Synthesis of Heparan Sulfate Oligosaccharides to Study Heavy Metals Binding and Adjuvant Activity

Abstract

Abstract: Heparan sulfate (HS) is a complex polysaccharide that is ubiquitous on all mammalian cell surfaces and extracellular matrices. The complexity of HS is encoded by broad sulfation patterns and uronic acid compositions. This pronounced structural heterogeneity of HS facilitates a wide range of biological activities. In the last two decades, carbohydrate chemists intensively pursued the synthesis of well-defined HS oligosaccharides to understand the structural-activity relationships (SARs). These studies revealed that sulfation codes and uronic acid variants synergistically modulate HS-protein interactions (HSPI). Furthermore, a growing number of studies associating the conformation plasticity of L-Iduronic acid, an uronic acid composition, provide a rationale to fine-tune the microenvironment to modulate the HSPI. In addition to traditional growth factors and chemokines binding, several non-canonical roles of HS are also reported, including HS binding to cell-penetrating peptides and metal ions. This thesis presents the synthesis of well-defined HS oligosaccharides and deciphers their binding affinity to heavy metal ions and toll-like receptors (TLRs). The thesis is divided into total of five chapters, and the summary of each chapter is outlined below. In Chapter 1, a brief introduction is given about the cell surface glycans and discussed the structural heterogeneity of heparan sulfate, its biosynthesis and the current insight into the structure-activity relationship (SARs) of specific uronic acid-composed HS with growth factors, chemokines and anticoagulants proteins. Chapter 2 describes an efficient total synthesis of HS tetrasaccharide composed of glucuronic acid. The key features of synthesis include the divergent strategy to obtain 3-O sulfated and 6-O-sulfate HS tetrasccharides and evaluated their binding with heavy metal ions, such as lead(II), cadmium(II) and mercury(II), which are a common toxic pollutant. Here, we immobilized HS tetrasaccharides on electrodes using electrografting technique and exposed to Pb2+, Cd2+, and Hg2+ ions and the impedimetric response was monitored by EIS. The systematic analysis based on a combination of electrochemical and surface characterization methods showed that chelation ability and binding preference of the HS tetrasaccharide are strongly dependent on their sulfation pattern and uronic acid composition. This study shows that electrochemical analysis and suface science tools provide valuable insight into the effect of structural components on heparan sulfate and heavy metal ions. 17 Chapter 3, describes the synthesis of a large pool of structurally well-defined HS and chondroitin sulfate (CS) oligosaccharides encapsulated fluorescent gold nanoparticles and study their cellular homing. We hypothesized that sulfation pattern and hexosamine composition might tune to nanoparticle homing in cancer and neural cell lines. Confocal imaging studies with different aggregation level of cancer cells showed no such significant disparity in the recognition pattern. While, U-87, a glioblastoma cell showed almost exclusive uptake of CS-E nanoprobe over HS-based nanoprobes. Whereas, primary neural cells of hippocampal and olfactory bulb showed enhanced homing of CS-E nanoprobe. Further, mechanistic studies revealed receptor CD44 and caveolae-mediated endocytosis of nanoparticles. These results open a new avenue for generating a combinatorial library of GAG-nanoprobe to target specific cell lines to design drug delivery vehicles. In chapter 4, the first systematic analysis of TLR4 activity of heparan sulfate and its mimics is reported. In addition, we describe the synthesis of glucosamine and uronic acid-based adjuvants, and investigate their TLR agonists activity. Chapter 5, describes the synthesis of spherical, rod-, and star-shaped gold nanostructures appended with CpG and imidazoquinoline ligands to evaluate shape-dependent immune cell uptake and immune modulation. Star shaped AuNPs showed good immune responses and moderate uptake rate with all TLR agonists in contrast to the rod and spherical-shaped AuNPs is the key finding. These results open a new avenue for generating AuNPs-based vaccines.