A tetrazole-based bioorthogonal reaction for protein functionalization and imaging in live cells
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Bioorthogonal chemistry has emerged as a powerful tool in probing biomolecular structure and function in living systems. Combined with recent developments in introducing novel chemical reporters into biomolecules site-selectively in vivo, bioorthogonal chemistry offers an unprecedented opportunity to monitor and expand biomolecular function in living systems. The objective of this thesis work is to develop a tetrazole-based photoinducible bioorthogonal reaction and apply it to image protein in live cells. Chapter 2 describes a photoinducible 1, 3-dipolar cycloaddition that allows for fast and residue-specific modification of engineered proteins carrying a diphenyltetrazole group. In a peptide tetrazole model study, the reaction was found to undergo a rapid photoinduced cycloreversion to generate a highly reactive nitrile imine dipolar (t 1/2 = 5.1 sec) which spontaneously cyclizes with acrylamide with a second-order rate constant of 11.0 M -1 s -1 . When the diaryltetrazole was introduced into lysozyme, we found that labeling by acrylamide, coumarin methacrylamide, and palmityl methacrylamide occurred selectively at the tetrazole sites of the modified lysozyme after 1 min photoinduction at 302 nm. The resulting cycloaddition products, pyrazolines, showed strong fluorescence in the wavelength region of 487-538 nm. In addition, a robust lipidation of green fluorescent protein was achieved by introducing photoreactive diaryltetrazoles to the C-terminus of EGFP through chemical ligation followed by irradiating the tetrazole-containing EGFP in the presence of a lipid dipolarophile in vitro. Taken together, this tetrazole-based photoinducible 1, 3-dipolar cycloaddition reaction represents a new and robust bioorthogonal reaction for selective protein modification in biological buffer. Chapter 3 describes the employment of the tetrazole-based, photoclick chemistry to selectively functionalize a genetically alkene-encoded protein inside E. coli cells. The reaction procedure was simple, straightforward, and nontoxic to E. coli cells. Additionally, fluorescent cycloadducts were formed, which enabled a facile monitoring of the reaction in vivo . The strategy to further optimize the tetrazole reactivity has also been put forth by systematically tuning the HOMO-lifting effect on nitrile imine dipoles. One of the optimized tetrazoles with the electron-donating methoxy substituent was found to label an alkene-encoded protein in less than 1 min inside E. coli cells. Chapter 4 describes a simple alkene tag, homoallylglycine (HAG), that can be co-translationally incorporated into a recombinant protein as well as into endogenous newly synthesized proteins in mammalian cells with high efficiency. In conjunction with a photoinduced tetrazole-alkene cycloaddition reaction (“photoclick chemistry”), this alkene tag further served as a bioorthogonal chemical reporter both for selective protein functionalization in vitro and for spatiotemporally controlled imaging of the newly synthesized proteins in live mammalian cells. Since the non-symmetrical spatial distribution of newly synthesized proteins in animal cells plays a central role in many cellular processes, this two-step metabolic alkene tagging–photo-controlled chemical functionalization approach may offer a potentially useful tool to study the role of the spatiotemporally regulated protein synthesis in mammalian cells. Chapter 5 describes a chemical lipidation model to study protein lipidations in live mammalian cells based on the bioorthogonal, photoinduced tetrazole-alkene cycloaddition reaction. The localization effect of photoinduced chemical lipidations on tetrazole conjugated EGFP both in organic solvent/PBS buffer mixture and in live HeLa cells have been demonstrated. This chemical strategy recapitulated some aspects of protein lipidation in vivo , e.g., the effect of lipid numbers on membrane association stability and the lipidation induced translocation into vesicles inside cells.