Search

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.

Isoleucyl-tRNA synthetase (IleRS) is a paradigm for understanding how specificity against smaller hydrophobic substrates evolved in both the synthetic and editing reactions. IleRS misactivates nonproteinogenic norvaline (Nva) and proteinogenic valine (Val), with a 200-fold lower efficiency than the cognate isoleucine (Ile). Translational errors are, however, prevented by IleRS hydrolytic editing. Nva and Val are both smaller than Ile by a single methylene group. How does the removal of one additional methylene group affects IleRS specificity? We found that the nonproteinogenic α-aminobutyrate (Abu) is activated 30-fold less efficiently than Nva and Val, indicating that the removal of the second methylene group comes with a lower penalty. As with Nva and Val, discrimination against Abu predominantly originated from a higher KM . To examine whether increased hydrophobicity could compensate for the loss of van der Waals interactions, we tested fluorinated Abu analogues. We found that fluorination further hampered activation by IleRS, and even more so by the evolutionary-related ValRS. This suggests that hydrophobicity is not a main driving force of substrate binding in these enzymes. Finally, a discrimination factor of 7100 suggests that IleRS is not expected to edit Abu. However, we found that the IleRS editing domain hydrolyzes Abu-tRNAIle with a rate of 40 s-1 and the introduction of fluorine did not slow down the hydrolysis. This raises interesting questions regarding the mechanism of specificity of the editing domain and its evolution. Understanding what shapes IleRS specificity is also of importance for reengineering translation to accommodate artificial substrates including fluorinated amino acids. ENZYMES: Isoleucyl-tRNA synthetase (EC 6.1.1.5), leucyl-tRNA synthetase (EC 6.1.1.4), valyl-tRNA synthetase (EC 6.1.1.9).

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.

Es existieren über 100 Hydrophobieskalen für Aminosäuren, die auf unterschiedlichen Ansätzen basieren und einheitlich in kondensierter Phase ermittelt werden. Ein Vergleich der Hydrophobiewerte aus verschiedenen Methoden und die Ermittlung ihrer relativen Rangfolge sind jedoch anspruchsvoll, da die Wechselwirkungen zwischen der Umgebung und der Aminosäure in jeder Methode spezifisch sind. Hier überwinden wir diese Einschränkung, indem wir die Eigenschaften von Aminosäuren in der Reinraumumgebung der Gasphase untersuchen. In der Gasphase bleiben standardmäßig entropische Beiträge des hydrophoben Effektes aus, und nur die Polarität der Seitenkette ist für die Selbstorganisation entscheidend. Dies ermöglicht die Ableitung einer neuartigen Hydrophobieskala, die ausschließlich auf der Interaktion einzelner Aminosäureeinheiten innerhalb des Clusters basiert und somit die Eigenschaften der Seitenketten besser abbildet. Dieses Prinzip kann für die Klassifizierung von nicht‐natürlichen Derivaten angewendet werden, wie hier für fluorierte Aminosäurevarianten gezeigt ist.

Abstract 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. Graphic Abstract

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.

The chemistry of fluorinated compounds was essentially established by humans. Yet, fluorinated molecules have been present in our daily lives for a long time, be it as a nonstick coating for pans or as liquid crystals that are used in every smartphone, or as an important component of herbicides, pesticides, and pharmaceuticals. However these compounds are also released into nature and interact with plants and bacteria. Little is known about how nature adapts to increasing exposure to fluorinated molecules. In this chapter we want to shed light on the paths that fluorine takes in the ecosphere. We will discuss what nature does with these molecules and how nature itself produces fluorinated molecules. This is important in the context of the development of sustainable concepts in chemistry in order to conserve resources and tackle climate change.

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.

The hexapeptide NFGAIL is a highly amyloidogenic peptide, derived from the human islet amyloid polypeptide (hIAPP). Recent investigations indicate that presumably soluble hIAPP oligomers are one of the cytotoxic species in type II diabetes. Here we use thioflavin T staining, transmission electron microscopy, as well as ion mobility-mass spectrometry coupled to infrared (IR) spectroscopy to study the amyloid formation mechanism and the quaternary and secondary structure of soluble NFGAIL oligomers. Our data reveal that at neutral pH NFGAIL follows a nucleation dependent mechanism to form amyloid fibrils. During the lag phase, highly polydisperse, polymorph, and compact oligomers (oligomer number n = 2-13) as well as extended intermediates (n = 4-11) are present. IR secondary structural analysis reveals that compact conformations adopt turn-like structures, whereas extended oligomers exhibit a significant amount of β-sheet content. This agrees well with previous molecular dynamic simulations and provides direct experimental evidence that unordered off-pathway NFGAIL aggregates up to the size of at least the 13-mer as well as partially folded β-sheet containing oligomers are coexisting.

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 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., F for formation: This study introduces systematic fluorination as a tool to investigate the nature of amyloid formation as shown for the model peptide NFGAIL. Modulation of hydrophobicity and the σ‐framework of the Phe residue trough fluorine and iodine led to different amyloid folding kinetics. TEM and SAXS studies revealed the presence of amyloid fibrils.

The classification of amino acids according to their intrinsic properties, such as the physico‐chemical properties and structure, yields fundamental insights into their role in interactions in biological processes. More than 100 hydrophobicity scales have been introduced, with each being based on a distinct condensed‐phase approach. However, a comparison of the hydrophobicity values gained from different techniques, and their relative ranking is not straightforward, as the interactions between the environment and amino acid are unique to each method. Here, we overcome this limitation by studying the properties of amino acids in the clean‐room environment of the gas phase. In the gas phase, entropic contributions from the hydrophobic effect are by default absent and only the side‐chain´s polarity dictates self‐assembly. This allows for the derivation of a novel hydrophobicity scale, which is solely based on the interaction between individual amino acid units within the cluster and thus more accurately reflects the intrinsic nature of a side‐chain. This principle can be further applied to classify non‐natural derivatives, as shown here for fluorinated amino acid variants.

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 N(ε)-(carboxyethyl)lysine (CEL) and N(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, m/z 142.1 and 187.1; CEL, m/z 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 m/z 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.

Recently, the structure of BAY 58-2667 bound to the Nostoc sp. H-NOX domain was published. On the basis of this structural information, we designed BAY 58-2667 derivatives and tested their effects on soluble guanylyl cyclase (sGC) activity. Derivative 20 activated sGC 4.8-fold more than BAY 58-2667. Co-crystallization of 20 with the Ns H-NOX domain revealed that the increased conformational distortion at the C-terminal region of αF helix containing 110-114 residues contributes to the higher activation triggered by 20.

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.

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.