Chemoenzymatic Assembly Of Mammalian O-Mannose Glycans

For many years,O-mannosylation of protein was considered as a specific modification only existing in fungi,until it was first identified in mammalian O-glycans in 1979.With the advancement of glycomics and glycoproteomics,scientists have found that this kind of glycosylation exist extensively in mammalian p'rotein modification.Current researchs show that up to 30%of brain O-glycans are O-mannose-initiated glycans and more than 50 kinds of O-mannosylated proteins have been identified.The O-mannosyl glycan is a type of O-glycan which is initiated by covalent attachment of mannose to the serine(Ser)and threonine(Thr)residues of proteins via ?-linkage,the mannose residue was then modified with N-Acetyl-glucosamine(GlcNAc)on different sites,O-mannose glycans are classed into three groups including Core M1?Core M2 and Core M3 according to their extension sites of core mannose.The O-mannose core structures can be further extended by the addition of galactose(Gal)residues,sialic acid terminals,fucose(Fuc)residues or glucuronic acid(GlcA)residues via different glycosidic linkage or be sulfonated and phosphorylated resulting in diverse and complex O-mannosyl glycans.The structural diversity of O-mannose glycans is closely related to their multiple biological functions.Although more than 50 o-mannosylated glycoproteins have been identified,current research has focused on o-mannose-modified ?-dystroglycan(?-DG),which is a key component of cell-surface membrane glycoptoeins,and an integral glycoprotein of the dystrophin-glycoprotein complex that links the intracellular cytoskeleton to extracellular matrix.Defects in O-mannosylation of a-DG results in varying severities of congenital muscular dystrophies(CMDs).Recent studies demonstrated that O-mannose glycans also serves as the receptor for infection of lymphocytic choriomeningitis virus(LCMV)and Lassa fever virus(LFV)to host cells,and contributes to the transfer and proliferation of cancer cells.O-Mannose glycans account for up to 30%of all O-linked glycans in mammalian brain tissue.It appears to play pivotal roles in the growth,development,repairment and signaling of central nervous system.Although over 20 O-mannose glycans have been well characterized,however,the biosynthetic pathway of O-mannose glycans,and the structure and function relationship of O-mannose glycans have not been fully understood.To solve the aforementioned problems,sufficient amount of structurally well-defined O-mannose glycan probes are highly desired to study the enzymes underlying the biosynthesis pathway of O-mannose glycan and its catalytic process,as well as decipher the functional glycan structures involved in multiple biological activities at molecular level.So far,the synthetic studies on O-mannose glycans mainly focused on the simple Core M1 structures using target-oriented synthesis strategy,which involved repeating protecting group manipulation and led to low total yield.In contrast,the previous enzymatic approaches are limited by the source of enzymes,low efficiency,restrict substrate specificity and low soluble expression level of the enzymes.Recently,a diversity-oriented chemoenzymatic strategy was developed in our lab,which provides an ease access to Core M1 O-mannose glycans.In this study,three enzyme modules applied for the synthesis of Core M1 O-mannose glycans were adopted for the chemoenzymatic synthesis of all the 63 Core M1 and Core M3 O-mannose glycans.This chemoenzymatic strategy features the gram-scale chemical synthesis of 5 judiciously designed core structures,and the diversity-oriented modification of the core structures with 3 enzyme modules to provide the rest 58 complex O-mannose glycans in a linear sequence that does not exceed 4 steps.This diversity-oriented chemoenzymatic strategy enables the first collective synthesis of all 63 Core M1 and Core M2 O-mannose glycans.The research was carried out from the following aspects:(1)Chemical preparation of 5 O-mannose glycan Core structuresA concise chemical approach was designed for the gram scale synthesis of 5 O-mannose glycan Core structures.Taking advantage of the reactivity difference of C2-and C6-OH of mannoside acceptor,a one-pot glycosylation approach was susscessfully applied for the parallel synthesis of 5 protected O-mannose glycan intermediates.After deprotection using a unified 3-steps procedure,5 core structures were achieved for further enzymatic diversification.(2)Enzymatic assembly of Core M1 O-mannose glycansThe synthesis of 7 Core M1 O-mannose glycans was achieved through stepwise enzymatic extension of a chemically synthesized C2-branched disaccharide using three enzyme modules.(3)Enzymatic assembly of C6-branched Core M1 O-mannose glycansThe synthesis of 7 C6-branched Core M1 O-mannose glycans was achieved through stepwise enzymatic extension of chemically synthesized C6-branched disaccharide by sequential glycosylation using three enzyme modules.(4)Enzymatic assembly of symmetric Core M2 O-mannose glycansThe 7 C2,C6-branched symmetric Core M2 structures were constructed from chemically synthesized symmetric trisaccharide by synchronous enzymatic extension at both C2-and C6-branched GlcNAc.(5)Enzymatic assembly of asymmetric Core M2 O-mannose glycansThe remaining 42 asymmetric Core M2 structures were generated from chemically synthesized 2 asymmetric tetrasaccharides by sequential glycosylation using the same three enzyme modules.All the 42 asymmetric Core M2 structures were synthesized for the first time.The innovations of the research including following parts:(1)Five core structures were obtained at gram-scale through concise designed one-pot chemical glycosylation utilizing two readily available donors and one acceptor and a unified deprotection procedure.(2)The collective synthesis of all 63 Core M1 and Core M2 O-mannose glycans were achieved for the first time using a diversity-oriented parallel enzymatic modular assembly strategy from 5 readily accessible core structures.