Bioorthogonal chemistries are powerful tools to investigate biomolecules in their native environments and provide a holistic understanding of cellular functions. These transformations can be performed in complex physiological settings without disturbing endogenous activity (i.e., they are “bioorthogonal”). While the spectrum of bioorthogonal reactions has grown over the last two decades, few reactions can be deployed in cells. Additionally, the majority of cell-compatible chemistries cannot be used together due to cross reactivity issues, largely prohibiting multi-component studies. Most applications have thus been restricted to detecting a single biological target, which is informative but cannot provide a complete picture of cellular function. A more holistic understanding may be possible via the development of compatible reactions to monitor multiple biomolecules simultaneously. Additionally, new methods to visualize biomolecule dynamics and identify biomolecule interaction networks would enable new biological pursuits. To address these limitations, I developed new chemical strategies to probe biomolecule targets in live cells using bioorthogonal cyclopropenone and phosphine reagents.Overall, this thesis focuses on the development of new chemical reactions for applications in cellular systems. In Chapter 1, I introduce the utilities and characteristics of a popular reagent for bioorthogonal applications, phosphines. I summarize the broad types of bioorthogonal chemistries of phosphine probes and their applications. In Chapter 2, I describe a new class of biocompatible reagents: cyclopropeniminium compounds. These probes are stable in aqueous solution and react with phosphines via a distinct mechanism. Due to reactivity differences, these probes were compatible with other existing transformations for multicomponent applications. In Chapter 3, I describe a fluorescence “turn-on” (i.e. fluorogenic) reaction of cyclopropenones and phosphines for deployment in live cells. Fluorescence enhancement was achieved via a regioselective activation and subsequent cyclization of non-emissive cyclopropenone reagents. Fluorogenic probes visualized biological targets in vitro and in live cells. The reaction was also found to be compatible with other fluorogenic transformations, and this orthogonality was leveraged to visualize multiple cellular targets simultaneously, a first for fluorogenic bioorthogonal chemistries. Chapter 4 discusses a chemical strategy to capture biomolecule interaction networks using cyclopropenone reporters. In this approach, cyclopropenones are activated to form electrophilic intermediates and transferred to nearby biomolecules. Proximity-mapping with cyclopropenone probes is readily translatable between biomolecule classes, and their utility for protein and metabolite interactome mapping is currently being explored.
In the future, these new bioorthogonal chemistries should enable investigations of ever more complex cellular experiments. Dynamic biological processes are currently challenging to visualize but are informative of biomolecule location and function. Additionally, an atlas of all cellular interactions would provide a holistic understanding of biomolecule roles and relationships. Cyclopropenone and phosphine reagents are poised to address these challenges. They are among the most biocompatible bioorthogonal probes and should be applicable in a range of physiological environments, including in vivo. Alongside their biocompatibility, their reactivity is versatile and should enable a range of applications, such as tagging, real-time visualization, crosslinking, and interactome mapping. Broadly, this thesis is a testament to the power of chemical reactivity to investigate living systems.