Structure-activity study of endomorphin-2 analogs with C-terminal modifications by NMR spectroscopy and molecular modeling

Article abstract of DOI:10.1016/j.bmc.2008.05.001

Endomorphin-2 (EM-2) is a putative endogenous μ-opioid receptor ligand. To get insight into the important role of C-terminal amide group of EM-2, we investigated herein a series of EM-2 analogs by substitution of the C-terminal amide group with -NHNH₂, -NHCH₃, -N(CH₃)₂, -OCH₃, -OCH₂CH₃, -OC(CH₃)₃, and -CH₂-OH. Their binding affinity and bioactivity were determined and compared. Despite similar (analogs 1, 4, and 7) or decreased (analogs 2, 3,5, and 6) μ affinity in binding assays, all analogs showed low guinea pig ileum (GPI) and mouse vas deferens (MVD) potencies compared to their parent peptide. Interestingly, as for analogs 2 and 3 (a single and double N-methylation of C-terminal amide), the potency order with the K_i (μ) values was 2 > 3; for the C-terminal esterified analogs 4-6, the potency order with the K_i (μ) values was 4 > 5 > 6. Thus, we concluded that the steric hindrance of C-terminus might play an important role in opioid receptor affinity. We further investigated the conformational properties of these analogs by 1D and 2D ¹H NMR spectroscopy and molecular modeling. Evaluating the ratios of cis- and trans-isomers, aromatic interactions, dihedral angles, and stereoscopic views of the most convergent conformers, we found that modifications at the C-terminal amide group of EM-2 affected these analog conformations markedly, therefore changed the opioid receptor affinity and in vitro bioactivity.

Full text of DOI:10.1016/j.bmc.2008.05.001

Bioorganic & Medicinal Chemistry 16 (2008) 6415–6422
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity study of endomorphin-2 analogs with C-terminal
modifications by NMR spectroscopy and molecular modeling
Chang-lin Wang a, , Jin-long Yao a, , Ye Yu a, Xuan Shao a, Yun Cui a, Hong-mei Liu a,
Lu-hao Lai a, Rui Wang a,b,
*
a Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Institute of Biochemistry and Molecular Biology, School of Basic Medical Science,
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, China
b State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University,
Kowloon, Hong Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Endomorphin-2 (EM-2) is a putative endogenous l-opioid receptor ligand. To get insight into the impor-
tant role of C-terminal amide group of EM-2, we investigated herein a series of EM-2 analogs by substi-
tution of the C-terminal amide group with –NHNH2, –NHCH3, –N(CH3)2, –OCH3, –OCH2CH3, –OC(CH3)3,
and –CH2–OH. Their binding affinity and bioactivity were determined and compared. Despite similar
(analogs 1, 4, and 7) or decreased (analogs 2, 3,5, and 6) l affinity in binding assays, all analogs showed
low guinea pig ileum (GPI) and mouse vas deferens (MVD) potencies compared to their parent peptide.
Interestingly, as for analogs 2 and 3 (a single and double N-methylation of C-terminal amide), the potency
order with the Ki (l) values was 2 > 3; for the C-terminal esterified analogs 4–6, the potency order with
the Ki (l) values was 4 > 5 > 6. Thus, we concluded that the steric hindrance of C-terminus might play an
important role in opioid receptor affinity. We further investigated the conformational properties of these
analogs by 1D and 2D 1H NMR spectroscopy and molecular modeling. Evaluating the ratios of cis- and
trans-isomers, aromatic interactions, dihedral angles, and stereoscopic views of the most convergent con-
formers, we found that modifications at the C-terminal amide group of EM-2 affected these analog con-
formations markedly, therefore changed the opioid receptor affinity and in vitro bioactivity.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 28 March 2008
Revised 30 April 2008
Accepted 1 May 2008
Available online 3 May 2008
Keywords:
Endomorphin-2
C-terminal modification
Conformation
NMR spectroscopy
Molecular modeling
1. Introduction
spacer in position 2, lipophilic and aromatic residues in positions 3
and 4 for the message, and amidation at the C-terminus for the ad-
The endogenous peptide ligands for l-opioid receptor, endo-
morphin-1 (Tyr-Pro-Trp-Phe-NH2, EM-1), and endomorphin-2
(Tyr-Pro-Phe-Phe-NH2, EM-2) were discovered in 1997 by Zadina
et al.1 Both endomorphins (EMs) exhibited the highest affinity for
l-opioid receptor and extraordinarily high selectivity relative to
d- and j-opioid receptor systems of all known opioid substances.1
They are also important model peptides to determine the struc-
ture–activity relationship based on their typical characteristics of
backbones and aromatic side chains.2,3
Naturally occurring opioid peptides consist of two distinct bio-
logically important parts, a N-terminal tri- or tetrapeptide frag-
ment, the message sequence, and the remaining C-terminal
fragment, the address sequence. Structure–activity studies showed
that the message sequence needed strict requirements for a good
interaction with opioid receptors. Important feathers are the pres-
ence of cationic amino group and a phenolic group in position 1, a
dress sequence, which seems to be determinant for peptide stabil-
ity.4,5 Thus, as for EM-2, there is a strict requirement for the amino
and phenolic groups of the Tyr1, an appropriate spacer (Pro2), and
an aromatic group (Phe3 and Phe4).6 According to the ‘message-ad-
dress’ concept,7 it is possible to consider that Tyr-Pro-Trp/Phe and
Phe-NH2 correspond to the message and address fragments,
respectively.6 These two sequences of EM-2 play an important role
in ligand recognition.6,8–11 Since the N-terminal tripeptide unit of
EM-2 is considered to be the ‘message’ fragments, their 3D struc-
tures may be important in the formation of bioactive conformation
and binding to opioid receptor, whereas, the C-terminal ‘address’
fragment may contribute to the stability of structure of the N-ter-
minal tripeptide unit, resulting in the biologically active form of
EM-2.11
A majority of bioactive peptides reveal their biological functions
by C-terminal a-amidation.12 As the deamination of such peptides
leads to considerable loss of activity, the amide group may be
strongly correlated with the bioactivity.13–15 C-Terminal amidation
would significantly associate with the bioactive conformation of a
bioactive peptide and its interaction with a receptor. Previous
* Corresponding author. Tel.: +86 931 8912567; fax: +86 931 8911255.
E-mail address: wangrui@lzu.edu.cn (R. Wang).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.001

6416
C. Wang et al. / Bioorg. Med. Chem. 16 (2008) 6415–6422
ing determining distances between two aromatic rings and the
dihedral angles of the backbone and side chains.
2. Results and discussion
2.1. Radioligand binding and in vitro bioactivity assays
The affinity and selectivity of EM-2 and all its analogs (charac-
terized in Table 1) were evaluated by radioligand binding assays
using rat brain membranes. In the binding assays, [H3]DAMGO
and [H3]DPDPE were used as l- and d-opioid receptor radioli-
gands, respectively. Their binding affinities for l- and d-opioid
receptor are summarized in Table 2. In the radioligand binding
assay, analog 1 in which the C-terminal amide group of EM-2
was modified into NHNH2 exhibited only slightly lower l affinity
than EM-2.19 A single and double N-methylation of C-terminal
amide of EM-2 (to give analogs 2 and 3) produced about 3- and
33-fold greater decrease in l affinity than EM-2, respectively. This
result indicated that N-methylation of C-terminal amide was not
liable to bind with l-opioid receptor. Moreover, previous studies
also reported that the EM-2 analogs, containing N-methylated
amino acids, displayed reduced l-opioid receptor affinity.20,21 In
previous study,19 our group changed the C-terminal amide group
of EM-2 into OCH3 to give analog 4 with high affinity, similar to
that of EM-2. In this study, we synthesized similar analogs 5 and
6 by changing the C-terminal amide into OCH2CH3 and OC(CH3)3,
respectively. Interestingly, the potency order with the Ki (l) val-
ues was 4 > 5 > 6, but the potency order with the Ki (d) values
was 6 > 5 > 4. Thus, we concluded that the increase in bulkiness
of the esterified group might decrease the binding of an analog
to l-opioid receptor, but increase the binding to d-opioid recep-
tor. The –ol derivatives of EMs, with the C-terminal amide to
alcohol conversion, exhibit high affinity and low intrinsic agonis-
tic efficiency in the pharmacological assays in vitro.9,10 Presently,
Figure 1. Schematic diagram of EM-2 and its analogs investigated in this study.
study has demonstrated that substitution of a carboxyl group for
the C-terminal amide group makes the molecular conformation
of EM-2 much flexible in DMSO solution. The C-terminal free acid
of EM-2 leads to a considerable loss of its binding affinity and ago-
nist activity for the l-opioid receptor.6,9,16 A similar decrease in
activity was observed for morphiceptin and its C-terminal free
acid.17 However, the d-opioid receptor affinity of EM-2 was in-
creased by the substitution of a carboxyl group for the C-terminal
amide group.16 Therefore, the differentiation between the l- and
d-opioid receptor selectivities of EM-2 is related to C-terminal
region.16 Nevertheless, the –ol derivatives of EMs, with the
C-terminal amide to alcohol conversion, are high-affinity and low
intrinsic efficiency agonists in the pharmacological assays in
vitro.9,10 Our previous study indicated that the C-terminal amide
to alcohol conversion produced different effects on the vasodepres-
sor activity of EM-1 and EM-2.8 A recent review18 reported that the
replacement of the carboxamide group of EM-2 by ester (COOMe)
or hydrazide (CONHNH2) group produced analogs with equal or
slightly improved l-affinity compared with EM-2.
As a possible clue to the biological and structural implications
of C-terminal amidation, we investigated herein the modifications
of C-terminal amide group of EM-2 by substitution of the C-termi-
nal amide group with –NHNH2, –NHCH3, –N(CH3)2, –OCH3,
–OCH2CH3, –OC(CH3)3, and –CH2–OH (Fig. 1). In the present study,
we evaluated and compared the opioid receptor affinity and selec-
tivity of these analogs by radioligand-binding assays and in vitro
bioactivity assays. We further investigated the conformational
properties of these analogs by 1D and 2D 1H NMR spectroscopy
and molecular modeling to reveal the conformational function of
the C-terminal group of these analogs. We compared the confor-
mations of EM-2 and its analogs generated by nuclear overhauser
effect (NOE) restraints in several steric structural analysis includ-
our result (analog 7) indicated similar high
(l) = 6.34 nM).
l affinity (Ki
We also evaluated the in vitro pharmacological activities using
isolated guinea pig ileum (GPI) for the l-opioid receptor and mouse
vas deferens (MVD) for the d-opioid receptor. The potencies of
these analogs to inhibit an electrically evoked neurotransmitter re-
lease and the resulting muscle contractions in GPI and MVD prep-
arations are summarized in Table 2. In the GPI assays, analogs 3
and 6 were almost devoid of activity. Furthermore, despite similar
or decreased l affinity in binding assays, analogs 1, 2, 4, 5, and 7
displayed about 4- to 14-fold lower potencies than their parent
peptide. In the MVD assays, only analog 3 showed a significant de-
crease in MVD potency.
Table 1
Analytical data of EM-2 and its analogs
RP-HPLC (tr)a
[a]D (°)
Mp (°C)
MS [M+H]+
b
Peptide No.
Sequence
Calculated
Foundc
EM-2
Tyr-Pro-Phe-Phe-NH2
10.365
9.086
À26
À24
À27
À28
À23
À32
À27
À31
130–132
142–145
137–139
121–124
108–110
108–110
126–128
92–94
571.3
586.3
586.3
599.3
586.3
600.3
628.3
558.3
572.0
587.2d
587.2
600.3
587.5d
601.2
629.3
559.4
1
2
3
4
5
6
7
Tyr-Pro-Phe-Phe-NHNH2
Tyr-Pro-Phe-Phe-NHCH3
Tyr-Pro-Phe-Phe-N(CH3)2
Tyr-Pro-Phe-Phe-OCH3
Tyr-Pro-Phe-Phe-OCH2CH3
Tyr-Pro-Phe-Phe-OC(CH3)3
Tyr-Pro-Phe-Phe-CH2OH
10.579
11.229
10.709
12.845
14.160
11.114
a
RP-HPLC elution on a Water Delta Park C18 column (3.9 Â 150 mm; Waters, Milford, MA). The solvents for analytical HPLC were as follows: A, 0.1% TFA in water; B, 0.1%
TFA in acetonitrile. The column was eluted at a flow rate of 0.6 ml/min with a linear gradient of A/B = 80:20 to A/B = 20:80 for 30 min and A/B = 20:80 to A/B = 80:20 for
5 min.
b
c = 0.3, MeOH (20 °C).
c
ESI-MS.
d
Values are cited from our group’s previous report.19

C. Wang et al. / Bioorg. Med. Chem. 16 (2008) 6415–6422
6417
Table 2
Opioid receptor binding affinities and in vitro pharmacological activity of EM-2 and its analogs; data are given as means SEM
Peptide No.
Ki (l) [nM]
Ki (d) [nM]
Ki (d)/Ki (l)
IC50 [nM]
GPI/MVD
GPI
9.67 0.84
MVD
Potency ratio
EM-2a
8.23 0.48
9.35 2.43
29.17 2.81
272.7 37.2
9.07 0.77
14.96 1.71
50.35 7.36
6.34 1.35
8360 1314
>10,000
8474 1558
>10,000
1016
25.8 5.2
185 79.7
0.37
0.71
2.5
0.66
1.1
1.0
40.5
1.0
1a
2
>1070
290.5
>36.7
>1102.5
196.5
4.9
132 42.4
136.2 31.8
1810 252.7
42.1 16.1
64.41 6.93
3002 1264
29.3 9.0
54.61 23.29
2723 545
37.2 15.0
63.75 6.06
74.12 5.16
29.17 21.44
3
4a
5
>10,000
2940 825.5
247 56.2
>10,000
6
7
>1577
a
Values are cited from our group’s previous report.19
Figure 2. The assignment of 1H NMR spectroscopy of [Phe4-CH2OH]-EM-2 (analog 7).
Table 3
2.2. NMR resonance assignments
The ratiosa of cis- and trans-isomers of EM-2 and its analogs
No.
Peptide
cis:trans
Conformational investigations of EM-2 and its analogs were
performed using standard one- and two-dimensional NMR spec-
troscopy in DMSO-d6 at 298 K. The 1H NMR spectra of analog 7
which had the highest l-opioid receptor affinity exhibited two sets
of conformers distinctly in the peaks of amide and aromatic pro-
tons (Fig. 2). The cis/trans isomers around the Tyr-Pro amide bond
were based on the characteristic sequential NOEs observed be-
tween Tyr1 CaH proton and Pro2 CaH/CdH protons, and the cis/trans
ratios of EM-2 and its analogs determined by the comparison of the
proton peak intensities are given in Table 3. The results indicated
that all the analogs had similar ratios (ꢀ1:1) in equilibrium be-
tween cis- and trans-isomers except analog 1. For analog 1, the ra-
tio of cis- and trans-isomers was 2:5. Nevertheless, it was reported
that EM-2 is in equilibrium between folded and open conformers
with cis/trans population ratio of 1:2 in DMSO-d6.6 A study with
C-terminal free acid of EM-2 in zwitterionic form indicated the
cis/trans population ratio of 1:2.16 It suggests that these modifica-
tions in C-terminal amide group of EM-2 distinguished from the
C-terminal free acid substitution might indicate a crucial structural
requisite of C-terminus in balancing the cis/trans ratio in solution,
since most of these analogs except analog 1 had higher ratios in
equilibrium between cis- and trans-isomers than that in EM-2.
All proton peak assignments were performed using a combina-
tion of connectivity information via scalar coupling in TOCSY
EM-2
EM-2
1:2
2:5
5:6
7:6
1:1
4:3
1:1
1:1
1
2
3
4
5
6
7
[Phe4-NHNH2]-EM-2
[Phe4-NHCH3]-EM-2
[Phe4-N(CH3)2]-EM-2
[Phe4-OCH3]-EM-2
[Phe4-OCH2CH3]-EM-2
[Phe4-OC(CH3)3]-EM-2
[Phe4-CH2OH]-EM-2
a
Determined by integration of 1H NMR.
experiments and sequential NOE network cross-peaks along the
peptide backbone protons. Following the protocol of structural
determination in solution, the chemical shifts of all analogs are
summarized in Supplementary Material. NOE cross-peak restraint
determined from ROESY spectra using correlation between signal
strength and interatomic distances was applied in restraint struc-
tural calculation. Based on the scalar defining the distance between
two protons bonded in the same carbon atom as 1.70 Å, the NOE
cross-peaks for the cis- and trans- isomers were classified as weak
(1.6–5.0 Å), medium (1.6–3.6 Å), and strong (1.6–2.9 Å). All NOE
cross-peaks of these EM-2 analogs were transferred for restraint
data. The NOE cross-peaks and intensities of EM-2 analogs are dis-
played in Supplementary Material.

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C. Wang et al. / Bioorg. Med. Chem. 16 (2008) 6415–6422
2.3. Structural calculation with NOE restraints
As the shorter distances indicate closer aromatic–aromatic
interactions, these isomers of analogs may exhibit more folded
structures compared with other ones. It is noted that the two most
percentage of aromatic interactions occurred in Phe3-Phe4 and
Tyr1-Phe4 of trans-isomer of analogs 2 and 4, respectively. There-
fore, the average distances were shorter related to other analogs.
Since few aromatic interactions were observed, the extended con-
formations were assumed to mainly exist in DMSO solution. For all
the analogs, only the trans-isomer of analog 4 had the shortest dis-
tances between the aromatic side chains, which may be responsi-
ble for the bow-shaped folded backbone structure.
Because of conformational flexibility, restrained molecular
dynamics was applied to determine conformation in the case of
peptides. We incorporated NOE restraints into theoretical confor-
mational analysis and molecular dynamics. To keep the peptide
bond preceding proline in the desired configuration (cis or trans),
the onefold torsional potential with the constant À100 and
100 kcal/mol was imposed to force the cis or the trans configura-
tion, respectively. For each analog, two sets of conformations were
generated, depending on the configuration of this peptide bond.
The cis and trans conformational ensembles of 10 least violated
structures of analog 7 are displayed in Figure 3, with mean back-
bone RMSD deviations 0.79 and 0.62 Å, respectively. The stereo-
scopic views of the most convergent conformers that belong to
the respective conformational groups are shown in Figure 4.
According to the conformational pattern of the backbone struc-
ture, all the NMR structures could be classified into two groups,
one was S- and reverse S-type, the other was bow-shaped type.
For analogs 2 and 3, they had the opposite type of backbone con-
formation regarding the cis and trans configurations, which were
S-type and bow-shaped type. The C-terminal esterified analogs
4–6, with C-terminus substituted by –OCH3, –OCH2CH3, and
–OC(CH3)3, took the S- or reverse S-type backbone except analog
4, which preferred bow-shaped backbone. As for the analog 7,
reverse S-type and bow-shaped type were preferred, compared
to the S- and reverse S-type of analog 1. Therefore, it suggested that
modifications in the C-terminus of EM-2 resulted in different
changes of the backbone conformation, and consequently showed
the binding affinities to l- and d-opioid receptor distinguished
considerably.
2.5. The torsions of backbone conformation and side-chain
disposition
The conformational requirements of all the peptides were
determined by the conformational constraint of the peptide back-
bone template (/ and w angles) and topographical constraint (v1
and v2) of the side chains. As seen in Table 5, the various C-termi-
nus modifications resulted in different spatial orientations of Tyr1,
Phe3, and Phe4 in cis- and trans-isomers. Notably, the third and
fourth phenyl rings were forced to point in nearly the opposite
direction in the trans-isomers of analog
4
(v13 = À163° and
v14 = À150°) relative to analogs 5 (v31 = 100°; v14 = 169°) and 6
(v13=66, v14 = 101°). Interestingly, for cis-isomers of these analogs,
the situations were the same. Therefore, the substitution groups of
C-terminus with –OCH3, –OCH2CH3, and –OC(CH3)3 determined
the spatial orientation of phenyl rings in the fourth position to some
extent. In accordance with the best binding affinity to l-opioid
receptor, the –OCH3 substituted group had the bow-shaped back-
bone conformation and different spatial orientation, both
distinguished from the latter two substitutions (–OCH2CH3 and
–OC(CH3)3). Therefore, we concluded that the modification in C-ter-
minus of EM-2, influenced the backbone conformation and side-
chain disposition of the fourth residue, and consequently changed
the binding affinity to the l- and d-opioid receptor, respectively.
2.4. Distances between aromatic rings
With the abundance of aromatic amino acids and flexibility,
EM-2 structures were supposed to be stabilized by the close pack-
ing of aromatic interactions instead of H-bonds.22,23 If the distance
of an aromatic ring pair between two centroids was less than 5.5 Å,
an interaction of aromatic–aromatic was assumed to exist.22,24 In
this case, we analyzed the conformations of aromatic side chains
and summarized the average distances of all analogs between
the centroids of aromatic rings (Table 4).
3. Conclusion
In the present study, to get insight into the important role of
C-terminal amide group of EM-2, we investigated a series of
EM-2 analogs by substitution of the C-terminal amide group with
Figure 3. Ensemble of the 10 most convergent and least violated structures of the family of cis- and trans-[Phe4-CH2OH]-EM-2 (analog 7) determined by DG calculations and
molecular dynamics with NOE distance restraints. The mean backbone RMSD of conformational clusters was labeled.

C. Wang et al. / Bioorg. Med. Chem. 16 (2008) 6415–6422
6419
Figure 4. Stereoscopic views of the most convergent and least violated structure of the cis-[Phe4-NHNH2]-EM-2 (A1), trans-[Phe4-NHNH2]-EM-2 (A2), cis-[Phe4-NHCH3]-EM-
2 (B1), trans-[Phe4-NHCH3]-EM-2 (B2), cis-[Phe4-N(CH3)2]-EM-2 (C1), trans-[Phe4-N(CH3)2]-EM-2 (C2), cis-[Phe4-OCH3]-EM-2 (D1), trans-[Phe4-OCH3]-EM-2 (D2), cis-[Phe4-
OCH2CH3]-EM-2 (E1), trans-[Phe4-OCH2CH3]-EM-2 (E2), cis-[Phe4-OC(CH3)3]-EM-2 (F1), trans-[Phe4-OC(CH3)3]-EM-2 (F2), cis-[Phe4-CH2OH]-EM-2 (G1), and trans-[Phe4-
CH2OH]-EM-2 (G2). The backbone and side-chain are represented by the thick and thin lines, respectively.
–NHNH2, –NHCH3, –N(CH3)2, –OCH3, –OCH2CH3, –OC(CH3)3, and
–CH2–OH. These analogs exhibited similar or reduced l-opioid
affinity in binding assays and showed low GPI and MVD potencies.
The N-methylation of C-terminal amide of EM-2 was not liable to
bind with l-opioid receptor since a single and double N-methyla-
tion of C-terminus decreased in l affinity. In addition, the increase
in bulkiness of the esterified group might decrease the binding of
an analog to l-opioid receptor. Thus, the steric hindrance of C-ter-
minus may play an important role in opioid receptor affinity. We
further investigated the conformational properties of these analogs
by 1D and 2D 1H NMR spectroscopy and molecular modeling. Eval-
uating the ratios of cis- and trans-isomers, aromatic interactions,
dihedral angles, and stereoscopic views of the most convergent
conformers, we found that modification at the C-terminal amide

6420
C. Wang et al. / Bioorg. Med. Chem. 16 (2008) 6415–6422
Table 4
and DMAP (0.8 mmol) at 0 °C. After 5 min, a solution of DCC
(2.4 mmol) in distilled CH2Cl2 (4 ml) was added to the mixture.
The mixture was stirred for 30 min at 0 °C and at room tempera-
ture overnight. DCU was then removed by filtration. Solvents
were then evaporated in vacuo. After removal of the solvents,
the residue was extracted with EtOAc and washed with 5% citric
acid, saturated NaHCO3, and saturated NaCl. The organic phase
was dried over Na2SO4, and evaporated in vacuo. Crude products
were purified by silica gel column chromatography (eluent: PE/
EtOAc 2:1) with a yield of 81% for Z-Phe-OCH2CH3 and 78% for
Z-Phe-OC(CH3)3.
Average distance (Å) between the centroids of aromatic rings in EM-2 analog clusters
of 100 conformations
Peptides
Tyr1-Phe3
Tyr1-Phe4
Phe3-Phe4
cis-EM-2a
trans-EM-2
7.11(11)b
8.54(7)
7.54(0)
11.33(0)
8.06(2)
11.57(0)
7.04(12)
10.14(0)
6.51(7)
7.53(9)
9.31(1)
9.90(1)
8.58(0)
9.52(8)
9.44(4)
10.86(0)
10.69(0)
8.69(1)
11.16(0)
12.91(0)
9.84(0)
11.89(0)
5.92(30)
12.75(0)
13.66(0)
12.20(0)
11.47(0)
11.45(0)
10.38(1)
7.82(0)
7.69(2)
8.42(1)
8.73(0)
7.32(11)
6.38(30)
9.11(0)
9.01(0)
9.33(0)
5.85(9)
8.67(0)
9.09(0)
8.86(1)
8.36(1)
8.15(9)
7.30(1)
cis-[Phe4-NHNH2]-EM-2 (1)
trans-[Phe4-NHNH2]-EM-2 (1)
cis-[Phe4-NHCH3]-EM-2 (2)
trans-[Phe4-NHCH3]-EM-2 (2)
cis-[Phe4-N(CH3)2]-EM-2 (3)
trans-[Phe4-N(CH3)2]-EM-2 (3)
cis-[Phe4-OCH3]-EM-2 (4)
trans-[Phe4-OCH3]-EM-2 (4)
cis-[Phe4-OCH2CH3]-EM-2 (5)
trans-[Phe4-OCH2CH3]-EM-2 (5)
cis-[Phe4-OC(CH3)3]-EM-2 (6)
trans-[Phe4-OC(CH3)3]-EM-2 (6)
cis-[Phe4-CH2OH]-EM-2 (7)
trans-[Phe4-CH2OH]-EM-2 (7)
4.1.3. General procedure for the synthesis of Boc-Phe-NHCH3/
Boc-Phe-N(CH3)2
To a stirred suspension of Boc-Phe-OH (2 mmol) and HOSu
(2.4 mmol) in anhydrous THF (10 ml), DCC (2.4 mmol) in THF
was added to the solution at 0 °C. The reaction mixture was stirred
for 30 min at 0 °C and 6 h at room temperature. After DCU was re-
moved by filtration, the remaining solution was added to methyl-
amine alcoholic solution (4 ml) or 30% dimethylamine aqueous
solution (8 ml) at 0 °C. The mixture was stirred for 30 min at 0 °C
and at room temperature overnight. Solvents were then evapo-
rated in vacuo. After removal of the solvents, the residue was ex-
tracted with EtOAc and washed with 5% citric acid, saturated
NaHCO3, and saturated NaCl. The organic phase was dried over
Na2SO4, and evaporated in vacuo. The residue was dissolved in
EtOH (10 ml), and distilled H2O (150 ml) was added to the solution
at 4 °C to obtain a precipitate, which was collected by filtration and
dried in vacuo. The yield of Boc-Phe-NHCH3 is 96%, and the yield of
Boc-Phe-N(CH3)2 is 77%.
10.64(1)
7.87(11)
8.25(11)
a
Data from Ref. 23.
The percentage of distance within 5.5 Å is given in bracket.
b
group of EM-2 affected these analog conformations markedly,
therefore changed the opioid receptor affinity and in vitro bioactiv-
ity. On the basis of our present study, these results gave the evi-
dence that the C-terminal residue was essential for high-affinity
binding of EMs to l-opioid receptor and for the development of
suitable l-opioid therapeutics.
4. Experimental
4.1. Peptide synthesis
4.1.4. Synthesis of Boc-Phe-Phe-OCH3
Boc-Phe-OH (5 mmol) was dissolved into anhydrous THF
(25 ml), then NMM 0.56 ml (5 mmol) and IBCF 0.67 ml (5 mmol)
were dropwise added to the solution under ice-salt bath below
À20 °C. The reaction mixture was stirred at À20 °C for 5 min to
form mixed acid anhydride. HClÁPhe-OCH3 (5 mmol) was dis-
solved into distilled DMF (10 ml), then NMM 0.56 ml (5 mmol)
was added to neutralize. After that the solution was added to
the mixed acid anhydride and the reaction mixture stirred at
room temperature overnight. Solvents were then evaporated in
vacuo. After removal of the solvents, the residue was extracted
with EtOAc and washed with 5% citric acid, saturated NaHCO3,
and saturated NaCl. The organic phase was dried over Na2SO4,
and evaporated in vacuo. The residue was crystallized from
EtOAc/PE with a yield of 96%.
4.1.1. Synthesis of HClÁPhe-OCH3
H-Phe-OH (10 mmol) was dissolved into absolute methanol
(40 ml), then distilled SOCl2 (10 ml) was dropwise added to the
solution under ice-salt bath below À20 °C. The reaction mixture
was stirred at room temperature for 1 h, and refluxed for 4 h. Sol-
vents were then evaporated in vacuo. The residue was crystallized
from abundant ice-cold petroleum ether (PE). The product was
washed with ether for several times to obtain the white powder.
4.1.2. General procedure for the synthesis of Z-Phe-OCH2CH3/Z-
Phe-OC(CH3)3
Absolute ethanol (0.7 ml) or tertiary butyl alcohol (1.2 ml) was
added to distilled CH2Cl2 (8 ml) containing Z-Phe-OH (2 mmol)
Table 5
Torsion angles of DG-calculated structures of EM-2 analogs used in the structure comparisona
Peptide
Tyr1
Pro2
Phe3
Phe4
W1
v11
U2
W2
U3
W3
v31
U4
W4
v41
cis-[Phe4-NHNH2]-EM-2 (1)
trans-[Phe4-NHNH2]-EM-2 (1)
cis-[Phe4-NHCH3]-EM-2 (2)
trans-[Phe4-NHCH3]-EM-2 (2)
cis-[Phe4-N(CH3)2]-EM-2 (3)
trans-[Phe4-N(CH3)2]-EM-2 (3)
cis-[Phe4-OCH3]-EM-2 (4)
À79
À121
38
2
À142
173
À180
106
152
91
À123
À86
180
À61
50
À113
49
À58
159
À76
65
À104
À163
149
100
3
À137
À66
180
54
78
98
180
68
145
89
—
—
—
—
—
À167
À108
À58
175
138
163
À131
À150
135
169
115
101
55
162
180
82
À54
À71
À49
À51
À87
À90
À120
À63
À84
À52
À85
À73
À57
À62
À81
À165
À165
147
À78
93
À180
À24
À85
À3
93
114
139
142
À176
164
82
152
81
80
165
93
À150
À169
99
135
127
170
155
112
À85
À20
178
À176
À60
À9
58
67
8
trans-[Phe4-OCH3]-EM-2 (4)
cis-[Phe4-OCH2CH3]-EM-2 (5)
trans-[Phe4-OCH2CH3]-EM-2 (5)
cis-[Phe4-OC(CH3)3]-EM-2 (6)
trans-[Phe4-OC(CH3)3]-EM-2 (6)
cis-[Phe4-CH2OH]-EM-2 (7)
trans-[Phe4-CH2OH]-EM-2 (7)
159
146
156
152
114
158
180
À102
À90
À107
À52
À127
À14
À70
94
À117
À115
—
À59
À79
À44
66
87
84
—
—
—
98
—
57
a
All angles reported in degrees.

C. Wang et al. / Bioorg. Med. Chem. 16 (2008) 6415–6422
6421
4.1.5. General procedure for the synthesis of Z-Phe-Phe-OCH2
CH3 /Z-Phe-Phe-OC(CH3)3
volume of 0.5 ml containing 300–500 lg/ml protein27 (protein con-
centration was determined by the method of Bradford28). In compe-
tition experiments, the following conditions were used for
incubations: [3H]DAMGO (0.5 nM), 1 h; and [3H]DPDPE (1 nM),
3 h.29 At first, [3H]DAMGO or [3H]DPDPE was added to membrane
protein suspension, then peptide samples of different concentra-
tions were rapidly added to separate suspension. Incubations were
started by the rapid addition of peptide samples to membrane sus-
pension in a rotating incubator at 25 °C and terminated by rapid vac-
uum filtration though GF/C filters using cell harvester. The filters
were washed thrice with 6 ml ice-cold buffer and then dried for
1 h at 80 °C. The radioactivity was measured by a Wallac Microbeta
1450 Trilux scintillation counter (GE Healthcare) after 12 h of incu-
bation in the scintillation cocktail. The extent of nonspecific binding
was determined in the presence of 10 lM naloxone. All experiments
were carried out in duplicate assays and repeated at least three
times. Affinity constants (Ki) were determined as described earlier.30
Z-Phe-OCH2CH3/Z-Phe-OC(CH3)3 (1 mmol) was dissolved in dis-
tilled MeOH (8 ml) containing Pd/C (80 mg) and H2, and the reac-
tion mixture was stirred at room temperature for 3 h. After the
catalyst Pd/C was removed by filtration, the solvent was evapo-
rated in vacuo, and the residue was dissolved in THF. The same
peptide-coupling procedure as for Boc-Phe-NHCH3/Boc-Phe-
N(CH3)2 was employed. Crude products were purified by silica
gel column chromatography (eluent: PE/EtOAc 2:1) with a yield
of 81% for Z-Phe-OCH2CH3 and 78% for Z-Phe-OC(CH3)3.
4.1.6. General procedure for the synthesis of Boc-Phe-Phe-NHCH3
/Boc-Phe-Phe-N(CH3)2
Boc-Phe-NHCH3/Boc-Phe-N(CH3)2 (1 mmol) was dissolved into
12.5 M HCl/EtOAc (1:4 v/v) and stirred for 2 h at room tempera-
ture. The pH of the solution was adjusted to 10 with 2 M NaOH.
The same peptide-coupling procedure as for Boc-Phe-NHCH3/
Boc-Phe-N(CH3)2 was employed. Crude products were purified by
silica gel column chromatography (eluent: PE/EtOAc 2:1).
4.3. In vitro bioactivity assays
In vitro opioid activities of peptides were tested in the GPI and
MVD bioassays as reported elsewhere.31 The GPI and MVD tissues
were mounted in a 10-ml bath that contained aerated (95% O2, 5%
CO2) Krebs–Henseleit solution at 37 and 36°C, respectively. Twitch
contractions were evoked by rectangular pulses with the following
parameters: 0.1-Hz, 50-V, 0.5-ms pulse width for GPI assay; pairs
(100-ms pulse distance) of rectangular impulses (1-ms pulse
width, 9 V/cm, i.e., supramaximal intensity) were repeated by
10 s for MVD assays. Isometric responses were recorded using a
train gauge transducer linked to a recorder system (model BL-
420 F, Taimeng Technology and Market Corporation of Chengdu,
China). Dose–response curves were constructed, and IC50 values
(concentration causing a 50% reduction of the electrically induced
twitches) were calculated graphically. The values are arithmetic
means of five to eight measurements.
4.1.7. General procedure for the synthesis of Boc-Phe-Phe-NH2/
Boc-Phe-Phe-CH2-OH
The pH of H-Phe-NH2ÁHCl/H-Phe-CH2-OHÁHCl was adjusted to
10 with 2 M NaOH. The same peptide-coupling procedure as for
Boc-Phe-NHCH3/Boc-Phe-N(CH3)2 was employed. Crude products
were purified by silica gel column chromatography.
4.1.8. Synthesis of Z-Tyr-Pro-OH
The pH of H-Pro-OH (10.4 mmol) was adjusted to 10 with 2 M
NaOH. The same peptide-coupling procedure as for Boc-Phe-
NHCH3/Boc-Phe-N(CH3)2 was employed. The residue was crystal-
lized from PE/EtOAc/HOAc (60:10:1). The yield was 70.3%.
4.1.9. General procedure for the synthesis of Tyr-Pro-Phe-Phe-
OCH3/Tyr-Pro-Phe-Phe-NHCH3/Tyr-Pro-Phe-Phe-N(CH3)2/Tyr-
Pro-Phe-Phe-OCH2CH3/Tyr-Pro-Phe-Phe-OC(CH3)3/Tyr-Pro-Phe-
Phe-NH2/Tyr-Pro-Phe-Phe-CH2-OH
4.4. NMR experiments
Deprotection of the Boc- and Z-group of C-terminal fragments
was performed with HCl/EtOAc and Pd/C, respectively. The same
peptide-coupling procedure as for Boc-Phe-NHCH3/Boc-Phe-
N(CH3)2 was employed. The crude protected tetrapeptides were
purified by silica gel column chromatography with a yield range
of 40–90%. The Z-group deprotection was performed by treatment
with Pd/C in MeOH containing H2 at room temperature. After 5 h
the solvent was evaporated in vacuo. The residue was easily puri-
fied by silica gel column chromatography.
Freeze–dried peptide samples were dissolved in dimethylsulf-
oxide-d6 (DMSO-d6) (99.9% isotopic purity; Cambridge Isotope Lab-
oratories, Andover, MA) at a concentration of 1–5 mg/500 lL. All
2D experiments were performed at 500 MHz on a Varian INOVA
NMR spectrometer with a constant temperature at 298 K. The
homonuclear correlation spectra, COSY, TOCSY, and ROESY were
obtained using standard pulse programs. The mixing times of 80
and 300 ms were used for TOCSY and ROESY spectra, respectively.
The 2D NMR matrixes were created and analyzed using the FE-
LIX 2004 computer program (Biosym Technologies Inc., San Diego,
VA). Each two-dimensional spectrum was acquired 1024 Â 1024
data matrix complex points in t1 and t2. The assignments of chem-
ical shifts were carried out using standard protein database and
custom unusual amino acid database built artificially. NOE cross-
peak restraints determined from ROESY spectra using correlation
between signal strength and interatomic distance were applied
in restraint structural calculation by the criteria of 1.70 Å between
two Cb protons.
4.1.10. Synthesis of Tyr-Pro-Phe-Phe-NHNH2
Z-Tyr-Pro-Phe-Phe-OCH3 (1.6 mmol) was dissolved into 5 ml of
absolute methanol, then H2N–NH2ÁH2O (0.6 ml) was dropwise
added to the solution. The mixture was stirred for 48 h at room
temperature until the precipitate was seen in the solution. Metha-
nol was evaporated in vacuo. The remainingss mixture was depos-
ited for 24 h. Then it was extracted with EtOAc. The extract was
washed with saturated NaCl to get the product with a yield of
85%. The Z-group deprotection was performed by treatment with
Pd/C in MeOH containing H2 at room temperature. After 5 h the
solvent was evaporated in vacuo. The residue was easily purified
by silica gel column chromatography.
4.5. Computational molecular modeling
All the molecular modeling calculations were performed on an
Origin 2000 workstation running the Irix 6.5 operation system (Sil-
icon Graphics Inc., Mountain View, CA, USA). The initial random
molecular structures were built for two isomers with x torsion
angles of Tyr1-Pro2 bond in allowance of 0° 10° and 180° 10°,
respectively. Then energy minimizations with the CVFF force field
(Accelrys Inc.) were carried out on Discover 98 module in the
4.2. Radioligand binding assays
Membranes were prepared from Wistar rat brain (without cere-
bellum) according to the literature methods.25,26 All binding exper-
iments were performed in 50 mM Tris–HCl buffer, pH 7.4, at a final

6422
C. Wang et al. / Bioorg. Med. Chem. 16 (2008) 6415–6422
Insight II 2000 package (Accelrys Inc.). The resulting coordinates
were applied in the generation of the distance-bound matrices. Cal-
culating by the standard protocol of the Distance Geometry (DG) II
package in NMR-Refine module of Insight II 2000 with NOE restraint
files exported from Felix2004. First, triangle-bound smoothing was
used to check out the correction of molecular structure by minimi-
zation. The force constant used for distance restraints was 50.0 kcal/
mol Å2. Second, the structures were used to generate in four dimen-
sions, then reduced to three dimensions with the EMBED algo-
rithm.32 Third, the assembly was optimized with a simulated
annealing (SA) step33 maintaining the distance constraints accord-
ing to the standard protocol of the DG II package. One hundred
structural ensembles were generated for every system.
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Acknowledgments
This work was supported by grants from the National Natural
Science Foundation of China (Nos. 20772052, 20621091, and
20525206), the Specialized Research Fund for the Doctoral Pro-
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the Chang Jiang Program of the Ministry of Education of China.
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.05.001.