dc.description.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.
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