which permit facile and immediate antifreeze film formation of a surface. Films may be formed by any method known in the art. Such methods may include spraying, dipping, washing, wiping, deposition by printing, or deposition by other means.

[0136] Applications for surface-bound films of the invention include anti-icing coatings for optical sensors and cameras, anti-frost layers on refrigerated surfaces, de-icing aids for small aerospace components, and others. More specifically, films of the invention may be used in the following exemplary fields:

[0137] Aerospace and aviation: small optically sensitive components (pitot tubes, sensors, camera domes), de-icing for unmanned aerial vehicles (UAVs), and others. Solar photovoltaics and mirrors: transparent films for reducing frost and lightblocking hoar can improve morning yield in cold regions. As PV panels demand transparency and long lifetimes, thin immobilized films combined with UV-stable protective overlayers may be employed.

[0138] Optics, cameras and LIDAR: clear protective films for surveillance cameras, automotive LIDAR / vision sensors, and scientific optics where fogging / frosting degrades performance.

[0139] Transportation: automotive windshields, train pantographs and sensors, and small glass or sensor components on vehicles.

[0140] Refrigeration and cold-chain equipment: evaporator coils, freezer doors, and display glass where frosting reduces efficiency. The films can lower ice adhesion, ease defrost cycles, and reduce mechanical cleaning.

[0141] Power infrastructure and utilities: instrument housings, small junction boxes and sensor heads on powerlines or poles.

[0142] Maritime and offshore sensors: hull sensor windows, sonar domes and exposed instrumentation on cold-region ships and buoys where biofouling and ice formation coexist. Combining films with anti-fouling chemistries offers dual benefits.

[0143] Scientific instruments and cryogenic systems: chamber ports, cryostat windows, and delicate surfaces in laboratory equipment where introducing bulk antifreeze is not possible and optical clarity is required.

[0144] Films of the invention may be formed using a variety of application tools and methods. For example, sprayable formulations may be used for field application. Dip-coating or spin-coating may be used for manufactured parts. Covalent immobilization during part fabrication may be used for durable anchoring. Also, composite approaches may be used, wherein a film of the invention is implemented beneath a thin, durable polymer overcoat that transmits activity but protects from abrasion, or films may be integrated with porous layers or soft elastomeric interlayers to reduce adhesion and replenish activity.

[0145] Notwithstanding the method application, the invention also contemplates a method for forming an anti-ice or antifreeze film on a target surface, the method comprising applying to the target surface a formulation comprising the peptides, as disclosed herein and allowing the peptide formulations to associates to the surface and self-assemble to form a continuous, adherent antifreeze film, wherein the formed film inhibits ice nucleation or reduces ice adhesion on the target surface.

[0146] In some embodiments, the method is for rendering the surface frost resistant.

[0147] The invention further provides a method of reducing ice formation or ice adhesion on an exterior surface exposed to freezing conditions, the method comprising applying a peptide formulation according to the invention to the exterior surface, whereby application of the peptide formulation forms an antifreeze film on the surface and reduces ice formation or adhesion during exposure to sub-freezing temperatures.

[0148] The invention further provides a kit for forming an antifreeze film on a surface, the kit comprising a solution of peptides according to the invention and instructions for forming an anti-ice or anti-freeze film or coating on a surface.

[0149] In some embodiments, the kit comprising a first container comprising a carrier- free of a lyophilized or concentrated form of the peptide formulation of the invention, a second container comprising a carrier suitable for reconstitution of the peptide formulation; and instructions for reconstitution and application of the peptide formulation to a target surface to form the antifreeze film.

[0150] The invention further provides:

[0151] A film bound to a surface and having a surface-binding face and an exposed face, said exposed face being an anti-ice or anti-freeze surface, wherein binding of the film to the surface comprises chemical association between a plurality of surface-binding moieties and surface localities, and wherein the film comprises a plurality of substantially perpendicularly aligned peptides having between 4 and 50 amino acids.

[0152] In some configurations of all films of the invention, each of the peptides comprising at least one surface-binding amino acid, at least one amino acid promoting self-assembly and an anti-ice or antifreeze amino acid.

[0153] In some configurations of all films of the invention, the at least one surfacebinding moiety is an amino acid having at least one atom or group of atoms capable of association to the surface.

[0154] In some configurations of all films of the invention, the at least one surfacebinding moiety is a silylated amino acid.

[0155] In some configurations of all films of the invention, the at least one atom or group of atoms capable of association to the surface is an oxygen atom or a group comprising same. In some configurations of all films of the invention, the group comprising the oxygen atom is a hydroxyl group.

[0156] In some configurations of all films of the invention, the hydroxyl group is a hydroxyaryl having two or three or four or five hydroxyl groups.

[0157] In some configurations of all films of the invention, the hydroxyaryl is a catecholbased group.

[0158] In some configurations of all films of the invention, the at least one surfacebinding moiety or amino acid is 3,4-dihydroxy-L-phenylalanin (DOPA), or a DOPA containing short peptide.

[0159] In some configurations of all films of the invention, the peptide comprises one or more DOPA amino acids.

[0160] In some configurations of all films of the invention, the peptide comprising a further an amino acid having a surface-binding atom or group, wherein the atom or group is or comprises S, N and O.

[0161] In some configurations of all films of the invention, the amino acid is selected from lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, and serine.

[0162] In some configurations of all films of the invention, the peptide comprising DOPA and at least one amino acid selected from lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, and serine.

[0163] In some configurations of all films of the invention, the peptide comprising the sequence DOPA-Lys- or Lys-DOPA-, wherein one or both of DOPA and Lys directly associate or interact with the surface.

[0164] In some configurations of all films of the invention, the at least one amino acid promoting self-assembly is same as the at least one surface-binding amino acid.

[0165] In some configurations of all films of the invention, the at least one amino acid promoting self-assembly is selected amongst amino acids comprising a functionality capable of interacting inter- or intramolecularly with functionalities present in its vicinity.

[0166] In some configurations of all films of the invention, the interaction is one or more of H-bond formations, TI~ TI stacking, and hydrophobic interactions.

[0167] In some configurations of all films of the invention, the at least one amino acid promoting self-assembly is selected from hydrophobic amino acids, amino acids having aromatic groups, or amino acids capable of generating intramolecular hydrogen bonding. In some configurations of all films of the invention, the at least one amino acid promoting self-assembly is selected from tyrosine, phenylalanine and tryptophan.

[0168] In some configurations of all films of the invention, the amino acid promoting self-assembly is phenylalanine, or a phenylalanine derivative.

[0169] In some configurations of all films of the invention, the peptide comprises at least one surface-binding amino acid selected from DOPA, Lys-DOPA- and DOPA-Lys, at least one amino acid promoting self-assembly selected from phenylalanine and phenylalanine derivatives and an anti-ice amino acid motif.

[0170] In some configurations of all films of the invention, the peptide is of a formula selected from:

[0171] DOPA-C

[0172] Lys-DOPA-Phe-C

[0173] Lys-DOPA-Phe-Phe-C

[0174] Lys-DOPA-Phe-C-Phe

[0175] Lys-DOPA-C-Phe-C

[0176] DOPA-Phe-C

[0177] DOPA-Phe-Phe-C wherein C is an anti-ice or antifreeze amino acid.

[0178] In some configurations of all films of the invention, the anti-ice or antifreeze amino acid is an amino acid sequence of a form Thr-x, x-Thr or Thr-x-Thr, wherein Thr is a threonine (T) and x is at least one amino acid.

[0179] In some configurations of all films of the invention, the peptide is of a form selected from:

[0180] DOPA-x-Thr-x

[0181] Lys-DOPA-Phe-x-Thr-x

[0182] Lys-DOPA-Phe-Phe-x-Thr-x

[0183] Lys-DOPA-Phe-x-Thr-x-Phe

[0184] Lys-DOPA-x-Thr-x-Phe-x-Thr-x

[0185] DOPA-Phe-x-Thr-x

[0186] DOPA-Phe-Phe-x-Thr-x

[0187] Lys-DOPA-Phe-Thr-x

[0188] Lys-DOPA-Phe-Phe-Thr-x

[0189] Lys-DOPA-Phe-Thr-x-Phe Lys-DOPA-C-Phe-Thr-x

[0190] Lys-DOPA-Phe-Thr-x

[0191] DOPA-Phe-Thr-x

[0192] DOPA-Phe-Phe-Thr-x

[0193] Lys-DOPA-Phe-Thr-x-Thr

[0194] Lys-DOPA-Phe-Phe-Thr-x-Thr

[0195] Lys-DOPA-Phe-Thr-x-Thr-Phe

[0196] Lys-DOPA-C-Phe-Thr-x-Thr

[0197] Lys-DOPA-Phe-Thr-x-Thr

[0198] DOPA-Phe-Thr-x-Thr

[0199] DOPA-Phe-Phe-Thr-x-Thr.

[0200] In some configurations of all films of the invention, the amino acid sequence x comprises between 5 and 20 amino acids, each selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine valine, pyrrolysine and selenocysteine; and amino acid analogs.

[0201] In some configurations of all films of the invention, the anti-ice or antifreeze amino acid motif is an amino acid sequence comprising a threonine-based motif, or is an amino acid sequence including:

[0202] -Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-Pro

[0203] Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-Pro-

[0204] Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro-

[0205] -Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro-

[0206] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-

[0207] - Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala- Al a-Glu-Leu

[0208] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-

[0209] - Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala-Glu-Leu

[0210] -Asp-Thr- Ala- Asp-Ala-Lys-Ala-Ala-Glu-Leu

[0211] Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-

[0212] - Asp-Thr- Al a- S er- Al a-Ly s- Al a- Ala-Glu-Leu, or

[0213] -Asp-Thr- Al a- Asp- Al a-Ly s- Al a- Ala- Al a-Leu .

[0214] In some configurations of all films of the invention, the peptide is one or more of Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr-Gly-Pro (SEQ ID NO 1), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2),

[0215] Lys-DOP A-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3),

[0216] Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 4), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 5), Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 6), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 7),

[0217] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 8), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 9), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 10), Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 11), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 12), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 13), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro-Pro-DOPA-Lys (SEQ ID NO 14), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Pro-DOPA-Lys (SEQ ID NO 15),

[0218] Lys-DOP A-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Pro-DOPA-Lys (SEQ ID NO 16),

[0219] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 17), Lys-DOP A-Pro-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala- Ala- Ala-Glu-Leu (SEQ ID NO 18), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 19), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 20), Lys-DOP A-Pro-Asp-Thr- Ala- Asp-Ala-Lys-Ala- Ala-Glu-Leu (SEQ ID NO 21), Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 22), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 23), and Lys-DOP A-Pro-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 24).

[0220] In some configurations of all films of the invention, the peptide is Lys-DOP A- Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 1), or Lys-DOP A-Phe- Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2), or Lys- DOP A- Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr -Gly-Pro (SEQ ID NO 3). In some configurations of all films of the invention, the surface is selected from a metal, a metal oxide, an activated glass, silanized surface, a polymeric surface, plastics, and a carbonaceous material.

[0221] A peptide composition is also provided, the composition comprising a plurality of peptides, each peptide comprises between 4 and 50 amino acids, of which at least one amino acid is a surface-binding group selected to bind to a surface, at least one amino acid is or comprises an anti-ice or an antifreeze motif that is selected to disrupt ice nucleation or that is selected to bind to ice crystal faces, and at least one amino acid is a self-assembly group that is selected to promote formation of a film on the surface, wherein the plurality of peptides being capable of forming, upon application to the surface from a formulation, an adherent anti-ice or antifreeze film reducing ice nucleation and / or ice adhesion on the surface.

[0222] In some configurations of all compositions of the invention, the composition is for forming a film on the surface, said film preventing ice formations on the surface; preventing ice adhesion to the surface; reducing a solidification temperature of water or a water-based medium coming into contact with the film; blocking water molecules from arranging into ice-like lattices at the interface with the surface; disrupting growth of ice crystals; and / or preventing or reducing interfacial contact of the surface with water.

[0223] In some configurations of all compositions of the invention, the composition is in a form of a solution comprising a carrier.

[0224] A method is provided for forming an anti-ice or antifreeze film on a surface, the method comprising applying to the surface a composition according to any embodiment of the invention and allowing peptides in said composition to associates to the surface and self-assemble to form a continuous, adherent antifreeze film, wherein the formed film inhibits ice nucleation or reduces ice adhesion on the target surface.

[0225] A method is provided of reducing ice formation or ice adhesion on an exterior surface exposed to freezing conditions, the method comprising applying a composition according to any embodiment of the invention to the exterior surface, whereby application of the composition forms an antifreeze film on the surface and reduces ice formation or adhesion during exposure to sub-freezing temperatures.

[0226] A peptide is provided that comprises at least one surface-binding amino acid, at least one amino acid promoting self-assembly and at least one an anti-ice or antifreeze amino acid, wherein the peptide comprises between 4 and 50 amino acids. In some configurations of all peptides of the invention, the peptide is for forming an anti-ice or antifreeze film on a surface.

[0227] In some configurations of all peptides of the invention, the peptide comprised DOPA and at least one amino acid selected from lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, and serine.

[0228] In some configurations of all peptides of the invention, the peptide comprising the sequence DOPA-Lys- or Lys-DOPA-, wherein one or both of DOPA and Lys directly associate or interact with the surface.

[0229] In some configurations of all peptides of the invention, the at least one amino acid promoting self-assembly is selected amongst amino acids comprising a functionality capable of interacting inter- or intramolecularly with functionalities present in its vicinity.

[0230] In some configurations of all peptides of the invention, the peptide comprises at least one surface-binding amino acid selected from DOPA, Lys-DOPA- and DOPA-Lys, at least one amino acid promoting self-assembly selected from phenylalanine and phenylalanine derivatives and an anti-ice amino acid motif.

[0231] In some configurations of all peptides of the invention, the peptide is of a formula selected from:

[0232] DOPA-C

[0233] Lys-DOPA-Phe-C

[0234] Lys-DOPA-Phe-Phe-C

[0235] Lys-DOPA-Phe-C-Phe

[0236] Lys-DOPA-C-Phe-C

[0237] DOPA-Phe-C

[0238] DOPA-Phe-Phe-C wherein C is an anti-ice or antifreeze amino acid.

[0239] In some configurations of all peptides of the invention, the anti-ice or antifreeze amino acid is a sequence of a form Thr-x, x-Thr or Thr-x-Thr, wherein Thr is a threonine (T) and x is at least one amino acid.

[0240] In some configurations of all peptides of the invention, the peptide being of a form selected from:

[0241] DOPA-x-Thr-x

[0242] Lys-DOPA-Phe-x-Thr-x

[0243] Lys-DOPA-Phe-Phe-x-Thr-x Lys-DOPA-Phe-x-Thr-x-Phe

[0244] Lys-DOPA-x-Thr-x-Phe-x-Thr-x

[0245] DOPA-Phe-x-Thr-x

[0246] DOPA-Phe-Phe-x-Thr-x

[0247] Lys-DOPA-Phe-Thr-x

[0248] Lys-DOPA-Phe-Phe-Thr-x

[0249] Lys-DOPA-Phe-Thr-x-Phe

[0250] Lys-DOPA-C-Phe-Thr-x

[0251] Lys-DOPA-Phe-Thr-x

[0252] DOPA-Phe-Thr-x

[0253] DOPA-Phe-Phe-Thr-x

[0254] Lys-DOPA-Phe-Thr-x-Thr

[0255] Lys-DOPA-Phe-Phe-Thr-x-Thr

[0256] Lys-DOPA-Phe-Thr-x-Thr-Phe

[0257] Lys-DOPA-C-Phe-Thr-x-Thr

[0258] Lys-DOPA-Phe-Thr-x-Thr

[0259] DOPA-Phe-Thr-x-Thr

[0260] DOPA-Phe-Phe-Thr-x-Thr.

[0261] In some configurations of all peptides of the invention, the anti-ice or antifreeze amino acid is an amino acid sequence comprising a threonine-based motif, or is an amino acid sequence including:

[0262] -Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-Pro

[0263] Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-Pro-

[0264] Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro-

[0265] -Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro-

[0266] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-

[0267] - Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala- Al a-Glu-Leu

[0268] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-

[0269] - Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala-Glu-Leu

[0270] -Asp-Thr- Ala- Asp-Ala-Lys-Ala-Ala-Glu-Leu

[0271] Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-

[0272] - Asp-Thr- Al a- S er- Al a-Ly s- Al a- Ala-Glu-Leu, or

[0273] -Asp-Thr- Al a- Asp- Al a-Ly s- Al a- Ala- Al a-Leu . In some configurations of all peptides of the invention, the peptide is one or more of

[0274] Lys-DOP A-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 1), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2),

[0275] Lys-DOP A-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3), Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr -Gly-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 4), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 5), Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 6), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 7),

[0276] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 8), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 9), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 10), Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 11), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 12), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 13), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro-Pro-DOPA-Lys (SEQ ID NO 14), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Pro-DOPA-Lys (SEQ ID NO 15),

[0277] Lys-DOP A-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Pro-DOPA-Lys (SEQ ID NO 16),

[0278] Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 17), Lys-DOP A-Pro-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala- Ala- Ala-Glu-Leu (SEQ ID NO 18), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 19), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 20), Lys-DOP A-Pro-Asp-Thr- Ala- Asp-Ala-Lys-Ala- Ala-Glu-Leu (SEQ ID NO 21), Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 22), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 23), and Lys-DOP A-Pro-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 24).

[0279] In some configurations of all peptides of the invention, the peptide is Lys-DOP A- Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 1), or Lys-DOPA-Phe- Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2), or Lys- DOPA- Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3).

[0280] BRIEF DESCRIPTION OF THE DRAWINGS

[0281] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0282] Figs. 1A-C. Analytical HPLC chromatograms of AFpepl at wavelengths of 220 nm (A) and 280 nm (B), and mass spectrometry analysis (C).

[0283] Figs. 2A-C. Analytical HPLC chromatograms of AFpep2 at wavelengths of 220 nm (A) and 280 nm (B), and mass spectrometry analysis (C).

[0284] Figs. 3A-C. Analytical HPLC chromatograms of AFpepCl at wavelengths of 220 nm (A) and 280 nm (B), and mass spectrometry analysis (C).

[0285] Figs. 4A-C. Analytical HPLC chromatograms of AFpepC2 at wavelengths of 220 nm (A) and 280 nm (B), and mass spectrometry analysis (C).

[0286] Figs. 5A-C. Analytical HPLC chromatograms of AFpepC3 at wavelengths of 220 nm (A) and 280 nm (B), and mass spectrometry analysis (C).

[0287] Figs. 6A-C. Analytical HPLC chromatograms of AFpepC4 at wavelengths of 220 nm (A) and 280 nm (B), and mass spectrometry analysis (C).

[0288] Figs. 7A-C. Analytical HPLC chromatograms of AFpepC5 at wavelengths of 220 nm (A) and 280 nm (B), and mass spectrometry analysis (C).

[0289] Figs. 8A-D. Morphological characterization of the peptide assemblies. (A and B) AFM and cryo-TEM images of AFPepl at a concentration of 5 mM; (C and D) AFM and cryo-TEM images of AFPep2 at a concentration of 5 mM.

[0290] Figs. 9A-F. AFM images of AFPepl at (A) 1 mM, (B) 0.2 mM, and (C) 0.1 mM concentrations, and AFPep2 at (D) 1 mM, (E) 0.2 mM, and (F) 0.1 mM concentrations.

[0291] Figs. 10A-F. Secondary structure characterization of the peptides. FT-IR spectra of the (A) AFPepl and (D) AFPep2. CD spectra of the (B) AFPepl and (E) AFPep2 at different temperatures. AlphaFol d2 calculated structures of AFPepl overlayed on the crystal structure of Rhagium inquisitor antifreeze protein (PDBid 4dt5 (C)) and the calculated structure of AFPep2 overlayed on the winter flounder antifreeze protein isoform hplc6 (PDBid Iwfa (F)) Figs. 11A-L. FT-IR and CD analysis of (A and B) AFPepCl, (C and D) AFPepC2, (E and F) AFPepC3, (G and H) AFPepC4, (I and J) AFPepC5, and (K and L) AFPepC6.

[0292] Figs. 12A-B. NMR TOCSY spectra of (A) AFPepl and (B) AFPep2, showing HN-Ha region and the unique picked peaks for3JHN-Hacoupling determination. Two HN-HP values were used for AFPep2 (not shown).

[0293] Figs. 13A-C. QCM analysis of the studied peptides (A) AFPepl, (B) AFPep2, and (C) Density of the AFPepl, AFPep2, and control peptides (AFPepCl- AFP epC6).

[0294] Figs. 14A-F. QCM-D measurements of (A) AFPC1, (B) AFPC2, (C) AFPC3, (D) AFPC4, (E) AFPC5, and (F) AFPC6.

[0295] Figs. 15A-C. Characterization of the peptides coating. (A) Contact angle of the silicon surface and coated silicone surface with AFPepl, AFPep2. (B) Coating thickness of the coated silicone surface with AFPepl, AFPep2 at different concentrations (0.1 mM, 0.2 mM, 1 mM, and 5 mM). The standard deviation (SD) was calculated based on data from three independent surfaces, each conducted in three locations. (C) Images of glass surfaces coated with AFPepl at 5 mM (on the left) and AFPep2 at 5 mM (on the right).

[0296] Fig. 16 Water contact angle values for the peptides AFPepCl -AFPep C6.

[0297] Fig. 17. Transmittance of glass surfaces coated with either AFPepl or AFPep2 at different concentrations (0.1 mM, 0.2 mM, 1 mM, and 5 mM). The standard deviation (SD) was calculated based on data from three independent surfaces conducted in three different areas.

[0298] Fig. 18A-C. Ice recrystallization inhibition analysis. (A) Microscopic images of ice crystals recrystallization at -8 °C in 45% sucrose solutions, including pure water, AFPepl, and AFPep2. (B) Ice recrystallization rate constant at zero ice fraction for AFPepl and AFPep2 as a function of concentration. (C) Ice recrystallization rate constant at zero ice fraction for sucrose 45 wt%, AFP type III at 2pm, and control peptides (AFPepCl -AFPepC6) at 2mM. The SD was calculated from data obtained from a minimum of three independent experiments.

[0299] Figs. 19A-C. Representative results for a cubic mean radius of ice crystals and ice volume fraction during IRI experiment for (A) water, (B) AFPepl, and (C) AFPep2.

[0300] Figs. 20A-H. HAADF-STEM images of (A) AFPepl, (B) AFPep2, (C) AFPepCl, (D) AFPepC2, (E) AFPepC3, (F) AFPepC4, (G) AFPepC5, and (H) AFPepC6.

[0301] Figs. 21A-C Thermal hysteresis measurement of AFPepl. A demonstration of the ice growth during slow cooling in 10 mM AFPepl. While cooling slowly from - 0.01 °C to -0.06 °C no growth is observed over 25 min, while sudden growth is observed over a few seconds, indicating the burst temperature. The difference between the burst temperature and the highest temperature without melting is the measure of the TH

[0302] Figs. 22A-B. (A and B) Fraction of frozen droplets at decreasing temperature for AFPepl and AFPep2, respectively.

[0303] Figs. 23A-D. Anti-freeze activity of peptide-coated surfaces. (A and B) Temperatures correspond to selected fractions of frozen droplets (10%, 30%, and 50%) for AFPepl and AFPep2, respectively. The standard error was calculated using data from at least three independent experiments. An ANOVA test followed by a Tukey-Kramer post hoc analysis was conducted to identify statistically significant differences between the Si2 surface and peptide-coated surfaces. Results were considered statistically significant at p <0.05 and are indicated with an asterisk (*). (C and D) Derivative of the fraction of frozen droplets for the control surfaces, AFPepl, and AFPep2, respectively.

[0304] DETAILED DESCRIPTION OF EMBODIMENTS

[0305] MATERIALS AND METHODS

[0306] Materials

[0307] Fmoc-DOPA(acetonide)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc- Gln(Trt)-Oh, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, and NH2-DOPA-Phe-Phe-OMe were purchased from GL Biochem (Shanghai, China). Fmoc-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Phe-OH, Fmoc-L-ASP(OtBu)-OH, rink amide AM resin, and ethyl cyanohydroxyiminoacetate (oxyma) were obtained from Matrix Innovation (Quebec, Canada). N-Fmoc-L-Ala was purchased from Thermo Fisher Scientific. N, N' -Diisopropylcarbodiimide (DIC), sodium dodecyl sulfate (SDS), sucrose, deuterium oxide (D2O), and alpha-cyano-4-hydroxycinnamic acid (a-Cyano) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Triisopropylsilane (TIPS) was purchased from TCI (Kita-Ku, Tokyo, Japan). Dimethylformamide (DMF), dichloromethane (DCM), diethyl ether, trifluoroacetic acid (TFA), piperidine, acetonitrile, and ultra-pure water LC / MS grade were purchased from Bio-Lab ltd (Jerusalem, Israel). Triple distilled water (TDW) was obtained by filtering distilled water through a Milli-Q water system (Millipore). Immersion Oil Type B was purchased from Cargille Laboratories (Cedar Grove, USA). AFP Type III, derived from the ocean pout (Macrozoarces americanus) fish, was produced through expression in E. coli. Substrates

[0308] Silicon wafers with a diameter of 10 cm were diced into 0.8 x 0.8 cm2pieces before use (7100 2 in. Pro-Vectus, ADT).

[0309] Synthesis of the peptides

[0310] The peptides were synthesized by using a Liberty blue microwave-assisted peptide synthesizer (CEM) utilizing standard Fmoc-SPPS chemistry. Rink amide resin was used and swelled in DMF for 30 minutes at ambient temperature before the synthesis. Fmoc deprotection was done with 5 ml of 20% piperidine solution in DMF for 1 minute at 90 °C. A double coupling of amino acids (0.2 mM) with DIC (1.0M) and Oxyma (1.0M) reagents for 8 minutes at 90 °C. Washes between deprotection and coupling steps involved DMF with a 5-second drain time, a total of 13 ml. Following the completion of the synthesis, the peptide resin underwent washing with DMF (three repetitions) and DCM (three repetitions), followed by drying under vacuum for 3 minutes. The peptide resin was suspended in a cleavage solution comprising a mixture of TFA / TDW / TIPS (95:2.5:2.5) totaling 20 ml and mixed for 3 h at ambient temperature. Then, the TFA was evaporated by bubbling nitrogen, and the crude peptide product was precipitated with diethyl ether. After centrifugation (10 minutes, 4000 rpm), diethyl ether was decanted, and the peptide was dissolved in TDW and subjected to lyophilization.

[0311] High-Performance Liquid Chromatography (HPLC)

[0312] The peptide purity was evaluated through analytical reversed-phase (RP) high- performance liquid chromatography (HPLC) using a Waters Alliance system with UV detection at 220 and 280 nm. The peptide was injected into the XSelect C18 column (3.5 pm, 130 A, 4.6 mm x 150 mm). The peptides were purified through preparative RP-HPLC by using an Ultimate 3000 HPLC system (Thermo-Fisher Scientific), equipped with a C18 LC column (10 pm, 110 A, 250 x 21.2 mm) and with UV detection at 220 and 280 nm. In both systems, peptide elution occurred through a linear gradient from 5% to 70% acetonitrile (with 0.1% TFA) in water (with 0.1% TFA). The flow rate was set at 1 ml / min and 30 °C for analytical RP-HPLC and 20 ml / min at room temperature for preparative RP-HPLC (Figs. 1-7). Mass Spectrometry (MS)

[0313] The fractions obtained during the RP-HPLC were analyzed using a mass spectrometer (Bruker MALDI-TOF MS Autoflex, Germany) in positive ion mode. The mass range analyzed was from 400 m / z to 2000 m / z. For the MALDI matrix, a 25 mM solution of a-Cyano was prepared in a solution consisting of 50% water / acetonitrile and 0.1% TFA. The peptide samples were mixed with the a-Cyano solution at a 1 : 1 volume ratio, placed on the MALDI plate, and then dried for analysis (Figs. 1-7).

[0314] Preparation of the Peptide Coating

[0315] Before peptide-coating, silicon surfaces (0.8 cm x 0.8 cm) underwent a series of cleaning treatments. Initially, they were exposed to a UVO chamber (Jelight Company, USA) for 10 min, followed by soaking in 2% SDS in TDW for 30 minutes. Subsequently, they were washed with TDW, dried using N2 gas, and subjected to a 30-second treatment with Oxygen / Plasma (Atto, Diener Electronic). The treated silicon surfaces (Sil) were then immersed in 200 pL of peptide solution (at concentrations of 5 mM, 1 mM, 0.2 mM, and 0.1 mM in LC / MS water) in 48 well plates and sealed overnight at room temperature. Following the peptide-coating process, the excess of the unattached peptide on the silicon surfaces was removed by pipetting 2 mL of LC / MS water, followed by drying under N2 gas. The coated surfaces were kept in a desiccator until the measurements were conducted.

[0316] Atomic force microscope (AFM)

[0317] The surface topography and roughness of the silicon surfaces were analyzed using Atomic Force Microscopy (AFM). The measurements were conducted on a Dimension Icon-XR SPM system (Bruker, USA) operating in tapping mode with an RTESP probe (F = 300 kHz, k = 42 N / m).

[0318] Cryo-Transmission Electron Microscopy (Cryo-TEM)

[0319] The peptide morphology was analyzed using cryo-TEM. Initially, a 300-mesh copper grid coated with a holey carbon film (Lacey substrate, Ted Pella, Ltd.) underwent pre-treatment via glow discharge (30 seconds in the plasma). Subsequently, a drop of peptide solution at a concentration of 5 mM in TDW was applied to the treated TEM grid. The specimens were rapidly vitrified by plunging into liquid ethane precooled with liquid nitrogen, maintaining controlled temperature and 100% relative humidity, utilizing a Vitrobot Mark IV. These vitrified samples were then transferred to a cryo specimen holder (Gatan model 626; Gatan Inc.) and imaged at -179°C using a Tecnai 12 G2Twin TEM (FEI), operated at an acceleration voltage of 120 kV in low-dose mode. Images were recorded using a 4Kx4K FEI Eagle CCD camera.

[0320] Circular dichroism (CD)

[0321] CD spectra were obtained using a J-1100 spectropolarimeter (JASCO, Tokyo, Japan) equipped with a 0.1 cm pathlength quartz cuvette for far-UV CD spectroscopy. Measurements were conducted at temperatures 20 °C, 10 °C, 0 °C, and -10 °C within the spectral range of 190 to 260 nm, with a step width of 0.1 nm. The samples were dissolved in TDW to achieve a final peptide concentration of 0.3 mM and were subsequently filtered using a 0.22 pm filter. For each sample, five spectra were gathered, averaged, and background- subtracted utilizing TDW as the baseline.

[0322] Fourier- transform infrared spectroscopy (FT-IR)

[0323] A 20 pL droplet of peptide solution, with a concentration of 5 mM in D2O, was deposited onto CaF2 plates and subsequently subjected to vacuum drying. The FT-IR spectra were acquired using a Nicolet 6700 FT-IR spectrometer equipped with a deuterated triglycine sulfate (DTGS) detector (Thermo Fisher Scientific, MA, USA). Spectra were collected over a range of 400 to 4000 cm-1with a resolution of 4 cm-1. To ensure precision in data, the spectra were averaged after 2000 scans.

[0324] Nuclear magnetic resonance (NMR)

[0325] NMR experiments were performed on a Bruker AVII 500 MHz spectrometer operating at the proton frequency of 500.13 MHz, using a 5-mm selective probe equipped with a self-shielded xyz-gradient coil at 13 °C. The transmitter frequency was set on the water signal. Two-dimensional spectra (TOCSY) were used to achieve the resolution of the HN-Ha peaks. Spectra were processed with TopSpin (Bruker Analytische Messtechnik GmbH) and NMRFAM SPARKY software was used to identify peak maxima. GraphPad online was used to perform an unpaired t-test to determine statistical significance. Peptide structure prediction

[0326] AlphaFold-Multimer-v2.0 was used to calculate the structures of the peptides. The relaxed highest-ranking structure is shown together with the predicted alignment error (PAE) plots.

[0327] Quartz Crystal Microbalance with Dissipation (QCM-D)

[0328] The adhesion of peptides to the silicon surface was examined using QCM-D (Q- sense, Biolin Scientific). Measurements were conducted in a flow module El system featuring SiCb sensors with a fundamental resonant frequency of 5 MHz (Qsense). Before each experiment, the silicon sensors were cleaned following the supplier's instructions. The experiments were carried out under flow-through conditions utilizing a digital peristaltic pump (IsmaTec Peristaltic Pump, IDEX). Initially, TDW was circulated into the sensor crystal chamber at a rate of 0.1 mL / min until a stable frequency and dissipation were achieved. Subsequently, the peptide solution, with a concentration of 0.6 mM dissolved in TDW, was injected into the sensor crystal chamber at the same rate for approximately 1 hour. Following peptide injection, the sensor was rinsed with TDW to remove non-adherent peptide. The adsorbed mass (Am) was then calculated using the Sauerbrey equation, utilizing the 7th overtones c&f

[0329] (1) Am = — where Af is frequency change during adsorption, C is a sensitivity constant characteristic of quartz crystal and equal to 17.7 ng cm’2Hz , and n is the overtone number.

[0330] Contact Angle Measurements

[0331] The water contact angle was measured using a Theta Lite Optical Tensiometer (Attension Theta, Finland). The volume of each drop was 1.0 pL. The measurements were averaged at three different locations on at least three independent surfaces.

[0332] Transmittance Measurements

[0333] The transmittance of peptide-coated glass surfaces at various concentrations (5 mM, 1 mM, 0.2 mM, and 0.1 mM) was measured using a spherical haze meter (Diffusion Systems Ltd, England). The uncoated glass was used as the reference. For each concentration, three surfaces were prepared, and their transmittance was measured and averaged.

[0334] Ellipsometer

[0335] The thickness of peptide-coated silicon surfaces at various concentrations (5 mM, 1 mM, 0.2 mM, and 0.1 mM) was evaluated by using an ellipsometer (J. A. Woollam, Lincoln, Nebraska, USA). The analysis was conducted at wavelengths ranging from 380 to 900 nm and at a 70° angle of incidence. The Cauchy dispersion model was employed to fit the thickness of the layers and refractive indices. Initially, the coefficients of the Cauchy equation were fixed for organic layers (An=1.45, Bn=0.01, and Cn=0), with allowance for an angle offset. Subsequently, these parameters were adjusted to achieve more precise values.

[0336] Ice recrystallization inhibition (IRI)

[0337] The sample was dissolved in LC / MS water and mixed with a 60 wt% sucrose solution to create a solution containing 45% sucrose. A 2 pL droplet of this solution was sandwiched between two glass coverslips and sealed with immersion oil to prevent evaporation. Then, it was positioned on a silver block within the Linkem cell (Linkam MDBCS 196 temperature-controlled cold stage, Linkam Scientific Instruments Ltd, UK). Subsequently, the temperature was rapidly decreased from room temperature to -40 °C at a rate of 10 °C / min, followed by a gradual warming to -8 °C at a rate of 5 °C / min. The sample was maintained at -8 °C for 40 minutes, during which images were captured every minute using a microscope (Olympus BX41) equipped with a Qlmaging EXi Aqua bioImaging microscopy camera. Each sample underwent at least three repetitions.

[0338] The average crystal area in each image (A) was analyzed by using Image J software. The average radius of the crystal (r) was calculated according to the following equation:

[0339] The ice volume fraction (Q) was calculated according to the following equation: (3) Q =Vicevice+vliq where vice is the total ice volume of the crystals and viiq is the total liquid volume.

[0340] The observed rate constant of recrystallization, ki(Q) was determined at a stable ice volume fraction (after 25-30 min) by calculating the slope using Lifshitz, Slyozov, and Wagner (LSW) equation: where ro is the initial mean crystal radius at time t=0 and r(t) is the ice crystal radius at time t.

[0341] By using the 4 and 5 equations, the rate constant of recrystallization scaled to Q=0 (kio ) was calculated. kdo is the apparent rate constant kd(Q) at Q=0, p is a scaling factor that considers a variable dependence on Q at different temperatures. For annealing at -8 °C, p=l .318 and kdo=O.65. a =1 is the ratio of the mean crystal radius r and the critical radius.

[0342] (6) kIO=kd°k,(Q)V 7 10kd(Q)

[0343] For the AFPepl and AFPep2, where the recrystallization rate was analyzed at different concentrations of these peptides, the sigmoid fit was used by the following equation:

[0344] Where kio(O) is the rate constant of 45% sucrose solution, scaled to Q = 0, Ci is the concentration in the inflection point of the curve, C is the peptide concentration, and s is the slope of the curve in the inflection region.

[0345] High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM)

[0346] The copper grids (200 mesh carbon coated with Formvar, Ted Pella, Inc.) were treated with O2 plasma for 30 seconds. Following this treatment, a 1.5 pL sample of peptide solution at a concentration of 0.2 mM in TDW was deposited onto the grids and allowed to dry under ambient conditions. Subsequently, the grids were examined using an analytical high-resolution SEM Apreo 2S (Thermo Fisher Scientific) equipped with a high-angle annular dark-field scanning transmission electron microscopy (HAADF- STEM) detector. The analysis was conducted at an operating voltage of 25 kV, a current of 0.2 nA, and a working distance of 10 mm.

[0347] Thermal hysteresis

[0348] Thermal hysteresis was evaluated using a custom-built, computer-controlled nanoliter osmometer. The apparatus featured a Peltier-based cooling block that housed a metal disc containing 500-micron diameter holes filled with immersion oil (type B). The cooling block incorporated Peltier coolers, and a thermistor was attached to the metal plate to enable precise temperature control, which was regulated by a driver managed via LabVIEW software. The entire assembly was mounted on a microscope, and the experimental procedure was recorded using a video CMOS camera (DMK 23UV024, The Imaging Source).

[0349] A solution containing peptides was injected into the immersion oil through a stretched glass capillary, forming droplets approximately 200 microns in diameter. The sample was cooled to a temperature below -25°C, triggering nucleation. The sample was then warmed until only a single ice crystal, measuring approximately 50 microns, remained. The melting temperature of this ice crystal was recorded.

[0350] The ice crystal was kept a few millikelvins below its melting point for a period of 10 minutes. After this, the temperature was decreased by 0.001°C every 20 seconds, corresponding to a cooling rate of 0.003°C per minute, until sudden crystal growth occurred. The thermal hysteresis gap (TH) was determined as the difference between the growth and melting temperatures. This measurement was repeated at least three times for each sample to ensure reliability.

[0351] Antifreeze activity

[0352] Antifreeze activity was assessed by recording droplet freezing using a Grant- Asymptote instrument (Grant Technologies, EF600M 106 cold stage, UK). A droplet freezing technique for the quantification of ice nucleation was used. Before testing, the silicon wafers were coated with either AFPepl or AFPep2, as described in the methods section. Two types of clean silicon wafers were used as controls. The first type, Sil, was prepared following the method described in the peptide coating preparation section. The second type, Si2, involved silicon wafers that were sonicated in ethanol for 15 minutes, washed with water, and then immersed in LC / MS water overnight.

[0353] In each run, forty silicon wafers were positioned atop a metal plate. Then, IpL droplets of LC / MS water were pipetted onto each wafer, and the plate was covered with a plastic lid. The system was isolated with a plastic box sealed with plastic foam, and recordings were captured through a hole at the top using a Microsoft webcam. The droplets, initially added at 15 °C, underwent a cooling gradient of 2 °C / min until reaching 0 °C, followed by a cooling gradient of 1 °C / min until -40 °C. The temperature changes of the cold plate were monitored using the accompanying Grant Asymptote User Software Suite (Version 3.0). The process was filmed throughout and during analysis, the number of frozen droplets was recorded. The results of each coating were then fitted with a negative sigmoid curve based on the following equation:

[0354] Where n(T) is the number of frozen droplets at a specific temperature, N is the total number of droplets (40 in each run), k is the droplet freezing rate factor, T is the temperature and Tso% is the temperature in which 50% of droplets were frozen.

[0355] The rate of freezing as a function of temperature was determined by calculating the derivative of equation 8 and utilizing the values of Tso%and k:

[0356] RESULTS AND DISCUSSION

[0357] Design and Synthesis of the Peptides

[0358] We designed two peptides to achieve antifreeze activity, both in solution and as a surface coating (

[0359] Table 1). The peptides include four elements: (I) the amino acid DOPA that enables the attachment to the surface, (II) the amino acid Lysine that further promotes the attachment of the peptide to the surface and improves the solubility of AFPepl, (III) diphenylalanine to allow the self-assembly of the peptides into a coating, and (IV) an antiice motif derived from the protein of either Rhagium inquisitor (

[0360] Table 1) or Pseudopleuronectes americanus (

[0361] Table 1). Additionally, control peptides (AFPepCl- AFPepC6) were also investigated to evaluate how these four elements affect both coating adhesion and antifreeze activity (

[0362] Table 1)

[0363] Table 1. The sequences of the designed peptides (all peptides contain an amide at the N- terminal and carboxylic acid at the C-terminal)

[0364] Peptide Coating Preparation

[0365] Peptide AFPepl or AFPep2 were dissolved in LC / MS water at varying concentrations (5 mM, 1 mM, 0.2 mM, and 0.1 mM) to optimize the coating and anti- freeze activity. A silicon surface was then immersed in the peptide solution overnight. Following this, the peptide-coated surfaces were thoroughly rinsed with LC / MS water ( )•

[0366] Anti-freeze peptide

[0367] Scheme 1

[0368] Initially, the peptide, denoted by red color, was added into the tube, followed by the addition of water. Subsequently, the peptide solution was vortexed and sonicated to enhance its solubility (step 1). The silicon surface, depicted by its gray surface, was then submerged in the peptide solution overnight (step 2). Following this immersion period, the surface was rinsed with LC / MS water and dried using nitrogen gas (step 3).

[0369] Peptide Characterization

[0370] AFM analysis revealed that at a concentration of 5 mM, AFPepl formed thin fibrils ranging in size from 10 to 900 nm (Fig. 8A). The length of fibrils decreased with the peptide concentration (Figs. 9A-C).

[0371] At a concentration of 5 mM, AFPPep2 formed fibrous structures (Fig. 8C). However, at 1 mM and 0.1 mM, the structures had a spherical shape (Figs. 9D, 9F), while at 0.2 mM they resembled the fibers observed at 5 mM. Cryo-TEM was utilized to analyze the assembled structures of AFPepl and AFPep2 in solution. Both peptides were dissolved at a concentration of 5 mM and exhibited the same morphology as observed by AFM on the surface (Figs. 8B and 8D). It is noteworthy that these structures appeared to be significantly longer in the solution phase.

[0372] CD and FT-IR spectroscopy were employed to assess the secondary structure of the peptides (Fig. 10). The FT-IR spectra of AFPepl exhibited a peak at 1632 cm'1(Fig. 10A) associated with a P-sheet structure typically found within the region 1620-1640 cm' f The CD spectra of AFP epl confirmed its secondary structure by a positive peak at 199 nm and a negative peak at 208 nm (Fig. 10C). The peaks at 222 nm and 228 nm are related to the aromatic side chains of Phenylalanine, DOPA, and Threonine. For the peptides AFPepCl, AFPepC2, and AFPepC3, all having the same antifreeze motif as the peptide AFP1, both FT-IR and CD analyses displayed peaks within the same range in the spectra, corresponding to a P-sheet structure as observed for AFPepl. The secondary structure of these peptides was identified as a P-sheet structure (Figs.llA-F). The lack of prominent peaks above 220 nm in the CD spectrum of AFPepCl can be ascribed to the absence of aromatic residues in its sequence, particularly Phenylalanine. The FT-IR spectra of AFPep2 had a peak at 1658 cm'1and a shoulder peak at 1688 cm'1associated with an a-helix structure (Fig. 10B). CD spectra analysis verified the secondary structure of the AFPep2 peptide, revealing two negative peaks at 227nm and 206 nm (Fig. 10C). The control peptide, AFPepC4, where Threonine residues were substituted with Alanine had a disordered structure according to FT-IR analysis, with a peak observed at 1651 cm'1(Fig. 11G). This structural characteristic was further validated by CD analysis that exhibited a negative peak at 207 nm (Fig. 11H). AFPC5, having the same sequence as AFPepC4 but without the amino acid Lysine, exhibited a peak at 1624 cm'1which can be ascribed to a P-sheet structure (Fig. 111). The peptide AFPepC6 displayed a P-turn structure, as indicated by a peak at 1666 cm'1in the FT-IR spectra (Fig. 11K). Additionally, the CD spectra showed a negative peak at 201 nm and a positive peak at 216 nm, further confirming the presence of a P-turn structure (Fig. 111). Notably, in the CD spectra, a decrease in temperature from 20 °C to -10 °C increased the intensity of peaks corresponding to an increase in secondary structure. This effect was observed at 200 nm and 208 nm for AFPepl and 206 nm and 227 nm for AFPep2.

[0373] For a higher structural characterization, 2D-NMR spectra (TOCSY) were acquired for the two peptides at 13 °C in water. Peak maxima were picked on the spectra for 11 resolved doublets in each of the HN-Ha regions, with two peaks from the HN-Hp region for the AFPep2 (Fig. 12). The average -Ha coupling of AFPepl was 8.05 Hz SD 0.46, and the corresponding value for AFPep2 was 7.51 Hz SD 0.56. The difference between these values is statistically significant according to an unpaired t-test with a two- tailed P-value of 0.0310, indicating that AFPepl is in a more extended structure than AFPep2. AlphaFold2 was used to calculate the structures of the two peptides. The calculated structure of AFPepl (residues 5-14) overlayed residues 108-117 of the crystal structure of Rhagium inquisitor antifreeze protein (PDBid 4dt5) with a backbone RMSD of 1.82 A (Fig. 10C). The backbone of the calculated structure of AFPep2 (residues 5- 16) overlayed residues 1-12 of the winter flounder antifreeze protein isoform hplc624(PDBid Iwfa) with an RMSD of 0.37 A (Fig. 10F).

[0374] To explore the real-time adhesion of the peptides to the surface, QCM-D analysis was performed. The peptide solution was circulated within a flow cell containing a silicone-coated QCM sensor. In an adhesion process, the frequency of the sensor decreases due to the growing mass of the adsorbed layer, while the dissipation increases due to the development of a film. The observed change in dissipation was approximately O. l x lO'6for all peptides, indicating the formation of a rigid film (Figs. 13A-B, and Fig. 14). This enables the use of the Sauerbrey equation, establishing a correlation between the change in frequency and the moles of the peptide adsorbed onto the sensor.

[0375] AFPepl and AFPep2 demonstrated the most substantial change in frequency, with a density of 0.28 nmol / cm2and 0.070 nmol / cm2, respectively, while the change in frequency for the other peptides (AFPepCl-AFPepC6) ranged from 0.0039 to 0.050 nmol / cm2(Fig. 13C). This suggests strong adhesion of both AFPepl and AFPep2 to the sensor. The improved binding to the surface of these two peptides compared with the rest of the studied peptides, can be attributed to the incorporation of the amino acid Lysine next to DOPA, which is known to enhance binding to the surface. AFPepC2 and AFPepC5, having the same peptide sequence as AFPepl but lacking the Lysine , demonstrated a relatively small change in frequency with a density of 0.033 nmol / cm2and 0.0039 nmol / cm2Respectively (Figs. 14B, 14E, and Fig. 13C). Moreover, the secondary structure of the peptide plays a role in influencing its binding to the surface. AFPepC4, having the same peptide sequence as AFPepl but with threonine substitution for alanine, disrupting the anti-ice motif, shows a relatively low change in frequency, with a density of 0.011 nmol / cm2(Fig. 14D). The relatively low adsorption can be attributed to the disordered structure of AFPepC4, as confirmed by FT-IR and CD analysis. The impact of the diphenylalanine residues on the formation of the peptide coating was seen through the AFPepCl, having the same peptide sequence as AFPepl but without the diphenylalanine residues while maintaining the same secondary structure. The absence of diphenylalanine residues in AFPepCl resulted in a relatively minor change in frequency with a density of 0.022 nmol / cm2(Fig. 14B). The significance of DOPA is evident in AFPepC3, which lacks this amino acid. This peptide exhibited a relatively small change in frequency with a density of 0.0040 nmol / cm2(Fig. 14C). These findings highlight the significance of including all three elements in the peptide sequence: Lysine, DOPA, and diphenylalanine, alongside the secondary structure.

[0376] To further confirm the peptide coating on the silicon surface and to evaluate the effect of peptide concentration on the coating, contact angle measurements were conducted on uncoated and peptide-coated silicone surfaces (Fig. 15A). A surface coated with AFPepl displayed an increase in the contact angle from 9±2° for a clean silicon surface to 33±1°, 32±0°, 36±5°, and 21±1° for concentrations of a peptide concentration of 5 mM, 1 mM, 0.2 mM, and 0.1 mM respectively. The coating formed by AFPep2 showed an increase in contact angle to 26±2°, 34±1°, 20±2°and 26±2° for 5 mM, 1 mM, 0.2 mM, and 0.1 mM, respectively. While both peptide coatings influenced the contact angle of the surface, there was no clear correlation between peptide concentration and contact angle. The control peptides (AFPepCl-AFPep6) exhibited an increase in contact angle compared to the silicon surface (Fig. 16). For control peptides AFPepC3-AFPepC5, this increase exceeded that observed with AFPepl, which can be attributed to the absence of charged amino acids (e.g. Lysine) or the replacement of the polar amino acid threonine with the hydrophobic amino acid alanine.

[0377] The peptide coating thickness was evaluated by using an ellipsometer. The analyzed measurements were based on fitting to the Cauchy dispersion model. The thickness of the coatings for both peptides at various concentrations ranged from 1.9-2.5 nm. Remarkably, the coating of AFPepl at a concentration of 5 mM exhibited the greatest thickness of 3.4 ± 0.4 nm (Fig. 5B).

[0378] To verify the transparency of the AFPepl and AFPep2 coating at different concentrations, transmittance measurements were conducted. Both peptide coatings demonstrated exceptionally high transmittance levels, surpassing 98.7%, indicating a notably transparent surface (Fig. 17 and Fig. 15C).

[0379] The rate of the ice crystal growth was evaluated by freezing the mixed sample of the peptide with sucrose solution. Afterwards, the frozen sample was warmed to the annealing temperature of -8 °C for 40 min and the crystal size was monitored over time. Fig. 18A shows the ice crystal growth in the presence of pure water, AFPepl, and AFPep2 at 2.2 mM. The control sample of pure water yielded a final crystal radius of 9±6 pm after 40 min. AFPepl demonstrated considerable inhibition of ice crystal growth, leading to a final radius of 4±2 gm. Furthermore, the presence of AFPepl in solution altered the shape of the ice crystals from disk-like to hexagonal plates, indicating a strong affinity of this peptide to adsorb onto basal and primary prism planes. This high affinity is attributed to the formation of supramolecular assemblies of regularly spaced threonine amino acids, forming the “TxT” motif. The hydroxyl groups of the two T-residues within each TxT- repeat mimic the arrangement of oxygen atoms in ice, aligning perfectly with the planes of ice and thereby reducing the temperature required for ice crystal growth. Although previous reports indicated that the anti-freeze segment of AFPep2 exhibits significant (IRI) activity, our findings do not demonstrate an inhibitory effect, resulting in a final radius of 9±5 gm. One possible explanation for this discrepancy is that AFPep2 was dissolved in pure water instead of a buffer solution, which might affect the solubility and aggregation of the peptide during the analysis.

[0380] To determine the recrystallization rate of ice crystal growth, the ice volume fraction was calculated throughout the entire experiment using Equation 3. Fig. 19 shows the results of the ice volume fraction for the water, AFPepl, and AFPep2. A linear growth function was used to fit the data (Fig. 19), suggesting that the kinetics of the ice recrystallization process are closely described by a bulk diffusion process of water molecules from smaller to larger ice crystals, consistent with LSW theory. Then, the ki(Q) was assessed by analyzing the slope of the graph depicting the crystal radius over time, using equation 4. The ice volume fraction, which influences the recrystallization rate, varied among the samples. To address this variation, we calculated the normalized recrystallization rate of crystal growth concerning the ice volume fraction (kio) using equations 5 and 6. The results of this analysis for AFPepl and AFPep2 at different concentrations are presented in Fig. 18B. For AFPepl, the data can be fit to a sigmoid curve using equation 7, and the inflection point in the curve, representing the inhibitor concentration, can be extracted. The inhibitor concentration for AFPepl is 0.5 ± 0.1 mM. Furthermore, the recrystallization endpoint which is the approximate concentration when kio equals zero was estimated from this curve. For AFPepl it was found to be approximately 2.3 mM. Based on the classification conducted previously on various IRI active materials, it is noted that the IRI activity of AFPepl is considered relatively effective among common antifreeze materials. The results of the kio for the AFP type III at 2.2 pM and for the control peptides at a concentration of 2.2 mM (AFPepl- AFPep6) are shown in Fig. 18C. The control peptides (AFPepCl- AFPepC3) having the anti-freeze sequence exhibited relatively higher values of kio compared to AFPepl. This higher kio could be related to the absence of key elements: diphenylalanine, crucial for promoting self-assembly into the long fibril structures observed in AFPepl, or lysine which is known to greatly enhance solubility. This reduced solubility might result in peptide aggregation and precipitation over time. The control peptides lacking an anti-freeze sequence (AFPepC4- AFPepC6) exhibited kio values closely resembling those of the pure water sample, as expected.

[0381] The effect of the formed peptide assemblies on the rate of ice crystal growth was analyzed using HAADF-STEM, with the resulting images shown in Fig. 20. Among all the controls with the anti-freeze sequence, only AFPepC2, which exhibited kio values close to those of AFPepl, displayed long fibril structures similar to those observed in AFPepl (Fig. 20D).

[0382] The thermal hysteresis (TH) of antifreeze peptides AFPepl and AFPep2 was determined using a nanoliter osmometer at a concentration of 2.2 mM. For AFPepl, a TH of 0.040 ± 0.002 °C was observed, indicating TH antifreeze activity. At 10 mM, a TH of 0.050 ± 0.002 °C was measured (Fig. 21). In contrast, no thermal hysteresis gap was observed for AFPep2 at the same concentration, nor for AFPepCl. The lack of TH for AFPepCl highlights the importance of the phenyl groups that contribute to the selfassembly of the peptide.

[0383] In the droplet freezing assay, droplets of IpL water were pipetted onto clean silicon wafers (Sil and Si2), as well as silicon wafers coated with either AFPepl or AFPep2 at a concentration of O.lmM, 0.2mM, ImM, and 5mM. The wafers were then frozen at a controlled rate until they reached -40 °C, with the entire process being filmed continuously. The changes in the frozen droplet fraction per temperature were then observed for each coating (Figs. 22 A and B). The observed results showed different freezing behavior: Si 1 froze at the highest temperature, followed by Si2, while the peptide coatings of both types (AFPep 1 and AFPep2) froze at a lower temperature. The first factor to inspect the antifreeze activity is the temperature at which 10% (Tio%), 30% (Tso%), and 50% (T5O%) of the droplets froze (Figs. 23A and B). For AFPepl coatings, Tio%ranged from -21.0±0.1 °C to -23.5±0.3 °C, and for AFPep2, they ranged from -21.4±0.6 °C to - 23.2±0.2 °C compared to Sil with -17±1 °C and Si2 with -19.4±0.5 °C. The AFPepl coatings at a concentration of 0.2mM and 5mM and AFPep2 coatings at a concentration of 0.2mM and ImM were statistically significant compared to Si2. For AFPepl, the T3o% of the coatings at O. lmM, 0.2mM, and 5mM were around -24.8 °C, while the ImM coating exhibited a higher temperature at -23.3±0.1 °C. AFPep2 coatings showed temperatures between -23.8±0.2 °C and -24.4±0.3 °C, with the 0. ImM coating having the lowest temperature. These results were compared to the clean wafers, which showed freezing temperatures of -21±1 °C for Sil and -22.4±0.6 °C for Si2, with statistically significant differences. The T5o% values were -22.6±0.8 °C for Sil and -23.3±0.8 °C for Si2. In contrast, the AFPepl coatings had T5o% values ranging from -24.5±0.3 °C to - 26.0±0.2 °C, and the AFPep2 coatings ranged from -24.3±0.2 °C to -25.2±0.2 °C. Both peptides exhibited the lowest temperatures at the O. lmM coating concentration, with all coatings showing statistically significant differences compared to Si2.

[0384] Table 2. Surface roughness of bare silicon surfaces (Sil and Si2) and peptides AFPepl and AFPep2 at different concentrations (5 mM, 1 mM, 0.2 mM, and 0.1 mM).

[0385] While it was expected that an increase in surface roughness would lower the antifreeze activity, the results exhibited a different trend. The AFPepl coatings, which had the highest roughness values (Table 2), also showed the highest antifreeze activity. It can be concluded that with no clear trend regarding the effect of contact angle or surface roughness, the antifreeze activity is primarily due to the peptide coating itself. Each surface's droplet freezing rate was calculated to further evaluate the antifreeze activity. A fitted curve was generated for each coating, considering the Tso% value. Then, the curve's derivative as a temperature function was calculated (Fig. 23C and D). The minimum point for the derivative is set at a temperature of Tso% and has a value of -k / 4. Generally, as Tso% decreases, the minimum point becomes lower, and the value of k increases. This trend is demonstrated by comparing Sil and Si2, as well as the peptide coatings with the controls (Sil and Si2). When comparing the two peptides, it was observed that the minimal rates for AFPepl were higher compared to AFPep2 even though the Tso% values were lower. For AFPepl, the minimum rates of the coatings ranged between -0.20 °C'1and -0.25 °C'1while the minimal rate of AFPep2 ranged between -0.35 °C'1and -0.46 °C'1. This corresponds to the higher antifreeze exhibited by AFPepl and can be explained by the presence of IRI activity for AFPepl which AFPep2 is lacking. The IRI activity causes slower ice crystal growth which could explain the higher resistance of AFPepl to the lowering temperatures. Moreover, It can be seen that both peptide coatings delayed drop freezing by approximately 5 °C compared to the control surfaces. Additionally, the coating of AFPepl O.lmM has showcased the lowest minimal growth rate while having the lowest Tso%, suggesting that this coating has the best antifreeze activity among the peptide coatings.

[0386] CONCLUSIONS

[0387] In this study, we designed two peptides incorporating anti-freeze motifs derived from two antifreeze proteins: one from the beetle Rhagium inquisitor and the other from the flounder fish Pseudopleuronectes americanus. The peptides, AFPepl and AFPep2, can spontaneously self-assemble in water into fibrous or spherical shapes on surfaces and fibrous structures in solution. The presence of the amino acids DOPA, Lysine, and diphenylamine, contributed to the formation of sustainable coating. Both peptides delayed the drop-freezing process, with AFPepl demonstrating better antifreeze activity than AFPep2. The antifreeze activity of AFPepl is attributed to the TxT-repeat, which mimics the arrangement of oxygen atoms in ice, allowing the protein to align perfectly with ice planes and inhibit ice crystal growth, thereby lowering the freezing point. The designed peptides hold great potential in various fields such as cryopreservation, anti-icing coatings, and the development of frost-resistant materials for industrial and biomedical applications. In the accompanying sequence listing, the following are the sequences: Lys-DOP A-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 1), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2), Lys-DOP A-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 4), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 5), Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 6), Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala- Ala- Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 7), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 8), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 9), Lys-DOPA-Phe-Phe- Asp-Thr- Ala- Asp- Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 10), Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 11), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 12), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 13), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro-Pro-DOPA-Lys (SEQ ID NO 14), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Pro-DOPA-Lys (SEQ ID NO 15), Lys-DOP A-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Pro-DOPA-Lys (SEQ ID NO 16), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 17), Lys-DOP A-Pro- Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala- Ala- Ala-Glu-Leu (SEQ ID NO 18), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 19), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 20), Lys-DOP A-Pro- Asp-Thr- Ala- Asp-Ala-Lys-Ala- Ala-Glu-Leu (SEQ ID NO 21), Asp-Thr- Ala- Asp-Ala-Lys- Ala- Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 22), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 23), Lys-DOP A-Pro- Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 24). DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 26) Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 25) Lys-DOP A-Phe-Phe-Pro- Ala-Gin- Ala-Gin- Ala-Ile-Ala-Gly-Pro (SEQ ID NO 27), and DOPA-Phe-Phe-Pro-Ala-Gln-Ala-Gln-Ala-Ile-Ala-Gly-Pro (SEQ ID NO 28).

Claims

CLAIMS:

1. A film bound to a surface and having a surface-binding face and an exposed face, said exposed face being an anti-ice or anti-freeze surface, wherein binding of the film to the surface comprises chemical association between a plurality of surface-binding moieties and surface localities, and wherein the film comprises a plurality of substantially perpendicularly aligned peptides having between 4 and 50 amino acids.

2. The film according to claim 1, wherein each of the peptides comprising at least one surface-binding amino acid, at least one amino acid promoting self-assembly and an anti-ice or antifreeze amino acid.

3. The film according to claim 1 or 2, wherein the at least one surface-binding moiety is an amino acid having at least one atom or group of atoms capable of association to the surface.

4. The film according to claim 1 or 2, wherein the at least one surface-binding moiety is a silylated amino acid.

5. The film according to claim 3, wherein the at least one atom or group of atoms capable of association to the surface is an oxygen atom or a group comprising same.

6. The film according to claim 5, wherein the group comprising the oxygen atom is a hydroxyl group.

7. The film according to claim 6, wherein the hydroxyl group is a hydroxyaryl having two or three or four or five hydroxyl groups.

8. The film according to claim 7, wherein the hydroxyaryl is a catechol-based group.

9. The film according to any one of the preceding claims, wherein the at least one surface-binding moiety or amino acid is 3,4-dihydroxy-L-phenylalanin (DOPA), or a DOPA containing short peptide.

10. The film according to claim 1, wherein the peptide comprises one or more DOPA amino acids.

11. The film according to any one of the preceding claims, the peptide comprising a further an amino acid having a surface-binding atom or group, wherein the atom or group is or comprises S, N and O.

12. The film according to claim 11, wherein the amino acid is selected from lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, and serine.

13. The film according to any one of the preceding claims, the peptide comprising DOPA and at least one amino acid selected from lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, and serine.

14. The film according to claim 13, comprising the sequence DOPA-Lys- or Lys- DOPA-, wherein one or both of DOPA and Lys directly associate or interact with the surface.

15. The film according to any one of the preceding claims, wherein the at least one amino acid promoting self-assembly is same as the at least one surface-binding amino acid.

16. The film according to any one of the preceding claims, wherein the at least one amino acid promoting self-assembly is selected amongst amino acids comprising a functionality capable of interacting inter- or intramolecularly with functionalities present in its vicinity.

17. The film according to claim 16, wherein the interaction is one or more of H-bond formations, TI~ TI stacking, and hydrophobic interactions.

18. The film according to claim 17, wherein the at least one amino acid promoting self-assembly is selected from hydrophobic amino acids, amino acids having aromatic groups, or amino acids capable of generating intramolecular hydrogen bonding.

19. The film according to claim 18, wherein the at least one amino acid promoting self-assembly is selected from tyrosine, phenylalanine and tryptophan.

20. The film according to claim 19, wherein the amino acid promoting self-assembly is phenylalanine, or a phenylalanine derivative.

21. The film according to any one of the preceding claims, wherein the peptide comprises at least one surface-binding amino acid selected from DOPA, Lys-DOPA- and DOPA-Lys, at least one amino acid promoting self-assembly selected from phenylalanine and phenylalanine derivatives and an anti-ice amino acid motif.

22. The film according to any one of the preceding claims, wherein the peptide is of a formula selected from:DOPA-CLys-DOPA-Phe-CLys-DOPA-Phe-Phe-CLys-DOPA-Phe-C-PheLys-DOPA-C-Phe-CDOPA-Phe-CDOPA-Phe-Phe-C wherein C is an anti-ice or antifreeze amino acid.

23. The film according to any one of the preceding claims, wherein the anti-ice or antifreeze amino acid is an amino acid sequence of a form Thr-x, x-Thr or Thr-x-Thr, wherein Thr is a threonine (T) and x is at least one amino acid.

24. The film according to claim 23, wherein the peptide is of a form selected from: DOPA-x-Thr-xLys-DOPA-Phe-x-Thr-xLys-DOPA-Phe-Phe-x-Thr-xLys-DOPA-Phe-x-Thr-x-PheLys-DOPA-x-Thr-x-Phe-x-Thr-xDOPA-Phe-x-Thr-xDOPA-Phe-Phe-x-Thr-xLys-DOPA-Phe-Thr-xLys-DOPA-Phe-Phe-Thr-xLys-DOPA-Phe-Thr-x-PheLys-DOPA-C-Phe-Thr-xLys-DOPA-Phe-Thr-xDOPA-Phe-Thr-xDOPA-Phe-Phe-Thr-xLys-DOPA-Phe-Thr-x-ThrLys-DOPA-Phe-Phe-Thr-x-ThrLys-DOPA-Phe-Thr-x-Thr-PheLys-DOPA-C-Phe-Thr-x-ThrLys-DOPA-Phe-Thr-x-ThrDOPA-Phe-Thr-x-ThrDOPA-Phe-Phe-Thr-x-Thr.

25. The film according to claim 24, wherein the amino acid sequence x comprises between 5 and 20 amino acids, each selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine valine, pyrrolysine and selenocysteine; and amino acid analogs.

26. The film according to claim 25, wherein the anti-ice or antifreeze amino acid motif is an amino acid sequence comprising a threonine-based motif, or is an amino acid sequence including:-Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-ProPro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-Pro-Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro--Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-- Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala- Al a-Glu-Leu Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-- Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala-Glu-Leu -Asp-Thr- Ala- Asp-Ala-Lys-Ala-Ala-Glu-Leu Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-- Asp-Thr- Al a- S er- Al a-Ly s- Al a- Ala-Glu-Leu, or-Asp-Thr- Al a- Asp- Al a-Ly s- Al a- Ala- Al a-Leu .

27. The film according to any one of the preceding claims, wherein the peptide is one or more ofLys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr-Gly-Pro (SEQ ID NO 1), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2),Lys-DOPA-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3),Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 4), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 5), Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 6),Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 7),Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 8), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 9), Lys-DOPA-Phe-Phe- Asp-Thr- Ala- Asp- Ala-Lys-Ala- Ala-Glu-Leu (SEQ ID NO 10), Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 11), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 12), Lys-DOP A-Phe-Phe- Asp-Thr- Ala- Asp-Ala-Lys-Ala- Ala- Ala-Leu (SEQ ID NO 13),Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro-Pro-DOPA-Lys (SEQ ID NO 14), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Pro-DOPA-Lys (SEQ ID NO 15), Lys-DOP A-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Pro-DOPA-Lys (SEQ ID NO 16), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 17), Lys-DOP A-Pro-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala- Ala- Ala-Glu-Leu (SEQ ID NO 18), Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 19), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 20), Lys-DOP A-Pro-Asp-Thr- Ala- Asp-Ala-Lys-Ala- Ala-Glu-Leu (SEQ ID NO 21), Asp-Thr- Ala- Asp-Ala-Lys- Ala- Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 22), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 23), and Lys-DOP A-Pro-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 24).

28. The film according to claim 1 or 27, wherein the peptide is Lys-DOP A-Phe-Phe- Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 1), or Lys-DOP A-Phe-Phe-Asp- Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2), or Lys-DOP A- Pro- Thr-Gln-Thr-Gln-Thr-Ile-Thr -Gly-Pro (SEQ ID NO 3).

29. The film according to any one of the preceding claims, wherein the surface is selected from a metal, a metal oxide, an activated glass, silanized surface, a polymeric surface, plastics, and a carbonaceous material.

30. A peptide composition comprising a plurality of peptides, each peptide comprises between 4 and 50 amino acids, of which at least one amino acid is a surface-binding group selected to bind to a surface, at least one amino acid is or comprises an anti-ice or an antifreeze motif that is selected to disrupt ice nucleation or that is selected to bind to ice crystal faces, and at least one amino acid is a self-assembly group that is selected to promote formation of a film on the surface, wherein the plurality of peptides being capable of forming, upon application to the surface from a formulation, an adherent antiice or antifreeze film reducing ice nucleation and / or ice adhesion on the surface.

31. The composition according to claim 30, for forming a film on the surface, said film preventing ice formations on the surface; preventing ice adhesion to the surface; reducing a solidification temperature of water or a water-based medium coming into contact with the film; blocking water molecules from arranging into ice-like lattices at the interface with the surface; disrupting growth of ice crystals; and / or preventing or reducing interfacial contact of the surface with water.

32. The composition according to claim 30 or 31, in a form of a solution comprising a carrier.

33. A method for forming an anti-ice or antifreeze film on a surface, the method comprising applying to the surface a composition according to any one of claims 28 to 30 and allowing peptides in said composition to associates to the surface and self-assemble to form a continuous, adherent antifreeze film, wherein the formed film inhibits ice nucleation or reduces ice adhesion on the target surface.

34. A method of reducing ice formation or ice adhesion on an exterior surface exposed to freezing conditions, the method comprising applying a composition according to any one of claims 30 to 32 to the exterior surface, whereby application of the composition forms an antifreeze film on the surface and reduces ice formation or adhesion during exposure to sub-freezing temperatures.

35. A peptide comprising at least one surface-binding amino acid, at least one amino acid promoting self-assembly and at least one an anti-ice or antifreeze amino acid, wherein the peptide comprises between 4 and 50 amino acids.

36. The peptide according to claim 35 for forming an anti-ice or antifreeze film on a surface.

37. The peptide according to claim 35 or 36, wherein the peptide comprised DOPA and at least one amino acid selected from lysine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, and serine.

38. The peptide according to claim 37, comprising the sequence DOPA-Lys- or Lys- DOPA-, wherein one or both of DOPA and Lys directly associate or interact with the surface.

39. The peptide according to any one of claims 35 to 38, wherein the at least one amino acid promoting self-assembly is selected amongst amino acids comprising a functionality capable of interacting inter- or intramolecularly with functionalities present in its vicinity.

40. The peptide according to any one of claims 35 to 39, wherein the peptide comprises at least one surface-binding amino acid selected from DOPA, Lys-DOPA- and DOPA-Lys, at least one amino acid promoting self-assembly selected from phenylalanine and phenylalanine derivatives and an anti-ice amino acid motif.

41. The peptide according to any one of claims 35 to 40, wherein the peptide is of a formula selected from:DOPA-CLys-DOPA-Phe-CLys-DOPA-Phe-Phe-CLys-DOPA-Phe-C-PheLys-DOPA-C-Phe-CDOPA-Phe-CDOPA-Phe-Phe-C wherein C is an anti-ice or antifreeze amino acid.

42. The peptide according to any one of claims 35 to 41, wherein the anti -ice or antifreeze amino acid is a sequence of a form Thr-x, x-Thr or Thr-x-Thr, wherein Thr is a threonine (T) and x is at least one amino acid.

43. The peptide according to claim 42, being of a form selected from:DOPA-x-Thr-xLys-DOPA-Phe-x-Thr-xLys-DOPA-Phe-Phe-x-Thr-xLys-DOPA-Phe-x-Thr-x-PheLys-DOPA-x-Thr-x-Phe-x-Thr-xDOPA-Phe-x-Thr-xDOPA-Phe-Phe-x-Thr-xLys-DOPA-Phe-Thr-xLys-DOPA-Phe-Phe-Thr-xLys-DOPA-Phe-Thr-x-PheLys-DOPA-C-Phe-Thr-xLys-DOPA-Phe-Thr-xDOPA-Phe-Thr-xDOPA-Phe-Phe-Thr-xLys-DOPA-Phe-Thr-x-ThrLys-DOPA-Phe-Phe-Thr-x-ThrLys-DOPA-Phe-Thr-x-Thr-PheLys-DOPA-C-Phe-Thr-x-ThrLys-DOPA-Phe-Thr-x-ThrDOPA-Phe-Thr-x-ThrDOPA-Phe-Phe-Thr-x-Thr.

44. The peptide according to any one of claims 35 to 43, wherein the anti -ice or antifreeze amino acid is an amino acid sequence comprising a threonine-based motif, or is an amino acid sequence including:-Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-ProPro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Gly-Pro-Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro--Pro-Thr-Gln- Thr-Gln-Thr -Ile-Thr -Pro-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-- Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala- Al a-Glu-Leu Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-- Asp-Thr- Al a- S er- Asp- Al a-Ly s- Al a- Ala-Glu-Leu -Asp-Thr- Ala- Asp-Ala-Lys-Ala-Ala-Glu-Leu Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-- Asp-Thr- Al a- S er- Al a-Ly s- Al a- Ala-Glu-Leu, or-Asp-Thr- Al a- Asp- Al a-Ly s- Al a- Ala- Al a-Leu .

45. The peptide according to any one of claims 35 to 44, wherein the peptide is one or more of:Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 1), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2),Lys-DOPA-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3),Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr -Gly-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 4), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 5), Lys-DOPA-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Phe-Phe-DOPA-Lys (SEQ ID NO 6), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 7),Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 8), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 9), Lys-DOPA-Phe-Phe- Asp-Thr- Ala- Asp- Ala-Lys-Ala- Ala-Glu-Leu (SEQ ID NO 10), Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Glu-Leu-Phe-Phe-DOPA-Lys (SEQ ID NO 11), Lys-DOPA-Phe-Phe-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 12), Lys-DOP A-Phe-Phe- Asp-Thr- Ala- Asp-Ala-Lys-Ala- Ala- Ala-Leu (SEQ ID NO 13),Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Gly-Pro-Pro-DOPA-Lys (SEQ ID NO 14), Pro-Thr-Gln-Thr-Gln-Thr -Ile-Thr -Pro-Pro-DOPA-Lys (SEQ ID NO 15), Lys-DOP A-Phe-Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Pro-Pro-DOPA-Lys (SEQ ID NO 16), Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 17), Lys-DOP A-Pro-Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala- Ala- Ala-Glu-Leu (SEQ ID NO 18), Asp-Thr- Ala-Ser- Asp-Ala-Lys-Ala-Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 19), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 20), Lys-DOP A-Pro-Asp-Thr- Ala- Asp-Ala-Lys-Ala- Ala-Glu-Leu (SEQ ID NO 21), Asp-Thr- Ala- Asp-Ala-Lys- Ala- Ala-Glu-Leu-Pro-DOPA-Lys (SEQ ID NO 22), Lys-DOP A-Pro-Asp-Thr-Ala-Ser-Ala-Lys-Ala-Ala-Glu-Leu (SEQ ID NO 23), and Lys-DOP A-Pro-Asp-Thr-Ala-Asp-Ala-Lys-Ala-Ala-Ala-Leu (SEQ ID NO 24).

46. The peptide according to claim 35 or 45, wherein the peptide is Lys-DOP A-Phe- Phe-Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 1), or Lys-DOP A-Phe-Phe- Asp-Thr-Ala-Ser-Asp-Ala-Lys-Ala-Ala-Ala-Glu-Leu (SEQ ID NO 2), or Lys-DOP A- Pro-Thr-Gln-Thr-Gln-Thr-Ile-Thr-Gly-Pro (SEQ ID NO 3).

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