摘要：SummaryN-Acetylglucosamine (GlcNAc) is an essential monosaccharide required in almost all organisms. Fluorescent labeling of the peptidoglycan (PG) onN-acetylglucosamine has been poorly explored. Here, we report on the labeling of the PG with a bioorthogonal handle on the GlcNAc. We developed a facile one-step synthesis of uridine diphosphateN-azidoacetylglucosamine (UDP-GlcNAz) using the glycosyltransferase OleD, followed byin vitroincorporation of GlcNAz into the peptidoglycan precursor Lipid II and fluorescent labeling of the azido group via click chemistry. In a PG synthesis assay, fluorescent GlcNAz-labeled Lipid II was incorporated into peptidoglycan by the DD-transpeptidase activity of bifunctional class A penicillin-binding proteins. We further demonstrate the incorporation of GlcNAz into the PG layer of OleD-expressed bacteria by feeding with 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP). Hence, our labeling method using the heterologous expression of OleD is useful to study PG synthesis and possibly other biological processes involving GlcNAc metabolismin vivo.Graphical abstractDisplay OmittedHighlights•Peptidoglycan consists of N-acetylglucosamine, N-acetylmuramic acid, and amino acids•We developed a one-step synthesis of azide-labeled UDP-N-acetylglucosamine•In vivo generated azide-labeled UDP-N-acetylglucosamine gets incorporated into peptidoglycan•Bacteria were fluorescently labeled on N-acetylglucosamine of peptidoglycanChemistry; Biosynthesis; Biomolecules; Glycobiology
