The importance of micelle-bound states for the bioactivities of bifunctional peptide derivatives for δ/μ opioid receptor agonists and neurokinin 1 receptor antagonists

Article abstract of DOI:10.1021/jm800389v

To provide new insight into the determining factors of membrane-bound peptide conformation that might play an important role in peptide-receptor docking and further biological behaviors, the dodecylphosphocholine (DPC) micelle-bound conformations of bifun

Full text of DOI:10.1021/jm800389v

6334
J. Med. Chem. 2008, 51, 6334–6347
The Importance of Micelle-Bound States for the Bioactivities of Bifunctional Peptide Derivatives
for δ/µ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists
Takashi Yamamoto,† Padma Nair,† Neil E. Jacobsen,† Peg Davis,‡ Shou-wu Ma,‡ Edita Navratilova,‡ Sharif Moye,‡
Josephine Lai,‡ Henry I. Yamamura,‡,3 Todd W. Vanderah,‡ Frank Porreca,‡ and Victor J. Hruby*,†
Department of Chemistry, UniVersity of Arizona, 1306 East UniVersity BouleVard, Tucson, Arizona 85721, Department of Pharmacology,
UniVersity of Arizona, 1501 North Campbell AVenue, Tucson, Arizona 85724
ReceiVed April 4, 2008
To provide new insight into the determining factors of membrane-bound peptide conformation that might
play an important role in peptide-receptor docking and further biological behaviors, the dodecylphospho-
choline (DPC) micelle-bound conformations of bifunctional peptide derivatives of δ-preferring opioid agonists
and NK1 antagonists (1: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF3)2; 2: Tyr-D-Ala-Gly-Phe-Met-
Pro-Leu-Trp-NH-3,5-Bzl(CF3)2; 3: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bzl) were determined based
on 2D NMR studies. Although the differences in the primary sequence were limited to the C-terminus, the
obtained NMR conformations were unexpectedly different for each compound. Moreover, their biological
activities showed different trends in direct relation to the compound-specific conformations in DPC micelles.
The important result is that not only were the NK1 antagonist activities different (the pharmacophore located
at the C-terminus)but the opioid agonist activities (this pharmacophore was at the structurally preserved
N-terminus) also were shifted, suggesting that a general conformational change in the bioactive state was
induced due to relatively small and limited structural modifications.
Introduction
a peptide should be important especially for their conformational
definitions.17
The importance of peptide and protein conformation has
become increasingly appreciated recently, as their biological
importance has been widely recognized. One notable example
is protein misfolding, which has been implicated in a large
number of diseases such as protein folding disorders (PFD).1-3
In the diseases known as amyloidoses,3 large quantities of
misfolded proteins or peptides undergo aggregation, resulting
in destroying brain cells and other tissues. These diseases, such
as Alzheimer’s disease and Parkinson’s disease, have high rates
of pathogenesis and give rise to the huge social problems all
over the world. The Alzheimer’s ꢀ-amyloid protein (AꢀP) is
the well-known offending substance whose conformational
change to the ꢀ-sheet oligomer and subsequent aggregation was
shown to enhance its neurotoxicity.3-5 Another important
misfolded protein is the cellular prion protein PrPC, whose
secondary structure was changed into a ꢀ-rich conformation
(PrPSc) to cause the prion diseases.6 Thus, many scientific efforts
have been made to seek the origin of such misfoldings.7-10
However, the full picture of their mechanisms is still largely
unknown.
Many G-protein coupled receptors (GPCRs), which are the
typical membrane-bound proteins, have their ligand binding sites
inornearthelipophilictrans-membrane(TM)domains.18-21Because
the docking event of such a receptor and a ligand must take place
near the membrane, ligand-membrane interactions should be
important and this research topic has been occasionally explored
for decades.22,23 In fact, it has been pointed out that the membrane
promotes ligand-receptor docking24-27 and that the ligand adop-
tion into membrane followed by membrane-catalyzed 2D search
should be more efficient than the three-dimensional ligand-receptor
binding through solvent space.12,13,23,24,28 Hence, understanding
ligand-membrane interactions and membrane-bound structures of
ligands is indispensable for further insight into their diverse
biological behaviors.12,13,28
The opioid and NK1a receptors are both GPCRs which have
significant importance in pain signal transmission, and their
a Abbreviations: Abbreviations used for amino acids and designation of
peptides follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J. Biol. Chem. 1972, 247, 977-983. The following additional
abbreviations are used: AcOH, acetic acid; Boc, tert-butyloxycarbonyl; BuOH,
butanol; Bzl, benzyl; BSA, bovine serum albumin; CCK, cholecystokinin; Cl-
HOBt, 1-hydroxy-6-chlorobenzotriazole; CHO, Chinese hamster ovary; DMEM,
Dulbecco’s modified Eagle’s medium; DCM, dichloromethane; DIEA, diiso-
propylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide;
DOR, δ opioid receptor; DPC, Dodecylphosphocholine; DPDPE, c[D-Pen2,D-
Pen5]-enkephalin; DQF-COSY, double quantum filtered correlation spectros-
copy; DAMGO, [D-Ala2,NMePhe4,Gly5-ol]-enkephalin; ESI, electrospray
ionization; Fmoc, fluorenylmethoxycarbonyl; GPI, guinea pig isolated ileum;
HCTU, 1H-benzotriazolium-1-[bis(dimethylamino)methylene]-5-chloro- hexaflu-
orophosphate-(1-),3-oxide; HEK, human embryonic kidney; HRMS; high-
resolution mass spectroscopy; LMMP, longitudinal muscle/myenteric plexus;
MOR, µ opioid receptor; MVD, mouse vas deferens; NK1, neurokinin-1;
NOESY, nuclear Overhauser enhancement; rmsd, root-mean-square deviation;
RP-HPLC, reverse phase high performance liquid chromatography; SAR,
structure-activity relationships; SEM, mean standard error; SPPS, solid phase
peptide synthesis; TFA, trifluoroacetic acid; TOCSY, total correlation spec-
troscopy; Trp-NH-3,5-Bzl(CF3)2, 3′,5′-(bistrifluoromethyl)-benzyl amide of
tryptophan; Trp-NH-Bzl, benzyl amide of tryptophan; Trp-O-3,5-Bzl(CF3)2,
3′,5′-(bistrifluoromethyl)-benzyl ester of tryptophan.
The conformation of small and linear peptides, such as the
endogenous opioid peptide enkephalin11 and its analogues,12,13
have been thought to have “random” conformations in aqueous
solution due to their high flexibility.14 However, in the presence
of trifluoroethanol (TFE) or membrane-mimicking surroundings,
they could have structured conformations because their second-
ary structural elements can be stabilized by the environmental
effects.12,13,15,16 Therefore, the circumstances surrounding such
* To whom correspondence should be addressed. Phone: (520)-621-6332.
Fax: (520)-621-8407. E-mail: hruby@email.arizona.edu.
†
Department of Chemistry, University of Arizona.
Department of Pharmacology, University of Arizona.
Dedicated to the memory of Professor Henry “Hank” I. Yamamura
‡
3
our dear friend, colleague and teacher who passed away peacefully on the
evening of September 4, 2008.
10.1021/jm800389v CCC: $40.75
2008 American Chemical Society
Published on Web 09/27/2008

δ/µ Opioid Receptor Agonists and NK1 Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6335
pig ileum (GPI) and mouse isolated vas deferens (MVD)
tissues.38,40,50,51
Results and Discussion
Peptide Synthesis. The peptide derivatives 1-3 (Figure 1)
were synthesized using previously published methods.40 Briefly,
Boc-Tyr(tBu)-D-Ala-Gly-Phe-Met-Pro-Leu-Trp(Boc)-OH was
synthesized using NR-Fmoc chemistry with HBTU as the
coupling reagent on a 2-chlorotrityl resin, followed by esteri-
fication or amidation of the protected C-terminal intermediate.
The esterification was performed employing cesium carbonate
to form the cesium salt of the protected peptide to react with
3′,5′-bis(trifluoromethyl)benzyl bromide, and standard EDC/Cl-
HOBt coupling chemistry with two equivalents of reactant amine
was used for the amidation. Final deprotection with the cleavage
cocktail (82.5% v/v TFA, 5% water, 5% thioanisole, 2.5% 1,2-
ethanedithiol, and 5% phenol to quench the highly stabilized
carbocations released from permanent protecting groups) gave
the final crude peptides, which were purified by RP-HPLC
(>99%) and characterized by analytical HPLC, 1H NMR,
HRMS, and TLC. The assignments of NMR resonances are
available in the Supporting Information.
Figure 1. Sequence of the bifunctional ligands synthesized and
examined.
ligand binding sites are found in or near the TM domains.20,21
The opioid receptors are classified into three subtypes (µ, δ,
and κ) and have endogenous peptides as their ligands such as
endomorphin, enkephalin, and dynorphin.20,29,30 It is well-known
that the 11-amino acid peptide substance P acts as an excitatory
and pronociceptive neurotransmitter of pain signaling through
the neurokinin-1 NK1 receptor.31-37 Recently, combining the
agonist effect at the opioid receptors together with the blocking
of signals through NK1 receptors has been reported to have
several merits for the analgesic effects such as enhanced potency
and the prevention of opioid-induced tolerance.31-37 Thus, we
have been developing in one molecule bifunctional peptide
derivatives that act as opioid agonists and NK1 antagonists, with
extensive discussions on their structure-activity relationships
(SAR), which were based on their primary sequences.38-40
However, their three-dimensional conformations in membrane
and compound-membrane interactions should also be important
for further understanding the biological activities of ligands both
in vitro and in vivo.
In the present article, we report our new findings that small
and local structural modifications induced large conformational
changes in membrane-mimicking dodecylphosphocholine (DPC)
micelles,41-46 together with a discussion of their relevance to
the biological activities. The starting point of our bifunctional
design was the successful C-terminal modifications on TY001
(Tyr1-D-Ala2-Gly3-Phe4-Pro5-Leu6-Trp7-O-3′,5′-Bzl(CF3)2) to
yield several potent bifunctional derivatives with better affinities
at the µ opioid receptors rather than at the δ receptor.40 Because
the selective agonists at the δ opioid receptor have analgesic
activity with fewer adverse effects, but less potency than
µ-selective drugs,47-49 a δ-selective opioid agonist with en-
hanced analgesic activity, which also has NK1 receptor antago-
nist activity, provides a novel way to find a potent drug candidate
for prolonged pain control. Thus, 1 (TY005: Tyr1-D-Ala2-Gly3-
Phe4-Met5-Pro6- Leu7-Trp8-O-3′,5′-Bzl(CF3)2), which has a Met5
as the key amino acid residue for the δ-opioid selectivity and
has been shown to have potent analgesic activities in vivo,38-40
was chosen as the current lead compound to find novel
δ-preferring bifunctional ligands with strong analgesic potency.
The C-terminus of 1 was modified to give two novel derivatives
2 (TY027: Tyr1-D-Ala2-Gly3-Phe4-Met5-Pro6-Leu7-Trp8-NH-
3′,5′-Bzl(CF3)2) and 3 (TY025: Tyr1-D-Ala2-Gly3-Phe4-Met5-
Pro6-Leu7-Trp8-NH-Bzl) (Figure 1).
Secondary Structure Analysis Based on Assigned 1H
NMR. NMR experiments were performed to obtain structural
information in DPC micelles as previously reported.45,46 Two-
dimensional NMR studies including TOCSY, DQF-COSY, and
NOESY, were performed on all three bifunctional peptide
derivatives 1-3 in pH 4.5 buffer (45 mM CD3CO2Na/HCl,
1 mM NaN3, 90% H2O/10% D2O) with 40-fold perdeuterated
DPC micelles. At concentrations above the critical micelle point,
DPC forms micelles with an aggregate number of 50-90,
corresponding to one or two peptide molecules per micelle.52
The obtained NOESY data showed the reasonably good
quality as seen in Figure 2. A total of 136, 155, and 184
nonredundant NOE restraints were used for 1, 2, and 3,
respectively, based on the NOESY cross-peak volumes including
sequential (50, 63, and 72, respectively), medium-range (2-4
residues; 31, 36, and 46, respectively), and long-range (1, 0,
and 3, respectively) restraints. The distribution of these restraints
along the peptide chain is shown in Figure 3D. Only one
dihedral angle restraint was used: the Leu ꢁ angle in 3. The
total numbers of restraints were 136, 155, and 185, respectively
(15.1, 17.2, and 20.4 per residue). The large numbers of NOEs
per residue for small and linear peptides suggest that the peptide
derivatives exist in well-defined conformations in the DPC
micelles.
3
The interresidual NOE connectivities and the JHN-HR cou-
pling constants of all of the peptide derivatives are illustrated
in Figure 3. The C-terminal benzyl moiety of 2 and 3 are
3
represented as residue 9. The JHN-HR coupling constants for
all residues in all three peptide derivatives were within the range
of 6-8 Hz except for Leu7 in 3. This is most likely due to
conformational averaging of the peptides in solution.53 The
observed NOE patterns, including dNN(i, i + 1), dRN(i, i + 1)
and some medium-range (i, i + 2 or 3) connectivities, suggest
the possibility of ꢀ-turn structures around residues 1-4 in all
three peptide derivatives as well as around residues 5-8 in 2.54
A few longer-range dRN(i, i + 3) and dRN(i, i + 4) connectivities
found in 3 indicate the existence of a helical structure in this
molecule, consistent with its HR CSI pattern (Figure 3C).54
Structural Calculations. The 20 structures with the lowest
total energies after rMD refinement were used to represent the
structure of the peptide derivatives in DPC micelles. Throughout
Our main intention for these structural modifications was to
seek induced conformational changes in the presence of lipid
media and to examine the influences of such changes on
manipulating bioactivities. Therefore, the NMR conformations
of 1-3 were determined in aqueous solution with membrane-
mimicking perdeuterated dodecylphosphocholine (DPC) mi-
celles. Fluorescence experiments as well as additional NMR
studies using paramagnetic agents also were performed to
determine the difference in membrane-compound interactions.
Moreover, the biological activities of 2 and 3 were extensively
evaluated using radioligand binding assays, GTPγS binding
assays, and tissue-based functional experiments using guinea
1
the H NMR studies, only one major rotamer was found for

6336 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Yamamoto et al.
Figure 2. Fingerprint (HN-HR) region of the NOESY spectrum of (A) 1, (B) 2, and (C) 3 in DPC micelles. Intraresidue HN-HR NOE cross-peaks
are labeled with residue numbers, and arrows indicate the connectivity path from N-terminal to C-terminal. X9 represents the cross-peaks derived
from the corresponding C-terminal HN and benzyl protons.
Figure 3. Diagram of HN-HR coupling constants, NOE connectivities, and HR chemical shift index (CSI) for (A) 1, (B) 2, and (C) 3. The HR CSI
was calculated using the random-coil values reported by Andersen et al.86,87 The residual interresidue NOE distance restraints of 1 (left), 2 (middle),
and 3 (right) (D). Each column shows the sequential (i, i + 1; open), medium-range (i, i + 2-4; hatched) and long-range restraints (i, i + >4;
filled), respectively. The residue Bzl or 9 stands for the respective C-terminal moieties.
peptides 1-3 and the populations of minor rotamers were all
negligible. The Met5-Pro6 bond of the major rotamers were
fixed in the trans configuration based on the observations of
5HR to Pro6 Hδ sequential NOEs together with the absence of
sequential 5HR-6HR NOEs in the structural calculations of 1-3.
On the basis of the observations, cis-isomers of peptides 1-3
have negligible population in the lipid-mimicking DPC micelles
and thus considered to have negligible effects on the biological
obtained low-energy structures did not correspond to a single
conformation under these experimental conditions. However,
the ensemble of an appropriate number of obtained structures
provides sufficient structural figures based on the “averaged”
or “dynamic” view of the conformations. Thus, statistics for
the 20 best structures were performed as shown in Table 1. The
average restraint violation energies were low (2.48, 2.95, and
1.13 kcal mol-1 for 1, 2, and 3, respectively), with average
maximum NOE distance violations of 0.17, 0.11, and 0.11 Å
with no dihedral angle violations. The 19 structures were aligned
3
behaviors. Because the experimental NOEs and JHN-HR were
the averaged values in the NMR experiment time course, the

δ/µ Opioid Receptor Agonists and NK1 Receptor Antagonists
Table 1. Structural Statistics
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6337
compd 1
compd 2
final 20
compd 3
final 20
final 20
structures
most stable
structure
most stable
structure
most stable
structure
structures
structures
rmsd from NOE dist restraints (Å)a
rmsd from backbone ꢁ angle restraints (deg)b
NOE dist restraints violations
>0.01 Å
0.025 ( 0.004
0.028
c
0.027 ( 0.004
0.016
c
0.016 ( 0.001
0.0 ( 0.0
0.016
0.0
c
c
13.9 ( 2.5
2.5 ( 1.4
0.17 ( 0.04
11
3
0.22
14.6 ( 1.5
3.9 ( 1.2
0.16 ( 0.02
14
3
0.13
14.2 ( 1.1
1.0 ( 0.0
0.11 ( 0.00
15
1
0.12
>0.1 Å
max dist violations (Å)
dihedral backbone angle violations
>0.1°
c
c
c
c
c
c
c
c
c
c
0 ( 0
0 ( 0
0 ( 0
0
0
0
>1°
c
c
max dihdral violations (deg)
rms deviation from ideal geometryd
bond length (Å)e
0.0061 ( 0.0004
2.14 ( 0.11
3.57 ( 0.63
0.0063
2.16
3.25
0.0052 ( 0.0002
1.78 ( 0.05
2.73 ( 0.40
9.08
2.95 ( 0.58
1.42 ( 0.08
12.8 ( 0.8
9.57 ( 1.61
0.74 ( 0.33
-12.23 ( 1.4
-11.5 ( 0.68
-0.01 ( 2.25
0.0052
1.72
2.90
0.0035 ( 0.00004
1.25 ( 0.003
1.54 ( 0.09
0.0035
1.25
1.45
bond valence angles (deg)f
out-of-plane angles (deg)g
AMBER energies (kcal mol-1
restrainth
)
2.48 ( 0.67
2.07 ( 0.22
19.28 ( 1.84
12.24 ( 1.9
1.63 ( 1.11
-11.65 ( 3.1
-9.6 ( 0.91
13.25 ( 2.12
2.78
2.20
19.49
9.54
1.15
-12.61
-9.93
9.08
2.41
1.40
11.89
7.99
0.61
-13.80
-11.84
-4.44
1.13 ( 0.07
1.68 ( 0.02
14.28 ( 0.22
14.52 ( 0.38
0.19 ( 0.03
-17.4 ( 0.75
-9.59 ( 0.41
3.14 ( 0.78
1.20
1.70
14.00
14.53
0.17
-18.41
-9.82
1.61
bond stretching
bond angles
dihedral angles
planarity
van der Waalsi
electrostaticj
total
atomic rmsd (Å): final 19 structures vs most stable structure
compd 1 compd 2
backbone
atoms (N, CR, C′) hydrogen atoms
compd 3
all non-
backbone atoms
all non-
hydrogen atoms
backbone atoms
all non-
(N, CR, C′)
(N, CR, C′)
hydrogen atoms
calculated on whole molecule
calculated only on 1-4 res.
calculated only on 5-8 res. and C-terminus
1.80 ( 0.47
1.11 ( 0.54
0.75 ( 0.26
2.72 ( 0.92
2.49 ( 1.12
1.82 ( 0.90
1.14 ( 0.43
1.05 ( 0.63
0.45 ( 0.38
2.09 ( 0.64
2.16 ( 0.98
1.02 ( 0.25
0.19 ( 0.20
0.14 ( 0.30
0.04 ( 0.01
0.84 ( 0.28
0.32 ( 0.66
0.76 ( 0.42
a The total number of NOE restraints were 136 for 1, 155 for 2, and 184 for 3, respectively. b Two backbone ꢁ angle restraints were applied only on 3.
c No restraints used. d Derived from the rMD calculations using the AMBER force field in DISCOVER. e The number of bond length were 160 for 1, 161
for 2, and 155 for 3, respectively. f The number of bond valence angles were 285 for 1, 287 for 2, and 275 for 3, respectively. g The number of out-of-plane
angles were 36 for 1, 36 for 2, and 37 for 3, respectively. h Calculated with force constants of 25 kcal mol-1 Å-2 and 100 kcal mol-1 rad-2 for the NOE
distance and dihedral angle restraints, respectively. i Calculated with the Lennard-Jones potential using the AMBER force field and a 12 Å cutoff. j Calculated
with a distance-dependent dielectric constant (ꢀ ) 4r).
with the most stable structure using all backbone atoms (Figure
4A), only the backbone atoms of residues 1-4 (Figure 4B), or
5-8 (Figure 4C). The backbone rmsd’s of the 19 structures
with respect to the most stable structure were 1.80, 1.14, and
0.19 Å for 1, 2, and 3, respectively, for all residues. The rmsd
values are significantly decreased if alignment is carried out
only on the backbone atoms of residues 5-8 (1: 0.75; 2: 0.45;
3: 0.04), indicating that the C-terminal half is much better
defined by the NMR restraints than the N-terminal half (residues
1-4). This may be due to greater flexibility in the N-terminal
portion. The decrease in rmsd going from a flexible ester (1) to
a more rigid amide (2) linkage at the C-terminus was expected,
but the much larger decrease resulting from removal of two
trifluoromethyl groups of 2 (3) was surprising.
2-5 for both 1 and 2, and all of them were classified as type
IV (“distorted”) ꢀ-turn by their backbone dihedral angles. A
second ꢀ-turn structure was found from Pro6 to the C-terminal
benzyl moiety (residue 9) in which the distance between the
CR(Pro6) and the benzylic carbon (CH2) of the C-terminus was
less than 7 Å in 17 and 19 of the best 20 structures for 1 and
2, respectively (Table 2). For peptide derivative 2, 6 of 19 turns
found in 2 were classified as type I ꢀ-turns, and a hydrogen
bond between the HN of residue 9 and the carbonyl oxygen
atom in Pro6 was observed in 9 of the 19 (Table 3). The
C-terminal ester (1) showed only distorted ꢀ-turns in this region,
with no hydrogen bonds, consistent with the larger backbone
rmsd observed for C-terminal half (0.75 Å vs 0.45 Å for 2).
These implied that the secondary structure elements were
consistent with the observed NOE connectivities (Figure 3).
Comparing the tandem ꢀ-turn structures of 1 and 2, the
peptide with no trifluoromethyl groups in the C-terminus (3)
showed significantly different structural properties. First, the
backbone of 3 had a well-defined helical structure, consistent
with the NOE connectivities and CSI values (Figure 4). It is
noteworthy that not only the C-terminal half-of 3, but also its
N-terminus was found to be quite structured (Table 1). Thus,
removal of two trifluoromethyl groups from the C-terminus led
to a drastic conformational change for the whole molecules from
tandem ꢀ-turn structures to a helical structure (Figure 4).
This well-defined structure of 3 was also confirmed by the
Ramachandran plot and angular order parameters (Figure 5).58
In Met-Enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5-OH) and Leu-
Enkephalin (Tyr1-Gly2-Gly3-Phe4-Leu5-OH), which form the
basis for the design of the N-terminal portion of peptide
sequence of 1-3, a ꢀ-turn structure was often found between
Tyr1 and Phe4 by several methods including X-ray crystal-
lography and NMR spectroscopy in environments which mimic
the membrane bilayer.55,56 In the case of peptide derivatives 1
and 2, a distance of less than 7 Å between the CR of D-Ala2
and the CR of Met5 was observed in 15 of the best 20 structures
for 1, and in all 20 structures for 2, whereas the ꢀ-turn between
Tyr1 and Phe4 were found in only 5 and 3 structures in the best
20 structures of 1 and 2, respectively (Table 2).57 This result
implied the existence of an alternative ꢀ-turn in the residues

6338 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Yamamoto et al.
Figure 4. Ensembles of the best 20 calculated structures in 40-fold DPC micelle/pH 4.5 buffer for (A) 1, (B) 2, and (C) 3 with the lowest restraint
energy, aligned on backbone atoms of residues (a) 1-8, (b) 1-4, and (c) 5-8, from N-terminal (up in the left image) to C-terminal (down). Only
backbone atoms were illustrated in (a) and (b) for easier comparison, and the most stable conformers (c) are shown with all non-hydrogen atoms.
Table 2. Number of Structures with Less than 7 Å Distance between R
Carbons of ith and (i + 3)th Residuesa
for 1 and 2, especially in the N-terminus where no structural
modification was made.
Tyr1-Phe4 D-Ala2-Met5 Gly3-Pro6 Met5-Trp8 Pro6-Bzl9
On the basis of the NMR structural analysis, it is clear that
the limited modifications at the C-terminal moiety gave rise to
several changes in the conformations of the peptide derivatives
1-3 in the presence of membrane-like DPC micelles. The
change of ester (1) into amide (2) resulted in a better-defined
conformation especially in the C-terminal portion of the peptide,
and a more rigid ꢀ-turn structural element with intramolecule
hydrogen-bond was found for residues 6-9 of 2. The removal
of the trifluoromethyl groups from the C-terminus of 3 induced
much larger changes in the three-dimensional structures, which
changed from a tandem ꢀ-turn (1 and 2) to a structured helical
conformation, with the smallest rmsd values for alignment at
residues 5-8 (Table 1), implying a large influence of the
trifluoromethyl groups on the conformations of the entire
molecules in the lipid environment. Because the structural
modifications were limited only to the C-terminus, it is not clear
how the simple electrostatic or steric changes of these modifica-
tions affects the conformations of the entire molecule. Because
the molar ratios of 1-3 were low compared to DPC, they
presumably form complexes with lipid molecules and thus
induce perturbations in the interaction between the compound
and micelles due to their unique C-terminus. It might be
suggested that the highly lipophilic and electronegative triflu-
oromethyl groups induced such a perturbation if these triflu-
oromethyl groups interact with the core of micelles, which
mostly consists of lipophilic and electrostatically neutral
hydrocarbon chains. The ester-to-amide substitution, in which
the oxygen atom (hydrogen-bond acceptor) was replaced to the
nitrogen with a proton to be a hydrogen-bond donor, also can
introduce some changes in the interactive mode between these
compounds and membrane-like micelles. Therefore, the interac-
tion of compound with DPC micelles might be compound-
specific, which is consistent with the different three-dimensional
conformations for each compound. To confirm these implica-
tions, further NMR experiments using paramagnetic agents as
residue
residue
residue
residue
residue
1
5
3
0
15
20
2
0
0
20
0
0
20
17
19
18
2
3b
a Out of the best 20 calculated structures. b Helical structure was found
in which no ꢀ-turn structures should not be defined according to the original
definition.57 Bzl stands for the cross-peaks derived from the corresponding
aromatic protons of benzyl moiety (residue 9).
Table 3. Observed Hydrogen Bondsa
molecule no.b
donor
acceptor distance (Å)c angle (deg)d
1
2
14
9
7
Leu7 HN
Met5
O
1.91 ( 0.07
2.16 ( 0.11
2.05 ( 0.11
2.04 ( 0.02
141.1 ( 6.0
158.5 ( 1.9
137.8 ( 8.1
132.3 ( 1.1
Bzl9 HNe Pro6
O
O
O
Gly3 HN
Trp8 HN
Tyr1
Met5
5
3
no hydrogen bond observed
a The hydrogen bonds which were observed in more than five structures
were listed. b The number of structures of the final 20 for which the listed
hydrogen bond is observed. c The distance is the mean proton-oxygen
distance ((SD) in the structures for which a hydrogen bond is observed.
d The angle is the mean N-H---O angle ((SD) in the structures for which
a hydrogen bond is observed. e Amide proton of C-terminal benzyl moiety.
In the Ramachandran plot of 3, only seven clear spots,
corresponding to the respective residues 2-8, were found.
Among them, only Gly3 has positive ꢁ angles in all of its 20
best structures. On the other hand, the corresponding Ram-
achandran plots for 1 and 2 showed more scattered views
together with positive ꢁ angles for Gly3 (10 structures in 1 and
3 structures in 2), Phe4 (3 structures in 1), Met5 (13 structures
in 1 and all 20 structures in 2), and Leu7 (1 structures in 1 and
6 structures in 2) in the seven L-amino acids and some of D-Ala2
(3 structures in 1 and 3 structures in 2) have negative ꢁ angles.
It is interesting that Met5 of 1 and 2, located between two
ꢀ-turns, had frequent positive ꢁ angles. As for the angular order
parameters, both the parameters for ꢁ and ψ angles in 3 were
close to 1 in all the residues, whereas 1 and 2 had smaller values
in some residues, implying a better-defined structure for 3 than

δ/µ Opioid Receptor Agonists and NK1 Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6339
Figure 5. The D-Ala2 (crosses), Gly3 (open circle), and Met5 with positive ꢁ angles (circled) were indicated in the Ramachandran ꢁ,ψ plots for
(A) 1, (B) 2, and (C) 3 for residues 2-7 of 20 final structures. Angular order parameters for ꢁ (D) and ψ (E) angles calculated from the 20 final
structures for 1 (open circles), 2 (filled squares), and 3 (crosses). For calculating the ψ angles of Trp8, noncarbonyl oxygen atoms of the C-terminal
ester (1) and nitrogen atoms of C-terminal amide (2 and 3) were used instead of N (i + 3), respectively.
Table 4. Solubility and Lipophilicity of Peptide Derivatives
ester (1) were less lipophilic than was the case for the C-terminal
amides (2 and 3), both of which had better-defined structures
in DPC micelles than 1 (Table 1). Therefore, the lipophilicity
near Trp8 was mostly influenced by ester-amide substitution
rather than the removal of trifluoromethyl groups, and the
lipophilic environment at the C-terminus might have some
responsibility for the structured conformations of the peptide
derivatives.
lipophilicity
a
no.
logD7.4
AlogPb
solubilityc (µg/mL)
1
2
3
>4.0
>4.0
3.6
5.74
5.45
3.97
<0.2
<0.2
1.1
a Logarithm of octanol/saline distribution coefficient in 0.05 N HEPES
buffer in 0.1 N NaCl solution. b Calculated with ALOGPS 2.1 software.
See refs 83, 84. c Solubility in 0.05 N HEPES buffer in 0.1 N NaCl solution.
Paramagnetic Broadening Studies on 1H NMR. To obtain
further information about the interaction between peptide
derivatives 1-3 and DPC micelles, we used a nitroxyl spin-
labeled 5-doxylstearic acid (5-DOXYL), and Mn2+ ions (MnCl2)
to induce selective broadening of NMR resonances close to the
paramagnetic probes.12,62 The cross-peaks of protons exposed
to an aqueous exterior are broadened or disappear due to the
paramagnetic effect of Mn2+, while cross-peaks of protons
located inside the micelles and close to the phosphate groups
of DPC are broadened by the free radical on the doxyl group,
which is bound to carbon 5 of the stearic acid. The paramagnetic
effects of these agents on the peptide resonances were studied
by comparing TOCSY spectra in the presence and absence of
the paramagnetic agents, and all peaks were classified into three
categories according to their sensitivities to the paramagnetic
agents: missed by 5-DOXYL only (in the micelle, but not deeply
buried), missed by both Mn2+ and 5-DOXYL (the proton is at
or near the surface of micelles) and preserved by either agent
(deeply buried in the micelle) (Figure 7 and Supporting
well as fluorescence experiments were performed to estimate
such differences in the membrane-compound interactions.
Fluorescence Study. It is well-known that the intrinsic
fluorescence spectrum of the indole ring in tryptophan shifts to
shorter wavelength (“blue-shifted”) as the polarity of the solvent
surrounding the tryptophan residue decreases, and this blue-
shift is a good index to monitor the lipophilicity of the
environment close to the tryptophan.59,60 The fluorescence of
Trp8 in 1-3 was measured in the presence and absence of DPC
micelles in order to examine the interaction between the peptides
and membrane-like micelles. The fluorescence spectra in DPC
micelles were compared to the spectra in the EtOH-buffer
solution (EtOH:pH 7.4 HEPES buffer ) 1:1), which was chosen
as the standard because the solubilities of the peptide derivatives
1-3 in aqueous media were too low to run the experiment
(Table 4).61 The emission spectra were obtained by excitation
at 290 nm to avoid excitement of the tyrosine residue.
Obvious blue-shifts of fluorescence spectra from the standard
solution were observed in all three peptide derivatives (6 nm,
1; 10 nm, 2; 10 nm, 3, respectively, Figure 6), implying that
the indole ring in Trp8 at the C-terminal of all peptides was
buried inside the micelles and that the peptides have strong
interactions with the lipophilic core of the micelles, at least the
C-terminus.
1
Information). In fact, none of the H resonances were missed
by Mn2+ only, indicating a strong association with the micelle
for all three derivatives.
For all three peptide derivatives 1-3, nearly all of the HN
related cross-peaks were categorized as sensitive to both Mn2+
and 5-DOXYL, implying that the peptide backbones are located
at or near the surface of micelles. On the other hand, most of
the side chain resonances were missed only by 5-DOXYL or
were nonsensitive to either agent. Thus, generally, the backbones
of 1-3 lie close to the surface with their side chains buried in
the micelles. However, there is one notable exception to this
general observation: the HN resonances of Met5 in 1-3 were
affected only by 5-DOXYL, implying that the protons are not
Because poorly water-soluble peptides 1-3 were easily
dissolved at millimolar concentration in the presence of micelles,
strong interactions between the peptide derivatives 1-3 and
micelles are also suggested (Table 4). Among compounds 1-3,
1 showed a smaller blue-shift than that of 2 and 3, suggesting
that the surroundings near the indole ring of Trp8 in C-terminal

6340 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Yamamoto et al.
Figure 6. Fluorescence spectra of (A) 1, (B) 2, (C) 3: the spectra were recorded at 500 µg/mL at 290 nm excitation in 40-fold DPC micelle/pH
7.4 HEPES buffer (solid line) or EtOH:pH 7.4 HEPES buffer ) 1:1 solution (broken line).
Figure 7. Typical example of the paramagnetic effects on TOCSY spectra. The aromatic region of 3 with DPC micelles (left column), with 200
µM Mn2+ (middle) and 5-DOXYL stearic acid (right). Preserved resonances (labeled) are in a phase not missed by the phase-specific radical probe
(Mn2+ or DOXYL). Spectra were compared from the same noise level. The full spectra for 1-3 are available in the Supporting Information.
Table 5. Binding Affinities of Bifunctional Peptides at δ/µ Opioid Receptors and NK1 Receptors
hDORa, [3H]DPDPEb
rMORa, [3H]DAMGOc
hNK1d, [3H]substance Pe
rNK1d, [3H]substance Pf
Ki(hNK1)/
Ki(rNK1)
g
g
g
g
no.
LogIC50
Ki (nM)
LogIC50
Ki (nM)
Ki(µ)/Ki(δ)
LogIC50
Ki (nM)
LogIC50
Ki (nM)
1h
2
3
-8.2 ( 0.06
-8.8 ( 0.07
-9.1 ( 0.09
2.8
-7.1 ( 0.11
-7.4 ( 0.05
-8.4 ( 0.03
36
16
1.8
1.4
13
24
4.1
0.54
-9.9 ( 0.25
-10.9 ( 0.10
-8.4 ( 0.42
0.084
0.0065
3.20
-9.0 ( 0.10
-7.6 ( 0.05
-5.6 ( 0.06
0.29
7.3
700
3.4
1100
220
0.66
0.44
2.6
biphalini
4
-8.8 ( 0.02
0.73
-6.4 ( 0.12
130
180
a Competition analyses were carried out using membrane preparations from transfected HN9.10 cells that constitutively expressed the δ and µ opioid
receptors, respectively. b Kd ) 0.45 ( 0.1 nM. c Kd ) 0.50 ( 0.1 nM. d Competition analyses were carried out using membrane preparations from transfected
CHO cells that constitutively expressed rat or human NK1 receptors. e Kd ) 0.40 ( 0.17 nM. f Kd ) 0.16 ( 0.03 nM. g The logIC50 ( standard errors are
expressed as logarithmic values determined from the nonlinear regression analysis of data collected from two independent experiments performed in duplicate
(four independent experimental values per drug concentration). The Ki values are calculated using the Cheng and Prusoff equation to correct for the concentration
of the radioligand used in the assay. h See ref 38. i See ref 85.
exposed to the surface of the micelles. Moreover, the side-chain
cross-peaks of Met5 in only 1 were missed by either paramag-
netic agent, indicating that Met5 side chain of 1 was exposed
to the surface of the micelle, whereas those of 2 and 3 were
sensitive only to 5-DOXYL, indicating a different orientation
in the side-chain of Met5.
interaction between compound and membrane-like micelles, as
suggested from the NMR conformations of 1-3.
Biological Activities. Finally, to evaluate the biological
influences of conformations in the presence of membrane-
mimicking micelles and compound-micelles interactions, the
bioactivities of peptide derivatives 2 and 3 were evaluated using
well-established assay systems (Tables 5 and 7; the details of
assay systems are found in the Experimental Section and
references herein).38,40,50,51
Compared to the subnanomolar-level affinities of the C-
terminal ester derivative 1 (Ki ) 0.29 nM), the C-terminal amide
derivative 2 was 25 times less potent but still showed binding
affinities in the nanomolecular range for the rNK1 receptor
(Ki ) 7.3 nM) (Table 5). The binding affinity at the rNK1 of 3,
which had no trifluoromethyl group in the C-terminal benzyl
moiety, was decreased to a Ki ) 700 nM. However, in the
functional assay using the GPI to examine their antagonist
activities, the Ke value for 3 (10 nM) was similar to that for 2
(9.9 nM) and better than that for 1 (25 nM) (Table 7). Several
factors, such as pharmacokinetic differences in the different
assay systems, might be responsible for this inconsistency, but
one good explanation should be provided by the known species
difference between rat and guinea pig NK1 receptors.63 It is
The sensitivity of side-chain protons to broadening by
5-DOXYL provides insight into their portioning in depth in the
micelles. The cross-peaks of two different aromatic protons
(para and ortho) in the C-terminal benzyl moiety were
eliminated in 1 and 2 by 5-DOXYL, but the resonance of 3
was preserved in spite of the hydrophobic trifluoromethyl group
in 1 and 2. On the other hand, the cross-peaks of the aromatic
protons of Trp8 were unaffected in 1 but were sensitive to
5-DOXYL for 2 and 3. Therefore, the C-terminal benzyl moiety
(1 and 2) and Trp8 (2 and 3) appear to be located close to the
phosphate moiety of DPC micelles, but the Trp8 of 1 and the
C-terminus of 3 were rather deeply buried into the micelles.
The important implication from these paramagnetic experi-
ments was that all the compounds (1-3) showed different
orientations at each C-terminus in DPC micelles. Thus, both of
the modifications, the ester-to-amide substitution and the
removal of trifluoromethyl groups, were shown to change the

δ/µ Opioid Receptor Agonists and NK1 Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6341
Table 6. Opioid Agonist Functional Activities in [35S]GTPγS Binding Assays
hDORa
rMORa
b
b
no.
Log EC50
EC50 (nM)
Emax (%)c
Log EC50
EC50 (nM)
Emax (%)c
1d
2
3
-8.5 ( 0.21
-8.1 ( 0.11
-8.6 ( 0.13
-9.0 ( 0.17
-8.8 ( 0.25
2.9
8.6
2.6
1.1
1.6
48
58
52
83
69
-7.5 ( 0.09
-8.2 ( 0.17
-7.7 ( 0.18
32
7.0
21
46
55
47
biphalin
DPDPE
DAMGO
-7.4 ( 0.19
37
150
a Expressed from HN9.10 cell. b The log EC50 ( standard error are logarithmic values determined from the nonlinear regression analysis of data collected
from two independent experiments performed in duplicate (four independent experimental values per drug concentration). c Net total bound/basal binding ×
100. d See ref 38.
Table 7. Functional Assay Result for Bifunctional Peptide Ligands at
Opioid and Substance P Receptors
trifluoromethyl groups led to further increased affinity at both
the DOR (Ki ) 0.44 nM) and the rMOR (Ki ) 1.8 nM) with
4.1-fold δ-selectivity. The binding affinity results correlated well
with the in vitro GTPγS binding assays and the functional assays
using GPI and MVD tissues (Table 6 and 7).
opioid agonist
substance P GPI
MVD (δ),
GPI (µ),
antagonist,
Ke (nM)b
no.
IC50 (nM)a
IC50 (nM)a
1c
2
3
22 ( 1.2
15 ( 2.0
4.8 ( 0.35
2.7 ( 1.5
360 ( 130
490 ( 29
61 ( 9.6
8.8 ( 0.3
25 ( 8.8
10 ( 2.1
9.9 ( 2.8
Here, 2 showed potent activity in the MVD assay with the
best δ selectivity (IC50 ) 15 nM in MVD and 490 nM in GPI).
The IC50 value of 3 in the GPI assay (IC50 ) 61 nM) was a
large increase from those of 1 and 2, with the best IC50 value
in the MVD assay (4.8 nM). Therefore, 3 was found to be a
bifunctional peptide derivative possessing potent agonist activi-
ties for both δ and µ opioid receptors together with a nanomolar
level hNK1 antagonist activity. On the other hand, 2 was
characterized as a very potent hNK1 antagonist with potent and
δ selective opioid agonist activities, which also has nanomolar
level affinity at the rNK1 receptor. Thus, 2 can be considered
as a promising candidate for a potent analgesic drug and it is
currently being tested in several animal models using rats and
will be published as a separate biological paper. Though both
2 and 3 have different biological profiles, they are expected to
be potent analgesics for pain control in humans.
biphalin
4
250 ( 87
a Concentration at 50% inhibition of muscle concentration at electrically
stimulated isolated tissues. b Inhibitory activity against the substance P
induced muscle contraction in the presence of 1 µM naloxone. Ke:
concentration of antagonist needed to inhibit substance P to half-its activity.
c See ref 38.
well-known that the human NK1 receptor has higher homology
to the guinea pig NK1 receptor than to the rat or mouse NK1,
and some NK1 antagonists have a large species difference.63
In fact, 3 showed a 220 times better Ki value at the hNK1
receptor (3.2 nM) compared to the value at the rNK1 receptor.
A smaller difference was found in 1, whose Ki value for the
hNK1 was 0.084 nM (3.4-fold species difference). Surprisingly,
2, which is the C-terminal benzyl amide with two trifluoromethyl
groups, showed the largest difference between the affinities at
the rNK1 and at the hNK1 (1100-fold) and the Ki value for the
hNK1 receptor was 6.5 pM, suggesting that this analogue would
be very potent in humans.
Therefore, substitution of the C-terminal ester for an amide
gave rise to increased species difference at the human and rat
NK1 receptors. This substitution changed hydrogen-bonding
interactions and planarity of the C-terminus, which might induce
a conformational change in the lipidic surroundings as found
in the NMR conformations (Figure 4). The summation of these
changes could explain the activity shifts at the NK1 receptors.
The existence of two trifluoromethyl groups at the C-terminus
plays an important role in the affinities for both rNK1 and hNK1
receptors. Because the 3′,5′-dimethyl-substituted analogue of
Ac-Trp-O-3′,5′-Bzl(CF3)2 (4, L-732,138)21 was reported to have
16-fold reduced affinity for the human NK1 receptor as
compared to 4,21 one good explanation of the influence by
trifluoromethyl introduction was that a more electronegative
phenyl ring at the C-terminus might be preferred by the NK1
receptors.64 Another possibility is that the induced conforma-
tional changes to a well-defined helical structure due to the
trifluoromethyl groups interfered with the binding of the
compound at the NK1 receptor.
It should be noted that 2, which had the better-defined
conformation in DPC micelles than 1, showed increased opioid
activities, although the primary sequences for opioid agonist
pharmacophore were exactly the same for both of 1 and 2
(Figure 1). Moreover, 3, which also had the same sequence for
opioid pharmacophore at the N-terminus, showed much im-
proved opioid activities versus 1 and 2. As discussed above, 3
had a structured helical conformation in DPC micelles, whereas
1 and 2 showed ꢀ-rich conformations. Moreover, the interactions
between the compounds and the membranes were different for
compounds 1-3.
Consequently, these results show that even small and limited
structural modification in the ligand could induce relatively large
conformational changes from a ꢀ-structure to a helical confor-
mation in membrane-mimicking DPC micelles, together with
significantly shifted biological activities. In addition, the reported
data suggests that the removal of trifluoromethyl groups from
the C-terminus induced much larger changes in the three-
dimensional conformations than the substitution of the ester to
amide, implying a large influence of lipophilic and electroneg-
ative trifluoromethyl groups on the conformations of whole
molecules in a lipid media. The importance of this article is
that such a small and local structural modification in the
C-terminus might affect on the bioactive states not only at the
C-terminus (NK1 pharmacophore) but also at the N-terminal
(opioid pharmacophore, which was preserved for all three
derivatives), and the observed differences of the conformations
in the membrane-like micelles could indicate their relevance to
the changes in their bioactive form. These findings provide
significant information regarding the nature of peptide drug-
membrane interactions as well as the specific driving force to
For the δ and µ opioid agonist activities, 2 showed 24-fold
δ selectivity, as expected from the existence of Met5,38 with
4-fold higher affinity at the hDOR (Ki ) 0.66 nM) and two
times higher affinity at the rMOR (Ki ) 16 nM) than 1. It is
interesting that a small modification at the C-terminus, which
is far from the opioid agonist pharmacophore, can influence the
opioid activities so much (Table 5). The elimination of two

6342 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Yamamoto et al.
(3H, m), 2.01 (3H, s), 2.42-2.47 (2H, m), 2.67 (1H, d-d, J ) 3.6,
13.2 Hz), 2.75 (1H, d-d, J ) 9.6, 13.8 Hz), 2.87 (1H, d-d, J ) 4.2,
13.2 Hz), 2.94 (1H, d-d, 2.4, 13.2 Hz), 3.04 (1H, d-d, 8.4, 15.0
Hz), 3.14 (1H, d-d, 4.8, 15.0 Hz), 3.48-3.53 (1H, m), 3.54-3.63
(2H, m), 3.68 (1H, d-d, J ) 5.4, 16.8 Hz), 4.15 (1H, d-d, J ) 6.0,
12.0 Hz), 4.20-4.30 (2H, m), 4.32 (1H, d-d, 3.6, 7.8 Hz),
4.48-4.55 (2H, m), 4.58 (1H, J ) 7.2, 14.4 Hz), 6.83 (2H, d, J )
7.8 Hz), 6.92 (1H, d, J ) 8.4 Hz), 7.12 (2H, d, J ) 7.8 Hz),
7.14-7.24 (7H, m), 7.30 (1H, t, J ) 7.8 Hz), 7.47 (1H, s),
7.87-7.92 (2H, m), 7.98-8.08 (4H, m), 8.30 (1H, d, J ) 7.2 Hz).
MS (ESI): 1262 (M + Na)+.
H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF3)2 ·
TFA (1). Boc-Tyr(tBu)-D-Ala-Gly-Phe-Met-Pro-Leu-Trp(Boc)-OH
(2.0 g, 1.61 mmol) and 3,5-bis(trifluoromethyl)benzyl bromide (1.24
g, 4.02 mmol) were dissolved in DMF (8 mL). Cesium carbonate
(1.05 g, 3.22mmol) was added to the solution at r.t. After stirring
for 2 h, saturated aqueous sodium bicarbonate (200 mL) was added
to the solution and extracted with ethyl acetate (200 mL) three times.
The combined organic phases were washed with 5% aqueous citrate
and saturated aqueous sodium chloride (200 mL each) and dried
over sodium sulfate. The solvent was evaporated off, and the crude
peptide was precipitated in cold petroleum ether (45 mL) and
centrifuged two times and dried under reduced pressure. The
obtained protected peptide was treated with 82.5% v/v TFA, 5%
water, 5% thioanisole, 2.5% 1,2-ethanedithiol, and 5% phenol (10
mL, 1 h). The crude peptide was precipitated out by the addition
of chilled diethyl ether (45 mL) to give white precipitates. The
suspension was centrifuged for 20 min at 7000 rpm, then the liquid
was decanted. The crude peptides were washed with diethyl ether
(2 × 45 mL), and after the final centrifugation, the peptides were
dried under vacuum (2 h). The resulting white residues (2.48 g,
quantitative) were dissolved in a 3:1 mixture of acetonitrile and
distilled water (5 mL) and the insoluble impurities were removed
by passing the solutions through syringe filters (Gelman Laboratory,
Ann Arbor, MI, Acrodisc 13 mm syringe filter with 0.45 µM PTFE
membrane). Final purification was accomplished by preparative RP-
HPLC and then lyophilized.
use conformations to manipulate bioactivities, both of which
can be a guide to new aspects of modern medicinal chemistry
to understand the folding of bioactive peptides or proteins.
Experimental Section
Materials. All amino acid derivatives and coupling reagents were
purchased from EMD Biosciences (Madison, WI), Bachem (Tor-
rance, CA), SynPep (Dublin, CA), and Chem Impex International
(Wood Dale, IL). 2-Chlorotrityl resin was acquired from Iris Biotech
GmbH (Marktredwitz, Germany). Perdeuterated DPC was pur-
chased from C/D/N Isotopes (Quebec, Canada). ACS grade organic
solvents were purchased from VWR Scientific (West Chester, PA),
and other reagents were obtained from Sigma-Aldrich (St. Louis,
MO) and used as obtained. The polypropylene reaction vessels
(syringes with frits) were purchased from Torviq (Niles, MI). Myo-
[2-3H(N)]-inositol, [tyrosyl-3,5-3H(N)] D-Ala2-MePhe4-Glyol5-en-
kephalin (DAMGO), [tyrosyl-2,6-3H(N)]-[2-D-penicillamine, 5-D-
penicillamine] enkephalin (DPDPE), [3H]-substance P, and
[35S]-guanosine 5′-(γ-thio) triphosphate were purchased from
Perkin-Elmer (Wellesley, MA). Bovine serum albumin (BSA),
protease inhibitors, Tris, and other buffer reagents were obtained
from Sigma (St. Louis, MO). Culture medium (MEM, DMEM, and
IMDM), penicillin/streptomycin, and fetal calf serum (FCS) were
purchased from Invitrogen (Carlsbad, CA).
Boc-Tyr(tBu)-D-Ala-Gly-Phe-Met-Pro-Leu-Trp(Boc)-OH. The
peptide was synthesized manually by the NR-Fmoc solid-phase
methodology using HBTU as the coupling reagents as previously
reported.40 2-Chlorotrityl resin (2.0 g, 1.56 mmol/g) was placed
into a 50 mL polypropylene syringe with the frit on the bottom
and swollen in DMF (20 mL) for 1 h. The resin was washed with
DMF (3 × 15 mL) and then with DCM (3 × 15 mL). Fmoc-
Trp(Boc)-OH (1.2 equiv) was dissolved in 30 mL of DCM, and
then DIEA (5 equiv) was added. The reaction mixture was
transferred into the syringe with the resin then shaken for 2 h. The
resin was washed three times with DMF (15 mL) and three times
with DCM (15 mL) and then with DMF (3 × 15 mL). The Fmoc
protecting group was removed by 20% piperidine in DMF (1 × 2
min and 1 × 20 min). The deprotected resin was washed with DMF
(3 × 15 mL), DCM (3 × 15 mL), and then with DMF (3 × 15
mL). Fmoc-Leu-OH (3 equiv) and HBTU (2.9 equiv) were
dissolved in 30 mL of DMF, then DIEA (6 equiv) was added. The
coupling mixture was transferred into the syringe with the resin,
then shaken for 2 h. All other amino acids, Pro, Met, Phe, Gly,
D-Ala, and Tyr, were consecutively coupled using the procedures
described above, using the TNBS test (all the amino acids except
for Met) or chloranil test (only for Met) to check the extent of
coupling. In case of a positive test result, the coupling was repeated
until a negative test result was obtained. The resulting batch of the
resin-bound protected Boc-Tyr(tBu)-D-Ala-Gly-Phe-Met-Pro-Leu-
Trp(Boc) was carefully washed with DMF (3 × 15 mL), DCM
(3 × 15 mL), DMF (3 × 15 mL), and DCM (3 × 15 mL) and
dried under reduced pressure. The peptide was cleaved off the solid
support with 1% v/v TFA in DCM (30 mL) for 30 min, and most
of the organic solvent was removed under reduced pressure. The
obtained crude peptides were precipitated out by the addition of
chilled petroleum ether (45 mL) to give a white precipitate. The
suspensions were centrifuged for 20 min at 7000 rpm, and then
the liquid was decanted off. The crude peptides were washed with
petroleum ether (2 × 50 mL), and after the final centrifugation,
the peptides were dried under vacuum (2 h) to obtain the title
compound (2.89 g, 74.8%). The purity of the final products (93.6%)
was checked by analytical RP-HPLC using a Hewlett-Packard 1100
system (230 nm) on a reverse phase column (Waters NOVA-Pak
C-18 column, 3.9 mm × 150 mm, 5 µm, 60Å). The peptide was
eluted with a linear gradient of aqueous CH3CN/0.1% CF3CO2H
(10-90% in 40 min) at a flow rate of 1.0 mL/min. The crude
peptide was used for next reactions without further purification.
1H NMR (DMSO-d6): 0.79 (3H, d, J ) 6.6 Hz), 0.84 (3H, d, J )
6.6 Hz), 1.10 (3H, d, J ) 6.6 Hz), 1.24 (9H, s), 1.26 (9H, s),
1.38-1.41 (2H, m), 1.61 (10H, s), 1.70-1.77 (3H, m), 1.80-1.94
H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3,5-Bzl(CF3)2 ·
TFA (2). Boc-Tyr(tBu)-D-Ala-Gly-Phe-Met-Pro-Leu-Trp(Boc)-OH
(3.0 g, 2.42 mmol) and Cl-HOBt (428 mg, 2.66 mmol) were
dissolved in DMF (10 mL). 3,5-Bistrifluoromethylbenzyl amine
(1.17 g, 4.84 mmol) and EDC (508 mg, 2.66 mmol) were added to
the solution at r.t. and stirred until the starting material was not
detected in TLC and then saturated aqueous sodium bicarbonate
(250 mL) was added. The reaction mixture was extracted with ethyl
acetate (250 mL) three times. The combined organic phases were
washed with 5% aqueous citrate and saturated aqueous sodium
chloride (250 mL each) and then dried over sodium sulfate. The
solvent was evaporated, and the crude peptide was precipitated in
cold petroleum ether (45 mL). The product was twice dispersed in
cold petroleum ether, centrifuged and decanted, and then dried under
reduced pressure. The obtained protected peptide was treated with
82.5% v/v TFA, 5% water, 5% thioanisol, 2.5% 1,2-ethanedithiol,
and 5% phenol (1.5 mL, 1 h). The crude peptide was precipitated
out by the addition of chilled diethyl ether (45 mL) to give a white
precipitate. The resulting peptide suspension was centrifuged for
20 min at 7000 rpm, and the liquid was decanted. The crude peptide
was washed with diethyl ether (2 × 45 mL), and after a final
centrifugation, the peptides were dried under vacuum (2 h). The
resulting white residue (3.42 g, quantitative) was dissolved in a
3:1 mixture of acetonitrile and distilled water (5 mL), and the
insoluble impurities were removed by passing the solutions through
syringe filters (Gelman Laboratory, Acrodisc 13 mm syringe filter
with 0.45 µM PTFE membrane). Final purification was ac-
complished by preparative RP-HPLC. The pure title compound was
obtained after lyophilization.
H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bzl · TFA (3).
The title peptide was prepared using the same method as described
for H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NH-3,5-Bzl(CF3)2 ·TFA (2).
The crude peptide was obtained quantitatively.

δ/µ Opioid Receptor Agonists and NK1 Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6343
Characterization of Peptides. Preparative RP-HPLC was
performed on Waters Delta Prep 4000 with Waters XTerra C-18
column (19 mm × 250 mm, 10 µm, a linear gradient of 33-53%
or 40-60% acetonitrile/0.1% TFA at a flow rate of 15.0 mL/min).
The purified peptides were characterized by HRMS, TLC, analytical
negligible. The Met5-Pro6 bond of the major rotamers were fixed
in the trans configuration based on the observations of 5HR to Pro6
Hδ sequential NOEs together with the absence of sequential
5HR-6HR NOEs in the structural calculations of 1-3. The volumes
of the assigned cross-peaks in the 2D NOESY spectrum were
converted into upper distance bounds of 3.0, 3.8, 4.8, or 5.8 Å.
For overlapping cross-peaks, the distance categories was increased
by one or two levels depending on the qualitative estimate of the
extent of overlap. Pseudoatoms were created for nonstereospecifi-
cally assigned methylene protons with a correction of 1.0 Å applied
to their upper bound distances. In addition to the distance
1
HPLC, and H-1D-NMR. Sequential assignment of proton reso-
nances was achieved by 2D-TOCSY NMR experiments.65-69 High-
resolution MS were taken in the positive ion mode using ESI
methods at the University of Arizona Mass Spectrometry Facility.
TLC was performed on aluminum sheets coated with a 0.2 mm
layer of silica gel 60 F254 Merck using the following solvent
systems: (1) CHCl3:MeOH:AcOH ) 90:10:3, (2) EtOAc:n-BuOH:
water:AcOH ) 5:3:1:1, and (3) n-BuOH:water:AcOH ) 4:1:1. TLC
chromatograms were visualized by UV light and by ninhydrin spray
followed by heating (hot plate). Analytical HPLC was performed
on a Hewlett-Packard 1100 or Hewlett-Packard 1090m with Waters
NOVA-Pak C-18 column (3.9 mm × 150 mm, 5 µm, 60 Å) or
Vydac 218TP104 C-18 column (4.6 mm × 250 mm, 10 µm, 300
Å). 1H-1D-NMR spectra were obtained on Bruker DRX-500 or
DRX-600 spectrometer. 2D-TOCSY NMR spectra were performed
on a Bruker DRX-600 spectrometer equipped with a 5 mm Nalorac
triple-resonance single-axis gradient probe. The NMR experiments
were conducted in DMSO-d6 solution at 298 K. Spectra were
referenced to residual solvent protons as 2.49 ppm. The processing
of NMR data was performed with the XwinNmr software (Bruker
BioSpin, Fremont, CA). In the TOCSY experiments, the TPPI
mode70 with MLEV-17 Mixing Sequence65 were used with a
mixing time of 62.2 ms, at a spin-lock field of 8.33 kHz. TOCSY
spectra were acquired with 2k complex pairs in t2 and 750 or 1024
FIDs using a 90°-shifted sine-squared window function in both
dimensions and zero filling to a final matrix size of 2048 (F2) ×
1024(F1) points.
3
constraints, ꢁ dihedral angle constraints derived from JHN-HR
3
coupling constants were set to between -90 and 40° for JHN-HR
3
< 6 Hz and to between -150 and -90° for JHN-HR > 8 Hz.
Dihedral angle constraints of 180° ( 5° for peptide bonds (ω) were
also used to maintain the planarity of these bonds.
Simulated annealing molecular dynamics analysis was done for
all the peptides to obtain an ensemble of NMR structures using
the NOE-derived distance constraints and dihedral angle (ꢁ)
constraints using the DGII74 program within the software package
Insight II 2000 (Accelrys Inc., San Diego, CA). Solvent was not
explicitly included in the calculations. All the embedded structures
successfully passed the simulated annealing step and were mini-
mized using the consistent valency force field (CVFF) (Accelrys
Inc.). The 50 structures with the lowest penalty function were further
refined by two rounds of restrained molecular dynamics (rMD)
using the all-atom AMBER force field with additional parameters
for fluorine atom75-78 using the standalone DISCOVER ver. 2.98
program (Accelrys Inc.). A 12.0 Å cutoff for nonbonded interactions
and a distance-dependent dielectric constant (4r) were used. All
amide bonds were constrained to trans conformation by a 100 kcal
mol-1 rad-2 energy penalty. The distance constraints and dihedral
angles (ꢁ) constraints were applied with a force constant of 25
kcal mol-1 Å-2 and 100 kcal mol-1 rad-2 were applied, respec-
tively. After 100 steps of steepest descents minimization and 1000
steps of conjugate gradient minimization on the initial structures,
an rMD equilibration at 500 K was performed for 1.5 ps, during
which a scale factor of 0.1 was applied to the experimental restraint
force constants. During the next 2 ps, full values of the experimental
restraint force constants were applied. A further 1 ps rMD
simulation was run at 500 K, and the system was then cooled to 0
K over 3 ps. After another 1 ps at 0 K, 100 cycles of steepest
descents and 2000 steps of conjugate gradient minimization were
performed. The final 20 structures with the lowest energies were
used for the analysis. All calculations were performed on a Silicon
Graphics Octane computer.
NMR Spectroscopy in DPC Amphipathic Media.45,46 All
NMR spectra were recorded on a Bruker DRX600 600 MHz
spectrometer with 5 mm Nalorac triple-resonance single-axis
gradient probe. The peptide concentration for the NMR experiments
varied from 3.5 to 4 mM. The samples were prepared by dissolving
the peptide in 0.5 mL of 45 mM sodium acetate-d3 buffer (pH 4.5)
containing 40 equiv of dodecylphosphocholine-d38 and 1 mM
sodium azide (90% H2O/10% D2O), followed by sonication for 5
min. Two-dimensional double quantum filtered correlation (DQF-
COSY), nuclear Overhauser effect71 (NOESY), and total correlation
spectra66 (TOCSY) were acquired using standard pulse sequences
and processed using XwinNmr and Felix 2000 (Accelrys Inc., San
Diego, CA). The mixing time for NOESY spectra was 450 ms. All
2D spectra were acquired in the TPPI mode with 2k or 1k complex
data points in t2 and 750 real data points in t1, and the spectral
processing used 90°-shifted sine bell window functions in both
dimensions. For suppressing the H2O signal, the 3-9-19
WATERGATE pulse sequence was used.72 Experiments were
conducted at 310 K and referenced to the H2O shift (4.631 ppm).
Coupling constants (3JNH-HR) were measured from 2D DQF-COSY
spectra by analysis of the fingerprint region. The matrix rows of
each of the upper and lower halves of a cross-peak were summed
to give an antiphase 1D spectrum, which was fitted using a
5-parameter Levenberg-Marquardt nonlinear least-squares proto-
col73 to a general antiphase doublet. The analysis yielded two
independent determinations of the J coupling and line width for
each cross-peak, one from the upper half and one from the lower
half, and the one with the better fitted curve was used for structure
calculations. Cross-peak volumes for determination of distance
restraints were measured using the Felix 2000 software. In the
radical experiment using Mn2+, a stock solution of 5 mM MnCl2
was prepared and added to the sample to achieve a total concentra-
tion of 200 µM in Mn2+. The DPC micelles with 5-DOXYL stearic
acid were prepared as the same procedure with about 1 mg mL-1
of 5-DOXYL stearic acid but sonicating for 30 min.
Fluorescence Emission Spectra. The Fluorescence spectra were
recorded on a Cary Eclipse fluorescence spectrometer (Varian,
Darmstadt, Germany). Emission spectra were obtained by excitation
at 290 nm and recorded in the wavelength range of 310-420 nm
with continuous stirring at 25 °C. A scan step was 1 nm and scan
speed was 120 nm min-1. Excitation and emission bandwidths were
set at 5 and 10 nm, respectively. The peptide concentration was
500 µM in HEPES buffer (10 mM HEPES, 150 mM NaCl, 1 mN
NaN3, 0.1 mM EDTA, pH ) 7.40) with 40-fold DPC or standard
solution (EtOH:HEPES buffer ) 1:1).61 At least two scans were
accumulated and averaged for each spectrum.
Octanol/Saline Distribution (logD7.4).38 HEPES buffer (0.05
M HEPES buffer in 0.1 M NaCl, pH 7.4, 500 µL) was added to
2 mg of peptide and mixed with 500 µL of 1-octanol. The sample
was shaken at r.t. for 12 h to allow equilibrating. The sample was
centrifuged at 6500 rpm in a VanGuard V6500 (GlaxoSmithKline,
Research Triangle Park, NC) for 15 min. The layers were separated
and each layer was centrifuged once again. The peptide concentra-
tions in the obtained layers were analyzed by HPLC (30-70% of
acetonitrile containing 0.1% TFA within 20 min and up to 95%
within additional 5 min, 1 mL/min, 230 nm, Vydac 218TP104 C-18
Conformational Structure Determination. The methods used
for structure calculations have been described previously.45,46
Throughout the 1H NMR studies, only one major rotamer for 1-3
was found and the populations of minor rotamers were all
column). The logarithm of 1-octanol/saline distribution (logD7.4
)
was calculated as the ratio of peptide concentration in the 1-octanol
and saline phases.

6344 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Yamamoto et al.
Solubility. HEPES buffer (0.05 M HEPES buffer in 0.1 M NaCl,
pH 7.4, 500 µL) was added to 1 mg of peptide. The sample was
vortexed for 30 s, sonicated for 5 min, shaken at r.t. for 2 h, and
then allowed to be stayed overnight to equilibration. The sample
was filtrated with an Acrodisc Syringe Filter (13 mm, 0.45 µm pore,
PTFE membrane, Pall Life Sciences, East Hills, NY). The peptide
concentration of the obtained filtrate was analyzed by HPLC
(30-70% of acetonitrile containing 0.1% TFA within 20 min and
up to 95% within an additional 5 min, 1 mL/min, 230 nm, Vydac
218TP104 C-18 column).
Cell Lines. For opioid receptors, the cDNA for the human δ
opioid receptor (DOR) was a gift from Dr. Brigitte Kieffer
(IGBMC, Illkirch, France). The cDNA for the rat µ opioid
receptor (MOR) was a gift from Dr. Huda Akil (University of
Michigan, MI). Stable expression of the rat MOR (pCDNA3)
and the human DOR (pCDNA3) in the neuroblastoma cell line
HN9.10 were achieved with the respective cDNAs by calcium
phosphate precipitation followed by clonal selection in neomycin.
Expression of the respective receptors was initially verified and
the level of expression periodically monitored by radioligand
saturation analysis (see below). All cells were maintained at 37
°C, with a 95% air/5% CO2 humidified atmosphere in a Forma
Scientific incubator in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% bovine serum albumin (BSA) and 100 U
mL penicillin/100 µg mL streptomycin. For NK1 receptor, the
hNK1/CHO and rNK1/CHO cell lines were obtained from Dr.
James Krause (University of Washington Medical School, St.
Louis, MI). Expression of the receptor was verified as previously
described by Krause et al.79 All cells were maintained at a 37
°C, 95% air and 5% CO2, humidified atmosphere, in a Forma
Scientific incubator in Ham’S F12 with 2.5 mM HEPES, 10%
fetal bovine serum, and 100 U mL penicillin/100 µg mLstrep-
tomycin/500 µg mL Geneticin.
Radioligand Labeled Binding Assays. For opioid receptors,
crude membranes were prepared as previously described80 from
the transfected cells that express the MOR or the DOR. The
protein concentration of the membrane preparations was deter-
mined by the Lowry method, and the membranes were stored at
-80 °C until use. Membranes were resuspended in assay buffer
(50 mM Tris, pH 7.4, containing 50 µg/mL bacitracin, 30 µM
bestatin, 10 µM captopril, 100 µM phenylmethylsulfonylfluoride
(PMSF), 1 mg/mL BSA). For saturation analysis, six concentra-
tions of tritiated [D-Ala2,NMePhe4,Gly5-ol]-enkephalin ([3H]DAM-
GO) (0.02-6 nM, 47.2 Ci/mmol), or six concentrations of
tritiated c[D-Pen2,D-Pen5]-enkephalin ([3H]DPDPE) (0.1-10nM,
44 Ci/mmol) were each mixed with 200 µg of membranes from
MOR or DOR expressing cells, respectively, in a final volume
of 1 mL. For competition analysis, 10 concentrations of a test
compound were each incubated with 50 µg of membranes from
MOR or DOR expressing cells and the Kd concentration of
[3H]DAMGO (1.0 nM, 50 Ci/mmol), or of [3H]DPDPE (1.0 nM,
44 Ci/mmol), respectively. Naloxone at 10 µM was used to define
the nonspecific binding of the radioligands in all assays. All
assays were carried out in duplicates and were repeated. The
samples were incubated in a shaking water bath at 25 °C for
3 h, followed by rapid filtration through Whatman grade GF/B
filter paper (Gaithersburg, MD) presoaked in 1% polyethylene-
imine, washed 4 times each with 2 mL of cold saline, and the
radioactivity determined by liquid scintillation counting (Beck-
man LS5000 TD).
For the NK1 receptors, competition binding assays for the NK1
receptors were carried out on crude membranes prepared from
transfected CHO cells expressing the human or rat NK1 receptor,
respectively. Ten concentrations of a test compound were each
incubated with 50-100 µg of membrane homogenate and 0.3-0.4
nM [3H] substance P (135 Ci/mmol, Perkin-Elmer) in 1 mL final
volume of assay buffer (50 mM Tris, pH 7.4, containing 5 mM
MgCl2, 50 µg/mL bacitracin, 30 µM bestatin, 10 µM captopril, 100
µM phenylmethylsulfonylfluoride (PMSF) at 25 °C for 20 min.
Substance P at 10 µM was used to define the nonspecific binding.
Membrane concentrations used in the assay were within the tissue
linearity range. The [3H] substance P concentration was selected
based on the saturation binding experiments, which showed a high
affinity binding with Kd values of 0.40 ( 0.17 for hNK1 and 0.16
( 0.03 nM for rNK1, respectively. The incubation times correspond
to the binding equilibrium as determined from the kinetics experi-
ments. The incubation was stopped by rapid filtration through a
GF/B glass filter (Brandel Inc.) presoaked in 0.5% polyethylene-
imide, followed by four washes with 2 mL of ice-cold saline using
a Brandel Harvester apparatus. The filter-bound radioactivity was
measured by liquid scintillation counting (Beckman LS 6000SC).
Log IC50 values were determined with standard errors from
nonlinear regression analysis of at least two independent experi-
ments performed in duplicates using GraphPad Prizm 4 software
(GraphPad, San Diego, CA).
For competition analysis using [3H]DAMGO, [3H]DPDPE or
[3H]substance P values were calculated from the IC50 by the Cheng
and Prusoff equation.
[35S]GTPγS Binding Assay. The method was carried out as
previously described.80 Membrane preparation (10 µg) to a final
volume of 1 mL of incubation mix (50 mM Hepes, pH 7.4, 1 mM
EDTA, 5 mM MgCl2, 30 µM GDP, 1 mM dithiothreitol, 100 mM
NaCl, 0.1 mM PMSF, 0.1% BSA, 0.1 nM [35S]GTPγS) was added
along with various concentrations, in duplicates or triplicates, of
the test drug and incubated for 90 min at 30 °C in a shaking water
bath. Reactions were terminated by rapid filtration through What-
man GF/B filters (presoaked in water), followed by four washes
with 4 mL of ice-cold wash buffer (50 mM Tris, 5 mM MgCl2,
100 mM NaCl, pH 7.4). The radioactivity was determined by liquid
scintillation counting as above. Basal level of [35S]GTPγS binding
was defined as the amount bound in the absence of any test drug.
Nonspecific binding was determined in the presence of 10 µM
unlabeled GTPγS. Total binding was defined as the amount of
radioactivity bound in the presence of the test drug. The effect of
the drug at each concentration on [35S]GTPγS binding was
calculated as a percentage by the following equation: [total bound-
basal]/[basal-nonspecific] × 100. Data were expressed as log EC50
( standard error from at least two independent experiments
performed in duplicate analyzed by nonlinear regression analysis
using GraphPad Prism4.
Guinea Pig Isolated Ileum Assay. The in vitro tissue bioassays
were performed as described previously.38 Male Hartley guinea
pigs under ether anesthesia were sacrificed by decapitation and
a nonterminal portion of the ileum removed and the longitudinal
muscle with myenteric plexus (LMMP) was carefully separated
from the circular muscle as described previously.81 These tissues
were tied to gold chains with suture silk and mounted between
platinum wire electrodes in 20 mL organ baths at a tension of
1 g and bathed in oxygenated (95:5 O2:CO2) Kreb’s bicarbonate
buffer at 37 °C and stimulated electrically (0.1 Hz, 0.4 ms
duration) at supramaximal voltage. Following an equilibration
period, compounds were added cumulatively to the bath in
volumes of 14-60 µL until maximum inhibition was reached.
A baseline for PL-017 was constructed to determine tissue
integrity and allow calculation of antagonist activity before
opioid analogue testing began. If no agonist activity was
observed at 1 µM, a repeat PL-017 dose-response curve was
constructed to test for antagonist qualities.
All substance P antagonist compound testing was performed in
the presence of 1 µM naloxone to block opioid effects on
unstimulated tissue. Two minutes after naloxone was added to the
bath, the test compound was added. Four minutes after naloxone
was added, the test dose of substance P was added to the bath, the
peak height was noted, and the tissues were washed. Agonist activity
of the analogue was also observed during this period. Testing
stopped at 1 mM concentrations of the test compound.
Mouse Isolated Vas Deferens (MVD) Assay. The in vitro tissue
bioassay was performed as described previously.38 Male ICR
mice under ether anesthesia were sacrificed by cervical disloca-
tion and the vasa deferentia removed. Tissues were tied to gold
chains with suture silk and mounted between platinum wire
electrodes in 20 mL organ baths at a tension of 0.5 g and bathed

δ/µ Opioid Receptor Agonists and NK1 Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6345
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in oxygenated (95:5 O2:CO2) magnesium free Kreb’s buffer at
37 °C and stimulated electrically (0.1 Hz, single pulses, 2.0 ms
duration) at supramaximal voltage as previously described.82
Following an equilibration period, compounds were added to
the bath cumulatively in volumes of 14-60 µL until maximum
inhibition was reached. Response to an IC50 dose of DPDPE
(10 nM) was measured to determine tissue integrity before
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Analysis of the GPI and MVD assays. For opioid data analysis,
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Acknowledgment. The work was supported by a grant from
the USDHS, National Institute on Drug Abuse, DA-13449. We
thank Dr. Guangxin Lin and Dr. Jinfa Ying for kind assistance
and advice with the NMR measurements and structure calcula-
tions, Dr. Robin Polt for fruitful discussion, especially about
the paramagnetic NMR experiments, Dr. John J. Osterhout for
use of the fluorescence spectrometer, Magdalena Kaczmarska
for culturing cells, and the University of Arizona Mass
Spectrometry Facility for the mass spectra measurements. We
express appreciation to Margie Colie and Brigid Blazek for
assistance with the manuscript.
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Structure, dynamics, and insertion of a chloroplast targeting peptide
in mixed micelles. Biochemistry 2000, 39, 8219–8227.
Supporting Information Available: The 1H NMR, HPLC, and
MS data of the peptide derivatives 1-3 are provided. This material
is available free of charge via the Internet at http://pubs.acs.org.
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