A tailored CoA ligase-N-acyltransferase cascade for on-DNA amide bond formation gives access to broad substrate scope.

DNA-encoded chemical library (DEL) technology is a powerful tool in early-stage drug discovery. Although widely applied in industry and academia, challenges persist in generating DELs with high quality and chemical diversity. Low yields in building-block incorporation, limited selectivity 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 under mild conditions. Targeting amide bond formation, we designed a cascade of complementary coenzyme A ligases and rationally tailored N-acyltransferases to access a broad amide scope on-DNA (>120 examples), identifying structural elements that optimize the biocatalysts' DNA compatibility in the process. Integrating the enzymatic cascade with chemical synthesis led to the construction of a diverse DEL without damage to the DNA barcode, highlighting the biocatalysts' applicability for early scaffold construction and late-stage functionalization.