Mechanism-inspired development of a chemoselective click-based bio-orthogonal reaction platform

背景-后基因组时代为生物学家提供了丰富的信息和基因组测序工具，以加深我们对基础生物学和疾病的理解。然而，在基因和蛋白质表达之间的联系脱钩的生物过程中，询问仍然是极其困难的。一个相关的例子是出现在细胞表面的健康细胞和癌细胞之间的糖基化状态的变化。一种用于询问这些变化的新兴工具是在目标生物分子中新陈代谢地并入“化学报告者”，并获得高度特异的“生物正交”化学反应，这些化学反应最好只与化学报告人组反应。这种方法成功的关键是快速、生物相容和化学选择性的生物正交反应的可用性。在化学选择性生物正交标记之后结合两个化学记者是这种标记和修饰策略的扩展，它提供了标记纤维素中两种不同生物分子的机会。此外，在单个生物分子中加入不同的化学报告分子，然后进行双差异标记，是探索单个生物分子动力学的一种新方法。目前，如果需要离散标记的产品(即没有混合的产品)，使用现有的生物正交策略实现这一点是具有高度挑战性的。以前的工作-我们的合作团队最近发现芳香族氰胺作为铜催化的炔叠氮环加成(CuAAC)反应的一种优秀试剂。这些炔类替代品需要的铜催化剂显著减少，是迄今为止报道的唯一能够在连续的两步CuAAC过程中进行化学选择性控制的炔类试剂。项目目标-本项研究的主要目标是将芳香族肌胺开发成一个强大的新的生物正交反应平台，用于前列腺癌细胞中糖蛋白的化学选择性标记。该项目的具体目标是：(I)从机理上了解肌动蛋白在CuAAC反应中的化学选择性增强，并有可能跨越其他生物正交反应类别。(Ii)为生物分子的化学选择性修饰创造条件。(Iii)探索肌动蛋白作为前列腺癌细胞糖蛋白标记剂的用途。

Background - The post-genomic era has provided biologists with a wealth of information and -omic sequencing tools to further our understanding of fundamental biology and disease. However, it is still exceedingly difficult to interrogate biological processes where the link between gene and protein expression is decoupled. A pertinent exemplar of this is changes in the glycosylation state between healthy and cancerous cells presented on the cell surface. An emerging tool used to interrogate these changes is the metabolic incorporation of 'chemical reporters' within a target biomolecule and access to highly specific 'bio-orthogonal' chemical reactions, which preferably react only with the chemical reporter group. Key to the success of this approach is the availability of bio-orthogonal reactions that are fast, biocompatible, and chemoselective.The incorporation of two chemical reporters followed by chemoselective bio-orthogonal labelling is an extension of this 'tag and modify' strategy, which offers the opportunity to label two different biomolecules in cellulo. Additionally, the incorporation of different chemical reporters within a single biomolecule followed by dual differential labelling is a nascent approach to probe the dynamics of a single biomolecule. At present, this is highly challenging to achieve with current bio-orthogonal strategies if discretely tagged products (i.e., without a mixture of products) are required.Previous work - Our collaborative team has recently identified the utility of aromatic ynamines as a superior reagent for Cu-catalyzed alkyne-azide cycloaddition (CuAAC) reactions. These alkyne surrogates require significantly less Cu catalyst are the only alkyne reagents reported to date which enable chemoselective control in a sequential two-step CuAAC process.Project Objective - The principal objective of this studentship is to develop aromatic ynamines into a powerful new bio-orthogonal reaction platform for the chemoselective tagging of glycoproteins in prostate cancer cells. The specific aims of the project are to:(i) gain a mechanistic understanding of the enhanced chemoselectivity of ynamines in CuAAC reactions, and potentially across other bio-orthogonal reaction classes.(ii) establish conditions for chemoselective modification of biomolecules.(iii) explore the utility of ynamines as glycoprotein tagging agents of prostate cancer cells.