Enzymatic Synthesis of Lacto-N-Difucohexaose I Which Binds to Helicobacter pylori

Helicobacter pylori is known to bind with sugar chains possessing Lewis b structure. We are trying to combine oligosaccharides containing Lewis b sugar chain to water insoluble polysaccharide through some linker. Lacto-N-difucohexaose I (LNDFH I; Fucα1→2Galβ1→3[Fucα1→4]GlcNAcβ1→3Galβ1→4Glc) fits for that purpose, since it consists of Lewis b tetrasaccharide and lactose whose d-glucose residue can be utilized as a linker. We thus developed a method to synthesize this hexaose enzymatically. First, β-1,3-N-acetylglucosaminyltransferase (β-1,3-GnT) was partially purified from bovine blood by an established method. Using this enzyme preparation, d-GlcNAc was attached to the d-galactose residue of lactose with a β-1,3-linkage to produce lacto-N-triose II at 44% yield. The low yield was thought to be due to contaminating N-acetylglucosaminidase that would have hydrolyzed the product, lacto-N-triose II. Next, d-galactose was attached by transglycosylation using ortho-nitrophenyl β-d-galactopyranoside as a donor with the aid of recombinant β-1,3-galactosidase from Bacillus circulans to generate lacto-N-tetraose (LNT) at 22% yield. l-Fucose was then linked to the d-galactose residue of LNT via an α-1,2-linkage using recombinant human fucosyltransferase I (FUT1) expressed in a baculovirus system (71% yield). The obtained pentasaccharide was subsequently incubated with GDP-β-l-fucose and commercial fucosyltransferase III (FUT3) to attach l-fucose to the d-GlcNAc residue of LNT with an α-1,4-linkage. After purification with an activated carbon column chromatography, 1.7mg of LNDFH I was obtained (85% yield). We thus produced LNDFH I over four enzymatic steps with a yield of 6%.