A Rapid and Sensitive MicroPlate Assay (MPSA) Using an Alkyne‐Modified CMP‐Sialic Acid Donor to Evaluate Human Sialyltransferase Specificity

International audience ; Human sialyltransferases primarily utilize CMP‐Sias, especially transferring Neu5Ac from CMP‐Neu5Ac to various acceptors. Advances in chemical biology have led to the synthesis of novel CMP‐Sia donors suitable for bioorthogonal reactions in cell‐based assays. However, the compatibility of these donors with all human enzymes remains uncertain. We synthesized a non‐natural CMP‐Sia donor with an alkyne modification on the N‐acyl group of Neu5Ac, which was effectively used by human ST6Gal I and ST3Gal I. A sensitive MicroPlate Sialyltransferase Assay (MPSA) was developed and expanded to a panel of 13 human STs acting on glycoproteins. All assayed enzymes tolerated CMP‐SiaNAl, allowing for the determination of kinetic parameters and turnover numbers. This study enhances the biochemical characterization of human sialyltransferases and opens new avenues for developing sialyltransferase inhibitors.