Chemoenzymatic Synthesis Of Human Milk Oligosaccharide Lsta And Haemophilus Parasuis Capsular Polysaccharide Repeating Units Related To Strains Serovar 5

This thesis contains two aspects of chemoenzymatic synthesis that are human milk oligosaccharides LSTa and Haemophilus parasuis capsular polysaccharide repeating units related to strains serovar 5.1 Chemoenzymatic synthesis of human milk oligosaccharides LSTaHuman milk oligosaccharides (HMOs) are complex glycans that are present at very high concentrations in human milk, following lactose and lipids, with concentrations ranging in 20-25 g/L in colostrum. HMOs are known as "the first prebiotic", which play important roles in the early development of the infant digestive system, and improving the immune system and the ecological balance of newborns. Previous studies have shown that human milk oligosaccharides have various biological functions, including the role of prebiotics that are maintaining intestinal flora balance, resisting pathogen infection、viruses and tumor cell invasion, adjusting the immune response and promoting early development of the neonatal brain etc. Moreover, there are other mechanisms of biological functions and features becoming sugar hotspot areas.Nowadays, more than 100 structurally well characterized HMOs have been isolated and indentified, which share a common core structure: lacto-N-tetrasaccharide (Galβ1-3GlcNAcβ1-3Galβ1-4Glc, LNT). Most of the components of HMOs are derived by α-sialylated and/or α-fucosylated at various sites to generate array of complex human milk glycans. Because of their biological significances and structural complexity, it is imperative to acquire the sufficient amount of homomgeneous HMOs to carry out their biological functions and develop their research in the field of food and medicine, such as artificial milk formula. In recent years, several elegant chemical synthesis, microbial and coupled enzymatic approaches and chemoenzymatic synthesis have been developed for the synthesis of LNT and its derivatives. However, chemical synthesis often encounters many steps, demanding operating conditions, and the very low total yield while enzymatic approaches and chemoenzymatic synthesis need to address the problems of the source of enzymes, protein less, adaptive narrow substrate and separation and purification of complex issues. Therefore, a simple and practical approach for the preparation of lacto-N-biose donor and lactoside acceptor building blocks is still highly desirable for convergent synthesis of LNT and their derivatives.Recently, we have developed an efficient one-pot two-enzyme approach for the synthesis of β1-3-linked galactosides using a recombinant Escherichia coli galactokinase (EcGalK) and a novel D-galactosyl-β1-3-N-acetyl-D-hexosamine phosphorylase cloned from Bifidobacterium infantis (BiGalHexNAcP). Herein we report a convergent chemoenzymatic approach for the highly efficient synthesis of human milk tetrasaccharide LNT containing a 3-azidopropyl spacer that can be sialylated to afford the common sialyllacto-N-tetraose a (LSTa). The Lacto-N-tetraose represents one of the most common core structures, which can be exploited through enzymatic sialylation and/or fucosylation to prepare a wide array of complex human milk oligosaccharides.The main achievements of our project include the following aspects:(1) The readily accessed Lacto-N-biose and lactoside building blocks can be used as key intermediates for the enzymatic or chemical generating various sialylated and/or fucosylated building blocks for the convergent chemical synthesis of diverse HMOs.(2) A highly efficient "one-pot two-enzyme oligosaccharide synthesis strategy" was successfully developed for expeditious synthesis of lacto-N-tetraose to obtain on preparative-scale and lacto-N-tetraose was sialylated for LSTa by "one-pot three-enzyme" catalytic system.(3) In contrast to common oligosaccharide chemoenzymatic synthetic approach, we either use an enzymatic produced building block for chemical synthesis, or use a chemical synthesized building block as a substrate for further enzymatic synthesis. Our approach takes advantage of enzymatically synthesized building blocks for chemical synthesis with additional enzymatic modifications.2 Chemoenzymatic synthesis of Haemophilus parasuis capsular polysaccharide repeating units related to strains serovar 5Haemophilus parasuis (Hps), a Gram-negative bacterium requiring a source of nicotinamide adenine dinucleotide (NAD) and a member of the family Pasteurellaceae, is a colonizer of the upper respiratory tract of pigs as a commensal but is also often found associated with pneumonia and the etiological agent of Glasser’s disease. Glasser’s disease depends on invasion of the bacteria into the systemic compartment and is characterized by fibrinous polyserositis, polyarthritis, meningitis, acute pneumonia without polyserositis and acute septicaemia. It imposes a significant economic and welfare burden on the global pig industry, resulting in a high demand for the use of expensive antimicrobials and mortality of animals in acute form of the disease. Classically, Haemophilus parasuis strains have been classified by serotyping, using soluble antigens (mainly surface polysaccharides and lipopolysaccharides). Wherein the capsular polysaccharide and lipopolysaccharide play an important role in bacterial infection of host cells, but the specific pathogenic mechanisms are unclear. Among the 15 known serovars, serotype 5 (to which Nagasaki strain belongs) shows highly virulent and is one of the most prevalent serotypes, but the virulence factors of Hps remain to be studied.The capsule structures of serovars 5 were recently published, which contained the same main chain with a unique disaccharide repeating unit of β-glucose-6P and 2,4-diacetamido-2,4,6-trideoxy-D-galactopyranose, substituted with α-Neu5R-3-a-Gal-1-P in the serovar 5 strain, where R represents an N-acetyl or N-glycolyl group. This novel and unique molecular structure has no relevant synthesis report, so we decided to synthesize the capsule for basic theoretical oriented study of potential virulence factors identified structural features and available pathogenic mechanisms. Firstly, we obtained deoxy sugar acceptor that is prevalent in bacterial and can be synthesized from β-thiomannoside, which were combined with different glucoside donors according to various efficient catalytic systems to afford the core disaccharide block of tetrasaccharide repeating unit. Meanwhile, other disaccharide blocks can be acquired by "one-pot three-enzyme" sialylated catalytic system and chemical conversions. The two disaccharide blocks were connected via phosphodiester bond to obtain fully protected tetrasaccharides, which were global deprotected successfully to obtain target tetrasaccharide repeating unit. The target molecules were functionalized with a pentenyl handle at the reducing terminus to allow for further attachment to carrier proteins or develop a potential broad-spectrum carbohydrate-based vaccine on Haemophilus parasuis.The main achievements of our project include the following aspects:(1) A convergent chemoenzymatic synthesis of unique tetrasaccharide repeating unit modified by two sialic acid forms from Haemophilus parasuis was reported for the first time, which provided an important structure basis to study Haemophilus virulence factors and pathogenic mechanisms.(2) We firstly developed the chemoenzymatic approach to synthesize coexistence molecules of phosphodiester and two forms of sialic acid, which will become a powerful strategy for the synthesis of oligosaccharides.(3) Novel oligosaccharides were synthesized via phosphodiester bond connecting galactose and glucose for the first time.(4) We have achieved and tried various glycosylation with different glucoside donors and the rare deoxy sugar acceptor.(5) We achieved the efficient synthesis of common deoxy sugar in bacteria, which provided a strong experimental basis for subsequent chemical modification of bacteria deoxy sugar moiety.(6) We used a "one-pot three-enzyme" sialylated catalytic system to solve classical challenge about sialic acid and completed the synthesis of molecular diversity according to different forms of sialic acid.