Sequential One-pot Multienzyme (OPME) Synthesis Of Lacto-N-Neotetraose And Its Derivatives

Human milk oligosaccharides (HMOs) are the third largest solid component of human milk following lactose and lipids. Lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3 Galβ1-4Glc, LNnT) is the essential component and one of the two core structures (lacto-N-tetraose, Galβ1-3GlcNAcβ1-3Galβ1-4Glc, LNT and LNnT) of HMOs, which can be a-sialylated and/or a-fucosylated at various positions to generate a series of complex HMOs. So far, over 100 structurally well characterized HMOs have been isolated which exhibit significant differences in composition and complexity from those of any other mammal.There is convincing evidence that HMOs exert broad beneficial effects for the breast-fed infants, such as encouraging the growth of gut beneficial organisms, protection from viral infections, prevention of pathogen adhesion and modulating the immune responses. Several HMOs including LNnT have been evaluated in the clinical trials for possible use in infant formula and foods. In addition, LNnT and its sialyl and/or fucosyl derivatives are also the common components of various O-glycans and N-glycans which mediate cell-cell adhesion and signal transduction in many physiological and pathogenic processes. For example, Lewis x (Lex) and sialyl Lewis x (sLex) carbohydrate ligands of selectins play important roles in the inflammatory response. Moreover, several LNnT derived epitopes such as Le" pentasaccharide and sLex hexasaccharide containing tumor-associated carbohydrate antigens (TACAs) were found overexpressed in various cancer cells, and have been used as ideal antigens for the development of TACA-based cancer vaccines.The biological importance and structure complexity of LNnT and its sialyl and/or fucosyl epitopes have attracted considerable attention from the synthetic community in the past two decades. Many chemical and enzymatic approaches have been explored for the synthesis of LNnT and its sialyl and fucosyl derivatives, such as linear synthesis, automated solid-phase assembly, one-pot synthesis, enzymatic synthesis, chemoenzymatic synthesis and whole-cell fermentation. However, traditional chemical synthesis is usually time consuming and labor intensive due to the iterative "protection-glycosylation-deprotection" steps and the extensive purification procedures which are necessary to achieve high purity and structural homogeneity. On the other hand, enzymatic synthesis is limited by the sources and strict substrate specificity of the enzymes used. Besides, due to the difficulty of product isolation, whole-cell fermentation is also not the best choice. Therefore, a practical preparative-scale synthesis of LNnT and its sialyl and fucosyl derivatives is still highly desired.To solve the above problems, this thesis developed a sequential one-pot multienzyme (OPME) system for the synthesis of LNnT and its sialyl and/or fucosyl derivatives. The research was mainly carried out from the following aspects:(1) The core structure of LNnT was synthesized by OPME 1 (β1-3-N-acetylglucosaminyltransferase, HPLgtA) and OPME 2 (β1-4-galactosyl-transferase, NmLgtB) systems initiated from lactoside.(2) OPEM 3 (a2-3-sialyltransferase, PmST1 M144D) and OPEM 4 (al-3-fucosyltransferase, Hp1,3FucTA66) was sequentially exploited for the synthesis of biological significant Lex or sLex containing structures initiated from LNnT.(3) Finally, without any further manipulation, selective a2-6-sialylation of the internal galactose of LNnT was achieved by OPEM 5 (a2-6-sialyltransferase, Pd2,6ST), just changing the sequence of the enzymatic glycosylation.In summary, tetrasaccharide LNnT, sialyl LNnT (3"’-sLNnT), Lex pentasaccharide (LNFP-III), sLex hexasaccharide (3"’-sLNFP-III) and the internally sialylated LNnT derivatives(6’-sLNnT. DSLNnT and 6’-sLNFP-III) were successively prepared in preparative-scale using the sequential one-pot multienzyme approach. Among them, derivatives of 6’-sLNnT, DSLNnT and 6’-sLNFP-III were synthesized for the first time.The main novelties were listed as follows:(1) A sequential one-pot multienzyme strategy was developed for the large-scale synthesis of the middle galactose sialylated LNnT derivatives for the first time.(2)Selective a2-6-sialylation of the internal galactose of LNnT was achieved by just changing the sequence of the enzymatic glycosylation for the first time.