Differential receptor binding characteristics of consecutive phenylalanines in μ-opioid specific peptide ligand endomorphin-2

Article abstract of DOI:10.1016/j.bmc.2007.03.009

Endogenous opioid peptides consist of a conserved amino acid residue of Phe³ and Phe⁴, although their binding modes for opioid receptors have not been elucidated in detail. Endomorphin-2, which is highly selective and specific for the μ opioid receptor, possesses two Phe residues at the consecutive positions 3 and 4. In order to clarify the role of Phe³ and Phe⁴ in binding to the μ receptor, we synthesized a series of analogs in which Phe³ and Phe⁴ were replaced by various amino acids. It was found that the aromaticity of the Phe-β-phenyl groups of Phe³ and Phe⁴ is a principal determinant of how strongly it binds to the receptor, although better molecular hydrophobicity reinforces the activity. The receptor binding subsites of Phe³ and Phe⁴ of endomorphin-2 were found to exhibit different structural requirements. The results suggest that [Trp³]endomorphin-2 (native endomorphin-1) and endomorphin-2 bind to different receptor subclasses.

Full text of DOI:10.1016/j.bmc.2007.03.009

Bioorganic & Medicinal Chemistry 15 (2007) 3883–3888
Differential receptor binding characteristics of consecutive
phenylalanines in l-opioid specific peptide ligand endomorphin-2
Takeshi Honda, Naoto Shirasu, Kaname Isozaki, Michiaki Kawano, Daiki Shigehiro,
Yoshiro Chuman,à Tsugumi Fujita,§ Takeru Nose and Yasuyuki Shimohigashi*
Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences,
Kyushu University, Fukuoka 812-8581, Japan
Received 6 January 2007; revised 2 March 2007; accepted 3 March 2007
Available online 12 March 2007
Abstract—Endogenous opioid peptides consist of a conserved amino acid residue of Phe3 and Phe4, although their binding modes
for opioid receptors have not been elucidated in detail. Endomorphin-2, which is highly selective and specific for the l opioid recep-
tor, possesses two Phe residues at the consecutive positions 3 and 4. In order to clarify the role of Phe3 and Phe4 in binding to the
l receptor, we synthesized a series of analogs in which Phe3 and Phe4 were replaced by various amino acids. It was found that the
aromaticity of the Phe-b-phenyl groups of Phe3 and Phe4 is a principal determinant of how strongly it binds to the receptor,
although better molecular hydrophobicity reinforces the activity. The receptor binding subsites of Phe3 and Phe4 of endomor-
phin-2 were found to exhibit different structural requirements. The results suggest that [Trp3]endomorphin-2 (native endomor-
phin-1) and endomorphin-2 bind to different receptor subclasses.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
(Phe), an aromatic amino acid, often plays a crucial role
in neuropeptides in their receptor binding and activa-
tion.1 Elucidation of the interaction mode of such Phe
residues appears to be one of the definite goals to clari-
fying the molecular mechanism of receptor activation.
The interaction of Phe is usually characterized by the
term ‘hydrophobic’, the intrinsic meaning of which is
rather obscure. We have postulated a means of differen-
tiating such a hydrophobic interaction of Phe, replacing
the residue with a series of amino acids having
side-chain varieties.2 Furthermore, the presence
of the edge-to-face CH/p interaction was demonstrated
between the Phe-phenyl group of thrombin receptor-
tethered ligand peptide and the receptor aromatic
group,3–5 and the face-to-face p/p stacking interaction
The presence of a bioactive conformation in neuropep-
tides is prerequisite to specific receptor interactions,
and these interactions are stabilized by distinct struc-
tural elements binding to the receptor. Phenylalanine
Abbreviations: Boc, tert-butyloxycarbonyl; BSA, bovine serum albu-
min; Cha, cyclohexylalanine; DMF, N,N-dimethylformamide; End-1,
endomorphin-1; End-2, endomorphin-2; HBTU, 2-(1H-benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, 1-hy-
droxybenzotriazole; MALDI-TOF, matrix-assisted laser desorption
ionization time-of-flight; MBHA, p-methylbenzhydrylamine; Nle,
norleucine; NMP, N-methylpyrrolidone; (F5)Phe, pentafluorophenyl-
alanine; RP-HPLC, reversed-phase high performance liquid chroma-
tography; RT, retention time; TFA, trifluoroacetic acid; Tris,
tris(hydroxymethyl)aminomethane.
1
was proven by X-ray crystallographic analysis and H
NMR analysis between the Phe-phenyl group of inhibi-
tor peptide and the His-imidazole group of the enzyme
chymotrypsin.6,7
Keywords: Endomorphins; Opioid peptide; Phenylalanine; Structure–
activity relationships.
*
Corresponding author. Tel./fax: +81 92 642 2584; e-mail:
shimoscc@mbox.nc.kyushu-u.ac.jp
Opioid peptides induce analgesia by interacting with
opioid receptors such as the endogenous l, d, and j sub-
types. It is generally accepted that the N-terminal por-
tion of opioid peptides such as enkephalins,
endorphins, and dynorphins is the message sequence
for binding to opioid receptors, and that Tyr at position
1 and Phe at positions 3 or 4 are essential for receptor
Present address: Nanotechnology Research Institute, National
Institute of Advanced Industrial Science and Technology (AIST),
Saga 841-0052, Japan.
à
Present address: Division of Chemistry, Graduate School of Science,
Hokkaido University, Sapporo 060-0810, Japan.
§
Present address: Department of Physiology, Faculty of Medicine,
Saga University, Saga 849-8501, Japan.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.009

3884
T. Honda et al. / Bioorg. Med. Chem. 15 (2007) 3883–3888
binding and activation.8–11 However, despite such a
residual importance, their role in the receptor interac-
tion has not necessarily been elucidated in detail.
Phe3 than for Phe4, suggesting their differential receptor
binding characteristics.
In our previous studies of a d-specific opioid peptide
named deltorphin II, Tyr-D-Ala-Phe-Glu-Val-Val-Gly-
NH2 isolated from African frog skin,12 the replacement
of Phe3-phenyl group by the cyclohexyl (–C6H12) or
n-propyl (–CH2CH2CH3) was found to retain the recep-
tor binding ability of the parent peptide.2 In contrast,
the same replacement of Phe4 of d-specific DSLET
(Tyr-D-Ser-Gly-Phe-Leu-Thr-OH, a synthetic analog of
endogenous opioid peptide enkephalin)13 did not sustain
the binding affinity, reducing by approximately 90% the
activity of DSLET.14 These results indicated that the
receptor interaction modes of Phe3 in deltorphin II
and Phe4 in DSLET residues are different from each
other.
2. Results
2.1. Peptide synthesis
Endomorphin-2 and its analogs were prepared by the
manual solid phase peptide synthesis method. Starting
from the MBHA resin, all the coupling reactions were
carried out by the HBTU/HOBt method.16 Completion
of the reaction was monitored by the Kaiser test.17 In
the syntheses of Phe3-substituted analogs, various Boc-
amino acids were introduced at the first coupling step
starting from Phe-preloaded MBHA resin, while this
was done directly for the MBHA resin in the syntheses
of Phe4-substituted analogs. Including native endomor-
phin-2, all 21 tetrapeptides were synthesized in an aver-
age yield of approximately 35%.
Endomorphin-1 (End-1: Tyr-Pro-Trp-Phe-NH2) and
endomorphin-2 (End-2: Tyr-Pro-Phe-Phe-NH2) are
endogenous ligands specific for the l receptor. Although
they were isolated from bovine brain,15 the genetic
sources have not yet been clarified. Among natural opi-
oid peptides, the sequences of endomorphins are unique,
having aromatic residues at the consecutive positions 3
and 4. It is thus intriguing to elucidate the mode of inter-
action between these aromatic amino acids and the
receptor. In the present study, in order to clarify the role
of Phe3 and Phe4 of endomorphin-2 in receptor binding,
we designed and synthesized a series of analogs in which
these Phe residues were replaced by various amino acids
(Fig. 1). When the activity profiles of each series of ana-
logs were compared, the same substitutions were found
to have different effects, with much stronger activity for
Analogs containing cyclohexylalanine (Cha) or penta-
fluorophenylalanine ((F5)Phe) were obtained in some-
what lower yields (about 25%). Although the exact
reason is not clear, their relatively high hydrophobicity
might bring about this lower recovery of the com-
pounds. Table 1 shows the analytical data of all the syn-
thesized tetrapeptides. The mass numbers measured
were coincident with the values calculated. The purity
of peptides was also verified by analytical HPLC, in
which all the peptides emerged as a single peak. Amino
acid analyses revealed a good coincidence of the number
of amino acid constituents.
The retention time in RP-HPLC usually exhibits the
characteristic structural nature of the peptide hydropho-
bicity. When the same substitution took place for the
Phe residues at both positions 3 and 4, the values of
the retention time of Phe3-substituted analogs were
slightly larger than those of Phe4-analogs (Fig. 2). This
difference was particularly prominent for Phe ! Ala
substitutions; that is, RT = 20.74 min for [Ala3]End-2,
and RT = 12.86 min for [Ala4]End-2. These findings
might indicate that the removal of Phe3-phenyl exposes
the peptide in the structure to the solvent more than
expected.
2.2. Receptor binding affinities of Phe3-substituted endo-
morphin-2 analogs
The binding affinity of peptides was evaluated by mea-
suring the ability to displace the radio-labeled ligand
specific for either l or d opioid receptor. Radio-labeled
ligands utilized were tritium-labeled enkephalin analog
[3H]DAGO for l receptor and tritium-labeled frog-skin
peptide [3H]deltorphin II for d receptor. From the anal-
ysis of the obtained dose–response curves, the IC50 val-
ues of the synthetic peptides were estimated, and Table 2
summarizes these values together with the selectivity
expressed by the ratio of IC50(d) versus IC50(l).
Figure 1. The structures of endomorphin-2 and the side-chains of the
amino acid residues at positions 3 and/or 4. Each box classifies a series
of amino acids with the side-chains such as aliphatic, basic, and
aromatic groups.
The importance of Phe-phenyl in endomorphin-2 can be
explored by assessing the activity profiles of the set of

T. Honda et al. / Bioorg. Med. Chem. 15 (2007) 3883–3888
3885
Table 1. Physical constants of synthetic endomorphin-2 and its Phe3- and Phe4-substituting analogs
Peptidesa
Phe3-substituents
MSc (Found/calcd)
Phe4-substituents
MSc (Found/calcd)
HPLCb (tR)
HPLCb (tR)
Phe (End-2)
Ala
Val
30.30
20.74
22.56
28.12
29.67
37.92
37.35
12.71
23.61
33.04
11.19
571.63/571.93
495.80/495.58
523.80/523.64
538.28/537.66
538.27/537.66
577.52/577.73
661.62/661.64
561.55/561.65
587.89/587.68
610.80/610.72
553.01/552.68
30.30
12.86
20.98
26.88
28.37
36.42
37.24
9.27
571.63/571.93
495.86/495.58
523.80/523.64
537.84/537.66
537.89/537.66
577.54/577.73
661.63/661.64
561.66/561.65
587.88/587.68
610.91/610.72
552.83/552.68
Leu
Nle
Cha
(F5)Phe
His
Tyr
20.60
32.16
9.04
Trp
Lys
a Peptides are designated by the amino acid residues at position 3 or 4 of endomorphin-2.
b Retention time (tR) of RP-HPLC was measured on an analytical column (Cica-Merck, LiChrospher 100 RP-18 (5l): 4 · 250 mm) with a linear
gradient of 0.1% aqueous trifluoroacetic acid (A solution) and acetonitrile containing 20% A solution (B solution).
c Values express the mass number (m/z) of (M+H)+ by MALDI-TOF-MS.
Table 2. Opioid receptor binding affinity and selectivity of endomor-
phin-2 and its Phe3-substituting analogs
Peptidesa
(Phe3-substituents)
IC50 (nM)
d receptor
Selectivityb
l receptor
Phe (End-2)
Ala
Val
3.46 1.21 10,300 2770 2980
3810 159
2650 339
926 28.3 >1 mM
Unboundc
>1 mM
(l)d
(l)
Leu
Nle
(l)
230 17.7 15,200 4950 66.1
148 16.3 3150 156 21.3
11.7 0.503 11,700 1010 1000
Cha
(F5)Phe
His
Tyr
4490 70.7 >1 mM
(l)
5.20
1100 113
1.89 0.731 3580 211
5720 326
Trp
Lys
DAGOe
1890
Inactive
643
>1 mM
Unboundc
1.13 0.263 727 109
Figure 2. The relationship between the binding affinity and the
hydrophobicity of endomorphin-2 and its analogs. The pA values of
endomorphin-2 analogs with the substituents at positions 3 (closed
circle) and 4 (closed triangle) are shown together with that of Phe3/
Phe4 (closed square) of native endomorphin-2. Two solid lines were
estimated by the least-square method using values of coordinates of
analogs with the substituents at positions 3 (His3, Ala3, Val3, Tyr3,
Leu3, Nle3, and Cha3) and 4 (Lys4, His4, Ala4, Val4, Tyr4, Leu4, Nle4,
and Trp4), respectively. The pA value of Lys3-analog is smaller than 3,
and thus it was eliminated from this plotting.
a Peptides are designated by the amino acid residues at position 3 of
endomorphin-2 (Tyr-Pro-/Xaa/-Phe-NH2).
b Receptor selectivity was estimated by calculating the ratio of the IC50
values for l receptor versus those for d receptor.
c ‘Unbound’ exhibits that tracer ligands ([3H]DAGO and [3H]deltor-
phin II) are not replaced with 10 mM of the synthetic peptides.
d (l) means ‘extremely or considerably weak selectivity for l receptor’.
e DAGO is a l-specific opioid peptide with the sequence of Tyr-D-Ala-
Gly-(N-Me)Phe-Gly-ol.
analogs in which the Phe residue is replaced by various
amino acids. We used amino acids with a variety of sub-
stituents at the side-chain b-position (Fig. 1). When Phe
was replaced by Ala at position 3, eliminating the b-phe-
nyl group, the resulting [Ala3]End-2 exhibited a drasti-
cally reduced binding affinity (IC50 = 3810 nM) as
compared with endomorphin-2 (IC50 = 3.46 nM). The
presence and absence of Phe3-phenyl cause the activity
difference of three-order magnitude, indicating the criti-
cal importance of the Phe3-phenyl group in binding to
the l receptor.
(43-fold) in the receptor binding affinity (Cha3; IC50
=
148 nM) as compared with endomorphin-2, indicating
that the presence of p-electrons and/or the rigid planar-
ity of Phe3-phenyl are key structural characteristics for
interaction with the receptor. Thus, a series of endomor-
phin-2 analogs containing aromatic amino acids such as
His, Tyr, Trp, and (F5)Phe were further examined.
Histidine (His) possesses a five-membered aromatic ring
(imidazole group), and Tyr has a phenol ring. Both are
rather hydrophilic as compared with Phe. On the other
hand, Trp is more hydrophobic than Phe, and its b-in-
dole group is distinctly larger than Phe-phenyl.
(F5)Phe is nearly isosteric with Phe, while it has an en-
hanced hydrophobicity and the inverse quadrupole mo-
ment.18 All the aromatic amino acids were introduced at
Cha possesses the saturated substituent (cyclohexyl
group) of the phenyl group. Cha is nearly isosteric with
Phe, but lacks the aromaticity and the quadrupole
moment associated with an aromatic ring. The Phe !
Cha replacement at position 3 resulted in a sharp drop

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T. Honda et al. / Bioorg. Med. Chem. 15 (2007) 3883–3888
Table 3. Opioid receptor binding affinity and selectivity of endomor-
phin-2 and its Phe4-substituting analogs
position 3 of endomorphin-2 instead of Phe. Neither His
nor Tyr substitutions resulted in retainment of the par-
ent binding affinity of endomorphin-2. [His3]End-2 and
[Tyr3]End-2 were extremely weak to bind to the l recep-
tor. [His3]End-2 exhibited only 0.08% activity of native
endomorphin-2, and [Tyr3]End-2 showed 0.3% activity.
It is obvious that His and Tyr cannot compensate
Phe3 for binding to the receptor.
Peptidesa
(Phe4-substituents)
IC50 (nM)
Selectivityb
l receptor
d receptor
10,300 2770 2980
Phe (End-2)
Ala
Val
3.46 1.21
85.4 13.5
76.5 3.39
62.0 9.66
33.9 5.73
917 181
Unboundc
>1 mM
>1 mM
>1 mM
(l)d
(l)
(l)
(l)
Leu
Nle
In contrast, [Trp3]End-2 was found to be extremely po-
tent as compared to these analogs. It should be noted
that this analog is more potent than native endomor-
phin-2 (about 180%). [Trp3]End-2 is native endomor-
phin-1, and Trp at position 3 appears to preferentially
interact with the l receptor than Phe. Replacement with
another aromatic amino acid (F5)Phe resulted in a drop
in activity, showing an approximately threefold weaker
affinity than endomorphin-2.
Cha
11,400 4070 21.3
1000
(F5)Phe
His
Tyr
2.74 0.511 8040 1610
138 70.7
62.2 13.9
87.1 10.0
408 95.5
>1 mM
54,500 3200 5.20
(l)
Trp
Lys
DAGOe
>1 mM
Unboundc
(l)
(l)
643
1.13 0.263 727 109
a Peptides are designated by the amino acid residues at position 4 of
endomorphin-2 (Tyr-Pro-Phe-/Xaa/-NH2).
b Receptor selectivity was estimated by calculating the ratio of the IC50
values for l receptor versus those for d receptor.
Non-aromatic alkyl amino acids were also incorporated
at position 3. These included Val, Leu, and Nle, with the
isopropyl, isobutyl, and butyl groups, respectively, at
the b-position in the side-chain. It was previously found
that replacement of the Phe3-phenyl group in deltorphin
II by alkyl groups fully retains the receptor binding.2
However, any substitutions with the same amino acid
at position 3 of endomorphin-2 were found to reduce
drastically the binding affinity; that is, 0.13% with
Val3, 0.37% with Leu3, and 1.50% with Nle3 substitu-
tions. Although the binding activities of these analogs
are quite weak, there is a clear tendency for the binding
affinities of these analogs to be dependent upon the size
of the alkyl groups of the substituents and the molecular
hydrophobicity of peptides.
c ‘Unbound’ exhibits that tracer ligands ([3H]DAGO and [3H]deltor-
phin II) are not replaced with 10 mM of the synthetic peptides.
d (l) means ‘extremely or considerably weak selectivity for l receptor’.
e DAGO is a l-specific opioid peptide with the sequence of Tyr-D-Ala-
Gly-(N-Me)Phe-Gly-ol.
Non-aromatic alkyl amino acids were also incorporated
at position 4. Neither Val-, Leu-, nor Nle-substitution
resulted in retainment of the binding affinity of endo-
morphin-2. These substitutions reduced the affinity of
endomorphin-2 to 4.5% with Val4, 5.6% with Leu4,
and 10% with Nle4.
3. Discussion
2.3. Receptor binding affinities of Phe4-substituted endo-
morphin-2 analogs
Endomorphin-2 has a characteristic structure in which
the aromatic amino acid Tyr is connected to the Phe-
Phe aromatic dipeptide. The connecting unit is Pro,
which usually creates a bent conformation and is impor-
tant in the determination and stabilization of the struc-
tures of endomorphins.19 The interrelationship between
Tyr and Phe-Phe might be crucial to recognizing the
receptor, and the binding site would independently be
constructed specifically for these aromatic amino
acids.15,20–22 The importance of aromatic–aromatic
interactions such as Tyr1Phe3, Tyr1Phe4, and Phe3Phe4
in the association between the receptor and ligand rec-
ognition has recently been reported.19 In order to eluci-
date the interaction mode of C-terminal consecutive Phe
residues, we evaluated the receptor activity of a series of
Phe3 or Phe4-substituted analogs. It is particularly
important to assess the structural importance of each
Phe residue to gain insight into the characteristic recep-
tor activity. The present results clearly indicate that the
b-phenyl groups of Phe3 and Phe4 are critical to binding
to the l receptor, and the aromaticity of Phe is a key
structural characteristic essential for this binding ability.
Table 3 summarizes the results of the binding affinity of
endomorphin-2 analogs with Phe4-substituents for the
l and d opioid receptors. The elimination of Phe-phenyl
at position 4 also resulted in a drastic drop in activity.
[Ala4]End-2 showed a 25-fold drop in binding affinity
(IC50 = 85.4 nM). Clearly, the Phe4-phenyl group is
essential for binding to the l receptor. [Cha4]End-2 also
exhibited a drastically reduced (about 270-fold) binding
affinity (IC50 = 917 nM), indicating the importance of
the aromaticity of Phe4-phenyl.
These results led to an assay to examine the analogs con-
taining a series of aromatic amino acids at position 4.
The Phe ! His and Phe ! Tyr replacements resulted
in activity decreases (40-fold and 18-fold, respectively).
Surprisingly, Phe ! Trp replacement brought about a
drastic activity drop (ca. 25-fold). [Trp4]End-2 showed
very weak (ca. 4% of endomorphin-2) binding to the
l receptor, but this was not the case at position 3, as
mentioned above. Eventually, [Trp3]End-2 is much more
potent (ca. 1900-fold) than [Trp4]End-2. It is clear that
Trp cannot compensate for the loss of Phe4 for binding
to the receptor. In contrast, another aromatic amino
acid (F5)Phe at position 4 did not change the activity
of Phe-containing native endomorphin-2.
To show the importance of the aromaticity, not the
hydrophobicity, we analyzed the interrelationship be-
tween the receptor binding affinities and the HPLC
retention time of endomorphin-2 analogs (Fig. 2).

T. Honda et al. / Bioorg. Med. Chem. 15 (2007) 3883–3888
3887
The retention time in RP-HPLC is usually characteristic
in revealing the molecular hydrophobicity of peptides. It
is clear that there is a rough linearity among a series of
analogs with substitutions at positions 3 and 4, suggest-
ing that the HPLC retention time correlates to the
molecular hydrophobicity. When comparing the activity
profiles of each series of endomorphin-2 analogs with
Phe3- and Phe4-substituents (Fig. 2), it is obvious that
the substitutions much more strongly affect the Phe3 res-
idue than the Phe4 residue. The line showing activity–
hydrophobicity interrelationships for Phe3-substituents
demonstrates a much lower activity region than for
Phe4-substituents, indicating that the receptor activities
of analogs with Phe3-substituents are much weaker than
those with Phe4-substituents.
Indeed, it has been proposed that endomorphin-1 and
endomorphin-2 may act through two distinct l1- and
l2-opioid receptor subclasses, respectively.21,23,24 We
used rat brain membrane preparations for receptor
binding assay in the present study. If such subclasses
were present, both of them would be contained together.
Thus, a combined result of receptor affinities would be
obtained for each analog. Since the two series of analogs
with the substituents at positions 3 and 4, namely,
(Xaa3-Phe4) and (Phe3-Xaa4), were aligned on different
lines, their structural requirements appear to be distinct
from each other. It should be noted that both native
endomorphins have the Phe residue at position 4. The
present results strongly suggest that endomorphins dis-
tinguish putative l-receptor binding subsites by amino
acids Phe and Trp at position 3. In other words, there
would be two distinct l opioid receptor subclasses in
rat brain.
It should be noted that the linearities between this
molecular hydrophobicity and receptor binding activity
are not complete. [Phe3,Phe4]End-2 (endomorphin-2 it-
self), [Trp3, Phe4]End-2 (endomorphin-1 itself),
[(F5)Phe3]End-2, and [(F5)Phe4]End-2 do not show such
linearity, exhibiting much more enhanced activity than
analogs. These results suggest that the receptor binding
sites for both Phe3 (or Trp3) and Phe4 (or (F5)Phe) are
constructed just for these aromatic amino acids, not
for hydrophobic amino acids.
4. Experimental
4.1. Materials
The Boc derivatives of fluorine-containing phenyl-
alanines were prepared as described,25–27 and all other
Boc-amino acids were purchased. p-Methylbenzhydryl-
amine (MBHA) resin, N-methylpyrrolidone (NMP),
dichloromethane, diisopropylethylamine, trifluoroacetic
acid (TFA), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetram-
ethyluronium hexafluorophosphate (HBTU), and
1-hydroxybenzo-triazole (HOBt) were also purchased.
N,N-dimethylformamide (DMF) and acetonitrile were
purchased from Kanto Chemical (Tokyo). Anhydrous
hydrogen fluoride (HF), p-cresol, Tris–HCl, bacitracin,
and bovine serum albumin (BSA) were purchased from
Hashimoto Kasei Co. (Osaka), Kishida Chemical Co.
(Osaka), Nacalai Tesque Co. (Kyoto), Sigma (St. Louis,
MO, USA), and Wako Pure Chemical Co. (Osaka),
respectively. All other chemicals were of the best grade
available.
The substitutions of Phe4 with Trp and Cha showed
unexpectedly diminished receptor affinity (Table 3).
Although the affinity of [Cha3]End-2 lay on the Phe3-cor-
relation line, as shown in Fig. 2, [Cha4]End-2 was extre-
mely weaker than expected from the Phe4-correlation
line. With the Cha4-side-chain it might be hard to retain
the interaction with certain receptor sites in the receptor.
For Trp-substitutions, [Trp3]End-2 was found to increase
the affinity as compared to parent endomorphin-2. This
trend is not surprising because [Trp3]End-2 is a natural
peptide ligand for the l receptor, namely, endomor-
phin-1. At position 3 of endomorphins, Trp appears to
be much more favorable than Phe. However, the fact
that the same substitution at position 4 drastically de-
creased the binding affinity is somehow surprising, since
the Trp4 residue may conserve both the aromaticity and
high hydrophobicity. This result is consistent with the
case of Phe4 ! Trp replacement in endomorphin-1.15
Tyr-Pro-Trp-Trp-NH2 was extremely weak, approxi-
mately 100-fold weaker than endomorphin-1. The in-
verse preference of the l-receptor for Trp and Phe in
endomorphins would be brought about by their differ-
ences in size, aromaticity, and perhaps hydrophobicity.
4.2. Peptide synthesis
All peptides were synthesized by the method of manual
solid phase synthesis. Amino acids were protected at
their amino group with the Boc group, and the side-
chain protecting groups were 2,6-dichlorobenzyl for
Tyr, 2-chlorobenzyloxycarbonyl for Lys, benzyloxym-
ethyl for His, and formyl for Trp. To obtain C-terminal
peptide amides, MBHA resin was utilized. The peptide
amides synthesized were endomorphin-2 (YP/Phe/Phe)
and its analogs: that is, YP/Ala/F, YP/Val/F, YP/Leu/
F, YP/Nle/F, YP/Cha/F, YP/Lys/F, YP/(F5)Phe/F, YP/
His/F, YP/Tyr/F, YP/Trp/F (corresponding to endo-
morphin-1), YPF/Ala, YPF/Val, YPF/Leu, YPF/Nle,
YPF/Cha, YPF/Lys, YPF/(F5)Phe, YPF/His, YPF/
Tyr, and YPF/Trp. Coupling reactions were carried
out by using HBTU/HOBt in a mixture of NMP and
DMF (1:2, v/v) for 30 min.16 Each coupling reaction
was checked by means of a ninhydrin test for
completion.17
It has not previously been clear whether endomorphin-1
and endomorphin-2 play different roles under physiolog-
ical conditions, and whether they interact with different
receptor subclasses. For such discrimination, the present
results definitely help to differentiate the structural char-
acteristics of the receptors. The Trp-indole ring is larger
than the Phe-phenyl ring, and the receptor-binding site
specific for Phe3 in endomorphin-2 would be just the
actual size of the Phe side-chain, but not the Trp side-
chain. On the other hand, the receptor-binding site spe-
cific for Trp3 in endomorphin-1 should be the size of the
Trp side-chain in order to accept the Trp-indole group.

3888
T. Honda et al. / Bioorg. Med. Chem. 15 (2007) 3883–3888
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Peptides were liberated from the resin by treatment with
anhydrous liquid HF containing 10% p-cresol at 0 ꢁC
for 1 h. The products were purified first by gel filtration
on a column (2.0 · 100 cm) of Sephadex G-15 (Pharma-
cia, Uppsala, Sweden) eluted with 30% AcOH and then
by preparative reversed-phase high performance liquid
chromatography (RP-HPLC) (Cica-Merck, LiChro-
spher RP-18 (e) (5l): 25 · 250 mm). The elution condi-
tions employed for RP-HPLC were as follows: solvent
system, 0.1% aqueous TFA (A solution) and acetonitrile
containing 20% A solution (B solution); flow rate,
3 ml/min; temperature, 25 ꢁC; and UV detection,
230 nm. Elution was carried out with 5% B solution
for the first 5 min and then with a linear concentration
gradient of B solution, 20–60% for 40 min.
The purity was verified by analytical RP-HPLC
(LiChrospher RP-18 (5l): 4 · 250 mm), using the same
conditions except for a flow rate of 0.7 ml/min. Amino
acid analyses of peptides were carried out by RP-HPLC
of phenylthiocarbamoyl derivatives of amino acids using
a PICO-TAGTM system (Waters, Milford, MA) after
hydrolysis in a constant-boiling hydrochloric acid at
110 ꢁC for 24 h. Mass spectra of peptides were measured
on a mass spectrometer VoyagerTM DE-PRO (PerSeptive
Biosystems, Framingham, MA) with the method of ma-
trix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectroscopy.
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4.3. Receptor binding assays
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essentially as described previously.28 Membranes were
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[3H]DAGO (55.3 Ci/mmol, DuPont/NEN Research
Products, Wilmington, DE) for l receptors and [3H]del-
torphin II (49.5 Ci/mmol, Amersham, Buckinghamshire,
UK) for d receptors. Each tube containing the mem-
brane preparations, synthetic peptides, and 0.25 nM
respective tritium-labeled ligand was incubated at room
temperature for 60 min in Tris–HCl buffer (pH 7.55)
containing 0.1% BSA. Bacitracin (100 lg/ml) was added
as an enzyme inhibitor. After incubation, solutions were
filtered by glass fiber filters (GF/B; Whatman, Clifton,
NJ) and washed twice with 10 mM Tris–HCl buffer
(pH 7.55, 4 ml). Filters were placed in scintillation vials
containing a 4-ml scintillation cocktail (Scintisol EX-H;
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ALLFIT.29
´
19. Leitgeb, B.; Toth, G. Eur. J. Med. Chem. 2005, 40, 674.
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References and notes
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2. Honda, T.; Shirasu, N.; Chuman, Y.; Okada, K.; Fujita,
T.; Nose, T.; Shimohigashi, Y. Bull. Chem. Soc. Jpn. 2000,
73, 2549.