Applications of Click Chemistry in Glycobiology

描述(申请人提供)：细胞表面多聚糖是细胞-细胞和细胞-基质相互作用的主要决定因素。细胞表面糖基化的变化标志着癌症和炎症的发生。在细胞内，它们可以调节转录、翻译和蛋白质运输。然而，在描述多糖功能的分子基础方面进展相当缓慢。这在一定程度上是因为，与其他生物聚合物不同，葡聚糖的生物合成既不受模板驱动，也不受直接转录控制。因此，用常规的遗传和生物化学方法来阐明多糖的功能及其与疾病的相关性，所产生的信息有限。该项目的长期目标是实现点击化学--一组强大、可靠和选择性的反应--作为糖生物学基础研究的通用工具。 第一个授权期(K99)的主要目标是在Carolyn Bertozzi教授(导师)的实验室中启动在蛋白质糖基化研究中应用点击化学的步骤。目的1利用基于点击化学的生物正交反应制备具有治疗价值的均一糖蛋白。基因编码的醛标签将用于位点特异性糖基化。目标2是利用糖树枝状大分子功能化的碳纳米管设计和构建特定的凝集素和抗体传感器。 结合第一个授权期的研究经验和知识，我将在下一个授权期(R00)在两个新的方向上拓展点击化学的应用。目标3是利用酶模板原位点击化学发现/开发糖生物合成和加工酶的小分子抑制剂。为了证明原理，我选择O-β-N-乙酰氨基葡萄糖转移酶(OGT)作为第一靶点。我计划开发片段文库，用于筛选OGT的自组装抑制剂。鉴于过度的O-GlcNAc修饰与胰岛素抵抗引发的高血糖之间的相关性，所开发的化合物可能会在糖尿病治疗中应用。目标4是用合成的非天然底物截取含有生物正交功能基团的糖的生物合成途径，如叠氮化合物和炔烃。同时，我还将开发基于点击化学的新的选择性反应，以便随后在活细胞中进行检测。 众所周知，多糖参与了许多正常和疾病的过程。这一系列的实验将促进我们对糖的生物合成和与这些疾病状态相关的碳水化合物-蛋白质相互作用的理解。这些研究也可能为治疗干预提供新的途径。我预计在这些研究中开发的新的化学工具将在生物医学研究中有广泛的应用。

DESCRIPTION (provided by applicant): Cell surface glycans are major determinants of cell-cell and cell-matrix interactions. Changes in cell surface glycosylation mark the onset of cancer and inflammation. Inside the cell, they can regulate transcription, translation as well as protein trafficking. Progress toward delineating the molecular basis of glycan function, however, has been rather slow. This is partly due to the fact that the biosynthesis of glycans, unlike other biopolymers, is neither template-driven nor under direct transcriptional control. Therefore, conventional genetic and biochemical approaches for elucidating glycan function, and its relevance to disease, have yielded limited information. The long-term goal of this project is to implement click chemistry--a set of powerful, reliable and selective reactions--as a general tool for fundamental studies of glycobiology. The major objective of the first granting period (K99) is to initiate steps for applying click chemistry in studies of protein glycosylation in the lab of Professor Carolyn Bertozzi (the mentor). Aim 1 intends to use click chemistry-based bioorthogonal reactions to prepare homogeneous glycoproteins with therapeutic value. A genetically encoded aldehyde tag will be used for site-specific glycosylation. Aim 2 is to design and build specific lectin and antibody sensors using glycodendrimer-functionalized carbon nanotubes. With the experience and knowledge gained from the research in the first granting period, I will expand the applications of click chemistry in two new directions in the next granting period (R00). Aim 3 is to discover/ develop small molecule inhibitors of glycan biosynthetic and processing enzymes using enzyme-templated in situ click chemistry. For proof of principle, I chose O-beta-N-acetylglucosaminyl-transferase (OGT) as the first target. I plan to develop fragment libraries that will be screened for self-assembled inhibitors of OGT. Given the correlation of excessive O-GlcNAc modification with insulin resistance-triggered hyperglycemia, the compounds developed may have applications in diabetes therapy. Aim 4 is to intercept glycan biosynthetic pathways with synthetic unnatural substrates bearing bioorthogonal functional groups, such as azides and alkynes. In parallel, I will also develop new selective reactions based on click chemistry for their subsequent detection in live cells. Glycans are known to participate in many normal and disease processes. This series of experiments will advance our understanding of glycan biosynthesis and carbohydrate-protein interactions related to these disease states. These studies may also offer new avenues for therapeutic intervention. I anticipate that the new chemical tools developed in these studies will have broad applications in biomedical research.