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.

Keywords: chirality; d-amino acids; halogenation; hydrogels; peptides; phenylalanine; self-assembly.

Chart 1. Phe-Phe Compounds Studied for Self-Assembly

Figure 1

Photographs of supramolecular hydrogels of compounds 1–6 at their mgc and hydrogel stability over 7 days (i.e., no transition to other phases).

Figure 2

TEM micrographs with, underneath, the corresponding fibril diameter distribution (n = 100) of self-assembled 1–6 at their mgc before (A–F) and after (G–L) thermoreversion.

Figure 3

CD spectra for 1–6 hydrogels in the assembled state (A) and upon heating-induced disassembly at 70–80 °C (B).

Figure 4

Single-crystal XRD structures of homochiral 1(44) (silver) and heterochiral 2 (cyan, CCDC 2016375) peptide nanotubes. (A) Side-view of stacked peptides revealed opposite screw-sense for 1 (silver) and 2 (cyan), dictated by the chirality of the N-terminal amino acids. (B) Top-view of nanotube inner cavities defined by the projection six peptide molecules arranged head-to-tail for both 1 (silver) and 2 (cyan), but only in 2 do the two Phe side chains interact face to face intramolecularly. (C) Top-view of the nanotubes identified by the projection of 18 peptide molecules for 1 (silver) showed hierarchical bundling stabilized by aromatic zippers; instead, a space-fill view of heterochiral 2 (cyan) revealed the outer diameter of a double layer of peptide molecules to agree with the diameter of fibrils measured by TEM.

Figure 5

(A) Single-crystal XRD structure (CCDC 2016373) of fluorinated heterochiral 5 revealed analogous packing to heterochiral 2 (compare with Figure 4C) with an intermediate level of bundling to generate 27 nm-wide fibrils as observed by TEM. (B,C) Powder XRD on microcrystals grown on hydrogels of 3–4 confirmed packing analogous to 5. (B) Fibrils of 3 had a diameter of 11–12 nm by TEM, corresponding to the packing of 19 water-channels. (C) Thermoreversion stabilized 4 nm wide fibrils for both 3 and 4, corresponding to a double-layer of peptides around a water channel as revealed by the single-crystal XRD of 5.

Figure 6

Single-crystal XRD structure of iodinated 6 (CCDC 2016374). (A) Hydrogen bonding and ionic interactions (dotted lines) defined hydrophilic layers (light blue), whereas iodine (purple sphere) allowed interdigitation of Phe side chains in hydrophobic layers (yellow). (B) Side-view of amphiphilic peptide stacks. Iodine atoms are located 4.9 Å from each other. (C) Space-fill view of peptide layers shown in panel A to show the tight supramolecular packing. (D) Side-view of peptide stacks reveals peptide backbones perpendicular to the preferential growth direction of the crystals.

Figure 7

(A) Raman spectra of iodinated 6 in the crystal, gel, and powder forms. The dotted orange line marks the C–I stretch. (B) Raman spectra of compounds 2–6 in the gel form. * denotes significant differences that were inferred to arise from supramolecular organization.

Figure 8

(A–L) Live (green)/dead (red) fluorescence (left,) and bright-field (right) microscopy images of fibroblasts cultured on hydrogels 1–6. Scale bars = 100 μm. (M) Quantitative MTT assay on keratinocytes incubated with peptides in solution.