Project Summary/Abstract This proposal aims to substantially advance two frontier research areas, Molecular Prosthetics and Automated Small Molecule Synthesis, and thereby have a major impact on human health. The first half targets the development of a new class of molecular prosthetics that replace missing biochemical reactions. Our lab has recently demonstrated in animals and in people that small molecules can replace the function of deficient proteins, thus operating as prostheses on the molecular scale. We further illuminated specific mechanisms that permit imperfect functional mimicry to be sufficient for substantial physiology restoration due to the inherent robustness of living systems. In this proposal we will launch a new research direction to find small molecules that replace deficient enzymes. There are more than 100 human diseases linked to loss of enzyme function, and we will specifically target three examples: Ornithine Transcarbamylase Deficiency, Argininosuccinate Lyase Deficiency, and Hereditary Tyrosinemia Type 1. These diseases have two features in common that we propose render them susceptible to this approach: 1) a high concentration of the substrate for the missing enzyme, or an upstream precursor, builds up in the blood and/or tissues, and 2) the corresponding substrate has unique chemical reactivity. We hypothesize that these two features will permit imperfect small molecule reagents to serve as effective functional surrogates for missing enzymes in human blood plasma and in mouse models. The second half targets new molecular lego kits for automated synthesis of two functionally-rich classes of natural products: lipids and polycyclic terpenoids. Both classes perform many biological and industrial functions, yet remain inherently challenging to synthesize. Building on our recent development of a fully automated lego-like platform for small molecule synthesis, we plan to create a lego kits for on-demand preparation of both classes of compounds. Parallel retrosynthetic analysis showed that preparation of ~130 known polyunsaturated fatty acids requires only 26 building blocks, which we will prepare. We will also develop two new Csp3-C bond forming methods to iteratively assemble building blocks, and a Nextgen synthesis machine to render this process fully automated. We will apply this platform to better understand molecular prosthetic ion channels. We also plan to combine modular lego-like construction of linear molecules with cyclization-promoting self-assembled resorcinarene capsules to create a highly efficient and flexible lego kit for polycyclic terpenoids. To this end, we will define new structure-cyclization relationships, characterize their underpinnings, and leverage them to achieve efficient syntheses of complex polycyclic terpenoid natural products. Collectively these studies will push the frontiers of chemical biology and organic synthesis and open new frontiers for medicine.