RESUMO
Azido sugars hold great promise as substrates in numerous click-chemistry applications. However, the synthesis of activated azido sugars is limited by cost and complexity. Conventional chemical activation methods are intricate and time-consuming. In response, we have developed a process for the large-scale production of UDP-6-azido-GalNAc through enzymatic nucleotide sugar synthesis on a gram scale. Our optimization strategies encompassed refining the process parameters of an enzyme cascade featuring NahK from Bifidobacterium longum and AGX1 from Homo sapiens. Using the repetitive-batch-mode technology, we synthesized up to 2.1â g of UDP-6-azido-GalNAc, achieving yields up to 97 % in five consecutive batch cycles using a single enzyme batch. The synthesis process demonstrated to have total turnover numbers (TTNs) between 4.4-4.8â g of product per gram of enzyme (gP/gE) and STYs ranging from 1.7-2.4â g per liter per hour (g*L-1*h-1). By purification of a product solution pool containing 2.6â g (4.1â mmol) UDP-6-azido-GalNAc, 2.1â g (2,122.1â mg) UDP-6-azido-GalNAc (sodium salt) with a purity of 99.96 % (HPLC) were obtained. The overall recovery after purification was 81 % (3.32â mmol). Our work establishes a robust production platform for the gram-scale synthesis of unnatural nucleotide sugars, opening new avenues for applications in glycan engineering.