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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2022)

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.

    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2021)
    Gangliosides are biologically important sialic acid-containing glycolipids found commonly in human and other vertebrates. Isolation of pure gangliosides from cells or tissues is difficult, and chemical synthesis of gangliosides usually involves numerous steps with low synthetic yields. We report here a chemoenzymatic synthesis and purification protocol for two ganglioside cancer antigens, GM3 and GD3. One-pot multienzyme glycosylation reactions are used to sequentially prepare GM3 and GD3 sphingosines from chemically synthesized lactosyl sphingosine. A facile C18-cartridge purification procedure after each glycosylation reaction provides the desired pure glycosyl sphingosine product, which is readily acylated to form the target ganglioside. © 2021 Wiley Periodicals LLC. Basic Protocol: Chemoenzymatic synthesis and purification of GM3 and GD3 gangliosides Support Protocol: Monitoring reactions by thin-layer chromatography.
      Cover page: Chemoenzymatic Synthesis and Facile Purification of Gangliosides.
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2022)
      Innovation in process development is essential for applying biocatalysis in industrial and laboratory production of organic compounds, including beneficial carbohydrates such as human milk oligosaccharides (HMOs). HMOs have attracted increasing attention for their potential application as key ingredients in products that can improve human health. To efficiently access HMOs through biocatalysis, a combined substrate and process engineering strategy is developed, namely multistep one-pot multienzyme (MSOPME) design. The strategy allows access to a pure tagged HMO in a single reactor with a single C18-cartridge purification process, despite the length of the target. Its efficiency is demonstrated in the high-yielding (71-91 %) one-pot synthesis of twenty tagged HMOs (83-155 mg), including long-chain oligosaccharides with or without fucosylation or sialylation up to nonaoses from a lactoside without the isolation of the intermediate oligosaccharides. Gram-scale synthesis of an important HMO derivative - tagged lacto-N-fucopentaose-I (LNFP-I) - proceeds in 84 % yield. Tag removal is carried out in high efficiency (94-97 %) without the need for column purification to produce the desired natural HMOs with a free reducing end. The method can be readily adapted for large-scale synthesis and automation to allow quick access to HMOs, other glycans, and glycoconjugates.
        Cover page: Substrate and Process Engineering for Biocatalytic Synthesis and Facile Purification of Human Milk Oligosaccharides
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2023)
        Large-scale synthesis of GM1, an important ganglioside in mammalian cells especially those in the nervous system, is needed to explore its therapeutic potential. Biocatalytic production is a promising platform for such a purpose. We report herein the development of process engineering and glycosyltransferase improvement strategies to advance chemoenzymatic total synthesis of GM1. Firstly, a new short route was developed for chemical synthesis of lactosylsphingosine from the commercially available Garner's aldehyde. Secondly, two glycosyltransferases including Campylobacter jejuni β1-4GalNAcT (CjCgtA) and β1-3-galactosyltransferase (CjCgtB) were improved on their soluble expression in E. coli and enzyme stability by fusing with an N-terminal maltose binding protein (MBP). Thirdly, the process for enzymatic synthesis of GM1 sphingosines from lactosylsphingosine was engineered by developing a multistep one-pot multienzyme (MSOPME) strategy without isolating intermediate glycosphingosines and by adding a detergent, sodium cholate, to the later enzymatic glycosylation steps. Installation of a desired fatty acyl chain to GM1 glycosphingosines led to the formation of target GM1 gangliosides. The combination of glycosyltransferase improvement with chemical and enzymatic process engineering represents a significant advance in obtaining GM1 gangliosides containing different sialic acid forms by total chemoenzymatic synthesis in a short route and with high efficiency.
          Cover page: Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2019)
          A sialyltransferase acceptor tagging and two-step enzymatic reaction strategy has been developed for multigram-scale chemoenzymatic synthesis of 2,7-anhydro-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac), a compound that can serve as a sole carbon source for the growth of Ruminococcus gnavus, a common human gut commensal. Different approaches of introducing hydrophobic UV-active tags to lactose as well-suited sialyltransferase acceptors have been explored and a simple two-step high-yield chemical synthetic procedure has been identified. The UV-active hydrophobic tag facilitates monitoring reaction progress and allows facile product purification by C18-cartridges. A two-step enzyme-catalyzed reaction procedure has been established to combine with C18 cartridge-based purification process for high-yield production of the desired product in multigram scales with the recycled use of chromophore-tagged lactoside starting material and sialoside intermediate. This study demonstrated an environmentally friendly highly-efficient synthetic and purification strategy for the production of 2,7-anhydro-Neu5Ac to explore its potential functions.
            Cover page: A substrate tagging and two-step enzymatic reaction strategy for large-scale synthesis of 2,7-anhydro-sialic acid
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2021)
            Carbohydrate-protein conjugates have diverse applications. They have been used clinically as vaccines against bacterial infection and have been developed for high-throughput assays to elucidate the ligand specificities of glycan-binding proteins (GBPs) and antibodies. Here, we report an effective process that combines highly efficient chemoenzymatic synthesis of carbohydrates, production of carbohydrate-bovine serum albumin (glycan-BSA) conjugates using a squarate linker, and convenient immobilization of the resulting neoglycoproteins on carboxylate-coated fluorescent magnetic beads for the development of a suspension multiplex array platform. A glycan-BSA-bead array containing BSA and 50 glycan-BSA conjugates with tuned glycan valency was generated. The binding profiles of six plant lectins with binding preference towards Gal and/or GalNAc, as well as human galectin-3 and galectin-8, were readily obtained. Our results provide useful information to understand the multivalent glycan-binding properties of human galectins. The neoglycoprotein-immobilized fluorescent magnetic bead suspension multiplex array is a robust and flexible platform for rapid analysis of glycan and GBP interactions and will find broad applications.
              Cover page: A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2021)
              Gangliosides are sialic acid-containing glycosphingolipids that have been found in the cell membranes of all vertebrates. Their important biological functions are contributed by both the glycan and the ceramide lipid components. GM3 is a major ganglioside and a precursor for many other more complex gangliosides. To obtain structurally diverse GM3 gangliosides containing various sialic acid forms and different fatty acyl chains in low cost, an improved process was developed to chemically synthesize lactosyl sphingosine from an inexpensive l-serine derivative. It was then used to obtain GM3 sphingosines from diverse modified sialic acid precursors by an efficient one-pot multienzyme sialylation system containing Pasteurella multocida sialyltransferase 3 (PmST3) with in situ generation of sugar nucleotides. A highly effective chemical acylation and facile C18-cartridge purification process was then used to install fatty acyl chains of varying lengths and different modifications. The chemoenzymatic method represents a powerful total synthetic strategy to access a library of structurally defined GM3 gangliosides to explore their functions.
                Cover page: Chemoenzymatic Total Synthesis of GM3 Gangliosides Containing Different Sialic Acid Forms and Various Fatty Acyl Chains
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