An integrated workflow for real-time chemoenzymatic CMP-sialic acid preparation and a bioorthogonal microplate sialyltransferase assay (MPSA)

Sialylation at the terminal ends of cell-surface glycoproteins and glycolipids plays a pivotal role in dictating cellular interactions, human health, and pathological processes. This crucial modification is mediated by a family of 20 distinct human sialyltransferases (STs) whose exquisite substrate specificities and stereochemistry make them high-priority therapeutic targets. Consequently, robust techniques to detect and quantify ST activity are critical for translational research and inhibitor screening. To address this need, we present a detailed protocol for the chemo-enzymatic synthesis of a C5 alkyne-functionalized CMP-Sia donor substrate tailored for bioorthogonal click chemistry. This synthesis is coupled with the development of a simple, highly sensitive, and reproducible microplate-based assay designed to perform in-depth enzymatic characterization of individual ST activities. The protocol eliminates common throughput limitations by streamlining substrate preparation and optimizing microplate detection metrics. Our results demonstrate that this assay provides exceptional sensitivity and consistency across diverse ST isoforms. Ultimately, this unified method establishes one of the most robust, easily replicable, and high-throughput platforms currently available for ST profiling and small-molecule inhibitor screening.