DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

细胞内指示剂和离子传输研究的发展

• 批准号: 6106707
• 负责人: Robert E London
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6106707
• 关键词: anomer; biomaterial development /preparation; calcium indicator; calcium metabolism; cations; chemical synthesis; cytoplasm; deoxyglucose; fluorine; glucose analog; intracellular transport; ion transport; laboratory rabbit; magnesium; membrane transport proteins; nuclear magnetic resonance spectroscopy; sarcoplasmic reticulum; uracil analog

Cellular cations play fundamental roles in hormonal signaling, and are also involved in the mediation of cell injury. The development of intracellular indicators for cytosolic cations and other parameters of interest has had a major impact on the field of cell biology. Our goals in this area have included the development of more sensitive intracellular calcium indicators, and more selective intracellular magnesium indicators. During the past year, we have worked on the development of a new and general route towward the synthesis of fluorescent chelators related to BAPTA [bis(o-aminophenoxy)ethane-N,N,N?,N?-tetraacetic acid] or APTRA (o-aminphenol-N,N,O-triacetic acid), which are used for determining calcium and magnesium ion levels in cells. The key step is a palladium-catalyzed coupling of the chelating moiety to the fluorophore. Using this method, it becomes possible to more rapidly synthesize and screen indicators with a range of potential fluorophores. Coupling of triflate [4-(CF3SO3)C6H3NO2-2-(OBenzyl)] with several polyaromatic boron and tin species proceeds with excellent yields. Further elaboration of the 2-benzyloxy-nitrophenyl ring (reduction, followed by triple alkalyation with BrCH2COOMe and saponification) gives the desired APTRA chelators. The fluorescent response to magnesium of several of these molecules has been evaluated. In addition to the above studies, we have recently succeeded in developing a more magnesium selective chelator than the APTRA molecules referred to above. These fluorescent molecules exhibit an emission shift upon magnesium complexation, and are relatively insensitive to calcium ions. Derivatives more suitable for loading into cells are currently in preparation. In parallel with the probe development work, physiological studies utilizing NMR have continued in collaboration with Dr. Murphy?s group at NIEHS and Dr. Steenbergen?s group at Duke University. These studies have been aimed at understanding the ionic changes which occur during periods of stress, and the role that these changes play in causing irreversible injury. During the last year, NMR studies performed on perfused rat hearts indicated that differences in intracellular pH observed between pre-conditioned and non pre-conditioned hearts are not due to increased H+ extrusion in the former. Studies on the transport of the fluorinated nucleoside: 2'-fluoro-5-methyl-beta-L-arabinofuranosyl uracil (L-FMAU), were recently concluded.

细胞阳离子在激素调节中起着重要作用。 信号传导，并且还参与细胞损伤的介导。的 开发细胞溶质阳离子的细胞内指示剂， 其他感兴趣的参数对该领域产生了重大影响， 细胞生物学我们在这方面的目标包括发展 更敏感的细胞内钙指标，以及更多 选择性细胞内镁指示剂。在过去的一年里， 我们一直致力于开发一条新的通用路线， BAPTA相关荧光螯合剂的合成 [双（邻氨基苯氧基）乙烷-N，N，N？，N？四乙酸]或 APTRA（邻氨基苯酚-N，N，O-三乙酸），用于 测定细胞中的钙和镁离子水平。的关键步骤 是钯催化的螯合部分与 荧光团。使用这种方法，可以更快地 综合和筛选具有一系列潜力指标 荧光团。三氟甲磺酸酯偶联 [4-（CF 3SO 3）C6 H3 NO2 -2-（OBenzyl）]与多个多环芳烃 硼和锡物质以优异的产率进行。进一步 2-苄氧基-硝基苯基环的制备（还原， 然后用BrCH 2COOMe进行三重碱解， 皂化）得到所需的APTRA螯合剂。荧光 这些分子中的几种对镁的反应已经被 评估。除上述研究外，我们最近 成功地开发了一种比 上述APTRA分子。这些荧光 分子在镁络合时表现出发射位移， 并且对钙离子相对不敏感。衍生产品more 目前正在制备适合于装载到细胞中的药物。 在 与探针开发工作、生理学研究 继续与墨菲博士合作S 和斯滕贝尔根博士他是杜克大学的一个研究小组的成员。 这些研究的目的是为了了解 在压力时期发生的，以及这些作用， 这些变化会造成不可逆的伤害。在过去的一年里， 对灌注的大鼠心脏进行的NMR研究表明， 在预处理的细胞之间观察到的细胞内pH的差异 而非预处理的心脏不是由于增加的H+ 挤压在前。含氟化合物迁移的研究 核苷：2 '-氟-5-甲基-β-L-阿拉伯呋喃糖基尿嘧啶 （L-FMAU），最近完成。