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Small interfering RNAs (siRNAs) are potent weapons for gene silencing, with an opportunity to correct defective genes and stop the production of undesirable proteins, with many applications in central nervous system (CNS) disorders. However, successful delivery of siRNAs to the brain parenchyma faces obstacles such as the blood-brain barrier (BBB), brain tissue penetration, and targeting of specific cells. In addition, siRNAs are unstable under physiological conditions and are susceptible to protein binding and enzymatic degradation, necessitating a higher dosage to remain effective. To address these issues and advance siRNA delivery, we report the development of covalently conjugated hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimer-siRNA conjugates, demonstrated with a siRNA against GFP (siGFP) conjugate (D-siGFP) utilizing glutathione-sensitive linkers. This allows for precise nucleic acid loading, protects the payload from premature degradation, delivers the siRNA cargo into cells, and achieves significant GFP knockdown

Supramolecular hydrogels formed by noncovalent self-assembly of low molecular weight (LMW) agents are promising next-generation biomaterials. Thixotropic shear response and mechanical stability are two emergent properties of hydrogels that are critical for biomedical applications including drug delivery and tissue engineering in which injection of the hydrogel will be necessary. Herein, we demonstrate that the emergent thixotropic properties of supramolecular phenylalanine-derived hydrogels are dependent on the conditions in which they are formulated. Specifically, hydrogels formed from fluorenylmethoxycarbonyl (Fmoc) modified phenylalanine derivatives, 3-fluorophenylalanine (Fmoc-3F-Phe) and pentafluorophenylalanine (Fmoc-F5-Phe), were characterized as a function of gelation conditions to examine how shear response and mechanical stability properties correlate to mode of gelation. Two distinct methods of gelation were compared. First, spontaneous self-assembly and gelation was triggered by a solvent exchange method in which a concentrated solution of the gelator in dimethylsulfoxide was diluted in water. Second, gelation was promoted by dissolution of the gelator in water at basic pH followed by gradual pH adjustment from basic to mildly acidic by the hydrolysis of glucono-delta-lactone. Hydrogels formed under solvent exchange conditions were mechanically unstable and poorly shear-responsive whereas hydrogels formed by gradual acidification were temporally stable and had highly shear-responsive viscoelastic character. These studies confirm that gelation environment and mechanism have a significant influence on the emergent properties of supramolecular hydrogels and offer insight into how gelation conditions can be used to tune hydrogel properties for specific applications.

Supramolecular hydrogels formed by self-assembly of low molecular weight (LMW) compounds have been identified as promising materials for applications in tissue engineering and regenerative medicine. In many cases, the relationship between the chemical structure of the gelator and the emergent hydrogel properties is poorly understood. As a result, empirical screening strategies instead of rational design approaches are often relied upon to tune the emergent properties of the gels. Herein, we describe a novel strategy to identify improved phenylalanine (Phe) derived gelators using a focused empirical approach. Fluorenylmethoxycarbonyl (Fmoc) protected Phe derivatives are a privileged class of gelators that spontaneously self-assemble into fibrils that entangle to form a hydrogel network upon dissolution into water. However, the Fmoc group has been shown to have toxicity drawbacks for potential biological applications, requiring the identification of new N-terminal modifications that promote efficient self-assembly but lack the shortcomings of the Fmoc group. We previously discovered that fibrils in Fmoc-p-nitrophenylalanine (Fmoc-4-NO(2)-Phe) hydrogels transition to crystalline microtubes after several hours by a mechanism that involves the hierarchical assembly and fusion of the hydrogel fibrils. We hypothesized that this hierarchical crystallization behavior could form the basis of a screening approach to identify alternative N-terminal functional groups to replace Fmoc in Phe-derived LMW gelators. Specifically, screening N-terminal modifying groups for 4-NO(2)-Phe that stabilize the hydrogel state by preventing subsequent hierarchical crystallization would facilitate empirical identification of functional Fmoc replacements. To test this approach, we screened a small series of 4-NO(2)-Phe derivatives with various N-terminal modifying groups to determine if any provided stable LMW supramolecular hydrogels. All but one of the 4-NO(2)-Phe derivatives assembled into crystalline forms. Only the 1-naphthaleneacetic acid (1-Nap) 4-NO(2)-Phe derivative self-assembled into a stable hydrogel network. Additional Phe derivatives were modified by N-terminal 1-Nap-Phe groups to confirm the general potential of 1-Nap as a suitable replacement for Fmoc, and all derivatives formed stable hydrogels under similar conditions to their Fmoc-Phe counterparts. These results illustrate the potential of this approach to identify next-generation Phe-derived LMW gelators with improved emergent properties.

Self-assembling peptides are extensively exploited as bioactive materials in applications such as regenerative medicine and drug delivery despite the fact that their relatively weak noncovalent interactions often render them susceptible to mechanical destruction and solvent erosion. Herein, we describe how covalent cross-linking enhances the mechanical stability of self-assembling π-conjugated peptide hydrogels. We designed short peptide-chromophore-peptide sequences displaying different reactive functional groups that can form cross-links with appropriately modified bifunctional polyethylene glycol (PEG)-based small guest molecules. These peptides self-assemble into one-dimensional fibrillar networks in response to pH in the aqueous environment. The cross-linking reactions were promoted to create a secondary network locked in place by covalent bonds within the physically cross-linked (preassembled) π-conjugated peptide strands. Rheology measurements were used to evaluate the mechanical modifications of the network, and the chemical changes that accompany the cross-linking were further confirmed by infrared spectroscopy. Furthermore, we modified these cross-linkable π-conjugates by incorporating extracellular matrix (ECM)-derived Ile-Lys-Val-Ala-Val (IKVAV) and Arg-Gly-Asp (RGD) bioactive epitopes to support human neural stem and progenitor cell (hNSCs) differentiation. The hNSCs undergo differentiation into neurons on IKVAV-derived π-conjugates while RGD-containing peptides failed to support cell attachment. These findings provide significant insight into the biochemical and electronic properties of π-conjugated peptide hydrogelators for creating artificial ECM to enable advanced tissue-engineering applications.

Experimental details, characterization data for all compounds, additional spectra, and x-ray crystal data

Cationic amyloid fibrils, including the Semen Enhancer of Virus Infection (SEVI), have recently been described in human semen. Simple methods for quantitating these fibrils are needed to improve our understanding of their biological function. We performed high-throughput screening to identify molecules that bind SEVI, and identified a small molecule (8E2), that fluoresced brightly in the presence of SEVI and other cationic fibrils. 8E2 bound SEVI with almost 40-fold greater affinity than thioflavin-T, and could efficiently detect high molecular weight fibrils in human seminal fluid.

Supramolecular hydrogels derived from the self-assembly of organic molecules have been exploited for applications ranging from drug delivery to tissue engineering. The relationship between the structure of the assembly motif and the emergent properties of the resulting materials is often poorly understood, impeding rational approaches for the creation of next-generation materials. Aromatic π-π interactions play a significant role in the self-assembly of many supramolecular hydrogelators, but the exact nature of these interactions lacks definition. Conventional models that describe π-π interactions rely on quadrupolar electrostatic interactions between neighboring aryl groups in the π-system. However, recent experimental and computational studies reveal the potential importance of local dipolar interactions between elements of neighboring aromatic rings in stabilizing π-π interactions. Herein, we examine the nature of π-π interactions in the self- and coassembly of Fmoc-Phe-derived hydrogelators by systematically varying the electron-donating or electron-withdrawing nature of the side chain benzyl substituents and correlating these effects to the emergent assembly and gelation properties of the systems. These studies indicate a significant role for stabilizing dipolar interactions between neighboring benzyl groups in the assembled materials. Additional evidence for specific dipolar interactions is provided by high-resolution crystal structures obtained from dynamic transition of gel fibrils to crystals for several of the self-assembled/coassembled Fmoc-Phe derivatives. In addition to electronic effects, steric properties also have a significant effect on the interaction between neighboring benzyl groups in these assembled systems. These findings provide significant insight into the structure-function relationship for Fmoc-Phe-derived hydrogelators and give cues for the design of next-generation materials with desired emergent properties.

Regio- and diastereoselective reactions of a homoproline enolate enable the synthesis of novel extended dipeptide surrogates. Bicyclic carbamate 9 and fused beta-lactam scaffold 11 were prepared from L-pyroglutamic acid via substrate-controlled electrophilic azidation. Synthesis of orthogonally protected hexahydropyrrolizine, hexahydropyrrolizinone, and hexahydropyrroloazepinone dipeptide surrogates relied on allylation of proline derivative 5, followed by Curtius rearrangement to introduce the N-terminal carbamate group. A total of six azabicycloalkane derivatives were evaluated for conformational mimicry of extended dipeptides by a combination of X-ray diffraction and molecular modeling. Analysis of putative backbone dihedral angles and N- to C-terminal dipeptide distances indicate that compounds (alpha'S)-14b and 21 approximate the conformation of dipeptides found in beta-sheets, while tripeptide mimic 28 is also highly extended in the solid state. Structural data suggest that ring size and relative stereochemistry have a profound effect on the ability of these scaffolds to act as beta-strand mimetics and should inform the design of related conformational probes.

Hydrogel fibril and crystal formation are related self-assembly processes that provide materials with distinct emergent properties. The relationship between fibril and crystal growth is poorly understood, and efforts to engineer controlled hydrogelation vs crystallization via small molecule self-assembly currently depend on empirical approaches. Herein, we report the dynamic transition of self-assembled hydrogel fibrils of a phenylalanine (Phe) derivative, Fmoc-p-nitrophenylalanine (Fmoc-4-NO2-Phe), to crystalline microtubes. As has been shown with other Fmoc-Phe derivatives, Fmoc-4-NO2-Phe spontaneously self-assembles into amyloid-like fibrils that form an entangled hydrogel network when suspended in water. However, Fmoc-4-NO2-Phe fibrils uniquely transform over time into crystalline microtubes. Hydrogel fibrils appear to be a kinetic state with microtube crystals more thermodynamically favored. This dynamic transition from fibril to crystal has enabled a high-resolution structural analysis of the packing orientation of these self-assembled materials. Taking cues from this structural analysis, we demonstrate a rational strategy for stabilization of the kinetic Fmoc-4-NO2-Phe hydrogel fibrils. These results represent significant advances in our understanding of the dynamic nature of self-assembly processes and in our ability to rationally engineer these processes to provide materials with desired emergent properties.

Self-assembled supramolecular peptide hydrogels are important biomaterials that have been designed as matrices for tissue engineering and regenerative medicine [1-4]. Hydrogels derived from low molecular weight dipeptides or functionalized amino acids are a subset of self-assembled peptide hydrogels that have garnered significant recent interest [5-7]. Serious effort has been directed toward engineering low molecular weight hydrogelators that rival peptides in terms of their emergent viscoelastic and biochemical properties. Biochemical cell signaling motifs are often incorporated into supramolecular peptide hydrogels to facilitate cell adhesion, migration, and differentiation. The fibronectin-derived Arg-Gly-Asp (RGD) motif has been frequently incorporated into these types of materials to enable cell adhesion by interacting with integrin receptors. Herein, we discuss two-component supramolecular hydrogels that functionally bind cell surface integrins in a fibronectin-like manner without the explicit display of the RGD peptide [8]. These hydrogels are composed of Fmoc-3F-Phe-Asp-OH (1) and Fmoc-3F-PheArg-NH2 (2) dipeptides (Figure 1) that coassemble to form a fibril network. These fibrils display Asp and Arg at the surface to spatially mimic the Asp and Arg residues in the RGD motif. NIH 3T3 fibroblast cells adhere to the hydrogel surface via integrin binding in a demonstration of the fibronectin-like biochemical behavior of these materials.

Peptide and low molecular weight amino acid-based materials that self-assemble in response to environmental triggers are highly desirable candidates in forming functional materials with tunable biophysical properties. In this paper, we explore redox-sensitive self-assembly of cationic phenylalanine derivatives conjugated to naphthalene diimide (NDI). Self-assembly of the cationic Phe-NDI conjugates into nanofibrils was induced in aqueous solvent at high ionic strength. Under reducing conditions, these self-assembled Phe-NDI conjugate fibrils underwent a morphological change to non-fibril aggregates. Upon reoxidation, the initially observed fibrils were reformed. The study herein provides an interesting strategy to effect reversible switching of the structure of supramolecular materials that can be applied to the development of sophisticated stimulus-responsive materials.

Peptide self-assembly leading to cross-β amyloid structures is a widely studied phenomenon because of its role in amyloid pathology and the exploitation of amyloid as a functional biomaterial. The self-assembly process is governed by hydrogen bonding, hydrophobic, aromatic π-π, and electrostatic Coulombic interactions. A role for aromatic π-π interactions in peptide self-assembly leading to amyloid has been proposed, but the relative contributions of π-π versus general hydrophobic interactions in these processes are poorly understood. The Ac-(XKXK)(2)-NH(2) peptide was used to study the contributions of aromatic and hydrophobic interactions to peptide self-assembly. Position X was globally replaced by valine (Val), isoleucine (Ile), phenylalanine (Phe), pentafluorophenylalanine (F(5)-Phe), and cyclohexylalanine (Cha). At low pH, these peptides remain monomeric because of repulsion of charged lysine (Lys) residues. Increasing the solvent ionic strength to shield repulsive charge-charge interactions between protonated Lys residues facilitated cross-β fibril formation. It was generally found that as peptide hydrophobicity increased, the required ionic strength to induce self-assembly decreased. At [NaCl] ranging from 0 to 1000 mM, the Val sequence failed to assemble. Assembly of the Phe sequence commenced at 700 mM NaCl and at 300 mM NaCl for the less hydrophobic Ile variant, even though it displayed a mixture of random coil and β-sheet secondary structures over all NaCl concentrations. β-Sheet formation for F(5)-Phe and Cha sequences was observed at only 20 and 60 mM NaCl, respectively. Whereas self-assembly propensity generally correlated to peptide hydrophobicity and not aromatic character the presence of aromatic amino acids imparted unique properties to fibrils derived from these peptides. Nonaromatic peptides formed fibrils of 3-15 nm in diameter, whereas aromatic peptides formed nanotape or nanoribbon architectures of 3-7 nm widths. In addition, all peptides formed fibrillar hydrogels at sufficient peptide concentrations, but nonaromatic peptides formed weak gels, whereas aromatic peptides formed rigid gels. These findings clarify the influence of aromatic amino acids on peptide self-assembly processes and illuminate design principles for the inclusion of aromatic amino acids in amyloid-derived biomaterials.

Two configurationally stable carbon-based analogues of pyochelin have been prepared from Boc-pyroglutamic acid-tert-butyl ester in 11 and 13 steps. Introduction of the amino group was achieved by a highly diastereoselective electrophilic azidation reaction to afford novel bis-alpha-amino acid proline derivatives.