Chemical genetics seeks to elucidate biological function by using small molecules to systematically perturb biological networks. In order to find small molecule probes for all gene products of interest, structurally complex and diverse collections of compounds are needed. Diversity-oriented synthesis (DOS) is one approach to generating such collections of small molecules, wherein simple starting materials are transformed into structurally complex and diverse products in few synthetic steps. The work presented in this dissertation describes a boronic ester annulation (BEA) strategy for DOS which generates functionally rich unsaturated boronate intermediates that can be differentiated into a variety of skeletally diverse products. Synthetic efforts yielded 123 compounds in 10-50 mg quantities which were submitted for high-throughput screening in the Chemical Biology Program of the Broad Institute of Harvard and MIT. The results of cellular profiling experiments with BEA compounds in 89 cell-based screens are presented, and preliminary data on relationships between chemical structure and assay performance are discussed. In addition, a new synthetic methodology is described, employing cyclic allylboronates as allylation reagents to generate 1,3-diols and α-hydroxy-γ-butyrolactones.