Short Antimicrobial Lipo‐α/γ‐AA Hybrid Peptides

The last two decades have seen the rise of antimicrobial peptides (AMPs) to combat emerging antibiotic resistance. Herein we report the solid‐phase synthesis of short lipidated α/γ‐AA hybrid peptides. This family of lipo‐chimeric peptidomimetics displays potent and broad‐spectrum antimicrobial activity against a range of multi‐drug resistant Gram‐positive and Gram‐negative bacteria. These lipo‐α/γ‐AA hybrid peptides also demonstrate high biological specificity, with no hemolytic activity towards red blood cells. Fluorescence microscopy suggests that these lipo‐α/γ‐AA chimeric peptides can mimic the mode of action of AMPs and kill bacterial pathogens via membrane disintegration. As the composition of these chimeric peptides is simple, therapeutic development could be economically feasible and amenable for a variety of antimicrobial applications.     

Design of a Short Thermally Stable α‐Helix Embedded in a Macrocycle

Although helices play key roles in peptide–protein and protein–protein interactions, the helical conformation is generally unstable for short peptides (10–15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain‐to‐side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non‐hydrogen‐bonded amide groups to solvent. Here, we develop a “capped‐strapped” peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end‐capping interactions. We have designed a ten‐residue capped‐strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and T_m≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X‐ray crystallography revealed a novel quaternary structure with implications for foldamer design.     

Gold(I)‐Catalyzed Tandem Alkoxylation/Lactonization of γ‐Hydroxy‐α,β‐Acetylenic Esters

The formation of 4‐alkoxy‐2(5H)‐furanones was achieved via tandem alkoxylation/lactonization of γ‐hydroxy‐α,β‐acetylenic esters catalyzed by 2 mol% of [2,6‐bis(diisopropylphenyl)imidazol‐2‐ylidine]gold bis(trifluoromethanesulfonyl)imidate [Au(IPr)(NTf₂)]. The economic and simple procedure was applied to a series of various secondary propargylic alcohols allowing for yields of desired product of up to 95%. In addition, tertiary propargylic alcohols bearing mostly cyclic substituents were converted into the corresponding spiro derivatives. Both primary and secondary alcohols reacted with propargylic alcohols at moderate temperatures (65–80 °C) in either neat reactions or using 1,2‐dichloroethane as a reaction medium allowing for yields of 23–95%. In contrast to [Au(IPr)(NTf₂)], reactions with cationic complexes such as [2,6‐bis(diisopropylphenyl)imidazol‐2‐ylidine](acetonitrile)gold tetrafluoroborate [Au(IPr)(CH₃CN)][BF₄] or (μ‐hydroxy)bis{[2,6‐bis(diisopropylphenyl)imidazol‐2‐ylidine]gold} tetrafluoroborate or bis(trifluoromethanesulfonyl)imidate – [{Au(IPr)}₂(μ‐OH)][X] (X=BF₄, NTf₂) – mostly stop after the alkoxylation. Analysis of the intermediate proved the exclusive formation of the E‐isomer which allows for the subsequent lactonization.     

Folding and Unfolding of Two Mixed α/β Peptides

We present a molecular dynamics simulation study of two peptides containing α‐ and β‐amino acid residues. According to experiment, the two peptides differ in the dominant fold when solvated in methanol: one shows a helical fold, the other a β hairpin. The simulations at 300 and 340 K were done by starting from a NMR spectroscopic model structure and from an extended (denatured) structure. The typical structural features of the two peptides are reproduced and a folding/unfolding equilibrium is observed on the nanosecond timescale at 300 K. Analysis of proton–proton NOE distance bounds and backbone ³J coupling constants gives results consistent with the experimental data. We conclude that our simulations are complementary to the experiments by providing detailed information on the conformational distributions.     

Arginine Arrangement of Bacteriophage λ N‐Peptide Plays a Role as a Core Motif in GNRA Tetraloop RNA Binding

A simple α‐helical N‐model‐peptide was designed to investigate the role of the arginine‐rich motif of bacteriophage λ N‐peptide in selective binding with boxB RNA. The five‐arginine arrangement of native N‐peptide was retained; all other residues were replaced with alanine. In vitro selection of RNA (30 random‐nucleotide region) was carried out with N‐model‐peptide immobilized on a 27 MHz quartz‐crystal microbalance (QCM). Selected RNAs were evaluated on the same QCM plate to obtain binding constants (K_a=10⁷–10⁸ M ^?1). Many selected RNAs contained GNR(N)A‐type loops (similar to the boxB RNA motif recognized by the native N‐peptide). Fragments and minimal RNAs containing the GNRA‐type loop also bound to N‐model‐peptide (K_a=10⁶–10⁷ M ^?1). The RNA recognition specificity of the peptide was studied by changing the “closing” U–A base pair and one base in the tetraloop of the RNA aptamers, and by peptide mutations (18th residue of N‐model‐peptide). It was concluded that the five‐arginine arrangement of the peptide performs selective recognition of the GNRA tetraloop and GNR(N)A pentaloop RNA structures, and that substitution of another functional amino acid residue at the 18th position in N‐peptide adds the recognition ability for a loop‐RNA sequence.     

Copper(I)‐Catalyzed Stereoselective Synthesis of (E)‐α‐Alkynyl‐α,β‐unsaturated Esters from a Terminal Alkyne,Diazoesters and Aldehydes

A new synthetic method for α‐alkynyl‐α,β‐unsaturated esters is presented herein. The method is based on a copper(I)‐catalyzed three‐component reaction of a terminal alkyne, diazoesters and aldehydes. The reaction is featured by mild conditions, high yields and excellent stereoselectivity. Cu(I) carbene migratory insertion is proposed as the key step in the transformation.

     

Reconstructing the Discontinuous and Conformational β1/β3‐Loop Binding Site on hFSH/hCG by Using Highly Constrained Multicyclic Peptides

Making peptide‐based molecules that mimic functional interaction sites on proteins remains a challenge in biomedical sciences. Here, we present a robust technology for the covalent assembly of highly constrained and discontinuous binding site mimics, the potential of which is exemplified for structurally complex binding sites on the “Cys‐knot” proteins hFSH and hCG. Peptidic structures were assembled by Ar(CH₂Br)₂‐promoted peptide cyclizations, combined with oxime ligation and disulfide formation. The technology allows unprotected side chain groups and is applicable to peptides of different lengths and nature. A tetracyclic FSH mimic was constructed, showing >600‐fold improved binding compared to linear or monocyclic controls. Binding of a tricyclic hCG mimic to anti‐hCG mAb 8G5 was identical to hCG itself (IC₅₀=260 vs. 470 pM ), whereas this mimic displayed an IC₅₀ value of 149 nM for mAb 3468, an hCG‐neutralizing antibody with undetectable binding to either linear or monocyclic controls.     

Targeting EphA2‐Sam and Its Interactome: Design and Evaluation of Helical Peptides Enriched in Charged Residues

The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam–Sam interactions. Because EphA2‐Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues. Several peptide sequences with high predicted helical propensities were designed, and detailed conformational analyses were conducted by diverse techniques including NMR, CD, and molecular dynamics (MD) simulations. Interaction studies were also performed by NMR, surface plasmon resonance (SPR), and microscale thermophoresis (MST) and led to the identification of two peptides capable of binding to the first Sam domain of Odin. These molecules represent early candidates for the generation of efficient Sam domain binders and antagonists of Sam–Sam interactions involving EphA2.     

Alpha‐Synuclein Binds to the Inner Membrane of Mitochondria in an α‐Helical Conformation

The human alpha‐Synuclein (αS) protein is of significant interest because of its association with Parkinson's disease and related neurodegenerative disorders. The intrinsically disordered protein (140 amino acids) is characterized by the absence of a well‐defined structure in solution. It displays remarkable conformational flexibility upon macromolecular interactions, and can associate with mitochondrial membranes. Site‐directed spin‐labeling in combination with electron paramagnetic resonance spectroscopy enabled us to study the local binding properties of αS on artificial membranes (mimicking the inner and outer mitochondrial membranes), and to evaluate the importance of cardiolipin in this interaction. With pulsed, twofrequency, double‐electron electron paramagnetic resonance (DEER) approaches, we examined, to the best of our knowledge for the first time, the conformation of αS bound to isolated mitochondria.     

Asymmetric Epoxidation of α,β‐Unsaturated Aldehydes in Aqueous Media Catalyzed by Resin‐Supported Peptide‐ Containing Unnatural Amino Acids

The enantio‐ and diastereoselective epoxidation of α,β‐unsaturated aldehydes in aqueous media was realized using a resin‐supported peptide catalyst. Introducing the hydrophobic and bulky unnatural amino acid 3‐(1‐pyrenyl)alanine into the peptide sequence was effective for enhancing the reaction rate and enantioselectivity.     

1,2,3‐Triazolium‐Based Cationic Amphipathic Peptoid Oligomers Mimicking Antimicrobial Helical Peptides

Amphipathic cationic peptoids (N‐substituted glycine oligomers) represent a promising class of antimicrobial peptide mimics. The aim of this study is to explore the potential of the triazolium group as a cationic moiety and helix inducer to develop potent antimicrobial helical peptoids. Herein we report the first solid‐phase synthesis of peptoid oligomers incorporating 1,2,3‐triazolium‐type side chains and their evaluation against Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. Several triazolium‐based oligomers, even of short length, selectively kill bacteria over mammalian cells. SEM visualization of S. aureus cells treated with a dodecamer and a hexamer reveals severe cell membrane damage and suggests that the longer oligomer acts by pore formation.     

Efficient Photochemical Synthesis of Peptide‐α‐Phenylthioesters

Low yields and substantial epimerization of peptide‐α‐thioesters often compromise the overall efficiency of native chemical ligation (NCL). Peptide arylthioesters are more reactive than peptide alkylthioesters in NCL, but are also more difficult to handle due to their propensity to hydrolyze, and are therefore often generated in situ. However, pre‐prepared peptide arylthioesters are required for some NCL applications. Here we present a 7‐nitroindoline‐based photochemical method that generates protected peptide phenylthioesters under neutral reaction conditions via their activated esters from photoreactive peptide precursors in high isolated yields, and with low levels of epimerization. This method is fully compatible with Fmoc‐strategy solid‐phase peptide synthesis. Global deprotection with trifluoroacetic acid furnishes peptide phenylthioesters for NCL. Photoreactive peptide precursors can also be converted into their hydrazides in two steps by this method.     

Dicer Nuclease‐Promoted Production of Let7a‐1 MicroRNA Is Enhanced in the Presence of Tryptophan‐Containing Amphiphilic Peptides

A library of Trp‐containing amphiphilic peptides was synthesized and screened for the ability to bind to pre‐miRNA targets. Two members of this family, peptides Ac‐WKKLLKWLKKLLKLAG‐NH₂ ( 2 b ) and Ac‐WKKLLKWLKKLLDabLAG‐NH₂ ( 4 b ) were found to have nanomolar binding affinities to pre‐let7a‐1. Peptides 2 b and 4 b caused an increase in the in vitro Dicer cleavage of pre‐let7a‐1. This observation was confirmed by a cell‐based assay, the results of which show an up to 50 % increase in Dicer activity. Enhanced expression of let7a‐1 promoted by the peptides results in specific reductions of target mRNAs. The results of a microarray study show that lower amount of fluctuating genes are generated in the presence of 2 b or 4 b , relative to that from exogenous delivery of let7a‐1. Because peptides 2 b and 4 b promote enhanced formation of mature let7a‐1 only at the endogenous miRNA level, this specifically controls genes related to let7a‐1.