Rationales Design von polyfluorierten und enzymatisch abbaubaren Biomaterialien auf Peptidbasis

Amphiphilic peptide-based biomaterials are of great interest for pharmaceutical and biomedical applications and mainly associated with pronounced biocompatibility and biodegradability. In fact, introducing fluorine-containing amino acids into peptides & proteins offers an unique opportunity to enhance their biophysical properties such as membrane permeability. Through its influence on hydrophobicity and polarity, the degree of fluorination dictates the extent of fluorine-specific interactions on peptide folding and stability, intermolecular interactions, and biological activity. The first study of this doctoral thesis describes the folding, self-assembly, and hydrogelation of single-strand amphipathic peptides with different degrees of fluorination on the amino acid side chains by the iterative incorporation of monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). A combination of experimental and theoretical approaches proved a higher degree of side chain fluorination to promote β-sheet formation and the rheological stability of peptide-based hydrogels in physiological conditions, whereas secondary structure formation was inhibited at a low fluorine content due to fluorine-induced polarity. In a follow-up study, the selective modification of antimicrobial peptides (AMPs) by fluorinated amino acids was investigated. A β-hairpin-forming peptide motif, whose amphipathic structure enables the targeted disruption of bacterial cell membranes, was therefore examined. Extensive MIC screening with Gram-negative and Gram-positive bacteria confirmed highly fluorinated amino acids such as trifluoroethylglycine (TfeGly) or pentafluoropropylglycine (PfpGly) to strengthen the bioactivity of the AMPs through enhanced intrinsic hydrophobicity without causing a simultaneous increase in toxic & hemolytic properties. Numerous studies on the singular incorporation of fluorinated amino acids have been published to date, whereas synthetic peptides with larger or exclusive amounts of these building blocks remained unexplored. That drove the motivation for the herein-described development and characterization of so-called "fluoropeptides". In brief, β-sheet to α-helix or fluorine-induced PPII-helix transitions were observed in SDS-supplemented buffer (pH 7.4). In situ SEIRAS experiments with POPC:POPG-based membrane models functioned to investigate the fluoropeptide’s lipid insertion and (re)folding. Thus, the highest α-helical secondary structure content was found for the nonfluorinated homooligopeptide and decreased in the order of tri-, di-, and mono-fluorination of the side chains. An important focus of this doctoral thesis was the evaluation of biodegradability for especially higher polyfluorinated sequences. In fact, all peptides prepared in this work could be hydrolyzed by various proteases regardless of the fluorine content. In cooperation with the University College Dublin, first data on the microbial digestion of fluorinated peptides and individual amino acids could be generated. The enzyme-catalyzed cleavage of the C-F bond on the side chain for both kind of substrates was, for instance, proven by detection of released fluoride ions in solution. The results of this work will contribute to the rational design and potential application of polyfluorinated peptides, whose enzymatic degradability is going to be of great interest for the future development of fluorinated biomaterials.Amphiphile peptidbasierte Biomaterialien sind vom großen Interesse für pharmazeutische und biomedizinische Anwendungen und überzeugen zumeist durch ihre Biokompatibilität und Bioabbaubarkeit. Die Einführung von fluorhaltigen Aminosäuren in Peptide & Proteine bietet hierbei die einzigartige Möglichkeit, ihre biophysikalischen Eigenschaften wie etwa die Membranpermeabilität zu verstärken. Insbesondere der Fluorierungsgrad spielt eine entscheidende Rolle, da er durch seinen Einfluss auf die Hydrophobie und Polarität die Gesamtheit fluor-spezifischer Wechselwirkungen auf die Peptidfaltung und -stabilität, intermolekularen Wechselwirkungen und biologische Aktivität steuern kann. Die erste Studie dieser Doktorarbeit beschreibt die Faltung, Selbstassemblierung und Hydrogelierung von einzelsträngigen amphipathischen Peptiden mit unterschiedlichen Fluorierungsgraden der Aminosäureseitenketten durch den iterativen Einbau von Monofluorethylglycin (MfeGly), Difluorethylglycin (DfeGly) und Trifluorethylglycin (TfeGly). Mittels einer Kombination aus experimentellen und theoretischen Ansätzen konnte gezeigt werden, dass bei physiologischen Bedingungen ein höherer Fluorierungsgrad die Bildung von β-Faltblattstrukturen und die rheologische Stabilität der peptid-basierten Hydrogele fördert, jedoch diese Sekundärstruktur von Peptiden mit niedrigem Fluorgehalt durch die fluor-induzierte Polarität inhibiert wird. In einer weiteren Studie wurde die gezielte Modifizierung der biologischen Eigenschaften antimikrobieller Peptide (AMP) durch den Einbau fluorierter Aminosäuren untersucht. Hierzu wurde ein β-Hairpin bildendes Peptidmotiv ausgewählt, dessen amphipathische Struktur die zielgerichtete Disruption bakterieller Zellmembrane ermöglicht. Die ermittelten minimalen Hemmkonzentrationen (MHK) gegen verschiedene Gram-negative und Gram-positive Bakterien zeigen, dass hochfluorierte Aminosäuren wie Trifluorethylglycin (TfeGly) und Pentafluorpropylglycin (PfpGly) die Bioaktivität antimikrobieller Peptide durch Erhöhung der intrinsischen Hydrophobie selektiv verstärken können, ohne eine gleichzeitige Zunahme toxischer & hämolytischer Eigenschaften zu verursachen. Zahlreiche Studien zum singulären Einbau fluorierter Aminosäuren wurden bis dato veröffentlicht, während synthetische Peptide mit größeren bzw. ausschließlichen Mengen dieser Bausteine unerforscht blieben. Dies war die Motivation zur Entwicklung und Charakterisierung sogenannter "Fluoropeptide". In SDS-beinhaltenden Puffer (pH 7.4) wurden, unter anderem, Übergänge von β-Faltblatt Strukturen zu α-Helices oder Fluor-induzierte PPII-Helices beobachtet. In-situ SEIRAS-Studien mit POPC:POPG-basierten Membranmodellen dienten zum Studium der Lipidinsertion und (Rück-)-Faltung der Fluoropeptide in Abhängigkeit zum gesamten Fluoranteil. Hierbei wurde der höchste Gehalt an α-helikaler Sekundärstruktur für das nichtfluorierte Homooligopeptid bestimmt, welcher in der Reihenfolge der Tri-, Di- und Monofluorierung der Seitenkette abnahm. Ein wichtiger Schwerpunkt dieser Doktorarbeit war die Bewertung der biologischen Abbaubarkeit für insbesondere höher polyfluorierte Sequenzen. Tatsächlich konnten alle in dieser Arbeit hergestellten Peptide unabhängig vom Fluorgehalt durch verschiedene Proteasen hydrolysiert werden. In Zusammenarbeit mit dem University College Dublin konnten zudem erste Daten zum mikrobiellen Verdau fluorierter Peptide und Aminosäuren generiert werden. Die enzymkatalysierte Spaltung der C-F-Bindung an der Seitenkette für beide Substratarten wurde beispielsweise durch den Nachweis von freigesetzten Fluorid-Ionen in Lösung nachgewiesen. Die Ergebnisse dieser Arbeit werden zum rationalen Design und potenzieller Anwendbarkeit neuartiger polyfluorierter Peptide beitragen, deren enzymatische Abbaubarkeit von großem Interesse für die künftige Entwicklung fluorhaltiger Biomaterialien sein wird

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

Fine-tuning the antimicrobial activity of β-hairpin peptides with fluorinated amino acids

Antimicrobial peptides (AMPs) possess bactericidal activity against a variety of pathogens depending on an overall balance of positively charged and hydrophobic residues. Selective fluorination of peptides serves to fine-tune the intrinsic hydrophobicity and that could improve AMP bioactivity without affecting the sequence length. Only a few studies have focused on the impact of this unique element on antimicrobial potency and came to somewhat contractionary results. Moreover, the influence of fluorinated amino acids on peptide proteolysis is yet not fully understood. In this work, we tackle the link between side chain fluorination and both antimicrobial activity and proteolytic stability for two series of amphiphilic β-hairpin peptides. In particular, a synergy between antimicrobial activity and peptide hydrophobicity was determined. All peptides were found to be barely hemolytic and non-toxic. Most surprisingly, the fluorinated peptides were susceptible to enzymatic degradation. Hence, the distinctive properties of these polyfluorinated AMPs will serve for the future design of peptide-based drugs

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 β-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

The Impact of Halogenated Phenylalanine Derivatives on NFGAIL Amyloid Formation

The hexapeptide hIAPP(22-27)(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 beta-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

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

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 β-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 smallangle 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

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 several different syntheses have been published in recent decades, obtaining fluorinated amino acids on a gram-scale still poses a challenge. Furthermore, the described pathways to obtain fluorinated amino acids are based on different synthetic strategies, making a uniform approach from similar starting materials highly interesting. Chiral Ni(II) complexes were introduced as powerful tools in the synthesis of non-canonical amino acids. In this work, we present a strategy for the synthesis of a diverse range of fluorinated amino acids from the corresponding Ni(II) complex on a gram-scale from which the products can be obtained in enantiopure form (>94%ee). In addition, we describe syntheses for alkyl iodide building blocks which 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