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theses

Multimodal ligands can be used to achieve selective clearance of impurities in a single chromatography step by having multiple modes of interaction between the ligand and the targeted protein analyte. One strategy for designing effective multimodal ligands is to establish a large library of chemically diverse ligands and utilize high-throughput screening methods to test each ligands effectiveness. However, this method becomes ineffective as the number of ligands in the library increases, so other strategies should also be utilized. A strategy used to create a smaller but, more effective library, is to design ligands that closely mimic protein-ligand binding interactions found in natural systems. One such example would be design ligands that mimic the multimodal interactions between proteins and complex glycans. However, limited research has been conducted to design and synthesize multimodal glycan-based ligands (or glycoligands) for protein chromatography or other similar applications.
Here, we investigate the chemoenzymatic synthesis of glycan-based multimodal glycoligands using a glycosynthase enzyme engineered from a native glucuronidase belonging to the family 2 glycosyl hydrolase. To the best of our knowledge, this work signifies the first reported attempt to chemoenzymatically synthesize a glucuronide with a bio-orthogonal aldehyde functional group allowing it to be covalently attached to a suitable resin or support. The four specific objectives that were completed to accomplish this goal were; (i) use in-silico docking simulations to establish and justify a library of hydrophobic alcohol acceptors containing an aldehyde functional group, (ii) synthesize and purify 1-Deoxy-1-fluoro-α-D-glucopyranuronic acid using TEMPO oxidation and anion exchange chromatography, (iii) generate a library of glycosynthase mutants from the E.coli glucuronidase gene, uidA, and express, purify and characterize the wild-type and nucleophilic mutant enzymes, (iv) run glycoligand synthesis assays with an active glycosynthase mutant, activated glucopyranuronic acid donor and a library of alcohol acceptors to confirm if the engineered uidA glycosynthases can synthesize the desired multimodal glycoligands. Our results led further credence to our initial hypothesis and in-silico studies, that uidA glycosynthases can synthesize glucuronides with aldehyde containing bio-orthogonal functional groups that can then serve as potential multimodal ligands for protein chromatography. We also discuss future research directions into how we can utilize additional glycosynthases enzymes to create more complex multimodal charged glycans by using our currently synthesized glycoligand product as substrate.

ETD_10345

M.S.

Includes bibliographical references

doi:10.7282/t3-fbyh-je93

ETD graduate

The author owns the copyright to this work.