Fluorescence‐Quenched Mycoloyl‐Arabinofuranoside Analogs: Fluorogenic Probes for Mycobacterial Outer Membrane‐Degrading Enzymes

Mycobacteria, including the tuberculosis pathogen Mycobacterium tuberculosis, are enclosed by a highly complex cell envelope with an outer membrane, or mycomembrane, which provides extraordinary protection from antibiotics and other stresses. The inner leaflet of the mycomembrane consists of arabinogalactan-linked mycolate (AGM), which is an enormous glycoconjugate comprising mycolic acids esterified to terminal D-arabinofuranosyl residues of an underlying arabinogalactan-peptidoglycan complex, also referred to as the mycoloyl-arabinogalactan-peptidoglycan (mAGP) complex. Whereas AGM biosynthesis is comparatively well characterized, less is known about AGM degradation by endogenous or exogenous factors. To facilitate studies on AGM breakdown by hydrolytic enzymes, here we synthesized fluorescence resonance energy transfer (FRET)-based mono- and disaccharide probes that mimic fragments of AGM and are designed to fluoresce upon cleavage. We devised a synthetic route that established the glycolipid core with the desired regio- and stereochemistry and allowed late-stage selective functionalization of the core with a FRET pair. Our data show that the intact FRET-AGM probes exist in a fluorescence-quenched state, but when exposed to lysin B (LysB), an AGM-degrading mycobacteriophage hydrolase with therapeutic relevance, the probes were activated through lipid ester hydrolysis, thereby generating fluorescence signal. FRET-AGM probes were activated by known mycomembrane glycolipid hydrolases, but not by several other types of hydrolases, demonstrating specificity. FRET-AGM probes may be useful in the future for identifying novel AGM hydrolases and quantitatively monitoring the activity of AGM hydrolases, which could provide insights into mycomembrane degradative processes and aid in tuberculosis therapeutic development.