Chemo-enzymatic Synthesis Of Sugar Nucleotides And Large Scale Production Of Sialyglycopeptides

Carbohydrate (including monosaccharides, oligosaccharides and polysaccharides) and glycoconjugate exist in organism widely. As an important structural components and information molecular, carbohydrate plays a crucial role in many biological processes. The function of sugar chain is closely linked to its structure, some changes of the sugar chain will cause a specific physiological response. The investigation of structure-activity relationship of sugar chain has become the research hotspots in glycobiology and biomedicine.Homogeneous glycoproteins and glycopeptides carrying structurally well-defined glycans are indispensable materials for the structural and functional investigation of glycoproteins. Due to the heterogeneity of the natural glycoconjugate in organism, the separation of glycoconjugate with homogeneous sugar chain from natural product has become very difficult. Therefore, the development of approaches for producing these glycoproteins and glycopeptides has become a research hopspot in glycobiology. At the present time, these glycoconjugate are abtained from syntheticism. Chemical approaches of synthesizing glycoconjugate have been developed. Due to its tedious process, strict conditions and low yield, the application of these methods has been restricted. On the contrary, the enzymatic synthesis using glycosyltransferases and endo-glycosidase has become the fast growing strategy in synthesizing glycoconjugate.This thesis is divided into two main parts:one was synthesizing various sugar nucleotides (Chapter2and3) which could be used as glycosyl donors of glycosyltransferases to synthesize glycoconjugate; two was separating SGP, which could be used as glycosyl donors of endo-glycosidases to synthesize glycoconjugate.In the synthesis of sugar chain with glycosyltransferases, sugar nucleotides which act as glycosyl donor are necessary. Structurally, sugar nucleotides are componds in which the anomeric carbon of monosaccharide is connected to nucleoside diphosphate or nucleoside monophosphate. Functionally, sugar nucleotides are active forms of monosaccharides. Sugar nucleotides as glycosyl donor of glycosyltransferases play a significant role in the synthesis of glycoconjugate. Therefore, the development of efficient approaches to prepare various kinds of sugar nucleotides are significant for the researchment of biochemisty and biological medicine. Besides, the sugar nucleotides library could be further used either to investigate carbohydrate metabolic pathways or to synthesize glycoconjugate with unnatural sugars.In the in vitro synthesis of sugar nucleotides, the most simple method is using kinase and pyrophosphorylase which exist in salvage pathway. Monosaccharides will first be phosphorylated to the form of sugar-1-phosphate, which will subsequently be catalyzed to sugar nucleotides by pyrophosphorylase. Therefore, the synthesis of sugar-1-P is the first step in the synthesis of sugar nucleotides.One goal of this paper is developing an enzymatic approach to synthesize Gal/Glc-1-P and their analoges, and then synthesize UDP-Gal/Glc and their analoges by enzymatic method. In Chapter2, galactokinase from Streptococcus pneumoniae TIGR4(GalKSpe4) was expressed and purified, then we chose34monosaccharides to investigate the substrate specificity of this enzyme and detect the yield of each product. Experimental results showed that GalKSpe4has a wide substrate adaptability. Among these34sugars,14were converted to their corresponding sugar-1-Ps. It was active to analogs with C2-, C4-and C6-modifications. The catalytic activity decreased gradually with the larger steric hindrance of C2-position. If two positions changed simultaneously in Gal structure, GalKSpe4would loss its activity. Besides, the configuration of sugars was very important for the identification ability of GalKSpe4. Compared with GalKs previously reported, GalK from Streptococcus pneumoniae TIGR4could synthesize Glc-1-P (25%) and GalNAc-1-P (68%), more surprisingly, GalKSpe4could synthesize L-Man-1-P (43%). Then, several important sugar-1-P were synthesized in preparation scale and purified by silica gel chromatography. Besides, the structure of these products were charaterized by NMR spectra.In Chapter3, the UDP-Glc pyrophosporylase from E. coli (GalU) was expressed and purified by Ni-column and gel feltration chromatography. We used14monosaccharides which could be used as GalKSpe4substrates, GalKSpe4and GalU to synthesize various sugar nucleotides in one-pot reaction. The experimental results demonstrated that, among14monosaccharides,9could be converted to their corresponding sugar nucleotides. For Gal analoges which had some changes in C2-position, the utilization rate of2-N3-Gal could reach78%, and GalNAc reached32%,2-DeO-Gal only reached15%. For Gal analoges which had some changes in C4-and C6-positions, the conversion rate decreased with the larger steric hindrance. Besides, UDP-Glc was synthesized in preparation scale by one-pot reaction, and isolated by ion exchange and gel filtration chromatography. We have set up a complete system to prepare sugar nucleotides as well.At present time, using transfructosylation activity of endo-glycosidase to synthesize glycoproteins and glycopeptides with homogeneous sugar chain is an efficient synthetic strategy. In this strategy, complete homogeneous sugar chain is the necessary glycosyl donor. Sialylglycopeptide (SGP) is an N-linked glycopeptide which carries an intact disialyl-biantennary complex-type sialyloligosaccharide. It can be used as a glycan donor for the synthesis of N-GlcNAc-containing glycopeptides or glycoproteins. With the wide use of SGP in the modification and synthesis of glycopeptides and glycoproteins, production of SGP now attracts extensive attention in the glycobiological fields. At the present time, the most efficient method for SGP production was reported by Seko and colleagues. This method requires7complicated steps, has a high cost, and is time-consuming. Therefore, the development of a more efficient scheme for the production of SGP from hen egg yolks is important for the structure-activity relationship studies and biomedical applications of glycoproteins.In Chapter4, one aim was to explore a convenient and efficient isolation approach for SGP separation. In our separation method, GCC, instead of G-25gel filtration and anion-and cation-exchangers, was used to desalt and remove hydrophilic impurities. The original seven separation steps were simplified to three and the separation conditions were optimized. A total of680mg of SGP was obtained from100eggs with our method. Subsequently the chemical structure and purity of the isolated SGP were measured. The results demonstrated that the molecular weight, chemical shift values of1H NMR, peptide sequence and glycosylation site were consistent with the expected structure. Furthermore, the product purity was over95%as determined by HPLC. In conclusion, we developed an efficient and time-and cost-saving tool for the large scale production of SGP, which is useful for various biomedical and glycobiological studies.