| Sialic acids are often the platform for orthogonal chemical functionalization on cell surfaces. This is accomplished via techniques such as metabolic glycoengineering, yet the ability of sialidases to remodel unnatural sialic acids incorporated within the glycome is unknown. Their substrate specificities toward unnatural sugars could be a significant factor in the efficacy of glycocalyx functionalization efforts. In addition, orthogonal modifications made to the glycome may conceivably impede or activate sialidase function in cells, depending on their ligand permissivity, thus affecting cellular behaviors such as protein recruitment and signaling cascades. Finally, interest in designing sialidase inhibitors specific for each isoform in humans, or that act preferentially on viral neuraminidases and not on human ones, is increasing and requires a better understanding of the structural determinants of activity in these enzymes. New probes are needed to understand sialidase activity in disease states, their role in glycoengineered sialic acid presentation, and their substrate specificities.;To address these needs, we reported the synthesis and utility of a panel of alpha-ketosides consisting of C5-modified sialic acids linked to a 4-methylumbelliferyl (4MU) aglycone as probes of sialidase activity in the human leukemic cell lines HL-60 and Jurkat. These sialic acids bore fluorinated, bulky and hydrophobic side chains to explore the binding pocket topologies of cell-surface sialidase isoforms. We found cell-surface sialidases in these cell lines capable of hydrolyzing fluorogenic glycosides, exhibiting a wide range of enzyme activity that was side chain dependent. These data gave valuable insight into the flexibility of the yet-uncharacterized binding pockets of these enzymes. We also assessed SAR between cell-surface sialidases and the bioorthogonal side chains most commonly employed in metabolic glycoengineering efforts, including ketones and alkynes. In order to validate our approach in the cellular context, corresponding N-acyl mannosamine derivatives were synthesized for incorporation and native presentation on cell-surface glycans in order to assess sialidase activity. Finally, virtual ligands representing our glycosides were built using known carbohydrate conformations in efforts to further understand the structural determinants of the sialidase activities observed.;Our work supports our hypothesis that human sialidase activity is indeed a significant factor to be considered in metabolic glycoengineering efforts and highlights the importance of further study into these widely influential enzymes. |
