Polyglutamic acids as potent antifreeze molecules

Abstract

The present disclosure is concerned with peptides comprising alanine residues and glutamic acid residues in a particular ratio (e.g., from about 1 : 1 to about 4; I ). wherein the peptide has a minimum chain length, such as, for example a chain length of at least 15 or at least 20 amino acid residues. The disclosed peptides beneficially inhibit ice crystal formation, and, therefore, offer utility in a wide range of applications, including, but not limited to biomedical cryopreservation. food technology, agriculture, cosmetics, and building materials. Thus, the disclosed peptides can be formulated into a composition (e.g.. a cryoprotectant composition, an agricultural formulation, a cosmetic composition) or a food product, or, alternatively, can be attached or coated onto a surface for use in structural applications. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

Description

Atorney Docket No.2110L0486P1POLYGLUTAMIC ACIDS AS POTENT ANTIFREEZE MOLECULESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U. S. Application No. 63 / 733,687, filed on December 13, 2024, the contents of which are incorporated herein by reference in their entireties.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under grant no. R35 GM147262 awarded by the National Institutes of Health and grant no. 2300012 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND

[0003] Worldwide, animal species have adapted strategies to survive sub-freezing temperatures (DeVries, A. L.; Wohlschlag, D. E. 1969 Science 163 (3871). 1073-1075; Scholander, P. F.; et al., 1957 J. Cell. Comp. Physiol. 49 (1), 5-24; Gordon, M. S.; et al., 1962 Biol. Bull., 122 (1), 52-62; Graham, L. A.; Davies, P. L. 2005 Science 310 (5747), 461-461). A particularly interesting example is the convergent evolution of antifreeze glycoproteins (AFGPs) in polar fish (Chen, L.; et al., 1997 Proc. Natl. Acad. Sci. U. S. A. 94 (8), 3817-3822), which are the most potent ice-binding molecules discovered to-date (Budke, C.; et al., 2014 Cryst. Growth Des. 14 (9), 4285-4294; Knight, C. A.; et al., 1984 Nature, 308 (5956), 295-296). Protein binding to embry onic ice cry stals modifies their shape and growth rate, resulting in small crystals, lower plasma freezing point, and prevention of mechanical damage to cells and tissues by large crystals.

[0004] There is broad demand for antifreeze molecules for applications in food technology', agriculture, fisheries, coatings, building materials, and in the petroleum industry' (Eskandari, A.; et al., 2020 Biomolecules 10 (12), 1-18; Voets, I. K., 2017 Soft Matter 13 (28), 4808-4823). Additionally, there is a great need for non-toxic ice-recrystallization inhibitors (IRI) to improve post-thaw tissue viability and function in biomedical cryopreservation (Bojic, S.; etAttorney Docket No.21101.0486P1al.. 2021 BMC Biology p 56; Brockbank, K. G. M.; et al., 2011 Vitr. Cell. Dev. Biol. -Anim. 47 (3), 210-217). For decades, AFGPs have been explored in the cry opreservation of tissues and even whole organs (Bojic, S.; et al., 2021 BMC Biology p 56; Brockbank, K. G. M.; et al., 2011 Vitr. Cell. Dev. Biol. - Anim. 47 (3), 210-217; Robles, V.; et al., 2019 Biomolecules 9 (5), 181; Amir, G.; et al., 2004 Ann. Thorac. Surg. 77 (5), 1648-1655) and have even been utilized as texture-improving ice cream additives (Meldolesi, A. GM 2009 Nat. Biotechnol. 27 (8), 682-682). However, the expense and impracticably of harvesting these specialized proteins from polar fish has limited widespread use. Dimethylsulfoxide and glycerol are still standard cellular cryoprotectants despite well known-toxic effects (Best, B. P. 2015 Rejuvenation Res. 18 (5), 422-436; Verheijen, M.; et al., 2019 Sci. Rep. 9 (1), 4641).

[0005] A wide variety of short peptides and polymer alternatives have been explored as AFGP mimics (Voets, I. K. 2017 Soft Matter, 13 (28), 4808-4823; Eniade, A.; Ben, R. N. 2001 Biomacromolecules 2 (2), 557-561; Wilkinson, B. L.; et al., 2012 Angew. Chemie - Int. Ed., 51 (15). 3606-3610; Eniade, Aet al.. 2001 Bioconjug. Chem., 12 (5). 817-823: Tseng. P. H.; et al., 2001 Chem. - A Eur. J. 7 (3), 585-590. Urbanczyk, M.; et al., 2017 Amino Acids, 49 (2), 209-222; Deller, R. C.; et al., 2014 Nat. Commun. 5, 3244; Judge, N.; et al., 2023 Biomacromolecules. 24 (6), 2459-2468; Graham, B.; et al., 2017 Angew. Chemie 56 (50), 15941-15944; Ben, R. N.; et al., 1999 Org. Lett. 1 (11). 1759-1762; Liu, S.; Ben, R. N.2005 Org. Lett. 7 (12), 2385-2388; Tsuda, T.; Nishimura, S. I. 1996 Chem. Commun. No.24, 2779-2780; Huang, M. L.; et al., 2012 Proc. Natl. Acad. Sci. U. S. A., 109 (49), 19922-19927; Filira, F.; et al., 1990 Int. J. Biol. Macromol. 12 (1), 41-49; Tachibana, Y.; et al., 2004 Angew. Chemie - Int. Ed.. 43 (7), 856-862; Tachibana. Y.; et al., 2002 Tetrahedron 58 (51), 10213-10224). However, despite efforts to develop AFGP mimics, none have achieved comparable potency to AFGPs. Therefore, there remains a need for bioinspired synthetic polymers that effectively inhibit the growth of ice crystals.SUMMARY

[0006] In accordance with the purpose(s) of the invention, as embodied and broadly described herein, the invention, in one aspect, relates to peptides comprising alanine residues and glutamic acid residues in a particular ratio (e.g., from about 3:2 to about 4: 1). As detailed herein, the peptide has a particular chain length, such as, for example a chain length of at least 15 or at least 20 amino acid residues. The disclosed peptides beneficially inhibit ice crystal formation, and, therefore, offer utility in a wide range of applications, including,Attorney Docket No. 21101.0486P1but not limited to biomedical cryopreservation, food technology', agriculture, cosmetics, and building materials. Thus, the disclosed peptides can be formulated into a composition (e.g., a cry oprotectant composition, an agricultural formulation, a cosmetic composition) or a food product, or, alternatively, can be attached to a surface for use in structural applications.

[0007] Thus, disclosed are peptides comprising a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality' of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 1: 1 to about 4:1, and wherein the peptide has a chain length of at least 15 amino acid residues.

[0008] Also disclosed are peptides comprising a plurality of alanine residues and a plurality7of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%, wherein the ratio of the plurality7of alanine residues to the plurality7of glutamic acid residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 20 amino acid residues.

[0009] Also disclosed are cryoprotectant compositions comprising an effective amount of a disclosed peptide and one or more selected from: (a) a non-antifreeze protein; (b) a microbe; (c) a cell component; and (d) a cell.

[0010] Also disclosed are food products comprising a disclosed peptide.

[0011] Also disclosed are agriculture compositions comprising a disclosed peptide.

[0012] Also disclosed are solid or semi-solid support comprising a surface covalently attached to a residue of a disclosed peptide.

[0013] Also disclosed are cosmetic compositions comprising a disclosed peptide.

[0014] Also disclosed are methods of inhibiting ice crystal formation in a sample, the method comprising contacting the sample with an effective amount of a disclosed peptide.

[0015] Also disclosed are kits comprising a disclosed peptide and one or more selected from: (a) a biological material; (b) a food product; (c) an agricultural product; (d) a solid or semisolid support; and (e) a cosmetic.

[0016] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skillAttorney Docket No. 21101.0486P1in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.BRIEF DESCRIPTION OF THE FIGURES

[0017] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.

[0018] FIG. 1A and FIG. IB show representative design and synthesis of antifreeze polymer panel.

[0019] FIG. 2A-F shows representative polypeptide structural characterization.

[0020] FIG.3A-M shows representative data illustrating antifreeze activity assays.

[0021] FIG.4A-G shows representative data illustrating cytocompatibility. biodegradation, and application of (ALEL)nin various freeze / thaw protection settings.

[0022] FIG.5A-D show representative ATR-FTIR of reactions.

[0023] FIG.6A-D show representative NMR spectra of antifreeze polymers.

[0024] FIG. 7 shows a representative CD spectra of (ALEL)nin PBS at 500 pg / mL (n = 50, 100, 150. and 200).

[0025] FIG.8 shows representative data of variable temperature CD spectra of wfAFPI in PBS at 500 pg / mL.

[0026] FIG.9 shows representative data illustrating ice recrystallization inhibition assay for (ALEL)n.

[0027] FIG. 10 shows representative data illustrating ice recrystallization inhibition assay of frozen dairy product.

[0028] FIG. 11A and FIG. 11B show representative data illustrating ice recrystallization inhibition assay data for (ALEL)3o at 100 pg / mL in PBS.Attorney Docket No. 21101.0486P1

[0029] FIG. 12 shows representative data of ice recrystallization inhibition assay for (ALKL)50 at varying concentrations in PBS.

[0030] FIG. 13A-C show representative data of ice recry stallization inhibition assay and dynamic ice shaping assay for (ALEL)5o prepared by Method A and Method B.

[0031] FIG. 14 shows representative data illustrating ice recrystallization inhibition assay for (ADED)50.

[0032] FIG. 15 shows representative data of quantified IRI of (ALEL)nat 5 mg / mL in PBS after 40 minutes of crystal growth.

[0033] FIG. 16 shows representative data illustrating ice recrystallization inhibition assay for (ADZLED / L)5o.

[0034] FIG. 17 shows representative data illustrating ice recrystallization inhibition assay for (VLEL)50.

[0035] FIG. 18 shows representative data illustrating ice recrystallization inhibition assay for wfAFPI after heating to 95 °C for 10 minutes.

[0036] FIG. 19A and FIG. 19B show representative SEC / MALS traces in DPBS indicating unimodal distribution of (ALEL)so made by polymerization method A and polymerization method B.

[0037] FIG. 20A and FIG. 20B show representative SEC / MALS traces in DPBS for Protease K and Pepsin.

[0038] FIG. 21 shows representative data for stacked SEC / MALS traces in DPBS of ProK, control copolypeptide (ADED)so, and copolypeptide (ADED)5o treated with active enzy me for 24 hrs at 37 °C.

[0039] FIG. 22 shows representative data illustrating ice recrystallization inhibition (IRI) activity of exemplary7peptides as determined via splat assays.

[0040] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.Attorney Docket No. 21101.0486P1DETAILED DESCRIPTION

[0041] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0042] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0043] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0044] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be constmed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein may be different from the actual publication dates, which can require independent confirmation.Attorney Docket No. 21101.0486P1A. DEFINITIONS

[0045] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide,” “a glycosyl moiety,” or “a residue” includes mixtures of two or more such peptides, glycosyl moities, or residues, and the like.

[0046] As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of and “consisting essentially of.”

[0047] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0048] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity7or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0049] References in the specification and concluding claims to parts by w eight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or articleAttorney Docket No. 21101.0486P1for which a part by weight is expressed. Thus, in a composition containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the composition.

[0050] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0051] As used herein, “thermal hysteresis’' or “TH” is intended to refer to the difference between the temperature at which ice crystals grow (the freezing temperature, Tf) and the temperature at which they melt (the melting point, Tm).

[0052] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0053] As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result. For example, in the case of a cryoprotectant composition, the “effective amount” can refer to the amount of the peptide that must be present in the composition in order to minimize, prevent, or otherwise delay ice crystal formation. Thus, in various aspects, the effective amount is the amount of the peptide required to reduce crystal mean grain size (MGS) by at least about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, or more than about 75%. In various further aspects, the effective amount is the amount of the peptide required such that the majority of the ice ciy sials present in the sample are hexagonal, square, and / or amorphous crystals (as opposed to spicular crystals). In various further aspects, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more than 90% of the ice cry stals present in the sample are hexagonal, square, and / or amorphous crystals.

[0054] As used herein, “kit” means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may beAttorney Docket No. 21101.0486P1supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0055] As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form, which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.

[0056] As used herein, the term “derivative"’ refers to a peptide having a structure derived from the structure of a parent peptide (e.g.. a peptide disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed peptides, or to induce, as a precursor, the same or similar activities and utilities as the claimed peptides. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent peptide.

[0057] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects.Attorney Docket No. 21101.0486P1unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0058] In defining various terms, “A1,” “A2,” “A3,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0059] The term “aliphatic” or “aliphatic group,” as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spirofused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. Aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0060] The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alky l” group is an alkyl group containing from one to six (e.g.. from one to four) carbon atoms. The term alkyl group can also be a Cl alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, Cl -CIO alky l, and the like up to and including aCl-C24 alkyl.

[0061] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by7identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyd” or “haloalky 1” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine.Alternatively, the term “monohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e.g. fluorine, chlorine, bromine, or iodine. The term “polyhaloalkyl” specifically refers to an alkyl group that is independently substituted withAttorney Docket No.21101.0486P1two or more halides, i.e. each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon. The term “alkoxyalkyd” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term ‘‘aminoalkyl” specifically refers to an alkyl group that is substituted with one or more amino groups. The term “hydroxyalkyl” specifically refers to an alkyl group that is substituted with one or more hydroxy groups. When “alkyd” is used in one instance and a specific term such as “hydroxyalkyl” is used in another, it is not meant to imply that the term “alkyd” does not also refer to specific terms such as “hydroxyalkyl” and the like.

[0062] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyd” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyd can be referred to as, e.g., an “alkylcycloalkyd.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalky 1,” and a specific term, such as “alkydcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0063] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalky l groups include, but are not limited to, cyclopropy 1, cyclobuty 1, cyclopentyd, cyclohexy 1, norbomyl, and the like. The term “heterocycloalkyl” is a ty pe of cycloalky l group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyd group can be substituted or unsubstituted. The cycloalky l group and heterocycloalkyd group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0064] The term “polyalky dene group” as used herein is a group having two or more CH2 groups linked to one another. The polyalkydene group can be represented by the formula — (CH2)a—, where “a” is an integer of from 2 to 500.

[0065] The terms “alkoxy” and “alkoxy!” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as — OA1where A1is alkyd or cycloalkyd as defined above. “Alkoxy” also includes polymers of alkoxyAttorney Docket No. 21101.0486P1groups as just described; that is, an alkoxy can be a poly ether such as — OA1— OA2or — OA1— (OA2)a— OA3, where “a” is an integer of from 1 to 200 and A1, A2, and A3are alkyl and / or cycloalkyl groups.

[0066] The term “alkenyl’' as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond.Asymmetric structures such as (A1A2)C=C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0067] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, z.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbomenyl, and the like. The term “heterocycloalkenyf’ is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0068] The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alky l, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl. cycloalkynyl, ar l, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0069] The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon tripleAttorney Docket No. 21101.0486P1bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,"’ where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to. nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alky l, cycloalkyd, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0070] The term “aromatic group” as used herein refers to a ring structure having cyclic clouds of delocalized n electrons above and below the plane of the molecule, where the n clouds contain (4n+2) n electrons. A further discussion of aromaticity’ is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “Aromaticity,” pages 477-497, incorporated herein by reference. The term “aromatic group” is inclusive of both ary l and heteroaryl groups.

[0071] The term “ary l"’ as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The ary l group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkeny l, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, — NEE, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” In addition, the aryl group can be a single ring structure or comprise multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carboncarbon bond. For example, biaryl can be two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0072] The term “aldehyde” as used herein is represented by the formula — C(O)H.Throughout this specification “C(O)” is a short hand notation for a carbonyl group, i.e., C=O.

[0073] The terms “amine” or “amino” as used herein are represented by the formula — NA1A2, where A1and A2can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl.Attorney Docket No.21101.0486P1cycloalkenyL alkynyl, cycloalkynyL aryl, or heteroaryl group as described herein. A specific example of amino is — NH2.

[0074] The term “alkydamino” as used herein is represented by the formula — NH(-alkyl) where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, penty lamino group, isopentylamino group, (tert-penty l)amino group, hexylamino group, and the like.

[0075] The term “dialkylamino” as used herein is represented by the formula — N(-alkyl)2 where alkyl is a described herein. Representative examples include, but are not limited to, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)amino group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

[0076] The term “carboxylic acid” as used herein is represented by the formula — C(O)OH.

[0077] The term “ester” as used herein is represented by the formula — OC(O)A’ or — C(O)OA', where A1can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyL aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula — (A1O(O)C-A2-C(O)O)a— or — (A1O(O)C-A2-OC(O))a—, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyL aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound or peptide having at least two carboxylic acid groups with a compound or peptide having at least two hydroxyl groups.

[0078] The term “ether” as used herein is represented by the formula A1OA2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyL alkynyl, cycloalkynyL aryl, or heteroary 1 group described herein. The term “polyether” as used herein is represented by the formula — (A1O-A2O)a—, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyL alkynyl, cycloalkynyL ary l, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of poly ether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.Attorney Docket No.21101.0486P1

[0079] The terms “hate,” “halogen,’7or “halide” as used herein can be used interchangeably and refer to F, Cl, Br, or I.

[0080] The terms “pseudohalide,” “pseudohalogen,” or “pseudohalo” as used herein can be used interchangeably and refer to functional groups that behave substantially similar to halides. Such functional groups include, by way of example, cyano, thiocyanate, azido, trifluoromethyl, tri fluorometh oxy, perfluoroal kyl, and perfluoroalkoxy groups.

[0081] The term “heteroalkyl” as used herein refers to an alkyd group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quatemized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0082] The term “heteroary 1” as used herein refers to an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, where N-oxides, sulfur oxides, and dioxides are permissible heteroatom substitutions. The heteroaryl group can be substituted or unsubstituted. The heteroaryl group can be substituted with one or more groups including, but not limited to, alky l, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. Heteroaryl groups can be monocyclic, or alternatively fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, jV-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl. pyrazinyl, benzofuranyl. benzodioxolyl. benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further not limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, bcnzo|c / | oxazolyl, benzo[< / ]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[1.2-b] pyridazinyl, imidazo[l,2-a]pyrazinyl, benzo[c][l,2,5]thiadiazolyl, benzofc] [l,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0083] The terms “heterocycle” or “heterocyclyl” as used herein can be used interchangeably and refer to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term is inclusive of, but not limited to, “heterocycloalkyl,” “heteroaryl,” “bicyclic heterocycle” and “polycyclic heterocycle.” Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole,Attorney Docket No. 21101.0486P1pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including, 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole, including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including 1, 2,3,4-tetrazole and 1,2.4, 5 -tetrazole, pyridazine, pyrazine, triazine, including 1.2.4-triazine and 1,3,5-triazine, tetrazine, including 1.2.4.5 -tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group can also be a C2 heterocyclyl, C2-C3 heterocyclyl, C2-C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, and the like up to and including a C2-C 18 heterocyclyl. For example, a C2 heterocyclyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazet l. oxiranyl, thiiranyl, and the like. Alternatively, for example, a C5 heterocyclyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, and the like. It is understood that a heterocyclyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocyclyl ring.

[0084] The term “bicyclic heterocycle" or “bicyclic heterocyclyl’" as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl encompasses ring systems wherein an aromatic ring is fused with another aromatic ring, or wherein an aromatic ring is fused with anon-aromatic ring. Bicyclic heterocyclyl encompasses ring systems wherein a benzene ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms or wherein a pyridine ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[ 1,5 -a] pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl. 1,3-benzodioxolyl. 2,3-dihydro-l,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, lH-pyrazolo[4,3-c]pyridin-3-yl; lH-pyrrolo[3,2-b]pyridin-3-yl; and 1H-pyrazolo[3,2-b]pyridin-3-yl.

[0085] The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems. The heterocycloalkyl ring-systems include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can beAttorney Docket No. 21101.0486P1substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0086] The term “hydroxyl” or “hydroxyl” as used herein is represented by the formula — OH.

[0087] The term “ketone” as used herein is represented by the formula A1C(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0088] The term “azide” or “azido” as used herein is represented by the formula — N3.

[0089] The term “nitro” as used herein is represented by the formula — NO2.

[0090] The term “nitrile” or “cyano” as used herein is represented by the formula — CN.

[0091] The term “silyl” as used herein is represented by the formula — SiA1A2A3, where A1. A2, and A3can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0092] The term “sulfo-oxo” as used herein is represented by the formulas — S(O)A1, — S(O)2A’, — OS(O)2A’, or — OS(O)2OA’, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl. cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification “S(O)” is a short hand notation for S=O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2A1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfone” as used herein is represented by the formula A1S(O)2A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfoxide” as used herein is represented by the formula A1S(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as descnbed herein.

[0093] The term “thiol” as used herein is represented by the formula — SH.

[0094] “R1,” “R2,” “R3,” “Rn,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or,Attorney Docket No. 21101.0486P1alternatively, the first group can be pendant (z.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyd group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

[0095] As described herein, peptides of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogen of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible peptides. In is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (z.e., further substituted or unsubstituted).

[0096] The term “stable,” as used herein, refers to peptides that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain aspects, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0097] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)0-4R°: -(CH2)o-40R0; -0(CH2)o-4R°, -0-(CH2)O4C(O)ORO; -(CH2)O4CH(ORO)2; -(CH2)O4SRO; -(CH2)0-4Ph, which may be substituted with R°; -(CH2)o40(CH2)O iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)0-4O(CH2)0-1-pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)o4N(R°)2: -(CH2)o4N(R°)C(O)R°; -N(R°)C(S)R°; -(CH2)O4N(RO)C(O)NR°2; -N(RO)C(S)NR°2; -(CH2)O4N(RO)C(O)ORO; -N(R°)N(R°)C(O)R°; -N(R°)N(RO)C(0)NRO2; -N(R°)N(R°)C(O)OR°; -(CH2)O4C(O)R°; -C(S)R°; -(CH2)O-4C(O)OR°; -(CH2)O4C(O)SR°; -(CH2)O4C(O)OSIR°3; -(CH2)O4OC(O)R°; -OC(O)(CH2)0-4SR°; -SC(S)SR°; -(CH2)0 4SC(O)R°; -(CH2)0-4C(O)NRO2: -C(S)NRO2; -C(S)SR°; -(CH2)O-4OC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2)O4SSRO; -(CH2)O4S(O)2RO; -(CH2)O4S(O)2ORO; -(CH2)0-4OS(O)2RO; -S(O)2NRO2; -(CH2)O4S(O)RO; -N(RO)S(O)2NR°2; -N(RO)S(O)2R°; -N(OR°)R°; -C(NH)NRO2; -P(O)2RO;-P(O)R°2; -OP(O)R°2; -OP(O)(OR°)2; -SiR°3; — (C1-4straight or branched alkylene)O-N(R°)2;Attorney Docket No. 21101.0486P1or -(Ci-4 straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, Ci-6 aliphatic, -CH2PI1, -0(CH2)o iPh, -CH2-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or ary l mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0098] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)O2R*, -(haloR*), -(CH2)o 2OH, -(CH2)0 2OR*, -(CH2)o 2CH(OR*)2; -O(haloR’), -CN, -N3, -(CH2)O2C(O)R*, -(CH2)O2C(O)OH, -(CH2)O2C(O)OR*. -(CH2)O2SR", -(CH2)O 2SH, -(CH2)O 2NH2, -(CH2)O2NHR‘, -(CH2)O2NR*2, -NO2, -SiR%, -OSIR*3, -C(O)SR* -(Ci 4 straight or branched alky lene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by ’halo" is substituted only with one or more halogens, and is independently selected from Ci 4 aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently7selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0099] Suitable divalent substituents on a saturated carbon atom of an ‘'optionally substituted” group include the following: =0, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*. =NOR*, -O(C(R*2))2 3O-, or -S(C(R*2))2-3S-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently7selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an ‘'optionally substituted” group include: -O(CR*2)23O-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0100] Suitable substituents on the aliphatic group of R include halogen. -Re, -(haloR*), -OH, -OR’, -O(haloR’), -CN, -C(O)OH, -C(O)OR’, -NH2, -NHR’, -NR’2, or -Attorney Docket No. 21101.0486P1N02. wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci-4 aliphatic, -CH2PI1, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0101] Suitable substituents on a substitutable nitrogen of an "‘optionally substituted’7group include -Rt; -NRt2, -C(O)Rt, -C(O)ORt, -C(O)C(O)Rt, -C(O)CH2C(O)Rt, -S(O)2Rf, -S(O)2NRt2, -C(S)NR‘;2. -C(NH)NRt2, or -N(Rt)S(O)2Rt; wherein each Rtis independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of Rt, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0102] Suitable substituents on the aliphatic group of RTare independently halogen, -R*, -(haloR*), OH, OR*, O(haloR*), CN, C(O)OH, C(O)OR*, NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo'’ is substituted only with one or more halogens, and is independently C1-4aliphatic, -CH2Ph, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0103] The term “leaving group’’ refers to an atom (or a group of atoms) with electron withdrawing ability' that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include halides and sulfonate esters, including, but not limited to, triflate, mesylate, tosylate, and brosylate.

[0104] The terms “hydrolysable group” and “hy drolysable moiety” refer to a functional group capable of undergoing hydrolysis, e.g., under basic or acidic conditions. Examples of hydrolysable residues include, without limitation, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, “Protective Groups in Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience, 1999).

[0105] The phrase “residue of a peptide” as used with respect to the phrase “a surface covalently attached to a residue of a disclosed peptide,” means the portion of the peptide that remains after the peptide is covalently attached to, for example, the surface. Thus, forAttorney Docket No. 21101.0486P1example, the residue of the peptide can refer to the peptide minus an atom or group of atoms at the N-terminus of the peptide such as, for example, a proton.

[0106] The term “organic residue’’ defines a carbon-containing residue, i.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove. Organic residues can contain various heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen, nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc. Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

[0107] Peptides described herein can contain one or more double bonds and. thus, potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless stated to the contrary', the invention includes all such possible isomers, as well as mixtures of such isomers.

[0108] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g, each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Peptides described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[0109] Many organic compounds (e.g, peptides) exist in optically active forms having the ability' to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-)Attorney Docket No.21101.0486P1or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Ingold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0110] When the disclosed peptides contain one chiral center, the peptides exist in two enantiomeric forms. Unless specifically stated to the contrary', a disclosed peptide includes both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixture. The enantiomers can be resolved by methods known to those skilled in the art, such as formation of diastereoisomeric salts that may be separated, for example, by cry stallization (see, CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation by David Kozma (CRC Press, 2001)); formation of diastereoisomeric derivatives or complexes that may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzy matic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by7one of the separation procedures described above, a further step can liberate the desired enantiomeric form. Alternatively, specific enantiomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.Attorney Docket No. 21101.0486P1

[0111] Designation of a specific absolute configuration at a chiral carbon in a disclosed peptide is understood to mean that the designated enantiomeric form of the peptides can be provided in enantiomeric excess (e.e.). Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%, for example, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%. Tn one aspect, the designated enantiomer is substantially free from the other enantiomer. For example, the “R” forms of the peptides can be substantially free from the “S” forms of the peptides and are, thus, in enantiomeric excess of the “S” forms. Conversely. “S” forms of the peptides can be substantially free of “R” forms of the peptides and are, thus, in enantiomeric excess of the “R” forms.

[0112] When a disclosed peptide or compound has two or more chiral carbons, it can have more than two optical isomers and can exist in diastereoisomeric forms. For example, when there are two chiral carbons, the peptide or compound can have up to four optical isomers and two pairs of enantiomers ((S, S) / (R, R) and (R, S) / (S, R)). The pairs of enantiomers (e.g., (S, S) / (R, R)) are mirror image stereoisomers of one another. The stereoisomers that are not mirror-images (e.g.. (S, S) and (R, S)) are diastereomers. The diastereoisomeric pairs can be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above. Unless otherwise specifically excluded, a disclosed peptide or compound includes each diastereoisomer of such peptides or compounds and mixtures thereof.

[0113] The peptides according to this disclosure may form prodrugs at hydroxyl or amino functionalities using alkoxy, amino acids, etc., groups as the prodrug forming moieties. For instance, the hydroxymethyl position may form mono-, di- or triphosphates and again these phosphates can form prodrugs. Preparations of such prodrug derivatives are discussed in various literature sources (examples are: Alexander et al., J. Med. Chem. 1988, 31, 318; Aligas-Martin et al., PCT WO 2000 / 041531, p. 30). The nitrogen function converted in preparing these derivatives is one (or more) of the nitrogen atoms of a compound of the disclosure.

[0114] “Derivatives” of the peptides disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, solvates and combinations thereof. The “combinations” mentioned in this context are refer to derivativesAtorney Docket No. 21101.0486P1falling within at least two of the groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, and solvates. Examples of radio-actively labeled forms include peptides labeled with tritium, phosphorous-32, iodine-129, carbon-11, fluorine- 18, and the like.

[0115] Peptides described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed peptides can be isotopically-labeled or isotopically-substituted peptides identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into peptides of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,170,35S,18F and36Cl, respectively. Isotopically labeled peptides of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0116] The peptides described in the invention can be present as a solvate. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate. The peptides can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution. In this connection, one, two, three or any arbitrary number of solvent or water molecules can combine with the peptides according to the invention to form solvates and hydrates. Unless stated to the contrary, the invention includes all such possible solvates.

[0117] The term “co-crystal” means a physical association of two or more molecules which owe their stability through non-covalent interaction. One or more components of this molecular complex provide a stable framew ork in the crystalline lattice. In certain instances, the guest molecules are incorporated in the crystalline lattice as anhydrates or solvates, see e.g. ‘“Crystal Engineering of the Composition of Pharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a New Path to Improved Medicines?” Almarasson,et. al., The Royal Society of Chemistry, 1889-1896, 2004. Examples of co-crystals include p-toluenesulfonic acid and benzenesulfonic acid.

[0118] It is also appreciated that certain peptides described herein can be present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form.Attorney Docket No. 21101.0486P1H Hketo form enol form amide form imidic acid form

[0119] Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. As another example, pyrazoles can exist in two tautomeric forms, TV1-unsubstituted, 3-A3and A1-unsubstituted, 5-A3as shown below.A4A4N-N N-NH HUnless stated to the contrary, the invention includes all such possible tautomers.

[0120] It is known that chemical substances form solids that are present in different states of order, which are termed polymorphic forms or modifications. The different modifications of a polymorphic substance can differ greatly in their physical properties. The compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.

[0121] In some aspects, a structure of a peptide can include a group represented by a formula:which is understood to be equivalent to a formula:f?n(a)Rn(d)wherein n is typically an integer. That is, R” is understood to represent five independent substituents, R"(a), Rn(b), R”(c), R”(d), R"(e). By “independent substituents,’' it is meant that each R substituent can be independently defined. For example, if in one instance R"(a)is halogen, then R”(b)is not necessarily halogen in that instance.Attorney Docket No. 21101.0486P1

[0122] Certain materials, peptides, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed peptides and compositions are either available from commercial suppliers such as Aldrich Chemical Co.. (Milwaukee, Wis.), Acros Organics (Moms Plains, N. J.). Strem Chemicals (Newburyport, MA), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods know n to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and supplemental volumes (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0123] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order.Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0124] Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular peptide is disclosed and discussed and a number of modifications that can be made to a number of molecules including the peptides are discussed, specifically contemplated is each and every combination and permutation of the peptide and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class ofAttorney Docket No.21101.0486P1molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

[0125] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety' of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.B. PEPTIDES

[0126] In one aspect, disclosed are peptides comprising alanine residues and glutamic acid in a particular ratio (e.g, from about 3:2 to about 4:1), wherein the peptide has a minimum chain length, such as, for example a chain length of at least 15 or at least 20 amino acids. As detailed elsewhere herein, the disclosed peptides beneficially inhibit ice crystal formation, and, therefore, offer utility in a wide range of applications, including, but not limited to biomedical cry opreservation, food technology, agriculture, cosmetics, and building materials.

[0127] In one aspect, the disclosed peptides exhibit inhibition of ice crystal formation in a sample, as further described herein. As would be understood by one of ordinary skill in the art, inhibition of ice crystal formation can be measured by, for example, ice recrystallization inhibition (IRI) and reduction in crystal mean grain size (MGS). Thus, in various aspects, the disclosed peptides reduce crystal MGS by at least about 10%, about 20%, about 25%. about 30%, about 40%, about 50%, about 60%, about 65%. about 70%, about 75%, or more than about 75%. In various further aspects, the disclosed peptides alter the shape of ice crystals that are formed in the sample such that the majority of the ice crystals present in the sample are hexagonal, square, and / or amorphous crystals (as opposed to spicular crystals). In various further aspects, at least 55%, at least 60%, at least 65%, at leastAttorney Docket No. 21101.0486P170%, at least 75%, at least 80%, at least 85%, at least 90%, or more than 90% of the ice crystals present in the sample are hexagonal, square, and / or amorphous crystals.

[0128] In one aspect, the disclosed peptides are useful in delaying, preventing, or otherwise decreasing ice crystal formation in a sample, as further described herein.

[0129] It is contemplated that each disclosed derivative can be optionally further substituted. It is also contemplated that any one or more derivative can be optionally omitted from the invention. It is understood that a disclosed peptide can be provided by the disclosed methods. It is also understood that the disclosed peptides can be employed in the disclosed methods of using.1. STRUCTURE

[0130] In one aspect, disclosed are peptides comprising a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 1: 1 to about 4:1, and wherein the peptide has a chain length of at least 15 amino acid residues.

[0131] In one aspect, disclosed are peptides comprising a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 20 amino acid residues.

[0132] In various aspects, the peptide consists essentially of the plurality of alanine residues and the plurality' of glutamic acid residues.

[0133] In various aspects, the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%. In a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 75 wt%. In a still further aspect, the plurality’ of alanine residues is present in the peptide in an amount of from about 55 wt% to about 72 wt%. In yet a further aspect, the plurality’ of alanine residues is present in the peptide in an amount of from about 55 wt% to about 70Attorney Docket No. 21101.0486P1wt%. In an even further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 68 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 66 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 60 wt%. In an even further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 58 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 58 wt% to about 80 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 60 wt% to about 80 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 62 wt% to about 80 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 64 wt% to about 80 wt%. In an even further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 66 wt% to about 80 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 68 wt% to about 80 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 70 wt% to about 80 wt%. In an even further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 72 wt% to about 80 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 74 wt% to about 80 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 76 wt% to about 80 wt%. In an even further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 78 wt% to about 80 wt%.

[0134] In various aspects, the plurality' of alanine residues is present in the peptide in an amount of from about 60 wt% to about 75 wt%. In a further aspect, the plurality' of alanine residues is present in the peptide in an amount of from about 60 wt% to about 72 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 60 wt% to about 70 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 60 wt% to about 68 wt%. In an even further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 60 wt% to about 66 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 60 wt% to about 64 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in anAttorney Docket No. 21101.0486P1amount of from about 60 wt% to about 62 wt%. In an even further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 62 wt% to about 75 wt%. In a still further aspect, the plurality' of alanine residues is present in the peptide in an amount of from about 64 wt% to about 75 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 66 wt% to about 75 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 68 wt% to about 75 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 70 wt% to about 75 wt%. In an even further aspect, the plurality’ of alanine residues is present in the peptide in an amount of from about 72 wt% to about 75 wt%. In a still further aspect, the plurality7of alanine residues is present in the peptide in an amount of from about 62 wt% to about 72 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 64 wt% to about 70 wt%. In an even further aspect, the plurality7of alanine residues is present in the peptide in an amount of from about 66 wt% to about 68 wt%.

[0135] In various aspects, the plurality’ of alanine residues is present in the peptide in an amount of about 60 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of about 61 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of about 62 wt%. In an even further aspect, the plurality of alanine residues is present in the peptide in an amount of about 63 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of about 64 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of about 65 wt%. In even further aspect, the plurality of alanine residues is present in the peptide in an amount of about 66 wt%. In an even still further aspect, the plurality' of alanine residues is present in the peptide in an amount of about 67 wt%. In an even yet further aspect, the plurality of alanine residues is present in the peptide in an amount of about 68 wt%. In a further aspect, the plurality of alanine residues is present in the peptide in an amount of about 69 wt%. In a still further aspect, the plurality of alanine residues is present in the peptide in an amount of about 70 v %. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of about 71 wt%. In even further aspect, the plurality of alanine residues is present in the peptide in an amount of about 72 wt%. In an even still further aspect, the plurality7of alanine residues is present in the peptide in an amount of about 73 wt%. In an even yet further aspect, the plurality of alanineAttorney Docket No.21101.0486P1residues is present in the peptide in an amount of about 74 wt%. In a further aspect, the plurality of alanine residues is present in the peptide in an amount of about 75 wt%.

[0136] In various aspects, the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%. In a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 38 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 36 wt%. In yet a further aspect, the plurality' of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 34 wt%. In an even further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 32 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 30 wt%. In yet a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 28 wt%. In an even further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 26 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 24 wt%. In yet a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 22 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 22 wt% to about 40 wt%. In yet a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 24 wt% to about 40 wt%. In an even further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 26 wt% to about 40 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 28 wt% to about 40 wt%. In yet a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 30 wt% to about 40 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 32 wt% to about 40 wt%. In yet a further aspect, the plurality’ of glutamic acid residues is present in the peptide in an amount of from about 34 wt% to about 40 wt%. In an even further aspect, the plurality' of glutamic acid residues is present in the peptide in an amount of from about 36 wt% to about 40 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of from about 38 wt% to about 40 wt%.Attorney Docket No. 21101.0486P1

[0137] In various aspects, the plurality of glutamic acid residues is present in the peptide in an amount of about 20 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 21 wt%. In yet a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 22 wt%. In an even further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 23 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 24 wt%. In yet a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 25 wt%. In even further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 26 wt%. In an even still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 27 wt%. In an even yet further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 28 wt%. In a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 29 wt%. In a still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 30 wt%. In yet a further aspect, the plurality' of glutamic acid residues is present in the peptide in an amount of about 31 wt%. In even further aspect, the plurality' of glutamic acid residues is present in the peptide in an amount of about 32 wt%. In an even still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 33 wt%. In an even yet further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 34 wt%. In a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 35 wt%. In a still further aspect, the plurality' of glutamic acid residues is present in the peptide in an amount of about 36 wt%. In yet a further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 37 wt%. In even further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 38 wt%. In an even still further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 39 wt%. In an even yet further aspect, the plurality of glutamic acid residues is present in the peptide in an amount of about 40 wt%.

[0138] In various aspects, the ratio of the plurality’ of alanine residues to the plurality of glutamic acid residues is from about 1: 1 to about 4:1. In various aspects, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4:1. In a further aspect, the ratio of the plurality of alanine residues to the plurality’ of glutamic acid residues is from about 19:11 to about 4:1. In a still further aspect, the ratio ofAttorney Docket No. 21101.0486P1the plurality of alanine residues to the plurality of glutamic acid residues is from about 2: 1 to about 4:1. In a yet further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 21:9 to about 4:1. In an even further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 11:4 to about 4:1. In an even still further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 23:7 to about 4:1. In an even yet further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 23:7 to about 4:1. In a further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 23:7. In a still further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 11:4. In a yet further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 21:9. In an even further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 2:1. In an even still further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 19: 11. In an even yet further aspect, the ratio of the plurality' of alanine residues to the plurality of glutamic acid residues is from about 19: 11 to about 23:7. In a further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 2: 1 to about 11:4. In a still further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 21:9 to about 11:4.

[0139] In various aspects, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is about 3:2. In a further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is about 19: 11. In a still further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is about 2:1. In a yet further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is about 21:9. In an even further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is about 11:4. In an even still further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is about 23:7. In an even yet further aspect, the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is about 4:1.

[0140] In various aspects, the peptide has a chain length of at least 20 amino acid residues. In a further aspect, the peptide has a chain length of at least 30 amino acid residues. In a still further aspect, the peptide has a chain length of at least 40 amino acid residues. In aAttorney Docket No.21101.0486P1yet further aspect, the peptide has a chain length of at least 50 amino acid residues. In an even further aspect, the peptide has a chain length of at least 60 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 70 amino acid residues. In an even yet further aspect, the peptide has a chain length of at least 80 amino acid residues. In a further aspect, the peptide has a chain length of at least 90 amino acid residues. In a still further aspect, the peptide has a chain length of at least 100 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 110 amino acid residues. In an even further aspect, the peptide has a chain length of at least 120 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 130 amino acid residues. In even yet further aspect, the peptide has a chain length of at least 140 amino acid residues. In a further aspect, the peptide has a chain length of at least 150 amino acid residues. In a still further aspect, the peptide has a chain length of at least 160 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 170 amino acid residues. In an even further aspect, the peptide has a chain length of at least 180 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 190 amino acid residues. In an even yet further aspect, the peptide has a chain length of at least 200 amino acid residues. In a further aspect, the peptide has a chain length of at least 210 amino acid residues. In a still further aspect, the peptide has a chain length of at least 220 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 230 amino acid residues. In an even further aspect, the peptide has a chain length of at least 240 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 250 amino acid residues. In even yet further aspect, the peptide has a chain length of at least 260 amino acid residues. In a further aspect, the peptide has a chain length of at least 270 amino acid residues. In a still further aspect, the peptide has a chain length of at least 280 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 290 amino acid residues. In an even further aspect, the peptide has a chain length of at least 300 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 310 amino acid residues. In an even yet further aspect, the peptide has a chain length of at least 320 amino acid residues. In a further aspect, the peptide has a chain length of at least 330 amino acid residues. In a still further aspect, the peptide has a chain length of at least 340 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 350 amino acid residues. In an even further aspect, the peptide has a chain length of at least 360 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 370 amino acid residues. In evenAttorney Docket No.21101.0486P1yet further aspect, the peptide has a chain length of at least 380 amino acid residues. In a further aspect, the peptide has a chain length of at least 390 amino acid residues. In a still further aspect, the peptide has a chain length of at least 400 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 410 amino acid residues. In an even further aspect, the peptide has a chain length of at least 420 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 430 amino acid residues. In an even yet further aspect, the peptide has a chain length of at least 440 amino acid residues. In a further aspect, the peptide has a chain length of at least 450 amino acid residues. In a still further aspect, the peptide has a chain length of at least 460 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 470 amino acid residues. In an even further aspect, the peptide has a chain length of at least 480 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 490 amino acid residues. In even yet further aspect, the peptide has a chain length of at least 500 amino acid residues.

[0141] In various aspects, the peptide has a chain length of from about 15 amino acid residues to about 500 amino acid residues. In various aspects, the peptide has a chain length of from about 20 amino acid residues to about 500 amino acid residues. In a further aspect, the peptide has a chain length of from about 30 amino acid residues to about 500 amino acid residues. In a still further aspect, the peptide has a chain length of from about 50 amino acid residues to about 500 amino acid residues. In a yet further aspect, the peptide has a chain length of from about 100 amino acid residues to about 500 amino acid residues. In an even further aspect, the peptide has a chain length of from about 200 amino acid residues to about 500 amino acid residues. In an even still further aspect, the peptide has a chain length of from about 300 amino acid residues to about 500 amino acid residues. In an even yet further aspect, the peptide has a chain length of from about 20 amino acid residues to about 400 amino acid residues. In a further aspect, the peptide has a chain length of from about 20 amino acid residues to about 300 amino acid residues. In a still further aspect, the peptide has a chain length of from about 20 amino acid residues to about 200 amino acid residues. In a yet further aspect, the peptide has a chain length of from about 20 amino acid residues to about 100 amino acid residues. In an even further aspect, the peptide has a chain length of from about 20 amino acid residues to about 50 amino acid residues. In an even still further aspect, the peptide has a chain length of from about 30 amino acid residues to about 400 amino acid residues. In even yet further aspect, the peptide has a chain length of from about 50 amino acid residues to about 300 amino acid residues. In a further aspect, the peptide hasAttorney Docket No. 21101.0486P1a chain length of from about 30 residues to about 70 residues. In a still further aspect, the peptide has a chain length of from about 70 amino acid residues to about 100 amino acid residues. In a yet further aspect, the peptide has a chain length of from about 100 amino acid residues to about 150 amino acid residues. In an even further aspect, the peptide has a chain length of from about 150 amino acid residues to about 200 amino acid residues. In an even still further aspect, the peptide has a chain length of from about 200 amino acid residues to about 250 amino acid residues. In even yet further aspect, the peptide has a chain length of from about 250 amino acid residues to about 300 amino acid residues. In a further aspect, the peptide has a chain length of from about 300 amino acid residues to about 350 amino acid residues. In a still further aspect, the peptide has a chain length of from about 350 amino acid residues to about 400 amino acid residues. In a yet further aspect, the peptide has a chain length of from about 400 amino acid residues to about 450 amino acid residues. In an even further aspect, the peptide has a chain length of from about 450 amino acid residues to about 500 amino acid residues. In an even still further aspect, the peptide has a chain length of from about 150 amino acid residues to about 250 amino acid residues. In even yet further aspect, the peptide has a chain length of from about 175 amino acid residues to about 225 amino acid residues. In a further aspect, the peptide has a chain length of from about 20 amino acid residues to about 40 amino acid residues. In a still further aspect, the peptide has a chain length of from about 30 amino acid residues to about 50 amino acid residues. In a yet further aspect, the peptide has a chain length of from about 40 amino acid residues to about 60 amino acid residues. In an even further aspect, the peptide has a chain length of from about 50 amino acid residues to about 70 amino acid residues. In an even still further aspect, the peptide has a chain length of from about 60 amino acid residues to about 80 amino acid residues. In even yet further aspect, the peptide has a chain length of from about 70 amino acid residues to about 90 amino acid residues. In a further aspect, the peptide has a chain length of from about 80 amino acid residues to about 100 amino acid residues.

[0142] In various aspects, the peptide has a number average molecular weight (Mn) of at least about 1,000. In a further aspect, the peptide has a number average molecular weight (Mn) of at least about 2,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of at least about 3,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 4,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of at least about 5,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of at least aboutAttorney Docket No. 21101.0486P16,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 7,000. In a further aspect, the peptide has a number average molecular weight (Mn) of at least about 8,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of at least about 9,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 10.000. In an even further aspect, the peptide has a number average molecular weight (Mn) of at least about 11,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of at least about 12,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 13.000. In a further aspect, the peptide has a number average molecular weight (Mn) of at least about 14,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of at least about 15,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 20,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of at least about 25,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of at least about 30,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 35,000. In a further aspect, the peptide has a number average molecular weight (Mn) of at least about 40,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of at least about 45.000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 50,000.

[0143] In various aspects, the peptide has a number average molecular weight (Mn) of from about 1,000 to about 50,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 3,000 to about 45.000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 5,000 to about 40,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 10,000 to about 35,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of from about 15,000 to about 30,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of from about 20,000 to about 25,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of from about 1,000 to about 3,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 1,000 to about 5,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 1,000 to about 10,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 1,000 to about 15,000. In an even further aspect, the peptide has a numberAttorney Docket No.21101.0486P1average molecular weight (Mn) of from about 1.000 to about 20,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of from about 1,000 to about 25,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of from about 1,000 to about 30,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 1.000 to about 35,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 1,000 to about 40,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 1,000 to about 45,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of from about 1.000 to about 50,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of from about 45,000 to about 50,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of from about 40,000 to about 50,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 35,000 to about 50,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 30,000 to about 50,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 25,000 to about 50,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of from about 20,000 to about 50,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of from about 15,000 to about 50,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of from about 10,000 to about 50,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 5,000 to about 50,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 3.000 to about 50,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 3,000 to about 45,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of from about 5,000 to about 40,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of from about 10,000 to about 30,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of from about 15,000 to about 25,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 18,000 to about 23,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 19,000 to about 21,000. In various aspects, the peptide has a degree of polymerization (DP) of at least about 20. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 30. In a still further aspect, the peptide has a degree of polymerization (DP) of at leastAttorney Docket No. 21101.0486P1about 40. In a yet further aspect the peptide has a degree of polymerization (DP) of at least about 50. In an even further aspect, the peptide has a degree of polymerization (DP) of at least about 60. In an even still further aspect, the peptide has a degree of polymerization (DP) of at least about 70. In an even yet further aspect, the peptide has a degree of polymerization (DP) of at least about 80. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 90. In a still further aspect, the peptide has a degree of polymerization (DP) of at least about 100. In a yet further aspect, the peptide has a degree of polymerization (DP) of at least about 110. In an even further aspect, the peptide has a degree of polymerization (DP) of at least about 120. In an even still further aspect, the peptide has a degree of polymerization (DP) of at least about 130. In even yet further aspect, the peptide has a degree of polymerization (DP) of at least about 140. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 150. In a still further aspect, the peptide has a degree of polymerization (DP) of at least about 160. In a yet further aspect, the peptide has a degree of polymerization (DP) of at least about 170. In an even further aspect, the peptide has a degree of polymerization (DP) of at least about 180. In an even still further aspect, the peptide has a degree of polymerization (DP) of at least about 190. In an even yet further aspect, the peptide has a degree of polymerization (DP) of at least about 200. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 210. In a still further aspect, the peptide has a chain length of at least 220 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 230 amino acid residues. In an even further aspect, the peptide has a degree of polymerization (DP) of at least about 240. In an even still further aspect, the peptide has a degree of polymerization (DP) of at least about 250. In an even yet further aspect, the peptide has a degree of polymerization (DP) of at least about 260. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 270. In a still further aspect, the peptide has a chain length of at least 280 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 290 amino acid residues. In an even further aspect, the peptide has a degree of polymerization (DP) of at least about 300.

[0144] In various aspects, the peptide has a degree of polymerization (DP) from about 20 to about 500. In a further aspect, the peptide has a degree of polymerization (DP) from about 20 to about 450. In a still further aspect, the peptide has a degree of polymerization (DP) from about 20 to about 400. In a yet further aspect, the peptide has a degree of polymerization (DP) from about 20 to about 350. In an even further aspect, the peptide has a degree of polymerization (DP) from about 20 to about 300. In an even still further aspect, theAttorney Docket No. 21101.0486P1peptide has a degree of polymerization (DP) from about 20 to about 250. In an even yet further aspect, the peptide has a degree of polymerization (DP) from about 20 to about 200. In a further aspect, the peptide has a degree of polymerization (DP) from about 20 to about 150. In a still further aspect, the peptide has a degree of polymerization (DP) from about 20 to about 100. In a yet further aspect, the peptide has a degree of polymerization (DP) from about 20 to about 50. In an even further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 500. In an even still further aspect, the peptide has a degree of polymerization (DP) from about 50 to about 500. In an even yet further aspect, the peptide has a degree of polymerization (DP) from about 100 to about 500. In a further aspect, the peptide has a degree of polymerization (DP) from about 150 to about 500. In a still further aspect, the peptide has a degree of polymerization (DP) from about 200 to about 500. In a yet further aspect, the peptide has a degree of polymerization (DP) from about 250 to about 500. In an even further aspect, the peptide has a degree of polymerization (DP) from about 300 to about 500. In an even still further aspect, the peptide has a degree of polymerization (DP) from about 350 to about 500. In an even yet further aspect, the peptide has a degree of polymerization (DP) from about 400 to about 500. In a further aspect, the peptide has a degree of polymerization (DP) from about 450 to about 500. In a still further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 450. In a yet further aspect, the peptide has a degree of polymerization (DP) from about 50 to about 400. In an even further aspect, the peptide has a degree of polymerization (DP) from about 100 to about 300. In an even still further aspect, the peptide has a degree of polymerization (DP) from about 50 to about 100. In an even yet further aspect, the peptide has a degree of polymerization (DP) from about 100 to about 150. In a further aspect, the peptide has a degree of polymerization (DP) from about 150 to about 200. In a still further aspect, the peptide has a degree of polymerization (DP) from about 200 to about 250. In a yet further aspect, the peptide has a degree of polymerization (DP) from about 250 to about 300. In an even further aspect, the peptide has a degree of polymerization (DP) from about 300 to about 350. In an even still further aspect, the peptide has a degree of polymerization (DP) from about 350 to about 400. In an even yet further aspect, the peptide has a degree of polymerization (DP) from about 400 to about 450. In a further aspect, the peptide has a degree of polymerization (DP) from about 100 to about 330.

[0145] In various aspects, the peptide further comprises one or more residues of a hydrophilic amino acid selected from serine, threonine, arginine, histidine, lysine, andAttorney Docket No. 21101.0486P1aspartic acid. In a further aspect, the residue of a hydrophilic amino acid is serine. In a still further aspect, the residue of a hydrophilic amino acid is threonine. In yet a further aspect, the residue of a hydrophilic amino acid is arginine. In an even further aspect, the residue of a hydrophilic amino acid is histidine. In an even still further aspect, the residue of a hydrophilic amino acid is lysine. In yet an even further aspect, the residue of a hydrophilic amino acid is aspartic acid.

[0146] In various aspects, the peptide further comprises a plurality of threonine residues. In a further aspect, the plurality of threonine residues is present in the peptide in an amount up to about 30 wt%. Thus, in various aspects, the plurality of threonine residues is present in the peptide in an amount up to about 25 wt%, up to 20 wt%, up to 15 wt%, up to 10 wt%, or up to 5 wt%.

[0147] In various aspects, the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of from 15 amino acid residues to 500 amino acid residues.

[0148] In various aspects, the peptide has a structure represented by a formula:wherein n is an integer selected from 15 to about 500; wherein the ratio of x to y is of from about 1: 1 to about 1:4, and wherein the sum of x and y is 1, or a pharmaceutically acceptable salt thereof.

[0149] In various aspects, the peptide has a structure represented by a formula:Attorney Docket No. 21101.0486P1wherein n is an integer selected from 20 to about 400; wherein the ratio of x to y is of from about 2:3 to about 1:4, and wherein the sum of x and y is 1, or a pharmaceutically acceptable salt thereof.

[0150] In various aspects, the peptide has a structure represented by a formula:wherein z is an integer from 0 wt% to about 30 wt%, or a pharmaceutically acceptable salt thereof.

[0151] As would be appreciated by one of ordinary skill in the art, the ratio of x and y or of x and z or of y and z represents the relative proportion of the recited monomer residues in the peptide and does not specify a specific sequence of residues in the peptide. The peptide can be random, semi-random, alternating, blocky, or a combination thereof.

[0152] In various aspects, x is from about 0.10 to about 0.4. Thus, in various aspects, x is from about 0.10 to about 0.35, about 0.10 to about 0.30, about 0.10 to about 0.25, about 0.10 to about 0.20, about 0.10 to about 0.15. about 0.15 to about 0.40, about 0.20 to about 0.40, about 0.25 to about 0.40. about 0.30 to about 0.40, about 0.35 to about 0.40, about 0.15 to about 0.35, or about 0.20 to about 0.30. In various further aspects, x is from about 0.15 to about 0.3.

[0153] In various aspects, x is from about 0.20 to about 0.40. In a further aspect, x is from about 0.22 to about 0.40. In a still further aspect, x is from about 0.24 to about 0.40. In a yet further aspect, x is from about 0.26 to about 0.40. In an even further aspect, x is from about 0.28 to about 0.40. In an even still further aspect, x is from about 0.30 to about 0.40. In an even yet further aspect, x is from about 0.32 to about 0.40. In a further aspect, x is from about 0.34 to about 0.40. In a still further aspect, x is from about 0.36 to about 0.40. In a yet further aspect, x is from about 0.38 to about 0.40. In an even further aspect, x is from about 0.20 to about 0.38. In an even still further aspect, x is from about 0.20 to about 0.36. In an even yet further aspect, x is from about 0.20 to about 0.34. In a further aspect, x is from about 0.20 to about 0.32. In a still further aspect, x is from about 0.20 to about 0.30. In a yet further aspect, x is from about 0.20 to about 0.28. In an even further aspect, x is from aboutAttorney Docket No. 21101.0486P10.20 to about 0.26. In an even still further aspect x is from about 0.20 to about 0.24. In an even yet further aspect, x is from about 0.20 to about 0.22.

[0154] In various aspects, x is about 0.20. In a further aspect, x is about 0.21. In a still further aspect, x is about 0.22. In a yet further aspect, x is about 0.23. In an even further aspect, x is about 0.24. In an even still further aspect, x is about 0.25. In an even yet further aspect, x is about 0.26. In a further aspect, x is about 0.27. In a still further aspect, x is about 0.28. In a yet further aspect, x is about 0.29. In an even further aspect, x is about 0.30. In an even still further aspect, x is about 0.31. In an even yet further aspect, x is about 0.32. In a further aspect, x is about 0.33. In a still further aspect, x is about 0.34. In a yet further aspect, x is about 0.35. In an even further aspect, x is about 0.36. In an even still further aspect, x is about 0.37. In an even yet further aspect, x is about 0.38. In a further aspect, x is about 0.39. In a still further aspect, x is about 0.40.

[0155] In various aspects, y is from about 0.60 to about 0.80. In a further aspect, y is from about 0.62 to about 0.80. In a still further aspect, y is from about 0.64 to about 0.80. In a yet further aspect, y is from about 0.66 to about 0.80. In an even further aspect, y is from about 0.68 to about 0.80. In an even still further aspect, y is from about 0.70 to about 0.80. In an even yet further aspect, y is from about 0.72 to about 0.80. In a further aspect, y is from about 0.74 to about 0.80. In a still further aspect, y is from about 0.76 to about 0.80. In a yet further aspect, y is from about 0.78 to about 0.80. In an even further aspect, y is from about 0.60 to about 0.78. In an even still further aspect, y is from about 0.60 to about 0.76. In an even yet further aspect, y is from about 0.60 to about 0.74. In a further aspect, y is from about 0.60 to about 0.72. In a still further aspect, y is from about 0.60 to about 0.70. In a yet further aspect, y is from about 0.60 to about 0.68. In an even further aspect, y is from about 0.60 to about 0.66. In an even still further aspect, y is from about 0.60 to about 0.64. In an even yet further aspect, y is from about 0.60 to about 0.62.

[0156] In various aspects, y is about 0.60. In a further aspect, y is about 0.61. In a still further aspect, y is about 0.62. In a yet further aspect, y is about 0.63. In an even further aspect, y is about 0.64. In an even still further aspect, y is about 0.65. In an even yet further aspect, y is about 0.66. In a further aspect, y is about 0.67. In a still further aspect, y is about 0.68. In a yet further aspect, y is about 0.69. In an even further aspect, y is about 0.70. In an even still further aspect, y is about 0.71. In an even yet further aspect, y is about 0.72. In a further aspect, y is about 0.73. In a still further aspect, y is about 0.74. In a yet further aspect, y is about 0.75. In an even further aspect, y is about 0.76. In an even still furtherAttorney Docket No. 21101.0486P1aspect y is about 0.77. In an even yet further aspect, y is about 0.78. In a further aspect, y is about 0.79. In a still further aspect, y is about 0.80.

[0157] In various aspects, z is an integer from about 0 to about 0.3. Thus, in various aspects, z is an integer of from about 0 to about 0.25, about 0 to about 0.2, about 0 to about 0.15, about 0 to about 0.1, about 0 to about 0.05, about 0.05 to about 0.3. about 0.1 to about 0.3, about 0.15 to about 0.3, about 0.2 to about 0.3, about 0.25 to about 0.3, about 0.05 to about 0.25, or about 0.1 to about 0.2. In various further aspects, z is an integer of from about 0.05 to about 0.2.

[0158] In various aspects, the ratio of x to y is about 1: 1 to about 4:1. In various aspects, the ratio of x to y is about 3: 2 to about 4:1. In a further aspect, the ratio of x to y is about 19: 11 to about 4:1. In a still further aspect, the ratio of x to y is about 2: 1 to about 4:1. In a yet further aspect, the ratio of x to y is about 21:9 to about 4:1. In an even further aspect, the ratio of x to y is about 11:4 to about 4:1. In an even still further aspect, the ratio of x to y is about 23: 7 to about 4:1. In an even yet further aspect, the ratio of x to y is about 23: 7 to about 4:1. In a further aspect, the ratio of x to y is about 3:2 to about 23:7. In a still further aspect, the ratio of x to y is about 3:2 to about 11:4. In a yet further aspect, the ratio of x to y is about 3:2 to about 21:9. In an even further aspect, the ratio of x to y is about 3:2 to about 2:1. In an even still further aspect, the ratio of x to y is about 3:2 to about 19: 11. In an even yet further aspect, the ratio of x to y is about 19: 11 to about 23:7. In a further aspect, the ratio of x to y is about 2: 1 to about 11:4. In a still further aspect, the ratio of x to y is about 21:9 to about 11:4.

[0159] In various aspects, the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0160] In various aspects, the peptide has a structure represented by a formula:Attorney Docket No. 21101.0486P1or a pharmaceutically acceptable salt thereof.

[0161] In various aspects, the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0162] In various aspects, the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0163] In various aspects, the peptide has a structure represented by a formula:wherein R20is selected from -OR31, -NHR32, -N?, a protein tag, a sortase recognition sequence, a sugar residue, and a structure:Attorney Docket No. 21101.0486P1wherein R31and R32is selected from hydrogen, -CttyPh. C1-C8 alkyl, C2-C8 alkyne, C1-C8 azide, tetrazinyl, cyclooctynyl, norbomenyl. and -(CH2CH2O)mCH3, and wherein m is an integer selected from 1 to 100, or a pharmaceutically acceptable salt thereof.

[0164] In various aspects, the peptide has a structure represented by a formula:wherein R20is selected from -OR31. -NHR32, -N3, a protein tag, a sortase recognition sequence, a sugar residue, and a structure:wherein R31and R32are selected from hydrogen, -CltyPh, C1-C8 alkyl, C2-C8 alkyne, C1-C8 azide, tetrazinyl, cyclooctynyl, norbomenyl, and -(CltyCItyO^CHs, and wherein m is an integer selected from 1 to 100, or a pharmaceutically acceptable salt thereof.

[0165] In various aspects, the peptide has a structure represented by a formula:Attorney Docket No. 21101.0486P1or a pharmaceutically acceptable salt thereof.

[0166] In various aspects, the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0167] In various aspects, the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0168] In various aspects, the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0169] In one aspect, n is an integer selected from 15 to 500. In various further aspects, n is an integer selected from 20 to 400. In a further aspect, n is an integer selected from 20 to 350. In a still further aspect, n is an integer selected from 20 to 300. In a yet further aspect, n is an integer selected from 20 to 250. In an even further aspect, n is anAttorney Docket No.21101.0486P1integer selected from 20 to 200. In an even still further aspect, n is an integer selected from 20 to 150. In an even yet further aspect, n is an integer selected from 20 to 100. In a further aspect, n is an integer selected from 20 to 50. In a still further aspect, n is an integer selected from 20 to 30. In a yet further aspect, n is an integer selected from 30 to 400. In an even further aspect, n is an integer selected from 50 to 400. In an even still further aspect, n is an integer selected from 100 to 400. In a further aspect, n is an integer selected from 150 to 400. In a still further aspect, n is an integer selected from 200 to 400. In a yet further aspect, n is an integer selected from 250 to 400. In an even further aspect, n is an integer selected from 300 to 400. In an even still further aspect, n is an integer selected from 350 to 400. In an even yet further aspect, n is an integer selected from 50 to 350. In a further aspect, n is an integer selected from 100 to 300. In a still further aspect, n is an integer selected from 150 to 250. In a yet further aspect, n is an integer selected from 20 to 40. In an even further aspect, n is an integer selected from 30 to 50. In an even still further aspect, n is an integer selected from 40 to 60. In an even yet further aspect, n is an integer selected from 60 to 80. In a further aspect, n is an integer selected from 80 to 100. In a still further aspect, n is an integer selected from 100 to 120. In ayet further aspect, n is an integer selected from 120 to 140. In an even further aspect, n is an integer selected from 140 to 160. In an even still further aspect, n is an integer selected from 160 to 180. In an even yet further aspect, n is an integer selected from 180 to 200. In a further aspect, n is an integer selected from 200 to 220. In a still further aspect, n is an integer selected from 220 to 240. In a yet further aspect, n is an integer selected from 240 to 260. In an even further aspect, n is an integer selected from 260 to 280. In an even still further aspect, n is an integer selected from 280 to 300. In an even yet further aspect, n is an integer selected from 300 to 320. In a further aspect, n is an integer selected from 320 to 340. In a still further aspect, n is an integer selected from 340 to 360. In ayet further aspect, n is an integer selected from 360 to 380. In an even further aspect, n is an integer selected from 380 to 400.

[0170] In one aspect, m is an integer selected from 1 to 100. In a further aspect, m is an integer selected from 2 to 100. In a still further aspect, m is an integer selected from 5 to 100. In ayet further aspect, m is an integer selected from 10 to 100. In an even further aspect, m is an integer selected from 20 to 100. In an even still further aspect, m is an integer selected from 30 to 100. In an even yet further aspect, m is an integer selected from 40 to 100. In a further aspect, m is an integer selected from 50 to 100. In a still further aspect, m is an integer selected from 60 to 100. In ayet further aspect, m is an integer selected from 70 toAttorney Docket No. 21101.0486P1100. In an even further aspect, m is an integer selected from 80 to 100. In an even still further aspect, m is an integer selected from 90 to 100. In an even yet further aspect, m is an integer selected from 1 to 90. In a further aspect, m is an integer selected from 1 to 80. In a still further aspect, m is an integer selected from 1 to 70. In a yet further aspect, m is an integer selected from 1 to 60. In an even further aspect, m is an integer selected from 1 to 50. In an even still further aspect, m is an integer selected from 1 to 40. In an even yet further aspect, m is an integer selected from 1 to 30. In a further aspect, m is an integer selected from 1 to 20. In a still further aspect, m is an integer selected from 1 to 10. In a yet further aspect, m is an integer selected from 1 to 5. In an even further aspect, m is an integer selected from 5 to 20. In an even still further aspect, m is an integer selected from 20 to 30. In an even yet further aspect, m is an integer selected from 30 to 40. In a further aspect, m is an integer selected from 40 to 50. In a still further aspect, m is an integer selected from 50 to 60. In a yet further aspect, m is an integer selected from 60 to 70. In an even further aspect, m is an integer selected from 70 to 80. In an even still further aspect, m is an integer selected from 80 to 90.

[0171] In various aspects, m is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In a further aspect, m is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. In a still further aspect, m is an integer selected from 1, 2, 3. 4, 5, 6, 7. and 8. In a yet further aspect, m is an integer selected from 1, 2, 3, 4, 5, 6, and 7. In an even further aspect, m is an integer selected from 1, 2, 3, 4, 5, and 6. In an even still further aspect, m is an integer selected from 1, 2, 3, 4, and 5. In an even yet further aspect, m is an integer selected from 1, 2, 3, and 4. In a further aspect, m is an integer selected from 1, 2, and 3. In a still further aspect, m is an integer selected from 1 and 2.a. R20GROUPS

[0172] In one aspect, R20is selected from -OR31, -NHR32, -N3, a protein tag, a sortase recognition sequence, a sugar residue, and a structure:Attorney Docket No.21101.0486P1

[0173] In various aspects, R20is selected from -OR31, -NHR32, -N3, and a structure:

[0174] In a further aspect, R20is selected from -OR31and -NHR32. In a still further aspect, R20is -OR31. In a yet further aspect, R20is -NHR32.

[0175] In various aspects, R20is -N3.

[0176] In various aspects, R20is the protein tag. As used herein, the term "protein tag” refers to a peptide sequence located at the N-terminus of the peptide (e.g.. a peptide as disclosed herein) for a specific purpose. For example, a fluorescence tag can be used to give visual readout on the peptide. Exemplary fluorescence tags include, but are not limited to, green fluorescent protein (GFP) and red fluorescent protein. Alternatively, a protein tag can be used to allow for specific enzymatic modification (such as biotinylation by a biotin ligase) or chemical modification (such as coupling to other protein). In various further aspects, the protein tag is selected from a polyglutamate tag, a polyarginine tag, a calmodulin-tag, CBP, FLAG, GST, HA, HBH, MBP, Myc, poly His, S-tag, SUMO, TAP, TRX, and V5.

[0177] In various aspects, R20is the sortase recognition sequence. As used herein, the term "sortase recognition sequence” refers to a sequence located at the N-terminus of the peptide (e.g., a peptide as disclosed herein) that is recognized by a sortase (a bacterial transpeptidase) and is subsequently cleaved. A sortase recognition sequence can be used, for example, to allow for sortase-mediated ligation to generate site-specifically modified proteins utilizing sortase transpeptides. In various further aspects, the sortase recognition sequence is selected from LPXTG (SEQ ID NO: 1 ), LPXTGG (SEQ ID NO: 2), LPXTGGG (SEQ ID NO: 3), and LPXTGGGG (SEQ ID NO: 4), wherein X is a natural or unnatural amino acid. Examples of natural amino acids include, but are not limited to, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Examples of unnatural amino acids include, but are not limited to, hydroxyproline, beta-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, and pyroglutamic acid. In various further aspects, the sortaseAttorney Docket No.21101.0486P1recognition sequence is selected from LPETG (SEQ ID NO: 5), LPETGG (SEQ ID NO: 6), LPETGGG (SEQ ID NO: 7), and LPETGGGG (SEQ ID NO: 8).

[0178] In various aspects, R20is the sugar residue. As used herein, the term “sugar residue” refers to the portion of a monosaccharide or a polysaccharide that remains after the monosaccharide or the polysaccharide is covalently attached to, for example, the N-terminus of the peptide (e.g., a peptide as disclosed herein). Thus, in various aspects, the sugar residue can refer to the sugar minus an atom such as, for example, a proton. In various aspects, the sugar residue is a residue of a sugar selected from fructose, glucose, and lactose. In a further aspect, the sugar residue is a residue of a sugar selected from fructose and glucose. In a still further aspect, the sugar residue is a residue of a sugar selected from fructose and lactose. In a yet further aspect, the sugar residue is a residue of a sugar selected from glucose and lactose. In an even further aspect, the sugar residue is a fructose residue. In an even still further aspect, the sugar residue is a glucose residue. In an even yet further aspect, the sugar residue is a lactose residue.

[0179] In various aspects, R20is a structure:b. R31ND R32GROUPS

[0180] In various aspects, R31and R32are selected from hydrogen, -CH2Ph, C1-C8 alkyl, C2-C8 alkyne, C1-C8 azide, tetrazinyl, cyclooctynyl, norbomenyl, and -(CH2CH2O)mCH3. In a further aspect, R31and R32is selected from hydrogen, -CH2Ph, C1-C4 alkyd, C2-C4 alkyne. C1-C4 azide, tetrazinyl, cyclooctynyl, norbomenyl, and - (CH2CH2O)mCH3. In a still further aspect, R31and R32is selected from hydrogen, -CH2Ph, methyl, ethyl, propyl, isopropyl. -CH=CH, -CH2CH=CH, -CH2N3, -CH2CH2N3, -CEECEECEENs, tetrazinyl, cyclooctynyl, norbomenyl, and -(CEECEEC^mCEE. In a yet further aspect, R31and R32is selected from hydrogen, -CH2Ph, methyl, ethyl, -CH=CH. -CH2 3, -CH2CH2N3, tetrazinyl, cyclooctynyl, norbomenyl, and -(CH2CH2O)mCH3. In anAttorney Docket No. 21101.0486P1even further aspect, R31and R32is selected from hydrogen, -CttyPh. methyl, -CH2N3, tetrazinyl, cyclooctynyl, norbomenyl, and -(CH2CH2O)mCH3.

[0181] In various aspects, R31and R32is selected from hydrogen, CttyPh. C1-C8 alky l, and C2-C8 alkyne. In a further aspect, R'1andR'2is selected from hydrogen, -CH2PI1, C1-C4 alkyl, and C2-C4 alkyne. In a still further aspect, R31and R32is selected from hydrogen, -CFhPh, methyl, ethyl, propyl, isopropyl, -CH=CH, and -CH2CH=CH. In a yet further aspect, R31and R32is selected from hydrogen, -CH2PI1, and methyl. In an even further aspect, R31and R32is selected from hydrogen and methyl.

[0182] In various aspects, R31and R32is selected from hydrogen and C1-C8 azide. In afurther aspect, R31and R32is selected from hydrogen and C1-C4 azide. In a still further aspect, R31and R32is selected from hydrogen, -CH2N3, -CH2CH2N3, -CH2CH2CH2N3. In a yet further aspect, R31andR'2is selected from hydrogen, -CH2N3, -CH2CH2N3. In an even further aspect, R31and R32is selected from hydrogen and -CH2N3.

[0183] In various aspects, R31and R32is selected from hydrogen, tetrazinyl, cyclooctynyl, and norbomenyl. In a further aspect, R31and R32is selected from hydrogen and tetrazinyl. In a still further aspect, R31and R32is selected from hydrogen and cyclooctynyl. In a yet further aspect, R31and R32is selected from hydrogen and norbomenyl.

[0184] In various aspects, R31and R32is selected from hydrogen and -(CH2CH2O)mCH3

[0185] In various aspect, v is selected from hydrogen and C1-C8 alkyl. In a further aspect, R31and R32is selected from hydrogen and C1-C4 alkyl. In a still further aspect, R31and R32is selected from hydrogen, methyl, ethyl, propyl and isopropyl. In a yet further aspect, R31and R32is selected from hydrogen, methyl, and ethyl. In an even further aspect, R31and R32is selected from hydrogen and methyl.

[0186] In various aspects, R31and R32is hydrogen.2. EXAMPLE PEPTIDES

[0187] In one aspect, a peptide can be present as one or more of the following structures:Attorney Docket No. 21101.0486P1or a pharmaceutically acceptable salt thereof.

[0188] It is contemplated that one or more peptides can optionally be omitted from the disclosed invention.Attorney Docket No. 21101.0486P1

[0189] It is understood that the disclosed peptides can be used in connection with the disclosed methods, compositions, food products, solid or semi-solid supports, kits, and uses.

[0190] It is understood that pharmaceutically acceptable derivatives of the disclosed peptides can be used also in connection with the disclosed methods, compositions, food products, solid or semi-solid supports, kits, and uses. The pharmaceutically acceptable derivatives of the peptides can include any suitable derivative, such as pharmaceutically acceptable salts as discussed below, isomers, radiolabeled analogs, tautomers, and the like.C. METHODS OF MAKING A PEPTIDE

[0191] The peptides of the invention can be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature, exemplified in the experimental sections or clear to one skilled in the art. For clarity, examples having a single substituent are shown where multiple substituents are allowed under the definitions disclosed herein.

[0192] Reactions used to generate the peptides of the invention are prepared by¬ employing reactions as shown in the following Reaction Schemes, as described and exemplified below. In certain specific examples, the disclosed peptides can be prepared by Routes I-II, as described and exemplified below. The following examples are provided so that the invention might be more fully understood, are illustrative only, and should not be construed as limiting.1. ROUTE I

[0193] In one aspect, peptide analogs can be prepared as shown below.SCHEME 1A.O1.1Attorney Docket No. 21101.0486P1

[0194] Peptides are represented in generic form, wherein R is a carboxylic acid protecting group such as benzyd or tert-butyl and with other substituents as noted in compound and peptide descriptions elsewhere herein. A more specific non-limiting example of the synthesis shown in Scheme 1A is set forth below.SCHEME IB.R’ = -CH3or -CH2CH2CO2H1.8

[0195] In one aspect, compounds of type 1.8, and similar compounds, can be prepared according to reaction Scheme IB above. Thus, compounds of type 1.7 can be prepared by NCA copolymerization of an appropriate glutamic acid analogs, e.g., 1.5 as shown above, and an appropriate alanine-N-carboxy anhydride, e.g., 1.6 as shown above. Appropriate glutamic acid analogs and appropriate alanine-N-carboxy anhydrides are commercially available or prepared by methods known to one skilled in the art. Further, as w ould be understood by one of skill, degrees of polymerization can be readily tuned by altering theAttorney Docket No. 21101.0486P1monomer to initiator ratios, and amino acid compositions can be tuned via the ammo acid N-carboxy anhydride feed ratios, as further detailed herein. The polymerization is carried out in the presence of an appropriate catalyst, e.g, tetrakis(trimethylphosphine)cobalt, in an appropriate solvent, e.g, tetrahydrofuran, at an appropriate temperature, e.g., 0°C, in an appropriate solvent, e.g.. tetrahydrofuran (THF). Compounds of type 1.8 can be prepared by¬ deprotection of an appropriate protected glutamic acid analog, e.g., 1.7 as shown above. The deprotection is carried out in the presence of an appropriate deprotecting agent, e.g., trifluoroacetic acid, in an appropriate solvent system, e.g., methanol and water. As can be appreciated by one skilled in the art. the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 1.1, 1.2, and 1.3), can be substituted in the reaction to provide peptide analogs similar to Formula 1.4.2. ROUTE II

[0196] In one aspect, peptide analogs can be prepared as show n below.SCHEME 2A.Initiator

[0197] Peptides are represented in generic form, wherein R is a carboxylic acid protecting group such as benzy l or tert-butyl, wherein R’ is a -CH3 or a -CH2CH2CO2H group, and with other substituents as noted in compound and peptide descriptions elsewhereAttorney Docket No.21101.0486P1herein. A more specific non-limiting example of the synthesis shown in Scheme 2A is set forth below.SCHEME 2B.

[0198] In one aspect compounds of type 2.10, and similar compounds, can be prepared according to reaction Scheme 2B above. Thus, compounds of type 2.9 can be prepared by NCA copolymerization of an appropriate glutamic acid analogs, e.g., 2.6 as shown above, an appropriate alanine-N-carboxy anhydride, e.g., 2.7 as shown above, and an appropriate threonine analogs, e.g, 2.8 as shown above. Appropriate glutamic acid analogs, appropriate alanine-N-carboxy anhydrides, and appropriate threonine analogs are commercially available or prepared by methods known to one skilled in the art. Further, as would be understood by one of skill, degrees of polymerization can be readily tuned by altering the monomer to initiator ratios, and amino acid compositions can be tuned via the amino acid N-carboxy anhydride feed ratios, as further detailed herein. The polymerization is carried out in the presence of an appropriate catalyst, e.g., tetrakis(trimethylphosphine)cobalt, in an appropriate solvent, e.g., tetrahydrofuran, at an appropriate temperature, e.g., 0°C. in an appropriate solvent, e.g.. tetrahydrofuran (THF). Compounds of type 2.10 can be prepared by deprotection of an appropriate protected glutamic acid analog, e.g., 2.9 as shown above. The deprotection is carried out in the presence of an appropriate deprotecting agent, e.g., trifluoroacetic acid, in an appropriateAttorney Docket No. 21101.0486P1solvent system, e.g., methanol and water. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 2.1, 2.2, 2.3, and 2.4), can be substituted in the reaction to provide peptide analogs similar to Formula 2.5.D. COMPOSITIONS

[0199] As detailed herein, the disclosed peptides are useful in a wide range of applications, including, but not limited to biomedical cryopreservation, agriculture, and cosmetics. Thus, in various aspects, the disclosed peptides can be formulated into a composition (e.g., a cry oprotectant composition, an agricultural composition, a cosmetic composition) to facilitate use in these areas.

[0200] In various aspects, the disclosed composition reduces or inhibits ice crystal formation at a temperature of from about 0°C to about -20°C (e.g., at about 0, -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, or -20°C). In a further aspect, the disclosed composition reduces or inhibits ice crystal formation at a temperature of from about -20°C to about -40°C (e.g., at about -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, or -40°C). In a still further aspect, the disclosed composition reduces or inhibits ice cry stal formation at about -20°C. In yet a further aspect, the disclosed composition reduces or inhibits ice crystal formation at a temperature of from about -40°C to about -200°C (e.g., at about -40, -45, -50. -55, -60, -65, -70, -75, -80, -85, -90, -95, -100, -105, -1 10, -115, -120, -125, -130, -135, -140, -145, -150, -155, -160, -165, -170, -175, -180, -185, -190, -195, or-200°C). In an even further aspect, the disclosed composition reduces or inhibits ice crystal formation at about -196°C.

[0201] In various aspects, the disclosed composition reduces or inhibits ice crystal formation at a temperature from about 0°C to about -200°C, from about -10°C to about -190°C, from about -20°C to about -I80°C, from about -30°C to about -I70°C, from about -40°C to about -160°C, from about -50°C to about -150°C, from about -60°C to about -140°C, from about -70°C to about -140°C, from about -80°C to about -130°C, from about -90°C to about -120°C, or from about -100°C to about -110°C.

[0202] In various aspects, the disclosed composition comprises the peptide in a concentration of from about 100 nM to about 1,000 mM. In a further aspect, the disclosedAttorney Docket No. 21101.0486P1composition comprises the peptide in a concentration of from about 100 nM to about 250 nM, from about 250 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 pM. from about 1 pM to about 5 pM. from about 5 pM to about 25 pM, from about 25 pM to about 50 pM, from about 50 pM to about 100 pM, from about 100 pM to about 250 pM, from about 250 pM to about 500 pM, from about 500 pM to about 750 pM, from about 750 pM to about 1 mM, from about 1 mM to about 10 mM, from about 10 mM to about 50 mM, from about 50 mM to about 100 mM, from about 100 mM to about 250 mM, from about 250 mM to about 500 mM, from about 500 mM to about 750 mM, or from about 750 mM to about 1,000 mM. In a still further aspect, the disclosed composition comprises the peptide in a concentration of about 100 nM, about 1 pM, about 10 pM, about 100 pM, about 1 mM, about 10 mM, about 100 mM, or about 1,000 mM. In yet a further aspect, the disclosed composition comprises the peptide in a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20. 30, 40, 50, 60, 70, 80, 90, or 100 mM.

[0203] It is understood that the disclosed compositions can be prepared from the disclosed peptide. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.1. CRYOPROTECTANT COMPOSITIONS

[0204] In one aspect, disclosed are cry oprotectant compositions comprising an effective amount of a disclosed peptide and one or more selected from: (a) a non-antifreeze protein; (b) a microbe; (c) a cell component; and (d) a cell. In a further aspect, the peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 1: 1 to about 4:1, and wherein the peptide has a chain length of at least 15 amino acid residues. In a still further aspect, the peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues. wherein the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%, wherein the ratio of the plurality7of alanine residues to the plurality7of glutamic acid residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 20 amino acid residues,Attorney Docket No. 21101.0486P1and one or more selected from: (a) anon-antifreeze protein; (b) a microbe; (c) a cell component; and (d) a cell.

[0205] As detailed herein, the disclosed peptides are useful in preventing or reducing damage caused by ice creation and ice recrystallization in a composition requiring cryoprotection. Thus, in various aspects, the disclosed peptides can be formulated into a cryoprotectant composition. As used herein, the term “cryoprotectant” refers to the ability of a composition (e.g., a cryoprotectant composition as disclosed herein) to protect a sample such as, for example, a biological sample (e.g., a non-antifreeze protein, a microbe, a cell component, or a cell) from freezing and / or from damage that occurs during freezing (e.g, due to ice formation).

[0206] Cr opreservation is a process whereby a sample (e.g., a biological sample) is preserved by cooling to sub-zero temperatures. At these low temperatures, any biological activity, including the biochemical reactions is slowed or stopped. For cryopreservation to be useful, the preserved sample should retain the integrity and viability to a reasonable level at the time of harvest. Thus, the process of preserving cells or tissue should preferably not, in itself, severely damage or destroy for example the cells or tissue architecture. However, it is known that upon freezing cells or tissue that ice crystals may form.

[0207] In conventional cryopreservation techniques, the cells or tissue are placed in a storage solution, and then preserved by freezing. When the sample is to be used, it is thawed, and then placed in a cell culture medium. Cry opreservation protocols subject the cells to a multitude of stresses and insults throughout the process of cell harvesting, freezing, and thawing. These stresses and insults can cause irreversible damage to the cell. Therefore, in order for the cells or tissues to be preserved, cryoprotectant compositions are typically used to prevent damage due to freezing during the cooling or thawing process.

[0208] To effect the desired cryoprotectant properties, the disclosed cryoprotectant composition can be applied directly to the sample as a pre-treatment (e.g., before storing), applied at an additional timepoint during freezing, and / or prior to thawing. In various further aspects, the cryoprotectant composition can be applied to the sample before freezing. In various further aspects, the cry oprotectant composition can be applied to the sample during freezing. In various further aspects, the cryoprotectant composition can be applied to the sample after freezing but prior to thawing. In various further aspects, the cryoprotectant composition can be applied to the sample at multiple timepoints (e.g., before, during, and / or after freezing).Attorney Docket No.21101.0486P1

[0209] The disclosed cryoprotectant composition can be formulated as a non-freezing liquid (e., an aqueous solution or anon-aqueous solution), anon-freezing gel, anon-freezing hydrogel, or a non-freezing paste. The cryoprotectant composition can be hygroscopic, thermally conductive, and / or biocompatible. In various aspects, the cryoprotectant composition can be formulated to be acoustically transparent such as. for example, to allow ultrasound to pass through the cryoprotectant compositions such as, for example, a water-based gel as described in US 4,002,221 and US 4,459,854.

[0210] In various aspects, the cry oprotectant composition comprises a non-antifreeze protein. As used herein, the term "‘non- antifreeze protein"’ refers to proteins that are not recognized to have properties of ice recrystallization inhibition or ice crystal shaping. Thus in various aspects, the non-antifreeze protein is selected from an enzy me, a hormone, an antibody, a growth factor, a vaccination protein, a therapeutic protein, or a nutrient protein. Additional examples of a non-antifreeze protein include, but are not limited to, egg albumin, bovine serum albumin, human serum albumin, and gelatin.

[0211] In various aspects, the cryoprotectant composition comprises a microbe. As used herein, the term “microbe” means a microorganism that can exist in a single-celled form or as a colony of cells. Examples of microbes include, but are not limited to viruses, bacteria, archaea, fungi, protists, protozoa, algae, amoebas, and slime molds.

[0212] In various aspects, the cryoprotectant composition comprises a cell component. As used herein, the term “cell component” refers to the biomolecules and structures of which cells are composed. Examples of cell components include, but are not limited to, a nucleolus, a nucleus, a ribosome, a vesicle, a rough endoplasmic reticulum, a Golgi apparatus, a cytoskeleton, a smooth endoplasmic reticulum, a mitochondria, a vacuole, a cytosol, a lysosome, and a centriole.

[0213] In various aspects, the cry oprotectant composition comprises a cell. As used herein, the term “cell” means the smallest, most basic membrane-bound unit that contains the fundamental molecules of life. Examples of cells include, but are not limited to. stem cells, bone cells, blood cells, muscle cells, sperm cells, female egg cells, fat cells, and nerve cell. Additional examples of cells include, but are not limited to, liver tissue or hepatocytes, kidney, intestine, heart, pancreas, genitourinary cells (e.g., sperm cells, oocytes), corpus cavemosum cells (e.g.. smooth muscle corpus cavemosum cells, epithelial corpus cavemosum cells), urinary bladder cells, urethral cells, ureter cells, kidney cells, testicularAttorney Docket No. 21101.0486P1cells), bone marrow, primary cells, organoids, and other biological cells and tissues for cryopreservation.

[0214] The disclosed cry oprotectant composition can also contain various additives including, but not limited to, a freezing point depressant, a thickening agent, a pH buffer, a humectant, a surfactant, and / or other additives configured to provide the desired cryoprotectant properties to the composition.

[0215] In various aspects, the cryoprotectant composition further comprises a freezing point depressant. Examples of freezing point depressants include, but are not limited to, propylene glycol (PG), polyethylene glycol (PEG), polypropylene glycol (PPG), ethylene glycol, dimethyl sulfoxide (DMSO), combinations thereof, and other glycols. The freezing point depressant can also include ethanol, propanol, iso-propanol, butanol, and / or other suitable alcohol compounds. Certain freezing point depressants (e.g., PG, PPG, PEG, etc.) can also be used to improve spreadability of the cryoprotectant and to provide lubrication. As would be understood by one of ordinary skill in the art, the freezing point depressant can lower the freezing point of a sample (e.g., a biological sample) to from about 0°C to about -40°C. In various aspects, the freezing point of a solution can be lowered to from about -I0°C to about -20°C, to from about -I0°C to about -I8°C, or to from about -I0°C to about -15°C. In various aspects, the freezing point of a sample can be lowered to a temperature less than about 0°C, less than about -5°C, less than about -I0°C, less than about -12°C, less than about -15°C, less than about -16°C, less than about -17°C, less than about -I8°C, less than about -19°C, or less than about -20°C. For example, the freezing point depressant can lower the freezing point of a sample (e.g., a biological sample) to a temperature of less than about -20°C to about -25°C, less than about -20°C to about -30°C, less than about -25°C to about -35°C, or less than about -30°C to about -40°C.

[0216] In various aspects, the cry oprotectant composition further comprises a thickening agent. Examples of thickening agents include, but are not limited to, carboxyl polyethylene polymer, hydroxyethyl xylose polymer, carboxyl methylcellulose, hydroxyethyl cellulose (HEC), and / or other viscosity modifiers, and can be used to provide a viscosity in the range of about 1 cP to about 10,000 cP. In various aspects, the thickening agent can provide a viscosity in the range of about 4,000 cP to about 8,000 cP. In a further aspect, the thickening agent can provide a viscosity in the range of about 5,000 cP to about 7,000 cP. Other viscosities can be achieved, if needed or desired. In various embodiments, a cryoprotectant composition having a viscosity7in one or more of these ranges can readilyAttorney Docket No. 21101.0486P1adhere to a treatment device, the surface of the sample, the skin of a subject, and / or the interface between the treatment device and the skin of the subject during treatment.

[0217] In various aspects, the cry oprotectant composition further comprises a pH buffer. Examples of pH buffers include, but are not limited to, cholamine chloride, cetamide, glycine, tricine, glycinamide, bicine. and / or other suitable pH buffers. In various aspects, the pH buffer can help the cryoprotectant composition to have a consistent pH of from about 3.5 to about 11.5. In a further aspect, the pH is of from about 5 to about 9.5. In a still further aspect, the pH is of from about 6 to about 7.5. In yet a further aspect, the pH of the cryoprotectant composition is within ±2 or ±1 of the pH of the sample.

[0218] In various aspects, the cryoprotectant composition further comprises a humectant. Examples of humectants include, but are not limited to, glycerin, alkylene glycol, polyalkylene glycol, propylene glycol, glyceryl triacetate, polyols (e.g., sorbitol and / or maltitol), polymeric polyols (e.g., poly dextrose), quillaia, lactic acid, and / or urea. In various aspects, the humectant can promote the retention of water to prevent the cryoprotectant composition from drying out.

[0219] In various aspects, the cryoprotectant composition further comprises a surfactant. Examples of surfactants include, but are not limited to, sodium dodecyl sulfate, ammonium lauryl sulfate, sodium lauryl sulfate, alkyl benzene sulfonate, sodium lauryl ether sulfate, and other suitable surfactants. In various aspects, the surfactant can promote easy spreading of the cryoprotectant composition when an operator applies the cryoprotectant to a sample (e.g., a biological sample).

[0220] The cryoprotectant may also include other additives in addition to or in lieu of the composition components described above. For example, some of the embodiments of cryoprotectant compositions may also include a coloring agent, fragrance or perfume, emulsifier, stabilizer, an anesthetic agent, and / or other ingredient.2. AGRICULTURAL COMPOSITIONS

[0221] In one aspect, disclosed are agricultural compositions comprising a disclosed peptide. In a further aspect, the peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality' of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acidAttorney Docket No.21101.0486P1residues is from about 1: 1 to about 4:1, and wherein the peptide has a chain length of at least 15 amino acid residues. In a still further aspect, the peptide comprises a plurality of alanine residues and a plurality7of glutamic acid residues, wherein the plurality7of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 20 amino acid residues. In various aspects, the agricultural composition is a cryoprotectant agricultural composition.

[0222] As used herein, the term “agricultural compositions’’ refers to compositions used treat agriculture products to increase food production and decrease the amount damage caused by environmental stress due to low temperatures near freezing. Non-limiting examples of agriculture products include grains, feeds, soybeans, tree nuts, fruits, and vegetables.

[0223] Frost is a major environmental stress caused by low temperature combined with dew points below freezing points (< 0°C), posing substantial economic threat on plants. At the organ level, the damage occurs when water within plant tissues freezes, forming extracellular ice crystals that result in cell dehydration. Freezing-induced cellular dehydration is the predominant cause of damage in which the cell membranes are disrupted when the dehydration exceeds cell dehydration-tolerance. Even a brief frost event persisting only for a few hours can cause substantial damage to various crops. Thus, agricultural compositions such as, for example, agricultural compositions having cryoprotective properties can beneficially reduce, minimize, and otherwise prevent damage to plants due to frost.

[0224] Additionally, many tropical and subtropical species of plants are in danger of extinction due to climate change and (a) bi otic stress. Cryopreservation is a promising longterm technique to preserve the germplasms of these species. However, tropical species are temperature delicate. Thus, cryoprotectant agncultural compositions that can increase the explant’s tolerance to low temperatures and / or enable the plant cells to withstand freezing are desirable.

[0225] As detailed herein, the resistance of plants and plant tissue to frost and low temperatures, including subfreezing temperatures, can be increased via application of an agricultural composition (e.g., a disclosed agricultural composition). For example, the agricultural composition can be sprayed onto the plants to be treated using a plant sprayAttorney Docket No. 21101.0486P1apparatus suitable for spraying aqueous solutions. The plants to be treated are thoroughly sprayed so that all of the plant tissue surfaces are completely covered. Due to the size or shape of a plant, a single application may require two or more sprayings. Alternatively, the plant can be dipped directly into the composition.

[0226] In various aspects, the agricultural composition further comprises a non-ionic surfactant, which can help to ensure that the entire surface of the plant is coated with the compositions. Examples of non-ionic surfactants that may be useful for this purpose include, but are not limited to polyoxyethylene sorbitan monolaurate (Tween 20) and polyoxyethylene sorbitan monooleate (Tween 80). In various aspects, the agricultural composition comprises from about 0.01 wt% to about 0.5 wt%, 0.05 wt% to about 0.5 wt%, 0.1 wt% to about 0.5 wt%, 0.01 wt% to about 0.1 wt%, 0.01 wt% to about 0.05 wt%, or 0.05 wt% to about 0.1 wt% of the non-ionic surfactant.

[0227] Additional agents that can be used in the disclosed agricultural composition include, but are not limited to, organic / inorganic fertilizers, pesticides, plant hormones, growth regulators, other polymers, and various coating materials. In various aspects, the disclosed agricultural composition does not include a pesticide.

[0228] Although the disclosed agricultural composition can be applied to the plants immediately prior to exposure to freezing conditions, it is also envisioned that the composition can be applied much earlier such as, for example, from about 4 hours to about 1 week prior to exposure to freezing conditions. In various aspects, the disclosed agricultural composition can be applied more than once before onset of the freezing temperatures, the first application being made from about several days to about one week prior to the onset of freezing temperatures and constituting a conditioning application. The second (or subsequent) application is then made a sufficient period prior to the onset of freezing temperatures to permit absorption of the composition, e.g., at least about 4 hours.

[0229] In various aspects, the disclosed agricultural composition can be applied regularly such as, for example, on a yearly basis, on a monthly basis, on a weekly basis, or even on a daily basis, in order to minimize any damage that might be caused by a sudden occurrence of freezing temperatures. Alternatively, the disclosed agricultural composition can be applied immediately before the exposure to freezing temperatures if the plant can to tolerate a high concentration of the agent.3. COSMETIC COMPOSITIONSAttorney Docket No. 21101.0486P1

[0230] In one aspect disclosed are cosmetic compositions comprising a disclosed peptide. In a further aspect, the peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality' of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 1: 1 to about 4: 1, and wherein the peptide has a chain length of at least 15 amino acid residues. In a still further aspect, the peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality’ of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 20 amino acid residues.

[0231] When cosmetics containing an oil component and a fat component are frozen, water contained in the cosmetics can become crystallized. As a result, the oil component and the fat component are physically pressed and the structure thereof is destroyed, causing the quality of the cosmetics to become deteriorated. The degradation of quality and the like can be avoided by incorporation of a disclosed peptide (z.e., to form a disclosed cosmetic composition) since crystallization of water can be prevented and the structure of oil component and fat component can be maintained.

[0232] In addition, the disclosed cosmetic compositions can also be formulated to penetrate skin, thereby reducing the risk of ice nucleation and protecting the tissue's cells if ice formation does occur. Such applications can increase the resistance of human skin to frostbite, for example, at temperatures near or below 0°C, and can minimize the resulting damage if cellular freezing does occur.

[0233] Methods of applying a cosmetic composition are well-known in the art and include, for example, application by hand, by spraying, or in conjunction with an occlusive backing such as in an adhesive patch. The formulation should be sufficiently viscous to remain on the skin for an extended period of time, to allow for maximum protection to be achieved.

[0234] In various aspects, the cosmetic composition is a skin care product. Thus, in various further aspects, the cosmetic composition is atopical formulation. Examples ofAttorney Docket No.21101.0486P1topical formulations include, but are not limited to, creams, serums, gels, solutions, aerosols, ointments, sprays, lotions, and patches.

[0235] Topical cosmetic compositions can be co-formulated to include inhibitors of apoptosis or reperfusion injury, and additional cryoprotectants such as glycerol, dimethylsulfoxide (DMSO), and / or low molecular weight sugars. The formulation can also include a variety of optional additives including, but not limited to, wetting agents, fragrances, and / or preservatives.E. FOOD PRODUCTS

[0236] In one aspect, disclosed are food products comprising a disclosed peptide. In a further aspect, the peptide comprises a plurality of alanine residues and a plurality' of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 1:1 to about 4:1, and wherein the peptide has a chain length of at least 15 amino acid residues. In a still further aspect, the peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%, wherein the plurality' of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4: 1, and wherein the peptide has a chain length of at least 20 amino acid residues.

[0237] Frozen compositions, such as ice cream, typically incorporate a hardening step that involves quickly freezing the composition to obtain a desired frozen composition mouthfeel. Mouthfeel is affected by the size of ice cry stals within the frozen composition. Larger ice crystals impart a grainy mouthfeel. Consequently, rapid freezing results in smaller ice crystals and smoother frozen composition mouthfeel. Without the hardening step, liquid water in frozen composition compositions freezes at much slower rates and forms large ice crystals which impart unacceptably grainy mouthfeel to the frozen composition. In addition, during frozen storage, ice crystal size increases over time as disproportionation occurs and smaller cry stals melt and recry stallize onto larger ice crystals in a dynamic process resulting in pronounced iciness, giving the product an undesirable characteristic. Controlling the iceAttorney Docket No.21101.0486P1crystal size, whether by formulation, processing, distribution temperature control, or product age management is an objective of all frozen composition manufacturers in order to ensure a high quality7finished product.

[0238] Frozen fruits and vegetables produced according to traditional freezing methods suffer from a breakdown in the vegetable or fruit cell wall structure and, as such, have a low textural crispness. Crispness generally relates to the amount of water found in the cells of the vegetable or fruit, and translates into plant textural firmness upon mastication. Crispness is also a function of the structural integrity7of the cells. A crisp vegetable is typically imbibed with water, has an intact cell wall structure, and, as such, has a firm, crisp texture. Most importantly, crisp vegetables and fruits have a crunchy and firm texture. For example, turgid or crisp celery7will be crisp and crunchy; non-turgid or low crispness celery will be limp. When the cell walls break, water exits and crispness decreases. This means that the product will have a poor or mushy texture and will not retain suitable amounts of water. Typically, with slower freezing techniques, upon thawing, water leaches out of a vegetable or fruit product that has been frozen, resulting in a low texture limp product. As such, a product having fresh-like characteristics is not produced.

[0239] As detailed herein, the disclosed peptides can be incorporated into a food product, in order slow, reduce, or otherwise inhibit ice crystal growth processes that influence the size and shape characteristics of the resultant ice that is formed during regrowth, thereby minimizing potential freezing damage by preventing or inhibiting ice recrystallisation of the product upon freezing. Thus, in various aspects, the disclosed food product reduces or inhibits ice crystal formation at a temperature of from about 0°C to about -20°C (e.g., at about 0, -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, or -20°C). In a further aspect, the disclosed food product reduces or inhibits ice cry stal formation at a temperature of from about -20°C to about -40°C (e.g., at about -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, or -40°C). In a still further aspect, the disclosed food product reduces or inhibits ice crystal formation at about -20°C. In yet a further aspect, the disclosed food product reduces or inhibits ice cry stal formation at a temperature of from about -40°C to about -200°C (e.g., at about -40, -45, -50, -55, -60, -65. -70, -75, -80. -85, -90, -95. -100, -105, -110, -115, -120, -125, -130, -135, -140, -145, -150. -155, -160, -165. -170, -175, -180. -185, -190, -195, or -200°C). In an even further aspect, the disclosed food product reduces or inhibits ice crystal formation at about -196°C.Attorney Docket No.21101.0486P1

[0240] In various aspects, the disclosed food product reduces or inhibits ice crystal formation at a temperature from about 0°C to about -200°C, from about -10°C to about -190°C, from about -20°C to about -180°C, from about -30°C to about -170°C, from about -40°C to about -160°C, from about -50°C to about -150°C, from about -60°C to about -140°C, from about -70°C to about -140°C, from about -80°C to about -130°C, from about -90°C to about -120°C, or from about -100°C to about -110°C.

[0241] In various aspects, the disclosed food product comprises the peptide in a concentration of from about 100 nM to about 1,000 mM. In a further aspect, the disclosed food product comprises the peptide in a concentration of from about 100 nM to about 250 nM, from about 250 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 pM. from about 1 pM to about 5 pM, from about 5 pM to about 25 pM, from about 25 pM to about 50 pM, from about 50 pM to about 100 pM, from about 100 pM to about 250 pM, from about 250 pM to about 500 pM, from about 500 pM to about 750 pM, from about 750 pM to about 1 mM, from about 1 mM to about 10 mM, from about 10 mM to about 50 mM, from about 50 mM to about 100 mM, from about 100 mM to about 250 mM, from about 250 mM to about 500 mM, from about 500 mM to about 750 mM, or from about 750 mM to about 1,000 mM. In a still further aspect, the disclosed food product comprises the peptide in a concentration of about 100 nM, about 1 pM, about 10 pM, about 100 pM, about 1 mM, about 10 mM, about 100 mM, or about 1,000 mM. In yet a further aspect, the disclosed food product comprises the peptide in a concentration

[0242] In various aspects, the food product is selected from ice cream, yogurt, seafood, fruit, and a meat product. In a further aspect, the food product is ice cream. In a still further aspect, the food product is yogurt. In a yet further aspect, the food product is ice cream. In an even further aspect, the food product is seafood. In an even still further aspect, the food product is a fruit. In an even yet further aspect, the food product is a meat product.F. SURFACES

[0243] In one aspect, disclosed are solid or semi-solid support comprising a surface covalently attached to a residue of a disclosed peptide. In a further aspect, the peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality' of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality ofAttorney Docket No. 21101.0486P1alanine residues to the plurality of glutamic acid residues is from about 1: 1 to about 4:1, and wherein the peptide has a chain length of at least 15 amino acid residues. In a still further aspect, the peptide comprises a plurality' of alanine residues and a plurality' of glutamic acid residues. wherein the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%, wherein the ratio of the plurality' of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 20 amino acid residues.

[0244] The development of anti-icing, anti-frosting surfaces is of importance in building materials because surfaces covered with ice layers and frost layers often cause serious issues, such as poor visibility through the windshields of aircraft, trains and automobiles; poor visibility7of traffic lights and surveillance cameras; decreases in efficiency of the heat exchanger and power generation efficiency7of solar panels; breaking of power transmission lines in winter; and deterioration of the aerodynamic performance of aircraft wings.

[0245] As detailed herein, the disclosed peptides can be attached to a surface in order to prevent, inhibit, or otherwise delay the formation of ice on objects including, but not limited to, aircrafts or parts thereof, gas pipelines, windows, electrical equipment, drones, cables (e.g., power lines), mechanical equipment (e.g., car engines, gear systems, brake systems, etc.), and the like. Thus, in various aspects, the disclosed solid or semi-solid support reduces or inhibits ice cry stal formation at a temperature of from about 0°C to about -20°C (e.g, at about 0, -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11. -12, -13, -14. -15, -16, -17. -18, -19, or -20°C). In a further aspect, the disclosed solid or semi-solid support reduces or inhibits ice crystal formation at a temperature of from about -20°C to about -40°C (e.g., at about -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, or -40°C). In a still further aspect, the disclosed solid or semi-solid support reduces or inhibits ice crystal formation at about -20°C. In yet a further aspect, the disclosed solid or semi-solid support reduces or inhibits ice crystal formation at a temperature of from about -40°C to about -200°C (e.g., at about -40, -45, -50, -55, -60, -65, -70, -75, -80, -85, -90, -95, -100, -105, -110, -115, -120, -125, -130. -135, -140, -145, -150, -155, -160, -165, -170, -175, -180, -185, -190. -195, or -200°C). In an even further aspect, the disclosed solid or semi-solid support reduces or inhibits ice crystal formation at about -196°C.Attorney Docket No. 21101.0486P1

[0246] In various aspects, the disclosed surface reduces or inhibits ice crystal formation at a temperature from about 0°C to about -200°C, from about -10°C to about -190°C, from about -20°C to about -180°C, from about -30°C to about -170°C, from about -40°C to about -160°C, from about -50°C to about -150°C, from about -60°C to about -140°C, from about -70°C to about -140°C, from about -80°C to about -130°C, from about -90°C to about -120°C, or from about -100°C to about -110°C.

[0247] In various aspects, the disclosed solid or semi-solid support reduces or inhibits ice crystal formation at a temperature from about 0°C to about -200°C, from about -10°C to about -190°C, from about -20°C to about -180°C, from about -30°C to about -170°C, from about -40°C to about -160°C, from about -50°C to about -150°C, from about -60°C to about -140°C, from about -70°C to about -140°C, from about -80°C to about -130°C, from about -90°C to about -120°C, or from about -100°C to about -110°C.

[0248] Methods of covalently attaching a peptide to a surface or a solid or semi-solid support of a surface are well-known in the art. Alternatively, the peptide can be formulated into a composition, and coated onto the surface. See, e.g., Stawikowski and Fields (2002) Curr Protoc Protein Sci. Chapter: Unit-18.1, doi: 10.1002 / 0471140864.psl801s26.

[0249] In various aspects, attached is via covalent attachment to the surface. In various further aspects, attached is via coating a cryoprotectant composition comprising the peptide on the surface.

[0250] In various aspects, the solid or semi-solid support is a glass bead, a silica-based resin, a cellulosic resin, an agarose bead, a polysty rene bead, or a polyacrylamide resin.

[0251] In various aspects, the disclosed surface comprises the peptide in a concentration of from about 100 nM to about 1,000 mM. In a further aspect, the disclosed surface comprises the peptide in a concentration of from about 100 nM to about 250 nM, from about 250 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 pM. from about 1 pM to about 5 pM. from about 5 pM to about 25 pM, from about 25 pM to about 50 pM, from about 50 pM to about 100 pM, from about 100 pM to about 250 pM, from about 250 pM to about 500 pM, from about 500 pM to about 750 pM, from about 750 pM to about 1 mM, from about 1 mM to about 10 mM, from about 10 mM to about 50 mM, from about 50 mM to about 100 mM, from about 100 mM to about 250 mM, from about 250 mM to about 500 mM, from about 500 mM to about 750 mM, or from about 750 mM to about 1,000 mM. In a still further aspect, the disclosed surface comprises the peptide in a concentration of about 100 nM, about 1 pM, about 10 pM, about 100 pM, aboutAttorney Docket No.21101.0486P11 mM, about 10 mM, about 100 mM, or about 1,000 mM. In yet a further aspect, the disclosed surface comprises the peptide in a concentration.

[0252] In various aspects, the cry oprotectant composition comprises the peptide in a concentration of from about 100 nM to about 1,000 mM. In a further aspect, the cryoprotectant composition comprises the peptide in a concentration of from about 100 nM to about 250 nM, from about 250 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 pM. from about 1 pM to about 5 pM, from about 5 pM to about 25 pM, from about 25 pM to about 50 pM, from about 50 pM to about 100 pM, from about 100 pM to about 250 pM, from about 250 pM to about 500 pM, from about 500 pM to about 750 pM, from about 750 pM to about 1 mM, from about 1 mM to about 10 mM, from about 10 mM to about 50 mM, from about 50 mM to about 100 mM, from about 100 mM to about 250 mM, from about 250 mM to about 500 mM, from about 500 mM to about 750 mM, or from about 750 mM to about 1,000 mM. In a still further aspect, the cryoprotectant composition comprises the peptide in a concentration of about 100 nM, about 1 pM, about 10 pM, about 100 pM, about 1 mM, about 10 mM, about 100 mM, or about 1,000 mM. In yet a further aspect, the cryoprotectant composition comprises the peptide in a concentration.

[0253] In various aspects, the disclosed solid or semi-solid support comprises the peptide in a concentration of from about 100 nM to about 1,000 mM. In a further aspect, the disclosed solid or semi-solid support comprises the peptide in a concentration of from about 100 nM to about 250 nM, from about 250 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 pM, from about 1 pM to about 5 pM, from about 5 pM to about 25 pM, from about 25 pM to about 50 pM, from about 50 pM to about 100 pM, from about 100 pM to about 250 pM, from about 250 pM to about 500 pM, from about 500 pM to about 750 pM, from about 750 pM to about 1 mM, from about 1 mM to about 10 mM, from about 10 mM to about 50 mM, from about 50 mM to about 100 mM, from about 100 mM to about 250 mM, from about 250 mM to about 500 mM, from about 500 mM to about 750 mM, or from about 750 mM to about 1,000 mM. In a still further aspect, the disclosed solid or semi-solid support comprises the peptide in a concentration of about 100 nM, about 1 pM, about 10 pM, about 100 pM, about 1 mM, about 10 mM, about 100 mM, or about 1,000 mM. In yet a further aspect, the disclosed solid or semi-solid support comprises the peptide in a concentration.

[0254] In various aspects, the residue of the peptide comprises an N-terminus, and the N-terminus is covalently attached to the surface.Attorney Docket No.21101.0486P1

[0255] In various aspects, the residue of the peptide has a structure represented by a formula:wherein R20is selected from -OR31, -NHR32, -N3, a protein tag, a sortase recognition sequence, a sugar residue, and a structure:wherein R31and R32is selected from hydrogen, -CH2PI1, C1-C8 alkyl, C2-C8 alkyne, C1-C8 azide, tetrazinyL cyclooctynyl, norbomenyl, and -(CH2CH2O)mCH3, and wherein m is an integer selected from 1 to 100.

[0256] As used herein, the term ‘‘support” refers to a material or substrate (e.g., a surface) onto which a peptide or a residue of a peptide, as defined herein, adheres.Adherence can be, for example, via chemical bonding, immobilization, dispersion, or association. Typically, a support is a polymeric material such as a network polymeric material. Supports include glasses, semiconductor materials, ceramic materials, metal surfaces, and other substrates on which the peptide or the residue of the peptide, as defined herein, can adhere. Additional examples of supports include, but are not limited to, glass beads, silica-based resins, cellulosic resins, agarose beads, polystyrene beads, or polyacrylamide resins. The support can be solid or semi-solid (e.g., gel-like, such as a polymer support composed of hydrogel polymers) as further described herein.Attorney Docket No.21101.0486P1G. METHODS OF INHIBITING ICE CRYSTAL FORMATION IN A SAMPLE

[0257] In one aspect, disclosed are methods of inhibiting ice crystal formation in a sample, the method comprising contacting the sample with an effective amount of a disclosed peptide. In a further aspect, the peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 1: 1 to about 4:1, and wherein the peptide has a chain length of at least 15 amino acid residues. In a still further aspect, the disclosed peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%. wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 20 amino acid residues.

[0258] The methods described herein are suitable for use in any number of cryopreservation protocols. For example, in various aspects, the disclosed peptides, compositions, products, supports, and methods are useful for cry opreservation during supercoiling to high sub-zero temperatures (e.g., from about 0°C to about -20°C). In various further aspect, the disclosed peptides, compositions, products, supports, and methods are useful for cryopreservation during freezing protocols (e.g., from about -20°C to about -196°C). Freezing protocols are typically performed at a controlled rate (sometimes referred to as slow freezing) during at least part of the temperature reduction. For example, a biological sample or macromolecule can be contacted with a disclosed peptide, and the temperature can be reduced at a controlled rate (e.g., lowered at a rate of 1°C per minute) until the desired temperature is reached. Alternatively, the temperature can be reduced at a controlled rate until a desired temperature is reached (e.g., from about -80°C to about -180°C), and then the sample or macromolecule can be flash frozen (e.g., by immersing the sample or macromolecule in liquid nitrogen or placing the sample or macromolecule above liquid nitrogen). The disclosed peptide should preferably be contacted with the sample or macromolecule being cryopreserved prior to freezing, to ensure that the peptide is in contact with the sample. Were the peptide to contact the sample after freezing, penetration throughAttorney Docket No. 21101.0486P1the ice block to the sample may hinder the ability of the peptide act as a cryoprotectant for the sample.

[0259] In various further aspects, the disclosed peptides, compositions, products, supports, and methods are useful for cryogenic freezing protocols (e.g., from about -90°C to about -196°C). For example, a biological sample or macromolecule can be contacted with a disclosed peptide or composition, then plunged into liquid nitrogen or a stream of liquid nitrogen vapor in order to quickly freeze the sample without the formation of ice crystals. No ice lattice exists and so the water within the sample or macromolecule is in an amorphous or glass-like state. Therefore, damaging ice is not formed.

[0260] As would be readily appreciated by one of ordinary skill in the art, the concentrations and compositions of a peptide disclosed herein can be modified depending on the particular biological sample and / or macromolecule being cryopreserved and the particular cryopreservation protocol being employed. Thus, in various aspects, the disclosed composition comprises the peptide in a concentration of from about 100 nM to about 1,000 mM. In a further aspect, the disclosed composition comprises the peptide in a concentration of from about 100 nM to about 250 nM, from about 250 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 pM, from about 1 pM to about 5 pM, from about 5 pM to about 25 pM, from about 25 pM to about 50 pM, from about 50 pM to about 100 pM, from about 100 pM to about 250 pM, from about 250 pM to about 500 pM, from about 500 pM to about 750 pM, from about 750 pM to about 1 mM, from about 1 mM to about 10 mM, from about 10 mM to about 50 mM, from about 50 mM to about 100 mM, from about 100 mM to about 250 mM, from about 250 mM to about 500 mM, from about 500 mM to about 750 mM, or from about 750 mM to about 1,000 mM. In a still further aspect, the disclosed composition comprises the peptide in a concentration of about 100 nM, about 1 pM, about 10 pM, about 100 pM, about 1 mM, about 10 mM, about 100 mM, or about 1,000 mM. In yet a further aspect, the disclosed composition comprises the peptide in a concentration of about 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM.

[0261] In various aspects, the disclosed peptide and / or the disclosed composition reduces or inhibits ice crystal formation at a temperature of from about 0°C to about -20°C (e.g., at about 0, -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11. -12, -13, -14. -15, -16, -17. -18, -19, or -20°C). In a further aspect, the disclosed peptide and / or the disclosed composition reduces or inhibits ice crystal formation at a temperature of from about -20°C to about -40°C (e.g., at about -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31,Attorney Docket No.21101.0486P1-32, -33, -34, -35, -36, -37, -38, -39, or -40°C). In a still further aspect, the disclosed peptide and / or the disclosed composition reduces or inhibits ice crystal formation at about -20°C. In yet a further aspect, the disclosed peptide and / or the disclosed composition reduces or inhibits ice cry stal formation at a temperature of from about -40°C to about -200°C (e.g., at about -40. -45, -50, -55. -60, -65, -70. -75, -80, -85. -90, -95, -100, -105. -110, -115, -120, -125, -130, -135, -140, -145, -150, -155, -160, -165, -170, -175, -180, -185, -190, -195, or -200°C). In an even further aspect, the disclosed peptide and / or the disclosed composition reduces or inhibits ice cry stal formation at about -196°C.

[0262] In various aspects, the disclosed peptide and / or the disclosed composition reduces or inhibits ice crystal formation at a temperature from about 0°C to about -200°C, from about -10°C to about -190°C, from about -20°C to about -180°C, from about -30°C to about -170°C, from about -40°C to about -160°C, from about -50°C to about -150°C, from about -60°C to about -140°C, from about -70°C to about -140°C, from about -80°C to about -130°C, from about -90°C to about -120°C, or from about -100°C to about -110°C.

[0263] In various aspects, contacting is via application (e.g., application onto a sample or surface, topical application). In various further aspects, contacting is via coating. In various further aspects, contacting is via spraying or dipping.

[0264] In various aspects, contacting is via covalent attachment. For example, as detailed herein, in various aspects, the disclosed peptide can be covalently attached to a surface.

[0265] In various aspects, contacting is for a time period of at least 1 hour, at least 2 hours, at least 5 hours, at least 10 hours, at least 20 hours, at least 24 hours, or for longer than 24 hours. In various further aspects, contacting is for a time period of at least 1 day, 2 days, 4 days, 6 days, 7 days, or longer than 7 days.

[0266] In various aspects, the method involves repeated contacting steps. For example, in various aspects, the sample can be contacted with the peptide or composition at least once, at least twice, at least three time, at least four times, or more than four times.

[0267] In various aspects, the sample is a biological material. Examples of biological materials include, but are not limited to, a non- antifreeze protein (z.e., an enzyme, a hormone, an antibody’, a growth factor, a vaccination protein, a therapeutic protein, or a nutrient protein), a microbe (i.e., a virus, a bacteria, an archaea, a fungi, and a protists), a cell component (i.e. a nucleolus, a nucleus, a ribosome, a vesicle, a rough endoplasmic reticulum,Attorney Docket No. 21101.0486P1a Golgi apparatus, a cytoskeleton, a smooth endoplasmic reticulum, a mitochondria, a vacuole, a cytosol, a lysosome, and a centriole), a cell (e.g., a sperm cell, an egg matrix cell, an embry onic cell, a stem cell), a tissue i.e., epithelial, connective, nervous, or muscle), and an organ (i.e., heart, kidney, liver, ovary, cornea).

[0268] In various aspects, the sample is a food product. Examples of food products for which the disclosed method can be useful include, but are not limited to, ice cream, yogurt, seafood, fruit, and meat products.

[0269] In various aspects, the sample is an agricultural product. Examples of agriculture products for which the disclosed method can be useful include, but are not limited to, grains, feeds, soybeans, tree nuts, fruits, and vegetables.

[0270] In various aspects, the sample is a cosmetic. Examples of cosmetics for which the disclosed method can be useful include, but are not limited to, lips balms, hair products, makeup, nail products, soaps and lotions.

[0271] In various aspects, the method further comprises storing the biological material for a period of time.

[0272] In various aspects, storing is at a temperature of about 25 °C or less. In a further aspect, storing is at a temperature of about 20°C. In a still further aspect, storing is at a temperature of about 15°C. In a yet further aspect, storing is at a temperature of about 10°C. In an even further aspect, storing is at a temperature of about 5°C. In an even still further aspect, storing is at a temperature of about 0°C. In an even further aspect, storing is at a temperature of about -5°C. In an even yet further aspect, storing is at a temperature of about -10°C. In a further aspect, storing is at a temperature of about -15°C. In a still further aspect, storing is at a temperature of about -20°C. In a yet further aspect, storing is at a temperature of about -25°C. In an even further aspect, storing is at a temperature of about -30°C. In an even still further aspect, storing is at a temperature of about -35°C. In an even further aspect, storing is at a temperature of about -40°C. In an even yet further aspect, storing is at a temperature of about -45°C. In a further aspect, storing is at a temperature of about -50°C. In a still further aspect, storing is at a temperature of about -55°C. In a yet further aspect, storing is at a temperature of about -60°C. In an even further aspect, storing is at a temperature of about -65°C. In an even still further aspect, storing is at a temperature of about -70°C. In an even further aspect, storing is at a temperature of about -75°C. In an even yet further aspect, storing is at a temperature of about -80°C.Attorney Docket No.21101.0486P1

[0273] In various aspect, storing is at a temperature of about 5 °C or less. In a further aspect, storing is at a temperature of about 4°C. In a still further aspect, storing is at a temperature of about 3°C. In a yet further aspect, storing is at a temperature of about 2°C. In an even further aspect, storing is at a temperature of about 2°C. In an even still further aspect, storing is at a temperature of about 0°C. In an even further aspect, storing is at a temperature of about -1 °C. In an even yet further aspect, storing is at a temperature of about -2°C. In a further aspect, storing is at a temperature of about -3°C. In a still further aspect, storing is at a temperature of about -4°C. In a yet further aspect, storing is at a temperature of about -5°C.H. USE OF PEPTIDES

[0274] In one aspect, the invention relates to the use of a disclosed peptide or a product of a disclosed method. As detailed herein, the disclosed peptides are useful in preventing or reducing damage caused by ice creation and ice recrystallization and can be formulated into a composition (e.g., a cry oprotectant composition, an agricultural composition, a cosmetic composition) to facilitate use in these areas.

[0275] Also provided are the uses of the disclosed peptides and products. In one aspect, the invention relates to use of at least one disclosed peptide; or an acceptable salt thereof. In a further aspect, the peptide used is a product of a disclosed method of making.

[0276] In a further aspect, the use relates to a process for preparing a composition comprising an effective amount of a disclosed peptide or a product of a disclosed method of making, or an acceptable salt thereof, wherein an acceptable carrier is intimately mixed with an effective amount of the peptide or the product of a disclosed method of making.

[0277] It is understood that the disclosed uses can be employed in connection with the disclosed peptides, products of disclosed methods of making, methods, compositions (e.g., a cryoprotectant composition, an agricultural composition, a cosmetic composition), food products, solid and semi-solid supports, and kits.1. KITS

[0278] In one aspect, disclosed are kits comprising a disclosed peptide and one or more selected from: (a) a biological material; (b) a food product; (c) an agricultural product; (d) a solid or semi-solid support; and (e) a cosmetic. In a further aspect, the peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, whereinAttorney Docket No.21101.0486P1the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 1: 1 to about 4: 1, and wherein the peptide has a chain length of at least 15 amino acid residues. In a still further aspect, the disclosed peptide comprises a plurality of alanine residues and a plurality of glutamic acid residues, wherein the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%, wherein the plurality7of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 40 wt%. wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 20 amino acid residues; and one or more selected from: (a) a biological material; (b) a food product; (c) an agricultural product; (d) a solid or semi-solid support; and (e) a cosmetic.

[0279] In various aspects, the kit includes the biological material. In various further aspects, the biological material is selected from a non-antifreeze protein (e.g., an enzyme, a hormone, an antibody, a growth factor, a vaccination protein, a therapeutic protein, or a nutrient protein), a microbe (e.g., a virus, a bacteria, an archaea, a fungi, and a protists), a cell component (e.g. a nucleolus, a nucleus, a ribosome, a vesicle, a rough endoplasmic reticulum, a Golgi apparatus, a cytoskeleton, a smooth endoplasmic reticulum, a mitochondria, a vacuole, a cytosol, a lysosome, and a centriole), a cell (e.g., a sperm cell, an egg matrix cell, an embryonic cell, a stem cell), a tissue (e.g., epithelial, connective, nervous, or muscle), and an organ (e.g., heart, kidney, liver, ovary, cornea).

[0280] In various aspects the kit includes the food product. Examples of food products include, but are not limited to, ice cream, yogurt, seafood, fruit, and meat products.

[0281] In various aspects, the kit includes the agricultural product. Examples of agriculture products include, but are not limited to, grains, feeds, soybeans, tree nuts, fruits, and vegetables.

[0282] In various aspects, the kit includes the solid or semi-solid support. Examples of solid and semi-solid supports include glasses, semiconductor materials, ceramic materials, metal surfaces, and other substrates on which the peptide or the residue of the peptide, as defined herein, can adhere. Additional examples of solid and semi-solid supports include, but are not limited to, glass beads, silica-based resins, cellulosic resins, agarose beads, polystyrene beads, or polyacrylamide resins. The support can be solid or semi-solid (e.g.,Attorney Docket No. 21101.0486P1gel-like, such as a polymer support composed of hydrogel polymers) as further described herein.

[0283] In various aspects, the kit includes the cosmetic. Example of cosmetics include, but are not limited to, lips balms, hair products, makeup, nail products, soaps, and lotions.

[0284] In a further aspect, the disclosed peptide and the biological material are coformulated. In a still further aspect, the disclosed peptide and the food product are coformulated. In a yet further aspect, the disclosed peptide and the agricultural product are coformulated. In an even further aspect, the disclosed peptide and the solid or semi-solid support are co-formulated. In an even still further aspect, the disclosed peptide and the cosmetic are co-formulated.

[0285] All publications and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In the event of an inconsistency between the present disclosure and any publications or patent application incorporated herein by reference, the present disclosure controls.I. EXAMPLES

[0286] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the peptides, compositions, food products, solid and semi-solid supports, methods, and kits claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0287] The Examples are provided herein to illustrate the invention, and should not be construed as limiting the invention in any way.1. CHEMISTRY EXPERIMENTALSAttorney Docket No. 21101.0486P1a. INSTRUMENTATION AND GENERAL METHODS

[0288] Reactions were conducted under an inert atmosphere of N2, using oven-dried glassware unless otherwise stated. Hexanes and dichloromethane were purified by first purging with dry nitrogen, followed by passage through columns of activated 3A molecular sieves. THF was purified by first purging with dry nitrogen, followed by passage through columns of activated alumina. Commercial anhydrous DMF was purchased and stored over 3A molecular sieves. All oven dried glassware was dried at 120°C. Infrared spectra were recorded on a Bruker Alpha ATR-FTIR Spectrophotometer. All polymerizations were monitored for completion via ATR-FTIR. Deionized water (18 MQ-cm) was obtained by passing in-house deionized water through a Thermo Scientific MicroPure UV / UF purification unit. Tandem gel permeation chromatography / light scattering (GPC / LS) was performed on an Agilent 1260 Infinity liquid chromatograph pump equipped with a Wyatt DAWN HELEOS-II light scattering (LS) and Wyatt Optilab T-rEX refractive index (RI) detectors. Separations were achieved using 105, 104, and 103A Phenomenex Phenogel 5 pm columns using 0.10 M LiBr in DMF as the eluent at 60°C. All GPC / LS samples were prepared at concentrations of 3 mg / mL.1H NMR spectra were recorded on a Varian Mercury spectrometer (400 MHz) or a Bruker AVANCE NEO spectrometer (500 MHz) and are reported relative to deuterated solvent. Data for1H NMR are reported as follows: chemical shift (8 ppm), multiplicity, coupling constant (Hz) and integration. Data for13C NMR spectra are reported in chemical shift. Common solvent impurities found in spectra are labelled (Kramer, J. R.; Deming, T. J. Biomacromolecules 2010, 11, 3668-3672). CD measurements of the polypeptide solutions were recorded in quartz cells with a path length of 0.1 cm, on a JASCO J-1500 CD spectrophotometer.b. SYNTHESIS OF SMALL MOLECULESi. PREPARATION OF D- AND L-ALANINE NCA

[0289] D- and L-Alanine NCAs were synthesized according to previously published methods (Kohout, V. R.. et al., (2023) J. Am. Chem. Soc 145 (30), 16573-16583).Attorney Docket No. 21101.0486P1ii. PREPARATION OF D- AND L-GLUTAMIC ACID N CAS

[0290] y-tert-Butyl-L-glutamate NCA i / Bu-ELNCA), y-terCbutyl-D-glutamate NCA (7BU-EDNCA), L-Glutamic acid NCA (EL), benzyloxycarbonyl-L-lysine NCA (Z-KLNCA) were synthesized according to previously published methods (Kramer JR, et aL, (2015) Proc Natl Acad Sci U S A. 112(41): 12574-9. Epub 20150929; Kramer, J. R.; Deming, T. J. Biomacromolecules 2010, 11, 3668-3672; Kohout VR, et al., (2023) J Am Chem Soc.145(30): 16573-83). Glutamic acid was utilized in both free acid form and with various protecting groups (i.e. tert-butyl, benzyd).c. POLYPEPTIDE SYNTHESES AND MODIFICATIONSi. GENERAL PROCEDURE FOR PREPARATION OF STATISTICAL COPOLYMERS USING PROTECTED GLUTAMIC ACID NCA AND ALA NCAo R' = tBu or BenzylR = CH3or CH2CH2CO2H

[0291] Under inert atmosphere, AL, AD, tBu-EL, tBu-ED, or Z-KLNCAs were dissolved in anhydrous THF at concentrations of 50 mg / ml. NCAs were mixed at the desired ratio of 3: 1 ALor ADto tBu-EL, tBu-ED, or Z-KL. The (PMe.d-iCo catalyst was added in one shot via syringe at the desired monomer to initiator ratio. Aliquots were removed for analysis by ATR-FTIR. Polymerizations were complete in 1 and 3 hours for 50 and lOOmer, respectively, and overnight for 150mer and 200mer. tert- But l groups were removed by treatment with trifluoroacetic acid.Attorney Docket No. 21101.0486P1ii. GENERAL PROCEDURE FOR TBU PROTECTING GROUP REMOVAL TO YIELD (ALEL)N

[0292] (ALtBu-EL)n copoly peptides were dissolved in trifluoroacetic acid at a concentration of lOmg / mL and stirred at room temp for 1 hours. Trifluoroacetic acid was removed by rotary evaporation and polymers were taken up in water / NaHCCh at a concentration of 10 mg / mL. Samples were dialyzed for 3 days against MilliQ water using a IkDa MWCO membrane. The resulting product was a white solid (60-99% yield) depending on polymer length. Polymers with 100+ residues had -20% insoluble portion which was removed by centrifugation prior to further experimental work.iii. GENERAL PROCEDURE FOR BENZYLOXYCARBONYL PROTECTING GROUP REMOVAL TO YIELD (ALKL)NNHCbz

[0293] (ALZ-KL)ncopolypeptides were dissolved in trifluoroacetic acid and 5 eq. of 33% HBr / acetic acid was added. The reaction was stirred at room temp for 2 hours.Polymers were precipitated into ether and the liquid was decanted. Solids were taken up in water and dialyzed for 3 days against MilliQ water using a IkDa MWCO membrane. The resulting product was a white solid (85-99% yield). Polymers with 100+ residues had -20% insoluble portion which was removed by centrifugation prior to further experimental work.Attorney Docket No.21101.0486P1iv. GENERAL PROCEDURE FOR STATISTICAL COPOLYMERS PREPARED BY METHOD B USING PROTECTING-GROUP-FREE CONDITIONS AND HEXYLAMINE INITIATORo

[0294] ALand ELNCAs were dissolved in DMF at 250 mg / mL. Hexylamine solution at.077M in DMF was added at the desired M: I ratio, followed immediately by the addition of water to yield a 1:1 DMF:water solution. The final concentration of NCA was 90 mg / mL. Aliquots were removed for analysis by ATR-FTIR. Polymerizations were generally complete within 30 minutes. The polymerization solution was diluted with water and dialyzed against MilliQ water using a IkDa MWCO membrane. Insoluble solids were removed by centrifugation and the yield of aqueous soluble material was ca. 40%.

[0295] Nickel and iron complexes can be substituted for the cobalt complex.Nucleophiles including but not limited to primary, secondary, or tertiary amines, alkoxides can also be utilized as polymerization initiators.2. BIOLOGICAL EXPERIMENTALSa. CIRCULAR DICHROISM SPECTROSCOPY

[0296] Polymers were either dissolved in PBS or Milli-Q water. A wavelength of 214 nm, extinction coefficient of 2200 cm^M^and Beer’s law were used to determine unknown peptide concentration and normalize CD data. Samples were prepared at concentrations 500 pg / mL or 100 pg / mL. All spectra were recorded as an average of 3 scans. Molar ellipticity ([0]) was calculated using the equation [0] = (0) / (lO*c*l), where 0 is measured ellipticity (mdeg), c is concentration (M), and 1 is path length of the cuvette (cm). For plotting, molar ellipticity was represented as [0]* 10'3.Attorney Docket No. 21101.0486P1b. DYNAMIC ICE SHAPING

[0297] To observe dynamic ice shaping 10 uL of solution containing polypeptide in 1 x PBS was placed on a microscope slide and sandwiched between a cover slip. The stage was rapidly cooled at a rate of 10°C / min to -30°C to freeze the polymer solution. The stage was then slowly warmed to -2.5°C at a rate of 8°C / min. Then the stage was warmed to -1.8°C at a rate of 0.5°C / min. The stage temperature was then increased at a rate of 0.05°C / min to -1.5 to -1°C depending on the polypeptide solution to isolate individual crystals. The stage was then cooled at 0.02°C / min to -2 to -1.5°C to observe dynamic ice shaping. The stage was then toggled between melting and freezing rates to observe the ice crystal change as the temperature was increased and then decreased. Images of the single crystals were taken as the temperature was decreased to observe ice cry stal grow th.c. ICE RECRYSTALLIZATION INHIBITION ASSAYS

[0298] Using a micropipette. lOpL aliquots of copolypeptide solutions in PBS were dropped from 2 meters through a PVC pipe onto a precooled slide. The slide was cooled on an aluminum block resting in a bed of dry ice. The slide containing the ice splat was quickly moved to the temperature- controlled stage (Linkam LTS120, WCP, and T96 controller) precooled to -6.4°C. The stage chamber was purged with N2 to prevent condensation from growing on the ice splat. The ice splat was annealed for 40 minutes and images of the ice crystals were recorded at 0, 20, and 40 minutes using cross polarizers (MOTICAM S3, MOTIC BA310E LED Trinocular) to observe ice recrystallization inhibition.d. ICE RECRYSTALLIZATION INHIBITION ASSAY WITH FROZEN DAIRY PRODUCT

[0299] Commercially available vanilla ice cream (Tillamook brand) was allowed to fully melt at ambient temperature. Aliquot were added directly to lyophilized copolypeptide to prepare solutions containing 0-1000 pg / mL (ALEL)so. Cooling splat assays were conducted using 4 pL aliquots, which were pipetted onto glass slides and placed on a cryostage. The glass slide was cooled to -30°C at a cooling rate of 30°C / min and equilibrated for 15 minutes (Piao Z, et al., (2021) ACS Macro Lett. 10(11): 1436-42). Then, the temperature was increased to -6°C at a heating rate of l°C / min. The ice splat was annealed for 40 minutes and images were acquired.Attorney Docket No. 21101.0486P1e. QUANTIFICATION OF ICE RECRYSTALLIZATION INHIBITION ACTIVITY

[0300] For all polypeptide solutions, Image J (Fiji) was used to analyze the mean grain size (MGS) of the ice crystals. For each cooling splat, three images were taken of different areas of the crystal. Briefly, ice crystals were traced by hand to determine the MGS. Statistical analysis showed that the 150pm x 150pm region of the image resulted in an MGS that is representative of the entire population. We randomly selected the region of the image to analyze. In all cases, three images were collected for each splat assay, and the ice cry stal areas for each image were averaged. The average and standard deviations of the three images are presented for each splat.f. CELL VIABILITY ASSAYS

[0301] HEK 293 cells were plated at a density of 10,000 cells / well in a 96 well plate. The cells were incubated at 37°C in 5% CO2 for 24 hours to allow the cells to adhere to the plate. Cells were then treated with polymer dissolved in complete media (DMEM with 10% FBS supplemented with 1% penicillin-streptomycin and 1% L-glutamine) for a final polymer concentration of 0.02, 0.2, or 2.0 mg / mL. Additionally, other wells of cells were treated with 100-X Triton to kill cells as a positive control or media to serve a negative control. The treated cells were again incubated at 37°C in 5% CO2 for 24 hours. The cells were then dosed with lOuL of CCK-8 solution (Dojindo) and incubated at 37°C in 5% CO2 for 3 hours. The absorbance at 450 nm of the treated cells was measured using a BioTek Synergy HTK multimode reader.g. PROTEASE DEGRADATION ASSAY

[0302] Protease K (ProK) was purchased from ThermoFisher (Cat# AM2542). Pepsin (Pep) was was purchased from ThermoFisher. Digestions with ProK were performed in lx PBS pH 7.4 buffer. Digestions with Pep were done in milliQ water at a pH of 2. All reactions were run at 3 mg / mL (ALEL)so or (ADED)5o, 10: 1 substrate: enzyme ratio, at 37 °C for 24 hours. After 24 hours, enzymes were denatured by heating the sample at 95 °C for 10 minutes.

[0303] ProK-treated solutions were filtered using a 0.22 pm PES filter and subjected to SEC / MALS / RI analysis in DPBS. The total polymer mass injected was 300 pg. Due to overlap in the Pep and polymer elution in SEC, Pep-treated samples were spin-filtered againstAttorney Docket No. 21101.0486P1a 3000 MWCO membrane to separate digested vs. intact polymer. The remaining solution in the spin filter was lyophilizedNMR in D2O.h. CRYOPROTECTION ASSAYS WITH LACTATE DEHYDROGENASE (LDH)

[0304] Type II LDH from rabbit muscle was acquired from Sigma Aldrich as an ammonium sulfate solution. Solutions of (ALEL)so and LDH were prepared with a final concentration of LDH at 5.0 units / mL and (ALEL)so at 0-1000 pg / mL. Freeze-thaw cycles were conducted using 200 pL samples which were placed on dry ice for 15 minutes to freeze, and thawed at 25°C in a water bath. LDH activity assays were conducted in triplicate on5 pL aliquots in a 96-well plate. A 200 pL solution of 63 mM NADH was prepared in PBS and a 500 pL solution of 10 mM sodium pyruvate was prepared in PBS. These solutions were added to 50mL of PBS (Park JK, et al., (2023) ACS Appl Mater Interfaces. 15(50):58092-102). 195 pL of this solution was added into each well, followed by the addition of 5 pL aliquots of freeze-thawed sample, or our unfrozen control samples. Absorbance values were measured at 0 minutes and 20 minutes. LDH activity % was calculated according to the following equation (eq 1). Ao and A20 represent absorbance values at 0 and 20 minutes, respectively.LDH Activity’ (%) = (A20 - Ao)SamPie / (A20 - AO)UFZ* 100 (eq 1)i. CRYOPROTECTION ASSAYS WITH ANTI-ENHANCED GREEN FLUORESCENT PROTEIN (AEGFP)

[0305] EGFP (contains His6 sequence, Cat. No.: 000033P) was purchased from Applied Biological Materials. Non-fat dry milk (Cat. No.: M0841) was purchased from LabScientific. For the primary antibody, a-EGFP (rabbit polyclonal) in PBS (Cat. No.:16118-T16) was purchased from SinoBiological. For the secondary7antibody, a-rabbit-IgG-H& L antibody (goat polyclonal, HRP conjugated) (Cat. No.: ab205718) was purchased from Abeam. QuantaBlu™ Fluorogenic Peroxidase Substrate Kit (Cat. No.: 15169) and Pierce™ Copper Coated High-Capacity Plates (Cat. No.: 15148) were purchased from ThermoFisher Scientific. (ALEL)so solutions were prepared in PBS at twice the final working concentration and was mixed with a-EGFP in a 1:1 ratio. Freeze-thaw cycles were conducted using 200 pL samples which were placed on dry ice for 10 minutes to freeze, and thawed at ambient temperature for 10 minutes. Solutions were stored at 4°C before use in the ELISA.Attorney Docket No. 21101.0486P1

[0306] EGFP was diluted to 250 ng / mL in PBS and immobilized onto copper-coated plates in 100 pL aliquots. The plate was incubated on a rocker at room temperature for 1 hr. The coating solution was removed, and each well was washed three times with 200 pL 0.05% PBST. (All following incubation and wash steps are performed in this manner if not otherwise specified.) Unoccupied binding sites were blocked with 5% milk in PBS with 15 min incubation. The blocking solution was removed and each well was washed. The EGFP was detected with the primary a-EGFP antibody diluted in PBS at 1:5000 with the dilution ratio relative to antibody (not to the mixture of antibody and polymer) with 1 hr incubation. The primary antibody solution was removed and each well was washed. The primary antibody was detected with the secondary antibody diluted 1: 1000 in PBS with 1 hr incubation. The secondary antibody solution was removed and each well was washed. The secondary' antibody was detected with 100 pL QuantaBlu working solution. Relative fluorescence units were acquired with on a Molecular Devices SpectraMax M2 microplate reader at 325 nm excitation wavelength and 420 nm emission wavelength. The data was plotted in Microsoft Excel.j. CRYOPROTECTION ASSAYS WITH TRASTUZUMAB (ANTI-HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR 2, (HER2))

[0307] HER2 (contains His6 sequence, Cat. No.: HE2-H5225-100 pg) was purchased from Aero Biosystems. For the primary antibody, human pharmaceutical grade trastuzumab (HERCEPTIN®. Roche) was commercially sourced from the University of Utah Pharmacy. For the secondary antibody, a-Human-IgG antibody (peroxidase conjugated, goat polyclonal) (Cat. No.: 609-1302) was purchased from Rockland. Trastuzumab was exchanged into 1 mM EDTA in PBS with Amicon® Ultra 30 kDa MWCO spin filters (Merck Millipore, Cat. No.: UFC203024) and tested for concertation with protein A280 on a NanoDrop™ 2000 spectrophotometer (147,000 Da, 225,000 M^cm’1). (ALEL)so solution in PBS with 1 mM EDTA w as combined with trastuzumab solution to yield a final antibody concentration of 21 mg / mL and (ALEL)5o 0-500 pg / mL. Freeze-thaw' cycles were conducted using 200 pL samples which were placed on dry ice for 10 minutes to freeze, and thawed at ambient temperature for 10 minutes. Solutions were stored at 4°C before use in the ELISA.

[0308] HER2 was diluted to 2 pg / mL in PBS and immobilized onto copper-coated plates in 100 pL aliquots. The plate was incubated on a rocker at room temperature for 2 hrs. The coating solution was removed, and each well was washed three times. UnoccupiedAttorney Docket No.21101.0486P1binding sites were blocked with 5% milk in PBS with 15 min incubation. The blocking solution was removed and each well was washed. HER2 was detected with trastuzumab diluted in PBS to 1 mg / mL with 1 hr incubation. The trastuzumab solution was removed and each well was washed. Trastuzumab was detected with the secondary antibody diluted 1:20000 in PBS with 1 hr incubation. The secondary antibody solution was removed and each well was washed. The secondary antibody was detected with 100 pL QuantaBlu working solution. Relative fluorescence units were acquired with on a Molecular Devices SpectraMax M2 microplate reader at 325 nm excitation wavelength and 420 nm emission wavelength. The data was plotted in Microsoft Excel.3. DISCUSSION

[0309] Fully synthetic polymers were designed that efficiently inhibit ice crystal growth. Ultra-potent, ultra-economical, antifreeze polymers composed entirely of natural amino acids (FIG. 1A) are described. FIG. 1A shows the chemical structures, cartoon depiction, and properties of the antifreeze polymers. The polypeptides inhibit ice cry stal coarsening at pg concentration, are non-toxic, and are biodegradable. Mechanistic insights toward the function of these antifreeze polymers by systematic variation of conformation, charge, and hydrophobicity were provided. Proof-of-concept experiments were demonstrated for both biomedical and food industry7applications.

[0310] Due to the toxicity and low potency of glycols, glycerol. DMSO, and polysaccharides, bio-friendly antifreezes are preferred. Nature has already addressed this challenge via evolution of proteins that protect extremophile organisms from freezing damage. Antifreeze proteins and glycoproteins (AF(G)Ps) have been identified in fish, insects, plants, bacteria, and fungi adapted to survive sub-zero temperatures (Voets, I. K. (2017) Soft Matter 13 (28), 4808-4823; Bar Dolev, M.; et al.. (2016) Annu. Rev. Biochem.85, 515-542; Devries, A. L. (1982) Comp. Biochem. Physiol. — Part A Physiol. 73 (4), 627-640; Graham, L. A.; Davies, P. (2005) Science (80-. ) 310 (5747), 461-461; DeVries, A. L.; Wohlschlag, (1969) Science (80-. ) 163 (3871), 1073-1075; Scholander, P. F.; et al., (1957) J. Cell. Comp. Physiol. 49 (1), 5-24; Gordon, M. S.; et al, (1962) Biol. Bull. 122 (1), 52-62; Duman, J. G.; Olsen, T. M. (1993) Cryobiology 30 (3), 322-328; Hashim, N. H. F.; et al., (2013) Extremophiles 17 (1), 63-73). Despite residing in ca. 55 M liquid water, AF(G)Ps bind specifically to water ordered in solid ice. Binding of the proteins to embry onic ice crystals modifies crystal shape, restricts growth, and lowers solution freezing points, thusAttorney Docket No. 21101.0486P1preventing tissue damage (Meister, K.; et al., (2018) J. Am. Chem. Soc. 140 (30), 9365-9368; Berger, T.; et al., (2019) J. Am. Chem. Soc. 141 (48), 19144-19150; DeVries, A. L.; et al., (1970) J. Biol. Chem. 245 (11), 2901-2908; DeVries, A. L. (1971) Science 172 (3988), 1152- 1155). Remarkably, AF(G)Ps exhibit such activities at only pg concentrations (Eskandari, A.; et al.. (2020) Biomolecules 10 (12), l-18Voets. I. K. 2017 Soft Matter. 13 (28), 4808-4823; Bojic, S.; et al., 2021 BMC Biology p 56; Brockbank, K. G. M.; et al., 2011 Vitr. Cell. Dev. Biol. - Anim. 47 (3), 210-217; Meldolesi, A. GM 2009 Nat. Biotechnol. 27 (8), 682-682).

[0311] AF(G)Ps evolved in a variety of sequences and structures, including globular, a-helical. (3-helical, and glycosylated PPII (polyproline type II) helical (Eskandari, A.; et al., (2020) Biomolecules 10 (12); Olijve, L. L. C.; et aL, (2016) Proc. Natl. Acad. Sci. 113 (14), 3740-3745). a-Helical type 1 antifreeze proteins (AFP1) and AFGPs are both relatively simple, consisting of alanine-rich sequences. AFPls are 3.3-4.5 kDa with hydrophobic residues (A, L) comprising ca. 70% of the sequence with the balance being neutral / polar (T, S, N) or charged (D, E, K, R) residues (Sicheri, F.; Yang, D. S. C. (1995) Nature 375 (6530), 427-431; Haymet, A. D. J.; et al., (1999) J. Am. Chem. Soc. 121 (5), 941-948;Mahatabuddin, S.; et al., (2017) Sci. Rep. 7). PPII helical AFGPs have a similar Ala content with the balance being mainly glycosylated Thr (DeVries, A. L; et al., (1970) J. Biol. Chem.245 (11). 2901-2908; Chen, L.; et al., (1997) Proc. Natl. Acad. Sci. U. S. A. 94 (8), 3817-3822). Broadly, the mechanism of action for both is generally accepted to be an adsorptioninhibition model, where proteins adsorb to the ice surface and impede grow th by inhibiting approach of liquid water (Raymond, J. A.; DeVries, A. L. (1977) Proc. Natl. Acad. Sci. U. S. A. 74 (6). 2589-2593; Liu. K.; et al.. (2016) Proc. Natl. Acad. Sci. U. S. A. 113 (51), 14739- 14744; Ben, R. N. (2001) ChemBioChem 2 (3), 161-166). However, molecular details of binding events have been elusive. A variety of competing data and theories have been proposed for the roles of specific residues (Meister, K.; et al., (2018) J. Am. Chem. Soc. 140 (30), 9365-9368; Berger, T.; et al., (2019) J. Am. Chem. Soc. 141 (48), 19144-19150;Sicheri, F.; Yang, D. S. C. (1995) Nature 375 (6530), 427-431; Raymond, J. A.; DeVries, A. L. (1977) Proc. Natl. Acad. Sci. U. S. A. 74 (6), 2589-2593; Tsuda, S.; et al., (2020) Biomolecules 10 (3), 423; Pandey, P.; et al., (2019) Phys. Chem. Chem. Phys. 21 ('!'), 3903- 3917; Tomczak, M. M.; et al., (2003) FEBS Lett. 551 (1-3), 13-19; Chou, K. C. (1992) J. Mol. Biol. 223 (2), 509-517; Haymet, A. D. J.; et al., (1998) FEBS Lett. 430 (3). 301-306; Jorgensen, H.; et al., (1993) Protein Eng. Des. Sci. 6 (1), 19-27; Wen, D.; Laursen, R. A. (1993) J. Biol. Chem. 268 (22), 16401-16405; Ebbinghaus, S.; et al., (2010) J. Am. Chem.Attorney Docket No.21101.0486P1Soc. 132 (35), 12210-12211). Computational work shed light on the potential evolutionary drivers of the conserved Ala-content and the residues’ role in ice-binding. These data indicated that Ala methyl groups specifically nest into ice-surface cavities and, driven by the entropy of dehydration, provide a driving force for binding (Mochizuki, K.; Molinero, V. (2018) J. Am. Chem. Soc. 140 (14), 4803-4811; Jorov, A.; et al., (2664) Protein Set. 13 (6)). This model was experimentally corroborated via observation of decreasing IRI acti ity with decreasing Ala content in synthetic Ala / glyco-Thr copolypeptides (Del eray, A. C.; et al., (2024) Chem. Mater. 6 (7), 3424-3434).

[0312] Despite their desirable properties, AF(G)Ps have not found broad application because harvest from extremophile organisms is expensive and laborious. Furthermore, purification challenges typically render the products mixtures of isoforms and with potential contamination with strong allergens (Olijve, L. L. C.; et al., (2016) Proc. Natl. Acad. Sci. 113 (14), 3740-3745). Recombinant synthesis has been achieved but the purified proteins are prohibitively expensive (MyBioSource AnpAFP recombinant protein. https: / / www.mybiosource.com / recombinant-protein / antifreeze-protein / 1201572 (accessed 2024-09-19); CUSABio Recombinant Antarctomyces psychrotrophicus Antifreeze protein. https: / / www. cusabio. com / Recombinant-Protein / Recombinant-Antarctomy ces-psychrotrophicus-Antifreeze-protein-X10S-partial-12935153.html (accessed 2024-09-19); CUSABio Recombinant Pseudopleuronectes americanus Ice-structuring protein 3. https: / / www. cusabio. com / Recombinant-Protein / Recombinant-Pseudopleuronectes-americanus-Ice-structuring-protein-3— B16D-B26D-B27D-12936774.html (accessed 2024-09-19). Therefore, diverse materials have been explored as AF(G)P mimics including peptides, peptoids, polyvinylalcohol, cellulose, surfactants, quantum dots, and even inorganic materials like graphene oxide. Yet, orders of magnitude higher concentration are required for IRI activity as compared to native A(G)FPs and many of these materials require complex syntheses. Additionally, the use of non-natural building blocks with poor or unknown biodegradability is undesirable in many applications. The need remains for an antifreeze material with excellent IRI activity and biocompatibility balanced with cost economy.

[0313] To fill this gap, our lab and another recently explored / V-carboxyanhydride (NCA)-derived synthetic copolymers of Ala and glycosylated Thr, glycosylated Hyp, succinylated Thr, or Lys, all of which demonstrated some level of IRI activity (Deleray, A. C.; et al., (2024) Chem. Mater. 36 (7), 3424-3434; Park, J. K.; et al., (2023) ACS Appl. Mater. Interfaces 15 (50; McPartlon, T. J.; et al., (2024) Biomacromolecules 25 (6), 3325-Attorney Docket No. 21101.0486P13334; Piao. Z.; et al.. (2021) A CS Macro Lett. 10 (11), 1436-1442; Piao, Z; et al., (2021) Biomacromolecides; Park, S.; et al., (2022) A CS Appl. Polym. Mater. 4 (4), 2896-2907). However, these structures require multi-step syntheses with protecting group manipulations, which is a barrier to scalability. Some also relied on post- polymerization modification chemistry with incomplete functionalization. Further, it is well known that polymers and peptides with a high density of cationic groups, such as Lys, are cytotoxic due to disruption of the cellular plasma membrane (Weiss, A. M.; et al., (2023 Macromolecules 56 (18), 7286-7299; Lv, H.; et al., (2006) J. Control. Release 114 (1), 100-109; Reineke, T. M.; et al., (2012) In Polymer Science: A Comprehensive Reference, 10 Volume Set,' 2012; Vol. 9, pp 497-527). Considering the established role of hydrophobic Ala in AF(G)Ps and these mimics, we sought to conserve this design feature while pursuing our goal of economical, biodegradable, non-toxic antifreeze materials.

[0314] Poly Ala is not water soluble and therefore is not an antifreeze candidate. Hydrophilic groups are required to solubilize Ala-rich structures and might also affect ordering of liquid water approaching the ice surface or affect inter- or intra-chain interactions. Glu is particularly attractive as the hydrophilic component since polyGlu is biocompatible, biodegradable, non-immunogenic, and has been extensively applied in biomedicine (Zhang, Y.; et al.. (2022) Biomolecules 12 (5), 636). It is notable that Glu and Ala are extremely economical building blocks with human-consumption grade material available for purchase on the bulk public market (i.e. Amazon) for ca. $5 / kg. By comparison, recombinant AFPs are commercially available for $2000-$5000 / mg. AFPs sourced from fish blood are less expensive at ca. $400 / g, but are known to contain impurities (Olijve, L. L. C.; et al., (2016) Proc. Natl. Acad. Sci. 113 (14), 3740-3745). Finally, a recent report noted preparation of polyGlu viaNCA polymerization, which is a rapid and scalable technique used commercially, without the need for protecting groups (Tian, Z. Y.; et al., (2021) Nat.Commun. 12 (1), 5810; Campos-Garcia, et al., (2017) Sci. Rep. 7 (1), 1-12; Deming, T. J. (2000) J. Polym. Sci. Part A Polym. Chem. 38 (17), 3011-3018; Cheng, J.; Deming, T. J. (2012) Top. Curr. Chem..

[0315] Two NCA polymerization methods were explored to obtain statistical copolypeptides (FIG. IB): Method A) copolymerization of tert-butyl (tBu) protected L-Glu (EL) NCA with L-Ala (AL) NCA using (PMesfi-iCo initiator in anhydrous tetrahydrofuran (THF) under inert atmosphere; and Method B) protecting-group-free open air copolymerization of ELand ALNCAs using hexylamine initiator in 1: 1Attorney Docket No. 21101.0486P1water: dimethylformamide (DMF). In Method A), the cobalt initiator is highly sensitive to oxygen, moisture, and labile protons but offers fast initiation kinetics with suppression of side reactions, enabling controlled and living polymerizations and yielding high molecular weight (MW) polymers of low dispersity (Deming, T. J. (2000) J. Polym. Sci. Part A Polym. Chem.38 (17), 3011-3018; Deming, T. J. (1997) Nature 390 (6658), 386-389). In Method B), primary amine initiators typically result in chains of higher dispersity and limited chain length, but are potentially more tolerant to oxygen, moisture, and labile protons (i.e. free acid on Glu)(Tian, Z. Y.; et al., (2021) Nat. Commun. 12 (1), 5810; Yali Hu, et al., (2022) Natl. Sci. Rev. 9). Though the tBu group is readily removed by brief treatment with trifluoroacetic acid (TFA), avoidance of protecting groups lowers the cost of starting materials, saves researcher time, and is greener overall. Both methods were explored to access a variety of chain lengths for the materials since antifreeze activity is known to increase with MW in native and synthetic AFGPs (Deleray, A. C.; et al., (2024) Chem. Mater, 36 (7), 3424-3434; Urbanczyk, M.; et al., (2017) Amino Acids 49 (2), 209-222;; McPartlon, T. J.; et al., (2024) Biomacromolecules 25 (6), 3325-3334; Ekpo, M. D.; et al., (2022) Int. J. Mol. Sci. 23 (5), 2639).

[0316] A1', E1', and tBu-E1' NCAs were prepared in ca. 90% yield by treatment of the corresponding amino acids with either triphosgene (or phosgene solution) in anhydrous THF, as previously reported (Tian, Z. Y.; et al., (2021) Nat. Commun. 12 (1), 5810: Kohout, V. R.: et al., (2023) J. Am. Chem. Soc. 145 (30), 16573-16583; Kramer, J. R.; Deming, T. J. (2010) Biomacromolecules 11 (12), 3668-3672; Tian, Z.; et al., (2021) Nat. Commun. 12, 5810). For tBu-ELand ELNCAs, acid scavenger epichlorohydrin was utilized in the reaction.Conveniently, NCAs were amenable to crystallization and preparation on multi-gram scale. NCAs were combined, and polymerization initiated by either hexylamine or (PMes^Co (vide supra). Based on the Ala content of natural winter flounder AFP1 (wfAFPl) and notothenoid AFGPs, a ratio of 3: 1 AL: ELwas chosen. Additionally, polypeptides with varying ratios of ALto glycosylated Thr were previously explored and found that reduction to 50% ALeliminated all antifreeze activity but polypeptides with >80% Ala were not soluble in water (Deleray, A. C.; et al., (2024) Chem. Mater, 36 ), 3424-3434).

[0317] Polymerization reaction progress w as monitored by attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR). Reaction times were rapid (i.e. <60 min.) for both methodologies. In all cases, complete monomer consumption was evidenced by disappearance ofNCA carbonyl stretches at 1856 and 1779 cm"1andAttorney Docket No. 21101.0486P1emergence of peptide carbonyl stretches at 1656 and 1546 cm4. Polymers containing tBu-ELwere deprotected by treatment with TFA for 30 minutes. All copolypeptides were converted to the sodium salts by treatment with aqueous sodium bicarbonate, purified by spin filtration, and lyophilized. (ALo.75-^ta / -ELo.25)n copolypeptides, abbreviated hereafter as (ALEL)n, were characterized by ATR-FTIR,1H NMR, and size exclusion chromatography coupled to multiangle light scattering and refractive index (SEC / MALS / RI) in Dulbecco’s phosphate buffered saline (DPBS). Materials prepared using both polymerization methods were spectroscopically identical and had unimodal distributions (FIG. 2A, FIG. 2B, FIG. 5A-D, FIG. 6A-D, FIG19A, and FIG. 19B). Representative polymer characterization data can be found in Table 1TABLE 1.Polypeptide Abbrev. Mn|a|M„|b|DPM 0[<i] Initiator(L-GlU025-. Sta / - (ALEL)30 2.925 3,791 40 1.10 (PMe3)4Co[e]L-Alao.75)30(L-GlU0.25-5'tot- (ALEL)50 4,635 4,498 48 1.11 (PMe3)4Co[eIL-Alao.75)50(L-Gluo25-.sta / - (ALEL)IOO 8,910 6,524 72 1.16 (PMe3)4Co[e]L-Alao.75)ioo(L-Gluo25-.staf- (ALEL)150 13,185 13,180 150 1.14 (PMes)4Co[elL-Alao.75)i5o(L-Lyso25-5tof- (ALKL)50 4,623 (PMe3)4Co[elL-Alao.75)5o(L-Gluo25-.sta / - (ALEL)50 '4,376 Hexylamine111L-Alao.75)50Representative polymerization data for preparation of Ala-rich statistical copolypeptides.[a]Theoretical number average molecular weight, Mn. Observed Mnas determined by SEC / MALS / RI in DPBS|c|Observed degree of polymerization, DP.,dlPolymer dispersity, D, as determined by SEC / MALS / RI. [e] (PMe3)4Co[6]initiator reactions were conducted using tBu-ELand ALin anhydrous THF under N2, 20°C.[f|Hexylamine initiator reactions were conducted using ELand ALin 1:1 DMF: water in open atmosphere 20°C.Attorney Docket No. 21101.0486P1

[0318] Referring to FIG. 5A-D. ATR-FT1R of representative reactions are shown. FIG. 5A shows ATR-FTIR indicating the disappearance of / Bu-E1NCA and the formation of (ALtBu-EL)50. FIG.5B shows ATR-FTIR indicating the disappearance of the tBu group (ALtBu-EL)so and the formation of (ALEL)5o. FIG. 5C shows ATR-FTIR indicating the disappearance of Z-KLNCA and the formation of (ALZKL)5o. FIG. 5D shows ATR-FTIR indicating the disappearance of the Z protecting group from (ALZ-KL)so and the formation of (ALKL)50.

[0319] Referring to FIG. 6A-D, NMR spectra of representative antifreeze polymers are shown. FIG. 6A shows (ALEL)so 'H-NMR in D2O. FIG. 6B shows (VLEL)so 'H-NMR in D2O. FIG. 6C shows (GLEL)5O 'H-NMR in D2O. FIG. 6D shows (AD / LED / L)5o 'H-NMR in D2O.

[0320] Referring to FIG19A and FIG. 19B, representative SEC / MALS traces DPBS indicating unimodal distribution is shown. FIG19A shows SEC / MALS traces DPBS for (ALEL)5o made by polymerization method A. FIG19B shows SEC / MALS traces DPBS for (ALEL)50made by polymerization method B.

[0321] Despite the need for the tBu protecting group, anhydrous solvent, and inert atmosphere, the cobalt initiator was found advantageous in that it could readily tune copolypeptide degree of polymerization (DP) to 200+ amino acids by variation of the monomerinitiator ([M]:[I]), and polymers had superbly low dispersities (D). Higher molecular weights were not explored since it was found that copolypeptides with DP>200 had reduced aqueous solubility. Since IRI activity increases with molecular weight in native AFGPs, and previously reported glycopolymers (Deleray. A. C.; et al., (2024) Chem. Mater, 36 (7), 3424-3434; Urbahczyk, M.; et al., (2011) Amino Acids 49 (2), 209-222;; McPartlon, T. J.; et al., (2024) Biomacromolecules 25 (6), 3325-3334; Ekpo, M. D.; et al., (2022) Int. J. Mol. Sci. 23 (5), 2639), were prepared (ALEL)nfrom 30-200 residues. With amine-initiators, reproducibly copolypeptides of ca. 50 residues could be prepared with an [M]:[I] of 50:1, but DP did not increase linearly with higher ratios. However, the method is still attractive since it can be conducted in open air, in mixed aqueous solvent, and without protecting groups. Thus, synthesis could be streamlined and the cost of building blocks is reduced. Overall, the NCA method offers a rapid, scalable, economical, and convenient route to synthesize antifreeze polymers free of potential contamination with strong allergens.

[0322] Besides chain length, additional structure-function information was sought to optimize and rationalize activity. Therefore, variation of structural elements was exploredAttorney Docket No. 21101.0486P1including hydrophobic residue identity, conformation, chirality, and charge. To this end, D-Ala (AD) and D-Glu (ED) NCAs were prepared and polymerized to probe activity of mirror image (ADED)so or racemic (AL / DEL / D)5o. These structures possess identical hydrophobicity and charge as (ALEL)so but should form a-helices of opposite handedness for the former variation or disordered conformations for the latter. To probe the effect of charge, N-&-carbobenzyloxy-L-lysine (Z-K) NCA with ALNCA were prepared and polymerized, which after removal of the Z-group with HBr, yielded cationic (ALKL)so. Finally, using the previously described NCA chemistry', copolypeptide hydrophobicity' was altered by substituting the ALresidues for Vai, Leu, or Gly to prepare (VLEL)so, (LLEL)SO, or (GLEL)so. (VLEL)SO was soluble in water, however, it was found that (LLEL)5O and (GLEL)5o had very poor aqueous solubility and therefore were not advanced as antifreeze candidates.a. CHARACTERIZATION OF AFP CONFORMATION

[0323] ATR-FTIR and circular dichroism spectroscopy (CD) were utilized to examine the secondary structures of our antifreeze candidates. Both techniques utilize the energy and intensity of light absorption by peptide bonds to reveal characteristics about bond orientation. ATR-FTIR spectra were obtained as thin films cast from water while CD spectra were obtained in PBS. Both methodologies clearly indicate (ALEL)nadopts a classic, right-handed a-helix (RHa-heli\). As shown in FIG. 2A and FIG. 2B, amide I and II (C=O stretching and N-H bending) peak frequencies were observed between 1640-1660 cm’1and 1540-1550 cm’1, which are characteristic of peptides in helical or disordered conformations (Byler, D. M.; et al., (1986) Biopolymers 25, 469-487; Jackson, M.; et al., (1995) Crit. Rev. Biochem. Mol. Biol. 30, 95-120; Murphy, B.; et al., (2014) Curr. Pharm. Biotechnol. 15 (9), 880-889). Amide q^jr* and n ^n* transitions observed by CD also indicated a classicRHa-helical conformation (FIG. 2D) (van Stokkum, I. H.; et al., (1990) Anal. Biochem. 191 (1), 110-118; Provencher, S. W.; et al., (1981) Biochemistry 20 (1), 33-37; Chemes, L. B.; et al., (2012) Methods Mol. Biol. 2012, 895, 387-404; Lopes, J. L. S.; et al., (2014) Protein Sci. 23 (12), 1765-1772). Similar to other polypeptide materials, helical stability increased with chain length (FIG. 7)(Detwiler, R. E.; et al.. (2021) J. Am. Chem. Soc. 143 (30), 11482-11489; Kramer, J. R.; et al., (2015) Proc. Natl. Acad. Sci. 112 (41). 12574-12579). FIG. 7 shows CD spectra of (ALEL)nin PBS at 500 pg / mL (n = 50, 100, 150, and 200). The stability' of theRHa-helix increased at low temperature as evidenced by the increased amide absorptions at 208 and 222 nm at 4°C as compared to 20°C.Attorney Docket No. 21101.0486P1

[0324] Charge had little effect on conformation andRHa-helices were observed for both (ALEL)5O and (ALKL)so (FIG. 2D). These data concur with a previous report noting a-helical conformation for statistical copolymers of Ala / Lys (Piao, Z.; et al., (2021) ACS Macro Lett. 70 (11), 1436-1442). As a benchmark, native winter flounder (wf) AFP1 which, as expected, exhibitedRIIa-helical character was examined. By contrast, the spectra of (VLEL)so was characteristic of disordered proteins (Chemes, L. B.; et al., (2012) Methods Mol. Biol. 895, 387-404; Miles, A. J.; et al., (2023) Commun. Biol. 6 (1), 823). This aligns with prior data ranking Vai as possessing lower helical propensity as Ala (Gregoret, L. M.; et al., (1998) Fold. Des. 3 (2), 119-126; Pace. C. N.; et al., (1998) Biophys. J. 75 (1), 422-427; Raman, S. S.; et al., (2008) J. Phys. Chem. B 112 (30), 9100-9104). Due to the equal but opposite rotation of light, the spectrum of mirror image (ADED)5o was a perfect mirror image of (ALEL)50's spectrum indicating a left-handed a-helix (LHa-helix) (FIG. 2E). The spectrum of racemic (AD / LED L)5o showed no absorbance minima or maxima since the equal mixtures of D and L stereochemistry amino acids inhibit formation of secondary structures.

[0325] Many coating, food, and biomedical applications in which antifreezes could be utilized require heat processing for sterilization, annealing, or mixing. Therefore, the stabi 1 i ty of the (A' E' lsoRHa-helix to heat denaturation was investigated. The CD spectrum of (ALEL)5o in PBS buffer was obtained at 4°C. The sample was then subjected to heating at 95°C, and then re-cooled to 4°C with examination by CD at each step (FIG. 2F). Without wishing to be bound by theory, since the spectra before and after heating were essentially identical, it was conclude that the conformation of (ALEL)so is thermally stable and this material is therefore attractive for applications requiring a thermal processing step. By contrast, some classes of natural antifreeze proteins are known to either fully or partially denature with heat, resulting in loss of antifreeze activity (Ma, Q.; et al., (2022) Biosci. Biotechnol. Biochem. 86 (5), 635-645). Indeed, partial denaturation was seen for wfAFPl subjected to the same conditions (FIG. 8). FIG. 8 shows variable temperature CD spectra of wfAFPl in PBS at 500 pg / mL. The sample was exposed to the following temperature series: 4°C 95°C 4°C.

[0326] Referring to FIG. 2A-F, data illustrating polypeptide structural characterization is shown. FIG. 2A shows FTIR traces of tBu-ELNC A as compared to (ALEL)5O. FIG.2B shows FTIR traces of E1,NCA as compared to (ALEL)5o. FIG. 2C-E show CD spectra of polypeptides in PBS buffer at 20°C. Data in FIG. 2C indicates length dependence helical propensity of (ALEL)n. Data in FIG. 2D reveals that (ALEL)so and (ALKL)50 adopt right-handed a-helices similar to that of winter flounder AFP1 but (VLEL)so isAttorney Docket No. 21101.0486P1disordered. Data in FIG.2E indicates that mirror-image (ADED)5o adopts a left-handed a-helix while racemic (AL / DEL / D)so is disordered. FIG. 2F shows CD spectra in PBS buffer of (ALEL)50at 4°C, 95°C, and at 4°C after heating demonstrating that the structure is thermally stable and, by comparison to 20°C that helical order increases slightly as the solution approaches freezing.b. ICE-BINDING AND ANTIFREEZE ACTIVITY ASSAYS

[0327] In contrast to vitrification agents like DMSO and glycols, natural AF(G)Ps exert their activity by binding to specific faces of embryonic ice crystals, influencing their overall shape and size. Unique characteristics of various AF(G)Ps, as well as techniques to measure antifreeze activity, have been reported and recently reviewed (Baskaran, A.: et al., (2021) Int. J. Biol. Macromol. 189, 292-305; Gharib. G.; et al., (2022) Front. Bioeng.Biotechnol. 9, 770588; Delesky, E. A.; et al., (2022) iScience 25 (5), 104286). Three properties are characteristic of ice-binding proteins: 1) IRI, where ice recrystallization via Ostwald ripening is limited, resulting in a reduction in crystal mean grain size (MGS); 2) shaping of single crystals into characteristic morphologies; and 3) thermal hysteresis (TEI) effects, which involve a non-colligative freezing point depression and gap between solution freezing and melting points. The copolypeptide panel were investigated for these characteristic properties.

[0328] IRI activity assays were conducted via the cooling splat assay and observation of crystal growth over time using cryostage microscopy (Deleray, A. C.; et al., (2024) Chem. Mater, 36 (7), 3424-3434; Knight, C. A.; et al., (1984) Nature 308 (5956), 295-296; Biggs, C. I.; et al., (2019) Macromol. Biosci. 19 (7), 1900082). The controls were PBS, DMSO, and wfAFPl. Ice crystal MGS was quantified using image analysis software or manual sizing in cases where crystals were too small for software recognition. Data were obtained from 3 separate images, using 150 pm x 150 pm regions with a minimum of 75 crystals analyzed, which was determined to yield statistically significant representative data (Deleray, A. C.; et al., (2024) Chem. Mater, 36 (7), 3424-3434). The average and standard deviation are presented for each experiment and statistical significance was determined with a one-way ANOVA and post-hoc Tukey test. Representative IRI images are shown in FIG. 3A-E. Quantified data are presented in FIG. 3F-H and FIG. 15.

[0329] The IRI activity of (ALEL)nwas examined at varied chain lengths from 30-200 residues and at varied concentrations from 50 pg / mL-5 mg / mL. Data for the 50mer andAttorney Docket No. 21101.0486P1200mer are shown in FIG. 3C, FIG. 3F, and FIG. 3G. Data for other lengths is shown in FIG. 9. FIG. 9 shows ice recrystallization inhibition assay for (ALEL)nwhere images show¬ crystal size at 0 minutes and 40 minutes of crystal growth. Polymer concentrations are 5 mg / mL in PBS. Scale bar is 200 gm. Near total IRI w as observed at concentrations of 100 gg / mL and higher for copolypeptides with 50 or more residues, and essentially no enlargement of ice crystals was observed over the 40-minute observation period. Compared to the PBS control, copolypeptides with 50+ residues reduced ice crystal MGS by 91-94% at 100 gg / mL (FIG. 3F and FIG. 3G). By comparison, DMSO at 1000-fold higher concentration (100 mg / mL) resulted in only a 45% reduction. Lower molecular weight 30mer chains were slightly less potent, offering an 83% reduction in MGS at 100 gg / mL (FIG. 11A and FIG. 11B). FIG. 11A shows ice recrystallization inhibition assay for (ALEL)3o at 100 gg / mL in PBS at 0 minutes and 40 minutes of crystal growth (scale bar is 200 gm). FIG. 11B shows Quantified IRI of (ALEL)3o at 100 gg / mL in PBS after 40 minutes of crystal growth. Standard deviation (** indicates p < 0.01). At 50 gg / mL, reduced IRI activity (60-62% MGS reduction) was observed for both the 50mer and 200mer. IRI activity w as not detectable by 10 gg / mL (FIG. 12). FIG. 12 shows ice recry stallization inhibition assay for (AI'KL)5O at varying concentrations in PBS, after 0 minutes and 40 minutes of crystal growth. Scale bar is 200 gm. That the longer chain did not have higher activity was notable since this was observed in our previously reported Ala-rich glycopolymers and has been reported for native AFGPs (Deleray, A. C.; et al., (2024) Chem. Mater, 36 (7), 3424-3434; McPartlon, T. J.; et al., (2024) Biomacromolecules 25 (6), 3325-3334). Without wishing to be bound bytheory, it was speculated that ice-binding mechanisms, including entropic effects, differ between these materials. Ultimately, the lack of dependence was found to be an advantageous result since the cleaner, greener, protecting-group-free method could be used to reproducibly prepare chains of 50 residues.

[0330] FIG. 13A-C show ice recry stallization inhibition assay and dynamic ice shaping assay for (ALEL)5o prepared by Method A and Method B. FIG. 13A shows ice recrystallization inhibition assay for Method A, cobalt complex-initiated, (ALEL)5o dissolved at varying concentrations in PBS, at 0 minutes and 40 minutes of crystal growth. Scale bar is 200 pm. FIG. 13B shows ice recry stallization inhibition assay for Method B, hexylamine-initiated, (ALEL)5o at varied concentrations in PBS at 0 minutes and 40 minutes of crystal growth. Scale bar is 200 gm. FIG. 13C shows dynamic ice shaping assay for (ALEL)so at 500Attorney Docket No. 21101.0486P1pg / mL in PBS, derived from polymerization Method A or B. Structures bind and shape ice in an equivalent fashion. Scale bars are 100 pm.

[0331] The IRI properties of the copolypeptide panel facilitate insights into structurefunction relationships in these materials, where conformation and structural order appear to play an essential role. IRI activity was completely abrogated with the conformationally-disordered (ADLED / L)5o, even at 10x the concentration at which (ALEL)so achieves total IRI (FIG. 3E, and FIG. 3H). Racemic (AD / LED / L)5o is composed of the exact chemical moieties as the highly activeRIIa-helical (ALEL)5o but cannot form an ordered helix due to the mixture of stereocenters. Interestingly, enantiomeric (ADED)5o, which adopts a mirror imageLHa-helix, had essentially identical IRI activity to that of (ALEL)5o (FIG. 3H and FIG. 14). These results align with prior work where solid phase peptide synthesis was used to prepare enantiomeric wfAFPl, which had equal activity of that of the L-conformer (Wen, D.; et al., (1993) FEBS Lett. 317 (1-2)). There was also no statistical significance between the activity of anionic (ALEL)5o and cationic (ALKL)so, both of which adopt aR[Ia-helix. Finally, the Ala-to-Val substitution in (VLEL)so, whose conformation it was determined is disordered, resulted in a ca. 50% loss of IRI activity (FIG. 17). FIG. 16 shows ice recrystallization inhibition assay for racemic (AD / I'ED / I')5o at 1000 pg / mL in PBS at 0 minutes and 40 minutes of crystal growth (Scale bar is 200 pm). FIG. 17 shows ice recrystallization inhibition assay for (VLEL)5o at 1000 pg / mL in PBS at 0 minutes and 40 minutes of crystal growth (Scale bar is 200 pm). FIG. 18 shows ice recrystallization inhibition assay for wfAFPl after heating to 95 °C for 10 minutes. Sample is 100 pg / mL in PBS and images are after 0 minutes and 40 minutes of crystal growth (Scale bar is 200 pm).

[0332] From these collective IRI data, it was concluded that ordered presentation of Ala methyl groups from the helical peptide backbone is required for function, that both cationic and anionic hydrophilic groups are tolerated, and that natural L-stereochemistry is not required. It was also conclude that, since these statistical copolypeptides are inherently composed of a randomized mixture of sequences, no specific sequence is required for icebinding to occur. These data concur with a previous report noting a lack of IRI activity for a statistical copolymer of Ala / Asp, which adopted a disordered conformation (Piao, Z.; et al., (2021) ACS Macro Lett. 10 (11). 1436-1442). Additionally, these data align with prior works from our lab on Ala-rich glycopolypeptides. These also had no specific sequence, but rather were statistical mixtures of Ala and glycosylated Thr or Hyp (Deleray, A. C.; et al., (2024)Attorney Docket No.21101.0486P1Chem. Mater, 36 (7). 3424-3434; McPartlon, T. J.; et al.. (2024) Biomacromolecules 25 (6).3325-3334).

[0333] The ability of our IRI-active structures to shape ice crystals was explored, which is a classic feature of ice-binding proteins. Dynamic ice shaping experiments, which involve isolation of single crystals, were performed on a cryostage microscope. For solutions of (ALEL)50in PBS, hexagonal crystals were observed identical to those formed in the presence of native Ala-rich wfAFPl and AFGPs that bind to the primary and secondary prism planes of ice (FIG. 3I)(Meister, K.; et al., (2018) J. Am. Chem. Soc. 140 (30), 9365-9368; Olijve, L. L. C.; et al., (2016) Proc. Natl. Acad. Set. 113 (14), 3740-3745; Budke, C.; et al., (2014) Cryst. Growth Des. 14 (9), 4285-4294; Knight, C. A.; et al., (1991) Biophys. J. 59 (2), 409-418; Knight, C. A.; et al., (1993) Biophys. J. 64 (1), 252-259). Since the copolypeptides are also Ala-rich, it was presumed (ALEL)5o also binds to the prism planes of ice. By contrast, ice crystals formed from PBS solution lacking antifreeze agent were round and amorphous.

[0334] Aside from interaction with ordered water molecules on the ice surface, the copolypeptide can interact with liquid water and dissolved ions. It was considered that charged groups within the copolypeptide could form ionic bonds with charged buffer components, which might in turn affect rate of approach of liquid w ater to the ice surface or affect binding of the copolypeptide to ice. Therefore, ice shaping properties of anionic (ALEL)5O VS. cationic (ALKL)so w as examined. PBS, w hich contains anionic phosphate groups, or tris(hydroxymethyl)aminomethane (TBS), which contains cationic amine groups w ere utilized. In all cases a standard concentration of 0.1M NaCl w as used. For both (ALEL)5o and (ALKL)50, copolypeptide-buffer pairs of opposite charge resulted in predominantly hexagonal and bipyramidal crystal morphologies (FIG. 3K and FIG. 3L). Curiously, the like-charge buffer-copolypeptide pairs had differing results. Cationic (ALKL)so with cationic TBS resulted in a reduction of the ice-shaping effects observed in anionic PBS, and mainly amorphous crystals were observed (FIG. 3L and FIG. 3M). In contrast, ice shaping abilities of anionic (ALEL)5O were not strongly affected by buffer. In both PBS and TBS, crystals were predominantly hexagonal and bipyramidal (FIG. 3J and FIG. 3K). For FIG.3J-M, wide view imaging was selected to show many crystals within the same field and allow observation of trends. It is noted that due to slight differences in nucleation time, crystals are captured in varied stages of growth. Therefore, it is not expected that they be of uniform shape and size.Attorney Docket No. 21101.0486P1

[0335] Referring to FIG. 3A-M, Antifreeze activity assays are shown. FIG. 3A-E show representative IRI splat assay imaging after 40 minutes of crystal growth for FIG. 3A negative control, PBS; FIG.3B positive control, wfAFPl, 100 pg / mL; FIG.3C (ALEL)5O,1OO pg / mL; FIG. 3D (VLEL)so,l mg / mL; FIG. 3E (AD / LED / L)5o, 1 mg / mL. Scale bars are 200 pm.FIG. F-H show quantified IRI assay data as % MGS relative to PBS for FIG. 3F (ALEL)so;FIG. 3G (ALEL)2OO at varied concentrations; FIG.3H (AD / LED / L)5o, (ALEL)so, (ADED)so, (ALKL)50, and (VLEL)so. Standard deviation; ** indicates p < 0.01. and I) side-by-side comparison of to various polypeptide compositions and controls; mean and standard deviation, ** indicates p < 0.01. FIG.3I-M show dynamic ice shaping experiments comparing anionic (ALEL)5o to (ALKL)so at 0.5 mg / mL in anionic PBS buffer vs. cationic TBS buffer. Scale bars are 100 pm. Referring to FIG. 14, ice recrystallization inhibition assay for (ADED)50at varied concentrations in PBS at 0 minutes and 40 minutes of crystal growth (Scale bar is 200 pm) is shown. Referring to FIG. 15. Quantified IRI of (ALEL)nat 5 mg / mL in PBS after 40 minutes of crystal growth (Standard deviation; ** indicates p < 0.01) is shown.

[0336] Without wishing to be bound by theory, one possible explanation for the difference in the like-pair charge systems could be increased stability of intermolecular interactions for (ALKL)so. The Lys sidechain has two additional methylene groups as compared to Glu, and thus increased side chain hydrophobicity. This in turn affects hydration sphere, intrachain packing of amino acid side chains along the helix, and potentially interhelix clustering. In TBS, (ALKL)5o might have stronger inter-chain interactions than (ALEL)so in PBS, thus reducing its solution concentration and ability to bind to the prism plane of ice. Alternatively, cooperative interactions between ions, copolypeptides, and the ice surface could be at play. In any case, we conclude that (ALEL)so is an attractive antifreeze agent in that it maintains its ice-binding capability in different buffers, which increases its potential applications.

[0337] Finally, TH assays were conducted to determine the freezing and melting points of (ALEL)50solutions but no activity was observed. The high IRI activity but lack of TH in the copolypeptide is a reasonable finding since some plant AFPs, as well as synthetic materials like polyvinylalcohol, also have undetectable TH but robust IRI activity (Biggs, C. L; et al., (2019) Macromol. Biosci. 19 (7). 1900082: Hassas-Roudsari, M.; Goff, H. D. (2012) Food Res. Int. 46 (1), 425-436). Ultimately, this was not consider a detriment of the materials since essentially all applications involve conditions far below the solution freezing point (i.e.,Attorney Docket No. 21101.0486P1-20-80°C). While TH activity is extremely important for creatures like polar fish residing in waters cooled to ca. -2°C, IRI activity becomes the more important property for damage prevention in coatings, foods, and biomedicine. Without wishing to be bound by theory, (ALEL)nis an attractive cryoprotectant due to the combination of high IRI activity, thermal and buffer stability, and cost / ease of synthesisc. APPLICATIONS

[0338] Antifreezes are needed to prevent damage from ice coarsening in diverse settings including building materials, transportation, agriculture, foods, and biomedicine (Mochida, K.; et al., (1987) Bull. Environ. Contain. Toxicol. 38 (1); Regulska, M.; et al., (2010) Pharmacol. Reports 62 (6)). Common cryoprotectants glycols and DMSO have well known toxic effects, as well as potential negative effects on the environment. Without wishing to be bound by theory, it was hypothesized that the antifreeze copolypeptide would be well tolerated by living tissues and would be biodegradable by natural proteases since it is based entirely on the natural amino acids Glu and Ala. To test this idea, the cytotoxicity was characterized and in vitro biodegradation of (ALEL)5o.

[0339] Cytocompatibility of ( A’E' hs was examined in a model human cell line currently in broad laboratory use (human embryonic kidney (HEK) 293 cells). For comparison, the cytotoxicity of cationic (ALKL)?5 was also quantified. CCK8 viability assays were conducted after 24 hours of treatment with copolypeptides at concentrations ranging from 20-2000 pg / mL. The high concentration in this experiment is far in excess of material needed for use antifreeze agents since IRI activity was observed at concentrations as low as 50 pg / mL and total IRI at 100 pg / mL. However, the high concentration of 2000 pg / mL was selected to emulate potential bioaccumulation scenarios where concentrations could become higher. Cells were treated with PBS as a negative control and Triton X-100 as a positive control. At the highest concentration tested, which exceeded the necessary concentration for IRI activity by 20-fold, no statistically significant effects on cellular viability were observed for anionic (ALEL)?5 (FIG. 4A). In contrast, cationic (ALKL)?5 resulted in cytotoxicity that increased with concentration. These data again highlight (ALEL)nas an attractive antifreeze agent that would mitigate the toxicity concerns of current cryogenic agents.

[0340] For applications in foods and biomedicine, it is typically desirable that additives and stabilizers biodegrade via known pathways into well-tolerated byproducts. Considering that (ALEL)5o is composed entirely of natural amino acids, it would be a substrateAttorney Docket No.21101.0486P1for natural proteases. To test this hypothesis, (ALEL)so was subjected to proteinase K (ProK) and pepsin (Pep). ProK is a broad-spectrum serine protease that preferentially cleaves peptide bonds adjacent to aliphatic and aromatic amino acids (Rawlings, N. D.; Salvesen, G. (2013) Handb. Proteolytic Enzym. 1-3), while Pep is an aspartic protease that has broad-spectrum activity with a preference for aromatic or carboxylic L-amino acids. Pep is found in the mammalian gastrointestinal system and is enzymatically most active at low pH (Ahn, J.; et al., (2013) Biochim. Biophys. Acta - Proteins Proteomics 1834 (6; Palashoff, M. (2008) Dep. Chem. Chem. Biol.). In certain applications, long material lifetimes are needed (i.e. coatings) and biodegradation is actually undesirable. Peptides based on D-amino acids are not typically protease substrates (Kohout, V. R.; et al., (2023) J. Am. Chem. Soc. 145 (30), 16573-16583). Without wishing to be bound by theory, it was hypothesized that IRI-active mirror-image (ADED)50would be degradation-resistant and therefore a good candidate for such applications.

[0341] Copolypeptides (ALEL)so and (ADED)so were incubated with Pep or ProK for 24 hours at an enzyme: substrate ratio of 1: 10. Enzymes were quenched by heat treatment and samples were analyzed by SEC / MALS / RI, gel electrophoresis, or1H NMR. Data for ProK with (ALEL)5O is show n in FIG. 4B and additional data is shown in FIG. 21, Disappearance of the unimodal (ALEL)50 SEC signal after ProK treatment clearly indicates the material is a protease substrate. By contrast, (ADED)5o was resistant to enzymatic degradation as indicated by retention of spectral signatures after the 24-hour enzyme incubation period (FIG. 21). For Pep, the SEC elution time of the enzyme had considerable overlap with that of (ALEL)5o. Therefore, enzy me-treated polymers w ere examined by NMR after separation of proteolyzed fragments from intact polymer by 3kD cutoff spin filtration. Complete disappearance of signal was again observed for the L-copolypeptide, indicating it is a digestible substrate. MW characterization by gel electrophoresis was also attempted but the low MWs of (ALEL)so and (ADED)50(ca. 4500 Da) rendered differentiation of bands of intact vs. digested material difficult. Overall, L-stereochemistry (ALEL)so is a substrate for natural proteases and should not bioaccumulate but the minor image (ADED)5o is degradation resistant and could be utilized in situations where longevity is desired.

[0342] Referring to FIG. 20A and FIG. 20B, SEC / MALS traces in DPBS for Protease K (FIG. 20A) and Pepsin (FIG. 20B) is shown.

[0343] To probe the application of (ALEL)so as a cryoprotective agent, proof-of-concept experiments were conducted in both biomedical and food applications. In biomedicine, cryogenic storage is commonly used to extend the shelflife of biologicalAttorney Docket No.21101.0486P1therapeutics including mRNA vaccines and protein-based drugs such as insulin, antibodies, and enzymes. Yet, the exposure of biomolecules to freeze / thaw cycles (F / T) can result in loss of function via mechanisms such as conformational changes, aggregate formation, adsorption to the ice-liquid interface, and pH changes due to buffer crystallization (Pabari, R. M.; et al., (2011) Pharmaceutics 3 (3); Chang. B. S.: et al., (1996) J. Pharm. Sci. 85 (12): Barnard. J. G; et al., (2011),7. Pharm. Sci. 100 (2)). Major suppliers of both research-grade and pharmaceutical-grade antibodies and enzy mes commonly recommend avoidance of F / T cycles. Despite the risk, repeated F / T cycles are often needed during drug manufacturing and storage or to improve drug loading in liposomal formulations (Zhang, W.; et al., (2015) Pharm. Res. 32 (4); Costa, A. P.; et al., (2014) Pharm. Res. 31 (1)). Further, accidental malfunction in temperature-controlled systems during cold-chain-transport or facility’ storage can lead to expensive loss of material. The application of (ALEL)5o to cryogenic storage of protein therapeutics was explored.

[0344] For model protein therapeutics, two readily-assayed antibodies were selected and one enzyme for examination of function after F / T. For antibodies, mouse polyclonal antienhanced green fluorescent protein (aEGFP) and trastuzumab (HERCEPTIN®) were chosen. aEGFP, which binds to enhanced green fluorescent protein, is an essential and widely used research laboratory tool for protein localization and visualization. Trastuzumab, which binds the human epidermal growth factor receptor 2 protein (HER2), is a lifesaving therapeutic monoclonal antibody currently in use in humans. For a model enz me, lactate dehydrogenase (LDH) was chose since this is a common blood marker of tissue damage, is known to suffer functional loss after freezing, and has commercially available activity assays.

[0345] Commercially-sourced LDH was supplied in PBS buffer and was used directly in F / T assays with or without supplementation with (ALEL)so. Enzymatic activity was obtained via colorimetric assay and data are normalized to the activity of untreated LDH never exposed to F / T. Samples were flash frozen, and thawed at ambient temperature. Data after 8 F / T cycles is shown in FIG. 4C, while data for other cycle numbers are in the SI. The activity’ of LDH with no (ALEL)so steadily declined with each F / T (see SI). By 8 F / T cycles, enzymatic activity was essentially undetectable. There was no statistical difference upon addition of (ALEL)5o below the IRI-active concentration (10 pg / mL). However, supplementation of the LDH solution with (ALEL)so at its lower limit of IRI activity, 50 pg / mL, resulted in ca. 60% retention of activity. It was found that 100 pg / mL (ALEL)so was sufficient to fully protect the enzyme from freezing damage and that activity over 8 F / TAttorney Docket No. 21101.0486P1cycles was similar to enzyme that had never been frozen. It was noted that a prior report of a polymeric F / T stabilizer for LDH was utilized at 50-fold higher concentration (50 mg / mL) than our materials (Park, J. K.; et al., (2023) ACSAppl. Mater. Interfaces 15 (50)).

[0346] To investigate antibody stability to F / T in the presence of (ALEL)so, aEGFP or trastuzumab was combined with increasing concentrations of copolypeptide. Commercial aEGFP was supplied in PBS buffer and used directly. Commercial pharmaceutical-grade trastuzumab is supplied with additives a,a-trehalose dihydrate, L-histidine HC1 monohydrate, L-histidine, and polysorbate 20. To isolate the stabilizing effects of (ALEL)5o, these additives were removed by spin fdtration against ethylenediaminetetraacetic acid in PBS buffer.Solutions were subjected to varied F / T cycles and then assayed for retention of binding functionality. The function of both antibodies was assayed by enzyme linked immunosorbent assays (ELISAs). Antibody binding targets EFGP or HER2 were attached to copper-coated plates via His6 sequences. Data were normalized to the binding levels of the antibody solutions prior to F / T and plotted as percent RFU change.

[0347] After just one F / T cycle, antibody aEGFP suffered ca. 35% loss of binding function (FIG.4D). Addition of 100 pg / mL (ALEL)so was sufficient to fully protect the antibody from freezing damage. No further effect was observed by increasing to 500 pg / mL. Surprisingly, additional F / T cycles did not further damage the function of this antibody, even up to 20 F / Ts. Trastuzumab was remarkable stable to F / T and therefore cryoprotective effects of (ALEL) so were not observed (FIG. 4E). These results were surprising since the manufacturer of trastuzumab specifically recommends against F / T (Roche. Herceptin Storage, Preparation and Administration Recommendations).

[0348] Stability of antibodies is likely dependent upon the unique structures of origin species, and isotype. No published data for stability of aEGFP was found. However, due to trastuzumab’s pharmaceutical status, changes in conformation and aggregation after F / T were investigated in multiple studies (Pabari, R. M.; et al., (2011) Pharmaceutics 2011, 3 (3); Mohamed, H. Eet al., (2018) Pharm. Biomed. Anal. 150; Gao, Y.; et al., (2023) Pharm. Dev. Techno! 28 (3-4); Dash, R.; et al., (2021) J. Pharm. Biomed. Anal. 201). In vitro binding function after F / T was also reported in one study (Dash, R.; et al., (2021) J. Pharm. Biomed. Anal. 201). Minor increase in aggregation with concomitant decrease in SEC peak was observed after 3 F / T cycles, but functionality for binding was not altered. The binding study was conducted at 1 mg / mL antibody, but the pharmaceutically relevant formulation is 21 mg / mL and aggregation events increase with concentration. Therefore, the trastuzumabAttorney Docket No. 21101.0486P1study was conducted at 21 mg / mL. As shown in Fig. 4E, a minor enhancement of binding was observed in the presence of (ALEL)so. Since a similar enhancement for LDH was seen, (ALEL)50could be acting as a non-specific inhibitor of aggregate species. In any case, it is clear that (ALEL)so does not interfere with enzymatic or binding activity in these cases, and can be used as a simple and natural stabilizer for a variety of functional proteins.

[0349] As proof-of-concept for food applications, the cryoprotective activity of (ALEL)50in a commercial frozen dairy product was explored. The formation of large ice crystals is considered undesirable in ice cream and fish AFPs have been of interest as a food additive (Meldolesi, A. GM 2009 Nat. Biotechnol. 27 (8), 682-682). However, the cost associated with recombinant or animal-sourced AFPs has been a limiting factor. Additionally, some consumers are put off by the concept of fish byproducts. (ALEL)so could offer an economical, readily digestible, and animal-free alternative. To test this, commercial ice cream was melted and supplemented with varying concentrations of (ALEL)so. Upon refreezing and conduction of IRI splat assays, we found substantial ice crystal growth in the untreated sample. However, for samples supplemented with (ALEL)so, ice crystal coarsening was strongly inhibited. Data for the lowest IRI-active concentration is shown in Fig. 4F-H, and other concentrations are shown in FIG. 10. These results concur with a prior report utilizing an Ala / Lys copolymer for IRI in ice cream (Piao, Z.; et al., (2021) ACS Macro Lett. 10 (11), 1436-1442). Neither cytotoxicity nor degradation data were reported, and concentrations utilized for IRI activity were 10-fold higher than for our materials.

[0350] Referring to FIG. 4A-G, cytocompatibility, biodegradation, and application of (ALEL)nin various freeze / thaw protection settings are shown. FIG. 4A shows HEK 293 cell viability as determined by CCK8 assay following 24-hour incubation with anionic (ALEL)?5 or cationic (ALKL)?5 at the indicated concentrations; live control is media alone while dead control is Triton X-100. FIG. 4B shows SEC / MALS trace in DPBS of (ALEL)so before and after treatment with proteinase K indicating biodegradability of (ALEL)so. FIG. 4C shows enzymatic activity of LDH after 8 F / T cycles with addition of varied concentration of (ALEL)50. FIG.4D shows Binding activity of otEGFP after 0, 1, or 20 F / T cycles with addition of varied concentration of ( ALEL)so. FIG. 4E shows binding activity of trastuzumab after 0, 1, or 20 F / T cycles with addition of varied concentration of (ALEL)50. FIG.4F shows quantified IRI activity of (ALEL)so in a frozen dairy product, as % MGS relative to the untreated control. FIG. 4G shows IRI image of control frozen dairy' product FIG. 4H shows IRI image of (ALEL)5o supplemented sample.Attorney Docket No.21101.0486P1

[0351] Development of ice binding polymers inspired by the structure of antifreeze proteins found in polar fish was reported. These polymers are based entirely on the natural amino acids alanine and glutamic acid, are non-toxic to living cells, and are digestible by natural proteases including those found in the human gastrointestinal system. The polypeptides efficiently inhibited ice crystal growth at microgram concentrations, similar to that of natural fish antifreeze proteins. Shaping of ice crystals characteristic of prism plane binding was also observed. Structural characterization indicated that helical conformation plays a role in antifreeze activity, but helical handedness does not. Mirror-image polypeptides were equally active, and offer an option for long-lived materials resistant to protease degradation. The antifreeze polymers were applied in proof-of-concept experiments to prevent freezing damage in two classes of therapeutic proteins, antibodies and enzymes. The polymers were effective to 8+ freeze-thaw cycles and did not interfere with enzyme activity or antibody binding. Ice crystal growth could also be inhibited in a model food technology application. The antifreeze polypeptides reported here are prepared from ultra-cheap materials in a relatively expedient and green manner, rendering them attractive for broad applications in biomedicine, foods, agriculture, coatings and more.4. ADDITIONAL EXEMPLARY POLYPEPTIDES

[0352] Additional polypeptides prepared are shown in Table 1.TABLE 1.Compound Structure6Cty.0(Alao.7-^-Gluo.i5-5-Thro.15)50uf / H iix 1 / OHH II 0.15 llL 0 LH3 0 / 0 15J nAttorney Docket No.21101.0486P1Compound StructureAi-10ZI '(Alao.7-s-Gluo.2o-s-Thro.io)5o / / OH0yO " nIZy y yo= — e( O 0 N—^.0, Z I(Al ao.7-^-Gluo.25-^'-Thro.50)50 [ ((H S YHJ / OH H.(NL H A Q 0-7 r CuH3 °-26VH Q II / 05nJ. REFERENCES

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Claims

Attorney Docket No. 21101.0486P1CLAIMSWhat is claimed is:

1. A peptide comprising a plurality of alanine residues and a plurality of glutamic acid residues,wherein the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%,wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%,wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 1: 1 to about 4:1, andwherein the peptide has a chain length of at least 15 amino acid residues.

2. The peptide of claim 1, wherein the peptide consists essentially of the plurality of alanine residues and the plurality of glutamic acid residues.

3. The peptide of claim 1 or claim 2, wherein the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%.

4. The peptide of any one of claims 1 to 3, wherein the plurality of alanine residues is present in the peptide in an amount of from about 60 wt% to about 75 wt%.

5. The peptide of any one of claims 1 to 4, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4:1.

6. The peptide of any one of claims 1 to 4, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is about 3:2.

7. The peptide of any one of claims 1 to 4, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is about 4:1.

8. The peptide of any one of claims 1 to 7, wherein the peptide has a chain length of at least 20 amino acid residues.Attorney Docket No.21101.0486P19. The peptide of any one of claims 1 to 7, wherein the peptide has a chain length of at least 30 amino acid residues.

10. The peptide of any one of claims 1 to 7, wherein the peptide has a chain length of at least 40 amino acid residues.

11. The peptide of any one of claims 1 to 7, wherein the peptide has a chain length of from 15 amino acid residues to 500 amino acid residues.

12. The peptide of any one of claims 1 to 7, wherein the peptide has a chain length of from 20 amino acid residues to 400 amino acid residues.

13. The peptide of any one of claims 1 to 7, wherein the peptide has a chain length of from 30 amino acid residues to 300 amino acid residues.

14. The peptide of any one of claims 1 to 7, wherein the peptide has a chain length of from about 30 amino acid residues to about 70 amino acid residues.

15. The peptide of any one of claims 1 to 7, wherein the peptide has a chain length of from about 40 amino acid residues to about 60 amino acid residues.

16. The peptide of any one of claims 1 to 15, wherein the peptide has a number average molecular weight (Mn) of at least about 1,000.

17. The peptide of any one of claims 1 to 15, wherein the peptide has a number average molecular weight (Mn) of at least about 5,000.

18. The peptide of any one of claims 1 to 15, wherein the peptide has a number average molecular weight (Mn) of at least about 10,000.

19. The peptide of any one of claims 1 to 15, wherein the peptide has a number average molecular weight (Mn) of at least about 15,000.

20. The peptide of any one of claims 1 to 15, wherein the peptide has a number average molecular weight (Mn) of from about 1,000 to about 40,00021. The peptide of any one of claims 1 to 15, wherein the peptide has a number average molecular weight (Mn) of from about 10,000 to about 30,000.Attorney Docket No. 21101.0486P122. The peptide of any one of claims 1 to 21, wherein the peptide has a degree of polymerization (DP) of at least about 20.

23. The peptide of any one of claims 1 to 21, wherein the peptide has a degree of polymerization (DP) of at least about 50.

24. The peptide of any one of claims 1 to 21, wherein the peptide has a degree of polymerization (DP) of at least about 100.

25. The peptide of claim 1, wherein the plurality of alanine residues is present in the peptide in an amount of from about 55 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acid residues is from about 3:2 to about 4: 1, and wherein the peptide has a chain length of at least 20 amino acid residues.

26. The peptide of any one of claims 1 to 25, wherein the peptide further comprises one or more residues of a hydrophilic amino acid selected from serine, threonine, arginine, histidine, lysine, and aspartic acid.

27. The peptide of any one of claims 1 to 25, wherein the peptide further comprises a plurality of threonine residues.

28. The peptide of claim 27, wherein the plurality of threonine residues is present in the peptide in an amount up to about 30 wt%.

29. The peptide of claim 27, wherein the plurality of threonine residues is present in the peptide in an amount up to about 20 wt%.

30. The peptide of claim 27, wherein the plurality of threonine residues is present in the peptide in an amount up to about 10 wt%.

31. The peptide of claim 27, wherein the plurality of alanine residues is present in the peptide in an amount of from about 50 wt% to about 80 wt%, wherein the plurality of glutamic acid residues is present in the peptide in an amount of from about 20 wt% to about 50 wt%, wherein the ratio of the plurality of alanine residues to the plurality of glutamic acidAttorney Docket No.21101.0486P1residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of from 15 amino acid residues to 500 amino acid residues.

32. The peptide of claim 1, wherein the peptide has a structure represented by a formula:wherein n is an integer selected from 15 to 500;wherein the ratio of x to y is of from about 1: 1 to about 1:4, andwherein the sum of x and y is 1,or a pharmaceutically acceptable salt thereof.

33. The peptide of claim 32, wherein the ratio of x to y is of from about 2:3 to about 1:4.

34. The peptide of claim 32, wherein x is from about 0.10 to about 0.4.

35. The peptide of claim 32, wherein x is from about 0.15 to about 0.3.

36. The peptide of claim 32, wherein x is from about 0.20 to about 0.4.

37. The peptide of claim 32, wherein x is about 0.25.

38. The peptide of any one of claims 32 to 37. wherein y is from about 0.60 to about 0.80.

39. The peptide of any one of claims 32 to 37, wherein y is about 0.75.

40. The peptide of any one of claims 32 to 37. wherein y is about 0.70.

41. The peptide of claim 32, wherein the peptide has a structure represented by a formula:nAttorney Docket No. 21101.0486P1or a pharmaceutically acceptable salt thereof.

42. The peptide of claim 32, wherein the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

43. The peptide of claim 32, wherein the peptide has a structure represented by a formula selected from:or a pharmaceutically acceptable salt thereof.

44. The peptide of claim 32, wherein the peptide has a structure represented by a formula:wherein R20is selected from -OR31, -NHR32, -Ns, a protein tag, a sortase recognition sequence, a sugar residue, and a structure:Attorney Docket No. 21101.0486P1wherein R31and R32are selected from hydrogen, -CH2PI1, C1-C8 alkyl, C2-C8 alkyne, C1-C8 azide, tetrazinyl, cyclooctynyl, norbomenyl, and -(CH2CH2O)mCH3, and wherein m is an integer selected from 1 to 100,or a pharmaceutically acceptable salt thereof.

45. The peptide of claim 44, wherein the protein tag is selected from CBP, FLAG, GST, HA, HBH, MBP, Myc, poly His, S-tag, SUMO, TAP, TRX, and V5.

46. The peptide of claim 44 or claim 45, wherein the sortase recognition sequence is selected from LPXTG (SEQ ID NO: 1), LPXTGG (SEQ ID NO: 2), LPXTGGG (SEQ ID NO: 3), and LPXTGGGG (SEQ ID NO: 4), wherein X is a natural or unnatural amino acid.

47. The peptide of claim 46, wherein the natural amino acid is selected from serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

48. The peptide of claim 46, wherein the natural amino acid is selected from glutamic acid, aspartic acid, arginine, histidine, and lysine.

49. The peptide of claim 46, wherein the unnatural amino acid is selected from hydroxyproline, beta-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, and pyroglutamic acid.

50. The peptide of any one of claims 44 to 49, wherein the sortase recognition sequence is selected from LPETG (SEQ ID NO: 5), LPETGG (SEQ ID NO: 6), LPETGGG (SEQ ID NO: 7), and LPETGGGG (SEQ ID NO: 8).

51. The peptide of any one of claims 44 to 49, wherein the sugar residue is a residue of a sugar selected from fructose, glucose, and lactose.

52. The peptide of any one of claims 44 to 49, wherein R20is selected from -OR31, -NR32, -N3, and a structure:Attorney Docket No. 21101.0486P153. The peptide of claim 44, wherein the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

54. The peptide of claim 44, wherein the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

55. The peptide of claim 44, wherein the peptide has a structure represented by a formula selected from:or a pharmaceutically acceptable salt thereof.

56. The peptide of claim 32, wherein the peptide has a structure represented by a formula:Attorney Docket No.21101.0486P1wherein z is an integer from 0 wt% to about 30 wt%,or a pharmaceutically acceptable salt thereof.

57. The peptide of claim 56, wherein z is an integer of from 1 wt% to about 30 wt%.

58. The peptide of claim 56, wherein x is from about 0.10 to about 0.4.

59. The peptide of claim 56, wherein x is from about 0.15 to about 0.3.

60. The peptide of any one of claims 56 to 59, wherein y is from about 0.60 to about 0.80.

61. The peptide of any one of claims 56 to 59, wherein y is about 0.70.

62. The peptide of claim 56, wherein the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

63. The peptide of claim 56, wherein the peptide has a structure represented by a formula:wherein R20is selected from -OR31, -NHR32, -Ns, a protein tag, a sortase recognition sequence, a sugar residue, and a structure:Attorney Docket No. 21101.0486P1wherein R31and R32are selected from hydrogen. -CH2PI1. C1-C8 alky l. C2-C8 alkyne, C1-C8 azide, tetrazinyl, cyclooctynyl, norbomenyl, and -(CH2CH2O)mCH3, and wherein m is an integer selected from 1 to 100,or a pharmaceutically acceptable salt thereof.

64. The peptide of claim 63, wherein the protein tag is selected from CBP, FLAC GST. HA, HBH, MBP, Myc, poly His, S-tag, SUMO. TAP. TRX, and V5.

65. The peptide of claim 63 or claim 64, wherein the sortase recognition sequence is selected from LPXTG (SEQ ID NO: 1), LPXTGG (SEQ ID NO: 2), LPXTGGG (SEQ ID NO: 3), and LPXTGGGG (SEQ ID NO: 4), wherein X is a natural or unnatural amino acid.

66. The peptide of claim 65, wherein the natural amino acid is selected from serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

67. The peptide of claim 65, wherein the natural amino acid is selected from glutamic acid, aspartic acid, arginine, histidine, and lysine.

68. The peptide of claim 65, wherein the unnatural amino acid is selected from hydroxyproline, beta-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, and pyroglutamic acid.

69. The peptide of any one of claims 63 to 68, wherein the sortase recognition sequence is selected from LPETG (SEQ ID NO: 5), LPETGG (SEQ ID NO: 6), LPETGGG (SEQ ID NO: 7), and LPETGGGG (SEQ ID NO: 8).

70. The peptide of any one of claims 63 to 68, wherein the sugar residue is a residue of a sugar selected from fructose, glucose, and lactose.Attorney Docket No. 21101.0486P171. The peptide of any one of claims 63 to 68, wherein R20is selected from -OR31, -NR32, -N3, and a structure:

72. The peptide of claim 44, wherein the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

73. A cryoprotectant composition comprising an effective amount of the peptide of any one of claims 1 to 72 and one or more selected from:(a) a non-antifreeze protein;(b) a microbe;(c) a cell component; and(d) a cell.

74. The cryoprotectant composition of claim 73, wherein the non-antifreeze protein is selected from an enzyme, a hormone, an antibody, a growth factor, a vaccination protein, a therapeutic protein, or a nutrient protein.

75. A food product comprising the peptide of any one of claims 1 to 72.

76. The food product of claim 75, wherein the food product is selected from ice cream, yogurt, seafood, fruit, and a meat product.Attorney Docket No. 21101.0486P177. An agricultural composition comprising the peptide of any one of claims 1 to 72.

78. A solid or semi-solid support comprising a surface covalently attached to a residue of the peptide of any one of claims 1 to 72.

79. The solid or semi-solid support of claim 78, wherein the residue of the peptide comprises an N-terminus, and wherein the N-terminus is covalently attached to the surface.

80. The solid or semi-solid support of claim 78 or claim 79, wherein the residue of the peptide has a structure represented by a formula:wherein R20is selected from -OR31, -NHR32, -Ns, a protein tag, a sortase recognition sequence, a sugar residue, and a structure:wherein R31and R32are selected from hydrogen, -CH2PI1, C1-C8 alkyl, C2-C8 alkyne, C1-C8 azide, tetrazinyl, cyclooctynyl, norbomenyl, and -(CH2CH2O)mCH3, and wherein m is an integer selected from 1 to 100.

81. The solid or semi-solid support of any one of claims 78 to 80, wherein the support is a glass bead, a silica-based resin, a cellulosic resin, an agarose bead, a polystyrene bead, or a polyacrylamide resin.

82. A cosmetic composition comprising the peptide of any one of claims 1 to 72.Attorney Docket No. 21101.0486P183. A method of inhibiting ice crystal formation in a sample, the method comprising contacting the sample with an effective amount of the peptide of any one of claims 1 to 72.

84. The method of claim 83, wherein the sample is a biological material.

85. The method of claim 83, wherein the biological material is selected from a nonantifreeze protein, a microbe, a cell component, a cell, a tissue, and an organ.

86. The method of claim 85, wherein the cell is selected from a sperm cell, an egg matrix cell, an embryonic cell, and a stem cell.

87. The method of claim 83, wherein the sample is a food product.

88. The method of claim 83, wherein the sample is an agricultural product.

89. The method of claim 83, wherein the sample is a solid or semi-solid support.

90. The method of claim 83, wherein the sample is a cosmetic.

91. The method of any one of claims 83 to 90. further comprising storing the biological material for a period of time.

92. The method of claim 91, wherein storing is at a temperature of about 25 °C or less.

93. The method of claim 91, wherein storing is at a temperature of about 4 °C or less.

94. A kit comprising the peptide of any one of claims 1 to 72 and one or more selected from:(a) a biological material;(b) a food product;(c) an agricultural product;(d) a solid or semi-solid support; and(e) a cosmetic.

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