Chemoenzymatic Synthesis of Genetically-Encoded Multivalent Liquid N-glycan Arrays
Creators: Lin, Chih Lan
Publisher: name: University of Alberta Library
Year: 2024

<p>In the first chapter of this thesis, I review different microarrays displaying from pure to mixtures of glycans. I describe chemo-enzymatically synthesized or mixed glycans were used for building glycan arrays. And I review different methods of late-stage modification to build glycan arrays on glass surface, on liposome, on DNA, and on cell surface. <br>
In Chapter 2, I describe chemoenzymatic remodeling of glycans displays on M13 phage. Using this approach, I produce a genetically-encoded liquid glycan array (LiGA) of biantennary complex type N-glycans. The core divergent intermediate in this chapter is azido-functionalized sialylglycosyl-asparagine derived from egg yolk. I ligated it to phage functionalized with 50–1000 copies of dibenzocyclooctyne  to produce a divergent phage-displayed glycan. This glycan can be trimmed by glycosidases and extended by glycosyltransferases to yield a library of phages with different N-glycans. Post-reaction analysis by MALDI-TOF MS provided a rigorous approach to confirm N-glycan structure and mean density, both of which were encoded in the bacteriophage DNA. This N-glycan LiGA was probed with fifteen glycan-binding proteins (GBPs), including CD22 or DC-SIGN expressed on live cells, revealing an optimal structure/density combination for GBPs recognition. Injection of the LiGA into mice identified glycoconjugates with structures and avidity necessary for enrichment in specific organs.<br>
In Chapter 3, I have evaluated the rate of the modification of glycans by a fucosyltransferase Hp1,3FT and a sialyltransferase Pd2,6ST across hundreds of diverse substrates and 7 orders of magnitude in reactivity. Transfer of nucleotide analogs functionalized with a biotin tag, such as GDP-FucAz-biotin, led to labeling of glycans displayed on the M13 bacteriophage. The fraction of the biotinylated phages can be measured by streptavidin capture and the reaction rate can be inferred from this capture. Calibration studies using phage modified by LacNAc (Gal-β-(14)-GlcNAc) and the fucosyltransferase Hp1,3FT or sialyltransferase Pd2,6ST with relaxed donor specificity showed a reliably detectable change in biotinylation as concentration of GDP-FucAz-biotin and Hp1,3FT was varied by 10 orders of magnitude or Pd2,6ST was varied by 4 orders of magnitude. We demonstrate that Liquid Glycan Arrays (LiGA) can be used for high-throughput acceptor specificity screening of glycosyltransferase as well as the efficient detection of reaction conversion rate across million-fold dynamic range.<br>
In Chapter 4, I describe the initial exploration of creating gradient density display of glycan on M13 phage. Neuraminidase and terminal Neu5Ac-(a26)-Lac were used to test the quenching condition for the cleavage progress. We did model study of cleavage of glycans on phage by neuraminidase and interrupted this cleavage at various time points between 0% conversion and 100 % conversion. The reaction interrupted at a specific time yields a specific residual density of sialic acids. Interrupted reaction can be applied to a library of phage, each displaying an identical glycan at an identical density and yield gradient of different density of sialic acids displayed on a constant density of glycan on phage.</p>