Novel Biotinylated Phenylarsonous Acids as Bifunctional Reagents for Spatially Close Thiols:  Studies on Reduced Antibodies and the Agonist Binding Site of ReducedTorpedoNicotinic 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.

An improved synthesis of arsenic–biotin conjugates

“…In fact, the reaction with biotinoyl chloride is a reasonable option, but even here this reported procedure using the chloride afforded 5 in only 14 % yield. 18 Despite the fact that p-aminophenylarsine oxide (3) exists as hydrate, 21 with time it probably no longer exists as a monomer. Other aryl arsenoxides have been reported to form an eight membered As 4 O 4 ring and to exist as cycles (Ar-AsO) 4 .…”

“…The 1 H-NMR and MS were in complete agreement with the data reported. 18 Solutions of arsenoxides exist as hydrates and can be deactivated over time, presumably by oxidation of arsenic from the trivalent to the pentavalent state. 20 To prevent this, the As-(OH) 2 group of 5 can be protected by reaction with dithiols.…”

Arsenic Binding to Proteins

“…276 Apart from GASO-B, three novel biotinylated PAO compounds with spacers of varying length were also synthesized as bifunctional reagents to examine spatially close thiols in Torpedo nicotinic receptors. 277 …”

Direct Binding of Arsenic Trioxide to AMPK and Generation of Inhibitory Effects on Acute Myeloid Leukemia Precursors

“…This compound has been previous used to isolated arsenic binding proteins as it will bind to thiol groups (6, 8, 37, 38). After lysis in phospho-lysis buffer, 500 μg of protein was diluted in buffer and the arsenic-protein complexes were then isolated with streptavidin agarose beads by tumbling at 4°C overnight (Invitrogen Life Technologies).…”