Agonist vs antagonist behavior of δ opioid peptides containing novel phenylalanine analogues in place of Tyr1

Article abstract of DOI:10.1021/jm9004913

The novel phenylalanine analogues 4′-[N-((4′-phenyl)phenethyl) carboxamido]phenylalanine (Bcp) and 2′,6′-dimethyl-4′-[N- ((4′-phenyl)phenethyl)carboxamido]phenylalanine (Dbcp) were substituted for Tyr¹ in the δ opioid antagonist TIPP (H-Tyr-Tic

Full text of DOI:10.1021/jm9004913

J. Med. Chem. 2009, 52, 6941–6945 6941
DOI: 10.1021/jm9004913
Agonist vs Antagonist Behavior of δ Opioid Peptides Containing Novel
Phenylalanine Analogues in Place of Tyr1
Irena Berezowska,† Nga N. Chung,† Carole Lemieux,† Brian C. Wilkes,† and Peter W. Schiller*,†,§
†Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West,
§
Montreal, Quebec H2W 1R7, Canada, and Departement de Pharmacologie, Universite de Montreal, Montreal, Quebec, Canada
ꢀ
ꢀ
ꢀ
Received April 17, 2009
The novel phenylalanine analogues 40-[N-((40-phenyl)phenethyl)carboxamido]phenylalanine (Bcp) and
20,60-dimethyl-40-[N-((40-phenyl)phenethyl)carboxamido]phenylalanine (Dbcp) were substituted for
Tyr1 in the δ opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH; Tic = tetrahydroisoquinoline-3-carbo-
xylic acid). Unexpectedly, [Bcp1]TIPP was a potent, selective δ opioid agonist, whereas [Dbcp1]TIPP
retained high δ antagonist activity. Receptor docking studies indicated similar binding modes for the
two peptides except for the biphenylethyl moiety which occupied distinct receptor subsites. The
dipeptide H-Dbcp-Tic-OH was a highly selective δ antagonist with subnanomolar δ receptor affinity.
Introduction
Onthebasisofearlystructure-activity relationship(SARa)
studies performed with Met- and Leu-enkephalin (for a re-
view, see ref 1), it was prematurely concluded that all structur-
al modifications of the Tyr1 phenolic ring in opioid peptides
would be detrimental to opioid activity, a belief that was held
for nearly 2 decades. In a key discovery, Mosberg and
colleagues showed in 1992 that dimethylation at the 20,60-
positions of the Tyr1 residue of a cyclic enkephalin analogue,
Figure 1. Structural formulas of L-Bcp and L-Dbcp.
as achieved by substitution of 20,60-dimethyltyrosine (Dmt),
ish μ receptor binding affinity.7 The latter finding prompted
increased opioid agonist potency by 1-2 orders of magni-
tude.2 The most plausible explanation for this potency in-
the design and synthesis of opioid peptide analogues in which
the Tyr1 hydroxyl function is replaced by a ((40-phenyl)phe-
crease is that the two methyl groups may engage in additional
nethyl)carboxamido group, which was achieved by substitu-
binding interactions with residues in the receptor binding site.
A more recent, interesting finding, independently reported by
tion of 40-[N-((40-phenyl)phenethyl)carboxamido]phenylala-
nine (Bcp, Figure 1).8 The cyclic enkephalin analogue H-Bcp-
c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 turned out to be a po-
tent, μ-selective agonist, indicating that the large bipheny-
lethyl substituent not only was tolerated at the μ opioid
receptor but likely enhanced binding affinity by interacting
with a receptor subsite.
two groups,3,4 was that substitution of a carboxamido
(-CONH2) group for the Tyr1 phenolic hydroxyl group in
opioid peptides, as achieved by replacement of the tyrosine
with p-carboxamidophenylalanine, resulted in compounds
that retained high opioid activity. Replacement of the phe-
nolic hydroxyl group in non-peptide opiates with the
-CONH2 group had previously been shown to result in
compounds retaining high μ receptor binding affinity,5,6 and
introduction of a (40-phenyl)phenethyl substituent at the
-CONH2 group of 8-carboxamidocyclazocine did not dimin-
δ opioid peptide antagonists containing 1,2,3,4-tetrahy-
droisoquinoline-3-carboxylic acid (Tic) in the 2-position of
the peptide sequence, the so-called TIP(P) peptides, were first
reported in 1992.9 The two prototypes, TIPP (H-Tyr-Tic-Phe-
Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) displayed high
δantagonist activity and high δreceptor selectivity. Subsequent
SAR studies led to a number of TIPP-derived δ antagonists
with subnanomolar δ receptor binding affinity (for a review,
see ref 10), including H-Dmt-Tic-Phe-Phe-OH (DIPP). The
dipeptide H-Tyr-Tic-OH was shown to be a weak δ opioid
antagonist, whereas H-Dmt-Tic-OH showed low nano-
molar δ antagonist activity and high δ receptor selectivity.11
Here, we describe the synthesis and opioid activity profiles of
analogues of TIP(P) peptides, in which the Tyr1 residue
was replaced by Bcp or by the novel Tyr analogue 20,60-dime-
thyl-40-[N-((40-phenyl)phenethyl)carboxamido]phenylalanine
(Dbcp) (Figure 1).
*To whom correspondence should be addressed. Phone: þ1-514-987-
5576. Fax: þ1-514-987-5513. E-mail: schillp@ircm.qc.ca.
a Abbreviations: Bcp, 40-[N-((40-phenyl)phenethyl)carboxamido]-
phenylalanine; Cha, cyclohexylalanine; DAMGO, H-Tyr-D-Ala-Gly-
Phe(NMe)-Gly-ol; Dbcp, 20,60-dimethyl-40-[N-((40-phenyl)phenethyl)-
carboxamido]phenylalanine; DIC, 1,3-diisopropylcarbodiimide; DIEA,
N,N-diisopropylethylamine; DIPP, H-Dmt-Tic-Phe-Phe-OH; Dmt,
20,60-dimethyltyrosine; DPDPE, H-Tyr-c[D-Pen-Gly-Phe-D-Pen]OH;
DPPF, 1,10-bis(diphenylphosphino)ferrocene; DSLET, H-Tyr-D-Ser-
Gly-Phe-Leu-Thr-OH; GPI, guinea pig ileum; HBTU, O-(benzotria-
zol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate; HOBt,
1-hydroxybenzotriazole; MVD, mouse vas deferens; SAR, structure-
-activity relationships; Tic, tetrahydroisoquinoline-3-carboxylic acid;
TAPP, H-Tyr-D-Ala-Phe-Phe-NH2; TIPP, H-Tyr-Tic-Phe-Phe-OH;
TMH, transmembrane helix.
r
2009 American Chemical Society
Published on Web 10/14/2009
pubs.acs.org/jmc

6942 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Berezowska et al.
Table 1. Opioid Receptor Binding Affinities of Bcp1- and Dbcp1-Analogues of TIPP Peptidesa
compd
peptide
Kiδ (nM)
Kiμ (nM)
Kiκ (nM)
Ki ratio δ/μ/κ
1
H-Bcp-Tic-Phe-Phe-OH
H-Bcp-Tic-Phe-OH
H-Bcp-Tic-OH
0.605 ( 0.058
0.536 ( 0.057
0.646 ( 0.088
2.00 ( 0.19
87.7 ( 8.0
173 ( 10
2270 ( 210
323 ( 16
334 ( 64
142 ( 41
615 ( 31
1720 ( 50
>1000
270 ( 74
1580 ( 720
4760 ( 200
1240 ( 50
1660 ( 60
597 ( 73
2750 ( 70
>1000
1/145/446
2
1/323/2950
1/3510/7370
1/162/620
3
4
H-Bcp-TicΨ[CH2NH]Phe-Phe-OH
H-Bcp-TicΨ[CH2NH]Cha-Phe-OH
H-Dbcp-Tic-Phe-Phe-OH
H-Dbcp-Tic-OH
H-Tyr-Tic-Phe-Phe-OH (TIPP)b
H-Tyr-Tic-OH
H-Dmt-Tic-Phe-Phe-OHb
H-Dmt-Tic-OHb
H-Cdp-Tic-Phe-Phe-OH
5
3.31 ( 0.32
1/101/502
1/181/762
6
0.783 ( 0.046
0.825 ( 0.107
1.22 ( 0.07
242 ( 28
0.248 ( 0.046
1.84 ( 0.24
7
1/745/3330
1/1410/>820
8
9
>10000
>1000
>1000
10
11
12
141 ( 24
1360 ( 160
2210 ( 490
1/569/>4030
1/739/>543
1/4410/>2000
0.501 ( 0.060
>1000
a Values represent the mean of three to six determinations. bData taken from Schiller et al.10
Table 2. Opioid Agonist or Antagonist Activities of Bcp1- and Dbcp1-Analogues of TIPP Peptidesa
MVD
GPI
compd
peptide
IC50 (nM)
Keδ (nM)
IC50 (nM)
Keμ (nM)
b
b
1
H-Bcp-Tic-Phe-Phe-OH
H-Bcp-Tic-Phe-OH
H-Bcp-Tic-OH
3.42 ( 0.36
223 ( 37 (IC40
458 ( 73 (IC35
)
)
2
18.7 ( 1.2
17.8 ( 0.7
3
5380 ( 380
b
b
b
4
H-Bcp-TicΨ[CH2NH]Phe-Phe-OH
H-Bcp-TicΨ[CH2NH]Cha-Phe-OH
H-Dbcp-Tic-Phe-Phe-OH
H-Dbcp-Tic-OH
11.8 ( 1.6 (IC40
32.0 ( 8.2 (IC40
)
)
1460 ( 310 (IC40)
b
5
721 ( 78 (IC35)
6
3.07 ( 0.51
1.76 ( 0.30
4.80 ( 0.20
263 ( 40
0.196 ( 0.035
6.55 ( 0.27
0.627 ( 0.058
373 ( 71
2910 ( 570
7
8
H-Tyr-Tic-Phe-Phe-OH (TIPP)c
H-Tyr-Tic-OH
>10000
inactive
769 ( 142
inactive
9
inactive
10
11
12
H-Dmt-Tic-Phe-Phe-OHc
H-Dmt-Tic-OHc
H-Cdp-Tic-Phe-Phe-OH
inactive
3070 ( 260
a Values represent the mean of three to six determinations. bPartial agonist. cData taken from Schiller et al.10
Boc-Bcp-OH was synthesized by activating the hydroxyl
group of Boc-Tyr-OMe as the triflate, followed by carbonyla-
tion with carbon monoxide in the presence of potassium
acetate, bis(diphenylphosphino)ferrocene (DPPF), and palla-
dium acetate, as described by Wang et al.,12 affording Boc-
Phe(40-COOH)-OMe in good yield. Subsequent reaction with
2-(4-biphenyl)ethylamine using O-(benzotriazol-1-yl)- N,N,N0,
N0-tetramethyluronium hexafluorophosphate (HBTU) as cou-
pling agent followed by ester hydrolysis with 2 N NaOH yielded
the target compound. Boc-Dbcp-OH was prepared in an
analogous manner, starting with Boc-Dmt-OEt. Peptides were
synthesized by standard solid-phase methods, were purified by
preparative HPLC, and were obtained in >95% purity.
In the GPI assay, peptide 2 was characterized as a weak
μ partial agonist. Further C-terminal truncation led to the
dipeptide H-Bcp-Tic-OH (3) which also displayed subnano-
molar δ receptor binding affinity and extraordinarily high
δ receptor selectivity. In the MVD assay, compound 3 was a
δ antagonist with a similar low efficacy δ partial agonist
component as peptide 2. The 375-fold higher δ receptor
binding affinity of H-Bcp-Tic-OH (Kiδ = 0.646 ( 0.088
nM) as compared to H-Tyr-Tic-OH (9, Kiδ = 242 ( 28
nM) indicates that the biphenylethylcarboxamido group con-
tributes in a major way to the binding interaction energy of 3.
The two pseudopeptides containing a reduced peptide bond
between the 2- and 3-position residues H-Bcp-TicΨ-
[CH2NH]Phe-Phe-OH (4) and H-Bcp-TicΨ[CH2NH]Cha-
Phe-OH (5, Cha = cyclohexylalanine) displayed 6- and 13-
fold lower δ receptor binding affinity than their respective
Tyr1-containing parent pseudopeptides, which are δ opioid
antagonists,10 and turned out to be nearly full δ agonists (e =
0.8) in the MVD assay with potencies 4- to 9-fold lower than
those of tetrapeptide 1. These results indicate that the altered
rotational mobility around the reduced peptide bond in these
two pseudopeptides may position the biphenylethylcarboxa-
mido group in a location at the δ receptor binding site that is
less favorable for optimal δ receptor binding and activation.
The tetrapeptide containing Dbcp (Figure 1) in place of
Tyr1, H-Dbcp-Tic-Phe-Phe-OH (6), showed subnanomolar
δ receptor binding affinity comparable to that of the Bcp1-
tetrapeptide 1 and similarly high δ vs μ and δ vs κ receptor
binding selectivity. However, unlike agonist 1, compound 6
retained high δ antagonist activity in the MVD assay and
was a 2-fold more potent antagonist than the TIPP parent
(8). H-Dbcp-Tic-Phe-Phe-OH (6) displayed 3-fold lower
Results and Discussion
The Bcp1-tetrapeptide analogue H-Bcp-Tic-Phe-Phe-OH
(1) showed about 2-fold higher δ receptor binding affinity
(Kiδ = 0.605 ( 0.058 nM) than the TIPP parent peptide
(δ antagonist) (8) and retained high δ receptor binding
selectivity (Table 1). Surprisingly, this compound turned out
to be a potent full δ opioid agonist (IC50 = 3.42 ( 0.36 nM) in
the MVD assay (Table 2). In the GPI assay, it displayed quite
weak μ partial agonist properties. The C-terminally truncated
tripeptide H-Bcp-Tic-Phe-OH (2) also showed subnanomolar
δ receptor binding affinity, as well as high δ vs μ and δ vs κ
binding selectivity. Unlike peptide 1, however, the Bcp1-
δ
tripeptide was a δ antagonist (Ke = 18.7 ( 1.2 nM) with
low efficacy δ partial agonist activity (maximal inhibition of
contractions = 15%) in the MVD assay. The low efficacy
partial agonist component may explain the discrepancy be-
tween the Keδ and Kiδ values determined for this compound.

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6943
Figure 2. Receptor docking studies. [Bcp1]TIPP bound to the δ receptor in the active state: (a) front view; (b) top view. [Dbcp1]TIPP bound to
the δ receptor in the inactive state: (c) front view; (d) top view. The peptide ligands are in magenta, and the two methyl groups of Dbcp are in
yellow. Selected residues of the receptor are in green, red (D128), or cyan (Y129 and W274). The hydrogen bonds between D128 and the amino
group of Bcp or Dbcp are indicated as white dashed lines.
δ receptor binding affinity than H-Dmt-Tic-Phe-Phe-OH (10)
and 1.5-fold lower δ affinity than H-Cdp-Tic-Phe-Phe-OH
(12; Cdp = 40-carboxamido-20,60-dimethylphenylalanine).
Similarly, in the MVD assay, 6 also showed 15- and 5-fold
decreases in δ antagonist activity (Ke) compared to 10 and 12,
respectively. These comparisons indicate that the presence of
the biphenylethyl substituent in H-Dbcp-Tic-Phe-Phe-OH
does not cause an increase in δ receptor binding affinity. It
is also possible that the interaction of the biphenylethyl
substituent with the receptor may slightly alter the positioning
of the 20,60-dimethylphenylalanine moiety at the receptor,
resulting in the observed decrease in δ receptor binding
affinity and δ antagonist activity. H-Dbcp-Tic-Phe-Phe-OH
(6) has the samelowμ receptor binding affinity as H-Dmt-Tic-
Phe-Phe-OH (10) but interestingly 15-fold higher μ affinity
than H-Cdp-Tic-Phe-Phe-OH (12). In agreement with the
result of the μ receptor binding assay, 6 also showed about
8-fold higher μ antagonist activity than 12 in the GPI assay.
This result indicates that the biphenylethyl substituent of 6
does strengthen binding to the μ receptor in the inactive state
to some extent. On the other hand, 10 behaves as a weak
agonist at the μ receptor.
The dipeptide H-Dbcp-Tic-OH (7) also displayed subna-
nomolar δ opioid receptor binding affinity (Kiδ = 0.825 (
0.107 nM), very high δ receptor binding selectivity, and
subnanomolar δ antagonist activity (Keδ = 1.76 ( 0.30 nM)
in the MVD assay. In comparison with H-Dmt-Tic-OH
(11),10,11 it showed 2-fold higher δ receptor binding affinity
and 4-fold higher δ antagonist activity. Thus, unlike in the
case of tetrapeptide 6, the biphenylethyl moiety of 7 strength-
ens binding to the δ receptor to some extent. This result can be
explainedwiththe differentbindingmodeofthe biphenylethyl
moiety in the dipeptide compared to the tetrapeptide (see
below). Compound 7 also displayed about 2-fold higher
μ receptor binding affinity than 11, and, consequently, these
two dipeptides have about the same high δ vs μ receptor
selectivity (Table 1). With the exception of a slightly elevated
molecular weight (MW = 576), the physicochemical proper-
ties of H-Dbcp-Tic-OH are in agreement with Lipinski’s rule
of five, and this compound may turn out to be of interest as a
pharmacological tool. Unlike peptides 2 and 3, which have a
weakδ partialagonistcomponent, 6 and 7 showed no δ opioid
agonist activity in the MVD assay at concentrations up to
10 μM.

6944 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Berezowska et al.
Studies of flexible docking of ligands to the δ opioid
receptor were performed using Mosberg’s models of the
receptor in the inactive and the activated state. The tetrapep-
tides H-Bcp-Tic-Phe-Phe-OH (1, δ agonist) and H-Dbcp-Tic-
Phe-Phe-OH (6, δ antagonist) were docked to the activated
and the inactive forms of the δ receptor, respectively
(Figure 2). In general, a comparison of the ligand-receptor
interactions of agonist 1 bound to the active form of the
receptor with those of antagonist 6 bound to the inactive
receptor form revealed that many of the interactions involved
the same receptor residues, with notable exceptions. In both
cases, the N-terminal amino group of the ligand formed a salt
bridge with Asp128 of the receptor. The Phe4 residue of the two
peptides bound to their respective receptor forms occupied
˚
only slightly different receptor regions, 2-3 A apart. While in
both cases interactions with Leu200 and Trp284 are seen, the
Phe4 side chain of 1 also interacts with Gln201 and Ser204 in the
active form of the receptor. The Phe3 residue in both peptides
interacts with Val281, Trp284, and Leu300 and, additionally,
withCys303 inthe case of agonist 1 and withIle304 inthe case of
antagonist 6. The Tic2 residue in the two ligands also occupies
a very similar receptor site, interacting with Met199 and
Leu300, and in addition, this residue in the agonist interacts
with Cys303, while in the antagonist it is close to Ile304. In both
ligands the phenyl ring of the phenylalanine portion of the 1-
position residue interacts with Tyr129, Met132, Phe133, and
Trp274 and additionally with Ile277 in the case of the antago-
nist.
Figure 3. Conformations of H-Bcp-Tic-OH (3, yellow), H-Dbcp-
Tic-Phe-Phe-OH (6, green), and H-Dbcp-Tic-OH (7, red) bound to
the δ receptor in the inactive state.
region as the phenyl rings of Phe3 and Phe4 in the tetrapeptide,
particularly with residues Leu200 and Trp284. These interac-
tions may explain the increased δ receptor binding affinity of
H-Dbcp-Tic-OH compared to H-Dmt-Tic-OH.
Conclusions
Replacement of the Tyr1 residue in TIPP peptides with a
Bcp or Dbcp residue resulted in a general increase in δ opioid
receptor binding affinity. All Bcp1- or Dbcp1- tetra-, tri-, and
dipeptide analogues displayed similar subnanomolar δ re-
ceptor binding affinity and high δ receptor binding selectiv-
ity. The dipeptide H-Dbcp-Tic-OH (7) is a low molecular
weight δ opioid antagonist with subnanomolar δ receptor
binding affinity and extraordinarily high δ receptor selectiv-
ity and represents a novel pharmacological tool. The ob-
servation that [Bcp1]TIPP (1) is a δ opioid agonist, whereas
[Dbcp1]TIPP (6) is a δ antagonist, is of great significance.
The flexible docking studies performed with these two
tetrapeptides indicated that the large biphenylethyl group
contained in the Bcp residue of the δ agonist interacts with an
accessory binding site of the activated receptor distinct from
the binding site of the biphenylethyl group of the Dbcp
residue of the δ antagonist bound to the receptor in the
inactive state. This finding demonstrates that the differential
orientation of a large hydrophobic substituent introduced at
an appropriate site of a ligand may result in the induction of
or interaction with a distinct active or inactive receptor
conformation. In the two docked dipeptide antagonists,
the biphenylethyl moiety assumes yet another very different
orientation, permitting it to interact with a receptor region
similar to that with which the Phe3 and Phe4 residues of the
tetrapeptide antagonist (6) interact. These results indicate
that the δ opioid receptor can accommodate the bipheny-
lethyl moiety contained in the 1-position residue of TIPP
peptides in a number of different binding modes that pro-
mote the interaction of the ligand with the receptor in an
activated or an inactive state. In future studies we will replace
the biphenylethyl group in the Bcp1- and Dbcp1-TIPP
analogues described here with a variety of substituents
containing large aromatic moieties that might interact with
different hydrophobic subsites at the δ receptor, in an effort
to further explore the agonist vs antagonist behavior of this
class of δ opioid receptor ligands.
Interestingly, the biphenylethyl substituent in agonist 1 and
antagonist 6 interacts with different receptor regions and are
˚
seen to be about 4-5 A apart from each other when the two
receptor complexes are compared. While the biphenyl moiety
of both ligands interacts with Lys214 and Phe218 of the
receptor, it differentially interacts with Glu201, Ser204
,
,
Asp210, and Thr211 in the case of the agonist and with Ile183
Thr213, and Val217 in the case of the antagonist. The different
positioning of the biphenyl moiety in the agonist- vs antag-
onist-receptor complexes appears to be due to additional
hydrophobic interactions of the 20,60-dimethyl groups of
Dbcp1 in the antagonist with Tyr127 and Trp274 of the receptor
in the inactive state and is reflected in different dihedral angles
for receptor-bound peptide 1 (Ψ1 = 89°, χ1 = 177°, χ2 = 69°)
vs receptor-bound peptide 6 (Ψ1 = 132°, χ1 = 161°, χ2 =
89°).
Flexible docking of the dipeptide antagonists H-Bcp-Tic-
OH (3) and Dbcp-Tic-OH (7) to the δ receptor in the inactive
state was also performed. A superposition of the receptor-
bound conformations of the dipeptide antagonists3 and 7 and
the tetrapeptide antagonist 6 is shown in Figure 3. As in the
case of the tetrapeptide, the N-terminal amino group of the
two dipeptides forms a salt bridge with Asp128 of the receptor.
The Tic2 residue and the phenyl ring of the phenylalanine
portion of the 1-position residue occupy the same receptor
binding subsites as the corresponding moieties in the tetra-
peptide. However, in comparison with the 20,60-dimethylphe-
nyl moiety in docked tetrapeptide 6, that same moiety in the
docked dipeptides 3 and 7 has a twisted orientation. As a
consequence of this twist, the interaction of one of the 20,60-
dimethyl groups in dipeptide 7 with Trp274, as seen in the case
of the tetrapeptide, is lost, whereas one of the methyl groups
still interacts with Tyr129 as in the case of the tetrapeptide.
Most interestingly, the biphenyl moiety of the dipeptides
assumes a completely different orientation compared to the
one in the tetrapeptide and interacts with the same receptor

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6945
References
Experimental Section
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Carboxamido)phenylalanine is a Surrogate for Tyrosine in Opioid
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Peptide Synthesis. Peptides wereprepared bythe manual solid-
phase technique using Boc protection for the R-amino group and
DIC/HOBt or HBTU/DIEA as coupling agents. Peptides 1, 2, 4,
5, 6, and 12 were assembled on a polystyrene-divinylbenzene
(1%) resin (100-200mesh) (Boc-Phe-resin, 0.65 equiv/g, Bachem
Bioscience, King of Prussia, PA). Forthe synthesis of dipeptides3
and 7 a Boc-Tic-resin (0.35 equiv./g) was prepared according to
the Gisin method,13 using a Merrifield resin (D-2120, 100-200
mesh, 1.05 mM/g Bachem Bioscience). Peptide assembly was
carried out according to a published protocol.14 To introduce the
reduced peptide bond between the Tic2 and Phe3 (or Cha3)
residues, a reductive alkylation reaction15 between 2-Boc-
1,2,3,4-tetrahydroisoquinoline-3-aldehyde14 and the amino
group of the resin-bound H-Phe-Phe- or H-Cha-Phe- dipeptide
was performed as follows. 2-Boc-1,2,3,4-tetrahydroisoquinoline-
3-aldehyde (2 equiv) in DMF containing 1% AcOH was added to
the resin. Sodium cyanoborohydride (5.0 equiv) was then added
portionwise over a period of 40 min, and the reaction was allowed
to continue for 3 h. After the resin was washed, deprotection and
coupling of the Boc-Bcp-OH residue were performed according
tothe standardprotocol. Peptideswerecleaved fromthe resinand
deprotected by HF/anisole treatment in the usual manner. After
evaporation of the HF, the resin was extracted three times with
Et2O and subsequently three times with glacial AcOH. The
peptides were obtained in solid form through lyophilization of
the acetic acid extracts. Peptides were purified by preparative
reversed phase HPLC. Analytical parameters are listed in Sup-
porting Information.
Theoretical Conformational Analyses. All calculations were
performed using the molecular modeling software SYBYL,
version 7.0 (Tripos Associates). The standard Tripos force field
was used for energy calculations and a dielectric constant of 1
was used. The TIPP analogues were constructed as previously
described,16,17 using the standard fragment library in SYBYL,
and were subjected to 300 ps of molecular dynamics simulation.
Conformations were sampled every 10 ps and were minimized,
and the lowest-energy conformation from the dynamics simula-
tion was used as the starting structure for flexible docking to the
receptor. The resulting overall conformations of the Bcp1- and
Dbcp1-peptide analogues were similar to the previously pro-
posed δ receptor-bound conformations of TIPP peptides.16,17
Models of the δ opioid receptor in the inactive and in the
activated state, constructed by Mosberg et al. by homology
modeling based on the crystal structure of rhodopsin (http://
mosberglab.phar.umich.edu/resources/), were used in the dock-
ing studies. Flexible docking was performed using the software
(9) Schiller, P. W.; Nguyen, T. M.-D.; Weltrowska, G.; Wilkes, B. C.;
Marsden, B. J.; Lemieux, C.; Chung, N. N. Differential Stereo-
chemical Requirements of μ vs. δ Opioid Receptors for Ligand
Binding and Signal Transduction: Development of a Class of
Potent and Highly δ-Selective Peptide Antagonists. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 11871–11875.
(10) Schiller, P. W.; Weltrowska, G.; Berezowska, I.; Nguyen, T. M.-D.;
Wilkes, B. C.; Lemieux, C.; Chung, N. N. The TIPP Opioid Peptide
Family: Development of δ Antagonists, δ Agonists, and Mixed μ
Agonist/δ Antagonists. Biopolymers 1999, 51, 411–425.
(11) Salvadori, S.; Attila, M.; Balboni, G.; Bianchi, C.; Bryant, S. D.;
Crescenzi, O.; Guerrini, R.; Picone, D.; Tancredi, T.; Temussi, P.
A.; Lazarus, L. H. δ Opioidmimetic Antagonists: Prototypes for
Designing a New Generation of Ultraselective Opioid Peptides.
Mol. Med. 1995, 1, 678–689.
(12) Wang, W.; Obeyesekere, N. U.; McMurray, J. S. Stereospecific
Synthesis of 4-Carboxyphenylalanine and Derivatives for Use in
Fmoc-Based Solid-Phase Peptide Synthesis. Tetrahedron Lett.
1996, 37, 6661–6664.
(13) Gisin, B. F. The Preparation of Merrifield-Resins through Total
Esterification with Cesium Salts. Helv. Chim. Acta 1973, 56, 1476–
1482.
(14) Schiller, P. W.; Weltrowska, G.; Nguyen, T. M.-D.; Wilkes, B. C.;
Chung, N. N.; Lemieux, C. TIPP[Ψ]: A Highly Potent and Stable
Pseudopeptide δ Opioid Receptor Antagonist with Extraordinary
δ Selectivity. J. Med. Chem. 1993, 36, 3182–3187.
(15) Sasaki, Y.; Coy, D. H. Solid Phase Synthesis of Peptides Contain-
ing the CH2NH Peptide Bond Isostere. Peptides 1987, 8, 119–121.
(16) Wilkes, B. C.; Schiller, P. W. Comparative Analysis of Various
Proposed Models of the Receptor-Bound Conformation of TIP-
(P)-Related δ Opioid Antagonists. Biopolymers 1995, 37, 391–400.
(17) Wilkes, B. C.; Nguyen, T. M.-D.; Weltrowska, G.; Carpenter, K.
A.; Lemieux, C.; Chung, N. N.; Schiller, P. W. The Receptor-
Bound Conformation of H-Tyr-Tic-(Phe-Phe)-OH-Related δ-
Opioid Antagonists Contains All Trans Peptide Bonds. J. Pept.
Res. 1998, 51, 386–394.
€
program GLIDE (Schrodinger LLC). Each of the resulting
ligand-receptor complexes was minimized using the conjugate
gradient approach.18 Molecular dynamics simulations of 100 ps
at 300 K were performed in order to assess the stability of each
complex. In each case no significant change in the complex
structure was observed during the simulation.
Acknowledgment. This work was financially supported by
the U.S. National Institutes of Health (Grant DA-004443)
and the Canadian Institutes of Health Research (Grant MOP-
89716).
Supporting Information Available: Experimental details and
refs 19-28. This material is available free of charge via the
Internet at http://pubs.acs.org.
(18) Powell, M. J. D. Restart Procedures for the Conjugate Gradient
Method. Math. Programming 1977, 12, 241–254.