Novel biotinylated phenylarsonous acids as bifunctional reagents for spatially close thiols: studies on reduced antibodies and the agonist binding site of reduced Torpedo nicotinic receptors.

Abstract

We synthesized three novel organoarsenicals as prototype bifunctional reagents for spatially close thiols, N-(4-arsenosophenyl) hexahydro-2-oxo-(3aS,4S,6aR)-1H-thieno[3, 4-d]imidazole-4-pentamide (1), 2-[4-[(4-arsenosophenyl)amino]-1, 4-dioxobutyl] hydrazide, (3aS,4S,6aR)-hexahydro-2-oxo- 1H-thieno[3, 4-d] imidazole-4-pentanoic acid (2), and [4-[[12-[[5-[(3aS,4S, 6aR)-hexahydro-2-oxo-1H-thieno[3, 4-d]imidazol-4-yl]-1-oxopentyl]amino]-1-oxododecyl]amino]phe nyl]-arso nous acid (3) containing both biotin and arsenic with intervening varying length spacers extending from 2 to 15 A beyond biotin bound to streptavidin. Conceptually, the arsenical group can form a stable, covalent ring structure with appropriately spaced thiols and thereby anchor the reagent to a macromolecule, while biotin allows for the detection of the reagent-macromolecule complex via avidin binding. Because the alpha-subunits of all characterized nicotinic receptors contain an easily reducible disulfide bond between adjacent cysteine residues, the reduced alpha-subunit is an attractive site for labeling. Compounds 1-3 all simultaneously bound streptavidin and dithiols, and all three decreased the number of [125I]alpha-bungarotoxin-binding sites in reduced Torpedo nicotinic receptors (IC50s 10-300 nM). Moreover, arsenylation of the receptors prevented their reoxidation with dithio-bis(nitrobenzoic acid), was reversible with 2,3-dimercaptopropanesulfonic acid, and protected the receptor from irreversible alkylation by bromoacetylcholine. However, in no case did 1-3 allow simultaneous binding to reduced nicotinic receptors and to [125I]streptavidin, although 3 alone allowed simultaneous labeling of a spatially close dithiol located in reduced antibodies.