Chemoenzymatic Synthesis of Glycosaminoglycan Oligos

描述（由申请人提供）：糖胺聚糖（GAGs）是一类结构复杂、高度硫酸化、多分散的线性多糖。肝素、硫酸肝素、硫酸软骨素、硫酸皮肤素和透明质酸都是这个家族的成员。肝素是研究最广泛的GAG，是丝氨酸蛋白酶抑制剂的主要激活剂，最近肝素和其他GAG已被证明在调节细胞生长和细胞间相互作用中起重要作用。全世界每年从动物组织中生产近300吨肝素，用作抗凝血剂。肝素与抗凝血酶III结合，使其成为凝血丝氨酸蛋白酶的有效抑制剂。与抗凝血酶III结合的五糖序列已被化学合成，合成步骤为60步，产率< 0.25%。尽管这种合成具有挑战性，但这种五糖目前已被用于治疗。由其他gag组成的低聚糖的合成报道很少。提出了利用多糖裂解酶和合酶合成GAG寡糖的化学酶法。这些酶首先在主要研究者和共同研究者的实验室中分离和表征。这些合成寡糖与一些gag结合蛋白的相互作用将被评估。

DESCRIPTION (provided by applicant): 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 include: 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.