Construction and structural characterization of versatile lactosaminoglycan-related compound library for the synthesis of complex glycopeptides and glycosphingolipids

We have established a facile and efficient protocol for the preparative-scale synthesis of various compound libraries related to lactosaminoglycans: cell surface oligosaccharides composed of N-acetyllactosamine as a repeating disaccharide unit, based on chemical and enzymatic approaches. Substrate specificity and feasibility of a bacterial glycosyltransferase, Neisseria meningitidis β1,3-N- acetylglucosaminyltransferase (LgtA), were investigated in order to synthesize various key intermediates suited for the construction of mammalian O-glycopeptides and glycosphingolipids containing poly-N-acetyllactosamine structures. Recombinant LgtA exhibited the highest glycosyltransferase activity with strongly basic conditions (pH = 10, glycine-NaOH buffer) and a broad range of optimal temperatures from 20 to 30 °C. Interestingly, it was found that LgtA discriminates L-serine and L-threonine and functions both as a core-1 β1,3-N-acetylglucosaminyltransferase and core-2 β1,3-N- acetylglucosaminyltransferase toward Fmoc-Ser derivatives, while LgtA showed only core-2 β1,3-N-acetylglucosaminyltransferase activity in the presence of Fmoc-Thr derivatives. Combined use of LgtA with human β1,4- galactosyltransferase allowed for controlled sugar extension reactions from synthetic sugar amino acids and gave synthetic lactosaminoglycans, such as a decasaccharide derivative, Galβ(1 → 4)GlcNAcβ(1 → 3)Galβ(1 → 4)GlcNAcβ(1 → 3)Galβ(1 → 4)GlcNAcβ(1 → 3)Galβ(1 → 4)GlcNAcβ(1 → 6)[Galβ(1 → 3)]GalNAcα1 → Fmoc-Ser-OH (6), and a dodecasaccharide derivative, Galβ(1 → 4)GlcNAcβ(1 → 3)Galβ(1 → 4)GlcNAcβ(1 → 3)Galβ(1 → 4)GlcNAcβ(1 → 6)[Galβ(1 → 4)GlcNAcβ(1 → 3)Galβ(1 → 4)GlcNAcβ(1 → 3)Galβ(1 → 3)]GalNAcα1 → Fmoc-Ser-OH (9). A partially protected pentasaccharide intermediate, GlcNAcβ(1 → 3)Galβ(1 → 4)GlcNAcβ(1 → 6)[Galβ(1 → 3)]GalNAcα1 → Fmoc-Thr-OH (11), was applied for the microwave-assisted solid-phase synthesis of a MUC1-related glycopeptide 19 (MW = 2610.1). The findings suggest that this sugar extension strategy can be employed for the modification of lactosyl ceramide mimetic polymers to afford convenient precursors for the synthesis of various glycosphingolipids.