Synthesis of Hyper-branched Oligosaccharide Epitopes from Glycoprotein GP72 of T. cruzi and Evaluation of their Binding to the WIC26.29 Monoclonal Antibody
Creators: Allas, Mikel Jason
Publisher: name: University of Alberta Library
Year: 2024

<p>Chagas disease is caused by the parasitic protozoan Trypanosoma cruzi, which is transmitted to humans mostly by contact with insects called ‘kissing bugs’. The presence of an immunogenic glycoprotein, GP72, on the cell surface of T. cruzi has a key role in the parasite’s life cycle. A monoclonal antibody (WIC29.26) that recognizes the unusual glycan portion of GP72 prevents the transformation of T. cruzi to its human infectious form suggesting that antibodies against this glycan may be an effective vaccine against the disease. This 13-residue glycan has a highly unusual structure of six different monosaccharides in seven different linkages, as well as two highly-branched residues, a fucose and a xylose. This research is focused on the synthesis of this glycan structure and fragments, which were to be used to probe the binding specificity of WIC29.26 mAb. The presence of the two ‘hyper-branched’ sugar residues is anticipated to pose a significant challenge in synthesizing the glycan structure.<br>
In Chapter 2, I describe my work on the synthesis of these fragments and the strategies employed. The hexasaccharide and heptasaccharide fragments each contain the ‘hyper-branched’ fucose and xylose residues, respectively. The synthesis of the hexasaccharide fragment was accomplished using a ‘pendulum’ glycosylation sequence while the heptasaccharide was accomplished using a ‘clockwise’ glycosylation sequence.<br>
In Chapter 3, I present my work describing the attempted synthesis of the whole tridecasaccharide glycan epitope of GP72. Unfortunately, multiple attempts to synthesize the whole glycan fragment were unsuccessful. The most significant progress achieved was the successful synthesis of an undecasaccharide intermediate which was synthesized from a linear synthesis starting from an octasaccharide acceptor. Further efforts, using a range of different approaches, to synthesize the tridecasaccharide by an [11+2] glycosylation was futile. Due to the limited and depleted amount of intermediates and the number of steps required to synthesize them, I have decided to finish my work at this point, but have proposed other synthetic routes to be investigated in the future.<br>
In Chapter 4, I report my work on the binding analysis performed between the smaller GP72 glycan fragments synthesized in Chapter 2 and the monoclonal antibody WIC29.26. These analyses were done using Bio-layer Interferometry (BLI) on an OctetRed96 machine. A dot blot assay was also performed to qualitatively analyze binding between the glycans and the mAb. However, the binding analyses performed did not show any significant binding between the glycans and the mAb. There is a possibility that these fragments adopt conformations that are not the same as in the whole glycan epitope found in the glycoprotein. Another possible reason could be the absence of the phosphate moieties in the synthetic glycans tested that are present in the native glycan.</p>