Synthesis of 4-azido sialic acid for testing against Siglec-7 and in metabolic oligosaccharide engineering†

An important approach for tracking and visualizing sialic acid-containing glycans involves using sialic acid reporters functionalized with bioorthogonal handles. More specifically, metabolic oligosaccharide engineering (MOE) commonly employs monosaccharides with an alkyne or azide handle for incorporation into cellular glycans, followed by a subsequent click reaction to elaborate with a biotin or fluorophore handle. For sialic acid, this has been carried out extensively, with an azide or alkyne appended to the C5 N-acetamido group being the most common location for the handle. However, circumstances may require the handle to be at different positions and, to date, the C7 and C9 positions have been shown to work to varying degrees. Herein, we synthesized protected 4AzNeu5Ac that could be incorporated into cellular glycans nearly as efficiently as Neu5Az and targeted with DBCO-biotin through strain promoted azide–alkyne cycloaddition. Owing to the good incorporation of 4AzNeu5Ac into cellular glycans, we followed up this ability by first synthesizing the deprotected form of 4AzNeu5Ac, using a thioglycoside to lock the anomeric center during deprotection of the acetyl groups. Activation of 4AzNeu5Ac to CMP-4AzNeu5Ac then enabled the use of this donor by human sialyltransferase ST3GAL1 to transfer CMP-4AzNeu5Ac to β-Galp-(1→3)-α-GalpNAc. With purified α-4AzNeup5Ac-(2→3)-β-Galp-(1→3)-α-GalpNAc in hand, we tested it as a ligand for Siglec-7 and found that the C4-Az modification is tolerated, opening future possibilities to exploit this position to generate high affinity and selective ligands. These findings expand the repertoire of metabolic oligosaccharide engineering agents and show that azide modifications are tolerated at the C4 position of sialic acid.