Synthetic and chemical biology studies of pseudaminic acid and acinetaminic acid

Abstract

As important components of the bacterial cell external glycans, bacterial non-2-ulosonic acids are unique for their chemical structural diversity and their biological roles in nature. They are widely distributed in multiple Gram-negative bacteria, such as Pseudomonas aeruginosa, Acinetobacter baumannii, Campylobacter jejuni, Helicobacter pylori, etc, and play a potential role in host–bacteria interactions. They are also identified as virulence factors of many multidrug-resistant pathogens with clinical importance. However, the accurate pathogenic mechanism of non-2-ulosonic acids is still less understood. Pseudaminic acid and acinetaminic acid are two important members of the non-2-ulosonic acid family. Considering the structural similarity of them to the eukaryote sialic acids, they may also participate the intercellular recognition within the bacterial colonies. More importantly, they may interact with host cell surface via binding to the sialic acid-binding immunoglobulin-type lectins (Siglecs) or other receptors, which possibly lead to the adhesion, infection and immune escape. However, the structural complexity of pseudaminic acid and acinetaminic acid makes them difficult to be obtained either by isolation from natural sources or by chemical syntheses reported previously, which limits the further study of their biological functions and developing related therapeutic approaches. To synthesize pseudaminic acid and its functionalized analogues with differently modified acyl groups on C5 and C7 positions, a de novo synthetic strategy starting from commercially available L-allo-threonine was developed, giving ~10% yield in 17 steps. Based on the synthesis of pseudaminic acid analogues, we investigated the pseudaminic acid enzyme in the biosynthetic pathway and probed substrate specificity. At the same time, we also tried to validate potential pseudaminic acid glycosyltransferase, KpsS1, via screening these pseudaminic acid analogues to different glycan acceptors with KpsS1. The synthesized pseudaminic acid analogues were also conjugated to carrier protein CRM197 using the ortho-phthalaldehyde chemistry to obtain Pse-CRM197 conjugates as vaccine candidates. These conjugates were proved to stimulate high immune responses in mice, which protected the immunized mice from infections caused by Acinetobacter baumannii. Furthermore, an alkyne-diazirine modified hexosamine 2,4,6-trideoxy-2,4-diamino-L-altrose, the biosynthetic precursor of pseudaminic acid, was synthesized and used to profile pseudaminic acid interactors in host cells and investigate the potential pseudaminic acid glycosyltransferase via metabolic labeling. The powerful de novo synthetic strategy enabled efficient access to pseudaminic acid and its biosynthetic intermediates, which could support further biological studies on the pseudaminic acid and related glycans. In addition, a new synthetic approach towards the acinetaminic acid thioglycoside donor was also developed successfully, which can be further applied to the synthesis of acinetaminic acid containing oligosaccharide and glycoconjugate vaccines.