Chemoenzymatic Synthesis Of Disialyl Tetrasaccharide Epitopes

Di-sialylated ganglioside tetrasaccharide epitopes Neu5Aca2-3Galβ1-3(Neu5Aca2-6)GalNAc widely distribute on the outermost position of cell-surface and play important roles in many physiological and pathological processes. The disialyl tetrasaccharide is found to be the major O-glycan of glycophorin (heavily glycosylated erythrocyte membrane glycoprotein), which prevents the red cells from aggregation and mediates many physiological processes. It is also the special O-glycan of mucin MUC Ⅱ which overexpressed in tumor cells. The disialyl tetrasaccharide is a special component at the non-reducing end of gangliosides GD1α, GT1aa, and GQ1bα and the minimal binding epitope for high-affinity myelin-associated glycoprotein (MAG) ligands. It is also the only O-glycan of erythropoietin (EPO).It is of great value to synthesize the di-sialyl tetrasaccharide in large quantities in order to study its functions at the molecular levels. Developing an efficient and practical synthesis approach will promote the process of drug discovery of lead compounds. However, these sialosides related to its complexity and instability make it difficult to separatie and purify. In order to evaluate its biological functions and significance at the molecular levels, developing an simple and efficient synthetic approach for synthesis of disialyl tetrasaccharides and their derivatives is imperative.Several elegant chemical or chemoenzymatic synthetic methods for these disialyl tetrasaccharides and related antagonists have been reported. However, strategies for pure chemical synthesis must address a host of issues, including tedious blocking and deprotection manipulations, low yield and low stereoselectivities. Furthermore, for the unique structure of nine-carbon sugar N-acetylneuraminic acid, the formation of the glycosidic bond is still considered one of the most difficult problems in synthesizing carbohydrates.Two distinct sialyltransferases (a2-3-sialyltransferase, PmST1and a2-6-sialyltransferase, Pd2-6ST) are required to introduce two N-acetylneuraminic acid (Neu5Ac) to the C3’and C6positions of Galβ1-3GalNAc disaccharide core. Thus far, only a few recombinant N-acetylgalactosamine a2-6sialyltransferases (ST6GalNAc) from mammalian sources have been employed to the synthesis of Neu5Aca2-6GalNAc sequence. A recombinant α2-6-sialyltransferase from chicken (chST6GalNAc I) and a recombinant a2-3-sialyltransferase from porcine (pST3Gal I) have been successfully utilized for enzymatic production of di-sialyl TF-antigen.However, two major problems encountered in these approaches:(1) Mammalian sialyltransferases are type II transmembrane protein and it is diffcult to realize high yield expression using existing technologies.(2) These mammalian sialyltransferases have strict acceptor substrates specificity. For example, the two mammalian sialyltransferases in related report only have activity for glycopeptide substrates.In contrast to mammalian sialyltransferases, several bacterial sialyltransferases that have been cloned and expressed in E. coli can be produced in sufficient amounts and conveniently purificated, owning remarkable expression amount and promiscuous substrate specificities. So, considering the full advantages of chemical synthesis and enzymatic synthesis, we herein report a chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes and their derivatives. In order to solve these problems, we carried out the research from the following aspects:(1) One-pot multienzyme (OPME) systemWe have efficiently synthesized β1-3linked galactosides,α2-3sialosides and a2-6sialosides using OPME system developed by our collaborators.(2) Synthesis of disialyl tetrasaccharide epitopes in glycorandomizationRandom sialylation of disaccharide Galβ1-3GalNAc and trisaccharide Neu5Aca2-3Galβ1-3GalNAc were investigated. Our previous finding showed that both terminal Gal and GalNAc can be recognized by Pd2-6ST to form Neu5Aca2-6Gal and Neu5Aca2-6GalNAc, respectively. Pd2-6ST was able to add Neu5Ac to both C6-OH of the internal GalNAc and C6’-OH of the terminal Gal.(3) Regioselective sialylation of conformationally constrained trisaccharide acceptors To circumvent the issue of substrate promiscuity of Photobacterium damselae (Pd2-6ST), we herein report a chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes and their derivatives through regioselective sialylation of conformationally constrained trisaccharide acceptors by utilizing a bacteria a2-6-sialyltransferase from Photobacterium damselae (Pd2-6ST).(4) Chemoenzymatic synthesis of disialylated tetrasaccharide derivativesPrevious structure-activity relationship (SAR) studies of MAG and disialyl tetrasaccharide epitopes have demonstrated that modification of Neu5Ac by introducing hydrophobic substituents at the C9position in the Neu5Aca2-3GalNAc sequence can significantly increase the binding affinity of the glycan and MAG. Encouraged by these results, chemoenzymatic synthesis of disialyl tetrasaccharide epitope containing a non-natural sialic acid9N3Neu5Aca2-3-linked to the Gal was carried out using the efficient lactone method described above. Meanwhile, the method has the general applicability. A similar high efficiency was achieved for the chemoenzymatic synthesis of disialyl tetrasaccharide containing the sialic acid N-glycolylneuraminic acid (Neu5Gc) and related analogues.In summary, we herein report a chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes and their derivatives through regioselective sialylation of conformationally constrained trisaccharide acceptors by utilizing bacteria Pasteurella multocida a2-3-sialyltransferase (PmST1) and Photobacterium damselae α2-6-sialyltransferase (Pd2-6ST). This strategy provides a new route for easy access of disialyl tetrasaccharide epitopes and their derivatives, solving the problems exsiting in chemical and chemoenzymatic methods. The application of similar stratigies can be explored for other substrates and for other acceptor substrate promiscuous enzymes. The research results are provided with originity and significance, revealing promising application.The main conclusions in this paper were as follows:(1) This manuscript investigated the substrate selectivities of a2-6-sialyltransferase on sialylation of Galpl-3GalNAcβProN3,Galβ1-3GalNAcaProN3,Neu5Aca2-3Galβ1-3GalNAcβProN3xNeu5Aca2-3Galβ1-3GalNAcaProN3,9N3Neu5Aca2-3Galβ1-3GalNAcβProN3,Neu5Gca2-3Galβ1-3GalNAcβProN3,Neu5Aca2-3Galβ1-3GalSEt and trisaccharide lactone and found the characteristics of substrate specificities of Pd2-6ST.(2) To circumvent the issues with the promiscuous substrate specificities of bacterial sialyltransferases Pd2-6ST and low yield in synthesis of natural disialyl tetrasaccharide, we herein report a chemical controlled strategy for the synthesis of disialyl tetrasaccharide through changing the promiscuous substrate specificities of Pd2-6ST. This strategy binds flexibilities of chemical synthesis and efficiency of enzymatic synthesis. In addition, bacterial sialyltransferases can be produced in sufficient amounts in convenient bacterial expression systems and easy purification. Therefore, this strategy provides a new route for easy access to disialyl complex oligosaccharide.(3) The chemoenzymatic synthesis of disialyl tetrasaccharide epitope containing the non-naturalsialic acid9N3Neu5Aca2-3-linked to the Gal using the efficient lactone method was firstly described. The9N3-group can be used as a chemical handle for easy derivatization to expand compound libraries and also lead to the development of lead compounds.(4) The α2-3-linked trisaccharide containing N-glycolylneuraminic acid (Neu5Gc), a nonhuman sialic acid form with an additional hydroxyl group at C5-NHAc, was also compatible with the regioselective sialylation approach. The general applicability of the method can be further explored for other substrates and for other acceptor substrate promiscuous enzymes.