It has recently been shown that the binding site of SAHase, an enzyme critical in the replication mechanism of viruses, is quite flexible and exhibits a large difference between the "open" and "closed" conformations, thus presenting an obstacle towards design efforts. As a possible solution to this dilemma, we have strategically designed and synthesized a series of structurally innovative nucleosides possessing a heteroaromatic purine ring split into its two components (for example, an imidazole and pyrimidine ring), thereby conferring additional degrees of conformational freedom and torsional flexibility to the ligand. As a result, these molecular "chameleons" can adapt to the environment of the flexible binding site in order to maximize and complement structural interactions, without losing the integrity of the crucial contacts involved in the enzyme's mechanism of action. The synthesis of several proximal analogues is presented herein.

Flexible enzymes are notoriously a bane to structure-based drug design and discovery efforts. This is because no single structure can accurately capture the vast array of conformations that exist in solution and many are subject to ligand-associated structural changes that are difficult to predict. Glutamate racemase (GR) – an antibiotic drug discovery target involved in cell wall biosynthesis – is one such enzyme that has eluded basic structure-based drug design and discovery efforts due to these flexibility issues. In this study, our focus is on overcoming the impediment of unpredictable ligand-associated structural changes in GR drug discovery campaigns. The flexibility of the GR active site is such that it is capable of accommodating ligands with very different structures. Though these ligands may bind to the same pocket, they may associate with quite dissimilar conformations where some are more favorable for complexation than others. Knowledge of these changes is invaluable in guiding drug discovery efforts, indicating which compounds selectively associate with more favorable conformations and are therefore better suited for optimization and providing starting structures to guide structure-based drug design optimization efforts. In this study, we develop a mutant GR possessing a genetically encoded non-natural fluorescent amino acid in a region remote from the active site whose movement has been previously observed to correlate with active site changes. With this mutant GR, we observe a differential fluorescence pattern upon binding of two structurally distinct competitive inhibitors known to associate with unique GR conformations – one to a favorable conformation with a smaller, less solvated active site and the other to an unfavorable conformation with a larger, more solvated active site. A concomitant computational study ascribes the source of this differential fluorescence pattern to ligand-associated conformational changes resulting in changes to the local environment of the fluorescent residue. Therefore, this mutant permits the elucidation of valuable structural information with relative ease by simply monitoring the fluorescence pattern resulting from ligand binding, which indicates whether the ligand has bound to a favorable or unfavorable conformation and offers insight into the general structure of this conformation.