Expediting chemoenzymatic access to bacterial 'sialic acid-like' sugars via mapping of wild type sialic acid aldolase enzyme substrate tolerance and promiscuity.

Bacterial 'sialic-acid-like' sugars, including legionaminic acid and pseudaminic acid, are critical for the virulence and immune evasion of numerous human pathogens, including pan-drug resistant Acinetobacter baumannii and global gut pathogens Campylobacter jejuni and Helicobacter pylori. Despite this importance, a detailed understanding of the role these sugars play in pathogenesis is hampered by the challenging synthesis of their 9-deoxy nonulosonic acid backbones. Herein, we expedite access to these complex glyco-architectures through exploration of the biocatalytic potential of sialic acid aldolase enzymes (also known as N-acetyl neuraminate lyases). By mapping the substrate tolerance and promiscuity of a library of wild-type aldolases, we identified the enzyme from Corynebacterium glutamicum (Cg) as a highly effective biocatalyst for the synthesis of 9-deoxy 'sialic acid-like' sugars, particularly Leg5Ac7Ac. Notably, the Cg aldolase synthetically outperformed established orthologs, highlighting its utility for preparative scale syntheses. Furthermore, we demonstrate that stereochemical limitations in these transformations can be bypassed through the use of N-acetyl to azide substitution, enabling chemoenzymatic access to pseudaminic acid architectures with the potential for late stage N-diversification. In summary, this study establishes a powerful, regioselective tool for the efficient production of complex bacterial carbohydrates and deeper exploration of their glycobiology.