Although it is widely accepted that HS is an information-rich polymer, the oligosaccharide structures that mediates particular biological interactions has been defined in only a few cases. This problem stems from the structural complexity of HS, which in turn, arises from a complex biosynthetic pathway. Lack of structure-activity relationship for HS-binding proteins makes it difficult to understand the physiology and pathology of HS at a molecular level and greatly complicates harnessing its therapeutic potential. As a continuation of our modular synthesis of HS, we have employed an anomeric aminopentyl linker protected by a benzyloxycarbonyl group modified by a perfluorodecyl tag for our synthesis, which enables the purification of polar intermediates by fluorous solid phase extraction. It also facilitate the repeated glycosylations to drive reactions to completion. Then, we have employed both bottom-up and top-down approach for the identification of HS ligand for lectins. In the first approach, the sulfation pattern was diversified at a late stage during the synthesis via per-sulfation, selective sulfation, de-sulfation and enzymatic 3-O-sulfation. Same strategy was applied to three other tetrasaccharides generating a library of 50 compounds. Microarray study with those compounds confirmed the essential role of 2-O-sulfate for FGF2 binding. Further, we found configuration of the uronic acid is also important. As compounds with 2-O-sulfate on glucuronic acid does not show any binding. The microarray data also indicated: by increasing the number of sulfate, their affinity with most lectins increased accordingly, which might correlate with the biological function of highly sulfated HS. Selectivity was observed for tetrasaccharides with intermediate sulfation level. With longer sequences or a more diversified library, we might get a better picture for their binding preferences. On the other hand, we also explored the top down approach combining enzymatic digestion, affinity column separation, mass spectrometry assisted structure identification, chemical synthesis, binding study via SPR to identify HS ligand for ROBO1. A unique octasaccharide sequence with a KD value of 3.5 M was identified.