Dual Strain-Promoted Alkyne–Nitrone Cycloadditions for Simultaneous Labeling of Bacterial Peptidoglycans

Bioorthogonal chemistry has been applied to study a multitude of biological processes in complex environments through incorporation and detection of small functional groups. However, few reactions are known to be compatible with each other to allow for studies of more than one biomolecule simultaneously. Here we describe a dual labeling method wherein two stereoelectronically contrasting nitrone tags are incorporated into bacteria peptidoglycan and detected via strain-promoted alkyne-nitrone cycloaddition (SPANC) simultaneously. Furthermore, we show orthogonality with the azide functionality broadening the potential for simultaneous biomolecular target labeling in less accommodating metabolic pathways. We also demonstrate the simultaneous labeling of two different food-associated bacteria, L. innocua (a model for the food-born pathogen L. monocytogenes) and L. lactis (a fermentation bacterium). The ability to monitor multiple processes and even multiple organisms concurrently through nitrone/nitrone or nitrone/azide incorporation strengthens the current bioorthogonal toolbox and gives rise to robust duplex labeling of organisms to potentiate the studies of rapid biological phenomena.