MULTIFUNCTIONAL GLYCO-ENGINEERED PLATFORMS: FROM NANOCLUSTER TARGETING TO PROTEOGLYCAN MODULATION AND RATIONAL DESIGN OF SMALL MOLECULE HEPARAN SULFATE ANTAGONISTS

Abstract

Heparan sulfate (HS) is a negatively charged polysaccharide ubiquitously expressed on the surface of mammalian cells and within the extracellular matrix. Its sequence-specific interactions, governed by distinct patterns of sulfation and uronic acid composition, enable binding to a wide range of proteins, including growth factors, chemokines, and viral or bacterial envelope proteins. These interactions are critically involved in key biological processes such as microbial pathogenesis and tumor progression. Although substantial progress has been made in the chemical synthesis of structurally defined HS oligosaccharides, their translational applications remain insufficiently explored. This thesis presents three distinct biological applications of HS and its synthetic mimetics. (a) In the first section, ultrasmall fluorescent glycogold nanoclusters were synthesized and functionalized with sulfated oligo-iduronic acid ligands (I34). These nanoclusters exhibit high affinity and selectivity for the HB-EGF receptor over FGF2, enabling direct imaging of EGFR-mediated cancer cell homing in both two-dimensional (2D) and three-dimensional (3D) cell culture systems. This provides a novel glycan-based nanoplatform for targeted imaging in oncology. (b) The second part focuses on the synthesis of proteoglycan mimetics incorporating di-, tetra-, and hexasaccharide analogues of HS. These mimetics were employed for the systematic engineering of cancer and normal cell surfaces, followed by analysis of cytokine secretion profiles upon treatment with peripheral blood mononuclear cells (PBMCs) and murine macrophage cell line PMJ2-PC. Our findings indicate that L-iduronic acid-based mimetics with high levels of sulfation significantly enhance cytokine secretion relative to their D-glucuronic acid-containing counterparts. Furthermore, we investigated the functional role of 3-O-sulfated HS in muscle stem cells (MuSCs), focusing mainly on MuSC proliferation. (c) The final segment describes the rational design and synthesis of a library of trisaccharide HS mimetics bearing guanidinium functionalities. These small molecules were engineered to disrupt HS–protein interactions, with a particular emphasis on their application as antidotes for anticoagulant agents such as heparin and fondaparinux. Incorporation of guanidine moieties promoted conformational shifts from 4C1 to 2S0. These guanidine-modified mimetics exhibited potent sub-micromolar antagonist activity, low cytotoxicity in both malignant and non-malignant cell lines, and demonstrated specific carbohydrate–carbohydrate interactions as confirmed by NMR spectroscopy. Collectively, this work introduces a versatile suite of glyco-engineered platforms encompassing cancer-targeted imaging, immune and muscle cell regulation, and anticoagulant reversal. These findings not only contribute to a deeper understanding of HS structure–function relationships but also pave the way for novel therapeutic and diagnostic applications across oncology, regenerative medicine, and hematology.