Chemoenzymatic Synthesis Of Histo-Blood Group Antigen-Associated Complex Glycans

Glycans are ubiquitous components of all living organisms that play important roles in various life processes.Histo-blood group antigens that exist in the form of glycans have attracted wide attention from the synthetic community owing to their diverse structures and meaningful biological functions.Initially,blood group antigens were considered to be antigens only presented on the surface of red blood cells,and their functional studies mainly focused on serology.In the past decades,accumulated evidence disclosed that histo-blood group antigens are widely expressed in many other cells,tissues and body fluids in the form of glycoproteins,glycolipids and free oligosaccharides.The structural diversity of histo-blood group antigens bestowed on them various important biological functions.Histo-blood group antigens play important roles in blood transfusion and blood typing.They act as direct receptors for signal molecules on the surface of cells,directly involved in cell recognition,adhesion,intercellular signal transduction,immune response,inflammation,autoimmune disease,aging,abnormal proliferation and metastasis of cancer cells,as well as pathogen infection.Although histo-blood group antigens have a number of important biological functions,their structural diversity and synthetic challenges have made the acquisition of these complex glycans an obstacle restricting further study of their structures and biological functions.Therefore,exploiting new methods to quickly and efficiently obtain glycans library with these structures is a research hotspot in recent years.This thesis focused on the synthesis of histo-blood group antigens-associated complex glycans.The first part is the systematic synthesis of histo-blood group Sd~a antigens using chemoenzymatic strategy.The second part is the enzymatic synthesis of N-glycans containing different histo-blood group antigen determinants.Which were described as follows:1.Chemoenzymatic synthesis of histo-blood group Sd~a antigensSimilar to some other histo-blood group antigens such as ABH(O),Sd~a blood group antigens are widely present in erythrocytes,tissues and body fluids in the form of glycans.They are involved in many physiological and pathological processes such as cancer,spinal cord injury,malaria infection and neuromuscular diseases.Due to the structural complexity and diversity of Sd~a blood group antigens,the biosynthetic pathway of Sd~a antigens and their biological functions have not been well elucidated.A few previous chemical synthesis mainly focused on the simple Sd~a antigens which require multiple protection and deprotection steps.The overall process is complicated and cumbersome,and the total yield is low.Therefore,the previous chemical approaches are not suitable for the diversity-oriented synthesis of Sd~a antigens.The known enzymatic synthesis generally used enzymes from mammals.The enzymatic strategies are limited by the availability as well as low catalytic efficiency and strict substrate specificity of mammalian enzymes.So it is difficult to apply to the large-scale synthesis of Sd~a antigens.To solve these problems,we developed a chemoenzymatic strategy,which combined the flexibility of chemical synthesis and high efficiency of enzymatic construction of challenging glycosidic linkages to complete the systematic synthesis of 15 complex histo-blood group Sd~a antigens.Details of this project were described as follows:(1)Seven enzymatic assembly modules were designed for the construction of six different glycosidic linkages.Each enzyme module contains the corresponding sugar nucleotide generating enzyme(s)and a glycosyltransferase for the synthesis of corresponding glycosidic linkage from simple monosaccharide donor precursor and appropriate acceptor in one step.(2)Starting from 6 chemically synthesized Oglycans core 2,core 3,type 1,type 2,core 1 and lacto-N-neotetraoside intermediates,12 complex Sd~a antigens including type 1 Sd~a antigens(?-1,?-2),type 2 Sd~a antigens(?-3,?-4),core 1 Sd~a antigens(?-5,?-6),core 2 Sd~a antigens(?-7,?-8),core 3 Sd~a antigens(?-9,?-10),paragloboside Sd~a antigens(?-11??-2)and 3 hybrid Sd~a antigens of A-Sd~a antigen(?-13),B-Sd~a antigen(II-14),H-Sd~a antigen(?-15)were systematically synthesized.2.Enzymatic synthesis of histo-blood group-associated N-glycansN-Glycosylation of proteins is an important post-translational modification.The structures of N-glycans found in nature are very complex and diverse.The structure diversity is mainly due to the diversity of monosaccharide building blocks and their various connection modes as well as different branching patterns.The structural diversity of N-glycan endows its numerous biological functions.N-glycans are involved in a wide range of biological processes,such as protein folding and degradation,inhibition of glycoprotease activity,cell adhesion and transportation,cell signaling,fertilization and embryogenesis,pathogen recognition and immune responses.N-glycans containing histo-blood group epitopes on the cell surface are often associated with cancer,atherosclerosis and many other diseases.Therefore,elucidating the structure and function of N-glycan containing histo-blood group antigen epitopes is of great significance for understanding the physiological and pathological processes,as well as for developing the diagnostic and therapeutic strategies of related diseases.N-glycan,which widely exists in mammals,has become the focus of carbohydrate scientists because of its complex structure and challenge in both chemical and enzymatic synthesis.During the past decades,various strategies including chemical methods,chemoenzymatic methods,and solid-phase synthesis have been developed for the synthesis of N-glycan.However,few of the reported synthesis targeted complex N-glycan structures containing histo-blood group antigens.To tackle these problems,we developed a multienzyme tandem reaction for the synthesis of histo-blood group antigen-associated complex N-glycan,etails of this project were described as follows:(1)Sialylglycopeptide containing N-glycan extracted from egg yolk powder,was enzymatic digested,desialylated with neuraminidase,and treated with galactosidase LacZ to afford the symmetric biantennary N-glycan nonasaccharide and asymmetric biantennary N-glycan octasaccharide.(2)Using 8 different glycosyltransferases and nucleoside activated sugar donors of UDP-Gal,UDP-GalNAc,UDP-GlcNAc,CMP-Neu5Ac,GDP-Fuc,the symmetric and asymmetric biantennary N-glycan nonasaccharide and octasaccharide were elaborated to a series of complex N-glycans containing ABH,Lex,Ley,poly-LacNAc and sialylated LacNAc epitopes.Innovation(1)A bacterial ?1,4-N-acetylgalactosyltransferase CjCgtA was successfully applied to the synthesis of ?1,4-GalNAc linkage of Sd~a epitope for the first time.Previously,only mammalian glycosyltransferase B4GALNT2 is known can be used for the synthesis of this ?1,4-Ga1NAc linkage.Combination of multi-enzyme modular assembly and chemical synthesis,the systematic synthesis of 15 complex and hybrid Sd~a antigens was completed for the first time.(2)A multienzyme tandem reaction was developed for the synthesis of histo-blood group antigens-associated complex N-glycans using 8 different glycosyltransferases.A general approach for the large-scale preparation of key symmetric N-glycan nonasaccharide and asymmetric N-glycan octasaccharide intermediates from sialylglycopeptide extracted from egg yolk powder was achieved via enzymatic digestion of peptide,desialylation,and selective degalactosylation.(3)Bacteria-derived glycosyltransferases and sugar nucleotide generating enzymes used in this study have promiscuous substrate specificity and high catalytic efficiency.Therefore,repeated protective group manipulation and purification steps in chemical synthesis are avoided,and this strategy also can be adapted for the large-scale synthesis of other types of complex glycans.