Enzymatic Synthesis And Application Of N-Acetylglucosamine/N-Acetylgalactosamine Sugar Nucleotide And Their Analogs

Carbohydrates (or glycans, including monosaccharides, oligosaccharides and polysaccharides) and glycoconjugates are prevalent in all living organisms ranging from bacteria to plants and animals. They are not only essential structural components, but also exhibit irreplaceable roles as informative molecules in a variety of vital biological processes. Specific changes in glycan profiles have been associated with certain disease states such as inflammation and tumor, indicating their protential application in clinical diagnosis and their possibility as targets in pharmaceutical development.N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) are two common N-acetylhexosamines. They are essential components of bacterial cell walls and glycosaminoglylcans, and are prevalent in the core structures of glycans in glycoproteins and glycolipids. They also affect inter-molecular (e.g. O-GlcNAcylation of proteins is involved in regulating signaling pathways) and intercellular (e.g. glycolipids, which have GalNAc residues, are involved in cellular interaction, differentiation, and other processes) interactions during various metabolic processes. Studying oligosaccharides, polysaccharides and glycoconjugates that contain these two monosaccharides will assist us in understading physiological and pathological processes as well as developing pharmaceuticals.Bacterial surfaces are coated with complex polysaccharides, among which lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs) are both covalently linked to cell surfaces. They are both composed of many repeating units, have diverse structures, and are vital virulence factors of disease-causing bacteria. Some bacteria have CPSs that have the same or similar structures with those polysaccharides from human beings. Therefore, studying LPSs and CPSs and their biosynthetic pathways will help us prevent or cure diseases caused by bacterial infection, and produce polysaccharides without pathogenic virus contamination with low cost as well.In order to study the biological roles of glycans, we need to obtain carbohydrates or glycoconjugates with homogeneous and defined structures. The structures of glycans in carbohydrates or glyconconjugates isolated from biological sources have microheterogeneity. Chemical synthesis of glycans is tedious and often has low yields. In contrast, glycosyltransferases in the biological synthetic pathway of glycans can efficiently form regio-specific and stereo-specific glycosidic bonds. Therefore, synthesis of structure-defined glycans by glycosyltransferases (mainly Leloir-type glycosyltransferases) has become an attractive alternative to the chemical synthesis. However, chemical synthesis is superior in generating unnatural glycans.These natural compound derivatives with various structural modifications can be potential targets in drug development. Some unnatural sugars contain groups that are easy for further detection, and will facilitate research in carbohydrate metabolism. When the flexibility of chemical synthesis and efficiency and specificity of enzymatic synthesis are combined, as in chemo-enzymatic synthesis, highly efficient synthesis of carbohydrate analogs becomes possible, and it has become an essential strategy widely used in glycobiology.Sugar nucleotides, also known as active sugars, are constructed by linking the anomeric carbon of monosaccharides to nucleoside diphosphates or nucleoside monophosphates. They are sugar donors of glycosyltransferases, and are essential precursors in biological synthesis of glycans and glycoconjugates. Analogs of natural sugar nucleotides may be inhibitor to enzymes, or can be used for analyzing the glycoconjugate biosynthetic pathways. Thus, preparation of natural and unnatural sugar nucleotides with high efficiency is significant to biological chemistry and medicinal chemistry. The de novo pathway of sugar nucleotide synthesis in living organisms has fewer steps and are easy to handle, hence is commonly used in enzymatic synthesis of sugar nucleotides.UDP-GlcNAc and UDP-GalNAc are the sugar donors for GlcNAc and GalNAc, respectively. In living organisms, both of them can be synthesized through a few pathways, including the salvage pathway. In salvage pathway, GalNAc can be converted into UDP-GalNAc by GalNAc 1-kinase and UDP-GalNAc pyrophosphorylase, in which all enzymes used in the previous reports are from mammals. However, three enzymes (including GlcNAc kinase, GlcNAc phosphomutase and UDP-GlcNAc pyrophosphorylase) are needed to convert GlcNAc into UDP-GlcNAc. The lack of GlcNAc 1-kinase made it difficult to enzymatically synthesize GlcNAc-1-P, which is the key intermediate in UDP-GlcNAc synthesis, and led to its high price. It also restricted enzymatic synthesis of GlcNAc-1-P analogs and the research based on GlcNAc-1-P and its analogs. Sugar nucleotides are also expensive. Their analogs which are important in research are not commercially available yet. Therefore, synthesizing sugar nucleotides and their analogs in a preparative scale is an research interest in glycobiology.One aim of this thesis is to enzymatically synthesize GlcNAc/GalNAc-1-P and their analogs, and to further enzymatically synthesize UDP-GlcNAc/GalNAc and their analogs. N-acetylhexosamine 1-kinase (NahK) from Bifidobacterium longum is the first GlcNAc 1-kinase reported in literature, which can form GlcNAc-1-P from GlcNAc by one-step phosphorylation. It has similar reaction rates towards GlcNAc and GalNAc, and can be considered as a 'GalNAc 1-kinase' as well, and is the first bacterial enzyme that has this activity. It also shows activity towards ManNAc, illustrating its relaxed substrate specificity. In Chapter 2, NahK was cloned and overexpressed in Escherichia coli BL21(DE3) with a C-terminal His6-tag. After purification by Ni-column and gel filtration chromatography, the purity of recombinant NahK was higher than 95%. With the consideration of steric effect of side chains, the configuration and deoxidization of hydroxyl groups, and the ease for further modification, we designed GlcNAc/GalNAc analogs with structural modifications at C2-, C3-, C4- and C6- positions and assayed enzymatic activity of NahK towards them. Including GlcNAc and GalNAc,25 sugars were assayed, among which 19 were converted to the corresponding sugar-1-Ps and the products were isolated by silica gel chromatography. The yields of 14 sugar-1-Ps were higher than 60%. Besides the GlcNAc/GalNAc analogs, ATP analogs bearing S atom at y site was also assayed in NahK-catalyzed reaction, and the yields of products were comparative to the yields when ATP was used as phospho donor, demonstrating that S atom did not affect NahK-catalyzed reaction very much. By NahK-mediated reaction and silica gel chromatography, the cost of synthesizing GlcNAc-1-P has been reduced dramatically, and the broad substrate specificity of NahK has enabled its use in preparation of GlcNAc/GalNAc-1-P analogs, laying a foundation for enzymatic synthesis of UDP-GlcNAc/GalNAc analogs.In Chapter 3, the UDP-GlcNAc pyrophosphorylase from E. coli (GlmU) and the UDP-GalNAc pyrophosphorylase from Homo sapiens (AGX1) were cloned and expressed separately in E. coli with N-terminal His6-tag. The two enzymes were active after purification by Ni-column and gel feltration chromatography, but they exhibited different substrate specificity. GlmU, whose natural substrate is GlcNAc-1-P, showed activity towards most sugar-1-P analogs with GlcNAc-type configuration. Even acylamino group at C2-position was replaced, after elongation of reaction duration, sugar nucleotides were still synthesized with moderate yields. However, its activity towards sugar-1-P analogs with GalNAc-type configuration was limited. It was active only to some analogs with C4- and C6-modifications. AGX1, which has higher activity towards GalNAc-1-P than GlcNAc-1-P, were active towards most GlcNAc/GalNAc-1-P analogs used in experiments. In addition, sugar-l-P(S)s were converted into their corresponding sugar nucleotides by GlmU, and the products can be used in studying glycosyltransferases. By ion exchange and gel filtration chromatography, we isolated UDP-GlcNAc/GalNAc and their analogs, which can be used to study the characteristics of enzymes such as glycosyltransferases and to prepare glycan analogs. We have set up a relatively easy strategy to prepare unnatural sugar nucleotides as well.To study the glycosyltransferases, the traditional methods involve radioactive or fluorescent labeling and isolating the molecules before detection. In Chapter 4, we established a new method (termed SAMDI MS) for analyzing glycosyltransferases. It combines self-assembled monolayers with MALDI-TOF MS, and no labeling work is needed. It is fast, and with only tiny amount of samples, it can analyze the reactions catalyzed by glycosyltransferases qualitatively or quantitatively through the mass spectra of reaction mixtures. We used Neisseria meningitidis?1,3-GlcNAc-transferase (LgtA) as an example, and analyzed its substrate specificity and kinetics study towards donor substrates by SAMDI MS. The success of this assay demonstrated the prospect of SAMDI MS in analyzing glycosyltransferases.GlcNAc/GalNAc are prevalent in many biologically important glycoconjugates (e.g. glycoproteins, proteoglycans, and glycosaminoglycans). Increasing research interests are focusing on replacing GlcNAc/GalNAc residues in polysaccharides with chemically modified unnatural sugars and modulating the function of these glycans. In Chapter 5, 2-ketoGlc (GlcNAc analogs) was supplemented into the medium of E. coli. By detection with chemically selective reagent, sugars bearing ketone groups were detected both on bacterial surface and in the core oligosaccharide of LPS, indicating the incorporation of 2-ketoGlc into E. coli cell surface polysaccharides through in vivo metabolism. Thus, we have shown that besides fucose analogs, other specific monosaccharide analogs can also be incorporated into cell surface polysaccharides. However, since a few metabolic pathways are possibly used by 2-ketoGlc, we did not identified the exact residue that was modified in the core oligosaccharide. Hyaluronan (HA) is widely used in cosmetics and pharmaceuticals. Its modification is often achieved chemically. The CPS of Pasteurella multocida (Type A) has HA structure. We tried to use the soluble part of P. multocida hyaluronan synthase as catalyst to synthsize HA derivatives from unnatural sugar nucleotide bearing bioorthoganol group (UDP-2-ketoGlc). However, probably due to the low sensitivity of detecting method, or because UDP-2-ketoGlc could not be used by PmHAS(T), no ketone groups were detected in HA product. Further work is still underway.In summary, this thesis established a strategy of enzymatic synthesis and chromatographical preparation of UDP-GlcNAc/GalNAc and their analogs with two enzymes from bacteria and one enzyme from human. Sugar nucleotide analogs were prepared, and were used in characterization of a bacterial GlcNAc-transferase and synthesis of bacterial polysaccharides. We also established a new strategy for glycosyltransferase characterization without labeling and isolating the target molecules.