Design and Synthesis of Triazabutadiene-based Fluorogenic Probes for Tyrosine Specific Labeling of Proteins

Chemical labeling is an important tool for understanding protein structure and function. Biological research often requires the use of molecular labels that are covalently attached to facilitate detection or purification of the labeled protein and its binding partners. Although the number of probes have been developed for labeling of specific residues of proteins is substantial, there is still a need for new reagents with better reactivity, and selectivity. Moreover, these chemical probes should be able to label the protein of interest under mild biologically relevant conditions. Aryl diazonium salts have been utilized for selective modification of tyrosine residues. However, most diazonium compounds need to be generated in situ under strongly acidic conditions due to their instability1. Our group has previously shown that triazabutadienes can be used as precursors that can generate diazonium under mild acidification2 or photo-irradiation3. Current reported systems for bioconjugation of tyrosine require an additional step for fluorescent labeling4. To address this issue and reduce background fluorescence that is associated with fluorescent labeling, coumarin triazabutadiene-based fluorogenic probes were synthesized and tested for tyrosine specific labeling of proteins under mild acidic condition or photo-irradiation. Furthermore, a coumarin triazabutadiene-based cross-linker was synthesized with an azide functionality that can be used to attached the coumarin triazabutadiene warhead onto the surface of a protein. Upon the activation of the triazabutadiene group, by light or lowering the pH, this system can generate a coumarin diazonium salt on the surface of the protein. Such a system can find application in the study of protein-protein interactions and virus-protein interactions. A cyclooctyne triazabutadiene was synthesized to attach a cyclooctyne group on the tyrosine residues of proteins in biologically relevant pH, and 3-azido 7-hydroxy coumarin was made as a fluorogenic partner of the cyclooctyne triazabutadiene. It was demonstrated that this system can label tyrosine residue followed by a copper-free click reaction with the azido coumarin fluorophore. This system has been tested on model proteins and can be consider as one the first fluorogenic triazabutadiene systems that can be utilized for labeling of tyrosine under mild conditions. In conclusion, this dissertation demonstrates progress in developing fluorescent and fluorogenic triazabutadienes systems for labeling of tyrosine residues of proteins as well as fluorophore triazabutadiene cross-linker that can be used for studying protein-protein interaction, and virus-protein interactions. These systems offer a convenient tool to those wishing to study proteins, protein-protein interactions, and virus-protein interactions.Release after 9-Jan-201