Antifreeze glycopolypeptides

Abstract

The present disclosure is concerned with peptides comprising alanine residues and glycosylated residues (e.g., glycosylated hydroxypropline residues, a combination of glycosylated hydroxyproline residues and glycosylated threonine residues) in a particular ratio (e g., from about 3:2 to about 4:1), wherein the peptide has a minumum chain length, such as, for example a chain length of at least 30 ammo 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 cry opreservation, 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

Attorney Docket No.21101.0476P1 ANTIFREEZE GLYCOPOLYPEPTIDES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U.S. Application No. 63 / 554,742, filed onFebruary 16, 2024, the contents of which are incorporated herein by reference in their entirety. STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under R35 GM147262 awarded bythe National Institutes of Health, and 2300012 awarded by the National Science Foundation. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] This Sequence Listing submitted February 14, 2025 as a xml file named“21101.0476P1.xml,” created on February 13, 2025, and having a size of 8,192 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND

[0004] Worldwide, animal species have adapted strategies to survive sub-freezingtemperatures (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 embryonic ice crystals 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.

[0005] There is broad demand for antifreeze molecules for applications in food technology,agriculture, fisheries, coatings, building materials, and in the petroleum industry (Eskandari,Attorney Docket No.21101.0476P1 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) toimprove post-thaw tissue viability and function in biomedical cryopreservation (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). For decades, AFGPs have been explored in the cryopreservation 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., 2019Biomolecules 9 (5), 181; Amir, G.; et al., 2004 Ann. Thorac. Surg. 77 (5), 1648–1655) andhave even been utilized as texture-improving ice cream additives (Meldolesi, A. GM 2009 Nat. Biotechnol.27 (8), 682–682). However, the expense and impracticality 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).

[0006] A wide variety of short peptides and polymer alternatives have been explored asAFGP 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. Urbańczyk, 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

[0007] In accordance with the purpose(s) of the invention, as embodied and broadlydescribed herein, the invention, in one aspect, relates to peptides comprising alanine residues and glycosylated residues (e.g., glycosylated hydroxypropline residues, a combination ofAttorney Docket No.21101.0476P1 glycosylated hydroxyproline residues and glycosylated threonine 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 30 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 to a surface for use in structural applications.

[0008] Thus, disclosed are peptides comprising a plurality of alanine residues and a pluralityof glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues.

[0009] Also disclosed are cryoprotectant compositions comprising an effective amount of adisclosed 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 surfaces attached (e.g., covalently attached or via coating on thesurface) to 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 methodcomprising 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 semi- solid support; and (e) a cosmetic.

[0016] While aspects of the present invention can be described and claimed in a particularstatutory 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 stateAttorney Docket No.21101.0476P1 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 thisspecification, illustrate several aspects and together with the description serve to explain the principles of the invention.

[0018] FIG. 1 shows a representative chemical structure and cartoon representation of anative AFGP (left) and an exemplary synthetic AFGP as disclosed herein (right).

[0019] FIG. 2 shows a representative scheme for the preparation of galactose-hydroxyprolineconjugates and conversion to NCA monomers.

[0020] FIG. 3 shows a representative scheme for the preparation of an antifreeze polymerpanel by NCA polymerization.

[0021] FIG. 4 shows representative IRI activity of various proline-based homo- and co-polypeptide structures.

[0022] FIG. 5 shows a representative Hyp0.33-s-Ala0.66)150 ice splat images at 0 minutes(left) and 40 (right) minutes.

[0023] FIG. 6 shows a representative MGS of ice crystals for synthetic polymers at 1mg / mLat time 40 minutes.

[0024] FIG. 7 shows a representative Hyp200 ice splat image at 0 minutes (left) and 40(right) minutes.

[0025] FIG. 8 shows representative Hyp200 homopolymer MGS data compared to PBScontrol at 1mg / mL.

[0026] FIG. 9 shows representative IRI activity of (GalHyp0.66-s-Ala0.33)n glycopolypeptidesof varied chain lengths.

[0027] FIG. 10 shows representative shaping of ice crystals by (GalHyp0.66-s-Ala0.33)nglycopolypeptides of varied chain lengths.

[0028] FIG. 11 shows a representative (GalHyp0.33-s-Ala0.66)103 copolymer full size image ofice shaping.Attorney Docket No.21101.0476P1

[0029] FIG. 12 shows a representative (GalHyp0.33-s-Ala0.66)155 copolymer full size image ofice shaping.

[0030] FIG. 13 shows a representative (GalHyp0.33-s-Ala0.66)234 copolymer full size image ofice shaping.

[0031] FIG. 14 shows a representative (GalHyp0.33-s-Ala0.66)312 copolymer full size image ofice shaping.

[0032] FIG. 15A and FIG. 15B show representative aqueous circular dichroism data forGalHyp containing polypeptides at 20 °C

[0033] FIG. 16 shows a representative CD spectra of (GalHyp0.33-s-Ala0.66)155 at varioustemperatures

[0034] FIG. 17 shows representative HEK 293 cell viability as determined by CCK8 assayfollowing 24-hour incubation with (GalHyp0.66-s-Ala0.33)150at the indicated concentrations.

[0035] FIG. 18 shows a representative 1H NMR spectra of Z-GalOAc4Hyp-OBn in CDCl3.

[0036] FIG. 19 shows a representative 13C NMR spectra of Z-GalOAc4Hyp-OBn in CDCl3.

[0037] FIG. 20 shows a representative HSQC plot of Z-GalOAc4Hyp-OBn in CDCl3.

[0038] FIG. 21 shows a representative 1H NMR spectra of GalOAc4-Hyp-Boc in CDCl3.

[0039] FIG. 22 shows a representative 13C NMR spectra of GalOAc4-Hyp-Boc in CDCl3.

[0040] FIG. 23 shows a representative HSQC plot of GalOAc4-Hyp-Boc in CDCl3.

[0041] FIG. 24 shows a representative 1H NMR spectra of GalOAc4-Hyp NCA in CDCl3.

[0042] FIG. 25 shows a representative 13C NMR spectra of GalOAc4-Hyp NCA in CDCl3.

[0043] FIG. 26 shows a representative HSQC plot of GalOAc4-Hyp NCA in CDCl3.

[0044] FIG. 27 shows a representative 1H NMR spectra of polyGalOAc4-Hyp in CDCl3.

[0045] FIG. 28 shows a representative 1H NMR spectra of polyGalOH4-Hyp in D2O.

[0046] FIG. 29 shows a representative 1H NMR spectra of PEG88-(Gal-OH-Hyp0.33-s-Ala0.66)155 in D2O.

[0047] FIG. 30 shows a representative 1H NMR spectra of PEG88-(GalOAc4-Hyp0.33-s-Ala0.66)234 in CDCl3.

[0048] FIG. 31A and FIG. 31B show representative ATR-FTIR spectra showing thedisappearance of GalHyp-OAc4 NCA and the formation of polyGalHyp-OAC4.

[0049] FIG. 32A and FIG. 32B show representative ATR-FTIR spectra showing thedisappearance of the acetyl protecting groups on the copolymer of ((GalHyp-OAc4)0.33-s- Ala0.66)n to form copolymer ((GalHyp-OH)0.33-s-Ala0.66)n.

[0050] FIG. 33 shows a representative Hyp0.33-s-Ala0.66) ice splat images at 0 minutes(left) and 40 (right) minutes for glycopolypeptides of varied chain lengths.Attorney Docket No.21101.0476P1

[0051] FIG. 34 shows a representative Hyp0.33-s-Ala0.66) ice splat images at 0 minutes(left) and 40 (right) minutes for various proline-based homo- and co-polypeptide structures.

[0052] Additional advantages of the invention will be set forth in part in the descriptionwhich 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. DETAILED DESCRIPTION

[0053] The present invention can be understood more readily by reference to the followingdetailed description of the invention and the Examples included therein.

[0054] Before the present compounds, compositions, articles, systems, devices, and / ormethods 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.

[0055] While aspects of the present invention can be described and claimed in a particularstatutory 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.

[0056] Throughout this application, various publications are referenced. The disclosures ofthese publications in their entireties are hereby incorporated by reference into this applicationAttorney Docket No.21101.0476P1 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 construed 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.A. DEFINITIONS

[0057] As used in the specification and the appended claims, the singular forms “a,” “an” and“the” include plural referent.s 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.

[0058] As used in the specification and in the claims, the term “comprising” can include theaspects “consisting of” and “consisting essentially of.”

[0059] 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.

[0060] As used herein, the terms “about” and “at or about” mean that the amount or value inquestion 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, reflectingAttorney Docket No.21101.0476P1 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 quantity or 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.

[0061] References in the specification and concluding claims to parts by weight of aparticular 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 article for 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.

[0062] A weight percent (wt. %) of a component, unless specifically stated to the contrary, isbased on the total weight of the formulation or composition in which the component is included.

[0063] As used herein, “thermal hysteresis” or “TH” is intended to refer to the differencebetween the temperature at which ice crystals grow (the freezing temperature, Tf) and the temperature at which they melt (the melting point, Tm).

[0064] As used herein, the terms “optional” or “optionally” means that the subsequentlydescribed 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.

[0065] As used herein, the terms “effective amount” and “amount effective” refer to anamount 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 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 least 70%, 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.Attorney Docket No.21101.0476P1

[0066] As used herein, “kit” means a collection of at least two components constituting thekit. 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 be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0067] As used herein, “instruction(s)” means documents describing relevant materials ormethodologies 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.

[0068] As used herein, the term “derivative” refers to a peptide having a structure derivedfrom 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.

[0069] As used herein, the term “substituted” is contemplated to include all permissiblesubstituents 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”Attorney Docket No.21101.0476P1 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, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0070] In defining various terms, “A1,” “A2,” “A3,” and “A4” are used herein as genericsymbols 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.

[0071] The term “aliphatic” or “aliphatic group,” as used herein, denotes a hydrocarbonmoiety 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.

[0072] The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbongroup of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s- 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 alkyl” 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 C1 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, C1-C10 alkyl, and the like up to and including a C1-C24 alkyl.

[0073] Throughout the specification “alkyl” is generally used to refer to both unsubstitutedalkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine.Attorney Docket No.21101.0476P1 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 with two 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 “alkoxyalkyl” 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 “alkyl” 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 “alkyl” does not also refer to specific terms such as “hydroxyalkyl” and the like.

[0074] This practice is also used for other groups described herein. That is, while a termsuch as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” 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 “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0075] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composedof at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl 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 heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl 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.

[0076] The term “polyalkylene group” as used herein is a group having two or more CH2groups linked to one another. The polyalkylene group can be represented by the formula — (CH2)a—, where “a” is an integer of from 2 to 500.Attorney Docket No.21101.0476P1

[0077] The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl or cycloalkylgroup bonded through an ether linkage; that is, an “alkoxy” group can be defined as —OA1where A1is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether 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.

[0078] The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbonatoms 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.

[0079] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” 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.

[0080] The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atomswith 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, 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.Attorney Docket No.21101.0476P1

[0081] The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ringcomposed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. 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, 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.

[0082] The term “aromatic group” as used herein refers to a ring structure having cyclicclouds of delocalized π electrons above and below the plane of the molecule, where the π clouds contain (4n+2) π 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 aryl and heteroaryl groups.

[0083] The term “aryl” as used herein is a group that contains any carbon-based aromaticgroup including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, ─NH2, 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 carbon- carbon 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.

[0084] 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.

[0085] 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.21101.0476P1 cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is ─NH2.

[0086] The term “alkylamino” 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, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, and the like.

[0087] The term “dialkylamino” as used herein is represented by the formula —N(-alkyl)2where 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.

[0088] The term “carboxylic acid” as used herein is represented by the formula —C(O)OH.

[0089] The term “ester” as used herein is represented by the formula —OC(O)A1 or —C(O)OA1, 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.

[0090] The term “ether” as used herein is represented by the formula A1OA2, where A1 andA2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl 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, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0091] The terms “halo,” “halogen,” or “halide” as used herein can be used interchangeablyand refer to F, Cl, Br, or I.Attorney Docket No.21101.0476P1

[0092] The terms “pseudohalide,” “pseudohalogen,” or “pseudohalo” as used herein can beused interchangeably and refer to functional groups that behave substantially similar to halides. Such functional groups include, by way of example, cyano, thiocyanato, azido, trifluoromethyl, trifluoromethoxy, perfluoroalkyl, and perfluoroalkoxy groups.

[0093] The term “heteroalkyl” as used herein refers to an alkyl group containing at least oneheteroatom. 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 quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0094] The term “heteroaryl” as used herein refers to an aromatic group that has at least oneheteroatom 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, alkyl, 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, N-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, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0095] The terms “heterocycle” or “heterocyclyl” as used herein can be used interchangeablyand 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, pyrazole, 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-Attorney Docket No.21101.0476P1 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-C18 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, dihydrodiazetyl, 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.

[0096] The term “bicyclic heterocycle” or “bicyclic heterocyclyl” as used herein refers to aring 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 a non-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-1,4-benzodioxinyl, 3,4-dihydro-2H- chromenyl, 1H-pyrazolo[4,3-c]pyridin-3-yl; 1H-pyrrolo[3,2-b]pyridin-3-yl; and 1H- pyrazolo[3,2-b]pyridin-3-yl.

[0097] The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturatedor 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 be substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.Attorney Docket No.21101.0476P1

[0098] The term “hydroxyl” or “hydroxyl” as used herein is represented by the formula —OH.

[0099] 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.

[0100] The term “azide” or “azido” as used herein is represented by the formula —N3.

[0101] The term “nitro” as used herein is represented by the formula —NO2.

[0102] The term “nitrile” or “cyano” as used herein is represented by the formula —CN.

[0103] 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.

[0104] The term “sulfo-oxo” as used herein is represented by the formulas —S(O)A1,—S(O)2A1, —OS(O)2A1, or —OS(O)2OA1, 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 described herein.

[0105] The term “thiol” as used herein is represented by the formula —SH.

[0106] “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, alternatively, the first group can be pendant (i.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 alkyl group. Alternatively, the amino group canAttorney Docket No.21101.0476P1 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.

[0107] As described herein, peptides of the invention may contain “optionallysubstituted” 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 (i.e., further substituted or unsubstituted).

[0108] The term “stable,” as used herein, refers to peptides that are not substantiallyaltered 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.

[0109] Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen; –(CH2)0–4R^; –(CH2)0–4OR^; - O(CH2)0-4Ro, –O–(CH2)0–4C(O)OR°; –(CH2)0–4CH(OR^)2; –(CH2)0–4SR^; –(CH2)0–4Ph, which may be substituted with R°; –(CH2)0–4O(CH2)0–1Ph 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)0–4N(R^)2; –(CH2)0–4N(R^)C(O)R^; – N(R^)C(S)R^; –(CH2)0–4N(R^)C(O)NR^2; -N(R^)C(S)NR^2; –(CH2)0–4N(R^)C(O)OR^; – N(R^)N(R^)C(O)R^; -N(R^)N(R^)C(O)NR^2; -N(R^)N(R^)C(O)OR^; –(CH2)0–4C(O)R^; – C(S)R^; –(CH2)0–4C(O)OR^; –(CH2)0–4C(O)SR^; -(CH2)0–4C(O)OSiR^3; –(CH2)0–4OC(O)R^; –OC(O)(CH2)0–4SR–, SC(S)SR°; –(CH2)0–4SC(O)R^; –(CH2)0–4C(O)NR^2; –C(S)NR^2; – C(S)SR°; -(CH2)0–4OC(O)NR^2; -C(O)N(OR^)R^; –C(O)C(O)R^; –C(O)CH2C(O)R^; –C(NOR^)R^; -(CH2)0–4SSR^; –(CH2)0–4S(O)2R^; –(CH2)0–4S(O)2OR^; –(CH2)0–4OS(O)2R^; –S(O)2NR^2; -(CH2)0–4S(O)R^; -N(R^)S(O)2NR^2; –N(R^)S(O)2R^; –N(OR^)R^; – C(NH)NR^2; –P(O)2R^; -P(O)R^2; -OP(O)R^2; –OP(O)(OR^)2; SiR^3; –(C1–4 straight or branched alkylene)O–N(R^)2; or –(C1–4 straight or branched alkylene)C(O)O–N(R^)2, wherein each R^ may be substituted as defined below and is independently hydrogen, C1–6aliphatic, –CH2Ph, –O(CH2)0–1Ph, -CH2-(5-6 membered heteroaryl ring), or a 5–6–Attorney Docket No.21101.0476P1 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 aryl mono– or bicyclic ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0110] Suitable monovalent substituents on R^ (or the ring formed by taking twoindependent occurrences of R^ together with their intervening atoms), are independently halogen, –(CH2)0–2R^, –(haloR^), –(CH2)0–2OH, –(CH2)0–2OR^, –(CH2)0–2CH(OR^)2; -O(haloR^), –CN, –N3, –(CH2)0–2C(O)R^, –(CH2)0–2C(O)OH, –(CH2)0–2C(O)OR^, –(CH2)0–2SR^, –(CH2)0–2SH, –(CH2)0–2NH2, –(CH2)0–2NHR^, –(CH2)0–2NR^2, –NO2, –SiR^3, –OSiR^3, -C(O)SR^,–(C1–4straight or branched alkylene)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 C1–4aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 5–6–membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R^ include =O and =S.

[0111] Suitable divalent substituents on a saturated carbon atom of an “optionallysubstituted” group include the following: =O, =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 independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: –O(CR*2)2–3O–, wherein each independent occurrence of R*is selected from hydrogen, C1–6aliphatic 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.

[0112] Suitable substituents on the aliphatic group of R* include 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 C1–4 aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 5–6–Attorney Docket No.21101.0476P1 membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0113] Suitable substituents on a substitutable nitrogen of an “optionally substituted”group include –R†, –NR†2, –C(O)R†, –C(O)OR†, –C(O)C(O)R†, –C(O)CH2C(O)R†, – S(O)2R†, -S(O)2NR†2, –C(S)NR†2, –C(NH)NR†2, or –N(R†)S(O)2R†; wherein each R†is independently hydrogen, C1–6aliphatic 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 R†, 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.

[0114] Suitable substituents on the aliphatic group of R† are 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, –O(CH2)0–1Ph, or a 5–6–membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0115] The term “leaving group” refers to an atom (or a group of atoms) with electronwithdrawing 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.

[0116] The terms “hydrolysable group” and “hydrolysable moiety” refer to afunctional 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).

[0117] The phrase “residue of a peptide” as used with respect to the phrase “a surfacecovalently 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, for example, 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.Attorney Docket No.21101.0476P1

[0118] The term “organic residue” defines a carbon-containing residue, i.e., a residuecomprising 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.

[0119] 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.

[0120] Unless stated to the contrary, a formula with chemical bonds shown only assolid 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.

[0121] Many organic compounds (e.g., peptides) exist in optically active formshaving 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 l or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 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 specificAttorney Docket No.21101.0476P1 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.

[0122] When the disclosed peptides contain one chiral center, the peptides exist intwo 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 crystallization (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 enzymatic 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 by one 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.

[0123] Designation of a specific absolute configuration at a chiral carbon in adisclosed 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%,Attorney Docket No.21101.0476P1 greater than 95%, greater than 98%, or greater than 99%. In 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.

[0124] When a disclosed peptide or compound has two or more chiral carbons, it canhave 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.

[0125] The peptides according to this disclosure may form prodrugs at hydroxyl oramino 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.

[0126] “Derivatives” of the peptides disclosed herein are pharmaceutically acceptablesalts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, solvates and combinations thereof. The “combinations” mentioned in this context are refer to derivatives falling 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.

[0127] Peptides described herein comprise atoms in both their natural isotopicabundance and in non-natural abundance. The disclosed peptides can be isotopically-labeledAttorney Docket No.21101.0476P1 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,17O,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.

[0128] The peptides described in the invention can be present as a solvate. In somecases, 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.

[0129] The term “co-crystal” means a physical association of two or more moleculeswhich owe their stability through non-covalent interaction. One or more components of this molecular complex provide a stable framework 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, O., et. al., The Royal Society of Chemistry, 1889-1896, 2004. Examples of co-crystals include p- toluenesulfonic acid and benzenesulfonic acid.

[0130] It is also appreciated that certain peptides described herein can be present asan equilibrium of tautomers. For example, ketones with an α-hydrogen can exist in an equilibrium of the keto form and the enol form.

[0131] Likewise, amides with an N-hydrogen can exist in an equilibrium of the amideform and the imidic acid form. As another example, pyrazoles can exist in two tautomeric forms, N1-unsubstituted, 3-A3and N1-unsubstituted, 5-A3as shown below.Attorney Docket No.21101.0476P1 Unless stated to thetautomers.

[0132] It is known that chemical substances form solids that are present in differentstates 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.

[0133] In some aspects, a structure of a peptide can include a group represented by aformula: ,which is understood to be equivalent to ,wherein n is typically an integer. to represent five independent substituents, Rn(a), Rn(b), Rn(c), Rn(d), Rn(e). By “independent substituents,” it is meant that each R substituent can bedefined. For example, if in one instance Rn(a)is halogen, then Rn(b)is not necessarily halogen in that instance.

[0134] Certain materials, peptides, compositions, and components disclosed hereincan 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 (Morris Plains, N.J.), Strem Chemicals (Newburyport, MA), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known 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 (JohnAttorney Docket No.21101.0476P1 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).

[0135] Unless otherwise expressly stated, it is in no way intended that any method setforth 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.

[0136] Disclosed are the components to be used to prepare the compositions of theinvention 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 of molecules 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.Attorney Docket No.21101.0476P1

[0137] 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

[0138] In one aspect, disclosed are peptides comprising alanine residues andglycosylated residues (e.g., glycosylated hydroxypropline residues, a combination of glycosylated hydroxyproline residues and glycosylated threonine residues) in a particular ratio (e.g., from about 3:2 to about 4:1), wherein the peptide has a minumum chain length, such as, for example a chain length of at least 30 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 cryopreservation, food technology, agriculture, cosmetics, and building materials.

[0139] In one aspect, the disclosed peptides exhibit inhibition of ice crystal formationin 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 least 70%, 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.

[0140] In one aspect, the disclosed peptides are useful in delaying, preventing, orotherwise decreasing ice crystal formation in a sample, as further described herein.

[0141] It is contemplated that each disclosed derivative can be optionally furthersubstituted. 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 disclosedAttorney Docket No.21101.0476P1 methods. It is also understood that the disclosed peptides can be employed in the disclosed methods of using. 1. STRUCTURE

[0142] In one aspect, disclosed are peptides comprising a plurality of alanine residuesand a plurality of glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues.

[0143] In various aspects, the peptide consists essentially of the plurality of alanineresidues and the plurality of glycosylated residues.

[0144] In various aspects, the plurality of alanine residues is present in the peptide inan amount of from about 60 wt% to about 70 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 68 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 66 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 64 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 62 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 70 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 plurality of alanine residues is present in the peptide in an amount of from about 66 wt% to about 70 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 70 wt%. In yet a further aspect, the plurality of alanine residues is present in the peptide in an amount of from about 62 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 64 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 65 wt% to about 67 wt%.

[0145] In various aspects, the plurality of alanine residues is present in the peptide inan amount of about 60 wt%. In a still further aspect, the plurality of alanine residues isAttorney Docket No.21101.0476P1 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 wt%.

[0146] In various aspects, the plurality of glycosylated residues are a combination ofglycosylated hydroxyproline residues and glycosylated threonine residues. In a further aspect, the peptide does not contain more than eleven Ala-Ala-Thr repeats. In a further aspect, the peptide does not contain more than ten Ala-Ala-Thr repeats. In a still further aspect, the peptide does not contain more than nine Ala-Ala-Thr repeats. In yet a further aspect, the peptide does not contain more than eight Ala-Ala-Thr repeats. In an even further aspect, the peptide does not contain more than seven Ala-Ala-Thr repeats. In a still further aspect, the peptide does not contain more than six Ala-Ala-Thr repeats. In yet a further aspect, the peptide does not contain more than five Ala-Ala-Thr repeats. In an even further aspect, the peptide does not contain more than four Ala-Ala-Thr repeats. In a still further aspect, the peptide does not contain more than three Ala-Ala-Thr repeats. In yet a further aspect, the peptide does not contain more than two Ala-Ala-Thr repeats. In an even further aspect, the peptide does not contain more than one Ala-Ala-Thr repeats. In an even still furher aspect, the peptide does not contain any Ala-Ala-Thr repeats.

[0147] In various aspects, the plurality of glycosylated residues are glycosylatedhydroxyproline residues.

[0148] In various aspects, the plurality of glycosylated residues are glycosylated withone or more of αGal, βGal, αGalNAc, βGalNAc, βGal(1→3)αGalNAc, αLac, and βLac. In a further aspect, the plurality of glycosylated residues are glycosylated with one or more of αGal, βGal, αGalNAc, βGalNAc, andr βGal(1→3)αGalNAc. In a further aspect, the plurality of glycosylated residues are glycosylated with one or more of αGal, βGal, αGalNAc, βGalNAc, αLac, and βLac. In a still further aspect, the plurality of glycosylated residues areAttorney Docket No.21101.0476P1 glycosylated with one or more of αGal, βGal, βGal(1→3)αGalNAc, αLac, or βLac. In yet a further aspect, the plurality of glycosylated residues are glycosylated with one or more of αGal, βGal, and βGal(1→3)αGalNAc. In an even further aspect, the plurality of glycosylated residues are glycosylated with one or more of αGalNAc, βGalNAc, and βGal(1→3)αGalNAc. In a still further aspect, the plurality of glycosylated residues are glycosylated with one or more of βGal(1→3)αGalNAc, αLac, and βLac. In yet a further aspect, the plurality of glycosylated residues are glycosylated with one or more of αGal, βGal, αGalNAc, and βGalNAc. In an even further aspect, the plurality of glycosylated residues are glycosylated with one or more of αGal, βGal, αLac, and βLac. In a still further aspect, the plurality of glycosylated residues are glycosylated with one or more of αGalNAc, βGalNAc, αLac, and βLac. In yet a further aspect, the plurality of glycosylated residues are glycosylated with one or more of αGal and βGal. In an even further aspect, the plurality of glycosylated residues are glycosylated with one or more of αGalNAc and βGalNAc. In an even still further aspect, the plurality of glycosylated residues are glycosylated with βGal(1→3)αGalNAc. In a yet even further aspect, the plurality of glycosylated residues are glycosylated with one or more of αLac and βLac.

[0149] In various aspects, the ratio of the plurality of alanine residues to the pluralityof glycosylated 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 glycosylated residues is from about 19:11 to about 4:1. In a still further aspect, the ratio of the plurality of alanine residues to the plurality of glycosylated 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 glycosylated 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 glycosylated 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 glycosylated 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 glycosylated 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 glycosylated 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 glycosylated 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 glycosylated 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 glycosylated 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 ofAttorney Docket No.21101.0476P1 glycosylated 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 glycosylated 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 glycosylated 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 glycosylated residues is from about 21:9 to about 11:4.

[0150] In various aspects, the ratio of the plurality of alanine residues to the pluralityof glycosylated residues is about 3:2. In a further aspect, the ratio of the plurality of alanine residues to the plurality of glycosylated residues is about 19:11. In a still further aspect, the ratio of the plurality of alanine residues to the plurality of glycosylated residues is about 2:1. In a yet further aspect, the ratio of the plurality of alanine residues to the plurality of glycosylated residues is about 21:9. In an even further aspect, the ratio of the plurality of alanine residues to the plurality of glycosylated residues is about 11:4. In an even still further aspect, the ratio of the plurality of alanine residues to the plurality of glycosylated residues is about 23:7. In an even yet further aspect, the ratio of the plurality of alanine residues to the plurality of glycosylated residues is about 4:1.

[0151] In various aspects, the peptide has a chain length of at least 30 amino acidresidues. In a further aspect, the peptide has a chain length of at least 40 amino acid residues. In a still further aspect, the peptide has a chain length of at least 50 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 60 amino acid residues. In an even further aspect, the peptide has a chain length of at least 70 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 80 amino acid residues. In an even yet further aspect, the peptide has a chain length of at least 90 amino acid residues. In a further aspect, the peptide has a chain length of at least 100 amino acid residues. In a still further aspect, the peptide has a chain length of at least 110 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 120 amino acid residues. In an even further aspect, the peptide has a chain length of at least 130 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 140 amino acid residues. In even yet further aspect, the peptide has a chain length of at least 150 amino acid residues. In a further aspect, the peptide has a chain length of at least 160 amino acid residues. In a still further aspect, the peptide has a chain length of at least 170 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 180 amino acid residues. In an even further aspect, the peptide has a chain length of at least 190 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 200 amino acid residues. In anAttorney Docket No.21101.0476P1 even yet further aspect, the peptide has a chain length of at least 210 amino acid residues. In a further aspect, the peptide has a chain length of at least 220 amino acid residues. In a still further aspect, the peptide has a chain length of at least 230 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 240 amino acid residues. In an even further aspect, the peptide has a chain length of at least 250 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 260 amino acid residues. In even yet further aspect, the peptide has a chain length of at least 270 amino acid residues. In a further aspect, the peptide has a chain length of at least 280 amino acid residues. In a still further aspect, the peptide has a chain length of at least 290 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 300 amino acid residues. In an even further aspect, the peptide has a chain length of at least 310 amino acid residues. In an even still further aspect, the peptide has a chain length of at least 320 amino acid residues. In an even yet further aspect, the peptide has a chain length of at least 330 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 even yet 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.

[0152] In various aspects, the peptide has a chain length of from about 30 amino acidresidues to about 500 amino acid residues. In a further aspect, the peptide has a chain length of from about 50 amino acid residues to about 500 amino acid residues. In a still furtherAttorney Docket No.21101.0476P1 aspect, the peptide has a chain length of from about 100 amino acid residues to about 500 amino acid residues. In a yet further aspect, the peptide has a chain length of from about 200 amino acid residues to about 500 amino acid residues. In an even further aspect, the peptide has a chain length of from about 300 amino acid residues to about 500 amino acid residues. In an even still further aspect, the peptide has a chain length of from about 40 amino acid residues to about 500 amino acid residues. In an even yet further aspect, the peptide has a chain length of from about 30 amino acid residues to about 400 amino acid residues. In a further aspect, the peptide has a chain length of from about 30 amino acid residues to about 300 amino acid residues. In a still further aspect, the peptide has a chain length of from about 30 amino acid residues to about 200 amino acid residues. In a yet further aspect, the peptide has a chain length of from about 30 amino acid residues to about 100 amino acid residues. In an even further aspect, the peptide has a chain length of from about 30 amino acid residues to about 50 amino acid residues. In an even still further aspect, the peptide has a chain length of from about 50 amino acid residues to about 400 amino acid residues. In even yet further aspect, the peptide has a chain length of from about 100 amino acid residues to about 300 amino acid residues. In a further aspect, the peptide has a chain length of from about about 50 residues to about 100 residues. In a still further aspect, the peptide has a chain length of from about 100 amino acid residues to about 150 amino acid residues. In a yet further aspect, the peptide has a chain length of from about 150 amino acid residues to about 200 amino acid residues. In an even further aspect, the peptide has a chain length of from about 200 amino acid residues to about 250 amino acid residues. In an even still further aspect, the peptide has a chain length of from about 250 amino acid residues to about 300 amino acid residues. In even yet further aspect, the peptide has a chain length of from about 300 amino acid residues to about 350 amino acid residues. In a further aspect, the peptide has a chain length of from about 350 amino acid residues to about 400 amino acid residues. In a still further aspect, the peptide has a chain length of from about 400 amino acid residues to about 450 amino acid residues. In a yet further aspect, the peptide has a chain length of from about 450 amino acid residues to about 500 amino acid residues. In an even further aspect, the peptide has a chain length of from about 125 amino acid residues to about 275 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.

[0153] In various aspects, the peptide has a number average molecular weight (Mn) ofat least about 3,000. In a further aspect, the peptide has a number average molecular weightAttorney Docket No.21101.0476P1 (Mn) of at least about 4,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of at least about 5,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 6,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of at least about 7,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of at least about 8,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 9,000. In a further aspect, the peptide has a number average molecular weight (Mn) of at least about 10,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of at least about 11,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 12,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of at least about 13,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of at least about 14,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 15,000. In a further aspect, the peptide has a number average molecular weight (Mn) of at least about 20,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of at least about 25,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 30,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of at least about 35,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of at least about 40,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of at least about 45,000. In a further aspect, the peptide has a number average molecular weight (Mn) of at least about 50,000.

[0154] In various aspects, the peptide has a number average molecular weight (Mn) offrom about 3,000 to about 50,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 5,000 to about 45,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 10,000 to about 40,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 15,000 to about 35,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of from about 20,000 to about 30,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of from about 3,000 to about 5,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of from about 3,000 to about 10,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 3,000 to about 15,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 3,000 to about 20,000. InAttorney Docket No.21101.0476P1 a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 3,000 to about 25,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of from about 3,000 to about 30,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of from about 3,000 to about 35,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of from about 30,000 to about 40,000. 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 45,000 to about 50,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 40,000 to about 50,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of from about 35,000 to about 50,000. In an even still further aspect, the peptide has a number average molecular weight (Mn) of from about 30,000 to about 50,000. In an even yet further aspect, the peptide has a number average molecular weight (Mn) of from about 25,000 to about 50,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 20,000 to about 50,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 15,000 to about 50,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 10,000 to about 50,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of from about 5,000 to about 50,000. 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 12,000 to about 28,000. In a further aspect, the peptide has a number average molecular weight (Mn) of from about 14,000 to about 25,000. In a still further aspect, the peptide has a number average molecular weight (Mn) of from about 15,000 to about 23,000. In a yet further aspect, the peptide has a number average molecular weight (Mn) of from about 18,000 to about 22,000. In an even further aspect, the peptide has a number average molecular weight (Mn) of from about 19,000 to about 21,000.

[0155] In various aspects, the peptide has a degree of polymerization (DP) of at leastabout 30. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 40. In a still further aspect, the peptide has a degree of polymerization (DP) of at least about 50. In a yet further aspect, the peptide has a degree of polymerization (DP) of at least about 60. In an even further aspect, the peptide has a degree of polymerization (DP) of at least about 70. In an even still further aspect, the peptide has a degree of polymerization (DP) of at least about 80. In an even yet further aspect, the peptide has a degree of polymerization (DP)Attorney Docket No.21101.0476P1 of at least about 90. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 100. In a still further aspect, the peptide has a degree of polymerization (DP) of at least about 112. In a yet further aspect, the peptide has a degree of polymerization (DP) of at least about 120. In an even further aspect, the peptide has a degree of polymerization (DP) of at least about 130. In an even still further aspect, the peptide has a degree of polymerization (DP) of at least about 140. In even yet further aspect, the peptide has a degree of polymerization (DP) of at least about 150. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 160. In a still further aspect, the peptide has a degree of polymerization (DP) of at least about 170. In a yet further aspect, the peptide has a degree of polymerization (DP) of at least about 180. In an even further aspect, the peptide has a degree of polymerization (DP) of at least about 190. In an even still further aspect, the peptide has a degree of polymerization (DP) of at least about 200. In an even yet further aspect, the peptide has a degree of polymerization (DP) of at least about 210. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 220. In a still further aspect, the peptide has a chain length of at least 230 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 240 amino acid residues. In an even further aspect, the peptide has a degree of polymerization (DP) of at least about 250. In an even still further aspect, the peptide has a degree of polymerization (DP) of at least about 260. In an even yet further aspect, the peptide has a degree of polymerization (DP) of at least about 270. In a further aspect, the peptide has a degree of polymerization (DP) of at least about 280. In a still further aspect, the peptide has a chain length of at least 290 amino acid residues. In a yet further aspect, the peptide has a chain length of at least 300 amino acid residues.

[0156] In various aspects, the peptide has a degree of polymerization (DP) from about30 to about 500. In a further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 450. In a still further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 400. In a yet further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 350. In an even further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 300. In an even still further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 250. In an even yet further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 200. In a further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 150. In a still further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 100. In a yet further aspect, the peptide has a degree of polymerization (DP) from about 30 to about 50. In an even further aspect, the peptide has a degree of polymerizationAttorney Docket No.21101.0476P1 (DP) from about 50 to about 500. In an even still further aspect, the peptide has a degree of polymerization (DP) from about 100 to about 500. In an even yet further aspect, the peptide has a degree of polymerization (DP) from about 150 to about 500. In a further aspect, the peptide has a degree of polymerization (DP) from about 200 to about 500. In a still further aspect, the peptide has a degree of polymerization (DP) from about 250 to about 500. In a yet further aspect, the peptide has a degree of polymerization (DP) from about 300 to about 500. In an even further aspect, the peptide has a degree of polymerization (DP) from about 350 to about 500. In an even still further aspect, the peptide has a degree of polymerization (DP) from about 400 to about 500. In an even yet further aspect, the peptide has a degree of polymerization (DP) from about 450 to about 500. In a further aspect, the peptide has a degree of polymerization (DP) from about 50 to about 450. In a still further aspect, the peptide has a degree of polymerization (DP) from about 100 to about 400. In a yet further aspect, the peptide has a degree of polymerization (DP) from about 150 to about 350. In an even further aspect, the peptide has a degree of polymerization (DP) from about 200 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.

[0157] In various aspects, the peptide has a structure represented by a formula:,Attorney Docket No.21101.0476P1 wherein n is an integer selected from 15 to about 250; wherein x is selected from 40 to about 20%; wherein y is selected from about 60 to about 80%; wherein R1is a glycosyl moiety having a structure represented by a formula selected from: ;wherein R10and R11are selected from ‒OH and ‒NHAc, wherein the ratio of y to x is of from about 3:2 to about 4:1, and wherein the sum of x and y is at least 100 residues, or a pharmaceutically acceptable salt thereof.

[0158] In various aspects, x is 6 or greater and y is 18 or greater. In a further aspect,x is 10 or greater and y is 30 or greater. In a still further aspect, x is 20 or greater and y is 60 or greater. In a yet further aspect, x is 30 or greater and y is 90 or greater. In an even further aspect, x is 40 or greater and y is 120 or greater. In an even still further aspect, x is 50 or greater and y is 150 or greater. In an even yet further aspect, x is 60 or greater and y is 180 or greater. In a further aspect, x is 70 or greater and y is 210 or greater. In a still further aspect,Attorney Docket No.21101.0476P1 x is 80 or greater and y is 240 or greater. In a yet further aspect, x is 90 or greater and y is 270 or greater. In an even further aspect, x is 100 or greater and y is 300 or greater. In an even still further aspect, x is 110 or greater and y is 330 or greater. In an even yet further aspect, x is 120 or greater and y is 360 or greater.

[0159] In various aspects, x is from 6 to 120 and y is from 18 to 360. In a furtheraspect, x is from 10 to 120 and y is from 30 to 360. In a still further aspect, x is from 20 to 120 and y is from 60 to 360. In a yet further aspect, x is from 30 to 120 and y is from 90 to 360. In an even further aspect, x is from 40 to 120 and y is from 120 to 360. In an even still further aspect, x is from 50 to 120 and y is from 150 to 360. In an even yet further aspect, x is from 60 to 120 and y is from 180 to 360. In a further aspect, x is from 70 to 120 and y is from 210 to 360. In a still further aspect, x is from 80 to 120 and y is from 240 to 360. In a yet further aspect, x is from 90 to 120 and y is from 270 to 360. In an even further aspect, x is from 110 to 120 and y is from 330 to 360. In an even still further aspect, x is from 6 to 110 and y is from 18 to 330. In an even yet further aspect, x is from 6 to 100 and y is from 18 to 300. In a further aspect, x is from 6 to 90 and y is from 18 to 270. In a still further aspect, x is from 6 to 80 and y is from 18 to 240. In a yet further aspect, x is from 6 to 70 and y is from 18 to 210. In an even further aspect, x is from 6 to 60 and y is from 18 to 180. In an even still further aspect, x is from 6 to 50 and y is from 18 to 150. In an even yet further aspect, x is from 6 to 40 and y is from 18 to 120. In a further aspect, x is from 6 to 30 and y is from 18 to 90. In a still further aspect, x is from 6 to 20 and y is from 18 to 60. In a yet further aspect, x is from 6 to 10 and y is from 18 to 30. In an even further aspect, x is from 6 to 60 and y is from 18 to 180. In an even still further aspect, x is from 6 to 50 and y is from 18 to 150. In an even yet further aspect, x is from 6 to 40 and y is from 18 to 120. In a further aspect, x is from 20 to 120 and y is from 60 to 180.

[0160] In various aspects, the ratio of y to x is about 3:2 to about 4:1. In a furtheraspect, the ratio of y to x is about 19:11 to about 4:1. In a still further aspect, the ratio of y to x is about 2:1 to about 4:1. In a yet further aspect, the ratio of y to x is about 21:9 to about 4:1. In an even further aspect, the ratio of y to x is about 11:4 to about 4:1. In an even still further aspect, the ratio of y to x is about 23:7 to about 4:1. In an even yet further aspect, the ratio of y to x is about 23:7 to about 4:1. In a further aspect, the ratio of y to x is about 3:2 to about 23:7. In a still further aspect, the ratio of y to x is about 3:2 to about 11:4. In a yet further aspect, the ratio of y to x is about 3:2 to about 21:9. In an even further aspect, the ratio of y to x is about 3:2 to about 2:1. In an even still further aspectthe ratio of y to x is about 3:2 to about 19:11. In an even yet further aspect, the ratio of y to x is about 19:11 toAttorney Docket No.21101.0476P1 about 23:7. In a further aspect, the ratio of y to x is about 2:1 to about 11:4. In a still further aspect, the ratio of y to x is about 21:9 to about 11:4.

[0161] In various aspects, the peptide has a structure represented by a formula:R1O or a pharmaceutically

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

[0163] In various aspects, the peptide has a structure represented by a formula:or a pharmaceutically acceptable salt thereof.

[0164] In various aspects, the peptide has a structure represented by a formula:Attorney Docket No.21101.0476P1 wherein R20is selected froma sortase recognition sequence, a sugar residue, and a structure: ,wherein R31 and R32 isalkyl, C2-C8 alkyne, C1-C8azide, tetrazinyl, cyclooctynyl, norbornenyl, and –(CH2CH2O)mCH3, and wherein m is aninteger selected from 1 to 100.

[0165] In various aspects, the peptide has a structure represented by a formula:R1O H ,or a pharmaceutically acceptable salt thereof.

[0166] In various aspects, the peptide has a structure represented by a formula:Attorney Docket No.21101.0476P1 OH OH O H , or a pharmaceutically

[0167] In various aspects, the peptide has a structure represented by a formula:OH OH H or a pharmaceutically

[0168] In one aspect, n is an integer selected from 15 to 250. In a further aspect, n isan integer selected from 15 to 200. In a still further aspect, n is an integer selected from 15 to 150. In a yet further aspect, n is an integer selected from 15 to 100. In an even further aspect, n is an integer selected from 15 to 50. In an even still further aspect, n is an integer selected from 50 to 250. In an even yet further aspect, n is an integer selected from 100 to 250. In a further aspect, n is an integer selected from 150 to 250. In a still further aspect, n is an integer selected from 200 to 250. In a yet further aspect, n is an integer selected from 50 to 100. In an even further aspect, n is an integer selected from 100 to 150. In an even still further aspect, n is an integer selected from 150 to 200.

[0169] In one aspect, m is an integer selected from 1 to 100. In a further aspect, m isan integer selected from 2 to 100. In a still further aspect, m is an integer selected from 5 to 100. In a yet 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 isAttorney Docket No.21101.0476P1 an integer selected from 60 to 100. In a yet further aspect, m is an integer selected from 70 to 100. 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.

[0170] 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. R1 GROUPS

[0171] In one aspect, R1 is a glycosyl moiety having a structure represented by aformula selected from: ,Attorney Docket No.21101.0476P1 .by aformula selected from: , ,Attorney Docket No.21101.0476P1 .by a formula selected from: .by a formula selected from: ,Attorney Docket No.21101.0476P1 .represented by a formula selected from: , .

[0176] an even a arepresented by a formula selected from:Attorney Docket No.21101.0476P1 .represented by a formula selected from: , .by aformula selected from: ,Attorney Docket No.21101.0476P1 .by a formula selected from: .by a formula selected from: .an even a arepresented by a formula selected from:Attorney Docket No.21101.0476P1 , .represented by a formula selected from: .represented by a formula selected from: ,Attorney Docket No.21101.0476P1 .by aformula selected from: .

[0185] representedby a formula selected from: .

[0186] by a formula selected from: .

[0187] an even a arepresented by a formula selected from:Attorney Docket No.21101.0476P1 .by aformula selected from: .b. R10 AND R11 GROUPS

[0189] In one aspect, R10 and R11 ares elected from ‒OH and ‒NHAc. In a furtheraspect, each of R10and R11is ‒OH. In a still further aspect, each of R10and R11is ‒NHAc.

[0190] In various aspects, R10 is selected from ‒OH and ‒NHAc. In a further aspect,R10is ‒OH. In a still further aspect, R10is ‒NHAc.

[0191] In various aspects, R11 is selected from ‒OH and ‒NHAc. In a further aspect,R11is ‒OH. In a still further aspect, R11is ‒NHAc. c. R20 GROUPS

[0192] In one aspect, R20 is selected from ‒OR31, ‒NHR32, ‒N3, a protein tag, asortase recognition sequence, a sugar residue, and a structure: .

[0193] In various aspects, R20 is selected from ‒OR31, ‒NHR32, ‒N3, and a structure:Attorney Docket No.21101.0476P1 .

[0194] In a furtherand ‒NHR32. In a still furtheraspect, R20is ‒OR31. In a yet further aspect, R20is ‒NHR32.

[0195] In various aspects, R20 is ‒N3.

[0196] In various aspects, R20 is the protein tag. As would 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 visueal 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.

[0197] In various aspects, R20 is the sortase recognition sequence. As used herein, theterm “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.21101.0476P1 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).

[0198] In various aspects, R20 is the sugar residue. As used herein, the term “sugarresidue” 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.

[0199] In various aspects, R20 is a structure:. 3d. R 1 AND

[0200] In various aspects, R31 and R32 are selected from hydrogen, ‒CH2Ph, C1-C8alkyl, C2-C8 alkyne, C1-C8 azide, tetrazinyl, cyclooctynyl, norbornenyl, and –(CH2CH2O)mCH3. In a further aspect, R31 and R32 is selected from hydrogen, ‒CH2Ph, C1-C4alkyl, C2-C4 alkyne, C1-C4 azide, tetrazinyl, cyclooctynyl, norbornenyl, and –(CH2CH2O)mCH3. In a still further aspect, R31 and R32 is selected from hydrogen, ‒CH2Ph,methyl, ethyl, propyl, isopropyl, ‒CH≡CH, ‒CH2CH≡CH, ‒CH2N3, ‒CH2CH2N3, ‒CH2CH2CH2N3, tetrazinyl, cyclooctynyl, norbornenyl, and –(CH2CH2O)mCH3. In a yetfurther aspect, R31 and R32 is selected from hydrogen, ‒CH2Ph, methyl, ethyl, ‒CH≡CH,‒CH2N3, ‒CH2CH2N3, tetrazinyl, cyclooctynyl, norbornenyl, and –(CH2CH2O)mCH3. In anAttorney Docket No.21101.0476P1even further aspect, R31 and R32 is selected from hydrogen, ‒CH2Ph, methyl, ‒CH2N3,tetrazinyl, cyclooctynyl, norbornenyl, and –(CH2CH2O)mCH3.

[0201] In various aspects, R31 and R32 is selected from hydrogen, ‒CH2Ph, C1-C8alkyl, and C2-C8 alkyne. In a further aspect, R31 and R32 is selected from hydrogen, ‒CH2Ph,C1-C4 alkyl, and C2-C4 alkyne. In a still further aspect, R31 and R32 is selected fromhydrogen, ‒CH2Ph, methyl, ethyl, propyl, isopropyl, ‒CH≡CH, and ‒CH2CH≡CH. In a yetfurther aspect, R31 and R32 is selected from hydrogen, ‒CH2Ph, and methyl. In an evenfurther aspect, R31 and R32 is selected from hydrogen and methyl.

[0202] In various aspects, R31 and R32 is selected from hydrogen and C1-C8 azide. Ina further aspect, R31 and R32 is selected from hydrogen and C1-C4 azide. In a still furtheraspect, R31 and R32 is selected from hydrogen, ‒CH2N3, ‒CH2CH2N3, ‒CH2CH2CH2N3. In ayet further aspect, R31 and R32 is selected from hydrogen, ‒CH2N3, ‒CH2CH2N3. In an evenfurther aspect, R31 and R32 is selected from hydrogen and ‒CH2N3.

[0203] In various aspects, R31 and R32 is selected from hydrogen, tetrazinyl,cyclooctynyl, and norbornenyl. In a further aspect, R31 and R32 is selected from hydrogen andtetrazinyl. In a still further aspect, R31 and R32 is selected from hydrogen and cyclooctynyl.In a yet further aspect, R31 and R32 is selected from hydrogen and norbornenyl.

[0204] In various aspects, R31 and R32 is selected from hydrogen and –(CH2CH2O)mCH3.

[0205] In various aspect, v is selected from hydrogen and C1-C8 alkyl. In a furtheraspect, R31 and R32 is selected from hydrogen and C1-C4 alkyl. In a still further aspect, R31and R32 is selected from hydrogen, methyl, ethyl, propyl and isopropyl. In a yet furtheraspect, R31 and R32 is selected from hydrogen, methyl, and ethyl. In an even further aspect,R31 and R32 is selected from hydrogen and methyl.

[0206] In various aspects, R31 and R32 is hydrogen.2. EXAMPLE PEPTIDES

[0207] In one aspect, a peptide can be present as one or more of the followingstructures:Attorney Docket No.21101.0476P1 , , , ,or a pharmaceutically acceptable salt thereof.

[0208] It is contemplated that one or more peptides can optionally be omitted fromthe disclosed invention.Attorney Docket No.21101.0476P1

[0209] It is understood that the disclosed peptides can be used in connection with thedisclosed methods, compositions, food products, solid or semi-solid supports, kits, and uses.

[0210] It is understood that pharmaceutically acceptable derivatives of the disclosedpeptides 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

[0211] The peptides of the invention can be prepared by employing reactions asshown 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.

[0212] Reactions used to generate the peptides of the invention are prepared byemploying 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

[0213] In one aspect, substituted glycosylated polyhydroxyproline analogs can beprepared as shown below. SCHEME 1A.Attorney Docket No.21101.0476P1

[0214] acidprotecting group such as benzyl, PG2is an amine protecting group such as benzyl or carbobenzyloxy, R’ is an alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl 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 1B.Attorney Docket No.21101.0476P1prepared according to reaction Scheme 1B above. Thus, compounds of type 1.10 can be prepared by glycosyl coupling of an appropriate hydroxyl protected substituted aminoacid, e.g., 1.8 as shown above, using an appropriate protected glycosyl residue, e.g., 1.9 as shown above. Appropriate protected hydroxyl substituted amino acids and appropriate protected glycosyl residues are commercially available or prepared by methods known to one skilled in the art. The coupling is carried out in the presence of an appropriate Lewis acid, e.g., boron trifluoride diethyl etherate, in an appropriate solvent, e.g., dichloromethane, at an appropriate temperature, e.g., 0 °C to room temperature, for an appropriate amount of time, e.g., overnight. Compounds of type 1.11 can be prepared by deprotection of an appropriate protected amino acid, e.g., 1.10 as shown above. The deprotection reaction is carried out in the presence of an appropriate deprotecting reagent, e.g., hydrogen gas, in the presence of an appropriate catalyst, e.g., 10 % palladium on carbon, in an appropriate solvent, e.g., methanol. Compounds of type 1.13 can be prepared by reaction of an appropriate dialkyl dicarbonate, e.g., 1.12 as shown above, with an appropriate amino acid, e.g., 1.11 as shown above. Appropirate dialkyl dicarbonates are commercially available or prepared by methods known to one skilled in the art. The reaction is carried out in the presence of an appropriate base, e.g., sodium bicarbonate, in an appropriate solvent system, e.g., tetrahydrofuan and water, at an appropriate temperature, e.g., 0 °C. Compounds of type 1.14 can be prepared by cyclization of an appropriate carbamate protected amino acid, e.g., 1.13 as shown above. The reaction is carried out in the presence of an appropriate carboxyl activating agent, e.g., triphosgene, and an appropriate base, e.g., triethylamine, in the presence of an appropriate chloride scavenger, e.g., epichlorohydrin, in an appropriate solvent, e.g., tetrahydrofuan, at an appropriate temperature, e.g., room temperature. 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 typeAttorney Docket No.21101.0476P11.1, 1.2, 1.3, 1.4, 1.5, and 1.6), can be substituted in the reaction to provide glycosylatedpolyhydroxyproline analogs similar to Formula 1.7. 2. ROUTE II

[0216] In one aspect, peptide analogs can be prepared as shown below.SCHEME 2A.

[0217] incompound and peptide descriptions elsewhere herein. A more specific non-limiting example of the synthesis shown in Scheme 2A is set forth below. SCHEME2B. OAc OAc O 1. (PMe3)4Co,

[0218] In one aspect, compounds of type 2.7, and similar compounds, can be preparedaccording to reaction Scheme 2B above. Thus, compounds of type 2.7 can be prepared by NCA copolymerization of an appropriate glycosylated polyhydroxyproline analogs, e.g., 2.5Attorney Docket No.21101.0476P1 as shown above, and an alanine-N-carboxy anhydride, e.g., 2.4 as shown above. Alanine-N- carboxy anhydride is commercially available or prepared by methods known to one skilled in the art. 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 catalyst, e.g., tetrakis(trimethylphosphine)cobalt, in an appropriate solvent, e.g., tetrahydrofuran, at an appropriate temperature, e.g., 0 °C. Subsequent acetyl group deprotection was carried out in the presence of an appropriate base, e.g., potassium carbonate, 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 2.1, 2.2, and 2.3), can be substituted in the reaction to provide peptide analogs similar to Formula 2.4.D. COMPOSITIONS

[0219] As detailed herein, the disclosed peptides are useful in a wide range ofapplications, 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 cryoprotectant composition, an agricultural composition, a cosmetic composition) to facilitate use in these areas.

[0220] In various aspects, the disclosed composition reduces or inhibits ice crystalformation 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 crystal 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, −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 composition reduces or inhibits ice crystal formation at about −196 °C.Attorney Docket No.21101.0476P1

[0221] In various aspects, the disclosed composition reduces or inhibits ice crystalformation 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.

[0222] In various aspects, the disclosed composition comprises the peptide in aconcentration 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 μM, from about 1 μM to about 5 μM, from about 5 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 100 μM, from about 100 μM to about 250 μM, from about 250 μM to about 500 μM, from about 500 μM to about 750 μM, from about 750 μM 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 μM, about 10 μM, about 100 μM, 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.

[0223] It is understood that the disclosed compositions can be prepared from thedisclosed peptide. It is also understood that the disclosed compositions can be employed in the disclosed methods of using. 1. CRYOPROTECTANT COMPOSITIONS

[0224] In one aspect, disclosed are cryoprotectant compositions comprising aneffective 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 glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 toAttorney Docket No.21101.0476P1 about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues, and one or more selected from: (a) a non-antifreeze protein; (b) a microbe; (c) a cell component; and (d) a cell.

[0225] As detailed herein, the disclosed peptides are useful in preventing or reducingdamage 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).

[0226] Cryopreservation is a process whereby a sample (e.g., a biological sample) ispreserved 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.

[0227] In conventional cryopreservation techniques, the cells or tissue are placed in astorage solution, and then preserved by freezing. When the sample is to be used, it is thawed, and then placed in a cell culture medium. Cryopreservation 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.

[0228] To effect the desired cryoprotectant properties, the disclosed cryoprotectantcomposition 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 cryoprotectant 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.21101.0476P1

[0229] The disclosed cryoprotectant composition can be formulated as a non-freezingliquid (e.g., an aqueous solution or a non-aqueous solution), a non-freezing gel, a non- 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.

[0230] In various aspects, the cryoprotectant composition comprises a non-antifreezeprotein. 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 enzyme, 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.

[0231] In various aspects, the cryoprotectant composition comprises a microbe. Asused 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.

[0232] In various aspects, the cryoprotectant composition comprises a cellcomponent. 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.

[0233] In various aspects, the cryoprotectant composition comprises a cell. As usedherein, the term “cell” means the smallest, most basic 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 cavernosum cells (e.g., smooth muscle corpus cavernosum cells, epithelial corpus cavernosum cells), urinary bladder cells, urethral cells, ureter cells, kidney cells, testicular cells), bone marrow, primary cells, organoids, and other biological cells and tissues for cryopreservation.Attorney Docket No.21101.0476P1

[0234] The disclosed cyroprotectant composition can also contain various additivesincluding, 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.

[0235] In various aspects, the cryoprotectant composition further comprises a freezingpoint 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 −10 °C to about −20 °C, to from about −10 °C to about −18 °C, or to from about −10 °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 −10 °C, less than about −12 °C, less than about −15 °C, less than about −16 °C, less than about −17 °C, less than about −18 °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.

[0236] In various aspects, the cryoprotectant composition further comprises athickening 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 viscosity in one or more of these ranges can readily adhere 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.Attorney Docket No.21101.0476P1

[0237] In various aspects, the cryoprotectant composition further comprises a pHbuffer. 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.

[0238] In various aspects, the cryoprotectant composition further comprises ahumectant. 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., polydextrose), 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.

[0239] In various aspects, the cryoprotectant composition further comprises asurfactant. 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).

[0240] The cryoprotectant may also include other additives in addition to or in lieu ofthe 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

[0241] In one aspect, disclosed are agricultural compositions comprising a disclosedpeptide. In a further aspect, the peptide comprises a plurality of alanine residues and a plurality of glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues. In various aspects, the agricultural composition is a cryoprotectant agricultural composition.Attorney Docket No.21101.0476P1

[0242] As used herein, the term “agricultural compositions” refers to compositionsused 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.

[0243] Frost is a major environmental stress caused by low temperature combinedwith dewpoints 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.

[0244] Additionally, many tropical and subtropical species of plants are in danger ofextinction due to climate change and (a)biotic stress. Cryopreservation is a promising long- term technique to preserve the germplasms of these species. However, tropical species are temperature delicate. Thus, cryoprotectant agricultural compositions that can increase the explant’s tolerance to low temperatures and / or enable the plant cells to withstand freezing are desirable.

[0245] As detailed herein, the resistance of plants and plant tissue to frost and lowtemperatures, 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 spray apparatus 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.

[0246] In various aspects, the agricultural composition further comprises a non-ionicsurfactant, 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 0.5Attorney Docket No.21101.0476P1 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.

[0247] Additional agents that can be used in the disclosed agricultural compositioninclude, 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.

[0248] Although the disclosed agricultural composition can be applied to the plantsimmediately 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.

[0249] In various aspects, the disclosed agricultural composition can be appliedregularly 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 COMPOSITIONS

[0250] In one aspect, disclosed are cosmetic compositions comprising a disclosedpeptide. In a further aspect, the peptide comprises a plurality of alanine residues and a plurality of glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues.

[0251] 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 theAttorney Docket No.21101.0476P1 quality of the cosmetics to become deteriorated. The degradation of quality and the like can be avoided by incorporation of a disclosed peptide (i.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.

[0252] In addition, the disclosed cosmetic compositions can also be formulated topenetrate 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.

[0253] Methods of applying a cosmetic composition are well-known in the art andinclude, 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.

[0254] In various aspects, the cosmetic composition is a skin care product. Thus, invarious further aspects, the cosmetic composition is a topical formulation. Examples of topical formulations include, but are not limited to, creams, serums, gels, solutions, aerosols, ointments, sprays, lotions, and patches.

[0255] Topical cosmetic compositions can be co-formulated to include inhibitors ofapoptosis 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

[0256] In one aspect, disclosed are food products comprising a disclosed peptide. Ina further aspect, the peptide comprises a plurality of alanine residues and a plurality of glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues.Attorney Docket No.21101.0476P1

[0257] Frozen compositions, such as ice cream, typically incorporate a hardening stepthat involves quickly freezing the composition to obtain a desired frozen composition mouthfeel. Mouthfeel is affected by the size of ice crystals 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 crystals melt and recrystallize onto larger ice crystals in a dynamic process resulting in pronounced iciness, giving the product an undesirable characteristic. Controlling the ice crystal 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 quality finished product.

[0258] Frozen fruits and vegatables produced according to traditional freezingmethods 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 integrity of 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 celery will 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.

[0259] As detailed herein, the disclosed peptides can be incorporated into a foodproduct, 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 crystalAttorney Docket No.21101.0476P1 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 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 food product reduces or inhibits ice crystal formation at about −196 °C.

[0260] In various aspects, the disclosed food product reduces or inhibits ice crystalformation 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.

[0261] In various aspects, the disclosed food product comprises the peptide in aconcentration 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 μM, from about 1 μM to about 5 μM, from about 5 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 100 μM, from about 100 μM to about 250 μM, from about 250 μM to about 500 μM, from about 500 μM to about 750 μM, from about 750 μM 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 μM, about 10 μM, about 100 μM, 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

[0262] 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.Attorney Docket No.21101.0476P1F. SURFACES

[0263] In one aspect, disclosed are surfaces attached to a disclosed peptide. In afurther aspect, the disclosed peptide comprises a plurality of alanine residues and a plurality of glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues.

[0264] In one aspect, disclosed are solid or semi-solid supports comprising a surfaceattached (e.g., covalently attached or coated) to a residue of a disclosed peptide. In a further aspect, the disclosed peptide comprises a plurality of alanine residues and a plurality of glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues.

[0265] The development of anti-icing, anti-frosting surfaces is of importance inbuilding 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 visibility of traffic lights and surveillance cameras; decreases in efficiency of the heat exchanger and power generation efficiency of solar panels; breaking of power transmission lines in winter; and deterioration of the aerodynamic performance of aircraft wings.

[0266] As detailed herein, the disclosed peptides can be attached to a surface in orderto 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 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 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,Attorney Docket No.21101.0476P1 −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.

[0267] In various aspects, the disclosed surface reduces or inhibits ice crystalformation 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.

[0268] In various aspects, the disclosed solid or semi-solid support reduces or inhibitsice 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.

[0269] Methods of covalently attaching a peptide to a surface or a solid or semi-solidsupport 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.ps1801s26.

[0270] In various aspects, attached is via covalent attachment to the surface. Invarious further aspects, attached is via coating a cyroprotectant composition comprising the peptide on the surface.

[0271] In various aspects, the surface is a surface of a solid or semi-solid support, andwherein the support is a glass bead, a silica-based resin, a cellulosic resin, an agarose bead, a polystyrene bead, or a polyacrylamide resin.

[0272] In various aspects, the disclosed surface comprises the peptide in aconcentration 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 750Attorney Docket No.21101.0476P1 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 100 μM, from about 100 μM to about 250 μM, from about 250 μM to about 500 μM, from about 500 μM to about 750 μM, from about 750 μM 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 μM, about 10 μM, about 100 μM, about 1 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.

[0273] In various aspects, the cryoprotectant composition comprises the peptide in aconcentration 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 μM, from about 1 μM to about 5 μM, from about 5 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 100 μM, from about 100 μM to about 250 μM, from about 250 μM to about 500 μM, from about 500 μM to about 750 μM, from about 750 μM 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 μM, about 10 μM, about 100 μM, 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.

[0274] In various aspects, the disclosed solid or semi-solid support comprises thepeptide 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 μM, from about 1 μM to about 5 μM, from about 5 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 100 μM, from about 100 μM to about 250 μM, from about 250 μM to about 500 μM, from about 500 μM to about 750 μM, from about 750 μM 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 aboutAttorney Docket No.21101.0476P1 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 μM, about 10 μM, about 100 μM, 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.

[0275] In various aspects, the residue of the peptide comprises an N-terminus, and theN-terminus is covalently attached to the surface.

[0276] In various aspects, the residue of the peptide has a structure represented by aformula: , 1wherein R is a glycosyl by a formula: , ,Attorney Docket No.21101.0476P1 ;the ratio of y to x is of from about 3:2 to about 4:1, and wherein the sum of x and y is at least 30 residues, or a pharmaceutically acceptable salt thereof.

[0277] In various aspects, the residue of the peptide has a structure represented by aformula: wherein R20is selected fromtag, a sortase recognition sequence, a sugar residue, and a structure: ,wherein R31 and R32 is selected from hydrogen, ‒CH2Ph, C1-C8 alkyl, C2-C8 alkyne, C1-C8azide, tetrazinyl, cyclooctynyl, norbornenyl, and –(CH2CH2O)mCH3, and wherein m is aninteger selected from 1 to 100.

[0278] As used herein, the term “support” refers to a material or substrate (e.g., asurface) 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, metalAttorney Docket No.21101.0476P1 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.G. METHODS OF INHIBITING ICE CRYSTAL FORMATION IN A SAMPLE

[0279] In one aspect, disclosed are methods of inhibiting ice crystal formation in asample, the method comprising contacting the sample with an effective amount of a disclosed peptide. In a further aspect, the disclosed peptide comprises a plurality of alanine residues and a plurality of glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues.

[0280] The methods described herein are suitable for use in any number ofcryopreservation protocols. For example, in various aspects, the disclosed peptides, compositions, products, supports, and methods are useful for cryopreservation during supercooling 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.21101.0476P1 the ice block to the sample may hinder the ability of the peptide act as a cryoprotectant for the sample.

[0281] 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.

[0282] As would be readily appreciated by one of ordinary skill in the art, theconcentrations 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 μM, from about 1 μM to about 5 μM, from about 5 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 100 μM, from about 100 μM to about 250 μM, from about 250 μM to about 500 μM, from about 500 μM to about 750 μM, from about 750 μM 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 μM, about 10 μM, about 100 μM, 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.

[0283] In various aspects, the disclosed peptide and / or the disclosed compositionreduces 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, −32, −33, −34, −35, −36, −37, −38, −39, or −40 °C). In a still further aspect, the disclosedAttorney Docket No.21101.0476P1 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 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 peptide and / or the disclosed composition reduces or inhibits ice crystal formation at about −196 °C.

[0284] In various aspects, the disclosed peptide and / or the disclosed compositionreduces 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.

[0285] In various aspects, contacting is via application (e.g., application onto asample or surface, topical application). In various further aspects, contacting is via coating. In various further aspects, contacting is via spraying or dipping.

[0286] In various aspects, contacting is via covalent attachment. For example, asdetailed herein, in various aspects, the disclosed peptide can be covalently attached to a surface.

[0287] In various aspects, contacting is for a time period of at least 1 hour, at least 2hours, 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.

[0288] In various aspects, the method involves repeated contacting steps. Forexample, 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.

[0289] In various aspects, the sample is a biological material. Examples of biologicalmaterials include, but are not limited to, a non-antifreeze protein (i.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, 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,Attorney Docket No.21101.0476P1 an embryonic cell, a stem cell), a tissue (i.e., epithelial, connective, nervous, or muscle), and an organ (i.e., heart, kidney, liver, ovary, cornea).

[0290] In various aspects, the sample is a food product. Examples of food productsfor which the disclosed method can be useful include, but are not limited to, ice cream, yogurt, seafood, fruit, and meat products.

[0291] In various aspects, the sample is an agricultural product. Examples ofagriculture products for which the disclosed method can be useful include, but are not limited to, grains, feeds, soybeans, tree nuts, fruits, and vegetables.

[0292] In various aspects, the sample is a cosmetic. Examples of cosmetics for whichthe disclosed method can be useful include, but are not limited to, lips balms, hair products, makeup, nail products, soaps and lotions.

[0293] In various aspects, the method further comprises storing the biologicalmaterial for a period of time.

[0294] In various aspects, storing is at a temperature of about 25 ℃ or less. In afurther aspect, storing is at a temperature of abour 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 abour 5 °C. In an even still further aspect, storing is at a temperature of abour 0 °C. In an even further aspect, storing is at a temperature of abour -5 °C. In an even yet further aspect, storing is at a temperature of abour -10 °C. In a further aspect, storing is at a temperature of abour -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 abour - 30 °C. In an even still further aspect, storing is at a temperature of abour -35 °C. In an even further aspect, storing is at a temperature of abour -40 °C. In an even yet further aspect, storing is at a temperature of abour -45 °C. In a further aspect, storing is at a temperature of abour -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 abour -65 °C. In an even still further aspect, storing is at a temperature of abour -70 °C. In an even further aspect, storing is at a temperature of abour -75 °C. In an even yet further aspect, storing is at a temperature of abour -80 °C.

[0295] In various aspect, storing is at a temperature of about 5 ℃ or less. In a furtheraspect, storing is at a temperature of abour 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 abour 2 °C. In an even still furtherAttorney Docket No.21101.0476P1 aspect, storing is at a temperature of abour 0 °C. In an even further aspect, storing is at a temperature of abour -1 °C. In an even yet further aspect, storing is at a temperature of abour -2 °C. In a further aspect, storing is at a temperature of abour -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

[0296] In one aspect, the invention relates to the use of a disclosed peptide or aproduct 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 cryoprotectant composition, an agricultural composition, a cosmetic composition) to facilitate use in these areas.

[0297] Also provided are the uses of the disclosed peptides and products. In oneaspect, 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.

[0298] In a further aspect, the use relates to a process for preparing a compositioncomprising 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.

[0299] It is understood that the disclosed uses can be employed in connection withthe 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

[0300] In one aspect, disclosed are kits comprising a disclosed peptide and one ormore 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 glycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues; andAttorney Docket No.21101.0476P1 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.

[0301] In various aspects, the kit includes the biological material. In various furtheraspects, 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).

[0302] In various aspects the kit includes the food product. Examples of foodproducts include, but are not limited to, ice cream, yogurt, seafood, fruit, and meat products.

[0303] In various aspects, the kit includes the agricultural product. Examples ofagriculture products include, but are not limited to, grains, feeds, soybeans, tree nuts, fruits, and vegetables.

[0304] In various aspects, the kit includes the solid or semi-solid support. Examplesof 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., gel-like, such as a polymer support composed of hydrogel polymers) as further described herein.

[0305] In various aspects, the kit includes the cosmetic. Example of cosmeticsinclude, but are not limited to, lips balms, hair products, makeup, nail products, soaps, and lotions.

[0306] In a further aspect, the disclosed peptide and the biological material are co-formualated. In a still further aspect, the disclosed peptide and the food product are co- formualated. In a yet further aspect, the disclosed peptide and the agricultural product are co- formualated. In an even further aspect, the disclosed peptide and the solid or semi-solid support are co-formualated. In an even still further aspect, the disclosed peptide and the cosmetic are co-formulated.Attorney Docket No.21101.0476P1

[0307] All publications and patent applications cited in this specification are hereinincorporated 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

[0308] The following examples are put forth so as to provide those of ordinary skill inthe 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.

[0309] The Examples are provided herein to illustrate the invention, and should notbe construed as limiting the invention in any way. 1. CHEMISTRY EXPERIMENTALSa. INSTRUMENTATION AND GENERAL METHODS

[0310] Reactions were conducted under an inert atmosphere of N2, using oven-driedglassware unless otherwise stated. Hexanes and dichloromethane were purified by first purging with dry nitrogen, followed by passage through columns of activated 3Å 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 3Å 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 MΩ-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-Attorney Docket No.21101.0476P1 II light scattering (LS) and Wyatt Optilab T-rEX refractive index (RI) detectors. Separations were achieved using 105, 104, and 103Å Phenomenex Phenogel 5 μm 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 (δ 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. (2S,4R)-DIBENZYL 4-HYDROXYPYRROLIDINE-1,2-DICARBOXYLATE (Z-HYP-OBN)

[0311] (2S,4R)-Dibenzyldicarboxylate (Z-Hyp-OBn) wassynthesized by modifying a published procedure (D. Lee, et al., (2012) J. Am. Chem. Soc., 134, 8260-8267). N-Cbz-4-hydroxyproline (10.0 g, 37.8 mmol) was dissolved in DMF.11.46 g K2CO3was added (2.2 eq, 83.0 mmol) followed by 0.56 g NaI (3.7 mmol , 0.1eq). The reaction fask was put under N2 and 19.34g of BnBr (3.0 eq, 113 mmol) was added dropwise. The reaction stirred at RT overnight. The reaction was diluted with 90mL EtOAc, and washed 8x with 100 mL H2O. Following this, 1 × 100 mL brine wash and then the organic layer was dried over Na2SO4. The product was purified via column chromatography using 1:1 EtOAc:Hexanes. Like fractions were combined and concentrated yielding 12.0g (90.0% yield).Attorney Docket No.21101.0476P1 ii. N-Α-(CARBOBENZYLOXY)-4-O-(2,3,4,6-TETRA-O-ACETYL-D-GALACTOPYRANOSYL)-L-PROLINE BENZYL ESTER(Z- GALOAC4HYP-OBN) AcO OAc O AcO

[0312] N-α-O-acetyl-D-galactopyranosyl)-L-proline benzyl ester (Z-GalOAc4Hyp-OBn): GalOAc5and Z-Hyp-OBn coupling was performed by modifying published procedures. Anhydrous DCM (196 mL, 0.1M) was added to a flask containing GalOAc5(19.15 g, 2.5 eq.) and Z-Pro-OBn (7.0 g, 1.0 eq). The solution was cooled to 0 °C and BF3-Et2O (8.36 mL, 3.0 eq.) was added. The reaction was allowed to warm to RT and went overnight. Upon completion, the reaction was diluted with DCM and quenched with aqueous sat. NaHCO3. The organic layer was washed 3× with aqueous sat. NaHCO3, 2× aqueous sat. NaCl, and the organic layer was dried over sodium sulfate, filtered through cotton, and concentrated. The crude product was purified using column chromatography (1:1 Hexanes:EtOAc)(2 columns were required to separate the material). The fractions were analyzed with TLC (1:1 Hexanes:EtOAc, 10% sulfuric acid in ethanol stain). Like fractions were combined and concentrated resulting in 7.6g (57% yield). FIG.18 shows1H NMR of Z-GalOAc4Hyp-OBn. FIG.19 shows13C NMR of Z-GalOAc4Hyp-OBn. FIG.20 shows HSQC of Z- GalOAc4Hyp-OBn.Attorney Docket No.21101.0476P1 iii. O-(2,3,4,6-TETRA-O-ACETYL-D-GALACTOPYRANOSYL)-L-PROLINE(GALOAC4HYPZ-GALOAC4HYP-OBN) AcO OAc O AcO O

[0313] O-(2,3,4,6-tetra-O--L-proline (GalOAc4Hyp Z-GalOAc4Hyp-OBn) (3.8g, 1eq.) was dissolved in MeOH (221 mL, 0.025M) and added to a flask containing 10% Pd / C (0.76 g, 20% starting material mass) under H2. The reaction was stirred vigorously for 3 hours. The reaction progress was monitored by TLC (1:3 Hexanes:EtOAc + 1% acetic acid, phosphomolybdic acid stain). After 3 hours, the reaction was filtered through cotton and a 0.45 μm filter to remove the Pd / C. The reaction was then concentrated, resulting in a white solid.1H NMR analysis confirmed the disappearance of aromatic protons belonging to theBn protecting groups. The product was used directly in the next reaction without further purification. iv. N-Α-(BUTOXYCARBONYL)-O-(2,3,4,6-TETRA-O-ACETYL-D-GALACTOPYRANOSYL)-L-PROLINE (BOC- GALOAC4HYP) GALOAC4HYP) AcO OAc

[0314] N-α- - O-acetyl-D-galactopyranosyl)-L-proline (Boc-GalOAc4Hyp) GalOAc4Hyp) (2.35g, 1 eq.) was added to a 1:1 mixture of THF / water (43.1 mL) and cooled to 0 °C. NaHCO3(5.35 g, 12.5 eq) and BocO2(5.56 g, 5 equiv) were added consecutively at increased molar equivalents. The reaction was allowed to warm to RT and stirred overnight. The reaction progress was monitored by TLC (1:3 hexanes:EtOAc + 1% acetic acid, phosphomolybdic acid stain). Upon completion, theAttorney Docket No.21101.0476P1 reaction was washed 3x with Et2O to remove excess BocO2. The aqueous layer was cooled to 0 °C and acidified to pH 4-5 using acetic acid. The aqueous layer was extracted 3× with DCM and the combined organic layers dried over sodium sulfate. The organic layers were concentrated yielding a white solid. The crude product was purified using column chromatography (1:3 Hexanes:EtOAc +1% acetic acid). Fractions were analyzed by TLC (1:3 Hexanes:EtOAc + 1% acetic acid, phosphomolybdic acid) and combined to produce a white solid (1.8g, 58% yield over two steps). BocO2 was weighed out in a capped glass vial. FIG.21 shows1H NMR of Boc-GalOAc4Hyp. FIG.22 shows13C NMR of Boc- GalOAc4Hyp. FIG.23 shows HSQC of Boc-GalOAc4Hyp. v. O-((2,3,4,6-TETRA-O-ACETYL)-D-GALACTOPYRANOSE))-L-PROLINE-N-CARBOXYANHYDRIDE (GALOAC4HYP NCA) AcO OAc O O O

[0315] The cyclization ofwas completed by modifying publishmethods (Yali Hu, et al., (2022) National Science Review, 9, 8, August). The Boc- GalOAc4Hyp (0.66g, 1 eq) was dissolved in anhydrous THF (11.76 mL, 0.1M). Triphosgene (0.175 g, 0.5 eq) was added and the solution was cooled to 0 °C. Epichlorohydrin (0.46 mL, 5 eq) was added to the solution followed by distilled TEA (0.0445 mL, 1.1 equiv) was added dropwise. The reaction was stirred for 24 hours, and reaction progress was monitored by ATR-FTIR. After 24 hours the reaction was filtered through cotton to remove TEA-HCl salts. The reaction solution was evaporated under reduced pressure, before being sequestered in a tandem solvent trap system cooled by liquid N2.The traps were immediately quenched with ammonium hydroxide. The crude product was purified using anhydrous silica chromatography with 5% to 10% to 15% THF in DCM. The collected fractions were analyzed by ATR-FTIR. NCA containing fractions were combined resulting in 0.41g of white crystalline solid (73% yield). FIG.24 shows1H NMR of GalOAc4Hyp NCA. FIG.25 shows13C NMR of GalOAc4Hyp NCA. FIG.26 shows HSQC of GalOAc4Hyp NCA.Attorney Docket No.21101.0476P1 c. POLYPEPTIDE SYNTHESES AND MODIFICATIONSi. GENERAL PROCEDURE FOR PREPARATION OFPOLYGALOAC4HYP(PGALHYP)WITH AZIDO-LEU AMIDO- AMIDATE NICKEL CATALYST (1)

[0316] (Detwiler, R. E.; et al., (2021) J. Am. Chem. Soc. 143, 11482–11489). All polymerizationreactions were performed in an N2-filled glove box. NCAs were dissolved in DMF / THF at 50 mg / ml. GalHyp NCA was added in one shot via syringe at the desired monomer to initiator ratio. The reaction was removed from the glovebox in a bomb tube and heated at 50 °C and polymerization progress was monitored by ATR-FTIR where NCA carbonyl absorbances disappeared and amide bonds appeared (FIG.31A and FIG.31B). Referring to FIG.31A and FIG.31B, ATR-FTIR spectra showing the disappearance of GalHyp-OAc4 NCA and the formation of polyGalHyp-OAC4are shown. Polymerizations were complete overnight up to 50MI and 3 days at 100MI. Aliquots were removed for analysis. Acetylated hydroxyPro polymers were analyzed by SEC / MALS / RI as previously described while alanine containing polymers were endcapped and analyzed by1H NMR as previously described, since these were insoluble in our SEC / MALS / RI mobile phase of DMF (Yali Hu, et al., (2022) National Science Review, 9, 8, August). Representative spectra are shown in the spectral data section. Reactions were precipitated into 1mM HCl in diethyl ether (pH~3) to remove nickel species. Polymers were collected by centrifugation and dried under reduced pressure prior to deprotection of acetyl groups. Polymers underwent dialysis with a 2Kda membrane in MIlliQwater for 4 days. FIG. 27 shows representative 1H NMR of polyGalOAc4-Hyp in CDCl3.Attorney Docket No.21101.0476P1 ii. GENERAL PROCEDURE FOR STATISTICAL COPOLYMERSdissolved in THF at concentrations of 50 mg / ml. NCA’s were mixed at the desired ratio. The catalyst was added in one shot via syringe at the desired monomer. Acetyl groups were deprotected using previously stated conditions and purified via dialysis. (GalHyp0.33-s- Ala0.66)npolymers with lengths greater than 150 residues, had ~10% insoluble portion after recovery and that was centrifuged down and the soluble portion was recovered. FIG.30 shows representative1H NMR of PEG88-(GalOAc4-Hyp0.33-s-Ala0.66)234in CDCl3. iii. GENERAL METHOD FOR THE DEACETYLATION OF ACO-PROTECTED POLYMERS

[0318] The polypeptide was suspended in 0.25M K2CO3 in 1:1 MeOH:H2O andstirred overnight. The deacetylated polymer in solution was transferred to a 2000 MWCO dialysis tubing. FIG.32A and FIG.32B show representative ATR-FTIR spectra showing the disappearance of the acetyl protecting groups on the copolymer of ((GalHyp-OAc4)0.33-s- Ala0.66)nto form copolymer ((GalHyp-OH)0.33-s-Ala0.66)n. The concentrate was recovered,frozen, and lyophilized. FIG. 28 shows representative 1H NMR of polyGalOH4-Hyp in D2O.FIG.29 shows representative1H NMR of PEG88-Hyp0.33-s-Ala0.66)155in D2O. 2. BIOLOGICAL EXPERIMENTALSAttorney Docket No.21101.0476P1 a. GENERAL METHODS FOR SAFGP BIOLOGICAL AND ICE BINDINGASSAYSi. GENERAL METHOD FOR OBSERVING DYNAMIC ICE SHAPING

[0319] To observe dynamic ice shaping 10 uL of solution containing polypeptide in1X 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 crystal growth (Deleray, A. C. et al. BioRxiv 2023.09.07.556704). ii. GENERAL METHOD FOR COOLING SPLAT ASSAYS

[0320] 10 μL of solution containing polypeptide in PBS was dropped from 2 metersthrough a PVC pipe onto a precooled slide using a micropipette. 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 N2to 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 (Deleray, A. C.et al. BioRxiv 2023.09.07.556704).iii. GENERAL METHOD FOR QUANTIFYING ICERECRYSTALLIZATION INHIBITION

[0321] For all polypeptide solutions, Image J (Fiji) was used to analyze the meangrain size (MGS) of the ice crystals using our previously reported method (Deleray, A. C. et al. BioRxiv 2023.09.07.556704). 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.Attorney Docket No.21101.0476P1 Statistical analysis showed that the 150 μm × 150 μm region of the image resulted in a MGS that is reprehensive of the entire population. The region of the image to analyze was randomly selected. In all cases, three images were collected for each splat assay, and the ice crystal areas for each image were averaged. The average and standard deviation of the three images are presented for each splat and statistical significance was determined with a one- way ANOVA and post-hoc Tukey tests, where ns indicates not significant, * indicates p<0.05 and ** p<0.01. iv. GENERAL METHOD FOR DETERMINING CELL VIABILITYWITH A CCK8 ASSAY

[0322] 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% CO2for 24 hours to allow the cells to adhere to the plate. The 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, 2.0 mg / mL. Additionally, other wells of cells are treated with 100-X Triton to kill cells for 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 10 uL of CCK-8 solution (Dojindo) and incubated at 37 °C in 5% CO2for 3 hours. The absorbance at 450 nm of the treated cells was measured after the 3 hours of incubation using a BioTek Synergy HTK multi-mode reader. b. CIRCULAR DICHROISM

[0323] Polymers were either dissolved in PBS or Milli-Q water. A wavelength of 214nm, extinction coefficient of 2200 cm-1M-1and Beer’s law were used to determine unknown peptide concentration and normalize CD data. Samples were prepared at concentrations 0.1 mg / mL. All spectra were recorded as an average of 3 scans. The molar ellipticity ([θ]) was calculated using the equation [θ] = (θ) / (10*c*l), where θ is measured ellipticity (mdeg), c is concentration (M), and l is path length of the cuvette (cm). For plotting, molar ellipticity was represented as [θ]*10-3. 3. DISCUSSION

[0324] Copolypeptides were prepared composed of Ala and various Pro derivatives.Synthetic antifreeze glycoproteins via NCA polymerization were prepared. Galactosylated-Attorney Docket No.21101.0476P1 hydroxyproline (GalHyp) is a suitable substitute for the disaccharide-Thr residue and enables facile preparation of potent synthetic AFGP mimics (FIG.1). Referring to FIG.1, a chemical structure and cartoon representation of native AFGPs as compared to synthetic mimics based on statistical copolymers of galactosylated-Pro and Ala is shown.

[0325] NCA polymerization is an attractive route to obtain polypeptides since themethod is rapid, high yielding, scalable, and allows precise tuning of chain length up to high molecular weights (Deleray, A. C.; Kramer, J. R. (2022) Biomacromolecules , 23 (3), 1453– 1461; Kramer, J. R.; et al., (2015) Proc. Natl. Acad. Sci. , 112 (41), 12574–12579; Campos- García, V. R.; et al., (2017) Sci. Rep. , 7 (1), 1–12. Compared to solid phase peptide synthesis, substantially higher molecular weights can be achieved, which has been shown to dramatically improve activity in both natural and synthetic AFGPs (Knight, C. A.; et al., (1984) Nature 308 (5956), 295–296; Deleray, A.C.; et al., (2023) bioRxiv; Berger, T.; et al., (2019) J. Am. Chem. Soc.141 (48), 19144–19150; DeVries, A. L. (1971) Science 1971, 172(3988), 1152–1155; DeVries, A. L. (1986) Methods Enzymol. 127 (C), 293–303). Themethod is much more scalable than recombinant protein production methods, and while recombinant non-glycosylated AFPs have been achieved, AFGPs have so far resisted all efforts (Eskandari, A.; et al., (2020) Biomolecules 10 (12), 1–18; Voets, I. K. (2017) Soft Matter 13 (28), 4808–4823; Biggs, C. I.; et al., (2017) Nature Communications December p 1546).

[0326] GalHyp was prepared in one step by coupling of commercially availableperacetylated galactose and N-carbobenzyloxy-hydroxyproline-O-benzyl using boron trifluoride etherate as a Lewis acid (FIG.2). The conjugate was isolated as a mixture of 1:1 α:β anomers that were not separated. Amino acid protecting groups were quantitatively removed by hydrogenolysis over palladium on carbon. GalHyp NCA was formed using conditions optimized for Pro NCA cyclization via t-butoxycarbonyl-Pro with triphosgene and triethylamine. GalHyp NCA was obtained in excellent yield after purification by anhydrous flash column chromatography on dry silica in the fume hood (Detwiler, R. E.; et al., (2021) J.R. J. Am. Chem. Soc. 143, 11482–11489). Ala NCA was prepared according to publishedmethods. Since polyPro and densely-hydroxylated polyvinylalcohol have also been explored as antifreeze polymers, Pro NCA and AcOHyp NCAs were prepared in order to examine polypeptides structurally related to GalHyp but that vary in hydroxyl density and potentially conformation.

[0327] NCA polymerization was initiated using (PMe3)4Co for polypeptidescontaining Ala or with Ni-complexes for polypeptides containing only Pro-based structuresAttorney Docket No.21101.0476P1 since these complexes bypass the need for the NCA nitrogen proton (FIG.3, Table 1)(Deleray, A. C.; Kramer, J. R. (2022) Biomacromolecules 23, 1453– 1461). Degrees ofpolymerization were readily tuned by altering the monomer to initiator ratios, and amino acid compositions were tuned via the NCA feed ratios. Polymer yields were quantitative and chain lengths were determined by end-group analysis as previously described, and copolypeptide samples are listed in Table 1. Glycan acetate protecting groups were quantitatively removed after polymerization by treatment with K2CO3 in methanol:water. Since polyPro is reported to have IRI activity, Pro homopolymers were prepared along with copolymers of varied GalHyp densities up to 100% GalHyp. Since polyhydroxylated PVA has also shown IRI activity, polyHyp was prepared from AcOPro NCA where the acetate groups were removed post-polymerization as previously described. Hyp:Ala 1:2 was also prepared to compare the effects of a single vs. the four hydroxyls of GalHyp:Ala 1:2. All chains were prepared at a similar length of ca.100 residues and all samples were purified by dialysis against Milli-Q water. Table 1 shows the antifreeze polymer panel nomenclature, molar masses, and chain lengths. TABLE 1. Polypeptide Mn[a]DP[b]Attorney Docket No.21101.0476P1 Polypeptide Mn[a]DP[b]we g t, Mn, was determned by SEC / MALS / RI or 1H NMR as described in prior publications.47,50– 52 [b] DP = degree of polymerization. e. ANTIFREEZE ACTIVITY ASSAYS

[0328] Native AFGPs bind irreversibly to embryonic ice crystals, inhibiting prism-face growth and influencing macroscopic crystal shape and size (FIG.4A, left top panel). This action results in an overall reduction in crystal mean grain size (MGS), i.e., IRI, and shaping of single crystals into characteristic hexagonal and bipyramidal structures (Meister, K.; et al., (2018) J. Am. Chem. Soc.140 (30), 9365–9368). To quantitate MGS and observe crystal shaping, cryostage microscopy and cooling splat assays were used (Knight, C. A.; et al., (1984) Nature 308 (5956), 295–296; Deleray, A.C.; et al., (2023) bioRxiv). Images were collected of the ice wafers at time zero and after 40 minutes of ice crystal growth. Particle size was quantified from 3 separate ice crystal images, 150 μm × 150 μm regions with a minimum of 75 crystals analyzed, and using image analysis software or manual sizing of crystals too small for software recognition. The average and standard deviation are presentedAttorney Docket No.21101.0476P1 for each experiment and statistical significance was determined with a one-way ANOVA and post-hoc Tukey test. Activity for our various polypeptides in PBS were compared to PBS alone.

[0329] IRI activity for the various (co)polypeptide structures is shown in FIG. 4. Atidentical solution concentrations of 1 mg / mL in PBS, the 40-minute MGS values for Pro100, Hyp75, and GalHyp103were not statistically different from PBS alone. However, the 40- minute MGS for ice crystals formed in the presence of 1 mg / mL (GalHyp0.33-s-Ala0.66)103 was reduced by 61% from that of PBS alone (from ca.4052 to 1588 µM2). FIG.4 shows IRI activity of various proline-based homo- and co-polypeptide structures. All samples were prepared at 1 mg / mL in PBS and ice crystals were imaged after 40 minutes of growth. Chain lengths were (GalHyp0.33-s-Ala0.66)103, Pro100, Hyp75, GalHyp100 (Scale bars are 200 µm). Clearly, Ala residues play a major role in IRI activity since the GalHyp homopolymer showed no discernable activity. IRI data for the copolypeptide (Hyp0.33-s-Ala0.66)100 was attempted to be collected, however, the sample had very poor aqueous solubility. Centrifugation revealed an insoluble portion totaling 50% of the mass. The IRI activity of the soluble portion was measured, which was relatively minor (FIG.5 and FIG.6).

[0330] FIG. 5 shows the (Hyp0.33-s-Ala0.66)150 ice splat images at 0 minutes (left)and 40 (right) minutes. FIG.6 shows the MGS of ice crystals for synthetic polymers at 1 mg / mL at time 40 minutes. (GalHyp0.33-s-Ala0.66)103 is significant to all other polymers. No significance between GalHyp100, (Hyp0.33-s-Ala0.66)150, and PBS. Pro100 is not significant to GalHyp100, (Hyp0.33-s-Ala0.66)150, and PBS. Hyp75 is not significant to GalHyp100, (Hyp0.33-s-Ala0.66)150, and PBS. Dissolved in 1 mg / mL PBS. Scale bar is 200 µM.

[0331] Preparation of a (Pro0.33-s-Ala0.66)100 yielded a material that was entirelyinsoluble in water. A higher molecular weight Hyp200 was also examined and though activity was slightly increased from that of the 75mer (FIG.7 and FIG.8), the IRI effects were poor compared to those of the GalHyp:Ala copolymer. Clearly, the biomimetic glycosylated copolymer with the native Ala content was the most promising antifreeze polymer candidate. FIG.7 shows Hyp200 ice splat image at 0 minutes (left) and 40 minutes (right). Dissolved in 1 mg / mL PBS. Scalebar is 200 µM. FIG.8 shows Hyp200homopolymer MGS data compared to PBS control at 1 mg / mL in PBS.

[0332] Since length-dependent function has been clearly established in native AFGPs,as well as disaccharide-Thr based mimics, GalHyp was prepared containing chains from 100– 300 residues and with the native content of 66% Ala. A further 37% increase in IRI activityAttorney Docket No.21101.0476P1 was observed with the increase in chain length from 100 to 150 residues, as evidenced by the reduction in MGS when comparing the two (FIG.9). Referring to FIG.9, the IRI activity of (GalHyp0.66-s-Ala0.33)n glycopolypeptides of varied chain lengths is shown. All samples were prepared at 1 mg / mL in PBS and ice crystals were imaged after 40 minutes of growth (Scale bar is 200 µm. ** p < 0.01). The effect plateaued however, and further increasing the chain length to 225 or 300 residues did not result in any statistically significant increase in IRI activity. The 150, 225, and 300mers all offered a remarkable ca.80% reduction in MGS.

[0333] The ice-shaping properties of the (GalHyp0.33-s-Ala0.66)n polymers was alsoexamined since hexagonal shaping has been linked to ice-binding and IRI activity and since antifreeze materials resulting in spicular crystals that can cause cellular damage are not optimal. Ice shaping data is shown in FIG.10 and full-size images are shown in FIG.11 to FIG.14. Specifically, FIG.10 shows shaping of ice crystals by (GalHyp0.66-s-Ala0.33)nglycopolypeptides of varied chain lengths. FIG.11 shows (GalHyp0.33-s-Ala0.66)103 copolymer full size image of ice shaping at 1 mg / mL in PBS. FIG.12 shows (GalHyp0.33-s- Ala0.66)155 copolymer full size image of ice shaping at 1 mg / mL in PBS. FIG.13 shows (GalHyp0.33-s-Ala0.66)234 copolymer full size image of ice shaping at 1 mg / mL in PBS. FIG.14 shows (GalHyp0.33-s-Ala0.66)312 copolymer full size image of ice shaping at 1 mg / mL in PBS.All samples were prepared at 1 mg / mL in PBS (scale bars are 100 µm). Hexagonal, square, and amorphous crystals were the predominant structures observed, indicating the polymers indeed bind to the surface of specific faces of embryonic ice crystals resulting in directional growth. Since spicular crystals were not the predominant structures observed, the polymers have excellent potential for applications in cryobiology in addition to applications in foods and agriculture. f. POLYPEPTIDE CONFORMATIONAL ANALYSIS

[0334] Spectroscopic data indicates native AFGPs adopt an extended structure similarto the left-handed PPII helix. The extended helical structure has been proposed to play a role in the ice-binding mechanism. Therefore, to understand the secondary structures and IRI mechanisms of the various copolypeptides, analyses was conducted by circular dichroism (CD) spectroscopy. This technique relies on the absorption of light by peptide bonds. The wavelengths at which the absorptions occur, and their intensities, reveal characteristics about the orientation of those bonds Distinct CD signatures have been shown for the η→π* and π→π* transitions of PPII, intrinsically disordered, sheet, or α-helicalAttorney Docket No.21101.0476P1 (Greenfield, N. J. (2007) Nat. Protoc.1 (6), 2876–2890; Kelly, S. M. M. M.; Price, N. C. C. C. (2000) Curr. Protein Pept. Sci.1 (4), 349–384; van Stokkum, I. H.; et al., (1990) Anal. Biochem.191 (1), 110–118). In organic solvent, polyPro adopts a compact helical structure with 3.3 residues per turn and cis bonds, termed a PPI helix, while in aqueous solution the trans-bond PPII structure with 3 residues per turn is favored (Traub, W.; Shmueli, U. (1963) Nature 198 (4886), 1165–1166; Cowan, P. M.; et al., (1955) J. Chem. Inf. Model.176, 1062; Steinberg, I. Z.; et al., (1960) J. Am. Chem. Soc.1960, 82 (20), 5263–5279; Harrington, W. F.; Sela, M. (1958) Biochim. Biophys. Acta 27, 24–41; Kurtz, J.; et al., (1956) Nature 178, 1066–1067; Steinberg, I. Z.; et al., (1958) Biochim. Biophys. Acta 1958, 28, 647–648; Dukor, R. K.; et al., (1996) Biospectroscopy 2 (2), 83–100). The PPI vs. PPII ratio is not only affected by solvent, but also by substituents at the 4-position of the proline ring. The two conformations can be easily differentiated by their absorption ellipticities. The PPI n→π* results in a moderate negative minimum at ~230 nm and a strong maximum at ~212 forthe π →π*. The PPII form has a minor positive maximum at ~226 nm and strong negative minimum at ~206 nm.

[0335] Prior structural work on glycosides of Hyp focused mainly on Hyp-richglycoproteins that are the major proteinaceous components of higher plant walls, such as Gp1 and extension (Ferris, P. J.; et al., (2001) Biochemistry 40 (9), 2978–2987; Liang, R.; et al., (2020) Front. Plant Sci.11, 1207). Predominant glycan structures are β-galactosides and α- arabinosides of varied chain lengths. CD spectra of these proteins indicated PPII structures, however, the specific effects of glycosylation were unclear since these natural peptides and proteins are typically isolable in only minuscule amounts and / or are heterogeneous in nature (Taylor, C. M.; et al., (2012) Glycobiology. pp 757–767). A 2010 study by Owens et al. compared the CD structures of acetyl-capped 9-mers of Pro, Hyp, and β-D-GalHyp (Owens, N. W.; et al., (2010) J. Am. Chem. Soc.132 (14)). The CD spectra of all three peptides were typical of a PPII helix, however, the glycosylated helices had substantially greater thermal stability. Molecular dynamics simulations indicated that the Gal units interact with both the peptide backbone and one another to result in the increased stability. Interestingly, later work by Huang et al. on β-D-GalHyp-containing collagen mimetic peptides reported the glycans slightly destabilized the collagen triple helices, but did not reduce their refolding rate (Huang, P. W.; et al., (2016) Amino Acids 48 (12), 2765–2772). It should be noted that interpretation of structural effects in short peptides is convoluted by their small size. Since the PPII helix contains 3 residues per turn, a 9-mer can only form three helical turns.Attorney Docket No.21101.0476P1

[0336] CD spectra of the high molecular weight GalHyp chains were obtained, alongwith spectra of GalHyp:Pro statistical copolypeptides so that whether or not there were any perturbations in structure resulting from increasing glycosylation density could be observed (FIG.15A) All data are reported in molar ellipiticity, which normalizes for samples of differing concentrations. As expected, all structures revealed spectra characteristic of PPII conformations. Increasing the density of statistically distributed GalHyp from 20% to 60% to 100% resulted in slight increases in characteristic PPII absorbances in the CD spectra, aligning well with prior data (vide supra).

[0337] The CD spectra of the (GalHyp0.33-s-Ala0.66)n were in stark contrast to those ofGalHyp and (GalHypx-s-Proy)n(FIG.15B). The Ala-containing polypeptide spectra are highly characteristic of those of known intrinsically disordered peptides (Miles, A. J.; et al., (2023) Commun. Biol.6 (1), 823; Chemes, L. B.; et al., (2012) Methods Mol. Biol.895, 387– 404). FIG.15A and FIG.15B show aqueous circular dichroism data for GalHyp containing polypeptides at 20 °C. Samples were typically prepared at 0.1 mg / mL and data was obtained in MillQ water for those in FIG.15A while those in FIG.15B were obtained in PBS buffer. Without wishing to be bound by theory, this was surprising since native AFGPs containing ca.66% Ala and 33% disaccharide-Thr, and prior synthetic analogs of identical compositions, adopted PPII-type structures. CD spectra was also obtained at varied temperatures to see if a different conformation is adopted at low temperatures (FIG.16). Specifically, FIG.16 shows CD spectra of (GalHyp0.33-s-Ala0.66)155 at various temperatures wherein the polymer was dissolved at 0.1mg / mL in PBS. The spectra at 4 °C was still characteristic of an intrinsically disordered structure, though the absorbances were slightly increased from those at 40 °C and 80 °C. Ultimately, the PPII structure is not required for ice-binding since these disordered structures had high IRI activity. g. CYTOCOMPATIBILITY STUDIES

[0338] For future biomedical applications, the structures were examined forcytocompatibility. Various similar synthetic glycopolypeptides have been shown to be non- toxic and well tolerated by human cells (Deleray, A.C.; et al., (2023) bioRxiv; Wardzala, C. L.; et al., (2022) Front. Cell Dev. Biol.10, 1–13; Kohout, V. R.; et al., (2023) J. Am. Chem. Soc. , 145 (30), 16573–16583). Therefore, toxicity or proliferation were not expected to be impacted the sAFGPs. To verify this, CCK8 assays were conducted in human embryonic kidney (HEK) 293 cells. HEK293 cells were selected as a model cell line in broad laboratoryAttorney Docket No.21101.0476P1 use. Viability was measured after 24 hours of treatment with sAFGPs of concentrations ranging from 0.2 – 2.0 mg / mL. Cells were treated with PBS as a negative control and Triton X-100 as a positive control (FIG.17). FIG.17 shows HEK 293 cell viability as determined by CCK8 assay following 24-hour incubation with (GalHyp0.66-s-Ala0.33)150at the indicated concentrations; live control is media alone and Triton X-100 was a positive control for dead cells; standard deviation; ** indicates p < 0.01. At the highest concentration tested, which exceeded the necessary concentration for IRI activity, there were no statistically significant effects on cellular viability observed. Considering that cryogenic applications typically would expose cells to antifreezes at room temperatures for only short durations and sub-freezing temperatures thereafter, compatibility for 24 hours at 37 °C is in excess of likely exposure. From these data, it was concluded that these structures are well tolerated by live human cells and promising for biomedical applications.

[0339] Refering to FIG. 6, HEK 293 cell viability as determined by CCK8 assayfollowing 24-hour incubation with (GalHyp0.66-s-Ala0.33)150at the indicated concentrations; live control is media alone and Triton X-100 was a positive control for dead cells; standard deviation; ** indicates p < 0.01. h. CONCLUSIONS

[0340] Bioinspired synthetic polymer that strongly inhibits the growth of ice crystalswere prepared. This structure, based on galactosylated hydroxyproline and alanine, is well- tolerated by human cells and is composed entirely of natural building blocks. Chain length affected ice recrystallization inhibition activity at lower molecular weight but plateaued above 150 residues. Natural antifreeze glycoproteins adopt an extended polyproline II-type helical structure. While polymers of galactosylated hydroxyproline adopt this structure, compositions with 66% alaine, as found in native fish antifreeze glycoproteins, were disordered. However, the native conformation is not required for antifreeze activity. Polyproline was previously explored as an antifreeze polymer; however, in side-by-side comparisons activity was very poor compared to disclosed structures. Without wishing to be bound by theory, considering the labor and expense required to harvest natural antifreeze glycoproteins, access to efficient synthetics is expected to play important roles in biomedicine, foods, agriculture, coatings, and building materials.Attorney Docket No.21101.0476P1J. REFERENCES

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[0392] Kramer, J. R.; Onoa, B.; Bustamante, C.; Bertozzi, C. R. Chemically TunableMucin Chimeras Assembled on Living Cells. Proc. Natl. Acad. Sci.2015, 112 (41), 12574– 12579. https: / / doi.org / 10.1073 / pnas.1516127112.

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[0403] Kurtz, J.; Berger, A.; Katchalski, E. Mutarotation of Poly-L-Proline. Nature1956, 178, 1066–1067. https: / / doi.org / 10.1038 / 1781066a0.Attorney Docket No.21101.0476P1

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[0413] Wardzala, C. L.; Clauss, Z. S.; Kramer, J. R. Principles of GlycocalyxEngineering with Hydrophobic-Anchored Synthetic Mucins. Front. Cell Dev. Biol.2022, 10, 1–13. https: / / doi.org / 10.3389 / fcell.2022.952931.

[0414] Kohout, V. R.; Wardzala, C. L.; Kramer, J. R. Mirror Image Mucins and ThioMucins with Tunable Biodegradation. J. Am. Chem. Soc.2023, 145 (30), 16573–16583. https: / / doi.org / 10.1021 / jacs.3c03659.Attorney Docket No.21101.0476P1

[0415] It will be apparent to those skilled in the art that various modifications andvariations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

Attorney Docket No.21101.0476P1 CLAIMS What is claimed is:

1. A peptide comprising a plurality of alanine residues and a plurality ofglycosylated residues selected from glycosylated hydroxyproline residues and a combination of glycosylated hydroxyproline residues and glycosylated threonine residues, wherein the ratio of the plurality of alanine residues to the plurality of glycosylated residues is from about 3:2 to about 4:1, and wherein the peptide has a chain length of at least 30 amino acid residues.

2. The peptide of claim 1, wherein the peptide consists essentially of the plurality ofalanine residues and the plurality of glycosylated residues.

3. The peptide of claim 1 or claim 2, wherein the plurality of alanine residues is presentin the peptide in an amount of from about 60 wt% to about 70 wt%.

4. The peptide of claim 1 or claim 2, wherein the plurality of alanine residues is presentin the peptide in an amount of about 66 wt%.

5. The peptide of any one of claims 1 to 4, wherein the plurality of glycosylated residuesare a combination of glycosylated hydroxyproline residues and glycosylated threonine residues.

6. The peptide of claim 5, wherein the peptide does not contain more than eleven Ala-Ala-Thr repeats.

7. The peptide of any one of claims 1 to 6, wherein the plurality of glycosylated residuesare glycosylated hydroxyproline residues.

8. The peptide of any one of claims 1 to 7, wherein the plurality of glycosylated residuesare glycosylated with one or more of αGal, βGal, αGalNAc, βGalNAc, βGal(1→3)αGalNAc, αLac, or βLac.

9. The peptide of any one of claims 1 to 8, wherein the ratio of the plurality of alanineresidues to the plurality of glycosylated residues is about 3:2.Attorney Docket No.21101.0476P110. The peptide of any one of claims 1 to 8, wherein the ratio of the plurality of alanineresidues to the plurality of glycosylated residues is about 4:1.

11. The peptide of any one of claims 1 to 10, wherein the peptide has a chain length of atleast 50 amino acid residues.

12. The peptide of any one of claims 1 to 10, wherein the peptide has a chain length of atleast 100 amino acid residues.

13. The peptide of any one of claims 1 to 10, wherein the peptide has a chain length of atleast 150 amino acid residues.

14. The peptide of any one of claims 1 to 10, wherein the peptide has a chain length offrom 30 amino acid residues to 500 amino acid residues.

15. The peptide of any one of claims 1 to 10, wherein the peptide has a chain length offrom 100 residues to 300 residues.

16. The peptide of any one of claims 1 to 10, wherein the peptide has a number averagemolecular weight (Mn) of at least about 3,000.

17. The peptide of any one of claims 1 to 10, wherein the peptide has a number averagemolecular weight (Mn) of at least about 5,000.

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

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

20. The peptide of any one of claims 1 to 10, wherein the peptide has a number averagemolecular weight (Mn) of from about 3,000 to about 50,000.

21. The peptide of any one of claims 1 to 10, wherein the peptide has a number averagemolecular weight (Mn) of from about 10,000 to about 30,000.

22. The peptide of any one of claims 1 to 10, wherein the peptide has a number averagemolecular weight (Mn) of from about 14,000 to about 25,000.Attorney Docket No.21101.0476P123. The peptide of any one of claims 1 to 10, wherein the peptide has a degree ofpolymerization (DP) of at least about 30.

24. The peptide of any one of claims 1 to 10, wherein the peptide has a degree ofpolymerization (DP) of at least about 50.

25. The peptide of any one of claims 1 to 10, wherein the peptide has a degree ofpolymerization (DP) of at least about 100.

26. The peptide of any one of claims 1 to 10, wherein the peptide has a degree ofpolymerization (DP) of at least about 150.

27. The peptide of any one of claims 1 to 10, wherein the peptide has a degree ofpolymerization (DP) from about 100 to about 330.

28. The peptide of any one of claims 1 to 27, wherein the peptide has a structurerepresented by a formula: ,wherein n is an integer wherein R1is a glycosyl moiety having a structure represented by a formula selected from: ,Attorney Docket No.21101.0476P1 ;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 at least 30, or a acceptable salt thereof.

29. The peptide of claim [00157], wherein x is 6 or greater and y is 18 or greater.

30. The peptide of claim [00157], wherein x is 10 or greater and y is 30 or greater.

31. The peptide of claim [00157], wherein x is from 20 to 120 and y is from 60 to 180.

32. The peptide of claim [00157], wherein the sum of x and y is at least 100.

33. The peptide of any one of claims 28 to 32, wherein R1 is a glycosyl moiety having astructure represented by a formula selected from:Attorney Docket No.21101.0476P1 .

34. The35. The peptide of any one of claims 28 to 32, wherein R10 is ‒OH.

36. The peptide of any one of claims 28 to 35, wherein the ratio of x to y is about 2:3 toabout 1:4.

37. The peptide of any one of claims 28 to 36, wherein the peptide has a structurerepresented by a formula: R1O , or a pharmaceutically38. The peptide of any one of claims 28 to 36, wherein the peptide has a structurerepresented by a formula: ,or a pharmaceutically39. The peptide of any one of claims 28 to 36, wherein the peptide has a structurerepresented by a formula:Attorney Docket No.21101.0476P1 , or a pharmaceutically40. The peptide of any one of claims 28 to 36, wherein the peptide has a structurerepresented by a formula: , wherein R20is selectedtag, a sortase recognition sequence, a sugar residue, and a structure: . wherein R31 and R32C1-C8 alkyl, C2-C8 alkyne,C1-C8 azide, tetrazinyl, cyclooctynyl, norbornenyl, and –(CH2CH2O)mCH3, and wherein m is an integer selected from 1 to 100.

41. The peptide of claim 40, wherein the protein tag is selected from CBP, FLAG< GST,HA, HBH, MBP, Myc, poly His, S-tag, SUMO, TAP, TRX, and V5.Attorney Docket No.21101.0476P142. The peptide of claim 40 or claim 41, wherein the sortase recognition sequence isselected 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.

43. The peptide of claim 42, 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.

44. The peptide of claim 42, wherein the natural amino acid is selected from glutamicacid, aspartic acid, arginine, histidine, and lysine.

45. The peptide of any one of claims 42 to 44, wherein the unnatural amino acid isselected from hydroxyproline, beta-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, and pyroglutamic acid.

46. The peptide of claim 40 or claim 41, wherein the sortase recognition sequence isselected from LPETG (SEQ ID NO: 5), LPETGG (SEQ ID NO: 6), LPETGGG (SEQ ID NO: 7), and LPETGGGG (SEQ ID NO: 8).

47. The peptide of any one of claims 40 to 46, wherein the sugar residue is a residue of asugar selected from fructose, glucose, and lactose.

48. The peptide of any one of claims 40 to 46, wherein R20 is selected from ‒OR31,‒NR32, ‒N3, and a structure: .

49. The peptide of anypeptide has a structurerepresented by a formula:Attorney Docket No.21101.0476P1 R1O H , or a pharmaceutically50. The peptide of any one of claims 40 to 46, wherein the peptide has a structurerepresented by a formula: OH OH H , or a pharmaceutically51. The peptide of any one of claims 40 to 46, wherein the peptide has a structurerepresented by a formula: OH OH H ,or a pharmaceutically52. A cryoprotectant composition comprising an effective amount of the peptide of anyone of claims 1 to 51or an acceptable salt thereof, and one or more selected from: (a) a non-antifreeze protein;Attorney Docket No.21101.0476P1 (b) a microbe;(c) a cell component; and(d) a cell.

53. The cryoprotectant composition of claim [0009], wherein the non-antifreeze protein isselected from an enzyme, a hormone, an antibody, a growth factor, a vaccination protein, a therapeutic protein, or a nutrient protein.

54. A food product comprising the peptide of any one of claims 1 to 51.

55. The food product of claim 54, wherein the food product is selected from ice cream,yogurt, seafood, fruit, and a meat product.

56. An agricultural composition comprising the peptide of any one of claims 1 to 51.

57. A surface attached to the peptide of any one of claims 1 to 51.

58. The surface of claim 57, wherein attached is via covalent attachment to the surface.

59. The surface of claim 57, wherein attached is via coating a cyroprotectant compositioncomprising the peptide on the surface.

60. The surface of any one of claims 57 to 59, wherein the surface is a surface of a solidor semi-solid support, and wherein the support is a glass bead, a silica-based resin, a cellulosic resin, an agarose bead, a polystyrene bead, or a polyacrylamide resin.

61. A cosmetic composition comprising the peptide of any one of claims 1 to 51.

62. A method of inhibiting ice crystal formation in a sample, the method comprisingcontacting the sample with an effective amount of the peptide of any one of claims 1 to 51.

63. The method of claim 62, wherein the sample is a biological material.

64. The method of claim 63, wherein the biological material is selected from a non-antifreeze protein, a microbe, a cell component, a cell, a tissue, and an organ.

65. The method of claim 64, wherein the cell is selected from a sperm cell, an egg matrixcell, an embryonic cell, and a stem cell.Attorney Docket No.21101.0476P166. The method of claim 62, wherein the sample is a food product.

67. The method of claim 62, wherein the sample is an agricultural product.

68. The method of claim 62, wherein the sample is a solid or semi-solid support.

69. The method of claim 62, wherein the sample is a cosmetic.

70. The method of claim 62, further comprising storing the biological material for aperiod of time.

71. The method of claim 70, wherein storing is at a temperature of about 25 ℃ or less.

72. The method of claim 70, wherein storing is at a temperature of about 4 ℃ or less.

73. A kit comprising the peptide of any one of claims 1 to 51 and one or more selectedfrom: (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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