Introducing Multivalency into DNA-Encoded Chemical Libraries through a Calix[4]arene Scaffold

DNA-encoded chemical libraries (DECLs) comprise collections of synthetic small molecules covalently linked to unique DNA tags that function as PCR-amplifiable identification barcodes.1 In recent years, DECL technology has gained popularity in drug discovery because, unlike conventional High-Throughput Screening techniques, it enables the simultaneous interrogation of all library members through affinity-based selection against solid phase-immobilized targets.2 Moreover, DECLs are typically constructed using split-and-pool synthetic approaches, allowing the generation of libraries containing thousands to billions of compounds. Together, these features facilitate the rapid identification and validation of ligands for pharmaceutically relevant targets. Two principal design strategies have been explored in DECL development. One approach focuses on the generation of Lipinski’s rule-of-five–compliant small molecules suitable for binding to proteins with well-defined pockets (e.g., kinases and phosphatases).3,4 Alternatively, libraries containing larger ligands, such as macrocyclic peptides, have been developed to target extended or relatively flat protein surfaces.5 While most reported DECLs encode monovalent ligands, here we describe the design and synthesis of the first multivalent-display DECL (Calix-DEL), based on a calix[4]arene macrocyclic scaffold. This library was designed to investigate multivalent binding effects—including enhanced avidity, specificity, and selectivity—and to explore ligand discovery against challenging or “undruggable” protein targets. The library synthesis begins with an asymmetrically substituted calix[4]arene modified at the lower rim with an alkyne functionality for DNA conjugation via CuAAC. The upper rim was functionalized using a novel strategy that includes Pd-catalyzed carboxylation followed by amide coupling with BocNH–Lys–OMe. Installation of four lysine units at the upper rim provides reactive sites for diversification with 35 amines and 150 carboxylic acids through amide bond formation, generating a 5250-member library. In parallel, a control library (mLys-DEL) based on a monovalent lysine-modified phenol scaffold was synthesized to assess the impact of multivalency on target binding. The two libraries were synthesized, purified, isolated and screened against several proteins of interest using affinity-based selection experiments.