Synthesis and Bioorthogonal Applications of Enamine N-oxides

Abstract

This dissertation describes the enamine N-oxide functional group and outlines their synthesis and biological applications. Two bioorthogonal applications are discussed herein: 1) a method to activate endogenous protein function is described in Chapter 2, and 2) a platform for small molecule target identification is described in Chapters 3 and 4. Chapter 3 describes the initial platform involving dihalogenated enamine N-oxides while Chapter 4 introduces additional chemical strategies and reagents that aim to further advance the utility of the platform.

In Chapter 1, I first provide an introductory overview of enamine N-oxides including their synthesis, reactivity, and other biologically relevant properties that provide precedence for subsequent chapters.

In Chapter 2, I introduce a method to perform bioorthogonal activation of protein function through a tandem retro-Cope elimination/Cope elimination reaction cascade. The method relies on a bioorthogonal cleavage reaction initiated by the hydroamination of cyclooctynes by N,N-dialkylhydroxylamines and featuring an enamine N-oxide as a key intermediate. A method to activate temporarily caged protein function utilizing unnatural amino acid incorporation is also described.

In Chapter 3, I discuss the development of a target identification platform where dihalogenated enamine N-oxides are employed as a caged reactive group in affinity-based label transfer reagents for small molecule target identification. This platform (Bioorthogonally activated reactive species, BARS) was validated through various chemical and biological experiments, including those employing mass spectrometry-based chemoproteomics. Importantly, the method was benchmarked against existing methods such as photoaffinity labeling (PAL).

Finally, Chapter 4 describes additional enamine N-oxide-based reagents that extend the utility of the BARS platform to applications beyond those outlined in Chapter 3. In particular, strategies to minimize the size of the probe compounds and generate a dissociative reactive species are described. Additionally, an unprecedented mechanism of reactive species formation was discovered serendipitously, and that this mechanism can be used for further target discovery. Taken together, the findings of Chapters 3 and 4 provide a generalizable and modular platform for using enamine N-oxides in small molecule target identification applications.