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

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

C–H-Functionalization logic guides the synthesis of a carbacyclopamine analog

The chemical synthesis of carbacyclopamine analog 2, a cyclopamine analog with an all-carbon E-ring, is reported. The use of C–H-functionalization logic and further metal-catalyzed transformations allows for a concise entry to this new class of acid-stable cyclopamine analogs

Exploring the locking stage of NFGAILS amyloid fibrillation via transition manifold analysis

We demonstrate the application of the transition manifold framework to the late-stage fibrillation process of the NFGAILS peptide, a amyloidogenic fragment of the human islet amyloid polypeptide (hIAPP). This framework formulates machine learning methods for the analysis of multi-scale stochastic systems from short, massively parallel molecular dynamical simulations. We identify key intermediate states and dominant pathways of the process. Furthermore, we identify the optimally timescale-preserving reaction coordinate for the dock-lock process to a fixed pre-formed fibril and show that it exhibits strong correlation with the mean native hydrogen-bond distance. These results pave the way for a comprehensive model reduction and multi-scale analysis of amyloid fibrillation processes

Approaches to Obtaining Fluorinated α-Amino Acids

Fluorine does not belong to the pool of chemical elements that nature uses to build organic matter. However, chemists have exploited the unique properties of fluorine and produced countless fluoro-organic compounds without which our everyday lives would be unimaginable. The incorporation of fluorine into amino acids established a completely new class of amino acids and their properties, and those of the biopolymers constructed from them are extremely interesting. Increasing interest in this class of amino acids caused the demand for robust and stereoselective synthetic protocols that enable straightforward access to these building blocks. Herein, we present a comprehensive account of the literature in this field going back to 1995. We place special emphasis on a particular fluorination strategy. The four main sections describe fluorinated versions of alkyl, cyclic, aromatic amino acids, and also nickel-complexes to access them. We progress by one carbon unit increments. Special cases of amino acids for which there is no natural counterpart are described at the end of each section. Synthetic access to each of the amino acids is summarized in form of a table at the end of this article with the aim to make the information easily accessible to the reader

Cyclopamine analogs bearing exocyclic methylenes are highly potent and acid-stable inhibitors of hedgehog signaling

The chemical synthesis and biological evaluation of new cyclopamine analogs bearing exocyclic methylenes in different positions is described. Bis-exo-cyclopamine 6 was identified as a potent inhibitor of the Gli1-dependent luciferase expression in Shh-LIGHTII cells. An extension of this study to F-ring-modified structures shows the necessity of a rigidly positioned nitrogen atom for bioactivity as well as the presence of the C21 methyl group for acid stability and bioactivity

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

Position-dependent impact of hexafluoroleucine and trifluoroisoleucine on protease digestion

Rapid digestion by proteases limits the application of peptides as therapeutics. One strategy to increase the proteolytic stability of peptides is the modification with fluorinated amino acids. This study presents a systematic investigation of the effects of fluorinated leucine and isoleucine derivatives on the proteolytic stability of a peptide that was designed to comprise substrate specificities of different proteases. Therefore, leucine, isoleucine, and their side-chain fluorinated variants were site-specifically incorporated at different positions of this peptide resulting in a library of 13 distinct peptides. The stability of these peptides towards proteolysis by α-chymotrypsin, pepsin, proteinase K, and elastase was studied, and this process was followed by an FL-RP-HPLC assay in combination with mass spectrometry. In a few cases, we observed an exceptional increase in proteolytic stability upon introduction of the fluorine substituents. The opposite phenomenon was observed in other cases, and this may be explained by specific interactions of fluorinated residues with the respective enzyme binding sites. Noteworthy is that 5,5,5-trifluoroisoleucine is able to significantly protect peptides from proteolysis by all enzymes included in this study when positioned N-terminal to the cleavage site. These results provide valuable information for the application of fluorinated amino acids in the design of proteolytically stable peptide-based pharmaceuticals

Systematic Evaluation of Fluorination as Modification for Peptide-Based Fusion Inhibitors against HIV-1 Infection

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics

Sensitive and Site-Specific Identification of Carboxymethylated and Carboxyethylated Peptides in Tryptic Digests of Proteins and Human Plasma

Glycation refers to a nonenzymatic post-translational modification formed by the reaction of amino groups and reducing sugars. Consecutive oxidation and degradation can produce advanced glycation end products (AGEs), such as <i>N</i>^ε-(carboxyethyl)­lysine (CEL) and <i>N</i>^ε-(carboxymethyl)­lysine (CML). Although CEL and CML are considered to be markers of arteriosclerosis, diabetes mellitus, and aging, the modified proteins and the exact modification sites are mostly unknown due to their low frequency and a lack of enrichment strategies. Here, we report characteristic fragmentation patterns of CML- and CEL-containing peptides and two modification-specific reporter ions for each modification (CML, <i>m</i>/<i>z</i> 142.1 and 187.1; CEL, <i>m</i>/<i>z</i> 156.1 and 201.1). The protocol allowed sensitive and selective precursor ion scans to detect the modified peptides in complex sample mixtures. The corresponding <i>m</i>/<i>z</i> values identified eight CEL/CML-modification sites in glycated human serum albumin (HSA) by targeted nano-RPC–MS/MS. The same strategy revealed 21 CML sites in 17 different proteins, including modified lysine residues 88 and 396 of human serum albumin, in a pooled plasma sample that was obtained from patients with type 2 diabetes mellitus