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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 (2024)

Carbohydrates are one of the most abundant organic molecules in nature. The complex structures existing in nature lead to diverse biological functions. Sialic acids (Sias) are a family of nine-carbon acidic monosaccharides. They are the common terminal sugars of glycoconjugates on cell surfaces and are the key components of molecules recognized by various carbohydrate-binding proteins including those from immune cells, bacteria, and viruses.

Neuraminidases (NAs) or sialidases are a large family of glycosidases catalyzing the cleavage of sialic acid. Bacterial and viral NAs play a significant role in the infection of hosts. Annual influenza virus infections continue to be a significant burden to healthcare systems and the economy, which can be eased by the development of rapid and precise influenza virus detection tools and therapeutics. 2,3-Dehydro-N-acetylneuraminic acid (Neu5Ac2en) is a transition state analog-based inhibitor against NAs and its 4-amino- and 4-guanidino- (Zanamivir) derivatives have shown to be highly efficient and selective against influenza virus NAs. However, the functions of the corresponding sialosides containing a 4-N-substituted sialic acid are underexplored. We have developed an efficient chemoenzymatic method to access a library of sialosides containing a diverse array of 4-N-substituted sialic acids. High-throughput substrate specificity studies showed that 4-azido-sialic acid, in addition to 4-O-acetyl analog, is selectively cleaved off by influenza A and B NAs. On the other hand, binding assays have indicated that sialosides containing 4-amino- or 4-guanidino-sialic acid are selective inhibitors and affinity ligands for influenza NAs. These 4-N-substituted substrate analogs of NAs thus have diverse potential applications in substrate profiling and affinity purification of influenza virus NAs for diagnostics and improving the development of the NA components of influenza virus vaccines. This study is described in Chapter 2.

In nature, Sias are structurally diverse and have variations at the functional groups linked to the C-5 and modifications at the hydroxyl groups on one or more sites at the C-4, -5, -7, -8, and/or -9 positions. O-Acetylation is a common Sia modification that blocks or is required by specific molecular recognition events. However, O-acetyl groups in sialoglycans are generally unstable towards even small pH changes and they are usually sensitive to esterases. To overcome the structure instability issues of O-acetylated Sias and to explore their diagnostic and therapeutic potentials, we have developed a strategy to replace the O-acetyl group in Sia with a more stable N-acetyl group.

4-O-Acetylated derivatives have been found in horses and guinea pigs but not in pigs or humans. 4-O-Acetyl Neu5Ac (Neu4,5Ac2)-containing oligosaccharides have been found to be the dominant components of the acidic milk oligosaccharides (MOSs) of monotremes such as echidnas and platypus. Its stable 4-N-acetyl analog 4NAcNeu5Ac has been chemically synthesized. In addition, its analog 4-amino-Neu5Ac (4NH2Neu5Ac) was synthesized and used as a chemoenzymatic synthon for constructing a library of 2–3- and 2–6-linked 4NAcNeu5Ac-containing sialosides using a highly efficient in a stepwise one-pot multienzyme (StOPMe) strategy. The obtained sialoglycans were used as stable probes in high-throughput multiplex bead assays. The results showed a general tolerance of 4-N-acetyl modification on sialoside by influenza virus hemagglutinins (HAs), which implies the potential of these sialoside ligands in detecting and purifying influenza HAs and virions. This study is described in Chapter 3. Sialosides containing 7-, and/or 9-O-acetylation, as well as 8-O-acetylated Neu5Ac have also been understudied due to their instability. In order to have a better understanding of their biological functions, the corresponding N-acetylated sialosides were synthesized by my colleagues. I have participated in and contributed to carrying out high-throughput sialidase substrate specificity assays as well as kinetic studies using the sialoside probes with 7- or 8-N-acetylated, or 7,9-di-N-acetylated Neu5Ac to explore their biological functions. This study is described in Chapter 4.

In summary, during my Ph.D. studies, I have dedicated my efforts to exploring the synthesis and functional studies of sialosides with modified sialic acids, especially those with modifications at C-4, and their application in influenza virus research. These research and results have demonstrated that these sialyl glycans are useful and convenient tools to explore the biological functions of those naturally occurring counterparts. I sincerely hope that my research can be applied to the battlefield of human fighting against the flu.

    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2020)
    O-Acetylated sialic acid has been found in the Neisseria meningitidis serogroup W (NmW) capsular polysaccharide (CPS) and is a required structural component of clinically used NmW CPS-based polysaccharide and polysaccharide-conjugate vaccines. The role of sialic acid O-acetylation in NmW CPS, however, is not clearly understood. This is partially due to the lack of a precise control of the percentage and the location of O-acetylation which is labile and susceptible to migration. We explore chemoenzymatic synthetic strategies for preparing N-acetylated analogues of O-acetylated NmW CPS oligosaccharides which can serve as structurally stable probe mimics. Substrate specificity studies of NmW CPS polymerase (NmSiaDW) identified 4-azido-4-deoxy-N-acetylmannosamine (ManNAc4N3) and 6-azido-6-deoxy-N-acetylmannosamine (ManNAc6N3) as suitable chemoenzymatic synthons for synthesizing N-acetyl analogues of NmW CPS oligosaccharides containing 7-O-acetyl-N-acetylneuraminic acid (Neu5,7Ac2) and/or 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). The synthesis was achieved by NmSiaDW-dependent sequential one-pot multienzyme (OPME) strategy with in situ generation of the corresponding sugar nucleotides from simple monosaccharides or derivatives to form N3-oligosaccharides which were converted to the desired NAc-oligosaccharides by an efficient one-step chemical transformation.
      Cover page: A Chemoenzymatic Synthon Strategy for Synthesizing N‑Acetyl Analogues of O‑Acetylated N. meningitidis W Capsular Polysaccharide Oligosaccharides
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2022)
      A human sialyltransferase ST3GAL II (hST3GAL II) was successfully expressed in Escherichia coli as an active soluble fusion protein with an N-terminal maltose-binding protein (MBP) and a C-terminal hexa-histidine tag. It was used as an efficient catalyst in a one-pot multienzyme (OPME) sialylation system for high-yield production of the glycans of ganglioside GM1b and highly sialylated brain gangliosides GD1a and GT1b. Further sialylation of GM1b and GD1a glycans using a bacterial α2-8-sialyltransferase in another OPME sialylation reaction led to the formation of the glycans of GD1c and brain ganglioside GT1a, respectively. The lower reverse glycosylation activity of the recombinant hST3GAL II compared to its bacterial sialyltransferase counterpart simplifies the handling of enzymatic synthetic reactions and has an advantage for future use in automated chemoenzymatic synthetic processes.
        Cover page: One‐pot Multienzyme (OPME) Chemoenzymatic Synthesis of Brain Ganglioside Glycans with Human ST3GAL II Expressed in E. coli
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2023)
        Sialidases or neuraminidases are sialic-acid-cleaving enzymes that are expressed by a broad spectrum of organisms, including pathogens. In nature, sialic acids are monosaccharides with diverse structural variations, but the lack of novel probes has made it difficult to determine how sialic acid modifications impact the recognition by sialidases. Here, we used a chemoenzymatic synthon strategy to generate a set of α2-3- and α2-6-linked sialoside probes that contain 7-N-acetyl or 7,9-di-N-acetyl sialic acid as structure mimics for those containing the less stable naturally occurring 7-O-acetyl- or 7,9-di-O-acetyl modifications. These probes were used to compare the substrate specificity of several sialidases from different origins. Our results show that 7-N-acetyl sialic acid was readily cleaved by neuraminidases from H1N1 and H3N2 influenza A viruses, but not by sialidases of human or bacterial origin, thereby indicating that the influenza enzymes possess a distinctive and more promiscuous substrate binding pocket.
          Cover page: Sialosides Containing 7‑N‑Acetyl Sialic Acid Are Selective Substrates for Neuraminidases from Influenza A Viruses
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2017)
          Although great success has been achieved in asymmetric fluoroalkylation reactions via nucleophilic or electrophilic processes, the development of asymmetric radical versions of this type of reactions remains a formidable challenge because of the involvement of highly reactive radical species. Here we report a catalytic asymmetric radical aminoperfluoroalkylation and aminodifluoromethylation of alkenes with commercially available fluoroalkylsulfonyl chlorides as the radical sources, providing a versatile platform to access four types of enantioenriched α-tertiary pyrrolidines bearing β-perfluorobutanyl, trifluoromethyl, difluoroacetyl and even difluoromethyl groups in excellent yields and with excellent enantioselectivity. The key to success is not only the introduction of the CuBr/chiral phosphoric acid dual-catalytic system but also the use of silver carbonate to suppress strong background and side hydroamination reactions caused by a stoichiometric amount of the in situ generated HCl. Broad substrate scope, excellent functional group tolerance and versatile functionalization of the products make this approach very practical and attractive.
            Cover page: Catalytic asymmetric radical aminoperfluoroalkylation and aminodifluoromethylation of alkenes to versatile enantioenriched-fluoroalkyl amines.
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2020)
            Although great success has been achieved in catalytic asymmetric hydroamination of unactivated alkenes using transition metal catalysis and organocatalysis, the development of catalytic desymmetrising hydroamination of such alkenes remains a tough challenge in terms of attaining a high level of stereocontrol over both remote sites and reaction centers at the same time. To address this problem, here we report a highly efficient and practical desymmetrising hydroamination of unactivated alkenes catalysed by chiral Brønsted acids with both high diastereoselectivity and enantioselectivity. This method features a remarkably broad alkene scope, ranging from mono-substituted and gem-/1,2-disubstituted to the challenging tri- and tetra-substituted alkenes, to provide access to a variety of diversely functionalized chiral pyrrolidines bearing two congested tertiary or quaternary stereocenters with excellent efficiency under mild and user-friendly synthetic conditions. The key to success is indirect activation of unactivated alkenes by chiral Brønsted acids via a concerted hydroamination mechanism.
              Cover page: Desymmetrization of unactivated bis-alkenes via chiral Brønsted acid-catalysed hydroamination.
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2017)
              The process of follicular development involves communications between oocyte and surrounding granulosa cells. FURIN is a member of the family of proprotein convertases that is involved in the activation of a large number of zymogens and proproteins by cleavage at its recognition motif. To investigate the functions of FURIN in female fertility, furinflox/flox (furfl/fl) mice were crossed with Zp3-Cre mice and Gdf9-Cre, respectively, to achieve oocyte-specific disruption of FURIN. Here we report for the first time that FURIN is dispensable for primordial follicle maintenance and activation but important for early secondary follicular development, as ablation of FURIN in oocytes caused failure of follicle development beyond the type 4 and/or 5a follicles in mutant mice, resulting in increased number of early secondary follicles and the severely decreased number of mature follicles, thus anovulation and infertility. We also found that the developmental arrest of early secondary follicles might be rooted in the loss of the mature form of ADAMTS1 (85-kDa prodomain truncated) and compromised proliferation of granulosa cells in mutant mice. Taken together, our data highlight the importance of FURIN in follicle development beyond the early secondary follicle stage and indicate that compromised FURIN function leads to follicular dysplasia and female infertility in mice.
                Cover page: Oocyte-specific deletion of furin leads to female infertility by causing early secondary follicle arrest in mice
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