Design and Synthesis of New Chemical Probes and Their Applications to Understanding Protein Structure and Function by NMR

了解蛋白质的结构和功能，包括蛋白质-蛋白质和蛋白质-小分子相互作用的机制，是具有挑战性的，需要使用一系列复杂的技术，包括X射线晶体学，NMR和cryoEM。最近，我们设计了一种功能强大的体外核磁共振分析，在一个复杂的测定中使用单个碳-13示踪剂监测蛋白质结合的生物合成转化，而无需任何纯化。本项目的目的是建立在这个令人兴奋的基础上，并扩展这个基础技术，为科学界的广泛应用。我们将着重于阐明酶的作用机制，以促进生物催化剂的发展，用于清洁和有效的合成生物活性靶标（重点是抗生素）和蛋白质构象的分析，包括，例如，本项目将设计和合成具有选择性结合碳-13标记的核磁共振探针.同位素标记的化合物的合成是具有挑战性的，这是由于合适标记的起始材料的有限可用性及其成本。因此，需要有效的合成策略。这些探针将被连接到蛋白质或用作研究中的系统的小分子组分。然后通过高场NMR光谱进行评估。将研究用氘取代邻近碳-13标记的质子以帮助锐化大型系统的NMR信号的益处。

Understanding protein structure and function, including the mechanisms of protein-protein and protein-small molecule interactions, is challenging and requires the use of a range of sophisticated techniques including X-ray crystallography, NMR and cryoEM. Recently we designed a powerful in vitro NMR assay to monitor protein-bound biosynthetic transformations using a single carbon-13 tracer in a complex assay without any need for purification.The aim of this project is to build upon this exciting foundation and expand this basic technique for wide application by the scientific community. We will focus on the elucidation of enzyme mechanisms to facilitate the development of biocatalysts for use in the clean and efficient synthesis of biological active targets (with a focus on antibiotics) and analysis of the conformation of proteins, including, for example, a-synuclein which is implicated in Parkinson's disease.This project will involve the design and synthesis of the NMR probes with selective incorporation of carbon-13 label(s). Syntheses of isotopically labelled compounds are challenging, due to the limited availability of suitably labelled starting materials and their costs. Hence efficient synthetic strategies are required. These probes will either be ligated to the protein or used as the small molecule component of the system under study. Assessment will then be conducted by high field NMR spectroscopy. The benefits of replacing protons adjacent to the carbon-13 label with deuterium to help to sharpen the NMR signals of large systems will be investigated.