UC Irvine

Bioorthogonal chemistries are widely used to image and profile biomolecules in their native environments. While many bioorthogonal reactions have been reported in recent years, most cannot be used inside cells owing to reagent instabilities. Additionally, there are few reactions that can be used concurrently for multi-component labeling. Many of the most popular bioorthogonal reagents exhibit significant cross- reactivities. To address these limitations, I developed new classes of bioorthogonal reagents, including cyclopropenones, cyclopropenethiones, and functionalized phosphines.

In Chapter 1, I introduce the overall strategy involved in designing and optimizing new bioorthogonal reagents. I also highlight some of the current needs in bioorthogonal reactivity and recent advances that have been made in these areas. In Chapter 2, I describe the optimization of bioorthogonal cyclopropenones for use in intracellular environments. I synthesized a panel of disubstituted cyclopropenones and identified scaffolds that are inert to thiols and other biological nucleophiles. These substituted cyclopropenones react efficiently with a wide array of functionalized phosphines to form covalent adducts. I further developed cyclopropenone amino acids that can be used for recombinant protein production and labeling applications. Chapter 3 showcases the investigation of cyclopropenethiones as more rapidly reacting heteroanalogs of cyclopropenones. Cyclopropenethione-phosphine ligations proceed at markedly improved rates, and cyclopropenethiones are suitable for use in complex biological environments. Finally, in Chapter 4, I discuss the use of cyclopropenones as chemically activatable protein crosslinkers. Using a model split protein system, I demonstrated that cyclopropenones can serve as electrophilic traps to form covalent adducts between interacting biomolecules.

Overall, this thesis describes the optimization of cyclopropenones, cyclopropenethiones, and phosphines for use in biological environments. The stability and highly tunable nature of these reagents make them useful for a wide range of applications, including protein conjugation, cellular imaging, and biomolecule trapping. The unique reactivity of these reagents also presents new opportunities for multicomponent labeling. Based on their biocompatibility and versatility, I anticipate that the cyclopropenone and cyclopropenethione will be broadly adopted for applications in cells and living systems.