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

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.

Egg yolk sialylglycopeptide: purification, isolation and characterization of N-glycans from minor glycopeptide species

“…In brief, for each α-2,6-linked sialic acid, a glycan will exhibit a mass shift of +28.03 Da whereas one α-2,3-linked sialic acid will produce a mass shift of −0.95 Da (Scheme S1†). 32…”

“…Organic & Biomolecular Chemistry sialic acid, a glycan will exhibit a mass shift of +28.03 Da whereas one α-2,3-linked sialic acid will produce a mass shift of −0.95 Da (Scheme S1 †). 32 The selectivity of the mass changes due to EEA was verified using glycan standards of known sialylation (Fig S2 †). ESI-MS/ MS fragmentation of α-2,6 linked sialic acid after EEA produced a fragment of 686.35 m/z whereas α-2,3 linked sialic acid produced a fragment of 656.33 which is consistent with ethyl esterification and amidation respectively 33 (Fig S5 †).…”

High-Throughput Glycomic Methods

“…To avoid this, a derivatization approach is applied (Figure 3) and dependent on the chemistry a distinction can be made between the differently linked sialic acids as a mass difference is introduced (esterification or dimethylamidation). 25…”

“…In the first step, α2,3-linked sialic acids react with the adjacent galactose to form a lactone, and the α2,6-linked sialic acids form a stable dimethylamide. The second step involves the addition of ammonia, with the lactone undergoing aminolysis, thereby transforming the carboxylic acid into a stable amide . (B) Illustration how an N -glycan attached to a protein (via an asparagine) is cleaved using the enzyme PNGase F. (C) Common procedures that are performed at the reducing end of the glycan: fluorescence detection can be enabled by introducing a label with a fluorophore, or MS ionization can be improved by adding a permanent positive charge (e.g., hydrazide labeling) or introducing a tertiary amine (e.g., carbamate chemistry), which could also allow the simultaneous analysis of glycans from different samples through the incorporation of stable isotopes (e.g., TMT-labeling).…”

Applications and continued evolution of glycan imaging mass spectrometry

“…Relevant to N‐glycan IMS are the strategies that take advantage of the differential chemical properties between α2,3‐ and α2,6‐linked anomers to stabilize each as a distinct moiety. In amidation syntheses developed by the Wuhrer group, α2,3‐ and α2,6‐linked sialic acid residues are modified with amide and dimethyl amine functional groups, respectively, by sequential incubations with 1‐ethyl‐3(3‐dimethylaminopropyl)carbodiimide (EDC)/1‐hydroxybenzotriazol (HOBt) and ammonia (Holst et al, 2016b; Pongracz et al, 2021) (Figure 7A). These reactions are adapted to use directly on tissue in situ before traditional MALDI‐IMS N‐glycan workflows.…”