Single-mutations at the galactose-binding site of enzymes GalK, GalU, and LgtC enable the efficient synthesis of UDP-6-azido-6-deoxy-d-galactose and azido-functionalized Gb3 analogs.

This article focuses on the enzymatic synthesis of azido-functionalized globotriaosylceramide (Gb3) analogs to facilitate the study of membrane organization in Anderson–Fabry disease (AFD), a genetic disorder characterized by lysosomal accumulation of Gb3 due to deficient α-galactosidase A activity. The authors engineered mutants of three enzymes—galactokinase (GalK), UTP–glucose-1-phosphate uridylyltransferase (GalU), and α-1,4-galactosyltransferase LgtC—from Streptococcus pneumoniae and Neisseria meningitidis to efficiently produce UDP-6-azido-6-deoxy-d-galactose (UDP-6AzGal), an unnatural sugar nucleotide donor, and subsequently synthesize azido-Gb3 analogs. These enzyme variants demonstrated significantly improved catalytic efficiency compared to wild-type enzymes, enabling one-pot synthesis of azido-globotriose and lyso-azido-Gb3 with conversion yields up to 90% and 70%, respectively. The azido-functionalized Gb3 analogs produced may serve as chemical reporters for bioorthogonal labeling and as precursors for further glycosphingolipid modifications, providing tools for investigating Gb3-related pathologies such as AFD.