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

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，以进一步加深我们对基础生物学和疾病的理解。然而，询问基因和蛋白质表达之间的联系是分离的生物学过程仍然非常困难。这方面的一个相关例子是在细胞表面呈现的健康细胞和癌细胞之间的糖基化状态的变化。用于询问这些变化的新兴工具是在目标生物分子中代谢结合“化学报告”，并获得高度特异性的“生物正交”化学反应，这些化学反应最好只与化学报告组发生反应。这种方法成功的关键是快速、生物相容性和化学选择性的生物正交反应的可用性。结合两个化学报告，然后是化学选择性生物正交标记，是这种“标记和修改”策略的延伸，它提供了标记纤维素中两种不同生物分子的机会。此外，在单个生物分子中结合不同的化学报告，然后进行双微分标记，是一种探索单个生物分子动力学的新兴方法。目前，如果需要离散标记的产品（即没有产品的混合物），那么用当前的生物正交策略来实现这一目标是极具挑战性的。以前的工作-我们的合作团队最近确定了芳香动力胺作为cu催化的炔叠氮环加成（CuAAC）反应的优越试剂的效用。这些炔代物需要更少的Cu催化剂，是迄今为止报道的唯一能够在连续两步CuAAC过程中进行化学选择控制的炔试剂。项目目标：本项目的主要目标是将芳香胺发展成为一种强大的新型生物正交反应平台，用于前列腺癌细胞中糖蛋白的化学选择标记。该项目的具体目标是：(i)获得在CuAAC反应中增强的动力学选择性的机制理解，并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）探索动力胺作为前列腺癌细胞糖蛋白标记剂的效用。

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.