Synthesis of Highly Branched Chlorella Virus N-glycans

Abstract

Chloroviruses are large, dsDNA-containing viruses that live in terrestrial waters. Like many viruses, they produce N-linked glycoproteins. However, the glycan structures on chlorovirus N-linked glycoproteins differ from all others identified to date. Chloroviruses, also differ from other viruses, in that they do not use host biosynthetic machinery to make their N-linked glycans; they do so using the carbohydrate-processing enzymes they produce. The structures of these unusual glycans were reported in 2013 by De Castro and coworkers at the University of Napoli in Italy. These complex molecules feature a core ‘hyper-branched’ fucose residue in which every hydroxyl group is glycosylated. Methods for the chemical synthesis of chlorovirus N-glycans would provide molecules for biological and biochemical studies leading to an increased understanding of their function and assembly. This thesis will focus on developing synthetic approaches to access chlorovirus N-glycans and, in turn, provide new insights for the synthesis of the highly branched oligosaccharides. In Chapter 2, I describe the development of a synthetic approach to assemble these complex structures. The synthesis of the N-glycan isolated from ATCV-1, which contains the simplest structural motif of all chloroviruses, was accomplished using a “counter-clockwise” assembly sequence. In Chapter 3, I detail the extension of this “counter-clockwise” assembly approach to two of the most complex chlorovirus N-glycans among those characterized. The synthesis of the nonasaccharide N-glycan from virus PBCV-1 was successful. However, the synthesis of the NY-2A1 N-glycan, also a nonasaccharide, was not, due to the failure in introducing a tetrasaccharide unit onto the ‘hyper-branched’ fucose moiety. Based on this work, I conclude that the developed “counter-clockwise” assembly approach is applicable for the synthesis of most chlorovirus N-glycans reported to date, except NY-2A1. In Chapter 4, I report my work to synthesize various probes for understanding the biosynthesis of the PBCV-1 N-glycan. This involved the synthesis of molecules that are expected to be substrates for two glycosyltransferases (A064R and A075L).