Oxidative three-component on-DNA click reactions using a convenient HILIC-LC-MS analysis method

DNA-encoded library (DEL) technology is a transformative tool for drug discovery that enables the rapid synthesis and screening of billions of compounds. New DNA-compatible methods are needed to expand bioactive chemical space; however, oxidative transformations present significant challenges for on-DNA chemistry. Herein, we report a suite of mild on-DNA oxidative three-component click (OTC) reactions, where in situ-generated 1,3-dipoles are trapped by DNA-conjugated alkenes and alkynes. The chemistry furnishes N-heterocycles in good-to-excellent yields, including isoxazolines, isoxazoles, pyrazolines, and pyrazoles of diverse substitution patterns, while preserving the integrity of the DNA barcodes. As traditional ion-pairing reversed-phase liquid chromatography-mass spectrometry (IP-RP LC-MS) could not be used on our shared departmental instrumentation, we developed an MS-friendly hydrophilic interaction chromatography (HILIC) method to facilitate analyses of small DNA-containing fragments. This work provides a practical framework for other academic labs to evaluate new methods for on-DNA chemistries without the need for dedicated instrumentation.