Project Summary The long-term goal of our research program is to develop chemistry that can facilitate the synergetic interaction between biomolecules and small molecules to achieve site-selective transformations in a biological context. The objective of this application is to develop the chemistry of N-oxides and integrate them into biological systems that stand to benefit from reversible covalent modifications by prosthetic agents. N-oxides are versatile reagents with a minimalist design that encapsulate both ligation and release properties. They are synthesized through a rapid bioorthogonal hydroamination reaction and disassembled under similarly bioorthogonal conditions through reduction. This chemistry allows the transient, yet controlled, attachment of small molecules to proteins or proteins to small molecules and enables the temporary use of functional modules that are not normally present in the active form of a protein or small molecule effector. The specific aims of the project are: 1) Development of reactive species for target identification applications; 2) Development of proximity labeling tools for interactome studies; 3) Development of biologically compatible directing group chemistry. In the first aim, we will develop reactive chemical species that can functional proteins and use them to identify the molecular target of a bioactive small molecule. In the second aim, we will develop new catalysts for proximity labeling. In the third aim, we will develop chemical methods and reagents for the reversible covalent functionalization of proteins. We use the chemistry to introduce small molecules or enzymes that can site-specifically modify proteins of interest with native post-translational modifications. The approach we take is innovative because we make use of a unique pair of chemical reactions that enables the rapid and complete bioorthogonal ligation and cleavage of two components in either a single step or in two discrete and independent steps. This contrasts with existing bioorthogonal tools designed either to ligate or to cleave but not to do both separately. The research we propose is significant because it provides a new method for discovering proteins that interact in either an on-target or off- target manner with a lead drug candidate, for studying the interactome of a protein complex, and for accessing homogeneous preparations of native post-translationally modified proteins for biochemical and biophysical studies. Most importantly, the fundamental chemistry we explore and the tools that we create will expand the functional repertoire of chemistry that can be performed on biological systems and pave the way to new discoveries.