Regioselective Enzymatic Galactosylation Enabled Divergent Synthesis of Asymmetrical Biantennary Human Milk Oligosaccharides

Human milk oligosaccharides (HMOs) are essential nutritional and bioactive components of human breast milk, conferring numerous beneficial effects on neonatal health. The HMO repertoire comprises over 200 unique glycan structures, and more than half of them are branched oligosaccharides. Owing to the structural diversity and complexity, the synthesis of branched HMOs remains challenging, which significantly hinders their functional study. We herein present a highly efficient biomimetic approach for the rapid and scaled-up chemoenzymatic synthesis of asymmetrical biantennary HMOs. Taking advantage of the inherent regioselectivity of a panel of bacterial galactosyltransferases, two asymmetrical branched pentasaccharide intermediates were obtained through the regioselective enzymatic β1,3- or β1,4-galactosylation of a readily available chemically synthesized biantennary symmetrical tetrasaccharide precursor. The two asymmetrical pentasaccharide intermediates were further diversified by sequential multienzyme cascade reactions to afford a library of over 20 structurally well-defined asymmetrical biantennary lacto-N-hexaose (LNH) and lacto-N-neo-hexaose (LNnH) series HMOs in less than 5 linear steps.