Structure-activity relationships of novel endomorphin-2 analogues with N-O turns induced by α-aminoxy acids

Article abstract of DOI:10.1016/j.bmcl.2005.04.050

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH₂, EM-2) is a putative endogenous μ-opioid receptor ligand. To study the structure-activity relationship against its receptor, we introduced N-O turns into EM-2 and got the analogues with potent affinities for μ-opioid receptor. Our results indicated that N-O turn structures at the Pro²-aminoxy-Phe ³ position of EM-2 analogues played important roles for their affinities. These novel analogues with N-O turns provided a new approach to develop potent analgesics related to EM-2.

Full text of DOI:10.1016/j.bmcl.2005.04.050

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2986–2989
Structure–activity relationships of novel endomorphin-2
analogues with N–O turns induced by a-aminoxy acids
Jie Wei,a Xuan Shao,a Maozhen Gong,a Beibei Zhu,a Yuxin Cui,c
Yanfeng Gaoa and Rui Wanga,b,
*
aDepartment of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China
bKey Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University,
Beijing 100083, China
cMedical and Health Analysis Center, Peking University, Beijing 100083, China
Received 6 March 2005; revised 17 April 2005; accepted 22 April 2005
Abstract—Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2, EM-2) is a putative endogenous l-opioid receptor ligand. To study the
structure–activity relationship against its receptor, we introduced N–O turns into EM-2 and got the analogues with potent affinities
for l-opioid receptor. Our results indicated that N–O turn structures at the Pro2-aminoxy-Phe3 position of EM-2 analogues played
important roles for their affinities. These novel analogues with N–O turns provided a new approach to develop potent analgesics
related to EM-2.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Opioid peptides have been conjectured as secondary ef-
fect-free pharmacological tools to be used in place of
morphine,1–3 because of their roles as endogenous anal-
gesics in mammals.4–7 However, the inherent conforma-
tional flexibility of the opioid peptides has hampered
numerous attempts to accurately assess the bioactive
structures. Significant efforts have been devoted to the
elucidation of the opioid receptor-bound structures
Figure 1. The primary structures of N–O turn and EM-2.
through systematic studies of structurally constrained
peptides8 or peptidomimetics.9 The goals of these efforts
are to study the structure–activity relationships against
their receptors and to develop potent analgesics without
oxy acid induces a strong eight-membered-ring hydrogen
the well-known side effects of morphine. Several studies
bond formed between adjacent amino acid residues (the
have suggested that turn structure is the potential bio-
logically active structure of opioid peptides.10–14
N–O turn). Furthermore, this turn structure is side-chain
independent, and gives rigid and predictable secondary
structure.17 However, to the best of our knowledge, there
are no reports of introducing N–O turns into opioid pep-
Up to now, many unnatural building blocks have been
designed to create novel foldamers whose oligomers or
tides. Most of the work has been limited to the studies of
the structures of N–O turns. It is interesting to investi-
polymers are able to adopt well-defined structures.15,16
Among them, an important novel turn mimic is N–O
gate whether the N–O turns are still favored when side
turn, which is induced by a-aminoxy acid (H2N–O–
CHR–CO2H) (Fig. 1). In the peptide backbone, a-amin-
chains are introduced to a-aminoxy acids in opioid pep-
tides, and to probe whether these analogues have affini-
ties for their opioid receptors. Also, the turn structures
of opioid peptides can be simulated by this method.
Keywords: Endomorphin-2; Opioid peptide; N–O turn; a-Aminoxy
acid; Simulation.
To demonstrate our concept, we chose endomorphin-2
(EM-2) as a target peptide (Fig. 1). Endomorphin-1,
*
Corresponding author. Tel.: +86 931 8912567; fax: +86 931
8912561; e-mail: wangrui@lzu.edu.cn
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.050

J. Wei et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2986–2989
2987
H-Tyr-Pro-Trp-Phe-NH2, and endomorphin-2, H-Tyr-
Pro-Phe-Phe-NH2 were isolated from the bovine brain
and human brain, which showed high affinities and
selectivities for l-opioid receptor (MOR).18 Among opi-
oid peptides, endomorphins (EMs) are regarded as the
most effective endogenous analgesics that are released
in response to pain stimuli. It is often convenient to
study the corresponding peptidomimetics or peptide
analogues by modifying the original structure of
EMs.19–22 The studies have indicated that a turn struc-
ture at the Pro2–Phe3 position is formed, which can be
recognized by MOR, although EM-2 has a random con-
formation in water. The turn structure is EM-2ꢀs biolog-
ically active structure.10 Thus, it is very important to
simulate the turn structure and understand the struc-
ture–activity relationships between EM-2 and MOR.
These studies can help us to find highly bioactive EM-
2 analogues. Herein, we report a novel synthesis of a ser-
ies of EM-2 analogues with different a-aminoxy acids by
conventional solution phase method.
Figure 2. Summary of the NOEs observed (n, NOE) in the NOESY
spectra of analogues 5a–d and 6a–d in DMSO at rt. 5a, R = Bn(L); 5b,
R = Bn(D); 5c, R = CH3(D); 5d, R = H; 6a, R = Bn(L); 6b, R = Bn(D);
6c, R = CH3(D); 6d, R = H.
All the a-aminoxy acids were synthesized using the
method which had been recently developed.23 The syn-
thetic steps used to prepare EM-2 analogues 5a–5d are
shown in Scheme 1. Initially after tert-butyl deprotec-
tion of a-aminoxy acid 1, the resulting acid was coupled
to HClÆPhe-NH2 by N-methylmorpholine (NMM) and
isobutyl chloroformate (IBCF) to give dipeptide 3. The
phthaloyl (Pht) of 3 was removed by hydrazine hydrate;
then the resulting amine was coupled to Boc-Tyr-Pro-
OH under the same coupling conditions of preparing
dipeptide 3 to give 4. After removal of the Boc-group
from 4, we got final compound 5. We synthesized EM-
2 analogues 6a–d (Fig. 2) with the similar methods of
5. Furthermore, to illustrate the importance of N–O
turns in these analogues, we synthesized EM-2 ana-
logues 9a–b with oxime links (Ca1–CH@N–O–Ca2).
Analogues 9a–b did not form N–O turn structures be-
cause of lacking the O atoms to function as acceptors
in hydrogen bonding. The synthetic steps used to pre-
pare 9a–b are shown in Scheme 2. Dipeptide 3 was cou-
pled to Boc-Pro-H aldehyde24 in the presence of AcONa
Scheme 2. Total synthesis of EM-2 analogues 9a and 9b. a: R = Bn(L),
b: R = Bn(D). Reagents and conditions: (i) NH2–NH2ÆH2O, MeOH, rt,
2 h; (ii) Boc-Pro-H aldehyde, AcONa, EtOH, anhydrous Na2SO4, rt,
overnight; (iii) 50% TFA, 0 ꢁC, 2 h; (iv) NMM, IBCF, Boc-Tyr-OH,
overnight; (v) 50% TFA, 0 ꢁC, 2 h.
and anhydrous Na2SO4 to give compound 7. After Boc
deprotection of 7, the resulting amine was coupled to
Boc-Tyr-OH by NMM and IBCF to give 8. After re-
moval of the Boc-group from 8, we got final compound
9. All the final products were TFA salts and were puri-
fied by preparative reversed-phase HPLC. The purity of
all peptides was greater than 95%. The analytical data
are listed in Table 1.
We initially investigated the structural features of novel
EM-2 analogues using FT-IR. For the IR spectra of
5a–d and 6a–d, the ratio of hydrogen-bonded and non-
hydrogen-bonded NH stretching bands reflected the po-
sition of the conformational equilibrium. The analogues
of 5a–d and 6a–d displayed exclusively peaks at 3195–
3302 cmꢀ1 that corresponded to a hydrogen-bonded
NH stretching band.23,25 However, the analogues of 9a
and 9b displayed peaks at 3397 and 3401 cmꢀ1 that cor-
responded to no hydrogen-bonded NH stretching band
(Table 1).
The clear evidence supporting analogues 5a–d and 6a–d
adopting N–O turn structures was obtained from analy-
sis of the 2D NOESY NMR spectra (Fig. 2). NOE be-
tween amide NH and N–OAaCa-H, and NOE between
oxyamide NH and N–OAaCa-H showed the formation
Scheme 1. Total synthesis of EM-2 analogues 5a–d. a–d: R = Bn(L),
Bn(D), CH3(D), H. Reagents and conditions: (i) 50% TFA, 0 ꢁC, 2 h;
(ii) NMM, IBCF, HClÆPhe-NH2, overnight; (iii) NH2–NH2ÆH2O,
MeOH, rt, 2 h; (iv) NMM, IBCF, Boc-Tyr-Pro-OH, overnight; (v)
50% TFA, 0 ꢁC, 2 h.

2988
J. Wei et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2986–2989
Table 1. Analytical data of EM-2 analogues
20
½aꢁD (ꢁ)
Code
EM-2 analogues sequence
MS [M+H]+
IR (cmꢀ1 c
)
Calcd
Found
5a
5b
5c
5d
6a
6b
6c
6d
9a
9b
Tyr-Pro-(L-aminoxy Phe)-PheNH2
Tyr-Pro-(D-aminoxy Phe)-PheNH2
Tyr-Pro-(D-aminoxy Ala)-PheNH2
Tyr-Pro-(aminoxy Gly)-PheNH2
Tyr-(L-aminoxy Phe)-Phe-PheNH2
Tyr-(D-aminoxy Phe)-Phe-PheNH2
Tyr-(D-aminoxy Ala)-Phe-PheNH2
Tyr-(aminoxy Gly)-Phe-PheNH2
Tyr-Pro=(L-aminoxy Phe)-PheNH2
Tyr-Pro=(D-aminoxy Phe)-PheNH2
588.2817
588.2817
512.2504
498.2347
638.2973
638.2973
562.2660
548.2504
572.28
588.2823a
588.2816a
512.2469a
498.2333a
638.2969a
638.2988a
562.2663a
548.2499a
572.2b
ꢀ42 (c = 0.5, MeOH)
+11 (c = 0.5, MeOH)
ꢀ24 (c = 0.125, MeOH)
ꢀ42 (c = 0.5, MeOH)
ꢀ15 (c = 0.5, MeOH)
+40 (c = 0.5, MeOH)
+40 (c = 0.5, MeOH)
+21 (c = 0.5, MeOH)
ꢀ32 (c = 0.5, MeOH)
+43 (c = 0.5, MeOH)
3200
3207
3208
3206
3302, 3207
3195
3302, 3205
3193
3397
572.28
572.2b
3401
a ESI-TOF-MS.
b FAB-MS.
c FT-IR data of EM-2 analogues between 3100 and 3500 cmꢀ1 in MeOH at room temperature (1 mM solutions).
of N–O turn structures.23,25 Combining with IR and
NMR analysis, we concluded that the N–O turns were
still favored in EM-2 analogues. However, in the struc-
tures of analogues 9a and 9b there were no N–O turn
structures in the oxime links.
analogues acted as l-opioid receptor agonists and weak
d-opioid receptor agonists (data are not shown here).
The results are the means of at least three experiments.
From the opioid receptor binding data shown in Table
2, we got the following results. The l-opioid receptor
(MOR) binding affinities of 5a and 5b, which formed
N–O turns at the Pro2-aminoxy-Phe3 position, were
about 10-fold better than that of 9a and 9b. Compounds
9a and 9b did not form N–O turn structures at the Pro2-
aminoxy-Phe3 position. Compounds 5a and 5b both
remained good selectivities for MOR over d-opioid
receptor (d/l). But 6a–d, which formed N–O turns at
the Tyr1-aminoxy-Aa2 position, decreased the MOR
affinities of 5a–b by more than 20-fold. At the same
time, 6a–d decreased the MOR affinities of 9a–b by more
than 2-fold. Hence, we got the structure–activity rela-
tionships between these analogues and affinities for
MOR. First, the N–O turns at the Tyr1-aminoxy-Aa2
position in analogues 6a–d were the unfavorable struc-
tures for MOR affinities. Second, the N–O turns at the
Pro2-aminoxy-Phe3 position in analogues 5a–b were
the most favorable structures for MOR affinities in the
three types of these analogues, although 5a–b decreased
the MOR affinities of EM-2. Based on these results, we
thought that when EM-2 bind to MOR, it is likely to
form a turn structure at the Pro2-Phe3 position, and
hardly forms a turn structure at the Tyr1-Pro2 position.
To determine the affinities toward the l- and d-opioid
receptors, the opioid receptor binding assays were per-
formed in 50 mM Tris–HCl buffer, pH 7.4, at a final vol-
ume of 0.5 mL containing 250–400 lg of protein
(Synaptosomal brain membrane P2 was prepared from
Wistar rats). In competition experiments, the following
conditions were used for incubations: [3H]DAMGO
(0.5 nM, 25 ꢁC, 1 h), [3H]DPDPE (1 nM, 25 ꢁC, 3 h).
Nonspecific binding was determined in the presence of
10 lM naxolone. Kd values of [3H]DAMGO and
[3H]DPDPE were 0.533 and 2.75 nM, respectively. Ki
values were calculated according to the equation of
Cheng and Prusoff.26 The Ki values of EM-2 in
[3H]DAMGO and [3H]DPDPE assays agreed with Toth
and co-workers27 and Okada et al.,21b respectively. The
data are listed in Table 2. The data shown in Table 2 are
the means of at least three experiments. Functional bio-
activities of these analogues were evaluated in guinea pig
ileum (GPI) and mouse vas deferens (MVD) assays,
respectively. Stand compounds (EM-2 for the GPI and
deltorphin I for the MVD) were assayed in each prepa-
ration to permit estimation of relative potencies. The
Table 2. Binding affinity in competition with [3H]DAMGO and [3H]DPDPE in rat brain membranes
EM-2 analogs
[3H]DAMGO (l) Ki SE (lM)
[3H]DPDPE (d) Ki SE (lM)
Selectivity (d/l)
EM-2
5a
5b
(8.23 0.5) · 10ꢀ3
0.434 0.07
0.6 0.07
>10 (38%a)
>10 (35%a)
>10 (22%a)
na
>10 (40%a)
>10 (34%a)
4.8 0.05
8.36 1.3
1016
>23
>17
—
>10 (15%a)
>10 (30%a)
>10 (30%a)
>10 (21%a)
>10 (18%a)
>10 (30%a)
>10 (35%a)
>10 (13%a)
19.8 2.3
5c
5d
—
—
6a
6b
—
—
6c
6d
—
9a
9b
4.1
9
4.04 0.03
37.1 3.5
na: no activity.
a Percent decrease of maximum binding at 10 lM peptide.

J. Wei et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2986–2989
2989
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These results might help us to increase the binding affin-
ities of EM-2 by introducing better turn structures into
the Pro2-Phe3 position of EM-2, and to develop the
structure–activity relationships between EM-2 and
MOR.
In conclusion, it was the first time a-aminoxy acids were
introduced into opioid peptides. A series of novel EM-2
analogues containing N–O turns were designed and syn-
thesized. Their N–O turn structures were proved by
NMR and FT-IR. Some of these analogues had potent
affinities for MOR. The affinities of the analogues that
formed N–O turns at the Pro2-aminoxy-Phe3 position
were much higher than that of the analogues that
formed N–O turns at the Tyr1-aminoxy-Aa2 position.
We developed the structure–activity relationships be-
tween the MOR affinities and the positions of N–O
turns. The relationships suggest that when EM-2 binds
to MOR, it is likely to form turn structure at the Pro2-
Phe3 position. According to these relationships, we can
develop more potent analgesics with less side effects re-
lated to EM-2. Further studies are currently in progress
in our laboratory to investigate the relationships be-
tween the structures of the different analogues and
receptor affinities, in particular the turn structures and
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We thank the National Natural Science Foundation of
China (20372028, 20472026), the Ministry of Science and
Technology of China (2002CCC00600, 2003AA2Z3540),
the Ministry of Education of China, and the Chinese
Academy of Sciences for financial support in the form of
grants.
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