A Unified Framework for the Incorporation of Bioorthogonal Compound Exposure Probes within Biological Compartments

Compartmentalization is a crucial facet of many biological systems, and key aspects of cellular processes rely on spatial segregation within the cell. While many drug targets reside in specific intracellular compartments, the tools available for assessing compound exposure are generally limited to whole-cell measurements. To address this gap, we recently developed a bioorthogonal chemistry-based method to assess compartment-specific compound exposure and demonstrated its use in Gram-negative bacteria. To expand the applicability of this approach, we report here novel bioorthogonal probe modalities which enable diverse probe incorporation strategies. The probes we developed utilize a cleavable thiocarbamate linker to connect localizing elements such as metabolic substrates to a cyclooctyne moiety which enables the detection of azide-containing molecules. Adducts between the probe and azide-bearing compounds can be recovered and affinity purified after exposure experiments, thus facilitating the mass-spectrometry based analysis used to assess compound exposure. The bioorthogonal system reported here thus provides a valuable new tool for interrogating compartment-specific compound exposure in a variety of biological contexts while retaining a simple and unified sample preparation and analysis workflow.