The bioorthogonal chemical reporter strategy provides a method for selectively labeling biomolecules with detectable probes. This strategy relies on the incorporation of a unique reporter into a biomolecule, followed by a covalent ligation with a secondary reagent. This approach can be used to visualize or retrieve biomolecules in complex environments. Over the past decade, the bioorthogonal chemical reporter strategy has been successfully applied to label and study various biomolecules in complex systems. However, the scope of this method has been hindered by a lack of reactions that are compatible with each other. Most of the popular bioorthogonal reagents to date cross react, precluding multicomponent imaging studies and other applications. To address this issue and expand the scope of the chemical reporter strategy, new reagents and chemistries were developed and their reactivities were analyzed.

In Chapter 1, I highlight the most commonly used bioorthogonal reactions, discuss the reactivity of the different reactions, and emphasize areas of improvements and sources of inspiration for thinking about bioorthogonal reactions. In Chapter 2, I focus on the development of new candidate reactions for biomolecule labeling. More specifically, the reactivity of 1,3- disubstituted and 3,3-disubstituted cyclopropenes with tetrazines and nitrile imines is discussed.3,3-Disubstituted cyclopropenes were shown to exhibit orthogonal reactivity with tetrazines, but still react robustly with nitrile imine probes. In Chapter 3, I discuss the development of 1,2,4- triazine as a novel diene for bioorthogonal labeling applications. 1,2,4-Triazines were synthesized via an expedient route, and their reactivity and stability were analyzed. 1,2,4- Triazines were shown to exhibit robust stability and selective reactivity with trans-cyclooctene over other commonly used strained dienophiles. Additionally, a non-canonical amino acid comprising the triazine motif was synthesized and successfully used to tag model proteins. In Chapter 4, the reactivity profile of the triazine scaffold is discussed. I analyzed the reactivity of isomeric 1,2,4-triazines with a panel of commonly used bioorthogonal reagents. A combination of steric and electronic perturbations was applied to the development of cycloaddition reactions that could be used simultaneously.

Collectively, this thesis explores novel chemical reagents for bioorthogonal chemistry. New chemical reagents that exhibit mutually orthogonal reactivity are developed and used to label biomolecules. Looking ahead, the chemistries described here will advance the scope of the chemical reporter strategy for multicomponent studies.