Chemoenzymatic Synthesis of Highly Complex Glycans

复杂的碳水化合物或聚糖参与几乎每一个生理或病理过程。糖科学发展的一个主要障碍是缺乏纯的和结构明确的碳水化合物和糖缀合物。这些化合物通常以低浓度和微不均匀形式存在，使其分离和表征非常复杂。在许多情况下，定义明确的寡糖只能通过化学或酶促方法获得。尽管取得了进展，但复杂寡糖的化学合成仍然非常耗时。对更有效方法的需求刺激了化学-酶促方法的发展，其中合成寡糖前体被一系列糖基转移酶修饰。这种方法的严重局限性在于它仅提供容易获得对称支化寡糖的途径。将开发一种新的化学酶合成方法来生产高度复杂的对称和不对称分支/V-连接的聚糖。该方法将利用核心五糖，该核心五糖在关键分支位置被正交保护基团修饰，使得可以通过化学糖基化选择性地连接独特的糖部分。将以这样的方式选择附加物，使得所得脱保护化合物的触角可以通过糖基转移酶独特地延伸，以产生大量的不对称多触角聚糖。此外，项目1中发现的酶特异性将用于选择性修饰，以提供否则无法合成的寡糖。新的化学-酶法将用于制备在人卵母细胞上发现的涉及精卵结合的一组寡糖。此外，将制备探针以鉴定识别SLE“的精子的聚糖结合蛋白。新方法也将用于合成在猪原代呼吸道上皮细胞上发现的N-聚糖，这些细胞与流感病毒感染有关。这些化合物将用于结合研究，以揭示有关聚糖拓扑结构以及人类和猪感染性的信息。最后，合成聚糖将用作复杂生物混合物中聚糖鉴定的标准品。最终，这些研究可能会导致识别各种疾病的新生物标志物。

Complex carbohydrates or glycans are involved in almost every physiological or pathological process. A major obstacle to advances in Glcyoscience is the lack of pure and structurally well-defined carbohydrates and glycoconjugates. These compounds are often found in low concentrations and in microheterogeneous forms greatly complicating their isolation and characterization. In many cases, well-defined oligosaccharides can only be obtained by chemical- or enzymatic approaches. Despite progress, the chemical synthesis of complex oligosaccharides remains very time consuming. The need for more efficient approaches has stimulated the development of chemo-enzymatic methods in which a synthetic oligosaccharide precursor is modified by a range of glycosyltransferases. A serious limitation of such an approach is that it only provides readily access to symmetrically branched oligosaccharides. A novel chemoenzymatic synthetic approach will be developed to produce highly complex symmetrically and asymmetrically branched /V-linked glycans. The approach will exploit a core pentasaccharide that at key branching positions is modified by orthogonal protecting groups that make it possible to selectively attach unique saccharide moieties by chemical glycosylation. The appendages will be selected in such a way that the antenna of the resulting deprotected compounds can be uniquely extended by glycosyltransferases to give large numbers of asymmetrical multi-antennary glycans. Furthermore, enzyme specificities uncovered in project 1 will be exploited for selective modifications to provide oligosaccharides that otherwise cannot be synthesized. The new chemo-enzymatic methodology will be employed to prepare a panel oligosaccharides found on human oocytes that have been implicated in sperm-egg binding. Furthermore probes will be prepared to identify the glycan binding protein of spermazoae that recognizes SLe''. The new methodology will also be employed to synthesize N-glycans found on primary swine respiratory epithelial cells that have been implicated in influenza virus infections. The compounds will be used in binding studies to uncover information about glycan topology and human and swine infectivity. Finally, the synthetic glycans will be employed as standard for glycan identification in complex biological mixtures. Ultimately, these studies may lead to the identification of novel biomarkers for various diseases.