Nelson, Jr., Shawn Daniel2019-08-082018-112018-09-172018Nelson, Jr., Shawn Daniel. 2018. Synthesis and rapid biological annotation of single-enantiomer small molecules. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.http://nrs.harvard.edu/urn-3:HUL.InstRepos:41121192Small molecules play key roles in biological processes and have been suggested to be the “missing link” in the central dogma of molecular biology. Discovery campaigns facilitated by the high-throughput screening of millions of small molecules have led to the development of new probes and therapeutics. This process may be improved by incorporating small-molecule libraries enriched in bioactives with minimal performance redundancy. Our understanding of which small molecules to make and how to measure their biological performance—toward discerning mechanisms of action—remains a high priority for discovering modulators of traditionally challenging targets. One approach to synthesizing performance-diverse libraries has been to leverage modern, asymmetric synthetic organic chemistry methods to access skeletally and stereochemically complex small molecules. This thesis describes three synthetic strategies and two small-molecule annotation methods to further explore this hypothesis. A pilot study underpinned by the pairing of single-enantiomer 1,2-amino alcohols with bis-electrophiles afforded an array of chiral fragments (MW < 300 Da) for fragment-based ligand discovery experiments. Next, two pathways, one based upon bicyclic enones derived from a single-enantiomer homopropargyl alcohol, and the other upon triazolodiazepines derived from L-serine, furnished additional scaffolds. With small molecules derived from these scaffolds, a previously described phenotypic assay, “cell painting,” was optimized and used to measure small-molecule-induced morphology changes (i.e., profiles) in human cancer cell lines in “real time.” Newly measured profiles can be correlated to those of well-annotated small molecules to suggest whether a small molecule is acting by a known or potentially novel mechanism of action. Next, as a complement to cell painting, a high-throughput, barcoded RNA-sequencing assay was investigated. The resulting sequencing data provided gene expression modulation fingerprints for many individual small-molecule perturbations in one multiplexed experiment. These “de-risked” synthetic pathways and biological performance measurements have contributed to a growing collection of performance-diverse small molecules for probe and drug discovery.application/pdfenchemistrysynthesischemical biologyThesis or Dissertation2019-08-080000-0002-5923-5192