Heparin is an anticoagulant that has been in use since the 1930's and has a current annual market value of about $3-4 billion. It is a heavily sulfated form of heparan sulfate, a polysaccharide found on the surfaces of almost types of animal cells. Both are based on a repeating unit of -uronic acid linked to -N-acetylglucosamine. In the cell these polysaccharides are modified by a series of enzymes to yield heterogeneous, highly anionic chains that bind a large variety of proteins. The anticoagulant activity of heparin is largely derived from its action on the serine protease inhibitr antithrombin (AT). Heparin bound to AT inhibits the coagulation cascade enzymes factor Xa and factor IIa (thrombin). Inhibition of factor Xa only requires a pentasaccharide sequence, but inhibition of thrombin requires a sixteen-monosaccharide sequence to bind AT and thrombin simultaneously. The synthesis of heparin in this size range is essentially impossible, requiring it to be derived from animal sources. This leads to the problem of contamination, as well as difficulties in obtaining pure samples for structure-activity studies. To overcome these difficulties, Dr. Jian Liu's laboratory at UNC Chapel Hill has developed a chemoenzymatic method capable of synthesizing heparin oligosaccharides from 5-10 residues, the size for binding a single protein. We will build heparin mimetic capable of binding multiple proteins by linking chemoenzymatically synthesized oligosaccharides. The Huisgen click reaction yields a 1,2,3-triazole linkage from an azido group and a terminal alkyne and will be used to link the oligosaccharide components. We began this project by developed an enzymatic method for adding an N-azidoacetyl glucosamine to the nonreducing end of an oligosaccharide. The reducing end will display a terminal alkyne on a flexible linker. We will use this method to assemble three heparin mimetics consisting of combinations of the AT-binding and thrombin-binding heparin sequences. We will evaluate the affinity to antithrombin by affinity coelectrophoresis and assess anti-IIa activity with a chromogenic substrate method. Pharmocodynamics and the adverse effect known as heparin-induced thrombocytopenia (HIT) will be tested in a murine model.