Novel Biotinylated Phenylarsonous Acids as Bifunctional Reagents for

Bioconjugate Chem. 1999, 10, 629−637

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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 R. Moaddel, A. Sharma, T. Huseni, G. S. Jones, Jr.,† R. N. Hanson, and R. H. Loring* Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115. Received December 28, 1998; Revised Manuscript Received March 22, 1999

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[3,4d]imidazol-4-yl]-1-oxopentyl]amino]-1-oxododecyl]amino]phenyl]-arsonous acid (3) containing both biotin and arsenic with intervening varying length spacers extending from 2 to 15 Å 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 R-subunits of all characterized nicotinic receptors contain an easily reducible disulfide bond between adjacent cysteine residues, the reduced R-subunit is an attractive site for labeling. Compounds 1-3 all simultaneously bound streptavidin and dithiols, and all three decreased the number of [125I]Rbungarotoxin-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.

INTRODUCTION

Nicotinic receptors play an important role in vertebrate neuromuscular and ganglionic transmission and are believed to be important in the pharmacology of nicotine dependence (1). In addition, nicotinic agonists are currently being investigated for use as therapeutic agents in the treatment of Alzheimer’s disease, Parkinson’s disease, Tourette’s syndrome, and ulcerative colitis (1). Muscle nicotinic receptors are pentameric membrane proteins consisting of two R1-subunits, and one β-, γ-, and δ-subunits. Multiple subtypes of neuronal nicotinic receptors are known, and all subtypes contain at least one kind of R-subunit. Currently, eight different neuronal R-subunits (R2-R9) and at least 3 neuronal β-subunits (β2-β4) are known (2). Since R-subunits from both muscle and nerve have an easily reducible disulfide bond between adjacent cysteine residues located within 0.9 nm of the agonist-binding site (3), the reduced R-subunit is an attractive site for labeling all known subtypes of nicotinic receptors. Bifunctional reagents that could label the agonist-binding site disulfide and give some spatial information relative to the rest of the receptor molecule would be very useful. Of all multimeric receptors, the Torpedo nicotinic receptor is the best characterized, since it is so abundant in electroplax tissue. The Torpedo receptor has two agonist-binding sites located at the interface between the * To whom correspondence should be addressed. † Current address: RSP Amino Acid Analogues Inc., Worcester, MA 01605.

R-δ and the R-γ subunits (4, 5). Both sites bind R-bungarotoxin (BGT), but a variety of evidence suggests that these two sites are not equivalent. The R-γ site binds D-tubocurarine with high affinity (6) and is easily reduced with dithiothreitol (DTT)1 and alkylated with bromoacetylcholine (BAC), while the R-δ site shows low affinity for D-tubocurarine and is not as easily reduced or alkylated. Monoclonal antibodies are available from a variety of laboratories that discriminate between these two sites [referred to as the A and B sites, respectively (7)], indicating that the interfaces have different conformations. On the other hand, Unwin (8-10) has visualized Torpedo nicotinic receptors to a 9 Å resolution and suggests that two similar pits about 20 Å deep are the agonist-binding sites. Although one of those pits is located near the area mapped by an antibody against the R-γ binding site (11), there is no information as to where the corresponding disulfide bond is located relative to the three-dimensional structure of the receptor. The Torpedo nicotinic receptor is the best characterized, but there are still questions about the location and structure of the agonist-binding sites. Thus, we chose this receptor as our initial target. We previously reported on the utility of simple trivalent organoarsenicals, which formed stable adducts with 1 Abbreviations: APA, p-aminophenylarsinedichloride hydrochloride; BAC, bromoacetylcholine; BGT, R-bungarotoxin; BSA, bovine serum albumin; DMPS, 2,3-dimercaptopropanesulfonic acid; DTNB, 5,5′-(dithiobis)-nitrobenzoic acid; DTT, dithiothreitol; IAA, iodoacetamide; MPB, maleimidopropionylbiocytin; NEM, N-ethylmaleimide; NHS, N-hydroxysuccinimide; SA-HRP, streptavidin-horseradish peroxidase conjugate.

10.1021/bc9801575 CCC: $18.00 © 1999 American Chemical Society Published on Web 05/29/1999

630 Bioconjugate Chem., Vol. 10, No. 4, 1999

reduced nicotinic receptors (12), from both Torpedo electroplax (ganglionic), and chick retina (neuronal). The findings were consistent with covalent As-S bond formation between trivalent arsenic and the adjacent cysteines generated upon reduction of the receptor. The decrease in [125I]BGT binding upon arsenylation of receptors reduced with DTT was long-lasting but reversible upon treatment with the competitive dithiol, 2,3-dimercaptopropane sulfonic acid (DMPS). Monothiols such as 2-mercaptomethanesulfonic acid are not effective reagents to reverse the effects of arsenylation (13), and arsenylation prevented irreversible alkylation by BAC. Thus, our preliminary results suggested that trivalent arsenic could be used to anchor labeling groups in the Torpedo agonistbinding site. These novel bifunctional reagents would be potentially useful tools for receptor localization and quantification studies and mapping the nicotinic receptor and, thereby, provide a more rational basis for the design of agonists. p-Aminophenylarsine oxide2 was envisioned as a potentially useful starting material in our design strategies. The commercial sources were solubilized with HCl or could be freshly synthesized by reducing p-arsanilic acid (14). Here, we describe the synthesis of three novel biotinylated arsenical compounds with varying length spacers, demonstrate that these reagents are bifunctional for dithiols and streptavidin and that they each block [125I]BGT binding when covalently attached to Torpedo nicotinic receptors. These compounds were then used to deduce information about the location of the disulfide in the Torpedo agonist-binding site, and one was found to be potentially useful as a labeling agent for antibodies. MATERIALS AND METHODS

Chemicals and Reagents. p-Aminophenyl arsine oxide, p-arsanilic acid, and all other chemical precursors were obtained from Aldrich Chemical Co. (Milwaukee, WI) unless otherwise noted. Biotin, streptavidin, goat anti-rat IgG agarose beads, 4-chloro-1-naphthol, and β-galactosidase were purchased from Sigma Chemical (St. Louis, MO). Solvents and other materials, chemicals, and reagents were all analytically pure or of reagent grade and used without further purification. TLC (Whatman silica gel 60 F-254 plates; 0.25 mm layer thickness) and PTLC (Analtech Uniplate silica gel GF plates; 20 × 20 cm; 1 mm layer thickness) were used. Maleimidopropionylbiocytin (MPB) was purchased from Molecular Probes (Eugene, OR), streptavidin-horseradish peroxidase (SA-HRP) from Bio-Rad (Richmond, CA), and the peptide corresponding to residues 181-198 of the Torpedo nicotinic R-subunit (YRGWKHVYYTCCPDTPPY) obtained from Chiron Mimetopes (Raleigh, NC). Mem2 The authors are aware that the terms arsine oxide/arsenoxide are archaic, and chemically incorrect, but these are the terms most widely used in the literature for this class of compounds which are sold as such commercially. The structures of members of this class of compounds reported in the literature, typically have not been convincingly established, but are probably not monomeric, particularly the commercial samples, since they are not soluble in the same solvents as the freshly synthesized compounds. For that reason, we distinguish between starting materials that were obtained commercially or freshly synthesized. See ref 36 for descriptions of oligomers. Also, in laser desorption MS of iodophenylarsine oxide, sodium adducts of various oligomeric species were observed (37). However, by all tests at our disposal including MS, the freshly synthesized products we made appear monomeric. Finally, p-aminophenylarsine oxide is no longer commercially available and now must be freshly made (14).

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branes rich in Torpedo nicotinic receptors were a generous gift of Dr. Jonathan Cohen, as was the monoclonal antibody mAb210 from Dr. Jon Lindstrom. R-Bungarotoxin (BGT; Biotoxins Inc., St. Cloud, FL) was radioiodinated using Iodogen (Pierce Chemical Co., Rockford IL) as previously described (15). 125I-Labeled streptavidin was produced similarly, and specific activities of both radioligands ranged 300-500 Ci/mmol determined by isotope dilution (16). Equipment. Melting points were determined with a Thomas-Hoover melting point apparatus. The NMR spectra were recorded with a Varian-300 spectrometer, and IR spectra (KBr) were recorded on a Perkin-Elmer 1600 FTIR spectrometer. MS and MS/MS data were obtained on a VG Quattro triple quadrupole mass spectrometer (Micromass Instruments, Beverly, MA) in the positive electrospray (ESI) mode. For product ion analyses by collision induced dissociation (CID), high-purity argon gas was used as the collision gas and a cell pressure of ≈1.0 X 10-3 mbar was maintained. A collision energy of 30 eV was employed for all product ion experiments. Elemental analyses were performed by Atlantic Microlab, Inc. (Norcross, GA). Chemical Synthesis. N-(4-Arsenosophenyl) hexahydro-2-oxo-(3aS,4S,6aR)-1H-thieno[3,4-d]imidazole-4-pentamide (1) (212391-23-6]).3 Commercial p-aminophenyl arsine oxide (92 mg, 0.5 mmol) was dissolved in water (1 mL) containing 1 M equiv of HCl. After filtration, the filtrate was diluted with 2 vol of acetone, followed by addition of sodium carbonate (3 M equiv). A suspension of biotinoyl chloride (0.5 mmol) in 3 mL of acetone was added dropwise to this solution. After 48 h, the white precipitate was collected by filtration and was purified bypreparativeTLConsilicagelusing10%methanol:dichloromethane as the eluant. The product (32 mg, 14% yield) tested positive for both biotin and the trivalent arsenic. mp 223-224 °C; TLC (10% methanol:dichloromethane) R×a6 ) 0.6. Anal. Calcd for C16H22AsN3O4S, 0.5H2O: C, 44.03; H, 5.31; N, 9.63. Found: C, 44.34; H, 5.33; N, 9.51. 1H NMR (CH OH-d ): δ 7.67 (d,2H; J ) 8.5 Hz), 7.60 3 4 (d,2H; J ) 8.6 Hz), 4.49 (m, 1H), 4.30 (m,1H), 2.93 (dd,1H), 2.70 (d,1H), 2.41 (t,2H), 1.75 (m,4H), 1.66 (m,1H), 1.51 (m,2H). IR (KBr): 750-660 cm-1 (As-O-As); 1686 cm-1 (CdO, ureide, amide); 730, 640 cm-1 (As-O). Molecular weight was confirmed by electrospray mass spectrometry.2 C16H22AsN3O4S (M + H+) Calcd: 428.1. Found: 428. p-(N-Carbomethoxypropionyl) Aminophenyl Arsonous Acid (4). Commercial p-aminophenylarsine oxide (366 mg, 2 mmol) was placed in 8 mL of anhydrous DMF to which 1 M HCl in ether (2 mL, 1 equiv) was added. The precipitate was removed by filtration, and triethylamine was added (310 mg, 3 mmol) to the filtrate. The mixture was stirred, filtered, and 3-carbomethoxypropionyl chloride (150 mg, 1 mmol) in 1 mL of anhydrous DMF was added dropwise to the filtrate. After 24 h, the precipitated 3-carbomethoxy propionic acid was removed by filtration and the filtrate was evaporated to dryness under nitrogen. The oily residue was triturated with a small amount of water, and the precipitate was collected via filtration. The crude product was purified by column chromatography on silica gel using chloroform:methanol:triethylamine (95:5:2) as the eluant. The desired product was isolated as a yellow oil (41 mg, 13% yield). 1H NMR (CH3OH-d4): δ 7.66 (d,2H, J ) 8.7 Hz) 7.57 (d,2H, J ) 8.7 3 The naming of compounds was provided by CAS services. CAS Registry Numbers: compound 1, 212391-23-6; compound 2, 212391-25-8; compound 3, 212391-26-9.

Phenylarsonous Acids as Reagents for Spatially Close Thiols

Hz), 3.80 (s,3H), 2.70 (s,4H). Molecular weight was confirmed by electrospray mass spectrometry. C11H12AsNO4 (M + H+) Calcd: 297.9. Found: 297.8. p-(N-Carbomethoxypropionyl) Aminophenyl Arsanilic Acid. p-Arsanilic acid (217 mg, 1 mmol) was placed in 3 mL of methanol and 1 mL of THF. To this solution, 3-carbomethoxypropionyl chloride (150 mg, 1 mmol) was added dropwise. After 24 h, the solution was taken to dryness. The residue was dissolved in methanol and purified by normal-phase preparative TLC with the eluant chloroform:methanol:ethyl acetate (65:5:5). The desired product was isolated as yellow crystals (222 mg, 67% yield). mp 179-180 °C. 1H NMR (CH3OH-d4): δ 7.88 (d,2H; J ) 9.0 Hz), 7.49 (d,2H; J ) 9.0 Hz), 3.80 (s, 4H), 2.61 (s,3H). Molecular weight was confirmed by electrospray mass spectrometry. C11H13O6NAsNa+ (M+H+) Calcd: 354.0. Found: 354.7. p-(N-Hydrazidopropionyl) Aminophenyl Arsonous Acid (5). A solution consisting of 12 mL of hydrazine hydrate (85%, 10.5 g, 0.2 mol) in 25 mL of ethanol was brought to a gentle reflux. A solution of p-(N-carbomethoxypropionyl) aminophenyl arsonous acid (60 mg, 0.20 mmol) in 4 mL of methanol was added dropwise to the stirred refluxing solution. After 2 h, the solution was concentrated and the resultant oil was purified by column chromatography on silica gel using chloroform:methanol: ethyl acetate (50:5:5) as the eluant (32 mg, 52% yield). IR (KBr) (CdO) amide 1652 cm-1, (CdO) hydrazide 1588 cm-1, N-H stretch(amide) 3290 cm-1. Molecular weight was confirmed by electrospray mass spectrometry. C10H14AsN3O4 (M + H+) Calcd: 316.2. Found: 316.4. 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) (212391-25-8). p-(N-Hydrazidopropionyl) aminophenyl arsonous acid (93 mg, 0.3 mmol) was placed in a flask containing 1 mL of DMSO. Biotinyl-NHS (68 mg, 0.2 mmol) in 1 mL of DMF was added to this solution and stirred at ambient temperature for 16 h, and then the products were evaporated to dryness. The residue was triturated with methanol, and the precipitate was collected by filtration (23 mg, 21% yield). 1H NMR (DMSO-d6): δ 7.71 (d,2H, J ) 7.50 Hz), 7.64 (d,2H, J ) 7.95 Hz), 6.43 (d,1H; J ) 19.32), 4.31 (m,1H), 4.12 (m,1H), 3.55(d,2H), 3.20 (s,2H), 3.11 (t,1H), 2.81 (dd,1H), 2.60 (m,2H), 2.50 (s,2H), 2.3 (m,1H), 2.13 (m,2H), 1.2-1.8 (m,5H). Molecular weight was confirmed by negative ion electrospray mass spectrometry. C20H28AsN5O6S (M - H+) Calcd: 540.5. Found: 540.6. N-Biotinylaminododecanoic Acid (6). A solution of biotinyl-NHS (170 mg, 0.5 mmol) (17) in 10 mL of DMF was added dropwise to a heated solution (60 °C) of 12aminododecanoic acid (129 mg, 0.6 mmol) in 6 mL of 0.2 M NaHCO3. The resulting solution was stirred at 60 °C for 4 h and then at ambient temperature for 16 h. The resulting precipitate was collected, washed sequentially with water and 0.01 M HCl, and then dried under vacuum. 12-Biotinylaminododecanoic acid was obtained (61 mg, 0.16 mmol) in 28% yield. mp 216-217 °C (Lit. mp 217 °C) (18). [4-[[12-[[5-[(3aS,4S,6aR)-Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]-1-oxopentyl]amino]-1-oxododecyl]amino]phenyl]-arsonous Acid (3) (212391-26-9). N,N′Carbonyldiimidazole (32.4 mg, 0.2 mmol) was added to a solution of N-biotinyl-aminododecanoic acid (88 mg, 0.2 mmol) in 5 mL of DMF at 95 °C. The reaction was maintained at 95 °C until the evolution of carbon dioxide ceased. The solution was stirred for 1 h at ambient temperature, during which time a precipitate formed. A solution of freshly prepared p-aminophenyl arsine oxide

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(74 mg, 0.4 mmol) in 5 mL of acetone was added to the stirred reaction mixture. The solution was heated at 50 °C for 5 days, cooled to room temperature and filtered. The filtrate was evaporated to dryness and triturated with ether. The precipitate was collected by filtration and stirred in hot 2-propanol. The insoluble product was collected by filtration and dried to give 32 mg, 25% yield. mp 172-173 °C. Anal. Calcd for C28H43AsN4O4S‚TEA: C, 57.77; H, 8.16; N, 10.34. Found: C, 57.77; H, 8.26; N, 9.9. 1H NMR (DMSO-d6): δ 7.63 (d,2H, J ) 7.4 Hz), 7.54 (d,2H, J ) 7.8 Hz), 6.40 (d,1H; J ) 20.52), 4.30 (m,1H), 4.13 (m,1H), 2.9-3.2 (m,4H), 2.83 (dd,2H), 2.6 (d,1H), 2.29 (t,2H), 2.19 (t,1H), 2.04 (t,2H), 1.2-1.8 (m,24H). Molecular weight was confirmed by electrospray mass spectrometry. C28H43AsN4O4S (M + H+) Calcd: 607.6. Found: 607.4. TLC (75 CHCl3:5CH3OH:5EtOAc) Rf ) 0.1. Assays. Quenching Effects of Compounds 1-3 on the Ellman Assay (19) of 2,3-Dimercaptopropanesulfonic Acid. Varying concentrations (range 0-74 µM) of compounds 1-3 or p-aminophenyldichloro-arsine hydrochloride (APA) were incubated with DMPS (50 µM final concentration) for 10 min. Tris buffer (pH 8.1; 0.3 mM final concentration) and 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB; 200 µM final concentration) were added to a final volume of 1.5 mL, and absorbance at 412 nm was recorded. Confirmation of the Biotin Moiety in Compounds 1-3. Varying concentrations (range 0-1 mM) of 1-3 or d-biotin were added to avidin complexed with 4-hydroxyazobenzene-2-carboxylic acid (HABA). Absorbance at 500 nm was measured and plotted as the mean and range of duplicate samples, according to the method of Green (20). In addition, a dimethylaminocinnamaldehyde spray reagent (21) was used to qualitatively confirm the presence of biotin on TLC plates. Displacement of [125I]R-Bungarotoxin Binding from Torpedo Nicotinic Receptors. Solubilized Torpedo receptors were immunoprecipitated with mAb210 or mAb35 coupled to goat anti-rat IgG agarose beads in phosphatebuffered saline (PBS; 145 mM NaCl and 10 mM sodium phosphate, pH 7.2) containing 1% Triton X-100 (13). After reduction by 2 mM DTT for 20 min, receptors were treated with varying concentrations (range 1 nM-100 µM) of either the biotinylated arsenicals or APA or with 30 µM BAC. After reoxidation with 1 mM DTNB for 20 min, the remaining BGT-binding sites were assayed. Nonspecific binding was determined in the presence of 1 µM unlabeled BGT. In other experiments, receptors were treated with 1 mM DMPS for 20 min to remove bound arsenicals. Staining of Dot-Blotted Torpedo Peptide and β-Galactosidase. Varying concentrations of Torpedo peptide or β-galactosidase in Burnette’s buffer (22) (20 mM Tris, 150 mM glycine, and 20% methanol, pH 8.3) were applied under vacuum to PVDF membranes (Polyscreen, NENDupont, Boston, MA) using a Schleicher and Schuell dotblotter. PVDF membranes were cut into strips and blocked for 45 min at RT with 3% bovine serum albumin (BSA) in PBS. The individual strips were then treated with MPB or compounds 1 or 2 dissolved in PBS and then treated with SA-HRP and stained with 4-chloronaphthol as indicated by Bio-Rad. The strips were reassembled and digitally scanned using a UMAX Astra 1200 S scanner and Adobe Photoshop software. [125I]Streptavidin Binding. Quadruplicate goat anti-rat IgG-coated agarose beads were treated in 1.5 mL centrifuge tubes with 2 mM DTT in PBS, varying concentrations of compounds 1-3 (usually 10 µM), and then treated with 1 mM DTNB for 20 min each treatment.


Moaddel et al.

Scheme 1

Scheme 2

The beads were washed between treatments and three times afterward by centrifuging for 5 s, aspirating the supernatant, and resuspending in 1 mL of PBS. [125I]Streptavidin (1 nM) was added to each sample in 0.1 mL of PBS with 1 mg/mL bovine serum albumin (BSA). After incubation at RT with mixing for 2 h, unbound streptavidin was removed by washing three times (1 mL at 4 °C) with PBS/BSA. Nonspecific binding was established by incubating with 100 nM streptavidin. Samples were counted in a Wizard γ-counter. Molecular Modeling. Molecular modeling was performed on a Silicon Graphics O2 system running Quanta 96 (Molecular Simulations, Inc.) to determine interatomic distances. Molecular structures of 1-3 were generated in their most extended conformation, and the length was measured from the carboxy group of the biotin to the arsenic group. RESULTS AND DISCUSSION

The use of biotinylated bifunctional reagents to characterize macromolecules is well documented (23); however, the incorporation of a p-aminophenylarsonous acid moiety is unprecedented. Although this compound is isosteric to p-aminophenylboronic and p-aminobenzoic acid, which are well characterized, the presence of the arsonous acid group created unique synthetic challenges. Unlike the carboxylic and boronic acids, the arsonyl group can be oxidized to a higher valence which is unreactive toward the targeted vicinal dithiol moiety. Therefore, synthetic transformations and isolation methods required care to avoid oxidation. Second, the synthetic transformations required that the arsonous acid remain in its unprotected state as opposed to the boronate or carboxylate esters which could be hydrolyzed at a later stage of the synthesis. Consequently, the chemical transformations and isolation steps were performed in the presence of a polar, ionizable functionality that compromised the

reactivity of the para-amino group and complicated chromatographic separations. The syntheses of the target compounds are shown in Schemes 1-3. Preparation of the simplest biotinylated arsenical 1 was achieved using classical SchottenBaumann chemistry. Although the isolated yield was low (14%), it was substantially better than methods employing the biotin-activated esters [N-hydroxysuccinimide (NHS), or pentafluorophenyl] or a diimide conjugating reagent [dicylohexylcarbodiimide, 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide]. The free arsonous acid exerted a dominating effect on the chromatographic behavior of the product, necessitating a polar solvent to elute it from the column. Compound 2 was prepared in three steps from p-aminophenylarsonous acid. Acylation of p-aminophenyl arsonous acid with 3-carbomethoxypropionyl chloride (13% yield) followed by hydrazinolysis (52%) and acylation with biotinyl NHS ester gave the final product (21%). The intermediates were purified by column chromatography and the final product by crystallization. Compound 3 was prepared in two steps beginning with the reaction of biotinyl NHS ester with 12aminododecanoic acid. The intermediate was converted to its acyl imidazolide with 1,1′-carbonyldiimidazole and reacted with p-aminophenylarsonous acid (Scheme 3). The crude product was recrystallized to give the final derivative in a 25% yield. Compounds 1-3 were characterized by various instrumental methods. In addition to the absorptions expected for the biotin-containing compounds, the infrared spectra contained strong absorptions in the 660-750 cm-1 region which were characteristic of the As-O stretching frequencies. The 1H NMR spectra were consistent with the proposed structures, confirming the presence of biotin, phenyl, and spacer groups. The NMR data also corroborated the valence of the arsenic group. Comparison of the NMR spectra of p-(N-carbomethoxypropionyl)



Figure 1. SA-HRP labeling with Compound 1 or MPB. Varying concentrations of Torpedo peptide (0-30 µg) or β-galactosidase (0-7 µg) in Burnette’s buffer (22) were applied under vacuum to PVDF as described in the Materials and Methods. The individual strips were then treated with either 100 µM MPB or 1 (20 µM) in PBS, and then treated with SA-HRP and stained with 4-chloronaphthol and H2O2. The Torpedo peptide samples in the second column were further treated with 1 mM DMPS for 20 min, while the peptide samples in the third column were pretreated with 1 mM NEM and 1 mM IAA for 20 min. Compound 1 labeled only the untreated Torpedo peptide but did not bind with the thiols on β-galactosidase (fourth column). In contrast, MPB, which will react with single thiols and is irreversibly bound, labeled both β-galactosidase and Torpedo peptide that was posttreated with DMPS. Scheme 3

aminophenyl arsonous acid and the corresponding arsanilic acid demonstrates a clear and distinctive difference in the chemical shift of the protons ortho to the arsenic group, occurring at 7.66 ppm in the former and 7.88 ppm in the latter. Electrospray ionization mass spectrometry was also valuable in confirming the molecular structure of the products. The full-scale mass spectrum for 1 gave major ions at m/z 478, 464, 450 (100%), 428, 424, and 410.

Assignments could be made for each ion and were consistent with the proposed structure. Mass spectra analysis for each of the other products were equally revealing. Elemental analyses also supported the proposed structure assignments. Preliminary Characterization of the Compounds. The trivalent nature of arsenic in 1-3 was inferred from the results of an assay in which the Ellman response of DMPS to DTNB was quenched in a concentration-


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Figure 2. Compound 2 labels dot-blotted Torpedo peptides. Triplicate samples of 30 µg of Torpedo peptide blotted onto PVDF membranes were incubated for 20 min with either compound 2 (10 µM, top row) or MPB (100 µM, bottom row) and then stained. Further treatment with 1 mM DMPS for 20 min removed compound 2 but not MPB (second column), while prior treatment with either 1 mM APA (third column) or 1 mM NEM and 1 mM IAA (fourth column) blocked labeling by both compounds.

dependent fashion (not shown). Compounds 1-3 also gave a positive color reaction on TLC with p-dimethylaminocinnamaldehyde spray reagent, which has been used as a qualitative assay for the presence of biotin (21), and were equipotent with biotin in their ability to displace HABA from avidin (not shown). To demonstrate that compound 1 could simultaneously bind to streptavidin while covalently linked to a dithiol, reduced Torpedo peptide was blotted onto PVDF membranes at varying concentrations and then treated with either 1 (20 µM) or the sulfhydryl labeling agent MPB (100 µM) (Figure 1). Staining with compound 1 but not MPB was blocked if the samples were subsequently treated with the bissulfhydryl containing DMPS (1 mM). Pretreatment with 1 mM N-ethylmaleimide (NEM) and 1 mM iodoacetamide (IAA) substantially blocked the labeling of the peptide by both 1 and MPB. β-Galactosidase is a protein that binds well to PVDF membranes and has 14 free thiols per molecule, none of which are spatially close (24). MPB strongly stains β-galactosidase, while 1 shows no staining above the background seen for Torpedo peptide treated with NEM and IAA (Figure 1). This result demonstrates the selectivity of the arsenical for spatially close dithiols. The staining effects of 1 on Torpedo peptide are concentration dependent (data not shown) and the lowest concentration that stains peptide above background is 30 nM. Compound 2 also demonstrated labeling of Torpedo peptide that could be blocked by subsequent treatment with DMPS, simultaneous addition of 1 mM APA, or pretreatment with NEM and IAA (Figure 2). The simultaneous binding of 3 to spatially close dithiols and [125I]streptavidin was demonstrated independently (see below). Displacement of [125I]R-Bungarotoxin Binding from Torpedo Nicotinic Receptor. In a previous study, we showed that aromatic trivalent arsenicals, e.g., p-APA, decreased the number of [125I]BGT-binding sites in reduced Torpedo nicotinic receptors to the same level seen after alkylation with BAC (12). Furthermore, arsenylation of Torpedo receptors prevented their reoxidation with DTNB, was reversible with DMPS, and protected the receptor from irreversible alkylation by BAC. Com-

Figure 3. Effects of arsenylation on the binding of [125I]BGT to reduced Torpedo nicotinic receptors: 1 (b); 2 (9); 3 (2); control treatment (1), DTT reduction followed by oxidation with DTNB ([); DTT reduction, alkylation with 30 µM BAC, followed by oxidation with DTNB (`).

pounds 1-3 displayed similar behavior and blocked [125I]BGT binding to DTT-treated Torpedo receptors to the same extent as alkylation with BAC (Figure 3). In other experiments (not shown), treatment of nonreduced receptors with 1-3 or reoxidation of DTT-treated receptors with DTNB before adding 1-3 prevented the blockade of [125I]BGT binding by these compounds. As we previously demonstrated with APA (12, 13), DMPS reverses the effects of 1-3 (Figure 4). Furthermore, adding compounds 1-3 to DTT-treated receptors before BAC and then treating with DMPS to remove the arsenicals restores [125I]BGT binding. These data suggest that 1-3 and BAC are competing for the same site, and since BAC and other affinity-labeling agents attach to the reduced


Figure 4. Compounds 1-3 are reversible with DMPS and protect [125I]BGT binding sites against irreversible alkylation with BAC. Solubilized Torpedo receptors were immunoprecipitated using agarose beads as outlined in the Materials and Methods. After reduction with 2 mM DTT, 10 µM 1-3 (black bars, diagonally striped bars, and striped bars, respectively) all blocked [125I]BGT binding to about the same extent as did 30 µM BAC. However, unlike BAC (not shown), the effects of 1-3 were completely reversed by treatment with 1 mM DMPS. Furthermore, DMPS reversed the effects of 1-3 even after the application of BAC.

disulfide in the agonist binding site of nicotinic receptors (25, 26), we infer that 1-3 also react with the reduced disulfide in the agonist site. [125I]Streptavidin Binding to Arsenylated Macromolecules. In no case did 1-3 simultaneously block [125I]BGT binding to Torpedo receptors and bind [125I]streptavidin (data not shown) if the arsenylating reagents were first added to DTT-reduced receptors and then the receptors were solubilized in Triton and immunoprecipitated. However, we observed significant [125I]streptavidin binding to 3 reacted to DTT-treated antibody-coated beads. Figure 5 demonstrates that this effect is only observed for DTT-treated antibody-coated beads treated with 3 but not 1 or 2, and that DMPS treatment significantly removes this binding. In other experiments (not shown), 3 did not label antibody-coated beads if the beads were not treated with DTT or if DTT-treated beads were reoxidized with DTNB before the addition of 3. Since we had previously shown that APA forms a very stable complex with Torpedo receptors (12), we investigated the stability of 3 complexed with antibodies (Figure 6). We observed no change in the binding of [125I]streptavidin to beads treated with 3 over a period of 24 h, but DMPS could be used at any time to substantially remove the labeling agent. The disulfide bonds located in the hinge regions of IgG isotypes are known to be important for the effector functions of these molecules, such as complement activa-


Figure 5. Compound 3, but not 1 or 2, labels DTT-treated antibody-coated beads. Quadruplicate samples (20 µL packed beads) of goat-anti rat IgG beads were treated for 10 min with 1 mM DTT before the DTT was removed, and then the beads were treated an additional 10 min with the compounds shown. All beads were then treated with 1 mM DTNB to oxidize any unreacted thiols. The beads were then incubated for 2 h in 1 nM [125I]streptavidin and then washed three times in PBS before counting. Nonspecific binding was determined in quadruplicate samples incubated in the presence of 100 nM unlabeled streptavidin.

tion (27). It has been known for over 20 years that gentle reduction of the interheavy chain disulfides in the hinge region yields an antibody that can strongly bind antigen but not C1q (28), the trigger for the complement cascade. Furthermore, electron microscopic (29), gel filtration (30), and immunological and X-ray scattering data (31) all show conformational changes in immunoglobulins upon reduction of the interheavy chain disulfide bonds. This site is a well-established target for antibody-labeling agents, and we infer that this is the most likely site of labeling by 3 since Fab fragments are not labeled (data not shown). One potential advantage of arsenical-based labeling agents is that they could maintain the conformation of the immunoglobulins better than other reagents, since the arsenic interposes itself between spatially close dithiols and reestablishes an approximation of a disulfide bond. In addition, our data suggests that arsenical-based chromatography may be useful in purifying immunoglobulins (32). Tsigelny et al. (33) modeled the extracellular domains of the nicotinic receptor based on homology with copperbinding proteins and current site-directed mutagenesis and affinity-labeling data. In their model, the disulfide bond in the agonist-binding site (Cys192-193) is highly exposed on the outer surface of the R-subunit. Unless glycosylation of the R-subunits prevents streptavidin binding to 3 due to unforseen steric block, the location of the disulfide in the agonist-binding site in that model does not seem to be very likely. Our results might suggest that the disulfide bond in the agonist-binding site is located greater than 15 Å from the surface of the receptor. Two recent reports by Fairclough et al. (11, 34) appear to contradict that interpretation. They used a monoclonal antibody (mAb 383C) that binds to a peptide epitope corresponding to amino acids 187-199 on the Torpedo R-subunit, with


Moaddel et al.

molecule. If this is the case, then one might expect that arsenical-based reagents should abruptly stop working when some critical distance of the biotin spacer has been reached. Finally, the spacer groups are fairly hydrophobic, and it is possible that the biotin is too closely associated with hydrophobic groups in the receptor molecule to be recognized by streptavidin. We plan to address these last two issues, by using spacer groups that are longer, more rigid and hydrophilic. However, the data using 1-3 on antibody-coated beads clearly indicates that the length of the biotin spacer group is critical and that once the biotin group extends sufficiently far from the molecular surface, streptavidin binding ensues. ACKNOWLEDGMENT

Figure 6. The complex of compound 3 and reduced antibodies is stable up to 24 h, but can be dissociated at any time by treating with DMPS. DTT-treated goat anti-rat IgG agarose beads were then either incubated with PBS [(-) left bars) or with 10 mM 3 (in PBS for 10 min, center and right bars) at time zero. At different times, immediately (clear bars), 6 (crosshatched bars) and 24 (reversed crosshatched bars) h later, the beads were then treated with either 1 mM DTNB (left and center bars) or 1 mM DMPS (right bars) for 10 min, and then assayed for specific [125I]streptavidin binding. All samples were in quadruplicate with nonspecific binding determined in the presence of 100 nM unlabeled streptavidin.

specificity for the A site (the R-R interface). This antibody bound to intact receptors, and mapped to a site very near one of the two pits suggested by Unwin (11) to be the agonist-binding sites [but at some distance from where Tsigelny et al. place the disulfide bond in their model (33)]. We propose two possible explanations for this apparent difference in results using these two techniques. First, monoclonal antibodies are well-known to induce conformational changes in the proteins to which they bind. For instance, a monoclonal antibody to avidin is reported to not only dissociate tetrameric avidin into monomers but also to cause the avidin to release bound biotin (18). Using fluorescence resonance energy transfer (FRET), Valenzuela et al. (35) report that binding of a different antibody [mAb A6 against the A site of Torpedo receptors (7)] induced the other agonist-binding site (the R-δ interface) to move about 30 Å closer to the membrane than observed for receptors without the bound antibody. This implies that monoclonal antibodies that selectively bind to one of the two agonist-binding sites can potentially induce major conformational changes. Also, the fact that an antibody binds suggests that the epitope is at least transiently exposed in one conformation of the receptor. It is possible that monoclonal antibodies can “trap” the receptor into a conformation that is relatively rare but in which the epitope including the disulfide bond is much more exposed than in other conformations. Second, our labeling method requires breaking the disulfide bond normally forming an eightatom ring in the agonist binding site, and reforming a nine-atom ring structure including the arsenic atom. This could also induce an abnormal conformation. In addition, the protein folding could point our arsenicals with the biotin groups going into, rather than out of, the receptor

This work was supported by a grant from the Smokeless Tobacco Research Council, Inc., and by NIH NS22472. We thank John Lindstrom for the generous gift of mAb210, Jonathan Cohen for Torpedo membranes, and Ban-Ahn Khaw for a sample of Fab fragments. We thank Jack Cunniff and Jeannine Delaney for the mass spectra data and XiaoGong Zhang and Abdullah Baaj for some of the preliminary binding data. Igor Tsigelny generously provided us with a file specifying the atomic locations of his model for the extracellular domains of the Torpedo nicotinic receptor. We thank William N. Green for helpful discussions. LITERATURE CITED (1) Williams, M., Sullivan, J. P., and Arneric, S. P. (1994) Neuronal Nicotinic Acetylcholine Receptors. Drug News Perspect. 7, 205-223. (2) McGehee, D. S., and Role, L. W. (1995) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu. Rev. Physiol. 57, 521-546. (3) Karlin, A. (1993) Structure of nicotinic acetylcholine receptors. Curr. Opin. Neurobiol. 3, 299-309. (4) Pedersen, S. E., and Cohen, J. B. (1990) d-Tubocurarine binding sites are located at R-gamma and R-δ subunit interfaces of the nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. U.S.A. 87, 2785-2789. (5) Martin, M., Czajkowski, C., and Karlin, A. (1996) The contributions of aspartyl residues in the acetylcholine receptor gamma and δ subunits to the binding of agonists and competitive antagonists. J. Biol. Chem. 271, 13497-13503. (6) Neubig, R. R., and Cohen, J. B. (1979) Equilibrium binding of 3H-tubocurarine and 3H-acetylcholine by Torpedo postsynaptic membranes: Stoichiometry and ligand interactions. Biochemistry 18, 5464-5475. (7) Dowding, A. J., and Hall, Z. W. (1987) Monoclonal antibodies specific for each of the two toxin binding sites of Torpedo acetylcholine receptors. Biochemistry 26, 6372-6381. (8) Unwin, N. (1993) Nicotinic acetylcholine receptor at 9 Å resolution. J. Mol. Biol. 229, 1101-1124. (9) Unwin, N. (1995) Acetylcholine receptor channel imaged in the open state. Nature 373, 37-43. (10) Unwin, N. (1996) Projection structure of the nicotinic acetylcholine receptor: Distinct conformations of the R subunits. J. Mol. Biol. 257, 586-596. (11) Fairclough, R. H., Twaddle, G. M., Gudipati, E., Stone, R. J., Richman, D. P., Burkwall, D. A., and Josephs, R. (1998) Mapping the mAb 383C epitope to alpha 2(187-199) of the Torpedo acetylcholine receptor on the three-dimensional model. J. Mol. Biol. 282, 301-315. (12) Loring, R. H., Dou, Y.-M., Lane, W., Jones, G. S., Jr., and Stevenson K. J. (1992) Aromatic trivalent arsenicals: Covalent yet reversible reagents for the agonist binding site of nicotinic receptors. Mol. Brain Res. 15, 113-120. (13) Dou, Y., McHugh, T., Lane, W. V., Rossant, C. J., and Loring, R. H. (1994) Interactions of dithiols with p-aminophenyldichloroarsine and nicotinic acetylcholine receptors. J. Biol. Chem. 269, 20410-20416.

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BC9801575

Novel Biotinylated Phenylarsonous Acids as Bifunctional Reagents for

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