Construction and Structural Characterization of Versatile Lactosaminoglycan-Related Compound Library for the Synthesis of Complex Glycopeptides and Glycosphingolipids

摘要

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 deg C.Interestingly,it was found that LgtA discriminates L-serine and L-threonine and functions both as a core-1 beta 1,3-N-acetylglucosaminyltransferase and core-2 beta1,3-N-acetylglucosaminyltransferase toward Fmoc-Ser derivatives,while LgtA showed only core-2 beta 1,3-N-acetylglucosaminyltransferase activity in the presence of Fmoc-Thr derivatives.Combined use of LgtA with human beta 1,4-galactosyltransferase allowed for controlled sugar extension reactions from synthetic sugar amino acids and gave synthetic lactosaminoglycans,such as a decasaccharide derivative,Gal beta(1 ->4)GlcNAc beta(1 ->3)Gal beta (1 ->4)GlcNAc beta (1 ->3)Gal beta (1 ->4)GlcNAc beta (1 ->3)Gal beta(1 ->4)GlcNAc beta (l ->6)[Gal beta (l ->3)]GalNAc alpha 1->Fmoc-Ser-OH (6),and a dodecasaccharide derivative,Gal beta(l - 4)GlcNAc beta(l - 3)Gal beta(l ->4)GlcNAc beta(l ->3)Gal beta(l - >4)-GlcNA beta(l ->6)[Gal beta(l ->4)GlcNAc beta (l ->3)Ga]betal ->4)GlcNAc beta(l ->3)Galbeta (l ->3)]GalNAc alpha 1 ->Fmoc-Ser-OH (9).A partially protected pentasaccharide intermediate,GlcNAc beta(l ->3)Gal beta (1 ->4)-GlcNAc beta(1 ->6)[Gal beta (l ->3)]GalNAc alpha 1 ->Fmoc-Thr-OH (11),was applied for the microwave-assisted solid-phase synthesis of a MUCl-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.