Structure optimization of lipopeptide assemblies for aldol reactions in an aqueous medium.

Four amphiphilic peptides were synthesized, characterized, and evaluated regarding their efficiency in the catalysis of direct aldol reactions in water. The lipopeptides differ by having a double lipid chain and a guanidinium pyrrole group functionalizing one Lys side chain. All the samples are composed of the amino acids l-proline (P), l-arginine (R), or l-lysine (K) functionalized with the cationic guanidiniocarbonyl pyrrole unit (GCP), l-tryptophan (W), and l-glycine (G), covalently linked to one or two long aliphatic chains, leading to surfactant-like designs with controlled proline protonation state and different stereoselectivity. Critical aggregation concentrations (cac) were higher in the presence of the GCP group, suggesting that self-assembly depends on charge distribution along the peptide backbone. Cryogenic Transmission Electron Microscopy (Cryo-TEM) and Small Angle X-ray Scattering (SAXS) showed a rich polymorphism including spherical, cylindrical, and bilayer structures. Molecular dynamics simulations performed to assess the lipopeptide polymorphs revealed an excellent agreement with core-shell arrangements derived from SAXS data and provided an atomistic view of the changes incurred by modifying head groups and lipid chains. The resulting nanostructures behaved as excellent catalysts for aldol condensation reactions, in which superior conversions (>99%), high diastereoselectivities (ds = 94 : 6), and enantioselectivities (ee = 92%) were obtained. Our findings contribute to elucidate the effect of nanoscale organization of lipopeptide assemblies in the catalysis of aldol reactions in an aqueous environment.

Sono-Assembly of the [Arg-Phe]4 Octapeptide into Biofunctional Nanoparticles.

High-frequency ultrasound treatment is found to be a one-pot green technique to produce peptide-based nanostructures by ultrasound assisted self-assembly of oligopeptides. [Arg-Phe]4 octapeptides, consisting of alternating arginine (Arg/R) and phenylalanine (Phe/F) sequences, were subjected to 430 kHz ultrasound in aqueous solution in the absence of any external agents, to form [RF]4 nanoparticles ([RF]4-NPs), ~220 nm in diameter. A comprehensive analysis of the obtained nanoparticles demonstrated that the aromatic moieties of the oligopeptides can undergo oxidative coupling to form multiple oligomeric species, which then self-assemble into well-defined fluorescent nanoparticles. [RF]4-NPs were functionalized with polyethylene glycol (PEGylated) to improve their colloidal stability. Unlike the parent peptide, the PEGylated [RF]4-NPs showed limited cytotoxicity towards MDA-MB-231 cells. Furthermore, the intracellular trafficking of PEGylated [RF]4-NPs was investigated after incubation with MDA-MB-231 cells to demonstrate their efficient endo-lysosomal escape. This work highlights that the combined use of ultrasonic technologies and peptides enables easy fabrication of nanoparticles, with potential application in drug delivery.

Polymorphism of asymmetric catalysts based on amphiphilic lipopeptides in solution.

The self-assembly of model [P]RWG lipopeptides (P: l-proline, R: l-arginine, W: l-tryptophan, G: l-glycine), containing one or two aliphatic octadecyl (C18) chains in water and cyclohexanone/water solutions was examined. The self-assembly of mixtures of these RWG and PRWG lipopeptides was also investigated. These materials presented a similar critical aggregation concentration of ∼4.0 × 10-4 wt% and were characterized by unordered secondary structures with some ß-sheet content. TEM and cryo-TEM revealed the presence of mainly nanotape structures with micelles observed for systems rich in PRWG(C18H37). Analysis of detailed SAXS form factor measurements revealed the presence of bilayers 3-4 nm thick while the PRWG(C18H37) micelles have a core radius of approximately 3 nm, and a shell thickness of 2 nm. For the cyclohexanone/water systems polymorphs containing cluster aggregates (with radius of 0.25 nm to 0.50 nm) and some elongated structures (with radius of 5.7 nm to 26.1 nm) were seen. Longer structures were formed with the increase of the proline-containing lipopeptide content. The catalytic activity of these peptides was assessed using a model nitro-aldol reaction. The concentration of water in the reaction system influenced the conversion, higher content promoted better efficiency for the water systems, but the opposite was observed for the cyclohexanone/water samples.

Self-assembled gold nanoparticles and amphiphile peptides: a colorimetric probe for copper(II) ion detection.

Morphological, spectroscopic and scattering studies of the self-assembly and aggregation process of hybrids containing gold nanoparticles (AuNPs) and the amyloid peptides [RF]4 and P[RF]4 (where R = arginine; F = phenylalanine; P = proline) in aqueous solution were performed. Two methodologies were tested for the AuNP nucleation, using sodium borohydride (NaBH4) or epigallocatechin gallate (EGCG) as a reducing agent. This led to remarkable distinct modes of assembly, AuNP decorated fibrils with NaBH4 reduction or isolated AuNPs with EGCG reduction. For both methodologies, the presence of spherical AuNPs was observed by plasmonic resonance bands in absorption spectra at ∼520 nm. Zeta potential measurements confirmed stable systems, with a similar aggregation state. Circular dichroism spectra revealed an antiparallel ß-sheet conformation of the peptides. The transmission electron microscopy (TEM) images showed the coexistence of nanometer fibers and globular nanoparticles with 20 nm size. The small-angle X-ray scattering (SAXS) results show that the NaBH4 systems presented large cylindrical structures, while with increasing P[RF]4 content, a decrease in radius was observed. However, the EGCG-AuNPs were characterized by spherical particles, with a radius of 10-20 nm. Also, the colorimetric efficiency of the hybrids in the capture of Cu2+ ions in solution was monitored. Raman spectroscopy data confirmed the conformation/structure of self-assembled samples. Moreover, there are indications for a surface-enhanced Raman spectroscopy (SERS) effect for Cu2+ sites. The set of results indicates that these systems could act as a promising sensitive metal concentration probes.

Amyloid Peptide Mixtures: Self-Assembly, Hydrogelation, Nematic Ordering, and Catalysts in Aldol Reactions.

Morphological, spectroscopic, and scattering studies of the self-assembly and aggregation of mixtures of [RF]4 and P[RF]4 peptides (where R = arginine; F = phenylalanine; P = proline), in solution and as hydrogels, were performed to obtain information about polymorphism. CD data confirmed a ß-sheet secondary structure in aqueous solution, and TEM images revealed nanofibers with diameters of ∼10 nm and micrometer lengths. SAXS curves were fitted using a mass fractal-component and a long cylinder shell form factor for the liquid samples, and only a long cylinder shell form factor for the gels. Increasing the P[RF]4 content in the systems leads to a reduction in cylinder radius and core scattering density, suggesting an increase in packing of the peptide molecules; however, the opposite effect is observed for the gels, where the scattering density is higher in the shell for the systems containing higher P[RF]4 content. These compounds show potential as catalysts in the asymmetric aldol reactions, with cyclohexanone and p-nitrobenzaldehyde in aqueous media. A moderate conversion (36.9%) and a good stereoselectivity (69:31) were observed for the system containing only [RF]4. With increasing P[RF]4 content, a considerable decrease of the conversion was observed, suggesting differences in the self-assembly and packing factor. Rheological measurements were performed to determine the shear moduli for the soft gels.

Self-Assembly, Nematic Phase Formation, and Organocatalytic Behavior of a Proline-Functionalized Lipopeptide.

The self-assembly of the amphiphilic lipopeptide PAEPKI-C16 (P = proline, A = alanine, E = glutamic acid, K = lysine, I = isoleucine, and C16 = hexadecyl) was investigated using a combination of microscopy, spectroscopy, and scattering methods and compared to that of C16-IKPEAP with the same (reversed) peptide sequence and the alkyl chain positioned at the N-terminus and lacking a free N-terminal proline residue. The catalytic activity of these peptides was then compared using a model aldol reaction system. For PAEPKI-C16, the cryo-TEM images showed the formation of micrometer-length fibers, which by small-angle X-ray scattering (SAXS) were found to have radii of 2.5-2.6 nm. Spectroscopic analysis shows that these fibers are built from ß-sheets. This behavior is in complete contrast to that of C16-IKPEAP, which forms spherical micelles with peptides in a disordered conformation [Hutchinson J. Phys. Chem. B 2019, 123, 613]. In PAEPKI-C16, spontaneous alignment of fibers was observed upon increasing pH, which was accompanied by observed birefringence and anisotropy of SAXS patterns. This shows the ability to form a nematic phase, and unprecedented nematic hydrogel formation was also observed for these lipopeptides at sufficiently high concentrations. SAXS shows retention of an ultrafine (1.7 nm core radius) fibrillar network within the hydrogel. PAEPKI-C16 with free N-terminal proline shows enhanced anti:syn diastereoselectivity and better conversion compared to C16-IKPEAP. The cytotoxicity of PAEPKI-C16 was also lower than that of C16-IKPEAP for both fibroblast and cancer cell lines. These results highlight the sensitivity of lipopeptide properties to the presence of a free proline residue. The spontaneous nematic phase formation by PAEPKI-C16 points to the high anisotropy of its ultrafine fibrillar structure, and the formation of such a phase at low concentrations in aqueous solution may be valuable for future applications.

Self-Assembly of a Catalytically Active Lipopeptide and Its Incorporation into Cubosomes.

The self-assembly and biocatalytic activity of the proline-functionalized lipopeptide PRW-NH-C16 are examined and compared to that of the related PRW-O-C16 lipopeptide, which differs in having an ester linker between the lipid chain and tripeptide headgroup instead of an amide linker. Lipopeptide PRW-NH-C16 self-assembles into spherical micelles above a critical aggregation concentration, similar to the behavior of PRW-O-C16 reported previously [B. M. Soares et al. Phys. Chem. Chem. Phys., 2017, 19, 1181-1189]. However, PRW-NH-C16 shows an improved catalytic activity in a model aldol reaction. In addition, we explore the incorporation of the biocatalytic lipopeptide into lipid cubosomes. SAXS shows that increasing lipopeptide concentration leads to an expansion of the monoolein cubosome lattice spacing and a loss of long-range cubic order as the lipopeptide is encapsulated in the cubosomes. At higher loadings of lipopeptide, reduced cubosome formation is observed at the expense of vesicle formation. Our results show that the peptide-lipid chain linker does not influence self-assembly but does impart an improved biocatalytic activity. Furthermore, we show that lipopeptides can be incorporated into lipid cubosomes, leading to restructuring into vesicles at high loadings. These findings point the way toward the future development of bioactive lipopeptide assemblies and slow release cubosome-based delivery systems.