Visible light activatable diazo-coumarin probes for fluorescent labeling of endogenous proteins in living cells

Abstract

Fluorescence technologies for characterizing and manipulating proteins in their native environment are fundamental to biological discoveries and mechanistic interrogation. Genetic engineering approaches have been routinely employed to visualize protein-of-interest (POIs) in living cells. However, the considerably large size of fusion proteins (15‒30 kDa) and challenges of incorporation of unnatural amino acids in mammalian cells hinder their applications. Most importantly, these methods can only be applied to cell types that are amenable to genetic manipulation, and the POIs would no longer be endogenously expressed. Selective labeling of endogenous proteins in living cells can be accomplished by using small molecule probes, with minimal perturbations to the cellular functions. However, the reported labeling methods are restricted to proteins containing nucleophilic residues adjacent to the binding sites. Using the concept of photoaffinity labeling, this limitation can be alleviated by the generation of highly reactive intermediates, such as carbenes and nitrenes, upon UV irradiation. Yet, the prerequisite of using cytotoxic UV irradiation has hindered this approach to be live cell compatible. Presented in this thesis are the design and synthesis of diazo-coumarin probes that can be photo-activated by visible light to uncage reactive carbene species from the diazo group, which can subsequently undergo bond insertion reactions. Several diazo-coumarin analogs have been examined, and 4-trifluoromethyldiazocoumarin has been found to be the most efficient. Under blue LED light irradiation, proteins-of-interest are efficiently captured to minimize the photo-toxicity of UV light to cells. Moreover, the fluorescence of coumarins is turned on concurrent to the labeling, enabling highly sensitive report of the labeled proteins in intact living cells, without the need for incorporating external fluorophores using the “click chemistry”. With a benzenesulfonamide incorporated as a specific ligand, photo-activated diazo-coumarin probes have efficiently labeled carbonic anhydrases (CAs) as model proteins. Both recombinant and endogenous CA-II can be fluorescently labeled in a highly selective manner. Subcellular localization of endogenous CA-II can be precisely traced by live cell fluorescence imaging, while labeled CA-II can be reliably quantified in multiple cell types by flow cytometry. A JQ-1 containing probe has also been synthesized in an attempt to target the nucleus-locating bromodomain-containing proteins (BRDs). An azide bearing handle has also been introduced into the probe design that may facilitate the enrichment of low abundant proteins using copper(I) catalyzed azide-alkyne cycloaddition reaction (CuAAC). Despite several designs and modifications, mixed results have been obtained in live cell experiments. Valuable insights in terms of the probe design have been acquired, which may facilitate the future application of this strategy. In conclusion, this thesis represents the first example of visible light induced covalent labeling of endogenous proteins in living cells. It is believed that this strategy has advanced a novel bioorthogonal approach for covalent labeling of biomolecules with fluorogenic probes in live cell studies, thus opens up exciting opportunities for biological discoveries and mechanistic interrogation in chemical biology.