Pseudoproline-containing analogues of morphiceptin and endomorphin-2: Evidence for a Cis Tyr-Pro amide bond in the bioactive conformation

Article abstract of DOI:10.1021/jm000332e

Analogues of the opioid peptides [D-Phe³]morphiceptin (H-Tyr-Pro-D-Phe-Pro-NH₂) and endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH₂) containing the pseudoproline (ψPro) (4R)-thiazolidine-4-carboxylic acid (Cys[ψ^R1,R2pro])

Full text of DOI:10.1021/jm000332e

3896
J . Med. Chem. 2001, 44, 3896-3903
P seu d op r olin e-Con ta in in g An a logu es of Mor p h icep tin a n d En d om or p h in -2:
Evid en ce for a Cis Tyr -P r o Am id e Bon d in th e Bioa ctive Con for m a tion
Michael Keller,†,§ Christophe Boissard,† Luc Patiny,† Nga N. Chung,‡ Carole Lemieux,‡ Manfred Mutter,*,† and
Peter W. Schiller*,‡
Institute of Organic Chemistry, BCH-Dorigny, University of Lausanne, CH-1015 Lausanne, Switzerland, and
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West,
Montreal, Quebec, Canada H2W 1R7
Received August 2, 2000
Analogues of the opioid peptides [D-Phe3]morphiceptin (H-Tyr-Pro-D-Phe-Pro-NH2) and
endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) containing the pseudoproline (ΨPro) (4R)-thiazoli-
dine-4-carboxylic acid (Cys[ΨR1,R2pro]) or (4S)-oxazolidine-4-carboxylic acid (Ser[ΨR1,R2pro]) in
place of Pro2 were synthesized. The pseudoproline ring in these compounds was either
unsubstituted (R1, R2 ) H) or dimethylated (R1, R2 ) CH3) at the 2-C position. 2-C-dimethylated
pseudoprolines are known to be quantitative or nearly quantitative inducers of the cis
conformation around the Xaai-1-Xaai[ΨCH ,CH pro] imide bond. All dihydropseudoproline-
containing analogues (R1, R2 ) H) showed good µ opioid agonist potency in the guinea pig
ileum (GPI) assay, high µ receptor binding affinity in the rat brain membrane binding assay,
3
3
1
and, like their parent peptides, excellent µ receptor binding selectivity. H NMR spectroscopic
analysis of the Cys[ΨH,Hpro]2- and Ser[ΨH,Hpro]2-containing analogues in DMSO-d6 revealed
that they existed in a conformational equilibrium around the Tyr-Xaa[ΨH,Hpro] peptide bond
with cis/ trans ratios of 40:60 and 45:55, respectively. The dimethylated thiazolidine- and
oxazolidine-containing [D-Phe3]morphiceptin- and endomorphin-2 analogues (R1, R2 ) CH3)
all retained full µ agonist potency in the GPI assay and displayed µ receptor binding affinities
in the nanomolar range and high µ receptor selectivity. As expected, no conformers of the latter
analogues with a trans conformation around the Tyr-Xaa[ΨCH ,CH pro] imide bond were detected
by 1H NMR spectral analysis, indicating that in these compounds the cis conformation is highly
predominant (>98%). These results represent the most direct evidence obtained so far to indicate
that morphiceptin and endomorphin-2 have the cis conformation around the Tyr-Pro peptide
bond in their bioactive conformations.
3
3
In tr od u ction
the two isomers cannot be isolated at room temperature.
The difference in energy (∆G°) between the cis and trans
conformers of a Tyr-Pro dipeptide unit is quite low,
typically ∼2-4 kcal/mol. Therefore, it is quite possible
that the minor, cis conformer might bind to the receptor
and that its relatively higher energy might be paid for
by the ligand-receptor interaction energy. After binding
of the cis conformers initially present in solution, the
cis/trans equilibrium would be continuously re-estab-
lished, thus permitting the binding of newly generated
cis conformers to the receptor in a relatively slow
binding process. Therefore, even when the trans/cis ratio
is quite high in solution, such as reported for endomor-
phin-1 (trans/cis ratio of 75:25 in DMSO5), the receptor-
bound peptide might nonetheless have the cis confor-
mation at the Tyr1-Pro2 peptide bond.
Direct experimental determinations of the conforma-
tion of a receptor-bound peptide are not yet feasible.
However, it is possible to rule out the trans isomer of a
Pro-containing peptide as the receptor-bound species
through substitution of the proline with an appropriate
proline analogue capable of forcing the peptide bond in
question into the cis conformation. 2-C-dimethylated
pseudoproline9,10 or proline11 analogues have previously
been shown to be quantitative or nearly quantitative
cis inducers. The pseudoprolines are structurally de-
rived from cysteine, threonine, or serine and contain a
Morphiceptin (H-Tyr-Pro-Phe-Pro-NH2)2 and the en-
domorphins [endomorphin-1 (H-Tyr-Pro-Trp-Phe-NH2)
and endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2)]3 are opi-
oid peptide agonists with high selectivity for µ opioid
receptors. These peptides contain a Pro residue in the
2-position of the peptide sequence and, consequently,
cis-trans isomerization occurs at the Tyr1-Pro2 peptide
bond.4,5 The question arises whether morphiceptin and
the endomorphins adopt the cis or the trans configura-
tion at this peptide bond when bound to the receptor.
The results of 1H NMR spectroscopic studies performed
with morphiceptin6 and endomorphin-15,7 in aqueous
solution indicated the existence of a cis/ trans equilib-
rium with a predominance of the trans isomer in both
cases. However, conformational studies of these peptides
free in solution do not permit any definite conclusions
with regard to their receptor-bound conformation, even
if they indicate that one isomer is predominant. Cis/
trans isomerization around the peptide bond preceding
a Pro residue in a peptide is a dynamic process,8 and
* Corresponding author [telephone (514) 987-5576; fax (514) 987-
5513; e-mail schillp@ircm.qc.ca].
† University of Lausanne.
§ Present address: Chemistry Department, Imperial College of
Science, Technology and Medicine, South Kensington SW7 2AY, U.K.
‡ Clinical Research Institute of Montreal.
10.1021/jm000332e CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/03/2001

Analogues of Morphiceptin and Endomorphin-2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3897
serine in the presence of formaldehyde. For the prepa-
ration of the dimethyloxazolidine-containing dipeptide
Fmoc-Tyr(All)-Ser(ΨMe,Mepro)-OH, Fmoc-Tyr(All)-F was
coupled to serine benzyl ester and the resulting dipep-
tide was then reacted with dimethoxypropane in the
presence of pyridinium p-toluenesulfonate (PPTS). Sub-
sequent catalytic hydrogenation yielded the desired
product. For the solid-phase syntheses of the final
tetrapeptide amides the C-terminal dipeptide segments
were first assembled on a Sieber-resin20 using 9-fluo-
renylmethyloxycarbonyl-protected amino acids and ben-
zotriazol-1-yloxytris(pyrrolidinophosphonium-hexaflu-
orophosphate) (PyBOP) as coupling agent. The N-termi-
nal dipeptide segments were then coupled to the resin-
bound dipeptides using the same coupling agent. After
removal of the Tyr1 allyl protecting group with Pd-
[PPh3]4, the peptides were cleaved from the resin with
2% TFA in CH2Cl2 and were purified by reversed-phase
HPLC.
Op ioid R ecep t or Bin d in g Assa ys a n d in Vit r o
Bioa ssa ys. Binding affinities of compounds for µ and
δ opioid receptors were determined by displacing,
respectively, [3H]DAMGO and [3H]DSLET from rat
brain membrane binding sites, and κ opioid receptor
binding affinities were measured by displacement of
[3H]U69,593 from guinea pig brain membrane binding
sites. For the determination of their in vitro opioid
activities, analogues were tested in bioassays based on
inhibition of electrically evoked contractions of the GPI
and MVD. The GPI assay is usually considered as being
representative for µ receptor interactions, even though
the ileum does also contain κ receptors. In the MVD
assay opioid effects are primarily mediated by δ recep-
tors, but µ and κ receptors also exist in this tissue.
F igu r e 1. Pseudoprolines Xaa(ΨR1,R2pro) are obtained by
cyclocondensation reaction of aldehydes or ketones with Xaa
) Cys, Ser, or Thr. X ) S, R ) H: Cys-derived thiazolidines.
X ) O, R ) H: Ser-derived oxazolidines. X ) O, R ) CH3:
Thr-derived oxazolidines. R1, R2 ) H, CH3, aryl.
sulfur or oxygen atom in place of the -CH2- mo¨ıety in
the 1-C position of the proline ring structure (Figure
1). Pseudoprolines were originally introduced for use in
a novel, orthogonal protection strategy for Cys, Thr, and
Ser in peptide synthesis.10 In addition, they were
successfully used to prevent secondary structure forma-
tion during solid-phase peptide synthesis of difficult
peptides.12 In a number of studies with model peptides,
the incorporation of pseudoprolines was shown to lower
the transition-state barrier (∆G‡) of the cis f trans
isomerization of the peptide bond preceding the pseu-
doproline residue,13 to quantitatively fixate a cis peptide
bond in short peptides,14 to induce an all-cis conforma-
tion of a cyclic tripeptide,15 and to lock in oligopeptides
in an all-cis polyproline I helix.16 Furthermore, the use
of 2-C-substituted pseudoprolines permitted the modu-
lation of the physicochemical and biological properties
of the cyclic peptide cyclosporin C,17 as well as the
production of a monoclonal antibody recognizing the
consensus loop tip GPGR of HIV glycoprotein 120 in its
cis-Gly-Pro conformation.18
In the present paper, we describe analogues of endo-
morphin-2 and of the potent D-Phe3-analogue of mor-
phiceptin6,19 in which the Pro2 residue was replaced with
2-C-dimethylated pseudoprolines known to be quantita-
tive or nearly quantitative cis-inducers9,13 (compounds
5, 7, 9, and 11). In case these peptides turned out to
retain opioid activity, this would constitute strong
evidence to indicate that the receptor-bound conforma-
tion of [D-Phe3]morphiceptin and endomorphin-2 has
the cis conformation at the Tyr1-Pro2 peptide bond. In
addition, we prepared analogues of these two peptides,
in which the sterically demanding methyl substituents
at the 2-C position of the pseudoproline ring were
replaced by two hydrogen atoms (Figure 1) (compounds
4, 6, 8, and 10). 2-C-dihydro-oxazolidine and thiazolidine
differ from native proline by the exchange of the
γ-methylene group in the proline ring with an oxygen
or a sulfur atom, respectively. They have previously
been shown to induce substantial structural flexibility
around the amide bond to the preceding amino acid due
to decreased cis-trans activation barriers.13 Therefore,
it was expected that the conformational adaptation of
these analogues to the µ receptor would be facilitated.
Ch em istr y. The N-terminal dipeptide segments of
analogues 4-11 were synthesized in solution, and the
final tetrapeptide amides were prepared by using the
solid-phase technique. The protected, thiazolidine-
containing dipeptides Fmoc-Tyr(All)-Cys(ΨH,Hpro)-OH
and Fmoc-Tyr(All)-Cys(ΨMe,Me pro)-OH were prepared
by coupling Fmoc-Tyr(All)-F with Cys(ΨH,Hpro)-OH and
Cys(ΨMe,Mepro)-OH, respectively. The dihydro-oxazoli-
dine-containing dipeptide Fmoc-Tyr(All)-Ser(ΨH,Hpro)-
OH was prepared by reacting Fmoc-Tyr(All)-F with
Resu lts a n d Discu ssion
In the GPI assay the dihydrothiazolidine-containing
[D-Phe3]morphiceptin analogue 4 showed slightly higher
potency than the parent peptide [D-Phe3]morphiceptin
and was 3 times as potent as [Leu5]enkephalin (Table
1). In agreement with these functional assay data,
analogue 4 displayed ∼4-fold higher µ receptor affinity
in the rat brain membrane binding assay than the
parent compound (Table 2). It bound very weakly to δ
opioid receptors (Kiδ ) 2450 nM) and, thus, retained
high µ versus δ receptor selectivity (Kiδ/Kiµ ) 286),
similar to that of [D-Phe3]morphiceptin. Analysis of the
1H NMR spectrum of 4 in DMSO-d6 solution revealed
that this compound exists in a conformational equilib-
rium around the Tyr-Pro amide bond with a cis/trans
ratio of 40:60 (Table 3). This ratio is similar to the cis/
trans ratio (39:61) around that same peptide bond
observed in the parent peptide [D-Phe3]morphiceptin.6
Introduction of two methyl substituents at the 2-C
position of the Pro2 ring in peptide 4 resulted in a
compound, H-Tyr-Cys(ΨMe,Mepro)-D-Phe-Pro-NH2 (5),
that showed only ∼3-4-fold lower µ agonist potency and
1
µ receptor binding affinity than 4. Inspection of the H
NMR resonances of the 2-C dimethyl groups of the Cys-
(ΨMe,Mepro) residue in DMSO-d6 revealed that this
compound assumed almost exclusively the cis conforma-
tion (>98%) around the Tyr-Cys(ΨMe,Mepro) peptide
bond. This result together with the observed high opioid
activity clearly indicates that analogue 5 must have the

3898 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Ta ble 1. GPI and MVD Assay of Opioid Peptide Analogues
Keller et al.
GPI
MVD
MVD/GPI
IC50 ratio
a
a
no.
compd
IC50 (nM)
rel potency
IC50 (nM)
rel potency
H-Tyr-Pro-D-Phe-Pro-NH2
109 ( 16
74.5 ( 9.3
246 ( 12
470 ( 41
860 ( 135
2.26 ( 0.33
3.30 ( 0.41
1.00 ( 0.05
0.523 ( 0.046
0.286 ( 0.045
594 ( 77
190 ( 14
1420 ( 4
1020 ( 80
851 ( 178
0.0192 ( 0.0025
0.0600 ( 0.0044
0.00803 ( 0.00002
0.0112 ( 0.0009
0.0134 ( 0.0028
5.45
2.55
5.77
2.17
0.990
4
5
6
7
H-Tyr-Cys(ΨH,Hpro)-D-Phe-Pro-NH2
H-Tyr-Cys(ΨMe,Mepro)-D-Phe-Pro-NH2
H-Tyr-Ser(ΨH,Hpro)-D-Phe-Pro-NH2
H-Tyr-Ser(ΨMe,Mepro)-D-Phe-Pro-NH2
H-Tyr-Pro-Phe-Phe-NH2
7.71 ( 1.47
22.2 ( 2.6
502 ( 27
41.7 ( 1.7
305 ( 37
31.9 ( 6.1
15.3 ( 1.8
138 ( 40
0.745 ( 0.088
1.98
6.22
8
9
10
11
H-Tyr-Cys(ΨH,Hpro)-Phe-Phe-NH2
H-Tyr-Cys(ΨMe,Mepro)-Phe-Phe-NH2
H-Tyr-Ser(ΨH,Hpro)-Phe-Phe-NH2
H-Tyr-Ser(ΨMe,Mepro)-Phe-Phe-NH2
11.1 ( 1.3
0.0826 ( 0.0239
0.490 ( 0.026
5.9 ( 0.24
0.807 ( 0.098
PA (26%)b
151 ( 37
606 ( 65
0.0755 ( 0.0185
0.0188 ( 0.0020
3.62
1.99
[Leu5]enkephalin
246 ( 39
1
11.4 ( 1.1
1
0.0463
a
b
Mean of three determinations ( SEM. Partial agonist (maximal inhibition of electrically evoked contractions ) 26%).
Ta ble 2. Binding Affinities of Opioid Peptide Analogues at µ and δ Receptors in Rat Brain Homogenates
[3H]DAMGO [3H]DSLET
rel potency
<0.000253
no.
compd
Kiµ (nM)a
35.2 ( 6.3
rel potency
Kiδ (nM)a
>10000
2450 ( 200
>10000
>10000
Kiδ/ Kiµ
H-Tyr-Pro-D-Phe-Pro-NH2
0.268 ( 0.048
8.57 ( 0.70 1.10 ( 0.09
>284
286
>265
>170
>104
4
5
6
7
H-Tyr-Cys(ΨH,Hpro)-D-Phe-Pro-NH2
0.00103 ( 0.00008
<0.000253
H-Tyr-Cys(ΨMe,Mepro)-D-Phe-Pro-NH2 37.7 ( 4.5
0.250 ( 0.030
0.160 ( 0.016
0.0977 ( 0.0163 >10000
H-Tyr-Ser(ΨH,Hpro)-D-Phe-Pro-NH2
H-Tyr-Ser(ΨMe,Mepro)-D-Phe-Pro-NH2 96.5 ( 16.1
58.9 ( 5.9
<0.000253
<0.000253
H-Tyr-Pro-Phe-Phe-NH2
2.06 ( 0.19 4.58 ( 0.42
6.41 ( 0.22 1.47 ( 0.05
50.4 ( 2.2 0.187 ( 0.008
3940 ( 460
1980 ( 590
0.000642 ( 0.000075 1910
8
9
H-Tyr-Cys(ΨH,Hpro)-Phe-Phe-NH2
H-Tyr-Cys(ΨMe,Mepro)-Phe-Phe-NH2
0.00128 ( 0.00038
<0.000253
309
>198
>509
>241
>10000
10 H-Tyr-Ser(ΨH,Hpro)-Phe-Phe-NH2
11 H-Tyr-Ser(ΨMe,Mepro)-Phe-Phe-NH2
7.86 ( 1.11 1.20 ( 0.17
>4000
>10000
<0.000632
<0.000253
46.9 ( 5.8
0.201 ( 0.025
[Leu5]enkephalin
9.43 ( 2.07
1
2.53 ( 0.35
1
0.268
a
Mean of three determinations ( SEM.
Ta ble 3. Analytical Data of Morphiceptin and Endomorphin-2
Analogues 4-11
ratio of 45:55 was determined in DMSO-d6, similar to
the ratio found for the dihydrothiazolidine-containing
analogue (4) and for the parent peptide. The dimethyl-
oxazolidine-containing [D-Phe3]morphiceptin analogue
(7) displayed somewhat lower µ agonist potency than
the corresponding dimethylthiazolidine analogue in the
GPI assay but, importantly, was still a full µ agonist.
It also showed somewhat lower µ receptor affinity but
retained high µ receptor selectivity. As was the case
with analogue 5, the Tyr-Ser(ΨMe,Mepro) peptide bond
in compound 7 was found to be almost exclusively in
the cis conformation (>98%,Table 3).
Similar results were obtained with the compounds of
the endomorphin-2 series. However, the dihydrooxazo-
lidine-containing analogue (10) displayed only 2-fold
lower µ agonist potency in the GPI assay than the
dihydrothiazolidine-containing analogue (8) and almost
equal µ receptor binding affinity. The cis/trans ratios
for the Tyr-Pro peptide bond in compounds 8 and 10
in DMSO-d6 were found to be exactly the same as those
observed with the corresponding [D-Phe3]morphiceptin
analogues (4 and 6). Interestingly, the dimethyloxazo-
lidine-containing endomorphin-2 analogue (11) turned
out to have slightly higher µ agonist potency and µ
receptor binding affinity than the dimethylthiazolidine-
containing analogue (9). These results are in contrast
to those obtained with the corresponding dimethylated
pseudoproline-containing analogues of [D-Phe3]mor-
phiceptin, because compound 5 is a more potent µ ago-
nist than compound 7. These differences may be ex-
plained with slightly different modes of receptor binding
between the [D-Phe3]morphiceptin- and endomorphin-2
analogues.
cis/transa
∆G° b
(kcal/mol)
no.
compd
(%)
4
5
6
7
8
9
H-Tyr-Cys(ΨH,Hpro)-D-Phe-Pro-NH2
H-Tyr-Cys(ΨMe,Mepro)-D-Phe-Pro-NH2
H-Tyr-Ser(ΨH,Hpro)-D-Phe-Pro-NH2
H-Tyr-Ser(ΨMe,Mepro)-D-Phe-Pro-NH2
H-Tyr-Cys(ΨH,Hpro)-Phe-Phe-NH2
H-Tyr-Cys(ΨMe,Mepro)-Phe-Phe-NH2
40:60
>98
-0.242
2.32
-0.120
2.32
-0.242
2.32
-0.120
2.32
45:55
>98
40:60
>98
45:55
>98
10 H-Tyr-Ser(ΨH,Hpro)-Phe-Phe-NH2
11 H-Tyr-Ser(ΨMe,Mepro)-Phe-Phe-NH2
a
Determined by integration of the 1H NMR resonances of the
2-C protons (compounds 4, 6, 8, and 10). In the case of compounds
5, 7, 9, and 11 inspection of the resonances of the 2C-dimethyl
groups of the pseudoproline residue indicated a high predominance
b
of the cis conformation (>98%) in all cases. Calculated from
equation ∆G°cis-trans ) -ln K × RT, where R is the universal gas
constant, T the temperature (300 K), and K the equilibrium
constant cis/trans.
cis conformation around the amide bond between the
first and second residues when bound to the µ receptor.
As was the case with the parent peptide, compound 5
showed no δ receptor binding affinity at concentrations
up to 10 µM and, thus, also was µ receptor-selective (Kiδ/
Kiµ > 265, Table 2).
In comparison with the parent peptide [D-Phe3]-
morphiceptin, the dihydrooxazolidine-containing ana-
logue (6) was a ∼3 times less potent µ agonist in the
GPI assay, had about half the affinity for µ receptors
in the binding assay, and showed a similarly high
preference for µ receptors over δ receptors. Thus, com-
pound 6 was a ∼6 times less potent µ agonist than the
corresponding Cys(ΨH,Hpro)2 analogue. For the Tyr-
Ser(ΨH,Hpro) peptide bond in compound 6 a cis/trans

Analogues of Morphiceptin and Endomorphin-2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3899
F igu r e 2. 1H NMR spectra of compounds 10 (A and B) and 11 (C and D) in DMSO at 25 °C. The minor isomer is represented
with a prime (′).
F igu r e 3. ROESY spectrum of 10 in CD3CN at -20 °C. The characteristic NOE between 1HR and 2HR indicates that the major
conformer has a cis amide bond between residues 1 and 2. The NOE between 2′Hδ and 1′HR indicates that in the minor conformer
this same amide bond is in the trans conformation.
As in the case of the [D-Phe3]morphiceptin analogues
5 and 7, the conformation of the peptide bond between
Tyr and the dimethylated pseudoproline residue in the
endomorphin-2 analogues 9 and 11 was found to be
predominantly cis (>98%, Table 3). The claim of a highly
predominant cis conformation was documented further
in the case of compound 11 as an illustrative example
Under these conditions the cis/trans ratio of 10 was 70:
30. The ROESY spectrum of 10 shows the characteristic
NOE between 1HR and 2HR, indicating a cis amide bond
between residues 1 and 2 in the major conformer, and
an NOE between 2′Hδ and 1′HR, indicating a trans
amide bond in the minor conformer (Figure 3). On the
other hand, in the ROESY spectrum of 11 only the NOE
between 1HR and 2HR is observed (Figure 4). Taken
together, these results clearly indicate that the amide
bond between the Tyr and Ser(ΨMe,Mepro) residues in
11 almost exclusively assumes the cis conformation.
Like the [D-Phe3]morphiceptin analogues (4-7), all
endomorphin-2 analogues (8-11) displayed high prefer-
ence for µ receptors over δ receptors. None of the
[D-Phe3]morphiceptin and endomorphin-2 analogues
1
by presenting 1-D H NMR and 2-D ROESY spectra of
11 in comparison with 10. Whereas a doubling of
resonances is clearly evident in the 1-D spectrum of 10
in DMSO-d6, a single set of resonances is observed with
11 (see Figure 2 and Experimental Section). The ROESY
spectra were obtained in CD3CN at -20 °C to prevent
most of the exchange between the cis and trans con-
formers that was observed at higher temperature.

3900 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Keller et al.
F igu r e 4. ROESY spectrum of 11 in CD3CN at -20 °C. The characteristic NOE between 1HR and 2HR indicates that the amide
bond between residues 1 and 2 is in the cis conformation.
described here showed significant binding affinity for κ
opioid receptors at concentrations up to 10 µM. Thus,
compounds 4-11 are all very selective µ opioid agonists.
In general, the dihydropseudoproline-containing ana-
logues showed slightly lower µ agonist potency and µ
receptor binding affinity than the corresponding Pro-
containing parent peptides with the exception of com-
pound 4, which was found to be a more potent µ agonist
than its parent. The transition state energies (∆G‡) for
the cis/trans isomerization of the Xaa-ΨPro unit in
peptides containing a dihydropseudoproline are typically
1-3 kcal/mol lower than those of the corresponding
peptides with a native proline residue.13 Because the
present results indicate that the [D-Phe3]morphiceptin
and endomorphin-2 analogues have the cis conformation
around the Tyr1-ΨPro2 peptide bond in their bioactive
conformation, one would expect that the dihydroproline-
containing analogues should display higher potency
than the Pro-containing parent peptides because of the
lower ∆G‡ for the transition from the trans to the cis
configuration. This turned out to be the case for the
dihydrothiazolidine-containing [D-Phe3]morphiceptin ana-
logue (4), but not for the other dihydropseudoproline-
containing morphiceptin or endomorphin-2 analogues.
Other factors that may be responsible for these differ-
ences in potency may include direct effects of the
heteroatom in the pseudoproline ring or slightly differ-
ent receptor binding modes of the various dihydropseu-
doproline-containing morphiceptin or endomorphin-2
analogues.
site. On average, the dimethylated analogues have only
a ∼5-fold lower µ receptor binding affinity than the
corresponding dihydro analogues. Assuming that the
trans conformer were the bioactive one, it would be
expected that the cis conformer should either be inactive
or have a µ receptor affinity reduced by several orders
of magnitude as compared to that of the trans con-
former. This expectation is based on the fact that the
spatial disposition of the two important pharmacophoric
moieties, the Tyr residue and the Phe3 side chain, in
the cis conformer is very different from that in the trans
conformer. Therefore, the obtained results represent
strong evidence in favor of a receptor-bound conforma-
tion with a cis amide bond between the first two
residues.
Con clu sion s
The four [D-Phe3]morphiceptin and endomorphin-2
analogues containing a dimethylated pseudoproline
residue in place of Pro2 (compounds 5, 7, 9, and 11) all
are full µ opioid agonists showing good potency in the
GPI assay and µ receptor binding affinities in the
nanomolar range in the binding assay. As expected, 1H
NMR spectral analysis revealed that the peptide bond
between the tyrosine and the dimethylated pseudopro-
line residue in these four compounds assumed almost
exclusively the cis conformation. Taken together, these
results represent the most direct evidence obtained so
far to indicate that the conformation of the Tyr-Pro
peptide bond of [D-Phe3]morphiceptin and endomor-
phin-2 in their receptor-bound conformation is cis. This
finding is in agreement with studies on the pharmaco-
logical and conformational properties of morphiceptin
analogues containing either cis-2-aminocyclopentan-
ecarboxylic acid or NR-methylalanine in place of Pro2,
which had led to the suggestion that the cis conforma-
tion around the Tyr-Pro amide bond is required for the
opioid activity of these morphiceptin analogues.6,21 The
All [D-Phe3]morphiceptin and endomorphin-2 ana-
logues containing a dimethylated pseudoproline residue
showed somewhat lower µ agonist potency and lower µ
receptor binding affinity than the corresponding dihy-
dropseudoproline-containing analogues and the corre-
sponding parent peptides. These reduced potencies are
most likely the result of steric interference caused by
the two 2-C-methyl substituents at the receptor binding

Analogues of Morphiceptin and Endomorphin-2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3901
(C) F m oc-Tyr (All)-Cys(ΨMe,Mep r o)-OH. Fmoc-Tyr(All)-F
(1 g, 2.25 mmol) and N-methylmorpholine (700 µL, 3 equiv)
results of a conformational study recently performed
with endomorphin-1 in reversed micelles of bis(2-eth-
ylhexyl)sulfosuccinate sodium salt were also interpreted
in favor of a model of the µ receptor-bound conformation
with a cis Tyr-Pro amide bond.7
were dissolved in 15 mL of DMF. After addition of Cys(ΨMe,Me
-
pro)-OH28 (1.32 g, 2.25 mmol), the solution was stirred for 16
h. At the end of the reaction, 50 mL of EtOAc was added and
the solution was washed with citric acid (5%, 25 mL, two times)
and water (25 mL, two times). The organic phase was dried
over MgSO4, and after filtration, the solvent was removed
under reduced pressure. Purification of the dipeptide was
achieved by flash chromatography over silica using CHCl3/
MeOH/AcOH (100:10:1, v/v) as eluent. Five hundred and ten
milligrams (39%) of pure Fmoc-Tyr(All)-Cys(ΨMe,Mepro)-OH
was obtained: HPLC (C18, 50-100% B, 20 min) tR ) 15.02 min;
MS-ESI, (m/z) 587.7 [M + H]+.
Syn th esis of Oxa zolid in e-Con ta in in g Dip ep tid es. (A)
F m oc-Tyr (All)-Ser (ΨH,Hp r o)-OH. Serine (11.05 g, 105 mmol)
was dissolved in aqueous Na2CO3 (2.5 N, 32 mL), and the
solution was cooled in an ice bath. A solution of formaldehyde
(37%, 19.3 mL) was added dropwise, and the solution was kept
at 4 °C for 16 h. Fmoc-Tyr(All)-F (2.7 g, 6.1 mmol) dissolved
in 84 mL of acetone was then added to the solution. After 3 h
of intensive stirring at room temperature, the product was
extracted twice with EtOAc (100 mL). The organic layer was
dried over MgSO4 and the solvent evaporated in vacuo. The
resulting solid was purified by flash chromatography over
silica (CHCl3/MeOH/AcOH, 100:5:0.5) to afford 2.4 g (75%) of
pure, white product in solid form: HPLC (C18, 0-100% B, 30
min) tR ) 25.7 min; MS-ESI, (m/z) 543.2 [M + H]+.
(B) F m oc-Tyr (All)-Op F . Fmoc-Tyr(All)-OH (7 g, 15.8
mmol) was dissolved in a mixture of 30 mL of CH2Cl2 and 4
mL of THF. Dicyclohexylcarbodiimide (DCC; 3.58 g, 1.1 equiv)
and 2,3,4,5,6-pentafluorophenol (3.2 g, 1.1 equiv) were then
added to the solution. A heavy precipitate formed. After
completion of the reaction, the suspension was filtered over
Celite and the solvent evaporated in vacuo to afford 6.9 g
(0.011 mol, 72%) of a pure, white solid: HPLC (C18, 0-100%
B, 30 min) tR ) 33.4 min; MS-ESI, (m/z) 610.2 [M + H]+.
(C) F m oc-Tyr (All)-Ser -OBzl. To a solution of Fmoc-Tyr-
(All)-OpF (5.6 g, 9.18 mmol) in 30 mL of CH2Cl2 were added
3.61 g (1.7 equiv) of H-Ser-OBzl and 1.77 mL (1.7 equiv) of
N-methylmorpholine. The solution was stirred at room tem-
perature for 16 h. After completion of the reaction, citric acid
(5%, 2 × 30 mL) was added. The organic layer was dried over
MgSO4 and evaporated in vacuo. The resulting solid was
dissolved in EtOAc, and the dipeptide was precipitated by the
addition of pentane. After filtration, a 95% pure product was
obtained as a white solid (5.41 g, 35%): HPLC (C18, 0-100%
B, 30 min) tR ) 28.4 min; MS-ESI, (m/z) 621.4 [M + H]+.
Exp er im en ta l Section
Gen er a l Meth od s. All solvents were obtained from Fluka,
Buchs, Switzerland, and were used without further purifica-
tion. Assembly of the peptides was carried out by the manual
solid-phase method using the Sieber resin (Novabiochem,
La¨ufelfingen, Switzerland). N-R-Fmoc amino acids (Alexis
Corp., La¨ufelfingen, Switzerland) were used throughout. 2-C-
dihydrothiazolidine was purchased from Fluka. Reversed-
phase HPLC was performed on a Waters 600 SL instrument,
using columns packed with Vydac Nucleosil 300 Å 5 µm C18
particles. Analytical columns (250 × 4.6 mm) were operated
at a flow rate of 1 mL/min and preparative columns (250 × 21
mm) at a flow rate of 18 mL/min, with UV monitoring at 214
nm. Solvent A was water (purified on a Milli-Q ion exchange
cartridge) containing 0.09% TFA, and solvent B was acetoni-
trile HPLC-R containing 10% water and 0.09% TFA (Biosolve,
Valkenswaard, The Netherlands).
1H NMR spectra were recorded at 400 MHz in DMSO-d6 at
25 °C or in CD3CN at -20 °C on a Bruker AM 400 MHz spec-
trometer using tetramethylsilane as internal standard at a
concentration of 10 mg/mL. The assignment of all resonances
and the determination of the conformation of the Tyr-ΨPro
peptide bond were based on DQF-COSY, HOHAHA, and
ROESY experiments. DQF-COSY experiments were done
using the phase cycle described by Derma et al.22 Two-
dimensional HOHAHA experiments23 were carried out using
MLEV-1724,25 and a mixing time of 50 ms. ROESY experi-
ments26 were performed using a mixing time of 200 ms. All
two-dimensional spectra were obtained using 4K data points
in the f2 domain and 512 points in the f1 domain. The data
were processed using SwaN-MR software.27 A zero-filling in
the f1 dimension and a square sine-bell window shifted by 90°
in both dimensions (0° for f2 in the case of the DQF-COSY)
were applied prior to two-dimensional Fourier transformation.
The protons were not assigned diastereoselectively. The as-
signment corresponds to the residue number followed by H
and the position in the side chain (R, â, γ, δ, ꢀ, ...). For ΨPro,
a nomenclature similar to that generally used for proline was
employed. In the case of dimethylpseudoproline the methyl
groups on the δ carbon were named Meδ1 and Meδ2. Coupling
constants are given in hertz. Mass spectra were obtained by
electron spray ionization (MS-ESI) on a Finnigan LC 710 mass
spectrometer.
Syn th esis of Th ia zolid in e-Con ta in in g Dip ep tid es. (A)
F m oc-Tyr (All)-F . Fmoc-Tyr(All)-OH (4.00 g, 8.98 mmol) and
pyridine (0.8 mL, 1 equiv) were dissolved in 80 mL of CH2Cl2.
Addition of cyanuric fluoride (6.5 mL, 8 equiv) resulted in a
white suspension. After completion of this reaction, the reac-
tion flask was cooled in an ice bath, and then water (50 mL)
was added dropwise to destroy the excess of activating reagent.
The suspension was filtered over Celite and the organic layer
dried with MgSO4. After evaporation of the solvent, the product
was obtained as a white powder (3.2 g, 80%). The purity of
the product was assessed by analytical HPLC after transfor-
mation of the fluoride to the methyl ester using MeOH/pyridine
(10 min): HPLC (C18, 0-100% B, 30 min) tR ) 29.1 min;
MS-ESI, (m/z) 444.2 [M + H]+.
(D) F m oc-Tyr (All)-Ser (ΨMe,Mep r o)-OBzl. Fmoc-Tyr(All)-
Ser-OBzl (2.9 g, 4.67 mmol) was dissolved in THF/CH2Cl2 (1:
1, v/v, 60 mL), and 2,2-dimethoxypropane (4.1 mL, 7.1 equiv)
was added to the solution. p-Toluene pyridinium sulfonic acid
(PPTS; 0.59 g, 0.5 equiv) was added, and the solution was then
heated under reflux for 16 h. After completion of the reaction,
all solvent was evaporated and the residue was taken up in
MeOH and purified by flash chromatography over silica
(CHCl3/MeOH/AcOH, 100:0.5:0.5). Pure, solid product (3.53 g,
70%) was obtained: HPLC (C18, 0-100% B, 30 min) tR ) 20.2
min; MS-ESI, (m/z) 661.2 [M + H]+.
(E ) F m oc-Tyr -Ser (ΨMe,Mep r o)-OBzl. Fmoc-Tyr(All)-Ser-
(ΨMe,Mepro)-OBzl (3.1 g, 4.67 mmol) was dissolved in a mixture
of 30 mL of CH2Cl2 and 9 mL of THF, and SiPhH3 (4.1 mL,
7.1 equiv) was added to the solution. The reaction mixture was
stirred under N2 for 10 min, and Pd(PPh3)4 was then added
still under N2. Deprotection was complete after 16 h of stirring
under nitrogen. After removal of all solvent, the remaining
black oil was purified by flash chromatography over silica
(B) Fm oc-Tyr (All)-Cys(ΨH,Hpr o)-OH. Fmoc-Tyr(All)-F (1.89
g, 4.25 mmol) and N-methylmorpholine (1.4 mL, 3 equiv) were
dissolved in 15 mL of DMF. After addition of Cys(ΨH,Hpro)-
OH (1.7 g, 4.78 mmol), the solution was stirred for 16 h. At
the end of the reaction the DMF was evaporated and the
residual was taken up in 3 mL of EtOAc. Purification of the
dipeptide was achieved by flash chromatography over silica
using EtOAc/MeOH/AcOH (100:10:0.5, v/v) as eluent. Three
hundred milligrams (13%) of pure Fmoc-Tyr(All)-Cys(ΨH,Hpro)-
OH was obtained: HPLC (C18, 0-100% B, 20 min) tR ) 12.32
min; MS-ESI, (m/z) 559.2 [M + H]+.
(MeOH) to afford 1.2 g (44%) of a white solid: HPLC (C18
,
0-100% B, 30 min) tR ) 15.7 min; MS-ESI, (m/z) 621.5 [M +
H]+.
(F ) F m oc-Tyr -Ser (ΨMe,Mep r o)-OH. Fmoc-Tyr-Ser(ΨMe,Me
-
pro)-OBzl (1.2 g, 1.93 mmol) was dissolved in 20 mL of MeOH,
and 250 mg of Pd/C catalyst and two drops of AcOH were
added to the solution under N2. The solution was then stirred

3902 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Keller et al.
for 2 h under an H2 atmosphere. After completion of the
reaction, the solution was filtered over Celite and the solvent
was evaporated in vacuo. Pure product (0.95 g, 93%) was
were resolved in both conformations and served as a basis for
the calculation of the cis/trans ratio.
(M) H-Tyr -Cys(ΨMe,Mep r o)-P h e-P h e-NH2 (9): HPLC (C18
,
10-100%, 20 min) tR ) 15.02 min; MS-ESI, (m/z) 618.6 [M +
obtained in solid form: HPLC (C18, 0-100% B, 30 min) tR
)
1
H]+; H NMR (DMSO-d6) one set of signals, attributed to the
7.8 min; MS-ESI, (m/z) 531.1 [M + H]+.
(G) Solid -P h a se P ep tid e Syn th esis. Sieber resin20 (0.26
g; 0.56 mmol/g loading) was successively washed for 10 min
with MeOH (10 mL), toluene (10 mL), and CH2Cl2 (10 mL)
before swelling in DMF (1 h). Couplings were carried out in
DMF, using Fmoc-amino acids (3 equiv), DIEA (5 equiv), and
PyBOP (3 equiv) as coupling agent. Coupling time was 2 h.
The ninhidrin test29 was used to check for completion of the
coupling reactions. Fmoc deprotection was performed with
piperidine (20% in DMF, 3 × 10 min). After assembly of the
C-terminal dipeptide H-Xaa-Yaa- (Xaa ) Phe, D-Phe; Yaa )
Phe, Pro) on the resin, coupling of the N-terminal dipeptide
(3 equiv) was carried out in DMF with PyBOP (230 mg, 3
equiv) as coupling agent in the presence of DIEA for 2 h. The
Tyr side chain in the dipeptides was protected with the allyl
group, except in the case of the 2-C-dimethyloxazolidine-
containing dipeptide, where the Tyr side chain was left
unprotected. The allyl group of the resin-bound tetrapeptides
was removed by reaction with Pd[P(Ph)3]4 (22.5 mg, 0.1 equiv)
and SiH3Ph (430 µL, 24 equiv) in CH2Cl2 under N2 for 15 min.
After removal of the final Fmoc group, peptides were cleaved
from the resin by treatment with 5% (v/v) TFA in CH2Cl2 (3 ×
10 min). To the resulting, slightly red solution was added 10
mL of toluene, and then all solvent was evaporated. Purifica-
tion of the tetrapeptides was achieved by reversed-phase
HPLC, using a gradient of 10-100% B (30 min).
cis conformation of the Tyr-ΨPro peptide bond, 7.60 (d, 1H,
J ) 8, 3 or 4HN), 7.52 (d, 1H, J ) 8, 4 or 3HN), 7.15-7.22 (m,
10H, 3Hδ+3Hꢀ+3Hú+4Hδ+4Hꢀ+4Hú), 7.08 (d, 2H, J ) 8.4,
1Hδ), d, 2H, J ) 7.3, 1Hꢀ), 6.67 (s, 1H, CONH2 syn), 6.05 (s,
1H, CONH2 anti), 4.64 (d×d, 1H, J ) 5.27, 1HR), 4.53 (d×d,
1H, J ) 5.32, 4HR), 4.01 (m, 1H, 3HR), 3.94 (d×d, 1H, J )
6.92, 2HR), 3.05 (m, 4H, 1Hâ+2Hâ), 2.86 (m, 4H, 3Hâ+4Hâ),
1.74 (s, 3H, 2Meδ1), 1.71 (s, 3H, 2Meδ2).
(N) H-Tyr -Ser (ΨH,Hp r o)-P h e-P h e-NH2 (10): HPLC (C18
,
0-100% B, 30 min) tR ) 16.2 min; MS-ESI, (m/z) 574.2 [M +
H]+; 1H NMR (CD3CN, -20 °C) trans/cis 30:70 (major isomer)
8.21 (3HN), 7.84 (4HN), 7.63 (1HN), 7.03 (1Hδ), 6.89 (CONH2),
6.78 (1Hꢀ), 6.36 (CONH2), 5.15 (2Hδ1), 4.77 (2Hδ2), 4.60 (4HR),
4.36 (3HR), 3.92 (1HR), 3.61 (2Hâ1), 3.55 (2Hâ2), 3.39 (2HR), 3.07
(4Hâ1), 3.02 (1Hâ), 2.86 (4Hâ2), 2.77 (3Hâ); (minor isomer) 7.41
(3HN), 7.35 (4HN), 7.28 (1HN), 7.01 (1Hδ), 6.75 (1Hꢀ), 6.63
(CONH2), 6.32 (CONH2), 4.98 (2Hδ1), 4.51 (4HR), 4.44 (3HR),
4.43 (2HR), 4.29 (2Hδ2), 4.13 (1HR), 4.06 (2Hâ1), 3.80 (2Hâ2), 3.13
(4Hâ1), 3.00 (3Hâ1), 2.94 (1Hâ), 2.91 (4Hâ2), 2.85 (3Hâ2); impor-
tant peaks in DMSO-d6 at 25 °C trans/cis 64:36 (major isomer)
5.20 (2Hδ1), 4.52 (2HR), 4.21 (1HR), 4.21 (2Hδ2), 4.14 (2Hâ1), 3.57
(2Hâ2), 2.85 (1Hâ); (minor isomer) 4.91 (2Hδ1), 4.76 (2Hδ2), 3.83
(2Hâ1), 3.69 (2HR), 3.68 (2Hâ2), 3.29 (1HR), 2.84 (1Hâ).
(O) H-Tyr -Ser (ΨMe,Mep r o)-P h e-P h e-NH2 (11): HPLC (C18,
0-100% B, 30 min) tR ) 17.1 min; MS-ESI, (m/z) 602.1 [M +
1
H]+; H NMR (DMSO-d6) one set of signals, attributed to the
(H) H-Tyr -Cys(ΨH,Hp r o)-D-P h e-P r o-NH2 (4): HPLC (C18
,
cis conformation of the Tyr-ΨPro peptide bond, 8.36 (d, 1H,
3HN), 8.20 (d, 1H, 4HN), 7.37 (s, 1H, CONH2), 7.15-7.30 (m,
10H, 3Hδ+3Hꢀ+3Hú+4Hδ+4Hꢀ+4Hú), 7.11 (s, 1H, CONH2),
6.92 (d, 2H, 1Hδ), 6.71 (d, 2H, 1Hꢀ), 4.62 (m, 1H, 3HR), 4.48
(m, 1H, 4HR), 3.81 (m, 2H, 2Hâ2+2HR), 3.73 (d×d, 1H, 2Hâ1),
3.07 (m, 1H, 1HR), 3.04 (m, 2H, 3Hâ2+4Hâ2), 2.83 (m, 2H, 1Hâ),
1.86 (m, 2H, 3Hâ1+4Hâ1), 1.50 (s, 3H, 2Meδ2), 1.34 (s, 3H,
2Meδ1); 1H NMR (CD3CN, -20 °C) 7.93 (1HN), 7.76 (4HN),
7.74 (3HN), 7.06 (1Hδ), 6.80 (1Hꢀ), 6.80 & 6.21 (CONH2), 4.59
(4HR), 4.39 (3HR), 3.89 (1HR), 3.68 (2Hâ), 3.52 (d, J ) 5.0, 2HR),
3.05 (4Hâ1), 3.01 (1Hâ), 3.01 (3Hâ1), 2.81 (3Hâ2), 2.77 (4Hâ2),
1.51 (s, 3H, 2Meδ1), 1.43 (s, 3H, 2Meδ2).
(P ) In Vitr o Bioa ssa ys a n d Op ioid Recep tor Bin d in g
Assa ys. The GPI30 and MVD31 bioassays were carried out as
reported in detail elsewhere.32,33 A log dose-response curve
was determined with [Leu5]enkephalin as standard for each
ileum and vas preparation, and IC50 values of the compounds
being tested were normalized according to a published proce-
dure.34
Opioid receptor binding studies were performed as described
in detail elsewhere.32 Binding affinities for µ and δ receptors
were determined by displacing, respectively, [3H]DAMGO
(Multiple Peptide Systems, San Diego, CA) and [3H]DSLET
(Multiple Peptide Systems) from rat brain membrane binding
sites, and κ opioid receptor affinities were measured by dis-
placement of [3H]U69,593 (Amersham) from guinea pig brain
membrane binding sites. Incubations were performed for 2 h
at 0 °C with [3H]DAMGO, [3H]DSLET, and [3H]U69,593 at
respective concentrations of 0.72, 0.78, and 0.80 nM. IC50
values were determined from log dose-displacement curves,
and Ki values were calculated from the obtained IC50 values
by means of the equation of Cheng and Prusoff,35 using values
of 1.3, 2.6, and 2.9 nM for the dissociation constants of [3H]-
DAMGO, [3H]DSLET, and [3H]U69,593, respectively.
10-100% B, 40 min) tR ) 16.93 min; MS-ESI, (m/z) 524.0 [M
+ H]+; 1H NMR (DMSO-d6) a detailed attribution of all signals
was not possible due to overlap of the signals of the cis and
trans ω-Tyr-ΨPro conformers. However, certain resonances
such as the ortho/meta protons of the phenyl ring of tyrosine
were nicely resolved in both conformations and served as a
basis for the calculation of the cis/trans ratio.
(I) H-Tyr -Cys(ΨMe,Mep r o)-D-P h e-P r o-NH2 (5): HPLC (C18,
10-100%, 20 min) tR ) 13.0 min; MS-ESI, (m/z) 568.6 [M +
1
H]+; H NMR (DMSO-d6) one set of signals, attributed to the
cis conformation of the Tyr-ΨPro peptide bond, 7.48 (d, 1H,
3HN), 7.27-7.35 (m, 5H, 3Hδ+Hꢀ+Hú), 7.12 (d, 2H, J ) 8.29,
1Hδ), 6.83 (d, 2H, J ) 8.33, 1Hꢀ), 6.75 (s, 1H, CONH2 syn),
6.53 (s, CONH2 anti), 4.67 (d×d, 1H, J ) 7.56/7.58, 3HR), 4.18
(m,1H,2HR),3.64 (m,2H,1HR+4HR),3.00 (m,6H,4Hâ+4Hδ+2Hâ),
2.95 (m, 2H, 1Hâ), 2.93 (d×d, 2H, 3Hâ), 1.8 (m, 2H, 4Hγ), 1.76
(s, 3H, 2Meδ1), 1.68 (s, 3H, 2Meδ2).
(J ) H-Tyr -Ser (ΨH,Hp r o)-D-P h e-P r o-NH2 (6): HPLC (C18
,
0-100% B, 30 min) tR ) 15.5 min; MS-ESI, (m/z) 524.0 [M +
1
H]+; H NMR (DMSO-d6) a detailed attribution of all signals
was not possible due to overlap of the signals of the cis and
trans ω-Tyr-ΨPro conformers. However, certain resonances
such as the ortho/meta protons of the phenyl ring of tyrosine
were well resolved in both conformations and served as a basis
for the calculation of the cis/trans ratio.
(K) H-Tyr -Ser (ΨMe,Mep r o)-D-P h e-P r o-NH 2 (7): HPLC
(C18, 0-100% B, 30 min) tR ) 16.9 min; MS-ESI, (m/z) 551.9
1
[M + H]+; H NMR (CD3CN) one set of signals, attributed to
the cis conformation of the Tyr-ΨPro peptide bond, 7.47 (d,
1H, 3HN), 7.27-7.35 (m, 5H, 3Hδ+Hꢀ+Hú), 7.14 (d, 2H, 1Hδ),
6.83 (d, 2H, 1Hꢀ), 6.78 (large resonance, 2H, CONH2), 4.68 (q,
1H, 3HR), 4.19 (d×d, 1H, 4HR), 3.92 (d×d, 1H, 2Hâ1), 3.81 (m,
2H, 2HR + 2Hâ2), 3.68 (m, 1H, 1HR), 3.65 (m, 1H, 4Hδ), 3.14
(m, 1H, 4Hâ1), 2.98 (m, 1H, 1Hâ), 2.01 (m, 1H, 4Hâ2), 1.86 (m,
2H, 4Hâ1+4Hγ2), 1.63 (m, 1H, 4Hγ1), 1.55 (s, 3H, 2Meδ1), 1.41
(s, 3H, 2Meδ2).
Ack n ow led gm en t. This work was supported by the
Swiss National Science Foundation (grant to M.M.) and
the Medical Research Council of Canada (Grant MT-
5655 to P.W.S.).
(L) H-Tyr -Cys(ΨH ,H p r o)-P h e-P h e-NH2 (8): HPLC (C18
,
0-100% B, 30 min) tR ) 15.5 min; MS-ESI, (m/z) 590.0 [M +
1
H]+; H NMR (DMSO-d6) a detailed attribution of all signals
Refer en ces
was not possible due to overlap of the signals of the cis and
trans ω-Tyr-ΨPro conformers. However, certain resonances
such as the ortho/meta protons of the phenyl ring of tyrosine
(1) Symbols and abbreviations are in accordance with recommenda-
tions of the IUPAC-IUB J oint Commission on Biochemical
Nomenclature: Nomenclature and Symbolism for Amino Acids

Analogues of Morphiceptin and Endomorphin-2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3903
and Peptides Biochem. J . 1984, 219, 345-373. The other
abbreviations are as follows: All, allyl; DAMGO, H-Tyr-D-Ala-
Gly-NRMePhe-Gly-ol; DCC, N,N′-dicyclohexylcarbodiimide; DIEA,
diisopropylethylamine; DSLET, H-Tyr-D-Ser-Gly-Phe-Leu-Thr-
OH; Fmoc, 9-fluorenylmethyloxycarbonyl; GPI, guinea pig ileum;
MS-ESI, electron spray ionization mass spectrometry; MVD,
mouse vas deferens; PPTS, pyridinium p-toluenesulfonate; Py-
BOP, benzotriazol-1-yloxytris(pyrrolidinophosphonium-hexafluo-
rophosphate); TFA, tetrafluoroacetic acid; THF, tetrahydrofuran;
U69,593, (5R,7R,8â)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-
[4.5]dec-8-yl]benzeneacetamide.
(18) Wittelsberger, A.; Keller, M.; Scarpellino, L.; Patiny, L.; Acha-
Orbea, H.; Mutter, M. Pseudoprolines: Targeting a Cis Confor-
mation in a Mimetic of the gp120 Loop of HIV-1. Angew. Chem.,
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(20) Sieber, P. A New Acid-labile Anchor Group for the Solid-Phase
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(21) Yamazaki, T.; Pro¨bstl, A.; Schiller, P. W.; Goodman, M. Biological
and Conformational Studies of [Val4]morphiceptin and [D-Val4]-
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Carboxylic Acid as a Peptidomimetic for Proline. Int. J . Pept.
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(22) Derome, A.; Williamson, M. Rapid-Pulsing Artifacts in Double-
Quantum-Filtered COSY. J . Magn. Reson. 1990, 88, 177-185.
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Via Two-Dimensional Homonuclear Hartmann-Hahn Spectros-
copy. J . Am. Chem. Soc. 1985, 107, 2820-2821.
(24) Bax, A.; Davis, D. G. MLEV-17-Based Two-Dimensional Homo-
nuclear Magnetization Transfer Spectroscopy. J . Magn. Reson.
1985, 65, 355-360.
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