Abstract

Among a series of N,N-dimethyl-2-chloro-2-phenethylamines, tile prototype molecule carrying an unsubstituted phenyl ring has the unique property of irreversibly inactivating erythrocyte acetylcholinesterase when acetylcholine is the substrate. The active species responsible for this irreversible inhibition is the corresponding N,N-dimethyl-2-phenylaziridinium ion (DPA) which is generated spontaneously and quantitatively from the 2-chloroamine progenitor at neutral pH. The quaternary ion nature of DPA would allow for reversible addition complex formation with the anionic site of the enzyme and would precede slow alkylation of a nucleophile at that level. A significant stereoselectivity of the enzyme for the levo-isomer of DPA was demonstrated. Such optical selectivity is characteristic of the active surfaces of enzymes in general and of acetylcholinesterase in particular. When the phenyl ring carries substituents such as a 3'-hydroxy, 3'-methoxy, 3'-bromo, 4'-bromo, 4'-methoxy, or 3',4'-dibromo, only a reversible addition complex forms. The inability of these substituted analogs of DPA to alkylate the enzyme bears no relationship to the chemical reactivity of the aziridinium rings, but suggests rather that only the unsubstituted phenyl (as in DPA) can induce in the anionic compartment the type of fit conditioning covalent bond formation. In agreement with the view that DPA interacts with the anionic site of the enzyme, the competitive inhibitor tetramethylammonium iodide was shown to be effective in retarding irreversible inactivation. Neither prolonged dialysis nor incubation with 2-pyridine-aldoxime methochloride (PAM) can reactivate the enzyme after treatment with DPA. No correlation exists between the mode of interaction of DPA and its analogs with acetylcholinesterase and their effects at the adrenergic α-receptor level. The unique properties of DPA suggest applications in the determination of the active sequence at the anionic binding site of acetylcholinesterase.