Biodegradation of Amphipathic Fluorinated Peptides Reveals a New Bacterial Defluorinating Activity and a New Source of Natural Organofluorine Compounds.

Three peptides comprising mono-, di-, and tri-fluoroethylglycine (MfeGly, DfeGly, and TfeGly) residues alternating with lysine were digested by readily available proteases (elastase, bromelain, trypsin, and proteinase K). The degree of degradation depended on the enzyme employed and the extent of fluorination. Incubation of the peptides with a microbial consortium from garden soil resulted in degradation, yielding fluoride ions. Further biodegradation studies conducted with the individual fluorinated amino acids demonstrated that the degree of defluorination followed the sequence MfeGly > DfeGly > TfeGly. Enrichment of the soil bacteria employing MfeGly as a sole carbon and energy source resulted in the isolation of a bacterium, which was identified as Serratia liquefaciens. Cell-free extracts of this bacterium enzymatically defluorinated MfeGly, yielding fluoride ion and homoserine. In silico analysis of the genome revealed the presence of a gene that putatively codes for a dehalogenase. However, the low overall homology to known enzymes suggests a potentially new hydrolase that can degrade monofluorinated compounds. 19F NMR analysis of aqueous soil extracts revealed the unexpected presence of trifluoroacetate, fluoride ion, and fluoroacetate. Growth of the soil consortium in tryptone soya broth supplemented with fluoride ions resulted in fluoroacetate production; thus, bacteria in the soil produce and degrade organofluorine compounds.

Introducing Aliphatic Fluoropeptides: Perspectives on Folding Properties, Membrane Partition and Proteolytic Stability.

A de novo designed class of peptide-based fluoropolymers composed of fluorinated aliphatic amino acids as main components is reported. Structural characterization provided insights into fluorine-induced alterations on ß-strand to α-helix transition upon an increase in SDS content and revealed the unique formation of PPII structures for trifluorinated fluoropeptides. A combination of circular dichroism, fluorescence-based leaking assays and surface enhanced infrared absorption spectroscopy served to examine the insertion and folding processes into unilamellar vesicles. While partitioning into lipid bilayers, the degree of fluorination conducts a decrease in α-helical content. Furthermore, this study comprises a report on the proteolytic stability of peptides exclusively built up by fluorinated amino acids and proved all sequences to be enzymatically degradable despite the degree of fluorination. Herein presented fluoropeptides as well as the distinctive properties of these artificial and polyfluorinated foldamers with enzyme-degradable features will play a crucial role in the future development of fluorinated peptide-based biomaterials.

Cyanine Dye Coupling Mediates Self-assembly of a pH Sensitive Peptide into Novel 3D Architectures.

Synthetic multichromophore systems are of great importance in artificial light harvesting devices, organic optoelectronics, tumor imaging and therapy. Here, we introduce a promising strategy for the construction of self-assembled peptide templated dye stacks based on coupling of a de novo designed pH sensitive peptide with a cyanine dye Cy5 at its N-terminus. Microscopic techniques, in particular cryogenic TEM (cryo-TEM) and cryo-electron tomography technique (cryo-ET), reveal two types of highly ordered three-dimensional assembly structures on the micrometer scale. Unbranched compact layered rods are observed at pH 7.4 and two-dimensional membrane-like assemblies at pH 3.4, both species displaying spectral features of H-aggregates. Molecular dynamics simulations reveal that the coupling of Cy5 moieties promotes the formation of both ultrastructures, whereas the protonation states of acidic and basic amino acid side chains dictates their ultimate three-dimensional organization.

Gram-Scale Asymmetric Synthesis of Fluorinated Amino Acids Using a Chiral Nickel(II) Complex.

Fluorinated amino acids play an important role in the field of peptide and protein engineering. Although numerous syntheses have been published in recent decades, strategies that allow routine access to fluorinated amino acids on a gram-scale have been poorly described. Furthermore, the described pathways that gain fluorinated amino acids are based on different synthetic strategies, making a uniform approach that uses similar starting materials highly beneficial. Chiral Ni(II) complexes were introduced as powerful tools in the synthesis of noncanonical amino acids. In this work, we present a strategy for the synthesis of a diverse range of fluorinated amino acids based on the corresponding Ni(II) complex from which the products can be obtained in enantiopure form (99% ee) on a gram-scale. In addition, we describe an optimized procedure for the synthesis of alkyl iodide building blocks that are required for the alkylation reactions with the corresponding Ni(II) complex. Finally, we characterized the synthesized fluorinated amino acids with regard to their hydrophobicity and α-helix propensity.

Rational design of amphiphilic fluorinated peptides: evaluation of self-assembly properties and hydrogel formation.

Advanced peptide-based nanomaterials composed of self-assembling peptides (SAPs) are of emerging interest in pharmaceutical and biomedical applications. The introduction of fluorine into peptides, in fact, offers unique opportunities to tune their biophysical properties and intermolecular interactions. In particular, the degree of fluorination plays a crucial role in peptide engineering as it can be used to control the characteristics of fluorine-specific interactions and, thus, peptide conformation and self-assembly. Here, we designed and explored a series of amphipathic peptides by incorporating the fluorinated amino acids (2S)-4-monofluoroethylglycine (MfeGly), (2S)-4,4-difluoroethylglycine (DfeGly) and (2S)-4,4,4-trifluoroethylglycine (TfeGly) as hydrophobic components. This approach enabled studying the impact of fluorination on secondary structure formation and peptide self-assembly on a systematic basis. We show that the interplay between polarity and hydrophobicity, both induced differentially by varying degrees of side chain fluorination, does affect peptide folding significantly. A greater degree of fluorination promotes peptide fibrillation and subsequent formation of physical hydrogels in physiological conditions. Molecular simulations revealed the key role played by electrostatically driven intra-chain and inter-chain contact pairs that are modulated by side chain fluorination and give insights into the different self-organization behaviour of selected peptides. Our study provides a systematic report about the distinct features of fluorinated oligomeric peptides with potential applications as peptide-based biomaterials.

The Impact of Halogenated Phenylalanine Derivatives on NFGAIL Amyloid Formation.

The hexapeptide hIAPP22-27 (NFGAIL) is known as a crucial amyloid core sequence of the human islet amyloid polypeptide (hIAPP) whose aggregates can be used to better understand the wild-type hIAPP's toxicity to ß-cell death. In amyloid research, the role of hydrophobic and aromatic-aromatic interactions as potential driving forces during the aggregation process is controversially discussed not only in case of NFGAIL, but also for amyloidogenic peptides in general. We have used halogenation of the aromatic residue as a strategy to modulate hydrophobic and aromatic-aromatic interactions and prepared a library of NFGAIL variants containing fluorinated and iodinated phenylalanine analogues. We used thioflavin T staining, transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) to study the impact of side-chain halogenation on NFGAIL amyloid formation kinetics. Our data revealed a synergy between aggregation behavior and hydrophobicity of the phenylalanine residue. This study introduces systematic fluorination as a toolbox to further investigate the nature of the amyloid self-assembly process.