Supramolecular and covalent collagen mimetic hydrogels and nanostructures with cation-Π interactions

Abstract

Disclosed herein are peptides compounds which are capable of forming collagen mimetic biomaterials. In some embodiments, the presently disclosed peptides may be stabilized by cation-pi interactions or by covalent interactions involving levodopa residues. In some embodiments, the present disclosure provides higher order structures formed from the peptides disclosed herein, including homotrimers, heterotrimers, and hydrogels.

Description

DESCRIPTIONSUPRAMOLECULAR AND COVALENT COLLAGEN MIMETIC HYDROGELS AND NANOSTRUCTURES WITH CATION-Π INTERACTIONSFederal Funding Statement5 This invention was made with government support under Grant Nos.1842494 and 2203937 awarded by the National Science Foundation. The government has certain rights in the invention.Background of the Invention

[0001] This application claims the benefit of United States Provisional Patent 10 Application No.63 / 700,393, filed September 27, 2024, the entirety of which is incorporated herein by reference.Reference to a Sequence Listing

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing 15 XML, created on September 11, 2025, is named RICEP0159WO.xml and is 72,244 bytes in size.I. Field of the Invention

[0003] The present invention relates generally to the field of chemistry, particularly 20 organic chemistry, biochemistry, medicinal chemistry, and biomedical engineering. More particularly, it concerns compositions for mimicking collagen.II. Description of Related Art

[0004] Fibrous collagens are comprised of amino acid sequences typically 1,000 residues in length. This leads to difficulty recreating synthetic molecules that adopt their 25 fibrous nature or hallmark characteristics, such as D-banding periodicity. Using collagen mimetic peptides, much shorter amino acid sequences can supramolecularly assemble into higher-order structures. However, a dearth of examples of these fibrous collagen mimetics that utilize canonical amino acids and their supramolecular interactions has limited applications.Synthesizing collagen mimetic fibrils with supramolecular assembly has remained a difficult 30 challenge because of the reliance on passive mechanisms that mediate self-assembly, unlike the natural collagen’s active, enzymatically guided assembly (Revell et al., 2021; Taguchi et1al., 2007) and the peptides that polymerize into higher order structures that exhibit characteristics, such as D-banding of natural collagen is also difficult because of the disparity in molecular weight and the relative simplicity of peptides versus natural collagens (Kotch & Raines, 2006; Hu et al., 2022). Therefore, there remains a need for compositions that are useful 5 for mimicking fibrous collagen

[0005] This invention was funded in part by the Robert A. Welch Foundation under Welch Grant No. C-2118.2Summary

[0006] The present disclosure provides peptide compositions for mimicking collagen.

[0007] In one aspect, the present disclosure provides peptides between 33 and 48 amino acids in length, wherein said peptide comprises a first region, a second region and a third 5 region, wherein:the first region is of the formula (PX1G)n, wherein:P is proline;X1is independently selected from amino acids with a positive formal charge at neutral pH; and10 G is glycine;n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14;the second region is of the formula (POG)m; whereinP is proline;O is 4-hydroxyproline;15 G is glycine;m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14;the third region is of the formula (Y1OG)o ; whereineach Y1is independently selected from aromatic amino acids, anionic amino acids, and levodopa, and further wherein at least one Y1 is an aromatic 20 amino acid;O is 4-hydroxyproline;G is glycine; ando is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; ora peptide between 31 and 48 amino acids in length comprising a fifth region, a sixth region, 25 and a seventh region, wherein:the fifth region is of the formula (POG)p, wherein:P is proline;O is 4-hydroxyproline;G is glycine;30 p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11;the sixth region of between three and twenty-four amino acids in length, each amino acid residue being independently selected from among proline, 4-3hydroxyproline, glycine, an amino acid having a reactive amine group, and levodopa; andthe seventh region is of the formula (POG)q; whereinP is proline;5 O is 4-hydroxyproline;G is glycine;q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

[0008] In some embodiments, the peptide comprises a first region, a second region and a third region, wherein:10 the first region is of the formula (PX1G)n, wherein:P is proline;X1is independently selected from amino acids with a positive formal charge at neutral pH; andG is glycine;15 n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14;the second region is of the formula (POG)m; whereinP is proline;O is 4-hydroxyproline;G is glycine;20 m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14;the third region is of the formula (Y1OG)o ; whereinwherein each Y1is independently selected from aromatic amino acids, anionic amino acids, or levodopa, and further wherein at least one Y1 is an aromatic amino acid;25 O is 4-hydroxyproline;G is glycine; ando is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0009] In some embodiments, X1 is selected from among arginine and lysine. In some embodiments, at least one X1 is arginine. In some embodiments, at least one X1 is lysine. In 30 some embodiments, n is 2, 3, 4, 5, or 6. In some embodiments, n is 4. In some embodiments, n is 2. In some embodiments, the first region of the peptide is (PKG)2(PRG)2 (SEQ ID NO: 1) or (PKGPRG)2 (SEQ ID NO: 2). In some embodiments, the first region of the peptide is (PKG)2(PRG)2 (SEQ ID NO: 1). In some embodiments, the first region of the peptide is4(PKGPRG)2 (SEQ ID NO: 2). In some embodiments, m is 2, 3, 4, 5, or 6. In some embodiments, m is 4.

[0010] In some embodiments, each Y1 is independently selected from among aspartic acid, glutamic acid, tyrosine, tryptophan, phenylalanine, histidine, and levodopa. In some 5 embodiments, each Y1 is independently selected from among aspartic acid, glutamic acid, tyrosine, tryptophan, histidine, and phenylalanine. In some embodiments, at least one Y1is aspartic acid. In some embodiments, at least one Y1 is aspartic acid. In some embodiments, at least one Y1is tyrosine. In some embodiments, at least one Y1is tryptophan. In some embodiments, at least one Y1 is phenylalanine. In some embodiments, at least one Y1 is 10 levodopa, at least one Y1is aspartic acid, and at least one Y1is an aromatic amino acid such as tyrosine, tryptophan, phenylalanine, and histidine. In some embodiments, at least one Y1 is levodopa, at least one Y1is glutamic acid, and at least one Y1is an aromatic amino acid such as tyrosine, tryptophan, phenylalanine, and histidine. In some embodiments, at least one Y1 is levodopa. In some embodiments, at least one Y1is histidine. In some embodiments, Y1is 15 independently selected from aspartic acid and tyrosine. In some embodiments, Y1 is independently selected from aspartic acid and tryptophan. In some embodiments, Y1is independently selected from aspartic acid and phenylalanine. In some embodiments, Y1 is independently selected from aspartic acid and histidine. In some embodiments, Y1is independently selected from aspartic acid and phenylalanine. In some embodiments, Y1 is 20 independently selected from glutamic acid and tyrosine. In some embodiments, Y1is independently selected from glutamic acid and tryptophan. In some embodiments, Y1 is independently selected from among glutamic acid and histidine. In some embodiments, Y1is independently selected from glutamic acid and phenylalanine. In some embodiments, o is 2, 3, 4, 5, or 6. In some embodiments, o is 2. In some embodiments, o is 4.25

[0011] In some embodiments, the third region is further defined as (Y1anOG)s(Y1arOG)t, (Y1arOG)s(Y1anOG)t or (Y1anOGY1arOG)o, or (Y1arOGY1anOG)o; wherein:Y1an is an anionic amino acid;Y1ar is an aromatic amino acid;o is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; and30 the sum of s and t is equal to o as defined above.

[0012] In some embodiments, the third region is (DOG)2(FOG)2 (SEQ ID NO: 3), (DOGFOG)2 (SEQ ID NO: 4), (FOGDOG)2 (SEQ ID NO: 5), (DOG)2(WOG)2 (SEQ ID NO: 6), (DOGWOG)2 (SEQ ID NO: 7), (WOGDOG)2 (SEQ ID NO: 8), (DOG)2(YOG)2 (SEQ ID NO: 9), (DOGYOG)2 (SEQ ID NO: 10), or (YOGDOG)2 (SEQ ID NO: 11).5

[0013] In some embodiments, the third region is (DOG)2(FOG)2 (SEQ ID NO: 3), (DOGFOG)2(SEQ ID NO: 4), or (FOGDOG)2(SEQ ID NO: 5). In some embodiments, the third region is (DOG)2(WOG)2 (SEQ ID NO: 6), (DOGWOG)2 (SEQ ID NO: 7), or (WOGDOG)2(SEQ ID NO: 8). In some embodiments, the third region is (DOG)2(YOG)2(SEQ 5 ID NO: 9), (DOGYOG)2 (SEQ ID NO: 10), or (YOGDOG)2 (SEQ ID NO: 11).

[0014] In some embodiments, the first region is located at the C-terminus of the peptide. In some embodiments, the third region is located at the N-terminus of the peptide. In some embodiments, the second region is located between the first region of the peptide and the third region of the peptide.10

[0015] In some embodiments, the peptide further comprises a fourth region of the formula (POG)r ; whereinP is proline;O is 4-hydroxyproline;G is glycine; and15 r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0016] In some embodiments, wherein r is 2, 3, 4, 5, or 6. In some embodiments, r is 4. In some embodiments, the fourth region is located at the C-terminus of the peptide.

[0017] In some embodiments, the peptide has at least 90% sequence identity with (PKG)2(PRG)2(POG)4(DOG)2(FOG)2 (SEQ ID NO: 12). In some embodiments, the peptide 20 has at least 90% sequence identity with (PKG)2(PRG)2(POG)4(DOG)2(WOG)2(SEQ ID NO:13). In some embodiments, the peptide has at least 90% sequence identity with (PKG)2(PRG)2(POG)4(DOG)2(YOG)2(SEQ ID NO: 14). In some embodiments, the peptide has at least 90% sequence identity with (POG)4(PKG)2(PRG)2(POG)2(DOG)2(YOG)2 (SEQ ID NO: 15). In some embodiments, the peptide has at least 90% sequence identity with 25 (PKGPRG)2(POG)4(DOG)2(YOG)2 (SEQ ID NO: 16). In some embodiments, the peptide has at least 90% sequence identity with (PKGPRG)2(POG)4(DOGYOG)2 (SEQ ID NO: 17). In some embodiments, the peptide has at least 90% sequence identity with (PKGPRG)2(POG)4(YOGDOG)2 (SEQ ID NO: 18). In some embodiments, the peptide is (PKG)2(PRG)2(POG)4(DOG)2(FOG)2 (SEQ ID NO: 12). In some embodiments, the peptide is 30 (PKG)2(PRG)2(POG)4(DOG)2(WOG)2 (SEQ ID NO: 13). In some embodiments, the peptide is (PKG)2(PRG)2(POG)4(DOG)2(YOG)2 (SEQ ID NO: 14). In some embodiments, the peptide is (POG)4(PKG)2(PRG)2(POG)2(DOG)2(YOG)2 (SEQ ID NO: 15). In some embodiments, the peptide is (PKGPRG)2(POG)4(DOG)2(YOG)2 (SEQ ID NO: 16). In some embodiments, the6peptide is (PKGPRG)2(POG)4(DOGYOG)2 (SEQ ID NO: 17). In some embodiments, the peptide is (PKGPRG)2(POG)4(YOGDOG)2(SEQ ID NO: 18).

[0018] In some embodiments, the peptide comprises:a fifth region of the formula (POG)p, wherein:5 P is proline;O is 4-hydroxyproline;G is glycine; andp is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;a sixth region of between and twenty seven six amino acids in length, each 10 amino acid residue being independently selected from among proline,4-hydroxyproline, glycine, an amino acid having a reactive amine group, and levodopa; anda seventh region of the formula (POG)q; whereinP is proline;15 O is 4-hydroxyproline;G is glycine;q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0019] In some embodiments, the peptide sequence is suitable for presenting an axial interaction between a lysine residue and a levodopa residue in a hydrogel formed from the 20 peptide. In some embodiments, the peptide sequence is suitable for presenting a lateral interaction between a lysine residue and a levodopa residue in a hydrogel formed from the peptide. In some embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 3. In some embodiments, the sixth region comprises at least one levodopa residue. In some embodiments, the sixth region comprises at least one levodopa residue and at least one amino acid having a 25 reactive amine group. In some embodiments, the sixth region comprises at least one levodopa residue and at least one lysine. In some embodiments, the sixth region is of the formula POGX2Y2G (SEQ ID NO: 19); wherein X2 is levodopa and Y2 is an amino acid having a reactive amine group. In some embodiments, the sixth region is of the formula POGX2KG (SEQ ID NO: 20); wherein X2 is levodopa. In some embodiments, the sixth region is of the 30 formula PY2GX2OG (SEQ ID NO: 21); wherein X2 is levodopa and Y2 is an amino acid having a reactive amine. In some embodiments, the sixth region is of the formula PKGX2OG (SEQ ID NO: 22); wherein X2 is levodopa.

[0020] In another aspect, the present disclosure provides hydrogel compositions formed from any of the peptides described above7

[0021] In another aspect, the present disclosure provides compositions comprising a first peptide, a second peptide, and a third peptide, wherein:the first peptide is 20 to 40 amino acids in length and comprises at least 3 amino acid residues having a positive formal charge at neutral pH and at least 5 1 aromatic amino acid residue;the second peptide is 20 to 40 amino acids in length and comprises at least 1 amino acid residue having a positive formal charge at neutral pH, at least 1 amino acid residue having a negative formal charge at neutral pH, and at least 1 aromatic amino acid residue; and10 the third peptide is 20 to 40 amino acids in length and comprises at least 1 amino acid residue having a positive formal charge at neutral pH, at least 2 amino acid residues having a negative formal charge at neutral pH, at least 1 aromatic amino acid residue, and at least 1 levodopa residue.

[0022] In some embodiments, the first peptide comprises 5, 6, 7, 8, 9, or 10 amino acids 15 having a positive formal charge at neutral pH. In some embodiments, the first peptide comprises 8 amino acids having a positive formal charge at neutral pH. In some embodiments, the first peptide has a net positive formal charge. In some embodiments, the first peptide has a net formal charge of between +5 and +10. In some embodiments, the first peptide has a net formal charge of +8. In some embodiments, the second peptide has a net formal charge between 20 -5 and +5. In some embodiments, the second peptide has a negative net formal charge. In some embodiments, the second peptide has a positive net formal charge. In some embodiments, the second peptide has a net formal charge between +2 and +4. In some embodiments, the second peptide has a net formal charge of +3. In some embodiments, the third peptide has a net formal charge between -5 and +5. In some embodiments, the second peptide has a positive net formal 25 charge. In some embodiments, the third peptide has a negative net formal charge. In some embodiments, the second peptide has a net formal charge between -3 and -5. In some embodiments, the second peptide has a net formal charge of -4. In some embodiments, the first peptide has at least 90% sequence identity with PKGROGPKGFOGYOGPRGROGKKGPRGPOG (SEQ ID NO: 23). In some embodiments, 30 the second peptide has at least 90% sequence identity with SKGDOGPOGDRGPKGPOGYKGPOGDKGFRG (SEQ ID NO: 24). In some embodiments, the third peptide has at least 90% sequence identity with PDGDRGPRGPOGYOGDDGPEGXOGPPGDOG (SEQ ID NO: 25). In some embodiments, the first peptide is PKGROGPKGFOGYOGPRGROGKKGPRGPOG (SEQ ID NO: 23). In8some embodiments, the second peptide is SKGDOGPOGDRGPKGPOGYKGPOGDKGFRG (SEQ ID NO: 24). In some embodiments, the third peptide is PDGDRGPRGPOGYOGDDGPEGXOGPPGDOG (SEQ ID NO: 25).

[0023] Other objects, features and advantages of the present disclosure will become 5 apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Note that simply because a particular compound is ascribed to one 10 particular generic formula doesn’t mean that it cannot also belong to another generic formula.9Brief Description of The Figures

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed 5 description.

[0025] FIGS.1A-1D show intrahelical versus interhelical assembly mechanisms of collagen mimetic peptides. FIG.1A: The hierarchical assembly of collagen commences with polyproline type II (PPII) helices assembling into a right-handed triple helix. These helices then associate into fibrils. FIG.1B: Tessellation utilizes symmetry rules to pair all theoretically 10 possible interactions between each PPII strand of the triple helix (black horizontal arrows) (SEQ ID NO: 47). FIG. 1C: Sticky-ended triple helix assembly relies on intrahelical electrostatic interactions between the leading to middle and middle to trailing PPII strands (black horizontal arrows) (SEQ ID NO: 46). This assembly strategy leaves unpaired sets of electrostatic interactions on the leading and trailing strands (gray vertical arrows) which can 15 interact interhelically. FIG.1D: Blunt-ended assembly of triple helices has been proposed to assemble through electrostatic interactions. Each triple helix is assembled, and then all available charge pairs form interhelical interactions (gray vertical arrows) (SEQ ID NO: 45).

[0026] FIGS.2A-2H show quantification of cation-π pairs stabilization to triple helical structure in fiber-forming collagen mimetic peptides. FIG.2A. Rendering of axial pairwise 20 interactions with supramolecular interaction distances, from left to right: lysine-aspartate charge pair, an arginine-tyrosine, arginine-phenylalanine, and arginine-tryptophan cation-π interactions. Black lines denote the supramolecular interaction between amino acid side chains.FIG.2B. A representative sequence of each peptide’s substitution scheme (SEQ ID NO: 48). Underlined amino acids correlate to the nomenclature. FIG. 2C. Circular dichroism (CD) 25 wavelength scans for phenylalanine-substituted FD2F2FIG. 2D. CD scans for tryptophan- substituted FD2W2 FIG.2E. CD scans for tyrosine-substituted FD2Y2 FIG.2F. CD thermal unfolding curves for phenylalanine-substituted FD2F2 FIG.2G. CD thermal unfolding curves for tryptophan-substituted FD2W2 FIG. 2H. CD thermal unfolding curves for tyrosine- substituted FD2Y2.30

[0027] FIGS.3A-3C show inverted Eppendorf® tubes of D2X2 substituted CMPs at 2% w / v in MQ H2O. (FIG.3A) FD2Y2 inverted Eppendorf® tubes. The pH 4.5 solution is a hydrogel and retains bubbles. (FIG.3B) FD2F2 inverted Eppendorf® tubes. (FIG.3C) FD2W210inverted Eppendorf® tubes. Peptide samples were prepared in pH 4.5, 7.4, and 9.0 in MQ H2O, respectively. Precipitation is observed as pairwise interactions strengthen at higher pH.

[0028] FIGS.4A-4C show CD Thermal Melts of D2X2 substituted CMPs at pH 4.5 and 7.4 while being monitored at 225 nm. (FIG.4A) FD2Y2thermal melt spectra (FIG.4B) 5 FD2F2 thermal melt spectra (FIG.4C) FD2W2 thermal melt spectra.

[0029] FIGS.5A-5E show characterization data for FD2Y2supramolecular assemblies at varying pH values. FIG.5A. A schematic of fiber formation at varying pH for FD2Y2. When the temperature (T) is below the Tm, supramolecular fibrillogenesis occurs. As electrostatic and cation-π interactions are enhanced near neutral pH, fibers begin to aggregate. FIG. 5B.10 Scanning electron microscopy (SEM) of FD2Y2at pH 4.5. The scale bar is 3µm. FIG.5C. Cryo- EM of FD2Y2, which forms a hydrogel at pH 4.5, shows a fibrous network. The scale bar is 40 nm. FIG.5D. Scanning electron microscopy (SEM) of FD2Y2at pH 7.4. The scale bar is 3µm.FIG.5E. Cryo-EM of FD2Y2, which remains viscous but starts to precipitate, shows a clustering of the fibrous assembly as pairwise interaction strengths increase. The scale bar is 200 nm. 15

[0030] FIGS.6A-6D show data related to a phosphate buffer screen of FD2Y2 at pH 7.4 (FIG.6A) Cryo-EM of FD2Y2in 10 mM phosphate buffer. Blank areas were present as there was a higher amount of association from the ion-containing buffer. (FIG.6B) Cryo-EM of FD2Y2in H2O. Scale bars are 200 nm. (FIG.6C) SAXS data for FD2Y2in both H2O (MQ) and phosphate buffer. (PB) (FIG.6D) Extrapolated dimensions of fibers in solution from SAXS 20 data.

[0031] FIGS.7A & 7B show SEM of FD2Y2 at pH 7.4. (FIG.7A) A zoomed-out SEM micrograph of 2 % (w / v) of FD2Y2. (FIG.7B) A zoomed-in SEM micrograph of 2 % (w / v) of FD2Y2 showing distinct clustering of the collagen mimetic fibers.

[0032] FIGS.8A-8D show a comparison of FDY2and FDY2(SEQ ID NOs: 49 and 50).25 FIG.8A. Sequence comparison of the two most stable staggers of FD2Y2 and FDY2. The FDY2 substation scheme results in a relatively larger energy gap. Deconvolved pairwise interaction strengths are at neutral pH (Cole et al., 2022; Zheng et al., 2019). FIG.8B. CD scan of FDY2 shows PPII character FIG.8C. First derivative of a thermal melt curve acquired on CD. FIG.8D. Sheer sweep of FDY2 and FD2Y2 hydrogel.30

[0033] FIGS.9A-9F show circular dichroism and electron microscopy of F0 (FIG.9A) CD spectra of F0. (FIG.9B) CD thermal melt curves of F0 monitored at 225 nm. (FIG.9C) CD first-order derivative of F0 thermal melt curves. (FIG.9D) Cryo-EM pf F0 fibers show multiple fibers associating at pH 7.4. The scale bar is 0.4 nm. (FIG.9E) SEM of F0 shows a fibrous matrix. (FIG.9F) Zoomed micrograph of F0’s fibrous matrix.11

[0034] FIGS.10A-10F show alternating interactions yield triple helical hydrogel and D-banding. FIG.10A. Sequence of FDY2with highlightedaminoacid substitutions FIG.10B.CD scan of FDY2 shows PPII character FIG.10C. First derivative of a thermal melt curve acquired on CD. FIG.10D. Sheer sweep of FDY2hydrogel FIG.10E. Cryo-EM of D-banding 5 fibers. The scale bar is 0.500 nm. FIG.10F.2D reconstruction of D- banding classes.

[0035] FIG.11 shows possible stagger comparisons of FD2Y2and FDY2(SEQ ID NOs: 49 and 50). Left: FD2Y2 staggers between strands show that the energy gap is in comparison with stagger (1) and staggers (2) and (5). Right: FDY2staggers between strands show that the energy gap is in comparison with stagger (1) and staggers (2), (3) and (6), but the energy 10 difference between the most stable (1) is greater than that for FD2Y2and its competing staggers.Relative pairwise interactions were deconvolved at pH 7.4.

[0036] FIGS.12A-12F show that the antiparallel association of triple helices leads to nanotube and nanosheets at neutral pH. FIG. 12A: Triple helices can also interact in an antiparallel manner, which satisfies a set of pairwise interactions at both the N and C-termini.15 This association is hypothesized, without being bound by theory, to drive tubular or sheet-like structures. FIG.12B: Comparison of the propensity of FDY2and FYD2(SEQ ID NOs: 50 and 51) to associate in a parallel or antiparallel manner with the three most stable shifts for each association paradigm. FIG. 12C: Cryo-EM of FDY2shows unilamellar and multi-lamellar nanotubes at pH 7.4. FIG.12D: Representative Cryo-EM of FDY2 at pH 7.4 shows nanosheet 20 formation. Scale bars are 100 nm. FIG.12E: A bar graph of 2.0 wt % (v / v) samples of FDY2and FYD2 after seven days shows pH-dependent rates of precipitation. FIG.12F: A bar graph of FDY2and FYD2zeta potentials at varying pH demonstrates a difference in overall charge of the microfibrils, nanotubes and nanosheets.

[0037] FIGS.13A & 13B show inverted Eppendorf® tubes of FDY2and FYD2at 2% 25 w / v in MQ H2O. (FIG. 13A) FDY2 inverted Eppendorf® tubes. The pH 4.5 solution is a hydrogel and retains bubbles. (FIG.13B) FD2F2 inverted Eppendorf® tubes. Peptide samples were prepared in pH 4.5, 7.4, and 9.0 MQ H2O, respectively. Precipitation is observed in all FYD2 samples. FDY2 is a hydrogel at pH 4.5 and solvated.

[0038] FIGS. 14A & 14B shows possible triple helix orientation to explain 8+2 30 stacking of FDY2 (FIG.14A) Hydrophobic slice of the central POG region in FDY2 compared with a slice from the experimental Cryo- EM data. This correlates to the lighter band of the D- band observed in FIG.10. (FIG.14B) Compared with a slice from the experimental Cryo-EM data, the overlap slice of the terminal regions in FDY2. This proposes a theoretical 16+4 packing12in the overlap region corresponding to the dark portion observed in the D-banding. The triple helical model was prepared using AlphaFold3. (Abramson, 2024).

[0039] FIGS.15A-15C show FYD2 supernatant characterization by circular dichroism. (FIG.15A) CD spectra of FYD2supernatant at various pH values. (FIG.15B) CD thermal melt 5 curves of FYD2 supernatant at various pH values. (FIG.15C) CD thermal spectra first-order derivative of FYD2supernatant at various pH values. All samples also had precipitate, which could lead to inaccuracies in calculated MRE.

[0040] FIGS.16A-16D show circular dichroism spectra of homotrimers and FDopa. (a) KGDopaO CD spectra of supramolecular (supra) and covalently captured species. (b) 10 OGDopaK CD spectra of supra and covalently captured species. (c) AGDopaO CD spectra of supra and covalently captured species. (d) FDopa CD spectra of supra and covalently captured. Supra spectra collected in 1 mM bicarbonate buffer at pH 4.5. Covalently captured spectra collected in 1 mM bicarbonate buffer at pH 9.5.

[0041] FIGS.17A-17I show the reaction kinetics of OGDopaK (FIG.17A, FIG.17B, 15 FIG.17C), KGDopaO (FIG.17D, FIG.17E, FIG.17F) and AGDopaO (FIG.17G, FIG.17H, FIG.17I) homotrimers at basic pH monitored by circular dichroism. (FIG.17A) CD thermal melt curves for OGDopaK from the supramolecular assembly to 72 hrs of covalent capture at pH 9.5. (FIG.17B) OGDopaK CD thermal reverse melts demonstrate a change in hysteresis (FIG.17C) First-order derivatives of OGDopaK containing peptides, where the minima denote 20 the transition temperature (Tm). (FIG. 17D) CD thermal melt curves for KGDopaO from supramolecular to 72 hrs. (FIG. 17E) KGDopaO CD thermal reverse melts demonstrate a change in hysteresis, where MRE recovery is greatest at 72 hrs (FIG. 17F) First-order derivatives of KGDopaO containing peptides, where the minima denote the Tm and stabilization of 48.5 °C at 72 hrs. (FIG.17G) CD thermal melt curves for AGDopaO from supramolecular 25 to 72 hrs. (FIG.17H) AGDopaO CD thermal reverse melts demonstrate a little to no change in hysteresis (FIG. 17I) First-order derivatives of AGDopaO containing peptides, where the minima denote the Tm. For CD, raw data are presented as points, and lines are a 3rd-order Savitsky-Golay fit (Savitzy & Golay, 1964).

[0042] FIGS. 18A-18D show higher order characterization of homotrimers with 30 MALDI ToF MS in linear positive mode. (FIG.18A) KGDopaO spectra shows clear dimer (expected 4590.9 m / z), trimer (expected 6886.3 m / z) and tetramer (expected 9181.7 m / z) formation. (FIG.18B) OGDopaK spectra shows clear dimer (expected 4590.9 m / z), trimer (expected 6886.3 m / z), tetramer (expected 9181.7 m / z) and possible +2 ionization states of higher-order species (e.g., pentameric, heptameric). (FIG. 18C) AGDopaO spectra shows13dimer formation (expected 4475.6 m / z). (FIG.18D) ABC- Dopa shows a mass correlating to the dimer formation within the range of the B – C dimer (expected 6061 m / z).

[0043] FIGS. 19A-19D illustrate pairwise interaction geometry and oxidation of levodopa (Dopa) to o-benzoquinone. (FIG.19A) A schematic showing Dopa oxidation and 5 then the subsequent possible Schiff Base and Michael Addition reactions with a primary amine.Dopa-Dopa crosslinking is also a possible product. (FIG. 19B) The prototypical collagen sequence necessary for different interaction geometries with lateral occurring between Yaa (top sequence) and Xaa of the same triplet (left X of central sequence) or axial occurring between Yaa (top sequence) and Xaa of the subsequent triplet (right X of central sequence). Top 10 sequence is SEQ ID NO: 58; central sequence is SEQ ID NO: 59; bottom sequence is SEQ ID NO: 60 (FIG. 19C) A lateral presentation of Lys and Dopa (blue and teal, respectively; connected with dotted line from Lys at right to Dopa at left). (FIG.19D) An axial presentation of Lys and Dopa (blue and magenta, respectively; connected with dotted line from Lys at left to Dopa at right). Amino acids were substituted from PDB ID: 3B0S15

[0044] FIGS.20A-20D show the UV-Vis spectra of homotrimers and an ABC-type Heterotrimer. (FIG. 20A) KGDopaO spectra after reacting at pH = 9.5 compared to the supramolecular homotrimer (pH = 4.5). (FIG.20B) OGDopaK spectra at pH = 9.5 compared to the supramolecular homotrimer (pH = 4.5). (FIG. 20C) AGDopaO spectra at pH = 9.5 compared to the supramolecular homotrimer (pH = 4.5). A distinct peak at ca. 495 nm 20 correlates to the distinct red color. (FIG.20D) ABC-22 Dopa spectra at pH = 4.5 and 9.5.

[0045] FIG.21 shows Eppendorf® tubes of homotrimer and heterotrimer solutions. From left to right: KGDopaO, OGDopaK, ABC-Dopa (pH = 7.4) and AGDopaO at 3mM and pH = 9.5.

[0046] FIGS. 22A-22C show KGDopaO 6,9 Gly deletion characterization with 25 circular dichroism. (FIG. 22A) Sequence comparison of KGDopaO and KGDopaO with glycine deletions at positions 6 and 9 (SEQ ID NOs: 52 and 53). Deleted glycines are underlined in the original sequence of KGDopaO at top. (FIG.22B) CD spectra of covalently captured and supramolecular KGDopaO 6,9 Gly deletion. (FIG.22C) CD thermal melts of covalently captured and supramolecular KGDopaO 6,9 Gly deletion. There is a lack of strong 30 thermal transition for both samples. All samples were covalently captured at pH= 9.5 and recorded after twenty-five days.

[0047] FIGS. 23A-23F show molecular dynamics plots of distance correlation between Lys and Dopa in axial and lateral interaction geometries within KGDopaO. (FIG.23A) Plotted frequencies of distances and correlated energy minimized structure between the14ζ-amine of Lys and the center of the benzyl ring for each pair of interacting amino acids in an axial geometry. (FIG.23B) Plotted frequencies of distances and correlated energy minimized structure between the Lys ζ-amine and Michael-addition site (Cδ2) of the benzyl ring for each pair of interacting amino acids in an axial geometry. (FIG. 23C) Plotted frequencies of 5 distances and correlated energy minimized structure between the Lys ζ-amine and Schiff Base sites (C^1 and Cζ) of the benzyl ring for each pair of interacting amino acids in an axial geometry. (FIG. 23D) Plotted frequencies of distances and correlated energy minimized structure between the Lys ζ-amine and the center of the benzyl ring for each pair of interacting amino acids in a lateral geometry. (FIG.23E) Plotted frequencies of distances and correlated 10 energy minimized structure between the Lys ζ-amine and Michael-addition site (Cδ2) of the benzyl ring for each pair of interacting amino acids in a lateral geometry. (FIG.23F) Plotted frequencies of distances and correlated energy minimized structure between the Lys ζ-amine and Schiff Base sites (C^1 and Cζ) of the benzyl ring for each pair of interacting amino acids in an axial geometry. Maxima of each distribution are denoted next to each line, where axial 15 interactions are generally in closer proximity than lateral interactions.

[0048] FIGS. 24A-24C show the angle characterization of cation-π interactions in KGDopaO. (FIG 24A) Lateral cation- π interaction with distance and angle correlations. (FIG 24B) Axial cation-π interaction. (FIG 24C) Axial versus lateral interaction angle population curves.20

[0049] FIGS.25A-25D show covalent capture of a complex heterotrimer. (FIG 25A) Stabilizing pairwise interactions are linked. The relative interaction strength of these stabilizing pairwise interactions are, in descending interaction strength: axial charge pairs (K – D), cation- π pairs (R – Y) and lateral charge pairs (D – R) (SEQ ID NOs: 54-56). In dark background and underlined is the axial Lys – Dopa pairwise interaction. (FIG 25B) Circular dichroism (CD) 25 spectra of each unary, binary and ternary mixtures. All supra samples are at pH = 4.5, and covalent capture was carried out at pH = 7.4 (FIG 25C) CD thermal melt curves of all equivalent possible mixtures of the ABC-type heterotrimer at 0.3 mM in 1 mM phosphate buffer. (FIG 25D) First derivatives of the CD thermal melt unfolding curves.

[0050] FIGS.26A & 26B show that HSQC NMR confirms a unique ABC-composition 30 is maintained upon covalent capture. (FIG.26A) Overlay of each HSQC of monomers A (dark blue), B (purple) and C (light blue). The supramolecular ABC mixture (black) shows corresponding peaks to each monomer and three distinct trimer peaks (Tx) which can be correlated with their respective strands based upon previous assignment (Cole et al., 2024). All spectra were acquired at pH = 4.5. (FIG.26B) The covalently captured sample, adjusted to pH15= 7.4, is overlaid with the HSQC spectra the supramolecular ABC mixture. After 120 hrs, the only observed peak shift is the TCsignal, which is adjacent to the covalent bond forming with C-22-Dopa. All spectra were acquired at 2.7 mM of total peptide, 10 % D2O (v / v) in 9 mM phosphate buffer and at 30 °C.5

[0051] FIGS. 27A-27D show sequence design and rheological characterization of FDopa. (FIG.27A) Sequence of polymerizing FDopa(SEQ ID NO: 57). (FIG.27B) Proposed polymerization mechanism of a sticky-ended peptide. (FIG.27C) FDopa inverted Eppendorf® tubes at pH 4.5, 7.4 and 9.0. (FIG.27D) Rheology of pH 4.5 hydrogel of FDopa.

[0052] FIGS. 28A-28I show covalently stabilization of fibrous collagen mimetic 10 assemblies through Lys-Dopa interactions. (FIG.28A) Sequence of FDopa(SEQ ID NO: 57) and reaction scheme of covalently stabilized fibrils with Dopa covalent capture. (FIG.28B) Cryogenic electron microscopy (Cryo-EM) of supramolecular assemblies of FDopaat pH= 4.5, the scale bar is 100 nm. (FIG.28C) Scanning electron microscopy (SEM) of FDopa at pH = 4.5 also shows a fibrous composition; the scale bar is1 µm. (FIG. 28D) Scattering curve of 15 supramolecular FDopa fibers at 5 °C and pH = 4.5 modeled as 400 nm long elliptical cylinder scattering objects. (FIG.28E) SAXS curves of the supramolecular fibers from 5-60 °C show a decrease in fiber signal with increasing temperature until the fibers are completely disassembled at ca.40 °C. (FIG.28F) Cryo-EM of covalently captured fibrous assemblies of FDopa at pH = 9.5, the scale bar is 100 nm. (FIG.28G) SEM of FDopa at pH = 9.5 retains a 20 fibrous composition, and the scale bar is 1 µm. (FIG.28H) Scattering curve of covalently captured FDopa fibers at 5 °C and pH =9.5 modeled as 400 nm long elliptical cylinder scattering objects showing that the major diameter doubles compared to the supramolecular fibers. (FIG.28I) Scattering curves of covalently captured FDopa fibers show only a minimal decrease in fiber signal from 5-60 °C25

[0053] FIGS.29A & 29B show Scattering and CD melt comparison of FDopa. (FIG.29A) Scattering melting curves taken by plotting the average intensity from FIG.28D / FIG.28H. (FIG.29B) FDopa CD melts demonstrates that PPII signal is not directly related to fiber stability.16Description of Illustrative Embodiments

[0054] Disclosed herein are novel collagen mimetic biomatrices. The biomatrices disclosed herein are formed from shorter amino acid sequences that facilitate assembly into higher order which are known in the art to be difficult to access. The presently disclosed 5 biomatrices harness charge pairs and cation-π interactions to facilitate the polymerization of a variety of amino acid sequences to access distinct compositions and morphologies. In some embodiments, the biomatrices are formed from an amino acid sequence comprising at least one aromatic amino acid, such as tyrosine. In some embodiments, the biomatrices disclosed herein comprise cation-π interactions. In some embodiments, the biomatrices disclosed herein are 10 formed substantially from canonical amino acids. In some embodiments, the biomatrices disclosed herein exhibit D-banding. In some embodiments, the biomatrices are formed at neutral pH. In some embodiments, the biomatrices are formed at basic pH. In some embodiments, the biomatrices are formed at acidic pH.I. Fibrous Proteins15

[0055] Fibrous proteins comprise most of the extracellular matrix (ECM) by mass (Frantz et al., 2010). Fibronectin, laminin, elastins and collagens are interwoven to provide structural support, template cellular growth and regulate cellular processes (Saraswathibhatla et al., 2023, Mouw et al., 2014). The most abundant of these proteins is collagen, which is classified into over twenty-eight different types and serves as a component for over forty collagen-like proteins (Kadler et al., 2008).20 Fibrous collagens typically comprise types I, II, III, V and XI (Ricard-Blum, 2011). While the fibrous assembly of the ECM is interdependent, understanding collagen fiber assembly is important because of its role in deleterious diseases such as osteogenesis imperfecta (OI) (Forlino & Marini, 2016), Ehler’s Danlos Syndrome (Pyeritz, 2000), aging (Henderson et al., 2020) and many cancers (Marino & Weeraratna, 2020). The hierarchical assembly of collagen fibrils commences with the 25 folding of a triple helix. Three monomer strands of three left-handed polyproline type II (PPII) secondary structures subsequently nucleate, propagate down the axis, and wind into an assembled right-handed supercoiled triple helix (Buevich & Baum, 2002; Cole et al., 2024). The canonical sequence of these PPII strands is a Xaa-Yaa-Gly repeat, where Xaa is frequently proline (P), and Yaa is 4-hydroxyproline (O). The glycine (G) in the third position is crucial for an axial hydrogen bond 30 and is also sterically favorable for triple helix formation (Shoulders & Raines, 2009). In addition, the optimized hydrogen bonding network imparted by the glycine also gives rise to a stagger of a single amino acid between the leading, middle and trailing strands, which defines a register between each strand. Pairwise interactions can occur between adjacent strands (Persikov et al., 2002). Intrahelical17pairwise interactions have been used to control triple helix design (Walker et al., 2021; Fallas et al., 2009), but higher-order assembly known to the art is still limited in design principles.

[0056] The most successful approach for designing supramolecular hierarchically assembling collagen mimetics has been employing electrostatic interactions between cationic 5 amino acids, such as lysine or arginine, and anionic amino acids, glutamate or aspartate. By utilizing the charge pairs of arginine (R) and glutamate (E), a fiber-forming collagen mimetic peptide (CMP) with the sequence (PRG)4(POG)4(EOG)4(SEQ ID NO: 45) was the first reported D-Banding CMP fibril. The periodicity was reported to be around 18 nm and hypothesized to assemble with a blunt-ended mechanism as shown in FIG.1A (Rele et al., 10 2007). Lysine (K) and aspartate (D) charge pairs stabilize triple helices. Therefore, the substitution of the R – E charge pair to K – D in (PKG)4(POG)4(DOG)4(SEQ ID NO: 26) (F0), as shown in FIG.1B, led to the first synthetic fibrous collagen mimetic hydrogel (O’Leary et al., 2011). The fiber design was proposed to interact in a sticky-ended manner, leading to interstrand interactions and unpaired sets of cationic and anionic regions that could interact 15 inter-helically (Sarkar et al., 2014). In FIG.1C, another proposed assembly mechanism that utilizes K – D electrostatic interactions resulted in successful fibrillogenesis and hydrogel formation by satisfying all theoretical pairwise inter- actions in an intrahelical manner (Tanrikulu et al, 2016; Tanrikulu et al., 2023). The deployment of electrostatic charge pairs has also led to the formation of various collagen mimetic peptide-based assemblies, including 20 nanotubes and nanosheets depending on the pH of the solution (Jiang et al, 2014; Merg et al., 2019). However, there has yet to be a comprehensive study that probes how all of these assembly mechanisms are intertwined; here, the present disclosure provides a means of probing such assembly paradigms with cation-π interactions.

[0057] Cation-π pairwise interactions occur between cationic (e.g., arginine) and 25 aromatic (e.g., tyrosine) to drive triple helix assembly. Using both electrostatic and cation-π pairs, the sequential nuances in generating fibrous collagen and other nanostructures, such as sheets and tubes, can be examined. Described in the sections that follow are peptides FD2F2, FD2W2 and FD2Y2 that facilitate a screen for improved cation-π interaction. In some embodiments, the compositions are formed from amino acid sequences comprising tyrosine, 30 such that tyrosine serves as the aromatic residue in a cation-π pair. Also described herein are compositions formed from amino acid sequences comprising an alternating substitution scheme (FDY2). In some embodiments, the present compositions are formed from amino acid18sequences that form hydrogels at acidic pH. In some embodiments, the present compositions are formed from amino acid sequences that form nanotubes at neutral pH.

[0058] Nature utilizes post-translational modifications (PTMs) to modify protein structures and to direct biological processes (Lee et al., 2023; Doll & Burlingame, 2015). Post- 5 translational modifications in the proteome include phosphorylation, glycosylation, farnesylation, hydroxylation and many others (Walsh et al., 2005). These crucial chemical modifications occur on both globular and fibrous proteins. The fibrous proteins of the extracellular matrix (ECM) rely on PTMs to guide structure, assembly, and function (Winkler et al., 2020; Ricard-Blum, 2011). Collagen, the most abundant protein in the ECM, relies 10 heavily on PTMs to guide folding and maintain its correct conformation both in vitro and in vivo (Shoulders & Raines, 2009).

[0059] Crucial PTMs in collagen include hydroxylation and cross-linking. Hydroxylation of pro- line at the 4th position is essential in providing stability to collagen’s triple helical backbone by stabilizing the imino ring’s exo pucker (Shoulders et al., 2008; 15 Hentzen et al., 2020). Other PTMs essential to collagen assembly are covalent cross-links.Collagen is a relatively unstable molecule at body temperature (Leikina et al., 2002). To overcome this inherent low stability, collagen cross-links with itself and other ECM proteins. Active enzymatic cross-linking can involve lysyl oxidases (LOX) and the class of lysyl- oxidase likes (LOXLs), which leads to the oxidative deamination of a ζ-amine of Lys to the 20 aldehyde-containing allylysine that can condense with each other or other Lys and hydroxylysine residues (Aronoff et al., 2021). There are also a variety of less-known interactions, such as sulfilimines, which are important covalent cross-links for collagen type IV (Vanacore et al., 2009).

[0060] It has been reported that collagen type I, the most abundant form of fibrous 25 collagens, contains levodopa (Dopa) residues which are implicated in the scavenging of oxidative radicals (Zapp et al., 2020; Kurth et al., 2023). Dopa is known to readily undergo oxidation to a benzoquinone under neutral and basic conditions (Foster, 1950). These reactive quinone species can form Dopa–Dopa covalent crosslinks or react with nucleophiles, such as primary amines, to form Schiff Bases of Michael Addition products (FIG.16). Covalent 30 bonds between Dopa and Lys are observed in proteins, including the collagen-related enzymes LOX and LOXL, which possess a lysl tyroslquinone (LTQ) cross-link between a Dopa and Lys residue in the enzyme’s active site (Moore et al., 2007; Zhang et al., 2018; Moon et al., 2014).19

[0061] Collagen mimetic peptides are short, synthetic sequences of amino acids that assemble into the right-handed triple helical tertiary structure of natural collagens (Brodsky & Persikov, 2005). Collagens have the canonical repetitive amino acid motif of Xaa-Yaa- Glycine, where the Xaa is typically proline, and Yaa is 4-hydroxyproline. The abundance of 5 imino acids leads to rigidity in the three left-handed polyproline II (PPII) that wind into a right-handed superhelix with a periodic symmetry (Shoulders & Raines, 2009, Okuyama et al., 2012). The glycine is sterically non-imposing and facilitates an inter-strand hydrogen bonding network between its amide protons and the carbonyl of the Xaa of the adjacent strand. This leads to a single amino acid offset and defines the register of the triple helix 10 between distance leading, middle and trailing strands (Brodsky & Ramshaw, 1997). When the amino acid sequences of all three strands are equivalent, the triple helices are referred to as homotrimers, while triple helices containing nonequivalent strands can be either A2B or ABC heterotrimers (Fallas et al., 2012; Xu et al., 2011).

[0062] The winding of the triple helix leads to distinct presentations of the amino acid 15 side chains in a pairwise manner. These pairwise interactions have two known geometries: axial and lateral (FIG.19B, FIG.19C). In most cases, the axial is more geometrically accessible than the lateral and has been used extensively in collagen mimetic peptide design (Persikov et al., 2002). It is also known from both molecular dynamics and experimental evidence that charge pairs, (Persikov et al., 2002) amide-π (Walker et al., 2021) and cation-π 20 (Chiang et al., 2017; Cole et al., 2022) pairwise interactions can effectively stabilize the triple helix. By achieving close proximity, pairwise interactions can also undergo various chemical modifications to create a synthetic, interstrand covalent bond (Hentzen et al., 2017). For example, the close interaction distance between residues in a charge-pair interaction allows for proximity-directed amidation and isopeptide bond formation, typically between 25 lysine and glutamate (Li et al., 2019), which enhances the thermal stability of the PPII helix through covalent capture (Cole et al., 2023) akin to disulfide bonds (Tanrikulu & Raines, 2014; DiChiara et al., 2018) However, this synthesis requires the use of activating reagents, like hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and has been reported with modest yields (Hulgan et al., 2020).30 II. Compounds of the Present Invention

[0063] The compounds of the present invention (also referred to as “compounds of the present disclosure”) are shown, for example in the summary of the invention section, in the examples section, and in the claims. They may be made using the synthetic methods outlined20in the Examples section. These methods can be further modified and optimized using the principles and techniques of organic chemistry. Such principles and techniques are taught, for example, in Smith, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, (2013), which is incorporated by reference herein. In addition, the synthetic methods 5 may be further modified and optimized for preparative, pilot- or large-scale production, either batch or continuous, using the principles and techniques of process chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in Anderson, Practical Process Research & Development – A Guide for Organic Chemists (2012), which is incorporated by reference herein.10

[0064] All the compounds of the present invention may in some embodiments be used for the prevention and treatment of one or more diseases or disorders discussed herein or otherwise. In some embodiments, one or more of the compounds characterized or exemplified herein as an intermediate, a metabolite, and / or prodrug, may nevertheless also be useful for the prevention and treatment of one or more diseases or disorders. As such unless explicitly stated 15 to the contrary, all the compounds of the present invention are deemed “active compounds” and “therapeutic compounds” that are contemplated for use as active pharmaceutical ingredients (APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for 20 protecting the public health by assuring the safety, effectiveness, quality, and security of human and veterinary drugs, vaccines and other biological products, and medical devices.

[0065] In some embodiments, the compounds of the present invention have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, more 25 metabolically stable than, more lipophilic than, more hydrophilic than, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than, and / or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.

[0066] Compounds of the present invention may contain one or more asymmetrically-30 substituted carbon, nitrogen, sulfur, or phosphorus atom and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual21diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present invention can have the S or the R configuration. In some embodiments, the present compounds may contain two or more atoms which have a defined stereochemical orientation.5

[0067] Chemical formulas used to represent compounds of the present invention will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given compound, and regardless of which one is most prevalent, all 10 tautomers of a given chemical formula are intended.

[0068] In addition, atoms making up the compounds of the present invention are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of 15 carbon include13C and14C.

[0069] In some embodiments, compounds of the present invention function as prodrugs or can be derivatized to function as prodrugs. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in 20 prodrug form. Thus, the invention contemplates prodrugs of compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Accordingly, prodrugs include, for example, compounds described herein in which 25 a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a patient, cleaves to form a hydroxy, amino, or carboxylic acid, respectively.

[0070] In some embodiments, compounds of the present invention exist in salt or non- salt form. With regard to the salt form(s), in some embodiments the particular anion or cation forming a part of any salt form of a compound provided herein is not critical, so long as the 30 salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0071] It will be appreciated that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These22complexes are known as “solvates.” Where the solvent is water, the complex is known as a “hydrate.” It will also be appreciated that many organic compounds can exist in more than one solid form, including crystalline and amorphous forms. All solid forms of the compounds provided herein, including any solvates thereof are within the scope of the present invention.5 III. Chemical Definitions

[0072] The use of the word “a” or “an,” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0073] Throughout this application, the term “about” is used to indicate that a value 10 includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects or patients.

[0074] An “active ingredient” (AI) or active pharmaceutical ingredient (API) (also referred to as an active compound, active substance, active agent, pharmaceutical agent, agent, biologically active molecule, or a therapeutic compound) is the ingredient in a pharmaceutical 15 drug that is biologically active.

[0075] The term “between” as used herein is inclusive. Therefore, any range disclosed as between value A and value B includes both A and B.

[0076] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” 20 “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[0077] The term “conservative amino acid substitution” or “conservative substitution” refers to a substitution that does not substantially affect the function or properties of a peptide 25 or protein, such as the ability of a peptide to adopt a particular conformation, secondary structure, or supramolecular structure. The term conservative substitution also includes the use of a substituted in place of an unsubstituted parent amino acid. Deletions or additions which alter, add, or delete a single amino acid or small amount of amino acids (for instance less than 5% or less than 1%) in an encoded sequence are conservative substitutions where the alteration 30 results in a peptide with similar properties to the parent peptide. The following six groups are examples of amino acids that are considered to be non-limiting examples of conservative substitutions:1) Alanine (A), Serine (S), Threonine (T);232) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and5 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0078] The term “effective,” as that term is used in the specification and / or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound 10 which, when administered to the patient or subject, is sufficient to effect such treatment or prevention of the disease as those terms are defined below.

[0079] An “excipient” is a pharmaceutically acceptable substance formulated along with the active ingredient(s) of a medication, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, to bulk up 15 the composition (thus often referred to as “bulking agents,” “fillers,” or “diluents” when used for this purpose), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include pharmaceutically acceptable versions of antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and 20 vehicles. The main excipient that serves as a medium for conveying the active ingredient is usually called the vehicle. Excipients may also be used in the manufacturing process, for example, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The suitability of an excipient will 25 typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.

[0080] The term “hydrate” when used as a modifier to a compound means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecules associated with each compound molecule, such as in solid 30 forms of the compound.

[0081] As used herein, the term “IC50” refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or24chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.

[0082] An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the 5 configuration of those atoms in three dimensions differs.

[0083] As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non- limiting examples of human patients are adults, juveniles, infants and fetuses.10

[0084] "Peptides" as used herein refers to amino acid-containing polymeric compounds, and is meant to encompass polymeric compounds, regardless of length or post- translational modification. As used herein, “peptides” comprise naturally occurring and non- naturally occurring amino acids, polymeric compounds comprising a mixture of naturally- occuring amino acids and non-naturally occurring amino acids, oligomers (oligopeptides), 15 cyclic polymers (cyclic peptides), polypeptides, and proteins, as well as peptide mimetics. A peptide has an amino terminal end (N-terminal) and a carboxy terminal (C-terminal) end. The peptides may be obtained by chemical synthesis or be produced from a genetically encoded source (e.g., recombinant source). Peptides can range in molecular weight, and can be from 200 Da to 10 kDa or greater in molecular weight. A “residue” refers to an amino acid, including 20 naturally occurring amino acids and non-naturally occurring amino acids, incorporated into a peptide by an amide bond or amide bond mimetic. “Peptide” is used interchangeably herein with polypeptide or protein.

[0085] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound 25 medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0086] “Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired 30 pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2- naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1- carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic25mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic 5 acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which 10 may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of 15 this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable.Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0087] A “pharmaceutically acceptable carrier,” “drug carrier,” or simply “carrier” is a 20 pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and / or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and / or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery 25 to the specific target sites. Examples of carriers include: liposomes, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.

[0088] A “pharmaceutical drug” (also referred to as a pharmaceutical, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical 30 product, medicinal product, medicine, medication, medicament, or simply a drug, agent, or preparation) is a composition used to diagnose, cure, treat, or prevent disease, which comprises an active pharmaceutical ingredient (API) (defined above) and optionally contains one or more inactive ingredients, which are also referred to as excipients (defined above).26

[0089] “Prevention” or “preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and / or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which 5 may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[0090] “Prodrug” means a compound that is convertible in vivo metabolically into an active pharmaceutical ingredient of the present invention. The prodrug itself may or may not have activity in its prodrug form. For example, a compound comprising a hydroxy group may 10 be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound.Non-limiting examples of suitable esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, phosphates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, 15 benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates, and esters of amino acids. Similarly, a compound comprising an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.

[0091] The term “sequence identity” as used herein refers to the similarity between amino acid sequences. Sequence identity is frequently measured in terms of percentage 20 identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a peptide will possess a relatively high degree of sequence identity when aligned using standard methods known in the art. As used herein, reference to “at least 90% identity” or similar language refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 25 100% identity” to a specified reference sequence.

[0092] A “stereoisomer” or “optical isomer” is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. “Enantiomers” are stereoisomers of a given compound that are mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers of a 30 given compound that are not enantiomers. Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer. In organic compounds, the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and27inorganic compounds. A molecule can have multiple stereocenters, giving it many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), the total number of hypothetically possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry 5 frequently have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. It is contemplated that that for any stereocenter or axis of chirality for which 10 stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures. As used herein, the phrase “substantially free from other stereoisomers” means that the composition contains ≤ 15%, more preferably ≤ 10%, even more preferably ≤ 5%, or most preferably ≤ 1% of another stereoisomer(s).15

[0093] “Treatment” or “treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and / or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and / or symptomatology), and / or (3) effecting any 20 measurable decrease in a disease or symptom thereof in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[0094] In some embodiments, the present disclosure provides peptides comprising one or more amino acids having a reactive amine group. As used herein, an amino acid having a reactive amine group possesses an optionally substituted amino group of the form -NH2that is 25 capable of reacting with a particular compound or functional group. As used herein, the reactive amine group is capable of reacting with a particular compound or functional group when the amino acid of which it forms a part is located within a polymer, such as a peptide. The amino acid having a reactive amine group may be naturally occurring or non-naturally occurring. In some embodiments, the reactive amine group is of the formula -NR1R2, wherein R1 and R2 are 30 independently hydrogen, alkyl, or cycloalkyl as those terms are defined below. A non-limiting example of an amino acid having a reactive amine group is lysine.

[0095] The term “alkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups −CH3(Me), −CH2CH3(Et), −CH2CH2CH3(n-Pr or propyl),28−CH(CH3)2 (i-Pr,iPr or isopropyl), −CH2CH2CH2CH3 (n-Bu), −CH(CH3)CH2CH3 (sec-butyl), −CH2CH(CH3)2(isobutyl), −C(CH3)3(tert-butyl, t-butyl, t-Bu ortBu), and −CH2C(CH3)3(neo- pentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of 5 attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups −CH2− (methylene), −CH2CH2−, −CH2C(CH3)2CH2−, and −CH2CH2CH2− are non-limiting examples of alkanediyl groups. The term “alkylidene” refers to the divalent group =CRR′ in which R and R′ are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include: =CH2, 10 =CH(CH2CH3), and =C(CH3)2. An “alkane” refers to the class of compounds having the formula H−R, wherein R is alkyl as this term is defined above.

[0096] The term “cycloalkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forming part of one or more non- aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than 15 carbon and hydrogen. If more than one ring is present, the rings may be fused, bridged, or spirocyclic. Non-limiting examples include: −CH(CH2)2(cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to a carbon atom of the non-aromatic ring structure. The term “cycloalkanediyl” refers to a divalent saturated 20 aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double ortriple bonds, and no atoms other than carbon and hydrogen. The group is a non- limiting example of cycloalkanediyl group. A “cycloalkane” refers t compoundshaving the formula H−R, wherein R is cycloalkyl as this term is defined above.

[0097] The term “alkenyl” refers to a monovalent unsaturated aliphatic group with a 25 carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: −CH=CH2(vinyl), −CH=CHCH3, −CH=CHCH2CH3, −CH2CH=CH2 (allyl), −CH2CH=CHCH3, and −CH=CHCH=CH2. The term “alkenediyl” refers to a divalent unsaturated aliphatic group, with two carbon atoms as 30 points of attachment, a linear or branched acyclic structure, at least one nonaromatic carbon- carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups −CH=CH−, −CH=C(CH3)CH2−, −CH=CHCH2−, and −CH2CH=CHCH2− are non-limiting examples of alkenediyl groups. It is noted that while the29alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms “alkene” and “olefin” are synonymous and refer to the class of compounds having the formula H−R, wherein R is alkenyl as this term is defined above. Similarly, the terms “terminal alkene” and “α-olefin” are synonymous and refer 5 to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.

[0098] The term “alkynyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the 10 term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups −C≡CH, −C≡CCH3, and −CH2C≡CCH3 are non-limiting examples of alkynyl groups. An “alkyne” refers to the class of compounds having the formula H−R, wherein R is alkynyl.

[0099] The term “aryl” refers to a monovalent unsaturated aromatic group with an 15 aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structures, each with six ring atoms that are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon 20 number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, −C6H4CH2CH3(ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e.g., 4-phenylphenyl). The term “arenediyl” refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one 25 or more six-membered aromatic ring structures, each with six ring atoms that are all carbon, and wherein the divalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are 30 connected with a covalent bond. Non-limiting examples of arenediyl groups include:30

[0101] An “arene” refers to the class of compounds having the formula H−R, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples 5 of arenes.

[0102] The term “aralkyl” refers to the monovalent group −alkanediyl−aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.

[0103] The term “heteroaryl” refers to a monovalent aromatic group with an 10 aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the 15 rings are fused; however, the term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroaryl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl, isoxazolyl, methylpyridinyl, oxazolyl, oxadiazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, 20 quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term “N-heteroaryl” refers to a heteroaryl group with a nitrogen atom as the point of attachment. A “heteroarene” refers to the class of compounds having the formula H−R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.

[0104] The term “heterocycloalkyl” refers to a monovalent non-aromatic group 25 with a carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the non-aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may 30 be fused, bridged, or spirocyclic. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring31atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, 5 tetrahydropyridinyl, pyranyl, oxiranyl, and oxetanyl. The term “N-heterocycloalkyl” refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group.

[0105]

[0106] The above definitions supersede any conflicting definition in any 10 reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the invention in terms such that one of ordinary skill can appreciate the scope and practice the present invention.IV. Examples15

[0107] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the example which follows represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of 20 the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1: Cation-π pairwise interactions for Peptide Collagen Mimetics A. Screening Pairwise Interactions for Supramolecular Assembly25

[0108] Electrostatic charge pairs have been well-studied in both single triple helices (Persikov et al., 2002; Walker et al., 2021) and higher-order assemblies. The role of charge pairs in directing assembly, without being bound by theory, has pointed to lysine- aspartic acid (K – D) as the ideal charge pair and is rendered in FIG.2A, where arginine (R) and glutamate (E) amino acids possess fewer degrees of freedom due to an extra hydrogen 30 bond between the backbone of the triple helix and the side chain of the amino acid and form a weaker pairwise interaction when compared with K – D (Persikov et al., 2002; Fallas et al., 2012).32

[0109] Cation-π interactions are known to be important supramolecular interactions and can be stabilizing to a triple helix, but are also largely dependent on the conform and average distance (typically less than 5 ˚A) between the amino acids.Additionally, sterically encumbered aromatic groups are more destabilizing to the triple helix 5 than electrostatic charge pairs (FIG.2A) when not paired with its counteraction (Cole et al., 2022; Zheng et al., 2019). Cation-π interactions prefer to have arginine as the cation because of delocalized charge of the δ-guanidinium ion can interact with the π-electrons of the aromatic group (Gallivan & Dougherty, 1999). The renderings of the three possible arginine- containing cation-π interactions in a triple helix, as well as a representative interaction 10 distance, are presented in FIG.2A. To design polymerizing triple helices, there is a need to optimize the offset of the pairwise interactions, similar to previous designs used in DNA and coiled-coil assemblies, to yield sticky-ended characteristics (Kotch & Raines, 2006). To this end, with the substitution scheme shown in FIG.2B, the relative stability of each triple helical assembly was screened by monitoring the polyproline type II (PPII) structures with 15 circular dichroism (CD). For each fibrous system, FD2F2, FD2W2 and FD2Y2, the PPII signal increased (FIGS.2C-2E) from acidic to the near neutral pH. All peptides had a precipitate at pH 9.0 (see FIG.3). This may be due, without being bound by theory, to changes in the protonation states of the charged amino acids (lysine-Nζ, pKa≈ 9.3 – 13.237 and arginine-N- guanidinium, pKa ≈ 12.0 – 13.838) All thermal unfolding experimental results are listed in 20 Table 1 and the thermal stabilities are defined by the minimum of the derivative (Tm) of each CD folding curve from FIG.4. The melt derivative curves are presented in FIGS.2F-2H. At both pH 4.5 and 7.4 FD2Y2had the highest thermal stability and a gain of +8.0 °C as the pH increased from pH 4.5 to 7.4. With pH adjustment, FD2W2 also gained +12.0 °C and FD2F2 increased +6.0 °C. As the pH is adjusted to 7.4, the lysine – aspartate interactions contribute 25 more to the thermal stability of the triple helix than at pH 4.5. With this assumption, though without being bound by theory, it is clear that the aromatic groups show the same destabilizing effect in the fibrous systems as they do in the triple helix, where tryptophan (W) is the most destabilizing, then phenylalanine is the least destabilizing (Cole et al., 2022). Due to the highest thermal stability and tyrosine’s ability to promote self-assembly (Jang et al., 30 2014), FD2Y2 was further investigated with microscopy.33)C°(e2)GetdaerOYti2 ) 2 2puis) )^( 2 caemsiG GO G)ermorOF W O GY O) 2 ) 2P*e43^ ^G G)rehci () 2 () 2^() 2D() 2YwO O, snDrG G G G YaO O ^ DG W O O G,oil 4D(t4D(D(P(O O FY(auc )r) ) 4 ) 4 ) 2rtiGO G G G GD(O O R) 4^() 4citnaecO G G nCmoD P( (P P Py( ( (oO Ocr 4 22 2 2) )) ) )b eP Pl( (A2 2G GG G G p) )d ^ee KO R R R,)G Gmcni aP(P P P P( ( ( (R Rn4D4 2 2 2s)) ) ) )m e P P( llurGG G G G G GedeAq K K K K O K Kt.ee nP P P P P P P i s( ( ( ( ( ( ( lSeDresvrda uncyti eUli–vibts a:antviOoSirl eQ nNa E 6 7 8 9 0 1 2d ADIS 2 2 2 2 3 3 3rme,)redrRh,o-TtKse r( idfdilt eop elhe tTbp −2fP 2 22 –F W Y Y 2 2 o:m 2 2 2 2 Y Ds a0 D D D D D Ya1n S F F F F F F FeolmiitbniaaT C mB. Analysis of Collagen in Mimetic Fibrils of FD2Y2

[0110] Collagen mimetic peptide fibrillogenesis, like natural collagen (Harris et al, 2013; Giubertoni et al., 2024), is dependent on a variety of factors in the aqueous 5 environment (Giubertoni et al., 2024), including factors like concentration (O’Leary et al., 2011), ionic strength (Tanrikulu et al, 2016) and pH (Li et al., 2009). At a concentration of 2% w / v and in MQ H2O, FD2Y2was found to be soluble at pH 4.5. The sample was confirmed to be fibrous with scanning electron microscopy (SEM) (FIG. 5B) and further confirmed with cryo-EM to have D-periodic fibers with a periodicity of ∼55-60 ˚A as presented in FIG.5C.10

[0111] Characterization for FD2Y2 at pH 7.4 in FIG.5B and FIG.5E showed the fibers aggregating. Additionally, when dissolved in 10 mM phosphate buffer, the fibers appeared to slightly thicken and shorten from 40 nm to 20 nm, as confirmed with both Cryo- EM and SAXS (FIG.6), which has previously been known in the art (Xu et al., 2012). The triple helices at pH 7.4 are more thermally stable (FIG.2F), which could, without being bound 15 by theory, give rise to a different nucleation and subsequent polymerization pathway and lead to the observed clustering. The fibrous system of FD2Y2 was then compared with the previously established F0(O’Leary et al., 2011) and a tessellating collagen mimetic peptide with cation-π interactions (FD2Y2−T) (Tanrikulu et al., 2016).

[0112] Further analysis of F0shows fibers that possessed distinct 20 morphological differences compared to FD2Y2. F0 fibers rely only on electrostatic charge pairs between lysine and aspartate. Circular dichroism showed asymmetry in the thermal unfolding curves but an overall gain in thermal stability as the pH increased. Cryo-EM revealed fibers of varying diameters and lacking periodicity, and SEM also supports the distinctness of this assembly as compared with FD2Y2.25

[0113] Next, FD2Y2was compared with a tessellating scheme that incorporated cation-π interactions (FD2Y2−T). FIG.7 shows the proposed assembly mechanism of FD2Y2−T; the main feature of this peptide is that, without being bound, there are theoretically no interhelical interactions. FD2Y2−T appears distinct from previous designs in the fact that the adjustment of pH, from 4.5 to 7.4, did not substantially increase the thermal stability.30 Additionally, the samples possessed higher solubility at even pH 9.0 and a marked increase in thermal stability (+9.0 °C) compared to pH 7.4. Cryo-EM (FIG.7E, FIG.7F) analysis reveals that there were two distinct assemblies at pH 4.5: fibrous and aggregated. The fibers were notably smaller in diameter compared to FD2Y2and F0. The aggregation may occur, without35being bound by theory, because tyrosine inhibits assembly due to its propensity to destabilize the helix and the fact that it is sterically larger than aspartic acid. In conclusion, these data suggest, again without being bound, that the resulting fibers from F0, FD2Y2−T and FD2Y2 are distinct collagen mimetic peptide assemblies at pH 4.5.5 C. Enhancing D-Periodicity in CMP Fibers with Alternating Interactions

[0114] In the supramolecular assembly of peptides, small, subtle changes to amino acid sequences can change the structure (Lampel et al., 2017). To probe how alternating triplets of each pairwise interaction can impact assembly, an alternating fiber design termed FDY2 was designed. According to the proposed model of triple helix10association for FD2Y2 and FDY2 provided in FIG.8A, the two most thermally stable staggers by pairwise interaction deconvolution (Cole et al., 2022; Zheng et al., 2019) are represented. While noting that the pairwise interaction values were deconvolved at roughly neutral pH and are intrahelical rather than in blunt-ended interhelical assemblies, which leads to discrepancies in absolute thermal stability values, these values can be useful for establishing 15 a relative model. All possible staggers are also diagrammed in FIG.9.

[0115] Both FD2Y2 and FDY2 have the propensity to form two cation-π and two charge pairs in their most thermally stable staggers, resulting in a stability of 2 × 7.3 °C (K – D) + 2 × 6.2°C (R – Y) = 27.0 °C. The second most stable staggers are markedly different. For FD2Y2, there is a total stabilizing effect of 7.3 °C (K – D) + 3.8 °C (K – Y) + 6.2 °C (R – 20 Y) = 17.3°C, a difference of -9.7 °C when compared to the most stable stagger. And for FDY2, there is a total stabilizing effect of 2 × 3.8 °C (K – Y) + 1.3 °C (R – D) = 8.9 °C, a difference of -18.1 °C when compared to the most stable stagger. This naively supports that the most favored assembly of FDY2 is substantially more likely than FD2Y2, which in turn should result in higher periodicity and less heterogeneity for FDY2.25

[0116] Discrepancies between the two systems were also observed upon comparison of the circular dichroism of FD2Y2and FDY2. FDY2had a higher MRE value in FIG.8B, while FD2Y2 had a higher thermal stability. The higher thermal stability of FD2Y2 in FIG.8C may be, without being bound by theory, due to its ability to adopt multiple staggers, resulting in heterogeneity and ultimately a higher degree of satisfied pairwise interactions. 30 Rheological characterization of FD2Y2and FDY2in FIG.8D suggested, without being bound by theory, a weak collagen mimetic peptide hydrogel. The hydrogel of FD2Y2 sheared at roughly 8% strain, wherein the storage modulus before the 8% drop is 39 Pa in the linear range, and the loss modulus is 9 Pa. FDY2 also formed a relatively weak hydrogel at pH 4.5,36and the loss and storage moduli are approximately 32 Pa and 5 Pa, respectively (FIG.8D). Samples that were prepared above pH 4.5 did not yield hydrogels.

[0117] With a model of possible staggers established and both secondary and macromolecular characterization of FDY2, the collagen mimetic hydrogel sample was then 5 analyzed with cryo-electron microscopy (Cryo-EM). Micrographs in FIG.10A show distinct fibers with D-banding characteristics, a hallmark of natural collagen. Further refinement of the D-banding into 2D class averages in FIG.10B show alternating periodicity and individual triple helices. A 3D reconstruction into a proposed electron density map in FIG.10C shows a periodicity of roughly 67 ˚A, which is 1 / 10 of the 670 ˚A periodicity of natural human 10 collagen (Habelitz et al., 2002; Cisneros et al., 2006).

[0118] Triple helices are proposed to have roughly a 10 / 3V or 7 / 2V symmetry, depending on the density of proline-rich amino acid residues (Shoulders & Raines, 2009; Okuyama et al., 2006). This symmetry defines the axial height of a helix and rise for each triplet in the sequence. For a 10 / 3V triple helix, the rise of each triplet is 7.8 ˚A, 15 which would yield a height of rough 102 ˚A. If calculated using the 7 / 2V model, with a rise of 8.57 ˚A per triplet, the helix would be roughly 94 ˚A. In FIG.8D, it can be seen that the volumes could easily overlay with a 93 ˚A triple helix. The periodicity of 67 ˚A is nearly 2 / 3 the length of the helix, which could encompass either the cationic / POG sequence (PKGPRG)2(POG)4 (SEQ ID NO: 33) triplets or the anionic and aromatic / POG regions 20 (POG)4(DOGYOG)2(SEQ ID NO: 34). This would lead to the last or first four triplets being unpaired, which could interact with the next set of helices. This observation was further supported by the apparent arrangement of non-overlapping regions, which appeared to have a central dimer sub-unit surrounded by eight triple helices (see FIG.10E), termed an 8+2 structure. At the overlapping region, four central sub-units are possibly surrounded by sixteen 25 outer triple helices in a 16+4 arrangement (FIG.10F). Models are proposed in in FIG.11. See also FIG.14. This is also observed in cartilage fibrils, where the triple helices are hypothesized to be arranged in a 10+4 bundling structure (Holmes & Kadler, 2006). These data also demonstrate, without being bound, that supramolecular assembly can be used to create collagen mimetics that are relatively uniform in their lateral packing and may be useful 30 for improving understanding of how lateral packing is curtailed in natural collagens (Revell et al., 2021).37D. Formation of collagen mimetic peptide nanotubes and nanosheets

[0119] To further probe how pairwise interactions are presented in the triple helix, FYD2 was synthesized. This peptide flipped the presentation of the aromatic triplet (YOG) and the anionic triplet (DOG) while keeping the cationic region constant (PKGPRGPKGPRG 5 (SEQ ID NO: 35)), as outlined in FIG. 12A. For samples of FYD2 precipate formation was observed at every pH (see FIG.13). Using the tyrosine tags of FYD2, FDY2and FD2Y2, a mass balance was performed of each peptide due to each sample, despite having a remarkably similar amino acid composition, different solubilities in water. The results, summarized in Table 2, indicated that the rationale was for FYD2 to have attenuated pairwise interactions (K – Y and R 10 – D). These assemblies formed single-layer and multi-layer nanotubes.38de.tm2rni8 8 2 2 ooI2. .0.0.0.0.02.0fno p 7 1 1 1 1 1 1epcitta it rticea pleicmorersio peF2 a)hletsGgseitOoIorY2p) 22nd^( e)) ; 2y G)d 9nGG:OH 3oG2 2OOit) )rPOF W Yo.G Gu) l^ ^ ^( ( (D(f2 2 2 2oO O) ) )Y)yYs,DcG G G G^rGanO O O O WGeOe, lD D D Pd( ( (O( 4Y4 4 4 2F)c) ) ) )n D ^ (( (eaG4 4G G G G) )cispO ReeO O OtG GatD P( (P P Pa( ( (O O4 2D2 2 2m) )) ) )ciP P( (H2 2oG GrG G G d) )K npe O R R R AG GicPd(P P P P( ( ( (^R R4n –4 2 2 2n),) ) ) )P P) ;ealGu G G G G G GeDsq K K K K K K O(geecP P P P P P P( ( ( ( ( ( (osdrenden yilu hrqe aedfSnoseUnd –oiit stan:peoiOmrPnN ogfADne iI ,)htQ RmrE 6 7 8 9 1 2 0s,o S 2 2 2 2 3 3 3etKFo(r ne edlodbikbFr:–aTs2−m2 2 -n2)e2 2 2l)o ke F W Y Yb Yi Dtc2 2 2 2mea aD Y( (a 0 D D D Dh Tcn F F F F F F F C

[0120] While there is no precedent for interaction geometries in fibers, alternating the sequence to FYD2would result in an exchange of the charge pair and cation-π partners. These pairwise interactions stabilize a triple helix slightly, with K – Y being only 5 3.8 °C and R – D pairs contributing only 1.3 °C in gained stability. These lesser interactions created a sheet-like morphology at pH 7.4. While both of these morphologies have been observed with charge pair containing CMPs (Merg et al., 2019; Merg et al., 2020), the control of their shape by pairwise interaction shifting has not been observed. Circular dichroism spectra for FYD2supernatant are provided in FIG.15.10 E. Experimental(i) Peptide Synthesis and Preparation

[0121] Peptides were synthesized using standard Fmoc solid phase peptide synthesis on a pre- loaded Gly-Wang resin. Fmoc-protected amino acids and resin were purchased from EMD Chemicals, and 2-(1H-7-azabenzotriazol-1yl)-1,1,3,3-tetramethyl 15 uranium hexafluorophosphate methanaminium (HATU) was purchased from P3Bio. All chemicals not otherwise specified were purchased from Sigma-Aldrich. A mixture of 25% v / v piperidine mixture in dimethylformamide (DMF) was used for deprotecting steps.Coupling was performed using HATU and diisopropylethylamine (DiEA) in DMF at 1:4:4:6 (resin / amino acid / HATU / DiEA, respectively). Acetylation of the N-terminus was performed 20 twice with an excess of acetic anhydride and DiEA in dichloromethane. Cleavage was performed with 7.5% (v / v) scav- engers (triisopropylsilane, H2O, and anisole) in trifluoroacetic acid (TFA).

[0122] Excess TFA was removed after cleavage from the resin by a nitrogen flow. The crude peptide was triturated twice with cold diethyl ether. Crude peptides were25dissolved in H2O to a 15 mg / mL concentration. This was sonicated and then filtered with a 0.3- micron syringe filter before purification by reverse-phase high-pressure liquid chromatography (HPLC) with water and acetonitrile with 0.05% TFA at a gradient of 0.85% per minute on a Waters XC18 column. Samples were roto-evaporated to remove acetonitrile and then lyophilized. Matrix-assisted laser desorption ionization-time of flight mass spectrometry 30 (MALDI-ToF MS) (Bruker Instruments) was used to confirm the peptide mass.(ii) Circular Dichroism40

[0123] Circular dichroism (CD) spectroscopy was performed on a Jasco J- 810 spectropolarimeter (Tokyo, Japan) with a Peltier temperature-controlled stage. Peptide samples were prepared at a 3 mM working concentration and diluted with MQ water to 0.3 mM for melts. Any tryptophan (W) containing peptide was diluted to 0.015 mM due to its 5 innate absorption in CD. After preparation, 200 µL of the sample was transferred to a quartz cuvette of 0.1 cm path length. Wavelength scans were performed between 180 and 250 nm to confirm the triple helical secondary structure. The maximum, which falls near 225 nm, is followed as the temperature increases from 5 to 65 °C at 10 °C / h. Using a first-degree Savitzky–Golay smoothing algorithm, the first derivative curve was calculated for melting 10 curves. The minimum of the first derivative is defined as the melting temperature (Tm). The MRE was calculated as previously reported.(iii) Rheology

[0124] Rheological testing was conducted on an AR-G2 rheometer (TA Instruments, New Castle, DE) with a 12 mm parallel plate.70 µL of peptide was pipetted 15 onto the stage, and the parallel plate was lowered to a gap of 550 µm. Any excess gel was scraped away before the gap was lowered to 500 µm, and oil was applied to prevent evaporation during testing.(iv) Scanning Electron Microscopy

[0125] Samples were dehydrated in a series of ethanol:water mixtures (30%, 20 50%, 60%, 70%, 80%, 90%, 2 × 100% (v / v)). Samples were placed in each bath for 10 min to dehydrate the peptide samples and then further dried using a Leica EM CPD300 Critical Point Dryer (Leica Biosystems, Deer Park, IL). Dried samples were then sputtered with 10 nm of gold to enhance conductivity with a Denton Desk V Sputter System (Denton Vacuum, Moorestown, NJ). Sputtered samples were then imaged on a Helios NanoLab 660 Scanning 25 Electron Microscope (FEI Company, Hillsboro, OR) at 1 kV and 25 pA.(v) Cryo Electron Microscopy

[0126] Fibrous samples were added to Lacey Carbon CU 300 mesh grids and were frozen with a VitrobotTM Mark IV plunge freezer. Prior to freezing, the grids were subject to Glow dis- charge treatment using an Electron Microscopy Sciences GloQube plasma 30 cleaner system. Grids were imaged on a Titan Krios equipped with a K3 direct electron detector at 105,000x magnification (0.82 As / pixel). Cryo-EM reconstruction and image processing of raw movies and subsequent image processing and reconstruction steps were handled in cryoSPARC. For reconstruction of the FDY2 filaments, helical reconstruction, and single- 41particle reconstruction were separately attempted. For the single-particle reconstruction, particles belonging to good 2D classes of filaments, were subject to ab initio reconstruction where a volume with the 67 ˚A periodicity was apparent. The ab initio volume and its corresponding particles were then used as inputs into a homogeneous refinement job with a 5 mask created from the ab initio. The structure had an apparent C2 symmetry, so a subsequent homogenous refinement job imposing this point group symmetry was run.(vi) UV-Vis Mass Balance

[0127] Samples were heated at 85°C for 30 minutes on heatblock to ensure all precipitates were dissolved. Absorbance values were measured at 280 nm using a Thermo 10 Scientific™ NanoDrop™ 2000 Spectrophotometer. Day 0 measurements were collected immediately after heating and were stored at 4°C. On day 7, samples were centrifuged at 10000 rpm for 2 minutes, and the supernatant was measured. The percentage of insolubility was calculated using the following equation: ((1 – AbsDay7) / AbsDay0 ) x 100=% Insoluble.Example 2: Covalent Stabilization of Collagen Mimetic Triple Helices and Assemblies 15 by Dopa Cross-linkingA. Covalent Capture of Collagen Homotrimers

[0128] In another aspect, the present disclosure provides stabilized triple helix structures. To investigate if cross-linking between Lys and Dopa residues could stabilize a triple helix, two peptides were synthesized: OGDopaK and KGDopaO. Two control peptides 20 were also synthesized, termed AGDopaO and KGDopaO6,9GlyDeletion, to investigate the propensity for side reactions, including the self-reactivity of the o-benzoquinone (Table 3). In OGDopaK, leading to middle (L-to-M) and middle to trailing (M-to-T) strands’ interactions between Lys and Dopa are both in the lateral geometry. Meanwhile, the trailing to leading (T- to- L) strands are too distant and expected to be non-interacting. The substitution scheme of 25 KGDopaO presents the Lys and Dopa with an axial interaction in the L-to-M and M-to-T, while the T-to-L is a lateral interaction.47 After synthesis and folding at 3 mM (pH 4.5), it was observed that all three homotrimers formed with a positive PPII signal at 225 nm as observed with circular dichroism (CD) (FIG.16).42)Ha2cH 2N- iN- H) 3b) 3N- G M) 3OGP(m0O G O GP(O1P(nG’XiY G O GY5.X93 G X P 4 O G) 2=P YP O) ) 3P He3c()pd)n G G G ne OP O- P OPaC uC q( (-(- M(e s c A crA ceAm3uttap daeCrtutnpeala:cv O yoN ltDnC IehlatiQ vEwS 63 73 83ocnsoeiltp azimlaib nobSatitmeSle T d Dae yl rpcG n9,u6Sah Ka Oa Op a Oa–Tm n o po pE o p:e D o C m G D D D3a G G GCeln O K A K=ba TmTa∆

[0129] By monitoring the CD thermal unfolding curves, the covalent stabilization of the triple helix by oxidation induced by a switch to pH 9.5 and subsequent covalent bond formation between the Lys and Dopa was measured. The oxidation of Dopa to 5 an o-benzoquinone can be accomplished at basic pH while simultaneously enhancing the nucleophilicity of the ζ-amine of Lys (lysine-Nζ, pKa ≈ 9.3 – 13.248). The thermal transition temperature (Tm) is defined by the minimum of the first derivative of the thermal unfolding curve. The supramolecular and covalently captured triple helix Tm values are summarized in Table 3.10

[0130] The presentation of Lys – Dopa interaction in the lateral geometry is accomplished in the OGDopaK homotrimer. There was a steady increase in thermal stability (FIG.17A). Refolding curves in FIG.17B show that the covalently captured homotrimer after 72 hrs had the highest recovery. However, the asymmetry of the first-order derivative melt curves in FIG.17C suggests, without being bound by theory, heterogeneity in the 15 covalently captured samples. This decreased cooperativity is also supported by the MALDI- MS data in FIG.18B, showed peaks correlating to dimer, trimer, tetramer and possibly higher-order species. Nevertheless, there was a 19.0 °C stabilization for OGDopaK when compared with the supramolecular triple helix.

[0131] Next, the propensity of stabilization for an axial interaction in the 20 homotrimer KGDopaO was investigated. The cation-π supramolecular interaction and subsequent covalent capture is favored in the axial conform and supported by the characterization of KGDopaO (FIGS.17D-17F). The supramolecular triple helix had a 9.5 °C difference in stability when compared with OGDopaK and agrees with previous studies on cation-π interactions in triple helices, demonstrating a preference for the axial geometry (Cole 25 et al., 2022). After covalent capture at pH 9.5, MALDI ToF MS showed dimer, trimer, and tetramer peaks (see FIG.18A). The Tm of KGDopaO triple helix gained a remarkable 48.5 °C. For comparison, previous interstrand stabilization attempts with cysteine and homocysteine were either destabilizing or negligible (Tanrikulu, 2014), and isopeptide amide bond formation provided a gain of 43.5°C (Cole et al., 2023).30

[0132] Next, the cross-linking experiment was repeated for two additional peptides, AGDopaO and KGDopaO6,9GlyDeletion, to further confirm the axial interaction is preferred for covalent stabilization. AGDopaO, which was synthesized to account for possible Dopa-Dopa cross- linking interactions (FIG.19A), showed a trivial gain in stability44of 2.0 °C. In addition, there was a lack of change in the hysterses of the reverse melts presented in FIG.17H, suggesting, without being bound by theory, that Dopa-Dopa covalent interactions are negligible in stabilizing the triple helix, despite some amount of dimer formation being observed with MALDI ToF MS (FIG.18C). It was also noticeable that a Lys 5 – Dopa containing peptides exhibited a yellow solution with a corresponding UV-Vis peak at roughly 355 nm (see FIG.20A, FIG.20B, AND FIG.21). In contrast, AGDopaO had a UV- Vis peak maximum that red-shifted to 495 nm in FIG.20C, and the solution was visually red, further confirming that KGDopaO and AGDopaO reactions are distinct.

[0133] KGDopaO6,9GlyDeletion was also used as a control to understand random 10 polymerization events between Lys and Dopa. Glycine, as already stated, is crucial for an interstrand hydrogen bond in natural collagen (Brodsky & Perskikov, 2005) and collagen mimetic peptides (Long et al., 1993). Removal of two glycines at positions six and nine prevented triple helix folding, which was not improved upon raising the pH to initiate covalent capture (FIG.22B, FIG 22C). This supports the idea, without being bound by 15 theory, that the templating of the triple helix, especially in the axial case, is essential for promoting stabilization. These results prompted a further probe into the nuances of the supramolecular interaction’s geometry and the distribution of interactions between Lys with various positions in the o-benzoquinone to form either a Schiff Base or Michael Addition product.20 B. Cation-π Interaction Propensity with Molecular Dynamics

[0134] The supramolecular assembly of the collagen triple helix can be used to guide covalent capture of the resulting tertiary structure. To leverage this supramolecular templating for Lys – Dopa covalent capture, understanding how the pairwise cation-π interactions between Dopa and Lys influence triple helical stabilization is crucial. Like the 25 covalent bond formation of cysteine (DiChiara et al., 2018), homocysteine (Tanrikulu & Raines, 2014), and charge pair isopeptide bond formation (Hulgan et al., 2020), the cation-π pair-wise interaction of Lys – Dopa is mediated, in part, by the distance of their supramolecular interactions (Steiner & Koellner, 2001).

[0135] By utilizing molecular dynamics simulations, the interaction 30 propensity was observed for both an axial interaction, which occurs from the L-to-M and M- to-T, and the lateral interaction, from the T-to-L strands, in the homotrimer of KGDopaO. The interaction distance between the center of the ring of Dopa and the amine of Lys are correlated in FIG.23Afor the axial interaction. With an ideal interaction distance for pairwise45cation-π interactions being less than 5 ˚A, it was observed that the axial interaction had a significant amount of the population density within this cutoff. In FIG.23D, the lateral interaction shows a marked shift towards a greater distance, which is consistent with previous literature (Cole et al., 2022).5

[0136] Covalent bonding between Lys and Dopa is possible through Michael Addition or the formation of a Schiff Base (FIG.19A). Thus, the distance between the nucleophilic amine of Lys and each carbon of Dopa was correlated. The Michael Addition product is possible through the reaction of the Lys primary amine with the Cδ2 position of the ring (FIG.23B, FIG.23E). Once again, it was observed, without being bound, that the 10 axial interactions would have a greater likelihood of being within 5 ˚A of this addition site.The angle (θ) between the normal of the center of the aromatic ring and the Lys ζ-amine for the cation-π interaction was also measured, with 0° and 180° representing the normal vector, and 90° correlated with the edge of the aromatic ring. In lateral cases, the angle was closer to 90° with population maxima at 59.58° and 118.69° (see FIG.24A). Axial interactions were 15 more likely to be located over the ring with maxima from the L-to-M strands having angles of 22.67° and 156.94° and M-to-T strands 22.63° and 157.20° (FIG.24B, FIG.24C).

[0137] The propensity to form a secondary ketimine (Schiff Base) at one of the ketone functional groups was also investigated with molecular dynamics by plotting the normalized frequency between each of the possible addition sites (C^1 or Cζ) of Dopa to the 20 Nζ-amine of Lys. In FIG.23C and FIG 23F there is again illustrated a preference for an axial interaction over a lateral interaction. Compared with the Michael Addition axial plot in FIG.23B, the Cζ had higher counts; however, simulations suggest, without being bound by theory, that both the Michael Addition product and Schiff Base can form in this system. These molecular dynamics data indicate that the axial over lateral geometry is more accessible for 25 covalent capture via Dopa oxidation, consistent with the CD thermal stability data in FIG.17.C. Covalent Capture of a Complex Heterotrimer

[0138] With homotrimer kinetics formally characterized with both experimental and computational techniques, the Lys – Dopa interaction was then incorporated into a complex ABC-type heterotrimer. Covalent capture strategies have proven successful in 30 creating discrete triple helices with greater thermal stability (Li et al., 2019), enhanced integrin binding (Peterson et al., 2022) and elucidated type I collagen’s heterotrimeric strand registration (Jalan et al., 2020). ABC-type heterotrimers, where the leading, middle and trailing strands are nonequivalent, are particularly challenging to stabilize because of the need for a46definite register (Walker et al., 2021; Islami et al., 2023). To confirm that an ABC-type triple helix could be selectively stabilized through Lys – Dopa reagent-free covalent capture, the ABC-type heterotrimer presented in Table 4 was synthesized. The variety of pairwise interactions promoting heterotrimer formation include axial, lateral charge pairs and axial 5 cation-π interactions (FIG. 25A). This heterotrimer sequence was based-on a known design and crystal structure (PDB ID: 8TW0) where the amino acid in position 22 of the C strand (C- 22), denoted with an X, was previously an aspartic acid (D) (Cole et al., 2024). The C-22 position was substituted with Dopa to form an axial cation-π interaction.Table 4: ABC-type Heterotrimer Sequence for Covalent Capture SEQ ID NO: Monomer Sequence10*isotopically labeled amino acids (HT3 A: G21; HT3 B: K2, G21; HT3 C: G21)

[0139] To validate the unique composition of an ABC-type heterotrimer, CD thermal melts were performed on each ternary, binary and unary mixture. Referring to Table 15 5, it can be seen that none of the unary solutions had observable thermal transitions, but the binary mixtures all folded to varying extents. Each binary (AB, AC and BC) and ternary mixture (ABC-Supra and ABC-Covalent) demonstrated a positive CD signal at roughly 225 nm and a negative signal around 198 nm (FIG.25B). In addition, it was evident in both the melt and melt derivative spectra that the ABC, ternary mixture was uniquely stable with a Tm20 = 40.0 °C (FIG.25B, FIG.25C). The specificity of the ABC-type heterotrimer is the difference in Tmbetween the most stable species (ABC) and the second most stable species (AB). The supramolecular ABC- type heterotrimer, therefore, has a specificity of 22.5 °C. This high specificity suggested a unique composition for the ternary mixture. Due to the potential disruption of heterotrimer folding under basic conditions, we covalently captured the folded 25 heterotrimer at pH 7.4. Visually, the sample turned yellow, suggesting oxidation at neutral conditions. In addition, the covalently captured ABC-type heterotrimer increased the Tm by 5.5°C to a stability of 45.5 °C after 72 hrs (FIG.25D).47Table 5: Tmof Binary and Ternary Mixtures for an ABC-type HeterotrimeraNo strong thermal transition observed bSupra spectra were collected at pH = 4.5. CCC was 5 performed at 3 mM, pH = 7.4 in 10 mM phosphate buffer and spectrum was collected after 72 hours.

[0140] To confirm that the unique ABC composition and register were not compromised by covalent bond formation a 2D (1H – 15N) heteronuclear sequential quantum 10 coherence (HSQC) NMR study of the ABC-type heterotrimer was performed and compared it against the supramolecular assembly (Buevich & Baum, 2001). Each strand was labelled with an 15N isotopic tag at the 21st glycine (See Table 4). It was hypothesized, without being bound by the theory, that the B-20-Lys – C-22-Dopa interaction would alter the chemical shift of the C-21-Gly position and possibly impact the chemical shifts of all tagged amino 15 acids if the triple helix was significantly compromised. When overlaying the HSQC spectra of the ABC supramolecular assembly at pH = 4.5 (FIG.26A), it was observed that there were three peaks correlating to the free monomer strand and three trimer peaks. These trimer peaks (Tx, where x = A, B or C) closely resemble the shifts of the previously synthesized ABC-type heterotrimer.5520

[0141] Next, the pH of the ABC mixture was adjusted to pH = 7.4 and spectra were acquired at six hours, to match CD, and 120 hrs (5 days). Notably, no additional peaks were observed, but the peak that corresponds to the C-strand in the ABC-type heterotrimer did shift upfield in both the 1H and 15N dimensions. The B-strand of the trimer (TB) also had a minor shift after covalent capture. Both peaks were consistent from the 6 hours to 120 hours 25 time points, suggesting that the triple helix is not compromised up to five days. The TA strand’s chemical shifts held constant upon covalent capture. These NMR data confirm that the ABC- type heterotrimer is a unique register and composition and the ABC-type heterotrimer is maintained after covalent capture.48D. Covalent Stabilization of Polymerized Collagen Mimetic Fibers

[0142] With covalent capture characterized in homotrimeric and heterotrimeric collagen mimetic peptides, interhelical interactions stabilized with Lys – Dopa covalent capture were investigated. Cross-links provide essential stability to interhelical 5 interactions in vivo.6 Fibrillogenesis of collagen is a complicated assembly process involving different sub-types of collagen, glycosylation, and active enzyme-mediated folding (Fratzl & Weinkamer, 2007). Utilizing supramolecular interactions, such as charge pairs (O’Leary et al., 2011), polymerizing collagen mimetic peptides have been shown to form higher-order structures. While these collagen mimetic peptides (CMPs) only employ 33 – 48 amino acid 10 residues compared to the roughly 1,000 residues in natural collagen Type I, biomimetic properties such as D-banding have been conserved (Rele et al., 2007). These CMP fibers utilize a sticky-ended61 or symmetric fiber assembly mechanisms (Tanrikulu et al., 2016, Tanrikulu et al., 2024).

[0143] To further show the utility of this covalent capture method, Dopa was 15 substituted into the sticky-ended polymerizing collagen scheme that contains the sequence of (PKG)4(POG)4(DOG)2XOGDOG (SEQ ID NO: 42) (FIG.27A, FIG.27B). When X = aspartic acid (D), this relatively short polypeptide polymerizes into a nanoscale, fibrous assembly that forms a hydrogel when utilizing only charge pairs (O’Leary et al., 2011).When the supramolecular cation-π interaction was incorporated, where X = Dopa and termed 20 FDopa, the sample demonstrated pH dependence and formed a hydrogel at pH 4.5. In contrast, at pH 7.4 and 9.0, the immediate oxidation caused precipitate to form (FIG.27C). At pH 4.5, a 2% w / v solution of FDopain H2O was prepared and formed a hydrogel. The hydrogel had a storage modulus of 640 Pa and loss modulus of 90 Pa and was linearly viscoelastic up to 6.5% strain (FIG.27D). Subjecting the hydrogel to higher strain resulted in a reduction of the 25 storage modulus, indicating that the hydrogel is shear thinning. At a strain of ca.10%, the loss modulus exceeds the storage modulus, and the material begins to flow like a liquid, losing its hydrogel character.

[0144] Using cryo-electron microscopy (Cryo EM), the collagen mimetic polymeric mesh of the supramolecular hydrogel at pH 4.5 was visualized (FIG. 28A).30 Subsequently, the system was covalently captured by adjusting the hydrogel to pH 9.5. Upon pH adjustment, the hydrogel character of the material was irreversibly lost due to the covalent capture reaction. In contrast to the thin fibers observed in supramolecular FDopa, a bundling of the fibrous assemblies into thick fibers was observed by Cryo EM (FIG.28A, FIG.28E). Due49to insolubility from covalent capture, scanning electron microscopy (SEM) was utilized to successfully confirm the presence of fibrous structures at both pH values using previously established methods (FIG.28B, FIG.28F) (O’Leary et al., 2011).

[0145] To measure the thermal stabilization of the covalently captured FDopa5 fibers, small-angle X-ray scattering (SAXS) experiments were performed from 5-60 °C on both the supramolecular and covalently captured fibers. SAXS is able to report on the average macromolecular structure of the fibers in situ as opposed to Cryo EM, which requires samples to be frozen, and CD, which can only be used to investigate the secondary structure and cannot report on the presence of fibers directly.10

[0146] Scattering curves of both the supramolecular and covalently captured samples at 5 °C confirmed that both formed fibers that could be modeled as elliptical cylinders (FIG.28C. FIG.28G, Table 6). Both the supramolecular and covalently captured fibers have a minor diameter of 2.2 nm, but the covalently captured system had an average major diameter that was 2-fold larger (6.6 vs 13.2 nm). These results are consistent with Cryo 15 EM, which shows that the fibers have a higher propensity to bundle together upon being covalently captured. These samples were then gradually heated to 60 °C, and SAXS measurements were taken every 5 °C to monitor changes in the fiber structure. Upon heating, the supramolecular fibers started to disappear, as seen as a temperature-dependent decrease in the scattering intensity (FIG.28D). By 40 °C, the scattering curve was consistent with the 20 presence of disordered peptide, suggesting, without being bound by theory, that the supramolecular fibers were completely disassembled at this temperature. The covalently captured fibers, in contrast, were much more resistant to heat, and only a modest decrease in scattering intensity was observed over the entire temperature range (FIG.28H).Table 6: Fitting parameters for SAXS fits of FDopaSample FDopaSupra FDopaCCScale6 × 10−43 × 10−4axis ratio 3 6length (nm) 4 × 1024 × 102SLD Peptide (˚A−2) 1.27 × 1.27 ×10−510−5SLD Solvent (˚A−2) 9.44 × 9.44 ×10−610−650

[0147] The average scattering intensity plotted as a function of temperature shows that the supramolecular fibers have a Tmof 25 °C, as determined by finding the inflection point of the sigmoidal curve fit (FIG.29A). The Tm for the covalently captured system could not be accurately calculated because the fibers did not completely disassemble over the 5 investigated temperature range. The thermal stability of the supramolecular FDopa fibers was further investigated by conducting a CD temperature sweep, which resulted in a Tmof 18°C (FIG.29B). Taken together, these data provide evidence that the Lys – Dopa covalent capture scheme can stabilize higher-order CMP fiber assemblies.E. Materials and Methods10 (i) Peptide Synthesis

[0148] Peptides were synthesized on a solid phase low-loading rink amide MBHA resin (0.35 mmol / gram) using standard Fmoc-protected amino acids to obtain peptides with C-termini amidation. For FDopa, the peptide was synthesized on preloaded Gly- Wang resin, resulting in a free C-terminus. For amino acid deprotection, 25% (v / v) piperidine 15 in dimethylformamide (DMF) was used. Coupling steps were performed with 2-(1H-7- azabnzotriazol-1yl)-1,1,3,3- tetramethyl uranium hexafluorophosphate methanaminium (HATU) and diisopropylethylamine (DiEA) in DMF at 1:4:4:6 equivalents of resin:amino acids:HATU:DiEA. Acetylation of the N-terminus was done using a double addition of excess acetic anhydride and DiEA in dichloromethane (DCM). Peptides were cleaved from 20 the resin with 7.5% v / v scavengers (tri-isopropylsilane, H2O, ethanedithiol and anisole) in trifluoroacetic acid (TFA). While FDopa was not acetylated, resulting in an unprotected N- terminus after TFA cleavage. The TFA mixture was evaporated under a stream of nitrogen gas, and cold diethyl ether was used to precipitate the crude peptide. The crude peptide was centrifuged, the ether was decanted, and the wash was repeated. Peptides were dissolved in 25 H2O and filtered for purification. The peptides were purified via reverse-phase high- performance liquid chromatography (HPLC), with the binary solvent system being water and acetonitrile, each with 0.05% TFA, at a gradient of 0.7% / min on a 19 x 250 mm XC-18 column. The samples were rotovapped to remove acetonitrile and then frozen before lyophilization. Liquid chromatography on an Agilent Pursuit 5 Diphenyl 150 x 2.0 mm 30 column with tandem mass spectrometry (LC-MS) confirmed the peptide mass and purity (Agilent, California, USA). Pure, lyophilized peptides were prepared at a working concentration in their respective buffers. All peptide concentrations were determined by mass.51(ii) Covalent Capture

[0149] To prepare peptides for covalent capture, all peptides were dissolved at pH 4.5 and annealed at 85 °C for 15 mins. Once the peptide solutions cooled to ambient temperature, the samples were left to fold for 24 hrs at 4 °C. All covalent capture reactions 5 were performed at a peptide concentration of 3 mM at pH 9.5 for a minimum of 3 days.Buffer and temperature reaction conditions varied as needed between peptide systems to ensure the supramolecular triple helix remained stable (Table 7). For homotrimer reactions, peptides were dissolved at 6 mM in water at pH 4.5, annealed, and allowed to fold for at least 24 hours. After a triple helical signal was confirmed with circular dichroism, the sample was 10 mixed in a 1:1 volume ratio with 20 mM bicarbonate buffer (pH 9.5) to initiate the covalent capture reaction. The pH was adjusted as needed with small volumes of NaOH to ensure the pH remained at 9.5 post-dilution. For heterotrimer reactions, the folded ABC system in 10 mM phosphate buffer dissolved at a total peptide concentration of 3 mM (1mM of each constituent peptide of the heterotrimer). Heterotrimer samples were adjusted to pH 7.4 to 15 initiate the reaction. Covalent capture of fiber-forming CMPs was performed in water without any additional buffer, and the pH was adjusted to 9.5 to initiate the reaction. The reactions were characterized by matrix-assisted laser desorption ionization (MALDI) TOF MS with a 1% (w / v) matrix of α-cyano-4-hydroxycinnamic acid (CHCA) in 50:50 acetonitrile:H2O or by UV-Vis. For UV-Vis characterization, homotrimer and heterotrimer samples were diluted 20 to 0.15 mM for total peptide concentration and in both cases of supramolecular and covalently captured peptides. Upon dilution, the absorbance measurements were carried out in a quartz cuvette on a UV-Vis Spectropho- tometer Evolution 220 (Thermo Scientific, USA).Table 7: Reaction Conditions for Covalent Capture25(iii) Circular Dichroism

[0150] All circular dichroism (CD) data were collected on a J-810 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a Peltier temperature controller.52Spectrum measurement was collected at 5 °C. Heterotrimer and homotrimer samples were diluted into a 1 mm cuvette with fresh MQ H2O for a final peptide concentration of 0.3 mM and 1 mM buffer with a pH = 4.5. The melting curves were collected from 5 °C to 85 °C with a heating rate of 10 °C / hour. To monitor refolding, the sample solution was kept at 85 °C for 5 30 min, and then the reverse temperature gradient was applied back to 5 °C. The first-order derivatives of the melting curves were calculated with the Savitsky-Golay smoothing algorithm, and the temperature where the minimum derivative value appears was defined as the melting temperature (Tm) (Savitsky & Golay, 1964). The mean residue ellipticity (MRE) value was then calculated with the equation, MRE = (θ × m) / (c × l × nr × 10) where θ represents 10 the experimental ellipticity in millidegrees (mDeg), m is the molecular weight of the peptide (g / mol), c is the peptide concentration (milligrams / milliliter), l is the path length of the cuvette (cm) and nr is the number of amino acid residues in the peptide.(iv) Molecular Dynamics Simulations

[0151] All-atom molecular dynamics (MD) simulations were performed to 15 estimate the distance between the Lys side chain’s nitrogen and Dopa’s benzene ring. The initial collagen structure was obtained from the Protein Data Bank (PDB ID: 3B0S) (Okuyama et al., 2012). By using DLPacker,37 a molecular packing software, energy-minimized R- groups for both Lys and Dopa were incorporated into the structure at the 12th and 14th positions. The simulation was prepared using the CHARMM-GUI (Jo et al., 2008; Lee et al., 20 2016). The cubic simulation box had a 12 nm side and approximately 118,000 atoms. The solution was comprised of 39198 TIP3P water molecules and 150 mM NaCl counter-ions. Gromacs (Berendsen et al., 1995; Bekker et al., 1993; Abraham et al., 2015) was used to carry out MD simulations and was parameterized with a Charmm36 force field (Huang et al., 2013). The simulations employed periodic boundary conditions in all directions, and electrostatic 25 interactions were treated by using the particle-mesh Ewald method (Darden et al., 1993;Essmann et al., 1995). The simulation system was minimized and thermally equilibrated under an NVT ensemble. After thermal equilibration, we carried out pressure equilibration under an NPT ensemble. Equilibration was performed using a 1 fs simulation step. After equilibration, we increased the simulation step to 2 fs and ran the production MD for 150 ns. The coordinates 30 of the simulation system were extracted every 100 ps. Visual Molecular Dynamics (VMD)46 software was used to analyze the simulation data.(v) NMR53

[0152] The isotopically-labeled ABC-type heterotrimer peptides were prepared at 2.7 mM in 9 mM phosphate buffer, 10 mM trimethylsilyl propanoic acid (TSP), and 90%H2O / 10%D2O. All characterizations were performed on a Bruker NEO 600 MHz High- Performance digital NMR with a helium-cooled inverse TCI probe at 30 °C. All 1H-15N 5 HSQC data was collected using the same set of parameters, including 1024 x 128 complex points, 8 scans, and 16 dummy scans. A spectral window of 35 ppm was used for the nitrogen dimension and 16 ppm for the hydrogen dimension. The nitrogen carrier frequency was set at 117 ppm, and the proton carrier frequency was set to match the water signal. The 90-degree pulse was calibrated separately for each sample. Raw NMR data was processed using TopSpin 10 4.3.0 software (Bruker, Mass., USA).(vi) Cryo Electron Microscopy

[0153] FDopawas prepared at 2.00% w / v in MQ H2O (pH 4.5 and 9.5). Before plunging, samples were diluted to 0.10% w / v in fresh MQ water. Samples were prepared on 200 mesh lacey carbon grids.3 µL of each sample were plunged into liquid ethane with a Leica 15 EM GP automatic plunge freezer (Leica Microsystems, Wetzlar, Germany). The plunging chamber was set to have the following conditions: a temperature of 20 °C and a humidity of 90%. After preparation, samples were examined at the National Center for High-Resolution Electron Microscopy on a JEM-2200FS (JOEL, Tokyo, Japan) transmission electron microscopy instrument using an F416.0 camera (TVIPS, Gauting, Germany) with an 20 accelerator voltage of 200 kV. Zero-loss images were acquired using Serial EM in a low-dose mode.(vii) Scanning Electron Microscopy

[0154] FDopa was prepared at 2.00% w / v in MQ H2O (pH 4.5 and 9.5). Samples were placed into Porous Spec Pots (Electron Microscopy Sciences, Hatfield, PA) and were 25 dehydrated using a series of ethanol in MQ water dilutions (30, 50, 60,70, 80, 90%, 2 × 100%) for 10 min each. The samples were then critical point dried using a Leica EM CPD300 (Leica Biosystems, Deer Park, IL), followed by sputter coating with 5 µm of gold using a Denton Desk V Sputter System (Denton Vacuum, Moorestown, NJ). SEM was done using a Helios NanoLab 660 Scanning Electron Microscope (FEI Company, Hillsboro, OR) and all 30 micrographs were obtained at 2 kV and 25 pA.(viii) Scattering

[0155] Small-angle X-ray scattering measurements were performed using the laboratory-based Xeuss 3.0 instrument (Xenocs, France) at The Center for Scattering Methods54(CSM) in the Faculty of Science, Lund University, equipped with an X-ray source producing a photon beam with a wavelength of 1.34 ˚A. The scattering patterns were recorded with an Eiger2 R1M 2D- detector (Dectris) and azimuthally integrated using the XSACT program available with the equipment, creating 1D scattering curves. The radially averaged intensity, 5 I(q), is given as a function of the scattering vector q=4π sinθ / λ , where λ is the wavelength and2θ is the scattering angle. The sample capillaries were put in a Peltier-type multi-sample holder, heating the samples from 5 to 60 °C in 5 °C steps. Samples were equilibrated for a minimum of 10 minutes at each temperature before scattering measurements were taken. The fitted scattering curves at 5 °C were fit using SasView 5.0.5 (fitting parameters shown in Table 6).10 (ix) ATR-FTIR

[0156] Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR-FTIR) was used to analyze the secondary structure of DOPA-containing peptides and controls using a NicoletTM iS20 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA). Each sample was dried on the window with a stream of nitrogen gas and measured 15 using 30 scans at a resolution of 4 cm−1. Intensities were normalized to the highest intensity of each sample.(x) Rheology

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Claims

What Is Claimed Is:

1. A peptide between 33 and 48 amino acids in length, wherein said peptide comprises a first region, a second region and a third region, wherein:the first region is of the formula (PX1G)n, wherein:P is proline;X1 is independently selected from amino acids with a positive formal charge at neutral pH; andG is glycine;n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14;the second region is of the formula (POG)m ; whereinP is proline;O is 4-hydroxyproline;G is glycine;m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14;the third region is of the formula (Y1OG)o; whereineach Y1 is independently selected from the group consisting of aromatic amino acids, anionic amino acids, and levodopa, and further wherein at least one Y1 is an aromatic amino acid;O is 4-hydroxyproline;G is glycine; ando is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; or a peptide between 31 and 48 amino acids in length comprising a fifth region, a sixth region, and a seventh region, wherein:the fifth region is of the formula (POG)p, wherein:P is proline;O is 4-hydroxyproline;G is glycine;p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11;the sixth region of between three and twenty-four amino acids in length, each amino acid residue being independently selected from among proline, 4-hydroxyproline, glycine, an amino acid having a reactive amine group, and levodopa; andthe seventh region is of the formula (POG)q; wherein65P is proline;O is 4-hydroxyproline;G is glycine;q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.2 The peptide according to claim 1, wherein X1 is arginine or lysine.

3. The peptide according to either claim 1 or 2, wherein each Y1 is independently aspartic acid, glutamic acid, tyrosine, tryptophan, histidine, or phenylalanine.

4. The peptide according to any one of claims 1-3, wherein each Y1 iseitheraspartic acid and tyrosine, aspartic acid and tryptophan, aspartic acid and phenylalanine, glutamic acid and tyrosine, glutamic acid and tryptophan, or glutamic acid and phenylalanine.

5. The peptide according to anyone of claims 1-4, wherein the third region of the peptide has at least 90 % sequence identity with (DOG)2(FOG)2(SEQ ID NO: 3), (DOGFOG)2(SEQ ID NO: 4), (FOGDOG)2 (SEQ ID NO: 5), (DOG)2(WOG)2 (SEQ ID NO: 6), (DOGWOG)2(SEQ ID NO: 7), (WOGDOG)2(SEQ ID NO: 8), (DOG)2(YOG)2(SEQ ID NO: 9), (DOGYOG)2 (SEQ ID NO: 10), or (YOGDOG)2 (SEQ ID NO: 11).

6. The peptide according to any one of claims 1-5, wherein the peptide further comprises a fourth region of the formula (POG)r; whereinP is proline;O is 4-hydroxyproline;G is glycine; andr is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

7. The peptide according to claim 1, wherein the peptide has at least 90% sequence identity with (PKG)2(PRG)2(POG)4(DOG)2(FOG)2 (SEQ ID NO: 12) (PKG)2(PRG)2(POG)4(DOG)2(WOG)2 (SEQ ID NO: 13) (PKG)2(PRG)2(POG)4(DOG)2(YOG)2 (SEQ ID NO: 14) (POG)4(PKG)2(PRG)2(POG)2(DOG)2(YOG)2 (SEQ ID NO: 15) (PKGPRG)2(POG)4(DOG)2(YOG)2 (SEQ ID NO: 16), (PKGPRG)2(POG)4(DOGYOG)2 (SEQ ID NO: 17), or (PKGPRG)2(POG)4(YOGDOG)2 (SEQ ID NO: 18).

8. The peptide according to claim 1, wherein the sixth region is of the formula POGX2Y2G or PY2GX2OG; wherein:66X2is levodopa; andY2 is an amino acid having a reactive amine group.

9. The peptide according to either claim 1 or claim 8, wherein the sixth region has at least 90 % sequence identity with POGX2KG (SEQ ID NO: 20) or PKGX2OG (SEQ ID NO: 22); wherein X2 is levodopa.

10. The peptide according to any one of claims 1, 8, or 9, wherein the peptide has at least 90% sequence identity with (POG)3POGX2KG(POG)3(SEQ ID NO: 43) or (POG)3PKGX2OG(POG)3 (SEQ ID NO: 44); wherein X2 is levodopa.

11. A hydrogel composition formed from a peptide according to any one of claims 1-10 or mixtures thereof.

12. A composition comprising a first peptide, a second peptide, and a third peptide, wherein:the first peptide is 20 to 40 amino acids in length and comprises at least 3 amino acid residues having a positive formal charge at neutral pH and at least 1 aromatic amino acid residue;the second peptide is 20 to 40 amino acids in length and comprises at least 1 amino acid residue having a positive formal charge at neutral pH, at least 1 amino acid residue having a negative formal charge at neutral pH, and at least 1 aromatic amino acid residue; andthe third peptide is 20 to 40 amino acids in length and comprises at least 1 amino acid residue having a positive formal charge at neutral pH, at least 2 amino acid residues having a negative formal charge at neutral pH, at least 1 aromatic amino acid residue, and at least 1 levodopa residue.

13. The composition of claim 12, wherein the first peptide has a net positive formal charge.

14. The composition of claim 13, wherein the first peptide has a net formal charge of +5, +6, +7, +8, +9, or +10.

15. The composition according to any one of claims 12-14, wherein the second and third peptides both have a net formal charge between -5 and +5.

16. The composition according to any one of claims 12-15, wherein the second peptide has a positive net formal charge.6717. The composition according to claim 16, wherein the second peptide has a net formal charge of +2, +3, or +4.

18. The composition according to any one of claims 12-15, wherein the third peptide has a negative net formal charge.

19. The composition according to claim 18, wherein the third peptide has a net formal charge of -3, -4, or -5.

20. The composition according to claim 19, wherein the composition comprises a peptide having at least 90% sequence identity with PKGROGPKGFOGYOGPRGROGKKGPRGPOG (SEQ ID NO: 23), a peptide having at least 90% sequence identity with SKGDOGPOGDRGPKGPOGYKGPOGDKGFRG (SEQ ID NO: 24), and a peptide having at least 90% sequence identity with PDGDRGPRGPOGYOGDDGPEGXOGPPGDOG (SEQ ID NO: 25).68

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