Small Molecule Probes for Chemical Biology

这项提案概述了一个多样化的计划，以推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正交探针，利用成像质谱仪同时监测多糖、蛋白质、DNA和肽聚糖的合成。在这些探针的道路上，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，我们将产生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的途径，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标记这些修饰的多糖用于检测。*我们的目标是用一种新型的苯丙氨酸类似物来跟踪细菌蛋白质的生产，这种类似物含有一种在蛋白质合成过程中被吸收和并入的碲。当被用来监测全球蛋白质合成时，掺入碲之后，可以使用最近开发的一种名为质谱仪的原子质谱仪技术。碲还为重组蛋白质的分析提供了有用的工具，碲中心将为X射线结晶学提供重原子和核磁共振活性核。将研究修饰后的氨基酸在重组蛋白中的掺入效果。此外，还将探索碲的生物正交化学。*使用成像质量细胞术，我们将应用我们新的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，将有可能监测何时何地合成多糖、肽聚糖、DNA和蛋白质。这种高度参数化的分析将是成像质量细胞术如何与多个生物正交化学相结合以了解复杂生物事件的第一个例子。*总体上，这项建议涵盖了大量的科学领域，涉及合成、酶学、生物物理研究和细菌检测。这项研究将为学生在化学和生物的交界处整合技能和解决问题提供一个出色的训练场地。**

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. **