Harnessing the Catalytic Promiscuity of Hydrolases to Promote the Three‐Component Reactions on DNA

DNA‐encoded libraries (DELs) are a powerful technology increasingly used in drug discovery for screening lead molecules. The key to success is dependent on the chemical space covered by DELs. Enzymes can catalyze complex chemical reactions under mild conditions, making them highly attractive for constructing structurally diverse DELs. However, traditional enzymatic transformations have been considered unsuitable for DEL construction due to their narrow substrate scope, leading to slow progress in this field. Here, we challenge this conventional perception by introducing the catalytic promiscuity of hydrolases into three‐component reactions on DNA. We successfully performed three enzyme‐promoted coupling reactions directly on DNA: the Aza‐Diels–Alder reaction, the Biginelli reaction, and the Mannich reaction.These reactions bring N‐heterocyclic bridged rings, pyrimidines, and α‐branched amine‐based nitrogen‐containing pharmacophores to DELs. Importantly, the entire coupling process on DNA tags does not require the use of harmful metals or stoichiometric organic catalysts, nor does it involve additional immobilization of the DNA strand. Using green and inexpensive hydrolases, the reactions can proceed directly in aqueous mixed solutions. Due to the mildness of enzyme‐catalyzed reaction conditions, all three reactions are highly compatible with DNA tags.