Reporter affinity labels: An o-phthalaldehyde derivative of β- naltrexamine as a fluorogenic ligand for opioid receptors

Full text of DOI:10.1021/jm000138s

© Copyright 2000 by the American Chemical Society
Volume 43, Number 13
J une 29, 2000
Communications to the Editor
Rep or ter Affin ity La bels: An
o-P h th a la ld eh yd e Der iva tive of
â-Na ltr exa m in e a s a F lu or ogen ic Liga n d
for Op ioid Recep tor s
Bertrand Le Bourdonnec,† Rachid El Kouhen,‡
Mary M. Lunzer,† Ping Y. Law,‡ Horace H. Loh,‡ and
Philip S. Portoghese*,†
Department of Medicinal Chemistry, College of Pharmacy,
and Department of Pharmacology, School of Medicine,
University of Minnesota, Minneapolis, Minnesota 55455
Received March 23, 2000
Affinity labels derived from opioid ligands have
provided a useful approach to aid in the pharmacologic
and structural characterization of opioid receptors.1
Electrophilic affinity labels have been particularly use-
ful in this regard. Because the location of covalently
bound residues can be determined through site-directed
mutagenesis and peptide mapping,2,3 the binding locus
of the ligand can be identified, as exemplified by
â-funaltrexamine4 (â-FNA, 1) which has been reported
to bind covalently to Lys233 of the µ receptor.5
Here we describe a unique type of opioid receptor
affinity label 2 that offers an advantage over conven-
tional affinity labels, in that it cross-links neighboring
lysine and cysteine residues through a highly selective
mechanism6 (Figure 1). We have named such ligands
“reporter affinity labels”, because cross-linking leads to
a fluorescent, covalently bound product whose fluores-
cence suggests the involvment of the ꢀ-amino group of
lysine and the sulfhydryl group of cysteine in the
formation of an isoindole moiety. Another feature of this
type of reporter affinity label is that cross-linking
neighboring residues should restrict the translational
mobility of the tethered ligand to a greater degree than
an affinity label that is bound through only one residue.
Consequently, modeling of the pharmacophore recogni-
tion locus of a specifically cross-linked ligand should,
in principle, require less effort.
The chemical basis for the design of 2 is derived from
the known reactivity of o-phthalaldehyde (OPTA) with
primary amines and thiols to form fluorescent iso-
indoles.6-8 In fact, the facility and selectivity of this
reaction has found utility in the analysis of amino acids
using OPTA as a fluorogenic reagent in the presence of
a thiol.9 Also, OPTA has proven to be useful in probing
the catalytic site of different enzymes.10 Given that
opioid receptors contain multiple lysine and cysteine
residues in the upper region of the transmembrane
domain, it is possible a similar reaction can occur if such
receptor-based residues are in proper juxtaposition to
the OPTA moiety of 2 in the reversibly bound state.
Compound 2 (in its hydrated dihydroxyphthalan
form) has been synthesized in seven steps from 3,4-
dimethylbenzoic acid and â-naltrexamine (3).11 Synthon
8, which was required for coupling to â-naltrexamine
(3), was prepared as follows (Scheme 1): 3,4-Dimeth-
ylbenzoic acid was converted to its tetrabromo derivative
4 in the presence of N-bromosuccinimide and benzoyl
peroxide.12 Treatment of 4 with a hot aqueous solution
* To whom correspondence should be addressed. Tel: 1-612-624-
9174. Fax: 1-612-626-6891. E-mail: porto001@tc.umn.edu.
† Department of Medicinal Chemistry.
‡ Department of Pharmacology.
10.1021/jm000138s CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/10/2000

2490 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13
Communications to the Editor
Sch em e 1a
the carboxylic acid 6. Coupling of 6 with glycine ethyl
ester in the presence of 1-hydroxybenzotriazole and
dicyclohexylcarbodiimide gave the amido ester 7 which
was saponified to afford the key intermediate 8. Cou-
pling of â-naltrexamine (3) with 8 afforded the corre-
sponding amide 9 (Scheme 2). Hydrolysis of 9 under
acidic conditions gave the hydrochloric salt of 2 as the
hydrated dihydroxyphthalan.13
Receptor binding of 2 was investigated in membranes
from CHO cells stably expressing µ (83 fmol/mg protein),
δ (51 fmol/mg protein), and κ (36 fmol/mg protein) opioid
receptors. Compound 2 inhibited [3H]diprenorphine
14
binding to µ, δ, and κ receptors with apparent Ki
values of 0.45 ( 0.12, 2.57 ( 0.46, and 0.70 ( 0.11 nM,
respectively. Pretreatment of µ, δ, and κ receptors with
low (1 nM) or high (1 µM) concentrations of 2 (25 °C, 1
h incubation in HEPES buffer, pH ) 7.5) followed by
extensive washing reduced the binding of [3H]diprenor-
phine in a concentration-dependent fashion (Figure 2).
In contrast, pretreatment of opioid receptors with nalox-
one (1 µM) followed by washing did not reduce radioli-
gand binding. These results demonstrated that 2 binds
irreversibly to µ, δ, and κ opioid receptors. The in vitro
pharmacological profile of 2 was investigated on the
electrically stimulated guinea-pig ileal longitudinal
muscle15 (GPI) preparation. Incubation of the GPI with
2 (20 nM) for 20 min followed by thorough washing led
to a parallel shift of the morphine dose-response curve
a
Reagents and conditions: (a) N-bromosuccinimide, benzoyl
peroxide, CCl4, reflux, 10 h, 48%; (b) Na2CO3, H2O, 60 °C, 4 h,
80%; (c) HO(CH2)2OH, p-TsOH, benzene, Dean-Stark, 15 h, 67%;
(d) HCl‚H2NCH2CO2C2H5, N(C2H5)3, DCC, HOBt, THF, rt, 10 h,
90%; (e) (1) NaOH, H2O, rt, 5 h, (2) HCl (1 N), 75%.
of sodium carbonate followed by acidic hydrolysis gave
3,4-diformylbenzoic acid (5).12 Bis-acetalization of 5 with
ethylene glycol under Dean-Stark conditions afforded
F igu r e 1. Mechanism for isoindole formation. The reaction of the OPTA moiety with a primary amine (NH2 group of lysine)
affords a carbinolamine (I), which then dehydrates to give the highly reactive protonated imine (II). This, in turn, is rapidly
attacked by a neighboring nucleophile (SH group of cysteine) to form an intermediate (III) which undergoes facile dehydration to
yield the fluorescent isoindole.

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13 2491
Sch em e 2a
a
Reagents and conditions: (a) 8, DCC, HOBt, THF, rt, 12 h, 64%; (b) HCl (1 N), N2, acetone, rt, 7 days, 38%.
F igu r e 3. Antinociceptive effect of morphine (112 µmol/kg,
sc) in mice pretreated with a single icv dose of 2 (1.2 nmol) at
indicated time periods in the mouse tail-flick assay. At least
10 animals were used at each time period to determine the
percentage of antinociception.
) 7.5), the fluorescence intensity rapidly (30 s) increased
relative to the autofluorescence of untreated cells.
Nonspecific staining was evaluated by incubation of 2
(1 µM) with nontransfected CHO cells. In this case, the
fluorescent distribution of total and nonspecific staining
overlapped (data not shown). To reduce the nonspecific
staining, the reaction of 2 with the cell surface was
quenched after 1 min incubation by adding a mixture
of lysine and mercaptoethanol at relatively high con-
centrations (10 µM), followed by several washings. This
permitted us to visualize the specific fluorescent stain-
ing of µ opioid receptors (Figure 4).
F igu r e 2. Irreversible binding of 2 to µ, δ, and κ opioid
receptors. Membranes of CHO cells stably transfected with µ,
δ, and κ opioid receptors were pretreated with or without 2 (1
nM or 1 µM) or with naloxone (1 µM, control) at 25 °C for 1 h.
Free receptor sites were determined in the presence of [3H]-
diprenorphine (1 nM) before and after washing. The values
represent means ( SE of three independent experiments
performed in triplicate.
In conclusion, the fluorogenic OPTA derivative of
â-naltrexamine (2) binds irreversibly and with high
affinity to the cloned µ, δ, and κ opioid receptors. The
in vitro and in vivo pharmacological experiments have
provided additional evidence of covalent association of
2 with µ opioid receptors. The fluorescence generation
that accompanied binding suggests that 2 has reacted
with neighboring lysine and cysteine residues of the µ
opioid receptor thereby generating an isoindole fluoro-
phore via the pathway outlined in Figure 1. Given that
the specific generation of fluorescence reports the
covalent association with neighboring lysine and cys-
teine residues, reporter affinity labels offer an advan-
tage over conventional electrophilic affinity labels (e.g.,
Michael acceptors, isothiocyanates, nitrogen mustards)
that are not as chemically selective and are nonfluoro-
genic. Furthermore, the participation of two neighboring
residues (lysine and cysteine), in principle, affords the
opportunity to more accurately localize the interaction
of the pharmacophore of 2 with the receptor. Site-
directed mutagenesis experiments are currently in
to the right (IC50 ratio ) 191).16 These data indicated
that the observed antagonism by 2 was irreversible.
The in vivo activity of 2 was evaluated using the
mouse tail-flick antinociceptive assay.17 The data show
that pretreatment of mice with 2 (1.2 nmol, icv) pro-
duced a time-dependent antagonism of morphine-
induced antinociception (Figure 3). This antagonism was
observed at 1 h and lasted at least 5 days after
administration of 2 with a maximal effect occurring at
2 h. This potent and long-lasting antagonism is similar
to that observed for â-FNA.17
While the aforementioned data indicate irreversible
binding of 2 to opioid receptors, it did not reveal whether
the covalent association between 2 and opioid receptors
was due to the formation of a fluorescent isoindole
adduct. This was accomplished with a Becton Dickinson
Facs Vantage equipped with a multiwavelength UV
laser for excitation using a band-pass filter of 530 ( 30
nm.18 When CHO cells with stably expressed µ opioid
receptors were incubated with 2 in HEPES buffer (pH

2492 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13
Communications to the Editor
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Ack n ow led gm en t. We thank Michael Powers and
J anet Peller for capable technical assistance. We also
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Institute on Drug Abuse.
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