Highly Reactive Hydrazone Chemistry: Orthogonal Modification in Cellular Contexts

描述(申请人提供)：我们建议的研究旨在将一种古老但有用的生物正交化反应--肼和肟的形成--发展成一种现代的、高效的和高度通用的生物功能化工具。我们将克服一些 这一反应过去的重要局限性包括在生物pH下反应速度慢和稳定性低，并增加了目前的正交反应所不具备的新的功能性质和能力。在我们的前期工作中，我们已经开发了多种新的有机催化剂，这些催化剂是目前存在的最好的肼和肟形成催化剂，使反应速度加快了数量级。重要的是，我们已经证明了这种催化剂可以加速RNA碱基的甲醛加合物的逆转，例如在福尔马林固定的组织中发现的那些。此外，我们还确定了醛和肼的重要结构特征，这些特征导致了特别高的反应速度，甚至超过了紧张的炔环加成反应。此外，我们还展示了一种新型荧光(“暗区”)标记剂的原理证明。我们建议的项目将专门针对开发新的、高活性的自催化醛和肼的目标，以将反应速度加快数量级，使它们比现代的正交环加成反应更有效。此外，我们还将建立新型的预合肼类化合物作为交换试剂，用于将醛或肼标记在感兴趣的生物分子上。此外，我们将开发非常高效、低毒、细胞渗透性的催化剂，以实现快速的细胞内生物结合。最后，我们的催化剂将被用来逆转福尔马林固定组织中的交联链，解锁临床上重要的RNA和蛋白质信息。这项工作测试了新的机制假说，以加速生成的肼/肟的反应性。它将引入几个新的化学设计概念，包括自催化“超快”反应物、“暗区”荧光试剂和用于超分辨率成像的硫酮标记。这项工作之所以重要，是因为它采用了一种广泛使用的、生物医学上重要的反应，并使其更加有效和有用。我们的实验将开发出现存最快的联氨和肟类反应物，并将开发出比目前已知的任何催化剂都更有效的催化剂。如果成功，这项研究将使以前无法完成的细胞实验成为可能，并将促进从数百万存储的组织样本中恢复具有临床重要性的分子信息。

DESCRIPTION (provided by applicant): Our proposed research is aimed at developing a venerable but useful bioorthogonal reaction - hydrazone and oxime formation - into a modern, efficient, and highly versatile tool for biological functionalization. We will overcome some of the important limitations of this reaction in the past, including slow rates at biological pH and low stability, and add to it new functional properties and capabilities that current orthogonal reactions do not have. In our preliminary work we have developed multiple new organocatalysts that are by far the best catalysts in existence for hydrazone and oxime formation, speeding the reaction by orders of magnitude. Importantly, we have shown that such catalysts can speed the reversal of formaldehyde adducts of RNA bases, such as those found in formalin-fixed tissue. In addition, we have identified important structural features in aldehyde and hydrazines that lead to especially high reaction rates, surpassing even those of strained alkyne cycloadditions. Further, we have demonstrated proof of principle for a new class of fluorogenic ("DarkZone") labeling agents. Our proposed project will specifically address a goal of developing new, highly reactive self-catalyzing aldehydes and hydrazines to accelerate reaction rates by orders of magnitude, making them even more efficient than modern orthogonal cycloadditions. In addition, we will establish novel preformed hydrazones as exchange reagents for labeling both aldehydes or hydrazines on biomolecules of interest. Further, we will develop exceptionally efficient, low-toxicity, cell-permeable catalysts to enable rapid intracellular bioconjugations. Finally, our catalysts will be used to reverse crosslinks in formalin-fixed tissues, unlocking clinically important RNA and protein-based information. This work tests novel mechanistic hypotheses for accelerating reactivity in hydrazone/oxime formation. It will introduce several new chemical design concepts, including self-catalyzing "ultrafast" reactants, "DarkZone" fluorogenic reagents, and hydrazone labels for superresolution imaging. The work is important because it takes a widely used, biomedically important reaction and makes it much more efficient and useful. Our experiments will develop the fastest hydrazine and oxime reactants in existence, and will develop catalysts that are more efficient than any known to date. If successful, the research will enable cellular experiments that could not be done before, and will facilitate the recovery of clinically important molecular information from millions of stored tissue specimens.