Chemical synthesis of the N-glycosylated receptor binding domain from SARS-CoV-2 and development of COVID-19 vaccine candidates

Abstract

Chemical synthesis has the potential to generate homogeneous glycoproteins with well-defined and modifiable glycan structures at designated sites, offering a solution to the heterogeneity problem of glycoproteins obtained through biological approaches. In this thesis, we demonstrated that the conserved N-glycosylation sequon (Asn-Xaa-Ser/Thr) of glycoproteins can serve as a general site for performing Ser/Thr ligation (STL) to achieve N-linked glycoprotein synthesis. To this end, we developed an N+2 strategy to prepare the corresponding glycopeptide salicylaldehyde (SAL) esters for STL, and we showed that the STL at the sequon was not affected by the steric hindrance brought about by large-sized glycan structures. In Chapter 2, the effectiveness of the strategy was showcased by the synthesis of glycopeptides chosen from the sequences of Erythropoietin, Interleukin 4, Interleukin 7, Interleukin 9, and Interleukin 15. The strategy was further demonstrated by the total synthesis of the N-glycosylated receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein. This approach provides a unique and universal means to generate homogeneous glycoproteins bearing glycans with both site-specificity and structure-specificity. Our goal is to develop vaccine candidates using pure synthetic N-glycoprotein as vaccine components to provide efficient protection. We will further investigate how glycosylation specifically affects RBD based vaccines, which will offer novel mimetic epitopes and effective approaches for vaccine design. In Chapter 3, the N+2 strategy was applied to prepare the glycopeptide SAL esters whose sequences are derived from RBD of S protein. STL was applied at the sequon (Asn-Xaa-Ser/Thr) to synthesize the homogeneous N-linked glycopeptide-SpyTag. Via introducing a SpyCatcher into the VLP, the glycopeptides with SpyTag could readily form complexes with VLP-SpyCatcher through covalent linkages. Thus, two VLP-platform COVID-19 vaccine candidates containing a single well-defined structural epitope via the SpyTag/SpyCatcher system were developed. The immunogenicity of the two VLP-platform vaccine candidates was evaluated using mice as experimental animal models, which indicated their capability to elicit promising antigen-specific immune responses. Additionally, the protective efficacy of the two VLP-platform vaccine candidates will be evaluated. Exploring the structure-activity relationship of S RBD glycopeptides in more depth could lead to the optimization of their parameters like sugar composition and density, as well as peptide length, resulting in optimal immunogenicity and effectiveness. In summary, the findings documented in this thesis will contribute to the field of glycoprotein chemical synthesis and inspire further development and application of efficient and versatile synthetic strategies. Our advancements in chemical synthesis techniques and successful synthesis of N-glycosylated S RBD, as well as the development of COVID-19 vaccine candidates, will contribute to the understanding of glycoprotein/peptide structure-function relationships and their potential applications in drug discovery and vaccine development.