Biosynthetic incorporation for visualizing bacterial glycans

Cell-surface glycans are central to many biological processes, yet methods for their site-selective modification are limited. Strategies for interrogating the structure and function of proteins have been enabled by chemoselective reactions of sidechain functionality for covalent modification, capture, or imaging. However, unlike protein sidechains, glycan building blocks lack distinguishing reactivity. Moreover, glycans are not primary gene products, so encoding glycan variants through genetic manipulation is challenging. Reactive functional groups can be introduced into glycans through metabolic engineering, which involves the generation of modified nucleotide-sugar building blocks. Lipid-linked building blocks, which are also used in glycan biosynthesis, have the advantage that they can be delivered directly to glycosyltransferases to function as surrogate substrates. This process, termed "biosynthetic incorporation," takes advantage of the properties of bacterial glycosyltransferase: they are selective for the products they generate yet promiscuous in their donor preferences. We describe how this strategy can be implemented to label arabinofuranose-containing glycans on the surface of mycobacterial cells. We anticipate that this platform can be expanded to develop chemoselective labeling agents for other important bacterial monosaccharides.