Radioiodinated Azide and Isothiocyanate Derivatives of Cocaine for

Apr 15, 2005 - Two novel N-substituted-3β-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transp...
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Bioconjugate Chem. 2005, 16, 644−649

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Radioiodinated Azide and Isothiocyanate Derivatives of Cocaine for Irreversible Labeling of Dopamine Transporters: Synthesis and Covalent Binding Studies John R. Lever,†,* Mu-Fa Zou,‡ M. Laura Parnas,§ Romain A. Duval,† Sara E. Wirtz,| Joseph B. Justice,| Roxanne A. Vaughan,§ and Amy Hauck Newman‡ Departments of Radiology, and Medical Pharmacology and Physiology, University of MissourisColumbia, Columbia, Missouri 65212 and Research Service, Harry S. Truman Veterans Administration Medical Center, Columbia, Missouri 65201, Medicinal Chemistry Section, National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland 21224, Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, North Dakota 58203, and Department of Chemistry, Emory University, Atlanta, Georgia 30322. Received November 19, 2004; Revised Manuscript Received February 15, 2005

Two novel N-substituted-3β-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One contains an iodoaryl azide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were prepared in a one-flask procedure by electrophilic radioiodination of the corresponding aniline under no-carrier-added conditions, followed either by diazotization and treatment with sodium azide, or by addition of thiophosgene under basic conditions. Specifically, (-)-N-[4-(3[125I]iodo-4-azidophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane ([125I]MFZ-2-24) and (-)N-[4-(3-[125I]iodo-4-isothiocyanophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane ([125I]MFZ 3-37) were synthesized. Isolation by reversed-phase HPLC and solid-phase extraction gave good average yields of [125I]MFZ-2-24 (67%, n ) 5) and [125I]MFZ-3-37 (45%, n ) 3) with high radiochemical purities (96-99%) and specific radioactivities (>2000 mCi/µmol). The utility of the radioligands was demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding to the full-length DAT.

INTRODUCTION

The dopamine transporter (DAT)1 is an 80 kDa phosphoprotein, thought to have 12 transmembrane domains (TMs), that is regulated by protein kinases, controls synaptic dopamine concentrations, and plays a pivotal role in locomotion (1, 2). Inhibition of dopamine uptake also is recognized as a critical determinant of the reinforcing properties and abuse liability of cocaine and other stimulants (3, 4). Thus, the identification of thera* Address correspondence to John R. Lever, Ph.D., Department of Radiology, M292 Medical Sciences Bldg., University of MissourisColumbia, One Hospital Dr., Columbia, MO 65212. Phone: 573-814-6000 ext. 3686. FAX: 573-814-6551. E-mail: [email protected]. † University of MissourisColumbia and Harry S. Truman Veterans Administration Medical Center. ‡ National Institute on Drug Abuse. § University of North Dakota. | Emory University. 1 Abbreviations: AD-96-129, 4-[2-(diphenylmethoxy)ethyl]-1[(4-azido-3-iodophenyl)methyl]piperidine; DAT, dopamine transporter; DEEP, 1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3iodophenyl)ethyl]piperazine; DTT, dithiothreitol; GA-2-34, N-[n-butyl-4-(4-azido-3-iodophenyl)]-4,4-difluoro-3-(diphenylmethoxy)tropane; GBR 12 ,909, 1-{2-[bis(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine; HEK, human embryonic kidney; IP, immunoprecipitated; MFZ-2-24, (-)-N-[4-(3-iodo-4azidophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane; MFZ 3-37, (-)-N-[4-(3-iodo-4-isothiocyanophenyl)butyl]2β-carbomethoxy-3β-(4-chlorophenyl)tropane; RTI-82, 3-(4chlorophenyl)tropane-2-carboxylic acid, 4-azido-3-iodophenylethyl ester; TM, transmembrane domain; WIN 35, 428, (-)-2-carbomethoxy-3-(4-fluorophenyl)tropane.

peutic DAT inhibitors that block the actions of stimulant drugs of abuse with minimal disruption of normal dopaminergic function is a topic of current interest (5, 6). In fact, dopamine, cocaine, amphetamine, and other structural classes of DAT ligands bind to recognition sites that seem to be at least partially discrete based upon functional, chimera, and mutagenesis studies. Accordingly, we have been developing irreversible ligands to assess and differentiate the features of DAT binding domains at the molecular level. These ligands include several radioiodinated photoaffinity probes: [125I]RTI-82 (7, 8), a tropane alkaloid congener of cocaine; [125I]DEEP (7,9) and [125I]AD-96-129 (10) which are based upon GBR12,909; and [125I]GA-2-34 (11), a benztropine with key pharmacophores drawn from both of the other structural classes (Figure 1). Studies to date confirm that distinct regions of the DAT primary sequence are labeled covalently depending upon ligand framework. In brief, [125I]RTI-82 becomes incorporated specifically in TMs 4-6, [125I]DEEP and [125I]GA-2-34 preferentially attach to TMs 1-2, and [125I]AD-96-129 labels TMs 1-2 and 4-6 to about the same extent (12, 13). The data for [125I]AD96-129 suggests that TMs 1-2 and 4-6 should be close together in the three-dimensional structure of the DAT and, by comparison to the results from [125I]DEEP, demonstrates the sensitivity of DAT binding to modest structural modifications (13). Interestingly, [125I]GA-2-34 and [125I]RTI-82 attach covalently to different TMs even though they both have a tropane ring system (12). This finding is consistent with work showing that benztropines exhibit potent DAT binding but do not display a behavioral profile like that

10.1021/bc0497214 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/15/2005

Radioiodinated Cocaine Derivatives for DAT Labeling

Figure 1. Structures of the cocaine and GBR 12 909 scaffolds, the DAT photoaffinity labels whose sites of incorporation have been identified, and the new irreversible ligands, [125I]MFZ-224 and [125I]MFZ-3-37.

of cocaine (14, 15) and comparative molecular field analyses indicating different DAT binding requirements for benztropines and cocaine (16, 17). As discussed by Zou et al. (18), [125I]GA-2-34 and [125I]RTI-82 might bind to discrete DAT recognition sites or might bind to the same site but covalently label different domains because of differing spatial orientations of the aryl azide substituents (cf. Figure 1). Therefore, a complementary RTI-82 analogue, MFZ-2-24, was prepared where the N-methyl was replaced by a butyl linker to an aryl azide moiety as found in GA-2-34. Nonradioactive MFZ-2-24 displayed high, reversible binding affinity (IC50 33 nM vs [3H]WIN35,428) for the human DAT expressed in mammalian cell line HEK 293 and the structurally related isothiocyanate, MFZ-3-37, covalently bound to human DAT in washout experiments (18). In the present study, we report the synthesis of [125I]MFZ-2-24, MFZ-3-37, and [125I]MFZ-3-37 (Figure 1). We also demonstrate, by photoaffinity and direct labeling of rat striatal DAT, their potential as tools for molecular characterization of DAT binding domains. EXPERIMENTAL PROCEDURES

General. Fully characterized, nonradioactive samples of tropane alkaloids were prepared as previously described (18). Other chemicals and solvents were the best grade available and were used as received from commercial sources. No-carrier-added [125I]NaI was obtained from Amersham Corp. (1 mCi/10 µL; NaOH solution, pH 8-12). HPLC equipment consisted of a Rheodyne 7125 injector, Waters 510EF pumps, Waters 490 UV absorbance detector (λ 254 nm), a flow-through NaI(Tl) crystal scintillation detector comprised of EG&G Ortec components, Shimadzu CR-3A integrating recorders, and a Rainan Dynamax system for digital recording. Waters C-18 Nova-Pak columns, protected by matching guard columns, were used for preparative and analytical HPLC. Activated Waters SEP-PAK Light t-C-18 cartridges were employed for solid-phase extraction. Radioactivity was

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measured with a radioisotope dose calibrator (Capintec CRC-15W) using similar counting geometries or correction factors for each reading. 1H and 13C NMR spectra were recorded on a Bruker AC-300 instrument. Proton chemical shifts are reported as parts per million (δ) relative to internal tetramethylsilane (δ ) 0.00 ppm). Carbon chemical shifts are reported as parts per million (δ) relative to deuterated chloroform (δ ) 77.0 ppm). Infrared spectra were recorded as a neat film on NaCl plates using a Perkin-Elmer 1600 FTIR spectrometer. Microanalyses were performed by Atlantic Microlab, Inc. (Norcross, GA) and agree within 0.4% of calculated values. (-)-N-[4-(3′-Iodo-4′-isothiocyanophenyl)butyl]-2βcarbomethoxy-3β-(4-chlorophenyl)tropane (MFZ-337). (-)-N-[4-(3′-Iodo-4′-aminophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane (18) (0.24 g, 0.43 mmol) was dissolved in a biphasic mixture of CHCl3 (25 mL) and water (10 mL) containing NaHCO3 (0.16 g). The mixture was cooled to 0 °C, and freshly distilled thiophosgene (0.56 mmol, 1.3 equiv) was added with vigorous stirring. After 3 h, the reaction was brought to ambient temperature, the layers were separated, and the aqueous phase was extracted with CHCl3 (3 × 10 mL). The combined organic extracts were dried (K2CO3) and concentrated in vacuo. The residue was purified by flash column chromatography (CHCl3/MeOH/NH4OH; 98:2:1) to afford 0.22 g (84%) of product that was converted to the salt with methanolic HCl. [R]25D -22.7 (c 1.1, CHCl3); 1 H NMR (CDCl3) δ 7.60 (s, 1H), 7.26-7.10 (m, 6H), 3.68 (m, 1H), 3.46 (s, 3H), 3.35 (m, 1H), 3.00-2.82 (m, 2H), 2.60-2.50 (m, 3H), 2.27-1.95 (m, 4H), 1.78-1.55 (m, 5H), 1.37 (m, 2H); 13C NMR (CDCl3) δ 26.0, 28.8, 34.2, 34.5, 35.3, 51.4, 523.2, 53.6, 61.8, 63.2, 94.4, 127.0, 128.4, 129.1, 129.7, 131.8, 132.9, 139.6, 142.2, 144.2, 172.2; IR (neat): 2076 (br, s), 1744.7(s) cm-1; Anal. (C26H28N2O2ClIS‚HCl‚2H2O) C, H, N. (-)-N-[4-(3′-[125I]Iodo-4′-azidophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane ([125I]MFZ2-24). In a representative procedure, (-)-N-[4-(4′-aminophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane (18) (50 µL, 3.0 mM) in NaOAc buffer (pH 4.0; 0.3 M) containing MeOH (33%, v/v) was added to a glass vial and sealed with a Teflon-faced septum. [125I]NaI (20 µL, 2.09 mCi; ca. 1.0 nmol) was added, followed by aqueous N-chloro-4-toluenesulfonamide (Chloramine-T) trihydrate (10 µL, 3.5 mM). The mixture was incubated for 30 min at ambient temperature, chilled in an ice/methanol bath, and then diluted with cold HOAc (50 µL, 3.0 M) followed by cold NaNO2 (25 µL, 0.5 M). After 15 min at -10 °C, NaN3 (25 µL, 0.5 M) was added, and the mixture was allowed to warm to ambient temperature and stand for 15 min before a Na2S2O5 (5 µL, 50 mM) quench. The contents were withdrawn by syringe, and combined with MeOH (2 × 100 µL) rinses of the reaction vessel for HPLC purification using a C-18 column (Waters Nova-Pak, radial compression module, 8 × 100 mm, 6 µm). Using a mobile phase of MeOH (21%), CH3CN (21%), and a water (58%) solution containing Et3N (2.1% v/v) and HOAc (2.8% v/v) at a flow rate of 5 mL/min, [125I]MFZ-2-24 eluted with a retention time (tR ) 34.3 min, k′ ) 56) corresponding to nonradioactive MFZ-2-24 and was resolved from radioactive and nonradioactive side products. The radioligand was collected in a 22.5 mL volume, diluted to 50 mL with distilled water, and passed through an activated solid-phase extraction cartridge that was flushed with water (2.5 mL), to remove salts, and then with air. Elution of the cartridge with MeOH (1.1 mL) provided 1.49 mCi (71%) of [125I]MFZ-2-24. Analytical

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Lever et al.

Scheme 1. Synthesis of [125I]MFZ-2-24

Figure 2. HPLC chromatogram for isolation of [125I]MFZ-224.

HPLC of 0.20 mCi aliquots, taken from the HPLC mobile phase after the preparative collection, used the same HPLC column with a mobile phase of MeOH (30%), CH3CN (30%) and the aqueous mixture (40%) at 4 mL/min. [125I]MFZ-2-24 (tR ) 5.1 min, k′ ) 12) had no radiochemical impurities, and specific radioactivities up to 2104 mCi/ µmol were calculated based upon comparisons with standard samples. The relation of mass to the HPLC UV response was validated by a six-point linear curve (r2 ) 0.99) for nonradioactive MFZ-2-24 that bracketed the region of interest (0.036-1.8 nmol). Only minor decomposition (ca. 5%) was noted after storage at -20 °C in the dark for 19 weeks. (-)-N-[4-(3′-[125I]Iodo-4′-isothiocyanophenyl)butyl]2β-carbomethoxy-3β-(4-chlorophenyl)tropane ([125I]MFZ-3-37). As described above, N-[4-(4′-aminophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane (50 µL, 3.0 mM) was treated with [125I]NaI (25 µL, 2.81 mCi) and Chloramine-T trihydrate (10 µL, 7.0 mM) for 30 min at ambient temperature and then chilled in an ice/ methanol bath. The mixture was made basic with NaHCO3 (50 µL, 0.5 M), and thiophosgene (1.14 µL, 15 µmol) in CHCl3 (50 µL) was added. After vortex mixing, the vessel was incubated at -20 °C for 120 h in a freezer and then warmed to ambient temperature. A homogeneous solution was obtained by adding 1.0 mL of the mobile phase to be used for HPLC: MeOH (25%), CH3CN (25%), and a water (50%) solution containing Et3N (2.1% v/v) and HOAc (2.8% v/v). Using a flow rate of 8 mL/min and a semipreparative C-18 column (Waters Nova-Pak, 7.8 mm × 30 cm, 6 µm), [125I]MFZ-3-37 eluted with an appropriate retention time (tR ) 53.8 min, k′ ) 52), and was resolved from radioactive and nonradioactive side products. The radioligand (1.90 mCi) was collected in a 40 mL volume, diluted to 133 mL with distilled water, and passed through an activated solidphase extraction cartridge that was flushed with water (2.5 mL), and then with air. Elution of the cartridge with MeOH (1.5 mL) provided 1.53 mCi (54%) of [125I]MFZ3-37. Analytical HPLC (Waters Nova-Pak, radial compression module, 8 × 100 mm, 6 µm) using a mobile phase of MeOH (30%), CH3CN (30%), and a water (40%) solution containing Et3N (2.1% v/v) and HOAc (2.8% v/v) at a flow rate of 4 mL/min showed a high degree of chemical and radiochemical purity (>96%) for the radioligand (tR ) 10.6 min, k′ ) 26). The specific radioactivity was estimated as 2000 mCi/µmol based upon comparisons with standard samples of nonradioactive material. The

MeOH was removed by evaporation under a stream of argon at 45 °C, and replaced with CH3CN. Covalent Labeling and Immunoprecipitation of DAT. Covalent labeling and immunoprecipitation of DAT with [125I]MFZ-2-24 and [125I]MFZ-3-37 were performed, with minor modifications, as previously described for other irreversible labels (12, 13). Briefly, male Sprague Dawley rats were decapitated, and striatal tissue was removed from the brain, weighed, and homogenized (Polytron) in sucrose-phosphate (SP) buffer (10 mM sodium phosphate, 0.32 M sucrose; pH 7.4). Homogenates were centrifuged (20 000g, 12 min; 4 °C), and the pellet was washed twice and resuspended to 20 mg/mL, original wet weight, in ice-cold SP buffer. Radioligands were added to the membrane suspension at a final concentration of 5 nM, and then incubated for 2 h at 0 °C. Aryl azide [125I]MFZ-2-24 was covalently attached to DAT by irradiating the sample with ultraviolet light for 45 s. This step was omitted for the isothiocyanate [125I]MFZ-3-37. Membranes were washed twice with SP buffer, and the final pellet was resuspended to 20 mg/mL in sample buffer (50 mM Tris-HCl, pH 6.8, containing 2% sodium dodecyl sulfate (SDS), 0.60 M dithiothreitol (DTT), 30% glycerol, and 0.001% bromphenol blue) for immunoprecipitation and electrophoresis. DTT and other reducing agents were omitted from samples labeled with [125I]MFZ-3-37. The solubilized DATs were immunoprecipitated with antiserum 16 as previously described in detail (12, 13). Total binding and immunoprecipitated samples were subjected to electrophoresis on a 10% SDS-polyacrylamide gel, followed by autoradiography using Hyperfilm MP film for 1 to 4 days at -80 °C. Animals were housed and cared for in accordance with guidelines established by the Institutional Animal Care and Use Committees, and the National Institutes of Health. RESULTS AND DISCUSSION

The radiosynthesis of [125I]MFZ-2-24 proved straightforward (Scheme 1). We followed the three-step, one-flask methodology developed by Wilson (9) for production of [125I]DEEP that has been applied successfully to several radioiodinated photoaffinity labels for the DAT (8, 10, 11). In brief, electrophilic radioiodination of the aniline (18) was accomplished by treatment with [125I]NaI under nocarrier-added conditions at ambient temperature for 30 min using chloramine-T as oxidant at pH 4. The incorporation of radioiodine was >90%. Acidification with HOAc, followed by treatment with NaNO2 at -10 °C, formed the diazonium salt in situ. A solution of NaN3 was added and the mixture warmed to ambient temperature to liberate the aryl cation. Trapping of this reactive intermediate by azide concluded the sequence. One major radioiodinated product was formed that displayed a chromatographic profile on reversed-phase, ion-pair HPLC (tR ) 34.3 min, k′ ) 56; Figure 2) that was identical to that of a characterized sample of

Radioiodinated Cocaine Derivatives for DAT Labeling

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Scheme 2. Synthesis of MFZ-3-37

Figure 3. HPLC chromatogram for isolation of [125I]MFZ-337.

Scheme 3. Synthesis of [125I]MFZ-3-37

nonradioactive MFZ-2-24 prepared as previously described (18). Conditions for purification on an analytical column were chosen so the [125I]MFZ-2-24 was resolved fully from radioactive and nonradioactive side products. The major nonradioactive materials were assigned as the corresponding azide (tR ) 13.7 min, k′ ) 22) and chloroazide (tR ) 23.2 min, k′ ) 38) taking into consideration the synthetic route, relative HPLC retention profiles and data from previous studies (8, 9). Model reactions conducted without radioiodine showed that the putative azide was the sole product in the absence of chloramineT, and that the proportion of the putative chloroazide grew with increasing amounts of chloramine-T. The amount of chlorination is relatively small and is a common side reaction when N-chloroamine oxidants are used for radioiodinations (19). Over a series of runs, isolated yields of [125I]MFZ-2-24 ranged from 58 to 73%, and averaged 67% (n ) 5). Specific radioactivities were near the theoretical value (2175 mCi/µmol) for the radioiodine employed. Calculations were based upon comparisons of HPLC UV absorbance peak areas for aliquots of [125I]MFZ-2-24 having known radioactivity to that of standards of nonradioactive material. The [125I]MFZ-2-24 was radiochemically pure by HPLC, and found to be stable in buffered methanol during prolonged storage (>2 half-lives) at -20 °C in the dark. To expand the scope of possible DAT labeling experiments, we also chose a radioiodinated isothiocyanate as

a target ligand. A tropane-based isothiocyanate with appropriate properties would not require photoactivation for irreversible DAT binding and would covalently label nucleophilic amino acid residues in close proximity to the binding site(s). As shown in Scheme 2, we prepared MFZ3-37, the isothiocyanate congener of MFZ-2-24, by treatment of the known iodoaniline (18) with thiophosgene in a biphasic mixture of CHCl3 and aqueous NaHCO3. Extractive work up, followed by column chromatography and formation of the HCl salt, gave MFZ-3-37 in 84% yield. Despite the high yields often noted for this transformation on a macroscopic scale, yields at the no-carrier-added level can be quite low, and there are relatively few examples of radiolabeled isothiocyanates. For instance, de Costa and colleagues obtained only a 13% yield for conversion of a tritiated aniline to an isothiocyanate for irreversible radiolabeling of kappa-opioid receptors (20). Moreover, isothiocyanate and thiourea functionalities are known to interfere with various radiohalogenation techniques (21). Even with a highly activated substrate, p-methoxyphenyl isothiocyanate, carefully optimized yields for chloramine-T-promoted radioiodination were only 1520%, and the reaction required ultrasonication for 2 h (22). Nonetheless, the radioiodinated p-methoxyphenyl isothiocyanate developed by Dewanjee et al. proved useful for conjugation with oligonucleotides (22). Thus, there are certain general constraints on the preparation of radiolabeled isothiocyanates. However, our present case logically calls for simple radioiodination of an activated aromatic system as the first step, and we further planned to minimize synthetic manipulations over the two-step process to reduce mechanical losses. In practice, we developed a one-flask method where electrophilic radioiodination (vide supra) of the anilino precursor was followed by basification of the chilled reaction mixture with aqueous NaHCO3 and treatment with a solution of thiophosgene in CHCl3 at -20 °C for 120 h (Scheme 3). After warming to room temperature, the biphasic mixture was made homogeneous by adding the mobile phase to be used for semipreparative, reversed-phase HPLC. The major radioactive material, assigned as [125I]MFZ-3-37, eluted with an appropriate retention time (tR ) 53.8 min, k′ ) 52) and was resolved from radioactive and nonradioactive side products (Figure 3). The two major nonradioactive materials observed are likely to be the isothiocyanate (tR ) 24.6 min, k′ ) 23) and the chlorinated isothiocyanate (tR ) 45.0 min, k′ ) 43) (vide supra). The radioligand was isolated in up to 54% yield with high specific radioactivity and >96% radiochemical purity. The average yield for three runs was 45%; thus, sufficient [125I]MFZ-3-37 is available for various DAT studies. An aprotic solvent, acetonitrile, was used for extended storage to minimize potential hydrolysis. With radioligands in hand, we turned our attention to a set of experiments designed to demonstrate covalent

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Figure 4. Irreversible labeling of rat striatal DAT with [125I]MFZ-2-24 and [125I]MFZ-3-37. Membranes were incubated with the indicated compounds and irradiated in the case of [125I]MFZ2-24. The solubilized proteins were subjected to electrophoresis directly (Total) or immunoprecipitated (IP) with serum 16, followed by autoradiography. The arrow indicates the migration of the DAT at about 80 kDa based upon molecular mass standards.

radiolabeling of rat striatal DAT. Incubation of membranes with either [125I]MFZ-2-24 followed by ultraviolet irradiation, or with [125I]MFZ-3-37 treatment alone, resulted in incorporation of the radioligands into membrane proteins (Figure 4). Levels of total binding similar to that displayed by the aryl azide, [125I]MFZ-2-24, are routinely found for other DAT photoaffinity labels (7, 1113). Much higher levels of total binding, involving numerous proteins, were noted for [125I]MFZ-3-37. This result is in accord with the more indiscriminate nature of covalent labeling of an isothiocyanate to various biological nucleophiles. For both radioligands, immunoprecipitation of the solubilized membranes with an antibody, serum 16, that is highly specific for amino acids 42 to 59 in the Nterminal DAT tail (23, 24) resulted in the extraction of a single radioactive band. This band migrates at about 80 kDa, a molecular mass appropriate for the full-length rat striatal DAT. Other studies using a nonimmunogenic serum (24) that does not precipitate the radiolabeled DAT band, as well as pharmacology studies using specific peptide blockers, are consistent with the identity of the radiolabeled protein as the DAT (data not shown). Together, the results demonstrate that both [125I]MFZ2-24 and [125I]MFZ-3-37 become attached covalently to the DAT and can be used as tools to help elucidate DAT binding domains. In fact, recent trypsin proteolysis and detailed epitope-specific immunoprecipitation studies show that [125I]MFZ-2-24 is complementary to [125I]RTI82 and primarily labels N-terminal TMs 1-2 accompanied by only a small fraction,