Synthesis and properties of NBD-n-acylcholines, fluorescent analogs of acetylcholine

We have synthesized a homologous series of fluorescent analogs of acetylcholine, N-7-(4-nitrobenzo-2-oxa-1,3-diazolyl)-omega-amino-n-alkanoic acid beta (N,N,N-trialkylammonium) ethylesters (NBD-n-acylcholines) and report here on their physiological and biochemical properties. All NBD-n-acylcholines trimethylated at the cholinergic nitrogen are agonists of acetylcholine at the frog neuromuscular junction. Their potencies in depolarizing frog muscle cells decrease with decreasing chain length. The affinities of binding to the purified receptor from Electrophorus electricus also decrease with decreasing chain length with a large drop in affinity for the derivatives n = 4 and n = 3. The rate constants of association to acetylcholine receptor and to acetylcholine esterase are of the order of 10(8) M-1 S-1 and do not vary significantly with the chain length of the NBD-n-acylcholines. In contrast, the dissociation rate constants decrease with increasing chain length. The quenching of fluorescence of NBD-n-acylcholines accompanying binding to purified receptor and esterase from E. electricus appears to be due to the formation of a hydrogen bond between the omega-amino group as donor and an unidentified acceptor group in a hydrophobic pocket of the protein. With their advantageous fluorescence properties, their simple pharmacology, and their clear structure-function relationships, these compounds are useful tools for the study of cholinergic mechanisms.