Irreversible inhibitors of nicotinic acetylcholine receptors: isolation and structural characterization of the biologically active solvolysis products of bipinnatin-A and bipinnatin-C.

The lophotoxins irreversibly inhibit nicotinic acetylcholine receptors by covalent modification of Tyr190 in the alpha-subunits of the receptor. Previous studies have shown that the naturally occurring lophotoxin analogs bipinnatin-A, -B, and -C are actually inactive protoxins and that their ability to irreversibly inhibit nicotinic receptors is enhanced by preincubation in buffer. However, the ability of lophotoxin to irreversibly inhibit nicotinic receptors does not appear to be enhanced by preincubation in buffer. These observations led to the current effort to isolate and determine the structures of biologically active bipinnatins. Disappearance of the lophotoxins from solution followed a simple first-order exponential decay function. Lophotoxin, however, was approximately 40-fold more stable then bipinnatin-A, -B, or -C. Solvolysis of the bipinnatins, but not of lophotoxin, resulted in production of an equimolar amount of acetic acid at a rate similar to the rate of solvolysis, suggesting that the initial event in solvolysis of these toxins involves hydrolysis of an acetate ester. Proton NMR and fast-atom bombardment mass spectroscopy were used to confirm the structures of the active solvolysis products of bipinnatin-A and -C. Their structures and the relative pH insensitivity of the solvolysis reaction suggest that biological activation of the bipinnatins may proceed through an SN1 type of substitution reaction involving elimination of acetate followed by reaction of a carbocation intermediate with solvent.