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Native chemical ligation enables the chemical synthesis of proteins. Previously, thiol-containing auxiliary groups have been used to extend the reaction scope beyond N-terminal cysteine residues. However, the N-benzyl-type auxiliaries used so far result in rather low reaction rates. Herein, a new N^α-auxiliary is presented. Consideration of a radical fragmentation for cleavage led to the design of a new auxiliary group which is selectively removed under mildly basic conditions (pH 8.5) in the presence of TCEP and morpholine. Most importantly and in contrast to previously described auxiliaries, the 2-mercapto-2-phenethyl auxiliary is not limited to Gly-containing sites and ligations succeed at sterically demanding junctions. The auxiliary is introduced in high yield by on-resin reductive amination with commercially available amino acid building blocks. The synthetic utility of the method is demonstrated by the synthesis of two antimicrobial proteins, DCD-1L and opistoporin-2.

A radical change of the auxiliary design enables extended native chemical peptide ligation at sterically demanding ligation junctions. The 2-mercapto-2-phenethyl group is small and flexible and avoids α-branching. The auxiliary is introduced by reductive amination on a solid support and removed under mildly basic conditions in a radical desulfurization triggered fragmentation reaction.

Resistance to glycopeptide antibiotics, the drugs of choice for life-threatening bacterial infections, is on the rise. In order to counter the threat of glycopeptide-resistant bacteria, we report development of a new class of semi-synthetic glycopeptide antibiotics, which not only target the bacterial membrane but also display enhanced inhibition of cell-wall biosynthesis through increased binding affinity to their target peptides. The combined effect of these two mechanisms resulted in improved in vitro activity of two to three orders of magnitude over vancomycin and no propensity to trigger drug resistance in bacteria. In murine model of kidney infection, the optimized compound was able to bring bacterial burden down by about 6 logs at 12 mg kg⁻¹ with no observed toxicity. The results furnished in this report emphasize the potential of this class of compounds as future antibiotics for drug-resistant Gram-positive infections.

Glycopeptide antibiotics: Newly developed glycopeptide antibiotics demonstrated high activity against multidrug-resistant bacteria and showed the ability to stall bacterial resistance development because of incorporation of a novel membrane-disrupting mechanism and enhanced inhibition of the cell-wall synthesis. The results emphasize that this multipronged approach could be used clinically to develop the next generation of glycopeptide antibiotics.

Bacteria remodel peptidoglycan structure in response to environmental changes. Many enzymes are involved in peptidoglycan metabolism; however, little is known about their responsiveness in a defined environment or the modes they assist bacteria to adapt to new niches. Here, we focused in peptidoglycan enzymes that intracellular bacterial pathogens use inside eukaryotic cells. We identified a peptidoglycan enzyme induced by Salmonella enterica serovar Typhimurium in fibroblasts and epithelial cells. This enzyme, which shows γ-D-glutamyl-meso-diaminopimelic acid D,L-endopeptidase activity, is also produced by the pathogen in media with limited nutrients and in resting conditions. The enzyme, termed EcgA for endopeptidase responding to cessation of growth’, is encoded in a S. Typhimurium genomic island absent in Escherichia coli. EcgA production is strictly dependent on the virulence regulator PhoP in extra- and intracellular environments. Consistent to this regulation, a mutant lacking EcgA is attenuated in the mouse typhoid model. These findings suggest that specialised peptidoglycan enzymes, such as EcgA, might facilitate Salmonella adaptation to the intracellular lifestyle. Moreover, they indicate that readjustment of peptidoglycan metabolism inside the eukaryotic cell is essential for host colonisation.

Many enzymes direct peptidoglycan metabolism but little it is known about their regulation. This study describes a novel peptidoglycan D,L-endopeptidase of Salmonella enterica that is up-regulated when the pathogen resides inside eukaryotic cells and whose expression is controlled positively by the virulence regulator PhoP. This enzyme, encoded in a genomic island, contributes to infection in vivo. Peptidoglycan remodelling taking place during colonization of the eukaryotic intracellular niche plays therefore an important role in Salmonella pathogenicity.

Bacteria possess complex and varying cell walls with many surface exposed proteins. Sortases are responsible for the covalent attachment of specific proteins to the peptidoglycan of the cell wall of Gram-positive bacteria. Sortase A of Staphylococcus aureus, which is seen as the archetypal sortase, has been shown to be essential for pathogenesis and has therefore received much attention as a potential target for novel therapeutics. Being widely present in Gram-positive bacteria, it is likely that other Gram-positive pathogens also require sortases for their pathogenesis. Sortases have also been shown to be of significant use in a range of industrial applications. We review current knowledge of the sortase family in terms of their structures, functions and mechanisms and summarize work towards their use as antibacterial targets and microbiological tools.

Sortases are responsible for the covalent attachment of proteins to the gram-positive cell wall. Staphylococcus aureus Sortase A has been shown to be required for pathogenesis and has therefore garnered considerable attention as a potential drug target. Here we review current structural, functional and mechanistic knowledge of sortases and efforts to target them therapeutically and exploit them industrially.

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Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine-reactive probes that target surface-exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site-specifically labeled product. Z₃₂BPA was synthesized by solid-phase peptide synthesis and further functionalized to give alkyne-Z₃₂BPA and azide-Z₃₂BPA for Cu^I-catalyzed cycloaddition, as well as DBCO-Z₃₂BPA for Cu-free strain-promoted cycloaddition. The Z₃₂BPA variants were conjugated to the human IgG1 antibody trastuzumab and site-specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2-expressing cells in immunofluorescence microscopy.

Two ways to click to the antibody: By equipping the IgG-binding Z domain with a photoactivatable probe, an antibody can be site-specifically, covalently labeled in the Fc region. Additionally, introducing bioorthogonal reactive groups into the Z domain provides a versatile labeling strategy.