Constraining and Modifying Peptides Using Pd-Mediated Cysteine Allylation.

Over the past decades, several strategies for inducing and stabilizing secondary structure formation in peptides have been developed to increase their proteolytic stability and their binding affinity to specific interaction partners. Here, we report how our recently introduced chemoselective Pd-catalyzed cysteine allylation reaction can be extended to stapling and how the resulting alkene-containing staples themselves can be further modified to introduce additional probes into such stabilized peptides. The latter is demonstrated by introducing a fluorophore as well as a PEG moiety into different stapled peptides using bioorthogonal thiol-ene and Diels-Alder reactions. Furthermore, we investigated structural implications of our allyl staples when used to replace conformationally relevant disulfide bridges. To this end, we chose a selective binder of integrin α3 ß1 (LXY3), which is only active in its cyclic disulfide form. We replaced the disulfide bridge by different stapling reagents in order to increase stability and binding affinity towards integrin α3 ß1 .

Site-selective photocatalytic functionalization of peptides and proteins at selenocysteine.

The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide.

Expressed Protein Selenoester Ligation.

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.

Expressed Protein Selenoester Ligation.

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.

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.

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.

An Intrinsic Hydrophobicity Scale for Amino Acids and Its Application to Fluorinated Compounds.

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 the 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 polarity of the side chain dictates the self-assembly. This allows for the derivation of a novel hydrophobicity scale, which is based solely 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.

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.

Coiled-coils in phage display screening: insight into exceptional selectivity provided by molecular dynamics.

Involved in numerous key biological functions, protein helix-helix interactions follow a well-defined intermolecular recognition pattern. The characteristic structure of the α-helical coiled-coil allows for the specific randomization of clearly defined interaction partners within heteromeric systems. In this work, a rationally designed heterodimeric coiled-coil was used to investigate potential factors influencing the sequence selectivity in interhelical interactions.

Synthesis of enantiomerically pure (2S,3S)-5,5,5-trifluoroisoleucine and (2R,3S)-5,5,5-trifluoro-allo-isoleucine.

A practical route for the stereoselective synthesis of (2S,3S)-5,5,5-trifluoroisoleucine (L-5-F3Ile) and (2R,3S)-5,5,5-trifluoro-allo-isoleucine (D-5-F3-allo-Ile) was developed. The hydrophobicity of L-5-F3Ile was examined and it was incorporated into a model peptide via solid phase peptide synthesis to determine its α-helix propensity. The α-helix propensity of 5-F3Ile is significantly lower than Ile, but surprisingly high when compared with 4'-F3Ile.