Enabling synthesis and biological studies of homogeneous heparan sulfate and chondroitin sulfate glyco-polypeptides and proteoglycans

蛋白聚糖（PG）普遍存在于哺乳动物细胞表面和细胞外基质中。 矩阵PG由硫酸乙酰肝素或硫酸软骨素糖胺聚糖链组成 通过四糖接头共价连接到核心蛋白。PG扮演重要角色 在许多生物过程中。然而，由于天然存在的PG的异质性， 纯化明确的结构以研究结构和活性是非常具有挑战性的 关系传统上，PG的生物学功能被认为是由 连接的糖胺聚糖链有证据表明，核心蛋白可能会显着 影响聚糖活性。在最后一次授权期间，均质硫酸乙酰肝素和 已经合成了硫酸软骨素糖肽， 分子第一次。然而，合成需要总共超过100个化学步骤， 完成一个糖肽与硫酸乙酰肝素链，这限制了合成产量。 此外，在目前的合成策略下，PG中的一些共同结构特征 是不可访问的。为了克服这些挑战，在此更新应用程序中， 生物合成酶将被利用来大大扩展PG合成的能力。在aim中 1、关键酶将产生的四糖键合区与核心结合 肽，并延长聚糖链。自动化合成策略将进一步发展， 加快合成。目的二是建立合成糖多聚体的连接策略， 具有多个聚糖链的肽。这将提供一个强有力的工具，延长多肽 骨架，并探测的假设，核心蛋白可以调节聚糖的活动。在 目的3完成硫酸软骨素蛋白聚糖bikunin样糖蛋白的合成。 Bikunin是一种被批准用于治疗可能危及生命的败血症的药物， 机制目前尚不清楚。在结构明确的合成bikunin的帮助下， 将建立bikunin抗炎活性的分子机制， 展示了合成的力量， 这些复杂分子的性质。

Proteoglycans (PGs) are ubiquitous on mammalian cell surfaces and in the extracellular matrix. PGs are made up of heparan sulfate or chondroitin sulfate glycosaminoglycan chains covalently attached to the core protein through tetrasaccharide linkers. PGs play important roles in many biological processes. However, due to the heterogeneity of naturally existing PGs, it is extremely challenging to purify well-defined structures to study the structure and activity relationship. Traditionally, the biological functions of PGs are believed to be dictated by the glycosaminoglycan chains attached. Evidence is emerging that the core protein may significantly impact the glycan activities. During the last grant period, homogeneous heparan sulfate and chondroitin sulfate glycopeptides have been synthesized, providing access to these complex molecules for the first time. However, the synthesis required total over 100 chemical steps to complete a glycopeptide with a heparan sulfate chain, which limited the synthetic output. Furthermore, with the current synthetic strategy, some of the common structural features in PGs are not accessible. To overcome these challenges, in this renewal application, the power of the biosynthetic enzymes will be harnessed to greatly expand the capability for PG synthesis. In aim 1, the key enzymes will be produced to generate the tetrasaccharide linkage region with the core peptide, and to extend the glycan chain. Automated synthesis strategy will be developed to further expedite the synthesis. In aim 2, ligation strategies will be established to synthesize glyco poly- peptides with multiple glycan chains. This will provide a powerful tool to extend the poly-peptide backbone, and to probe the hypothesis that the core protein can modulate glycan activities. In aim 3, synthesis of a chondroitin sulfate proteoglycan bikunin like glycoprotein will be completed. Bikunin is an approved drug to treat the potentially life-threatening sepsis conditions, and its mechanism is currently unclear. Aided by the structurally well-defined synthetic bikunin, the molecular mechanisms of the anti-inflammatory activities of bikunin will be established, demonstrating the power of synthesis in expanding the understanding of the interesting biological properties of these complex molecules.