Synthesis and rapid biological annotation of single-enantiomer small molecules

Abstract

Small 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.