Synthesis of a naphthylvinylpyridine derivative and its use for affinity chromatography of choline acetyltransferase

N-(10-carboxy)decamethylene-4(1-naphthylvinyl)pyridinium chloride, a derivative of the choline acetyltransferase (CAT) inhibitor naphthylvinylpyridine (NVP) was synthesized and used as a ligand for affinity chromatography of choline acetyltransferase. The preparation of this inhibitor included the quaternization of naphthylvinylpyridine with 11-Br-undecanoic acid methyl ester to obtain N-(10-carbomethoxy)decamethylene-4-(1-naphthylvinyl)pyridinium bromide, followed by hydrolysis to free the carboxylic group. This inhibitor (C₁₁-NVP⁺) had a potency comparable to that of N-methyl-4(1-naphthylvinyl) pyridinium iodide (C₁-NVP⁺) which is the most potent derivative of NVP but which lacks a functional group for conjugation to Sepharose. The C₁₁-NVP⁺ was then bound through the carboxylic group to aminoalkyl Sepharose by a carbodiimide promoted condensation reaction. Interaction of CAT with the inhibitor retarded its elution from a column of Sepharose-C₁₁-NVP⁺ and permitted the purification of the enzyme to electrophoretic homogeneity starting from a preparation in which CAT represented about 20% of the total proteins. Conventional procedures of protein purification had previously been unsuccessful in isolating the enzyme in pure form. Inhibition studies showed that CAT could exhibit either a "high" or a "low" sensitivity to inhibition by naphthylvinylpyridine and its derivatives (I₅₀ with C₁-NVP⁺ = 0.57 μm or 5.2 μm). A direct relationship existed between the sensitivity of CAT to these inhibitors and the retention of the enzyme by the affinity column.