Conformational analysis and mu-opioid receptor affinity of short peptides, endomorphin models in a low polarity solvent.

Article abstract of DOI:10.1039/b306161m

Peptide carbamates containing the sequence H-Pro-Trp-PheNH2 showed in CDCl3 restricted conformations stabilized by the presence of a gamma-turn. To test the reliability of the peptides as endomorphin conformational models, we measured the affinities for mu-receptors labelled with [3H]-DAMGO. In particular, Cbz-Pro-Trp-PheNH2 displayed a nanomolar affinity.

Full text of DOI:10.1039/b306161m

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Conformational analysis and ꢀ-opioid receptor affinity of short
peptides, endomorphin models in a low polarity solvent
a
a
a
b
Giuliana Cardillo,* Luca Gentilucci,* Alessandra Tolomelli, Ahmed R. Qasem,
b
b
Santi Spampinato* and Maria Calienni
a
Dipartimento di Chimica “G. Ciamician”, C.S.F.M., Università di Bologna, via Selmi 2,
4
0126-Bologna, Italy. E-mail: cardillo@ciam.unibo.it; gentil@ciam.unibo.it;
Fax: ϩ39 0512099456
b
Dipartimento di Farmacologia, Università di Bologna, via Irnerio 48, 40126-Bologna, Italy.
E-mail: spampi@biocfarm.unibo.it
Received 30th May 2003, Accepted 10th July 2003
First published as an Advance Article on the web 28th July 2003
Peptide carbamates containing the sequence H-Pro-Trp-PheNH showed in CDCl restricted conformations
2
3
stabilized by the presence of a γ-turn. To test the reliability of the peptides as endomorphin conformational models,
3
we measured the affinities for µ-receptors labelled with [ H]-DAMGO. In particular, Cbz-Pro-Trp-PheNH displayed
2
a nanomolar affinity.
Introduction
benzyl carbamate, was synthesized. We also studied the effect
of the introduction of a glycinol residue at the C-terminus in
Endomorphin-1 (H-Tyr-Pro-Trp-PheNH ) and endomorphin-2
2
compound
3 (Boc-Pro-Trp-Phe-Glyol), the same residue
(
H-Tyr-Pro-Phe-PheNH ) are endogenous opioid peptides
11
2
present in DAMGO (Tyr--Ala-Gly-N-methyl-Phe-Gly-ol),
a
1
2
with high affinity for the µ-receptor. Their discovery in the
mammalian brain encouraged the application of natural and
synthetic peptides as analgesics instead of morphine.
prototypic µ-opioid agonist. To test the reliability of the peptide
analogues as endomorphin-1 models, we evaluated the affinity
towards µ-opioid receptors.
2,3
They both show in the second position the presence of
proline, which is known to play a key role in the formation
of secondary structures in peptides. Indeed, the cis–trans con-
formational equilibrium of the peptide bond preceding the
prolyl residue in solution is an important process in protein
Finally, the influence on both conformation and affinity of
the carbamate substituent was investigated by comparison
α
of 1–3 with the N -benzyl derivative 4 (PhCH -Pro-Trp-
2
PheNH ), which maintains an aromatic hydrophobic group in
2
the N-terminal region, but does not present a carbonyl moiety
to be involved in a hydrogen bond.
4
folding. This equilibrium strongly depends on the amino acid
sequence and the solvent.
The study of structure–activity relationships of µ-receptor
agonists and antagonists is the subject of increasing interest, as
it could clarify the structural and conformational require-
Results and discussion
Peptides 1, 2, 3, and endomorphin-1, have been synthesized in
solution following the conventional Boc chemistry, using
EDCI/HOBt as condensing agents. Boc deprotection with
trifluoroacetic acid (TFA) in CH Cl gave TFA peptide salts,
which were used without purification. After each coupling,
5
ments for ligand–receptor interaction. Several papers reported
1
2
investigations on the bioactive conformation of endomorphins
and endomorphin derivatives. On the basis of spectroscopic
analysis in polar solvents such as DMSO or water, or in
membrane-mimetic environments, such as SDS micelles or
AOT reverse micelles, predominant reverse conformations have
2
2
peptides were purified by flash-chromatography over silica-gel.
1
The H NMR spectrum of 1, 2, and 3 in CDCl solution
3
6
13
been proposed, not revealing the presence of intramolecular
(0.01 M) resulted as a sharp set of unique resonances.
hydrogen-bonds.
As a part of our ongoing interest in this field, we have
investigated the possibility that tripeptide carbamates, designed
as endomorphin analogues, could assume a compact bioactive
conformation stabilized by the presence of a hydrogen bond.
Herein we report the synthesis of peptide 1 (Cbz-Pro-Trp-
Strong evidence for the intramolecular hydrogen-bond stab-
ilization in chloroform was provided by variable temperature
(VT) NMR, and IR experiments.
7
The unambiguous assignment of the resonance sets of Phe
and Trp in each compound was performed by HMBC-NMR
analysis (heteronuclear multiple bond correlation). The
correlation observed between Hα and Hβ with the quaternary
indole or with the quaternary phenyl carbons, allowed
PheNH ), where the tyrosine present in endomorphin-1 was
2
changed with a benzyl carbamate group. This functionality
was introduced with the aim to mimic the aromatic group of
1
attribution of the side chain H-NMR signals to Trp or Phe,
tyrosine and to increase the peptide solubility in CDCl . We
respectively.
3
supposed that this non-competitive solvent could be considered
a suitable environment to simulate the hydrophobic core of a
The preferential conformation assumed by 1 in CDCl was
analyzed by IR, CD and H NMR spectroscopy.
3
1
8
receptor. It is expected that folded structures stabilized by
The IR absorption spectrum of 1 in CDCl (0.01 M) showed
3
9
Ϫ1
Ϫ1
hydrogen bonds may be favored in this apolar solvent. Our
comparable intense bands at 3479 cm , at 3384 cm and at
3325 cm in the NH stretching region. The first one is clearly
Ϫ1
purpose was to verify if a compact structure, stabilized by
a hydrogen bond, could interact with the opioid receptor,
associated with a non hydrogen-bonded group, while the other
two bands can be ascribed to NH amide protons hydrogen-
10
although lacking a cationic amine and a phenolic function,
9d,14
which are commonly considered necessary to manifest bio-
logical activity through interaction with this kind of receptor.
In order to evaluate the role of the Cbz group, compound 2
bonded in a folded conformation (Fig. 1).
The above con-
clusion is supported by the spectrum in the C᎐O stretching
᎐
region, which showed a smooth, broad band at 1692–1672
Ϫ1
(
Boc-Pro-Trp-PheNH ), with tert-butyl carbamate replacing
cm , consisting of a non hydrogen-bonded amide and a
2
3
010
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 1 0 – 3 0 1 4
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3

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the addition of DMSO-d (0 to 2%, for NH-Phe ∆δ = Ϫ0.003
6
ppm, for NH-Trp ∆δ = Ϫ0.014 ppm; 2 to 5%, for NH-Phe
9d,19
∆
δ = 0.006 ppm, for NH-Trp ∆δ = 0.039 ppm).
Finally, DPFG-NOE (Double Pulse Field Gradient Nuclear
Overhauser Effect) experiments showed strong signals due to
the proximity of αNH-Trp and NH-Phe, αNH-Trp and Hα-Pro
and a signal of moderate intensity between Hα-Pro and
NH-Phe. Furthermore, a signal of moderate intensity was
recorded between αNH-Trp and the hydrogen on C2 of the
indole heterocycle.
These data provided evidence that αNH-Trp and NH-Phe in
CDCl are involved in a seven-membered ring γ-turn and a
3
9d
ten-membered ring β-turn, respectively, in equilibrium.
A
comprehensive evaluation of the spectroscopic data reported
above indicates for peptide 1 a folded conformation, as reported
in Fig. 1.
Fig. 1 NH region of the infrared absorption spectrum of peptide 1,
and conformation in CDCl3.
In an almost similar way, compound 2 (0.01 M solution in
urethane carbonyl. The noticeable lowering of the frequency
CDCl ) showed in the IR absorption spectrum an intense band
of the C᎐O stretching mode of the urethane group is an
3
᎐
Ϫ1
at 3477 cm , and a comparatively less intense absorbance from
unequivocal point in favor of the involvement of this carbonyl
Ϫ1
14b
3390 to 3323 cm , in the hydrogen-bonded NH region (Fig. 3).
in the intramolecular bond.
The CD spectrum of 1 measured in CH OH could be
3
indicative of a reverse turn, for the presence of the typical
15
negative band at 220 nm; however, no definitive conclusions
could be deduced for the conformation of 1 in CDCl , for the
3
different behaviour of short and flexible peptides in the differ-
ent solvents.
1
The down field H NMR chemical shifts of αNH-Trp (6.63
ppm) and NH-Phe (6.98 ppm) suggest their involvement in
16
13
intramolecular hydrogen bonds. Furthermore, the C chem-
ical shifts of C(β) and C(γ) of the Pro residue are very sensitive
17
to the conformation of the preceding peptide bond. A differ-
ence of 4–6 ppm is indicative of a trans conformation while a
difference of 8-10 ppm is expected for a cis conformation.
Peptide 1 showed a difference of 5.2 ppm between C(β) and
C(γ) of Pro, confirming a trans conformation of the Pro-amide
bond.
Fig. 3 NH region of the infrared absorption spectrum of peptide 2,
and conformation in CDCl3.
The VT (variable temperature) NMR analysis was in agree-
14a,18
ment with the formation of hydrogen bonds.
In a 0.01 M
1
The H NMR spectrum of 2 in CDCl displayed the same
3
solution in CDCl , over the range of 296–326 K, both NH-Phe
3
trend as 1, and exhibited a set of unique signals. The resonance
of αNH-Trp was scarcely sensitive to increasing temperature,
showing a strong hydrogen bond involvement (Fig. 4, ∆δ/∆T =
and αNH-Trp resonances were scarcely sensitive to increasing
temperature, typical behaviour of protons involved in hydrogen
Ϫ1
bonds (Fig. 2, for NH-Phe ∆δ/∆T = 0.45 ppb K , for NH-Trp
Ϫ1
Ϫ1
Ϫ0.5 ppb K ), while NH-Phe was more sensitive, indicating a
∆
δ/∆T = Ϫ0.55 ppb K ). In contrast, the resonances of the two
Ϫ1
not completely bonded proton (Fig. 4, ∆δ/∆T = Ϫ2.4 ppb K ).
protons of the primary amide group were strongly sensitive
Ϫ1
Also in this case NOE experiments showed, besides the obvious
signals, a strong interaction of αNH-Trp and NH-Phe with
Hα-Pro (Fig. 3).
(
Fig. 2, ∆δ/∆T around Ϫ5 ppb K ), indicating that they do not
participate in hydrogen bonds.
Fig. 2 VT NMR data for 1 in CDCl solution.
3
Fig. 4 VT NMR data for 2 in CDCl solution.
3
Further clues as to the participation of αNH-Trp and
The last compound 3 showed a similar behaviour in solution
NH-Phe in hydrogen bonds were obtained with the exam-
ination of the dependence of chemical shift of the H NMR
as 2. The IR absorption spectrum of 3 in CDCl (0.01 M)
3
1
confirmed the formation of a compact structure, exhibiting a
Ϫ1
resonances in CDCl upon the addition of a small amount of
narrow absorbance at 3475 cm , a more intense broad band
3
Ϫ1
Ϫ1
DMSO-d as competitive solvent. The NH-Phe and αNH-Trp
at 3385 cm and a weak third broad signal around 3350 cm
6
signals showed chemical shifts which were scarcely modified by
(Fig. 5). Under identical conditions the same peaks were present
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 1 0 – 3 0 1 4
3011

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Table 1 Affinities and Hill slopes of ligands 1–4, DAMGO, and
3
endomorphin-1 for [ H]-DAMGO binding sites in rat brain membranes
Compounds
K /nM
i
IC /nM
50
nH
DAMGO
Endomorphin-1
1
2
3
4
1.64 (±0.33)
0.14 (±0.05)
3.44 (±0.11)
250 (±13)
> 10
200 (±15)
9.89 (±0.67)
4.80 (±0.21)
103 (±10)
500 (±50)
nd
0.9 (±0.1)
0.8 (±0.1)
0.6 (±0.2)
0.7 (±0.2)
nd
1.05 (±0.1)
3
13800 (±200)
nd = not determined. Means ± S.E. of three experiments performed in
triplicate.
Fig. 5 NH region of the infrared absorption spectrum of peptide 3,
and conformation in CDCl3.
under the usual conditions. The unambiguous assignment of
the resonance sets was performed by HMBC-NMR analysis.
As could be expected, on the basis of VT NMR, no hydrogen
bond was deduced for 4. A series of NOE experiments
indicated a conformation which strongly differed from that
of peptides 1–3. Besides the obvious signals, a strong NOE
in the Glyol O-acetylated derivative of 3, confirming that none
of the above peaks could be attributed to the OH stretch.
The VT (variable temperature) NMR analysis was in agree-
ment with the formation of a hydrogen bond. The chemical
α
shifts of αNH-Trp showed a small temperature dependence
between a proline N -benzylic proton and a Trp methylene
Ϫ1
(
∆
Fig. 6, ∆δ/∆T = 0.64 ppb K ), while NH-Phe (Fig. 4,
proton allowed placement of the indolyl group in proximity to
Ϫ1
α
δ/∆T = Ϫ1.9 ppb K ), and NH-Glyol (Fig. 6, ∆δ/∆T = Ϫ1.8
the N -benzylic substituent of proline.
Ϫ1
ppb K ), were more sensitive to increasing temperature. The
latter amide protons participate in hydrogen bonds more
Since the information obtained by NMR analysis was not
sufficient to determine the preferred conformation assumed by
4, we calculated its more stable geometry (Fig. 7) by means of
20
weakly than those involving NH-Trp. In addition, the narrow
IR absorbance at 3475 cm could be attributed to the non
hydrogen-bonded amide NH-Glyol group.
Ϫ1
22
molecular mechanics computations. The minimum-energy
conformation compatible with NOEs was calculated by means
of AMBER minimization of a conformation set generated by a
Monte Carlo procedure, introducing the estimated distances
furnished by NOEs as restraints.
Fig. 6 Chemical shift data for peptide 3 as a function of temperature
in CDCl3.
Fig. 7 Conformation of 4 in CDCl solution.
3
To evaluate if the carbamate-peptides might be suitable
endomorphin-1 models for conformational analysis, we tested
the biological relevance of 1, 2, 3 and 4 and we compared their
affinities for µ-opioid receptors with that of endomorphin-1
and DAMGO.
The NH-Phe and NH-Trp signals showed chemical shifts
which were scarcely modified by the addition of a small amount
of a competitive solvent (DMSO, 0 to 2%, for NH-Phe ∆δ =
Ϫ0.056 ppm, for NH-Trp ∆δ = Ϫ0.036 ppm; 2 to 5%, for
NH-Phe ∆δ = 0.031 ppm, for NH-Trp ∆δ = 0.075 ppm), while
the NH-Glyol signal was more sensitive (DMSO, 0 to 2%, ∆δ =
Ϫ0.095 ppm; 2 to 5%, ∆δ = 0.080 ppm).
3
The affinities for µ-receptors labelled with [ H]-DAMGO
were measured through binding assays performed on rat
7c,23
brain membranes (Table 1).
The K values measured for
i
Further useful information was deduced from NOE experi-
ments, that showed a strong interaction between αNH-Trp
and Hα-Pro and a medium interaction between NH-Phe and
Hα-Pro (Fig. 5).
DAMGO and for endomorphin-1 were in agreement with the
literature.
The results in Table 1 showed that, while the Cbz-tripeptide 1
1,10,11
had a K value in the nanomolar scale, peptides 2 and 3 main-
i
All these data collectively indicate for compounds 2 and 3
tained only a poor affinity for µ-receptors, even though the
three peptides displayed very similar conformations. These
results suggest that the substitution of Tyr for Cbz group gave a
peptide which still retained a certain ability to bind receptors.
The low affinity measured for 2 and 3 could be attributed to the
presence of the bulky tert-butyl group in place of the aromatic
substituent which is present in endomorphin-1 and in 1.
The affinity of 4 for µ-opioid receptors, measured under the
same conditions as reported for 1–3, is rather poor (Table 1).
Since 4 carries an aromatic substituent at the proline, the low
affinity measured could be attributed to the different conform-
ation adopted with respect to 1–3, which can be ascribed for the
most part to the absence of the intramolecular hydrogen-bonds.
a strong γ-turn between αNH-Trp and carbamate C᎐O, and
᎐
a weaker β-turn between NH-Phe and carbamate C᎐O, in
᎐
equilibrium. Therefore, it can be deduced that the conformation
of 2 and 3 shows a stronger preference for the γ-turn with
respect to the β-turn.
Apparently, trimers 2 and 3, containing the bulky t-butyl
carbamate, showed a less compact structure than benzyl
carbamate 1.
Finally, peptide 4 (PhCH -Pro-Trp-PheNH ) was designed
2
2
to evaluate the importance of the effect of carbamate carbonyl
α
on conformations. It was prepared by coupling N -benzyl-
21
proline and the TFA salt of the dipeptide H-Trp-PheNH2
3
012
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 1 0 – 3 0 1 4

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Conclusion
(dimethylamino) propyl]-3-ethylcarbodiimide (1.5 equiv.), in a
: 1 mixture of CHCl and DMF at 0 ЊC and under nitrogen
atmosphere. After 8 h, the solvent was evaporated at reduced
pressure, and the residue was dissolved in EtOAc. The solution
9
3
In conclusion, we have prepared the carbamate-tripeptides 1, 2,
α
3
and N -benzyl tripeptide 4, and we have examined their
conformational behaviour in CDCl . By way of the analysis
3
was washed with 0.5 M HCl, sat. NaHCO , and brine. The
3
reported above, we observed for 1, 2, and 3, a similar preferred
conformation, showing the Cbz or Boc group close to the Phe
aromatic substituent. Peptide 4 showed a completely different
conformation, mainly because of the absence of the intra-
molecular hydrogen bond. In compounds 1, 2 and 3, a trans
Pro-amide bond configuration was stabilized by the formation
of a γ-turn involving αTrp-NH and Boc or Cbz carbonyl. For
the Cbz peptide 1, a β-turn involving Phe-NH and the same
carbonyl was also possible.
Interestingly, peptide 1 showed a good affinity for the
µ-receptors, giving the opportunity to probe the biologically
active conformation of peptides that potentially adopt a reverse
turn conformation.
organic layer was dried over Na SO , and solvent was removed
2
4
at reduced pressure. Peptides were obtained pure by flash-
chromatography over silica-gel (EtOAc : MeOH 96 : 4) with
yields from 60 to 90%.
N-tert-Butyloxycarbonyl group deprotection was performed
by treatment with 30% TFA in CH Cl at r.t. After 45 min the
2
2
solvent was evaporated at reduced pressure and the resulting
TFA peptide salt, obtained in quantitative yield, was used
without purification for the next coupling.
Cbz-Pro-Trp-PheNH (1). IR (CDCl ) ν 3695, 3595, 3479,
2
3
3
1
1
384, 3325, 3155, 1818, 1792, 1680 br., 1460, 1387, 1301, 1255,
Ϫ1
1
215, 1162, 1096 cm ; H-NMR (CDCl ) δ 1.60–1.80 (m, 2H),
3
.85–2.20 (m, 2H), 2.86 (dd, J = 11.1, 14.7 Hz, 1H), 2.85–3.07
(
(
m, 1H), 3.13 (dd, J = 6.6, 14.4 Hz, 1H), 3.18–3.27 (m, 2H), 3.44
dd, J = 4.0, 15.0 Hz, 1H), 4.03 (dd, J = 4.8, 9.3 Hz, 1H),
Experimental section
General remarks
4.47–4.55 (m, 1H), 4.56 (d, J = 12.9 Hz, 1H), 4.88–4.98 (m, 1H),
5
.01 (d, J = 12.9 Hz, 1H), 5.45 (s, 1H), 6.30 (s, 1H), 6.63
Unless stated otherwise, chemicals were obtained from com-
mercial sources and used without further purification. CH Cl
was distilled from P O . Flash chromatography was performed
on Merck silica gel 60 (230–400 mesh), and solvents were
(
(
d, J = 6.3 Hz), 6.87 (s, 1H), 6.98 (d, J = 8.1 Hz, 1H), 7.09–7.48
2
2
13
m, 14H), 7.69 (s, 1H); C-NMR (CDCl ) δ 24.8, 29.6, 36.7,
3
2
5
4
1
1
6.8, 53.7, 55.7, 67.3, 111.5, 116.4, 116.7, 116.8, 117.1, 120.0,
22.4, 126.4, 127.1, 128.3, 128.6, 129.1, 135.7, 138.3, 158.1,
simply distilled. NMR Spectra were recorded with a Mercury
65.2, 171.3, 173.9; FAB-MS [Mϩ1]: 580.4; calcd. for 1: 579.2.
1
spectrometer (Oxford magnet) at 400 ( H NMR) and at 75
20
[α]D = Ϫ86.9 (c 0.4, CHCl ).
13
3
MHz ( C NMR). Chemical shifts are reported as δ values
1
relative to the solvent peak of CDCl set at δ = 7.27 ( H NMR)
3
Boc-Pro-Trp-PheNH (2). IR (CDCl ) ν 3477, 3390, 3323,
158, 1816, 1793, 1671, 1470, 1382, 1095 cm ; H-NMR
13
2
3
or δ = 77.0 ( C NMR). Infrared absorptions were recorded
with an FT-IR Nicolet 210 spectrophotometer. Optical activity
measurements were performed with a Perkin-Elmer 343 polar-
Ϫ1
1
3
(
CDCl ) δ 1.40 (s, 9H), 1.60–1.80 (m, 2H), 1.87–2.20 (m, 2H),
3
2
1
3
.78 (dd, J = 11.1, 14.7 Hz, 1H), 2.95–3.05 (m, 1H), (dd, J = 6.6,
4.4 Hz, 1H), 3.21–3.35 (m, 2H), 3.41 (dd, J = 4.0, 14.7 Hz, 1H),
.98 (dd, J = 5.0, 9.3 Hz, 1H), 4.54 (q, J = 5.1 Hz, 1H), 5.00
imeter. DPFG-NOE spectra were recorded in CDCl at r.t.
3
HMBC spectra were recorded in CDCl3 at r.t., selecting a
spin coupling constant of 8 Hz. The FAB-mass instrument
employed was a Micromass ZMD spectrometer equipped with
single quadrupole analyzer and a Z-spray ionspray source
outfitted with a 50-mm deactivated fused Si capillary connected
to a Harvard Apparatus pump 11 for sample injection. Data
acquisition and spectra analysis were conducted with Masslynx
(
m, 1H), 5.44 (s, 1H), 6.46 (s, 1H,), 6.72 (d, J = 6.6 Hz, 1H), 6.82
(
s, 1H), 6.97 (d, J = 8.0 Hz, 1H), 7.10–7.50 (m, 9H), 8.11 (s, 1H);
13
C-NMR (CDCl ) δ 24.5, 27.5, 28.1, 29.7, 36.6, 47.1, 55.8, 61.0,
3
8
1
0.7, 109.5, 111.7, 117.7, 119.9, 122.3, 123.7, 126.8, 128.2,
28.8, 135.9, 137.8, 156.2, 173.7, 173.8. FAB-MS [Mϩ1]: 546.6;
20
calcd. for 2: 545.3. [α]D = Ϫ86.4 (c 0.3, CHCl ).
3
3
.3 software running on a Digital Equipment Corp. Personal
Computer. Nitrogen was used both as desolvation and
nebulizer gas. Desolvation temperature was set at 200 ЊC and
capillary voltage at 3.0 kV. Analytical HPLC was performed on
an HP Series 1100, with an HP Hypersil ODS column (4.6-µm
particle size, 100 Å pore diameter, 250 mm), DAD 215.8 nm.
Homogenates were centrifuged in Beckman J6B and Beckman
J2-21 centrifuges. Radioactivity was measured by liquid scintil-
Boc-Pro-Trp-Phe-Glyol (3). IR (CDCl ) ν 3689, 3602, 3475,
385, 3350 br., 1818, 1800, 1680, 1534, 1467 cm ; H-NMR
3
Ϫ1
1
3
(
2
(
(
CDCl ) δ 1.33 (s, 9H), 1.58–1.94 (m, 4H), 2.15–2.23 (m, 1H),
3
.70 (dd, J = 11.4, 14.0 Hz, 1H), 3.08–3.18 (m, 2H), 3.18–3.34
m, 2H), 3.59 (dd, J = 3.2, 14.0 Hz, 1H), 3.62–3.73 (m, 4H), 3.99
dd, J = 4.8, 8.8 Hz, 1H), 4.56 (q, J = 4.6 Hz, 1H), 5.18 (m, 1H),
.22 (s, 1H), 6.60 (d, J = 6.8 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H),
.00 (t, J = 4.4 Hz, 1H), 7.11–7.56 (m, 5H), 7.81 (s, 1H);
6
α
lation spectrometry using a Beckman apparatus. N -Benzyl-
proline was prepared according to the literature (see below).
7
13
C-NMR (CDCl ) δ 24.5, 25.2, 28.1, 29.9, 36.9, 43.2, 47.1, 52.7,
3
5
1
1
6.2, 61.2, 61.5, 81.1, 108,1, 111.8, 116.8, 120.1, 122.3, 123.8,
26.4, 127.5, 128.2, 128.8, 135.8, 138.0, 155.7, 171.1, 171.3,
74.5; FAB-MS [Mϩ1]: 592.2; calcd. for 3: 591.3. [α]D = Ϫ111
ꢁ
21
N -Benzylproline
20
A suspension of proline (0.87 g, 7.56 mmol) and KOH (1.27 g,
7.0 mmol) in isopropanol (15 mL) was heated at 40 ЊC while
(
c 0.4, CHCl ).
2
3
stirring. After 10 min, BnCl (1.0 mL, 8.7 mmol) was added, and
the mixture was stirred at 40 ЊC for 8 h. The mixture was
1
PhCH -Pro-Trp-PheNH (4). H-NMR (CDCl ) δ 1.49–1.62
2
2
3
(
m, 2H), 1.89–2.12 (m, 2H), 2.45–2.55 (m, 1H), 2.64 (t, J = 8.0
Hz, 1H), 2.77 (dd, J = 7.2, 10.0 Hz, 1H), 2.85 (dd, J = 6.4, 10.0
Hz, 1H), 2.98 (dd, J = 4.2, 10.2 Hz, 1H), 3.00–3.12 (m, 2H), 3.14
d, J = 12.9 Hz, 1H), 3.51 (d, J = 12.9 Hz, 1H), 4.50 (q, J = 6.9
Hz, 1H), 4.56 (q, J = 6.3 Hz, 1H), 5.64 (s, 1H), 6.19 (s, 1H), 6.63
d, J = 7.8 Hz, 1H), 6.86–7.18 (m, 11H), 7.29 (d, J = 7.8 Hz, 2H),
filtered, and the solid was washed twice with CHCl , twice with
3
acetone, and the filtrate was concentrated at reduced pressure.
The resulting solid was re-crystallized from MeOH and ether,
giving benzylproline (1.40 g, 90%) as a white solid. Spectro-
(
21
scopic analysis was in agreement with the literature.
(
7
.53 (d, J = 7.8 Hz, 2H), 7.94 (d, J = 7.5 Hz, 1H), 9.42 (s, 1H);
Synthesis and characterization of the peptides
1
3
C-NMR (CDCl ) δ 24.7, 28.8, 30.7, 39.0, 54.2, 54.7, 55.5, 60.5,
3
As a general procedure, the peptide coupling was performed by
stirring overnight the TFA salt of the amino amide, the Boc or
Cbz protected amino acid (1.2 equiv.), triethylamine (3 equiv.),
68.1, 110.2, 112.4, 119.9, 122.5, 124.8, 126.0, 127.6, 128.1,
128.9, 129.3, 130.2, 137.9, 138.2, 139.5, 173.1, 175.4, 176.8.
FAB-MS [Mϩ1]: 536.4; calcd. for 4: 535.3. [α]D = Ϫ63 (c 0.4,
20
1
-hydroxy-1H-benzotriazole (1.5 equiv.), the HCl salt of 1-[3-
CHCl ).
3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 1 0 – 3 0 1 4
3013

View Article Online
L. Gentilucci, P. Melchiorre and S. Spampinato, Bioorg. Med. Chem.
Lett., 2000, 10, 2755–2758; (c) G. Cardillo, L. Gentilucci,
A. Tolomelli, M. Calienni, A. R. Qasem and S. Spampinato, Org.
Biomol. Chem., 2003, 1, 1498–1502; (d ) M. Royo, F. Sgarzi,
J. Ferrara-Sinfreu, F. Albericio, L. Gentilucci and G. Cardillo,
J. Pept. Sci., 2002, 8(suppl.), 180.
Acknowledgements
We thank ISOF-CNR, MURST (Cofin 2002 and 60%), and the
University of Bologna (funds for selected topics ЉSintesi e
Caratterizzazione di Biomolecole per la Salute UmanaЉ) for
financial support.
8
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c) D. Picone, A. D’Ursi, A. Motta, T. Tancredi and P. A. Temussi,
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23 [ H]-DAMGO as µ-specific radioligand, 1 nM, specific activity 64 Ci
Ϫ1
Ϫ1
7
mmol , K = 0.485 nM, B
= 48 fmol mg protein; incubation
d
max
buffer: 50 mM TRIS, 0.1% BSA pH 7.4 at 4 ЊC.
3
014
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 0 1 0 – 3 0 1 4