Chemoenzymatic Synthesis Of Fluorinated GM3 Carbohydrate And Its Immunological Evaluation

Tumor-associated carbohydrate antigens are an important marker on the surface of tumor cells.GM3,a sialylated trisaccharide antigen,is one of the most abundant TACAs in several types of tumors,such as malignant melanoma and neuroectodermal tumors.GM3 is an endogenous substance with poor immunogenicity and cannot trigger the T cell-mediated immunity that is necessary for cancer immunotherapy.It is also easily hydrolyzed by endogenous glycosidases and lost the integrity of the antigen,thereby reducing its immunogenicity.In order to solve this problem,modification of the GM3 antigen structure has become an effective solution.In this study,fluorine atoms was introduced into the natural structure of GM3,aiming to using the characteristics of fluorine atom itself,such as,enhancing the immunogenicity,metabolic stability,fat solubility,and bioavailability.In order to increase the immunogenicity of the antigen,four GM3 derivatives in this paper were also covalently coupled to carrier proteins.Due to the low reactivity of the fluorinated sugars,the glycation reaction needs to be carried out under severe reaction conditions,which leads to low synthesis efficiency and yield,hindering the development of vaccines.In order to solve the bottleneck problem of difficult preparation of GM3 and its fluorinated derivatives,a recombinant E.coli-derived aldolase(Pm.Aldolase),CMP-sialic acid synthetase(NmCSS)and Pasteurella multocida α2,3-sialyltransferase(PmST1)-catalyzed "one-pot three enzymes" reaction strategy was developed for efficient and rapid preparation of GM3 and its fluorinated derivatives.The details were as follows:(1)Lactose receptor LacβProN3 containing propyl azide at the reducing end was prepared in 47%yield by 6 step reaction using lactose as starting material;(2)A series of mannosamine derivatives were prepared as precursors required for the enzymatic reaction;(3)GM3 and its derivatives were afforded in 77～91%yield by "one-pot three enzymes"reaction with mannosamine derivatives as donors and LacβProN3 as the receptor;(4)A series of glycoconjugates were successfully prepared by coupling the glycoantigen with CRM 197 through a bifunctional group DSA.Also,the sugar loading of the glycoconjugates were measured by colorimetry;(5)A mouse immune experiment was carried out to determine the titer level of IgG antibody in serum.The experimental results show that the change of the sialic acid type in the sugar antigen,which is,Neu5Gc replacing Neu5Ac can enhance the antigen immunogenicity;the IgG titer of V3 is 11200,the results show that the introduction of fluorine atoms can significantly enhance the immunogenicity of natural antigens.At the same time,a large change in antigen structure,namely the introduction of a benzene ring at the C5 position of sialic acid,the IgG titer of vaccine V4 is 16000.The results show that the greater the structural modification of the natural sugar antigen,the stronger the immunogenicity.The results of the paper provide theoretical basis and data supports for further design and synthesis of GM3 tumor vaccine.