PROJECT SUMMARY/ABSTRACT The human body contains two million or so distinct proteins. Chemistry harbors the potential to endow these proteins with attributes desirable for biomedicine. The proposed research 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. The strategy is to O-alkylate protein carboxyl groups by using a tuned diazo compound, generating esters that are analogous to those in 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 in vitro and in cellulo (Aim 1). Esterification will then be used to deliver proteins for tumor suppression (Aim 2), genome editing (Aim 3), and anti-viral activity (Aim 4). The work relies on methods and ideas from organic chemistry, enzymology, chemical biology, and related fields, and is designed to establish a new paradigm for developing chemotherapeutic agents that are capable of addressing numerous unmet medical needs.