Synthesis of Gly-ψ[(Z)CFCH]-Phe, a Fluoroalkene Dipeptide Isostere, and Its Incorporation into a Leu-enkephalin Peptidomimetic

Article abstract of DOI:10.1021/acschemneuro.6b00163

A new Leu-enkephalin peptidomimetic designed to explore the hydrogen bond acceptor ability of the third peptide bond has been prepared and studied. This new analog is produced by replacing the third amide of Leu-enkephalin with a fluoroalkene. An efficient and innovative synthesis of the corresponding dipeptide surrogate Fmoc-Gly-ψ[(Z)CFCH]-Phe-OH is described. The key step involves the alkylation of a tin dienolate from the less hindered face of its chiral sulfonamide auxiliary derived from camphor. Once its synthesis was complete, its incorporation into the peptidomimetic sequence was achieved on a solid support with chlorotrityl resin following the Fmoc strategy. The peptidomimetic was characterized using competition binding with [¹²⁵I]-deltorphin I on membrane extracts of HEK293 cells expressing the mouse delta opioid receptor (DOPr) and based on its abilities to inhibit the electrically induced contractions of the mouse vas deferens and to activate the ERK1/2 signaling pathway in DRGF11/DOPr-GFP cells. Together with our previous observations, our findings strongly suggest that the third amide bond of Leu-enkephalin primarily acts as a hydrogen bond acceptor in DOPr. Consequently, this amide bond can be successfully replaced by an ester, a thioamide, or a fluoroalkene without greatly impacting the binding or biological activity of the corresponding analogs. The lipophilicity (LogD_7.4) of the active analog was also measured. It appears that fluoroalkenes are almost as efficient at increasing the lipophilicity as normal alkenes.

Full text of DOI:10.1021/acschemneuro.6b00163

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Article
Synthesis of Gly-#[(Z)CF=CH]-Phe, a fluoroalkene dipeptide
isostere and its incorporation into a Leu-enkephalin peptidomimetic
Jean-Francois Nadon, Kristina Rochon, Sébastien Grastilleur, Guillaume Langlois, Thi
Thanh Hà Dao, Véronique Blais, Brigitte Guérin, Louis Gendron, and Yves L. Dory
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00163 • Publication Date (Web): 20 Oct 2016
Downloaded from http://pubs.acs.org on October 22, 2016
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Synthesis of Glyꢀψ[(Z)CF=CH]ꢀPhe, a fluoroalkene
dipeptide isostere, and its incorporation into a Leuꢀ
enkephalin peptidomimetic
Jean-François Nadona, Kristina Rochonb, Sébastien Grastilleurb, Guillaume Langloisa, Thi Thanh Hà
Daoa, Véronique Blaisb, Brigitte Guérind,e*, Louis Gendronb,c,e,f*, and Yves L. Dorya*
a Laboratoire de synthèse supramoléculaire, Département de chimie, Faculté des sciences
b Département de pharmacologieꢀphysiologie,
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c Département d’anesthésiologie et
d Département de médecine nucléaire et radiobiologie, Faculté de médecine et des sciences de la santé
e Centre de recherche du CHUS
f Quebec Pain Research Network
Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke,
3001, 12e avenue Nord, Sherbrooke, Québec, J1H 5N4, Canada
* Correspondence should be addressed to: Brigitte.Guerin2@USherbrooke.ca;
Louis.Gendron@USherbrooke.ca;
Yves.Dory@USherbrooke.ca
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
TITLE RUNNING HEAD: Replacement of the third peptide bond of Leuꢀenkephalin by a fluoroalkene.
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ABSTRACT
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A new Leuꢀenkephalin peptidomimetic designed to explore the hydrogen bond acceptor ability of the
third peptide bond has been prepared and studied. This new analog is produced by replacing the third
amide of Leuꢀenkephalin with a fluoroalkene. An efficient and innovative synthesis of the
corresponding dipeptide surrogate FmocꢀGlyꢀψ[(Z)CF=CH]ꢀPheꢀOH is described. The key step
involves the alkylation of a tin dienolate from the less hindered face of its chiral sulfonamide auxiliary
derived from camphor. Once its synthesis was complete, its incorporation into the peptidomimetic
sequence was achieved on a solid support with chlorotrityl resin and following the Fmoc strategy. The
peptidomimetic was characterized using competition binding with [125I]ꢀdeltorphin I on membrane
extracts of HEK293 cells expressing the mouse delta opioid receptor (DOPr) and based on its abilities to
inhibit the electrically induced contractions of the mouse vas deferens and to activate the ERK1/2
signaling pathway in DRGF11/DOPrꢀGFP cells. Together with our previous observations, our findings
strongly suggest that the third amide bond of Leuꢀenkephalin primarily acts as a hydrogen bond
acceptor in DOPr. Consequently, this amide bond can be successfully replaced by an ester, a thioamide,
or a fluoroalkene without greatly impacting the binding or biological activity of the corresponding
analogs. The lipophilicity (LogD7.4) of the active analog was also measured. It appears that
fluoroalkenes are almost as efficient at increasing the lipophilicity as normal alkenes.
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Keywords: Delta opioid receptor, enkephalin, amide bonds, fluoroalkene, lipophilicity, Hꢀbonds
INTRODUCTION
Opioids, which are widely used for treating moderate to severe pain, are currently the most powerful
class of analgesics and, unlike nonsteroidal antiꢀinflammatory drugs (NSAIDs), are effective against
neuropathic pain.1,2 The major drawback of this class of compounds is undoubtedly the large array of
unwanted effects accompanying acute and chronic treatments (e.g., sedation, constipation, nausea,
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tolerance, dependence, addiction and respiratory depression). These effects are commonly associated
with the activation of the mu opioid receptor (MOPr), a G proteinꢀcoupled receptor (GPCR) that acts as
the biological target of most clinically used opioids, including morphine.3,4 In animal studies, it has been
observed that the activation of the delta opioid receptor (DOPr), another opioid receptor subtype (the
third one being the kappa opioid receptor, KOPr) produces analgesia in preclinical chronic pain
models.5 Furthermore, the administration of DOPr agonists leads to less addiction and a smaller loss of
gastrointestinal motility than the administration of MOPr agonists, and it has been postulated that the
selective activation of DOPr could produce analgesia devoid of the undesirable effects commonly
associated with opioids.6 Indeed, SNCꢀ80, a nonꢀpeptide selective DOPr agonist, produces analgesia and
antidepressant effects while causing limited constipation, addiction or respiratory depression.5 However,
its therapeutic use is prevented by the receptorꢀmediated convulsions that are often observed with a
variety of nonꢀpeptide DOPr agonists.7
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Discovered in 1975 by Hughes and Kosterlitz,8,9 Leuꢀenkephalin is a pentapeptide (TyrꢀGlyꢀGlyꢀPheꢀ
Leu) known to activate DOPr.10 Despite presenting a good pharmacodynamic profile, this peptide does
not produce analgesia when injected intravenously due to its poor pharmacokinetic properties. Indeed,
because of their low lipophilicity, enkephalins cannot readily cross the bloodꢀbrain barrier to reach the
opioid receptors in the central nervous system.11 When injected intraꢀcerebroventricularly, however,
Leuꢀenkephalin produces analgesic effects. Unfortunately, the analgesic effect of Leuꢀenkephalin has a
very short duration (t1/2 ≈ 2 min) because it is rapidly degraded by various peptidases (EC 3.4.24.11
enkephalinase and EC 3.4.11.2 alanyl aminopeptidase, among others).12,13 These weaknesses are largely
due to the peptidic nature of the enkephalins. Indeed, the peptide bonds (amide) that link the amino
acids constituting these peptides are considered highly hydrophilic, making peptides generally unable to
cross the bloodꢀbrain barrier and thus the target of peptidase enzymes. These two characteristics make
these peptide bonds a strategic target for improving the pharmacokinetic profile of Leuꢀenkephalin. In
the past, bioisosteres have been used extensively to obtain peptide analogs, called peptidomimetics,14,15
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in which the amide bonds have been replaced by other functional groups, imparting the molecule with
an improved pharmacokinetic profile while retaining its biological activity.16
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By systematically replacing the amide bonds in Leuꢀenkephalin with various dipeptide isosteres, we and
others have previously found that the first amide bond can be substituted by a transꢀalkene dipeptide
isostere,11 that the second can be substituted by a thioamide,17 and that the fourth can be substituted
either by a thioamide,17 an ester13 or an Nꢀmethyl amide dipeptide isostere13 without a major loss of
affinity or activity toward DOPr. These observations suggest that the first amide bond acts as neither a
hydrogen bond donor nor an acceptor, that the second plays a donor role, and that the fourth is an
acceptor. Regarding the third amide bond, we observed that its substitution by an ester leads to some
losses in affinity (~ 5ꢀfold) and activity.13 However, the fact that it appears to be somewhat tolerated
(and that substitution by a thioamide is less tolerated)17 could indicate that a hydrogen bond acceptor is
important at this position. Substitution by an Nꢀmethylamide at the same position led to a considerable
loss of activity, which may be due to the steric hindrance caused by the addition of a methyl group.13 To
further ascertain the acceptor role of the third amide, it may prove interesting to substitute it by another
mimetic acting as a hydrogen bond acceptor that is small enough to avoid a loss of activity.
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Geometrically, fluoroalkenes are isosteres for amide bonds. In an amide bond, the lone pair of electrons
on the nitrogen is delocalized in the π system of the carbonyl, which provides the CꢀN bond with a
strong doubleꢀbond character. The bond length, angles, and rigidity are very similar parameters in
fluoroalkenes and in amide bonds.18 Furthermore, the fluorine atom is able to mimic, to a certain extent,
the oxygen atom of the carbonyl because they have similar van der Waals radii (1.47 Å for fluorine and
1.52 Å for oxygen). Thus, they are, to some degree, able to participate in hydrogen bonds as acceptors,
although the interaction can be much weaker than with amides. Fluoroalkenes cannot act as hydrogen
bond donors. Like many amide bioisosteres, they disturb αꢀhelix and βꢀturn structures.15,18,19
Fluoroalkenes acting as amide bioisosteres have been used in the synthesis of substance P analogs that
present activity similar to that of the parent peptide and in the synthesis of dipeptidase inhibitors.20,21 In
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addition, fluoroalkenes are considered highly lipophilic, which makes them very interesting isosteres for
enhancing membrane permeability.
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Although many methods have been reported for the synthesis of fluoroalkene dipeptide isosteres
containing a glycine,22ꢀ24 the obtained compounds were either Glyꢀψ[(Z)CF=CH]ꢀGly or, in one case,
racemic Pheꢀψ[(Z)CF=CH]ꢀGly. Thus far, no method has been reported for the synthesis of
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enantiomerically pure Glyꢀψ[(Z)CF=CH]ꢀPhe, the dipeptide isostere required for synthesizing a Leuꢀ
enkephalin peptidomimetic in which the third amide bond has been substituted with a fluoroalkene. A
widely used and versatile method for the synthesis of fluoroalkene dipeptide isosteres (FADIs) involves,
as a key step, the copperꢀmediated reduction of a conjugated alkene followed by a oneꢀpot alkylation.
The addition of a chiral auxiliary (such as one derived from camphor) allows the synthesis of L and D
amino acid dipeptide isosteres.25 This method has led to the synthesis of various fluoroalkene dipeptide
isosteres, namely, ValꢀGly, ValꢀAla, ValꢀDꢀAla, ValꢀPhe, ValꢀDꢀPhe, ValꢀAsp, ValꢀDꢀAsp and ValꢀDꢀ
Tyr.25
In this study, we describe the use of copperꢀmediated reduction followed by a oneꢀpot alkylation of an
alkene to synthesize a suitably Nꢀprotected GlyꢀPhe fluoroalkene 2 (Figure 1).
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Figure 1. Relationship between Leuꢀenkephalin and its fluoroalkene peptidomimetic 1 that have a
similar Hꢀbond acceptor ability at the third amide position. The structure of the required Fmocꢀprotected
FADI 2 is also shown.
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In addition to applying the method for the synthesis of an FADI that has not been previously
synthesized, we replaced hexamethylphosphoramide (HMPA), which has been proven to be
carcinogenic, with 1,3ꢀdimethylꢀ3,4,5,6ꢀtetrahydroꢀ2(1H)ꢀpyrimidinone (DMPU), a solvent with similar
characteristics.26 Furthermore, we introduced Glyꢀψ[(Z)CF=CH]ꢀPhe into a Leuꢀenkephalin
peptidomimetic 1. The lipophilicity (LogD7.4) of this analog was measured, and its binding affinity and
its ability to activate DOPr were also investigated.
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RESULTS AND DISCUSSION
Chemistry
The Fmocꢀprotected fluoroalkene dipeptide isostere of GlyꢀPhe, FmocꢀGlyꢀψ[(Z)CF=CH]ꢀPhe 2, was
synthesized using liquidꢀphase chemistry before being incorporated into the Leuꢀenkephalin
peptidomimetic 1 using solidꢀphase peptide synthesis. The condensation of benzotriazole,
paraformaldehyde and benzylamine using a DeanꢀStark apparatus led to the formation of the two
isomers of 1,2,3ꢀbenzotriazolylꢀN,Nꢀdibenzylmethanamine as an inseparable mixture 3, which was
contaminated with benzotriazole (Scheme 1).27 A Reformatskyꢀtype reaction between ethyl
bromodifluoroacetate and mixture 3 afforded compound 4.28
Scheme 1. Synthesis of ethyl 3ꢀ(tertꢀbutoxycarbonylamino)ꢀ2,2ꢀdifluoropropanoate 5.
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Mixture 3 was not purified before this last step because the benzotriazole moiety acts as a leaving group
in the Reformatskyꢀtype reaction, and both isomers are able to play this role and lead to the same final
product. Furthermore, because the reaction is irreversible, the presence of benzotriazole, a reaction
product, in the starting mixture is not problematic. Compound 3 was therefore used directly after several
washes.
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The dibenzylated amine 4 was then hydrogenated using a palladium/carbon catalyst with tertꢀ
butoxycarbonyl anhydride to yield the Bocꢀprotected amine 5.29 Because the use of methanol (as
described in the literature)29 as a solvent led to a mixture of methyl and ethyl esters of 5 (detected by 1H
NMR and LCꢀMS), ethanol was used in place of methanol, thereby avoiding transesterification.
To allow the stereoselective alkylation necessary for the synthesis of Glyꢀψ[(Z)CF=CH]ꢀPhe, the
camphorꢀbased chiral auxiliary 8 was synthesized in two steps (Scheme 2), with a global yield of 85%.
Thus, (1S)ꢀ(ꢀ)ꢀcamphorꢀ10,2ꢀsultam was deprotonated with sodium hydride and then alkylated using
chloroacetyl chloride.30 The resulting compound 7 then reacted with neat triethyl phosphite to yield
phosphonoacetate 8.31
Scheme 2. Synthesis of the conjugated alkene 9.
Ester 5 was reduced to the corresponding aldehyde (with DIBAL) before undergoing Hornerꢀ
WadsworthꢀEmmons olefination with phosphonoacetate 8 to yield the conjugated alkene 9.19 Lithium
chloride was used to help stabilize the phosphonoacetate enolate and thus increase the acidity of the α
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protons to the carbonyl, thereby facilitating their removal using a weak base such as N,Nꢀ
diisopropylethylamine (DiPEA).32 The conjugated alkene 9 was consecutively reduced and alkylated (in
a oneꢀpot reaction) to yield the deconjugated fluoroalkene 10 (Scheme 3).19 As the final step of the
synthesis, hydrolysis of the chiral auxiliary followed by deprotection of the Bocꢀprotected amine and its
subsequent protection with Fmoc yielded carboxylic acid 2 with an overall yield of 14% from ethyl
bromodifluoroacetate.19
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Scheme 3. Synthesis of the Fmocꢀprotected GlyꢀPhe fluoroalkene dipeptide isostere 2.
The reduction of the conjugated alkene 9 occurs in two consecutive singleꢀelectron transfers (SETs) by
Me2CuLi (Scheme 4).19 The resulting anion 11 then undergoes the elimination of a fluorine atom,
leading to dienolate 12. Studies performed with methyl iodide have indicated that this type of lithium
dienolate is rather unreactive. Similar to HMPA, DMPU is a solvent known to activate the nucleophilic
properties of lithium reagents.33 DMPU was therefore used to trap the lithium cation and enable its
substitution with a tin atom. The tin dienolate 13 then attacked benzyl bromide from its less hindered
face (Figure 2) to yield the desired product 10 as a single isomer. The stereochemistry of compound 10
was confirmed by Xꢀray diffraction (Figure 3).
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Scheme 4. Mechanism of the oneꢀpot reduction/alkylation.
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Figure 2. 3D representation of tin dienolate 13, showing its preferential face (top) for nucleophilic
attack. The Boc group is at the front position of the molecule, whereas the chiral camphor region is at
the back.
Figure 3. Crystal structure of compound 10. ORTEP representation with 25% probability level for
heavy atoms.
The synthesis of the Leuꢀenkephalin peptidomimetic was performed via solidꢀphase peptide synthesis
(SPPS) using 2ꢀchlorotrityl chloride resin (CTC, Scheme 5). FmocꢀLeuꢀOH was coupled to CTC in the
presence of DiPEA. The second, third and fourth couplings were conducted using compound 2, Fmocꢀ
GlyꢀOH and FmocꢀTyr(tBu)ꢀOH, respectively, with N,Nꢀdiisopropylcarbodiimide (DiC) and 1ꢀ
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hydroxybenzotriazole (HOBt) as coupling agents. Each coupling was followed by washes with
DMF/MeOH/DCM and Fmoc deprotection using 20% piperidine (pip) in DMF.34 Cleavage from the
resin and deprotection of the phenol hydroxyl were conducted using a mixture of TFA, TiPS and water,
yielding the Leuꢀenkephalin fluoroalkene peptidomimetic 1 HꢀTyrꢀGlyꢀGlyꢀψ[(Z)CF=CH]ꢀPheꢀLeuꢀOH
with a yield of 85%, as calculated from the substitution level of the resin (measured by UV absorbance).
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Scheme 5. Solidꢀphase peptide synthesis of the Leuꢀenkephalin fluoroalkene peptidomimetic 1.
Binding properties and activity at DOPr
Following the synthesis of the Leuꢀenkephalin peptidomimetic 1, its affinity for DOPr and its
lipophilicity were evaluated. The affinities of Leuꢀenkephalin and of the peptidomimetic 1 toward DOPr
were measured by competitive binding assays using iodinated deltorphin I ([125I]ꢀdeltorphin I) on
membrane preparations obtained from HEK293 cells expressing a FLAGꢀtagged version of the mouse
DOPr (Figure 4). For all other peptidomimetics, competitive binding assays were performed with
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tritiated deltorphin II ([3H]ꢀdeltorphin II) on membrane preparations obtained from GH3 cells (note that
Leuꢀenkephalin was also tested in this assay in our previous publications with similar results).11,13
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Although compound 1 is still able to bind DOPr (Ki = 43 +/ꢀ 14 nM), the substitution of the third amide
bond by a fluoroalkene is accompanied by a slight (6x) decrease in affinity compared to Leuꢀenkephalin
(Ki = 6.9 +/ꢀ 2.3 nM). However, note that compound 1 shows a considerably higher affinity (>10x) than
a peptidomimetic in which the third amide bond has been replaced by an alkene (Ki = 587 +/ꢀ 19 nM).11
These results suggest that the fluoroalkene isostere and the third amide bond of Leuꢀenkephalin act as a
hydrogen bond acceptor. These findings are in agreement with previous results, where the substitution
of the third amide bond of Leuꢀenkephalin by an ester13 or a thioamide isostere17 also led to highꢀ
affinity DOPr ligands with Ki values of 34 +/ꢀ 17 nM and 47 +/ꢀ 17 nM, respectively. In summary, Leuꢀ
enkephalin and its ester, fluoroalkene, thioamide, and alkene isosteres at the third amide position have
their affinities ranked in approximately the same order as their hydrogen bond acceptor abilities18
(Figure 4), strongly supporting the hypothesis that the third peptide bond interacts with a hydrogen bond
donor group inside the binding pocket of DOPr. This same peptide bond does not appear to partner with
an acceptor because the ester and the fluoroalkene still retain a reasonable affinity for the receptor,
although they have no donor ability. Moreover, the thioamide, which is a considerably stronger
hydrogen bond donor that the amide,18 does not show increased affinity.
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As shown in Figure 5, peptidomimetic 1 also induces timeꢀ and concentrationꢀdependent
phosphorylation of the ERK1/2 signaling pathway. The phosphorylation of ERK1/2 reached a peak at 5
min of stimulation (Fig. 5A), and the effect was significant at 1 µM and 10 µM (Fig. 5B and C). The
results of this assay suggest that the ability of peptidomimetic 1 to activate DOPr is similar to that of
Leuꢀenkephalin. The mouse vas deferens (MVD) assay was also used to further quantify the potency of
peptidomimetic 1. In this assay, the concentrations of Leuꢀenkephalin and peptidomimetic 1 necessary
to produce a 50% inhibition (EC50) of the MVD contractions induced by electric field stimulation were
found to be 30 +/ꢀ 2 nM and 226 +/ꢀ 21 nM, respectively. The efficacy (Emax) of peptidomimetic 1 was
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identical to that of Leuꢀenkephalin. Indeed, at 10 µM, peptidomimetic 1 produced a maximal inhibition
of the electric fieldꢀinduced MVD contractions. These results confirm that although the affinity of
peptidomimetic 1 is slightly lower than that of Leuꢀenkephalin, this compound is fully active on DOPr.
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Figure 4. Structures and affinities (Ki) of five Leuꢀenkephalin analogs, including 1, that have peptide
bond surrogates at the third amide position.
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Figure 5. Compound 1 induces a rapid, transient, and doseꢀdependent phosphorylation (activation) of
ERK1/2. A; Western blot analyses of phosphorylated ERK1/2 (pERK1 and pERK2) after 5 to 60 min of
stimulation of DRGF11/DOPrꢀGFP cells with compound 1 (1 µM) or 5 min of stimulation with Leuꢀ
enkephalin (LE; 1 µM) are shown. The total amount of ERK1/2 was analyzed (bottom blot) and used as
a loading control. Images are representative of 3 independent experiments. B; Compound 1 produces a
doseꢀdependent phosphorylation of ERK1/2 after 5 min of stimulation. Images are representative of 4
independent experiments. C; Ratiometric quantification (ratio of pERK/ERK signals) of the doseꢀ
dependent phosphorylation of ERK1/2 induced by compound 1 is illustrated (n = 4). Data are expressed
as foldꢀincrease over control +/ꢀ standard error of the mean (S.E.M.). Oneꢀway ANOVA followed by
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KruskalꢀWallis multiple comparison test; * p < 0.05 and ** p < 0.01 compared to control, nonꢀ
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stimulated cells.
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Lipophilicity
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The lipophilicity of compound 1 was measured using the LogD7.4 (Table 1),13 which represents the
logarithm of the ratio of the compound’s concentrations in octanol and in aqueous solution at
physiological pH (7.4) after being shaken in a mixture of both solvents and is given by the equation
LogD7.4 = Log ([Xoctanol] / [Xwater])
where [Xoctanol] represents the compound’s concentration in the octanol phase and [Xwater] represents the
concentration in the aqueous phase.
Table 1. LogD7.4 values for Leuꢀenkephalin and peptidomimetics containing alkene and fluoroalkene
peptide bond isosteres at the third amide position.
Peptidomimetic
LogD7.4
TyrꢀGlyꢀGlyꢀPheꢀLeu
TyrꢀGlyꢀGly//PheꢀLeu
ꢀ0.954 ± 0.006
ꢀ0.065 ± 0.003
ꢀ0.109 ± 0.007
TyrꢀGlyꢀGlyF//PheꢀLeu (1)
Gly//Phe and GlyF//Phe stand for Glyꢀψ[(E)CH=CH]ꢀPhe and Glyꢀψ[(Z)CF=CH]ꢀPhe, respectively.
The substitution of the third amide bond by a fluoroalkene leads to a 0.845 increase in LogD7.4, which is
0.044 lower than the value for the compound in which one amide bond has been substituted by an
alkene bond (represented by TyrꢀGlyꢀGly//PheꢀLeu).11 The LogD7.4 of compound 1 is still substantially
lower than the minimum suggested value (2) obtained by a statistical analysis of drugs that show good
permeability toward the bloodꢀbrain barrier.35
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Conclusions
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9
The asymmetric synthesis of the GlyꢀPhe fluoroalkene dipeptide isostere (Glyꢀψ[(Z)CF=CH]ꢀPhe) has
been accomplished using a oneꢀpot reduction of a conjugated alkene followed by an enantioselective
alkylation of the resulting tin dienolate bearing a camphorꢀbased chiral auxiliary, with DMPU as a
solvent. The dipeptide isostere was then incorporated into a Leuꢀenkephalin peptidomimetic using solidꢀ
phase peptide synthesis (SPPS). The affinity of peptidomimetic 1 for DOPr was slightly lower than that
of Leuꢀenkephalin, although the loss of affinity was less than one order of magnitude, supporting the
hypothesis that a hydrogen bond acceptor is necessary at the third peptide position for optimal
complementarity with DOPr. The peptidomimetic also showed greater lipophilicity than Leuꢀ
enkephalin, thus demonstrating that the substitution of amide bonds by fluoroalkenes in peptides can be
a good approach to improve their pharmacokinetic profiles.
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To pursue research on Leuꢀenkephalin peptidomimetics in the same vein, it would be interesting to
sequentially and systematically substitute all the peptide’s amide bonds (1, 2 and 4) by a fluoroalkene.
The method used in this paper involves a chiral auxiliary for the formation of a chiral center on the Cꢀ
terminus amino acid of the dipeptide isostere. Another method that uses Lꢀphenylglycine (or its
enantiomer) for the formation of the Nꢀterminus amino acid’s chiral center36 could be used to synthesize
the TyrꢀGly and PheꢀLeu fluoroalkene dipeptide isosteres. The substitution of amides 1 and 4 could be
of particular interest because we have shown that the former can be substituted by an alkene and the
latter by a hydrogen bond acceptor (ester, thioamide, or NꢀMe amide), both without a substantial loss of
affinity or activity for DOPr.11,13
EXPERIMENTAL SECTION
Chemistry
Commercialꢀgrade reagents (SigmaꢀAldrich and VWR International for organic synthesis and Chemꢀ
Impex International for SPPS) were used without further purification. When necessary, the solvents
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(Fisher Scientific) were purified and dried prior to use. Peptide purification by HPLC was performed
using preparative reverseꢀphase HPLC, detecting at 280 nm, with a VYDAC 218TP C18 column
(Grace Discovery Sciences, SalaberryꢀdeꢀValleyfield, Canada) and using an acetonitrile (MeCN)
gradient in a 0.1% TFA aqueous solution (from 0% to 60%) with a flow rate of 7 mL/min over 2 h. The
purity of all fractions was analyzed using an Agilent 1100 series analytical HPLC, detecting at 214 nm,
with a Phenomenex 5 µm, 4.6 × 100 mm C18 column using a gradient of MeCN in 0.1% TFA aqueous
solution. Fractions with a purity greater than 95% were pooled and freezeꢀdried. Optical rotation
measurements were performed using a PerkinꢀElmer 241 Polarimeter and are reported in units of 10ꢀ1
deg cm2 gꢀ1. Infrared spectra were recorded on a PerkinꢀElmer Spectrum 1600 FTIR. 1H and 13C NMR
spectra were recorded in deuterated solvents on Bruker AC 300 NMR and Bruker AscendTM 400
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spectrometers. The H and 13C NMR chemical shifts are reported in ppm (parts per million). The
residual solvent peaks were 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 ZABꢀ2F or MALDIꢀTOF or on an ESIꢀQꢀTof (Maxis).
Ethyl 3ꢀ(dibenzylamino)ꢀ2,2ꢀdifluoropropanoate (4)
A solution containing benzotriazole (18.9 g, 159 mmol), dibenzylamine (30.5 mL, 159 mmol) and
paraformaldehyde (4.77 g, 159 mmol) in toluene (400 mL) was heated under reflux with a DeanꢀStark
trap until the theoretical amount of water (2.86 mL) was collected. The solvent was evaporated under
reduced pressure, and the residue was dissolved in ethyl acetate and then washed six times with 1 M
aqueous NaOH. The organic phase was dried (MgSO4) and concentrated under reduced pressure, and
the resulting residue was triturated with diethyl ether to yield a mixture of isomers (1Hꢀ1,2,3ꢀ
benzotriazolꢀ1ꢀylꢀN,Nꢀdibenzylmethanamine
and
2Hꢀ1,2,3ꢀbenzotriazolꢀ2ꢀylꢀN,Nꢀ
dibenzylmethanamine) 3 as a white solid (35.6 g, 68%), which was used in the next step without
further purification.
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TMSCl (13.8 mL, 108.5 mmol) was added to a suspension of zinc dust (14.2 g, 217 mmol) in anhydrous
THF (100 mL) under an argon atmosphere. After stirring for 10 min, BrCF2CO2Et (8.97 mL, 70.0
mmol) was slowly added; the reaction appeared to be slightly exothermic. After stirring for 10 min, a
mixture of 1Hꢀ and 2Hꢀ1,2,3ꢀbenzotriazolꢀ1ꢀylꢀN,Nꢀdibenzylmethanamine 3 (35.6 g, 108.5 mmol) in
anhydrous THF (150 mL) was added. The resulting solution was stirred at room temperature for 3 h,
after which saturated aqueous NaHCO3 (150 mL) was added, and the mixture was then filtered using
diatomaceous earth. The aqueous phase was extracted with EtOAc (3 × 200 mL). The combined organic
phases were washed with 1 N aqueous HCl, dried (MgSO4) and then concentrated under reduced
pressure. The crude product was purified using dryꢀcolumn chromatography on silica gel, eluting with
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ethyl acetate and hexane (1:19) to yield the title compound (11.4 g, 50%) as a colorless oil. H NMR
(300 MHz, CDCl3) δ (ppm): 7.34ꢀ7.25 (m, 10H), 4.18 (q, 2H, J=7.0 Hz), 3.68 (s, 4H), 3.15 (t, 2H,
13
J=13.0 Hz), 1.22 (t, 3H, J=7.0 Hz). C NMR (75 MHz, CDCl3) δ (ppm): 163.8 (t, J=32 Hz), 138.1,
ꢀ1
129.2, 128.3, 127.3, 116.2 (t, J=253 Hz), 62.7, 58.5, 55.2 (t, J=26 Hz), 13.8. IR (NaCl) ν (cm ) 3029,
2834, 1767, 1660, 1448, 1057. Exact mass: calculated for C19H21F2NO2Na+: 356.1433, found:
356.1436.
Ethyl 3ꢀ(tertꢀbutoxycarbonylamino)ꢀ2,2ꢀdifluoropropanoate (5)
A solution containing ethyl 3ꢀ(dibenzylamino)ꢀ2,2ꢀdifluoropropanoate (4) (11.1 g, 33.4 mmol) and
Boc2O (11.1 g, 50.9 mmol) in EtOH (350 mL) was added to Pd/C (10%, 2.13 g, 2.00 mmol). Gaseous
hydrogen (five balloons of approximatively 15 cm in diameter, for a total of approximatively 9 L) was
bubbled through the solution while stirring vigorously, and the suspension was stirred overnight at room
temperature under a hydrogen atmosphere. The mixture was filtered using diatomaceous earth and
rinsed with EtOH. The solvent was evaporated under reduced pressure, and the crude product was
purified using flash chromatography on silica gel, eluting with ethyl acetate and hexane (3:17 to 1:3 to
1
7:13) to yield the title compound (6.72 g, 80%) as a colorless oil. H NMR (300 MHz, CDCl3) δ (ppm):
4.97 (br, 1H), 4.29 (q, 2H, J=7.0 Hz), 3.72 (td, 2H, J=13.5, 7.0 Hz), 1.40 (s, 9H), 1.32 (t, 3H, J=7.0 Hz).
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13
C NMR (75 MHz, CDCl3) δ (ppm): 163.0 (t, J=32 Hz), 155.3, 113.3 (t, J=252 Hz), 80.3, 63.2, 43.4
1
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3
4
ꢀ1
(t, J=28 Hz), 28.1, 13.8. IR (NaCl) ν (cm ) 3572ꢀ3201 (br), 2980, 2941, 1767, 1705, 1516, 1247, 1157,
1124. Exact mass: calculated for C10H17F2NO4Na+: 276.1018, found: 276.1022.
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Chloroacetylꢀ(S)ꢀcamphorꢀ10,2ꢀsultam (7)
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To a solution of (1S)ꢀ(ꢀ)ꢀcamphorꢀ10,2ꢀsultam (5.58 g, 25.9 mmol) in DCM (235 mL) was slowly added
NaH (60% suspension in mineral oil, 1.04 g, 25.9 mmol), and the suspension was stirred for 30 min at
room temperature under an argon atmosphere. The temperature was lowered to ꢀ60 °C, and chloroacetyl
chloride (2.48 mL, 31.07 mmol) was added dropwise to the mixture, which was then stirred overnight at
room temperature. Saturated aqueous ammonium chloride solution and water were added to the mixture,
and the layers were separated. The aqueous phase was extracted twice with DCM. The combined
organic layers were washed with saturated aqueous sodium bicarbonate and then dried using anhydrous
MgSO4, and the solvent was evaporated under reduced pressure. The crude product was purified using
flash chromatography on silica gel, eluting with ethyl acetate and hexane (1:7) to yield the title
1
compound (6.40 g, 85%) as a crystalline white solid. mp 131ꢀ132 °C. H NMR (300 MHz, CDCl3) δ
(ppm): 4.50 (s, 2H), 3.92 (dd, 1H, J=7.5, 5.0 Hz), 3.53 (d, 1H, J=14.0 Hz), 3.46 (d, 1H, J=14.0 Hz),
13
2.22ꢀ2.05 (m, 2H), 1.99ꢀ1.87 (m, 3H), 1.48ꢀ1.37 (m, 2H), 1.14 (s, 3H), 0.98 (s, 3H). C NMR (75
MHz, CDCl3) δ (ppm): 165.6, 65.4, 52.6, 49.2, 47.9, 44.5, 42.4, 38.0, 32.7, 26.4, 20.7, 19.8. IR (NaCl)
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ꢀ1
ν (cm ) 3750ꢀ3020 (br), 3005, 2962, 1655, 1325. [α] D ꢀ113 (c=9.06, CHCl3). Exact mass: calculated
for C12H18ClNO3SNa+: 314.0588, found: 314.0600.
Diethylphosphonylacetylꢀ(S)ꢀcamphorꢀ10,2ꢀsultam (8)
A solution containing chloroacetylꢀ(S)ꢀcamphorꢀ10,2ꢀsultam (7) (6.25 g, 21.4 mmol) in triethyl
phosphite (95 mL, 554 mmol) was heated overnight under reflux under an argon atmosphere, and the
triethyl phosphite was removed under reduced pressure. The crude product (still containing some
triethyl phosphite) was purified using flash chromatography on silica gel, eluting with ethyl acetate and
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1
hexane (2:3 to 1:1 to 3:2) to yield the title compound (8.42 g, 100%) as a colorless oil. H NMR (300
MHz, CDCl3) δ (ppm): 4.24ꢀ4.08 (m, 4H), 3.85 (dd, 1H, J=7.5, 5.0 Hz), 3.59ꢀ3.36 (m, 3H), 3.17 (1H,
dd, J= 22.5, 15.5 Hz), 2.19ꢀ1.97 (m, 2H), 1.96ꢀ1.82 (m, 3H), 1.46ꢀ1.20 (m, 8H), 1.14 (s, 3H), 0.93 (s,
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9
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3H). C NMR (75 MHz, CDCl3) δ (ppm): 163.6 (d, J=7 Hz), 65.2, 62.8 (d, J=6 Hz), 62.6 (d, J=6 Hz),
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52.9, 48.1 (d, J=38 Hz), 44.6, 38.2, 35.8, 34.0, 32.8, 26.5, 20.7, 19.9, 16.4 (d, J=4 Hz), 16,3 (d, J=3 Hz).
ꢀ1
IR (NaCl) ν (cm ) 3757ꢀ3024 (br), 2985, 1638, 1325, 1051, 1018. Exact mass: calculated for
C16H28NO6PSNa+: 416.1267, found: 416.1278. [α]20D ꢀ60.5 (c=7.54, CHCl3).
(E)ꢀ5ꢀ(tertꢀButoxycarbonyl)amino)ꢀ4,4ꢀdifluoropentꢀ2ꢀenoylꢀ(S)ꢀcamphorꢀ10,2ꢀsultam (9)
To a solution of ethylꢀ3ꢀ(tertꢀbutoxycarbonylamino)ꢀ2,2ꢀdifluoropropanoate (5) (4.86 g, 19.2 mmol) in
anhydrous DCM (200 mL) at ꢀ78 °C under an argon atmosphere was slowly added diisobutylaluminum
hydride (1 M in toluene, 30.8 mL, 30.8 mmol). The reaction was closely monitored by thinꢀlayer
chromatography on silica gel, and after 40 min, saturated aqueous citric acid and water were added to
the mixture, which was then extracted with diethyl ether (3 × 200 mL). The organic phase was washed
with brine and dried using anhydrous magnesium sulfate, and the solvent was evaporated under reduced
pressure to yield the aldehyde, which was used in the next step without further purification.
To a solution of diethylphosphonylacetylꢀ(S)ꢀcamphorꢀ10,2ꢀsultam (8) (8.31 g, 21.1 mmol) in MeCN
(140 mL) at 0 °C under an argon atmosphere was added LiCl (1.06 g, 25.0 mmol) and DiPEA (4.35 mL,
25.0 mmol). After stirring for 30 min at 0 °C, a solution of the above aldehyde in MeCN (45 mL) was
added, and the mixture was stirred for 2 h at 0 °C and overnight at room temperature. Saturated aqueous
ammonium chloride was added, and the mixture was extracted with diethyl ether (3 × 200 mL). The
organic phase was washed with brine and dried using anhydrous magnesium sulfate, and the solvent was
evaporated under reduced pressure. The crude product was purified using flash chromatography on
silica gel, eluting with ethyl acetate and hexane (1:3 to 7:13) to yield the title compound (7.03 g, 82%)
1
as a colorless oil. H NMR (300 MHz, CDCl3) δ (ppm): 7.01 (dt, 1H, J=15.5, 2.0 Hz), 6.93ꢀ6.81 (m,
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1H), 4.81 (t, 1H, J=6.0 Hz), 3.94 (dd, 1H, J=6.5, 6.5 Hz), 3.64 (td, 2H, J=13.5, 6.5 Hz), 3.53 (d, 1H,
J=13.8 Hz), 3.46 (d, 1H, J=13.8 Hz), 2.16ꢀ2.07 (m, 2H), 2.01ꢀ1.82 (m, 3H), 1.53ꢀ1.29 (m, 2H), 1.43 (s,
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9H), 1.16 (s, 3H), 0.98 (s, 3H). C NMR (75 MHz, CDCl3) δ (ppm): 162.1, 155.4, 137.5 (t, J=26 Hz),
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8
125.4 (t, J=8 Hz), 118.4 (t, J=242 Hz), 80.3, 65.1, 53.0, 48.7, 47.8, 45.2, 44.7, 38.3, 32.9, 28.2, 26.4,
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20.8, 19.9. IR (NaCl) ν (cm ) 3493ꢀ3288 (br), 2963, 1705, 1683, 1515, 1331, 1163, 1135. Exact mass:
calculated for C20H30F2N2O5SNa+: 471.1736, found: 471.1746. [α]20D ꢀ55.0 (c=7.64, CHCl3).
(2R,Z)ꢀ2ꢀBenzylꢀ5ꢀ(Nꢀ(tertꢀButoxycarbonyl)ꢀamino)ꢀ4ꢀfluoropentꢀ3ꢀenoylꢀ(S)ꢀcamphorꢀ10,2ꢀ
sultam (10)
To a suspension of CuI (3.43 g, 18.0 mmol) in anhydrous THF (55 mL) was slowly added a solution of
MeLi.LiBr complex in diethyl ether (1.5 M, 24.0 mL, 36.0 mmol), using a syringe that was cooled with
dry ice for 1 min. The resulting mixture was stirred for 10 min at 0 °C. At this point, the solution has to
be colorless (or very slightly pink, but not yellowish, even slightly) for the next step to be successful.
The temperature was lowered to ꢀ78 °C, and a solution of (E)ꢀ5ꢀ(tertꢀButoxycarbonyl)amino)ꢀ4,4ꢀ
difluoropentꢀ2ꢀenoylꢀ(S)ꢀcamphorꢀ10,2ꢀsultam (9) (1.96 g, 4.38 mmol) in anhydrous THF (82 mL) was
added dropwise. After stirring for 30 min at ꢀ78 °C, DMPU (8.67 mL, 72.0 mmol) was added to the
mixture. After stirring for 30 min at ꢀ78 °C, a solution of triphenyltin chloride (3.48 g, 9.02 mmol) in
anhydrous THF (82 mL) was added to the mixture. After stirring for 30 min at ꢀ40 °C, benzyl bromide
(4.28 mL, 36.0 mmol) was slowly added to the mixture, which was then stirred overnight at ꢀ40 °C. Due
to the sensitive nature of the reaction, it should not be monitored by TLC until the next day. The
reaction was quenched by the addition of saturated aqueous ammonium chloride and 28% aqueous
ammonium hydroxide (1:1). The mixture was then stirred at room temperature for 30 min, after which it
was extracted with diethyl ether (3 × 200 mL). The organic phase was washed with brine and dried
using anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The crude
product was purified using flash chromatography on silica gel, eluting with ethyl acetate and hexane
(3:7) to yield the title compound (1.45 g, 64%) as a white solid (all the following data have been
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1
recorded from the solid collected after chromatography). mp 54ꢀ58 °C H NMR (300 MHz, CDCl3) δ
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(ppm): 7.27ꢀ7.06 (m, 5H), 4.97 (dd, 1H, J=35.5, 9.5 Hz), 4.69 (br, 1H), 4.45ꢀ4.37 (m, 1H), 3.83ꢀ 3.59
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(m, 3H), 3.41 (d, 1H, J=14.0 Hz), 3.34 (d, 1H, J=14.0 Hz), 3.09 (dd, 1H, J=13.0, 8.0 Hz), 2.77 (dd, 1H,
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J=13.0, 7.5 Hz), 2.08ꢀ1.70 (m, 5H), 1.42 (s, 9H), 1.32ꢀ1.14 (m, 2H), 0.87 (s, 3H), 0.73 (s, 3H).
C
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NMR (75 MHz, CDCl3) δ (ppm): 172.2, 156,9 (d, J=261 Hz), 155.3, 137.4, 129.5, 128.2, 126.7, 104.0
(d, J=11 Hz), 79.8, 65.1, 53.0, 48.2, 47.6, 44.6, 43.1, 41.1 (d, J=33 Hz), 40.3, 38.3, 32.8, 28.3, 26.4,
ꢀ1
20.5, 19.8. IR (NaCl) ν (cm ) 3773ꢀ2874 (br), 2097, 1638, 1325, 1163. Exact mass: calculated for
C27H37FN2O5SNa+: 543.2299, found: 543.2310. [α]20D ꢀ57.0 (c=6.75, CHCl3).
(R,Z)ꢀ5ꢀ(((9HꢀFluorenꢀ9ꢀyl)methoxy)carbonylamino)ꢀ2ꢀbenzylꢀ4ꢀfluoropentꢀ3ꢀenoic acid (2)
To a solution of (2R,Z)ꢀ2ꢀbenzylꢀ5ꢀ(Nꢀ(tertꢀbutoxycarbonyl)ꢀamino)ꢀ4ꢀfluoropentꢀ3ꢀenoylꢀ(S)ꢀcamphorꢀ
10,2ꢀsultam (10) (117 mg, 0.225 mmol) in THF (3.3 mL) and H2O (0.6 mL) was added 30% H2O2 (133
ꢀL, 1.30 mmol) and LiOH (0.45 mL, 1 N in water, 0.45 mmol). The mixture was stirred at room
temperature for 2.5 h, and 1 N aqueous HCl was added until a pH of 2 was reached. The mixture was
then extracted with EtOAc (2 × 10 mL). The organic layers were combined and dried using anhydrous
MgSO4, and the solvent was evaporated under reduced pressure. The crude residue was then dissolved
in DCM (10 mL) and TFA (2.7 mL). The solution was stirred at room temperature for 30 min, the
solvent was evaporated under reduced pressure, and the resulting residue was dissolved in MeCN (6.7
mL), DMF (6.0 mL) and H2O (0.7 mL). At a temperature of 0 °C, FmocꢀOSu (106 mg, 0.315 mmol)
and Et3N (62.8 ꢀL, 0.450 mmol) were added, and the mixture was stirred overnight at room
temperature. Aqueous 1 N HCl was added until a pH of 2 was reached, and the mixture was extracted
with EtOAc (3 × 10 mL). The organic layers were combined and dried using anhydrous MgSO4, and the
solvent was evaporated under reduced pressure. The crude product was purified by flash
chromatography on silica gel, eluting with EtOAc, hexane and acetic acid (35:65:1 to 40:60:1 to
45:55:1) to yield the title compound (65 mg, 65%) as a white solid. mp 80ꢀ84 °C 1H NMR (300 MHz,
CDCl3) δ (ppm): 7.77 (d, 2H, J=7.5 Hz), 7.61 (d, 2H, J=7.5 Hz), 7.41 (dd, 2H, J=7.5, 7.5 Hz), 7.32 (dd,
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2H, J=7.5, 7.5 Hz), 7.26ꢀ7.09 (m, 5H), 6.2ꢀ5.4 (br, 1H), 4.99ꢀ4.71 (m, 2H), 4.44 (d, 2H, J=7.0 Hz), 4.21
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(t, 1H, J=7.0 Hz), 3.87ꢀ3.75 (m, 3H), 3.12 (dd, 1H, J=13.5, 6.5 Hz), 2.83 (dd, 1H, J=13.5, 8.0 Hz).
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NMR (75 MHz, CDCl3) δ (ppm): 177.8, 156.8 (d, J=259 Hz), 156.2, 143.7, 141.3, 137.7, 129.1, 128.4,
127.8, 127.1, 126.7, 125.0, 120.1, 104.6 (d, J=12 Hz), 67.1, 47.1, 42.3, 41.5 (d, J=30 Hz), 38.3. IR
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(NaCl) ν (cm ) 3694ꢀ3103 (br), 3063, 3024, 1711, 1521, 1443, 1247, 749, 738. Exact mass: calculated
for C27H24FNO4Na+: 468.1582, found: 468.1593. [α]20D ꢀ29.3 (c=6.58, CHCl3).
(S)ꢀ2ꢀ((R,Z)ꢀ5ꢀ(2ꢀ((S)ꢀ2ꢀAminoꢀ3ꢀ(4ꢀhydroxyphenyl)propanamido)acetamido)ꢀ2ꢀbenzylꢀ4ꢀ
fluoropentꢀ3ꢀenamido)ꢀ4ꢀmethylpentanoic acid (1)
HꢀTyrꢀGlyꢀGlyF//PheꢀLeuꢀOH
CTC resin loading: The loading of the resin was determined by UV quantification of Fmoc release
following a first coupling with FmocꢀLeuꢀOH (see details in the next paragraph). An aliquot (precise
mass M ~ 10 mg) of resin was dried under vacuum. A mixture of piperidine (1 mL) and DMF (1 mL)
was added, and the suspension was agitated in a shaker for 30 min. A portion of the supernate (0.5 mL)
was diluted in DCM (4.5 mL), and the UV absorbances (at 290 nm and 301 nm) of the resulting solution
were recorded (A1290 and A1301). The reference absorbances (A0290 and A0301) were measured in the same
way from a blank solution consisting of a mixture of piperidine (0.25 mL), DMF (0.25 mL) and DCM
(4.5 mL). The loading of the resin (mmol.g−1) was then calculated as the average of the two values
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[20,000 × (A1 – A0290) / (5,800 × M)] and [20,000 × (A1 – A0301) / (7,800 × M)], where M is the
quantity of the resin aliquot in mg.
SPPS: A small amount of 2ꢀchlorotrityl chloride resin (285 mg, loading of 0.38 mmol/g) was used.
FmocꢀLeuꢀOH (165 mg, 0.456 mmol), DiPEA (517 ꢀL, 2.85 mmol) (the resin was stirred for 5 min
with the first 207 ꢀL, 1.14 mmol, before adding the remaining 310 ꢀL, 1.71 mmol) and DCM (5 mL)
were used in the first coupling. The free sites were subsequently protected using MeOH (1 mL), DiPEA
(0.25 mL) and DCM (4 mL) and agitating in a shaker for 1 h. Fmoc deprotection was performed using
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piperidine and DMF (1:4), agitating for 45 min in a shaker. The same Fmoc deprotection procedure was
applied after each coupling using the following Fmocꢀprotected amino acids: compound 2, FmocꢀGlyꢀ
OH and FmocꢀTyr(tBu)ꢀOH. (R,Z)ꢀ5ꢀ(((9HꢀFluorenꢀ9ꢀyl)methoxy)carbonylamino)ꢀ2ꢀbenzylꢀ4ꢀ
fluoropentꢀ3ꢀenoic acid (2) (288 mg, 0.648 mmol), DiC (102 ꢀL, 0.648 mmol), HOBt.H2O (99 mg,
0.648 mmol) and DMF (5 mL) were used in the second coupling. FmocꢀGlyꢀOH (321 mg, 1.08 mmol),
DiC (169 ꢀL, 1.08 mmol), HOBt.H2O (165 mg, 1.08 mmol) and DMF (5 mL) were used in the third
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coupling. FmocꢀTyr(tBu)ꢀOH (496 mg, 1.08 mmol), DiC (169 ꢀL, 1.08 mmol), HOBt.H2O (165 mg,
1.08 mmol) and DMF (5 mL) were used in the fourth coupling. After removal of the Nꢀterminal Fmoc
group, cleavage of the final peptide off the resin was performed in a glass vial using TFA, water and
TiPS (95:2.5:2.5) and agitating for 1.5 h. After cleavage, the mixture was filtered on cotton into a large
amount of water (20 mL). The remaining TFA was evaporated in vacuo, and the aqueous solution was
1
freezeꢀdried. HPLC purification yielded the title peptide as a white solid (51 mg, 85%). H NMR (400
MHz, D2O) δ (ppm): 7.34 (t, 2H, J=7.1 Hz), 7.30ꢀ7.23 (m, 3H), 7.17 (d, 2H, J=8.6 Hz), 6.89 (d, 2H,
J=8.6 Hz), 4.98 (dd, 1H, J=36.2, 9.3 Hz), 4.29 (dd, 1H, J=8.1, 7.9 Hz), 4.25 (t, 1H, J= 7.9 Hz), 3.88 (m,
4H), 3.76 (q, 1H, J= 7.9 Hz), 3.15 (d, 2H, J=7.4 Hz), 3.01 (dd, 1H, J=13.6, 8.1 Hz), 2.85 (dd, 1H,
1
J=13.6, 7.4 Hz), 1.65ꢀ1.45 (m, 3H), 0.88 (d, 3H, J=6.3 Hz), 0.82 (d, 3H, J=6.2 Hz). H NMR (300
MHz, DMSOꢀd6) δ (ppm): 12.42 (br, 1H), 9,33 (s, 1H), 8.70 (t, 1H, J=5.5 Hz), 8.24ꢀ8.20 (m, 2H), 8.02
(br, 3H), 7.23ꢀ7.10 (m, 5H), 7.02 (d, 2H, J=8.5 Hz), 6.67 (d, 2H, J=8.5 Hz), 4.91 (dd, 1H, J=38.0, 9.5
Hz), 4.15ꢀ4.08 (m, 1H), 3.95 (br, 1H), 3.84ꢀ3.66 (m, 4H), 3.59ꢀ3.51 (m, 1H), 3.00ꢀ2.90 (m, 2H), 2.77
(dd, 1H, J=14.0, 8.5 Hz), 2.55 (dd, 1H, J=14.0, 8.5 Hz), 1.62ꢀ1.37 (m, 3H), 0.84 (d, 3H, J=6.5 Hz), 0.78
13
(d, 3H, J=6.5 Hz). C NMR (101 MHz, D2O) δ (ppm): 176.2, 175.3, 170.5, 169.7, 156.3 (d, J=258
Hz), 155.2, 138.4, 130.8, 129.1, 128.5, 126.7, 125.4, 115.9, 105.1 (d, J=12 Hz), 54.5, 51.4, 43.3, 42.3,
13
39.2 (d, J=30 Hz), 37.7, 35.9, 24.3, 22.1, 20.5. C NMR (75 MHz, CD3OD) δ (ppm): 174.4, 172.4,
168.9, 168.5, 157.9, 156.2 (d, J=255 Hz), 139.2, 130.9, 129.5, 128.4, 126.5, 125.2, 115.8, 105.9 (d,
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J=11 Hz), 54.2, 50.8, 43.0, 42.2, 40.5 (d, J=30 Hz), 39.2, 36.7, 24.7, 23.2, 21.8. F NMR (377 MHz,
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ꢀ1
D2O) δ (ppm): ꢀ75.59 (s, 3F from TFA), 112.51 (dt, J=36.2, 12.7 Hz, 1F). IR (NaCl) ν (cm ) 3748,
2971, 2932, 2364, 2339, 1654, 1565. Exact mass: calculated for C29H37FN4O6Na+: 579.2589, found:
579.2604. [α]20D ꢀ1.5 (c=3.47, MeOH).
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Cell culture
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GH3 (a rat pituitary tumor cell line) and HEK293 (human embryonic kidney) cells stably expressing the
mouse DOPr and DRGF11 (mouse neuroblastoma / embryonic rat dorsal root ganglia neuron
hybridoma) cells stably expressing mouse DOPrꢀeGFP were grown at 37 °C in DMEM supplemented
with 10% fetal bovine serum and 10 IU penicillin, 100 mg/L streptomycin and 2 mM glutamine. The
cells were maintained in a humidified atmosphere of 95% air and 5% CO2.11
Competitive binding assays
We evaluated the affinity (Ki) of the different compounds for DOPr using membrane extracts from
either GH3 (thioamide) or HEK293 cells (Leuꢀenkephalin and compound 1) expressing the mouse
DOPr.
Leuꢀenkephalin and compound 1: HEK293 cells grown to confluence in 150 mm Petri dishes were
frozen at ꢀ80 °C until use. On the day of the experiment, the cells were submitted to a heat shock by
placing the Petri dishes at 37 °C for 60 sec before returning to ice. The cells were then harvested in iceꢀ
cold buffer A (trisꢀHCl 20 mM, MgCl2 5 mM, NaCl 150 mM, pH 7.4) using a cell scraper and
centrifuged at 3200 g for 15 min at 4 °C. The pellet containing the membrane extract was resuspended
in 1 mL of buffer A. The protein concentration was determined with BioꢀRad DC™ Protein Assay
reagents (BioꢀRad Laboratories, Mississauga, ON, Canada), and the pellet was further diluted in buffer
A containing 0.1% BSA and 0.01% bacitracin and distributed in 96ꢀwell plates. [125I]ꢀDeltorphin I
(specific activity: ~2000 Ci/mmol) was used to determine the binding affinity of Leuꢀenkephalin and
peptidomimetic 1 in a competitive binding assay. Experiments were performed using a membrane
concentration of 150ꢀ200 µg of proteins/mL and 200 000 cpm of the radiolabeled ligand. Nonꢀspecific
binding was determined using 10 µM nonꢀradioactive deltorphin II. Incubations were performed for 60
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min at 37 °C with increasing concentrations of compound ranging from 10 µM to 1 pM, and the
reaction was stopped by filtration using iceꢀcold buffer A on filtered 96ꢀwell plates. Filters were placed
in 5 mL tubes, and the radioactivity was determined using a Wizard2 Automatic Gamma Counter
(PerkinElmer Canada). Data were analyzed using a nonlinear fitting analysis (SigmaPlot 7.0), and Ki
values were calculated from the IC50 determination using the ChengꢀPrusoff equation37, Ki = IC50 / (1 +
([L]/Kd)), where Ki represents the inhibition constant, IC50 represents the required concentration to
inhibit 50% of the radioligand binding, [L] represents the radioligand concentration and Kd represents
the dissociation constant of the radioligand (Kd for [125I]ꢀdeltorphin I was determined experimentally =
0.9 nM). Ki are expressed as the mean +/ꢀ S.E.M. from 3 independent experiments, each performed in
duplicate.
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Thioamide: After being harvested in phosphateꢀbuffered saline, the GH3 cells were obtained by
centrifugation. The resulting pellet was resuspended in a 10 mM potassium phosphate buffer pH 7.2
(buffer B) and centrifuged for 10 min at 40,000 g. The cell pellet was resuspended in buffer B and left
on ice for 20 min. The suspension was then centrifuged three times at 800 g for 5 min, the supernatants
were saved, and the pellets were resuspended in buffer B. The supernatants were pooled and centrifuged
for 10 min at 40,000 g. The resulting pellet was finally resuspended in buffer B supplemented with 0.32
M sucrose and 5 mM EDTA and stored at ꢀ80 °C until use. The protein concentration was determined
with BioꢀRad DC™ Protein Assay reagents. [3H]ꢀDeltorphin II (specific activity: 30ꢀ60 Ci/mmol;
PerkinElmer, Waltham, MA, USA) was used to determine the binding affinity of the thioamide in a
competitive binding assay. Experiments were performed using a membrane concentration of 100 µg of
proteins/mL and a concentration of the radiolabeled ligand near the Kd value previously obtained in
saturation binding assays (1 nM). Nonꢀspecific binding was determined using 10 µM nonꢀradioactive
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 for 60 min at 37 °C, and the reaction was
stopped by filtration using iceꢀcold buffer B 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
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Canada, Inc., Mississauga, ON, Canada), and the radioactivity was determined using a Beckman Coulter
LSꢀ6500 scintillation counter (Beckman Coulter Canada, Inc.). Data were analyzed using a nonlinear
fitting analysis (SigmaPlot 7.0), and Ki values were calculated as described above from the IC50
determination using the ChengꢀPrusoff equation. For [3H]ꢀdeltorphin II, the dissociation constant (Kd)
was 1 nM. Ki are expressed as the mean +/ꢀ S.E.M. from 3 independent experiments, each performed in
duplicate.
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Mouse vas deferens assay
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 118.1, KCl 4.7, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25 and Dꢀglucose 11.1, gassed with
95% O2 and 5% CO2 and kept at 37 °C. Longitudinal contractions were recorded isometrically at a
constant tension of 0.5 g using a Radnoti force displacement transducer (Radnoti, LLC, Monrovia, CA,
USA), amplified by a Powerlab 8/30 and displayed on LabChart 8 (AD Instruments, Colorado Springs,
CO, USA). Electric stimulation trains were generated by a Grass S48 square pulse stimulator (Astroꢀ
Med, Inc.) interfaced through a stimulus power booster (StimuꢀSplitter II; MedꢀLab Instruments,
Loveland, CO, USA) and connected to a universal coil platinum electrode (Radnoti, LLC, Monrovia,
CA, USA). Trains were repeated every 20 sec. The trains consisted of 6 impulses of 20 V for 1 ms with
9 ms intervals. A volume of 150 µL of the tested ligand was added to the 15 mL bath to obtain final
concentrations ranging from 10ꢀ11 to 10ꢀ5 M. After recording for 5 min, the preparation was washed
twice with 15 mL of fresh medium. As a control, Leuꢀenkephalin was added at a final concentration of
10ꢀ5 M. The data were analyzed with a nonlinear fitting analysis, and the concentration required to
produce a 50% inhibition (EC50) of the contraction was determined. Data are expressed as the mean +/ꢀ
S.E.M. of 4 different MVDs.
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ERK1/2 phosphorylation
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ERK1/2 phosphorylation/activation was measured using DRGF11 cells stably expressing green
fluorescent protein (GFP)ꢀtagged mDOPr (DRGF11/DOPrꢀGFP).11 After 2ꢀ3 days in culture, the cells
were stimulated for periods ranging from 5 to 60 min with 1 µM of either Leuꢀenkephalin or compound
1 or for 5 min with increasing concentrations (10ꢀ9ꢀ10ꢀ5 M) of compound 1. At the end of the incubation,
the cells were washed with Hanks’ buffered saline (HBS; (mM): NaCl 130, KCl 3.5, CaCl2·2H2O 2.3,
MgCl2·6H2O 0.98, HEPES 5, EGTA 0.5) and stabilized for 10 min in iceꢀcold HBS containing 0.1 µM
staurosporine, 1 mM sodium orthovanadate and Complete™ protease inhibitor (Roche Diagnostics,
Laval, Canada). The cells were then lysed for 30 min in 50 mM HEPES pH 7.8 containing 1% Triton Xꢀ
100, 0.1 µM staurosporine, 1 mM sodium orthovanadate, and Complete™ protease inhibitor. The
lysates were then centrifuged at 13,500 g for 10 min at 4 °C, and the supernatants were stored at ꢀ20 °C.
Proteins (15 µg per sample) were separated on 10% SDSꢀpolyacrylamide gels and transferred onto
PVDF membranes. Membranes were then blocked for 1 h in trisꢀbuffered saline (TBS) containing 1%
gelatin and incubated for 2 h with rabbit polyclonal antiꢀphosphorylated p42/44mapk (1:1000; #9101, Cell
Signaling Technology, Danvers, MA, USA) or antiꢀp42/44mapk (1:1000; #9102, Cell Signaling
Technology) antibodies. After three washes with TBS containing 0.05% Tween 20 (TBSꢀT), the
membranes were incubated for 1 h with horseradish peroxidaseꢀconjugated antiꢀrabbit secondary
antibodies (1:2000; #NA934, GE Healthcare, Mississauga, ON, Canada), and the proteins were detected
using an enhanced chemiluminescence detection system (Amersham ECL western blotting detection
reagents, GE Healthcare Life Sciences). Signal intensities were obtained by densitometry using NIH
ImageJ software (1.47v).
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Log D7.4 determination
The distribution coefficient (LogD) was determined using a modified version of the shakeꢀflask method.
Before the experiment, octanol and phosphate buffer (PBS pH 7.4) were mixed together for 24 h to
allow the saturation of each solution. The mixture was allowed to rest, and the phases were separated
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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, and the vial was then mechanically shaken for
30 min. The mixture was allowed to rest for 15 min or until the phase separation was complete. Aliquots
of both phases were collected and injected into an HPLC (10 ꢁL of each aliquot was injected in an
Agilent 1100 series HPLC, column: Agilent Eclipse XD8 Cꢀ18 column, 250 mm × 4.6 mm, 5 ꢁm;
solvent A, 0.1% TFA in water; solvent B 0.1% TFA in acetonitrile; 10ꢀ70% B in A over 20 min; flow
rate: 0.2 mL/min; UV detection at 210 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).
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ACKNOWLEDGMENTS
This work was supported by grants #MOPꢀ102612 and MOPꢀ136871 from the Canadian Institute for
Health Sciences (CIHR), awarded to BG, YLD and LG. KR was the recipient of the 2009 Dominicoꢀ
Regoli/Institut de Pharmacologie de Sherbrooke fellowship and the 2010 Centre d’Excellence en
Neurosciences de Sherbrooke (CENUS)/Institut de Pharmacologie de Sherbrooke fellowship,
respectively. KR was also the recipient of a Master studentship from the Fonds de Recherche Québec –
Santé (FRQS). LG is the recipient of a Seniorꢀsalary support from the FRQS. BG, YLD and LG are
members of the FRQSꢀfunded Centre de Recherche du CHUS and of the Institut de Pharmacologie de
Sherbrooke. LG is a member of CENUS and the FRQSꢀfunded Quebec Pain Research Network (QPRN)
and Réseau québécois de recherche sur le Médicament (RQRM). The authors thank Dr. Richard
Howells (Rutgers UniversityꢀNew Jersey Medical School, Newark, NJ, USA) for providing the
HEK293 cells expressing the mouse DOPr. The authors also thank Dr. Brian Holleran for the iodination
of the deltorphin I and Dr. Jerome Côté for his technical assistance with the MVD assay.
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ACS Chemical Neuroscience
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PDF file containing the following information: 1H NMR spectra for compounds 1, 2, 4, 5, and 7ꢀ10; 13C
NMR spectra for compounds 1, 2, and 10; 19F NMR spectrum for compound 1; HPLCꢀUV
chromatograms of compound 1 and crystal structure report for compound 10. Crystallographic
information file (cif) for compound 10. These files are available free of charge via the internet at: To be
completed by the journal.
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(4) Kieffer, B. L. (1999) Opioids: first lessons from knockout mice. Trends Pharmacol Sci 20, 19ꢀ26.
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