Date of Award
Doctor of Philosophy (PhD)
Peng George Wang
FUANCTION: Deamidation has been recognized as a common spontaneous pathway of protein degradation and its rate is sequence-dependent. Deamidation is a prevalent concern in pharmaceutical industry, causing the reduction of protein/peptide drug efficacy and shelf-life in several cases. Therefore, it is critical to discover means of controlling the deamidation rate of protein and peptide drugs. Deamidation of physiological proteins is also related to several human diseases, which is considered as a ‘timer’ for the diseases. In this thesis, it was demonstrated that deamidation is prevented by the naturally occurring glycosylation of Asn. All of the non-glycosylated peptides have different half-lives ranging from one to twenty days through incubation. Deamidation reaction was significantly reduced by the introduction of N-linked glycosylation. RNase B was used to demonstrate that glycoprotein has elongated deamidation half-life than non-glycosylated protein RNase A. In this thesis, N-linked glycosylation was also applied on a therapeutic peptide and it was found that the N-linked glycosylation not only prevented its deamidation but also increased its potency.
SYNTHESIS: A cation exchange assisted binding-elution (BE) strategy for enzymatic synthesis of human milk oligosaccharides (HMOs) was developed. An amino linker was used to provide the cation ion under acidic condition which can be readily bound to cation exchange resin and then eluted off by saturated ammonium bicarbonate. Ammonium bicarbonate in the collections was easily removed by vacuum evaporation. This strategy circumvented the incompatible issue between glycosyltransferases and solid support or large polymers, and no purification was needed for intermediate products. With current approach, polyLacNAc backbones of HMOs and fucosylated HMOs were synthesized smoothly. His-tag facilitated enzymatic synthesis of oligosaccharides was developed for the purpose of automation of enzymatic synthesis of oligosaccharides. His-tag with six histine residues was successfully synthesized and enzymatic substrates were conjugated onto his-tag with a squarate linker readily. Two examples of oligosaccharides, polyLacNAc and Heparosan, were successfully synthesized to demonstrate this His-tag technology.
APPLICATION: A method of how to determine the oxidation degree of dextran as a drug carrier has been established. LNFPIII was successfully conjugated onto oxidized dextran with specific loading numbers by a double reduction amination reaction.
Zhu, Hailiang, "Glycosylation: Function, Synthesis and Application." Dissertation, Georgia State University, 2018.