Synthesis and evaluation of n,n-dialkyl enkephalin-based affinity labels for δ opioid receptors

Article abstract of DOI:10.1021/jm000123u

To develop affinity labels for δ opioid receptors based on peptide antagonists, the Phe⁴ residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib²,Aib³]leucine enkephalin (ICI-174,864) were substituted with either Ph

Full text of DOI:10.1021/jm000123u

J . Med. Chem. 2000, 43, 3941-3948
3941
Syn th esis a n d Eva lu a tion of N,N-Dia lk yl En k ep h a lin -Ba sed Affin ity La bels for δ
Op ioid Recep tor s
Dean Y. Maeda,† J ane E. Ishmael,# Thomas F. Murray,‡ and J ane V. Aldrich*,†
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201,
Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602,
and College of Pharmacy, Oregon State University, Corvallis, Oregon 97331
Received March 13, 2000
To develop affinity labels for δ opioid receptors based on peptide antagonists, the Phe4 residues
of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib2,Aib3]leucine enkephalin (ICI-174,864)
were substituted with either Phe(p-NCS) or Phe(p-NHCOCH2Br). A general synthetic method
was developed for the conversion of small peptide substrates into potential affinity labels. The
target peptides were synthesized using Phe(p-NH2) and a Boc/Fmoc orthogonal protection
strategy which allowed for late functional group conversion of a p-amine group in the peptides
to the desired affinity labeling moieties. A key step in the synthesis was the selective
deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl
trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand
binding experiments in Chinese hamster ovary (CHO) cells expressing δ or µ opioid receptors.
The δ receptor affinities of the N,N-dibenzylleucine enkephalin analogues were 2.5-10-fold
higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the
para position of Phe4 decreased binding affinity at both δ and µ receptors in standard radioligand
binding assays; the one exception was N,N-dibenzyl[Phe(p-NCS)4]leucine enkephalin (2) which
exhibited a 2-fold increase in affinity for δ receptors (IC50 ) 34.9 nM) compared to N,N-
dibenzylleucine enkephalin (IC50 ) 78.2 nM). The decreases in µ receptor affinities were greater
than in δ receptor affinities so that all of the analogues tested exhibited significantly greater
δ receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested,
only N,N-dibenzyl[Phe(p-NCS)4]leucine enkephalin (2) exhibited wash-resistant inhibition of
radioligand binding to δ receptors. To our knowledge, 2 represents the first peptide-based
affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As
a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for
the further study of δ opioid receptors.
In tr od u ction
reward pathways,10 and antagonists may help attenuate
alcoholism11,12 and cocaine addiction.13 Antagonists at
the δ receptor also exhibit immunosuppressive effects14
and may represent a novel way to treat autoimmune
disorders and organ rejection in transplant patients.15
Although considerable research has been performed on
δ opioid receptors, there is still much to be learned about
δ receptors and their physiological roles.
Affinity labels are ligands which bind to receptors in
a nonequilibrium manner and can be useful as phar-
macological tools.16 Nonpeptide affinity labels that have
been used in the study of δ opioid receptors include
fentanyl isothiocyanate (FIT),17 cis-(+)-3-methyl-
fentanyl isothiocyanate (SUPERFIT),18 and naltrin-
dole isothiocyanate (NTII).19 Peptide-based affinity
labels for δ opioid receptors include [D-Ala2,Cys6]-
enkephalin (DALCE)20 and its S-3-nitropyridinesul-
fenyl (Npys) protected derivative [D-Ala2,Cys(Npys)6]-
leucine enkephalin,21 [D-Ala2,D-Leu5]enkephalin chlo-
romethyl ketone (DALECK),22 and, most recently, Tyr-
D-Ser-Gly-Phe-Leu-Thr-NH-Gly-maleoyl.23
For centuries, humans have used the extract of the
opium poppy, Papaver somniferum, for the alleviation
of pain. Although morphine (the major constituent of
opium) and many other synthetic analogues are used
clinically, these analgesics have also been associated
with serious side effects including physical dependence,
respiratory depression, and tolerance.1,2 Opioids interact
with three major opioid receptor types (mu (µ), kappa
(κ), delta (δ)), and many of the undesired side effects
associated with opioid usage have been associated with
activation of the µ receptor.3 Activation of the κ receptor
also exhibits side effects including dysphoria, diuresis,
and sedation. Because of these factors, there is consid-
erable interest in studying δ receptor ligands for the
alleviation of pain and other therapeutic implications.4
Interest in the δ receptor is not limited to its potential
for mediating analgesia. Subanalgesic doses of δ ago-
nists have been shown to potentiate morphine antinoci-
ception,5,6 and δ antagonists are effective at decreasing
the development of tolerance and dependence to
morphine.7-9 The δ opioid system interacts with many
The irreversible nature of an affinity label offers
several advantages over a reversible ligand for certain
receptor studies. Opioid affinity labels have been used
to visualize the distribution of δ receptors in the rat
neostriatum24 and for the long-term blockade of opioid
receptor activation.25 Recently, affinity labels have been
* To whom correspondence should be addressed. Tel: 410-706-6863.
Fax: 410-706-0346. E-mail: jaldrich@umaryland.edu.
† University of Maryland.
‡ University of Georgia.
#
Oregon State University.
10.1021/jm000123u CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/03/2000

3942 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Maeda et al.
used to investigate the binding requirements of the δ
opioid receptor. Liu-Chen and co-workers have identified
the region of the δ opioid receptor responsible for the
irreversible binding of SUPERFIT,26 thereby gaining
new information about possible residues involved in
receptor/ligand interactions. The affinity labels cur-
rently in use for δ opioid receptors are based on both
peptide agonists and nonpeptidic ligands (both agonists
and antagonists). Due to potential differences in binding
modes between peptides and nonpeptides and agonists
versus antagonists, affinity labels based on δ-selective
peptide antagonists may yield additional structural
information about the binding site of δ opioid receptors.
We previously attempted to develop affinity labels
based on δ-selective peptide antagonists by introducing
a nitrogen mustard into N-terminal dialkylated en-
kephalins. In an initial study of the structure-activity
relationships (SAR) for N-terminal substituents on
leucine enkephalin, the N,N-dibenzyl derivative exhib-
ited the highest potency and selectivity for the δ opioid
receptor.27 Therefore in a subsequent attempt to prepare
peptide-based affinity labels, the Phe4 residues of this
pentapeptide and the related δ-selective peptide an-
tagonist N,N-diallyl[Aib2,Aib3]leucine enkephalin (ICI-
174,864, Aib ) 2-aminoisobutyric acid)28 were substi-
tuted with melphalan (Mel), the para-substituted
nitrogen mustard analogue of phenylalanine.29 Mel was
sufficiently stable to be directly incorporated into the
growing peptide chain. Unfortunately, neither of the
Mel-containing peptides synthesized exhibited signifi-
cant wash-resistant antagonism in the mouse vas def-
erens (MVD) smooth muscle assay. The labeling process
is thought to occur in two discrete steps: first, reversible
binding of the affinity label to the receptor occurs,
followed by covalent linkage with the receptor.16 The
lack of irreversible binding of the Mel-containing pep-
tides might be due to potential problems in both steps.
The large, sterically demanding nitrogen mustard moi-
ety of the Mel residue may have had a deleterious effect
on the reversible binding of the synthesized peptides.
In addition, the relatively low reactivity of the aromatic
nitrogen mustard may have hindered the process of
covalent bond formation with the receptor.
F igu r e 1. Target enkephalin-based affinity labels and related
peptides.
cells stably transfected with the mouse δ and rat µ
opioid receptors.31
Syn th etic Str a tegy
In the synthetic scheme for these potential affinity
labels, a major consideration was the strategy for intro-
ducing the electrophilic groups. In an earlier study, the
nitrogen mustard of Mel lent itself very well to standard
conditions of solution-phase peptide synthesis and could
be directly incorporated into the growing peptide chain
with no special protection or reaction conditions.29
Unfortunately, the more reactive isothiocyanate and
bromoacetamide moieties were not expected to be stable
to multiple peptide couplings and certain functional
group deprotections. Therefore a synthetic scheme was
devised that would allow functional group conversion
late in the peptide synthesis. p-Nitrophenylalanine was
chosen as the precursor of the peptide-based affinity
labels because the nitro group can be reduced to an
amine which can then be transformed into a number of
labeling moieties, including the aforementioned isothio-
cyanate and bromoacetamide. Late reduction of the
nitro group to the amine was not an option in the
synthesis of these target peptides due to the susceptibil-
ity of the N-terminal substituents to hydrogenolysis.
An important aspect for these syntheses was the
selective deprotection of an amine group in the presence
of other protecting groups. Because the tert-butyl pro-
tecting group is easily and effectively removed using
trifluoroacetic acid (TFA), this protecting group was
chosen for semipermanent protection of the peptides due
to the mild conditions required for final deprotection.
This then required a transient protecting group for the
p-amino functionality which could be selectively depro-
tected to allow electrophilic functional group introduc-
tion. Our laboratory initially explored the use of some
hyperacid labile protecting groups, such as Bpoc (1-
methyl-1-(4-biphenyl)ethoxycarbonyl), Ppoc (2-triphe-
nylphosphonoisopropoxycarbonyl), and Ddz (2-(3,5-
dimethoxyphenyl)-2-propyloxycarbonyl), for these syn-
theses. These protecting groups are generally introduced
as the azides or active esters, but unfortunately these
reagents were not reactive enough to protect the aniline
functionality of Phe(p-NH2) (unpublished results).
We then explored possible orthogonal protection
strategies using base-labile protecting groups for the
p-amino group in conjunction with the semipermanent
To further investigate N,N-dialkylated enkephalin
derivatives as potential affinity labels, we have incor-
porated more reactive and less sterically demanding
functional groups at the para position of Phe4. A second
goal in the syntheses of these peptide derivatives was
the development of a general methodology flexible
enough for the incorporation of various labeling moieties
into a target peptide. The functional groups incorporated
in these analogues were the isothiocyanate and the
bromoacetamide groups. While these groups have shown
their effectiveness in various nonpeptidic affinity labels
for opioid receptors,17-19,30 prior to this study they have
not been incorporated into opioid peptide derivatives.
The target peptides for this study included the p-
isothiocyanate and p-bromoacetamide derivatives of
N,N-dibenzylleucine enkephalin and ICI-174,864 (see
Figure 1). The amine-containing derivatives were also
prepared as reversible control compounds for the wash-
ing procedures used in the assay for wash-resistant
inhibition of binding. The assays for inhibition of
radioligand binding under both reversible and nonequi-
librium conditions used Chinese hamster ovary (CHO)

N,N-Dialkyl Enkephalin-Based Affinity Labels
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3943
Sch em e 1a
a
Conditions: (i) iBuOCOCl, NMM; (ii) HCl‚H-Leu-OtBu, NMM (81%); (iii) NH4HCO2, 10% Pd/C (94%); (iv) Fmoc-Cl, 10% NaHCO3,
dioxane (80%); (v) TMS-OTf, toluene (97%).
tert-butyl-type groups. One such group is the 9-fluore-
nylmethoxycarbonyl (Fmoc) protecting group,32 which
is removed through â-elimination using an organic base,
most commonly piperidine. This protection strategy
allowed for the deprotection of the p-amine on phenyl-
alanine near the end of the synthesis, and therefore the
electrophilic groups would not be subjected to a lengthy
synthetic process.
Since the target peptides share a common C-terminal
dipeptide fragment, the synthetic scheme involved a
convergent [3+2] fragment condensation of the common
C-terminal dipeptide fragment to the two different
N-terminal fragments to yield the desired precursor
pentapeptides. The electrophilic groups were then in-
troduced into the protected peptides, followed by final
TFA deprotection to yield the desired peptides.
The N-terminal tripeptides were prepared using
previously described methods29 and coupled to the
C-terminal dipeptide 10 using either iBuOCOCl (for
N,N-dibenzyl-Tyr-Gly-Gly-OH) or pivaloyl chloride36 (for
N,N-diallyl-Tyr-Aib-Aib-OH) to provide pentapeptides
11 and 16, respectively (Scheme 2). Next the Fmoc
group was removed to expose the p-amine for electro-
phile introduction. Tris(2-aminoethylamine) (TAEA),
which was developed by Carpino and co-workers37 to
simplify the removal of the dibenzofulvene byproduct
during solution-phase synthesis, was used to remove the
Fmoc group from 11 and 16 to yield the protected
pentapeptide amines 12 and 17, respectively.
The penultimate step in the synthetic strategy was
the incorporation of the reactive functional groups into
the protected pentapeptides (Scheme 3). During the
synthesis of the dibenzylleucine enkephalin derivatives,
functional group conversion of the amine to the bro-
moacetamide initially used bromoacetyl chloride. Ex-
tended reaction times resulted in halide exchange and
led to the isolation of chloroacetamide 14, which was
identified by high-resolution mass spectrometry. This
problem was circumvented in subsequent syntheses
through the use of bromoacetyl bromide. The isothio-
cyanates 13 and 18 were formed by reaction of 12 and
17 with thiophosgene, respectively. Final deprotection
of the peptide derivatives using TFA and anisole as a
tert-butyl cation scavenger yielded the target peptides
1-6 for pharmacological evaluation.
Syn th esis
The synthesis of the potential affinity labels began
with preparing the common C-terminal dipeptide frag-
ment 10. Initial attempts to prepare 10 through the
selective p-amine protection of Phe(p-NH2) with the
Fmoc group using pH control33 resulted in a compound
with poor solubility, and attempts to protect Boc-Phe-
(p-NH2)-OH with the Fmoc group resulted in low yields.
Therefore derivatization at the dipeptide stage was
pursued. The synthesis (Scheme 1) began with the
isobutyl chloroformate-mediated coupling of Boc-Phe-
(p-NO2)-OH to HCl•H-Leu-OtBu to give dipeptide 7 in
81% yield. Reduction of the p-nitro group by catalytic
transfer hydrogenation34 provided the amine derivative
8 in near quantitative yield, and protection of the
resulting amine using Fmoc-Cl under standard condi-
tions gave the fully protected dipeptide derivative 9 in
80% yield.
P h a r m a cologica l Eva lu a tion
These final peptides were subjected to radioligand
binding assays under standard conditions using cloned
δ and µ receptors expressed in CHO cells and [3H]Tyr-
cyclo[D-Pen-Gly-Phe-D-Pen] (DPDPE) and [3H]Tyr-D-
Ala-Gly-MePhe-Leu-Gly-ol (DAMGO), respectively (Table
1).31 In all cases, the dibenzyl derivatives exhibited
higher (2.5-10-fold) affinity for δ opioid receptors than
the corresponding diallyl derivatives, while the diallyl
derivatives displayed higher δ selectivity by virtue of
greater decreases in binding to µ opioid receptors. It was
In a key step of the synthesis, the Boc group was
removed in the presence of a tert-butyl ester. This
selective deprotection was achieved using 1 equiv of
trimethylsilyl trifluoromethansulfonate (TMS-OTf) in
toluene.35 After addition of the reagent, the product 10
(as the triflate salt) precipitated out of solution as a gel.

3944 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Maeda et al.
Sch em e 2
Sch em e 3a
Substitution at the para position of Phe4 on the two
enkephalin derivatives had varying effects on affinity.
For the dibenzyl derivatives, substitution at the para
position of Phe4 generally caused a decrease in δ
receptor binding affinity; the notable exception was the
isothiocyanate derivative 2, which exhibited the highest
δ receptor affinity of all of the peptides tested. Substitu-
tion at the para position was better tolerated by the
diallyl derivatives, as the isothiocyanate- and bromoac-
etamide-containing peptides demonstrated 2-fold higher
affinity for the δ receptor than the unsubstituted parent
compound, N,N-diallyl[Aib2,Aib3]leucine enkephalin.
Although the amine is the smallest substituent, peptides
1 and 4 displayed the lowest affinity in their respective
series, indicating that the decreases in affinity at the δ
receptor for the other analogues were not due to steric
bulk alone. Within a series, the large acetamide groups
in peptides 3, 3a , and 6 caused a decrease in µ binding
affinity, thereby increasing the δ selectivity of these
compounds with respect to either the isothiocyanate (2
and 5) or the amine (1 and 4) derivatives. The different
substituent effects, particularly the different effects of
the same substituent in the two series, may suggest
subtle differences in the interactions between these two
series of peptides and the δ receptor.
The peptides were then examined for wash-resistant
inhibition of [3H]DPDPE binding to δ receptors (Figure
2). The experiments involved incubation of the test
compounds with CHO cell membranes for 90 min at
room temperature, followed by washing the membranes
via centrifugation, decanting, and resuspension in fresh
buffer. The efficiency of the washing procedure was
examined following incubation of the membranes with
a high concentration (3 µM) of the amine-containing
peptide 1, which cannot bind covalently to the receptors.
These initial studies indicated that a series of five
washes was sufficient to remove the majority of this
peptide without an appreciable loss of protein from the
CHO cell membranes. The other peptide derivatives
were initially assessed for wash-resistant inhibition of
binding at a concentration of 1 µM to identify com-
pounds which deserved further evaluation. The results
after the washing procedure indicated that binding
a
Conditions: (i) 10% anisole/TFA; (ii) thiophosgene, DIPEA; (iii)
bromoacetyl chloride, DIPEA, overnight at rt; (iv) bromoacetyl
bromide, DIPEA, 2 h at 0 °C.
Ta ble 1. Binding Affinities of Peptides 1-6 under Standard
Assay Conditions
IC50
IC50 ( SEM (nM)
ratio
compd
δ
µ
µ/δ
N,N-dibenzyl-Leu
enkephalin
1
2
78.2 ( 1.1
1600 ( 1100
20
486 ( 1
34.9 ( 3.3
149 ( 3
145 ( 4
703 ( 1
23000 ( 1300
1900 ( 1300
9100 ( 1300
19000 ( 1200
18900 ( 1100
47
54
61
131
27
3
3a (p-NHCH2Cl)
N,N-diallyl[Aib2,Aib3]Leu
enkephalin (ICI-174,864)
4
5
6
1394 ( 1
361 ( 3
392 ( 3
87700 ( 1900
33400 ( 1300
>100000
63
92
>255
interesting to note that N,N-dibenzylleucine enkephalin
exhibited a 10-fold greater affinity for δ receptors than
the widely used ICI-174,864. This is in contrast to
earlier studies29 in which N,N-diallyl[Aib2,Aib3]leucine
enkephalin was reported to be more potent than N,N-
dibenzylleucine enkephalin in antagonizing the effects
of [D-Ala2,D-Leu5]enkephalin (DADLE) in the MVD
smooth muscle assay. This reversal in rank order
potency suggests differences between the δ receptors
present in the MVD and cloned receptor binding assays,
and may be due to δ receptor subtypes.38,39

N,N-Dialkyl Enkephalin-Based Affinity Labels
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3945
(Figure 3). These data suggest that the isothiocyanate-
containing dibenzyl compound 2 may be useful as an
affinity label in the study of δ opioid receptors.
Con clu sion s
Utilizing a convergent [3+2] synthetic strategy, po-
tential affinity labels based on the δ-selective peptide
antagonists N,N-dibenzylleucine enkephalin27 and ICI-
174,86428 were prepared. An Fmoc group was used to
protect a p-amine functionality on Phe4 prior to deriva-
tization to the isothiocyanate or bromoacetamide groups.
A key step in the synthesis was the selective removal
of a Boc group in the presence of a tert-butyl ester, which
was accomplished with an equimolar amount of TMS-
OTf in toluene. The orthogonal Boc/Fmoc protection
strategy utilized in these syntheses should prove to be
a versatile method for the preparation of a wide variety
of affinity labels based on small peptides.
The derivatized peptides 1-6 displayed a wide range
of affinities for δ opioid receptors, with IC50 values
varying from 34 nM to 1.4 µM under standard radioli-
gand binding assay conditions. Despite the wide range
of affinities, all of the peptides were selective for δ over
µ opioid receptors. Substitution at the para position of
Phe of the N,N-dibenzyl enkephalin analogues generally
decreased binding affinity at the δ opioid receptor, but
isothiocyanate and bromoacetamide functionalization
increased δ opioid receptor binding for the ICI-174,864
derivatives. This would suggest that the differences in
N-terminal and backbone substitution between the N,N-
dibenzyl and ICI-174,864 derivatives resulted in subtle
differences in their interactions with δ opioid receptors.
F igu r e 2. Wash-resistant inhibition of binding. Results are
percent [3H]DPDPE binding ((SEM) following incubation with
peptides 1-6 or NTI and subsequent washing. All compounds
were tested at 1.0 µM, except for compound 1, which was tested
at 3 µM.
Of the compounds tested, only the isothiocyanate-
containing derivative of N,N-dibenzylleucine enkephalin
(2) exhibited wash-resistant inhibition of binding to
CHO cells expressing δ receptors. The corresponding
ICI-174,864 derivative 5 did not exhibit wash-resistant
inhibition of binding, despite its structural similarity
to 2. The affinity labeling process begins with reversible
binding of the ligand to the receptor, followed by
covalent bond formation if a receptor-based nucleophile
is in proper alignment with the electrophilic moiety.
Different spatial orientations during binding, which may
be due to differences in the N-terminal and backbone
substitutions in 5, may either decrease reversible bind-
ing to the receptor and/or prohibit proper alignment of
the isothiocyanate with a receptor-based nucleophile.
From these observations, enkephalin analogue 2
emerges as an affinity label selective for the δ opioid
receptor. Peptide 2 represents the first peptide-based
affinity label to utilize an isothiocyanate group as the
electrophilic labeling moiety for opioid or any receptors.
Further pharmacological studies with 2 in conjunction
with other δ-selective affinity labels may provide further
insight on the binding requirements and physiological
roles of δ opioid receptors. These studies are ongoing in
our laboratory.
F igu r e 3. Dose-dependent wash-resistant inhibition of [3H]-
DPDPE binding by compound 2. Results are percent [3H]-
DPDPE binding ((SEM) following incubation with compound
2 (tested at the concentrations in parentheses) or 1.0 µM N,N-
dibenzylleucine enkephalin (DBLE).
levels of [3H]DPDPE for the treated membranes were
close to those for untreated membranes, except for the
membranes treated with the isothiocyanate-containing
dibenzyl compound 2. In these membranes, [3H]DPDPE
binding was only approximately 19% of untreated
control membrane. This level of wash-resistant inhibi-
tion of binding was close to that observed following
treatment with naltrindole 5′-isothiocyanate (NTII) at
a similar concentration (Figure 2). Since the affinity of
N,N-dibenzylleucine enkephalin for δ receptors is simi-
lar to that observed for 2 under standard binding assay
conditions, N,N-dibenzylleucine enkephalin was used as
the control compound in subsequent examination of 2
for wash-resistant inhibition of binding. N,N-Diben-
zylleucine enkephalin and 2 were examined for wash-
resistant inhibition of binding at concentrations which
should give comparable levels of receptor saturation (1.0
and 0.44 µM, respectively). The washing procedure
effectively removed 1.0 µM N,N-dibenzylleucine en-
kephalin, while preincubation with 0.44 µM peptide 2
still resulted in 76% wash resistant inhibition of [3H]-
DPDPE binding (Figure 3). Even following preincuba-
tion with 100 nM 2, [3H]DPDPE binding was only
approximately 60% of untreated control membranes
Exp er im en ta l Section
All amino acids except for Boc-Phe(p-NO2) and Aib were
purchased from Sigma Chemical Co. (St. Louis, MO). Synthetic
reagents and the amino acids Boc-Phe(p-NO2) and Aib were
purchased from Aldrich Chemical Co. (Milwaukee, WI). Gen-
eral laboratory solvents were obtained from Mallinckrodt

3946 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Maeda et al.
Baker Inc. (Phillipsburg, NJ ), and HPLC-grade solvents were
obtained from Burdick and J ackson (Muskegon, MI).
Syntheses were monitored using thin-layer chromatography
using Kieselgel 60 F254 silica plates of 0.20 mm thickness
purchased from EM Science (Gibbstown, NJ ). Intermediates
were purified by flash chromatography using 230-400 mesh,
60 Å silica gel also purchased from EM Science.
NMR analyses were done on an Bruker AC300 instrument
at the Department of Chemistry, Oregon State University,
Corvallis, OR. The molecular weights of the amino acid
derivatives and peptides were determined by fast atom
bombardment (FAB) mass spectrometry in the postitive mode
on a Kratos MS50RFTC in the Environmental Health Sciences
Center at Oregon State University, Corvallis, OR.
NR-ter t-Bu tyloxyca r bon yl-L-p-a m in o(9-flu or en ylm eth -
oxyca r bon yl)p h en yla la n yl-L-leu cin e ter t-Bu tyl Ester (9).
Ten percent Na2CO3 was added to a solution of 8 (1.10 g, 2.40
mmol) in dioxane (10 mL) to adjust the pH to 8. A solution of
9-fluorenylmethyl chloroformate (0.56 g, 2.2 mmol) was added,
and the pH was maintained using additional 10% Na2CO3; a
light, white precipitate formed immediately. The reaction was
allowed to proceed overnight. The resultant suspension was
then diluted with H2O (10 mL) and the precipitate filtered and
washed with 0.1 N HCl and H2O. The crude solid (1.53 g,
105%) was dried and recrystallized from MeOH/H2O to yield
1.29 g (80%) of 9 as a white solid: Rf (EtOAc) ) 0.69; HPLC
(column A) tR ) 43.7 min; mp 207-210 °C; [R]D ) -6.2° (c )
1
0.45, MeOH); H NMR (CDCl3) δ ) 0.88 (d, 3H, J ) 3.3 Hz),
0.89 (d, 3H, J ) 8.9 Hz), 1.40 (s, 9H), 1.41 (s, 9H), 1.51 (m,
3H), 3.01 (d, 2H, J ) 6.5 Hz), 4.25 (t, 2H, J ) 6.5 Hz), 4.31(m,
1H), 4.41 (m, 1H), 4.51 (d, 2H, J ) 6.6 Hz), 6.22 (d, 1H, J )
7.9 Hz), 6.56 (s, 1H), 7.12 (d, 2H, J ) 8.2 Hz), 7.31 (t, 4H, J )
7.5 Hz), 7.40 (t, 2H, J ) 7.3 Hz), 7.59 (d, 2H, J ) 7.2 Hz), 7.76
(d, 2H, J ) 7.4 Hz); FAB-MS [M + H]+ 672.3 (calcd 672.4).
The purity of intermediates and the final peptides were
determined by high-performance liquid chromatography (HPLC)
analysis. The system consisted of a Beckman model 431A
HPLC using either a Vydac (C4, 300 Å, 5 µm, 4.6 × 250 mm,
column A) or a Zorbax Protein Plus (C3, 300 Å, 10 µm, 4.6 ×
250 mm, column B) analytical column. The compounds were
eluted using a linear gradient of 0-75% B over 50 min at a
flow rate of 1.5 mL/min and detected at 214 nm; solvent A
was aqueous 0.1% TFA and solvent B was acetonitrile contain-
ing 0.1% TFA.
Isobu tyl Ch lor ofor m a te-Med ia ted P ep tid e Cou p lin g
(Meth od I).40 Under an N2 atmosphere, a 0.25 M solution of
the carboxyl component (1.0 equiv) in dry tetrahydrofuran
(THF) was cooled to -15 °C (dry ice/MeOH) and neutralized
with one equivalent of N-methylmorpholine (NMM). A sto-
ichometric amount of isobutyl chloroformate was then added,
followed 90 s later by a 0.5 M solution of the amine component
and NMM (each 1.0 equiv) in N,N-dimethylformamide (DMF).
The reaction mixture was kept at -15 °C for 0.5 h and then
allowed to warm to room temperature. When the reaction was
complete, the THF was evaporated and the residue suspended
in EtOAc. The organic layer was washed with H2O, 5% KHSO4,
H2O, 5% NaHCO3, H2O, and then finally with saturated NaCl.
The organic layer was then dried over MgSO4 and the EtOAc
evaporated to give the crude peptide.
L-p-Am in o(9-flu or en ylm eth oxyca r bon yl)p h en yla la n yl-
L-leu cin e ter t-Bu tyl Ester (10). A solution of TMS-OTf in
toluene (0.358 M, 4.77 mL, 1.66 mmol) was added dropwise
to a solution of 9 (1.10 g, 1.70 mmol) in CH2Cl2 (15 mL). The
product (as the triflate salt) began to precipitate out as a
gelatinous mass following addition. After 4 h, the gel was
filtered, washed with additional CH2Cl2, and then dried to give
0.94 g (97%) of intermediate 10 as a white solid: Rf (EtOAc)
) 0.68; HPLC (column A) tR ) 24.3 min; [R]D ) -14.5° (c )
1
1.00, MeOH); H NMR (CDCl3) δ ) 0.90 (d, 6H, J ) 3.3 Hz),
1.45 (s, 9H), 1.57 (m, 3H), 1.78 (broad s, 1H), 4.25 (t, 1H, J )
Hz), 4.45 (m, 1H), 4.56 (d, 2H, J ) Hz), 6.65 (s, 1H), 7.12 (d,
2H, J ) 8.2 Hz), 7.31 (t, 4H, J ) 7.5 Hz), 7.40 (t, 2H, J ) 7.3
Hz), 7.59 (d, 2H, J ) 7.2 Hz), 7.76 (d, 2H, J ) 7.4 Hz); FAB-
MS [M + H]+ 572.3 (calcd 572.3).
N,N-Dib en zyl-O-ter t-b u t yl-L-t yr osylglycylglycyl-L-p -
a m in o(9-flu or en ylm eth oxyca r bon yl)p h en yla la n yl-L-leu -
cin e ter t-Bu tyl Ester (11). The tripeptide N,N-dibenzyl-O-
tert-butyl-L-tyrosylglycylglycine27 (310 mg, 0.7 mmol) was
coupled to 10 as described in method I, except that DMF was
used instead of THF/DMF as the solvent. The crude material
was purified by flash silica gel chromatography using EtOAc
as the eluent to give 0.57 g (74%) of 11 as a foam: Rf (EtOAc)
) 0.43; HPLC (column A) tR ) 42.3 min; [R]D ) -8.63° (c )
1.2, CHCl3); FAB-MS [M + H]+ 1085.7 (calcd 1085.6).
F in a l Dep r otection w ith TF A (Meth od II). The peptide
was treated with a 10% anisole/TFA for 1 h at room temper-
ature. The TFA was then evaporated and the product precipi-
tated with cold ether. The precipitate was washed five times
with cold ether, and then dried in vacuo.
NR-ter t-Bu tyloxyca r bon yl-L-p-n itr op h en yla la n yl-L-leu -
cin e ter t-Bu tyl Ester (7). N-tert-Butyloxycarbonyl-L-p-nitro-
phenylalanine (1.18 g, 3.80 mmol) was coupled to L-leucine tert-
butyl ester hydrochloride (0.85 g, 3.80 mmol) according to
method I to give 1.48 g (81%) of 7 as a white solid: Rf (EtOAc)
) 0.68; Rf [butanol/acetic acid/water, (BAW) 3:1:1] ) 0.87;
HPLC (column A) tR ) 37.5 min; mp 161-163 °C; [R]D ) -15.9°
N,N-Dib en zyl-O-ter t-b u t yl-L-t yr osylglycylglycyl-L-p -
a m in op h en yla la n yl-L-leu cin e ter t-Bu tyl Ester (12). Com-
pound 11 (269 mg, 0.25 mmol) was dissolved in CH2Cl2 (2.0
mL) and TAEA (1.8 mL, 12.5 mmol) was then added. A white
precipitate formed immediately. After 3 h, the solvent was
removed in vacuo, and the residue was dissolved in EtOAc (30
mL). The EtOAc layer was washed with saturated NaCl, 1 M
phosphate buffer at pH 5.5 (3 × 10 mL), and saturated NaCl
and then dried over MgSO4. The solvent was removed in vacuo,
and the crude residue was applied to a flash silica gel column
with EtOAc as the eluent to give 160 mg (75%) of 12 as a clear
glass: Rf (EtOAc) ) 0.14; Rf [CHCl3/MeOH/acetic acid, (CMA)
1
(c ) 0.94, MeOH); H NMR (CDCl3) δ ) 0.88 (d, 3H, J ) 1.7
Hz), 0.91 (d, 3H, J ) 1.4 Hz), 1.39 (s, 9H), 1.41 (s, 9H), 1.55
(m, 3H), 3.09 (dd, 2H, J ) 6.7 Hz, 14.3 Hz), 4.10 (m, 2H), 5.08
(d, 1H), 6.21 (d, 1H), 7.35 (d, 2H, J ) 8.7 Hz), 8.12 (d, 2H, J )
8.7 Hz); FAB-MS [M + H]+ 480.1 (calcd 480.3).
NR-ter t-Bu t yloxyca r b on yl-L-p -a m in op h en yla la n yl-L-
leu cin e ter t-Bu tyl Ester (8). Ammonium formate (1.5 g, 24.5
mmol) was added to a solution of 7 (1.30 g, 2.70 mmol) in
MeOH (10 mL). Once the ammonium formate had dissolved,
a suspension of 10% Pd/C (0.26 g) in MeOH/H2O (1:1, 1 mL)
was then added dropwise. After 0.5 h, the reaction mixture
was filtered through Celite, and the MeOH evaporated. The
residue was then dissolved in EtOAc (15 mL), and the organic
layer was washed with H2O (10 mL) and saturated NaCl (10
mL) and then dried over MgSO4. The EtOAc was evaporated
to yield dipeptide 8 as a white solid (1.15 g, 94%): Rf (EtOAc)
95:5:1] ) 0.18; Rf (BAW, 3:1:1) ) 0.67; HPLC (column A) tR
)
34.0 min; FAB-MS [M + H]+ 863.5 (calcd 863.5).
N,N-Dib en zyl-O-ter t-b u t yl-L-t yr osylglycylglycyl-L-p -
isot h iocya n a t op h en yla la n yl-L-leu cin e ter t-Bu t yl E st er
(13). Compound 12 (37 mg, 0.04 mmol) was dissolved in CH2-
Cl2 (0.5 mL). N,N-Diisopropylethylamine (DIPEA; 6.5 µL, 0.08
mmol) was added to the solution, followed by thiophosgene (6.5
µL, 0.08 mmol). After 4 h, the solvent was removed under a
stream of N2, and the crude residue was applied to silica gel
flash chromatography with EtOAc as the eluent to give 38 mg
(94%) of 13 as a clear glass: Rf (EtOAc) ) 0.48, Rf (CMA 95:
5:1) ) 0.28; HPLC (column A) tR ) 41.2 min; FAB-MS [M +
H]+ 905.8 (calcd 905.5).
) 0.57; Rf (EtOAc/hexane, 9:1) ) 0.52; HPLC (column A) tR
)
27.2 min; mp 158-160 °C; [R]D ) -19.0° (c ) 1.04, MeOH);
1H NMR (CDCl3) δ ) 0.88 (d, 3H, J ) 2.1 Hz), 0.90 (d, 3H, J
) 1.8 Hz), 1.39 (s, 9H), 1.42 (s, 9H), 1.54 (m, 3H), 2.94 (d, 2H,
J ) 6.5 Hz), 4.24 (broad s, 1H), 4.41 (m, 1H), 4.95 (broad s,
1H), 6.28 (d, 1H, J ) 8.4 Hz), 6.65 (d, 2H, J ) 8.4 Hz), 6.98 (d,
2H, J ) 8.3 Hz); FAB-MS [M + H]+ 450.2 (calcd 450.3).
N,N-Dib en zyl-O-ter t-b u t yl-L-t yr osylglycylglycyl-L-p -
ch lor oa ceta m id op h en yla la n yl-L-leu cin e ter t-Bu tyl Ester
(14). Compound 12 (77 mg, 0.09 mmol) was dissolved in CH2-
Cl2 (1.0 mL) and chilled to 0 °C in an ice water bath. DIPEA

N,N-Dialkyl Enkephalin-Based Affinity Labels
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3947
(31 µL, 0.18 mmol) was added to the solution, followed by
bromoacetyl chloride (11 µL, 0.14 mmol) A thick white vapor
formed upon addition of the bromoacetyl chloride, and the
solution quickly turned a dark color. After 1 h at 0 °C, the
solution was allowed to warm to room temperature and the
reaction continued overnight. The solvent was then removed
under an N2 stream, and the residue was applied to silica gel
flash chromatography with EtOAc as the eluent to give 49 mg
(54%) of 14 as a clear glass: HPLC (column A) tR ) 37.0 min;
FAB-MS [M + H]+ 939.5 (calcd 939.5); high-resolution FAB-
MS (calcd for C52H69ClN6O8) 939.4787, found 939.4778.
N,N-Dib en zyl-O-ter t-b u t yl-L-t yr osylglycylglycyl-L-p -
br om oa ceta m id op h en yla la n yl-L-leu cin e ter t-Bu tyl Ester
(15). Compound 12 (21 mg, 0.02 mmol) was dissolved in CH2-
Cl2 (0.5 mL) and chilled to 0 °C in an ice water bath. DIPEA
(8.5 µL, 0.05 mmol) was added to the solution, followed by
bromoacetyl bromide (3.2 µL, 0.04 mmol). After 2 h at 0 °C,
the solution was allowed to warm to room temperature for 1
h. The solvent was then removed in vacuo, and the residue
was purified by silica gel flash chromatography with EtOAc
as the eluent to give 16 mg (91%) of 15 as a glass: Rf (EtOAc)
) 0.62; HPLC (column A) tR ) 37.8 min; FAB-MS [M + H]+
985.4 (calcd 985.0);
N,N-Diben zyl-L-tyr osylglycylglycyl-L-p-a m in op h en yl-
a la n yl-L-leu cin e (1). Compound 12 (20.0 mg, 0.02 mmol) was
deprotected using method II to yield 16.0 mg (72%) of 1 as a
white solid: HPLC (column A) tR ) 46.6 min (94% purity);
FAB-MS [M + H]+ 751.4 (calcd 751.4).
N ,N -Dib e n zyl-L -t y r o sy lg ly c y lgly c y l-L -p -iso t h io c y-
a n a top h en yla la n yl-L-leu cin e (2). Compound 13 (38.0 mg,
0.04 mmol) was deprotected using method II to yield 15.4 mg
(41%) of 2 as a white solid: HPLC (column A) tR ) 32.0 min
(95% purity); FAB-MS [M + H]+ 793.3 (calcd 793.3).
N,N-Diben zyl-L-tyr osylglycylglycyl-L-p-ch lor oa ceta m i-
d op h en yla la n yl-L-leu cin e (3a ). Compound 14 (20.0 mg, 0.02
mmol) was deprotected using method II to yield 19.1 mg (95%)
of 3a as a white solid: HPLC (column A) tR ) 27.4 min (87%
purity); FAB-MS [M + H]+ 827.4 (calcd 827.4).
N,N-Diben zyl-L-tyr osylglycylglycyl-L-p-br om oa ceta m i-
d op h en yla la n yl-L-leu cin e (3). Compound 15 (20.0 mg, 0.02
mmol) was deprotected using method II to yield 14.6 mg (73%)
of 3 as a white solid: HPLC (column A) tR ) 27.8 min (98%
purity); FAB-MS [M + H]+ 873.3 (calcd 873.3).
N,N-Dia llyl-O-ter t-bu tyl-L-tyr osyl-r-a m in oisobu tyr yl-
r-aminoisobutyryl-L-p-amino(9-fluorenylmethoxycarbonyl)-
p h en yla la n yl-L-leu cin e ter t-Bu tyl Ester (16). N,N-
Diallyl-O-tert-butyl-L-tyrosyl-R-aminoisobutyryl-R-amino-
isobutyric acid (250 mg, 0.48 mmol) was dissolved in toluene
(2 mL) and cooled in an ice bath. Triethylamine (166 µL, 1.19
mmol) was added to the solution, followed by trimethylacetyl
chloride (60 µL, 0.48 mmol). The reaction was kept at 0 °C for
1 h and then allowed to warm to room temperature overnight.
The reaction mixture was then filtered and concentrated by
rotary evaporation. The dipeptide fragment 10 (175 mg, 0.24
mmol) was dissolved in EtOAc and the solution washed with
5% NaHCO3, H2O, and saturated NaCl, dried over Na2SO4 and
evaporated to yield 132 mg (0.23 mmol) of L-p-amino(9-
fluorenylmethoxycarbonyl)phenylalanine-L-leucine tert butyl
ester as the free amine. A solution of the free amine in toluene
(1 mL) was then added to the anhydride intermediate, and
the reaction was left at room temperature overnight. Following
the standard workup, the crude product was applied to a silica
gel flash column with EtOAc/hexane (4:1) as the eluent to yield
140 mg (57%) of 16 as a foam: Rf (EtOAc) ) 0.61; HPLC
(column B) tR ) 37.2 min; FAB-MS [M + H]+ 1041.6 (calcd
1041.6).
N,N-Dia llyl-O-ter t-bu tyl-L-tyr osyl-r-a m in oisobu tyr yl-
r-a m in oisob u t yr yl-L-p -isot h iocya n a t op h en yla la n yl-L-
leu cin e ter t-Bu tyl Ester (18). Compound 17 (15.0 mg, 0.02
mmol) was treated as described in the preparation of 13 to
yield 13.3 mg (80%) of 18 as a clear glass: Rf (EtOAc) ) 0.54;
HPLC (column B) tR ) 25.0 min; FAB-MS [M + H]+ 861 (calcd
861.5).
N,N-Dia llyl-O-ter t-bu tyl-L-tyr osyl-r-a m in oisobu tyr yl-
r-a m in oisobu tyr yl-L-p-br om oa ceta m id op h en yla la n yl-L-
leu cin e ter t-Bu tyl Ester (19). Compound 17 (14.0 mg, 0.02
mmol) was treated as described in the preparation of 15 to
yield 9.1 mg (58%) of 19 as a clear glass: HPLC (column B) tR
) 40.9 min; FAB-MS [M + H]+ 941 (calcd 941.5).
N,N-Diallyl-L-tyr osyl-r-am in oisobu tyr yl-r-am in oisobu -
tyr yl-L-p-a m in op h en yla la n yl-L-leu cin e (4). Compound 17
(9.9 mg, 0.01 mmol) was deprotected as described in method
II to yield 8.9 mg (78%) of 4 as a white solid: HPLC (column
B) tR ) 22.5 min (96% purity); FAB-MS [M + H]+ 707.4 (calcd
707.4).
N,N-Diallyl-L-tyr osyl-r-am in oisobu tyr yl-r-am in oisobu -
tyr yl-L-p-isoth iocya n a top h en yla la n yl-L-leu cin e (5). Com-
pound 18 (13.0 mg, 0.01 mmol) was deprotected as described
in method II to yield 9.1 mg (70%) of 5 as a white solid: HPLC
(column B) tR ) 33.3 min (98% purity); FAB-MS [M + H]+
749.3 (calcd 749.4).
N,N-Dia llylt yr osyl-r-a m in oisob u t yr yl-r-a m in oisob u -
tyr yl-L-p-br om oacetam idoph en ylalan yl-L-leu cin e (6). Com-
pound 19 (9.0 mg, 0.01 mmol) was deprotected as described
in method II to yield 8.5 mg (98%) of 6 as a white solid: HPLC
(column B) tR ) 40.9 min (98% purity); FAB-MS [M + H]+
829.3 (calcd 829.3).
Bin d in g Assa ys. Radioligand binding assays of the poten-
tial affinity label derivatives were performed with Chinese
hamster ovary (CHO) cells stably transfected with either
mouse δ or rat µ opioid receptors.31 Cells were harvested 48-
72 h following plating in 50 mM Tris buffer, pH 7.4, at 4 °C
and homogenized using a Dounce homogenizer. The homoge-
nate was then centrifuged at 45000g for 10 min at 4 °C. The
pellet was washed twice by resuspension and recentrifugation
as in the previous step. The pellet was resuspended in 50 mM
Tris buffer, pH 7.4, at 4 °C to yield a protein concentration of
30-60 µg/mL. Incubations were performed for 90 min at 22
°C with [3H]DPDPE and [3H]DAMGO for δ and µ receptors,
respectively. The concentrations of [3H]DPDPE and [3H]-
DAMGO ranged from 0.5 to 0.8 nM in all binding assays.
These radioligand concentrations approximate the KD values
of DPDPE and DAMGO for these receptors (0.5 and 0.64 nM,
respectively). Binding assays were carried out in mixtures
containing 100 µg of membrane protein, 3 mM Mg2+, and
peptidase inhibitors (10 µM bestatin, 30 µM captopril, and 50
µM L-leucyl-L-leucine) in a final volume of 2 mL 50 mM Tris
buffer, pH 7.4, at 22 °C. Nonspecific binding was determined
in the presence of 10 µM unlabeled DPDPE and DAMGO for
the δ and µ binding assays, respectively. The reactions were
terminated by rapid filtration over Whatman GF/B glass fiber
filters using a Brandel M48-R cell harvester, then washed with
5 × 2 mL of ice-cold Tris buffer. The filters were presoaked
for at least 2 h in 0.5% polyethylenimine to decrease nonspe-
cific binding. The filter disks were then placed in minivials
with 4 mL of Cytoscint (ICN Radiochemicals) and allowed to
elute for at least 6 h before counting in a Beckman LS 6800
scintillation counter. IC50 values were then derived from
nonlinear regression analysis of competition curves using nine
concentrations of the peptides; results are reported as (SEM
of 3-4 experiments.
Wa sh -Resista n t Bin d in g Assa ys. Potential affinity label
derivatives for the δ opioid receptor were examined for wash-
resistant inhibition of binding to opioid receptors. CHO cell
membranes expressing δ receptors were incubated in the
absence or presence of the enkephalin derivatives for 90 min
at room temperature. The homogenates were then centrifuged
at 40000g for 15 min at 4 °C and the pellet was resuspended
in 7 mL of 50 mM Tris buffer, pH 7.4, at 4 °C. The centrifuga-
tion and resuspension steps were repeated four times as the
N,N-Dia llyl-O-ter t-bu tyl-L-tyr osyl-r-a m in oisobu tyr yl-
r-am in oisobu tyr yl-L-p-am in oph en ylalan yl-L-leu cin e ter t-
Bu tyl Ester (17). Compound 16 (221 mg, 0.21 mmol) was
deprotected using TAEA as described in the preparation of 12.
The crude peptide was applied to a flash silica gel column with
EtOAc as the eluent to yield 158 mg (91%) of 17 as a clear
glass: Rf (EtOAc) ) 0.25; HPLC (column B) tR ) 35.0 min;
FAB-MS [M + H]+ 819.6 (calcd 819.5).

3948 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Maeda et al.
(19) Portoghese, P. S.; Sultana, M.; Takemori, A. E. Naltrindole 5′-
Isothiocyanate: A Nonequilibrium, Highly Selective δ Opioid
Receptor Antagonist. J . Med. Chem. 1990, 33, 1547-1548.
(20) Bowen, W. D.; Hellewell, S. B.; Kelemen, M.; Huey, R.; Stewart,
D. Affinity Labeling of Delta-opiate Receptors Using [D-Ala2,-
Leu5,Cys6]enkephalin. Covalent Attachment via Thiol-disulfide
Exchange. J . Biol. Chem. 1987, 262, 13434-13439.
(21) Matsueda, R.; Yasunaga, T.; Kodama, H.; Kondo, M.; Costa, T.;
Shimohigashi, Y. Design and Synthesis of Highly Specific and
Selective Enkephalin Analogue Containing S-Npys-Cysteine for
δ Opioid Receptors. Chem. Lett. 1992, 1259-1262.
(22) Pelton, J . T.; J ohnston, R. B.; Balk, J .; Schmidt, C. J .; Roche, E.
B. Synthesis and Biological Activity of Chloromethyl Ketones
of Leucine Enkephalin. Biochem. Biophys. Res. Commun. 1980,
97, 1391-1398.
(23) Szatmari, I.; Orosz, G.; Medzihradszky, K.; Borsodi, A. Affinity
Labeling of Delta Opioid Receptors by an Enkephalin-derivative
Alkylating Agent, DSLET-Mal. Biochem. Biophys. Res. Commun.
1999, 265, 513-519.
(24) Pasquini, F.; Bochet, P.; Garbay-J aureguiberry, C.; Roques, B.
P.; Rossier, J .; Beaudet, A. Electron Microscopic Localization of
Photoaffinity-labeled Delta Opioid Receptors in the Neostriatum
of the Rat. J . Comput. Neurol. 1992, 326, 229-244.
(25) Sayre, L. M.; Takemori, A. E.; Portoghese, P. S. Alkylation of
Opioid Receptor Subtypes by R-Chlornaltrexamine Produces
Concurrent Irreversible Agonistic and Irreversible Antagonistic
Activities. J . Med. Chem. 1983, 26, 503-506.
washing protocol. After the fifth resuspension, the homogenate
was recentrifuged and the final pellet resuspended in 50 mM
Tris buffer, pH 7.4, at 4 °C. These final CHO cell membrane
homogenates were then subjected to radioligand binding
assays as described above. The radioligand binding to mem-
branes treated with the enkephalin analogues were then
expressed as percent binding of untreated control membranes
((SEM). The data for the initial screening of the peptides and
N,N-dibenzylleucine enkepahlin are the average of at least two
experiments, and the data for the subsequent evaluation of
peptide 2 are the average of three experiments.
Ack n ow led gm en t. The authors thank Brian Arbo-
gast for obtaining the mass spectral data and Rodger
Kohnert for his help in NMR interpretation. This
research was supported by the National Science Foun-
dation (Grant IBN-9023149) and the National Institute
of Drug Abuse (Grant DA10035). Additional financial
assistance from the ACS Medicinal Chemistry Division
in the form of a predoctoral fellowship (D.Y.M.) is also
greatly appreciated.
Refer en ces
(26) Zhu, J .; Yin, J .; Law, P.-Y.; Claude, P. A.; Rice, K. C.; Evans, C.
J .; Chen, C.; Yu, L.; Liu-Chen, L.-Y. Irreversible Binding of cis-
(+)-3-Methylfentanyl Isothiocyanate to the δ Opioid Receptor
and Determination of Its Binding Domain. J . Biol. Chem. 1996,
271, 1430-1434.
(27) Aldrich Lovett, J .; Portoghese, P. S. N, N-Dialkylated Leucine
Enkephalins as Potential Delta Opioid Receptor Antagonists.
J . Med. Chem. 1987, 30, 1144-1149.
(28) Cotton, R.; Giles, M. G.; Miller, L.; Shaw, J . S.; Timms, D. ICI-
174,864: A Highly Selective Antagonist for the Opioid Delta
Receptor. Eur. J . Pharmacol. 1984, 97, 331-332.
(29) Aldrich Lovett, J .; Portoghese, P. S. Synthesis and Evaluation
of Melphalan-Containing N,N-Dialkylenkephalin Analogues as
Irreversible Antagonists of the Delta Opioid Receptor. J . Med.
Chem. 1987, 30, 1668-1674.
(30) Archer, S.; Seyed-Mozaffari, A.; Osei-Gyimah, P.; Bidlack, J . M.;
Abood, L. G. 14-â-(2-Bromoacetamido)morphine and 14-â-(2-
Bromoacetamido)morphinone. J . Med. Chem. 1983, 26, 1775-
1777.
(31) Arttamangkul, S.; Ishmael, J . E.; Murray, T. F.; Grandy, D. K.;
DeLander, G. E.; Kieffer, B. L.; Aldrich, J . V. Synthesis and
Opioid Activity of Conformationally Constrained Dynorphin A
Analogues. 2. Conformational Constraint in the “Address”
Sequence. J . Med. Chem. 1997, 40, 1211-1218.
(32) Carpino, L. A.; Han, G. Y. The 9-Fluorenylmethoxycarbonyl
Amino-Protecting Group. J . Org. Chem. 1972, 37, 3404-3409.
(33) Fahrenholz, F.; Thierauch, K.-H. Synthesis of p-Amino-L-Phe-
nylalanine Derivatives with Protected p-Amino Group for Prepa-
ration of p-Azido-L-Phenylalanine Peptides. Int. J . Pept. Protein
Res. 1980, 15, 323-330.
(34) Anwar, M. K.; Spatola, A. F. An Advantageous Method for the
Rapid Removal of Hydrogenolyzable Protecting Groups Under
Ambient Conditions; Synthesis of Leucine Enkephalin. Synthesis
1980, 929-932.
(1) Aldrich, J . V. Analgesics. In Burger’s Medicinal Chemistry and
Drug Discovery, 5th ed.; Wolff, M. E., Ed.; J ohn Wiley and
Sons: New York, 1996; Vol. 3, pp 321-441.
(2) Olson, G. A.; Olson, R. D.; Vaccarino, A. L.; Kastin, A. J .
Endogenous Opiates: 1997. Peptides 1998, 19, 1791-1843.
(3) Kieffer, B. L. Opioids: First Lessons from Knockout Mice. Trends
Pharmacol. Sci. 1999, 20, 19-25.
(4) Rapaka, R.; Porreca, F. Development of Delta Opioid Peptides
as Nonaddicting Analgesics. Pharm. Res. 1991, 8, 1-7.
(5) He, L.; Lee, N. M. Delta Opioid Receptor Enhancement of Mu
Opioid Receptor-induced Antinociception in Spinal Cord. J .
Pharmacol. Exp. Ther. 1998, 285, 1181-1186.
(6) Heyman, J . S.; Vaught, J . L.; Mosberg, H. I.; Haaseth, R. C.;
Porreca, F. Modulation of Mu-mediated Antinociception by Delta
Agonists in the Mouse: Selective Potentiation of Morphine and
Normorphine by [D-Pen2,D-Pen5]enkephalin. Eur. J . Pharmacol.
1989, 165, 1-10.
(7) Abdelhamid, E. E.; Sultana, M.; Portoghese, P. S.; Takemori,
A. E. Selective Blockage of Delta Opioid Receptors Prevents the
Development of Morphine Tolerance and Dependence in Mice.
J . Pharmacol. Exp. Ther. 1991, 258, 299-303.
(8) Fundytus, M. E.; Schiller, P. W.; Shapiro, M.; Weltrowska, G.;
Coderre, T. J . Attenuation of Morphine Tolerance and Depen-
dence with the Highly Selective Delta-Opioid Receptor Antago-
nist TIPP[psi]. Eur. J . Pharmacol. 1996, 286, 105-108.
(9) Hepburn, M. J .; Little, P. J .; Gingras, J .; Kuhn, C. M. Differential
Effects of Naltrindole on Morphine-Induced Tolerance and
Physical Dependence in Rats. J . Pharmacol. Exp. Ther. 1997,
281, 1350-1356.
(10) Herz, A. Opioid Reward Mechanisms: a Key Role in Drug Abuse?
Can. J . Physiol. Pharmacol. 1998, 76, 252-258.
(11) Froehlich, J . C.; Badia-Elder, N. E.; Zink, R. W.; McCullough,
D. E.; Portoghese, P. S. Contribution of the Opioid System to
Alcohol Aversion and Alcohol Drinking Behavior. J . Pharmacol.
Exp. Ther. 1998, 287, 284-292.
(12) Gianoulakis, C. Implications of Endogenous Opioids and Dopam-
ine in Alcoholism- Human and Basic Science Studies. Alcohol
Alcoholism 1996, 31, 33-42.
(13) Suzuki, T.; Mori, T.; Funada, M.; Misawa, M.; Nagase, H.
Attenuation of the Discriminative Stimulus Properties of Cocaine
by δ-Opioid Receptor Antagonists. Eur. J . Pharmacol. 1994, 263,
207-211.
(14) Cheido, M.; Idova, G.; Devoino, L. Involvement of Delta-opioid
Receptors in Immunosuppression. Int. J . Neurosci. 1996, 84,
195-203.
(15) Arakawa, K.; Akami, T.; Okamoto, M.; Oka, T.; Nagase, H.;
Matsumoto, S. The Immunosuppressive Effect of Delta-opioid
Receptor Antagonists on Rat Renal Allograft Survival. Trans-
plantation 1992, 53, 951-953.
(16) Takemori, A. E.; Portoghese, P. S. Affinity Labels for Opioid
Receptors. Annu. Rev. Pharmacol. Toxicol. 1985, 25, 193-223.
(17) Rice, K. C.; J acobson, A. E.; Burke, T. R. J .; Bajwa, B. S.; Streaty,
R. A.; Klee, W. A. Irreversible Ligands with High Selectivity
Toward δ or µ Opiate Receptors. Science 1983, 220, 314-316.
(18) Burke, T. R.; J acobson, A. E.; Rice, K. C.; Silverton, J . V.;
Simonds, W. F.; Streaty, R. A.; Klee, W. A. Probes for Narcotic
Receptor Mediated Phenomena. 12. cis-(+)-3-Methylfentanyl
Isothiocyanate, a Potent Site-directed Acylating Agent for δ
Opioid Receptors. Synthesis, Absolute Configuration, and Recep-
tor Enantioselectivity. J . Med. Chem. 1986, 29, 1087-1093.
(35) Vorbruggen, H.; Kroliklewicz, K. Selective Acidic Cleavage of
the tert-Butoxycarbonyl Group. Angew. Chem., Int. Ed. Engl.
1975, 14, 818.
(36) Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis;
Springer-Verlag: New York, 1984.
(37) Carpino, L. A.; Sadat-Aalaee, D.; Beyermann, M. Tris(2-amino-
ethyl)amine as a Substitute for 4-(Aminomethyl)piperidine in
the Fmoc/Polyamine Approach to Rapid Peptide Synthesis. J .
Org. Chem. 1990, 55, 1673-1675.
(38) Wild, K. D.; Carlisi, V. J .; Mosberg, H. I.; Bowen, W. D.;
Portoghese, P. S.; Sultana, M.; Takemori, A. E.; Hruby, V. J .;
Porreca, F. Evidence for
a Single Functional Opioid Delta
Receptor Subtype in the Mouse Isolated Vas Deferens. J .
Pharmacol. Exp. Ther. 1993, 264, 831-838.
(39) Zaki, P. A.; Bilsky, E. J .; Vanderah, T. W.; Lai, J .; Evans, C. J .;
Porreca, F. Opioid Receptor Types and Subtypes- the Delta
Receptor as a Model. Annu. Rev. Pharmacol. Toxicol. 1996, 36,
379-401.
(40) Meienhofer, J . The Mixed Carbonic Anhydride Method of Peptide
Synthesis. In The Peptides; Udenfriend, S., Meienhofer, J ., Eds.;
Academic Press: New York, 1979; Vol. 1, pp 263-315.
J M000123U