Heterochirality and Halogenation Control Phe-Phe Hierarchical Assembly

Abstract

Diphenylalanine is an amyloidogenic building block that can form a versatile array of supramolecular materials. Its shortcomings, however, include the uncontrolled hierarchical assembly into microtubes of heterogeneous size distribution and well-known cytotoxicity. This study rationalized heterochirality as a successful strategy to address both of these pitfalls and it provided an unprotected heterochiral dipeptide that self-organized into a homogeneous and optically clear hydrogel with excellent ability to sustain fibroblast cell proliferation and viability. Substitution of one l -amino acid with its d -enantiomer preserved the ability of the dipeptide to self-organize into nanotubes, as shown by single-crystal XRD analysis, whereby the pattern of electrostatic and hydrogen bonding interactions of the backbone was unaltered. The effect of heterochirality was manifested in subtle changes in the positioning of the aromatic side chains, which resulted in weaker intermolecular interactions between nanotubes. As a result, d -Phe- l -Phe self-organized into homogeneous nanofibrils with a diameter of 4 nm, corresponding to two layers of peptides around a water channel, and yielded a transparent hydrogel. In contrast with homochiral Phe-Phe stereoisomer, it formed stable hydrogels thermoreversibly. d -Phe- l -Phe displayed no amyloid toxicity in cell cultures with fibroblast cells proliferating in high numbers and viability on this biomaterial, marking it as a preferred substrate over tissue-culture plastic. Halogenation also enabled the tailoring of d -Phe- l -Phe self-organization. Fluorination allowed analogous supramolecular packing as confirmed by XRD, thus nanotube formation, and gave intermediate levels of bundling. In contrast, iodination was the most effective strategy to augment the stability of the resulting hydrogel, although at the expense of optical transparency and biocompatibility. Interestingly, iodine presence hindered the supramolecular packing into nanotubes, resulting instead into amphipathic layers of stacked peptides without the occurrence of halogen bonding. By unravelling fine details to control these materials at the meso- and macro-scale, this study significantly advanced our understanding of these systems.

Nanoscale Assembly of Functional Peptides with Divergent Programming Elements

“…ThT assay on the tripeptides was performed as previously described, using a concentration for each peptide of 20 mM, or 24 mM for 2a / 2b , and 6 and 24 h as time points for self-assembly. 26 For the Aβ(1–42) fibrillization inhibition studies, Aβ(1–42) was monomerized for 2 h in HFIP (0.5 mM); then HFIP was evaporated under an argon flow and left overnight under vacuum. Then, it was dissolved in DMSO at 0.3 mM and stored at −20 °C.…”

“…MTT assays were performed as previously described. 26 For live/dead assays, NIH3T3 fibroblasts (10,000 cells per well, 30 μL of DMEM + 10% fetal serum albumin, and 2% antimycotic and antibiotic from GIBCO) were added to the microwells of an uncoated μ-Slide for Angiogenesis (Ibidi, Germany) and cultured at 37 °C, 5% CO 2 for 24 h, according to the manufacturers’ instructions. A stock solution of 3a (4 mg/mL in 10 mM PBS, pH 7.4) was diluted to the desired concentration (0.1, 0.2, 0.5, or 1 mM) and added to the wells.…”

Peptide–Protein Interactions: From Drug Design to Supramolecular Biomaterials

“…While the design and understanding of the self-assembly of bare self-assembling scaffolds has advanced in recent years, the decoration with bioactive ligands often leads to unexpected changes in the self-assembly. The unforeseen changes in the self-assembling structures upon ligand functionalization, including chirality, are a beautiful new entry to expand the scope of sizes, stabilities, and shapes of self-assembling systems [ 55 , 56 , 127 ]. These changes at the same time provide more molecular insights into these fascinating bioactive supramolecular architectures.…”

“…In particular, the analysis showed that the formation of halogen bonds [ 167 ] stabilizes the supramolecular structures, allowing further possibilities for a fine control of the amyloid nanostructure [ 168 ]. Halogenation proved useful also to achieve fine control over the hierarchical assembly of heterochiral Phe-Phe to yield homogenous fibrils and hydrogels, although no halogen bonding was involved in this case [ 56 ]. Interestingly, heterochirality per se allowed substituting inter- with intra-molecular interactions, thus stabilizing 4 nm-wide nanotubes composed of two peptide layers around an inner water channel, and overall alleviating the homochiral Phe-Phe microtubes toxicity observed in cell culture [ 56 ].…”

“…Thus, the self-assembling processes are influenced by several factors: (i) amino acid sequence, (ii) the degree of hydrophobicity, (iii) the length of the peptides, and (iv) the self-assembling time [ 21 ]. These new materials based on SAPs involve synthetic scaffolds capable of containing multiple cell-interactive components and often yielding hydrogels [ 22 ] able to mimic the extracellular matrix [ 23 ] for applications in a variety of biomedical fields [ 24 ]; some examples as reported in Table 1 [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 ].…”