Heterochirality Restricts the Self-Assembly of Phenylalanine Dipeptides Capped with Highly Aromatic Groups.

The influence of stereochemistry on the self-assembly of phenylalanine (Phe) dipeptides bearing aromatic fluorenyl groups at both the N- and C-termini (Fmoc, OFm) has been investigated. For this purpose, Fmoc-d-Phe-l-Phe-OFm and Fmoc-l-Phe-l-Phe-OFm have been examined considering a wide variety of solvents, which differ in dielectric constant and volatility. Results reveal that replacement of l-Phe by d-Phe has a major impact on the self-assembly propensities, restricting drastically the structural diversity and polymorphism shown by the homochiral dipeptide. Thus, the analogous heterochiral dipeptide shows a great propensity to form micro/nanofibers, independently of the environmental conditions. Theoretical calculations revealed that the stability of antiparallel disposition is much higher (a factor of ca. 15) for Fmoc-d-Phe-l-Phe-OFm than that for Fmoc-l-Phe-l-Phe-OFm, which has been attributed to the hydrophobic core formed in the former. Overall, results suggest that control of the backbone chirality is a potent and versatile strategy to drive and finely tune the self-assembly propensities of highly aromatic peptides.

Revisiting the Self-Assembly of Highly Aromatic Phenylalanine Homopeptides.

Diphenylalanine peptide (FF), which self-assembles into rigid tubular nanostructures, is a very short core recognition motif in Alzheimer's disease ß-amyloid (Aß) polypeptide. Moreover, the ability of the phenylalanine (F or Phe)-homopeptides to self-assemble into ordered nanostructures has been proved. Within this context it was shown that the assembly preferences of this family of compounds is altered by capping both the N- and C-termini using highly aromatic fluorenyl groups (i.e., fluorenyl-9-methoxycarbonyl and 9-fluorenylmethyl ester, named Fmoc and OFm, respectively). In this article the work performed in the field of the effect of the structure and incubation conditions on the morphology and polymorphism of short (from two to four amino acid residues) Phe-homopeptides is reviewed and accompanied by introducing some new results for completing the comparison. Special attention has been paid to the influence of solvent: co-solvent mixture used to solubilize the peptide, the peptide concentration and, in some cases, the temperature. More specifically, uncapped (FF, FFF, and FFFF), N-capped with Fmoc (Fmoc-FF, Fmoc-FFF, and Fmoc-FFFF), C-capped with OFm (FF-OFm), and doubly capped (Fmoc-FF-OFm, Fmoc-FFF-OFm, and Fmoc-FFFF-OFm) Phe-homopeptides have been re-measured. Although many of the experienced assembly conditions have been only revisited as they were previously reported, other experimental conditions have been examined by the first time in this work. In any case, pooling the effect of highly aromatic blocking groups in a single study, using a wide variety of experimental conditions, allows a perspective of how the disappearance of head-to-tail electrostatic interactions and the gradual increase in the amount of π-π stacking interactions, affects the morphology of the assemblies. Future technological applications of Phe-homopeptides can be envisaged by choosing the most appropriate self-assemble structure, defining not only the length of the peptide but also the amount and the position of fluorenyl capping groups.

Amyloid fibrils from organic solutions of an amphiphilic dipeptide.

Non-hydrated organic solutions of a diphenylalanine amphiphile blocked at the C-terminus with a fluorenylmethyl ester and stabilized at the N-terminus with a trifluoroacetate have been used to prepare amyloid fibrils. The solvent used to prepare the stock solution together with the co-solvent added enables regulation of the characteristics of the fibrils, which is important for their use in technological applications.

Amyloid-like Fibrils from a Diphenylalanine Capped with an Aromatic Fluorenyl.

The self-assembly behavior of a diphenylalanine amphiphile blocked at the C-terminus with a 9-fluorenylmethyl ester and stabilized at the N-terminus with a trifluoroacetate (TFA) anion, TFA·FF-OFm, has been examined. At low peptide concentration (0.5 mg/mL), long amyloid-like fibrils, which come from the fusion of two or more helical ribbons and/or thinner fibrils, organized in bundles or as individual entities are detected. Microbeam synchrotron radiation infrared spectroscopy has shown that TFA·FF-OFm molecules in amyloid-like fibrils arrange, forming antiparallel ß-sheets. Alteration of the experimental conditions to prioritize the thermodynamic contribution with respect to the kinetic one in the self-assembly process inhibits the organization of amyloid-like structures in favor of the formation of conventional fibrous structures. On the basis of experimental observations, a structural model where the individual antiparallel ß-sheets are oriented in parallel has been proposed for TFA·FF-OFm amyloid-like fibrils.

Self-assembly of diphenylalanine with preclick components as capping groups.

Alkyne and azide, which are commonly used in the cycloaddition reaction recognized as "click chemistry", have been used as capping groups of two engineered diphenylalanine (FF) derivatives due to their ability to form weak intermolecular interactions (i.e. dipole-π and π-π stacking). In Poc-FF-N3, alkyne and azide act as N- and C-terminal capping groups, respectively, while such positions are exchanged in N3-FF-OPrp. The self-assembly of such two synthesized peptides has been extensively studied in their "pre-click" state, considering the influence of three different factors: the peptide concentration, the polarity of the medium, and the nature of the substrate. Poc-FF-N3 assembles into microfibers that, depending on the medium and the substrate, can aggregate hierarchically in supramolecular structures with different morphologies. The most distinctive one corresponds to very stable birefringent dendritic-like microstructures, which are derived from the ordered agglomeration of microfibers. These branched supramolecular structures, which are observed under a variety of conditions, are relatively uncommon in short FF sequences. At the molecular level, Poc-FF-N3 organizes in antiparallel ß-sheets stabilized by N-HO intermolecular hydrogen bonds and re-enforced by weak interactions between the azide and alkyne groups of neighbouring molecules. In contrast, N3-FF-OPrp exhibits a very poor tendency to organize into structures with a well-defined morphology. Theoretical calculations on model complexes indicate that the tendency of the latter peptide to organize into small amorphous agglomerates is due to its poor ability to form specific intermolecular interactions in comparison with Poc-FF-N3. The implications of the weak interactions induced by the alkyne and azide groups, which strengthen peptidepeptide hydrogen bonds and π-ladders due to the stacked aromatic phenyl side groups, are discussed.

Diversity and Hierarchy in Supramolecular Assemblies of Triphenylalanine: From Laminated Helical Ribbons to Toroids.

Microstructures from small phenylalanine-based peptides have attracted great attention lately because these compounds are considered to be a new class of tunable materials. In spite of the extensive studies on uncapped diphenylalanine and tetraphenylalanine peptides, studies on the self-assembly of uncapped triphenylananine (FFF) are very scarce and nonsystematic. In this work, we demonstrate that FFF assemblies can organize in a wide number of well-defined supramolecular structures, which include laminated helical-ribbons, leaflike dendrimers, doughnut-, needle-, and flower-shapes. These organizations are produced by the attractive or repulsive interactions between already formed assemblies and therefore can be controlled through the choice of solvents used as the incubation medium. Thus, the formation of the desired supramolecular structures is regulated through the protonation/deprotonation of the terminal groups, the polarity of the incubation medium, which affects both peptide···solvent interactions and the cavity solvation energy (i.e., solvent···solvent interactions), and the steric interactions between own assemblies that act as building blocks. Finally, the ß-sheet disposition in the latter structural motifs has been examined using both theoretical calculations and Fourier transform infrared spectroscopy. Results indicate that FFF molecules can adopt both parallel and antiparallel ß-sheets. However, the former one is the most energetically favored because of the formation of π-π stacking interactions between the aromatic rings of hydrogen-bonded strands.

Effect of Solvent Choice on the Self-Assembly Properties of a Diphenylalanine Amphiphile Stabilized by an Ion Pair.

A diphenylalanine (FF) amphiphile blocked at the C terminus with a benzyl ester (OBzl) and stabilized at the N terminus with a trifluoroacetate (TFA) anion was synthetized and characterized. Aggregation of peptide molecules was studied by considering a peptide solution in an organic solvent and adding pure water, a KCl solution, or another organic solvent as co-solvent. The choice of the organic solvent and co-solvent and the solvent/co-solvent ratio allowed the mixture to be tuned by modulating the polarity, the ionic strength, and the peptide concentration. Differences in the properties of the media used to dissolve the peptides resulted in the formation of different self-assembled microstructures (e.g. fibers, branched-like structures, plates, and spherulites). Furthermore, crystals of TFA⋅FF-OBzl were obtained from the aqueous peptide solutions for X-ray diffraction analysis. The results revealed a hydrophilic core constituted by carboxylate (from TFA), ester, and amide groups, and the core was found to be surrounded by a hydrophobic crown with ten aromatic rings. This segregated organization explains the assemblies observed in the different solvent mixtures as a function of the environmental polarity, ionic strength, and peptide concentration.

Hierarchical self-assembly of di-, tri- and tetraphenylalanine peptides capped with two fluorenyl functionalities: from polymorphs to dendrites.

Homopeptides with 2, 3 and 4 phenylalanine (Phe) residues and capped with fluorenylmethoxycarbonyl and fluorenylmethyl esters at the N-terminus and C-terminus, respectively, have been synthesized to examine their self-assembly capabilities. Depending on the conditions, the di- and triphenylalanine derivatives self-organize into a wide variety of stable polymorphic structures, which have been characterized: stacked braids, doughnut-like shapes, bundled arrays of nanotubes, corkscrew-like shapes and spherulitic microstructures. These highly aromatic Phe-based peptides also form incipient branched dendritic microstructures, even though they are highly unstable, making their manipulation very difficult. Conversely, the tetraphenylalanine derivative spontaneously self-assembles into stable dendritic microarchitectures made of branches growing from nucleated primary frameworks. The fractal dimension of these microstructures is ∼1.70, which provides evidence for self-similarity and two-dimensional diffusion controlled growth. DFT calculations at the M06L/6-31G(d) level have been carried out on model ß-sheets since this is the most elementary building block of Phe-based peptide polymorphs. The results indicate that the antiparallel ß-sheet is more stable than the parallel one, with the difference between them growing with the number of Phe residues. Thus, the cooperative effects associated with the antiparallel disposition become more favorable when the number of Phe residues increases from 2 to 4, while those of the parallel disposition remained practically constant.

Self-Assembly of Tetraphenylalanine Peptides.

Three different tetraphenylalanine (FFFF) based peptides that differ at the N- and C-termini have been synthesized by using standard procedures to study their ability to form different nanoassemblies under a variety of conditions. The FFFF peptide assembles into nanotubes that show more structural imperfections at the surface than those formed by the diphenylalanine (FF) peptide under the same conditions. Periodic DFT calculations (M06L functional) were used to propose a model that consists of three FFFF molecules defining a ring through head-to-tail NH3(+)⋅⋅⋅(-)OOC interactions, which in turn stack to produce deformed channels with internal diameters between 12 and 16 Å. Depending on the experimental conditions used for the peptide incubation, N-fluorenylmethoxycarbonyl (Fmoc) protected FFFF self-assembles into a variety of polymorphs: ultra-thin nanoplates, fibrils, and star-like submicrometric aggregates. DFT calculations indicate that Fmoc-FFFF prefers a parallel rather than an antiparallel ß-sheet assembly. Finally, coexisting multiple assemblies (up to three) were observed for Fmoc-FFFF-OBzl (OBzl = benzyl ester), which incorporates aromatic protecting groups at the two peptide terminals. This unusual and noticeable feature is attributed to the fact that the assemblies obtained by combining the Fmoc and OBzl groups contained in the peptide are isoenergetic.