Diversity-Oriented Enzymatic Synthesis of Human Milk Oligosaccharides Bearing a 3-Fucosyllactose Core

A notable subset of human milk oligosaccharides (HMOs) bearing an α1,3-fucosylated lactose (3FL) moiety at the reducing end constitutes one of the most abundant and representative classes of HMOs, referred to as 3FL-related HMOs (3FL-HMOs). Despite their significant biological relevance, the systematic synthesis of 3FL-HMOs has remained challenging due to their structural diversity and complexity, which has hindered comprehensive functional studies. Herein, we report a highly efficient enzymatic modular assembly strategy for the collective synthesis of 32 3FL-HMOs, encompassing 5 core backbones and 11 distinct terminal epitopes. Notably, this approach employs a sialic acid-assisted, site-specific α1,3-fucosylation strategy to enable the precise installation of diverse fucosylation patterns on oligosaccharide scaffolds.