A tailored enzyme cascade facilitates DNA-encoded library technology and gives access to a broad substrate scope

DNA-encoded chemical library (DEL) technology has emerged as a powerful tool in early-stage drug discovery. Although the methodology is widely applied in industry and academia, major challenges persist in generating DELs with high quality and chemical diversity. Low yields in building-block incorporation, limited stereo-, regio-, and chemoselectivity, and, most importantly, DNA damage from harsh reaction conditions compromise library quality, reduce signal-to-noise in affinity selections, and ultimately hinder drug discovery. Here, we show that tailored enzymes can be harnessed for the effective construction of molecular diversity on DNA, opening new avenues for the generation of high-quality and diverse DELs under mild conditions. Targeting amide bond formation, we engineered a cascade of complementary CoA ligases and rationally tailored N-acyltransferases (NATs) to access a broad amide scope on-DNA (> 120 examples), identifying transferable structural motifs that optimize DNA-compatibility of the biocatalysts in the process. The successful integration of the biocatalytic steps with chemical synthesis led to the construction of a diverse DEL without damage to the DNA barcode and underscored the broad utility of the enzymatic cascade, enabling applications in both early scaffold construction and late-stage functionalization.