There is evidence to indicate that the Asp residue in the third transmembrane helix (TMH) of opioid receptors forms a salt bridge with the positively charged nitrogen of endogenous and exogenous opioid ligands. To further examine the role of this electrostatic interaction in receptor binding and activation, we synthesized "carba"-analogues of a published fentanyl analogue containing a 3-(guanidinomethyl)-benzyl group in place of the phenyl moiety attached to the ethylamido group (C. Dardonville et al., Bioorg. Med. Chem. 2006, 14, 6570-6580 (1)), in which the piperidine ring nitrogen was replaced with a carbon. As expected, the resulting cis and trans isomers (8a and 8b) showed reduced mu and kappa opioid receptor binding affinities as compared to 1 but, surprisingly, retained opioid full agonist activity with about half the potency of leucine-enkephalin in the guinea pig ileum assay. In conjunction with performed receptor docking studies, these results indicate that the electrostatic interaction of the protonated nitrogen in the piperidine ring of fentanyl analogues with the Asp residue in the third TMH is not a conditio sine qua non for opioid receptor activation.