Synthesis and Characterization of Azido Aryl Analogs of IBNtxA for Radio-Photoaffinity Labeling Opioid Receptors in Cell Lines and in Mouse Brain

Article abstract of DOI:10.1007/s10571-020-00867-6

Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice variants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate the analgesic action of novel opioid drugs such as 3′-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that IBNtxA is a potent analgesic effective in a spectrum of pain models but lacks many side-effects associated with traditional opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efficiently radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously, as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specific ligand–protein contacts and its signaling complexes.

Full text of DOI:10.1007/s10571-020-00867-6

Cellular and Molecular Neurobiology
https://doi.org/10.1007/s10571-020-00867-6
ORIGINAL RESEARCH
Synthesis and Characterization of Azido Aryl Analogs of IBNtxA
for Radio‑Photoafnity Labeling Opioid Receptors in Cell Lines
and in Mouse Brain
Steven G. Grinnell2,3 · Rajendra Uprety2 · Andras Varadi2,3 · Joan Subrath2 · Amanda Hunkele2
Ying Xian Pan1,2 · Gavril W. Pasternak1,2,3,4 · Susruta Majumdar5
·
Received: 20 February 2020 / Accepted: 5 May 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and
fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice vari-
ants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate
the analgesic action of novel opioid drugs such as 3′-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that
IBNtxA is a potent analgesic efective in a spectrum of pain models but lacks many side-efects associated with traditional
opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow
photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may
comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efciently
radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously,
as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated
handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specifc ligand–protein contacts
and its signaling complexes.
Keywords Photoafnity labeling · Mu opioid receptor complex · Opioid · IBNtxA · Click · Radioligand binding
Steven G. Grinnell and Rajendra Uprety have contributed equally
Introduction
to this work.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10571-020-00867-6) contains
supplementary material, which is available to authorized users.
Mu opioid receptors (MOR-1) are members of the G-protein
coupled receptors (GPCR) family. The gene encoding them,
Oprm1, generates a range of receptors due to alternative
splicing (Pasternak and Pan 2013). As the repertoire of opi-
oid targets has expanded, questions have arisen regarding
their detail structure and nature of specifc protein–protein
interactions responsible for their biological action. A major
focus in many of these studies has been the identifcation and
isolation of these targets. One of the robust approaches is to
use afnity labeling tools (Bochet et al. 1988; Takayanagi
et al. 1982). Afnity labeling is a classical biochemical tech-
nique in which a high afnity ligand bearing a reactive group
is covalently coupled within its binding pocket (Sumranjit
and Chung 2013; Wofsy et al. 1962). Photoafnity labeling
enables a formation of covalent cross-link between the target
and the ligand upon brief exposure to ultraviolet (UV) light
and the technique can be used to map the binding pocket of
the ligand. Rationally designed opioid photoafnity probes
* Steven G. Grinnell
Steven.vasquez.grinnell@gmail.com
* Susruta Majumdar
susrutam@email.wustl.edu
1
Department of Neurology, Memorial Sloan-Kettering Cancer
Center, New York, NY, USA
2
Molecular Pharmacology and Chemistry Program, Memorial
Sloan-Kettering Cancer Center, New York, NY, USA
3
Neuroscience Graduate Program, Weill Cornell Graduate
School of Medical Sciences, New York, NY, USA
4
Pharmacology Graduate Program, Weill Cornell Graduate
School of Medical Sciences, New York, NY, USA
5
Center for Clinical Pharmacology, St Louis College
of Pharmacy and Washington University School of Medicine,
St Louis, MO, USA
Vol.:(0123456789)
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Cellular and Molecular Neurobiology
with a bio-conjugation handle such as alkyne can be radi-
olabeled that provides a way to follow the target during puri-
fcation. Next, the alkyne handle can be used to conjugate,
through CLICK chemistry, with biotin or peptide epitopes
to facilitate the purifcation of the target protein (Best 2009;
Thirumurugan et al. 2013). CLICK chemistry utilizes the
highly selective coupling of allylazides with alkynes at room
temperature under biologically acceptable conditions. Using
CLICK methodology after covalent coupling of an afn-
ity label to its target permits the attachment of a variety of
structures that might have interfered with the ability of the
afnity probe to label its target. Here, we describe synthe-
sis and in vitro characterization of two new high afnity,
high-specifc activity, photoactivatable aryl azidoaryl opi-
oid derivatives, closely related analogs of IBNtxA (Grinnell
et al. 2014; Majumdar et al. 2011a, b); one of which has an
alkyne moiety suitable to CLICK chemistry for photoafnity
probes. Both probes 10 and 17 have high afnity and speci-
fcity at opioid receptors and efciently radio-photolabeled
mMOR-1 as well as mouse brain.
mammalian expression vector. The resulting plasmid,
mDOR-1/pcDNA3.1-Hygro was used to transfect Chinese
hamster ovary (CHO-K1, CCL-61, ATCC) cells by Lipo-
fectAMINE reagent (Invitrogen). Stable transformants
were obtained 2 weeks after selection with hygromycin
and screened with [3H]DPDPE binding assay. These cell
lines have been previously used in our previous publications
(Majumdar et al. 2011a, 2012; Varadi et al. 2016).
Animals
Adult male CD-1 and C57Bl/6 (6–8 weeks old) mice were
purchased from Charles River Laboratories. MOR Exon
11 knockout animals were derived as previously described
(Pan et al. 2003) and backcrossed at least 10 generations
on a C57Bl6/J background. Opioid receptor triple knock-
out (TKO) animals used were generated in the laboratory
of John Pintar (Clarke et al. 2001; Schuller et al. 1999; Zhu
et al. 1999) and maintained on an inbred 129S6 background.
All mice were maintained on a 12-h light/dark cycle with
Purina rodent chow and water available ad libitum. Mice
were housed in groups of fve until sacrifce by cervical dis-
location. All animal studies were approved by the Institu-
tional Animal Care and Use Committee of Memorial Sloan-
Kettering Cancer Center and performed in accordance with
the National Institutes of Health Guide for the Care and Use
of Laboratory Animals in an Association for Assessment and
Accreditation of Laboratory Animal Care accredited facility.
Methods
Chemicals
Drugs were obtained from the Research Technology branch
of the National Institute on Drug Abuse (Rockville, MD),
Tocris Bioscience (Bristol UK), or Cayman Chemical (Ann
Arbor, MI).[35S]-GTPγS and Na125I were purchased from
Perkin Elmer (Waltham, MA). Miscellaneous chemicals and
bufers were obtained from Sigma Aldrich (St. Louis, MO).
IBNtxA, IBNalA and 125I-BNtxA were synthesized and
structures were confrmed as previously described (Majum-
dar et al. 2011a, 2012). All non radioactive compounds were
dissolved in DMSO and diluted with water for conducting
assays. The fnal concentration of DMSO while conducting
the assay was<1%.
Receptor Binding Studies
125I-IBNtxA and 125I-labeled photoafnity ligand binding
assays were carried out in the homogenate of CHO cells sta-
bly expressing mMOR-1, mDOR-1, or mKOR-1 receptors,
prepared as described previously (Majumdar et al. 2011a),
with 3–10 µg protein in a volume of 1 mL of 50 mM KPO4
bufer, pH 7.4. Triple knockout mouse brain membrane
homogenates were prepared and binding carried out as previ-
ously described (Majumdar et al. 2011b) with 200 µg protein
in 1 mL of 50 mM KPO4 with 5 mM MgSO4 bufer. 6TM/
E11 competition binding assays using 125I-BNtxA (0.15 nM)
were carried out in whole brain membrane homogenates
(0.5 ml; 0.5 mg protein) at 25 °C in potassium phosphate
bufer (50 mM; pH 7.4) with magnesium sulfate (5 mM) for
90 min in the presence of CTAP, U50488H and DPDPE, all
at 200 nM, to block traditional opioid binding sites. Specifc
binding was defned as the diference between total bind-
ing and nonspecifc binding, determined in the presence of
levallorphan (8 μM) (). Ki values were determined by the
following the equation: Ki = (IC50)/(1+S) where S=(con-
centration of radioligand)/(K, of radioligand) according to
the previous report (Cheng and Prusof 1973; Chou 1974).
Cell Lines
Chinese hamster ovary (CHO-K1, ATCC® CCL-61™)
cells were bought from ATCC. SK-N Be(2)C cells (CCL-
2268) obtained from ATCC and CHO-K1 cells were main-
tained in F12 medium supplemented with 10% fetal bovine
serum and minimum Eagle’s medium supplemented with
10% fetal bovine serum and F12:nonessential amino acids,
respectively, in an atmosphere of 5% CO2 at 37 °C. The
mMOR-1 and mKOR-1 stable line generation used proto-
cols as described previously (Pan 2003). The cDNA frag-
ment containing the full-length mDOR-1 generated by
PCR using a mDOR-1/SKII plasmid as the template was
subcloned into pcDNA3.1-Hygro vector (Invitrogen), a
1 3

Cellular and Molecular Neurobiology
Nonspecifc binding was determined in the presence of
levallorphan (1 µM) and specifc binding is reported. For
competition and saturation assays, binding was carried out
for 90 min at 25 °C to reach equilibrium, then terminated by
fltration through Whatman GF/C Glass fber flters soaked
in 0.5% polyethyleneimine for at least 15 min prior to fl-
tration to minimize nonspecifc binding to the flters. Fil-
ters were then washed with 3 × 2 mL cold bufer (50 mM
Tris–HCl, pH 7.4), cut out, and counted on a Perkin Elmer
Wizard 1470 gamma counter.
by scintillation spectroscopy in a Tri-Carb 2900TR counter
(Perkin Elmer Life and Analytical Sciences). Basal binding
was determined in the presence of GDP and the absence
of drug. Data were normalized to 100 nM DAMGO for
mMOR-1. EC50 and %Emax values were calculated by non-
linear regression analysis (GraphPad Prism, San Diego, CA).
Photoafnity Labeling
The indicated 125I-labeled photoafnity ligand was incubated
with 0.5 mg-2 mg protein (mMOR-1) at a fnal concentration
of 0.3–0.5 nM in a volume of 2 mL 50 mM KPO4 or 50 mM
KPO4/5 mM MgSO4 bufer, pH 7.4, in microcentrifuge tubes
for 90 min at 25 °C to reach equilibrium. Tubes were then
centrifuged for 10 min at room temperature at 20,000×g to
separate free ligand and rapidly resuspended in 2 mL ice-
cold KPO4 bufer and transferred to an acrylic cuvette (Nüm-
brecht, Germany). Photolysis was initiated in a Rayonet
photoreactor (RPR-200, Southern New England Ultraviolet
Company, Branford CT) equipped with 13 RPR-3500 bulbs
for irradiation at 350 nm for 5 min. The photolysate was
then transferred back to a microcentrifuge tube, centrifuged
at 4 °C for 10 min, and the supernatant discarded. The pellet
was then solubilized at 4 °C with either denaturing (radio-
immuno precipitation assay bufer, (RIPA)) or nondenatur-
ing (0.2–0.25wt% MNG-3 in 1 × Tris bufered saline; Chae
et al. 2010) bufer with EDTA-free HALT protease inhibitor
cocktail (containing AEBSF HCl, Aprotinin, Bestatin, E-64,
Leupeptin, Pepstatin A; ThermoFisher Scientifc, Waltham,
MA). The solubilizate was then clarifed by centrifugation
for 10 min at 4 °C at 20,000×g.
Specifc Binding of IBzA
At mMOR-1, 7.5 µg total protein per tube, a maximum of
86.4% of total binding was specifc, decreasing to 73.6%
at the highest concentration shown as specific binding
approached saturation. At mKOR-1, 10 µg total protein per
tube, a maximum of 82.6% of total binding was specifc,
decreasing to 59.2% at the highest concentration shown as
specifc binding approached saturation.
Specifc Binding of Alkynyl‑IBzA.
At mMOR-1, 2.5 µg total protein per tube, a maximum of
62.1% of total binding was specifc, decreasing to 32.4%
at the highest concentration shown as specific binding
approached saturation. At mKOR-1, 7.5 µg total protein per
tube, a maximum of 62.4% of total binding decreasing to
39.8% at the highest concentration shown as specifc binding
approached saturation.
Dissociation assays were performed as above except that
after 90 min of binding to reach equilibrium, 10 µM leval-
lorphan was added at various time points up to 4 h later
to compete radioligand binding and all tubes fltered at the
same time. 10 µM levallorphan was used to defne nonspe-
cifc binding and specifc binding is reported.
The clarifed solubilizate was either directly run on a 10%
SDS-PAGE gel (BioRad, Berkeley, CA) at 300 V for 19 min
following incubation at room temperature for 30 min with
Laemmli sample bufer and 5% β-mercaptoethanol or sub-
jected to further purifcation. BioRad Precision Plus molecu-
lar weight markers were used, with MagicMark XP western
blot molecular weight markers. Gels were transferred to
PVDF in a BioRad Transblot semi-dry transfer device for
7 min at 2.5A and apposed to Koday Biomax MR flm with a
Kodak Biomax Intensifying Screen at − 80 °C for exposure
from 3 h to 8 days.
[35S]GTPγS Functional Assay
[35S]GTPγS binding was performed on membranes prepared
from stably opioid receptor transfected cells in the presence
and absence of the indicated compound for 60 min at 30 °C
in the assay bufer (50 mM Tris–HCl, pH 7.4, 3 mM MgCl2,
0.2 mM EGTA, and 10 mM NaCl) containing 0.05 nM [35S]
GTPγS; 2 μg/ml each leupeptin, pepstatin, aprotinin, and
bestatin; and 30 µM guanosine diphosphate (GDP), as pre-
viously described (Bolan et al. 2004). After the incubation,
the reaction was fltered through glass-fber flters (Whatman
Schleicher & Schuell, Keene, NH) and washed three times
with 3 mL of ice-cold bufer (50 mM Tris–HCl, pH 7.4)
on a semiautomatic cell harvester. Filters were transferred
into vials with 3 mL of Liquiscint (National Diagnostics,
Atlanta, GA), and the radioactivity in vials was determined
Wheat germ agglutinin (WGA) affinity purification
was performed according to the manufacturer’s proto-
col (Vector Labs, Burlingame, CA). Briefly, 25 µL of a
slurry of WGA-agarose beads was transferred to a micro-
centrifuge tube containing 20 volumes of nondenaturing
MNG-3 (maltose lauryl neopentyl glycol, Affymetrix,
Santa Clara, CA) buffer and washed 3 times with 20 vol-
umes of fresh non denaturing MNG-3 buffer solution. The
clarified solubilizate was added and rotated for 30 min at
room temperature before 3 washes with 30 volumes of
fresh nondenaturing buffer. The target was then eluted
1 3

Cellular and Molecular Neurobiology
twice using N-acetylglucosamine elution solution (Vector
Labs), run through a 7 kDa MWCO Zeba spin desalting
column equilibrated with nondenaturing buffer, and con-
centrated on a 10 kDa NMWL Amicon Ultra 0.5 spin con-
centrator cartridge to the desired final volume. The final
product was incubated at room temperature for 30 min
with Laemmli sample buffer and 5% β-mercaptoethanol
and separated by SDS-PAGE as above.
per manufacturer’s protocol, then transferred to PVDF and
apposed to flm with an intensifying screen as above.
Synthesis of compounds
All chemicals were purchased from Sigma Aldrich Chemi-
cals and Alfa Aesar, and were used without further puri-
fcation. Reaction mixtures were purifed by Silica Flash
chromatography on E. Merck 230–400 mesh silica gel 60
using a Teledyne ISCO CombiFlash Rf instrument with UV
detection at 280 nm and 254 nm. RediSep Rf silica gel nor-
mal phase columns were used with a gradient of MeOH in
DCM or EtOAc in hexanes. The yields reported are isolated
yields. IR spectra were recorded on a Bruker Optics Tensor
27 FTIR spectrometer with peaks reported in cm–1. NMR
spectra were recorded on Bruker Avance III 500, Avance III
600 with DCH CryoProbe instruments. NMR spectra were
processed with MestReNova software (ver. 6.1.1.). Chemi-
cal shifts are reported in parts per million (ppm) relative to
residual solvent peaks rounded to the nearest 0.01 for proton
and 0.1 for carbon (CDCl3 1H: 7.26, 13C: 77.1; CD3OD 1H:
3.31, 13C: 49.0; DMSO-d6 13C: 39.5). Peak multiplicity is
reported as follows: s—singlet, d—doublet, t—triplet, q—
quartet, m—multiplet. Coupling constants (J) are expressed
in Hz. Mass spectra were obtained at the MSKCC Analytical
Core Facility on a Waters Acuity SQD LC–MS by electro-
spray (ESI) ionization. High resolution mass spectra were
obtained on a Waters Acuity Premiere XE TOF LC–MS by
electrospray ionization. Accurate masses are reported for
the molecular ion [M+H]+. HPLC: Waters 1525 Binary
Pump, Waters 2489 UV/vis Detector, Waters XBridge C18
column (5 μm × 150 × 4.6 mm), mobile phase: solvent A:
water with 0.1% TFA; aolvent B: acetonitrile with 0.1%
TFA. Gradient: 5–95% acetonitrile/water. Flow rate: 1 ml/
min. A reversed-phase HPLC using a Perkin Elmer LC pump
series 200 and a 785A UV/vis detector (214 nM) was used.
A Varian microsorb MV 100 − 5 reversed-phase column
(5 μm × 4.6 mm × 250 mm) with the mobile phases being
0.1% TFA in water and 0.1% TFA in ACN with a gradient
elution at a fow rate of 1 mL/min was used.
Immunoafnity purifcation was also performed under
both denaturing and nondenaturing conditions, accord-
ing to manufacturer’s protocols. Solubilizate was incu-
bated with rabbit polyclonal antibody directed against the
C-terminal tail of the mu opioid receptor (C-20, Santa
Cruz Biotechnology, Dallas, TX) according to the manu-
facturer’s suggested dilution relative to the protein added,
rotating at 4 °C for 1 h—overnight. Antibody–receptor
complexes were then pulled down with Protein A-agarose
(EzView Red, Sigma Aldrich) for 1–2 h and equilibrated
according to manufacturer’s protocols. Beads were washed
2–3 times with 30 volumes bufer until no radioactiv-
ity could be detected in the wash with a survey meter.
For denaturing conditions, beads were eluted for 30 min
with 6.5 M urea, 150 mM DTT, and 1 × Laemmli sample
bufer at room temperature and separated by SDS-PAGE
as above. For native conditions, beads were eluted 2 times
with 100 µg/mL MOR C-20 blocking peptide (Santa Cruz
Biotechnology) for 30 min at room temperature. The elu-
ate was then concentrated in a 100 kDa NMWL Millipore
Biomax spin concentrator.
Blue Native PAGE
Blue native polyacrylamide gel electrophoresis was per-
formed according to manufacturer’s protocols (Life Tech-
nologies, Carlsbad, CA), based on the method of Schägger
and von Jagow. Briefy, samples were prepared by adding
glycerol (10% fnal concentration) and Coomassie Brilliant
Blue G-250 (0.05–0.0625% fnal concentration). Samples
were then loaded onto a 4–16% native gel (Life Technolo-
gies) and separated for 105–115 min at 150 V. Gels for
imaging were destained in 40% MeOH/10% Acetic Acid
solution for 15 min, followed by 8% Acetic acid for 1hour,
then imaged on a Chemidoc MP (BioRad). Gels for auto-
radiography were transferred to PVDF and apposed to flm
as above.
Ethyl 4‑amino‑3‑iodobenzoate (2)
Concentrated sulfuric acid (a catalytic amount, one drop)
was added to a stirred solution of ethyl 4-amino benzo-
ate 1 (1 g, 6.05 mmol) dissolved in acetonitrile (17 mL) at
room temperature. N-iodosuccinimide (1.5 g 6.66 mmol)
was added to the reaction mixture. After stirring for 2 h
at rt, the reaction mixture was quenched with phosphate
bufer (pH7.4, 17 mL). Then, the product was extracted in
EtOAc (50 mL). The EtOAc layer was dried over anhydrous
Na2SO4, fltered, concentrated under reduced pressure; and
dried under high vacuum to get desired product 2 (1.38 g;
2D Blue Native/SDS-PAGE was performed by excising
a lane from a BN-PAGE gel and incubating at room tem-
perature in 1% SDS/1% β-mercaptoethanol with rocking for
30 min. The highest molecular weight portion of the lane
was trimmed to make a 6 cm strip for insertion into a Life
Technologies NuPage Novex 12% Bis–Tris gel with a 2D
well. Gels were run with MOPS-SDS bufer for 1 h at 200 V
1 3

Cellular and Molecular Neurobiology
yield 79%). 1H NMR (500 MHz, CDCl3): δ=8.32 (s, 1H),
7.81 (d, J=5.1 Hz, 1H), 6.69 (d, J=4.2 Hz, 1H), 4.51 ( br,
2H), 4.31 ( m, 2H), 1.36 ( t, J=7.1 Hz, 3H).
with 5%EtOAc in hexanes (15 mL); and the mixture was
fltered through celite. The fltrate was concentrated under
reduced pressure and the residue was purifed by silica col-
umn chromatography (ISCO, 4 g) using 0–1% EtOAc in
hexanes. The desired product fractions (26–27 tubes) were
combined, concentrated under reduced pressure; and dried
under high vacuum to get a colorless oil 5 (18 mg; yield
25%). 1H NMR (500 MHz, CDCl3): δ=8.07 (s, 1H), 8.01
(d, J=5.1 Hz, 1H), 7.16 (d, J=4.1 Hz, 1H), 4.38–4.34 (m,
2H), 1.54–1.40 (m, 6H), 1.39–1.30 (m, 10H), 1.13–1.09 (m,
5H), 0.2–0.87 (m 9H).
Ethyl 4‑azido‑3‑iodobenzoate (3)
Concentrated hydrochloric acid (4 mL, 12 M) was added
drop wise to the stirred solution of ethyl amino benzoate 2
(1.1 g, 3.8 mmol) in MeOAc (12 mL) at rt. over a period of
2 min. The reaction mixture was cooled to 4 °C. Aqueous
solution (2.5 mL) of NaNO2 (780 mg, 11.3 mmol) was added
to the mixture and the reaction continued for 45 min in dark
(covered with an aluminum foil) at the temperature. Then,
NaN3 (784 mg, 12.1 mmol) was added to the reaction mix-
ture in portions for period of 15 min. After stirring the reac-
tion mixture for additional 15 min, the reaction mixture was
quenched with phosphate bufer (pH 7, 25 mL). Then, the
product was extracted in EtOAc (50 mL). The EtOAc layer
was dried over anhydrous Na2SO4, fltered, concentrated
under reduced pressure; and dried under high vacuum to get
desired product 3 (1.0 g; yield 83%). 1H NMR (500 MHz,
CDCl3): δ=8.45 (s, 1H), 8.05 (d, J=5.1 Hz, 1H), 7.16 (d,
J=4.1 Hz, 1H), 4.36 ( m, 2H), 1.39 ( t, J=7.1 Hz, 3H).
4‑Azido‑3‑(tributylstannyl)benzoic Acid (6)
Aqueous potassium hydroxide (0.03 mmol, 200 µL) was
added to the stirred solution of tributyl tin azido ethyl ester
5 (10 mg, 0.02 mmol) dissolved in MeOH (1 mL) at rt. The
reaction mixture was heated to 60 °C overnight. TLC indi-
cated the completion of ester hydrolysis. Then, the reaction
mixture was cooled to rt and concentrated under the reduced
pressure. Then, the residue was diluted with DCM (20 mL),
and saturated solution of NH4Cl (10 mL) was added into
it. The DCM layer was separated and was washed with
brine (10 mL), dried over anhydrous Na2SO4; and fltered.
The fltrate was concentrated under reduced pressure and
dried under high vacuum to get colorless oil 6 (7 mg; yield
77%).1H NMR (500 MHz, CDCl3): δ=8.13 (s, 1H), 8.08 (d,
J=4.1 Hz, 1H), 7.19 (d, J=4.1 Hz, 1H), 1.15–1.34 (m, 5H),
1.32–1.29 (m, 8H), 1.14–1.11 (m, 5H), 0.93–0.87 (m, 9H).
4‑Azido‑3‑iodobenzoic acid (4)
Aqueous solution (500 µL) of lithium hydroxide (22 mg,
0.94 mmol) was added to the stirred solution of ethyl ester 3
(100 mg, 0.31 mmol) dissolved in MeOH: THF (1:1) mixture
(1 mL) at rt. The reaction mixture was continued overnight.
TLC indicated the completion of ester hydrolysis. Then, the
reaction mixture was concentrated under reduced pressure
to remove organic solvents and the remaining content was
diluted with water (5 mL). The aqueous layer was acidifed
(pH3) with dilute HCl and diluted with brine (10 mL). Then,
the product was extracted in DCM (2×10 mL), the DCM
layer was dried over anhydrous Na2SO4, and fltered. The
fltrate was concentrated under reduced pressure and dried
under high vacuum to get brown solid 4 (89 mg; quanti-
tative yield; used without further purifcation for the next
N‑((4R,4aS,7R,7aR,12bS)‑3‑Allyl‑4a,9‑dihydroxy‑2,3,4,4a,
5,6,7,7a‑octahydro‑1H‑4,12‑methanobenzofuro[3,2‑e]
isoquinolin‑7‑yl)‑4‑azido‑3‑(tributylstannyl)benzamide (9)
DIPEA (27.0 µL, 0.154 mmol) and ally amine 8 (10.0 mg,
0.030 mmol) were added to the stirred solution of azide acid
6 (14.0 mg, 0.030 mmol) dissolved in DMF (700 µL) at rt
under Argon atmosphere. The reaction mixture was cooled
to 0 °C and HATU (11.7 mg, 0.030 mmol) was added to the
reaction mixture. After stirring the reaction mixture for 3 h
at 0 °C to rt., the reaction mixture was poured into EtOAc
(20 mL) and was washed with brine (7×15 mL). The EtOAc
layer was dried over Na2SO4, fltered; and concentrated
under reduced pressure. Then, the residue was redissolved
in MeOH (2 mL), and treated with NaOMe in methanol
(0.5 M, 150 µL), for one hour at rt. Finally, the reaction
mixture was concentrated under reduced pressure, and the
residue was purifed by silica gel column chromatography
(ISCO, 4 g) using 5–10% MeOH in DCM. The desired prod-
uct fractions were concentrated under reduced pressure and
dried under high vacuum to get desired product 9 (10 mg;
1
step). H NMR (500 MHz, CDCl3): δ = 8.52 (s, 1H), 8.11
(d, J=5.1 Hz, 1H), 7.20 (d, J=4.1 Hz, 1H).
Ethyl 4‑azido‑3‑(tributylstannyl)benzoate (5)
Bis(tributyltin) (320 µL, 0.63 mmol) was added to the stirred
solution of 3-iodo 4-azido ethyl ester 4 (50 mg, 0.15 mmol)
dissolved in dry dioxane (2 mL) under argon atmosphere at
rt. Pd (PPh3)4 (18 mg, 0.015 mmol) was added to the mixture
at rt; and the reaction mixture was heated to refux overnight.
TLC (using silica plate with developing solvent 1% EtOAc
in hexanes) indicated the complete consumption of starting
material. Then, the reaction mixture was cooled to rt, diluted
1
Yield 45%); H NMR (600 MHz, CDCl3) δ 7.97–7.75
(m, 2H), 7.32 (s, 1H), 7.16 (d, J = 8.1 Hz, 1H), 6.75 (d,
1 3

Cellular and Molecular Neurobiology
J=8.1 Hz, 1H), 6.59 (d, J=8.1 Hz, 1H), 5.81 (s, 1H), 5.21
(d, J=25.5 Hz, 2H), 4.52 (s, 1H), 4.29 (s, 1H), 3.21–3.06
(m, 3H), 2.97 (s, 1H), 2.70–2.53 (m, 2H), 2.27–2.21 (m,
2H), 1.66 (d, J=33.9 Hz, 4H), 1.57–1.47 (m, 6H), 1.37–1.27
2.29 mmol, 0.05 eq.) in dry DCM (190 mL) was added and
38 °C under Ar for 16 h. The cooling to rt, the mixture was
fltered and washed with cold DCM, then water. The solid
was dissolved in a mixture of water and conc. aq. HCl (7:
1, 480 mL) at 40 °C. The aqueous layer was washed with
DCM, then it was basifed to pH ~ 9 with ammonia. The
resulting solid was fltered and dried under high vacuum to
(m, 6H), 1.16–1.10 (m, 6H), 0.89 (t, J=7.4 Hz, 12H). 13
C
NMR (151 MHz, CDCl3) δ 149.25, 143.60, 136.34, 135.07,
130.54, 129.10, 119.36, 116.80, 93.14, 70.32, 62.59, 60.57,
57.93, 49.61, 47.20, 38.77, 31.75, 29.17, 27.47, 22.82, 14.29,
13.89, 10.36. HRMS: m/z calcd for C38H53N5O4Sn [M+H]+:
764.3198; found 764.3212; purity>95% by HPLC.
1
yield 11 as a light orange solid (10.98 g; Yield: 83%). H
NMR (600 MHz, Methanol-d4) δ 6.65 (d, J=8.1 Hz, 1H),
6.62 (d, J=8.0 Hz, 1H), 4.69 (s, 1H), 3.13 (d, J=6.1 Hz,
1H), 3.10–2.98 (m, 3H), 2.80 (dd, J=13.4, 4.5 Hz, 1H), 2.57
(td, J = 13.0, 3.6 Hz, 1H), 2.50 (td, J = 12.4, 4.6 Hz, 1H),
2.26–2.18 (m, 1H), 1.88–1.77 (m, 1H), 1.62 (td, J = 14.2,
3.4 Hz, 1H), 1.39 (dd, J=12.3, 3.5 Hz, 1H).
N‑((4R,4aS,7R,7aR,12bS)‑3‑Allyl‑4a,9‑dihydroxy‑2,3,4,4a,
5,6,7,7a‑octahydro‑1H‑4,12‑methanobenzofuro[3,2‑e]
isoquinolin‑7‑yl)‑4‑azido‑3‑iodobenzamide (10)
DIPEA (11.2 µL, 0.064 mmol) and allyl amine 8 (7.0 mg,
0.021 mmol) were added to the stirred solution of azide
acid 4 (12.3 mg, 0.042 mmol) dissolved in DMF (700 µL)
at rt under an argon atmosphere. The reaction mixture was
cooled to 0 °C and HATU (16.3 mg, 0.064 mmol) was added
to the reaction mixture. After stirring the reaction mixture
for 2 h at 0 °C to rt., the reaction mixture was poured into
EtOAc (20 mL) and was washed with brine (7× 15 mL).
The EtOAc layer was dried over Na2SO4, fltered; and con-
centrated under reduced pressure. Then, the residue was
redissolved in MeOH (2 mL), and treated with NaOMe in
methanol (0.5 M, 150 µL), for one hour at rt. Finally, the
reaction mixture was concentrated under reduced pressure,
and the residue was purifed by silica gel column chroma-
tography (ISCO, 4 g) using 5–10% MeOH in DCM. The
desired product fractions were concentrated under reduced
pressure and dried under high vacuum to get desired prod-
uct 10 (8.0 mg; Yield 64%); 1H NMR (600 MHz, CDCl3)
δ=7.84 (dd, J=8.3, 2.0 Hz, 1H), 7.44 (d, J=8.9 Hz, 1H),
7.09 (d, J = 8.3 Hz, 1H), 6.61 (dd, J = 57.6, 8.1 Hz, 2H),
5.90–5.69 (m, 1H), 5.25–5.11 (m, 2H), 4.64 (d, J=5.9 Hz,
1H), 4.06–4.03 (m, 1H), 3.18–3.07 (m, 3H), 2.97 (q, J=8.4,
7.0 Hz, 1H), 2.67–2.51 (m, 2H), 2.19 (d, J= 7.4 Hz, 2H),
1.99–1.89 (m, 1H), 1.69–1.65 (m, 1H), 1.62–1.56 (m, 1H),
1.52–1.41 (m, 2H), 1.29–1.21 (m, 1H). 13C NMR (151 MHz,
CDCl3) δ=142.82, 139.08, 132.29, 130.67, 128.61, 119.50,
117.99, 117.72, 92.68, 87.59, 70.48, 62.53, 57.92, 50.91,
47.39, 43.73, 31.59, 29.35, 23.45, 22.85, 14.35. HRMS: m/z
calcd for C26H26N5O4I [M+H]+: 600.1108; found 600.1084;
purity>97% by HPLC.
(4R,4aS,7aR,12bS)‑4a,9‑Dihy‑
droxy‑3‑(prop‑2‑yn‑1‑yl)‑2,3,4,4a,5,6‑hexa‑
hydro‑1H‑4,12‑methanobenzofuro[3,2‑e]isoquino‑
lin‑7(7aH)‑one (12)
Sodium carbonate (370.4 mg, 3.49 mmol) and propargyl
bromide (389 µL, 3.49 mmol, 80% in toluene) were added
to a stirred solution of noroxymorphone 11 (1 g, 3.48 mmol)
dissolved in anhydrous DMF (5 mL) at rt. under an argon
atmosphere. The reaction mixture was heated to 90 °C
overnight. Mass spectrometry of the crude reaction mixture
indicated the completion of the reaction. The solvent was
removed under reduced pressure and the residue was redis-
solved in EtOAc (50 mL). The EtOAc layer was washed
with brine (5 ×30 mL), dried over anhydrous Na2SO4, fl-
tered, and concentrated under reduced pressure. The crude
product was purifed by silica gel column chromatography
(ISCO-combifash) using 1–5% MeOH in DCM. The desired
product fractions were concentrated under reduced pressure
and dried under high vacuum to obtain of-white solid 12
(897 mg; yield 78%). 1H NMR (600 MHz, CDCl3 +MeOH)
δ=7.92–7.76 (m, 1H), 7.26 (dd, J=8.5, 4.4 Hz, 1H), 7.15 (t,
J=7.2 Hz, 1H), 5.25–5.06 (m, 1H), 3.95 (dd, J=8.3, 4.0 Hz,
2H), 3.80–3.65 (m, 2H), 3.65–3.49 (m, 1H), 3.25–3.10 (m,
2H), 2.99–2.78 (m, 4H), 2.54–2.39 (m, 1H), 2.20–2.12 (m,
2H).
(4R,4aS,7S,7aR,12bS)‑3‑(Prop‑2‑yn‑1‑yl)‑1,2,3,4,5,6,7,7a
‑octahydro‑4aH‑4,12‑methanobenzofuro[3,2‑e]isoquino‑
line‑4a,7,9‑triol (13)
(4R,4aS,7aR,12bS)‑4a,9‑Dihydroxy‑2,3,4,4a,5,6‑hex‑
ahydro‑1H‑4,12‑methanobenzofuro[3,2‑e]isoquino‑
lin‑7(7aH)‑one (11)
K-Selectride (5.2 mL, 5.22 mmol, 1 M in THF) was
slowly added to a stirred solution of ketone 12 (850 mg,
2.61 mmol) dissolved in anhydrous THF (20 mL) at 0 °C
under an argon atmosphere and the reaction was continued
for 30 min at the temperature. Then, the reaction mix-
ture was warmed to rt. and continued overnight. Mass
spectrometry of the crude reaction mixture indicated the
Dissolved naloxone (7, 15 g, 45.82 mmol) in dry DCM
(110 mL), then a solution of N,N-dimethyl barbituric
acid (4.29 g, 27.49 mmol, 0.6 eq.) and Pd(PPh3)4 (2.65 g,
1 3

Cellular and Molecular Neurobiology
completion of the reaction. The reaction mixture was
cooled to 0 °C and water (10 mL) was slowly added to
quench the reaction. The reaction mixture was stirred
for 10 min at rt. Then, the solvent was removed under
reduced pressure and the remaining content was diluted
with brine (30 mL). The aqueous layer was extracted with
DCM (5 × 30 mL). The combined DCM layer was dried
over anhydrous Na2SO4, fltered, and concentrated under
reduced pressure. The crude product was purifed by sil-
ica gel column chromatography (ISCO-combifash) using
1–5% MeOH in DCM. The desired product fractions were
combined, concentrated under reduced pressure; and dried
under high vacuum to obtain white solid 13 (730 mg; yield
85%). 1H NMR (600 MHz, CDCl3) δ = 6.71 (d, J = 8.1 Hz,
1H), 6.55 (d, J=8.2 Hz, 1H), 4.67 (d, J=4.6 Hz, 1H), 4.26
(d, J = 11.2 Hz, 1H), 3.34 ( d, J = 3.3 Hz, 2H), 3.22–2.97
(m, 2H), 2.72–2.56 (m, 2H), 2.38 (td, J = 12.1, 3.7 Hz,
1H), 2.27 (t, J = 2.5 Hz, 1H), 1.79–1.56 (m, 3H), 1.46
(ddd, J = 14.6, 8.6, 3.2 Hz, 1H), 1.21–1.03 (m, 1H).
(d, J = 8.3 Hz, 1H), 4.70 (s, 2H), 4.06 (ddd, J = 12.9, 8.3,
4.4 Hz, 1H), 3.39 (s, 2H) 3.24–3.10 (m, 2H), 2.81 (dd,
J = 13.3, 2.8 Hz, 1H), 2.74–2.58 (m, 2H), 2.42–2.21 (m,
3H), 1.79–1.68 (m, 1H), 1.54–1.41 (m, 3H).
(4R,4aS,7R,7aR,12bS)‑7‑Amino‑3‑(prop‑2‑yn‑1‑yl)‑1,2,3,
4,5,6,7,7a‑octahydro‑4aH‑4,12‑methanobenzofuro[3,2‑e]
isoquinoline‑4a,9‑diol (15)
The cis-2-penten-1-ol (2.2 mL, 21.03 mmol) and hydra-
zine hydrate (819 µl, 13.15 mmol; 50% hydrazine solution)
were added to a stirred solution of opiate phthalimides 14
(600 mg, 1.34 mmol) dissolved in MeOH (16 mL) at rt. and
the reaction was continued overnight. Mass spectrometry
of the crude reaction mixture indicated the completion of
the reaction. The volatilities were removed under reduced
pressure and the reaction mixture was treated with dilute
acetic acid (20 mL, 1.5 M) and fltered. The fltrate was
basifed (pH10) with aqueous ammonia (10% NH4OH), and
the mixture was extracted with DCM (3×20 mL). The com-
bined DCM layer was dried over anhydrous Na2SO4, fltered,
and concentrated under reduced pressure. The crude product
was purifed by silica gel column chromatography (ISCO-
combifash) using 5–15% MeOH in DCM. The desired prod-
uct fractions were combined, concentrated under reduced
pressure; and dried under high vacuum to obtain white solid
2‑((4R,4aS,7R,7aR,12bS)‑4a,9‑Dihydroxy‑3‑(prop‑2‑yn‑1
‑yl)‑2,3,4,4a,5,6,7,7a‑octahydro‑1H‑4,12‑methanobenzo‑
furo[3,2‑e]isoquinolin‑7‑yl)isoindoline‑1,3‑dione (14)
Phthalimide (594.7 mg, 4.04 mmol) and triphenylphos-
phine (1.06 g, 4.04 mmol) were added to a stirred solution
of opiate alcohol 13 (661 mg, 2.02 mmol) dissolved in
anhydrous THF (16 mL) at rt. under an argon atmosphere.
A solution of diisopropyl azodicarboxylate (795.8 μl,
4.042 mmol) in toluene (1.5 mL) was added drop wise to
the reaction mixture and the reaction was continued over-
night. Water (3 mL) was added to the reaction mixture and
the resulting in mixture was stirred for 10 more minutes.
The volatilities were removed under reduced pressure.
Aqueous citric acid (2%, 18 mL) and dilute HCl ( 0.1 M,
6 mL) were added to the crude mixture and the result-
ing aqueous layer was washed with EtOAc (3 × 20 mL)
to remove excess of reagent and the by other products.
Then, the aqueous layer was basifed (pH10) with aqueous
ammonia (10% NH4OH), and the mixture was extracted
with DCM (3 × 20 mL). The combined DCM layer was
dried over anhydrous Na2SO4, fltered, and concentrated
under reduced pressure. The crude product was purifed
by silica gel column chromatography (ISCO-combifash,
220 g) using 20% hexanes in EtOAc (six column volume;
to elute impurities) and then with 3–10% MeOH in DCM
to elute the desired product. The desired product fractions
were combined, concentrated under reduced pressure;
and dried under high vacuum to obtain of-white solid 14
(646 mg; yield 70%). 1H NMR (600 MHz, CDCl3) δ=7.85
(dd, J = 5.4, 3.0 Hz, 2H), 7.72 (dd, J = 5.5, 3.0 Hz, 2H),
6.77 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H), 5.18
1
15 (353.8 mg; Yield 82%). H NMR (600 MHz, CDCl3)
δ=6.63 (d, J=8.1 Hz, 1H), 6.56 (s, 1H), 4.26 (d, J=7.0 Hz,
1H), 3.35 (d, J=2.5 Hz, 2H), 3.16–3.00 (m, 2H), 2.67–2.52
(m, 3H), 2.31–2.09 (m, 3H), 1.79 (tt, J=9.7, 4.6 Hz, 2H),
1.62 (dt, J=13.3, 3.3 Hz, 1H), 1.46–1.34 (m, 2H).
4‑Azido‑N‑((4aS,7R,12bS)‑4a,9‑dihydroxy‑3‑(prop‑2‑y
n‑1‑yl)‑2,3,4,4a,5,6,7,7a‑octahydro‑1H‑4,12‑methano‑
benzofuro[3,2‑e]isoquinolin‑7‑yl)‑3‑(tributylstannyl)
benzamide (16)
DIPEA (8.0 µL, 0.046 mmol) and alkyne amine 5 (5.5 mg,
0.017 mmol) were added to the stirred solution of azide
acid 6 (7 mg, 0.015 mmol) dissolved in DMF (300 µL) at
rt under an argon atmosphere. The reaction mixture was
cooled to 0 °C and HATU (6.4 mg, 0.017 mmol) was added
to the reaction mixture. After stirring the reaction mixture
for 2 h at 0 °C to rt., the reaction mixture was poured into
EtOAc (20 mL) and was washed with brine (5 × 15 mL).
The EtOAc layer was dried over Na2SO4, fltered; and con-
centrated under reduced pressure. Then, the residue was
redissolved in MeOH (2 mL), and treated with NaOMe in
methanol (0.5 M, 150 µL), for one hour at rt. Finally, the
reaction mixture was concentrated under reduced pressure,
and the residue was purifed by silica gel column chromatog-
raphy (ISCO, 4 g) using 0–5% MeOH in DCM. The desired
product fractions (21–22 tubes) were concentrated under
1 3

Cellular and Molecular Neurobiology
reduced pressure and dried under high vacuum to get color-
less viscous oil 16 (5.1 mg; Yield 45%); 1H NMR (600 MHz,
CDCl3) δ 7.85–7.80 (m, 2H), 7.16 (d, J=8.2 Hz, 1H), 6.75
(d, J=8.1 Hz, 1H), 6.59 (d, J=8.1 Hz, 1H), 5.34 (s, 1H),
5.02 (s, 1H), 4.53 (d, J=5.0 Hz, 1H), 4.28 (d, J=4.8 Hz,
1H), 3.42–3.31 (m, 2H), 3.18 (d, J=18.0 Hz, 2H), 2.74–2.61
(m, 2H), 2.39–2.20 (m, 3H), 1.84 (s, 1H), 1.67 (s, 2H),
1.60–1.49 (m, 7H), 1.33 (dt, J=14.6, 7.3 Hz, 7H), 1.15–1.08
(m, 6H), 0.89 (t, J = 7.3 Hz, 9H). 13C NMR (151 MHz,
CDCl3) δ=166.62, 149.26, 143.60, 135.10, 130.54, 129.08,
119.40, 117.69, 116.80, 93.12, 70.40, 62.64, 49.65, 47.05,
44.25, 29.24, 27.48, 23.05, 13.90, 10.36. HRMS: m/z calcd
for C38H51N5O4Sn [M+H]+: 762.3041; found 762.3058;
purity>99% by HPLC.
Results and Discussion
Chemistry
The design of new radioiodinated photoafnity labels, 10
and 17 is based on the potent opioid analgesic compounds
IBNtxA and its allyl analog IBNalA (Fig. 1) that we had
reported before (Majumdar et al. 2012). In case of IBNalA
analog, the N-cyclopropyl methyl (CPM) group of IBNtxA
was swapped with a N-allyl group commonly seen in the
naloxone scafold. This small change in structure led to
greater selectivity in binding assays for mMOR-1 as well
as 6TM/E11 over mDOR-1 when compared to IBNtxA
(see Table 1).
We designed our afnity label namely IBzA 10, incor-
porating an azido group ortho to the 3′-iodo group of
IBNalA and alkynyl-IBzA 17, where the N-allyl group
of 10 is substituted with a N-alkynyl group so that it is
available for click chemistry approaches later on for fol-
low up work.
4‑Azido‑N‑((4R,4aS,7R,7aR,12bS)‑4a,9‑dihydroxy‑3‑(prop‑
2‑yn‑1‑yl)‑2,3,4,4a,5,6,7,7a‑octahydro‑1H‑4,12‑methanobe
nzofuro[3,2‑e]isoquinolin‑7‑yl)‑3‑iodobenzamide (17)
DIPEA (11.2 µL, 0.064 mmol) and alkyne amine 15 (7.0 mg,
0.021 mmol) were added to the stirred solution of azide acid
4 (12.3 mg, 0.042 mmol) dissolved in DMF (700 µL) at rt
under an argon atmosphere. The reaction mixture was cooled
to 0 °C and HATU (16.3 mg, 0.064 mmol) was added to the
reaction mixture. After stirring the reaction mixture for 2 h
at 0 °C to rt., the reaction mixture was poured into EtOAc
(20 mL) and was washed with brine (7×15 mL). The EtOAc
layer was dried over Na2SO4, fltered; and concentrated
under reduced pressure. Then, the residue was redissolved in
MeOH (2 mL), and treated with NaOMe in methanol (0.5 M,
150 µL), for one hour at rt. Finally, the reaction mixture
was concentrated under reduced pressure, and the residue
was purifed by silica gel column chromatography (ISCO,
4 g) using 4–10% MeOH in DCM. The desired product frac-
tions were concentrated under reduced pressure and dried
under high vacuum to get desired product 17 (8.2 mg; yield
66%); 1H NMR (600 MHz, CDCl3) δ=7.84 (d, J=8.3, Hz,
1H), 7.38 (d, J=8.9 Hz, 1H), 7.17–7.03 (m, 1H), 6.67 (d,
J=8.1 Hz, 1H), 6.57 (d, J=8.2 Hz, 1H), 6.21 (br, 1H), 5.04
(br, 1H), 4.63 (d, J=5.9 Hz, 1H), 4.06 (dt, J=9.2, 4.9 Hz,
1H), 3.37 (d, J=2.4 Hz, 2H), 3.27–3.07 (m, 2H), 2.74–2.56
(m, 2H), 2.40–2.14 (m, 4H), 1.94 (dt, J=14.2, 4.8 Hz, 1H),
1.71 (dt, J=13.9, 5.1 Hz, 1H), 1.64–1.57 (m, 1H), 1.57–1.51
(m, 1H), 1.50–1.45 (m, 1H). 13C NMR (151 MHz, CDCl3)
δ=144.79, 142.87, 139.55, 139.07, 132.28, 130.51, 128.59,
119.54, 118.00, 117.79, 92.69, 87.60, 77.37, 73.45, 70.53,
62.58, 50.82, 47.21, 31.40, 29.27, 23.39, 23.16. HRMS: m/z
calcd for C26H24N5O4I [M+H]+: 598.0951; found 598.0937;
purity>95% by HPLC.
The synthesis of the azido aryl photoaffinity opioid
probes, namely IBzA 10, alkynyl-IBzA 17 and the stan-
nylated precursors to the corresponding radiolabelled 125I
compounds 18 and 19 was proceeded with naloxone as a
starting material (Scheme 1). 4-Azido-3-iodobenzoic acid
(4) and 4-azido-3-(tributylstannyl)benzoic acid (6) were
prepared from ethyl-4-amino benzoate 1. Iodination of 1
with N-iodosuccinimide and subsequent diazotization of
the amine 2 in acidic conditions followed by nucleophilic
substitution with an azide resulted in ethyl 4-azido-3-iodo-
benzoate 3. The 3-iodo functionality in 3 was converted to
tributylstannyl moiety in 5 using a palladium catalyst. Alka-
line hydrolysis of esters 3 and 5 furnished the correspond-
ing acids 4 and 6, respectively. Naloxone (7) was used as a
starting material to prepare azido-opiate probes (9, 10, 16,
17) (Scheme 1). First, 7 was converted to β-naloxamine (8)
using our published method (Majumdar et al. 2011a). Deal-
lylation of 7 using a Pd(PPh3)4 (Giguere et al. 2012) was
employed to obtain noroxymorphone (11). Compound 11
was in turn converted to the N-propargylamine analog 15 in
four steps. N-alkylation of 11 with propargyl bromide was
followed by stereoselective reduction of the ketone using
K-Selectride to yield the α-alcohol 13 (Brossi et al. 1982).
The Mitsunobu reaction was employed to convert α-alcohol
13 to β-phthalimide 14 (Váradi et al. 2015). Then, removal
of the phthalimide moiety in 14 using hydrazine hydrate in
methanol gave the desired N-propargyl β–amine 15. Finally,
coupling of N-propargyl β–amine 15 or β-naloxamine 8 with
acids 4 or 6 using HATU as a coupling reagent aforded the
desired azidoaryl probes 9, 10, 16, 17 and. Then, the reaction
of the tributyltin precursors 9 or 16 with chloramine-T and
Na125I and their semi-preparative purifcation using reverse
1 3

Cellular and Molecular Neurobiology
Fig. 1 Chemical structures of
IBNtxA, IBNalA and photoaf-
fnity ligands synthesized (IBzA
10, alkynyl-IBzA 17)
Table 1 Radioligand ligand
Compound
mMOR-1
mDOR-1
mKOR-1
Triple KO
6TM/E11
binding (Ki, nM) assay
IBzA 10
0.48 0.02
0.25 0.12
0.11 0.02
0.22 0.12
8.3 0.7
3.5 0.4
0.24 0.1
2.55 0.18
0.70 0.20
0.92 0.29
0.03 0.001
0.08 0.06
4.1 [3.5–6.4]
0.89 [0.71–1.1]
0.12 0.04
nd
4.64 0.2
11.97 1.63
0.16 0.04
0.25 0.12
Alkynyl-IBzA 17
IBNtxAa
IBNalAa
Each photoafnity ligand with increasing concentrations was incubated with [125I]-IBNtxA and membranes
homogenates from either CHO cells stably expressing mMOR-1, mDOR-1, or mKOR-1, or 6TM/E11 assay
(with mMOR-1, mKOR-1 and mDOR-1 blockers added to mouse brain) or triple knockout mouse brain.
Results are from at least 3 independent replications and are presented as mean SEM except for TKO
mouse brain which is a single determination reported as Ki [95% Confdence interval]
nd not determined
aValues from literature (Majumdar et al. 2012)
phase HPLC by following the literature procedure aforded
Pharmacology
[
125I]-IBzA 18 and [125I]-Alkynyl-IBzA 19 (Majumdar et al.
2011a).
Both drugs IBzA 10 and Alkynyl-IBzA 17 displayed a high
binding afnity for mMOR-1 and mKOR-1 (mouse kappa
opioid receptor transfected in CHO cells) in competition
assays, however with a slightly lower afnity for mDOR-1
(mouse delta opioid receptors transfected in CHO cells) by
about 10- to 15-fold (Table 1). This kind of selectivity for
mMOR-1, mKOR-1 and 6TM/E11 over the mDOR-1 was
Two aryl azide photoaffinity labels were synthe-
sized by amide bond coupling of 4-azido-3-iodobenzoic
acid and 6-β-naloxonamine (IBzA) or N-propargyl-6-β-
nornaloxonamine (Alkynyl-IBzA) and their structures vali-
dated (Fig. 1).
1 3

Cellular and Molecular Neurobiology
I
)
NH2
NH2
N3
N3
N3
N3
I
I
I
SnBu3
SnBu3
a
b
c
e
79%
83%
100%
77%
1
2
3
4
5
6
O
OEt
O
OEt
O
OEt
O
OH
O
OEt
O
OH
d
25%
HO
O
OH
6
, f
O
II
)
N
64%
Bu3Sn
N3
HO
HO
N
H
9
i-k
O
OH
O
OH
HO
N
N
4
, f
H2N
O
O
OH
8
45%
7
O
N
I
N
H
N3
10
III
)
HO
HO
HO
HO
HO
g
h
i
j
k
O
OH
O
OH
NH
O
OH
O
OH
O
OH
7
83%
78%
85%
70%
82%
O
N
N
N
N
H2N
O
O
HO
N
11
12
13
14
15
O
HO
IV
V
)
)
HO
HO
O
OH
6
, f
O
N
O
OH
O
OH
45%
l
Bu3Sn
N3
N
O
O
H
N
N
Bu3Sn
N3
125I
N3
16
R
R
N
H
N
H
15
HO
9
18;
R = allyl
R = allyl
18%
19; 30 %
4
, f
O
OH
16
R = propargyl
R = propargyl
O
66%
N
I
N
H
N3
17
Scheme 1 Reagents and conditions: (a) N-iodosuccinimide, Conc.
H2SO4, acetonitrile, 2 h, rt; (b) Conc. HCl, methyl acetate, aq.
NaNO2, 45 min, 4 °C, NaN3, 30 min, rt; (c) aq. LiOH, MeOH/THF
(1:1), 16 h, rt; (d) Bis(tributyltin), Pd(PPh3)4, dioxane, 16 h, refux;
(e) aq. KOH, MeOH, 16 h, 60 °C; (f) 4 or 6, HATU, N,N-diisopropy-
lethylamine (DIEA), DMF, 2 h, 0 °C to rt; (g) N,N-dimethylbarbituic
acid, Pd(PPh3)4, DCM, 38 °C, 16 h; (h) Na2CO3, propargyl bromide,
DMF, 16 h, 90 °C; (i) K-Selectride, THF, 30 min, 0 °C; (j) phthal-
imide, Ph3P, THF, diisopropyl azodicarboxylate (DIAD), 16 h, rt; (k)
hydrazine hydrate, cis-2-penten-1-ol, MeOH, 16 h, rt; (l) Chloramine-
T, Na125I, MeOH/HOAc/H2O
seen previously using IBNalA as well. The mMOR-1 afni-
ties of both afnity labels were similar to that of IBNtxA and
IBNalA. However, in contrast to both IBNtxA and IBNalA,
both afnity labels had lower afnity for 6TM/E11 sites and
neither ligand showed any selectivity for 6TM/E11 over the
classical opioid receptors. To summarize, the incorpora-
tion of 4′-azido group in the aryl ring of IBNalA or IBNtxA
led to a diminished 6TM/E11 afnity while retaining high
afnity at classical cloned opioid receptors. This unantici-
pated diminished afnity and selectivity at 6TM/E11 lim-
ited their utilization for characterizing this site even though
these probes were designed for that purpose. We thereby
focused on characterizing the classical opioid receptors and
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Cellular and Molecular Neurobiology
Table 2 [35S] GTPγS functional assay
its complexes in particular focusing on mMOR-1 by using
these novel photoafnity labels.
Compound
mMOR-1
In [35S]GTPγS functional assays, both IBzA 10 and alky-
nyl-IBzA 17 were full agonists at MOR-1 (Table 2). Results
were found comparable to that of parent compounds IBNtxA
and IBNalA in terms of agonistic potency. No eforts were
made to characterize these probes at mKOR-1 and mDOR-1
because a limited literature is available on protein complexes
of both these subtypes and the efciency of photochemical
coupling of these afnity labels with mKOR-1 in our hands
was poor (as described later).
EC50 (nM)
Emax (%)
IBzA 10
0.86 0.24
1.1 0.1
19
0.49 0.12
1.4 0.3
122
103
100
101
3
3
Alkynyl-IBzA 17
DAMGO
IBNtxA
7
3
IBNalA
75 3.5
Efcacy data were obtained using agonist induced stimulation of
[35S]GTPγS binding assay. Efcacy is represented as EC50 SEM
The 125I-radiolabeled versions of these drugs bound to
mMOR-1 or mKOR-1 receptors stably expressed in CHO
cells with high afnity (Fig. 2). The afnity was greater
(nM) and percent maximal stimulation (Emax
) SEM relative to
standard agonist DAMGO (at mMOR-1) at 1000 nM; Results are
from 3 independent replications and are presented as mean SEM
A
B
mMOR-1
mMOR-1
20000
15000
10000
5000
0
15000
10000
5000
0
0.00
0.05
0.10
0.15
0.20
0.00
0.05
0.10
0.15
0.20
[IBzA] (nM)
[Alkynyl-IBzA] (nM)
D
C
mKOR-1
mKOR-1
15000
40000
30000
20000
10000
0
10000
5000
0
0.0
0.1
0.2
0.3
0.0
0.1
0.2
0.3
[IBzA] (nM)
[Alkynyl-IBzA] (nM)
Fig. 2 Saturation binding assays.[125I]-IBzA or [125I]-Alkynyl-IBzA
were incubated at the indicated concentrations with membrane
homogenate from cells stably transfected with mMOR-1 or mKOR-
1. Curves were ft by nonlinear regression using Graphpad Prism.
Each study was replicated 3 times and mean values of KD SEM of
3–4 independent replications are presented. There was no signifcant
diference between the KD values of the drugs at either mMOR-1 or
mKOR-1. a IBzA, 0.036 0.006 nM at mMOR-1vs, b Alkynyl-IBzA,
0.051 0.013 at mMOR-1. c IBzA, 0.053 0.002 nM at mKOR-1 vs,
d Alkynyl-IBzA, 0.056 0.001 nM at mKOR-1
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Cellular and Molecular Neurobiology
A
B
IBzA
Alkynyl-IBzA
100
10
1
100
10
1
°
°
°
0 C
0 C
25 C
°
25 C
0
50
100
150
200
250
0
50
100
150
200
250
Time (min)
Time (min)
Fig. 3 Dissociation kinetics of binding. Each drug (0.1 nM) was
incubated for 1.5 h with mMOR-1/CHO membranes at 25 °C. Lev-
allorphan (10 µM) was then added at the indicated time points
(5–240 min) after the 1.5 h incubation to determine the kinetics of
drug dissociation. Results are pooled from 3 independent replicates
with similar results and are expressed as mean [95% confdence inter-
vals]. a The kinetics of dissociation for 125I-IBzA at 25 °C. Data were
best ft by a 2-phase exponential decay model (p<0.0001 vs 1-phase
decay model, Extra sum of squares F-test), with 53% [51–56%] dis-
sociating rapidly and 47% dissociating very slowly. τ1/2 Fast: 4.6 min
[4.1–5.2 min], τ1/2 Slow: 81 min [75–89 min]. At 0 °C, the fraction of
binding which dissociated slowly was increased to 83% [77–90%]
and the dissociation half-life increased to t1/2 Slow =244 min [207–
299 min]. b At 25 °C 125I-alkynylIBzA dissociation was best ft by
a 2-phase exponential decay model (p<0.0001 vs 1-phase decay
model, Extra sum of squares F-test), with 53% [49–56] dissociat-
ing rapidly and the remainder very slowly. τ1/2 Fast =5.2 min [4.4–
6.5 min], τ1/2 Slow =144 min [126–169 min]. At 0 °C, the fraction of
binding which dissociated slowly was increased to 87% [82–92%]
and the dissociation half-life increased to τ1/2 Slow =389 min [321–
487 min]
than anticipated based upon the competition studies, raising
the possibility of two diferent afnity states of the recep-
tor. This was confrmed in dissociation experiments that
revealed the presence of both a rapidly and slowly dissociat-
ing component based upon curve ftting of a two-state model
(Fig. 3). With both 125I-ligands, approximately half the bind-
ing at 25 °C dissociated rapidly and half slowly. Lowering
the temperature to 0 °C increased the percentage of slowly
dissociating binding to approximately 85%. Since the cell
lines expressed a single opioid receptor protein, it seemed
likely that the two components in the dissociation curves
represented agonist/antagonist conformations of the recep-
tor. To assess this possibility, we examined the efects of the
GTP analog GppNHp, which lowers agonist binding afnity
(Childers and Snyder 1978). Inclusion of GppNHp virtually
eliminated the slowly dissociating component, implying that
the radioligands labeled both agonist and antagonist receptor
conformations (data not shown).
Both compounds photoaffinity labeled opioid recep-
tors. In mMOR-1-expressing CHO cells, 125I-BzA and
125I-alkynyl-IBzA labeled bands of approximately 65–70 kD
(Figs. 4a, 5a), consistent with those from literature (Huang
et al. 2015; Lupp et al. 2011). Labeling was found specifc,
as shown by the absence of the band in the nonspecifc lane
from tissue in which labeling was carried out in the presence
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Cellular and Molecular Neurobiology
◂Fig. 5 Photoafnity labeling with [125I]-Alkynyl-IBzA in mMOR-1/
CHO, mKOR-1/CHO, and Mouse Brain. The indicated tissues were
photolabeled with [125I]-Alkynyl-IBzA and separated by SDS-PAGE.
All results have been independently replicated and are representa-
tive fgures. T total; NS nonspecifc. a mMOR-1/CHO. Photola-
beled mMOR-1 ran as a difuse band of approximately 68–75 kDa.
b mMOR-1 Western Blot. Probing membrane from a with MOR-1
antibody confrmed the presence of mMOR-1in both total and non-
specifc lanes. c PNGaseF treatment. eglycosylation with PNGase
F reduced the molecular weight to the predicted molecular weight
of 44 kDa. d mKOR/CHO. Following Wheat Germ afnity enrich-
ment, photolabeled mKOR-1 ran as a difuse band from 50–57 kDa.
e SK-N-BE(2)C neuroblastoma. mMOR-1 were photolabeled in a
natively-expressing cell line, appearing as a difuse band from about
70–77 kDa. f C57 Mouse Brain. mMOR-1 in mouse brain ran with a
lower apparent molecular weight than observed in cell lines, approxi-
mately 57–65 kDa
The expression level of native MOR-1 in mouse brain and
SK-N-Be(2)C cells is far lower than in stably transfected cell
lines, requiring the addition of an immunoafnity enrich-
ment step. For MOR-1, we immunopurifed the receptor
using an antibody raised against an epitope from exon 4
of Oprm1. 125I-alkynyl-IBzA specifcally labeled a band
in mouse brain with 60–70 kDa (Fig. 4c), a size slightly
smaller than those seen in mMOR-1-expressing CHO cells
(Fig. 4a) and SK-N-Be(2)C that endogenously express mu
opioid receptors (Fig. 5e). This is may be due to diferent
glycosylation states of the receptor, as previously reported
(Huang et al. 2008). 125I-BzA and 125I-alkynyl-IBzA pho-
tolabeled mKOR-1 receptors with signifcantly lower ef-
ciency, requiring enrichment of the receptor with wheat
germ agglutinin afnity chromatography prior to SDS-PAGE
(Figs. 4b, 5d). The labeled receptor ran as a difuse band
from about 50–57 kDa (Figs. 4b, 5d), consistent with the
early report (Li et al. 2007).
of levallorphan (1 µM). When using levallorphan (1 µM)
that blocks 125I-alkynyl-IBzA binding, the receptor is still
present at equivalent protein levels in both total and nonspe-
cifc lanes, as shown in the Western blot (Fig. 5b). Cleavage
of N-linked sugars from the receptor with PNGaseF yielded
a photolabeled species with the predicted molecular weight
of the mu receptor’s amino acid sequence alone, 44 kDa
(Fig. 5c).
The MOR-1 associates with many proteins to form a
protein complex. We explored mMOR-1-containing pro-
tein complex using a blue native PAGE approach that
permits the evaluation of the receptor complex in rela-
tive native states (Schagger et al. 1994; Schagger and
Fig. 6 BN-PAGE of Photoafnity labeled mMOR-1. a mMOR-1/
CHO and mouse brain membranes were photolabeled with IBzA
and immunoprecipitated under nondenaturing conditions before sep-
aration by blue native PAGE to yield a single difuse band with an
approximate molecular weight of 200–220 kDa. b mMOR-1/CHO
membranes were photolabeled with Alkynyl-IBzA and resolved
by BN-PAGE with no enrichment; a simple wheat germ agglutinin
enrichment step was added to visualize the species in mouse brain
tissue photolabeled with Alkynyl-IBzA. Results have each been inde-
pendently replicated and are representative autoradiograms
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Cellular and Molecular Neurobiology
Fig. 7 B2D BN-PAGE/SDS-PAGE. After photolabeling and BN-
PAGE separation, a gel lane was excised and run on a denaturing
and reducing SDS-PAGE gel in an orthogonal direction. The higher
molecular weight band observed by BN-PAGE migrated at approxi-
mately 70–75 kDa following SDS-PAGE, consistent with our previ-
ous observations for the mu receptor. Results have each been inde-
pendently replicated and are representative autoradiograms. a IBzA
photolabeled mMOR-1/CHO membranes, immunoprecipitated under
non-denaturing conditions. b Alkynyl-IBzA photolabeled mMOR-1/
CHO membranes with no enrichment step
von Jagow 1991). On blue native PAGE, 125I-BzA and
125I-alkynyl-IBzA both specifcally labeled bands with
molecular weights of approximately 200 kDa in mMOR-
1-expressing CHO cells and mouse brain (Fig. 6). A sec-
ond dimension SDS-PAGE under denaturing conditions
showed that the radiolabelled bands with 200 kDa were
separated into bands with 70–75 kDa in size (Fig. 7) that
were similar to those seen in one-dimension SDS-PAGE
(Figs. 4, 5). These results suggest that mMOR-1 exists as
a protein complex with ~ 200 kDa in size under the condi-
tions we used. The composition of the complex, however,
remains unclear. MOR-1 may form homodimers based
upon its published crystal structure (Manglik et al. 2012)
and may contribute to the complex, although these need to
be validated. Perhaps Sigma1 receptors (~ 25 kDa) could
also be included within the complex since they tend co-
immunoprecipitate with MOR-1 (Kim et al. 2010). It will
be interesting to further explore mMOR-1-containing pro-
tein complex using IP-crosslinking-proteomics approach.
In conclusion, we have synthesized two photoafnity
labels targeting mMOR-1 and mKOR-1. Future studies will
utilize the N-propargyl group on alkynyl-IBzA to CLICK
arms to purify the target. The ability to keep the receptor
complex intact, as shown by blue native PAGE suggests
that it may be feasible to purify the complex and defne its
components.
Author contributions SG and SM jointly supervised this work. SGG,
RU, AV, JS and AH performed research and provided tool compounds.
SGG, RU, YXP, GWP and SM analyzed data. SGG, RU, GWP and
SM wrote the paper with input from all authors. All authors read and
approved the fnal manuscript.
Funding SM is supported by funds from NIH Grants DA045884 and
W81XWH-17-1-0256 (Ofce of the Assistant Secretary of Defense
for Health Afairs through the Peer Reviewed Medical Research Pro-
gram) and start-up funds from Center for Clinical Pharmacology, St.
Louis College of Pharmacy and Washington University to SM. This
research was funded in part through the NIH/NCI Cancer Center Sup-
port Grant P30 CA008748. Studies were also supported by NIDA
grants DA042888 and DA046714 (YXP) and DA048379, DA007242
and DA006241 (GWP/YXP).
Compliance with Ethical Standards
Conflict of interest GWP, YXP and SM are co‐founders of Sparian
BioSciences. All authors report no other biomedical fnancial interests
or potential conficts of interest.
Ethical Approval All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed. This
article does not contain any studies with human participants or animals
performed by any of the authors.
1 3

Cellular and Molecular Neurobiology
parafn-embedded tissues. Regul Pept 167:9–13. https://doi.
org/10.1016/j.regpep.2010.09.004
References
Majumdar S, Burgman M, Haselton N, Grinnell S, Ocampo J, Paster-
nak AR, Pasternak GW (2011a) Generation of novel radiolabeled
opiates through site-selective iodination. Bioorg Med Chem Lett
21:4001–4004. https://doi.org/10.1016/j.bmcl.2011.05.008
Majumdar S et al (2011b) Truncated G protein-coupled mu opioid
receptor MOR-1 splice variants are targets for highly potent
opioid analgesics lacking side efects. Proc Natl Acad Sci USA
108:19778–19783. https://doi.org/10.1073/pnas.1115231108
Majumdar S et al (2012) Synthesis and evaluation of aryl-naloxamide
opiate analgesics targeting truncated exon 11-associated mu opi-
oid receptor (MOR-1) splice variants. J Med Chem 55:6352–6362.
https://doi.org/10.1021/jm300305c
Best MD (2009) Click chemistry and bioorthogonal reactions: unprec-
edented selectivity in the labeling of biological molecules. Bio-
chemistry 48:6571–6584
Bochet P, Icard-Liepkalns C, Pasquini F, Garbay-Jaureguiberry C,
Beaudet A, Roques B, Rossier J (1988) Photoafnity labeling of
opioid d receptors with an iodinated azido-ligand: [125I]D-Thr2,
pN3Phe4, Leu5]enkephalyl-Thr6. Mol Pharmacol 34:436–443
Bolan EA, Pan YX, Pasternak GW (2004) Functional analysis of
MOR-1 splice variants of the mouse mu opioid receptor gene
Oprm. Synapse 51:11–18. https://doi.org/10.1002/syn.10277
Brossi A, Hsu FL, Rice KC (1982) A simplified synthesis of
(+)-4-hydroxy-N-methylmorphinan-6-one. J Organ Chem
47:5214–5216. https://doi.org/10.1021/jo00147a039
Manglik A et al (2012) Crystal structure of the mu-opioid receptor
bound to a morphinan antagonist. Nature 485:321–326. https://
doi.org/10.1038/nature10954
Chae PS et al (2010) Maltose-neopentyl glycol (MNG) amphiphiles
for solubilization, stabilization and crystallization of membrane
proteins. Nat Methods 7:1003–1008. https://doi.org/10.1038/
nmeth.1526
Pan YX (2003) Expression of opioid receptors in mammalian cell lines.
Methods Mol Med 84:17–28
Pasternak GW, Pan Y-X (2013) Mu opioids and their receptors: evo-
lution of a concept. Pharmacol Rev 65:1257–1317. https://doi.
org/10.1124/pr.112.007138
Cheng Y-C, Prusof WH (1973) Relationship between the inhibition
constant (K1) and the concentration of inhibitor which causes
50 percent inhibition (I50) of an enzymatic reaction. Biochem
Pharmacol 22:3099–3108
Schagger H, von Jagow G (1991) Blue native electrophoresis for isola-
tion of membrane protein complexes in enzymatically active form.
Anal Biochem 199:223–231
Childers SR, Snyder SH (1978) Guanine nucleotides diferentiate ago-
nist and antagonist interactions with opiate receptors. Life Sci
23:759–762
Schagger H, Cramer WA, von Jagow G (1994) Analysis of molecu-
lar masses and oligomeric states of protein complexes by blue
native electrophoresis and isolation of membrane protein com-
plexes by two-dimensional native electrophoresis. Anal Biochem
217:220–230
Chou T-C (1974) Relationships between inhibition constants and
fractional inhibition in enzyme-catalyzed reactions with diferent
numbers of reactants, diferent reaction mechanisms, and diferent
types and mechanisms of inhibition. Mol Pharmacol 10:235–247
Clarke S, Chen ZP, Hsu MS, Pintar J, Hill R, Kitchen I (2001) Quanti-
tative autoradiographic mapping of the ORL1, m-, d- and kappa-
receptors in the brains of knockout mice lacking the ORL1 recep-
tor gene. Brain Res 906:13–24
Schuller AG et al (1999) Retention of heroin and morphine-6 beta-
glucuronide analgesia in a new line of mice lacking exon 1 of
MOR-1. Nat Neurosci 2:151–156. https://doi.org/10.1038/5706
Sumranjit J, Chung SJ (2013) Recent advances in target characteri-
zation and identification by photoaffinity probes. Molecules
18:10425–10451. https://doi.org/10.3390/molecules180910425
Takayanagi I, Shibata R, Miyata N, Hirobe M (1982) Photoafnity
labeling of opioid receptor with morphine-7,8-oxide (morphine
epoxide). J Pharmacol Methods 7:185–192
Giguere JR, McCarthy KE, Reisch HA, Sandoval S, Stymiest JL (2012)
Transition metal-catalyzed processes for the preparation of N-allyl
compounds and use thereof. US patent US9624232B2
Grinnell SG, Majumdar S, Narayan A, Le Rouzic V, Ansonof M,
Pintar JE, Pasternak GW (2014) Pharmacologic characteriza-
tion in the rat of a potent analgesic lacking respiratory depres-
sion, IBNtxA. J Pharmacol Exp Ther 350:710–718. https://doi.
org/10.1124/jpet.114.213199
Thirumurugan P, Matosiuk D, Jozwiak K (2013) Click chemistry for
drug development and diverse chemical-biology applications.
Chem Rev 113:4905–4979. https://doi.org/10.1021/cr200409f
Váradi A et al (2015) Synthesis and characterization of a dual
kappa-delta opioid receptor agonist analgesic blocking cocaine
reward behavior. ACS Chem Neurosci 6:1813–1824. https://doi.
org/10.1021/acschemneuro.5b00153
Huang P et al (2008) Brain region-specifc N-glycosylation and lipid
rafts association of the rat mu opioid receptor. Biochem Biophys
Res Commun 365:82–88
Huang P, Chen C, Liu-Chen LY (2015) Detection of mu opioid recep-
tor (MOPR) and its glycosylation in rat and mouse brains by
western blot with anti-muC, an afnity-purifed polyclonal anti-
MOPR antibody. Methods Mol Biol 1230:141–154. https://doi.
org/10.1007/978-1-4939-1708-2_11
Varadi A et al (2016) Mitragynine/corynantheidine pseudoindoxyls as
opioid analgesics with mu agonism and delta antagonism, which
do not recruit beta-arrestin-2. J Med Chem 59:8381–8397. https
://doi.org/10.1021/acs.jmedchem.6b00748
Wofsy L, Metzger H, Singer SJ (1962) Afnity labeling-a general
method for labeling the active sites of antibody and enzyme mol-
ecules. Biochemistry 1:1031–1039
Kim FJ, Kovalyshyn I, Burgman M, Neilan C, Chien CC, Pasternak
GW (2010) Sigma 1 receptor modulation of G-protein-coupled
receptor signaling: potentiation of opioid transduction independ-
ent from receptor binding. Mol Pharmacol 77:695–703. https://
doi.org/10.1124/mol.109.057083
Zhu YX et al (1999) Retention of supraspinal delta-like analgesia and
loss of morphine tolerance in d opioid receptor knockout mice.
Neuron 24:243–252
Li JG, Chen C, Liu-Chen LY (2007) N-Glycosylation of the human
kappa opioid receptor enhances its stability but slows its traf-
fcking along the biosynthesis pathway. Biochemistry 46:10960–
10970. https://doi.org/10.1021/bi700443j
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
Lupp A, Richter N, Doll C, Nagel F, Schulz S (2011) UMB-3, a novel
rabbit monoclonal antibody, for assessing mu-opioid recep-
tor expression in mouse, rat and human formalin-fixed and
1 3