Glycosylation plays a vital role in cellular functions, yet the synthesis of complex mucin‐type O ‐glycans such as disialyl galacto‐ N ‐biose (DSGNB) remains significant challenge due to structural complexity and enzyme specificity. A modular chemoenzymatic strategy employing bacterial glycosyltransferases BiGalHexNAcP, LgtD, Cst‐I, and an engineered Psp2,6ST A366G for the regioselective synthesis of DSGNB analogs is reported. A panel of GalNR‐based acceptors ( 11 – 14 ), varying in anomeric configuration, aglycone, and amine protection, enables sequential one‐pot β1,3‐galactosylation to generate diverse GNB derivatives. BiGalHexNAcP exhibits strong preference for α‐GalNR with allyl linkers and N ‐acetyl‐protection, whereas LgtD shows broader tolerance. Subsequent α2,3‐sialylation with Cst‐I and CMP‐Neu5Ac affords monosialyl GNBs (MSGNB, 2 – 10 ) in 80%–100% yield within 1–2 h. Final α2,6‐sialylation with Psp2,6ST furnishes DSGNB ( 1b ) regio‐ and stereoselectively from β‐ O ‐allyl MSGNB 3 (93% yield), while the α‐ O ‐allyl acceptor 2 produces a DSGNB:iso‐DSGNB mixture (2:1). Notably, β‐ O ‐benzyl aglycones further enhance regioselectivity to 6:1. Molecular docking provides mechanistic insight, showing that in β‐ O ‐allyl MSGNB 3 , the GalNAc C6‐OH is positioned closer to catalytic Asp232 than in the α‐ O ‐linked analog. The method also enables the synthesis of disialyl T‐antigen in 70% yield, demonstrating a broadly applicable platform for constructing well‐defined O ‐GalNAc glycans with potential applications in biomedical research and therapeutic development.
Chien et al. (Thu,) studied this question.