A novel photoaffinity ligand for the dopamine transporter based on pyrovalerone

Article abstract of DOI:10.1016/j.bmc.2009.04.057

Non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored in contrast to tropane-based compounds such as cocaine. In order to fill this knowledge gap, a ligand was synthesized in which the aromatic ring of pyrova

Full text of DOI:10.1016/j.bmc.2009.04.057

Bioorganic & Medicinal Chemistry 17 (2009) 3770–3774
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel photoaffinity ligand for the dopamine transporter based
on pyrovalerone
a
a
a
b
David J. Lapinsky a, , Shaili Aggarwal , Yurong Huang , Christopher K. Surratt , John R. Lever ,
*
James D. Foster c, Roxanne A. Vaughan c
a Division of Pharmaceutical Sciences, Duquesne University Mylan School of Pharmacy, 600 Forbes Avenue, Pittsburgh, PA 15282, USA
b Departments of Radiology, Medical Pharmacology and Physiology, Radiopharmaceutical Sciences Institute and Ellis Fischel Cancer Center, University of Missouri—Columbia,
Harry S. Truman Veterans, Administration Medical Center, 800 Hospital Drive, Columbia, MO 65201, USA
c Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored in
contrast to tropane-based compounds such as cocaine. In order to fill this knowledge gap, a ligand was
synthesized in which the aromatic ring of pyrovalerone was substituted with a photoreactive azido
group. The analog 1-(4-azido-3-iodophenyl)-2-pyrrolidin-1-yl-pentan-1-one demonstrated appreciable
binding affinity for the DAT (Ki = 78 18 nM), suggesting the potential utility of a radioiodinated version
in structure-function studies of this protein.
Received 22 January 2009
Revised 22 April 2009
Accepted 24 April 2009
Available online 3 May 2009
Keywords:
Pyrovalerone
Ó 2009 Elsevier Ltd. All rights reserved.
Photoaffinity labeling
Dopamine transporter
Cocaine
1. Introduction
ligands continue to remain pertinent towards mapping substrate
and inhibitor DAT binding sites at the amino acid level. The chem-
Despite decades of committed research, no FDA approved med-
ications are clinically available to combat psychostimulant abuse
and addiction.1 Pharmacological and behavioral studies indicate
that the dopamine transporter (DAT) is the brain receptor chiefly
responsible for the reward/reinforcing properties associated with
amphetamines2 and cocaine.3 There are a plethora of structurally
heterogeneous ligands that are known to bind to the DAT and inhi-
bit the uptake of dopamine;4 however, details regarding the trans-
port inhibition mechanism and the discrete ligand binding pockets
remain poorly understood. As a result, the synthesis of compounds
towards elucidating conformational states and structural elements
associated with the DAT, namely via probing the interactions of
substrates and inhibitors with this protein, remains an important
objective in the search for psychostimulant abuse therapeutics.
Results from structure–activity relationship (SAR) studies and
site-directed mutagenesis experiments imply that structurally dis-
parate inhibitors bind to different domains or binding sites within
the DAT.5–7 Additionally, it has been suggested that the binding of
inhibitors to distinct DAT domains could affect their behavioral
profile in cocaine abuse animal models.6 As a result, radiolabeled
(3H, 125I) affinity (–NCS) and photoaffinity (–N3) irreversible
ical development of DAT irreversible ligands to date has predomi-
nantly focused on tropane-based ligands8–16 and their
conformationally flexible piperidine17 and piperazine18–22 analogs.
[
125I]-MFZ-2-24 (1, Fig. 1), an irreversible tropane-based cocaine
analog, demonstrated covalent ligation via photoaffinity labeling
to the more intracellular-proximal half of transmembrane domain
(TM) 1 within a 13-amino acid sequence.23 However, [125I]-RTI-82
(2), which possesses the same tropane pharmacophore found in
MFZ-2-24 but features the photoreactive azide moiety anchored
off the ester rather than the tropane nitrogen, demonstrated
N3
I
N3
N3
Me
N
N
I
O
O
I
N
N
OMe
O
Cl
Cl
O
MFZ-2-24 (1)
RTI-82 (2)
DEEP (3)
* Corresponding author. Tel.: +1 412 396 6069; fax: +1 412 396 5593.
E-mail address: lapinskyd@duq.edu (D.J. Lapinsky).
Figure 1. Representative examples of tropane- and piperazine-based DAT photo-
affinity ligands.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.057

D. J. Lapinsky et al. / Bioorg. Med. Chem. 17 (2009) 3770–3774
3771
incorporation into TM6.24 Collectively these probes provide evi-
HCl, H2O
reflux
dence of the close 3-D proximity of DAT TMs 1 and 6. Additionally,
piperazine analog [125I]-DEEP (3), a conformationally flexible ana-
log of the tropane-based benztropine class of DAT inhibitors,6 also
demonstrated incorporation into TM 1-2.25
N
N
NHAc
NH2
O
O
7
8
Structurally heterologous non-tropane-based compounds have
received significantly less attention versus tropane-based ligands
in terms of their development into DAT irreversible probes. In an ef-
fort to expand the arsenal of complementary chemical probes useful
for characterizing DAT 3-D structure, we report herein the design,
synthesis, and preliminary labeling studies of a photoaffinity probe
based on pyrovalerone (5, Fig. 2), a modestly selective inhibitor of
the dopamine transporter over the norepinephrine transporter with
little effect upon serotonin trafficking.
Our interest in pyrovalerone stems from its structural resem-
blance to bupropion (4), a well-known drug marketed as a smok-
ing-cessation agent (Zyban) and antidepressant (Wellbutrin).
More recently, bupropion as a dual norepinephrine and dopamine
reuptake inhibitor has attracted significant attention as a pharma-
cotherapeutic for methamphetamine dependence.1,26 However,
determination of the DAT conformational states and binding sites
for bupropion and structurally related compounds is in its in-
fancy.27 With respect to development of this structural class of
inhibitors into potential DAT photoaffinity probes, it is demon-
strated herein that pyrovalerone displays markedly higher binding
affinity than bupropion at the DAT. Additionally, structure–activity
relationship studies performed by Meltzer and colleagues indicate
that pyrovalerone’s aromatic ring is able to tolerate a wide range of
substitutions in terms of retaining appreciable DAT affinity.28 Thus,
the present study seeks to develop pyrovalerone into a compact
DAT photoaffinity probe for providing high resolution structural
information regarding its binding site. This work also further ex-
plores the therapeutic potential of its analogs.
ICl, AcOH
rt
1.) NaNO2,
CF3CO2H,0 C
I
I
N
N
N3
NH2
2.) NaN3, 0 C
O
O
9
6
Scheme 1. Synthesis of target compound 6.
bupropion (4) and pyrovalerone (5) were also synthesized28,29
and pharmacologically evaluated for comparison to the novel
compounds. Replacement of the 4-Me group in pyrovalerone with
4-NHAc slightly reduced DAT binding affinity.28 However, hydroly-
sis of the amide to the corresponding aniline results in a compound
(8) with high DAT affinity comparable to pyrovalerone. Addition of
the 3-I group resulted in ꢀ6-fold decrease in binding affinity for
the DAT while replacing the aniline NH2 with the N3 group further
decreased affinity by 2.5-fold. The 78 nM DAT affinity for target
compound 6 was sixfold higher than bupropion yet 10-fold less
than pyrovalerone, retaining substantial DAT affinity that justified
its further development into a potential DAT photoaffinity probe.
Uptake inhibition IC50 values (Table 1) were typically 3–4-fold
higher than the binding Ki values for each compound (using the
Cheng–Prusoff equation, conversion of uptake inhibition constants
from IC50 to Ki did not significantly change the value, allowing for
direct comparison of binding and uptake results). This 3–4-fold
shift was previously observed with rDAT/CHO cells in this labora-
tory for WIN 35,428, cocaine, mazindol, and methylphenidate.7
Interestingly, compound 8 displayed essentially the same value
for binding and uptake inhibition (Table 1), a pattern previously
seen for benztropine and the related compounds GBR-12,909 and
rimcazole.7 Cocaine and benztropine have been suggested to occu-
py nonidentical DAT sites or conformations;5–7 the present result
may imply that compound 8 also interacts with the DAT in a fash-
ion different from the other compounds in Table 1.
2. Results and discussion
2.1. Chemistry
Target compound 6 was prepared in six overall steps via syn-
thetic methodology common to the construction of DAT photo-
affinity ligands (Scheme 1). First, N-[4-(2-pyrrolidin-1-yl-
pentanoyl)phenyl acetamide (7) was synthesized via Friedel–Crafts
2.3. Radiosynthesis
acylation,
a-bromination, and pyrrolidine displacement as previ-
ously described.28 Amide hydrolysis of 7 under acidic conditions
provided the aniline pyrovalerone derivative 8 in moderate yield
(43%). Regioselective electrophilic aromatic iodination was then
achieved using ICl in acetic acid as previously described.10 Finally,
conversion of 9 to target azido–iodo analog 6 was accomplished in
good yield (93%) via diazotization with nitrous acid and displace-
ment with sodium azide.
Given that 6 demonstrated reasonably high DAT affinity, and
that wash-resistant binding experiments on nonradioactive azido
compounds frequently give false positives in the assessment of
covalent attachment,14
[
125I]-6 was directly synthesized. The
radioiodo compound could then be used to determine if photoacti-
vation produced covalent ligation to the DAT. As shown in Scheme
2, a one-flask synthesis of [125I]-6 was performed using methodol-
ogy previously described in detail for the preparation of radioio-
dinated cocaine analogs as DAT photoaffinity labels.11 Briefly,
electrophilic radioiodination of 8 with [125I]-NaI (1.67 mCi) under
no-carrier-added conditions using Chloramine-T as the oxidant
was followed by diazotization and subsequent treatment with so-
dium azide. Although this sequence ending with reversed-phase
HPLC isolation provided [125I]-6 in only 20% isolated yield, high
2.2. Pharmacology
With pyrovalerone derivatives 6–9 in hand, ligand affinities (Ki
values) were determined for inhibition of [3H]-WIN-35,428 (a co-
caine analog) binding to hDAT in N2A neuroblastoma cells. [3H]-
Dopamine uptake inhibition potencies in the same cells under
the same conditions were also determined (Table 1). Racemic
purity (>99%) and high specific activity (1946 mCi/lmol) were
achieved. The radioligand exhibited a chromatographic profile
identical to that of non-radioactive 6 (Fig. 3) and showed good sta-
bility upon prolonged storage at À20 °C in the dark (92% radio-
chemical purity after 25 days). Figure 3A shows the preparative
HPLC trace where [125I]-6 (tR = 20.0 min) was well resolved from
radioactive and non-radioactive side-products. The major non-
radioactive materials are assigned as the azide (tR = 6.2 min) and
chloroazide (tR = 13.0 min) congeners based upon model studies
Cl
I
HN
N
N
N3
O
O
O
Bupropion (4)
Pyrovalerone (5)
6
Figure 2. Structural relationship between bupropion (4), pyrovalerone (5), and
target photoaffinity compound 6.

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D. J. Lapinsky et al. / Bioorg. Med. Chem. 17 (2009) 3770–3774
incorporation of [125I]-6 into the DAT (Fig. 4). For both samples,
photolabeling was blocked by cocaine, suggesting that [125I]-6 la-
bels the DAT as opposed to a nonspecific cell surface site.30
It should be noted that in addition to a photoreactive azide
functional group, compound 6 also contains a phenyl ketone struc-
tural motif. The carbonyl of such a group could also result in elec-
tron disproportionation upon photolysis, as such chemistry is
traditionally displayed by benzophenone functional groups in
photoaffinity labeling.31 Thus, the involvement of this carbonyl
carbon (vs the presumed nitrene resulting from azide photolysis)
remains a possibility with respect to covalent ligation to the DAT.
Additionally, successful adduction of [125I]-6 to the DAT sug-
gests that this non-tropane ligand tolerates direct substitution of
a photoreactive azido group on the aromatic ring of the inhibitor
scaffold. This contrasts with designs of tropane-based irreversible
compounds in which the azide is placed at a distance (usually via
a linker) from the inhibitor pharmacophore in order to achieve suc-
cessful protein labeling.10 Thus, compact irreversible ligands based
on a pyrovalerone scaffold may offer the advantage of a shorter
tether between compound functional groups and DAT residues in
or near the inhibitor binding site.
Table 1
Inhibition of [3H]-WIN 35,428 binding and [3H]-dopamine uptake of compounds at
hDAT N2A neuroblastoma cells
Compound #
Aromatic
substituent
[3H]-WIN binding
[3H]-DA uptake
inhibition IC50 (nM)
a
inhibition Kia (nM)
4 (Bupropion)
5 (Pyrovalerone)
6
7
8
9
3-Cl
441 174
1567 716
4-Me
8
2
32
8
4-N3-3-I
4-NHAc
4-NH2
78 18
264 78
30.2 2.0b
67.9 8.4b
5
1
7
2
4-NH2-3-I
28
8
175 59
a
Each Ki or IC50 value represents data from at least three independent experi-
ments with each data point on the curve performed in duplicate.
b
From Ref. 28.
1.) [125I]-NaI, Chloramine-T
125I
2.) HOAc / NaNO2
N
N
NH2
N3
3.) NaN3
O
O
125I]-6
8
[
Scheme 2. Synthesis of [125I]-6.
3. Conclusion
conducted in the presence and in the absence of Chloramine-T. Fig-
ure 3B shows co-elution of purified [125I]-6 with a fully character-
ized sample of non-radioactive 6 under the same reversed-phase
HPLC conditions.
We have synthesized and pharmacologically evaluated a novel
photoaffinity ligand (6) based on the non-tropane DAT inhibitor pyr-
ovalerone. This is the first example of a DAT irreversible ligand based
on the 2-substituted aminopentanophenone class of monoamine
uptake inhibitors, thus representing an important contribution to
the growing battery of probes for characterizing DAT function and
3-D structure. There is evidence that DAT inhibitors bind to noniden-
tical DAT sites or conformations,5–7 suggesting that novel crosslink-
ing agents based on this structural class may yield new monoamine
transporter structure-function information. Ligand 6 bound with
relatively high affinity to the DAT and its 125I analog was shown to
bind covalently to rDAT and hDAT expressed in cultured cells. It
should be noted that the S-isomer of pyrovalerone has been found
to be the more biologically active compound;28 thus, attempts to
2.4. DAT photoaffinity labeling
We next turned our attention to preliminary photoaffinity
labeling of DAT with [125I]-6. LLC-PK1 cells expressing rDAT and
HEK 293 cells expressing 6Xhis-hDAT were photoaffinity labeled
with [125I]-6 in the absence or presence of 100
l
M (À)-cocaine.
The cells were then solubilized and the lysates were subjected to
immunoprecipitation followed by SDS–PAGE and autoradiography
according to previously described procedures.30 Labeled proteins
of ꢀ80 kDa were obtained from each lysate, demonstrating the
Figure 3. (A) Reversed-phase HPLC chromatogram for isolation of [125I]-6. The radioligand exhibited an appropriate retention time based on the non-radioactive standard and
was well resolved from radioactive and non-radioactive side-products. (B) Co-elution of purified [125I]-6 and a standard sample of 6 under the same HPLC conditions used for
preparative work.

D. J. Lapinsky et al. / Bioorg. Med. Chem. 17 (2009) 3770–3774
3773
was then extracted with CHCl3 (X3) and the combined organic lay-
ers were dried (MgSO4), filtered, and concentrated in vacuo to pro-
vide 620 mg of iodo aniline 9 (70%). Rf = 0.2 (hexanes/EtOAc/Et3N,
80:18:2). 1H NMR (CDCl3, 400 MHz) d 8.47 (d, 1H, J = 1.9 Hz),
7.98 (dd, 1H, J = 1.9, 8.5 Hz), 6.71 (d, 1H, J = 8.5 Hz), 4.65 (br s,
2H), 3.75 (m, 1H), 2.66 (m, 2H), 2.55 (m, 2H), 1.91–1.84 (m, 1H),
1.78–1.70 (m, 5H), 1.29–1.20 (m, 2H), 0.87 (t, 3H, J = 7.3 Hz). 13C
NMR (CDCl3, 100 MHz) d 198.0, 150.9, 140.7, 130.7, 128.8, 113.0,
82.8, 68.8, 51.3, 33.5, 23.4, 19.3, 14.3. HRMS calcd for C15H21IN2O-
Na+ 395.0591, found 395.0582.
Figure 4. Photoaffinity labeling of DAT with [125I]-6. Cells expressing rDAT or 6X-
his-hDAT were photoaffinity labeled with [125I]-6 in the absence or presence of
100
lM (À)-cocaine. Cells were solubilized and DATs were immunoprecipitated
with DAT antibody 16 (rDAT) or anti-his antibody (hDAT) followed by SDS–PAGE
and autoradiography.
4.3. 1-(4-Azido-3-iodophenyl)-2-pyrrolidin-1-yl-pentan-1-one
(6)
resolve racemic 6 into its enantiomerically pure components are
underway toward obtaining a specific and improved DAT photo-
affinity probe. Future directions include development of additional
DAT irreversible ligands based on the 2-substituted aminopentan-
ophenone structural class, their pharmacological characterization,
and detailed elucidation of DAT binding domains for comparison
to established tropane-based probes.
A 0 °C solution of 9 (248 mg, 0.67 mmol) in trifluoroacetic acid
(3.3 mL) was treated with NaNO2 (113 mg, 1.64 mmol). The mix-
ture was stirred in the dark for 45 min at 0 °C, then carefully trea-
ted with NaN3 (532 mg, 8.18 mmol) and Et2O (3.3 mL). The
reaction was stirred in the dark at 0 °C for 2 h, then diluted with
H2O and Et2O. The organic layer was separated, washed with brine,
dried (MgSO4), filtered, and concentrated. Chromatography (hex-
anes/EtOAc/Et3N, 80:18:2) provided 247 mg of azido iodo
6
4. Experimental
(93%). Rf = 0.37 (hexanes/EtOAc/Et3N, 80:18:2). 1H NMR (CDCl3,
400 MHz) d 8.59 (d, 1H, J = 1.9 Hz), 8.27 (dd, 1H, J = 1.9, 8.4 Hz),
7.18 (d, 1H, J = 8.4 Hz), 3.73 (m, 1H), 2.67 (m, 2H), 2.54 (m, 2H),
1.92–1.82 (m, 1H), 1.80–1.70 (m, 5H), 1.28–1.17 (m, 2H), 0.88 (t,
3H, J = 7.3 Hz). 13C NMR (CDCl3, 100 MHz) d 198.2, 146.0, 140.8,
134.4, 130.2, 117.9, 87.6, 69.9, 60.4, 51.1, 32.5, 23.4, 21.0, 19.4,
14.2. HRMS calcd for C15H20IN4O+ 399.0676, found 399.0669.
All solvents and chemicals were purchased from Aldrich Chem-
ical Co. or Fisher Scientific and used without further purification.
All column chromatography was performed using Fisher S826-25
silica gel sorbent (70–230 mesh) and eluting solvent mixtures as
specified. Thin-layer chromatography (TLC) was performed using
TLC Silica Gel 60 F254 plates obtained from EMD Chemicals, Inc.
and compounds were visualized under UV light. Proportions of sol-
vents used for TLC are by volume. The 1H and 13C NMR spectra
were recorded on a Bruker 400 MHz spectrometer. Chemical shifts
for 1H and 13C NMR spectra are reported as parts per million (d
ppm) relative to tetramethylsilane (0.00 ppm) as an internal stan-
dard. Coupling constants are measured in hertz. HRMS samples
were analyzed by positive ion electrospray on a Bruker 12 Tesla
APEX -Qe FTICR-MS with an Apollo II ion source. Chemical names
follow IUPAC nomenclature. A radioisotope dose calibrator (Capin-
tec CRC-15W) was used for radioactivity measurements and simi-
lar counting geometries were employed for each determination.
4.4. Pharmacology
[3H]-WIN-35,428 binding inhibition assays were performed with
N2A neuroblastoma cells stably transfected with human wildtype
DAT cDNA. Cell monolayers were grown to confluence at 37 °C, 5%
CO2 in 24 well plates with Opti-MEM media supplemented with
10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin,
and 100 lg/ml G-418 (all from Fisher Scientific). Confluent mono-
layers were first washed 2 Â 1 ml with 22 °C ‘KRH buffer’ (25 mM
HEPES, pH 7.3, 125 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM
MgSO4, 1.2 mM KH2PO4, and 5.6 mM glucose) supplemented with
50 mM ascorbic acid (KRH/AA). The washed and aspirated monolay-
ers were incubated with 500 ml [3H]-WIN-35,428(1 nM) and nonra-
4.1. 1-(4-Aminophenyl)-2-pyrrolidin-1-yl-pentan-1-one (8)
dioactive competitor (0.1 nM–10
by removal by aspiration and 2 Â 1 ml KRH/AA washes. Non-specific
binding was determined by using 10 M mazindol as the competi-
tor. Monolayers were solubilized by incubation with 1 ml 1% SDS
at 22 °C for 1 h with gentle shaking. Cell lysateswere transferred into
vials containing 5 ml ScintiSafe fluid (Fisher), mixed thoroughly by
inversion and vortexing, and analyzed via scintillation counting for
determination of remaining tritium radioactivity.
lM) for 15 min at 22 °C, followed
A solution of N-[4-(2-pyrrolidin-1-yl-pentanoyl)phenyl]acet-
amide28 (7, 183 mg, 0.63 mmol) in 1 M aq HCl (7 ml) was refluxed
for 15 h. The mixture was cooled to room temperature, diluted
with water, carefully alkalinized with K2CO3, and extracted with
EtOAc. The organic layer was washed with brine, dried (MgSO4), fil-
tered, concentrated, then chromatographed (hexanes/EtOAc/Et3N,
60:38:2) to provide 67 mg of aniline 8 as a yellow oil (43%).
Rf = 0.24 (hexanes/EtOAc/Et3N, 60:38:2). 1H NMR (CDCl3,
400 MHz) d 7.89 (d, 2H, J = 8.7 Hz), 6.56 (d, 2H, J = 8.8 Hz), 4.53
(br s, 2H), 3.78 (m, 1H), 2.60 (m, 2H), 2.48 (m, 2H), 1.82 (m, 1H),
1.67 (m, 5H), 1.17 (m, 2H), 0.77 (t, 3H, J = 7.3 Hz). 13C NMR (CDCl3,
100 MHz) d 199.3, 151.9, 131.1, 126.9, 113.5, 68.1, 51.3, 33.9, 23.2,
19.3, 14.3. HRMS calcd for C15H23N2O+ 247.1805, found 247.1804.
l
[3H]-Dopamine uptake inhibition assays were conducted identi-
cal to the [3H]-WIN 35,428 binding inhibition assays described
above with the exceptions that 10 nM [3H]-dopamine replaced
the WIN radioligand and the nonradioactive inhibitor drug was
added 10 min before the 5 min [3H]-dopamine uptake interval
commenced.
Ki values for nonlinear regression of [3H]-WIN-35,428 displace-
ment curves were determined with GraphPad Prism 5.0 (GraphPad,
La Jolla, CA). The algorithm converts Ki values from IC50 values
using the Cheng–Prusoff equation: Ki = IC50/1 + [Ligand]/Kd.32 The
regression best fit the data points using one site competitive bind-
ing curves. Irreversible binding was not otherwise addressed in
these assays (it is unknown whether the time course employed is
adequate for covalent linkage, nor was UV irradiation employed).
4.2. 1-(4-Amino-3-iodophenyl)-2-pyrrolidin-1-yl-pentan-1-one
(9)
ICl (426 mg, 2.62 mmol) was added to a solution of 8 (577 mg,
2.34 mmol) in glacial AcOH (41.5 mL). The mixture was stirred
for 16 h at room temperature then concentrated to dryness. The
residue was partitioned between H2O and CHCl3 and the aqueous
layer was basified with satd aq NaHCO3 to pH 9. The aqueous layer

3774
D. J. Lapinsky et al. / Bioorg. Med. Chem. 17 (2009) 3770–3774
4.5. [125I]-1-(4-Azido-3-iodophenyl)-2-pyrrolidin-1-yl-pentan-
1-one ([125I]-6)
described previously23,24 using antiserum 16 generated against
amino acids 42–59 of rDAT or anti-his monoclonal antibody (Sig-
ma) for his-tagged hDAT. Immunoprecipitated samples were sepa-
rated on 4–20% SDS-polyacrylamide gels followed by
autoradiography using HyperfilmTM MP film for 1–4 days at –
80 °C.
Aniline 8 (40
treated with no-carrier-added [125I]-NaI (20
by N-chloro-4-toluenesulfonamide (Chloramine-T) trihydrate
(10 L, 7.0 mM). After 30 min at ambient temperature the mixture
was cooled to À5 °C, and treated sequentially with cold HOAc
(100 L, 3.0 M) and NaNO2 (30 L, 0.5 M). After 15 min, sodium
azide (30 L, 0.5 M) was added and the mixture allowed to warm
to ambient temperature over 20 min. The reaction mixture was
then quenched with Na2S2O5 (10 L, 50 mM) and taken up in a syr-
inge along with a rinse (200 L) of the vessel with HPLC mobile
lL, 5.0 mM) in NaOAc buffer (pH 4.0; 0.2 M) was
lL, 1.67 mCi) followed
l
Acknowledgments
l
l
l
This work was funded by a Hunkele Dreaded Disease Award
(D.J.L.), the Mylan School of Pharmacy at Duquesne University
(D.J.L.), and NIH Grants DA16604 (C.K.S.) and DA15175 (R.A.V.).
We thank NIDA Drug Supply for contribution of certain nonradio-
active DAT ligand compounds.
l
l
phase: MeOH (16.5%), CH3CN (16.5%) and an aqueous solution
(67%) containing Et3N (2.1% v/v) and HOAc (2.8% v/v). The HPLC
system was equipped with a UV absorbance detector (280 nm), a
flow-through radioactivity detector, and a Waters C-18 Nova-Pak
References and notes
column (radial compression module, 8 Â 100 mm, 4
lm). Using a
flow rate of 3 mL/min, radioactive material (tR = 20.0 min) corre-
sponding to 6 was resolved from non-radioactive and radioactive
side products. [125I]-6 was collected (6.5 mL), diluted to 30 mL with
distilled water, and passed through an activated (EtOH/water) so-
lid phase extraction cartridge (Waters Sep-Pak Light t-C-18) that
was flushed with water (2.0 mL) to remove residual salts, and then
with air. The cartridge retained >98% of the radioactivity and then
was fully eluted with EtOH (1.5 mL) to give [125I]-6 (0.33 mCi; 20%)
as a concentrated solution. The material co-eluted with 6 under the
HPLC conditions described above and displayed 99% radiochemical
purity. After 25 days of storage at À20 °C in the dark, 92% radio-
chemical purity was observed by HPLC. The radioligand was
accompanied by a single, less lipophilic, decomposition product
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(8%). A specific radioactivity of 1946 mCi/lmol was determined
for [125I]-6 using HPLC methods to determine the mass associated
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tentatively assigned as the azide (tR = 6.2 min) and the chloroazide
(tR = 13.0 min) based upon HPLC analyses of model reactions.
These were performed as described above without radioiodine. In
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the azide, and then again with NaNO2, NaN3 and varying amounts
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4.6. DAT photoaffinity labeling
LLC-PK1 cells expressing rDAT and HEK-293 cells expressing
6Xhis hDAT were grown to 90% confluency in 6-well plates. Med-
ium was removed and 1 ml of [125I]-6 prepared in KRH buffer was
added to a final concentration of 30 nM for 1 h at 4 °C. For pharma-
cological displacement, 100
l
M (À)-cocaine was included in the
binding mixture. Cells were irradiated with shortwave ultraviolet
light (254 nm, Fotodyne UV Lamp model 3-6000) for 45 s at a dis-
tance of 15–20 mm to photoactivate the radioligand. Medium was
removed and the cells were washed twice with 1 ml of ice-cold
KRH buffer. The photolabeled cells were lysed with 0.6 ml/well
RIPA buffer (50 mM NaF, 2 mM EDTA, 125 mM Na3PO4, 1.25% Tri-
ton X-100, and 1.25% sodium deoxycholate) for 15 min at 0 °C, fol-
lowed by centrifugation at 20,000 g for 15 min at 4 °C. The
supernatant fraction was transferred to clean tubes for immuno-
precipitation. Lysates were subjected to immunoprecipitation as
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