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

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 (AfKdnase) 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.