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.

唾液酸普遍存在于脊椎动物的糖组中，并且通常以 α2,3- 或 α2,6- 连接的方式对聚糖进行封端，具有不同的生物学作用。连接特异性唾液酸表征越来越多地通过质谱法进行，并借助差异唾液酸衍生化来区分连接异构体。通常，在此类衍生反应的第一步中，在羧基活化剂和催化剂存在下，α2,3-连接的唾液酸与亚末端单糖缩合形成内酯，而α2,6-连接的唾液酸形成酰胺或酯衍生物。在第二步中，内酯被转化为酰胺衍生物。值得注意的是，内酯中间体在所报道的反应中的结构和作用仍然不明确，因此尚不清楚α2,3-连接的唾液酸的酰胺化在多大程度上依赖于内酯的直接氨解，而不是内酯水解和随后的酰胺化。在本报告中，我们通过对简单和复杂的聚糖标准品应用受控反应条件，使用质谱法揭示了内酯中间体在 α2,3-连接唾液酸酰胺化中的作用。结果明确表明，在常见的唾液酸衍生化方案中，先前的内酯形成是α2,3-连接的唾液酸有效的、键特异性酰胺化的先决条件，该酰胺化主要通过直接氨解进行。此外，核磁共振波谱证实，在唾液酸乳糖标准品上仅形成C2内酯中间体。这些见解使得未来可以为特定于连锁的唾液酸衍生化开发更合理的方法。