Substrate recognition of the membrane-associated sialidase NEU3 requires a hydrophobic aglycone.

The human neuraminidases (NEU) consist of a family of four isoforms (NEU1-NEU4). Members of this enzyme family are proposed to have important roles in health and disease through regulation of the composition of cellular sialosides. The NEU3 isoform is a membrane-associated enzyme that cleaves glycolipid substrates. However, few reports have examined the substrate specificity of the enzyme for non-natural substrates. We report here a series of 11 synthetic trisaccharides that feature modifications of the aglycone or the Neu5Ac residue of an octyl ß-sialyllactoside. The time course of substrate cleavage by NEU3 was monitored using an electrospray ionization mass spectrometry assay to obtain relative rates (k(rel)). We observed that NEU3 substrate activity was directly dependent upon the hydrophobicity of the aglycone but had no apparent requirement for features of the ceramide headgroup. We also observed that trisaccharides with incorporated azide groups in the Neu5Ac residue at either C9 or the N5-Ac position were substrates, and in the case of the N5-azidoacetyl derivative, the activity was superior to that of GM3. However, the incorporation of larger aryl groups was tolerated only at C9, but not at N5-Ac. We propose a two-site model for enzyme recognition, requiring interaction at both the Neu5Ac residue and the hydrophobic aglycone.

A modular synthesis of alkynyl-phosphocholine headgroups for labeling sphingomyelin and phosphatidylcholine.

A general route to phospho- and sphingolipids that incorporate an alkyne in the phosphocholine headgroup is described. The strategy preserves the ammonium functionality of the phosphocholine and can be easily modified to introduce desired functional groups at the N-acyl chain. The targets accessible with this strategy provide a bioorthogonal handle for postsynthetic introduction of fluorophores or other labeling agents with aqueous phase chemistry. We report the synthesis of sphingomyelin derivatives that incorporate a fluorophore and an alkyne. The modified sphingolipids retain activity as substrates for sphingomyelinase, making these compounds viable probes of enzymatic activity. Importantly, the strategy allows modification of the lipid across the phosphodiester, making the alkyne a potential probe of sphingomyelinase activity.