A tetrazole-based bioorthogonal reaction for protein functionalization and imaging in live cells

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 (t1/2 = 5.1 sec) which spontaneously cyclizes with acrylamide with a second-order rate constant of 11.0 M -1s-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.