Synthesis and Functional Characterization of Novel Chimeric Vaccine Adjuvants & Chemo-Enzymatic Approaches to Synthesize Heparan Sulfate Oligosaccharides
Authors:   , Zhang Liangwei 
Published: February 2024

The work described in this thesis focuses on the development of novel chimeric vaccine adjuvants and the development of novel controlled chemo-enzymatic methods to synthesize a diverse library of heparan sulfate oligosaccharides for structure-activity study.

In chapter 1, a general introduction is given on vaccines and vaccine adjuvants. chapter 2 introduces the biosynthesis of heparan sulfate and the development of the chemical and chemo-enzymatic methods for the synthesis of heparan sulfate oligosaccharides.

Chapter 3 describes the chemical synthesis and functional characterization of novel chimeric NOD2/TLR2 agonists. Different pattern recognition receptor (PRR) agonists can synergize and balance each other's immunomodulatory activities. It was hypothesized that covalent attachment of two PAMPs, for example TLR-2 and NOD2 agonists, will ensure that immune cells are being exposed to both, resulting in efficient cross-talk of signal transduction pathways and in synergistic immune activation. In this chapter, a study presents an assessment of the potential synergistic effects of NOD2 and TLR-2 stimulation by chimeric ligands on the induction of proinflammatory cytokine secretion in vitro. The chapter highlights the rationale for design, the chemical synthesis of the individual agonists targeting NOD2 receptor and TLR-2 receptor, respectively. Six chimeric NOD2/TLR-2 agonists were synthesized and characterized by using a copper(I)-catalyzed click reaction, followed by cellular experimental analysis on mouse bone marrow-derived macrophages, showing that the chimeric agonists were significantly more effective than the individual ligands in inducing TNF-α and IL-6 release.

Chapter 4 and Chapter 5 describe the chemical synthesis of 6-O modification tetrasaccharide precursors and further enzymatic diversification on these precursors to access to heparan sulfate oligosaccharides. In chapter 4, a controlled chemo-enzymatic approach has been developed for the preparation of diverse libraries of heparan sulfate (HS) oligosaccharides. It employs a chemically synthesized oligosaccharide having a tert-butyloxycarbonyl (Boc) functionality at a GlcN residue, which in unanticipated manners influences the site of modification by NST, C5-Epi/2-OST and 6-OST1/6-OST3. Because the Boc group is slowly removed even at room temperature in water, making it difficult to carry out multiple enzymatic modifications. Therefore, Boc protecting group is not an ideal removable handle for further enzymatic modification. In chapter 5, former controlled chemo-enzymatic approaches that employ methyl group and tert-butyloxycarbonyl (Boc) have been developed for the preparation of diverse libraries of heparan sulfate (HS) oligosaccharides. However, the unremovable methyl ether seriously limit the synthetic flexibility. Also, the Boc-protected substrate is labile in water, which makes the reaction uncontrollable and also lead to difficulties with purification. In this chapter, the goal is to develop a methodology to prepare an oligosaccharide that has an allyl ether at C-6 of GlcN for controlled enzymatic modification. After removal of the masking allyl group, the sites blocked to 6-OST and C5-epi/2-OST would be available again for modification by these enzymes.