title: Construction and structural characterization of versatile lactosaminoglycan-related compound library for the synthesis of complex glycopeptides and glycosphingolipids.
abstract: 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 beta1,3-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 degrees C. Interestingly, it was found that LgtA discriminates L-serine and L-threonine and functions both as a core-1 beta1,3-N-acetylglucosaminyltransferase and core-2 beta1,3-N-acetylglucosaminyltransferase toward Fmoc-Ser derivatives, while LgtA showed only core-2 beta1,3-N-acetylglucosaminyltransferase activity in the presence of Fmoc-Thr derivatives. Combined use of LgtA with human beta1,4-galactosyltransferase allowed for controlled sugar extension reactions from synthetic sugar amino acids and gave synthetic lactosaminoglycans, such as a decasaccharide derivative, Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 6)[Galbeta(1 --> 3)]GalNAcalpha1 --> Fmoc-Ser-OH (6), and a dodecasaccharide derivative, Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 6)[Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 3)]GalNAcalpha1 --> Fmoc-Ser-OH (9). A partially protected pentasaccharide intermediate, GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 6)[Galbeta(1 --> 3)]GalNAcalpha1 --> 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.
authors.affiliation: Division of Advanced Chemical Biology, Graduate School of Advanced Life Science, Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021, Japan.
authors.forename: Kentarou
authors.initials: K
authors.lastname: Naruchi
authors.forename: Tomoki
authors.initials: T
authors.lastname: Hamamoto
authors.forename: Masaki
authors.initials: M
authors.lastname: Kurogochi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hinou
authors.forename: Hiroki
authors.initials: H
authors.lastname: Shimizu
authors.forename: Takahiko
authors.initials: T
authors.lastname: Matsushita
authors.forename: Naoki
authors.initials: N
authors.lastname: Fujitani
authors.forename: Hirosato
authors.initials: H
authors.lastname: Kondo
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000606:Amino Sugars / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D005609:Free Radicals / Q000737:chemistry; D006020:Glycopeptides / Q000138:chemical synthesis* / Q000737:chemistry; D006028:Glycosphingolipids / Q000138:chemical synthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D013237:Stereoisomerism
chemical_list: D000606:Amino Sugars; D005609:Free Radicals; D006020:Glycopeptides; D006028:Glycosphingolipids; D011134:Polysaccharides; C039448:lactosaminoglycan