WO2012166706A2

WO2012166706A2 - Supramolecular nanofibers and hydrogels based on oligopeptides functionalized with nucleobases

Info

Publication number: WO2012166706A2
Application number: PCT/US2012/039822
Authority: WO
Inventors: Bing Xu
Original assignee: Brandeis University
Priority date: 2011-05-31
Filing date: 2012-05-29
Publication date: 2012-12-06
Also published as: WO2012166706A3

Abstract

Described herein are nucleopeptide compounds that include a nucleobase and an oligopeptide. The compounds may self-assemble to form supramolecular hydrogels. Also, the compounds may be used as a platform to examine specific biological functions (e.g., binding to DNA and RNA) of a dynamic supramolecular system that is able to interact with both proteins and nucleic acids. Other uses include: methods of growing cells and methods of delivering a substance to a cell.

Description

Supramolecular Nanofibers and Hydrogels Based on Oligopeptides Functionalized with Nucleobases

RELATED APPLICATIONS

This application claims the benefit of priority to United States Provisional Patent Application serial number 61/491,547, filed May 31, 2011, the contents of which are hereby incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under DMR 0820492 awarded by the National Institutes of Health and the National Science Foundation. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Nucleopeptides are a class of molecules that contain both nucleobases and amino acids, which have considerable biological and biomedical importance. Naturally occurring nucleopeptides, such as willardiine-containing nucleopeptides and peptidyl nucleosides, are antibiotics. A number of unnatural nucleobase-containing peptides, such as peptide nucleic acids (PNA), have applications in biology and biomedicine (e.g., as analogues of DNA). Such biological significance renders nucleopeptides useful molecules for studying biology.

Additionally, supramolecular hydrogels, resulting from molecular self-assembly of nucleopeptides in water, have exhibited considerable promise for applications in biomedicine due to their inherent biocompatibility and biodegradability.

SUMMARY OF THE INVENTION

In certain embodiments, the invention relates to a hydrogelator of Formula I

I

wherein, independently for each occurrence,

guaninyl, adeninyl, thyminyl, uracilyl, or an oligonucleic acid; R is H or alkyl; R¹ is H, alkyl, alkylthioalkyl, aralkyl, heteroaralkyl, hydroxyaralkyl, phosphorylated aralkyl, H0₂C-alkyl, or guanidinylalkyl;

R² is H, alkyl, -OR, or -NR₂; and

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In certain embodiments, the invention relates to a supramolecular structure comprising a plurality of any one of the aforementioned hydrogelators.

In certain embodiments, the invention relates to a hydrogel, comprising, consisting essentially of, or consisting of a plurality of any one of the aforementioned hydrogelators; and water.

In certain embodiments, the invention relates to a hydrogel, comprising, consisting essentially of, or consisting of a plurality of any one of the aforementioned supramolecular structures; and water.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts (a) the molecular structures and simulated 3D shapes of exemplary hydrogelators and corresponding precursors based on nucleopeptides; and (b) a schematic showing self-assembly.

Figure 2 depicts (a) an exemplary synthetic route to hydrogelator 1A and precursor 2A based on adenine; and (b) an illustration of the dephosphorylation process catalyzed by alkaline phosphatase (ALP) that converts 2A to 3A, resulting in nanofibers and a hydrogel.

Figure 3 depicts the molecular structures of, and exemplary synthetic routes to, nucleopeptides IT, 2T, 1G, 2G, 1C and 2C.

Figure 4 depicts a summary of the preparation conditions used, and properties of the nucleopeptide hydrogelators and corresponding supramolecular nanofibers and hydrogels. ^aThe thin nanofibers (3C) have low quantity and coexist with nanoparticles, thus failing to produce a hydrogel.

Figure 5 depicts transmission electron micrographs of the hydrogels formed from 1A, 1G, IT, 1C, 3A, 3G, 3T and the solution of 3C (scale bar = 100 nm).

Figure 6 depicts the 1H NMR spectrum of nucleopeptide hydrogelator 2A, and ³¹P NMR spectra before and after the addition of alkaline phosphatase (ALP) to 2A.

Figure 7 depicts the 1H NMR spectrum of nucleopeptide hydrogelator 2G, and ³¹P NMR spectra before and after the addition of alkaline phosphatase (ALP) to 2G.

Figure 8 depicts the 1H NMR spectrum of nucleopeptide hydrogelator 2T, and ³¹P NMR spectra before and after the addition of alkaline phosphatase (ALP) to 2T. Figure 9 depicts the 1H NMR spectrum of nucleopeptide hydrogelator 2C, and ³¹P NMR spectra before and after the addition of alkaline phosphatase (ALP) to 2C.

Figure 10 depicts CD spectra of (a) the hydrogels formed by hydrogelators 1A, 1G, IT and 1C, respectively; and (b) the hydrogels formed by 3A, 3G, and 3T, respectively, and the solution of 3C.

Figure 11 depicts the strain dependence of the dynamic storage moduli (C) and loss moduli (G") of (a) the hydrogels formed by hydrogelators 1A, 1G, IT, 1C, respectively; and (b) the hydrogels formed by hydrogelator 3A, 3G, 3T, respectively, and the solution of 3C.

Figure 12 depicts the calculated fiber width dependences of the stabilization energies of 1A, 1G, IT and 1C, respectively.

Figure 13 depicts (a) a comparison of the widths of fibers of hydrogels 1A, 1C, 1G and IT, based on transmission electron micrographs and molecular mechanical calculations; (b) 72 h cell viability test at concentrations of, from left to right, 10 μΜ, 50 μΜ, 100 μΜ, 200 μΜ, and 500 μΜ of 1Α, 1C, IT and 1G; (c) 72 h cell viability test at concentrations of, from left to right, 10 μΜ, 50 μΜ, 100 μΜ, 200 μΜ, and 500 μΜ of 2Α, 2C, 2T and 2G; and (d) optical images of HeLa cells on the surface 0 h and 20 h after the creation of scratch- wound in the presence of hydrogel 3T (by adding 27.7 mM of 3T to the media).

Figure 14 depicts optical images of HeLa cells on the surface 0 h and 20 h after the creation of scratch-wound in the medium without the presence of the hydrogel of 3T.

Figure 15 depicts the time-dependent course of the digestions of hydrogelators of

IT, 2T, 2C, 2G, 2A, 3T, 3C, 3A, and 3G by proteinase K.

DETAILED DESCRIPTION OF THE INVENTION

OVERVIEW

In certain embodiments, the invention relates to a nucleopeptide compound, comprising, consisting essentially of, or consisting of a nucleobase; and an oligopeptide.

In certain embodiments, the invention relates to the use of a nucleopeptide as a biomaterial. In certain embodiments, the biomaterial may be used as a platform to examine specific biological functions (e.g., binding to DNA and RNA) of a dynamic supramolecular system that is able to interact with both proteins and nucleic acids.

In certain embodiments, the invention relates to a hydrogel formed by an enzymatic reaction upon a nucleopeptide of the invention. In certain embodiments, the invention relates to a hydrogel formed from a nucleopeptide of the invention upon a change in pH. In certain embodiments, the invention relates to a soft, biocompatible material, comprising, consisting essentially of, or consisting of a nucleopeptide.

HYDROGELATOR DESIGN, SYNTHESIS, AND DISCUSSION

As shown in Figure la, the connection of a nucleobase (adenine, guanine, thymine, or cytosine) to a dipeptide segment (Phe-Phe), which is prone to self-assembly, affords a series of nucleopeptides (1) ("hydrogelators") that self-assemble in water to form nanofibers and produce hydrogels at the concentration of 2.0 wt% and pH around 5. The conjugation of a tyrosine phosphate to 1 yields another group of nucleopeptides, precursors 2, which undergo catalytic dephosphorylation to generate hydrogelators 3 that result in supramolecular nanofibers and hydrogels at low concentration (2.0 wt%) and physiological pH.

Figure 2a shows a synthetic route exemplified by the process for making the hydrogelators based on adenine. Following the procedures reported by Nieddu for making nucleobase acetic acids, we first synthesized bis(tert-butyloxycarbonyl) (bis-Boc) protected adenine, (A^-fos-Boc-adenine- -y -acetic acid (4). After being activated by N- hydroxysuccinimide (NHS), 4 reacts with L-Phe to afford 5, which undergoes the same NHS activation and phenylalanine coupling to give the key intermediate 6. Subsequent removal of the Boc-protecting groups with trifluoro acetic acid (TFA) yields the nucleopeptides (1A) in 47% total yield. 1A self-assembles to form nanofibers with a diameter of 16 nm (Figure 5) and results in a hydrogel at a concentration of 2.0 wt% and pH of 5.0. Encouraged by this data, we used the NHS-activated intermediate 6 to react with L-Tyr-phosphate to obtain 7, which forms precursor 2A after the deprotection of the Boc groups. Figure 2b illustrates the dephosphorylation process of precursor 2A catalyzed by an enzyme, which leads to a translucent hydrogel of nucleopeptide 3A (Figure 4) at the physiological pH. A ³¹P NMR study confirms that precursor 2 A completely transforms into hydrogelator 3 A 12 h after the addition of alkaline phosphatase (ALP) (Figure 6), and the TEM images (Figure 5) of the negative stained hydrogel of 3A reveals nanofibers with a width of 20 nm, confirming that nanofibers of 3A act as a matrix to sustain the hydrogel (with a storage modulus around 2082 Pa at 2.0 wt%).

The formation of the nanofibers from 1A and 3A indicates that the direct attachment of a purine or pyrimidine base to a small peptide is a valid approach to designing hydrogelator nucleopeptides. To examine the generality of this approach, we used synthetic procedures similar to those in Figure 2a to produce nucleopeptides consisting of other nucleobases (G, T, or C), and examined their abilities to form nanofibers and hydrogels. As revealed by TEM (Figure 5), hydrogelators 1G, IT, and 1C self-assemble to form nanofibers with a width of 15, 9, and 10 nm, respectively, and the nanofibers entangle to trap water and result in the hydrogels (Figure 4) at a concentration of 2.0 wt% and pH 5.0.

Like 2A, precursors 2G and 2T, at 2.0 wt% and pH 7.4, upon the addition of alkaline phosphatase (ALP, 10 U), turn into hydrogelators 3G and 3T, respectively. This enzymatic conversion leads to the formation of nanofibers of 3G and 3T, and results in the corresponding hydrogels shown in Figure 4. TEM reveals that the diameters of the nanofibers of 3G (14 nm) and 3T (9 nm) are similar to those of the nanofibers of 1G and IT, respectively. At a concentration of 2.0 wt% and pH 7.4, 3C self-assembles to afford both nanoparticles (11 nm) and short, thin nanofibers (4 nm in diameter and about 200 nm long), but fails to form well-defined nanofiber networks that produce a hydrogel.

We measured the rheological properties of the hydrogels to gain further insight into their characteristics. As shown in Figure 4, the hydrogel of 1G exhibits the highest storage modulus (12613 Pa), the hydrogels of 1A and IT possess relatively high storage moduli of 8090 Pa and 6346 Pa, respectively, and the hydrogel of 1C has the lowest storage modulus (26 Pa). The storage moduli of the hydrogels of 3G and 3T are 682 Pa and 2.9 Pa, respectively, indicating that the hydrogel of 3T possesses much weaker mechanical strength than those of the hydrogels 3A and 3G (Figure 4). The relatively high storage moduli of hydrogels of 1A, 1G, 3A, and 3G may stem from the fact that purine bases favor the formation of Hoogsteen base pairing, in addition to the strong π-π interaction found in purine nucleobases that contain two fused five- and six-member heterocyclic rings. Moreover, the lower storage moduli of the hydrogels of 3 as compared to those of the hydrogels of 1 suggest that the presence of tyrosine may reduce the efficiency of the non-covalent interactions required for the stabilization of self-assembled nanostructures, resulting in the relatively weak viscoelastic properties of those hydrogels.

We used circular dichroism (CD) spectroscopy to study the superstructures in the gel phase of the nanofibers of self-assembled nucleopeptides. The CD spectra of the hydrogels of 1 show a positive peak near 195 nm and a negative peak around 210 nm (Figure 10), suggesting these nucleopeptides arrange into β-sheet-like configurations. The CD spectra of the hydrogels of 3 A, 3G, and 3T display a positive peak near 195 nm and a negative peak around 210 nm, also suggesting that these nucleopeptides adopt a β-sheet-like configuration. The CD spectrum of a solution of 3C exhibits a positive peak near 203 nm and a negative peak around 215 nm, which is red-shifted with respect to the absorbances found in a typical β-sheet. The red-shifted β-sheet signal is likely indicative of a twisted structure as opposed to the standard planar β-sheet; an increase in β-sheet twisting causes disorder and may result in short nanofibers and nanoparticles, which, in turn, leads to weak mechanical strength. Overall, the signals indicating a β-sheet configuration (i.e., transitions at 195 nm-225 nm) of 1 are stronger than those of 3. This corresponds with the observed storage moduli trends - the storage moduli of hydrogels based on 1 are larger than those of hydrogels based on 3.

We also used molecular mechanical (MM) calculations to simulate the width of the nanofibers of 1. As shown in Figure 13 a, the simulated widths of the nanofibers are 15 nm, 16 nm, 9 nm and 11 nm for nucleopeptides 1A, 1G, IT and 1C, respectively. The calculated values correlate well with the observed values (Figure 5). According to simulation, the thicker width of nanofibers in the hydrogels of 1A and 1G likely result from the formation of Hoogsteen base pairing by adenine or guanine nucleobases. In addition, the MM calculations support the theory that the molecules self-assemble into a β-sheet-like structure.

To verify the biocompatibility of the hydrogelators, we added hydrogelator 1 or precursor 2 into the culture of HeLa cells and measured the proliferation of the cells. According to the MTT assay shown in Figure 13, after being incubated with the 500 μΜ of hydrogelator (1A, IT, or 1C) or the precursor (2A, 2T, or 2C) for 72 hours, the cell viability remained at 100%. Although the cell viability decreases slightly when the cells are incubated with 500 μΜ of 1G or 2G for 72 hours, the IC₅₀ is still > 500 μΜ. These results support the notion that nucleopeptides 1, 2, and 3 are biocompatible.

We also used a simple wound-healing assay to examine the capability of the nanofibers and hydrogels of 3 to serve as a material for maintaining cell-matrix interaction. As shown in Figure 13d, the presence of the hydrogel of 3T in cell culture has little inhibitory effect on the migration of cells, further supporting the biocompatibility of 3.

DEFINITIONS

For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

In order for the present invention to be more readily understood, certain terms and phrases are defined below and throughout the specification. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. In addition, polymers of the present invention may also be optically active. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and ^-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

EXEMPLARY HYDROGELATORS OF THE INVENTION

In certain embodiments, the invention relates to a hydrogelator of Formula I

I

wherein, independently for each occurrence,

guaninyl, adeninyl, thyminyl, uracilyl, or an oligonucleic acid; R is H or alkyl;

R¹ is H, alkyl, alkylthioalkyl, aralkyl, heteroaralkyl, hydroxyaralkyl, phosphorylated aralkyl, H0₂C-alkyl, or guanidinylalkyl;

R² is H, alkyl, -OR, or -NR₂; and

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In certain embodiments, the invention relates to any one of the aforementioned

hydrogelators, wherein is cytosinyl. In certain embodiments, the invention relates to

any one of the aforementioned hydrogelators, wherein is guaninyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein

adeninyl. In certain embodiments, the invention relates to any one of the

aforementioned hydrogelators, wherein is thyminyl. In certain embodiments, the

invention relates to any one of the aforementioned hydrogelators, wherein is uracilyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein

an oligonucleic acid. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R is H.

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is H.

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is alkyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is methyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is ethyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is propyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is isopropyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is butyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is isobutyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is sec-butyl.

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is alkylthioalkyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is CH₃-S-CH₂CH₂-.

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is aralkyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is benzyl.

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is heteroaralkyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is indolyl-CI¾-. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is hydroxyaralkyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is hydroxybenzyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is 4-hydroxybenzyl.

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is phosphorylated aralkyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is I¾P0₄-benzyl. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R¹ is 4-H₂P0₄-benzyl.

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein at least one instance of R¹ is aralkyl, hydroxyaralkyl, or phosphorylated aralkyl.

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R² is -OR. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein R² is -OH.

In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein n is 1. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein n is 2. In certain embodiments, the invention relates to any one of the aforementioned hydrogelators, wherein n is 3.

In certain embodiments, the invention relates to a compound selected from the group consisting of:





EXEMPLARY SUPRAMOLECULAR STRUCTURES OF THE INVENTION

In certain embodiments, the invention relates to any one of the aforementioned supramolecular structures, wherein the supramolecular structure is in the form of nanofibers. In certain embodiments, the average diameter of the nanofibers is about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, or about 25 nm. In certain embodiments, the nanofibers are crosslinked. In certain diameters, the nanofibers are substantially straight. In certain embodiments, the nanofibers are bent. In certain embodiments, the nanofibers form bundles of nanofibers. In certain embodiments, the nanofibers are about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, or about 300 nm in length. In certain embodiments, the nanofibers are greater than about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, or about 300 nm in length. EXEMPLARY HYDROGELS OF THE INVENTION

In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel is formed from a solution of the hydrogelators in water. In certain embodiments, the hydrogelator is present in an amount from about 0.5% to about 4% by weight. In certain embodiment, the hydrogelator is present in an amount of about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, or about 4.0% by weight.

In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel is formed from a solution of the hydrogelators in water. In certain embodiments, the temperature of the solution is about 20 °C, about 25 °C, or about 30 °C.

In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel is formed by decreasing the pH of the solution of hydrogelators in water. In certain embodiments, the pH at which the supramolecular structure is formed is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, about 5.5, about 5.0, about 4.5, or about 4.0.

In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel is formed by the addition of an enzyme to the solution of hydrogelators in water. In certain embodiments, the enzyme is a phosphatase. In certain embodiments, the enzyme is alkaline phosphatase.

In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel has a critical strain value of from about 0.2% to about 10.0%. In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel has a critical strain value of about 0.2%>, about 0.3%>, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.2%, about 1.4%, about 1.6%, about 1.8%, about 2.0%, about 2.2%, about 2.4%, about 2.6%, about 2.8%, about 3.0%, about 3.2%, about 3.4%, about 3.6%, about 3.8%, about 4.0%, about 4.2%, about 4.4%, about 4.6%, about 4.8%, about 5.0%, about 5.2%, about 5.4%, about 5.6%, about 5.8%, about 6.0%, about 6.2%, about 6.4%, about 6.6%, about 6.8%, about 7.0%, about 7.2%, about 7.4%, about 7.6%, about 7.8%, about 8.0%, about 8.2%, about 8.4%, about 8.6%, about 8.8%, about 9.0%, about 9.2%, about 9.4%, about 9.6%, about 9.8%, or about 10%.

In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel has a storage modulus of from about 2.0 Pa to about 14.0 KPa. In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel has a storage modulus of about 2.0 Pa, about 2.1 Pa, about 2.2 Pa, about 2.3 Pa, about 2.4 Pa, about 2.5 Pa, about 2.6 Pa, about 2.7 Pa, about 2.8 Pa, about 2.9 Pa, about 3.0 Pa, about 3.1 Pa, about 3.2 Pa, about 3.3 Pa, about 3.4 Pa, about 3.5 Pa, about 3.6 Pa, about 3.7 Pa, about 3.8 Pa, about 3.9 Pa, about 4.0 Pa, about 5.0 Pa, about 10 Pa, about 15 Pa, about 20 Pa, about 25 Pa, about 30 Pa, about 35 Pa, about 40 Pa, about 45 Pa, about 50 Pa, about 100 Pa, about 150 Pa, about 200 Pa, about 250 Pa, about 300 Pa, about 350 Pa, about 400 Pa, about 450 Pa, about 500 Pa, about 550 Pa, about 600 Pa, about 650 Pa, about 700 Pa, about 750 Pa, about 800 Pa, about 850 Pa, about 900 Pa, about 950 Pa, about 1.0 KPa, about 1.5 KPa, about 2.0 KPa, about 2.5 KPa, about 3.0 KPa, about 3.5 KPa, about 4.0 KPa, about 4.5 KPa, about 5.0 KPa, about 5.5 KPa, about 6.0 KPa, about 6.5 KPa, about 7.0 KPa, about 7.5 KPa, about 8.0 KPa, about 8.5 KPa, about 9.0 KPa, about 9.5 KPa, about 10.0 KPa, about 10.5 KPa, about 11.0 KPa, about 11.5 KPa, about 12.0 KPa, about 12.5 KPa, about 13.0 KPa, about 13.5 KPa, or about 14.0 KPa.

In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel is substantially biocompatible. In certain embodiments, the invention relates to any one of the aforementioned hydrogels, wherein the hydrogel is substantially biostable.

EXEMPLARY METHODS OF THE INVENTION

In certain embodiments, the invention relates to a method of growing cells, comprising contacting a plurality of cells with any one of the aforementioned supramolecular structures or any one of the aforementioned hydrogels. In certain embodiments, the cells are engineered tissue cells. In certain embodiments, the invention relates to a method of delivering a substance to a cell, comprising

contacting the substance with any one of the aforementioned supramolecular structures or any one of the aforementioned hydrogels, thereby forming a substance-hydrogel delivery vehicle; and

contacting the substance-hydrogel delivery vehicle and a cell.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substance is a drug. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substance is a protein. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substance is a gene. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substance is siRNA. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substance is microRNA. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substance is a second cell.

In certain embodiments, the invention relates to a method of binding a nucleic acid, comprising

contacting a nucleic acid with any one of the aforementioned supramolecular structures or any one of the aforementioned hydrogels.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nucleic acid binding is selective nucleic acid binding.

In certain embodiments, the invention relates a method of separating a protein from a substance, comprising

contacting a mixture with any one of the aforementioned supramolecular structures or any one of the aforementioned hydrogels, wherein the mixture comprises a protein.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the mixture comprises at least two proteins.

In certain embodiments, the invention relates to a method of treating or preventing a viral infection, comprising

administering to a mammal in need thereof a therapeutically effective amount of any one of the aforementioned hydrogelators. In certain embodiments, the invention relates to a method of treating or preventing cancer, comprising

administering to a mammal in need thereof a therapeutically effective amount of any one of the aforementioned hydrogelators.

In certain embodiments, the invention relates to a method of preventing adhesion of an organism or a cell to a surface, comprising

contacting the surface with any one of the aforementioned supramolecular structures or any one of the aforementioned hydrogels.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1 - Materials, Techniques, and General Procedures

Chemical reagents and solvents were used as received from commercial sources. 1H,

¹³C, and ³¹P NMR spectra were obtained on a Varian Unity Inova 400 NMR spectrometer, CD on a JASCO J-810 spectrometer, LC-MS on a Waters Acouity ultra Performance LC with a Waters MICROMASS detector, TEM on a Morgagni 268 transmission electron microscope.

Example 2 - Synthetic Methods

Figure 3 depicts five synthetic schemes for various compounds of the invention. Synthesis of Bis-Boc-Adenine-Phe (5). Bis-Boc adenine acetic acid (393.4 mg, 1 mmol) and NHS (126.5 mg, 1.1 mmol) were dissolved in 30 mL of THF, and DCC (226.6 mg, 1.1 mmol) was added to the above solution with stirring. After the reaction, the mixture was stirred at room temperature overnight, and the resulting solid was filtered off. The filtrate was evaporated under reduced pressure to dryness to afford the crude product for the next reaction without purification.

L-Phenylalanine (166 mg, 1 mmol) and Na₂C0₃ (84.8 mg, 0.8 mmol) were dissolved in 20 mL of water with stirring, and the solution of crude product (dissolved in 30 mL THF) was added. The resulted reaction mixture was stirred at room temperature for 24 h. After evaporation of the organic solvent, the residue was redissovled in 30 mL of water and acidified with hydrochloric acid to pH 2-3. The white precipitate was filtered off and purified by column chromatography over silica gel using chloroform/methanol as the eluents to afford compound 5 (443 mg, 82% ) for next step reaction. 1H NMR (400 MHz, DMSO-<¾): δ 8.80 (s, 1H), 8.66 (b, 1H), 8.50 (s, 1H), 7.27-7.17 (m, 5H), 5.03 (dd, J = 20.0 Hz, 24.0 Hz, 2H), 4.37 (m, 1H), 3.08 (dd, J = 4.0, 12.0 Hz, 1H), 2.92 (dd, J = 8.0, 12.0 Hz, 1H), 1.37 (s, 18H) ppm.

Synthesis of Bis-Boc-Adenine-Phe-Phe (6). Compound 5 (540 mg, 1 mmol) and

NHS (126.5 mg, 1.1 mmol) were dissolved in THF (30 mL), and DCC (226.6 mg, 1.1 mmol) was added to the above solution with stirring. After the reaction mixture was stirred at room temperature for 12 h, the resulting solid was filtered off. Then the filtrate was evaporated under reduced pressure to dryness. The crude product was used for the next step reaction without purification.

L-Phenylalanine (166 mg, 1 mmol) and Na₂C0₃ (84.8 mg, 0.8 mmol) were dissolved in water (20 mL) with stirring, and the solution of crude product (dissolved in 30 mL of THF) was added. The resulted reaction mixture was stirred at room temperature for 24 h. After evaporation of the organic solvent, the residue was redissovled in 30 mL of water and acidified with hydrochloric acid to pH 2-3. The white precipitate was filtered off and purified by column chromatography over silica gel using chloroform/methanol as the eluents to afford compound 6 (488 mg, 80% ). 1H NMR (400 MHz, DMSO-<¾): δ 8.78 (s, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.49 (d, J = 8.0 Hz, 1H), 8.42 (s, 1H), 7.24-7.10 (m, 10H), 4.96 (dd, J = 16.0, 28.0 Hz, 2H), 4.61-4.56 (m, 1H), 4.46-4.40 (m, 1H), 3.09-2.99 (m, 2H), 2.91 (dd, J = 8.0, 12.0 Hz, 1H), 2.75 (dd, J= 8.0, 12.0 Hz, 1H), 1.37 (s, 18H) ppm.

Synthesis of Adenine-Phe-Phe (1A). 0.5 mmol of compound 6 (344 mg) was dissolved in 10 mL of 90% Trifluoroacetic acid in water and stirred at room temperature for 2 h. The reaction mixture was concentrated by vacuum and the white solid was purified by using HPLC with water-acetonitrile as eluent (from 8:2 to 4:6) to afford the product (1A) in 73% yield. 1H NMR (400 MHz, OMSO-d₆): δ 8.63 (d, J = 8.0 Hz, 1H), 8.51 (d, J = 8.4 Hz, 1H), 8.29 (s, 1H), 8.12 (s, 1H), 7.24-7.18 (m, 10H), 4.85 (dd, J = 29.6, 16.4 Hz, 2H), 4.60- 4.55 (m, 1H), 4.46-4.41 (m, 1H), 3.06-2.96 (m, 2H), 2.90 (dd, J = 16.4, 10.0 Hz, 1H), 2.74 (dd, J = 13.6, 4.8 Hz, 1H). ¹³C NMR (400 MHz, DMSO-<¾): δ 37.2, 38.4, 45.9, 54.3, 54.5, 118.4, 127.1, 127.2 128.8, 128.9, 129.7, 129.9, 138.0, 144.8, 147.3, 149.5, 152.0, 166.1, 171.5, 173.4. MS: calcd M⁺=487.51, obsd (M+l)⁺=488.51.

Synthesis of Adenine-Phe-Phe- Tyr-phosphate (2A). Compound 6 (687.7 mg, 1 mmol) and NHS (126.5 mg, 1.1 mmol) were dissolved in THF (30 mL), and DCC (226.6 mg, 1.1 mmol) was added to the above solution with stirring. After the reaction, the mixture was stirred at room temperature for 12 h, and the resulted solid was filtered off. The filtrate was evaporated under reduced pressure to dryness. The crude product was used for the next reaction without purification.

L-Tyrosine-phosphate (261.17 mg, 1 mmol) and Na₂C0₃ (212 mg, 2 mmol) were dissolved in water (20 mL) with stirring, and the solution of crude product (dissolved in 30 mL of THF) was added. The resulted reaction mixture was stirred at room temperature for 24 h. After evaporation of the organic solvent, the residue was redissovled in 30 mL of water and acidified with hydrochloric acid to pH 2. The white precipitate was filtered off and treated with 90% trifluoroacetic acid in water for 2 h. Then the mixture was concentrated by vacuum and purified by using HPLC with water-acetonitrile as eluent (from 8:2 to 5:5) to afford the product (2A) in 51% yield. 1H NMR (400 MHz, DMSO-<¾): δ 8.51 (d, J = 8.0 Hz, 1H), 8.32-8.26 (m, 1H), 8.13 (s, 1H), 7.96 (s, 1H), 7.62 (s, 1H), 7.26-7.06 (m, 14H), 4.78 (dd, J = 30.0, 16.8 Hz, 2H), 4.52-4.41 (m, 3H), 3.04-2.68 (m, 6H). ¹³C NMR (400 MHz, OMSO-d₆): δ 37.7, 38.6, 38.8, 45.8, 54.1, 54.5, 118.5, 120.4, 126.9, 127.2, 128.6, 128.8, 130.0, 130.8, 133.5, 137.8, 138.5, 144.4, 148.4, 150.0, 150.9, 153.0, 166.2, 171.0, 173.5. MS: calcd M⁺=730.66, obsd (M+Na)⁺=753.66.

Synthesis of Thymine-Phe (8). Thymine acetic acid (184 mg, 1 mmol) and NHS (126.5 mg, 1.1 mmol) were dissolved in 20 mL of DMF, and DCC (226.6 mg, 1.1 mmol) was added to the above solution with stirring. After the reaction, the mixture was stirred at room temperature overnight, and the resulted solid was filtered off. The filtrate was evaporated under reduced pressure to dryness, and the crude product was used in the next reaction without purification.

L-Phenylalanine (166 mg, 1 mmol) and Na₂C0₃ (84.8 mg, 0.8 mmol) were dissolved in 20 mL of water with stirring, and the solution of crude product (dissolved in 20 mL DMF) was added. The resulted reaction mixture was stirred at room temperature for 24 h. The reaction mixture was vacuum-dried, then 30 mL of water was added and acidified to pH=3. The resulted product was obtained by filtration, washed with water, and then dried in vacuum. The white solid was purified by using HPLC with water-acetonitrile as eluent (from 8:2 to 4:6) to afford the product (8) in 78% yield for next step reaction. 1H NMR (400 MHz, DMSC ¾): δ 8.56-8.54 (m, 1H), 7.33-7.20 (m, 6H), 4.45-4.40 (m, 1H), 4.19 (dd, J = 16.0, 28.0 Hz, 2H), 3.04 (dd, J=4.0, 12.0 Hz, 1H), 2.89 (dd, J = 8.0, 16.0 Hz, 1H), 1.73 (s, 3H) ppm. Synthesis of Thymine-Phe-Phe (IT). Compound 8 (331 mg, 1 mmol) and NHS (126.5 mg, 1.1 mmol) were dissolved in 20 mL of DMF, and DCC (226.6 mg, 1.1 mmol) was added to the above solution with stirring. After the reaction mixture was stirred at room temperature overnight, the resulted solid was filtered off, and the filtrate was evaporated under reduced pressure to dryness. The crude product was used in the next reaction without purification.

L-Phenylalanine (166 mg, 1 mmol) and Na₂C0₃ (84.8 mg, 0.8 mmol) were dissolved in 20 mL of water with stirring, and the solution of crude product (dissolved in 20 mL DMF) was added. The resulted reaction mixture was stirred at room temperature for 24 h. The reaction mixture was vacuum-dried, then 30 mL of water was added and the mixture was acidified to pH=3. The resulted product was obtained by filtration, washed with water, and then dried in vacuum. Compound IT (white powder) was collected with 76% yield (364 mg). 1H NMR (400 MHz, DMSC ¾): δ 8.41-8.37 (m, 1H), 7.29-7.18 (m, 10H), 4.57- 4.52 (m, 1H), 4.43-4.38 (m, 1H), 4.23 (dd, J = 16.8, 28.4 Hz, 2H), 3.06-2.89 (m, 3H), 2.72 (dd, J = 9.6, 15.2 Hz, 1H), 1.71 (s, 3H). ¹³C NMR (400 MHz, DMSC ¾): δ 12.6, 38.4, 49.5, 55.0, 55.6, 108.6, 126.6, 126.9, 128.5, 128.7, 130.0, 130.1, 138.5, 139.3, 142.8, 151.6, 165.0, 167.3. MS: calcd M⁺=478.50, obsd (M+l)⁺=479.50.

Synthesis of Thymine-Phe-Phe-Tyr-phosphate (2T). Compound IT (478.5 mg, 1 mmol) and NHS (126.5 mg, 1.1 mmol) were dissolved in DMF (30 mL), and DCC (226.6 mg, 1.1 mmol) was added to the above solution with stirring. After the reaction mixture was stirred at room temperature for 12 h, the resulted solid was filtered off, and the filtrate was evaporated under reduced pressure to dryness. The crude product was used for the next reaction without purification.

L-Tyrosine-phosphate (261.17 mg, 1 mmol) and Na₂C0₃ (212 mg, 2 mmol) were dissolved in water (20 mL) with stirring, and the solution of crude product (dissolved in 30 mL of DMF) was added. The resulted reaction mixture was stirred at room temperature for 24 h. The reaction mixture was vacuum-dried, then 30 mL of water was added and the mixture was acidified to pH~2.0. The resulted product was isolated by filtration, washed with water, and then dried in vacuum. The white solid was purified by using HPLC with water-acetonitrile as eluent (from 8:2 to 5:5) to afford the product (2T) in 53% yield (382 mg). 1H NMR (400 MHz, DMSO-<¾): δ 8.29 (dd, J = 9.2, 32.0 Hz, 1H), 7.26-7.06 (m, 14H), 4.56-4.42 (m, 3H), 4.23 (d, J = 4.8 Hz, 2H), 3.03-2.88 (m, 4H), 2.81-2.67 (m, 2H), 1.71 (s, 3H). ¹³C NMR (400 MHz, OMSO-d₆): δ 12.6, 36.6, 38.1, 38.3, 49.6, 54.4, 55.6, 108.5, 120.4, 126.9, 128.7, 130.0, 130.5, 138.2, 138.4, 142.9, 151.6, 165.1 , 167.2, 171.2, 171.9, 173.3. MS: calcd M⁺=721.65, obsd (M+Na)⁺=744.65.

Synthesis of Bis-Boc-Guanine-Phe (9). Compound 9 was synthesized by following the procedures described in synthesis of compound 5 except replacing the bis-Boc adenine acetic acid with bis-Boc guanine acetic acid. Compound IGa (white powder) was collected with 81% yield (462 mg). 1H NMR (400 MHz, DMSO-<¾): δ 8.71 (d, J = 8.0 Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 7.31-7.19 (m, 5H), 4.91-4.79 (m, 2H), 4.44 (m, 1H), 3.06-3.01 (m, 2H), 2.94-2.88 (m, 2H), 1.34 (s, 18H) ppm.

Synthesis of Bis-Boc-Guanine-Phe-Phe (10). Compound 10 was synthesized by following the procedures described in synthesis of compound 6 except replacing the bis-Boc adenine acetic acid with bis-Boc guanine acetic acid. Compound 10 (white powder) was collected with 75% yield (528 mg). 1H NMR (400 MHz, OMSO-d₆): δ 8.52 (d, J = 8.0 Hz, 1H), 8.45 (s, 1H), 7.93 (s, 1H), 7.23-7.17 (m, 10H), 4.83-4.70 (m, 2H), 4.56 (s, 1H), 4.40 (s, 1H), 3.08-2.99 (m, 2H), 2.92-2.71 (m, 2H), 1.33 (s, 18H) ppm.

Synthesis of Guanine-Phe-Phe (1G). Compound 1G was synthesized by following the procedures described in synthesis of compound 1 A except replacing the bis-Boc adenine acetic acid with bis-Boc guanine acetic acid. Compound 1G (white powder) was collected with 58% yield (292 mg). 1H NMR (400 MHz, DMSO-<¾): δ 8.56-8.51 (m, 2H), 7.71 (s, 1H), 7.26-7.18 (m, 10H), 4.70 (s, 2H), 4.60-4.54 (m, 1H), 4.45-4.40 (m, 1H), 3.09-2.96 (m, 2H), 2.93-2.87 (m, 1H), 2.76-2.70 (m, 1H). ¹³C NMR (400 MHz, OMSO-d₆): δ 37.3, 38.7, 45.7, 54.3, 54.4, 127.0, 127.2, 128.7, 128.9, 129.8, 130.0, 138.1 , 138.2, 166.3, 171.5, 173.4. MS: calcd M⁺=503.51 , obsd (M+l)⁺=504.51.

Synthesis of Guanine-Phe-Phe- Tyr-phosphate (2G). Compound 2G was synthesized by following the procedures described in synthesis of compound 2A except replacing the bis-Boc adenine acetic acid with bis-Boc guanine acetic acid. Compound 2G (white powder) was collected with 51% yield (381 mg). 1H NMR (400 MHz, DMSO-<¾): δ 8.53 (t, J = 8.0 Hz, 1H), 8.42 (s, 1H), 8.27 (t, J = 8.0 Hz, 1H), 7.61 (s, 1H), 7.26-7.06 (m, 14H), 4.66-4.36 (m, 5H), 3.07-2.67 (m, 6H). ¹³C NMR (400 MHz, DMSO-<¾): δ 22.9, 37.3, 38.2, 44.4, 45.7, 46.3, 54.4, 54.6, 55.6, 1 13.2, 120.1 , 127.0, 128.8, 129.8, 130.9, 132.8, 138.1 , 138.4, 139.0, 151.3, 154.8, 166.2, 171.0, 171.5, 171.9, 173.4. MS: calcd M⁺=746.66, obsd (M+Na)⁺=769.66.

Synthesis of Bis-Boc-Cytosine-Phe (12). Compound 12 was synthesized by following the procedures described in synthesis of compound 5 except replacing the bis-Boc adenine acetic acid with bis-Boc cytosine acetic acid. Compound 12 (white powder) was collected with 83% yield (429 mg). 1H NMR (400 MHz, DMSO-<¾): δ 8.29 (s, 1H), 8.01 (d, J= 4.0 Hz, 1H), 7.22-7.16 (m, 5H), 6.79 (d, J= 8.0 Hz, 1H), 4.58-4.41 (m, 2H), 4.27 (s. 1H), 1.49 (s, 18H) ppm.

Synthesis of Bis-Boc-Cytosine-Phe-Phe (13). Compound 13 was synthesized by following the procedures described in synthesis of compound 6 except replacing the bis-Boc adenine acetic acid with bis-Boc cytosine acetic acid. Compound 13 (white powder) was collected with 78% yield (518 mg). 1H NMR (400 MHz, OMSO-d₆): δ 8.51 (d, J = 8.0 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.29-7.16 (m, 10H), 6.77 (d, J = 8.0 Hz, 1H), 4.58-4.38 (m, 4H), 3.07-2.71 (m, 4H), 1.48 (s, 18H) ppm

Synthesis of Cytosine-Phe-Phe (1C). Compound 1C was synthesized by following the procedures described in synthesis of compound 1A except replacing the bis-Boc adenine acetic acid with bis-Boc cytosine acetic acid. Compound 1C (white powder) was collected with 61% yield (283 mg). 1H NMR (400 MHz, DMSO-<¾): δ 8.87 (s, 1H), 8.48 (d, J = 7.6 Hz, 2H), 8.16 (s, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.30-7.16 (m, 10H), 5.91 (d, J = 8.4 Hz, 1H), 4.59-4.54 (m, 1H), 4.45-4.41 (m, 3H), 3.01-2.90 (m, 3H), 2.75-2.70 (m, 1H). ¹³C NMR (400 MHz, OMSO-d₆): 5 36.7, 37.8, 50.4, 53.7, 93.1, 126.4, 126.5, 128.1, 128.3, 129.2, 129.4, 137.6, 149.7, 161.6, 166.1, 170.9, 172.8. MS: calcd M⁺=463.49, obsd (M+l)⁺=464.49.

Synthesis of Cytosine-Phe-Phe-Tyr-phosphate (2C). Compound 2C was synthesized by following the procedures described in synthesis of compound 2A except replacing the bis-Boc adenine acetic acid with bis-Boc cytosine acetic acid. Compound 2C (white powder) was collected with 51% yield (360 mg). 1H NMR (400 MHz, DMSO-<¾): δ 8.54-8.42 (m, 2H), 8.24-8.19 (m, 1H), 7.62 (s, 1H), 7.26-7.06 (m, 14H), 5.85 (d, J = 7.2 Hz, 1H), 4.54-4.45 (m, 5H), 3.04-2.66 (m, 6H). ¹³C NMR (400 MHz, DMSO-<¾): δ 36.5, 37.9, 51.4, 54.4, 54.8, 93.8, 120.6, 126.9, 128.7, 129.9, 130.6, 138.2, 149.0, 151.7, 158.5, 170.9, 173.2. MS: calcd M⁺=706.64, obsd (M+Na)⁺=729.64.

Example 3 - Gelation Triggered by Alkaline Phosphatase

We dissolved 6.0 mg of precursor 2 in 300 μΐ, of water at pH = 7.4 to make a clear solution, then followed by adding 10 unit of alkaline phosphatase in 1 μΐ_^ to afford a translucent hydrogel.

Figure 6, figure 7, figure 8, and figure 9 depict the ³¹P NMR spectra of hydrogelators 2A, 2G, 2T, and 2C before and after the addition of alkaline phosphatase (ALP). Example 4 - Circular Dichroism (CD) Spectroscopy

CD spectra were recorded (185-350 nm) using a JASCO 810 spectrometer under a nitrogen atmosphere. The hydrogels (0.2 mL, 2.0 wt %) were placed evenly on the 1 mm thick quartz curvet and scanned with 0.5 nm interval.

Figure 10 depicts CD spectra of various hydrogels of the invention.

Example 5 - Rheological Measurements

Rheological tests were conducted on TA ARES G2 rheometer (with TA Orchestrator Software). 25 mm parallel plate was used during the experiment. 0.5 mL of hydrogel sample was placed on the parallel plate.

i) Dynamic Strain Sweep Test

Test range (0.1 to 10% strain, frequency = 10 rads^"1), 10 points per decade. Sweep mode is "log" and temperature was carried out at 25 °C.

ii) Critical strain determination

The critical strain (γο) value was determined from the storage-strain profiles of the hydrogel sample. The strain applied to the hydrogel sample increased from 0.1 to 100% (10 rad/s and 25 °C). Over a certain strain, a drop in the elastic modulus was observed, and the strain amplitude at which storage moduli just begins to decrease by 5%> from its maximum value was determined and taken as a measure of the critical strain of the hydrogels, which correspond to the breakdown of the crosslinked network in the hydrogel sample.

Example 6 - Simulation of the width of the nanofibers by molecular mechanical (MM) calculation

Molecular mechanics (MM) calculations were carried out to simulate the nanofibers of nucleopeptides with different diameters using the Dreiding Force Field as implemented in the molecular modeling programs (Accelrys Inc., San Diego, CA, USA). The initial crystal parameters of nucleopeptides were obtained from NapFF crystal structure. Then, the crystal structures of nucleopeptides were optimized by MM method. We determined the crystal growth habit of the nucleopeptide nanofibers by employing the Bravais-Friedel-Donnay- Harker (BFDH) method. We found all growth habits of the nanofibers are in the order of A>B>C axes. Accordingly, we fixed the long axis (A axis) to 33 unit cells and varied the widths (B axis) of the nanofibers to calculate the stabilization energy of the nanofibers. The width dependences of the stabilization energies are shown in Figure 12. Nonlinear curve fittings were carried out by three exponential functions

y = y₀ + A_xe ¹ + A₂e ² + A₃e ^

where A_n and b_n coefficients are calculated by the iterative method. Based on this method, we obtained four y₀ (i.e., the stabilization energy with infinity width). We fixed IT to 9 nm as a reference and then we can calculate the energy difference as the scaling factor based on yo of IT. According to this reference energy, we can estimate fiber diameters for other nucleopeptide nanofibers. Finally, we found that the simulated fiber diameters of nucleopeptides are in good agreement with the experimental data observed by TEM.

Example 7 - Wound-Healing Assay

HeLa cells were re-suspended in 10 cm tissue culture dish after washing cells once with PBS. 0.8 mL 0.25 % trypsin containing 0.1 % EDTA was then added, and the cells were re-suspended with 1.6 mL complete medium. 5000 cells (in 100 μΐ, medium) were plated into each vial on a 96 well plate to create a confluent monolayer. After adherent for 24 hr, a wound was created by scraping the cell monolayer with a p200 pipet tip. The cells were washed once with 100 of complete medium to remove flowing cells and replace with 100 of complete medium. 0 hr image was acquired as a reference point. The medium was replaced with 100 μΐ_^ of medium containing 27.7 mM of hydrogel 3T and the plate was incubate at 37 °C, 5 % C0₂ for 20 h. 0-h and 20-h images were acquired at the match photographed region. Figure 13d depicts optical images of HeLa cells on the surface O h and 20 h after the creation of scratch-wound in the medium in the presence of hydrogel 3T. Figure 14 depicts the control (no hydrogel).

Example 8 - Biostability

1 mg of each compound was dissolved in 5 mL HEPES buffer at pH=7.5. Then proteinase K was added in concentration 3.2 units/mL and incubated at 37 °C for 24 h, then 100 μΐ_^ of sample were taken out each time and analyzed by HPLC. Figure 15.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications cited herein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

I claim:

1. A hydrogelator of Formul

I

wherein, independently for each occurrence,

R² is H, alkyl, -OR, or -NR₂; and

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, or 20.

2. The hydrogelator of claim 1 , wherein is cytosinyl.

3. The hydrogelator of claim 1 , wherein is guaninyl.

4. The hydrogelator of claim 1 , wherein is adeninyl.

5. The hydrogelator of claim 1 , wherein is thyminyl.

6. The hydrogelator of claim 1 , wherein is uracilyl.

7. The hydrogelator of claim 1 , wherein

is an oligonucleic acid.

8. The hydrogelator of any one of claims 1-7, wherein R is H.

9. The hydrogelator of any one of claims 1-8, wherein R¹ is H.

10. The hydrogelator of any one of claims 1-8, wherein R¹ is alkyl.

11. The hydrogelator of any one of claims 1-8, wherein R¹ is methyl.

12. The hydrogelator of any one of claims 1-8, wherein R¹ is ethyl.

13. The hydrogelator of any one of claims 1-8, wherein R¹ is propyl.

14. The hydrogelator of any one of claims 1-8, wherein R¹ is isopropyl.

15. The hydrogelator of any one of claims 1-8, wherein R¹ is butyl.

16. The hydrogelator of any one of claims 1-8, wherein R¹ is isobutyl.

17. The hydrogelator of any one of claims 1-8, wherein R¹ is sec-butyl.

18. The hydrogelator of any one of claims 1-8, wherein R¹ is alkylthioalkyl.

19. The hydrogelator of any one of claims 1-8, wherein R¹ is CH₃-S-CH₂CH₂-.

20. The hydrogelator of any one of claims 1-8, wherein R¹ is aralkyl.

21. The hydrogelator of any one of claims 1-8, wherein R¹ is benzyl.

22. The hydrogelator of any one of claims 1-8, wherein R¹ is heteroaralkyl.

23. The hydrogelator of any one of claims 1-8, wherein R¹ is indolyl-CH₂-.

24. The hydrogelator of any one of claims 1-8, wherein R¹ is

25. The hydrogelator of any one of claims 1-8, wherein R¹ is hydroxy aralkyl.

26. The hydrogelator of any one of claims 1-8, wherein R¹ is hydroxybenzyl.

27. The hydrogelator of any one of claims 1-8, wherein R¹ is 4-hydroxybenzyl.

28. The hydrogelator of any one of claims 1-8, wherein R¹ is phosphorylated aralkyl.

29. The hydrogelator of any one of claims 1-8, wherein R¹ is f^PC -benzyl.

30. The hydrogelator of any one of claims 1-8, wherein R¹ is 4-H₂P0₄-benzyl.

31. The hydrogelator of any one of claims 1-8, wherein at least one instance of R¹ is aralkyl, hydroxyaralkyl, or phosphorylated aralkyl.

32. The hydrogelator of any one of claims 1-8, wherein R¹ is HC^C-alkyl.

33. The hydrogelator of any one of claims 1-8, wherein R¹ is HO₂C-CH₂-.

34. The hydrogelator of any one of claims 1-8, wherein R¹ is guanidinylalkyl.

35. The hydrogelator of any one of claims 1-8, wherein R¹ is guanidinyl-CH₂CH₂CH₂-.

36. The hydrogelator of any one of claims 1-35, wherein R² is -OR.

37. The hydrogelator of any one of claims 1-35, wherein R² is -OH.

38. The hydrogelator of any one of claims 1-37, wherein n is 1.

39. The hydrogelator of any one of claims 1-37, wherein n is 2.

40. The hydrogelator of any one of claims 1-37, wherein n is 3.

41. The hydrogelator of any one of claims 1-37, wherein n is 4.

42. The hydrogelator of any one of claims 1-37, wherein n is 5.

43. The hydrogelator of any one of claims 1-37, wherein n is 6.





30

46. A supramolecular structure, comprising a plurality of hydrogelators of any one of claims 1-45.

47. The supramolecular structure of claim 46, wherein the supramolecular structure is in the form of nano fibers.

48. The supramolecular structure of claim 46, wherein the supramolecular structure is in the form of nanofibers; and the average diameter of the nanofibers is about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, or about 25 nm.

49. The supramolecular structure of claim 46, wherein the supramolecular structure is in the form of nanofibers; and the nanofibers are crosslinked.

50. The supramolecular structure of claim 46, wherein the supramolecular structure is in the form of nanofibers; and the nanofibers are substantially straight.

51. The supramolecular structure of claim 46, wherein the supramolecular structure is in the form of nanofibers; and the nanofibers are bent.

52. The supramolecular structure of claim 46, wherein the supramolecular structure is in the form of nanofibers; and the nanofibers form bundles of nanofibers.

53. The supramolecular structure of claim 46, wherein the supramolecular structure is in the form of nanofibers; and the nanofibers are about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, or about 300 nm in length.

54. The supramolecular structure of claim 46, wherein the supramolecular structure is in the form of nanofibers; and the nanofibers are greater than about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, or about 300 nm in length.

55. A hydrogel, comprising a plurality of hydrogelators of any one of claims 1-45; and water.

56. A hydrogel, comprising a plurality of supramolecular structures of any one of claims 56-54; and water.

57. The hydrogel of any one of claims 55-56, wherein the hydrogel is formed from a solution of the hydrogelators in water.

58. The hydrogel of claim 57, wherein the hydrogelator is present in an amount from about 0.5% to about 4% by weight.

59. The hydrogel of claim 57, wherein the hydrogelator is present in an amount of about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, or about 4.0% by weight.

60. The hydrogel of claim 57, wherein the temperature of the solution is about 20 °C, about 25 °C, or about 30 °C.

61. The hydrogel of claim 57, wherein the hydrogel is formed by decreasing the pH of the solution of hydrogelators in water.

62. The hydrogel of claim 61, wherein the pH at which the supramolecular structure is formed is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, about 5.5, about 5.0, about 4.5, or about 4.0.

63. The hydrogel of claim 57, wherein the hydrogel is formed by the addition of an enzyme to the solution of hydrogelators in water.

64. The hydrogel of claim 63, wherein the enzyme is a phosphatase.

65. The hydrogel of claim 63, wherein the enzyme is alkaline phosphatase.

66. The hydrogel of any one of claim 55-65, wherein the hydrogel has a critical strain value of from about 0.2% to about 10.0%.

67. The hydrogel of any one of claim 55-65, wherein the hydrogel has a critical strain value of about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about

0.8%, about 0.9%, about 1.0%, about 1.2%, about 1.4%, about 1.6%, about 1.8%, about 2.0%, about 2.2%, about 2.4%, about 2.6%, about 2.8%, about 3.0%, about 3.2%, about 3.4%, about 3.6%, about 3.8%, about 4.0%, about 4.2%, about 4.4%, about 4.6%, about 4.8%, about 5.0%, about 5.2%, about 5.4%, about 5.6%, about 5.8%, about 6.0%, about 6.2%, about 6.4%, about 6.6%, about 6.8%, about 7.0%, about 7.2%, about 7.4%, about 7.6%, about 7.8%, about 8.0%, about 8.2%, about 8.4%, about 8.6%, about 8.8%, about 9.0%, about 9.2%, about 9.4%, about 9.6%, about 9.8%, or about 10%.

68. The hydrogel of any one of claim 55-67, wherein the hydrogel has a storage modulus of from about 2.0 Pa to about 14.0 KPa.

69. The hydrogel of any one of claim 55-67, wherein the hydrogel has a storage modulus of about 2.0 Pa, about 2.1 Pa, about 2.2 Pa, about 2.3 Pa, about 2.4 Pa, about 2.5 Pa, about 2.6 Pa, about 2.7 Pa, about 2.8 Pa, about 2.9 Pa, about 3.0 Pa, about 3.1 Pa, about 3.2 Pa, about 3.3 Pa, about 3.4 Pa, about 3.5 Pa, about 3.6 Pa, about 3.7 Pa, about 3.8 Pa, about

3.9 Pa, about 4.0 Pa, about 5.0 Pa, about 10 Pa, about 15 Pa, about 20 Pa, about 25 Pa, about 30 Pa, about 35 Pa, about 40 Pa, about 45 Pa, about 50 Pa, about 100 Pa, about 150 Pa, about 200 Pa, about 250 Pa, about 300 Pa, about 350 Pa, about 400 Pa, about 450 Pa, about 500 Pa, about 550 Pa, about 600 Pa, about 650 Pa, about 700 Pa, about 750 Pa, about 800 Pa, about 850 Pa, about 900 Pa, about 950 Pa, about 1.0 KPa, about 1.5 KPa, about 2.0 KPa, about 2.5 KPa, about 3.0 KPa, about 3.5 KPa, about 4.0 KPa, about 4.5 KPa, about 5.0 KPa, about 5.5 KPa, about 6.0 KPa, about 6.5 KPa, about 7.0 KPa, about 7.5 KPa, about 8.0 KPa, about 8.5 KPa, about 9.0 KPa, about 9.5 KPa, about 10.0 KPa, about 10.5 KPa, about 11.0 KPa, about 11.5 KPa, about 12.0 KPa, about 12.5 KPa, about 13.0 KPa, about 13.5 KPa, or about 14.0 KPa.

70. The hydrogel of any one of claim 55-69, wherein the hydrogel is substantially biocompatible.

71. The hydrogel of any one of claim 55-70, wherein the hydrogel is substantially biostable.

72. A method of growing cells, comprising contacting a plurality of cells with a supramolecular structure of any one of claims 46-54 or a hydrogel of any one of claim 55-71.

73. The method of claim 72, wherein the cells are engineered tissue cells.

74. A method of delivering a substance to a cell, comprising

contacting the substance with a supramolecular structure of any one of claims 46-54 or a hydrogel of any one of claim 55-71, thereby forming a substance-hydrogel delivery vehicle; and

contacting the substance-hydrogel delivery vehicle and a cell.

75. The method of claim 74, wherein the substance is a drug.

76. The method of claim 74, wherein the substance is a protein.

77. The method of claim 74, wherein the substance is a gene.

78. The method of claim 74, wherein the substance is siR A.

79. The method of claim 74, wherein the substance is microRNA.

80. The method of claim 74, wherein the substance is a second cell.

81. A method of binding a nucleic acid, comprising

contacting a nucleic acid with a supramolecular structure of any one of claims 46-54 or a hydrogel of any one of claim 55-71.

82. The method of claim 81, wherein the nucleic acid binding is selective nucleic acid binding.

83. A method of separating a protein from a substance, comprising

contacting a mixture with a supramolecular structure of any one of claims 46-54 or a hydrogel of any one of claim 55-71, wherein the mixture comprises a protein.

84. The method of claim 83, wherein the mixture comprises at least two proteins.

85. A method of treating or preventing a viral infection, comprising

administering to a mammal in need thereof a therapeutically effective amount of hydrogelator of any one of claims 1-45.

86. A method of treating or preventing cancer, comprising

administering to a mammal in need thereof a therapeutically effective amount of a hydrogelator of any one of claims 1-45.

87. A method of preventing adhesion of an organism or a cell to a surface, comprising contacting the surface with a supramolecular structure of any one of claims 46-54 or a hydrogel of any one of claim 55-71.