Glycosaminoglycans (GAGs) are a family of structurally complex, highly sulfated, polydisperse, linear polysaccharides. Heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and hyaluronic acid are all members of this family. Heparin, the most widely studied GAG, is a major activator of serine protease inhibitors and more recently heparin and other GAGs have been shown to be important in the regulation of cell growth and cell-cell interaction. Nearly 300 metric tons of heparin are produced worldwide each year from animal tissue and used as an anticoagulant. Heparin binds to antithrombin III making it a potent inhibitor of coagulation serine proteases. The pentasaccharide sequence binding to antithrombin III, has been chemically synthesized in >60 synthetic steps and in < 0.25% yield. Despite this challenging synthesis, this pentasaccharide is currently being used therapeutically. There have been few reports of the synthesis of the oligosaccharide comprising of the other GAGs. The chemoenzymatic synthesis of GAG oligosaccharides is proposed using polysaccharide lyases and synthases. These classes of enzymes were first isolated and characterized in the laboratories of the principal investigator and co-investigator. The interaction of these synthetic oligosaccharides to a number of GAG-binding proteins will be evaluated. There are four specific aims in the current proposal.
These specific aims i nclude: 1 .Oligosaccharides will be prepared using polysaccharide lyases for evaluation as substrates in a variety of glycosylation reactions directed at the synthesis of GAG oligosaccharides. 2. Synthases will be used to extend GAG oligosaccharide acceptors using both standard UDP monosaccharide donors and novel, partially protected UDP monosaccharide donors. 3. RTILS will be used as solvents for the chemical and enzymatic glycosylation of polysaccharide lyase-prepared oligosaccharides. 4. The interaction of the synthesized oligosaccharides will be evaluated using a variety of GAG-binding proteins. GAG oligosaccharides are being synthesized to evaluate critical structural elements required for selective, high affinity interaction with protein binding partners. Three-dimensional structures identify potential bonding networks, but this information alone cannot predict definitively the major binding forces. Only the synthesis of analogs can afford the structural insights for biological understanding leading to new therapeutic agents.
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