Deciphering Structure-Functional Relationship of Heparan Sulfate using Iduronic Acid Glycans

Abstract

Heparan Sulfate (HS), a member of the glycosaminoglycan family, is composed of repeating units of α(1-4) linked D-glucosamine and uronic acid residues (L-iduronic acid and D-glucuronic acid) having diverse N-sulfation and O-sulfation patterns. Their complex structure enables binding to a great number of proteins and facilitates the modulation of numerous biological processes. Despite rapid progress in the synthesis of structurally defined HS oligosaccharides, how conformation plasticity of L-IdoA directly contributes HS biological functions remains largely obscure. In my thesis, I have explored the role of L-Iduronic acid in conformation plasticity, molecular recognition and its biological activity. Chapter 1 highlights the structure-functional relationship of heparan sulfate in physiological and pathological conditions. More specifically, we discuss HS's structural diversity and emphasize their binding affinity to various proteins, including growth factors, chemokines, anticoagulants, and viral proteins. Finally, we also discuss modern techniques like HS microarray to profile HS-protein interactions. Chapter 2 deals with linear approach for the synthesis of oligo-iduronic acid (oligo-IdoA) derivative using IdoA-thiophenol as the donor and a β-L-idopyranosyl derivative as the acceptor. Sequential modifications of the L-Idose residue yielded oligo-IdoA derivatives in moderate overall yields. We have also discussed different strategy to synthesize the oligo-IdoA and its drawback in more details. We anticipate that the new set of HS mimics will enable systematic study of the role of IdoA conformation plasticity and, oligosaccharide secondary structures, thereby developing the ability to modulate their biological functions. Chapter 3 demonstrate tailor-made HS mimics to probe conformation plasticity of IdoA and to unravel regulatory sites of growth factors. NOE and vicinal 3JH-H coupling constants analysis of HS mimics, confirmed that 4-O-sulfation enhances the population of the 1C4 geometry at the corresponding ring. Interestingly, the 1C4 conformer becomes almost exclusive upon additional 2-O-sulfation, whereas the non-sulfated IdoA rings display the conformation equilibrium between the 1C4-, 4C1- and 2S0-forms. The HS mimics microarray data with various growth factors revealed key sulfation code, oligosaccharide chain length and conformation plasticity important to modulate the xiii binding affinity. Notably, HB-EGF displayed strong binding affinity to highly sulfated Iduronic acid trisaccharides (I-34), whereas 4-O-sulfated mono and di-iduronic acid (I-11 and I-21) showed clear difference in binding affinity with VEGF165.Furthermore, invitro assay showed marked difference in the blockage of endothelial cell proliferation. Taken together, sulfated oligo-Iduronic acids present encouraging consideration for therapeutic applications. Chapter 4 reports exhaustive microarray and SPR analysis of HS mimics with chemokines. Our data revealed that homeostatic and inflammatory chemokines displayed several cryptic binding pockets for HS mimics, which is significantly differ from one other and could be potential selective inhibitors for chemokines. Notably, I-45 turned out to be a potential small molecule inhibitor for CCR2/CCL2 mediated cancer cell invasion and metastasis. Taken together, HS mimics offer new therapeutic molecule for cancer and immuno-therapy. Chapter 5 reports the synthesis of novel amphiphilic heparan sulfate mimics in which highly sulfated L-Idose and L-Iduronic acid units are connected to cholestanol moiety with different oligosaccharide chain length. These molecules displayed strong binding to spike protein of SARS-CoV-2 and inhibited SARS-CoV-2 infection, making it possible to acts as a potential antiviral drug.