Systematic replacement of amides by 1,4-disubstituted[1,2,3]triazoles in Leu-enkephalin and the impact on the delta opioid receptor activity

Article abstract of DOI:10.1016/j.bmcl.2013.08.020

Using Cu(I)-catalyzed azide-alkyne cycloaddition in a mixed classical organic phase and solid phase peptide synthesis approach, we synthesized four analogs of Leu-enkephalin to systematically replace amides by 1,4-disubstituted[1,2,3]triazoles. The peptidomimetics obtained were characterized by competitive binding, contractility assays and ERK1/2 phosphorylation. The present study reveals that the analog bearing a triazole between Phe and Leu retains some potency, more than all the others, suggesting that the hydrogen bond acceptor capacity of the last amide of Leu-enkephalin is essential for the biological activity of the peptide.

Full text of DOI:10.1016/j.bmcl.2013.08.020

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5267–5269
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Systematic replacement of amides by 1,4-disubstituted[1,2,3]
triazoles in Leu-enkephalin and the impact on the delta opioid
receptor activity
Arnaud Proteau-Gagné a, Kristina Rochon b, Mélissa Roy b, Pierre-Julien Albert b, Brigitte Guérin c,d,
,
⇑
Louis Gendron b,d, , Yves L. Dory a,d,
⇑
⇑
a Laboratoire de chimie supramoléculaire, département de chimie, Université de Sherbrooke, 3001 12e av., Nord Sherbrooke, QC J1H 5N4, Canada
b Département de physiologie et biophysique, Université de Sherbrooke, 3001 12e av., Nord Sherbrooke, QC J1H 5N4, Canada
c Département de médecine nucléaire et de radiobiologie, Université de Sherbrooke, 3001 12e av., Nord Sherbrooke, QC J1H 5N4, Canada
d Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001 12e av., Nord Sherbrooke, QC J1H 5N4, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Using Cu(I)-catalyzed azide–alkyne cycloaddition in a mixed classical organic phase and solid phase
peptide synthesis approach, we synthesized four analogs of Leu-enkephalin to systematically replace
amides by 1,4-disubstituted[1,2,3]triazoles. The peptidomimetics obtained were characterized by
competitive binding, contractility assays and ERK1/2 phosphorylation. The present study reveals that
the analog bearing a triazole between Phe and Leu retains some potency, more than all the others,
suggesting that the hydrogen bond acceptor capacity of the last amide of Leu-enkephalin is essential
for the biological activity of the peptide.
Received 17 June 2013
Revised 25 July 2013
Accepted 5 August 2013
Available online 12 August 2013
Keywords:
Click chemistry
Delta opioid receptor
1,2,3 Triazole
Ó 2013 Elsevier Ltd. All rights reserved.
Leu-enkephalin
Peptidomimetics
Although Leu-enkephalin possesses poor selectivity (1 to 5Â)
for DOPr over the Mu opioid receptor (MOPr), it is considered to
be an endogenous DOPr ligand.1 Due to the rapid degradation by
enzymes and the poor hydrophilic character of Leu-enkephalin, a
lot of research activity has been done to design and synthesize pep-
tidomimetics of this peptide.
Since the discovery of the Cu(I)-catalyzed azide–alkyne cyclo-
addition known as ‘click chemistry’,2 various applications of this
robust reaction were published.3 In the field of peptidomimetics,
the use of [1,2,3]triazole as an amide surrogate has been applied
to create locked mimetics of both trans4 and cis5 amides producing
biologically active peptidomimetics. To a certain extend, the
molecular properties of the triazole and the amide groups are sim-
ilar6 (Fig. 1).
Both functions have hydrogen bond acceptor properties and a
similar dipolar moment.7 The triazole was rarely proved to be a
weak hydrogen bond donor.8 Incorporation of triazoles as amide
mimics substantially stretches the resulting peptidomimetics by
ꢀ1.2 Å. In several peptidomimetics this extension induced no sig-
nificant change to the biological activity.4,5 Since the amide bonds
in endogenous peptides are the primary site targeted by degrading
enzymes, their replacement by triazole groups represents an effi-
cient way to design analogs with enhanced biological half-life.7
This study presents a series of Leu-enkephalin (Tyr-Gly-Gly-
Phe-Leu) analogs obtained by systematic replacement of amides
by triazole groups (Fig. 2). The analogs were tested to determine
their biological activity at the delta opioid receptor (DOPr). It has
been hypothesized that selective DOPr agonists could lead to the
development of improved chronic pain medication,9,10 emphasiz-
ing the interest of this biological target.
In previous studies, we have shown that the systematic replace-
ment of amides in Leu-enkephalin by E-alkenes,11 esters12 and N-
Methyl amides12 can reveal important information regarding the
biological role of each amide in relevant analogs of Leu-enkephalin
and can therefore increase our understanding of the non-covalent
interactions between DOPr and its endogenous ligands. To our
knowledge the use of a triazole as a dipeptide isostere has never
been applied to the field of opioid peptidomimetics, although some
tetrazoles mimicking cis-amides have already been reported.5d
We used a mixed approach combining solid-phase synthesis
with classical organic synthesis for the preparation of Leu-enkeph-
alin analogs bearing a triazole. First, the azide moiety was prepared
in solution using known procedures13 (Scheme 1). Triflyl azide
⇑
Corresponding authors. Tel.: +1 819 564 5299.
E-mail addresses: Brigitte.Guerin2@usherbrooke.ca (B. Guérin), Louis.Gendro-
n@USherbrooke.ca (L. Gendron), Yves.Dory@USherbrooke.ca (Y.L. Dory).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.020

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A. Proteau-Gagné et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5267–5269
Figure 1. Comparison of the s-trans amide and the 1,4-disubstituted[1,2,3]triazole
dipeptide isostere.
Scheme 2. Synthesis of the alkyne moiety.
Scheme 3. Synthesis of the triazoles and Fmoc protection.
The azides 1–3 and the alkynes 4–6 were cross-coupled in
Cu(I)-catalyzed azide–alkyne cycloaddition reactions (Scheme 3).15
The resulting Boc protected building block 7 was used in solid
phase peptide synthesis as such without further modification due
to its N-terminal position in the Leu-enkephalin mimetic 14
(Fig. 2). For compounds 8–10, the Boc protective group was cleaved
and a Fmoc protection was introduced instead to give Fmoc pro-
tected11 building blocks 11–13 (Scheme 3). The synthesis of each
building block was achieved in good overall yields from their cor-
responding amino-alkyne and azide–acid precursors.
Afterward, the building blocks 7, 11–13 were used in solid
phase peptide synthesis with Fmoc methodology either on Tenta-
Gel S PHB resin to generate the mimetic 14 or on Wang resin to
produce the analogs 15–17 (Scheme 4). The loading onto the resin,
the benzoyl capping, the Fmoc deprotection, the HATU normal
peptide coupling and the resin cleavage were all carried out follow-
ing standard protocols.11,16 Whereas, the couplings of triazole
building blocks 7, 11–13 were done using DIC and HOBt. These
base free conditions are known to minimize racemization of tria-
zole dipeptide isosteres.15 After cleavage, the crude peptides were
purified on preparative reverse phase HPLC and all fractions over
95% in purity were combined. Analogs 14–17 (Fig. 2) were ob-
tained in yields ranging from 17% to 100%.
Figure 2. 1,4-Disubstituted[1,2,3]triazole analogs of Leu-enkephalin.
(N3Tf) was prepared first and then added to the amino acids to give
the azido acids 1–3.
Two different strategies were taken for the preparation of the
alkyne moieties (Scheme 2). Alkyne 4 was obtained in one
alkylation step from commercial di-tBu-imino-dicarboxylate.14 Al-
kynes 5 and 6 were obtained by partial reduction, with DIBAL, of
protected amino esters to yield the corresponding aldehydes,
followed by Seyferth–Gilbert homologation using Bestmann’s
reagent.5b It is worth noting that no epimerization of the chiral
center was observed during this step.
In order to evaluate the ability of each compound to bind DOPr,
we performed competitive binding assays using GH3/DOPr cell
membrane preparations. As shown in Table 1, the systematic
replacement of amides by triazoles did not produce peptidomimet-
ics with high retention of the parent peptide biological activity.
Among all compounds tested, analog 17 showed the highest
Scheme 1. Synthesis of the azide moiety.

A. Proteau-Gagné et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5267–5269
5269
evidenced by the decreased activity of the corresponding analogs
14–17. Those findings should be taken in consideration before
choosing triazoles as peptide bond surrogates, since their electron-
ics are noticeably different as well as their geometries. The strategy
to introduce triazole dipeptide isosteres in different parts of a pep-
tide sequence could be applied to other endogenous ligands.
Acknowledgments
This work was supported by grant #MOP-102612 from the
Canadian Institute for Health Sciences (CIHR) awarded to B.G.,
Y.L.D. and L.G. A.P.G. and K.R. were the recipients of the 2009 Dom-
inico-Regoli/Institut de Pharmacologie de Sherbrooke fellowship
and the 2010 Centre des Neurosciences/Institut de Pharmacologie
de Sherbrooke fellowship, respectively. A.P.G. and K.R. are, respec-
tively, the recipients of a PhD and a Master studentship from the
Fonds de Recherche Québec—Santé (FRQS). LG is the recipient of
a Junior 2-salary support from the FRQS. B.G., Y.L.D. and L.G. are
members of the FRQS-funded Centre de Recherche Clinique Éti-
enne-Le Bel and of the Institut de Pharmacologie de Sherbrooke.
L.G. is a member of the Centre des Neurosciences de Sherbrooke
as well as of the FRQS-funded Quebec Pain Research Network
(QPRN) and Réseau Québécois de Recherche sur le Médicament
(RQRM).
Supplementary data
Supplementary data (pharmacological characterization, experi-
mental procedures and spectral data) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2013.08.020.
Scheme 4. Solid phase synthesis of the Leu-enkephalin analogs: Reagents and
conditions: (a) Fmoc-Leu or 13, 2,6-diClBzCl, Pyr, DMF; (b) BzCl, Pyr, DMF; (c) Pip/
DMF (1:1); (d) Fmoc-AA (3 eq), HATU (3 eq), NMM (6 eq), DMF; (e) 7 or 11 or 12
(5 eq), DIC (5 eq), HOBt (5 eq), DMF, DCM; (f) TFA/TIPS/H2O (38:1:1).
References and notes
Table 1
Affinities and potencies of 1,4-disubstituted[1,2,3]triazole analogs of Leu-enkephalin
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15
16
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>1500
>1500
>1500
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a
The binding affinity (Ki) of each compound was determined by its ability to
inhibit the binding of [3H]-deltorphin II (competitive binding), a selective DOPr
agonist, to GH3/DOPr cell membrane extracts. Ki values are the means SEM of
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b
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ble 1) and to induce ERK1/2 phosphorylation (Fig. S1). Together,
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holding peptide segments in place but is also involved in biologi-
cally relevant interactions, possibly as a hydrogen bond acceptor.
The ꢀ1.2 Å extension (Fig. 1) induced by the triazole incorporation
might be involved in the significant loss of affinity.
In this study we presented a mixed synthetic approach combin-
ing solid-phase synthesis and classical organic synthesis to obtain
peptidomimetics with medium to high overall yields. Although ac-
tive peptidomimetics were obtained by triazole replacements,4,5
our study shows that this heterocycle is in no ways a viable bioiso-
stere for any of the four amides of Leu-enkephalin, as clearly
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