An integrated workflow for real-time chemoenzymatic CMP-sialic acid preparation and a bioorthogonal microplate sialyltransferase assay (MPSA)
Abstract
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.
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BibTeX
@article{lancel2026integrated,
title = {{An integrated workflow for real-time chemoenzymatic CMP-sialic acid preparation and a bioorthogonal microplate sialyltransferase assay (MPSA)}},
author = {Lucas Lancel and Sushmaa Dangudubiyyam and Clémence Simon and Hugo Segura and Christophe Biot and Yann Guérardel and Anne Harduin-Lepers and Cédric Lion},
journal = {Biology Methods \& Protocols},
year = {2026},
month = oct,
doi = {10.1093/biomethods/bpag058},
url = {https://doi.org/10.1093/biomethods/bpag058},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - An integrated workflow for real-time chemoenzymatic CMP-sialic acid preparation and a bioorthogonal microplate sialyltransferase assay (MPSA)
AU - Lucas Lancel
AU - Sushmaa Dangudubiyyam
AU - Clémence Simon
AU - Hugo Segura
AU - Christophe Biot
AU - Yann Guérardel
AU - Anne Harduin-Lepers
AU - Cédric Lion
PY - 2026
DA - 2026/10/14
JO - Biology Methods & Protocols
DO - 10.1093/biomethods/bpag058
UR - https://doi.org/10.1093/biomethods/bpag058
AB - 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.
SN - 2396-8923
ER -