Small Molecule Probes for Chemical Biology

该提案概述了一个多样化的计划，以促进我们在化学生物学方面的工作，重点是开发操纵和研究细菌的工具。我们的目标是开发一系列的正交探针，将允许监测多糖，蛋白质，DNA和肽聚糖的合成，同时，使用成像质量细胞术。在通往这些探针的道路上，将开发新的生物正交化学，酶抑制剂和碲的生物用途。 通过合成非天然单糖，这些单糖被认为是天然的，我们将生产针对特定代谢途径的化合物。我们假设，通过明智地选择靶向途径，将有可能标记由选定的细菌产生的多糖。我们将开发新的生物正交化学来标记这些修饰的多糖用于检测。 我们的目标是遵循细菌蛋白质的生产与一种新的苯丙氨酸类似物，含有碲吩，这是采取和蛋白质合成过程中纳入。当用于监测全球蛋白质合成时，碲的掺入可以通过最近开发的原子质谱技术（称为质谱细胞术）进行。碲吩还为重组蛋白的分析提供了有用的工具，碲中心将为X射线晶体学和NMR活性核提供重原子。将研究在重组蛋白中掺入修饰的氨基酸的影响。此外，还将探索碲吩的新型生物正交化学。 使用成像质量细胞仪，我们将应用我们的新多糖和蛋白质合成探针来回答有关细菌如何形成称为生物膜的多细胞群落的问题。通过使用一系列不同的标签，将有可能监测多糖，肽聚糖，DNA和蛋白质合成的位置和时间。这种高度参数化的分析将是成像质谱细胞术如何与多种生物正交化学相结合以了解复杂生物事件的第一个例子。 总的来说，这项建议包括大量的科学广度，涉及合成，酶学，生物物理研究和细菌测定。这项研究将为学生提供一个优秀的训练基地，以整合他们的技能和解决问题的化学和生物学的接口。

This proposal outlines a diverse program to further our work in chemical biology focusing on the development of tools to manipulate and study bacteria. We aim to develop a series of orthogonal probes that will allow monitoring of polysaccharide, protein, DNA and peptidoglycan synthesis, simultaneously, using imaging mass cytometry. On the path to these probes, new bioorthogonal chemistry, enzyme inhibitors and biological uses of tellurium will be developed. Through the synthesis of unnatural monosaccharides, which are recognized like their natural counterparts, we will produced compounds that target specific metabolic pathways. We hypothesize that through the judicious choice of the pathways targeted, it will be possible to label the polysaccharides produced by selected bacteria. We will develop new bioorthogonal chemistry to label these modified polysaccharides for detection. We aim to follow bacterial protein production with a novel phenylalanine analogue, containing a tellurophene, which is taken up and incorporated during protein synthesis. When used for monitoring global protein synthesis, the incorporation of tellurium can be followed by a recently developed atomic mass spectrometry technique called mass cytometry. The tellurophene also provides a useful tool for the analysis of recombinant proteins, the tellurium centre will provide a heavy atom for X-ray crystallography and an NMR active nucleus. The effect of incorporation of the modified amino acid in recombinant proteins will be studied. In addition, novel bioorthogonal chemistry at the tellurophene will be explored. Using imaging mass cytometry, we will apply our new polysaccharide and protein synthesis probes to answer questions about how bacteria form multicellular communities known as biofilms. Through employing a series of different tags it will be possible to monitor where and when polysaccharides, peptidoglycan, DNA and proteins are being synthesized. This highly parameterized assay will be the first example of how imaging mass cytometry can be coupled with multiple bioorthogonal chemistries to understand complex biological events. Overall this proposal encompasses substantial scientific breadth involving synthesis, enzymology, biophysical studies and bacterial assays. This research will provide an outstanding training ground for students to integrate their skills and solve problems at the interface of chemistry and biology.