Enzymatic synthesis of para-lacto-N-hexaose and its isomeric structures as well as those a1,2-fucosylated variants naturally occurring in human milk oligosaccharide (HMOs) was achieved using a sequential one-pot enzymatic system. Three glycosylation routes comprising bacterial glycosyltransferases and corresponding sugar-nucleotidegenerating enzymes were developed to facilitate efficient production of extended type-1 and type-2 N-acetyllactosamine (LacNAc) backbones and hybrid chains. Further fucosylation efficiency of two a1,2-fucosyltransferases on both type-1 and type-2 chains of the hexasaccharide was investigated to achieve practical synthesis of the fucosylated glycans. The availability of structurally defined HMOs offers a practical approach for investigating future biological applications.

In this study, we focused on the synthetic application of α1,3/4-fucosyltransferase obtained from Helicobacter pylori DSM 6709 (FucTIII) on l-fucose-containing glycans. By combining FucTIII with the sequential one-pot enzymatic system of human milk oligosaccharide (HMO) production, various fucosylated HMOs, such as lacto-N-fucopentose V (LNFP V), LNFP VI, lacto-N-difucohexaose II (LNDFH II), and lacto-N-neodifucohexaose II (LNnDFH II), were synthesized. Moreover, l-fucose-containing glycan synthesis of Lewis antigens, such as Lewis x, Lewis y, Lewis a, Lewis b, sialyl Lewis x, sialyl Lewis a, and their derivatives, was achieved. Enzyme kinetics proved that the catalytic efficiency (k cat/K m) of FucTIII on type-2 N-acetyl lactosamine (LacNAc) was 39 times higher than that on type-1 LacNAc. Furthermore, enzyme kinetics revealed that additional GlcNAc on the nonreducing end of the acceptors can enhance the catalytic efficiency of FucTIII on the glycan acceptors. Investigations of bacterial FucTs on substrate spectra indicate that future studies should study expansion of the inventory of biocatalysis for the synthesis of valuable glycans.

Bacteroides fragilis α1,3/4-fucosyltransferase (Bf13FT) was expressed in Escherichia coli and characterized as an α1,3/4-fucosyltransferase that can be used as a versatile catalyst for the synthesis of various fucosides, including Lex, Ley, blood group H1-antigen, 3FL, LNFP III, LNFP V, LNnFP V, LNDFH II, LNDFH III, IFLNH III, DF-pLNnH, and TF-pLNnH, and hybrid-type glycans, such as Ley-3FL and Ley-Lex-3FL. The preferential fucosylation activity on Fucα1,2LacNAc and LacNAc over Lac, which enabled programmable fucosylation, led to the one-pot synthesis of a KH-1 antigen.

In this study, we developed an efficient fluorous tagging strategy for site-selective enzymatic fucosylation/sialylation in which appending a readily removable auxiliary tag (SF 17 ) to the reducing end of an acceptor provided the regioselectivity control of a given wild-type fucosyltransferase/sialyltransferase. By combining this strategy with the sequential one-pot enzymatic system of HMO production, various fucosylated para-HMOs, such as LNFP I, LNFP II, LNFP III, LNFP V, LNDFH I, LNDFH II, TF-LNT, F-pLNH I, F-pLNH IV, DF-pLNH I isomer, DF-pLNH II, TF-pLNH I, TF-pLNH II, F-pLNnH, F-pLNnH I, DF-pLNnH, TF-pLNnH, and TetraF-pLNO, were synthesized. Furthermore, the SF 17 -tagged acceptors improved the reaction rates of the enzymatic glycan chain extension. Terminal α2,6-sialylation on SF 17 -tagged pLNnH was directly achieved using wild-type α2,6-sialyltransferase from Photobacterium damsela (Pd26ST). Moreover, enzyme kinetics revealed that the catalytic efficiency (k cat/K M) of α1,4-fucosylation of Bf13FT (α1,3/4-fucosyltransferase from Bacteroides fragilis) on SF 17 -tagged LNFP V acceptors could be enhanced by 55 times compared with tagging with an azidohexyl aglycone. We developed an efficient and reliable strategy for generating complex and diverse natural HMO libraries for functional gut microbiome studies. This SF 17 -assisted glycosylation strategy provides a practical approach to harness the extreme substrate flexibility offered by readily available bacterial glycosyltransferases, which enables the site-selective fucosylation/sialylation on para-HMOs.

Carbohydrate purification remains problematic due to the intrinsic diversity of structural isomers present in nature. Although liquid chromatography‐based techniques are suitable for analyzing or preparing most glycan structures acquired either from natural sources or through chemical or enzymatic synthesis, the separation of regioisomers or linkage isomers with a clear resolution remains challenging. Herein, a carbon dioxide supercritical fluid chromatography (SFC) method was devised to resolve 18 human milk glycosides: oligomers (disaccharides to hexasaccharides), fucosylated regioisomers (lacto‐N‐fucopentaose I, III, and V; lacto‐N‐neofucopentaose V; lacto‐N‐difucohexaose III; blood group H1 antigen; and TF‐LNnT), and connectivity isomers (lacto‐N‐tetraose/lacto‐N‐neotetraose and para‐lacto‐N‐hexaose/para‐lacto‐N‐neohexaose/type‐1 hexasaccharide). The analysis of these glycosides represents a major limitation associated with conventional carbohydrate analysis. The unprecedented resolution achieved by the SFC method indicates the suitability of this key technology for revealing complex human milk glycomes.