Preparation and evaluation at the delta opioid receptor of a series of linear Leu-enkephalin analogues obtained by systematic replacement of the amides

Article abstract of DOI:10.1021/cn4000583

Leu-enkephalin analogues, in which the amide bonds were sequentially and systematically replaced either by ester or N-methyl amide bonds, were prepared using classical organic chemistry as well as solid phase peptide synthesis (SPPS). The peptidomimetics were characterized using competition binding, ERK1/2 phosphorylation, receptor internalization, and contractility assays to evaluate their pharmacological profile over the delta opioid receptor (DOPr). The lipophilicity (LogD_7.4) and plasma stability of the active analogues were also measured. Our results revealed that the last amide bond can be successfully replaced by either an ester or an N-methyl amide bond without significantly decreasing the biological activity of the corresponding analogues when compared to Leu-enkephalin. The peptidomimetics with an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogues with enhanced stability. Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds.

Full text of DOI:10.1021/cn4000583

Research Article
pubs.acs.org/chemneuro
Preparation and Evaluation at the Delta Opioid Receptor of a Series
of Linear Leu-Enkephalin Analogues Obtained by Systematic
Replacement of the Amides
Kristina Rochon,†,⊥ Arnaud Proteau-Gagne,‡,⊥ Philippe Bourassa,† Jean-Francois Nadon,‡ Jerome Cote,§
̧
́ ̂ ́
́
Veronique Bournival,† Fernand Gobeil, Jr.,§ Brigitte Guerin,*,∥ Yves L. Dory,*,‡ and Louis Gendron*,†
́
́
†Dep
pharmacologie, and Dep
3001, 12e Avenue Nord, Sherbrooke, Queb
́
artement de Physiologie et Biophysique, ‡Laboratoire de Synthes
̀
e Supramolec
ire et Radiobiologie, Institut de Pharmacologie, Universite
ec J1H 5N4, Canada
́
ulaire, Dep
́
artement de Chimie, §Dep
de Sherbrooke,
́
artement de
∥
́
artement de Med
́
ecine Nuclea
́
́
́
S
* Supporting Information
ABSTRACT: Leu-enkephalin analogues, in which the amide
bonds were sequentially and systematically replaced either by
ester or N-methyl amide bonds, were prepared using classical
organic chemistry as well as solid phase peptide synthesis
(SPPS). The peptidomimetics were characterized using
competition binding, ERK1/2 phosphorylation, receptor
internalization, and contractility assays to evaluate their
pharmacological profile over the delta opioid receptor
(DOPr). The lipophilicity (LogD7.4) and plasma stability of
the active analogues were also measured. Our results revealed
that the last amide bond can be successfully replaced by either
an ester or an N-methyl amide bond without significantly
decreasing the biological activity of the corresponding analogues when compared to Leu-enkephalin. The peptidomimetics with
an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-
enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of
Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide
bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogues with enhanced stability.
Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds.
KEYWORDS: Delta opioid receptor, enkephalin, amide bonds, degradation, lipophilicity, H-bonds
human studies was prevented by the fact that the first
ur knowledge in the field of pain and analgesia has
Osignificantly improved over the past decades. The generation of small molecules selectively activating this receptor
produced nonlethal convulsions in rodents and nonhuman
primates.9,10 Recent advance in the field however revealed that
convulsions are not induced by all DOPr agonists.6,11,12 Indeed,
as of to date, at least three nonpeptide delta compounds have
reached clinical trials for the treatment of pain or depression.
Due to their relative selectivity (1× to 5×) for DOPr over
MOPr, their lack of MOPr-related unwanted effects, the
simplicity of their structure, and their lack of toxicity,
enkephalins (Tyr-Gly-Gly-Phe-Leu/Met) remain very attractive
for the development of drugable compounds. However, because
of their amide bonds, enkephalins are rapidly metabolized by
peptidases (half-life ∼ 2 min)13 and are unable to cross the
blood-brain barrier (BBB) to reach the opioid receptors located
in the central nervous system, that is, the receptors playing a
major role in analgesia.1
neuronal networks involved in pain processing are well-
known and are among the best-characterized pathways. The
investigation of these networks has led to the identification of
new putative targets to alleviate pain. Despite the discovery of
novel analgesic compounds for various targets, opioids are still
among the most commonly used drugs for the treatment of
moderate to severe pain. At least three major subtypes of opioid
peptide receptors are defined, namely, mu (MOPr), delta
(DOPr), and kappa (KOPr).1,2 In the clinic, commonly used
opioids, such as morphine and its analogues, preferentially
target and activate MOPr. The activation of this receptor is,
however, also responsible for most of the side-effects of opioids.
Several studies suggest that targeting DOPr may provide a
strategy for developing new therapies to alleviate chronic pain
without the usual adverse effects of narcotics.3−6 Based on these
studies, the hypothesis that selective DOPr activation would
lead to a better therapeutic profile is generally acknowledged.7,8
A number of useful DOPr agonists have been synthesized.
Unfortunately, the progression of DOPr agonists toward
Received: February 28, 2013
Accepted: May 7, 2013
© XXXX American Chemical Society
A
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In endogenous bioactive peptides, the amide acts principally
as a peptide chain holder. The amide bond is also frequently
involved in important interactions as an H-bond donor or
acceptor (or both). The nature of these interactions can be
intramolecular (to stabilize an active conformation) or
intermolecular (to promote binding with the receptor). In
medicinal chemistry, amide bonds can be replaced with
isosteric functions to (1) protect peptides from proteolysis,
(2) increase peptide rigidity to favor the bioactive conforma-
tion, and (3) increase the lipophilicity of peptides. The
exploitation of isosteric functions therefore represents an
alternative to generating analogues featuring enhanced
pharmacokinetic profiles.14
We previously reported the effects of the systematic
replacement of amide bonds in Leu-enkephalin with E-
alkenes.15 This strategy allowed us to demonstrate that the
sole replacement of the first amide bond by an E-alkene did not
affect the biological activity of Leu-enkephalin.15 Therefore, the
first amide bond of enkephalins does not appear to be involved
in either intramolecular or intermolecular interactions with
DOPr, whereas the three subsequent amide bonds probably
are. We now intend to extend our study to other amide
surrogates, namely, ester and N-methyl amide functions that
can be considered isosteric (i.e., having similar physical and/or
chemical properties) to the amide bond. The existing synthetic
analogues of Leu-enkephalins can be classified according to the
way that they are obtained. These synthetic methods are mostly
limited to the following four classes: (1) modification of the
side chains in Tyr, Gly, Phe, and Leu,16,17 (2) inversion of the α
chiral centers (e.g., DADLE),18 (3) rigidification of the
enkephalin backbone through side chain-to-tail19 or side
chain-to-side chain cyclization (e.g., DPDPE),20 and (4)
introduction of constrained turn mimetics within the main
chain.21 By comparison, few investigations have been devoted
to the four peptide bonds. It is likely that most amides are
involved either in binding to the receptor or in stabilizing the
active conformer of Leu-enkephalin via hydrogen bonding. To
our knowledge, systematic exploration of these important
bonds has been achieved thrice, with thioesters,22 4-
imidazolidinones,23 and alkenes.15 All of the other replacements
of amides have only been partial. Some replacements involve N-
methyl amides,24 triazoles and tetrazoles (via click chemistry),25
sulfonamides,26 phosphonamidates,27 ketomethylenes,28 and
retro-inverso amides.29,30
Indeed, both are devoid of hydrogen bond donor capabilities, a
useful aspect to exploring the hydrogen bonding patterns of
ligands of interest like Leu-enkephalin. It is however worth
noting that additional properties are introduced when a normal
secondary amide is turned into a tertiary amide or an ester.
Indeed, the N-methyl group introduces steric bulk, distorts the
planearity of the amide and restricts the conformational space
around the modified amide.32 Occurrence of the cis isomer for
tertiary amide bonds is also possible.33 The ester linkage
appears to be a better mimic of a peptide bond as it does not
suffer from that many geometric alterations.31,34 The Z
conformer is the most stable as in amides, although the partial
double bond character of the ester C(O)−O bond is not as
pronounced as in the amide C(O)−NH bond.34 Consequently,
esters are structurally more flexible. Despite these structural
differences, we rely on induced fit phenomena or conforma-
tional selection35 to sample out minimally modified ligands
with shapes approaching that of biologically active Leu-
enkephalin. In this study, we sequentially and systematically
replaced all of the amide bonds of Leu-enkephalin with either
an ester or an N-methyl amide. The Leu-enkephalin analogues
were then examined with respect to DOPr affinity, induction of
extracellular signal-regulated kinase 1 and 2 (ERK1/2)
phosphorylation, DOPr-internalization and inhibition of
mouse vas deferens (MVD) contraction. The ability of the
modified bonds to protect the synthesized analogues against
degradation in plasma and to increase their lipophilicity was
also assessed by measuring their half-life in diluted rat plasma
and their LogD7.4 values.
RESULTS AND DISCUSSION
■
Leu-enkephalin is considered an endogenous agonist for DOPr.
However, like most peptides, Leu-enkephalin has a poor
pharmacokinetic profile (i.e., short half-life and high hydro-
philicity), which significantly hampers its usefulness as a
drugable compound. A significant amount of work has been
conducted to better understand the role of each amino acid in
the biological activity of enkephalins. Because of their strong
hydrophilic nature and their sensitivity to protease degradation,
targeting amide bonds represents another strategy to improve
the pharmacokinetic profile of Leu-enkephalin and to better
understand the implications of these bonds in intra- and/or
intermolecular interactions. As we have previously shown, the
systematic and sequential replacement of amide bonds in Leu-
enkephalin with isosteric functions can help in understanding
their contributions to the biological activity of Leu-enkephalin
at DOPr.15 In a continuous effort to assign the biological role of
individual amide bonds in Leu-enkephalin and to increase the
stability and the lipophilicity of this peptide, we have
sequentially replaced all amide bonds first by an ester and
then by an N-methyl amide function. These functions were
selected because they are known to be isosteric to the amide
bond and because they can participate in hydrogen bonds but
only as hydrogen-bond acceptors,14 therefore offering a strategy
to evaluate the role of an amide as a hydrogen-bond donnor.
Chemistry. We have synthesized and thoroughly tested 8
Leu-enkephalin analogues (Figure 2). Four of these analogues
(1−4) contain an ester bond in place of an amide bond. The
abbreviations used to describe these analogues are based on the
names of the corresponding hydroxyl acid replacing the amino
acid: Glc, glycolic acid (glycine); Pla, phenyl lactic acid
(phenylalanine); Hic, hydroxy isocaproic acid (leucine). The
replacement of an amide bond with an ester (depsipeptides)
Because the N-methyl amide and ester functions have the
potential to act as hydrogen-bond acceptors and not as
hydrogen-bond donors (Figure 1),14 they can be introduced in
peptides to mimic some properties of the amide bond and to
study the roles of amide bonds in receptor binding and activity.
Nevertheless, no peptide bond surrogate is absolutely perfect;31
N-methyl amides and esters therefore have their drawbacks.
Figure 1. Illustration of the possible H-bonds. Possible hydrogen
bonds in amide, ester and N-methyl amide functions are illustrated
(red arrows). As opposed to the amide, the ester and the N-methyl
amide are unable to interact as a hydrogen-bond donor, although they
have the potential to accept a hydrogen bond.
B
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Figure 2. Chemical structures of compounds 1−8.
has occasionally led to more stable compounds.36,37 Fur-
thermore, few natural cyclic depsipeptides38,39 have anticancer
(e.g., Rhomidepsin/FK-22840), antibacterial, anti-inflammatory,
or antiviral (e.g., Valinomycin41) properties. Although less
common, linear depsipeptides, such as Dolastatin 15, an
anticancer drug,42 are also biologically active.
MOPr agonist, is by far one of the most widely used
pharmacological tools.
Synthesis. For analogues 1−4, a protected depsipeptide was
first synthesized and subsequently used in solid-phase peptide
synthesis (SPPS). The NMe series of compounds 5−7 were
synthesized from commercial substrates in SPPS. Compound 8
was synthesized in solution.
The other four analogues (5−8) contain an N-methyl amide
function in place of an amide, noted with −NMe− between the
two amino acids or Sar, sarcosine (N-methyl glycine).43 The
substitution of the normal secondary amides of Leu-enkephalin
by N-methyl amide functions may reveal a number of positive
effects. These more substituted amides can adopt cis
conformations,32,44 which are extremely rare in secondary
peptide bonds (Figure 1). For example, all known crystal
structures of Leu-enkephalin have all of their amides in a trans
conformation.45−50 Thus, these modified peptides could
provide a way to explore additional modes of binding to
DOPr. Among other benefits, increased selectivity toward
receptor subtypes,51 enhanced potency,52 and increased
permeability are also observed, the latter being a partial
consequence of NH masking in the amide linkage. Importantly,
N-methylated peptides have a tendency to cross the blood-
brain barrier more easily than their nonmethylated parents.53 In
the opioid field, the replacement of an amide with an N-methyl
amide has already proved to be a valuable strategy to modify
and improve peptide stability.54 The N-methyl amide bond-
containing peptidomimetics DAMME (FK 33-824) and TAPS
both have better resistance to peptidases when compared to
endogenous opioid peptides.55,56 Among the opioid peptides
containing an N-methyl amide, DAMGO, a highly selective
Starting from the amino acid, the corresponding alcool of
phenylalanine and leucine, 9 and 10, were synthesized.57 The
carboxylic acid was then protected in a benzyl ester,58
producing 11 and 12 in overall yields ranging from 39 to
66% (Scheme 1).
Scheme 1. Synthesis of Benzyl Esters 11 and 12
For all four protected depsipeptides, the ester bond was
created between a Boc protected amino acid 13−15 and an
alcohol 11, 12, and 16, in yields ranging from 91% to
quantitative.59 Hydrogenolysis of benzyl esters 17, 19, and 20
produced the related carboxylic acids 21−23 in yields ranging
from 87 to 99% (Scheme 2).59 Without further modifications,
protected depsipeptide 21 was used in the SPPS of analogue 1.
Cleavage of the Boc protecting group of 22 and 23 with TFA
and subsequent introduction of an Fmoc protecting group
C
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Scheme 2. Synthesis of Depsipeptides 18 and 21−23
produced 24 and 25 in yields ranging from 75% to quantitative
were purified on preparative reverse phase-HPLC (RP-HPLC),
and all fractions over 95% in purity were combined.
(Scheme 3).15
To avoid potential formation of diketopiperazine61 during
the Fmoc deprotection of Phe, the analogue 8 was synthesized
in solution. Starting from 13, the protected tripeptide 3015 was
obtained using two Gly-OMe couplings62 and two subsequent
hydrolyses of the resulting methyl esters (28 and 29).
Esterification of commercial amino acid 31 produced 32,
which was coupled to Boc-Phe 15 using HATU to yield 33.
Cleavage of the Boc protection of 33 gave dipeptide 34, which
was then coupled with 30. Hydrolysis of the methyl ester and
consecutive tBu cleavage with TFA produced the enkephalin
analog 8 (Scheme 6). The crude peptide was purified on
Scheme 3. Synthesis of Depsipeptides 24 and 25
The Boc protecting group of compound 18 was cleaved with
TFA, and the amine obtained was coupled to a tyrosine Pfp
ester60 to generate 26. Hydrogenolysis of the benzyl ester
produced 27 in an overall yield of 77% (Scheme 4).
Scheme 6. Synthesis of Analogue 8
Scheme 4. Synthesis of Depsipeptide 27
With all of the protected depsipeptides (21, 24, 25, and 27)
in hand, the SPPS of the desired enkephalin analogues was
conducted. Fmoc-Leu and 25 were coupled to the resin. The
standard Fmoc methodology procedures were used with Fmoc-
Phe, Fmoc-Gly, Fmoc-Tyr(tBu), 21, 24, and 27 to yield Leu-
enkephalin analogues 1−7 (Scheme 5). After SPPS, all peptides
preparative RP-HPLC, and all fractions over 95% in purity were
combined. HPLC purities of depsipeptides 1−8 are similar
under two set of solvent systems, indicating that no
Scheme 5. Solid Phase Synthesis of Enkephalin Analogues 1−7
D
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epimerization occurred during synthesis of the Leu-enkephalin
analogues (see the Supporting Information).
Binding Properties and Activity at DOPr. In order to
evaluate the ability of each compound to bind DOPr, we
performed competitive binding assays using GH3/DOPr cell
membrane preparations. Leu-enkephalin and compounds 1−8
inhibited the binding of 1 nM of [3H]-deltorphin II on DOPr in
a concentration-dependent manner (Supporting Information
Figure S1). Although Leu-enkephalin was found to have a
better affinity than compounds 1, 2, and 5−7 for DOPr,
replacing the fourth amide bond either by an ester bond
(Figure S1A; compound 4) or an N-methyl amide bond
(Figure S1B; compound 8) gave rise to peptidomimetics with
similar affinities to Leu-enkephalin (Table 1). Replacing the
third amide bond of Leu-enkephalin by an ester also retained
most of the affinity for DOPr (Table 1 and Figure S1A;
compound 3).
Table 1. Affinities of Leu-Enkephalin and Compounds 1−8
a
on DOPr
Figure 3. Leu-enkephalin and compounds 1−8 activate ERK1/2.
Following agonist stimulation of DOPr, ERK1/2 proteins are rapidly
and transiently phosphorylated (activated). (A) Western blot of
ERK1/2 phosphorylation (pERK1 and pERK2) after 5 min of
stimulation of DRGF11/DOPr-GFP cells with increasing concen-
trations (10−9−10−5 M) of Leu-enkephalin. (B) Densitometric
analyses of Western blot results (pERK compared to control)
obtained with increasing concentrations of Leu-enkephalin and
compounds 1−8.
and 8 to induce internalization of DOPr-GFP. At 1 μM, Leu-
enkephalin produced a robust internalization of DOPr visible as
soon as 5 min (not shown) following its application. As shown
in Figure 4, the internalization is almost complete after 30 min,
as revealed by the loss of fluorescence labeling at the membrane
and by the accumulation of vesicle-like puncta in the cytoplasm
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 three separate experiments
each performed in triplicate.
The activation of DOPr by an agonist induces rapid and
transient phosphorylation of extracellular signal-regulated
kinases 1 and 2 (ERK1/2). As previously described,
phosphorylation of ERK1/2 can be used as an indicator of
the ability of a compound to behave as an agonist.15 Because
the maximal effect of Leu-enkephalin appeared 5 min after
stimulation (not shown), the effects of Leu-enkephalin (Figure
3A) and of compounds 1−8 were evaluated after 5 min of
stimulation, with concentrations ranging from 10−9 to 10−5 M.
We found that for three compounds (Figure 3B; compounds 1,
4, and 8) the lowest concentration that produced a significant
phosphorylation of ERK1/2 was 10−7 M, which is similar to
Leu-enkephalin. Compounds 3 and 7 required a concentration
of 10−6 M, whereas for compound 2 the phosphorylation of
ERK1/2 was only visible when the compound was used at 10−5
M. Finally, in accordance with observations made by others
who showed that compounds 5 and 6 lack activity in the mouse
vas deferens assay,63 a concentration of 10−5 M is not sufficient
to induce a significant phosphorylation of ERK1/2.
Figure 4. Leu-enkephalin and compounds 4 and 8 induce DOPr
internalization. Stimulation of DOPr with an agonist rapidly induces its
internalization. Micrographs were taken before (control) and 30 min
after treatment of DRGF11/DOPr-GFP cells with 1 μM of Leu-
enkephalin or compound 4 or 8. All compounds induced robust
internalization of DOPr. Indeed, while the membrane labeling is
decreased, fluorescent vesicle-like structures are clearly visible inside
the cells stimulated with the agonists.
Most DOPr agonists induce a rapid internalization of the
receptor. We have recently reported that the internalization of a
fluorescent chimeric DOPr (DOPr-GFP) in transfected
DRGF11 cells can be used to screen for active compounds.15
We therefore decided to measure the ability of compounds 4
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of the cells. At 1 μM, compounds 4 and 8 also induced DOPr
internalization (Figure 4). The level of internalization induced
by these compounds was similar to the level observed with Leu-
enkephalin at the same concentration. As indicated in Table 2,
tPSAs of ∼160 Å2 that produced antinociceptive effects after
systemic administration.68
As previously stated, replacing an amide bond with an ester
or an N-methyl amide function has the potential to decrease
peptide sensitivity toward protease-mediated degradation. We
therefore assessed the stability of Leu-enkephalin and active
compounds 4 and 8 in rat plasma diluted in an equal volume of
saline. We used diluted plasma to decrease the rate of
degradation of Leu-enkephalin. As expected, Leu-enkephalin
was rapidly degraded (Figure 5). The LC-MS analysis revealed
Table 2. Potency of Compounds 4 and 8 in the Mouse Vas
a
Deferens Assay
compd
EC50 (nM)
Leu-enkephalin
74
1
5
4
8
140
35 14
a
Potency (EC50) of each compound was determined by evaluating
their ability to inhibit the electric-field induced contractions of the
mouse vas deferens. EC50 values are the means
separate experiments.
SEM of three
these analogues also potently inhibit electrically induced
contractions of the mouse vas deferens, with compound 8
exhibiting the greatest activity. Furthermore, at the highest
concentration tested (10−5 M), both analogues inhibited nerve-
mediated contractions to a degree comparable to Leu-
enkephalin (10−5 M), suggesting similar efficacy (not shown).
Hydrophobicity and Plasma Stability. Because our
underlying goal is to obtain analogues of Leu-enkephalin that
are able to cross the BBB, we assessed the lipophilicity of the
synthesized analogues by measuring some of their physico-
chemical properties (Table 3). The LogD7.4 values and
Figure 5. Analogue containing N-methyl replacing an amide bond is
more stable than Leu-enkephalin. Leu-enkephalin, compounds 4 and 8
were incubated in diluted rat plasma for 0−60 min. The amount of
intact peptide/analogue was then determined by HPLC and expressed
as the percentage of control. Experiments were performed in triplicate.
Error bars represent the standard error of the mean (SEM).
Table 3. Measured and Calculated Physicochemical
Properties
a
b
c
c
compd
tR (min)
LogD 7.4
cLogP
tPSA (Å2)
Leu-enkephalin
8.44
9.93
9.23
1.22
−0.886 0.030
−0.400 0.019
−0.363 0.004
−0.160 0.005
−0.851
−0.52
−0.230
0.571
199.95
197.15
191.16
52.93
that the major degradation products of Leu-enkephalin, that is,
Gly-Gly-Phe-Leu and Phe-Leu, appeared after 5 and 10 min of
incubation in rat plasma, respectively (Table 4). These
4
8
morphine
a
Retention time: RP-HPLC was performed using an Agilent Eclipse
Table 4. Plasma Stability of Enkephalin and Its Analogues
Plus C-18 column, 50 mm × 3.0 mm, 1.8 μm. Solvent A, 0.1% TFA in
water; solvent B, 0.1% TFA in acetonitrile; 2−98% B in A over 20 min;
flow rate, 0.4 mL/min. Partition coefficient measured using the
shake-flask method with PBS pH = 7.4. Calculated using
ChemBioDraw 12.0; tPSA, topological polar surface area.
half-life
a
degradation products (time for
b
b
compd
(min)
95% CI
first appearance)
c
Leu-enkephalin
4.6
3.3
[4.3−5.1]
Gly-Gly-Phe-Leu (5 min)
Phe-Leu (10 min)
4
[3.0−3.9]
Gly-Gly-Phe-OLeu (5 min)
Phe-OLeu (5 min)
Tyr-Gly-Gly-Phe (5 min)
Gly-Gly-Phe (20 min)
retention times were measured for Leu-enkephalin, compounds
4, 8, and morphine. The cLogP and the topological polar
surface area (tPSA) were also calculated for these compounds.
For morphine and Leu-enkephalin, we obtained LogD7.4 values
of −0.160 and −0.886, respectively. These values are similar to
8
10.7
[8.7−12.7] Gly-Gly-Phe-NMeLeu (5 min)
Phe-NMeLeu (20 min)
a
Half-life: The stability of each compound was determined using an
those previously reported by others for morphine (LogD7.4
=
Agilent 1100 series Symmetry C-18 HPLC column, 150 mm × 4.6
mm, 5 μm, heated at 30 °C, flow, 1.2 mL/min, start with 0.1% TFA in
water then 0 to 75% acetonitrile in 20 min, UV detection at 223 nm.
−0.0764) and Leu-enkephalin (LogD7.07 = −0.84565). The
experimental LogD7.4 and calculated cLogP values show
similarities. Our experimental data show an increased LogD7.4
for each isostere, indicating that our method is a valid way to
obtain analogues with increased hydrophobicity. Of all of the
modifications, the N-methyl amide led to compounds
possessing the highest experimental lipophilicity. The HPLC
retention time of a compound can be used to estimate its
lipophilic nature; however, using our HPLC method, the
retention times were not always in line with the experimental
LogD7.4. Compounds 4 and 8 displayed high tPSA values,
whereas CNS acting drugs usually exhibit smaller values.66,67
Nevertheless, there are some linear dermorphin analogues with
b
Degradation products: Degradation products in diluted rat plasma
were identified by LC-MS analysis.
fragments are also generated in vivo following cleavage of the
first and third amide bonds by aminopeptidase N and
enkephalinase.13 Replacing the fourth amide bond with an N-
methyl amide function (compound 8) significantly increased
the half-life of this peptidomimetic, possibly by increasing the
stability of the third amide bond (Figure 5 and Table 4). By
contrast, introducing an ester in lieu of the fourth amide bond
(compound 4) had no significant effect on the half-life of this
F
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NMR (300 MHz, CDCl3) δ (ppm) 7.36−7.24 (m, 5H), 4.53 (dd, 1H,
J = 4.5 et 7.0 Hz), 3.21 (dd, 1H, J = 4.5 et 14.0 Hz), 3.00 (dd, 1H, J =
7.0 et 14.0 Hz). 13C NMR (75 MHz, CDCl3) δ (ppm) 177.2, 138.5,
129.5, 128.6, 127.2, 70.9, 40.2. IR (NaCl) ν (cm−1) 3450−2395, 3368,
analog in diluted rat plasma when compared to Leu-enkephalin
(Figure 5). In fact, the ester replacement might even increase
the sensitivity to proteolysis, because the Tyr-Gly-Gly-Phe
fragment was also present in the LC-MS analysis soon after
incubation in plasma (Table 4), suggesting that the fourth
amide bond of Leu-enkephalin is less sensitive to degradation
than its ester analog.
+
2927, 1731, 1090. MS (m/e, rel intensity) 184 (MH4 , 23), 149 (9),
108 (26), 91 (100). Exact mass: calculated for C9H14N1O3, 184.0974;
found, 184.0974. [α]D20 −33.8 (c = 0.98, CHCl3):69 [α]D21 −26.9 (c =
1.00, acetone).
L-(−)-2-Hydroxyisocaproic Acid (H-Hica-OH) (10). L-Leucine
(18.1 g, 138 mmol) was dissolved in a sulfuric acid solution (1 N, 324
mL, 324 mmol), and the temperature was lowered to −5 °C. Sodium
nitrite (70.7 g, 1.02 mol) was dissolved in water (300 mL) and was
added dropwise to the mixture. The reaction was stirred at rt for 65 h.
Brine (300 mL) was added, and the crude mixture was extracted with
ethyl acetate (3 × 250 mL). The organic phases were combined and
dried (MgSO4). The title compound was obtained as a white solid
(14.6 g, 80%). 1H NMR (300 MHz, CDCl3) δ (ppm): 7.70−6.10 (br,
2H), 4.28 (t, 1H, J = 6.5 Hz), 2.00−1.70 (m, 1H), 1.62 (t, 2H, J = 7.0
Hz), 0.96 (d, 6H, J = 6.5 Hz). 13C NMR (75 MHz, CDCl3) δ (ppm):
180.4, 68.9, 43.1, 24.4, 23.2, 21.4. IR (NaCl) ν (cm−1): 3421, 2953,
CONCLUSION
■
In the current study, we demonstrated that the fourth amide of
Leu-enkephalin can be replaced by either an ester or an N-
methyl amide bond, two functions with hydrogen-bond
acceptor but no hydrogen-bond donor properties, without
significantly affecting the biological activity of the analogues
when compared to Leu-enkephalin. Taken together with our
previous observations showing that the replacement of the
fourth amide bond by an E-alkene impaired the binding and the
biological activity of the analogue,15 these findings reveal that
the hydrogen-bond acceptor properties of the fourth amide of
Leu-enkephalin are important for its biological activity on
DOPr. As expected, we also observed that the replacement of a
single amide bond by an ester or an N-methyl amide function
improves the lipophilicity of the analogues as compared to Leu-
enkephalin. Finally, compound 8 has an improved stability in
rat plasma. In conclusion, the replacement of the fourth amide
bond of Leu-enkephalin by an N-methyl amide function
appears as a promising strategy to increase the lipophilicity and
the stability of enkephalin analogues. Our results also show that
the systematic replacement of amide bonds by isosteric
functions represents an efficient way to design and synthesize
novel peptide analogues useful in studying the biological roles
of amide bonds, a strategy that could be helpful for the design
and the synthesis of novel Leu-enkephalin analogues.
+
2623, 1711, 1267. MS (m/e, rel intensity): 150 (MNH4 , 100), 99 (6),
85 (10). Exact mass: calculated for C6H16NO3, 150.1130; found,
150.1127. [α]D +6.0 (c = 0.15, CHCl3):70 [α]D +5.5 (c = 1.00,
CHCl3).
20
20
H-Pla-OBn (General Protocol for Benzyl Protection) (11).
Phenyllactic acid (5.90 g, 35.5 mmol), benzyl bromide (4.64 mL, 39.1
mmol), and triethylamine (4.31 g, 42.6 mmol) were dissolved in
acetone (50 mL). The reaction was refluxed for 16 h. The crude
mixture was filtered and concentrated under vacuum. The crude solid
was dissolved in ethyl acetate (100 mL) and washed with water (3 ×
50 mL). The aqueous phases were combined and were extracted with
ethyl acetate (3 × 50 mL). All the organic phases were combined and
dried (MgSO4). The crude mixture was purified by flash
chromatography on silica gel using EtOAc and hexanes (3:17). The
1
title compound was obtained as colorless oil (8.60 g, 95%). H NMR
(300 MHz, CDCl3) δ (ppm) 7.39−7.13 (m, 10H), 5.18 (s, 2H), 4.50
(dd, 1H, J = 4.5 et 6.5 Hz), 3.12 (dd, 1H, J = 4.5 et 14.0 Hz), 2.98 (dd,
1H, J = 6.5 et 14.0 Hz), 2.71 (d, 1H, J = 6.5 Hz). 13C NMR (75 MHz,
CDCl3) δ (ppm) 174.1, 136.2, 135.1, 129.6, 129.5, 128.7, 128.7, 128.4,
126.9, 71.3, 67.4, 40.5. IR (NaCl) ν (cm−1) 3486, 3027, 2950, 1740,
METHODS
■
Chemistry. For SPPS, commercial grade reagents were used
without further purification. When necessary, all solvents were purified
and dried prior to use. Optical rotation measurements were made on a
Perkin-Elmer 241 polarimeter and are quoted in units of 10−1 deg cm2
g−1. Infrared spectra were recorded on a Perkin-Elmer Spectrum 1600
FTIR instrument. 1H and 13C NMR spectra were recorded in
+
1454. MS (m/e, rel intensity) 274 (MH4 , 100), 228 (8), 108 (17).
Exact mass: calculated for C16H20N1O3, 274.1443; found, 274.1447.
20
[α]D −45.0 (c = 3.55, CHCl3).
Boc-Gly-Glc-OBn (General Protocol for Esterification) Glc:
Glycolic Acid (18). Boc-Gly-OH (4.60 g, 26.2 mmol), benzyl
glycolate (3.69 g, 22.2 mmol), and DMAP (271 mg, 2.20 mmol) were
dissolved in DCM (75 mL). DIC (4.12 mL, 26.2 mmol) was added
dropwise at 0 °C. The reaction was allowed to warm up to rt and was
stirred for 16 h. The reaction was filtered on Celite and adsorbed on
silica gel. The mixture was purified by flash chromatography on silica
gel using EtOAc and hexanes (3:7). The title compound was obtained
as a white solid (6.58 g, 91%). 1H NMR (300 MHz, CDCl3) δ (ppm)
7.38−7.34 (m, 5H), 5.19 (s, 2H), 5.01 (br, 1H), 4.72 (s, 2H), 4.04 (d,
2H, J = 5.5 Hz), 1.45 (s, 9H). 13C NMR (75 MHz, CDCl3) δ (ppm)
170.0, 167.3, 155.7, 134.9, 128.6, 128.5, 128.3, 79.9, 67.2, 61.0, 42.1,
28.3. IR (NaCl) ν (cm−1) 3380, 2965, 1701, 1219. MS (m/e, rel
1
deuterated solvents on a Bruker AC 300 NMR instrument. The H
and 13C NMR chemical shifts are reported in ppm (parts per million).
The residual solvent peaks have been used as an internal reference. All
coupling constants (J) are in Hertz. The abbreviations for the peak
multiplicities are as follows: s (singlet), d (doublet), dd (doublet of
doublets), t (triplet), q (quartet), qt (quintet), m (multiplet), and br
(broad). Mass spectra were recorded on a VG Micromass ZAB-2F, on
a MALDI-Tof, or on a ESI-Q-Tof (Maxis) instrument. HPLC
preparative purification was done on a VYDAC 218TP C18 column.
L-(−)-3-Phenyllactic Acid (H-Pla-OH) (9). L-Phenylalanine (15.0
g, 90.9 mmol) was dissolved in sulfuric acid (200 mL, 2 M), and
sodium bromide (22.7 g, 220 mmol) was added. The mixture was
stirred at −10 °C, and sodium nitrite (15.2 g, 220 mmol) was then
added slowly. The reaction was allowed to reach rt and was stirred for
3 h. The crude mixture was extracted with ethyl ether (3 × 75 mL).
The organic phases were pooled, concentrated under vacuum, and
purified by flash chromatography on silica gel using EtOAc and
hexanes (3:7). The bromide (17.0 g, 74.0 mmol) obtained was
immediately dissolved in water (100 mL), and sodium carbonate (8.65
g, 82.0 mmol) was added slowly. The reaction was refluxed for 4 h.
The resulting mixture was washed with ethyl ether. The aqueous phase
was acidified using HCl 1 N until pH = 2 and was extracted with ethyl
ether (3 × 50 mL). The combined organic phases were dried
(MgSO4). The crude product was crystallized using ethyl ether and
hexanes. A white crystalline solid was obtained (6.23 g, 41%). 1H
+
intensity) 341 (MH4 , 22), 285 (100), 223 (72), 91 (15). Exact mass:
calculated for C16H25N2O6, 341.1712; found, 341.1718.
Fmoc-Gly-Pla-OH (General Protocol for Fmoc Protection)
(24). Boc-Gly-Pla-OH (1.52 g, 3.80 mmol) was dissolved in DCM (8
mL) and TFA (2 mL). The solution was stirred for 1 h at rt. The
resulting mixture was concentrated under vacuum and dried under
vacuum using toluene (3 × 50 mL). The crude salt obtained was
dissolved in water (8 mL), and sodium carbonate (1.30 g, 15.2 mmol)
was added. A solution of Fmoc-Cl (1.03 g, 3.99 mmol) in THF (5
mL) was added slowly to the aqueous mixture at 0 °C. The reaction
was allowed to warm at rt and stirred for 16 h. The THF in the
mixture was evaporated under vacuum. The resulting mixture was
diluted in water (150 mL), acidified with 1 N HCl to reach pH 2, and
extracted using ethyl acetate (3 × 60 mL). The organic extract were
G
dx.doi.org/10.1021/cn4000583 | ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
1
combined, dried (MgSO4). The crude compound was purified by flash
chromatography on silica gel eluting with ethyl acetate, hexanes, and
acetic acid (40:59:1) to yield the title compound as a white solid (2.00
g, 100%). 1H NMR (300 MHz, CDCl3) δ (ppm) 7.75 (d, 2H, J = 7.5
Hz), 7.57 (d, 2H, J = 7.5 Hz), 7.39 (d, 2, J = 7.5 Hz), 7.32−7.19 (m,
7H), 5.33 (dd, 1H, J = 4.0 et 8.5 Hz), 5.24 (t, 1H, J = 5.0 Hz), 4.38 (t,
2H, J = 7.0 Hz), 4.20 (t, 1H, J = 7.0 Hz), 4.03 (d, 1H, J = 5.5 Hz), 3.96
(br, 1H), 3.26 (dd, 1H, J = 4.0 et 14.0 Hz), 3.13 (dd, 1H, J = 8.5 et
14.0 Hz). 13C NMR (75 MHz, CDCl3) δ (ppm) 177.7, 173.1, 169.8,
156.8, 143.8, 141.3, 135.6, 129.4, 128.7, 127.8, 127.3, 127.3, 125.2,
120.1, 73.5, 67.5, 47.1, 42.6, 37.1. IR (NaCl) ν (cm−1) 3577−2272,
(1:1) to yield the title compound as a white solid (2.92 g, 100%). H
NMR (300 MHz, CDCl3) δ (ppm) 7.13 (d, 2H, J = 8.5 Hz), 6.95 (d,
2H, J = 8.5 Hz), 5.06 (br, 1H), 4.39 (br, 1H), 4.75 (dd, 1H, J = 5.5,
18.5 Hz), 4.95 (dd, 1H, J = 5.0, 18.5 Hz), 3.76 (s, 3H), 3.15−2.90 (m,
2H), 1.44 (s, 9H), 1.36 (s, 9H). 13C NMR (75 MHz, CDCl3) δ (ppm)
172.7, 171.9, 155.2, 136.2, 129.6, 128.5, 128.4, 126.8, 79.7, 54.5, 52.1,
51.6, 38.9, 37.1, 30.9, 30.3, 28.2, 24.6, 23.76, 21.5. IR (NaCl) ν (cm−1)
3323, 2982, 1759, 1662, 1509, 1169. MS (m/e, rel intensity) 408 (M+,
2), 235 (100), 190 (48). Exact mass: calculated for C21H32N2O6,
408.2260; found, 408.2252. [α]20 +2.83 (c = 2.40, CHCl3).
Boc-Tyr(tBu)-Gly-OH (GenDeral Methyl Ester Hydrolysis
Protocol). Boc-Tyr(tBu)-Gly-OMe (2.32 g, 5.90 mmol) was dissolved
in THF (90 mL). LiOH (990 mg, 23.6 mmol) was dissolved in water
(10 mL) and was added to the reaction which was stirred for 16 h at rt.
The resulting mixture was concentrated under vacuum, diluted in H2O
(100 mL), and washed with ethyl ether (3 × 20 mL). The aqueous
phase was acidified with 1 N HCl to reach pH 4 and extracted with
ethyl acetate (3 × 50 mL). The organic extract were combined, dried
(MgSO4), and concentrated under vacuum to yield the title compound
as a white solid (2.20 g, 98%). 1H NMR (300 MHz, CDCl3) δ (ppm)
7.13 (d, 2H, J = 8.5 Hz), 6.95 (d, 2H, J = 8.5 Hz), 5.34 (br, 1H), 4.59
(br, 1H), 4.18−4.05 (m, 1H), 3.91 (dd, 1H, J = 4.0, 18.5 Hz), 3.02 (br,
2H), 1.40 (s, 9H), 1.35 (s, 9H). 13C NMR (75 MHz, CDCl3) δ (ppm)
172.0, 155.9, 154.0, 131.4, 129.7, 124.4, 80.7, 78.6, 55.5, 41.3, 38.2,
28.8, 28.2. IR (NaCl) ν (cm−1) 3375−2592, 3285, 2928, 2928, 1672,
1642, 1525, 1215, 1159. MS (m/e, rel intensity) 394 (M+, 1), 221
(100), 176 (95). Exact mass: calculated for C20H30N2O6, 394.2112;
+
3404, 3022, 2950, 1727, 1177. MS (m/e, rel intensity) 463 (MH4 , 38),
315 (28), 224 (93), 179 (100). Exact mass: calculated for
20
C26H27N2O6, 463.1869; found, 463.1879. [α]D −6.99 (c = 2.75,
CHCl3).
Boc-Tyr(tBu)-Gly-Glc-OBn (26). Boc-Gly-Glc-OBn (500 mg, 1.32
mmol) was dissolved in DCM (7 mL) and TFA (3 mL). The reaction
was stirred for 1 h at rt. The resulting mixture was concentrated under
vacuum and dried under vacuum using toluene (3 × 20 mL). The
crude product was dissolved in water (5 mL) and THF (10 mL).
Potassium carbonate (365 mg, 2.64 mmol) was added, and the mixture
was stirred at rt for 5 min. Boc-Tyr(tBu)-OPfp (664 mg, 1.32 mmol)
was dissolved in THF (20 mL) and added to the reaction. The mixture
was stirred for 2 h at rt. The crude mixture was diluted with ethyl
acetate (125 mL) and then washed with saturated aq NaHCO3 (75
mL) and water (75 mL). The organic phase was dried (MgSO4). The
crude mixture was purified by flash chromatography on silica gel
eluting with EtOAc and hexanes (from 1:3 to 2:3). The title
found, 394.2104. [α]20 +2.27 (c = 1.26, CHCl3).
D
1
General Method for All Peptide Synthesis on Solid Support
(Fmoc Methodology).15 The resins (Wang resin for the preparation
of peptide 1 and Tenta Gel PHB resin for peptides 2−7) were washed
using DMF (3×), iPrOH (3×), and DCM (3×), unless stated
otherwise. These washing were made after every loading, benzyl
protection, coupling, and Fmoc deprotection. Loading of the resin:71
The resin was placed in a sintered glass peptide synthesis vessel. The 2
equiv of the first amino acid, 2 equiv of 2,6-dichlorobenzoylchloride,
and 4 equiv of pyridine were added to the resin. The suspension was
agitated in a shaker for 16 h. The resin was then washed. All loadings
were quantified by UV quantitation of Fmoc release: An aliquot (10
mg) of resin was dried under vacuum; a piperidine (1 mL) and DMF
(1 mL) solution was added, and the suspension was agitated in a
shaker for 30 min; a portion of the solution (0.5 mL) was diluted in
DCM (4.5 mL) and was read with a UV spectrometer. Loading of the
resin (mmol/g) = (absorbance 301 nm × 103 × 20)/(7800 × weight
of the aliquot). The loadings obtained for each resin are mentioned.
After the initial loading,72 the remaining free sites were protected using
equal amounts of benzoyl chloride and pyridine (0.3 mL/g of resin) in
DMF; the solution was agitated in a shaker for 3 h. For all Fmoc
deprotections, the resin was treated with 50% piperidine in DMF and
the suspension was agitated in a shaker for 20 min. All three couplings
were performed using 3 equiv of protected amino acid and HBTU,
with 6 equiv of NMM in a minimum volume of DMF, and the
suspension was agitated in a shaker. All coupling procedures were
stopped after 16 h or after the Kaiser’s test result was negative. The
first coupling was done with Fmoc-Leu-OH, the second with Fmoc-
Phe-OH, the third and fourth with Fmoc-Gly-OH, and the fifth with
Fmoc-Tyr(tBu)-OH. The amount of modified amino acid (replacing
the previously stated amino acids) used in coupling for each peptide is
mentioned. All the final peptides were cleaved from their resin in a
glass vial, and the suspension was stirred for 1 h 30 min with a
magnetic stirrer. All cleavage solutions were done with 95% TFA, 2.5%
H2O, and 2.5% TIPS. For each gram of resin, 3 mL of cleavage
solution was used. After cleavage, the mixtures were filtered on cotton
and dropped in a large amount of water (20 mL). The remaining
solvents were concentrated under vacuum, and the aqueous solution
was frozen and lyophilized. Purification and purity requirements: All
crude peptides were purified using preparative reverse-phase HPLC,
detecting at 280 nm, with a VYDAC 218TP C18 column and using
ACN gradient in a 0.1% TFA aq solution (from 1:9 to 2:3), with a flow
rate of 5 mL/min over 1 h. The purity of all fractions was analyzed
compound was obtained as a white solid (640 mg, 81%). H NMR
(300 MHz, CDCl3) δ (ppm) 7.38−7.33 (m, 5H), 7.09 (d, 2H, J = 8.5
Hz), 6.91 (d, 2H, J = 8.5 Hz), 6.36 (br, 1H), 5.19 (s, 2H), 4.89 (br,
1H), 4.69 (s, 2H), 4.29 (br, 1H), 3.17 (qd, 2H, J = 19.0 et 5.0 Hz),
3.07−2.98 (m, 2H), 1.40 (s, 9H), 1.32 (s, 9H). 13C NMR (75 MHz,
CDCl3) δ (ppm) 171.8, 169.0, 167.1, 154.2, 134.9, 131.4, 129.7, 128.6,
128.6, 128.4, 124.3, 80.2, 67.2, 55.6, 40.9, 37.7, 28.8, 28.2. IR (NaCl) ν
(cm−1) 3340, 2989, 1752, 1690, 1160. MS (m/e, rel intensity) 543
(MH+, 23), 502 (19), 443 (100), 108 (18). Exact mass: calculated for
20
C29H39N2O8, 543.02706; found, 543.02700. [α]D +2.1 (c = 1.69,
CHCl3).
Boc-Tyr(tBu)-Gly-Glc-OH (General Protocol for Benzyl Ester
Hydrogenolysis) (27). Palladium 10% on activated charcoal (19 mg,
0.0180 mmol) was placed in a round-bottom flask, and ethyl acetate
was added slowly under an argon atmosphere. Hydrogen was bubbled
in the solution for 10 min. Boc-Tyr(tBu)-Gly-Glc-OBn (100 mg, 0.18
mmol) was dissolved in ethyl acetate (5 mL) and then added to the
palladium suspension. The reaction was stirred under a hydrogen
atmosphere for 1.5 h at rt. The reaction was filtered on Celite and
concentrated under vacuum. The title compound was obtained as a
1
white solid (90.5 mg, 95%). H NMR (300 MHz, CDCl3) δ (ppm)
7.08 (d, 2H, J = 8.5 Hz), 6.90 (d, 2H, J = 8.5 Hz), 6.76 (br, 1H), 5.16
(s, 2H), 4.67 (s, 2H), 4.52 (br, 1H), 4.17−4.06 (m, 2H), 3.01−2.87
(m, 2H), 1.36 (s, 9H), 1.32 (s, 9H). 13C NMR (75 MHz, CDCl3) δ
(ppm) 172.1, 169.9, 169.0, 156.0, 154.1, 131.3, 129.7, 124.3, 80.9, 60.9,
40.9, 37.9, 28.8, 28.2. IR (NaCl) δ (cm−1) 3914−2731, 3402, 2977,
1661, 1507. MS (m/e, rel intensity) 453 (MH+, 8), 409 (12), 294 (63),
277 (100). Exact mass: calculated for C22H33N2O8, 453.2237; found,
20
453.2250. [α]D +2.9 (c = 0.80, CHCl3).
Boc-Tyr(tBu)-Gly-OMe (General Protocol for Glycine Cou-
pling) (28). H-Gly-OMe (1.11 g, 8.85 mmol) was dissolved in 1 M
NaHCO3 (50 mL) and extracted with DCM (3 × 50 mL). The
combined organic phases were dried (MgSO4) and concentrated
under vacuum to a volume of 10 mL. Boc-Tyr(tBu)-OH (2.00 g, 5.90
mmol) and HOBt (160 mg, 1.18 mmol) were dissolved DCM (60
mL) and added to the solution. DIC (1.10 mL, 7.08 mmol) was added
dropwise, and the reaction was stirred at rt for 16 h. The crude mixture
was concentrated under vacuum and dissolved in ethyl ether (150
mL). The organic phase was washed with aqueous saturated NaHCO3
(2 × 100 mL), 0.5 N HCl (2 × 100 mL), and water (100 mL) and was
dried (MgSO4). The crude compound was purified by flash
chromatography on silica gel eluting with ethyl acetate and hexanes
H
dx.doi.org/10.1021/cn4000583 | ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
using an Agilent 1100 series analytical HPLC, detecting at 214 nm,
with a Phenomenex 5 μm 4.6.0 × 100 mm C18 column using method
A, ACN gradient in a 0.1% TFA aq solution (from 0:1 to 1:0) over 60
min, with a flow rate of 1 mL/min; method B, ACN gradient in a 20
mM triethylammonium phosphate pH = 2.5 aq solution (from 0:1 to
4:6) over 60 min, with a flow rate of 1 mL/min. The fractions with
purities of 95% or higher were combined, frozen, and lyophilized.
Following these methods, compounds 1−7 were prepared.
136.2, 131.1, 129.2, 128.8, 127.2, 125.1, 115.8, 54.7, 51.8, 51.6, 42.2,
41.8, 39.5, 37.2, 36.5, 35.3, 24.3, 22.2, 20.6. IR (NaCl) ν (cm−1)
3750−2850 (br), 1653, 1210, 1143. MS (m/e, rel intensity) 570 (MH+,
100), 304 (20), 282 (32), 214 (59). Exact mass: calculated for
C29H39N5O7, 570.2928; found, 570.2928. [α]20 +16.4 (c = 0.97,
D
MeOH).
H-Tyr-Gly-Sar-Phe-Leu-OH (6). An amount of 750 mg of Tenta Gel
S PHB resin with a loading of 0.250 mmol/g was used. Sarcosine (233
mg, 0.750 mmol) was used in the third coupling. The title peptide was
obtained as a white solid (55.3 mg, 52%). 1H NMR (300 MHz,
MeOH-d4) δ (ppm) 8.50−8.40 (m, 1H), 8.27−8.17 (m, 1H), 8.00−
7.91 (m, 1H), 7.26−7.17 (m, 5H), 7.09 (d, 2H, J = 8.5 Hz), 6.75 (d,
2H, J = 8.5 Hz), 4.82−4.71 (m, 1H), 4.43−4.40 (m, 1H), 4.23−3.57
(m, 5H), 3.20−3.12 (m, 2H), 2.97−2.74 (m, 2H), 2.86 (s, 3H), 1.69−
1.62 (m, 3H), 0.93 (d, 3H, J = 6.0 Hz), 0.89 (d, 3H, J = 6.0 Hz). 13C
NMR (75 MHz, MeOH-d4) δ (ppm) 174.3, 172.1, 169.5, 168.9, 168.8,
156.9, 137.0, 130.1, 129.0, 128.0, 126.3, 124.6, 115.4, 54.3, 50.7, 40.1,
37.8, 37.5, 36.2, 34.5, 33.9, 24.5, 22.0, 20.4. IR (NaCl) ν (cm−1)
3575−2665 (br), 1660, 1461, 1203, 1140. MS (m/e, rel intensity) 570
(MH+, 100), 304 (43), 282 (45), 214 (83) Exact mass: calculated for
H-Tyr-Gly-Glc-Phe-Leu-OH (1). An amount of 320 mg of resin with
a loading of 0.5 mmol/g was used. Boc-Tyr-Gly-Glc-OH (676 mg,
1.28 mmol) was used in the third coupling. The title peptide was
obtained as a white solid (58.4 mg, 66%). 1H NMR (300 MHz,
CD3OD) δ (ppm) 7.26−7.16 (m, 5H), 7.09 (d, 2H, J = 8.5 Hz), 6.76
(d, 2H, J = 8.5 Hz), 4.72 (dd, 1H, J = 5.0 et 9.5 Hz), 4.63 (d, 1H, J =
15.0 Hz), 4.42 (br, 2H), 3.20−3.11 (m, 2H), 2.98−2.90 (m, 2H),
1.65−1.59 (m, 3H), 0.90 (dd, 6H, J = 6.0 et 14.0 Hz). 13C NMR (75
MHz, CD3OD) δ (ppm) 174.3, 172.1, 169.5, 168.3, 167.8, 156.9,
136.7, 130.2, 129.0, 127.9, 126.4, 124.4, 115.4, 62.0, 54.5, 53.9, 50.8,
40.9, 40.1, 37.5, 36.4, 24.5, 21.9, 20.3. IR (NaCl) ν (cm−1) 3748−
2741, 3423, 2965, 1653, 1457. MALDI-TOF (m/e, rel intensity) 556.7
20
(MH+, 100). [α]D +14.9 (c = 0.94, CH3OH).
C29H39N5O7, 570.2928; found, 570.2929. [α]20 +3.0 (c = 1.82,
D
H-Tyr-Glc-Gly-Phe-Leu-OH (2). An amount of 750 mg of Tenta Gel
S PHB resin with a loading of 0.250 mmol/g was used. Boc-Tyr-Glc-
OH (296 mg, 0.750 mmol) was used in the fourth coupling. The title
MeOH).
H-Tyr-Gly-Gly-(NMe)Phe-Leu-OH (7). An amount of 600 mg of
Tenta Gel S PHB resin with a loading of 0.250 mmol/g was used.
Fmoc-(NMe)Phe-OH (250 mg, 0.600 mmol) was used in the second
coupling. The title peptide was obtained as a white solid (32.6 mg,
1
peptide was obtained as a white solid (46.3 mg, 44%). H NMR (300
MHz, CD3OD) δ (ppm) 8.40 (d, 1H, J = 8.0 Hz), 7.97 (d, 1H, J = 8.0
Hz), 7.24−7.14 (m, 5H), 7.08 (d, 2H, J = 8.5 Hz), 6.76 (d, 2H, J = 8.5
Hz), 4.76−4.64 (m, 2H), 4.41−4.31 (m, 2H), 3.97−3.67 (m, 3H),
3.10−3.02 (m, 2H), 2.90−2.82 (m, 1H), 1.67−1.59 (m, 3H), 0.91 (dd,
6H, J = 6.0 et 12.5 Hz). 13C NMR (75 MHz, CD3OD) δ (ppm) 174.3,
172.1, 169.3, 168.3, 167.8, 157.0, 136.8, 130.2, 128.9, 128.0, 126.3,
115.5, 115.4, 63.1, 61.2, 54.2, 53.9, 50.7, 41.4, 40.1, 37.5, 24.5, 24.5,
21.9, 20.4. IR (NaCl) ν (cm−1) 3600−2550, 3290, 2963, 1645, 1513.
MALDI-TOF (m/e, rel intensity) 556.9 (MH+, 100). [α]D20 −1.88 (c =
0.55, CH3OH).
1
38%). H NMR (300 MHz, D2O) δ (ppm) 7.33−7.21 (m, 5H), 7.12
(d, 2H, J = 8.5 Hz), 6.83 (d, 2H, J = 8.5 Hz), 5.22−5.17 (m, 1H),
4.47−4.28 (m, 1H), 4.19−4.15 (m, 1H), 4.01−3.79 (m, 4H), 3.30−
3.02 (m, 4H), 2.88 (s, 3H), 1.63−1.58 (m, 3H), 0.85 (d, 3H, J = 6.5
Hz) 0.81 (d, 3H, J = 6.5 Hz). 13C NMR (75 MHz, D2O) δ (ppm)
176.2, 172.1, 170.8, 170.5, 155.2, 136.8, 130.8, 129.2, 129.0, 128.7,
126.9, 125.4, 115.8, 59.6, 54.5, 51.4, 42.1, 41.1, 39.0, 35.9, 33.4, 24.4,
22.2, 20.3. IR (NaCl) ν (cm−1) 3610−2530 (br), 1669, 1203, 1139.
MS (m/e, rel intensity) 570 (MH+, 34), 301 (39), 158 (61), 141 (100).
Exact mass: calculated for C29H39N5O7, 570.2928; found, 570.2933.
H-Tyr-Gly-Gly-Pla-Leu-OH (3). An amount of 1.00 g of Tenta Gel S
PHB resin with a loading of 0.250 mmol/g was used. Fmoc-Gly-Pla-
OH (445 mg, 1.00 mmol) was used in the second coupling. A white
[α]20 −2.0 (c = 1.31, MeOH).
D
H-Tyr-Gly-Gly-Phe-(NMe)Leu-OH (8). LiOH (26.0 mg, 0.612
mmol) dissolved in H2O (4 mL) was added to Boc-Tyr(tBu)-Gly-
Gly-Phe-(NMe)Leu-OMe (113 mg, 0.153 mmol) dissolved in THF (4
mL). The mixture was stirred for 4 h at rt. The solvents were
evaporated under reduced pressure. This crude solid was immediately
dissolved in DCM (5 mL), TFA (2 mL), and TIPS (0.5 mL). The
reaction was stirred at rt for 2 h and was concentrated under vacuum.
The crude peptide was purified using preparative RP-HPLC with a
C18 column and using ACN gradient in a 0.1% TFA aq solution (from
1:9 to 2:3). The purity of all fractions was analyzed using an analytical
HPLC instrument, detecting at 214 nm, with a C18 column. All pure
fractions were combined, frozen, and lyophilized. The title compound
1
solid was obtained (60.5 mg, 43%). H NMR (300 MHz, CD3OD) δ
(ppm) 7.26−7.18 (m, 5H), 7.07 (d, 2H, J = 8.5 Hz), 6.74 (d, 2H, J =
8.5 Hz), 5.25 (dd, 1H, J = 4.0 et 8.0 Hz), 4.47−4.38 (m, 1H), 4.02−
3.77 (m, 5H), 3.17−3.07 (m, 3H), 2.94 (dd, 1H, J = 8.0 et 13.0 Hz),
1.61−1.56 (m, 3H), 0.86 (dd, 6H, J = 6.0 et 11.5 Hz). 13C NMR (75
MHz, CD3OD) δ (ppm) 174.2, 170.3, 170.0, 168.9, 168.7, 156.8,
135.9, 130.1, 129.3, 128.0, 126.5, 124.6, 115.4, 74.6, 54.6, 50.3, 41.5,
40.4, 39.9, 37.2, 36.2, 24.3, 22.0, 20.3. IR (NaCl) ν (cm−1) 3695−
2518, 3259, 2954, 1668, 1513. MALDI-TOF (m/e, rel intensity) 556.9
20
(MH+, 100). [α]D +9.7 (c = 0.99, CH3OH).
H-Tyr-Gly-Gly-Phe-Hica−OH (4). An amount of 1.00 g of Tenta Gel
R PHB resin with a loading of 0.100 mmol/g was used. Fmoc-Phe-
Hica-OH (251 mg, 0.500 mmol) was used in the initial coupling. The
1
was obtained as a white solid (18.1 mg, 21%). H NMR (300 MHz,
1
CD3OD) δ (ppm) 7.32−7.17 (m, 5H), 7.13 (d, 2H, J = 8.5 Hz), 6.80
(d, 2H, J = 8.5 Hz), 5.20−5.04 (m, 1H), 4.14−3.73 (m, 6H), 3.50−
3.41 (m, 1H), 3.29−3.11 (m, 2H), 3.04−2.92 (m, 2H), 2.98 (s, 3H),
1.79−1.68 (m, 2H), 1.54−1.40 (m, 1H), 0.90 (dd, 1H, J = 6.5, 11.5
Hz). 13C NMR (75 MHz, CD3OD) δ (ppm) 172.9, 169.9, 169.6,
169.4, 156.9, 136.5, 130.1, 129.0, 128.2, 126.5, 124.5, 115.5, 55.1, 54.8,
51.1, 42.5, 41.6, 37.0, 36.8, 36.3, 30.7, 24.5, 22.2, 20.4. IR (NaCl) ν
(cm−1) 3573−2502 (br), 3286, 3072, 2956, 1671, 1518, 1201, 1134.
MS (m/e, rel intensity) 570 (M+, 100), 552 (14). Exact mass: calculated
title peptide was obtained as a white solid (51.0 mg, 91%). H NMR
(300 MHz, CD3OD) δ (ppm) 7.29−7.16 (m, 5H), 7.07 (d, 2H, J = 8.5
Hz), 6.75 (d, 2H, J = 8.5 Hz), 5.03−4.97 (m, 1H), 4.74−4.69 (m, 1H),
4.02−3.73 (m, 5H), 3.11 (dd, 1H, J = 6.5 et 14.0 Hz), 3.00−2.88 (m,
2H), 1.82−1.63 (m, 3H), 0.92 (dd, 6H, J = 6.0 et 9.0 Hz). 13C NMR
(75 MHz, CD3OD) δ (ppm) 171.2, 169.9, 169.1, 156.8, 140.0, 136.9,
130.1, 128.8, 128.1, 126.4, 124.6, 115.4, 71.8, 54.7, 53.6, 41.9, 41.5,
39.5, 36.6, 36.3, 24.4, 22.0, 20.4. IR (NaCl) ν (cm−1) 3713−2418,
3281, 2954, 1663, 1513. MALDI-TOF (m/e, rel intensity) 557.3 (MH+,
20
20
for C29H39N5O7, 570.2928; found, 570.2934. [α]D −5.92 (c = 0.49,
100). [α]D +12.6 (c = 1.03, CH3OH).
CH3OH).
H-Tyr-Sar-Gly-Phe-Leu-OH (5). An amount of 750 mg of Tenta Gel
S PHB resin with a loading of 0.250 mmol/g was used. Sarcosine (233
mg, 0.750 mmol) was used in the fourth coupling. The title peptide
Cell Culture. GH3 cells (a somatomammotroph tumor cell line)
stably expressing the mouse DOPr (GH3/DOPr) and DRGF11 cells
(a fusion product of cells of mouse neuroblastoma cell line N18TG-2
with embryonic rat dorsal-root ganglion neurons) stably expressing
green fluorescent protein (GFP)-tagged DOPr (DRGF11/DOPr-
GFP) were grown at 37 °C in DMEM supplemented with 10% fetal
bovine serum and 50 mg/L gentamicin in a humidified atmosphere of
95% air and 5% CO2.15
1
was obtained as a white solid (47.8 mg, 45%). H NMR (300 MHz,
D2O) δ (ppm) 7.31−7.20 (m, 5H), 7.13 (d, 2H, J = 8.5 Hz), 6.84 (d,
2H, J = 8.5 Hz), 4.71−4.62 (m, 1H), 4.61−4.51 (m, 1H), 4.31−4.21
(m, 1H), 3.91−3.82 (m, 4H), 3.09−3.07 (m, 4H), 2.83 (s, 3H), 1.56−
1.54 (m, 3H), 0.84 (d, 3H, J = 6.0 Hz), 0.79 (d, 3H, J = 6.0 Hz). 13C
NMR (75 MHz, D2O) δ (ppm) 176.3, 172.8, 170.7, 170.5, 155.3,
I
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ACS Chemical Neuroscience
Research Article
DOPr-Induced ERK1/2 Activation. The DRGF11/DOPr-GFP
cells were stimulated for 5 min with increasing concentrations (10−9−
10−5 M) of the different compounds. The stimulation was terminated
by aspiration of the culture media and by the addition of ice-cold
Hank’s buffered saline (HBS) (130 mM NaCl, 3.5 mM KCl, 1.8 mM
CaCl2, 0.5 mM MgCl2, 2.5 mM NaHCO3, 5 mM HEPES, and 0.5 mM
EGTA). The cells were then stabilized with ice-cold HBS containing 2
mM Na3VO4, 0.1 μM staurosporine, and complete protease inhibitors
(Roche Diagnostics, Indianapolis, IN) for 10 min. Cell lysis was
performed using a 50 mM HEPES pH 7.8 solution containing 1%
Triton X-100, 2 mM Na3VO4, 0.1 μM staurosporine, and complete
protease inhibitors (Roche Diagnostics). Cell lysates were centrifuged
for 10 min at 13 500g and 4 °C. The supernatants were saved and
stored at −20 °C until use. Protein extracts (15 μg per sample) were
resolved with a 10% SDS-polyacrylamide gel and transferred on
polyvinylidene fluoride membranes. The membranes were then
blocked for 1 h using a Tris-buffered saline with 0.05% Tween 20
(TBS-T) containing 1% gelatin. After blocking, the membranes were
incubated for 2 h at room temperature with anti-phosphoERK1/2 or
anti-ERK1/2 rabbit antibodies (both 1:1000; Cell Signaling Technol-
ogy, Danvers, MA). The membranes were washed three times with
TBS-T and incubated for 1 h at room temperature with horseradish
peroxidase conjugated anti-rabbit antibody (1:2000; GE Healthcare
Life Sciences, Piscataway, NJ). Protein detection was performed using
an enhanced chemiluminescence detection kit (Amersham ECL
Western Blotting Detection Reagents from GE Healthcare Life
Sciences). Densitometric analyses of pERK1/2 were performed
using ImageJ, and the results were compared to the control.
Competitive Binding Assays. We evaluated the affinity of the
different compounds for DOPr with GH3/DOPr membrane extracts.
First harvested by a phosphate-buffered saline, cells were obtained by
centrifugation. The resulting pellets were resuspended in a 10 mM
potassium phosphate buffer pH 7.2 (buffer A) and centrifuged for 10
min at 40 000g. Cell pellets were resuspended in buffer A and left on
ice for 20 min. Then, cells were centrifuged three times at 800g for 5
min, the supernatants were saved, and the pellets were resuspended in
buffer A. Supernatants were pooled together and centrifuged for 10
min at 40 000g. The resulting pellet was finally resuspended in buffer A
supplemented with 0.32 M sucrose and 5 mM EDTA and stored at
−80 °C until use. Protein concentration was determined with Bio-Rad
DC Protein Assay reagents (Bio-Rad Laboratories, Mississauga, ON,
Canada).
scientific, Rockland, MA). Before cell stimulation with 1 μM of each
compound, 3 min of baseline recording were performed. The images
were obtained using a 60× objective and a QuantEM:512SC camera
(Photometrics, Tucson, AZ) at room temperature at intervals of 25 s
for 30 min.
Inhibition of the Mouse Vas Deferens Contraction. Mice were
anesthetized with isoflurane and sacrificed by a cervical dislocation.
Their vas deferens were dissected out, the semen was ejected, and the
tissues were bathed in modified Krebs solution (mM): NaCl 119.3,
KCl 4.7, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25, and D-glucose 11.1,
gassed with 95% O2 and 5% CO2 and kept at 34 °C. Longitudinal
contractions were recorded isometrically at a constant reting tension of
0.5 gwt by a force displacement transducer FT03, amplified by a
compact transducer amplifier P11T and displayed on a data acquisition
system dash 4u (Grass Technologies, Astro-Med, Inc., Brossard, QC,
Canada). Electric stimulation trains were generated by a Grass S88X
dual output square pulse stimulator (Astro-Med, Inc.) coupled to
universal coil platinum electrode (Radnoti, LLC, Monrovia, CA,) and
repeated every 20 s. They consisted of six impulsions of 20 V during 1
ms with 9 ms intervals. A volume of 100 μL of the tested ligand was
added in the 10 mL bath to obtain final concentrations ranging from
10−11 to 10−5 M. After 5 min of recording, the preparation was washed
twice with 10 mL of fresh medium. As a control, Leu-enkephalin was
added at a final concentration of 10−5 M. The data were analyzed with
nonlinear fitting analysis, and the concentration necessary to produce a
50% inhibition (EC50) of the contraction was determined.
LogD7.4 Determination. The determination of the distribution
coefficient (LogD) was performed using a modified version of the
shake-flask method. Before the experiment, octanol and phosphate
buffer (PBS pH 7.4) were mixed together for 24 h to allow saturation
of each solution. The mixture was allowed to rest, and the phases were
separated and used as solvents in the coefficient measurement. The
experiment was performed at room temperature using triplicates for
each measurement. Each peptide (0.1 mg) was placed in a vial to
which saturated PBS (1 mL) and octanol (0.5 mL) were added. The
vial was then shaken mechanically for 10 min. The mixture was
allowed to rest for 30 min or until phase separation was completed.
Aliquots of both phases were taken and injected in an HPLC
instrument (10 μL of each aliquot was injected in an Agilent 1100
series HPLC, column: Agilent Eclipse Plus C-18 column, 50 mm × 3.0
mm, 1.8 μm; solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in
acetonitrile; 2−98% B in A over 20 min; flow rate, 0.4 mL/min; UV
detection at 214 nm). The retention time of each peptide was already
known from the HPLC purity analysis of each peptide. The octanol
peak did not interfere with the experiment. The area under the curve
(AUC) of the corresponding peak was integrated for each phase
injected. The LogD for each peptide was calculated as follows: LogD7.4
= log10 (AUC octanol phase/AUC PBS phase).
[3H]-Deltorphin II (specific activity: 30−60 Ci/mmol; PerkinElm-
er, Waltham, MA) was used during competitive binding assays to
selectively target DOPr highly expressed in membrane extracts.
Experiments were performed using a membrane concentration of 100
μg of proteins/mL and a radiolabeled ligand concentration near the KD
value previously obtained in saturation binding assays (∼1 nM).
Nonspecific binding was determined using 10 μM Deltorphin II.
Experiments were conducted with Tris buffer solution pH 7.4 in 5 mL
polypropylene tubes for a final volume of 0.5 mL. Incubations were
performed during 60 min at 37 °C, and the reaction was stopped by
filtration using ice-cold assay buffer on a Whatman GF/C filter (GE
Healthcare Life Sciences). Filters were placed in vials containing 8 mL
of Ready Gel scintillation cocktail (Beckman Coulter Canada, Inc.,
Mississauga, ON, Canada), and the radioactivity was determined using
a Beckman Coulter LS-6500 scintillation counter (Beckman Coulter
Canada, Inc.). Data were analyzed using a nonlinear fitting analysis,
and Ki values were calculated from IC50 determination using the
Cheng-Prusoff equation.73
DOPr Internalization Assay. Using 35 mm glass bottom dishes
(MatTek Corporation, Ashland, MA), DRGF11/DOPr-GFP cells
were grown for 2−3 days in DMEM supplemented with 10% fetal
bovine serum. Prior to the assay, the culture media was replaced by
Earle’s buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.9 mM
MgCl2, 25 mM HEPES, 0.2% BSA, and 0.09% glucose). For each dish,
three different cells were chosen and observed with an IX81 Olympus
microscope (Olympus America, Center Valley, PA) equipped with a
CSU-X1 confocal scanner unit (Yokogawa Electric Corporation,
Newnan, GA) and a ProScan II motorized stage system (Prior
Plasma Stability. Plasma was prepared from two male Sprague−
Dawley rats (300−350 g; Charles River Laboratories, St-Constant,
Quebec, Canada). All animal procedures were approved by the Ethical
́
Committee for Animal Care and Experimentation of the Universite de
Sherbrooke (protocol #234-10) and were performed according to the
regulations of the Canadian Council on Animal Care (CCAC). Briefly,
rats were anesthetized with isoflurane (3% isoflurane with 97% medical
air) and exsanguinated using an 18G-11/2 needle connected to a 10
mL syringe. The blood was rapidly transferred to an EDTA-containing
tube and centrifuged at 1600g for 15 min at 4 °C. The plasma was then
stored at −80 °C in 750 μL aliquots until use. The stability of Leu-
enkephalin and its analogues was determined using a modified
procedure of a previously published protocol.74 In order to decrease
the peptide degradation and to allow comparisons between Leu-
enkephalin and its analogues, the experiments were performed in
plasma diluted to 50% with saline. Nondiluted plasma (25 μL) was
incubated at 37 °C for 15 min before the addition of the peptide
solution (25 μL). All peptide solutions consisted of a 100 μM dilution
of peptide in isotonic sodium chloride (0.9% w/v). After addition of
the peptide, the solution was vortexed. The eight aliquots were
incubated at 37 °C, and degradation was stopped at 0, 5, 10, 15, 20, 30,
40, and 60 min by the addition of methanol (100 μL). The resulting
J
dx.doi.org/10.1021/cn4000583 | ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
solutions were centrifuged at 13 000 rpm for 15 min at 4 °C. To 155
μL of the supernatant was added 5 μL of internal standard. The
internal standard solution consisted of 500 μM Fmoc-leucine in
methanol. Degradations were performed in triplicate, and the resulting
solutions were analyzed by HPLC (40 μL of solution was injected in
an Agilent 1100 series HPLC, column: Symmetry C18 5 μm 4.6 × 150
mm, heated at 30 °C, flow 1.2 mL/min, start with 0.1% TFA in water
then 0 to 75% acetonitrile in 20 min, UV detection at 223 nm). A
blank sample at the same dilution but containing no peptide was
injected to identify background peaks due to plasma. A standard
solution at the same dilution but containing no plasma was injected to
ensure that no peptides were lost in the plasma precipitate. The
percentage of nondegraded peptide was calculated by determining the
ratio between the area under the curve (AUC) of Fmoc-leucine and
the AUC of the tested peptide. For all points, the means of the ratios
for the triplicates were calculated, and the mean of the 0 min triplicate
was fixed at 100% peptide remaining. The AUC of the 0 min triplicate
and the standard solution (no plasma) in all cases were not
significantly different. For analysis of the appearance of peptide
fragments, the same solutions were injected in an LC-MS instrument
with the following method: 20 μL of solution was injected in an
Waters Alliance/Waters micromass ZQ LC-MS; column, X terra MS
C18 3.5 μM 2.1 × 50 mm, flow 1 mL/min, 0 to 50% acetonitrile in 8
min, mass detection between 150 and 700.
ACKNOWLEDGMENTS
■
The authors are grateful to Robert Dumaine and Philippe
Sarret for providing access to the Canadian Foundation for
Innovation (CFI)-funded confocal microscope.
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ASSOCIATED CONTENT
■
S
* Supporting Information
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1
HPLC analysis, and H NMR spectrum for the precursors and
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: Brigitte.Guerin2@USherbrooke.ca (B.G.); Yves.
Dory@USherbrooke.ca (Y.L.D.); Louis.Gendron@
USherbrooke.ca (L.G.).
Author Contributions
⊥K.R. and A. P.-G. have contributed equally to this work. F.B.,
B.G., Y.L.D., and L.G. designed the study and wrote the
manuscript. K.R., A.P.-G., P.B., J.-F.N., J.C., and V.B. performed
the experiments and wrote the manuscript.
Funding
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 Dominico-Regoli/Institut de Pharmacologie de
Sherbrooke fellowship and the 2010 Centre des Neuro-
sciences/Institut de Pharmacologie de Sherbrooke fellowship,
respectively. A.P.-G. and K.R. are, respectively, the recipients of
a Ph.D. and a Master studentship from the Fonds de Recherche
Quebec − Sante (FRQS). L.G. is the recipient of a Junior 2-
́ ́
salary support from the FRQS. F.G., B.G., Y.L.D., and L.G. are
members of the FRQS-funded Centre de Recherche Clinique
Etienne 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 Res
recherche sur le Medicament (RQRM).
Notes
The authors declare no competing financial interest.
́ ́ ́
eau quebecois de
́
K
dx.doi.org/10.1021/cn4000583 | ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
(15) Proteau-Gagne, A., Bournival, V., Rochon, K., Dory, Y., and
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