摘要
Free-reducing end glycans are essential tools in glycan-protein interaction studies, structural biology, and functional glycomics, owing to their native conformational flexibility and unmodified binding epitopes. Human milk oligosaccharides (HMOs) are bioactive glycans with growing applications in nutrition and therapeutics. While bacterial glycosyltransferases (GTs) are widely used in HMO synthesis due to their ease of recombinant expression in Escherichia coli, preparative-scale production requires sufficient amounts of active enzymes, which are often obtained through labor-intensive purification. Using crude cell lysates provides a practical alternative that avoids protein loss during purification, particularly for labile or membrane-bound GTs. However, crude lysates typically contain endogenous hydrolases that can degrade substrates and compromise synthetic efficiency. Here we present a sulfo-fluorous affinity tag (SF17) that enables beta-galactosidase-resistant glycan synthesis directly in crude lysates. SF17-tagged glycans show high stability, enhanced regioselectivity, and are readily purified via fluorous solid-phase extraction. This strategy facilitated preparative-scale access to diverse HMOs, including para-lacto-N-hexaose (pLNH), A-antigen derivatives, 2 '-fucosyllactose (2 '-FL), lactodifucotetraose (LDFT), disialyllacto-N-tetraose (DSLNT), and difucosyllacto-N-hexaose b (DF-LNH b). The SF17 tag is easily removed under mild acidic conditions, yielding pure free-reducing glycans suitable for downstream applications. This work provides a practical and scalable platform that addresses major enzymatic and purification bottlenecks in glycan synthesis.