Rapid Formation of Intramolecular Disulfide Bridges using Light: An Efficient Method to Control the Conformation and Function of Bioactive Peptides.

Disulfide bonds are widely found in natural peptides and play a pivotal role in stabilizing their secondary structures, which are highly associated with their biological functions. Herein, we introduce a light-mediated strategy to effectively control the formation of disulfides. Our strategy is based on 2-nitroveratryl (oNv), a widely used photolabile motif, which serves both as a photocaging group and an oxidant (after photolysis). We demonstrated that irradiation of oNv-caged thiols with UV light could release free thiols that are rapidly oxidized by locally released byproduct nitrosoarene, leading to a "break-to-bond" fashion. This strategy is highlighted by the in situ restoration of the antimicrobial peptide tachyplesin I (TPI) from its external disulfide-caged analogue TPI-1. TPI-1 exhibits a distorted structure and a diminished function. However, upon irradiation, the ß-hairpin structure and membrane activity of TPI were largely restored via rapid intramolecular disulfide formation. Our study proposes a powerful method to regulate the conformation and function of peptides in a spatiotemporal manner, which has significant potential for the design of disulfide-centered light-responsive systems.

Diversity in the Synthesis of Functionalized Cyclohexene Oxide Derivatives by a Cycloaddition-Fragmentation Sequence from Benzene Oxide.

A cycloaddition-fragmentation sequence from benzene oxide and a nitroso- or azo-dienophile was investigated as a tool for access to highly substituted cyclohexene oxide derivatives. Alkyl lithium-promoted fragmentation of the cycloadducts led to the cyclic derivatives after 1,4- or 1,2-addition of a second equivalent of the lithium reagent. New fragmentation processes were observed when using non-nucleophilic bases of highly hindered alkyl lithium reagents. All reactions proceeded with complete stereocontrol.

Using the Noncanonical Metallo-Amino Acid [Cu(II)(2,2'-Bipyridin-5-yl)]-alanine to Study the Structures of Proteins.

Genetic code expansion allows modification of the physical and chemical properties of proteins by the site-directed insertion of noncanonical amino acids. Here we exploit this technology for measuring nanometer-scale distances in proteins. (2,2'-Bipyridin-5-yl)alanine was incorporated into the green fluorescent protein (GFP) and used as an anchoring point for Cu(II) to create a spin-label. The incorporation of (2,2'-bipyridin-5-yl)alanine directly into the protein resulted in a high-affinity binding site for Cu(II) capable of outcompeting other binding positions in the protein. The resulting Cu(II)-spin label is very compact and not larger than a conventional amino acid. By using 94 GHz electron paramagnetic resonance (EPR) pulse dipolar spectroscopy we have been able to determine accurately the distance between two such spin-labels. Our measurements revealed that GFP dimers can adopt different quaternary conformations. The combination of spin-labeling using a paramagnetic nonconventional amino acid with high-frequency EPR techniques resulted in a sensitive method for studying the structures of proteins.

Customized Reversible Stapling for Selective Delivery of Bioactive Peptides.

We have developed a new concept for reversible peptide stapling that involves macrocyclization between two amino groups and decyclization promoted via dual 1,4-elimination. Depending on the trigger moiety, this strategy could be employed to selectively deliver peptides to either intracellular or extracellular targets. As a proof of concept, a peptide inhibitor targeting a lysine-specific demethylase 1 (LSD1) was temporarily cyclized to enhance its stability and ability to cross the cell membrane. Once inside the cells, the biologically active linear peptide was released under reducing environment. Moreover, we have developed reversibly stapled peptides using antimicrobial peptides (RStAMPs) whose bioactive helical conformation can be temporarily destabilized by stapling the peptide backbone. The resulting helix-distorted RStAMPs are nontoxic and highly resistant to protease hydrolysis, while at the infection site, RStAMPs can be rapidly activated by the overproduced H2O2 through the dual 1,4-elimination. The latter restored the helical structure of the native peptide and its antimicrobial activity. This work illustrates a highly valuable macrocyclization strategy for the peptide community and should greatly benefit the field of peptide delivery.

Polymyxin B-inspired non-hemolytic tyrocidine A analogues with significantly enhanced activity against gram-negative bacteria: How cationicity impacts cell specificity and antibacterial mechanism.

Naturally occurring cyclic antimicrobial peptides (AMPs) such as tyrocidine A (Tyrc A) and gramicidin S (GS) are appealing targets for the development of novel antibiotics. However, their therapeutic potentials are limited by undesired hemolytic activity and relatively poor activity against Gram-negative bacteria. Inspired by polycationic lipopeptide polymyxin B (PMB), the so called 'last-resort' antibiotic for the treatment of infections caused by multidrug-resistant Gram-negative bacteria, we synthesized and biologically evaluated a series of polycationic analogues derived from Tyrc A. We were able to obtain peptide 8 that possesses 5 positive charges exhibiting potent activities against both Gram-negative and Gram-positive bacteria along with totally diminished hemolytic activity. Intriguingly, antibacterial mechanism studies revealed that, rather than the 'pore forming' model that possessed by Tyrc A, peptide 8 likely diffuses membrane in a 'detergent-like' manner. Furthermore, when treating mice with peritonitis-sepsis, peptide 8 showed excellent antibacterial and anti-inflammatory activities in vivo.

Gramicidin-S-Inspired Cyclopeptidomimetics as Potent Membrane-Active Bactericidal Agents with Therapeutic Potential.

Antimicrobial peptides (AMPs) are promising antibacterial agents often hindered by their undesired hemolytic activity. Inspired by gramicidin S (GS), a well-known cyclodecapeptide, we synthesized a panel of antibacterial cyclopeptidomimetics using ß,γ-diamino acids (ß,γ-DiAAs). We observed that peptidomimetic CP-2 displays a bactericidal activity similar to that of GS while possessing lower side-effects. Moreover, extensive studies revealed that CP-2 likely kills bacteria through membrane disruption. Altogether, CP-2 is a promising membrane-active antibiotic with therapeutic potential.

Development of Therapeutic Gramicidin S Analogues Bearing Plastic ß,γ-Diamino Acids.

Gramicidin S (GS), one of the most widely investigated antimicrobial peptides (AMPs), is known for its robust antimicrobial activity. However, it is restricted to topical application due to undesired hemolytic activity. With the aim of obtaining nontoxic GS analogues, we describe herein a molecular approach in which the native GS ß-turn region is replaced by synthetic ß,γ-diamino acids (ß,γ-DiAAs). Four ß,γ-DiAA diastereomers were employed to mimic the ß-turn structure to afford GS analogues GS3-6, which exhibit diminished hemolytic activity. A comparative structural study demonstrates that the (ßR,γS)-DiAA is the most-stable ß-turn mimic. To further improve the therapeutic index (e. g., high antibacterial activity and low hemolytic activity) and to extend the molecular diversity, GS5 and GS6 were used as structural scaffolds to introduce additional hydrophobic or hydrophilic groups. We show that GS6K, GS6F and GS display comparable antibacterial activity, and GS6K and GS6F have significantly decreased toxicity. Moreover, antibacterial mechanism studies suggest that GS6K kills bacteria mainly through the disruption of the membrane.

Recent Advances in the Exploration of Therapeutic Analogues of Gramicidin S, an Old but Still Potent Antimicrobial Peptide.

Gramicidin S (GS), one of the oldest commercially used peptide antibiotics, is known for its robust antibacterial activity against both Gram-positive and Gram-negative bacterial strains. Although it was discovered well over 70 years ago, its clinical potential was limited to topical applications because of its high hemolytic activity. To overcome this side effect, significant efforts have been invested in the chase for GS analogues with high therapeutic index (e.g., high antimicrobial activity and low hemolytic activity) in the past decades. In this Perspective, the structural properties and biological profiles (including the recently discovered activities) of representative GS analogues designed by different approaches are described and analyzed. We also present how the general structure-activity relationships were established and how they could help in the design of more efficient GS analogues.

Unexpected dimerization of a tripeptide comprising a ß,γ-diamino acid.

We have previously reported the synthesis of enantiopure ß,γ-diamino acids and relevant short α/γ-peptide containing such building blocks. Complete nuclear magnetic resonance (NMR) studies, together with molecular modeling, highlighted the ability of a ß,γ-diamino acid to induce various intramolecular turns. In this paper, we describe for the first time the formation of a dimeric structure constituted by α/γ/α-tripeptide and stabilized by intermolecular interactions. A structural model is proposed based on extensive NMR measurements.

Oxidative coupling of enolates using memory of chirality: an original enantioselective synthesis of quaternary α-amino acid derivatives.

We describe here the first enantioselective oxidative heterocoupling of enolates. Our strategy relies on the memory of chirality concept and allows the stereocontrolled formation of quaternary centres on α-amino acid derivatives with an enantiomeric excess of up to 94%.

ß,γ-diamino acids as building blocks for new analogues of Gramicidin S: Synthesis and biological activity.

We describe here the synthesis and biological activity study of a pair of diastereomeric analogues of Gramicidin S using ß,γ-diamino acids as ß-turn mimic. The synthesis of the orthogonally protected ß,γ-diamino acids was achieved in 6 steps starting from d-alanine. The analogues were then synthesized in solution phase and on solid phase. Biological activity tests showed that, compared with Gramicidin S, both analogues exerted diminished hemolytic activity while they retained interesting antibacterial activity.

Base-Mediated Fragmentation of Bicyclic Dihydro-3,6-oxazines: Transformation of Nitroso Diels-Alder Cycloadducts.

Nitroso Diels-Alder cycloadditions of benzene oxide with various acyl-nitroso derivatives are described. Treatment of these cycloadducts with methyllithium results in a fast fragmentation reaction, leading to highly functionalized cyclic amino alcohols. The mechanism of the reaction and the role of the epoxide in the fragmentation process are investigated. The reaction proceeds via the formation of an unsaturated imine, which tautomerizes to an enamine if no neighboring epoxide is present.

Interfering peptides targeting protein-protein interactions: the next generation of drugs?

Protein-protein interactions (PPIs) are well recognized as promising therapeutic targets. Consequently, interfering peptides (IPs) - natural or synthetic peptides capable of interfering with PPIs - are receiving increasing attention. Given their physicochemical characteristics, IPs seem better suited than small molecules to interfere with the large surfaces implicated in PPIs. Progress on peptide administration, stability, biodelivery and safety are also encouraging the interest in peptide drug development. The concept of IPs has been validated for several PPIs, generating great expectations for their therapeutic potential. Here, we describe approaches and methods useful for IPs identification and in silico, physicochemical and biological-based strategies for their design and optimization. Selected promising in-vivo-validated examples are described and advantages, limitations and potential of IPs as therapeutic tools are discussed.

Frozen Chirality of Tertiary Aromatic Amides: Access to Enantioenriched Tertiary α-Amino Acid or Amino Alcohol without Chiral Reagent.

One of the fundamental and intriguing aspects of life is the homochirality of the essential molecules. In this field, the absolute asymmetric synthesis of α-amino acids is a major challenge. Herein, we report access, by chemical means, to tertiary α-amino acid derivatives in up to 96 % ee without using any chiral reagent. In our strategy, the dynamic axial chirality of tertiary aromatic amides is frozen in a crystal and is responsible for the stereoselectivity of the subsequent steps. Furthermore, we could control the configuration of the final product by manually sorting and selecting the initial crystals. Based on vibrational circular dichroism studies, we could rationalize the observed stereoselectivity.

Efficient synthesis of both diastereomers of ß,γ-diamino acids from phenylalanine and tryptophan.

We synthesized in a few steps both diastereomers of orthogonally protected ß,γ-diamino acids starting from L-phenylalanine or L-tryptophan. These final compounds are interesting building blocks for peptide synthesis and foldamer chemistry. The key step is a Blaise reaction performed under ultrasound conditions.

Β,γ-diamino acid: an original building block for hybrid α/γ-peptide synthesis with extra hydrogen bond donating group.

Using a ß,γ-diamino acid, several small hybrid α/γ peptides have been synthesized and their conformations investigated through extensive NMR studies and molecular dynamics. A tripeptide and a tetrapeptide have thus shown several hydrogen bonds in solution, including a 13-membered ring involving the ß-nitrogen.

Substrate control in enantioselective and diastereoselective aldol reaction by memory of chirality: a rapid access to enantiopure ß-hydroxy quaternary α-amino acids.

An asymmetric aldol reaction by memory of chirality is reported with a substrate control of stereoselectivity by aldehyde and rationalized. Starting from l-alanine, several diastereopure and enantioenriched ß-hydroxy quaternary α-amino acids have been obtained in three steps. The initial chirality of l-alanine is memorized through the dynamic axial chirality of tertiary aromatic amide.

Original ß,γ-diamino acid as an inducer of a γ-turn mimic in short peptides.

Original αγα tripeptides containing one ß,γ-diamino acid have been synthesized and their conformation determined by extensive NMR and molecular dynamic studies. These studies revealed the presence of a C(9) hydrogen bonded turn around the ß,γ-diamino acid which was stabilized by bulky side chains of the preceding residue. This turn can be considered as a mimic of the well-known γ-turn.