This study illustrates the utility of click chemistry in functionalizing triazabutadienes by allowing access to various applications both biological and material based. Triazabutadienes have been shown to trigger the release of highly reactive diazonium species in a pH dependent way when placed in acidic conditions. Electron-rich phenyl systems such as tyrosine residues have been shown to react with diazonium compounds to form stable azo bonds. Modification of these triazabutadiene motifs can functionalize them as linkers or impact solubility; which can allow for target specificity and mild cleavage of linker in order to liberate diazonium near site of interest. Incorporation of azide-alkyne cycloadditions onto these molecules will allow chemical functionalization and cross-linking properties. The 1,2,3-triazole triazabutadiene derivatives are synthesized via Huisgen 1,3-dipolar cycloaddition from alkynyl modifications on the triazabutadiene that are reacted with various azides that show substrate diversity.

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.

Labeling proteins with chemical tools is important for examining natural systems, discovering therapeutic agents and developing protein constructs. These methods offer simple but reliable chemistry to the study of peptides and proteins and thus have gained popularity among chemists and biologists. Despite the fact that the number of successful examples has been largely increased over the past decade, there is still an ongoing need for new reagents with better accessibility and reactivity. Diazonium ions are known to selectively react with tyrosine residues for more than a century. But the harsh condition required for diazotization makes it difficult to use this strategy in biological applications. To address this, bench-stable triazabutadienes are made to release diazonium ions upon mild acidification or photoirradiation. Based on our previous study, imidazole N-alkyl substituted triazabutadienes were synthesized and tested for diazonium ion-releasing rates. Surprisingly, the imidazole N-tert-butyl substituted triazabutadiene showed the fastest rate in neutral and basic aqueous solutions. A subsequent NMR study revealed that this rapid release of diazonium ions might be ascribed to the lack of intramolecular π-interactions. In addition, triazabutadienes can be rendered more basic upon photo-isomerization. A water-soluble triazabutadiene was shown to adjust the pH of aqueous solutions. These findings open up new opportunities in protein labeling with unprecedented ease. Moreover, a boronic acid-based photocrosslinker was synthesized to detect protein-protein interactions of glycoproteins. By incorporating benzophenone with a boronic acid and a terminal alkyne, this photocrosslinker is designed to capture the glycoprotein-substrate complex using the combination of photochemistry and bioorthogonal reactions. In conclusion, this dissertation demonstrates progress in developing new probes for protein labeling and protein-protein interactions. These newly developed strategies offer convenient alternatives to those wishing to explore protein activities.