Rawling, TristanMacDermott-Opeskin, HugoRoseblade, APazderka, CClarke, CBourget, KWu, XLewis, WNoble, Dr BenjaminGale, P AO'Mara, MeganCranfield, CMurray, M2022-08-012022-08-012041-6520http://hdl.handle.net/1885/270075Respiring mitochondria establish a proton gradient across the mitochondrial inner membrane (MIM) that is used to generate ATP. Protein-independent mitochondrial uncouplers collapse the proton gradient and disrupt ATP production by shuttling protons back across the MIM in a protonophoric cycle. Continued cycling relies on the formation of MIM-permeable anionic species that can return to the intermembrane space after deprotonation in the mitochondrial matrix. Previously described protonophores contain acidic groups that are part of delocalised p-systems that provide large surfaces for charge delocalisation and facilitate anion permeation across the MIM. Here we present a new class of protonophoric uncoupler based on aryl-urea substituted fatty acids in which an acidic group and a p-system are separated by a long alkyl chain. The aryl-urea group in these molecules acts as a synthetic anion receptor that forms intermolecular hydrogen bonds with the fatty acid carboxylate after deprotonation. Dispersal of the negative charge across the aryl-urea system produces lipophilic dimeric complexes that can permeate the MIM and facilitate repeated cycling. Substitution of the aryl-urea group with lipophilic electron withdrawing groups is critical to complex lipophilicity and uncoupling activity. The aryl-urea substituted fatty acids represent the first biological example of mitochondrial uncoupling mediated by the interaction of a fatty acid and an anion receptor moiety, via self-assembly.This study was supported by grants from the Australian National Health and Medical Research Council (1031686 and 1087248). PAG thanks the Australian Research Council (DP200100453 and DP180100612) for funding.application/pdfen-AUThis journal is © The Royal Society of Chemistry 2020https://creativecommons.org/licenses/by-nc/3.0/202010.1039/d0sc02777d2021-08-01Creative Commons Attribution-NonCommercial 3.0 Unported Licence