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A polyether antibiotic, ecteinamycin (1), was isolated from a marine Actinomadurasp., cultivated from the ascidian Ecteinascidia turbinata. 13C enrichment, high resolution NMR spectroscopy, and molecular modeling enabled elucidation of the structure of 1, which was validated on the basis of comparisons with its recently reported crystal structure. Importantly, ecteinamycin demonstrated potent activity against the toxigenic strain of Clostridium difficileNAP1/B1/027 (MIC = 59 ng/μL), as well as other toxigenic and nontoxigenic C. difficileisolates both in vitroand in vivo. Additionally, chemical genomics studies using Escherichia colibarcoded deletion mutants led to the identification of sensitive mutants such as trkAand kdpDinvolved in potassium cation transport and homeostasis supporting a mechanistic proposal that ecteinamycin acts as an ionophore antibiotic. This is the first antibacterial agent whose mechanism of action has been studied using E. colichemical genomics. On the basis of these data, we propose ecteinamycin as an ionophore antibiotic that causes C. difficiledetoxification and cell death viapotassium transport dysregulation.

Establishing the relative configuration of a bioactive natural product represents the most challenging part in determining its structure. Residual dipolar couplings (RDCs) are sensitive probes of the relative spatial orientation of internuclear vectors. We adapted a force field structure calculation methodology to allow free sampling of both Rand Sconfigurations of the stereocenters of interest. The algorithm uses a floating alignment tensor in a simulated annealing protocol to identify the conformations and configurations that best fit experimental RDC and distance restraints (from NOE and J-coupling data). A unique configuration (for rigid molecules) or a very small number of configurations (for less rigid molecules) of the structural models having the lowest chiral angle energies and reasonable magnitudes of the alignment tensor are provided as the best predictions of the unknown configuration. For highly flexible molecules, the progressive locking of their stereocenters into their statistically dominant Ror Sstate dramatically reduces the number of possible relative configurations. The result is verified by checking that the same configuration is obtained by initiating the locking from different regions of the molecule. For all molecules tested having known configurations (with conformations ranging from mostly rigid to highly flexible), the method accurately determined the correct configuration.