Date of Award
5-1-2026
Document Type
Thesis
Degree Name
M.S. in Pharmaceutical Science
First Advisor
Joshua Zhu
Second Advisor
Paul Boudreau
Third Advisor
David Colby
School
University of Mississippi
Relational Format
dissertation/thesis
Abstract
Chemokine-receptor interactions play a central role in immune regulation and disease pathogenesis, particularly through CCR5, a receptor engaged by chemokines such as CCL3, CCL4, and CCL5. Post-translational modifications of the CCR5 N-terminus—including tyrosine sulfation at positions 3, 10, 14, and 15, and O-glycosylation at serine 6—critically modulate chemokine recognition and selectivity. However, the impact of specific sulfation patterns on chemokine binding and the synergistic effects of combined sulfation and glycosylation remain incompletely understood. This thesis presents a two-project chemical approach to systematically investigate CCR5 post-translational modifications. In Project 1, a comprehensive 16-member library of CCR5???? sulfopeptides representing all possible combinations of tyrosine sulfation was synthesized using Fmoc-based solid-phase peptide synthesis with orthogonal protection strategies. Site-selective sulfation was achieved using a 2,2,2-trichloroethyl (TCE)-protected sulfuryl imidazolium reagent, enabling controlled installation of sulfate esters at designated positions. Following global deprotection and Pd-catalyzed TCE removal, all 16 peptides were purified by reverse-phase HPLC and characterized by high-resolution mass spectrometry, confirming site-specific sulfation patterns. The sulfated peptides were selectively labeled with fluorescein isothiocyanate (FITC) at a C-terminal lysine, generating fluorescent probes suitable for future biophysical binding assays. In Project 2, enzymatic glycan extension strategies were explored to access complex O-linked glyco-amino acids relevant to CCR5 modification.
In this work, a pre-synthesized Ser-Core6 glyco-amino acid as starting material, sequential enzymatic reactions with ?1,4-galactosyltransferase, ?1,3-fucosyltransferase, and ?2,3 sialyltransferase were employed to generate Ser-Core6-Gal-Fuc-Sia bearing the sialyl Lewis X (SLe?) tetrasaccharide epitope. The sialylation step proved most challenging, requiring multiple overnight incubations on small scale (2 mg) to achieve complete conversion and showing limited success in scale-up attempts. In a complementary effort, enzymatic glycan extension was used to generate defined O-glycosylated amino acids as building blocks for future CCR5 N-terminal constructs. Separately, chemically synthesized Core 2 glyco-amino acids enabled the synthesis and characterization of two model glycopeptides (17 and 18) derived from CCR5 and CCR1 N terminal sequences. Together, this work establishes a chemically well-defined toolkit comprising (i) a complete CCR5 sulfopeptide library, (ii) enzymatically generated O-glycosylated amino acids, and (iii) model glycopeptides bearing defined O-glycan structures. These tools provide a foundation for future biophysical studies aimed at elucidating how site-specific modifications govern CCR5-chemokine recognition and for rational design of CCR5-targeted therapeutics.
Recommended Citation
Ali Eldeen, Mostafa Mohammed Mohammed, "CCR5 Sulfopeptide Library and O-Glycosylated Amino Acids as Chemical Tools for Probing Chemokine Receptor Recognition" (2026). Electronic Theses and Dissertations. 8792.
https://egrove.olemiss.edu/etd/8792