Two classes of nicotinic cholinergic agonists, which vary in flexibility and electronegativity, have been synthesized, and their structural and dynamic properties have been studied with nuclear magnetic resonance (NMR) spectroscopy. Although the compounds are chemically identical except for the presence or absence of one cyclicizing C--C bond, single channel recording and radioligand binding studies have shown that the cyclic compounds are considerably more potent than the acyclic derivatives (McGroddy, K.A., A.A. Carter, M.M. Tubbert, and R.E. Oswald. 1993. Biophys. J. 64:325-338). Using one- and two-dimensional NMR spectroscopy, we have shown that these molecules exist in two distinct stable conformers, which differ in the orientation of the amide bond. The cyclic 1,1-dimethyl-4-trifluoroacetyl-piperazinium iodide and its trifluoromethyl derivative compounds are symmetric, and the two conformers are of equal energy. The acyclic N,N,N,N'-tetramethyl-N'-acetylethylene-diamine iodide (TED) and its trifluoromethyl derivative derivatives, however, populate two energetically unequal solution conformations. Using variable temperature NMR spectroscopy on these molecules and their uncharged precursors, we have characterized the energetics of amide bond isomerization and have distinguished steric and electrostatic contributions to the equilibrium between the two conformers. The more populated TED conformer has the amide methyl group trans to the carbonyl oxygen, and it is stabilized by an electrostatic attraction between the partially negative carbonyl oxygen and the positively charged quaternary amine nitrogen. As discussed in the accompanying paper (McGroddy, K.A., A.A. Carter, M.M. Tubbert, and R.E. Oswald. 1993. Biophys. J. 64:325-338), the differences in the stable solution structures of the TED derivatives and their interconversion kinetics may be of biological significance.

The relationship between the structure and function of a series of nicotinic cholinergic agonists has been studied using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy. The cyclic compound 1,1-dimethyl-4-acetylpiperazinium iodide and its trifluoromethyl analogue (F3-PIP) interact with nicotinic acetylcholine receptors (nAChRs) from both Torpedo electroplaque and BC3H-1 cells at lower concentrations than the acyclic derivatives, N,N,N,N'-tetramethyl-N'-acetylethylenediamine iodide and its fluorinated analogue (F3-TED). The magnitude of the difference in potencies depends on the type of measurement. In binding experiments, the differences between the two classes of compounds depends mainly on the conditions of the experiment. In measurements of the initial interaction with the nAChR, the PIP compounds have an affinity approximately one order of magnitude higher than that of the TED compounds. Longer incubations indicated that the PIP compounds were able to induce a time-dependent shift in receptor affinity consistent with desensitization, whereas the TED compounds were unable to induce such a shift. The activation of single channel currents by the cyclic compounds occurs at concentrations approximately two orders of magnitude lower than for the acyclic compounds, but the TED compounds exhibit a larger degree of channel blockade than the PIP compounds. Previous work (McGroddy, K.A., and R.E. Oswald. 1992. Biophys. J. 64:314-324) has shown that the TED compounds can exist in two energetically distinct conformational states related by an isomerization of the amide bond. 19F nuclear magnetic resonance experiments suggest that the higher energy population of the TED compounds may interact preferentially with the ACh binding sites on the nAChRs and that a significant fraction of the difference between the initial affinity of the PIP and TED compounds may be accounted for by the predominance in solution of a conformational state less able to interact with the ACh binding sites on nAChRs.

Molecular dynamics simulations have been performed on aqueous solutions of two chemically similar nicotinic cholinergic agonists in order to compare their structural and dynamical differences. The cyclic 1,1-dimethyl-4-acetylpiperazinium iodide (HPIP) molecule was previously shown to be a strong agonist for nicotinic acetylcholine receptors (McGroddy et al., 1993), while the acyclic N,N,N,N'-tetramethyl-N'-acetylethylenediamine iodide (HTED) derivative is much less potent. These differences were expected to arise from differences in the solution structures and internal dynamics of the two molecules. HPIP was originally thought to be relatively rigid; however, molecular dynamics simulations suggest that the acetyl portion of the molecule undergoes significant ring dynamics on a psec timescale. The less constrained HTED molecule is relatively rigid, with only one transition observed about any of the major dihedrals in four 100 psec simulations, each started from a different conformation. The average structures obtained from the simulations are very similar to the starting minimized structure in each case, except for the HTED simulation where a single rotation about the N-C-C-N(+) backbone occurred. In each case, HTED had three to five more water molecules in its primary solvation shell than HPIP, indicating that differences in the energetics of desolvation before binding may partially explain the increased potency of HPIP as compared to HTED.