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.

Public Health Relevance

Our proposed research is aimed at developing improved chemical reagents and methods for manipulating and labeling biological molecules such as proteins and nucleic acids. Our approach will take an old but well-known reaction (hydrazone formation), and redesign it to greatly enhance its performance and add new functional capabilities that current reactions do not offer. This work will provide valuable tools for biologits and biomedical scientists in imaging and studying biological molecules associated with human disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Lees, Robert G
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Stanford University
Schools of Arts and Sciences
United States
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K├Âlmel, Dominik K; Kool, Eric T (2017) Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis. Chem Rev 117:10358-10376
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Kietrys, Anna M; Kool, Eric T (2016) Epigenetics: A new methyl mark on messengers. Nature 530:423-4
Ji, Debin; Mohsen, Michael G; Harcourt, Emily M et al. (2016) ATP-Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling. Angew Chem Int Ed Engl 55:2087-91
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