Using Physical Chemistry To Differentiate Nicotinic from Cholinergic Agonists at the Nicotinic Acetylcholine Receptor

Abstract

Synthesis of α-hydroxy threonine (Tah, 2R, 3S-dihydroxy-butanoic acid) (1). L-Threonine (2.2 g, 18.5 mmol), suspended in 5 ml of water at -5 °C, was treated simultaneously with a solution of 1.38 g NaNO 2 (20 mmol) in 2 ml of water and 557 µl of concentrated H 2 SO 4 (10 mmol) in 1.5 ml H 2 O. The two solutions were added slowly while stirring so that the temperature remained between 0 °C and 5 °C. The reaction turned yellow upon addition. The solution was then stirred overnight at room temperature. The reaction mixture was concentrated, the mixture treated with 3 ml of EtOH, and the salts were filtered. The solution was concentrated. The material was dry loaded onto a flash silica gel column and run in 1:1 hexanes/ethyl acetate with 1% acetic acid to give 730 mg (38 %) of hydroxythreonine Synthesis of Tah cyanomethyl ester (2R, 3S-dihydroxy-butanoate cyanomethyl ester) (2).The hydroxy acid (385 mg, 3.21 mmol) was dissolved in 5.1 ml of ClCH 2 CN (80.1 mmol) and 1.2 ml Et 3 N (8.44 mmol). Upon stirring under Ar for 30 min, the solution turned yellow. A gradient flash silica gel column from 20% to 80% ethyl acetate/hexanes was run, and the isolated product was dried on vacuum to yield 50.9 mg (10%) of hydroxythreonine cyanomethyl ester: 1 H NMR (D 2 O) δ 1.27 (d, 3H, J= 6 Hz), 4.22 (m, 1H), 4.34 (d, 1H, J= 3 Hz), 5.01 (s,

Atypical nicotinic agonist bound conformations conferring subtype selectivity

“…This relationship between Ls-and Ac-AChBPs in neonicotinoid selectivity is clearly interpretable to that for vertebrate and insect nAChR subtypes. Finally, in nicotinoids, one fundamental bound conformation is conserved for all AChBP and nAChR subtypes (7)(8)(9)(10)23). The same agonist molecule can also adopt different binding orientations at other Cys-loop receptors, depending on the nature and position of aromatic amino acid side chains, that is, serotonin (5-HT) at 5-HT 3 versus MOD-1 receptors and ␥-aminobutyric acid (GABA) at GABA A versus GABA C receptors (24,25).…”

Mapping the elusive neonicotinoid binding site

“…Thus, the conformational flexibility of the loop C segment induced by ligand binding (7) appears to play an important role for interaction with the electronegative pharmacophore of the neonicotinoid. In sharp contrast to the neonicotinoid, an iminium cation of the nicotinoid DNIMI or DCTHIA, as with an ammonium moiety of nicotine or EPI, critically contacts the carbonyl oxygen of the loop B Trp-147 via hydrogen-bonding and secondarily makes van der Waals contact with the -electron of the Trp indole side chain (cationinteraction) (5,7,9,21). Amazingly, these two very different interactions of neonicotinoids and nicotinoids occur in the same binding pocket, accounting for the similarity of their structureactivity relationships (22,23).…”

Insight in nAChR subtype selectivity from AChBP crystal structures

“…Other studies making use of unnatural amino acids and featuring an nAChR with an ester bond between residues 143 and 144 (LsAChBP numbering) lead to substantially different conclusions [40,41]. These studies suggest that a negative charge in position 89 of the nAChR α subunit is not essential in receptor function, but rather that D89 would be responsible in shaping the binding site of the receptor through an extensive hydrogen bond network, hence correctly positioning the conserved tryptophan residue.…”

“…These studies suggest that a negative charge in position 89 of the nAChR α subunit is not essential in receptor function, but rather that D89 would be responsible in shaping the binding site of the receptor through an extensive hydrogen bond network, hence correctly positioning the conserved tryptophan residue. The loss of the planarity in the bond between residues 143 and 144 (LsAChBP numbering) through the substitution of the peptide bond by an ester [40,41], however introduces a further uncharacterized variable in the picture, and hence leaves this issue unresolved.…”