A Metabolic‐Tag‐Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria**

The general lack of permeability small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane hypothesized be principal determinant access antibiotics their molecular targets. We describe a novel assay that combines metabolic tagging peptidoglycan, which sits directly beneath mycomembrane, click chemistry test molecules, and fluorescent labeling chase step, measure permeation molecules. showed workflow was robust compatible with high-throughput analysis in mycobacteria by testing panel azide-tagged trend similar across two types some notable exceptions. anticipate this platform will lay foundation medicinal efforts understand improve uptake both existing drugs newly-discovered compounds into mycobacteria.