Fully‐Functionalized Natural Product Probes to Expand the Chemical Tractability of the Human Proteome

Abstract Photoaffinity (PA)‐based chemoproteomic methods have emerged as a powerful means for proteome‐wide mapping of ligandable proteins directly in cells, as well as the concatenated discovery of small molecule chemical probes. However, due to the relatively low throughput of screening small molecule libraries by proteomics and the synthetic burden incurred in installing the requisite photoactivatable functionality on each member, great priority must be placed on library design to maximize efficient exploration of biologically relevant chemical space and to ensure the identification of authentic binding interactions. To address this challenge, here we leverage the inherent complexity of natural products (NPs) to access a structurally unique series of diastereo/regioisomeric PA probes for ligand discovery in cells. Through semi‐synthesis, we exploit sp 3 ‐rich NP scaffolds and employ mass spectrometry (MS)‐based chemoproteomics to identify and quantify their interactions in human cells, uncovering topology, regio‐, and stereoselective ligands for functionally diverse proteins that currently lack reported chemical probes. Collectively, our findings highlight the potential of NP‐inspired chemoproteomic libraries to expand the boundaries of the ligandable proteome.