Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.

Department of Biochemistry Faculty of Science Charles University Hlavova 8 12843 Prague 2 Czech Republic

Department of Cell Biology Faculty of Science Charles University Viničná 7 12843 Prague 2 Czech Republic

Institute of Macromolecular Chemistry of the Czech Academy of Sciences Heyrovského n 2 16206 Prague 6 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo n 2 16610 Prague 6 Czech Republic

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