UC Davis

Carbohydrates are the most abundant organic molecules found in nature. The complex heterogeneity of carbohydrates in living systems, including glycoproteins, glycolipids, and unconjugated glycans is a direct result of the intrinsic characteristics of carbohydrate structures. The structural variability and complexity of glycans lead to a diverse array of biological functions. To illustrate the biological functions of carbohydrates, structurally defined compounds are needed. Various methods have been developed to obtain different types of carbohydrates, including extraction from natural sources, chemical synthesis, enzymatic synthesis, and chemoenzymatic synthesis. To access carbohydrates in a sustainable, environmentally friendly, and structure controllable way, I aimed to develop efficient chemoenzymatic synthesis and purification processes. I present my research effort below for two major classes of carbohydrates including human milk oligosaccharides (HMOs) and glycosphingolipids. Human milk oligosaccharides (HMOs) are breast milk’s third major solid component. They are indigestible glycans providing substantial benefits to breastfed newborn infants. There is an urgent need to access sufficient HMOs with defined structures to explore their functions and potential applications. To synthesize HMOs, lactose was chemically modified with a hydrophobic tag to simplify the purification process and the tag can be removed after purification. The lactose derivative LacNHCbz was used as an acceptor in multistep one-pot multienzyme (MSOPME) systems to synthesize different HMOs. In this study, twenty-one different HMOs with a size up to nonasaccharide have been synthesized by using MSOPME. This study is described in Chapter 2. A donor pre-generation process has also been developed for the MSOPME system to improve the synthetic process of HMOs. Sugar nucleotides needed for the reaction are pre-generated from the corresponding monosaccharides and nucleoside triphosphates and used for the glycosyltransferase-catalyzed reaction in a sequential manner. Combining the pre-generation process, MSOPME system, and the lactose derivative, HMOs can be synthesized in a shorter time with precise structure control as well as high yield and purity. All the possible fucosylated HMOs containing lacto-N-tetraose (LNT) or lacto-N-neotetraose (LNnT) as the core structure have been systematically synthesized. Sialylation has also been carried out using one-pot multienzyme (OPME) sialylation systems. This study is described in Chapter 3. Glycosphingolipids are biomolecules that each contains an oligosaccharide component conjugated with a hydrophobic lipid moiety. Gangliosides are sialic acid-containing glycosphingolipids that play a pivotal role in modulating ion channel function, receptor signaling, tumorigenesis, and cancer progression. They are attractive targets for studying brain diseases and cancer vaccines. One-pot multienzyme (OPME) systems were applied to synthesize two ganglioside sphingosines. The sphingosine was used as a hydrophobic tag to help the product purification from the enzymatic reaction mixture. With an efficient chemical acylation method, the sphingosine component of the glycosylsphingosines can be converted to the ceramide to form the desired glycosphingolipids with high yields. This study is described in Chapter 4. In summary, structurally defined compounds are needed to explore the essential biological functions of carbohydrate-containing biomolecular. Chemoenzymatic synthesis is an advanced method that can be applied to synthesize complex carbohydrates. During my Ph.D. study, I have been dedicating my efforts to improve the efficiency of chemoenzymatic synthesis and purification strategies. The improved methods have been applied to synthesize the human milk oligosaccharides and glycosphingolipids in the lab setting. Due to the easy procedures and high efficiency, I hope that some of the approaches can be adapted by different labs and even large-scale production in industry.