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

We synthesized three novel organoarsenicals as prototype bifunctional reage nts for spatially close thiols, N-(4-arsenosopheny) hexahydro-2-oxo-(3aS,4S ,6aR)-1H-thieno[3,4-d]imidazole-4-pentamide (1), 2-[4-[(4-arsenosophenyl)am ino]-1,4-dioxobutyl] hydrazide, (3aS,4S,GaR)-hexahydro-2-oxo- 1H-thieno[3,4 -d] imidazole-4-pentanoic acid (2), and [4-[[12-[[5-[(3aS,4S,GaR)-hexahydro -2-ox d]imidazol-4-yl]-1-oxopentyl]amino]-1-oxododecyl]amino]phenyl]-arsono us acid (3) containing both biotin and arsenic with intervening varying len gth spacers extending from 2 to 15 Angstrom beyond biotin bound to streptav idin. Conceptually, the arsenical group can form a stable, covalent ring st ructure with appropriately spaced thiols and thereby anchor the reagent to a macromolecule, while biotin allows for the detection of the reagent-macro molecule complex via avidin binding. Because the alpha-subunits of all char acterized nicotinic receptors contain an easily reducible disulfide bond be tween adjacent cysteine residues, the reduced a-subunit is an attractive si te for labeling. Compounds 1-3 all simultaneously bound streptavidin and di thiols, and all three decreased the number of [I-125]alpha- bungarotoxin-bi nding sites in reduced Torpedo nicotinic receptors (IC50s 10-300 nM). Moreo ver, arsenylation of the receptors prevented their reoxidation with dithio- bis(nitrobenzoic acid), was reversible with 2,3-dimercaptopropanesulfonic a cid, and protected the receptor from irreversible alkylation by bromoacetyl choline. However, in no case did 1-3 allow simultaneous binding to reduced nicotinic receptors and to [I-125]streptavidin, although 3 alone allowed si multaneous labeling of a spatially close dithiol located in reduced antibod ies.