Enzymatic Modular Synthesis And Automated Enzymatic Synthesis Of Oligosaccharides

Glycosylation,one of the important post-translational modifications of proteins,plays vital roles in many biological processes.Changes of glycoforms are highly related with cell status and the processes of many diseases.Thus,it is important to study the structures of glycans and their related functions.However,the synthesis of structure defined glycans in large scale is still a big challenge.Therefore,many studies focus on the development of convenient and efficient strategies to obtain structurally well-defined complex glycansThis work mainly focused on the synthesis of two type of functional glycans,poly-LacNAc and O-GalNAc glycans,with manual and automated synthesis strategies based on enzymatic modular synthesis systems,which were described as follows.1.Enzymatic modular synthesis of poly-LacNAc and its derivativesPoly-N-acetyllactosamine glycans are the ubiquitous components of various N-glycans,O-glycans and glycosphingolipids.They play pivotal roles in numerous biological processes,including inflammation,cell adhesion,and cancer cell metastasis.Due to the biological importance of poly-LacNAc derivatives,many chemical and enzymatic synthetic strategies have been developed.However,current chemical synthesis strategies suffer lower regio-and stereo-selectivity,multi-steps of protection and deprotection and low total yield.In contrast,current enzymatic synthesis strategies are hindered by the employment of expensive sugar nucleotides and limited glycosyltransferases.Thus,a practical preparative-scale synthesis of poly-LacNAc glycans with various lengths and their sialyl and/or fucosyl derivatives is highly needed.Herein,we report a robust and efficient enzymatic modular assembly strategy for the preparative-scale synthesis of poly-LacNAc derivatives.Details of this project were described as follows.(1)Four enzymatic synthesis modules for introducing GlcNAc,Gal,Fuc and Sia were designed.Accessible bacterial glycosyltransferases were employed in combination with corresponding sugar nucleotide generating enzymes,thus the expensive nucleotide activated sugar donors would be generated in situ which paves the way for preparative-scale synthesis of complex oligosaccharides.(2)With the employment of enzymatic synthesis modules,poly-LacNAc derivatives with different lengths were synthesized in preparative-scale.Additionally,by changing the sequence of enzymatic synthesis modules,we successfully obtained VIM-2 containing glycans.(3)Poly-LacNAc glycan array was printed and the binding specifities of glycans with GBPs were investigated by using the array.We found the glycan length and the postion of glycan epitope would affect the binding affinity of glycan with glycan binding proteins.2.Chemoenzymatic synthesis of O-GalNAc glycansO-GalNAc glycans(also termed as mucin O-glycans)represent one of the major components of mammalian glycocalyx.O-GalNAc glycans have 8 Cores,among them 5 Cores can be extended with various glycan determinants to form the repertoire of O-glycans.Synthesis of O-glycopeptides attached with simple O-glycans by solid-phase synthesis has been well developed.However,strategies to access large numbers of O-GalNAc glycans with various natural epitopes are still limited.Thus,it is an urgent need for the development of an efficient strategy for the synthesis of O-glycan library.To rapidly access structurally diverse O-GalNAc glycans,we developed a Core synthesis-enzymatic modular assembly strategy for the synthesis of 83 O-GalNAc glycan library.Details of this project were described as follows.(1)According to structures of different antigenic epitopes and characteristics of different types of glycosyltransferases,13 enzymatic synthesis modules were designed.(2)With the employment of 13 enzymatic synthesis modules,83 O-GalNAc glycan libraries containing different epitopes were obtained starting from 5 different Core structures.(3)O-GalNAc glycan array was printed and investigated with glycan binding proteins.The array assay showed that lectins that used for identification of O-GalNAc glycans had low affinity toward O-glycan and they mainly recognize some specific glycan epitopes on O-glycans,while glycan binding antibodies show high affinity than lectins.3.Machine-driven enzymatic oligosaccharide synthesisGlycans are mainly achieved by chemical synthesis and enzymatic synthesis.Incontrast to the solid sphase automatic synthesis of nucleic acid and peptide,the stepwise synthesis of glycans still a time consuming process.Therefore,it is urgent to develop simple and effective automatic approach for the synthesis of oligosaccharides.A machine-driven enzymatic synthesis strategy was developed and successfully applied for the assembly of complex poly-LacNAc glycans and other important glycans.Details of this work were described as follows.(1)A machine-driven enzymatic synthesis strategy was developed based on thermosensitive polymer PNIPAM and peptide synthesizer.(2)Using a panel of designed enzymatic synthesis modules,the automated synthesis system was successefully applied for the synthesis of complex oligosaccharides,such as GM1,ABO blood group antigens and poly-LacNAc glycans.4.Innovation(1)Preparative scale synthesis of poly-LacNAc and its sialylated and fucosylated derivatives through an enzymatic modular assembly strategy were achieved.The Lex tetramers were obtained for the first time.(2)Using designed enzymatic modular sequence,VIM-2 series glycans were synthesized.(3)A repertoire of 83 O-GalNAc glycans with different epitopes were synthesized by the employment of Core synthesis-enzymatic extension strategy for the first time.(4)A fully automated system is reported for oligosaccharides synthesis based on the use of a thermosensitive polymer and a commercially available peptide synthesizer,(5)Synthetic poly-LacNAc and O-GalNAc glycan microarrays were prepared for the first time.