Process Development for the Enzymatic Gram‐Scale Production of the Unnatural Nucleotide Sugar UDP‐6‐Azido‐GalNAc

Abstract

AbstractAzido 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 2.1 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 4.4–4.8 g of product per gram of enzyme (gP/gE) and STYs ranging from 1.7–2.4 g per liter per hour (g*L−1*h−1). By purification of a product solution pool containing 2.6 g (4.1 mmol) UDP‐6‐azido‐GalNAc, 2.1 g (2,122.1 mg) UDP‐6‐azido‐GalNAc (sodium salt) with a purity of 99.96 % (HPLC) were obtained. The overall recovery after purification was 81 % (3.32 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.