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Development of epoxide probes for protein labeling, and, Development of fluorescent reporter substrate probes for oxidoreductases.

机译：用于蛋白质标记的环氧化物探针的开发，以及用于氧化还原酶的荧光报告分子底物探针的开发。

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Development of epoxide probes for protein labeling. The research presented herein describes the development of new affinity probes for protein labeling based on an epoxide reactive group. Systematic screening revealed that an epoxide functionality possesses the special combination of stability and reactivity which renders it stable toward proteins in solution but reactive on the protein surface outside the active site (proximity-induced reactivity). Highly efficient and selective labeling of purified HCA II (human carbonic anhydrase II) was achieved. Importantly, the epoxide probe selectively labeled HCA II both in simple protein mixtures and in cellular extracts. The utility of our modular probe design was confirmed by an epoxide probe containing an adamantyl ligand instead of a benzenesulfonamide, which was constructed and shown to label P450cam efficiently and selectively in a proteomic context. In addition to being a promising platform for mild, selective in vitro protein labeling, this system has the potential to be an interesting and useful tool for proteomic analysis and exploration.;Development of fluorescent reporter substrates probes for oxidoreductases . Based on the rational design of fluorescent switches and exploration of enzymes promiscuity, the research presented herein describes the development of fluorogenic small molecule reporters for dehydrogenases and monoamine oxidases. Rational design of fluorescence switch based on PET and TICT quenching mechanism was exploited, novel oxidative deamination coupled with pyrrole-formation cascade reporting pathway was utilized. Systematic exploration of amino- and pyrrole-coumarin motifs was undertaken which led to the development of two systems with desired optical properties (fluorogenic probes). Ultimately, the ability of these compounds to serve as reporter substrate was demonstrated, most notably by the development of selective probe for MAO B. Moreover, the effectiveness of the diamine probes was demonstrated in context of intact mitochondria. These probes represent exciting leads for novel imaging probes that can be used in vivo.
机译：用于蛋白质标记的环氧化物探针的开发。本文介绍的研究描述了基于环氧化物反应基团的新型蛋白质标记亲和探针的开发。系统性筛选显示，环氧官能团具有稳定性和反应性的特殊组合，这使其对溶液中的蛋白质稳定，但在活性位点以外的蛋白质表面具有反应性（邻近诱导的反应性）。对纯化的HCA II（人碳酸酐酶II）进行了高效和选择性标记。重要的是，环氧探针在简单的蛋白质混合物和细胞提取物中均选择性标记了HCA II。我们的模块化探针设计的实用性通过包含金刚烷基配体而不是苯磺酰胺的环氧化物探针得以证实，该探针被构建并显示出在蛋白质组学背景下有效且选择性地标记P450cam的能力。除了作为温和的，选择性的体外蛋白质标记的有前途的平台外，该系统还可能成为蛋白质组学分析和探索的有趣且有用的工具。；氧化还原酶荧光报告底物探针的开发。基于荧光开关的合理设计和对酶混杂的探索，本文介绍的研究描述了用于脱氢酶和单胺氧化酶的荧光小分子报道分子的开发。利用基于PET和TICT猝灭机理的荧光开关的合理设计，利用新颖的氧化脱氨基结合吡咯-形成级联报告途径。进行了氨基和吡咯香豆素基序的系统探索，这导致了两个具有所需光学特性的系统（荧光探针）的发展。最终，证明了这些化合物用作报告底物的能力，最明显的是通过开发了针对MAO B的选择性探针。此外，在完整的线粒体中证明了二胺探针的有效性。这些探针代表了可用于体内的新型成像探针的令人兴奋的线索。

著录项

  • 作者

    Chen, Gong.;

  • 作者单位

    Columbia University.;

  • 授予单位 Columbia University.;
  • 学科 Biology Cell.;Chemistry Organic.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 391 p.
  • 总页数 391
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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