The growing importance of DNA-encoded chemical libraries (DECLs) as tools for the discovery of protein binders has sparked an interest for the development of efficient screening methodologies, capable of discriminating between high- and medium-affinity ligands. Here, we present a systematic investigation of selection methodologies, featuring a library displayed on single-stranded DNA, which could be hybridized to a complementary oligonucleotide carrying a diazirine photoreactive group. Model experiments, performed using ligands of different affinity to carbonic anhydrase IX, revealed a recovery of preferential binders up to 10%, which was mainly limited by the highly reactive nature of carbene intermediates generated during the photo-cross-linking process. Ligands featuring acetazolamide or p-phenylsulfonamide exhibited a higher recovery compared to their counterparts based on 3-sulfamoyl benzoic acid, which had a lower affinity toward the target. A systematic evaluation of experimental parameters revealed conditions that were ideally suited for library screening, which were used for the screening of a combinatorial DECL library, featuring 669 240 combinations of two sets of building blocks. Compared to conventional affinity capture procedures on protein immobilized on solid supports, photo-cross-linking provided a better discrimination of low-affinity CAIX ligands over the background signal and therefore can be used as a tandem methodology with the affinity capture procedures.

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New 5-azo substituted thiazole derivatives have been obtained, under mild conditions and in good yields, by the reaction between 2-N-pyrrolidinylthiazole and a series of aryldiazonium salts bearing, mainly in the para position, groups with different electronic effects. The NMR spectra of the products show broad signals of the methylene groups in the alpha position to the pyrrolidinyl nitrogen, suggesting a hindered rotation around the C2-N bond, of which a double bond character was also evidenced by X-ray diffraction analyses. The free energies of activation for the rotational processes have been obtained from (1)H NMR experiments and computer simulations at different temperatures and provided good correlation with the σ constants of the substituents on the 'remote' benzene ring. This represents an unexpected and peculiar result since the restricted rotation around the C2-N bond was shown to be influenced by a substituent situated very far away. 2,4-Di-N-pyrrolidinylthiazole showed a much greater reactivity than 2-N-pyrrolidinylthiazole with aryldiazonium salts.