Late stage modification of receptors identified from dynamic combinatorial libraries

Abstract

Small molecule receptors are attractive potential sensors of post-translational modifications, including methylated lysine and methylated arginine. Using dynamic combinatorial chemistry (DCC), our lab previously identified a suite of receptors that bind to Kme₃ with a range of affinities ranging from low micromolar to high nanomolar, each with a unique selectivity for Kme₃ over the lower methylation states. To enable these receptors to have broad application as Kme₃ sensors, we have developed a method for their late-stage modification, which we used to synthesize biotinylated derivatives of A₂B, A₂D, and A₂G in a single step. For our most attractive receptor for applications, A₂N, we needed to develop an alternative method for its selective functionalization, which we achieved by “activating” the carboxylic acids on the constituent monomer A or N by pre-functionalizing them with glycine (Gly). Using the resulting Gly-A and Gly-N monomers, we synthesized the novel A₂N variants A₂Gly-N, Gly-A₂N, and Gly-A₂Gly-N, which enabled the late stage biotinylation of A₂N wherever Gly was incorporated. Finally, we performed ITC and NMR binding experiments to study the effect that carboxylate spacing has on the affinity and selectivity of A₂Gly-N and Gly-A₂N for Kme_X guests compared to A₂N. These studies revealed the proximity of the carboxylates to play a complex role in the molecular recognition event, despite their positioning on the outside of the receptor.