Factors to Consider for Synthesis in 1536-Well Plates─An Amide Coupling Case Study for PROTAC Synthesis

Ultra high-throughput chemistry carried out in 1536-well plates is increasingly utilized for reaction optimization protocols and direct-to-biology (D2B) platforms, where nanomolar quantities of the final product are directly assessed for biochemical or cellular activity without purification. As their popularity increases, it is crucial that the synthesis of these molecules is reliable and reproducible. Research in our laboratories has identified several nuances of amide couplings when performed on the nanoscale that result in poor translation from 1536-well plates to batch-scale reactions. This case study presents a nanoscale amide coupling reaction to synthesize 700 PROTAC molecules, which identified a range of factors crucial to reaction success on the nanoscale, despite having no influence on reaction conversion in batch. This work presents a guide for high-throughput chemists to consider when working in 1536-well plates and their importance in drawing conclusions from nanoscale synthesis.