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2D NMR spectroscopy and J coupling constant analysis are applied to resolve the structure of two photoproducts of thymidylyl-(3′→5′)-thymidine. These products are cyclobutane type thymine dimers possessing the cis-syn (the predominant one) and trans-syn geometry. The cis-syn is formed in an ANTI-ANTI conformation about the N-glycosyl linkages and resembles the normal base-stacked configuration. The glycosidic conformation in solution of the 5′ terminal fragment differs from the crystal in which the less common SYN conformation is observed. In this isomer only the sugar pucker of the 3′ terminal fragment is changed substantially with respect to the dinucleotide. The trans-syn isomer is formed in a SYN-ANTI glycosidic conformation. In this isomer the sugar puckers of both deoxyribose rings are affected and a preference for a pure 2′-endo conformation is observed. [ABSTRACT FROM AUTHOR]

A ring-closing metathesis (RCM) - peptide coupling - ruthenium-catalyzed azide alkyne cycloaddition (RuAAC) strategy was developed to synthesize a tricyclic hexapeptide in which the side chain to side chain connectivity pattern resulted in a mimic with a topology that effectively mimics the bioactivity of vancomycin as a potent binder of the bacterial cell wall D-Ala-D-Ala dipeptide sequence and more importantly being an effective inhibitor of bacterial growth., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C 55 -P). In this study we describe the design and synthesis of new C 55 -P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C 55 -P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C 55 -P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

For canonical asparagine glycosylation, the primary amino acid sequence that directs glycosylation at specific asparagine residues is well-established. Here we reveal that a recently discovered bacterial enzyme EarP, that transfers rhamnose to a specific arginine residue in its acceptor protein EF-P, specifically recognizes a β-hairpin loop. Notably, while the in vitro rhamnosyltransferase activity of EarP is abolished when presented with linear substrate peptide sequences derived from EF-P, the enzyme readily glycosylates the same sequence in a cyclized β-hairpin mimic. Additional studies with other substrate-mimicking cyclic peptides revealed that EarP activity is sensitive to the method used to induce cyclization and in some cases is tolerant to amino acid sequence variation. Using detailed NMR approaches, we established that the active peptide substrates all share some degree of β-hairpin formation, and therefore conclude that the β-hairpin epitope is the major determinant of arginine-rhamnosylation by EarP. Our findings add a novel recognition motif to the existing knowledge on substrate specificity of protein glycosylation, and are expected to guide future identifications of rhamnosylation sites in other protein substrates., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

There is a constant need for new therapeutic compounds. Fungi have proven to be an excellent, but underexplored source for biologically active compounds with therapeutic potential. Here, we combine mycology, embryology and chemistry by testing secondary metabolites from more than 10,000 species of fungi for biological activity using developing zebrafish (Danio rerio) embryos. Zebrafish development is an excellent model for high-throughput screening. Development is rapid, multiple cell types are assessed simultaneously and embryos are available in high numbers. We found that 1,526 fungal strains produced secondary metabolites with biological activity in the zebrafish bioassay. The active compounds from 39 selected fungi were purified by liquid-liquid extraction and preparative HPLC. 34 compounds were identified by a combination of chemical analyses, including LCMS, UV-Vis spectroscopy/ spectrophotometry, high resolution mass spectrometry and NMR. Our results demonstrate that fungi express a wide variety of biologically active compounds, consisting of both known therapeutic compounds as well as relatively unexplored compounds. Understanding their biological activity in zebrafish may provide insight into underlying biological processes as well as mode of action. Together, this information may provide the first step towards lead compound development for therapeutic drug development.

Laspartomycin C is a lipopeptide antibiotic with activity against a range of Gram-positive bacteria including drug-resistant pathogens. We report the first total synthesis of laspartomycin C as well as a series of structural variants. Laspartomycin C was found to specifically bind undecaprenyl phosphate (C55-P) and inhibit formation of the bacterial cell wall precursor lipid II. While several clinically used antibiotics target the lipid II pathway, there are no approved drugs that act on its C55-P precursor.

A new divalent highly potent inhibitor of the Pseudomonas aeruginosa lectin and virulence factor LecA was prepared. It contains two thiourea linkages which were found to be in the Z,Z isomeric form. This brings the spacer into an elongated conformation required to bridge the two binding sites, which results in the chelating binding mode responsible for the high potency.

The bacterial adhesion lectin LecA is an attractive target for interference with the infectivity of its producer P. aeruginosa. Divalent ligands with two terminal galactoside moieties connected by an alternating glucose-triazole spacer were previously shown to be very potent inhibitors. In this study, we chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the LecA inhibitory potency of the molecules. Based on the binding mode, modifications were made to the spacer to enable additional spacer-protein interactions. The introduction of positively charged, negatively charged, and also lipophilic functional groups was successful. The compounds were good LecA ligands, but no improved binding was seen, even though altered thermodynamic parameters were observed by isothermal titration calorimetry (ITC).

The calcium-dependent lipopeptide antibiotics represent a promising new class of antimicrobials for use in combating drug-resistant bacteria. At present, daptomycin is the only such lipopeptide used clinically and displays potent antimicrobial activity against a number of pathogenic Gram-positive bacteria. Given the increasing need for new antibiotics, practical synthetic access to unnatural analogues of daptomycin and related antimicrobial lipopeptides is of value. We here report an efficient synthetic route combining solid- and solution-phase techniques that allows for the rapid preparation of daptomycin analogues. Using this approach, four such analogues, including two enantiomeric variants, were synthesized and their antimicrobial activities and hydrolytic stabilities evaluated.