Ketodeoxynonulosonic Acid Hydroxylase (Kdnase) Assisted Site‐Specific Enzymatic α2,6‐Sialylation

Comprehensive Summary Owing to its promiscuous substrate specificity and high catalytic efficiency, the bacterial α2,6‐sialyltransferase from Photobacterium damselae (Pd2,6ST) has been widely used for the synthesis of various α2,6‐linked sialosides. However, Pd2,6ST is not a suitable enzyme for the regioselective α2,6‐sialylation of complex acceptor substrates containing multiple galactose (Gal) and/or N ‐acetylgalactosamine (GalNAc) residues due to its promiscuous substrate specificity. In this study, a novel enzymatic substrate engineering strategy was developed to overcome this limitation by employing enzymatically introduced α2,6‐linked ketodeoxynonulosonic acid (Kdn) as temporary “protecting group” at the unwanted sialylation sites. The Kdn “protecting group” can be selectively removed by a ketodeoxynonulosonic acid hydrolase from Aspergillus fumigatus ( Af Kdnase) at appropriate stage without affecting coexisting sialic acid residues, such as N ‐acetylneuraminic acid (Neu5Ac) or N ‐glycolylneuraminic acid (Neu5Gc). This strategy provides a general and practical approach for the synthesis of complex sialosides, including sialylated poly‐LacNAc glycans, disialylated ganglioside glycan epitopes, and branched human milk oligosaccharides.