The use of photocatalysts and far-red light to induce bioorthogonal chemistry, enable pretargeted uncaging inside live cells, and generate reactive electrophiles for protein labeling
Date
2023
Authors
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Publisher
University of Delaware
Abstract
The focus of my research is the use of photocatalysts and far-red light to induce bioorthogonal chemistry for applications in ligand-directed uncaging and generation of reactive electrophiles. Biocompatibility is an important factor when developing photocatalytic methods. Singlet oxygen generation is a common side effect of photochemical reactions and can cause degradation to biomolecules. My work demonstrates the use of far-red light and biocompatible fluorophores as photocatalysts to activate dihydrotetrazine (DHTz) precursors for the tetrazine ligation, a powerful bioorthogonal reaction. ☐ Chapter 1 introduces the photocatalytic turn-on of the tetrazine ligation and my work in screening fluorophores as far-red light photocatalysts. Sila-rhodamine (SiR) derivatives were tested for their ability to catalyze the oxidation of DHTz for the turn-on of the tetrazine ligation. Using only 500 nM of catalyst, successful oxidation of DHTz was observed upon light irradiation (660 nm). SiR derivatives that maintained their light-absorbing, “open” form were more efficient than the parent SiR, which has an equilibrium between its “open” and “closed” forms. One of these photocatalysts was then tested for its biocompatibility. Successful activation of the tetrazine ligation was demonstrated on a model protein as a result of photocatalytic oxidation of a DHTz. Importantly, oxidative damage to the protein was not observed. These SiR photocatalysts are now commonly used in the Fox lab and efforts to design more efficient catalysts are ongoing. ☐ Described in Chapter 2 are ligand-directed catalysts for live-cell, photocatalytic activation of bioorthogonal uncaging chemistry. Catalytic groups are localized via a tethered ligand either to DNA or to tubulin, and red-light (660 nm) photocatalysis is used to initiate a cascade of DHTz-oxidation, intramolecular DielsAlder reaction, and elimination to release phenolic compounds. SiR dyes, more conventionally used as biological fluorophores, serve as photocatalysts that have high cytocompatibility and produce minimal singlet oxygen. Commercially-available conjugates of Hoechst dye (SiR-H) and docetaxel (SiR-T) are used to localize SiR to the nucleus and microtubules, respectively. Computation was used to assist the design of a new class of redox-activated photocage to release either phenol or n-CA4, a microtubule-destabilizing agent. In model studies, uncaging is complete within 5 min using only 2 µM of SiR and 40 µM of the photocage. In situ spectroscopic studies support a mechanism involving rapid intramolecular Diels-Alder reaction and a rate determining elimination step. In cellular studies, this uncaging process is successful at low concentration of both the photocage (25 nM) and the SiR-H dye (500 nM). Uncaging n-CA4 causes microtubule depolymerization and an accompanying reduction in cell area. Control studies demonstrate that SiR-H catalyzes uncaging inside the cell, and not in the extracellular environment. With SiR-T, the same dye serves as photocatalyst and the fluorescent reporter for microtubule depolymerization, and with confocal microscopy it was possible to visualize microtubule depolymerization in real time as the result of photocatalytic uncaging in live cells. Additional preliminary work is also described using alternative DHTz photocages and freely soluble photocatalysts for the uncaging of bioactive and fluorescent molecules. ☐ Photocatalytic protein labeling has become an increasingly studied area of chemical biology with direct implications in discovering new drug targets and uncovering protein-protein interactions. In Chapter 3, I describe my work developing n-alkylated DHTz as stable precursors of reactive electrophiles for protein labeling applications. I have been successful at modifying small molecule nucleophiles as well as labeling thiol-containing peptides and proteins with these probes. Structure and mechanistic elucidation studies revealed a Zincke-like mechanism with the alkyl handle and modified nucleophile still intact. Future work will include further mechanistic studies as well as localized protein labeling using a ligand-directed photocatalyst.
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Keywords
Live cells, Electrophiles, Protein labeling, Photocatalysts, Protein-protein interactions