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 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.
Moaddel
R
R
Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, USA.
Sharma
A
A
Huseni
T
T
Jones
G S
GS
Hanson
R N
RN
Loring
R H
RH
D000818:Animals; D001152:Arsenicals / Q000737:chemistry*; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D001710:Biotin / Q000737:chemistry*; D002038:Bungarotoxins / Q000378:metabolism; D005696:Galactosidases / Q000737:chemistry; D015151:Immunoblotting; D007202:Indicators and Reagents; D008958:Models, Molecular; D018722:Nicotinic Agonists / Q000737:chemistry* / Q000494:pharmacology; D010084:Oxidation-Reduction; D011978:Receptors, Nicotinic / Q000187:drug effects*; D019809:Streptavidin / Q000737:chemistry / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014101:Torpedo / Q000378:metabolism*
D001152:Arsenicals; D002038:Bungarotoxins; D007202:Indicators and Reagents; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; D013438:Sulfhydryl Compounds; C046652:phenylarsonous acid; D001710:Biotin; D019809:Streptavidin; D005696:Galactosidases