Modular microgel flow reactors for automated glycan synthesis with immobilized enzymes

Glycans are biomolecules that play roles in numerous biological processes and are becoming increasingly important for applications in biomedicine, nutrition, pharmacy, and research. Their structural diversity makes glycans attractive target molecules, but also poses a challenge in their synthesis. Compared to chemical synthesis, enzymatic glycan synthesis offers various advantages: high regio- and stereoselectivity, the use of mild reaction conditions, and better ecological and economic sustainability. Automated enzymatic glycan synthesis (AEGS) is an essential development step in making defined glycan structures efficiently accessible on a larger scale. Here, we present a modular Mi crogel continuous flow R eactor for A utomated and continuous G lycan synthesis with immobilized E nzymes (MiRAGE). The enzymes are immobilized in microgels, SpyT-agarose particles, or Ni 2+ -IDA beads using different strategies. The immobilized enzymes are integrated into reactor compartments, which are supplied with substrates via a continuous reaction buffer flow. A semipermeable membrane ensures that the enzymes as well as substrates and products are retained in the module, while by-products can be selectively removed via a crossflow stream. The modular design allows several enzymatic reaction steps to be coupled, automating the synthesis of complex glycan structures. To this end, we demonstrate the concept towards the synthesis of the human milk oligosaccharide LNFPIII, Lewis X , blood group-, and Galili epitopes. In addition, we compare the different immobilization techniques and reactor strategies in terms of consistency and yield. MiRAGE represents a robust, scalable, and versatile approach that not only enables the automated synthesis of complex glycans but can also be transferred to other biocatalytic processes. Key points • Immobilization of SpyCatcher-glycosyltransferases on SpyTag agarose and microgels • Embedment of immobilized glycosyltransferases in a continuous flow bioreactor • Enzymatic glycan synthesis with removal of by-products and retention of biocatalysts