Polymer-peptide bundlemers and assemblies

Abstract

Disclosed herein are polymer-peptide bundlemers and bundlemer-templated copolymers including a peptide bundle and one or more polymers or monomers. Polymer-peptide bundle assemblies that include two or more polymer-peptide bundles covalently bound to each other, wherein each of the two or more polymer-peptide bundles includes: (i) a peptide bundle including two or more peptides, and (ii) one or more polymers covalently bound at a N- or C-terminus of at least one peptide of the two or more polymer-peptide bundles and / or on a side chain of at least one peptide of the two or more polymer-peptide bundles are also disclosed. Methods of making any of the polymer-peptide bundle assemblies, bundlemers and bundlemer-templated copolymers are also disclosed herein.

Description

[0001] POLYMER-PEPTIDE BUNDLEMERS AND ASSEMBLIES

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 588,460 filed on October 06, 2023, and to U.S. Provisional Patent Application No. 63 / 588,355 filed on October 06, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under Grant No. 2003897 and 2011824 awarded by the National Science foundation (NSF) and Grant No. DE-SC0019355 awarded by the US Department of Energy. The government has certain rights in the invention.

[0006] FIELD

[0007] The present disclosure relates to polymer-peptide bundles and assemblies that can be used in the production of, for example, nanostructured films for membranes, fuel cells, batteries, chemical separators, water desalination devices, data storage, biomedical devices, drug delivery devices.

[0008] BACKGROUND

[0009] The ability to design, synthesize, and characterize exact nanoscale architectures inspired by natural proteins is an ongoing challenge in the materials field. Natural proteins exhibit exquisite combinations of structure and function by virtue of the folding of their amino acid sequences. For example, folded protein structures can provide specificity in the spatial display of chemical groups. Synthetic, i.e., non-natural, polymer materials, in contrast, lack such specificity in shape and chemical display but do provide limitless choices of chemical monomers and functionalization strategies for materials design. While natural proteins have rightfully served as long-standing inspiration for the design of assembled materials with controllable structures and sizes, methods to prescribe non-natural protein or peptide solution assembled structures made with synthetic peptide building blocks remain in their infancy.

[0010] The development and optimization of chemical strategies, such as modem polymerization techniques and click chemistry, have enabled better control over the design of nanoscale architectures, especially in regard to molecular distribution and connectivity. The combination of these synthesis strategies with biological materials capable of forming hierarchical assemblies, e.g., proteins, can enable the creation of a large range of complex supramolecular architectures.

[0011] Protein structures are composed of folded amino acid sequences and include both amorphous and structured moieties. Among the structured moieties found within proteins, the coiled coil is distinctive in its ability to form stable, rigid, isolated structures in solution. Following well-known design principles, de novo protein / peptide sequences can be synthesized, which form helical, amphophilic structures that subsequently self-assemble in water into coiled coils peptide bundles with specific oligomerization states.

[0012] The amino acid residues on the outer surface of these coiled coils can be modified to project chemical functional groups, such as click chemistry handles, at precise locations along the periphery. This allows for the modification of the coiled coil bundle unit, or 'bundlemer,' to present non-natural chemical interactions at its surface, promoting further assembly of these bundlemers.

[0013] While synthetic polymers lack the precise structural control of natural systems in terms of primary sequence and molar mass dispersity, they offer versatility in their range of monomer types, enabling diverse interactions. Minor variations in these interactions between different polymers can lead to phase separation, a phenomenon leveraged to create various nanoscale morphologies in block copolymers, where two or more polymers are covalently connected. Unfortunately, the alignment and registry of these morphological features often require significant processing and / or geometrical confinement.

[0014] Modem reversible deactivation radical polymerization (RDRP) techniques, such as atom transfer radical polymerization (ATRP), enable well-defined, low- dispersity polymers to be synthesized from surfaces. These techniques are compatible with a large range of vinyl monomers, utilize relatively inexpensive reagents, and have been used to impart polymer functionality onto a variety of macromolecular structures. The development of surface-initiate RDRP-compatible structures that are capable of hosting a wide array of polymers with controlled display of these polymers can provide alignment of polymer-grafted materials with new, novel morphologies.

[0015] Thus, to address the foregoing issues and provide the materials field with new, and novel structures that can be used in a wide variety of applications, we disclose herein polymer-peptide bundles and assemblies with improved alignments and morphological assemblies.

[0016] SUMMARY

[0017] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

[0018] Disclosed herein is a polymer-peptide bundlemer including (i.e., comprising) a peptide bundle including two or more peptides and one or more surface-initiating groups present at a N- or C-terminus of the peptide within a peptide bundle and / or on a side chain of at least one peptide in the peptide bundle, wherein the one or more surface-initiating groups enable polymerization from a surface of the peptide bundle and the polymerization is initiated either thermally, photochemically, or through redox initiation.

[0019] Disclosed herein is also a polymer-peptide bundlemer including a peptide bundle including two or more peptides and one or more monomers (or polymers) grafted at a N- or C-terminus of the peptide within the peptide bundle and / or on at least one side chain of a peptide in the peptide bundle, wherein the one or more monomers (or polymers) are connected to the peptide bundle by a linker moiety formed from a reaction between two complementary reactive functional groups.

[0020] Disclosed herein is a bundlemer-templated copolymer including a peptide bundle including two or more peptides, and two or more polymers covalently bound at the N- or C-terminus of a peptide within the peptide bundle and / or on a side chain of at least one peptide of the two or more peptides of the peptide bundle, wherein at least two polymers of the two or more polymers are compositionally different. Disclosed herein is a polymer-peptide bundlemer assembly that includes two or more polymer-peptide bundlemers covalently bound to each other, wherein each of the two or more polymer-peptide bundlemers includes: (i) a peptide bundle including two or more peptides, and (ii) one or more polymers covalently bound at a N- or C-terminus of at least one peptide of the two or more polymer-peptide bundlemers and / or on a side chain of at least one peptide of the two or more polymer-peptide bundlemers.

[0021] Disclosed herein is a method of making any polymer-peptide bundlemer including two or more peptides and one or more monomers (or polymers) grafted at a N- or C-terminus of the peptide within the bundle and / or on at least one side chain of a peptide in the peptide bundle disclosed herein, the method including one or more of (i) synthesizing or preparing a peptide bundle comprising at least two peptides; (ii) installing a surface-initiating group onto the peptide bundle, wherein the surface initiating group is connected to a linker moiety formed from a reaction between two complementary reactive functional groups; and (iii) thermally, photochemically, or through redox initiation of at least one monomer (or polymer) from the surface-initiating group.

[0022] Disclosed herein is a method of making a bundlemer-templated copolymer, the method including one or more of (i) preparing a peptide bundle including at least two peptides, (ii) installing one or more surface-initiating groups onto the peptide bundle, wherein the one or more surface initiating groups are connected to a linker moiety formed from a reaction between two complementary reactive functional groups, and / or (iii) reacting at least one monomer or polymer with the one or more surface-initiating groups.

[0023] Disclosed herein is a method of making a bundlemer-templated copolymer, the method including one or more of (i) adding one or more surface-initiating groups and / or one or more reactive functional groups to a peptide bundle at a N- or C-terminus of a peptide within the peptide bundle, and / or on a side chain of least one peptide of the peptide bundle, wherein the adding occurs either during or after peptide bundle synthesis, and / or (ii) performing at least one of the following steps: (a) contacting a first monomer or polymer with one or more surface-initiating groups to graft the first monomer or polymer at the N- or C-terminus of a peptide within the peptide bundle, and / or graft the first monomer or polymer on the side chain of least one peptide of the peptide bundle; and / or (b) contacting a second monomer or polymer having a complementary reactive functional group to the one or more reactive functional groups of the peptide bundle to graft the second monomer or polymer at the N- or C terminus of a peptide within the peptide bundle, and / or graft the second monomer or polymer on the side chain of least one peptide of the peptide bundle.

[0024] Disclosed herein is also a method of making any of the polymer-peptide bundlemer assemblies disclosed herein, the method including one or more of (i) copolymerizing two or more peptide bundles and (ii) grafting one or more polymers at a N- or C-terminus of at least one peptide of the two or more peptide bundles and / or on a side chain of at least one peptide of the two or more peptide bundles.

[0025] Disclosed herein is a method of making a polymer-peptide bundlemer assembly, the method including one or more of (i) covalently bonding two or more peptide bundles together, and / or (ii) grafting one or more polymers at a N- or C- terminus of a peptide within at least one peptide bundle and / or on a side chain of at least one peptide of the two or more peptide bundles.

[0026] Disclosed herein is an article including any one of the polymer-peptide bundlemer assemblies and / or bundlemer-templated copolymers disclosed herein.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Other features and advantages of the compositions, devices and methods disclosed herein will be apparent to those skilled in the art reading the following detailed description in conjugation with the exemplary embodiments illustrated in the drawings, wherein:

[0029] FIG. 1 depicts a representative illustration of an exemplary peptide bundle containing a complementary reactive functional group.

[0030] FIG. 2 depicts a representative illustration of a peptide bundle containing a surface initiating group.

[0031] FIG. 3 depicts a representative illustration of polymer-peptide bundle assembly synthesis that follows a grafting-from approach after peptide bundle copolymerization. Feature A is a polymer that has been grafted onto the polymer- peptide bundle assembly. Reaction MA is a photo-mediated ATRP polymerization reaction.

[0032] FIG. 4 depicts a representative illustration of a polymer-peptide bundle assembly synthesis following a grafting-from approach before peptide bundle copolymerization. Feature A is a polymer that has been grafted onto the polymerpeptide bundle assembly. Reaction MA is a photo-mediated ATRP polymerization reaction.

[0033] FIG. 5 depicts a representative illustration of a polymer-peptide bundle copolymer synthesis following a grafting-to approach after peptide bundle copolymerization. Feature B is a polymer that has been grafted onto the polymer- peptide bundle assembly.

[0034] FIG. 6 depicts a representative illustration of a polymer-peptide bundle copolymer synthesis following a grafting-to approach before peptide bundle copolymerization.

[0035] FIG. 7 depicts a representative illustration of a synthesis process that installs a surface initiating group onto a peptide bundle via a complementary reactive functional group.

[0036] FIG. 8 depicts a representative illustration of a polymer-peptide bundle copolymer synthesis following a subsequent grafting-from approach after peptide bundle copolymerization. Features A and B are polymers that have been grafted onto the polymer-peptide bundle assembly. Reactions MA and MB are photomediated ATRP polymerization reactions.

[0037] FIG. 9 depicts a representative illustration of a polymer-peptide bundlemer synthesis that produces a polymer-peptide bundlemer with different grafted polymers. Features A and C are polymers that have been grafted onto the polymer- peptide bundle. Reactions MA and Me are photo-mediated ATRP polymerization reactions.

[0038] FIG. 10 depicts a representative illustration of a polymer-peptide bundle assembly synthesis that uses different complementary reactive functional groups to produce a polymer-peptide bundle assembly with different grafted polymers. Features A and B are polymers that have been grafted onto the polymer-peptide bundle assembly. Reaction MA is a photo-mediated ATRP polymerization reaction. FIG. 11 depicts a representative illustration of a statistical polymer-peptide bundle assembly synthesis that produces a polymer-peptide bundle assembly with different grafted polymers. Features A and B are polymers that have been grafted onto the polymer-peptide bundle assembly. Reactions MA and MB are photomediated ATRP polymerization reactions.

[0039] FIG. 12 depicts a representative illustration of a block-copolymer polymer- peptide bundle assembly synthesis that produces a polymer-peptide bundle assembly with different grafted polymers. Features A and B are polymers that have been grafted onto the polymer-peptide bundle assembly. Reaction MA is a photomediated ATRP polymerization reaction. Features n and m indicate the number of each represented block-copolymer structure.

[0040] FIG. 13 depicts a representative illustration of a periodic polymer-peptide bundle assembly synthesis that produces a polymer-peptide bundle assembly with different grafted polymers. Features A and B are polymers that have been grafted onto the polymer-peptide bundle assembly. Reactions MA and Me are a photomediated ATRP polymerization reactions. Features n and m indicate the number of each represented block-copolymer structure. Feature p represents the total number of periodic structures in the assembly.

[0041] FIG. 14 depicts a representative illustration of a polymer-peptide bundle assembly synthesis method that involves click chemistry and ATRP.

[0042] FIG. 15 depicts the results of small molecule studies used to probe the efficiency of an exemplary Diels-Alder cycloaddition reaction. The LC-MS results (graph on right) show successful conjugation for the anticipated change in molecular weight of the species. LIPLC results (graph on left) show both the exo and endo isomers of the exemplary product.

[0043] FIG. 16 depicts TEM images of rigid rod structures from exemplary bundlemers.

[0044] FIG. 17 depicts polymerization data for exemplary grafted polymer-peptide bundlemers. (A) H-NMR data showing the appearance of the polymer peak. (B) GPC traces showing the ability to graft polymers with low dispersities of varoius sizes. (C) LCST measurements for each conjugate. FIG. 18 depicts negatively stained TEM images of exemplary bottlebrush polymers and a control sample. (A) and (B) are negatively stained TEM images of exemplary peptidic rigid rods synthesized via a Diels-Alder cycloaddition with 4 kDa PDMAEMA grafted onto the surface of the exemplary peptidic rigid rod. (C) Control sample consisting of bundlemer grafts with 4 kDa PDMAEMA without any chain-like structures.

[0045] FIG. 19 depicts a representative synthesis schematic of a bundlemer macroinitiator and subsequent grafting-from processes. (A) The bundlemer forming peptide amino acid sequence synthesized with a halide terminated amino acid side chain to produce a tetramer ATRP macroinitiator. (B) Bundlemer-DMAEMA grafts formed via the introduction of the ATRP activator (i.e., Cu(l)Br) to produce a fourarmed star-polymer architecture.

[0046] FIG. 20 depicts sequence and CD spectra of a BNDLE peptide with various side chains, measured over a range of temperatures to demonstrate its melting behavior and overall stability. A lysine is modified at position 13, resulting in CD spectra of the unmodified BNDLE (A), the BNDLE-Br (B), and BNDLE-KG-Br (C).

[0047] FIG. 21 depicts the characterization of a bundlemer-PDMAEMA conjugate.

[0048] (A) Room temperature CD spectra of the coiled coil structure after polymerization.

[0049] (B) GPC of the cleaved polymers shows three distinct molecular weights having a narrow dispersity of approximately 1 .3. (C) Light absorption at 490 nm confirming the presence of a molecular weight dependent LCST for the bundlemer-polymer conjugates. The reported Tm are the inflection points in the data and the images are of a specimen before and after phase separation.

[0050] DETAILED DESCRIPTION

[0051] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. The present disclosure relates to using base peptide bundle nanoparticles as substrates for polymer grafts. The resulting macromolecular material includes two or more polymer-peptide bundles with polymers covalently bonded at exact positions along the periphery of the polymer-peptide bundles. These macromolecular materials have some similarities to block copolymers, except that the polymers in the polymer-peptide bundles can be either the same or different, thus enabling new morphologies and better organized microphases with fewer morphological defects. The ability to specifically control the covalent attachments of the polymers to the peptide backbones allows for controlled directional display of polymer interactions, thereby providing access to complex morphologies that are not possible with just block copolymers.

[0052] As used herein, the term "grafting" refers to the act of covalently connecting a monomer or polymer to a peptide bundle.

[0053] As used herein, the term “polymer graft” refers to a polymer covalently connected to a side of another molecule, molecular assembly, particle, or surface. A polymer graft can have been formed using a grafting to, grafting from, or grafting through approach, which are all terms well known to one skilled in the art. In exemplary embodiments, a polymer graft is formed from covalently attaching a polymer to a peptide bundle using a synthetic approach such as, but not limited to, atom transfer radical polymerization. In exemplary embodiments, a polymer graft is formed by covalently attaching a polymer to a peptide bundle using a grafting to synthetic approach by linking the end group of a polymer to the side group of a peptide bundle using complementary reactive functional groups.

[0054] One aspect of the present disclosure is a polymer-peptide bundlemer including a peptide bundle including two or more peptides and one or more surfaceinitiating groups present at a N- or C-terminus of a peptide within the peptide bundle and / or on a side chain of at least one peptide in the peptide bundle, wherein the one or more surface-initiating groups enable polymerization from a surface of the peptide bundle and the polymerization is initiated thermally, photochemically, or through redox initiation.

[0055] As used herein, the term "peptide bundle" is used interchangeably with “bundlemer”, "peptide bundlemer" or "base-peptide bundle" and refers to two or more peptides or one peptide / protein that can form an ordered structure e.g., alpha helices) under physiologic conditions (e.g., in aqueous solution at pH 7.2, 22° C., under physiologically-relevant salt concentrations). Thus, a peptide bundle can be composed of multiple peptides which assemble into a desired structure. The peptide bundle can also be composed of a single peptide or protein sequence that assembles into a desired structure. The peptide bundle can include an amino acid coding for more than one peptide or protein that can fold into a coiled coil (for example zig zag'ing back and forth with looped ends) structure. The peptide bundles can assemble into helical structures and coiled coil structures (i.e., a- helical structures in which helices are wound around each other to form superhelical bundles). The ordered structures that peptide bundles can assume include, but are not limited to, helical and coiled-coil structures. Other ordered structures that the peptide bundles can assume and the methods for achieving these structures are well-known to those skilled in the art (e.g., see Derek N. Woolfson, The Design of Coiled-Coil Structures and Assemblies, Advances in Protein Chemistry, Academic Press, Volume 70, 2005, pages 79-112, ISSN 0065- 3233, ISBN 9780120342709).

[0056] As used herein, the term "polymer-peptide bundle" is used interchangeably with “polymer-peptide bundlemer”, and refers to a peptide bundle having one or more monomer or polymers covalently attached to constituent peptides of the peptide bundle. Each peptide of polymer-peptide bundle can be connected to one or more than one monomer or polymer and the monomers or polymers connected to each peptide can be the same or different. The polymer connected to each peptide can be a homopolymer, where the constituent monomers are of the same type, or a copolymer, where the constituent monomers and their distribution along the polymer backbone are different.

[0057] As used herein, the term “bundlemer polymer conjugate” refers to a peptide bundle having two or more monomers or polymers covalently attached to constituent peptides of the peptide bundle. Each peptide of polymer-peptide bundle can be connected to two or more monomers or polymers and the monomers or polymers connected to each peptide can be the same or different. The polymers connected to each peptide can be a homopolymer, where the constituent monomers are of the same type, or a copolymer, where the constituent monomers and their distribution along the polymer backbone are different.

[0058] As used herein, the term “bundlemer-templated copolymer” refers to a peptide bundle having two or more monomer or polymers covalently attached or conjugated to constituent peptides of the peptide bundle. Each peptide of polymerpeptide bundle can be connected to two or more monomers or polymers and at least two of the monomers or polymers connected to each peptide are compositionally different. At least one polymer connected to each peptide can be a homopolymer, where the constituent monomers are of the same type, or a copolymer, where the constituent monomers and their distribution along the polymer backbone are different.

[0059] As used herein, the term “surface-initiating group” refers to a functional group that enables polymerization from a peptide bundle surface. The polymerization can be thermally, photochemically, or redox initiated through thermochemical, photochemical, or through reduction-oxidation chemical processes, respectively, producing radicals or other active species that propagate via polymerization of monomers.

[0060] In exemplary embodiments, the polymer-peptide bundlemer includes at least one linker moiety.

[0061] As used herein, the term "linker moiety" refers to a structural group that either covalently links peptide bundles together into polymer-peptide bundlemer assemblies or links peptide bundles with (co)polymers and / or surface-initiating groups. The linker moiety can be formed by reacting complementary reactive functional groups on peptide bundles. The linker moiety can also link the peptide bundles and / or polymer-peptide bundlemer assemblies to a drug molecule, a RNA molecule, a biofunctional peptide, a protein, an enzyme, a polysaccharide, a glycosaminoglycan, a fluorescent dye, a semiconductive organic molecule, a catalyst or an inorganic nanoparticle.

[0062] As used herein, the term "complementary reactive functional groups" refers to reactive functional groups in which a first reactive functional group on a first moiety is capable of reacting with a second reactive functional group on a second moiety to form at least one covalent bond between the reactive functional groups. The first and the second moiety can be peptides and / or polymers. Reactions that allow for a first reactive functional group on a first moiety to covalently bond with a second reactive functional group on a second moiety include, but are not limited to, reactions that are characterized as click chemistry. Common click chemistry reactions that can be performed to covalently bond a first reactive functional group on a first moiety with a second reactive functional group on a second moiety are known to those skilled in the art (e.g., see “Click Chemistry in Materials Science”, WXi, TF Scott, CJ Kloxin, CN Bowman, Advanced Functional Materials 24 (18), 2572-2590).

[0063] In exemplary embodiments, the polymer-peptide bundlemer includes at least one linker moiety that is a product of a reaction between two complementary reactive functional groups. In exemplary embodiments, at least one of the complementary reactive functional groups is present at the N- or C-terminus of a peptide within a peptide bundle and / or on at least one peptide of the peptide bundle. In exemplary embodiments, at least one of the complementary reactive functional groups is on a side chain of at least one peptide of the peptide bundle. In exemplary embodiments, at least one of the complementary reactive functional groups comprises a maleimide and the other complementary reactive functional group comprises a furan. In exemplary embodiments, at least one of the complementary reactive functional groups includes an azide and the other complementary reactive functional group includes an alkyne. In exemplary embodiments, the at least one linker moiety is located between at least one peptide in the peptide bundle and at least one surface-initiating group. In exemplary embodiments, the at least one linker moiety includes a peptide, for example, a lysine-glycine (KG) peptide. The linker moiety can include other amino acids besides a lysine-glycine peptide so long as the peptide possesses a free amine from which these peptidic linkers can be added (e.g., via a peptide coupling reaction) or reacted with a click functional group (e.g., propiol ic acid) to which a surface-initiating group can be chemically attached (e.g., through a copper- catalyzed azide — alkyne cycloaddition rearciont). Other linker moieties can be hetereo- or homo-bifunctional crosslinkers that are commonly used in bioconjugation reactions. Examples of hetereo- or homo-bifunctional crosslinkers include, but are not limited to, polyethylene glycol or alkane units. Additional examples of hetereo- or homo-bifunctional crosslinkers can be found in “Bioconjugation Techniques” by Greg T. Hermanson, Academic Press, 3rdEd., September 2013, ISBN-13: 978-0123822390. The resulting functional group is then used as a chemical handle to attach a surface-initiating group.

[0064] In exemplary embodiments, the one or more surface-initiating groups have thermal-, photo-, or redox-active functionality that, once triggered, can initiate polymerization. In exemplary embodiments, the one or more surface-initiating groups include a functional group that can mediate a controlled radical polymerization selected from, but not limited to, bromoisobutyrate, an alkoxyamine, a dithiocarbamate, and a thiothiocarbonyl.

[0065] In exemplary embodiments, the polymer-peptide bundlemer includes one or more surface-initiating groups capable of initiating a reversible deactivation radical polymerization selected from, but not limited to, surface-initiated atom transfer radical polymerization, surface reversible addition-fragmentation chain transfer polymerization, surface-initiated nitroxide-mediate polymerization, and surface- initiated photoinitiator mediated polymerization.

[0066] As used herein, a "reversible deactivation radical polymerization" or RDRP is a polymerization technique that proceeds through an equilibrium of active and dormant growing polymer chains. This mechanism mitigates chain termination reactions, thereby producing low dispersity polymer changes. Common RDRP techniques include atom transfer radical polymerization (ATRP), reversible addition — fragmentation chain transfer (RAFT), and nitroxide-mediated polymerization (NMP).

[0067] In exemplary embodiments, the polymer-peptide bundlemer includes one or more surface-initiating groups for ATRP that include a halide selected from fluorine, chlorine, bromine and iodine.

[0068] In exemplary embodiments, the polymer-peptide bundlemer includes one or more surface-initiating groups that contain either a dithioester, a thiocarbamate, a xanthate for RAFT polymerization or an alkoxyamine for NMP.

[0069] Another aspect of the present disclosure is a bundlemer-templated copolymer including a peptide bundle including two or more peptides and two or more monomers (or polymers) grafted at a N- or C-terminus of a peptide within the peptide bundle and / or on at least one side chain of a peptide in the peptide bundle, wherein the two or more monomers (or polymers) are connected to the peptide bundle by a linker moiety formed from a reaction between two complementary reactive functional groups. In exemplary embodiments, the two or more monomers or polymers are different from each other. In exemplary embodiments, the polymerpeptide bundlemer has a coiled-coil structure.

[0070] In exemplary embodiments, the bundlemer-templated copolymer includes two or more monomers that form polymers belonging to a group selected from, but not limited to, polyolefins, polyacrylics, polyesters, polyethers, polythioethers, polyvinylhalides, polysilylenes, polyamides, polystyrenics, polyimides, polysulfones, polycarbonates, and copolymers thereof.

[0071] In exemplary embodiments, the bundlemer-templated copolymer includes at least three monomer or polymers, and at least two monomers or polymers are the same monomer or polymer.

[0072] In exemplary embodiments, the one or more monomers (or polymers) grafted onto the polymer-peptide bundlemer and / or the two or more monomers (or polymers) grafted onto the bundlemer-templated copolymer include a polymerizable functionality such as, but not limited to, a radical polymerizable methacrylate functional group. In other exemplary embodiments, the one or more monomers (or polymers) grafted onto the polymer-peptide bundlemer and / or the two or more monomers (or polymers) grafted onto the bundlemer-templated copolymer include a reactive functional group including a click chemistry functional handle such as, but not limited to, an azide for copper-catalyzed azide alkyne cycloaddition (CuAAC), a furan as a diene for the Diels-Alder (DA) cycloaddition, or a thiol for a thiol-Micheal addition reaction. Any suitable reactive functional group can be included on the one or more monomers (or polymers) grafted onto the polymer-peptide bundlemer and / or the two or more monomers (or polymers) grafted onto the bundlemer-templated copolymer, for example, an azide, a furan, a thiol, an alkyne or a maleimide.

[0073] In exemplary embodiments, the polymer-peptide bundlemer includes one or more of the same monomer (or polymer). In exemplary embodiments, all monomers (or polymers) grafted onto the polymer-peptide bundlemer are the same monomer (or polymer).

[0074] In exemplary embodiments, the polymer-peptide bundlemer and / or the bundlemer-templated copolymer has an aspect ratio from 2 nm by 4 nm (a single bundlemer containing peptides with, for example, 28 amino acids). Polymer-peptide bundle assemblies disclosed herein can have aspect ratios up to 2 nm by 10 microns or more. As the bundlemer assemblies can be very rigid, where the persistence length (the length over which curvilinear direction of the backbone remains correlated or, more simply, the length before the assembly begins to bend) can be as long as the bundlemer assembly itself. These dimensional characteristics promote the formation of liquid crystalline phases.

[0075] In exemplary embodiments, the polymer-peptide bundlemer has a coiled-coil structure.

[0076] Another aspect of the present disclosure is a bundlemer-templated copolymer including one or more of (i) a peptide bundle comprising two or more peptides, and / or (ii) two or more polymers covalently bound at the N- or C-terminus of a peptide within the peptide bundle and / or on a side chain of at least one peptide of the two or more peptides of the peptide bundle, wherein at least two polymers of the two or more polymers are compositionally different. In exemplary embodiments, the bundlemer-templated copolymer has a coiled coil structure.

[0077] In exemplary embodiments, at least one polymer of the two or more polymers of the bundlemer-templated copolymer is a homopolymer.

[0078] In exemplary embodiments, at least one polymer of the two or more polymers of the bundlemer-templated copolymer is a copolymer.

[0079] In exemplary embodiments, at least one polymer of the two or more polymers of the bundlemer-templated copolymer is different from the other polymers by at least one of: a nature of constituent monomer(s), a distribution of constituent monomer(s), or a combination of monomer(s) along a backbone of the bundlemer-templated copolymer.

[0080] In exemplary embodiments, the two or more polymers of the bundlemer- templated copolymer are covalently bound to side chains of two or more peptides of the peptide bundle and the bundlemer-templated copolymer has a multi-armed star-polymer architecture.

[0081] In exemplary embodiments, the peptide bundle of the bundlemer-templated copolymer is a homo-tetramer including four peptides, wherein each peptide includes two polymers covalently bound to a side chain of each peptide, thereby resulting in eight polymers covalently bound on sides of the homo-tetramer.

[0082] In exemplary embodiments, each of the two or more polymers of the bundlemer-templated copolymer is a polymer belonging to a group selected from, but not limited to, polyolefins, polyacrylics, polyesters, polyethers, polythioethers, polyvinylhalides, polysilylenes, polyamides, polystyrenics, polyimides, polysulfones, polycarbonates, and copolymers thereof.

[0083] Another aspect of the present disclosure is a method of making a bundlemer-templated copolymer including one or more of (i) preparing a peptide bundle including at least two peptides, (ii) installing one or more surface-initiating groups onto the peptide bundle, wherein the one or more surface initiating groups are connected to a linker moiety formed from a reaction between two complementary reactive functional groups, and / or (iii) reacting at least one monomer or polymer with the one or more surface-initiating groups.

[0084] In exemplary embodiments, the installing of the one or more surfaceinitiating groups onto the peptide bundle of the method includes one or more of: (i) installing one or more surface-initiating groups at a N- or C-terminus of a peptide within at least one peptide of the peptide bundle, and / or (ii) installing one or more surface-initiating groups on at least one side chain of at least one peptide of the peptide bundle.

[0085] In exemplary embodiments, the method includes (i) installing one or more additional surface-initiating groups onto the peptide bundle, wherein the one or more additional surface-initiating groups are different from surface-initiating groups previously installed onto the peptide bundle and are connected to a linker moiety formed from a reaction between two complementary reactive functional groups, and / or (ii) reacting at least one additional monomer or polymer with the one or more additional surface-initiating groups. In exemplary embodiments, the at least one additional monomer or polymer is different from the at least one monomer or polymer previously reacted with the one or more surface-initiating groups.

[0086] Another aspect of the present disclosure is a method of making a bundlemer-templated copolymer including one or more of (i) adding one or more surface-initiating groups and / or one or more reactive functional groups to a peptide bundle at a N- or C-terminus of a peptide within the peptide bundle, and / or on a side chain of least one peptide of the peptide bundle, wherein the adding occurs either during or after peptide bundle synthesis; and / or (ii) performing at least one of the following steps: (a) contacting a first monomer or polymer with one or more surface-initiating groups to graft the first monomer or polymer at the N- or C- terminus of a peptide within the peptide bundle, and / or graft the first monomer or polymer on the side chain of least one peptide of the peptide bundle; and / or (b) contacting a second monomer or polymer having a complementary reactive functional group to the one or more reactive functional groups of the peptide bundle to graft the second monomer or polymer at the N- or C terminus of a peptide within the peptide bundle, and / or graft the second monomer or polymer on the side chain of least one peptide of the peptide bundle.

[0087] In exemplary embodiments, the one or more surface-initiating groups added onto the peptide bundle include thermal-, photo-, or redox-active functionality that can initiate polymerization.

[0088] In exemplary embodiments, the one or more surface-initiating groups added onto the peptide bundle include a functional group to mediate a controlled radical polymerization, wherein the functional group is selected from, but not limited to, a bromoisobutyrate, an alkoxyamine, a dithiocarbamate, and a thiocarbonylthio, wherein the bromoisobutyrate mediates an atom transfer radical polymerization (ATRP), the alkoxyamine mediates a nitroxide mediated polymerization (NMP), the dithiocarbamate mediates a photoiniferter-mediated polymerization (PIMP), and the thiocarbonylthio mediates a reversible addition-fragmentation chain transfer (RAFT) polymerization.

[0089] In exemplary embodiments, the first monomer contacted with the one or more surface-initiating groups includes a methacrylate functional group as a radical polymerizable functionality. In exemplary embodiments, the one or more reactive functional groups added onto the peptide bundle include an azide, a furan, or a thiol.

[0090] In exemplary embodiments, the complementary reactive functional group of the second monomer or polymer includes an alkyne or a maleimide.

[0091] In exemplary embodiments, the step of adding one or more surface-initiating groups and / or one or more reactive functional groups includes selectively removing an amino acid protecting group at the end of the peptide synthesis, and then adding the one or more surface-initiating groups and / or one or more reactive functional groups for subsequent polymerization.

[0092] In exemplary embodiments, the step of contacting the second monomer or polymer having the complementary reactive functional group includes triggering a complementary reaction by heat, light, catalyst, or other activator.

[0093] In exemplary embodiments, the peptide bundle includes two or more different surface-initiating groups, and the method includes contacting a third monomer or polymer that is different from the first monomer or polymer with one of the two or more different surface-initiating groups to graft the third monomer or polymer at the N- or C-terminus of a peptide within the peptide bundle, and / or graft the third monomer or polymer on the side chain of least one peptide of the peptide bundle.

[0094] In exemplary embodiments, the peptide bundle includes two or more different reactive functional groups, and the method includes contacting a fourth monomer or polymer that is (i) different from the second monomer or polymer and (ii) has a complementary reactive functional group to one of the two or more different reactive functional groups to graft the fourth monomer or polymer at the N- or C-terminus of a peptide within the peptide bundle, and / or graft the fourth monomer or polymer on the side chain of least one peptide of the peptide bundle.

[0095] In exemplary embodiments, the contacting of the second monomer or polymer having the complementary reactive functional group to the one or more reactive functional groups of the peptide bundle occurs before the contacting of the first monomer or polymer with the one or more surface-initiating groups.

[0096] Another aspect of the present disclosure is a polymer-peptide bundlemer assembly that includes two or more polymer-peptide bundles covalently bound to each other, wherein each of the two or more polymer-peptide bundlemers includes: (i) a peptide bundle comprising two or more peptides, and (ii) one or more polymers covalently bound at a N- or C-terminus of a peptide within at least one peptide of the two or more polymer-peptide bundlemers and / or on a side chain of at least one peptide of the two or more polymer-peptide bundlemers.

[0097] As used herein, the term “polymer-peptide bundlemer assembly” is used interchangeably with "macromolecular assembly" and refers to polymer-peptide bundles that are covalently connected and can be the same or different. Peptide bundle units, not containing an attached polymer, can be part of the polymer- peptide bundlemer assembly. The connections of the peptide bundle units and / or polymer-peptide bundle units can be any copolymer sequence, such as alternating, statistical (random), gradient, periodic, or block, which are terms well known to those skilled in the art. A polymer-peptide bundlemer assembly can have a length of more than 1 nm, more than 5 nm, more than 10 nm, more than 50 nm, more than 100 nm, more than 1 pm, more than 5 pm, more than 10 pm, more than 1 mm or any subrange or specific length falling with the range of 1 nm to 1 mm.

[0098] In exemplary embodiments, the polymer-peptide bundlemer assembly includes at least one polymer-peptide bundlemer that is different from at least one other polymer-peptide bundlemer in the assembly by at least one of (a) the location of the polymer on the polymer-peptide bundlemer, (b) the number of polymers covalently attached to the polymer-peptide bundlemer, (c) the polymer composition, (d) the polymer molecular weight and its distribution, and / or (e) the peptides present in the peptide bundle.

[0099] In exemplary embodiments, at least one polymer-peptide bundlemer in the polymer-peptide bundlemer assembly is different from at least one other polymer- peptide bundlemer by at least two of (a), (b), (c), (d) and (e).

[0100] In exemplary embodiments, at least one polymer-peptide bundlemer in the polymer-peptide bundlemer assembly is different from at least one other polymer- peptide bundlemer by at least three of (a), (b), (c), (d) and (e).

[0101] In exemplary embodiments, at least one polymer-peptide bundlemer in the polymer-peptide bundlemer assembly is different from at least one other polymer- peptide bundlemer by at least four of (a), (b), (c), (d) and (e). In exemplary embodiments, at least one polymer-peptide bundlemer in the polymer-peptide bundlemer assembly is different from at least one other polymerpeptide bundlemer by all of (a), (b), (c), (d) and (e).

[0102] In exemplary embodiments, the polymer-peptide bundlemer assembly includes one or more base-peptide bundles, each including two or more basepeptides. In exemplary embodiments, the one or more base-peptide bundles are the same and / or are the same as the peptide bundles of the two or more polymer- peptide bundlemers in the assembly. In other exemplary embodiments, the one or more base-peptide bundles are different from each other and / or are different from the peptide bundles of the two or more polymer-peptide bundlemers in the assembly.

[0103] In exemplary embodiments, the polymer-peptide bundlemer assembly includes the two or more polymer-peptide bundlemers arranged in a copolymer configuration selected from the group including statistical, random, alternating, periodic, block or combinations thereof.

[0104] In exemplary embodiments, the polymer-peptide bundlemer assembly includes one or more base-peptide bundles and the two or more polymer-peptide bundlemers arranged in a copolymer configuration selected from the group including statistical, random, alternating, periodic, block or combinations thereof.

[0105] In exemplary embodiments, the polymer-peptide bundlemer assembly includes one or more base-peptide bundles, each base-peptide bundle comprising two or more base-peptides, wherein the one or more base-peptide bundles are the same as or different from at least one peptide bundle of the two or more polymer- peptide bundles.

[0106] In exemplary embodiments, the polymer-peptide bundlemer assembly includes the two or more polymer-peptide bundlemers and / or one or more base- peptide bundles, wherein the two or more polymer-peptide bundlemers and / or the one or more base-peptide bundles contain at least one of a linker moiety, a spacer moiety, or a combination thereof.

[0107] As used herein, the term "spacer moiety" refers to a molecular group that covalently links peptide bundles together into polymer-peptide bundlemer copolymers assemblies or links peptide bundles with (co)polymers or surface- initiating groups. The spacer moiety can possess two or more complementary reactive functional groups that are the same or different. A spacer moiety containing two linker moieties of the same type is known to one skilled in the art as a homo-bifunctional crosslinker, whereas a spacer moiety containing two linker moieties of a different type is known to one skilled in the art as a hetero-bifunctional crosslinker.

[0108] In exemplary embodiments, the at least one linker moiety, spacer moiety, or combination thereof is located at (i) a N- or C-terminus of a peptide within at least one peptide bundle of the two or more polymer-peptide bundlemers and / or (ii) a N- or C-terminus of a peptide within at least one of the one or more base-peptide bundles.

[0109] In exemplary embodiments, the at least one linker moiety, spacer moiety, or combination thereof is located on a side chain of (i) at least one peptide of the two or more polymer-peptide bundlemers and / or (ii) at least one peptide of at least one of the one or more base-peptide bundles.

[0110] In exemplary embodiments, the at least one linker moiety in the polymer- peptide bundlemer assembly is a product of a reaction between two complementary reactive functional groups.

[0111] In exemplary embodiments, at least one of the two complementary reactive functional groups is at (i) the N- or C-terminus of a peptide within at least one of the two or more polymer-peptide bundles and / or (ii) a N- or C-terminus of a peptide within at least one of the one or more base-peptide bundles.

[0112] In exemplary embodiments, at least one of the two complementary reactive functional groups is on a side chain of (i) at least one peptide of the two or more polymer-peptide bundles and / or (ii) at least one peptide of at least one of the one or more base-peptide bundles.

[0113] In exemplary embodiments, the polymer-peptide bundlemer assembly includes a linker moiety on at least one of the two or more polymer-peptide bundlemers, wherein the linker moiety is functionalized with either a drug molecule, a RNA molecule, a biofunctional peptide, a protein, an enzyme, a polysaccharide, a glycosaminoglycan, a fluorescent dye, a semiconductive organic molecule, a catalyst, an inorganic nanoparticle or a combination thereof. In exemplary embodiments, the polymer-peptide bundlemer assembly includes a spacer moiety that is a homo-bifunctional crosslinker or a heterobifunctional crosslinker, the spacer moiety including a central molecule flanked by a reaction product of two complementary reactive functional groups on two peptide bundles.

[0114] In exemplary embodiments, the polymer-peptide bundlemer assembly includes at least two polymer-peptide bundlemers, where each of the polymer- peptide bundlemers includes a polymer selected from, but not limited to, a polyolefin, a polyacrylic, a polyester, a polyether, a polythioether, a polyvinylhalide, a polysilylene, a polyamide, a polystyrenic, a polyimide, a polysulfone, a polycarbonate, and copolymers thereof.

[0115] Suitable examples of polymers that can be grafted onto the bundlemers and bundlemer assemblies disclosed herein include, but are not limited to, polyolefins such as polyethylene and polypropylene, polyacrylics such as poly(2- (dimethylamino)ethyl methacrylate), poly(oligo(ethylene glycol) methyl ether methacrylate), poly(hydroxyethyl methacrylate), poly(acrylic acid), poly(methacrylic acid), poly(methyl methacrylate), poly(acrylonitrile) and poly(acrylamide), polyesters such as polycaprolactone, polyglycolic acid, polylactic acid, polyhydroxyalkanoates, and polyethylene terephthalate, polyethers such as polyethylene oxide, polypropylene oxide, polyoxymethlyene, and polyphenylene oxide, polythioethers such as poly(phenylene sulfide), poly(thioether sulfone), and poly(thioether ether ketone), poly(vinyl halides) such as poly(vinyl chloride), poly(vinyl fluoride), and poly(vinylidene chloride), polysilylenes such as poly(methylsilylene), poly(methylphenylsilylene), poly(vinylmethylsilylene), and poly(phenylsilylene), polyamides such as nylon, proteins, and other peptides, polystrenics such as polystyrene and poly(styrene sulfonate), polyimides such as polyetherimide and poly(oxydiphenylene pyromellitimide), polysulfones such as poly(ether sulfone) and poly(phenylene sulfone), polycarbonates such as poly(4,4'- isopropylidenediphenylene carbonate), and copolymers thereof.

[0116] In exemplary embodiments, the polymer-peptide bundlemer assembly includes at least two polymer-peptide bundlemers and / or one or more base-peptide bundles, the at least two polymer-peptide bundlemers and / or one or more base- peptide bundles including at least one of a linker moiety, a spacer moiety, or a combination thereof covalently bonded at least one of the following locations: (i) on two or more polymer-peptide bundlemers, (ii) on one or more polymers or a surface initiating group at the N- or C-terminus of a peptide within the polymer-peptide bundlemers and / or the base-peptide bundles, and / or (iii) on one or more polymers or a surface initiating group on a side chain of at least one peptide of the polymer- peptide bundlemers and / or the base-peptide bundles.

[0117] In exemplary embodiments, the polymer-peptide bundlemer assembly includes one or more polymers covalently bound to at least one peptide bundle, where at least one polymer of the one or more polymers is a homopolymer.

[0118] In exemplary embodiments, the polymer-peptide bundlemer assembly includes one or more polymers covalently bound to at least one peptide bundle, where at least one polymer of the one or more polymers is a copolymer.

[0119] In exemplary embodiments, the polymer-peptide bundlemer assembly includes at least two polymers covalently bound to one or more peptide bundles, where the two polymers are the same.

[0120] In exemplary embodiments, the polymer-peptide bundlemer assembly includes at least two polymers covalently bound to one or more peptide bundles, wherein at least one polymer of is different from the other by at least one of nature of constituent monomer(s), distribution of the constituent monomer(s), and combination of monomers along the polymer backbone.

[0121] In exemplary embodiments, the polymer-peptide bundlemer assembly includes at least two polymers covalently bound to the side chains of two or more peptides of the polymer-peptide bundlemer assembly in a multi-armed star-polymer architecture. In other exemplary embodiments, the polymer-peptide bundlemer assembly includes a homo-tetramer peptide bundle containing four peptides, wherein each peptide includes two polymers covalently bound to a side chain of each peptide, thereby resulting in eight polymers covalently bound on the sides of the tetramer peptide bundle.

[0122] In exemplary embodiments, the polymer-peptide bundlemer assembly includes at least two polymer-peptide bundlemers and / or one or more base-peptide bundles having a coiled-coil structure. In exemplary embodiments, the entire polymer-peptide bundlemer assembly has a coiled-coil structure.

[0123] Another aspect of the present disclosure is a method of making any polymer-peptide bundlemer including two or more peptides and one or more monomers (or polymers) grafted at a N- or C-terminus of a peptide within the peptide bundle and / or on at least one side chain of a peptide in the peptide bundle disclosed herein, the method including one or more of (i) synthesizing or preparing a peptide bundle comprising at least two peptides; (ii) installing a surface-initiating group onto the peptide bundle, wherein the surface initiating group is connected to a linker moiety formed from a reaction between two complementary reactive functional groups; and (iii) thermally, photochemically, or redox initiation of at least one monomer (or polymer) with the surface-initiating group to produce a grafted polymer-peptide bundlemer.

[0124] In exemplary embodiments, the method includes selectively removing an amino acid protecting group at the end of the peptide bundle synthesis before installing the surface-initiating group for subsequent polymerization.

[0125] In exemplary embodiments, the step of thermally, photochemically, or redox initiation of at least one monomer (or polymer) includes triggering the reaction with heat, light, a catalyst, and / or with other activators.

[0126] In exemplary embodiments, the method of making any polymer-peptide bundlemer including two or more peptides and one or more monomers (or polymers) grafted at a N- or C-terminus of a peptide within the peptide bundle and / or on at least one side chain of a peptide in the peptide bundle disclosed herein includes (i) installing the at least one surface-initiating group at a N- or C- terminus of a peptide within at least one peptide of the peptide bundle, and / or (ii) installing the at least one surface-initiating group on at least one side chain of at least one peptide of the peptide bundle.

[0127] In exemplary embodiments, the method of making any polymer-peptide bundlemer including two or more peptides and one or more monomers (or polymers) grafted at a N- or C-terminus of a peptide within the peptide bundle and / or on at least one side chain of a peptide in the peptide bundle disclosed herein includes (i) installing one or more additional surface-initiating groups onto the peptide bundle, wherein the one or more additional surface-initiating groups are different from a surface-initiating group previously installed onto the polymerpeptide bundlemer and are connected to a linker moiety formed from a reaction between two complementary reactive functional groups; and / or (ii) thermally, photochemically, or through redox initiation reacting at least one additional monomer (or polymer) with the one or more additional surface-initiating groups. In exemplary embodiments, the at least one additional monomer (or polymer) is different from a monomer (or polymer) previously installed onto the polymer-peptide bundlemer.

[0128] Another aspect of the present disclosure is a method of making any of the polymer-peptide bundlemer assemblies disclosed herein, the method including one or more of (i) copolymerizing two or more peptide bundles and (ii) grafting one or more polymers at a N- or C-terminus of at least one peptide of the two or more peptide bundles and / or on a side chain of at least one peptide of the two or more peptide bundles.

[0129] In exemplary embodiments, the step of grafting one or more polymers on at least one peptide of the two or more peptide bundles comprises (i) grafting a first polymer to a first peptide either before or after the copolymerization step, (ii) grafting a second polymer to a second peptide after the step of grafting the first polymer, and / or (iii) optionally repeating steps (a) and (b) for a plurality of different types of polymers.

[0130] In exemplary embodiments, the copolymerizing step includes polymerizing at least two base-peptide bundles together or with at least one polymer-peptide bundle of the two or more polymer-peptide bundles. In exemplary embodiments, the polymerizing of at least two base-peptide bundles together or with at least one polymer-peptide bundle of the two or more polymer-peptide bundles is followed by grafting a polymer to the base-peptide bundle after the step of copolymerizing.

[0131] In exemplary embodiments, the method includes (i) adding a surface initiating group and / or a reactive functional group, during or after peptide synthesis, at a N- or C-terminus of at least one peptide of the two or more peptide bundles, and / or on a side chain of at least one peptide of the two or more peptide bundles, and (ii) performing at least one of the following steps: (a) contacting a monomer (or polymer) with the surface initiating group to graft the monomer (or polymer) at the N- or C-terminus of the at least one peptide of the two or more peptide bundles, and / or on at least one side chain of the at least one peptide of the two or more peptide bundles; and / or (b) contacting a monomer (or polymer) having a complementary reactive functional group with the reactive functional group of the at least one peptide of the two or more peptide bundles to thereby graft the monomer (or polymer) at the N- or C terminus of the at least one peptide of the two or more peptide bundles, and / or on the side chain of the at least one peptide of the two or more peptide bundles. In exemplary embodiments, at least one peptide bundle comprises two or more different surface initiating groups, and the method further comprises repeating step (a) with a monomer (or polymer) different from the first monomer (or polymer) grafted onto the peptide bundle. In other exemplary embodiments, at least one peptide bundle comprises two or more different reactive functional groups, and the method further comprises repeating step (b) to graft a plurality of different types of monomers (or polymers), each having a different complementary reactive group. In exemplary embodiments, step (b) is performed before step (a).

[0132] In exemplary embodiments, at least one polymer-peptide bundlemer of the assembly is synthesized via a grafting-to process. A grafting-to process includes the installation of a complementary reactive functional group, such as those used in click chemistry, onto a peptide bundle, followed by a chemical ligation reaction that involves reacting a complementary reactive functional group on the peptide bundle with a second complementary reactive functional group on a desired polymer to produce a covalent bond between the peptide bundle and the desired polymer. A representative illustration of a peptide bundle containing a complementary reactive functional group, which can be used in grafting-to processes, can be seen in FIG. 1.

[0133] In exemplary embodiments, the two or more peptide bundles are created / synthesized via a solid phase peptide synthesis (SPPS) process. In other exemplary embodiments, the two or more peptide bundles are created / synthesized via a recombinant expression process. In exemplary embodiments, at least one polymer-peptide bundlemer of the assembly is synthesized via a grafting-from process. A grafting-from process includes the installation of a surface initiating group onto a peptide bundle (e.g., either during a SPPS process or after peptide synthesis), followed by a polymerization reaction that involves the surface initiating group reacting with a desired monomer (or polymer) to form a covalent bond between the peptide bundle and the desired monomer (or polymer). A representative illustration of a peptide bundle containing a surface initiating group, which can be used in grafting-from processes, can be seen in FIG. 2.

[0134] Another aspect of the present disclosure is a method of making a polymerpeptide bundlemer assembly including one or more of covalently bonding two or more peptide bundles together, and / or grafting one or more polymers at a N- or C- terminus of a peptide within at least one peptide bundle and / or on a side chain of at least one peptide of the two or more peptide bundles.

[0135] In exemplary embodiments, the method includes one or more of (a) grafting a first polymer to a first peptide of at least one peptide bundle either before or after the covalent bonding step, (b) grafting a second polymer to a second peptide of at least one peptide bundle after the grafting of the first polymer, and / or optionally repeating steps (a) and (b) for a plurality of different types of polymers.

[0136] In exemplary embodiments, the method includes one or more of forming one or more intermediate polymer-peptide bundlemers, covalently bonding at least one base-peptide bundle with the polymer-peptide bundlemer, and / or optionally grafting a polymer to the at least one base-peptide bundle after the covalent bonding with the polymer-peptide bundlemer.

[0137] In exemplary embodiments, the method includes one or more of (i) adding at least one surface initiating group and / or at least one reactive functional group, during or after the covalent bonding of the two or more peptide bundles, at a N- or C-terminus of a peptide within at least one peptide of the two or more peptide bundles, and / or on a side chain of at least one peptide of the two or more peptide bundles, and (ii) performing at least one of the following steps: (a) contacting a monomer or polymer with the at least one surface initiating group to graft the monomer or polymer at the N- or C-terminus of the at least one peptide of the two or more peptide bundles, and / or on the side chain of the at least one peptide of the two or more peptide bundles; and / or (b) contacting a monomer or polymer having a complementary reactive functional group with the at least one reactive functional group of the at least one peptide of the two or more peptide bundles to thereby graft the monomer or polymer at the N- or C terminus of the peptide within the at least one peptide of the two or more peptide bundles, and / or on the side chain of the at least one peptide of the two or more peptide bundles. In exemplary embodiments, at least one peptide bundle includes two or more different surfaceinitiating groups and the method includes repeating step (a) with a different monomer or polymer. In exemplary embodiments, at least one peptide bundle includes two or more different reactive functional groups and the method includes repeating step (b) to graft a plurality of different types of monomers or polymers, each having a different complementary reactive group. In exemplary embodiments, step (b) is performed before step (a).

[0138] Another aspect of the present disclosure is an article including any one of the polymer-peptide bundlemer assemblies and bundlemers disclosed herein.

[0139] In exemplary embodiments, the article is a macromolecular material that can be used in a wide variety of applications, such as, but not limited to, nanostructured films for membranes, fuel cells, batteries, chemical separators, water desalination devices, data storage, biomedical devices, drug delivery devices, smart coatings in implants and diagnostic equipment, and cell scaffolds for tissue engineering. Many of the applications that are well suited for block copolymers are also well suited for macromolecular materials containing any one of the polymer-peptide assemblies disclosed herein. An exemplary added benefit of using any of the polymer-peptide assemblies disclosed herein in macromolecular materials is improved degrees of spatial control in the macromolecular materials, which enable enhanced morphological control over their assembly.

[0140] Examples

[0141] The present disclosure will be described in more detail with reference to the following Examples, which shows exemplary embodiments in accordance with the present disclosure. The present disclosure is not limited to these exemplary embodiments. Example 1 : SPPS Synthesis of Base Peptide Bundles

[0142] Coiled coil forming peptides having an amino acid sequence of SEQ. ID. NO. 1 (DEEIRRM AEEIRQM AERIQQM AEQIQWE A, denoted BNDLE1 ) and SEQ. ID. NO. 2 (DEKIKNM ADQIKHM AWMIDRM AEKIDRE A, denoted BNDLE2) were synthesized using standard Fmoc-protected solid phase peptide synthesis (SPPS) procedures. A 0.25 mmol scale synthesis on Rink Amide resin was carried out using 4-fold excess of Fmoc-protected amino acids. First, 0.2 M solutions of amino acids dissolved in N,N-dimethylformamide (DMF), 1 M oxyma in DMF, and 1 M N,N-diisopropylcarbodiimide (DIC) in DMF were prepared and placed accordingly on a peptide synthesizer. The peptides were synthesized via iterations of deprotection steps, using 20% piperidine in DMF at 75 °C for about one minute, followed by coupling steps, with sequential amino acid and coupling reagents, performed at 90 °C for two minutes per coupling step. Next, the N-termi were modified with appropriate reactive handles. BNDL1 was modified with furoyl chloride and BNDLE2 was modified with 4-maleimidobutyric acid to enable a 4+2 cycloaddition (i.e., a Diels-Alder) reaction between peptide bundles.

[0143] Example 2: Synthesis of a Peptide Bundle Containing a Surface Initiator for a Grafting-from Approach

[0144] Using a modified synthetic approach given in Example 1 , an Fmoc- Lys(Alloc)-OH amino acid was substituted at position 13 of BNDLE1 and orthogonally deprotected to create a branching point within the peptide. The Alloc protecting group was removed from the lysine using a deprotection solution of 0.025 M Pd(PPh3)4 in dichloromethane (DCM) with 0.5 M phenylsilane in DCM, revealing a free amine from where more amino acids can be added. Next, an amino acid armed linker possessing a lysine-glycine (KG) pair was synthesized at the branched point using standard Fmoc-protected SPPS procedures. Finally, alpha- bromoisobutyryl bromide (BIBB) was added to the resin as a solution containing 4 eq. BIBB and 4 eq. triethylamine (TEA) in anhydrous DCM to produce a peptide with a bromide end-group. The peptide was then cleaved from the resin by incubating the resin in a solution of (95 / 5 / 5) (v / v / v) trifluoroacetic acid (TFA), MilliQ water, triisopropylsilane with 5% (w / v) phenol for three hours at room temperature. The peptide TFA solution was then precipitated into chilled ethyl ether, washed three times, and dried under N2. The isolated peptides were then subjected to HPLC for purification and lyophilization. Circular dichroism (CD) spectroscopy was performed to confirm coiled coil-formation.

[0145] Example 3: Synthesis of a Peptide Bundle Containing a Complementary Reactive Functional Group for a Grafting-to Approach

[0146] Similar to Example 2, a modified SPPS approach based on Example 1 was used to create a peptide bundle for use in a grafting-to process. Specially, a Fmoc- Lys(Alloc)-OH amino acid was substituted at position 13 of BNDLE2 and orthogonally deprotected to create a branching point within the peptide. The Alloc protecting group was removed from the lysine using a deprotection solution of 0.025 M Pd(PPhs)4 in dichloromethane (DCM) with 0.5 M phenylsilane in DCM, thus revealing a free amine from where more amino acids can be added. Next, an amino acid armed linker containing a lysine-glycine (KG) pair was synthesized at the branched point using standard Fmoc-protected SPPS procedures. Finally, a propiolic acid coupled to the free amine of the KG pair, thereby providing an alkyne functional group for subsequent copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions with an azide functional synthetic target.

[0147] Example 4: Synthesis of a Polymer-Peptide Bundle Assembly using a Grafting-from Approach after Peptide Bundle Copolymerization

[0148] A representative illustration of this synthesis process can be seen in FIG. 3. The peptide bundle containing the bromide surface initiating group in Example 2 and the peptide bundle BNDLE2 from Example 1 were copolymerized in an aqueous solution and left to sit for 10 minutes so that rigid rods could properly form through a Diels-Alder reaction between a furan on one bundle and maleimide on the other, thereby producing an assembly with peptide bundles containing bromide surface initiating groups. Next, stoichiometric amounts of 2-(dimethylamino)ethyl methacrylate (D MAE MA) were added to 2 mL vials. Prior to addition to the vials, the DMAEMA was passed through alumina basica to remove any inhibitors that may be present in the monomer. The peptide bundle assembly and a catalytic solution containing 10 mM copper (II) bromide, 40 mM tris(2-pyridylmethyl) amine (TPMA), 300 mM sodium bromide (NaBr) and water were then added to the vials containing the DMAEMA. Precise amounts of water, peptide stock solution, and catalytic solution were added to vials such that there would be 1 eq. CuBr2, 4 eq. TPMA, and 30 eq. NaBr in each reaction vial, with a peptide dimer concentration of 2.5 mM. The vials were nitrogen purged for 15 minutes and then subjected to a photo-mediated ATRP polymerization reaction under 365 nm light at 2.5 mW cm-2 intensity for 2-8 hours. After the ATRP polymerization reaction, the reaction mixtures in the vials were passed through alumina neutral to remove the catalysts. The isolated samples were then placed in deuterated water to allow for NMR spectroscopy analysis. After NMR acquisition, the samples were dialyzed against Millipore water using 3.5 kDa MWCll tubing to remove excess catalyst, monomer, and salts.

[0149] Example 5: Synthesis of a Polymer-Peptide Bundle Assembly using a Grafting-from Approach before Peptide Bundle Copolymerization

[0150] A representative illustration of this synthesis process can be seen in FIG. 4. The peptide bundle containing the bromide surface initiating group in Example 2 was subjected to a photo-mediated ATRP polymerization reaction with DMAEMA. The conditions of the ATRP polymerization reaction were the same as those used in Example 4. After the ATRP polymerization reaction, a copolymer of the polymer- grafted peptide bundle product and the peptide bundle BNDL2 from Example 1 was prepared by placing both peptide bundles in an aqueous solution and mixing them for 10 minutes so that the peptide bundles could copolymerize with each other via a Diels-Alder reaction between a furan on one bundle and maleimide on the other. Once rigid rods of the peptide bundles properly formed, the samples were isolated and dialyzed against Millipore water using 3.5 kDa MWCll tubing to remove excess catalyst, monomer, and salts.

[0151] Example 6: Synthesis of a Polymer-Peptide Bundle Copolymer using a Grafting-to approach after Peptide Bundle Copolymerization

[0152] A representative illustration of this synthesis process can be seen in FIG. 5. A copolymer of the peptide bundle from Example 3 and the peptide bundle BNDL1 from Example 1 was prepared by stirring these two peptide bundles in water for 10 minutes until rigid rods properly formed via a Diels-Alder reaction between a furan on one bundle and maleimide on the other. Next, aqueous stock solutions of 200 mM copper sulfate, 400 mM tris-hydroxypropyltriazolylmethylamine (THPTA), and 1 M sodium ascorbate were prepared. A 40 microliter solution of the copolymerized peptide bundle (5 mM peptide) was prepared and a 160 microliter aqueous solution containing 5 eq. of PEG azide was added to the copolymerized peptide bundle solution. 1 microliter of a copper sulfate stock solution, 1 microliter of THPTA stock solution, and 2 microliters of the sodium ascorbate stock solution were added to the reaction mixture. The reaction mixture was stirred at 40°C for 48 hours. A grafted polymer-peptide bundle assembly was then recovered from the reaction mixture.

[0153] Example 7: Synthesis of a Polymer-Peptide Bundle Copolymer using a Grafting-to approach before Peptide Bundle Copolymerization

[0154] A representative illustration of this synthesis process can be seen in FIG. 6. The peptide bundle of Example 3 was reacted with an azide end functionalize PEG under conditions identical to those of Example 6. The resulting grafted polymer- peptide bundle product was then copolymerized with the peptide bundle BNDLE2 from Example 1. The peptide bundle copolymerization conditions were also identical to those of Example 6.

[0155] Example 8: Synthesis of a Peptide Bundle containing a Surface Initiator from a Complementary Reactive Functional Group

[0156] A representative illustration of this synthesis process can be seen in FIG. 7. A 2.5 mM solution of the peptide bundle from Example 3 was added to a 12.5 mM solution of 3-azidopropyl 2-bromo-2-methylpropanoate, copper sulfate and sodium ascorbate. The reaction mixture was stirred until completion. The resulting product was dialyzed against Millipore water to remove excess catalyst, small molecular azide, and salts. The production was confirmed using LCMS.

[0157] Example 9: Synthesis of a Polymer-Peptide Bundle Copolymer using a Subsequent Grafting-from Approach after Peptide Bundle Copolymerization

[0158] A representative illustration of this synthesis process can be seen in FIG. 8. The peptide bundle of Example 2 was copolymerized with the peptide bundle of Example 3. Polymerization from the bromide surface initiating groups on the bundle-peptide copolymer was achieved through atom-transfer radical polymerization from the surface, similar to the procedure in Example 4. The bromide end-group was then removed by reaction with a thioalkyl moiety. The remaining alkyne functional groups were reacted with 3-azidopropyl 2-bromo-2- methylpropanoate using a CuAAC reaction similar to the procedure from Example 8. Polymerization of the resulting bromide functional groups on the bundle-peptide copolymer was achieved through atom-transfer radical polymerization from the surface, similar to the procedure in Example 4, thereby producing a poly(oligo(ethylene oxide) monomethyl ether methacrylate) polymer attachment.

[0159] Example 10: Prophetic Example for Synthesis of a Polymer-Peptide Bundle Containing Multiple Grafted Polymers

[0160] The peptide bundle formed from Example 3 can undergo a photo-mediated ATRP polymerization reaction similar to that of Example 4 to produce a polymerpeptide bundle containing DMAEMA grafted onto the peptide bundle surface and reactive handles at the C and N termini of the bundle. The reactive handles can then undergo a click-chemistry reaction with surface-initiating groups containing complementary reactive groups, thereby producing a polymer-peptide bundlemer with surface-initiating groups at the C and N termini of the bundlemer and a grafted DMAEMA monomer. This bundlemer can then under subsequent photo-mediated ATRP polymerization reactions to graft different monomers or polymers onto the peptide bundle. A representative illustration of this synthesis process can be seen in FIG. 9.

[0161] Example 11 : Prophetic Example for Synthesis of a Polymer-Peptide Bundle Assembly Using Different Complementary Reactive Functional Groups

[0162] Modified versions of BNDLE1 and BNDLE2 can be synthesized via processes similar to those of Example 3 to create two peptide bundles with different complementary reactive functional groups. One of these different complementary reactive functional groups can then react directly with a monomer containing a complementary reactive functional group to produce a first grafted polymer-peptide bundle assembly. The first grafted polymer-peptide bundle assembly can then react with a surface-initiating group containing a reactive functional group that reacts with the other complementary reactive functional group on the first grafted polymer- peptide bundle assembly, thereby installing another point on the assembly that can react with a second monomer to produce a grafted polymer-peptide bundle assembly containing at least two different monomers. A representative illustration of this synthesis process can be seen in FIG. 10. Example 12: Prophetic Example for Synthesis of a Polymer-Peptide Bundle Assembly Using a Statistical Copolymerization Approach

[0163] Modified versions of BNDLE1 and BNDLE2 can be synthesized according to the methods of the above examples to produce versions where both BNDLE1 and BNDLE2 possess a surface-initiating group and a complementary reactive functional group (4 different peptide bundles in total). These bundles can then be randomly covalently bond together to form a random peptide bundle assembly. The produced random peptide bundle assembly can then undergo grafting processes described in the examples above to produce a polymer-peptide bundle assembly. A representative illustration of this synthesis process can be seen in FIG. 11 .

[0164] Example 13: Prophetic Example for Synthesis of a Polymer-Peptide Bundle Assembly Using a Block-Copolymer Copolymerization Approach

[0165] Modified versions of BNDLE1 and BNDLE2 can be synthesized according to the methods of the above examples to produce versions where both BNDLE1 and BNDLE2 possess a surface-initiating group and a complementary reactive functional group (4 different peptide bundles in total). These peptide bundles can then be covalently bonded together to produce block-copolymer structures. These block-copolymer structures can then be covalently bonded together to produce a peptide bundle assembly. The produced random peptide bundle assembly can then undergo grafting processes described in the examples above to produce a polymer-peptide bundle assembly. A representative illustration of this synthesis process can be seen in FIG. 12.

[0166] Polymer-peptide bundle assemblies formed from a block-copolymer copolymerization approach can possess at least two different block units and possess "m" units of one block and "n" units of another block, "n" and "m" are each integers and n+m can be 1 or larger, 100 or larger, or 10,000 or larger.

[0167] Example 14: Prophetic Example for Synthesis of a Polymer-Peptide Bundle Assembly Using a Periodic Copolymerization Approach

[0168] Periodic copolymers of polymer-peptide bundles can be synthesized similar to the block-copolymer method where the oligomer percussors are not made of a single polymer-peptide bundle type. For example, modified versions of BNDLE1 and BNDLE2 can be synthesized according to the methods of the above examples to produce versions where both BNDLE1 and BNDLE2 possess a surface-initiating group and a complementary reactive functional group (4 different peptide bundles in total). Some of these peptide bundles can undergo grafting processes to install monomers on their surfaces. Next, block structures of peptide bundles and polymer-peptide bundlemers can be formed by covalently bonding these bundles and bundlemers together. The block structures can then be covalently bonded together to form a polymer-peptide bundle assembly. Additional grafting reactions can then be performed to graft polymers on peptide bundles containing either surface initiating groups or complementary reactive functional groups. A representative illustration of this synthesis process can be seen in FIG. 13.

[0169] Polymer-peptide bundle assemblies formed from a periodic copolymerization approach can possess at least two different block units and possess "m" units of one block and "n" units of another block, "n", "m" and "p" are each integers and n+m can be 1 or larger, 100 or larger, or 10,000 or larger, p can = 125,000 / (m+n).

[0170] Example 15: Polymer-Peptide Bundle Assemblies Synthesized via Click Chemistry and ATRP

[0171] This example involves creating polymer-peptide bundle assemblies using a strategy that includes first synthesizing peptide bundles with chemical handles capable of forming covalent bonds with each other, and then utilizing click chemistry reactions to install polymers and / or monomers on the peptide bundles. A representative illustration of this strategy can be seen in FIG. 14.

[0172] METHODS:

[0173] Peptide Synthesis

[0174] Peptides (denoted BNDLE1 and BNDLE2) were synthesized on CEM Liberty Blue® peptide microwave synthesizer using the standard Fmoc-protected solid phase peptide synthesis (SPPS) procedure of Example 1. BNDL1 was modified with furoyl chloride and BNDLE2 was modified with 4-maleimidobutyric acid to enable a 4+2 cycloaddition (i.e., a Diels-Alder) reaction between peptide bundles.

[0175] For ATRP initiating peptides, a Fmoc-Lys(Alloc)-OH amino acid was substituted at position 13 of BNDLE1 and orthogonally deprotected to create a branching point within the peptide. The Alloc protecting group was removed from the lysine using a deprotection solution of 0.025 M Pd(PPhs)4 in dichloromethane (DCM) with 0.5 M phenylsilane in DCM, revealing a free amine from where more amino acids can be added. Next, an amino acid armed linker containing a lysineglycine (KG) pair was conjugated to the peptide at the branched point using a CEM Liberty Blue ® instrument. Finally, alpha-bromoisobutyryl bromide (BIBB) was added to the resin using 4 eq. BIBB and 4 eq. triethylamine (TEA) in anhydrous DCM. The peptide was then cleaved from the resin in a solution of (95 / 5 / 5) (v / v / v) trifluoroacetic acid (TFA), MilliQ water, triisopropylsilane with 5% (w / v) phenol for three hours at room temperature. The peptide TFA solution was then precipitated into chilled ethyl ether, washed three times, and dried under N2. Peptides were then subjected to HPLC for purification and lyophilization. Circular dichroism (CD) spectroscopy was performed to ensure the peptide still assemble into coiled coilbased bundlemers.

[0176] Diels-Alder (DA) cycloaddition polymerization of bundlemers to form rigid rods

[0177] Each lyophilized peptide was dissolved separately at 10 mM concentrations in Millipore water. The aqueous solutions were combined in a 1 .5 mL vial such that there was about a 1 :1 .1 peptide ratio, with the furan peptide in excess, and a final concentration of 5 mM per peptide. The vial was then placed on an Eppendorf ® Thermomixer C and left to react at 45 °C and 750 rpm for approximately 5-6 days. Samples were then transferred to 20 kDa MWCll dialysis tubing against a weak HCI solution (around pH 2) to inhibit aggregation and remove unreacted peptide.

[0178] PhotoA TRP from rigid rod surface

[0179] First, a stock catalytic solution was prepared consisting of 10 mM copper (II) bromide, 40 mM tris(2-pyridylmethyl) amine (TPMA), 300 mM sodium bromide (NaBr), and D2O. A peptidic rigid rod stock solution was prepared as well in D2O and left to sit for 10 minutes so that rigid rods could properly assemble. Next, stoichiometric amounts of 2-(dimethylamino)ethyl methacrylate was added to 2 mL autosampler vials. Prior to addition, the monomer was passed through alumina basica to remove any inhibitors. Precise amounts of deuteroxide, peptide stock solution, and catalytic solution were then added to vials such that there would be 1 eq. CuBr2, 4 eq. TPMA, and 30 eq. NaBr in each reaction autosampler vial, with a peptide dimer concentration of 2.5 mM. The samples were then nitrogen purged for 15 minutes, then allowed to stir while under 365 nm light at 2.5 mW cm-2intensity for 2-8 hours. After polymerization, samples were passed through alumina neutral to remove the catalyst and analyzed via NMR spectroscopy. After NMR acquisition, samples are dialyzed against Millipore water using 3.5 kDa MWCll tubing to remove excess catalyst, monomer, and salts, then lyophilized to yield dry white conjugate powder.

[0180] TEM analysis

[0181] Transmission electron microscopy (TEM) imaging was performed on a FEI TALOS F200C microscope with 200kV accelerating voltage. All the images were recorded via a FEI Ceta 16M camera. To prepare the sample grids, carbon-coated 200 mesh copper grids (CF200-Cu; Electron Microscopy Sciences, Inc.) were freshly treated by glow discharge using a glow discharge cleaning system (Pelco EasiGlow; Ted Pella, Inc.). Five microliters of the sample solution were applied to the plasma-treated grids for about 30-60 seconds, then the remaining liquid was blotted from the edge of the grids using filter papers. To negatively stain the grids, 5 mL of an aqueous solution of phosphotungstic acid (2% w / v; pH 6.4) was applied to the cast-film grids and incubated for about 40-60 seconds. Then the grids were blotted by filter paper to remove the remaining staining solution. The stained grids were allowed to sit for at least 20 min under ambient conditions before TEM observation.

[0182] LCST measurements

[0183] Solutions of conjugate samples (1 .5 mg / mL) were prepared in phosphate buffer saline (PBS) buffer at pH 8.0. Their absorbance was monitored via UV-Vis at 490 nm as a function of temperature from 30 °C to 80 °C. A temperature ramp of 1 °C / min was utilized and the LCST of each conjugate was determined as the sharp increase 58 in absorbance at 490 nm. Typically, samples were measured three times to find their average.

[0184] Experiment 1: The Diels-Alder Cycloaddition Bundlemer Polymerization The Diels-Alder (DA) cycloaddition was selected for bundlemer conjugation due to its lack of need for a catalyst, availability of inexpensive reactive handles, and compatibility with subsequent ATRP reactions. The DA reaction utilizes an electron rich diene and electron poor dienophile that will spontaneously react to form a substituted cyclohexene derivative. Furan and the maleimide groups are known reactive pairs within polymer chemistry, therefore, they were utilized in the DA reaction.

[0185] To first probe whether the DA reaction would work, an ARTP initiator (KGBr) was clicked onto the bundlemer. The Furan-BNDLE1-KGBr bundle was dissolved in Millipore water at 5 mM concentration, and 4-maleimidobutyric acid (2 eq.) was added. The solution was left to vortex at 45 °C and 750 rpm. After 4 days, the LC- MS of the reaction mixture revealed that almost all the Furan-BNDLE1-KGBr had reacted with the maleimide, as shown in FIG. 15. This was concluded by the expected change in the molecular weight of the species. There is a double peak seen in the LIPLC trace of this peptide, however integration of each peak leads to the same m / z results. It is assumed that these are different isomers (endo vs. exo) being formed from the cycloaddition that elute slightly differently from the LIPLC column.

[0186] After successful small molecule studies, the reaction was performed with assembled bundlemers by mixing a slight excess of the Furan-BNDLE1-KGBr bundlemer with the BNDLE2-maleimide bundlemer (1.1 : 1 ). The slight excess was employed due to the possibility of the double bond within the maleimide participating in subsequent ATRP reactions. The reaction went for approximately 5 days at 45°C, followed by analysis via LC-MS and TEM to assess the polymerization of bundles to form rigid rod architectures.

[0187] The reaction was originally attempted at a concentration of 2.5 mM for each peptide in Millipore water, but it was later found that a higher concentration of peptides in acidic conditions may be needed to improve the yield of the material synthesis. Examination in TEM did not reveal a high yielding polymerization, although LC-MS did show that some dimer was present for initial attempts using 2.5 mM peptide in Millipore water. An increase of concentration to 5 mM per peptide, however, did reveal a larger presence of dimer peptide after about 6 days of reacting, but TEM revealed only large aggregate species. Dialysis performed against a weak HCI solution (pH 2) with 20 kDa MWCll dialysis tubing was able to inhibit aggregation, and examination of this sample in TEM did reveal the anticipated rigid rod structures, shown in FIG. 16. It was hypothesized that the BNDLE2 peptide might be prone to aggregation since (i) this peptide was computationally designed to self-assemble into higher ordered structured lattices, and (ii) increased acidity can cause these bundles to be charged and disfavor lattice formation. Subsequent reactions were performed at a concentration of 5 mM of each peptide in Millipore water at pH 4, which consistently enabled the stepgrowth polymerization of bundles into nanorods, as evidenced by TEM images and LC-MS results. Once assembled, the supramolecular polymeric macro-initiating species were ready to undergo the photoATRP reaction to form bottlebrush nanorods.

[0188] Experiment 2: Grafting PDMAEMA from rod surface

[0189] Once the Diels Alder cycloaddition of bundle-bundle pairs yielded supramolecular rigid rod polymers, photoATRP was used to graft PDMAEMA from the rod backbone to synthesize an exemplary bottlebrush structure. Bundlemer rods were designed so that alternating bundles contained four initiating sites, thus allowing for the synthesis of precise, highly dense bottlebrush nanorods. There is a plug-and-play nature of the material design, meaning that the number and proximity of initiating sites is dependent on bundlemer starting material (e.g., tetrameric vs. trimeric coiled coil bundles). A photoATRP procedure that successfully synthesized bundlemer grafts was compatible with the rigid rods, and NMR analysis showed the presence of polymer at conversions around 60% given the amount of time allotted for the reaction. Higher conversions could be achieved with longer reaction times but were avoided as longer reactive times correspond to poorer dispersities. Polymerization results are shown in Table 1 and FIG. 17 as the appearance of the peak around 1 .0 ppm can be seen (this peak corresponds to the saturated alkyl peak for the PDMAEMA polymer).

[0190] Table 1 : Calculated Values for Grafted PDMAEMA

[0191] * Table values were experimentally derived for varying sized PDMAEMA chains grafted from peptidic rigid rod structures. Polymers were synthesized to target 4 kDa or 8 kDa PDMAEMA in size, corresponding to the size of a single peptide and double the size of a single peptide. Following NMR analysis, the samples were dialyzed in 3.5 kDA MWCll tubing against Millipore water to remove unreacted monomer, salt, and the catalytic species. The samples were then lyophilized prior to further analysis.

[0192] Bottlebrush nanorods were designed to be monodispersed, precise structures through utilization of living polymerization techniques. GPC analysis was conducted to evaluate the polydispersity of the PDMAEMA chains grown from the rigid rod backbone. Prior to analysis, the samples were digested via acid hydrolysis to prevent peptide assembly from effecting the polymer elution during GPC. Once the cleaved polymers were obtained, they were analyzed via GPC against PMMA standards, as shown in FIG. 17. This analysis revealed polymers with dispersities around 1 .3.

[0193] The synthetic polymer, PDMAEMA, was chosen due to its environmental responsiveness to changes in thermal, pH, and ionic conditions, which is imparted to the hybrid structure. PDMAEMA also exhibits a lower critical solution temperature (LCST), meaning that an increase in temperature causes the PDMAEMA chains to become dehydrated and collapse to shield from water, causing a two-phase region. The LCST behavior is highly dependent on the size of chains, concentration of sample, temperature, and pH of solution. At higher temperatures and more basic conditions, the chains are more likely to be deprotonated, causing this two-phase transition. However, at lower temperatures or acidic conditions, the chains are hydrated and expanded in solution, promoting solubility. The LCST behavior of PDMAEMA is well-documented and tends to be approximately 50 °C at pH 8.0. The LCST of the solution was determined by dissolving samples in phosphate buffer saline pH 8 and monitoring its absorbance at 490 nm as a function of temperature. A sharp increase in the absorbance denotes the temperature at which the LCST is occurring, as shown in FIG. 17. The appearance of the LCST indicates that the polymer is imparting its thermos- responsive properties onto the rigid rod peptides, which can be used for future functionalities, such as purification. The lyophilized samples were evaluated via TEM to determine the structure of synthesized bottlebrush nanorods (see FIG. 18). FIG. 18 shows long chain-like structures that are much thicker than rigid rods. It was hypothesized that the observed thickness can be attributed to the polymer chains exhibiting LCST behavior. Samples were dehydrated during the TEM sample prep process, which could have caused them to collapse into a phase separated region. This phase change could then cause chains to aggregate, leading to the observed TEM images. TEM images of a control sample of single bundlemer-grafts ( / .e. grafting from single bundles rather than grafting from rigid rod surface) was also obtained (see FIG 5c). These samples show many smaller structures, but no chain-like behavior. This suggests that the observed aggregated chain-like behavior is a result of the polymer grafted from the rigid rod surface.

[0194] Example 15 Conclusions

[0195] In conclusion, the experiments performed in Example 10 demonstrate that biomimetic bottlebrush nanorods with programmable responsive behavior can be successfully synthesized by the methods disclosed herein and characterized. The Diels-Alder cycloaddition synthesized peptidic rigid rod material, both with and without the ATRP initiating handle, as confirmed by LC-MS and TEM. A stimuli responsive polymer, PDMAEMA, was then grafted from the supramolecular rigid rod surfaces via photoATRP to form bottlebrush polymers. The conjugate material exhibited low dispersity and thermoresponsive behavior that could be exploited for future functionalities, such as purification. Finally, the presence of supramolecular bottlebrush polymers was confirmed by TEM imaging, suggesting that the inclusion of polymers does not induce the disassembly of supramolecular rigid rods

[0196] The manipulation of bundlemer building blocks to build more advanced architectures is crucial in the advancement of bio-inspired materials. Knowledge of the various combinations of synthetic strategies, such as click chemistry and living polymerization tools, is essential when designing synthetic schemes to build higher- ordered supramolecular structures of increased complexity. This example can serve as a starting point to demonstrate ways in which click chemistry can be incorporated along with ATRP to impart responsiveness to complex bundlemer nanostructures. Example 16: Thermo-responsive Coiled Coil Peptide-polymer Grafts

[0197] This example involves using a grafting-from approach to create a polymerpeptide bundlemers and assemblies. A representative illustration of this strategy can be seen in FIG. 19.

[0198] Materials

[0199] All reagents used in this Example were purchased from ThermoFisher Scientific unless otherwise stated. Fmoc-protected amino acids and Rink amide (0.56 meq / g) resin for peptide synthesis were purchased from ChemPep. Oxyma was purchased from CEM. Pallidium tetrakis triphenylphosphine (Pd(PPh3)4), acetic anhydride, N,N'-diisopropylcarbodiimide (DIC), and triethylamine (TEA) were purchased from Chemlmpex. Alumina basica, phenylsilane, trifluoroacetic acid (TFA), tris(2-pryidylmethyl) amine (TPMA), and tris(3- hydroxypropyltriazolylmethyl)amine (THPTA) were purchased from TCI America. Piperidine, dimethylacetamide (DMAc), copper (II) bromide, 2-bromoisobutyryl bromide (BIBB), lithium bromide (LiBr), / V, / V-dimethylaminoethyl methacrylate (DMAEMA), and poly- / V, / V-dimethylaminoethyl methacrylate, PDMAEMA, 10 kDa were purchased from Millipore Sigma. DMAEMA was passed through alumina basica column to remove the inhibitor prior to use, while all other reagents were used without further modification.

[0200] Measurements

[0201] Peptides were synthesized on a CEM Liberty Blue automated synthesizer and purified on a Waters HPLC equipped with a Waters C18 column. Bundlemer- PDMAEMA conjugates were monitored via H-NMR on a Broker AVIII 600 MHz NMR spectrometer. Purity of peptide samples were analyzed on a Waters Xevo G2-S QTof mass spectrometry instrument equipped with a Waters LIPLC for LC- MS / MS analysis with an ESI source. Peptide self-assembly was characterized via circular dichroism (CD) spectroscopy on a Jasco 1500 spectropolarimeter and UV- Vis measurements were taken either on a Agilent Cary UV-Vis Compact Peltier with a temperature-controlled cell holder or on a DeNovix DS-11 FX+ Nanodrop Spectrophotometer. Number and weight average molecular weights and the dispersity of polymer conjugates were determined using gel permeation chromatography (GPC) relative to a PMMA standard using a TOSOH EcoSEC Elite GPC System with an integrated Rl detector.

[0202] Synthesis of macro-initiator peptide

[0203] Peptides were synthesized on OEM Liberty Blue® microwave peptide synthesizer using standard Fmoc-protected solid phase peptide synthesis (SPPS) procedures. The peptides were synthesized using Rink Amide resin (ChemPep, 0.56 eq. / gram) on a 0.25 mmol scale using a 4-fold excess of Fmoc-protected amino acids. 0.2 M amino acids solutions were prepared in N,N dimethylformamide (DMF) and placed on the SPPS instrument. The peptide was then sequentially synthesized via iterations of Fmoc deprotection (20% piperidine in DMF) and amino acid coupling (0.2 M amino acid, 1 M oxyma, and 1 M DIC in DMF) steps at 90 °C for two minutes per coupling. Each step was followed by a DMF rinse. The N- terminus of the peptide was acetylated with a solution of 10% (v / v) acetic anhydride. The alloc-protecting group is removed from the lysine using a deprotection solution of 0.025 M Pd(PPhs) in dichloromethane (DCM) with 0.5 M phenylsilane in DCM for 12 minutes at 75 °C. Three iterations were performed to fully deprotect the amine. Next, the desired amino acid armed linker was conjugated to the peptide at the branched point. These reactions were performed on the CEM Liberty Blue ® instrument. Finally, BIBB was added to the resin using a 4 eq. BIBB and 4 eq. TEA in anhydrous DCM for three hours on ice while nitrogen purging.

[0204] Peptide cleavage

[0205] Once all on -resin modifications were complete, the peptide was liberated from the resin and its protecting groups cleaved using an acid cleavage cocktail. The peptide resin was first swelled in a solution of DCM for about ten minutes, followed by excessive washing steps with DCM, DMF, and MeOH to ensure all impurities were drained from the RV. A cleavage cocktail solution was prepared, consisting of 95: 2.5: 2.5 (v / v / v) solution of TFA: TIPS: H2O and 5% (w / v) of phenol. The additives acted as scavengers for the reaction to neutralize any cleaved protecting groups or other side reactions. The cleavage cocktail was slowly added to the peptide resin, and was allowed to vortex at room temperature for about three hours. After this time, the eluent was pipetted into chilled ethyl ether to precipitate out the peptide. It was then centrifuged, and the supernatant discarded to yield the crude peptide. Two additional ethyl ether washes were performed to remove excess TFA and other impurities from the crude peptide. The peptide was then dried, analyzed via LC-MS, and ready for HPLC purification.

[0206] Purification of peptides via HPLC

[0207] Crude peptides were subjected to purification via preparative scale RP- HPLC. The peptides were dissolved at about 15 mg / mL in a 90:10 (v / v) water: acetonitrile solution and sonicated at 50 °C. Once the peptides were fully dissolved, they were filtered through 0.2 micron PDTE filters prior to injection. Solutions of Millipore water and acetonitrile were used during HPLC, each with either 0.04% or 0.1 % TFA. The HPLC gradient used changes from around 40% acetonitrile to 55% acetonitrile at a 0.75% / min change flowing at 22.5 mL / min. The elutions were monitored using a UV-Vis detector set at 214 nm. Peptides eluted around 50% acetonitrile, and their fractions were analyzed via LC-MS, combined, evaporated, then lyophilized to yield a dry powder.

[0208] Determination of purity of peptides

[0209] Peptide purity was assessed via LC-MS. Results were obtained from a Waters Xevo G2-S QTof equipped with a Waters UPLC. The peptide solutions were prepared at a concentration around 10-30 pM in DI water.

[0210] PhotoATRP procedure: Photoinduced ATRP of from the bundlemer macroinitiator

[0211] Bundlemer-polymer conjugates were synthesized via photoinduced ATRP3. The monomer was passed through alumina basica to remove the inhibitor. Then a stock catalytic solution was prepared by adding calculated amounts of copper (II) bromide, TPMA, NaBr, and D2O to a 1 .5 mL vial. A peptide stock solution was prepared as well in D2O. Next, the monomer was added to 2 mL autosampler vials. Calculated amounts of D2O, peptide stock solution, and catalytic solution were then added to the vials. Catalytic solution was added such that there would be 1 eq. CuBr2, 4 eq. TPMA, and 30 eq. NaBr in each reaction autosampler vial, with a peptide concentration of 2.5 mM. The equivalence of DMAEMA was chosen based on the desired degree of polymerization and was either 50 eq., 100 eq., or 200 eq. The samples were then N2 purged for 15 minutes, then allowed to stir while under 365 nm light at 2.5 mW cm-2intensity for 1-6 hours. After polymerization, samples were passed through alumina neutral to remove the catalyst and analyzed via NMR spectroscopy. After NMR acquisition, samples were dialyzed against Millipore water using 3.5 kDa MWCll tubing to remove excess monomer and salts, then lyophilized to yield dry white conjugate powder.

[0212] PhotoA TRP procedure: Optimization of the A TRP procedure

[0213] PMDETA was originally used but was later changed to TPMA to receive lower and more consistent dispersity values. Experiments were also performed to confirm the livingness of the reaction, in having a linear growth trend. In this Example, the equivalency of each reagent is [1 : 100: 1 : 4: 30] peptide: DMAEMA: CuBr2: TPMA: NaBr. The reactions were irradiated with 365 nm light at 2.5 mW cm’2. The equivalency of copper was also evaluated. Experiments with no copper showed no conversion, whereas changing the amount of copper from 0.25 - 1 eq. showed little difference in the conversion. For this Example, the ratio of TPMA and NaBr was consistent with the amount of copper used, and the polymerizations were allowed to run for 2 hours.

[0214] NMR spectroscopy of the Bundlemer-polymer conjugate samples

[0215] The 1 H NMR spectra of synthesized conjugates were measured using either a Bruker AVIII 600 MHz or Bruker AVIII 400 MHz NMR spectrometer, equipped either with a 5-mm Bruker SMART probe or a Bruker BBFO probe, respectively. ATRP was performed in D2O allowing for easier NMR data acquisition. Samples were passed through alumina basica to remove catalyst immediately after the reaction, and then were analyzed via NMR prior to dialysis to determine conversion by relating the quantity of unreacted monomer and converted polymer. The peaks within the NMR spectra were then integrated to determine the conversion of each of the conjugates.

[0216] The conversion and subsequent Mn was calculated for each of the bundlemer-polymer conjugates immediately following ATRP. Conversions of 50% were targeted. The peaks around 5.5 ppm correspond to the two protons that belong to the vinyl monomer, whereas the peak around 1 ppm corresponded to the three protons belonging to the upfield shifted methyl group. The conversion was calculated as follows:

[0217] While the Mn is calculated as the conversion multiplied by the degree of polymerization and molecular weight of the monomer (157 Da).

[0218] Additional NMR analytical methods

[0219] As a secondary method to confirm the number average molecular weight of the species, lyophilized conjugates were compared against PDMAEMA, 10 kDa (Sigma Aldrich: SKU909866) to calculate their number average molecular weight using H-NMR. For these calculations, a recorded mass amount of conjugate and DMF were dissolved in 0.65 mL of D2O and were analyzed via H-NMR. Commercially purchased PDMAEMA was analyzed using this method as well and used as a standard to compare to the other values. The number of moles for each was calculated using DMF as an internal standard, then for the conjugates. The peak at around 1 ppm corresponding to the three protons on the polymer were compared to the peak at around 8 ppm corresponding to DMF.

[0220] The number of polymer moles was first calculated by comparing this number to the number of mmoles of DMF, as shown below:

[0221] This value was then used to calculate the Mn, by using the polymer mass weighed out divided by the calculated number of moles (i.e., Polymer, Mn =Polymer, mg / Polymer_mmole). For the Sigma Aldrich sample, this value was 130.6 Da. This number should be closer to 10,000 Da. The difference was assumed to be related to the difference in size of DMF to the polymer, causing differences in their relaxation times during NMR acquisition. Therefore, the mass should be 76.6 times larger than calculated for each conjugate sample (76.58=10000 / 130.6). Therefore, this number was multiplied by the Poly, Mn determined for the other samples to calculate an estimate of the Mn for each conjugate size. Determination of peptide stability via Circular Dichroism (CD)

[0222] Circular dichroism measurements for secondary and tertiary structure characterization were performed on a Jasco J1500 spectropolarimeter. Samples (peptide or conjugates) were prepared in Millipore water or phosphate buffer saline (PBS) pH 8 ranging in concentration from 0.1- 0.25 mM using a Hellma brand 1 mm path length quartz cuvette. CD spectra of each sample were measured three times and averaged by collecting data from 185-250 nm using a scanning speed of 50 nm / min and digital integration time (DIT) of 4 seconds. Temperature melt measurements were taken by monitoring the change in ellipticity at 222 nm from 10 °C to 90 °C at a temperature ramp of 20 °C / hour, with a two minute equilibration time at each temperature. CD signal was converted to mean residue ellipticity (MRE) utilizing the concentration of peptide or conjugate and the pathlength of the cuvette. The concentration of peptide was calculated by taking advantage of the absorption of the tryptophan residue at 280 nm and converting to concentration utilizing the Beer-Lambert Law.

[0223] Determination of concentration of peptides and conjugates via UV-Vis

[0224] Each of the peptides and conjugate samples subjected to CD analysis were examined via UV-Vis to determine their concentration for MRE determination. The peptide sequences have a tryptophan residue, which has a molar extinction coefficient of 5.6 mM-1cm-1at 280 nm. The UV absorption was measured at room temperature using a nanodrop with a path length of 10 mm from 800-200 nm a total of four times. The data was then observed to find a local maximum around 280 nm, and the absorbance here was taken and averaged. This absorbance was then used to determine the sample peptide concentration utilizing Beer Lambert’s Law.

[0225] Cleavage of the Grafted PDMAEMA from the Bundlemer-polymer Conjugate PDMAEMA was cleaved from the peptide. Conjugate samples (5-10 mg) dissolved in 6N HCI aq. (1-2 mL) were placed in an acid hydrolysis tube. After vacuum was pulled from the tube for about 5 minutes, the hydrolysis was performed at 110 °C for 24 hours while under an inert vacuum. The solution was then diluted with 2-4 mL of 1 mM NaOH to slightly neutralize it, and transferred to 1 kDa MWCU dialysis tubing, where it was dialyzed against Millipore deionized water for 24 hours. Samples were examined via CD and NMR spectrometry to confirm absence of peptide and presence of polymer, respectively. The cleaved polymer was then lyophilized to yield a dry white powder and analyzed via GPC.

[0226] GPC of the Cleaved Polymer

[0227] Solutions of cleaved polymer samples were prepared at 1 mg / mL using a solution of DMAc with 0.5wt% LiBr and allowed to vortex overnight. They were then filtered through 1 micron PDFE filters and their GPC chromatogram was acquired. Measurements were acquired using a TOSOH EcoSEC Elite GPC System with integrated Rl detector instrument running at 50 °C with a flow rate of 0.4 mL / min. Their Mn, Mw, and dispersity were calculated against PMMA standards.

[0228] Determination of LC ST of the Bundlemer-polymer Conjugate

[0229] Solutions of conjugate samples (1 .5 mg / mL) were prepared in PBS buffer at pH 8.0. Their absorbance was monitored via UV-Vis at 490 nm as a function of temperature from 30 °C to 80 °C. A temperature ramp of 1 °C / min was utilized and the LCST of each conjugate was determined as the sharp increase in absorbance at 490 nm. Each measurement was averaged over three replicates.

[0230] Discussion and Results of Example 16

[0231] The bundle forming peptide used in this Example was computationally designed de novo to form an antiparallel homotetrameric coiled coil. This peptide sequence follows a seven amino acid repeat pattern, where the first and fourth amino acids are alanine and isoleucine, which ultimately reside in the hydrophobic core of the bundlemer. The other amino acids are charged and were selected through an energy minimization routine. The coiled coil sequence chosen for this Example is shown in FIG. 19A. The tetrameric oligomerization state of this computational designed peptide bundle has been confirmed in past works through biophysical techniques, such as analytical ultracentrifugation and small angle neutron scattering, demonstrating that tetramers still form even at high peptide concentrations or with added salts. This bundle sequence has remarkable stability with respect to changes in temperature, pH, and ionic strength. This stability also enables the bundlemer to be viewed as a well-defined nanoparticle with exact display of chemistry defined by the primary sequence of the peptide, which can be used as a substrate from which one can perform polymerization. The peptide was synthesized via microwave SPPS, where one of the amino acid protecting groups was selectively removed and converted to an alkyl halide initiator. Specifically, a select lysine residue was added to the peptide, which contained an alloc protecting group that was selectively removed on resin using Pd to reveal a free amine. The amine was used as a branch point for subsequent modification and functionalization. While the middle of the sequence was selected ( / .e., position 13), SPPS allows for easy placement of the alkyl halide at any amino acid position in the sequence that resides on the exterior of the coiled coil. Initially, the alkyl halide initiator was added directly onto the lysine using 2-bromoisobutyrl bromide (BIBB). The peptide was liberated from the resin by acid treatment, which additionally removed the remaining amino acid protecting groups. After purification via HPLC and lyophilization, the coiled coil formation and aqueous solution stability of the purified peptide was assessed via circular dichroism (CD).

[0232] The addition of the BIBB initiator negatively affected the thermal stability of the bundlemer as demonstrated by a decrease helicity, characterized by a maximum at 195 and minima at 208 and 222 nm in the CD spectra (see FIG. 20), and decreased melting temperature. Specifically, the coiled coil underwent an alpha helical to Gaussian coil transition at 57.3 ± 0.7 °C, as determined by the inflection in the sigmodal fit of the MRE versus temperature at 222nm. The direct addition of the BIBB initiator both eliminated the ammonium charge, provided by the amine, and added a relatively hydrophobic tertiary alkyl halide species. These combined effects were speculated to negatively impact the assembly of the bundlemer, which was driven by hydrophobic interactions. While the bundlemer did maintain the coiled coil structure at room temperature, it was critical that the halide was readily accessible to participate in the photo-ATRP reaction. The synthetic route of the peptide readily enabled further modification to the amine branch point to incorporate additional amino acid functionality.

[0233] Improved thermal stability of the coiled coil was achieved by the inclusion of a lysine within the branch, which preserved the original positive charge and disfavored interactions of the branch side group with the hydrophobic core of the coiled coil. This new bundlemer construct, which also included a glycine spacer in the peptide branch (i.e., BNDLE-KG-Br), resulted in similar CD spectra to the original bundlemer structure shown in FIG. 20. The peptide remained assembled even after being held for several hours at 90 °C, supporting that it would remain stable during the polymerization process Given the stability and halide accessibility of the bundlemer, BNDLE-KG-Br was selected for all subsequent photo-ATRP reactions.

[0234] A grafting-from ATRP approach was performed on the assembled bundlemer under aqueous conditions, which was similar to strategies in which protein-polymer conjugates are generated to maintain biological function. Within an aqueous medium, there are several mechanisms associated with a loss of the ATRP control, which leads to poor dispersity. Since bromide anions have a high degree of solubility in water, the reversible dissociation of the halide anion from the deactivating complex (i.e., Cu(ll)Br2 in FIG. 19B), can lower the concentration of deactivating species and lead to uncontrolled polymerization. The addition of halide salts suppressed this dissociation. Additionally, activator regenerative ATRP methods can be implemented to reduce the Cu(ll) species back into the Cu(l) species, providing a competing reaction to Cu(ll) dissociation. Here, photoATRP is utilized, which additionally enabled the reaction to be more oxygen tolerate, to not require exogeneous radicals or high temperatures, and to be turned on and off (i.e., temporal control).

[0235] Suitable photoATRP reaction conditions were determined to polymerize 2- (dimethylamino)ethyl methacrylate from the bundlemer macroinitiator. Specifically, a solution was prepared consisting of 2.5 mM peptide, 2.5 mM copper (II) bromide, 10 mM tris(2-pyridylmethyl)amine (TPMA), and 75 mM sodium bromide. TPMA was used as a copper ligand and, as discussed, sodium bromide was added to suppress the dissociation of the Cu(ll)Br2 species. TPMA is a tripodal ligand that contains three picolyl substituents that help stabilize the copper in the deactivated state, thus leading to better polymerization control as compared to other ligands.

[0236] Monomer amounts were added based on desired degree of polymerization (50, 100, or 200 eq.). The reaction was performed in D2O to make subsequent NMR analysis easier. The samples were exposed to 365 nm light at 2.5 mW cm-2intensity to allow the reaction to proceed. The polymerization proceeded for 2 hours, reaching approximately 50% conversion (determined by NMR). While conversions as high as 90% were attainable at reaction times of 4-5 hours, lower conversions led to a lower molecular weight dispersity.

[0237] The target molar masses of 4, 8, and 16 kDa were selected to be similar to the peptide, twice the peptide, and four times the peptide (i.e., similar to the bundlemer) molecular weight, respectively. Upon optimization of reaction times, polymer conjugates having number average molar masses of 3.7, 8.0, 15.6 kDa were obtained (determined by NMR). The control reaction, where a peptide without a halide initiating species was used (unmodified BNDLE), exhibited no polymerization. After polymerization, dialysis, and lyophilization, the bundlemer- polymer conjugate exhibited the higher-order coiled coil structure at room temperature, as determined by the minima at 208 and 222 nm in the CD spectroscopy (see FIG. 21 A).

[0238] The polymer grafts were uniform in size as indicated by LC-MS and GPC. The purified peptide exhibited a single MW species. To isolate the molecular weight of the polymer product, the peptide was digested utilizing acid hydrolysis. GPC of the polymers, using 0.5wt% LiBr in dimethylacetamide as eluent and relative to a PMMA standard, revealed an estimated dispersity of around 1 .3 (see FIG. 21 B). This data indicated that the molecular weight can be rationally tuned by the stoichiometry of monomer and the reaction time, which ultimately influenced the bundlemer-PDMAEMA star responsiveness.

[0239] At temperatures above the LCST, the PDMAEMA grafted chains became dehydrated and collapsed into core-shell nanoparticles that precipitate to cause a phase change within the solution. The LCST of PDMAEMA was dependent on the temperature, pH, and ionic strength of the solution, as well as the molecular weight and concentration of the sample. Literature values for its LCST are well- documented and tend to be approximately 50 °C at pH 8.0. As the LCST of PDMAEMA decreased with increasing molecular weight, the phase change behavior could be controlled through molecular design of the conjugate, namely the original monomer-bundlemer stoichiometry and reaction time.

[0240] The LCST of each conjugate was determined by turbidity measurements by monitoring the absorbance of visible light as a function of temperature (see FIG. 21 C). Increasing the polymer conjugate chain length decreased the LCST of the solution. This demonstrates that the conjugate's responsiveness to changes in solution conditions, and the ability to control the architectures behavior simply by increasing the solution temperature. Moreover, this transition was reversible, with a decrease in temperature causing the chains to expand and resolubilize in their aqueous environment. CD spectroscopy revealed that bundlemer-polymer conjugates remained assembled throughout this heating and cooling cycle. The reversible nature of the LCST can be exploited for future functionalities, such as purification, and demonstrates success in imparting specific features of the synthetic polymer onto the bundlemer material.

[0241] The ability to introduce synthetic polymer side chains rationally and robustly into peptidic materials allowed for an immense expansion in peptide design possibilities. While this Example selected DMAEMA as the polymer, the versatility of this surface initiated photo-ATRP approach to bundlemer-polymer grafts is readily extended to a wide range of aqueous vinyl free-radical polymerizing systems, such as oligoethylene glycol methacrylate and / V-lsopropylacrylamide. The combination of being able to place ATRP initiators at precise position along the bundlemer periphery with extensive chemical diversity and interaction provided by synthetic polymers enables new materials discovery and fundamental exploration polymer interactions on a nanoparticle. The ability to precisely incorporate ATRP initiators into coiled coil peptide bundles coupled with the development of more user-friendly forms of ATRP, should lead to a surge of studies using this method to modify bundles or other peptidic structures to contain synthetic moieties.

[0242] It will be appreciated by those skilled in the art that the present disclosure can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The presently disclosed embodiments are therefore considered in all respects to be illustrative and not restricted. The scope of the disclosure is indicated by the appended claims rather than the foregoing description and all changes that come within the meaning and range and equivalence thereof are intended to be embraced therein.

Claims

CLAIMS:1 . A bundlemer-templated copolymer comprising:(i) a peptide bundle comprising two or more peptides, and(ii) two or more monomers or polymers grafted at a N- or C- terminus of a peptide within the peptide bundle and / or on at least one side chain of a peptide in the peptide bundle, wherein the two or more monomers or polymers are connected to the peptide bundle by a linker moiety formed from a reaction between two complementary reactive functional groups, wherein the two or more monomers or polymers are different from each other, and wherein the polymer-peptide bundlemer has a coiled-coil structure.

2. The bundlemer-templated copolymer according to claim 1 , wherein the two or more monomers grafted onto the bundlemer form polymers selected from polyolefins, polyacrylics, polyesters, polyethers, polythioethers, polyvinylhalides, polysilylenes, polyamides, polystyrenics, polyimides, polysulfones, polycarbonates, and copolymers thereof.

3. The bundlemer-templated copolymer according to claim 1 , wherein the bundlemer-templated copolymer comprises at least three monomer or polymers, and at least two monomers or polymers are the same monomer or polymer.

4. A bundlemer-templated copolymer comprising:(i) a peptide bundle comprising two or more peptides, and(ii) two or more polymers covalently bound at the N- or C-terminus of a peptide within the peptide bundle and / or on a side chain of at least one peptide of the two or more peptides of the peptide bundle, wherein at least two polymers of the two or more polymers are compositionally different, wherein the bundlemer-templated copolymer has a coiled coil structure.

5. The bundlemer-templated copolymer of claim 4, wherein at least one polymer of the two or more polymers is a homopolymer.

6. The bundlemer-templated copolymer of claim 4, wherein at least one polymer of the two or more polymers is a copolymer.

7. The bundlemer-templated copolymer of claim 4, wherein at least one polymer of the two or more polymers is different from the other polymers by at least one of: a nature of constituent monomer(s), a distribution of constituent monomer(s), or a combination of monomer(s) along a backbone of the bundlemer- templated copolymer.

8. The bundlemer-templated copolymer of claim 4, wherein the two or more polymers are covalently bound to side chains of two or more peptides of the peptide bundle and the bundlemer-templated copolymer has a multi-armed star- polymer architecture.

9. The bundlemer-templated copolymer of claim 4, wherein the peptide bundle is a homo-tetramer comprising four peptides, and wherein each peptide comprises two polymers covalently bound to a side chain of each peptide, thereby resulting in eight polymers covalently bound on sides of the homo-tetramer.

10. The bundlemer-templated copolymer according to claim 4, wherein each of the two or more polymers is a polymer selected from the group consisting of polyolefins, polyacrylics, polyesters, polyethers, polythioethers, polyvinylhalides, polysilylenes, polyamides, polystyrenics, polyimides, polysulfones, polycarbonates, and copolymers thereof.11 . A method of making the bundlemer-templated copolymer of claim 4, comprising:(i) preparing a peptide bundle comprising at least two peptides,(ii) installing one or more surface-initiating groups onto the peptide bundle, wherein the one or more surface initiating groups are connected to a linker moiety formed from a reaction between two complementary reactive functional groups; and(iii) reacting at least one monomer or polymer with the one or more surface-initiating groups.

12. The method according to claim 11 , wherein the installing of the one or more surface-initiating groups onto the peptide bundle comprises:(a) installing one or more surface-initiating groups at a N- or C- terminus of a peptide within at least one peptide of the peptide bundle, and / or(b) installing one or more surface-initiating groups on at least one side chain of at least one peptide of the peptide bundle.

13. The method according to claim 11 , wherein the method comprises:(iv) installing one or more additional surface-initiating groups onto the peptide bundle, wherein the one or more additional surface-initiating groups are different from the surface-initiating groups of (ii) and are connected to a linker moiety formed from a reaction between two complementary reactive functional groups; and(v) reacting at least one additional monomer or polymer with the one or more additional surface-initiating groups.

14. The method according to claim 13, wherein the at least one additional monomer or polymer of (v) is different from the at least one monomer or polymer of (iii).

15. A method of making the bundlemer-templated copolymer of claim 4, comprising:(i) adding one or more surface-initiating groups and / or one or more reactive functional groups to the peptide bundle at a N- or C-terminus of a peptide within the peptide bundle, and / or on a side chain of least one peptide of thepeptide bundle, wherein the adding occurs either during or after peptide bundle synthesis; and(ii) performing at least one of the following steps:(a) contacting a first monomer or polymer with one or more surface-initiating groups to graft the first monomer or polymer at the N- or C- terminus of a peptide within the peptide bundle, and / or graft the first monomer or polymer on the side chain of least one peptide of the peptide bundle; and / or(b) contacting a second monomer or polymer having a complementary reactive functional group to the one or more reactive functional groups of the peptide bundle to graft the second monomer or polymer at the N- or C terminus of a peptide within the peptide bundle, and / or graft the second monomer or polymer on the side chain of least one peptide of the peptide bundle.

16. The method according to claim 15, wherein the one or more surfaceinitiating groups comprise thermal-, photo-, or redox-active functionality that can initiate polymerization.

17. The method according to claim 15, wherein the one or more surfaceinitiating groups comprise a functional group to mediate a controlled radical polymerization, wherein the functional group is selected from a group consisting of a bromoisobutyrate, an alkoxyamine, a dithiocarbamate, and a thiocarbonylthio, wherein:(i) the bromoisobutyrate mediates an atom transfer radical polymerization (ATRP),(ii) the alkoxyamine mediates a nitroxide mediated polymerization (NMP),(iii) the dithiocarbamate mediates a photoiniferter-mediated polymerization (PIMP), and(iv) the thiocarbonylthio mediates a reversible additionfragmentation chain transfer (RAFT) polymerization.

18. The method according to claim 15, wherein the first monomer comprises a methacrylate functional group as a radical polymerizable functionality.

19. The method according to claim 15, wherein the one or more reactive functional groups is selected from a group consisting of an azide, a furan, and a thiol.

20. The method according to claim 15, wherein the complementary reactive functional group comprises an alkyne or a maleimide.21 . The method according to claim 15, wherein the step of adding one or more surface-initiating groups and / or one or more reactive functional groups further comprises: selectively removing an amino acid protecting group at the end of the peptide synthesis, and then adding the surface-initiating groups and / or one or more reactive functional groups for subsequent polymerization.

22. The method according to claim 15, wherein the step of contacting the second monomer or polymer having the complementary reactive functional group comprises: triggering a complementary reaction by heat, light, catalyst, or other activator.

23. The method according to claim 15, wherein the peptide bundle comprises two or more different surface-initiating groups, and the method further comprises: contacting a third monomer or polymer that is different from the first monomer or polymer with one of the two or more different surface-initiating groups to graft the third monomer or polymer at the N- or C-terminus of a peptide within the peptide bundle, and / or graft the third monomer or polymer on the side chain of least one peptide of the peptide bundle.

24. The method according to claim 15, wherein the peptide bundle comprises two or more different reactive functional groups, and the method further comprises: contacting a fourth monomer or polymer that is (i) different from the second monomer or polymer and (ii) has a complementary reactive functional group to one of the two or more different reactive functional groups to graft the fourth monomer or polymer at the N- or C-terminus of a peptide within the peptide bundle, and / or graft the fourth monomer or polymer on the side chain of least one peptide of the peptide bundle.

25. The method of claim 15, wherein the step (b) is performed before the step(a).

26. An article comprising the bundlemer-templated copolymer according to claim 1 or claim 4.

27. Use of the bundlemer-templated copolymer according to claim 1 or claim 4 for providing a macromolecular material selected from nanostructured films for membranes, fuel cells, batteries, chemical separators, water desalination devices, data storage, biomedical devices, drug delivery devices, smart coatings in implants and diagnostic equipment, and cell scaffolds for tissue engineering.

Images