Bioorthogonal chemistry enables researchers to study biomolecules in their native environments without perturbing endogenous cellular processes. Over the past two decades, significant strides have been made in developing new, and refining existing, bioorthogonal reactions. The continuously expanding toolbox has opened avenues for tackling increasingly complex biological questions. Despite these advances, limitations remain. The majority of bioorthogonal chemistries follow similar mechanisms, and thus cannot be used simultaneously in multicomponent labeling applications. Bioorthogonal chemistry has also seen little use outside of biomolecule labeling and pull-down applications. To push bioorthogonal chemistry into new directions, I took advantage of two l scaffolds previously developed in the lab: the 1,2,4-triazine and the cyclopropenone.

In Chapter 1, I summarize recent advances made in the field of bioorthogonal chemistry. I focus on how physical organic principles can guide the iterative refinement of existing bioorthogonal transformations. Along the way, I also highlight advances made in developing collections of bioorthogonal chemistries that can used in multicomponent labeling applications. In Chapter 2, I describe the use of bioorthogonal 1,2,4-triazines as surrogates for aromatic amino acid residues. These scaffolds are isosteric to phenylalanine, and in principle could be metabolized into the proteome by endogenous cellular machinery. The unique reactivity profile of 1,2,4-triazines also makes them strong candidates for developing mutually orthogonal bioorthogonal reactions. My efforts to develop a triplet set of bioorthogonal reactions is also highlighted. In Chapter 3, I discuss the use of bioorthogonal cyclopropenones as chemically triggered crosslinkers to capture biomolecule interactions. This reaction is robust enough to be conducted in the presence of bacterial cellular lysate. Chapter 4 showcases the use of the cyclopropenone as a synthon for accessing butenolide scaffolds. Butenolides are found in a variety of natural products, but existing routes to access them are not generalizable. The methodology is organocatalytic, and has a broad tolerance for functional groups and substitution patterns.

This thesis describes the use of bioorthogonal chemistry in underexplored applications. Outside of biomolecule labeling, these transformations can have utility in areas such as chemical crosslinking and synthetic methodology. Both the cyclopropenone and the 1,2,4-triazine are prime candidates for pushing the frontiers of bioorthogonal chemistry. These scaffolds exhibit remarkable stability in biological environments, and possess unique manifolds of reactivity that can be exploited in multiple settings outside of traditional biomolecule tagging.