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Abstract:
The introduction of automation in carbohydrate synthesis has paved the way towards getting faster access to complex, immunologically relevant glycans. Automated assembly enables the synthesis of oligomers and polymers (as long as 50 mers) to be accomplished in weeks, compared to months or even years when performed via conventional solution phase synthesis. However, the optimization of the entire process still lies in its infancy, and standard protocols for the automated synthesis of oligosaccharides are not yet available. The work in this thesis aims to develop optimized protocols for the automated assembly of complex glycans using the Glyconeer synthesizer, which was accomplished by firstly, optimizing the synthesis of building blocks. Since building block synthesis is very often the most laborious and time consuming step in the entire process of automation, ready availability of standard protocols for procuring multi-gram quantities of these monomers further reduces the time required to synthesize complex glycans via automated synthesis. Thereafter, with multi-gram quantities of building blocks in hand, the automated synthesis of glycans using the Glyconeer synthesizer were streamlined. Optimized protocols for automated glycan assembly of complex glycans were established by streamlining the variables associated with the glycosylation reaction, namely, temperature, number of glycosylation cycles and concentration of donor (in this case building blocks). Therefore, the AGA of Lewisx epitope was optimized to establish optimal glycosylation conditions for the building blocks (Chapter 3) on a 0.0125 mmol scale. With these optimized protocols, the synthesis was then scaled up to a 0.025 mmol scale to procure milligram quantities of the Lex epitope. Secondly, in order to establish the reproducibility of these protocols, the optimized synthetic protocols were applied to procure a library of Lewis antigens, namely, Lewisa, Lewisy and Lewisb antigens (Chapter 4). The successful synthesis of these antigens further confirmed the reproducibility of the established synthetic protocols. Thereafter, these synthetic protocols were then validated for synthesizing longer structures, which was achieved by synthesizing a library of poly-N-acetyl glucosamine oligomers, such as, tetramer, pentamer and hexamer (Chapter 5). The oligomers synthesized were then used to determine the substrate specificity of the glucosamine hydrolase from xii Pseudomonas aeruginosa. Finally, the boundaries of AGA were further pushed by synthesizing one of the most difficult linkage, namely, the beta mannose linkage via automation (Chapter 6), which makes the synthesis of complex N-glycans possible by automated synthesis. In a nutshell, the work performed in this thesis aims to overcome the challenge of gaining quick access to pure oligosaccharides by establishing standard protocols for synthesizing immunologically relevant glycans via automated glycan assembly, thereby significantly reducing the time required to synthesize these glycans.
