Synthesis of DNA-Encoded Macrocyclic Peptides via Nitrile-Aminothiol Click Reaction.

DNA-encoded library (DEL) technology holds exciting potential for discovering novel therapeutic macrocyclic peptides (MPs). Herein, we describe the development of a DEL-compatible peptide macrocyclization method that proceeds via intramolecular click-condensation between 3-(2-cyano-4-pyridyl)-l-alanine (Cpa) and an N-terminal cysteine. Cyclization takes place spontaneously in a buffered aqueous solution and affords the cyclized products in excellent yields. The reaction exhibits a broad substrate scope and can be employed to generate MPs of variable ring size and amino acid composition.

Oxadiazole-Containing Macrocyclic Peptides Potentiate Azole Activity against Pathogenic Candida Species.

Opportunistic pathogens of the genus Candida reign as the leading cause of mycotic disease and are associated with mortality rates greater than 40%, even with antifungal intervention. This is in part due to the limited arsenal of antifungals available to treat systemic fungal infections. Azoles have been the most widely deployed class of antifungal drug for decades and function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. However, their utility is compromised by their fungistatic nature, which favors the development of resistance. Combination therapy has the potential to confer enhanced efficacy as well as mitigate the evolution of resistance. Previously, we described the generation of structurally diverse macrocyclic peptides with a 1,3,4-oxadiazole and an endocyclic amine grafted within the peptide backbone. Importantly, this noncanonical backbone displayed high membrane permeability, an important attribute for compounds that need to permeate across the fungal cell wall and membrane in order to reach their intracellular target. Here, we explored the bioactivity of this novel chemical scaffold on its own and in combination with the azole fluconazole. Although few of the oxadiazole-containing macrocyclic peptides displayed activity against Candida albicans on their own, many increased the efficacy of fluconazole, resulting in a synergistic combination that was independent of efflux inhibition. Interestingly, these molecules also enhanced azole activity against several non-albicans Candida species, including the azole-resistant pathogens Candida glabrata and Candida auris This work characterizes a novel chemical scaffold that possesses azole-potentiating activity against clinically important Candida species.IMPORTANCE Fungal infections, such as those caused by pathogenic Candida species, pose a serious threat to human health. Treating these infections relies heavily on the use of azole antifungals; however, resistance to these drugs develops readily, demanding novel therapeutic strategies. This study characterized the antifungal activity of a series of molecules that possess unique chemical attributes and the ability to traverse cellular membranes. We observed that many of the compounds increased the activity of the azole fluconazole against Candida albicans, without blocking the action of drug efflux pumps. These molecules also increased the efficacy of azoles against other Candida species, including the emerging azole-resistant pathogen Candida auris Thus, we describe a novel chemical scaffold with broad-spectrum bioactivity against clinically important fungal pathogens.

Development of Endocyclic Control Elements for Peptide Macrocycles.

Synthetic methods that provide control over macrocycle conformation represent valuable tools for the discovery of bioactive molecules. Incorporation of heterocycles into cyclic peptides may offer a way to stabilize their solution conformations. Herein, we used N-(isocyanimino)triphenylphosphorane (Pinc) to install an oxadiazole ring and an endocyclic amine into peptide macrocycles. To elucidate the conformational effect of this constellation of functionalities, we performed synthesis, variable temperature NMR analysis, and NOE-based molecular dynamics simulation of a range of macrocycles in DMSO. As part of this study, we conducted experiments to compare macrocycle conformation in aqueous and DMSO solutions. The obtained solution structures suggest that the reduced amide bond/heterocycle (RAH) motif can stabilize macrocycle conformations in both water and DMSO, which addresses an enduring challenge in molecular design. The conformational effect of the RAH was used in an effort to mimic the biologically relevant secondary structure of MAdCAM-1. This resulted in the discovery of a novel α4ß7 integrin antagonist.

Oxadiazole grafts in peptide macrocycles.

Synthetic methods that provide control over macrocycle conformation and, at the same time, mitigate the polarity of peptide bonds represent valuable tools for the discovery of new bioactive molecules. Here, we report a macrocyclization reaction between a linear peptide, an aldehyde and (N-isocyanimino)triphenylphosphorane. This process generates head-to-tail cyclic peptidomimetics in a single step. This method is tolerant to variation in the peptide and aldehyde components and has been applied for the synthesis of 15-, 18-, 21- and 24-membered rings. The resulting peptide macrocycles feature a 1,3,4-oxadiazole and a tertiary amine in their scaffolds. This non-canonical backbone region acts as an endocyclic control element that promotes and stabilizes a unique intramolecular hydrogen-bond network and can lead to macrocycles with conformationally rigid turn structures. Oxadiazole-containing macrocycles can also display a high passive membrane permeability, an important property for the development of bioavailable peptide-based therapeutics.

Side-chain-to-tail cyclization of ribosomally derived peptides promoted by aryl and alkyl amino-functionalized unnatural amino acids.

A strategy for the production of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Two genetically encodable unnatural amino acids, bearing either an aryl or alkyl amino group, were investigated for their efficiency toward promoting the formation of medium to large-sized peptide macrocycles via intein-mediated side-chain-to-C-terminus cyclization. While only partial cyclization was observed with precursor proteins containing para-amino-phenylalanine, efficient peptide macrocyclization could be achieved using O-2-aminoethyl-tyrosine as the reactive moiety. Conveniently, the latter was generated upon quantitative, post-translational reduction of the azido-containing counterpart, O-2-azidoethyl-tyrosine, directly in E. coli cells. This methodology could be successfully applied for the production of a 12 mer cyclic peptide with enhanced binding affinity for the model target protein streptavidin as compared to the acyclic counterpart (KD: 5.1 µM vs. 22.4 µM), thus demonstrating its utility toward the creation and investigation of novel, functional macrocyclic peptides.

Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Aminothiol Unnatural Amino Acids.

A versatile method for orchestrating the formation of side chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner in vitro or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low-micromolar streptavidin binder (KD = 1.1 µM) from a library of cyclic peptides produced in Escherichia coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles.

Designer macrocyclic organo-peptide hybrids inhibit the interaction between p53 and HDM2/X by accommodating a functional α-helix.

We report the design of side-chain-to-tail linked organo-peptide hybrids incorporating an α-helical protein-binding motif. Using this strategy, macrocyclic inhibitors of the p53:HDM2 interaction displaying dual specificity against the HDMX homolog as well as increased proteolytic stability could be obtained.

Use of the visual range of detection to estimate effective sweep width for land search and rescue based on 10 detection experiments in North America.

OBJECTIVE: Standard-of-practice search management requires that the probability of detection (POD) be determined for each search resource after a task. To calculate the POD, a detection index (W) is obtained by field experiments. Because of the complexities of the land environment, search planners need a way to estimate the value of W without conducting formal experiments. We demonstrate a robust empirical correlation between detection range (Rd) and W, and argue that Rd may reliably be used as a quick field estimate for W. METHODS: We obtained the average maximum detection range (AMDR), Rd, and W values from 10 detection experiments conducted throughout North America. We measured the correlation between Rd and W, and tested whether the apparent relationship between W and Rd was statistically significant. RESULTS: On average we found W ≈ 1.645 × Rd with a strong correlation (R(2) = .827). The high-visibility class had W ≈ 1.773 × Rd (also R(2) = .867), the medium-visibility class had W ≈ 1.556 × Rd (R(2) = .560), and the low-visibility had a correction factor of 1.135 (R(2) = .319) for Rd to W. Using analysis of variance and post hoc testing, only the high- and low-visibility classes were significantly different from each other (P < .01). We also found a high correlation between the AMDR and Rd (R(2) = .9974). CONCLUSIONS: Although additional experiments are required for the medium- and low-visibility search objects and in the dry-domain ecoregion, we suggest search planners use the following correction factors to convert field-measured Rd to an estimate of the effective sweep width (W): high-visibility W = 1.8 × Rd; medium-visibility W = 1.6 × Rd; and low-visibility W = 1.1 × Rd.

Design, synthesis, and diversification of ribosomally derived peptide macrocycles.

Ring topologies are widespread structural motifs among biologically active peptides found in nature. The recurrence of this motif is linked to the inherent advantages resulting from backbone cyclization, which include increased resistance against proteolytic degradation, improved cell permeability, and tighter and more specific interaction with the respective biomolecular target. Inspired by these natural product topologies, a number of groups have recently focused on developing methodologies that hinge upon the chemical elaboration of ribosomally derived polypeptides toward the synthesis and diversification of macrocyclic peptide structures. In this review, we highlight recent advances in this emerging new area and discuss the opportunities created by these methods toward the discovery of new functional entities.

Emerging strategies to access peptide macrocycles from genetically encoded polypeptides.

Macrocyclic peptides have emerged as attractive molecular scaffolds for the development of chemical probes and therapeutics. In this synopsis, we highlight contemporary strategies to access peptide macrocycles from ribosomally produced polypeptides. Challenges that have been tackled in this area involve orchestrating the desired macrocyclization process in the presence of unprotected polypeptide precursors and expanding the functional space encompassed by these molecules beyond that of canonical amino acid structures. Applications of these methodologies for the discovery of bioactive molecules are also discussed.

Macrocyclization of organo-peptide hybrids through a dual bio-orthogonal ligation: insights from structure-reactivity studies.

Macrocycles constitute an attractive structural class of molecules for targeting biomolecular interfaces with high affinity and specificity. Here, we report systematic studies aimed at exploring the scope and mechanism of a novel chemo-biosynthetic strategy for generating macrocyclic organo-peptide hybrids (MOrPHs) through a dual oxime-/intein-mediated ligation reaction between a recombinant precursor protein and bifunctional, oxyamino/1,3-amino-thiol compounds. An efficient synthetic route was developed to access structurally different synthetic precursors incorporating a 2-amino- mercaptomethyl-aryl (AMA) moiety previously found to be important for macrocyclization. With these compounds, the impact of the synthetic precursor scaffold and of designed mutations within the genetically encoded precursor peptide sequence on macrocyclization efficiency was investigated. Importantly, the desired MOrPHs were obtained as the only product from all the different synthetic precursors probed in this study and across peptide sequences comprising four to 15 amino acids. Systematic mutagenesis of the "i-1" site at the junction between the target peptide sequence and the intein moiety revealed that the majority of the 20 amino acids are compatible with MOrPH formation; this enables the identification of the most and the least favorable residues for this critical position. Furthermore, interesting trends with respect to the positional effect of conformationally constrained (Pro) and flexible (Gly) residues on the reactivity of randomized hexamer peptide sequences were observed. Finally, mechanistic investigations enabled the relative contributions of the two distinct pathways (side-chain→C-end ligation versus C-end→side-chain ligation) to the macrocyclization process to be dissected. Altogether, these studies demonstrate the versatility and robustness of the methodology to enable the synthesis and diversification of a new class of organo-peptide macrocycles and provide valuable structure-reactivity insights to inform the construction of macrocycle libraries through this chemo-biosynthetic strategy.

Diverse organo-peptide macrocycles via a fast and catalyst-free oxime/intein-mediated dual ligation.

Macrocyclic Organo-Peptide Hybrids (MOrPHs) can be prepared from genetically encoded polypeptides via a chemoselective and catalyst-free reaction between a trifunctional oxyamino/amino-thiol synthetic precursor and an intein-fusion protein incorporating a bioorthogonal keto group.

Rhodium-mediated enantioselective cyclopropanation of allenes.

Reaction of monosubstituted allenes with aryldiazoacetate esters under dirhodium tetracarboxylate catalysis led to alkylidene cyclopropane products in 80-90% ee. Monosubstituted alkyl- and arylallene substrates gave 60-75% yield under standard conditions, while yields for 1,1-disubstituted allenes were significantly lower. Cyclopropanation of 1-methyl-1-(trimethylsilyl)allene proceeded in higher yield than other 1,1-disubstituted substrates, suggesting rate enhancement mediated by a significant beta-silicon effect.