Bioorthogonal Heptose Phosphate Probes for Chemical Profiling of Bacterial Glycans

Heptose sugars are essential components of Gram-negative bacterial lipopolysaccharide (LPS) and play emerging roles in innate immune activation. To enable functional studies of heptose biology, we developed a bioorthogonal metabolic probe, 7-azido-heptose-1-phosphate (7AzHep1P), which incorporates into the LPS inner core and generates a truncated yet detectable LPS structure. Using click chemistry, we visualized LPS remodeling in live bacteria and confirmed reporter incorporation across various LPS mutants. Notably, 7AzHep1P-labeled LPS preserved immunogenic activity, triggering cytokine release and macrophage activation. We further applied this probe for chemical glycoproteomics, identifying 133 O-heptosylated glycopeptides from 97 bacterial proteins. Beyond canonical serine and threonine sites, heptosylation was also detected on other residues, revealing unappreciated substrate diversity. Together, this strategy enables nongenetic, in situ profiling of heptose-linked biomolecules, offering a valuable tool for dissecting LPS biosynthesis, immune modulation, and glycosylation in bacterial systems.