Precipitation-first Synthesis and Deep Profiling of a 1,200-Member Acrylamide Library for Covalent Drug Discovery

Abstract

Screening libraries of low molecular weight (LMW) compounds are foundational in early-stage drug discovery. However, the parallel synthesis of milligram-scale large libraries -especially for covalent inhibitors -remains constrained by resourceintensive purification protocols, high solvent consumption, and limited scalability. Electrophilic fragments such as acrylamides are widely used to target nucleophilic cysteines in disease-relevant proteins, but practical and sustainable workflows to synthesize diverse covalent libraries in sufficient quantities and quality are lacking. Here, we report a high-throughput, chromatography-free workflow to synthesize and analyze 1,235 acrylamides using a modified Ugi four-component reaction (U-4CR) featuring ammonia and acrylic acid. This strategy exploits direct product precipitation to replace chromatographic purification steps with simple filtration, enabling synthesis at scale with common parallel lab instrumentation. Our protocol provides a sustainable and modular solution to library generation, especially suitable for covalent drug discovery. Using complementary analytical methods -¹H NMR, UPLC-MS-UV, and AEMS -we systematically evaluated compound purity, identity, and long-term stability. The large dataset was systematically processed using PeakSel® (live dashboard at https://peaksel.elsci.io/a/domling_group/post/8gY7CqUMVtb ), providing complementary information on compound quality and long-term stability. This platform delivers the largest fully profiled covalent electrophile library to date and offers a generalizable framework for sustainable library production and high-content analytical workflows. It complements emerging analytics-only approaches by offering a complete and green synthesis-to-analysis pipeline accessible with standard laboratory instrumentation.