PROJECT SUMMARY/ABSTRACT Approximately 80% of drugs in the pharmacopeia are small molecules. Half of those small molecules act on extracellular targets, and half act on intracellular targets. The remaining 20% of drugs are proteins, and virtually all of those proteins are antibodies, hormones, or enzymes that act on extracellular targets. The proposed research will enable proteins to act on intracellular targets. The proposed research program takes advantage of new chemical reactivity that is applicable in a physiological context. The overall goal is to develop a facile, general means to endow native proteins with the ability to enter the cytosol of human cells with desirable consequences. The strategy is to esterify protein carboxyl groups by using α-aryl-α-diazoamides. The basicity of these diazo compounds are tuned to enable the efficient esterification of carboxyl groups in water. The resulting esterified proteins are analogous to small- molecule prodrugs. Intracellular esterases make these modifications traceless, avoiding any compromise to proper function or potential for immunogenicity. “Protein esterification” has an uncharted landscape. Accordingly, work will begin by exploring fundamental attributes of esterified proteins, including the mechanism of cellular uptake and the enzymology of ester hydrolysis by cellular esterases. Esterification will then be used to deliver proteins for eliciting cytotoxicity, tumor suppression, and genome editing. The work relies on methods and ideas from chemical biology and allied fields, especially organic chemistry and enzymology. The research will establish a new paradigm for developing chemotherapeutic agents that are capable of addressing numerous unmet medical needs.