A One-pot Approach To Bio-synthesize Globotriose And Its Derivatives

Glycoconjugates are widely distributed in the nature. It is an indispensable element in organism and it is involved in numerous biological processes, such as signal transduction, cell recognition, fertilization and development. Glycoconjugates are closely related to pathological processes when the body is in abnormal physiological conditions. Moreover, in medical treatments and drug exploitations, glycoconjugates are extensively applied (clinical diagnosis of tumors and development of tumor vaccines, etc.).80%of glycoconjugates are in the form of glycolipids, among which, glycosphingolipids are the most common form of glycolipids and nearly all glycolipids in vertebrates are glycosphingolipids.Gb3(Globotriaosylceramide) belongs to the neutral glycosphingolipid family. The structural formula of Gb3is a-Gal(1â†'4)p-Gal(1â†'4)β-Glc(1â†'O-ceramide). The trisaccharide, globotriose (Galαl-4Galβ1-4Glc), is the carbohydrate portion of globotriosylceramide and it is engaged in many physiologic events. Recent studies have demonstrated that globotriose could not only be used in cancer therapies, but also be used as a crucial indicator in organism for susceptibility to HTV-1infection. In addition, many human pathogens initiate disease by binding their microbial adhesin proteins to sugar chains of glycoconjugates on host cell mucosal surfaces. For example, the Shiga toxin (Stx) produced from pathogenic strain (O157:H7), often causes diarrhea and even life-threatening hemolytic uremic syndrome (HUS) in humans, by binding to an intestinal cell surface glycan, globotriose. Then the local Stx is translocated across the intestinal barrier and is absorbed into the host’s circulation.There are mainly two parts in the thesis. The first part is the introduction. In the second part, we develop a method to synthesize globotriose and its derivatives in a one-pot fashion. Due to the importance of globotriose in preclinical research and drug development, we studied preparative scale synthesis of globotriose in vitro by one-pot muti-enzymatic system. This reaction system contains lactose (Lac), galactose (Gal), UTP, ATP, DTT and MgCl2in Tris-HCl buffer (pH7.5). Four enzymes were used in the system:SpGalK (galactokinase), SpGalU (UDP-glucose pyrophosphorylase), LgtC (α-1,4-galactotransferase) and commercially available PPase (inorganic pyrophosphatase). In this multi-enzymatic system, Gal is first phosphorylated at C1position by SpGalK. The Gal-1-phosphate is subsequently pyrophosphorylated by SpGalU. PPase is used to hydrolyze the by-product PPi (pyrophosphate), thus driving the reaction forward. With the catalysis of LgtC, the galactosyl is transferred from the resulting UDP-Gal (donor) to Lac (acceptor), generating the target product, globotriose. And the yield is more than95%. The reaction mixture was purified by activated charcoal chromatography and Bio-Gel P-2filtration. Mass Spectrometry (LCMS-IT-TOF) and Nuclear Magnetic Resonance (1H NMR and13C NMR,400MHz, D2O) were performed to confirm the target product. More importantly, the substrate specificity of SpGalk and SpGalU is highly promiscuous and various UDP-galactose derivatives obtained could be used as the donor substrates for LgtC. We used2-deoxyGal,4-deoxyGal or6-deoxyGal as the initial substrate instead of Gal. Finally, three globotriose analogs were achieved by this one-pot multi-enzyme system in our study. This one-pot system would play an important role in low-cost and large-scale synthesis of globotriose and its derivatives.