Enzymatic Modular Assembly Of Chimeric Blood Group Antigens

Chimeric blood group antigens are mainly found on the cell surface of erythrocyte,endothelial and epithelial cells,as well as in the human kidney,urogenital system,gastrointestinal tract and other tissues.The carbohydrate determinants of chimeric blood group antigens present on the cell surface in the form of glycoproteins or glycolipids.The structures of chimeric blood group antigens of ALey,BLey,ALeb and BLeb comprise the essential fragments of both ABO(H)blood group antigens and Lewis blood group antigens.For the biosynthesis of ALey and BLey characteristic pentasaccharides structures,the non-reducing end of type 2 disaccharides(Gal?1,4GlcNAc)are firstly extended to the A antigens or B antigens,then fucosylated modification was carried out at the C-3 of GlcNAc unit to form the ALey and BLey antigens pentasaccharides.The ALeb and BLeb characteristic pentasaccharides structures are biosynthesized from the non-reducing end of type 1 disaccharide(Gai?1,3GlcNAc).The type 1 disaccharides are converted into A antigens or B antigens,respectively,then the A or B antigens are fucosylated at the C-4 of GlcNAc unit to form the characteristic pentasaccharides of ALeb and BLeb.The carbohydrate determinants of chimeric blood group antigens can integrate with proteins or lipids to form glycoconjugates or exist as free oligosaccharides or glycans,which can mediate or regulate the interaction between cells and cells or cells and extracellular environment.It has been reported that can competitively bind with pathogenic microorganisms such as helicobacter pylori and norovirus.Owing to its important biological significances,the synthesis of chimeric blood group antigens has attracted extensive attentions in recent years.Due to the complexity of the chimeric blood group antigens,the synthesis of this glycans remains extremely challenging.So far,Only Bovin research group of the Russian Academy of Sciences has completed the chemical synthesis of these antigens in low yields.The synthesis of the carbohydrate determinants of chimeric blood group antigens using enzymatic methods are no challenging.How to site-specific fucosylation has always been a huge challenge for bacterially derived glycosyltransferases.Although chemically or enzymatically can synthesizes characteristically glycan chains.but no enzymatic synthesis pathway can accurately introduce fucose at multiple fucosylation sites of the glycan chains.In order to solve this problem of synthesis in low yield and site-specific fucosylation,An innovative synthesis strategy was developed in the thesis.Through reprogramming the enzymatic assembly line for site-specific fucosylation.This thesis mainly includes the following aspects:(1)Two di-sialylated octasaccharides were synthesized from LactosepProN3 disaccharide by sequential elongation using four enzyme modules.The glycan chains containing Type 1 and Type 2.Sialic acid as a temporary"protection group".The di-sialylated octasaccharides as key intermediates were converted into sialic acid-protected blood group H antigens by ?1,2-fucosylation using enzyme module 7(EM 7).(2)Using blood group H antigens as substrates,Four blood group A and B antigens decasaccharides were parallelly synthesized using enzyme module 8(EM 8)or enzyme module 9(EM 9)for N-acetyl-galactosaminylation or galactosylation at the non-reducing terminal of Gal.(3)Using sialic acid-protected blood group A and B antigens as substrates,Four sialic acid-protected chimeric blood group antigens of ALey,BLey,ALeb and BLeb undecasaccharides were parallelly synthesized using enzyme module 4(EM 4)or enzyme module 6(EM 6)for site-specific fucosylation.(4)After desialylation using sialidase,Four chimeric blood group antigens of ALey,BLey,ALeb and BLeb undecasaccharides were parallelly synthesized.The main innovative points of this thesis including the following two aspects:(1)An innovative synthesis strategy was developed in the thesis.Using reprogramming the enzymatic assembly line for site-specific fucosylation.Introducing the a2,6-sialylation assembly module to the synthesis of glycan chains although it has no need to synthesize skeletal glycans chains.The competitive reaction of a2,6-salivary acidification and a 1,3-fucosylation or a1,4-fucosylation was used to precisely control the modification site of fucosylation.The precise fucosylation of skeletal glycans chains were realized(2)Through the reprogramming of the enzymatic assembly of the glycans chains,Four complex chimeric blood group antigens of nonasaccharides was realized efficiently synthesis for the first time.