Heparan sulfate (HS) participates in a variety of biological functions and has been exploited for its ability to be utilized as a HS-based drug. Chemical synthesis of HS remains extremely challenging. Previous research has proven the feasibility of using a HS enzyme-based approach to synthesize HS structures with unique biological activities. Our central hypothesis is that all subsequent modifications following N-sulfation during HS biosynthesis are governed by the number and position of the GlcNS residue. In this dissertation, a fluorous affinity tag-assisted chemoenzymatic synthesis technique has been developed to build a HS octasaccharide library with defined N-sulfo glucosamine (GlcNS) positions. The HS backbone was synthesized by heparosan biosynthetic enzymes. N-acetyl glucosaminyl transferase from E.coli K5 (KfiA) was used to transfer either GlcNAc or GlcNTFA (N-trifluoroacetylglucosamine) residues to the growing chain. Heparosan synthase from pasteurella (PmHS2) was used to transfer the GlcUA residues. A selective de-trifluoroacetylation was performed because under these conditions, the GlcNTFA is labile and will be converted to glucosamine (GlcNH2) while the GlcNAc residue remains intact. The resultant GlcNH2 is then converted to a GlcNS residue by N-sulfotransferase (NST). N-sulfo-6-O-sulfo HS backbones with different 6-O-sulfation patterns and different sizes were also prepared. Furthermore, we prepared oligosaccharide capable of binding to antithrombin (AT), which correlates to HS anticoagulant activity. In this study, an AT-binding dodecasaccharide was prepared and its structure was proven. The continuation of this dissertation will allow us to not only investigate enzymatic approaches to synthesize HS-based anticoagulant drugs, but also develop a general method for synthesizing structurally defined HS oligosaccharides that could aid in the discovery of novel HS-based therapeutic agents.