| Lewis antigens belong to the family of fucosylated carbohydrate determinants or epitopes.They are common carbohydrate determinants and ubiquitous components of various cell surface glycoconjugates and secreted unconjugated glycans,which mediate or modulate interactions among cells and between cell and extracellular environment,thus play essential roles in many biological and pathological processes.The structural diversity and complexity of Lewis-related antigens bestow them a broad spectrum of significant biological functions.For example,Lex antigen,a stage-specific embryonic antigen(SSEA-1/CD15),is abundant in the poly-LacNAc chains on the embryo cell surface during tissue development.sLex and sLea antigens are ligands for selectins(P-,L-and E-selectin)for leukocytes rolling,cell signaling,and chemotaxis in inflammation.Lex,sLex,Ley,and sLea are common tumor-associated carbohydrate antigens(TACAs),and their high overexpression on a range of carcinomas is correlated with cancer progression and poor prognosis.These TACAs have been used as biomarkers for cancer clinical diagnosis and antigenic targets for the development of carbohydrate-based cancer immunotherapies.Furthermore,these Lewis-related antigens on the host cells have been used as receptors for various bacterial,viral,and parasitic infections,and many pathogens also express these antigens as molecular mimicry to escape from the host immune surveillance.Moreover,these Lewis-related antigens are common determinants for many well-characterized human milk oligosaccharides(HMOs),which are important for shaping the gut microbiota.Lewis antigens are a family of fucosylated N-acetyllactosamine(Galβ1,4GlcNAc,LacNAc or LN,a type-2 chain)or lacto-N-biose(Galβ1,3GlcNAc,LNB,a type-1 chain)structures.Lewis x(Lex),sialyl Lewis x(sLex),and Lewis y(Ley)share the same type-2 chain as their biosynthetic precursor,while Lewis a(Lea),sialyl Lewis a(sLea),and Lewis b(Leb)are biosynthesized from the type-1 chain.The Ley and Leb determinants with additional α1,3-linked N-acetylgalactosamine(GalNAc)or galactose(Gal)at the non-reducing end are the chimeric histo blood group antigens ALey,BLey.ALeb,and BLeb,respectively.These 10 Lewis antigens are common termini and among determinants on the poly-LacNAc structures which are a type of glycan chains with repeating β1,3-linked LacNAc or/and LNB units and are ubiquitous components of various O-glycans,N-glycans and glycolipids in human cells,parasites,and bacteria.Owing to their significant biological functions and great promises for biomedical applications,the synthesis of these structurally complex Lewis-related antigens has gained significant interests for decades.Since the first chemical synthesis of Lea antigen by the Lemieux group in 1975,numerous chemical or enzymatic strategies have been explored for the assembly of these complex Lewis-related antigens,such as one-pot chemical synthesis,automatic solid-phase synthesis,and one-pot multi-enzyme synthesis.Most recently,chemoenzymatic syntheses of Lewis antigen branched asymmetric N-glycans,O-glycans,and HMOs have also been achieved.Despite tremendous progresses on the chemical and chemoenzymatic synthesis of poly-LacNAc glycans and complex Lewis carbohydrate determinants in the past decades,the regioselective introduction of Lewis antigen on poly-LacNAc glycan chain is still a formidable challengeTraditional chemical synthesis of Lewis antigens is involved in multiple tedious protection-deprotection manipulations and the numerous purification procedures which waste more time and need the professional technology for researchers.Biosynthesis of Lewis antigens involves multiple fucosyltransferases that catalyze the fucosylation of poly-LacNAc carbohydrate backbone in a non-site-specific manner,thus generating heterogeneous and incomplete fucosylated Lewis antigen regioisomers.In this study,taking advantage of the substrate specificities of bacterial a2,6-sialyltransferase and al,3/4-fucosyltransferases,an a2,6-sialylation module was used to introduce a2,6-linked sialic acid to specific sites to protect them from fucosylation,thus precisely controlling enzymatic fucosylation of poly-LacNAc glycans in a site-specific manner.The sialic acids can be easily removed by sialidase after fucosylation to provide a variety of fucosylated poly-LacNAc glycans with defined fucosylation patternsThis thesis contains the following parts:(1)The substrate specificities of 4 bacterial glycosyltransferases,including aα1,3-fucosyltransferase from Helicobacter pylori(Hpαl,3-FucT),a bifunctionalα1,3/4-fucosyltransferase from Helicobacter pylori(Hpαl,3/4-FucT)，aα2,6-sialyltransferase from Photobacterium damselae(Pd2,6ST),aβ1,3-N-acetyl-glucosaminyltransferase from Helicobacter pylori(HpLgtA),were investigated.A reprogrammed enzymatic assembly line was developed for the site-specific fucosylation of complex poly-LacNAc glycans.(2)The reprogramming enzymatic assembly strategy was used for the site-specific al,3-fucosylation modification of type-2 poly-LacNAc glycans.Through sequential glycosylation with 4 enzyme modules and enzymatic desialylation,all 7 Lex antigens were synthesized,including 3 mono-fucosylated Lex antigens,3 fucosylated di-Lex antigens,and 1 fucosylated tri-Lex antigen.(3)The reprogramming enzymatic assembly strategy was explored for the site-specific α1,3-or α1,4-fucosylation modification of type-1 and type-2 hybrid glycan chains.Through sequential glycosylation with 7 enzyme modules and enzymatic desialylation,2 Lex antigens,2 Lea antigens,Leb antigen,Ley antigen,and 4 chimeric histo-blood group antigens with defined fucosylation pattern were synthesized.(4)To further highlight the power of this reprogramming enzymatic assembly strategy,5 enzyme modules and the desialylation were reprogrammed for the site-specific α1,3-fucosylation modification of the more complex poly-LacNAc carbohydrate backbone.Complex VIM-2 antigen dodecasaccharide,sialyl di-Lex antigen tridecasaccharide,and regioselectively di-fucosylated biantennary N-glycan pentadecasaccharide were successfully synthesized.The main novelties were listed as below:(1)The concept and strategy of organic synthesis was incorporated into the enzymatic synthesis of oligosaccharides.A reprogramming enzymatic assembly strategy was developed,which overcame the limitation of the oligosaccharide biosynthetic pathway,and provided the first generally applicable method for the precise synthesis of complex Lewis-related antigens.(2)The reprogramming enzymatic assembly strategy was successfully applied for the synthesis of a variety of fucosylated poly-LacNAc glycans with defined α1,3-or α1,4-fucosylation patterns.The general applicability and robustness of this reprogrammed enzymatic assembly line was exemplified in the synthesis of 18 complex Lewis antigens with a total of 8 enzyme modules for the construction of 8 different glycosidic linkages.This work provided standard compounds for the characterization and quantification of complex natural fucosylated oligosaccharides,and makes it possible to systematically study the biological functions of complex glycan families. |
PDF Full Text Request