Chemoenzymatic Synthesis Of Sialylated Thomsen-Friedenreich(TF) Antigens And Their Fluorinated Derivatives

Tumor-associated carbogydrate antigens are an important marker on the surface of tumor cells.It is also a main direction in the research and development of anti-tumor vaccine.In recent years,with the deeper understanding of TACAs,the importance of designing cancer vaccine through chemical synthesis has been gradually recognized.Therefore,obtaining TACAs with stable structure of is very important for the study of its biological activity and the development of anti-tumor vaccine.The Thomsen-Friedenreicht antigen is a di saccharide containing galactose,N-acetylgalactosamine residue and a erythrocyte antigen exposed to the action of a bacterial neuraminidase,in which GalNAcα-can be linked to the Ser or Thr residue of a mucin by an oxyside bond or an azoside bond.TF-Ag is a very important tumor target because it is expressed in breast cancer,colon cancer,bladder cancer,prostate cancer,liver and stomach cancer.Cell adhesion mediated by TF-Ag plays an important role in metastasis,it can stimulate the body to produce humoral immunity,thereby killing tumor cells,and can also block the diffusion ability of tumor cells.The first study of the tumor vaccine targeting TF-Ag was published by the Georg Spillier group in 1995.However,due to the immunological tolerance and the immunogenicity of natural Tn/sTn/TF and other antigens is weak,which can not stimulate the body to produce a strong immune response.In order to solve this problem,researchers began to modify and change the structure of natural tumor-associated sugar antigen.The results showed that the introduction of F atom can improve the immunogenicity of tumor vaccine.Although fluorinated TACAs can improve the immunogenicity of tumor vaccine,there are existing difficulties in chemical synthesis,such as complex operation,harsh conditions and low yield.Therefore,it is urgent to find a new strategy to obtain large quantities fluorinated TF antigens to study their activities.In this thesis,we successfully carried out a strategy of "one-pot three enzymes"system catalyzed by a recombinant E.coli-derived CMP-sialic acid synthetase and adolase,and sialyltransferase PmSTI derived from the source of the multocimultocida.The strategy can be used to synthesize TF antigen and its fluorinated derivatives in large quantities with high efficiency and selectivity.Firstly,using galactosamine hydrochloride as the starting material,the reductive terminal galactoside intermediate with 3-azidopropyl and 3-OH exposed as the receptor was synthesized by chemical method.Using 6-F substituted total acetyl galactosimide as donor,TF and 6-F-TF antigen were synthesized by chemical synthesis.Secondly,we modified the structure of the ManNAc at C-2 or C-6 to get ManGc,ManNHTFA,6-F-ManNAc,6-N3-ManNAc and other intermediates as precursors compounds for "one-pot three enzymes" method.Subsequently,the sTF antigen,sTF derivative and its lactone compounds were synthesized efficiently and rapidly by using the strategy of "one-pot three enzymes".Alsowe studied the stability of these compounds.Finally,the oligosaccharide-protein conjugates were successfully obtained by combining the oligosaccharides with BSA by squaric acid chemistry in order to facilitate the later activity study.The main achievements of this paper include the following aspects:(1)The sTF antigens and fluorinated sTF were synthesized successfully by using the "one pot three enzymes" system.(2)The firstly successful synthesis of the lactone structure of STF antigen and its derivatives by chemical method,which can prevent the Neu5Acα2-3Gal from free rotation,and provide the basis for further study of substrate specificity of sialyltransferase on thetrisaccharide level(3)In order to study the stability of fluorinated sTF,we study on the influence of the introduction of fluorine substituent on the stability of sialic acid glycoside bond under the action of sialic acid glycosidase hydrolysis.(4)The glycoprotein conjugate were obtained by connecting fluorinated sTF to BSA by squaric acid chemistry.It provides the basis for the immunogenicity study and cross recognition of antibody...