The structure and role of lactone intermediates in linkage-specific sialic acid derivatization reactions

Sialic acids occur ubiquitously throughout vertebrate glycomes and often endcap glycans in either alpha 2,3- or alpha 2,6-linkage with diverse biological roles. Linkage-specific sialic acid characterization is increasingly performed by mass spectrometry, aided by differential sialic acid derivatization to discriminate between linkage isomers. Typically, during the first step of such derivatization reactions, in the presence of a carboxyl group activator and a catalyst, alpha 2,3-linked sialic acids condense with the subterminal monosaccharides to form lactones, while alpha 2,6-linked sialic acids form amide or ester derivatives. In a second step, the lactones are converted into amide derivatives. Notably, the structure and role of the lactone intermediates in the reported reactions remained ambiguous, leaving it unclear to which extent the amidation of alpha 2,3-linked sialic acids depended on direct aminolysis of the lactone, rather than lactone hydrolysis and subsequent amidation. In this report, we used mass spectrometry to unravel the role of the lactone intermediate in the amidation of alpha 2,3-linked sialic acids by applying controlled reaction conditions on simple and complex glycan standards. The results unambiguously show that in common sialic acid derivatization protocols prior lactone formation is a prerequisite for the efficient, linkage-specific amidation of alpha 2,3-linked sialic acids, which proceeds predominantly via direct aminolysis. Furthermore, nuclear magnetic resonance spectroscopy confirmed that exclusively the C2 lactone intermediate is formed on a sialyllactose standard. These insights allow a more rationalized method development for linkage-specific sialic derivatization in the future.

Automated summary

Sialic acids are widely distributed throughout vertebrate glycomes and serve diverse biological roles by capping glycans at the terminal end of sugar chains. Mass spectrometry is increasingly used for linkage-specific sialic acid characterization, with the formation of lactones playing a crucial role in the efficient amidation of alpha 2,3-linked sialic acids.