Structure-Activity Relationships of the Peptide Kappa Opioid Receptor Antagonist Zyklophin

Article abstract of DOI:10.1021/jm501827k

The dynorphin (Dyn) A analogue zyklophin ([N-benzyl-Tyr¹-cyclo(d-Asp⁵,Dap⁸)]dynorphin A(1-11)NH₂) is a kappa opioid receptor (KOR)-selective antagonist in vitro, is active in vivo, and antagonizes KOR in the CNS

Full text of DOI:10.1021/jm501827k

Article
pubs.acs.org/jmc
Structure−Activity Relationships of the Peptide Kappa Opioid
Receptor Antagonist Zyklophin
Anand A. Joshi,†,§ Thomas F. Murray,‡ and Jane V. Aldrich*,†,∥
†Department of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas 66045, United States
‡Department of Pharmacology, School of Medicine, Creighton University, Omaha, Nebraska 68102, United States
ABSTRACT: The dynorphin (Dyn) A analogue zyklophin
([N-benzyl-Tyr1-cyclo(D-Asp5,Dap8)]dynorphin A(1−11)NH2)
is a kappa opioid receptor (KOR)-selective antagonist in vitro,
is active in vivo, and antagonizes KOR in the CNS after systemic
administration. Hence, we synthesized zyklophin analogues to
explore the structure−activity relationships of this peptide. The
synthesis of selected analogues required modification to
introduce the N-terminal amino acid due to poor solubility
and/or to avoid epimerization of this residue. Among the N-
terminal modifications, the N-phenethyl and N-cyclopropyl-
methyl substitutions resulted in analogues with the highest KOR
affinities. Pharmacological results for the alanine-substituted
analogues indicated that Phe4 and Arg6, but interestingly not the Tyr1 phenol, are important for zyklophin’s KOR affinity and that
Arg7 was important for KOR antagonist activity. In the GTPγS assay, while all of the cyclic analogues exhibited negligible KOR
efficacy, the N-cyclopropylmethyl-Tyr1 and N-benzyl-Phe1 analogues were 28- and 11-fold more potent KOR antagonists,
respectively, than zyklophin.
very low agonist potencies in the GPI assay.17 When these
structural modifications were combined, we obtained the Dyn
INTRODUCTION
■
While clinically used drugs acting at opioid receptors primarily
interact with mu opioid receptors (MOR), extensive research
has explored potential therapeutic applications of ligands for
other opioid receptors. Kappa opioid receptor (KOR)
A(1−11)NH2 analogue zyklophin (Figure 1) which exhibits
high selectivity for KOR and is a KOR antagonist in the
adenylyl cyclase assay.18
antagonists were initially used only as pharmacological tools,
but they have been recently explored for potential therapeutic
use in the treatment of depression,1−4 anxiety,4−6 and opiate
and cocaine addiction.2,4,7,8 The nonpeptide KOR antagonists
nor-binaltorphimine (norBNI) and JDTic [(3R)-7-hydroxy-N-
[(2S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-
1-yl]-3-methylbutan-2-yl]-1,2,3,4-tetrahydroisoquinoline-3-car-
boxamide] have been used extensively to study KOR
involvement in physiological processes and disease states,9
and JDTic was examined in a Phase I clinical trial.10 However,
these prototypical KOR-selective antagonists exhibit exception-
ally long KOR antagonist activity, lasting weeks after a single
dose,9 which complicates their use as pharmacological tools and
could impact their therapeutic application.
Our research group has designed and synthesized analogues
of the endogenous kappa opioid peptide dynorphin (Dyn) A as
KOR-selective peptide antagonists.11−13 In early studies on N-
terminal-alkylated analogues of [D-Pro10]Dyn A(1−11), we
found that despite its high affinity for KOR the N-benzyl
analogue displayed lower agonist potency than that of other N-
alkyl derivatives in the guinea pig ileum (GPI) assay14,15 and
that it produced partial agonism in the adenylyl cyclase assay.16
A series of (5,8) cyclic Dyn A(1−13)NH2 analogues with
varying ring sizes synthesized in our laboratory also displayed
Figure 1. Structures of Dyn A(1−11)NH2 and zyklophin (1).
Zyklophin was subsequently shown to also be a selective
KOR antagonist in vivo with a finite duration of KOR
antagonist activity (12−18 h) following systemic administra-
tion.19 Following peripheral (subcutaneous, s.c.) administra-
tion, zyklophin antagonized the antinociceptive activity of the
centrally administered KOR agonist U50,488, suggesting that
this peptide crossed the blood−brain barrier to act on KOR in
the CNS.19 This peptide also suppressed stress-induced
reinstatement of cocaine-seeking behavior in mice following
s.c. administration,19 suggesting its potential as a lead
compound for the development of therapeutics for the
treatment of cocaine addiction.
Received: November 25, 2014
Published: October 22, 2015
© 2015 American Chemical Society
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a
Table 1. Analogues of Zyklophin
a
Changes from zyklophin are shown in bold.
We are exploring the structure−activity relationships (SAR)
of zyklophin in order to examine its potential interactions with
KOR and to enhance its antagonist potency. We initially
prepared linear [N-benzyl-Tyr1]Dyn A(1−11)NH2 analogues 2
and 3 (Table 1) in which positions 5 and 8, respectively, were
modified to assess their individual contributions to the activity
of zyklophin. We expected that the D-amino acid in position 5
adjacent to the “message” sequence20 might affect the
orientation of pharmacophoric groups in the receptor binding
site and therefore could play a significant role in the antagonist
activity of zyklophin, whereas the residue in position 8 was not
expected to affect peptide efficacy. In this initial SAR study of
cyclic analogues, we made three types of modifications: to the
N-terminal group (analogues 4−8, Table 1), amino acid
substitutions in the sequence (analogues 9−14), and to the
cyclic constraint (analogues 15−17). We explored the effect of
different N-terminal alkyl substituents on the receptor affinities
and KOR activity of zyklophin based on the hypothesis that the
N-terminal alkyl group could affect the interactions of the rest
of the peptide with KOR and therefore the ability of zyklophin
to bind to this receptor without activating it. We investigated
the contribution of different residue side chains to the KOR
affinity and antagonist activity of zyklophin by performing an
alanine scan of the non-glycine residues of zyklophin (excluding
residues 5 and 8) up through residue 9 (Table 1). (Previous
studies of linear [N-benzyl-Tyr1]Dyn A(1−11)NH2 analogues
suggested that modifications in the C-terminus do not affect
peptide efficacy.21) In addition, to explore the importance of
the phenolic group of Tyr1 on the activity of zyklophin, we
synthesized the Phe1 analogue of zyklophin (14, Table 1).
Finally, we explored the role of ring size and residues involved
in the cyclic constraint on the KOR interaction of zyklophin.
RESULTS AND DISCUSSION
■
Synthesis. The synthesis of the zyklophin analogues
involved synthesizing the N-terminal N-alkyl amino acid
derivatives in solution and assembly of the peptides on the
solid support. Following solid-phase synthesis of the (2−11)
peptide fragments, the appropriate N-alkyl amino acid
derivatives were coupled to the N-terminus to obtain the
protected (1−11) peptides on resin. The final peptides were
then obtained by cleavage from the resin.
Amino Acid Synthesis. Except for N-allyl-Tyr, the N-alkyl
amino acid derivatives were synthesized by reductive amination
of the amino acid t-butyl ester with the appropriate aldehyde
and sodium triacetoxyborohydride to yield the N-alkyl amino
acid t-butyl ester, which was subsequently cleaved with 90%
TFA and 10% water to yield the N-alkyl amino acid (Scheme
1a). N-Allyl-Tyr (Scheme 1c) was prepared by alkylating Tyr-
Ot-Bu with 1 equiv of allyl bromide in the presence of N,N-
diisopropylethylamine (DIEA) in DMF. Using only 1 equiv of
allyl bromide minimized formation of the diallyl compound; the
desired monoallyl amino acid ester was separated from
unreacted starting material by silica gel flash column
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Scheme 1. Synthesis of Amino Acid Derivatives
chromatography using EtOAc/hexane. The t-butyl ester was
subsequently cleaved with 90% TFA and 10% water.
temperature. This avoided heating and potential racemization
of the amino acids during coupling (see below).
The resulting N-alkyl amino acids, obtained as TFA salts,
exhibited low solubility in DMF at room temperature and
required heating to >85 °C for solubilization (see below).
While microwave couplings are routinely performed at ≥75 °C,
racemization at elevated temperatures could be a potential issue
during coupling of the N-alkyl amino acids to the (2−11)
fragments. Initially, Fmoc protection of N-benzyl-Tyr-Ot-Bu by
treatment with Fmoc-Cl was examined, but after removal of the
t-butyl ester the resulting Fmoc-protected amino acid was not
soluble in DCM or DMF at room temperature. Therefore,
selected N-alkyl amino acid derivatives with low solubility,
namely N-CPM-Tyr-Ot-Bu (CPM = cyclopropylmethyl) and
N-benzyl-Phe-Ot-Bu, were protected with the Alloc (allylox-
ycarbonyl) group by treating with allyl chloroformate and
DIEA in DCM to afford the Alloc-N-alkyl amino acid esters
(Scheme 1b); in the case of N-CPM-Tyr-Ot-Bu the bis-Alloc
derivative was obtained. The t-butyl esters were subsequently
cleaved with 90% TFA/10% DCM to afford the Alloc-protected
amino acids (Scheme 1b), which, unlike the unprotected N-
alkyl amino acids, were readily soluble in DMF at room
Peptide Synthesis. The peptides were synthesized using
the Fmoc solid-phase synthetic strategy. The (2−11) peptide
fragments were assembled on a low-load poly(ethylene glycol)-
polystyrene (PEG-PS) resin containing the peptide amide
linker [PAL; 5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric
acid linker] using benzotriazol-1-yl-oxytripyrrolidino-
phosphonium hexafluorophosphate (PyBOP), 1-hydroxybenzo-
triazole (HOBt) (4 equiv each), and DIEA (10 equiv) in
DCM/DMF (1:1) to couple the Fmoc-amino acids (4 equiv)
to the growing peptide chain using a custom manual multiple
peptide synthesizer.22 The side chain protecting groups used
were 2,2,4,6,7-pentamethyl dihydrobenzofurane-5-sufonyl
(Pbf) for Arg and Boc for Lys.
For linear analogues 2 and 3 the (2−11) peptide precursors
were synthesized in a manner similar to the above procedure
with the side chains of D-Asn in peptide 2 and 2,3-
diaminopropionic acid (Dap) in peptide 3 protected by trityl
(Trt) and 4-methyltrityl (Mtt) groups, respectively. For peptide
3, following the synthesis of the (2−11) linear fragment, the
Mtt protecting group on Dap was selectively deprotected using
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Scheme 2. Solid-Phase Synthesis of Cyclic Peptides 5, 7, 9, and 15−17
3% TFA and 5% triisopropylsilane (TIS) in DCM, and the
resulting amine was acetylated using acetyl imidazole in DMF.
The (2−11) fragments of cyclic peptides 4−9 and 14−17
were synthesized by a similar approach (Scheme 2). The D-Asp
and Dap residues in positions 5 and 8 were protected with the
hyperacid-labile 2-phenylisopropyl (Pip) and Mtt groups,
respectively. Following the synthesis of the (5−11) fragments,
the Pip and Mtt groups were deprotected by 3% TFA and 5%
TIS in DCM. The selective deprotection of the Mtt group on
residue 8 was monitored by acetylation of the resulting free
amine by acetic anhydride or acetyl imidazole, followed by
cleavage of an aliquot of resin and HPLC analysis of the
resulting product. Subsequently, the carboxyl group of D-Asp5
and the amine of Dap8 were cyclized using 6-chloro-
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-
phosphate (PyClocK) and 1-hydroxy-7-azabenzotriazole
(HOAt) (4 equiv each) with DIEA (10 equiv) in a mixture
of DCM/DMF (1:1), generally for 20−24 h. The cyclizations
were monitored using the qualitative ninhydrin test.23 As
expected for syntheses performed on a low-load resin, there was
no evidence for cyclodimerization by mass spectrometry. Any
remaining unreacted free amine of Dap was acetylated by
treatment with acetyl imidazole and DIEA in DMF. Further
extension of the peptide up to Gly2 afforded the (2−11)
fragments of the cyclic peptides. For the synthesis of peptides
15 and 16 Dab(Mtt) (Dab = 2,3-diaminobutyric acid) and
Orn(Mtt) (Orn = ornithine) were incorporated in position 8,
respectively, and for peptide 17 L-Asp(Pip) was incorporated in
position 5. In the case of these three peptides the cyclizations
required longer reaction times (∼36 h) to go to completion.
Coupling of the N-Alkyl Amino Acids. The coupling of
the N-terminal amino acid derivatives to the resin-bound (2−
11) peptide sequences was carried out using PyClocK, HOAt,
and DIEA in DMF. The solutions of the N-alkyl amino acids
required elevated temperatures (80−85 °C for N-benzyl-Tyr
and N-benzyl-Ala and >100 °C for the N-allyl- and N-
phenethyl-Tyr derivatives) to obtain complete dissolution in
DMF. For N-benzyl-Tyr and N-benzyl-Ala the solutions were
cooled to room temperature prior to addition of the coupling
reagents and base, but the solutions of N-allyl- and N-
phenethyl-Tyr became turbid upon cooling; hence, the
coupling agents were added to these solutions while they
were still hot, followed by cooling the solution, addition of the
base, and coupling to the (2−11) fragment. In these cases
where the unprotected N-terminal N-alkyl amino acid was
coupled to the (2−11) peptides, there was no evidence of
incorporation of multiple N-alkyl amino acids (i.e., formation of
12-mer peptides); this is likely due to the steric hindrance of
the bulky secondary amine and the low (2-fold) excess of
amino acid used in these reactions. N-CPM-Tyr did not couple
to the (2−11) fragment, probably due to its minimal solubility
in DMF.
N-CPM-Tyr and N-benzyl-Phe were coupled as their N-
protected derivatives which are soluble in DMF at room
temperature. Following coupling of the unprotected N-benzyl-
Phe to the (2−11) fragment, the crude peptide 14 showed a
second peak in the HPLC (Figure 3a). ESI analysis of the
peptide mixture did not show formation of a higher molecular
weight species (i.e., the 12-mer peptide), suggesting that the
second peak resulted from N-benzyl-Phe undergoing race-
mization during activation of the amino acid or subsequent
coupling to the (2−11) fragment. To avoid potential
epimerization and enhance their solubility, the Alloc-protected
derivative of N-benzyl-Phe, as well as N-CPM-Tyr, was
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prepared as described above. Following coupling of the Alloc
derivatives to the (2−11) peptide fragment on the resin, the
Alloc group was removed by Pd(0) and phenylsilane.24 To
synthesize peptide 4 Fmoc-N-Me-Tyr(t-Bu) was coupled to the
(2−11) peptide fragment, followed by Fmoc deprotection. In
the case of peptide 8 Fmoc-Tyr(t-Bu) was coupled to the (2−
11) peptide fragment by the standard coupling procedure,
followed by Fmoc deprotection and subsequent acylation of the
N-terminal Tyr with benzoic anhydride in the presence of
DIEA in DMF.
Investigation of Potential Racemization of the N-
Terminal Residue of Zyklophin and Analogue 14 by
HPLC. Because dissolution of the N-alkyl amino acids required
elevated temperatures, the peptides were examined by HPLC
for potential epimerization of the N-terminal residue. To verify
that the diastereomeric peptides could be separated by HPLC,
we synthesized [N-benzyl-D-Tyr1]zyklophin. N-Benzyl-D-Tyr
was synthesized as described above (Scheme 1a) and coupled
to the (2−11) fragment to give the N-benzyl-D-Tyr isomer of
zyklophin. Although a reversed-phase HPLC (RP-HPLC)
system using a slow gradient of aqueous acetonitrile containing
TFA (10−25% over 30 min) did not adequately resolve the
diastereomeric mixture of zyklophin and [N-benzyl-D-Tyr1]-
zyklophin (Figure 2a), a solvent system using aqueous 0.09 M
triethylammonium phosphate (TEAP, pH 2.5)25 in acetonitrile
(1−21% over 40 min) did provide baseline resolution of the
diastereomers of zyklophin (Figure 2b), with the D-Tyr
diastereomer eluting before zyklophin. Zyklophin synthesized
by the standard procedure involving solubilization of N-benzyl-
Tyr at 85 °C did not show detectable epimerization when
analyzed using the TEAP solvent system (Figure 2c). This
HPLC solvent system was used to detect potential epimeriza-
tion of the N-terminal residue during the synthesis of other
zyklophin analogues. None of the peptides analyzed using this
system (Table 2) except for peptide 14 showed evidence of
epimerization of the N-terminal residue.
The Phe1 analogue of zyklophin 14, however, showed
evidence of epimerization of the N-terminal residue when
synthesized by the standard procedure in which N-benzyl-Phe
in DMF was dissolved at 75−80 °C prior to coupling to the
(2−11) peptide fragment. Analysis of the crude peptide in the
TEAP solvent system (Figure 3a) showed a mixture of two
peaks, peak A (tR = 18.3 min, 17.5%) and peak B (tR = 20.3
min, 82.5%), for the peptide synthesized by this standard
method. However, when synthesized using Alloc-N-benzyl-Phe,
peptide 14 did not show detectable epimerization of the N-
terminal residue; only peak B (tR = 20.3 min) was observed in
the chromatogram (Figure 3b).
Figure 2. Separation of a diastereomeric mixture containing [N-
benzyl-D-Tyr1]zyklophin and zyklophin using (a) a linear gradient of
aqueous acetonitrile containing 0.1% TFA (solvent A = aqueous 0.1%
TFA and solvent B = acetonitrile containing 0.1% TFA; 10−25%
solvent B over 30 min), and (b) a linear gradient of TEAP (solvent A
= aqueous 0.09 M TEAP, pH 2.5, and solvent B = acetonitrile; 1−21%
solvent B over 40 min). For the D-Tyr diastereomer of zyklophin tR =
17.3 min, and for zyklophin tR = 18.2 min. (c) Zyklophin (tR = 18.3
min) synthesized by the standard procedure and analyzed using the
TEAP solvent system showed no detectable epimerization.
In order to assess the individual contributions of the residues
in positions 5 and 8 of zyklophin, linear peptides 2 and 3 were
synthesized. Previously, we found that the linear zyklophin
analogue [N-benzyl-Tyr1,D-Asn5,Dap(Ac)8]Dyn A(1−11)NH2
with substitutions in both positions exhibited KOR affinity 2-
fold lower (Ki = 66.9 nM) than zyklophin and 8-fold lower than
Dyn A(1−11)NH2.18,21 When the peptides with a single
modification were evaluated we found that peptide 2 had 4-fold
lower KOR affinity (Ki = 412 nM) compared to zyklophin (Ki
= 95.2 nM), whereas peptide 3 exhibited 3-fold higher KOR
affinity (Ki = 28 nM). These results suggest that the residue in
position 5 had a much larger influence on the KOR affinity of
zyklophin than the amino acid in position 8. While zyklophin
has a D-Asp involved in the lactam linkage at position 5, peptide
3 has L-Leu in this position, the same as found in the
The peptides were purified by preparative RP-HPLC and
analyzed by ESI-MS and analytical RP-HPLC. The final purity
of all of the peptides by all methods was ≥98% (Table 2).
Pharmacological Results. The peptides were evaluated for
receptor affinities in radioligand binding assays using Chinese
hamster ovary (CHO) cells stably expressing opioid receptors
(Table 3). Zyklophin showed somewhat lower KOR affinity in
the radioligand binding assay than that obtained originally (Ki =
30.3 nM).18 Similarly, the KOR binding affinity of Dyn A(1−
11)NH2 (Ki = 2.6 0.3 nM) was also lower than that found
previously (Ki = 0.57
0.01 nM),18 possibly due to subtle
differences in assay conditions (e.g., differences in the
radioligand, filtration rate, etc.). The MOR affinity found here
for zyklophin was similar to that reported previously.18
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suggesting that Tyr in position 1, while making some
contribution to its affinity, is not critical for zyklophin’s binding
to KOR. Alanine-substituted analogues 10 (Ala4) and 11 (Ala6)
showed the largest decreases, 42- and 18-fold (Ki = 4010 and
1750 nM), respectively, in KOR affinity compared to that of
zyklophin, indicating that the Phe4 and the Arg in position 6 are
critical for maintaining the KOR affinity of zyklophin.
Interestingly, peptide 12 (Ala7) showed only a 3-fold decrease
in KOR affinity (Ki = 293 nM), a much smaller decrease than
seen for substitution of Arg6, suggesting that Arg7 makes only a
minor contribution to the KOR affinity of zyklophin. Analogue
13 (Ala9) displayed KOR affinity (Ki = 168 nM) within 2-fold
of zyklophin, indicating that this positively charged residue is
not important for the KOR affinity of zyklophin. Analogues 9−
13 all displayed low affinity for MOR (Ki ≥ 2 μM) that was
similar to zyklophin.
The alanine scan of Dyn A(1−13) performed previously26
revealed that within the Leu-enkephalin core of the peptide
Ala1 and Ala4 substitutions caused dramatic decreases in opioid
receptor affinities, indicating that Tyr1 and Phe4 residues are
critical for opioid receptor binding affinity. Outside the Leu-
enkephalin core of Dyn A(1−13) the Ala6 and Ala7 analogues
showed the largest decreases (4- to 7-fold) in binding affinities,
suggesting that Arg6 and Arg7 contribute to the opioid receptor
affinity of Dyn A(1−13). Ala9 and Ala11 analogues showed
relatively small (3-fold) decreases in opioid receptor binding
affinity, suggesting that these residues make minor contribu-
tions to the opioid receptor affinity of Dyn A(1−13).26
Figure 3. Chromatograms of peptide 14 synthesized by (a) the
standard procedure and (b) using Alloc-N-benzyl-Phe analyzed with
the TEAP solvent system.
Comparison of the alanine scan results for Dyn A(1−13) and
zyklophin reveals significant differences in the contributions of
some of the residues to the opioid receptor affinities of these
two peptides. Although Tyr1 was critical in maintaining the
opioid receptor affinity of Dyn A(1−13), it appeared to make a
relatively minor contribution to the KOR binding affinity of
zyklophin. Phe4, however, was critical for both the opioid
receptor affinity of Dyn A(1−13) and the KOR affinity of
zyklophin. Arg6 contributed to both the opioid receptor affinity
of Dyn A(1−13) and the KOR affinity of zyklophin, although
the impact of substituting Ala for Arg6 in zyklophin appeared to
be greater (18-fold decrease) than the substitution of the same
amino acid in Dyn A(1−13) (7-fold decrease). Arg7 appeared
to make a minor contribution to both the opioid receptor
affinity of Dyn A(1−13) and the KOR affinity of zyklophin,
whereas Arg9 did not appear to contribute significantly to the
binding affinities of either peptide. The differences in the results
for the alanine scan analogues of the two peptides suggest
differences in the binding interactions with opioid receptors.
Some differences in receptor interactions would be expected
since Dyn A(1−13) is a KOR agonist while zyklophin is a KOR
antagonist. Differences in the radioligand binding assays used to
evaluate the Ala-substituted analogues of the two peptides,
however, limit the comparisons that can be made. The Dyn
A(1−13) analogues were evaluated in radioligand binding
assays performed in rat brain homogenates, which contain
MOR and delta opioid receptor (DOR) but relatively low levels
of KOR,27 using the nonselective radioligand [3H]etorphine, so
that the affinities of the analogues for individual opioid
receptors cannot be assessed from the reported data.
endogenous Dyn A. Hence, the side chain of the residue at
position 5 and/or changes in the backbone conformation of the
peptide due to the different configuration at residue 5
influenced the KOR affinity of the zyklophin analogues.
While peptide 2 had low MOR affinity (Ki = 2440 nM),
peptide 3 had 28-fold higher MOR affinity (Ki = 157 nM) than
zyklophin (Ki = 4380 nM), suggesting that residue 5 is
important for the low MOR affinity of zyklophin. Both linear
analogues 2 and 3 displayed similar selectivities for KOR vs
MOR that were lower than that of zyklophin.
Sterically diverse alkyl groups ranging from methyl to
phenethyl were incorporated at the N-terminus to examine
their effect on the affinity, efficacy, and potency of the
zyklophin analogues. Peptides 4, 6, and 7 had similar to slightly
higher KOR affinities (Ki = 44−160 nM) compared to
zyklophin, suggesting that these N-alkyl modifications are
tolerated in the KOR binding site. The N-allyl analogue 5 had
the lowest KOR affinity (Ki = 326 nM) among the N-alkylated
analogues examined. The N-benzoyl derivative 8 showed only
somewhat lower (3.6-fold) KOR affinity (Ki = 346 nM) than
that of zyklophin, indicating that a basic secondary amine at the
N-terminus is not essential for maintaining the KOR affinity of
zyklophin analogues. All of these analogues displayed low MOR
affinity (Ki > 1 μM) similar to zyklophin (only the N-methyl
derivative 4 displayed increased MOR affinity (Ki = 1490 nM)).
The increased KOR affinity of analogues 6 and 7 resulted in
increases in their KOR vs MOR selectivities (Ki ratios (MOR/
KOR) = 245 and 137, respectively).
An alanine scan of zyklophin of the nonglycine residues up
through position 9, excluding the residues in positions 5 and 8
involved in the lactam, was also performed to identify residues
important for the KOR affinity of zyklophin. Unexpectedly,
Ala1 analogue 9 displayed only a 5-fold decrease in the KOR
affinity (Ki = 516 nM) compared to that of zyklophin,
The N-benzyl-Phe1 analogue 14 was also synthesized and
displayed similar KOR affinity (Ki = 69 nM) to that of
zyklophin, verifying that the phenol of the N-terminal Tyr1
does not contribute to the affinity of zyklophin for KOR. This
is in contrast to Dyn A(1−11)NH2 where substitution of Tyr1
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Table 2. Analytical Data for the Peptides
HPLC tR (min)
ESI-MS (m/z)
a
b
c
peptides
system 1 (% purity)
system 2 (% purity)
system 3 (% purity)
calculated
observed
2
15.4 (>99.5)
17.7 (99.4)
11.3 (>99.5)
12.1 (>99.5)
13.1 (>99.5)
18.0 (>99.5)
23.1 (>99.5)
13.1 (99.4)
14.2 (>99.5)
14.4 (99.0)
[M + 3H]3+ = 484.9
[M + 2H]2+ = 726.9
[M + 3H]3+ = 489.6
[M + 2H]2+ = 733.9
[M + 3H]3+ = 444.9
[M + 2H]2+ = 666.8
[M + 3H]3+ = 453.5
[M + 2H]2+ = 679.8
[M + 3H]3+ = 458.2
[M + 2H]2+ = 686.8
[M + 3H]3+ = 474.9
[M + 3H]3+ = 474.9
[M + 2H]2+ = 711.8
[M + 3H]3+ = 439.5
[M + 3H]3+ = 444.9
[M + 2H]2+ = 666.8
[M + 3H]3+ = 441.9
[M + 2H]2+ = 662.3
[M + 3H]3+ = 441.9
[M + 2H]2+ = 662.3
[M + 3H]3+ = 441.9
[M + 2H]2+ = 662.3
[M + 3H]3+ = 464.9
[M + 3H]3+ = 474.9
[M + 2H]2+ = 711.9
[M + 3H]3+ = 479.6
[M + 2H]2+ = 718.9
[M + 3H]3+ = 470.2
[M + 3H]3+ = 484.9
[M + 2H]2+ = 726.9
[M + 3H]3+ = 489.6
[M + 2H]2+ = 733.9
[M + 3H]3+ = 444.9
[M + 2H]2+ = 666.9
[M + 3H]3+ = 453.6
[M + 2H]2+ = 679.9
[M + 3H]3+ = 458.2
[M + 2H]2+ = 686.8
[M + 3H]3+ = 474.9
[M + 3H]3+ = 474.9
[M + 2H]2+ = 711.9
[M + 3H]3+ = 439.6
[M + 3H]3+ = 444.9
[M + 2H]2+ = 666.9
[M + 3H]3+ = 441.9
[M + 2H]2+ = 662.4
[M + 3H]3+ = 441.9
[M + 2H]2+ = 662.4
[M + 3H]3+ = 441.9
[M + 2H]2+ = 662.3
[M + 3H]3+ = 464.9
[M + 3H]3+ = 474.9
[M + 2H]2+ = 711.9
[M + 3H]3+ = 479.6
[M + 2H]2+ = 718.9
[M + 3H]3+ = 470.2
[M + 2H]2+ = 704.8
3
4
5
6
11.9 (>99.5)
13.5 (>99.5)
21.5 (98.9)
7
8
17.0 (>99.5)
18.1 (>99.5)
15.3 (>99.5)
23.3 (>99.5)
9
13.3 (>99.5)
10.1 (>99.5)
13.3 (>99.5)
8.0 (>99.5)
11.6 (>99.5)
10
11
12
13
15.1 (>99.5)
14.8 (99.0)
14.8 (>99.5)
18.5 (>99.5)
15.2 (98.9)
14.6 (>99.5)
14
15
17.0 (98.1)
15.6 (>99.5)
18.5 (>99.5)
20.1 (98.7)
15.3 (>99.5)
16
17
15.1 (>99.5)
13.2 (99.1)
18.1 (>99.5)
15.2 (99.0)
[M + 2H]2+ = 704.8
a
b
c
Aqueous acetonitrile containing 0.1% TFA; λ = 214 nm. Aqueous methanol containing 0.1% TFA; λ = 220 nm. Aqueous acetonitrile containing
0.09 M TEAP; λ = 214 nm. See the Experimental Section for details.
by Phe resulted in a 23-fold decrease in KOR affinity,28
demonstrating that the phenol of Tyr is critical for the KOR
affinity of this agonist. Peptide 14 also exhibited 3.5-fold higher
selectivity for KOR over MOR compared to that of zyklophin.
Changes in the lactam ring of zyklophin had a minor effect
on KOR and MOR affinities. Peptides 15 and 16 with larger
ring sizes than zyklophin had KOR affinities 2- to 4-fold lower
(Ki = 403 and 222 nM, respectively) than zyklophin. Peptide 17
containing L-Asp in position 5 also showed 4-fold lower KOR
affinity (Ki = 410 nM); thus, a change in the configuration of
this amino acid was reasonably well tolerated by KOR without a
large loss in affinity. Similar to zyklophin, analogues 15−17 all
displayed low (micromolar) affinity for MOR. However, the
KOR vs MOR selectivity varied depending on the analogue,
with analogues 15 and 17 exhibiting lower (6- to 8-fold) and
analogue 16 similar (37-fold) selectivities compared to that of
zyklophin (46-fold in these assays).
Only linear peptide 3 exhibited any appreciable affinity for
DOR (Ki = 2290 520 nM). All of the remaining peptides
exhibited minimal DOR affinity.
The efficacies of the zyklophin analogues were evaluated in
the agonist-stimulated GTPγS binding assay. All of the cyclic
peptides displayed negligible efficacy (<15%) when screened at
10 μM in this assay. Our hypothesis was that the N-terminal
alkyl group might shift the peptide in the KOR binding site,
thereby changing interactions of other parts of the peptide with
the receptor and preventing the peptide from activating the
receptor. Among the N-alkyl analogues, peptide 4 with an N-
methyl substitution was expected to cause minimal shift of the
peptide in its binding site and hence exhibit agonist activity, but
this was not the case. The negligible efficacy of peptides 4−8
and 15−17 in the GTPγS assay suggest that changes in the N-
terminal alkyl group, ring size, or residue 5 configuration did
not increase efficacy compared to that of zyklophin. As
expected, none of the alanine-substituted analogues exhibited
appreciable efficacy in this assay, nor did the Phe1 analogue 14.
Only linear peptide 3 exhibited partial agonist activity at KOR
in the GTPγS assay (26% efficacy compared to the full agonist
Dyn A(1−13)NH2, EC50 = 39.5 17.4 nM); these results are
consistent with those found for [N-benzyl-Tyr1]Dyn A(1−
11)NH2 in the adenylyl cyclase assay.21
The KOR antagonist potency of selected analogues was
analyzed in the GTPγS assay by Schild analysis against Dyn
A(1−13)NH2 as the KOR agonist29 (Figure 4 and Table 4).
The potency of zyklophin as a KOR antagonist in the GTPγS
assay was consistent with its KOR affinity, but it was somewhat
lower than that found previously in the adenylyl cyclase assay
(KB = 84 nM).21 The N-CPM peptide 6 showed potent
antagonism in this assay, 28-fold higher than zyklophin, and the
N-phenethyl peptide 7 exhibited reasonable antagonist potency.
The Phe1 peptide 14 also exhibited potent KOR antagonism,
consistent with its binding affinity, indicating that the Tyr
phenol is not required for the KOR antagonist potency of
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Table 3. KOR and MOR Affinities and Selectivities of
Zyklophin Analogues
Table 4. KOR Antagonist Potencies of Selected Zyklophin
Analogues in the Dyn A(1−13)NH2-Stimulated GTPγS
Assay
relative
ab
,
KOR Ki
(nM SEM)
KOR
MOR Ki
(nM SEM)
K
icd
,
peptide
KB (95% confidence interval), nM
a
b
a
peptide
2, D-Asn5
affinity
ratio
6, N-CPM
7, N-phenethyl
14, Phe1
12.1 (0.8−61)
412 60
0.23
3.3
2440 570
157 25
6
92.5 (9.0−6110)
29.7 (0.5−1320)
336 (180−535)
3, Dap(Ac)8
28.6 0.6
160 18
5
4, N-Me
0.60
0.29
2.1
1490 410
9
1, zyklophin
5, N-Allyl
6, N-CPM
7, N-phenethyl
8, N-benzoyl
9, Ala1
326 72
11 000 4000
10 900 2700
8470 560
10 600 1700
>10 000
34
245
137
31
>19
>2.5
7
a
KB values derived from Schild analysis in the GTPγS assay. Values for
44.4 16.3
62.0 19.9
346 21
the peptides are the average from 2 to 5 independent experiments. For
1.5
b
reference, the norBNI KB = 0.074 nM. Peptide 12 did not cause a
0.28
0.18
0.02
0.05
0.32
0.57
1.4
significant shift in the Dyn A(1−13)NH2 concentration−response
curve at concentrations up to 10 μM.
516 82
10, Ala4
4010 360
1750 540
293 118
168 50
>10 000 (2)
11 500 2600
>10 000
11, Ala6
12, Ala7
>34
15
164
8
was potential for epimerization of the N-terminal residue
because solutions of the N-alkyl amino acid required heating to
obtain complete dissolution. An HPLC solvent system that
could resolve the diastereomers of zyklophin was identified and
used to examine the peptides for potential racemization of the
N-terminal residue. Only in the case of N-benzyl-Phe was
evidence of epimerization detected. These results are not
surprising because elevated temperatures (typically 75 °C) are
routinely used in microwave-assisted amino acid couplings in
peptide synthesis and because phenylalanine is well known to
be prone to racemization. To avoid potential epimerization of
N-benzyl-Phe and to increase the solubility of N-CPM-Tyr the
Alloc derivatives, which were readily soluble at room temper-
ature, were prepared; this eliminated the epimerization of N-
benzyl-Phe and increased the yield of the N-CPM-Tyr1 peptide
6.
13, Ala9
2550 640
11 300 1980
3200 850
8260 1870
2610 780
4380 560
14, Phe1
68.7 18.1
403 91
15, Dab8
16, Orn8
0.24
0.42
0.23
1.0
222
5
37
6
17, L-Asp5
410 17
e
1, zyklophin
95.2 28.8
46
a
Ki values for KOR and MOR were obtained from n ≥ 3 independent
b
experiments except where noted in parentheses. Compared to
c
d
zyklophin (1.0). Ki (MOR)/Ki (KOR). The analogues exhibited
minimal DOR affinity (<20% inhibition of binding when screened
against DOR at 10 μM, Ki > 5 μM) except for 3 (Ki = 2290 520
e
nM). 95% confidence interval 30−160 nM (n = 10).
zyklophin. The Ala7 analogue 12 did not antagonize KOR,
consistent with its lower KOR affinity.
The in vitro pharmacological evaluation of the linear and the
cyclic zyklophin analogues provided initial SAR for the lead
peptide zyklophin. The receptor affinities of linear analogues 2
and 3 suggested that the residue at position 5 has a greater
influence on the opioid receptor affinities of zyklophin than
CONCLUSIONS
■
Various linear and cyclic analogues of zyklophin were
synthesized in order to examine the SAR of this peptide for
KOR interaction and antagonism. During the synthesis there
Figure 4. Schild regression analysis of the ability of zyklophin (1) and peptides 6, 7, and 14 to produce rightward shifts in the Dyn A(1−13)NH2
concentration−response relationships for stimulation of [35S]GTPγS binding. Data depicted were pooled from 2 to 5 experiments for each peptide.
The 95% confidence intervals for the KB values are listed in Table 4.
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Pierce (Rockford, IL). Benzaldehyde and sodium cyanoborohydride
were from Sigma-Aldrich; phenylacetaldehyde, allyl bromide, allyl
chloroformate, and TIS were from Acros Organics (Fairlawn, NJ).
Amino Acid Synthesis. General Procedure for N-Alkyl-Tyr-OH
Synthesis. The amino acid t-butyl ester (1 equiv) and NaBH(OAc)3
(1 equiv) were dissolved in anhydrous acetonitrile (SureSeal, 5−10
mL/mmol), followed by the addition of the corresponding aldehyde
(1.5 equiv) and a catalytic amount of glacial acetic acid (0.2 mL). The
reaction was performed under N2 for 12−18 h at room temperature
with continuous stirring. The reaction was monitored by TLC (1:2
EtOAc/hexane plus 1 drop of NEt3). After completion of the reaction
the acetonitrile was evaporated in vacuo, the pH was adjusted to 7.0
with saturated NaHCO3, and the aqueous layer was extracted with
EtOAc (3 × 10 mL). The combined EtOAc extracts were washed with
water, and the organic layer was dried (MgSO4) and evaporated under
reduced pressure to obtain crude N-alkyl-Tyr-Ot-Bu that was purified
by flash silica gel column chromatography (20−30% EtOAc in
hexane). The N-alkyl-Tyr-Ot-Bu was analyzed for purity by HPLC
using a gradient of 5−50% aqueous acetonitrile with 0.1% TFA over
45 min at a flow rate of 1 mL/min. The t-butyl ester on the purified N-
alkyl-Tyr-Ot-Bu was cleaved by 90% TFA plus 10% water (8−10 mL/
mmol) for 12 h. The reaction mixture was concentrated in vacuo, and
the desired product was obtained by precipitation with diethyl ether.
N-Benzyl-Tyr-OH (23a). Tyr-Ot-Bu (0.474 g, 2.0 mmol, 1 equiv)
was reacted with benzaldehyde and NaBH(OAc)3 in anhydrous
acetonitrile (12 mL) for 18 h. The crude product was isolated as
described above, and pure N-benzyl-Tyr-Ot-Bu (520 mg, 80%) was
obtained following flash silica gel column chromatography (25%
EtOAc/hexane) as an off-white amorphous solid: ESI-MS m/z [M +
H]+ 328.1913 (calcd), 328.1784 (observed); HPLC: tR 24.3 min
does residue 8. The L-configuration and/or the hydrophobic
side chain of Leu in position 5 of peptide 3 likely contributes to
the higher KOR and MOR affinities of this peptide. However,
the KOR affinity of cyclic analogue 17 with an L-Asp at position
5 suggested that the configuration of residue 5 has a minor
influence on the KOR affinity of cyclic zyklophin analogues.
The N-phenethyl and N-CPM substitutions resulted in a
slightly higher (1.5- to 2-fold) affinity for KOR than zyklophin,
whereas the other N-terminal modifications and ring variations
were also well tolerated. The basic amine at the N-terminus of
zyklophin is not necessary for maintaining the KOR affinity of
this peptide, as shown by the relatively modest reduction in
KOR affinity of the N-benzoyl analogue 8 compared to that of
zyklophin. These results are consistent with other peptide KOR
antagonists based on dynorphin identified in our labora-
tory.11−13 The pharmacological results for alanine-substituted
analogues indicate that Phe4 and Arg6 are critical for zyklophin’s
KOR affinity. Interestingly, the Tyr1 residue makes a relatively
minor contribution to the KOR affinity of zyklophin based on
the results for the N-benzyl-Ala1 analogue 9, and the similar
KOR affinity of the N-benzyl-Phe1 analogue 14 to that of
zyklophin indicates that the phenol of Tyr does not contribute
to the KOR affinity of zyklophin. Although the loss of KOR
affinity with Ala substitution of Phe4 is similar to that found for
Dyn A(1−13),26 the minimal effect of substitution by Ala or
Phe in position 1 is in marked contrast to the effects in Dyn
A(1−13) and Dyn A(1−11)NH2.26,28 Substitution of Ala at
postion 6 markedly decreased KOR affinity, whereas replace-
ment of residue 7 had a much smaller effect, indicating that a
basic residue in position 6 is much more important for the
KOR affinity of zyklophin than is one in position 7. As
expected, the Ala9 analogue displayed affinity comparable to
that of zyklophin, indicating that a basic residue at position 9 is
not important for maintaining the KOR affinity of zyklophin.
The results from the agonist-stimulated GTPγS assays
indicate that all of the zyklophin analogues synthesized in the
present study, except for the linear peptide 3, exhibited
negligible efficacy (<15% compared to that of full agonist Dyn
A(1−13)NH2) similar to zyklophin. Schild analysis of the
analogues with the highest KOR affinities found that the N-
phenethyl-Tyr1, N-CPM-Tyr1, and N-benzyl-Phe1 analogues
were more potent KOR antagonists than zyklophin in this
assay. These analogues appear to be promising compounds for
additional studies toward the potential development of potent
peptide KOR antagonists. Further studies of selected zyklophin
analogues are underway in our laboratory.
1
(purity >99.5%); H NMR (500 MHz, acetone-d6) δ 8.14 (s, 1H),
7.35−7.15 (m, 5H), 7.04 (d, J = 8.2 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H),
3.82 (d, J = 13.3 Hz, 1H), 3.64 (d, J = 13.3 Hz, 1H), 3.30 (t, J = 6.8
Hz, 1H), 2.88−2.71 (m, 2H), 1.38 (s, 9H); 13C NMR (126 MHz,
CDCl3) δ 171.90, 153.14, 136.83, 128.38, 126.42, 126.38, 125.96,
125.20, 113.40, 79.60, 60.52, 49.84, 36.56, 25.98.
N-Benzyl-Tyr-Ot-Bu (200 mg) was cleaved to the corresponding
acid using TFA as described above to yield N-benzyl-Tyr-OH (160
mg, 68%) as a white amorphous powder: ESI-MS m/z [M + H]+
272.1287 (calcd), 272.1227 (observed).
N-Benzyl-D-Tyr-OH (23b). D-Tyr-Ot-Bu (0.118 g, 0.5 mmol, 1
equiv) and NaBH(OAc)3 in anhydrous acetonitrile (5 mL) were
reacted with benzaldehyde for 18 h. Pure N-benzyl-D-Tyr-Ot-Bu (90
mg, 55%) was obtained following flash silica gel column chromatog-
raphy (20% EtOAc/hexane) as an off-white amorphous solid: ESI-MS
m/z [M + H]+ 328.1913 (calcd), 328.1890 (observed); 1H NMR (500
MHz, CDCl3) δ 7.35−7.17 (m, 5H), 6.97 (d, J = 8.4 Hz, 2H), 6.62 (d,
J = 8.5 Hz, 2H), 3.80 (d, J = 12.8 Hz, 1H), 3.66 (d, J = 12.8 Hz, 1H),
3.42 (t, J = 6.9 Hz, 1H), 2.95−2.77 (m, 2H), 1.39 (s, 1H); 13C NMR
(126 MHz, CDCl3) δ 172.36, 153.37, 137.44, 128.83, 126.84, 126.74,
126.67, 125.59, 113.75, 79.98, 60.98, 50.29, 37.03, 26.43.
N-Benzyl-D-Tyr-Ot-Bu (90 mg) was cleaved with TFA as described
above to yield N-benzyl-D-Tyr-OH (51 mg, 49%) as a white
amorphous powder: ESI-MS m/z [M + H]+ 272.1287 (calcd),
272.1251 (observed).
EXPERIMENTAL SECTION
■
Materials. Fmoc-protected PAL-PEG-PS resin was purchased from
Applied Biosystems (Foster City, CA). Standard Fmoc-protected
amino acids were obtained from EMD Biosciences (Gibbstown, NJ)
and Peptides International (Louisville, KY), Fmoc-D-Asp(Pip)-OH
was obtained from Bachem (King of Prussia, PA), and Fmoc-
Dap(Mtt)OH, Fmoc-Dab(Mtt)-OH, and Fmoc-Orn(Mtt)-OH were
obtained from EMD Biosciences. Fmoc-N-Me-Tyr(t-Bu)-OH was
obtained from APPTec (Louisville, KY), and Tyr-Ot-Bu was obtained
from EMD Biosciences. PyBOP and PyClocK were purchased from
EMD Biosciences. HOBt was purchased from Peptides International,
and DIEA was from Fisher Scientific (Fair Lawn, NJ). Piperidine,
THF, and anhydrous acetonitrile (SureSeal) were purchased from
Sigma-Aldrich (St. Louis, MO). All HPLC-grade solvents (acetonitrile,
DMF, DCM, EtOAc, hexane, and methanol) used for amino acid and
peptide synthesis or HPLC analysis were obtained from Fisher
Scientific. TFA for HPLC purification and analysis was purchased from
N-Phenethyl-Tyr-OH (24). Treatment of Tyr-Ot-Bu (0.474 g, 2.0
mmol, 1 equiv) with NaBH(OAc)3 and phenylacetaldehyde in
anhydrous acetonitrile (10 mL) for 16 h yielded pure N-phenethyl-
Tyr-Ot-Bu (452 mg, 66%) as an off-white oil following flash silica gel
column chromatography (25% EtOAc/hexane): ESI-MS m/z [M +
H]+ 342.2069 (calcd), 342.2004 (observed); HPLC: tR 27.9 min
1
(purity >99.5%); H NMR (500 MHz, acetone-d6) δ 8.13 (s, 1H),
7.32−7.11 (m, 5H), 7.04 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 8.5 Hz, 2H),
3.31 (dd, J = 6.4 and 7.6 Hz, 1H), 2.83−2.68 (m, 6H), 1.34 (s, 9H);
13C NMR (126 MHz, acetone-d6) δ 174.49, 156.89, 141.43, 131.36,
129.64, 129.18, 126.82, 115.77, 80.84, 64.77, 50.22, 39.67, 37.42,
28.25.
N-Phenethyl-Tyr-Ot-Bu (165 mg) was cleaved to afford the
corresponding acid 24 (125 mg, 65%) as a yellowish-white solid:
ESI-MS m/z [M + H]+ 286.1443 (calcd), 286.1432 (observed).
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N-Benzyl-Ala-OH (25). Ala-Ot-Bu·HCl (0.43 g, 2.4 mmol, 1.2
equiv) and benzaldehyde (0.2 mL, 2.0 mmol, 1 equiv) were dissolved
in CHCl3 (10 mL), followed by addition of a solution of NaBH(OAc)3
(0.63 g, 3 mmol, 1.5 equiv) in CHCl3 (5 mL). The reaction mixture
was stirred at rt for 12 h, water (20 mL) was added, and the aqueous
layer was separated and extracted with additional CHCl3 (2 × 20 mL).
The combined CHCl3 layers were washed with water (20 mL) and
dried over MgSO4, and the solvent was evaporated to obtain the
product, which was purified by flash silica gel column chromatography
(15% EtOAc/hexane) to give N-benzyl-Ala-Ot-Bu (220 mg, 39%) as a
white semisolid: ESI-MS m/z [M + H]+ 236.1651 (calcd), 236.1628
(observed); HPLC: tR 13.1 min (purity 95.8%); 1H NMR (500 MHz,
CDCl3) δ 7.39−7.23 (m, 5H), 3.82 (d, J = 12.7 Hz, 1H), 3.68 (d, J =
12.7 Hz, 1H), 3.27 (q, J = 7.0 Hz, 1H), 1.86 (s, 1H), 1.51 (s, 9H), 1.30
(d, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 175.37, 140.14,
128.59, 128.45, 127.19, 81.07, 56.86, 52.15, 28.29, 19.35.
J = 13.0 Hz, 1H), 3.73 (d, J = 13.0 Hz, 1H), 3.51 (dd, J = 7.6 and 6.6
Hz, 1H), 3.00 (dd, J = 13.6 and 6.5 Hz, 1H), 2.91 (dd, J = 13.6 and 7.7
Hz, 1H), 1.35 (s, 9H); 13C NMR (126 MHz, CDCl3) δ 172.82,
138.19, 136.99, 129.47, 128.53, 128.49, 128.31, 127.45, 126.70, 81.65,
62.23, 51.65, 39.21, 27.97.
Alloc-N-benzyl-Phe-OH (32). N-Benzyl-Phe-Ot-Bu (0.25 g, 0.8
mmol, 1 equiv) in anhydrous DCM (2 mL) was treated with allyl
chloroformate (0.169 mL, 1.6 mmol, 2 equiv) and DIEA (0.278 mL,
1.6 mmol, 2 equiv) for 12 h. Alloc-N-benzyl-Phe-Ot-Bu (208 mg,
65%) was isolated as a yellowish semisolid as described above for
Alloc-N-CPM-Tyr(Alloc)-Ot-Bu: ESI-MS (m/z) [M + Na]+ 418.1994
(calcd), 418.2047; HPLC: tR 18.7 min (25−95% aqueous acetonitrile
1
with 0.1% TFA over 35 min, purity 96.5%); H NMR (500 MHz,
CDCl3, 50 °C) δ 7.32−7.21 (m, 6H), 7.21−7.05 (m, 4H), 6.05−5.89
(m, 1H), 5.28 (m, 2H), 4.82−4.52 (m, 3H), 4.16 (m, 1H), 3.99−3.82
(m, 1H), 3.32 (dd, J = 14.0 and 5.8 Hz, 1H), 3.16 (dd, J = 14.0 and 5.8
Hz, 1H), 1.39 (s, 9H); 13C NMR (126 MHz, CDCl3) δ 169.47,
156.03, 138.33, 137.51, 132.81, 129.21, 128.33, 128.13, 127.07, 126.36,
117.50, 81.49, 66.26, 62.40, 51.77, 36.24, 27.86.
N-Benzyl-Ala-Ot-Bu (120 mg) was cleaved to afford the
corresponding acid as a yellowish white solid (75 mg, 50%): ESI-
MS m/z [M + H]+ 180.1025 (calcd), 180.1009 (observed).
N-CPM-Tyr-Ot-Bu (27). Tyr-Ot-Bu (0.237 g, 1.0 mmol, 1 equiv)
was reacted with cyclopropylcarboxaldehyde and NaBH(OAc)3 in
anhydrous acetonitrile (7 mL) for 12 h and purified by flash silica gel
column chromatography (30% EtOAc/hexane) to give N-CPM-Tyr-
Ot-Bu (160 mg, 55%) as a white powder: ESI-MS m/z [M + H]+
292.1913 (calcd), 292.1819 (observed); HPLC: tR 19.1 min (purity
The ester (120 mg) was cleaved with 90% TFA/10% DCM for 12 h
at rt and isolated as described above for 31 to yield acid 32 (100 mg,
98%) as a yellowish white semisolid: ESI-MS m/z [M + Na]+
362.1368 (calcd), 362.1347 (observed); HPLC: tR 12.1 min (25−
95% aqueous acetonitrile with 0.1% TFA over 35 min, purity >99.5%).
N-Allyl-Tyr-OH (34). Tyr-Ot-Bu (0.237 g, 1.0 mmol, 1 equiv) in
DMF (2 mL) was cooled to 0−4 °C, and DIEA (0.174 mL, 1.0 mmol,
1 equiv) was added, followed by slow addition of allyl bromide (0.086
mL, 1 mmol, 1 equiv). The progress of the reaction was monitored by
TLC (1:3 EtOAc/hexane plus 2 drops of NEt3). After 45 h at rt under
N2 a negligible amount of diallyl product was visible on TLC, although
a substantial amount of residual starting material was present. The
DMF was then removed in vacuo, and the crude product was purified
by flash silica gel column chromatography (22% EtOAc/hexane) to
give N-allyl-Tyr-Ot-Bu (80 mg, 29%) as a white solid powder: ESI-MS
(m/z) [M − t-Bu]+ 222.1130 (calcd), 222.1083 (observed); HPLC: tR
15.9 min (purity >99.5%); 1H NMR (500 MHz, acetone-d6) δ 8.19 (s,
1H), 7.05 (d, J = 8.1 Hz, 2H), 6.75 (d, J = 8.3 Hz, 2H), 5.82 (m, 1H),
5.15 (dd, J = 17.2 and 1.3 Hz, 1H), 5.01 (d, J = 10.2 Hz, 1H), 3.31 (t, J
= 6.9 Hz, 1H), 3.27 (dd, J = 14.2 and 5.9 Hz, 1H), 3.12 (dd, J = 14.2
and 5.9 Hz, 1H), 2.76 (dd, J = 13.5 and 6.9 Hz, 2H), 2.81 (dd, J = 13.5
and 6.9 Hz, 2H), 13C NMR (126 MHz, acetone-d6) δ 174.52, 156.89,
138.10, 131.35, 129.53, 115.85, 115.76, 80.94, 63.75, 51.09, 39.67,
28.26.
Pure N-allyl-Tyr-Ot-Bu (80 mg) was deprotected as described
above to obtain N-allyl-Tyr-OH (43 mg, 44%) as a white powder: ESI-
MS (m/z) [M + H]+ 222.1130 (calcd), 222.1060 (observed).
Peptide Synthesis. General Procedures for Solid-Phase Peptide
Synthesis. Synthesis of (2−11) Peptide Fragments. The peptides
were assembled on low-load Fmoc-PAL-PEG-PS resin (0.19 mmol/g).
Following removal of the Fmoc group from the resin (200 mg, 0.038
mmol) using 20% piperidine in DMF (2 × 20 min), the Fmoc-
protected amino acids (4 equiv, 0.152 mmol) were coupled using
PyBOP and HOBt (4 equiv of each, 0.152 mmol) as the coupling
reagents and DIEA (10 equiv, 0.38 mmol) as the base in DCM/DMF
(1:1, 3−4 mL) for 2 h (unless otherwise noted) on a manual multiple
peptide synthesis apparatus (CHOIR)22 to afford the linear fragments.
The side chains of Lys and Arg were protected by Boc and Pbf,
respectively, those of Dap, Dab, and Orn were protected by Mtt, and
those of D-Asp and L-Asp were protected by Pip.
1
97.4%); H NMR (500 MHz, acetone-d6) 8.05 (s, 1H), 6.97 (dd, J =
6.5 and 2.0 Hz, 2H), 6.65 (dd, J = 6.5 and 2.1 Hz, 2H), 3.22 (dd, J =
7.6 and 6.3 Hz, 1H), 2.79−2.67 (m, 1H), 2.66−2.59 (m, 1H), 2.38
(dd, J = 11.7 and 6.1 Hz, 1H), 2.21 (dd, J = 11.7 and 7.2 Hz, 1H), 1.26
(s, 9H), 0.82−0.72 (m, 1H), 0.35−0.27 (m, 2H), 0.05−0.01 (dd, J =
4.8 and 1.8 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 174.11, 155.11,
130.60, 128.73, 115.55, 81.53, 63.38, 53.28, 38.99, 28.23, 11.10, 3.86,
3.46.
Alloc-N-CPM-Tyr(Alloc)-OH (31). N-CPM-Tyr-Ot-Bu (0.08 g, 0.27
mmol, 1 equiv) in anhydrous DCM (2.5 mL) was treated with allyl
chloroformate (0.117 mL, 1.08 mmol, 4 equiv) and DIEA (0.189 mL,
1.08 mmol, 4 equiv) for 12 h at rt. The DCM was evaporated in vacuo,
EtOAc (10 mL) was added, and the solution was washed with water
(10 mL). Following back extraction of the aqueous layer with
additional EtOAc (10 mL), the combined EtOAc fractions were
washed with water (10 mL), dried (MgSO4), and evaporated in vacuo
to afford the product 29 (91 mg, 73%) as a yellowish white semisolid:
ESI-MS m/z [M + Na]+ 482.2155 (calcd), 482.2087 (observed);
HPLC tR 18.5 min (25−95% of aqueous acetonitrile with 0.1% TFA
1
over 35 min, purity 95.4%); H NMR (500 MHz, CDCl3, 50 °C) δ
7.22−7.14 (m, 2H), 7.07 (d, J = 8.5 Hz, 2H), 6.03−5.87 (m, 2H),
5.46−5.18 (m, 4H), 4.74−4.48 (m, 4H), 4.08 (m, 1H), 3.23 (m, 3H),
2.56 (m, 1H), 1.44 (s, 9H), 0.69 (m, 2H), 0.35 (m, 2H), 0.05 (m,
1H); 13C NMR (126 MHz, CDCl3) δ 169.97, 155.99, 153.61, 150.25,
136.79, 131.57, 130.48, 121.02, 119.34, 81.73, 69.17, 66.30, 63.35,
53.64, 29.86, 28.24, 10.34, 4.19, 3.83.
The ester was cleaved with 90% TFA/10% DCM for 12 h at room
temperature. The TFA was evaporated, and EtOAc (10 mL) and water
(10 mL) were added. The aqueous layer was extracted with EtOAc (10
mL), and the combined organic extracts were washed with water (10
mL), dried with MgSO4, and evaporated in vacuo to yield the product
(75 mg, 94%) as a yellowish white semisolid: ESI-MS m/z (M + K)+
442.1268 (calcd), 442.1250 (observed); HPLC: tR 12.05 min (25−
95% aqueous acetonitrile with 0.1% TFA over 35 min, purity 94.5%).
N-Benzyl-Phe-Ot-Bu (28). A solution of NaBH(OAc)3 (1.5 equiv)
in CHCl3 (2 mL) was added to a solution of Phe-Ot-Bu·HCl (0.618 g,
2.4 mmol, 1.2 equiv) and benzaldehyde (1 equiv) in CHCl3 (5 mL),
and the reaction mixture stirred at rt for 12 h. Water (20 mL) was
added, and the aqueous layer was extracted with additional CHCl3 (3
× 20 mL). The combined CHCl3 extracts were washed with water (20
mL) and dried over MgSO4, and the crude product was purified by
flash silica gel column chromatography (5−10% EtOAc/hexane) to
give 28 (335 mg, 44%) as a white semisolid: ESI-MS m/z [M + H]+
312.1964 (calcd), 312.1853 (observed); HPLC: tR 29.0 min (purity
99.1%); 1H NMR (500 MHz, CDCl3) δ 7.31−7.18 (m, 10H), 3.86 (d,
The (5−11) linear precursors of the cyclic peptides were
synthesized by this standard procedure. Selective deprotection of the
Pip and Mtt protecting groups on D-Asp and Dap, respectively, was
performed using 3% TFA and 5% TIS in DCM (3 × 10−15 min). The
cyclizations were performed using PyClocK and HOAt (4 equiv of
each) with DIEA (10 equiv) as the base in DCM/DMF (1:1, 4 mL for
200 mg of peptide−resin) for 24 h, with the coupling reagents
refreshed after every 8−12 h unless otherwise noted. The cyclizations
were monitored using the qualitative ninhydrin test.23 Any remaining
unreacted free amine of Dap was capped by treatment with acetyl
imidazole (16 equiv, 0.61 mmol) with DIEA (8 equiv) as the base in
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DMF (∼3 mL) for 30 min. Further extension of the peptide assembly
up to Gly2 afforded the cyclic (2−11) peptide fragments.
(2−11) peptide was performed with PyClocK (31 mg, 0.057 mmol, 2
equiv) and HOAt (8 mg, 0.057 mmol, 2 equiv) in the presence of
DIEA (6 equiv) for 1 h, followed by the subsequent removal of the
Alloc group (see below) to afford protected peptide 6 on the resin. For
peptide 14 Alloc-N-benzyl-Phe-OH (20 mg, 0.057 mmol, 2 equiv) was
dissolved in DMF (3 mL) at rt with PyClocK and HOAt (2 equiv of
each) and DIEA (6 equiv) and coupled to the (2−11) peptide−resin.
The coupling reaction was performed for 1 h, followed by subsequent
removal of the Alloc group (see below) to afford the protected peptide
14 on resin.
Coupling of the N-Terminal Amino Acid to the (2−11) Peptide.
Prior to coupling, complete dissolution of the N-alkyl-Tyr-OH (2
equiv) was achieved by heating at 80−120 °C in DMF (1 mL/0.02
mmol of N-alkyl amino acid), followed by cooling to rt (unless
otherwise indicated) and subsequent addition of PyClock and HOAt
(2 equiv of each) plus DIEA (6 equiv). The coupling reactions were
generally complete after 12 h, as indicated by the qualitative ninhydrin
test.
Final Deprotection of the Peptides. The peptides were cleaved
from the resin using Reagent B30 (87.5% TFA, 5% water, 5% phenol,
and 2.5% TIS, 4−5 mL/200 mg of resin). The solutions were filtered,
diluted with 10% aqueous acetic acid (10−15 mL), and extracted with
diethyl ether (3 × 10 mL). The ether extracts were back extracted with
10% acetic acid (10 mL); the combined aqueous solutions were
pooled and lyophilized to give the crude peptides.
Synthesis of Linear Peptides 2 and 3. The linear (2−11) fragments
were synthesized as described above under the general procedure
except that Fmoc-D-Asn(Trt)-OH and Fmoc-Dap(Mtt)-OH were
used in a 2-fold excess for the synthesis of peptides 2 and 3,
respectively. In addition, for peptide 3 the coupling of Fmoc-
Dap(Mtt)-OH to the peptide resin was performed using PyClocK and
HOAt (2 equiv of each) in the presence of DIEA (6 equiv) in DCM/
DMF (1:1, 4 mL). For peptide 3, the Mtt on Dap was selectively
deprotected by 3% TFA and 5% TIPS in DCM (3 × 10 min, 3−4 mL
each time), followed by acetylating with acetyl imidazole (16 equiv,
0.61 mmol) in the presence of DIEA (8 equiv) in DMF (3 mL) for 1
h. The coupling of N-benzyl-Tyr-OH (2 equiv) to the (2−11)
fragment was performed according to the general procedure described
above.
Alloc Deprotection of Peptides 6 and 14. The resin was swollen in
DCM (2 × 5 min), phenylsilane (24 equiv/g of resin) was added, and
the mixture was bubbled with N2 for 5 min. Tetrakis-
(triphenylphosphine)palladium (0.3 equiv) was added, and a septum
with a needle was fixed onto the reaction vessel to allow the evolved
carbon dioxide to escape. After shaking the reaction for 12 h, the
solution was drained, and the resin was subjected to a series of washes
as follows: DCM (4 × 1 min), THF (4 × 1 min), DCM (3 × 1 min),
DMF (3 × 1 min), 0.5% DIEA in DMF (3 × 2 min), 0.2 M sodium
diethyldithiocarbamate in DMF (3 × 15 min), DMF (3 × 2 min),
DCM (3 × 1 min), and methanol (3 × 1 min), followed by drying of
the resin.24,31
Synthesis of Cyclic Peptides 10−13. The synthesis of peptide 10
was performed according to the general procedure described above
with Ala incorporated in position 4. For peptides 11−13 the (5−11)
linear peptide fragments with Ala incorporated in the indicated
position were synthesized as described under the general procedure.
Following selective deprotection by 3% TFA and 5% TIPS (3 × 10
min) in DCM, the cyclization reactions were performed for 24 h using
PyClocK and HOAt (4 equiv each) in the presence of DIEA (6 equiv)
in DCM/DMF (1:1, 4 mL) with the reagents replaced after 12 h.
Following treatment with acetyl imidazole, the peptide chain was
extended up through Gly2 as described under the general procedure.
The coupling of N-benzyl-Tyr-OH to the (2−11) fragments followed
the general procedure as described above to obtain peptides 11−13.
Synthesis of Cyclic Peptides 15−17. For peptide 15 [D-
Asp(Pip)5,Dab(Mtt)8]Dyn A(5−11)NH2 was synthesized on resin
according to the general procedure described above, except that Fmoc-
Dab(Mtt)-OH (2 equiv) was coupling three times, for 2 h each during
the initial two couplings and overnight for the third coupling.
Subsequently, the Pip and Mtt protecting groups on D-Asp and Dab,
respectively, were selectively deprotected, and the peptide was cyclized
for 36 h as described above, with coupling agents replaced every 8 h.
Any unreacted free amine of Dab was acetylated as described above for
Dap. Further extension of the peptide chain up through Gly2 was
performed as described above under the general procedure. For
peptides 16 and 17 the (5−11) linear peptide fragments were
synthesized by the general procedure as described above. The Pip and
Mtt protecting groups on D-Asp and Orn, respectively (peptide 16),
and on L-Asp and Dap, respectively (peptide 17), were selectively
deprotected using 3% TFA and 5% TIS in DCM (3 × 10 min). The
peptides were cyclized for 36 h using PyClocK and HOAt (4 equiv of
each) in the presence of DIEA (6 equiv) in DCM/DMF (1:1, 4 mL)
with the reagents replaced every 12 h. Following treatment with acetyl
imidazole, the peptide chain was extended up through Gly2 as
described under the general procedure. Subsequent coupling of N-
benzyl-Tyr-OH to the corresponding (2−11) fragment was done as
described in the general procedure to afford peptides 15−17.
Purification and Analysis of the Peptides. The crude peptides
were purified by preparative reversed-phased HPLC on a Vydac C18
column (10 μ, 300 Å, 22 × 250 mm) equipped with a Vydac C18
guard cartridge using an LC-AD Shimadzu liquid chromatography
system equipped with an SPD-10A VP system controller and SPD-10A
VP UV−vis detector. For purification, a linear gradient of 5−40%
aqueous acetonitrile containing 0.1% TFA over 35 min, at a flow rate
of 18 mL/min, was used. The purifications were monitored at 214 nm.
The purity of the final peptides was verified on a Vydac 218-TP
column (5 μ, 300 Å, 4.6 × 50 mm) equipped with a Vydac guard
cartridge on a Shimadzu LC-10AT VP analytical HPLC liquid
chromatograph (equipped with an SCL-10A VP system controller
Synthesis of [N-Benzyl-D-Tyr1]zyklophin and Peptides 4−9 and
14. The (2−11) cyclic peptide fragment was synthesized according to
the general procedure described above. For the D-Tyr derivative of
zyklophin N-benzyl-D-Tyr (5.16 mg, 0.019 mmol, 2 equiv) was
dissolved in DMF (2 mL) by heating to 85 °C, and the solution was
cooled to room temperature, followed by addition of PyClocK, HOAt
(2 equiv of each), and DIEA (6 equiv) and coupling to the (2−11)
peptide for 12 h. In the case of peptide 5 N-allyl-Tyr-OH (20 mg,
0.076 mmol, 2 equiv) was dissolved in DMF (6 mL) by heating to 105
°C. Since the solution became turbid when it was cooled to ∼70 °C,
the coupling reagents were added to the amino acid solution at 105
°C. The solution was then cooled to rt, DIEA (6 equiv) was added,
and the activated amino acid was reacted with the (2−11) peptide
fragment for 12 h to obtain peptide 5. For peptide 7 complete
dissolution of N-phenethyl-Tyr-OH (33 mg, 0.114 mmol, 2 equiv) was
achieved by heating to 115 °C in DMF (10 mL). The solution was
cooled to ∼70 °C, followed by the addition to PyClocK and HOAt (2
equiv of each). Subsequently, the solution was cooled to rt, followed
by the addition of DIEA (6 equiv). The amino acid was reacted with
the (2−11) peptide fragment for 24 h, with reagents replaced after 12
h. In the synthesis of peptide 9 N-benzyl-Ala-OH (2 equiv) was heated
to 85 °C in DMF (3.5 mL), followed by cooling the solution to 60 °C
before adding PyClocK and HOAt (2 equiv of each) and DIEA (6
equiv). The coupling reaction was performed for 24 h, with the amino
acid and coupling reagents replaced after 12 h.
For peptide 4 Fmoc-N-Me-Tyr(t-Bu)-OH (54 mg, 0.114 mmol, 2
equiv) was dissolved in DMF (5 mL) at rt and coupled to the (2−11)
cyclic peptide using PyBOP (60 mg, 0.114 mmol, 2 equiv) and HOBt
(16 mg, 0.114 mmol, 2 equiv) in the presence of DIEA (6 equiv),
followed by subsequent removal of the Fmoc group. For peptide 8
Fmoc-Tyr(t-Bu)-OH (52 mg, 0.114 mmol, 4 equiv) was coupled to
the (2−11) peptide fragment in the presence of PyBOP (60 mg, 0.114
mmol, 4 equiv), HOBt (15 mg, 0.114 mmol, 4 equiv each), and DIEA
(10 equiv) in DCM/DMF (1:1, 3 mL) for 2 h. Following Fmoc
deprotection of Tyr, the N-terminus of the Tyr was reacted with
benzoic anhydride (25 mg, 0.114 mmol, 4 equiv) and DIEA (8 equiv)
in DMF (2−3 mL) overnight.
For peptide 6 Alloc-N-CPM-Tyr(Alloc)-OH (23 mg, 0.057 mmol, 2
equiv) was dissolved in DMF (3 mL) at rt, and the coupling to the
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and SPD-10A VP UV−vis detector) or on an Agilent 1200 series liquid
chromatography system equipped with a multiple wavelength UV−vis
detector. Three solvent systems were used for the analyses: (1) a linear
gradient of 5−50% solvent B (solvent A = aqueous 0.1% TFA; solvent
B = acetonitrile containing 0.1% TFA) over 45 min at a flow rate of 1
mL/min (system 1), (2) a linear gradient of 15−60% solvent B
(solvent A = aqueous 0.1% TFA; solvent B = methanol containing
0.1% TFA) over 45 min at a flow rate of 1.0 mL/min (system 2), and
(3) a linear gradient of 1−21% solvent B (solvent A = aqueous 0.09 M
TEAP, pH 2.5; solvent B = acetonitrile) over 40 min at a flow rate of 1
mL/min (system 3). The final purity of all peptides in all analytical
systems was ≥98% (see Table 2). Molecular weights of the
compounds were determined by ESI-MS using a time-of-flight mass
spectrometer analyzer (LCT premier, Waters, Milford, MA).
Pharmacological Evaluation. Radioligand Binding Assays.
Radioligand binding assays were performed as previously described17
using cloned rat KOR and MOR and mouse DOR stably expressed on
CHO cells. Incubations with isolated membrane protein were
performed in triplicate with concentrations of the peptides from 0.1
nM to 10 μM for 90 min in 50 mM Tris, pH 7.4, at 22 °C using
[3H]diprenorphine (Kd = 0.45 nM), [3H]DAMGO ([D-Ala2,N-Me-
Phe4,glyol]enkephalin, Kd = 0.49 nM), and [3H]DPDPE (cyclo[D-
Pen2,D-Pen5]enkephalin, Kd = 1.76 nM) as radioligands in assays for
KOR, MOR, and DOR, respectively (Bmax = 2.27, 2.20, and 1.45
pmol/mg protein, respectively). Nonspecific binding was determined
in the presence of 10 μM unlabeled Dyn A(1−13)NH2, DAMGO, and
DPDPE for KOR, MOR, and DOR, respectively. Reported Ki values
for the analogues are the mean SEM from at least three independent
experiments except where noted.
(3R)-7-hydroxy-N-[(2S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-
dimethylpiperidin-1-yl]-3-methylbutan-2-yl]-1,2,3,4-tetrahy-
droisoquinoline-3-carboxamide; KOR, kappa opioid receptor;
MOR, mu opioid receptor; Mtt, 4-methyltrityl; norBNI, nor-
binaltorphimine; Orn, ornithine; PAL, peptide amide linker;
Pbf, 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sufonyl; PEG-
PS, poly(ethylene glycol)-polystyrene; Pip, 2-phenylisopropyl;
PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
fluorophosphate; PyClocK, 6-chlorobenzotriazol-1-yl-oxytri-
pyrrolidinophosphonium hexafluorophosphate; RP-HPLC, re-
versed-phase HPLC; s.c., subcutaneous; TEAP, triethylammo-
nium phosphate; TIS, triisopropylsilane; Trt, trityl
REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*E-mail: janealdrich@ufl.edu. Phone: 352-273-8708. Fax: 352-
392-9455.
■
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Present Addresses
§(A.A.J.) Department of Pharmaceutics, Virginia Common-
wealth University, Richmond, Virginia 23298, United States.
∥(J.V.A.) Department of Medicinal Chemistry, University of
Florida, Gainesville, Florida 32610, United States.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors thank Dr. Zhengyu Cao, Bridget Leuschen, and
Stacey Sigmon at Creighton University for carrying out the
pharmacological studies. This research was supported by grant
R01 DA018832.
ABBREVIATIONS USED
■
Alloc, allyloxycarbonyl; CHO, Chinese hamster ovary; CPM,
cyclopropylmethyl; Dab, 2,3-diaminobutyric acid; DAMGO,
([D-Ala2,N-Me-Phe4,glyol]enkephalin; Dap, 2,3-diaminopro-
pionic acid; DIEA, N,N-diisopropylethylamine; DOR, delta
opioid receptor; DPDPE, cyclo[D-Pen2,D-Pen5]enkephalin; Dyn,
dynorphin; GPI, guinea pig ileum; HOAt, 1-hydroxy-7-
azabenzotriazole; HOBt, 1-hydroxybenzotriazole; JDTic,
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