Abstract
Sialic acids occur ubiquitously throughout vertebrate glycomes and often endcap glycans in either α2,3- or α2,6-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, α2,3-linked sialic acids condense with the subterminal monosaccharides to form lactones, while α2,6-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 α2,3-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 α2,3-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 α2,3-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.
Highlights
Protein glycosylation is a ubiquitous co- and post-translational modification, which has lately received considerable attention given its relevance in a multitude of biological processes [1, 2]
We present a set of experiments performed to unravel the role of the lactone intermediate in the amidation during linkage-specific sialic acid derivatization
We further present the structural characterization of the lactone intermediate formed with common linkage-specific sialic acid derivatization conditions
Summary
Protein glycosylation is a ubiquitous co- and post-translational modification, which has lately received considerable attention given its relevance in a multitude of biological processes [1, 2]. Glycosylation affects folding and solubility of glycoproteins, and changes in response to diverse environmental cues [3, 4]. Sialic acids are monosaccharides which are endcapping glycans, where they play important roles in either masking the glycoprotein from its surroundings or by mediating interaction with glycan-binding proteins [5]. In the presence of a carboxylic acid activator (such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; EDC [21]) and a catalyst (such as 1-hydroxybenzotriazole; HOBt [22]) α2,6-linked sialic acids react with added amines or alcohols to form amide or ester derivatives, respectively. Α2,3-linked sialic acids form lactones under the same conditions (Fig. 1) [14, 20]. Because of the limited stability of the lactones, a second reaction step is often included to convert them into amide products [15, 23]
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