Enzymatic synthesis of all 15 naturally occurring human ABH antigens was achieved using a diversity-oriented enzymatic modular assembly (EMA) strategy. Three enzyme modules were developed, each one-pot multienzyme module comprises a glycosyltransferase and one or two corresponding sugar nucleotide generating enzyme(s). These multienzyme cascade processes provide an efficient and convenient platform for collective synthesis of all 15 ABH antigens in three operationally simple steps from five readily available disaccharide acceptors and three simple free sugars as donor precursors.Keywords: ABH antigen; biocatalysis; blood group; diversity-oriented synthesis; enzymatic synthesis; glycosyltransferase;

Lacto-N-neotetraose and its sialyl and fucosyl derivatives including Lewis x (Lex) pentasaccharide, sialyl Lewis x (sLex) hexasaccharide and internally sialylated derivatives were enzymatically synthesized from readily available lactoside, commercially available uridine 5′-diphosphate-glucose (UDP-Glc) and the corresponding monosaccharides using a highly efficient sequential one-pot multienzyme (OPME) strategy. The OPME strategy which combines bacterial glycosyltransferases and sugar nucleotide generation enzymes provides easy access to the biologically important complex oligosaccharides at preparative scale. Moreover, the same OPME strategy can be used for the regioselective introduction of sialic acid to the internal galactose unit of LNnT in a designed glycosylation route by simply changing the glycosylation sequence.

The diversity-oriented chemoenzymatic synthesis of α-dystroglycan (α-DG) core M1 O-mannose glycans has been achieved via a three-step sequential one-pot multienzyme (OPME) glycosylation of a chemically prepared disaccharyl serine intermediate. The high flexibility and efficiency of this chemoenzymatic strategy was demonstrated for the synthesis of three more complex core M1 O-mannose glycans for the first time along with three previously reported core M1 structures.

•Chemical synthesis of unsymmetrical 3,6-branched Man5 oligosaccharide.•One-pot sequential glycosylation is superior to stepwise synthesis.•Three-component convergent synthesis.An expeditious three-component, one-pot sequential glycosylation protocol has been developed for the preparation of 3,6-branched unsymmetrical mannopentaose (Man5), employing a mannose trisaccharide donor, a mannose monosaccharide donor and a mannose monosaccharide acceptor. The high efficiency of this one-pot procedure was demonstrated by comparison study with a stepwise synthesis using the same three building blocks.

•Highly efficient one-pot two-enzyme large-scale synthesis of lacto-N-biose (LNB).•Convergent chemical synthesis of LNT using enzymatic generated LNB disaccharide building block.•Enzymatically synthesized building blocks for chemical synthesis with additional enzymatic modifications.A concise and practical chemoenzymatic synthesis of lacto-N-tetraose, a major component and one of the most common core structures of human milk oligosaccharides (HMOs) was reported. This convergent synthesis relies on the glycosylation of a readily available lactoside acceptor with a lacto-N-biose donor generated from a highly efficient one-pot two-enzyme synthesis.

A novel chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes found as the terminal oligosaccharides of GD1α, GT1aα, and GQ1bα is described. It relies on chemical manipulation of enzymatically generated trisaccharides as conformationally constrained acceptors for regioselective enzymatic α2–6-sialylation. This strategy provides a new route for easy access to disialyl tetrasaccharide epitopes and their derivatives.

Fluorinated Thomsen–Friedenreich (T) antigens were synthesized efficiently from chemically produced fluorinated monosaccharides using a highly efficient one-pot two-enzyme chemoenzymatic approach containing a galactokinase and a D-galactosyl-β1–3-N-acetyl-D-hexosamine phosphorylase. These fluorinated T-antigens were further sialylated to form fluorinated ST-antigens using a one-pot two-enzyme system containing a CMP-sialic acid synthetase and an α-2–3-sialyltransferase.

•Highly efficient enzymatic synthesis of human P1PK blood group P1 pentasaccharide antigen.•Three steps with an overall yield of 46% from 3 simple starting materials.•One-pot multienzyme sequential glycosylation with in situ generation of sugar nucleotide donors.The enzymatic synthesis of biologically important and structurally unique human P1PK blood group type P1 pentasaccharide antigen is described. This synthesis features a three-step sequential one-pot multienzyme (OPME) glycosylation for the stepwise enzymatic chain elongation of readily available lactoside acceptor with cheap and commercially available galactose and N-acetylglucosamine as donor precursors. This enzymatic synthesis provides an operationally simple approach to access P1 pentasaccharide and its structurally related Gb3 and P1 trisaccharide epitopes.

Fluorinated Thomsen–Friedenreich (T) antigens were synthesized efficiently from chemically produced fluorinated monosaccharides using a highly efficient one-pot two-enzyme chemoenzymatic approach containing a galactokinase and a D-galactosyl-β1–3-N-acetyl-D-hexosamine phosphorylase. These fluorinated T-antigens were further sialylated to form fluorinated ST-antigens using a one-pot two-enzyme system containing a CMP-sialic acid synthetase and an α-2–3-sialyltransferase.

The diversity-oriented chemoenzymatic synthesis of α-dystroglycan (α-DG) core M1 O-mannose glycans has been achieved via a three-step sequential one-pot multienzyme (OPME) glycosylation of a chemically prepared disaccharyl serine intermediate. The high flexibility and efficiency of this chemoenzymatic strategy was demonstrated for the synthesis of three more complex core M1 O-mannose glycans for the first time along with three previously reported core M1 structures.

Lacto-N-neotetraose and its sialyl and fucosyl derivatives including Lewis x (Lex) pentasaccharide, sialyl Lewis x (sLex) hexasaccharide and internally sialylated derivatives were enzymatically synthesized from readily available lactoside, commercially available uridine 5′-diphosphate-glucose (UDP-Glc) and the corresponding monosaccharides using a highly efficient sequential one-pot multienzyme (OPME) strategy. The OPME strategy which combines bacterial glycosyltransferases and sugar nucleotide generation enzymes provides easy access to the biologically important complex oligosaccharides at preparative scale. Moreover, the same OPME strategy can be used for the regioselective introduction of sialic acid to the internal galactose unit of LNnT in a designed glycosylation route by simply changing the glycosylation sequence.