Hyaluronan (HA) is a non-sulfated negatively charged linear polysaccharide, which is composed of 2,000-25,000 repeating units of disaccharides: [-D-glucuronic acid-â-1,3-N-acetyl-D-glucosamine-â-1,4-]. Tumor microenvironment contains different type of cells and ECM. HA and HA binding proteins are the major components of extraceullular matrix (ECM). CD44 is the primary receptor for HA. CD44, a single chain transmembrane protein, is composed of four parts: the N-terminal hyaluronan binding domain (HABD), the stem domain, the transmembrane domain and the C-terminal cytoplasmic-tail domain. Multivalent HA-CD44 interactions induce signaling pathways that promote tumor cell invasiveness, proliferation, survival and multidrug resistance (MDR). Multivalent HA-CD44 interactions are required for formation of constitutive signaling complexes. Replacement of the multivalent interactions with monovalent interactions by the treatment with hyaluronan oligosaccharide (sHA) causes disassembly of constitutive signaling complexes and attenuated signaling pathway. These finally lead to the inhibition of tumor cell proliferation and MDR. In this dissertation, the chemical synthesis of a hyaluronan decasaccharide (HA10) using the pre-activation based chemoselective glycosylation strategy is described. Assembly of large oligosaccharides is generally challenging due to the increased difficulties in both glycosylation and deprotection. Indeed, the same building blocks previously employed for hyaluronan hexasaccharide (HA6) synthesis failed to yield the desired HA10. After extensive experimentation, the HA10 backbone was successfully constructed with an overall yield of 37% from disaccharide building blocks. The trichloroacetyl group was used as the nitrogen protective group for the glucosamine units and the addition of trimethylsilyl triflate (TMSOTf) was found to be crucial to suppress the formation of trichloromethyl oxazoline side-product and enable high glycosylation yield. For deprotections, the combination of a mild basic condition and the monitoring methodology using 1H-NMR allowed the removal of all base-labile protective groups, which facilitated the generation of the fully deprotected HA10. Based on the co-crystal structure of CD44 HABD and hyaluronan octasaccharides (HA8), hyaluronan pentasaccharide (HA5) analogues were designed and synthesized. Due to synthetic difficulties, the initial design of Library A was abolished. By rotating S-C4 bond, new analogues, Library B and Library C, were generated. Eleven compounds in Library B and Library C were synthesized by a cutting edge method, and screened by inhibitory Enzyme-linked immunosorbent assay (ELISA). Finally, it was found that the aromatic group in analogue 56 contributes to the binding of 56 to CD44. And this interaction overcomes the loss of favorable enthalpy caused by the loss of H-bonds from COOH of glucronic acid 7 (GluUA7) and primary OH of N-Acetylglucosamine 8 (GlcNAc8). Although the binding affinity of 56 is only comparable to HA6 and less than HA8, this provides a new direction towards further design of novel HA inhibitors.
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